Patent Publication Number: US-2022235066-A1

Title: Oxabicycloheptane prodrugs

Description:
This application claims the benefit of U.S. Provisional Application No. 62/162,501, filed May 15, 2015, the contents of which are hereby incorporated by reference. 
    
    
     Throughout this application various publications are referenced. The disclosures of these documents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. 
     BACKGROUND OF THE INVENTION 
     Retinoids, metabolites of vitamin A, have been examined therapeutically against a variety of tumors, including gliomas (Yung et al. 1996). Nuclear receptor co-repressor (N-CoR) is closely associated with the retinoid receptor and is released upon ligand binding to the receptor (Bastien et al. 2004). By preventing the action of protein phosphatase-1 and protein phosphatase-2A (PP2A), anti-phosphatases increase the phosphorylated form of N-CoR and promote its subsequent cytoplasmic translocation (Hermanson et al. 2002). 
     The phosphatase inhibitor, cantharidin, has anti-tumor activity against human cancers of the liver (hepatomas) and of the upper gastrointestinal tract but is toxic to the urinary tract (Wang, 1989). Cantharidin acts as a protein phosphatase inhibitor, which prompted a more general interest in compounds with this type of chemical structure (Li and Casida 1992). Previously, it had been found that the simpler congener and its hydrolysis product (commercially available as the herbicide, Endothal) are hepatotoxic (Graziani and Casida, 1997). Binding studies have shown that the action of certain cantharidin homologs is direct on protein phosphatase-2A and indirect on protein phosphatase-1 (Honkanen et al., 1993; Li et al., 1993). 
     Of the known congeners of this type of compound, only the parent, cantharidin and its bis(normethyl)-derivative, norcantharidin, have seen any use as anti-cancer drug substances and only norcantharidin is used as an anti-neoplastic agent (Tsauer et al. 1997). 
     Despite these successes, few compounds of this type have been screened for anti-tumor or cytotoxic activity. Currently, there is a significant need to develop inhibitors of protein phosphatases that are more active, less toxic and more specific in action than the known substances mentioned above. In particular, the need is present for diseases such as high-grade malignant gliomas of children and adults. 
     Diffuse intrinsic pontine glioma (DIPG) is a non-operable cancer of the brainstem in children for which no treatment other than radiation has offered any extension of life, with survival with best care being about 12 months. Multiple trials of adjuvant chemotherapy have not significantly improved outcomes (Warren et al. 2011; Hawkins et al. 2011). There are about 300 new cases diagnosed annually in the United States. Glioblastoma multiforme (GBM) is an aggressive brain cancer occurring in about 20,000 adults annually in the US for which standard treatment (primary surgery, followed by 6-weeks of radiation plus temozolomide, followed by daily oral temozolomide) has only increased average lifespan from less than one year to about 18 months despite 50 years of testing experimental therapies (Stupp et al. 2009). There is an urgent need for new treatments of these gliomas. 
     Many chemotherapeutic agents used to treat cancer exhibit serious toxicity, resulting in unwanted side effects for patients and reducing efficacy by limiting the doses that can be safely administered. Prodrugs, which are converted to the active drug in vivo, can offer many advantages over parent drugs such as increased solubility, enhanced stability, improved bioavailability, reduced side effects, better selectivity and improved entry of the drug to certain tissues. Activation of prodrugs can involve many enzymes through a variety of mechanisms including hydrolytic activation (Yang, Y. et al. 2011). Enzymes involved in the hydrolytic activation of prodrugs include carboxylesterases and amidases. 
     Endothal is the common name for 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid. It is an inhibitor of PP2A, an enzyme present in both plants and animals that is involved in the dephosphorylation of proteins. Endothal is structurally similar to cantharidin, a chemical compound secreted by many species of blister beetle. Endothal is known as an active defoliant and potent contact herbicide used in many agricultural situations. It is considered effective as a pre-harvest desiccant and as a selective pre-emergence herbicide. Endothal has been tested against a limited number of human cancer cell lines (Thiery J. P. et al. 1999). 
     SUMMARY OF THE INVENTION 
     The present invention provides a compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
X is X is OR 1 , NR 2 R 3 , OH, O-alkyl, O-alkenyl, O-alkynyl, O-aryl, O-alkylaryl, O-heteroaryl,
 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylaryl, (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , O(C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             R 2  and R 3  are each independently H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , O(C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             R 17  is H, alkyl, hydroxyalkyl, alkenyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, C(O)O-t-Bu or —CH 2 CN; 
             R 18  is H or alkyl; 
             R 19  is (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , (C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 R 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 4  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 5  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 6  and R 7  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 8  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; and
 
Y is OR 9  or NR 10 R 11 ,
 
               
             
             wherein 
             R 9  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylaryl, (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             R 10  is H; and 
             R 11  is (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl, 
               
             
             wherein when Y is OR 9  where R 9  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylaryl, then 
             X is 
           
         
       
    
     
       
         
         
             
             
         
       
     
     and
         when X is       

     
       
         
         
             
             
         
       
     
     where R 17  is CH 3 , then X is other than —O(C 4  alkyl)-OP(O)(OEt) 2  or —NH(C 4  alkyl)-OP(O)(OEt) 2 ,
 
or a salt or ester of the compound.
 
     The present invention also provides a compound having the structure: 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 20  and R 21  are each independently H, alkyl, hydroxyalkyl, alkenyl, alkynyl, aryl, alkylaryl, or heteroaryl,
 
or a salt or ester of the compound.
 
           
         
       
    
     The present invention also provides a compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
X′ is OH, O(alkly) or NR 22 R 23 ;
         R 22  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, or heteroaryl;   R 23  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, or heteroaryl, or R 22  and R 23  combine to form an N-methylpiperazine;
 
Y′ is an anti-cancer agent A containing at least one amine nitrogen and the nitrogen on the anti-cancer agent covalently bonds directly to carbon γ, or
 
Y′ is an anti-cancer agent A containing at least one hydroxyl oxygen and the oxygen on the anti-cancer agent covalently bonds directly to carbon γ, or
       

     Y′ is 
     
       
         
         
             
             
         
       
         
         
           
             wherein A is an anti-cancer agent containing at least one carboxylic acid and the carbonyl carbon of the carboxylic acid on the anti-cancer agent covalently bonds directly to oxygen φ, and R 24  is H or alkyl,
 
or a salt or ester of the compound.
 
           
         
       
    
    
    
     
       BRIEF DESCRIPTION OF THE FIGURE 
         FIG. 1A : Concentration versus time curves of 153 in plasma following iv or po administration, and in liver and brain following iv administration of 153 to SD rats. 
         FIG. 1B : Concentration versus time curves of Endothal in plasma following iv or po administration, and in liver following iv administration of 153 to SD rats. 
         FIG. 1C : Concentration versus time curves of 157 in plasma following iv or po administration, and in, liver and brain following iv administration of 157 to SD rats. 
         FIG. 1D : Concentration versus time curves of Endothal in plasma following iv or po administration, and in liver following iv administration of 157 to SD rats. 
         FIG. 2A : Mean plasma and liver concentration-time profiles of 105 after IV dose of 1 mg/kg in SD rats (N=2/time point). 
         FIG. 2B : Mean plasma and liver concentration-time profile of Endothal after IV dose of 1 mg/kg 105 in male SD rats (N=2/time point). 
         FIG. 2C : Mean plasma and liver concentration-time profile of 105 and Endothal after an IV dose of 1 mg/kg 105 in male SD rats (N=2/time point). 
         FIG. 3A : Mean plasma, brain and liver concentration-time profile of 113 after IV or PO dose of 1.4 mg/kg in male SD rats (N=2/time point). 
         FIG. 3B : Mean plasma and liver concentration-time profile of Endothal after IV dose of 1.4 mg/kg 113 in male SD rats (N=2/time point) 
         FIG. 3C : Mean plasma and liver concentration-time profile of 100 after IV dose of 1.4 mg/kg 113 in male SD rats (N=2/time point) 
         FIG. 3D : Mean plasma, brain and liver concentration-time profile of 113, 100 and Endothal after IV or PO dose of 113 at 1.4 mg/kg in male SD rats (N=2/time point) 
         FIG. 4A : Concentration versus time curves of 100 in plasma following iv administration of 100 to SD rats. 
         FIG. 4B : Concentration versus time curves of 100 in brain following iv administration of 100 to SD rats. 
         FIG. 4C : Concentration versus time curves of 100 in liver following iv administration of 100 to SD rats. 
         FIG. 4D : Concentration versus time curves of endothal in plasma following iv administration of 100 to SD rats. 
         FIG. 4E : Concentration versus time curves of endothal in liver following iv administration of 100 to SD rats. 
         FIG. 5 : Summary of results of liver S9 stability study for LB151, LB100 POM and LB-100 Cabronate. 
         FIG. 6A : Chart showing formation of endothal in monkey liver S9 study. 
         FIG. 6B : Chart showing formation of endothal in human liver S9 study. 
         FIG. 6C : Chart showing formation of endothal in rat liver S9 study. 
         FIG. 6D : Chart showing formation of endothal in monkey liver S9 study. 
         FIG. 6E : Chart showing formation of endothal in human liver S9 study. 
         FIG. 6F : Chart showing formation of endothal in rat liver S9 study. 
         FIG. 6G : Chart showing formation of LB100 in monkey liver S9 study. 
         FIG. 6H : Chart showing formation of LB100 in human liver S9 study. 
         FIG. 6I : Chart showing formation of LB100 in rat liver S9 study. 
         FIG. 6J : Chart showing formation of LB100 in monkey liver S9 study. 
         FIG. 6K : Chart showing formation of LB100 in human liver S9 study. 
         FIG. 6L : Chart showing formation of LB100 in rat liver S9 study. 
         FIG. 7 : Summary of results of whole blood half-life studies for LB151, LB100 POM and LB-100 Cabronate. 
         FIG. 8A : Chart showing formation of endothal in Dog whole blood study. 
         FIG. 8B : Chart showing formation of endothal in Human whole blood study. 
         FIG. 8C : Chart showing formation of endothal in monkey whole blood study. 
         FIG. 8D : Chart showing formation of endothal in rat whole blood study. 
         FIG. 8E : Chart showing formation of LB100 in dog whole blood study. 
         FIG. 8F : Chart showing formation of LB100 in human whole blood study. 
         FIG. 8G : Chart showing formation of LB100 in monkey whole blood study. 
         FIG. 8H : Chart showing formation of LB100 in rat whole blood study. 
         FIG. 9 : Summary of results of MDCK-MDR1 permeability studies for LB151, LB100 POM and LB-100 Cabronate. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides a compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
X is X is OR 1 , NR 2 R 3 , OH, O-alkyl, O-alkenyl, O-alkynyl, O-aryl, O-alkylaryl, O-heteroaryl,
 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylaryl, (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , O(C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             R 2  and R 3  are each independently H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , O(C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             R 17  is H, alkyl, hydroxyalkyl, alkenyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, C(O)O-t-Bu or —CH 2 CN; 
             R 18  is H or alkyl; 
             R 19  is (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , (C 1 -C 4 alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 4  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 5  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 6  and R 7  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
               
             
             wherein each occurrence of R 8  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; and
 
Y is OR 9  or NR 10 R 11 ,
 
             wherein 
             R 9  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylaryl, (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             R 10  is H; and 
             R 11  is (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl, 
               
             
             wherein when Y is OR 9  where R 9  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylaryl, then 
             X is 
           
         
       
    
     
       
         
         
             
             
         
       
     
     and
         when X is       

     
       
         
         
             
             
         
       
     
     where R 17  is CH 3 , then X is other than —O(C 4  alkyl)-OP(O)(OEt) 2  or —NH(C 4  alkyl)-OP(O)(OEt) 2 ,
 
or a salt or ester of the compound.
 
     In some embodiments, Y is OR 9  or NR 10 R 11 ,
         wherein   R 9  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylaryl, (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 ,       

     
       
         
         
             
             
         
       
         
         
           
             R 10  is H; and 
             R 11  is (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl, 
               
             
           
         
       
    
     In some embodiments, X is OR 1  or NR 2 R 3 ,
         wherein R 1  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylaryl, (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , O(C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 ,       

     
       
         
         
             
             
         
       
         
         
           
             R 2  and R 3  are each independently H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , O(C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 4  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 5  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 6  and R 7  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 8  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl. 
               
             
           
         
       
    
     In some embodiments, X is OH, O-alkyl, O-alkenyl, O-alkynyl, O-aryl, O-alkylaryl, O-heteroaryl; and 
     Y is OR 9  or NR 10 R 11 ,
         wherein R 9  is (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 ,       

     
       
         
         
             
             
         
       
         
         
           
             R 10  is H; and 
             R 11  is (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 1 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl. 
               
             
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein each n=0-19, m=0-8 and o=0-6;
 
Y is OR 9  or NR 10 R 11 ,
         wherein R 9  is (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 ,       

     
       
         
         
             
             
         
       
         
         
           
             R 10  is H; and 
             R 11  is (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl. 
               
             
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein each n=0-19, m=0-8 and n=0-6; 
     Y is OR 9 , 
     
         
         
           
             wherein R 9  is 
           
         
       
    
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 Z is an amino acid substituent; and 
                 AA is an amino acid moiety. 
               
             
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     and 
     Y is OR 9 , 
     
         
         
           
             wherein R 9  is 
           
         
       
    
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 Z is an amino acid substituent; and 
                 AA is an amino acid moiety. 
               
             
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
         
         
           
             wherein 
             Each n=0-19, m=0-8 and n=0-6;
 
Y is NR 10 R 11 ,
 
wherein
 
           
         
       
    
     R 10  is H; and 
     R 11  is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
         
         
           
             wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             Z is an amino acid substituent; and 
             AA is an amino acid moiety. 
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     and
 
Y is NR 10 R 11 ,
 
wherein
 
     R 10  is H; and 
     R 11  is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
         
         
           
             wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             Z is an amino acid substituent; and 
             AA is an amino acid moiety. 
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
R 17  is H, alkyl, hydroxyalkyl, alkenyl or alkylaryl;
 
Y is OR 9  or NR 10 R 11 ,
         wherein R 9  is (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 ,       

     
       
         
         
             
             
         
       
         
         
           
             R 10  is H; and 
             R 11  is (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl. 
               
             
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
R 17  is H, alkyl, hydroxyalkyl, alkenyl or alkylaryl; and
 
     Y is OR 9 , 
     
         
         
           
             wherein R 9  is 
           
         
       
    
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 Z is an amino acid substituent; and 
                 AA is an amino acid moiety. 
               
             
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
R 17  is H, methyl, ethyl, CH 2 CH 2 OH, CH 2 (phenyl); and
 
     Y is OR 9 , 
     
         
         
           
             wherein R 9  is 
           
         
       
    
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 Z is an amino acid substituent; and 
                 AA is an amino acid moiety. 
               
             
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein 
     Y is OR 9 , 
     
         
         
           
             wherein R 9  is 
           
         
       
    
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 Z is an amino acid substituent; and 
                 AA is an amino acid moiety. 
               
             
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
R 17  is H, alkyl, hydroxyalkyl, alkenyl or alkylaryl; and
 
Y is NR 10 R 11 ,
 
wherein
 
     R 10  is H; and 
     R 11  is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
         
         
           
             wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             Z is an amino acid substituent; and 
             AA is an amino acid moiety. 
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
R 17  is H, methyl, ethyl, CH 2 CH 2 OH, CH 2 (phenyl); and
 
Y is NR 10 R 11 ,
 
wherein
 
     R 10  is H; and 
     R 11  is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
         
         
           
             wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             Z is an amino acid substituent; and 
             AA is an amino acid moiety. 
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
Y is NR 10 R 11 ,
 
wherein
 
     R 10  is H; and 
     R 11  is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
         
         
           
             wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 13  is, independently, H, alkyl, wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
             Z is an amino acid substituent; and 
             AA is an amino acid moiety. 
           
         
       
    
     In some embodiments, the compound wherein 
     X is OR 1  or NR 2 R 3 ,
         wherein R 1  is (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , (C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 ,       

     
       
         
         
             
             
         
       
         
         
           
             R 2  is H; and 
             R 3  is (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , (C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 4  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 5  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 6  and R 7  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 8  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl;
 
Y is OR 9  or NR 10 R 11 ,
 
               
             
             wherein R 9  is (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             R 10  is H; 
             R 11  is (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , O(C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl. 
               
             
           
         
       
    
     In some embodiments, the compound wherein 
     X is OR 1 , 
     
         
         
           
             wherein R 1  is (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , (C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 4  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 5  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 6  and R 7  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 8  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
               
             
           
         
       
    
     Y is OR 9 , 
     
         
         
           
             wherein (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl. 
               
             
           
         
       
    
     In some embodiments, the compound wherein 
     X is OR 1 , 
     
         
         
           
             wherein R 1  is (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , (C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl) P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 4  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 5  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 6  and R 7  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 8  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl;
 
Y is NR 10 R 11 ,
 
               
             
             wherein 
             R 10  is H; 
             R 11  is (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 2 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 2 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl. 
               
             
           
         
       
    
     In some embodiments, the compound wherein 
     X is NR 2 R 3 ,
         wherein   R 2  is H; and   R 3  is (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , (C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 ,       

     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 4  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 5  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 6  and R 7  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 8  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl;
 
Y is NR 10 R 11 ,
 
               
             
             wherein 
             R 10  is H; 
             R 11  is (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 14  and R 15  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 16  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl. 
               
             
           
         
       
    
     In some embodiments, the compound wherein 
     R 9  is 
     
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 9  is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 9  is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 9  is 
     
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 10  is H; and 
     R 11  is 
     
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 10  is H; and 
     R 11  is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 10  is H; and 
     R 11  is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 10  is H; and 
     R 11  is 
     
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 1  and R 9  are each, independently, 
     
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 1  and R 9  are each, independently, 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 1  and R 9  are each, independently, 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 1  and R 9  are each, independently, 
     
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 3  and R 11  are each, independently, 
     
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 3  and R 11  are each, independently, 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 3  and R 11  are each independently, 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In some embodiments, the compound wherein 
     R 3  and R 11  are each, independently, 
     
       
         
         
             
             
         
       
     
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein 
     R 9  is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein 
     R 11  is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein 
     R 9  is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In some embodiments the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein 
     R 11  is 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     
       
         
         
             
             
         
       
     
     wherein 
     R 11  is 
     
       
         
         
             
             
         
       
     
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
         
         
           
             wherein 
             R 18  is H or alkyl; 
             R 19  is (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , (C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 4  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 5  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 6  and R 7  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 8  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; and 
               
             
             R 9  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylaryl, (C 1 -C 4  alkyl)-O(CO)R 12 , (C 1 -C 4  alkyl)-O(CO)OR 12 , (C 1 -C 4  alkyl)-OP(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 12 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 12 ) 2 , (C 1 -C 4  alkyl)NR 12 R 13 , (C 1 -C 4  alkyl)NC(O)R 12 , (C 1 -C 4  alkyl)C(O)OR 12 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 12 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 12 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 12 R 13 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 13  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl,
 
or a salt or ester of the compound.
 
               
             
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
         
         
           
             wherein 
             R 18  is H or alkyl; 
             R 19  is (C 1 -C 4  alkyl)-O(CO)R 4 , (C 1 -C 4  alkyl)-O(CO)OR 4 , (C 1 -C 4  alkyl)-OP(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)OR 4 ) 2 , (C 1 -C 4  alkyl)-OP(O)(O(C 1 -C 4  alkyl)-O(CO)R 4 ) 2 , (C 1 -C 4  alkyl)NR 4 R 5 , (C 1 -C 4  alkyl)NC(O)R 4 , (C 1 -C 4  alkyl)C(O)OR 4 , (C 1 -C 4  alkyl)OC(O)aryl(C 1 -C 4  alkyl)P(O)(OR 4 ) 2 , (C 1 -C 4  alkyl)OC(O)(C 2 -C 4  alkenyl)CO 2 R 4 , (C 1 -C 4  alkyl)OC(O)(C 1 -C 4  alkyl)NH 2 , (C 1 -C 4  alkyl)C(O)NR 4 R 5 , 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein each occurrence of R 4  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 5  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 6  and R 7  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl; 
                 wherein each occurrence of R 8  is, independently, H, halogen, alkyl, alkenyl, alkynyl, aryl or heteroaryl; and 
               
             
             R 9  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, or alkylaryl. 
           
         
       
    
     In some embodiments, the compound wherein
         R 9  is H or alkyl.       

     In some embodiments, the compound wherein
         R 9  is —H,
           —CH 3      —CH 2 CH 3 ,   —CH 2 CH 2 CH 3 ,   —CH 2 CH 2 CH 2 CH 3 ,   —CH 2 CH 2 CH 2 CH 2 CH 3 ,   —CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ,   —CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ,   —CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ,   —CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ,   —CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 ,   —CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 , or   —CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH═CHCH 2 CH═CHCH 2 CH 2 CH 2 CH 2 CH 3 .   
               

     In some embodiments, the compound wherein
         R 18  is —H or —CH 3 ; and   R 19  is (C 1 -C 4  alkyl)-O(CO)R 4  or (C 1 -C 4  alkyl)-O(CO)OR 4 .       

     In some embodiments, the compound wherein
         R 18  is —H or —CH 3 ; and   R 19  is —CH 2 —O(CO)CH 3 , —CH(CH 3 )—O(CO)CH 3 , —CH 2 —O(CO)OCH 3 , —CH(CH 3 )—O(CO)OCH 3 .       

     In some embodiments, the compound wherein R 9  is 
     
       
         
         
             
             
         
       
     
     The present invention also provides a compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
R 17  is H, alkyl, hydroxyalkyl, alkenyl, alkenyl, alkynyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, C(O)O-t-Bu or —CH 2 CN;
 
Y is OR 9 , wherein R 9  is (C 1 -C 4  alkyl)-O(CO)R 12  or (C 1 -C 4  alkyl)-O(CO)OR 12 ,
         wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl.       

     In some embodiments, the compound wherein 
     R 17  is H, methyl, ethyl, CH 2 CH 2 OH, CH 2 (phenyl); and
 
Y is OR 9 , wherein R 9  is (C 1 -C 4  alkyl)-O(CO)R 12  or (C 1 -C 4  alkyl)-O(CO)OR 12 ,
         wherein each occurrence of R 12  is, independently, H, alkyl, alkenyl, alkynyl, aryl or heteroaryl.       

     In some embodiments, the compound wherein 
     R 1 , is H, methyl, ethyl, CH 2 CH 2 OH, CH 2 (phenyl); and
 
Y is OR 9 , wherein R 9  is (C 1 -C 4  alkyl)-O(CO)R 12  or (C 1 -C 4  alkyl)-O(CO)OR 12 ,
         wherein each occurrence of R 12  is an alkyl.       

     In some embodiments, the compound wherein 
     R 17  is methyl; and
 
Y is OR 9 , wherein R 9  is (C 1 -C 4  alkyl)-O(CO)R 12  or (C 1 -C 4  alkyl)-O(CO)OR 12 ,
         wherein each occurrence of R 12  is an alkyl.       

     In some embodiments, the compound having the structure 
     
       
         
         
             
             
         
       
     
     or a salt of the compound. 
     In some embodiments, the compound having the structure 
     
       
         
         
             
             
         
       
     
     or a salt of the compound. 
     
       
         
         
             
             
         
       
     
     or a salt of the compound. 
     The present invention also provides a compound having the structure: 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 20  and R 21  are each independently H, alkyl, hydroxyalkyl, alkenyl, alkynyl, aryl, alkylaryl, or heteroaryl,
 
or a salt or ester of the compound.
 
           
         
       
    
     In some embodiments, the above compound wherein R 20  and R 21  are each independently H, methyl, ethyl, CH 2 CH 2 OH, or CH 2 (phenyl). 
     In some embodiments, the above compound wherein R 20  and R 21  are both H. 
     In some embodiments, the above compound wherein R 20  and R 21  are both methyl. 
     The present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable carrier. 
     The present invention provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. 
     The present invention provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and an anticancer agent, and at least one pharmaceutically acceptable carrier. 
     In some embodiments, the pharmaceutical composition wherein the pharmaceutically acceptable carrier comprises a liposome. 
     In some embodiments, the pharmaceutical composition wherein the compound is contained in a liposome or microsphere, or the compound and the anti-cancer agent are contained in a liposome or microsphere. 
     The present invention also provides a method for in vivo delivery of endothal to a target cell in a subject, the method comprising administering to the subject a compound of the present invention, wherein one or two bonds in the compound are subject to in vivo hydrolytic cleavage in the subject, so as to thereby deliver endothal to the target cell in the subject. 
     In some embodiments of the above method, the compound has the structure 
     
       
         
         
             
             
         
       
         
         
           
             wherein one or both of bond α and bond β is subject to in vivo hydrolytic cleavage in the subject. 
           
         
       
    
     In some embodiments of the above method, the compound has the structure 
     
       
         
         
             
             
         
       
         
         
           
             wherein one or more of bonds χ, δ, ε, and ϕ are subject to in vivo hydrolytic cleavage in the subject. 
           
         
       
    
     In some embodiments of the above method, wherein the delivery of the endothal to the target cell in the subject is effective to treat a disease in the subject afflicted with the disease. 
     In some embodiments of the above method, wherein the disease is cancer. 
     In some embodiments of the above method, wherein the cancer is a breast cancer, colon cancer, large cell lung cancer, adenocarcinoma of the lung, small cell lung cancer, stomach cancer, liver cancer, ovary adenocarcinoma, pancreas carcinoma, prostate carcinoma, promylocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, colorectal cancer, ovarian cancer, lymphoma, non-Hodgkin&#39;s lymphoma or Hodgkin&#39;s lymphoma. 
     In some embodiments of the above method, wherein the cancer is a brain cancer. 
     In some embodiments of the above method, wherein the brain cancer is a glioma, pilocytic astrocytoma, low-grade diffuse astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, oligodendroglioma, ependymoma, meningioma, pituitary gland tumor, primary CNS lymphoma, medulloblastoma, craniopharyngioma, or diffuse intrinsic pontine glioma. 
     In some embodiments of the above method, further comprising administering to the subject an anti-cancer agent. 
     In some embodiments of the above method, wherein the anti-cancer agent is selected from x-radiation or ionizing radiation. 
     In some embodiments of the above method, wherein the target cell is a cancer cell. 
     In some embodiments of the above method, wherein the cancer cell is a breast cancer, colon cancer, large cell lung cancer, adenocarcinoma of the lung, small cell lung cancer, stomach cancer, liver cancer, ovary adenocarcinoma, pancreas carcinoma, prostate carcinoma, promylocytic leukemia, chronic myelocytic leuemia, acute lymphocytic leukemia, colorectal cancer, ovarian cancer, lymphoma, non-Hodgkin&#39;s lymphoma or Hodgkin&#39;s lymphoma cell. 
     In some embodiments of the above method, wherein the cancer cell is a brain cancer cell. 
     In some embodiments of the above method, wherein the brain cancer cell is a glioma, pilocytic astrocytoma, low-grade diffuse astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, oligodendroglioma, ependymoma, meningioma, pituitary gland tumor, primary CNS lymphoma, medulloblastoma, craniopharyngioma, or diffuse intrinsic pontine glioma cell. 
     In some embodiments of the above method, wherein the target cell is in the brain of the subject. 
     In some embodiments of the above method, wherein the endothal is delivered to a target cell in the brain of the subject. 
     In some embodiments of the above method, the hydrolytic cleavage of the α and/or β bond is facilitated by a carboxylesterase or an amidase in the subject. 
     In some embodiments of the above method, wherein the hydrolytic cleavage of the χ, δ, ε, and ϕ bond is facilitated by a carboxylesterase or an amidase in the subject. 
     The present invention also provides a compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
X′ is OH, O(alkly) or NR 22 R 23 ;
         R 22  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, or heteroaryl;   R 23  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, or heteroaryl, or R 22  and R 23  combine to form an N-methylpiperazine;
 
Y′ is an anti-cancer agent A containing at least one amine nitrogen and the nitrogen on the anti-cancer agent covalently bonds directly to carbon γ, or
 
Y′ is an anti-cancer agent A containing at least one hydroxyl oxygen and the oxygen on the anti-cancer agent covalently bonds directly to carbon γ, or
       

     Y′ is 
     
       
         
         
             
             
         
       
         
         
           
             wherein A is an anti-cancer agent containing at least one carboxylic acid and the carbonyl carbon of the carboxylic acid on the anti-cancer agent covalently bonds directly to oxygen φ, and R 24  is H or alkyl,
 
or a salt or ester of the compound.
 
           
         
       
    
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
A is an anti-cancer agent containing at least one amine nitrogen and the nitrogen on the anti-cancer agent covalently bonds directly to carbon γ, or A is an anti-cancer agent containing at least one hydroxyl oxygen and the oxygen on the anti-cancer agent covalently bonds directly to carbon γ.
 
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
A is an anti-cancer agent containing at least one amine nitrogen and the nitrogen on the anti-cancer agent covalently bonds directly to carbon γ, or A is an anti-cancer agent containing at least one hydroxyl oxygen and the oxygen on the anti-cancer agent covalently bonds directly to carbon γ.
 
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein 
     Q is NH or O; 
     R 22  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, or heteroaryl;
 
R 23  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, or heteroaryl, or
 
R 22  and R 23  combine to form an N-methylpiperazine;
 
R 24  is H or alkyl; and
 
A is an anti-cancer agent containing at least one carboxylic acid or primary amide and the carbonyl carbon of the carboxylic acid or primary amide on the anti-cancer agent covalently bonds directly to Q,
 
or a salt or ester of the compound.
 
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
R 22  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, or heteroaryl;
 
R 23  is H, alkyl, alkenyl, alkynyl, aryl, alkylaryl, or heteroaryl, or
 
R 24  is H or alkyl; and
 
A is an anti-cancer agent containing at least one carboxylic acid and the carbonyl carbon of the carboxylic acid on the anti-cancer agent covalently bonds directly to oxygen φ, or A is an anti-cancer agent containing at least one primary amide and the carbonyl carbon of the primary amide on the anti-cancer agent covalently bonds directly to nitrogen φ,
 
or a salt or ester of the compound.
 
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     wherein
 
R 24  is H or alkyl; and
 
A is an anti-cancer agent containing at least one carboxylic acid and the carbonyl carbon of the carboxylic acid on the anti-cancer agent covalently bonds directly to oxygen φ, or A is an anti-cancer agent containing at least one primary amide and the carbonyl carbon of the primary amide on the anti-cancer agent covalently bonds directly to nitrogen φ, or
 
or a salt or ester of the compound.
 
     In some embodiments, the above compound wherein A is adenine, emtricitabine, vapreotide, troxacitabine, triptorelin, trimetrexate glucuronate, trimetrexate, tipifarnib, tiazofurin, thioguanine, squalamine lactate, piritrexim isethionate, pentetreotide, pemetrexed, peldesine, oxaliplatin, nelarabine, mitoguazone, methyl aminolevulinate, methotrexate, melphalan, leuprolide, lanreotide, idarubicin, histamine, goderelin, gemtuzumab ozogamicin, gemcitabine, fludarabine, epirubicin, eflornithine, doxorubicin, decitabine, 5-aza-2′-deoxycytidine, daunorubicin, dactinomycin, cytarabine, clofarabine, cladribine, cliengtide, cetrorelix acetate, cetrorelix, bleomycin, azacitidine, aminolevulinic acid, aminogluthethimide, amifostine, abarelix, amifostine, abarelix, phentermine, corticorelin, metyrosine or monomethyl auristatin E (MMAE). 
     In some embodiments, the above compound wherein A is abarelix, azacitidine, bleomycin, broxuridine, capecitabine, cetrorelix, cetrorelix acetate, cladribine, clofarabine, cytarabine, dactinomycin, dasatinib, daunorubicin, decitabine, docetaxel, doxorubicin, dromostanolone propionate, emtricitabine, epirubicin, estramustine, etoposide, etoposide phosphate, fludarabine, fulvestrant, gemcitabine, gemtuzumab ozogamicin, goserelin, goserelin acetate, irinotecan, irinotecan hydrochloride, irofulven, lanreotide acetate, lanreotide, leuprolide, leuprolide acetate, mitobronitol, mitolactol, mitoxantrone, mitoxantrone hydrochloride, motexafin gadolinium, nelarabine, paclitaxel, patupilone, pentostatin, plicamycin, plitidepsin, porfimer, porfimer sodium, squalamine lactate, streptozocin, taxol, temsirolimus, tezacitabine, teniposide, tiazofurin, trabectedin, treosulfan, triptorelin, troxacitabine, valrubicin or zosuquidar trihydrochloride. 
     In some embodiments, the above compound wherein A is acitretin, aminolevulinic acid, bexarotene, carboplatin, cetrorelix acetate, chlorambucil, cilengitide, corticorelin, eflornithine, exisulind, fumagillin irinotecan, melphalan, methotrexate, metyrosine, pemetrexed, pentetreotide, phenylbutyrate, porfimer, sulindac, verteporfin ortemozolomide. 
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     or salt or ester of the compound. 
     In some embodiments, the compound having the structure: 
     
       
         
         
             
             
         
       
     
     or salt or ester of the compound. 
     The present invention also provides method for in vivo delivery of endothal and an anti-cancer agent to a cancer cell in a subject, the method comprising administering to the subject a compound of the present invention so as to thereby deliver endothal and the anti-cancer agent to the cancer cell in the subject. 
     In some embodiments of the above method, wherein the compound has the structure: 
     
       
         
         
             
             
         
       
     
     wherein bond η is subject to in vivo hydrolytic cleavage in the subject, so as to thereby deliver endothal and the anti-cancer agent to the cancer cell in the subject. 
     In some embodiments of the above method, wherein A is an anti-cancer agent containing at least one amine nitrogen and the nitrogen on the anti-cancer agent covalently bonds directly to carbon γ and the hydrolytic cleavage of the q bond is facilitated by an amidase in the subject. 
     In some embodiments of the above method, wherein A is an anti-cancer agent containing at least one hydroxyl oxygen and the oxygen on the anti-cancer agent covalently bonds directly to carbon γ and the hydrolytic cleavage of the η is facilitated by a carboxylesterase in the subject. 
     In some embodiments of the above method, wherein the compound has the structure: 
     
       
         
         
             
             
         
       
     
     wherein bonds κ and λ are subject to in vivo hydrolytic cleavage in the subject, so as to thereby deliver endothal and the anti-cancer agent to the cancer cell in the subject. 
     In some embodiments of the above method, wherein the compound has the structure: 
     
       
         
         
             
             
         
       
         
         
           
             wherein bonds κ and λ are subject to in vivo hydrolytic cleavage in the subject, so as to thereby deliver endothal and the anti-cancer agent to the cancer cell in the subject. 
           
         
       
    
     In some embodiments of the above method, wherein the hydrolytic cleavage of the κ bond and/or the λ bond is facilitated by a carboxylesterase or amidase in the subject. 
     In some embodiments of the above method, wherein the delivery of the endothal and the anti-cancer agent to the cancer cell in the subject is effective to treat a cancer in a subject afflicted with the cancer. 
     In some embodiments of the above method, wherein the cancer is a breast cancer, colon cancer, large cell lung cancer, adenocarcinoma of the lung, small cell lung cancer, stomach cancer, liver cancer, ovary adenocarcinoma, pancreas carcinoma, prostate carcinoma, promylocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, colorectal cancer, ovarian cancer, lymphoma, non-Hodgkin&#39;s lymphoma or Hodgkin&#39;s lymphoma. 
     In some embodiments of the above method, wherein the cancer is a brain cancer. 
     In some embodiments of the above method, wherein the brain cancer is a glioma, pilocytic astrocytoma, low-grade diffuse astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, oligodendroglioma, ependymoma, meningioma, pituitary gland tumor, primary CNS lymphoma, medulloblastoma, craniopharyngioma, or diffuse intrinsic pontine glioma. 
     In some embodiments of the above method, wherein the cancer cell is a breast cancer, colon cancer, large cell lung cancer, adenocarcinoma of the lung, small cell lung cancer, stomach cancer, liver cancer, ovary adenocarcinoma, pancreas carcinoma, prostate carcinoma, promylocytic leukemia, chronic myelocytic leuemia, acute lymphocytic leukemia, colorectal cancer, ovarian cancer, lymphoma, non-Hodgkin&#39;s lymphoma or Hodgkin&#39;s lymphoma cell. 
     In some embodiments of the above method, wherein the cancer cell is a brain cancer cell. 
     In some embodiments of the above method, wherein the brain cancer cell is a glioma, pilocytic astrocytoma, low-grade diffuse astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, oligodendroglioma, ependymoma, meningioma, pituitary gland tumor, primary CNS lymphoma, medulloblastoma, craniopharyngioma, or diffuse intrinsic pontine glioma cell. 
     In some embodiments of the above method, wherein the target cell is in the brain of the subject. 
     In some embodiments of the above method, wherein the endothal and anti-cancer agent are delivered to a cancer cell in the brain of the subject. 
     In some embodiments of the above method, the compound is co-administered with an anti-cancer agent. 
     The present invention provides a method of treating a subject afflicted with cancer comprising administering to the subject a therapeutically effective amount of the compound of the present invention. 
     The present invention provides a method of enhancing the anti-cancer activity of an anti-cancer agent in a subject afflicted with a cancer, comprising administering to the subject the compound of the present invention in an amount effective to enhance the anti-cancer activity of the anti-cancer agent. 
     The present invention provides a method of treating a subject afflicted with cancer comprising periodically administering to the subject: 
     a) an amount of the compound of the present invention or a pharmaceutically acceptable salt thereof, and
 
b) an anti-cancer agent,
 
wherein the amounts when taken together are more effective to treat the subject than when each agent at the same amount is administered alone.
 
     The present invention provides for the use of the compound of the present invention or a pharmaceutically acceptable salt thereof and an anti-cancer agent in the preparation of a combination for treating a subject afflicted with cancer wherein the amount of the compound and the amount of the anti-cancer agent are administered simultaneously or contemporaneously. 
     The present invention provides a pharmaceutical composition comprising an amount of the compound of the present invention or a pharmaceutically acceptable salt thereof for use in treating a subject afflicted with cancer as an add-on therapy or in combination with, or simultaneously, contemporaneously or concomitantly with an anti-cancer agent. 
     In some embodiments, the compound of the present invention or a pharmaceutically acceptable salt thereof for use as an add-on therapy or in combination with an anti-cancer agent in treating a subject afflicted with cancer. 
     In some embodiments, the compound of the present invention or a pharmaceutically acceptable salt thereof and an anti-cancer agent for the treatment of a subject afflicted with cancer wherein the compound and the anti-cancer agent are administered simultaneously, separately or sequentially. 
     In some embodiments, a product containing an amount of the compound of the present invention or a pharmaceutically acceptable salt thereof and an amount of an anti-cancer agent for simultaneous, separate or sequential use in treating a subject afflicted cancer. 
     In some embodiments, the compound of the present invention or a pharmaceutically acceptable salt thereof for use in treating cancer. 
     In some embodiments, the compound of the present invention or a pharmaceutically acceptable salt thereof in combination with an anti-cancer agent for use in treating cancer. 
     In some embodiments of any of the above methods, uses, pharmaceutical compositions, compounds or products, the cancer is breast cancer, colon cancer, large cell lung cancer, adenocarcinoma of the lung, small cell lung cancer, stomach cancer, liver cancer, ovary adenocarcinoma, pancreas carcinoma, prostate carcinoma, promylocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, colorectal cancer, ovarian cancer, lymphoma, non-Hodgkin&#39;s lymphoma or Hodgkin&#39;s lymphoma. 
     In some embodiments of any of the above methods, uses, pharmaceutical compositions, compounds or products, the cancer is brain cancer. 
     In some embodiments of any of the above methods, uses, pharmaceutical compositions, compounds or products, the brain cancer is a glioma, pilocytic astrocytoma, low-grade diffuse astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, oligodendroglioma, ependymoma, meningioma, pituitary gland tumor, primary CNS lymphoma, medulloblastoma, craniopharyngioma, or diffuse intrinsic pontine glioma. 
     In some embodiments of any of the above methods, uses, pharmaceutical compositions, compounds or products, the compound crosses the blood brain barrier of the subject. 
     In some embodiments of any of the above methods, uses, pharmaceutical compositions, compounds or products, the compound and/or a metabolite of the compound crosses the blood brain barrier of the subject. 
     The present invention provides a method of inhibiting proliferation or inducing apoptosis of a cancer cell in a human subject, comprising administering to the subject: 
     a) the compound of the present invention, or a salt of the compound, in an amount effective to inhibit the proliferation or to induce apoptosis of the cancer cell, and
 
b) an anti-cancer agent in an amount effective to inhibit the proliferation or to induce apoptosis of the cancer cell.
 
     The present invention provides a method of inhibiting proliferation or inducing apoptosis of a cancer cell in a human subject which overexpresses translationally controlled tumour protein (TCTP) comprising administering to the subject 
     a) the compound of the present invention, or a salt of the compound, in an amount effective to inhibit the proliferation or to induce apoptosis of the cancer cell, and
 
b) an anti-cancer agent in an amount effective to inhibit the proliferation or to induce apoptosis of the cancer cell.
 
     In some embodiments of the above methods, the cancer cell does not overexpress N-CoR. 
     In some embodiments of any of the above methods, uses, pharmaceutical compositions, compounds or products, the anti-cancer agent is selected from x-radiation or ionizing radiation. 
     In some embodiments of any of the above methods, uses, pharmaceutical compositions, compounds or products, the anti-cancer agent is selected from a DNA damaging agent, a DNA intercalating agent, a microtubule stabilizing agent, a microtubule destabilizing agent, a spindle toxin, abarelix, aldesleukin, alemtuzumab, alitertinoin, allopurinol, altretamine, amifostin, anakinra, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, actinomycin D, dalteparin sodium, darbepoetin alfa, dasatinib, daunorubicin, daunomycin, decitabine, denileukin, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, exulizumab, epirubicin, epoetin alfa, erlotinib, estramustine, etoposide phosphate, etoposide, VP-16, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gosereline acetate, histrelin acetate, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a, interferon alfa 2b, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovrin, leuprolide acetate, levamisole, lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine, mesna, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine, nofetumomab, oprelvekin, oxaliplatin, paclitaxel, palifermin, pamidronate, panitumumab, pegademase, pegaspargase, pegfilgrastim, peginterferon alfa 2b, pemetrexed disodium, pentostatin, pipobroman, plicamycin, mithramycin, porfimer sodium, procarbazine, quinacrine, rasburicase, rituximab, sargrmostim, sorafenib, streptozocin, sunitinib, sunitinib maleate, talc, tamoxifen, temozolomide, teniposide, VM-26, testolactone, thalidomide, thioguanine, G-TG, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin ATRA, uracil mustard, valrunicin, vinblastine, vincristine, vinorelbine, vorinostat, zoledronate, zoledronic acid, abraxane and brentuximab vedotin. 
     In some embodiments of any of the above methods, uses, pharmaceutical compositions, compounds or products, the subject is a human. 
     In some embodiments of any of the above methods, uses, pharmaceutical compositions, compounds or products, the cancer is any one of adrenocortical cancer, bladder cancer, osteosarcoma, cervical cancer, esophageal, gallbladder, head and neck cancer, lymphoma, Hodgkin&#39;s lymphoma, non-Hodgkin&#39;s lymphoma, renal cancer, melanoma, pancreatic cancer, rectal cancer, thyroid cancer, throat cancer, brain cancer, breast cancer, lung cancer, prostate cancer, melanoma, pancreatic cancer, colon cancer, large cell lung cancer, adenocarcinoma of the lung, small cell lung cancer, stomach cancer, liver cancer, ovary adenocarcinoma, pancreas carcinoma, prostate carcinoma, promylocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, colorectal cancer, ovarian cancer or hepatocellular carcinoma. 
     In one embodiment, a pharmaceutical composition comprising the compound of the present invention. In one embodiment, a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier. 
     In one embodiment of the method, the compound of the present invention inhibits PP2A activity in the subject. In one embodiment of the method, the compound of the present invention inhibits PP2A activity in the brain of the subject. In one embodiment of the method, the compound of the present invention crosses the blood brain barrier of the subject. 
     In some embodiments, the compounds of the present invention are ester derivatives of compound 100 and serve as pro-drugs of compound 100. 
     In some embodiments, the compounds of the present invention are ester derivatives of 100 and serve as pro-drugs that can be converted into 100 by serum esterases and/or brain esterases. 
     In some embodiments, the compounds of the present invention are derivatives of compound 100 and serve as pro-drugs of endothal. 
     In some embodiments, the compounds of the present invention are derivatives of compound 100 and serve as pro-drugs that can be converted into endothal by serum esterases and/or brain esterases. 
     In some embodiments, the compounds of the present invention are derivatives of compound 100 and serve as pro-drugs that cross the blood brain barrier and deliver endothal to the brain. 
     Administration of a pro-drug of endothal is more effective at delivering endothal to targets cells in a subject than administration of endothal itself. 
     The metabolic profile of endothal is such that administration of a pro-drug of endothal is more effective at delivering endothal to targets cells in a subject than administration of endothal itself. 
     In some embodiments, the method wherein the compound is first converted to compound 100 in vivo, which in turn is converted to endothal in vivo. 
     The compounds disclosed herein act as prodrugs of endothal, altering metabolism by masking one or two acid groups with an amide or an ester moiety. The design of the prodrug will result in reduced toxicity and increased systemic exposure of endothal in the subject. 
     In some embodiments of the delivery method, a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier. 
     As used herein, a “symptom” associated with a disease includes any clinical or laboratory manifestation associated with the disease and is not limited to what the subject can feel or observe. 
     As used herein, “treatment of the diseases”, “treatment of the injury” or “treating”, e.g. of a disease encompasses inducing inhibition, regression, or stasis of the disease or injury, or a symptom or condition associated with the disease or injury. 
     As used herein, “inhibition” of disease encompasses preventing or reducing the disease progression and/or disease complication in the subject. 
     As used herein, “overexpressing N-CoR” means that the level of the Nuclear receptor co-repressor (N-CoR) expressed in cells of the tissue tested are elevated in comparison to the levels of N-CoR as measured in normal healthy cells of the same type of tissue under analogous conditions. The nuclear receptor co-repressor (N-CoR) of the subject invention may be any molecule that binds to the ligand binding domain of the DNA-bound thyroid hormone receptor (T3R) and retinoic acid receptor (RAR) (U.S. Pat. No. 6,949,624, Liu et al.). Examples of tumors that overexpress N-CoR may include glioblastoma multiforme, breast cancer (Myers et al. 2005), colorectal cancer (Giannini and Cavallini 2005), small cell lung carcinoma (Waters et al 2004.) or ovarian cancer (Havrilesky et al. 2001). 
     As used herein, the term “amino acid moiety” or “AA” refers to any natural or unnatural amino acid including its salt form, ester derivative, protected amine derivative and/or its isomeric forms. Amino Acids comprise, by way of non-limiting example: Agmatine, Alanine Beta-Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Phenyl Beta-Alanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, and Valine. The amino acids may be L or D amino acids. The amino acid may be attached via the acid to form an ester linker or via the amine to form a secondary amine linker. 
     As used herein, the term “amino acid moiety” refers to H, OH, alkyl, benzyl, methyl, ethyl, propyl, butyl, isopropyl, isobutyl, —(CH 2 )C(O)NH 2 , —(CH 2 ) 2 C(O)NH 2 , —(CH 2 )C(O)OH, —(CH 2 ) 2 C(O)OH, —(CH 2 ) 5 C(O)OH, —CH(CH 3 ) CH 2 CH 3 , propyl, butyl, —(CH 2 CH 2 CH 2 ) NH 2 , —(CH 2 )SH, —(CH 2 CH 2 )SH, —(CH 2 ) SCH 3 , —(CH 2 CH 2 )SCH 3 , —(CH 2 CH 2 )OH, —(CH 2 )OH, —(CH 2 )-indole, —(CH 2 )-thiophene, —(CH 2 )-imidazole, —CH(OH)CH 3 , —CH(CH 3 )C(SH)(CH 3 ) 2 , —CH 2  (4-methoxyphenyl) or —(CH 2 ) 3 NHC(NH)NH 2 . 
     As used herein, “alkyl” is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. Thus, C 1 -C n  as in “C 1 -C n  alkyl” is defined to include groups having 1, 2 . . . , n−1 or n carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, isopropyl, isobutyl, sec-butyl and so on. An embodiment can be C 1 -C 20  alkyl, C 2 -C 20  alkyl, C 3 -C 20  alkyl, C 4 -C 20  alkyl and so on. An embodiment can be C 1 -C 30  alkyl, C 2 -C 30  alkyl, C 3 -C 30  alkyl, C 4 -C 30  alkyl and so on. “Alkoxy” represents an alkyl group as described above attached through an oxygen bridge. 
     The term “alkenyl” refers to a non-aromatic hydrocarbon radical, straight or branched, containing at least 1 carbon to carbon double bond, and up to the maximum possible number of non-aromatic carbon-carbon double bonds may be present. Thus, C 2 -C n  alkenyl is defined to include groups having 1, 2 . . . , n−1 or n carbons. For example, “C 2 -C 6  alkenyl” means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and at least 1 carbon-carbon double bond, and up to, for example, 3 carbon-carbon double bonds in the case of a C 6  alkenyl, respectively. Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl. As described above with respect to alkyl, the straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated. An embodiment can be C 2 -C 12  alkenyl, C 3 -C 12  alkenyl, C 2 -C 20  alkenyl, C 3 -C 20  alkenyl, C 2 -C 30  alkenyl, or C 3 -C 30  alkenyl. 
     The term “alkynyl” refers to a hydrocarbon radical straight or branched, containing at least 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present. Thus, C 2 -C n  alkynyl is defined to include groups having 1, 2 . . . , n−1 or n carbons. For example, “C 2 -C 6  alkynyl” means an alkynyl radical having 2 or 3 carbon atoms, and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms, and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms, and up to 3 carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl and butynyl. As described above with respect to alkyl, the straight or branched portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated. An embodiment can be a C 2 -C n  alkynyl. An embodiment can be C 2 -C 12  alkynyl or C 3 -C 2  alkynyl, C 2 -C 20  alkynyl, C 3 -C 20  alkynyl, C 2 -C 30  alkynyl, or C 3 -C 30  alkynyl. 
     As used herein, “aryl” is intended to mean any stable monocyclic or bicyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydro-naphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring. The substituted aryls included in this invention include substitution at any suitable position with amines, substituted amines, alkylamines, hydroxys and alkylhydroxys, wherein the “alkyl” portion of the alkylamines and alkylhydroxys is a C 2 -C n  alkyl as defined hereinabove. The substituted amines may be substituted with alkyl, alkenyl, alkynl, or aryl groups as hereinabove defined. 
     The alkyl, alkenyl, alkynyl, and aryl substituents may be unsubstituted or unsubstituted, unless specifically defined otherwise. For example, a (C 1 -C 6 ) alkyl may be substituted with one or more substituents selected from OH, oxo, halogen, alkoxy, dialkylamino, or heterocyclyl, such as morpholinyl, piperidinyl, and so on. 
     In the compounds of the present invention, alkyl, alkenyl, and alkynyl groups can be further substituted by replacing one or more hydrogen atoms by non-hydrogen groups described herein to the extent possible. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl. 
     The term “substituted” as used herein means that a given structure has a substituent which can be an alkyl, alkenyl, or aryl group as defined above. The term shall be deemed to include multiple degrees of substitution by a named substitutent. Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally. By independently substituted, it is meant that the (two or more) substituents can be the same or different. 
     Examples of substituent groups include the functional groups described above, and halogens (i.e., F, Cl, Br, and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl, n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, such as methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such as phenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) and p-trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy); heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto; sulfanyl groups, such as methylsulfanyl, ethylsulfanyl and propylsulfanyl; cyano; amino groups, such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; and carboxyl. Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally. By independently substituted, it is meant that the (two or more) substituents can be the same or different. 
     In the compounds of the present invention, the substituents may be substituted or unsubstituted, unless specifically defined otherwise. 
     In the compounds of the present invention, alkyl, heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl. 
     It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results. 
     As used herein, a “compound” is a small molecule that does not include proteins, peptides or amino acids. 
     As used herein, an “isolated” compound is a compound isolated from a crude reaction mixture or from a natural source following an affirmative act of isolation. The act of isolation necessarily involves separating the compound from the other components of the mixture or natural source, with some impurities, unknown side products and residual amounts of the other components permitted to remain. Purification is an example of an affirmative act of isolation. 
     “Administering to the subject” or “administering to the (human) patient” means the giving of, dispensing of, or application of medicines, drugs, or remedies to a subject/patient to relieve, cure, or reduce the symptoms associated with a condition, e.g., a pathological condition. The administration can be periodic administration. As used herein, “periodic administration” means repeated/recurrent administration separated by a period of time. The period of time between administrations is preferably consistent from time to time. Periodic administration can include administration, e.g., once daily, twice daily, three times daily, four times daily, weekly, twice weekly, three times weekly, four times weekly and so on, etc. 
     As used herein, “administering” an agent may be performed using any of the various methods or delivery systems well known to those skilled in the art. The administering can be performed, for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery, subcutaneously, intraadiposally, intraarticularly, intrathecally, into a cerebral ventricle, intraventicularly, intratumorally, into cerebral parenchyma or intraparenchchymally. 
     As used herein, “combination” means an assemblage of reagents for use in therapy either by simultaneous or contemporaneous administration. Simultaneous administration refers to administration of an admixture (whether a true mixture, a suspension, an emulsion or other physical combination) of the compound and the anti-cancer agent. The combination may be the admixture or separate containers that are combined just prior to administration. Contemporaneous administration refers to the separate administration, or at times sufficiently close together that a synergistic activity relative to the activity of either the alone is observed. 
     As used herein, “concomitant administration” or administering “concomitantly” means the administration of two agents given in close enough temporal proximately to allow the individual therapeutic effects of each agent to overlap. 
     As used herein, “add-on” or “add-on therapy” means an assemblage of reagents for use in therapy, wherein the subject receiving the therapy begins a first treatment regimen of one or more reagents prior to beginning a second treatment regimen of one or more different reagents in addition to the first treatment regimen, so that not all of the reagents used in the therapy are started at the same time. 
     The following delivery systems, which employ a number of routinely used pharmaceutical carriers, may be used but are only representative of the many possible systems envisioned for administering compositions in accordance with the invention. 
     Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA&#39;s). 
     Other injectable drug delivery systems include solutions, suspensions, gels. Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc). 
     Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprylactone. 
     Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc). 
     Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid). 
     Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. 
     Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA). 
     As used herein, “pharmaceutically acceptable carrier” refers to a carrier or excipient that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. It can be a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the subject. 
     The compounds used in the method of the present invention may be in a salt form. As used herein, a “salt” is a salt of the instant compounds which has been modified by making acid or base salts of the compounds. In the case of compounds used to treat an infection or disease, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the alkaline earth metal salts, sodium, potassium or lithium. The term “pharmaceutically acceptable salt” in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19). 
     As used herein, an “amount” or “dose” of an agent measured in milligrams refers to the milligrams of agent present in a drug product, regardless of the form of the drug product. 
     As used herein, the term “therapeutically effective amount” or “effective amount” refers to the quantity of a component that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. The specific effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. 
     Where a range is given in the specification it is understood that the range includes all integers and 0.1 units within that range, and any sub-range thereof. For example, a range of 77 to 90% is a disclosure of 77, 78, 79, 80, and 81% etc. 
     As used herein, “about” with regard to a stated number encompasses a range of +one percent to −one percent of the stated value. By way of example, about 100 mg/kg therefore includes 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, 100, 100.1, 100.2, 100.3, 100.4, 100.5, 100.6, 100.7, 100.8, 100.9 and 101 mg/kg. Accordingly, about 100 mg/kg includes, in an embodiment, 100 mg/kg. 
     It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, “0.2-5 mg/kg/day” is a disclosure of 0.2 mg/kg/day, 0.3 mg/kg/day, 0.4 mg/kg/day, 0.5 mg/kg/day, 0.6 mg/kg/day etc. up to 5.0 mg/kg/day. 
     Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention. 
     This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter. 
     EXPERIMENTAL DETAILS 
     Abbreviations 
     ACN—Acetonitrile; AUC last —Area under concentration-time curve from time 0 to the last quantifiable concentration; AUC INF —Area under concentration-time curve from time 0 to infinity; BQL—Below quantifiable limit; CL—Clearance; C max —Maximum plasma concentration; hr or Hr—Hour; IV Intravenous; kg—Kilogram; L—Liter; LC Liquid chromatography; LLOQ—Lower limit of quantification; MeOH Methanol; mg Milligram; MS—mass spectrometry; NH 4 OAc—Ammonium acetate; PK—Pharmacokinetics PO—Oral; SD Standard deviation; t 1/2 —Terminal half-life; T max —Time to reach maximum plasma concentration; V ss —Volume of distribution at steady-state 
     Materials and Methods 
     Representative Method for Preparation of Prodrugs: 
     
       
         
         
             
             
         
       
     
     A mixture of exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride (50.0 mmol) and the appropriate alkyl alcohol (110.0 mmol) in toluene is heated at 70-75° C. overnight. The reaction mixture is concentrated on rotary evaporator and the crude solid is triturated with 20 mL of isopropyl ether while heating, and filtered to give a solid. To the mixture of alkyl ester in methylene chloride is added N-hydroxybenzotriazole (5 mmol) followed by N-methylpiperazine (200 mmol) and EDC (75 mmol). The reaction mixture is stirred overnight at room temperature and evaporated to dryness. The product is purified by column chromatography and recrystallization. 
     
       
         
         
             
             
         
       
     
     A mixture of exo-3,6-Epoxy-1,2,3,6-tetrahydrophthalic anhydride (50.0 mmol) and the appropriate alkyl alcohol (110.0 mmol) in toluene is heated at 70-75° C. overnight. The reaction mixture is concentrated on rotary evaporator and the crude solid is triturated with 20 mL of isopropyl ether while heating, and filtered to give a solid. To the mixture of alkyl ester in methylene chloride is added N-hydroxybenzotriazole (5 mmol) followed by N-methylpiperazine (200 mmol) and EDC (75 mmol). The reaction mixture is stirred overnight at room temperature and evaporated to dryness. The product is purified by column chromatography and recrystallization. 
     
       
         
         
             
             
         
       
     
     To the mixture of the acid in methylene chloride is added TEA (1 mmol) followed by the acid (1 mmol) and Alkyl bromide (1.5 mmol). The reaction mixture is stirred overnight at room temperature and evaporated to dryness. The product is purified by column chromatography and recrystallization to afford the pure prodrug. 
     
       
         
         
             
             
         
       
     
     To the mixture of the acid in methylene chloride is added TEA (1 mmol) followed by the alkyl chloride (1.5 mmol). The reaction mixture is stirred overnight at room temperature and evaporated to dryness. The product is purified by column chromatography and recrystallization to afford the pure prodrug. 
     
       
         
         
             
             
         
       
     
     To the mixture of the acid in methylene chloride is added TEA (1 mmol) followed by the alkyl chloride (1.5 mmol). The reaction mixture is stirred overnight at room temperature and evaporated to dryness. The product is purified by column chromatography and recrystallization to afford the pure prodrug. 
     
       
         
         
             
             
         
       
     
     To the mixture of the acid in methylene chloride is added triethylamine (1 mmol) followed by the alkyl chloride (1 mmol). The reaction mixture is stirred overnight at room temperature and diluted with H 2 O. The aqueous phase is extracted (3×) with dichloromethane. The combined organic layer are then washed (3×) with saturated sodium bicarbonate. 
     The organic layer is then concentrated and purified by column chromatography and recrystallization to afford the pure prodrug. 
     Compound 100 has the structure: 
     
       
         
         
             
             
         
       
     
     Compound 105 has the structure: 
     
       
         
         
             
             
         
       
     
     Compound 113 has the structure: 
     
       
         
         
             
             
         
       
     
     Compound 151 has the structure: 
     
       
         
         
             
             
         
       
     
     Compound 153 has the structure: 
     
       
         
         
             
             
         
       
     
     Compound 157 has the structure: 
     
       
         
         
             
             
         
       
     
     Example 1. Pharmacokinetic Study of Compounds 153 and 157 
     The pharmacokinetic studies on 153, 157 and its metabolite endothal were conducted in SD rats. 153 at 1.25 mg/kg and 157 at 1.5 mg/kg were administrated via iv and po route into SD rats. The blood, liver and brain tissue samples were collected at predetermined times from rats. The LC/MS/MS methods were developed to determine 153, 157 and endothal in plasma, liver and brain samples. In the report, the concentrations of 153, 157 and endothal in plasma, liver and brain samples after iv dose were presented. The bioavailability of 153 and 157 was also calculated. Compound were diluted shortly before use in 4% sodium bicarbonate for sterile injection (this is the standard pediatric solution of NaHCO 3  with a pH of about 8.5). 
     A total of 30 female SD rats were assigned to this study as shown in the table below: 
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                   
               
               
                   
                   
                 Animal 
                   
                 Dose 
                 Volume 
                   
                   
               
               
                 Group 
                 Cpds 
                 number 
                 Route 
                 (mg/kg) 
                 (ml/kg) 
                 2 rats/Timepoint 
                 Sampling 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 Control  
                 2 
                   
                   
                   
                   
                   
               
               
                 2 
                 153 
                 12 
                 IV 
                 1.25 mg/kg 
                 5 ml/kg 
                 15 min, 1 hr, 2 hr, 6 hr 
                 Plasma, liver and brain 
               
               
                   
                   
                   
                   
                   
                   
                 10 hr, 24 hr 
                 tissue 
               
               
                 3 
                 157 
                 12 
                 IV 
                 1.5 mg/kg 
                 5 ml/kg 
                 15 min, 1 hr, 2 hr, 6 hr  
                 Plasma, liver and brain 
               
               
                   
                   
                   
                   
                   
                   
                 10 hr, 24 hr 
                 tissue 
               
               
                 4 
                 153 
                 2 
                 PO 
                 1.25 mg/kg 
                 5 ml/kg 
                 30 min, 1 hr, 2 hr, 6 hr  
                 Plasma 
               
               
                   
                   
                   
                   
                   
                   
                 10 hr, 24 hr 
                   
               
               
                 5 
                 157 
                 2 
                 PO 
                 1.5 mg/k9 
                 5 ml/kg 
                 30 min, 1 hr, 2 hr, 6 hr 
                 Plasma 
               
               
                   
                   
                   
                   
                   
                   
                 10 hr, 24 hr 
                   
               
               
                   
               
            
           
         
       
     
     Compound 153 was freshly prepared by diluting the drugs shortly before use in 4% sodium bicarbonate for sterile injection (this is the standard pediatric solution of NaHCO 3  with a pH of about 8.5). The final concentrations of 153 solutions were 0.25 mg/mL. The 153 solutions were administered via iv or po route at dose volume of 5 ml/kg according to the latest body weight. Compound 157 was freshly prepared by diluting the drugs shortly before use in 4% sodium bicarbonate for sterile injection (this is the standard pediatric solution of NaHCO 3  with a pH of about 8.5). The final concentrations of 153 solutions were 0.3 mg/mL. The 157 solutions were administered via iv or po route at dose volume of 5 ml/kg according to the latest body weight. 
     Twelve (12) female SD rats per group were dosed by iv with 153 or 157. The rats were fasted overnight prior to dosing, with free access to water. Foods were withheld for 2 hours post-dose. Blood, liver and brain tissue samples in two animals each group were collected at each time point, within 10% of the scheduled time for each time point. Two extra animals were used for analytic method development. 
     Blood (&gt;0.3 mL) were collected via aorta abdominalis in anaesthetic animals into tubes containing heparin at 15 min, 1, 2, 6, 10 and 24 hours after iv administration. Liver and brain tissues were collected immediately after animal death. The liver and brain tissues were excised and rinsed with cold saline to avoid blood residual. Upon collection, each sample was placed on ice and the blood samples were subsequently centrifuged (4° C., 11000 rpm, 5 min) to separate plasma. 
     The obtained plasma, liver and brain tissue samples were stored at −70° C. until LC-MS/MS analysis. 
     Two (2) female SD rats per group were dosed by po with 153 or 157. The rats were fasted overnight prior to dosing, with free access to water. Foods were withheld for 2 hours post-dose. Blood samples (&gt;0.3 mL) were collected via aorta abdominalis in anaesthetic animals into tubes containing heparin at 30 min, 1, 2, 6, 10 and 24 hours after po administration. 
     Preparation of Plasma, Liver and Brain Samples for Compound 153 
     Frozen unknown plasma samples were thawed at room temperature and vortexed thoroughly. With a pipette, 50 μL of plasma was transferred into a 1.5 mL Eppendorf tube. To each sample, 20 μL IS-D (for blank samples, 20 μL acetonitrile:water (1:1) was added) and 300 ul acetonitrile was added. The sample mixture was vortexed for approximately 3 min. After centrifugation at 10000 rpm for 5 min at 4° C., 100 μL of the upper layer was transferred to a new tube and added 200 μL 0.4% formic acid in water (pH 6.0). The mixture was vortexed for approximately 3 min before injected onto the LC/MS/MS system for analysis. 
     On the day of the assay, the frozen liver and brain samples were thawed unassisted at room temperature. An about 200 mg weighed sample of each thawed tissue was placed into a plastic tube with water (0.6 mL) to facilitate homogenization. Tissue processing was conducted using a homogenizer for approximately 1 min, 200 μl homogenate was transferred into a fresh Eppendorf tube. To each tube, 50 μL IS-D was added and mixed. Then 600 ul acetonitrile was added and the sample mixture was vortexed for approximately 3 min. After centrifugation at 10000 rpm for 5 min at 4° C., 400 μL of the upper layer was transferred to a new tube and evaporate the supernatant to dryness at 35° C. Reconstitute the residue with 200 μL of 0.4% formic acid in water (pH6.0), and vortex for 3 min, submit for LC-MS/MS analysis. 
     Preparation of Plasma, Liver and Brain Samples for Compound 157 
     Frozen unknown plasma samples were thawed at room temperature and vortexed thoroughly. With a pipette, 50 μL of plasma was transferred into a 1.5 mL Eppendorf tube. To each sample, 30 μL IS-D (for blank samples, 20 μL acetonitrile:water (1:1) was added) and 300 ul acetonitrile was added. The sample mixture was vortexed for approximately 3 min. After centrifugation at 10000 rpm for 5 min at 4° C., 100 μL of the upper layer was transferred to a new tube and added 200 μL 0.4% formic acid in water (pH6.0). The mixture was vortexed for approximately 3 min before injected onto the LC/MS/MS system for analysis. 
     On the day of the assay, the frozen liver and brain samples were thawed unassisted at room temperature. An about 200 mg weighed sample of each thawed tissue was placed into a plastic tube with water (0.6 mL) to facilitate homogenization. Tissue processing was conducted using a homogenizer for approximately 1 min, 100 μl homogenate was transferred into a fresh Eppendorf tube. To each tube, 50 μL IS-D was added and mixed. Then 500 ul acetonitrile was added and the sample mixture was vortexed for approximately 3 min. After centrifugation at 10000 rpm for 5 min at 4° C., 100 μL of the upper layer was transferred to a new tube and evaporate the supernatant to dryness at 35° C. Reconstitute the residue with 200 μL of 0.4% formic acid in water (pH 6.0), and vortex for 3 min, submit for LC-MS/MS analysis. 
     Preparation of Plasma, Liver and Brain Samples for Endothal 
     Frozen unknown plasma samples were completely thawed at room temperature and vortexed thoroughly. With a pipette, 50 μL of plasma was transferred into a 2.0 mL Eppendorf tube. 50 μL of 0.1N HCl and 800 μL ethyl acetate were added into each sample. The sample mixture was vortexed for approximately 3 min. After centrifugation at 10000 rpm for 5 min at 4° C., the 600 μl supernatant was transferred into a 1.5 mL Eppendorf tube. The precipitate were extracted with 800 μL ethyl acetate again and 600 μl supernatant was transferred into the same tube, and evaporated into dryness. The residue was reconstituted with 150 μL IS-D (for blank samples, 0.05% formic acid in acetonitrile), and vortexed for 3 min. submit for LC/MS/MS analysis. On the day of the assay, the frozen liver and brain tissues samples were thawed unassisted at room temperature. An about 200 mg weighed sample of each thawed tissue was placed into a plastic tube with water (0.6 mL) to facilitate homogenization. 150 μL of each homogenate was transferred into a fresh Eppendorf tube, 150 μL of 0.1N HCl and 800 μL of acetic ether were added into each homogenate sample. The sample mixture was vortexed and centrifuged at 10000 rpm for 5 min at 4° C. 600 μl supernatant was transferred into a 1.5 mL Eppendorf tube, the precipitate were extracted with 800 μL ethyl acetate again and 600 μl supernatant was transferred into the same tube, and evaporated into dryness. The residue was reconstituted with 200 μL IS-D (for blank samples, 0.05% formic acid in acetonitrile), and vortexed for 3 min. submit for LC/MS/MS analysis. 
     Preparation of Calibration Samples for Compound 153 
     1) Preparation of Calibration Samples for Plasma Samples Analysis 
     Calibration standards were prepared by spiking 25 μL of the 153 standard solutions into 25 μL of heparinized blank rat plasma. The nominal standard concentrations in mouse plasma were 2.00, 4.00, 10.0, 50.0, 100, 500, 900 and 1000 ng/mL. 
     2) Preparation of Calibration Samples for Liver and Brain Tissue Samples Analysis 
     In order to quantify 153 in liver and brain tissue samples, a calibration curve consisting of 8 standard samples was prepared, using the same blank tissue homogenate as sample matrix analyzed (final concentrations: 1.00, 2.00, 5.00, 25.0, 50.0, 250, 450 and 500 ng/g). 
     Preparation of Calibration Samples for Compound 157 
     1) Preparation of Calibration Samples for Plasma Samples Analysis 
     Calibration standards were prepared by spiking 25 μL of the 157 standard solutions into 25 μL of heparinized blank rat plasma. The nominal standard concentrations in mouse plasma were 0.500, 1.00, 2.50, 12.5, 25.0, 125, 225 and 250 ng/mL. 
     2) Preparation of Calibration Samples for Liver and Brain Tissue Samples Analysis 
     In order to quantify 157 in liver and brain tissue samples, a calibration curve consisting of 8 standard samples was prepared, using the same blank tissue homogenate as sample matrix analyzed (final concentrations: 0.500, 1.00, 2.50, 12.5, 25.0, 125, 225 and 250 ng/mL). 
     Preparation of Calibration Samples for Endothal 
     1) Preparation of Calibration Samples for Plasma Samples Analysis 
     Calibration standards were prepared by spiking 25 μL of the endothal standard solutions into 25 μL of heparinized blank rat plasma. The nominal standard concentrations in rat plasma were 20.0, 40.0, 100, 200, 400, 2000, 3600 and 4000 ng/mL. 
     2) Preparation of Calibration Samples for Liver Tissue Samples Analysis 
     In order to quantify endothal in liver tissue samples, a calibration curve consisting of 8 standard samples was prepared, using the same blank tissue homogenate as sample matrix analyzed (final concentrations: 20.0, 40.0, 100, 200, 400, 2000, 3600 and 4000 ng/g). 
     LC/MS/MS System 
     The analysis was performed using a LC-MS/MS system consisting of the following components: HPLC system: Shimadzu UFLC 20-AD XR; MS/MS system: API-5000 triple quadrupole mass spectrometer (Applied Biosystems); Data system: Watson LIMS version 7.2. 
     1) Chromatographic Conditions for Compound 153 
       
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Analytical column: 
                 Luna C18 5 μm, 50 × 2.0 mm 
               
               
                   
                 Mobile phase: 
                 A: 0.4% formic acid in water (pH 6.0) 
               
               
                   
                   
                 B: Acetonitrile 
               
               
                   
                 Injection volume: 
                 20~30 μL 
               
               
                   
                 Run Time: 
                 ~4.5 min 
               
               
                   
                 Flow Rate: 
                 0.5 mL/min 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                   
               
               
                 Time 
                 0 
                 0.5 
                 0.6 
                 2.0 
                 2.1 
                 3.0 
                 3.1 
                 4.5 
               
               
                   
               
             
            
               
                 % B 
                 15 
                 15 
                 45 
                 45 
                 95 
                 95 
                 15 
                 Stop 
               
               
                 Divert 
                 Waste 
                 MS 
                 MS 
                 MS 
                 MS 
                 Waste 
                   
                 Waste 
               
               
                 Valve 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Position 
               
               
                   
               
            
           
         
       
     
     2) Mass Spectrometric Conditions for Compound 153 
       
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Parameters 
                 153 
               
               
                   
                 Ion Spray (IS) 
                 5000 V 
               
               
                   
                 Curtain Gas (CUR) 
                  15 
               
               
                   
                 Temperature (TEM) 
                 500° C. 
               
               
                   
                 Entrance Potential (EP) 
                  10 
               
               
                   
                 Collision Gas (CAD) 
                  6 
               
               
                   
                 Collision Cell Exit Potential (CXP) 
                  15 
               
               
                   
                 Dwell Time (ms) 
                 100 
               
               
                   
                 Gas 1 
                  40 
               
               
                   
                 Gas 2 
                  40 
               
               
                   
                 Declustering potential (DP) 
                 120 
               
               
                   
                 Ionization Mode: 
                 (+) ESI 
               
               
                   
                   
               
            
           
         
       
     
     (CE): 
       
     
       
         
           
               
               
               
               
               
             
               
                   
                   
               
               
                   
                   
                 Precursor 
                 Product 
                   
               
               
                   
                   
                 ion 
                 ion 
                 CE 
               
               
                   
                 Compound 
                 (m/z) 
                 (m/z) 
                 (eV) 
               
               
                   
                   
               
             
            
               
                   
                 153 
                 311.1 
                 169.2 
                 30 
               
               
                   
                 Irbesartan (IS) 
                 429.4 
                 207.2 
                 30 
               
               
                   
                   
               
            
           
         
       
     
     1) Chromatographic Conditions for Compound 157 
       
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Analytical column: 
                 Luna C18 5 μm, 50 × 2.0 mm 
               
               
                   
                 Mobile phase: 
                 A: 0.4% formic acid in water (pH 6.0) 
               
               
                   
                   
                 B: Acetonitrile 
               
               
                   
                 Injection volume: 
                 10 μL 
               
               
                   
                 Run Time: 
                 ~4.5 min 
               
               
                   
                 Flow Rate: 
                 0.5 mL/min 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                   
                   
               
               
                   
                 Time 
                 0 
                 0.5 
                 2.0 
                 2.1 
                 3.0 
                 3.1 
                 4.0 
               
               
                   
                   
               
             
            
               
                   
                 % B 
                 45 
                 45 
                 45 
                 95 
                 95 
                 45 
                 Stop 
               
               
                   
                 Divert 
                 Waste 
                 MS 
                 MS 
                 MS 
                 Waste 
                   
                 Waste 
               
               
                   
                 Valve 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 Position 
               
               
                   
                   
               
            
           
         
       
     
     2) Mass Spectrometric Conditions for Compound 157 
       
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Parameters 
                 157 
               
               
                   
                 Ion Spray (IS) 
                 5000 V 
               
               
                   
                 Curtain Gas (CUR) 
                  15 
               
               
                   
                 Temperature (TEM) 
                 450° C. 
               
               
                   
                 Entrance Potential (EP) 
                  10 
               
               
                   
                 Collision Gas (CAD) 
                  6 
               
               
                   
                 Collision Cell Exit Potential (CXP) 
                  15 
               
               
                   
                 Dwell Time (ms) 
                 100 
               
               
                   
                 Gas 1 
                  40 
               
               
                   
                 Gas 2 
                  40 
               
               
                   
                 Declustering potential (DP) 
                 120 
               
               
                   
                 Ionization Mode: 
                 (+) ESI 
               
               
                   
                   
               
            
           
         
       
     
     (CE): 
       
     
       
         
           
               
               
               
               
               
             
               
                   
                   
               
               
                   
                   
                 Precursor 
                 Product 
                   
               
               
                   
                   
                 ion 
                 ion 
                 CE 
               
               
                   
                 Compound 
                 (m/z) 
                 (m/z) 
                 (eV) 
               
               
                   
                   
               
             
            
               
                   
                 157 
                 367.3 
                 251.0 
                 25 
               
               
                   
                 Verapamil (IS) 
                 455.1 
                 303.3 
                 25 
               
               
                   
                   
               
            
           
         
       
     
     1) Chromatographic Conditions for Endothal 
     Chromatographic separation was carried out at room temperature. 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Analytical column: 
                 Luna HILIC 5 μm, 100 × 2.0 mm 
               
               
                   
                 Mobile phase: 
                 A: 0.1% formic acid in water 
               
               
                   
                   
                 B: Acetonitrile 
               
               
                   
                 Injection volume: 
                 5 μL 
               
               
                   
                 Run Time: 
                 ~2.5 min 
               
               
                   
                 Flow Rate: 
                 0.6 mL/min 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                 Time 
                 0 
                 0.4 
                 2.0 
                 2.5 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 % B 
                 88 
                 88 
                 88 
                 Stop 
               
               
                 Divert Valve Position 
                 Waste 
                 MS 
                 Waste 
                 Waste 
               
               
                   
               
            
           
         
       
     
     2) Mass Spectrometric Conditions for Endothal 
       
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Parameters 
                 endothal 
               
               
                   
                   
               
             
            
               
                   
                 Ion Spray (IS) 
                 −4500 V 
               
               
                   
                 Curtain Gas (CUR) 
                 20 
               
               
                   
                 Temperature (TEM) 
                 450° C. 
               
               
                   
                 Entrance Potential (EP) 
                 −10 
               
               
                   
                 Collision Gas (CAD) 
                 6 
               
               
                   
                 Collision Cell Exit Potential (CXP) 
                 −10 
               
               
                   
                 Dwell Time (ms) 
                 150 
               
               
                   
                 Gas 1 
                 45 
               
               
                   
                 Gas 2 
                 45 
               
               
                   
                 Declustering potential (DP) 
                 −80 
               
               
                   
                 Ionization Mode: 
                 (−) ESI 
               
               
                   
                   
               
            
           
         
       
     
     (CE): 
       
     
       
         
           
               
               
               
               
               
             
               
                   
                   
               
               
                   
                   
                 Precursor 
                 Product 
                 CE 
               
               
                   
                 Compound 
                 ion (m/z) 
                 ion (m/z) 
                 (eV) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Endothal 
                 185 
                 141 
                 −30 
               
               
                   
                 PAH (IS) 
                 192.9 
                 149 
                 −20 
               
               
                   
                   
               
            
           
         
       
     
     Quantification 
     Quantification was achieved by the external standard method for 153, 157 and endothal. Concentrations of the test article were calculated using a weighted least-squares linear regression (W=/x 2 ). 
     Pharmacokinetic Interpretation 
     The pharmacokinetic parameters were evaluated using Watson LIMS (version 7.2), assuming a non-compartmental model for drug absorption and distribution.
         AUC 0-t  (AUC last ) is the area under the plasma concentration-time curve from time zero to last sampling time, calculated by the linear trapezoidal rule.   AUC 0-∞  (AUC INF ) is the area under the plasma concentration-time curve with last concentration extrapolated based on the elimination rate constant.       

     Results 
     The calibration curve of 153 in rat plasma was linear throughout the study in the range of 2.00-1000 ng/mL. The linear equation and the correlation coefficient of calibration curve is y=0.0252x+0.0127 and R 2 =0.9957. 
     The calibration curve of 100 in the tested tissues was linear throughout the study in the range of 1.00-500 ng/g. The linear equation and the correlation coefficient of calibration curve is y=0.0233x+0.0213 and R 2 =0.9939. 
     The calibration curve of 157 in rat plasma was linear throughout the study in the range of 0.50-250 ng/mL. The linear equation and the correlation coefficient of calibration curve is y=0.333x−0.0136 and R 2 =0.9986. 
     The calibration curve of 157 in the tested tissues was linear throughout the study in the range of 0.50-250 ng/g. The linear equation and the correlation coefficient of calibration curve is y=0.0467x+0.0034 and R 2 =0.9989. 
     The calibration curves of endothal in rat plasma were linear throughout the study in the range of 20.0-4000 ng/mL. The linear equation and the correlation coefficient of calibration curve is y=0.00155x−0.00162 and R 2 =0.9986. 
     The calibration curves of endothal in rat liver tissues were linear throughout the study in the range of 20.0-4000 ng/g. The linear equation and the correlation coefficient of calibration curve are y=0.00349x+0.0177 and R 2 =0.997. 
     Following single iv &amp; po administration of 153 to SD rats, plasma, liver and brain tissue concentrations of both 153 and endothal were determined by the LC/MS/MS method described above. The plasma, liver and brain tissue concentrations at each sampling time are listed in Tables 6.1-6.8 and  FIGS. 1A-1B . The calculated pharmacokinetic parameters are listed in Table 6.9-6.12. 
     153 was orally available at 1.25 mg/kg to SD rats, the C max  was 239 ng/mL, AUC was 164 ng·h/ml, and the BA is 55.41%. 
     The mean C max  in plasma was 557 ng/ml following iv administration of 153. The mean C max  in liver and brain were 762.0 ng/kg and 42.7 ng/kg, respectively. AUC last  in plasma was 295 ng·h/ml, with 500 ng·h/g in liver and 39.4 ng·h/g in brain, respectively. T 1/2  in plasma, liver and brain were 0.921 h, 0.626 h and 0.596 h, respectively. 
     As shown in Table 6.5-6.8 and  FIG. 1B , endothal was detectable in plasma and liver samples following single iv administration of 153 at 1.25 mg/kg, whereas not detectable in brain samples. The mean C max  in plasma and liver were 70.5 ng/ml and 2068 ng/ml, respectively. AUC last  in plasma and liver were 378 ng·h/ml and 10820 ng·h/g, respectively. T 1/2  in plasma and liver were 5.20 h and 2.79 h, respectively. 
     Following single iv &amp; po administration of 157 to SD rats, plasma, liver and brain tissue concentrations of both 157 and endothal were determined by the LC/MS/MS method described above. The plasma, liver and brain tissue concentrations at each sampling time are listed in Tables 6.13-6.20 and  FIG. 1C-1D . The calculated pharmacokinetic parameters are listed in Table 6.21-6.24. 157 was poorly orally available at 1.5 mg/kg to SD rats, the C max  was 6.14 ng/mL, AUC was 3.2 ng·h/ml, and the BA was 6.98%. 
     The mean C max  in plasma was 115 ng/ml following iv administration of 157 at 1.5 mg/kg to SD rats. The mean Cmax in liver and brain were 297 ng/kg and 60.0 ng/kg, respectively. AUC last  in plasma was 47.2 ng·h/ml, with 152 ng·h/g in liver and 24.6 ng·h/g in brain, respectively. T 1/2  in plasma, liver and brain were 0.391h, 0.813h and 0.162 h, respectively. 
     As shown in table 6.17-6.20 and  FIG. 1D , endothal was detectable in plasma and liver samples following single iv administration of 157 at 1.5 mg/kg, whereas endothal was not detectable in brain samples. The mean C max  in plasma and liver were 98.1 ng/ml and 3720 ng/ml, respectively. AUC last  in plasma and liver were 374 ng·h/ml and 15025 ng·h/g, respectively. T 1/2  in plasma and liver were 5.94 h and 2.61 h, respectively. 
     153 was orally available at 1.25 mg/kg to SD rats, the C max  was 239 ng/mL, AUC was 164 ng·h/ml, and the BA was 55.41%. The mean C max  in plasma was 557 ng/ml following iv administration of 153. The mean C max  in liver and brain were 762.0 ng/kg and 42.7 ng/kg, respectively. AUC last  in plasma was 295 ng·h/ml, with 500 ng·h/g in liver and 39.4 ng·h/g in brain, respectively. T 1/2  in plasma, liver and brain were 0.921 h, 0.626 h and 0.596 h, respectively. 
     Endothal was detectable in plasma and liver samples following single iv administration of 153 at 1.25 mg/kg. The mean C max  in plasma and liver were 70.5 ng/ml and 2068 ng/ml, respectively. AUC last  in plasma and liver were 378 ng·h/ml and 10820 ng·h/g, respectively. T 1/2  in plasma and liver were 5.20 h and 2.79 h, respectively. However, endothal was undetectable in brain tissue. 
     157 was poorly orally available at 1.5 mg/kg to SD rats, the C max  was 6.14 ng/mL, AUC was 3.2 ng·h/ml, and the BA was 6.98%. 
     The mean C max  in plasma was 115 ng/ml following iv administration of 157 at 1.5 mg/kg to SD rats. The mean C max  in liver and brain were 297 ng/kg and 60.0 ng/kg, respectively. AUC last  in plasma was 47.2 ng·h/ml, with 152 ng·h/g in liver and 24.6 ng·h/g in brain, respectively. T 1/2  in plasma, liver and brain were 0.391h, 0.813h and 0.162 h, respectively. 
     Endothal was detectable in plasma and liver samples following single iv administration of 157 at 1.5 mg/kg. The mean C max  in plasma and liver were 98.1 ng/ml and 3720 ng/ml, respectively. AUC last  in plasma and liver were 374 ng·h/ml and 15025 ng·h/g, respectively. T 1/2  in plasma and liver were 5.94 h and 2.61 h, respectively. However, endothal was undetectable in brain tissue. 
     
       
         
           
               
             
               
                 TABLE 6.1 
               
             
            
               
                   
               
               
                 Analytical data of 153 plasma concentration (ng/mL)  
               
               
                 in SD rats following PO administration. 
               
               
                 1.25 mg/kg Liver concentration (ng/g) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 0.25 
                 872 
                 652 
                 762 
                 155.6 
               
               
                   
                 1 
                 131 
                 121 
                 126 
                 7.1 
               
               
                   
                 2 
                 42 
                 41.2 
                 41.6 
                 0.6 
               
               
                   
                 6 
                 BLQ 
                 BLQ 
                 NA 
                 NA 
               
               
                   
                 10 
                 BLQ 
                 ND 
                 NA 
                 NA 
               
               
                   
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.2 
               
             
            
               
                   
               
               
                 Analytical data of 153 plasma concentration (ng/mL)  
               
               
                 in SD rats following iv administration. 
               
               
                 1.25 mg/kg Plasma concentration (ng/ml) 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 563 
                 550       
                 557 
                 9.2 
               
               
                 1 
                 58 
                 51.4    
                 54.7 
                 4.7 
               
               
                 2 
                 14.8 
                 13      
                 13.9 
                 1.3 
               
               
                 6 
                 1.04 
                 1.02  
                 1.03 
                 0 
               
               
                 10 
                 ND 
                 9.42* 
                 NA 
                 NA 
               
               
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
               
               
                 *Conc. was 9.42 ng/mL, which was abnormal and did not include in the calculation. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.3 
               
             
            
               
                   
               
               
                 Analytical data of 153 liver concentration (ng/g)  
               
               
                 in SD rats following iv administration. 
               
               
                 1.25 mg/kg Liver concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 872 
                 652 
                 762 
                 155.6 
               
               
                 1 
                 131 
                 121 
                 126 
                 7.1 
               
               
                 2 
                 42 
                 41.2 
                 41.6 
                 0.6 
               
               
                 6 
                 BLQ 
                 BLQ 
                 NA 
                 NA 
               
               
                 10 
                 BLQ 
                 ND 
                 NA 
                 NA 
               
               
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.4 
               
             
            
               
                   
               
               
                 Analytical data of 153 brain concentration (ng/g)  
               
               
                 in SD rats following iv administration. 
               
               
                 1.25 mg/kg Brain concentration (ng/g) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 0.25 
                 45 
                 40.3 
                 42.7 
                 3.3 
               
               
                   
                 1 
                 13.9 
                 14.3 
                 14.1 
                 0.3 
               
               
                   
                 2 
                 4.05 
                 4.75 
                 4.4 
                 0.5 
               
               
                   
                 6 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                 10 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.5 
               
             
            
               
                   
               
               
                 Analytical data of endothal plasma concentration 
               
               
                 (ng/ml) in SD rats following po administration of 153. 
               
               
                 Endothal plasma concentration (ng/ml) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 0.25 
                 41.4 
                 40.2 
                 40.8 
                 0.8 
               
               
                   
                 1 
                 53.6 
                 38.9 
                 46.3 
                 10.4 
               
               
                   
                 2 
                 34.5 
                 35.3 
                 34.9 
                 0.6 
               
               
                   
                 6 
                 25.8 
                 20.8 
                 23.3 
                 3.5 
               
               
                   
                 10 
                 BLQ 
                 ND 
                 NA 
                 NA 
               
               
                   
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.6 
               
             
            
               
                   
               
               
                 Analytical data of endothal plasma concentration (ng/ml) 
               
               
                 in SD rats following iv administration of 153. 
               
               
                 Endothal plasma concentration (ng/ml) 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 70.9 
                 63.8 
                 67.4 
                 5 
               
               
                 1 
                 57.1 
                 44.3 
                 50.7 
                 9.1 
               
               
                 2 
                 77.1 
                 56.1 
                 66.6 
                 14.8 
               
               
                 6 
                 42.2 
                 35.4 
                 38.8 
                 4.8 
               
               
                 10 
                 21.7 
                 BLQ 
                 NA 
                 NA 
               
               
                 24 
                 BLQ 
                 BLQ 
                 NA 
                 NA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.7 
               
             
            
               
                   
               
               
                 Analytical data of endothal liver concentration (ng/g) in 
               
               
                 SD rats following iv administration of 153. 
               
               
                 Endothal liver concentration (ng/g) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 0.25 
                 1524 
                 956 
                 1240 
                 401.6 
               
               
                   
                 1 
                 1836 
                 2012 
                 1924 
                 124.5 
               
               
                   
                 2 
                 1912 
                 2224 
                 2068 
                 220.6 
               
               
                   
                 6 
                 492 
                 980 
                 736 
                 345.1 
               
               
                   
                 10 
                 301 
                 256 
                 279 
                 31.8 
               
               
                   
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.8 
               
             
            
               
                   
               
               
                 Analytical data of endothal brain concentration (ng/g) in 
               
               
                 SD rats following iv administration of 153. 
               
               
                 Endothal brain concentration (ng/g) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 0.25 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                 1 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                 2 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                 6 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                 10 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.9 
               
             
            
               
                   
               
               
                 Main pharmacokinetic parameters of 153 in SD rats 
               
               
                 following iv or po administration. 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 Plasma 
                   
                   
                   
                   
                 MRT  
                   
                   
               
               
                   
                 PK 
                 C max   
                 T max   
                 AUC 
                 AUC  0-∞   
                 (0-t) 
                 T 1/2   
                 F 
               
               
                 Dosage 
                 Parameters 
                 ng/mL 
                 Hours 
                 ng * Hours/mL 
                 ng * Hours/mL 
                 Hours 
                 Hours 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1.25 mg/ 
                 1 
                 249 
                 0.5 
                 163 
                 163 
                 0.987 
                 0.33 
                   
               
               
                 kg (PO 
                 2 
                 229 
                 0.5 
                 164 
                 164 
                 1.04 
                 0.355 
                   
               
               
                 Group) 
                 Mean 
                 239 
                 0.5 
                 164 
                 164 
                 1.01 
                 0.343 
                 55.41 
               
               
                 1.25 mg/ 
                 1 
                 563 
                 0.25 
                 303 
                 303 
                 0.666 
                 0.907 
                   
               
               
                 kg (IV 
                 2 
                 550 
                 0.25 
                 288 
                 288 
                 0.647 
                 0.934 
                   
               
               
                 Group) 
                 Mean 
                 557 
                 0.25 
                 295 
                 296 
                 0.657 
                 0.921 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.10 
               
             
            
               
                   
               
               
                 Main pharmacokinetic parameters of 153 in liver &amp; brain 
               
               
                 of SD rats following iv or po administration. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Plasma 
                   
                   
                   
                   
                 MRT 
                   
               
               
                   
                   
                   
                 PK 
                 C max   
                 T max   
                 AUC 
                 AUC  0-∞   
                 (0-t) 
                 T 1/2   
               
               
                 TA 
                 Dosage 
                 Group 
                 Parameters 
                 ng/mL 
                 Hrs 
                 ng * Hrs/mL 
                 ng * Hrs/mL 
                 Hrs 
                 Hrs 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 End. 
                 153 
                 PO 
                 1 
                 53.6 
                 1 
                 189 
                 395 
                 2.8 
                 5.53 
               
               
                   
                 1.25 mg/kg 
                   
                 2 
                 40.2 
                 0.5 
                 169 
                 333 
                 2.72 
                 5.45 
               
               
                   
                   
                   
                 Mean 
                 46.9 
                 0.75 
                 179 
                 364 
                 2.76 
                 5.49 
               
               
                   
                 153 
                 IV 
                 1 
                 77.1 
                 2 
                 482 
                 618 
                 3.93 
                 4.37 
               
               
                   
                 1.25 mg /kg 
                   
                 2 
                 63.8 
                 0.25 
                 274 
                 581 
                 2.74 
                 6.02 
               
               
                   
                   
                   
                 Mean 
                 70.5 
                 1.13 
                 378 
                 600 
                 3.34 
                 5.2 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.11 
               
             
            
               
                   
               
               
                 Main pharmacokinetic parameters of Endothal in SD rats 
               
               
                 following single iv or po administration of 153. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 MRT 
                   
               
               
                   
                 PK 
                 C max   
                 T max   
                 AUC 
                 AUC  0-∞   
                 (0-t) 
                 T 1/2   
               
               
                 Group 
                 Parameters 
                 ng/mL 
                 Hours 
                 ng * Hours/mL 
                 ng * Hours/mL 
                 Hours 
                 Hours 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Liver 
                 1 
                 872 
                 0.25 
                 547 
                 547 
                 0.745 
                 0.609 
               
               
                 1.25 mg/kg 
                 2 
                 652 
                 0.25 
                 453 
                 453 
                 0.825 
                 0.643 
               
               
                 IV 
                 Mean 
                 762 
                 0.25 
                 500 
                 500 
                 0.785 
                 0.626 
               
               
                 Brain 
                 1 
                 45 
                 0.25 
                 39.2 
                 39.2 
                 0.934 
                 0.562 
               
               
                 1.25 mg/kg 
                 2 
                 40.3 
                 0.25 
                 39.5 
                 39.5 
                 1.01 
                 0.629 
               
               
                 IV 
                 Mean 
                 42.7 
                 0.25 
                 39.4 
                 39.35 
                 0.972 
                 0.596 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.12 
               
             
            
               
                   
               
               
                 Main pharmacokinetic parameters of Endothal in SD rats 
               
               
                 liver &amp; brain following single iv administration of 153. 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                 MRT 
                   
               
               
                   
                   
                 PK 
                 C max   
                 T max   
                 AUC 
                 AUC  0-∞   
                 (0-t) 
                 T 1/2   
               
               
                 TA 
                 Dosage 
                 Parameters 
                 ng/mL 
                 Hrs 
                 ng * Hrs/mL 
                 ng * Hrs/mL 
                 Hrs 
                 Hrs 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 End. 
                 153 
                 1 
                 1912 
                 2 
                 9528 
                 10800 
                 3.05 
                 3 
               
               
                   
                 1.25 mg/kg 
                 2 
                 2224 
                 2 
                 12112 
                 13100 
                 3.43 
                 2.57 
               
               
                   
                 (Liver 
                 Mean 
                 2068 
                 2 
                 10820 
                 11950 
                 3.24 
                 2.79 
               
               
                   
                 Group) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 153 
                 1 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                   
                 1.25 mg/kg 
                 2 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                   
                 (Brian 
                 Mean 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                   
                 Group) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.13 
               
             
            
               
                   
               
               
                 Analytical data of 157 plasma concentration (ng/mL)  
               
               
                 in SD rats following PO administration. 
               
               
                 1.5 mg/kg Plasma concentration (ng/ml) 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 0.5 
                 5.92 
                 6.35 
                 6.14 
                 0.3 
               
               
                 1 
                 1.48 
                 1.26 
                 1.37 
                 0.2 
               
               
                 2 
                 0.303 
                 0.194 
                 0.249 
                 0.1 
               
               
                 6 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                 10 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.14 
               
             
            
               
                   
               
               
                 Analytical data of 157 plasma concentration (ng/mL)  
               
               
                 in SD rats following iv administration. 
               
               
                 1.5 mg/kg Plasma concentration (ng/ml) 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 116 
                 114 
                 115 
                 1.4 
               
               
                 1 
                 2.67 
                 3.57 
                 3.12 
                 0.6 
               
               
                 2 
                 0.491 
                 0.556 
                 0.524 
                 0 
               
               
                 6 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                 10 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.15 
               
             
            
               
                   
               
               
                 Analytical data of 157 liver concentration (ng/g)  
               
               
                 in SD rats following iv administration. 
               
               
                 1.5 mg/kg Liver concentration (ng/g) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 0.25 
                 337 
                 257 
                 297 
                 56.6 
               
               
                   
                 1 
                 29.4 
                 17.6 
                 23.5 
                 8.3 
               
               
                   
                 2 
                 6.40 
                 9.72 
                 8.06 
                 2.3 
               
               
                   
                 6 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                 10 
                 ND 
                 BLQ 
                 NA 
                 NA 
               
               
                   
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.16 
               
             
            
               
                   
               
               
                 Analytical data of 157 brain concentration (ng/g)  
               
               
                 in SD rats following iv administration. 
               
               
                 1.5 mg/kg Brain concentration (ng/g) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 0.25 
                 60.0 
                 60.0 
                 60.0 
                 0.0 
               
               
                   
                 1 
                 1.99 
                 2.80 
                 2.40 
                 0.6 
               
               
                   
                 2 
                 BLQ 
                 BLQ 
                 NA 
                 NA 
               
               
                   
                 6 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                 10 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.17 
               
             
            
               
                   
               
               
                 Analytical data of endothal plasma concentration (ng/ml) 
               
               
                 in SD rats following po administration of 157. 
               
               
                 Endothal plasma concentration (ng/ml) 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 93.5 
                 65.4 
                 79.5 
                 19.9 
               
               
                 1 
                 91.8 
                 150 
                 121 
                 41.2 
               
               
                 2 
                 142 
                 68.9 
                 105 
                 51.7 
               
               
                 6 
                 22.7 
                 31.9 
                 27.3 
                 6.5 
               
               
                 10 
                 BLQ 
                 BLQ 
                 NA 
                 NA 
               
               
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.18 
               
             
            
               
                   
               
               
                 Analytical data of endothal plasma concentration (ng/ml) 
               
               
                 in SD rats following iv administration of 157. 
               
               
                 Endothal plasma concentration (ng/ml) 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 76.4 
                 53.4 
                 64.9 
                 16.3 
               
               
                 1 
                 113 
                 83.2 
                 98.1 
                 21.1 
               
               
                 2 
                 91.5 
                 45.7 
                 68.6 
                 32.4 
               
               
                 6 
                 47.7 
                 45 
                 46.4 
                 1.9 
               
               
                 10 
                 BLQ 
                 BLQ 
                 NA 
                 NA 
               
               
                 24 
                 BLQ 
                 BLQ 
                 NA 
                 NA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.19 
               
             
            
               
                   
               
               
                 Analytical data of endothal liver concentration (ng/g) in 
               
               
                 SD rats following iv administration of 157. 
               
               
                 Endothal liver concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 3676 
                 3536 
                 3606 
                 99.0 
               
               
                 1 
                 3124 
                 3764 
                 3444 
                 452.5 
               
               
                 2 
                 2484 
                 2272 
                 2378 
                 149.9 
               
               
                 6 
                 1000 
                 1076 
                 1038 
                 53.7 
               
               
                 10 
                 218 
                 344 
                 281 
                 89.1 
               
               
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.20 
               
             
            
               
                   
               
               
                 Analytical data of endothal brain concentration (ng/g) in 
               
               
                 SD rats following iv administration of 157. 
               
               
                 Endothal brain concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                 1 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                 2 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                 6 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                 10 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                 24 
                 ND 
                 ND 
                 NA 
                 NA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.21 
               
             
            
               
                   
               
               
                 Main pharmacokinetic parameters of 157 in SD rats 
               
               
                 following iv or po administration. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 Plasma 
                   
                   
                   
                   
                 MRT 
                   
                   
               
               
                   
                   
                 PK 
                 C max   
                 T max   
                 AUC 
                 AUC  0-∞   
                 (0-t) 
                 T 1/2   
                 F 
               
               
                 Dosage 
                 Group 
                 Parameters 
                 ng/mL 
                 Hrs 
                 ng * Hrs/mL 
                 ng * Hrs/mL 
                 Hrs 
                 Hrs 
                 % 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 1.5 mg/kg 
                 PO 
                 1 
                 5.92 
                 0.5 
                 3.4 
                 3.4 
                 0.988 
                 0.437 
                   
               
               
                   
                   
                 2 
                 6.35 
                 0.5 
                 3 
                 3 
                 0.903 
                 0.37 
                   
               
               
                   
                   
                 Mean 
                 6.14 
                 0.5 
                 3.2 
                 3.2 
                 0.946 
                 0.404 
                 6.78 
               
               
                   
                 IV 
                 1 
                 116 
                 0.25 
                 47.1 
                 47.1 
                 0.333 
                 0.409 
                   
               
               
                   
                   
                 2 
                 114 
                 0.25 
                 47.3 
                 47.3 
                 0.349 
                 0.373 
                   
               
               
                   
                   
                 Mean 
                 115 
                 0.25 
                 47.2 
                 47.2 
                 0.341 
                 0.391 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.22 
               
             
            
               
                   
               
               
                 Main pharmacokinetic parameters of 157 in SD rats liver &amp; 
               
               
                 brain following iv administration. 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                 MRT 
                   
               
               
                   
                   
                 PK 
                 C max   
                 T max   
                 AUC 
                 AUC  0-∞   
                 (0-t) 
                 T 1/2   
               
               
                 Dosage 
                 Tissues 
                 Parameters 
                 ng/mL 
                 Hrs 
                 ng * Hrs/mL 
                 ng * Hrs/mL 
                 Hrs 
                 Hrs 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1.5 mg/kg 
                 Liver 
                 1 
                 337 
                 0.25 
                 168 
                 168 
                 0.531 
                 0.455 
               
               
                   
                   
                 2 
                 257 
                 0.25 
                 136 
                 136 
                 0.647 
                 1.17 
               
               
                   
                   
                 Mean 
                 297 
                 0.25 
                 152 
                 152 
                 0.589 
                 0.813 
               
               
                   
                 Brain 
                 1 
                 60 
                 0.25 
                 24.2 
                 24.2 
                 0.305 
                 0.153 
               
               
                   
                   
                 2 
                 60 
                 0.25 
                 25 
                 25 
                 0.323 
                 0.17 
               
               
                   
                   
                 Mean 
                 60 
                 0.25 
                 24.6 
                 24.6 
                 0.314 
                 0.162 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.23 
               
             
            
               
                   
               
               
                 Main pharmacokinetic parameters of Endothal in SD rats 
               
               
                 following single iv &amp; po administration of 157. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Plasma 
                   
                   
                   
                   
                 MRT 
                   
               
               
                   
                   
                   
                 PK 
                 C max   
                 T max   
                 AUC 
                 AUC  0-∞   
                 (0-t) 
                 T 1/2   
               
               
                 TA 
                 Dosage 
                 Group 
                 Parameters 
                 ng/mL 
                 Hours 
                 ng * Hours/mL 
                 ng * Hours/mL 
                 Hours 
                 Hours 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Endothal 
                 157 
                 PO 
                 1 
                 142 
                 2 
                 492.6 
                 542 
                 2.15 
                 1.51 
               
               
                   
                 (1.25 
                   
                 2 
                 150 
                 1 
                 365 
                 481 
                 2.32 
                 2.51 
               
               
                   
                 mg/kg) 
                   
                 Mean 
                 146 
                 1.5 
                 429 
                 512 
                 2.24 
                 2.01 
               
               
                   
                 157 
                 IV 
                 1 
                 113 
                 1 
                 452 
                 733 
                 2.52 
                 4.08 
               
               
                   
                 (1.25 
                   
                 2 
                 83.2 
                 1 
                 297 
                 803 
                 2.85 
                 7.8 
               
               
                   
                 mg/kg) 
                   
                 Mean 
                 98.1 
                 1 
                 374 
                 768 
                 2.69 
                 5.94 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6.24 
               
             
            
               
                   
               
               
                 Main pharmacokinetic parameters of Endothal in SD rats 
               
               
                 liver &amp; brain following single iv administration of 157. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                   
                 MRT 
                   
               
               
                   
                   
                   
                 PK 
                 C max   
                 T max   
                 AUC 
                 AUC  0-∞   
                 (0-t) 
                 T 1/2   
               
               
                 TA 
                 Dosage 
                 Tissues 
                 Parameters 
                 ng/mL 
                 Hrs 
                 ng * Hrs/mL 
                 ng * Hrs/mL 
                 Hrs 
                 Hrs 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Endothal 
                 157 
                 Liver 
                 1 
                 3676 
                 0.25 
                 14759 
                 15500 
                 2.97 
                 2.28 
               
               
                   
                 (1.25 
                   
                 2 
                 3764 
                 1 
                 15292 
                 16700 
                 3.12 
                 2.94 
               
               
                   
                 mg/kg 
                   
                 Mean 
                 3720 
                 0.625 
                 15025 
                 16100 
                 3.05 
                 2.61 
               
               
                   
                 IV) 
                 Brain 
                 1 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                   
                   
                   
                 2 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                   
                   
                   
                 Mean 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                   
               
            
           
         
       
     
     Example 2. Pharmacokinetic Study of Compound 105 
     The purpose of this study was to determine the pharmacokinetics parameters of 105 and endothal in plasma and liver following single intravenous administration of 105 to male SD rats. 105 was dissolved in 4% NaHCO 3  in saline for IV administration. The detailed procedure of dosing solution preparation was presented in Appendix I. 
     Animal Source: 
       
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Species 
                 Gender 
                 Vendor 
                 Certificate No. 
               
               
                   
               
             
            
               
                 SD rats 
                 Male 
                 SLAC Laboratory 
                 SCXK (SH) 2007-0005 
               
               
                   
                   
                 Animal Co. LTD 
               
               
                   
               
            
           
         
       
     
     Thirteen (13) animals were placed on the study. The animals in IV arm were free access to food and water. One extra animal was used for blank liver and plasma generation (5 mL per animal). The resulting blank liver and plasma was then applied to the development of bioanalytical method and sample bioanalysis for the entire study. 
     In-Life Study Design 
       
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                   
               
               
                   
                 Body 
                   
                 Route 
                 Dose 
                 Dose 
                 Dose 
                   
               
               
                 Treatment 
                 Weight 
                 No. of 
                 of 
                 Level* 
                 Conc. 
                 Volume 
                   
               
               
                 Group 
                 (g) 
                 Animals 
                 Admin. 
                 (mg/kg) 
                 (mg/mL) 
                 (mL/kg) 
                 Time points 
               
               
                   
               
             
            
               
                 1 
                 220-255 
                 12 
                 IV 
                 1 
                 1 
                 1 
                 Sampling at 0.25, 1, 
               
               
                   
                   
                   
                   
                   
                   
                   
                 2, 6, 10 and 24 hr 
               
               
                   
                   
                   
                   
                   
                   
                   
                 post dose. 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Terminally collect 
               
               
                   
                   
                   
                   
                   
                   
                   
                 plasma and liver 
               
               
                   
                   
                   
                   
                   
                   
                   
                 samples from the 
               
               
                   
                   
                   
                   
                   
                   
                   
                 same animal. 
               
               
                   
               
               
                 *Dose was expressed as free base of 105. 
               
            
           
         
       
     
     Dosing, Sampling, Sample Processing and Sample Storage 
     The IV injection was conducted via foot dorsal vein. Animals were free access to food and water before dose. 
     The animal is restrained manually. Approximately 150 μL of blood/time point is collected into sodium heparin tube via cardiac puncture for terminal bleeding (anesthetized under carbon dioxide). Blood sample will be put on ice and centrifuged to obtain plasma sample (2000 g, 5 min under 4° C.) within 10 minutes. 
     The animal will be euthanized with carbon dioxide inhalation. Open abdominal cavity with scissor to expose internal organs. Hold the carcass in an upright position and allow the organs to fall forward. Cut the connective tissues and remove the organs. Then the organs are rinsed with cold saline, dried on filtrate paper, placed into a screw-top tube and weighed, snap frozen by placing into dry-ice immediately. 
     Plasma and liver samples were stored at approximately −80° C. until analysis. The backup samples will be discarded after three weeks after in-life completion unless requested. The unused dosing solutions will be discarded within three weeks after completion of the study 
     LC-MS-MS Analysis Analytical Method for 105 
       
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 Instrument 
                 UPLC/MS-MS-010 (API-4000) 
               
               
                 Matrix 
                 SD rat plasma and liver homogenate 
               
               
                 Analyte(s) 
                 Compound 105 
               
               
                 Internal 
                 Dexamethasone 
               
               
                 standard(s) 
                   
               
               
                 MS 
                 ESI: Positive ion 
               
               
                 conditions 
                 MRM detection 
               
               
                   
                 LB-105: [M + H] +  m/z 283.3→ 265.2 
               
               
                   
                 Dexamethasone: [M + H] +  m/z 393.3 ® 373.1 
               
               
                   
                 Mobile Phase A: H 2 0-0.1% FA-5 mM NH 4 OAc 
               
               
                   
                 Mobile Phase B: ACN 
               
            
           
           
               
               
               
            
               
                   
                 Time (min) 
                 Mobile Phase B (%) 
               
               
                   
                 0.20 
                  2.00 
               
               
                   
                 1.00 
                 95.0  
               
               
                   
                 1.60 
                 95.0  
               
               
                   
                 1.61 
                  2.00 
               
               
                   
                 2.20 
                 stop 
               
            
           
           
               
               
            
               
                   
                 Column: ACQUITY UPLC HSS T3 (2.1 × 50 mm,  
               
               
                   
                 1.8 μm) 
               
               
                   
                 Flow rate: 0.60 mL/min 
               
               
                   
                 Column temperature: 60° C. 
               
               
                   
                 Retention time: 
               
               
                   
                 LB-105: 0.97 min 
               
               
                   
                 Dexamethasone: .1.25 min 
               
               
                 HPLC 
                 For plasma samples: An aliquot of 30 μL sample 
               
               
                 conditions 
                 was added with 100 μL IS (Dexamethasone, 100 
               
               
                   
                 ng/mL in ACN). The mixture was vortexed for 10 
               
               
                   
                 min at 750 rpm and centrifuged at 6000 rpm for 
               
               
                   
                 10 min. An aliquot of 3 μL supernatant was 
               
               
                   
                 injected for LC-MS/MS analysis. 
               
               
                   
                 For diluted samples: An aliquot of 3 μL plasma 
               
               
                   
                 sample was diluted with 27 μL blank plasma. 
               
               
                   
                 The following processing procedure was the 
               
               
                   
                 same as those un-diluted plasma samples. 
               
               
                   
                 For all the samples preparation, allow 
               
               
                   
                 calibration, quality control, blanks, and test 
               
               
                   
                 samples to thaw at 4° C. (nominal). And keep 
               
               
                   
                 each step on an ice bath or at 4° C. 
               
               
                 Calibration 
                 10.00-3000 ng/mL for LB-105 in SD rat plasma 
               
               
                 curve 
                 and liver homogenate. 
               
               
                   
               
            
           
         
       
     
     LC-MS-MS Analysis Analytical Method for Endothal 
       
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 Instrument 
                 UPLC/MS-MS-015 (API-5500, Q-trap) 
               
               
                 Matrix 
                 SD rat plasma and liver homogenate 
               
               
                 Analyte(s) 
                 Endothal 
               
               
                 Internal 
                 Diclofenac 
               
               
                 standard(s) 
                   
               
               
                 MS conditions  
                 ESI: Negative ion 
               
               
                   
                 MRM detection 
               
               
                   
                 Endothal: [M − H]− m/z 184.9 → 141.0 
               
               
                   
                 Diclofenac: [M − H]− m/z 294.2 → 249.9 
               
               
                   
                 Mobile Phase A: H 2 0-0.1% FA-5 mM NH 4 OAc 
               
               
                   
                 Mobile Phase B: ACN 
               
            
           
           
               
               
               
            
               
                   
                 Time (min) 
                 Mobile Phase B (%) 
               
               
                   
                 0.40 
                  2.00 
               
               
                   
                 1.00 
                 85.0  
               
               
                   
                 1.50 
                 85.0  
               
               
                   
                 1.51 
                  2.00 
               
               
                   
                 2.00 
                 stop 
               
            
           
           
               
               
            
               
                   
                 Column: ACQUITY UPLC HSS T3 (2.1 × 50 mm,  
               
               
                   
                 1.8 μm) 
               
               
                   
                 Flow rate: 0.60 mL/min 
               
               
                   
                 Column temperature: 60° C. 
               
               
                   
                 Retention time: 
               
               
                   
                 Endothal: 0.87 min 
               
               
                   
                 Diclofenac: 1.28 min 
               
               
                 HPLC 
                 For plasma samples: 
               
               
                 conditions 
                 An aliquot of 30 μL sample was added with 100 μL IS 
               
               
                   
                 (Diclofenac, 100 ng/mL in ACN). The mixture was 
               
               
                   
                 vortexed for 10 min at 750 rpm and centrifuged at 
               
               
                   
                 6000 rpm for 10 min. An aliquot of 3 μL supernatant 
               
               
                   
                 was injected for LC-MS/MS analysis. 
               
               
                   
                 For liver homogenate samples: 
               
               
                   
                 The liver samples were homogenized with 3 volumes 
               
               
                   
                 (v/w) of homogenizing solution PBS (pH 7.4) for 2 
               
               
                   
                 mins. An aliquot of 30 μL tissue homogenate sample 
               
               
                   
                 was added with 100 μL IS (Diclofenac, 100 ng/mL in 
               
               
                   
                 ACN). Vortex at 750 rpm for 10 min and centrifuged 
               
               
                   
                 at 6000 rpm for 10 min. An aliquot of 3 μL 
               
               
                   
                 supernatant was injected for LC-MS/MS analysis. 
               
               
                   
                 For all the samples preparation, allow calibration, 
               
               
                   
                 quality control, blanks, and test samples to thaw at 
               
               
                   
                 4° C. (nominal). And keep each step on an ice bath or 
               
               
                   
                 at 4° C. 
               
               
                 Calibration 
                 20.00-3000 ng/mL for Endothal in SD rat plasma and 
               
               
                 curve 
                 liver homogenate.. 
               
               
                   
               
            
           
         
       
     
     Pharmacokinetic Analysis 
     Software: The PK parameters were determined by non-compartmental model of non-compartmental analysis tool, Pharsight Phoenix WinNonlin® 6.2 software. 
     “BQL” rule: Concentration data under 80% of LLOQ (LLOQ=10.00 ng/mL in rat plasma and liver homogenate for 105, and 20.00 ng/mL for Endothal) was replaced with “BQL” and excluded from graphing and PK parameters estimation. Concentration data within 80%-120% of LLOQ was considered within normal instrumental variation and presented in the results. 
     Terminal t 1/2  calculation: Time points were automatic selected by “best fit” model for terminal half life estimation as the first option. Manual selection was applied when “best fit” could not well define the terminal phase. 
     Clinical Observations 
     The concentration-time data and pharmacokinetic parameters of 105 and Endothal in rat plasma and liver after IV administration were listed in Tables 7.1 to 7.8, and illustrated in  FIGS. 2A to 2C . 
     
       
         
           
               
             
               
                 TABLE 7.1 
               
             
            
               
                   
               
               
                 Individual and mean plasma concentration-time data of 105 
               
               
                 after an IV dose of 1 mg/kg in male SD rats 
               
            
           
           
               
               
               
            
               
                 Time (hr) 
                 Individual 
                 Mean (ng/mL) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 0.25 
                 1930 
                 1530 
                 1730 
               
               
                 1 
                 263 
                 228 
                 246 
               
               
                 2 
                 45.2 
                 21.5 
                 33.4 
               
               
                 6 
                 BQL 
                 BQL 
                 BQL 
               
               
                 10 
                 BQL 
                 BQL 
                 BQL 
               
               
                 24 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
               
               
                 LLOQ of 105 in plasma sample is 10.0 ng/mL. 
               
               
                 ULOQ of 105 in plasma sample is 3000 ng/mL. 
               
               
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7.2 
               
             
            
               
                   
               
               
                 Individual and mean liver concentration-time data of 105 
               
               
                 after an IV dose of 1 mg/kg in male SD rats 
               
            
           
           
               
               
               
            
               
                 Time (hr) 
                 Individual 
                 Mean (ng/g) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 0.25 
                 1070 
                 988 
                 1029 
               
               
                 1 
                 576 
                 446 
                 511 
               
               
                 2 
                 99.2 
                 131 
                 115 
               
               
                 6 
                 BQL 
                 BQL 
                 BQL 
               
               
                 10 
                 BQL 
                 BQL 
                 BQL 
               
               
                 24 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
               
               
                 The liver sample is homogenized with 3 volumes (v/w) of homogenizing solution (PBS PH 7.4). 
               
               
                 Liver concentration = liver homogenate conc. × 4, assuming 1 g wet liver tissue equals to 1 mL. 
               
               
                 LLOQ of 105 in liver homogenate sample is 10.0 ng/mL. 
               
               
                 ULOQ of 105 in liver homogenate sample is 3000 ng/mL. 
               
               
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7.3 
               
             
            
               
                   
               
               
                 Liver-plasma concentration ratio of 105 after an IV dose 
               
               
                 of 1 mg/kg in male SD rats 
               
            
           
           
               
               
               
            
               
                 Time (hr) 
                 Individual 
                 Mean 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 0.25 
                 0.554 
                 0.646 
                 0.600 
               
               
                 1 
                 2.19 
                 1.96 
                 2.07 
               
               
                 2 
                 2.19 
                 6.09 
                 4.14 
               
               
                 6 
                 NA 
                 NA 
                 NA 
               
               
                 10 
                 NA 
                 NA 
                 NA 
               
               
                 24 
                 NA 
                 NA 
                 NA 
               
               
                   
               
               
                 NA: Not Applicable 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7.4 
               
             
            
               
                   
               
               
                 Individual and mean plasma concentration-time data of 
               
               
                 Endothal after an IV dose of 1 mg/kg 105 in SD rats 
               
            
           
           
               
               
               
            
               
                 Time (hr) 
                 Individual 
                 Mean (ng/mL) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 0.25 
                 263 
                 188 
                 226 
               
               
                 1 
                 69.7 
                 45.2 
                 57.5 
               
               
                 2 
                 23.2 
                 BQL 
                 23.2 
               
               
                 6 
                 BQL 
                 BQL 
                 BQL 
               
               
                 10 
                 BQL 
                 21.9 
                 21.9 
               
               
                 24 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
               
               
                 LLOQ of Endothal in plasma sample is 20.0 ng/mL. 
               
               
                 ULOQ of Endothal in plasma sample is 3000 ng/mL. 
               
               
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7.5 
               
             
            
               
                   
               
               
                 Individual and mean liver concentration-time data of 
               
               
                 Endothal after an IV dose of 1 mg/kg 105 in SD rats 
               
            
           
           
               
               
               
            
               
                 Time (hr) 
                 Individual 
                 Mean (ng/g) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 0.25 
                 475 
                 462 
                 469 
               
               
                 1 
                 541 
                 386 
                 464 
               
               
                 2 
                 151 
                 304 
                 228 
               
               
                 6 
                 76.9 
                 163 
                 120 
               
               
                 10 
                 70.0 
                 156 
                 113 
               
               
                 24 
                 BQL 
                 63.8 
                 63.8 
               
               
                   
               
               
                 The liver sample is homogenized with 3 volumes (v/w) of homogenizing solution (PBS PH 7.4). 
               
               
                 Liver concentration = liver homogenate conc. × 4, assuming 1 g wet liver tissue equals to 1 mL. 
               
               
                 LLOQ of Endothal in liver homogenate sample is 20.0 ng/mL. 
               
               
                 ULOQ of Endothal in liver homogenate sample is 3000 ng/mL. 
               
               
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7.6 
               
             
            
               
                   
               
               
                 Liver-plasma concentration ratio of Endothal after an IV 
               
               
                 dose of 1 mg/kg 105 in SD rats 
               
            
           
           
               
               
               
            
               
                 Time (hr) 
                 Individual 
                 Mean 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 0.25 
                 1.81 
                 2.46 
                 2.13 
               
               
                 1 
                 7.76 
                 8.54 
                 8.15 
               
               
                 2 
                 6.51 
                 NA 
                 6.51 
               
               
                 6 
                 NA 
                 NA 
                 NA 
               
               
                 10 
                 NA 
                 7.12 
                 7.12 
               
               
                 24 
                 NA 
                 NA 
                 NA 
               
               
                   
               
               
                 NA: Not Applicable 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7.7 
               
             
            
               
                   
               
               
                 Mean Pharmacokinetics Parameters of 105 after an IV dose 
               
               
                 of 1 mg/kg in male SD rats 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Dosing 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 Route 
                 AUC (0-t)   
                 AUC (0-∞)   
                 t 1/2z   
                 T max   
                 C max   
                 CL 
                 V SS   
                 MRT INF   
                 AUC last-liver / 
               
               
                 Matrix 
                 (Dose) 
                 h * ng/mL 
                 h * ng/mL 
                 hr 
                 hr 
                 ng/mL 
                 L/hr/kg 
                 L/kg 
                 hr 
                 AUC last-plasma   
               
               
                   
               
               
                 Plasma 
                 IV (1 
                 1511 
                 1526 
                 0.309 
                 NA 
                 NA 
                 0.655 
                 0.215 
                 0.328 
                 NA 
               
               
                 Liver 
                 mg/kg) 
                 1019 
                 NA 
                 NA 
                 0.25 
                 1029 
                 NA 
                 NA 
                 NA 
                 67.4 
               
               
                   
               
               
                 NA: Not Applicable 
               
            
           
         
       
     
                     TABLE 7.8                  Mean Pharmacokinetics Parameters of Endothal after an IV       dose of 1 mg/kg 105 in male SD rats                                                 Dosing                                   Route   AUC (0-t)     AUC (0-∞)     t 1/2     T max     C max     AUC last-liver /       Matrix   (Dose)   h * ng/mL   h * ng/mL   hr   hr   ng/mL   AUC last-plasma                                                       Plasma   IV (1 mg/kg)   355   673   10.1   0.250   226   NA       Liver       3152   4896   19.0   0.250   469   888               NA: Not Applicable            
IV-1 mg/kg 105
 
     After an IV dose of 105 at 1 mg/kg in male SD rats, concentration of 105 in rat plasma declined with a terminal half life (T 1/2 ) of 0.309 hours. The area under curve from time 0 to last time point (AUC last ) and from time 0 to infinity (AUC INF ) were 1511 and 1526 hr*ng/mL respectively. The total clearance CL and volume of distribution at steady state V ss  were 0.655 L/hr/kg and 0.215 L/kg, respectively. 
     The mean values of C max  in liver was 1029 ng/g and corresponding T max  value was 0.25 hr. The mean value of AUC (0-last)  was 1019 ng/g*hr. AUC (0-t)  ratio of liver over plasma was 67.4. 
     Endothal 
     Following intravenous administration of 1 mg/kg 105 to Male SD rats, concentration of Endothal in rat plasma declined with a terminal half-life (T 1/2 ) of 10.1 hours. The area under curve from time 0 to last time point (AUC last ) and from time 0 to infinity (AUC INF ) were 355 and 673 hr*ng/mL respectively. The mean values of C max  and T max  in plasma were 226 ng/mL and 0.25 hr, respectively. 
     The mean values of C max  in liver was 469 ng/g and corresponding T max  value was 0.25 hr. The mean value of AUC (0-last)  and AUC (0-∞)  were 3152 and 4896 ng/g*hr, respectively. AUC (0-t)  ratio of liver over plasma was 888. 
     Example 3. Pharmacokinetic Study of Compound 113 
     The purpose of this study was to determine the pharmacokinetics parameters of 113, 100 and Endothal following single intravenous (IV) or oral (PO) administrations of 113 to male SD rats. 113 was dissolved in 4% NaHCO 3  in saline for IV administration. The detailed procedure of dosing solution preparation was presented in Appendix I. 
     Animal Source 
       
                                         Species   Gender   Vendor   Certificate No.                  SD rats   Male   SLAC Laboratory   SCXK (SH)               Animal Co. LTD   2007-0005                    
15 animals were placed on the study. The animals in IV arm were free access to food and water. For PO dose group, the animals were fasted overnight prior to dosing and the food was resumed 4 hours postdose.
 
     One extra animal was used for blank liver, brain and plasma generation (5 mL per animal). The resulting blank liver, brain and plasma were then applied to the development of bioanalytical method and sample bioanalysis for the entire study. 
     In-Life Study Design 
       
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                   
               
               
                   
                 Body 
                   
                 Route 
                 Dose 
                 Dose 
                 Dose 
                   
               
               
                 Treatment 
                 Weight 
                 No. of 
                 of 
                 Level* 
                 Conc. 
                 Volume 
                   
               
               
                 Group 
                 (g) 
                 Animals 
                 Admin. 
                 (mg/kg) 
                 (mg/mL) 
                 (mL/kg) 
                 Time points 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 275- 
                 12 
                 IV 
                 1.4 
                 1.4 
                 1 
                 Sampling at 0.25, 1, 
               
               
                   
                 295 
                   
                   
                   
                   
                   
                 2, 6, 10 and 24 hr 
               
               
                   
                   
                   
                   
                   
                   
                   
                 post dose. Terminally 
               
               
                   
                   
                   
                   
                   
                   
                   
                 collect plasma, brain 
               
               
                   
                   
                   
                   
                   
                   
                   
                 and liver samples from 
               
               
                   
                   
                   
                   
                   
                   
                   
                 the same animal. 
               
               
                 2 
                 275- 
                 2 
                 PO 
                 1.4 
                 0.14 
                 10 
                 Sampling at 0.25, 1, 
               
               
                   
                 295 
                   
                   
                   
                   
                   
                 2, 6, 10 and 24 hr 
               
               
                   
                   
                   
                   
                   
                   
                   
                 post dose. Serial 
               
               
                   
                   
                   
                   
                   
                   
                   
                 bleeding from the same 
               
               
                   
                   
                   
                   
                   
                   
                   
                 animal for plasma 
               
               
                   
                   
                   
                   
                   
                   
                   
                 only. 
               
               
                   
               
               
                 *Dose was expressed as free base of 113. 
               
            
           
         
       
     
     Dosing, Sampling, Sample Processing and Sample Storage 
     The IV injection was conducted via foot dorsal vein. PO via oral gavage. 
     Blood collection: The animal is restrained manually. Approximately 200 μL of blood/time point is collected into sodium heparin tube via cardiac puncture for terminal bleeding (anesthetized under carbon dioxide). Blood sample will be put on ice and centrifuged to obtain plasma sample (2000 g, 5 min under 4° C.) within 10 minutes. 
     Liver collection: The animal will be euthanized with carbon dioxide inhalation. Open abdominal cavity with scissor to expose internal organs. Hold the carcass in an upright position and allow the organs to fall forward. Cut the connective tissues and remove the organs. Then the organs are rinsed with cold saline, dried on filtrate paper, placed into a screw-top tube and weighed, snap frozen by placing into dry-ice immediately. 
     Brain collection: Make a mid-line incision in the animals scalp and retract the skin. Using small bone cutters and rongeurs, remove the skull overlying the brain. Remove the brain using a spatula and rinse with cold saline, dried on filtrate paper, placed into a screw-top tube and weighed, snap frozen by placing into dry-ice immediately. Brain tissue will be homogenized for 2 min with 3 volumes (v/w) of homogenizing solution (PBS pH 7.4) right before analysis. Plasma, brain and liver samples were stored at approximately −80° C. until analysis. The backup samples will be discarded after three weeks after in-life completion unless requested. The unused dosing solutions will be discarded within three weeks after completion of the study. 
     LC-MS-MS Analysis Analytical Method for 113 
       
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 Instrument 
                 UPLC/MS-MS-010 (API-4000) 
               
               
                 Matrix 
                 SD rat plasma, brain and liver homogenate 
               
               
                 Analyte(s) 
                 113 
               
               
                 Internal 
                 Dexamethasone/Propranolol 
               
               
                 standard(s) 
                   
               
               
                 MS conditions 
                 ESI: Positive ion 
               
               
                   
                 MRM detection 
               
               
                   
                 LB-113: [M + H] +  m/z 399.1→ 251.2 
               
               
                   
                 Dexamethasone:[M + H] +  m/z 393.3 ® 373.1 
               
               
                   
                 Propranolol: [M + H] +  m/z 260.2 → 116.1 
               
               
                   
                 Mobile Phase A: H 2 0-0.1% FA-5 mM NH 4 OAc 
               
               
                   
                 Mobile Phase B: ACN 
               
            
           
           
               
               
               
            
               
                   
                 Time (min)  
                 Mobile Phase B (%) 
               
               
                   
                 0.20 
                  2.00 
               
               
                   
                 0.60 
                 95.0  
               
               
                   
                 1.20 
                 95.0  
               
               
                   
                 1.21 
                  2.00 
               
               
                   
                 1.80 
                 stop 
               
            
           
           
               
               
            
               
                   
                 Column: ACQUITY UPLC HSS T3 (2.1 × 50 mm,  
               
               
                   
                 1.8 μm) 
               
               
                   
                 Flow rate: 0.60 mL/min 
               
               
                   
                 Column temperature: 60° C. 
               
               
                   
                 Retention time: 
               
               
                   
                 LB-113: 0.95 min 
               
               
                   
                 Dexamethasone: .1.02 min 
               
               
                   
                 Propranolol: 0.92 min 
               
               
                 HPLC 
                 For plasma samples: 
               
               
                 conditions 
                 An aliquot of 30 μL sample was added with 100 μL IS 
               
               
                   
                 (Dexamethasone, 100 ng/mL and Propranolol, 50 ng/mL  
               
               
                   
                 in ACN). The mixture was vortexed for 10 min at 750  
               
               
                   
                 rpm and centrifuged at 6000 rpm for 10 min. An aliquot  
               
               
                   
                 of 1 μL supernatant was injected for LC-MS/MS analysis. 
               
               
                   
                 For diluted plasma samples: 
               
               
                   
                 An aliquot of 3 μL plasma sample was diluted with 27 μL  
               
               
                   
                 blank plasma. The following processing procedure was  
               
               
                   
                 the same as those un-diluted plasma samples. 
               
               
                   
                 For brain homogenate samples: 
               
               
                   
                 The brain samples were homogenized with 3 volumes  
               
               
                   
                 (v/w) of homogenizing solution PBS (pH 7.4) for 2 mins.  
               
               
                   
                 An aliquot of 30 μL tissue homogenate sample was added  
               
               
                   
                 with 100 μL IS (Dexamethasone, 100 ng/mL and  
               
               
                   
                 Propranolol, 50 ng/mL in ACN). Vortex at 750 rpm for 10  
               
               
                   
                 min and centrifuged at 6000 rpm for 10 min. An aliquot  
               
               
                   
                 of 1 μL supernatant was injected for LC-MS/MS analysis. 
               
               
                   
                 For liver homogenate samples: 
               
               
                   
                 The liver samples were homogenized with 3 volumes  
               
               
                   
                 (v/w) of homogenizing solution PBS (pH 7.4) for 2 mins.  
               
               
                   
                 An aliquot of 30 μL tissue homogenate sample was added  
               
               
                   
                 with 100 μL IS (Dexamethasone, 100 ng/mL and  
               
               
                   
                 Propranolol, 50 ng/mL in ACN). Vortex at 750 rpm for  
               
               
                   
                 10 min and centrifuged at 6000 rpm for 10 min. An  
               
               
                   
                 aliquot of 1 μL supernatant was injected for LC-MS/MS  
               
               
                   
                 analysis. 
               
               
                   
                 For all the samples preparation, allow calibration, quality 
               
               
                   
                 control, blanks, and test samples to thaw at 4° C.  
               
               
                   
                 (nominal). And keep each step on an ice bath or at 4° C. 
               
               
                 Calibration 
                 1.00-3000 ng/mL for LB-113 in SD rat plasma, brain and  
               
               
                 curve 
                 liver homogenate. 
               
               
                   
               
            
           
         
       
     
     LC-MS-MS Analysis Analytical Method for Endothal 
       
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 Instrument 
                 UPLC/MS-MS-015 (API-5500, Q-trap) 
               
               
                 Matrix 
                 SD rat plasma, brain and liver homogenate 
               
               
                 Analyte(s) 
                 Endothal 
               
               
                 Internal 
                 Diclofenac 
               
               
                 standard(s) 
                   
               
               
                 MS 
                 ESI: Negative ion 
               
               
                 conditions 
                   
               
               
                   
                 MRM detection 
               
               
                   
                 Endothal: [M − H]− m/z 184.9 → 141.0 
               
               
                   
                 Diclofenac: [M − H]− m/z 294.2 → 249.9 
               
               
                   
                 Mobile Phase A: H 2 O-0.1% FA-5 mM NH 4 OAc 
               
               
                   
                 Mobile Phase B: ACN 
               
            
           
           
               
               
               
            
               
                   
                 Time (min) 
                 Mobile Phase B (%) 
               
               
                   
                 0.40 
                 2.00 
               
               
                   
                 1.00 
                 85.0 
               
               
                   
                 1.50 
                 85.0 
               
               
                   
                 1.51 
                 2.00 
               
               
                   
                 2.00 
                 stop 
               
            
           
           
               
               
            
               
                   
                 Column: ACQUITY UPLC HSS T3 (2.1 × 50 mm, 1.8 μm)  
               
               
                   
                 Flow rate: 0.60 mL/min  
               
               
                   
                 Column temperature: 60° C.  
               
               
                   
                 Retention time:  
               
               
                   
                 Endothal: 0.87 min  
               
               
                   
                 Diclofenac : 1.28 min  
               
               
                 HPLC  
                 For plasma samples:  
               
               
                 conditions  
                 An aliquot of 30 μL sample was added with 100 μL IS  
               
               
                   
                 (Diclofenac, 100 ng/mL in ACN). The mixture was vortexed  
               
               
                   
                 for 10 min at 750 rpm and centrifuged at 6000 rpm for 10  
               
               
                   
                 min. An aliquot of 3 μL supernatant was injected for LC-  
               
               
                   
                 MS/MS analysis. 
               
               
                   
                 For brain homogenate samples:  
               
               
                   
                 The brain samples were homogenized with 3 volumes (v/w) of  
               
               
                   
                 homogenizing solution PBS (pH 7.4) for 2 mins. An aliquot of  
               
               
                   
                 30 μL tissue homogenate sample was added with 100 μL IS  
               
               
                   
                 (Diclofenac, 100 ng/mL in ACN). Vortex at 750 rpm for 10  
               
               
                   
                 min and centrifuged at 6000 rpm for 10 min. An aliquot of 3  
               
               
                   
                 μL supernatant was injected for LC-MS/MS analysis.  
               
               
                   
                 For liver homogenate samples:  
               
               
                   
                 The liver samples were homogenized with 3 volumes (v/w) of  
               
               
                   
                 homogenizing solution PBS (pH 7.4) for 2 mins. An aliquot of  
               
               
                   
                 30 μL tissue homogenate sample was added with 100 μL IS  
               
               
                   
                 (Diclofenac, 100 ng/mL in ACN). Vortex at 750 rpm for 10  
               
               
                   
                 min and centrifuged at 6000 rpm for 10 min. An aliquot of 3  
               
               
                   
                 μL supernatant was injected for LC-MS/MS analysis.  
               
               
                   
                 For all the samples preparation, allow calibration, quality  
               
               
                   
                 control, blanks, and test samples to thaw at 4° C. (nominal).  
               
               
                   
                 And keep each step on an ice bath or at 4° C.  
               
               
                 Calibration  
                 20.00-3000 ng/mL for Endothal in SD rat plasma, brain and  
               
               
                 curve  
                 liver homogenate. 
               
               
                   
               
            
           
         
       
     
     LC-MS-MS Analysis Analytical Method for Compound 100 
       
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 Instrument 
                 UPLC/MS-MS-010 (API-4000) 
               
               
                 Matrix 
                 SD rat plasma, brain and liver homogenate 
               
               
                 Analyte(s) 
                 100 
               
               
                 Internal 
                 Diclofenac/Propranolol 
               
               
                 standard(s) 
                   
               
               
                 MS conditions 
                 ESI: Positive ion 
               
               
                   
                 MRM detection 
               
               
                   
                 LB-100: [M + H] +  m/z 269.3→ 101.1 
               
               
                   
                 Diclofenac: [M + H] +  m/z 296.0 @ 250.3 
               
               
                   
                 Propranolol: [M + H] +  m/z 260.2 → 116.1 
               
               
                   
                 Mobile Phase A: H 2 O-0.1% FA-5 mM NH 4 OAc 
               
               
                   
                 Mobile Phase B: ACN 
               
            
           
           
               
               
               
            
               
                   
                 Time (min) 
                 Mobile Phase B (%) 
               
               
                   
                 0.20 
                 15.0 
               
               
                   
                 1.60 
                 98.0 
               
               
                   
                 3.10 
                 98.0 
               
               
                   
                 3.11 
                 15.0 
               
               
                   
                 5.00 
                 stop 
               
            
           
           
               
               
            
               
                   
                 Column: Agilent Eclipse XDB-C18 (4.6 × 150 mm,  
               
               
                   
                 5 μm) 
               
               
                   
                 Flow rate: 0.80 mL/min  
               
               
                   
                 Column temperature: 40° C.  
               
               
                   
                 Retention time:  
               
               
                   
                 LB-100 : 1.75 min  
               
               
                   
                 Diclofenac: 3.56 min  
               
               
                   
                 Propranolol: 2.77 min  
               
               
                 HPLC  
                 For plasma samples:  
               
               
                 conditions 
                 An aliquot of 30 μL sample was added with 100 μL IS  
               
               
                   
                 (Diclofenac, 100 ng/mL and Propranolol, 50 ng/mL  
               
               
                   
                 in ACN). The mixture was vortexed for 10 min at 750  
               
               
                   
                 rpm and centrifuged at 6000 rpm for 10 min. An  
               
               
                   
                 aliquot of 5 μL supernatant was injected for LC-MS/ 
               
               
                   
                 MS analysis.  
               
               
                   
                 For brain homogenate samples:  
               
               
                   
                 The brain samples were homogenized with 3 volumes  
               
               
                   
                 (v/w) of homogenizing solution PBS (pH 7.4) for 2 mins.  
               
               
                   
                 An aliquot of 30 μL tissue homogenate sample was added  
               
               
                   
                 with 100 μL IS (Diclofenac, 100 ng/mL and Propranolol,  
               
               
                   
                 50 ng/mL in ACN). Vortex at 750 rpm for 10 min and  
               
               
                   
                 centrifuged at 6000 rpm for 10 min. An aliquot of 5 μL  
               
               
                   
                 supernatant was injected for LC-MS/MS analysis.  
               
               
                   
                 For liver homogenate samples:  
               
               
                   
                 The liver samples were homogenized with 3 volumes  
               
               
                   
                 (v/w) of homogenizing solution PBS (pH 7.4) for 2  
               
               
                   
                 mins. An aliquot of 30 μL tissue homogenate sample was  
               
               
                   
                 added with 100 μL IS (Diclofenac, 100 ng/mL and  
               
               
                   
                 Propranolol, 50 ng/mL in ACN). Vortex at 750 rpm for  
               
               
                   
                 10 min and centrifuged at 6000 rpm for 10 min. An  
               
               
                   
                 aliquot of 5 μL supernatant was injected for LC-MS/MS  
               
               
                   
                 analysis. 
               
               
                   
                 For all the samples preparation, allow calibration,  
               
               
                   
                 quality control, blanks, and test samples to thaw at 4° C.  
               
               
                   
                 (nominal). And keep each step on an ice bath or at 4° C.  
               
               
                 Calibration  
                 3-3000 ng/mL for LB-100 in SD rat plasma;  
               
               
                 curve  
                 6-3000 ng/mL for LB-100 in SD rat brain and liver  
               
               
                   
                 homogenate. 
               
               
                   
               
            
           
         
       
     
     Pharmacokinetic Analysis 
     Software: The PK parameters were determined by non-compartmental model of non-compartmental analysis tool, Pharsight Phoenix WinNonlin® 6.2 software. 
     “BQL” rule: Concentration data under 80% of LLOQ (LLOQ=1.00 ng/mL in rat plasma, brain and liver homogenate for 113. LLOQ=20.00 ng/mL in rat plasma, brain and liver homogenate for Endothal. LLOQ=3.00 ng/mL for 100 in rat plasma, 6.00 ng/mL for 100 in rat brain and liver homogenate) was replaced with “BQL” and excluded from graphing and PK parameters estimation. Concentration data within 80%-120% of LLOQ was considered within normal instrumental variation and presented in the results. 
     Terminal t 1/2  calculation: Time points were automatic selected by “best fit” model for terminal half life estimation as the first option. Manual selection was applied when “best fit” could not well define the terminal phase. 
     Results 
     No abnormal clinical symptom was observed after IV and PO administrations. 
     The concentration-time data and pharmacokinetic parameters of 113, 100 and Endothal in rat plasma, brain and liver after IV or PO administrations were listed in Tables 8.1 to 8.19, and illustrated in  FIGS. 3A-3D . 
     
       
         
           
               
             
               
                 TABLE 8.1 
               
             
            
               
                   
               
               
                 Individual and mean plasma concentration-time data of 113 
               
               
                 after an IV dose of 1.4 mg/kg in male SD rats 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Time 
                   
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 (hr) 
                 Individual 
                 Mean (ng/mL) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 
                 173 
                 193 
                 183 
               
               
                   
                 1 
                 10.8 
                 9.96 
                 10.4 
               
               
                   
                 2 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 6 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 10 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 24 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                   
               
               
                   
                 LLOQ of 113 in plasma sample is 1.00 ng/mL. 
               
               
                   
                 ULOQ of 113 in plasma sample is 3000 ng/mL. 
               
               
                   
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.2 
               
             
            
               
                   
               
               
                 Individual and mean plasma concentration-time data of 113 
               
               
                 after a PO dose of 1.4 mg/kg in male SD rats 
               
            
           
           
               
               
               
               
            
               
                   
                 Time (hr) 
                 Individual 
                 Mean (ng/mL) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 
                 18.3 
                 17.0 
                 17.7 
               
               
                   
                 1 
                 4.61 
                 8.56 
                 6.59 
               
               
                   
                 2 
                 BQL 
                 2.15 
                 2.15 
               
               
                   
                 6 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 10 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 24 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                   
               
               
                   
                 LLOQ of 113 in plasma sample is 1.00 ng/mL. 
               
               
                   
                 ULOQ of 113 in plasma sample is 3000 ng/mL. 
               
               
                   
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.3 
               
             
            
               
                   
               
               
                 Individual and mean liver concentration-time data of 113  
               
               
                 after an IV dose of 1.4 mg/kg in male SD rats  
               
            
           
           
               
               
               
               
            
               
                   
                 Time (hr) 
                 Individual 
                 Mean (ng/g) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 
                 55.5 
                 36.9 
                 46.2 
               
               
                   
                 1 
                 14.6 
                 11.8 
                 13.2 
               
               
                   
                 2 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 6 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 10 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 24 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                   
               
               
                   
                 The liver sample is homogenized with 3 volumes (v/w) of homogenizing solution (PBS PH 7.4). 
               
               
                   
                 Liver concentration = liver homogenate conc. x 4, assuming 1 g wet liver tissue equals to 1 mL. 
               
               
                   
                 LLOQ of 113 in liver homogenate sample is 1.00 ng/mL. 
               
               
                   
                 ULOQ of 113 in liver homogenate sample is 3000 ng/mL. 
               
               
                   
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.4 
               
             
            
               
                   
               
               
                 Liver-plasma concentration ratio of 113 after an IV dose  
               
               
                 of 1.4 mg/kg in male SD rats  
               
            
           
           
               
               
               
               
            
               
                   
                 Time (hr) 
                 Individual 
                 Mean 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 
                 0.321 
                 0.191 
                 0.256 
               
               
                   
                 1 
                 1.35 
                 1.18 
                 1.27 
               
               
                   
                 2 
                 NA 
                 NA 
                 NA 
               
               
                   
                 6 
                 NA 
                 NA 
                 NA 
               
               
                   
                 10 
                 NA 
                 NA 
                 NA 
               
               
                   
                 24 
                 NA 
                 NA 
                 NA 
               
               
                   
                   
               
               
                   
                 NA: Not Applicable 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.5 
               
             
            
               
                   
               
               
                 Individual and mean brain concentration-time data of 113  
               
               
                 after an IV dose of 1.4 mg/kg in male SD rats  
               
            
           
           
               
               
               
               
            
               
                   
                 Time (hr) 
                 Individual 
                 Mean (ng/g) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 
                 86.2 
                 94.5 
                 90.4 
               
               
                   
                 1 
                 5.80 
                 6.42 
                 6.11 
               
               
                   
                 2 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 6 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 10 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 24 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                   
               
               
                   
                 The brain sample is homogenized with 3 volumes (v/w) of homogenizing solution (PBS PH 7.4). 
               
               
                   
                 Brain concentration = brain homogenate conc. x 4, assuming 1 g wet brain tissue equals to 1 mL. 
               
               
                   
                 LLOQ of 113 in brain homogenate sample is 1.00 ng/mL. 
               
               
                   
                 ULOQ of 113 in brain homogenate sample is 3000 ng/mL. 
               
               
                   
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.6 
               
             
            
               
                   
               
               
                 Brain-plasma concentration ratio of 113 after an IV dose 
               
               
                 of 1.4 mg/kg in male SD rats 
               
            
           
           
               
               
               
               
            
               
                   
                 Time (hr) 
                 Individual 
                 Mean 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 
                 0.498 
                 0.490 
                 0.494 
               
               
                   
                 1 
                 0.537 
                 0.645 
                 0.591 
               
               
                   
                 2 
                 NA 
                 NA 
                 NA 
               
               
                   
                 6 
                 NA 
                 NA 
                 NA 
               
               
                   
                 10 
                 NA 
                 NA 
                 NA 
               
               
                   
                 24 
                 NA 
                 NA 
                 NA 
               
               
                   
                   
               
               
                   
                 NA: Not Applicable 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.7 
               
             
            
               
                   
               
               
                 Individual and mean plasma concentration-time data of  
               
               
                 Endothal after an IV dose of 1.4 mg/kg 113 in SD rats  
               
            
           
           
               
               
               
               
            
               
                   
                 Time (hr) 
                 Individual 
                 Mean (ng/mL) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 
                 24.9 
                 61.2 
                 43.1 
               
               
                   
                 1 
                 41.6 
                 36.1 
                 38.9 
               
               
                   
                 2 
                 43.3 
                 17.4 
                 30.4 
               
               
                   
                 6 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 10 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 24 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                   
               
               
                   
                 LLOQ of Endothal in plasma sample is 20.0 ng/mL. 
               
               
                   
                 ULOQ of Endothal in plasma sample is 3000 ng/mL. 
               
               
                   
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.8 
               
             
            
               
                   
               
               
                 Individual and mean liver concentration-time data of  
               
               
                 Endothal after an IV dose of 1.4 mg/kg 113 in SD rats  
               
            
           
           
               
               
               
               
            
               
                   
                 Time (hr) 
                 Individual 
                 Mean (ng/g) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 
                 727 
                 988 
                 858 
               
               
                   
                 1 
                 902 
                 1230 
                 1066 
               
               
                   
                 2 
                 998 
                 795 
                 897 
               
               
                   
                 6 
                 526 
                 477 
                 502 
               
               
                   
                 10 
                 288 
                 157 
                 223 
               
               
                   
                 24 
                 66.9 
                 68.8 
                 67.9 
               
               
                   
                   
               
               
                   
                 The liver sample is homogenized with 3 volumes (v/w) of homogenizing solution (PBS PH 7.4). 
               
               
                   
                 Liver concentration = liver homogenate conc. x 4, assuming 1 g wet liver tissue equals to 1 mL. 
               
               
                   
                 LLOQ of Endothal in liver homogenate sample is 20.0 ng/mL. 
               
               
                   
                 ULOQ of Endothal in liver homogenate sample is 3000 ng/mL. 
               
               
                   
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.9 
               
             
            
               
                   
               
               
                 Liver-plasma concentration ratio of Endothal after an IV  
               
               
                 dose of 1.4 mg/kg 113 in SD rats 
               
            
           
           
               
               
               
               
            
               
                   
                 Time (hr) 
                 Individual 
                 Mean 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 
                 29.2 
                 16.1 
                 22.7 
               
               
                   
                 1 
                 21.7 
                 34.1 
                 27.9 
               
               
                   
                 2 
                 23.0 
                 45.7 
                 34.4 
               
               
                   
                 6 
                 NA 
                 NA 
                 NA 
               
               
                   
                 10 
                 NA 
                 NA 
                 NA 
               
               
                   
                 24 
                 NA 
                 NA 
                 NA 
               
               
                   
                   
               
               
                   
                 NA: Not Applicable 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.10 
               
             
            
               
                   
               
               
                 Individual and mean brain concentration-time data of  
               
               
                 Endothal after an IV dose of 1.4 mg/kg 113 in SD rats  
               
            
           
           
               
               
               
               
            
               
                   
                 Time (hr) 
                 Individual 
                 Mean (ng/g) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 
                 BQL 
                 SQL 
                 BQL 
               
               
                   
                 1 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 2 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 6 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 10 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 24 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                   
               
               
                   
                 The brain sample is homogenized with 3 volumes (v/w) of homogenizing solution (PBS PH 7.4). 
               
               
                   
                 Brain concentration = brain homogenate conc. x 4, assuming 1 g wet brain tissue equals to 1 mL. 
               
               
                   
                 LLOQ of Endothal in brain homogenate sample is 20.0 ng/mL. 
               
               
                   
                 ULOQ of Endothal in brain homogenate sample is 3000 ng/mL. 
               
               
                   
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.11 
               
             
            
               
                   
               
               
                 Brain-plasma concentration ratio of Endothal after an IV  
               
               
                 dose of 1.4 mg/kg 113 in SD rats  
               
            
           
           
               
               
               
               
            
               
                   
                 Time (hr) 
                 Individual 
                 Mean 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 
                 NA 
                 NA 
                 NA 
               
               
                   
                 1 
                 NA 
                 NA 
                 NA 
               
               
                   
                 2 
                 NA 
                 NA 
                 NA 
               
               
                   
                 6 
                 NA 
                 NA 
                 NA 
               
               
                   
                 10 
                 NA 
                 NA 
                 NA 
               
               
                   
                 24 
                 NA 
                 NA 
                 NA 
               
               
                   
                   
               
               
                   
                 NA: Not Applicable 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.12 
               
             
            
               
                   
               
               
                 Individual and mean plasma concentration-time data of 100  
               
               
                 after an IV dose of 1.4 mg/kg 113 in SD rats  
               
            
           
           
               
               
               
               
            
               
                   
                 Time (hr) 
                 Individual 
                 Mean (ng/mL) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 
                 510 
                 598 
                 554 
               
               
                   
                 1 
                 273 
                 170 
                 222 
               
               
                   
                 2 
                 135 
                 45.3 
                 90.2 
               
               
                   
                 6 
                 3.25 
                 BQL 
                 3.25 
               
               
                   
                 10 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 24 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                   
               
               
                   
                 LLOQ of 100 in plasma sample is 3.00 ng/mL. 
               
               
                   
                 ULOQ of 100 in plasma sample is 3000 ng/mL. 
               
               
                   
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.13 
               
             
            
               
                   
               
               
                 Individual and mean liver concentration-time data of 100  
               
               
                 after an IV dose of 1.4 mg/kg 113 in SD rats  
               
            
           
           
               
               
               
               
            
               
                   
                 Time (hr) 
                 Individual 
                 Mean (ng/g) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0.25 
                 2090 
                 1700 
                 1895 
               
               
                   
                 1 
                 1360 
                 690 
                 1025 
               
               
                   
                 2 
                 425 
                 306 
                 366 
               
               
                   
                 6 
                 23.8 
                 21.8 
                 22.8 
               
               
                   
                 10 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
                 24 
                 BQL 
                 BQL 
                 SQL 
               
               
                   
                   
               
               
                   
                 The liver sample is homogenized with 3 volumes (v/w) of homogenizing solution (PBS pH 7.4). 
               
               
                   
                 Liver concentration = liver homogenate conc. x 4, assuming 1 g wet liver tissue equals to 1 mL. 
               
               
                   
                 LLOQ of 100 in liver homogenate sample is 6.00 ng/mL. 
               
               
                   
                 ULOQ of 100 in liver homogenate sample is 3000 ng/mL. 
               
               
                   
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.14 
               
             
            
               
                   
               
               
                 Liver-plasma concentration ratio of 100 after an IV dose of  
               
               
                 1.4 mg/kg 113 in SD rats 
               
            
           
           
               
               
               
            
               
                 Time (hr) 
                 Individual 
                 Mean 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 0.25 
                 4.10 
                 2.84 
                 3.47 
               
               
                 1 
                 4.98 
                 4.06 
                 4.52 
               
               
                 2 
                 3.15 
                 6.75 
                 4.95 
               
               
                 6 
                 7.32 
                 NA 
                 7.32 
               
               
                 10 
                 NA 
                 NA 
                 NA 
               
               
                 24 
                 NA 
                 NA 
                 NA 
               
               
                   
               
               
                 NA: Not Applicable 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.15 
               
             
            
               
                   
               
               
                 Individual and mean brain concentration-time data of 100 
               
               
                 after an IV dose of 1.4 mg/kg 113 in SD rats 
               
            
           
           
               
               
               
            
               
                 Time (hr) 
                 Individual 
                 Mean (ng/g) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 0.25 
                 BQL 
                 BQL 
                 BQL 
               
               
                 1 
                 BQL 
                 BQL 
                 BQL 
               
               
                 2 
                 BQL 
                 BQL 
                 BQL 
               
               
                 6 
                 BQL 
                 BQL 
                 BQL 
               
               
                 10 
                 BQL 
                 BQL 
                 BQL 
               
               
                 24 
                 BQL 
                 BQL 
                 BQL 
               
               
                   
               
               
                 The brain sample is homogenized with 3 volumes (v/w) of homogenizing solution (PBS PH 7.4). 
               
               
                 Brain concentration = brain homogenate conc. × 4, assuming 1 g wet brain tissue equals to 1 mL. 
               
               
                 LLOQ of 100 in brain homogenate sample is 6.00 ng/mL. 
               
               
                 ULOQ of 100 in brain homogenate sample is 3000 ng/mL. 
               
               
                 BLQ: Below Limit of Quantitation 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.16 
               
             
            
               
                   
               
               
                 Brain-plasma concentration ratio of 100 after an IV dose 
               
               
                 of 1.4 mg/kg 113 in SD rats 
               
            
           
           
               
               
               
            
               
                 Time (hr) 
                 Individual 
                 Mean 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 0.25 
                 NA 
                 NA 
                 NA 
               
               
                 1 
                 NA 
                 NA 
                 NA 
               
               
                 2 
                 NA 
                 NA 
                 NA 
               
               
                 6 
                 NA 
                 NA 
                 NA 
               
               
                 10 
                 NA 
                 NA 
                 NA 
               
               
                 24 
                 NA 
                 NA 
                 NA 
               
               
                   
               
               
                 NA: Not Applicable 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.17 
               
             
            
               
                   
               
               
                 Mean Pharmacokinetics Parameters of 113 after an IV dose of 1.4 mg/kg in male SD rats 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Dosing 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 Route 
                 AUC (0-t)   
                 AUC (0-∞)   
                 t 1/2   
                 T max   
                 C max   
                 CL 
                 V ss   
                 MRT INF   
                 F 
                 AUC last-liver (brain) / 
               
               
                 Matrix 
                 (Dose) 
                 h * ng/mL 
                 h * ng/mL 
                 hr 
                 hr 
                 ng/mL 
                 L/hr/kg 
                 L/kg 
                 hr 
                 % 
                 AUC last-plasma   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Plasma 
                 PO 
                 15.7 
                 NA 
                 NA 
                 0.25 
                 17.7 
                 NA 
                 NA 
                 NA 
                 10.1 
                 NA 
               
               
                   
                 (1.4 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 mg/kg) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Plasma 
                 IV 
                 155 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 Liver 
                 (1.4 
                 28.1 
                 NA 
                 NA 
                 0.25 
                 46.2 
                 NA 
                 NA 
                 NA 
                 NA 
                 18.1 
               
               
                 Brain 
                 mg/kg) 
                 47.5 
                 NA 
                 NA 
                 0.25 
                 90.4 
                 NA 
                 NA 
                 NA 
                 NA 
                 30.6 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8.18 
               
             
            
               
                   
               
               
                 Mean Pharmacokinetics Parameters of Endothal after 
               
               
                 an IV dose of 1.4 mg/kg 113 in male SD rats 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Dosing 
                   
                   
                   
                   
                   
                   
               
               
                   
                 Route 
                 AUC (0-t)   
                 AUC (0-∞)   
                 t 1/2   
                 T max   
                 C max   
                 AUC last-liver / 
               
               
                 Matrix 
                 (Dose) 
                 h * ng/mL 
                 h * ng/mL 
                 hr 
                 hr 
                 ng/mL 
                 AUC last-plasma   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Plasma 
                 IV 
                 70.7 
                 NA 
                 NA 
                 0.25 
                 43.1 
                 NA 
               
               
                 Liver 
                 (1.4 
                 8086 
                 8678 
                 6.04 
                 1 
                 1066 
                 11438 
               
               
                 Brain 
                 mg/kg) 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                   
               
            
           
         
       
     
                     TABLE 8.19                  Mean Pharmacokinetics Parameters of 100 after       an IV dose of 1.4 mg/kg 113 in male SD rats                                                 Dosing                                   Route   AUC (0-t)     AUC (0-∞)     t 1/2z     T max     C max     AUC last-liver /       Matrix   (Dose)   h * ng/mL   h * ng/mL   hr   hr   ng/mL   AUC last-plasma                                                       Plasma   IV   703   707   0.825   0.25   554   NA       Liver   (1   2804   2834   0.934   0.25   1895   399       Brain   mg/kg)   NA   NA   NA   NA   NA   NA                    
IV-1.4 mg/kg 113
 
     After an IV dose of 113 at 1.4 mg/kg in male SD rats, the area under curve from time 0 to last time point (AUC last ) was 155 hr*ng/mL. 
     The mean values of C max  in liver was 46.2 ng/g and corresponding T max  value was 0.25 hr. The mean value of AUC (0-last)  was 28.1 ng/g*hr. AUC (0-t)  ratio of liver over plasma was 18.1. 
     The mean values of C max  in brain was 90.4 ng/g and corresponding T max  value was 0.25 hr. The mean value of AUC (0-last)  was 47.5 ng/g*hr. AUC (0-t)  ratio of liver over plasma was 30.6. 
     PO-1.4 mg/kg 113 
     After a PO dose of 113 at 1.4 mg/kg, the C max  value in rat plasma was 17.7 ng/mL, and corresponding mean T max  value was 0.250 hr. The area under curve from time 0 to last time point AUC last  was 15.7 hr*ng/mL. After the IV dose of 1.4 mg/kg and the PO dose of 1.4 mg/kg, the bioavailability of this compound in SD rat was estimated to be 10.1%. 
     Endothal 
     Following intravenous administration of 1.4 mg/kg 113 to Male SD rats, the area under curve from time 0 to last time point (AUC last ) was 70.7 hr*ng/mL. The mean values of C max  and T max  in plasma were 43.1 ng/mL and 0.25 hr, respectively. 
     The mean values of C max  in liver was 1066 ng/g and corresponding T max  value was 1.00 hr. The mean value of AUC (0-last)  and AUC (0-∞)  were 8086 and 8678 ng/g*hr, respectively. AUC (0-t)  ratio of liver over plasma was 11438. 
     Compound 100 
     The mean values of C max  and T max  in plasma were 554 ng/mL and 0.25 hr, respectively. The mean value of AUC (0-last)  and AUC (0-∞)  were 703 ng/mL*hr and 707 ng/mL*hr, respectively. 
     The mean values of C max  in liver was 1895 ng/g and corresponding T max  value was 0.25 hr. The mean value of AUC (0-last)  and AUC (0-∞)  were 2804 ng/g*hr and 2834 ng/g*hr, respectively. AUC (0-t)  ratio of liver over plasma was 399. 
     Example 4. Pharmacokinetic Study of Compound 151 
     A pharmacokinetic study of 151 was conducted in SD rats. The study consisted of two dose levels at 1.0 (iv) and 10 (oral) mg/kg. The blood samples were collected at predetermined times from rats and centrifuged to separate plasma. An LC/MS/MS method was developed to determine the test article in plasma samples. The pharmacokinetic parameters of 151 following iv and oral administration to SD rats were calculated. The absolute bioavailability was evaluated. 
     Study Design 
     A total of 5 male SD rats were assigned to this study as shown in the table below: 
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                   
                 Number  
                   
                 Dose 
                 Dose 
               
               
                   
                 of rats 
                 Route of 
                 level 
                 volume 
               
               
                 Groups 
                 (male) 
                 administration 
                 (mg/kg) 
                 (ml/kg) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 3 
                 oral 
                 10 
                 10 
               
               
                 2 
                 2 
                 iv 
                 1.0 
                 5.0 
               
               
                   
               
            
           
         
       
     
     Dose Preparation and Dose Administration 
     151 (MW 282.34, purity 99.2%, lot no. 20110512) was prepared by dissolving the article in PBS (pH 7.4) on the day of dosing. The final concentration of the test article was 0.2 mg/mL for iv administration and 1.0 mg/mL for oral administration. The test article solutions were administered using the most recent body weight for each animal. 
     Sample Collection 
     Blood (approximately 0.3 mL) were collected via orbital plexus into tubes containing sodium heparin at 0.25, 0.5, 1, 2, 3, 5, 7, 9, and 24 hours after oral administration; at 5 min, 15 min, 0.5, 1, 2, 3, 5, 7, 9 and 24 hours after iv administration. Samples were centrifuged for 5 min, at 4° C. with the centrifuge set at 11,000 rpm to separate plasma. The obtained plasma samples were stored frozen at a temperature of about −70° C. until analysis. 
     Preparation of Plasma Samples 
     Frozen plasma samples were thawed at room temperature and vortexed thoroughly. With a pipette, an aliquot (30 μL) of plasma was transferred into a 1.5-mL conical polypropylene tube. To each sample, 160 μL of acetonitrile were added. The samples were then vigorously vortex-mixed for 1 min. After centrifugation at 11000 rpm for 5 min, a 15 μL aliquot of the supernatant was injected into the LC-MS/MS system for analysis. 
     Preparation of Calibration Samples 
     Calibration standards were prepared by spiking 30 μL of the 151 standard solutions into 30 μL of heparinized blank rat plasma. The nominal standard concentrations in the standard curve were 1.00, 3.00, 10.0, 30.0, 100, 300, 1000 and 3000 ng/mL. 
     LC/MS/MS System 
     The analysis was performed using an LC-MS/MS system consisting of the following components—HPLC system: Agilent 1200 series instrument consisting of G1312B vacuum degasser, G1322A binary pump, G1316B column oven and G1367D autosampler (Agilent, USA); MS/MS system: Agilent 6460 triple quadrupole mass spectrometer, equipped with an APCI Interface (Agilent, USA); Data system: MassHunter Software (Agilent, USA). 
     Chromatographic Conditions 
     Chromatographic separation was carried out at room temperature—Analytical column: C 8  column (4.6 mm×150 mm I.D., 5 μm, Agilent, USA); Mobile phase: Acetonitrile:10 mM ammonium acetate (75:25, v/v); Flow rate: 0.80 mL/min; Injection volume: 15 μL. 
     Mass Spectrometric Conditions 
     The mass spectrometer was operated in the positive mode. Ionization was performed applying the following parameters: gas temperature, 325° C.; vaporizer temperature, 350° C.; gas flow, 4 L/min; nebulizer, 20 psi; capillary voltage, 4500 V; corona current, 4 μA. 151 was detected using MRM of the transitions m/z 283→m/z 123 and m/z 283→m/z 251, simultaneously. The optimized collision energies of 25 eV and 10 eV were used for m/z 123 and m/z 251, respectively. 
     Quantification 
     Quantification was achieved by the external standard method. Concentrations of the test article were calculated using a weighted least-squares linear regression (W=1/x 2 ). 
     Pharmacokinetic Interpretation 
     The pharmacokinetic parameters were evaluated using WinNonlin version 5.3 (Pharsight Corp., Mountain View, Calif., USA), assuming a non-compartmental model for drug absorption and distribution.
         AUC 0-t  is the area under the plasma concentration-time curve from time zero to last sampling time, calculated by the linear trapezoidal rule.   AUC 0-∞  is the area under the plasma concentration-time curve from time zero extrapolating to infinity.   T 1/2  is the elimination half-life associated with the terminal (log-linear) elimination phase, which is estimated via linear regression of time vs. log concentrations.   CL is the total body clearance.   V ss  is the volume of distribution at steady-state.       

     Calibration Curve for Plasma Samples 
     The calibration curve for L151 in rat plasma was linear throughout the study in the range of 1.00-3000 ng/mL. The linear regression equation of the calibration curve was y=885.6448 x+791.9622, r 2 =0.9927, where y represents the peak area of 151 and x represents the plasma concentrations of 151. 
     Plasma Concentrations of 151 in SD Rats 
     Following iv (1.0 mg/kg) and oral (10 mg/kg) administration of 151 to SD rats, plasma concentrations of the test articles were determined by the LC/MS/MS method described above. The plasma concentrations at each sampling time are listed in Tables 9.1 and 9.2. 
     Interpretation of Pharmacokinetics 
     The major pharmacokinetic parameters of 151 in plasma are summarized in Tables 9.3 and 9.4. Following oral administration of 10 mg/kg to SD rats (n=3), 151 was rapidly absorbed with peak plasma concentration occurring at 0.5 h after dose. The elimination of 151 was fast with mean half-life of 1.26 h. Following iv administration of 1.0 mg/kg (n=2), the elimination half-life of 151 was 0.89 h. The mean clearance of 151 from rat plasma and the volume of distribution at steady state were 859 ml/h/kg and 736 ml/kg. Based on the exposure (AUC 0-∞) , the absolute bioavailability (F) of 151 was 54.6% following oral administration at 10 mg/kg to SD rats. 
     
       
         
           
               
             
               
                 TABLE 9.1 
               
             
            
               
                   
               
               
                 Analytical data of 151 plasma concentration (ng/mL) 
               
               
                 in SD rats following PO administration at 10 mg/kg. 
               
            
           
           
               
               
            
               
                 Rat 
                 Time (h) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 No. 
                 0.25 
                 0.50 
                 1.0 
                 2.0 
                 3.0 
                 5.0 
                 7.0 
                 9.0 
                 24 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 2231 
                 2451 
                 2204 
                 1100 
                 521 
                 125 
                 42.6 
                 52.1 
                 BLQ 
               
               
                 2 
                 2029 
                 3934 
                 2581 
                 1237 
                 660 
                 99.4 
                 20.7 
                 38.2 
                 BLQ 
               
               
                 3 
                 2731 
                 3343 
                 2538 
                 1582 
                 794 
                 192 
                 68.0 
                 66.1 
                 BLQ 
               
               
                 Mean 
                 2330 
                 3243 
                 2441 
                 1306 
                 658 
                 139 
                 43.8 
                 52.1 
                   
               
               
                 SD 
                 361 
                 747 
                 206 
                 248 
                 136 
                 48 
                 23.6 
                 13.9 
               
               
                   
               
               
                 BLQ: Below the lower limit of quantification 1.00 ng/mL. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9.2 
               
             
            
               
                   
               
               
                 Analytical data of 151 plasma concentration (ng/mL) 
               
               
                 in SD rats following IV administration at 1.0 mg/kg. 
               
            
           
           
               
               
            
               
                 Rat 
                 Time (h) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 No. 
                 0.083 
                 0.250 
                 0.50 
                 1.0 
                 2.0 
                 3.0 
                 5.0 
                 7.0 
                 9.0 
                 24 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 4 
                 1677 
                 1160 
                 760 
                 381 
                 95.8 
                 39.6 
                 9.75 
                 12.2 
                 BLQ 
                 BLQ 
               
               
                 5 
                 1301 
                 949 
                 607 
                 314 
                 103 
                 28.1 
                 3.63 
                 1.83 
                 2.01 
                 BLQ 
               
               
                 Mean 
                 1489 
                 1055 
                 683 
                 348 
                 99.6 
                 33.8 
                 6.69 
                 7.02 
                 1.00 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9.3 
               
             
            
               
                   
               
               
                 The main pharmacokinetic parameters of 151 in SD 
               
               
                 rats following PO administration at 10 mg/kg. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Rat 
                 Tmax 
                 Cmax 
                 AUC 0-t   
                 AUC 0-∞   
                 T 1/2   
                 MRT 
                 F 
               
               
                 No. 
                 (ng/ml) 
                 (ng/ml) 
                 (ng · h/ml) 
                 (ng · h/ml) 
                 (h) 
                 (h) 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 0.50 
                 2451 
                 5399 
                 5499 
                 1.33 
                 1.86 
                   
               
               
                 2 
                 0.50 
                 3934 
                 6423 
                 6484 
                 1.10 
                 1.62 
                   
               
               
                 3 
                 0.50 
                 3343 
                 7199 
                 7328 
                 1.35 
                 1.95 
                   
               
               
                 Mean 
                 0.50 
                 3243 
                 6340 
                 6437 
                 1.26 
                 1.81 
                 54.6 
               
               
                 SD 
                 0.00 
                 747 
                 903 
                 916 
                 0.14 
                 0.17 
                   
               
               
                 CV (%) 
                 0.0 
                 23.0 
                 14.2 
                 14.2 
                 11.0 
                 9.4 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9.4 
               
             
            
               
                   
               
               
                 The main pharmacokinetic parameters of 151 in SD 
               
               
                 rats following IV administration at 1.0 mg/kg. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Rat 
                 AUC 0-t   
                 AUC 0-∞   
                 T 1/2   
                 MRT 
                 V ss   
                 CL 
               
               
                 No. 
                 (ng · h/ml) 
                 (ng · h/ml) 
                 (h) 
                 (h) 
                 (ml/kg) 
                 (ml/h/kg) 
               
               
                   
               
               
                 4 
                 1293 
                 1309 
                 0.91 
                 0.91 
                 696 
                 764 
               
               
                 5 
                 1045 
                 1047 
                 0.87 
                 0.81 
                 775 
                 955 
               
               
                 Mean 
                 1169 
                 1178 
                 0.89 
                 0.86 
                 736 
                 859 
               
               
                   
               
            
           
         
       
     
     LB100 concentrations of the LB151 plasma samples were also measured and pharmacokinetic parameters were calculated. LB151 was converted to LB100 (see Tables 9.5-9.8). 
     
       
         
           
               
             
               
                 TABLE 9.5 
               
             
            
               
                   
               
               
                 Plasma Concentrations of 100 after PO administration 
               
               
                 of 10 mg/kg 151 to SD rat (ng/mL) 
               
            
           
           
               
               
               
            
               
                   
                 Rat 
                 Time (h) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Group 
                 No. 
                 0.25 
                 0.50 
                 1.0 
                 2.0 
                 3.0 
                 5.0 
                 7.0 
                 9.0 
                 24 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 PO-10 
                 1 
                 966 
                 1426 
                 882 
                 734 
                 236 
                 81.1 
                 37.9 
                 31.6 
                 BLQ 
               
               
                 mg/kg 
                 2 
                 522 
                 1489 
                 1141 
                 645 
                 396 
                 79.4 
                 20.3 
                 22.5 
                 BLQ 
               
               
                   
                 3 
                 1056 
                 1439 
                 1447 
                 963 
                 624 
                 185 
                 56.0 
                 39.6 
                 BLQ 
               
               
                   
                 Mean 
                 848 
                 1451 
                 1156 
                 781 
                 419 
                 115 
                 38.1 
                 31.3 
                   
               
               
                   
                 SD 
                 286 
                 33 
                 283 
                 164 
                 195 
                 61 
                 17.9 
                 8.6 
               
               
                   
               
               
                 BLQ: Below the lower limit of quantification 10.0 ng/mL 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9.6 
               
             
            
               
                   
               
               
                 Plasma Concentrations of 100 after iv administration 
               
               
                 of 1.0 mg/kg 151 to SD rat (ng/mL) 
               
            
           
           
               
               
               
            
               
                   
                 Rat 
                 Time (h) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Group 
                 No. 
                 0.083 
                 0.25 
                 0.5 
                 1.0 
                 2.0 
                 3.0 
                 5.0 
                 7.0 
                 9.0 
                 24 
               
               
                   
               
               
                 IV-1 
                 4 
                 646 
                 345 
                 308 
                 257 
                 125 
                 32.2 
                 10.2  
                 BLQ 
                 BLQ 
                 BLQ 
               
               
                 mg/kg 
                 5 
                 430 
                 239 
                 231 
                 182 
                 114 
                 33.3 
                 BLQ 
                 BLQ 
                 BLQ 
                 BLQ 
               
               
                   
                 Mean 
                 538 
                 292 
                 270 
                 219 
                 120 
                 32.7 
                 5.10 
               
               
                   
               
               
                 BLQ: Below the lower limit of quantification 10.0 ng/ml. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9.7 
               
             
            
               
                   
               
               
                 PK parameters of 100 after PO administration 
               
               
                 of 10 mg/kg 151 to SD rat 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Rat 
                 T max   
                 C max   
                 AUC 0-t   
                 AUC 0-∞   
                 T 1/2   
                 MRT 
               
               
                 Group 
                 No. 
                 (h) 
                 (ng/ml) 
                 (ng · h/ml) 
                 (ng · h/ml) 
                 (h) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 PO-10 
                 1 
                 0.50 
                 1426 
                 2795 
                 2862 
                 1.45 
                 2.06 
               
               
                 mg/kg 
                 2 
                 0.50 
                 1489 
                 3006 
                 3046 
                 1.25 
                 1.96 
               
               
                   
                 3 
                 1.00 
                 1447 
                 4309 
                 4391 
                 1.43 
                 2.29 
               
               
                   
                 Mean 
                 0.67 
                 1454 
                 3370 
                 3433 
                 1.38 
                 2.10 
               
               
                   
                 SD 
                 0.29 
                 32 
                 820 
                 835 
                 0.11 
                 0.17 
               
               
                   
                 CV (%) 
                 43.3 
                 2.2 
                 24.3 
                 24.3 
                 8.1 
                 8.1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9.8 
               
             
            
               
                   
               
               
                 PK parameters of 100 after iv administration 
               
               
                 of 1.0 mg/kg 151 to SD rat 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Rat 
                 T max   
                 C max   
                 AUC 0-t   
                 AUC 0-∞   
                 T 1/2   
                 MRT 
               
               
                 Group 
                 No. 
                 (h) 
                 (ng/ml) 
                 (ng · h/ml) 
                 (ng · h/ml) 
                 (h) 
                 (h) 
               
               
                   
               
               
                 IV-1 
                 4 
                 0.083 
                 646 
                 681 
                 694 
                 0.88 
                 1.16 
               
               
                 mg/kg 
                 5 
                 0.083 
                 430 
                 481 
                 526 
                 0.93 
                 1.27 
               
               
                   
                 Mean 
                 0.083 
                 538 
                 581 
                 610 
                 0.91 
                 1.21 
               
               
                   
               
            
           
         
       
     
     Example 5. Pharmacokinetic Study of Compound 100 
     The pharmacokinetic studies on 100 and its metabolite endothal were conducted in SD rats. 100 was administrated via iv route at 0.5, 1.0 and 1.5 mg/kg into SD rats. The blood, liver and brain tissue samples were collected at predetermined times from rats. The LC/MS/MS methods were developed to determine 100 and endothal in plasma, liver and brain samples. In the report, the concentrations of 100 and endothal in plasma, liver and brain samples were presented. 
     Sample Collection 
     Twelve (12) female SD rats per group were dosed by iv with 100. The rats were fasted overnight prior to dosing, with free access to water. Foods were withheld for 2 hours post-dose. Blood, liver and brain tissue samples in two animals each group were collected at each time point, within 10% of the scheduled time for each time point. Two extra animals were used for analytic method development. Blood (&gt;0.3 mL) were collected via aorta abdominalis in anaesthetic animals into tubes containing heparin at 15 min, 1, 2, 6, 10 and 24 hours after iv administration. Liver and brain tissues were collected immediately after animal death. The liver and brain tissues were excised and rinsed with cold saline to avoid blood residual. Upon collection, each sample was placed on ice and the blood samples were subsequently centrifuged (4° C., 11000 rpm, 5 min) to separate plasma. The obtained plasma, liver and brain tissue samples were stored at −70° C. until LC-MS/MS analysis. 
     Pharmacokinetic Interpretation 
     The pharmacokinetic parameters were evaluated using WinNonlin version 5.3 (Pharsight Corp., Mountain View, Calif., USA), assuming a non-compartmental model for drug absorption and distribution. AUC 0-t  (AUC last ) is the area under the plasma concentration-time curve from time zero to last sampling time, calculated by the linear trapezoidal rule. AUC 0-∞  (AUC INF)  is the area under the plasma concentration-time curve with last concentration extrapolated based on the elimination rate constant. 
     Plasma, Liver and Brain Tissue Concentrations of Test Articles in SD Rats 
     Following single iv administration of 100 to SD rats, plasma, liver and brain tissue concentrations of both 100 and endothal were determined by the LC/MS/MS method described above. The plasma, liver and brain tissue concentrations at each sampling time are listed in Tables 10.1-10.6 and  FIG. 4A-4D . The calculated pharmacokinetic parameters are listed in Table 10.7-10.8. 100 could pass through blood-brain barrior (BBB) following iv administration at 0.5, 1.0 and 1.5 mg/kg to SD rats. The mean C max  in plasma was 1110˜3664 ng/ml. The mean C max  in liver and brain were 586˜2548 ng/kg and 17.4˜43.5 ng/kg, respectively. AUC Last  in plasma was 695.8˜7399.6 ng·h/ml, with 758.6˜9081.0 ng·h/g in liver and 10.8˜125.5 ng·h/g in brain, respectively. T 1/2  in plasma, liver and brain were 0.31˜2.20 h, 0.78˜2.01h and 1.67˜1.93 h, respectively. 
     As shown in Table 10.4-10.6 and  FIG. 4D-4E , endothal was detectable in plasma and liver samples following single iv administration of 100 at 0.5, 1.0 and 1.5 mg/kg, and the concentrations in plasma and liver increased with dose level of 100, whereas endothal was not detectable in brain samples. The mean C max  in plasma and liver were 577-1230 ng/ml and 349-2964 ng/ml, respectively. AUC last  in plasma and liver were 546-4476 ng·h/ml and 2598-18434 ng·h/g, respectively. T 1/2  in plasma and liver were 6.25-7.06 h and 4.57-10.1h, respectively. 
     Following single iv administration, the mean C max  of 100 in plasma was 1110˜3664 ng/ml and T 1/2  in plasma was 0.31˜2.20 h. AUC last  in pasma was 695.8˜7399.6 ng·h/ml, and AUC increased proportionally with the dose level of 100. Following single iv administration, 100 was both detectable in liver and brain tissue samples. The concentration of 100 in liver samples was much higher than that in brain samples at same sampling time point, but 100 in liver and brain tissues was both below limit of quantification 24 hours after iv administration. Following single iv administration of 100, endothal was detectable and stay a long time in plasma and liver tissue. The mean C max  in plasma and liver were 577-1230 ng/ml and 349-2964 ng/ml, respectively. AUC last  in plasma and liver were 546-4476 ng·h/ml and 2598-18434 ng·h/g, respectively. T 1/2  in plasma and liver were 6.25-7.06 h and 4.57-10.1h, respectively. However, endothal was undetectable in brain tissue. 
     
       
         
           
               
             
               
                 TABLE 10.1 
               
             
            
               
                   
               
               
                 Analytical data of 100 plasma concentration (ng/mL) in SD 
               
               
                 rats following iv administration. 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
            
               
                 0.5 mg/kg Plasma concentration (ng/ml) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 1000 
                 1219 
                 1110 
                 154.68 
               
               
                 1 
                 192 
                 103 
                 148 
                 62.78 
               
               
                 2 
                 25.8 
                 19.4 
                 22.6 
                 4.58 
               
               
                 6 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 10 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 24 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
            
           
           
               
            
               
                 1.0 mg/kg Plasma concentration (ng/ml) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 2118 
                 2648 
                 2383 
                 374.46 
               
               
                 1 
                 354 
                 595 
                 474 
                 170.92 
               
               
                 2 
                 1030 
                 239 
                 634.4 
                 559.22 
               
               
                 6 
                 3.27 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 10 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 24 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
            
           
           
               
            
               
                 1.5 mg/kg Plasma concentration (ng/ml) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 3779 
                 3548 
                 3664 
                 162.94 
               
               
                 1 
                 1758 
                 2273 
                 2015 
                 364.20 
               
               
                 2 
                 1314 
                 1104 
                 1209 
                 148.70 
               
               
                 6 
                 263 
                 519 
                 391 
                 180.40 
               
               
                 10 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 24 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10.2 
               
             
            
               
                   
               
               
                 Analytical data of 100 liver concentration (ng/g) in SD 
               
               
                 rats following iv administration. 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
            
               
                 0.5 mg/kg Liver concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 520 
                 651 
                 586 
                 92.76 
               
               
                 1 
                 695 
                 123 
                 459 
                 333.91 
               
               
                 2 
                 109 
                 148 
                 128 
                 27.06 
               
               
                 6 
                 BLQ 
                 4.80 
                 BLQ 
                 N/A 
               
               
                 10 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 24 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
            
           
           
               
            
               
                 1.0 mg/kg Liver concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 1299 
                 1442 
                 1371 
                 101.47 
               
               
                 1 
                 865 
                 682 
                 773 
                 129.61 
               
               
                 2 
                 1318 
                 398 
                 858 
                 650.73 
               
               
                 6 
                 13.9 
                 5.73 
                 9.83 
                 5.81 
               
               
                 10 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 24 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
            
           
           
               
            
               
                 1.5 mg/kg Liver concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 1980 
                 1709 
                 1844 
                 191.66 
               
               
                 1 
                 2144 
                 2953 
                 2548 
                 571.97 
               
               
                 2 
                 2404 
                 1585 
                 1995 
                 579.17 
               
               
                 6 
                 407 
                 536 
                 471 
                 91.77 
               
               
                 10 
                 BLQ 
                 5.25 
                 BLQ 
                 N/A 
               
               
                 24 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10.3 
               
             
            
               
                   
               
               
                 Analytical data of 100 brain concentration (ng/g) in SD 
               
               
                 rats following iv administration. 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
            
               
                 0.5 mg/kg Brain concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 15.3 
                 19.5 
                 17.42 
                 3.02 
               
               
                 1 
                 6.31 
                 4.77 
                 5.54 
                 1.09 
               
               
                 2 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 6 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 10 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 24 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
            
           
           
               
            
               
                 1.0 mg/kg Brain concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 21.9 
                 45.8 
                 33.90 
                 16.90 
               
               
                 1 
                 16.3 
                 8.05 
                 12.20 
                 5.84 
               
               
                 2 
                 24.3 
                 6.60 
                 15.40 
                 12.49 
               
               
                 6 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 10 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 24 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
            
           
           
               
            
               
                 1.5 mg/kg Brain concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 46.9 
                 40.1 
                 43.49 
                 4.82 
               
               
                 1 
                 28.2 
                 36.9 
                 32.56 
                 6.18 
               
               
                 2 
                 27.2 
                 24.1 
                 25.66 
                 2.16 
               
               
                 6 
                 4.23 
                 6.77 
                 5.50 
                 1.79 
               
               
                 10 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 24 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10.4 
               
             
            
               
                   
               
               
                 Analytical data of endothal plasma concentration (ng/g) 
               
               
                 in SD rats following iv administration. 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
            
               
                 0.5 mg/kg Endothal plasma concentration (ng/ml) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 355 
                 798 
                 576 
                 313.25 
               
               
                 1 
                 104 
                 59.5 
                 81.75 
                 31.47 
               
               
                 2 
                 44.6 
                 28.1 
                 36.35 
                 11.67 
               
               
                 6 
                 20.3 
                 BLQ 
                 20.3 
                 N/A 
               
               
                 10 
                 48.1 
                 25.3 
                 36.70 
                 16.12 
               
               
                 24 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
            
           
           
               
            
               
                 0.1 mg/kg Endothal plasma concentration (ng/ml) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 1310 
                 1150 
                 1230 
                 113.14 
               
               
                 1 
                 164 
                 456 
                 310 
                 206.48 
               
               
                 2 
                 699 
                 213 
                 456 
                 343.65 
               
               
                 6 
                 33.6 
                 38.2 
                 35.90 
                 3.25 
               
               
                 10 
                 32.9 
                 31.8 
                 32.35 
                 0.78 
               
               
                 24 
                 29.4 
                 22.0 
                 25.70 
                 5.23 
               
            
           
           
               
            
               
                 1.5 mg/kg Endothal plasma concentration (ng/ml) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 1610 
                 745 
                 1177 
                 611.65 
               
               
                 1 
                 760 
                 458 
                 609 
                 213.55 
               
               
                 2 
                 539 
                 600 
                 569.50 
                 43.13 
               
               
                 6 
                 373 
                 444 
                 408.50 
                 50.20 
               
               
                 10 
                 22.3 
                 33.1 
                 27.70 
                 7.64 
               
               
                 24 
                 21.5 
                 34.1 
                 27.80 
                 8.91 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10.5 
               
             
            
               
                   
               
               
                 Analytical data of endothal liver concentration (ng/g) 
               
               
                 in SD rats following iv administration of 100. 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
            
               
                 0.5 mg/kg Endothal liver concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 316 
                 382 
                 349 
                 46.67 
               
               
                 1 
                 256 
                 131 
                 193.50 
                 88.39 
               
               
                 2 
                 168 
                 273 
                 220.50 
                 74.25 
               
               
                 6 
                 85.8 
                 112 
                 98.90 
                 18.53 
               
               
                 10 
                 129 
                 118 
                 123.50 
                 7.78 
               
               
                 24 
                 32.0 
                 36.4 
                 34.20 
                 3.11 
               
            
           
           
               
            
               
                 1.0 mg/kg Endothal liver concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 768 
                 1320 
                 1044 
                 390.32 
               
               
                 1 
                 1380 
                 618 
                 999 
                 538.82 
               
               
                 2 
                 1530 
                 542 
                 1036 
                 698.62 
               
               
                 6 
                 298 
                 241 
                 269.50 
                 40.31 
               
               
                 10 
                 151 
                 94.2 
                 122.60 
                 40.16 
               
               
                 24 
                 66.6 
                 115 
                 90.80 
                 34.22 
               
            
           
           
               
            
               
                 1.5 mg/kg Endothal liver concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 2298 
                 2160 
                 2229 
                 97.58 
               
               
                 1 
                 2874 
                 2976 
                 2925 
                 72.12 
               
               
                 2 
                 2952 
                 2226 
                 2589 
                 513.36 
               
               
                 6 
                 1686 
                 1326 
                 1506 
                 254.56 
               
               
                 10 
                 137 
                 329 
                 233 
                 135.76 
               
               
                 24 
                 75.0 
                 52.1 
                 63.55 
                 16.19 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10.6 
               
             
            
               
                   
               
               
                 Analytical data of endothal brain concentration (ng/g) in 
               
               
                 SD rats following iv administration of 100. 
               
            
           
           
               
               
               
               
               
            
               
                 Time (hr) 
                 Rat 1 
                 Rat 2 
                 Mean 
                 SD 
               
               
                   
               
            
           
           
               
            
               
                 0.5 mg/kg Endothal brain concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 1 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 2 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 6 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 10 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 24 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
            
           
           
               
            
               
                 1.0 mg/kg Endothal brain concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 1 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 2 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 6 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 10 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 24 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
            
           
           
               
            
               
                 1.5 mg/kg Endothal brain concentration (ng/g) 
               
            
           
           
               
               
               
               
               
            
               
                 0.25 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 1 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 2 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 6 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 10 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                 24 
                 BLQ 
                 BLQ 
                 BLQ 
                 N/A 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10.7 
               
             
            
               
                   
               
               
                 Main pharmacokinetic parameters of 100 in SD rats following iv administration. 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 Dose of 
                   
                   
                   
                 Cmax 
                 AUClast 
                 AUCINF 
                   
               
               
                   
                 LB-100 
                   
                 T½ 
                 Tmax 
                 ng/ml 
                 ng · h/ml 
                 ng · h/ml 
                 MRT 
               
               
                 Analyte 
                 mg/kg 
                 Tissue 
                 h 
                 h 
                 or ng/g 
                 or ng · h/g 
                 or ng · h/g 
                 h 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 100 
                 0.5 
                 Brain 
                 / 
                 0.25 
                 17.4 
                 10.8 
                 / 
                 / 
               
               
                   
                   
                 Liver 
                 0.78 
                 0.25 
                 586 
                 758.6 
                 902.2 
                 1.17 
               
               
                   
                   
                 Plasma 
                 0.31 
                 0.25 
                 1110 
                 695.8 
                 706.0 
                 0.45 
               
               
                   
                 1.0 
                 Brain 
                 1.67 
                 0.25 
                 33.9 
                 35.3 
                 72.5 
                 2.68 
               
               
                   
                   
                 Liver 
                 0.79 
                 0.25 
                 1371 
                 3526.5 
                 3537.7 
                 1.51 
               
               
                   
                   
                 Plasma 
                 0.99 
                 0.25 
                 2383 
                 1923.5 
                 2830.2 
                 1.57 
               
               
                   
                 1.5 
                 Brain 
                 1.93 
                 0.25 
                 43.5 
                 125.5 
                 140.8 
                 2.57 
               
               
                   
                   
                 Liver 
                 2.01 
                 1.0 
                 2548 
                 9081.0 
                 10449.1 
                 2.90 
               
               
                   
                   
                 Plasma 
                 2.20 
                 0.25 
                 3664 
                 7399.6 
                 8641.4 
                 2.82 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10.8 
               
             
            
               
                   
               
               
                 Main pharmacokinetic parameters of Endothal in SD 
               
               
                 rats following single iv administration of 100. 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 Dose of 
                   
                   
                   
                 Cmax 
                 AUClast 
                 AUCINF 
                   
               
               
                   
                 LB-100 
                   
                 T½ 
                 Tmax 
                 ng/ml 
                 ng · h/ml 
                 ng · h/ml 
                 MRT 
               
               
                 Analyte 
                 mg/kg 
                 Tissue 
                 h 
                 h 
                 or ng/g 
                 or ng · h/g 
                 or ng · h/g 
                 h 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Endothal 
                 0.5 
                 Brain 
                 / 
                 / 
                 / 
                 / 
                 / 
                 / 
               
               
                   
                   
                 Liver 
                 10.1  
                 0.25 
                  349 
                 2598 
                 3095 
                 7.90 
               
               
                   
                   
                 Plasma 
                 6.65 
                 0.25 
                  577 
                  546 
                  828 
                 2.96 
               
               
                   
                 1.0 
                 Brain 
                 / 
                 / 
                 / 
                 / 
                 / 
                 / 
               
               
                   
                   
                 Liver 
                 6.10 
                 0.25 
                 1425 
                 6673 
                 7370 
                 6.14 
               
               
                   
                   
                 Plasma 
                 7.06 
                 0.25 
                 1230 
                 2487 
                 2750 
                 4.38 
               
               
                   
                 1.5 
                 Brain 
                 / 
                 / 
                 / 
                 / 
                 / 
                 / 
               
               
                   
                   
                 Liver 
                 4.57 
                 0.25 
                 2964 
                 18434  
                 18850  
                 4.54 
               
               
                   
                   
                 Plasma 
                 6.25 
                 0.25 
                 1178 
                 4476 
                 4730 
                 4.57 
               
               
                   
               
            
           
         
       
     
     Endothal concentrations of the 100 plasma samples were measured and pharmacokinetic parameters were calculated. LB100 was converted to endothal. 
     Example 6. Administration of Compound 
     Compounds 100, 105, 113, 153 and 157 are PP2A inhibitors. The present invention provides analogues of 100, 105, 113, 153 and 157, which are inhibitors of PP2A in vitro in human cancer cells and in xenografts of human tumor cells in mice when given parenterally in mice. These compounds inhibit the growth of cancer cells in mouse model systems. The analogues of 100, 105, 113, 153 and 157 are intraperitoneally administered to mice and PP2A activity is measured in the liver and brain. The analogues of 100, 105, 113, 153 and 157 reduce PP2A activity in the liver and brain. 
     An amount of any one of the compounds of the present invention is administered to a subject afflicted with brain cancer. The amount of the compound is effective to treat the subject. 
     An amount of any one of the compounds of the present invention is administered to a subject afflicted with diffuse intrinsic pontine glioma. The amount of the compound is effective to treat the subject. 
     An amount of any one of the compounds of the present invention is administered to a subject afflicted with glioblastoma multiforme. The amount of the compound is effective to treat the subject. 
     An amount of any one of the compounds of the present invention is administered to a subject afflicted with brain cancer. The amount of the compound is effective to cross the blood brain barrier of the subject and treat the subject. 
     An amount of any one of the compounds of the present invention is administered to a subject afflicted with diffuse intrinsic pontine glioma. The amount of the compound is effective to cross the blood brain barrier of the subject and treat the subject. 
     An amount of any one of the compounds of the present invention is administered to a subject afflicted with glioblastoma multiforme. The amount of the compound is effective to cross the blood brain barrier of the subject and treat the subject. 
     Example 7. Administration of Compound in Combination with an Anti-Cancer Agent 
     An amount of any one of the compounds of the present invention in combination with an anti-cancer agent is administered to a subject afflicted with brain cancer. The amount of the compound is effective to enhance the anti-cancer activity of the anti-cancer agent. 
     An amount of any one of the compounds of the present invention in combination with ionizing radiation, x-radiation, docetaxel or temozolomide is administered to a subject afflicted with brain cancer. The amount of the compound is effective to enhance the anti-cancer activity of the ionizing radiation, x-radiation, docetaxel or temozolomide. 
     An amount of any one of the compounds of the present invention in combination with an anti-cancer agent is administered to a subject afflicted with diffuse intrinsic pontine glioma or glioblastoma multiforme. The amount of the compound is effective to enhance the anti-cancer activity of the anti-cancer agent. 
     An amount of any one of the compounds of the present invention in combination with ionizing radiation, x-radiation, docetaxel or temozolomide is administered to a subject afflicted with diffuse intrinsic pontine glioma or glioblastoma multiforme. The amount of the compound is effective to enhance the anti-cancer activity of the ionizing radiation, x-radiation, docetaxel or temozolomide. 
     Example 8. Endothal Prodrugs 
     As demonstrated in the data contained herein Compounds 105, 113, 153 and 157 are metabolized to endothal in vivo. The analogues of 105, 113, 153 and 157 contained herein are also metabolize to endothal in vivo and act as prodrugs of endothal. The edothal dimer analogs contained herein are also metabolized to endothal in vivo and act as prodrugs of endothal. 
     In addition, while not wishing to be bound to a theory, it is believed that the prodrugs of the present application allow for targeted delivery of endothal to specific cells, i.e. cancer cells, in a subject. Direct administration of endothal is undesirable due to toxicity. The prodrugs provide improved absorption leading to greater bioavailability of the active compound. 
     An amount of any one of the compounds of the present invention is administered to a subject afflicted with cancer. The amount of the compound is effective to deliver endothal to cancers cells in the subject. 
     An amount of any one of the compounds of the present invention is administered to a subject afflicted with brain cancer. The amount of the compound is effective to deliver endothal to brain cancers cells in the subject. 
     An amount of any one of the compounds of the present invention is administered to a subject afflicted with diffuse intrinsic pontine glioma or glioblastoma multiforme. The amount of the compound is effective to deliver endothal to diffuse intrinsic pontine glioma cells or glioblastoma multiforme cells in the subject. 
     An amount of any one of the compounds of the present invention is administered to a subject afflicted with brain cancer. The amount of the compound is effective to deliver endothal across the blood brain barrier of the subject. 
     Example 9. Dual Endothal/Chemotherapeutic Agent Prodrug 
     As demonstrated in the data contained herein Compounds 105, 113, 153 and 157 are metabolized to endothal in vivo. The analogues of 105, 113, 153 and 157 contained herein are also metabolized to endothal in vivo and act as dual endothal/chemotherapeutic agents prodrugs. The dual prodrugs contained herein are also metabolized to endothal in vivo and act as prodrugs of endothal. However, the metabolism to endothal simultaneously releases a chemotherapeutic agent. 
     In addition, while not wishing to be bound to a theory, it is believed that the dual prodrugs of the present application allow for targeted delivery of endothal and a chemotherapeutic agent to specific cells, i.e. cancer cells, in a subject. Direct administration of endothal and/or a chemotherapeutic agent is undesirable due to toxicity. 
     In addition, while not wishing to be bound to a theory, it is believed that the dual prodrugs of the present application allow for targeted delivery of endothal and a chemotherapeutic agent to specific cells, i.e. cancer cells, in a subject. Furthermore, the dual prodrugs have the advantage of having two bioactive compounds combined into one drug (novel structure). That structures alone have their own advantages, e.g., improved absorption leading to greater bioavailability of either constituent. Furthermore, direct administration of endothal and/or a chemotherapeutic agent could be undesirable due to either&#39;s intrinsic toxicity. 
     Example 10. Synthesis of LB-100 POM Ester and LB-100 Carbonate 
     LB-100 POM Ester 
     
       
         
         
             
             
         
       
     
     To a solution of LB-100 (106 mg, 0.4 mmol) in DMF (5 mL) is added Cs 2 CO 3  (386 mg, 1.2 mmol) at room temperature. After stirring for 5 min., chloromethyl pivalate (178 mmg, 1.2 mmol) is added. The resulting mixture is stirred at room temperature overnight. Water (10 ml) is added, the mixture is extracted with ethyl acetate (5×10 ml). The organic phase is dried over MgSO 4 , filtered and the solvent is removed. The residue is titrated with hexane, and filtered to give white solid (103 mg, 68% yield).  1 H NMR (CDCl 3 ) 1.20 (s, 9H), 1.52 (d, 2H), 1.84 (d, 2H), 2.28-2.52 (m, 7H), 2.88 (d, 1H), 3.16 (d, 1H), 3.36-3.52 (m, 3H), 3.72 (m, 1H), 4.80 (s, 1H), 5.00 (s, 1H), 5.68 (d, 1H), 5.72 (d, 1H). 
     LB-100 Carbonate 
     
       
         
         
             
             
         
       
     
     To a solution of LB-100 (150 mg, 0.56 mmol) in DMF (5 mL) is added Cs 2 CO 3  (546 mg, 1.7 mmol) at room temperature. After stirring for 5 min., chloromethyl ethyl carbonate (232 mmg, 1.7 mmol) is added. The resulting mixture is stirred at room temperature overnight. Water (10 ml) is added, the mixture is extracted with ethyl acetate (5×10 ml). The organic phase is dried over MgSO 4 , filtered and the solvent is removed. The residue is titrated with hexane, and filtered to give white solid (124 mg, 60% yield).  1 HNMR (CDCl3) 1.23 (t, 3H), 1.52 (d, 2H), 1.84 (d, 2H), 2.28-2.52 (m, 7H), 2.84 (d, 1H), 3.18 (d, 1H), 3.36-3.52 (m, 3H), 3.72 (m, 1H), 4.20 (q, 2H), 4.80 (s, 1H), 5.00 (s, 1H), 5.62 (d, 1H), 5.80 (d, 1H). 
     Example 12. Administration of LB-100 Carbonate or LB-100 POM 
     An amount of LB-100 Carbonate or LB-100 POM is administered to a subject afflicted with brain cancer. The amount of the compound is effective to treat the subject. 
     An amount of LB-100 Carbonate or LB-100 POM is administered to a subject afflicted with diffuse intrinsic pontine glioma. The amount of the compound is effective to treat the subject. 
     An amount of LB-100 Carbonate or LB-100 POM is administered to a subject afflicted with glioblastoma multiforme. The amount of the compound is effective to treat the subject. 
     An amount of LB-100 Carbonate or LB-100 POM is administered to a subject afflicted with brain cancer. The amount of the compound is effective to cross the blood brain barrier of the subject and treat the subject. 
     An amount of LB-100 Carbonate or LB-100 POM is administered to a subject afflicted with diffuse intrinsic pontine glioma. The amount of the compound is effective to cross the blood brain barrier of the subject and treat the subject. 
     An amount of LB-100 Carbonate or LB-100 POM is administered to a subject afflicted with glioblastoma multiforme. The amount of the compound is effective to cross the blood brain barrier of the subject and treat the subject. 
     An amount of LB-100 Carbonate or LB-100 POM in combination with an anti-cancer agent is administered to a subject afflicted with brain cancer. The amount of the compound is effective to enhance the anti-cancer activity of the anti-cancer agent. 
     An amount of LB-100 Carbonate or LB-100 POM in combination with ionizing radiation, x-radiation, docetaxel or temozolomide is administered to a subject afflicted with brain cancer. The amount of the compound is effective to enhance the anti-cancer activity of the ionizing radiation, x-radiation, docetaxel or temozolomide. 
     An amount of LB-100 Carbonate or LB-100 POM in combination with an anti-cancer agent is administered to a subject afflicted with diffuse intrinsic pontine glioma or glioblastoma multiforme. The amount of the compound is effective to enhance the anti-cancer activity of the anti-cancer agent. 
     An amount of LB-100 Carbonate or LB-100 POM in combination with ionizing radiation, x-radiation, docetaxel or temozolomide is administered to a subject afflicted with diffuse intrinsic pontine glioma or glioblastoma multiforme. The amount of the compound is effective to enhance the anti-cancer activity of the ionizing radiation, x-radiation, docetaxel or temozolomide. 
     Example 13. LB-100 Carbonate and LB-100 POM Prodrugs 
     As demonstrated in the data contained herein LB-100 Carbonate and LB-100 POM are metabolized to endothal in vivo and act as prodrugs of endothal. In addition, while not wishing to be bound to a theory, it is believed that the prodrugs of the present application allow for targeted delivery of endothal to specific cells, i.e. cancer cells, in a subject. Direct administration of endothal is undesirable due to toxicity. The prodrugs provide improved absorption leading to greater bioavailability of the active compound. 
     An amount of LB-100 Carbonate or LB-100 POM is administered to a subject afflicted with cancer. The amount of the compound is effective to deliver endothal to cancers cells in the subject. 
     An amount of LB-100 Carbonate or LB-100 POM is administered to a subject afflicted with brain cancer. The amount of the compound is effective to deliver endothal to brain cancers cells in the subject. 
     An amount of LB-100 Carbonate or LB-100 POM is administered to a subject afflicted with diffuse intrinsic pontine glioma or glioblastoma multiforme. The amount of the compound is effective to deliver endothal to diffuse intrinsic pontine glioma cells or glioblastoma multiforme cells in the subject. 
     An amount of LB-100 Carbonate or LB-100 POM is administered to a subject afflicted with brain cancer. The amount of the compound is effective to deliver endothal across the blood brain barrier of the subject. 
     Example 14. Liver and Whole Blood Assays 
     The stability (whole blood, liver S9, SGF, SIF, and PBS buffer) and MDCK-MDR1 monolayer permeability of LB100, LB-100 Carbonate and LB-100 POM were evaluated. 
     Analytical Method Development 
     The analyte signal was optimized for each compound by ESI positive or negative ionization mode. An MS2 scan or an SIM scan was used to optimize the fragmenter voltage and a product ion analysis was used to identify the best fragment for analysis, and the collision energy was optimized using a product ion or MRM scan. An ionization ranking was assigned indicating the compound&#39;s ease of ionization. 3.3 Sample Analysis (Chemical Stability, Whole Blood Stability, and S9 Stability Assays) 
     Sample Analysis (Chemical Stability, Whole Blood Stability, and S9 Stability Assays) 
     Samples were analyzed by LC-MS/MS using a SCIEX QTrap 5500 mass spectrometer coupled with an Agilent 1290 HPLC Infinity series, a CTC PAL chilled autosampler, all controlled by Analyst software. After separation on a C18 reverse phase HPLC column (Acquity UPLC HSS T3, 1.8, 2.1×50 mm) using an acetonitrile-water gradient system, peaks were analyzed by mass spectrometry (MS) using ESI ionization in MRM mode. 
     Sample Analysis (MDCK-MDR1 Permeability Assay) 
     Samples were analyzed by LC/MS/MS using an Xevo II mass spectrometer coupled with an Acquity HPLC and a CTC PAL chilled autosampler, all controlled by MassLynx (Waters). After separation on a C18 reverse phase HPLC column (Waters Acquity UPLC HSS T3 1.8 um 1×50 mM) using an acetonitrile-water gradient system, peaks were analyzed by mass spectrometry (MS) using ESI ionization in MRM mode. 
     HPLC Gradient (Chemical Stability, Whole Blood Stability, and S9 Stability Assays) 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Time 
                 Flow rate 
                 % A 
                 % B 
               
               
                 (min) 
                 (mL/min) 
                 Mobile Phase 
                 Mobile Phase 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 0.05 
                 0.6 
                 100 
                 0 
               
               
                 1.0 
                 0.6 
                 5 
                 95 
               
               
                 1.40 
                 0.6 
                 5 
                 95 
               
               
                 1.41 
                 0.6 
                 100 
                 0 
               
               
                 1.8 
                 0.6 
                 100 
                 0 
               
               
                   
               
               
                 Solution A: H 2 O with 0.1% Formic acid; 
               
               
                 Solution B: Acetonitrile with 0.1% Formic acid 
               
            
           
         
       
     
     HPLC Gradient (MDCK-MDR1 Permeability) 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Time 
                 Flow rate 
                 % A 
                 % B 
               
               
                 (min) 
                 (mL/min) 
                 Mobile Phase 
                 Mobile Phase 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 0.00 
                 0.600 
                 99.9 
                 0.1 
               
               
                 0.01 
                 0.600 
                 99.9 
                 0.1 
               
               
                 1.0 
                 0.600 
                 5 
                 95 
               
               
                 1.4 
                 0.600 
                 99.9 
                 0.1 
               
               
                 1.8 
                 0.600 
                 99.9 
                 0.1 
               
               
                   
               
               
                 Solution A: H 2 O with 0.1% Formic acid; 
               
               
                 Solution B: Acetonitrile with 0.1% Formic acid 
               
            
           
         
       
     
     Chemical Stability: Experimental Conditions 
     
       
         
           
               
               
               
               
               
               
             
               
                   
               
               
                   
                 Test 
                   
                   
                 Reference 
                 Analytical 
               
               
                 Test Article 
                 conc. 
                 Test conditions 
                 Incubation 
                 compounds 
                 method 
               
               
                   
               
             
            
               
                 LB-151 
                 5 μM 
                 PBS buffer (pH 7.4) 
                 0, 1, 2, 
                 omeprazole 
                 LC-MS/MS 
               
               
                 LB-100 POM Ester 
                   
                 SGF (pH 1.2) 
                 and 4 hrs  
                 warfarin 
                   
               
               
                 LB-100 Carbonate 
                   
                 SIF (pH 6.5) 
                 (37° C.) 
               
               
                   
               
            
           
         
       
     
     Experimental Procedure: The compound was incubated in duplicate with either PBS buffer (pH 7.4), SGF (pH 1.2) or SIF (pH 6.5) at 37° C. At the indicated times, an aliquot was removed from each experimental reaction and mixed with three volumes of ice-cold Stop Solution (methanol containing propranolol/diclofenac/bucetin as analytical internal standards). Stopped reactions were incubated for ten minutes at −20° C. The samples were centrifuged, and the supernatants were analyzed by LC/MS/MS to quantitate the remaining parent as well as the formation of metabolites. Data was converted to % remaining by dividing by the time zero concentration value. Data were fit to a first-order decay model to determine half-life. 
     Liver S9 Stability: Experimental Conditions 
     Experimental Procedure: Test agent is incubated in duplicate with liver S9 at 37° C. The reaction contains liver S9 protein in 100 mM potassium phosphate, 2 mM NADPH, 3 mM MgCl 2 , pH 7.4. A control is run for each test agent omitting NADPH to detect NADPH-free degradation. At indicated times, an aliquot is removed from each experimental and control reaction and mixed with an equal volume of ice-cold Stop Solution (methanol, containing internal standard propranolol). Stopped reactions are incubated for 10 minutes at −20° C. Samples are centrifuged to remove precipitated protein, and supernatants are analyzed by LC/MS/MS to quantitate remaining parent and the formation of metabolites. Data are reported as % remaining by dividing by the time zero concentration value. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                   
               
               
                 Test 
                 Test 
                 S9 
                 Protein 
                   
                 Reference 
                 Analytical 
               
               
                 Article 
                 Conc 
                 Species 
                 Conc 
                 Incubation 
                 Compound 
                 Method 
               
               
                   
               
             
            
               
                 LB-151 
                 1 μM 
                 Rat, monkey, 
                 1.0 mg/mL 
                 0, 1, 2, and 4 hr 
                 verapamil 
                 LC-MS/MS 
               
               
                 LB-100 POM Ester 
                   
                 and human 
                   
                 (37° C.) 
                 warfarin 
                   
               
               
                 LB-100 Carbonate 
               
               
                   
               
            
           
         
       
     
     Whole Blood Stability: Experimental Conditions 
     Experimental Procedure: The stock solution was first diluted in acetonitrile at a concentration that is 100x of the desired final concentration. It was incubated in duplicate with whole blood at 37° C. At indicated times, an aliquot was removed from each experimental and control reaction and mixed with three volumes of ice-cold Stop Solution (methanol containing propranolol as internal standard). Stopped reactions were incubated at least ten minutes at −20° C. The samples were centrifuged to remove precipitated protein, and the supernatants were analyzed by LC-MS/MS to quantitate the remaining parent and the formation of metabolites. 
     Data were converted to % remaining by dividing by the time zero concentration value. Data were fit to a first-order decay model to determine half-life. 
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                   
                 Test 
                   
                   
                 Analytical 
               
               
                 Test Article 
                 Conc. 
                 Species 
                 Incubation 
                 Method 
               
               
                   
               
             
            
               
                 LB-151 
                 5 μM 
                 rat, dog,  
                 0, 1, 2,  
                 LC-MS/MS 
               
               
                 LB-100  
                   
                 monkey, 
                 and 4 hr  
                   
               
               
                 POM Ester 
                   
                 and human 
                 (37° C.) 
                   
               
               
                 LB-100  
                   
                   
                   
                   
               
               
                 Carbonate 
               
               
                   
               
            
           
         
       
     
     MDCK-MDR1 Permeability: Experimental Conditions 
     Experimental Procedure: MDCK-MDR1 cells grown in tissue culture flasks are trypsinized, suspended in medium, and the suspensions were applied to wells of a Millipore 96 well plate. The cells are allowed to grow and differentiate for three weeks, feeding at 2-day intervals. For Apical to Basolateral (A→B) permeability, the test agent is added to the apical (A) side and amount of permeation is determined on the basolateral (B) side; for Basolateral to Apical (B→A) permeability, the test agent is added to the B side and the amount of permeation is determine on the A side. The A-side buffer contains 100 μM Lucifer yellow dye, in Transport Buffer (1.98 g/L glucose in 10 mM HEPES, 1× Hank&#39;s Balanced Salt Solution) pH 7.4, and the B-side buffer is Transport Buffer at pH 7.4. MDCK-MDR1 cells are incubated with these buffers for 2 hr, and the receiver side buffer is removed for analysis by LC/MS/MS (using propranolol as an analytical internal standard). To verify the MDCK-MDR1 cell monolayers are properly formed, aliquots of the cell buffers are analyzed by fluorescence to determine the transport of the impermeable dye Lucifer Yellow. Any deviations from control values are reported. 
     Masa Spectrometry Method Development: MS/MS 
     Metabolites of LB-151 (LB-100, Endothal, and Endothal methyl ester), LB-100 Carbonate (LB-100 and Endothal) and LB-100 POM (LB-100 and Endothal) were monitored. 
     
       
         
           
               
               
               
               
               
               
             
               
                   
               
               
                   
                   
                 ESI 
                 Precursor 
                 Product 
                 Ionization 
               
               
                   
                 MW 
                 Polarization 
                 m/z 
                 m/z 
                 Classification 
               
               
                   
               
             
            
               
                 Test Article 
                   
                   
                   
                   
                   
               
               
                 LB-151 
                 282.34 
                 positive 
                 283.15 
                 251.153 
                 1 
               
               
                 LB-100 POM Ester 
                 382.46 
                 positive 
                 383.196 
                 251.17 
                 1 
               
               
                 LB-100 Carbonate 
                 370.4 
                 positive 
                 371.153 
                 251.137 
                 1 
               
            
           
           
               
               
            
               
                 Monitored Metabolites 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 LB-100 
                 268.31 
                 positive 
                 269.171 
                 251.138 
                 1 
               
               
                 Endothall  
                 186.16 
                 negative 
                 184.986 
                 140.92 
                 1 
               
               
                 Endothall methyl ester 
                 200.19 
                 negative 
                 198.94 
                 110.89 
                 1 
               
               
                   
               
               
                 1 = highly ionizable, 
               
               
                 2 = intermediate, 
               
               
                 3 = pooly ionizable 
               
               
                 m/z: mass-to-charge ratio of analyte 
               
            
           
         
       
     
     In the liver S9 Stability study, metabolites LB-100 and endothall were observed in both LB-100 carbonate and LB-100 POM ester in the presence and absence of NADPH (cross species), suggesting that these metabolites were formed by non-NADPH dependent enzymes (e.g. esterases and amidases). No metabolites were observed in LB-151 samples (see  FIG. 5 ). The LB-100 carbonate and LB-100 POM ester metabolites were studied in rat, dog, monkey and human (see  FIG. 6 ). 
     In the whole blode half-life study, formation of endothall and LB-100 were observed in LB-100 carbonate and LB-100 POM ester (cross species). No metabolites were detected in LB-151 (see  FIG. 7 ). In the whole blood metabolite study, LB-100 carbonate and LB-100 POM ester were metabolized to endothall and LB-100 in rat, dog, monkey and human (see  FIG. 8 ). 
     In the MDCK-MDR1 permeability study, no metabolites were observed in all samples (see  FIG. 9 ). 
     DISCUSSION 
     Inhibition of PP2A interferes with multiple aspects of the DNA damage repair (DDR) mechanisms and with exit from mitosis. These mechanisms sensitize cancer cells to cancer treatments that cause acute DNA injury. Compound 100 (see U.S. Pat. No. 7,998,957 B2) has anti-cancer activity when used alone (Lu el al. 2009a) and significantly potentiates in vivo, without observable increase in toxicity, the anti-tumor activity of standard cytotoxic anti-cancer drugs including temozolomide (Lu et al. 2009b, Martiniova et al. 2010), doxorubicin (Zhang et al. 2010), and docetaxel. 100 was recently approved for Phase I clinical evaluation alone and in combination with docetaxel and is in clinical trial. 
     Compound 100 is a serine-threonine phosphatase inhibitor that potentiates the activity of standard chemotherapeutic drugs and radiation. The mechanism of potentiation is impairment of multiple steps in a DNA-damage repair process and inhibition of exit from mitosis. Compound 100 has been shown to potentiate the activity of temozolomide, doxorubicin, taxotere, and radiation against a variety of human cancer cell lines growing as subcutaneous xenografts. Compound 100 treatment yields a radiation dose enhancement factor of 1.45. Mice bearing subcutaneous (sc) xenografts of U251 human GBM cells were treated with compound 100 intraperitoneally together with radiation, each given daily for 5 days×3 courses. The drug/radiation combination was no more toxic that radiation alone and eliminated 60% of the xenografts (6 months plus follow-up). The remaining 40% of xenografts treated with the combination recurred two months later than xenografts treated with radiation alone. Wei et al. (2013) showed that inhibition of PP2A by compound 100 enhanced the effectiveness of targeted radiation in inhibiting the growth of human pancreatic cancer xenografts in an animal model. Thus, 100 would seem to be an ideal agent to combine with radiation to treat localized cancers such as brain tumors. 
     Compound 100 is highly effective against xenografts of human gliomas in combination with temozolomide and/or radiation. Compound 100, which has an IC 50  of 1-3 μM for a broad spectrum of human cancer cell lines, is a highly water soluble zwitterion that does not readily pass the blood brain barrier (BBB) as determined in rats and non-human primates. GLP toxokinetic studies of compound 100 given intravenously daily×5 days were performed in the rat and dog. The major expected toxicities at clinically tolerable doses expected to inhibit the target enzyme, PP2A, in vivo (3-5 mg/m 2 ) are reversible microscopic renal proximal tubule changes and microscopic alterations in epicardial cells. It is of interest that fostriecin, a natural-product selective inhibitor of PP2A, was evaluated given iv daily for 5 days in phase I trials several years ago. Dose limiting toxicity was not achieved before the studies were terminated for lack of a reliable drug supply. In those studies, the major toxicities were reversible non-cumulative increases in serum creatinine and hepatic enzymes. 
     Compound 100 is considered stable relative to verapamil in the presence of mouse, rat, dog, monkey, and human microsomes. Compound 100 is poorly absorbed from or broken down in the gut so that little is present in plasma after oral administration. In glp studies in the male and female Sprague Dawley rat, the PK parameters for compound 100 given by slow iv bolus daily×5 days were also dose dependent and comparable on day 1 and day 4. The values for female rats after drug at 0.5, 0.75, and 1.25 mg/kg on day 4 were respectively: C o  (ng/ml) 1497, 2347, and 3849; AUC last  (ng·h/ml) 452, 691, and 2359; SC AUC last  (ng·h/ml) 17.7, 54.0, and 747; DN AUC last  904, 921, and 1887; AUC* (ng·h/ml) 479, 949, and 2853; % AUC* Extrapolated 5.6, 27, and 17; T 1/2  (h) 0.25, 0.59, and 1.8; Cl (mL/h/kg) 1045, 790, 438 (MALE 1071, 1339, 945); V z  (ml/kg) 378, 677, and 1138. In GLP studies in the male and female dog, the toxicokinetic parameters for compound 100 given iv over 15 minutes daily for 5 days were dose dependent and comparable on day 1 and day 4. The values for the female dogs on after drug at 0.15, 0.30, and 0.50 mg/kg on day 4 were respectively: C o  (ng/ml) 566, 857, and 1930; AUC last  (ng·h/ml) 335, 1020, and 2120; Can (ng/ml) 370, 731, 1260; T max  (hr) 0.25, 0.35, and 0.25; and, T 1/2  (h) 0.47, 0.81, and 1.2 (IND No. 109,777: compound 100 for Injection). Inhibition of the abundant PP2A in circulating white blood cells (isolated by Ficoll-Hypaque) has been shown to be dose dependent in the rat following slow iv administration of 100 at 0.375, 0.75, and 1.5 mg/kg resulting 9, 15 and 25% inhibition, respectively. 
     The methyl ester of 100, compound 151, which has an oral bioavailability of about 60% versus 1% for compound 100, was given by mouth to rats. Compound 151 treatment resulted in substantial levels of compound 100 in the plasma with an apparently much greater half life compared with 100 given intravenously. 
     Based on the data contained in Examples 8-11, compounds 105, 113, 153 and 157 are converted to endothal in the plasma when administered to rats. Accordingly, compounds 105, 113, 151, 153 and 157 and derivative thereof are useful as prodrugs of endothal. The compounds of the present application contain different substituents which are cleaved in vivo when administered to a subject thereby releasing endothall. These compound contain X or Y groups which are more efficiently cleaved in vivo. 
     Diffuse Intrinsic Pontine Glioma (DIPG) is a uniformly fatal brain tumor of children for which no standard treatment other that radiation is available. Pediatric neurooncologists believe it is appropriate to treat even previously untreated patients on an investigational protocol that offers a new approach. There has been no advance in overall survival in Glioblastoma Multiforme (GBM) patients since the definite but marginal improvement shown years ago by the addition of temozolomide to radiation after surgery. Recurrent GBM is often treated with Avastin as second line therapy but following relapse after Avastin, experimental treatment is the standard. Of interest concerning inhibition of PP2A in brain tumors is the recent report that increased levels of PP2A are present in GBM and that patients with the highest levels of PP2A in their gliomas have the worst prognosis (Hoffstetter et al., 2012). 
     As shown in PK and PD studies presented herein, LB-100 itself enters tissues and is converted in part to endothall in tissues. As an inhibitor of the purified target protein of LB-100, protein phosphatase PP2A, endothal is potent with an IC 50  of ˜90 nM. In vivo, endothall has a longer half-life that LB-100 on the order of 6 hours compared to about 1 hour or less for LB-100. Thus LB-100 is both an active anti-cancer agent in itself and by its in vivo conversion to endothal increases the effective duration of inhibition of the intended target, PP2A, in tissue. A half-life of several hours of activity is clinically more desirable that much shorter durations. Modification of substituents of LB-100 provide opportunities to further enhance the clinical usefulness of LB-100 by for example improving oral absorption, uptake into specific organs bearing the disease process, for example, the brain, and further modifying the the rate of conversion for effective delivery of parent compound and/or endothall to tissue. 
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