Abstract:
There is disclosed a Dolastatin derivative, conjugated to an antibody, comprising a Dolastatin derivative moiety of Formula IV.

Description:
CROSS REFERENCE TO PRIOR APPLICATION 
       [0001]    This patent application claims priority to pending U.S. provisional patent application 62/108,894 filed 28 Jan. 2015. 
     
    
     TECHNICAL FIELD 
       [0002]    The present disclosure provides antibody drug conjugates (Formula I) comprising a Dolastatin derivative moiety of Formula II as the drug component. 
       BACKGROUND 
       [0003]    Dolastatins, such as natural product Dolastatin 10, and its synthetic derivatives Monomethyl Auristatin E (MMAE) and Monomethyl Auristatin F (MMAF) are products that show potent antineoplastic and tubulin inhibitory property. Because of their high toxicity, the direct use of Dolastatins as therapeutic agents has not been effective. Instead, they were conjugated to an antibody for targeted delivery to kill cancer cells. 
         [0000]    
       
                 
         
             
             
         
       
     
       SUMMARY 
       [0004]    The present disclosure provides a compound comprising a Dolastatin derivative moiety of Formula IV: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein Y is OH, or NH 2 ,
 
R 4  is OH, NH 2 , F, Cl, Br, I, OR 5 , wherein R 5  is C1-C4 alkyl.
 
         [0005]    The present disclosure further provides an antibody drug-conjugate having the structure of Formula I: 
         [0000]      AbL 1 -L 2 -D) n    (I)
 
         [0000]    or a pharmaceutically acceptable salt thereof,
 
wherein:
 
Ab is a monoclonal antibody
 
L 1  is a connector
 
L 2  is a linker
 
D is an active agent having the structure of Formula II
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein Y is O, or NH, the wavy line indicates the point of attachment,
 
X is —CH 2 N 3  or
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein R is C1-C8 alkyl, C3-C6 cyclic alkyl, aryl or heteroaryl.
 
n is an integer from 1-8.
 
         [0006]    Preferably, L 2  is selected from the group consisting of an amino acid, peptide, —(CH 2 ) n —, —(CH 2 CH 2 O) n —, p-aminobenzyl (PAB), Val-Cit (Citrulline)-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, or combinations thereof. Preferably, -L 1 -L 2  is selected from the group consisting of 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    Preferably, Ab-L 1 -L 2  is selected from the group consisting of 
         [0000]    
       
                 
         
             
             
         
       
     
         [0007]    The present disclosure further provides a method for synthesizing an antibody drug-conjugate having the structure of Formula I: 
         [0000]      AbL 1 -L 2 -D) n    (I)
 
         [0000]    or a pharmaceutically acceptable salt thereof,
 
wherein:
 
Ab is a monoclonal antibody
 
L 1  is a connector
 
L 2  is a linker
 
D is an active agent having the structure of Formula II
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein Y═O, or NH, the wavy line indicates the point of attachment
 
X is —CH 2 N 3  or
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein R is C1-C8 alkyl, C3-C6 cyclic alkyl, aryl or heteroaryl.
 
n is an integer from 1-8, comprising
 
reacting a compound of formula III with a Lys on an Ab
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein G is selected from the group consisting of —F, —Cl, —Br, —I, —N 3 , —OR, SR, —ONRR, RC(═O)O—, and RSO 2 —O—; and
 
R is optionally substituted alkyl, or optionally substituted aryl.
 
m=0, or 1.
 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0008]      FIG. 1  shows a single dose of conjugate 16 administered to BALB/c nude mice (n=8) by intravenous administration. 
           [0009]      FIG. 2  shows a single dose of conjugate 16 administered to BALB/c nude mice (n=8) by intravenous administration. 
           [0010]      FIG. 3  shows pictures of the mice 35 days after treatment. 
           [0011]      FIG. 4A  shows in vitro activity of ADC-23 (anti-Her2 antibody) in a group of tumor cell lines. 
           [0012]      FIG. 4B  shows in vitro activity of ADC-16 (anti-Her2 antibody) in a group of tumor cell lines. 
           [0013]      FIG. 5  shows in vivo efficacy of ADC-65, ADC-23 and ADC-19 in various xenograft tumor models. 
           [0014]      FIGS. 6A and 6B  shows a single dose of conjugate 16 and 19 administered to BALB/c nude mice (n=8) by intravenous administration. 
       
    
    
     DETAILED DESCRIPTION 
       [0015]    The present disclosure provides compounds and conjugates, such as ADC (antibody drug conjugates), wherein a linker moiety that is peptide based has an attaching point at its C terminal which reacts with either Cys or Lys on an antibody in a controlled fashion. For Lys conjugation, for example, the DAR (drug antibody ratio) is 2. The DAR (drug antibody ratio) of the majority of conjugate is 4, when conjugation occurred on Cys. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Examples of structures of drug-linker moieties for Lys conjugation onto an antibody. 
               
             
          
           
               
                 Com- 
                   
               
               
                 pound 
                   
               
               
                 ID 
                 Structures 
               
               
                   
               
               
                 1 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 2 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 4 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 62 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Examples of structures of drug-linker compounds (for Cys conjugation) to be conjugated onto a hinge region of an IgG class antibody. 
               
             
          
           
               
                   
                 Com- 
                   
               
               
                   
                 pound 
                   
               
               
                   
                 ID 
                 Structures 
               
               
                   
                   
               
               
                   
                  6 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                  7 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                  8 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                  9 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 13 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 63 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Examples of structures of antibody (Ab)-drug conjugates. 
               
             
          
           
               
                   
                 Com- 
                   
               
               
                   
                 pound 
                   
               
               
                   
                 ID 
                 Structures 
               
               
                   
                   
               
               
                   
                 16 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 17 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 19 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 64 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 21 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 22 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 23 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 24 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 28 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 65 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
             
          
         
       
     
       Definitions 
       [0016]    Abbreviations are defined as follows:
   Ac Acetyl   aq. Aqueous   BOC or Boc tert-Butoxycarbonyl   BrOP bromo tris(dimethylamino) phosphonium hexafluorophosphate   Bu n-Butyl   ° C. Temperature in degrees Centigrade   Cit Citrulline   DCM methylene chloride   DEPC Diethylcyanophosphonate   DIC diisopropylcarbodiimide   DIEA Diisopropylethylamine   DMA N,N′-Dimethylacetamide   DMF N,N′-Dimethylformamide   EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide   Et Ethyl   EtOAc Ethyl acetate   Eq Equivalents   Fmoc 9-Fluorenylmethoxycarbonyl   g Gram(s)   h Hour (hours)   HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate   HOBT N-Hydroxybenzotriazole   HOSu N-Hydroxysuccinimide   HPLC High-performance liquid chromatography   LC/MS Liquid chromatography-mass spectrometry   Me Methyl   MeOH Methanol   MeCN Acetonitrile   mL Milliliter(s)   MS mass spectrometry   PAB p-aminobenzyl   RP-HPLC reverse phase HPLC   rt room temperature   t-Bu tert-Butyl   TEA Triethylamine   Tert, t tertiary   TFA Trifluoracetic acid   THF Tetrahydrofuran   TLC Thin-layer chromatography   μL Microliter(s)   
 
       General Synthesis Procedure— 
       [0057]    Formation of an activated ester (e.g. NHS) from an acid An acid was dissolved in DCM (methylene chloride) and DMF (N,N′ dimethyl formamide) was added to aid dissolution if necessary. N-hydroxysuccinimide (1.5 eq) was added, followed by EDC.HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (1.5 eq). The reaction mixture was stirred at room temperature for 1 h until most of the acid was consumed. The progress of the reaction was monitored by RP-HPLC. The mixture was then diluted with DCM and washed successively with citric acid (aq. 10%) and brine. The organic layer was dried and concentrated to dryness. The crude product was optionally purified by RP-HPLC or silica gel column chromatography. 
       Example 1 
     Preparation of Compound 1 
       [0058]    
       
                 
         
             
             
         
       
     
         [0059]    To a crude solution of compound 47 (0.1 mmol) in THF (3 mL) was added a solution of piperidine 4-carboxylic acid (60 mg) in sat. aq. NaHCO 3  (1 mL). The mixture was stirred at room temperature for 30 min, then acidified with 1N aq. HCl to pH=4-5. The reaction mixture was concentrated and the residue was purified by reverse phase HPLC to give compound 1 as a white powder after lyophilization (68 mg). MS m/z 1020.7 (M+H). 
       Example 2 
     Preparation of Compound 2 
       [0060]    
       
                 
         
             
             
         
       
     
         [0061]    Compound 52 (185 mg, 0.2 mmol) was dissolved in DCM/DMF (5/1, v/v, 5 mL). 
         [0062]    EDC.HCl (0.5 mmol) and HOSu (0.3 mmol) were added. The mixture was stirred at room temperature for 30 min. HPLC analysis confirmed that all of compound 52 was consumed. The reaction was diluted with DCM (50 mL) and washed with brine. The organic layer was concentrated to 1 mL and diluted with acetonitrile/water (6/4, v/v, 3 mL). A solution of pyrrolidine 3-carboxylic acid (60 mg) in sat. aq. NaHCO 3  (1 mL) was added and the mixture was stirred at room temperature for 10 min. The reaction was acidified with HOAc and concentrated. The crude product was purified by RP-HPLC to give compound 2 (138 mg, 68%). MS m/z 1020.6 (M+H). 
       Example 3 
     Preparation of Compound 4 
       [0063]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
       Preparation of Compound 38: 
       [0064]    To compound 37 (261 mg, 0.52 mmol) in 6 mL of DMF was added HATU (217 mg, 0.57 mmol), DIEA (362 μL, 2.08 mmol), and amine 36 (213 mg, 0.52 mmol). The mixture was stirred for 30 min, then 400 μL of piperidine was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 38 (171 mg, 60%). MS m/z 548.3 (M+H). 
       Preparation of Compound 40: 
       [0065]    To compound 39 (37 mg, 0.15 mmol) in 4 mL of DMF was added HATU (59 mg, 0.15 mmol), DIEA (108 μL, 0.6 mmol), and amine 38 (102 mg, 0.15 mmol). The mixture was stirred for 30 min, then evaporated to dryness. The residue was dissolved in 2 mL of DCM, then 1 mL of TFA was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 40 (94 mg, 78%). MS m/z 673.4 (M+H). 
       Preparation of Compound 4: 
       [0066]    To compound 41 (85 mg, 0.12 mmol) in 2 mL of DMF was added HATU (48 mg, 0.12 mmol), DIEA (83 μL, 0.48 mmol), and amine 40 (94 mg, 0.12 mmol). The mixture was stirred for 30 min, then a solution of 90 mg of NaOH in 1 mL of water was added and stirred for 30 min. The mixture purified by HPLC to give compound 4 (86 mg, 58%). MS m/z 1239.7 (M+H). 
       Example 4 
     Preparation of Compound 6 
       [0067]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
       Preparation of Compound 46: 
       [0068]    To compound 41 (1000 mg, 1.67 mmol) in 20 mL of DMF was added HATU (640 mg, 1.68 mmol), DIEA (870 μL, 5.00 mmol), and amine 45 (535 mg, 1.67 mmol). The mixture was stirred for 30 min, then evaporated and purified by HPLC to give compound 46 (1140 mg, 70%). MS m/z 865.5 (M+H). 
       Preparation of Compound 47: 
       [0069]    To compound 46 (500 mg, 0.57 mmol) in 10 mL of DMA was added bis(p-nitropenyl)carbonate (210 mg, 0.69 mmol), and DIEA (35 μL, 0.2 mmol). The mixture was stirred for 18 h, then 100 mL of ether was added and the precipitate was collected by filtration to give compound 47 (500 mg, 85%). MS m/z 1030.6 (M+H). 
       Preparation of Compound 49: 
       [0070]    To compound 47 (125 mg, 0.12 mmol) in 4 mL of DMF was added HOBt (7 mg, 0.05 mmol), DIEA (21 μL, 0.12 mmol), and amine 48 (40 mg, 0.12 mmol). The mixture was stirred for 16 h, then 200 μL of piperidine was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 49 (72 mg, 60%). MS m/z 1005.6 (M+H). 
       Preparation of Compound 6: 
       [0071]    To compound 49 (30 mg, 0.027 mmol) in 2 mL of DCM was added DIEA (15 μL, 0.086 mmol), DIEA (50 μL, 0.288 mmol), and anhydride 50 (19 mg, 0.027 mmol). The mixture was stirred for 30 min, then evaporated and purified by HPLC to give compound 6 (32 mg, 88%). MS m/z 1347.5 (M+H). 
       Example 5 
     Preparation of Compound 7. 
       [0072]    
       
                 
         
             
             
         
       
     
         [0000]    Compound 7 was synthesized from compound 49 (0.1 mmol) and anhydride 63 (0.1 mmol) as described for the synthesis of compound 6. Yield: 79%. MS m/z 1296.8 (M+H). 
       Example 6 
       [0073]    
       
                 
         
             
             
         
       
     
       Preparation of Compound 8. 
       [0074]    To a solution of compound 47 (0.1 mmol) in THF (3 mL) was added a solution of compound 64 (0.15 mmol, 67 mg) in acetonitrile/water (1/1, v/v, 1 mL), followed by DIEA (50 μL). After 30 min, the reaction was acidified and concentrated. The residue was purified by reverse phase HPLC to give compound 8 as a white solid (87 mg). MS m/z 1243.6 [M+H]+. 
       Example 7 
     Preparation of Compound 9. 
       [0075]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
       Preparation of Compound 52: 
       [0076]    To compound 46 (120 mg, 0.12 mmol) in 3 mL of DMF was added K 2 CO 3  (118 mg, 0.85 mmol), and bromoacetate 51 (35 mg, 0.18 mmol). The mixture was stirred for 16 h, then evaporated. The residue was dissolved in 2 mL of DCM, filtered, and 2 mL of TFA was added. After 20 min the mixture was evaporated and purified by HPLC to give compound 52 (92 mg, 83%). MS m/z 923.5 (M+H). 
       Preparation of Compound 53: 
       [0077]    To compound 52 (92 mg, 0.1 mmol) in 2 mL of DMF was added HATU (38 mg, 0.1 mmol), DIEA (70 μL, 0.4 mmol), and boc-hydrazine (15 mg, 0.12 mmol). The mixture was stirred for 30 min, then evaporated to dryness. The residue was dissolved in 2 mL of DCM, then 1 mL of TFA was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 53 (82 mg, 78%). MS m/z 937.5 (M+H). 
       Preparation of Compound 9: 
       [0078]    To compound 54 (53 mg, 0.156 mmol) in 2 mL of DCM was added DIC (10 mg, 0.078 mmol) and stirred for 10 min. Then DIEA (54 μL, 0.312 mmol) and amine 53 (82 mg, 0.078 mmol) was added and the mixture was stirred for 15 min. The mixture was evaporated and purified by HPLC to give compound 9 (62 mg, 63%). MS m/z 1260.5 (M+H). 
       Example 8 
       [0079]    Preparation of Compound 13 
         [0000]    
       
                 
         
             
             
         
       
     
         [0080]    To compound 37 (130 mg, 0.26 mmol) in 3 mL of DMF was added HATU (110 mg, 0.29 mmol), DIEA (175 μL, 1 mmol), and amine 36 (110 mg, 0.27 mmol). The mixture was stirred for 30 min, then concentrated to dryness. The residue was then treated with TFA/DCM (1/4, v/v, 5 mL) for 30 min. The mixture was evaporated and purified by HPLC to give compound 66 (108 mg, 65%). MS m/z 670.5 (M+H). 
         [0081]    To compound 41 (85 mg, 0.12 mmol) in 2 mL of DMF was added HATU (48 mg, 0.12 mmol), DIEA (83 μL, 0.48 mmol), and amine 66 (94 mg, 0.12 mmol). The mixture was stirred for 30 min, then piperidine (0.2 mL) was added and stirred for 30 min. The mixture was concentrated and purified by HPLC to give compound 67 (87 mg, 63%). MS m/z 1028.7 (M+H). 
         [0082]    To a solution of compound 67 (57 mg, 0.05 mmol) and acid 68 (22 mg) in DCM/DMF (3/1, v/v, 4 mL) was added PyBrOP (0.055 mmol) and DIEA (35 μL). The mixture was stirred at room temperature for 30 min and then concentrated to about 2 mL. The residue was purified by reverse phase HPLC to give compound 13 (41 mg). MS m/z 1425.7 (M+H). 
       Example 9 
       [0083]    This example provides the results of EC50 assays of the designated drug conjugated antibodies measured in vitro in specified cells. The antibody used was an anti-HER2 IgG class of antibody. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                   
                 MDA- 
                 MDA- 
                 MDA-MB- 
               
               
                   
                 SBKR3  
                 HCC1954 
                 SKOV-3 
                 BT474 
                 MB-453 
                 MB-175 
                 361  
               
               
                 Conjugate 
                 (Her2+++) 
                 (Her2+++) 
                 (Her2+++) 
                 (Her2+++) 
                 (Her2++) 
                 (Her2+) 
                 (Her2+++) 
               
             
          
           
               
                 ID 
                 EC50 [nM] 
               
               
                   
               
             
          
           
               
                 16 
                 0.040 
                 0.138 
                 0.405 
                 0.423 
                 1.195 
                 3.635 
                   
               
               
                 17 
                 0.106 
                 0.237 
                 0.334 
                 0.623 
                 26.42 
                 20.08 
                   
               
               
                 19 
                 0.156 
                 0.193 
                 0.340 
                 0.232 
                 3.946 
                   
                 0.640 
               
               
                 21 
                 0.3432 
                 0.1788 
                   
                 1.065 
                   
                 0.4904 
                 0.1326 
               
               
                 22 
                 0.06349 
                 0.04926 
                   
                 0.346 
                 0.137 
                 0.2628 
                 0.04987 
               
               
                 23 
                 0.04644 
                 0.03678 
                   
                 0.345 
                 0.118 
                 0.2095 
                 0.04657 
               
               
                 65 
                 0.158 
                 0.117 
                   
                 0.100 
                 4.762 
               
               
                   
               
             
          
         
       
     
       Example 10 
       [0084]    This example shows in vivo efficacy of ADC 16 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model.  FIG. 1  shows a single dose of conjugate 16 administered to BALB/c nude mice by intravenous administration. There were 8 mice in each group and total 6 groups of mice were studied: 3 groups were injected with T-DM1 (Trastuzumab—DM1 conjugate) at different doses; 2 groups were injected with ADC 16 at different doses; and one vehicle control. All the drugs were administered in the same manner (single dose). A single dose of ADC-16 iv. at 1 mg/kg or 3 mg/kg outperformed T-DM1 at 3 mg/kg or 10 mg/kg respectively. 3 mg/kg ADC-16 completely inhibited tumor growth up to 100 days. 
       Example 11 
       [0085]    This example shows in vivo safety of ADC 16 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model.  FIG. 2  shows a single dose of conjugate 16 administered to BALB/c nude mice by intravenous administration. There were 8 mice in each group and total 7 groups of mice were studied: 3 groups were injected with T-DM1 (Trastuzumab—DM1 conjugate) at different doses; 3 groups were injected with ADC 16 at different doses; and one vehicle control. All the drugs were administered in the same manner (single dose). A single dose of ADC-16 iv. at 1 mg/kg, 3 mg/kg or 10 mg/kg did not retard body weight gain. The difference of the body weights between T-DM1 and ADC-16 groups were caused by the difference of tumor weight.  FIG. 3  shows pictures of the mice 35 days after treatment. 
       Example 12 
       [0086]    This example ( FIG. 4A ) shows ADC-23 induces equivalent or stronger anti-proliferative activity in breast cancer cell lines, compared to MMAE conjugates. In these studies, the cells were all treated with either ADC-23 or MMAE conjugates for 3 d. IC50 is determined as the concentration that showed 50% inhibition of cell growth. 
       Example 13 
       [0087]    This example ( FIG. 4B ) shows ADC-16 induces equivalent or stronger anti-proliferative activity in breast cancer cell lines, compared to MMAE conjugates. In the above studies, the cells were all treated with either ADC-16 or MMAE conjugates for 3 d. IC50 is determined as the concentration that showed 50% inhibition of cell growth. 
       Example 14 
       [0088]    This example ( FIG. 5 ) shows the in vivo efficacy of ADC-65, ADC-23 and ADC-19 in LoVo (Colon), MDA-MB-468 (Breast), BxPC-3 (Pancreatic), PA-1 (Ovarian) and H1975 NSCLC xenograft nude mice. All ADCs were given as single dose via iv. at indicated concentrations. The ADCs tested outperformed MMAF in most cases at the same level, and completely inhibited tumor growth by single dose. 
       Example 15 
       [0089]    This example shows in vivo safety and efficacy of ADC 19 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model.  FIGS. 6A and 6B  shows a single dose of conjugate 19 administered to BALB/c nude mice by intravenous administration. There were 8 mice in each group and total 3 groups of mice were studied: 1 group of mice was injected with ADC 16; 1 group of mice was injected with ADC 19; and one vehicle control. All the drugs were administered in the same manner (single dose). A single dose of ADC-19 iv. at 2 mg/kg was comparable to that of ADC-16 at the same dose and completely inhibited tumor growth up to 49 days and did not retard body weight gain that was comparable to ADC-16. 
       Example 16 
       [0090]    This example shows the general conjugation procedure for synthesizing antibody drug conjugates 16, 17, 19, and 64. To a solution of 0.5-50 mgs/mL of antibody in buffer at pH 6.0-9.0 with 0-30% organic solvent, was added 0.1-10 eq of activated drug linker conjugate (1, or 2, 
         [0000]    
       
                 
         
             
             
         
       
     
       Definitions 
       [0091]    Abbreviations are defined as follows:
   Ac Acetyl   aq. Aqueous   BOC or Boc tert-Butoxycarbonyl   BrOP bromo tris(dimethylamino) phosphonium hexafluorophosphate   Bu n-Butyl   ° C. Temperature in degrees Centigrade   Cit Citrulline   DCM methylene chloride   DEPC Diethylcyanophosphonate   DIC diisopropylcarbodiimide   DIEA Diisopropylethylamine   DMA N,N′-Dimethylacetamide   DMF N,N′-Dimethylformamide   EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide   Et Ethyl   EtOAc Ethyl acetate   Eq Equivalents   Fmoc 9-Fluorenylmethoxycarbonyl   g Gram(s)   h Hour (hours)   HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate   HOBT N-Hydroxybenzotriazole   HOSu N-Hydroxysuccinimide   HPLC High-performance liquid chromatography   LC/MS Liquid chromatography-mass spectrometry   Me Methyl   MeOH Methanol   MeCN Acetonitrile   mL Milliliter(s)   MS mass spectrometry   PAB p-aminobenzyl   RP-HPLC reverse phase HPLC   rt room temperature   t-Bu tert-Butyl   TEA Triethylamine   Tert, t tertiary   TFA Trifluoracetic acid   THF Tetrahydrofuran   TLC Thin-layer chromatography   μL Microliter(s)   
 
       General Synthesis Procedure— 
       [0132]    Formation of an activated ester (e.g. NHS) from an acid An acid was dissolved in DCM (methylene chloride) and DMF (N,N′ dimethyl formamide) was added to aid dissolution if necessary. N-hydroxysuccinimide (1.5 eq) was added, followed by EDC.HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (1.5 eq). The reaction mixture was stirred at room temperature for 1 h until most of the acid was consumed. The progress of the reaction was monitored by RP-HPLC. The mixture was then diluted with DCM and washed successively with citric acid (aq. 10%) and brine. The organic layer was dried and concentrated to dryness. The crude product was optionally purified by RP-HPLC or silica gel column chromatography. 
       Example 1 
     Preparation of Compound 1 
       [0133]    
       
                 
         
             
             
         
       
     
         [0134]    To a crude solution of compound 47 (0.1 mmol) in THF (3 mL) was added a solution of piperidine 4-carboxylic acid (60 mg) in sat. aq. NaHCO 3  (1 mL). The mixture was stirred at room temperature for 30 min, then acidified with 1N aq. HCl to pH=4-5. The reaction mixture was concentrated and the residue was purified by reverse phase HPLC to give compound 1 as a white powder after lyophilization (68 mg). MS m/z 1020.7 (M+H). 
       Example 2 
     Preparation of Compound 2 
       [0135]    
       
                 
         
             
             
         
       
     
         [0136]    Compound 52 (185 mg, 0.2 mmol) was dissolved in DCM/DMF (5/1, v/v, 5 mL). EDC.HCl (0.5 mmol) and HOSu (0.3 mmol) were added. The mixture was stirred at room temperature for 30 min. HPLC analysis confirmed that all of compound 52 was consumed. The reaction was diluted with DCM (50 mL) and washed with brine. The organic layer was concentrated to 1 mL and diluted with acetonitrile/water (6/4, v/v, 3 mL). A solution of pyrrolidine 3-carboxylic acid (60 mg) in sat. aq. NaHCO 3  (1 mL) was added and the mixture was stirred at room temperature for 10 min. The reaction was acidified with HOAc and concentrated. The crude product was purified by RP-HPLC to give compound 2 (138 mg, 68%). MS m/z 1020.6 (M+H). 
       Example 3 
     Preparation of Compound 4 
       [0137]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
       Preparation of Compound 38: 
       [0138]    To compound 37 (261 mg, 0.52 mmol) in 6 mL of DMF was added HATU (217 mg, 0.57 mmol), DIEA (362 μL, 2.08 mmol), and amine 36 (213 mg, 0.52 mmol). The mixture was stirred for 30 min, then 400 μL of piperidine was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 38 (171 mg, 60%). MS m/z 548.3 (M+H). 
       Preparation of Compound 40: 
       [0139]    To compound 39 (37 mg, 0.15 mmol) in 4 mL of DMF was added HATU (59 mg, 0.15 mmol), DIEA (108 μL, 0.6 mmol), and amine 38 (102 mg, 0.15 mmol). The mixture was stirred for 30 min, then evaporated to dryness. The residue was dissolved in 2 mL of DCM, then 1 mL of TFA was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 40 (94 mg, 78%). MS m/z 673.4 (M+H). 
       Preparation of Compound 4: 
       [0140]    To compound 41 (85 mg, 0.12 mmol) in 2 mL of DMF was added HATU (48 mg, 0.12 mmol), DIEA (83 μL, 0.48 mmol), and amine 40 (94 mg, 0.12 mmol). The mixture was stirred for 30 min, then a solution of 90 mg of NaOH in 1 mL of water was added and stirred for 30 min. The mixture purified by HPLC to give compound 4 (86 mg, 58%). MS m/z 1239.7 (M+H). 
       Example 4 
     Preparation of Compound 6 
       [0141]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
       Preparation of Compound 46: 
       [0142]    To compound 41 (1000 mg, 1.67 mmol) in 20 mL of DMF was added HATU (640 mg, 1.68 mmol), DIEA (870 μL, 5.00 mmol), and amine 45 (535 mg, 1.67 mmol). The mixture was stirred for 30 min, then evaporated and purified by HPLC to give compound 46 (1140 mg, 70%). MS m/z 865.5 (M+H). 
       Preparation of Compound 47: 
       [0143]    To compound 46 (500 mg, 0.57 mmol) in 10 mL of DMA was added bis(p-nitropenyl)carbonate (210 mg, 0.69 mmol), and DIEA (35 μL, 0.2 mmol). The mixture was stirred for 18 h, then 100 mL of ether was added and the precipitate was collected by filtration to give compound 47 (500 mg, 85%). MS m/z 1030.6 (M+H). 
       Preparation of Compound 49: 
       [0144]    To compound 47 (125 mg, 0.12 mmol) in 4 mL of DMF was added HOBt (7 mg, 0.05 mmol), DIEA (21 μL, 0.12 mmol), and amine 48 (40 mg, 0.12 mmol). The mixture was stirred for 16 h, then 200 μL of piperidine was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 49 (72 mg, 60%). MS m/z 1005.6 (M+H). 
       Preparation of Compound 6: 
       [0145]    To compound 49 (30 mg, 0.027 mmol) in 2 mL of DCM was added DIEA (15 μL, 0.086 mmol), DIEA (50 μL, 0.288 mmol), and anhydride 50 (19 mg, 0.027 mmol). The mixture was stirred for 30 min, then evaporated and purified by HPLC to give compound 6 (32 mg, 88%). MS m/z 1347.5 (M+H). 
       Example 5 
     Preparation of Compound 7. 
       [0146]    
       
                 
         
             
             
         
       
     
         [0000]    Compound 7 was synthesized from compound 49 (0.1 mmol) and anhydride 63 (0.1 mmol) as described for the synthesis of compound 6. Yield: 79%. MS m/z 1296.8 (M+H). 
       Example 6 
       [0147]    
       
                 
         
             
             
         
       
     
       Preparation of Compound 8. 
       [0148]    To a solution of compound 47 (0.1 mmol) in THF (3 mL) was added a solution of compound 64 (0.15 mmol, 67 mg) in acetonitrile/water (1/1, v/v, 1 mL), followed by DIEA (50 μL). After 30 min, the reaction was acidified and concentrated. The residue was purified by reverse phase HPLC to give compound 8 as a white solid (87 mg). MS m/z 1243.6 [M+H]+. 
       Example 7 
     Preparation of Compound 9. 
       [0149]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
       Preparation of Compound 52. 
       [0150]    To compound 46 (120 mg, 0.12 mmol) in 3 mL of DMF was added K 2 CO 3  (118 mg, 0.85 mmol), and bromoacetate 51 (35 mg, 0.18 mmol). The mixture was stirred for 16 h, then evaporated. The residue was dissolved in 2 mL of DCM, filtered, and 2 mL of TFA was added. After 20 min the mixture was evaporated and purified by HPLC to give compound 52 (92 mg, 83%). MS m/z 923.5 (M+H). 
       Preparation of Compound 53: 
       [0151]    To compound 52 (92 mg, 0.1 mmol) in 2 mL of DMF was added HATU (38 mg, 0.1 mmol), DIEA (70 μL, 0.4 mmol), and boc-hydrazine (15 mg, 0.12 mmol). The mixture was stirred for 30 min, then evaporated to dryness. The residue was dissolved in 2 mL of DCM, then 1 mL of TFA was added and stirred for 10 min. The mixture was evaporated and purified by HPLC to give compound 53 (82 mg, 78%). MS m/z 937.5 (M+H). 
       Preparation of Compound 9: 
       [0152]    To compound 54 (53 mg, 0.156 mmol) in 2 mL of DCM was added DIC (10 mg, 0.078 mmol) and stirred for 10 min. Then DIEA (54 μL, 0.312 mmol) and amine 53 (82 mg, 0.078 mmol) was added and the mixture was stirred for 15 min. The mixture was evaporated and purified by HPLC to give compound 9 (62 mg, 63%). MS m/z 1260.5 (M+H). 
       Example 8 
       [0153]    Preparation of Compound 13 
         [0000]    
       
                 
         
             
             
         
       
     
         [0154]    To compound 37 (130 mg, 0.26 mmol) in 3 mL of DMF was added HATU (110 mg, 0.29 mmol), DIEA (175 μL, 1 mmol), and amine 36 (110 mg, 0.27 mmol). The mixture was stirred for 30 min, then concentrated to dryness. The residue was then treated with TFA/DCM (1/4, v/v, 5 mL) for 30 min. The mixture was evaporated and purified by HPLC to give compound 66 (108 mg, 65%). MS m/z 670.5 (M+H). 
         [0155]    To compound 41 (85 mg, 0.12 mmol) in 2 mL of DMF was added HATU (48 mg, 0.12 mmol), DIEA (83 μL, 0.48 mmol), and amine 66 (94 mg, 0.12 mmol). The mixture was stirred for 30 min, then piperidine (0.2 mL) was added and stirred for 30 min. The mixture was concentrated and purified by HPLC to give compound 67 (87 mg, 63%). MS m/z 1028.7 (M+H). 
         [0156]    To a solution of compound 67 (57 mg, 0.05 mmol) and acid 68 (22 mg) in DCM/DMF (3/1, v/v, 4 mL) was added PyBrOP (0.055 mmol) and DIEA (35 μL). The mixture was stirred at room temperature for 30 min and then concentrated to about 2 mL. The residue was purified by reverse phase HPLC to give compound 13 (41 mg). MS m/z 1425.7 (M+H). 
       Example 9 
       [0157]    This example provides the results of EC50 assays of the designated drug conjugated antibodies measured in vitro in specified cells. The antibody used was an anti-HER2 IgG class of antibody. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                   
                 MDA- 
                 MDA- 
                 MDA-MB- 
               
               
                   
                 SBKR3  
                 HCC1954 
                 SKOV-3 
                 BT474 
                 MB-453 
                 MB-175 
                 361  
               
               
                 Conjugate 
                 (Her2+++) 
                 (Her2+++) 
                 (Her2+++) 
                 (Her2+++) 
                 (Her2++) 
                 (Her2+) 
                 (Her2+++) 
               
             
          
           
               
                 ID 
                 EC50 [nM] 
               
               
                   
               
             
          
           
               
                 16 
                 0.040 
                 0.138 
                 0.405 
                 0.423 
                 1.195 
                 3.635 
                   
               
               
                 17 
                 0.106 
                 0.237 
                 0.334 
                 0.623 
                 26.42 
                 20.08 
                   
               
               
                 19 
                 0.156 
                 0.193 
                 0.340 
                 0.232 
                 3.946 
                   
                 0.640 
               
               
                 21 
                 0.3432 
                 0.1788 
                   
                 1.065 
                   
                 0.4904 
                 0.1326 
               
               
                 22 
                 0.06349 
                 0.04926 
                   
                 0.346 
                 0.137 
                 0.2628 
                 0.04987 
               
               
                 23 
                 0.04644 
                 0.03678 
                   
                 0.345 
                 0.118 
                 0.2095 
                 0.04657 
               
               
                 65 
                 0.158 
                 0.117 
                   
                 0.100 
                 4.762 
               
               
                   
               
             
          
         
       
     
       Example 10 
       [0158]    This example shows in vivo efficacy of ADC 16 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model.  FIG. 1  shows a single dose of conjugate 16 administered to BALB/c nude mice by intravenous administration. There were 8 mice in each group and total 6 groups of mice were studied: 3 groups were injected with T-DM1 (Trastuzumab—DM1 conjugate) at different doses; 2 groups were injected with ADC 16 at different doses; and one vehicle control. All the drugs were administered in the same manner (single dose). A single dose of ADC-16 iv. at 1 mg/kg or 3 mg/kg outperformed T-DM1 at 3 mg/kg or 10 mg/kg respectively. 3 mg/kg ADC-16 completely inhibited tumor growth up to 100 days. 
       Example 11 
       [0159]    This example shows in vivo safety of ADC 16 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model.  FIG. 2  shows a single dose of conjugate 16 administered to BALB/c nude mice by intravenous administration. There were 8 mice in each group and total 7 groups of mice were studied: 3 groups were injected with T-DM1 (Trastuzumab—DM1 conjugate) at different doses; 3 groups were injected with ADC 16 at different doses; and one vehicle control. All the drugs were administered in the same manner (single dose). A single dose of ADC-16 iv. at 1 mg/kg, 3 mg/kg or 10 mg/kg did not retard body weight gain. The difference of the body weights between T-DM1 and ADC-16 groups were caused by the difference of tumor weight.  FIG. 3  shows pictures of the mice 35 days after treatment. 
       Example 12 
       [0160]    This example ( FIG. 4A ) shows ADC-23 induces equivalent or stronger anti-proliferative activity in breast cancer cell lines, compared to MMAE conjugates. In these studies, the cells were all treated with either ADC-23 or MMAE conjugates for 3 d. IC50 is determined as the concentration that showed 50% inhibition of cell growth. 
       Example 13 
       [0161]    This example ( FIG. 4B ) shows ADC-16 induces equivalent or stronger anti-proliferative activity in breast cancer cell lines, compared to MMAE conjugates. In the above studies, the cells were all treated with either ADC-16 or MMAE conjugates for 3 d. IC50 is determined as the concentration that showed 50% inhibition of cell growth. 
       Example 14 
       [0162]    This example ( FIG. 5 ) shows the in vivo efficacy of ADC-65, ADC-23 and ADC-19 in LoVo (Colon), MDA-MB-468 (Breast), BxPC-3 (Pancreatic), PA-1 (Ovarian) and H1975 NSCLC xenograft nude mice. All ADCs were given as single dose via iv. at indicated concentrations. The ADCs tested outperformed MMAF in most cases at the same level, and completely inhibited tumor growth by single dose. 
       Example 15 
       [0163]    This example shows in vivo safety and efficacy of ADC 19 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model.  FIGS. 6A and 6B  shows a single dose of conjugate 19 administered to BALB/c nude mice by intravenous administration. There were 8 mice in each group and total 3 groups of mice were studied: 1 group of mice was injected with ADC 16; 1 group of mice was injected with ADC 19; and one vehicle control. All the drugs were administered in the same manner (single dose). A single dose of ADC-19 iv. at 2 mg/kg was comparable to that of ADC-16 at the same dose and completely inhibited tumor growth up to 49 days and did not retard body weight gain that was comparable to ADC-16. 
       Example 16 
       [0164]    This example shows the general conjugation procedure for synthesizing antibody drug conjugates 16, 17, 19, and 64. To a solution of 0.5-50 mgs/mL of antibody in buffer at pH 6.0-9.0 with 0-30% organic solvent, was added 0.1-10 eq of activated drug linker conjugate (1, or 2, or 3, or 4, or 5, or 62) in a manner of portion wise or continuous flow. The reaction was performed at 0-40° C. for 0.5-50 hours with gentle stirring or shaking, monitored by HIC-HPLC. The resultant crude ADC product underwent necessary down-stream steps of desalt, buffet changes/formulation, and optionally, purification, using the state-of-art procedures. The ADC product was characterized by HIC-HPLC, SEC, RP-HPLC, and optionally LC-MS. 
       Example 17 
       [0165]    This example shows a general conjugation procedure for synthesizing antibody drug conjugates 21, 22, 23, 24, 28, and 65. To a solution of antibody, 0.5-50 mgs/mL, in a certain buffet at pH 5.0-9.0, such as PBS, was added 0.5-100 eq of reducing agent such as TCEP and DTT. The reduction was performed at 0-40° C. for 0.5-40 hours with gentle stirring or shaking, and then the reducing agent was removed by column or ultrafiltration. To the reduced antibody, 0.5-50 mgs/mL, in a certain buffet at pH 5.0-9.0, such as PBS, with 0-30% of organic co-solvent such as DMA, was added 0.5-10 eq of the drug-linker reactant (selected from compound 6-15, or 63). The reaction was conducted at 0-40° C. for 0.5-40 hours with gentle stirring or shaking, monitored by HIC-HPLC. The resultant crude ADC product underwent necessary down-stream steps of desalt, buffet changes/formulation, and optionally, purification, using the state-of-art procedures. The final ADC product was characterized by HIC-HPLC, SEC, RP-HPLC, and optionally LC-MS.