PATENT ABSTRACT
The present invention is directed to novel macrocyclic compounds of formula (I) and their pharmaceutically acceptable salts, hydrates or solvates: 
                                 
wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , n 1 , m, p Z 1 , Z 2 , and Z 3  are as describe in the specification. The invention also relates to compounds of formula (I) which are antagonists of the motilin receptor and are useful in the treatment of disorders associated with this receptor and with or with motility dysfunction.

PATENT DESCRIPTION
RELATED APPLICATION INFORMATION 
     This application is a continuation under 35 U.S.C. §120 of U.S. patent application Ser. No. 12/273,638, filed Nov. 19, 2008, currently pending, which is a continuation of U.S. patent application Ser. No. 10/872,142, filed Jun. 18, 2004 and issued as U.S. Pat. No. 7,521,420, which claims the benefit of U.S. Patent Application Ser. No. 60/479,223, filed Jun. 18, 2003. The disclosure of each application is incorporated herein by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to novel conformationally-defined macrocyclic compounds, pharmaceutical compositions comprising same and intermediates used in their manufacture. More particularly, the invention relates to macrocyclic compounds that have been demonstrated to selectively antagonize the activity of the motilin receptor. The invention further relates to macrocyclic compounds useful as therapeutics for a range of gastrointestinal disorders, in particular those in which malfunction of gastric motility or increased motilin secretion is observed, such as hypermotilinemia, irritable bowel syndrome and dyspepsia. 
     BACKGROUND OF THE INVENTION 
     A number of peptide hormones are involved in the control of the different functions in the gastrointestinal (GI) tract, including absorption, secretion, blood flow and motility (Mulvihill, et al. in  Basic and Clinical Endocrinology,  4 th  edition, Greenspan, F. S.; Baxter, J. D., eds., Appleton &amp; Lange: Norwalk, Conn., 1994, pp 551-570). Since interactions between the brain and GI system are critical to the proper modulation of these functions, these peptides can be produced locally in the GI tract or distally in the CNS. 
     One of these peptide hormones, motilin, a linear 22-amino acid peptide, plays a critical regulatory role in the GI physiological system though governing of fasting gastrointestinal motor activity. As such, the peptide is periodically released from the duodenal mucosa during fasting in mammals, including humans. More precisely, motilin exerts a powerful effect on gastric motility through the contraction of gastrointestinal smooth muscle to stimulate gastric emptying, decrease intestinal transit time and initiate phase III of the migrating motor complex in the small bowel (Itoh, 1, Ed.,  Motilin , Academic Press: San Diego, Calif., 1990, ASIN: 0123757304; Nelson, D. K.  Dig. Dis. Sci.  1996, 41, 2006-2015; Peeters, T. L.; Vantrappen, G.; Janssens, J.  Gastroenterology  1980, 79, 716-719). 
     Motilin exerts these effects through receptors located predominantly on the human antrum and proximal duodenum, although its receptors are found in other regions of the GI tract as well (Peeters, T. L.; Bormans, V.; Vantrappen, G.  Regul. Pept.  1988, 23, 171-182). Therefore, motilin hormone is involved in motility of both the upper and lower parts of the GI system (Williams et al.  Am. J. Physiol.  1992, 262, G50-G55). In addition, motilin and its receptors have been found in the CNS and periphery, suggesting a physiological role in the nervous system that has not yet been definitively elucidated (Depoortere, I.; Peeters, T. L.  Am. J. Physiol.  1997, 272, G994-999 and O&#39;Donohue, T. L et al.  Peptides  1981, 2, 467-477). For example, motilin receptors in the brain have been suggested to play a regulatory role in a number of CNS functions, including feeding and drinking behavior, micturition reflex, central and brain stem neuronal modulation and pituitary hormone secretion (Itoh, Z. Motilin and Clinical Applications.  Peptides  1997, 18, 593-608; Asakawa, A.; Inui, A.; Momose, K.; et al., M.  Peptides  1998, 19, 987-990 and Rosenfeld, D. J.; Garthwaite, T. L.  Physiol. Behav.  1987, 39, 753-756). Physiological studies have provided confirmatory evidence that motilin can indeed have an effect on feeding behavior (Rosenfeld, D. J.; Garthwaite, T. L.  Phys. Behav.  1987, 39, 735-736). 
     The recent identification and cloning of the human motilin receptor (WO 99/64436) has simplified and accelerated the search for agents which can modulate its activity for specific therapeutic purposes. 
     Due to the critical and direct involvement of motilin in control of gastric motility, agents that either diminish (hypomotility) or enhance (hypermotility) the activity at the motilin receptor, are a particularly attractive area for further investigation in the search for new effective pharmaceuticals towards these indications. 
     Peptidic agonists of the motilin receptor, which have clinical application for the treatment of hypomotility disorders, have been reported (U.S. Pat. Nos. 5,695,952; 5,721,353; 6,018,037; 6,380,158; 6,420,521, U.S. Appl. 2001/0041791, WO 98/42840; WO 01/00830 and WO 02/059141). Derivatives of erythromycin, commonly referred to as motilides, have also been reported as agonists of the motilin receptor (U.S. Pat. Nos. 4,920,102; 5,008,249; 5,175,150; 5,418,224; 5,470,961; 5,523,401, 5,554,605; 5,658,888; 5,854,407; 5,912,235; 6,100,239; 6,165,985; 6,403,775). 
     Antagonists of the motilin receptor are potentially extremely useful as therapeutic treatments for diseases associated with hypermotility and hypermotilinemia, including irritable bowel syndrome, dyspepsia, gastroesophogeal reflux disorders, Crohn&#39;s disease, ulcerative colitis, pancreatitis, infantile hypertrophic pyloric stenosis, diabetes mellitus, obesity, malabsorption syndrome, carcinoid syndrome, diarrhea, atrophic colitis or gastritis, gastrointestinal dumping syndrome, postgastroenterectomy syndrome, gastric stasis and eating disorders leading to obesity. 
     A variety of peptidic compounds have been described as antagonists of the motilin receptor (Depoortere, I.; Macielag, M. J.; Galdes, A.; Peeters, T. L.  Eur. J. Pharmacol.  1995, 286, 241-247; U.S. Pat. Nos. 5,470,830; 6,255,285; 6,586,630; 6,720,433; U.S. 2003/0176643; WO 02/64623). These peptidic antagonists suffer from the known limitations of peptides as drug molecules, in particular poor oral bioavailability and degradative metabolism. 
     Cyclization of peptidic derivatives is a method employed to improve the properties of a linear peptide both with respect to metabolic stability and conformational freedom. 
     Cyclic molecules tend to be more resistant to metabolic enzymes. Such cyclic tetrapeptide motilin antagonists have been reported (Haramura, M. et al  J. Med. Chem.  2002, 45, 670-675, U.S. 2003/0191053; WO 02/16404). 
     Other motilin antagonists, which are non-peptidic and non-cyclic in nature have also been reported (U.S. Pat. Nos. 5,972,939; 6,384,031; 6,392,040; 6,423,714; 6,511,980; 6,624,165; 6,667,309; U.S. 2002/0111484; 2001/041701; 2002/0103238; 2001/0056106, 2002/0013352; 2003/0203906 and 2002/0002192) 
     The macrocyclic motilin antagonists of the present invention comprise elements of both peptidic and non-peptidic structures in a combination which has not been pursued for this application previously. 
     Indeed, the structural features of antagonists of the present invention are different. In particular, within the known motilin antagonists which are cyclic peptides, it was found that such derivatives containing D-amino acids were devoid of activity. In contrast, for the tripeptidomimetic compounds of the present invention, the D-stereochemistry is required for two of the three building elements. 
     The motilin antagonists of the present invention are also distinct from the prior art in that they comprise a tether element to fulfill the dual role of controlling conformations and providing additional sites for interaction either through hydrophobic interactions, hydrogen bonding or dipole-dipole interactions. 
     SUMMARY OF THE INVENTION 
     In a first aspect, the present invention is directed to compounds of formula (I): 
                                
and pharmaceutically acceptable salts, hydrates or solvates thereof wherein:
 
     Z 1 , Z 2  and Z 3  are independently selected from the group consisting of O, N and NR 10 , wherein R 10  is selected from the group consisting of hydrogen, lower alkyl, and substituted lower alkyl; 
     R 1  is independently selected from the group consisting of lower alkyl substituted with aryl, lower alkyl substituted with substituted aryl, lower alkyl substituted with heteroaryl and lower alkyl substituted with substituted heteroaryl; 
     R 2  is hydrogen; 
     R 3  is independently selected from the group consisting of alkyl and cycloalkyl with the proviso that when Z 1  is N, R 3  can form a four, five, six or seven-membered heterocyclic ring together with Z 1 ; 
     R 4  is hydrogen; 
     R 5  and R 6  are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl and substituted heteroaryl, with the proviso that at least one of R 5  and R 6  is hydrogen; 
     X is selected from the group consisting of O, NR 8 , and N(R 9 ) 2   + ;
         wherein R 8  is selected from the group consisting of hydrogen, lower alkyl, substituted lower alkyl, formyl, acyl, carboxyalkyl, carboxyaryl, amido, sulfonyl, sulfonamido and amidino; and   R 9  is selected from the group consisting of hydrogen, lower alkyl, and substituted lower alkyl;       

     m, n 1  and p are independently selected from 0, 1 or 2; and 
     T is a bivalent radical of formula II:
 
—U—(CH 2 ) d —W—Y—Z—(CH 2 ) e —  (II)
         wherein d and e are independently selected from 0, 1, 2, 3, 4 or 5;   wherein U is bonded to X of formula (I) and is —CH 2 — or —C(═O)—;   wherein Y and Z are each optionally present;   W, Y and Z are independently selected from the group consisting of: —O—, —NR 28 —, —S—, —SO—, —SO 2 —, —C(═O)—O—, —O—C(═O)—, —C(═O—NH—, —NH—C(═O)—, —SO 2 —NH—, —NH—SO 2 —, —CR 29 R 30 —, —CH═CH— with a configuration Z or E, and —C≡C—, or from a ring structure independently selected from the group       

                                
wherein any carbon atom contained within said ring structure, can be replaced by a nitrogen atom, with the proviso that if said ring structure is a monocyclic ring structure, it does not comprise more than four nitrogen atoms and if said ring structure is a bicyclic ring structure, it does not comprise more than six nitrogen atoms;
         G 1  and G 2  each independently represent a covalent bond or a bivalent radical selected from the group consisting of —O—, —NR 41 —, —S—, —SO—, —SO 2 —, —C(═O)—, —O—C(═O)—, —C(═O)NH—, —NH—C(═O)—, —SO 2 —NH—, —NH—SO 2 —, —CR 42 R 43 —, —CH═CH— with a configuration Z or E, and —C≡C—; with the proviso that G 1  is bonded closer to U than G 2 ;   K 1 , K 2 , K 3 , K 4 , K 6 , K 15  and K 16  are independently selected from the group consisting of O, NR 44  and S;   f is selected from 1, 2, 3, 4, 5 or 6;   R 31 , R 32 , R 38 , R 39 , R 48  and R 49  are independently selected from hydrogen, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamido; and   R 33 , R 34 , R 35 , R 36 , R 37 , R 47 , R 50  and R 51  are independently selected from hydrogen, halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamido.       

     In a second aspect, the invention also proposes compounds of formula (1) which are antagonists of the motilin receptor. 
     In a third aspect, the invention proposes a method of treating a disorder associated with the motilin receptor or motility dysfunction in humans and other mammals, comprising administering a therapeutically effective amount of a compound of formula (1). 
     While the invention will be described in conjunction with example embodiments, it will be understood that it is not intended to limit the scope of the invention to such embodiment. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included as defined by the appended claims. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Preferably in formula (I), as depicted hereinabove, R 1  is selected from the group consisting of —(CH 2 ) q R 11 , and —CHR 12 R 13  
         wherein q is 0, 1, 2 or 3; and   R 11  and R 12  are independently selected from a ring structure from the following group:       

     
       
                 
         
             
             
         
      
         
         
           
             wherein any carbon atom in said ring structure can be replaced a nitrogen atom, with the proviso that if said ring structure is a monocyclic ring structure, it does not comprise more than four nitrogen atoms and if said ring structure is a bicyclic ring structure, it does not comprise more than six nitrogen atoms; 
             A 1 , A 2 , A 3 , A 4  and A 5  are each optionally present and are independently selected from the group consisting of halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamido; 
             B 1 , B 2 , B 3 , and B 4  are independently selected from NR 14 , S or O, wherein R 14  is selected from the group consisting of hydrogen, alkyl, substituted alkyl, formyl, acyl, carboxyalkyl, carboxyaryl, amido, sulfonyl and sulfonamido; 
             R 13  is as defined for as R 11  and R 12  or is selected from the group comprising lower alkyl, substituted lower alkyl, hydroxy, alkoxy, aryloxy, amino, carboxy, carboxyalkyl, carboxyaryl, and amido.
 
wherein A 1 , A 2 , A 3 , A 4  and A 5  are most preferably selected from halogen, trifluororomethyl, C 1-6  alkyl or C 1-6 alkoxy.
 
           
         
       
    
     Preferably, R 11 , R 12  and R 13  are selected from the group consisting of: 
                                
wherein R a  and R b  are chosen from the group consisting of Cl, F, CF 3 , OCH 3 , OH, and C(CH 3 ) 3  and CH 3 .
 
     Also preferably, R 3  in formula (I), is selected from the group consisting of:
         —(CH 2 ) s CH 3 , —CH(CH 3 )(CH 2 ) t CH 3 , —CH(OR 15 )CH 3 , —CH 2 SCH 3 —CH 2 CH 2 SCH 3 , —CH 2 S(═O)CH 3 , —CH 2 CH 2 S(═O)CH 3 , —CH 2 S(═O) 2 CH 3 , —CH 2 CH 2 S(═O) 2 CH 3 , —(CH 2 ) u CH(CH 3 ) 2 , —C(CH 3 ) 3 , and —(CH 2 ) y —R 21 , wherein:
           s and u are independently selected from 0, 1, 2, 3, 4 or 5;   t is independently selected from 1, 2, 3 or 4;   y is selected from 0, 1, 2, 3 or 4;   R 15  is selected from the group consisting of hydrogen, alkyl, substituted alkyl, formyl and acyl;   R 21  is selected from a ring structure selected from the following group:   
               

     
       
                 
         
             
             
         
      
         
         
           
             wherein any carbon atom in said ring structure can be replaced by a nitrogen atom, with the proviso that if said ring structure is a monocyclic ring structure, it does not comprise more than four nitrogen atoms and if said ring structure is a bicyclic ring structure, it does not comprise more than six nitrogen atoms; 
             z is selected from 1, 2, 3, 4 or 5; 
             E 1 , E 2  and E 3  are each optionally present and are independently selected from the group consisting of halogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, cyano, nitro, mercapto, sulfinyl, sulfonyl and sulfonamido; and J is optionally present and is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl and sulfonamido. 
           
         
       
    
     The tether portion (T) of formula (I) is preferably selected from the group consisting of: 
                                
wherein L 1  is O, NH or NMe; L 2  is CH or N; L 3  is CH or N; L 4  is O or CH 2 ; L 5  is CH or N L 6  is CR 52 R 53  or O; R 46  is H or CH 3 ;
 
R 52 , R 53 , R 54 , R 55 , R 56  and R 57  are independently selected from hydrogen, lower alkyl, substituted lower alkyl, hydroxy, alkoxy, aryloxy, amino, and oxo; or R 52  together with R 53  or R 54  together with R 55  or R 58  together with R 57  can independently form a three to seven-membered cyclic ring comprising carbon, oxygen, sulfur and for nitrogen atoms;
 
(X) is the site of a covalent bond to X in formula (I); and
 
(Z 3 ) is the site of a covalent bond to Z 3  in formula (I).
 
     In a particularly preferred embodiment of the invention, there are provided compounds of formula (I) wherein m, n and p are 0, X, Z 1 , Z 2  and Z 3  are NH and R 2 , R 4  and R 5  are hydrogen, represented by formula (III): 
     
       
                 
         
             
             
         
      
     
     According to another aspect of the invention, there are provided compounds of formula (I) wherein when Z 1  is a nitrogen atom, R 3  forms a four, five, six or seven-membered heterocyclic ring together with Z 1 , represented by formula (IV): 
                                
wherein said heterocyclic ring may contain a second nitrogen atom, or an oxygen, or sulfur atom;
 
n 2  is selected from 0, 1, 2 or 3
 
R 7  is optionally present and is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, alkoxy, aryloxy, oxo, amino, halogen, formyl, acyl, carboxy, carboxyalkyl, carboxyaryl, amido, carbamoyl, guanidino, ureido, amidino, mercapto, sulfinyl, sulfonyl and sulfonamide.
 
     It is to be understood, that in the context of the present invention, the terms amino, guanidine, ureido and amidino encompass substituted derivatives thereof as well. 
     Preferably, the invention provides a method of treating a disorder associated with hypermotility or hypermotilinemia in humans and other mammals comprising administering a therapeutically effective amount of a compound of formula (I). 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  depicts Scheme 1 presenting a general synthetic strategy to conformationally-defined macrocycles of the present invention. 
         FIG. 2  depicts the standard procedure for the synthesis of tether T8 of Example 16. 
         FIG. 3  depicts the standard procedure for the synthesis of tether T9 of Example 17. 
         FIG. 4  depicts the standard procedure for the synthesis of Ddz-propargylamine of Example 18. 
         FIG. 5A  depicts the standard procedure for the synthesis of tether T10 of Example 19. 
         FIG. 5B  depicts the second synthetic route to tether T10 of Example 19. 
         FIG. 6  depicts the standard procedure for the synthesis of Tether T11 of Example 20. 
         FIG. 7  depicts the standard procedure for the synthesis of tether T12 of Example 26. 
         FIG. 8  depicts the procedure for synthesis of PPh 3 -DIAD adduct of Example 29-C. 
         FIG. 9  depicts the standard procedure for attachment of tethers via reductive amination of Example 30. 
         FIG. 10  depicts the standard procedure for the synthesis of tether T28 of Example 32. 
         FIG. 11  the standard procedure for the synthesis of tether T32 of Example 36. 
         FIGS. 12A ,  12 B depict the standard procedure for the synthesis of tether T33a and T33b of Example 37. 
         FIG. 13  depicts the standard procedure for the synthesis of tether T34 of Example 38. 
         FIG. 14  depicts the standard procedure for the synthesis of tether T35 of Example 39. 
         FIG. 15  depicts the standard procedure for the synthesis of tether T36 of Example 40. 
         FIG. 16  depicts the standard procedure for the synthesis of tether T37 of Example 41. 
         FIG. 17  depicts the standard procedure for the synthesis of tether T38 of Example 42. Chiral T38 can be accessed through the use of asymmetric synthesis methods, resolution or chiral chromatography techniques available in the literature. 
       HPLC (standard gradient) t R =8.46 min 
       Chiral material can be accessed by starting with the chiral epoxide. For example, the (S)-isomer of T38 was constructed in 89% overall yield from (S)-propylene oxide. 
         FIG. 18  depicts the standard procedure for the synthesis of tether T39 of Example 43. Chiral T39 can be accessed through the use of asymmetric synthesis methods, resolution or chiral chromatography techniques available in the literature. 
         FIG. 19  depicts the standard procedure for the synthesis of tether T40 of Example 44. Chiral T40 can be accessed through the use of asymmetric synthesis methods, resolution or chiral chromatography techniques available in the literature. 
         FIG. 20  depicts the standard procedure for the synthesis of tether T41 of Example 45. 
         FIG. 21  depicts the standard procedure for the synthesis of tether T42 of Example 46. 
         FIG. 22  depicts Scheme 2 of the thioester strategy for macrocyclic compounds of the present invention. 
         FIG. 23  depicts the competitive binding curve for compound 8. 
         FIG. 24  depicts the competitive binding curve for compound 11 
     
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     Although preferred embodiments of the present invention have been described in detail herein and illustrated in the accompanying structures, schemes and tables, it is to be understood that the invention is not limited to these precise embodiments and that various changes and modifications may be effected therein without departing from the scope or spirit of the present invention. 
     Specifically preferred compounds of the present invention, include, but are not limited to: 
     
       
                 
         
             
             
         
      
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
     In addition to the preferred tethers (T) illustrated previously, other specific tethers employed for compounds of the invention are shown hereinbelow: 
     
       
                 
         
             
             
         
      
       
                 
         
             
             
         
       
     
     In a preferred embodiment, the present invention is directed to a method of treating irritable bowel syndrome, dyspepsia, Crohn&#39;s disease, gastroesophogeal reflux disorders, ulcerative colitis, pancreatitis, infantile hypertrophic pyloric stenosis, carcinoid syndrome, malabsorption syndrome, diarrhea, diabetes mellitus, obesity, postgastroenterectomy syndrome, atrophic colitis or gastritis, gastric stasis, gastrointestinal dumping syndrome, celiac disease and eating disorders leading to obesity in humans and other mammals comprising administering a therapeutically effective amount of a compound of formula (I). 
     Synthetic Methods 
     A. General Information 
     Reagents and solvents were of reagent quality or better and were used as obtained from various commercial suppliers unless otherwise noted. DMF, DCM and THF used are of DriSolv® (EM Science, now EMD Chemicals, Inc., part of Merck KgaA, Darmstadt, Germany) or synthesis grade quality except for (i) deprotection, (ii) resin capping reactions and (iii) washing. NMP used for the amino acid (AA) coupling reactions is of analytical grade. DMF was adequately degassed by placing under vacuum for a minimum of 30 min prior to use. Tyr(3tBu) was synthesized following the method reported in JP2000 44595. Cpa was made using literature methods (Tetrahedron: Asymmetry 2003, 14, 3575-3580) or obtained commercially. Boc- and Fmoc-protected amino acids and side chain protected derivatives, including those of N-methyl and unnatural amino acids, were obtained from commercial suppliers or synthesized through standard methodologies known to those in the art. Ddz-amino acids were either synthesized by standard procedures or obtained commercially from Orpegen (Heidelberg, Germany) or Advanced ChemTech (Louisville, Ky., USA). Bts-amino acids were synthesized as described in Example 6. Hydroxy acids were obtained from commercial suppliers or synthesized from the corresponding amino acids by literature methods. Analytical TLC was performed on pre-coated plates of silica gel 60F254 (0.25 mm thickness) containing a fluorescent indicator. The term “concentrated/evaporated under reduced pressure” indicates evaporation utilizing a rotary evaporator under either water aspirator pressure or the stronger vacuum provided by a mechanical oil vacuum pump as appropriate for the solvent being removed. “Dry pack” indicates chromatography on silica gel that has not been pre-treated with solvent, generally applied on larger scales for purifications where a large difference in R f  exists between the desired product and any impurities. For solid phase chemistry processes, “dried in the standard manner” is that the resin is dried first in air (1 h), and subsequently under vacuum (oil pump usually) until full dryness is attained (˜30 min to O/N). 
     B. Synthetic Methods for Building Blocks of the Invention 
     Example 6 
     Standard Procedure for the Synthesis of Bts-Amino Acids 
     
       
                 
         
             
             
         
      
     
     To a solution of the amino acid or amino acid derivative (0.1 mol, 1.0 eq) in 0.25 N sodium hydroxide (0.08 mol, 0.8 eq) with an initial pH of approximately 9.5 (pH meter) at rt, solid Bts-Cl (0.11 mol, 1.1 eq) was added in one portion. The resulting suspension was stirred vigorously for 2-3 d. The pH of the reaction should be adjusted with 5.0 N sodium hydroxide as required to remain within the range 9.5-10.0 during this time. Typically, the pH has to be adjusted every 20-30 min during the first 5 h. Once the pH stops dropping, it is an indication that the reaction is almost complete. This can be confirmed by TLC (EtOAc:MeOH, 95:5). Upon completion, the reaction mixture was washed with Et 2 O. Washing is continued until the absence of non-polar impurities in the aqueous layer is confirmed by TLC (typically 3×100 mL). The aqueous solution was then cooled to 0° C., acidified to pH 2.0 with 1 N HCl until no additional cloudiness forms, and extracted with EtOAc (3×100 mL). Alternatively, a mixture of DCM and EtOAc may be used as the extraction solvent, depending on the solubility of the product obtained from different amino acids or derivatives. Note that DCM cannot be used solely as solvent because of the emulsion formed during extraction. The combined organic phases were washed with brine (2×150 mL), dried over MgSO 4 , filtered and evaporated under reduced pressure. DCM (1×) and hexanes (2×) were evaporated from the residue in order to ensure complete removal of the EtOAc and give the desired compound as a solid in 55-98% yield. 
     The following are modifications that have proven useful for certain amino acids: 
     Gly, Ala, D-Ala, β-Ala and GABA: Use 1.5 eq of amino acid per eq of Bts-Cl, in order to prevent dibetsylation. 
     Met: Carry out the reaction under N2 to prevent oxidation. 
     Gln and Asn: Due to the solubility of Bts-Gln and Bts-Asn, the work-up required is modified from the standard procedure: Upon completion of the reaction, the reaction mixture was washed with diethyl ether. Washing is continued until the absence of non-polar impurities in the aqueous layer is confirmed by TLC (typically 3×100 mL). The aqueous phase was then cooled to 0° C. and acidified to pH 2.0 with 6 N HCl. 6 N HCl was employed to minimize the volume of the solution due to the water solubility of Bts-Gln and Bts-Asn. (They are, in contrast, difficult to dissolve in DCM, EtOAc or chloroform.) The solution was maintained at 0° C. for 10 min and the product was collected by filtration as a white precipitate. The solid was washed with cold water (1×), cold brine (2×) and water (1×, 25° C.). The pH of this wash was taken, if it is not approximately 4, the solid was washed again with water. Finally, the solid was washed with cold EtOAc, then with cold Et 2 O (2×), and finally dried under vacuum (oil pump) (83-85% yield). 
     C. General Synthetic Strategy to Conformationally-Defined Macrocycles of the Present Invention 
     The compounds of Formula I can be synthesized using traditional solution synthesis techniques or solid phase chemistry methods. In either, the construction involves four phases: first, synthesis of the building blocks, including one to four moieties, comprising recognition elements for the biological target receptor, plus one tether moiety, primarily for control and definition of conformation. These building blocks are assembled together, typically in a sequential fashion, in a second phase employing standard chemical transformations. The precursors from the assembly are then cyclized in the third stage to provide the macrocyclic structures. Finally, a post-cyclization processing stage involving removal of protecting groups and optional purification then provides the desired final compounds (see  FIG. 1 ). This method has been previously disclosed in WO 01/25257 and U.S. patent application Ser. No. 09/679,331. A general synthetic strategy is shown in  FIG. 1 . 
     D. Procedures for the Synthesis of Representative Tethers of the Present Invention 
     The important tether component required for compounds of the invention are synthesized as described in WO01/25257, U.S. Provisional Pat. Appl. Ser. No. 60/491,248 or herein. A standard procedure for the synthesis of tether B is shown in  FIG. 2 .
     Step T8-1: Chlorotrimethylsilane (116 mL, 0.91 mol, 1.5 eq) was added to a suspension of 2-hydroxycinnamic acid (100 g, 0.61 mol, 1.0 eq) in MeOH (500 mL, HPLC grade) over 30 min at 0° C. The resulting mixture was stirred at rt O/N. The reaction was monitored by TLC (EtOAc/MeOH:98/2). Heating the reaction mixture in a hot water can accelerate the process if necessary. After the reaction was completed, the reaction mixture was evaporated under reduced pressure to afford methyl 2-hydroxycinnamate as a white solid (108.5 g) in quantitative yield. The identity of this intermediate compound is confirmed by NMR. This reaction can be carried out on larger (kg) scale with similar results   Step T8-2: 3,4-Dihydro-2H-pyran (DHP, 140 mL, 1.54 mol, 2.52 eq) was added dropwise to 2-bromoethanol (108 mL, 1.51 mol, 2.5 eq) in a 2 L three-neck flask with mechanical stirring at 0° C. over 2 h. The resulting mixture was stirred for additional 1 h at rt. Methyl 2-hydroxycinnamate from Step T8-1 (108 g, 0.61 mol, 1.0 eq), potassium carbonate (92.2 g, 0.67 mol, 1.1 eq), potassium iodide (20 g, 0.12 mol, 0.2 eq) and DMF (300 mL, spectrometric grade) were added to the above flask. The reaction mixture was stirred at 70° C. (external temperature) for 24 h. The reaction was monitored by TLC (DCM/Et 2 O: 95/5). The reaction was allowed to cool to rt and Et 2 O (450 mL) was added. The inorganic salts were removed by filtration and washed with Et 2 O (3×50 mL). The filtrate was diluted with hexanes (400 mL) and washed with water (3×500 mL), dried over MgSO 4 , filtered and the filtrate evaporated under reduced pressure. The crude ester (desired product and excess Br—C 2 H 4 —OTHP) was used for the subsequent reduction without further purification.   Step T8-3: DIBAL (1.525 L, 1.525 mol, 2.5 eq, 1.0 M in DCM) was added slowly to a solution of the above crude ester from Step T8-2 (0.61 mol based on the theoretical yield) in anhydrous DCM (610 mL) at −35° C. with mechanical stirring over 1.5 h. The resulting mixture was stirred for 1.5 h at −35° C., then 1.5 h at 0° C. The reaction was monitored by TLC (hexlEtOAc: 50/50). When complete, Na 2 SO 4 .10 H 2 O (100 g, 0.5 eq) was slowly added; hydrogen evolution was observed, when it subsided water was added (100 mL). The mixture was warmed to rt and stirred for 10 min, then warmed to 40° C. with hot water and stirred under reflux for 20 min. The mixture was cooled to rt, diluted with DCM (600 mL), and the upper solution decanted into a filter. The solid that remained in the flask was washed with dichloromethane (5×500 mL) with mechanical stirring and filtered. The filtrate from each wash was checked by TLC, and additional washes performed if necessary to recover additional product. In an alternative work-up procedure, after dilution with DCM (600 mL), the mixture was filtered. The resulting solid was then continuously extracted with 0.5% TEA in dichloromethane using a Soxhlet extractor. Higher yield was typically obtained by this alternative procedure, although it does require more time. The filtrate was concentrated under reduced pressure and the residue purified by dry pack (EtOAc/hex/Et 3 N: 20/80/0.5) to give the product alcohol as a yellowish oil (yield: 90%). The identity and purity were confirmed by NMR.   Step T8-4: To a mixture of the allylic alcohol from Step T8-3 (28 g, 0.100 mol, 1.0 eq) and collidine (0.110 mol, 1.1 eq) in 200 mL of anhydrous DMF under N 2  was added anhydrous LiCl (4.26 g, 0.100 mol, 1.0 eq.) dissolved in 100 mL of anhydrous DMF. The mixture was then cooled to 0° C., and MsCl (12.67 g, 0.110 mol, 1.1 eq., freshly distilled over P 2 O 5 ), was added dropwise. The reaction was allowed to warm to it and monitored by TLC (3:7 EtOAc/hex). When the reaction was complete, NaN 3  (32.7 g, 0.500 mol, 5.0 eq.) was added. The reaction mixture was stirred at rt O/N with progress followed by NMR. When the reaction was complete, the mixture is poured into an ice-cooled water bath, and extracted with diethyl ether (3×). The combined organic phases were then washed sequentially with citrate buffer (2×), saturated sodium bicarbonate (2×), and finally with brine(1×). The organic layer was dried with MgSO 4 , filtered and the filtrate concentrated under reduced pressure. The allylic azide was obtained in 90% combined yield, and was of sufficient quality to use as such for the following step.   Step T8-5: PPh 3  (25.9 g, 0.099 mol, 1.5 eq) was added at 0° C. to a solution of the allylic azide from Step T8-4 (20.0 g, 0.066 mol, 1.0 eq.) in 100 mL of THF. The solution was stirred for 30 min at 0° C. and 20 h at rt. Water (12 mL) was then added and the resulting solution was heated at 60° C. for 4 h. The solution was cooled to rt, 2N HCl (15 mL) added and the mixture stirred for 90 min at 50° C. The separated organic phase was extracted with 0.05 N HCl (2×100 mL). The combined aqueous phase was washed with Et 2 O (5×150 mL) and toluene (4×150 mL) (more extraction could be necessary, follow by TLC), which were combined and back-extracted with 0.05 N HCl (1×100 mL). This acidic aqueous phase from back-extraction was combined with the main aqueous phase and washed with ether (5×150 mL) again. The pH of the aqueous phase was then adjusted to 8-9 by the addition of sodium hydroxide (5 N). Care must be exercised to not adjust the pH above 9 due to the reaction conditions required by the next step. The aqueous phase was concentrated under reduced pressure (aspirator, then oil pump) or lyophilized to dryness. Toluene (2×) was added to the residue and then also evaporated under reduced pressure to remove traces of water. The crude product (desired amino alcohol along with inorgnic salt) was used for the next reaction without further purification.   Step T8-6: A mixture of the crude amino alcohol from Step T8-5 (0.5 mol based on the theoretical yield), Ddz-OPh (174 g, 0.55 mol, 1.1 eq) and Et 3 N (70 mL, 0.5 mol, 1.0 eq) in DMF (180 mL) was stirred for 24 h at 50° C. Additional DMF is added if required to solubilize all materials. The reaction was monitored by TLC (hex/EtOAc:50/50, ninhydrin detection). After the reaction was complete, the reaction mixture was diluted with Et 2 O (1.5 L) and water (300 mL). The separated aqueous phase was extracted with Et 2 O (2×150 mL). The combined organic phase was washed with water (3×500 mL) and brine (1×500 mL), dried over MgSO 4 , filtered and the filtrate concentrated under reduced pressure. The layers were monitored by TLC to ensure no product was lost into the aqueous layer. If so indicated, perform one or more additional extractions with Et 2 O of the aqueous phase to recover this material. The crude product was purified by dry pack (recommended column conditions: EtOAc/hex/Et 3 N: 35/65/0.5 to 65/35/0.5) to give the tether Ddz-T8 as a pale yellow syrup (yield: ˜40%). The identity and purity of the product was confirmed by NMR.   

       1 H NMR (DMSO-d 6 ): 1.6 ppm (s, 6H, 2×CH3), 3.6-3.8 ppm (wide s, 10H, 2×OCH 3 , 2×OCH 2 ), 3.95 ppm (triplet, 2H, CH 2 N), 6-6.2 ppm (m, 2H, 2×CH), 6.2-6.5 ppm (m, 3H, 3×CH, aromatic), 6.6-7.6 ppm (m, 5H, aromatic). 
     A standard procedure for the synthesis of tether T9 is shown in  FIG. 3 . 
     Tether T9 can also be synthesized from T8 by reduction as in step T9-3 or with other appropriate hydrogenation catalysts known to those in the art. 
     A standard procedure for the synthesis of Ddz propargylamine is shown in  FIG. 4 . 
     In a dried three-neck flask, a solution of propargylamine (53.7 g, 0.975 mol, 1.5 eq) in degassed DMF (Drisolv, 388 mL) was treated with Ddz-N 3  (170.9 g, 0.65 mol, 1.0 eq), tetramethylguanidine (TMG, 81.4 mL, 0.65 mol, 1.0 eq) and DIPEA (113.1 mL, 0.65 mol, 1.0 eq) and stirred at 50° C., O/N. The reaction was monitored by TLC (conditions: 25/75 EtOAc/hex. R f : 0.25; detection: UV, ninhydrin). Upon completion, DMF was evaporated under reduced pressure until dryness and the residue dissolved in Et 2 O (1 L). The organic solution was washed sequentially with citrate buffer (pH 4.5, 3×), saturated aqueous sodium bicarbonate (2×), and brine (2×), then dried with MgSO 4 , filtered and the filtrate evaporated under reduced pressure. A pale orange solid was obtained. This solid was triturated with 1% EtOAc in hex, then collected by filtration and dried under vacuum (oil pump) to provide the desired product (153.4 g, 85.2%). 
     A standard procedure for the synthesis of tether T10 is shown in  FIG. 5A . 
     Two alternative routes to this tether have been developed. The first synthetic approach proceeded starting from the commercially available monobenzoate of resorcinol (T10-0). Mitsunobu reaction under standard conditions with the protected amino alcohol from Example 9, followed by saponification of the benzoate provided T10-1 in good yield after recrystallization. Alkylation of the phenol with 2-bromoethanol using the optimized conditions shown permitted the desired product Ddz-T10 to be obtained after dry pack purification in 42% yield. 
     A second synthetic route to T10 is shown in  FIG. 5B . 
     From resorcinol, two successive Mitsunobu reactions are conducted with the appropriate two carbon synthons illustrated, themselves derived from 2-aminoethanol and ethylene glycol, respectively, through known protection methodologies. Lastly, deprotection of the silyl ether, also under standard conditions provided Boc-T10. 
     Although the yields in the two methods are comparable, the first required less mechanical manipulation and is preferred for larger scales. 
     A standard procedure for the synthesis of tether T11 is shown in  FIG. 6 . 
     A standard procedure for the synthesis of tether T12 is shown in  FIG. 7 . 
     In a 3-L flame-dried three-neck flask, a solution of (aminomethyl)phenylthiobenzyl alcohol (12-0, 96 g, 0.39 mol) in degassed DMF (1 L, 0.4 M) was prepared. To this was added DdzN 3  (0.95 eq), followed by TMG (0.39 mol, 49 mL). The reaction was stirred for 10 min, then DIPEA (68 mL, 0.39 mol) added. The mixture was heated at 50° C. under N 2  until TLC indicated no DdzN 3  remained (48 h typically). (TLC eluent: EtOAc:Hex 50:50; detection: ninhydrin). Upon completion, to the reaction mixture was added 3 L citrate buffer and the separated aqueous layer extracted with Et 2 O (3×1500 mL). The combined organic phase was washed sequentially with citrate buffer (2×200 mL), water (2×200 mL) and brine (2×200 mL). The organic layer was dried over MgSO 4 , filtered and the filtrate evaporated under reduced pressure. A dark orange oil was obtained, which was purified by dry-pack. For this procedure, the oil was first dissolved in EtOAc:Hex:DCM:TEA (20:80:1:0.5, v/v/v/v). At this point, a little extra DCM was sometimes required to ensure complete dissolution. The solution was loaded onto the column, then the column eluted with EtOAc:Hex:DCM:Et 3 N (20:80:1:0.5) until all the impurities were separated out as indicated by TLC, paying particular attention to that closest to the desired product. The elution was then continued with EtOAc:Hex:Et 3 N 30:70:0.5 (v/v/v) and finally with EtOAc:hexanes:Et 3 N (50:50:0.5) to elute the desired product. After removal of the solvent from the fractions containing the product under reduced pressure, the residue was dissolved in the minimum amount of DCM, a three-fold larger volume of hexanes added, then the solvents again evaporated under reduced pressure. This treatment was repeated until an off-white foam was obtained. The latter solidified while drying under vacuum (oil pump). Alternatively, the material yielded a solid after sequential concentration with DCM (1×) and hexanes (2×). Tether Ddz-T12 was obtained as an off-white solid (85-90% yield). 
     Example 29 
     Standard Procedure for Attachment of Tethers Utilizing the Mitsunobu Reaction 
     Example 29-A 
     Using PPh 3 -DIAD Isolated Adduct 
     To a 0.2 M solution of the appropriate tether (1.5 eq) in THF or THF-toluene (1:1) was added the PPh 3 -DIAD (pre-formed by mixing equivalent amounts of the reagents and isolated by evaporation of solvent, see Example 29-C) adduct (1.0 eq.). The resultant mixture was manually agitated for 10 sec (the solution remained turbid), then added to the resin. Alternatively, the resin was added to the solution. The reaction suspension was agitated O/N (after ˜5 min the mixture becomes limpid). The resin was filtered and washed 2×DCM, 1× toluene, 1×EtOH, 1× toluene, 1×(DCM/MeOH), 1×(THF/MeOH), 1×(DCM/MeOH), 1×(THF/MeOH), 2×DCM, then dried in the standard manner. 
     Example 29-B 
     Using “PPh 3 -DIAD In Situ Procedure” 
     To a 0.2 M solution of the appropriate tether (4 eq) in THF or THF-toluene (1:1) was added triphenylphosphine (4 eq). The resultant mixture was manually shaken until a homogenous solution was obtained, then added to the resin. Alternatively, the resin (or IRORI™ MiniKans® (NEXUS Biosystems, Poway, Calif.), miniaturized microreactors, containing resin) was added to the solution. To this suspension was then added DIAD (3.9 eq) and the reaction agitated O/N. Note: Since the reaction is exothermic, for larger scales, the reaction should be cooled in an ice bath. In addition, an appropriate vent must be supplied to allow any pressure build-up to be released. The resin was filtered and washed DCM (2×), toluene (1×), EtOH (1×), toluene (1×), DCM/MeOH (1×), 1×THF/MeOH (1×), DCM/MeOH (1×), THF/MeOH (1×), 2×DCM, then dried in the standard manner. 
     A procedure for the synthesis of PPh 3 -DIAD adduct is shown in  FIG. 8 . 
     DIAD (1 eq) was added dropwise to a well-stirred solution of triphenylphosphine (1 eq) in THF (0.4 M) at 0° C. under nitrogen. The mixture was then maintained at 0° C. with stirring for 30 min. The white solid obtained was collected by filtration (use medium sized fritted filters), washed with cold anhydrous THF until the washes were colorless, and lastly washed once with anhydrous Et 2 O. The white solid product was then vacuum-dried (oil pump) and stored under nitrogen. (Note: The PPh 3 -DIAD adduct can be made in larger than immediately required quantity and stored under nitrogen; it is very important to store this reagent under anhydrous conditions.) 
     Example 30 
     Standard Procedure for Attachment of Tethers via Reductive Amination as Shown in FIG.  9   
     In certain instances, the Mitsunobu process of Example 29 cannot be applied or is not efficient for incorporation of the tether. Hence, reductive amination has been developed as an alternative that can be employed for tether incorporation as illustrated hereinbelow for one of the preferred tethers. Similar chemistry can be used to incorporate other tethers of the present invention. 
     The Tether (30-2) with the amine protected as its Ddz derivative was efficiently oxidized to the corresponding aldehyde 30-2 using SO 3 ·pyr in DMSO-Et 3 N-DCM. This aldehyde (0.14 mmol, 56 mg, 1.5 eq based upon loading of resin support) was dissolved in a 1:3 mixture of TMOF-MeOH (DriSolv, 4 mL) at rt. To this was added the resin containing the tripeptide (30-1, as its trifluoroacetic acid salt from the deprotection of the terminal amine), the mixture was agitated briefly to wet the resin, and then borane-pyridine complex (as the commercially available 8 M solution, 23 μL, 2 eq) was introduced to the suspension. The reaction was agitated O/N, then the resin filtered, washed with DCM (2×), THF (1×), DCM/MeOH [3:1] (1×), THF/MeOH [3:1] (1×), DCM (2×) and dried in the standard manner. Care must be taken to ensure that the desired resin bound product 30-3 is not contaminated with the dialkylated material. However, even if the reaction does not proceed to completion or if a small amount of the dialkylation side product is present, the material is of sufficient purity for the macrocyclization reaction. 
     A standard procedure for the synthesis of tether T28 is shown in  FIG. 10 . 
     Henry reaction of 2-hydroxybenzaldehyde 28-0 provided 28-1 in 79% yield. This was followed by reduction first with sodium borohydride, then with catalytic hydrogenation, to give the amine, which was then protected as its Boc derivative, 28-2. Yields of these first two steps were lower on larger scales. Alkylation of 28-2 with the TBDMS ether of 2-bromoethanol, itself synthesized by standard methods, gave 28-3 in 74% yield. Deprotection of the silyl ether under standard conditions yielded the desired protected tether, Boc-T28. Alternative use of ethylene carbonate for the phenol alkylation to avoid the protection/deprotection steps, gave 73% yield. 
     A standard procedure for the synthesis of tether T32 is shown in  FIG. 11 . 
     A standard procedure for the synthesis of tether T33a and T33b is shown in  FIGS. 12A and 12B . 
     The construction to the (R)-isomer of this tether (T33a) was accomplished from 2-iodophenol (33-0) and (S)-methyl lactate (33-A). Mitsunobu reaction of 33-0 and 33-A proceeded with inversion of configuration in excellent yield to give 33-1. Reduction of the ester to the corresponding alcohol (33-2) also occurred in high yield and was followed by Sonagashira reaction with Ddz-propargylamine. The alkyne in the resulting coupling product, 33-3, was reduced with catalytic hydrogenation. Workup with scavenger resin provided the desired product, Ddz-T33a. 
     The synthesis of the (S)-enantiomer (Ddz-T33b) was carried out in an identical manner in comparable yield starting from (R)-methyl lactate (33-B). See  FIG. 12B . 
     Standard procedures for the synthesis of various tethers are shown in the figures: tether T34 ( FIG. 13 ), tether T35 ( FIG. 14 ), tether T36 ( FIG. 15 ), tether T37 ( FIG. 16 ), tether T38 ( FIG. 17 ), tether T39 ( FIG. 18 ), tether T40 ( FIG. 19 ), tether T41 ( FIG. 20 ) and tether T42 ( FIG. 21 ). 
     E. Examples of Synthetic Strategies for the Macrocyclic Compounds of the Invention 
       FIG. 22  presents a scheme depicting a thioester strategy for macrocyclic compounds of the present invention. 
     It should be noted that one or more of the amino acids indicated can be replaced by corresponding hydroxy acids and coupled to the next building block utilizing methods known to those in the art. 
     Example 47 
     Standard Procedure for Macrocyclization with Thioester Linker 
     The resin containing the cyclization precursor is combined in an appropriate vessel with pre-washed MP-carbonate resin [Argonaut Technologies, Foster City, Calif., commercially supplied MP-carbonate resin was treated with 3×THF (1 L per 400 g) and dried O/N at 30° C. in a vacuum oven] (1.4 to 1.6 eq relative to the initial loading of the synthesis resin). A 0.2 M DIPEA solution in THF was then added to the combined resins (1 mL/60 mg MP-carbonate resin) and the suspension agitated O/N at rt. Subsequently, the resin was filtered and rinsed 2×THF. The combined filtrates are collected together in an appropriate vessel, then the volatile contents evaporated under reduced pressure [in addition to the standard methods, solvent can also be removed in vacuo using centrifugal evaporation (ThermoSavant Discovery®, SpeedVac® or comparable) (Thermo Electron Corporation, Waltham, Mass.)] to provide the crude macrocycles. 
     Example 48 
     Standard Procedure for Silver-Assisted Macrocyclization with Thioester Linker 
     Except for the cyclization itself and subsequent work-up, this procedure is identical to that of Example 47. The resin containing the cyclization precursor was combined in an appropriate vessel with pre-washed MP-carbonate resin [Argonaut Technologies, commercially supplied MP-carbonate resin was treated with THF (3×, 1 L per 400 g) and dried O/N at 30° C. in a vacuum oven] (1.4 to 1.6 eq relative to the initial loading of the synthesis resin). To this was added THF (1 mL per 100 mg resin) and silver trifluoroacetate (1 eq relative to the initial loading of the resin). Finally, an amount of DIPEA sufficient to obtain a 0.2 M solution was added. The reaction mixture was agitated at rt O/N. The solution was then filtered and the resins washed 2×THF. The filtrates are collected together in an appropriate vessel, then evaporated under reduced pressure [(the volatile contents could also be removed in vacuo using centrifugal evaporation (ThermoSavant Discovery®, SpeedVac® or comparable)] to provide the crude macrocycles. For this procedure, silver trifluoroacetate should be stored in a dessicator between uses. In addition, it is recommended to use a new bottle of THF (or a bottle that has been recently opened under N 2  or Ar) to minimize formation of silver oxide. 
     Additionally, a ring-closing metathesis (RCM) strategy, as developed by Grubbs et al. can also be used to access some of the macrocyclic compounds of the invention (see for example U.S. Pat. No. 5,811,515; Grubbs, R. H. et al.  J. Org. Chem.  2001, 66, 5291-5300; Fürstner, A. Angew. Chem. Int. Ed. 2000, 39, 3012-3043). 
     To access certain derivatives of compounds of the present invention, additional reactions from those in the general scheme were required. For some, it was advantageous to react the functionality to be derivatized prior to the formation of the macrocyclic ring. The cyclic structure can restrict access of reagents to that functionality. For example, in the synthesis of N-methyl and N-acyl derivatives of macrocycles, where the secondary nitrogen atom of the ring is the site of derivatization, the reaction is preferred to be performed prior to the application of the appropriate cyclization protocol. 
     In other cases, for example the derivatization of side chain functionality, the reaction was best performed after formation of the macrocyclic ring. For example, further reaction of amino moieties on side chains examples was typically efficiently done by reaction of the partially protected macrocycle. In this manner, acylation, sulfonylation, alkylation (via reductive amination), guanidine and urea formation were performed via standard methods. 
     Table 1, hereinbelow, shows a representative, but by no means exclusive, summary of the chemical synthesis of several representative compounds of the invention. 
                                                                                 TABLE 1                   Synthesis of Representative Compounds of the Present Invention                                Tether   Additional           AA 1     AA 2     AA 3     Tether   Attachment   Steps                    1   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nva   Ddz-T8   Example 29   none       2   Bts-D-Phe   Boc-D-Val   Boc-Nva   Boc-T8   Example 29   none       3   Bts-D-Phe   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       4   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nva   Ddz-T9   Example 29   none       5   Bts-D-Tyr(tBu)   Boc-D-Ala   Boc-Nva   Ddz-T8   Example 29   none       6   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Met   Ddz-T8   Example 29   none       7   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nle   Ddz-T8   Example 29   none       8   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Phe   Ddz-T8   Example 29   none       9   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Val   Ddz-T8   Example 29   none       10   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Leu   Ddz-T9   Example29   none       11   Bts-D-2-Nal   Boc-D-Val   Boc-Nva   Boc-T8   Example 29   none       12   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Abu   Ddz-T8   Example 29   none       13   Bts-D-Phe   Boc-D-Val   Boc-Leu   Boc-T9   Example 29   none       14    Bts-D-2-Nal   Boc-D-Val   Boc-Leu   Boc-T9   Example 29   none       15   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       16   Bts-D-Phe(4Cl)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       17   Bts-D-Trp(Boc)   Boc-D-Val   Boc-Nva   Ddz-T9   Example 29   none       18   Bts-D-Tyr(tBu)   Boc-D-2-Abu   Boc-Nva   Ddz-T9   Example 29   none       19   Bts-D-Phe(4F)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       20   Bts-D-Phe   Boc-D-Val   Boc-Leu   Boc-T8   Example 29   none       21   Bts-D-2-Nal   Boc-D-Val   Boc-Leu   Boc-T8   Example 29   none       22   Bts-D-Tyr(OMe)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       23    Bts-D-1-Nal   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       24   Bts-D-2-Thi   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       25   Bts-D-Phe(2Cl)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       26   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Cpa   Ddz-T9   Example 29   none       27    Bts-D-4-Thz   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       28    Bts-D-3-Pal   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       29   Bts-D-Tyr(tBu)   Boc-D-Val    Ddz-Dap(Boc)   Ddz-T9   Example 29   none       30   Bts-D-Tyr(tBu)   Hnva(THP)   Boc-Nva   Ddz-T9   Example 29   none       34   Bts-D-Tyr(tBu)   Ddz-D-Tyr(tBu)   Boc-Nva   Ddz-T8   Example 29   None       38   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Ala   Ddz-T8   Example 29   none       39   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-□-Ala   Ddz-T8   Example 29   none       40   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Gly   Ddz-T8   Example 29   none       41   Bts-D-Tyr(tBu)   Boc-DPhe   Boc-Nva   Ddz-T8   Example 29   none       52   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Phg   Ddz-T8   Example 29   none       55   Bts-D-Tyr(tBu)   Ddz-D-Val    Ddz-Lys(Boc)   Ddz-T8   Example 29   none       56   Bts-D-Tyr(tBu)   Ddz-D-Val    Ddz-Orn(Boc)   Ddz-T8   Example 29   none       57   Bts-D-Tyr(tBu)   Ddz-D-Val    Ddz-Ser(tBu)    Ddz-T8   Example 29   none       58   Bts-D-Tyr(tBu)   Ddz-D-Val    Ddz-Tyr(tBu)   Ddz-T8   Example 29   none       59   Bts-D-Tyr(tBu)   Ddz--D-Val   Ddz-Trp(Boc)    Ddz-T8   Example 29   none       60   Bts-D-Tyr(tBu)   Boc-D-Val    Boc-Tyr(OMe)   Ddz-T8   Example 29   none       65   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nva   Ddz-T2   Example 29   none       71   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nva   Ddz-T10   Example 29   none       72   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-2-Nal   Ddz-T8   Example 29   none       76   Bts-D-Tyr(tBu)   Boc-D-2-Nal   Boc-Nva   Ddz-T8   Example 29   none       77   Bts-D-Tyr(tBu)   Boc-D-Nle   Boc-Nva   Ddz-T8   Example 29   none       80   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Ile   Ddz-T8   Example 29   none       85   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-D-Nva   Ddz-T8   Example 29   none       87   Bts-D-Bip    Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       88   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nva   Ddz-T9   Example 29   none       89   Bts-D-Hfe    Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       90   Bts-D-Dip   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       91   Bts-D-Tyr(tBu)   Boc-D-Nva   Boc-Nva   Ddz-T9   Example 29   none       92   Bts-D-Tyr(tBu)   Boc-D-Tle   Boc-Nva   Ddz-T9   Example 29   none       96   Bts-D-Tyr(tBu)   Boc-β-Ala   Boc-Nva   Ddz-T9   Example 29   none       97   Bts-D-Tyr(tBu)   Boc-D-Chg   Boc-Nva   Ddz-T9   Example 29   none       98   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nva   Ddz-T18   Example 29   none       99   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nva   Ddz-T15   Example 29   none       109   Bts-D-Tyr(tBu)   Boc-D-Val   Ddz-Dab(Boc)    Ddz-T9   Example 29   none       110   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nva   Ddz-T11   Example 29   none       111   Bts-D-Tyr(tBu)   Boc-D-Val   Hval(THP)   Ddz-T9   Example 29   none       112   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nva   Ddz-T9   Example 29   none       120   Bts-D-Tyr(tBu)   Boc-D-Pro   Boc-Nva   Ddz-T8   Example 29   none       121   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nva   Ac-T8-NH2   Example 29   none       122   Boc-D-3-Pal   Boc-D-Val   Boc-Nva   Boc-T9   Example 30   none       123   Boc-D-2-Pal   Boc-D-Val   Boc-Nva   Boc-T9   Example 30   none       124   Boc-D-4-Pal   Boc-D-Val   Boc-Nva   Boc-T9   Example 30   none       125   Bts-D-Tyr(tBu)   Boc-D-Cpg   Boc-Nva   Boc-T9   Example 29   none       126   Bts-D-Tyr(tBu)   Boc-D-Val    Boc-NMeLeu   Boc-T9   Example 29   none       127   Boc-D-His(Mts)   Boc-D-Val   Boc-Nva   Boc-T12   Example 30   none       128   Bts-D-Tyr(OMe)   Boc-D-Val   Boc-Leu   Boc-T9   Example 29   none       129   Bts-D-1-Nal   Boc-D-Val   Boc-Leu   Boc-T9   Example 29   none       130   Bts-D-2-Thi   Boc-D-Val   Boc-Leu   Boc-T9   Example 29   none       131   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Leu   Boc-T9   Example 29   none       132   Bts-D-Phe(4Cl)   Boc-D-Val   Boc-Leu   Boc-T9   Example 29   none       133   Bts-D-Phe(4F)   Boc-D-Val   Boc-Leu   Boc-T9   Example 29   none       134   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Leu   Boc-T2   Example 29   none       135   Bts-D-Tyr(OMe)   Boc-D-Val   Boc-Leu   Boc-T11   Example 29   none       136   Bts-D-1Nal    Boc-D-Val   Boc-Leu   Boc-T11   Example 29   none       137   Bts-D-2-Thi   Boc-D-Val   Boc-Leu   Boc-T11   Example 29   none       138   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Leu   Boc-T11   Example 29   none       139   Bts-D-Phe(4Cl)   Boc-D-Val   Boc-Leu   Boc-T11   Example 29   none       140   Bts-D-Phe(4F)   Boc-D-Val   Boc-Leu   Boc-T11   Example 29   none       141   Bts-D-Tyr(OMe)   Boc-D-Val   Boc-Cpa   Boc-T9   Example 29   none       142   Bts-D-1-Nal   Boc-D-Val   Boc-Cpa   Boc-T9   Example 29   none       143   Bts-D-2-Thi   Boc-D-Val   Boc-Cpa   Bac-T9   Example 29   none       144   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Cpa   Boc-T9   Example 29   none       145   Bts-D-Phe(4Cl)   Boc-D-Val   Boc-Cpa   Boc-T9   Example 29   none       146   Bts-D-Phe(4F)   Boc-D-Val   Boc-Cpa   Boc-T9   Example 29   none       147   Bts-D-Tyr(OMe)   Boc-D-Val   Boc-Cpa   Boc-T11   Example 29   none       148   Bts-D-1-Nal   Boc-D-Val   Boc-Cpa   Boc-T11   Example 29   none       149   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Cpa   Boc-T11   Example 29   none       150   Bts-D-Phe(4Cl)   Boc-D-Val   Boc-Cpa   Boc-T11   Example 29   none       151   Bts-D-Phe(4F)   Boc-D-Val   Boc-Cpa   Boc-T11   Example 29   none       152   Bts-D-Tyr(OMe)   Ddz-D-Val    Ddz-Dap(Boc)   Ddz-T9   Example 29   none       153   Bts-D-1-Nal    Ddz-D-Val   Ddz-Dap(Boc)   Ddz-T9   Example 29   none       154   Bts-D-2-Thi    Ddz-D-Val    Ddz-Dap(Boc)   Ddz-T9   Example 29   none       155   Bts-D-Phe(3Cl)   Ddz-D-Val    Ddz-Dap(Boc)   Ddz-T9   Example 29   none       156   Bts-D-Phe(4Cl)   Ddz-D-Val    Ddz-Dap(Boc)   Ddz-T9   Example 29   none       157   Bts-D-Phe(4F)   Ddz-D-Val    Ddz-Dap(Boc)    Ddz-T9   Example 29   none       158   Bts-D-Phe(3Cl)   Ddz-D-Val    Ddz-Dap(Boc)   Ddz-T11   Example 29   none       159   Bts-D-Tyr(But)   Boc-D-Ile   Boc-Nva   Boc-T9   Example 29   none       160   Bts-D-Tyr(But)   Boc-D-alloIle   Boc-Nva   Boc-T9   Example 29   none       161   Boc-D-Phe(4CH2NHFmoc)   Boc-D-Val   Boc-Nva   Boc-T9   Example 30   none       162   Bts-D-Phe(2Me)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       163   Bts-D-Phe(3Me)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       164   Bts-D-Phe(4Me)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       165   Bts-D-Phe(3OMe)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       166   Bts-D-Phe(2OMe)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       167   Bts-D-3-benzothienyl   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       168   Bts-D-3-Thi   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       169   Bts-D-□-HomoPhe(3Cl)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       170   Bts-D-Phe(3,4diCl)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       171   Bts-D-Phe(3,4diF)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       172   Bts-D-Phe(3,4diOMe)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       173   Bts-D-1Nal    Hnva(THP)   Boc-Nva   Boc-T9   Example 29   none       174   Bts-D-Tyr(OMe)   Hnva(THP)    Boc-Nva   Boc-T9   Example 29   none       175   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nva   Boc-T33b   Example 29   none       176   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nva   Boc-T33a   Example 29   none       177   Bts-D-Tyr(tBu)   Boc-D-Val   Boc-Nva   Boc-T28   Example 29   none       178   Bts-D-Tyr(OMe)   Ddz-D-Val    Ddz-Ser(tBu)   Ddz-T9   Example 29   none       179   Bts-D-1-Nal   Ddz-D-Val    Ddz-Ser(tBu)   Ddz-T9   Example 29   none       180    Bts-D-2-Thi   Ddz-D-Val    Ddz-Ser(tBu)   Ddz-T9   Example 29   none       181   Bts-D-Phe(3Cl)   Ddz-D-Val    Ddz-Ser(tBu)   Ddz-T9   Example 29   none       182   Bts-D-Phe(4Cl)   Ddz-D-Val    Ddz-Ser(tBu)   Ddz-T9   Example 29   none       183   Bts-D-Phe(4F)   Ddz-D-Val   Ddz-Ser(tBu)   Ddz-T9   Example 29   none       184   Bts-D-1-Nal   Ddz-D-Val    Ddz-Dap(Boc)   Ddz-T11   Example 29   none       185   Bts-D-Phe(4Cl)   Ddz-D-Val    Ddz-Dap(Boc)   Ddz-T11   Example 29   none       186   Ddz-D-Tyr(tBu)   Ddz-D-Val    Ddz-His(Mts)   Ddz-T9   Example 30   none       187   Bts-D-Phe(3CF3)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       188   Bts-D-Phe(3F)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       189   Bts-D-Phe(4NO2)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       190   Bts-D-3-benzothienyl   Boc-D-Val   Boc-Cpa   Boc-T9   Example 29   none       191   Bts-D-Phe(3OMe)   Boc-D-Val   Boc-Cpa   Boc-T9   Example 29   none       192   Bts-D-Phe(3,4diCl)   Boc-D-Val   Boc-Cpa   Boc-T9   Example 29   none       193   Bts-D-Phe(3,4diF)   Boc-D-Val   Boc-Cpa   Boc-T9   Example 29   none       194   Bts-D-Tyr(OMe)   Boc-D-Val   Boc-Nva   Boc-T34   Example 29   none       195   Bts-D-Tyr(OMe)   Boc-D-Val   Boc-Nva   Boc-T38   Example 29   none       196   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Cpa    Ddz-T32(Boc)   Example 29   none       197   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Cpa   Boc-T34   Example 29   none       198   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Cpa   Bae-T38   Example 29   none       199   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Cpa   Boc-T41   Example 29   none       200   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Cpa   Boc-T8   Example 29   none       201   Bts-D-1-Nal   Boc-D-Val   Boc-Nva   Boc-T8   Example 29   none       202   Bts-D-Phe(3OMe)   Boc-D-Val   Boc-Nva   Boc-T8   Example 29   none       203   Bts-D-Phe(4Cl)   Ddz-D-Val   Ddz-Dap(Boc)    Ddz-T9   Example 29   acetylation       204   Bts-D-Phe(4Cl)   Ddz-D-Val   Ddz-Dap(Boc)    Ddz-T9   Example 29    guanidinylation       205   Bts-DPhe(3Cl)   Boc-D-Val   Boc-NMeLeu   Boc-T9   Example 29   none       206   Bts-D-Phe(4Cl)   Ddz-D-Val   Ddz-Dap(Boc)   Ddz-T9   Example 29   mesylation       207   Bts-D-Phe(4Cl)   Ddz-D-Val    Ddz-Dap(Boc)   Ddz-T9   Example 29   TMS-                               isocyanate                               followed by                               dilute acid       208   Bts-D-Tyr(tBu)   Ddz-D-Val   Ddz-Dap(Boc)   Ddz-T9   Example 29    guanidinylation       209   Bts-D-Tyr(tBu)   Ddz-D-Val   Ddz-Dap(Boc)    Ddz-T9   Example 29   acetylation       210   Bts-D-Tyr(tBu)   Ddz-D-Val   Ddz-Dap(Boc)    Ddz-T9   Example 29   reductive                               amination with                               acetone       211   Bts-D-Phe(4Cl)   Ddz-D-Val    Ddz-Dap(Boc)   Ddz-T9   Example 29   reductive                               amination with                               excess                               formaldehyde       212   Bts-D-Phe(4Cl)   Ddz-D-Val   Ddz-Dap(Boc)   Ddz-T9   Example 29   reductive                               amination with                               acetone       213   Bts-D-Tyr(3,5dil)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       214   Bts-D-Tyr(OMe)   Boc-D-Val   Boc-Hse(Bzl)   Boc-T9   Example 29   hydrogenolysis                               for protecting                               group removal       215   Bts-D-Tyr(tBu)   Ddz-D-Val   Ddz-Dap(Boc)   Ddz-T9   Example 29   reductive                               amination with                               excess                               formaldehyde       216   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Cpa   Boc-T40   Example 29   none       217   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Cpa   Boc-T36   Example 29   none       218   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Nva   Boc-T39   Example 29   none       219   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Nva   Boc-T37   Example 29   none       220   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Nva   Boc-T39   Example 29   none       221   Bts-D-Phe(3Cl)   Boc-D-Val   Boc-Nva   Boc-T35   Example 29   none       222   Bts-D-Tyr(3tBu)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   none       223   Bts-D-Tyr(But)   Boc-D-Val   Boc-Nva   Boc-T9   Example 29   acetylation       224   Bts-D-1-Nal   Boc-D-Val   Boc-Leu   Boc-T9   Example 29   reductive                               amination with                               formaldehyde       225   Bts-D-1-Nal   Boc-D-Val   Boc-Leu   Boc-T9   Example 29   acetylation       226   Bts-D-1-Nal   Boc-D-Val   Boc-Leu   Boc-T9   Example 29   reductive                               amination with                               aldehyde       227   Bts-D-1-Nal   Boc-D-Val   Boc-Leu   Boc-T9   Example 29   reductive                               amination with                               benzaldehyde               Notes       Any amino acid or tether designated as the Boc derivative could be substituted with the corresponding Ddz derivative.            
D. Analytical Data for Selected Compounds of the Invention
 
       1 H and  13 C NMR spectra were recorded on a Varian Mercury 300 MHz spectrometer (Varian, Inc., Palo Alto, Calif.) and are referenced internally with respect to the residual proton signals of the solvent. Information about the conformation of the molecules in solution can be determined utilizing appropriate two-dimensional NMR techniques known to those skilled in the art. HPLC purifications were run on a Waters Xterra® MS C18 column, using the Waters FractionLynx® system (Waters Corporation, Milford, Mass.). Automated medium pressure chromatographic purifications were performed on an Isco CombiFlash® 16× system with disposable silica or C18 cartridges that permitted up to sixteen (16) samples to be run simultaneously (Teledyne Isco, Inc., Lincoln, Nebr.). MS spectra were recorded on a Waters Micromass® Platform II or ZQ™ system. HRMS spectra were recorded with a VG Micromass ZAB-ZF spectrometer. Chemical and biological information were stored and analyzed utilizing the ActivityBase® database software (ID Business Solutions Ltd., Guildford, Surrey, UK). 
     General Methods for Analytical HPLC Analyses 
     HPLC analyses are performed on a Waters Alliance® system 2695 running at 1 mL/min using an Xterra MS C18 column 4.6×50 mm (3.5 μm). A Waters 996 PDA provided UV data for purity assessment (Waters Corporation, Milford, Mass.). An LCPackings (Dionex Corporation, Sunnyvale, Calif.) splitter (50:40:10) allowed the flow to be separated in three parts. The first part (50%) went to a Micromass® Platform II MS equipped with an APCI probe for identity confirmation. The second part (40%) went to an evaporative light scattering detector (ELSD, Polymer Laboratories, now part of Varian, Inc., Palo Alto, Calif., PL-ELS-1000™) for purity assessment and the last portion (10%) to a chemiluminescence nitrogen detector (CLND, Antek® Model 8060, Antek Instruments, Houston, Tex., part of Roper Industries, Inc., Duluth, Ga.) for quantitation and purity assessment. Data was captured and processed utilizing the most recent version of the Waters Millenium® software package (Milford, Mass.). 
     An example LC method suitable for compounds of the present invention uses MeOH as solvent A, H 2 O as solvent B and 1% TFA/H 2 O as solvent D. Initial mobile-phase composition is 5% A, 85% B and 10% D. Details of the standard gradient method are shown below: 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Time 
                 A % 
                 B % 
                 D % 
                 Curve 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 0.00 
                 5 
                 85 
                 10 
                 6 
               
               
                 1.00 
                 5 
                 85 
                 10 
                 6 
               
               
                 6.00 
                 50 
                 40 
                 10 
                 6 
               
               
                 9.00 
                 50 
                 40 
                 10 
                 6 
               
               
                 14.00 
                 90 
                 0 
                 10 
                 6 
               
               
                 17.00 
                 90 
                 0 
                 10 
                 6 
               
               
                 17.50 
                 5 
                 85 
                 10 
                 6 
               
               
                 20.00 
                 5 
                 85 
                 10 
                 6 
               
               
                   
               
             
          
         
       
     
     Compounds 2-6, 8-10, 56, 65 and 144 are as defined in Table (3), hereinbelow. 
     Compound 2 
     Yield: 12 mg pure macrocycle was obtained (CLND quantification). 
       1 H NMR (300 MHz, DMSO-d 6 ) δ 8.83 (m, 1H); 8.53 (m, 1H); 7.63 (m, 1H); 7.4-7.08 (m, 7H); 7.00-6.84 (m, 2H); 6.60 (d, 15 Hz, 1H); 6.41 (dt, 15 Hz, 5.4 Hz, 1H); 4.35 (m, 1H); 4.25-4.05 (m, 3H); 3.94 (dt, 1H, 6 Hz, 15 Hz); 3.79 (dd, 1H, 3.6 Hz, 8.4 Hz); 3.60 (m, 1H); 3.52-3.40 (bd, 1H); 3.22-3.06 (m, 4H); 1.88 (m, 2H); 1.54-1.28 (m, 2H); 1.25 (d, 3H, 4.8 Hz); 1.22 (d, 3H, 2.7 Hz); 0.92-0.80 (m, 6H). 
     HRMS calc. for C 30 H 40 N 4 O 4 : 520.3049. found 520.3057±0.0016 
     HPLC [standard gradient method (refers to that presented in General Methods for Analytical HPLC Analyses)] t R =9.55 min. 
     Compound 4 
     Yield: 12 mg pure macrocycle was obtained (CLND quantification). 
       1 H NMR (300 MHz, DMSO-d 6 ) δ 9.35 (b, 1H); 8.98 (b, 1H); 5.52 (d, 1H, 8.4 Hz); 8.38 (b, 1H); 7.25 (b, 1H); 7.13-7.07 (m, 4H); 6.86 (t, 2H, 7.5 Hz); 6.57 (d, 2H, 8.7 Hz); 4.33 (b, 1H); 4.21-4.02 (m, 3H); 3.78 (dd, 1H, 3.3 Hz; 8.1 Hz); 3.65-3.54 (m, 1H); 3.31-3.23 (m, 1H); 3.13-3.02 (m, 4H); 2.78-2.2.28-2.18 (m, 1H); 2.0-1.80 (m, 2H); 1.50-1.30 (m, 3H); 1.25 (d, 3H, 4.5 Hz); 1.22 (d, 3H, 4.5 Hz); 1.01 (d, 3H, 6.6 Hz); 0.90 (d, 3H, 6.6 Hz); (t, 3H, 7.5 Hz). 
       13 C NMR (75.5 MHz, DMSO-d 6 ) δ 172.22; 171.37; 157.77; 157.44; 156.04; 131.76; 130.80; 130.70; 127.88; 121.82; 115.83; 111.71; 62.13; 60.62; 54.21; 52.81; 47.13; 42.47; 33.31; 29.69; 29.30; 28.61; 20.36; 19.44; 18.72; 17.60; 13.97. 
     HRMS calc. for C 30 H 42 N 4 O 6 : 538.3155. found: 538.3145±0.0016 
     HPLC (standard gradient) t R =8.12 min. 
     Compound 5 
     Yield: 17 mg pure macrocycle was obtained (CLND quantification). 
       1 H NMR (300 MHz, DMSO-d 6 ) δ 9.02 (b, 1H); 8.47 (d, 1H, 8.4 Hz); 7.7 (b, 1H); 7.58 (d, 1H, 5.4 Hz); 7.28 (dd, 1H, 7.8 Hz, 0.8 Hz); 7.20 (t, 1H, 9.0 Hz, 0.8 Hz); 7.14 (d, 2H, 8.4 Hz); 6.98-6.91 (m, 3H); 6.66 (d, 8.7 Hz); 6.63 (d, 1H, 15.0 Hz); 6.43 (dt, 1H, 6.0 Hz, 15.0 Hz); 4.28-3.86 (m, 6H); 3.60-3.40 (m, 2H); 3.22-3.12 (m, 1H0; 3.05 (d, 2H, 5.4 Hz); 1.92-1.80 (m, 1H); 1.56-1.40 (m, 1H); 1.36-1.20 (m, 2H); 1.25 (d, 3H, 6.6 Hz); 0.84 (t, 3H, 7.2 Hz). 
       13 C NMR (75.5 MHz, DMSO-d 6 ) δ 172.54; 171.86; 158.97; 158.56; 127.39; 155.84; 131.62; 129.73; 129.20; 129.02; 128.43; 126.30; 124.51; 122.01; 115.85; 112.88; 61.23; 52.90; 51.23; 47.08; 42.66; 36.13; 33.30; 21.14; 19.57; 17.07; 14.14; 11.49. 
     HRMS calc. for C 26 H 36 N 4 O 6 : 508.2685. found: 508.2681±0.0015 
     HPLC (standard gradient) t R =7.67 min. 
     Compound 6 
     Yield: 16 mg pure macrocycle was obtained (CLND quantification). 
       1 H NMR (300 MHz, DMSO-d 6 ) δ 9.37 (b, 1H); 8.87 (b, 1H); 8.61 (d, 1H, 8.7 Hz); 7.62 (b, 1H); 7.27 (d, 1H, 7.8 Hz); 7.21 (t, 1H, 8.4 Hz); 7.14 (d, 2H, 8.4 Hz); 6.98-6.87 (m, 3H); 6.64 (d, 2H, 8.1 Hz); 6.70 (d, 1H, 15.6 Hz); 6.39 (dt, 1H, 6.3 Hz, 15.6 Hz); 4.44-4.36 (m, 1H); 4.34-4.08 (m, 2 Hz); 4.45-3.92 (dt, 1H, 6.9 Hz, 15.6 Hz); 3.74 (dd, 1H, 3.6 Hz, 8.4 Hz); 3.54-3.26 (m, 3H); 3.22-3.02 (m, 3H); 2.60-2.36 (m, 4H); 2.24-2.14 (m, 1H); 2.02 (s, 3H); 1.96-1.89 (m, 1H); 1.80-1.66 (m, 1H); 1.01 (d, 3H, 6.3 Hz); 0.90 (d, 3H, 6.6 Hz). 
       13 C NMR (75.5 MHz, DMSO-d 6 ) δ 171.51; 171.26; 158.90; 158.49; 157.38; 155.86; 131.63; 129.82; 129.21; 128.86; 128.63; 126.21; 121.98; 115.83; 112.83; 62.11; 61.06; 51.97; 47.10; 42.78; 30.91; 30.67; 29.34; 20.37; 19.39; 15.06. 
     HRMS calc. for C 30 H 40 N 4 O 6 S: 568.2719; found: 568.2711±0.0017 
     HPLC R t  (general method) 7.92 min. 
     Compound 8 
     Yield: 27 mg pure macrocycle was obtained (CLND quantification). 
       1 H NMR (300 MHz, DMSO-d 6 ) δ 9.05 (b, 1H); 8.43 (b, 1H); 8.34 (d, 1H, 9.3 Hz); 7.40 (b, 1H); 6.97 (d, 1H, 7.5 Hz); 6.92-6.74 (m, 9H); 6.67-6.54 (m, 2H); 6.33-6.25 (m, 3H); 6.10 (dt, 1H, 5.7 Hz, 16.2 Hz); 4.22 (dt, 1H, 0.9 Hz, 12 Hz); 3.94-6.66 (m, 4H); 3.30 (dd, 1H, 3.6 Hz, 7.8 Hz); 3.24 (m, 1H); 3.18 (m, 1H); 2.85-2.68 (m, 3H); 2.44-2.23 (m, 2H); 1.32 (o, 1H, 7.5 Hz); 0.97-0.89 (m, 1H); 0.42 (d, 3H, 6.6 Hz); 0.01 (d, 3H, 6.6 Hz). 
       13 C NMR (75.5 MHz, DMSO-d 6 ) δ 171.20; 157.35; 155.88; 139.12; 131.61; 130.87; 129.74; 129.21; 128.77; 128.88; 126.85; 126.19; 121.97; 115.82; 112.84; 62.04; 61.10; 55.07; 50.01; 47.09; 42.85; 37.42; 29.11. 
     HRMS calc, For C 34 H 42 N 4 O 6 : 586.3155. found: 586.3145±0.0017 
     HPLC R t  (general method) 9.34 min. 
     Compound 9 
     Yield: 17 mg pure macrocycle was obtained (CLND quantification). 
       1 H NMR (300 MHz, DMSO-d 6 ) δ 9.39 (b, 1H); 8.83 (b, 1H); 8.29 (d, 1H, 9.3 Hz); 7.62 (b, 1H); 7.28 (d, 1H, 6.6 Hz); 7.20 (t, 1H, 6.9 Hz); 7.12 (d, 2H, 7.8 Hz); 6.98-6.91 (m, 2H); 6.63 (d, 2H, 8.4 Hz); 6.58 (d, 1H, 16.2 Hz); 6.40 (dt, 1H, 5.7 Hz, 16.2 Hz); 4.29-4.13 (m, 3H); 4.03-3.92 (m, 2H); 3.52 (m, 1H); 3.15-3.05 (m, 3H); 2.45-2.37 (m, 1H); 1.96-1.88 (m, 1H); 1.25 (dd, 2H, 4.5 Hz; 6 Hz); 1.01 (d, 3H, 6.3 Hz); 0.91 (d, 3H, 6.6 Hz); 0.86 (d, 3H, 7.2 Hz); 0.81 (d, 3H, 6.6 Hz). 
       13 C NMR (75.5 MHz, DMSO-d 6 ) δ 171.85; 171.17; 157.37; 155.87; 131.59; 129.88; 129.18; 128.97; 128.78; 128.51; 126.16; 121.97; 115.83; 112.85; 61.55; 61.18; 58.15; 54.22; 47.08; 42.89; 36.32; 29.35; 29.00; 20.34; 19.56; 18.73; 17.44. 
     HRMS calc. for C 30 H 40 N 4 O 6  536.2998; found: 536.2990±0.0017. 
     HPLC (standard gradient) t R =8.15 min. 
     Compound 10 
     Yield: 24 mg pure macrocycle was obtained (CLND quantification). 
       1 H NMR (300 MHz, DMSO-d 6 ) δ 9.33 (b, 1H); 8.82 (b, 1H); 8.56 (d, 1H, 8.3 Hz); 7.60 (b, 1H); 7.27 (d, 2H, 7.8 Hz); 7.20 (t, 1H, 7.8 Hz); 7.13 (d, 2H, 8.4 Hz); 6.95 (t, 2H, 7.8 Hz); 6.64 (d, 2H, 8.4 Hz); 6.57 (d, 1H, 15.4 Hz); 6.38 (dt, 1H, 15.4 Hz, 5.8 Hz); 4.26-4.10 (m, 3H); 3.96 (dt, 1H, 5.4 Hz, 8.4 Hz); 3.77 (dd, 1H, 3.7 Hz, 7.8 Hz); 3.51-3.24 (m, 3H); 3.18-3.02 (m, 3H); 1.90 (h, 1H, 6.4 Hz); 1.73-1.54 (m, 2H); 1.45 (dt, 1H, 6.7 Hz, 0.9 Hz); 0.99 (d, 3H, 6.6 Hz); 0.89 (d, 3H, 6.3 Hz); 0.87 (d, 3H, 6.0 Hz); 0.80 (d, 3H, 6.3 Hz). 
       13 C NMR (75.5 MHz, DMSO-d 6 ) δ 172.23; 171.17; 157.37; 155.88; 131.62; 129.82; 129.19; 128.95; 128.59; 126.24; 121.99; 115.84; 112.88; 64.23; 61.98; 61.14; 51.43; 61.14; 51.43; 47.07; 42.81; 29.38; 24.85; 24.11; 21.00; 20.32; 19.30. 
     HRMS calc. for C 31 H 42 N 4 O 5  550.3155; found: 550.3150±0.0016. 
     HPLC (standard gradient) t R =8.91 min. 
     Compound 56 
     Yield: 16 mg pure macrocycle was obtained (CLND quantification). 
       1 H NMR (300 MHz, DMSO-d 6 ) δ 9.39 (b, 1H); 8.90 (b, 1H); 8.67 (d, 1H, 8.4 Hz); 7.74 (b, 4H); 7.29-7.08 (m, 4H); 6.99-6.87 (m, 2H); 6.64 (d, 2H, 8.1 Hz); 6.61 (d, 1H, 16.5 Hz); 6.40 (dt, 1H, 5.7 Hz, 16.5 Hz); 4.40-4.06 (m, 4H); 4.02-3.95 (m, 1H); 3.79 (dd, 1H, 3.6 Hz, 7.8 Hz); 3.55-3.30 (m, 2H); 3.16-3.05 (m, 3H); 2.82-2.69 (m, 2H); 2.02-1.85 (m, 2H); 1.64-1.43 (m, 3H); 1.29-1.23 (m, 1H); 1.01 (d, 3H, 6.3 Hz); 0.91 (d, 3H, 6.3 Hz); 0.86-0.84 (m, 2H). 
     HPLC (standard gradient) t R =5.71 min. 
     Compound 65 
     Yield: 17 mg pure macrocycle was obtained (CLND quantification). 
       1 H NMR (300 MHz, DMSO-d 6 ) δ 9.60 (b, 1H); 9.39 (b, 1H); 8.88 (b, 1H); 8.70 (d, 1H, 7.5 Hz); 8.57 (d, 1H, 4.2 Hz); 7.27 (t, 6 Hz); 6.96 (d, 2H, 8.4 Hz); 6.66 (d, 2H, 8.4 Hz); 5.78-5.68 (m, 1H); 5.42-5.33 (m, 1H); 3.96-3.89 (m, 1H); 3.80-3.57 (m, 5H); 3.41-3.34 (m, 1H); 3.10-2.90 (m, 1H); 2.78-2.66 (m, 1H); 2.21-2.10 (m, 1H); 2.06-1.93 (m, 1H); 1.70-1.60 (m, 1H); 1.52-1.41 (m, 1H); 1.39-1.26 (m, 1H); 1.25 (d, 3H, 4.8 Hz); 1.23 (d, 3H, 4.5 Hz); 0.83 (dd, 3H, 3 Hz, 4.5 Hz). 
       13 C NMR (75.5 MHz, DMSO-d 6 ) δ 172.68; 172.63; 159.15; 158.73; 157.38; 15725; 130.89; 124.99; 116.03; 62.51; 62.12; 54.29; 49.27; 42.47; 32.77; 30.43; 28.85; 20.46; 19.59; 18.72; 17.39; 13.90; 13.09. 
     HRMS calc. for C 24 H 36 N 4 O 4 : 444.2736; found: 444.2726±0.0013 
     HPLC (standard gradient) t R =6.80 min. 
     Compound 144  1 H NMR (300 MHz, CD 3 OD) δ 7.4 (m, 1H); 7.27 (d t , 1H, 1.5 Hz, 6.6 Hz); 7.22-7.14 (m, 2H); 7.08-6.98 (m, 2H); 6.78 9t, 2H, 6.6 Hz); 4.45-4.39 (m, 2H); 4.15 (d, 2H, 8.1 Hz); 7.74 (d, 1H, 9.3 Hz); 3.54 (d, 1H, 10.8 Hz); 3.35-3.22 (m, 2H); 3.20 (q, 1H, 1.5 Hz); 2.82-2.71 (m, 1H); 2.61-2.55 (m, 1H); 2.21-2.11 (m, 1 h); 2.02-1.94 (m, 1H); 1.74-1.40 (m, 5H); 1.04 (d, 3H, 6.6 Hz); 0.93 (d, 3H, 6.6 Hz); 0.74-0.64 9m, 1H); 0.45-0.28 (m, 2H); 0.15-0.08 (m, 1H); 0.06-0.02 (m, 1H). 
       13 C NMR (75.5 MHz, CD 3 OD) δ 173.29; 172.14; 167.51; 155.47; 134.86; 134.81; 130.38; 130.31; 128.81; 128.25; 127.44; 121.63; 110.39; 107.71; 105.02; 67.10; 66.66; 62.81; 62.06; 60.10; 53.99; 41.44; 36.07; 31.91; 30.01; 29.18; 28.94; 27.79; 23.68; 23.15; 19.08; 18.25; 8.17; 4.98; 3.16. 
     HRMS: calc. for C 31 H 41 N 4 O 4 C1568.2816; found 568.2802±0.0017 
     F. Mass Spectral Data for Selected Compounds of the Invention 
                                                                 TABLE 2                   Analysis of selected compounds of the invention                    Molecular Weight   Monoisotopic   M + H           Molecular Formula   (calculated)   Mass   Found                        1   C30H40N4O5   536.7   536   537       2   C30H40N4O4   520.7   520   521       3   C30H42N4O4   522.7   522   523       4   C30H42N4O5   538.7   538   539       5   C28H36N4O5   508.6   508   509       6   C30H40N4O5S   568.7   568   569       7   C31H42N4O5   550.7   550   551       8   C34H42N4O5   586.7   586   587       9   C30H40N4O5   536.7   536   537       10   C31H42N4O5   550.7   550   551       11   C34H44N4O4   572.7   572   573       12   C29H38N4O5   522.6   522   523       13   C31H44N4O4   536.7   536   537       14   C35H46N4O4   586.8   586   587       15   C30H41N4O4Cl   557.1   556   557       16   C30H41N4O4Cl   557.1   556   557       17   C32H43N5O4   561.7   561   562       18   C29H40N4O5   524.7   524   525       19   C30H41N4O4F   540.7   540   541       20   C31H42N4O4   534.7   534   535       21   C35H44N4O4   584.7   584   585       22   C31H44N4O5   552.7   552   553       23   C34H44N4O4   572.7   572   573       24   C28H40N4O4S   528.7   528   529       25   C30H41N4O4Cl   557.1   556   557       26   C31H42N4O5   550.7   550   551       27   C27H39N5O4S   529.7   529   530       28   C29H41N5O4   523.7   523   524       29   C28H39N5O5   525.6   525   526       30   C30H41N3O6   539.7   539   540       34   C34H40N4O6   600.7   600   601       38   C28H36N4O5   508.6   508   509       39   C28H36N4O5   508.6   508   509       40   C27H34N4O5   494.6   494   495       41   C34H40N4O5   584.7   584   585       52   C33H38N4O5   570.7   570   571       55   C31H43N5O5   565.7   565   566       56   C30H41N5O5   551.7   551   552       57   C28H36N4O6   524.6   524   525       58   C34H40N4O6   600.7   600   601       59   C36H41N5O5   623.7   623   624       60   C35H42N4O6   614.7   614   615       65   C24H36N4O4   444.6   444   445       71   C29H40N4O6   540.7   540   541       72   C38H42N4O5   634.8   634   635       76   C38H42N4O5   634.8   634   635       77   C31H42N4O5   550.7   550   551       80   C31H42N4O5   550.7   550   551       85   C30H40N4O5   536.7   536   537       87   C36H46N4O4   598.8   598   599       88   C34H50N4O5   594.8   594   595       89   C31H44N4O4   536.7   536   537       90   C36H46N4O4   598.8   598   599       91   C30H42N4O5   538.7   538   539       92   C31H44N4O5   552.7   552   553       96   C28H38N4O5   510.6   510   511       97   C33H46N4O5   578.7   578   579       98   C24H39N5O4   461.6   461   462       99   C24H39N5O4   461.6   461   462       109   C29H41N5O5   539.7   539   540       110   C29H41N5O5   539.7   539   540       111   C30H41N3O6   539.7   539   540       112   C31H44N4O5   552.7   552   553       120   C30H38N4O5   534.6   534   535       121   C32H45N5O6   595.7   595   596       122   C31H43N4O4Cl   571.2   570   571       123   C29H41N5O4   523.7   523   524       124   C29H41N5O4   523.7   523   524       125   C30H40N4O5   536.7   536   537       126   C32H46N4O5   566.7   566   567       127   C30H38N6O3S   562.7   562   563       128   C32H46N4O5   566.7   566   567       129   C35H46N4O4   586.8   586   587       130   C29H42N4O4S   542.7   542   543       131   C31H43N4O4Cl   571.2   570   571       132   C31H43N4O4Cl   571.2   570   571       133   C31H43N4O4F   554.7   554   555       134   C25H37N4O3Cl   477.0   476   477       135   C31H45N5O5   567.7   567   568       136   C34H45N5O4   587.8   587   588       137   C28H41N5O4S   543.7   543   544       138   C30H42N5O4Cl   572.1   571   572       139   C30H42N5O4Cl   572.1   571   572       140   C30H42N5O4F   555.7   555   556       141   C32H44N4O5   564.7   564   565       142   C35H44N4O4   584.7   584   585       143   C29H40N4O4S   540.7   540   541       144   C31H41N4O4Cl   569.1   568   569       145   C31H41N4O4Cl   569.1   568   569       146   C31H41N4O4F   552.7   552   553       147   C31H43N5O5   565.7   565   566       148   C34H43N5O4   585.7   585   586       149   C30H40N5O4Cl   570.1   569   570       150   C30H40N5O4Cl   570.1   569   570       151   C30H40N5O4F   553.7   553   554       152   C29H41N5O5   539.7   539   540       153   C32H41N5O4   559.7   559   560       154   C26H37N5O4S   515.7   515   516       155   C28H38N5O4Cl   544.1   543   544       156   C28H38N5O4Cl   544.1   543   544       157   C28H38N5O4F   527.6   527   528       158   C27H37N6O4Cl   545.1   544   545       159   C31H44N4O5   552.7   552   553       160   C31H44N4O5   552.7   552   553       161   C31H45N5O4   551.7   551   552       162   C31H44N4O4   536.7   536   537       163   C31H44N4O4   536.7   536   537       164   C31H44N4O4   536.7   536   537       165   C31H44N4O5   552.7   552   553       166   C31H44N4O5   552.7   552   553       167   C32H42N4O4S   578.8   578   579       168   C28H40N4O4S   528.7   528   529       169   C31H43N4O4Cl   571.2   570   571       170   C30H40N4O4Cl2   591.6   590   591       171   C30H40N4O4F2   558.7   558   559       172   C32H46N4O6   582.7   582   583       173   C34H43N3O5   573.7   573   574       174   C31H43N3O6   553.7   553   554       175   C31H44N4O5   552.7   552   553       176   C31H44N4O5   552.7   552   553       177   C29H40N4O5   524.7   524   525       178   C29H40N4O6   540.7   540   541       179   C32H40N4O5   560.7   560   561       180   C26H36N4O5S   516.7   516   517       181   C28H37N4O5Cl   545.1   544   545       182   C28H37N4O5Cl   545.1   544   545       183   C28H37N4O5F   528.6   528   529       184   C31H40N6O4   560.7   560   561       185   C27H37N6O4Cl   545.1   544   545       186   C31H40N6O5   576.7   576   577       187   C31H41N4O4F3   590.7   590   591       188   C30H41N4O4F   540.7   540   541       189   C30H41N5O6   567.7   567   568       190   C33H42N4O4S   590.8   590   591       191   C32H44N4O5   564.7   564   565       192   C31H40N4O4Cl2   603.6   602   603       193   C31H40N4O4F2   570.7   570   571       194   C32H48N6O6   612.8   612   613       195   C32H46N4O5   566.7   566   567       196   C32H43N6O4Cl   611.2   610   611       197   C32H45N6O5Cl   629.2   628   629       198   C32H43N4O4Cl   583.2   582   583       199   C27H39N4O6Cl   551.1   550   551       200   C31H39N4O4Cl   567.1   566   567       201   C34H42N4O4   570.7   570   571       202   C31H42N4O5   550.7   550   551       203   C30H40N5O5Cl   586.1   585   586       204   C29H40N7O4Cl   586.1   585   586       205   C32H45N4O4Cl   585.2   584   585       206   C29H40N5O6SCl   622.2   621   622       207   C29H39N6O5Cl   587.1   586   587       208   C29H41N7O5   567.7   567   568       209   C30H41N5O6   567.7   567   568       210   C31H45N5O5   567.7   567   568       211   C30H42N5O4Cl   572.1   571   572       212   C31H44N5O4Cl   586.2   585   586       213   C30H40N4O5I2   790.5   790   791       214   C30H42N4O6   554.7   554   555       215   C30H43N5O5   553.7   553   554       216   C32H43N4O4Cl   583.2   582   583       217   C31H40N4O4FCl   587.1   586   587       218   C31H43N4O4Cl   571.2   570   571       219   C30H40N4O4Cl2   591.6   590   591       220   C31H43N4O4F   554.7   554   555       221   C30H40N4O4FCl   575.1   574   575       222   C34H50N4O5   594.8   594   595       223   C32H44N4O6   580.7   580   581       224   C36H48N4O4   600.8   600   601       225   C37H48N4O5   628.8   628   629       226   C39H49N5O4S   683.9   683   684       227   C42H52N4O4   676.9   676   677               Notes       1. Molecular formulas and molecular weights (MW) are calculated automatically from the structure via ActivityBase ® software (IDBS, Guildford, Surrey, UK) or, for MW only, from the freeware program Molecular Weight Calculator v. 6.32       2. M + H obtained from LC-MS analysis using the General Method as described       3. All analyses conducted on material after preparative HPLC purification            
Biological Methods and Results
 
     The compounds of the present invention were evaluated for their ability to interact at the human motilin receptor utilizing a competitive radioligand binding assay as described in Method B1. Further characterization of the interaction can be performed utilizing the functional assays described in Methods B2, B3 and B4. Some of these methods can be conducted, if so desired, in a high throughput manner to permit the simultaneous evaluation of many compounds. Other assays have also been described that are suitable for HTS, such as that based upon the stable expression of a synthetic gene for the human motilin receptor. 
     Results for the examination of representative compounds of the present invention using Method B1 are presented in Table 3. The binding activity is listed as ranges with the following levels: A=0.001-0.10 μM; B=0.10-1.0 μM; C=1.0-10.0 μM. In addition, the assay results of two additional compounds using this Method are shown below. As can be observed, this demonstrates the activity of a representative bicyclic compound of Formula IV of the invention, which resulted from incorporation of D-proline as the second recognition building block. Significantly, the lack of binding activity obtained with compound 121, which is the linear analogue of compound 1 (K i =level B), illustrates the critical importance of the cyclic structure to attaining the desired interaction. 
     
       
                 
         
             
             
         
      
     
     Competitive binding curves for two representative compounds of the invention (Compounds 8 and 11) are presented in  FIG. 23  and  FIG. 24 , respectively. 
     For determination of functional significance of the binding, the compounds are preferably tested in the Aequorin assay as described in Method B2, although the procedure of Method B3 is also applicable. As can be seen from the data presented in Table 4, the representative compounds examined act as antagonists at the motilin receptor and are devoid of angonist activity at the concentrations studied. The functional activity is listed as ranges with the following levels: A=0.001-0.10 μM; B=0.10-1.0 μM. The higher sensitivity of the assay of Method B2, almost 100 times that of Method C, makes it the preferred one for this assessment. This is evident in the EC 50  values obtained in each for the positive angonist standard, motilin. Additionally, Method B2 measures the actual signaling event, which makes it more relevant to the effect that is desired, whereas the assay of Method B3 simply measures GTP turnover. 
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Demonstration of Antagonist Activity at the Motilin Receptor 
               
             
          
           
               
                   
                 Aequorin (Method B2) 1   
                   
               
             
          
           
               
                 Compound 
                 Binding (K i ) 
                 IC 50   
               
               
                   
               
               
                 142 
                 A 
                 B 
               
               
                 149 
                 A 
                 B 
               
               
                 167 
                 A 
                 A 
               
               
                 168 
                 A 
                 A 
               
               
                 212 
                 A 
                 A 
               
               
                 Motilin 
                 0.6 
                 not applicable 
               
               
                 (human, porcine) 2   
               
               
                   
               
               
                   1 Activity is listed as ranges with the following levels: A = 0.001-0.10 μM; B = 0.10-1.0 μM 
               
               
                   2 Human and porcine motilin are the same peptide. 
               
             
          
         
       
     
     In addition, a common and scientifically-accepted ex vivo assay for the measurement of angonist or antagonist activity at the motilin receptor is the contraction of rabbit duodenum or other gastrointestinal smooth muscle tissue. A2-A4  Agonists are defined as compounds that induce &gt;50% contraction relative to the motilin peptide, whereas antagonists are defined as compounds that cause &gt;50% inhibition of the response to motilin. Compounds of the present invention have shown significant antagonist activity in this assay. For example, compound 144 exhibited a pA 2 =6.95, while compound 165 had a pA 2 =7.17, as calculated from the Schild plots of the response obtained at various concentrations as described in Method B4. 
     Gastric motility is generally measured in the clinical setting as the time required for gastric emptying and subsequent transit time through the GI tract. Gastric emptying scans are well known to those skilled in the art an, briefly, comprise use of an oral contrast agent, such as barium, or a radiolabeled meal. Solid and liquids can be measured independently. A test food or liquid is radiolabeled with an isotope ( 99m Tc) and after ingestion or administration, transit time through the GI tract and gastric emptying are measured by visualization using gamma cameras. These studies are performed before and after the administration of the therapeutic agent to quantify the efficacy of the compound. 
     Example Method B1 
     Competitive Radioligand Binding Assay (Motilin Receptor) 
     Materials: 
     
         
         
           
             Membranes were prepared from CHO cells stably transfected with the human motilin receptor and utilized at a quantity of 1.5 μg/assay point. [PerkinElmer™ SignalScreen® Product #6110544, PerkinElmer, Inc., Wellesley, Mass.] 
             [ 125 I]-Motilin (PerkinElmer, #NEX-378); final concentration: 0.04-0.06 nM 
             Motilin (Bachem™, #H-4385, Bachem Bioscience Inc., King of Prussia, Pa.); final concentration: 1 μM 
             Multiscreen® Harvest plates-GF/B (Millipore™, #MAHFB1H60, Billerica, Mass.) 
             Deep-well polypropylene titer plate (Beckman Coulter™, #267006, Fullerton, Calif.) 
             TopSeal-A™ (PerkinElmer, #6005185, Wellesley, Mass.) 
             Bottom seal (Millipore™, #MATAHOP00, Billerica, Mass.) 
             MicroScint-0™ (PerkinElmer, #6013611, Wellesley, Mass.) 
             Binding Buffer: 50 mM Tris-HCl (pH 7.4), 10 mM MgCl 2 : 1 mM EDTA, 0.1% BSA
 
Assay Volumes:
 
             150 μL of membranes diluted in binding buffer 
             10 μL of compound diluted in binding buffer 
             10 μL of radioligand ([ 125 I]-Motilin) diluted in binding buffer 
           
         
       
    
     Final Test Concentrations (N=11) for Compounds:
         10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005 μM.
 
Compound Handling:
       

     Compounds were provided frozen on dry ice at a stock concentration of 10 mM diluted in 100% DMSO and stored at −20° C. until the day of testing. On the test day, compounds were allowed to thaw at room temperature and than diluted in assay buffer according to the desired test concentrations. Under these conditions, the maximum final DMSO concentration in the assay was 0.5%. 
     Assay Protocol: 
     In deep-well plates, diluted cell membranes (1.5 μg/mL) are combined with 10 μL of either binding buffer (total binding, N=5), 1 μM motilin (non-specific binding, N=3) or the appropriate concentration of test compound. The reaction is initiated by addition of 10 μl of [ 125 I]-motilin (final conc. 0.04-0.06 nM) to each well. Plates are sealed with TopSeal-A, vortexed gently and incubated at room temperature for 2 hours. The reaction is arrested by filtering samples through pre-soaked (0.3% polyethyleneimine, 2 h) Multiscreen Harvest plates using a Tomtec® Harvester (Tomtec, Hamden, Conn.)), washed 9 times with 500 μL of cold 50 mM Tris-HCl (pH 7.4), and than plates are air-dried in a fumehood for 30 minutes. A bottom seal is applied to the plates prior to the addition of 25 μL of MicroScint-0™ to each well. Plates are then sealed with TopSeal-A® and counted for 30 sec per well on a TopCount® Microplate Scintillation and Luminescence Counter (PerkinElmer, Wellesley, Mass.) where results are expressed as counts per minute (cpm). 
     Data are analyzed by GraphPad™ Prism (GraphPad Software, San Diego, Calif.) using a variable slope non-linear regression analysis. K i  values were calculated using a K d  value of 0.16 nM for [ 125 I]-motilin (previously determined during membrane characterization). 
               D   max     =     1   -                 test   ⁢           ⁢   concentration   ⁢           ⁢   with   ⁢           ⁢   maximal   ⁢           ⁢   displacement     -               non   ⁢     -     ⁢   specific   ⁢           ⁢   binding               total   ⁢           ⁢   binding     -     non   ⁢     -     ⁢   specific   ⁢           ⁢   binding         ×   100             
where total and non-specific binding represent the cpm obtained in the absence or presence of 1 μM motilin, respectively.
 
     Example Method B2 
     Aequorin Functional Assay (Motilin Receptor) 
     Materials:
         Membranes were prepared using AequoScreen™ (EUROSCREEN, Belgium) cell lines expressing the human motilin receptor (cell line ES-380-A; receptor accession #AF034632). This cell line is constructed by transfection of the human motilin receptor into CHO-K1 cells co-expressing G α16  and the mitochondrially targeted Aequorin (Ref #ES-WT-A5).   Motilin (Bachem™, #H-4385, Bachem Bioscience Inc., King of Prussia, Pa.)   Assay buffer: DMEM-F12 (Dulbeccoe&#39;s Modified Eagles Medium) with 15 mM HEPES and 0.1% BSA (pH 7.0)   Coelenterazine (Molecular Probes™, Leiden, The Netherlands)       

     Final Test Concentrations (N=5) for Compounds:
         10, 3.16, 1, 0.316, 0.1 μM.
 
Compound Handling:
       

     Compounds were provided as dry films at a quantity of approximately 1.2 μmol in pre-formatted 96-well plates. Compounds were dissolved in 100% DMSO at a concentration of 10 mM and stored at −20° C. until further use. Daughter plates were prepared at a concentration of 500 μM in 30% DMSO with 0.1% BSA and stored at −20° C. until testing. On the test day, compounds were allowed to thaw at room temperature and than diluted in assay buffer according to the desired test concentrations. Under these conditions, the maximum final DMSO concentration in the assay was 0.6%. 
     Cell Preparation: 
     Cells are collected from culture plates with Ca 2+  and Mg 2+ -free phosphate buffered saline (PBS) supplemented with 5 mM EDTA, pelleted for 2 minutes at 1000×g, resuspended in assay buffer (see above) at a density of 5×10 6  cells/mL and incubated overnight in the presence of 5 μM coelenterazine. After loading, cells were diluted with assay buffer to a concentration of 5×10 5  cells/mL. 
     Assay Protocol: 
     For angonist testing, 50 μl of the cell suspension was mixed with 50 μl of the appropriate concentration of test compound or motilin (reference angonist) in 96-well plates (duplicate samples). The emission of light resulting from receptor activation was recorded using the Functional Drug Screening System 6000 ‘FDSS 6000’ (Hamamatsu Photonics K.K., Japan). 
     For antagonist testing, an approximate EC80 concentration of motilin (i.e. 0.5 nM; 100 μL) was injected onto the cell suspension containing the test compounds (duplicate samples) 15-30 minutes after the end of angonist testing and the consequent emission of light resulting from receptor activation was measured as described in the paragraph above. 
     Results are expressed as Relative Light Units (RLU). Concentration response curves were analyzed using GraphPad™ Prism® (GraphPad Software, San Diego, Calif.) by non-linear regression analysis (sigmoidal dose-response) based on the equation E=E max /(1+EC 50 /C)n where E is the measured RLU value at a given angonist concentration (C), E max  is the maximal response, EC 50  is the concentration producing 50% stimulation and n is the slope index. For angonist testing, results for each concentration of test compound were expressed as percent activation relative to the signal induced by motilin at a concentration equal to the EC 80  (i.e. 0.5 nM). For antagonist testing, results for each concentration of test compound were expressed as percent inhibition relative to the signal induced by motilin at a concentration equal to the EC 80  (i.e. 0.5 nM). 
     Example Method B3: FlashPlate® Motilin [ 35 S]-GTPγS Functional Assay 
     Materials: 
     
         
         
           
             Membranes were prepared from CHO cells stably transfected with the human motilin receptor and utilized at a quantity of 1.5 μg/assay point.
           [PerkinElmer™ SignalScreen® Product #6110544, PerkinElmer, Inc. Wellesley, Mass.]   
         
             GTPγS Guanosine 5′-[γ-thio]triphosphate tetralithium salt (Sigma, #G-8634, Sigma-Aldrich, St. Louis, Mo.) 
             [ 35 S]-GTPγS (PerkinElmer, #NEX-030H) 
             Motilin (Bachem™, #H-4385, Bachem Bioscience Inc., King of Prussia, Pa.) 
             96-well FlashPlate® white polystyrene microplates (PerkinElmer, #SMP200, Wellesley, Mass.) 
             Deep-well polypropylene titer plate (Beckman Coulter™, #267006, Fullerton, Calif.) 
             TopSeal-A™ (PerkinElmer, #6005185, Wellesley, Mass.) 
             Assay Buffer: 50 mM Tris (pH 7.4), 100 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA, 1 μM GDP, 0.1% BSA
 
Assay Volumes:
 
             25 μL of compound diluted in assay buffer 
             25 μL of assay buffer (angonist assay) or 0.6 μM motilin (0.1 μM final concentration) diluted in assay buffer (antagonist assay) 
             100 μL of [ 35 S]-GTPγS diluted in assay buffer 
           
         
       
    
     Final Test Concentrations (N=12) for Compounds:
         50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01 μM.
 
Compound Handling:
       

     Compounds were provided frozen on dry ice at a stock concentration of 10 mM diluted in 100% DMSO and stored at −20° C. until the day of testing. On the test day, compounds were allowed to thaw at room temperature and than diluted in assay buffer according to the desired test concentrations. Under these conditions, the maximum final DMSO concentration in the assay was 0.5%. 
     Assay Protocol: 
     CHO membranes were immobilized into 96-well FlashPlate® microplates. Test compound, GTPγS, motilin and [ 35 S]-GTPγS were combined in each well according to the Assay Volumes described above. 
     For the assay to measure angonist activity, an additional 25 μl of buffer was added to each well in addition to 25 μL of either buffer (basal value, N=4), 1 μM (final conc.) motilin (E max  value, N=3), 25 μM (final conc.) GTPγS (non-specific value, N=4), or the appropriate concentration of test compound (N=3). 
     For the assay to measure antagonist activity, an additional 25 μL of either buffer (unstimulated control) or motilin (0.1 μM final conc.) is added to each well, in addition to either 25 μL of buffer (basal value, N=3), 1 μM (final conc.) motilin (E max  value, N=3), 25 μM (final conc.) GTPγS (non-specific value, N=4), or the appropriate concentration of test compound (N=3). 
     The reaction is initiated by addition of 100 mL of [ 35 S]-GTPγS to each well. Each plate is sealed (TopSeal-AT™) and incubated in the dark at room temperature for 150 min. Then, plates are counted for 30 seconds per well on the TopCount® NXT. 
     Data were analyzed by GraphPad™ Prism® 3.0 (GraphPad Software, San Diego, Calif.) using non-linear regression analysis (sigmoidal dose-response) for the calculation of IC 50 /EC 50  values. 
     
       
         
           
             
               
                 
                   E 
                   max 
                 
                 ⁡ 
                 
                   ( 
                   agonist 
                   ) 
                 
               
               ⁢ 
               
                   
               
               ⁢ 
               or 
               ⁢ 
               
                   
               
               ⁢ 
               
                 
                   D 
                   max 
                 
                 ⁡ 
                 
                   ( 
                   antagonist 
                   ) 
                 
               
             
             = 
             
               
                 
                   Top 
                   - 
                   Bottom 
                 
                 Bottom 
               
               × 
               100 
             
           
         
       
     
     Where Top and Bottom correspond to the top and bottom values of the dose-response curve calculated by GraphPad™ Prism®). 
     Example Method B4 
     Rabbit Duodenum Contractility Assay 
     Duodenal segments were vertically suspended in organ chambers of 10 mL filled with Krebs buffer and connected to an isotonic force transducer, with a preload of 1 g. After a stabilization period, the muscle strips were challenged with 10 −4 M acetylcholine and washed. This was repeated until a stable maximal contraction was obtained (2-3 times), with an interval of at least 20 minutes. 
     After a stable base line was reached, test compounds were added to the bath. After 15 min incubation, a dose response to motilin was recorded by adding logarithmically increasing concentrations of motilin to the bath (final concentration 10 −9  to 10 −6  M). A blank experiment (no test compound present) was also performed. At the end of the dose response curve, a supramaximal dose of acetylcholine (10 −4  M) was given and this response was used as a reference (100% contraction). 
     The results of experiments at different concentrations of test compound were combined and analyzed to derive the pA 2  value from the Schild plot. 
     It is appreciated that although specific experimental methods have been described herein for the purposes of illustration, various modifications to these experimental methods as well as alternate methods of experimentation may be used without departing from the scope of this invention. 
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Binding activity of selected compounds 
               
             
          
           
               
                   
                 R 1   
                 R 3   
                 R 6   
                 T 
                 K i   1,2   
               
               
                   
               
             
          
           
               
                 1 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 2 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 3 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 4 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 5 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 CH3 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 6 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 7 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 8 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 9 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 10 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 11 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 12 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 13 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 14 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 15 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 16 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 17 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 18 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 19 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 20 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 21 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 22 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 23 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 24 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 25 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 26 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 27 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 28 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 29 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 30 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 34 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 38 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 CH 3   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 39 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 H 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 40 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 H 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 41 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 52 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 55 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 56 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 57 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 58 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 59 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 60 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 65 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 71 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 72 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 76 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 77 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 80 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 85 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 H 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 87 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 88 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 89 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 90 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 91 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 92 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 96 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 H 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 97 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 98 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 99 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 109 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 110 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 111 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 112 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 122 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 123 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 124 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 125 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 126 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 127 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 128 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 129 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 130 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 131 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 132 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 133 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 134 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 135 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 136 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 137 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 138 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 139 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 140 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 141 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 142 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 143 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 144 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 145 
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 146 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 147 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 148 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 149 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 150 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 151 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 152 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 153 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 154 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 155 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 156 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 157 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 158 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 159 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 160 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 161 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 162 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 163 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 164 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 165 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 166 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 167 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 168 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 169 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 170 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 171 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 172 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 173 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 174 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 175 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 176 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 177 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 178 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 179 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 180 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 181 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 182 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 183 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 184 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 185 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 186 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 187 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 188 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 189 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 190 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 191 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 192 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 193 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 194 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 195 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 196 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 197 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 198 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 199 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 200 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 201 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 202 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 203 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 204 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 205 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 206 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 207 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 208 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 209 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 210 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 211 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 212 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 213 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 214 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 215 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 216 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 217 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 218 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 219 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 220 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 221 
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 222 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 A 
               
               
                   
               
               
                 223 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 224 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 225 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 226 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 C 
               
               
                   
               
               
                 227 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 B 
               
               
                   
               
               
                 Notes 
               
               
                 Radioligand competitive binding assays performed using Method B1 
               
               
                 Values reported as ranges: A = 0.001-0.100 μM; B = 0.100-1.0 μM; C = 1.0-10.0 μM 
               
               
                 X is NH except for: 
               
               
                 Compound 223 and 225, X is: 
               
               
                                           
Compound 224, X is NMe 
               
               
                 Compound 226, X is: 
               
               
                                           
Compound 227, X is 
               
               
                                           
Z 1 , Z 2  and Z 3  are NH except for compounds 30, 173 and 174 and where Z1 is O and compound 111 where Z 2  is O. 
               
               
                 R 2 , R 4  and R 5  are hydrogen except for compound 85 where it is: 
               
               
                                           
m, n 1  and p are zero.