IMMUNOMODULATORS

Abstract

The present disclosure provides novel macrocyclic peptides which inhibit the PD-1/PD-L1 and PD-L1/CD80 protein/protein interaction, and thus are useful for the amelioration of various diseases, including cancer and infectious diseases.

Claims

1. A compound of formula (I) ##STR00081## or a pharmaceutically acceptable salt thereof, wherein: A is selected from a bond, ##STR00082## wherein: denotes the point of attachment to the carbonyl group and denotes the point of attachment to the nitrogen atom; z is 0, 1, or 2; w is 1 or 2; n is 0 or 1; m is 1 or 2; m′ is 0 or 1; p is 0, 1, or 2; R.sup.x is selected from hydrogen, amino, hydroxy, and methyl; R.sup.14 and R.sup.15 are independently selected from hydrogen and methyl; and R.sup.z is selected from hydrogen and —C(O)NHR.sup.16; wherein R.sup.16 is selected from hydrogen, —CHR.sup.17C(O)NH.sub.2, —CHR.sup.17C(O)NHCHR.sup.18C(O)NH.sub.2, and —CHR.sup.17C(O)NHCHR.sup.18C(O)NHCH.sub.2C(O)NH.sub.2; wherein R.sup.17 is selected from hydrogen and —CH.sub.2OH and wherein R.sup.18 is selected from hydrogen and methyl; R.sup.v is hydrogen or a natural amino acid side chain; Q is selected from ##STR00083## wherein R.sup.b is defined below, and a five- or six-membered ring containing one, two, or three double bonds and optionally containing one, two, or three heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the ring is optionally substituted with one, two, three, or four substituents independently selected from C.sub.1-C.sub.6alkoxycarbonyl, C.sub.1-C.sub.3alkyl, amino, C.sub.1-C.sub.3alkylamino, carboxy, C.sub.1-C.sub.3dialkylamino, halo, and haloC.sub.1-C.sub.3alkyl; U is selected from ##STR00084## wherein R.sup.k is defined below, and a five- or six-membered ring containing one, two, or three double bonds and optionally containing one, two, or three heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the ring is optionally substituted with one, two, three, or four substituents independently selected from C.sub.1-C.sub.6alkoxycarbonyl, C.sub.1-C.sub.3alkyl, amino, C.sub.1-C.sub.3alkylamino, carboxy, C.sub.1-C.sub.3dialkylamino, halo, and haloC.sub.1-C.sub.3alkyl; provided that at least one of Q and U is a five- or six-membered ring containing one, two, or three double bonds and containing one, two, or three heteroatoms independently selected from nitrogen, oxygen, and sulfur; R.sup.c, R.sup.f, R.sup.h, R.sup.i, R.sup.m, and R.sup.n are hydrogen; R.sup.a, R.sup.e, R.sup.j, and R.sup.k, are each independently selected from hydrogen and methyl; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, and R.sup.13 are independently selected from a natural amino acid side chain and an unnatural amino acid side chain or form a ring with the corresponding vicinal R group as described below; R.sup.e and R.sup.k can each form a ring with the corresponding vicinal R group and the atoms to which they are attached selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy; R.sup.b is methyl or, R.sup.b and R.sup.2, together with the atoms to which they are attached, form a ring selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy; R.sup.d is hydrogen or methyl, or, R.sup.d and R.sup.4, together with the atoms to which they are attached, can form a ring selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, hydroxy, and phenyl; R.sup.g is hydrogen or methyl or R.sup.g and R.sup.7, together with the atoms to which they are attached, can form a ring selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, benzyl optionally substituted with a halo group, benzyloxy, cyano, cyclohexyl, methyl, halo, hydroxy, isoquinolinyloxy optionally substituted with a methoxy group, quinolinyloxy optionally substituted with a halo group, and tetrazolyl; and wherein the pyrrolidine and the piperidine ring are optionally fused to a cyclohexyl, phenyl, or indole group; and R.sup.l is methyl or, R.sup.l and R.sup.12, together with the atoms to which they are attached, form a ring selected from azetidine and pyrollidine, wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, and hydroxy.

2. A compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: Q is selected from ##STR00085## and a five-membered ring containing one or two double-bonds and containing one, two, three, or four nitrogen atoms; and U is selected from ##STR00086## and a five-membered ring containing one or two double-bonds and containing one, two, three, or four nitrogen atoms.

3. A compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein A is ##STR00087##

4. A compound of claim 3, or a pharmaceutically acceptable salt thereof, wherein z and w are each 1; R.sup.14 and R.sup.15 are hydrogen; and R.sup.z is —C(O)NHR.sup.16; wherein R.sup.16 is —CHR.sup.17C(O)NH.sub.2.

5. A compound of claim 4 wherein R.sup.1 is phenylC.sub.1-C.sub.3alkyl wherein the phenyl is optionally substituted with hydroxy; R.sup.2 is C.sub.1-C.sub.7alkyl or, R.sup.b and R.sup.2, together with the atoms to which they are attached, form a morpholine or piperidine ring; R.sup.3 is selected from —CH.sub.2CO.sub.2H and —CH.sub.2C(O)NH.sub.2; R.sup.4 and R.sup.d, together with the atoms to which they are attached, form a pyrrolidine ring; R.sup.5 is selected from —CH.sub.2NH.sub.2 and —CH.sub.2(imidazolyl); R.sup.6 is selected from C.sub.1-C.sub.7alkyl, —CH.sub.2CH.sub.2C(O)NH.sub.2, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2, and —CH.sub.2CH.sub.2CO.sub.2H; R.sup.7 is hydrogen or R.sup.7 and R.sup.9, together with the atoms to which they are attached, form a pyrroldine ring optionally substituted with a hydroxy group; R.sup.8 is —(CH.sub.2)indolyl; R.sup.9 is selected from aminomethyl, hydroxymethyl, —CH.sub.2CH.sub.2NH.sub.2, and CH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2; R.sup.10 is selected from —(CH.sub.2)indolyl, —(CH.sub.2)naphthyl, and —(CH.sub.2)benzothienyl, each optionally substituted with —CH.sub.2CO.sub.2H; R.sup.11 is C.sub.1-C.sub.7alkyl; and R.sup.12 is C.sub.1-C.sub.7alkyl.

6. A compound of formula (II) ##STR00088## or a pharmaceutically acceptable salt thereof, wherein: A is selected from a bond, ##STR00089## wherein: denotes the point of attachment to the carbonyl group and denotes the point of attachment to the nitrogen atom; n is 0 or 1; R.sup.14 and R.sup.15 are independently selected from hydrogen and methyl; and R.sup.16 is selected from hydrogen, —CHR.sup.17C(O)NH.sub.2, —CHR.sup.17C(O)NHCHR.sup.18C(O)NH.sub.2, and —CHR.sup.17C(O)NHCHR.sup.18C(O)NHCH.sub.2C(O)NH.sub.2; wherein R.sup.17 is selected from hydrogen and —CH.sub.2OH and wherein R.sup.18 is selected from hydrogen and methyl; Q is a five- or six-membered ring containing one, two, or three double bonds and optionally containing one, two, or three heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the ring is optionally substituted with one, two, three, or four substituents independently selected from C.sub.1-C.sub.6alkoxycarbonyl, C.sub.1-C.sub.3alkyl, amino, C.sub.1-C.sub.3alkylamino, carboxy, C.sub.1-C.sub.3dialkylamino, halo, and haloC.sub.1-C.sub.3alkyl; R.sup.a, R.sup.f, R.sup.j, R.sup.k, R.sup.l, and R.sup.m are hydrogen; R.sup.b and R.sup.c are methyl; R.sup.g is selected from hydrogen and methyl; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.9, R.sup.10, R.sup.11, and R.sup.12 are independently selected from a natural amino acid side chain and an unnatural amino acid side chain or form a ring with the corresponding vicinal R group as described below; R.sup.20 is hydrogen; R.sup.8 is selected from a natural amino acid side chain, an unnatural amino acid chain, or can form a ring with the corresponding vicinal R group as described below, or, alternatively, R.sup.8 can form a three- to six-membered carbocyclic ring with R.sup.20; R.sup.d is selected from hydrogen and methyl, or, R.sup.d and R.sup.4, together with the atoms to which they are attached, form a ring selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, halomethyl, and hydroxy; R.sup.e is selected from hydrogen and methyl, or, R.sup.e and R.sup.5, together with the atoms to which they are attached, form a ring selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, halomethyl, and hydroxy; and R.sup.h is selected from hydrogen and methyl, or, R.sup.h and R.sup.8, together with the atoms to which they are attached, form a ring selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, halomethyl, and hydroxy.

7. A compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein Q is a five-membered ring containing one or two double-bonds and containing one, two, three, or four nitrogen atoms.

8. A compound of claim 7, or a pharmaceutically acceptable salt thereof, wherein A is ##STR00090##

9. A compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 and R.sup.2 are phenylC.sub.1-C.sub.3alkyl; R.sup.3 is C.sub.1-C.sub.7alkyl; R.sup.4 is hydrogen; R.sup.5 is carboxymethyl; R.sup.6 is C.sub.1-C.sub.7alkyl; R.sup.7 is phenylC.sub.1-C.sub.3alkyl; R.sup.8 is phenylC.sub.1-C.sub.3alkyl wherein the phenyl is substituted with hydroxy, or, alternatively, R.sup.8 and R.sup.20 form a six-membered carbocyclic ring; R.sup.9 is hydrogen; R.sup.10 is —(CH.sub.2)indolyl; R.sup.11 is phenylC.sub.1-C.sub.3alkyl substituted with hydroxy; and R.sup.12 is C.sub.1-C.sub.7alkyl.

10. A compound selected from: Example 1001 (SEQ ID NO: 6); Example 1002 (SEQ ID NO: 7); Example 1003 (SEQ ID NO: 8); Example 1004 (SEQ ID NO: 9); Example 1005 (SEQ ID NO: 10); Example 1006 (SEQ ID NO: 11); Example 1007 (SEQ ID NO: 12); Example 1010 (SEQ ID NO: 13); Example 1012 (SEQ ID NO: 14); Example 1013 (SEQ ID NO: 15); Example 1014 (SEQ ID NO: 16); Example 1050 (SEQ ID NO: 17); Example 1051 (SEQ ID NO: 18); Example 1052 (SEQ ID NO: 19); Example 1053 (SEQ ID NO: 20); Example 1054 (SEQ ID NO: 21); Example 1055 (SEQ ID NO: 22); Example 1056 (SEQ ID NO: 23); Example 1057 (SEQ ID NO: 24); Example 1058 (SEQ ID NO: 25); Example 1059 (SEQ ID NO: 26); Example 1060 (SEQ ID NO: 27); Example 1061 (SEQ ID NO: 28); Example 1100 (SEQ ID NO: 29); Example 1101 (SEQ ID NO: 30); Example 1102 (SEQ ID NO: 31); Example 1103 (SEQ ID NO: 32); Example 1104 (SEQ ID NO: 33); Example 1105 (SEQ ID NO: 34); Example 1106 (SEQ ID NO: 35); Example 1107 (SEQ ID NO: 36); Example 1108 (SEQ ID NO: 37); Example 1109 (SEQ ID NO: 38); Example 1110 (SEQ ID NO: 39); Example 1111 (SEQ ID NO: 40); Example 1113 (SEQ ID NO: 41); Example 1114 (SEQ ID NO: 42); Example 3001 (SEQ ID NO: 44); Example 3002 (SEQ ID NO: 44); Example 3003 (SEQ ID NO: 45); or a pharmaceutically acceptable salt thereof.

11. A compound which is Example 1115 (SEQ ID NO: 43).

12. A method of enhancing, stimulating, and/or increasing the immune response in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of claim 1 or a therapeutically acceptable salt thereof.

13. The method of claim 12 further comprising administering an additional agent prior to, after, or simultaneously with the compound of claim 1 or a therapeutically acceptable salt thereof.

14. The method of claim 13 wherein the additional agent is an antimicrobial agent, an antiviral agent, a cytotoxic agent, and/or an immune response modifier.

15. The method of claim 13 wherein the additional agent is an HDAC inhibitor.

16. The method of claim 13 wherein the additional agent is a TLR7 and/or TLR8 agonist

17. A method of inhibiting growth, proliferation, or metastasis of cancer cells in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount a compound of claim 1 or a therapeutically acceptable salt thereof.

18. The method of claim 17 wherein the cancer is selected from melanoma, renal cell carcinoma, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, castration-resistant prostate cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, squamous cell carcinoma of the head and neck, carcinomas of the esophagus, gastrointestinal tract and breast, and hematological malignancies.

19. A method of treating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of claim 1 or a therapeutically acceptable salt thereof.

20. The method of claim 19 wherein the infectious disease is caused by a virus.

21. The method of claim 20 wherein the virus is selected from HIV, Hepatitis A, Hepatitis B, Hepatitis C, herpes viruses, and influenza.

22. A method of treating septic shock in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of claim 1 or a therapeutically acceptable salt thereof.

23. A method blocking the interaction of PD-L1 with PD-1 and/or CD80 in a subject, said method comprising administering to the subject a therapeutically effective amount of a compound of claim 1 or a therapeutically acceptable salt thereof.

Description

EXAMPLES

[0321] The abbreviations used in the present application, including particularly in the illustrative schemes and examples which follow, are well-known to those skilled in the art. Some of the abbreviations used are as follows: min or mins for minutes; h or hr or hrs for hours; RT or rt for room temperature or retention time (context will dictate); sat. for saturated; TFA for trifluoroacetic acid; DMF for N,N-dimethylformamide; DCM for dichloromethane; Fmoc for 9-fluorenylmethyloxycarbonyl; HATU for (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate); DIEA or DIPEA for diisopropylethylamine; NMP for N-methylpyrrolidone; EDC for 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; DMSO for dimethylsulfoxide; MeOH for methanol; EtOAc for ethyl acetate; Et.sub.3N for triethylamine; MeCN or ACN for acetonitrile; DIAD for diethyl azodicarboxylate; and TMSI for trimethylsilyl iodide.

[0322] Analytical Data:

[0323] Mass Spectrometry: “ESI-MS(+)” signifies electrospray ionization mass spectrometry performed in positive ion mode; “ESI-MS(−)” signifies electrospray ionization mass spectrometry performed in negative ion mode; “ESI-HRMS(+)” signifies high-resolution electrospray ionization mass spectrometry performed in positive ion mode; “ESI-HRMS(−)” signifies high-resolution electrospray ionization mass spectrometry performed in negative ion mode. The detected masses are reported following the “m/z” unit designation. Compounds with exact masses greater than 1000 were often detected as double-charged or triple-charged ions.

[0324] Analysis LCMS Condition A:

[0325] Column: BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: water with 0.05% TFA; Mobile Phase B:Acetonitrile with 0.05% TFA; Temperature: 50° C.; Gradient: 2% B to 98% B over 2 minutes, then a 0.5 minutes hold at 98% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

[0326] Analysis LCMS Condition B:

[0327] Column: BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.05% TFA; Mobile Phase B: 95:5 acetonitrile:water with 0.05% TFA; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.11 mL/min.

[0328] Analysis LCMS Condition C:

[0329] Column: BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: water with 0.2% Formic Acid and 0.01% TFA; Mobile Phase B: Acetonitrile with 0.2% Formic acid an 0.01% TFA; Temperature: 50° C.; Gradient: 2% B to 80% B over 2 minutes, 80% B to 98% B over 0.1 minute then a 0.5 minutes hold at 98% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

[0330] Analysis LCMS Condition D:

[0331] Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.11 mL/min; Detection: UV at 220 nm.

[0332] Analysis LCMS Condition E:

[0333] Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.11 mL/min; Detection: UV at 220 nm.

[0334] Analysis LCMS Condition F:

[0335] Column: Waters Xbridge C18, 2.1×50 mm; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 35° C.; Gradient: 0-100% B over 4 minutes, then a 1-minute hold at 100% B; Flow: 4 mL/min; Detection: UV at 220 nm.

[0336] Analysis LCMS Condition G:

[0337] Finnigan LTQ Mass Spectrometer; column: Phenomenex Jupiter C4, 1×50 mm; Mobile Phase A: 1% formic acid in water; Mobile Phase B: 0.1% formic acid in acetonitrile; Temperature: 30° C.; Gradient: 1% B, 1 min. hold; 1-95% B over 3 min., then a 3-min. hold at 95% B; Flow: 0.15 mL/min.

[0338] Analysis LCMS Condition H:

[0339] Column: Waters BEH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.5-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm.

[0340] Analysis LCMS Condition I:

[0341] Column: Waters BEH C18, 2.0×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 methanol:water with 10 mM ammonium

acetate; Mobile Phase B: 95:5 methanol:water with 10 mM ammonium acetate; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.5-minute hold at 100% B; Flow: 0.5 mL/min; Detection: UV at 220 nm.

[0342] Analysis HPLC Condition A:

[0343] Column: YMC Pack ODS-AQ 3 um 150×4.6 mm Mobile Phase A: water with 0.1% TFA; Mobile Phase B: Acetonitrile with 0.1% TFA; Temperature: 60° C.; Gradient: from 35% B to 80% B over 25 min.; Flow: 1 mL/min; Detection: UV at 217 nm.

[0344] Analysis HPLC Condition B:

[0345] Column: YMC Pack ODS-AQ 3 um 150×4.6 mm Mobile Phase A: water with 0.1% TFA; Mobile Phase B: Acetonitrile with 0.1% TFA; Temperature: 60° C.; Gradient: from 25% B to 75% B over 25 min.; Flow: 1 mL/min; Detection: UV at 217 nm.

[0346] Analysis HPLC Condition C:

[0347] Column: YMC Pack ODS-AQ 3 um 150×4.6 mm Mobile Phase A: water with 0.1% TFA; Mobile Phase B: Acetonitrile with 0.1% TFA; Temperature: 60° C.; Gradient: from 20% B to 70% B over 25 min.; Flow: 1 mL/min; Detection: UV at 217 nm.

[0348] Analysis HPLC Condition D:

[0349] Column: YMC Pack ODS-AQ 3 um 150×4.6 mm Mobile Phase A: water with 0.1% TFA; Mobile Phase B: Acetonitrile with 0.1% TFA; Temperature: 60° C.; Gradient: from 15% B to 65% B over 25 min.; Flow: 1 mL/min; Detection: UV at 217 nm.

[0350] Analysis HPLC Condition E:

[0351] Column: YMC Pack ODS-AQ 3 um 150×4.6 mm Mobile Phase A: water with 0.1% TFA; Mobile Phase B: Acetonitrile with 0.1% TFA; Temperature: 60° C.; Gradient: from 25% B to 60% B over 20 min.; Flow: 1.25 mL/min; Detection: UV at 217 nm.

[0352] Analysis HPLC Condition F:

[0353] Column: YMC Pack ODS-AQ 3 um 150×4.6 mm Mobile Phase A: water with 0.1% TFA; Mobile Phase B: Acetonitrile with 0.1% TFA; Temperature: 60° C.; Gradient: from 25% B to 65% B over 20 min.; Flow: 1.25 mL/min; Detection: UV at 217 nm.

[0354] Analysis HPLC Condition G:

[0355] Column: Sunfire C18 3.5 um, 3.0×150 mm; Mobile Phase A: 5:95 acetonitrile:water with 0.05% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.05% trifluoroacetic acid; temperature: 50° C.; Gradient: 10-100% B over 12 minutes, then a 3-minute hold at 100% B; Flow: 1 mL/min; Detection: UV at 220 nm.

[0356] Analysis HPLC Condition H:

[0357] Column: Xbridge Phenyl 3.5×150 um, Mobile Phase A: 5:95 acetonitrile:water with 0.05% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.05% trifluoroacetic acid; temperature: 50° C.; Gradient: 10-100% B over 12 minutes, then a 3-minute hold at 100% B; Flow: 1 mL/min; Detection: UV at 220 nm.

[0358] Analysis HPLC Condition I:

[0359] Column: Phenomenex Luna 5u C18(2) 150×4.6 mm; mobile Phase A: water with 0.1% triflouroacetic acid, mobile Phase B: acetonitrile with 0.1% triflouroactic acid, Gradient 5-100% B over 20 min, then a 5 minute hold at 100% B; Flow 1 mL/min, Detection: UV at 220.

[0360] Analysis HPLC Condition J:

[0361] Column: Phenomenex Luna 5u C18(2) 150×4.6 mm; mobile Phase A: water with 0.1% triflouroacetic acid, mobile Phase B: acetonitrile with 0.1% triflouroactic acid, Gradient 10-100% B over 20 min, then a 5 minute hold at 100% B; Flow 1 mL/min, Detection: UV at 220.

[0362] General Procedures:

[0363] Prelude Method A:

[0364] All manipulations were performed under automation on a Prelude peptide synthesizer (Protein Technologies). All procedures unless noted were performed in a 10 or 45 mL polypropylene tube fitted with a bottom frit. The tube connects to the Prelude peptide synthesizer through both the bottom and the top of the tube. DMF and DCM can be added through the top of the tube, which washes down the sides of the tube equally. The remaining reagents are added through the bottom of the tube and pass up through the frit to contact the resin. All solutions are removed through the bottom of the tube. “Periodic agitation” describes a brief pulse of N.sub.2 gas through the bottom frit; the pulse lasts approximately 5 seconds and occurs every 30 seconds. Amino acid solutions were generally not used beyond three weeks from preparation. DMF=dimethylformamide; DIC=N,N′-diisopropylcarbodiimide; HOAt=1-hydroxy 7-azabenzotriazole; Sieber=Fmoc-amino-xanthen-3-yloxy, where “3-yloxy” describes the position and type of connectivity to the polystyrene resin. The resin used is Merrifield polymer (polystyrene) with a Sieber linker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol/g loading. Common amino acids used are listed below with side-chain protecting groups indicated inside parenthesis. Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

[0365] The procedures of “Prelude Method A” describe an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. This scale corresponds to approximately 140 mg of the Sieber-Merrifield resin described above. All procedures can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin-swelling procedure, described below as “Resin-swelling procedure”. Coupling of amino acids to a primary amine N-terminus used the “Single-coupling procedure” described below.

[0366] Coupling of Fmoc-N-methyl amino acids and coupling to a secondary amine N-terminus used the “Secondary amine-coupling procedure” described below. Coupling of chloroacetyl group to the N-terminus of the peptide is described by the “Chloroacetyl chloride coupling procedure” or “Chloroacetic acid coupling procedure” detailed below.

[0367] Resin-Swelling Procedure:

[0368] To a 40 mL polypropylene solid-phase reaction vessel was added Merrifield:Sieber resin (140 mg, 0.100 mmol). The resin was washed three times as follows: to the reaction vessel was added DMF (5.0 mL) and DCM (5.0 mL), upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 10 minutes before the solvent was drained.

[0369] Single-Coupling Procedure:

[0370] To the reaction vessel containing resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperdine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 60 seconds before the solution was drained through the frit. To the reaction vessel was added a solution of the amino acid and HOAt (0.2M in DMF, 5.0 mL, 10 eq), then DIC (0.2M in DMF, 5.0 mL, 10 eq). The mixture was periodically agitated for 60 min, then the reaction solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically agitated for 10 minutes, then the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0371] Secondary Amine-Coupling Procedure:

[0372] To the reaction vessel containing resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperdine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 60 seconds before the solution was drained through the frit. To the reaction vessel was added a solution of the amino acid and HOAt (0.2M in DMF, 5.0 mL, 5 eq), then DIC (0.2M in DMF, 5.0 mL, 5 eq). The mixture was periodically agitated for 300 min, then the reaction solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically agitated for 10 minutes, then the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0373] Chloroacetyl Chloride Coupling Procedure:

[0374] To the reaction vessel containing the resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added 3.0 mL of a solution of DIPEA (4.0 mmol, 0.699 mL, 40 eq), and chloroacetyl chloride (2.0 mmol, 0.160 mL, 20 eq) in DMF. The mixture was periodically agitated for 12 to 18 hours, then the solution was drained. The resin was washed successively three times as follows: for each wash, DMF (4.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained. The resin was washed successively four times as follows: for each wash, DCM (4.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained.

[0375] Prelude Method B:

[0376] All manipulations were performed under automation on a Prelude peptide synthesizer (Protein Technologies). All procedures were performed in a 10 or 45 mL polypropylene tube fitted with a bottom frit. DMF and DCM can be added through the top of the tube, which washes down the sides of the tube equally. The remaining reagents are added through the bottom of the tube and pass up through the frit to contact the resin. All solutions are removed through the bottom of the tube. “Periodic agitation” describes a brief pulse of N.sub.2 gas through the bottom frit; the pulse lasts approximately 5 seconds and occurs every 30 seconds. Amino acid solutions were generally not used beyond three weeks from preparation. DMF=dimethylformamide; HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium; DIPEA=diisopropylethylamine; Sieber=Fmoc-amino-xanthen-3-yloxy, where “3-yloxy” describes the position and type of connectivity to the polystyrene resin. The resin used is Merrifield polymer (polystyrene) with a Sieber linker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol/g loading. Common amino acids used are listed below with side-chain protecting groups indicated inside parenthesis.

[0377] Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

[0378] The procedures of “Prelude Method B” describe an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. This scale corresponds to approximately 140 mg of the Sieber-Merrifield resin described above. All procedures can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin-swelling procedure, described below as “Resin-swelling procedure”. Coupling of amino acids to a primary amine N-terminus used the “Single-coupling procedure” described below. Coupling of amino acids to a secondary amine N-terminus used the “Secondary amine-coupling procedure” described below. Coupling of chloroacetyl group to the N-terminus of the peptide is described by the “Chloroacetyl chloride coupling procedure” or “Chloroacetic acid coupling procedure” detailed below.

[0379] Resin-Swelling Procedure:

[0380] To a 40 mL polypropylene solid-phase reaction vessel was added Merrifield:Sieber resin (140 mg, 0.100 mmol). The resin was washed (swelled) three times as follows: to the reaction vessel was added DMF (5.0 mL) and DCM (5.0 mL), upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 10 minutes before the solvent was drained through the frit.

[0381] Single-Coupling Procedure:

[0382] To the reaction vessel containing resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperdine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 60 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 5.0 mL, 10 eq), then HCTU (0.2M in DMF, 5.0 mL, 10 eq), and finally DIPEA (0.8M in DMF, 2.5 mL, 20 eq). The mixture was periodically agitated for 30 minutes, then the reaction solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically agitated for 10 minutes, then the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0383] Double-Coupling Procedure:

[0384] To the reaction vessel containing resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperdine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 60 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 5.0 mL, 10 eq), then HCTU (0.2M in DMF, 5.0 mL, 10 eq), and finally DIPEA (0.8M in DMF, 2.5 mL, 20 eq). The mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit. The resin was washed successively 3 times with DMF (4.0 mL) through the top of the vessel and the resulting mixture was periodically agitated for 60 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 5.0 mL, 10 eq), then HCTU (0.2M in DMF, 5.0 mL, 10 eq), and finally DIPEA (0.8M in DMF, 2.5 mL, 20 eq). The mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0385] Secondary Amine-Coupling Procedure:

[0386] To the reaction vessel containing resin from the previous step was added piperdine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperdine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 10 eq), then HCTU (0.2M in DMF, 2.5 mL, 10 eq), and finally NMM (0.8M in DMF, 1.5 mL, 12 eq). The mixture was periodically agitated for 12 hrs, then the reaction solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0387] Chloroacetyl Chloride Coupling Procedure A:

[0388] To the reaction vessel containing the resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added 3.0 mL of a solution of DIPEA (4.0 mmol, 0.699 mL, 40 eq), and chloroacetyl chloride (2.0 mmol, 0.160 mL, 20 eq) in DMF. The mixture was periodically agitated for 12 to 18 hours, then the solution was drained through the frit. The resin was washed successively three times as follows: for each wash, DMF (4.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, CH.sub.2Cl.sub.2 (2.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit.

[0389] Chloroacetic Acid Coupling Procedure B:

[0390] To the reaction vessel containing the resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added DMF (2.0 mL), chloroacetic acid (1.2 mmol, 113 mg, 12 eq), and N,N′-Diisopropylcarbodiimide (1.2 mmol, 0.187 mL, 12 eq). The mixture was periodically agitated for 12 to 18 hours, then the solution was drained through the frit. The resin was washed successively three times as follows: for each wash, DMF (4.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, CH.sub.2Cl.sub.2 (2.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit.

[0391] Prelude Method C:

[0392] All manipulations were performed under automation on a Prelude peptide synthesizer (Protein Technologies). All procedures unless noted were performed in a 10 or 45 mL polypropylene tube fitted with a bottom frit. The tube connects to a the Prelude peptide synthesizer through both the bottom and the top of the tube. DMF and DCM can be added through the top of the tube, which washes down the sides of the tube equally. The remaining reagents are added through the bottom of the tube and pass up through the frit to contact the resin. All solutions are removed through the bottom of the tube. “Periodic agitation” describes a brief pulse of N.sub.2 gas through the bottom frit; the pulse lasts approximately 5 seconds and occurs every 30 seconds. Amino acid solutions were generally not used beyond three weeks from preparation. HATU solution were used within 5 days of preparation. DMF=dimethylformamide; HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium; HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; DIPEA=diisopropylethylamine; Sieber=Fmoc-amino-xanthen-3-yloxy, where “3-yloxy” describes the position and type of connectivity to the polystyrene resin. The resin used is Merrifield polymer (polystyrene) with a Sieber linker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol/g loading. Common amino acids used are listed below with side-chain protecting groups indicated inside parenthesis.

[0393] Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

[0394] The procedures of “Prelude Method C” describe an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. This scale corresponds to approximately 140 mg of the Sieber-Merrifield resin described above. All procedures can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin-swelling procedure, described below as “Resin-swelling procedure”. Coupling of amino acids to a primary amine N-terminus used the “Single-coupling procedure” described below. Coupling of amino acids to a secondary amine N-terminus used the “Secondary amine-coupling procedure” described below. The Final wash of the Resin used the “Final Wash procedure” described below.

[0395] Resin-Swelling Procedure:

[0396] To a 40 mL polypropylene solid-phase reaction vessel was added Merrifield:Sieber resin (140 mg, 0.100 mmol). The resin was washed (swelled) three times as follows: to the reaction vessel was added DMF (5.0 mL) and DCM (5.0 mL), upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 10 minutes before the solvent was drained through the frit.

[0397] Single-Coupling Procedure:

[0398] To the reaction vessel containing resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperdine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 60 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 5.0 mL, 10 eq), then HATU (0.2M in DMF, 5.0 mL, 10 eq), and finally DIPEA (0.8M in DMF, 2.5 mL, 20 eq). The mixture was periodically agitated for 60 minutes, then the reaction solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically agitated for 10 minutes, then the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0399] Secondary Amine-Coupling Procedure:

[0400] To the reaction vessel containing resin from the previous step was added piperdine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperdine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 5 eq), then HATU (0.2M in DMF, 2.5 mL, 5 eq), and finally DIPEA (0.8M in DMF, 1.5 mL, 12 eq). The mixture was periodically agitated for 300 minutes, then the reaction solution was drained through the frit. The resin was twice washed as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 5 eq), then HATU (0.2M in DMF, 2.5 mL, 5 eq), and finally DIPEA (0.8M in DMF, 1.5 mL, 12 eq). The mixture was periodically agitated for 300 minutes, then the reaction solution was drained through the frit. The resin was twice washed as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically agitated for 10 minutes, then the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0401] Custom Amino Acids-Coupling Procedure:

[0402] To the reaction vessel containing resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperdine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 0.5 to 2.5 mL, 1 to 5 eq), then HATU (0.2M in DMF, 0.5 to 2.5 mL, 1 to 5 eq), and finally DIPEA (0.8M in DMF, 0.5 to 1.5 mL, 4 to 12 eq). The mixture was periodically agitated for 60 minutes to 600 minutes, then the reaction solution was drained through the frit. The resin was twice washed as follows: for each wash, DMF (2.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically agitated for 10 minutes, then the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0403] Final Wash Procedure:

[0404] The resin was washed successively two times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DCM (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0405] Chloroacetic Acid Coupling Procedure:

[0406] Note Manuel step. To the reaction vessel containing the resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was shaken at Room temperature for 5 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was agitated before the solution was drained through the frit. To the reaction vessel was added DMF (2.0 mL), chloroacetic acid (1.2 mmol, 113 mg, 12 eq), and N,N′-Diisopropylcarbodiimide (1.2 mmol, 0.187 mL, 12 eq). The mixture was shaken at room temperature for 12 to 18 hours, then the solution was drained through the frit. The resin was washed successively three times as follows: for each wash, DMF (4.0 mL) was added to top of the vessel and the resulting mixture was agitated for 90 seconds before the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, CH.sub.2Cl.sub.2 (4.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit.

[0407] Prelude Method D:

[0408] All manipulations were performed under automation on a Prelude peptide synthesizer (Protein Technologies). All procedures unless noted were performed in a 10 or 45 mL polypropylene tube fitted with a bottom frit. The tube connects to a the Prelude peptide synthesizer through both the bottom and the top of the tube. DMF and DCM can be added through the top of the tube, which washes down the sides of the tube equally. The remaining reagents are added through the bottom of the tube and pass up through the frit to contact the resin. All solutions are removed through the bottom of the tube. “Periodic agitation” describes a brief pulse of N.sub.2 gas through the bottom frit; the pulse lasts approximately 5 seconds and occurs every 30 seconds. Amino acid solutions were generally not used beyond three weeks from preparation. HATU solution were used within 5 days of preparation. DMF=dimethylformamide; HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium; HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; DIPEA=diisopropylethylamine; Sieber=Fmoc-amino-xanthen-3-yloxy, where “3-yloxy” describes the position and type of connectivity to the polystyrene resin. The resin used is Merrifield polymer (polystyrene) with a Sieber linker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol/g loading. Common amino acids used are listed below with side-chain protecting groups indicated inside parenthesis.

[0409] Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

[0410] The procedures of “Prelude Method D” describe an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. This scale corresponds to approximately 140 mg of the Sieber-Merrifield resin described above. All procedures can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin-swelling procedure, described below as “Resin-swelling procedure”. Coupling of amino acids to a primary amine N-terminus used the “Single-coupling procedure” described below. Coupling of amino acids to a secondary amine N-terminus used the “Secondary amine-coupling procedure” described below. The Final wash of the Resin used the “Final Wash procedure” described below.

[0411] Resin-Swelling Procedure:

[0412] To a 40 mL polypropylene solid-phase reaction vessel was added Merrifield:Sieber resin (140 mg, 0.100 mmol). The resin was washed (swelled) three times as follows: to the reaction vessel was added DMF (5.0 mL) and DCM (5.0 mL), upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 10 minutes before the solvent was drained through the frit.

[0413] Single-Coupling Procedure:

[0414] To the reaction vessel containing resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperdine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 60 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25 mL, 2.5 eq), then HATU (0.2M in DMF, 1.25 mL, 2.5 eq), and finally DIPEA (0.8M in DMF, 0.75 mL, 5 eq). The mixture was periodically agitated for 30 minutes, then the reaction solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically agitated for 15 minutes, then the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0415] Secondary Amine-Coupling Procedure:

[0416] To the reaction vessel containing resin from the previous step was added piperdine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperdine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25 mL, 2.5 eq), then HATU (0.2M in DMF, 1.25 mL, 2.5 eq), and finally DIPEA (0.8M in DMF, 0.75 mL, 5 eq). The mixture was periodically agitated for 30 minutes, then the reaction solution was drained through the frit. The resin was twice washed as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino (0.2M in DMF, 1.25 mL, 2.5 eq), then HATU (0.2M in DMF, 1.25 mL, 2.5 eq), and finally DIPEA (0.8M in DMF, 0.75 mL, 5 eq). The mixture was periodically agitated for 30 minutes, then the reaction solution was drained through the frit. The resin was twice washed as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically agitated for 15 minutes, then the solution was drained through the frit. The resin was twice washed as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically agitated for 15 minutes, then the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0417] Final Wash Procedure:

[0418] The resin was washed successively two times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DCM (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0419] Chloroacetic Acid Coupling Procedure:

[0420] Note Manuel step. To the reaction vessel containing the resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was shaken at Room temperature for 5 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was agitated before the solution was drained through the frit. To the reaction vessel was added DMF (2.0 mL), chloroacetic acid (1.2 mmol, 113 mg, 12 eq), and N,N′-Diisopropylcarbodiimide (1.2 mmol, 0.187 mL, 12 eq). The mixture was shaken at room temperature for 12 to 18 hours, then the solution was drained through the frit. The resin was washed successively three times as follows: for each wash, DMF (4.0 mL) was added to top of the vessel and the resulting mixture was agitated for 90 seconds before the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, CH.sub.2Cl.sub.2 (4.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit.

[0421] CEM Method A:

[0422] All manipulations were performed under automation on a CEM Liberty microwave peptide synthesizer (CEM Corporation). All procedures unless noted were performed in a 30 or 125 mL polypropylene tube fitted with a bottom frit to a CEM Discovery microwave unit. The tube connects to a the CEM Liberty synthesizer through both the bottom and the top of the tube. DMF and DCM can be added through the top and bottom of the tube, which washes down the sides of the tube equally. All solutions are removed through the bottom of the tube except while transferring resin from the top. “Periodic bubbling” describes a brief bubbling of N.sub.2 gas through the bottom frit. Amino acid solutions were generally not used beyond three weeks from preparation. HATU solution were used within 5 days of preparation. DMF=dimethylformamide; HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium; HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; DIPEA=diisopropylethylamine; Sieber=Fmoc-amino-xanthen-3-yloxy, where “3-yloxy” describes the position and type of connectivity to the polystyrene resin. The resin used is Merrifield polymer (polystyrene) with a Sieber linker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol/g loading. Other Common resins Such as Rink, ChloroTrityl, or other acid sensitive linkers can be employed in the synthesis, Seiber amide resin is used unless otherwise noted in specific examples. Common amino acids used are listed below with side-chain protecting groups indicated inside parenthesis.

[0423] Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Orn(Boc)-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

[0424] The procedures of “CEM Method A” describe an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. This scale corresponds to approximately 140 mg of the Sieber-Merrifield resin described above. All procedures can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin-swelling procedure, described below as “Resin-swelling procedure”. Coupling of amino acids to a primary amine N-terminus used the “Single-coupling procedure” described below. Coupling of amino acids to a secondary amine N-terminus used the “Secondary amine-coupling procedure” described below. Coupling of chloroacetyl group to the N-terminus of the peptide is described by the “Chloroacetyl chloride coupling procedure” or “Chloroacetic acid coupling procedure” detailed above.

[0425] Resin-Swelling Procedure:

[0426] To 50 mL polypropylene conical tube was added Merrifield:Sieber resin (140 mg, 0.100 mmol). Then DMF (7 mL) was added to the tube followed by DCM (7 mL). The resin was then transferred to the reaction vessel from top of the vessel. The procedure is repeated additionally two times. DMF (7 mL) was added followed by DCM (7 mL). The resin was allowed to swell with N.sub.2 bubbling from the bottom of the reaction vessel for 15 minutes before the solvent was drained through the frit.

[0427] Standard Coupling Procedure:

[0428] To the reaction vessel containing resin from the previous step was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5 eq), and DIPEA (2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N.sub.2 bubbling for 5 minutes at 75° C. for all amino acids, except Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH which are coupled at 50° C., the reaction solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically bubbled for 2 minutes at 65° C., then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. The resulting resin was used directly in the next step.

[0429] Double-Couple Coupling Procedure:

[0430] To the reaction vessel containing resin from the previous step was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5 eq), and DIPEA (2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N.sub.2 bubbling for 5 minutes at 75° C. for all amino acids, except Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH which are coupled at 50° C., the reaction solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5 eq), and DIPEA (2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N.sub.2 bubbling for 5 minutes at 75° C. for all amino acids, except Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH which are coupled at 50° C., the reaction solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically bubbled for 2 minutes at 65° C., then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. The resulting resin was used directly in the next step.

[0431] Secondary Amine Coupling Procedure:

[0432] To the reaction vessel containing resin from the previous step was added a solution of 5% piperazine and 0.1 M HOBt in DMF (7 mL). The mixture was periodically agitated for 3 minutes at 75° C. and then the solution was drained. This procedure was repeated one more time. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 5 eq), HCTU (0.5M in DMF, 1.0 mL, 5 eq), and DIPEA (2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N.sub.2 bubbling for 5 minutes at 75° C. for all amino acids (50° C. for Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH), followed by 6 hrs with no heating. After draining, the resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically bubbled for 2 minutes at 65° C., then the solution was drained. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. The resulting resin was used directly in the next step.

[0433] Custom Amino Acids-Coupling Procedure:

[0434] To the reaction vessel containing resin from the previous step was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added the amino acid solution (1.25 mL to 5 mL, 2.5 eq to 10 eq) containing HATU (2.5 eq to 10 eq), and finally DIPEA (2M in NMP, 0.5 mL to 1 mL, 20 eq). The mixture was mixed by N.sub.2 bubbling for 5 minutes to 2 hours at 25° C. to 75° C., then the reaction solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically bubbled for 2 minutes at 65° C., then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. The resulting resin was used directly in the next step.

[0435] Symphony Method A:

[0436] All manipulations were performed under automation on a Symphony peptide synthesizer (Protein Technologies). All procedures unless noted were performed in a Symphony polypropylene tube fitted with a bottom frit. The tube connects to a the Symphony peptide synthesizer through both the bottom and the top of the tube. All Solvents, DMF, DCM, amino acids and reagents are added through the bottom of the tube and pass up through the frit to contact the resin. All solutions are removed through the bottom of the tube. “Periodic agitation” describes a brief pulse of N.sub.2 gas through the bottom frit; the pulse lasts approximately 5 seconds and occurs every 15 seconds. Amino acid solutions were generally not used beyond three weeks from preparation. HATU solution were used within 5 days of preparation. DMF=dimethylformamide; HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium; HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; NMM=n-Methyl morpholine; DIPEA=diisopropylethylamine; Sieber=Fmoc-amino-xanthen-3-yloxy, where “3-yloxy” describes the position and type of connectivity to the polystyrene resin. The resin used is Merrifield polymer (polystyrene) with a Sieber linker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol/g loading. Other common Acid sensitive resins can also be used in the synthesis such as Rink or functionalized Chloro trityl Resin. Common amino acids used are listed below with side-chain protecting groups indicated inside parenthesis.

[0437] Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

[0438] The procedures of “Symphony Method A” describes an experiment performed on a 0.050 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. This scale corresponds to approximately 70 mg of the Sieber-Merrifield resin described above. All procedures can be scaled beyond 0.050 mmol scale by adjusting the described volumes by the multiple of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin-swelling procedure, described below as “Swelling procedure”. Coupling of amino acids to a primary amine N-terminus used the “Standard-coupling procedure” described below. Coupling of amino acids to a secondary amine N-terminus used the “Double-coupling”, custom amino acids are coupled via a manual Blank addition of the amino acid “Blank coupling” described below.

[0439] Swelling Procedure:

[0440] To a Symphony polypropylene solid-phase reaction vessel was added Merrifield: Sieber resin (70 mg, 0.050 mmol). The resin was washed (swelled) three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 10 minutes before the solvent was drained through the frit. To the reaction vessel was added piperdine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25 mL, 5 eq), then HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed with DMF (6.25 mL) added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25 mL, 5 eq), then HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. The resulting resin was used directly in the next step.

[0441] Standard-Coupling Procedure:

[0442] The resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit. The resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25 mL, 5 eq), then HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed with DMF (6.25 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25 mL, 5 eq), then HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed successively three times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0443] Secondary Amine-Coupling Procedure:

[0444] The resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit. The resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25 mL, 5 eq), then HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 300 minutes, then the reaction solution was drained through the frit. The resin was washed with DMF (6.25 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25 mL, 5 eq), then HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 300 minutes, then the reaction solution was drained through the frit. The resin was washed successively three times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0445] Custom Amino Acids-Coupling Procedure:

[0446] The resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit. The resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The synthesis was paused by the Symphony software to add to the reaction vessel manually the custom amino acid (0.2M in DMF, 1.25 mL, 5 eq), then restart automation: to add HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 300 minutes, then the reaction solution was drained through the frit. The resin was washed six times as follows with DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the Ac.sub.2O/DIPEA/DMF (v/v/v 1:1:3 2.5 mL) the mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed successively three times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0447] Symphony Method B:

[0448] All manipulations were performed under automation on a Symphony peptide synthesizer (Protein Technologies). All procedures unless noted were performed in a Symphony polypropylene tube fitted with a bottom frit. The tube connects to a the Symphony peptide synthesizer through both the bottom and the top of the tube. All Solvents, DMF, DCM, amino acids and reagents are added through the bottom of the tube and pass up through the frit to contact the resin. All solutions are removed through the bottom of the tube. “Periodic agitation” describes a brief pulse of N2 gas through the bottom frit; the pulse lasts approximately 5 seconds and occurs every 15 seconds. Amino acid solutions were generally not used beyond three weeks from preparation. HATU solution were used within 5 days of preparation. DMF=dimethylformamide; HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium; HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; NMM=n-Methyl morpholine; DIPEA=diisopropylethylamine; Sieber=Fmoc-amino-xanthen-3-yloxy, where “3-yloxy” describes the position and type of connectivity to the polystyrene resin. The resin used is Merrifield polymer (polystyrene) with a Sieber linker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol/g loading. Other common Acid sensitive resins can also be used in the synthesis such as Rink or functionalized Chloro trityl Resin. Common amino acids used are listed below with side-chain protecting groups indicated inside parenthesis.

[0449] Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

[0450] The procedures of “Symphony Method B” describes an experiment performed on a 0.050 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. This scale corresponds to approximately 70 mg of the Sieber-Merrifield resin described above. All procedures can be scaled beyond 0.050 mmol scale by adjusting the described volumes by the multiple of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin-swelling procedure, described below as “Swelling procedure”. Coupling of amino acids to a primary amine N-terminus used the “Standard-coupling procedure” described below. Coupling of amino acids to a secondary amine N-terminus used the “Secondary amine-coupling procedure B”, Custom amino acids are coupled via a manual Blank addition of the amino acid “Custom amino acids-coupling procedure” described below, and ChloroAcetyl Anhydride is added to the final position of the sequence using the “final capping procedure” described below.

[0451] Swelling Procedure:

[0452] To a Symphony polypropylene solid-phase reaction vessel was added Merrifield:Sieber resin (70 mg, 0.050 mmol). The resin was washed (swelled) three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 10 minutes before the solvent was drained through the frit. To the reaction vessel was added piperdine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25 mL, 5 eq), then HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed with DMF (6.25 mL) added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25 mL, 5 eq), then HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. The resulting resin was used directly in the next step.

[0453] Standard-Coupling Procedure:

[0454] The resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit. The resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25 mL, 5 eq), then HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit. The resin was washed 6 times as follows: DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added Ac2O/DIPEA/DMF (v/v/v 1:1:3 2.5 mL) the mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0455] Secondary Amine-Coupling Procedure:

[0456] The resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit. The resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25 mL, 5 eq), then HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit. The resin was washed with DMF (6.25 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (0.2M in DMF, 1.25 mL, 5 eq), then HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit. The resin was washed successively three times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added Ac2O/DIPEA/DMF (v/v/v 1:1:3 2.5 mL) the mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0457] Custom Amino Acids-Coupling Procedure:

[0458] The resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit. The resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The System was paused by the system for the manual addition of the custom amino acid to the reaction vessel (0.2M in DMF, 1.25 mL, 5 eq), then the automation was restarted to add to the reaction vesicle HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit. The resin was washed 6 times as follows: DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added Ac2O/DIPEA/DMF (v/v/v 1:1:3 2.5 mL) the mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resulting resin was used directly in the next step.

[0459] Final Capping Procedure:

[0460] The resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N.sub.2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit. The resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added NMM (0.8M in DMF, 1.25 mL, 10 eq) followed by the addition of the Chloroacetic anhydride (0.4M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit. The resin was washed with DMF (6.25 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added NMM (0.8M in DMF, 1.25 mL, 10 eq) followed by the addition of the Chloroacetic anhydride (0.4M in DMF, 1.25 mL, 10 eq). The mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit. The resin was washed 6 times as follows: DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added Ac2O/DIPEA/DMF (v/v/v 1:1:3 2.5 mL) the mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit. The resin was washed successively six times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DCM (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. The resulting resin was then dried with a stream of Nitrogen for 10 mins.

[0461] Global Deprotection Method A:

[0462] All manipulations were performed manually unless noted otherwise. The procedure of “Global Deprotection Method A” describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. A “deprotection solution” was prepared using trifluoroacetic acid: water:triisopropylsilane:dithiothreitol (92.5:2.5:2.5:2.5 v:v:v:w). The resin was removed from the reaction vessel and transferred to a 25 mL syringe equipped with a frit. To the syringe was added the “deprotection solution” (5.0 mL). The mixture was mixed in a shaker for 85 min. The solution was filtered through, concentrated and diluted in diethyl ether (30 mL). The precipitated solid was centrifuged for 3 minutes. The supernatant solution was decanted and the solid was resuspended diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was suspended diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was dried under high vacuum. The crude peptide was obtained as a white to off-white solid.

[0463] Global Deprotection Method B:

[0464] All manipulations were performed manually unless noted otherwise. The procedure of “Global Deprotection Method B” describes an experiment performed on a 0.04 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. The procedure can be scaled beyond 0.04 mmol scale by adjusting the described volumes by the multiple of the scale. A “deprotection solution” was prepared using trifluoroacetic acid:triisopropylsilane (96:4; v:v). The resin was removed from the reaction vessel and transferred to a 10 mL syringe equipped with a frit. To the syringe was added the “deprotection solution” (2.0-3.0 mL). The mixture was mixed in a shaker for 1 h or 1.5 h. The solution was filtered through, washed with deprotection solution (0.5 mL), concentrated and diluted in diethyl ether (30 mL). The precipitated solid was centrifuged for 3 minutes. The supernatant solution was decanted and the solid was resuspended diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was suspended diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was dried under high vacuum. The crude peptide was obtained as a white to off-white solid.

[0465] Global Deprotection Method C:

[0466] All manipulations were performed manually unless noted. The procedure of “Global Deprotection Method C” describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. A “deprotection solution” was prepared using trifluoroacetic acid:triisopropylsilane:dithiothreitol (95:2.5:2.5 v:v:w). The resin was removed from the reaction vessel and transferred to a Bio-Rad tube. To the Bio-Rad tube was added the “deprotection solution” (4.0 mL). The mixture was mixed in a shaker for 60 minutes. The solution was filtered through and diluted in diethyl ether (30 mL). The precipitated solid was centrifuged for 3 minutes. The supernatant solution was decanted and the solid was resuspended diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was suspended diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was dried under high vacuum. The crude peptide was obtained as a white to off-white solid.

[0467] Global Deprotection Method D:

[0468] All manipulations were performed manually unless noted otherwise. The procedure of “Global Deprotection Method B” describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. A “deprotection solution” was prepared using trifluoroacetic acid:triisopropylsilane:dithiothreitol (94:3:3 v:v:w). The resin was removed from the reaction vessel and transferred to a 25 mL syringe equipped with a frit. To the syringe was added the “deprotection solution” (5.0 mL). The mixture was mixed in a shaker for 5 minutes. The solution was filtered through and diluted in diethyl ether (30 mL). The precipitated solid was centrifuged for 3 minutes. The supernatant solution was decanted and the solid was resuspended diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was suspended diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was dried under high vacuum. The crude peptide was obtained as a white to off-white solid.

[0469] Global Deprotection Method E:

[0470] All manipulations were performed manually unless noted. The procedure of “Global Deprotection Method E” describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Fmoc Gly-ClTrt linker bound to the resin. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. A “deprotection solution” was prepared using trifluoroacetic acid:triisopropylsilane:dithiothreitol (95:2.5:2.5 v:v:w). The resin was removed from the reaction vessel and transferred to a Bio-Rad tube. To the Bio-Rad tube was added the “deprotection solution” (2.0 mL). The mixture was mixed in a shaker for 3 minutes. The solution was filtered, and collected in a Centrifuge tube. To the Bio-Rad tube was added the “deprotection solution” (2.0 mL). The mixture was mixed in a shaker for 3 minutes. The solution was filtered, and collected in a Centrifuge tube. To the Bio-Rad tube was added the “deprotection solution” (2.0 mL). The mixture was mixed in a shaker for 3 minutes. The solution was filtered, and collected in a Centrifuge tube. The solution in the Centrifuge tube was allowed to stand for 60 minutes. The collected solution was then diluted with diethyl ether (30 mL), and precipitate formed. The precipitated solid was centrifuged for 3 minutes. The supernatant solution was decanted and the solid was resuspended diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was suspended diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was dried under high vacuum. The crude peptide was obtained as a white to off-white solid.

[0471] Global Deprotection Method F:

[0472] All manipulations were performed manually unless noted. The procedure of “Global Deprotection Method F” describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Rink linker bound to the resin. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. A “deprotection solution” was prepared using trifluoroacetic acid:triisopropylsilane:dithiothreitol (95:2.5:2.5 v:v:w). The resin was removed from the reaction vessel and transferred to a 6 mls Bio-Rad tube. To the Bio-Rad was added the “deprotection solution” (4.0 mL). The mixture was mixed in a shaker for 90 minutes. The solution was filtered through and diluted in diethyl ether (30 mL). The precipitated solid was centrifuged for 3 minutes. The supernatant solution was decanted and the solid was resuspended diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was suspended diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was dried under high vacuum. The crude peptide was obtained as a white to off-white solid.

[0473] Cyclization Method A

[0474] All manipulations were performed manually unless noted otherwise. The procedure of “Cyclization Method A” describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin that was used to generate the peptide. This scale is not based on a direct determination of the quantity of peptide used in the procedure. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. The crude peptide solids were dissolved in a solution of acetonitrile:aqueous 8M Guanidine/50 mM TRIS (1:3) (pH 8.6) (7 mL:18 mL or similar ratio), and the solution was then adjusted to pH=8.5-9.0 using aq NaOH (1.0M), if necessary. The solution was then mixed using a shaker for 12 to 18 hours. The reaction solution was concentrated and the residue was then dissolved in acetonitrile:water. This solution was subjected to reverse-phase HPLC purification to afford the desired cyclic peptide.

[0475] Cyclization Method C:

[0476] All manipulations were performed manually unless noted. The procedure of “Cyclization Method C” describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin that was used to generate the peptide. This scale is not based on a direct determination of the quantity of peptide used in the procedure. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. The crude peptide solids were dissolved in a solution of acetonitrile:aqueous 0.1M ammonium bicarbonate buffer (11 mL:24 mL or similar ratio), and the solution was then carefully adjusted to pH=8.5-9.0 using aq NaOH (1.0M). The solution was then mixed using a shaker for 12 to 18 hours. The reaction solution was concentrated and the residue was then dissolved in acetonitrile:water. This solution was subjected to reverse-phase HPLC purification to afford the desired cyclic peptide.

[0477] Cyclization Method D:

[0478] All manipulations were performed manually unless noted. The procedure of “Cyclization Method D” describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin that was used to generate the peptide. This scale is not based on a direct determination of the quantity of peptide used in the procedure. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. The crude peptide solids were dissolved in a solution of acetonitrile:aqueous 0.1M ammonium bicarbonate buffer (11 mL:24 mL), and the solution was then carefully adjusted to pH=8.5-9.0 using aq NaOH (1.0M). The solution was then mixed with stirring for 12 to 18 hours. The reaction solution was concentrated and the residue was then dissolved in acetonitrile:water. This solution was subjected to reverse-phase HPLC purification to afford the desired cyclic peptide.

[0479] Cyclization Method E:

[0480] All manipulations were performed manually unless noted. The procedure of “Cyclization Method E” describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin that was used to generate the peptide. This scale is not based on a direct determination of the quantity of peptide used in the procedure. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. The crude peptide solids were dissolved in a solution of aqueous 6M guanidine HCl buffer (15 mL), the solution was then mixed with stirring for 12 to 18 hours. The reaction solution was concentrated and 15 mL of DMSO was added to the residue affording a slurry which was filtered. This filtered solution was subjected to reverse-phase HPLC purification to afford the desired cyclic peptide.

[0481] Manual Coupling Procedure A:

[0482] To Bio-Rad reaction vessel containing resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically shaken for 5 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was shaken for 60 seconds before the solution was drained through the frit. To the reaction vessel was added the amino acid (1.2-10 equivalents) typical (0.2M in DMF, 2.5 mL, 5 eq), then HATU (1.210 equivalents) typical (0.2M in DMF, 2.5 mL, 5 eq), and finally DIPEA (2.4-20 equivalents) typical (0.8M in DMF, 1.25 mL, 10 eq). The mixture was shaken for 60 minutes to 18 hours, then the reaction solution was drained through the frit. The resin was washed successively four times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was shaken for 60 seconds before the solution was drained through the frit.

[0483] N-Methylation On-Resin Method A.

[0484] (Turner, R. A.; Hauksson, N. E.; Gipe, J. H.; Lokey, R. S. Org. Lett. 2013, 15(19), 5012-5015):

[0485] All manipulations were performed manually unless noted. The procedure of “N-methylation on-resin Method A” describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin that was used to generate the peptide. This scale is not based on a direct determination of the quantity of peptide used in the procedure. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale.

[0486] The resin was transferred into a 25 mL fritted syringe. To the resin was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was shaken for 3 min. and then the solution was drained through the frit. The resin was washed 3 times with DMF (4.0 mL). To the reaction vessel was added piperdine:DMF (20:80 v/v, 4.0 mL). The mixture was shaken for 3 min. and then the solution was drained through the frit. The resin was washed successively three times with DMF (4.0 mL) and three times with DCM (4.0 mL). The resin was suspended in DMF (2.0 mL) and ETHYL TRIFLUOROACETATE (0.119 ml, 1.00 mmol), 1,8-DIAZABICYCLO[5.4.0]UNDEC-7-ENE (0.181 ml, 1.20 mmol). The mixture was placed on a shaker for 60 min. The solution was drained through the frit. The resin was washed successively three times with DMF (4.0 mL) and three times with DCM (4.0 mL).

[0487] The resin was washed three times with dry THF (2.0 mL) to remove any residual water. In an oven dried 4.0 mL vial was added THF (1.0 mL) and TRIPHENYLPHOSPHINE (131 mg, 0.500 mmol) on dry 4 Å molecular sieves (20 mg). The solution was transferred to the resin and diisopropyl azodicarboxylate (0.097 mL, 0.5 mmol) was added slowly. The resin was stirred for 15 min. The solution was drained through the frit and the resin was washed with three times with dry THF (2.0 mL) to remove any residual water. In an oven dried 4.0 mL vial was added THF (1.0 mL), TRIPHENYLPHOSPHINE (131 mg, 0.500 mmol) on dry 4 Å molecular sieves (20 mg). The solution was transferred to the resin and diisopropyl azodicarboxylate (0.097 mL, 0.5 mmol) was added slowly. The resin was stirred for 15 min. The solution was drained through the frit. The resin was washed successively three times with DMF (4.0 mL) and three times with DCM (4.0 mL). The resin was suspended in Ethanol (1.0 mL) and THF (1.0 mL), and SODIUM BOROHYDRIDE (37.8 mg, 1.000 mmol) was added. The mixture was stirred for 30 min. and drained. The resin was washed successively three times with DMF (4.0 mL) and three times with DCM (4.0 mL).

[0488] Microcleavage A:

[0489] To a small <10 mg sample of resin is added 2 drops of TIS and 1 mL of Triflouroacetic acid, shake at rt. After 1 h, remove a small aliquot and dilute with 0.5 mL Acetonitrile, filter, and obtain analysis by LC-MS.

(S)-methyl 2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)propanoate

[0490] ##STR00013##

[0491] (S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanoic acid (500 mg, 1.670 mmol) and HATU (699 mg, 1.837 mmol) were dissolved into CH.sub.2Cl.sub.2 (10 mL) and DMF (4 mL). The mixtures were allowed to stir at RT for 10 mins before (S)-methyl 2-aminopropanoate, HCl (245 mg, 1.754 mmol) and Hunig'sBase (1.167 mL, 6.68 mmol) in 1 mL of DMF was added. The mixtures were allowed to stir at RT for 2 hours at which time LC-MS showed completed conversion. Quench with 3 ml 1N HCl. Poured into 150 mL of EA, washed with 10% LiCl twice. The organic layer was then concentrated. The crude materials were purified on 50 g GOLDC18 column. 10%-100% SolB gradient in 22 mins. SolA: Water/TFA; SolB: ACN/TFA

[0492] Fractions containing the desired product were combined and dried via centrifugal evaporation to provide the title compound (600 mg, 86%).

[0493] Analysis LCMS Condition A: Retention time=0.88 min; ESI-MS(+) m/z 385.1 (M+H).

(S)-methyl 2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-phenylethyl)-1H-tetrazol-1-yl)propanoate

[0494] ##STR00014##

[0495] A pressure vial was charged with (S)-methyl 2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)propanoate (600 mg, 1.561 mmol) and Diphenyl-2-pyridylphosphine (1644 mg, 6.24 mmol) in THF (20 ml) under N.sub.2. Purged the solution with N.sub.2 before DIAD (1.214 ml, 6.24 mmol) was added dropwise. Diphenylphosphoryl azide (1.682 ml, 7.80 mmol) was then added slowly. The mixtures were heated to 50° C. and allowed to stir at 50° C. for 14 hours at which time LC-MS showed desired product peak. Cooled down to RT and the mixtures were poured into 80 mL EtOAc, washed with cold 1N HCl, sat. NaHCO.sub.3 and brine. The organic layer was then concentrated and the obtained material was purified by RediSep Column: Silica 80 g Gold; Flow Rate: 60 ml/min; gradient 0%-45% SolB in 15 mins; Solvent: A1 hexane; Solvent: B1 ethyl acetate.

[0496] Fractions containing the desired product were combined and dried via centrifugal evaporation to provide the title compound (290 mg)

[0497] Analysis LCMS Condition A: Retention time=1.0 min; ESI-MS(+) m/z 410.1 (M+H).

(S)-2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-phenylethyl)-1H-tetrazol-1-yl)propanoic acid

[0498] ##STR00015##

[0499] (S)-methyl 2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-phenylethyl)-1H-tetrazol-1-yl)propanoate (290 mg, 0.425 mmol) were dissolved into THF (4 mL) and MeOH (1 mL) cosolvent. To this solution was added LiOH (1.275 mL, 2.55 mmol) in two portions. LC-MS shows completed conversion after stirring at RT for 2 hours. Quenched the reaction with 1N HCl, adjusted pH to 1-2. Extracted with EtOAc twice. The organic layer was then dried over Na.sub.2SO.sub.4 and concentrated to provide 285 mg crude materials. No purification was performed.

[0500] Analysis LCMS Condition A: Retention time=0.83 min; ESI-MS(+) m/z 396.3 (M+H).

(S)-2-(5-((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-phenylethyl)-1H-tetrazol-1-yl)propanoic acid

[0501] ##STR00016##

[0502] (S)-2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-phenylethyl)-1H-tetrazol-1-yl)propanoic acid (285 mg, 0.721 mmol) was dissolved into MeOH (4 ml) under N.sub.2. Purged the system with N.sub.2 before Pd/C (77 mg, 0.072 mmol) was added. The mixtures were allowed to stir at RT under H.sub.2 balloon for overnight. Filtered out catalyst using a plug of diatomaceous earth (Celite®), washed with methanol and the solvent was removed.

[0503] The above residues were dissolved into acetonitrile (4 mL) and Water (2 mL). Cooled down to 0° C. and to the solution was added Na.sub.2CO.sub.3 (1.802 ml, 3.60 mmol) followed by (9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (267 mg, 0.793 mmol) in 1 mL CH.sub.3CN. The reaction mixtures were allowed to warm to RT in 2 hours. Acidified with 1N HCl to pH2 and extracted with EtOAc. The organic layer was then dried over Na.sub.2SO.sub.4 and concentrated. The obtained crude materials were purified on reverse phase HPLC. Column: Axia Luna C18 30*100 mm; SolA: 95% H.sub.2O/5% ACN/0.05% TFA; SolB: 95% ACN/5% H.sub.2o/0.05% TFA; gradient 10%-100% SolB in 12 mins.

[0504] Fractions containing the desired product were combined and dried via centrifugal evaporation to provide the title compound (90 mg).

[0505] Analysis LCMS Condition A: Retention time=0.97 min; ESI-MS(+) m/z 484.0 (M+H).

(R)-methyl 2-((S)-3-(benzo[b]thiophen-3-yl)-2-(((benzyloxy)carbonyl)amino)propanamido)hexanoate

[0506] ##STR00017##

[0507] To a solution of (S)-2-(((benzyloxy)carbonyl)amino)-3-(4-(tert-butoxy)phenyl)propanoic acid (2.24 g, 6.03 mmol) and (S)-methyl 2-aminopropanoate hydrochloride (0.850 g, 6.09 mmol) in DCM (30.2 ml) at 0° C. was added solid HATU (2.408 g, 6.33 mmol) after 15 mins added N-methylmorpholine (1.326 ml, 12.06 mmol), and allowed to warm to RT overnight. Added 1 N HCl 20 mls and stirred 10 min. Poured into separatory funnel, separated layers, and extracted aqueous phase 2× additional with DCM (30 ml). Combined organic layers dried over Na.sub.2SO.sub.4, filtered and stripped to oil.

[0508] Purified Material on Silica Gel using Hexanes to Ethyl acetate 0 to 100% on Isco 80 G column produces 1.91 grams.

[0509] Analysis LCMS Condition E: retention time 1.05 min 483.4 (M+1); .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 8.46 (1H, d, J=7.46 Hz), 7.96 (2H, t, J=8.62 Hz), 7.57 (1H, d, J=8.68 Hz), 7.19-7.49 (6H, m), 4.95 (m, 2H) 4.38-4.57 (1H, m), 4.28 (1H, td, J=8.01, 5.50 Hz), 3.53-3.76 (3H, s), 3.32 (2H, s), 3.12-3.24 (1H, m), 3.06 (1H, dd, J=14.67, 10.15 Hz), 1.50-1.80 (2H, m), 1.29 (3H, br. s.), 0.75-0.98 (3H, m).

(2R)-methyl 2-(5-((S)-2-(benzo[b]thiophen-3-yl)-1-(((benzyloxy)carbonyl)amino)ethyl)-4H-1,2,3-triazol-4-yl)hexanoate

[0510] ##STR00018##

[0511] A single neck round bottom flask equipped with a nitrogen line and a magnetic stirrer was charged with (S)-methyl 2-((S)-3-(benzo[b]thiophen-3-yl)-2-(((benzyloxy)carbonyl)amino)propanamido)hexanoate (1.91 g, 3.96 mmol) and Diphenyl-2-pyridylphosphine (3.13 g, 11.87 mmol) then THF (25 ml) under N.sub.2. Purged the solution with N.sub.2 for 5 mins before DIAD (2.309 ml, 11.87 mmol) was added dropwise. Diphenylphosphoryl azide (3.41 ml, 15.83 mmol) was then added via an addition funnel. Reaction stirred at RT under a nitrogen atmosphere for 18 hours. Removed THF to thick oil, dissolved thick oil in ethyl acetate 100 mL and added 100 mL of 1N HCl. Extracted aqueous phase 2 additional times with 50 mL ethyl acetate. Combined organic dried over Na.sub.2SO.sub.4 filtered and stripped to an oil.

[0512] ISCO 220 gram Silica; Hexanes to Ethyl acetate gradient; isolated desired material in 0.556 grams. Material was taken on crude as a mixture.

[0513] Analysis LCMS Condition E: retention time 1.1 min 508.4 (M+1).

(S)-2-(5-((S)-2-(benzo[b]thiophen-3-yl)-1-(((benzyloxy)carbonyl)amino)ethyl)-1H-tetrazol-1-yl)hexanoic acid

[0514] ##STR00019##

[0515] To (S)-methyl 2-(5-((S)-2-(benzo[b]thiophen-3-yl)-1-(((benzyloxy)carbonyl)amino)ethyl)-1H-tetrazol-1-yl)hexanoate (0.566 g, 1.115 mmol) was added MeOH (2.79 ml), and THF (2.79 ml). Mixture stirred until in solution then added lithium hydroxide (1.673 ml, 3.35 mmol) and stirred 15 mins at RT. Added 1N HCl to make slightly acidic pH 6 and extracted result 3× with 25 mL Ethyl acetate. Dried combined organics with Na.sub.2SO.sub.4, filtered, and removed solvent to a colorless oil, which was taken on without purification. LCMS Condition E: retention time=0.99 mins 494.4 (M+1).

(S)-2-(5-((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(benzo[b]thiophen-3-yl)ethyl)-1H-tetrazol-1-yl)hexanoic acid

[0516] ##STR00020##

[0517] To (S)-2-(5-((S)-2-(benzo[b]thiophen-3-yl)-1-(((benzyloxy)carbonyl)amino)ethyl)-1H-tetrazol-1-yl)hexanoic acid (0.55 g, 1.114 mmol) dissolved in acetonitrile (12 ml) cooled in an ice water bath was added at 0° C. iodotrimethylsilane (0.465 ml, 3.34 mmol). Stirred ½ hour at 0° C. then warmed to RT for ½ hour. Added 1N HCl and extracted 4× with 20 mL ethyl acetate. Dried organic layer over Na.sub.2SO.sub.4, filtered, and stripped to a Yellow Brown Oil. Purification on ISCO with 80 gram Gold silica column, produced 0.126 grams white solid.

[0518] LCMS Condition E: retention time=0.99 mins 582.5 (M+1)

(S)-methyl 2-((S)-2-(((benzyloxy)carbonyl)amino)-3-(4-(tert-butoxy)phenyl)propanamido)propanoate

[0519] ##STR00021##

[0520] To a solution of (S)-2-(((benzyloxy)carbonyl)amino)-3-(4-(tert-butoxy)phenyl)propanoic acid (2.24 g, 6.03 mmol) and (S)-methyl 2-aminopropanoate hydrochloride (0.850 g, 6.09 mmol) in DCM (30.2 ml) at 0° C. was added solid HATU (2.408 g, 6.33 mmol). After 15 mins N-methylmorpholine (1.326 ml, 12.06 mmol) was added and the mixture was allowed to warm to RT, overnight. Added 1 N HCl 30 ml and extracted 3× with dichloromethane (30 mL), combined organics were dried over Na.sub.2SO.sub.4, filtered, and stripped to a wax. The residue was purified via normal phase chromatography, ISCO 80 grams silica, hexanes to ethyl acetate gradient, produced 1.3 grams.

[0521] LCMS Condition E: retention time=0.97 mins 457.5 (M+1); .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 7.40-7.31 (m, 5H), 7.14-7.05 (m, J=8.1 Hz, 2H), 6.97-6.84 (m, 2H), 5.31 (s, 1H), 5.11 (s, 2H), 4.49 (s, 1H), 3.72 (s, 3H), 1.57 (br. s., 2H), 1.38-1.29 (m, 12H).

(S)-methyl 2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-(4-(tert-butoxy)phenyl)ethyl)-1H-tetrazol-1-yl)propanoate

[0522] ##STR00022##

[0523] In a single neck round bottom flask equipped with a Nitrogen Line, magnetic stirrer and charged with (S)-methyl 2-((S)-2-(((benzyloxy)carbonyl)amino)-3-(4-(tert-butoxy)phenyl)propanamido)propanoate (1.77 g, 3.88 mmol), was added Diphenyl-2-pyridylphosphine (3.05 grams, 11.64 mmol) in THF (25 ml) under N.sub.2. Purged the solution with N.sub.2 before DIAD (2.261 ml, 11.63 mmol) was added dropwise. Diphenylphosphoryl azide (3.34 ml, 15.51 mmol) was then added slowly. Reaction stirred at RT 2.5 hours. The mixture was heated to 55° C. and allowed to stir at 50° C. for 20 hours. Cooled to RT and pour into 40 mL ethyl acetate, washed with cold 1N HCl, sat. NaHCO.sub.3, and brine. Removed organics to a thick oil. The residue was purified on ISCO 80 gram column; 0 to 100% Ethyl acetate in Hexanes, produced 2.24 grams of a mixture which was taken on without further purification.

[0524] LCMS Condition D: retention time=1.01 mins 482.5.

(S)-2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-(4-(tert-butoxy)phenyl)ethyl)-1H-tetrazol-1-yl)propanoic acid

[0525] ##STR00023##

[0526] To (S)-methyl 2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-(4-(tert-butoxy)phenyl)ethyl)-1H-tetrazol-1-yl)propanoate (2.24 g, 4.65 mmol) was added MeOH (11.63 ml), and THF (11.63 ml). Mixture stirred until in solution then lithium hydroxide was added (6.98 ml, 13.96 mmol). Mixture stirred 15 mins at RT. Added 1N HCl to make slightly acidic pH 6 and extracted result 3× with 25 mL Ethyl acetate. Dried combined organics with Na.sub.2SO.sub.4, filtered and removed solvent to provide an oil which was taken on to the next step without purification.

[0527] LCMS Condition D: retention time=0.93 mins 468.4.

(S)-2-(5-((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(4-(tert-butoxy)phenyl)ethyl)-1H-tetrazol-1-yl)propanoic acid

[0528] ##STR00024##

[0529] (S)-2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-(4-(tert-butoxy)phenyl)ethyl)-1H-tetrazol-1-yl)propanoic acid (3.4 g, 7.27 mmol) was dissolved into 20 mL MeOH under N.sub.2. To this solution was added Pd/C (500 mg, 0.470 mmol). Degassed the solution before switching to H.sub.2 balloon. The mixtures were allowed to stir under H.sub.2 for 4 hours. LC-MS showed >95% conversion. Filtered out catalyst, removed solvent to provide crude material.

[0530] The above material was dissolved in THF (8 mL), Acetonitrile (6 mL) and 3 mL water were added. Cooled down to 0° C. in an ice water bath, added Na.sub.2CO.sub.3 (2.312 g, 21.82 mmol) followed by (9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (2.70 g, 8.00 mmol) in portions. Stirred overnight at RT. Added 1 N HCl to make slightly acidic, pH 5. Extracted result 4× with 25 mL Ethyl acetate. Combined extracts dried over Na.sub.2SO.sub.4, filtered and removed solvent to provide a yellow oil. ISCO 220 gram column, with a Hexanes to Ethyl Acetate gradient provides 0.403 grams

[0531] LCMS Condition D: retention time=1.2 mins 556.5

[0532] Analysis HPLC condition H: retention time 11.34 min 94%

[0533] Analysis HPLC condition G: retention time 12.39 min 93.9%

[0534] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.9-13.4 (s, 1H), 8.33 (d, J=8.0 Hz, 1H), 7.86 (d, J=7.5 Hz, 2H), 7.71-7.59 (m, 1H), 7.57 (d, J=7.5 Hz, 1H), 7.47-7.34 (m, 2H), 7.34-7.16 (m, 3H), 6.92-6.75 (m, 2H), 5.72-5.47 (m, 1H), 5.3-5.05 (m, 1H), 4.27-4.02 (m, 3H), 3.25 (dd, J=13.8, 10.3 Hz, 1H), 3.10 (dd, J=14.1, 4.5 Hz, 1H), 1.99-1.71 (m, 2H), 1.65 (d, J=7.0 Hz, 1H), 1.29-1.11 (m, 9H).

tert-butyl 3-((S)-2-(((benzyloxy)carbonyl)amino)-3-(((S)-1-methoxy-1-oxohexan-2-yl)amino)-3-oxopropyl)-1H-indole-1-carboxylate

[0535] ##STR00025##

[0536] HATU (30.0 g, 79 mmol) was added to a stirred solution of (S)-2-(((benzyloxy)carbonyl)amino)-3-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)propanoic acid (28.8 g, 65.7 mmol) in CH.sub.2Cl.sub.2 (274 ml) and DMF (132 ml) at RT. Reaction mixer was stirred at RT for 10 min. Then added (S)-methyl 2-aminohexanoate hydrochloride (14.32 g, 79 mmol) and DIPEA (40.2 ml, 230 mmol) in 2 mL DMF at RT. Reaction mixed at RT for 1½h. Reaction mixture was washed with 1.5N HCl and 10% LiCl solution and extracted with dichloromethane. Organic layers were dried over Na.sub.2SO.sub.4 and concentrated. Crude material was purified by ISCO normal phase chromatography. Compound was eluted in 20% Ethyl acetate/Pet ether.

[0537] LCMS Condition D: retention time 2.23 min 566.4 (M+1)

[0538] Analysis HPLC condition G: retention time 23.97 min 97.9%.

tert-butyl 3-((S)-2-(((benzyloxy)carbonyl)amino)-2-(1-((S)-1-methoxy-1-oxohexan-2-yl)-1H-tetrazol-5-yl)ethyl)-1H-indole-1-carboxylate

[0539] ##STR00026##

[0540] To a stirred solution of tert-butyl 3-((S)-2-(((benzyloxy)carbonyl)amino)-3-(((S)-1-methoxy-1-oxohexan-2-yl)amino)-3-oxopropyl)-1H-indole-1-carboxylate (10 g, 17.68 mmol) and triphenylphosphine (13.91 g, 53.0 mmol) in THF (80 mL) under N.sub.2. Purged the solution with N.sub.2 before DIAD (10.31 mL, 53.0 mmol) was added drop wise. Diphenyl phosphorazidate (13.33 mL, 61.9 mmol) was then added slowly. The reaction mixture were heated 75° C. and allowed to stirred at 75° C. for 24 hours. The reaction mixture was cooled to RT and poured into 500 ml Ethyl Actate, washed with 2N HCl, saturated NaHCO.sub.3 and brine. The organic layer was then concentrate and crude material obtained. The crude material was purified by normal phase chomatography to produce tert-butyl 3-((S)-2-(((benzyloxy)carbonyl)amino)-2-(1-((S)-1-methoxy-1-oxohexan-2-yl)-1H-tetrazol-5-yl)ethyl)-1H-indole-1-carboxylate (6 g, 9.65 mmol, 54.6% yield)

[0541] LCMS Condition D: retention time 2.54 min 591.3 (M+1).

[0542] Analysis HPLC condition K: retention time 2.58 min 95.7%.

(S)-2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-1H-tetrazol-1-yl)hexanoic acid

[0543] ##STR00027##

[0544] A stirred solution of tert-butyl 3-((S)-2-(((benzyloxy)carbonyl)amino)-2-(1-((S)-1-methoxy-1-oxohexan-2-yl)-1H-tetrazol-5-yl)ethyl)-1H-indole-1-carboxylate (6 g, 10.16 mmol) in THF (33.6 ml) and MeOH (8.40 ml) was treated with LiOH (15.24 ml, 30.5 mmol) and stirred for 3 h at rt. The reaction mixture was acidified with 1N HCl and extracted with ethyl acetate. Organic layer dried and concentrated to get crude. The crude product purified by combi flash to produce (S)-2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-1H-tetrazol-1-yl)hexanoic acid (3.5 g, 5.89 mmol, 58.0% yield)

[0545] LCMS Condition D: retention time 2.41 min 477.2 (M+1).

(S)-2-(5-((S)-1-amino-2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-1H-tetrazol-1-yl)hexanoic acid

[0546] ##STR00028##

[0547] To a stirred solution of(S)-2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-1H-tetrazol-1-yl)hexanoic acid (3.5 g, 6.07 mmol) in MeOH (56.7 ml) under N.sub.2 was added Pd/C (1.292 g, 1.214 mmol). Degassed the solution before switching to H.sub.2 The mixture was allowed to stirred under H.sub.2 for 7 hours at RT. The reaction mixture filtered through diatomaceous earth (Celite®) and bed washed with methanol, and concentrated to get (S)-2-(5-((S)-1-amino-2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-1H-tetrazol-1-yl)hexanoic acid (1.8 g, 3.69 mmol, 42.6% yield).

[0548] LCMS Condition D: retention time 2.13 min 441.2 (M+1).

(S)-2-(5-((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-1H-tetrazol-1-yl)hexanoic acid

[0549] ##STR00029##

[0550] To a stirred solution of (S)-2-(5-((S)-1-amino-2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-1H-tetrazol-1-yl)hexanoic acid (1.8 g, 4.07 mmol) in Acetonitrile (24.88 ml) and Water (12.44 ml) cooled to 0° C., was added Na.sub.2CO.sub.3 (6.51 ml, 13.02 mmol) followed by FMOC-OSU (1.647 g, 4.88 mmol) and stirred at RT for 4 h. The reaction mixture was quenched with 1N HCl and pH adjusted to 1-2. Extracted with ethyl acetate and concentrated to get crude oil. The crude compound purified by reveres phase combi flash to get (S)-2-(5-((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)ethyl)-1H-tetrazol-1-yl)hexanoic acid 0.88 gram.

[0551] LCMS Condition D: retention time 2.25 min 666.6 (M+1).

[0552] Analysis HPLC condition H: retention time 16.2 mins 92%.

[0553] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.45 (d, J=8.0 Hz, 1H), 8.02 (d, J=8.5 Hz, 1H), 7.84 (d, J=7.0 Hz, 2H), 7.76 (d, J=8.0 Hz, 1H), 7.66 (s, 1H), 7.60-7.49 (m, 2H), 7.40-7.26 (m, 3H), 7.26-7.14 (m, 2H), 5.16 (d, J=10.5 Hz, 1H), 4.18-4.04 (m, 2H), 3.51-3.36 (m, 2H), 3.17 (s, 2H), 2.28 (br. s., 1H), 1.63-1.46 (m, 9H), 1.19 (d, J=7.0 Hz, 2H), 1.07 (d, J=8.0 Hz, 1H), 0.75-0.63 (m, 2H).

(S)-2-(((benzyloxy)carbonyl)amino)-3-(naphthalen-1-yl)propanoic acid

[0554] ##STR00030##

[0555] Benzyl carbonochloridate (1.081 mL, 7.67 mmol) and NaOH (8.36 mL, 8.36 mmol) were dropped at the same time to a stirred solution of (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (1.5 g, 6.97 mmol) in THF (7 mL) and 7 ml of 1N NaOH at 0° C. The mixtures were allowed to stir at RT for 2 hours at which time LC-MS showed desired product peak Acidified with 1N HCl and extracted with EtOAc. Removed EtOAc and the obtained crude materials were purified by RediSep Column: Silica 80 g

[0556] Flow Rate: 60 ml/min.

[0557] Solvent: A dichloromethane; Solvent: B 20% MeOH/80% DCM.

[0558] Gradient 0%-50% SolB in 20 mins.

[0559] Fractions containing the desired product were combined and dried via centrifugal evaporation to provide the title compound (2.21 g, 89%).

[0560] Analysis LCMS Condition A: Retention time=0.98 min; ESI-MS(+) m/z 350.08 (M+H).

(S)-methyl 2-((S)-2-(((benzyloxy)carbonyl)amino)-3-(naphthalen-1-yl)propanamido)hexanoate

[0561] ##STR00031##

[0562] (S)-2-(((benzyloxy)carbonyl)amino)-3-(naphthalen-1-yl)propanoic acid (2.1 g, 6.01 mmol) and HATU (2.74 g, 7.21 mmol) were dissolved into CH.sub.2Cl.sub.2 (25 ml) and DMF (12 mL). The mixtures were allowed to stir at RT for 10 min before (S)-methyl 2-aminohexanoate, HCl (1.310 g, 7.21 mmol) and Hunig'sBase (3.67 ml, 21.04 mmol) in 2 ml of DMF were added. The mixtures were allowed to stir at RT for 2 hours at which time LC-MS showed completed conversion. Quenched with 10 ml 1N HCl. The mixtures were poured into 250 ml of EtOAc, washed with 10% LiCl twice, Brine once. The organic layer was then dried over Na.sub.2SO.sub.4 and concentrated. The crude materials were purified by RediSep Column: Silica 220 g

[0563] Flow Rate: 150 ml/min

[0564] Equilibration Volume: 1.0 CV

[0565] Solvent: A1 hexane; Solvent: B1 ethyl acetate

[0566] Gradient 0%-40% Sol B in 15 mins.

[0567] Fractions containing the desired product were combined and dried via centrifugal evaporation to provide the title compound (1.86 g, 65%).

[0568] Analysis LCMS Condition A: Retention time=1.14 min; ESI-MS(+) m/z 477.1 (M+H).

(S)-methyl 2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-(naphthalen-1-yl)ethyl)-1H-tetrazol-1-yl)hexanoate

[0569] ##STR00032##

[0570] A pressure vial was charged with (S)-methyl 2-((S)-2-(((benzyloxy)carbonyl)amino)-3-(naphthalen-1-yl)propanamido)hexanoate (1.8 g, 3.78 mmol) and Diphenyl-2-pyridylphosphine (2.98 g, 11.33 mmol) in THF (35 ml) under N.sub.2 was purged with N.sub.2 before DIAD (2.203 ml, 11.33 mmol) was added dropwise. Diphenylphosphoryl azide (2.85 ml, 13.22 mmol) was then added slowly. The mixtures were heated to 55° C. and allowed to stir at 55° C. for 18 hours at which time LC-MS showed desired product peak. Cooled down to RT and poured into 200 ml EtOAc, washed with 2N HCl, sat. NaHCO.sub.3 and brine. The organic layer was then concentrated and the obtained material was purified byRediSep Column: Silica 330 g Gold

[0571] Flow Rate: 200 ml/min

[0572] Equilibration Volume: 3.0 CV

[0573] Solvent: A1 hexane; Solvent: B1 ethyl acetate

[0574] Gradient 0%-40% SolB in 15 mins.

[0575] Fractions containing the desired product were combined and dried via centrifugal evaporation to provide the title compound (533 mg, 28%)

[0576] Analysis LCMS Condition A: Retention time=1.18 min; ESI-MS(+) m/z 502.2 (M+H).

(S)-2-(5-((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(naphthalen-1-yl)ethyl)-1H-tetrazol-1-yl)hexanoic acid

[0577] ##STR00033##

[0578] (S)-methyl 2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-(naphthalen-1-yl)ethyl)-1H-tetrazol-1-yl)hexanoate (533 mg, 1.063 mmol) was dissolved into THF (8 mL) and MeOH (2 mL) cosolvent. To this solution was added LiOH (1.594 mL, 3.19 mmol) in two portions. LC-MS showed completed conversion after stirring at RT for 2 hours. Product peak came at 0.99/2.0 min. Quenched the reaction with 1N HCl, adjusted pH to 1-2. Extracted with EtOAc. The organic layer was then dried over Na.sub.2SO.sub.4 and concentrated to provide crude materials.

[0579] The above obtained materials were dissolved into 10 ml MeOH under N.sub.2. To this solution was added Pd/C (170 mg, 0.159 mmol). Degassed the solution before switching to H.sub.2 balloon. The mixtures were allowed to stir under H.sub.2 for 7 hours at which time LC-MS showed >95% conversion. Filtered out catalyst and removed the solvent. The obtained residues were dissolved into Acetonitrile (6 mL) and water (3 ml). Cooled down to 0° C., added Na.sub.2CO.sub.3 (1.594 mL, 3.19 mmol) followed by (9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (394 mg, 1.169 mmol). The mixtures were allowed to stir at RT for 3 hours at which time LC-MS showed completed conversion. Quenched with 1N HCl, adjusted to pH to 1-2 and extracted with EtOAc. The organic layer was then concentrated under vacuum and the obtained crude materials were purified byRediSep Column: Silica 40 g

[0580] Flow Rate: 40 ml/min

[0581] Equilibration Volume: 5.0 CV

[0582] Solvent: A dichloromethane

[0583] Solvent: B 20% MeOH/DCM

[0584] Gradient 0%-50% SolB in 15 mins.

[0585] Fractions containing the desired product were combined and dried via centrifugal evaporation to provide the title compound (429 mg, 63%, 3 steps)

[0586] Analysis LCMS Condition A: Retention time=1.18 min; ESI-MS(+) m/z 576.2 (M+H)

[0587] .sup.1H NMR (400 MHz, DMSO-d.sub.6) □ 14.15-13.65 (s, 1H), 8.47 (d, J=8.5 Hz, 1H), 8.05 (s, 1H), 7.95-7.89 (m, 1H), 7.89-7.76 (m, 3H), 7.63-7.49 (m, 5H), 7.43-7.32 (m, 3H), 7.26 (d, J=6.5 Hz, 1H), 7.23 (d, J=8.0 Hz, 1H), 5.51-5.31 (m, 1H), 5.31-5.18 (m, 1H), 4.17-4.04 (m, 3H), 3.81-3.71 (m, 2H), 2.24 (br. s., 2H), 2.85-2.1 (m, 2H), 1.23-1.08 (m, 2H), 0.75-0.62 (m, 3H).

(S)-benzyl (1-oxo-3-phenylpropan-2-yl)carbamate

[0588] ##STR00034##

[0589] To a mixture of (S)-benzyl (1-hydroxy-3-phenylpropan-2-yl)carbamate (0.457 g, 1.6 mmol) in DCM (6 mL) was added Dess-Martin Periodinane (0.814 g, 1.920 mmol). After 2 hour, LCMS showed completed conversion after stirring at RT for 6 hours. The reaction mixture was diluted with dichloromethane and washed with cold 1N NaOH. The organic layer was dried with MgSO.sub.4, filtered and concentrated. Used in next step without further purification.

[0590] Analysis LCMS Condition A: Retention time=0.85 min; ESI-MS(+) m/z 284.0 (M+H).

(S)-benzyl (1-phenylbut-3-yn-2-yl)carbamate

[0591] ##STR00035##

[0592] Starting materials (S)-benzyl (1-oxo-3-phenylpropan-2-yl)carbamate (400 mg, 1.412 mmol) were dissolved into dry MeOH (8 mL). Cooled down to 0° C. and then added K.sub.2CO.sub.3 (390 mg, 2.82 mmol) followed by Dimethyl (1-diazo-2-oxopropyl)phosphonate (0.318 mL, 2.118 mmol). The mixtures were allowed to warm to RT slowly and stirred at for 3 hours. LC-MS showed partial conversion. The desired product peak was at 0.97/2.0 min. Reaction stirred at RT for overnight, at which time LC-MS showed completed conversion. The mixtures were poured into 0.5N HCl, extracted with ethyl acetate. The organic layer was then dried over Na.sub.2SO.sub.4 and concentrated. The crude materials were then purified on 24 g ISCO column.

[0593] SolA: CH.sub.2Cl.sub.2; SolB: 20% MeOH/CH.sub.2Cl.sub.2. gradient 0-50% SolB in 15 mins. 50% SolB hold 10 mins.

[0594] Fractions containing the desired product were combined and dried via centrifugal evaporation to provide the title compound (240 mg, 61%).

[0595] Analysis LCMS Condition A: Retention time=0.96 min; ESI-MS(+) m/z 280.1 (M+H).

(S)-tert-butyl 2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-phenylethyl)-1H-1,2,3-triazol-1-yl)propanoate

[0596] ##STR00036##

[0597] Starting materials (S)-benzyl (1-phenylbut-3-yn-2-yl)carbamate (85 mg, 0.304 mmol) and (S)-tert-butyl 2-azidopropanoate (208 mg, 1.217 mmol) were dissolved into toluene (4 mL). Purged with N.sub.2 before Ru-catalyst [113860-07-4](16.54 mg, 0.015 mmol) was added. The mixtures were allowed to stir at 100° C. for 16 hours at which time LC-MS showed completed conversion. Removed solvent. The obtained residues were purified on reverse phase HPLC Column: 30*100 mm Luna

[0598] SolA: 95% H2O/5% CH.sub.3CN/0.1% TFA; SolB: 5% H2O/95% CH.sub.3CN/0.1% T.sub.FA Fractions containing the desired product were combined and dried via centrifugal evaporation to provide the title compound (56 mg, 41%).

[0599] Analysis LCMS Condition A: Retention time=1.01 min; ESI-MS(+) m/z 451.1 (M+H)

(S)-2-(5-((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-phenylethyl)-1H-1,2,3-triazol-1-yl)propanoic acid

[0600] ##STR00037##

[0601] Starting materials (S)-tert-butyl 2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-phenylethyl)-1H-1,2,3-triazol-1-yl)propanoate (142 mg, 0.315 mmol) were dissolved into MeOH (4 ml) under N.sub.2. Purged the system with N.sub.2 before Pd/C (33.5 mg, 0.032 mmol) was added. The mixtures were allowed to stir at RT under H.sub.2 balloon for 7 hours, at which time LC-MS showed completed conversion to intermediate (S)-tert-butyl 2-(5-((S)-1-amino-2-phenylethyl)-1H-1,2,3-triazol-1-yl)propanoate. Filtered out catalyst and removed the solvent. The obtained residues were dissolved into Acetonitrile (4 mL) and Water (2 mL). Cooled down to 0° C. and to the solution was added N.sub.2CO.sub.3 (0.788 ml, 1.576 mmol), followed by (9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (117 mg, 0.347 mmol) in 1 ml CH.sub.3CN. The reaction mixtures were allowed to warm to RT in 2 hours. LC-MS showed starting materials were consumed. Acidified with 1N HCl to pH2 and extracted with ethyl acetate. The organic layer was then dried over Na.sub.2SO.sub.4 and concentrated. The obtained materials were then treated with 20% TFA in CH.sub.2Cl.sub.2 (5 ml) at RT for 7 hours. LC-MS showed completed conversion. The obtained crude materials were purified on reverse phase HPLC.

[0602] Column: 30*100 mm Luna

[0603] SolA: 95% H2O/5% CH.sub.3CN/0.1% TFA; SolB: 5% H2O/95% CH.sub.3CN/0.1% TFA

[0604] Fractions containing the desired product were combined and dried via centrifugal evaporation to provide the title compound (68 mg, 44% 3 steps).

[0605] Analysis LCMS Condition A: Retention time=0.95 min; ESI-MS(+) m/z 483.1 (M+H).

Intermediate Resin A: (9H-fluoren-9-yl)methyl ((S)-1-(((S)-4-amino-1-((S)-2-(((5R,8S,11S,14S,17S,20S,23S,29S,32S)-17,23-bis((1H-indol-3-yl)methyl)-1-amino-11,14-dibutyl-8-(3-guanidinopropyl)-20-(hydroxymethyl)-33-(1H-imidazol-5-yl)-29-isobutyl-5-(mercaptomethyl)-12,15,27-trimethyl-1,4,7,10,13,16,19,22,25,28,31-undecaoxo-3,6,9,12,15,18,21,24,27,30-decaazatritriacontan-32-yl)carbamoyl)pyrrolidin-1-yl)-1,4-dioxobutan-2-yl)amino)-1-oxopropan-2-yl)(methyl)carbamate Seiber amide resin

[0606] ##STR00038##

[0607] To a 125 mL reactor was added Sieber resin (1410 mg, 1 mmol), and the vessel was placed on the CEM Liberty microwave peptide synthesizer. The following procedures were then performed sequentially:

“CEM Method A: Resin-swelling procedure” was followed;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Gly-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Cys(Trt)-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Arg(Pbf)-OH;
“CEM Method A: Double-couple Coupling procedure” was followed with Fmoc-[N-Me]Nle-OH;
“CEM Method A: Double-couple Coupling procedure” was followed with Fmoc-[N-Me]Nle-OH;
“CEM Method A: Double-couple Coupling procedure” was followed with Fmoc-Trp(Boc)-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Ser(tBu)-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Trp-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Sar-OH;
“CEM Method A: Double-couple Coupling procedure” was followed with Fmoc-Leu-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-His(Trt)-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Pro-OH;
“CEM Method A: Double-couple Coupling procedure” was followed with Fmoc-Asn(tBu)-OH;

[0608] The resulting resin was transferred to a 100 mL polypropylene tube equipped with a frit and washed with DCM (40 mL×3). Finally, the resin was dried under vacuum and used as a intermediate for synthesis.

[0609] Microcleavage A was followed.

[0610] Analysis LCMS Condition A: Retention time=1.0 min, ESI-MS(+) m/z 966.6 (M+2H+1CO.sub.2).

Preparation of Example 1001 and Example 1002

[0611] ##STR00039##

[0612] Starting with Intermediate Resin A the resulting Resin was treated with the following procedures performed sequentially:

“Manual Coupling procedure A” was followed for(S)-2-(5-((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-phenylethyl)-1H-tetrazol-1-yl)propanoic acid
“Chloroacetic acid coupling procedure B”

“Global Deprotection Method C”

“Cyclization Method D”

Example 1001, Isomer 1

[0613] The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Gradient: 20-60% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 2.1 mg, and its estimated purity by LCMS analysis was 95%.

[0614] Analysis LCMS Condition D: Retention time=1.57 min; ESI-MS(+) m/z 932.15 (M+2H)

[0615] Analysis LCMS Condition E: Retention time=1.44 min; ESI-MS(+) m/z 932.35 (M+2H).

##STR00040##

Example 1002, Isomer 2

[0616] The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Gradient: 20-60% B over 25 minutes, then a 5-minute hold at 60% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 1.5 mg, and its estimated purity by LCMS analysis was 99%.

[0617] Analysis LCMS Condition D: Retention time=1.79 min; ESI-MS(+) m/z 932.15 (M+2H).

[0618] Analysis LCMS Condition E: Retention time=1.66 min; ESI-MS(+) m/z 931.85 (M+2H).

Preparation of Example 1003 and Example 1004

[0619] ##STR00041##

[0620] Started with Intermediate Resin A. The resulting Resin was treated with the following procedures performed sequentially:

“Manual Coupling procedure A” was followed for (S)-2-(5-((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-phenylethyl)-1H-1,2,3-triazol-1-yl)propanoic acid.
“Chloroacetic acid coupling procedure B”

“Global Deprotection Method C”

“Cyclization Method D”

[0621] General Synthetic sequences “Chloroacetic acid coupling procedure B”, “Global Deprotection Method C”, and “Cyclization Method D” were then followed.

Example 1003, isomer 1

[0622] The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 20-65% B over 25 minutes, then a 5-minute hold at 65% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 0.9 mg, and its estimated purity by LCMS analysis was 100%.

[0623] Analysis LCMS Condition D: Retention time=1.54 min; ESI-MS(+) m/z 931.85 (M+2H)

[0624] Analysis LCMS Condition E: Retention time=1.41 min; ESI-MS(+) m/z 931.90 (M+2H).

##STR00042##

Example 1004, Isomer 2

[0625] The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 20-60% B over 25 minutes, then a 5-minute hold at 60% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 1.9 mg, and its estimated purity by LCMS analysis was 96%.

[0626] Analysis LCMS Condition D: Retention time=1.55 min; ESI-MS(+) m/z 931.90 (M+2H)

[0627] Analysis LCMS Condition E: Retention time=1.42 min; ESI-MS(+) m/z 931.60 (M+2H).

Preparation of Example 1005

[0628] ##STR00043##

[0629] Example Tetrazole 1005 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method A: Resin-swelling procedure”, “Symphony Method A: Standard-coupling procedure”, “Symphony Method A: Secondary amine-coupling procedure B”, “Chloroacetic acid coupling procedure B”, “Global Deprotection Method C”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: 25-65% B over 25 minutes, then a 10-minute hold at 65% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 3.6 mg, and its estimated purity by LCMS analysis was 98%.

[0630] Analysis LCMS Condition D: Retention time=1.83 min; ESI-MS(+) m/z 937.9 (M+2H)

[0631] Analysis LCMS Condition E: Retention time=1.76 min; ESI-MS(+) m/z 938.0 (M+2H).

Preparation of Example 1006

[0632] ##STR00044##

[0633] Example Tetrazole 1006 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method A: Resin-swelling procedure”, “Symphony Method A: Standard-coupling procedure”, “Symphony Method A: Secondary amine-coupling procedure B”, “Chloroacetic acid coupling procedure B”, “Global Deprotection Method C”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 15-65% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 3.1 mg, and its estimated purity by LCMS analysis was 96%.

[0634] Analysis LCMS Condition D: Retention time=1.69 min; ESI-MS(+) m/z 959.3 (M+2H).

Preparation of Example 1007

[0635] ##STR00045##

[0636] Example Tetrazole 1007 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method A: Resin-swelling procedure”, “Symphony Method A: Standard-coupling procedure”, “Symphony Method A: Secondary amine-coupling procedure B”, “Chloroacetic acid coupling procedure B”, “Global Deprotection Method C”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 15-65% B over 25 minutes, then a 10-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 1.4 mg, and its estimated purity by LCMS analysis was 100%.

[0637] Analysis LCMS Condition D: Retention time=1.71 min; ESI-MS(+) m/z 943.4 (M+2H)

[0638] Analysis LCMS Condition E: Retention time=1.69 min; ESI-MS(+) m/z 943.8 (M+2H).

Preparation of Example 1010

[0639] ##STR00046##

[0640] Example 1010 was prepared on Rink Resin following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method A: Resin-swelling procedure”, “Symphony Method A: Standard-coupling procedure”, “Symphony Method A: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method A: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18 300, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 25-70% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 25-70% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 1.9 mg, and its estimated purity by LCMS analysis was 98%.

[0641] Analysis LCMS Condition D: Retention time=1.73 min; ESI-MS(+) m/z 954.0 (M+2H).

[0642] Analysis LCMS Condition E: Retention time=1.67 min; ESI-MS(+) m/z 954.2 (M+2H).

Preparation of Example 1012

[0643] ##STR00047##

[0644] Example 1012 was prepared on Rink Resin following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method A: Resin-swelling procedure”, “Symphony Method A: Standard-coupling procedure”, “Symphony Method A: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method A: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 15-65% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 3.2 mg, and its estimated purity by LCMS analysis was 100%.

[0645] Analysis LCMS Condition D: Retention time=1.51 min; ESI-MS(+) m/z 940.5 (M+2H).

[0646] Analysis LCMS Condition E: Retention time=1.44 min; ESI-MS(+) m/z 940.3 (M+2H).

Preparation of Example 1013

[0647] ##STR00048##

[0648] Example 1013 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Prelude Method B: Resin-swelling procedure”, “Prelude Method B: Standard-coupling procedure”, “Prelude Method B: Secondary amine-coupling procedure”, “Prelude Method B: Custom amino acids-coupling procedure”, “Prelude Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-65% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 20-65% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 3.2 mg, and its estimated purity by LCMS analysis was 100%.

[0649] Analysis LCMS Condition D: Retention time=1.54 min; ESI-MS(+) m/z 949.0 (M+2H).

[0650] Analysis LCMS Condition E: Retention time=1.50 min; ESI-MS(+) m/z 949.3 (M+2H).

Preparation of Example 1014

[0651] ##STR00049##

[0652] Example 1014 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Prelude Method B: Resin-swelling procedure”, “Prelude Method B: Standard-coupling procedure”, “Prelude Method B: Secondary amine-coupling procedure”, “Prelude Method B: Custom amino acids-coupling procedure”, “Prelude Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-m particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-65% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 15-60% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 4.8 mg, and its estimated purity by LCMS analysis was 100%.

[0653] Analysis LCMS Condition D: Retention time=1.43 min; ESI-MS(+) m/z 914.9 (M+2H).

[0654] Analysis LCMS Condition E: Retention time=1.43 min; ESI-MS(+) m/z 914.9 (M+2H).

Preparation of Example 1050

[0655] ##STR00050##

[0656] Example 1050 was prepared on Rink Resin following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 10-65% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 2.0 mg, and its estimated purity by LCMS analysis was 98%.

[0657] Analysis LCMS Condition D: Retention time=1.47 min; ESI-MS(+) m/z 940.3 (M+2H).

[0658] Analysis LCMS Condition E: Retention time=1.42 min; ESI-MS(+) m/z 940.6 (M+2H).

Preparation of Example 1051

[0659] ##STR00051##

[0660] Example 1051 was prepared on Rink Resin following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-65% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 1.9 mg, and its estimated purity by LCMS analysis was 95%.

[0661] Analysis LCMS Condition D: Retention time=1.43 min; ESI-MS(+) m/z 914.8 (M+2H).

[0662] Analysis LCMS Condition E: Retention time=1.44 min; ESI-MS(+) m/z 915.2 (M+2H).

Preparation of Example 1052

[0663] ##STR00052##

[0664] Example 1052 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 10-65% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 2.4 mg, and its estimated purity by LCMS analysis was 93%.

[0665] Analysis LCMS Condition D: Retention time=1.58 min; ESI-MS(+) m/z 967.0 (M+2H).

[0666] Analysis LCMS Condition E: Retention time=1.52 min; ESI-MS(+) m/z 966.8 (M+2H).

Preparation of Example 1053

[0667] ##STR00053##

[0668] Example 1053 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-65% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 4.3 mg, and its estimated purity by LCMS analysis was 91%.

[0669] Analysis LCMS Condition D: Retention time=1.38 min; ESI-MS(+) m/z 936.5 (M+2H).

[0670] Analysis LCMS Condition E: Retention time=1.48 min; ESI-MS(+) m/z 936.1 (M+2H).

Preparation of Example 1054

[0671] ##STR00054##

[0672] Example 1054 was prepared following the general synthetic sequence described for the preparation of Example 1001, composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method D”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 10-65% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 15-60% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 2.2 mg, and its estimated purity by LCMS analysis was 100%.

[0673] Analysis LCMS Condition D: Retention time=1.44 min; ESI-MS(+) m/z 929.0 (M+2H).

[0674] Analysis LCMS Condition E: Retention time=1.39 min; ESI-MS(+) m/z 929.0 (M+2H).

Preparation of Example 1055

[0675] ##STR00055##

[0676] Example 1055 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method D”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 15-65% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 20-60% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 1.1 mg, and its estimated purity by LCMS analysis was 93%.

[0677] Analysis LCMS Condition D: Retention time=1.50 min; ESI-MS(+) m/z 907.4 (M+2H).

[0678] Analysis LCMS Condition E: Retention time=1.49 min; ESI-MS(+) m/z 907.9 (M+2H).

Preparation of Example 1056

[0679] ##STR00056##

[0680] Example 1056 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Custom amino acids-coupling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-m particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 40-80% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 10.8 mg, and its estimated purity by LCMS analysis was 100%.

[0681] Analysis LCMS Condition H: retention time=1.41 min.; ESI-MS(+) m/z 937.4 (M+2H).

[0682] Analysis LCMS Condition I: retention time=2.03 min.; ESI-MS(+) m/z 936.6 (M+2H).

Preparation of Example 1057

[0683] ##STR00057##

[0684] Example 1057 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Symphony Method B: Custom amino acids-coupling procedure”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 45-85% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 5-45% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 3.3 mg, and its estimated purity by LCMS analysis was 97%.

[0685] Analysis LCMS Condition H: retention time=1.68 min.; ESI-MS(+) m/z 634.4 (M+3H).

[0686] Analysis LCMS Condition I: retention time=2.35 min.; ESI-MS(+) m/z 947.0 (M+2H).

Preparation of Example 1058

[0687] ##STR00058##

[0688] Example 1058 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Symphony Method B: Custom amino acids-coupling procedure”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammoniumacetate; Gradient: 50-90% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 5.7 mg, and its estimated purity by LCMS analysis was 97%.

[0689] Analysis LCMS Condition H: retention time=1.81 min.; ESI-MS(+) m/z 986.9 (M+2H).

[0690] Analysis LCMS Condition I: retention time=2.50 min.; ESI-MS(+) m/z 986.8 (M+2H).

Preparation of Example 1059

[0691] ##STR00059##

[0692] Example 1059 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Symphony Method B: Custom amino acids-coupling procedure”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 15-55% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 3.6 mg, and its estimated purity by LCMS analysis was 94%.

[0693] Analysis LCMS Condition H: retention time=1.61 min.; ESI-MS(+) m/z 934.4 (M+2H).

[0694] Analysis LCMS Condition I: retention time=3.01 min.; ESI-MS(+) m/z 934.3 (M+2H).

Preparation of Example 1060

[0695] ##STR00060##

[0696] Example 1060 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Symphony Method B: Custom amino acids-coupling procedure”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 40-80% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 1.1 mg, and its estimated purity by LCMS analysis was 97%.

[0697] Analysis LCMS Condition H: retention time=1.52 min.; ESI-MS(+) m/z 960.9 (M+2H).

[0698] Analysis LCMS Condition I: retention time=2.81 min.; ESI-MS(+) m/z 960.7 (M+2H).

Preparation of Example 1061

[0699] ##STR00061##

[0700] Example 1061 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Symphony Method B: Custom amino acids-coupling procedure”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 50-100% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 20-60% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 14.4 mg, and its estimated purity by LCMS analysis was 96%.

[0701] Analysis LCMS Condition H: retention time=1.54 min.; ESI-MS(+) m/z 956.3 (M+2H).

[0702] Analysis LCMS Condition I: retention time=2.84 min.; ESI-MS(+) m/z 956.8 (M+2H).

Preparation of Example 1100

[0703] ##STR00062##

[0704] Example 1100 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 15-65% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 5.9 mg, and its estimated purity by LCMS analysis was 99%.

[0705] Analysis LCMS Condition D: Retention time=1.53 min; ESI-MS(+) m/z 893.2 (M+2H).

[0706] Analysis LCMS Condition E: Retention time=1.51 min; ESI-MS(+) m/z 893.3 (M+2H).

Preparation of Example 1101

[0707] ##STR00063##

[0708] Example 1101 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 35-75% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 3.2 mg, and its estimated purity by LCMS analysis was 98%.

[0709] Analysis LCMS Condition H: Retention time=1.404 min; ESI-MS(+) m/z 930.85 (M+2H).

[0710] Analysis LCMS Condition I: Retention time=2.715 min; ESI-MS(+) m/z 930.90 (M+2H).

Preparation of Example 1102

[0711] ##STR00064##

[0712] Example 1102 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 35-75% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 19.0 mg, and its estimated purity by LCMS analysis was 98%.

[0713] Analysis LCMS Condition H: Retention time=1.796 min; ESI-MS(+) m/z 941.20 (M+2H).

[0714] Analysis LCMS Condition I: Retention time=2.389 min; ESI-MS(+) m/z 940.95 (M+2H).

Preparation of Example 1103

[0715] ##STR00065##

[0716] Example 1103 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 15-55% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 1.3 mg, and its estimated purity by LCMS analysis was 98%.

[0717] Analysis LCMS Condition H: Retention time=1.60 min; ESI-MS(+) m/z 932.1 (M+2H).

[0718] Analysis LCMS Condition I: Retention time=2.70 min; ESI-MS(+) m/z 932.8 (M+2H).

Preparation of Example 1104

[0719] ##STR00066##

[0720] Example 1104 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 35-75% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 1.8 mg, and its estimated purity by LCMS analysis was 95%.

[0721] Analysis LCMS Condition H: Retention time=1.537 min; ESI-MS(+) m/z 961.65 (M+2H).

[0722] Analysis LCMS Condition I: Retention time=2.988 min; ESI-MS(+) m/z 961.10 (M+2H).

Preparation of Example 1105

[0723] ##STR00067##

[0724] Example 1105 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 methanol:water with 0.1% trifluoroacetic acid; Gradient: 40-80% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-55% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 3.9 mg, and its estimated purity by LCMS analysis was 92%.

[0725] Analysis LCMS Condition H: Retention time=1.622 min; ESI-MS(+) m/z 935.80 (M+2H).

[0726] Analysis LCMS Condition I: Retention time=3.125 min; ESI-MS(+) m/z 935.25 (M+2H).

Preparation of Example 1106

[0727] ##STR00068##

[0728] Example 1106 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 40-80% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 15-55% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 6.5 mg, and its estimated purity by LCMS analysis was 97%.

[0729] Analysis LCMS Condition H: Retention time=1.419 min; ESI-MS(+) m/z 964.80 (M+2H).

[0730] Analysis LCMS Condition I: Retention time=2.875 min; ESI-MS(+) m/z 946.95 (M+2H).

Preparation of Example 1107

[0731] ##STR00069##

[0732] Example 1107 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 15-55% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 40-80% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 0.8 mg, and its estimated purity by LCMS analysis was 100%.

[0733] Analysis LCMS Condition H: Retention time=1.624 min; ESI-MS(+) m/z 939.80 (M+2H).

[0734] Analysis LCMS Condition I: Retention time=3.090 min; ESI-MS(+) m/z 939.85 (M+2H).

Preparation of Example 1108

[0735] ##STR00070##

[0736] Example 1108 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 15-55% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 1.0 mg, and its estimated purity by LCMS analysis was 100%.

[0737] Analysis LCMS Condition H: Retention time=1.702 min; ESI-MS(+) m/z 942.90 (M+2H).

[0738] Analysis LCMS Condition I: Retention time=3.245 min; ESI-MS(+) m/z 942.95 (M+2H).

Preparation of Example 1109

[0739] ##STR00071##

[0740] Example 1109 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 15-55% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 2.5 mg, and its estimated purity by LCMS analysis was 100%.

[0741] Analysis LCMS Condition I: Retention time=2.73 min; ESI-MS(−) m/z 913.6 (M-2H).

Preparation of Example 1110

[0742] ##STR00072##

[0743] Example 1110 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 40-80% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 4.4 mg, and its estimated purity by LCMS analysis was 94%.

[0744] Analysis LCMS Condition H: Retention time=1.494 min; ESI-MS(+) m/z 936.15 (M+2H).

[0745] Analysis LCMS Condition I: Retention time=2.966 min; ESI-MS(+) m/z 935.80 (M+2H).

Preparation of Example 1111

[0746] ##STR00073##

[0747] Example 1111 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 45-85% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 19.8 mg, and its estimated purity by LCMS analysis was 99%.

[0748] Analysis LCMS Condition H: Retention time=1.56 min; ESI-MS(+) m z 933.4 (M+2H).

[0749] Analysis LCMS Condition I: Retention time=2.71 min; ESI-MS(+) m z 933.4 (M+2H).

Preparation of Example 1113

[0750] ##STR00074##

[0751] Example 1113 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 45-85% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 4.2 mg, and its estimated purity by LCMS analysis was 98%.

[0752] Analysis LCMS Condition H: Retention time=1.53 min; ESI-MS(+) m/z 926.1 (M+2H).

[0753] Analysis LCMS Condition I: Retention time=2.61 min; ESI-MS(+) m/z 925.8 (M+2H).

Preparation of Example 1114

[0754] ##STR00075##

[0755] Example 1114 was prepared following the general synthetic sequence described for the preparation of Example 1001 of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 45-85% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mM ammonium acetate; Gradient: 15-55% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 11.0 mg, and its estimated purity by LCMS analysis was 98%.

[0756] Analysis LCMS Condition H: Retention time=1.57 min; ESI-MS(+) m/z 925.9 (M+2H).

[0757] Analysis LCMS Condition I: Retention time=2.65 min; ESI-MS(+) m/z 925.8 (M+2H).

Preparation of Example 1115

[0758] ##STR00076##

[0759] Example 1115 was prepared following the general synthetic sequence described for the preparation of Example 1001 composed of the following general procedures: “Symphony Method B: Resin-swelling procedure”, “Symphony Method B: Standard-coupling procedure”, “Symphony Method B: Secondary amine-coupling procedure”, “Manual Coupling procedure A”, “Symphony Method B: Final capping procedure”, “Global Deprotection Method F”, and “Cyclization Method D”. The crude material was purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammonium acetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammonium acetate; Gradient: 45-90% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The material was further purified via preparative LC/MS with the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 10-50% B over 30 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of the product was 1.2 mg, and its estimated purity by LCMS analysis was 100%.

[0760] Analysis LCMS Condition H: Retention time=1.60 min; ESI-MS(+) m/z 938.3 (M-2H).

[0761] Analysis LCMS Condition I: Retention time=2.80 min; ESI-MS(+) m/z 940.3 (M+2H).

[0762] Analytical Data:

[0763] Mass Spectrometry: “ESI-MS(+)” signifies electrospray ionization mass spectrometry performed in positive ion mode; “ESI-MS(−)” signifies electrospray ionization mass spectrometry performed in negative ion mode; “ESI-HRMS(+)” signifies high-resolution electrospray ionization mass spectrometry performed in positive ion mode; “ESI-HRMS(−)” signifies high-resolution electrospray ionization mass spectrometry performed in negative ion mode. The detected masses are reported following the “m/z” unit designation. Compounds with exact masses greater than 1000 were often detected as double-charged or triple-charged ions.

[0764] High resolution mass spectrometry (HRMS) analyses were performed on a Fourier Transform Orbitrap mass spectrometer (Exactive, Thermo Fisher Scientific, San Jose, Calif.) using positive or negative electrospray ionization operating at 25,000 resolution (full width at half height maximum, FWHM). The instrument was calibrated daily according to manufacturer's specifications resulting in mass accuracy errors <5 ppm. The operating software, Xcalibur, was used to calculate theoretical mass-to-charge values and to process the obtained data.

[0765] Analysis LCMS Condition A:

[0766] Column: BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: water with 0.05% TFA; Mobile Phase B:Acetonitrile with 0.05% TFA; Temperature: 50° C.; Gradient: 2% B to 98% B over 1 min., then a 0.5 min. hold at 98% B; Flow: 0.8 mL/min; Detection: UV at 220 nm.

[0767] Analysis LCMS Condition D:

[0768] Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.11 mL/min; Detection: UV at 220 nm.

[0769] Analysis LCMS Condition E:

[0770] Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.11 mL/min; Detection: UV at 220 nm.

[0771] General Procedures:

[0772] CEM MethodA:

[0773] All manipulations were performed under automation on a CEM Liberty microwave peptide synthesizer (CEM Corporation). All procedures unless noted were performed in a 30 or 125 mL polypropylene tube fitted with a bottom frit to a CEM Discovery microwave unit. The tube connects to a the CEM Liberty synthesizer through both the bottom and the top of the tube. DMF and DCM can be added through the top and bottom of the tube, which washes down the sides of the tube equally. All solutions are removed through the bottom of the tube except while transferring resin from the top. “Periodic bubbling” describes a brief bubbling of N.sub.2 gas through the bottom frit. Amino acid solutions were generally not used beyond three weeks from preparation. HATU solution was used within 5 days of preparation. DMF=dimethylformamide; HCTU=2-(6-Chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium; HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; DIPEA=diisopropylethylamine; Sieber=Fmoc-amino-xanthen-3-yloxy, where “3-yloxy” describes the position and type of connectivity to the polystyrene resin. The resin used is Merrifield polymer (polystyrene) with a Sieber linker (Fmoc-protected at nitrogen); 100-200 mesh, 1% DVB, 0.71 mmol/g loading. Common amino acids used are listed below with side-chain protecting groups indicated inside parenthesis. Fmoc-Ala-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Bzt-OH; Fmoc-Cys(Trt)-OH; Fmoc-Dab(Boc)-OH; Fmoc-Dap(Boc)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Gly-OH; Fmoc-His(Trt)-OH; Fmoc-Hyp(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Lys(Boc)-OH; Fmoc-Nle-OH; Fmoc-Met-OH; Fmoc-[N-Me]Ala-OH; Fmoc-[N-Me]Nle-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Sar-OH; Fmoc-Ser(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Trp(Boc)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH.

[0774] The procedures of “CEM Method A” describe an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. This scale corresponds to approximately 140 mg of the Sieber-Merrifield resin described above. All procedures can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. Prior to amino acid coupling, all peptide synthesis sequences began with a resin-swelling procedure, described below as “Resin-swelling procedure”. Coupling of amino acids to a primary amine N-terminus used the “Single-coupling procedure” described below. Coupling of amino acids to a secondary amine N-terminus used the “Secondary amine-coupling procedure” described below. Coupling of chloroacetyl group to the N-terminus of the peptide is described by the “Chloroacetyl chloride coupling procedure” or “Chloroacetic acid coupling procedure” detailed above.

[0775] Resin-Swelling Procedure:

[0776] To 50 mL polypropylene conical tube was added Merrifield:Sieber resin (140 mg, 0.100 mmol). Then DMF (7 mL) was added to the tube followed by DCM (7 mL). The resin was then transferred to the reaction vessel from top of the vessel. The procedure is repeated additionally two times. DMF (7 mL) was added followed by DCM (7 mL). The resin was allowed to swell with N.sub.2 bubbling from the bottom of the reaction vessel for 15 minutes before the solvent was drained through the frit.

[0777] Standard Coupling Procedure:

[0778] To the reaction vessel containing resin from the previous step was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5 eq), and DIPEA (2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N.sub.2 bubbling for 5 minutes at 75° C. for all amino acids, except Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH which are coupled at 50° C., the reaction solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically bubbled for 2 minutes at 65° C., then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. The resulting resin was used directly in the next step.

[0779] Double-Couple Coupling Procedure:

[0780] To the reaction vessel containing resin from the previous step was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5 eq), and DIPEA (2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N.sub.2 bubbling for 5 minutes at 75° C. for all amino acids, except Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH which are coupled at 50° C., the reaction solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added the amino acid (0.2M in DMF, 2.5 mL, 5 eq), HATU (0.5M in DMF, 1.0 mL, 5 eq), and DIPEA (2M in NMP, 0.5 mL, 10 eq). The mixture was mixed by N.sub.2 bubbling for 5 minutes at 75° C. for all amino acids, except Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH which are coupled at 50° C., the reaction solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically bubbled for 2 minutes at 65° C., then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. The resulting resin was used directly in the next step.

[0781] Custom Amino Acids-Coupling Procedure:

[0782] To the reaction vessel containing resin from the previous step was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added a solution of piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added the amino acid solution (1.25 mL to 5 mL, 2.5 eq to 10 eq) containing HATU (2.5 eq to 10 eq), and finally DIPEA (2M in NMP, 0.5 mL to 1 mL, 20 eq). The mixture was mixed by N.sub.2 bubbling for 5 minutes to 2 hours at 25° C. to 75° C., then the reaction solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added a solution of acetic anhydride:DIEA:DMF (10:1:89 v/v/v, 5.0 mL). The mixture was periodically bubbled for 2 minutes at 65° C., then the solution was drained through the frit. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. The resulting resin was used directly in the next step.

[0783] Chloroacetyl Chloride Coupling Procedure A:

[0784] To the reaction vessel containing the resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added 3.0 mL of a solution of DIPEA (4.0 mmol, 0.699 mL, 40 eq), and chloroacetyl chloride (2.0 mmol, 0.160 mL, 20 eq) in DMF. The mixture was periodically agitated for 12 to 18 hours, then the solution was drained through the frit. The resin was washed successively three times as follows: for each wash, DMF (4.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, CH.sub.2Cl.sub.2 (2.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit.

[0785] Chloroacetic Acid Coupling Procedure A:

[0786] To the reaction vessel containing the resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit. To the reaction vessel was added DMF (2.0 mL), chloroacetic acid (1.2 mmol, 113 mg, 12 eq), and N,N′-Diisopropylcarbodiimide (1.2 mmol, 0.187 mL, 12 eq). The mixture was periodically agitated for 12 to 18 hours, then the solution was drained through the frit. The resin was washed successively three times as follows: for each wash, DMF (4.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit. The resin was washed successively four times as follows: for each wash, CH.sub.2Cl.sub.2 (2.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit.

[0787] Global Deprotection Method B:

[0788] All manipulations were performed manually unless noted. The procedure of “Global Deprotection Method B” describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. A “deprotection solution” was prepared using trifluoroacetic acid:triisopropylsilane:dithiothreitol (94:3:3 v:v:w). The resin was removed from the reaction vessel and transferred to a 25 mL syringe equipped with a frit. To the syringe was added the “deprotection solution” (5.0 mL). The mixture was mixed in a shaker for 5 minutes. The solution was filtered through and diluted in diethyl ether (30 mL). The precipitated solid was centrifuged for 3 minutes. The supernatant solution was decanted and the solid was resuspended in diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was suspended in diethyl ether (25 mL). The suspension was centrifuged for 3 minutes. The supernatant was decanted and the remaining solid was dried under high vacuum. The crude peptide was obtained as a white to off-white solid.

[0789] Cyclization Method C:

[0790] All manipulations were performed manually unless noted. The procedure of “Cyclization Method C” describes an experiment performed on a 0.100 mmol scale, where the scale is determined by the amount of Sieber linker bound to the resin that was used to generate the peptide. This scale is not based on a direct determination of the quantity of peptide used in the procedure. The procedure can be scaled beyond 0.100 mmol scale by adjusting the described volumes by the multiple of the scale. The crude peptide solids were dissolved in a solution of acetonitrile:aqueous 0.1M ammonium bicarbonate buffer (11 mL:24 mL), and the solution was then carefully adjusted to pH=8.5-9.0 using aq NaOH (1.0M). The solution was then mixed using a shaker for 12 to 18 hours. The reaction solution was concentrated and the residue was then dissolved in acetonitrile:water. This solution was subjected to reverse-phase HPLC purification to afford the desired cyclic peptide.

Preparation of (S)-2-(5-((S)-1-((((9H-fluoren-9-yl)methoxy) carbonyl)amino)-2-phenylethyl)-1H-tetrazol-1-yl)-3-phenylpropanoic acid [Fmoc-Pheψ[CN.SUB.4.]-L-Phe-OH]

[0791] ##STR00077##

[0792] Step 1:

[0793] was treated with 1-Hydroxy-7-azabenzotriazole (1.120 g, 8.23 mmol) and EDC (1.577 g, 8.23 mmol) and stirred at rt for 15 h. The mixture was washed with saturated NaHCO.sub.3 (2×100 mL), 1 M HCl (2×50 mL) and brine (50 mL), dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure to give (S)-benzyl 2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)-3-phenylpropanoate (3.2 g, 5.96 mmol, 87% yield) as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.42-7.11 (m, 21H), 4.96 (d, J=2.0 Hz, 4H), 4.63-4.45 (m, 1H), 4.06-3.91 (m, 2H), 3.34-2.96 (m, 3H), 2.85 (br. s., 4H).

[0794] Step 2 (Schroeder, G. M.; Marshall, S.; Wan, H.; Purandare, A. V. Tetrahedron Lett. 2010, 51, 1404-1406.):

[0795] A pressure vial was charged with (S)-benzyl 2-((S)-2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)-3-phenylpropanoate (1.0 g, 1.864 mmol) and Diphenyl-2-pyridylphosphine (3.17 g, 7.45 mmol) in THF (9.32 ml) under N.sub.2. The solution was purged with N.sub.2 before DIAD (1.449 ml, 7.45 mmol) was added dropwise. The reaction was allowed to stir for 5 mins. Diphenylphosphoryl azide (1.606 ml, 7.45 mmol) was then added dropwise over 30 mins. After the addition was completed and there was no more nitrogen formation, the sealed tube was closed and the reaction is allowed to stir at 55° C. for 4 hours.

[0796] The reaction was cooled down to RT and poured into ethyl acetate (200 mL), washed with cold 1N HCl (100 mL), sat. NaHCO.sub.3 (100 mL) and brine (100 mL). The organic layer dried over anhydrous sodium sulfate, filtered and concentrated to give an orange oil. Purification by silical gel chormatography using 100% hexanes to 60% Ethyl acetate in hexanes as eluent afforded 660 mg of (S)-benzyl 2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-phenylethyl)-1H-tetrazol-1-yl)-3-phenylpropanoate with low purity. Second purification by preparative HPLC using the following conditions: Column: Phenomenex Luna 5u C18(2) 100A 250×21.2 mm AXIA packed (10-100 mg)#520551-2. Mobile Phase A: 0.1 TFA in water; Mobile Phase B: 0.1% TFA in acetonitrile; Gradient: 50-80% B over 20 minutes, then a 10-minute gradient 95% B; Flow: 15 mL/min; Detection: UV at 220 nm. Fractions containing the desired product were combined and dried via centrifugal evaporation. (S)-benzyl 2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-phenylethyl)-1H-tetrazol-1-yl)-3-phenylpropanoate was obtained as a beige hygroscopic solid in 60.0 mg yield in 70% purity by LC/MS analysis. Analysis condition A: Retention time=1.82 min; ESI-MS(+) m/z 562.5 (M+H).

[0797] Step 3:

[0798] A solution of (S)-benzyl 2-(5-((S)-1-(((benzyloxy)carbonyl)amino)-2-phenylethyl)-1H-tetrazol-1-yl)-3-phenylpropanoate (60 mg, 0.107 mmol) in MeOH (534 μl) was hydrogenated over 10% Palladium on Carbon (22.74 mg, 0.021 mmol) using hydrogen from a latex balloon for 2 h. The catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to give a white gummy solid. The product was dissolved in MeCN (2 mL) and water (2 mL) and Et.sub.3N (100 μL) and treated with FMOC-OSu (36.0 mg, 0.107 mmol). The mixture was stirred at rt for 15 h. The reaction mixture was diluted with EtOAc (50 mL), washed with 1 M HCl (2×20 mL) and brine (20 mL), dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure to give Fmoc-Pheψ[CN.sub.4]-L-Phe-OH [(S)-2-(5-((S)-1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-phenylethyl)-1H-tetrazol-1-yl)-3-phenylpropanoic acid] (42 mg, 0.075 mmol, 70% yield) as a yellow solid. Product purity was estimated to be 64% by LC/MS analysis.

[0799] Analysis LCMS condition A: Retention time=1.70 min; ESI-MS(+) m/z 560.5 (M+H).

Preparation of 2-(5-(1-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)cyclohexyl)-1H-tetrazol-1-yl)acetic acid [Fmoc-Acc.SUB.6.ψ[CN.SUB.4.]-Gly-OH

[0800] ##STR00078##

[0801] Step 1.

[0802] A solution of 2-(5-(1-(benzylamino)cyclohexyl)-1H-tetrazol-1-yl)acetic acid (Chembridge; CAS No. 915920-47-7) (0.500 g, 1.585 mmol) in MeOH (7.48 ml) was hydrogenated over 10% Palladium on Carbon (0.675 g, 0.634 mmol) using hydrogen from a latex balloon for 16 h. The catalyst was removed by filtration on diatomaceous earth (Celite®) with DCM washing and the resulting solution was concentrated under reduced pressure.

[0803] Step 2.

[0804] To a solution of the crude amino acid from Step 1 in Acetonitrile (3.74 ml), Water (3.74 ml) and Triethylamine (0.221 ml, 1.585 mmol), cooled to 0° C., was added (9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (0.535 g, 1.585 mmol) and the reaction mixture was stirred at 0° C. for 4 h. The solution was diluted in EtOAc, washed with 1N HCl and brine, dried over anhydrous Na.sub.2SO.sub.4 and concentrated to a yellow oil under reduced pressure. This was purified by flash chromatography using a gradient from 0-100% EtOAc in hexanes. The column was flushed with 30% MeOH/DCM to completely elute the product. The fractions containing the desired product were pooled and concentrated under reduced pressure, to yield 116 mg (16% overall yield) of the desired product as a brown solid. Product purity was estimated to be near 100% by LC/MS analysis.

[0805] Analysis LCMS condition A: Retention time=1.45 min; ESI-MS(+) m/z 448.0 (M+H).

Preparation of Examples 3001 and 3002

[0806] ##STR00079##

[0807] Examples 3001 and 3002 were prepared following the general synthetic sequence described below.

[0808] To a 50 mL polypropylene tube was added Sieber resin (140 mg, 0.100 mmol), and the tube was placed on the CEM Liberty microwave peptide synthesizer.

[0809] The following procedures were then performed sequentially:

“CEM Method A: Resin-swelling procedure” was followed;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Gly-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Cys(Trt)-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Leu-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Tyr(tBu)-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Tyr(tBu)-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Sar-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Tyr(tBu)-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-[N-Me]Phe-OH;
“CEM Method A: Custom amino acids-coupling procedure” was followed with Fmoc-Val-OH using 10 eq for 2 hours at 75° C.;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Asp(OtBu)-OH;
“CEM Method A: Standard coupling procedure” was followed with Fmoc-Sar-OH;
“CEM Method A: Custom amino acids-coupling procedure” was followed with Fmoc-[N-Me]Nle-OH using 5 eq for 10 min;
“CEM Method A: Custom amino acids-coupling procedure” was followed with Fmoc-Pheψ[CN.sub.4]-L-Phe-OH using 5 eq for 10 min; “Chloroacetyl chloride coupling procedure A” was followed, “Global Deprotection Method B” was followed and “Cyclization Method C” was followed.

[0810] Example 3001, Isomer 1. The crude material was purified via preparative LC/MS with the following conditions: Column: Phenomenex Luna 5u C18(2) 250×21.2 AXIA, 100A Ser.#520221-1; Mobile Phase A: 0.1% TFA in water; Mobile Phase B: 0.1% TFA in acetonitrile; Gradient: 35-75% B over 50 minutes, then a 5-minute gradient up to 95% B; Flow: 15 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation and lyophilization. The yield of Example 3001, Isomer 1 was 1.5 mg, and its estimated purity by HPLC analysis was 82.0% using the following conditions: Column: Phenom Kinetex 2.6u C18(2) 2.1×50 mm Ser.#515561-57; Mobile Phase A: 0.025% Ammonium Acetate in 5% methanol/water; Mobile Phase B: Acetonitrile/water (4:1); Gradient: 30-95% B over 20 minutes at 60° C.; Flow: 1 mL/min. Detection UV: 217 nm.

[0811] Analysis LCMS condition A: Retention time=1.50 min; ESI-MS(+) m/z 904.5 (M+2H).

[0812] A second isomer, Isomer 2, was isolated from the above preparative LC/MS conditions. The yield of Example 3002, Isomer 2 was 1.6 mg, and its estimated purity by HPLC analysis was 89.6% using the following conditions: Column: Phenom Kinetex 2.6u C18(2) 2.1×50 mm Ser.#515561-57; Mobile Phase A: 0.025% Ammonium Acetate in 5% methanol/water; Mobile Phase B: Acetonitrile/water (4:1); Gradient: 30-95% B over 20 minutes at 60° C.; Flow: 1 mL/min. Detection UV: 217 nm.

[0813] Analysis LCMS condition A: Retention time=1.52 min; ESI-MS(+) m/z 904.5 (M+2H).

Preparation of Example 3003

[0814] ##STR00080##

[0815] Example 3003 was prepared following the general synthetic sequence described for the preparation of Example 3001, composed of the following general procedures: “CEM Method A: Resin-swelling procedure”, “CEM Method A: Standard coupling procedure”, “CEM Method A: Custom amino acids-coupling procedure”, “Chloroacetic acid coupling procedure A”, “Global Deprotection Method B”, and “Cyclization Method C”. Fmoc-[2-(5-(1-aminocyclohexyl)-1H-tetrazol-1-yl)]-OH [Fmoc-Acc.sub.6ψ[CN.sub.4]-Gly-OH was coupled after the Trp residue using a “CEM Method A: Custom amino acids-coupling procedure”.

[0816] The crude material was purified via preparative LC/MS using the following conditions: Column: XBridge C18 300, 19×250 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1% trifluoroacetic acid; Gradient: 30-70% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product were combined and dried via centrifugal evaporation. The yield of product was 0.37 mg, and its estimated purity by LCMS analysis was 82% using “Analysis LCMS conditions D and E”.

[0817] Analysis LCMS condition D: Retention time=1.66 min; ESI-MS(+) m/z 885.5 (M+2H).

[0818] Analysis LCMS condition E: Retention time=1.87 min; ESI-MS(+) m/z 885.4 (M+2H).

[0819] ESI-HRMS(+) m/z:

[0820] Calculated: 884.9371 (M+2H).

[0821] Found: 884.9343 (M+2H).

Methods for Testing the Ability of Macrocyclic Peptides to Compete for the Binding of Pd-1 to Pd-L1 Using Homogenous Time-Resolved Fluorescence (HTRF) Binding Assays

[0822] The ability of the macrocyclic peptides of the present disclosure to bind to PD-L1 was investigated using a PD-1/PD-L1 Homogenous Time-Resolved Fluorescence (HTRF) binding assay.

[0823] Methods

[0824] Homogenous Time-Resolved Fluorescence (HTRF) Assays of Binding of Soluble PD-1 to Soluble PD-L1. Soluble PD-1 and soluble PD-L1 refers to proteins with carboxyl-end truncations that remove the transmembrane-spanning regions and are fused to heterologous sequences, specifically the Fc portion of the human immunoglobuling G sequence (Ig) or the hexahistidine epitope tag (SEQ ID NO: 3) (His). All binding studies were performed in an HTRF assay buffer consisting of dPBS supplemented with 0.1% (w/v) bovine serum albumin and 0.05% (v/v) Tween-20. For the PD-1-Ig/PD-L1-His binding assay, inhibitors were pre-incubated with PD-L1-His (10 nM final) for 15 m in 4 μl of assay buffer, followed by addition of PD-1-Ig (20 nM final) in 1 μl of assay buffer and further incubation for 15 m. PD-L1 fusion proteins from either human, cynomologous macaques, mouse, or other species were used. HTRF detection was achieved using europium crypate-labeled anti-Ig monoclonal antibody (1 nM final) and allophycocyanin (APC) labeled anti-His monoclonal antibody (20 nM final). Antibodies were diluted in HTRF detection buffer and 5 μl was dispensed on top of binding reaction. The reaction was allowed to equilibrate for 30 minutes and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer. Additional binding assays were established between PD-1-Ig/PD-L2-His (20 and 5 nM, respectively), CD80-His/PD-L1-Ig (100 and 10 nM, respectively) and CD80-His/CTLA4-Ig (10 and 5 nM, respectively). Binding/competition studies between biotinylated Compound No. 71 and human PD-L1-His were performed as follows. Macrocyclic peptide inhibitors were pre-incubated with PD-L1-His (10 nM final) for 60 minutes in 4 μl of assay buffer followed by addition of biotinylated Compound No. 71 (0.5 nM final) in 1 μl of assay buffer. Binding was allowed to equilibrate for 30 minutes followed by addition of europium crypated labeled Streptavidin (2.5 pM final) and APC-labeled anti-His (20 nM final) in 5 μl of HTRF buffer. The reaction was allowed to equilibrate for 30 m and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer.

[0825] Recombinant Proteins. Carboxyl-truncated human PD-1 (amino acids 25-167) with a C-terminal human Ig epitope tag [hPD-1 (25-167)-3S-IG] and human PD-L1 (amino acids 18-239) with a C-terminal His epitope tag [hPD-L1(19-239)-tobacco vein mottling virus protease cleavage site (TVMV)-His] were expressed in HEK293T cells and purified sequentially by recombinant Protein A affinity chromatography and size exclusion chromatography. Human PD-L2-His (Sino Biologicals), CD80-His (Sino Biologicals), CTLA4-Ig (RnD Systems) were all obtained through commercial sources.

[0826] Sequence of Recombinant Human PD-1-Ig

TABLE-US-00001 hPD1(25-167)-3S-IG (SEQ ID NO: 1) 1 LDSPDRPWNP PTFSPALLVV TEGDNATFTC SFSNTSESFV LNWYRMSPSN 51 QTDKLAAFPE DRSQPGQDCR FRVTQLPNGR DFHMSVVRAR RNDSGTYLCG 101 AISLAPKAQI KESLRAELRV TERRAEVPTA HPSPSPRPAG QFQGSPGGGG 151 GREPKSSDKT HTSPPSPAPE LLGGSSVFLF PPKPKDTLMI SRTPEVTCVV 201 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 251 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 301 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLVSKLTVD 351 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

[0827] Sequence of Recombinant Human PD-L1-TVMV-His(PD-L1-His)

TABLE-US-00002 hPDL1(19-239)-TVMV-His (SEQ ID NO: 2) 1 PTVTVPKDLY VVEYGSNMTI ECKFPVEKQL DLAALIVYWE MEDKNIIQFV 51 HGEEDLKVQH SSYRQRARLL KDQLSLGNAA LQITDVKLQD AGVYRCMISY 101 GGADYKRITV KVNAPYNKIN QRILVVDPVT SEHELTCQAE GYPKAEVIWT 151 SSDHQVLSGK TTTTNSKREE KLFNVTSTLR INTTTNEIFY CTFRRLDPEE 201 NHTAELVIPE LPLAHPPNER TGSSETVRFQ GHHHHHH

[0828] The results are shown in Table 1. As shown, the macrocyclic peptides of the present disclosure demonstrated potent inhibition of PD-1-Ig binding activity to PD-L1-TVMV-His (PD-L1-His). Ranges are as follows: A=0.10-10 μM; B=0.01-0.099 μM; C=0.005-0.0099 μM.

TABLE-US-00003 TABLE 1 Example Number HTRF IC50 (μM) Example 1001 A Example 1002 0.20 Example 1003 A Example 1004 A Example 1005 A Example 1006 A Example 1007 A Example 1010 A Example 1012 0.03 Example 1013 B Example 1014 C Example 1050 A Example 1051 A Example 1052 B Example 1053 B Example 1054 A Example 1055  0.008 Example 1056 A Example 1057 A Example 1058 B Example 1059 B Example 1060 0.02 Example 1061 A Example 1100 A Example 1101 A Example 1102 A Example 1103 A Example 1104 B Example 1105 A Example 1106 A Example 1107 A Example 1108 A Example 1109 B Example 1110 A Example 1111 2.16 Example 1113 A Example 1114 A Example 3001 A Example 3002 A Example 3003 0.59

[0829] It will be evident to one skilled in the art that the present disclosure is not limited to the foregoing illustrative examples, and that it can be embodied in other specific forms without departing from the essential attributes thereof. It is therefore desired that the examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing examples, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.