METHODS OF MAKING INCRETIN ANALOGS

Abstract

Intermediate compounds are disclosed for making incretin analogs, or pharmaceutically acceptable salts thereof. In addition, methods are disclosed for making incretin analogs by coupling from two to four of the intermediate compounds herein via hybrid liquid solid phase synthesis or native chemical ligation.

Claims

1. A method of making an incretin analog of SEQ ID NO:6, the method comprising the step of: coupling, via hybrid liquid solid phase synthesis, four intermediate compounds selected from the groups consisting of: a. SEQ ID NOS:7, 8, 9 and 10, b. SEQ ID NOS:7, 11, 12, and 10, and c. SEQ ID NOS:7, 13, 14 and 10.

2. A method of making an incretin analog of SEQ ID NO:6, the method comprising the step of: coupling, via hybrid liquid solid phase synthesis, three intermediate compounds selected from the groups consisting of: a. SEQ ID NOS:7, 13 and 15, b. SEQ ID NOS:16, 17 and 10, c. SEQ ID NOS:18, 12 and 10, and d. SEQ ID NOS:7, 45 and 10.

3. A method of making an incretin analog of SEQ ID NO:6, the method comprising the step of: coupling, via hybrid liquid solid phase synthesis, two intermediate compounds selected from the groups consisting of: a. SEQ ID NOS:15 and 19 and b. SEQ ID NOS:18 and 20.

4. An intermediate compound comprising: any one of SEQ ID NOS:7 to 28, 30 to 54 and 56 to 62, or a pharmaceutically acceptable salt thereof.

5-37. (canceled)

38. A method of making an incretin analog of SEQ ID NO:29, the method comprising the step of: coupling, via hybrid liquid solid phase synthesis, intermediate compounds selected from the groups consisting of: a. SEQ ID NOS:7, 62, 42 and 31, b. SEQ ID NOS:43, and 44.

Description

EXAMPLES

[0131] The following non-limiting examples are offered for purposes of illustration, not limitation.

Peptide and Polypeptide Synthesis

Example 1: Solid Phase Peptide Synthesis of Intermediate Compound 1

[0132] Intermediate Compound 1 (SEQ ID NO:7), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Sieber resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 1.

TABLE-US-00001 TABLE 1 SPPS Conditions for Example 1. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 2 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 3 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 0% Pip/DMF 6 hr, rt 4 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 3-4* Alternatively use Fmoc-L-Pro-Pro-OH dimer instead of step 3 & 4 5 2 × 30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 6 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 7 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 8 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 9 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 10  2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt

[0133] Fmoc Deprotection, Fragment Cleavage and Isolation: Fragment on Sieber resin is stirred twice with 10 V of 20% piperidine/DMF for 20-30 min, then washed six times with 10 V of DMF. The de-Fmoced fragment on Sieber resin is swelled twice using 10 V DCM for 10-20 min. A reactor with resin is cooled to about 15° C., and 20 V of 5% TFA/DCM is charged to the reactor and then stirred for 2 hr under nitrogen maintaining the temperature at about 15° C. The resin is filtered and washed with 3×10 V of DCM. All the filtrates are combined together. DCM is removed from the resulting solution under reduced pressure while maintaining the internal temperature at ≤20° C. to 22.5 V residual volume. MTBE (25 V) is charged to the solution, and DCM/MTBE solvents are again removed under reduced pressure while maintaining the internal temperature at ≤20° C. to 22.5 V residual volume. Addition of MTBE/distillation operation is repeated until residual concentration of fragment in supernatant has not reached <0.11 wt %. Then, the resulting slurry is filtered while maintaining temperature at about 15° C. To the cake, 14 V of fresh MTBE is added, is stirred for 30 min at about 15° C., and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 35° C.

Example 2: Solid Phase Peptide Synthesis of Intermediate Compound 2

[0134] Intermediate Compound 2 (SEQ ID NO:8), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Gly-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 2.

TABLE-US-00002 TABLE 2 SPPS Conditions for Example 2. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.5 AA/2.5 PyBOP/5.0 DIPEA 20% Pip/DMF 4 hr, rt 2 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 3 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 4 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 5 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 6 2 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 7 2 × 20-30 min Fmoc-L-Phe-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt

[0135] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 10 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen at about 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine, and then 5 V of DMSO is added to the filtrate. Resin treatment with 1% TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM, and stirred for 10-15 min. All the filtrates and wash are combined. The fragment solution is concentrated under vacuum to 6-10 V maintaining temperature at ≤35° C. (residual DCM concentration ≤15%). DMSO solution of the fragment is added to 11-15 V of H.sub.2O over 2-6 hr period (<1 L/min) at about 25° C. The formed slurry of precipitated fragment is stirred for 30-40 min at about 25° C. and then filtered. The resulting solid is suspended in 8-12 V of H.sub.2O at about 25° C., is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 40° C.

Example 3: Solid Phase Peptide Synthesis of Intermediate Compound 3

[0136] Intermediate Compound 3 (SEQ ID NO:9), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Ala-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 3.

TABLE-US-00003 TABLE 3 SPPS Conditions for Example 3. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-Aib-OH 2.5 AA/2.5 PyBOP/5.0 DIPEA 20% Pip/DMF 4 hr, rt 2 2 × 20-30 min Fmoc-L-Gln(Trt)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 3 2 × 20-30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 4 2 × 20-30 min Fmoc-L-Lys(t- 1.5 AA/1.65 DIC/1.5 Oxyma 20% Pip/DMF BuOOC—(CH.sub.2).sub.18—COO- 4 hr, rt γ-L-Glu-AEEA)* 5 2 × 20-30 min Fmoc-L-Lys(Boc)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 6 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt *Fmoc-L-Lys(t-BuOOC—(CH.sub.2).sub.18—COO-γ-L-Glu-AEEA)

##STR00015##

[0137] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 5 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen maintaining temperature at about 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine. Resin treatment with 1% TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM and stirred for 10-15 min. All the filtrates and wash are combined, and the resulting mixture cooled ≤20° C. The fragment solution is concentrated under vacuum to 2-4 V maintaining temperature at ≤20° C. 5 V of ACN is added to the solution, and residual DCM removed under vacuum (residual DCM concentration ≤X %) maintaining temperature at ≤20° C. An ACN solution of the fragment is added to 5 V of ice-cold H.sub.2O over 2-6 hr period (<1 L/min) maintaining temperature at about 0° C. The resulting slurry of precipitated fragment is stirred for 30-40 min at about 0° C. and then is filtered at about 0° C. The resulting solid is suspended in 3-5 V of H.sub.2O at about 25° C., is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 40° C.

Example 4: Solid Phase Peptide Synthesis of Intermediate Compound 4

[0138] Intermediate Compound 4 (SEQ ID NO:10), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Leu-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 4.

TABLE-US-00004 TABLE 4 SPPS Conditions for Example 4. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings:DMF  1 2 × 20-30 min Fmoc-L-2-Me-Ile- 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF OH 12-18 hr, rt capping procedure  2 3 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure  3 2 × 20-30 min Fmoc-L-Ser(t-Bu)- 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF OH 4-6 hr, rt  4 2 × 20-30 min Fmoc-L-Tyr(t-Bu)- 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF OH 4-6 hr, rt  5 2 × 20-30 min Fmoc-L-Asp(t-Bu)- 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF OH 4-6 hr, rt  6 2 × 10 min Fmoc-L-Ser(t-Bu)- 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% OH 4-6 hr, rt Pip/DMF  7 2 × 10 min Fmoc-L-Thr(t-Bu)- 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% OH 4-6 hr, rt Pip/DMF  8 2 × 10 min Fmoc-L-Phe(t-Bu)- 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% OH 4-6 hr, rt Pip/DMF  9 2 × 10 min Fmoc-L-Thr(t-Bu)- 2.0 AA/2.2 DIC/2.2 Oxyma 5% Oxyma/20% OH 4-6 hr, rt Pip/DMF 10 2 × 10 min Boc-L-Tyr(t-Bu)- 1.5 AA/1.65 DIC/1.5 Oxyma 5% Oxyma/20% Aib-L-Gln(Trt)-Gly- 4-6 hr, rt Pip/DMF OH* *structure for Boc-L-Tyr(t-Bu)-Aib-L-Gln(Trt)-Gly-OH is as follows: [00016]

[0139] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 5 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen maintaining temperature at about 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine. Resin treatment with 1% with TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM, and is stirred for 10-15 min. All the filtrates and wash are combined, and the resulting mixture is cooled to ≤20° C. The fragment solution is concentrated under vacuum to 2-4 V maintaining temperature at ≤20° C. 2 V of DMSO is added to the solution, and residual DCM removed under vacuum (residual DCM concentration ≤5%) maintaining temperature at ≤20° C. A DMSO solution of the fragment is added to 7-9 V of ice-cold H.sub.2O over 2-6 hr (<1 L/min), while maintaining temperature at about 0° C. The resulting slurry of precipitated fragment is stirred for 30-40 min at about 0° C. and then is filtered at about 0° C. The resulting solid is suspended in 3-5 V of H.sub.2O at about 25° C., is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at 40° C.

Example 5: Solid Phase Peptide Synthesis of Intermediate Compound 5

[0140] Intermediate Compound 5 (SEQ ID NO:11), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Gly-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 5.

TABLE-US-00005 TABLE 5 SPPS Conditions for Example 5. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.5 AA/2.5 PyBOP/5.0 DIPEA 20% Pip/DMF 4 hr, rt 2 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 3 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 4 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 5 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 6 2 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 7 2 × 20-30 min Fmoc-L-Phe-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 8 2 × 20-30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt

[0141] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 10 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen at about 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine, and then 5 V of DMSO is added to the filtrate. Resin treatment with 1% TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM and is stirred for 10-15 min. All the filtrates and wash are combined. The fragment solution is concentrated under vacuum to 6-10 V maintaining temperature at ≤35° C. (residual DCM concentration ≤15%). DMSO solution of the fragment is added to 11-15 V of H.sub.2O over 2-6 hr (<1 L/min) at about 25° C. The formed slurry of precipitated fragment is stirred for 30-40 min at about 25° C. and then is filtered. The resulting solid is suspended in 8-12 V of H.sub.2O at about 25° C., is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 40° C.

Example 6: Solid Phase Peptide Synthesis of Intermediate Compound 6

[0142] Intermediate Compound 6 (SEQ ID NO:12), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Aib-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 6.

TABLE-US-00006 TABLE 6 SPPS Conditions for Example 6. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-L-Gln(Trt)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 2 2 × 20-30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 3 2 × 20-30 min Fmoc-L-Lys(t- 1.5 AA/1.65 DIC/1.5 Oxyma 20% Pip/DMF BuOOC—(CH.sub.2).sub.18—COO- 4 hr, rt γ-L-Glu-AEEA) 4 2 × 20-30 min Fmoc-L-Lys(Boc)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 5 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt

[0143] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 5 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen maintaining temperature at about 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine. Resin treatment with 1% with TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM and is stirred for 10-15 min. All the filtrates and wash are combined, and the resulting mixture cooled to ≤20° C. The fragment solution is concentrated under vacuum to 2-4 V maintaining temperature at ≤20° C. 5 V of ACN is added to the solution, and residual DCM is removed under vacuum (residual DCM concentration ≤15%) maintaining temperature at ≤20° C. An ACN solution of the fragment is added to 5 V of ice-cold H.sub.2O over 2-6 hr (<1 L/min), while maintaining temperature at about 0° C. The resulting slurry of precipitated fragment is stirred for 30-40 min at about 0° C. and then is filtered at about 0° C. The resulting solid is suspended in 3-5 V of H.sub.2O at about 25° C., is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 40° C.

Example 7: Solid Phase Peptide Synthesis of Intermediate Compound 7

[0144] Intermediate Compound 7 (SEQ ID NO:13), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Gly-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 7.

TABLE-US-00007 TABLE 7 SPPS Conditions for Example 7. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.5 AA/2.5 PyBOP/5.0 DIPEA 20% Pip/DMF 4 hr, rt 2 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 3 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 4 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt, 5 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 6 2 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 7 2 × 20-30 min Fmoc-L-Phe-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 8 2 × 20-30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 9 2 × 20-30 min Fmoc-Aib-OH 2.0 AA/2.2 PyBOP/4.0 DIEA 20% Pip/DMF 6-10 hr, rt 10 2 × 20-30 min Fmoc-L-Gln(Trt)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure

[0145] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 10 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen at 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine, and then 5 V of DMSO is added to the filtrate. Resin treatment with 1% TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM and is stirred for 10-15 min. All the filtrates and wash are combined. The fragment solution is concentrated under vacuum to 6-10 V maintaining temperature at ≤35° C. (residual DCM concentration ≤15%). DMSO solution of the fragment is added to 11-15 V of H.sub.2O over 2-6 hr (<1 L/min) at about 25° C. The formed slurry of precipitated fragment is stirred for 30-40 min at about 25° C. and then is filtered. The resulting solid is suspended in 8-12 V of H.sub.2O at about 25° C., is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 40° C.

Example 8: Solid Phase Peptide Synthesis of Intermediate Compound 8

[0146] Intermediate Compound 8 (SEQ ID NO:14), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Ala-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 8.

TABLE-US-00008 TABLE 8 SPPS Conditions for Example 8. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-L-Lys(t- 1.5 AA/1.65 DIC/1.5 Oxyma 20% Pip/DMF BuOOC—(CH.sub.2).sub.18—COO- 4 hr, rt γ-L-Glu-AEEA) 2 2 × 20-30 min Fmoc-L-Lys(Boc)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 3 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt

[0147] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 5 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen maintaining temperature at 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine. Resin treatment with 1% with TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM and is stirred for 10-15 min. All the filtrates and wash are combined, and the resulting mixture cooled to ≤20° C. The fragment solution is concentrated under vacuum to 2-4 V maintaining temperature at ≤20° C. 5 V of ACN is added to the solution, and residual DCM is removed under vacuum (residual DCM concentration ≤15%) maintaining temperature at ≤20° C. An ACN solution of the fragment is added to 5 V of ice-cold H.sub.2O over 2-6 hr (<1 L/min), while maintaining temperature at about 0° C. The resulting slurry of precipitated fragment is stirred for 30-40 min at about 0° C. and then is filtered at about 0° C. The resulting solid is suspended in 3-5 V of H.sub.2O at about 25° C., is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 40° C.

Example 9: Solid Phase Peptide Synthesis of Intermediate Compound 9

[0148] Intermediate Compound 9 (SEQ ID NO:15), or a pharmaceutically acceptable salt, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Ala-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 9.

TABLE-US-00009 TABLE 9 SPPS Conditions for Example 9. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-L-Lys(t- 1.5 AA/1.65 DIC/1.5 Oxyma 20% Pip/DMF BuOOC—(CH.sub.2).sub.18—COO- 4 hr, rt γ-L-Glu-AEEA) 2 2 × 20-30 min Fmoc-L-Lys(Boc)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 3 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 4 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 5 3 × 20-30 min Fmoc-L-2-Me-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 6 3 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 7 2 × 20-30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 8 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 9 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 10 2 × 10 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 11 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 12 2 × 10 min Fmoc-L-Phe(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 13 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.2 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 14 2 × 10 min Boc-L-Tyr(t-Bu)- 1.5 AA/1.65 DIC/1.5 Oxyma 5% Oxyma/20% Aib-L-Gln(Trt)- 4-6 hr, rt Pip/DMF Gly-OH

[0149] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 5 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen maintaining temperature at 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine. Resin treatment with 1% with TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM and is stirred for 10-15 min. All the filtrates and wash are combined, and the resulting mixture cooled to ≤20° C. The fragment solution is concentrated under vacuum to 2-4 V maintaining temperature at ≤20° C. 5 V of ACN is added to the solution, and residual DCM is removed under vacuum (residual DCM concentration ≤15%) maintaining temperature at ≤20° C. An ACN solution of the fragment is added to 5 V of ice-cold H.sub.2O over 2-6 hr (<1 L/min), while maintaining temperature at about 0° C. The resulting slurry of precipitated fragment is stirred for 30-40 min at about 0° C. and then is filtered at about 0° C. The resulting solid is suspended in 3-5 V of H.sub.2O at about 25° C., is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 40° C.

Example 10: Solid Phase Peptide Synthesis of Intermediate Compound 10

[0150] Intermediate Compound 10 (SEQ ID NO:16), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Sieber resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 10.

TABLE-US-00010 TABLE 10 SPPS Conditions for Example 10. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 2 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 3 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 4 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 3-4* Alternatively use coupling of Fmoc-L-Pro-Pro-OH dimer instead of step 3 & 4 5 2 × 30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 6 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 7 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 8 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 9 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 10 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 11 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 10-11* Alternatively use coupling of Fmoc-Gly-Gly-OH dimer instead of step 3 & 4 12 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.5 AA/2.5 PyBOP/5.0 DIPEA 20% Pip/DMF 4 hr, rt 13 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 14 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 15 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 16 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 17 2 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 18 2 × 20-30 min Fmoc-L-Phe-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt

[0151] Fmoc Deprotection, Fragment Cleavage and Isolation: Fragment on Sieber resin is stirred twice with 10 V of 20% piperidine/DMF for 20-30 min, then washed six times with 10 V of DMF. The de-Fmoced fragment on Sieber resin is swelled twice using 10 V DCM for 10-20 min. Reactor with resin is cooled to about 15° C. 20 V of 5% TFA/DCM is charged to the reactor and is stirred for 2 hr under nitrogen maintaining temperature at about 15° C. The resin is filtered and is washed with 3×10 V of DCM. All the filtrates are combined together. DCM is removed from the resulting solution under reduced pressure while maintaining internal temperature at ≤20° C. to 22.5 V residual volume. MTBE (25 V) is charged to the solution and DCM/MTBE solvents are again removed under reduced pressure while maintaining temperature at ≤20° C. to 22.5 V residual volume. Addition of MTBE/distillation operation is repeated until residual concentration of fragment in supernatant does not reached <0.11 wt %. Then, the resulting slurry is filtered while maintaining temperature at about 15° C. To the cake, 14 V of fresh MTBE is added, and the slurry is stirred for 30 min at about 15° C. and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 35° C.

Example 11: Solid Phase Peptide Synthesis of Intermediate Compound 11

[0152] Intermediate Compound 11 (SEQ ID NO:17), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Aib-2-CTC resin (0.6-0.9 mmol/g) with the conditions set forth below in Table 11.

TABLE-US-00011 TABLE 11 SPPS Conditions for Example 11. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-L-Gln(Trt)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 2 2 × 20-30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 3 2 × 20-30 min Fmoc-L-Lys(t- 1.5 AA/1.65 DIC/1.5 Oxyma 20% Pip/DMF BuOOC—(CH.sub.2).sub.18—COO- 4 hr, rt γ-L-Glu-AEEA) 4 2 × 20-30 min Fmoc-L-Lys(Boc)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 5 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt

[0153] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 5 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen maintaining temperature at about 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine. Resin treatment with 1% with TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM and is stirred for 10-15 min. All the filtrates and wash are combined, and the resulting mixture cooled to ≤20° C. The fragment solution is concentrated under vacuum to 2-4 V maintaining temperature at ≤20° C. 5 V of ACN is added to the solution, and residual DCM is removed under vacuum (residual DCM concentration ≤X %) maintaining temperature at ≤20° C. An ACN solution of the fragment is added to 5 V of ice-cold H.sub.2O over 2-6 hr (<1 L/min), while maintaining temperature at about 0° C. The resulting slurry of precipitated fragment is stirred for 30-40 min at about 0° C. and then is filtered at about 0° C. The resulting solid is suspended in 3-5 V of H.sub.2O at about 25° C., is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 40° C.

Example 12: Solid Phase Peptide Synthesis of Intermediate Compound 12

[0154] Intermediate Compound 12 (SEQ ID NO:18), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Sieber resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 12.

TABLE-US-00012 TABLE 12 SPPS Conditions for Example 12. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 2 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 3 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 4 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 3-4* Alternatively use coupling of Fmoc-L-Pro-Pro-OH dimer instead of step 3 & 4 5 2 × 30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 6 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 7 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 8 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 9 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 10 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 11 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 10-11* Alternatively use coupling of Fmoc-Gly-Gly-OH dimer instead of step 3 & 4 12 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.5 AA/2.5 PyBOP/5.0 DIPEA 20% Pip/DMF 4 hr, rt 13 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 14 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 15 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 16 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 17 2 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 18 2 × 20-30 min Fmoc-L-Phe-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 19 2 × 20-30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt

[0155] Fmoc Deprotection, Fragment Cleavage and Isolation: Fragment on Sieber resin is stirred twice with 10 V of 20% piperidine/DMF for 20-30 min, then washed six times with 10 V of DMF. The de-Fmoced fragment on Sieber resin is swelled twice using 10 V DCM for 10-20 min. Reactor with resin is cooled to about 15° C. 20 V of 5% TFA/DCM is charged to the reactor and is stirred for 2 hr under nitrogen maintaining temperature at about 15° C. The resin is filtered and is washed with 3×10 V of DCM. All the filtrates are combined together. DCM is removed from the resulting solution under reduced pressure while maintaining internal temperature at ≤20° C. to 22.5 V residual volume. MTBE (25 V) is charged to the solution, and DCM/MTBE solvents are again removed under reduced pressure while maintaining temperature at ≤20° C. to 22.5 V residual volume. Addition of MTBE/distillation operation is repeated until residual concentration of fragment in supernatant has not reached <0.11 wt %. Then, the resulting slurry is filtered while maintaining temperature at about 15° C. To the cake, 14 V of fresh MTBE is added, and the slurry is stirred for 30 min at about 15° C. and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 35° C.

Example 13: Solid Phase Peptide Synthesis of Intermediate Compound 13

[0156] Intermediate Compound 13 (SEQ ID NO:19), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Sieber resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 13.

TABLE-US-00013 TABLE 13 SPPS Conditions for Example 13. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 2 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 3 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 4 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 3-4* Alternatively use coupling of Fmoc-L-Pro-Pro-OH dimer instead of step 3 & 4 5 2 × 30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 6 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 7 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 8 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 9 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 10 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 11 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 10-11* Alternatively use coupling of Fmoc-Gly-Gly-OH dimer instead of step 3 & 4 12 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.5 AA/2.5 PyBOP/5.0 DIPEA 20% Pip/DMF 4 hr, rt 13 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 14 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 15 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 16 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 17 2 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 18 2 × 20-30 min Fmoc-L-Phe-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 19 2 × 20-30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 20 3 × 20-30 min Fmoc-Aib-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 8-12 hr, rt capping procedure 21 3 × 20-30 min Fmoc-L-Gln(Trt)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure

[0157] Fmoc Deprotection, Fragment Cleavage and Isolation: Fragment on Sieber resin is stirred twice with 10 V of 20% piperidine/DMF for 20-30 min, then washed six times with 10 V of DMF. The de-Fmoced fragment on Sieber resin is swelled twice using 10 V DCM for 10-20 min. Reactor with resin is cooled to about 15° C. 20 V of 5% TFA/DCM is charged to the reactor and is stirred for 2 hr under nitrogen maintaining temperature at about 15° C. The resin is filtered and is washed with 3×10 V of DCM. All the filtrates are combined together. DCM is removed from the resulting solution under reduced pressure while maintaining temperature at ≤20° C. to 22.5 V residual volume. MTBE (25 V) is charged to the solution, and DCM/MTBE solvents are again removed under reduced pressure while maintaining temperature at ≤20° C. to 22.5 V residual volume. Addition of MTBE/distillation operation is repeated until residual concentration of fragment in supernatant has not reached <0.11 wt %. Then, the resulting slurry is filtered while maintaining temperature at about 15° C. To the cake, 14 V of fresh MTBE is added, and the slurry is stirred for 30 min at about 15° C. and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 35° C.

Example 14: Solid Phase Peptide Synthesis of Intermediate Compound 14

[0158] Intermediate Compound 14 (SEQ ID NO:20), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Aib-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 14.

TABLE-US-00014 TABLE 14 SPPS Conditions for Example 14. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-L-Gln(Trt)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 2 2 × 20-30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 3 2 × 20-30 min Fmoc-L-Lys(t- 1.5 AA/1.65 DIC/1.5 Oxyma 20% Pip/DMF BuOOC—(CH.sub.2).sub.18—COO- 4 hr, rt γ-L-Glu-AEEA) 4 2 × 20-30 min Fmoc-L-Lys(Boc)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 5 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 6 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 7 3 × 20-30 min Fmoc-L-2-Me-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 8 3 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 9 2 × 20-30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 10 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 11 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 12 2 × 10 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 13 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 14 2 × 10 min Fmoc-L-Phe(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 15 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.2 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 16 2 × 10 min Boc-L-Tyr(t-Bu)- 1.5 AA/1.65 DIC/1.5 Oxyma 5% Oxyma/20% Aib-L-Gln(Trt)- 4-6 hr, rt Pip/DMF Gly-OH

[0159] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 5 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen maintaining temperature at about 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine. Resin treatment with 1% with TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM and is stirred for 10-15 min. All the filtrates and wash are combined, and the resulting mixture cooled to ≤20° C. The fragment solution is concentrated under vacuum to 2-4 V maintaining temperature at ≤20° C. 5 V of ACN is added to the solution, and residual DCM is removed under vacuum (residual DCM concentration ≤X %) maintaining temperature at ≤20° C. An ACN solution of the fragment is added to 5 V of ice-cold H.sub.2O over 2-6 hr (<1 L/min), while maintaining temperature at about 0° C. The resulting slurry of precipitated fragment is stirred for 30-40 min at about 0° C. and then is filtered at about 0° C. The resulting solid is suspended in 3-5 V of H.sub.2O at about 25° C., is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 40° C.

Example 15: Solid Phase Peptide Synthesis of Intermediate Compound 15

[0160] Intermediate Compound (SEQ ID NO:21), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Aib-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 15.

TABLE-US-00015 TABLE 15 SPPS Conditions for Example 15. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-L-Gln(Trt)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 2 2 × 20-30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 3 2 × 20-30 min Fmoc-L-Lys(PG)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6-8 hr, rt 4 2 × 20-30 min Fmoc-L-Lys(Boc)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 5 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 6 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 7 3 × 20-30 min Fmoc-L-2-Me-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 8 3 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 9 2 × 20-30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 10 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 11 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 12 2 × 10 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 13 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 14 2 × 10 min Fmoc-L-Phe(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 15 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.2 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 16 2 × 10 min Boc-L-Tyr(t-Bu)- 1.5 AA/1.65 DIC/1.5 Oxyma 5% Oxyma/20% Aib-L-Gln(Trt)- 4-6 hr, rt Pip/DMF Gly-OH

[0161] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 5 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen maintaining temperature at about 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine. Resin treatment with 1% with TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM and is stirred for 10-15 min. All the filtrates and wash are combined, and the resulting mixture cooled to ≤20° C. The fragment solution is concentrated under vacuum to 2-4 V maintaining temperature at ≤20° C. 5 V of ACN is added to the solution, and residual DCM is removed under vacuum (residual DCM concentration ≤X %) maintaining temperature at ≤20° C. An ACN solution of the fragment is added to 5 V of ice-cold H.sub.2O over 2-6 hr (<1 L/min), while maintaining temperature at about 0° C. The resulting slurry of precipitated fragment is stirred for 30-40 min at about 0° C. and then is filtered at about 0° C. The resulting solid is suspended in 3-5 V of H.sub.2O at about 25° C., is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 40° C.

Example 16: Solid Phase Peptide Synthesis of Intermediate Compound 16

[0162] Intermediate Compound 16 (SEQ ID NO:22), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Ala-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 16.

TABLE-US-00016 TABLE 16 SPPS Conditions for Example 16. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-L-Lys(PG)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 2 2 × 20-30 min Fmoc-L-Lys(Boc)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 3 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 4 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 5 3 × 20-30 min Fmoc-L-2-Me-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 6 3 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 7 2 × 20-30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 8 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 9 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 10 2 × 10 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 11 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 12 2 × 10 min Fmoc-L-Phe(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 13 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.2 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 14 2 × 10 min Boc-L-Tyr(t-Bu)- 1.5 AA/1.65 DIC/1.5 Oxyma 5% Oxyma/20% Aib-L-Gln(Trt)- 4-6 hr, rt Pip/DMF Gly-OH

[0163] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 5 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen maintaining temperature at about 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine. Resin treatment with 1% with TFA/DCM followed by filtrate neutralization is repeated two more times. The Resin is washed with 3 V of DCM and is stirred for 10-15 min. All the filtrates and wash are combined, and the resulting mixture cooled to ≤20° C. The fragment solution is concentrated under vacuum to 2-4 V maintaining temperature at ≤20° C. 5 V of ACN is added to the solution, and residual DCM is removed under vacuum (residual DCM concentration ≤X %) maintaining temperature at ≤20° C. An ACN solution of the fragment is added to 5 V of ice-cold H.sub.2O over 2-6 hr (<1 L/min), while maintaining temperature at 0° C. The resulting slurry of precipitated fragment is stirred for 30-40 min at about 0° C. and then is filtered at about 0° C. The resulting solid is suspended in 3-5 V of H.sub.2O at about 25° C., is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 40° C.

Example 17: Hybrid Liquid Solid Phase Peptide Synthesis of Intermediate Compound 17

[0164] Intermediate Compound 17 (SEQ ID NO:23), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Aib-2-CTC-hydrazine resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 17.

TABLE-US-00017 TABLE 17 SPPS Conditions for Example 17. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-L-Gln(Trt)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 2 2 × 20-30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 h, rt 3 2 × 20-30 min Fmoc-L-Lys(t- 1.5 AA/1.65 DIC/1.5 Oxyma 20% Pip/DMF BuOOC—(CH.sub.2).sub.18—COO- 4 hr, rt γ-L-Glu-AEEA) 4 2 × 20-30 min Fmoc-L-Lys(Boc)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 5 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 6 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 7 3 × 20-30 min Fmoc-L-2-Me-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 8 3 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 9 2 × 20-30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 10 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 11 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 12 2 × 10 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 13 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 14 2 × 10 min Fmoc-L-Phe(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 15 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.2 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 16 2 × 10 min Boc-L-Tyr(t-Bu)- 1.5 AA/1.65 DIC/1.5 Oxyma 5% Oxyma/20% Aib-L-Gln(Trt)- 4-6 hr, rt Pip/DMF Gly-OH

[0165] The fragment on resin is swelled with DCM (3×10 V) using filter reactor. Deprotection cocktail is prepared by mixing 10 V of TFA, 0.4 V of TIPS, 0.4 V H.sub.2O and 0.3 Weight V of DTT and is stirred until homogeneous. Cocktail is added to the resin, and the resulting slurry is stirred for 3 hr at rt. The resin is filtered and is washed with DCM (2×3 V). Then, the resulting filtrates are combined and cooled to about −10° C., and 75 V of MTBE is added slowly. The resulting slurry is filtered and is washed with 2×10 V of MTBE. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid.

[0166] Crude peptide hydrazide is dissolved in 30 V of ligation buffer (6 M guanidine hydrochloride and 0.2 M sodium hydrogen phosphate monobasic buffer, pH 3.35) and cooled to about −15° C. 1 M sodium nitrite solution (5.0-10.0 equiv) is added to the hydrazide solution and is allowed to stir for 10 min at about −15° C. After 10 min, 2,2,2-trifluoroethanethiol (20.0 equiv, pH 7.0) is added to the peptidyl azide generated from the oxidation of the peptide hydrazide. The pH of the reaction mixture is adjusted to 7.0 with 5 N sodium hydroxide solution. Thiolysis of the peptidyl azide is allowed to run for 1 hr, and then the resulting peptide thioester is used directly in ligation chemistry or purified via reverse phase chromatography (see, Huang et al. (2014) Tetrahedron 70:2951-2955).

Example 18: Solid Phase Peptide Synthesis of Intermediate Compound 18

[0167] Intermediate Compound 18 (SEQ ID NO:24), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Sieber resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 18.

TABLE-US-00018 TABLE 18 SPPS Conditions for Example 18. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 2 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 3 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 4 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 3-4* Alternatively use coupling of Fmoc-L-Pro-Pro-OH dimer instead of step 3 & 4 5 2 × 30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 6 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 7 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 8 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 9 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 10 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 11 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 10-11* Alternatively use coupling of Fmoc-Gly-Gly-OH dimer instead of step 3 & 4 12 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.5 AA/2.5 PyBOP/5.0 DIPEA 20% Pip/DMF 4 hr, rt 13 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 14 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 15 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 16 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 17 2 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 18 2 × 20-30 min Fmoc-L-Phe-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 19 2 × 20-30 min Fmoc-L-Cys(Trt)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt

[0168] Cleavage and Deprotection: The fragment on resin is swelled with DCM (3×10 V) using filter reactor. Deprotection cocktail is prepared by mixing 10 V of TFA, 0.4 V of TIPS, 0.4 V H.sub.2O and 0.3 Weight V of DTT and is stirred until homogeneous. Cocktail is added to the resin, and the resulting slurry is stirred for 3 hr at rt. The resin is filtered and is washed with DCM (2×3 V). Then, the resulting filtrates are combined and cooled to about −10° C., and 75 V of MTBE is added slowly. The resulting slurry is filtered and is washed with 2×10 V of MTBE. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid.

Example 19: Hybrid Liquid Solid Phase Peptide Synthesis of Intermediate Compound 19

[0169] Intermediate Compound 19 (SEQ ID NO:25), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-L-Lys(t-BuOOC-(CH.sub.2).sub.18-COO-γ-L-Glu-AEEA)-Lys-2-CTC-hydrazine resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 19.

TABLE-US-00019 TABLE 19 SPPS Conditions for Example 19. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-L-Lys(Boc)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 2 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 3 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 4 3 × 20-30 min Fmoc-L-2-Me-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 5 3 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 6 2 × 20-30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 7 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 8 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 9 2 × 10 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 10 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 11 2 × 10 min Fmoc-L-Phe(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 12 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.2 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 13 2 × 10 min Boc-L-Tyr(t-Bu)- 1.5 AA/1.65 DIC/1.5 Oxyma 5% Oxyma/20% Aib-L-Gln(Trt)- 4-6 hr, rt Pip/DMF Gly-OH

[0170] The fragment on resin is swelled with DCM (3×10 V) using filter reactor. Deprotection cocktail is prepared by mixing 10 V of TFA, 0.4 V of TIPS, 0.4 V H.sub.2O and 0.3 Weight V of DTT and is stirred until homogeneous. Cocktail is added to the resin, and the resulting slurry is stirred for 3 hr at rt. The resin is filtered and is washed with DCM (2×3 V). Then, the resulting filtrates are combined and cooled to about −10° C., and 75 V of MTBE is added slowly. The resulting slurry is filtered and is washed with 2×10 V of MTBE. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid.

[0171] Crude peptide hydrazide is dissolved in 30 V of the ligation buffer (6 M guanidine hydrochloride and 0.2 M sodium hydrogen phosphate monobasic buffer, pH 3.35) and cooled to about −15° C. 1 M sodium nitrite solution (5.0-10.0 equiv) is added to the hydrazide solution and is allowed to stir for 10 min at about −15° C. After 10 min, 2,2,2-trifluoroethanethiol (20.0 equiv, pH 7.0) is added to the peptidyl azide generated from oxidizing the peptide hydrazide. The pH of the reaction mixture is adjusted to 7.0 with 5 N sodium hydroxide solution. Thiolysis of the peptidyl azide is allowed to run for 1 hr, and then the resulting peptide thioester is used directly in ligation chemistry or purified via reverse phase chromatography (see, Huang (2014)).

Example 20: Solid Phase Peptide Synthesis of Intermediate Compound 20

[0172] Intermediate Compound 20 (SEQ ID NO:26), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Sieber resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 20.

TABLE-US-00020 TABLE 20 SPPS Conditions for Example 20. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 2 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 3 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 4 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6 hr, rt 3-4* Alternatively use coupling of Fmoc-L-Pro-Pro-OH dimer instead of step 3 & 4 5 2 × 30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 6 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 7 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 8 2 × 30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 9 2 × 30 min Fmoc-L-Pro-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 10 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 11 2 × 30 min Fmoc-Gly-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 10-11* Alternatively use coupling of Fmoc-Gly-Gly-OH dimer instead of step 3 & 4 12 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.5 AA/2.5 PyBOP/5.0 DIPEA 20% Pip/DMF 4 hr, rt 13 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 14 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 15 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 16 2 × 20-30 min Fmoc-L-Glu(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 17 2 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 18 2 × 20-30 min Fmoc-L-Phe-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 19 2 × 20-30 min Fmoc-L-Ala-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 20 3 × 20-30 min Fmoc-Aib-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 8-12 hr, rt capping procedure 21 3 × 20-30 min Fmoc-L-Gln(Trt)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 22 2 × 20-30 min Fmoc-L-Cys(Trt)-OH AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt

[0173] Cleavage and Deprotection: The fragment on resin is swelled with DCM (3×10 V) using filter reactor. Deprotection cocktail is prepared by mixing 10 V of TFA, 0.4 V of TIPS, 0.4 V H.sub.2O and 0.3 Weight V of DTT and is stirred until homogeneous. Cocktail is added to the resin, and the resulting slurry is stirred for 3 hr at rt. The resin is filtered and is washed with DCM (2×3 V). Then, the resulting filtrates are combined and cooled to about −10° C., and 75 V of MTBE is added slowly. The resulting slurry is filtered and is washed with 2×10 V of MTBE. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid.

Example 21: Hybrid Liquid Solid Phase Peptide Synthesis of Intermediate Compound 21

[0174] Intermediate Compound 21 (SEQ ID NO:27), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Lys(Boc)-2-CTC-hydrazine resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 21.

TABLE-US-00021 TABLE 21 SPPS Conditions for Example 21. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-L-Lys(PhAc)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 2 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 3 2 × 20-30 min Fmoc-L-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 4 3 × 20-30 min Fmoc-L-2-Me-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 5 3 × 20-30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt capping procedure 6 2 × 20-30 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 7 2 × 20-30 min Fmoc-L-Tyr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 8 2 × 20-30 min Fmoc-L-Asp(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-6 hr, rt 9 2 × 10 min Fmoc-L-Ser(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 10 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma, 5% Oxyma/20% 4-6 h, rt. Pip/DMF 11 2 × 10 min Fmoc-L-Phe(t-Bu)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 12 2 × 10 min Fmoc-L-Thr(t-Bu)-OH 2.0 AA/2.2 DIC/2.2 Oxyma 5% Oxyma/20% 4-6 hr, rt Pip/DMF 13 2 × 10 min Boc-L-Tyr(t-Bu)- 1.5 AA/1.65 DIC/1.5 Oxyma 5% Oxyma/20% Aib-L-Gln(Trt)- 4-6 hr, rt Pip/DMF Gly-OH

[0175] The fragment on resin is swelled with DCM (3×10 V) using filter reactor. Deprotection cocktail is prepared by mixing 10 V of TFA, 0.4 V of TIPS, 0.4 V H.sub.2O and 0.3 Weight V of DTT and is stirred until homogeneous. Cocktail is added to the resin, and the resulting slurry is stirred for 3 hr at rt. The resin is filtered and is washed with DCM (2×3 V). Then, the resulting filtrates are combined and cooled to about −10° C., and 75 V of MTBE is added slowly. The resulting slurry is filtered and is washed with 2×10 V of MTBE. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid.

[0176] Crude peptide hydrazide is dissolved in 30 V of the ligation buffer (6 M guanidine hydrochloride and 0.2 M sodium hydrogen phosphate monobasic buffer, pH 3.35) and cooled to about −15° C. 1 M sodium nitrite solution (5.0-10.0 equiv) is added to the hydrazide solution and is allowed to stir for 10 min at about −15° C. After 10 min, 2,2,2-trifluoroethanethiol (20.0 equiv, pH 7.0) is added to the peptidyl azide generated by oxidizing the peptide hydrazide. The pH of the reaction mixture is adjusted to 7.0 with 5 N sodium hydroxide solution. Thiolysis of the peptidyl azide runs for 1 hr, and then the resulting peptide thioester is used directly in ligation chemistry or purified via reverse phase chromatography (see, Huang (2014)).

Example 22: Solid Phase Peptide Synthesis of Intermediate Compound 22

[0177] Intermediate Compound 22 (SEQ ID NO:28), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Gly-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 22.

TABLE-US-00022 TABLE 22 SPPS Conditions for Example 22. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 30 min Fmoc-L-Gln(Trt)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6-9 hr, rt 2 2 × 30 min Fmoc-L-Aib-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4-8 hr, rt 3 2 × 30 min Boc-L-Tyr(Boc)-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 12-18 hr, rt

[0178] Cleavage and Deprotection: Tetramer on CTC resin is swelled using DCM (5-10 V) for 2×30 min. 3.5 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen maintaining temperature at about 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine. Resin treatment with 1% TFA/DCM followed by filtrate neutralization is repeated four more times. The resin is washed with 3.5 V of DCM and is stirred for 5-10 min. All the filtrates and washes are combined. The fragment solution is concentrated under vacuum to 1.5 V maintaining temperature at ≤35° C. 5 V of IPAc is added to the solution, and residual IPAc/DCM solvents are removed under vacuum to 1.5 V maintaining temperature at ≤40° C. Addition of 5 V of IPAc and distillation under vacuum are repeated to produce 3.5 V of the final fragment solution. This solution is then washed with 3×2 V of 5.0% NaCl solution, and then IPAc is removed to 1.5 V under reduced pressure maintaining temperature at ≤40° C. 4-5 V heptane is added to the solution at 40° C. Then, temperature is reduced to about 15° C., and the resulting slurry is stirred for 30 min. The IPAc/heptane solvents are removed to 3.5 V under reduced pressure maintaining temperature at ≤40° C. The heptane charge and distillation are repeated, and the resulting slurry of precipitated fragment is cooled to about 20° C., is filtered, is washed with 2 V of heptane, and the resulting solid is dried at about 35° C.

Example 23: Solid Phase Peptide Synthesis of Intermediate Compound 23

[0179] ##STR00017##

or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Leu-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 23.

TABLE-US-00023 TABLE 23 SPPS Conditions for Example 23. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 30 min Fmoc-L-2-Me-Leu-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 6-9 hr, rt 2 2 × 30 min Fmoc-L-Ile-OH 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 16-18 hr, rt

[0180] Cleavage and Deprotection: Tetramer on CTC resin is swelled using DCM (5-10 V) for 2×30 min. 3.5 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen maintaining temperature at about 25° C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine. Resin treatment with 1% TFA/DCM followed by filtrate neutralization is repeated four more times. The resin is washed with 3.5 V of DCM and is stirred for 5-10 min. All the filtrates and washes are combined. The fragment solution is concentrated under vacuum to 1.5 V maintaining temperature at ≤35° C. 5 V of IPAc is added to the solution, and residual IPAc/DCM solvents are removed under vacuum to 1.5 V maintaining temperature at ≤40° C. Addition of 5 V of IPAc and distillation under vacuum are repeated to produce 3.5 V of the final fragment solution. This solution is then washed with 3×2 V of 5.0% NaCl solution, and IPAc is removed to 1.5 V under reduced pressure maintaining temperature at ≤40° C. 4-5 V heptane is added to the solution at about 40° C. Then, temperature is reduced to about 15° C., and the resulting slurry is stirred for 30 min. The IPAc/heptane solvents are removed to 3.5 V under reduced pressure maintaining temperature at ≤40° C. The heptane charge and distillation are repeated, the resulting slurry of precipitated fragment is cooled to about 20° C., is filtered and washed with 2 V of heptane, and the resulting solid is dried at about 35° C.

Example 24: Liquid Phase Peptide Synthesis of Intermediate Compound 24

[0181] ##STR00018##

or a pharmaceutically acceptable salt thereof, can be synthesized by coupling of H-L-2-Me-Leu and Fmoc-L-Ile-OH using standard coupling chemistry in solution followed by work up and isolation.

Example 25: Solid Phase Peptide Synthesis of Fatty Acid Moiety (Compound 25)

[0182] ##STR00019##

or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-PEG-2-CTC resin (loading factor 0.6-1.1 mmol/g) with the conditions set forth below in Table 24.

TABLE-US-00024 TABLE 24 SPPS Conditions for Example 25. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 20-30 min Fmoc-γ-Glu-Ot-Bu 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt 2 2 × 20-30 min t-BuOOC—(CH.sub.2).sub.18—COO 2.0 AA/2.2 DIC/2.0 Oxyma 20% Pip/DMF 4 hr, rt

Example 26: Hybrid Liquid Solid Phase Synthesis of Incretin Analog from Four Intermediate Compounds Via Chemical Conjugation

[0183] Coupling Protocol: The incretin analog of SEQ ID NO:6 can be made by coupling SEQ ID NOS:7, 8, 9 and 10 via HLSPS. Briefly, a solution of SEQ ID NO:7 (1.05-1.30 mmol) and a solution of SEQ ID NO:8 (1.00 mmol) in 30-40 V of DMSO/ACN (70:30) are coupled using PyBOP, HATU or PyOXim reagent (1.30-2.00 mmol) and DIEA (4.00-5.00 mmol) at rt. The mixture is stirred at rt for 2-4 hr. Then, 10 equivalents of DEA are added, and the mixture is stirred for 4 hr. The mixture is quenched with 20 V of 15-20% brine solution, then an additional 10 V of water is added and is stirred for 10 min. The resulting slurry is filtered, and the solid is washed with 3×10 V of water. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid.

[0184] Next, a solution of the coupled SEQ ID NOS:7+8 (1.00 mmol) and a solution of SEQ ID NO:9 (1.05-1.30 mmol) in 30-40 V of DMSO/ACN (70:30) are coupled using PyBOP, HATU or PyOXim reagent (1.30-2.00 mmol) and DIEA (4.00-5.00 mmol) at rt. The mixture is stirred at rt for 2-4 hr. Then, 10 equivalents of DEA are added, and the mixture is stirred for 2-4 hr. The mixture is quenched with 20 V of 15-20% brine solution, then an additional 10 V of water is added and is stirred for 10 min. The resulting slurry is filtered, and the solid is washed with 3×10 V of water. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid.

[0185] Next, a solution of the coupled SEQ ID NOS:7+8+9 (1.00 mmol) and a solution of SEQ ID NO:10 (1.20-1.30 mmol) in 30-40 V of DMSO/ACN (70:30) are coupled using PyBOP, HATU or PyOXim reagent (1.50-2.00 mmol) and DIEA (4.00-5.00 mmol) at rt. The mixture is stirred at rt for 3-4 hr. The mixture is quenched with 20 V of 15-20% brine solution, and then an additional 10 V of water is added and is stirred for 10 min. The resulting slurry is filtered, and the solid is washed with 3×10 V of water. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid.

[0186] Global Deprotection: Deprotection cocktail is prepared by mixing 10 V of TFA, 2 V of DCM, 0.4 V of TIPS, 0.4 V H.sub.2O and 0.3 weight V of DTT and is stirred until homogeneous. The cocktail is cooled to about 15° C., and then solid, coupled SEQ ID NOS:7+8+9+10 is added, and the resulting reaction mixture is warmed to rt and is stirred for 3 hr at rt. The mixture is cooled to about −10° C., and 75 V of MTBE is added slowly. The resulting slurry is filtered and is washed with 2×10 V of MTBE. The solid is dried in a vacuum dryer (40° C.) to yield product SEQ ID NO:6 as a white solid.

Example 27: Hybrid Liquid Solid Phase Synthesis of Incretin Analog from Four Intermediate Compounds Via Chemical Conjugation

[0187] Here, the incretin analog of SEQ ID NO:6 is made by coupling SEQ ID NOS:7, 11, 12 and 10 via convergent solid-phase peptide synthesis (CSPPS) essentially as described in Example 26 for coupling SEQ ID NOS:7, 8, 9 and 10.

Example 28: Hybrid Liquid Solid Phase Synthesis of Incretin Analog from Four Intermediate Fragments Via Chemical Conjugation

[0188] Here, the incretin analog of SEQ ID NO:6 is made by coupling SEQ ID NOS:7, 13, 14 and 10 via CSPPS essentially as described in Example 26 for coupling SEQ ID NOS:7, 8, 9 and 10.

Example 29: Hybrid Liquid Solid Phase Synthesis of Incretin Analog from Three Intermediate Fragments Via Chemical Conjugation

[0189] The incretin analog of SEQ ID NO:6 can be made by coupling SEQ ID NOS:7, 13 and 15 via CSPPS. Briefly, a solution of SEQ ID NO:7 (1.05-1.30 mmol) and a solution of SEQ ID NO:13 (1.00 mmol) in 30-40 V of DMSO/ACN (70:30) are coupled using PyBOP, HATU or PyOXim reagent (1.30-2.00 mmol) and DIEA (4.00-5.00 mmol) at rt. The mixture is stirred at rt for 2-4 hr. Then, 10 equivalents of DEA are added, and the mixture is stirred for 4 hr. The mixture is quenched with 20 V of 15-20% brine solution, and then an additional 10 V of water is added and is stirred for 10 min. The resulting slurry is filtered, and the solid is washed with 3×10 V of water. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid.

[0190] Next, a solution of the coupled SEQ ID NOS:7+13 (1.00 mmol) and a solution of SEQ ID NO:15 (1.20-1.30 mmol) in 30-40 V of DMSO/ACN (70:30) are coupled using PyBOP, HATU or PyOXim reagent (1.50-2.00 mmol) and DIEA (4.00-5.00 mmol) at rt. The mixture is stirred at rt for 2-4 hr. Then, 10 equivalents of DEA are added, and the mixture is stirred for 2-4 hr. The mixture is quenched with 20 V of 15-20% brine solution, and then an additional 10 V of water is added and stirred for 10 min. The resulting slurry is filtered, and the solid is washed with 3×10 V of water. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid.

[0191] Global Deprotection: Deprotection cocktail is prepared by mixing 10 V of TFA, 2 V of DCM, 0.4 V of TIPS, 0.4 V H.sub.2O and 0.3 weight V of DTT and is stirred until homogeneous. The cocktail is cooled to about 15° C., and then solid, coupled SEQ ID NOS:7+13+15 is added, and the resulting reaction mixture is warmed to rt and is stirred for 3 hr at rt. The mixture is cooled to about −10° C., and 75 V of MTBE added slowly. The resulting slurry is filtered and is washed with 2×10 V of MTBE. The solid is dried in a vacuum dryer (40° C.) to yield product SEQ 6 as a white solid.

Example 30: Hybrid Liquid Solid Phase Synthesis of Incretin Analog from Three Intermediate Fragments Via Chemical Conjugation

[0192] The incretin analog of SEQ ID NO:6 can be made by coupling SEQ ID NOS:16, 9 and 10 via CSPPS. Briefly, a solution of SEQ ID NOS:16 (1.00 mmol) and a solution of SEQ ID NO:9 (1.05-1.30 mmol) in 30-40 V of DMSO/ACN (70:30) are coupled using PyBOP, HATU or PyOXim reagent (1.30-2.00 mmol) and DIEA (4.00-5.00 mmol) at rt. The mixture is stirred at rt for 3-4 hr. The mixture is quenched with 20 V of 15-20% brine solution, and then an additional 10 V of water is added and is stirred for 10 min. The resulting slurry is filtered, and the solid is washed with 3×10 V of water. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid.

[0193] Next, a solution of the coupled SEQ ID NOS:16+9 (1.00 mmol) and a solution of SEQ ID NO:10 (1.20-1.30 mmol) in 30-40 V of DMSO/ACN (70:30) are coupled using PyBOP, HATU or PyOXim reagent (1.50-2.00 mmol) and DIEA (4.00-5.00 mmol) at rt. The mixture is stirred at rt for 2-4 hr. Then, 10 equivalents of DEA are added, and the mixture is stirred for 2-4 hr. The mixture is quenched with 20 V of 15-20% brine solution, and then an additional 10 V of water is added and is stirred for 10 min. The resulting slurry is filtered, and the solid is washed with 3×10 V of water. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid.

[0194] Global Deprotection: Deprotection cocktail is prepared by mixing 10 V of TFA, 2 V of DCM, 0.4 V of TIPS, 0.4 V H.sub.2O and 0.3 weight V of DTT and is stirred until homogeneous. The cocktail is cooled to about 15° C., and then solid, coupled SEQ ID NOS:16+9+10 is added, and the resulting reaction mixture is warmed to rt and is stirred for 3 hr at rt. The mixture is cooled to about −10° C., and 75 V of MTBE added slowly. The resulting slurry is filtered and is washed with 2×10 V of MTBE. The solid is dried in a vacuum dryer (40° C.) to yield product SEQ 6 as a white solid.

Example 31: Hybrid Liquid Solid Phase Synthesis of Incretin Analog from Three Intermediate Fragments Via Chemical Conjugation

[0195] Here, the incretin analog of SEQ ID NO:6 is made by coupling SEQ ID NOS:18, 12 and 10 via CSPPS essentially as described in Example 30 for coupling SEQ ID NOS:16, 9 and 10.

Example 32: Hybrid Liquid Solid Phase Synthesis of Incretin Analog from Two Intermediate Fragments Via Chemical Conjugation

[0196] The incretin analog of SEQ ID NO:6 can be made by coupling SEQ ID NOS:19 and 15 via CSPPS. Briefly, a solution of SEQ ID NOS:18 (1.00 mmol) and a solution of SEQ ID NO:15 (1.20-1.30 mmol) in 30-40 V of DMSO/ACN (70:30) are coupled using PyBOP, HATU or PyOXim reagent (1.50-2.00 mmol) and DIEA (4.00-5.00 mmol) at rt. The mixture is stirred at rt for 2-4 hr. The mixture is quenched with 20 V of 15-20% brine solution, and then an additional 10 V of water is added and is stirred for 10 min. The resulting slurry is filtered, and the solid is washed with 3×10 V of water. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid.

[0197] Global Deprotection: Deprotection cocktail is prepared by mixing 10 V of TFA, 2 V of DCM, 0.4 V of TIPS, 0.4 V H.sub.2O and 0.3 weight V of DTT and is stirred until homogeneous. The cocktail is cooled to about 15° C., and then solid, coupled SEQ ID NOS:19+15 is added, and the resulting reaction mixture is warmed to rt and is stirred for 3 hr at rt. The mixture is cooled to about −10° C., and 75 V of MTBE added slowly. The resulting slurry is filtered and is washed with 2×10 V of MTBE. The solid is dried in a vacuum dryer (40° C.) to yield product SEQ ID NO:6 as a white solid.

Example 33: Hybrid Liquid Solid Phase Synthesis of Incretin Analog from Two Intermediate Fragments Via Chemical Conjugation

[0198] Here, the incretin analog of SEQ ID NO:6 is made by coupling SEQ ID NOS:18 and 20 via CSPPS essentially as described in Example 32 for coupling of SEQ ID NOS:15 and 19.

Example 34: Hybrid Liquid Solid Phase Synthesis of Incretin Analog from Two Intermediate Fragments Via Chemical Conjugation

[0199] Here, the incretin analog of SEQ ID NO:6 is made by coupling SEQ ID NOS:21 and 18 or SEQ ID NOS:22 and 19 via CSPPS essentially as described in Example 32 for coupling of SEQ ID NOS:15 and 19 with one exception that after the coupling of the two fragments, the protecting group on Lys17 is selectively removed via chemical transformation (conditions depend on the nature of the group) and then selectively acylated with the fatty acid side chain followed by global deprotection.

Example 35: Hybrid Liquid Solid Phase Synthesis of Incretin Analog from Two Intermediate Fragments Via Native Chemical Ligation

[0200] The incretin analog of SEQ ID NO:6 can be made by coupling SEQ ID NOS:23 and 24 via native chemical ligation. Briefly, a peptide thioester of SEQ ID NOS:23 is dissolved in 30-50 V of ligation buffer (6 M guanidine hydrochloride and 0.2 M sodium hydrogen phosphate monobasic buffer, pH 7.04). N-terminal cysteine-containing peptide fragment SEQ ID NOS:24 (0.9-0.95 equiv) is added to the thioester solution. 40 equiv of 2,2,2-trifluoroethanethiol (pH 7.16) and 20 equiv of tris(2-carboxyethyl)phosphine (pH 7.0) are added to the reaction mixture, and the pH is adjusted to 7.0 with 5 N sodium hydroxide solution. The reaction is allowed to stir at room temperature for 24 hours, and the resulting solution is used directly in reverse phase purification.

Example 36: Hybrid Liquid Solid Phase Synthesis of Incretin Analog from Two Intermediate Fragments Via Native Chemical Ligation

[0201] Here, the incretin analog of SEQ ID NO:6 can be made by coupling SEQ ID NOS:25 and 26 via CSPPS essentially as described in Example 35 for coupling SEQ ID NOS:23 and 24.

Example 36: Synthesis of Compound 35

[0202] ##STR00020##

[0203] Dichloromethane (7.5 L, 15.0 vol.) is added to a 20 L four-necked flask at 15-30° C., and 18-(tert-butoxy)-18-oxooctadecanoic acid (500.5 g, 1.0 eq. 1.35 mol) and N-hydroxysuccinimide (185.6 g, 1.2 eq., 1.61 mol) are added at 15-30° C. to obtain a suspension. The reaction mixture is cooled to 0-10° C. and charged with N-ethyl-N′-carbodiimide (338.4 g, 1.3 eq., 1.77 mol) in one portion to obtain a solution. The reaction mixture is washed three times with 8 volumes of semi-saturated brine. Compound 35 is used directly in the next step to make Compound 36.

[0204] Alternate Method for Compound 35

[0205] 18-(tert-butoxy)-18-oxooctadecanoic acid (20 g, 53.431 mmol, 99 mass %), N,N′-Disuccinimidyl carbonate (1.2 equiv., 64.117 mmol, 99.6 mass %) and 4-dimethylaminopyridine (0.2 equiv., 1.31 g, 10.7 mmol, 100 mass %) are charged into a 1000 mL baffled, jacketed-reactor equipped with an overhead agitator. Ethyl acetate (800 mL, 40 volumes) is added and the resultant slurry is stirred overnight (18-24 h) at ambient temperature (18° C.-23° C.). .sup.1H-NMR of the crude sample after 24 h generally shows 97-99% reaction completion. The batch is extracted with de-ionized water (3×82 mL). The organic layer is concentrated in vacuo to ˜160 mL. Ethyl acetate (200 mL) is added to the reaction and the crude reaction solution reduced to ˜180 mL in vacuo at 50° C. The solution is transferred to a jacketed filter and cooled slowly to 3° C., while being agitated. The reaction is then held at 3-5° C. for 1 h. The solids are filtered, washed with cold ethyl acetate (18 mL) and dried under vacuum (7 in of Hg) to give Compound 35 as a solid (22.6 g, 90.5% yield, 99.17% HPLC-CAD).

Example 37: Synthesis of t-BuO-C18-Glu-1-OtBu (Compound 36)

[0206] ##STR00021##

[0207] Compound 35 is added to a solution of (4S)-4-amino-5-tert-butoxy-5-oxo-pentanoic acid (H-Glu-1-OtBu) (289 g, 1.14 eq., 1 mol) in dichloromethane (2.5 L, 5.0 vol.) in a 20 L four-necked flask at 15-30° C. The reactor is then charged with diisopropylethylamine (230 g, 1.5 eq., 1.78 mol) at 15-30° C., to get a solution. Once Preparation 1<0.5%, the reaction is continued with the next step. The organic phase is washed with 2% aqueous solution of KHSO.sub.4 (4 g/g×3) and concentrated to 1-2 vol. under vacuum at T<50° C. and <−0.08 MPa. Acetonitrile (8 vol.) is charged to the reactor and concentrated to 1-2 vol. at <60° C. Acetonitrile (8 vol.) is charged to the reactor again and concentrated to 5-6 vol. at <60° C. The concentrate is cooled to 40-50° C., stirred for 0.5-1 h and then cooled to 15-30° C. for 2-4 h. The slurry is filtered, washed with acetonitrile (3 vol.) and dried under N.sub.2 to give Compound 36 as a solid (659.5 g, 86.3% yield, 99.1% LCAP).

[0208] Alternate Method for Compound 36

[0209] Compound 35 (50 g, 104.8 mmol, 98 mass %), (4S)-4-amino-5-tert-butoxy-5-oxo-pentanoic acid H-Glu-1-OtBu (H-Glu-1-OtBu) (25.7 g, 126 mmol, 99.3 mass %) is charged into a 1 L baffled, jacketed-reactor equipped with an overhead agitator and a thermocouple. Acetonitrile (500 mL, 10 V) is used to wash the solids down the funnel into the reaction vessel. Diisopropylethylamine (22 mL, 126 mmol, 99.75 mass %) is then added to the reaction. The reaction is heated to 40° C. and allowed to stir for 18 h. After reaction completion is confirmed by .sup.1H-NMR/HPLC-CAD, acetic acid (7.2 mL, 130 mmol, 100 mass %) and water (215 mL, 11934.6 mmol, 100 mass %) is charged into the reaction and stirred at 30-35° C. for 1 h. The batch is transferred to a jacketed filter, equipped with an overhead agitator and cooled to −20° C. Solids begin to crystallize out of solution at Tr=2° C. and Tj=−9° C. The solids are held at this temperature for 1 h. De-ionized water (8.6 V, 460 mL) is then poured into this batch and the filter warmed to 0° C. The solids are filtered and dried under high vacuum at 40° C. to give Compound 36 as a solid (55.5 g, 95.3% yield, 99.62% HPLC-CAD).

Example 38: Synthesis of t-BuO-C18-Glu-1-OtBu-5-ONSu (Compound 37)

[0210] ##STR00022##

[0211] Acetonitrile (12.0 vol.) is added to a 20 L four-necked flask at 15-30° C. Compound 36 (500.4 g, 1.0 eq., 0.90 mol) and N,N′-Disuccinimidyl carbonate (278.5 g, 1.2 eq., 1.09 mol) are added to the flask at 15-30° C. to obtain a suspension. 4-dimethylaminopyridine (11.0 g 0.1 equiv. 0.09 mol) is added in one portion to obtain a solution. Water (1.6 Kg) is added to the over 0.5-1 h. The mixture is cooled to 0-10° C. for 1-2 h, filtered, washed with acetonitrile (2 vol. 0-10° C.) and dried under N.sub.2 to give Compound 37 as a solid (536.0 g, 91.3% yield 100.0% LCAP).

[0212] Alternate Synthesis of Compound 37

[0213] Compound 36 (55 g, 98.96 mmol), N,N′-Disuccinimidyl carbonate (31 g, 121 mmol, 99.6 mass %), 4-dimethylaminopyridine (1.22 g, 9.89 mmol, 99 mass %) are charged into 1 L baffled, jacketed-reactor equipped with an overhead agitator and a thermocouple. Acetonitrile (660 mL, 12 V) is added to the reaction vessel. The reaction is stirred at 24° C. for 4 h. After reaction completion is confirmed by .sup.1H-NMR/HPLC-CAD, the reaction solution is transferred to a 1000 mL beaker and de-ionized water (180 mL) is added to the beaker equipped with a magnetic stir bar. Solids crashed out of the reaction as the solution is stirred. The reaction slurry is cooled overnight in the refrigerator (2-10° C.). The solids are filtered and the filter cake, washed with 125 mL cold (2-10° C.) acetonitrile. The solids are dried under high vacuum at 40° C. for 24 h forming Compound 37 (59.3 g, 91.8% yield, 99.61% HPLC-CAD).

Example 39: Synthesis of t-BuO-C18-Glu-1-OtBu-5-(AEEA).SUB.2 .(Compound 38)

[0214] ##STR00023##

[0215] Dichloromethane (7.5 L, 15.0 vol.) is added to a 20 L four-necked flask in one portion at 15-30° C. followed by (AEEA).sub.2 (261 g, 1.1 eq., 0.85 mol), Compound 37 (501 g, 1.0 eq., 0.77 mol) and diisopropylethylamine (1.5 eq.) at 15-30° C. Once Compound 37 is <0.5%, the reaction is continued with the next step. The reaction mixture is then concentrated to 5-6 vol. under vacuum at T<30° C., P<−0.08 MPa. Ethyl acetate is added to the crude product (5 vol.) and concentrated under vacuum at T<50° C., P<−0.08 MPa. Ethyl acetate (10 vol.) is added to the concentrate and washed with 2% aqueous KHSO.sub.4 (5 g/g×5-6) solution and concentrated to 1.2 vol. under vacuum at T<40° C. and P<−0.0 8 MPa. Dimethylformamide is added to the concentrate (3 g/g vol.) to give the product Compound 38 as a pale yellow solution (2.4 Kg, 92.7% yield, 98.7% LCAP).

[0216] Alternate Synthesis of Compound 38

[0217] (AEEA).sub.2 (27.6 g, 1.1 equiv., 85.0 mmol, 95 mass %), N-methyl-N-trimethylsilylacetamide (30 mL, 2 equiv., 200 mmol, 90 mass %) and ethyl acetate (230 mL) are added to a 500 mL flask, equipped with a thermocouple and a magnetic stir bar. The reaction is stirred for 3 h at 18-23° C. After 3 h, Compound 37 (50 g, 76.58 mmol, 100 mass %) and ethyl acetate (130 mL) are added to the reaction flask and stirred for 2 h at 18-23° C. After reaction completion is confirmed by .sup.1H-NMR/HPLC-CAD, the reaction solution is transferred to a separatory funnel and the organic layer washed with 2% KHSO.sub.4 solution (100 mL×3) and 2% NaCl solution (100 mL×6). The organic layer is concentrated, and the resulting oil is dried under high vacuum at 50° C. for 24 h to give Compound 38 as a waxy solid at −20° C. (66.32 g, 94.9% potency by Q-NMR, 99.53% HPLC-CAD).

Example 40: Native Chemical Ligation

[0218] ##STR00024##

[0219] Synthesis of Compound 39

[0220] Fmoc-hydrazine-2-chlorotrityl resin (1.16 g, 0.85 mmol) is swollen on a Symphony X synthesizer with 2×10 mL DMF for 20 min each. Fmoc deprotection is performed with 3×10 mL 20% piperidine/DMF for 30 min each. The resin is then washed with 5×10 mL DMF.

[0221] (2S)-6-[[2-[2-[2-[[2-[2-[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid (Compound 38, 2.13 g, 1.78 mmol, 2.1 equiv) and TNTU (0.715 g, 1.96 mmol, 2.31 equiv) is dissolved in about 20.5 mL of DMF, and N,N-diisopropylethylamine (0.57 mL, 3.27 mmol, 3.85 equiv) is added to the solution. The solution is allowed to mix for 15 min on a rotatory mixer. After 15 min, the solution of pre-activated Compound 38 is added to the resin and the coupling is allowed to run for 8 hours. Then, the resin is washed with 5×10 mL DMF, 5×10 mL DCM and dried for 8 hours on the synthesizer. The resin loading is determined to be 0.45 mmol/g by quantitative NMR.

Example 41: Synthesis of Compound 40 (SEQ ID NO:58)

[0222] About 1.10 g each of Compound 39 (loading value: 0.45 mmol/g) are added in two 40 mL reactor vessels and swollen with 2×20 mL DMF for 20 min each. SEQ ID NO:58 is synthesized using standard SPPS protocols.

[0223] Deprotection: 4×9 mL of 20% v/v piperidine in DMF, 30 minutes each.

[0224] Couplings: 3 equivalents of amino acid, 3 equivalents of OXYMA and 3.3 equivalents of DIC are used for amino acid coupling.

[0225] During SPPS, the resin is washed with 5×9 mL DMF with 1 min N.sub.2 mix after each coupling and the final iteration of fmoc deprotection. At the end of the peptide hydrazide synthesis, the resin is washed with DCM with N.sub.2 mixing. The resin is dried on the peptide synthesizer.

[0226] Global Deprotection and Cleavage

[0227] 45 mL of the cleavage cocktail made with 2.5% w/v dithiothreitol (DTT), 2.5% v/v water, 2.5% v/v triisopropylsilane (TIPS) and 92.5% trifluoroacetic acid (TFA) is added to the dried resin (4.2 g) in a 500 mL three-necked round bottom flask and stirred for about 3 hours. The resin is filtered and washed with 2×2.5 mL TFA. The filtrate is poured into 350 mL cold MTBE and the peptide precipitated out immediately. The filtration flask is washed with 2×2.5 mL TFA and poured into the cold MTBE. It is cooled down to −20° C. for half an hour and then centrifuged. The peptide precipitate is then washed twice with 300 mL MTBE and centrifuged. The peptide precipitate is dried in a vacuum oven at 27° C. for about 14 hours. About 2.75 g of the crude Compound 40 is obtained after drying [Expected (mass+2H.sup.+)/2=1356.2257, observed (mass+2H.sup.+)/2=1356.2245].

Example 42: Synthesis of Compound 41 (SEQ ID NO:59)

[0228] About 0.50 mmol of Compound 41 (SEQ ID NO:59) is synthesized on Sieber amide resin by standard SPPS protocols similar to the synthesis of Compound 40, SEQ ID NO:58.

[0229] Global Deprotection and Cleavage: 25 mL of the cleavage cocktail made with 5% w/v dithiothreitol (DTT), 2.5% v/v water, 2.5% v/v triisopropylsilane (TIPS) and 90% trifluoroacetic acid (TFA) is added to the dried resin (2.21 g) and mixed for 3 hours on a rotary mixer. The resin is filtered and washed with 2×2.0 mL TFA. The filtrate is poured into 175. mL cold MTBE and peptide precipitated out immediately. The filtration flask is washed with 2×2 mL TFA and is poured into the cold MTBE. It is cooled down to −20° C. for 30 min and then centrifuged. The peptide precipitate is washed twice with 150 mL MTBE and centrifuged. The peptide precipitate is dried in a vacuum oven at 27° C. for 14 hours. About 1.351 g of the crude Compound 41 (SEQ ID NO:58) is obtained after drying. It is purified by RP-HPLC on a Waters CSH C18 10 um column (10 mm×250 mm) at the ambient temperature with a linear gradient of 15-35% acetonitrile in water (0.1% TFA) over 23 min after 10% acetonitrile in water (0.1% TFA) for the first 3 min and 10-15% acetonitrile in water (0.1% TFA) from 3 to 5 min. About 650 mg of the purified Compound 41 is obtained [Expected (mass+2H.sup.+)/2=1138.5486, observed (mass+2H.sup.+)/2=1138.5458].

Example 43: Synthesis of Compound 42 (SEQ ID NO:60)

[0230] Compound 42 (Thioester synthesis), SEQ ID NO:60

[0231] Crude peptide hydrazide (Compound 40; SEQ ID NO:58, 118.2 mg, 0.044 mmol) is dissolved in 10 mL of the ligation buffer (6 M guanidine hydrochloride and 0.3 M sodium hydrogen phosphate monobasic, pH about 3.5). The solution is cooled to −15° C. in an acetone-ice bath. 0.3 mL of 1 M sodium nitrite solution (20.7 mg, 0.3 mmol, 6.8 equiv.) is added to the peptide hydrazide solution and allowed to stir for 15 min at −15° C. Meanwhile, 0.2 mL thiophenol is diluted to 1.1 mL with the ligation buffer (pH about 7.0). After 15 min, 1.1 mL of the thiophenol mixture is added to the peptide hydrazide solution to cause in-situ thiolysis of the peptidyl azide generated from Compound 40.

[0232] The pH of the reaction mixture is adjusted to about 7.0 with 5 N sodium hydroxide solution. Thiolysis of the peptidyl azide is allowed to run for 15 min to give Compound 42 (SEQ ID NO:60).

Example 44: Compound 43, SEQ ID NO:61 (Native Chemical Ligation with the Thioester Compound 42)

[0233] Compound 41 (SEQ ID NO:59 (75.4 mg, 0.033 mmol) is dissolved in 2 mL of the ligation buffer (pH about 7.0) in a scintillation vial and the solution is added to the crude thioester solution Compound 42 (SEQ ID NO:60). The vial is rinsed with 1 mL of the ligation buffer (pH about 7.0) and the rinse is added to the reaction mixture. 1.5 mL of tris(2-carboxyethyl)phosphine (TCEP, 0.25 M in the ligation buffer, pH about 7.0) and 1.0 mL of ascorbic acid solution (0.53 M in the ligation buffer, pH about 7.0) are added to the reaction mixture. The pH of the reaction mixture is adjusted to about 7.1 with 5 N NaOH solution and the solution turned clear. The reaction is complete in 9-10 hours to yield SEQ ID NO:61.

Example 45: Synthesis of SEQ ID NO:62 (Compound 44)

[0234]

TABLE-US-00025 TABLE 25 SPPS Conditions for SEQ ID NO: 62 (Compound 44). SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 5-30 min Fmoc-Gly-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 2 2 × 5-30 min Fmoc-Glu(OtBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 3 2 × 5-30 min Fmoc-Ile-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 4 2 × 5-30 min Fmoc-Leu-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 5 2 × 5-30 min Fmoc-2-MeTyr(tBu)-OH 1.5-3.0 AA/1.7-3.3 DIC/1.5-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 6 2 × 5-30 min Fmoc-D-Glu(OtBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 7 2 × 5-30 min Fmoc-Ile-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 8 2 × 5-30 min Fmoc-Phe-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 9 2 × 5-30 min Fmoc-Glu(OtBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 10 2 × 5-30 min Fmoc-Aib-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 11 2 × 5-30 min Fmoc-Gln(Trt)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt

[0235] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled twice using DCM. A reactor with resin is cooled to about 15° C., and 2% TFA/DCM (4 ml/g of resin) is charged to the reactor and then stirred for 15 minutes under nitrogen. 1% TFA/DCM (4 ml/g of resin) is then charged and allowed to stir for 15 minutes and after filtering this is repeated. The resin is filtered and washed with 3×10 V of DCM. All the filtrates are combined together and neutralized with DIPEA. DCM is removed from the resulting solution and brine is charged to precipitate fragment 2. Then, the resulting slurry is filtered while maintaining temperature at about 15° C. To the cake, 14 V of fresh MTBE is added, is stirred for 30 min at about 15° C., and then is filtered. Washing is repeated one more time, and the resulting light-yellow solid is dried at about 35° C.

Example 46: Synthesis of SEQ ID NO:42 (Compound 45)

[0236]

TABLE-US-00026 TABLE 26 SPPS Conditions for the synthesis of SEQ ID NO: 42 (Compound 45). SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 5-30 min 20% Fmoc-Ala-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 Pip/DMF Oxyma 4-15 hrs, rt 2 2 × 5-30 min 20% Fmoc-Lys(Mtt)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 Pip/DMF Oxyma 4-15 hrs, rt 3 2 × 5-30 min 20% Fmoc-Orn(Boc)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 Pip/DMF Oxyma 4-15 hrs, rt 4 2 × 5-30 min 20% Fmoc-Asp(OtBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 Pip/DMF Oxyma 4-15 hrs, rt 5 HFIP/DCM Compound 38 1.5-3.0 AA/1.7-3.3 DIC/1.5-3.0 Oxyma 4-15 hrs, rt [00025]

[0237] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled twice using DCM. A reactor with resin is cooled to about 15° C., and 20% HFIP/DCM (8 ml/g of resin) is charged to the reactor and then stirred for 60 minutes under nitrogen. 20% HFIP/DCM (4 ml/g of resin) is then charged and allowed to stir for 60 minutes. The resin is filtered and washed with 3×10 V of DCM. All the filtrates are combined and DCM and HFIP are removed from the resulting and heptane and ether is charged to precipitate fragment 3. Then, the resulting slurry is filtered while maintaining temperature at about 15° C. To the cake, 14 V of fresh MTBE is added, is stirred for 30 min at about 15° C., and then is filtered. Washing is repeated one more time, and the resulting orange solid is dried at about 35° C.

Example 47: Synthesis of SEQ ID NO:31 (Compound 28)

[0238]

TABLE-US-00027 TABLE 27 SPPS Conditions for the synthesis of SEQ ID NO: 31. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 5-30 min Fmoc-Leu-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 2 2 × 5-30 min Fmoc-2-MeLeu-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 3 2 × 5-30 min Fmoc-Ile-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 4 2 × 5-30 min Fmoc-Ser(tBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 5 2 × 5-30 min Fmoc-4Pal-OH 1.5-3.0 AA/1.7-3.3 DIC/1.5-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 6 2 × 5-30 min Fmoc-Asp(OtBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 7 2 × 5-30 min Fmoc-Ser(tBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 8 2 × 5-30 min Fmoc-Thr(tBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 9 2 × 5-30 min Fmoc-2-MePhe(2F)-OH 1.5-3.0 AA/1.7-3.3 DIC/1.5-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 10 2 × 5-30 min Fmoc-Thr(tBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 11 2 × 5-30 min Fmoc-Gly-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 12 2 × 5-30 min Fmoc-Glu(OtBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 13 2 × 5-30 min Fmoc-Tyr(tBu)-Aib- 1.5-3.0 AA/1.7-3.3 DIC/1.5-3.0 20% Pip/DMF OH Oxyma 4-15 hrs, rt 14 (Boc)2O

[0239] Fragment Cleavage and Isolation: Fragment on CTC resin is swelled twice using DCM. A reactor with resin is cooled to about 15° C., and 2% TFA/DCM (4 ml/g of resin) is charged to the reactor and then stirred for 15 minutes under nitrogen. 1% TFA/DCM (4 ml/g of resin) is then charged and allowed to stir for 15 minutes and after filtering this is repeated. The resin is filtered and washed with 3×10 V of DCM. All the filtrates are combined together and neutralized with DIPEA. DCM is removed from the resulting solution and brine is charged to precipitate fragment 2. Then, the resulting slurry is filtered while maintaining temperature at about 15° C. To the cake, 14 V of fresh MTBE is added, is stirred for 30 min at about 15° C., and then is filtered. Washing is repeated one more time, and the resulting off-white solid is dried at about 35° C.

Example 48: Synthesis of SEQ ID NO:43 (Compound 46)

[0240]

TABLE-US-00028 TABLE 28 Preparation of SEQ ID NO: 43 via SPPS: SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 5-30 min Fmoc-L-Ser(t-Bu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 2 2 × 5-30 min Fmoc-L-Pro-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 3 2 × 5-30 min Fmoc-L-Pro-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 4 2 × 5-30 min Fmoc-L-Pro-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 3-4* Alternatively use Fmoc-L-Pro-Pro-OH dimer instead of step 3 & 4 5 2 × 5-30 min Fmoc-L-Ala-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 6 2 × 5-30 min Fmoc-Gly-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 7 2 × 5-30 min Fmoc-L-Ser(t-Bu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 8 2 × 5-30 min Fmoc-L-Ser(t-Bu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 9 2 × 5-30 min Fmoc-L-Pro-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 10 2 × 5-30 min Fmoc-Gly-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 11 2 × 5-30 min Fmoc-Gly-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 12 2 × 5-30 min Fmoc-Glu(OtBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 13 2 × 5-30 min Fmoc-Ile-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 14 2 × 5-30 min Fmoc-Leu-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 15 2 × 5-30 min Fmoc-2-MeTyr(tBu)-OH 1.5-3.0 AA/1.7-3.3 DIC/1.5-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 16 2 × 5-30 min Fmoc-D-Glu(OtBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 17 2 × 5-30 min Fmoc-Ile-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 18 2 × 5-30 min Fmoc-Phe-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 19 2 × 5-30 min Fmoc-Glu(OtBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 20 2 × 5-30 min Fmoc-Aib-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 21 2 × 5-30 min Fmoc-Gln(Trt)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt

[0241] Fragment Cleavage and Isolation: Fragment on Sieber resin is swelled twice using 10 V DCM for 10-20 min. A reactor with resin is cooled to about 15° C., and washed sequentially with 6% TFA/DCM (5 ml/g of resin) for 15 min, 3% TFA/DCM (5 ml/g of resin) for 15 min, 1% TFA/DCM (10 ml/g of resin) for 5 min, 1% TFA/DCM (5 ml/g of resin) for 3 min and 1% TFA/DCM (2.5 ml/g of resin) for 3 min. The resin is filtered and washed with 3×10 V of DCM. All the filtrates are combined together. DCM is removed from the resulting solution under reduced pressure and reconstituted with EtOAc. Heptane is charged to the solution and the resulting slurry is filtered while maintaining temperature at about 15° C. To the cake, 14 V of fresh MTBE is added, is stirred for 30 min at about 15° C., and then is filtered. Washing is repeated one more time, and the resulting off-white solid is dried at about 35° C.

Example 49: Synthesis of SEQ ID NO:44 (Compound 47)

[0242]

TABLE-US-00029 TABLE 29 Preparation of SEQ ID NO: 44. SPPS Conditions Cycle Deprotection Amino Acid Solvent for Couplings: DMF 1 2 × 5-30 min Fmoc-Ala-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 2 2 × 5-30 min Fmoc-Lys(Mtt)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 3 2 × 5-30 min Fmoc-Orn(Boc)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 4 2 × 5-30 min Fmoc-Asp(OtBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 5 HFIP/DCM Compound 38 1.5-3.0 AA/1.7-3.3 DIC/1.5-3.0 Oxyma 4-15 hrs, rt 6 2 × 5-30 min Fmoc-Leu-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 7 2 × 5-30 min Fmoc-2-MeLeu-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 8 2 × 5-30 min Fmoc-Ile-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 9 2 × 5-30 min Fmoc-Ser(tBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 10 2 × 5-30 min Fmoc-4Pal-OH 1.5-3.0 AA/1.7-3.3 DIC/1.5-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 11 2 × 5-30 min Fmoc-Asp(OtBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 12 2 × 5-30 min Fmoc-Ser(tBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 13 2 × 5-30 min Fmoc-Thr(tBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 14 2 × 5-30 min Fmoc-2-MePhe(2F)—OH 1.5-3.0 AA/1.7-3.3 DIC/1.5-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 15 2 × 5-30 min Fmoc-Thr(tBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 16 2 × 5-30 min Fmoc-Gly-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 17 2 × 5-30 min Fmoc-Glu(OtBu)-OH 2.0-3.0 AA/2.2-3.3 DIC/2.0-3.0 20% Pip/DMF (H2O) Oxyma 4-15 hrs, rt 18 2 × 5-30 min Fmoc-Tyr(tBu)-Aib-OH 1.5-3.0 AA/1.7-3.3 DIC/1.5-3.0 20% Pip/DMF Oxyma 4-15 hrs, rt 19 (Boc)2O

[0243] Fragment Cleavage and Isolation: Fragment on Sieber resin is swelled twice using 10 V DCM for 10-20 min. A reactor with resin is cooled to about 15° C., and washed sequentially with 6% TFA/DCM (5 ml/g of resin) for 15 min, 3% TFA/DCM (5 ml/g of resin) for 15 min, 1% TFA/DCM (10 ml/g of resin) for 5 min, 1% TFA/DCM (5 ml/g of resin) for 3 min and 1% TFA/DCM (2.5 ml/g of resin) for 3 min. The resin is filtered and washed with 3×10 V of DCM. All the filtrates are combined together. DCM is removed from the resulting solution under reduced pressure and reconstituted with EtOAc. Heptane is charged to the solution and the resulting slurry is filtered while maintaining temperature at about 15° C. To the cake, 14 V of fresh MTBE is added, is stirred for 30 min at about 15° C., and then is filtered. Washing is repeated one more time, and the resulting off-white solid is dried at about 35° C.

Example 50: Preparation of SEQ ID NO:29

[0244] Hybrid Liquid Solid Phase Synthesis of SEQ ID NO:29 from Four Intermediate Fragments Via Chemical Conjugation.

[0245] SEQ ID NO:29 can be made by coupling SEQ ID NOs:7, 42, 31 and 62 via HLSPS. SEQ ID NO's:7 and 62 are coupled to produce SEQ ID NO:43. SEQ ID NO's:42 and 31 are coupled to produce SEQ ID NO:44. SEQ ID NO:43 and SEQ ID NO:44 are coupled to produce SEQ ID NO:29.

[0246] Briefly, a solution of SEQ ID NO:7 (1.00 mmol) and a solution of SEQ ID NO:62 (1.1 mmol) in 10 V of DMSO are coupled using PyBOP, DEPBT or EDC/HONB reagent (1.30-2.00 mmol) and DIEA (2 mmol) at rt. The mixture is stirred at rt until the reaction is deemed complete by HPLC. The mixture is quenched with 20 V of 15-20% brine solution, and then an additional 10 V of water is added and is stirred for 10 min. The resulting slurry is filtered, and the solid is washed with 3×10 V of water. The solid is dried in a vacuum dryer (40° C.) to yield SEQ ID NO:43 as an off-white solid.

[0247] Next, a solution of SEQ ID NO:31 (1.00 mmol) and a solution of SEQ ID NO:42 (1.1 mmol) in x V of DMSO are coupled using PyBOP, DEPBT or EDC/HONB reagent (1.30-2.00 mmol) and DIEA (2 mmol) at rt. The mixture is stirred at rt until the reaction is deemed complete by HPLC. The mixture is quenched with 20 V of 15-20% brine solution, and then an additional 10 V of water is added and is stirred for 10 min. The resulting slurry is filtered, and the solid is washed with 3×10 V of water. The solid is dried in a vacuum dryer (40° C.) to yield SEQ ID NO:44 as an off-white solid.

[0248] Finally, a solution of SEQ ID NO:43 (1.00 mmol) and a solution of SEQ ID NO:44 (1.0 mmol) in 17 V of THF are coupled using DEPBT reagent (1.30-2.00 mmol) and DIEA (1 mmol) at rt. The mixture is stirred at rt until the reaction is deemed complete by HPLC. The mixture is quenched with 20 V of water. The resulting slurry is filtered, and the solid is washed with 3×10 V of water. The solid is dried in a vacuum dryer (40° C.) to yield product as a white solid. The resulting peptide is deprotected by with a cocktail of TFA:TIPS:DTT:water (92.5:2.5:2.5:2.5) with 10 ml per g of starting material. After stirring for 3 hours at room temperature, the product is precipitated with 4 mL of 20% Heptane/MTBE per mL of cocktail while keeping the temperature below 30° C. The resulting slurry is filtered, and the solid is washed with 3×10 V of MTBE. The solid is dried in a vacuum dryer (40° C.) to yield product as an off-white solid.

Example 51: Alternative Synthesis of SEQ ID NO:7

[0249]

TABLE-US-00030 TABLE 30 The synthesis uses Fmoc-Sieber amide resin with a loading of 0.80 mmol/g. The general SPPS procedure is used with the following modifications: SPPS conditions Cycle Amino acid Solvent for couplings: DMF 1 Fmoc-L-Ser(t-Bu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 2.0 AA/2.2 DIC/2.0 Oxyma 4 h, rt. 2 Fmoc-L-Pro-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, Double coupling: (1.5 AA/1.65 DIC/1.5 Oxyma) × 2 3 + 16 h, rt. 3 Fmoc-L-Pro-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, Double coupling: (1.5 AA/1.65 DIC/1.5 Oxyma) × 2 3 + 16 h, rt. 4 Fmoc-L-Pro-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, Double coupling: (1.5 AA/1.65 DIC/1.5 Oxyma) × 2 3 + 16 h, rt. 5 Fmoc-L-Ala-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, Double coupling: (1.5 AA/1.65 DIC/1.5 Oxyma) × 2 3 + 16 h, rt. 6 Fmoc-Gly-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 2.0 AA/2.2 DIC/2.0 Oxyma 4 h, rt. 7 Fmoc-L-Ser(t-Bu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 2.0 AA/2.2 DIC/2.0 Oxyma 4 h, rt. 8 Fmoc-L-Ser(t-Bu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 2.0 AA/2.2 DIC/2.0 Oxyma 6 h, rt 9 Fmoc-L-Pro-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 2.0 AA/2.2 DIC/2.0 Oxyma 6 h, rt 10 Fmoc-Gly-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 2.0 AA/2.2 DIC/2.0 Oxyma 10 h, rt.

[0250] The resin bound SEQ ID NO:7 (54 g, ˜24.0 mmol) is treated with 2×300 mL (30 min each) of 20% Pip/DMF. 2) Wash with 6×300 mL of DMF followed by 5×300 mL of DCM. 3) Add 500 mL TFA/DCM (5/95, v/v) and stir for 2 h. 4) Filter the mixtures and wash with 500 mL of DCM, to give a total filtrate volume of 1000 mL. 5) Concentrate to ˜250 mL. 6) Charge 250 mL MTBE. 7) Repeat step 5-6 for 5-6 times. 8) Filter and collect wet cake, and dry in a vacuum oven at 33° C. overnight to produce SEQ ID NO:7 (18.3 g, 75% yield) of a white solid. Analysis of the isolated solid using UPLC (94.4 area %). LC-MS ([M+H].sup.+): 1020.58.

Example 52: Synthesis of SEQ ID NO:45 (Compound 48)

[0251]

TABLE-US-00031 TABLE 31 The synthesis uses 2-CTC resin with a loading of 0.80 mmol/g. The general SPPS procedure is used with the following modifications: SPPS conditions Cycle Amino acid Solvent for couplings: DMF 1 Fmoc-Gly-OH 3.0 AA/6.0 DIEA 4 h, rt. 2 Fmoc-L-Glu(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 6 h, rt. 3 Fmoc-L-Leu-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 4 Fmoc-L-Leu-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 5 Fmoc-L-Tyr(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 6 Fmoc-L-Glu(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 7 Fmoc-L-Ile-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 6 h, rt. 8 Fmoc-L-Phe-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 9 Fmoc-Ala-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 10 Fmoc-Aib-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 12 h, rt. 11 Fmoc-L-Gln(Trt)-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 18 h, rt. 12 Fmoc-L-Ala-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 13 Fmoc-L-Lys(t- 3 × 30 min De-Fmoc cycles, BuOOC—(CH.sub.2).sub.18—COO- 6 × 2 min post-dep washes, γ-L-Glu-AEEA) 2.0 AA/4.0 DIEA/2.0 PyBOP 16 h, rt. 14 Fmoc-L-Lys(Boc)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 8 h, rt. 15 Fmoc-L-Asp(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt.

SEQ ID NO:45 Soft Cleavage:

[0252] 1) Add resin bound SEQ ID NO:45 (4.0 g, ˜1.34 mmol) and charge 40 mL cleavage cocktail TFA/DCM (1/99, v/v/v). 2) Stir it for 10 min at rt. 3) Filter and collect the filtrate. 4) Neutralize the filtrate with 0.44 mL pyridine (1/1, mol/mol). 5) Repeat Steps 1-4 for 3 more times. 6) Concentrate the combined filtrate to dryness. 7) Dissolve the slurry with 10 mL of DMSO. 8) Charge the DMSO solution slowly to cold 100 mL water with stirring. 9) Filter and collect precipitation. 10) Reslurry with 50 mL water for 2 times. 11) Dry in vacuum overnight to produce SEQ ID NO:45 (2.5 g, 58% yield) of a white solid. Analysis of the isolated solid using UPLC (95.0 area %). LC-MS ([M+2H]2.sup.+/2): 1604.97.

Example 53: Alternative Synthesis of SEQ ID NO:10

[0253]

TABLE-US-00032 TABLE 32 The synthesis uses Fmoc-Leu-OH 2-CTC resin with a loading of 0.80 mmol/g. The general SPPS procedure is used with the following modifications: SPPS conditions Cycle Amino acid Solvent for couplings: DMF 1 Fmoc-L-αMe-Leu-OH 3.0 AA/6.0DIEA 18 h, rt 2 Fmoc-L-Ile-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 18 h, rt. 3 Fmoc-L-Ser(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 4 Fmoc-L-Tyr(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt 5 Fmoc-L-Asp(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4h, rt. 6 Fmoc-L-Ser(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 7 Fmoc-L-Thr(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 8 Fmoc-L-Phe-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt 9 Fmoc-L-Thr(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt 10 Fmoc-Gly-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 6 h, rt. 11 Fmoc-L-Gln(Trt)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 12 Fmoc-Aib-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 8 h, rt. 13 Boc-L-Tyr(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 16 h, rt.

[0254] Soft cleavage: 1) Add resin bound SEQ ID NO:10 (60 g, ˜40 mmol) and charge 600 mL cleavage cocktail TFA/DCM (1/99, v/v/v). 2) Stir it for 10 min at rt. 3) Filter and collect the filtrate. 4) Neutralize the filtrate with 6.6 mL pyridine (1/1, mol/mol). 5) Repeat Steps 1-4 for 3 more times. 6) Concentrate the combined filtrate to dryness. 7) Dissolve the slurry with 60 mL of DMSO. 8) Charge the DMSO solution slowly to cold 600 mL water with stirring. 9) Filter and collect precipitation. 10) Reslurry with 300 mL water for 2 times. 11) Dry in vacuum overnight to produce SEQ ID NO:10 (56.4 g, 125% yield) as a wet solid. Analysis of the isolated solid using UPLC (93.1 area %). LC-MS ([M+H].sup.+): 2341.52.

Example 54: Synthesis of SEQ ID NO:46 (Compound 49) by LPPS

[0255] To a 20 mL glass scintillation vial, add SEQ ID NO:7 (250 mg, 77.9 μmol), SEQ ID NO:45 (103 mg, 90.7 μmol), and DMSO (5 mL). Add DIEA (81 μL, 0.47 mmol) to this solution followed by PyAOP (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate) (100 mg, 192 μmol). Stir the reaction for 4 hours then, slowly add 35 mL cold water. Collect the precipitated product by filtration and subsequently wash with water (2×35 mL). Dry the wetcake under vacuum to obtain SEQ ID NO:46 (Compound 49) as a white solid (199 mg, 61% yield). UPLC: 46.2 area %.

Example 55: Synthesis of SEQ ID NO:47 (Compound 50) by LPPS

[0256] To a 20 mL glass scintillation vial, add SEQ ID NO:46 (500 mg, 120 μmol), followed by 2 mL MeCN (2 mL). Charge Et.sub.2NH (0.5 mL, 4.8 mmol), and stir for 4 h. Concentrate the solution to dryness. Charge 5 mL MeCN and concentrate to dryness again. Repeat MeCN addition and drying for 2-3 more times. Charge 1 mL of MeCN to dissolve the slurry, then charge the reaction solution slowly to 15 mL cold MTBE with stirring. Filter and collect precipitation, and reslurry with 10 mL MTBE for 2 times. Dry the wetcake under vacuum to obtain SEQ ID NO:47 (Compound 50) as a white solid (200 mg, 42% yield). UPLC: 75.3 area %. LC-MS [M+3H].sup.3+/3: 1331.60.

Example 56: Synthesis of SEQ ID NO:48 (Compound 51) by LPPS

[0257] To a 20 mL glass scintillation vial, add SEQ ID NO:10 (75 mg, 32.1 μmol), SEQ ID NO:47 (100 mg, 25.0 μmol), 1-Hydroxy-7-azabenzotriazole (HOAt; 5 mg, 36.8 μmol), and DMSO (2 mL). Add DIEA (30 μL, 173 μmol) to this solution followed by PyAOP (33 mg, 63 μmol). Stir the reaction for 5 hours then, slowly add 15 mL cold water. Collect the precipitated product by filtration and subsequently wash with water (3×10 mL). Dry the wetcake under vacuum to obtain SEQ ID NO:48 (Compound 51) as a white solid (120 mg, 76% yield). UPLC: 53.6 area %.

Example 57: Synthesis of SEQ ID NO:6 by Global Deprotection

[0258] Charge 1 mL cleavage cocktail solution TFA/H.sub.2O/TIPS/DTT (0.925/0.025/0.025/0.025, v/v/v/v), then a sample of SEQ ID NO:48 (76 mg, 12.0 μmol) is added to this mixture to provide a solution. The mixture is stirred at about ambient temperature for about 3 hours. Pour the reaction mixture to −15° C. MTBE (10 mL), stir the resulting suspension for about 30 min. Perform filtration through filter and wash the wetcake with MTBE (2×10 mL). The wet cake is dried at 35° C. in vacuo resulting in SEQ ID NO:6 (80 mg, 44.8 area %, 140% crude yield) as a wet solid. UPLC: 44.8 area %. LC-MS [M+3H].sup.3+/3: 1183.20.

Example 58: Alterative Synthesis of SEQ ID NO:11

[0259]

TABLE-US-00033 TABLE 33 The synthesis uses Fmoc-Gly-CTC resin with a loading of 0.835 mmol/g. The general SPPS procedure is used with the following modifications. SPPS conditions Cycle Amino acid Solvent for couplings: DMF 1 Fmoc-L-Leu-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 2 Fmoc-L-Leu-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 3 Fmoc-L-Tyr(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 4 Fmoc-L-Glu(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 5 Fmoc-L-Ile-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 6 Fmoc-L-Phe-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt. 7 Fmoc-Ala-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt.

[0260] Soft cleavage: 1) Add resin bound SEQ ID NO:11 (10.0 g, ˜4.4 mmol) and charge 100 mL cleavage cocktail TFA/DCM (1/99, v/v). 2) Stir it for 10 min at rt. 3) Filter and collect the filtrate. 4) Neutralize the filtrate with 1.1 mL pyridine (1/1, mol/mol). 5) Repeat Steps 1-4 for 3 more times. 6) Concentrate the combined filtrate to dryness. 7) Dissolve the slurry with 20 mL of DMSO. 8) Charge the DMSO solution slowly to cold 100 mL water with stirring. 9) Filter and collect precipitation. 10) Re-slurry with 100 mL water for 2 times. 11) Dry in vacuum overnight to produce SEQ ID NO:11 (4.01 g, 62% yield) of a white solid. Analysis of the isolated solid using UPLC (97.6 area %). LC-MS [M+H].sup.+: 1445.78.

Example 59: Alternative Synthesis of SEQ ID NO:18

[0261]

TABLE-US-00034 TABLE 34 The synthesis uses Fmoc-Sieber amide resin with a loading of 0.71 mmol/g. The general SPPS procedure is used with the following modifications: Cycle Amino acid Coupling reaction 1 Fmoc-L-Ser(t-Bu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 4 hours coupling 2 Fmoc-L-Pro-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 6 hours coupling 3 Fmoc-L-Pro-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 12 hours coupling 4 Fmoc-L-Pro-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 12 hours coupling 5 Fmoc-L-Ala-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 10 hours coupling 6 Fmoc-Gly-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 4 hours coupling 7 Fmoc-L-Ser(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 4 hours coupling 8 Fmoc-L-Ser(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 6 hours coupling 9 Fmoc-L-Pro-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 6 hours coupling 10 Fmoc-Gly-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 10 hours coupling 11 Fmoc-Gly-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 4 hours coupling 12 Fmoc-L-Glu(OtBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 4 hours coupling 13 Fmoc-L-Leu-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 6 hours coupling 14 Fmoc-L-Leu-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 6 hours coupling 15 Fmoc-L-Tyr(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 4 hours coupling 16 Fmoc-L-Glu(OtBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 4 hours coupling 17 Fmoc-L-Ile-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 6 hours coupling 18 Fmoc-L-Phe-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 4 hours coupling 19 Fmoc-L-Ala-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, DIC/oxyma/AA = 3.3/3/3 4 hours coupling

[0262] Soft cleavage: 1) After the final 2×30 min De-Fmoc cycles, charge the resin bound SEQ ID NO:18 (8.2 g, ˜ 3.1 mmol) to 40 mL cleavage cocktail TFA/HFIP/DCM (1/25/74, v/v/v), and stir it for 5 minutes at 25° C. 2) Filter and collect the filtrate and neutralize the filtrate with 0.44 mL pyridine (1/1, mol/mol). 3) Repeat the cleavage process for 2 more times. 4) Concentrate the combined filtrate to dryness. 5) Dissolve the slurry with 10 mL of DMSO and charge the DMSO solution slowly to 200 mL MTBE with stirring. 6) Filter and collect precipitation. 7) Re-slurry with 2 more time with 40 mL MTBE and filter the precipitate. 8) Dry the crude material in vacuum overnight to obtain 2.76 grams of crude (41.4% yield) with 92.5% purity by HPLC. LC-MS [M+2H].sup.2+/2: 1113.90.

Example 60: Alternative Synthesis of SEQ ID NO:20

[0263]

TABLE-US-00035 TABLE 35 The synthesis uses 2-CTC resin with a loading of 0.80 mmol/g. The general SPPS procedure is used with the following modifications. SPPS conditions Cycle Amino acid Solvent for couplings: DMF 1 Fmoc-Aib-OH 3.0 AA/6.0 DIEA 4 h, rt. Capping performed using 10 vol MeOH/DIEA/DMF, 5/15/80, v/v/v at rt 2 Fmoc-L-Gln(Trt)-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 18 h, rt. 3 Fmoc-Ala-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma 4 h, rt 4 Fmoc-L-Lys(t- 3 × 30 min De-Fmoc cycles, BuOOC—(CH.sub.2).sub.18—COO- PyBOP/DIEA/AA = γ-L-Glu-AEEA) 2/4/2 >16 hours coupling 5 Fmoc-Lys(Boc)-OH 2 × 30 min De-Fmoc cycles, 3.0 AA/3.3 DIC/3.0 Oxyma >8 hours coupling 6 Fmoc-Asp(OtBu)-OH 2 × 30 min De-Fmoc cycles, 3.0 AA/3.3 DIC/3.0 Oxyma >4 hours coupling 7 Fmoc-Leu-OH 2 × 30 min De-Fmoc cycles, 3.0 AA/3.3 DIC/3.0 Oxyma >4 hours coupling 8 Fmoc-L-αMe-Leu-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma >12 h, rt. 9 Fmoc-L-Ile-OH 3 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma >18 h, rt. 10 Fmoc-L-Ser(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma >4 h, rt. 11 Fmoc-L-Tyr(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma >4 h, rt 12 Fmoc-L-Asp(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma >4 h, rt. 13 Fmoc-L-Ser(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma >4 h, rt. 14 Fmoc-L-Thr(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma >4 h, rt. 15 Fmoc-L-Phe-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma >4 h, rt 16 Fmoc-L-Thr(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma >4 h, rt 17 Fmoc-Gly-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma >4 h, rt. 18 Fmoc-L-Gln(Trt)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma >4 h, rt. 19 Fmoc-Aib-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma >8 h, rt. 20 Boc-L-Tyr(tBu)-OH 2 × 30 min De-Fmoc cycles, 6 × 2 min post-dep washes, 3.0 AA/3.3 DIC/3.0 Oxyma >16 h, rt.

[0264] Soft cleavage: 1) Add resin bound SEQ ID NO:20 (5.7 g, ˜10 mmol) and charge 60 mL cleavage cocktail TFA/DCM (1/99, v/v/v). 2) Stir it for 10 min at rt. 3) Filter and collect the filtrate. 4) Neutralize the filtrate with 6.6 mL pyridine (1/1, mol/mol). 5) Repeat Steps 1-4 for 3 more times. 6) Concentrate the combined filtrate to dryness. 7) Dissolve the slurry with 30 mL of DMSO. 8) Charge the DMSO solution slowly to cold 300 mL water with stirring. 9) Filter and collect precipitation. 10) Re-slurry with 200 mL water for 2 times. 11) Dry in vacuum overnight to produce SEQ ID NO:20 (4.5 g, 63.4% yield) of a white solid. Analysis of the isolated solid using UPLC (99.4 area %). LC-MS [M+2H].sup.2+/2: 2053.39.

Example 61: Synthesis of SEQ ID NO:49 (Compound 52) by LPPS

[0265] To a 20 mL glass scintillation vial, charge SEQ ID NO:11 (1.0 eq, 145 mg), SEQ ID NO:7 (1.1 eq, 125 mg), and DMSO/MeCN (5 mL, 4/1, v/v) to dissolve all material. Add DIEA (3.0 eq, 0.055 mL) to the reaction mix followed by PyOxim (1.5 eq, 80 mg). Stir the reaction for 4 hours then, slowly add 40 ml of water with stirring. Collect the precipitated product by filtration and subsequently wash with water (2×40 mL). Dry the wet cake under vacuum to obtain crude solid SEQ ID NO:49 as a white solid (180 mg, 73.2% yield) with 89.5% purity by HPLC. LC-MS [M+2H].sup.2+/2: 1225.2.

Example 62: Synthesis of SEQ ID NO:18 by LPPS

[0266] To a 20 mL glass scintillation vial, add SEQ ID NO:49 (700 mg), followed by 8 mL DMSO (2 mL). Charge Et2NH (2.0 mL), and stir for 4 h. Concentrate the solution to dryness. Charge 60 mL cold MTBE with stirring. Filter and collect precipitation, and reslurry with 60 mL MTBE for 2 times. Dry the wetcake under vacuum to obtain SEQ ID NO:18 as a white solid (520 mg, 82% yield) with 82.3% purity by HPLC. LC-MS [M+2H].sup.2+/2: 1113.8.

Example 63: Synthesis of SEQ ID NO:48 by LPPS

[0267] To a 20 mL glass scintillation vial, add SEQ ID NO:20 (1.0 eq, 84 mg), SEQ ID NO:18 (1.1 eq, 50 mg), 1-Hydroxy-7-azabenzotriazole (HOAt; 1.0 eq, 3 mg), and DMSO (2 mL) to dissolve all materials. Add DIEA (6 eq, 21 μL) to this solution followed by PyAOP (2.5 eq, 22 mg) and mix for 6 hours. Charge additional PyAOP (1.0 eq, 9 mg) and DIEA (2.5 eq, 9 μL) and mix for 12 hours. Charge additional PyAOP (1.0 eq, 9 mg) and DIEA (2.5 eq, 9 μL) and mix for 6 hours. Charge the reaction solution slowly to cold water with stirring. Collect the precipitated product by filtration and subsequently wash with water 3 times (3×10 ml). Dry the product under vacuum to obtain white solid SEQ ID NO:48 (90 mg, 69.8% yield). UPLC: 81.9 area %.

Example 64: Global Deprotection of SEQ ID NO:48 to Produce SEQ ID NO:6

[0268] Global deprotection is carried out using the following procedure: 1) Charge 4 mL cleavage cocktail TFA/H.sub.2O/TIPS/DTT (0.925/0.025/0.025/0.025) into R1, followed by charging of SEQ ID NO:48 (180 mg). 2) Stir it for 3 hours at 20-30° C. 3) Pour the solution to chilled MTBE (30 mL). Stir the suspension for 0.5 hours. 4) Perform filtration through filter followed by MTBE washing (30 mL) twice. 5) Dry the wet cake under reduce pressure until constant weight. 6) Obtain 180 mg of dried crude obtained with 66.3% purity by HPLC.

Example 65: Native Chemical Ligation

[0269] Synthesis of Resin Compound 53:

##STR00026##

[0270] Fmoc-hydrazine-2-chlorotrityl resin (30.06 g, 25.5 mmol) is swollen in 300 mL DCM for 15 min. It is swollen with 2×400 mL DMF for 15 min each. Fmoc deprotection is performed with 3×400 mL 20% piperidine/DMF for 30 min each. The resin is then washed with 5×400 mL DMF. Fmoc-L-Lys(alloc)-OH (34.63 g, 76.5 mmol, 3.0 equiv) and HBTU (29.17 g, 76.9 mmol, 3.02 equiv) are dissolved in 400 mL of DMF. N, N-diisopropylethylamine (27 mL, 155 mmol, 6.08 equiv) is added to the amino acid solution. The solution is then added to the resin preparation XX and is allowed to stir for 6 hours. The resin is washed with 5×400 mL DMF, then 5×300 mL DCM and the resin is dried at 35° C. in a vacuum oven for about 16 hours. The resin loading is determined to be 0.52 mmol/g by quantitative NMR.

Example 66: Synthesis of Peptide Hydrazide SEQ ID NO:50 (Compound 54)

[0271] About 12.19 g of resin Compound 53 (5.4 mmol, loading value: 0.44 mmol/g) is swollen with 3×120 mL DMF for 15 min each. Peptide hydrazide (SEQ ID NO:50 is synthesized using standard SPPS as previously described.

Deprotection: 4×100 mL of 20% v/v piperidine in DMF, 20 minutes each.
Couplings: 3 equivalents of amino acid, 3 equivalents of OXYMA and 3.3 equivalents of DIC are used for amino acid coupling.
During the SPPS, the resin is washed with 5×120 mL DMF with 5 min N.sub.2 mix after each coupling and the final iteration of fmoc deprotection.
At the end of the peptide hydrazide synthesis, the resin is washed with DCM with N.sub.2 mixing. The resin is dried on the peptide synthesizer.

Alloc Deprotection and Sidechain Coupling:

[0272] The resin is washed with 5×120 mL DCM with 5 min stir. A solution of palladium tetrakis (500 mg, 0.43 mmol, 0.1 equiv) and phenylsilane (0.7 mL, 5.7 mmol, 1.02 equiv) is made in 75 mL DCM. It is added to the resin and stirred for 20 min. It is washed with 5×120 mL DCM and stirred for 5 min each. The alloc deprotection with Pd(PPh.sub.3).sub.4 and PhSiH.sub.3 is repeated twice.

[0273] The resin is washed with 5×120 mL DMF and stirred for 5 min each. TNTU (3.95 g, 10.82 mmol, 2 equiv) is dissolved in the DMF solution of (S)-13-(tert-butoxycarbonyl)-36,36-dimethyl-10,15,34-trioxo-3,6,35-trioxa-9,14-diazaheptatriacontanoic acid (Compound 25, 27 mL, 0.29 g/mL, 7.873 g, 10.8 mmol, 2 equiv) and this solution is made up to 75 mL with DMF. 3.8 mL of N, N-diisopropylethylamine (3.8 mL, 21.82 mmol, 4.0 mmol) is added to the solution of Compound 25 and stirred for 10 min. It is then added to the resin and stirred for 14 hours. The resin is then washed with 5×120 mL DMF (5 min stir) and 5×120 mL DCM (5 min stir). The resin is dried in a vacuum oven for about 16 hours at 35° C.

Global Deprotection and Cleavage:

[0274] 250 mL of the cleavage cocktail made with 2.5% w/v dithiothreitol (DTT), 2.5% v/v water, 2.5% v/v triisopropylsilane (TIPS) and 92.5% trifluoroacetic acid (TFA) is added to the dried resin (22.2 g) in a 500 mL three-necked round bottom flask and stirred for about 2.5 hours. The resin is filtered and washed with 2×7.5 mL TFA. The filtrate is poured into 1.40 L cold MTBE and the peptide precipitated out immediately. The filtration flask is washed with 2×5 mL TFA and poured into the cold MTBE. It is cooled down to −20° C. for half an hour and then centrifuged. The peptide precipitate is then washed twice with 300 mL MTBE and centrifuged. The peptide precipitate is dried in a vacuum oven at 27° C. for about 16 hours. About 9.9 g of the crude SEQ ID NO:50 is obtained after drying.

Example 67: Synthesis of the Thioester SEQ ID NO:51 (Compound 55)

[0275] Crude peptide hydrazide (SEQ ID NO:50, 3.65 g, 1.41 mmol) is dissolved in 250 mL of the ligation buffer (6 M guanidine hydrochloride and 0.1 M sodium hydrogen phosphate monobasic, pH about 7.0). The pH is adjusted to about 3.3 with 5 N HCl solution and the solution is cooled to −15° C. in an acetone-ice bath. 2.5 mL of 4.31 M sodium nitrite solution (742.7 mg, 10.8 mmol, 7.6 equiv.) is added to the peptide hydrazide solution and allowed to stir for 15 min at −15° C. Meanwhile, 4-mercaptophenol (1.052 g, 8.34 mmol) is suspended in 3 mL of the ligation buffer, pH is adjusted to about 7.0 with 5 N NaOH solution and made up to 10 mL with ligation buffer (6 M guanidine hydrochloride and 0.1 M sodium hydrogen phosphate monobasic, pH about 7.0). After 15 min, 7.5 mL of the 4-mercaptophenol is added to the peptide hydrazide solution to cause in-situ thiolysis of the peptidyl azide generated from SEQ ID NO:50.

[0276] The pH of the reaction mixture is adjusted to about 6.5 with 5 N sodium hydroxide solution. Thiolysis of the peptidyl azide is allowed to run for 15 min and the crude thioester mixture is purified by RP-HPLC on a Phenomenex Luna C18 10 μm column (30 mm×250 mm) at ambient temperature with a linear gradient of 30-55% acetonitrile in water over 25 min after 10% acetonitrile in water for the first 3 min and 10-30% acetonitrile in water from 3 min to 5 min with constant 5% ammonium acetate throughout the purification. This yields about 0.415 g of the peptide thioester (SEQ ID NO:51) [Expected (mass+2H.sup.+)/2=1342.7052, observed (mass+2H.sup.+)/2=1342.6958].

Example 68: Native Chemical Ligation to Synthesize SEQ ID NO:53

[0277] Aqueous solution of 6 M guanidine hydrochloride and 0.1 M sodium hydrogen phosphate monobasic (pH about 7.0) is the ligation buffer used in native chemical ligation. The buffer is degassed with nitrogen gas for 15 min. 4-mercaptophenol (193 mg, 1.5 mmol, 10 equiv), tris(2-carboxyethyl)phosphine (TCEP, 656.6 mg, 2.3 mmol, 15.3 equiv) and ascorbic acid (269 mg, 1.5 mmol, 10 equiv) are taken in a 3 neck-round bottom flask. The flask is under nitrogen gas. 41 mL of the ligation buffer is added to dissolve the reagents in the round bottom flask. The pH of the solution is adjusted to about 7.0 with 5 N NaOH solution. The peptide thioester SEQ ID NO:51 ((406.8 mg, 0.15 mmol) and the N-terminal cysteine fragment SEQ ID NO 52 (Compound 56) (326.5 mg, 0.15 mmol, 1 equiv) are added to the above solution. pH is adjusted to about 7.0 with 5 N NaOH solution. The reaction mixture is allowed to stir for about 10 hours under nitrogen. Most of the thioester SEQ ID NO:51 is consumed and hence, the reaction mixture is stored in a freezer at −20° C. for about 14 hours. SEQ ID NO:53 is purified by RP-HPLC on a Phenomenex Luna C18 10 μm column (30 mm×250 mm) at ambient temperature with a linear gradient of 30-50% acetonitrile in water over 25 min after 20% acetonitrile in water for the first 3 min and 20-30% acetonitrile in water from 3 min to 5 min with constant 5% ammonium acetate throughout the purification. This yields about 0.52 g of the cysteine analogue SEQ ID NO:53 [Expected (mass+3H.sup.+)/3=1587.8219, observed (mass+3H.sup.+)/3=1587.8198].

[0278] Photodesulfurization: Aqueous buffer of 3 M guanidine hydrochloride and 0.1 M sodium hydrogen phosphate monobasic (pH about 7.0) is freshly made. 0.5 mL solution of 7.64 mM tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate (2.86 mg, 0.004 mmol) is made in the buffer. Tris(2-carboxyethyl)phosphine (TCEP, 64.3 mg, 0.22 mmol) is suspended in the buffer and the pH is adjusted to about 7.0 with 5 N NaOH solution. This is diluted to 2 mL with the buffer. SEQ ID NO:53 (10 mg, 0.0021 mmol) is dissolved in 4 mL of the buffer in a 7 mL scintillation vial. Triphenylphosphine-3,3′,3″-trisulfonic acid trisodium salt (TPPTS, 231.2 mg, 0.41 mmol, 194 equiv) and 2-mercaptoethanesulfonic acid sodium salt (MESNa, 32.6 mg, 0.20 mmol, 95 equiv) are added to the solution of SEQ ID NO:6. 28 μL of tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate (0.00021 mmol, 0.1 equiv) and 20 μL of TCEP solution (0.0022 mol, 1.0 equiv) are added to the reaction mixture. The vial Is placed in a Penn Optical Coatings photoreactor ml and stirred at 459 RPM with 91% LED intensity. Fan is run at 3564 RPM to prevent heating of the reaction mixture. After about 3.5 hours, TCEP (0.40 mg, 0.7 equiv) is further added and the reaction mixture is stirred in the photoreactor for 16 hours. After 16 hours, the reaction is complete and more than 95% of the SEQ ID NO:53 is converted into SEQ ID NO:6.

[0279] Metal-free desulfurization: Aqueous solution of 6 M guanidine hydrochloride and 0.1 M sodium hydrogen phosphate monobasic (pH 7.0) is the buffer used for this reaction. The buffer is thoroughly purged with nitrogen gas for more than an hour. SEQ ID NO:53 (40.3 mg, 0.0085 mmol), 2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (27.7 mg, 0.0857 mmol, 10.1 equiv), L-glutathione reduced (L-GSH, 25.9 mg, 0.0843 mmol, 10 equiv) and tris(2-carboxyethyl)phosphine (TCEP, 36.5 mg, 0.1273 mmol, 15.0 equiv) are dissolved in 4 mL of the buffer. The reaction mixture is degassed again with nitrogen gas for about 2 min and the pH is adjusted to about 7.0 with 5 N NaOH. The solution is stirred under nitrogen at 45° C. for 12 hours and then at the room temperature for 8 hours. After 20 hours, most of the SEQ ID NO:53 is converted to the SEQ ID NO:6. [Expected (mass+3H.sup.+)/3=1604.5153, observed (mass+3H.sup.+)/3=1577.1581].

Example 69: Synthesis of SEQ ID NO:53 (Compound 57) by Native Chemical Ligation (NCL) Using SEQ ID NOs 52 and 54 Using Cysteinylprolyl Ester (CPE)

[0280] 3M Buffer solution: Guanidine hydrochloride (2.86 g, 30.0 mmol), sodium dihydrogen phosphate (0.24 g, 2.0 mmol) and tris(2-carboxyethyl)phosphine hydrochloride [TCEP] (0.0166 g, 0.0579 mmol) is weighed into a 15 mL centrifuge tube, dissolved in deionized water and made up to approximately 9.5 mL. The pH of the buffer solution is adjusted to ˜8.3 by adding 5N NaOH as required. If the pH is overshot, it is readjusted to ˜8.3 by addition of 1N HCl.

General Procedure for Ligation:

[0281] 1 mL of the buffer solution is added to a 5 mL scintillation vial containing pre-weighed SEQ ID NO:54 [CPE-peptide analogue] (0.01 g, 0.003 mmol, 94.69 mass %) and SEQ ID NO:52 [Cys-peptide] (0.0073 g, 0.0032 mmol, 96.9 mass %). The peptide fragments are fully dissolved in the 3M buffer solution by sonication. The pH of the solution is recorded and readjusted by addition of 5N NaOH to pH-8.30. If the pH is overshot, it is readjusted to pH-8.3 by addition of 1N HCl. The solution is then transferred to a HPLC vial and the sample monitored at different time points by ELTIVO at 37° C. (32° C. internal temperature). Complete conversion to SEQ ID NO:53 analogue (up to 69% by Q-Tof) is generally observed after 18 h.

[0282] 5M Buffer solution: Guanidine hydrochloride (4.78 g, 50.0 mmol), sodium dihydrogen phosphate (0.24 g, 2.0 mmol) and (tris(2-carboxyethyl)phosphine hydrochloride [TCEP] (0.0166 g, 0.0579 mmol) is weighed into a 15 mL centrifuge tube, dissolved in deionized water and made up to approximately 9.5 mL. The pH of the buffer solution is adjusted to ˜8.3 by adding 5N NaOH as required. If the pH is overshot, it is readjusted to ˜8.3 by addition of 1N HCl.

General Procedure for Ligation:

[0283] 3 mL of the buffer solution is added to a 5 mL scintillation vial containing pre-weighed SEQ ID NO:54 [CPE-peptide analogue] (0.01 g, 0.003 mmol, 94.69 mass %) and SEQ ID NO:52 [Cys-peptide] (0.0073 g, 0.0032 mmol, 96.9 mass %). The peptide fragments are fully dissolved in the 5M buffer solution by sonication. The pH of the solution is recorded and readjusted by addition of 5N NaOH to pH-8.30. If the pH is overshot, it is readjusted to pH-8.3 by addition of 1N HCl. The thiol [such as; MeSNa, thiophenol, hydroxythiophenol] (5 equiv.) is then added to the reaction solution. The pH is monitored again and further adjusted to pH-8.3 using 1N NaOH or 1N HCl as required. The solution is then transferred to a HPLC vial and the sample monitored at different time points HPLC at 37° C. (32° C. internal temperature). Complete conversion to SEQ ID NO:53 is generally observed after approximately 17 h (by HPLC).

Example 70: Synthesis of Fmoc-L-Pro-glycolic Acid-L-Val-OH (Compound 58)

[0284] ##STR00027##

Step 1 (Fmoc-L-Val-OH Coupling):

[0285] Prior to the first coupling, Fmoc-Rink amide AM resin (0.74 g/mmol, 1.35 g, 1.00 mmol) is charged to the reaction vessel. The resin is swelled with 3×10 ml of DMF for 15 minutes each, then deprotected with 3×10 ml of 20% piperidine/DMF (v/v) for 30 minutes each and washed with 5×10 ml of DMF for 1 minute each. A solution is prepared of (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoic acid (1.018 g, 3.00 mmol) and 1-hydroxybenzotriazole hydrate (0.74 g, 3.30 mmol, 60 mass %) in 10 ml of DMF. To this solution is added N, N′-diisopropylcarbodiimide (0.52 mL, 3.30 mmol) and the corresponding solution is added to the reaction vessel containing the swelled resin. The reaction is mixed for 1 hour at ambient temperature, then the liquid is drained. The resin is washed with 5×10 ml of DMF for 1 minute each and then forward processed to step 2.

Step 2 (Glycolic Acid Coupling):

[0286] The Fmoc group is removed by treatment of the resin from step 1 with 3×10 ml of 20% piperidine/DMF (v/v) for 30 minutes each and washed with 5×10 ml of DMF for 1 minute each. A solution is prepared of glycolic acid (228 mg, 3.00 mmol) and 1-hydroxybenzotriazole hydrate (353 mg, 2.31 mmol) in 10 ml of DMF. To this solution is added N, N′-diisopropylcarbodiimide (0.52 mL, 3.30 mmol) and the corresponding solution is added to each reactor. The reaction is mixed for 5 hours at ambient temperature, then the liquid is drained. The resin is washed with 5×10 ml of DMF for 1 minutes and then forward processed to step 3.

Step 3 (Fmoc-L-Pro-OH Coupling):

[0287] A solution is prepared of (2R)-1-(9H-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid (1.012 g, 3.00 mmol), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (1.25 g, 3.30 mmol), and N,N-diisopropylethylamine (0.87 mL, 5.00 mmol) in 10 ml of DMF. The solution is shaken for a few minutes, then transferred to the reaction vessel containing the resin. The reaction is mixed for 16 hours at ambient temperature, then the liquid is drained. The resin is washed with 5×10 ml of DMF for 1 minutes and 5×10 ml of dichloromethane for 2 minutes, then dried to constant weight to provide 1.719 g of the title compound on resin.

[0288] A 50 mg sample of the peptide is cleaved from the resin with 2.0 mL of a solution consisting of 92.5% TFA, 2.5% triisopropylsilane, 2.5% water, and 2.5% dithiothreitol (v/v/v/w). The mixture is agitated on a rotary mixer for 1.5 hours, diluted with 16 ml of 80:20 DMSO/acetonitrile (v/v), and filtered to remove the resin. The filtrate is analyzed by LC/MS and shown to contain 75.5 area % desired tripeptide and 16.8% of product containing multiple glycolic acid additions.

Example 71: Alternate Synthesis of Fmoc-L-Pro-Glycolic Acid-L-Val-OH

Step 1 (Fmoc-L-Val-OH Coupling):

[0289] Prior to the first coupling, Rink amide AM resin (0.74 g/mmol, 1.35 g, 1.00 mmol) is charged to the reactor vessel. Each resin is swelled with 3×10 ml of DMF for 20 minutes each, then deprotected with 3×10 ml of 20% piperidine/DMF (v/v) for 30 minutes each and washed with 5×10 ml of DMF for 1 minute each. A solution is prepared of (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoic acid (1.018 g, 3.00 mmol) and 1-hydroxybenzotriazole hydrate (353 mg, 2.31 mmol) in 10 ml of DMF. To this solution is added N, N′-diisopropylcarbodiimide (517 μL, 3.30 mmol) and the corresponding solution is added to the reaction vessel containing the swelled resin. The reaction is mixed for 4 hours at ambient temperature and then drained. The resin is washed with 5×10 ml of DMF for 1 minute each and forward processed to the next step.

Step 2 (Fmoc-Glycolic Acid Coupling):

[0290] The Fmoc group is removed by treatment with 3×10 ml of 20% piperidine/DMF (v/v) for 30 minutes each and washed with 5×10 ml of DMF for 1 minute each. A solution is prepared of 2-(9H-fluoren-9-ylmethoxycarbonyloxy)acetic acid (894.9 mg, 3.00 mmol) and 1-hydroxybenzotriazole hydrate (353 mg, 2.31 mmol) in 10 ml of DMF. To this solution is added N, N′-diisopropylcarbodiimide (517 μL, 3.30 mmol) and the corresponding solution is added to the reactor containing the resin. The reaction is mixed for 16 hours at ambient temperature and the liquid is drained. The resin is washed with 5×10 ml of DMF for 1 minute each and then forward processed to the next step.

Step 3 (Fmoc-L-Pro-OH Coupling):

[0291] The Fmoc group is removed by treatment with 3×10 ml of 20% piperidine/DMF (v/v) for 30 minutes each and washed with 5×10 ml of DMF for 1 minute each. A solution is prepared of (2S)-1-(9H-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid (1.012 g, 3.00 mmol), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (1.25 g, 3.30 mmol), and N,N-diisopropylethylamine (870 μL, 5.00 mmol) in 10 ml of DMF. The corresponding solution is added to the reactor containing the resin. The reaction is mixed for 8 hours at ambient temperature and then drained. The resin is washed with 5×10 ml of DMF for 1 minute each and 5×10 ml of dichloromethane for 1 minute, then dried to constant weight to provide 1.622 g of the title compound on resin.

[0292] A 50 mg sample of the peptide is cleaved from the resin with 2.0 mL of a solution consisting of 92.5% TFA, 2.5% triisopropylsilane, 2.5% water, and 2.5% dithiothreitol (v/v/v/w). The mixture is agitated on a rotary mixer for 1.5 hours, diluted with 16 ml of 80:20 DMSO/acetonitrile (v/v), and filtered to remove the resin. The filtrate is analyzed by LC/MS and shown to contain 84.93 area % desired tripeptide no detectable multiple glycolic acid additions.

Example 72: Synthesis of 2-(9H-fluoren-9-ylmethoxycarbonyloxy)acetic acid (Fmoc-Glycolic Acid) (Compound 59)

[0293] ##STR00028##

Step 1 (tert-butyl 2-(9H-fluoren-9-ylmethoxycarbonyloxy)acetate):

[0294] To a magnetically stirred solution of tert-butyl 2-hydroxyacetate (10.00 g, 71.90 mmol, 95 mass %) in 120 ml of dichloromethane in a 500 mL round bottomed flask is added pyridine (60 mL, 742.0 mmol) in one portion. The resulting solution is cooled to 0-5° C. in an ice bath. To this solution is added a solution of 9-fluorenylmethyl chloroformate (20.00 g, 77.30 mmol) in 60 ml of dichloromethane dropwise via dropping funnel over 30 minutes. By the time the addition is complete, a precipitate had formed in the reaction. The ice bath is removed and the reaction mixture is allowed to stir for 18 hours at ambient temperature. More precipitate formed during the additional stir time. The reaction mixture is concentrated under reduced pressure to a solid-oil residue to remove most of the pyridine and dichloromethane, and then re-dissolved in 200 ml of dichloromethane. The solution is washed with 2×100 ml of 1M aqueous sodium bisulfate solution followed by 2×100 ml of saturated brine solution. The organic layer is dried over magnesium sulfate and concentrated to 27.13 grams of a yellow oil that gradually solidified. The crude product is forward processed without purification in the next step.

Step 2 (2-(9H-fluoren-9-ylmethoxycarbonyloxy)acetic acid):

[0295] Tert-butyl 2-(9H-fluoren-9-ylmethoxycarbonyloxy)acetate (26.0 g, 73.40 mmol) from step 1 is dissolved in dichloromethane (200 mL). To the magnetically stirred solution is added trifluoroacetic acid (52 mL) followed by triisopropylsilane (13 mL). The resulting solution is stirred for 3 hours at ambient temperature. The solution is concentrated under reduced pressure to remove the dichloromethane and nearly all of the trifluoroacetic acid. The resulting viscous residue is treated gradually with 1000 mL of 5% aqueous sodium bicarbonate solution to prevent foaming and the aqueous solution is washed with 3×500 ml of methyl tert-butyl ether to remove residual triisopropylsilane. The aqueous solution is cooled to 0-5° C. and 300 ml of ethyl acetate are added. The biphasic mixture is acidified to ˜pH 2 with 40% aq. phosphoric acid, requiring about 75 ml of acid. After separating the layers, the organic layer is dried over magnesium sulfate and concentrated under reduced pressure to a viscous pale yellow oil. The oil is cooled to −20° C. in the freezer, which caused the material to completely solidify to a white solid. The solid is triturated with 75 ml of cold heptane and after sonication, a uniform white suspension formed. The solid is filtered off, washed with heptane, and dried in the vacuum oven overnight at 33° C. to afford 15.21 g (69.5% yield over two steps) of a white solid.

NMR (CDCl.sub.3) confirmed that the desired product has been isolated.

Example 73: Synthesis of Fmoc-Lys(Mtt)-Cys(Trt)-OH (Compound 60)

[0296] ##STR00029##

Step 1

[0297] (2S)-6-[[diphenyl(p-tolyl)methyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid (A, 15 g, 24.01 mmol), N,N′-disuccinimidyl carbonate (7.45 g, 29.0 mmol, 99.6 mass %), 4-dimethyl aminopyridine [DMAP] (0.3 g, 2 mmol, 99 mass %) is weighed into 250 mL flask, equipped with a stir bar. Ethyl acetate (225 mL, 2000 mmol, 100 mass %) is then added and the solution mixed at room temperature (21-24° C.) until a solution is obtained. The reaction is stirred for 18 hours or until completion of reaction is confirmed by LCMS/NMR. The reaction mixture is transferred to a separatory funnel, washed with deionized water (60 mL×3) and the organic layer concentrated to dryness on the rotary evaporator to obtain crude compound of step 1.

Step 2

[0298] To crude compound of step 1 (17.33 g, 24.01 mmol) in N,N-dimethylformamide (208 mL, 2690 mmol) is added N,N-diisopropylethylamine (5.03 mL, 28.8 mmol) and (2R)-2-amino-3-tritylsulfanyl-propanoic acid (9.6 g, 26 mmol). The reaction is stirred using magnetic stirring at room temperature (21-24° C.) for 18 hours or until completion of reaction is confirmed by LCMS/NMR. The reaction mixture is transferred to a separatory funnel, washed with 10% citric acid (120 mL×2) and extracted with dichloromethane (100 mL×5). The organic layer is washed with deionized water (100 mL×2) and the combined organic layers concentrated to dryness on the rotary evaporator at 48-50° C. to remove excess solvent. The crude Compound 60 is dissolved in Acetonitrile (30 mL) by sonication. The crude Compound 60 solution is added dropwise to cold acetonitrile: deionized water (3:2, 700 mL) while being stirred. The slurry is stirred at 0° C., overnight. The solid is filtered, washed with hexanes (70 mL) and dried in the vacuum oven at 40° C. to give the product Fmoc-Lys(Mtt)-Cys(Trt)-OH (Compound 60) (21.0 g, 76.8% yield corrected for potency by Q-NMR).

Example 74: Synthesis of Fmoc-L-Lys(mtt)-L-Cys(trt)-L-Pro-glycolic acid-L-Val-OH (Compound 61)

[0299] ##STR00030##

[0300] Prior to the coupling reaction, Fmoc-L-Pro-glycolic acid-L-Val-OH on resin from example 73 above (1.719 g, 1.00 mmol) is swelled with 3×15 ml of DMF for 20 minutes each, then deprotected with 4×15 ml of 20% piperidine/DMF (v/v) for 30 minutes each and washed with 5×15 ml of DMF for 1 minute each. A solution is made of (2R)-2-[[(2S)-6-[[diphenyl(p-tolyl)methyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-3-tritylsulfanyl-propanoic acid (2.28 g, 2.00 mmol, 85.24 mass %), hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt) (0.375 g, 2.30 mmol, 95 mass %), and N,N′-diisopropylcarbodiimide (0.41 ml, 2.60 mmol) in 10 ml of DMF. The corresponding solution is added to the reactor containing the resin. The reaction is mixed for 12 hours at ambient temperature and the liquid is drained. The resin is washed with 5×15 ml of DMF for 1 minute each and 5×15 ml of dichloromethane for 1 minute, then dried to constant weight to provide 1.973 g of the title compound on resin.

[0301] A 50 mg sample of the peptide is cleaved from the resin with 2.0 mL of a solution consisting of 92.5% TFA, 2.5% triisopropylsilane, 2.5% water, and 2.5% dithiothreitol (v/v/v/w). The mixture is agitated on a rotary mixer for 1.5 hours, diluted with 16 ml of 80:20 DMSO/acetonitrile (v/v), and filtered to remove the resin. The filtrate is analyzed by LC/MS and shown to contain 81.93 area % desired pentapeptide along with 2.91 area % of des-Proline and 3.83% of des-Valine.

Example 75: Synthesis of Fmoc-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-OH (SEQ ID NO:55)

[0302] The title compound is prepared using standard solid phase synthesis conditions (Fmoc-protected amino acids/ethyl cyanoglyoxylate-2-oxime (Oxyma)/N,N′-diisopropylcarbodiimide (DIC) as described below.

Solvent and Reagents Preparations:

[0303] Twenty L of DMF are charged to the solvent reservoir. Five L of 20% Piperidine/DMF (v/v) solution are charged to the deprotection reservoir. 600 mL of 0.660 M DIC solution is prepared using N,N′-diisopropylcarbodiimide (49.98 g, 396.0 mmol) and DMF and charged to the DIC/solvent reservoir. 500 ml of 0.750 M Oxyma solution is prepared using ethyl cyanoglyoxylate-2-oxime (53.29 g, 371.2 mmol) and DMF and is charged to the Oxyma/solvent reservoir. Sieber resin (0.71 mmol/g, 14.09 g, 10.00 mmol) is charged to the reactor. Prior to beginning the synthetic steps shown below, the resin is swelled with 3×180 ml of DMF for 20 minutes each and the Fmoc group is removed with 3×180 ml of 20% piperidine/DMF (v/v) for 30 minutes each.

Amino Acid Solution Preparations:

[0304] One hundred mL of 0.375 M FmocNH-L-Ala-OH solution is prepared from (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid (11.68 g, 37.52 mmol) and DMF and charged to the appropriate amino acid bottle. 200 mL of 0.375 M FmocNH-Gly-OH solution is prepared from 2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (22.30 g, 75.01 mmol) and DMF and charged to the appropriate amino acid bottle. 360 mL of 0.375 M FmocNH-L-Pro-OH solution is prepared from (2S)-1-(9H-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid (45.54 g, 135.0 mmol) and DMF and charged to the appropriate amino acid bottle. 280 mL of 0.375 M FmocNH-L-Ser(tBu)-OH solution is prepared from (2S)-3-tert-butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid (40.25 g, 105.0 mmol) and DMF and charged to the appropriate amino acid bottle.

Coupling Conditions:

[0305] Pro: 0.18 M, 3.0 equiv amino acid, 3.0 equiv Oxyma/3.3 equiv DIC, 30 minute pre-activation of activated ester solution, 6 hour coupling time at ambient temperature, 4×30 minute deprotection with 20% piperidine/DMF (v/v), 5×2 minute DMF washes post deprotection and post-coupling.

[0306] Ala (after Pro), Gly (after Pro): 0.18 M, 3.0 equiv amino acid, 3.0 equiv Oxyma/3.3 equiv DIC, 30 minute pre-activation of activated ester solution, 4 hour coupling time at ambient temperature, 4×30 minute deprotection with 20% piperidine/DMF (v/v), 5×2 minute DMF washes post deprotection and post-coupling.

[0307] All other couplings: 0.18 M, 3.0 equiv amino acid, 3.0 equiv Oxyma/3.3 equiv DIC, 30 minute pre-activation of activated ester solution, 4 hour coupling time at ambient temperature, 3×30 minute deprotection with 20% piperidine/DMF (v/v), 5×2 minute DMF washes post deprotection and post-coupling.

[0308] At the end of the synthesis, the resin is washed with 5×180 ml of DMF for 2 minutes each, followed by 5×180 ml of MTBE for 2 minutes each. The resin is removed from the reactor, transferred to a tared crystallization dish, and dried in vacuo at 40° C. to constant weight to provide 25.15 g of the title compound on resin. Based upon the mass of the resin starting material, the yield of peptide is 11.07 g (89%).

[0309] The Fmoc group is removed from a 251 mg sample of the peptide on resin by swelling the resin with 3×6 ml of DMF for 10 minutes, treating with 3×6 ml of 20% piperidine/DMF (v/v) for 30 minutes each, washing with 5×6 ml of DMF for 1 minute each, washing with 5×6 ml of dichloromethane for 1 minute each and drying to constant weight.

[0310] The deprotected product sample is cleaved from the resin by mixing on a rotary mixer in a 20 ml scintillation vial for 2 hours with 5 mL of TFA/TIS/H2O/DTT ([0.925 v:0.025 v:0.025 v]:0.025 w) solution. The resin is filtered and the resin wet cake is washed with 2 mL of neat TFA.

[0311] The resulting crude peptide is precipitated with 35 mL of cold MTBE, centrifuged, washed with 2×35 ml of MTBE, and dried in vacuo overnight at 33° C. to give 105.1 mg (94.9%) of the fully deprotected peptide. Analysis by UPLC showed 98.62 area % purity with no related substances over 0.30 area %. The loading of the peptide on resin is measured at 0.37 mmol/g vs theoretical loading of 0.37 mmol/g.

Example 75: Synthesis of H-Cys-Gln-Aib-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH.SUB.2 .SEQ ID NO:52 (Compound 62)

[0312] The title compound is prepared using standard solid phase synthesis conditions (Fmoc-protected amino acids/ethyl cyanoglyoxylate-2-oxime (Oxyma)/N,N′-diisopropylcarbodiimide (DIC) as described below.

Solvent and Reagents Preparations:

[0313] Forty L of DMF are charged to the solvent reservoir. 4 L of 20% Piperidine/DMF (v/v) solution are charged to the deprotection reservoir. 600 mL of 0.660 M DIC solution is prepared using N,N′-diisopropylcarbodiimide (49.98 g, 396.0 mmol) and DMF and charged to the DIC/solvent reservoir. 500 ml of 0.750 M Oxyma solution is prepared using ethyl cyanoglyoxylate-2-oxime (53.29 g, 371.2 mmol) and DMF and charged to the Oxyma/solvent reservoir. 9H-fluoren-9-ylmethyl N-[2-[(2S)-2-[[(1S)-2-[[(1S)-2-[[2-[[(1S)-2-[(2S)-2-[(2S)-2-[(2S)-2-[[(1S)-2-amino-1-(tert-butoxymethyl)-2-oxo-ethyl]carbamoyl]pyrrolidine-1-carbonyl]pyrrolidine-1-carbonyl]pyrrolidin-1-yl]-1-methyl-2-oxo-ethyl]amino]-2-oxo-ethyl]amino]-1-(tert-butoxymethyl)-2-oxo-ethyl]amino]-1-(tert-butoxymethyl)-2-oxo-ethyl]carbamoyl]pyrrolidin-1-yl]-2-oxo-ethyl]carbamate on Sieber resin (0.41 mmol/g, 1.22 g, 0.500 mmol) is charged to each of eleven reactors (total of 5.5 mmol of peptide on resin). Prior to beginning the synthetic steps shown below, the resin in each reactor is swelled with 3×10 ml of DMF for 20 minutes each then the Fmoc group is removed with 3×10 ml of 20% piperidine/DMF (v/v) for 30 minutes each and the resin is washed with 5×10 ml of DMF for 1 minute each.

Amino Acid Solution Preparations:

[0314] 1. 57 mL of 0.375 M FmocNH-L-Ala-OH solution is prepared from (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid (6.66 g, 21.38 mmol) and DMF and charged to the appropriate amino acid bottle.

[0315] 2. 103 mL of 0.375 M FmocNH-L-Glu(tBu)-OH solution is prepared from (2S)-5-tert-butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-pentanoic acid (16.44 g, 38.63 mmol) and DMF and charged to the appropriate amino acid bottle.

[0316] 3. 61 mL of 0.375 M FmocNH-L-Phe-OH solution is prepared from (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-phenyl-propanoic acid (8.83 g, 22.79 mmol) and DMF and charged to the appropriate amino acid bottle.

[0317] 4. 57 mL of 0.375 M FmocNH-Gly-OH solution is prepared from 2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (6.36 g, 21.38 mmol) and DMF and charged to the appropriate amino acid bottle.

[0318] 5. 57 mL of 0.375 M FmocNH-L-Ile-OH solution is prepared from (2S,3S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-pentanoic acid (7.55 g, 21.38 mmol) and DMF and charged to the appropriate amino acid bottle.

[0319] 6. 103 mL of 0.375 M FmocNH-L-Leu-OH solution is prepared from (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-4-methyl-pentanoic acid (13.65 g, 38.62 mmol) and DMF and charged to the appropriate amino acid bottle.

[0320] 7. 82 mL of 0.375 M FmocNH-L-Gln(trt)-OH solution is prepared from (2S)-5-(tert-butylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-pentanoic acid (13.05 g, 21.38 mmol) and DMF and charged to the appropriate amino acid bottle.

[0321] 8. 57 mL of 0.375 M FmocNH-L-Tyr(tBu)-OH solution is prepared from (2S)-3-(4-tert-butoxyphenyl)-2-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid (9.82 g, 21.38 mmol) and DMF and charged to the appropriate amino acid bottle.

[0322] 9. 57 mL of 0.375 M FmocNH-Aib-OH solution is prepared from 2-(9H-fluoren-9-ylmethoxycarbonylamino)-2-methyl-propanoic acid (6.96 g, 21.38 mmol) and DMF and charged to the appropriate amino acid bottle.

[0323] 10. 44 mL of 0.375 M FmocNH-L-Cys(trt)-OH solution is prepared from (2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanyl-propanoic acid (9.66 g, 16.50 mmol) and DMF and charged to the appropriate amino acid bottle.

Coupling Conditions:

[0324] Gln: 0.18 M, 3.0 equiv amino acid, 3.0 equiv Oxyma/3.3 equiv DIC, 30 minute pre-activation of activated ester solution, 18 hour coupling time at ambient temperature, 4×30 minute deprotection with 20% piperidine/DMF (v/v), 5×1 minute DMF washes post deprotection and post-coupling.

[0325] Aib, Ala, Leu-9: 0.18 M, 3.0 equiv amino acid, 3.0 equiv Oxyma/3.3 equiv DIC, 30 minute pre-activation of activated ester solution, 12 hour coupling time at ambient temperature, 4×30 minute deprotection with 20% piperidine/DMF (v/v), 5×1 minute DMF washes post deprotection and post-coupling.

[0326] Phe, Ile: 0.18 M, 3.0 equiv amino acid, 3.0 equiv Oxyma/3.3 equiv DIC, 30 minute pre-activation of activated ester solution, 8 hour coupling time at ambient temperature, 4×30 minute deprotection with 20% piperidine/DMF (v/v), 5×1 minute DMF washes post deprotection and post-coupling.

[0327] Cys: 0.18 M, 3.0 equiv amino acid, 3.0 equiv Oxyma/3.3 equiv DIC, no pre-activation of activated ester solution, 8 hour coupling time at ambient temperature, 4×30 minute deprotection with 20% piperidine/DMF (v/v), 5×1 minute DMF washes post deprotection and post-coupling.

[0328] All other couplings: 0.18 M, 3.0 equiv amino acid, 3.0 equiv Oxyma/3.3 equiv DIC, 30 minute pre-activation of activated ester solution, 4 hour coupling time at ambient temperature, 3×30 minute deprotection with 20% piperidine/DMF (v/v), 5×1 minute DMF washes post deprotection and post-coupling.

[0329] At the end of the synthesis, each resin is washed with 5×10 ml of DMF for 1 minute each, followed by 5×1 ml of dichloromethane for 1 minute each. The resins are dried to constant weight and combined to provide 24.394 g of the title compound on resin. The Fmoc group is removed from a 91.8 mg sample of the peptide on resin by swelling the resin with 3×4 ml of DMF for 15 minutes each, treating with 3×4 ml of 20% piperidine/DMF (v/v) for 30 minutes each, washing with 5×4 ml of DMF for 1 minute each, washing with 5×4 ml of dichloromethane for 1 minute each and drying to constant weight. The deprotected product sample is cleaved from the resin by mixing on a rotary mixer in a 20 ml scintillation vial for 2 hours with 5 mL of TFA/TIS/H.sub.2O/DTT ([0.925 v:0.025 v:0.025 v]:0.025 w) solution. The resin is filtered and the resin wet cake is washed with 2 mL of neat TFA. The resulting crude peptide is precipitated with 35 mL of cold MTBE, centrifuged, washed with 2×35 ml of MTBE, and dried in vacuo overnight at 33° C. to give 48.2 mg of the fully deprotected peptide. Analysis by UPLC showed 88.02 area % purity with no related substances over 1.0 area %. The remainder of the peptide is cleaved from the resin by mechanically stirring with 200 ml of a solution made up of 185 mL of trifluoroacetic acid, 5.0 mL of triisopropylsilane, 5.0 mL of water, 5.0 g of dithiothreitol in a 3-necked round bottomed flask for 2 hours at ambient temperature. The resin is removed by filtration on a fritted funnel and washed with 80 ml of TFA to give a total solution volume of approximately 280 mL. The peptide is precipitated by addition to 1400 ml of cold MTBE. After aging at −20° C. for 1 hour, the slurry is divided into six bottles and centrifuged. The resulting solids after centrifugation are combined into two bottles and each solid is washed twice with 250 ml of room temperature MTBE. The resulting white solid is dried overnight in the vacuum oven at 33° C. to give 20.07 g of crude peptide.

Example 76: Synthesis of Boc-Tyr(tBu)-Aib-Gln(trt)-Glu(tBu)-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Tyr(tBu)-Ser(tBu)-Ile-αMeLeu-Leu-Asp(tBu)-Lys(mtt)-Cys(trt)-Pro-glycolic acid-Val-NH.SUB.2 .(SEQ ID NO:56; Compound 63)

[0330] The title compound is prepared using standard solid phase synthesis conditions (Fmoc-protected amino acids/ethyl cyanoglyoxylate-2-oxime (Oxyma)/N,N′-diisopropylcarbodiimide (DIC).

Solvent and Reagents Preparations:

[0331] Forty L of DMF are charged to the solvent reservoir. 4 L of 20% Piperidine/DMF (v/v) solution are charged to the deprotection reservoir. 600 mL of 0.660 M DIC solution is prepared using N,N′-diisopropylcarbodiimide (49.98 g, 396.0 mmol) and DMF and charged to the DIC/solvent reservoir. 500 ml of 0.750 M Oxyma solution is prepared using ethyl cyanoglyoxylate-2-oxime (53.29 g, 371.2 mmol) and DMF and charged to the Oxyma/solvent reservoir. [2-[[(1S)-1-carbamoyl-2-methyl-propyl]amino]-2-oxo-ethyl] (2S)-1-[(2R)-2-[[(2S)-6-[[diphenyl(p-tolyl)methyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoyl]amino]-3-trityl sulfanyl-propanoyl]pyrrolidine-2-carboxylate on Rink Amide AM, Rink Amide MBHA or Sieber resin (0.500 mmol) is charged to each of eight reactors (total of 4.0 mmol of peptide on resin).

[0332] Prior to beginning the synthetic steps shown below, the resin in each reactor is swelled with 3×10 ml of DMF for 20 minutes each then the Fmoc group is removed with 4×10 ml of 20% piperidine/DMF (v/v) for 30 minutes each and the resin was washed with 5×10 ml of DMF for 1 minute each.

Amino Acid Solution Preparations:

[0333] 1. 57 mL of 0.375 M FmocNH-Aib-OH solution is prepared from 2-(9H-fluoren-9-ylmethoxycarbonylamino)-2-methyl-propanoic acid (6.96 g, 21.37 mmol) and DMF, then charged to the appropriate amino acid bottle.

[0334] 2. 103 mL of 0.375 M FmocNH-L-Asp(tBu)-OH solution is prepared from (2S)-4-tert-butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-butanoic acid (15.87 g, 38.63 mmol) and DMF, then charged to the appropriate amino acid bottle.

[0335] 3. 57 mL of 0.375 M FmocNH-L-Phe-OH solution is prepared from (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-phenyl-propanoic acid (8.28 g, 21.38 mmol) and DMF, then charged to the appropriate amino acid bottle.

[0336] 4. 57 mL of 0.375 M FmocNH-Gly-OH solution is prepared from 2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (6.36 g, 21.38 mmol) and DMF, then charged to the appropriate amino acid bottle.

[0337] 5. 57 mL of 0.375 M FmocNH-L-Ile-OH solution is prepared from (2S,3S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-pentanoic acid (7.55 g, 21.38 mmol) and DMF, then charged to the appropriate amino acid bottle.

[0338] 6. 57 mL of 0.375 M FmocNH-L-Lys(boc)-OH solution is prepared from (2S)-6-(tert-butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid (10.02 g, 21.38 mmol) and DMF, then charged to the appropriate amino acid bottle.

[0339] 7. 57 mL of 0.375 M FmocNH-L-Leu-OH solution is prepared from (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-4-methyl-pentanoic acid (7.55 g, 21.38 mmol) and DMF, then charged to the appropriate amino acid bottle.

[0340] 8. 57 mL of 0.375 M FmocNH-L-Gln(trt)-OH solution is prepared from (2S)-5-(tert-butylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-pentanoic acid (13.05 g, 21.38 mmol) and DMF, then charged to the appropriate amino acid bottle.

[0341] 9. 103 mL of 0.375 M FmocNH-L-Ser(tBu)-OH solution is prepared from (2S)-3-tert-butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid (14.81 g, 38.63 mmol) and DMF, then charged to the appropriate amino acid bottle.

[0342] 10. 103 mL of 0.375 M FmocNH-L-Thr(tBu)-OH solution is prepared from (2S,3R)-3-tert-butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)butanoic acid (15.35 g, 38.63 mmol) and DMF, then charged to the appropriate amino acid bottle.

[0343] 11. 57 mL of 0.375 M FmocNH-L-Tyr(tBu)-OH solution is prepared from (2S)-3-(4-tert-butoxyphenyl)-2-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid (9.82 g, 21.38 mmol) and DMF, then charged to the appropriate amino acid bottle.

[0344] 12. 57 mL of 0.375 M BocNH-L-Tyr(tBu)-OH solution is prepared from (2S)-2-(tert-butoxycarbonylamino)-3-(4-tert-butoxyphenyl)propanoic acid (7.21 g, 21.38 mmol) and DMF, then charged to the appropriate amino acid bottle.

[0345] 13. 57 mL of 0.375 M FmocNH-L-αMeLeu-OH solution is prepared from (2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-2,4-dimethyl-pentanoic acid (7.85 g, 21.37 mmol) and DMF, then charged to the appropriate amino acid bottle.

Coupling Conditions:

[0346] Boc-Tyr, Ile: 0.18 M, 3.0 equiv amino acid, 3.0 equiv Oxyma/3.3 equiv DIC, 30 minute pre-activation of activated ester solution, 18 hour coupling time at ambient temperature, 4×30 minute deprotection with 20% piperidine/DMF (v/v), 5×1 minute DMF washes post deprotection and post-coupling.

[0347] Aib, Gln, αMeLeu: 0.18 M, 3.0 equiv amino acid, 3.0 equiv Oxyma/3.3 equiv DIC, 30 minute pre-activation of activated ester solution, 12 hour coupling time at ambient temperature, 4×30 minute deprotection with 20% piperidine/DMF (v/v), 5×1 minute DMF washes post deprotection and post-coupling.

[0348] All other couplings: 0.18 M, 3.0 equiv amino acid, 3.0 equiv Oxyma/3.3 equiv DIC, 30 minute pre-activation of activated ester solution, 4 hour coupling time at ambient temperature, 3×30 minute deprotection with 20% piperidine/DMF (v/v), 5×1 minute DMF washes post deprotection and post-coupling. At the end of the synthesis, each resin is washed with 5×10 ml of DMF for 1 minute each, followed by 5×1 ml of dichloromethane for 1 minute each. The resins are dried to constant weight and forward processed to the next step. A sample from one of the reactors (˜80 mg) is cleaved from the resin by mixing on a rotary mixer in a 20 ml scintillation vial for 2 hours with 5 mL of TFA/TIS/H2O/DTT ([0.925 v:0.025 v:0.025 v]:0.025 w) solution. The resin is filtered and the resin wet cake is washed with 2 mL of neat TFA. The resulting crude peptide is precipitated with 35 mL of cold MTBE, centrifuged, washed with 2×35 ml of MTBE, and dried in vacuo overnight at 33° C. to give a sample of the fully deprotected peptide. Analysis by UPLC showed 59.8 area % purity.

Example 77: Synthesis of Tyr-Aib-Gln-Glu-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys(AEEA-AEEA-γGlu-C.SUB.20.-OH)-Cys-Pro-glycolic acid-Val-NH.SUB.2 .(SEQ ID NO:57; Compound 64)

Step 1 (Deprotection of mtt Protecting Group):

[0349] [2-[[(1S)-1-carbamoyl-2-methyl-propyl]amino]-2-oxo-ethyl] (2S)-1-[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-tert-butoxy-2-[[(2S)-2-[[(2S)-2-[[(2S,3 S)-2-[[(2S)-3-tert-butoxy-2-[[(2S)-2-[[(2S)-4-tert-butoxy-2-[[(2S)-3-tert-butoxy-2-[[(2S,3R)-3-tert-butoxy-2-[[(2S)-2-[[(2S,3R)-3-tert-butoxy-2-[[2-[[(2S)-2-[[2-[[(2S)-2-(tert-butoxycarbonylamino)-3-(4-tert-butoxyphenyl)propanoyl]amino]-2-methyl-propanoyl]amino]-5-oxo-5-(tritylamino)pentanoyl]amino]acetyl]amino]butanoyl]amino]-3-phenyl-propanoyl]amino]butanoyl]amino]propanoyl]amino]-4-oxo-butanoyl]amino]-3-(4-tert-butoxyphenyl)propanoyl]amino]propanoyl]amino]-3-methyl-pentanoyl]amino]-2,4-dimethyl-pentanoyl]amino]-4-methyl-pentanoyl]amino]-4-oxo-butanoyl]amino]-6-(tert-butoxycarbonylamino)hexanoyl]amino]-6-[[diphenyl(p-tolyl)methyl]amino]hexanoyl]amino]-3-tritylsulfanyl-propanoyl]pyrrolidine-2-carboxylate on Rink Amide AM, Rink Amide MBHA or Sieber resin (0.500 mmol) is charged to each of eight different reactors. Each resin is swelled with 3×10 ml of DCM for 15 min each and then treated with 1,1,1,3,3,3-hexafluoro-2-propanol, 30% in dichloromethane (v/v) (10 mL, 94.98 mmol) and mixed for one hour. The liquid is drained and the resin is again treated with 1,1,1,3,3,3-hexafluoro-2-propanol, 30% in dichloromethane (v/v) (10 mL, 94.98 mmol) and mixed for one hour. The liquid is again drained and the resin is washed with 5×10 ml of dichloromethane for 1 minute each, then 5×10 ml of DMF for 1 minute each and forward processed to the coupling reaction.

Step 2 (Coupling of Sidechain):

[0350] 2-[2-[2-[[(4S)-5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetic acid (6.41 g, 8.00 mmol, 91 mass %) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (4.16 g, 8.00 mmol) are dissolved in 72 mL of DMF. N,N-diisopropylethylamine (1.40 mL, 8.00 mmol) is added and the resulting solution is shaken for 1 minute, then one-eighth of the solution is added each resin in the reaction vessels and mixed for 16 hours. The liquid is drained and the resin is washed with 5×15 ml of DMF for 1 minute each, 5×15 ml of dichloromethane for 1 minute each, and then dried to constant weight to afford 15.66 g of the peptide on resin.

Step 3 (Cleavage and Global Deprotection of Peptide from Resin):

[0351] The peptide is cleaved from the resin by mechanically stirring with 160 ml of a solution made up of 148 mL of trifluoroacetic acid, 4.0 mL of triisopropylsilane, 4.0 mL of water, and 4.0 g of dithiothreitol in a 3-necked round bottomed flask for 2 hours at ambient temperature. The resin is removed by filtration on a fritted funnel and washed with 64 ml of TFA to give a total solution volume of approximately 224 mL. The peptide is precipitated by addition to 1120 ml of cold MTBE. After aging at −20° C. for 1 hour, the slurry is divided into four bottles and centrifuged. The resulting solids after centrifugation are combined into two bottles and each solid is washed twice with 250 ml of room temperature MTBE. The resulting solid is dried overnight in the vacuum oven at 33° C. to give 7.817 g of crude title compound.

Example 78: Purification of SEQ ID NO:6

[0352] The crude product (76.23 g) is dissolved in 3.05 L of 25% ACN/Water mixture (25 g/L crude concentration) in a 5 L reactor and stirred for 30 min. Remediate depsipeptide isomers by conversion using 28% ammonium hydroxide adjusting pH=9.0 and stir for 60 min, followed by a subsequent adjustment back to the acidic side (pH=2 using TFA). The final ACN content needs to be 30% to ensure solubility after the pH adjustment. Filter the crude prior to the first chromatography step.

[0353] First Chromatography Step: Column: DAC200, 200 mm×250 mm, stationary phase (YMC Triart C18, 10 um, 12 nm); Mobile phase A: 0.1% TFA in H.sub.2O; Mobile phase B: 100% ACN; detection at 230 nm; Injection volume: 3.5 L (by injection pump with the flow of 300 ml/min).

[0354] Gradient:

TABLE-US-00036 Time/min Flow/(ml/min) % A % B 0 1000 70 30 1 1000 70 30 52.5 1000 45 55

[0355] Collect fractions with desired product.

[0356] Second Chromatography Step: dilute with equal volume of H.sub.2O, and adjust pH to 6.5 using diluted ammonia. Column: DAC200, 200 mm×250 mm, stationary phase (YMC Triart C18, 10 um, 12 nm). Mobile phase A: 10 mM NH.sub.4HCO.sub.3 in H.sub.2O; Mobile phase B: 100% ACN; detection at 230 nm; Injection volume: 7.0 L (by injection pump with the flow of 300 ml/min).

[0357] Gradient:

TABLE-US-00037 Time/min Flow/(ml/min) % A % B 0 1000 70 30 1 1000 70 30 52.5 1000 45 55

[0358] Collect fractions with desired product.

[0359] Sodium Salt conversion step: Charge 1.76 g (44.0 mmol) NaOH dissolved in 200 ml H.sub.2O dropwise into the 7.2 L of the separated fraction and lyophilize. Obtain 38.02 g purified product (purity:98.0%).

Example 79: Purification of SEQ ID NO:29

[0360] The crude product is purified using a 20 cm column (4.8 kg Daiso C18-ODS-RPS, 10μ, 120 Å) and Mobile phase A: 0.1% TFA in H.sub.2O; Mobile phase B: 100% ACN; detection at 230 nm. The first purification step:

[0361] Gradient:

TABLE-US-00038 Time/min Flow/(ml/min) % A % B 0 600 80 20 3 600 70 30 75 600 45 55

[0362] Collect fractions with product of about 88% or greater purity, dilute 1:1 with H.sub.2O and adjust pH to 6.5 with 50% NH.sub.4OH.

[0363] Second chromatography step: use column in first step. Mobile phase A: 10 mM NH.sub.4HCO.sub.3 in H.sub.2O; Mobile phase B: 100% ACN; detection at 230 nm.

[0364] Gradient:

TABLE-US-00039 Time/min Flow/(ml/min) % A % B 0 600 80 20 45 600 50 50

[0365] Collect fractions with purity of about 97% or greater purity.

[0366] Convert to the sodium salt by adding 3 eq of NaOH followed by lyophilization.

SEQUENCES

[0367] The following amino acid sequences are referred to in the disclosure and are provided below for reference.

TABLE-US-00040 human GIP SEQ ID NO: 1 YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ human GLP-1 (7-36) amide SEQ ID NO: 2 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH.sub.2 human GCG SEQ ID NO: 3 HSQGTFTSDYSKYLDSRRAQDFVQWLMNT
SEQ ID NO:4—Incretin analog
YX.sub.2QGTFTSDYSIX.sub.13LDKX.sub.17AX.sub.19X.sub.20AFIEYLLX.sub.28X.sub.29GPSSX.sub.34APPPS, where X.sub.2 is Aib, X.sub.13 is L or αMeL, X.sub.17 is any amino acid with a functional group available for conjugation, and the functional group is conjugated to a C.sub.16-C.sub.22 fatty acid, X.sub.19 is Q or A, X.sub.20 is Aib, αMeK, Q or H, X.sub.28 is E or A, X.sub.29 is G or Aib, X.sub.34 is G or Aib, and the C-terminal amino acid is optionally amidated.

TABLE-US-00041 Incretin analog SEQ ID NO: 5 Y(Aib)QGTFTSDYSI(αMeL)LDKKAQ(Aib)AFIEYLLEGGPSSGAPP PS Incretin analog SEQ ID NO: 6 Y(Aib)QGTFTSDYSI(αMeL)LDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl)-(γGlu)-CO-(CH.sub.2).sub.18-CO.sub.2H)AQ(Aib)AFIE YLLEGGPSSGAPPPS-NH.sub.2

##STR00031## ##STR00032##

where R can be 2,2,2-trifluoroethyl.

##STR00033##

where R may be 2,2,2-trifluoroethyl.

##STR00034##

where R may be 2,2,2-trifluoroethyl.

##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050##

Where R is Pro-glycolic acid-Val or Pro-glycolic acid

##STR00051## ##STR00052## ##STR00053## ##STR00054##