Alcohols

Example 2

Tertiary propargylamine bridges were introduced into the peptide by initial incorporation of aza-propargylglycine and ε-N-alkyl-lysine residues into the GHRP-6 peptide sequence, followed by copper-catalyzed macrocyclization using an aldehyde linchpin. The A³-macrocyclization was examined immediately after introduction of the azapropargylglycine residue, as well as after completing the peptide sequence. To seek a diversity-oriented synthesis, two strategies were employed, in which an ε-N-alkyl-lysine residue was introduced respectively at the C-terminal and a central residue of the peptide sequence. With the ε-N-alkyl-lysine residue at the C-terminal, the macrocycle ring-size diversity was varied by azapropargyiglycine position scanning, in which the azapropargylglycyl residue was marched systematically to the N-terminal of the GHRP-6 sequence prior to macrocyclization with formaldehyde. With the ε-N-alkyl-lysine residue centred in the sequence, the influence of various & amino substituents was examined on macrocyclization.

The important step for the effective diversity-oriented synthesis of cyclic azapeptides by A³-macrocyclization was development of solid-phase methods to install the azapropargyiglycine residue and ε-N-alkyl-lysine residue into the peptide sequence prior to the copper-catalyzed macrocyclization using an aldehyde linchpin. The azapropargyiglycine can be inserted by submonomer synthesis of azapeptides on solid phase.^[13] The ε-N-alkylated lysine was prepared in solution and then coupled to the resin-bound peptide; however, solid-phase ε-N-alkylation of lysine was also performed by Mitsunobu chemistry on the corresponding ε-N-o-nitrobenzenesulfonyl (o-NBS) amine.^[20]

As a proof-of-concept of the A³-macrocyclization, cyclic azatripeptide 8 was pursued by placing ε-N-methyl lysine at the peptide C-terminal and inserting aza-propargyiglycine at the i+2 position. Prior to attachment to Rink amide resin, Fmoc-Lys(methyl, o-NBS)—OH 1 was synthesized from Boc-Lys-OH in solution. After Fmoc group removals and elongation with Fmoc-D-Phe-OH using DIC and HOBt, dipeptide 2a was acylated by the active carbazate prepared from benzophenone hydrazone and N,N′-disuccinimidyl carbonate (DSC) to provide semicarbazone 3a.^[14] Propargylation was performed using Cs₂CO₃ (300 mol %) and proparyl bromide (600 mol %) to furnish the aza-propargyiglycine 4a in good purity as accessed by LCMS analysis of a cleaved aliquot. After removal of the o-NBS-group with 2-mercaptoethanol and DBU, secondary ε-N-methylamine 5a was ready to test the A³-macrocyclization. Macrocycle 6a was prepared successfully by treating aza-peptide 5a with CuI (20 mol %) and 37% aqueous formaldehyde (600 mol %) in DMSO at rt for 24 h, as verified by LCMS analysis. Elongation of macrocycle 6a to cyclic GHRP-6 analog 8 was accomplished by removal of the semicarbazone with hydroxylamine hydrochloride in pyridine, acylation of the resulting semicarbazide 7a using the symmetric anhydride from treating Fmoc-Ala-OH with DIC, and standard solid-phase peptide synthesis, deprotection and resin cleavage. GHRP-6 macrocycle 8 was isolated in 3.5% overall yield after purification by preparative HPLC. Employing the same strategy, macrocycle 9 was obtained in 2.4% overall yield.

[Figure (not displayed)]

With macrocyclic GHRP-6 analogs 8 and 9 in hand, ring-size scope was investigated by systematically moving the azapropargylglycine residue towards the N-terminal of the sequence. Moreover, the ε-N-alkyl-lysine residue was prepared on solid phase by a method designed to expand the diversity of the ε-amine substituent. After coupling Fmoc-Lys(o-NBS)—OH 10^[19] to RINK amide resin and peptide elongation, semicarbazones 11a-d were synthesized. Chemoselective modification of the ε-N-o-(NBS)amine nitrogen was achieved by employing Mitsunobu chemistry to alkylate the former. Treatment of sulphonamide 11a-d with allyl alcohol, PPh₃, and diisopropyl azodicarboxylate (DIAD) provided selectively ε-N-(allyl)lysinyl peptides 12a-d as verified by LCMS analysis of cleaved aliquots. Subsequently, propargylation of semicarbazone was performed using Cs₂CO₃ (300 mol %) and proparyl bromide (600 mol %) to yield aza-propargylglycine peptides 13a-d. A³-Macrocyclization was then performed using the same conditions as discussed above to provide respectively 16-, 19, 21, and 24-membered macrocycles 15a-d as verified by LCMS analysis. After cyclization, semicarbazone removal, semicarbazide acylation, peptide elongation and resin cleavage were performed as described above to afford cyclic GHRP-6 analogs 17 and 18 after purification by preparative HPLC (Table 1). Coupling to semicarbazide macrocycles 16c and 16d was however unsuccessful in the syntheses of the corresponding cyclic GHRP-6 analogs. Steric hindrance inhibited apparently, the coupling to the semicarbazide of the larger ring-sizes. Semicarbazide 16d was however cleaved from resin to give cyclic aza-hexapeptide 19 with a N-terminal semicarbazide after purification by preparative HPLC.

[Figure (not displayed)]
[Figure (not displayed)]
[Figure (not displayed)]

[Figure (not displayed)]
[Figure (not displayed)]

Failure to elongate semicarbazides 16c and 16d after cyclization promoted investigation of a strategy featuring elongation of the complete linear peptide prior to A3-macrocyclization as the penultimate step before simultaneous deprotection and resin cleavage. Semicarbazone 13a was thus treated with hydroxylamine hydrochloride to liberate the semicarbazide 20a, and the linear peptide was elongated as described for its cyclic counterpart above. Aza-hexapeptide 21a was treated with DBU and 2-mercaptoethanol to selectively remove the o-NBS group. Subsequently, aza-hexapeptide 23a was effectively converted to macrocycle 17 using the standard A³-macrocyclization conditions. Resin cleavage gave cyclic azapeptide 17 in about 2-fold higher yield (1.2%) than the earlier approach, involving peptide elongation after cyclization.

Employing the peptide elongation/A³-macrocyclization approach, linear peptides 22b-d were also successfully converted into macrocyclic aza-GHRP-6 analogs 18, 24 and 25. Cyclic azapeptides 24 and 25 were respectively prepared with N-terminal alanine residues to avoid racemization during coupling to the semicarbazide with histidine, and to add an N-terminal basic amine that may favor biological activity.

The diversity of the ε-amine substituent was explored by the synthesis of cyclic azatetrapeptides 30-32 employing different alcohols as electrophiles in the Mitsunobu reaction: methanol, allyl alcohol and isopropyl alcohol. An ε-N-alkylated lysine was inserted in the peptide sequence to replace the tryptophan residue and an azapropargylglycine was placed at the i+3 position to replace the histidine residue in the GHRP-6 sequence. Cyclic analog 33 was synthesized with an additional alanine in the N-terminal for comparison with analog 31 to study the importance of the N-terminal basic amine.

[Figure (not displayed)]

Cyclic azapeptide GHRP-6 analogs were synthesized by the A³-macrocyclization method in yields and purities suitable for biological evaluation (Table 1).

Solution-Phase Chemistry

Ornithine Building Block Synthesis

[Figure (not displayed)]

Fmoc-Orn(o-NBS)—OH (RGO1):

Fmoc-Orn(Boc)-OH (2.02 g, 4.44 mmol) was dissolved in CH₂Cl₂ (30 mL) treated with TFA (20 mL) stirred at room temperature for 3 hours, and the volatiles were removed by rotary evaporation. The resulting yellow oil was co-evaporated with toluene to give a residue that was dissolved in THF (40 mL) and water (40 mL) and treated with iPr₂NEt (7.70 mL, 44.2 mmol) and o-NBSCl (1.13 g, 5.08 mmol) in one portion. The reaction was stirred at room temperature for 3 hours, diluted with EtOAc (100 mL) and sequentially washed with aqueous HCl (1 M, 100 mL×3), water (100 mL) and brine (100 mL). The organic layer was dried over MgSO₄ and the volatiles were removed by rotary evaporation to give sulfonamide RGO1 (2.4 g, quant.) as a light yellow solid. The amino acid was used without further purification.

¹H NMR (300 MHz, DMSO) δ 8.10 (t, J=5.6 Hz, 1H), 8.03-7.92 (m, 2H), 7.92-7.81 (m, 4H), 7.72 (d, J=7.4 Hz, 2H), 7.61 (d, J=8.0 Hz, 1H), 7.41 (t, J=7.2 Hz, 2H), 7.32 (t, J=7.1 Hz, 2H), 4.34-4.16 (m, 3H), 3.89 (td, J=8.7, 4.6 Hz, 1H), 2.90 (q, J=6.3 Hz, 2H), 1.73 (s, 1H), 1.65-1.43 (m, 3H). ¹³C NMR (75 MHz, DMSO) δ 173.7, 156.1, 147.8, 143.8, 140.7, 134.0, 132.7, 132.6, 129.4, 127.7, 127.1, 125.3, 124.4, 120.1, 65.6, 53.5, 46.7, 42.3, 27.9, 26.0. LCMS (10-90% MeOH containing 0.1% formic acid over 10 min) R_t=11.04 min. ESI-MS m/z calcd for C₂₆H₂₆N₃O₈S⁺ [M+H]⁺ 540.1, found 540.1. Melting point: 108-110° C.

Solid-Phase Chemistry

Fmoc-based peptide synthesis was performed using standard conditions (W. D. Lubell, J. W. Blankenship, G. Fridkin, and R. Kaul (2005) “Peptides.” Science of Synthesis 21.11, Chemistry of Amides. Thieme, Stuttgart, 713-809) on an automated shaker using polystyrene Rink amide resin. The loading was calculated from the UV absorbance for Fmoc-deprotection after the coupling of the first amino acid. Couplings of amino acids (3 equiv.) were performed in DMF using DIC (3 equiv.) and HOBt (3 equiv.) for 3-6 hours. Fmoc-deprotections were performed by treating the resin with 20% piperidine in DMF for 30 min. The resin was washed after each coupling and deprotection step sequentially with DMF (×3), MeOH (×3) THF (×3) and CH₂Cl₂ (×3).

Lysine as AA1

[Figure (not displayed)]

Fmoc-Lys(o-NBS)-Rink Amide Resin RGO7:

On Rink amide resin (3.00 g) in a syringe fitted with a Teflon™ filter, Fmoc removal was performed by treating the resin with a solution of 20% piperidine in DMF over 30 min. The resin was filtered and washed sequentially with DMF (×3), MeOH (×3) and CH₂Cl₂ (×3). Fmoc-Lys(o-NBS)—OH (1.62 g, 2.93 mmol) was dissolved in DMF (20 mL) and treated with DIC (0.7 mL, 4.52 mmol) and HOBt (611 mg, 4.52 mmol), stirred for 3 min. and added to the syringe containing the resin. The mixture was shaken for 14 hours. The resin was then filtered and sequentially washed with DMF (×3), MeOH (×3) and CH₂Cl₂ (×3). The resin was dried and the loading was measured at 0.345 mmol/g resin.

[Figure (not displayed)]

Fmoc-Lys(o-NBS, Allyl)-Rink Amide Resin RGO8:

Vacuum dried Fmoc-Lys(o-NBS)-resin RGO7 (0.441 mmol) was placed in a syringe fitted with a Teflon™ filter, suspended in THF (dry, 5 mL) and treated sequentially with solutions of allyl alcohol (206 μL, 3.03 mmol) in THF (dry, 1 mL), PPh₃ (397 mg, 1.51 mmol) in THF (dry, 1 mL), and DIAD (298 μL, 1.51 mmol) in THF (dry, 1 mL). The mixture in the syringe was shaken for 90 min. The resin was filtered and sequentially washed with DMF (×3), MeOH (×3), THF (×3) and CH₂Cl₂ (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete allylation: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=8.65 min. ESI-MS m/z calcd for C₃₀H₃₃N₄O₇S⁺ [M+H]⁺ 593.2, found 593.2.

[Figure (not displayed)]

Boc-Ala-D-Pra-Ala-Trp(Boc)-D-Phe-Lys(o-NBS, Allyl)-Rink Amide Resin RGO99:

LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=5.73 min. ESI-MS m/z calcd for C₄₆H₅₇N₁₀O₁₀S⁺ [M−2Boc+H]⁺941.4, found 941.4.

[Figure (not displayed)]

Boc-Ala-L-Pra-Ala-Trp(Boc)-D-Phe-Lys(o-NBS, Allyl)-Rink Amide Resin RGO100:

LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=5.77 min. ESI-MS m/z calcd for C₄₆H₅₇N₁₀O₁₀S⁺ [M−2Boc+H]⁺941.4, found 941.4.

[Figure (not displayed)]

Boc-Ala-D-Pra-D-Trp(Boc)-Ala-Trp-D-Phe-Lys(o-NBS, Allyl)-Rink Amide Resin RGO65:

LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=6.48 min. ESI-MS m/z calcd for C₅₇H₆₇N₁₂O₁₁S⁺ [M−3Boc+H]⁺1127.5, found 1127.5.

[Figure (not displayed)]

Boc-Ala-L-Pra-D-Trp(Boc)-Ala-Trp-D-Phe-Lys(o-NBS, Allyl)-Rink Amide Resin RGO66:

LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=6.66 min. ESI-MS m/z calcd for C₅₇H₆₇N₁₂O₁₁S⁺ [M−3Boc+H]⁺1127.5, found 1127.5.

[Figure (not displayed)]

Boc-Ala-D-Pra-Ala-Trp(Boc)-D-Phe-Lys(Allyl)-Rink Amide Resin RGO104:

o-NBS-protected hexapeptide RGO99 (˜600 mg, 0.156 mmol) in a syringe fitted with a Teflon™ filter was swollen in DMF (5 mL) and treated with DBU (210 μL, 1.40 mmol) and 2-mercaptoethanol (50 μL, 0.71 mmol). The mixture in the syringe was shaken for 1 h. The resin was filtered and sequentially washed with DMF (×3), MeOH (×3), THF (×3) and CH₂Cl₂ (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete o-NBS-removal: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=1.50 min. ESI-MS m/z calcd for C₄₀H₅₄N₉O₆⁺ [M−2Boc+H]⁺ 756.4, found 756.4.

[Figure (not displayed)]

Boc-Ala-L-Pra-Ala-Trp(Boc)-D-Phe-Lys(Allyl)-Rink Amide Resin RGO105:

o-NBS-protected hexapeptide RGO100 (˜600 mg, 0.14 mmol) in a syringe fitted with a Teflon™ filter was swollen in DMF (5 mL) and treated with DBU (210 μL, 1.40 mmol) and 2-mercaptoethanol (50 μL, 0.71 mmol). The mixture in the syringe was shaken for 1 h. The resin was filtered and sequentially washed with DMF (×3), MeOH (×3), THF (×3) and CH₂Cl₂ (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete o-NBS-removal: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=1.51 min. ESI-MS m/z calcd for C₄₀H₅₄N₉O₆⁺ [M−2Boc+H]⁺ 756.4, found 756.4.

[Figure (not displayed)]

Boc-Ala-D-Pra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Lys(allyl)-Rink Amide Resin RGO69:

o-NBS-protected heptapeptide RGO65 (˜300 mg, 0.10 mmol) in a syringe fitted with a Teflon™ filter was swollen in DMF (6 mL) and treated with DBU (150 μL, 1.00 mmol) and 2-mercaptoethanol (35 μL, 0.50 mmol). The mixture in the syringe was shaken for 1 h. The resin was filtered and sequentially washed with DMF (×3), MeOH (×3), THF (×3) and CH₂Cl₂ (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete o-NBS-removal: LCMS (20-80% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=4.79 min. ESI-MS m/z calcd for C₅₁H₆₄N₁₁O₇⁺ [M−3Boc+H]⁺ 942.5, found 942.5.

[Figure (not displayed)]

Boc-Ala-L-Pra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Lys(Allyl)-Rink Amide Resin RGO70:

o-NBS-protected heptapeptide RGO66 (˜300 mg, 0.09 mmol) in a syringe fitted with a Teflon™ filter was swollen in DMF (6 mL) and treated with DBU (130 μL, 0.87 mmol) and 2-mercaptoethanol (30 μL, 0.43 mmol). The mixture in the syringe was shaken for 1 h. The resin was filtered and sequentially washed with DMF (×3), MeOH (×3), THF (×3) and CH₂Cl₂ (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete o-NBS-removal: LCMS (20-80% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=5.05 min. ESI-MS m/z calcd for C₅₁H₆₄N₁₁O₇⁺ [M−3Boc+H]⁺ 942.5, found 942.5.

[Figure (not displayed)]

Cyclic Peptide MPE-110:

Hexapeptide resin RGO104 (˜600 mg, 0.156 mmol) was swollen in DMSO (6 mL) for 30 min in a syringe tube equipped with Teflon™ filter, and stopper, treated with CuI (5.0 mg, 0.03 mmol) and aqueous formaldehyde (70 μL, 0.94 mmol, 37% in H₂O), shaken on an automated shaker for 30 h, and filtered. After filtration, the resin was washed sequentially with AcOH/H₂O/DMF (5:15:80, v/v/v, ×3), DMF (×3), THF (×3), MeOH (×3), and DCM (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete conversion, and a peak with molecular ion consistent with cyclic hexapeptide MPE-110 was observed: MS m/z calcd for Ca₄₁H₅₄N₉O₆⁺ [M+H]⁺ 768.4, found 768.4.

Resin-bound cyclic peptide MPE-110 was deprotected and cleaved from the support using a freshly made solution of TFA/H₂O/TES (95:2.5:2.5, v/v/v, 5 mL) at rt for 2 h. The resin was filtered and rinsed with TFA (5 mL). The filtrate and rinses were concentrated until a crude oil persisted, from which a precipitate was obtained by addition of cold ether (10 mL). After centrifugation (1200 rpm for 10 min), the supernatant was removed and the crude peptide precipitate was taken up in aqueous MeOH (10% v/v) and freeze-dried prior to purification. The resulting light brown fluffy material was purified by preparative HPLC to give cyclic pentapeptide MPE-110 (2.0 mg, 2%) as white fluffy material.

LCMS analysis of cyclic peptide MPE-110 was performed using a linear gradient of a) 10-90% of MeOH containing 0.1% formic acid in H₂O (0.1% formic acid) over 10 min, then at 10% MeOH (0.1% formic acid) for 5 min, R_t=4.24 min; b) 10-90% MeCN containing 0.1% formic acid in H₂O containing 0.1% formic acid over 10 min, then at 10% MeCN (0.1% formic acid) for 5 min, R_t=1.70 min; HRMS m/z. calcd for C₄₁H₅₄N₉O₆⁺ [M+H]⁺ 768.4192, found 768.4176.

[Figure (not displayed)]

Cyclic Peptide MPE-111:

Hexapeptide resin RGO105 (˜600 mg, 0.14 mmol) was swollen in DMSO (6 mL) for 30 min in a syringe tube equipped with Teflon™ filter, and stopper, treated with CuI (5.0 mg, 0.03 mmol) and aqueous formaldehyde (60 μL, 0.84 mmol, 37% in H₂O), shaken on an automated shaker for 30 h, and filtered. After filtration, the resin was washed sequentially with AcOH/H₂O/DMF (5:15:80, v/v/v, ×3), DMF (×3), THF (×3), MeOH (×3), and DCM (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete conversion, and a peak with molecular ion consistent with cyclic hexapeptide MPE-111 was observed: MS m/z. calcd for C₄₁H₅₄N₉O₆⁺ [M+H]⁺ 768.4, found 768.4.

Resin-bound cyclic peptide MPE-111 was deprotected and cleaved from the support using a freshly made solution of TFA/H₂O/TES (95:2.5:2.5, v/v/v, 5 mL) at rt for 2 h. The resin was filtered and rinsed with TFA (5 mL). The filtrate and rinses were concentrated until a crude oil persisted, from which a precipitate was obtained by addition of cold ether (10 mL). After centrifugation (1200 rpm for 10 min), the supernatant was removed and the crude peptide precipitate was taken up in aqueous MeOH (10% v/v) and freeze-dried prior to purification. The resulting light brown fluffy material was purified by preparative HPLC to give cyclic hexapeptide MPE-111 (2.9 mg, 3%) as white fluffy material.

LCMS analysis of cyclic peptide MPE-111 was performed using a linear gradient of a) 10-90% of MeOH containing 0.1% formic acid in H₂O (0.1% formic acid) over 10 min, then at 10% MeOH (0.1% formic acid) for 5 min, R_t=4.50 min; b) 10-90% MeCN containing 0.1% formic acid in H₂O containing 0.1% formic acid over 10 min, then at 10% MeCN (0.1% formic acid) for 5 min, R_t=2.03 min; HRMS m/z. calcd for C₄₁H₅₄N₉O₆⁺ [M+H]⁺ 768.4192, found 768.4172.

[Figure (not displayed)]

Cyclic Peptide MPE-074:

Heptapeptide resin RGO69 (˜300 mg, 0.10 mmol) was swollen in DMSO (5 mL) for 30 min in a syringe tube equipped with Teflon™ filter, and stopper, treated with CuI (4.0 mg, 0.02 mmol) and aqueous formaldehyde (50 μL, 0.69 mmol, 37% in H₂O), shaken on an automated shaker for 29 h, and filtered. After filtration, the resin was washed sequentially with AcOH/H₂O/DMF (5:15:80, v/v/v, ×3), DMF (×3), THF (×3), MeOH (×3), and DCM (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete conversion, and a peak with molecular ion consistent with cyclic heptapeptide MPE-074 was observed: MS m/z calcd for C₅₂H₆₃N₁₁NaO₇⁺ [M+Na]⁺ 976.5, found 976.4.

Resin-bound cyclic peptide MPE-074 was deprotected and cleaved from the support using a freshly made solution of TFA/H₂O/TES (95:2.5:2.5, v/v/v, 5 mL) at rt for 2 h. The resin was filtered and rinsed with TFA (5 mL). The filtrate and rinses were concentrated until a crude oil persisted, from which a precipitate was obtained by addition of cold ether (10 mL). After centrifugation (1200 rpm for 10 min), the supernatant was removed and the crude peptide precipitate was taken up in aqueous MeOH (10% v/v) and freeze-dried prior to purification. The resulting light brown fluffy material was purified by preparative HPLC to give cyclic heptapeptide MPE-074 (0.7 mg, 1%) as white fluffy material.

LCMS analysis of cyclic peptide MPE-074 was performed using a linear gradient of a) 10-90% of MeOH containing 0.1% formic acid in H₂O (0.1% formic acid) over 10 min, then at 10% MeOH (0.1% formic acid) for 5 min, R_t=1.72 min; b) 10-90% MeCN containing 0.1% formic acid in H₂O containing 0.1% formic acid over 10 min, then at 10% MeCN (0.1% formic acid) for 5 min, R_t=4.24 min; HRMS m/z calcd for C₅₂H₆₃N₁₁NaO₇⁺ [M+Na]⁺ 976.4804, found 976.4817.

[Figure (not displayed)]

Cyclic Peptide MPE-075:

Heptapeptide resin RGO69 (˜300 mg, 0.09 mmol) was swollen in DMSO (5 mL) for 30 min in a syringe tube equipped with Teflon™ filter, and stopper, treated with CuI (3.0 mg, 0.02 mmol) and aqueous formaldehyde (50 μL, 0.69 mmol, 37% in H₂O), shaken on an automated shaker for 29 h, and filtered. After filtration, the resin was washed sequentially with AcOH/H₂O/DMF (5:15:80, v/v/v, ×3), DMF (×3), THF (×3), MeOH (×3), and DCM (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete conversion, and a peak with molecular ion consistent with cyclic heptapeptide MPE-075 was observed: MS m/z calcd for C₅₂H₆₄N₁₁O₇⁺ [M+H]⁺ 954.5, found 954.5.

Resin-bound cyclic peptide MPE-075 was deprotected and cleaved from the support using a freshly made solution of TFA/H₂O/TES (95:2.5:2.5, v/v/v, 5 mL) at rt for 2 h. The resin was filtered and rinsed with TFA (5 mL). The filtrate and rinses were concentrated until a crude oil persisted, from which a precipitate was obtained by addition of cold ether (10 mL). After centrifugation (1200 rpm for 10 min), the supernatant was removed and the crude peptide precipitate was taken up in aqueous MeOH (10% v/v) and freeze-dried prior to purification. The resulting light brown fluffy material was purified by preparative HPLC to give cyclic heptapeptide MPE-075 (1.5 mg, 2%) as a white fluffy material.

LCMS analysis of cyclic peptide MPE-075 was performed using a linear gradient of a) 10-90% of MeOH containing 0.1% formic acid in H₂O (0.1% formic acid) over 10 min, then at 10% MeOH (0.1% formic acid) for 5 min, R_t=1.89 min; b) 10-90% MeCN containing 0.1% formic acid in H₂O containing 0.1% formic acid over 10 min, then at 10% MeCN (0.1% formic acid) for 5 min, R_t=4.47 min; HRMS m/z calcd for C₅₂H₆₄N₁₁O₇⁺ [M+H]⁺ 954.4985, found 954.4973.

Ornithine as AA1

[Figure (not displayed)]

Fmoc-Orn(o-NBS)-Rink Amide Resin RGO3:

Rink amide resin (2.51 g) was placed in a syringe fitted with a Teflon™ filter. The Fmoc group was removed by treating the resin with a solution of 20% piperidine in DMF over 30 min. The resin was filtered and washed sequentially with DMF (×3), MeOH (×3) and CH₂Cl₂ (×3). Fmoc-Orn(o-NBS)—OH (1.33 g, 2.46 mmol) was dissolved in DMF (20 mL) and treated with DIC (0.57 mL, 3.68 mmol) and HOBt (494 mg, 3.66 mmol) and stirred for 3 min, before being transferred to the syringe containing the swollen resin, and the mixture was shaken for 14 hours. The resin was filtered and washed sequentially with DMF (×3), MeOH (×3) and CH₂Cl₂ (×3). The resin was dried and the loading was measured to 0.187 mmol/g resin.

[Figure (not displayed)]

Fmoc-Orn(o-NBS, Allyl)-Rink Amide Resin RGO4:

Vacuum dried Fmoc-Orn(o-NBS)-resin (0.362 mmol) was placed in a syringe fitted with a Teflon™ filter, suspended in THF (dry, 20 mL) and treated sequentially with solutions of allyl alcohol (250 μL, 3.68 mmol) in THF (dry, 1 mL), PPh₃ (482 mg, 1.84 mmol) in THF (dry, 2 mL) and DIAD (360 μL, 1.83 mmol) in THF (dry, 1 mL). The resin mixture in the syringe was shaken for 90 min. The resin was filtered and washed sequentially with DMF (×3), MeOH (×3), THF (×3) and CH₂Cl₂ (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete allylation: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=8.47 min. ESI-MS m/z calcd for C₂₉H₃₁N₄O₇S⁺ [M+H]⁺ 579.2, found 579.2.

[Figure (not displayed)]

Fmoc-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Orn(o-NBS, Allyl)-Rink Amide Resin RGO22:

LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=6.13 min. ESI-MS m/z calcd for C₄₈H₅₅N₁₀O₉S⁺ [M-Fmoc-2Boc+H]⁺ 947.4, found 947.3.

[Figure (not displayed)]

Fmoc-azaPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Orn(o-NBS, Allyl)-Rink Amide Resin RGO79:

N′-Propargyl-fluorenylmethylcarbazate (248 mg, 0.849 mmol, prepared by alkylation of fluorenylmethylcarbazate with propargyibromide as —N(R¹⁰)— described below) was dissolved in CH₂Cl₂ (dry, 40 mL) under argon atmosphere. The solution was cooled to 0° C., treated with a 20% solution of phosgene in toluene (1 mL, 1.87 mmol), warmed to rt, stirred 50 min, and the volatiles were removed by rotary evaporation. The residue was re-dissolved in CH₂Cl₂ (10 mL) and the volatiles were once again removed by rotary evaporation. The resulting white solid was dissolved in CH₂Cl₂ (dry, 7 mL) and added to the Fmoc-deprotected pentapeptide RGO22 in a syringe fitted with a Teflon™ filter. The mixture in the syringe was shaken for 28 h. The resin was filtered and washed sequentially with DMF (×3), MeOH (×3), THF (×3) and CH₂Cl₂ (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete coupling: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=8.26 min. ESI-MS m/z calcd for C₅₂H₅₉N₁₂O₁₀S⁺ [M-Fmoc-2Boc+H]⁺ 1043.4, found 1043.3.

[Figure (not displayed)]

Boc-Ala-azaPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Orn(o-NBS, Allyl)-Rink Amide RGO29:

Coupling onto the semicarbazide RGO79 was performed by using amino acid symmetric anhydrides that were generated in situ (J. Zhang, C. Proulx, A. Tomberg, W. D. Lubell, Org. Lett. 2013, 16, 298-301). The procedure was repeated twice on semicarbazide RGO79. Examination by LCMS of a cleaved resin sample (5 mg) showed complete coupling: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=6.39 min. ESI-MS m/z calcd for C₅₅H₆₄N₁₃O₁₁S⁺ [M−3Boc+H]⁺ 1114.5, found 1114.4.

[Figure (not displayed)]

Boc-Ala-azaPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Orn(Allyl)-Rink Amide RGO30:

o-NBS-protected hetapeptide RGO29 (˜1 g, 0.2 mmol) in a syringe fitted with a Teflon™ filter was swollen in DMF (6 mL) and DBU (300 μL, 2.01 mmol) and treated with 2-mercaptoethanol (70 μL, 1.00 mmol). The mixture in the syringe was shaken for 1 h. The resin was filtered and washed sequentially with DMF (×3), MeOH (×3), THF (×3) and CH₂Cl₂ (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete o-NBS-removal: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) R_t=4.49 min. ESI-MS m/z calcd for C₄₉H₆₁N₁₂O₇⁺ [M−3Boc+2Na]²⁺ 487.2, found 487.3.

[Figure (not displayed)]

Cyclic Azapeptide MPE-048:

Azaheptapeptide resin RGO30 (˜1 g, 0.2 mmol) was swollen in DMSO (8 mL) for 30 min in a syringe tube equipped with Teflon™ filter, and stopper, treated with CuI (7.0 mg, 0.04 mmol) and aqueous formaldehyde (90 μL, 1.2 mmol, 37% in H₂O), shaken on an automated shaker for 31 h, and filtered. After filtration, the resin was washed sequentially with AcOH/H₂O/DMF (5:15:80, v/v/v, ×3), DMF (×3), THF (×3), MeOH (×3), and DCM (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete conversion, and a peak with molecular ion consistent with cyclic azaheptapeptide MPE-048 was observed: MS m/z calcd for C₅₀H₆₁N₁₂O₇⁺ [M+H]⁺ 941.5, found 941.4.

Resin-bound cyclic azapeptide MPE-048 was deprotected and cleaved from the support using a freshly made solution of TFA/H₂O/TES (95:2.5:2.5, v/v/v, 5 mL) at rt for 2 h. The resin was filtered and rinsed with TFA (5 mL). The filtrate and rinses were concentrated until a crude oil persisted, from which a precipitate was obtained by addition of cold ether (10 mL). After centrifugation (1200 rpm for 10 min), the supernatant was removed and the crude peptide precipitate was taken up in aqueous MeOH (10% v/v) and freeze-dried prior to purification. The resulting light brown fluffy material was purified by preparative HPLC to give cyclic azaheptapeptide MPE-048 (1.3 mg, 1%) as white fluffy material.

LCMS analysis of cyclic peptide MPE-048 was performed using a linear gradient of a) 10-90% of MeOH containing 0.1% formic acid in H₂O (0.1% formic acid) over 10 min, then at 10% MeOH (0.1% formic acid) for 5 min, R_t=1.80 min; b10-90% MeCN containing 0.1% formic acid in H₂O containing 0.1% formic acid over 10 min, then at 10% MeCN (0.1% formic acid) for 5 min, R_t=4.30 min; HRMS m/z calcd for C₅₀H₆₀N₁₂O₇Na⁺ [M+Na]⁺ 963.4600, found 963.4573.

Synthesis of Cyclic Analogs MPE-189, MPE-201, MPE-202, and MPE-203

Synthesis of Carbazates 2 and 3

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To a well-stirred solution of fluorenylmethyl carbazate (1, 1 eq., 2.8 g, 11 mmol, prepared according to reference 1) and DIEA (2 eq., 2.85 g, 3.64 mL, 22 mmol) in dry DMF (280 mL) at 0° C., a solution of 3-bromopropyne (0.9 eq., 1.47 g, 1.07 mL, 9.91 mmol, 80 wt. % in toluene) in dry DMF (10 mL) was added drop-wise by cannula over 30 min. The cooling bath was removed. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. The volatiles were evaporated. The residue was partitioned between EtOAc and brine. The aqueous layer was separated and extracted with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered, and evaporated. The residue was purified by silica gel chromatography eluting with 40% EtOAc in hexane as solvent system to obtain N′-propargyl-fluorenylmethylcarbazate 3 (1.8 g, 62%), as white solid: R_f 0.42 (60% EtOAc); mp 148-149° C.; ¹H NMR (500 MHz, DMSO-d₆) δ 8.82 (s, 1H), 7.89 (d, J=7.5 Hz, 2H), 7.70 (d, J=7.4 Hz, 2H), 7.50-7.43 (m, 2H), 7.37-7.28 (m, 2H), 4.89 (q, J=4.6 Hz, 1H), 4.29 (d, J=6.9 Hz, 2H), 4.22 (t, J=6.1 Hz, 1H), 3.48 (s, 2H), 3.09 (t, J=2.3 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 156.7, 143.8, 140.7, 127.7 (2C), 127.1 (2C), 125.3 (2C), 120.1 (2C), 81.2, 74.2, 65.6 (2C), 46.6, 39.6 (2C). IR (neat) vmax/cm-1 3304, 3290, 2947, 1699, 1561, 1489, 1448, 1265, 1159, 1021; HRMS m/z calculated for C₁₈H₁₇N₂O₂ [M+H]⁺ 293.1285; found 293.1275.