Recent Reports of Solid-Phase Cyclohexapeptide Synthesis and Applications

Abstract

Macrocyclic peptides are privileged scaffolds for drug development and constitute a significant portion of macrocyclic drugs on the market today in fields spanning from infectious disease to oncology. Developing orally bioavailable peptide-based drugs remains a challenging task; however, macrocyclization of linear peptides can be an effective strategy to improve membrane permeability, proteolytic stability, oral bioavailability, and overall drug-like characteristics for this class. Significant advances in solid-phase peptide synthesis (SPPS) have enabled the efficient construction of macrocyclic peptide and peptidomimetic libraries with macrolactamization being performed on-resin or in solution phase. The primary goal of this review is to summarize solid-phase cyclohexapeptide synthesis using the on-resin and solution-phase macrocyclization methodologies published since 2013. We also highlight their broad applications ranging from natural product total synthesis, synthetic methodology development, and medicinal chemistry, to drug development and analyses of conformational and physiochemical properties.

Keywords: cyclohexapeptide; macrocyclization; macrolactamization; natural products; on-resin cyclization; solid-phase synthesis; solution-phase cyclization; structure–activity relationship; total synthesis.

Figure 1

(A). Illustration of solid-phase cyclohexapeptide synthesis. (B). Resins highlighted in this review.

Figure 2

Structures of naturally occurring nocardiamides A (1) and B (2).

Scheme 1

Solid-phase synthesis of nocardiamides A (1) and B (2) on 2-chlorotrityl chloride (2-CTC) resin. Reagents and conditions: (a) Fmoc-Tyr(^tBu)-OH; (b) HBTU, DIPEA, and DMF (r.t., 1.5 h); (c) MeOH (r.t., 0.5 h); (d) 20% piperidine/DMF (r.t., 20 min); (e) Fmoc-AA-OH, HBTU, DIPEA, and DMF (r.t., 0.5 h); (f) TFA/thioanisole/PhOH/1,2-ethanedithiol/H₂O (82.5/5/5/2.5/5) (r.t.; 31.1% yield for 3 and 47.8% for 4 after RP-HPLC purification); (g) HBTU, DIPEA, and DMF (r.t.; 7.2% yield for nocardiamide A (1) and 10.7% for nocardiamide B (2) after RP-HPLC purification).

Scheme 2

Solid-phase synthesis of cyclohexapeptide 7 on 2-chlorotrityl chloride (2-CTC) resin. Reagents and conditions: (a) 1. Fmoc-Gly-OH, DIPEA, and DCM/DMF (4/1) (r.t., 16 h); 2. MeOH (r.t., 20 min); (b) two treatments with 20% piperidine/DMF (r.t., 5 min); (c) Fmoc-Tyr(^tBu)-OH, HATU (0.5M), DIPEA, and DMF (70 °C, 5 min, microwave heating); (d) Fmoc-Gly-OH, HATU (0.5M), DIPEA, and DMF (70 °C, 5 min, microwave heating); (e) three treatments with 5% TFA in CH₃CN/H₂O (4:1) (r.t., 20 min, 85%); (f) PyBOP and DMF (r.t., 1 h; quantitative yield after purification by flash chromatography (silica gel)).

Scheme 3

Solid-phase synthesis of 9 and 10 on 2-chlorotrityl chloride (2-CTC) resin. Reagents and conditions: (a) Fmoc-Leu-OH, DIPEA, and DCM (r.t., overnight); (b) treated twice with 50% piperidine/DMF (r.t., 10 min); (c) Fmoc-Leu-OH, HATU, DIPEA, and DMF (r.t., 2 h); (d) TFA/TIPS/H₂O (95/2.5/2.5) (r.t., 2 h); (e) dropwise addition of linear peptide solution in DMF to solution of PyBOP, DIPEA, and DMF (over 3 h, r.t., overnight; 17% yield for 9 and 3% for 10 after preparative RP-HPLC purification).

Figure 3

Structures of naturally occurring cyclohexapeptides 11–14.

Scheme 4

Solid-phase peptide synthesis of 11 on trityl alcohol lantern. Reagents and conditions: (a) AcCl and DCM (r.t., 4 h); (b) Fmoc-D-Ala-OH, DIPEA, and DCM (r.t., 12 h); (c) 20% piperidine/DMF (r.t., 1 h); (d) Fmoc-AA-OH, DIC, HOBt, and DMF (r.t., 12 h); (e) 30% hexafluoroisopropanol (HFIP)/DCM (r.t., 1 h); (f) HATU, DIPEA, and DCM (r.t., 3 h); yields (over 13 steps): 29% (11), 41% (12), 39% (13), and 29% (14)).

Figure 4

Structures of aerucyclamides A–D.

Scheme 5

Solid-phase peptide synthesis of 15–21 on 2-chlorotrityl chloride (2-CTC) resin. Reagents and conditions: (a) Fmoc-AA-OH (or Fmoc-Thz-OH in the synthesis of 20 and 21), DIPEA, and DCM (r.t., 1 h, capped with MeOH, r.t., 0.5 h); (b) 1. deprotection: 20% piperidine/DMF (r.t., 2 × 5 min followed by 1 × 10 min); 2. coupling: Fmoc-AA-OH, HOAt, DIC, and DMF (r.t., 2 h); 3. steps 1–2 repeated; (c) cleavage: four treatments with 1% TFA/DCM (r.t., 3 min); (d) macrocyclization: (concentration of 1–5 mM) HBTU, DIPEA, DMAP, and DCM (r.t., 3–5 days; cyclization yields: 59% (15), 55% (16), 48% (17), 66% (18), 54% (19), 83% (20), and 40% (21)).

Figure 5

Structure of dichotomin A (22).

Scheme 6

Solid-phase peptide synthesis of dichotomin A (22) on 2-chlorotrityl chloride (2-CTC) resin. Reagents and conditions: (a) 1. Fmoc-Gly-OH (for 23) or Fmoc-Phe-OH (for 24), DIPEA, and DCM; 2. DCM/MeOH/DIPEA (17:2:1); (b) 10% piperidine/DMF; (c) Fmoc-AA-OH, HBTU, DIPEA, and NMP; (d) 20% HFIP/DCM; (e) (concentration of 1 mM) DMTMM·BF₄, DIPEA, and DMF (r.t., 3 h; yield of 25: 84% from 23 and 87% from 24)); (f) TFMSA/water (2:1) (r.t., 4 h, 33%); (g) NiCl₂, NaBH₄, and MeOH (0 °C, 0.5 h, 24%).

Figure 6

Structures of cyclohexapeptides 27 and 28.

Figure 7

Structures of dianthin G (29), “dicarba” analogue 30, and N-methyl analogues 31–35.

Scheme 7

Representative solid-phase peptide synthesis of 31 on aminomethyl polystyrene resin. Reagents and conditions: (a) Fmoc-Gly-O-HMMP-OH, DIC, DCM, and DMF (r.t., 4 h); (b) deprotection: 20% piperidine/DMF (r.t., 2 × 5 min); (c) coupling: Fmoc-AA-OH, HATU, DIPEA, and DMF (r.t., 45 min); (d) 2-nitrobenzenesulfonyl chloride, sym-collidine, and NMP (r.t., 2 × 15 min); (e) dimethyl sulfate, DBU, and NMP (r.t., 2 × 5 min); (f) 2-mercaptoethanol, DBU, and NMP (r.t., 2 × 5 min); (g) TFA/TIPS/H₂O (95/2.5/2.5) (r.t., 3 h); (h) HBTU, 6-Cl-HOBt, DIPEA, DCM, and DMF (r.t., 36 h; overall yields: 52% (29), 38% (31), 22% (32), 51% (33), 43% (34), 39% (35)).

Scheme 8

Solid-phase peptide synthesis of 42 on 2-chlorotrityl chloride (2-CTC) resin. Reagents and conditions: (a) Fmoc-Leu-OH, DIPEA, and DCM (r.t., 2 h); (b) 20% piperidine/DMF (r.t., 2 × 10 min); (c) Fmoc-AA-OH, HBTU, DIPEA, and NMP (r.t., 1 h); (d) 20% HFIP/DCM (r.t., 1 h); (e) (concentration of 1 mM) HATU, HOBt, DIPEA, and DMF (0 °C to r.t.; r.t., 3 days; cyclization yield: 68%); (f) TFA/TIPS/H₂O (95/2.5/2.5) (r.t., 3 h; yield of 42: 91%).

Figure 8

Structures of cyclohexapeptides 43–47.

Figure 9

Wollamides A (48), and B (42) and desotamide B (49).

Scheme 9

Solid-phase peptide synthesis of 42 on 2-chlorotrityl chloride (2-CTC) resin. Reagents and conditions: (a) Fmoc-Asn(Trt)-OH, DIPEA, and DCM (r.t., 3 h); (b) 25% 4-methylpiperidine/DMF; (c) Fmoc-Val-OH, DIC, HOBt, and DMF/DCM (1:1) (r.t., 4 h); (d) Fmoc-D-Leu-OH, DIC, HOBt, and DMF/DCM (1:1) (r.t., 4 h); (e) Fmoc-Leu-OH, DIC, HOBt, and DMF/DCM (1:1) (r.t., 4 h); (f) Fmoc-Trp(Boc)-OH, DIC, HOBt, and DMF/DCM (1:1) (r.t., 4 h); (g) Fmoc-D-Orn(Boc)-OH, DIC, HOBt, and DMF/DCM (1:1) (r.t., 4 h); (h) HFIP/DCM (1:4) (r.t., 0.5 h); (i) (concentration of 1 mM) HBTU, DIPEA, and DMF (r.t., 0.5 h; cyclization yield of 51: 72%); (j) TFA/TIPS/DCM (50:5:45) (r.t., 0.5 h, 42%).

Figure 10

Synthesis and structure–activity relationship (SAR) studies of cyclohexapeptides 48, 52, and 42.

Figure 11

Structures of cyclohexapeptides 53 and 54.

Scheme 10

Solid-phase synthesis of 53 and 54 on trityl resin. Reagents and conditions: (a) 20% 4-methylpiperidine/DMF (r.t., 2 × 5 min); (b) Fmoc-AA-OH, PyOxim, DIPEA, and DMF/NMP (1:1) (r.t., 45 min); (c) Pd(PPh₃)₄ and 10% piperidine/THF (r.t., 2 h); (d) HATU, HOBt, DIPEA, and DMF/NMP (1:1) (r.t., 18 h); (e) 10% TFA/DCM (r.t., 4 × 5 min); (f) LiO^tBu and THF (r.t., 0.5 h); (g) MeI and DMSO (r.t., 0.5 h).

Scheme 11

Solid-phase peptide synthesis of 59–62 on 2-chlorotrityl chloride (2-CTC) resin. Reagents and conditions: (a) 1. (for X=NH) Fmoc-Lys-OAll, DIPEA, and THF (r.t., 2 h); 2. DCM/MeOH/DIPEA (17:1:2) (r.t., 3 × 2 min); 1. (for X=O), Fmoc-Hln-OAll, pyridine, and DCM/DMF (1:1) (r.t., 64 h); 2. DCM/MeOH/DIPEA (17:1:2) (r.t., 3 × 2 min); (b) 1. deprotection: 20% piperidine, 0.1M HOBt, and DMF (microwave irradiation: 35 W, 75 °C, 30 s, followed by 44 W, 75 °C, 3 min); 2. coupling: Fmoc-AA-OH, HBTU, DIPEA, and DMF (microwave irradiation: 21 W, 75 °C, 5 min); 3. steps 1–2 repeated; (c) Pd(PPh₃)₄, and DCM/NMM/acetic acid (8:2:1) (r.t., 4 h); (d) 20% piperidine/DMF (r.t., 2 × 8 min); (e) HATU, DIPEA, and DMF (r.t., 4 h); (f) 2% N₂H₄ and DMF (r.t., 5–12 × 5 min); (g) 4-fluorobenzoyl chloride, NEt₃, and DCM (r.t., 2 h); (h) TFA/TES/H₂O (95/2.5/2.5) (r.t., 3 h); (i) TFA/TES/DCM (1:5:94) (r.t., 0.5 h); (j) Dess–Martin periodinane and DCM (r.t., 3 h); (k) TFA/DCM (9:1) (r.t., 1 h; overall yields: 80% (59), 30% (60), and 25% (61)).

Scheme 12

Solid-phase peptide synthesis (SPPS) of cyclohexapeptides 65a–d. Reagents and conditions: (a) Fmoc-D-Ser-OAll, BF₃·OEt₂, and dry TFA (r.t., 1 h); (b) SPPS for five cycles: deprotection: 20% piperidine/DMF; coupling: Fmoc-AA-OH, BOP, HOBt, DIPEA, and DMF; (c) Pd(PPh₃)₄ and PhSiH₃; (d) 20% piperidine/DMF; (e) PyBOP, HOBt, DIPEA, and DMF (r.t., 17 h); (f) 95% TFA (r.t., 2 h; yields: 15% (65a), 38% (65b), 63% (65c), and 13% (65d).

Scheme 13

Representative solid-phase peptide synthesis (SPPS) of 49 on Rink amide resin. Reagents and conditions: (a) 20% piperidine/DMF (r.t., 5 × 10 min); (b) Fmoc-Asp-OAll, HCTU, DIPEA, and DMF (r.t., 1 h); (c) SPPS: coupling: Fmoc-AA-OH, HCTU, DIPEA, and DMF (r.t., 1 h); deprotection: 20% piperidine/DMF (r.t.; 5 × 10 min); coupling/deprotection steps repeated. (d) Pd(PPh₃)₄, phenylsilane, and DCM; (e) PyAOP, HOAt, NMM, and NMP (r.t., 12 h); (f) TFA/H₂O/phenol/TIPS (88:5:5:2, v/v/v/v) (r.t., 2 h).

Figure 12

Structures of desotamide B (49), wollamide B (42), and cyclohexapeptide analogues 70–73.

Figure 13

Structures of cyclohexapeptides 74–78.

Scheme 14

Solid-phase peptide synthesis of 75–78 on 2-chlorotrityl chloride (2-CTC) resin. Reagents and conditions: (a) Fmoc-Glu-OAll, DIPEA, and DMF; (b) capped with MeOH; (c) deprotection: 20% piperidine/DMF; (d) coupling: Fmoc-AA-OH, HBTU, DIPEA, and DMF (r.t., 1–2 h); (e) Pd(PPh₃)₄ and 10% piperidine/THF (r.t., 3 h); (f) DIC, Cl-HOBt, and DMF/DCM (8:2) (r.t., overnight); (g) 1% TFA/DCM (r.t., 2–3 min; overall yield: 51% (76), 63% (77), 46% (78), and 80% (79)).