Two-Steps Hantzsch Based Macrocyclization Approach for the Synthesis of Thiazole Containing Cyclopeptides

Abstract

Macrocyclization via an efficient high-yielding solid-phase intramolecular thioalkylation reaction is described. The reaction of S-nucleophiles with newly generated N-terminal 4-chloromethyl thiazoles led to the desired macrocyclization products 5 in high purities and good overall yields.

Introduction

Conformational constraint by cyclization is a common approach used to restrict the flexibility of peptides and therefore is a valuable approach to study topographical requirements of receptors.¹ Cyclization of peptides can provide potent and selective ligands for receptors when appropriate conformational constraints are incorporated.¹ Furthermore cyclic peptides are often more stable to peptidases, and therefore they can have improved pharmacokinetic profiles and serve as promising lead compounds for further development.² Macrocycles are known for their broad range of activities including antitumor activities and antibiotic activities such as the structurally complex vancomycin family.³ Reported approaches on the solid-phase synthesis of macrocyclic compounds include intramolecular nucleophilic substitutions,^4,1a intramolecular amide formations,^5,1c disulfide formations,⁶ intramolecular Suzuki reactions,⁷ ring closing metathesis reactions⁸ and S_NAr displacement reactions.⁹ Of particular interest, thioalkylation reactions offer a facile and versatile approach to the synthesis of cyclic peptides.^4,10 Examples of described macrocyclizations via thioalkylation include the reaction of the thiol group of a C-terminal cysteine with N-terminal acetyl bromide or N-terminal benzyl bromide.^4,10 A conceptually different approach, wherein thioalkylation proceeds via Michael addition of a thiolate anion to an α,β-unsaturated ester, has been reported for the synthesis of cyclic thioether dipeptides. ¹¹

Results and Discussion

Herein, we describe an innovative thioalkylation approach toward the generation of macrocyclic peptides following the intramolecular nucleophilic substitution (S_N2) of N-terminus 4-chloro methyl thiazole peptides with the thiol group of cysteine. The final products are not entirely peptidic and the described newly generated macrocyclic compounds contain the thiazole ring, a pharmacophore present in many natural and synthetic products with a wide range of pharmacological activities that can be well illustrated by the large numbers of naturally occurring thiazole containing macrocyclic compounds ¹² and drugs in the market containing this function group.¹³

We have shown the feasibility of the proposed approach by the parallel synthesis of different thiazole containing macrocyclic peptides. Starting from resin-bound orthogonally protected Fmoc-Cys-(Trt)-OH 1, the thiomethyl thiazolyl macrocyclic peptidomimetics 5 were synthesized following stepwise Fmoc deprotection¹⁴ and standard repetitive Fmoc-amino-acid couplings yielding the linear tripeptide 2. The resulting N-terminal free amine was treated with Fmoc-isothiocyanate. Following Fmoc deprotection, the thioureas were treated with 1,3-dichloroacetone to afford following Hantzsch’s cyclocondensation¹⁵ the resulting resin-bound chloro methyl thiazolyl peptide 4. The Trt group was deprotected in the presence of 5%TFA in DCM and the resin was treated with a solution of CsOH-H₂O in DMF to undergo an S_N2 intramolecular cyclization. The resin was cleaved with HF/anisole and the desired thiazolyl thioether cyclic peptides 5 were obtained in good yield and high purity. An Ellman test for free thiols was performed on all samples. No free thiol was present in the crude material. The identity of the final products was confirmed by LC-MS and NMR spectroscopy.

Many reagents and techniques have been developed to facilitate the synthesis of cyclic peptides, for which the yield-limiting step is generally the cyclization reaction. Particularly, the cyclization of tetra-, penta-, and hexapeptides in the all L-configuration can be problematic, especially in the absence of beta-turn promoting structures such as glycine, proline, or a D-amino acid.¹⁶ Using the approach outlined in scheme 1, we first tested our approach by performing the parallel synthesis of various thiazole containing cyclic tetrapeptides and pentapeptides from all L-amino acids (Figure 1).

Scheme 1.

Reagents and conditions: (a) Solid phase peptide synthesis using Fmoc Chemistry; (b) FmocNCS (6 equiv) in DMF (0.3 M), RT, overnight; (c) 20% piperidine/DMF; (d) 1,3-dichloroacetone (10 equiv) in DMF (0.3 M), 70°C, overnight; (e) TFA/(Bu^t)₃SiH/DCM (5:5:90), 30 min; (f) Cs₂CO₃ in DMF overnight; (g) HF/anisole, 0°C, 90 min.

Figure 1.

We selected different amino acids for each of the position of diversities R₁, R₂ and R₃ for the synthesis of eleven tetrapeptides (5a-5k). Thus, we choose Phe, Tyr, Ser and Val for the position R₁, Phe, Tyr and Pro for the position R₂ and Tyr, Pro and Lys for the position R₃. Similarly, we selected three amino acids for each of the position of diversities R₁ (Tyr, Phe, Asp), R₃ (Tyr, Pro, Arg) and two amino acids for each of the position of diversities R₂ ( Tyr, Gly) and and R₄ (Tyr, Pro) for the synthesis of nine pentapeptides (5l-5t). As shown in table 1, high purities were obtained for all compounds. In all cases, the intramolecular thioalkylation reaction led to the desired cyclic monomers with negligible traces of dimerization. The NMR data show a clear singlet at 6.4 ppm which is specific to the proton on C-5 of the aminothiazole ring. The presented methodology is not limited to cyclic tetrapeptides and pentapeptides. It was successfully used for the synthesis of cyclic hexapeptides 5u-5v and heptapeptide 5w.

Table 1.

Synthesis of thiazole containing cyclic tetrapeptides and pentapeptides

Entry	Peptide	Puritya (%)	Yieldb
5a	Tyr-Phe-Phe	88	45
5b	Pro-Phe-Phe	85	38
5c	Tyr-Tyr-Phe	88	43
5d	Pro-Tyr-Phe	89	41
5e	Tyr-Phe-Tyr	87	40
5f	Pro-Phe-Tyr	76	29
5g	Tyr-Tyr-Tyr	83	38
5h	Pro-Tyr-Tyr	89	46
5i	Tyr-Pro-Ser	85	47
5j	Tyr-Tyr-Ser	86	43
5k	Lys-Tyr-Val	82	40
5l	Tyr-Tyr-Tyr-Phe	86	38
5m	Pro-Tyr-Tyr-Phe	90	39
5n	Tyr-Pro-Tyr-Phe	80	31
5o	Pro-Pro-Tyr-Phe	83	37
5p	Tyr-Tyr-Tyr-Tyr	82	31
5q	Pro-Tyr-Tyr-Tyr	85	35
5r	Tyr-Pro-Tyr-Tyr	80	34
5s	Pro-Pro-Tyr-Tyr	78	35
5t	Tyr-Arg-Gly-Asp	84	43
5u	Arg-Gly-Asp-Tyr-Tyr	82	40
5v	Phe-Tyr-Val-Ser-Ala	80	37
5w	Phe-Ala-Pro-Tyr-Ser-Phe	82	39

^aPurity of crude products: The products were run on a Vydac column, gradients 5 to 95% formic acid in ACN in 7 min. The purity was estimated on analytical traces at λ= 214 nm and 254 nm.

^bThe yields are based on the weight of purified products and are relatives to the initial loading of the resin (The purity of the purified compounds is higher than 95% for all the compounds).

Conclusion

We presented a new method for macrocyclization reaction via thioalkylation. The reaction of S-nucleophiles with newly generated N-terminal 4-chloromethyl thiazoles led to the desired macrocyclization products in high purities and good overall yields. The presented approach can be extended toward the synthesis of macrocyclic libraries where the cysteine residue can be placed anywhere in the peptide sequence, allowing for extension of the peptide beyond the cyclic link. As part of our drug discovery program, we are in the process of preparing a variety of thiazole containing macrocyclic libraries. The synthesis and the screening results will be reported elsewhere.

Experimental Section

Synthesis of resin-bound cysteine

A 100 mg sample of p-methylbenzhydrylamine hydrochloride (MBHA·HCl) resin (CHEM-IMPEX INTERNATIONAL 1.15 mequiv/g, 100-200 mesh, 1% DVB) was contained within a sealed polypropylene mesh bag. 23 bags (23 × 100 mg resin, 2.64 mmol) were put in a polyethylene bottle. Following the neutralization of resin with 500 ml of 5% diisopropylethylamine (DIEA) in dichloromethane (DCM), L-Fmoc-Cys(Trt)-OH (3 eq, 4.64 g, 7.93 mmol) was coupled using the conventional reagents hydroxybenzotriazole (HOBt, 1.07 g, 7.93 mmol) and diisopropylcarbodiimide (DIC, 1.16 ml, 7.93 mmol) in 300 ml anhydrous DMF overnight at room temperature. Completion of the coupling was monitored by the ninhydrin test.

General procedure for the solid-phase synthesis of resin-bound linear cyclic peptide 5a

One bag of resin 1 (100 mg, 0.115 mmol) was put into a small polyethylene bottle and the Fmoc group was deprotected with 15 ml 20% piperidine in DMF (2×10min). The resin was then washed with 15 ml DMF (3×) and 15 ml DCM (3×). L-Fmoc-Phe-OH (6 equiv, 0.267 g, 11.04 mmol) was coupled in the presence of hydroxybenzotriazole (HOBt, 6 equiv, 0.094 g, 11.04 mmol) and diisopropylcarbodiimide (DIC, 6 equiv, 0.101 ml, 11.04 mmol) in 15 ml anhydrous DMF for 2h at room temperature. The resin-bound dipeptide was washed with DMF (3×) and DCM (3×). Completion of the coupling was monitored by the ninhydrin test. The Fmoc group was deprotected with 15 ml 20% piperidine in DMF (2×10min) and followed by the coupling of L-Fmoc-Phe-OH (6 equiv, 0.267 g, 11.04 mmol) using the same reaction conditions. The Fmoc group was deprotected and the resin-bound tripeptide was coupled to L-Fmoc-Tyr(^tBu)-OH in the same conditions to yield following Fmoc deprotection the corresponding resin-bound protected linear peptide 2a.

The resulting N-terminal free amine of resin-bound linear peptide 2a was treated with Fmoc-isothiocyanate (6 equiv, 0.193 g, 11.04 mmol) in 15 ml DMF anhydrous overnight at room temperature. Following Fmoc deprotection with a solution of 20% piperidine in DMF, the resin-bound N-terminal thiourea was treated with 1,3-dichloroacetone (10 equiv, 0.145 g, 18.4 mmol) in DMF anhydrous overnight at 70 °C to afford following Hantzsch’s cyclocondensation the resulting resin-bound chloro methyl thiazolyl peptide 4a. The Trt group was deprotected in the presence of TFA/(Bu^t)₃SiH/DCM (5:5:90) for 30 min. The resin was washed with DCM (5×) and DIEA/DCM (5:95) and was treated overnight with a solution of Cs₂CO₃ (10 eq, 0.325 g) in 15 ml DMF at room temperature to undergo an S_N2 intramolecular thialkylation. The resin was cleaved with HF/anisole for 90 min at 0 °C, and the desired thiazolyl thioether cyclic peptides 5a was obtained following extraction with 95% acetic acid in water and lyophilization as a white powder (61.9 mg). The cyclic peptides 5a was purified by preparative reverse-phase HPLC.