Alkynylamide-mediated preparation of cyclic peptide compounds

Abstract

The application discloses a novel method for preparing cyclic peptide compounds, which uses straight-chain polypeptides or derivatives thereof without a protecting group at a C terminal and an N terminal as raw materials, and an alkyne amide compound as a condensing agent to stir in an organic solvent to obtain the target cyclic peptide compound, and a reaction formula is (1). The synthesis method provided by the application has the advantages of mild conditions, no need of any additive, no racemization in the reaction process, easy availability of raw materials, simple operation and wide industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of chemical biology, and in particular to a method for the efficient preparation of cyclic peptide compounds at room temperature without any additives or racemization. Background Technology

[0002] In recent years, as the development of small organic molecule drugs has become increasingly difficult, peptide and protein drugs and diagnostic reagents have attracted widespread attention from medicinal chemists due to their high targeting selectivity, easy absorption, and low toxicity. Peptides and proteins have become an important source for new drug development. However, peptide drugs have relatively large molecular weights and poor biomembrane penetration; moreover, they are inevitably easily recognized by proteolytic enzymes in the body and are rapidly degraded after entering the body, resulting in poor stability. These drawbacks limit the routes of administration for peptide drugs and severely restrict their development. Therefore, improving the stability of peptide drugs will be an important aspect of peptide drug development for the foreseeable future.

[0003] Cycling linear peptides is one of the most direct and convenient methods to improve the stability of peptide drugs. Cyclic peptides, due to the absence of exposed amino and carboxyl groups, reduce enzymatic degradation and significantly improve their stability in vivo; furthermore, their cyclic structure restricts their conformation, resulting in greater stability and higher receptor selectivity (Chem. Rev. 2017, 117, 8094-8128). Studies have shown that active linear peptides exhibit higher biological activity after partial or complete cyclization. In recent years, research on the cyclization of linear peptides has yielded significant results. Among existing cyclization methods, cyclic peptides using lactams as bridging structures are the most prevalent. Kent et al. reported a natural chemical linking method in 1994 (Science 1994, 266, 776-779), a synthetic strategy widely used in the synthesis of peptides and proteins. In 1997, Tam et al. applied the natural chemical linking method to intramolecular cyclization to synthesize the plant cyclic peptide Cyclopsychotride (CPT) (Tetrahedron Lett. 1997, 38, 5599-5602). This cyclization method is efficient, with high purity and yield, but the target cyclic peptide requires the presence of Cys residues. Studies have found that only 1.17% of proteins in nature contain Cys residues, which significantly limits the widespread use of natural chemical linking for cyclic peptide synthesis. Using condensing agents is another method to achieve linear peptide cyclization. Among the many reported condensing agents, common ones used for linear peptide cyclization include DCC, HBTU, HATU, and PyBOP. However, these condensing agents also have certain shortcomings: the byproduct (DCU) generated during the use of DCC has very low solubility in most organic solvents and is sometimes mixed in the product and difficult to remove completely. In addition, it can also cause diastereomerization due to racemization; HBTU and PyBOP will produce a large degree of racemization during use; HATU has too high a synthesis cost and is not suitable for large-scale use.

[0004] Therefore, developing a novel, efficient, and non-racemic condensing agent for the cyclization of linear peptides is an important aspect of peptide drug research. Summary of the Invention

[0005] The purpose of this invention is to solve the problems existing in the synthesis of cyclic peptides and to provide a simpler and more efficient method for preparing cyclic peptide compounds.

[0006] The present invention adopts the following technical solution:

[0007] A method for preparing cyclic peptide compounds, characterized in that: using linear peptides or their derivatives without protecting groups at the C-terminus and N-terminus obtained by solid-phase synthesis of peptides as raw materials, dichloromethane (DCM), dimethyl sulfoxide (DMSO), methanol or N,N-dimethylformamide (DMF) as solvents, and acetylacetamide compounds as condensing agents, the target cyclic peptide compound can be obtained by stirring the reaction at room temperature, and the reaction formula is (1).

[0008]

[0009] Wherein: Formula 1 represents a linear polypeptide or its derivative compound; Formula 2 represents an acetylacetamide compound; Formula 3 represents a cyclic peptide compound; EWG (electron-withdrawing group) is selected from alkylsulfonyl, arylsulfonyl, alkylyl, aryl, nitrile, nitro, etc.; R 1 Selected from alkyl or aryl, R 2 It is selected from aryl, alkyl, ynyl, alkenyl, etc.

[0010] The specific steps of the above preparation method are as follows: (1) Add linear peptide compound (0.005-0.05 mmol), acetylene amide condenser (0.05-0.5 mmol), and solvent (0.5-10 mL) to a clean reaction flask and stir at room temperature for 5-100 hours; (2) After the reaction is completed, separate and purify the cyclic peptide target compound by monitoring.

[0011] In this preparation method, the -OH, -CONH2, and -NH- groups on the side chains of the linear peptides do not need to be protected.

[0012] In this preparation method, a mixed solvent of dichloromethane (DCM) and dimethyl sulfoxide (DMSO) is the optimal reaction solvent.

[0013] In this preparation method, N-ethynyl-N-methylmethanesulfonamide exhibits the best reaction effect when used as a condensing agent.

[0014] The technical advantages of this invention are: it provides a novel method for preparing cyclic peptide compounds, which is characterized by mild conditions, no need for any additives, no racemization during the reaction, readily available raw materials, simple operation, and effective avoidance of dimerization products, thus having broad prospects for industrial application. Attached Figure Description

[0015] Figure 1 The HPLC monitoring results of Stylostatin 1 in Example 1 are shown.

[0016] Figure 2 The HPLC monitoring results of Phakel-listatin 13 in Example 2 are shown.

[0017] Figure 3 The HPLC tracking and monitoring results of Dichotomin G in Example 3 are shown.

[0018] Figure 4 The HPLC monitoring results of Phakel-listatin 12 in Example 4 are shown.

[0019] Figure 5 The HPLC tracking and monitoring results of Stelladelin D in Example 5 are shown. Detailed Implementation

[0020] The beneficial effects of the present invention will be described in detail below with reference to Examples 1 to 5, in order to help readers better understand the essence of the present invention, but should not be construed as limiting the implementation and scope of protection of the present invention.

[0021] In the following embodiments, unless otherwise specified, the experimental methods are conventional methods; the reagents and materials can be obtained commercially or prepared according to reported literature.

[0022] Example 1

[0023]

[0024] Synthesis of the natural cyclic peptide Stylostatin 1

[0025] In a clean 5 mL reaction flask, add H-Ala-Ile-Pro-Phe-Asn-Ser-Leu-OH (0.005 mmol), N-ethynyl-N-methylmethanesulfonamide (MYMsA) (0.05 mmol), dichloromethane (DCM) (0.9 mL), and dimethyl sulfoxide (DMSO) (0.1 mL). Stir at room temperature and monitor by HPLC. The peak eluted at 13.845 min [phenomenex C18, 250 × 4.6 mm, Buffer A (0.039% TFA and 10% water in acetonitrile), Buffer B (0.045% TFA in water), λ = 214 nm, flow rate 1 mL / min, 10% A → 100% A in 30 min]. The reaction was complete after 30 hours. The target product was obtained by separation and purification as a white solid with a yield of 73%.

[0026] Example 2

[0027]

[0028] Synthesis of the natural cyclic peptide Phakel-listatin 13

[0029] Add H-Gly-Pro-Thr to a clean 5mL reaction flask. t Bu)-Leu-Trp(Boc)-Pro-Phe-OH (0.005 mmol), N-ethynyl-N-methylmethanesulfonamide (MYMsA) (0.05 mmol), dichloromethane (DCM) (0.9 mL), and dimethyl sulfoxide (DMSO) (0.1 mL) were stirred at room temperature. HPLC monitoring showed an elution at 24.915 min [phenomenex C18, 250 × 4.6 mm, Buffer A (0.039% TFA and 10% water in acetonitrile), Buffer B (0.045% TFA in water), λ = 214 nm, flow rate 1 mL / min, 10% A → 100% A in 30 min]. The reaction was completed after 20 hours. The target product was obtained after separation and purification as a white solid with a yield of 63%.

[0030] Example 3

[0031]

[0032] Synthesis of the natural cyclic peptide Dichotomin G

[0033] In a clean 5 mL reaction flask, add H-Ser-Pro-Leu-Pro-Ile-Pro-Pro-Phe-Tyr-OH (0.005 mmol), N-ethynyl-N-methylmethanesulfonamide (MYMsA) (0.05 mmol), dichloromethane (DCM) (0.9 mL), and dimethyl sulfoxide (DMSO) (0.1 mL). Stir at room temperature and monitor by HPLC. The peak eluted at 18.108 min [phenomenex C18, 250 × 4.6 mm, Buffer A (0.039% TFA and 10% water in acetonitrile), Buffer B (0.045% TFA in water), λ = 214 nm, flow rate 1 mL / min, 10% A → 100% A in 30 min]. The reaction was complete after 35 hours. The target product was obtained by separation and purification as a white solid with a yield of 61%.

[0034] Example 4

[0035]

[0036] Synthesis of the natural cyclic peptide Phakel-listatin 12

[0037] In a clean 5 mL reaction flask, add H-Leu-Pro-Pro-Tyr-Ile-Pro-Pro-Ile-Phe-Thr-OH (0.005 mmol), N-ethynyl-N-methylmethanesulfonamide (MYMsA) (0.05 mmol), dichloromethane (DCM) (0.9 mL), and dimethyl sulfoxide (DMSO) (0.1 mL). Stir at room temperature and monitor by HPLC. The peak eluted at 18.126 min [phenomenex C18, 250 × 4.6 mm, Buffer A (0.039% TFA and 10% water in acetonitrile), Buffer B (0.045% TFA in water), λ = 214 nm, flow rate 1 mL / min, 10% A → 100% A in 30 min]. The reaction was completed after 40 hours. The target product was obtained by separation and purification as a white solid with a yield of 65%.

[0038] Example 5

[0039]

[0040] Synthesis of the natural cyclic peptide Stelladelin D

[0041] In a clean 5 mL reaction flask, add H-Ala-Ile-Gly-Val-Pro-Ser-Pro-Tyr-Phe-Pro-Ala-OH (0.005 mmol), N-ethynyl-N-methylmethanesulfonamide (MYMsA) (0.05 mmol), dichloromethane (DCM) (0.9 mL), and dimethyl sulfoxide (DMSO) (0.1 mL). Stir at room temperature and monitor by HPLC. The peak eluted at 14.831 min [phenomenex C18, 250 × 4.6 mm, Buffer A (0.039% TFA and 10% water in acetonitrile), Buffer B (0.045% TFA in water), λ = 214 nm, flow rate 1 mL / min, 10% A → 100% A in 30 min]. The reaction was completed after 90 hours. The target product was obtained by separation and purification as a white solid with a yield of 62%.

[0042] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing cyclic peptide compounds, characterized in that, In a solvent, using linear peptides or their derivatives without C-terminal and N-terminal protecting groups as raw materials, and acetylacetamide compounds as condensing agents, cyclic peptide compounds can be obtained by stirring the reaction for a period of time. The reaction formula is (1). ； Wherein: Formula 1 represents a linear polypeptide or its derivative; Formula 2 represents an acetylacetamide compound; Formula 3 represents a cyclic peptide compound; EWG (electron-withdrawing group) is selected from alkylsulfonyl, arylsulfonyl, alkylyl, aryl, nitrile, and nitro groups; R 1 Selected from alkyl, aryl, R 2 Selected from aryl, alkyl, ynyl, and alkenyl groups; In the formula, 1 represents H-Ala-Ile-Pro-Phe-Asn-Ser-Leu-OH, H-Gly-Pro-Thr( t Bu)-Leu-Trp(Boc)-Pro-Phe-OH, H-Ser-Pro-Leu-Pro-Ile-Pro-Pro-Phe-Tyr-OH, H-Leu-Pro-Pro-Tyr-Ile-Pro-Pro-Ile-Phe-Thr-OH or H-Ala-Ile-Gly-Val-Pro-Ser-Pro-Tyr-Phe-Pro-Ala-OH; where 3 in the corresponding formulas are natural cyclic peptides Stylostatin 1, Phakel-listatin 13, Dichotomin G, Phakel-listatin 12 or Stelladelin D, respectively. 、 、 、 、 ； The solvent is a mixture of dichloromethane and dimethyl sulfoxide; The acetylide compound is N-ethynyl-N-methylmethanesulfonamide; The reaction is carried out at room temperature.

2. The method according to claim 1, characterized in that, The method includes the following steps: (1) Add a linear polypeptide or its derivative without C-terminal and N-terminal protecting groups, an acetylacetamide compound and a solvent to a clean reaction flask and stir; (2) Cyclic peptide compounds were obtained by purifying after the reaction was completed through monitoring.

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