A method for preparing a cis-amide bioisosteric cyclic peptide compound

Abstract

This invention discloses a method for preparing a cyclic peptide compound containing a cisamide bioelectron isosteric cyclic peptide. The method involves introducing a thioamide unit at a specific position on the peptide backbone, making it a potential activatable reaction site. This site is selectively activated by a metal salt and undergoes an intramolecular condensation reaction with an acylhydrazine group located in the same peptide chain, generating a 1,2,4-triazole heterocyclic structure in situ. In a single reaction process, the cyclization of the peptide and the precise construction of the cisamide bioelectron isosteric group in the peptide backbone are achieved simultaneously.

Description

Technical Field

[0001] This invention belongs to the field of chemical engineering, and specifically relates to a method for preparing cyclic peptide compounds. Background Technology

[0002] Peptides and cyclic peptides have attracted widespread attention due to their ability to cover macromolecular interfaces and achieve highly selective binding in areas such as protein-protein interaction (PPI) regulation, molecular recognition, biological signal transduction, and drug discovery. However, linear peptides generally suffer from high conformational flexibility, poor in vivo stability, and susceptibility to protease degradation, which severely restricts their drug applications. To overcome these shortcomings, cyclization has been widely used in peptide structure modification. By introducing covalently closed cyclic structures, the conformational rigidity, metabolic stability, and target affinity of peptides can be significantly improved. Therefore, cyclic peptides have become an important branch of peptide drug development. Existing cyclic peptide construction techniques mainly rely on side-chain-side-chain, side-chain-head, or head-tail connections. The reaction sites are concentrated on the peptide side chains or terminal functional groups, with limited impact on the peptide backbone structure itself, making it difficult to achieve deep and programmable structural control at the scaffold level. Meanwhile, at the peptide backbone level, the cis-trans isomerism of amide bonds plays a decisive role in the overall conformation, turn formation, and intramolecular hydrogen bond network of peptide molecules. However, in natural peptides and conventionally synthesized polypeptides, due to steric hindrance and electronic effects, amide bonds exist almost entirely in the trans conformation, with a very low proportion of cis amides. Current techniques for regulating amide geometry mainly involve introducing proline or N-methylated amino acids at the monomer level to weaken the trans conformation preference, or pre-synthesizing non-natural amino acid monomers containing amide bioisosteres and then introducing them into the peptide chain via solid-phase peptide synthesis. However, these methods typically suffer from limitations such as limited conformational locking ability, cumbersome monomer synthesis steps, high cost, limited sites for introduction, and difficulty in achieving subsequent backbone editing, making it difficult to meet the needs of rapid structural optimization for complex peptide systems and peptide drugs.

[0003] Therefore, existing technologies urgently need a novel technical approach that can directly use the peptide backbone as the reaction object without relying on prefunctionalized amino acid monomers, and simultaneously achieve structural reprogramming of the peptide backbone and precise control of amide geometry during cyclization, thereby constructing conformationally controlled, highly stable, and application-potential cyclic peptide molecules. Summary of the Invention

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides the application of a linear polypeptide containing a thioamide bond and an acylhydrazine bond in the preparation of cyclic peptide compounds.

[0005] In some embodiments, the linear polypeptide has the structure described in Formula I. R1-(AA1) p -AA2-ψ(CS-NH)-(AA3) m -AA4(R2)-(AA5) q -R3 Formula I in, The amino acids corresponding to AA2 and AA4 are each independently selected from one of the natural amino acids; p and q are each independently selected from integers from 0 to 100; when p and q > 1, the amino acid corresponding to each AA1 or AA5 can be independently selected from either a synthetic amino acid or a natural amino acid. m is selected from integers from 1 to 100. When m>1, the amino acid corresponding to each AA3 can be independently selected from either synthetic amino acids or natural amino acids. R1 and R3 are independently selected from -Fmoc, -Boc, and -O, respectively. t Bu and -OBn; R2 is an acylhydrazide group (-NHNH2); The side chain functional groups of the amino acids corresponding to AA1, AA2, AA3, AA4 and AA5 are optionally protected by protecting groups.

[0006] In some embodiments, the protecting group is 9-fluorenyloxycarbonyl, tert-butyloxycarbonyl, tert-butyl ester, and benzyl ester.

[0007] In some embodiments, the natural or synthetic amino acids are all L-type.

[0008] In some embodiments, the natural amino acids include tryptophan, lysine, phenylalanine, methionine, threonine, valine, leucine, isoleucine, cysteine, serine, glycine, tyrosine, aspartic acid, asparagine, glutamic acid, glutamine, alanine, arginine, histidine, and proline.

[0009] In some embodiments, the amino acid corresponding to AA2 is selected from alanine, glycine, valine, leucine, serine, proline, tryptophan, methionine, and lysine, wherein the side chain functional group of tryptophan or lysine is optionally protected by a tert-butyloxycarbonyl group.

[0010] In some embodiments, the amino acid corresponding to AA3 is selected from alanine, phenylalanine, proline, and glycine.

[0011] In some implementations, the amino acid corresponding to AA4 is glutamic acid.

[0012] In some implementations, m is selected from an integer from 1 to 10.

[0013] In some implementations, m is 4.

[0014] In some implementations, Formula I includes the following structure: (1) Fmoc-Ala-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu (SEQ ID NO.1); (2) Fmoc-Val-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu (SEQ ID NO.2); (3) Fmoc-Leu-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu (SEQ ID NO.3); (4) Fmoc-Ser-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu (SEQ ID NO.4); (5) Fmoc- S Pro-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu (SEQ ID NO.5); (6) Fmoc-Trp(Boc)-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu (SEQ ID NO. 6); (7) Fmoc-Met-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu (SEQ ID NO.7); (8) Fmoc-Lys(Boc)-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu (SEQ ID NO.8).

[0015] In some embodiments, the cyclic peptide compound contains a cisamide bioelectron isostere.

[0016] In some embodiments, the cisamide bioelectron isostere has a 1,2,4-triazole heterocyclic structure.

[0017] In some embodiments, the cyclic peptide compound has the structure shown in Formula II:

[0018] Formula II in, R4 is selected from hydrogen, methyl, isopropyl, isobutyl, sec-butyl, pyrrolyl, benzyl, 2-methylthioethyl, hydroxymethyl, 1-hydroxyethyl, mercaptomethyl, p-hydroxybenzyl, 3-carbamoylpropyl, 2-carbamoylethyl, indolemethyl, carboxymethyl, 2-carboxyethyl, 4-aminobutyl and imidazolemethyl, wherein R4 is optionally substituted with tert-butoxycarbonyl or tert-butoxy. R5 is hydrogen, or R5 and R4 together with the atoms attached thereto form a 5-6 membered heterocyclic group, wherein the heterocyclic group includes at least one heteroatom selected from N, O or S; The amino acids corresponding to AA are selected from natural amino acids or synthetic amino acids; n is an integer between 1 and 100.

[0019] In some embodiments, the natural amino acid is selected from tryptophan, lysine, phenylalanine, methionine, threonine, valine, leucine, isoleucine, cysteine, serine, glycine, tyrosine, aspartic acid, asparagine, glutamic acid, glutamine, alanine, arginine, histidine, and proline.

[0020] In some embodiments, R4 is selected from hydrogen, methyl, isopropyl, isobutyl, tert-butyloxymethyl, indolemethyl, 2-methylthioethyl, tert-butyloxycarbonylaminobutyl.

[0021] In some implementations, R5 is hydrogen.

[0022] In some embodiments, R5 and R4 together with the atoms they are attached to form a 5-membered heterocyclic group, the heterocyclic group including an N atom; preferably, the 5-membered heterocyclic group is a pyrrolidinyl group.

[0023] In some implementations, n is 4.

[0024] In some embodiments, the amino acid corresponding to AA is selected from alanine, phenylalanine, prolyl, and glycine.

[0025] In some implementations, -(AA) n -For-Ala-Phe-Pro-Gly-.

[0026] In some embodiments, the cyclic peptide compound is selected from any one of the following compounds:

[0027]

[0028] .

[0029] A second aspect of the present invention provides a method for preparing a cyclic peptide compound, characterized in that the method involves an intramolecular cyclization reaction of a linear polypeptide having the structure of Formula I. R1-(AA1) p -AA2-ψ(CS-NH)-(AA3) m -AA4(R2)-(AA3) q -R3 Formula I The amino acids corresponding to AA2 and AA4 are each independently selected from one of the natural amino acids; p and q are each independently selected from integers from 0 to 100; when p and q > 1, the amino acid corresponding to each AA1 or AA5 can be independently selected from either a synthetic amino acid or a natural amino acid. m is selected from integers from 1 to 100. When m>1, the amino acid corresponding to each AA3 can be independently selected from either synthetic amino acids or natural amino acids. R1 and R3 are independently selected from -Fmoc, -Boc, and -O, respectively. t Bu and -OBn; R2 is an acylhydrazide group (-NHNH2); The side chain functional groups of the amino acids corresponding to AA1, AA2, AA3, AA4 and AA5 are optionally protected by protecting groups.

[0030] In some embodiments, the preparation method includes the steps of adding a metal salt and a reaction solvent.

[0031] In some embodiments, the metal salt is a silver salt. Preferably, the silver salt is selected from AgOAc, Ag2CO3, and AgNO3. More preferably, the silver salt is AgOAc.

[0032] In some embodiments, the amount of silver salt used is 0.05-0.5 mmol. Preferably, the amount of silver salt used is 0.01 mmol.

[0033] In some embodiments, the reaction solvent is dichloromethane or dimethyl sulfoxide.

[0034] In some embodiments, the amount of the reaction solvent used is 0.5-10 mL. Preferably, the amount of the reaction solvent used is 1 mL.

[0035] In some embodiments, the preparation method includes the step of an intramolecular condensation reaction between -ψ(CS-NH)- and -NHNH2 in a linear polypeptide of Formula I to generate a 1,2,4-triazole heterocyclic structure in situ.

[0036] In some embodiments, the reaction temperature of the internal cyclization reaction is room temperature.

[0037] In some embodiments, the reaction time for the cyclization reaction is 10-72 hours. Preferably, the reaction time for the cyclization reaction is 72 hours.

[0038] In some embodiments, the preparation method further includes the step of preparing a linear polypeptide of formula I.

[0039] In some embodiments, the method for preparing the I-type linear peptide includes solid-phase synthesis or liquid-phase synthesis.

[0040] In some embodiments, the solid-phase synthesis method uses Fmoc-hydrazino-2-chlorotriphenylmethyl resin as a solid-phase support to gradually construct linear peptide chains containing thioamide bonds through solid-phase peptide synthesis.

[0041] In some embodiments, the solid-phase synthesis method includes: pretreatment of Fmoc-hydrazino-2-chlorotriphenylmethyl resin; amino acid coupling; Fmoc deprotection; introduction of thioamide bonds; resin cleavage and obtaining linear peptides.

[0042] Pretreatment of Fmoc-hydrazino-2-chlorotriphenylmethyl resin; amino acid coupling; Fmoc deprotection; introduction of thioamide bonds; resin cleavage and obtaining linear peptides.

[0043] The beneficial effects of this invention are as follows: By introducing thioamide units as potential activation sites into the peptide backbone and inducing them to undergo intramolecular condensation reactions with acylhydrazine groups in the peptide chain under the action of metal salts, this invention can simultaneously achieve peptide cyclization and in-situ construction of cisamide bioisosteres in the peptide backbone in a single reaction process, thereby overcoming the technical bottleneck that the peptide backbone cannot directly participate in structural reprogramming in the prior art; this method can complete the later editing of the peptide backbone without relying on the pre-synthesis and introduction of complex non-natural amino acid monomers, which significantly simplifies the synthetic route and improves the flexibility of structural modification; it has good adaptability to different peptide sequences and ring sizes, mild reaction conditions, high site selectivity, good versatility and reproducibility, and is suitable for applications in chemical biology research and peptide drug development. Attached Figure Description

[0044] Figure 1 The HPLC chromatogram and mass spectrometry data of cyclic peptide 1 in Example 1 are shown.

[0045] Figure 2The HPLC chromatogram and mass spectrometry data of cyclic peptide 2 in Example 2 are shown.

[0046] Figure 3 The HPLC chromatogram and mass spectrometry data of cyclic peptide 3 in Example 3 are shown.

[0047] Figure 4 The HPLC chromatogram and mass spectrometry data of cyclic peptide 4 in Example 4 are shown.

[0048] Figure 5 The HPLC chromatogram and mass spectrometry data of cyclic peptide 5 in Example 5 are shown.

[0049] Figure 6 The HPLC chromatogram and mass spectrometry data of cyclic peptide 6 in Example 6 are shown.

[0050] Figure 7 The HPLC chromatogram and mass spectrometry data of cyclic peptide 7 in Example 7 are shown.

[0051] Figure 8 The HPLC chromatogram and mass spectrometry data of cyclic peptide 8 in Example 8 are shown.

[0052] Figure 9 This is the HPLC chromatogram of cyclic peptide 1 under different pH conditions. Detailed Implementation

[0053] The specific implementation of the present invention will be further described in detail below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0054] Preparation method of Formula I This method uses Fmoc-hydrazino-2-chlorotriphenylmethyl resin as a solid-phase support to progressively construct linear peptide chains containing thioamide bonds via solid-phase peptide synthesis. The specific steps are as follows: Step 1: Pretreatment of Fmoc-hydrazino-2-chlorotriphenylmethyl resin Fmoc-hydrazino-2-chlorotriphenylmethyl resin (Fmoc-NHNH-2-chlorotrityl chloride resin, nominal loading 1.08 mmol / g) was swelled in anhydrous dichloromethane (DCM, 5 mL) for 10 min, followed by washing with DCM (3 × 5 mL) and dimethylformamide (DMF, 3 × 5 mL) sequentially. The resin was then deprotected with a 20% piperidine / DMF solution at 25°C in three separate treatments for 3 min, 3 min, and 5 min respectively. After deprotection, the resin was washed sequentially with DMF (3 × 5 mL), DCM (3 × 5 mL), and DMF (3 × 5 mL) to obtain the free hydrazino-functionalized resin.

[0055] Step 2: Amino acid coupling Fmoc-protected glutamic acid (4.0 equivalents), N,N′-diisopropylcarbodiimide (DIC, 4.2 equivalents), and Oxyma (4.5 equivalents) were dissolved in DMF to prepare a coupling reaction solution. This reaction solution was added to the above resin, and the reaction was carried out with shaking at room temperature for 60 min. After the reaction was completed, the resin was washed sequentially with DMF (3×5 mL), DCM (3×5 mL), and DMF (3×5 mL).

[0056] Step 3: Fmoc Deprotection After the coupling reaction, the resin was deprotected from Fmoc using a 20% (v / v) piperidine / DMF solution at 25°C, in three separate treatments for 3 min, 3 min, and 5 min respectively. After deprotection, the resin was washed sequentially with DMF (3 × 5 mL), DCM (3 × 5 mL), and then with DMF (3 × 5 mL). Steps two and three can be repeated as needed to gradually extend the peptide chain, depending on the target peptide sequence.

[0057] Step 4: Introduction of thioamide bonds α-Thioacyloxyenamide (2.0 equivalents) was dissolved in DMF and added to the above resin. The mixture was shaken and reacted at room temperature for 60 min to introduce thioamide bonds at specific sites on the peptide chain. After the reaction was completed, the resin was washed sequentially with DMF (3×5 mL), DCM (3×5 mL), and DMF (3×5 mL).

[0058] Step 5: Resin cleavage and acquisition of linear peptides After completing all peptide chain construction steps, the resin was washed sequentially with DMF (3×5 mL), methanol (MeOH, 3×5 mL), and DCM (3×5 mL), and then dried under reduced pressure. A cleavage reagent system (TFE / acetic acid / dichloromethane = 1:3:6, v / v / v) was added to the dried resin, and the mixture was shaken at room temperature for 1 h to cleave the linear peptide chain from the resin. After the reaction, the resin was removed by filtration, and washed with a small amount of cleavage reagent (TFE / acetic acid / dichloromethane = 1:3:6, v / v / v), and the filtrates were combined. The resulting filtrate was slowly added dropwise to cooled anhydrous diethyl ether to precipitate the crude linear peptide product. After centrifugation and discarding the diethyl ether, the remaining solid was dissolved in an acetonitrile / water mixture and freeze-dried to obtain a linear peptidyl hydrazide product containing a thioamide structure for subsequent cyclization reactions.

[0059] Example 1 Synthesis of Cyclic Peptide 1

[0060] Add Fmoc-Ala-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O to a clean 4mL reaction flask. t Bu (0.005 mmol), AgOAc (0.01 mmol), and dichloroethane (1 mL) were stirred at room temperature and monitored by HPLC. The reaction was completed after 72 hours. The target product was obtained by HPLC separation and purification followed by lyophilization as a white solid, with a yield of 70%. The HPLC method included: The chromatographic column was a Chiral 5μm MD(2)-RH 4.6×250 mm; Mobile phase A was a 0.045% trifluoroacetic acid aqueous solution (v / v); mobile phase B was an acetonitrile solution containing 0.039% trifluoroacetic acid (v / v) and 10% water; gradient elution was performed, and the proportion of mobile phase B increased linearly from 35% to 80% within 30 minutes; the flow rate was 1.0 mL / min; The detection wavelength was 214 nm. The HPLC chromatogram of cyclic peptide 1 is shown below. Figure 1 As shown.

[0061] 1 H NMR (400 MHz, CHLOROFORM- D )δ 8.44 (dd, J = 8.7, 4.1 Hz, 1H), 8.14(d, J = 3.7 Hz, 1H), 7.65 (d, J = 7.5 Hz, 1H), 7.59 (d, J= 7.5 Hz, 1H), 7.37 –7.27 (m, 7H), 7.13 – 7.06 (m, 4H), 6.72 (d, J = 7.4 Hz, 1H), 6.17 (d, J = 6.5 Hz, 1H), 5.24 (q, J = 7.0 Hz, 1H), 4.82 (q, J = 7.1 Hz, 1H), 4.51 – 4.38 (m, 3H), 4.24 – 4.08 (m, 3H), 3.62 – 3.42 (m, 4H), 2.93 (dd, J = 12.8, 10.5 Hz, 1H), 2.81 – 2.59 (m, 4H), 2.10 – 2.00 (m, 5H), 1.68 (d, J = 7.2 Hz, 3H), 1.51 (d, J =7.2 Hz, 3H), 1.39 (s, 9H). 13 C NMR (100 MHz, CHLOROFORM-D)δ 171.4, 170.9, 170.2, 169.1, 168.3,156.7, 155.5, 152.9, 143.4, 142.8, 141.1, 141.1, 134.8, 129.3 (2C), 129.2(2C), 127.8, 127.7, 127.7, 127.2, 127.0, 124.9, 124.6, 120.0 (2C), 82.9,67.6, 60.8, 55.6, 52.1, 51.7, 47.0, 46.9, 44.0, 41.8, 37.7, 31.2, 31.0, 27.8(3C), 21.9, 20.9, 20.5, 16.9 ppm. HRMS (ESI), m / z calcd. for C 46 H 55 N8O8 + [M+H] + = 847.4137; found = 847.4124. Example 2 Synthesis of Cyclic Peptide 2

[0062] Add Fmoc-Val-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O to a clean 4mL reaction flask. t Bu (0.005 mmol), silver salt (AgOAc) (0.01 mmol), and dichloroethane (1 mL) were added and stirred at room temperature, with HPLC monitoring. The reaction was completed after 72 hours. The target product was obtained by HPLC separation and purification followed by lyophilization as a white solid, with a yield of 63%. The HPLC method included: The chromatographic column was a Jupiter® C18 liquid chromatography column, 4.6 × 250 mm, with a particle size of 5 μm; Mobile phase A was a 0.045% trifluoroacetic acid aqueous solution (v / v), and mobile phase B was an acetonitrile solution containing 0.039% trifluoroacetic acid (v / v) and 10% water. The proportion of mobile phase B was varied in a gradient of 40% → 50% → 60% → 100% over 50 minutes (gradient durations were 35 minutes + 10 minutes + 5 minutes respectively). The flow rate was 1.0 mL / min, and the detection wavelength was 214 nm. The HPLC chromatogram of cyclic peptide 2 is shown below. Figure 2 As shown.

[0063] 1 H NMR (400 MHz, CHLOROFORM- D )δ 8.51 (d, J = 8.8 Hz, 2H), 8.25 (dd, J =8.8, 4.2 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.58 (d, J = 7.6 Hz, 1H), 7.39 – 7.33(m, 2H), 7.30 – 7.24 (m, 5H), 7.06 (q, J = 6.9 Hz, 2H), 7.00 – 6.94 (m, 2H), 6.79 (d, J = 7.0 Hz, 1H), 5.35 (q, J = 6.7 Hz, 1H), 4.50 – 4.39 (m, 3H), 4.32(dd, J = 10.0, 6.8 Hz, 1H), 4.21 (d, J = 9.6 Hz, 1H), 4.12 (t, J = 7.8 Hz, 1H), 4.00 (t, J= 9.5 Hz, 1H), 3.60 – 3.38 (m, 4H), 3.08 – 2.91 (m, 2H), 2.86 – 2.60(m, 3H), 2.47 (dd, J = 10.9, 6.3 Hz, 1H), 2.11 – 1.85 (m, 3H), 1.70 – 1.65 (m,1H), 1.61 (d, J = 7.1 Hz, 3H), 1.28 (d, J = 6.4 Hz, 3H), 1.22 (s, 9H), 1.05 (dd, J = 14.0, 7.0 Hz, 1H), 0.82 (d, J = 6.5 Hz, 3H). 13 C NMR (100 MHz, CHLOROFORM- D )δ 171.3, 170.9, 170.3, 168.8, 166.4,157.8, 156.0, 153.8, 143.3, 142.6, 141.0, 140.9, 134.5, 129.2 (4C), 127.8(2C), 127.7, 127.1, 127.0, 125.4, 124.8, 119.9 (2C), 83.4, 68.2, 60.9, 55.7,53.5, 53.3, 51.4, 47.1, 46.5, 43.9, 37.6, 32.5, 30.9, 29.3, 27.5 (3C), 21.8,20.3, 20.1, 19.8, 16.3 ppm. HRMS (ESI), m / z calcd. for C 48 H 59 N8O8 + [M+H] + = 875.4450; found = 875.4456. Example 3 Synthesis of Cyclic Peptide 3

[0064] Add Fmoc-Leu-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O to a clean 4mL reaction flask. tBu (0.005 mmol), AgOAc (0.01 mmol), and dichloroethane (1 mL) were added and stirred at room temperature, with HPLC monitoring. The reaction was completed after 72 hours. The target product was obtained by HPLC separation and purification followed by lyophilization as a white solid, with a yield of 63%. The HPLC method included: The chromatographic column was a Jupiter® C18 liquid chromatography column, 4.6 × 250 mm, with a particle size of 5 μm; Mobile phase A was a 0.045% trifluoroacetic acid aqueous solution (v / v), and mobile phase B was an acetonitrile solution containing 0.039% trifluoroacetic acid (v / v) and 10% water. The proportion of mobile phase B was varied in a gradient of 35% → 47% → 80% → 100% over 50 minutes (gradient durations were 30 min + 15 min + 5 min respectively). The flow rate was 1.0 mL / min, and the detection wavelength was 214 nm. The HPLC chromatogram of cyclic peptide 3 is shown below. Figure 3 As shown.

[0065] 1 H NMR (400 MHz, CHLOROFORM- D )δ 8.48 – 8.40 (m, 1H), 8.29 (s, 1H),7.65 (dd, J = 7.4, 2.6 Hz, 1H), 7.59 (dd, J = 7.7, 2.5 Hz, 1H), 7.37 – 7.27 (m,7H), 7.15 – 7.02 (m, 4H), 6.72 (d, J = 7.0 Hz, 1H), 6.09 (s, 1H), 5.22 (q, J =7.7 Hz, 1H), 4.73 (q, J = 7.2 Hz, 1H), 4.51 – 4.35 (m, 3H), 4.24 – 4.22 (m,1H), 4.18 – 4.07 (m, 2H), 3.66 – 3.36 (m, 4H), 3.04 – 2.62 (m, 5H), 2.08 –1.89 (m, 4H), 1.52 (d, J = 7.0, 3H), 1.37 (s, 9H), 1.00 (d, J = 6.5, 3H), 0.95(d, J = 6.5, 3H). 13 C NMR (100 MHz, CHLOROFORM- D)δ 171.4, 170.9, 170.2, 169.0, 168.2,156.8, 155.2, 152.9, 143.3, 142.8, 141.1, 141.0, 134.8, 129.3 (2C), 129.1(2C), 127.8 (2C), 127.6, 127.1, 127.0, 124.9, 124.6, 120.0 (2C), 82.9, 67.6,60.8, 55.6, 52.1, 51.6, 47.0, 46.8, 44.5, 44.0, 43.8, 37.7, 30.9 (2C), 27.8(3C), 24.7, 22.4, 22.3, 21.8, 20.8, 16.8 ppm. HRMS (ESI), m / z calcd. for C 49 H 61 N8O8+[M+H]+ = 889.4607; found =889.4608. Example 4 Synthesis of Cyclic Peptide 4

[0066] Add Fmoc-Ser-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O to a clean 4mL reaction flask. t Bu (0.005 mmol), AgOAc (0.01 mmol), and dichloroethane (1 mL) were added and stirred at room temperature, with HPLC monitoring. The reaction was completed after 72 hours. The target product was obtained by HPLC separation and purification followed by lyophilization as a white solid, with a yield of 63%. The HPLC method included: The chromatographic column was a Jupiter® C18 liquid chromatography column, 4.6 × 250 mm, with a particle size of 5 μm; Mobile phase A was a 0.045% trifluoroacetic acid aqueous solution (v / v), and mobile phase B was an acetonitrile solution containing 0.039% trifluoroacetic acid (v / v) and 10% water. Gradient elution was performed with the proportion of mobile phase B varying from 40% to 70% to 100% over 45 minutes (gradient durations were 40 minutes + 5 minutes). The flow rate was 1.0 mL / min, and the detection wavelength was 214 nm or 254 nm. The HPLC chromatogram of cyclic peptide 4 is shown below. Figure 4 As shown.

[0067] 1H NMR (400 MHz, CHLOROFORM-D)δ 8.78 (d, J = 7.8 Hz, 1H), 8.31 – 8.28(m, 2H), 7.65 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.38 – 7.27 (m,7H), 7.10 – 7.00 (m, 4H), 6.81 (d, J = 6.9 Hz, 1H), 5.38 (q, J = 7.1 Hz, 1H),5.10 (q, J = 8.0 Hz, 1H), 4.50 – 4.42 (m, 2H), 4.39 – 4.19 (m, 3H), 4.17 –3.94 (m, 4H), 3.64 – 3.51 (m, 2H), 3.48 – 3.40 (m, 2H), 3.07 – 2.88 (m, 3H),2.82 – 2.63 (m, 2H), 2.06 – 1.95 (td, J = 16.0, 6.6 Hz, 3H), 1.73 – 1.68 (m,1H), 1.63 (d, J = 6.9 Hz, 3H), 1.25 (s, 9H), 1.10 (s, 9H). 13 C NMR (100 MHz, CHLOROFORM-D)δ 171.4, 170.9, 170.6, 168.9, 166.6,157.7, 155.8, 153.8, 143.3, 142.6, 141.0 (2C), 134.4, 129.2 (3C), 127.9,127.8 (2C), 127.7, 127.2, 127.1, 125.3, 124.8, 119.9 (2C), 83.6, 77.3, 77.0,76.7, 74.9, 68.5, 62.4, 61.0, 55.8, 53.9, 51.5, 47.5, 47.2, 46.6, 43.9, 37.6,30.9, 29.0, 27.6 (3C), 27.2 (3C), 21.8, 20.3, 15.8 ppm. HRMS (ESI), m / z calcd. for C 50 H 63 N8O9 + [M+H] += 919.4713; found = 919.4686. Example 5 Synthesis of Cyclic Peptide 5

[0068] Add Fmoc-Pro-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O to a clean 4mL reaction flask. t Bu (0.005 mmol), AgOAc (0.01 mmol), and dichloroethane (1 mL) were stirred at room temperature and monitored by HPLC. The reaction was completed after 72 hours. The target product was obtained by lyophilization after HPLC separation and purification, and the yield was 76%. The HPLC method included: The chromatographic column was a Jupiter® C18 liquid chromatography column, 4.6 × 250 mm, with a particle size of 5 μm; Mobile phase A was a 0.045% trifluoroacetic acid aqueous solution (v / v), and mobile phase B was an acetonitrile solution containing 0.039% trifluoroacetic acid (v / v) and 10% water; gradient elution was used, with the proportion of mobile phase B linearly increasing from 10% to 100% within 30 minutes; the flow rate was 1.0 mL / min; and the detection wavelength was 214 nm. The HPLC chromatogram of cyclic peptide 5 is shown below. Figure 5 As shown.

[0069] 1 H NMR (400 MHz, CHLOROFORM- D )δ 8.56 – 8.52 (m, 2H), 7.65 (d, J = 7.5Hz, 1H), 7.60 (d, J = 7.4 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.32 – 7.26 (m, 6H), 7.09 (d, J = 7.1 Hz, 4H), 6.65 (d, J = 7.4 Hz, 1H), 5.29 (d, J = 7.6 Hz, 1H), 4.84(d, J = 6.4 Hz, 1H), 4.54 – 4.44 (m, 2H), 4.37 – 4.32 (m, 1H), 4.25 – 4.20 (m,1H), 4.15 – 4.12 (m, 2H), 3.83 (q, J= 8.2 Hz, 1H), 3.70 – 3.63 (m, 1H), 3.59 –3.42 (m, 4H), 2.92 (t, J = 11.9 Hz, 1H), 2.80 – 2.57 (m, 5H), 2.38 (t, J = 7.5Hz, 2H), 2.11 – 2.06 (m, 3H), 2.01 – 1.95 (m, 3H), 1.49 (d, J = 6.5 Hz, 3H), 1.43 (s, 9H). 13 C NMR (100 MHz, CHLOROFORM- D )δ 171.5, 170.8, 170.1, 169.3, 168.9,155.1, 154.6, 152.9, 143.4, 143.1, 141.1 (2C), 134.9, 129.3 (2C), 129.2 (2C),127.7 (2C), 127.6, 127.1, 127.0, 124.8, 124.6, 120.0 (2C), 82.8, 67.8, 60.8,55.7, 52.1, 51.6, 50.9, 47.0 (2C), 46.6, 44.1, 37.7, 33.0, 32.2, 31.0, 27.9(3C), 24.9, 21.9, 21.2, 17.1 ppm. HRMS (ESI), m / z calcd. for C 48 H 57 N8O8 + [M+H] + = 873.4294; found = 873.4230. Example 6 Synthesis of Cyclic Peptide 6

[0070] Add Fmoc-Trp(Boc)-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O to a clean 4mL reaction flask. t Bu (0.005 mmol), AgOAc (0.01 mmol), and dichloroethane (1 mL) were stirred at room temperature and monitored by HPLC. The reaction was completed after 72 hours. The target product was obtained by HPLC separation and purification followed by lyophilization as a white solid, with a yield of 63%. The HPLC method included: The chromatographic column was a Jupiter® C18 liquid chromatography column, 4.6 × 250 mm, with a particle size of 5 μm; Mobile phase A was a 0.045% trifluoroacetic acid aqueous solution (v / v), and mobile phase B was an acetonitrile solution containing 0.039% trifluoroacetic acid (v / v) and 10% water; gradient elution was used, with the proportion of mobile phase B linearly increasing from 40% to 100% within 30 minutes; the flow rate was 1.0 mL / min; and the detection wavelength was 214 nm. The HPLC chromatogram of cyclic peptide 6 is shown below. Figure 6 As shown.

[0071] 1 H NMR (400 MHz, CHLOROFORM- D δ 8.25 (s, 2H), 8.10 (d, J = 8.2 Hz, 1H),7.92 (s, 1H), 7.66 (d, J = 7.6 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.41 – 7.28 (m,9H), 7.16 – 6.98 (m, 6H), 6.68 (d, J = 7.1 Hz, 1H), 5.13 (q, J = 7.8 Hz, 1H), 4.70 (q, J = 7.2 Hz, 1H), 4.40 – 4.34 (m, 3H), 4.16 – 4.06 (m, 3H), 3.59 (d, J =8.2 Hz, 2H), 3.49 – 3.35 (m, 5H), 3.01 – 2.95 (m, 1H), 2.89 – 2.83 (m, 2H), 2.61 – 2.53 (m, 2H), 2.48 – 2.43 (m, 1H), 2.01 (dd, J = 12.6, 6.3 Hz, 2H), 1.86(dd, J = 13.9, 7.0 Hz, 1H), 1.65 (s, 9H), 1.28 (s, 9H), 0.40 (d, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, CHLOROFORM- D)δ 171.3, 170.9, 170.1, 168.8, 167.8,156.7, 154.9, 152.9, 149.2, 143.4, 142.8, 141.1 (2C), 135.4, 134.9, 130.9,129.3 (2C), 129.2 (2C), 127.8, 127.7, 127.7, 127.1, 127.0, 125.0, 125.0,124.7, 124.6, 123.2, 120.0 (2C), 118.3, 115.5, 114.2, 84.2, 82.9, 67.8, 60.8,55.6, 52.1, 51.5, 47.4, 46.9, 46.8, 43.9, 37.8, 30.9, 30.8, 30.2, 28.1 (3C),27.8 (3C), 21.8, 20.4, 14.6 ppm. HRMS (ESI), m / z calcd. for C 59 H 68 N9O 10 + [M+H] + =1062.5084; found =1062.5084. Example 7 Synthesis of Cyclic Peptide 7

[0072] Add Fmoc-Met-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O to a clean 4mL reaction flask. t Bu (0.005 mmol), AgOAc (0.01 mmol), and dichloroethane (1 mL) were stirred at room temperature and monitored by HPLC. The reaction was completed after 72 hours. The target product was obtained by HPLC separation and purification followed by lyophilization as a white solid, with a yield of 62%. The HPLC method included: The chromatographic column was a Jupiter® C18 liquid chromatography column, 4.6 × 250 mm, with a particle size of 5 μm; Mobile phase A was a 0.045% trifluoroacetic acid aqueous solution (v / v), and mobile phase B was an acetonitrile solution containing 0.039% trifluoroacetic acid (v / v) and 10% water; gradient elution was used, with the proportion of mobile phase B linearly increasing from 10% to 100% within 30 minutes; the flow rate was 1.0 mL / min; and the detection wavelength was 214 nm. The HPLC chromatogram of cyclic peptide 7 is shown below. Figure 7 As shown.

[0073] 1H NMR (400 MHz, CHLOROFORM- D )δ 8.50 – 8.40 (m, 1H), 8.33 (s, 1H),7.66 (d, J = 7.4 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.35 – 7.26 (m, 6H), 7.12 –7.04 (m, 4H), 6.76 (d, J = 7.2 Hz, 1H), 6.41 (d, J = 7.3 Hz, 1H), 5.34 (q, J = 7.7Hz, 1H), 5.03 (q, J = 7.3 Hz, 1H), 4.52 – 4.39 (m, 3H), 4.25 (dd, J = 9.7, 4.7Hz, 1H), 4.15 – 4.08 (m, 2H), 3.64 – 3.43 (m, 4H), 3.06 – 2.50 (m, 7H), 2.44– 2.15 (m, 7H), 2.08 – 2.01 (m, 2H), 1.87 – 1.81 (m, 1H), 1.71 – 1.66 (m,1H), 1.53 (d, J = 7.0 Hz, 3H), 1.36 (s, 9H). 13 C NMR (100 MHz, CHLOROFORM- D )δ 171.4, 170.9, 170.2, 168.9, 168.2,156.8, 154.5, 153.3, 143.3, 142.7, 141.1, 141.0, 134.9, 129.3 (2C), 129.1(2C), 127.8, 127.7, 127.6, 127.1, 127.0, 124.9, 124.6, 120.0 (2C), 82.8,67.6, 60.8, 55.5, 52.4, 51.6, 46.9, 46.8, 44.5, 44.0, 37.8, 33.1, 30.9, 30.8,30.0, 27.8 (3C), 21.8, 20.8, 17.1, 15.0 ppm. HRMS (ESI),m / z calcd. for C48 H 59 N8O8S + [M+H] + = 907.4171; found = 907.4176. Example 8 Synthesis of Cyclic Peptide 8

[0074] Add Fmoc-Lys(Boc)-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O to a clean 4mL reaction flask. t Bu (0.005 mmol), AgOAc (0.01 mmol), and dichloroethane (1 mL) were stirred at room temperature and monitored by HPLC. The reaction was completed after 72 hours. The target product was obtained by HPLC separation and purification followed by lyophilization as a white solid, with a yield of 61%. The HPLC method included: The chromatographic column was a Jupiter® C18 HPLC column, 4.6 × 250 mm, with a particle size of 5 μm; mobile phase A was 0.045% trifluoroacetic acid aqueous solution (v / v); mobile phase B was an acetonitrile solution containing 0.039% trifluoroacetic acid (v / v) and 10% water; gradient elution was used, with the proportion of mobile phase B linearly increasing from 35% to 85% within 30 minutes; the flow rate was 1.0 mL / min; and the detection wavelength was 214 nm. The HPLC chromatogram of cyclic peptide 8 is shown below. Figure 8 As shown.

[0075] 1 H NMR (400 MHz, CHLOROFORM- D)δ 8.44 (dd, J = 8.9, 4.3 Hz, 1H), 8.26(s, 1H), 7.64 (d, J = 7.6 Hz, 1H), 7.58 (d, J = 7.7 Hz, 1H), 7.41 – 7.26 (m,8H), 7.13 – 7.04 (m, 4H), 6.72 (d, J = 7.3 Hz, 1H), 6.44 (s, 1H), 5.23 (q, J= 7.1 Hz, 1H), 4.68 – 4.61 (m, 2H), 4.50 – 4.42 (m, 2H), 4.36 – 4.32 (m, 1H),4.23 – 4.19 (m, 1H), 4.15 – 4.07 (m, 2H), 3.62 – 3.37 (m, 4H), 3.19 – 3.06(m, 2H), 2.97 – 2.62 (m, 5H), 2.26 – 1.99 (m, 8H), 1.80 – 1.75 (m, 1H), 1.70– 1.64 (m, 1H), 1.51 (d, J = 7.2 Hz, 3H), 1.42 (s, 9H), 1.36 (s, 9H). 13 C NMR (100 MHz, CHLOROFORM- D )δ 171.4, 170.9, 170.1, 169.0, 168.3,156.9, 156.1, 155.0, 152.9, 143.4, 142.8, 141.0 (2C), 134.8, 129.3 (2C),129.1 (2C), 127.7 (2C), 127.6, 127.1, 127.0, 125.0, 124.6, 119.9 (2C), 82.8,79.3, 67.6, 60.8, 55.5, 52.1, 51.6, 46.9, 46.8, 46.4, 44.0, 39.6, 37.7, 33.9,31.0, 30.9, 29.7, 28.4 (3C), 27.8 (3C), 23.2, 21.8, 20.8, 16.9 ppm. HRMS (ESI),m / z calcd. for C 54 H 70 N9O 10 + [M+H] +=1004.5240; found =1004.5251. Example 9 Stability of cyclic peptide compounds To verify the stability of the cyclic peptide described in this invention, cyclic peptide 1 was subjected to stability tests under different pH conditions. For example... Figure 9 As shown, HPLC results indicate that the cyclic peptide exhibits good stability in both acidic and alkaline environments. No significant degradation was observed after incubation for 96 hours in a 10 mM PBS (containing 10% acetonitrile) system at pH 2.8–10.6.

[0076] 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. Application of a linear polypeptide containing thioamide and hydrazide bonds in the preparation of cyclic peptide compounds.

2. The application according to claim 1, characterized in that, The linear polypeptide has the structure described in Formula I. R1-(AA1) p -AA2-ψ(CS-NH)-(AA3) m -AA4(R2)-(AA5) q -R3 Formula I in, The amino acids corresponding to AA2 and AA4 are each independently selected from one of the natural amino acids; p and q are each independently selected from integers from 0 to 100; when p and q > 1, the amino acid corresponding to each AA1 or AA5 can be independently selected from either a synthetic amino acid or a natural amino acid. m is selected from integers from 1 to 100. When m>1, the amino acid corresponding to each AA3 can be independently selected from either synthetic amino acids or natural amino acids. R1 and R3 can be independently selected from -Fmoc, -Boc, and -O, respectively. t Bu and -OBn; R2 is an acylhydrazide group (-NHNH2); The side chain functional groups of the amino acids corresponding to AA1, AA2, AA3, AA4 and AA5 are optionally protected by protecting groups.

3. The application according to claim 2, characterized in that, The amino acid corresponding to AA2 is selected from alanine, glycine, valine, leucine, serine, proline, tryptophan, methionine, and lysine; the amino acid corresponding to AA4 is glutamic acid.

4. The application according to claim 1, characterized in that, The linear polypeptide is selected from: (1)Fmoc-Ala-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu(SEQ ID NO.1); (2)Fmoc-Val-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu(SEQ ID NO.2); (3)Fmoc-Leu-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu(SEQ ID NO.3); (4)Fmoc-Ser-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Yes (SEQ ID NO.4)? (5)Fmoc- S Pro-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu(SEQ ID NO.5); (6)Fmoc-Trp(Boc)-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu(SEQ ID NO.6); (7)Fmoc-Met-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Yes (SEQ ID NO.7)? (8)Fmoc-Lys(Boc)-Ψ(CSNH)-Ala-Phe-Pro-Gly-Glu(NHNH2)-O t Bu(SEQ ID NO.8)。 5. The application according to claim 1, characterized in that, The cyclic peptide compound has the structure shown in Formula II: Formula II in, R4 is selected from hydrogen, methyl, isopropyl, isobutyl, sec-butyl, pyrrolyl, benzyl, 2-methylthioethyl, hydroxymethyl, 1-hydroxyethyl, mercaptomethyl, p-hydroxybenzyl, 3-carbamoylpropyl, 2-carbamoylethyl, indolemethyl, carboxymethyl, 2-carboxyethyl, 4-aminobutyl and imidazolemethyl, wherein R4 is optionally substituted with tert-butoxycarbonyl or tert-butoxy. R5 is hydrogen, or R5 and R4 together with the atoms attached thereto form a 5-6 membered heterocyclic group, wherein the heterocyclic group includes at least one heteroatom selected from N, O or S; The amino acids corresponding to AA are selected from natural amino acids or synthetic amino acids; n is an integer between 1 and 100.

6. The cyclic peptide compound according to claim 1, characterized in that, The cyclic peptide compound is selected from any one of the following compounds: 。 7. A method for preparing a cyclic peptide compound, characterized in that, The method involves preparing the product from a linear polypeptide having the structure of Formula I via an intramolecular cyclization reaction. R1-(AA1) p -AA2-ψ(CS-NH)-(AA3) m -AA4(R2)-(AA3) q -R3 Formula I in, The amino acids corresponding to AA2 and AA4 are each independently selected from one of the natural amino acids; p and q are each independently selected from integers from 0 to 100; when p and q > 1, the amino acid corresponding to each AA1 or AA5 can be independently selected from either a synthetic amino acid or a natural amino acid. m is selected from integers from 1 to 100. When m>1, the amino acid corresponding to each AA3 can be independently selected from either synthetic amino acids or natural amino acids. R1 and R3 are independently selected from -Fmoc, -Boc, and -O, respectively. t Bu and -OBn; R2 is an acylhydrazide group (-NHNH2); The side chain functional groups of the amino acids corresponding to AA1, AA2, AA3, AA4 and AA5 are optionally protected by protecting groups.

8. The preparation method according to claim 7, characterized in that, The preparation method includes the steps of adding a metal salt and a reaction solvent; Preferably, the metal salt is a silver salt; Preferably, the reaction solvent is dichloromethane or dimethyl sulfoxide.

9. The preparation method according to claim 7 or 8, characterized in that, The method includes the step of an intramolecular condensation reaction between -ψ(CS-NH)- and -NHNH2 in the linear polypeptide of Formula I, to generate a 1,2,4-triazole heterocyclic structure in situ.

10. The preparation method according to claim 7, characterized in that, The intramolecular cyclization reaction was carried out at room temperature; the reaction time was 10-72 hours.

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