CN105646675A - Coibamide A analog, synthesis method and applications thereof - Google Patents

Abstract

The present invention provides coyibatide A analogues and their synthesis method and application, the method comprising providing hydrazine resin; using an activator, solid-phase organic synthesis of any hydrazine resin-supported cyclization precursor in Formula A to Formula F; Carry out the cleavage reaction in the presence of an oxidizing agent, so that the polypeptide chain in the cyclization precursor loaded on the hydrazine resin is separated from the resin to obtain the polypeptide chain cyclization precursor; remove the protection on the nitrogen terminal of the obtained polypeptide chain cyclization precursor After grouping P _NH , a condensing agent is used to carry out ring closure reaction to obtain the compound shown in formula I, wherein the structural formulas of formula I and formula A to formula F are the same as those described in the description. The present invention also provides an isomer of coyibatide A shown in formula I-1-1, wherein the structural formula of formula I-1-1 is the same as that described in the description, and the isomer has good anti-cellular hyperactivity proliferative activity.

Description

Coyiba peptide A analogue and its synthesis method and application

technical field

The invention relates to a coyiba peptide A analogue and its synthesis method and application, belonging to the field of solid-phase organic synthesis.

Background technique

US20120028905A1 discloses a highly N-methylated natural cyclic ester peptide—coibamide A (coibamide A) that McPhail et al. isolated and purified from the Panama marine cyanobacterium Leptolyngbyasp. It has low nanomolar cytotoxicity, and has good tissue selectivity for breast, central nervous and ovarian cancer cells. Studies have shown that coibamideA inhibits the proliferation of cancer cells through a new mechanism of action. Therefore, coibamideA can be used as a lead of anticancer drugs with good prospects. However, due to the limited amount of coibamideA in nature, the separation and purification of coibamideA from algae requires complex processes, a large amount of organic solvents, and a long time, and the yield of coibamideA is very low, which cannot meet the needs of research and applications, such as The need for relevant pharmacodynamic evaluation of coibamideA cannot be met.

In 2014, HeWei (Total synthesis of proposed structure of coibamideA, ahighlyN-andO-methylatedcytotoxicmarinecyclodepsipeptide. Tetrahedron Lett. 2014, 55, 6109-12) reported for the first time a total synthesis method of coibamideA, which uses [(4+1)+3+3] fragment splicing Strategy, through a multi-step liquid phase reaction, synthesized coibamideA. However, this synthesis method requires a variety of condensation reagents, which takes a long time, and the synthesis efficiency is very low due to the racemization of amino acids in the polypeptide condensation process.

Compared with the time-consuming liquid-phase synthesis method, the solid-phase peptide synthesis method (SPPS) can make the synthesis of coibamideA simple and fast. In 2015, Nabika et al. (Synthesis and biological evaluation of the [D-MeAla ¹¹ ]-epimerofcoibamide A. Bio. Med. Chem. Lett. 2015, 25, 302-6.) used TCP resin as a solid phase carrier and synthesized a ring containing all amino acid units through the Fmoc strategy Precursor, after excising the resin with TFA/DCM (5:95, v/v), finally synthesize the isomer of coibamideA with isomerization of MeAla at the 11-position by performing macrolide reaction in solution. In the reaction, the cyclization yield is only 3.8%. Based on the substitution degree of the first amino group, the total yield of the isomers of coibamideA is only 1%.

In summary, the synthesis method of coibamideA provided by the prior art is time-consuming and has a low yield, and it is difficult to ensure that the synthesized molecule is the designed target molecule due to the racemization reaction occurring during the reaction. Therefore, it is an urgent technical problem to be solved in this field to develop an efficient and rapid method for synthesizing coibamideA or coibamideA isomers or analogs thereof to meet the needs of research and application.

Contents of the invention

One of the objectives of the present invention is to provide a method for synthesizing coibamide A analogs, which can quickly and efficiently synthesize the designed coibamide A analogs.

Another object of the present invention is to provide an isomer of coiba peptide A, which has good anti-cell overproliferation activity.

Another object of the present invention is to provide the application of the isomer of coiba peptide A.

In order to achieve the above object, on the one hand, the present invention provides a method for synthesizing coiba peptide A analogs, the coiba peptide A analogs have the structural formula shown in formula I:

In formula I, R ₁ , R ₂ , R ₃ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₁ , R ₁₃ , and R ₁₄ are each independently hydrogen, methyl or ethyl;

R is methoxybenzyl, _ethoxybenzyl , methylaminobenzyl or ethylaminobenzyl;

R ₆ and R ₁₀ are each independently n-butyl, isobutyl or sec-butyl;

R ₁₂ is methoxymethyl, ethoxymethyl, methoxyethyl or ethoxyethyl;

G is hydrogen, methyl,

Or amino protecting group; The amino protecting group is Boc, Fmoc, Cbz, Trt or Allyl;

R ₁₅ , R ₂₁ , and R ₂₂ are each independently n-propyl, isopropyl, isobutyl or sec-butyl;

R ₁₆ , R ₁₇ , R ₁₉ , R ₂₀ , R ₂₃ , R ₂₄ are each independently hydrogen, methyl, ethyl or amino protecting group;

R ₁₈ is each independently methoxymethyl, ethoxymethyl, methoxyethyl or ethoxyethyl;

Y is hydroxyl, methoxy, amino, methylamino or dimethylamino;

X is O, N or N( _CH3 );

Preferably, formula I has the structure shown in formula I-1:

More preferably, formula I-1 has the structure shown in formula I-1-1 or formula I-1-2:

The method comprises the steps of:

a. Provide hydrazine resin;

b. Using an activator, any hydrazine resin-loaded cyclization precursor in solid-phase organic synthesis formula A to formula F:

PNH in any one of formula A to formula F represents an amino protecting group; the amino protecting group is Boc, Fmoc, Cbz, Trt or Allyl, preferably Fmoc;

c. performing a cleavage reaction in the presence of an oxidant to separate the polypeptide chain in the hydrazine resin-loaded cyclization precursor obtained in step b from the resin to obtain a polypeptide chain cyclization precursor;

Preferably, the carbon terminal of the polypeptide chain cyclization precursor is a carboxyl group;

d. After removing the protective group PNH on the nitrogen terminal of the polypeptide chain cyclization precursor obtained in step c, a condensing agent is used to perform a cyclization reaction to obtain the compound shown in formula I.

The present invention finds that the cyclization precursor supported by the hydrazine resin of formula A to formula F in step b can effectively carry out the cyclization reaction after cleavage, and significantly improve the yield of synthetic coyibatide A analogs. When the method described above is applied to the synthesis of coyibatide A or its stereoisomers, the yield can be as high as 8% to 20% based on the degree of substitution of the first amino group, which is far superior to the coyiba peptide A provided by the prior art. Synthetic route of ebatide A or its stereoisomers.

Preferably, in step b of the method of the present invention, an activator is used to solid-phase organically synthesize the hydrazine resin-supported cyclization precursor represented by formula D;

More preferably, the cyclization precursor supported by the hydrazine resin shown in formula D-1 is solid-phase organic synthesis:

Further preferably, the solid-phase organic synthesis formula D-1-1 or the cyclization precursor supported by the hydrazine resin shown in the formula D-1-2:

According to a specific embodiment of the present invention, in the method of the present invention, the cyclization precursor supported by the hydrazine resin represented by formula D-1 is prepared as follows:

b-1. Using an activator, the main chain supported by the hydrazine resin shown in the solid-phase organic synthesis formula D-1-F1:

Preferably, the main chain supported by the hydrazine resin shown in the solid-phase organic synthesis formula D-1-1-F1 or D-1-2-F1:

The P _OH described in formula D-1-F1, formula D-1-1-F1 or formula D-1-2-F1 is a hydroxyl protecting group, preferably tert-butyl, trityl, allyl or benzyl ;

b-2. Using a deprotecting group agent to remove the hydroxyl protecting group P _OH on the main chain supported by the hydrazine resin obtained in step b-1;

b-3. Esterification reaction is carried out on the hydroxyl group exposed in step b-2 to obtain the structure shown in formula D-1-F2:

After removing the amino protecting group P _NH in the formula D-1-F2, the coupling is continued to form the cyclization precursor supported by the hydrazine resin shown in the formula D-1;

Preferably, an esterification reaction is carried out on the hydroxyl group exposed in step b-2 to obtain the structure shown in formula D-1-1-F2 or formula D-1-2-F2:

After removing the amino protecting group P _NH of formula D-1-1-F2 or formula D-1-2-F2, continue coupling to form the hydrazine resin shown in formula D-1-1 or formula D-1-2 Loaded cyclization precursor.

The present invention effectively synthesizes the hydrazine resin-supported ring shown in formula D-1 by selecting a specific resin and protecting group strategy so that step b-2 does not break the connection bond between the resin and the peptide chain under the condition of removing _POH . chemical precursors.

Those skilled in the art should know that when a series of amino acids with a defined configuration are used, coiba peptide A, or a coiba peptide A isomer of a certain configuration or its analog. For example, in step b-1, a series of amino acids with defined configurations are selected, and an activator is used to synthesize formula D-1-1-F2 in solid phase, and formula D-1-2 is formed in step b-3, and the cleavage Coyibatide A shown in the formula I-1-2 of the definite configuration disclosed in the prior art can be prepared after the cyclization; another example is to select a series of amino acids with a definite configuration in step b-1, and use an active agent to solidify Phase organic synthesis of the main chain formula D-1-1-F1, and the formation of formula D-1-1 in step b-3, after cleavage and ring closure, Ke of the definite configuration shown in formula I-1-1 can be prepared Isomers of stimulant peptide A.

According to a specific embodiment of the present invention, in step b-1, the reagents used are Fmoc-MeIle ⁷ -OH, Fmoc-MeSer(Me) ⁶ -OH, Fmoc-MeThr(P _OH ) ⁵ -OH, Fmoc-MeLeu ⁴ -OH, Fmoc-MeSer(Me) ³ -OH and Me ₂ Val ¹ -Hiv ² -OH; or

Use protected or unprotected part or all of the fragments selected from the following sequences as reagents: Me ₂ Val ¹ -Hiv ² -MeSer(Me) ³ -MeLeu ⁴ -MeThr(P _OH ) ⁵ -MeSer(Me) ⁶ -MeIle ⁷ .

Those skilled in the art should know that when the reagent used is Fmoc-MeIle ⁷ -OH, Fmoc-MeSer(Me) ⁶ -OH, Fmoc-MeThr(P _OH ) ⁵ -OH, Fmoc-MeLeu ⁴ -OH, Fmoc-MeSer (Me) ³ -OH and Me ₂ Val ¹ -Hiv ² -OH are actually coupled one by one on the hydrazine resin to form the main chain supported by the hydrazine resin. When the reagent used is protected or unprotected part or all of the fragments selected from the main chain sequence as the reagent, in fact, the fragments conforming to the main chain peptide sequence loaded on the hydrazine resin are sequentially coupled to the resin, the combination of the above fragments The form and the number are not limited, as long as the main chain supported by the hydrazine resin shown in the formula D-1-F1 can be formed, for example, coupling Fmoc-MeIle ⁷ -OH on the hydrazine resin from which the Fmoc group has been removed Afterwards, Fmoc-MeThr(P _OH ) ⁵ -MeSer(Me) ⁶ -OH, Fmoc-MeSer(Me) ³ -MeLeu ⁴ -OH and Me ₂ Val ¹ -Hiv ² -OH were sequentially coupled; Fmoc-MeLeu ⁴ -MeThr(P _OH ) ⁵ -MeSer(Me) ⁶ -OH and Me ² Val ¹ -Hiv ₂ ^-MeSer ( Me) ³ -OH.

According to a specific embodiment of the present invention, after step b-3 removes the amino protecting group P _NH in formula D-1-F2, the reagent used is Tyr(Me) ¹⁰ , MeLeu ⁹ or Ala ⁸ ; or

After removing the amino protecting group P _NH in the formula D-1-F2 in step b-3, a part or all of the fragments selected from the following sequences protected by the Fmoc or Boc group are reagents: Tyr(Me) ¹⁰ -MeLeu ⁹ - Ala ⁸ .

Those skilled in the art should know that when the reagent used is Tyr(Me) ¹⁰ , MeLeu ⁹ or Ala ⁸ protected by Fmoc or Boc group, the _formula D-1-1 in which the amino protecting group PNH is removed is actually -F2 are sequentially coupled one by one to form formula D-1. When the reagent used is a part or all of the fragments selected from the Tyr(Me) ¹⁰ -MeLeu ⁹ -Ala ⁸ sequence protected by the Fmoc or Boc group as the reagent, the fragments that conform to the sequence are actually coupled to the Except for the formula D-1-1-F2 of the amino protecting group P _NH , the combination form and number of the above fragments are not limited, as long as the formula D-1 can be formed.

According to a specific embodiment of the present invention, in step b or step b-1, the activator is DCC, DIC, HATU, HBTU, HCTU, HOAt, HOBt, PyBOP, BOP-Cl, phosgene, diphosgene A combination of one or more of triphosgene, preferably phosgene, diphosgene or triphosgene;

More preferably, in step b-1, the Fmoc group-protected monomer amino acid or amino acid fragment and Me ₂ Val ¹ -Hiv ² -OH to be linked are independently activated by using phosgene, diphosgene or triphosgene, The activation step includes: dissolving the monomer amino acid or amino acid fragment protected by the Fmoc group to be connected, or Me ₂ Val ¹ -Hiv ² -OH and phosgene, diphosgene or triphosgene in an inert solvent, adding an organic base, and reacting To obtain the corresponding monomer amino acid chloride or amino acid fragment acid chloride, or Me ₂ Val ¹ -Hiv ² -Cl, wherein, the moles of phosgene or diphosgene and triphosgene converted to phosgene are calculated, and the moles of phosgene The number is 1.0 to 2.0 times the mole number of the monomeric amino acid or amino acid fragment protected by the Fmoc group, or Me ₂ Val ¹ -Hiv ² -OH;

More preferably, in step b-3, phosgene, diphosgene or triphosgene are used to independently activate the Fmoc or Boc group protection monomer amino acid or amino acid fragment to be connected, and the activation step comprises: each independently activating the Fmoc Or Boc group protection monomer amino acid or amino acid fragment and phosgene, diphosgene or triphosgene are dissolved in inert solvent, add organic base, react to obtain corresponding monomer amino acid chloride or amino acid fragment acid chloride, wherein, with phosgene or will Diphosgene and triphosgene are converted into moles of phosgene, and the moles of phosgene are 1.0 to 2.0 times the moles of Fmoc or Boc group-protected monomeric amino acids or amino acid fragments; further preferably, in step b In -3, after the corresponding monomer amino acid chloride or amino acid fragment acid chloride is obtained by the reaction, HOAt is added immediately to convert the monomer amino acid chloride or amino acid fragment acid chloride into the corresponding ester; wherein, the molar number of HOAt is Fmoc or Boc group protection unit 0.5 to 1.0 times the molar number of amino acids or amino acid fragments;

Wherein, in step b-1 or step b-3, the organic base is one of collidine, diisopropylethylamine, pyridine, lutidine and 2-methylquinoline or various combinations;

The inert solvent is one or a combination of tetrahydrofuran, dioxane, diglyme and 1,3-dichloropropane.

The present invention found that when the monomeric amino acid is a protein amino acid (non-N-methylated amino acid), the acid chloride produced by BTC activation will have various side reactions, which will reduce the condensation efficiency, and the acid chloride will be converted into the corresponding -OAt by adding HOAt Esters can significantly improve the solid-state condensation efficiency of amino acids with large steric hindrance and poor nucleophilicity (such as amino acids with methyl and other substituents on nitrogen atoms), and can increase the condensation yield in a relatively short period of time. Rate.

According to a specific embodiment of the present invention, in step a, the hydrazine resin is a phenylhydrazine resin; more preferably, the substitution degree of the hydrazine resin is 0.1-1.0 mmol/g.

According to a specific embodiment of the present invention, in step c, the oxidizing agent is a combination of one or more of copper acetate, N-bromosuccinimide and oxygen;

In step d, the condensing agent is EDC·HCl, DCC, DIC, DMTMM ⁺ BF ₄ ^- , DMTMM ⁺ Cl ^- , HATU, HBTU, HCTU, HOAt, HOBt, BOP, BOP-Cl, PyBOP, PyAOP, A combination of one or more of DPPA, FDP and FDPP.

The role of the oxidizing agent in step c in the present invention is to oxidize the hydrazine bond into a diazene bond, and then under the action of a cleavage agent, such as water, the polypeptide chain in the cyclization precursor supported by the hydrazine resin obtained in step b Separation from the resin to obtain the cyclization precursor of the polypeptide chain. Those skilled in the art should know that this step can be oxidized first and then cut the resin, or the resin can be cleaved while oxidizing.

In another aspect, the present invention provides an isomer of coiba peptide A, which has a structure shown in formula I-1-1:

The research of the present invention finds that the isomer of coyibatide A has good anti-cell hyperproliferation activity.

Comparing the structure of coyiba peptide A shown in formula I-1-2 disclosed in the prior art with the structure of the isomer of coyiba peptide A shown in formula I-1-1, it can be seen that the coyiba peptide A The configuration of the 2-position carboxylic acid and the 11-position amino acid of the isomer of Bateptide A is changed, and the unexpected anti-cell proliferation activity is brought about by the change of the configuration.

In yet another aspect, the present invention provides the use of the isomer of coibatide A represented by formula I-1-1 in the preparation of a drug for treating hyperproliferative diseases;

Preferably, said hyperproliferative diseases include colon cancer, rectal cancer, brain tumor (preferably glioblastoma), lung cancer (preferably non-small cell lung cancer), epidermal squamous cell carcinoma, bladder cancer, pancreatic cancer, Breast cancer, ovarian cancer, cervical cancer, endometrial cancer, colorectal cancer, renal cell carcinoma, esophageal adenocarcinoma, esophageal squamous cell carcinoma, non-Hodgkin's lymphoma, liver cancer, skin cancer, thyroid cancer, head and neck cancer, One or more of prostate cancer, glioma and nasopharyngeal carcinoma; more preferably, the hyperproliferative disease is breast cancer and non-small cell lung cancer.

In summary, the present invention provides a rapid and efficient method for synthesizing the designed coiba peptide A analogues, and using this method, a coiba peptide A isomer with good anti-cell hyperproliferation is obtained .

Abbreviations in the present invention have the following definitions:

BTC: triphosgene; EDC·HCl: 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; DCC: N,N'-dicyclohexyl carboimide; DIC: N,N'-Diisopropylcarbodiimide; DMTMM ⁺ BF ₄ ^- : 4-(4,6-Dimethoxytriazin-2-yl)-4-methylmorpholine tetrafluoroborate ; DMTMM ⁺ Cl ^- : 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine chloride salt; HATU: O-(7-azobenzotriazole)-N ,N,N',N'-tetramethyluronium hexafluorophosphate; HBTU: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate; HCTU: 6-chloro Benzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate; HOAt: 1-hydroxy-7-azobenzotriazole; HOBt: 1-hydroxybenzotriazole; BOP : benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; BOP-Cl: bis(2-oxo-3-oxazolidinyl)phosphinyl chloride; PyBOP: Benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate; PyAOP: (3H-1,2,3-triazolo[4,5-b]pyridin-3-oxyl)tri- 1-pyrrolidinylphosphonium hexafluorophosphate; DPPA: diphenylphosphonium azide; FDP: pentafluorophenyldiphenylphosphate; FDPP: pentafluorophenyl diphenylphosphate; DMAP: 4-dimethylaminopyridine; TFA: trifluoroacetic acid ; TIS: triisopropylsilane; Fmoc: 9-fluorenylmethoxycarbonyl; Boc: tert-butoxycarbonyl; Cbz (Z): benzyloxycarbonyl; Trt: trityl; Allyl: allyloxycarbonyl;

Fmoc-MeIle ⁷ -OH: Fmoc-(L)-MeIle ⁷ -OH:

Fmoc-MeSer(Me) ⁶ -OH: Fmoc-(L)-MeSer(Me) ⁶ -OH:

Fmoc-MeThr(P _OH ) ⁵ -OH: Fmoc-(L)-MeThr(P _OH ) ⁵ -OH:

Fmoc-MeLeu ⁴ -OH: Fmoc-(L)-MeLeu ⁴ -OH:

Fmoc-MeSer(Me) ³ -OH: Fmoc-(L)-MeSer(Me) ³ -OH:

Me ₂ Val ¹ —Hiv ² -OH: Me ₂ Val ¹ -Hiv ² -Cl:

(L)-Me ₂ Val ¹ —(D)-Hiv ² -OH:

(L)-Me ₂ Val ¹ —(L)-Hiv ² -OH:

Tyr(Me) ¹⁰ : MeLeu ⁹ : Ala ⁸ :

Tyr(Me) ¹⁰ - MeLeu ⁹ - Ala ⁸ :

Fmoc-(D)-MeAla ¹¹ -OH: Fmoc-(L)-MeAla ¹¹ -OH:

Boc-(L)-Tyr(Me) ¹⁰ -OH: Fmoc-(L)-MeLeu ⁹ -OH:

Fmoc-(L)-Ala ⁸ -OH:

Me ₂ Val ¹ -Hiv ² -MeSer(Me) ³ -MeLeu ⁴ -MeThr(P _OH ) ⁵ -MeSer(Me) ⁶ -MeIle ⁷ :

Description of drawings

Fig. 1 is the basic flow diagram of the isomer of coiba peptide A shown in the preparation formula I-1-1 in Example 1;

Fig. 2 is the basic route diagram of the cyclization precursor formula 4 that embodiment 1 prepares phenylhydrazine resin load;

Fig. 3 is the basic route diagram of the isomer of coiba peptide A shown in formula I-1-1 prepared by the cyclization precursor formula 4 supported by phenylhydrazine resin in Example 1;

Fig. 4 is the ¹ HNMR figure of the cyclization precursor 6 prepared in embodiment 1;

Fig. 5 is the ¹³ CNMR figure of the cyclization precursor 6 prepared in embodiment 1;

Fig. 6 is the HRMS figure of the cyclized precursor 6 that embodiment 1 prepares;

Fig. 7 is the ¹ HNMR figure of the isomer of coiba peptide A shown in formula I-1-1 prepared in Example 1;

Fig. 8 is the ¹³ CNMR figure of the isomer of coiba peptide A shown in formula I-1-1 prepared in Example 1;

Fig. 9 is the HRMS chart of the isomer of coiba peptide A represented by formula I-1-1.

detailed description

In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solutions of the present invention are described in detail below in conjunction with specific embodiments and accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the present invention. the scope of the invention.

Example 1

This example provides isomers of coiba peptide A represented by formula I-1-1 and its preparation method.

Fig. 1 roughly shows the preparation method of the isomer of coibatide A shown in formula I-1-1: adopt the phenylhydrazine resin that provides Fmoc protection; After removing the Fmoc protecting group on the phenylhydrazine resin, synthesize benzene Main chain supported by hydrazine resin; removal of tert-butyl protecting group on (L)-MeThr ⁵ hydroxyl group; first amino acid (D)-MeAla ¹¹ connected to side chain by esterification reaction; synthesis of side chain; oxidation Cleaving the cyclization precursor supported by the hydrazine resin to prepare a polypeptide chain cyclization precursor with a carboxyl group at the carbon terminal; finally cyclization to obtain the isomer of coiba peptide A shown in formula I-1-1.

Specifically, the cyclization precursor 4 supported by phenylhydrazine resin was first synthesized according to the route shown in FIG. 2 .

(1) Preparation of main chain 1 supported by phenylhydrazine resin:

Add Fmoc-protected phenylhydrazine resin (400mg, 0.66mmol/g, 0.26mmol, purchased from Novabiochem) and 3mL CH ₂ Cl ₂ into a 10mL solid-phase reactor, swell the phenylhydrazine resin for 30min, and remove CH ₂ Cl ₂ , use 3mL of 20% piperidine/DMF solution to remove the Fmoc protecting group on the phenylhydrazine resin, after 10min, remove the reaction solution, and use 3mL of 20% piperidine/DMF solution to remove the Fmoc protecting group on the phenylhydrazine resin again, after 10min , remove the reaction solution, use DMF (4 × 3mL) to wash the resin, then wash the resin with anhydrous THF (2 × 3mL), and set aside; at the same time, 97mgFmoc-(L)-MeIle ⁷ -OH (0.26mmol) and 32mg Sanguang Gas (BTC, 0.11mmol) was dissolved in 2mL of anhydrous THF, and 420μL collidine (collidine, 3.17mmol) was slowly added dropwise to the solution, and a large amount of white precipitate was produced immediately after the reaction, and reacted for 3min after the addition, and the reaction solution Transfer to the phenylhydrazine resin obtained above to remove Fmoc, add 870μL DIEA (5.28mmol), and mix evenly by bubbling with _N2 . After condensation reaction for 30min, remove the reaction solution, wash the resin with DMF (4×3mL), and Wash the resin with 3 mL of anhydrous DMF; add 125 μL of acetic anhydride (1.32 mmol) and 2 mL of DMF solution containing 200 μL of DIEA (1.20 mmol) to the resin, react for 10 min, remove the reaction solution, and wash the resin with DMF (4×3 mL) to obtain Fmoc-(L)-MeIle ⁷ -phenylhydrazine resin, its degree of substitution is 0.30mmol/g (400mg, 0.12mmol, 1eq);

As shown in Figure 2, repeat the above deprotection and condensation steps, and continue to couple Fmoc-(L)-MeSer(Me) ⁶ -OH, Fmoc-(L)-MeThr(t-Bu ) ⁵ -OH, Fmoc-(L)-MeLeu ⁴ -OH, Fmoc-(L)-MeSer(Me) ³ -OH and (L)-Me ₂ Val ¹ —(D)-Hiv ² -OH until complete Main chain supported by phenylhydrazine resin 1((L)-Me ₂ Val ¹ －(D)-Hiv ² －(L)-MeSer(Me) ³ －(L)-MeLeu ⁴ －(L)-MeThr(t- Bu) ^5- (L)-MeSer(Me) ^6- (L)-MeIle ⁷ -phenylhydrazine resin) synthesis, wherein the steps of deprotection and condensation are basically the same, for example, in the preparation of Fmoc-(L)-MeIle After ⁷ -phenylhydrazine resin, use 3mL20% piperidine/DMF solution to remove the Fmoc protecting group on the Fmoc-(L)-MeIle ⁷ -resin, after 10min, remove the reaction solution, and again use 3mL20% piperidine/DMF solution The Fmoc protecting group on the phenylhydrazine resin was removed, and after 10 min, the reaction liquid was extracted, and the resin was washed with DMF (4×3 mL), and then washed with anhydrous THF (2×3 mL), and set aside. Simultaneous activation of each Fmoc-(L)-MeSer(Me) ⁶ -OH to be connected, the specific operation is: 4.0eq of Fmoc-(L)-MeSer(Me) ⁶ -OH to be connected and 1.64eq of triphosgene (BTC) was dissolved in 2mL of anhydrous THF, and 12eq collidine (collidine) was slowly added dropwise to the solution, and a large amount of white precipitates were produced immediately in the reaction, and the reaction was completed for 3 minutes, and the reaction solution was transferred to a place where the Fmoc protecting group was removed. (L)-NHMeIle ⁷ -phenylhydrazine resin, then add 20eq DIEA, N ₂ bubbling and mixing, condensation reaction 0.5 ~ 1h, after the chloranil reagent detects that the reaction is complete, remove the reaction solution, and use DMF (4 × 3mL) to wash the resin;

(2) Preparation of cyclization precursor 4 loaded on phenylhydrazine resin:

The main chain 1 loaded on the phenylhydrazine resin prepared above was washed with CH ₂ Cl ₂ (2×3ml), the CH ₂ Cl ₂ was extracted, and 3.0 mL of TFA/TIS/H ₂ O was added (v/v/v=95: 2.5:2.5) to remove the t-butyl protecting group t-Bu from the mixed solution, remove the solvent after 2 min, and add TFA/TIS/H ₂ O (volume ratio 95:2.5:2.5) mixed solution (3.0 mL) to react for 5 min , washed the resin with CH ₂ Cl ₂ (2×3ml) and DMF (4×3mL), and then washed the resin with 3mL of anhydrous DMF to obtain the main chain 2 loaded on the phenylhydrazine resin from which t-Bu was removed, and set aside. At the same time, 4.0eq of Fmoc-(D)-MeAla ¹¹ -OH and 2eqDCC were dissolved in 2mLCH ₂ Cl ₂ /DMF (v/v=1:1), stirred at 0°C for 2h, centrifuged to separate the white precipitate, and the reaction Transfer the solution to the main chain 2 supported by phenylhydrazine resin to remove t-Bu, add catalytic amount of DMAP, N ₂ bubbling and mixing, condensation reaction for 2.5h, remove the reaction liquid, wash with DMF (4×3mL) Resin, obtain product 3;

Use 3 mL of 20% piperidine/DMF solution to remove the Fmoc protecting group in 3, and after 10 min, wash the resin with DMF (4×3 mL), and then wash the resin with anhydrous DMF (2×3 mL), and set aside. At the same time, 4.0eq Boc-(L)-Tyr(Me) ¹⁰ -OH and 1.64eq triphosgene (BTC) were dissolved in 1mL anhydrous THF, and 12eq collidine was slowly added dropwise to the solution, and the reaction immediately produced A large number of white precipitates were added to the reaction system, 4.0eqHOAt and 1.5mLDIEA/DMF (15%, v/v) were added to the reaction system. After 3 minutes of reaction, the reaction solution was transferred to the product 3 from which Fmoc was removed, and N ₂ was bubbled and mixed. Condensation reaction for 0.5 to 1 hour, after the reaction was detected by the chloranil reagent, the reaction solution was removed, and the resin was washed with DMF (4×3mL);

Wash the resin with CH ₂ Cl ₂ (2×3ml), pump off CH ₂ Cl ₂ , add 3.0 mL of TFA/TIS/H ₂ O (v/v/v=95:2.5:2.5) mixed solution, and remove the Boc protecting group After 2 min, the solvent was removed, and 3.0 mL of TFA/TIS/H ₂ O (v/v/v=95:2.5:2.5) mixed solution was added again, and the reaction was carried out for 5 min, and CH ₂ Cl ₂ (2×3 mL) and DMF (4 ×3mL) to wash the resin, and then wash the resin with 3mL of anhydrous THF, set aside. At the same time, 4.0eq Fmoc-(L)-MeLeu ⁹ -OH and 1.64eq triphosgene (BTC) were dissolved in 2mL anhydrous THF, and 12eq collidine was slowly added dropwise to the solution, and a large amount of white precipitate was produced immediately in the reaction , after adding the reaction for 3 minutes, transfer the reaction solution to the phenylhydrazine resin obtained above for removing Boc, then add 20eq DIEA, mix well by bubbling with N ₂ , and perform the condensation reaction for 0.5-1h. After completion, the reaction solution was extracted, and the resin was washed with DMF (4×3mL);

Use 3 mL of 20% piperidine/DMF solution to remove the Fmoc protecting group in the phenylhydrazine resin obtained above. After 10 minutes, remove the reaction solution, and use 3 mL of 20% piperidine/DMF solution to remove the Fmoc protecting group on the phenylhydrazine resin for 10 minutes. Finally, the reaction solution was extracted, the resin was washed with DMF (4×3 mL), and then with anhydrous DMF (2×3 mL), and set aside. At the same time, 4.0eq Fmoc-(L)-Ala ⁸ -OH and 1.64eq triphosgene (BTC) were dissolved in 1mL anhydrous THF, and 12eq collidine (collidine) was slowly added dropwise to the solution, and a large amount of white precipitate was produced immediately in the reaction , add 4.0eqHOAt and 1.5mLDIEA/DMF (15%, v/v) to the reaction system, after reacting for 3min, transfer the reaction solution to the phenylhydrazine resin that removes Fmoc, N ₂ bubbling and mixing, condensation reaction After 0.5 to 1 hour, after the reaction was detected by the chloranil reagent, the reaction solution was pumped out, and the resin was washed with DMF (4×3 mL) to complete the synthesis of the cyclization precursor 4 supported on the phenylhydrazine resin.

(3) Synthesis of isomers of coiba peptide A shown in formula I-1-1

Synthesize the isomer of coiba peptide A shown in the formula I-1-1 according to the route shown in Figure 3:

Take out the cyclization precursor 4 loaded on the phenylhydrazine resin obtained in step (2), add 4mLCH ₂ Cl ₂ , 10mgCu(OAc) ₂ , 200μL pyridine and 200μL water, shake and react at room temperature for 24h, filter the resin, and wash with 20mLCH ₂ Cl ₂ to wash the resin. The organic phase was concentrated, and the residue was purified by preparative HPLC (10% acetonitrile—H ₂ O (containing 1% TFA) isocratic elution for 5 min, 10% to 100% acetonitrile—H ₂ O (containing 1% TFA) ) gradient elution for 25 min, retention time T _R =27 min), the product was collected and the solvent was evaporated to dryness under reduced pressure to obtain product 5 (27 mg, based on the degree of substitution of the first amino acid, the yield was 20%). HRMS (ESI) m/z: The theoretically calculated exact molecular weight of C ₈₀ H ₁₂₃ N ₁₀ O ₁₉ [M+H] ⁺ is 1527.8960, and the measured value is 1527.8963.

Add 1.5mL Et ₂ NH and 1.5mL CH ₃ CN to 15.3 mg of product 5 (0.01 mmol), stir the reaction at room temperature for 15 min, evaporate the solvent under reduced pressure, and purify the residue by preparative HPLC (10% acetonitrile—H ₂ O (containing 1% TFA) isocratic elution for 5 min, 10% to 100% acetonitrile-H ₂ O (containing 1% TFA) gradient elution for 25 min, retention time _TR = 20 min), the product was collected and freeze-dried to obtain a white The solid 6 is ready for use, and its ¹ HNMR, ¹³ CNMR and HRMS are shown in Figure 4, Figure 5 and Figure 6, respectively.

19.2 mg EDC·HCl (0.10 mmol), 13.6 mg HOAt (0.10 mmol), 70 μL DIEA (0.40 mmol) were dissolved in 30 mL CH ₂ Cl ₂ and cooled to 0° C. in an ice bath. The above white solid 6 was dissolved in CH ₂ Cl ₂ (2 mL), and slowly added dropwise to the reaction solution of EDC·HCl/HOAt/DIEA at 0°C. After 1 h of reaction, the ice bath was removed, and the reaction mixture was continued to stir at room temperature After reacting for 17 hours, the solvent was distilled off under reduced pressure, purified by preparative HPLC (10% acetonitrile—H ₂ O (containing 1% TFA) isocratic elution for 5 minutes, 10% to 100% acetonitrile—H ₂ O (containing 1% TFA) TFA) gradient elution for 25min, retention time T _R =25min), the product was collected and freeze-dried to obtain 7.2mg of the isomer of coyiba peptide A shown in formula I-1-1, with a yield of 60%. Based on the degree of substitution of the first amino acid, the total yield was 12%. The ¹ HNMR, ¹³ CNMR and HRMS are shown in Figure 7, Figure 8 and Figure 9, respectively.

[α] _D ²³ -58.4° (c0.10, CHCl ₃ );

IR: 3376, 2957, 1734, 1644, 1512, 1468, 1404, 1248, 1097, 757cm ^-1 ;

¹ HNMR (600MHz, CDCl ₃ ) δ0.83(d, J=6.6Hz, 3H), 0.88-0.99(m, 18H), 1.00(d, J=4.2Hz, 3H), 1.05-1.08(m, 9H ),1.11(m,1H),1.11(d,J=7.2Hz,3H),1.28(d,J=6.6Hz,3H),1.31(m,1H),1.36(m,2H),1.50(m ,2H),1.60(m,1H),1.68(m,1H),2.02(m,1H),2.05(m,1H),2.21(m,1H),2.34(brs,9H),2.75(s, 3H),2.84(m,2H),2.86(s,3H),2.89(m,3H),2.99(m,1H),3.04(s,3H),3.12(s,3H),3.15(s,3H ),3.30(s,3H),3.35(s,3H),3.53(m,1H),3.61(m,1H),3.65(dd,J=11.4,4.2Hz,1H),3.75(m,1H) ,3.77(s,3H),3.83(m,1H),3.90(m,1H),4.73(m,1H),5.00(d,J=5.5Hz,1H),5.11(m,1H),5.32- 5.38(m,2H),5.50(brs,1H),5.69(m,1H),6.00(brs,1H),6.35(brs,1H),6.63(brs,1H),6.68(brs,1H),6.77 (d, J=9.0Hz, 2H), 7.09(d, J=8.4Hz, 2H);

¹³ CNMR (100MHz, CDCl ₃ ) δ11.6, 12.9, 15.7, 17.9, 18.0, 18.4, 18.6, 19.5, 19.6, 21.2, 21.4, 23.2, 23.3, 24.2, 24.9, 25.3, 27.6, 28.9, 29.7, 29.9, 30.1,30.2,31.3,32.0,36.2,37.9,38.9,39.4,41.3,47.0,49.9,51.0,51.1,52.5,52.9,55.3,58.6,58.8,63.5,64.7,68.6,69.3,73.8,74.8,113.7, 128.4, 130.4, 158.6, 167.2, 168.7, 169.7, 170.0, 170.5, 171.2, 171.3, 171.5, 172.4ppm;

HRMS (ESI-TOF) m/z: The theoretically calculated C ₆₅ H ₁₁₁ N ₁₀ O ₁₆ [M+H] ⁺ precise molecular weight is 1287.8174, and the measured value is 1287.8176.

Example 2

This example provides coiba peptide A represented by formula I-1-2 and its preparation method.

The method for preparing formula I-1-2 is basically similar to the preparation of formula I-1-2 in Example 1, only (L)-Me ₂ Val ¹ -(L)-Hiv ² -OH is used to replace (L) in Example 1 )-Me ₂ Val ¹ -(D)-Hiv ² -OH, and replace Fmoc-(D)-MeAla ¹¹ -OH in Example 1 with Fmoc-(L)-MeAla ¹¹ -OH, and the rest of the steps are the same, During the experiment, the total yield was 8% based on the degree of substitution of the first amino acid.

[α] _D ²³ -128.9°(c0.7, CHCl ₃ );

IR: 2960, 2923, 1732, 1645, 1508, 1459, 1405, 1248, 1093, 1012, 756cm ^-1 ;

¹ HNMR(CDCl ₃ ,400MHz):δ0.62-1.15(m,36H),1.15-1.38(m,7H),1.40-1.51(m,2H),1.60-1.76(m,2H),2.00-2.10 (m,2H),2.12-2.20(m,1H),2.30-2.35(m,6H),2.65-3.40(m,28H),2.45(brs,1H),3.45-3.60(m,2H),3.60 -3.70(m,1H),3.70-3.86(m,6H),4.52-5.24(m,5H),5.34-5.60(m,2H),5.60-5.75(m,2H),5.77-5.80(m, 0.7H), 5.89(d, 0.3H, J=4.2Hz), 6.57(brs, 1H), 6.80-6.85(m, 2H), 6.97(brs, 1H), 7.10-7.20(m, 2H)ppm;

¹³ CNMR (CDCl ₃ , 100MHz): δ11.6, 13.5, 14.1, 15.9, 18.0, 18.1, 18.5, 18.6, 18.7, 19.1, 19.3, 19.6, 21.2, 21.5, 22.7, 23.5, 23.6, 24.2, 24.8, 25.4 ,27.7,27.8,28.5,28.8,29.7,29.9,30.1,30.8,30.9,31.0,31.9,32.0,33.4,36.4,38.4,40.0,40.1,41.3,46.5,50.1,50.9,52.1,52.6,55.0,55.1 ,55.2,55.3,58.7,58.8,58.9,64.1,64.7,68.7,68.9,69.0,74.0,74.1,74.5,74.6,113.9,114.2,114.4,130.2,130.4,158.8,169.6,169.7,1770.3.1, ,170.4,171.6,171.8ppm;

HRMS (ESI-TOF) m/z: The theoretically calculated exact molecular weight of C ₆₅ H ₁₁₁ N ₁₀ O ₁₆ [M+H] ⁺ is 1287.8174, and the measured value is 1287.8170.

Example 3 Anti-cell Transitional Proliferation Activity of Coyiba Peptide A Isomers Shown in Formula I-1-1

1) Preparation of drugs and reagents: add 1L of water to a bag of RPMI1640 medium, add 2g of sodium bicarbonate, 100,000 units of penicillin and 100mg of streptomycin, adjust the pH to 7.4, and filter it out with a 0.22μm sterile filter membrane. bacteria. 95mL culture medium plus 5mL inactivated newborn calf serum is the complete culture medium. Trypsin was made into a 0.25% solution with D-hanks buffer, and stored at 4°C after filter sterilization.

2) Preparation of sample solution to be tested: Accurately weigh 1.29 mg (1.0 μmol) of the isomer of coibamideA shown in formula I-1-1 prepared in Example 1, and add it to a sterilized 0.5 mL centrifuge tube 100 μL DMSO was added to prepare a 10 μM stock solution, which was stored at -40°C. Before use, take an appropriate amount and dilute it with complete culture solution to the corresponding concentration after thawing.

3) Cell culture and subculture: All cells were cultured adherently in cell culture flasks containing 10 mL of complete culture medium at 37° C., 5% CO ₂ , and saturated humidity. After the cells cover the bottom of the bottle, wash twice with sterilized D-hanks solution, add 0.25% trypsin to digest the cells for 2 minutes, pour off the trypsin, and after the cells can be completely detached by shaking gently, add 30ml of complete culture solution, and pipette The liquid tube blows away the cells, and divides them into 3 new cell culture flasks, and continues the culture.

4) Drug treatment: Take a bottle of cells that have just grown into a complete monolayer, collect the cells after trypsinization, blow evenly with a pipette, take two drops of cell suspension for Trypan Blue staining, and count the live cells under a microscope. Cell number (the number of dead cells shall not exceed 5%), adjust the cell number to 1×105 cells/ml with complete culture medium. Add 100 μL of cell suspension to each well of a 96-well cell culture plate, place the culture plate in a CO ₂ incubator and incubate for 12 hours, take out the culture plate and add 100 μL of complete culture solution containing different concentrations of samples to be tested in each well , so that the final drug concentrations were 1000, 100.0, 10.0, 1.0, 0.1, and 0.01 nM, respectively. Four parallel wells were set up for each concentration, and cells in four wells were added with complete culture medium without drugs as normal control wells. After adding the medicine, the culture plate was shaken and mixed evenly on a microplate shaker, and placed in a CO ₂ incubator to continue culturing for 72 hours. Take out the culture plate, add 20 μL of 5 mg/mL MTT solution to each well, shake and mix well, and continue to incubate for 4 hours. Discard the culture medium in each well, add 100 μL of DMSO to dissolve the formazan crystals, shake for 10 min to completely dissolve.

5) Determination of cytotoxic activity: the light absorption of each well was measured with a microplate reader for the half-inhibitory concentration, and the measurement wavelength was 490 nm. Calculate the inhibitory rate of the drug on cell proliferation according to the OD value of each well: inhibitory rate (%)=[1-OD (medicated group)/OD (negative control group)]×100%. According to the inhibition rate corresponding to the logarithm of the drug concentration, use MicrocalOrigin software to do linear regression, obtain the linear equation, and calculate the corresponding drug concentration when the inhibition rate is 50%, which is the half inhibitory concentration (IC ₅₀ ) of the test sample to tumor cells, and the obtained The data are shown in Table 1:

The IC value of the isomer of _coiba peptide A shown in table 1 formula I-1-1

It can be seen from Table 1 that the coiba peptide A represented by the formula I-1-1 synthesized by the present invention has good anti-cancer activity, especially anti-breast cancer cells and non-small cell cancer cells.

Claims (10)

1. the synthetic method of Yi Zhong Ke Yiba peptide A analogue, this Ke Yiba peptide A analogue has the structural formula shown in formula I:

In formula I, R₁��R₂��R₃��R₅��R₇��R₈��R₉��R₁₁��R₁₃��R₁₄It is hydrogen, methyl or ethyl independently of one another;

R₄For methoxy-benzyl, ethoxy benzyl, methylamino benzyl or ethylamino-benzyl;

R₆��R₁₀It is normal-butyl, isobutyl-or sec-butyl independently of one another;

R₁₂For methoxymethyl, ethoxyl methyl, methoxy ethyl or ethoxyethyl group;

G be hydrogen, methyl,

Or amino protecting group; This amino protecting group is Boc, Fmoc, Cbz, Trt or Allyl;

R₁₅��R₂₁��R₂₂It is n-propyl, sec.-propyl, normal-butyl, isobutyl-or sec-butyl independently of one another;

R₁₆��R₁₇��R₁₉��R₂₀��R₂₃��R₂₄It is hydrogen, methyl, ethyl or amino protecting group independently of one another;

R₁₈For methoxymethyl, ethoxyl methyl, methoxy ethyl or ethoxyethyl group;

Y is hydroxyl, methoxyl group, amino, methylamino-or two methylamino;

X is O, N or N (CH₃);

Preferably, formula I has the structure shown in formula I-1:

More preferably, formula I-1 has the structure shown in formula I-1-1 or formula I-1-2:

Described method comprises the steps:

A., hydrazine resin is provided;

B. activator is adopted, the cyclisation precursor that in Solid-phase organic synthesis formula A��formula F, any one hydrazine is resin-carried:

The P of formula A��formula F in any one_NHRepresent amino protecting group; This amino protecting group is Boc, Fmoc, Cbz, Trt or Allyl, it is preferable that Fmoc;

C. carrying out cleavage reaction under the existence of oxygenant, polypeptide chain in the cyclisation precursor making gained hydrazine in step b resin-carried and resin isolation, obtain polypeptide chain cyclisation precursor;

Preferably, the one of carbon tip of described polypeptide chain cyclisation precursor is carboxyl;

D. de-except the protecting group P on nitrogen end on gained polypeptide chain cyclisation precursor in step c_NHAfter, adopt condensing agent to carry out ring-closure reaction, obtain compound shown in formula I.

2. method according to claim 1, wherein, in stepb, adopts activator, and Solid-phase organic synthesis is such as formula the resin-carried cyclisation precursor of the hydrazine shown in D;

Preferably, Solid-phase organic synthesis is such as formula the resin-carried cyclisation precursor of the hydrazine shown in D-1:

More preferably, the cyclisation precursor that hydrazine shown in Solid-phase organic synthesis formula D-1-1 or formula D-1-2 is resin-carried:

3. method according to claim 2, wherein, the resin-carried cyclisation precursor of the hydrazine shown in formula D-1 prepares as follows:

B-1. activator is adopted, the main chain that the hydrazine shown in Solid-phase organic synthesis formula D-1-F1 is resin-carried:

Preferably, the main chain that hydrazine shown in Solid-phase organic synthesis formula D-1-1-F1 or D-1-2-F1 is resin-carried:

P described in formula D-1-F1, formula D-1-1-F1 or formula D-1-2-F1_OHFor hydroxyl protecting group, it is preferable that the tertiary butyl, trityl, allyl group or benzyl;

B-2. adopt and go protecting group agent to take off except the hydroxyl protecting group P on the resin-carried main chain of step b-1 gained hydrazine_OH;

B-3. the hydroxyl exposed in step b-2 carries out esterification with the structure shown in obtained formula D-1-F2:

De-except the amino protecting group P in formula D-1-F2_NHAfter, continue coupling and form the resin-carried cyclisation precursor of the hydrazine shown in formula D-1;

Preferably, the hydroxyl exposed in step b-2 carries out esterification with the structure shown in obtained formula D-1-1-F2 or formula D-1-2-F2:

The de-amino protecting group P except formula D-1-1-F2 or formula D-1-2-F2_NHAfter, continue coupling to form the resin-carried cyclisation precursor of the hydrazine shown in formula D-1-1 or formula D-1-2.

4. method according to claim 3, wherein, in step b-1, the reagent of employing is Fmoc-MeIle⁷-OH��Fmoc-MeSer(Me)⁶-OH��Fmoc-MeThr(P_OH)⁵-OH��Fmoc-MeLeu⁴-OH��Fmoc-MeSer(Me)³-OH and Me₂Val¹-Hiv²-OH; Or

The part or all of fragment being selected from following sequence protected or do not protect is adopted to be reagent: Me₂Val¹-Hiv²-MeSer (Me)³-MeLeu⁴-MeThr (P_OH)⁵-MeSer (Me)⁶-MeIle⁷��

5. method according to claim 3, wherein, takes off except the amino protecting group P in formula D-1-F2 in step b-3_NHAfterwards, the reagent of employing is the Tyr (Me) of Fmoc or Boc radical protection¹⁰��MeLeu⁹Or Ala⁸; Or

Take off except the amino protecting group P in formula D-1-F2 in step b-3_NHAfterwards, the part or all of fragment being selected from following sequence adopting Fmoc or Boc radical protection is reagent: Tyr (Me)¹⁰-MeLeu⁹-Ala⁸��

6. method according to any one of Claims 1 to 5, wherein, in step b or step b-1, described activator is one or more the combination in DCC, DIC, HATU, HBTU, HCTU, HOAt, HOBt, PyBOP, BOP-Cl, phosgene, trichloromethylchloroformate and triphosgene, it is preferable that phosgene, trichloromethylchloroformate or triphosgene;

More preferably, in step b-1, phosgene, trichloromethylchloroformate or triphosgene is adopted to activate the single amino acid of Fmoc radical protection to be connected or amino acid fragment and Me independently of one another₂Val¹-Hiv²-OH, activation step comprises: by the single amino acid of Fmoc radical protection to be connected or amino acid fragment, or Me₂Val¹-Hiv²-OH photoreactive gas, trichloromethylchloroformate or triphosgene are dissolved in inert solvent, add organic bases, are obtained by reacting corresponding single amino acid acyl chlorides or amino acid fragment acyl chlorides, or Me₂Val¹-Hiv²-Cl, wherein, is converted into the mole number after phosgene calculates with phosgene or by trichloromethylchloroformate, triphosgene, and the mole number of phosgene is single amino acid or the amino acid fragment of described Fmoc radical protection, or Me₂Val¹-Hiv²1.0��2.0 times of the mole number of-OH;

More preferably, in step b-3, adopt phosgene, trichloromethylchloroformate or triphosgene activate Fmoc or Boc radical protection single amino acid to be connected or amino acid fragment independently of one another, activation step comprises: independently of one another by Fmoc or Boc radical protection single amino acid or amino acid fragment photoreactive gas, trichloromethylchloroformate or triphosgene are dissolved in inert solvent, add organic bases, it is obtained by reacting corresponding single amino acid acyl chlorides or amino acid fragment acyl chlorides, wherein, with phosgene or by trichloromethylchloroformate, triphosgene is converted into the mole number after phosgene and calculates, the mole number of phosgene is 1.0��2.0 times of the mole number of Fmoc or Boc radical protection single amino acid or amino acid fragment, further preferably, in step b-3, after being obtained by reacting corresponding single amino acid acyl chlorides or amino acid fragment acyl chlorides, add HOAt immediately and make single amino acid acyl chlorides or amino acid fragment acyl chlorides change into corresponding ester,Wherein, the mole number of HOAt is 0.5��1.0 times of the mole number of Fmoc or Boc radical protection single amino acid or amino acid fragment;

Wherein, in step b-1 or step b-3, described organic bases is one or more the combination in trimethylpyridine, diisopropylethylamine, pyridine, lutidine and 2-toluquinoline;

Described inert solvent is one or more the combination in tetrahydrofuran (THF), two alkane, diglyme and 1,3-propylene dichloride.

7. method according to any one of Claims 1 to 5, wherein, in step a, described hydrazine resin is phenylhydrazine resin; More preferably, the substitution degree of described phenylhydrazine resin is 0.1��1.0mmol/g.

8. method according to any one of Claims 1 to 5, wherein, in step c, described oxygenant is that neutralized verdigris, N-bromine are for one or more the combination in succimide and oxygen;

In steps d, described condensing agent is EDC HCl, DCC, DIC, DMTMM⁺BF₄ ^-��DMTMM⁺Cl^-, one or more combination in HATU, HBTU, HCTU, HOAt, HOBt, BOP, BOP-Cl, PyBOP, PyAOP, DPPA, FDP and FDPP.

9. the isomer of Ke Yiba peptide A of method synthesis according to any one of claim 1��8, the isomer of this Ke Yiba peptide A has the structure shown in formula I-1-1:

10. the application of the isomer of Ke Yiba peptide A according to claim 9 in the medicine for the preparation of overmedication proliferative disease;

Preferably, wherein said excess proliferative disease comprises one or more in colon and rectum carcinoma, brain tumor, lung cancer, epidermis squama cancer, bladder cancer, carcinoma of the pancreas, mammary cancer, ovarian cancer, cervical cancer, carcinoma of endometrium, colorectal cancer, renal cell carcinoma, adenocarcinoma of esophagus, esophageal squamous cell carcinoma, non-Hodgkin lymphoma, liver cancer, skin carcinoma, thyroid carcinoma, head and neck cancer, prostate cancer, neurospongioma and nasopharyngeal carcinoma; More preferably, described excess proliferative disease is mammary cancer, nonsmall-cell lung cancer.