CN112159462B

CN112159462B - Coibatide A derivative and application thereof

Info

Publication number: CN112159462B
Application number: CN202011029160.0A
Authority: CN
Inventors: 房丽晶; 粟武; 姚贵阳; 王伟; 武春雷
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2017-12-13
Filing date: 2017-12-13
Publication date: 2022-03-08
Anticipated expiration: 2037-12-13
Also published as: CN109912697B; CN112159462A; CN109912697A

Abstract

The invention discloses a kobaypeptide A derivative and application thereof, and particularly discloses a kobaypeptide A or a derivative thereof, which has a structural formula shown in a formula I:

also disclosed is the use of a derivative of kobatide A in the manufacture of a medicament for the treatment of a hyperproliferative disease; and the use of test agents in cell localization studies.

Description

Coibatide A derivative and application thereof

The application is a divisional application of a Chinese patent application with the application number of 201711331700.9, the application date of 2017, 12 months and 13 days, and the invention name of the invention is "kobaypeptide A derivative and a preparation method and application thereof".

Technical Field

The invention relates to a kobatide A derivative and application thereof.

Background

Coibatide A (cobamide A) is a highly N-methylated natural cyclic depsipeptide. The structure is as follows:

natural cyclic lipopeptides which can be isolated and purified from the marine cyanobacteria Leptolyngbya sp. Panama are disclosed for the first time in US20120028905A 1. However, in this prior art, since the method of extracting a natural product is used to obtain the kobaypeptide A, the purification process is complicated, a large amount of solvent and a long time are required, and it cannot be used for scale-up production.

Research shows that the corynebeptide A inhibits cancer cell proliferation through a new action mechanism, has low nanomolar cytotoxicity on lung cancer, breast cancer, melanoma, leukemia, central nerve cancer and the like, and has good tissue selectivity on breast cancer, central nerve cancer and ovarian cancer cells. Therefore, the chrysotide A can be used as a lead of an anticancer drug with good prospect. However, since the amount of kobaypeptide a present in nature is very limited, scientists have proposed new strategies for the synthesis of kobaypeptide a.

For example, in 2014 He Wei (Total Synthesis of proposed structure of conjugate A, a high ly N-and O-methylated cytoxic amine cycle polypeptide. tetrahedron Lett.2014, 55, 610g-12) firstly reported a Total synthesis method of a Columbus peptide A isomer, which adopts a fragment splicing strategy of [ (4+1) +3+3] and synthesizes a presumed structure of Columbus peptide A through multi-step liquid phase reaction, but nuclear magnetic data thereof is not consistent with natural products reported by McPhail research group. Meanwhile, the synthesis method relates to a plurality of condensation reagents, column chromatography separation and purification of the intermediate are required, and the synthesis efficiency is low due to the racemization phenomenon of amino acid in the polypeptide condensation process.

Compared with a liquid phase synthesis method, the solid phase polypeptide synthesis method (SPPS) can be adopted to ensure that the synthesis of the kombu peptide A is simple, convenient and quick. In 2015, Nabika et al (Synthesis and biological evaluation of the [ D-MeAla ]¹¹]Epimer of coibamide A.Bio.Med.chem.Lett.2015, 25, 302-6.) Using TCP resin as solid support, a cyclized precursor containing all amino acid units was synthesized by Fmoc strategy, resin was cleaved with TFA/DCM (5: 95, v/v), and then reverse-esterified by macrocyclization in solutionIsomers of kobaypeptide a, in which MeAla at position 11 is isomerized, should be synthesized, and the yield of cyclization reaction is extremely low, only 3.8%, and the total yield of the isomers of kobaypeptide a synthesized is only 1% as calculated from the degree of substitution of the first amino group.

Furthermore, effective Synthesis and Stereochemical review of coibamide A.J.am.chem.Soc.2015, 137, 13488-91; CN201510648096.7, namely the subject group uses arylhydrazine resin as solid phase carrier and Melle^g-Ala⁸The intermediate is a cyclization site, BTC is a condensation reagent of all amido bonds, a solid-phase synthesis method of Fmoc-and Boc-strategies is jointly applied to obtain a cyclization precursor, and after resin is oxidized and cut off, acylation cyclization reaction is carried out in solution, so that the real structure of the kojiba peptide A is synthesized. Although the proposal takes hydrazine resin as a solid phase carrier to realize the synthesis of the kobaypeptide A and the analog thereof, the hydrazine resin is easy to generate acylation reaction in the condensation process of amino acid, so that partial peptide chain can not be cut from the resin, and the yield of the linear peptide is low.

In the Solid-Phase Total Synthesis of the deployed Structure of Coibamide A and Its Derivative: in high hy Methylated Cyclic depsipetides.eur.j.org.chem.2015.7043-52, Sable et al uses 2-CTC resin as solid phase carrier, firstly, double protected tyrosine is loaded on the resin by the hydroxyl group on tyrosine, the protecting group on tyrosine amino group is selectively removed, then the growth of peptide chain is realized by Fmoc-solid phase synthesis strategy, after the protecting group on tyrosine carboxyl group is removed, solid phase cyclization is carried out, the peptide chain is further prolonged, after the resin is cut, methylation reaction is carried out on the hydroxyl group in tyrosine in solution, thus obtaining the isomer of kobaypeptide A. However, although this scheme achieves solid phase cyclization, the methylation reaction still needs to be carried out in solution eventually, with low overall yield; in addition, because the spectrogram of the compound obtained by the route is inconsistent with that of the same compound obtained by other subject groups, the reaction process of the route has the problem of racemization of amino acid, and the technical scheme can not realize the synthesis of the kobaypeptide A.

In summary, the synthesis method of the kombu peptide a provided by the prior art is long in time consumption and low in yield, and due to racemization of amino acid in the reaction process, it is difficult to ensure that the synthesized molecule is the designed target molecule. Therefore, the development of a method for efficiently and rapidly synthesizing the kobaypeptide A or the isomer of the kobaypeptide A or the analogue thereof so as to meet the requirements of research and application is a technical problem to be solved in the field.

Disclosure of Invention

It is known from the prior art that although methods for synthesizing linear or cyclic polypeptides in solid or liquid phase are known in the prior art, the site of cyclization, the synthesis strategy and the method of the cyclic peptide containing ester bonds and N-methylated amide bonds greatly affect the synthesis efficiency and the configuration of the final product. The inventor finds that in the synthesis process, because a plurality of amido bonds in the kojiba peptide A and the derivative thereof are N methylated amido bonds, and the cyclic peptide structure contains ester bonds, when solid-phase synthetic resin is adopted for polypeptide synthesis, the DKP reaction is easy to occur when the Fmoc protecting group of Tyr is removed in the synthesis process of cyclized precursor polypeptide, so that the polypeptide chain is partially dropped, and the synthesis of the cyclized precursor polypeptide cannot be completed. Meanwhile, when esterification reaction is carried out on resin and the commonly used DCC/DMAP is taken as a condensation reagent, the reaction time is long, the efficiency is low, and better yield can be obtained by multiple times of condensation.

In order to solve the problems, the inventor of the invention can avoid partial dropping of a peptide chain and failure to obtain a cyclized precursor by controlling the site of cyclization in the synthesis of the kobaypeptide A and the derivative thereof, thereby greatly improving the synthesis efficiency and the yield.

Aiming at the problem of low esterification efficiency, the inventor of the invention uses triphosgene to convert amino acid into acyl chloride intermediate with high reaction activity in situ, and can greatly improve the esterification efficiency.

One aspect of the present invention provides a method for synthesizing cyclic depsipeptide with side tail in a ring shape, wherein the cyclic depsipeptide comprises an ester bond, and the amino group of the first amino acid on the side providing carboxyl terminal in the ester bond is a methylated amino acid, the method comprises the following steps,

1) taking a second-position amino acid on one side of the carboxyl terminal provided in the ester-forming bond as an initial amino acid, sequentially coupling the amino acids on solid-phase synthetic resin to obtain polypeptide chains, wherein the amino group of the second-position amino acid is protected by Fmoc, and the carboxyl group of the second-position amino acid is coupled with the solid-phase synthetic resin;

2) cleaving the resin to obtain a cyclized precursor polypeptide;

3) in a liquid phase, the cyclized precursor polypeptide is subjected to condensation coupling with a carboxyl group of a second-position amino acid on the side of the carboxyl terminal in an ester bond of the target depsipeptide ring and an amino group of a first-position amino acid on the side of the carboxyl terminal in the ester bond to obtain the cyclic depsipeptide.

In another aspect of the present invention, there is provided a process for the preparation of kojibapeptide a or a derivative thereof, which comprises the steps of:

1) taking the 10 th amino acid tyrosine of the kobaypeptide A as an initial amino acid, sequentially coupling the 10 th to 1 st amino acids on solid-phase synthetic resin by a solid-phase synthesis strategy, and coupling the 11 th amino acid alanine on a 5 th amino acid threonine side chain to obtain resin-polypeptide;

2) cleaving the resin to obtain a free cyclized precursor;

3) and (3) carrying out cyclization condensation reaction by taking the amino acid tyrosine at the 10 th position and the amino acid at the 11 th position as cyclization sites to obtain the kobaypeptide A or the derivative thereof.

In the technical scheme of the invention, the structure of the resin-polypeptide in the step 1) is as follows:

wherein R is₁＝-CH₃、-CH₂OC(CH₃)₃、-CH₂OH、-CH₂OCH₃；

R₂＝H、-CH₃；

R₃＝

-CO(CH₂)₃CH₃、H；

R₄＝-CH₃、-CH₂OCH₃、-CH₂OH、-CH₂OC(CH₃)₃、-CH₂CH₂CH₂CH₂NHBoc；

A＝O、-NH、-NCH₃；

R₅＝H、-CH₃、-COCH₂CH₂NH₂、-COCH₂CH₂NHCtz。

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ser⁶(Me)-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ser³(Me)-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH; removing the Fmoc protecting group from Val at position 1, methylating Val¹A terminal amino group; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removing the Fmoc protecting group.

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ser⁶(Me)-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ser³(Me)-OH、Fmoc-D-Val²-OH and L-Me₂-Val¹-OH; removing the Fmoc protecting group from Val at position 1, methylating Val¹A terminal amino group; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removing the Fmoc protecting group.

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ala⁶-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ala³-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH; removing the Fmoc protecting group from Val at position 1, methylating Val¹A terminal amino group; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removing the Fmoc protecting group.

In the technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ser⁶(Me)-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ser³(Me)-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH; removing the Fmoc protecting group of Val at the 1 st position, and continuing to couple Z-beta-Ala-OH; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removing the Fmoc protecting group.

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ser⁶(Me)-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-D-N-Me-Ser³(Me)-OH、Fmoc-L-N-Me-VaI¹-L-HIV²-OH; removing the Fmoc protecting group from Val at position 1, methylating Val¹A terminal amino group; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removing the Fmoc protecting group.

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-D-N-Me-Ala⁶-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-D-N-Me-Ala³-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH; removing the Fmoc protecting group from Val at position 1, methylating Val¹A terminal amino group; Fmoc-L-N-Me-Ala esterified and coupled in the Thr side chain at position 5¹¹-OH, removing the Fmoc protecting group.

In one embodiment of the invention, Fmo is coupled in sequence in step 1)c-L-Tyr¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ser⁶(Me)-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ser³(Me)-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH; removing the Fmoc protecting group from Val at position 1, methylating Val¹A terminal amino group; Fmoc-L-N-Me-Ala esterified and coupled in the Thr side chain at position 5¹¹-OH, removing the Fmoc protecting group.

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ser⁶(Me)-OH、Fmoc-D-N-Me-Thr⁵(tBu)-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ser³(Me)-OH、Fmoc-L-N-Me-Val¹-L-HIV²-OH; removing the Fmoc protecting group from Val at position 1, methylating Val¹A terminal amino group; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removing the Fmoc protecting group.

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ala⁶-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ala³-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH; removing the Fmoc protecting group of Val at the 1 st position; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removing the Fmoc protecting group.

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(tBu)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ser⁶(Me)-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ser³(Me)-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH; fmoc protection for removal of Val at position 1Yl, methylated Val¹A terminal amino group; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removal of the Fmoc protecting group;

and also comprises a step of removing the tBu protecting group after the cyclization in the step 3) is completed.

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ser⁶(tBu)-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ser³(tBu)-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH; removing the Fmoc protecting group from Val at position 1, methylating Val¹A terminal amino group; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removal of the Fmoc protecting group;

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-N-Me-Ser⁶(tBu)-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-N-Me-Ser³-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH; removing the Fmoc protecting group from Val at position 1, methylating Val¹A terminal amino group; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removing the Fmoc protecting group.

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ala⁶-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ala³-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH; removing the Fmoc protecting group of Val at the 1 st position, and continuing coupling Boc-beta-Ala-OH; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removal of the Fmoc protecting group;

and also comprises a procedure for removing Boc after the cyclization in the step 3) is finished.

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ser⁶(Me)-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ser³(Me)-OH、Fmoc-D-Val²-OH and L-Me₂Val¹-OH; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removing the Fmoc protecting group.

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ala⁶-OH、Fmoc-L-N-Me-Thr⁵-OH、Boc-L-N-Me-Leu⁴-OH; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removal of the Fmoc protecting group;

and further comprising a step of removal of Boc after the cyclization in step 3).

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ala⁶-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ser³(tBu)-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH; removing the Fmoc protecting group from Val at position 1, methylating Val¹A terminal amino group; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removing the Fmoc protecting group.

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ser⁶(Me)-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ser³(Me)-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH; removing the Fmoc protecting group from Val at position 1, methylating Val¹A terminal amino group; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removal of the Fmoc protecting group;

and a step of removing the tBu protecting group is further included after the cyclization in step 3).

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ala⁶-OH、Fmoc-L-N-Me-Thr⁵-OH、Boc-L-N-Me-Leu⁴-OH; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removing Fmoc protecting group, and carrying out condensation reaction with decanoic acid under the action of PyBOP/DIEA.

In one technical scheme of the invention, Fmoc-L-Tyr is sequentially coupled in the step 1)¹⁰(Me)-OH、Fmoc-L-N-Me-Leu⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷OH、Fmoc-N-Me-Lys⁶(Boc)-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-N-Me-Ala³-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH; removing the Fmoc protecting group from Val at position 1, methylating Val¹A terminal amino group; Fmoc-D-N-Me-Ala esterification coupling to Thr side chain at position 5¹¹-OH, removing the Fmoc protecting group.

In the technical scheme of the invention, Fmoc-L-N-Me-Val¹-D-HIV²The synthetic method of-OH is Fmoc-L-N-Me-Val¹Esterification of-OH and (S) -2-hydroxy-3-methyl-allyl butyrate with the aid of condensing agents (triphenylphosphine and DIAD), followed by Pd (PPh)₃)₄Reacting with DMBA to remove allyl protecting group to obtain Fmoc-L-N-Me-Val¹-D-HIV²-OH。

In the technical scheme of the invention, Fmoc-L-N-Me-Ser^3/6The synthesis method of (Me) -OH comprises the steps of adding NaHMDS into Boc-L-Ser-OH for complete reaction, dropwise adding methyl iodide for complete reaction, and reacting with ammonium chloride aqueous solutionQuenching, removing Boc protecting group from the obtained intermediate in DCM/TFA (v: v ═ 1: 1, 60ml), and reacting with Fmoc-Cl under basic condition to obtain Fmoc-protected Fmoc-L-N-Me-Ser^3/6(Me)-OH。

In the technical scheme of the invention, the method for synthesizing the linear peptide by the solid phase synthesis method in the step 1) comprises the steps of sequentially coupling Fmoc-protected N-methylated amino acid or non-N-methylated amino acid on solid phase synthesis resin, removing Fmoc protecting groups, and continuing coupling the N-methylated amino acid or the non-N-methylated amino acid until the linear peptide i is obtained; the N-methylated amino acid or non-N-methylated amino acid can be a single amino acid or a short peptide fragment of a plurality of amino acids.

In the technical scheme of the invention, the Fmoc protecting group removal method comprises the steps of removing by using 20% piperidine/DMF solution; preferably, a 20% piperidine/DMF solution is added to the peptide chain-resin and the suspension is shaken; the solution is filtered off with suction and 20% piperidine DMF solution, N is added again₂Bubbling and uniformly mixing; washed with DMF and then with anhydrous THF.

In the technical scheme of the invention, the condensation method for coupling the N-methylated amino acid to the resin or the polypeptide-resin by the amido bond comprises the steps of reacting the N-methylated amino acid with a condensing agent, adding an alkaline agent, adding the N-methylated amino acid activated by carboxyl into the deprotected resin or the polypeptide-resin, and reacting completely.

In embodiments of the invention, N-methylated amino acids include amino acids having one or two methyl groups on the alpha amino group, including but not limited to L-N-Me-Leu^4/9-OH、L-N-Me-Ile⁷-OH、L-N-Me-Ser^3/6(Me)-OH、L-N-Me-Thr⁵-OH、L N-Me-Val¹、L-N-Me-Ala^3/6-OH、D-N-Me-Thr⁵(tBu)-OH、、L-N-Me-Ser⁶(tBu)-OH、Me₂Val¹-OH and the like.

In the technical scheme of the invention, the condensation method for coupling the non-N-methylated amino acid to the resin or the polypeptide-resin by the amido bond comprises the steps of reacting the non-N-methylated amino acid with a condensing agent, adding an alkaline agent, adding the carboxyl activated non-N-methylated amino acid into the deprotected resin or the polypeptide-resin, and reacting completely.

In embodiments of the invention, non-N-methylated amino acids include amino acids that do not have a methyl group on the alpha amino group, including but not limited to L-Tyr¹⁰(Me)-OH、L-Ala⁸-OH、D-HIV²-OH、L-HIV²-OH、D-Val²-OH、Z-β-Ala-OH、、L-HIV²-OH and the like.

In the technical scheme of the invention, Fmoc-L-N-Me-Thr is used in step 1)⁵The condensation method for coupling-OH to resin or polypeptide-resin by amido bond is that Fmoc-L-N-Me-Thr⁵Adding PyAop and OxymaPure into-OH for carboxyl activation, adding an alkaline agent, and then adding carboxyl activated Fmoc-L-N-Me-Thr⁵-OH is added to the deprotected resin or polypeptide-resin and the reaction is completed.

In the solution of the invention, the N-methylated amino acid 11 (preferably N-Me-Ala) is esterified¹¹-OH, more preferably L-N-Me-Ala¹¹-OH or D-N-Me-Ala¹¹-OH) coupling to Thr⁵The side chain method comprises the steps of reacting N-methylated amino acid 11 with triphosgene, adding an alkaline agent, and then adding the N-methylated amino acid 11 activated by carboxyl into the polypeptide-resin to react completely.

In the invention, triphosgene is used as a condensation reagent for solid-phase synthesis esterification, and the reaction efficiency is higher than that of common condensation reagents such as DCC and EDC.

In the technical scheme of the invention, the conditions for cleaving the polypeptide and the resin are TFEA/HOAc/DCM, 2% TFA/DCM and the like.

In the technical scheme of the invention, the cyclized condensation method is to react the polypeptide with the condensing agent and the alkaline agent to the completion.

In the technical scheme of the invention, the condition for removing tBu and/or Boc protecting groups is TFA, preferably, the specific steps are that at 0 ℃, a cyclized product is added into TFA, the mixture is stirred and reacted for 30min at the same temperature, trifluoroacetic acid is removed by evaporation under reduced pressure, and the target compound is obtained by HPLC purification.

In the technical scheme of the invention, the condensing agent for the amido bond is EDC & HCl, EDIC, triphosgene (BTC), DCC, DIC, DMTMM⁺BF4^-、DMTMM⁺Cl^-One or more of HATU, HBTU, HCTU, HOAt, HOBt, BOP-Cl, PyBOP, PyAOP, OxymaPure, DPPA, FDP and FDPP.

In the technical scheme of the invention, the alkaline agent is one or a combination of more of collidine, Diisopropylethylamine (DIEA), pyridine, lutidine (DMAP), triethylamine (NMM) and 2-methylquinoline.

In the technical scheme of the invention, the solid phase synthetic resin is selected from 2-CTC resin, Wang resin, Rink resin and other resins with carboxyl at the tail end after resin cutting.

In the technical scheme of the invention, chloranil/acetaldehyde detection is adopted to confirm that the condensation reaction is complete, and in the reaction process, an object to be detected is taken and washed by DMF, 2% acetaldehyde solution and 2% chloranil solution are added, and the mixture is placed for 5 minutes at room temperature. If the resin turns green or blue, the reaction is not complete, otherwise the reaction is complete.

In the context of the present invention, the amino acids mentioned are to be understood in the broadest sense, including the L-configuration amino acids and also the D-configuration amino acids. It includes amino acids without any modification and amino or carboxyl or side chain protection with a protecting group. It encompasses both natural and synthetic amino acids, and even compounds comprising two reactive groups, amino and carboxyl, are to be understood, in particular in the context of the present invention HIV, i.e.2 hydroxy-3 methyl-butyric acid, a compound comprising two reactive groups, is to be understood as a pan-amino acid.

One aspect of the present invention provides a kobaypeptide a derivative having the structural formula shown in formula I:

wherein R is₁＝-CH₃、-CH₂OC(CH₃)₃、-CH₂OH、-CH₂OCH₃；

R₂＝H、-CH₃；

R₃＝

-CO(CH₂)₃CH₃、H；

A＝O、-NH、-NCH₃

R₅＝H、-CH₃、-COCH₂CH₂NH₂、-COCH₂CH₂NHCtz。

In a specific technical scheme of the invention, the chrysophatide A derivative has the structure of compounds 13-30 in the following table.

In another aspect, the invention provides the use of a chrysanthemumab a derivative according to the invention in the manufacture of a medicament for the treatment of hyperproliferative diseases, preferably wherein the hyperproliferative diseases comprise one or more of colon cancer, rectal cancer, brain tumor, lung cancer, epidermal squamous 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, prostate cancer, glioma and rhinostomata cancer; more preferably, the hyperproliferative disease is breast cancer or non-small cell lung cancer.

In another aspect, the invention provides an application of the derivative of the kobazaki peptide A in preparing a test agent for researching the kobazaki peptide A and the derivative thereof in cell localization research.

Abbreviations in the present invention have the following definitions:

BTC: triphosgene; EDC. HCI: 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride; DCC: n, N' -dicyclohexylcarbodiimide; DIC: n, N' -diisopropylcarbodiimide; DMTMM⁺BF₄ ^-: 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholinium tetrafluoroborate; DMTMM⁺Cl^-: 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine chloride salt; HATU: o- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate; HBTU: benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate; HCTU: 6-chlorobenzotriazole-1, 1, 3, 3-tetramethylurea hexafluorophosphate; HOAt: 1-hydroxy-7-azobenzotriazol; HOBt: 1-hydroxybenzotriazole; BOP: benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate; BOP-Cl: bis (2-oxo-3-oxazolidinyl) phosphoryl chloride; PyBOP: benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; PyAOP: (3H-1, 2, 3-triazolo [4, 5-b ]]Pyridine-3-oxy) tris-1-pyrrolidinyl phosphonium hexafluorophosphate; DPPA: diphenyl phosphorazidate; FDP: pentafluorophenyldiphenylphosphate; FDPP: pentafluorophenyl diphenyl phosphate; DMAP: 4-dimethylaminopyridine; TFA: trifluoroacetic acid; and (3) TIS: triisopropylsilane; fmoc: 9-fluorenylmethoxycarbonyl; boc: a tert-butoxycarbonyl group; o isxmayure: 2-oxime ethyl cyanoacetate; DIEA: diisopropylethylamine; DMAP: lutidine (I) and (II) dimethyl pyridine; NMM: triethylamine.

Advantageous effects

The method uses simple and cheap amino acid as a raw material, 2-CTC resin as a solid phase carrier, sequentially completes the synthesis of a main peptide chain and a side chain by selecting a proper cyclization site and a condensation reagent and applying a solid phase synthesis method of an Fmoc-strategy, cuts off the resin and then carries out acylation reaction in a solution to close the loop, and can efficiently, quickly and economically prepare the natural product of the kombu peptide A or an isomer or an analogue thereof, wherein the total yield of the synthetic natural product of the kombu peptide A is about 30 percent.

Drawings

FIG. 1 shows the synthesis of Compound 1 according to the invention (lower panel) and the synthesis of the kobaypeptide A of the prior art 4¹H NMR spectra (upper panel) are compared (400 MHz).

Figure 2 is the HRMS spectrum of compound 1.

FIG. 3 is a drawing of Compound 8¹H NMR spectrum.

FIG. 4 is a drawing of Compound 8¹³C NMR spectrum.

FIG. 5 is a drawing of Compound 9¹H NMR spectrum.

FIG. 6 is a drawing of Compound 9¹³C NMR spectrum.

FIG. 7 is a schematic representation of compound 12Fmoc-D-N-Me-Ser (Me) -OH¹H NMR spectrum.

FIG. 8 is a HRMS spectrum of compound 12Fmoc-D-N-Me-Ser (Me) -OH.

FIG. 9 is a drawing of Compound 13¹H NMR spectrum.

Figure 10 is the HRMS spectrum of compound 3.

FIG. 11 is a drawing of Compound 14¹H NMR spectrum.

FIG. 12 is a drawing of Compound 14¹³C NMR spectrum.

Figure 13 is the HRMS spectrum of compound 14.

Figure 14 is the HRMS spectrum of compound 15.

FIG. 15 is a drawing of Compound 16¹H NMR spectrum.

FIG. 16 is a drawing of Compound 16¹³C NMR spectrum.

Figure 17 is the HRMS spectrum of compound 16.

FIG. 18 is a drawing of Compound 17¹H NMR spectrum.

FIG. 19 is a drawing of Compound 17¹³C NMR spectrum.

Figure 20 is the HRMS spectrum of compound 17.

FIG. 21 is a drawing of Compound 18¹H NMR spectrum.

FIG. 22 is a drawing of Compound 18¹³C NMR spectrum.

Figure 23 is the HRMS spectrum of compound 18.

FIG. 24 is a drawing of Compound 20¹H NMR spectrum.

FIG. 25 is a drawing of Compound 20¹³C NMR spectrum.

Figure 26 is the HRMS spectrum of compound 20.

FIG. 27 is a drawing of Compound 21¹H NMR spectrum.

Figure 28 is the HRMS spectrum of compound 21.

FIG. 29 is of Compound 22¹H NMR spectrum.

Figure 30 is the HRMS spectrum of compound 22.

FIG. 31 is of Compound 23¹H NMR spectrum.

Figure 32 is the HRMS spectrum of compound 23.

Figure 33 is the HRMS spectrum of compound 24.

FIG. 34 is a drawing of Compound 25¹H NMR spectrum.

Figure 35 is the HRMS spectrum of compound 25.

FIG. 36 is a photograph of Compound 26¹H NMR spectrum.

Figure 37 is the HRMS spectrum of compound 26.

FIG. 38 is of Compound 27¹H NMR spectrum.

Figure 39 is the HRMS spectrum of compound 27.

FIG. 40 is a photograph of Compound 28¹H NMR spectrum.

Figure 41 is the HRMS spectrum of compound 28.

FIG. 42 is of Compound 29¹H NMRSpectra.

Figure 43 is the HRMS spectrum of compound 29.

FIG. 44 is a photograph of Compound 30¹H NMR spectrum.

Figure 45 is the HRMS spectrum of compound 30.

FIG. 46 is a flow chart of the preparation of Coibatide A.

Detailed Description

For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description of the embodiments of the present invention taken in conjunction with the accompanying drawings, which are included to illustrate and not to limit the scope of the present invention.

Example 1 Synthesis of Columba peptide A

(1) Synthesis of special amino acids:

1) compound 9Fmoc-L-N-Me-Val¹-D-HIV²-synthesis of OH:

DIAD (416. mu.L, 2.1mmol) was slowly added to Fmoc-L-N-Me-Val¹-OH (6) (707mg, 2mmol), (S) -2-hydroxy-3-methyl-allyl butyrate (7) (316mg, 2mmol) and triphenylphosphine (525mg, 2mmol) in tetrahydrofuran (10ml) were stirred at room temperature overnight, followed by thin layer chromatography, after completion of the reaction, the solvent was distilled off under reduced pressure to give a crude product, which was isolated by column chromatography (hexane/ethyl acetate) to give 8(907mg, 92%) as a colorless oily product.¹H NMR(400MHz，CDCl₃)δ0.73-1.10(m，12H)，2.30-2.20(m，2H)，2.92(s，1.26H)，2.95(s，1.77H)，4.20-4.72(m，6H)，4.85(d，J＝4.2Hz，0.45H)，4.89(d，J＝4.2Hz，0.55H)，5.17-5.25(m，1H)，5.28(dd，J＝2.8，1.4Hz，0.45H)，5.32(dd，J＝2.8，1.4Hz，0.55H)，5.87(ddt，J＝16.2，10.5，5.8Hz，1H)，7.30(t，J＝7.4Hz，2H)，7.37(t，J＝7.3Hz，2H)，7.65-7.58(m，2H)，7.74(d，J＝7.5Hz，2H)；¹³C NMR(100MHz，CDCl₃)δ16.9，18.5，18.6，19.5，27.1，29.7，30.1，63.0，63.5，65.3，67.4，76.7，118.5，119.7，124.6，124.8，126.8，127.4，131.3，141.0，143.6，156.7，168.6，170.3.HRMS(ESI-TOF)m/z：calcd.For C₂₉H₃₆NO₆[M+H]⁺ 494.2537found 494.2534.

Fmoc-L-N-Me-Val¹-D-HIV²-OH (9): weighed amounts of the above oily product 8(907mg, 1.84mmol) and tetrakis (triphenylphosphine) palladium (213mg, 0.184mmol) were dissolved in anhydrous tetrahydrofuran, and a solution of 1, 3-dimethylbarbituric acid (DMBA, 1.61g) in tetrahydrofuran (1ml) was slowly added dropwise to the solution. Stirring for one hour at room temperature, evaporating the solvent under reduced pressure, and separating the crude product by column chromatography to obtain a colorless oily product 9.¹H NMR(400MHz，CDCl₃)δ0.69-1.08(m，12H)，2.12-2.32(m，2H)，2.88(s，1.29H)，2.91(s，1.69H)，4.17(d，J＝10.6Hz，0.45H)，4.21-4.31(m，1H)，4.38-4.58(m，2H)，4.64(d，J＝10.3Hz，0.56H)，4.84(d，J＝4.0Hz，0.45H)，4.89(d，J＝4.0Hz，0.55H)，7.30(t，J＝7.4Hz，2H)，7.38(t，J＝7.4Hz，2H)，7.60(d，J＝7.7Hz，2H)，7.76(d，J＝7.5Hz，2H)；¹³C NMR(100MHz，CDCl₃)δ16.9，18.7，19.6，20.7，27.3，29.8，30.4，63.7，67.8，76.6，119.8，124.7，124.9，126.9，127.6，141.2，143.7，157.1，170.5，174.48；HRMS(ESI-TOF)m/z：calcd for C₂₆H₃₂NO₆[M+H]⁺ 454.2224found 454.2211.

2) Compound Fmoc-L-N-Me-Ser^3/6Synthesis of (Me) -OH (12):

adding sodium bis (trimethylsilyl) amide (NaHMDS) (2.0M, 37.5ml and 75mmol) dropwise into a tetrahydrofuran (100ml) solution dissolved with Boc-L-Ser-OH (10) (5.12g and 25mmol) at-78 ℃, stirring for 30min at-78 ℃, then adding iodomethane (11.36g and 5.0ml and 80mmol) dropwise, slowly heating to room temperature, stirring for 20h, quenching with a saturated ammonium chloride aqueous solution, extracting with dichloromethane, washing with water, washing with saturated common salt water, drying with anhydrous sodium sulfate, and distilling under reduced pressure to obtain a crude product 11.

Dissolving the crude product 11 in DCM/TFA (v: v ═ 1: 1, 60ml), stirring at 0 ℃ for 2h, distilling under reduced pressure to remove the solvent, diluting with 1, 4-dioxane (50ml), cooling to 0 ℃, slowly adding 10% sodium carbonate solution until the pH is 8-9, then adding 4g of Fmoc-Cl, stirring at room temperature for 10h, adding diluted hydrochloric acid under ice bath to adjust the pH to 3-4, extracting with ethyl acetate for three times, washing with water twice, washing with saturated common salt once, drying, distilling under reduced pressure to obtain a crude product, and separating by column chromatography to obtain a colorless oily compound 12.¹H NMR(400MHz，CDCl₃)δ2.96(s，1.5H)，3.05(s，3H)，3.27(s，1.5H)，3.39(s，3H)，3.58(dt，J＝18.3，8.0Hz，1H)，3.86(ddd，J＝14.8，10.5，6.2Hz，2H)，4.23(t，J＝5.8Hz，1H)，4.29(t，J＝7.1Hz，2H)，4.38-4.46(m，2H)，4.47-4.57(m，1H)，4.61(dd，J＝7.7，4.6Hz，0.5H)，4.99(dd，J＝7.8，4.3Hz，1H)，7.28-7.35(m，3H)，7.40(m，3H)，7.56(d，J＝7.4Hz，1H)，7.61(d，J＝7.4Hz，2H)，7.74(d，J＝7.7Hz，1H)，7.77(d，J＝7.5Hz，2H).HRMS(ESI-TOF)m/z：calcd for C₂₀H₂₂NO₅[M+H]⁺ 356.1493found 356.1490.

(2) Preparation of linear peptide 3:

400mg of 2-CTC resin (0.98mmol/g, 0.392mmol) were weighed into a 25ml solid phase synthesis tube, then 3ml dichloromethane was added and soaked for 20 minutes, CH was aspirated₂Cl₂. Weighing Fmoc-L-Tyr¹⁰(Me) -OH (73.48mg, 0.072mmol) and DIEA (200. mu.L) were dissolved in 3ml of anhydrous tetrahydrofuran, stirred for one minute and added to the resin. N is a radical of₂Bubbling and mixing evenly, after condensation reaction for 2h, washing the resin with DMF for four times, and washing the resin with anhydrous DMF once. Then 2ml of anhydrous DMF solution of acetic acid (125. mu.L, 1.32mmol) and DIEA (200. mu.L, 1.20mmol) were added, N₂Bubble well for 2h, then wash with DMF four times. The degree of substitution was determined to be 0.40mmol/g by Bienert's method.

Fmoc protecting group was removed from the above resin (400mg, 0.40mmol/g, 0.16mmol) using method A. Simultaneously selecting one of methods B, C or D according to the type of the desired amino acid to perform amino acid reductionCombining; Fmoc-L-N-Me-Leu condensation in sequence⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ser⁶(Me)-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ser³(Me)-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH. Repeating the deprotection and condensation steps until the synthesis of the linear peptide 3 is completed.

Wherein, the method A: fmoc-deprotecting

To the resin was added 3ml of 20% piperidine DMF solution and the suspension was shaken for 10 min. The solution is filtered off with suction and 3ml of 20% piperidine DMF solution, N₂Bubbling and mixing for 10 minutes. Washed 6 times with 3ml of analytically pure DMF and then once with 3ml of anhydrous THF.

The method B comprises the following steps: condensation of N-methylated amino acids

Dissolving N-methylated amino acid (4.0eq) and BTC (1.64eq) in 3ml of tetrahydrofuran, adding collidine (12eq) dropwise to the mixed solution, adding DIEA (20.0eq) and then adding to the deprotected resin, N₂Bubbling and mixing evenly until the color is not changed any more when detecting chloranil/acetaldehyde. Then washed four times with 3ml of analytically pure DMF.

The method C comprises the following steps: condensation of non-N-methylated amino acids

non-N-methylated amino acid (4.0eq) and BTC (1.60eq) were dissolved in 3ml of THF, collidine (12eq) was added dropwise to the mixed solution, and after stirring for one minute, 1ml of DMF solution in which HOAt (4.0eq) and DIEA (20eq) were dissolved was added and stirred for 1 minute. Then added to the deprotected resin, N₂Bubbling and mixing evenly until the color is not changed any more when detecting chloranil/acetaldehyde. Then washed four times with 3ml of analytically pure DMF.

The method D comprises the following steps: Fmoc-L-N-Me-Thr⁵-OH condensation

Fmoc-L-N-Me-Thr⁵-OH (4eq), PyAOP (4eq) and OxymaPure (4eq) were dissolved in 3ml of DMF, DIEA (8eq) was added, stirred at room temperature for two minutes and added to the deprotected resin, N₂Bubbling and mixing evenly until the color is not changed any more when detecting chloranil/acetaldehyde. Then washed four times with 3ml of analytically pure DMF.

(3) Preparation of Linear peptide 4Preparation of-Val¹Fmoc-removed post-up-NHCH₃Methylation procedure above:

fmoc protecting group was removed from resin 3 above using method A. The resin carrying the linear peptide 3 was substituted with CH₂Cl₂(3mL) twice, then add NaBH (OAc) to the resin₃(10eq) solution of DCE (3mL) and formaldehyde (10eq) solution of DCE (3mL), N₂After bubbling through for 2h, the mixture was washed twice with DCE (3mL) and MeOH (3mL), respectively, and twice with anhydrous THF (3mL) to obtain the linear peptide 4.

(4) Preparation of linear peptide 5:

and (3) carrying out solid-phase esterification reaction by using the method E, and then removing the Fmoc protecting group on the resin by using the method A.

By CH₂Cl₂(2X 3ml) Wash the resin, strip CH₂Cl₂A mixed solution (10.0mL) of TFEA/HOAc/DCM (1: 8) was added, and the reaction was stirred at room temperature for 24 hours. The resin was filtered off and CH was used₂Cl₂The resin was washed (20 mL). The organic phase was concentrated and the residue was purified by preparative HPLC, the product was collected, evaporated to remove the organic solvent and lyophilized to give linear peptide 5(114mg, 55%). hrms (esi) m/z: calcd for C₆₅H₁₁₃N₁₀O₁₇[M+H]⁺ Exact Mass：1305.8280，found 1305.8274.

Wherein, the method E is an esterification reaction on resin:

n-methylated amino acid (12eq) and BTC (4.8eq) were dissolved in 2ml of tetrahydrofuran, and collidine (36eq) was added dropwise to the mixed solution, followed by stirring for one minute and DIEA (60.0eq) was added. 2ml of tetrahydrofuran and DMAP (4eq) were added to the deprotected resin, followed by the reaction mixture of activated amino acids, giving a suspension N₂It was bubbled through and mixed well for 1h, washed four times with 3ml of analytically pure DMF.

(5) Preparation of a copaiba peptide A:

EDCI (19.2mg, 0.10mmol), HOAt (13.6mg, 0.10mmol), DIEA (70. mu.L, 0.40mmol) were dissolved in CH₂Cl₂In (30mL), the mixture was cooled to 0 ℃ under ice bath. Linear peptide 5(13mg, 0.01mmol) was dissolved in CH₂Cl₂(2mL), slowly added dropwise to EDCl/HOAt/DI at 0 deg.CAfter 1 hour of reaction, the ice bath was removed from the reaction mixture of EA, the reaction mixture was further stirred at room temperature for 48 hours, the solvent was evaporated under reduced pressure, and the product was purified by preparative HPLC, collected and lyophilized to give the product, copaiba peptide A (1) (overall yield: 7.2mg, 56%). [ alpha ] to]_D ²³-58.4°(c 0.10，CHCl₃)；IR(neat)：3376，2957，1734，1644，1512，1468，1404，1248，1097，757cm^-1；¹H NMR(600MHz，CDCl₃)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(br s，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(br s，1H)，5.69(m，1H)，6.00(br s，1H)，6.35(br s，1H)，6.63(br s，1H)，6.68(br s，1H)，6.77(d，J＝9.0Hz，2H)，7.09(d，J＝8.4Hz，2H)；¹³C NMR(100MHz，CDCl₃)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：calcd for C₆₅H₁₁₁N₁₀O₁₆[M+H]⁺ 1287.8174，found 1287.8176.

Example 2 Synthesis of a derivative of Coibatide A

Compound 13 was prepared by the same method as that used to prepare the kobaypeptide A in example 1, except that coupling Fmoc-L-N-Me-Val was performed¹-D-HIV²-OH substitution by sequential coupling of Fmoc-D-Val²-OH and L-Me₂-Val¹-OH, and omitting Val¹Fmoc-removed post-up-NHCH₃The methylation procedure above. Comprehensive yield: 54 percent.

EXAMPLE 3 Synthesis of Compound 14

Compound 14 was prepared by the same method as that for the preparation of kobatide A in example 1, except that Fmoc-L-N-Me-Ser was used³(Me) -OH substitution to Fmoc-L-N-Me-Ala³-OH; Fmoc-L-N-Me-Ser⁶(Me) -OH substitution to Fmoc-L-N-Me-Ala⁶-OH. Comprehensive yield: and 55 percent.

EXAMPLE 4 Synthesis of Compound 15

Compound 15 was prepared in the same manner as in example 1 except that Fmoc-L-N-Me-Val was added to the resulting linear peptide 3¹After the Fmoc protecting group is removed by the method A, the coupling of Z-beta-Ala-OH is continued by the method C, and Val is omitted¹Fmoc-removed post-up-NHCH₃The methylation procedure above. Comprehensive yield: 54 percent.

EXAMPLE 5 Synthesis of Compound 16

Compound 16 was prepared by the same method as that for the preparation of kobatide A in example 1, except that Fmoc-L-Me-Ser was used³(Me) -OH substitution to Fmoc-D-Me-Ser³(Me) -OH, and the (S) -2-hydroxy-3-methyl-allyl butyrate is replaced by (R) -2-hydroxy-3-methyl-allyl butyrate. Comprehensive yield: 50 percent.

EXAMPLE 6 Synthesis of Compound 17

Compound 17 was prepared by the same method as that for the preparation of kobatide A in example 1, except that Fmoc-L-N-Me-Ser was used³(Me) -OH substitution to Fmoc-D-N-Me-Ala³-OH; Fmoc-L-N-Me-Ser⁶(Me) -OH substitution to Fmoc-D-N-Me-Ala⁶-OH; and Fmoc-D-N-Me-Ala¹¹Replacement of-OH by Fmoc-L-N-Me-Ala¹¹-OH. Comprehensive yield: 52 percent.

EXAMPLE 7 Synthesis of Compound 18

Compound 18 was prepared by the same procedure as that used to prepare the kobatide A in example 1, except that Fmoc-D-N-Me-Ala was used¹¹Replacement of-OH by Fmoc-L-N-Me-Ala¹¹And (5) OH. Comprehensive yield: 50 percent.

EXAMPLE 8 Synthesis of Compound 19

Compound 19 was prepared by the same procedure as for the preparation of kobatide A in example 1, except that Fmoc-L-N-Me-Thr was used⁵(tBu) -OH by Fmoc-D-N-Me-Thr⁵(tBu) -OH; and converting (S) -2-hydroxy-3-methyl-allyl butyrate to (R) -2-hydroxy-3-methyl-allyl butyrate. Comprehensive yield: 49 percent.

EXAMPLE 9 Synthesis of Compound 20

Compound 20 can be prepared by the same method as that for preparing kobaypeptide A in example 1, except that Val is omitted¹Fmoc-removed post-up-NHCH₃The methylation procedure above; Fmoc-L-N-Me-Ser³(Me) -OH substitution to Fmoc-L-N-Me-Ala³-OH; and Fmoc-L-N-Me-Ser⁶(Me) -OH substitution to Fmoc-L-N-Me-Ala⁶-OH. Comprehensive yield: 48 percent.

EXAMPLE 10 Synthesis of Compound 21

Compound 21 was prepared by the same method as that for the preparation of kobaypeptide A in example 1, except that Fmoc-L-N-Tyr was used¹⁰(Me) -OH substitution to Fmoc-L-N-Tyr¹⁰(tBu) -OH and the resulting cyclisation product was subjected to a tBu-removal procedure as follows: at 0 ℃, the cyclized product is added into TFA, the reaction is stirred at the same temperature for 30min, the trifluoroacetic acid is evaporated under reduced pressure, and the target compound is obtained after HPLC purification. Comprehensive yield: and 47 percent.

EXAMPLE 11 Synthesis of Compound 22

Compound 22 was prepared by the same method as that for the preparation of kobatide A in example 1, except that Fmoc-N-Me-Ser was used³(Me) -OH substitution to Fmoc-N-Me-Ser³(tBu) -OH, Fmoc-N-Me-Ser⁶(Me) -OH substitution to Fmoc-N-Me-Ser⁶(tBu) -OH and the resulting cyclisation product was subjected to a tBu-removal procedure as follows: at 0 ℃, the cyclized product is added into TFA, the reaction is stirred at the same temperature for 30min, the trifluoroacetic acid is evaporated under reduced pressure, and the target compound is obtained after HPLC purification. Comprehensive yield: 44 percent.

EXAMPLE 12 Synthesis of Compound 23

Compound 23 was prepared by the same method as that for the preparation of kobatide A in example 1, except that Fmoc-N-Me-Ser was used³(Me) -OH substitution to Fmoc-N-Me-Ser³OH, Fmoc-N-Me-Ser⁶(Me) -OH substitution to Fmoc-N-Me-Ser⁶(tBu) -OH. Comprehensive yield: and 55 percent.

EXAMPLE 13 Synthesis of Compound 24

Synthesis of Compound 24 was carried out similarly to the preparation of Cabaptide A in example 1, except that in the linear peptide synthesis, Fmoc-L-N-Me-Leu was condensed in sequence by one of methods B, C or D according to the type of amino acid desired⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ala⁶-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ala³-OH、Fmoc-L-N-Me-Val¹-D-HIV²-OH and Boc- β -Ala-OH, and subjecting the cyclized product obtained to a Boc removal procedure as follows: the cyclized product was added to TFA (1ml) at 0 ℃, the reaction was stirred at the same temperature for 30min, and trifluoroacetic acid was evaporated under reduced pressure and purified by HPLC to give the objective compound. Comprehensive yield: 48 percent.

EXAMPLE 14 Synthesis of Compound 25

Compound 25 was prepared by a method similar to that of example 1 except that in the synthesis of the linear peptide, Fmoc-L-N-Me-Leu was condensed in sequence using one of methods B, C or D according to the type of amino acid desired⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ala⁶-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ala³-OH、Fmoc-D-Val²-OH and Me₂Val¹-OH. Comprehensive yield: 54 percent.

EXAMPLE 15 Synthesis of Compound 26

Compound 26 was prepared by a method similar to that of example 1 except that in the synthesis of the linear peptide, Fmoc-L-N-Me-Leu was condensed in sequence using one of methods B, C or D according to the type of amino acid desired⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ala⁶-OH、Fmoc-L-N-Me-Thr⁵-OH、Boc-L-N-Me-Leu⁴-OH and subjecting the cyclised product obtained to a procedure for Boc removal, in particular as follows: the cyclized product was added to TFA (1ml) at 0 ℃, the reaction was stirred at the same temperature for 30min, and trifluoroacetic acid was evaporated under reduced pressure and purified by HPLC to give the objective compound. Comprehensive yield: 46 percent.

EXAMPLE 16 Synthesis of Compound 27

Compound 27 was prepared by the same method as that for the preparation of kobaypeptide A in example 1, except that in the synthesis of a linear peptide, Fmoc-L-N-Me-Leu was condensed in sequence by selecting either one of methods B, C or D depending on the type of amino acid desired⁹-OH、Fmoc-L-Ala⁸-OH、Fmoc-L-N-Me-Ile⁷-OH、Fmoc-L-N-Me-Ala⁶-OH、Fmoc-L-N-Me-Thr⁵-OH、Fmoc-L-N-Me-Leu⁴-OH、Fmoc-L-N-Me-Ser³(tBu) -OH and Fmoc-L-N-Me-Val¹-D-HIV²-OH. Comprehensive yield: 52 percent.

EXAMPLE 17 Synthesis of Compound 28

The preparation method of the compound 28 is a procedure for removing tBu from the compound 27, and comprises the following specific steps: the cyclized product was added to TFA (1ml) at 0 ℃, the reaction was stirred at the same temperature for 30min, and trifluoroacetic acid was evaporated under reduced pressure and purified by HPLC to give the objective compound. Comprehensive yield: 46 percent.

EXAMPLE 18 Synthesis of Compound 29

The compound 29 is prepared by condensation reaction of compound 26 with decanoic acid under the action of PyBOP/DIEA and purification by HPLC to obtain the target compound. Comprehensive yield: 50 percent.

EXAMPLE 19 Synthesis of Compound 30

Compound 30 was prepared by the same method as that for the preparation of kobatide A in example 1, except that Fmoc-N-Me-Ser was used³(Me) -OH substitution to Fmoc-N-Me-Ala³OH, Fmoc-N-Me-Ser⁶(Me) -OH substitution to Fmoc-N-Me-Lys⁶(Boc) -OH. Comprehensive yield: 45 percent.

Anti-cell proliferation activity of the compound of example 3:

1) preparing medicines and reagents: 1L of water was added to a bag of RPMI1640 medium supplemented with 2g of sodium bicarbonate, 10 million units of penicillin and 100mg of streptomycin, the pH was adjusted to 7.4 and sterilized by filtration through a 0.22 μm sterile filter. And adding 5mL of inactivated newborn bovine serum into 95mL of culture medium to obtain the complete culture solution. Preparing trypsin into 0.25% solution with D-hanks buffer solution, filtering, sterilizing, and storing at 4 deg.C.

2) Preparing a sample solution to be detected: accurately weighing 1.0 μmol of Colibapeptide A (1) or its analogue, adding into sterilized 0.5mL centrifuge tube, adding 100 μ L DMSO to obtain 10 μ M stock solution, and freezing at-40 deg.C. Before use, the mixture is thawed and diluted with complete culture solution to corresponding concentration.

3) Cell culture and passage: the cells are cultured in a cell culture bottle containing 10mL of complete culture solution in a uniform adherence manner at 37 ℃ and 5% CO₂And culturing under saturated humidity. After the cells are full of the bottom of the bottle, washing the bottle twice by using a sterilized D-hanks solution, adding 0.25% trypsin to digest the cells for 2 minutes, pouring off the trypsin, after the cells can completely shed by shaking lightly, adding 30ml of complete culture solution, blowing the cells by using a pipette, subpackaging the cells in 3 new cell culture bottles, and continuing to culture.

4) And (3) drug treatment: taking a bottle of cells which just grow into a complete monolayer, collecting the cells after trypsinization, blowing and beating the cells uniformly by a pipette, taking two drops of cell suspension Trypan Blue (Trypan Blue) for staining, counting the number of living cells (the number of dead cells does not exceed 5 percent) under a microscope, and adjusting the number of the cells to 1 multiplied by 105 cells/ml by complete culture solution. Add 100. mu.L of cell suspension to each well of a 96-well cell culture plate, place the plate in CO₂Culturing in an incubator for 12 hours, taking out the culture plate, adding 100 μ L of complete culture solution containing samples to be tested with different concentrations into each well to make the final concentrations of the drugs respectively 1000, 100.0, 10.0, 1.0, 0.1 and 0.01nM, setting 4 parallel wells for each concentration, and setting 4 wells for adding the complete culture solution without the drugs as normal control wells. Adding the medicine, vibrating the culture plate on a microplate oscillator, mixing, and placing in CO₂The incubator was continued for 72 hours. The plate was removed, 20. mu.L of MTT solution (5 mg/mL) was added to each well, mixed by shaking, and cultured for another 4 hours. The culture medium was discarded from each well, and formazan crystals were dissolved by adding 100. mu.L of DMSO and then shaken for 10min to completely dissolve the crystals.

5) Determination of cytotoxic Activity: the half inhibitory concentration was measured by measuring the light absorption of each well with a microplate reader, and the measurement wavelength was 490 nm. And (3) calculating the inhibition rate of the drugs on cell proliferation according to the OD value of each well: inhibition (%) ([ 1-OD (additional drug group)/OD (negative control group)]X 100%. Performing linear regression with Microcal Origin software according to the inhibition rate corresponding to logarithm of drug concentration to obtain a linear equation, and calculating the drug concentration corresponding to 50% of the inhibition rate to obtain the half-Inhibitory Concentration (IC) of the sample to be tested on tumor cells₅₀) The data obtained are shown in table 1:

TABLE 1 IC of kobaypeptide A (1) and its analogs 13-30₅₀Value of

It can be seen from table 1 that the

compounds

14,15,23, and 27 synthesized by the present invention have good anti-cancer cell proliferation activity, especially anti-breast cancer cells and non-small cell cancer cells.

Claims

1. A kobaypeptide a derivative having the formula shown in the following table:

。

2. use of a chrysanthemi peptide a derivative according to claim 1 in the manufacture of a medicament for the treatment of a hyperproliferative disease;

wherein said hyperproliferative disease is colon cancer, pancreatic cancer, non-small cell lung cancer.