CN101280012B

CN101280012B - Exendin-4 active isomer and application thereof

Info

Publication number: CN101280012B
Application number: CN2008100864766A
Authority: CN
Inventors: 李惟; 陈洁; 王丽萍; 孔维; 王丽凤
Original assignee: CHANGCHUN BCHT BIOTECHNOLOGY Co Ltd
Current assignee: Changchun Beyel Pharmaceutical Co., Ltd.
Priority date: 2007-03-14
Filing date: 2008-03-13
Publication date: 2011-10-19
Anticipated expiration: 2028-03-13
Also published as: CN101280012A

Abstract

The invention relates to a new active isomer of Exendin-4 and the application thereof, as well as the application in the drugs for curing II diabetes, belonging to organic chemistry and chemical medicine fields.The peptide linkages of the Exendin-4 sequence are partially constructed by substituting the Alpha- amido or carboxyl of the amino acid and the Alpha-carboxyls or amidos of the adjacent amino acids with the non-Alpha- amidos or carboxyls of the corresponding amino acid. The active isomer of Exendin-4 and the modified compounds thereof are hydrolysed with enzyme in blood plasma under37DEG C, and the hydrolysates are processed with high pressure liquid chromatography(HPLC)analysis; compared with normal structures, the hydrolysates are of longer half life than the orginal structures. Experiemtns on medical effects of the active isomer of Exendin-4 show that the active isomer of Exendin-4 has the effect of remarkably reducing the blood suger of the laboratory animal.

Description

New active isomer of Exendin-4 and application thereof

Technical field

The invention belongs to organic chemistry and chemical medicine field, the particularly active isomer of exendin-4 polypeptide and modifier, and the application in the type ii diabetes medicine.

Background technology

Exendin-4 is by excretory one peptide species in the Gila monster sialisterium of Southwestern United Stares, contains 39 amino-acid residues, with people's GLP-1 53% homology is arranged, and the height affinity is arranged and have similar physiological function to the GLP-1 acceptor.

Glucagon-like-peptide-1 (glucagon-likepeptide1 GLP-1) is a kind of incretin, and GLP-1 can stimulate beta Cell of islet regeneration, promotes insulin secretion, the release of glucagon suppression, and the gastric emptying rate that slows down suppresses food intake.Its promoting insulin secretion carries out according to glucose level, so can reduce hypoglycemic incidence, and insensitive diabetes B patient still has blood sugar reducing function to other Drugs Promoting Insulin Secretions, GLP-1 can also alleviate diabetes B patient's body weight simultaneously, is the brand-new Remedies for diabetes of a class.Because GLP-1 is easily by pepx IV-Dipeptidylpeptidase-4 (DPP-IV) hydrolysis, be 1-2 minute plasma half-life, and it can't be used as clinical medicine.

Exendin-4 has the pharmacological actions such as effect, glucagon suppression release, triglyceride reducing level and delay gastric acid secretion activity that promote insulin secretion, and Exendin-4 can also promote the beta Cell of islet hyperplasia, stimulates β cell new life and suppress the apoptotic function of β in addition.In Mammals, can resist the hydrolysis of dipeptidyl peptidase.Transformation period in its blood plasma is longer than GLP-1.Therefore have a lot of potential advantages than GLP-1 aspect the treatment diabetes B.

The Exendin-4 of synthetic is developed as the novel blood sugar lowing medicine by Amylin company and the joint research of EliLilly company at present, by the drugs approved by FDA listing, be used for the fully diabetes B patient of controlling blood sugar of N1,N1-Dimethylbiguanide, sulfonylurea or N1,N1-Dimethylbiguanide and sulfonylurea combined utilization in April, 2005.

Because exendin-4 is polypeptide, as peptide medicament its have relatively poor membrane permeability, easily by enzyme hydrolysis, easy-clear, indissoluble is separated and is easily assembled in some cases, these characteristics have lower bioavailability when making the peptide class as oral therapeutic drug.As the polypeptide of outside administration, the susceptibility to proteolytic ferment in stomach and intestine, blood and other tissue makes its quick removing, has significantly reduced the effect in answering, is very important so improve the stability of peptide medicament.

Summary of the invention

The invention provides a kind of new Exendin-4 active isomer and application thereof, strengthen the stability of peptide hormone, the transformation period that prolongs it is to improve curative effect, this is that of peptide hormone generally needs the problem that solves, thus transform peptide hormone structure, to increase their in vivo stability, improve bioavailability be very important.

The present invention mainly modifies the structure of exendin-4, designed the bioactive peptide isomer, on the basis of exendin-4 original structure, with partial amino-acid in its aminoacid sequence with corresponding β-, gamma-amino acid replaces, and their activity, vitro stability measured, their pharmacological effect is studied.The result shows, inside and outside stability improves these isomer greatly on the former activated basis having kept, and this invention provides theoretical foundation for the research and the new drug development of exendin-4 activity mechanism.

Partial amino-acid is with its corresponding beta-amino acids, gamma-amino acid and the displaced aminoacid sequence of other amino acid in the aminoacid sequence that activity isomerism body structure of the present invention is exendin-4.The Exendin-4 native sequences is:

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

The aminoacid sequence that Exendin-4 active isomer of the present invention has is:

X-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Me t-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-As n-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂, wherein:

X is ethanoyl or hydrogen

The amino acid of position 3 can be: Glu, γ-Glu, β-Asp, β-Ala

The amino acid of position 4 can be: Gly, β-Ala

The amino acid of position 6 can be: Phe, β-Phe

The amino acid of position 7 can be: Thr, Ser

The amino acid of position 9 can be: Glu, γ-Glu, Asp, β-Asp

The amino acid of position 13 can be: Gln, Tyr, γ-Glu

The amino acid of position 17 can be: Gln, Tyr, γ-Glu

The amino acid of position 24 can be: Gln, Tyr, γ-Glu.

In the new active isomer sequence of Exendin-4 of the present invention, at least one or a plurality of peptide bond are by the β-carboxyl of Asp and the alpha-amino group of Leu, γ-the carboxyl of Glu and the alpha-amino group of Gly, γ-the carboxyl of Glu and the alpha-amino group of Glu, α-the carboxyl of Ser and the epsilon-amino of Lys, γ-the carboxyl of Glu and the alpha-amino group of Ala, γ-the carboxyl of Glu and the alpha-amino group of Trp, α-the carboxyl of Leu and the epsilon-amino of Lys, γ-the carboxyl of Glu and the alpha-amino group of β-Ala, the carboxyl of β-Phe and the alpha-amino group of Thr, the carboxyl of β-Arg and the alpha-amino group of Leu, the carboxyl of γ-Arg and the alpha-amino group of Leu.Compound can be included in the pharmaceutical carrier component.Formula Chinese and English abbreviation expression: His Histidine, Ala L-Ala, Glu L-glutamic acid, the Gly glycine, Thr Threonine, Phe phenylalanine, the Ser Serine, Asp aspartic acid, Val Xie Ansuan, the Leu leucine, Lys Methionin, Gln glutamine, the Met methionine(Met), Arg arginine, Ile Isoleucine, the Trp tryptophane, Asn l-asparagine, Pro proline(Pro).

The new active isomer of Exendin-4 of the present invention also comprises the Gln in the exendin-4 natural acid sequence replaced with Tyr, and rest part amino acid is with its corresponding beta-amino acids, the displaced aminoacid sequence of gamma-amino acid.

The present invention is preferred with the following compounds, but is not limited to listed compound.(X is ethanoyl or hydrogen)

One of The compounds of this invention (exendin-4 β-Asp9) aminoacid sequence is as follows:

X-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-βAsp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is respectively to be generated by the β-carboxyl of Asp and the alpha-amino group of Leu that a peptide bond is arranged.

The compounds of this invention two (exendin-4Tyr13 β-Asp9) aminoacid sequence is as follows:

X-Hi?s-Gly-Glu-Gly-Thr-Phe-Thr-Ser-βAsp-Leu-Ser-Lys-Tyr-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is respectively to be generated by the β-carboxyl of Asp and the alpha-amino group of Leu that a peptide bond is arranged, and the Gln of No. 13 positions is changed to Tyr.

Three (exendin-4 γ-Glu3,17,24) aminoacid sequence of The compounds of this invention is as follows:

X-His-Gly-γGlu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-γGlu-Ala-Val-Arg-Leu-Phe-Ile-γGlu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is respectively to be generated by the γ-carboxyl of Glu and the alpha-amino group of Gly that three peptide bonds are arranged, the γ-carboxyl of Glu and the alpha-amino group of Ala, and the γ-carboxyl of Glu and the alpha-amino group of Trp generate.

Four (exendin-4 Tyr13, γ-Glu3,17,24) aminoacid sequence of The compounds of this invention is as follows:

X-His-Gly-γGlu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Glu-γGlu-Ala-Val-Arg-Leu-Phe-Ile-γGlu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is respectively to be generated by the γ-carboxyl of Glu and the alpha-amino group of Gly that three peptide bonds are arranged, the γ-carboxyl of Glu and the alpha-amino group of Ala, and the γ-carboxyl of Glu and the alpha-amino group of Trp generate, and the Gln of No. 13 positions is changed to Tyr.

Five (exendin-4 γ-Glu3,17) aminoacid sequence of The compounds of this invention is as follows:

X-His-Gly-γGlu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-γGlu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is respectively to be generated by the γ-carboxyl of Glu and the alpha-amino group of Gly that two peptide bonds are arranged, and the γ-carboxyl of Glu and the alpha-amino group of Ala generate.

Six (exendin-4 Tyr13, γ-Glu3,17) aminoacid sequence of The compounds of this invention is as follows:

X-His-Gly-γGlu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Glu-γGlu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is respectively to be generated by the γ-carboxyl of Glu and the alpha-amino group of Gly that two peptide bonds are arranged, and the γ-carboxyl of Glu and the alpha-amino group of Ala generate, and the Gln of No. 13 positions is changed to Tyr.

Seven (exendin-4 γ-Glu17,24) aminoacid sequence of The compounds of this invention is as follows:

X-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-γGlu-Ala-Val-Arg-Leu-Phe-Ile-γGlu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is respectively by the γ-carboxyl of Glu and the alpha-amino group of Ala that three peptide bonds are arranged, and the γ-carboxyl of Glu and the alpha-amino group of Trp generate.

Eight (exendin-4 Tyr13, γ-Glu17,24) aminoacid sequence of The compounds of this invention is as follows:

X-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Glu-γGlu-Ala-Val-Arg-Leu-Phe-Ile-γGlu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is respectively by the γ-carboxyl of Glu and the alpha-amino group of Ala that three peptide bonds are arranged, and the γ-carboxyl of Glu and the alpha-amino group of Trp generate, and the Gln of No. 13 positions is changed to Tyr.

Nine (exendin-4 γ-Glu3,24) aminoacid sequence of The compounds of this invention is as follows:

X-His-Gly-γGlu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-γGlu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is respectively to be generated by the γ-carboxyl of Glu and the alpha-amino group of Gly that three peptide bonds are arranged, and the γ-carboxyl of Glu and the alpha-amino group of Trp generate.

Ten (exendin-4 Tyr13, γ-Glu3,24) aminoacid sequence of The compounds of this invention is as follows:

X-His-Gly-γGlu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-γGlu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is respectively to be generated by the γ-carboxyl of Glu and the alpha-amino group of Gly that three peptide bonds are arranged, and the γ-carboxyl of Glu and the alpha-amino group of Trp generate, and the Gln of No. 13 positions is changed to Tyr.

The compounds of this invention 11 (exendin-4 γ-Glu3) aminoacid sequence is as follows:

X-His-Gly-γGlu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is to be generated by the γ-carboxyl of Glu and the alpha-amino group of Gly that a peptide bond is arranged.

The compounds of this invention 12 (exendin-4 Tyr13, γ-Glu3) aminoacid sequence is as follows:

X-His-Gly-γGlu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is to be generated by the γ-carboxyl of Glu and the alpha-amino group of Gly that a peptide bond is arranged.The Gln of No. 13 positions is changed to Tyr.

The compounds of this invention 13 (exendin-4 γ-Glu17) aminoacid sequence is as follows:

X-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-γGlu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is to be generated by the γ-carboxyl of Glu and the alpha-amino group of Ala that a peptide bond is arranged.

The compounds of this invention 14 (exendin-4 Tyr13, γ-Glu17) aminoacid sequence is as follows:

X-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Glu-γGlu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is to be generated by the γ-carboxyl of Glu and the alpha-amino group of Ala that a peptide bond is arranged, and the Gln of No. 13 positions is changed to Tyr.

The compounds of this invention 15 (exendin-4 γ-Glu24) aminoacid sequence is as follows:

X-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-γGlu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is to be generated by the γ-carboxyl of Glu and the alpha-amino group of Trp that a peptide bond is arranged.

The compounds of this invention 16 (exendin-4 Tyrl3, γ-Glu24) aminoacid sequence is as follows:

X-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-γGlu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

It is to be generated by the γ-carboxyl of Glu and the alpha-amino group of Trp that a peptide bond is arranged, and the Gln of No. 13 positions is changed to Tyr.

Preferred compound of the present invention also comprises (X is ethanoyl or hydrogen):

17 of The compounds of this invention:

X-His-Gly-βAsp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

18 of The compounds of this invention:

X-His-Gly-γGlu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Asp-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

19 of The compounds of this invention:

X-His-Gly-Glu-βAla-Thr-Phe-Ser-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

20 of The compounds of this invention:

X-His-Gly-βAsp-Gly-Thr-Phe-Ser-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

21 of The compounds of this invention:

X-His-Gly-γGlu-βAla-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

22 of The compounds of this invention:

X-His-Gly-βAsp-Gly-Thr-βPhe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

23 of The compounds of this invention:

X-His-Gly-Glu-Gly-Thr-βPhe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

24 of The compounds of this invention:

X-His-Gly-Glu-Gly-Thr-βPhe-Ser-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

25 of The compounds of this invention:

X-His-Gly-βAsp-Gly-ThrβPhe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

26 of The compounds of this invention:

X-His-Gly-Glu-βAla-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

Among the present invention the preparation method of activity isomerism peptide with those skilled in the art the common Fmoc solid phase method of peptide synthesis synthetic.

Raw material and reagent: connecing peptide resin is Rink Amide mbha resin.Amino acid derivative is

Fmoc-Ser(But)-OH、Fmoc-Pro-oH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Arg(pbf)-OH、Fmoc-Val-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Thr(But)-OH、Fmoc-His(Trt)-OH、Fmoc-Asp-OtBu、Fmoc-Glu-OtBu、Fmoc-β-Ala-OH、Fmoc-β-Phe-OH。

The linked reaction condensing agent is benzene a pair of horses going side by side triazole-1-oxygen-three (dimethylamino) phosphorus hexafluorophosphate (BOP), 1-hydroxyl benzotriazole (HOBt), N-methylmorpholine (NMM).Deprotection agent is 20% piperidines/DMF solution.Cutting reagent is a trifluoroacetic acid.

Resin is used DMF swelling 30 minutes, and deprotection is 20 minutes then, washs with DMF; add amino acid derivative and condensing agent in the reactor, room temperature reaction 2 hours, washing; deprotection adds amino acid derivative and condensing agent again, repeats to connecting all amino acid.Add cutting reagent in the reactor again, room temperature reaction 1 hour, after-filtration, filtrate is precipitated in ether, gets crude product.

The purifying of thick product: adopt high performance liquid chromatography to carry out purifying.Elutriant is a trifluoroacetic acid aqueous solution, acetonitrile.Flow velocity is 20ml/min, and the monitoring wavelength is 214nm.

Exendin-4 active isomer of the present invention and modifier thereof in blood plasma 37 ℃ carry out enzymolysis, analyze hydrolysate with HPLC then, and compare with normal configuration, they all have prolongation than the original structure transformation period.

The present invention also comprises Exendin-4 active isomer and the application of modifier aspect Remedies for diabetes thereof.Pharmacodynamic experiment to the exendin-4 active isomer shows to have the effect of remarkable reduction laboratory animal blood sugar.

Embodiment

The following examples can illustrate in greater detail the present invention, but do not limit the present invention in any form.

Below among each embodiment, compound 17, compound 12, compound 18, compound 19, compound 20, X is a hydrogen in the compound 24; All the other compounds are that middle X is an ethanoyl.

The solid phase synthesis of embodiment 1 compound

1, linked reaction

Take by weighing 15.4g Rink Amide mbha resin, in the reaction flask of the automatic DNA synthesizer DNA of packing into, each amino acid derivative, coupling reagent, DMF are respectively charged in each solvent bottle, set response procedures, begin to synthesize.

Temperature of reaction is 25 ℃, and the linked reaction time is 1 hour, and the deprotection time is 30 minutes, and it is 200ml that coupling and deprotection reaction solvent add charge, and washing the resin number of times is 3min/ time * 5 times.

Last amino acid coupling is washed resin 5 times with methyl alcohol after finishing, and feeds nitrogen drying 30 minutes.The detection of coupling and remove-insurance reaction

Whether finish the back at coupling each time and deprotection and suspend reaction, it is complete to take out a few grainy resin detection reaction.Detection reagent is divided into three parts, and reagent A is the anhydrous ether solution of triketohydrindene hydrate, and reagent B is the ethanol solution of phenol, and reagent C is the pyridine solution of potassium cyanide.Resin number to be detected is put in the test tube, adds each two of mentioned reagent respectively, 120 ℃ of heating 5 minutes, if linked reaction is complete, then resin particle is a glassy yellow.If deprotection reaction is complete, then resin particle becomes blueness or red-purple.

2, deprotection and cut peptide-resin

The deprotection cutting reagent is that (92.5: 2.5: 2.5: 2.5), it was 300ml that the deprotection cutting reagent adds charge to TFA/EDT/ thioanisole/phenylmethylether, at N ₂Protection down, 25 ℃ of reactions 1 hour down, filter, collect filtrate.Resin cut 30 minutes again, merged to collect liquid.

3, precipitation

The precipitation reagent anhydrous diethyl ether 800ml that in reaction solution, adds precooling, precipitation is 20 minutes in-20 ℃, with 6000rpm centrifugal 8 minutes then, precipitation was washed secondary with the 100ml anhydrous diethyl ether again, vacuum-drying, weigh crude product 23.6g, slightly the peptide yield is 93.7%.

4, purification process

Exendin-4 active isomer crude product is mixed with the aqueous solution that concentration is 100mg/ml, sample introduction 10ml, and Mobile phase B from 28% to 35% gradient elution 70min collects the product peak.Acetonitrile is removed in the sample solution underpressure distillation of collecting, and vacuum freezedrying gets the pure product 3.05g of trifluoroacetic acid type Exendin-4 active isomer, and yield is 12.1%.

The mensuration of stability in the embodiment 2 external blood plasma

For measuring the vitro stability of each compound, we with sample in blood plasma 37 ℃ carry out enzymolysis, analyze hydrolysate with HPLC then, and compare with Exendin-4.

Method: 37 ℃ of water-bath activation of Freshman blood plasma 30 minutes, add sample again, the final concentration that makes sample is 50 μ g/ml, 37 ℃ of water-bath hydrolysis, every interval sampling in 1 minute kind 400 μ l add acetic acid 400 μ l termination reactions.Sample is asked and is calculated the transformation period with the concentration that HPLC detects hydrolysate.

Measurement result:

Sample

Transformation period (hour)

Exendin-4 compound one compound two compounds three compound Four Modernizations compounds five compounds six compounds seven compounds eight compounds nine compounds ten compounds 11 compounds 12 compounds 17 compounds 18 compounds 19 compounds 20 compounds 24

9.5 9.8 10.36 19.8 12.5 9.4 13.8 11.5 12.3 13.5 12.6 15.4 13.7 27.6 11.5 18.8 13.9 10.9

Conclusion: the vitro stability of each compound is significantly better than Exendin-4.

Half life determination experiment in embodiment 3 bodies

The Wistar rat, male and female half and half, body weight 230-280g, Changchun High-technology Medical Animal Experiment Research Center provides, conformity certification number: No. (2003) 004, lucky real kinoplaszm word.

Medicine: compound 17, compound 12, compound 19.

Method:

1, takes by weighing and tried peptide, use physiological saline solution.

2, healthy Wistar rat is 10, duration of test ad lib and drinking-water.With test-compound subcutaneous administration (0.1mg/kg), in administration 0,0.25,0.5,0.75,1,1.5,2,3,4,5,6 hour after the extracting vein blood 1ml of rat eye posterior vein place places the heparinization test tube, the centrifugal 10min of 15000rpm, separated plasma, 20 ℃ of preservations, to be measured.

3, sample filters with the good C18 solid-phase extraction column of activation, washes post twice again with water, removes the foreign protein in the blood plasma, with 90% acetonitrile solution, the 100 μ l that contain 0.01% trifluoroacetic acid sample is eluted then.Feed nitrogen in the sample and concentrate, the sample after concentrating is analyzed with HPLC-MS, and samples contg over time in the mensuration blood plasma.

Curve when drawing medicine with marker method is used Topfit2.2 computed in software sample in the intravital transformation period of rat.

Transformation period in each chemical combination object

Sample	Transformation period (hour)
		Exendin-4 compound 17 compounds 12 compounds 19	1.39±0.26 2.95±0.23 2.53±0.31 1.98±0.19

Conclusion: the body internal stability of each compound is significantly better than Exendin-4.

Embodiment 4 cell proliferation experiment

1, the preparation of cell suspension

Rat is put to death, open the abdominal cavity and take out pancreas, add Hanks liquid, the pancreas tissue is shredded adding collagenase vibration 5 minutes, clean the back with Hanks and add Krebs-Ringer bicarbonate damping fluid, the collection pancreas islet. the pancreas islet of collecting is washed 1 time with PBS, be incubated 20 minutes with the PBS salts solution in 37 degrees centigrade. then pancreas islet is blown and beaten into individual cells, cell is positioned in the ice bath, adds nutrient solution, treat that cell attachment can be used for experiment.

2, reaction system

In 96 orifice plates, every porocyte concentration is 10 with the β cell inoculation ⁷About, the medicine that adds optimal concentration was cultivated after 68 hours, added MTT again, continue to cultivate 4 hours. take out 96 orifice plates then, in 2500r centrifugal 5 minutes, abandon supernatant, every hole adds 100 microlitre dimethyl sulfoxide (DMSO), measures absorption value down in the 570nm wavelength, calculates proliferation rate.

Sample	SR (%)
		Exendin-4 compound one compound two compounds three compound Four Modernizations compounds five	60.32 59.87 61.02 74.07 70.53 62.13

Compound six compounds seven compounds eight compounds nine compounds ten compounds 11 compounds 12 compounds 17 compounds 18 compounds 19 compounds 20 compounds 24

69.87 65.23 70.84 59.96 71.25 67.22 63.56 77.98 68.56 67.21 66.26 60.88

Conclusion: the influence of each compound on cell proliferation and Exendin-4 are quite or be better than Exendin-4.

Embodiment 5 effect experiments

Each compound causes the hypoglycemic activity of diabetic mice to streptozocin (STZ), and to the influence of mouse islets β cell quantity.

1, test materials

Cleaning level mouse body weight 18～20g, male and female half and half are selected in this test for use.

2, test method

2.1 test grouping and administration

This test grouping situation is blank group, model control group, Exendin-4 group, each sample sets.The blank group gives water for injection; Model group gives STZ (50mg/kg); Exendin-4 and compound one～compound 24 give sample 50mgkg in the sample sets ^-1

Each group gave water for injection or sample continuous 10 days by above-mentioned dosage subcutaneous injection, and model group and sample sets are at beginning abdominal cavity filling in 5 days streptozocin.

2.2 observation index

In about 8 o'clock of the 1st～10 day, 12,15,18, the 21 day morning of test mouse tail vein is got blood respectively, carry out rapid blood sugar and measure.

After the off-test, put to death mouse and take out pancreas, weigh.After remainder pancreas is fixed in BouinShi liquid, carry out the specific stain of aldehyde-fuchsin β cell.The conventional dehydration of the pancreatic tissue that fixes embedding, section dewaxes to water, and with acidifying potassium permanganate liquid oxidation 5 minutes, 2% oxalic acid liquid bleaching 2～3 minutes, flowing water dashed 5 minutes, went into aldehyde-fuchsin liquid 15～30 minutes, ethanol dehydration, dimethylbenzene is transparent, the neutral gum sealing.The beta cell spy dyes back kytoplasm red-purple, writes down the positive β cell quantity in the complete pancreas islet on all stained of every mouse, averages as the cell counting of β in every mouse islets.

3, data processing

Data with Expression adopts the SPSS statistical software to carry out the t check, estimates significance with P＜0.05.

4, test-results

4.1 each sample is to the influence of blood glucose in diabetic mice

Behind each sample of injection, mouse blood sugar is obviously than diabetic model group mouse low (P＜0.05) continuously, and model group blood sugar after the 3rd day begins obvious rising (comparing P＜0.05 with the blank group), to off-test.Each sample sets began to play a role after treatment on the 4th day, obviously descended with model group comparison blood sugar, the results are shown in following table.

Time (my god)

1

2

3

4

5

6

7

The blank group

7.05 ±1.10

7.69 ±0.88

6.99 ±0.65

6.56 ±1.02

7.39 ±0.95

7.12 ±1.35

7.96 ±0.82

Model group

7.36 ±0.82

7.75 ±1.92

8.69 ±0.46

9.24 ±1.74

11.05 ±1.31

12.58 ±1.66

12.98 ±0.83

Exendin-4

6.36 ±0.58

7.91 ±0.74

7.58 ±0.62

8.01 ±1.42

8.55 ±0.50

9.82 ±1.07

9.66 ±1.0

Compound one

7.09 ±0.44

7.62 ±0.79

7.36 ±1.62

7.91 ±0.42

8.31 ±0.47

9.49 ±0.98

9.47 ±1.00

Compound two

7.36 ±0.23

7.62 ±0.87

7.96 ±1.62

8.54 ±1.02

8.62 ±1.50

9.63 ±1.06

9.85 ±1.0 ^*

Compound three

7.25 ±0.23

7.36 ±0.37

7.45 ±1.60

7.14 ±1.02

7.84 ±1.04

8.33 ±1.39

8.75 ±1.09

Compound four

7.52 ±0.21

7.49 ±0.35

7.65 ±0.37

7.74 ±1.47

8.14 ±1.15

8.52 ±1.51

8.91 ±1.83

Compound five

7.14 ±0.29

7.69 ±0.39

7.44 ±0.63

7.95 ±1.28

8.19 ±1.51

9.06 ±1.11

9.46 ±1.02

Compound six

6.96 ±0.48

7.54 ±0.84

7.25 ±0.84

8.02 ±1.53

8.24 ±0.47

9.57 ±1.04

9.47 ±1.02

Compound seven

7.36 ±0.62

7.31 ±0.48

7.98 ±0.65

8.67 ±1.54

8.86 ±0.54

9.76 ±1.45

9.55 ±1.43

Compound eight

7.87 ±0.45

7.36 ±0.87

7.97 ±0.46

7.54 ±1.45

8.09 ±1.02

8.23 ±1.43

8.34 ±1.32

Compound nine

6.94 ±0.87

7.06 ±0.56

7.47 ±0.47

8.09 ±1.5

8.34 ±0.42

9.06 ±1.55

9.22 ±1.02

Compound ten

7.23 ±0.43

7.38 ±0.85

7.47 ±0.36

7.55 ±1.46

7.64 ±1.0

7.92 ±1.42

8.14 ±1.76

Compound 11

7.38 ±0.68

7.53 ±0.63

7.33 ±0.25

7.49 ±1.09

7.45 ±1.09

8.79 ±1.04

9.09 ±1.66

Compound 12

7.24 ±1.01

7.52 ±0.21

7.47 ±0.84

7.85 ±1.44

8.77 ±1.84

9.20 ±1.34

9.22 ±1.32

Compound 17

7.01 ±0.36

7.36 ±0.41

7.39 ±0.88

7.74 ±1.47

7.84 ±1.1

8.01 ±1.22

8.25 ±1.19

Compound 18

7.66 ±0.33

7.24 ±0.82

7.66 ±0.97

8.25 ±0.99

8.88 ±0.46

9.95 ±1.2

9.38 ±1.24

Compound 19

7.09 ±0.68

7.24 ±0.45

7.32 ±0.58

7.37 ±1.32

7.48 ±1.54

8.85 ±1.44

9.22 ±1.74

Compound 20

7.03 ±0.36

7.32 ±0.68

7.57 ±1.08

7.28 ±0.89

8.25 ±0.57

9.0 ±1.28

9.35 ±1.54

Compound 24

7.35 ±0.77

7.62 ±0.88

7.87 ±0.96

8.43 ±1.11

8.42 ±1.34

9.57 ±1.37

9.20 ±1.09

Time (my god)

8

9

10

12

15

18

21

The blank group

7.67± 0.63

7.58 ±0.54

7.35 ±1.07

7.79 ±0.24

7.25 ±1.48

7.63 ±0.28

7.96 ±0.45

Model group

13.22. ±1.55

15.21. ±0.24

16.07. ±0.72

16.37. ±0.95

17.32. ±0.48

18.07 ±1.23

19.24. ±0.76

Exendin-4

10.04 ±1.83

12.47 ±1.85

12.06 ±1.44

13.28 ±1.23

13.54 ±2.00

15.87 ±2.01

15.69 ±1.50

Compound one

10.36 ±0.86

12.87 ±1.06

13.25 ±1.22

13.89 ±1.54

14.04 ±1.31

15.99 ±1.31

16.09 ±1.62

Compound two

10.54 ±0.69

12.08 ±1.45

12.76 ±1.57

13.21 ±1.03

13.77 ±1.07

15.53 ±1.61

15.82 ±1.33

Compound three

9.26 ±0.80

9.87± 1.43

10.76 ±1.62

12.08 ±1.49

12.35 ±1.49

13.71 ±1.01

14.06 ±1.38

Compound four

9.86 ±1.42

10.02 ±1.09

11.29 ±1.84

13.35 ±1.09

13.44 ±2.01

14.28 ±1.91

14.96 ±1.65

Compound five

9.99± 0.34

11.72 ±0.45

12.36 ±1.07

13.01 ±1.42

13.77 ±1.24

14.93 ±0.69

15.38 ±1.37

Compound six

10.08 ±1.23

10.36 ±1.19

11.07 ±1.48

12.95 ±1.13

13.18 ±2.31

13.97 ±1.84

14.56 ±0.76

Compound seven

10.29 ±1.31

11.35 ±1.45

12.07 ±1.32

12.93 ±1.71

13.23 ±1.14

14.62 ±1.39

15.08 ±1.28

Compound eight

8.79 ±1.24

9.32± 1.43

10.09 ±0.84

11.35 ±1.33

12.84 ±1.13

13.67 ±1.00

13.96 ±1.37

Compound nine

9.04± 1.35

10.97 ±1.37

11.86 ±1.38

13.02 ±1.82

13.48 ±1.02

14.78 ±1.19

15.21 ±1.34

Compound ten

8.96 ±1.80

9.33± 1.29

10.05 ±1.04

11.88 ±1.54

12.13 ±1.37

13.05 ±1.41

13.96 ±1.57

Compound 11

9.87 ±1.62

10.57 ±1.38

11.24 ±1.85

12.18 ±1.63

13.44 ±1.37

14.57 ±1.28

14.97 ±1.76

Compound 12

10.09

11.17

12.24

13.00

13.86

14.88

15.27

±1.34

±1.45

±1.71

±1.34

±1.54

±1.69

±1.19

Compound 17

8.96 ±1.81

9.07± 1.35

10.25 ±1.33

11.78 ±1.37

12.12 ±1.73

12.87 ±1.08

13.76 ±1.85

Compound 18

10.22 ±1.65

10.86 ±1.37

11.47 ±1.85

12.69 ±1.04

13.58 ±2.17

14.07 ±1.84

14.79 ±1.76

Compound 19

9.82 ±1.04

10.24 ±1.49

11.57 ±1.21

12.08 ±1.84

13.49 ±1.96

13.98 ±1.24

14.25 ±1.68

Compound 20

9.75 ±1.46

10.28 ±1.37

11.56 ±1.41

12.11 ±1.34

13.85 ±1.73

14.47 ±1.68

14.55 ±1.49

Compound 24

9.85± 1.69

11.98 ±1.35

12.78 ±1.65

13.64 ±1.23

14.28 ±1.37

15.31 ±1.46

15.67 ±1.42

Compare P＜0.05 with model group

4.2 pancreas weight

After putting to death animal, weighing pancreas weight is calculated pancreas weight/body weight.The result shows that model group animal pancreatic weight/weight index obviously descends, and sample treatment group pancreas weight/weight index and model group increase more to some extent, the results are shown in following table.

Sample is to the influence of diabetic mice pancreas weight

Group

Number of animals (only)

Pancreas weight/body weight (mg/g)

Blank group model group Exendin-4 group compound one compound two compounds three compound Four Modernizations compounds five compounds six compounds seven compounds eight compounds nine compounds ten compounds 11

10 10 10 10 10 10 10 10 10 10 10 10 10 10

2.45±0.21 1.26±0.52 2.09±0.33 2.32±0.25 2.19±0.38 1.86±0.47 1.96±0.38 2.11±0.54 2.19±0.43 2.04±0.47 1.78±0.52 1.85±0.31 2.14±0.25 2.00±0.18

Compound 12 compounds 17 compounds 18 compounds 19 compounds 20 compounds 24

10 10 10 10 10 10

2.24±0.33 2.06±0.41 1.82±0.39 2.02±.024 2.25±0.31 2.03±0.25

Compare P＜0.05 with model group

4.3 beta Cell of islet counting

The result of β cell shows in the counting pancreas islet, the β cell quantity that the model group pancreas islet includes secretory granules obviously descends than the blank group, after the treatment of Exendin-4 active isomer, the beta Cell of islet quantity that contains secretory granules the results are shown in following table obviously more than model group.

Each sample is to the influence of diabetic mice beta Cell of islet quantity

Group	Number of animals (only)	The beta Cell of islet counting
			Blank group model group Exendin-4 group compound one compound two compounds three compound Four Modernizations compounds five compounds six compounds seven compounds eight compounds nine compounds ten compounds 11 compounds 12 compounds 17 compounds 18 compounds 19 compounds 20 compounds 24	10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10	67.2±16.5 8.6±4.7 27.9±11.5 28.3±11.2 25.5±9.4 26.2±12.5 25.9±11.3 27.4±12.9 26.8±11.2 27.1±15.5 28.0±11.1 27.4±13.6 26.9±11.8 27.1±14.5 25.8±12.3 26.0±11.7 27.2±11.3 25.9±11.9 26.8±12.4 27.2±13.6

^*Compare P＜0.05 with model group

Can effectively control streptozocin inductive mouse blood sugar by each sample of this test subcutaneous injection increases, the blood sugar of sample sets mouse is starkly lower than model control group during the administration, and the blood sugar reducing function of Exendin-4 and active isomer thereof still continues the long period after drug withdrawal.

Each sample is to the reduction effect and promotion beta Cell of islet hyperplasia of blood glucose in diabetic mice, and it is relevant to increase beta Cell of islet quantity.Model control group mice pancreatic weight/weight index is lower than the normal control group in this research, but obviously increases through each sample treatment class index.Aldehyde-fuchsin specific stain result shows, each sample treatment group beta Cell of islet number showed increased.

The determination experiment of stability in the outer blood plasma of synthesis, half life determination experiment in the body, cell proliferation experiment and effect experiment draw: the compound of following aminoacid sequence has more pharmaceutical use.Be respectively:

Compound 17:

His-Gly-βAsp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂。

Compound 12:

His-Gly-γGlu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂。

Compound 19:

His-Gly-Glu-βAla-Thr-Phe-Ser-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂。

Claims

1. new active isomer of Exendin-4 is characterized in that its aminoacid sequence is:

X ₁-His-Gly-X ₂-Gly-Thr-Phe-Thr-Ser-X ₃-Leu-Ser-Lys-Tyr-Met-Glu-Glu-

Glu-Ala-Val-Arg-Leu-Phe-Ile-X ₄-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-

Gly-Ala-Pro-Pro-Pro-Ser-NH ₂；

Wherein: X ₁Be ethanoyl or hydrogen,

X ₂Be: Glu or γ-Glu or β-Asp;

X ₃Be: Asp or γ-Glu or β-Asp;

X ₄Be: Glu or γ-Glu;

At least comprise a kind of β or γ amino acid in its aminoacid sequence, its position is X ₂, X ₃, X ₄

2. the new active isomer of Exendin-4 as claimed in claim 1 is characterized in that its aminoacid sequence is:

His-Gly-βAsp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Glu-

Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-

Pro-Pro-Pro-Ser-NH ₂。

3. the new active isomer of Exendin-4 as claimed in claim 1 is characterized in that its aminoacid sequence is:

His-Gly-γGlu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Tyr-Met-Glu-Glu-

Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-

Pro-Pro-Pro-Ser-NH ₂。

4. the application in the medicine of preparation treatment diabetes as claim 1 or the new active isomer of 2 or 3 described Exendin-4.