RU2823623C1 - Exenatide analogue - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: present invention relates to biotechnology and can be used in medicine. Invention discloses a new peptide analogue of Exenatide. In comparison with the natural hormone GLP1 and exenatide, the analogue according to the present invention has an increased half-life of the subject.

EFFECT: invention can be used in treating diseases associated with disorders of biological function of hormone GLP-1, in particular in treating metabolic diseases, for example diabetes (type I or II), dyslipidemia, impaired glucose tolerance, etc.

6 cl, 4 tbl, 8 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of Chinese Patent Application No. 201910908468.3, filed with the National Intellectual Property Administration of China on September 25, 2019, entitled "ANALOGUE OF EXENATIDE", which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The present invention relates to an exenatide analogue and its use, and the analogue is a glucagon-like peptide-1 (GLP-1) analogue.

BACKGROUND OF THE ART

[0003] Among non-communicable diseases, diabetes ranks third in prevalence after cardiovascular and cerebrovascular diseases and tumors. The World Health Organization (WHO) predicts that by 2030, the number of people suffering from diabetes in the world will exceed 360 million, of which more than 90% will be patients with type II diabetes. GLP-1, a secretin secreted by intestinal L cells, has functions such as stimulating insulin secretion, inhibiting glucagon release, stimulating islet β-cell proliferation, inducing islet β-cell regeneration, preventing islet β-cell apoptosis, improving insulin sensitivity and increased glucose utilization, which plays an important role in the occurrence and development of type II diabetes mellitus. In patients with type II diabetes mellitus, the “incretin effect” is impaired, which mainly manifests itself in the fact that the increase in GLP-1 concentration after meals is less than in normal people, but the stimulation of insulin secretion and hypoglycemic effects are not significantly impaired. Therefore, GLP-1 can be used as an important target for the treatment of type II diabetes. In addition, GLP-1 function is dependent on glucose concentrations, and its hypoglycemic properties are the basis and guarantee of clinical safety, eliminating concerns that existing diabetes drugs and treatment regimens may cause severe hypoglycemia in patients. Thus, GLP-1 has broad application prospects in the field of diabetes treatment.

[0004] However, the clinical application of GLP-1 also faces enormous challenges. GLP-1 produced by the human body is very unstable and is easily broken down in the body by dipeptidyl peptidase IV (DPP-IV). GLP-1 has a short plasma lifetime, which limits its clinical use. In addition, many patients with type II diabetes do not respond well to daily injections, so the development of safe and effective GLP-1 analogues that can be administered once a week holds great promise.

SUMMARY OF THE INVENTION

[0005] The present invention provides an exenatide analogue and its use, and the analogue is a glucagon-like peptide-1 (GLP-1) analogue.

[0006] To achieve the above object, the present invention firstly provides a compound having structure I, a pharmaceutically acceptable salt thereof, a solvate thereof, a chelate thereof or a non-covalent complex thereof, a prodrug thereof or any mixture thereof.

AA1-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-AA2(R)-AA3

Structure I

[0007] AA1 in structure I is:

,

X ₁ and X ₂ are each independently selected from the group consisting of H,

CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , CH(CH ₂ CH ₃ ) ₂ , C(CH ₂ CH ₃ ) ₃ , CH(CH ₂ CH ₂ CH ₃ ) ₂ , C(CH ₂ CH ₂ CH ₃ ) ₃ , CH(CH(CH ₃ )) ₂ , C(CH(CH ₃ )) ₃ ,

CH ₂ CH ₃ , CH ₂ CH(CH ₃ ) ₂ , CH ₂ C(CH ₃ ) ₃ , CH ₂ CH(CH ₂ CH ₃ ) ₂ , CH ₂ C(CH ₂ CH ₃ ) ₃ , CH ₂ CH(CH ₂ CH ₂ CH ₃ ) ₂ , CH ₂ C(CH ₂ CH ₂ CH ₃ ) ₃ , CH ₂ CH(CH(CH ₃ )) ₂ , and CH ₂ C(CH(CH ₃ )) ₃ ;

[0008] AA2 from structure I is selected from the group consisting of Lys, Dah, Orn, Dab and Dap;

[0009] AA3 from structure I is NH ₂ or OH; And

[0010] R from structure I is HO ₂ C(CH ₂ ) _n1 CO-(γGlu) _n2 -(PEG _n3 (CH2) _n4 CO) _n5 -,

n1 represents an integer from 10 to 20,

n2 represents an integer from 1 to 5,

n3 represents an integer from 1 to 30,

n4 is an integer from 1 to 5, and

n5 represents an integer from 1 to 5.

[0011] The present invention also relates to a pharmaceutical composition containing a compound of the present invention, as well as the use of a pharmaceutical composition of the present invention in the manufacture of a medicament for treating a disease.

[0012] Preferably, the pharmaceutical composition is used in the manufacture of a medicament for the treatment of a disease, wherein the disease is selected from the group consisting of type II diabetes, impaired glucose tolerance, type I diabetes, obesity, hypertension, metabolic syndrome, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, coronary heart disease, cardiovascular disease, stroke, irritable bowel syndrome, dyspepsia, gastric ulcers, fibrotic liver disease, fibrotic pulmonary disease and combinations thereof.

[0013] Preferably, the pharmaceutical composition is used in the manufacture of a medicament for the treatment of type II diabetes with delayed efficacy and/or for the prevention of exacerbation of type II diabetes.

[0014] Preferably, the pharmaceutical composition is used in the manufacture of a medicament to reduce food intake, reduce β-cell apoptosis, enhance β-islet function, increase β-cell number, and/or restore β-cell glucose sensitivity.

[0015] The present invention also provides a method for regulating blood glucose levels in the body by administering the compound to a subject in need thereof.

[0016] More aspects of the present invention are described in more detail below, or some of them may be illustrated in embodiments of the present invention.

[0017] Unless otherwise indicated, the amounts of various components and reaction conditions used herein can be interpreted as “approximate” or “approximate” in any case. Accordingly, unless otherwise stated, the numerical parameters given below and in the claims are approximate parameters, and different numerical parameters may be obtained due to differences in standard errors in the respective experimental conditions.

[0018] When there is disagreement or doubt between the chemical structural formula and the chemical name of a compound herein, the chemical structural formula is used to accurately identify the compound. A compound described herein may contain one or more chiral centers and/or double bonds and the like, and may also exist as stereoisomers, including double bond isomers (such as geometric isomers), optically active enantiomers, or diastereomers. Accordingly, any chemical structure within the scope of this specification, whether part or all of a structure containing similar structures as defined above, includes all possible enantiomers and diastereomers of the compound, including any pure stereoisomers (such as pure geometric isomers, pure enantiomers or pure diastereomers) and any mixture of these isomers. These racemic and stereoisomeric mixtures can also be further separated into their constituent enantiomers or stereoisomers by those skilled in the art using various separation techniques or chiral molecular synthesis techniques.

[0019] Compounds having structure I include, but are not limited to, optical isomers, racemates, and/or mixtures of these compounds. In the above case, a pure enantiomer or diastereomer, such as an optical isomer, can be obtained by asymmetric synthesis or resolution of racemates. Resolution of racemates can be achieved by various methods, such as conventional recrystallization with a resolving agent or chromatography. In addition, compounds having structure I also include cis and/or trans isomers with double bonds.

[0020] The compound of the present invention includes, but is not limited to, compounds with structure I and all various pharmaceutically acceptable forms. The various pharmaceutically acceptable forms include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs of the foregoing, and any mixture of the foregoing.

[0021] The compound having structure I of the present invention has stable properties, is poorly degraded by dipeptidyl peptidase IV (DPP-IV) in the body, is a long-acting GLP-I analogue, and has a significant hypoglycemic effect.

DETAILED DESCRIPTION

[0022] The present invention discloses a glucagon-like peptide-1 (GLP-1) analog and its use, and those skilled in the art can study the contents of the present invention and appropriately improve the relevant parameters to achieve the objects of the invention. It should be emphasized that all such substitutions and modifications will be obvious to those skilled in the art and are considered to be included in the present invention. The method of the present invention has been described by way of preferred embodiments, and it is obvious that persons concerned can achieve the objects of the invention and practice the methods of the present invention through changes or appropriate modifications and combinations of the compound and production method described herein without deviating from the contents, essence and the scope of the present invention.

[0023] The names corresponding to the abbreviations used in the present invention are given in the following table:

Reduction Name Reduction Name Fmoc 9-fluorenyl-methoxycarbonyl OtBu Tert-butoxy tBu Tert-butyl Boc Tert-butoxycarbonyl Trt Trityl Pbf (2,3-dihydro-2,2,4,6,7-pentamethylbenzofuran-5-yl)sulfonyl Ala Alanin Leu Leucine Arg Arginine Lys Lysine Asn Asparagine Met Methionine Asp Aspartic acid Phe Phenylalanine Cys Cysteine Pro Proline Gln Glutamine Ser Serin Glu Glutamic acid Thr Threonine Gly Glycine Trp Tryptophan His Histidine Tyr Tyrosine Ile Isoleucine Val Valin Dap 2,3-diaminopropionic acid Dab 2,4-diaminobutyric acid Orn Ornithine Dah 2,7-diaminoheptanoic acid Dhser Dehydroxyserine Dhthr Dehydroxythreonine Dhval 2,3-didehydrovaline

Example 1: Getting connection 1

[0024] The production method includes: producing a peptide on a resin by a solid-phase polypeptide synthesis method, then cleaving the peptide from the resin to obtain a crude product, and finally purifying the crude product to obtain a pure product; wherein the step of producing a peptide on a resin by a solid-phase polypeptide synthesis method consists of sequentially adding a protected amino acid or fragment according to a predetermined sequence to a carrier resin using a solid-phase coupling reaction to produce a peptide on the resin.

In the above production method, the amount of Fmoc-protected amino acid or protected amino acid fragment is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total number of moles of the loaded resin.

[0026] In the above production method, the degree of substitution of the carrier resin is 0.2-1.0 mmol/g resin, preferably 0.3-0.5 mmol/g resin.

[0027] As a preferred embodiment of the present invention, the solid phase coupling synthesis method includes: deprotecting an Fmoc protected amino acid on a resin obtained in the previous step, which is then subjected to a coupling reaction with the next protected amino acid. Fmoc deprotection time is 10-60 minutes, preferably 15-25 minutes. The coupling reaction time is 60-300 minutes, preferably 100-140 minutes.

[0028] The coupling reaction requires the addition of a condensing reagent, and the condensing reagent is selected from the group consisting of DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 2-(7-aza- 1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate and O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate; preferably N,N-diisopropylcarbodiimide. The molar amount of the condensing reagent is 1.2-6 times, preferably 2.5-3.5 times the total number of moles of amino groups in the amino resin.

[0029] The coupling reaction requires the addition of an activating agent, and the activating agent is selected from the group consisting of 1-hydroxybenzotriazole and N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The amount of activating agent is 1.2-6 times, preferably 2.5-3.5 times the total number of moles of amino groups in the amino resin.

[0030] As a preferred embodiment of the present invention, the Fmoc deprotection reagent is a PIP/DMF (piperidine/N,N-dimethylformamide) mixed solution, the piperidine content of the mixed solution being 10-30% (V). The amount of Fmoc removing agent is 5-15 ml per gram of amino resin, preferably 8-12 ml per gram of amino resin.

[0031] Preferably, the peptide-containing resin is subjected to acid hydrolysis to cleave the peptide from the resin and remove the side chain protecting group, yielding a crude product.

[0032] In addition, preferably, the agent used for acid hydrolysis of the peptide on the resin is a mixture of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) and water, and the volume percentage of each component in the mixture is: TFA 80- 95%, EDT 1-10%, and the rest is water.

[0033] More preferably, the volume percentage of each component in the mixed solvent is: TFA 89-91%, EDT 4-6%, and the balance being water. The optimal volume percentage of each component in the mixed solvent is: TFA 90%, EDT 5%, and the balance being water.

[0034] The amount of acid hydrolyzing agent is 4-15 ml of acid hydrolyzing agent per gram of peptide on resin; preferably, 7-10 ml of acidic hydrolyzing agent per gram of peptide on resin is required.

The digestion time using an acid hydrolyzing agent is 1-6 hours at room temperature, preferably 3-4 hours.

In addition, the crude product is purified by high performance liquid chromatography and freeze-dried to obtain a pure product.

1. Peptide synthesis on resin

[0037] The protected amino acids shown in the table below were sequentially coupled to Rink's amide BHHA resin as a carrier resin by deprotecting Fmoc and coupling to produce a peptide on the resin. The protected amino acids used in this example are as follows:

The procedure for joining a joint stock company, No. Protected amino acid 1 Fmoc-Lys(Alloc) 2 Fmoc-Ser(tBu) 3 Fmoc-Pro 4 Fmoc-Pro 5 Fmoc-Pro 6 Fmoc-Ala 7 Fmoc-Gly 8 Fmoc-Ser(tBu) 9 Fmoc-Ser(tBu) 10 Fmoc-Pro eleven Fmoc-Gly 12 Fmoc-Gly 13 Fmoc-Asn(Trt) 14 Fmoc-Lys(Boc) 15 Fmoc-Leu 16 Fmoc-Trp(Boc) 17 Fmoc-Glu(OtBu) 18 Fmoc-Ile 19 Fmoc-Phe 20 Fmoc-Leu 21 Fmoc-Arg(pbf) 22 Fmoc-Val 23 Fmoc-Ala 24 Fmoc-Glu(OtBu) 25 Fmoc-Glu(OtBu) 26 Fmoc-Glu(OtBu) 27 Fmoc-Met 28 Fmoc-Gln(Trt) 29 Fmoc-Lys(Boc) thirty Fmoc-Ser(tBu) 31 Fmoc-Leu 32 Fmoc-Asp(OtBu) 33 Fmoc-Ser(tBu) 34 Fmoc-Thr(tBu) 35 Fmoc-Phe 36 Fmoc-Thr(tBu) 37 Fmoc-Gly 38 Fmoc-Glu(OtBu) 39 Fmoc-Dhthr 40 Boc-His(Trt) Side chain -1 Fmoc-AEEA Side chain -2 Fmoc-AEEA Side chain -3 Fmoc-γGlu-OtBu Side chain -4 Mono-tert-butyl octadecanedioate Note Dhthr: dehydroxythreonine

[0038] (1) Attaching the first protected amino acid in the main chain

[0039] 0.03 mol of the first protected amino acid and 0.03 mol of HOBt were dissolved in the appropriate amount of DMF. Then, 0.03 mol of DIC was slowly added to the solution of the protected amino acid in DMF while stirring, stirred, and reacted at room temperature for 30 minutes to obtain an activated solution of the protected amino acid for later use.

[0040] 0.01 mol of Rink's amide MBHA resin (degree of substitution was about 0.4 mmol/g) was deprotected with 20% PIP/DMF solution for 25 minutes, washed and filtered to obtain a deprotected resin. protecting group Fmoc.

[0041] The activated solution of the first protected amino acid was added to the deprotected Fmoc resin for a coupling reaction for 60-300 minutes, washed and filtered to obtain a resin containing the first protected amino acid.

[0042] (2) Linking protected amino acids 2 to 40 in the main chain

[0043] The above-mentioned corresponding protected amino acids 2 to 40 were sequentially coupled in the same manner as described above for linking the first protected amino acid in the main chain, obtaining a resin containing 40 amino acids in the main chain.

[0044] (3) Linking the first protected amino acid in the side chain

[0045] 0.03 mol of the protected 1st side chain amino acid and 0.03 mol of HOBt were dissolved in the appropriate amount of DMF. 0.03 mol DIC was slowly added to a solution of the protected amino acid in DMF with stirring, mixed and allowed to react at room temperature for 30 minutes to obtain an activated solution of the protected amino acid.

[0046] 2.5 mmol of tetrakistriphenylphosphine palladium and 25 mmol of phenylsilane were dissolved in an appropriate amount of dichloromethane, deprotected for 4 hours, washed and filtered to obtain an Alloc deprotected resin for subsequent use.

[0047] The activated solution of the protected 1st amino acid from the side chain was added to the deprotected Alloc resin for a coupling reaction for 60-300 minutes, washed and filtered to obtain a resin containing the protected 1st amino acid from the side chain.

[0048] (4) Linking to the 2nd to 4th protected amino acids in the side chain

[0049] The 2nd to 4th protected amino acids corresponding to the side chain and the monoprotected fatty acid were sequentially coupled in the same manner as described above for coupling the first protected amino acid in the backbone to form a peptide on the resin.

2. Obtaining the crude product

[0050] A release agent with a volume ratio of TFA:water:EDT=95:5:5 (10 ml release agent/g resin) was added to the above peptide resin, mixed uniformly, and the reaction was carried out under stirring at room temperature. temperature for 3 hours. The reaction mixture was filtered through a sand-core funnel, the filtrate was collected, and the resin was washed three times with a small amount of TFA. The filtrates were combined, concentrated under reduced pressure, precipitated by adding anhydrous ether, washed three times with anhydrous ether, the liquid was removed and the residue was dried to obtain the crude product as an off-white powder.

3. Obtaining a pure product

[0051] Water was added to the above crude product and stirred. The pH of the solution was adjusted to 8.0 with aqueous ammonia until the crude product was completely dissolved. The solution was filtered through a 0.45 μm mixed microporous filter membrane for subsequent purification.

[0052] Purification was performed using high performance liquid chromatography. The chromatographic column packing material for purification was C18 10 µm reverse phase chromatography silica gel. The mobile phase system was 0.1%TFA/water-0.1%TFA/acetonitrile. The flow rate in the 30mm*250mm chromatography column was 20 ml/min. A gradient system was used for elution and application was carried out in a purification cycle. The crude product solution was applied to a chromatography column and then eluted with the mobile phase. The eluate containing the main peak was collected and evaporated to remove acetonitrile, yielding a purified intermediate concentrate.

[0053] The purified intermediate concentrate was filtered through a 0.45 μm membrane for subsequent use. High performance liquid chromatography was used for salt exchange. The mobile phase system was 1% acetic acid in water/acetonitrile. The chromatographic column packing material for purification was C18 10 µm reverse phase chromatography silica gel. The flow rate of the 30mm*250mm chromatography column was 20 ml/min (the corresponding flow rate can be adjusted depending on the parameters of the chromatography column). The method of gradient elution and cyclic application of the sample was used. The sample was applied to a chromatography column and then eluted with the mobile phase. The eluates were collected and the spectrum was analyzed, observing the change in absorbance. The eluate containing the major salt exchange peak was collected and purity determined using analytical liquid chromatography. The eluate containing the major salt exchange peak was combined and concentrated under reduced pressure to give an aqueous-acetic solution of the pure product, which was then lyophilized to give 7.7 g of purified product with a purity of 95.8%, a total yield of 15.2% and molecular weight weighing 5056.2 (100% M+H).

Example 2: Getting connection 2

[0054] The preparation method was the same as in Example 1, and the following protected amino acids were used:

The procedure for joining a joint stock company, No. Protected amino acid 1 Fmoc-Lys(Alloc) 2 Fmoc-Ser(tBu) 3 Fmoc-Pro 4 Fmoc-Pro 5 Fmoc-Pro 6 Fmoc-Ala 7 Fmoc-Gly 8 Fmoc-Ser(tBu) 9 Fmoc-Ser(tBu) 10 Fmoc-Pro eleven Fmoc-Gly 12 Fmoc-Gly 13 Fmoc-Asn(Trt) 14 Fmoc-Lys(Boc) 15 Fmoc-Leu 16 Fmoc-Trp(Boc) 17 Fmoc-Glu(OtBu) 18 Fmoc-Ile 19 Fmoc-Phe 20 Fmoc-Leu 21 Fmoc-Arg(pbf) 22 Fmoc-Val 23 Fmoc-Ala 24 Fmoc-Glu(OtBu) 25 Fmoc-Glu(OtBu) 26 Fmoc-Glu(OtBu) 27 Fmoc-Met 28 Fmoc-Gln(Trt) 29 Fmoc-Lys(Boc) thirty Fmoc-Ser(tBu) 31 Fmoc-Leu 32 Fmoc-Asp(OtBu) 33 Fmoc-Ser(tBu) 34 Fmoc-Thr(tBu) 35 Fmoc-Phe 36 Fmoc-Thr(tBu) 37 Fmoc-Gly 38 Fmoc-Glu(OtBu) 39 Fmoc-Dhval 40 Boc-His(Trt) Side chain -1 Fmoc-AEEA Side chain -2 Fmoc-AEEA Side chain -3 Fmoc-γGlu-OtBu Side chain -4 Mono-tert-butyl octadecanedioate Note Dhval: 2,3-didehydrovaline

[0055] 6.2 g of purified product was obtained with a purity of 95.8%, an overall yield of 12.2% and a molecular weight of 5070.6 (100% M+H).

Example 3: Making Compound 3

[0056] The preparation method was the same as in Example 1, and the following protected amino acids were used:

The procedure for joining a joint stock company, No. Protected amino acid 1 Fmoc-Lys(Alloc) 2 Fmoc-Ser(tBu) 3 Fmoc-Pro 4 Fmoc-Pro 5 Fmoc-Pro 6 Fmoc-Ala 7 Fmoc-Gly 8 Fmoc-Ser(tBu) 9 Fmoc-Ser(tBu) 10 Fmoc-Pro eleven Fmoc-Gly 12 Fmoc-Gly 13 Fmoc-Asn(Trt) 14 Fmoc-Lys(Boc) 15 Fmoc-Leu 16 Fmoc-Trp(Boc) 17 Fmoc-Glu(OtBu) 18 Fmoc-Ile 19 Fmoc-Phe 20 Fmoc-Leu 21 Fmoc-Arg(pbf) 22 Fmoc-Val 23 Fmoc-Ala 24 Fmoc-Glu(OtBu) 25 Fmoc-Glu(OtBu) 26 Fmoc-Glu(OtBu) 27 Fmoc-Met 28 Fmoc-Gln(Trt) 29 Fmoc-Lys(Boc) thirty Fmoc-Ser(tBu) 31 Fmoc-Leu 32 Fmoc-Asp(OtBu) 33 Fmoc-Ser(tBu) 34 Fmoc-Thr(tBu) 35 Fmoc-Phe 36 Fmoc-Thr(tBu) 37 Fmoc-Gly 38 Fmoc-Glu(OtBu) 39 Fmoc-Dhthr 40 Boc-His(Trt) Side chain -1 Fmoc-PEG ₅ CH ₂ COOH Side chain -2 Fmoc-γGlu-OtBu Side chain -3 Mono-tert-butyl octadecanedioate Note Dhthr: dehydroxythreonine

[0057] 8.9 g of purified product was obtained with a purity of 98.5%, an overall yield of 17.6% and a molecular weight of 5043.2 (100% M+H).

Example 4: Making Compound 4

[0058] The preparation method was the same as in Example 1, and the following protected amino acids were used:

The procedure for joining a joint stock company, No. Protected amino acid 1 Fmoc-Lys(Alloc) 2 Fmoc-Ser(tBu) 3 Fmoc-Pro 4 Fmoc-Pro 5 Fmoc-Pro 6 Fmoc-Ala 7 Fmoc-Gly 8 Fmoc-Ser(tBu) 9 Fmoc-Ser(tBu) 10 Fmoc-Pro eleven Fmoc-Gly 12 Fmoc-Gly 13 Fmoc-Asn(Trt) 14 Fmoc-Lys(Boc) 15 Fmoc-Leu 16 Fmoc-Trp(Boc) 17 Fmoc-Glu(OtBu) 18 Fmoc-Ile 19 Fmoc-Phe 20 Fmoc-Leu 21 Fmoc-Arg(pbf) 22 Fmoc-Val 23 Fmoc-Ala 24 Fmoc-Glu(OtBu) 25 Fmoc-Glu(OtBu) 26 Fmoc-Glu(OtBu) 27 Fmoc-Met 28 Fmoc-Gln(Trt) 29 Fmoc-Lys(Boc) thirty Fmoc-Ser(tBu) 31 Fmoc-Leu 32 Fm oc-Asp(OtBu) 33 Fmoc-Ser(tBu) 34 Fmoc-Thr(tBu) 35 Fmoc-Phe 36 Fmoc-Thr(tBu) 37 Fmoc-Gly 38 Fmoc-Glu(OtBu) 39 Fmoc-Dhval 40 Boc-His(Trt) Side chain -1 Fmoc-PEG ₅ CH ₂ COOH Side chain -2 Fmoc-γGlu-OtBu Side chain -3 Mono-tert-butyl octadecanedioate Note Dhval: 2,3-didehydrovaline

[0059] 5.2 g of purified product was obtained with a purity of 96.3%, an overall yield of 10.3% and a molecular weight of 5057.6 (100% M+H).

Example 5: Getting compound 5

[0060] The preparation method was the same as in example 1, and the following protected amino acids were used:

The procedure for joining a joint stock company, No. Protected amino acid 1 Fmoc-Lys(Alloc) 2 Fmoc-Ser(tBu) 3 Fmoc-Pro 4 Fmoc-Pro 5 Fmoc-Pro 6 Fmoc-Ala 7 Fmoc-Gly 8 Fmoc-Ser(tBu) 9 Fmoc-Ser(tBu) 10 Fmoc-Pro eleven Fmoc-Gly 12 Fmoc-Gly 13 Fmoc-Asn(Trt) 14 Fmoc-Lys(Boc) 15 Fmoc-Leu 16 Fmoc-Trp(Boc) 17 Fmoc-Glu(OtBu) 18 Fmoc-Ile 19 Fmoc-Phe 20 Fmoc-Leu 21 Fmoc-Arg(pbf) 22 Fmoc-Val 23 Fmoc-Ala 24 Fmoc-Glu(OtBu) 25 Fmoc-Glu(OtBu) 26 Fmoc-Glu(OtBu) 27 Fmoc-Met 28 Fmoc-Gln(Trt) 29 Fmoc-Lys(Boc) thirty Fmoc-Ser(tBu) 31 Fmoc-Leu 32 Fm oc-Asp(OtBu) 33 Fmoc-Ser(tBu) 34 Fmoc-Thr(tBu) 35 Fmoc-Phe 36 Fmoc-Thr(tBu) 37 Fmoc-Gly 38 Fmoc-Glu(OtBu) 39 Fmoc-Dhthr 40 Boc-His(Trt) Side chain -1 Fmoc-PEG ₁₀ CH ₂ COOH Side chain -3 Fmoc-γGlu-OtBu Side chain -4 Mono-tert-butyl octadecanedioate Note Dhthr: dehydroxythreonine

[0061] 5.6 g of purified product was obtained with a purity of 97.6%, an overall yield of 10.6% and a molecular weight of 5263.8 (100% M+H).

Example 6: Making Compound 6

[0062] The preparation method was the same as in Example 1, and the following protected amino acids were used:

The procedure for joining a joint stock company, No. Protected amino acid 1 Fmoc-Lys(Alloc) 2 Fmoc-Ser(tBu) 3 Fmoc-Pro 4 Fmoc-Pro 5 Fmoc-Pro 6 Fmoc-Ala 7 Fmoc-Gly 8 Fmoc-Ser(tBu) 9 Fmoc-Ser(tBu) 10 Fmoc-Pro eleven Fmoc-Gly 12 Fmoc-Gly 13 Fmoc-Asn(Trt) 14 Fmoc-Lys(Boc) 15 Fmoc-Leu 16 Fmoc-Trp(Boc) 17 Fmoc-Glu(OtBu) 18 Fmoc-Ile 19 Fmoc-Phe 20 Fmoc-Leu 21 Fmoc-Arg(pbf) 22 Fmoc-Val 23 Fmoc-Ala 24 Fmoc-Glu(OtBu) 25 Fmoc-Glu(OtBu) 26 Fmoc-Glu(OtBu) 27 Fmoc-Met 28 Fmoc-Gln(Trt) 29 Fmoc-Lys(Boc) thirty Fmoc-Ser(tBu) 31 Fmoc-Leu 32 Fm oc-Asp(OtBu) 33 Fmoc-Ser(tBu) 34 Fmoc-Thr(tBu) 35 Fmoc-Phe 36 Fmoc-Thr(tBu) 37 Fmoc-Gly 38 Fmoc-Glu(OtBu) 39 Fmoc-Dhval 40 Boc-His(Trt) Side chain -1 Fmoc-PEG ₁₀ CH ₂ COOH Side chain -2 Fmoc-γGlu-OtBu Side chain -3 Mono-tert-butyl octadecanedioate Note Dhval: 2,3-didehydrovaline

[0063] 4.5 g of purified product was obtained with a purity of 97.1%, an overall yield of 8.5% and a molecular weight of 5277.6 (100% M+H).

Example 7: Activity Detection

1. Method

[0064] GLP-1R, primarily present on the surface of islet β cells, is a G protein-coupled receptor (GPCR). When stimulated with a specific agonist, GLP-1R can activate the intracellular adenylate cyclase pathway, increase cAMP levels and ultimately lead to the production and release of insulin. When a stable cell line expressing GLP-1R is stimulated with the test agonist, the level of intracellular cAMP will rapidly increase, and the relative light units (RLU) after cell stimulation at each dose are determined by chemiluminescence to calculate the EC ₅₀ of the agonist. This activity determination method is commonly used to analyze the activity of GLP-1 receptor agonists in China and around the world.

[0065] The stable cell line CHO-K1 expressing GLP-1R was used in the experiment. Cells were stimulated with different concentrations of agonist, relative light units were measured after cells were stimulated with each dose, and the biological activity of the agonist was obtained.

2. Results

[0066] The results are shown in the table below.

Connection, no. Biological activity (%) Connection 1 52.64 Connection 2 18.14 Connection 3 82.92 Connection 4 20.25 Connection 5 46.48 Connection 6 14.51

Example 8 Preliminary determination of pharmacokinetic properties

[0067] Each compound was tested in two administration groups. SD rats were used, 4 males and 4 females in each group, for a total of 8 rats.

[0068] Tail vein intravenous injection group: dose was 1 mg/kg. Blood samples were collected from the ophthalmic vein of rats before administration (0 h) and 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 96 h and 144 h after administration. Plasma samples were collected after centrifugation.

[0069] Subcutaneous administration group: the dose was 1 mg/kg. Blood samples were collected from the ophthalmic vein of rats before administration (0 h) and 1 h, 2 h, 3 h, 4 h, 8 h, 24 h, 48 h, 96 h and 144 h after administration. Plasma samples were collected after centrifugation.

[0070] Concentrations of the corresponding compounds in SD rat plasma samples were determined using LC/MS. After intravenous and subcutaneous administration, the half-life of the compounds in SD rats administered the compound subcutaneously (SC) is given in the following table:

Connection, no. t _1/2 (h) Connection 1 8.9 Connection 2 8.4 Connection 3 8.7 Connection 4 8.9 Connection 5 10.5 Connection 6 10.1

Claims (18)

1. Exenatide analogue for agonistic stimulation of the glucagon-like peptide-1 (GLP-1) receptor, having structure I: AA1-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-AA2(R)-AA3 Structure I, where AA1 in structure I is: , X ₁ and X ₂ are each independently selected from the group consisting of H, CH ₃ , CH(CH ₃ ) ₂ , C(CH ₃ ) ₃ , CH(CH ₂ CH ₃ ) ₂ , C(CH ₂ CH ₃ ) ₃ , CH(CH ₂ CH ₂ CH ₃ ) ₂ , C(CH ₂ CH ₂ CH ₃ ) ₃ , CH(CH(CH ₃ )) ₂ , C(CH(CH ₃ )) ₃ , CH ₂ CH ₃ , CH ₂ CH(CH ₃ ) ₂ , CH ₂ C(CH ₃ ) ₃ , CH ₂ CH(CH ₂ CH ₃ ) ₂ , CH ₂ C(CH ₂ CH ₃ ) ₃ , CH ₂ CH(CH ₂ CH ₂ CH ₃ ) ₂ , CH ₂ C(CH ₂ CH ₂ CH ₃ ) ₃ , CH ₂ CH(CH(CH ₃ )) ₂ and CH ₂ C(CH(CH ₃ )) ₃ ; AA2 from structure I is selected from the group consisting of Lys, Dah, Orn, Dab and Dap; AA3 from structure I is NH ₂ or OH; And R from structure I is HO ₂ C(CH ₂ ) _n1 CO-(γGlu) _n2 -(PEG _n3 (CH2) _n4 CO) _n5 -, n1 represents an integer from 10 to 20, n2 represents an integer from 1 to 5, n3 represents an integer from 1 to 30, n4 is an integer from 1 to 5, and n5 represents an integer between 1 and 5. 2. An exenatide analogue according to claim 1, which is a pharmaceutically acceptable salt thereof, a solvate thereof, or any mixture thereof. 3. Use of an exenatide analogue according to claim 1 or 2 for the treatment of a disease, where the disease is selected from the group consisting of type II diabetes, impaired glucose tolerance, type I diabetes, obesity, dyslipidemia and combinations thereof. 4. Use of an exenatide analogue according to claim 3 for the treatment of type II diabetes with delayed effectiveness and/or prevention of exacerbation of type II diabetes. 5. Use of an exenatide analogue according to claim 1 or 2 to reduce food consumption. 6. The use of an exenatide analogue according to claim 1 or 2 to reduce β-cell apoptosis, enhance the function of islet β-cells, increase the number of β-cells and/or restore the sensitivity of β-cells to glucose.