Double-agonist compound

Abstract

The invention relates to the field of medicine synthesis, and discloses a GCG-GLP-1 dual agonist compound. The GCG-GLP-1 dual-agonist compound provided by the invention is used for preparing a pharmaceutical composition for treating diseases, and the application of the pharmaceutical composition in preparing a medicine for treating at least one of the following diseases, the diseases include type II diabetes mellitus, impaired glucose tolerance, type I diabetes mellitus, obesity, hypertension, metabolic syndrome, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, coronary heart disease, cardiovascular disease, stroke, inflammatory bowel syndrome and / or dyspepsia or gastric ulcer, hepatic fibrosis disease and pulmonary fibrosis disease.

Description

Technical Field

[0001] The present invention relates to a GLP-1 / GCG dual agonist compound and its use. The compound is a dual agonist compound of glucagon (GCG) receptor and glucagon-like peptide-1 (GLP-1) receptor. Background Art

[0002] GLP-1 is a 37 amino acid peptide that stimulates insulin secretion, protects pancreatic beta cells, and inhibits glucagon secretion, gastric emptying, and food intake, leading to weight loss. GLP-1 is known as an incretin; incretin receptor signaling plays a key physiologically relevant role in glucose homeostasis. In normal physiology, GLP-1 is secreted from the intestine after a meal. These incretins enhance the physiological response to food, including satiety, insulin secretion, and nutrient disposal.

[0003] The most common side effects of GLP-1 analogs are the inability to achieve full glycemic control and weight loss, while GCG alone has very modest glucose-lowering ability in patients with type 2 diabetes. Native GLP-1 can be rapidly inactivated by the ubiquitous protease DPP IV and can therefore only be used for short-term metabolic control.

[0004] GCG, also known as glucagon, anti-insulin or insulin B, is a hormone secreted by the α cells of the pancreas of vertebrates along with insulin. It opposes insulin and plays a role in increasing blood sugar.

[0005] New studies have shown that GLP-1 / GCG receptor dual agonist compounds not only have better blood sugar control, but also have the effects of significantly reducing body weight and treating non-alcoholic fatty liver disease. The purpose of the present invention is to provide a GLP-1 / GCG receptor dual agonist compound with better activity. Summary of the invention

[0006] The present invention provides a GLP-1 / GCG dual agonist compound and its use. The compound is a dual agonist compound of glucagon-like peptide-1 (GLP-1) receptor and glucagon (GCG) receptor.

[0007] In one aspect, the present invention provides a compound represented by structural formula I: His-Aib-Gln-Gly-Thr-AA1-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys((CO(CH 2 ) n1 PEG n2 ) n3 -(AA2) n4-CO(CH 2 ) n5 -COOH)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AA3 Structural formula Ⅰ in: AA1 is selected from any one of Phe, αMePhe, and αMePhe(2F); AA2 is selected from any one of γGlu, δAad, εApm, and ζAsu; AA3 is selected from any one of amino and hydroxyl groups; n1=1-5, n2=1-30, n3=0 or =1-5, n4=0 or =1-5, n5=10-30;

[0008] Optionally, the GLP-1 / GCG dual agonist compound is characterized in that AA1 is selected as Phe, AA2 is selected as γGlu, AA3 is selected as amino, n1=1-5, n2=1-30, n3=0 or =1-5, n4=0 or =1-5, n5=10-30; preferably, AA1 is selected as Phe, AA2 is selected as γGlu, AA3 is selected as amino, n1=1, n2=5, n3=1, n4=1, n5=18;

[0009] Optionally, the GLP-1 / GCG dual agonist compound is characterized in that AA1 is selected as αMePhe, AA2 is selected as γGlu, AA3 is selected as amino, n1=1-5, n2=1-30, n3=0 or =1-5, n4=0 or =1-5, n5=10-30; preferably, AA1 is selected as αMePhe, AA2 is selected as γGlu, AA3 is selected as amino, n1=1, n2=5, n3=1, n4=1, n5=18;

[0010] Optionally, the GLP-1 / GCG dual agonist compound is characterized in that AA1 is selected as αMePhe, AA2 is selected as δAad, AA3 is selected as amino, n1=1-5, n2=1-30, n3=0 or =1-5, n4=0 or =1-5, n5=10-30; preferably, AA1 is selected as αMePhe, AA2 is selected as δAad, AA3 is selected as amino, n1=1, n2=5, n3=1, n4=1, n5=18;

[0011] Optionally, the GLP-1 / GCG dual agonist compound is characterized in that AA1 is selected as αMePhe (2F), AA2 is selected as γGlu, AA3 is selected as amino, n1=1-5, n2=1-30, n3=0 or =1-5, n4=0 or =1-5, n5=10-30; preferably, AA1 is selected as αMePhe (2F), AA2 is selected as γGlu, AA3 is selected as amino, n1=1, n2=5, n3=1, n4=1, n5=18;

[0012] Optionally, the GLP-1 / GCG dual agonist compound is characterized in that AA1 is selected as αMePhe (2F), AA2 is selected as δAad, AA3 is selected as amino, n1=1-5, n2=1-30, n3=0 or =1-5, n4=0 or =1-5, n5=10-30; preferably, AA1 is selected as αMePhe (2F), AA2 is selected as δAad, AA3 is selected as amino, n1=1, n2=5, n3=1, n4=1, n5=18;

[0013] Optionally, the above-mentioned GLP-1 / GCG dual agonist compound is characterized in that the GLP-1 / GCG dual agonist compound comprises a pharmaceutically acceptable salt, chelate or non-covalent complex formed by the compound and a precursor of the compound, or any mixture of the above forms.

[0014] A second aspect of an embodiment of the present invention provides a use of the GLP-1 / GCG dual agonist compound as described in any one of the first aspects above in the preparation of a pharmaceutical composition for treating a disease.

[0015] Optionally, the pharmaceutical composition for treating a disease is characterized in that the pharmaceutical composition is used in the preparation of a medicament for treating at least one of the following diseases, the diseases comprising 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, inflammatory bowel syndrome and / or indigestion or gastric ulcer, liver fibrosis and pulmonary fibrosis.

[0016] Preferably, the pharmaceutical composition is used in the preparation of a drug for treating type II diabetes mellitus with delayed efficacy and / or preventing the deterioration of type II diabetes mellitus.

[0017] Preferably, the pharmaceutical composition is used in the preparation of a drug for reducing food intake, reducing β-cell apoptosis, increasing pancreatic β-cell function, increasing β-cell mass and / or restoring glucose sensitivity to β-cells.

[0018] The present invention further provides a method for regulating blood sugar in vivo by administering the compound to a subject.

[0019] More contents involved in the present invention are described in detail below, or some of them can also be experienced in the embodiments of the present invention.

[0020] Unless otherwise indicated, the quantities of different components and reaction conditions used herein may be interpreted as "roughly" or "approximately" in any case. Accordingly, unless otherwise specified, the numerical parameters cited below and in the claims are approximate parameters, and different numerical parameters may be obtained under respective experimental conditions due to different standard errors.

[0021] In this article, when there is a disagreement or ambiguity between the chemical formula and the chemical name of a compound, the chemical formula is used to accurately define the compound. The compounds described herein may contain one or more chiral centers, and / or double bonds and structures such as these, and may also exist as stereoisomers, including double bond isomers (such as geometric isomers), optical enantiomers or diastereomers. Accordingly, any chemical structure within the scope of the description herein, whether it contains the above-mentioned similar structures in part or in its entirety, includes all possible enantiomers and diastereomers of the compound, including any single stereoisomer (such as a single geometric isomer, a single enantiomer or a single diastereomer) and any mixture of these isomers. These racemic isomers and mixtures of stereoisomers can also be further separated into their constituent enantiomers or stereoisomers by those skilled in the art using continuous separation techniques or chiral molecule synthesis methods.

[0022] The compounds of formula I include, but are not limited to, optical isomers, racemates and / or other mixtures of these compounds. In the above cases, single enantiomers or diastereomers, such as optically active isomers, can be obtained by asymmetric synthesis or racemate resolution. The resolution of the racemate can be achieved by different methods, such as conventional recrystallization with a resolving agent, or by chromatographic methods. In addition, the compounds of formula I also include cis and / or trans isomers with double bonds.

[0023] The compounds of the present invention include, but are not limited to, compounds of formula I and all of their pharmaceutically acceptable forms. The pharmaceutically acceptable forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above substances, and any mixtures of the above forms.

[0024] The compound shown in structural formula I provided by the present invention is stable in nature and is not easily degraded by dipeptidyl peptidase IV (DPP-IV) in the body. It is a GCG / GLP-I dual agonist analogue and has significant blood sugar and body weight reduction effects. DETAILED DESCRIPTION

[0025] The present invention discloses a GCG / GLP-1 analog and its use. Those skilled in the art can refer to the content of this article and appropriately improve the relevant parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the compounds and preparation methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0026] The Chinese names corresponding to the English abbreviations involved in the present invention are shown in the following table: Abbreviations Chinese name Abbreviations Chinese name Fmoc 9-Fluorenylmethoxycarbonyl oeLh tert-Butoxy tBu Tert-butyl Boc tert-Butyloxycarbonyl Trt Trityl Pbf (2,3-Dihydro-2,2,4,6,7-pentamethylbenzofuran-5-yl)sulfonyl Ala Alanine Leu Leucine Arg Arginine Lys Lysine Asn Asparagine Phe Phenylalanine Asp Aspartic acid Pro Proline Cys Cysteine Ser Serine Gln Glutamine Thr Threonine Glu Glutamate Trp Tryptophan Gly Glycine Tyr Tyrosine His Histidine Val Valine Ile Isoleucine Ada 2-Aminoadipic acid Aib GABA Apm 2-Aminopimelate 5-Ava 5-Aminovaleric acid Asu 2-aminosuberic acid

[0027] Example 1 Preparation of Compounds

[0028] The preparation method comprises: the starting resin is Rink Amide MBHA resin, the peptide resin is prepared by solid phase peptide synthesis method, the peptide resin is then subjected to acid hydrolysis to obtain a crude product, and finally the crude product is purified to obtain a pure product; wherein the step of preparing the peptide resin by solid phase peptide synthesis method is to sequentially access the corresponding protected amino acids or fragments in the polypeptide sequence on the carrier resin by solid phase coupling synthesis method to prepare the peptide resin.

[0029] In the above preparation method, the amount of the Fmoc-protected amino acid or protected amino acid fragment is 1.2 to 6 times the total molar number of the resin fed, preferably 2.5 to 3.5 times.

[0030] In the above preparation method, the substitution value of the carrier resin is 0.2-1.0 mmol / g resin, and the preferred substitution value is 0.3-0.5 mmol / g resin.

[0031] As a preferred embodiment of the present invention, the solid phase coupling synthesis method is: the protected amino acid-resin obtained in the previous step is deprotected from the Fmoc protecting group and then coupled with the next protected amino acid. The deprotection time of the Fmoc deprotection is 10 to 60 minutes, preferably 15 to 25 minutes. The coupling reaction time is 60 to 300 minutes, preferably 100 to 140 minutes.

[0032] The coupling reaction requires the addition of a condensation reagent, which is selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate or O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate; preferably N,N-diisopropylcarbodiimide. The molar amount of the condensation reagent is 1.2 to 6 times the total molar number of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0033] The coupling reaction requires the addition of an activation reagent, which is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The amount of the activation reagent is 1.2 to 6 times the total molar number of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0034] As a preferred embodiment of the present invention, the Fmoc-removing reagent is a PIP / DMF (piperidine / N,N-dimethylformamide) mixed solution, wherein the mixed solution contains 10-30% (V) piperidine. The amount of the Fmoc-removing reagent is 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.

[0035] Preferably, the peptide resin is subjected to acid hydrolysis to simultaneously remove the resin and the side chain protecting groups to obtain a crude product:

[0036] Further preferably, the acid hydrolysis agent used in the acid hydrolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) and water, and the volume ratio of the mixed solvent is: TFA is 80-95%, EDT is 1-10%, and the balance is water.

[0037] More preferably, the volume ratio of the mixed solvent is: TFA is 89-91%, EDT is 4-6%, and the balance is water. Optimally, the volume ratio of the mixed solvent is: TFA is 90%, EDT is 5%, and the balance is water.

[0038] The amount of the acid hydrolysis agent is 4 to 15 mL per gram of peptide resin; preferably, 7 to 10 mL per gram of peptide resin.

[0039] The time for lysis using an acid hydrolysis agent is 1 to 6 hours at room temperature, preferably 3 to 4 hours.

[0040] Furthermore, the crude product was purified by high performance liquid chromatography and freeze-dried to obtain a pure product.

[0041] 1. Synthesis of peptide resin

[0042] Rink Amide BHHA resin was used as a carrier resin, and the peptide resin was prepared by coupling with the protected amino acids corresponding to the polypeptide sequence in sequence through Fmoc removal and coupling reaction.

[0043] (1) Insertion of the first protected amino acid in the main chain

[0044] Take 3 mmol of the first protected amino acid and 3 mmol of HOBt, dissolve them in an appropriate amount of DMF; take another 3 mmol of DIC, slowly add it to the protected amino acid DMF solution under stirring, and react with stirring at room temperature for 30 minutes to obtain an activated protected amino acid solution for use.

[0045] Take 1 mmol of Rink amide MBHA resin (substitution value is about 0.4 mmol / g), use 20% PIP / DMF solution to deprotect for 25 minutes, wash and filter to obtain the resin without Fmoc.

[0046] The activated first protected amino acid solution is added to the Fmoc-free resin, and the coupling reaction is carried out for 60 to 300 minutes. The resin containing one protected amino acid is filtered and washed to obtain.

[0047] (2) Insertion of main chain protective amino acids

[0048] The same method as described above for inserting the first protected amino acid of the main chain is used to sequentially insert the protected amino acids corresponding to the polypeptide sequence to obtain a resin containing main chain amino acids.

[0049] (3) Insertion of the first protected amino acid in the side chain

[0050] Take 0.03 mol of the first protected amino acid of the side chain and 0.03 mol of HOBt, dissolve them in an appropriate amount of DMF; take another 0.03 mol of DIC, slowly add it to the protected amino acid DMF solution under stirring, and react with stirring at room temperature for 30 minutes to obtain an activated protected amino acid solution.

[0051] Take 2.5 mmol of tetrakistriphenylphosphine palladium and 25 mmol of phenylsilane, dissolve them in an appropriate amount of dichloromethane, deprotect for 4 hours, filter and wash, and obtain the de-Allocated resin for use.

[0052] The activated first side chain protected amino acid solution is added to the de-Allocated resin, and the coupling reaction is carried out for 60 to 300 minutes. The resin containing the first side chain protected amino acid is obtained by filtering and washing.

[0053] (4) Insertion of side chain protected amino acids

[0054] The same method as described above for inserting the first protected amino acid in the main chain was used to sequentially insert the corresponding protected amino acid and the mono-protected fatty acid in the side chain to obtain a peptide resin.

[0055] 2. Preparation of crude product

[0056] Take the above peptide resin, add a cleavage reagent with a volume ratio of TFA: water: EDT = 95:5:5 (cleavage reagent 10 mL / g resin), stir evenly, and react at room temperature for 3 hours. Filter the reaction mixture using a sand core funnel, collect the filtrate, wash the resin with a small amount of TFA 3 times, combine the filtrate and concentrate under reduced pressure, add anhydrous ether to precipitate, wash the precipitate with anhydrous ether 3 times, and dry to obtain an off-white powder as the crude product.

[0057] 3. Preparation of pure product

[0058] Take the above crude product, add water and stir, adjust the pH to 8.0 with ammonia water until it is completely dissolved, filter with a 0.45μm filter membrane and set aside.

[0059] The product was purified by high performance liquid chromatography. The chromatographic filler used for purification was 10 μm reverse phase C18. The mobile phase system was 0.1% TFA / water solution-0.1% TFA / acetonitrile solution. The flow rate of the 30 mm*250 mm chromatographic column was 20 mL / min. A gradient system was used for elution and cyclic injection purification. The crude product solution was loaded on the chromatographic column, the mobile phase elution was started, the main peak was collected and the acetonitrile was evaporated to obtain a purified intermediate concentrate.

[0060] The purified intermediate concentrate was filtered through a 0.45 μm filter membrane for later use, and the salt was exchanged by high performance liquid chromatography. The mobile phase system was 1% acetic acid / water solution-acetonitrile, the chromatographic filler for purification was 10 μm reverse phase C18, and the flow rate of the 30 mm*250 mm chromatographic column was 20 mL / min (the corresponding flow rate can be adjusted according to the different specifications of the chromatographic column); gradient elution and cyclic loading method were used, the sample was loaded on the chromatographic column, the mobile phase elution was started, the spectrum was collected, the change of absorbance was observed, the main peak of salt exchange was collected and the purity was detected by analytical liquid phase, the main peak solutions of salt exchange were combined, concentrated under reduced pressure, and the pure acetic acid aqueous solution was obtained, and the pure peptide was freeze-dried to obtain pure peptide.

[0061] The following compounds were synthesized using the above method: Compound Sequence structure Reference substance OXM3 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys( AEEA-AEEA-γGlu-20 alkanedioic acid)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-NH2 Compound 1 <![CDATA[His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(COCH 2 PEG 5 -γGlu-20 alkane diacid)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 > Compound 2 <![CDATA[His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(COCH 2 PEG 5 -δAad-20-(5-Ava)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 ]]> Compound 3 <![CDATA[His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(COCH 2 PEG 5 -εApm-20μm-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 ]]> Compound 4 <![CDATA[His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(COCH 2 PEG 5 -ζAsu-20 alkane diacid)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 > Compound 5 <![CDATA[His-Aib-Gln-Gly-Thr-αMePhe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(PEG 5 CH 2 CO-γGlu-20-amino-2-amino)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-NH 2 ]]> Compound 6 <![CDATA[His-Aib-Gln-Gly-Thr-αMePhe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(COCH 2 PEG 5 -δAad-20-(5-Ava)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 ]]> Compound 7 <![CDATA[His-Aib-Gln-Gly-Thr-αMePhe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(COCH 2 PEG 5 -εApm-20μm-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 ]]> Compound 8 <![CDATA[His-Aib-Gln-Gly-Thr-αMePhe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(COCH 2 PEG 5 -ζAsu-20-alkanedioic acid)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 > Compound 9 <![CDATA[His-Aib-Gln-Gly-Thr-αMePhe(2F)-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(PEG 5 CH 2 CO-γGlu-20-amino-2-amino)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-NH 2 ]]> Compound 10 <![CDATA[His-Aib-Gln-Gly-Thr-αMePhe(2F)-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(COCH 2 PEG 5 -δAad-20-(5-Ava)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 ]]> Compound 1 <![CDATA[His-Aib-Gln-Gly-Thr-αMePhe(2F)-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(COCH 2 PEG 5 -εApm-20μm-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 ]]> Compound 12 <![CDATA[His-Aib-Gln-Gly-Thr-αMePhe(2F)-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(COCH 2 PEG 5 -ζAsu-20-alkanedioic acid)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 > Compound 13 <![CDATA[His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(AEEA-AEEA-γGlu-20 alkane diacid)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 <!-- 6 -->]]> Compound 14 <![CDATA[His-Aib-Gln-Gly-Thr-αMePhe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(AEEA-AEEA-γGlu-20-alkanedioic acid)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 > Compound 15 <![CDATA[His-Aib-Gln-Gly-Thr-αMePhe(2F)-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys(AEEA-AEEA-γGlu-20 alkane diacid)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 >

[0062] Example 2 Activity determination

[0063] 1. GLP-1 activity determination method

[0064] GLP-1R, under the stimulation of its specific agonist, can activate the intracellular adenylate cyclase pathway, increase the cAMP level, and ultimately lead to the production and release of insulin. By stimulating the cell line stably transfected with GLP-1R with the test substance, the intracellular cAMP level of the cell increases rapidly. The relative light unit (RLU) after each dose of cell stimulation is measured by chemiluminescence, and then the EC50 of the agonist is calculated. This activity determination method is currently a common GLP-1 receptor agonist activity detection method at home and abroad.

[0065] The CHO-K1 cell line stably expressing GLP-1R was used to stimulate the stably transfected cells with different concentrations of agonists. The EC of the agonist was calculated by measuring the relative light units after stimulation at each dose. 50 value.

[0066] 2. GCG activity determination method

[0067] GCG-R, under the stimulation of its specific agonist, can activate the intracellular adenylate cyclase pathway, increase the cAMP level, and ultimately lead to the production and release of insulin. By stimulating the cell line stably transfected with GCG-R with the test substance, the intracellular cAMP level of the cell increases rapidly. The relative light unit (RLU) after each dose of cell stimulation is measured by chemiluminescence method, and then the EC50 of the agonist is calculated. This activity determination method is currently a common GCG receptor agonist activity detection method at home and abroad.

[0068] The CHO-K1 cell line stably expressing GCG-R was used to stimulate the stably transfected cells with different concentrations of agonists. The EC of the agonist was calculated by measuring the relative light units after stimulation at each dose. 50 value.

[0069] 3. Measurement results Compound <![CDATA[GLP-1 activity

EC 50 (pmol)

EC 50 (nmol)

[0070] The experimental results show that while maintaining the CGC activity, the GLP-1 activity of the compounds in the examples is greatly improved.

[0071] Example 3 Determination of preliminary pharmacokinetic properties

[0072] Among the above compounds, the principles of their long-acting modification are basically the same, so the two compounds with the best activity were selected for preliminary pharmacokinetic property verification tests.

[0073] The test animals were male cynomolgus monkeys. Two cynomolgus monkeys were used for each compound, and the dose was 0.2 mg / kg. Blood was collected from the vein before drug administration (0h) and 1h, 2h, 3h, 4h, 8h, 12h, 18h, 24h, 48h, 96h, 144h, and 168h after administration. The plasma samples were separated by centrifugation, and the blood drug concentrations of the corresponding compounds in the plasma samples were determined by liquid chromatography-mass spectrometry. The half-life of the compound after subcutaneous (SC) administration is shown in the table below: Compound <![CDATA[t 1 / 2 (h)]]> Compound 6 124.6 Compound 10 136.1

[0074] The preliminary pharmacokinetic properties experimental results of cynomolgus monkeys showed that the subcutaneous administration half-life of compound 6 and compound 10 was greater than 100 hours, which fully met the requirements of long-acting drug delivery.

Claims

1. A GLP-1 / GCG dual agonist compound having structural formula I: His-Aib-Gln-Gly-Thr-AA1-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Lys-Ala-Lys((CO(CH2) n1 PEG n2 ) n3 -(AA2) n4 -CO(CH2) n5 -COOH)-Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AA3 Structural formula Ⅰ in: AA1 is selected from any one of Phe, αMePhe, and αMePhe(2F); AA2 is selected from any one of γGlu, δAad, εApm, and ζAsu; AA3 is selected from any one of amino and hydroxyl groups; n1=1-5, n2=1-30, n3=0 or =1-5, n4=0 or =1-5, n5=10-30.

2. The GLP-1 / GCG dual agonist compound according to claim 1, characterized in that The AA1 is selected as Phe, AA2 is selected as γGlu, AA3 is selected as amino, n1=1-5, n2=1-30, n3=0 or =1-5, n4=0 or =1-5, and n5=10-30.

3. The GLP-1 / GCG dual agonist compound according to claim 2, characterized in that: The AA1 is selected as Phe, AA2 is selected as γGlu, AA3 is selected as amino, n1=1, n2=5, n3=1, n4=1, and n5=18.

4. The GLP-1 / GCG dual agonist compound according to claim 1, characterized in that The AA1 is selected as αMePhe, AA2 is selected as γGlu, AA3 is selected as amino, n1=1-5, n2=1-30, n3=0 or =1-5, n4=0 or =1-5, and n5=10-30.

5. The GLP-1 / GCG dual agonist compound according to claim 4, characterized in that: The AA1 is selected as αMePhe, the AA2 is selected as γGlu, the AA3 is selected as amino, n1=1, n2=5, n3=1, n4=1, and n5=18.

6. The GLP-1 / GCG dual agonist compound according to claim 1, characterized in that The AA1 is selected as αMePhe, AA2 is selected as δAad, AA3 is selected as amino, n1=1-5, n2=1-30, n3=0 or =1-5, n4=0 or =1-5, and n5=10-30.

7. The GLP-1 / GCG dual agonist compound according to claim 6, characterized in that: The AA1 is selected as αMePhe, AA2 is selected as δAad, AA3 is selected as amino, n1=1, n2=5, n3=1, n4=1, and n5=18.

8. The GLP-1 / GCG dual agonist compound according to claim 1, characterized in that: The AA1 is selected as αMePhe (2F), AA2 is selected as γGlu, AA3 is selected as amino, n1=1-5, n2=1-30, n3=0 or =1-5, n4=0 or =1-5, and n5=10-30.

9. The GLP-1 / GCG dual agonist compound according to claim 8, characterized in that: The AA1 is selected as αMePhe(2F), the AA2 is selected as γGlu, the AA3 is selected as amino, n1=1, n2=5, n3=1, n4=1, and n5=18.

10. The GLP-1 / GCG dual agonist compound according to claim 1, characterized in that: The AA1 is selected as αMePhe(2F), AA2 is selected as δAad, AA3 is selected as amino, n1=1-5, n2=1-30, n3=0 or =1-5, n4=0 or =1-5, and n5=10-30.

11. The GLP-1 / GCG dual agonist compound according to claim 10, characterized in that: The AA1 is selected as αMePhe(2F), AA2 is selected as δAad, AA3 is selected as amino, n1=1, n2=5, n3=1, n4=1, and n5=18.

12. The GLP-1 / GCG dual agonist compound according to claims 1-11, comprising a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex of the compound, a prodrug based on the compound, or any mixture of the above forms.

13. The GLP-1 / GCG dual agonist compound according to claims 1-12, for use in preparing a pharmaceutical composition for treating a disease.

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition is used in the preparation of a medicament for treating at least one of the following diseases, wherein the diseases include 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, inflammatory bowel syndrome and / or dyspepsia or gastric ulcer, liver fibrosis and pulmonary fibrosis.