Tri-agonist compound

Abstract

The invention relates to the field of medicine synthesis, and discloses a GLP-1 / GIP / GCG tri-agonist compound. The GLP-1 / GIP / GCG three-agonist compound is used for preparing a medicine composition for treating diseases, and the application of the medicine composition in preparing a medicine for treating at least one of the following diseases, namely, the GLP-1 / GIP / GCG three-agonist compound, and the GLP-1 / GIP / GCG three-agonist compound. 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 / GIP / GCG triple agonist compound and its use. The compound is a glucagon-like peptide-1 (GLP-1), human glucose-dependent insulinotropic polypeptide (GIP), and glucagon (GCG) triple agonist modified compound. Background Art

[0002] The most common side effects of GLP-1 compounds are that administration fails to achieve full-effect blood glucose control and weight loss, while alone, GIP has a very modest glucose-lowering ability in type 2 diabetes patients. Both native GIP and GLP-1 can be rapidly inactivated by the ubiquitously present protease DPP IV, and thus can only be used for short-term metabolic control.

[0003] GIP is a 42-amino acid gastrointestinal regulatory peptide, which plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic β-cells in the presence of glucose and protecting pancreatic β-cells. GLP-1 is a 37-amino acid peptide, which stimulates insulin secretion, protects pancreatic β-cells, and inhibits glucagon secretion, gastric emptying, and food intake, resulting in weight loss. GIP and GLP-1 are called incretins; incretin receptor signaling plays a key physiologically relevant role in glucose homeostasis. In normal physiology, GIP and GLP-1 are secreted from the intestine after a meal, and these incretins enhance the physiological responses to food, including satiety, insulin secretion, and nutrient disposal.

[0004] GCG is also called glucagon or anti-insulin or insulin B. It is a hormone secreted by the α-cells of the islets of Langerhans in the pancreas of vertebrates along with insulin. It acts in opposition to insulin and plays a role in increasing blood glucose.

[0005] New research shows that GLP-1 / GIP / GCG triple receptor agonist compounds not only have better blood glucose control, but also have the effects of significantly reducing body weight and treating non-alcoholic fatty liver. The purpose of the present invention is to provide new compounds with better biological activity for clinical use. Summary of the Invention

[0006] The present invention provides a GLP-1 / GIP / GCG triple agonist compound and its use. The compound is a glucagon-like peptide-1 (GLP-1), human glucose-dependent insulinotropic polypeptide (GIP), and glucagon (GCG) triple agonist compound.

[0007] In the first aspect of the embodiment of the present invention, a GLP-1 / GIP / GCG triple agonist compound is provided: Tyr-Aib-Gln-Gly-Thr-AA1-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp- Lys-Lys((CO(CH2) n1 PEG n2 ) n3 -(AA2) n4 -CO(CH2) n5 -COOH)-Ala-Gln-Aib-Ala- Phe-Ile-Glu-Tyr-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AA3 Structural formula Ⅰ Wherein: AA1 is selected from any one of αMePhe and αMePhe(2F); AA2 is selected from any one of γGlu, δAad, εApm, and ζAsu; AA3 is selected from any one of amino group and hydroxyl group; n1 = 1 - 5, n2 = 1 - 30, n3 = 0 or = 1 - 5, n4 = 0 or = 1 - 5, n5 = 10 - 30;

[0008] Optionally, the GLP-1 / GIP / GCG triple agonist compound is characterized in that AA1 is selected as αMePhe, AA2 is selected as γGlu, AA3 is selected as amino group, 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 group, n1 = 1, n2 = 2, n3 = 1, n4 = 1, n5 = 18;

[0009] Optionally, the GLP-1 / GIP / GCG triple agonist compound is characterized in that AA1 is selected as αMePhe, AA2 is selected as δAad, AA3 is selected as amino group, 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 group, n1 = 1, n2 = 2, n3 = 1, n4 = 1, n5 = 18;

[0010] Optionally, the GLP-1 / GIP / GCG triple 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 = 2, n3 = 1, n4 = 1, n5 = 18;

[0011] Optionally, the GLP-1 / GIP / GCG triple 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 = 2, n3 = 1, n4 = 1, n5 = 18;

[0012] Optionally, the above-mentioned GLP-1 / GIP / GCG triple agonist compound is characterized in that the triple 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.

[0013] The second aspect of the embodiments of the present invention provides an application of the GLP-1 / GIP / GCG triple agonist compound as described in any one of the above first aspects in the preparation of a pharmaceutical composition for treating diseases.

[0014] Optionally, the pharmaceutical composition for treating diseases is characterized in that the pharmaceutical composition for treating diseases is used for at least one of the following diseases, including 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 disease and pulmonary fibrosis disease.

[0015] Unless otherwise indicated, the quantities and reaction conditions used herein to represent different components can be interpreted as "substantially" or "approximately" in any case. Correspondingly, unless specifically specified, the numerical parameters cited in the following and the claims are approximate parameters, and different numerical parameters may be obtained under their respective experimental conditions due to different standard errors.

[0016] In this article, when there are discrepancies or ambiguities between the chemical structural formula and the chemical name of a compound, the compound is precisely defined by its chemical structural formula. The compounds described herein may contain one or more chiral centers, and / or double bonds and structures of this kind, and there may also be stereoisomers, including isomers of double bonds (such as geometric isomers), enantiomers or diastereomers. Correspondingly, any chemical structure within the scope described herein, whether partially or wholly containing the above-mentioned similar structures, includes all possible enantiomers and diastereomers of this compound, including any single stereoisomer (such as a pure geometric isomer, a pure enantiomer or a pure diastereomer) and any mixture of these isomers. These racemic isomers and mixtures of stereoisomers can be further separated into their component enantiomers or stereoisomers by those skilled in the art using separation techniques or chiral molecule synthesis methods.

[0017] The compounds of Structural Formula I include, but are not limited to, the optical isomers, racemates and / or other mixtures of these compounds. In the above cases, a single enantiomer or diastereomer, such as an optically active isomer, can be obtained by asymmetric synthesis or racemate resolution methods. Racemate resolution can be achieved by different methods, such as conventional recrystallization with a resolving agent or chromatography. In addition, the compounds of Structural Formula I also include cis and / or trans isomers with double bonds.

[0018] The compounds described in the present invention include, but are not limited to, the compounds shown in Structural Formula I and all their different pharmaceutically available forms. These different pharmaceutically available forms of the compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above substances and any mixture of the above forms.

[0019] The compounds shown in Structural Formula I provided by the present invention have stable properties and are highly active GLP-1 / GIP / GCG triple receptor agonist compounds, having significant effects on reducing blood sugar and body weight. Detailed implementation mode

[0020] The present invention discloses a GLP-1 / GIP / GCG triple receptor agonist compound and its uses. Those skilled in the art can draw on the content of this article and appropriately modify relevant parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods of the present invention have been described through preferred embodiments, and it is obvious that relevant personnel can make changes or appropriate alterations and combinations to 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.

[0021] The corresponding Chinese names of the English abbreviations involved in the present invention are shown in the following table: English Abbreviation Chinese Name English Abbreviation Chinese Name Fmoc 9-Fluorenylmethoxycarbonyl OtBu tert-Butoxy tBu tert-Butyl Boc tert-Butoxycarbonyl Trt Triphenylmethyl 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 Glutamic Acid Trp Tryptophan Gly Glycine Tyr Tyrosine His Histidine Val Valine Ile Isoleucine Ada 2-Aminoadipic Acid Aib Aminoisobutyric Acid Apm 2-Aminoheptanedioic Acid 5-Ava 5-Aminovaleric Acid Asu 2-Aminooctanedioic Acid

[0022] Example 1 Preparation of the compound

[0023] The preparation method includes: preparing a peptide resin by solid-phase peptide synthesis method, acid-hydrolyzing the peptide resin to obtain a crude product, and finally purifying the crude product to obtain a pure product; wherein the step of preparing the peptide resin by solid-phase peptide synthesis method is to sequentially connect the corresponding protected amino acids or fragments in the polypeptide sequence onto the carrier resin by solid-phase coupling synthesis method to prepare the peptide resin:

[0024] In the above preparation method, the amount of the Fmoc-protected amino acid or protected amino acid fragment used is 1.2 to 6 times the total molar amount of the charged resin; preferably 2.5 to 3.5 times.

[0025] In the above preparation method, the substitution value of the carrier resin is 0.2 to 1.0 mmol / g resin, and the preferred substitution value is 0.3 to 0.5 mmol / g resin.

[0026] As a preferred embodiment of the present invention, the solid-phase coupling synthesis method is: after the protected amino acid-resin obtained from the previous reaction removes the Fmoc protecting group, it is then coupled with the next protected amino acid. The deprotection time for removing the Fmoc protection is 10 to 60 minutes, preferably 15 to 25 minutes. The coupling reaction time is 60 to 300 minutes, preferably 100 to 140 minutes.

[0027] The coupling reaction described above requires the addition of a condensing reagent, which is selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazol-1-yloxytris-pyrrolidinophosphonium hexafluorophosphate, 2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate, or O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate; preferably N,N-diisopropylcarbodiimide. The molar amount of the condensing reagent used is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total molar amount of amino groups in the amino resin.

[0028] The coupling reaction described above requires the addition of an activating reagent, which is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The amount of the activating reagent used is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total molar amount of amino groups in the amino resin.

[0029] As a preferred embodiment of the present invention, the reagent for removing the Fmoc protection is a mixed solution of PIP / DMF (piperidine / N,N-dimethylformamide), and the piperidine content in the mixed solution is 10 to 30% (V). The amount of the reagent for removing the Fmoc protection used is 5 to 15 mL per gram of amino resin, preferably 8 to 12 mL per gram of amino resin.

[0030] Preferably, the peptide resin is acid-hydrolyzed to simultaneously remove the resin and side-chain protecting groups to obtain a crude product:

[0031] More preferably, the acid-hydrolyzing agent used for acid-hydrolyzing 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 to 95%, EDT is 1 to 10%, and the balance is water.

[0032] Even more preferably, the volume ratio of the mixed solvent is: TFA is 89 to 91%, EDT is 4 to 6%, and the balance is water. Most preferably, the volume ratio of the mixed solvent is: TFA is 90%, EDT is 5%, and the balance is water.

[0033] The amount of the acid-hydrolyzing agent used is 4 to 15 mL of the acid-hydrolyzing agent per gram of peptide resin; preferably, 7 to 10 mL of the acid-hydrolyzing agent per gram of peptide resin.

[0034] The time for cleavage using the acid-hydrolyzing agent is 1 to 6 hours at room temperature, preferably 3 to 4 hours.

[0035] Furthermore, the crude product is purified by high performance liquid chromatography and freeze-dried to obtain a pure product.

[0036] 1. Synthesis of peptide resin

[0037] Using Rink Amide BHHA resin as the carrier resin, through de-Fmoc protection and coupling reactions, the protected amino acids corresponding to the polypeptide sequence were successively coupled to obtain the peptide resin.

[0038] (1) Incorporating the first protected amino acid into the main chain

[0039] Take 0.03 mol of the first protected amino acid and 0.03 mol of HOBt, and dissolve them in an appropriate amount of DMF; separately take 0.03 mol of DIC, and slowly add it to the DMF solution of the protected amino acid under stirring. Stir and react at room temperature for 30 minutes to obtain the activated protected amino acid solution for standby.

[0040] Take 0.01 mol of Rink amide MBHA resin (substitution value about 0.4 mmol / g), and deprotect it with 20% PIP / DMF solution for 25 minutes. Wash and filter to obtain the de-Fmoc resin.

[0041] Add the activated first protected amino acid solution to the de-Fmoc resin, carry out the coupling reaction for 60 - 300 minutes, filter and wash to obtain the resin containing one protected amino acid.

[0042] (2) Incorporating the protected amino acids into the main chain

[0043] Using the same method as incorporating the first protected amino acid into the main chain above, successively incorporate the protected amino acids corresponding to the respective polypeptide sequences to obtain the resin containing the main chain amino acids.

[0044] (3) Incorporating the first protected amino acid into the side chain

[0045] Take 0.03 mol of the first protected amino acid of the side chain and 0.03 mol of HOBt, and dissolve them in an appropriate amount of DMF; separately take 0.03 mol of DIC, and slowly add it to the DMF solution of the protected amino acid under stirring. Stir and react at room temperature for 30 minutes to obtain the activated protected amino acid solution.

[0046] Take 2.5 mmol of tetrakis(triphenylphosphine)palladium and 25 mmol of phenylsilane, dissolve them in an appropriate amount of dichloromethane, deprotect for 4 hours, filter and wash to obtain the de-Alloc resin for standby.

[0047] Add the activated first protected amino acid solution of the side chain to the de-Alloc resin, carry out the coupling reaction for 60 - 300 minutes, filter and wash to obtain the resin containing the first protected amino acid of the side chain.

[0048] (4) Incorporating other protected amino acids or mono-protected fatty acids into the side chain

[0049] Using the same method as above for introducing the first protected amino acid into the main chain, the corresponding protected amino acids of the side chain and the mono-protected fatty acid were introduced successively to obtain the peptide resin.

[0050] 2. Preparation of the crude product

[0051] Take the above peptide resin, add a cleavage reagent with a volume ratio of TFA∶water∶EDT = 95∶5∶5 (10 mL of the cleavage reagent per gram of resin), stir evenly, stir and react at room temperature for 3 hours. The reaction mixture was filtered through a fritted funnel, and the filtrate was collected. The resin was washed 3 times with a small amount of TFA. After combining the filtrates, they were concentrated under reduced pressure, precipitated with anhydrous ether, and the precipitate was washed 3 times with anhydrous ether. After drying by suction, a white powder was obtained, which was the crude product.

[0052] 3. Preparation of the pure product

[0053] Take the above crude product, add water and stir. Adjust the pH to 8.0 with ammonia water until it is completely dissolved, and filter through a 0.45 μm filter membrane for standby.

[0054] Purification was carried out by high performance liquid chromatography. The chromatographic packing material for purification was reversed-phase C18 with a particle size of 10 μm. The mobile phase system was 0.1% TFA / aqueous solution - 0.1% TFA / acetonitrile solution. The flow rate of a 30 mm×250 mm chromatographic column was 20 mL / min. Gradient elution was used, and cyclic injection purification was carried out. The crude product solution was loaded onto the chromatographic column, the mobile phase was started for elution, and the main peak was collected. After evaporating acetonitrile, a concentrated solution of the purified intermediate was obtained.

[0055] The concentrated solution of the purified intermediate was filtered through a 0.45 μm filter membrane for standby. Salt exchange was carried out by high performance liquid chromatography. The mobile phase system was 1% acetic acid / aqueous solution - acetonitrile. The chromatographic packing material for purification was reversed-phase C18 with a particle size of 10 μm. The flow rate of a 30 mm×250 mm chromatographic column was 20 mL / min (the flow rate can be adjusted according to different specifications of the chromatographic column); gradient elution and cyclic loading methods were used. The sample was loaded onto the chromatographic column, the mobile phase was started for elution, the chromatogram was collected, the change in absorbance was observed, the main peak of salt exchange was collected and its purity was detected by analytical liquid chromatography. The solutions of the main peaks of salt exchange were combined, concentrated under reduced pressure to obtain an aqueous solution of pure acetic acid, and freeze-dried to obtain the pure peptide.

[0056] The following compounds were synthesized by the above method: Compound Sequence Structure Positive Control Retatrutide Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-γGlu -20-alkanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 Compound 1 Tyr-Aib-Gln-Gly-Thr-αMePhe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-γGlu-20-alkanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 Compound 2 Tyr-Aib-Gln-Gly-Thr-αMePhe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-δAad-20-alkanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 Compound 3 Tyr-Aib-Gln-Gly-Thr-αMePhe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-εApm-20-alkanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 Compound 4 Tyr-Aib-Gln-Gly-Thr-αMePhe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-ζAsu-20-alkanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 Compound 5 Tyr-Aib-Gln-Gly-Thr-αMePhe(2F)-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-γGlu-20-alkanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 Compound 6 Tyr-Aib-Gln-Gly-Thr-αMePhe(2F)-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-δAad-20-alkanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 Compound 7 Tyr-Aib-Gln-Gly-Thr-αMePhe(2F)-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-εApm-20-alkanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 Compound 8 Tyr-Aib-Gln-Gly-Thr-αMePhe(2F)-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-ζAsu-20 alkanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 Compound 9 Tyr-Aib-Gln-Gly-Thr-αMePhe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-AEEA-γGlu-20 alkanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 Compound 10 <![CDATA[Tyr-Aib-Gln-Gly-Thr-αMePhe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(COCH2PEG5-γGlu-20-alkanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2]]> Compound 11 Tyr-Aib-Gln-Gly-Thr-αMePhe(2F)-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(AEEA-AEEA-γGlu-20 alkanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 Compound 12 <![CDATA[Tyr-Aib-Gln-Gly-Thr-αMePhe(2F)-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp-Lys-Lys(COCH2PEG5-γGlu-20-alkanedioic acid)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-(5-Ava)-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2]]>

[0057] Example 2 Determination of activity

[0058] 1. GLP-1 activity determination method

[0059] Upon stimulation by its specific agonist, GLP-1R 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 cells can be rapidly increased. The relative light units (RLU) after stimulation of the cells at each dose are measured by chemiluminescence method, and then the EC50 of the agonist is calculated. This activity assay method is the commonly used GLP-1 receptor agonist activity detection method at home and abroad.

[0060] Using the CHO-K1 cell line stably expressing GLP-1R, the stably transfected cells are stimulated with agonists at different concentrations. By measuring the relative light units after stimulation of the cells at each dose, the EC 50 value of the agonist is calculated.

[0061] 2. GIP Activity Assay Method

[0062] Upon stimulation by its specific agonist, GIPR 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 GIPR with the test substance, the intracellular cAMP level of the cells can be rapidly increased. The relative light units (RLU) after stimulation of the cells at each dose are measured by chemiluminescence method, and then the EC50 of the agonist is calculated. This activity assay method is the commonly used GIP receptor agonist activity detection method at home and abroad.

[0063] Using the CHO-K1 cell line stably expressing GIPR, the stably transfected cells are stimulated with agonists at different concentrations. By measuring the relative light units after stimulation of the cells at each dose, the EC 50 value of the agonist is calculated.

[0064] 3. GCG Activity Assay Method

[0065] Upon stimulation by its specific agonist, GCG-R 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 cells can be rapidly increased. The relative light units (RLU) after stimulation of the cells at each dose are measured by chemiluminescence method, and then the EC50 of the agonist is calculated. This activity assay method is the commonly used GCG receptor agonist activity detection method at home and abroad.

[0066] Using the CHO-K1 cell line stably expressing GCG-R, the stably transfected cells are stimulated with agonists at different concentrations. By measuring the relative light units after stimulation of the cells at each dose, the EC 50 value of the agonist is calculated.

[0067] 4. Measurement Results

[0068] The measurement results are shown in the following table: Compound <![CDATA[GLP-1

EC 50 (pmol)

EC 50 (pmol)

EC 50 (pmol)

[0069] The experimental results show that while maintaining the activity of GLP-1, the activity of compound GIP in the examples is increased by different degrees, and the activity of GCG is greatly increased.

[0070] Example 3 Determination of preliminary pharmacokinetic properties

[0071] Among the above compounds, the principle of long-acting modification is basically the same. Therefore, the two compounds with the best activity are selected for the verification test of preliminary pharmacokinetic properties.

[0072] The test animals are cynomolgus monkeys, male. Two cynomolgus monkeys are used for each compound, and the drug is administered subcutaneously at a dose of 0.2 mg / kg. Blood samples are taken intravenously before drug administration (0 h) and at 1 h, 2 h, 3 h, 4 h, 8 h, 12 h, 18 h, 24 h, 48 h, 96 h, 144 h, and 168 h after drug administration. Plasma samples are separated by centrifugation, and the plasma concentrations of the corresponding compounds in the plasma samples are measured by liquid chromatography-tandem mass spectrometry. The half-lives of the compounds after subcutaneous (SC) administration are shown in the following table: Compound <![CDATA[t 1 / 2 (h)]]> Compound 2 107.6 Compound 6 115.2

[0073] The experimental results of the preliminary pharmacokinetic properties of cynomolgus monkeys show that the half-lives of subcutaneous administration of compound 2 and compound 6 are both greater than 100 hours, fully meeting the requirements of long-acting drug administration.

Claims

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

2. The GLP-1 / GIP / GCG triple agonist compound according to claim 1, wherein AA1 is selected as αMePhe, AA2 is selected as γGlu, AA3 is selected as amino group, n1 = 1 - 5, n2 = 1 - 30, n3 = 0 or = 1 - 5, n4 = 0 or = 1 - 5, n5 = 10 - 30.

3. The GLP-1 / GIP / GCG triple agonist compound according to claim 2, wherein AA1 is selected as αMePhe, AA2 is selected as γGlu, AA3 is selected as amino group, n1 = 1, n2 = 2, n3 = 1, n4 = 1, n5 = 18.

4. The GLP-1 / GIP / GCG triple agonist compound according to claim 1, wherein AA1 is selected as αMePhe, AA2 is selected as δAad, AA3 is selected as amino group, n1 = 1 - 5, n2 = 1 - 30, n3 = 0 or = 1 - 5, n4 = 0 or = 1 - 5, n5 = 10 - 30.

5. The GLP-1 / GIP / GCG triple agonist compound according to claim 4, characterized in that, AA1 is selected as αMePhe, AA2 is selected as δAad, AA3 is selected as amino group, n1 = 1, n2 = 2, n3 = 1, n4 = 1, n5 = 18.

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

7. The GLP-1 / GIP / GCG triple agonist compound according to claim 6, characterized in that, AA1 is selected as αMePhe(2F), AA2 is selected as γGlu, AA3 is selected as amino group, n1 = 1, n2 = 2, n3 = 1, n4 = 1, n5 = 18.

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

9. The GLP-1 / GIP / GCG triple agonist compound according to claim 8, wherein AA1 is selected as αMePhe(2F), AA2 is selected as δAad, AA3 is selected as amino group, n1 = 1, n2 = 2, n3 = 1, n4 = 1, n5 = 18.

10. The GLP-1 / GIP / GCG triple agonist compound according to any one of claims 1-9, characterized in that, The triple agonist compound includes a medicinal salt, chelate or non-covalent complex formed by the compound, and a precursor of the compound, or any mixture of the above forms.

11. Use of a GLP-1 / GIP / GCG triple agonist compound according to any one of claims 1 - 10 in the preparation of a pharmaceutical composition for treating diseases.

12. The pharmaceutical composition for treating diseases according to claim 11, wherein The pharmaceutical composition for treating diseases is used for at least one of the following diseases, which 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 indigestion or gastric ulcer, liver fibrosis disease and pulmonary fibrosis disease.