Long-acting GLP-1 / GLP-2 dual agonist compound

Abstract

The present invention relates to the field of pharmaceutical synthesis and discloses a long-acting GLP-1 / GLP-2 dual agonist compound. The GLP-1 / GLP-2 dual agonist compound described in the present invention is used to manufacture a pharmaceutical composition for treating at least one disease selected from the group consisting of type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, metabolic syndrome, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, coronary artery disease, cardiovascular disease, stroke, inflammatory bowel disease, irritable bowel syndrome, indigestion or gastric ulcer, liver fibrosis disease and pulmonary fibrosis.

Description

[Technical field]

[0001] This application claims priority based on Chinese patent application No. 202210150559.7, filed with the China Patent Office on February 18, 2022, for the invention entitled "Long-acting GLP-1 / GLP-2 dual agonist compound," the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a long-acting GLP-1 / GLP-2 dual agonist compound and its use. The compound is a kind of dual incretin peptidomimetic compound that activates glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) receptors. [Background technology]

[0003] 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.

[0004] Glucagon-like peptide-2 (GLP-2) is produced and secreted by endocrine cells in the small and large intestine, where its amino terminus is rapidly dissolved and inactivated under the action of dipeptidyl peptidase IV in the circulation, and metabolized by the kidney.

[0005] GLP-2 promotes the growth and development of intestinal mucosa and the proliferation of intestinal crypt epithelial cells, inhibits apoptosis of intestinal mucosal epithelial cells and crypt cells, and induces an increase in small intestinal villi and intestinal weight. It also regulates the activity of intestinal brush border membrane enzymes, maintains the integrity of the small intestinal mucosal epithelium, and promotes repair after small intestinal damage, so it is expected to be applied to animal nutrition.

[0006] Chinese Patent CN201780074629.2 reports that certain GLP-1 / GLP-1 compounds exhibit both GLP-1 and GLP-2 activity, and these compounds have good therapeutic effects on gastrointestinal diseases. The present invention aims to seek derivatives with higher possible biological activity that can reduce clinical usage and corresponding side effects. Summary of the Invention

[0007] The present invention provides a long-acting GLP-1 / GLP-2 dual agonist compound and its use, which is a type of dual incretin peptidomimetic compound that agonizes the receptors of glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2).

[0008] To achieve the above objects, the present invention provides a compound represented by the following structural formula I, and also provides a pharma- ceutically acceptable salt, solvate, chelate or non-covalent complex of said compound, a drug precursor based on said compound, or any mixture of these forms. His-AA1-Glu-Gly-AA2-AA3-AA4-Ser-Glu-Leu-Ala-Thr-AA5-Leu-Asp-AA6(R)-AA7-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-AA8-AA9-Lys-Ile-Thr-Asp-AA10 [Structural formula I] In the formula I, AA1 is Aib, Gly, Acpr, Acp, Acpe, or Ach; AA2 in formula I is Ser or Thr; AA3 in structural formula I is Phe, αMePhe, or αMePhe(2F); AA4 in formula I is Ser or Thr; AA5 in formula I is Ile, Aib, or αMeLeu; AA6 in structural formula I is Lys, Dap, Dab, Orn, Dah, Dao, Asp [NH(CH2)m NH], Glu[NH(CH2) m NH], Ada[NH(CH2) m NH], Apm[NH(CH2) m NH], or Asu[NH(CH2) m NH] (wherein m is an integer from 2 to 10); AA7 in formula I is Gln, Glu, or Leu; AA8 in formula I is Ala or Gln; AA9 in formula I is His or Thr; AA10 in formula I is NH2 or OH; R in structural formula I is HO2C(CH2) n1 CO-(AA11) n2 -(PEG n3 (CH2) n4 CO) n5 - and (where R is HO2C(CH2) n1 CO-(AA11) n2 Does not contain -(PEG2CH2CO)2- n1 is an integer from 10 to 20, n2 is an integer from 1 to 5, n3 is an integer from 1 to 30, n4 is an integer from 1 to 5; n5 is an integer from 1 to 5, n6 is an integer from 1 to 10; AA11 is γGlu, εLys, β-Ala, γ-aminobutyric acid, or 5-Ava.

[0009] The invention also provides pharmaceutical compositions comprising the compounds of the invention and the use of pharmaceutical compositions of the compounds of the invention in the manufacture of a medicament for treating a disease.

[0010] In a preferred embodiment, the pharmaceutical composition is for use in the manufacture of a medicament for treating at least one disease selected from the group consisting of type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, metabolic syndrome, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, coronary artery disease, cardiovascular disease, stroke, inflammatory bowel disease, irritable bowel syndrome, dyspepsia or gastric ulcer, hepatic fibrosis disease and pulmonary fibrosis.

[0011] More details regarding the present invention are described in detail below, or partly understood in the embodiments of the present invention.

[0012] Unless otherwise indicated, the quantities used to refer to various components, reaction conditions in this specification can be construed in any case as being "approximate" or "about." Accordingly, unless expressly specified otherwise, the numerical parameters recited below and in the claims are approximate parameters, and different numerical parameters may be obtained due to standard errors for different experimental conditions.

[0013] In the present specification, if there is any difference or doubt in the chemical structure and chemical name of a compound, the compound is appropriately defined by the chemical structure. The compounds described herein may contain one or more chiral centers and / or double bonds and similar structures, and may have stereoisomers, including double bond isomers (e.g., geometric isomers), optically rotatory enantiomers or diastereomers. And, any chemical structure within the scope described herein, when it contains the above similar structures in part or in the overall structure, includes all possible enantiomers and diastereomers of the compound, and also includes any simple stereoisomers (e.g., simple geometric isomers, simple enantiomers, or simple diastereomers) and any mixture of these isomers. These racemates and stereoisomeric mixtures can be further resolved into their component enantiomers or stereoisomers by those skilled in the art using various separation techniques or methods of chiral molecule synthesis.

[0014] 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 (e.g., optical isomers) or diastereomers, e.g., optical rotatory isomers, can be obtained by asymmetric synthesis or racemate resolution. Resolution of racemates can be achieved by various methods, e.g., recrystallization with reagents used in conventional resolutions, or chromatographic methods. Additionally, the compounds of formula I include cis and / or trans isomers of the double bonds.

[0015] The compounds according to the present invention include, but are not limited to, the compounds represented by structural formula I and all pharma- ceutically acceptable forms thereof, including various pharma- ceutically acceptable salts, solvates, complexes, chelates, non-covalent conjugates, drug precursors based on said substances, and any mixtures of these forms. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention discloses a long-acting GLP-1 / GLP-2 dual agonist compound and its use, and those skilled in the art can realize it by appropriately improving the relevant parameters while referring to the contents of this specification. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and are all considered to be included in the present invention. The method of the present invention is described by the preferred embodiment, but it is obvious to those skilled in the art to modify or appropriately change and combine the compounds and preparation methods described herein to realize and apply the technology of the present invention without departing from the content, spirit and scope of the present invention.

[0017] The terms and their abbreviations relevant to the present invention are set out in the following table. [Table 1] EXAMPLES

[0018] Example 1 Preparation of Compound The preparation method includes preparing a peptide resin by solid-phase polypeptide synthesis, further hydrolyzing the peptide resin with an acid to obtain a crude product, and finally purifying the crude product to obtain a purified product. In the step of preparing the peptide resin by solid-phase polypeptide synthesis, corresponding protected amino acids or fragments in the peptide sequence are sequentially coupled onto a carrier resin by solid-phase coupling synthesis to prepare the peptide resin.

[0019] In the above production method, the amount of the Fmoc-protected amino acid or protected amino acid fragment is 1.2 to 6 times, and preferably 2.5 to 3.5 times, the total number of moles of the charged resin. In the above manufacturing method, the substitution value of the carrier resin is 0.2 to 1.0 mmol / g resin, and preferably 0.3 to 0.5 mmol / g resin.

[0020] In a preferred embodiment of the present invention, in the solid phase coupling synthesis method, the protected amino acid-resin obtained in the previous reaction step is subjected to a coupling reaction with the next protected amino acid after removing the Fmoc protecting group. The time for the Fmoc deprotection is 10 to 60 minutes, preferably 15 to 25 minutes. The time for the coupling reaction is 60 to 300 minutes, preferably 100 to 140 minutes.

[0021] The above coupling reaction requires the addition of a condensing agent. The condensing agent is one selected from the group consisting of DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, (benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate. N,N-diisopropylcarbodiimide is preferred. The molar amount of the condensing agent is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total number of moles of amino in the amino resin.

[0022] The above coupling reaction requires the addition of an activator. The activator is 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The dosage of the activator is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total mole number of amino in the amino resin.

[0023] In a preferred embodiment of the present invention, the Fmoc deprotecting agent is a mixed solution of PIP / DMF (piperidine / N,N-dimethylformamide) containing 10-30% (V) of piperidine. The dosage of the Fmoc deprotecting agent is 5-15 mL per 1 g of amino resin, and preferably 8-12 mL per 1 g of amino resin.

[0024] Preferably, the peptide resin is hydrolyzed with an acid and the resin and side chain protecting groups are removed to obtain the crude product. More preferably, the acid decomposition agent used for the acid decomposition of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), and water, the volume ratio of which is 80-95% TFA, 1-10% EDT, and the remaining amount being water.

[0025] The volume ratio of the mixed solvent is more preferably 89-91% TFA, 4-6% EDT, and the remainder water, and most preferably 90% TFA, 5% EDT, and the remainder water.

[0026] The amount of the acid decomposition agent is 4 to 15 mL per 1 g of peptide resin, and preferably 7 to 10 mL per 1 g of peptide resin. The decomposition time with an acid decomposition agent is 1 to 6 hours, preferably 3 to 4 hours, at room temperature. The crude product is then purified by high performance liquid chromatography and freeze-dried to obtain a purified product.

[0027] 1. Synthesis of Peptide Resin Using Rink Amide BHHA resin as a carrier resin, the peptide was sequentially coupled to protected amino acids corresponding to the peptide sequence by Fmoc deprotection and coupling reaction to obtain a peptide resin.

[0028] (1) The first protected amino acid of the main chain was bound 0.03 mol of the first protected amino acid and 0.03 mol of HOBt were dissolved in an appropriate amount of DMF. 0.03 mol of DIC was slowly added to the DMF solution of the protected amino acid while stirring, and the mixture was reacted at room temperature for 30 minutes while stirring to obtain a solution of the activated protected amino acid.

[0029] 0.01 mol of Rink amide MBHA resin (substitution value approximately 0.4 mmol / g) was deprotected with 20% PIP / DMF solution for 25 minutes, filtered, and washed to obtain the Fmoc-free resin. The activated first protected amino acid solution was added to the Fmoc-depleted resin, and the coupling reaction was carried out for 60 to 300 minutes, followed by filtration and washing to obtain a resin containing one protected amino acid.

[0030] (2) A protected amino acid was attached to the main chain. Using the same method as that used for coupling the first main chain protected amino acid, the protected amino acids corresponding to the corresponding peptide sequence were sequentially coupled to obtain a resin containing main chain amino acids.

[0031] (3) The first protected amino acid in the side chain was attached. 0.03 mol of the first protected amino acid of the side chain and 0.03 mol of HOBt were dissolved in an appropriate amount of DMF. 0.03 mol of DIC was slowly added to the DMF solution of the protected amino acid while stirring, and the reaction was carried out at room temperature for 30 minutes while stirring to obtain a solution of the activated protected amino acid.

[0032] 2.5 mmol of tetrakis(triphenylphosphine)palladium and 25 mmol of phenylsilane were dissolved in an appropriate amount of dichloromethane, deprotected for 4 hours, filtered, and washed to obtain the Alloc-free resin. The solution of the first protected amino acid on the side chain after activation was added to the Alloc-free resin, and a coupling reaction was carried out for 60 to 300 minutes, followed by filtration and washing to obtain a resin containing the first protected amino acid on the side chain.

[0033] (4) Another protected amino acid or mono-protected fatty acid in the side chain is bound Using the same method as that used for binding the first protected amino acid of the main chain above, the corresponding protected amino acid and mono-protected fatty acid were sequentially bound to the side chain to obtain a peptide resin.

[0034] 2. Manufacturing of crude products The peptide resin was taken, and a decomposition agent with a volume ratio of TFA:water:EDT=95:5:5 (10 mL of decomposition agent per 1 g of resin) was added thereto, stirred uniformly, and reacted with stirring at room temperature for 3 hours. The reaction mixture was filtered through a sand core filter funnel, the filtrate was collected, the resin was washed with a small amount of TFA three times, the filtrate was combined and then concentrated under reduced pressure, precipitated by adding anhydrous ethyl ether, precipitated three times with anhydrous ethyl ether, filtered by suction and dried to obtain a crude product as a neutral white powder.

[0035] 3. Manufacturing of refined products The crude product was taken, and water was added thereto and stirred, and the pH was adjusted to 8.0 with aqueous ammonia until the crude product was completely dissolved. The solution was filtered through a 0.45 μm composite microporous membrane to purify it. For purification, high performance liquid chromatography was adopted, using 10μm reversed phase C18 as the purification chromatography filler, 0.1% TFA / water solution-0.1% TFA / acetonitrile solution as the mobile phase system, and the flow rate of the 30mm x 250mm chromatography column was 20mL / min, and purification was performed using gradient elution and cyclic sample injection mode. The crude product solution was injected into the chromatography column, the elution of the mobile phase was started, the main peak was collected, and the acetonitrile was evaporated, after which a concentrated solution of the purified intermediate was obtained.

[0036] After filtering the purified intermediate concentrate through a 0.45 μm membrane filter, salt exchange was carried out using high performance liquid chromatography, the mobile phase system was 1% acetic acid / aqueous solution-acetonitrile, the purification chromatography filler was 10 μm reversed phase C18, and the flow rate of the 30 mm x 250 mm chromatography column was 20 mL / min (the corresponding flow rate can be adjusted according to the column specifications). The sample was injected into the chromatography column using gradient elution and cyclic sample injection mode, the elution of the mobile phase was started, the spectrum was collected, the change in absorbance was observed, the main peak of salt exchange was collected, and the purity was detected by liquid chromatography analysis. The main peak solution of salt exchange was combined and then concentrated under reduced pressure to obtain the purified acetic acid aqueous solution, which was then freeze-dried to obtain the purified peptide.

[0037] The following compounds were synthesized using the above method. [Table 2]

[0038] Example 2 Measurement of GLP-1 activity 1.Measurement method Under stimulation of its specific agonist, GLP-1R can activate the intracellular adenylate cyclase pathway to increase cAMP levels, resulting in the production and release of insulin. By stimulating a cell line stably transfected with GLP-1R with a test substance, the intracellular cAMP level is rapidly increased. Then, the relative light units (RLU) after stimulating the cells with each dose are measured by chemiluminescence, and the EC50 of the agonist is calculated. This activity measurement method is a method for measuring GLP-1 receptor agonist activity that is widely used around the world.

[0039] Using a CHO-K1 cell line stably expressing GLP-1R, we stimulated the stably transfected cells with different concentrations of agonists and measured the relative luminescence of cells stimulated at each dose, thereby determining the EC 50 The value was calculated.

[0040] 2.Measurement results The measurement results are shown in the table below. [Table 3]

[0041] Example 3 Measurement of GLP-2 activity 1.Measurement method Under the stimulation of its specific agonist, GLP-2R can activate the intracellular adenylate cyclase pathway to increase cAMP levels, resulting in the production and release of insulin. By stimulating a cell line stably transfected with GLP-2R with a test substance, the intracellular cAMP level is rapidly increased. Then, the relative light units (RLU) after stimulating the cells with each dose are measured by chemiluminescence, and the EC50 of the agonist is calculated. This activity measurement method is a method for measuring GLP-2 receptor agonist activity that is used worldwide.

[0042] Using a CHO-K1 cell line stably expressing GLP-2R, the EC50 values ​​of agonists were determined by stimulating the stably transfected cells with different concentrations of agonists and measuring the relative luminescence of cells stimulated at each dose. 50 The value was calculated.

[0043] 2.Measurement results The measurement results are shown in the table below. [Table 4]

[0044] Example 3: Measurement of preliminary drug metabolism characteristics Cynomolgus monkeys were used as experimental animals. There were two male cynomolgus monkeys in each compound group. The compound was administered subcutaneously at a dose of 0.1 mg / kg. Blood samples were collected from the vein before 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 administration. The plasma samples were centrifuged, and the blood concentrations of the corresponding compounds in the plasma samples were measured by liquid chromatography-mass spectrometry. The half-lives of the compounds administered subcutaneously (SC) are shown in the table below. [Table 5]

Claims

1. A compound having the following structural formula I: His-AA1-Glu-Gly-AA2-AA3-AA4-Ser-Glu-Leu-Ala-Thr-AA5-Leu-Asp-AA6(R)-AA 7-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-AA8-AA9-Lys-Ile-Thr-Asp-AA10 [Structural formula I] (wherein AA1 in structural formula I is Aib, Gly, Acpr, Acp, Acpe, or Ach; AA2 in formula I is Ser or Thr; AA3 in structural formula I is Phe, αMePhe, or αMePhe(2F); AA4 in formula I is Ser or Thr; AA5 in structural formula I is Ile, Aib, or αMeLeu; AA6 in structural formula I is Lys, Dap, Dab, Orn, Dah, Dao, Asp [NH(CH ２ ) ｍ NH], Glu[NH(CH ２ ) ｍ NH], Ada[NH(CH ２ ) ｍ NH], Apm[NH(CH ２ ) ｍ NH], or Asu[NH(CH ２ ) ｍ NH], where m is an integer from 2 to 10; AA7 in structural formula I is Gln, Glu, or Leu; AA8 in formula I is Ala or Gln; AA9 in structural formula I is His or Thr; AA10 in structural formula I is NH ２ or OH, R in formula I is HO ２ C (CH ２ ) ｎ１ CO-(AA11) ｎ２ -(PEG ｎ３ (CH ２ ) ｎ４ CO) ｎ５ - and Here, R is HO ２ C (CH ２ ) ｎ１ CO-(AA11) ｎ２ -(PEG ２ CH ２ CO) ２ - does not include, n1 is an integer from 10 to 20, n2 is an integer from 1 to 5, n3 is an integer from 1 to 30, n4 is an integer from 1 to 5, n5 is an integer from 1 to 5, n6 is an integer from 1 to 10, AA11 is γGlu, εLys, β-Ala, γ-aminobutyric acid, or 5-Ava.

2. The compound of claim 1, characterized in that it is in the form of a member selected from the group consisting of a pharma- ceutically acceptable salt, solvate, chelate or non-covalent complex of said compound, a drug precursor based on said compound, and any mixture thereof.

3. 13. Use of the compound according to claim 1 in the manufacture of a long-acting GLP-1 / GLP-2 dual agonist.

4. 13. Use of a compound according to claim 1 in the manufacture of a medicament for treating at least one disease selected from the group consisting of type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, metabolic syndrome, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, coronary artery disease, cardiovascular disease, stroke, inflammatory bowel disease, irritable bowel syndrome, dyspepsia or gastric ulcer, hepatic fibrosis disease, and pulmonary fibrosis.

5. A pharmaceutical composition comprising a compound according to claim 1.

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