A long-acting dual agonist compound

Abstract

The present application relates to the field of medicine synthesis, disclose a long-acting dual agonist compound. The tirzepatide derivative described in the application is used for preparing a pharmaceutical composition for treating diseases, the use of the pharmaceutical composition in the preparation of a medicine for treating at least one of the following diseases, 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 indigestion or gastric ulcer, liver fibrosis disease and pulmonary fibrosis disease.

Description

Technical Field

[0001] This invention relates to a long-acting dual agonist compound and its use, which is a class of dual incretin peptide mimics that stimulate human glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors. Background Technology

[0002] GIP is a 42-amino acid gastrointestinal regulatory peptide that 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 that stimulates insulin secretion, protects pancreatic β-cells, and inhibits glucagon secretion, gastric emptying, and food intake, leading to weight loss. GIP and GLP-1 are collectively known as incretins; incretin receptor signaling plays a crucial physiological role in glucose homeostasis. In normal physiology, GIP and GLP-1 are secreted from the intestines after meals. These incretins enhance physiological responses to food, including satiety, insulin secretion, and nutrient processing.

[0003] The most common side effect of GLP-1 compounds is that administration does not achieve full glycemic control and weight loss, while GIP alone has a very modest glucose-lowering capacity in patients with type 2 diabetes. Both natural GIP and GLP-1 can be rapidly inactivated by the ubiquitous protease DPP IV, and therefore can only be used for short-term metabolic control.

[0004] WO 2013 / 164483 and WO 2014 / 192284 reported that certain GIP / GLP-1 compounds exhibited activities of both GIP and GLP-1, and found that these compounds could achieve better glycemic control and weight loss.

[0005] Tirzepatide is a GIP / GLP-1 compound. Due to its relatively low biological activity, the clinical dosage is relatively high. The purpose of this invention is to seek derivatives with higher biological activity to reduce the clinical dosage and corresponding side effects. Summary of the Invention

[0006] This invention provides a long-acting dual agonist compound and its use, which is a dual incretin peptide mimic compound that stimulates the human glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors.

[0007] To achieve the above objectives, the present invention first provides a compound shown in structure I, a pharmaceutically usable salt, solvate, chelate or non-covalent complex of the compound, a drug precursor based on the compound, or any mixture thereof.

[0008]

[0009] In structure I, AA1 can be Aib, Acpr, Acp, Acpe, or Ach;

[0010] In structure I, AA2 is either Phe or N. α -Me-Phe;

[0011] In structure I, AA3 can be Lys, Orn, Dab, or Dap;

[0012] In structure I, AA4 can be Lys, Orn, Dab, or Dap;

[0013] In structure I, AA5 is either Phe or 1-Nal;

[0014] In structure I, AA6 is either NH2 or OH;

[0015] In structure I, R stands for HO2C(CH2). n1 CO-(γGlu) n2 -(PEG n3 (CH2) n4 CO) n5 -; or HO2C(CH2) n1 CO-(γGlu) n2 -(AA4) n6 -:

[0016] Where: n1 is an integer from 10 to 20;

[0017] n2 is an integer from 1 to 5;

[0018] n3 is an integer from 1 to 30;

[0019] n4 is an integer from 1 to 5;

[0020] n5 is an integer from 1 to 5;

[0021] n6 is an integer from 1 to 10;

[0022] AA4 could be Gly, Ser, or Glu.

[0023] The present invention also provides pharmaceutical compositions comprising compounds according to the present invention, and provides pharmaceutical uses of pharmaceutical compositions comprising compounds according to the present invention for the preparation of a medicine for treating diseases.

[0024] Preferably, the pharmaceutical composition is used in the preparation of a medicament for treating at least one of the following diseases: 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.

[0025] Preferably, the pharmaceutical composition is used in the preparation of a medicament for treating delayed efficacy of type 2 diabetes and / or preventing the exacerbation of type 2 diabetes.

[0026] Preferably, the pharmaceutical composition is used in the preparation of a medicament for reducing food intake, reducing β-cell apoptosis, increasing pancreatic β-cell function, increasing β-cell clusters and / or restoring glucose sensitivity to β-cells.

[0027] The present invention further provides a method for administering the compound to a treatment subject to regulate blood glucose levels.

[0028] Further details of this invention are described in detail below, or some of them may be experienced in the embodiments of this invention.

[0029] Unless otherwise specified, the quantities of different components and reaction conditions used herein are to be interpreted as "approximate" or "about". Accordingly, unless otherwise specified, the numerical parameters cited below and in the claims are approximate parameters, and different numerical parameters may be obtained under their respective experimental conditions due to different standard errors.

[0030] In this document, when there is disagreement or ambiguity regarding the chemical structure and name of a compound, the compound is defined precisely by its chemical structure. The compounds described herein may contain one or more chiral centers, and / or double bonds and similar structures, and may also exist as stereoisomers, including isomers of double bonds (e.g., geometric isomers), optical enantiomers, or diastereomers. Accordingly, any chemical structure within the scope of this description, whether partially or entirely containing similar structures, includes all possible enantiomers and diastereomers of the compound, including any single stereoisomer (e.g., a single geometric isomer, a single enantiomer, or a single diastereomer) and any mixture of these isomers. Mixtures of racemic and stereoisomers can be further separated into enantiomers or stereoisomers of their constituent parts by those skilled in the art using continuous separation techniques or chiral molecule synthesis methods.

[0031] Compounds of Formula I include, but are not limited to, 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 by racemic resolution. Racemic resolution can be achieved by various methods, such as conventional recrystallization with a resolving agent or by chromatographic methods. Additionally, compounds of Formula I also include cis and / or trans isomers with double bonds.

[0032] The compounds described in this invention include, but are not limited to, the compounds shown in structural formula I and all their various pharmaceutically available forms. These pharmaceutically available forms of the compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, drug prodrugs based on the above-described substances, and any mixtures of these forms.

[0033] The compound shown in Structure I provided by this invention is stable and not easily degraded by dipeptidyl peptidase IV (DPP-IV) in the body. It is a GIP / GLP-1 dual agonist compound with significant hypoglycemic and weight-reducing effects. Detailed Implementation

[0034] This invention discloses a GIP / GLP-1 compound and its uses. Those skilled in the art can refer to this document and appropriately modify the relevant parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method of this invention has been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the compounds and preparation methods described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0035] The Chinese names corresponding to the English abbreviations used in this invention are shown in the table below:

[0036]

[0037] Example 1: Preparation of Compound 1

[0038] Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys(PEG5CH2CO-γGlu-20 alkanedioic acid)-Ile- Ala-Gln-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0039] The preparation method includes: preparing peptide resin using a solid-phase polypeptide synthesis method, followed by acid hydrolysis of the peptide resin to obtain a crude product, and finally purification of the crude product to obtain a pure product; wherein the solid-phase polypeptide synthesis method for preparing peptide resin involves sequentially inserting the corresponding protected amino acids or fragments from the following sequences onto a carrier resin via a solid-phase coupling synthesis method to prepare the peptide resin:

[0040] In the above preparation method, the amount of Fmoc-protected amino acid or protected amino acid fragment is 1.2 to 6 times the total molar amount of the resin added; preferably 2.5 to 3.5 times.

[0041] 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.

[0042] As a preferred embodiment of the present invention, the solid-phase coupling synthesis method is as follows: the protected amino acid-resin obtained in the previous step is deprotected by the Fmoc protecting group and then coupled with the next protected amino acid. The deprotection time for the Fmoc protecting group is 10-60 minutes, preferably 15-25 minutes. The coupling reaction time is 60-300 minutes, preferably 100-140 minutes.

[0043] The coupling reaction requires the addition of a condensing agent, which is selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, benzotriazol-N,N,N',N'-tetramethylurea hexafluorophosphate, or O-benzotriazol-N,N,N',N'-tetramethylurea tetrafluoroborate; preferably N,N-diisopropylcarbodiimide. The molar amount of the condensing agent is 1.2 to 6 times the total molar amount of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0044] The coupling reaction requires the addition of an activating agent, which is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The amount of activating agent used is 1.2 to 6 times the total molar number of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0045] As a preferred embodiment of the present invention, the reagent for removing Fmoc protection is a PIP / DMF (piperidine / N,N-dimethylformamide) mixed solution, wherein the mixed solution contains 10-30% (V) piperidine. The amount of the Fmoc removal reagent is 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.

[0046] Preferably, the peptide resin is acid-hydrolyzed to remove both the resin and the side-chain protecting groups, yielding the crude product:

[0047] More preferably, the acid hydrolysate used during the acid hydrolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethylenedithiol (EDT) and water, with the volume ratio of the mixed solvent being: TFA 80-95%, EDT 1-10%, and the remainder being water.

[0048] More preferably, the volume ratio of the mixed solvent is: TFA 89-91%, EDT 4-6%, and the balance is water. Most preferably, the volume ratio of the mixed solvent is: TFA 90%, EDT 5%, and the balance is water.

[0049] The amount of acid hydrolysant used is 4 to 15 mL per gram of peptide resin; preferably, 7 to 10 mL per gram of peptide resin.

[0050] The pyrolysis time using the acid hydrolysate is 1 to 6 hours at room temperature, preferably 3 to 4 hours.

[0051] Furthermore, the crude product was purified by high performance liquid chromatography and lyophilized to obtain the pure product.

[0052] 1. Synthesis of peptide resins

[0053] Using Rink Amide BHHA resin as the carrier resin, peptide resins were prepared by sequentially coupling with the protected amino acids shown in the table below via Fmoc deprotection and coupling reactions. The protected amino acids used in this example are shown below:

[0054]

[0055]

[0056] (1) Integrating the first protected amino acid in the main chain

[0057] 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; take another 0.03 mol of DIC, and slowly add it to the DMF solution of the protected amino acid while stirring. Stir and react at room temperature for 30 minutes to obtain the activated protected amino acid solution for later use.

[0058] Take 0.01 mol of Rink amide MBHA resin (substitution value approximately 0.4 mmol / g), protect it with 20% PIP / DMF solution for 25 minutes, wash and filter to obtain Fmoc-free resin.

[0059] The activated solution of the first protected amino acid was added to the resin that had been de-Fmoc-treated, and the coupling reaction was carried out for 60–300 minutes. After filtration and washing, a resin containing one protected amino acid was obtained.

[0060] (2) Incorporate the 2nd to 39th protected amino acids into the main chain.

[0061] Using the same method as described above for adding the first protected amino acid to the main chain, the corresponding second to 39th protected amino acids were sequentially added to obtain a resin containing 39 amino acids in the main chain.

[0062] (3) Add the first protected amino acid to the side chain

[0063] Take 0.03 mol of the first protected amino acid on the side chain and 0.03 mol of HOBt, and dissolve them in an appropriate amount of DMF; take another 0.03 mol of DIC, and slowly add it to the DMF solution of the protected amino acid while stirring. Stir and react at room temperature for 30 minutes to obtain the activated protected amino acid solution.

[0064] Take 2.5 mmol of tetrakis(triphenylphosphine)palladium and 25 mmol of phenylsilane, dissolve them in an appropriate amount of dichloromethane, remove the protective layer for 4 hours, filter and wash to obtain the alloc-free resin for later use.

[0065] The activated side-chain first protected amino acid solution was added to the de-Allocated resin, and the coupling reaction was carried out for 60-300 minutes. After filtration and washing, a resin containing the first protected amino acid in the side chain was obtained.

[0066] (4) Add other protected amino acids or monoprotected fatty acids to the side chain.

[0067] Using the same method as described above for adding the first protected amino acid to the main chain, the corresponding protected amino acids and monoprotected fatty acids of the side chains are sequentially added to obtain peptide resin.

[0068] 2. Preparation of crude product

[0069] Take the above peptide resin and add a lysis reagent with a volume ratio of TFA:water:EDT = 95:5:5 (10 mL of lysis reagent per gram of resin). Stir well and react at room temperature for 3 hours. Filter the reaction mixture using a sintered funnel and collect the filtrate. Wash the resin three times with a small amount of TFA. Combine the filtrates and concentrate under reduced pressure. Add anhydrous diethyl ether to precipitate the precipitate and wash it three times with anhydrous diethyl ether. Dry the precipitate to obtain a white powder, which is the crude product.

[0070] 3. Preparation of pure products

[0071] Take the above crude product, add water and stir, adjust the pH to 8.0 with ammonia until completely dissolved, filter the solution through a 0.45μm mixed microporous membrane, and purify for later use;

[0072] Purification was performed using high performance liquid chromatography (HPLC). The chromatographic packing material was a 10 μm reversed-phase C18 column, and the mobile phase system was 0.1% TFA / water solution-0.1% TFA / acetonitrile solution. The flow rate of the 30 mm x 250 mm column was 20 mL / min. Gradient elution was used, and the sample was injected repeatedly for purification. The crude product solution was loaded into the column, the mobile phase was started for elution, and the main peak was collected. After acetonitrile was removed, the purified intermediate concentrate was obtained.

[0073] The purified intermediate concentrate was filtered through a 0.45 μm filter membrane and set aside. High-performance liquid chromatography (HPLC) was used for salt exchange. The mobile phase system was 1% acetic acid / water solution-acetonitrile. The chromatographic packing material was a 10 μm reversed-phase C18 column (30 mm x 250 mm) with a flow rate of 20 mL / min (the flow rate can be adjusted according to different column specifications). Gradient elution and cyclic loading were used. The sample was loaded into the column, the mobile phase was started for elution, the chromatogram was collected, and the absorbance change was observed. The salt-exchanged main peak was collected and its purity was determined by analytical liquid chromatography. The salt-exchanged main peak solutions were combined, concentrated under reduced pressure to obtain pure acetic acid aqueous solution, and freeze-dried to obtain 8.1 g of pure product with a purity of 98.8% and an overall yield of 16.9%. The molecular weight was 4800.6 (100% M+H).

[0074] Example 2 Preparation of Compound 2

[0075] Tyr-Aib-Glu-Gly-Thr-N α -Me-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys(PEG5CH2CO-

[0076] γGlu-20 alkanedioic acid)-Ile-Ala-Gln-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-

[0077] Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0078] The preparation method is the same as in Example 1, and the protected amino acids used are shown in the table below:

[0079]

[0080]

[0081] 5.4 g of pure product was obtained, with a purity of 98.1% and a total yield of 11.2%. The molecular weight was 4814.6 (100% M+H).

[0082] Example 3 Preparation of Compound 3

[0083] Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys(AEEA-AEEA-γGlu-20 alkanedioic acid)-Ile- Ala-Gln-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0084] The preparation method is the same as in Example 1, and the protected amino acids used are shown in the table below:

[0085]

[0086]

[0087] 7.5g of pure product was obtained, with a purity of 98.0% and a total yield of 15.6%. The molecular weight was 4813.6 (100% M+H).

[0088] Example 4: Preparation of Compound 4

[0089] Tyr-Aib-Glu-Gly-Thr-N α -Me-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys(AEEA-AEEA-

[0090] γGlu-20 alkanedioic acid)-Ile-Ala-Gln-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-

[0091] Gly-Ala-Pro-Pro-Pro-Ser-NH2

[0092] The preparation method is the same as in Example 1, and the protected amino acids used are shown in the table below:

[0093]

[0094]

[0095] 4.7g of pure product was obtained, with a purity of 97.3% and a total yield of 9.7%. The molecular weight was 4827.6 (100% M+H).

[0096] Example 5: Determination of GLP-1 activity

[0097] 1. Measurement Method

[0098] GLP-1R, when stimulated by its specific agonist, can activate the intracellular adenylate cyclase pathway, increase cAMP levels, and ultimately lead to insulin production and release. Cell lines stably transfected with GLP-1R were stimulated with the analyte, resulting in a rapid increase in intracellular cAMP levels. The relative light units (RLU) after each dose of stimulation were measured using a chemiluminescence method, and the EC50 of the agonist was calculated. This activity assay is currently a widely used method for detecting GLP-1 receptor agonist activity both domestically and internationally.

[0099] Using the CHO-K1 cell line stably expressing GLP-1R, the stable cells were stimulated with different concentrations of agonists. The EC50 values ​​of the agonists were obtained by measuring the relative light units after each dose of stimulation, with Tirzepatide as a control.

[0100] 2. Measurement Results

[0101] The measurement results are shown in the table below:

[0102]

[0103] Example 6 Determination of GIP activity

[0104] 1. Measurement Method

[0105] GIPR, when stimulated by its specific agonist, activates the intracellular adenylate cyclase pathway, increases cAMP levels, and ultimately leads to insulin production and release. Cell lines stably transfected with GIPR were stimulated with the analyte, resulting in a rapid increase in intracellular cAMP levels. The relative light units (RLU) after each dose of stimulation were measured using a chemiluminescence method, and the EC50 of the agonist was calculated. This activity assay is currently a widely used method for detecting GIP receptor agonist activity both domestically and internationally.

[0106] Using the CHO-K1 cell line that stably expresses GIPR, the stable cells were stimulated with different concentrations of agonists. The EC50 value of the agonists was obtained by measuring the relative light units after each dose of stimulation, with Tirzepatide as a control.

[0107] 2. Measurement Results

[0108] The measurement results are shown in the table below:

[0109]

[0110] Example 7 Preliminary PK Test

[0111] The experimental animals were cynomolgus monkeys. The drug was administered subcutaneously at a dose of 0.2 mg / kg. Blood samples were collected venously before administration (0 h) and after each administration, at 5 min, 10 min, 30 min, 45 min, 1 h, 2 h, 3 h, 6 h, 8 h, 24 h, 32 h, 48 h, 72 h, 96 h, 144 h, and 192 h. Plasma samples were separated by centrifugation, and the plasma concentrations of the corresponding compounds in the plasma samples were determined by liquid chromatography-mass spectrometry (LC-MS) to obtain preliminary pharmacokinetic parameters for subcutaneous (SC) administration of the compounds.

[0112]

Claims

1. A long-acting dual agonist compound having the following structural formula: Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys(AEEA-AEEA-γGlu-20 alkanedioic acid)-Ile-A la-Gln-Lys-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2.

2. A pharmaceutically usable salt formed from the compound of claim 1.

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