CN113967213A - Composition containing phenyl substituted ACC inhibitor and application thereof - Google Patents

Abstract

The invention belongs to the field of pharmaceutical preparations, relates to a composition containing a phenyl substituted ACC inhibitor and application thereof, and particularly relates to (R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) containing a formula (I)Yl) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d]Pyrimidin-3 (4H) -yl) benzoic acid or solvates or crystalline pharmaceutical compositions thereof. The pharmaceutical composition provided by the invention not only can be completely dissolved, but also has stable preparation property under the conditions of high humidity, high temperature, illumination, acceleration and long-term test, has good absorption property and high relative bioavailability,

Description

Composition containing phenyl substituted ACC inhibitor and application thereof

Technical Field

The invention belongs to the field of pharmaceutical preparations, relates to a composition containing a phenyl-substituted ACC inhibitor and application thereof, and particularly relates to a composition containing (R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazole-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidine-3 (4H) -yl) benzoic acid (also named as (R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazole-2-yl) benzoic acid ) -2, 4-dioxo-1, 4-dihydrothieno [2,3-d ] pyrimidin-3 (2H) -yl) benzoic acid) or a solvate or crystalline pharmaceutical composition thereof.

Background

Acetyl-coa carboxylase (ACC), a biotinidase that catalyzes the reaction of acetyl-coa to malonyl-coa, is the rate-limiting step in the first stage of fatty acid synthesis. In mammals, ACC exists as two tissue-specific isozymes, with ACC1 being present predominantly in lipogenic tissues, such as liver and fat, and ACC2 being present predominantly in oxygenated tissues, such as liver, heart and skeletal muscle. ACC1 and ACC2 are encoded by independent genes, and although present different cellular distributions, share 75% overall amino acid sequence identity. In the liver, Fatty Acid (FA) synthesis and elongation are malonyl-coa catalyzed by ACC1 to acetyl-coa production, leading to triglyceride formation and Very Low Density Lipoprotein (VLDL) production. Malonyl-coa formed from ACC2 functions to regulate FA oxidation in heart and skeletal muscle with limited ability to synthesize fatty acids [ Tong L, hartwood HJ jr.j Cell biochem.2006,99(6): 1476-.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are considered as two manifestations of liver metabolic abnormalities, which are currently the most common chronic liver disease, and the incidence rate thereof is rising year by year. NASH among them may further progress to cirrhosis and liver cancer, possibly causing death due to liver disease. At present, an effective treatment strategy is lacked, the existing treatment medicines still comprise an insulin sensitizer and an antioxidant (such as vitamin E) represented by thiazolidinediones, and in addition, the treatment effects of the medicines comprise lipid-lowering medicines, angiotensin receptor antagonists, polyunsaturated fatty acids and the like, and the treatment effects are very limited. In several studies at present, ACC1 and ACC2 are considered as promising drug targets for the treatment of NAFLD and NASH [ Geraldine Harriman, Jerney Greenwood, Sathesh Bhat, et al Proc Natl Acad Sci U.S. A.2016,113(13): E1796-E1805 ].

There has been some progress and research foundation for drug research targeting ACC pathway, de novo (de novo) synthesis of fat in liver cells can be inhibited by inhibiting ACC1 and ACC2, and the treatment regimen can significantly reduce liver fat content and degree of cirrhosis, while earlier reducing liver fibrosis marker levels. Another study showed that simultaneous inhibition of ACC1 and ACC2 reduced the ability to regenerate FA in tumor tissues, and had the effect of inhibiting tumor cell growth [ Svensson RU, Parker SJ, Eichner LJ, et al. Nat Med.2016,22(10): 1108-. However, there is still a need to develop more excellent ACC inhibitors in order to obtain more active and safer drugs for treating ACC mediated event related diseases such as fibrotic diseases, metabolic diseases, tumors and proliferative diseases.

Disclosure of Invention

The invention discloses a phenyl substituted ACC inhibitor, the structure of which is shown in the following formula (I), and the chemical name of the phenyl substituted ACC inhibitor is (R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazole-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidine-3 (4H) -yl) benzoic acid (hereinafter referred to as the compound of the formula (I)):

the research of the inventors of the present invention finds that the compound of formula (I) or a hydrate, solvate or crystal thereof has good inhibitory activity on ACC, and is very promising as a therapeutic agent for ACC expression-related diseases with higher therapeutic effects and fewer side effects, such as fibrotic diseases, metabolic diseases, cancer or tissue proliferative diseases.

Suitable formulations comprising a compound of formula (I) or an isomer thereof or a pharmaceutically acceptable salt thereof have not been reported so far. It is well known in the art that many problems are faced in the formulation of such formulations, such as long term stability of the formulation, control of the relevant substances, absorption and bioavailability of the drug, etc. These problems are determined by a number of factors, such as the many factors that affect absorption of the drug, including dissolution or release of the drug from the formulation, dissolution of the drug under physiological conditions, and permeability in the gastrointestinal tract.

Therefore, there is a need for research into compounds of formula (I) or derivatives thereof to provide suitable formulations that meet the clinical requirements.

The inventors of the present invention have found that the compound of formula (I) is practically insoluble in water or ethanol; almost insoluble in 0.1mol/L hydrochloric acid solution, Caco-2 cells showed moderate permeability, which makes the compound of formula (I) poorly absorbed in vivo.

It is an object of the present invention to provide a pharmaceutical composition comprising (a) a compound of formula (I) or a solvate or crystal thereof; and (b) a disintegrating agent, the pharmaceutical composition can be completely dissolved out and has high bioavailability.

It is another object of the present invention to provide a pharmaceutical formulation comprising the pharmaceutical composition of the present invention.

It is a further object of the present invention to provide the use of a pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment and/or prevention of a disease associated with ACC expression.

Aiming at the above purpose, the invention provides the following technical scheme:

in one aspect of the invention, there is provided a pharmaceutical composition comprising:

(a) a compound of formula (I) or a solvate or crystal thereof,

and (b) a disintegrant.

In some specific embodiments, the compound of formula (I) or its solvate or crystal thereof in the pharmaceutical composition of the present invention is present in an amount of about 1% to 80% (w/w), preferably about 2% to 50% (w/w), and more preferably about 3% to 20% (w/w), based on the amount of the compound of formula (I).

In some specific embodiments, the pharmaceutical composition of the present invention comprises a compound of formula (I) or a solvate or a crystallization and a disintegrant thereof, wherein the disintegrant is selected from one or more of sodium carboxymethyl starch (abbreviated as CMS-Na), crospovidone, croscarmellose sodium (abbreviated as CCNa), and low substituted hydroxypropyl cellulose (abbreviated as L-HPC), preferably from one or more of sodium carboxymethyl starch, croscarmellose sodium, and low substituted hydroxypropyl cellulose, and further preferably from low substituted hydroxypropyl cellulose. In some embodiments, the disintegrant croscarmellose sodium of the present invention includes, but is not limited to, croscarmellose sodium SD 711. It is known in the art that, within a certain range, as the disintegrating dosage increases, the disintegration of the capsule and the dissolution of the drug are promoted, but, beyond a certain amount, the disintegration-promoting effect gradually weakens, and even hinders the dissolution of the drug. Therefore, the content and the use method of the disintegrant need to be studied so that the best disintegration effect of the disintegrant is achieved. In some embodiments, the present invention provides pharmaceutical compositions wherein the disintegrant is present in an amount of about 1% to 30% (w/w), preferably 2% to 10% (w/w), more preferably 3% to 5% (w/w), based on the total weight of the pharmaceutical composition. The inventors of the present invention found that the dissolution of the compound of formula (I) can be significantly improved by using the pharmaceutical composition of the present invention containing the above disintegrant, and that the preparation is stable in properties and contains few impurities under high humidity, high temperature, light and accelerated and long-term test conditions.

In some embodiments, the pharmaceutical compositions provided herein further comprise a lubricant, i.e., the pharmaceutical compositions provided herein comprise a compound of formula (I) or a solvate or crystal thereof, a disintegrant and a lubricant, wherein the disintegrant has the definition described above and the lubricant is selected from one or more of magnesium stearate, calcium stearate, stearic acid, zinc stearate, sodium stearyl fumarate, talc, polyethylene glycol, glyceryl behenate, glyceryl monostearate, glyceryl palmitostearate, sodium lauryl sulfate and hydrogenated vegetable oils. More preferably, the lubricant is selected from one or more of magnesium stearate, calcium stearate, zinc stearate, stearic acid, polyethylene glycol, talc, sodium stearyl fumarate, and glyceryl behenate. In some embodiments of the present invention, the present invention provides a pharmaceutical composition wherein the lubricant is present in an amount of about 0% to about 10% (w/w), preferably about 0.1% to about 5% (w/w), more preferably about 0.3% to about 3% (w/w), and even more preferably about 0.4% to about 1% (w/w), based on the total weight of the pharmaceutical composition.

In some embodiments, the pharmaceutical compositions provided herein further comprise a filler, i.e., the pharmaceutical compositions provided herein comprise a compound of formula (I) or a solvate or crystal thereof, a disintegrant, a lubricant, and a filler, wherein the disintegrant and lubricant have the definitions described above, and the filler is selected from one or more of microcrystalline cellulose (MCC), powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, Mannitol (MAN), starches, Lactose (LAC), sugars, celluloses, and inorganic salts. In some embodiments, the pharmaceutical compositions of the present invention comprise two or more fillers, one of which is selected from microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate and pregelatinized starch, and the other fillers are selected from one or more of mannitol, starches, lactose, sugars, celluloses and inorganic saltsAnd (4) seed preparation. More preferably, the other filler is selected from one or more of starch, dextrates, glucose, lactose, dextrin, fructose, maltose, sorbitol and mannitol. In some embodiments, the filler lactose of the present invention includes, but is not limited to, lactose

100. In some embodiments, the filler of the present invention is a microcrystalline cellulose type including, but not limited to, microcrystalline cellulose PH101, microcrystalline cellulose VIVAPUR 102. In some embodiments of the present invention, the filler in the pharmaceutical composition provided by the present invention is about 0% to 90% (w/w), preferably about 20% to 85% (w/w), and more preferably about 50% to 85% (w/w), based on the total weight of the pharmaceutical composition. In some embodiments, the pharmaceutical compositions of the present invention further comprise lactose and microcrystalline cellulose, wherein the weight ratio of lactose to microcrystalline cellulose is from about 1:0.25 to about 1:8, preferably from about 1:0.5 to about 1:6, and more preferably from about 1:1 to about 1: 4.

In some embodiments, the pharmaceutical composition provided by the present invention further comprises a wetting agent, i.e. the pharmaceutical composition provided by the present invention comprises a compound of formula (I) or a solvate or crystal thereof, a disintegrant, a lubricant, a filler and a wetting agent, wherein the disintegrant, the lubricant and the filler have the definitions described above, and the wetting agent is water.

In some preferred embodiments, the pharmaceutical composition of the present invention consists of a compound of formula (I) or a solvate or crystal thereof, a disintegrant, a lubricant, and a filler, wherein the compound of formula (I) or the solvate or crystal thereof comprises about 1% to 80% (w/w) of the total weight of the pharmaceutical composition, the disintegrant comprises about 1% to 30% (w/w) of the total weight of the pharmaceutical composition, the lubricant comprises about 0% to 10% (w/w) of the total weight of the pharmaceutical composition, and the filler comprises about 0% to 90% (w/w) of the total weight of the pharmaceutical composition, based on the amount of the compound of formula (I). Further preferably, the pharmaceutical composition of the present invention consists of a compound of formula (I) or a solvate or crystal thereof, a disintegrant, a lubricant and a filler, wherein the compound of formula (I) or the solvate or crystal thereof accounts for about 2% to 50% (w/w) of the total weight of the pharmaceutical composition, the disintegrant accounts for about 2% to 10% (w/w) of the total weight of the pharmaceutical composition, the lubricant accounts for about 0.1% to 5% (w/w) of the total weight of the pharmaceutical composition, and the filler accounts for about 20% to 85% (w/w) of the total weight of the pharmaceutical composition, based on the amount of the compound of formula (I). Still further preferably, the pharmaceutical composition of the present invention consists of a compound of formula (I) or a solvate or crystal thereof, a disintegrant, a lubricant and a filler, wherein the compound of formula (I) or the solvate or crystal thereof accounts for about 3% to 20% (w/w) of the total weight of the formulation, the disintegrant accounts for about 3% to 5% (w/w) of the total weight of the pharmaceutical composition, the lubricant accounts for about 0.4% to 1% (w/w) of the total weight of the pharmaceutical composition, and the filler accounts for about 50% to 85% (w/w) of the total weight of the pharmaceutical composition, based on the amount of the compound of formula (I).

The invention also provides a pharmaceutical preparation comprising the pharmaceutical composition of the invention.

In some preferred embodiments, the pharmaceutical formulation of the present invention is a tablet, granule, powder, sustained release formulation, pill, capsule, lozenge or cachet. In a specific embodiment, the sustained release agent is a sustained release pellet. In a specific embodiment, the pharmaceutical formulation of the present invention is a tablet, capsule or granule.

In some preferred embodiments, the present invention provides a pharmaceutical formulation comprising a pharmaceutical composition of the present invention, the pharmaceutical composition consisting of a compound of formula (I) or a solvate or crystal thereof, a disintegrant, a lubricant, and a filler, wherein the compound of formula (I) or the solvate or crystal thereof comprises about 1% to 80% (w/w) of the total weight of the pharmaceutical composition, the disintegrant comprises about 1% to 30% (w/w) of the total weight of the pharmaceutical composition, the lubricant comprises about 0% to 10% (w/w) of the total weight of the pharmaceutical composition, and the filler comprises about 0% to 90% (w/w) of the total weight of the pharmaceutical composition, based on the amount of the compound of formula (I). Further preferably, the pharmaceutical composition of the present invention consists of a compound of formula (I) or a solvate or crystal thereof, a disintegrant, a lubricant and a filler, wherein the compound of formula (I) or the solvate or crystal thereof accounts for about 2% to 50% (w/w) of the total weight of the pharmaceutical composition, the disintegrant accounts for about 2% to 10% (w/w) of the total weight of the pharmaceutical composition, the lubricant accounts for about 0.1% to 5% (w/w) of the total weight of the pharmaceutical composition, and the filler accounts for about 20% to 85% (w/w) of the total weight of the pharmaceutical composition, based on the amount of the compound of formula (I). Still further preferably, the pharmaceutical composition of the present invention consists of a compound of formula (I) or a solvate or crystal thereof, a disintegrant, a lubricant and a filler, wherein the compound of formula (I) or the solvate or crystal thereof accounts for about 3% to 20% (w/w) of the total weight of the formulation, the disintegrant accounts for about 3% to 5% (w/w) of the total weight of the pharmaceutical composition, the lubricant accounts for about 0.4% to 1% (w/w) of the total weight of the pharmaceutical composition, and the filler accounts for about 50% to 85% (w/w) of the total weight of the pharmaceutical composition, based on the amount of the compound of formula (I).

In another aspect, the invention provides a method for preparing the composition of the invention, comprising mixing the components of the pharmaceutical composition of the invention.

In some embodiments, the present invention provides a method of preparing a pharmaceutical formulation of the present invention, said formulation comprising a compound of formula (I) or a solvate or a crystal and a disintegrant thereof, said method comprising: a step of mixing the compound of formula (I) or a solvate or crystal thereof and a disintegrant, followed by granulation. In some specific embodiments, according to the method for preparing a pharmaceutical formulation of the present invention, the disintegrant is selected from one or more of carboxymethyl starch sodium, crospovidone, croscarmellose sodium, or low substituted hydroxypropyl cellulose.

In some preferred embodiments, the present invention provides a process for the preparation of a pharmaceutical formulation of the invention, said formulation comprising a compound of formula (I) or a solvate or crystal thereof, a filler and a disintegrant, said process comprising: mixing the compound of formula (I) or its solvate or crystal with filler and disintegrant, and granulating. In some specific embodiments, according to the method for preparing a pharmaceutical formulation of the present invention, the filler is selected from one or more of microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, mannitol, starches, lactose, sugars, celluloses, and inorganic salts; the disintegrant is selected from one or more of carboxymethyl starch sodium, crospovidone, croscarmellose sodium or low-substituted hydroxypropyl cellulose.

In some preferred embodiments, the present invention provides a process for the preparation of a pharmaceutical formulation of the invention, said formulation comprising a compound of formula (I) or a solvate or crystal thereof, a lubricant and a disintegrant, said process comprising: mixing the compound of formula (I) or its solvate or crystal with filler and disintegrant, and granulating. In some specific embodiments, according to the method of preparing a pharmaceutical formulation of the present invention, the lubricant is selected from one or more of magnesium stearate, calcium stearate, zinc stearate, stearic acid, polyethylene glycol, talc, sodium stearyl fumarate, glyceryl behenate; the disintegrant is selected from one or more of carboxymethyl starch sodium, crospovidone, croscarmellose sodium or low-substituted hydroxypropyl cellulose.

In other preferred embodiments, the present invention provides a process for the preparation of a pharmaceutical formulation of the invention, said formulation comprising a compound of formula (I) or a solvate or crystal thereof, a filler, a disintegrant and a lubricant, said process comprising: mixing the compound of formula (I) or its solvate or crystal with filler, disintegrant and lubricant, and granulating. In some specific embodiments, according to the method for preparing a pharmaceutical formulation of the present invention, the filler is selected from one or more of microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, mannitol, starches, lactose, sugars, celluloses, and inorganic salts; the disintegrant is selected from one or more of carboxymethyl starch sodium, crospovidone, croscarmellose sodium or low-substituted hydroxypropyl cellulose; the lubricant is one or more selected from magnesium stearate, calcium stearate, stearic acid, zinc stearate, sodium stearyl fumarate, pulvis Talci, polyethylene glycol, glyceryl behenate, glyceryl monostearate, glyceryl palmitostearate, sodium lauryl sulfate and hydrogenated vegetable oil.

In other preferred embodiments, the present invention provides a process for the preparation of a pharmaceutical formulation of the invention, said formulation comprising a compound of formula (I) or a solvate or crystal thereof, a filler, a disintegrant, a lubricant and a wetting agent, said process comprising: mixing the compound of formula (I) or its solvate or crystal with filler, disintegrant, lubricant and wetting agent, and granulating. In some specific embodiments, according to the method for preparing a pharmaceutical formulation of the present invention, the filler is selected from one or more of microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, mannitol, starches, lactose, sugars, celluloses, and inorganic salts; the disintegrant is selected from one or more of carboxymethyl starch sodium, crospovidone, croscarmellose sodium or low-substituted hydroxypropyl cellulose; the lubricant is one or more selected from magnesium stearate, calcium stearate, stearic acid, zinc stearate, sodium stearyl fumarate, pulvis Talci, polyethylene glycol, glyceryl behenate, glyceryl monostearate, glyceryl palmitostearate, sodium lauryl sulfate and hydrogenated vegetable oil; the wetting agent is water.

In other preferred embodiments, the present invention provides a method of preparing a pharmaceutical formulation of the present invention, the method comprising: mixing the compound of formula (I) or its solvate or crystal with filler, disintegrant, lubricant and wetting agent, granulating, and canning.

In other preferred embodiments, the present invention provides a method of preparing a pharmaceutical formulation of the present invention, the method comprising: mixing the compound of formula (I) or its solvate or crystal with filler, disintegrant, lubricant and wetting agent, granulating, and tabletting.

In other preferred embodiments, the present invention provides a method of preparing a pharmaceutical formulation of the present invention, the method comprising: if necessary, the compound of formula (I) or a solvate or crystal thereof may be mixed with a filler, a disintegrant, a lubricant, a wetting agent, etc. and subjected to stirring granulation, extrusion granulation, rotary granulation, spray one-step granulation, etc., or may be subjected to direct dry granulation. In addition, the medicine can also be prepared by adopting a micro-pill medicine feeding mode. In addition, the granules may be granulated and pulverized as necessary. Further, the raw materials can be tableted into tablets or directly filled into capsules as required; optionally adding disintegrating agent, filler, lubricant, binder, antioxidant, colorant, etc., and making into tablet or capsule.

In some specific embodiments, the present invention provides a method of preparing a pharmaceutical formulation of the present invention, the method comprising:

(1) uniformly mixing a compound of formula (I) or a solvate or crystal thereof, a disintegrant and optional filler and a lubricant;

(2) optionally adding a proper amount of wetting agent into the mixed material in the step (1) for wet granulation;

(3) drying and finishing the granules in the step (2);

(4) optionally, uniformly mixing the particles obtained in the step (3) with a filler and a lubricant;

(5) and (4) preparing the mixed material in the step (4) into a preparation.

In some specific embodiments, the present invention provides a method of preparing a pharmaceutical formulation of the present invention, the method comprising:

(1) sieving the compound of formula (I) or solvate or crystal thereof, disintegrant and optional filler and lubricant, adding into wet granulator according to the amount of prescription, and mixing;

(2) adding a proper amount of wetting agent into the mixed material obtained in the step (1) for wet granulation;

(3) drying the granules obtained in the step (2) in a drying device;

(4) optionally, adding the granules, the filling agent and the lubricant in the step (3) into a replaceable barrel mixer for uniform mixing;

(5) and (4) pouring the mixed material in the step (4) into a capsule filling machine to fill capsules.

In some specific embodiments, the present invention provides a method of preparing a pharmaceutical formulation of the present invention, the method comprising:

(1) sieving the compound of formula (I) or solvate or crystal thereof, disintegrant and optional filler and lubricant, adding into wet granulator according to the amount of prescription, and mixing;

(2) adding a proper amount of wetting agent into the mixed material obtained in the step (1) for wet granulation;

(3) drying the granules obtained in the step (2) in a drying device;

(4) optionally, adding the granules, the filling agent and the lubricant in the step (3) into a replaceable barrel mixer for uniform mixing;

(5) and (4) pouring the mixed material in the step (4) into a tablet machine to be pressed into tablets.

A further aspect of the invention provides the use of a composition of the invention in the manufacture of a medicament for the treatment and/or prevention of a disease associated with ACC expression, such as a fibrotic disease, a metabolic disease, a tumor and a proliferative disease, wherein the fibrotic disease is selected from liver fibrosis, wherein the metabolic disease is selected from obesity, diabetes, non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, wherein the tumor and proliferative disease is selected from liver cancer, kidney cancer, lung cancer, breast cancer, melanoma, papillary thyroid tumors, cholangiocarcinoma, colon cancer, ovarian cancer, malignant lymphoma, carcinomas and sarcomas of the bladder, prostate and pancreas, and primary and recurrent solid tumors of the skin, colon, thyroid and ovary.

In a preferred embodiment, the present invention provides a method for treating and/or preventing ACC expression-related diseases and a use thereof in the preparation of a medicament for treating and/or preventing ACC expression-related diseases, wherein the fibrotic disease is selected from liver fibrosis, wherein the metabolic disease is selected from obesity, diabetes, non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, wherein the neoplastic and proliferative diseases are selected from liver cancer, kidney cancer, lung cancer, breast cancer, melanoma, papillary thyroid tumors, cholangiocarcinoma, colon cancer, ovarian cancer, malignant lymphoma, carcinomas and sarcomas of the bladder, prostate and pancreas, and primary and recurrent solid tumors of the skin, colon, thyroid and ovary.

In the pharmaceutical compositions of the invention, any form or amorphous form of the compound of formula (I) may be employed, resulting in formulations with very good stability. In other embodiments, the pharmaceutical compositions of the present invention are prepared using crystalline forms of the compound of formula (I).

The pharmaceutical composition provided by the invention can be completely dissolved out in vitro, has few impurities and good stability, and can simplify storage and transportation conditions and the like.

Description of the terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "solvate" refers to a form of the compound of the present invention which forms a complex in a solid or liquid state by coordination with a solvent molecule, and includes hydrates, ethanolates, acetonitriles, and the like.

The term "crystalline" refers to the various solid forms formed by the compounds of the present invention, including crystalline forms, amorphous forms. The "hydrogen", "carbon" and "oxygen" in the compounds of the present invention include all isotopes thereof. Isotopes are understood to include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include protium, tritium, and deuterium, and isotopes of carbon include¹³C and¹⁴c, isotopes of oxygen including¹⁶O and¹⁸o, and the like.

The term "API" refers to (R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoic acid.

Detailed Description

The following representative examples are intended to better illustrate the present invention and are not intended to limit the scope of the present invention. The materials used in the following examples are all commercially available unless otherwise specified.

Example 1: preparation of (R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoic acid

Step 1: preparation of ethyl 2- (3- (3- (ethoxycarbonyl) phenyl) ureido) -4-methylthiophene-3-carboxylate

Methylene chloride (79.5kg), ethyl 2-amino-4-methylthiophene-3-carboxylate (6.0kg) and N, N' -carbonyldiimidazole (5.777kg) were charged into a 100L reactor, and reacted overnight at 20 to 25 ℃ under nitrogen. After the reaction, triethylamine (3.605kg) and ethyl-3-aminobenzoate methanesulfonate (9.309kg) were added at 25 ℃ and reacted overnight at 20-25 ℃. After the reaction is finished, slowly adding the reaction solution into heptane in batches, stirring for 0.5-1 h after the reaction solution is added, centrifuging, and rinsing with water; pulping the wet product with water at 10-20 deg.C for 2 hr, centrifuging, and rinsing with water. The solid was air dried at 70. + -. 5 ℃ to give the title compound in a total of 9.68kg at 79.40% yield.

Step 2: preparation of 3- (5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoic acid

1, 4-dioxane (33.08kg) and ethyl 2- (3- (3- (ethoxycarbonyl) phenyl) ureido) -4-methylthiophene-3-carboxylate (3.2kg) were added to a 100L reactor, the temperature was raised to 50. + -. 5 ℃ and stirred until all ethyl 2- (3- (3- (ethoxycarbonyl) phenyl) ureido) -4-methylthiophene-3-carboxylate was dissolved, potassium tert-butoxide (2.38kg) was added and the reaction was carried out at 50. + -. 5 ℃ for 0.5 h. And after the reaction is finished, adding purified water, reacting for 0.5h, stopping the reaction, slowly adding hydrochloric acid in batches, adjusting the pH to 2-3, crystallizing for more than 1h at 35 +/-5 ℃, then crystallizing for more than 8h at 15 +/-5 ℃, centrifuging, and rinsing with water to obtain a solid. The above charge, reaction and work-up steps were repeated in two further 100L reaction vessels. The solids obtained in three times were combined and air dried at 70 ℃ for 8h or more to give the title compound in a total of 7.00kg, 90.90% yield.

And step 3: preparation of ethyl 3- (5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoate

In a 100L reactor, N-dimethylformamide (39.7kg), 3- (5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoic acid (6.98kg) and N, N' -carbonyldiimidazole (5.62kg) were added and reacted at 35. + -. 5 ℃ for 2 hours under nitrogen. Anhydrous ethanol was added to the reaction mixture, and the mixture was reacted at 35. + -. 5 ℃ overnight. After the reaction is finished, the reaction solution is slowly added into water in batches, and stirred for more than 3 hours at 15 +/-5 ℃. Centrifuging, rinsing with water, and air drying at 70 deg.C for more than 8 hr. The title compound was obtained in a total of 6.62kg with a yield of 96.79%.

And 4, step 4: preparation of ethyl 3- (6-bromo-5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoate

Adding N, N-dimethylacetamide (41.38kg) and ethyl 3- (5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoate (2.945kg) into a 100 reaction kettle, cooling to-20 ℃ after solid is dissolved, dropwise adding a solution of N, N-dimethylacetamide (13.79kg) of N-bromosuccinimide (1.587kg), keeping the temperature not higher than-20 ℃, and stirring for 0.5H after the dropwise adding is finished. After the reaction is finished, heating until the solid is dissolved, dropwise adding purified water, controlling the temperature to be not higher than 30 ℃, after the dropwise adding is finished, crystallizing for more than 3 hours at the temperature of 10-15 ℃, centrifuging, and rinsing with water to obtain the solid. The above charge, reaction and work-up steps were repeated in a further 100L reactor. The solids from both were combined and dried by air blast at 70 ℃ for more than 8h to give the crude title compound in a total of 6.105kg with 83.92% yield.

Adding 1, 4-dioxane (33.08kg) and a crude product (3.05kg) of ethyl 3- (6-bromo-5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoate into a 100L reaction kettle, heating to 85 ℃, dropwise adding purified water (13.70kg) after solid is dissolved, maintaining the temperature and stirring for 15-30 min after dropwise adding, filtering while hot, crystallizing at 15 +/-5 ℃ for 1H, centrifuging, and rinsing with water; refluxing and pulping wet product with methanol for 1h, crystallizing at 15 + -5 deg.C for 1h, centrifuging, and rinsing with methanol to obtain solid. The above charge, reaction and work-up steps were repeated in a further 100L reactor. The solids obtained in both steps were combined and dried by air blow at 70 ℃ for 8h or more to give the title compound in a total of 5.05kg with a yield of 82.80%.

And 5: preparation of ethyl (R) -3- (6-bromo-1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoate

1-methyl-2-pyrrolidone (29.390kg) and 3- (6-bromo-5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2, 3-d) were charged in a 50L vertical jacketed reaction kettle]Ethyl pyrimidin-3 (4H) -yl) benzoate (2.859kg), (R) -4- (2-bromo-1- (2-methoxyphenyl) ethoxy) tetrahydro-2H-pyran (2.000kg), and potassium carbonate (1.450kg) were stirred on, with nitrogen protection, heated on, with the temperature controlled at 120-130 ℃. After the reaction is completed, the temperature of the reaction solution is reduced to 25 +/-5 ℃. Adding methyl tert-butyl ether and water, stirring for 20-30min, standing for layering, removing water layer, and retaining organic layer. The organic layers were combined, water (28.600kg) was added, stirred, allowed to stand to separate layers, the aqueous layer was removed, and the organic layer was retained. The above charge, reaction and work-up steps were repeated in a further 50L reactor. The two organic layers were combined, concentrated under reduced pressure, rinsed with ethanol to give a solid which was air dried at 50 + -5 deg.C to give the title compound in a total of 2.530kg with 61.87% yield. ESI-MS [ M + H ]]⁺m/z:643.1。

Step 6: preparation of ethyl (R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoate

In a 10L four-necked reaction flask, 1, 4-dioxane (6.460kg), (R) -ethyl 3- (6-bromo-1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoate (1.250kg), 2-dicyclohexylphosphonium-2 ',4',6' -triisopropylbiphenyl (0.557kg), tris (dibenzylideneacetone) dipalladium (0.267kg) and 2- (tri-n-butylstannyl) oxazole (0.769kg) were charged, replaced with nitrogen gas for 3 to 5 times, heated and stirred with being turned on, reflux for 30min and stop the reaction. Filtering the reaction solution at 60-100 deg.C, removing solid, and collecting filtrate. The above charge, reaction steps and post-treatment steps were repeated in an additional 10L four-necked reaction flask. The two filtrates were combined and concentrated under reduced pressure. The solid was rinsed with ethanol and dried by air blow at 50 + -5 deg.C to give 2.090kg of crude title compound.

Adding methanol (4.100kg), ethyl acetate (4.680kg) and the crude product (2.085kg) obtained in the previous step into a 50L reaction kettle, heating, refluxing and stirring, adding mercaptosilica gel (0.100kg) when the temperature is 70 +/-5 ℃, stirring for 30min, then carrying out suction filtration while the solution is hot, transferring the filtrate into the 50L reaction kettle, cooling to 0-10 ℃, crystallizing for more than 4 hours, filtering, leaching the filter cake with methanol (0.395kg) to obtain a solid, and carrying out forced air drying on the solid at 60 +/-5 ℃ for more than 12 hours to obtain 1.310kg of the title compound with the yield of 53.30%. ESI-MS [ M + H ]]⁺m/z:632.2。

And 7: preparation of (R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoic acid

Methanol (4.27kg) and tetrahydrofuran (14.4kg) were added to a 100L reaction kettle, ethyl (R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoate (2.156kg) was added, stirred and dissolved, then a solution of lithium hydroxide (0.131kg) in water (5.390kg) was added, the temperature was controlled at 0 to 10 ℃, and after the dropwise addition, the reaction was stirred at room temperature. After the reaction is completed, cooling the reaction solution to 0-5 ℃, adding hydrochloric acid (1.08kg), adjusting the pH to 1-2, centrifugally filtering the precipitated solid, washing a filter cake with water (5.000kg), and drying by air blowing at 60 +/-5 ℃ for more than 12 hours. The title compound was obtained in a total of 2.025kg with a yield of 98.4%.

And 8: purification of (R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoic acid

Ethanol (13.880kg) was added to a 100L reactor and the (R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2, 3-d) prepared in the above step was added]Pyrimidin-3 (4H) -yl) benzoic acid (1.000kg), dissolved by heating with stirring, and added

Stirring Thiol (0.050kg) for 20min, adding active carbon (0.050kg), stirring for 20min, and filtering. Transferring the filtrate into a 50L reaction kettle, cooling to 10 ℃, stirring for crystallization, and filtering to obtain a solid. Forced air drying at 70 + -5 deg.C gave 0.825kg of the title compound in 82.5% yield.¹H NMR(400MHz,DMSO-d₆)δ13.11(s,1H),8.25(s,1H),8.04(d,1H),7.83(d,1H),7.67(t,1H),7.52(t,2H),7.42(s,1H),7.32(t,1H),7.04(dd,2H),5.35-5.32(m,1H),4.17-4.14(m,1H),4.04-3.98(m,1H),3.80(s,3H),3.62-3.59(m,2H),3.44-3.41(m,1H),3.31-3.24(m,2H),2.79(s,3H),1.72-1.65(m,2H),1.30-1.24(m,2H)。ESI-MS[M+H]⁺m/z:604.2。

Examples 2 to 17

Examples 2 to 17 shown in tables 1 to 3 are formulations prepared using the compound of formula (I) (API for short) prepared in example 1 of the present invention and various disintegrants, fillers, wetting agents, lubricants and the like.

TABLE 1

TABLE 2

Examples	8	9	10	11
					Unit of	mg/tablet	mg/tablet	mg/tablet	mg/tablet
Batch size	100 pieces	100 pieces	100 pieces	100 pieces
					API	20	20	20	20
Microcrystalline cellulose PH101	62.4	62.4	62.4	60.9
					Mannitol 160C	62.4	62.4	62.4	60.9
Croscarmellose sodium SD711	/	/	4.5	7.5
					Sodium carboxymethyl starch Vivastar P	/	4.5	/	/
Low substituted hydroxypropyl cellulose LH-21	4.5	/	/	/
					Magnesium stearate	0.7	0.7	0.7	0.7
Water (W)	Proper amount of	Proper amount of	Proper amount of	Proper amount of

TABLE 3

Examples 2-17 were prepared as follows:

(1) API, microcrystalline cellulose PH101 and lactose were weighed in accordance with the recipes of examples 2 to 17 shown in tables 1 to 3

100. Mannitol 160C, croscarmellose sodium SD711, magnesium stearate, carboxymethyl starch sodium Vivastar P, and low substituted hydroxypropyl cellulose LH-21, sieving with 60 mesh sieve, shaking, mixing, and premixing;

(2) adding a proper amount of wetting agent into the mixed material in the step (1), manually sieving the mixed material by a 20-mesh sieve, and granulating;

(3) drying the particles in the step (2) in an oven at the temperature of 60 ℃, and screening the dried particles through a 24-mesh screen for dry granulation;

(4) and (4) tabletting the mixed material obtained in the step (3) by using a DP30A single-punch tablet press.

Examples 18 to 19

Examples 18-19 shown in table 4 are formulations made using the compound of formula (I) (API) prepared in example 1 of the present invention and various disintegrants, fillers, wetting agents, lubricants, etc.

TABLE 4

The preparation of examples 18 to 19 is as follows:

(1) API and lactose were weighed according to the recipes for examples 18 to 19 shown in Table 4

100. Respectively sieving croscarmellose sodium SD711, microcrystalline cellulose VIVAPUR102 and magnesium stearate with a 60-mesh sieve for pretreatment;

(2) premixing by using an experimental multifunctional wet mixing granulator of G10 GYITET, setting premixing parameters as stirring 5r/s and cutting 10r/s, and pre-treating lactose in step (1)

100 and API are mixed for 2min, then the cross-linked sodium carboxymethyl cellulose SD711 pretreated in the step (1) is added and continuously mixed for 2 times, each time is 2min, and the mixture is mixed for 6 min;

(3) carrying out wet granulation on the granules in the step (2) by adopting a Xinyite G10 experimental multifunctional wet mixing granulator, adding 252G of wetting agent into 10000 tablets in batches with the specification of 5mg, adding liquid for about 2min, and granulating for 30s after liquid adding is finished; the liquid adding amount of 10000 tablets with the specification of 20mg is 268g, the liquid adding time is about 2min, the granules are granulated for 30s after the liquid adding is finished, and the stirring speed is 5r/s and the cutting speed is 15 r/s;

(4) drying the granules obtained in the step (3) in an oven at the temperature of 60 +/-5 ℃, controlling the moisture to be below 3.0 percent, and finishing the drying;

(5) finishing the dry finished particles in the step (4) by using a P-100 finishing machine, wherein the aperture of a screen is 1.0mm, and the rotating speed is 100 rpm;

(6) adopting a Nano SRH30 experimental tank-changing mixer to carry out total mixing, firstly adding the granules obtained in the step (5) after size stabilization and the microcrystalline cellulose VIVAPUR102 obtained in the step (1) after pretreatment into a total mixing tank, wherein the frequency is 50Hz, the rotating speed is 2000rpm, and the mixing time is 10min, and then adding the magnesium stearate obtained in the step (1) after pretreatment into the total mixing tank, wherein the frequency is 50Hz, the rotating speed is 60rpm, and the mixing time is 3 min;

(7) and (4) tabletting the mixed material obtained in the step (6) by using a ZP10A rotary tablet press, wherein the tabletting hardness is controlled to be 3-9 kg.

Experimental example 1: EXAMPLES 2 to 19 measurement of elution Properties

Dissolution test method: dissolution test (second method of 0931 in the four general guidelines of the pharmacopoeia of China 2015).

The method comprises the following specific operations: respectively using pH1.2-1.0% SDS, pH4.0-0.5% SDS, water-0.5% SDS, pH6.8-0.05% SDS, 900ml as dissolving medium; dissolution conditions: the temperature is 37 +/-0.5 ℃, the paddle method is adopted, and the rotating speed is 50 rpm; sampling volume is 5 ml; sampling time: 5min, 10min, 15min, 20min, 30min, 45min, 60 min; according to the measurement of high performance liquid chromatography (China pharmacopoeia 2015 edition four parts general rules 0512), an Agilent Eclipse Plus C18 chromatographic column (4.6X 100mm, 3.5 mu m) is used as a chromatographic column, 0.1% phosphoric acid water solution-acetonitrile (35:65) is used as a mobile phase, the flow rate is 1mL/min, the detection wavelength is 310nm, and the column temperature is 30 ℃. Precisely measuring 10 μ L of each of the test solution and the reference solution, respectively injecting into a high performance liquid chromatograph, recording chromatogram, and calculating dissolution amount of each granule by peak area according to external standard method, wherein the dissolution rate is shown in tables 5, 6 and 7.

TABLE 5

TABLE 6

TABLE 7

The experimental results show that: the dissolution rate of the embodiment of the invention in an acid medium is too slow, no obvious difference is seen in each part when the dissolution rate in water is 30min, and the embodiment has better dissolution rate in SDS with the pH value of 6.8-0.05 percent; the preparations obtained in examples 2 to 19 were all sufficiently dissolved out.

Experimental example 2: stability of

The preparation prepared in the above examples is coated and packaged, and the packaging materials are polyvinyl chloride solid medicinal dark brown hard tablet (specification 250mm multiplied by 0.30mm) and medicinal aluminum foil (specification 250 multiplied by 0.024 mm). Under the packaging, the inner package (the inner package is polyvinyl chloride solid medicinal dark brown hard sheet (dark brown) and medicinal aluminum foil) is removed, and the package is respectively irradiated under high temperature (60 deg.C), high humidity (75% +/-5% and 92.5% +/-5%), strong light (illuminance 4500Lux, near ultraviolet energy 90 μ w/cm)²) Standing for 5 days, 10 days and 30 days under the condition; under the condition of the package, the preparation is placed for 6 months under accelerated test conditions (40 ℃ plus or minus 2 ℃ and RH 75% + orminus 5%), samples are respectively taken for one time in 1 month, 2 months, 3 months and 6 months, and the preparation is placed under long-term test conditions (30 ℃ plus or minus 2 ℃ and RH 65% + orminus 5%), samples are taken for one time every 3 months for detection, and the detection result shows that the preparation has stable property, few impurities and no obvious change of various indexes.

From the above experimental results, it can be seen that the (R) -3- (1- (2- (2-methoxyphenyl) -2- ((tetrahydro-2H-pyran-4-yl) oxy) ethyl) -5-methyl-6- (oxazol-2-yl) -2, 4-dioxo-1, 2-dihydrothieno [2,3-d ] pyrimidin-3 (4H) -yl) benzoic acid preparation containing the disintegrant of the present invention has complete dissolution, and has stable preparation properties under high humidity, high temperature, light and accelerated and long-term test conditions, less impurities, no obvious change in each index, and meets the clinical medication requirements; the prepared compound of the formula (I) has good absorption property and high relative bioavailability.

Experimental example 3: in vitro acetyl-CoA carboxylase (ACC) inhibition assay

1 materials of the experiment

1.1 Compounds

The control compound is the compound ND-630 disclosed in patent WO2013/071169, example I-181, which is currently the most promising drug in the clinic for such diseases, with the chemical name (2- [1- [2- (2-methoxyphenyl) -2- (oxacyclohex-4-yloxy) ethyl ] -5-methyl-6- (1, 3-oxazol-2-yl) -2, 4-dioxo-1H, 2H,3H, 4H-thieno [2,3-d ] pyrimidin-3-yl ] -2-methylpropionic acid), prepared with reference to the methods described in patent WO2013/071169 and identified by hydrogen and mass spectrometry.

Preparation of compound: the compounds of the invention and control compounds prepared in example 1 above were each prepared at 10mM in DMSO, and were used in the assay at 1000nM, 333.3nM, 111.1nM, 37.1nM, 12.3nM, 4.12nM, 1.37nM, 0.46nM, 0.15nM, 0.05nM in a 3-fold dilution from 1000 nM.

1.2 Primary reagents

HEPES buffer from Invitrogen; MgCl₂Potassium citrate buffer solution, DTT, acetyl-CoA and NaHCO₃Purchased from Sigma company; BRIJ-35 available from MERCK corporation; ACC1 and ACC2 enzymes were both purchased from BPS bio; ADP-Glo^TMThe kinase kit was purchased from Promega.

1.3 consumables and instruments:

96-well polypropylene plates were purchased from Nunc; oscillators are available from QILINBEIER; centrifuge is available from Eppendorf; 384 well whiteboards and Envision 2104 plate readers are available from Perkin Elmer.

2 method of experiment

2.1. Reagent preparation

1 × reaction buffer (pH 7.4): with HEPES (1M), MgCl₂Preparing enzyme activity reaction buffer solution from stock solutions of (1M), BRIJ-35 (10%), potassium citrate buffer solution (1M), BSA (10mg/mL) and DTT (500 mM): comprising HEPES (50mM), MgCl₂(2mM), BRIJ-35 (0.01%), potassium citrate buffer solution (2mM), BSA (50. mu.g/mL), and DTT (2 mM).

ACC enzyme Activity test

1) ACC1 enzyme activity test

Add 4.5 μ L of 2.2 × ACC1 enzyme (2nM) working solution to 384-well plates; then 0.5. mu.L of different concentrations of the compound was added and incubated for 15min at room temperature.

2 Xsubstrate (40. mu.M ATP, 20. mu.M acetyl CoA, 60mM NaHCO3) was prepared with the buffer prepared in 2.1; add 5. mu.L of 2 Xsubstrate to 384 well plates and incubate for 30min at room temperature; then adding 10 mu L ADP-Glo reagent, incubating for 40min at room temperature, and stopping reaction; finally 20. mu.L of enzyme detection reagent was added, incubated at room temperature for 40min, and the fluorescence signal (RLU) was read using an Envision 2104 instrument.

2) ACC2 enzyme activity test

Add 4.5 μ L of 2.2 × ACC2 enzyme (1.1nM) working solution to 384-well plates; then 0.5. mu.L of different concentrations of the compound was added and incubated for 15min at room temperature.

2 Xsubstrate (40. mu.M ATP, 40. mu.M acetyl CoA, 24mM NaHCO) was prepared with buffer prepared in 2.1₃) (ii) a Add 5. mu.L of 2 Xsubstrate to 384 well plates and incubate for 30min at room temperature; then adding 10 mu L ADP-Glo reagent, incubating for 40min at room temperature, and stopping reaction; finally 20. mu.L of enzyme detection reagent was added, incubated at room temperature for 40min, and the fluorescence signal (RLU) was read using an Envision 2104 instrument.

3 Experimental data processing

Negative control group: vehicle containing 5% DMSO; positive control group: containing 100nM ND-630. The data for each concentration and positive and negative controls were averaged and the standard deviation calculated. Percent inhibition was calculated from the formula: inhibition (100%) < 100 × (RLU)_{Negative control}-RLU_{Compound (I)})/(RLU_{Negative control}-RLU_{Positive control}). Inhibition data IC was calculated for each compound using non-linear regression equation fitting₅₀The nonlinear regression equation is Y ═ lowest + (highest-lowest)/(1 +10 + lowest)^{((LogIC50 -X)×HillSlope)}) Where X is the logarithm of the concentration of the compound and Y is the percentage inhibition (100%).

4 results of the experiment

TABLE 8

The results of the above experiments show that the compounds of the present invention have good inhibitory activity against both ACC1 and ACC 2.

Experimental example 4 evaluation of liver distribution in rat

1. Experimental Material

1.1 animals

Male SD rats, SPF grade, purchased from shanghai sierpickika laboratory animals ltd; 220- > 250g, license number: SCXK (Shanghai) 2013-0016. An adaptation period of 2-3 days was given before the experiment. Fasting is carried out for 8-12 h before administration, water is supplied after administration for 2h, and food is supplied after 4 h.

1.2 test Compounds

The compound of formula (I) of the invention of example 1 and the control compound ND-630.

1.3 instruments

API model 4000 triple quadrupole LC MS and Analyst QS A01.01 chromatography workstation were purchased from AB SCIEX, USA; Milli-Q ultrapure water was purchased from Millipore corporation; CF16R XII bench-top high speed refrigerated centrifuge from Hitachi; the Qilinbeier Vortex-5 oscillator was purchased from IKA, Germany; the electric heating constant temperature water bath kettle is purchased from China electric appliance, Inc. of Changzhou; electric pipettors were purchased from Thermo corporation, usa; microanalysis balances were purchased from Shanghai Meltrier, Inc.

2. Experimental methods

2.1 preparation of test drugs

Weighing 6mg (calculated by free alkali) of a test compound, adding the test compound into 20mL of ethanol-PEG 400-normal saline (10:30:60), vortexing for 2min, performing ultrasonic treatment for 3min, and preparing a test solution with the concentration of 0.3mg/mL for oral administration; taking 100 mu L of the test solution, diluting the solution to a constant volume of 10ng/mL by using methanol, preparing a reference substance with equal concentration, performing HPLC (high performance liquid chromatography) sample loading detection on the concentrations of the test solution and the reference solution, and calculating the accuracy of the test solution.

2.2 sample Collection

SD rats were given 3mg/kg of test compound orally in a single dose at a volume of 10mL/kg, and after administration, rats were bled at 0.25h, 1h and 4h carotid artery, respectively, euthanized to remove liver, immediately collected liver and blood (anticoagulated with heparin sodium), and placed on ice.

2.3 liver sample processing and analysis

Weighing 0.4g liver, cutting, homogenizing in 2mL 75% methanol-water, centrifuging (centrifugation condition: 8000rpm/min, 5min, 4 deg.C), transferring supernatant, freezing, redissolving before sample injection, centrifuging, collecting supernatant, and analyzing the content of compound in the supernatant sample by LC-MS/MS.

2.4 plasma sample processing and analysis

The collected whole blood sample is placed in an ice box, centrifuged within 30min (centrifugation conditions: 8000rpm/min, 5min, 4 ℃) and 100 mu L of upper plasma is transferred, 300 mu L of methanol is added for precipitation, the mixture is oscillated and centrifuged, a mobile phase is added for dilution, a supernatant is taken, and the content of the compound in the supernatant sample is analyzed by LC-MS/MS.

3 results of the experiment

TABLE 9

The higher the concentration of the compound in the liver, the higher the potency for treating liver diseases, the better the therapeutic effect at the same dose, and the higher the liver/plasma ratio, indicating that the stronger the target organ selectivity of the test compound, the better the safety of the compound may be. From the results, the compound has higher distribution in the liver, and good liver selectivity and targeting (liver/blood ratio is more than 50), so the compound is expected to become a more effective and safer medicament for treating metabolic liver diseases such as fatty liver, non-alcoholic fatty liver disease (NASH) and the like.

Experimental example 5: in vitro human liver stellate cell LX-2 activation inhibition experiment

1 materials of the experiment

1.1 Compounds preparation examples of the compounds of formula (I) according to the invention and a control compound ND-630, each compound being formulated in DMSO to give 60mM, ready for use.

1.2 cell lines

Human hepatic stellate cell LX-2, established by professor Xuelung in the center for liver disease of West Neishan medical college, USA, and stored in Shanghai institute for liver disease.

1.3 Primary reagents

DMEM medium, FBS, pancreatin, phosphate buffer (DPBS) and penicillin-streptomycin diabody were purchased from GIBCO, usa; recombinant human TGF-. beta.1 cytokine (PeproTech Co., Cat. No.: 100-21); TransZOL Up Plus RNA extraction kit (gold full scale, cat # ER 501-01); cDNA reverse transcription kit (Whole formula gold, cat # AH 341-01); 5 XSSYBR Green qPCR kit (QuantiNovaTM, cat # 154045739).

1.4 consumables and instruments:

CKX41 inverted microscope, Olympus, Japan, multifunctional microplate reader, Molecular Devices, U.S.A., Thermo Nano Drop 2000 nucleic acid quantitation analyzer, ABI 9700PCR amplification apparatus, ABI 7500PCR quantitation, Thermo high speed centrifuge (MEGAFUGE 8); fully automatic ice making machines (snow, IMS-30).

2 method of experiment

2.1. Reagent preparation

DMSO stock solutions of the compounds of the examples of the present invention and the control compounds were diluted to 30. mu.M, 10. mu.M, and 3. mu.M in this order with the medium. TGF-. beta.1 was dissolved to 1. mu.g/mL in 10mM citric acid buffer as formulated with the kit according to the PeproTech kit instructions for use.

LX-2 cell treatment

2X 10 after passage of LX-2 cells⁵The cells were plated at a density of 2mL in 6-well plates in DMEM (2 mL/5% CO) containing 10% FBS₂The cells were cultured in an incubator at 37 ℃ and recorded as Day 1. After 24h (Day2), the confluency reached 70-80%, the old medium was discarded and the cells were starved with serum-free DMEM. The old culture medium is discarded in Day3, and the culture medium or the culture medium containing drugs with different concentrations is added to continue the incubation culture, so that the culture medium is divided into a control group (serum-free DMEM culture medium), a TGF beta 1 group, a TGF beta 1+ compound group and a TGF beta 1 working concentration of 10 ng/mL. After 24h of drug action (Day4), the cell supernatant was aspirated and the cells were washed 1 time with pre-chilled 1 × PBS for total RNA extraction.

2.3. Total RNA extraction

2.3.1 sample pretreatment: adding 1mL of TransZOL Up reagent into each hole of a 6-hole cell culture plate, horizontally placing for a moment to ensure that a lysate is uniformly distributed on the cell surface and lyses cells, blowing the cells by using a pipette gun to ensure that the cells completely fall off, transferring the lysate into a 2mL RNase free centrifuge tube, and repeatedly blowing and sucking until the lysate does not have obvious precipitation.

2.3.2 extraction step: the procedure was performed according to the instructions of the TransZOL Up Total RNA extraction kit.

2.4. Total RNA concentration and purity determination

mu.L of total RNA was put in a NanoVue spectrophotometer to detect absorbance at A260nm wavelength, and the RNA concentration was calculated. The purity of the RNA sample is calculated according to the ratio of the light absorption values of 260nm and 280nm (A260/A280), the ratio is in the range of 1.8-2.1, and the RNA is proved to be free from pollution and degradation, so that the RNA can be used for subsequent tests.

Synthesis of cDNA

Diluting the extracted RNA to the corresponding concentration of 0.1-0.5 mug (Total RNA is less than or equal to 1 mug) according to the equal mass, and testing the mass of the Total RNA in the reverse transcription system of each sample to be 500 ng.

The following procedures were performed according to the reverse transcription kit (TransScript II All-in-One First-Strand cDNA Synthesis kit, Lot: AH 341-1). The synthesized cDNA was stored at-70 ℃ for further use.

The above system was mixed gently, the above system liquid was put into ABI 9700PCR instrument, and the following procedure was set: 50 ℃ X15 min → 85 ℃ X5 s → 4 ℃ X10 min, and the obtained cNDA is stored at-20 ℃ or used immediately.

Real-time PCR reaction

The Real-Time PCR primers used:

PCR sample system:

after sample application, the mixture was gently mixed, centrifuged, and the PCR tube was placed in a PCR instrument, and the procedure was as follows.

Setting the PCR instrument in a circulating way:

each sample is provided with 2 multiple wells, a PCR instrument sets a program, and the relative expression quantity of the target gene is calculated.

3 Experimental data processing

The threshold of the Real Time PCR result was automatically set by the Real Time PCR detector system, and the relative content of the Col1a1 gene was calculated as follows.

Δ Ct (drug treatment group Col1a1 gene) ═ avg.ct (drug treatment group Col1a1 gene) -avg.ct (drug treatment group GAPDH gene);

Δ Ct (TGF group Col1a1 gene) ═ avg.ct (TGF group Col1a1 gene) -avg.ct (TGF group GAPDH gene);

Δ Ct (control Col1a1 gene) ═ avg.ct (control Col1a1 gene) -avg.ct (control GAPDH gene);

Δ Δ Ct ═ average of Δ Ct (TGF group/drug treatment group Col1a1 gene) - Δ Ct (control group Col1a1 gene); the relative content calculation formula of the Col1A1 gene is as follows: RQ ═ 2^-ΔΔCt

The relative quantitative result is automatically analyzed by an ABI 7500 real-time quantitative fluorescent PCR instrument.

4 results of the experiment

TABLE 10 calculation of relative content of Col1A1 Gene

Compound (I)	Inhibition ratio (%)
		Example 1	82.11
ND-630	42.06

Collagen 1 is a key signaling factor in the process of liver fibrosis, and its expression is represented by the content of Col1A1 gene. Experimental results show that the compound has obvious inhibition activity on expression of the Collagen 1 gene of LX-2 cells induced by TGF-beta 1. Compared with ND-630, the compound of the invention has stronger inhibitory activity on the fibrosis form of the liver cells, and can be used for ACC-mediated fibrosis diseases, proliferative diseases and the like.

Experimental example 6 evaluation of NASH and hepatic fibrosis drug effects induced by HFD-CCL4

The method comprises the steps of firstly inducing animal liver steatosis by High Fat Diet (HFD), and then inducing liver inflammation, necrosis and hepatic fibrosis by carbon tetrachloride (CCL4), wherein the process and pathological phenomena of the model are similar to those of human NASH diseases. The objective of this experiment was to evaluate the efficacy of the compounds of the invention in the NASH model of HFD-CCL 4-induced C57BL/6 mice, with ND-630 as a control compound. HFD-CCL4 was induced for 10 weeks, drug intervention was performed for 4 weeks, and the effect of the drug on NASH and hepatic fibrosis was observed.

1. Experimental Material

1.1 instruments

A dehydrator Leica HistoCore PEARL; a paraffin embedding machine Leica HistoCore Arcadia C & H; paraffin slicer Leica RM 2235; automatic stain Leica ST 5020; scanner HAMAMATSU NANO Zoomer S210; SR staining analysis software Visiopharm VIS 6.6.0.2516.

1.2 animals

C57BL/6 mice (male, 18-20g) were purchased from Wei Tony Hua, Inc., Beijing. Experimental animal feeding all experimental procedures were approved by the KCI animal use and welfare committee (IACUC). The mouse feeding conditions were as follows: the temperature is 20-25 ℃, the humidity is 40-70%, and the light and shade alternation time is 12 hours/12 hours. Bedding was changed 2 times per week.

2. Experimental methods

2.1 Compounds preparation of the inventionThe compound of formula (I) of example 1 and ND-630 were prepared using PEG200:0.2M Na₂HPO₄-NaH₂PO₄The buffer solution (35:65) is diluted to 0.3mg/mL, 1mg/mL, 3mg/mL for administration and ready to use.

2.2 animal Molding

HFD-CCL4 induced the C57BL/6 mouse NASH model: after the animals are adaptively raised in a central SPF barrier of a KCI experimental animal for 3-7 days, the animals are replaced with HFD feed for raising, and the raising period is 10 weeks. At the end of week 6 of HFD feeding, HFD groups were randomized according to animal body weight, 10 per group, and CCL4 was administered orally (three times a week, 9-10 am) for 4 weeks. Detailed modeling method a male C57BL/6 mouse NASH model induced by HFD-CCL4 was established according to the established method of KCI, and the modeling agent was Olive Oil + CCL4 solution (prepared by KCI). The remaining 10 animals were given normal maintenance feed with concomitant rearing as normal control animals.

Animals were divided into a normal control group, HFD-CCL4 model group (model group), and compound group (compound group of formula (I) of example 1 of the present invention, ND-630 group).

2.3 dosing regimens for Compounds

After the end of the 6 th week of HFD feeding, gavage administration of the compound of formula (I) of example 1 of the present invention and ND-630 was started once a day for 4 weeks, and the administration was ended at the 10 th week. The doses of the compound of formula (I) of example 1 of the present invention and ND-630 group were each 30 mg/kg/d.

2.4 Experimental sample Collection

The next day after the end of the last dose, i.e., 48 hours after the last dose of CCL4, animals in each group were fasted for six hours and were euthanized according to KCI standard operating procedures. Dissecting animals according to KCI animal dissection experiment operating procedures, collecting livers after the animals are perfused into the whole body by low-temperature PBS, and rapidly freezing part of animal livers (the same liver lobe on the left side of each animal is fixedly selected) by liquid nitrogen and storing at the low temperature of-80 ℃. And fixing the rest animal liver with formalin (the volume ratio of the liver to the fixing solution is 1:10), and performing related pathological correlation detection.

2.5 hematoxylin-eosin staining

Liver left leaf was fixed with 10% formalin, embedded with paraffin, and prepared into 5 μm sections for hematoxylin-eosin (H & E) staining. Hematoxylin-eosin staining reflects the degree of tissue inflammation, fat deposition, vacuolar degeneration and tissue fibrosis, and semi-quantitative analysis is performed on the degree of lesion.

2.6 Tianlang scarlet staining

The liver tissue was sliced to 5 μm, dried for 2h, rehydrated, stained with Tianlang scarlet (Beijing Haidechun, cat # 26357) for 30min at room temperature, and then dehydrated and mounted for image analysis. The pathological sections were scanned 200 × fold with Aperio ScanScope CS2 (lycra), and the scanned pictures were opened in the Aperio ImageScope program to remove blood vessels, leaving the target image for analysis with Color resolution v9 algorithm. The portion of fibrosis stained red was identified as a positive signal using the software and the percentage of fibrosis calculated.

3. Statistical analysis

Data are expressed as mean ± sem. The significance analysis used students t-test, one way ANOVA or two way ANOVA and post-hoc Dunnett's test.

4. Results of the experiment

4.1 hepatic steatosis

When experimental animals are fed with high-fat diet for 10 weeks, compared with normal control group, the degree of liver steatosis of the model group is obviously deepened. The compound group (30mg/kg/d) of example 1 showed no significant loss of steatosis compared with the model group, no difference from the normal control group, and significant superiority to the ND-630 group (30 mg/kg/d). The results are shown in Table 11.

TABLE 11 hepatic steatosis

Therefore, the compound has a certain therapeutic effect on a mouse NASH model induced by HFD-CCL 4; in histopathology, liver steatosis can be effectively reduced compared with a model group.

Although the present invention has been described in detail above, those skilled in the art will appreciate that various modifications and changes can be made to the present invention without departing from the spirit and scope of the invention. The scope of the invention is not to be limited by the above detailed description but is only limited by the claims.

Claims (10)

1. A pharmaceutical composition comprising:

(a) a compound of formula (I) or a solvate or crystal thereof,

and (b) a disintegrant.

2. The pharmaceutical composition according to claim 1, wherein the compound of formula (I) is in crystalline form or amorphous form.

3. The pharmaceutical composition according to claim 1 or 2, wherein the disintegrant is one or more of sodium starch glycolate, crospovidone, croscarmellose sodium and low substituted hydroxypropyl cellulose.

4. A pharmaceutical composition according to any one of claims 1 to 3, further comprising a lubricant, preferably said lubricant is selected from one or more of magnesium stearate, calcium stearate, stearic acid, zinc stearate, sodium stearyl fumarate, talc, polyethylene glycol, glyceryl behenate, glyceryl monostearate, glyceryl palmitostearate, sodium lauryl sulphate and hydrogenated vegetable oils.

5. The pharmaceutical composition according to any one of claims 1 to 4, further comprising a filler, preferably the filler is selected from one or more of microcrystalline cellulose, powdered cellulose, magnesium carbonate, calcium sulfate dihydrate, pregelatinized starch, mannitol, starches, lactose, sugars, celluloses and inorganic salts.

6. The pharmaceutical composition according to any one of claims 1 to 5, further comprising a wetting agent, preferably the wetting agent is water.

7. The pharmaceutical composition according to any one of claims 1-6, consisting of a compound of formula (I) or a solvate or crystal thereof, a disintegrant, a lubricant, and a filler, wherein the compound of formula (I) or the solvate or crystal thereof comprises about 1% to 80% (w/w) of the total weight of the pharmaceutical composition, the disintegrant comprises about 1% to 30% (w/w) of the total weight of the pharmaceutical composition, the lubricant comprises about 0% to 10% (w/w) of the total weight of the pharmaceutical composition, and the filler comprises about 0% to 90% (w/w) of the total weight of the pharmaceutical composition, based on the amount of the compound of formula (I).

8. A pharmaceutical formulation comprising the pharmaceutical composition of any one of claims 1-7, preferably said pharmaceutical formulation is a tablet, granule, powder, sustained release, pill, capsule, lozenge or cachet.

9. A method of preparing the pharmaceutical formulation of claim 8, the method comprising:

(1) uniformly mixing a compound of formula (I) or a solvate or crystal thereof, a disintegrant and optional filler and a lubricant;

(2) optionally adding a proper amount of wetting agent into the mixed material in the step (1) for wet granulation;

(3) drying the granules of step (2);

(4) optionally, uniformly mixing the particles obtained in the step (3) with a filler and a lubricant;

(5) and (4) preparing the mixed material in the step (4) into a preparation.

10. Use of the pharmaceutical composition according to any one of claims 1 to 7 or the pharmaceutical preparation according to claim 8 for the manufacture of a medicament for the treatment and/or prevention of a disease associated with ACC expression.