CN107903252B

CN107903252B - Triazole derivative and application thereof in preparation of drugs for treating liver diseases

Info

Publication number: CN107903252B
Application number: CN201711047043.5A
Authority: CN
Inventors: 许勇; 范昭泽; 于静
Original assignee: Wuhan Jiuzhou Yumin Medical Technology Co ltd
Current assignee: Wuhan Jiuzhou Yumin Medical Technology Co ltd
Priority date: 2017-10-31
Filing date: 2017-10-31
Publication date: 2020-04-24
Anticipated expiration: 2037-10-31
Also published as: CN107903252A

Abstract

The invention discloses a triazole compound for treating liver diseases. The compound is a compound shown as a formula I, and pharmaceutically acceptable salt, hydrate, solvate or metabolite thereof. The compound can be used for preparing medicaments for treating and/or preventing liver diseases.

Description

Triazole derivative and application thereof in preparation of drugs for treating liver diseases

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a triazole derivative and application thereof in preparing a medicine for treating liver diseases.

Background

Liver diseases are common diseases and frequently encountered diseases in China, and comprise fatty liver, various acute and chronic hepatitis, hepatic fibrosis, cirrhosis, liver cancer and other series diseases, and the liver diseases are mostly caused by excessive obesity, medicines, excessive drinking, hepatitis B and hepatitis C virus infection and the like. Estimated by the world health organization, the number of deaths due to various types of hepatitis is approximately 140 to 150 thousands per year, with the 7 th among various diseases. And the world health organization warns that hepatitis will soon surpass aids as a 6 th leading health killer in humans. China has become the world with the greatest social costs for hepatitis, cirrhosis and liver cancer. According to the statistics of the disease prevention and control center in China: more than 100 ten thousand patients with newly increased viral hepatitis are reported every year in China, wherein the proportion of hepatitis B patients is maintained at about 80 percent; the number of hepatitis B virus surface antigen carriers in the whole country is more than 1.2 hundred million, which accounts for 8-10% of the total population in the whole country and approximately 1/3% of the total number of hepatitis B virus carriers in the whole world; and nearly 100 million people are infected with new hepatitis B every year.

According to the internal network of rice, the total market scale of the liver disease medicines in China has increased from 233.28 million yuan in 2010 to 537.005 million yuan in 2015, the total market scale of the liver disease medicines in China in 2014 and 2015 is 459.61 million yuan and 537.05 million yuan respectively, and the growth rate is 16.85%; the overall size of the liver disease treatment drug market is expected to maintain an annual growth rate of 15-20%.

In particular, no medicine is currently on the market in China for the Fatty liver (Fatty liver) indication. The seal oil omega-3 polyunsaturated fatty acid of Jinnukang organisms in Zhejiang is applied to market and is in the registration stage. In addition, obeticholic acid (obeticholic acid) was approved by the FDA in the united states on day 5-27 of 2016, the first approved indication for the treatment of primary biliary cirrhosis, and its indications for non-alcoholic fatty liver disease are currently in phase 3 clinical in the united states and europe and in phase 2 clinical studies in japan. For Liver fibrosis (Liver fibrosis), a total of 46 drugs for this indication have been developed: the number of the clinical samples is 2, 1 registration, 2 clinical samples at the stage 3, 8 clinical samples at the stage 2, 4 clinical samples at the stage 1, 28 clinical samples before clinical samples and 1 study stop. The two marketed drugs are gamma-interferon of a compound star medicine and a traditional Chinese medicine capsule for strengthening the body resistance and removing blood stasis of Shanghai Huanghai medicine respectively. For Cirrhosis (Cirrhosis), through further screening, 2 drugs with market data and subdivided indications of Primary Biliary Cirrhosis (PBC) are ursodeoxycholic acid and obeticholic acid respectively. In addition, the number of the treatment or diagnosis medicines related to hepatocellular carcinoma or liver tumor which are marketed at home and abroad is 14: doxorubicin, iodine [131I ] methacyline, cisplatin, miplatin, 18F-fluorocholine chloride, DW-166HC, Immunsell-LC, recombinant human interleukin-2, arsenious acid, disodium gadoxetate, neat stastine, gadofosamine, pirarubicin, and sorafenib. Wherein 5 of the 14 drugs except miboplatin, 18F-fluoro choline chloride, DW-166HC, Immunsell-LC and netstastin ester do not enter the China market, and the other 9 drugs are already on the market in China. 2 of the 9 medicaments (gadoxetate disodium and gadoform amine) are used for medical imaging diagnosis, and only sorafenib is used for targeted therapy of liver cancer at present, but the adverse reactions of liver cancer patients taking sorafenib are reported to be more.

In order to achieve better treatment effect for liver diseases such as fatty liver, liver cirrhosis, liver fibrosis, liver cancer and the like, and to better meet the clinical and market demands, development of safer and more efficient drugs for treating liver diseases is urgently needed.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a novel triazole compound for solving the defects that the existing indications have no proper treatment medicines and the existing clinical medicines are overcome, and the compound can be used for treating and/or preventing liver diseases such as fatty liver, liver cirrhosis, hepatic fibrosis, liver cancer and the like.

The invention provides a compound shown as a formula I, and pharmaceutically acceptable salts, hydrates, solvates and metabolites thereof,

wherein, R is₁Is selected from phenyl, pyridyl, cyclohexyl or cyclopentyl, each of which is optionally substituted with 1-3R₄Substitution; or R₁Is optionally substituted by 1-2R₄Or phenyl substituted cyclopropyl; preferably R₁Is selected from 1 to 3R₄Substituted phenyl;

the R is₂Is selected from C_1-4Alkyl, halo C_1-4Alkyl, unsubstituted C_3-8Cycloalkyl, or optionally substituted C_3-8A cycloalkyl group; preferably R₂Is selected from cyclopropyl, cyclobutyl, or cyclopentyl;

the R is₃Is selected from H, C_1-10Alkyl radical, C_3-8Cycloalkyl radical, C_2-10Alkenyl radical, C_2-10Alkynyl, aryl, heteroaryl or heterocyclyl, all substituents being optionally substituted by 1, 2 or 3 substituents selected from halogen, oxo, C_1-6Alkyl radical, C_3-8Cycloalkyl, heterocyclyl, phenyl, phenoxy, halogen, -CN, -O-R₅、-C(O)-R₅、-OC(O)-R₅-C(O)-O-R₅、-N(R₅)-C(O)-O-R₆、-N(R₅)-C(O)-R₆、-N(R₅)-C(O)-N(R₅)(R₆) and-C (O) -N (R)₅)(R₆) Wherein alkyl, cycloalkyl, heterocyclyl, phenyl, and phenoxy are optionally substituted with 1, 2, or 3 substituents selected from C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_1-6Alkoxy, hydroxy, and halogen;

the R is₄Is selected from halogen, C_1-6Alkyl, halo C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkoxy, -SR₇Or cyclopropyl;

the R is₅And R₆Independently selected from hydrogen, C_1-6Alkyl, or C_3-8A cycloalkyl group; or R₅And R₆When taken together with the nitrogen to which they are attached form a heterocyclic ring;

according to some embodiments of the invention, R is preferred₃Is selected from H, C_1-10Alkyl, or C_3-8A cycloalkyl group;

according to some embodiments of the invention, R is more preferred₃Is selected from H, methyl, ethyl, isopropyl, tert-butyl, or cyclopropyl;

the R is₇Is selected from C_1-6Alkyl, preferably R₇Is selected from methyl, ethyl, isopropyl, or tert-butyl;

x is selected from O or NH, preferably X is selected from O.

Thus, throughout this specification, the skilled person will be able to refer to the R in the compounds of formula I₁～R₇And X and substituents thereof are selected to provide a stable compound of formula I or a pharmaceutically acceptable salt, hydrate, solvate, or metabolite thereof as described in embodiments of the invention.

According to an embodiment of the present invention, the compound of formula I according to the present invention is any one of the following compounds:

the compound of formula I of the invention can be prepared according to conventional chemical synthesis methods in the field, and the steps and conditions thereof can refer to the steps and conditions of similar reactions in the field. The reaction solvent used in each reaction step described in the present invention is not particularly limited, and any solvent that can dissolve the starting materials to some extent and does not inhibit the reaction is included in the present invention. Further, many equivalents, substitutions, or equivalents in the art to which this invention pertains, as well as different proportions of solvents, solvent combinations, and solvent combinations described herein, are deemed to be encompassed by the present invention.

The invention provides a method for preparing a compound shown in a formula I, which comprises the following synthetic route:

the carboxylic acid of formula I-a is reacted with the amine of formula I-b under conditions suitable to form an amide. For example, (2- (7-aza-1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium Hexafluorophosphate (HATU) and a base, typically N-methylmorpholine, are added to a mixture of a compound of formula I-a and a compound of formula I-b in an inert solvent such as N, N-Dimethylformamide (DMF) and the mixture is maintained at about room temperature for about 3 to 18 hours.

The compounds of the invention may also be isolated and purified according to standard techniques well known to those skilled in the art. One particularly useful technique, such as in the purification of compounds, is preparative liquid chromatography, which uses mass spectrometry as a means of detecting pure compounds flowing from a chromatographic column.

Preparative LC-MS is a standard efficient method for purifying small organic molecules, such as the compounds described herein. The Liquid Chromatography (LC) and Mass Spectrometry (MS) methods can be modified to allow better crude separation and to improve MS detection of the sample. Optimization of preparative gradient LC methods involves changing the column, volatile eluent and modulators and gradients. These methods are well known in the art of optimizing preparative LC-MS methods, which are employed to purify compounds. Such methods are described in the following documents: rosentretere u, Huber u.; an Optimal fraction collecting in preliminary LC/MS; j CombChem; 2004; 159-64 and Leister W, Strauss K, Wisnoski D, ZHao Z, Lindsley C, Development of a custom high-throughput predictive chromatography/mass spectrometer platform for the predictive purification and analytical analysis of compound libraries; j Comb chem.; 2003; 5 (3); 322-9.

The compound of formula I of the present invention can be used as an active ingredient, and can be added with pharmaceutical additives such as carriers or excipients to prepare a preparation. Any of the conventionally generally employed forms such as orally administered preparations in a form suitable for absorption from the digestive tract, e.g., tablets, granules, capsules, liquid preparations for internal use, parenterally administered preparations such as injections, suppositories, patches, and transdermal preparations such as pastes, solid preparations, and liquid preparations, and the like, can be suitably used, and from the viewpoint of the flow property, the storage property, and the like, it is necessary to dissolve a solid solvent in a suitable solvent at the time of use. In addition, in order to improve the bioavailability and stability of the present compound, a drug delivery system including a preparation technique such as microencapsulation, micronization or inclusion may be used.

The invention provides a pharmaceutical composition comprising a compound of formula I, a pharmaceutically acceptable salt, hydrate, solvate, or metabolite thereof, and a pharmaceutically acceptable adjuvant; the compound of formula I, a pharmaceutically acceptable salt, hydrate, solvate, or metabolite thereof, can be used in a therapeutically effective amount.

While it is possible for a compound of formula I as described herein to be administered as the active compound alone, it is preferred to present it as a pharmaceutical composition (e.g., formulation) comprising at least one active compound of the invention and one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilizers, preservatives, lubricants or other materials well known to those skilled in the art, and optionally other therapeutic or prophylactic agents. Thus, the present invention also provides a pharmaceutical composition as defined above and a process for the preparation of a pharmaceutical composition, which process comprises admixing at least one active compound as defined above with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilizers or other materials as described herein.

In the pharmaceutical composition, the compound of formula I, a pharmaceutically acceptable salt, hydrate, solvate, or metabolite thereof, may be used in a therapeutically effective amount.

The pharmaceutical excipients can be those widely used in the field of pharmaceutical production. The excipients are used primarily to provide a safe, stable and functional pharmaceutical composition and may also provide methods for dissolving the active ingredient at a desired rate or for promoting the effective absorption of the active ingredient after administration of the composition by a subject.

The invention also provides application of the compound shown in the formula I, pharmaceutically acceptable salt, hydrate, solvate or metabolite thereof in preparing medicines for treating and/or preventing liver diseases. The liver diseases include but are not limited to fatty liver, liver cirrhosis, liver fibrosis, liver cancer and the like.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is standard in the art to which the claimed subject matter belongs. In case there are multiple definitions for a term, the definitions herein control. When referring to a URL or other identifier or address, it should be understood that such identifier may change and that particular information on the internet may change, but equivalent information may be found by searching the internet. The reference demonstrates that such information is available and publicly disseminated.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Furthermore, the term "comprising" is open-ended and not closed-ended.

The present invention employs, unless otherwise indicated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques or pharmacological detection, and the various steps and conditions may be referred to those conventional in the art. Unless otherwise indicated, the present invention employs standard nomenclature for analytical chemistry, organic synthetic chemistry, and medicinal chemistry, as well as standard laboratory procedures and techniques. In some cases, standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and drug delivery, and treatment of patients.

The term "pharmaceutically acceptable" as used herein is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from the compounds of the present invention found to have particular substituents, with relatively nontoxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like; and salts of organic acids including acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like; also included are salts of amino acids (e.g., arginine, etc.), and salts of organic acids such as glucuronic acid (see Berge et al, "Pharmaceutical salts," Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds of the invention contain both basic and acidic functionalities and can thus be converted to any base or acid addition salt. Preferably, the neutral form of the compound is regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms by certain physical properties, such as solubility in polar solvents.

The term "pharmaceutically acceptable salts" as used herein pertains to derivatives of the compounds of the present invention wherein the parent compound is modified by salification with an acid or by salification with a base. Examples of pharmaceutically acceptable salts include, but are not limited to: inorganic or organic acid salts of bases such as amines, alkali metal or organic salts of acid groups such as carboxylic acids, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound, for example, salts formed with non-toxic inorganic or organic acids. Conventional non-toxic salts include, but are not limited to, those derived from inorganic or organic acids selected from the group consisting of 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptose, gluconic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, hydroiodide, hydroxynaphthalene, isethionic acid, lactic acid, lactose, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, glycolic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannin, tartaric acid, and p-toluenesulfonic acid.

The "pharmaceutically acceptable salts" of the present invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, such salts are prepared by the following method: prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid, in water or an organic solvent or a mixture of the two. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in polycrystalline or amorphous form.

The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, a compound such as tritium (3H), iodine-125 (125I) or C-14(14C) may be labeled with a radioisotope. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

The term "effective amount" or "therapeutically effective amount" with respect to a drug or pharmacologically active agent refers to a sufficient amount of the drug or agent that is non-toxic but achieves the desired effect. For oral dosage forms of the invention, an "effective amount" of one active agent in a composition is the amount required to achieve the desired effect when combined with another active agent in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient and also on the particular active substance, and an appropriate effective amount in an individual case can be determined by a person skilled in the art according to routine tests.

The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

The term "comprising" is open-ended, i.e. includes the elements indicated in the present invention, but does not exclude other elements.

According to the embodiment of the invention, the triazole compound is convenient to prepare and low in production cost.

According to the embodiment of the invention, the triazole compound can obviously inhibit ballooning swelling of hepatic cells with NASH pathological state and reduce the area rate of CD68 positive cells. Has good curative effect on non-alcoholic steatohepatitis (NASH), and the effect is superior to that of the existing medicine.

According to the embodiment of the invention, the triazole compound disclosed by the invention can reduce the degeneration and necrosis degree of rat hepatocytes, can obviously reduce the fibroplasia degree of rats, has an obvious anti-hepatic fibrosis effect, and is small in dosage and high in safety.

According to the embodiment of the invention, the triazole compound has anti-liver cancer activity, and has good inhibition effect on the growth of human liver cancer cell strains HepG2, Hep3b and SMMC-7721.

According to the embodiment of the invention, the triazole compound has no obvious inhibition effect on an hERG channel in a cardiac hERG experiment, and shows good cardiac safety.

Therefore, the compound can be used as a therapeutic drug for liver diseases, and is used for treating and/or preventing liver diseases, such as fatty liver, liver cirrhosis, liver fibrosis, liver cancer and other liver diseases. The triazole compound can be used for preparing a medicament for treating and/or preventing liver diseases.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The embodiment of the invention provides a compound shown in formula I or a pharmaceutically acceptable salt, hydrate, solvate or metabolite thereof, a method and an intermediate for preparing the compound shown in formula I or the pharmaceutically acceptable salt, hydrate, solvate or metabolite thereof, a pharmaceutical composition and application of the compound in preparing a medicament.

EXAMPLE 1 preparation of Compound I-1

Compound I-a1(440mg, 1.05mmol), compound I-b1(203mg, 1.0mmol), (2- (7-aza-1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium Hexafluorophosphate (HATU) (456mg, 1.2mmol) and N-methylmorpholine (13. mu.L, 1.2mmol) were added to DMF and stirred at 70 ℃ for 8H, the solvent was removed by concentration under reduced pressure, the residue was suspended in acetonitrile and the solid product was isolated by filtration, washed with water (80mL), acetonitrile (80mL), acetone (80mL) and dried under vacuum to give 374mg of a white-like solid (yield 62%), which was product Compound I-1 (LC/MS: [ M + H ] (M + H)]⁺605)。

Examples 2-5 preparation of Compounds I-2 to I-5

The compounds I-2 to I-5 were prepared in a similar manner to example 1, except that the starting compounds used were different. After the reaction is finished, separating the products to respectively obtain compounds I-2 to I-5, and verifying the products by LC/MS.

Compound I-2 (LC/MS: [ M + H ]]⁺523)。

Compound I-3 (LC/MS: [ M + H ]]⁺568)。

Compound I-4 (LC/MS: [ M + H ]]⁺565)。

Compound I-5 (LC/MS: [ M + H ]]⁺549)。

EXAMPLE 6 Effect of the Compounds of the invention in the NASH model of mice with LDL receptor Gene knockout

The present invention discusses a role in high fat/high cholesterol diet (Western diet) loaded LDL receptor knockout mice (Yoshimatsu m. et al, int.j.exp.path, 85, 335-43(2004)) known to have the pathological condition characteristic of NASH and the onset of hepatitis.

Animals were used: male LDL receptor knockout mice were used in the experiments. During 13 weeks from about 8 weeks of age, they were allowed to freely ingest a high fat/high cholesterol diet, which was subject to NASH. Mice were subjected to a high fat/high cholesterol diet load and blood sampling and body weight measurements were performed 1 week later.

Groups were divided into the following groups in such a way that there was no difference in plasma lipid and body weight between groups: a control group (0.5% aqueous methylcellulose solution), a group to which 0.25mg/kg of the compound represented by the formula I-1 of the present invention is administered (the drug group of the present invention), and a group to which 100mg/kg of fenofibrate is administered.

Administration of the drug: the administration volume was 5ml/kg body weight, 1 time per 1 day by oral administration, 0.5% methylcellulose aqueous solution was administered to the control group, and the compound represented by formula I-1 of the present invention and a liquid medicine of fenofibrate were administered to the drug group of the present invention and the fenofibrate administration group, respectively. The administration period was 12 weeks.

Observation and inspection methods: after the administration, the liver was excised under anesthesia with pentobarbital sodium (50mg/kg), fixed with paraformaldehyde, and then hematoxylin-eosin stained specimens and CD68 immunostained specimens were prepared. Balloon-like enlargement of hepatocytes was scored using hematoxylin-eosin stained specimens under blind examination conditions according to the following criteria (Kleiner et al, Hepatology 41, 1313-21, 2005), see table 1. The area rate of CD 68-positive cells in the liver under blind examination was determined by an image analysis system (WinROOF).

TABLE 1

Grade	Scoring
		Balloon-free type swollen cells	0
Balloon-like enlargement with few cells	1
		Balloon-like swelling with multiple or significant cells	2

The results show that: (1) when the drug group of the present invention was administered at 0.25mg/kg, significant suppression of balloon-like enlargement of hepatocytes was confirmed. In the group administered with 100mg/kg of fenofibrate, the inhibition of balloon-like enlargement was confirmed, but it was not statistically significant. (2) The area rate of CD68 positive cells in the liver was significantly reduced (reduction rate 85.5%) by the administration of 0.25mg/kg of the drug group of the present invention. And the reduction rate of the CD68 positive cell area rate of the fenofibrate group is 75%.

The results show that: the drug group of the invention is superior to fenofibrate in balloon swelling effect of hepatic cells for inhibiting NASH pathological state and reduction of CD68 positive cell area rate.

Example 7 use of the Compounds of formula I of the present invention in the treatment of KK-A with a methionine/choline deficiency of the dietary burden^yEffect in non-alcoholic steatohepatitis (NASH) model in mice

This experiment examined the effects in KK-Ay mice (Nakano s. et al, Hepatol res., 38(10), 1026-39, 2008) on MCD diet loaded mice known to develop fatty liver, a pathological condition characteristic of NASH, by subjecting the experimental animals to a methionine/choline deficient diet (MCD diet) load.

Animals were used: male KK-Ay mice were used in the experiment and were allowed to freely ingest the MCD diet starting at about 12 weeks of age for NASH to develop.

Comprises the following components: the group was divided into the following groups in such a way that there was no difference in body weight between the groups: a normal diet group, a control group (MCD diet load), a group to which 0.25mg/kg of the compound represented by the formula I-2 of the present invention was administered (group of the present invention), and a group to which bezafibrate was administered at 60 mg/kg.

Administration of the drug: by mixed feeding administration. The control group was allowed to freely ingest the MCD diet containing no drug, the group of the present invention was allowed to freely ingest the MCD diet containing 0.00025% of the compound represented by formula I-2, and the group administered bezafibrate was allowed to freely ingest the MCD diet containing 0.06% bezafibrate. The dosing period was 16 weeks.

The observation and inspection method comprises the following steps: after the administration, the liver was removed under anesthesia with pentobarbital sodium (50mg/kg), fixed with paraformaldehyde, and then hematoxylin-eosin stained specimens were prepared. Fatty liver scores (steatoscore) were evaluated under blind examination. The Grade of fat deposition (Grade) was observed at 100-fold magnification, and fatty liver was scored at 0 to 3 according to the following criteria, as shown in table 2.

TABLE 2

Grade	Scoring
		Less than 5 percent	0
More than 5 percent and less than 33 percent	1
		More than 33 percent and less than 66 percent	2
Over 66 percent	3

As a result: in the group of the present invention, all the examples had a fatty liver score of 0, i.e., the fatty deposits were almost completely disappeared. In the group administered with 60mg/kg of bezafibrate, although the fat deposition had been suppressed to the same extent as in the normal diet group, the fat deposition did not reach the extent that the fat deposition was almost completely disappeared as in the present invention group. It is thus clear that the compounds of formula I according to the invention inhibit fat deposition, one of the nasH pathologies, more strongly than bezafibrate. The results show that: hair brushKK-A of MCD diet load of a Compound of formula I in model animals of NASH^yIn mice, a significant reduction in fatty deposits of the liver was shown. Therefore, the compound of the present invention is useful as a prophylactic and/or therapeutic agent for non-alcoholic fatty liver disease, particularly highly severe non-alcoholic steatohepatitis, in mammals including humans. The compound shown in the formula I can be used for preparing medicines for treating non-alcoholic steatohepatitis

(NASH) preventive/therapeutic agent, and the pharmaceutical effect thereof is superior to that of the existing drug bezafibrate.

Example 8 Effect of the Compound of formula I of the present invention on the degree of liver fibroplasia in liver fibrosis rats

SPF-grade Wistar rats 46 with body weights of 190-220 g and half male and female. Rats were randomly divided into 4 groups of a normal control group, a model control group, a positive control group, and the drug group of the present invention. Except for normal control group, the first rat of other groups was injected with pure CCl subcutaneously₄5ml/kg body weight, followed by subcutaneous injection of 40% CCl₄Peanut oil solution 3ml/kg body weight, every 3 days for 1 time, total 6 weeks. Feeding normal solid feed, and freely drinking water.

After molding, the following doses of the respective test substances were administered: according to the weight, the positive medicine rat gavage colchicine 5 mg.kg^-1·d^-1The drug group of the invention rats were gavaged with 1 mg/kg of the compound of the formula I-4 of the invention^-1·d^-1The rats of the normal control group and the model control group were given the same volume of distilled water. Gavage is carried out for 1 time every day for 10 weeks, each group is fasted for 12 hours after the last administration, weighed, anesthetized by pentobarbital sodium intraperitoneal injection, the liver of the right lobule of a rat is taken, liver tissue with the thickness of about 0.5 cm is cut, fixed by neutral formaldehyde buffer solution, embedded by paraffin, and sliced to the thickness of 5 mu m. The pathological tissue photos are taken under a light microscope, and the change condition of the proliferation degree of the fibrous tissues is observed. By observing pathological sections, the following findings are found:

normal control group: the liver lobule structure in the liver tissue of the rat is clear and complete, the liver cell cord is arranged orderly, and the liver cell necrosis, inflammatory cell infiltration and fibroplasia are avoided;

model control group: liver tissues of rats can be seen with hepatic cell arrangement disorder, hepatic cell vacuolation-like degeneration and necrosis, a large amount of fibrous tissues in a sink area are proliferated and dispersed in inflammatory cells for infiltration, and most of the visible fibrous tissues of rats extend to hepatic lobules to separate the hepatic lobules to form false lobules;

a positive drug group: the degeneration and necrosis of the liver cells of the rats are reduced, and false lobules can be seen in part of the rats;

the medicine group of the invention is: the degeneration and necrosis of the liver cells of the rats are reduced, and pseudo lobules can be seen in part of the rats.

The fibrous tissue proliferation was most severe with "++++" and "-" no fibrous proliferation observed under a light microscope. The liver tissue section of the normal control group rat shows no liver fibrosis, and the fiber hyperplasia of the model group is obvious, which indicates that the model is successfully made. The specific results are shown in Table 3.

TABLE 3 grading comparison of tissue collagen fiber proliferation degree of rats in each group

The results show that: the drug group of the invention reduces the degeneration and necrosis degree of rat liver cells and can obviously reduce the fibroplasia degree of rats. The medicine of the compound shown in the formula I has obvious anti-hepatic fibrosis effect. In addition, the compound shown in the formula I-4 has stronger hepatic fibrosis resistance than colchicine, and has smaller administration dosage than colchicine and high safety.

Experimental example 11: MTT method is adopted to evaluate the growth inhibition effect of the compound shown as the formula I-5 on human liver cancer cell strains

1. The method comprises the following steps: cells in log phase of growth: human liver cancer cell strains HepG2, Hep3b and SMMC-7721 at a ratio of 1.5 × 10⁴The concentrations were plated in 96-well plates. The original culture medium is aspirated after 24h of cell culture adherence. The test is divided into a blank control group and a drug treatment group. Replacing the blank group with 1640 culture medium containing 10% fetal bovine serum; the drug treatment group contained 100. mu.M, 50. mu.M, 10. mu.M, 1. mu.M, 0.1. mu.M concentrationsM, 0.01. mu.M and 0.001. mu.M of Houttuynoid C. After 48h incubation, MTT was added at a concentration of 5mg/mL and CO was added₂Culturing in an incubator for 4h, then absorbing 100 μ L of supernatant along the upper part of the culture, adding 100 μ L of LDMSO, standing in the dark for 10min, measuring the absorbance (wavelength 570nm) with a microplate reader (Sunrise), and calculating the survival of cells according to the absorbance, with 6 duplicate wells for each treatment. Cell survival rate (%). DELTA.OD_{Drug treatment}/ΔOD_{Blank control}×100。

2. As a result: the compounds shown in the formula I-5 have the effects on human liver cancer cell strains HepG2, Hep3b and

the growth of SMMC-7721 has significant inhibitory effect. IC of the compound for inhibiting growth of human hepatoma cell strains HepG2, Hep3b and SMMC-7721₅₀The values are respectively: 9nM, 19nM, 26 nM.

The examples show that the compound shown in the formula I has good inhibition effect on the growth of human liver cancer cell strains HepG2 and SMMC-7721. Therefore, the compound shown in the formula I has the anti-liver cancer activity and can be used for preparing anti-liver cancer medicines.

Example 20 cardiac hERG experiments

The influence of the compound on the hERG potassium ion channel is detected by using a manual patch clamp method, and the results show that the compound disclosed in the embodiments 1-5 of the invention has no effect of inhibiting the hERG current to IC when the compound is at the highest test concentration (30 mu M)₅₀I.e. IC₅₀Are all made of>30 μ M. Thus, it was demonstrated that the test compounds described in examples 1-5 of the present invention did not significantly inhibit hERG channel at the concentrations tested in this assay. All 5 samples showed good cardiac safety within the scope of this test. Amitriptyline (Amitriptyline) is one of the most widely used tools for blocking hERG current, and therefore, it was used as a positive control in this study, its IC₅₀The concentration was 3.16. mu.M (note: the IC50 for hERG current inhibition by positive control Amitriptyline in this study was 3.16. mu.M. this result is in agreement with the results reported in the literature (Block of the HERG human cardiac K + channel by the anti-reactive drug therapy. British Journal of Pharmacol)Jo, SH et al, (2000)). This indicates that the results of this experiment are authentic.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example" or "some examples" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims

1. A compound shown as a formula I or a pharmaceutically acceptable salt thereof, wherein the compound shown as the formula I is any one of the following compounds:

2. a pharmaceutical composition comprising a compound of formula I as defined in claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant; the compound of formula I or a pharmaceutically acceptable salt thereof may be used in a therapeutically effective amount.

3. Use of a compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 2 for the preparation of a medicament for the treatment and/or prevention of liver diseases.

4. The use of claim 3, wherein the liver disease is fatty liver, cirrhosis, liver fibrosis, or liver cancer.