CN110627774A

CN110627774A - Anti-hepatic fibrosis compound, preparation method and application

Info

Publication number: CN110627774A
Application number: CN201910978213.4A
Authority: CN
Inventors: 艾观华; 彭维杰; 刘宇政; 罗丹; 冷红文; 古超伦; 赵林; 赵芙蓉; 刘玉婷; 李婷婷
Original assignee: Jiangxi Academy Of Medical Sciences; Nanchang University; Gannan Medical University
Current assignee: Jiangxi Academy Of Medical Sciences; Nanchang University; Gannan Medical University
Priority date: 2019-10-15
Filing date: 2019-10-15
Publication date: 2019-12-31

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an anti-hepatic fibrosis compound, a preparation method and application, wherein the structural formula is as follows:the medicine of the present invention can obviously improve hepatic fibrosis, improve liver function indexes, reduce the accumulation of extracellular matrixes such as collagen in the extracellular matrix, reduce the degree of hepatic fibrosis, and inhibit the formation and the development of hepatic fibrosis.

Description

Anti-hepatic fibrosis compound, preparation method and application

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an anti-hepatic fibrosis compound, a preparation method and application.

Background

Hepatic fibrosis is a process of diffuse excessive deposition of liver extracellular matrix (ECM) caused by various pathogenic factors, and is a common pathological pathway for most chronic liver diseases. Further progression of liver fibrosis can lead to cirrhosis of the liver and possibly even further hepatocellular carcinoma. Research proves that hepatic fibrosis can be reversed, and no definite anti-hepatic fibrosis treatment medicine is found clinically at present, so that the search for a treatment medicine capable of effectively reversing hepatic fibrosis has important clinical significance. Autotaxin (ATX) is a secreted glycoprotein, has phosphodiesterase and lysophospholipase D activities, and clinical case reports show that ATX in serum is positively correlated with hepatic fibrosis degree, and clinical studies also show that patients with viral infection (HBV and HCV) have increased serum ATX level, liver cirrhosis and liver cancer incidence; preliminary mechanism research shows that the active product LPA of ATX can activate hepatic stellate cells and promote collagen secretion. Therefore, ATX is also considered to be a potent serum marker of liver fibrosis and a target for developing anti-liver fibrosis drugs.

Research on autotaxin inhibitors has progressed, late rain sprout, etc., in central south medicine 2016, 9, 14 th, 9 th, and various ATX inhibitors are described, 1, ATX inhibitors of fatty alcohol phosphatidic acid structure, including enteron (darmstoff), cyclic phosphatidic acid (cPA), α -halomethylenephosphonic acid, alkyl aromatic methylenephosphonic acid, and inhibitors of tyrosine backbone structure, each of which consists of 1 electron-rich polar phosphate moiety and 1 hydrophobic chain; 2. small molecule ATX inhibitors including ATX inhibitors having a long linear or flexible structure, ATX inhibitors of rigid structural molecules, and metal ion chelating molecule ATX inhibitors; 3. other structures of ATX inhibitors.

Disclosure of Invention

The invention aims to solve the technical problem of providing an anti-hepatic fibrosis compound, a preparation method and application.

The invention comprises an anti-hepatic fibrosis compound, namely 2- ({5- [1- (4-chlorophenoxy) ethyl ] -4-phenyl-4H-1, 2, 4-triazol-3-yl } thio) -N- (1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazol-4-yl) acetamide, with the following structural formula:

the invention provides a preparation for resisting hepatic fibrosis, which comprises a compound for resisting hepatic fibrosis, and the preparation is tablets, coated tablets, sugar-coated pills, hard and soft gelatin capsules, solutions, emulsions, suspensions, nasal sprays, suppositories, ointments, gels or injections.

The preparation for resisting hepatic fibrosis can be a medicinal salt of a compound for resisting hepatic fibrosis, and can also be used in the form of a medicinal preparation. The pharmaceutical formulation may be administered internally in the following manner: such as orally (for example in the form of tablets, coated tablets, dragees, hard and soft gelatine capsules, solutions, emulsions or suspensions), nasally (for example in the form of nasal sprays) or rectally (for example in the form of suppositories) or topically ocularly (for example in the form of solutions, ointments, gels or water-soluble polymer inserts). Administration can also be effected parenterally, such as intramuscularly, intravenously or intraocularly (for example in the form of sterile injectable solutions).

The anti-hepatic fibrosis preparation can be processed with pharmaceutically inert, inorganic or organic adjuvants, and can be used for preparing tablet, coated tablet, sugar-coated pill, hard gelatin capsule, injection or topical preparation. Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts and the like can be used, for example, as such auxiliary materials for tablets, dragees and hard gelatine capsules.

Suitable excipients for soft gelatine capsules are, for example, vegetable oils, waxes, fats, semi-solid substances, liquid polyols and the like. Suitable adjuvants for the preparation of solutions and syrups are, for example, water, polyols, sucrose, invert sugar, glucose and the like. Suitable excipients for injection solutions are, for example, water, alcohols, polyols, glycerol, vegetable oils and the like. Suitable excipients for suppositories are, for example, natural or hardened oils, waxes, fats, semi-solid or liquid polyols and the like. Suitable adjuvants for topical ocular formulations are, for example, cyclodextrins, mannitol, or many other carriers and excipients known in the art. In addition, the pharmaceutical preparations may contain preservatives, solubilizers, viscosity-increasing substances, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for regulating the osmotic pressure, buffers, masking agents or antioxidants; they may also contain other therapeutically valuable substances.

The invention provides a preparation method of an anti-hepatic fibrosis compound, which comprises the following steps:

adding 4-chloroacetylamidoantipyrine, 5- (1- (4-chlorophenoxy) ethyl) -4-phenyl-4H-1, 2, 4-triazole-3-thiol and potassium carbonate into acetone, refluxing, filtering, evaporating filtrate to dryness, and recrystallizing residue to obtain compound for resisting hepatic fibrosis.

The preparation method of the 4-chloracetyl amino antipyrine comprises the steps of adding chloracetyl chloride and triethylamine into a dichloromethane solution containing the 4-amino antipyrine, and stirring at room temperature; then adding ice water into the solution for extraction, taking an organic layer, washing the organic layer by using a hydrochloric acid solution, then washing by using a saturated sodium bicarbonate aqueous solution, and then washing by using brine; after drying, removing the solvent to obtain a crude product; recrystallizing the crude product to obtain the 4-chloroacetylaminoaantipyrine.

The preparation method of the 5- (1- (4-chlorophenoxy) ethyl) -4-phenyl-4H-1, 2, 4-triazole-3-thiol comprises the steps of dissolving 2- (2- (4-chlorophenoxy) propionyl) -N-phenylhydrazine thiocarboxamide in a sodium hydroxide solution, heating and refluxing, cooling, adjusting the pH value to 2-3, filtering, and drying to obtain the 5- (1- (4-chlorophenoxy) ethyl) -4-phenyl-4H-1, 2, 4-triazole-3-thiol.

The 2- (2- (4-chlorophenoxy) propionyl) -N-phenylhydrazine thiocarboxamide is prepared by adding 2- (4-chlorophenoxy) propionylhydrazine and phenyl isothiocyanate to absolute ethyl alcohol, refluxing, cooling the mixture to room temperature, filtering, and concentrating the filtrate to obtain 2- (2- (4-chlorophenoxy) propionyl) -N-phenylhydrazine thiocarboxamide.

The preparation method of the 2- (4-chlorophenoxy) propionylhydrazine comprises the steps of dissolving 2- (4-chlorophenoxy) propionate in ethanol, and then adding a hydrazine water solution; the mixture was then stirred under reflux, concentrated in vacuo, and washed to give 2- (4-chlorophenoxy) propionylhydrazide.

The preparation method of the 2- (4-chlorophenoxy) propionic ester comprises the steps of dissolving the 2- (4-chlorophenoxy) propionic acid in methanol, and then slowly adding 98% concentrated sulfuric acid; stirring under reflux, cooling to room temperature, concentrating in vacuo, and dissolving the residue in dichloromethane, washing, drying, and removing the solvent to give 2- (4-chlorophenoxy) propionate.

The specific reaction route is as follows:

the invention provides application of an anti-hepatic fibrosis compound in preparing an ATX inhibitor, an anti-hepatic fibrosis preparation or a liver cancer treatment drug. Anti-hepatic fibrosis compounds when used as ATX inhibitors, anti-hepatic fibrosis preparations or drugs for treatment of liver cancer, the dosage may vary within wide ranges and will of course be adapted to the individual requirements in each particular case. Generally, in case of oral administration, a daily dose of about 0.1mg to 20mg per kg body weight, preferably about 0.5mg to 4mg per kg body weight (e.g. about 300mg per person), preferably divided into 1-3 individual doses which may e.g. consist of the same amount, should be suitable. In the case of topical administration, the formulation may contain from 0.001% to 15% by weight of the drug, and the required dose (which may be 0.1-25mg) may be administered by a single dose per day or week, or by multiple doses (2 to 4) per day, or by multiple doses per week. However, it will be clear that, when explicitly indicated, the upper or lower limits given herein may be exceeded.

The compound is an inhibitor specially aiming at human ATX enzyme, can well act on target enzyme ATX, regulates the in vivo LPA level by inhibiting ATX so as to play a role in preventing or treating hepatic fibrosis, and determines the IC of the compound through immunofluorescence detection₅₀The value is 43.05 mu M, and the compound has better anti-hepatic fibrosis effect as shown in figures 3-5.

The compound is an ATX inhibitor, can inhibit lysophospholipase D from catalyzing and generating lysophosphatidic acid LPA by taking Lysophosphatidylcholine (LPC) as a substrate through inhibiting the activity of the lysophospholipase D, can be used as a regulator of LPA level and related signal transduction, is a specific inhibitor of ATX, can inhibit the generation or further aggravation of hepatic fibrosis through inhibiting the activity of the ATX, and has good clinical application prospect.

The compound can well improve the hepatic fibrosis of mice through mouse anti-fibrosis efficacy tests, and has a bright prospect in developing medicaments for treating the hepatic fibrosis in the future.

Drawings

FIG. 1 is a mass spectrum of the compound.

FIG. 2 shows the compound¹H-NMR spectrum.

FIG. 3 shows CC1 used alone₄Sirius red staining pattern of mouse liver tissue section of mouse liver injury model.

FIG. 4 shows the use of high dose (30 mg. kg)^-1·d^-1) Inventive drug treatment CC1₄Sirius red staining pattern of mouse liver tissue section of mouse liver injury model.

FIG. 5 shows the use of a low dose (15 mg. kg)^-1·d^-1) Inventive drug treatment CC1₄Sirius red staining pattern of mouse liver tissue section of mouse liver injury model.

Detailed Description

Example 1 preparation process conditions:

preparation of 2- (4-chlorophenoxy) propionic acid (2)

4-chlorophenol (12.80g, 100.0mmol) was dissolved in 150mL acetonitrile, followed by the addition of potassium carbonate (41.40g, 300.0 mmol). The mixture was stirred at 50 ℃ for 30 minutes, then 2-bromopropionic acid (15.20g, 100.0mmol) was added. The mixture was stirred at reflux overnight. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated in vacuo. The residue was added to water and the pH was adjusted to 3 with 1N HCl and the resulting mixture was extracted with DCM (3 × 100 mL). The organic layer was washed with brine (100mL), dried over MgSO4, and concentrated in vacuo to give 17.10g of a colorless oil. The yield thereof was found to be 85%.

2.2 preparation of methyl (4-chlorophenoxy) propionate (3)

2- (4-chlorophenoxy) propionic acid was dissolved in 150mL of methanol, and 10mL of concentrated sulfuric acid (98 wt%) was slowly added. The mixture was then stirred at reflux overnight. After cooling to room temperature, the mixture was concentrated in vacuo and the residue was dissolved in DCM and washed with water (3 × 100mL) and again with saturated NaHCO₃The aqueous solution (100mL) was washed with brine (100 mL). With MgSO₄After drying, the solvent was removed to give 17.30g of a colorless oil, which was used in the next step without further purification. The yield thereof was found to be 95%.

3.2 preparation of (4-chlorophenoxy) propionylhydrazide (4)

2- (4-chlorophenoxy) propionate was dissolved in 150mL of ethanol, and then 15mL of an aqueous solution of hydrazine (80 wt% in water) was added. The mixture was then stirred at reflux for 3 hours. The mixture was then concentrated in vacuo and the residue was washed with ethanol. The obtained dried white solid was used to obtain 13.05g of 2- (4-chlorophenoxy) propionylhydrazide. The yield thereof was found to be 75%.

4.2 preparation of 2- (2- (4-chlorophenoxy) propionyl) -N-phenylhydrazine thiocarboxamide (5)

2- (4-chlorophenoxy) propionohydrazide (13.05g, 60.9mmol) and phenyl isothiocyanate (8.22g, 60.9mmol) were placed in 120mL of anhydrous ethanol and refluxed for 5 hours on a steam bath. The mixture was cooled to room temperature and then filtered. The filtrate was concentrated to give 18.15g of a light brown oil, which was used in the next step without further purification.

5.5- (1- (4-chlorophenoxy) ethyl) -4-phenyl-4H-1, 2, 4-triazole-3-thiol (6) 18.15g of 2- (2- (4-chlorophenoxy) propionyl) -N-phenylhydrazine thiocarboxamide were dissolved in 100ml of 2N sodium hydroxide solution and heated under reflux for 3 hours. Cooled, acidified to pH2-3 with 1N HCl and filtered to collect a white solid. After drying, 13.80g of a white solid are obtained. The yield thereof was found to be 76%.

Preparation of 2-chloro-N- (1, 5-dimethyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazol-4-yl) acetamide (8)

Chloroacetyl chloride (9.04g, 80mmol) and triethylamine (8.08g, 80mmol) were added to 150mL of a solution containing 4-aminoantipyrine (16.24g, 80mmol) in DCM and stirred at room temperature overnight. Then, a volume of ice water was added to the solution for extraction, the organic layer was taken and washed first with 1N HCl solution (3X 100mL) and then with saturated NaHCO₃The aqueous solution (100mL) was washed, then brine (100mL) was added. With MgSO₄After drying, the solvent was removed to obtain 25.15g of a green solid. The crude product was recrystallized from DCM/MTBE to give 13.83g of a white solid. The yield thereof was found to be 62%.

7. Preparation of 2- ((5- (1- (4-chlorophenoxy) ethyl) -4-phenyl-4H-1, 2, 4-triazol-3-yl) thio) -N- (1, 5-dimethyl-3) oxo-2-phenyl-2, 3-dihydro-1H-pyrazol-4-yl) acetamide (9)

A mixture of 4-chloroacetylamidoantipyrine (8.42g, 30.2mmol), 5- (1- (4-chlorophenoxy) ethyl) -4-phenyl-4H-1, 2, 4-triazole-3-thiol (10.00g, 30.2mmol) and anhydrous potassium carbonate (8.33g, 60.4mmol) were added to 150mL of acetone, refluxed for 4 hours, filtered and the filtrate evaporated to dryness. The residue was recrystallized from MeOH/MTBE to give 7.05g of a white solid. The yield thereof was found to be 40%.

As shown in FIGS. 1-2, FIG. 1 is the mass spectrum of the compound, and FIG. 2 is the mass spectrum of the compound¹H-NMR spectrum.

Nuclear magnetic spectrum data of¹H-NMR(400MHz，DMSO-d6):δ＝1.58(3H,d,J＝6.0Hz),2.07(3H,s), 3.04(3H,s),4.10(2H,s),5.54(1H,q),6.76(3H,d,J＝8.8Hz),7.24(3H,d,J＝9.2Hz),7.23–7.53 (10H,m),9.48(1H,s)。

Experimental example 1 human ATX enzyme inhibition assay

1. ATX inhibition was measured by fluorescence quenching assay using the specifically labeled substrate Amplex Red reagent, which was obtained by purchasing the kit.

2. The assay working solution was prepared as follows:

2.1 assay buffer (50mM Tris-HCl, 140mM NaCl, 5mM KCl, 1mM CaCl₂、1mM MgCl₂、 0.01％Triton-X-100、pH8.0；

2.2ATX solution: stock solutions of ATX (mouse ATX- β from nano Biological) (48nM, diluted in assay buffer) were diluted to a final concentration of 2nM in assay buffer at the time of assay;

2.3LPC substrate solution: LPC (16:0) substrate solid was weighed and diluted to 250nM final concentration in assay buffer.

2.4 detection method: ATX was detected using the Amplex Red kit, each reaction containing 50. mu.M Amplex Red reagent, 1U/mL HRP, 0.1U/mL choline oxidase, 250nM LPC and the amount of ATX diluted with 1 Xreaction buffer, and the reaction was incubated at 37 ℃ for 1 hour. Fluorescence was measured using a fluorescence microplate reader with an excitation wavelength of 530. + -. 12.5nm and an emission wavelength of 590. + -. 17.5 nm.

2.5A series of solutions of the compound of the present invention diluted with DMSO to 10mM, 8mM, 6.4mM, 5.12mM, 4.10mM, 3.28mM, 2.62mM, 2.10mM, 1.68mM, 1.34mM, 1.07mM were added to 2. mu.L to 2.4, respectively, of the kit solutionThe change in fluorescence values after addition of the compound was determined by the method of 2.4, and the IC of the compound was calculated from these data₅₀The value was 43.05. mu.M.

Example 2 study of drug efficacy

1. Experimental materials and animals

1.1 drugs and reagents: glutamic-pyruvic transaminase determination kit (R1, R2) (MIVDRAY kit), glutamic-oxaloacetic transaminase determination kit R1, R2) (MIVDRAY kit), carbon tetrachloride (analytically pure), and rapeseed oil is prepared into 20% rapeseed oil solution before use.

1.2 Experimental animals: clean-grade Kunming mice, weighing 18-22g, Hunan Slek Jingda laboratory animals Co.

1.3 Experimental instruments: MIVDRAY BS-380 model full-automatic biochemical analyzer, EX 125DZH model electronic analytical balance (Aohaus instruments, Inc.), HC30L8R model high-speed refrigerated frozen centrifuge (Anhui Zhongzhou scientific instruments, Inc.).

1.4 tested drugs and treatment methods: taking the compound obtained in the preparation of example 1, the high dose group was diluted to 1.5mg/ml with 5% Tween 80 (diluted with 0.01mol/L PBS) and the low dose group was diluted to 0.75mg/ml with 5% Tween 80 (diluted with 0.01mol/L PBS).

2. Experimental methods

2.1 pairs of CC1₄Effects on hepatic fibrosis in mice

2.1.1 mouse CCl₄Establishing a model for inducing hepatic fibrosis injury, and injecting 20% CC1 into abdominal cavity of each group except normal group₄Rapeseed oil solution (5. mu.L/g body weight) was injected twice a week.

2.2.2 taking 48 male ICR mice, randomly dividing into 4 groups, normal group, model group, high dose group (30 mg. kg. group)^-1·d^-1) Low dose group (15 mg. kg)^-1·d^-1)。

2.2.3 Normal group: no drug administration, no model making, and normal diet and drinking water; model group: intraperitoneal injection of 20% CC1 alone₄Rapeseed oil solution; administration group (high, low): besides the molding, the corresponding reagent solution prepared under item 1.4 should be injected into the abdominal cavity. This experiment givesThe drug combination is administered once a day and 6 times a week from the 14 th day of molding, and orbital bleeding and mice are sacrificed on the 34 th day of molding for a total of 33 days and 20 days for the normal group, the model group and the administration group. Centrifuging at 3000r/min for 10min after blood collection, collecting supernatant, and determining CC1 by kit operation method₄ALT and AST contents in the blood serum of the liver injury mouse. The mice were sacrificed after blood collection, the livers were rapidly removed, the surrounding connective tissues were removed and fixed in 4% paraformaldehyde, paraffin-embedded, 4 μm-thick serial sections were stained with sirius red, and the condition of the collagen fibers in the liver tissues was observed under an optical microscope.

3. Results of the experiment

3.1 Effect of the Compounds on liver function in mice

The compound pair CC1₄The results of the serum indices for the mouse liver function influencing test are shown in the following table

TABLE 1 serum index data table for effect test of compound on mouse liver function

The results show that the liver function of a mouse in the carbon tetrachloride modeling group is obviously reduced, the detection results of the blood glutamic-pyruvic transaminase and the glutamic-oxalacetic transaminase are obviously increased, and the compound can reduce the level of serum transaminase and has a protective effect on the liver function.

3.2 this Compound on CCL₄Induced effects of liver histomorphology in mice

As shown in FIGS. 3-5, the staining patterns of the liver tissue of mice with the compound at different doses in the model group showed significant reticular fibrosis. Compared with the model group mouse, the high-dose group and the low-dose group which are administrated have the advantages that the pathological condition of the liver is obviously improved, and the proliferation of the liver fibrous connective tissue is obviously reduced. The gold standard of the tissue section shows that the compound has exact prevention effect on the formation of hepatic fibrosis of the mice.

Claims

1. An anti-hepatic fibrosis compound, characterized by the structural formula:

2. a preparation for combating liver fibrosis, comprising a compound according to claim 1 in the form of tablets, coated tablets, dragees, hard and soft gelatine capsules, solutions, emulsions, suspensions, nasal sprays, suppositories, ointments, gels or injections.

3. A method for preparing the anti-hepatic fibrosis compound of claim 1, comprising the steps of:

4. The method for preparing a compound according to claim 3, wherein the 4-chloroacetylamidoantipyrine is prepared by adding chloroacetyl chloride and triethylamine to a dichloromethane solution containing 4-aminoantipyrine, and stirring at room temperature; then adding ice water into the solution for extraction, taking an organic layer, washing the organic layer by using a hydrochloric acid solution, then washing by using a saturated sodium bicarbonate aqueous solution, and then washing by using brine; after drying, removing the solvent to obtain a crude product; recrystallizing the crude product to obtain the 4-chloroacetylaminoaantipyrine.

5. The method for preparing a hepatic fibrosis-resistant compound according to claim 3, wherein the 5- (1- (4-chlorophenoxy) ethyl) -4-phenyl-4H-1, 2, 4-triazole-3-thiol is prepared by dissolving 2- (2- (4-chlorophenoxy) propionyl) -N-phenylhydrazine thiocarboxamide in sodium hydroxide solution, heating under reflux, cooling to pH2-3, filtering, and drying to obtain 5- (1- (4-chlorophenoxy) ethyl) -4-phenyl-4H-1, 2, 4-triazole-3-thiol.

6. The method for preparing a compound according to claim 5, wherein the 2- (2- (4-chlorophenoxy) propionyl) -N-phenylhydrazine thiocarboxamide is prepared by adding 2- (4-chlorophenoxy) propionylhydrazine and phenyl isothiocyanate to absolute ethanol, refluxing, cooling the mixture to room temperature, filtering, and concentrating the filtrate to obtain 2- (2- (4-chlorophenoxy) propionyl) -N-phenylhydrazine thiocarboxamide.

7. The method for preparing a compound according to claim 6, wherein the 2- (4-chlorophenoxy) propionohydrazide is prepared by dissolving 2- (4-chlorophenoxy) propionate in ethanol, and adding hydrazine in water; the mixture was then stirred under reflux, concentrated in vacuo, and washed to give 2- (4-chlorophenoxy) propionylhydrazide.

8. The method for preparing a compound according to claim 7, wherein the 2- (4-chlorophenoxy) propionic acid ester is prepared by dissolving 2- (4-chlorophenoxy) propionic acid in methanol, and slowly adding 98% concentrated sulfuric acid; stirring under reflux, cooling to room temperature, concentrating in vacuo, and dissolving the residue in dichloromethane, washing, drying, and removing the solvent to give 2- (4-chlorophenoxy) propionate.

9. Use of the anti-hepatic fibrosis compound of claim 1 in preparing an ATX inhibitor, an anti-hepatic fibrosis preparation or a medicament for treating liver cancer.