Diphenyltriazine compound, preparation method therefor and use thereof

Abstract

The present application relates to the technical field of chemical medicine, and particularly to a diphenyltriazine compound, a preparation method therefor and a use thereof. The diphenyltriazine compound does not have a PGI2 backbone, has strong selectivity and affinity for a PGI2 receptor, and has good target selectivity as compared with a PGI2 analogue. Experiments have proven that the in-vivo efficacy of the compound is superior to that of Selexipag, and the toxicity of the compound is significantly lower than that of Selexipag. Therefore, the compound can be used as a pulmonary arterial hypertension therapeutic drug having a higher clinical value and development prospect.

Description

A diphenyltriazine compound and its preparation method and application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application CN202410169645.1 with an application date of February 6, 2024 and Chinese patent application CN202410701022.4 with an application date of May 31, 2024, and this application cites the full text of the above-mentioned Chinese patent applications. Technical Field

[0003] The present application belongs to the technical field of chemical medicines, and specifically relates to a diphenyltriazine compound, a preparation method thereof, and an application thereof. Background Art

[0004] Pulmonary arterial hypertension (PAH) is a disease characterized by vasospasm, intimal hyperplasia, and remodeling of the pulmonary arterioles. This proliferation and remodeling leads to a progressive increase in pulmonary vascular resistance, ultimately causing right heart failure and ultimately death. PAH is ranked the third most common cardiovascular disease, second only to hypertension and coronary heart disease in prevalence. It has become a serious public health concern threatening human health and well-being, and is included in the World Health Organization's global monitoring of major chronic diseases.

[0005] PGI2 is a substance produced in vivo from arachidonic acid via prostaglandin H2 (PGH2). PGI2 deficiency can cause pulmonary hypertension. Currently, marketed PGI2 receptor agonists include epoprostenol, beraprost, and iloprost, all of which are PGI2 analogs. However, due to the very short biological half-life of PGI2 and its poor target selectivity, it is difficult to separate the intended effect from other effects, making adverse reactions more likely. Selexipag is currently the only PGI2 receptor agonist that does not have a PGI2 skeleton but exhibits excellent selectivity for the PGI2 receptor and demonstrated efficacy. It has been approved for marketing in multiple countries for the treatment of adult pulmonary hypertension. Its specific therapeutic effect is stronger and more long-lasting than other drugs with similar mechanisms, but its high price undoubtedly adds a significant financial burden to patients with pulmonary hypertension who require long-term treatment. In addition, although selexipag has relatively good specificity, it still has obvious adverse reactions, such as headache, facial flushing, nausea, vomiting, etc. For patients who need long-term medication, the cumulative damage caused by the drug to the body will reduce their health level and quality of life to varying degrees. Technical issues

[0006] In response to the above problems, the present application provides a diphenyltriazine compound, a preparation method and application thereof. The compound has good affinity for PGI2 receptors and very low adverse reactions, avoiding the shortcomings of current PGI2 analogs and selexipag. Technical Solutions

[0007] In order to achieve the above application objectives, this application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a diphenyltriazine compound or a pharmaceutically acceptable salt, stable isotope derivative, isomer, and mixture thereof, the structural formula of which is shown in Formula I:

[0009] In a second aspect, the present application also provides a method for preparing the above-mentioned diphenyltriazine compounds, which specifically comprises the following operations:

[0010] The compound of formula II, methylsulfonamide, 4-dimethylaminopyridine (DMAP) and carbodiimide (EDCI) are mixed in dichloromethane, heated to 35-45°C for reaction for 2-3 hours, then cooled to 20-40°C. The resulting reaction solution is washed with purified water and then with hydrochloric acid solution, and then dehydrated to remove the solvent to obtain the product.

[0011] In conjunction with the second aspect, the equivalent ratio of the methanesulfonamide to the compound of formula II is ≥ 1. Preferably, the equivalent ratio of the methanesulfonamide to the compound of formula II is ≥ 1.3.

[0012] In conjunction with the second aspect, the equivalent ratio of the 4-dimethylaminopyridine to the compound of formula II is ≥ 1. Preferably, the equivalent ratio of the 4-dimethylaminopyridine to the compound of formula II is ≥ 1.1.

[0013] In conjunction with the second aspect, the ratio of the equivalents of the carbodiimide to the equivalents of the compound of formula II is ≥ 1. Preferably, the ratio of the equivalents of the carbodiimide to the equivalents of the compound of formula II is greater than ≥ 1.1.

[0014] In conjunction with the second aspect, the amount of dichloromethane used can be sufficient to ensure that all reactants are dissolved therein, and this application does not impose any limitation on this.

[0015] In combination with the second aspect, the concentration of the hydrochloric acid solution is 1.8 to 2.2 M (ie, mol / L).

[0016] In conjunction with the second aspect, the dehydration can be carried out by drying with anhydrous sodium sulfate. Anhydrous sodium sulfate can also be replaced by other desiccants that can dehydrate the organic phase without affecting the reactants.

[0017] In conjunction with the second aspect, the compound represented by the structural formula II can be prepared by the following method, which specifically comprises the following steps:

[0018] S1. Mix glacial acetic acid, benzil, semicarbazide hydrochloride and purified water, heat to 100-110°C, keep warm for 2-2.5 hours, then cool to 30-40°C, add purified water, stir and react at 20-30°C for 0.5-1 hour, separate the solid and liquid, place the resulting solid phase in ethyl acetate, reflux for 2-3 hours, cool and separate the solid and liquid to obtain intermediate II-1;

[0019] S2, phosphorus oxychloride and the intermediate II-1 are mixed, the temperature is raised to 80-85 ° C, stirred to dissolve, and the reaction is kept warm for 1-1.5 hours. After removing the solvent, a mixed solvent of toluene and isopropanol is added to the resulting product, stirred and dispersed, and solid-liquid separation is performed to obtain intermediate II-2;

[0020] S3. Add intermediate II-2 and 4-(isopropylamino)butanol to the reaction flask in sequence, raise the temperature to 140-150°C, keep the temperature for reaction for 12-15 hours, cool to room temperature, pour the reaction solution into water, add ethyl acetate for extraction, wash the organic phase with saturated sodium chloride aqueous solution and dry, remove the solvent, and purify the resulting product by silica gel column chromatography to obtain intermediate II-3;

[0021] S4, mixing the intermediate II-3 with dichloromethane, cooling to 0-10°C, adding Dess-Martin reagent, reacting at 0-10°C for 12-15 hours, washing the resulting reaction solution with a saturated sodium bicarbonate solution, then washing with a saturated sodium chloride aqueous solution, drying the organic phase, removing the solvent, and purifying the resulting product by silica gel column chromatography to obtain intermediate II-4;

[0022] S5, triethyl phosphoacetate and tetrahydrofuran were mixed, the temperature was lowered to 0-10°C, sodium hydride was added and the reaction was carried out for 1-1.5 hours, the intermediate II-4 was added at 0-5°C, the temperature was raised to room temperature and the reaction was carried out for 2-3 hours, water was added dropwise to the reaction solution to quench the reaction, and then at least 80% of tetrahydrofuran was removed by concentration. Ethyl acetate was added to the concentrate for extraction, the organic phase was washed with saturated sodium chloride aqueous solution and dried, the solvent was removed, and the resulting product was purified by silica gel column chromatography to obtain intermediate II-5;

[0023] S6, mixing the intermediate II-5, anhydrous ethanol and palladium carbon, replacing with hydrogen, reacting at room temperature for 5 to 8 hours, removing the palladium carbon, and removing the solvent from the resulting reaction solution to obtain intermediate II-6;

[0024] S7. Mix the intermediate II-6, tetrahydrofuran, sodium hydroxide and purified water, reflux for 2 to 4 hours, concentrate to remove tetrahydrofuran, add purified water and ethyl acetate, stir evenly, stand for separation, retain the aqueous phase, add hydrochloric acid to the aqueous phase to adjust the pH to 3 to 5, add methyl tert-butyl ether for extraction, dry the organic phase and remove the solvent to obtain a compound with the structural formula shown in Formula II.

[0025] Preferably, in step S1 of the preparation method, the ratio of the equivalents of the semicarbazide hydrochloride to the equivalents of the benzil is greater than 1. Preferably, the ratio of the equivalents of the semicarbazide hydrochloride to the equivalents of the benzil is ≥1.4.

[0026] Preferably, in step S1 of the preparation method, the volume ratio of the glacial acetic acid to the purified water is 2 to 3:1.

[0027] Preferably, in step S1 of the preparation method, the amount of glacial acetic acid and purified water used is sufficient to ensure that all reactants are dissolved therein, and this application does not impose any limitation on this.

[0028] Preferably, in step S2 of the preparation method, the mass ratio of the phosphorus oxychloride to the intermediate II-1 is ≥6.5.

[0029] Preferably, in step S2 of the preparation method, the volume ratio of toluene to isopropanol in the mixed solvent of toluene and isopropanol is 1:2.5-3.5.

[0030] Preferably, in step S3 of the preparation method, the equivalent ratio of the 4-(isopropylamino)butanol to the intermediate II-2 is 3 to 5:1.

[0031] Preferably, in step S3 of the preparation method, purification is performed by 200-mesh silica gel column chromatography, and the eluent is a dichloromethane-methanol mixture with a volume ratio of 30:1.

[0032] Preferably, in step S4 of the preparation method, the equivalent ratio of the Dess-Martin reagent to the intermediate II-3 is 1.5 to 3:1.

[0033] Preferably, in step S4 of the preparation method, purification is performed by 200-mesh silica gel column chromatography, and the eluent is a mixture of n-hexane and ethyl acetate in a volume ratio of 2:1.

[0034] Preferably, in step S5 of the preparation method, the equivalent ratio of the triethyl phosphoacetate to the intermediate II-4 is 1 to 2:1.

[0035] Preferably, in step S5 of the preparation method, the equivalent ratio of the sodium hydride to the intermediate II-4 is 1 to 2:1.

[0036] Preferably, in step S5 of the preparation method, purification is performed using a 200-mesh silica gel column, and the eluent is a mixture of n-hexane and ethyl acetate in a volume ratio of 5:1.

[0037] Preferably, in step S6 of the preparation method, the mass of the palladium carbon is 5% to 20% of the mass of the intermediate II-5.

[0038] In a third aspect, the present application also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, an excipient, and the above-mentioned diphenyltriazine compound or its pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof as an active ingredient.

[0039] In combination with the third aspect, the dosage form of the pharmaceutical composition is a pharmaceutically acceptable dosage form.

[0040] For example, the above dosage forms include conventional dosage forms such as tablets, granules, capsules, powders or injections.

[0041] In a fourth aspect, the present application also provides the use of the above-mentioned diphenyltriazine compounds or their pharmaceutically acceptable salts, stable isotope derivatives, isomers and mixtures thereof or the above-mentioned pharmaceutical compositions in the preparation of drugs for treating or preventing pulmonary hypertension, pulmonary arterial hypertension, chronic thrombotic pulmonary arterial hypertension, Fontane disease and pulmonary hypertension associated with Fontane disease, sarcoidosis and pulmonary hypertension associated with sarcoidosis.

[0042] The above application is preferably used in the preparation of a drug for treating or preventing pulmonary hypertension, pulmonary arterial hypertension or chronic thrombotic pulmonary arterial hypertension.

[0043] In a fifth aspect, the present application also provides the use of the above-mentioned diphenyltriazine compounds or their pharmaceutically acceptable salts, stable isotope derivatives, isomers and mixtures thereof or the above-mentioned pharmaceutical compositions in the preparation of drugs for treating or preventing peripheral circulatory disorders, connective tissue diseases, chronic kidney diseases including glomerulonephritis and diabetic nephropathy at any stage, diseases involving organ or tissue fibrosis, respiratory diseases, ulcer prevention, digital ulcers, diabetic gangrene, or diabetic foot ulcers. Beneficial effects

[0044] The diphenyltriazine compound provided in this application does not have a PGI2 skeleton and has strong selectivity and affinity for the PGI2 receptor, showing good target selectivity compared to PGI2 analogs. Experimental verification shows that the compound has superior in vivo efficacy to selexipag and far less toxicity than selexipag. Therefore, the compound may be a more clinically valuable and promising drug for the treatment of pulmonary arterial hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG1 is a diagram of Example 1 of the present application 1 H-NMR spectrum;

[0046] FIG2 is a block diagram of the embodiment 1 of the present application. 13 C-NMR spectrum;

[0047] FIG3 is a HPLC spectrum in Example 1 of the present application;

[0048] Figure 4 shows the EC of compound A for IP, EP1, EP2, EP3, EP4, DP, FP, and TP targets in Example 2 of the present application. 50 picture;

[0049] Figure 5 shows the EC of Selexipag for IP, EP1, EP2, EP3, EP4, DP, FP, and TP targets in Example 2 of the present application. 50 picture;

[0050] Figure 6 shows the effects of Compound A and Selexipag on the proliferation of human pulmonary artery smooth muscle cells in Example 2 of the present application;

[0051] FIG7 shows the effects of Compound A and Selexipag on rat pulmonary artery ring tension in Example 2 of the present application;

[0052] FIG8 shows the effect of oral administration of Compound A and Selexipag on the survival rate (%) of rats with pulmonary hypertension in Example 2 of the present application (Note: Survival rate (%) = number of surviving animals in a group / total number of animals in a group × 100%);

[0053] Figure 9 shows the effect of oral administration of Compound A and Selexipag on right ventricular systolic pressure (mmHg) in rats with pulmonary hypertension in Example 2 of the present application (*** indicates P ≤ 0.001 compared with the model control group);

[0054] Figure 10 shows the effect of oral administration of Compound A and Selexipag on the right heart hypertrophy index (%) in rats with pulmonary hypertension in Example 2 of the present application (* indicates P ≤ 0.05 compared with the model control group, *** indicates P ≤ 0.001 compared with the model control group);

[0055] Figure 11 is a comparison of the percentage (%) of pulmonary arteriolar media thickness in each group of animals in Example 2 of the present application (*** indicates P ≤ 0.001 compared with the model control group);

[0056] Figure 12 is a lung pathology image of each group of rats in Example 2 of the present application, wherein Figure A is the normal control group, lung, HE staining, 200×; Figure B is the model control group, lung, HE staining, 200×; Figure C is the low-dose group of the test sample, lung, HE staining, 200×; Figure D is the medium-dose group of the test sample, lung, HE staining, 200×; Figure E is the high-dose group of the test sample, lung, HE staining, 200×; Figure F is the positive control group, lung, HE staining, 200×. Modes for Carrying Out the Invention

[0057] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0058] the term

[0059] In the preparation methods of the compounds of this application, "eq" in chemistry means equivalent. This concept is very critical in stoichiometry and chemical reactions, especially when it comes to the molar ratio of substances. For example, if 0.3 mol (1 eq) of A is used in a reaction, and the amount of B used is 6 times that of A, that is, 6 eq, then the amount of B used is 1.8 mol. This representation method helps to make it more convenient and accurate when calculating the molar ratio of substances in chemical reactions.

[0060] In this application document, "eq" and "equivalent" are calculated on a molar basis, and each step may use its own independent equivalent standard for the convenience of calculation and testing.

[0061] PGI2 receptor agonists are currently commonly used drugs for the treatment of PAH, but they have a short biological half-life, poor target selectivity, and are prone to adverse reactions. Selexipag does not have a PGI2 skeleton and has relatively good target selectivity, but it still has certain adverse reactions. The present application provides a diphenyltriazine compound, the structural formula of which is shown in Formula I:

[0062] This compound does not have a PGI2 skeleton and has strong target selectivity. The results of in vivo pharmacodynamic experiments have demonstrated that the compound is more effective than selexipag, and the results of toxicology experiments have demonstrated that the compound is far less toxic than selexipag. It has higher clinical value and broader development prospects in the treatment of pulmonary arterial hypertension.

[0063] As defined herein, "isomers" refer to compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers." Stereoisomers include optical isomers, geometric isomers, and conformational isomers.

[0064] The compounds of the present invention may exist as optical isomers. Depending on the configuration of the substituents around the chiral carbon atom, these optical isomers are "R" or "S" configurations. Optical isomers include enantiomers and diastereomers. Methods for preparing and separating optical isomers are known in the art.

[0065] The compounds of the present invention may also exist as geometric isomers. The present invention contemplates various geometric isomers and mixtures thereof resulting from the distribution of substituents around carbon-carbon double bonds, carbon-nitrogen double bonds, cycloalkyl groups, or heterocyclic groups. Substituents around carbon-carbon double bonds or carbon-nitrogen bonds are designated as Z or E configurations, and substituents around cycloalkyl groups or heterocyclic rings are designated as cis or trans configurations.

[0066] "Isotopes" include all isotopes of atoms present in the compounds of the present invention. Isotopes include those atoms having the same atomic number but different mass numbers. Examples of isotopes suitable for incorporation into the compounds of the present invention are hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, for example, but not limited to, 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 35 S. 18 F and 36 Cl. Isotope-labeled compounds of the present application can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the accompanying Examples using appropriate isotope-labeled reagents in place of non-isotope-labeled reagents. Such compounds have various potential uses, for example as standards and reagents in determining biological activity. In the case of stable isotopes, such compounds have the potential to advantageously alter biological, pharmacological, or pharmacokinetic properties.

[0067] "Pharmaceutically acceptable salts" or "pharmaceutically acceptable salts" refer to salts made from pharmaceutically acceptable bases or acids, including inorganic bases or acids and organic bases or acids. In the case where the compounds of the present application contain one or more acidic or basic groups, the present application also includes their corresponding pharmaceutically acceptable salts. Therefore, the compounds of the present application containing acidic groups can exist in salt form and can be used according to the present application, for example, as alkali metal salts, alkaline earth metal salts or as ammonium salts, exemplified by sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines, such as ethylamine, ethanolamine, triethanolamine or amino acids. The compounds of the present application containing basic groups can exist in salt form and can be used according to the present application in the form of addition salts thereof with inorganic or organic acids. The example of suitable acid comprises hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-methyl benzenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid and other acid well known by persons skilled in the art.If the compound of the application contains acidic and basic groups in molecule simultaneously, the application also comprises inner salt or betaine except mentioned salt form.Each salt can obtain by conventional method well known by persons skilled in the art, for example, by making these and organic or inorganic acid or alkali contact or by with other salt anion exchange or cation exchange in solvent or dispersant.

[0068] A "pharmaceutical composition" refers to a composition comprising one or more compounds described herein, or pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives, isomers, and mixtures thereof, as well as other components such as pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0069] The solution of this application is described below through specific embodiments.

[0070] Example 1

[0071] This embodiment provides a diphenyltriazine compound, a preparation method thereof, and structural characterization.

[0072] Preparation method:

[0073] 1. Preparation of the compound shown in Formula II

[0074] The intermediate shown in Formula II (chemical name: 6-((5,6-diphenyl-1,2,4-triazin-3-yl)(isopropyl)amino)hexanoic acid) has the following synthesis route:

[0075] The specific synthesis method is as follows.

[0076] (1) Add glacial acetic acid (2.5 L), benzil (500 g, 1 eq), semicarbazide hydrochloride (889 g, 1.5 eq) and purified water (1 L) to the reaction flask in sequence, stir evenly, heat to 100-105 ° C and keep warm for 2.5 h, then cool to 30-40 ° C, add purified water, stir and react at 20-30 ° C for 1 h, filter, place the filter cake in ethyl acetate, heat and reflux with stirring for 3 h, cool to room temperature and filter, and vacuum dry the filter cake to obtain 502 g of intermediate II-1 (chemical name: 5,6-diphenyl-1,2,4-triazine-3-ol).

[0077] (2) Phosphorus oxychloride (2 L) and intermediate II-1 (500 g, 1 eq) were added to the reaction flask in sequence, the temperature was raised to 80-85°C, the solution was stirred and the reaction was carried out at this temperature for 1.5 h. After the phosphorus oxychloride was concentrated and removed, a mixed solvent of toluene (500 mL) and isopropanol (1500 mL) was added, and the solid was dispersed by stirring at room temperature. The solid was filtered and dried in vacuo to obtain 350 g of intermediate II-2 (chemical name: 3-chloro-5,6-diphenyl-1,2,4-triazine).

[0078] (3) To the reaction flask, add intermediate II-2 (100 g, 1 eq) and 4-(isopropylamino)butanol (171.5 g, 3.5 eq) in sequence, raise the temperature to 140-150°C, keep the temperature for reaction for 14 h, cool to room temperature, pour the reaction solution into water, add ethyl acetate for extraction, wash the organic phase three times with saturated sodium chloride aqueous solution, combine the organic phases, dry over anhydrous sodium sulfate, and concentrate to dryness. The concentrate is purified by silica gel column (the stationary phase of the silica gel column is 200 mesh silica gel, the eluent (mobile phase) is dichloromethane: methanol = 30:1), and 71 g of intermediate II-3 (chemical name: 4-((5,6-diphenyl-1,2,4-triazin-3-yl)(isopropyl)amino)butan-1-ol) is obtained.

[0079] (4) Add intermediate II-3 (70 g, 1 eq) and dichloromethane (700 ml) to the reaction flask, cool to 0-10°C, add Dess-Martin reagent (163.8 g, 2 eq) in batches, and keep at 0-10°C for 14 h. Stop the reaction, wash the reaction solution twice with saturated sodium bicarbonate solution, then with saturated sodium chloride solution, dry the organic phase with anhydrous sodium sulfate, and concentrate to dryness. The concentrate is purified by silica gel column (the stationary phase of the silica gel column is 200 mesh silica gel, and the eluent is n-hexane:ethyl acetate = 2:1) to obtain 41.3 g of intermediate II-4 (chemical name: 4-((5,6-diphenyl-1,2,4-triazin-3-yl)(isopropyl)amino)butyraldehyde).

[0080] (5) Add triethyl phosphoacetate (29.8 g, 1.2 eq) and tetrahydrofuran (400 ml) to the reaction flask, cool to 0-10°C, add sodium hydride (4.4 g, 1 eq), stir for 1 h, add intermediate II-4 (40 g, 1 eq) at 0-5°C, warm to room temperature after addition and react for 2.5 h, add 50 ml of water dropwise to the reaction solution to quench the reaction, concentrate to remove most (more than 80%) of tetrahydrofuran, and remove tetrahydrofuran. To the concentrate of the fumarate was added 400 ml of ethyl acetate for extraction, the organic phase was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to dryness. The concentrate was purified by silica gel column (the stationary phase of the silica gel column was 200 mesh silica gel, and the eluent was n-hexane: ethyl acetate = 5:1) to obtain 21.3 g of intermediate II-5 (chemical name: 6-((5,6-diphenyl-1,2,4-triazin-3-yl)(isopropyl)amino)-2-hexenoic acid ethyl ester).

[0081] (6) 21 g of intermediate II-5, 2.1 g of 10% palladium carbon, and anhydrous ethanol (210 ml) were added to the reaction flask, replaced with hydrogen, and reacted at room temperature for 7 h. The palladium carbon was removed by filtration, and the filtrate was concentrated to dryness to obtain 20.2 g of intermediate II-6 (chemical name: 6-((5,6-diphenyl-1,2,4-triazin-3-yl)(isopropyl)amino)hexanoic acid ethyl ester).

[0082] (7) To the reaction flask were added intermediate II-6 (20 g, 1 eq), tetrahydrofuran (200 ml), purified water (20 ml), and sodium hydroxide (7.4 g, 4 eq) in sequence. The temperature was raised to reflux and the reaction was continued for 2 h. The tetrahydrofuran was removed by concentration. Purified water (100 ml) and ethyl acetate (100 ml) were added and stirred evenly. The mixture was allowed to stand for separation. The aqueous phase was retained. Hydrochloric acid was added to the aqueous phase to adjust the pH to 3-5. Methyl tert-butyl ether was added for extraction. The organic phase was dried over anhydrous sodium sulfate and then concentrated to dryness under reduced pressure to obtain 16.8 g of the compound represented by formula II. The H NMR spectrum data are as follows: 1 H-NMR (500MHz, CDCl3): δ: 7.510~7.493 (m, 2H), 7.475~7.448 (m, 2H), 7.396~7.364 (m, 1H), 7.317~7.269 (m, 5H), 5.141~ 5.070(m, 1H), 3.592(m, 2H), 2.407~2.362(m, 2H), 1.779~1.707(m, 4H), 1.506~1.445(m, 2H), 1.322~1.308(m, 6H), ppm.

[0083] 2. Preparation of the compound shown in Formula I

[0084] To a reaction flask, add the compound of Formula II (1 g, 1 eq), dichloromethane (20 ml), methylsulfonamide (353 mg, 1.5 eq), DMAP (362 mg, 1.2 eq), and EDCI (569 mg, 1.2 eq). The reaction mixture was heated to 35-45°C for 3 h, then cooled to room temperature. The resulting reaction solution was washed once with purified water, once with 2M hydrochloric acid solution, and once again with purified water. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness to yield 650 mg of the compound of Formula I (chemical name: 6-((5,6-diphenyl-1,2,4-triazin-3-yl)(isopropyl)amino)-N-(methylsulfonyl)hexanamide).

[0085] The structural characterization diagrams of the compound represented by Formula I are shown in Figures 1, 2 and 3, and the specific data are as follows:

[0086] 1 H-NMR (500MHz, CDCl3): δ: 10.144 (br, 1H), 7.480~7.497 (m, 2H), 7.447~7.466 (m, 2H), 7.398 (m, 1H), 7.262~7.321 (m, 5H), 5.075 (m, 1H), 3.622 (m, 2H), 3.236 (s, 3H), 2.370 ~ 2.399 (t, 2H), 1.854 ~ 1.716 (m, 4H), 1.460 ~ 1.487 (m, 2H), 1.292 ~ 1.306 (m, 6H), ppm.

[0087] 13 C NMR: (500MHz, CDCl3): δ: 20.41, 23.89, 25.71, 36.13, 41.36, 41.67, 46.66, 77.2 5, 128.21, 128.30, 129.16, 129.69, 130.22, 136.14, 136.59, 147.75, 158.84ppm.

[0088] High-resolution mass spectrometry: [M+1] + Measured value (m / z): 482.2210.

[0089] HPLC spectrum: the retention time of the main peak is 5.520 minutes.

[0090] The elemental analysis results are shown in Table 1:

[0091] Table 1 Elemental analysis results

[0092] The above structural characterization results indicate that the compound has the structural formula shown in Formula I:

[0093] Example 2

[0094] This example provides pharmacodynamic experiments and results of the compound represented by Formula I (hereinafter referred to as "Compound A").

[0095] 1. Evaluation of the effects of compound A on IP, EP1, EP2, EP3, EP4, DP, FP, and TP targets

[0096] This study used cell lines stably expressing IP, EP1, EP2, EP3, EP4, DP, FP, and TP receptors. The HTRF cAMP and HTRF IP1 methods were used to study the functional activities of compound A and Selexipag against eight targets, IP, EP1, EP2, EP3, EP4, DP, FP, and TP, based on changes in cell signal intensity. The EC values ​​in the corresponding concentration-effect curves were calculated. 50 Value (EC 50 It usually refers to the dose required for an exogenous substance to cause a 50% change in a certain biological effect of the body, which means the drug concentration that causes 50% of individuals to be effective), see Figure 4.

[0097] The results showed that compound A had an agonistic effect on the IP target under the initial detection conditions of 10000nM, and the absolute EC 50 The value was 0.48nM; there was no obvious agonist effect on EP1, EP2, EP3, EP4, DP, FP and TP targets under the initial detection condition of 10000nM, and the absolute EC 50 The values ​​were all greater than 10000 nM.

[0098] As a comparison, this example also tested the effects of Selexipag on IP, EP1, EP2, EP3, EP4, DP, FP, and TP targets. The results showed that under the initial detection conditions of 10,000 nM, Selexipag had an agonist effect on IP, EP2, and DP targets, with an absolute EC 50 The values ​​were 0.27nM, 9754.34nM and 2736.01nM respectively; there was no obvious agonist effect on EP1, EP3, EP4, FP and TP targets under the initial detection condition of 10000nM, and the absolute EC 50 The values ​​were all greater than 10000 nM, see Figure 5.

[0099] The above results indicate that: The agonist activity of compound A towards the IP receptor is weaker than that of Selexipag, but its target selectivity is stronger than that of Selexipag. Therefore, it can be reasonably speculated that the adverse reactions similar to those caused by the over-activation of the IP receptor by Selexipag, such as headache, flushing, nausea, vomiting and other side effects, are less for compound A, and the adverse reactions caused by the activation of other prostacyclin receptors, such as muscle pain, are also less.

[0100] II. Effects of Compound A on the Proliferation of Human Pulmonary Artery Smooth Muscle Cells

[0101] Human pulmonary artery smooth muscle cells were cultured using a special smooth muscle medium, and the medium was placed statically in a cell culture incubator at a temperature of 37 °C, a CO2 concentration of 5%, and saturated humidity. Human pulmonary artery smooth muscle cells were seeded at 6×10 3 / well onto a 96-well cell culture plate and cultured in the 96-well plate (final volume: 100 μl). After 24 hours, they were starved with serum-free medium for 24 hours. Then, compound A and Selexipag were serially diluted with a medium containing hPDGF-BB at a final concentration of 20 ng / ml (serial dilution concentrations were 10000, 3000, 1000, 300, 100, 30, 10, 3, 1, 0.3, 0.1 nM). The medium in the wells of the cell culture plate was removed, and the serially diluted test substances were added to the wells of the cell culture plate, with a volume of 100 μl. The CTG method was used to study the inhibitory effects of the test substances compound A and Selexipag on the proliferation of human pulmonary artery smooth muscle cells, and their half-maximal inhibitory concentration IC 50 was fitted to evaluate the potential of the test substances for treating pulmonary hypertension.

[0102] The results showed (see Figure 6): The IC 50 values of compound A and Selexipag for the inhibitory effect on PDGF-induced proliferation of pulmonary artery smooth muscle cells were 9.65 and 18.69 μM, respectively. The inhibitory effect of compound A was positively correlated with the compound concentration at a concentration of 0.3 - 10 μM.

[0103] In summary, compound A and Selexipag showed effective inhibitory effects on PDGF-induced proliferation of pulmonary artery smooth muscle cells. The IC 50 value of the inhibitory effect of compound A < Selexipag, indicating that the inhibitory effect of compound A is stronger than that of Selexipag.

[0104] III. Effects of Compound A on the Tension of Rat Pulmonary Artery Rings

[0105] After anesthetizing the SD rat, the heart and lungs were found and freed, and immersed in oxygenated perfusion fluid. After washing away the blood, the freed heart and lungs were placed in a clean, oxygenated incubation fluid. The pulmonary artery was found and freed under a microscope (be careful not to pull the pulmonary artery during this process), the blood remaining in the endothelium of the pulmonary artery was removed, and a vascular ring with an inner diameter of about 2mm to 3mm was prepared. The vascular ring was hung on the hook of the tension transducer (be careful not to damage the endothelial cells), and the pulmonary artery branch was immersed in the liquid of the incubation tank. The basic tension of the pulmonary artery branch was slightly adjusted to about 0.8 to 1g and stabilized for 1 to 2 hours. After the basic tension of the pulmonary artery ring is stable, high K + After the incubation medium induced an increase in the pulmonary artery ring tension and stabilized, incubation medium containing different concentrations of test compounds was added, including compound A (1μM, 5μM, 10μM, 20μM, 60μM) and Selexipag (5μM, 10μM, 20μM, 40μM, 60μM), N=5, and the changes in pulmonary artery ring tension were recorded. After the maximum efficacy of the drug was observed, the incubation medium was replaced with the basic incubation medium to restore the pulmonary artery ring tension to the basic level.

[0106] The results showed (see Figure 7): Compound A and Selexipag were able to inhibit high K to varying degrees at the five concentrations tested. + induced pulmonary artery vasoconstriction in rats, half maximal inhibitory concentration IC 50 The inhibitory efficiency of the compounds is 11.15μM and 23.97μM respectively. This shows that the order of the inhibitory efficiency of the compounds from high to low is: Compound A > Selexipag. In summary, both Compound A and Selexipag can effectively inhibit high K + Compound A inhibited pulmonary artery vasoconstriction more than selexipag.

[0107] IV. Pharmacological efficacy of compound A on MCT-induced pulmonary hypertension model rats

[0108] Sixty-five rats were randomly divided into six groups: a normal control group, a model control group, a low-dose test article group, a medium-dose test article group, a high-dose test article group, and a positive control group. The normal control group consisted of 10 rats, and the remaining five groups consisted of 11 rats each. Except for the normal control group, all other groups were treated with monocrotaline (60 mg / kg, 5 ml / kg) to establish a model. The normal control group received an intraperitoneal injection of sodium chloride injection (5 ml / kg). Approximately 2 hours after the animals were administered the modeling agent, all other groups began receiving drug administration.

[0109] The normal control group and the model control group were gavaged with 0.5% sodium carboxymethylcellulose (CMC-Na), the low-dose test sample group was gavaged with 0.15 mg / kg compound A, the medium-dose test sample group was gavaged with 0.5 mg / kg compound A, and the high-dose test sample group was gavaged with 1.5 mg / kg compound A. The positive control group was gavaged with 1 mg / kg selexipag, twice a day, for 19 consecutive days.

[0110] The body weight changes of the rats were recorded and the survival rate was observed. The mean pulmonary artery pressure of the rats was measured by right cardiac catheterization. The pathological changes of right ventricular and lung tissues were observed by HE staining. The percentage of medial thickness and right ventricular hypertrophy index were calculated.

[0111] The results show:

[0112] (1) Effect of Compound A on Body Weight in Rats with Pulmonary Hypertension

[0113] After the animals were given the modeling agent, their body weight was significantly lower than that of the normal control group. As the experimental period prolonged, the average body weight of the animals in each treatment group increased steadily, while the body weight of the animals in the model control group decreased significantly, as shown in Table 2.

[0114] Table 2 Effects of Compound A and Selexipag administered orally on body weight (g) in rats with pulmonary hypertension Note: Compared with the model control group, “*” indicates P ≤ 0.05, “**” indicates P ≤ 0.01, and “***” indicates P ≤ 0.001.

[0115] (2) Compound A reduces the mortality rate of rats with pulmonary hypertension

[0116] During the experiment, the survival rate of the animals in the model control group was 82%, and no animals died in the other groups, with a survival rate of 100%, as shown in Figure 8.

[0117] (3) Compound A dose-dependently reduced right ventricular systolic pressure, right ventricular hypertrophy index, and pulmonary arteriolar media thickness in rats

[0118] Compared with the right ventricular systolic pressure (RVSP, mmHg) of the model control group, the right ventricular systolic pressure of the animals in the low-dose test article group (0.15 mg / kg), the medium-dose test article group (0.5 mg / kg), the high-dose test article group (1.5 mg / kg), and the positive control group (1 mg / kg) were significantly reduced, with the maximum reductions being approximately 15%, 33%, 43%, and 33%, respectively, and all showing statistically significant differences (P ≤ 0.001), as shown in Figure 9 and Table 3.

[0119] Table 3 Effects of Compound A and Selexipag on pulmonary artery blood pressure (mmHg) in rats with pulmonary hypertension Note: Compared with the model control group, “***” indicates P ≤ 0.001.

[0120] Compared with the model control group, Compound A at doses of 0.15 mg / kg, 0.5 mg / kg, and 1.5 mg / kg significantly reduced the right ventricular hypertrophy index (RV / (LV+S)) in rats, with maximum reductions of 17%, 27%, and 29%, respectively. Selexipag at a dose of 1 mg / kg reduced the right ventricular hypertrophy index in rats by 26%, as shown in Figure 10 and Table 4.

[0121] Table 4 Effects of Compound A and Selexipag administered orally on right ventricular hypertrophy index (%) in rats with pulmonary hypertension Note: Compared with the model control group, “*” indicates P ≤ 0.05, and “***” indicates P ≤ 0.001.

[0122] Compared with the model control group, the percentage of membrane thickness (MT) was significantly reduced at the 0.5 mg / kg and 1.5 mg / kg doses of Compound A, as well as in the positive control group (P ≤ 0.001). The percentage of membrane thickness in the 0.15 mg / kg Compound A group did not show a significant difference compared with the model control group. Compared with the 1.5 mg / kg Compound A group, the percentage of membrane thickness in the positive control group was slightly reduced, as shown in Figure 11 and Table 5.

[0123] Table 5 Effects of Compound A and Selexipag administered orally on the percentage (%) of pulmonary artery media thickness in rats with pulmonary hypertension Note: Compared with the model control group, “***” indicates P ≤ 0.001.

[0124] (4) Compound A alleviates lung tissue pathological damage in rats with pulmonary hypertension

[0125] The animals in the model control group and the drug-treated group were observed under a microscope, and some pulmonary and bronchial lesions caused by the pulmonary hypertension model were observed, such as alveolar foamy macrophage infiltration, alveolar hemorrhage, alveolar fibrinoid exudation, congestion, and pulmonary arteriolar media hypertrophy / luminal stenosis. In terms of comprehensive lung lesions, the incidence and severity of lung lesions in animals treated with Compound A and the positive control were reduced to a certain extent compared to the model control group. In addition, the severity of lesions in the positive control group was reduced to a certain extent compared to the high, medium, and low doses of the test article groups, as shown in Figure 12.

[0126] The above results show that under the experimental conditions, compound A at doses of 0.15 mg / kg, 0.5 mg / kg, and 1.5 mg / kg, and selexipag at a dose of 1 mg / kg, both administered orally twice daily for 19 consecutive days, for a total of 38 doses, significantly reduced pulmonary artery pressure, right heart hypertrophy index, and pulmonary artery medial thickness percentage in rats with monocrotaline-induced pulmonary hypertension, increased survival rate, and had a certain improvement effect on lung tissue lesions, showing a dose-response relationship. The effect of compound A at a dose of 0.5 mg / kg was close to that of the positive control, selexipag (1 mg / kg).

[0127] In summary, the diphenyltriazine compounds of the present application, or their pharmaceutically acceptable salts, stable isotope derivatives, isomers, and mixtures thereof, have significant effects in the treatment or prevention of diseases such as pulmonary hypertension, pulmonary arterial hypertension, and chronic thrombotic pulmonary hypertension. Furthermore, they have significant effects in treating Fontan disease and Fontan disease-associated pulmonary hypertension, and sarcoidosis and sarcoidosis-associated pulmonary hypertension.

[0128] Based on the same mechanism, the diphenyltriazine compounds of the present application or their pharmaceutically acceptable salts, stable isotope derivatives, isomers and mixtures thereof have therapeutic or preventive effects on peripheral circulatory disorders (e.g., chronic arterial occlusion, intermittent claudication, peripheral embolism, vibration syndrome, Raynaud's disease).

[0129] Based on the same mechanism as above, in terms of connective tissue diseases, for example, systemic lupus erythematosus, scleroderma, mixed connective tissue disease, vasculitis syndrome have a therapeutic or preventive effect. In terms of arteriosclerosis and thrombosis, there is a therapeutic or preventive effect, for example, acute cerebral thrombosis, pulmonary embolism, etc. In the thrombocytopenia caused by dialysis, the disease involving organ or tissue fibrosis has a preventive or therapeutic effect, for example, nephropathy such as tubulointerstitial nephritis. In respiratory system diseases, for example, interstitial pneumonia, (idiopathic) pulmonary fibrosis, chronic obstructive pulmonary disease, digestive system diseases (for example, cirrhosis of the liver, viral hepatitis, chronic pancreatitis and sclerosing gastric cancer), and anti-ulcer, finger ulcer, diabetic gangrene, or diabetic foot ulcer, there is a preventive or therapeutic effect.

[0130] Example 3

[0131] This example provides toxicological experiments and results of the compound represented by Formula I (hereinafter referred to as "Compound A").

[0132] 1. Acute toxicity test of compound A in rats by oral gavage

[0133] Twenty-four healthy Sprague-Dawley rats, half male and half female, were randomly divided into four groups based on body weight. Group 1 received the vehicle (0 mg / kg), while Groups 2-4 received low-, medium-, and high-dose Compound A at doses of 100, 300, and 1000 mg / kg, respectively. All animals received a single oral gavage dose of 10 ml / kg on Day 1 and were observed for 7 days.

[0134] One female rat in Group 4 became moribund on Day 2 and was subsequently euthanized.

[0135] Detailed clinical observations revealed that the female animals in the high-dose (1000 mg / kg) Compound A group experienced loss of righting reflex, cold skin to touch, disheveled fur, and near-death symptoms on Day 2, symptoms believed to be toxic reactions caused by Compound A. Two female rats in the medium-dose (300 mg / kg) Compound A group also experienced red nasal discharge on Day 2. This nasal discharge only appeared on Day 2 and subsequently returned to normal, with no apparent dose-related effect. This is believed to be related to Compound A, but not a toxic reaction. Compared to their body weight during the acclimation period, the body weight of the female animals in the high-dose (1000 mg / kg) Compound A group decreased on Day 2, a toxic reaction believed to be caused by Compound A. Food consumption and gross anatomical observations of the animals in all dose groups showed no significant abnormalities. The maximum tolerated dose (MTD) of SD rats following a single oral gavage of Compound A was less than 1000 mg / kg.

[0136] The above-mentioned toxicity symptoms are generally consistent with those observed in rats after administration of selexipag. However, Compound A did not cause severe toxicity symptoms such as tail blackening or loss in rats. The non-lethal dose of selexipag in rats is 250 mg / kg, and the approximate lethal dose is 500 mg / kg. Combined with efficacy data, the safe dose range of Compound A in rats is greater than that of selexipag, and the maximum tolerated dose of Compound A in rats is higher than that of selexipag.

[0137] 2. Toxicity study of compound A in rats after repeated administration for 4 weeks

[0138] 160 healthy SD rats (SPF grade), half male and half female, were randomly divided into 8 groups according to sex and body weight. Groups 1 to 4 were toxicity study groups (including solvent control group, low-dose group, medium-dose group, and high-dose group), with 15 animals per sex in each group; Groups 5 to 8 were toxicity study groups (including solvent control group, low-dose group, medium-dose group, and high-dose group), with 5 animals per sex in each group. Each group of animals was orally gavaged with different doses of compound A for 4 consecutive weeks, with the dosage being 0 mg / kg (solvent), 50 mg / kg, 150 mg / kg, and 500 mg / kg, respectively. The dosage volume was 10 ml / kg, and the administration was once a day.

[0139] During the experiment, animal behavior and physical signs were observed and recorded. Body weight and food intake were measured weekly during the dosing and recovery periods for Groups 1 to 4. Fundus examinations were performed at the end of dosing and at the end of the recovery period. Clinical pathology examinations (including hematology, coagulation, serum biochemistry, and urinalysis) were performed on animals scheduled for autopsy at the end of dosing and the recovery period. Before collecting clinical pathology samples, all animals scheduled for autopsy were fasted overnight (≥10 hours). Urine was collected for approximately 12 hours one day in advance for urinalysis. Gross anatomical examinations, organ weights, and clinical pathology examinations were performed on animals scheduled for autopsy at the end of dosing and at the end of the recovery period.

[0140] No animal died during the test. The no observed adverse effect level (NOAEL) of compound A was 50 mg / kg. After repeated administration at the NOAEL dose for 28 days, the AUC of compound A in male and female animals was (0-24) The NOAEL of Selexipag is 6 mg / kg. After repeated administration at the NOAEL dose for 28 days, the AUC of Selexipag in male and female animals was 3784 h×ng / ml (male) and 7016 h×ng / ml (female). (0-24) They are 10h×ng / ml (male) and 40h×ng / ml (female) respectively.

[0141] Compound A doses ≥500 mg / kg caused salivation and increased thyroid volume. Doses ≥150 mg / kg caused weight loss, decreased red blood cell (RBC), hemoglobin (HGB), and hematocrit (HCT), increased RET / RET%, elevated alanine aminotransferase (ALT), increased urine volume, increased liver weight, and histopathologically revealed hypertrophy of the distal endocrine cells of the male pituitary, thyroid follicular cells, and liver cells. All of these changes resolved or showed a recovery trend by the end of the recovery period. Among these changes, salivation was only observed in some animals on some testing days and disappeared after drug withdrawal. In the medium-dose (150 mg / kg) group, weight changes were minimal, as were decreases in RBC, HGB, and HCT. The increased RET / RET% was considered a compensatory effect, and urine volume increased, but no other abnormalities were observed. These effects are considered non-adverse reactions related to Compound A. Food consumption, ophthalmological examinations, and coagulation tests in all groups showed no toxic effects related to Compound A.

[0142] 3. Genotoxicity test of compound A

[0143] Genotoxicity test procedure: Test compound A and selexipag were diluted serially into eight concentrations at a final concentration of 1000 μg / well. A 6-well plate incorporation method was used, with two wells treated in parallel. Negative (DMSO) and positive controls were also included. Parallel experiments were performed with or without a metabolic activation system (±S9). After 48-72 hours of incubation, the test samples were observed for precipitation and background plaque growth, and the number of revertant colonies in each well was counted. The test results are shown in Tables 6-9.

[0144] Positive result determination

[0145] A result is considered positive if one or both of the following criteria are met:

[0146] 1) In at least one strain, the number of revertant colonies shows a dose-dependent increase with or without metabolic activation, and the number of revertant colonies is 2 times or more that of the negative control group.

[0147] 2) With or without metabolic activation, the number of revertant colonies in one or more dose groups increases significantly and reproducibly, and the number of revertant colonies is 2 times or more that of the negative control group.

[0148] After the test sample is tested with two test strains, as long as one of the test strains is positive, regardless of whether S9 mixed solution is added or not, the test sample can be determined to be a mutagen.

[0149] Negative result judgment

[0150] If the test results show that there is no dose-dependent increase in the number of revertant colonies of each test strain, and the peak value of the number of revertant colonies in each dose group of all strains does not exceed 2 times that of the negative control group, the test article can be determined to be a non-mutagenic agent.

[0151] The results showed that under ±S9 conditions, non-interfering precipitation was observed for TA98 and TA100 strains at a final Selexipag concentration of ≥250 μg / well; non-interfering precipitation was only observed at a final Compound A concentration of 1000 μg / well. Under ±S9 conditions, a reduction in background plaques was observed for TA98 and TA100 strains at a final Selexipag concentration of ≥250 μg / well; no abnormal background plaques were observed at any concentration of Compound A. For TA98 and TA100 strains, under ±S9 conditions, the number of revertant colonies in each Compound A and Selexipag concentration group did not reach twice that of the negative control group, and there was no concentration-effect relationship, so the test results were considered negative.

[0152] Therefore, Compound A and Selexipag were not genotoxic.

[0153] Table 6. Results of the 6-well plate screening test of bacterial reverse mutation of compound A (TA98 strain)

[0154] Remark:

[0155] Background plaque: T0 normal.

[0156] Solubility of test / reference substance: P0 normal / no precipitation; P1 non-interfering precipitation under the microscope.

[0157] *: The number of reverse mutant colonies in the positive control group was 3 times higher than that in the negative control group.

[0158] Table 7 Results of the 6-well plate screening test of bacterial reverse mutation of compound A (TA100 strain)

[0159] Remark:

[0160] Background plaque: T0 normal.

[0161] Solubility of test / reference substance: P0 normal / no precipitation; P1 non-interfering precipitation under the microscope.

[0162] *: The number of reverse mutant colonies in the positive control group was 3 times higher than that in the negative control group.

[0163] Table 8. Results of the 6-well plate screening test for bacterial reverse mutations in Selexipag (TA98 strain)

[0164] Remark:

[0165] NA: Not counted.

[0166] Background plaque: T0 normal; T1 background plaque slightly reduced; T2 background plaque moderately reduced; T3 background plaque severely reduced;

[0167] The background plaques of T4 disappeared.

[0168] Solubility of test / reference substance: P0 normal / no precipitation; P1 non-interfering precipitation under the microscope.

[0169] a : The positive drugs when S9 was not added and added were 2-nitrofluorene (0.4 μg / well) and 2-aminoanthracene (0.6 μg / well), respectively.

[0170] *: The number of reverse mutant colonies in the positive control group was 3 times higher than that in the negative control group.

[0171] Table 9 Results of the initial screening test of Selexipag bacterial reverse mutation in 6-well plates (TA100 strain)

[0172] Remark:

[0173] NA: Not counted.

[0174] Background plaque: T0 is normal; T1 background plaque is slightly reduced; T3 background plaque is severely reduced; T4 background plaque disappears.

[0175] Solubility of test / reference substance: P0 normal / no precipitation; P1 non-interfering precipitation under the microscope.

[0176] a : The positive drugs when S9 was not added and added were sodium azide (0.4 μg / well) and 2-aminoanthracene (0.6 μg / well), respectively.

[0177] *: The number of reverse mutant colonies in the positive control group was 3 times higher than that in the negative control group.

[0178] In summary, the NOAEL for Compound A in rats is 100 times the effective dose, while the NOAEL for selexipag is 6 times the effective dose, indicating that Compound A has a greater safety margin than selexipag. Furthermore, compared to selexipag, repeated administration of Compound A for 4 weeks did not cause severe adverse reactions. These results demonstrate that Compound A is safer than selexipag.

[0179] Example 4

[0180] This embodiment provides a tablet for treating pulmonary arterial hypertension, the preparation method of which is as follows: using the compound represented by Formula I as the active ingredient, and preparing the tablet according to conventional processes with tablet excipients. The excipients generally include diluents, binders, disintegrants, etc. to meet the tablet preparation process and product quality.

[0181] This embodiment also provides other dosage forms of the pharmaceutical composition for treating pulmonary arterial hypertension, including granules, capsules, powders, and injections, all of which are prepared using conventional preparation processes in the art.

[0182] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A diphenyltriazine compound or a pharmaceutically acceptable salt, stable isotope derivative, isomer, and mixture thereof, characterized in that: Its structural formula is shown in Formula I:

2. The method for preparing the diphenyltriazine compound according to claim 1, wherein The specific steps include: The compound of formula II, methylsulfonamide, 4-dimethylaminopyridine and carbodiimide are mixed in dichloromethane, heated to 35-45°C for reaction for 2-3 hours, then cooled to 20-40°C, and the resulting reaction solution is washed with purified water and then with hydrochloric acid solution, and then dehydrated to remove the solvent to obtain the product; 3. The preparation method according to claim 2, characterized in that The equivalent ratio of the methylsulfonamide to the compound of formula II is ≥1; and / or The equivalent ratio of the 4-dimethylaminopyridine to the compound of formula II is ≥1; and / or The ratio of the equivalent weight of the carbodiimide to the equivalent weight of the compound having the structural formula shown in Formula II is ≥1.

4. The preparation method according to claim 2, characterized in that The preparation method of the compound represented by the structural formula II specifically comprises the following steps: S1. Mix glacial acetic acid, benzil, semicarbazide hydrochloride and purified water, heat to 100-110°C, keep warm for 2-2.5 hours, then cool to 30-40°C, add purified water, stir and react at 20-30°C for 0.5-1 hour, separate the solid and liquid, place the resulting solid phase in ethyl acetate, reflux for 2-3 hours, cool and separate the solid and liquid to obtain intermediate II-1; S2, phosphorus oxychloride and the intermediate II-1 are mixed, the temperature is raised to 80-85 ° C, stirred to dissolve, and the reaction is kept warm for 1-1.5 hours. After removing the solvent, a mixed solvent of toluene and isopropanol is added to the resulting product, stirred and dispersed, and solid-liquid separation is performed to obtain intermediate II-2; S3. Add intermediate II-2 and 4-(isopropylamino)butanol to the reaction flask in sequence, raise the temperature to 140-150°C, keep the temperature for reaction for 12-15 hours, cool to room temperature, pour the reaction solution into water, add ethyl acetate for extraction, wash the organic phase with saturated sodium chloride aqueous solution and dry, remove the solvent, and purify the resulting product by silica gel column chromatography to obtain intermediate II-3; S4, mixing the intermediate II-3 with dichloromethane, cooling to 0-10°C, adding Dess-Martin reagent, reacting at 0-10°C for 12-15 hours, washing the resulting reaction solution with a saturated sodium bicarbonate solution, then washing with a saturated sodium chloride aqueous solution, drying the organic phase, removing the solvent, and purifying the resulting product by silica gel column chromatography to obtain intermediate II-4; S5, triethyl phosphoacetate and tetrahydrofuran were mixed, the temperature was lowered to 0-10°C, sodium hydride was added and the reaction was carried out for 1-1.5 hours, the intermediate II-4 was added at 0-5°C, the temperature was raised to room temperature and the reaction was carried out for 2-3 hours, water was added dropwise to the reaction solution to quench the reaction, and then at least 80% of tetrahydrofuran was removed by concentration. Ethyl acetate was added to the concentrate for extraction, the organic phase was washed with saturated sodium chloride aqueous solution and dried, the solvent was removed, and the resulting product was purified by silica gel column chromatography to obtain intermediate II-5; S6, mixing the intermediate II-5, anhydrous ethanol and palladium carbon, replacing with hydrogen, reacting at room temperature for 5 to 8 hours, removing the palladium carbon, and removing the solvent from the resulting reaction solution to obtain intermediate II-6; S7. Mix the intermediate II-6, tetrahydrofuran, sodium hydroxide and purified water, reflux for 2 to 4 hours, concentrate to remove tetrahydrofuran, add purified water and ethyl acetate, stir evenly, stand for separation, retain the aqueous phase, add hydrochloric acid to the aqueous phase to adjust the pH to 3 to 5, add methyl tert-butyl ether for extraction, dry the organic phase and remove the solvent to obtain a compound with the structural formula shown in Formula II.

5. The preparation method according to claim 4, characterized in that In step S1, the equivalent ratio of the semicarbazide hydrochloride to the benzil is greater than 1; and / or In step S1, the volume ratio of the glacial acetic acid to the purified water is 2 to 3:1; and / or In step S2, the mass ratio of the phosphorus oxychloride to the intermediate II-1 is ≥ 6.5; and / or In step S2, the volume ratio of toluene to isopropanol in the mixed solvent of toluene and isopropanol is 1:2.5-3.5; and / or In step S3, the equivalent ratio of the 4-(isopropylamino)butanol to the intermediate II-2 is 3 to 5; and / or In step S3, purification is performed using a 200-mesh silica gel column chromatography, with a dichloromethane-methanol mixture in a volume ratio of 30:1 as the eluent; and / or In step S4, the equivalent ratio of the Dess-Martin reagent to the intermediate II-3 is 1.5 to 3:1; and / or In step S4, purification is performed by 200 mesh silica gel column chromatography, and the eluent is a mixture of n-hexane and ethyl acetate in a volume ratio of 2:1; and / or In step S5, the equivalent ratio of the triethyl phosphoacetate to the intermediate II-4 is 1 to 2:1; and / or In step S5, the equivalent ratio of the sodium hydride to the intermediate II-4 is 1 to 2:1; and / or In step S5, purification is performed by 200 mesh silica gel column chromatography, and the eluent is a mixture of n-hexane and ethyl acetate in a volume ratio of 5:1; and / or In step S6, the mass of the palladium carbon is 5% to 20% of the mass of the intermediate II-5.

6. A pharmaceutical composition, characterized in that The invention comprises a pharmaceutically acceptable carrier, an excipient, and the compound according to claim 1 or its pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof as an active ingredient.

7. The pharmaceutical composition according to claim 6, wherein The dosage form of the pharmaceutical composition is a pharmaceutically acceptable dosage form.

8. The pharmaceutical composition according to claim 7, wherein The dosage forms include tablets, granules, capsules, powders or injections.

9. Use of the diphenyltriazine compound according to claim 1 or its pharmaceutically acceptable salt, stable isotope derivative, isomer and mixture thereof, or the pharmaceutical composition according to any one of claims 6 to 8 in the preparation of a medicament for treating or preventing pulmonary arterial hypertension, Fontane disease and pulmonary hypertension associated with Fontane disease, sarcoidosis and pulmonary hypertension associated with sarcoidosis.

10. The use according to claim 9, characterized in that Including use in preparing drugs for treating or preventing pulmonary hypertension.

11. The use according to claim 10, characterized in that The pulmonary hypertension is chronic thrombotic pulmonary hypertension.

12. Use of the diphenyltriazine compound according to claim 1 or its pharmaceutically acceptable salts, stable isotope derivatives, isomers and mixtures thereof, or the pharmaceutical composition according to any one of claims 6 to 8, in the preparation of a medicament for treating or preventing peripheral circulatory disorders, connective tissue diseases, chronic kidney diseases including glomerulonephritis and diabetic nephropathy at any stage, diseases involving organ or tissue fibrosis, digital ulcers, diabetic gangrene or diabetic foot ulcers.

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