Diphenylpyrazine compounds and compositions, applications and preparation methods thereof

Abstract

The present invention belongs to the technical field of medical uses of small molecule compounds, and relates to diphenylpyrazine compounds and compositions thereof, applications and preparation methods. It mainly relates to the use of diphenylpyrazine compounds and compositions thereof in the preparation of drugs for inhibiting platelet aggregation, and specifically relates to the use of diphenylpyrazine compounds and compositions thereof in the preparation of drugs for treating cardiovascular and cerebrovascular diseases and other vascular thrombosis and embolic diseases related to platelet aggregation. The present invention confirms through in vitro platelet aggregation experiments that the diphenylpyrazine compounds exhibit extremely strong inhibitory effects on both AA-induced platelet aggregation and ADP-induced platelet aggregation, have lower toxicity, and have outstanding clinical advantages.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application CN202311824286.0, filed on December 27, 2023, and cites the full text of the aforementioned Chinese patent application. Technical Field

[0003] The present invention belongs to the field of medical technology, and specifically relates to diphenylpyrazine compounds and compositions, applications and preparation methods thereof, and mainly relates to applications in the preparation of drugs for inhibiting platelet aggregation. Background Art

[0004] Cardiovascular disease is the number one killer that threatens human health. Among cardiovascular diseases, the death toll from ischemic heart disease alone exceeds the death toll from all cancers. The common pathological basis of acute coronary syndrome and ischemic stroke is arterial thrombosis caused by abnormal platelet activation, and antiplatelet therapy is effective. Rupture of atherosclerotic plaques is a common cause of abnormal platelet activation, and stent implantation within 3-6 hours of onset has an immediate effect. As a foreign body in a blood vessel, the stent itself has the risk of activating platelets and inducing thrombosis. After stent implantation, antiplatelet drugs need to be taken long-term or even lifelong to prevent thrombosis within the stent, and there is a huge clinical demand for antiplatelet drugs.

[0005] Clinically available antiplatelet drugs primarily fall into five categories: cyclooxygenase inhibitors (e.g., aspirin), P2Y12 receptor antagonists (e.g., clopidogrel, prasugrel), phosphodiesterase inhibitors (e.g., cilostazol), fibrinogen receptor antagonists, and thrombin receptor PAR1 antagonists. However, these five categories of antiplatelet drugs also have clinical limitations: oral antiplatelet drugs such as aspirin and clopidogrel have a slow onset of action; most oral antiplatelet drugs, such as aspirin and clopidogrel, have a mild antiplatelet effect; and antiplatelet therapy cannot completely prevent clinical thrombotic events. Furthermore, patients may experience "aspirin resistance" and "clopidogrel resistance," and those with significant responses are prone to bleeding side effects.

[0006] Selexipag, a selective prostaglandin (PGI2) receptor agonist, clinically reduces the symptoms of pulmonary hypertension by dilating the pulmonary arteries and inhibiting platelet aggregation. In addition to its use in treating pulmonary hypertension, selexipag also has the potential to act as a platelet aggregation inhibitor. However, selexipag has poor coagulation efficacy and significant adverse reactions, such as headache, facial flushing, nausea, and vomiting. For patients who require long-term medication, the cumulative damage to the body caused by the drug will, to varying degrees, reduce their health and quality of life. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a platelet aggregation inhibitor with stronger inhibitory effect and its composition, application and preparation method. The inhibitor and its composition have outstanding performance in inhibiting platelet aggregation and have greater clinical development potential than existing representative drugs.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a diphenylpyrazine compound represented by formula (I) or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer and mixture thereof.

[0010]

[0011] The second aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective dose of a diphenylpyrazine compound represented by formula (I) or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer, and mixture thereof.

[0012] Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0013] The third aspect of the present invention provides the diphenylpyrazine compound represented by the above formula (I) or its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer and mixture thereof, as well as the use of the above pharmaceutical composition in the preparation of drugs for inhibiting platelet aggregation.

[0014] Furthermore, the inhibition of platelet aggregation is inhibition of AA-induced platelet aggregation.

[0015] Furthermore, the inhibition of platelet aggregation is inhibition of ADP-induced platelet aggregation.

[0016] The fourth aspect of the present invention is to provide the diphenylpyrazine compound represented by the above formula (I) or its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer and mixture thereof, as well as the use of the above pharmaceutical composition in the preparation of anticoagulant drugs.

[0017] The fifth aspect of the present invention is to provide the diphenylpyrazine compounds represented by the above formula (I) or their pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers and mixtures thereof, as well as the use of the above pharmaceutical compositions in the preparation of drugs for preventing or treating cardiovascular and cerebrovascular diseases related to platelet aggregation, other vascular thrombosis and embolic diseases.

[0018] Furthermore, the platelet aggregation-related cardiovascular and cerebrovascular diseases, other vascular thrombotic and embolic diseases mainly include but are not limited to cerebral thrombosis, cerebral infarction, myocardial infarction, heart failure, pulmonary embolism, thromboangiitis obliterans, chronic arterial occlusive disease, central retinal vein thrombosis, etc. Other vascular thrombotic and embolic diseases refer to vascular thrombotic and embolic diseases other than cardiovascular and cerebrovascular diseases.

[0019] The sixth aspect of the present invention is to provide the diphenylpyrazine compound represented by the above formula (I) or its pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer and mixture thereof, as well as the use of the above pharmaceutical composition in the preparation of drugs for extracorporeal circulation protection of platelets, post-arterial angiography treatment or post-vascular reconstruction treatment.

[0020] Platelet aggregation inhibitors primarily inhibit platelet aggregation. Platelet aggregation is a key step in thrombosis. Once platelets aggregate, they can trigger a thrombosis, leading to vascular obstruction. Therefore, platelet aggregation inhibitors can prevent thrombosis and effectively reduce the risk of thrombotic diseases.

[0021] The diphenylpyrazine compounds represented by formula (I) above, or their pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers, and mixtures thereof, as well as the pharmaceutical compositions described above, can stimulate prostacyclin receptors, increase the cAMP content in platelets, convert more free calcium into calcium storage granules, render platelets inert, and inhibit primary platelet aggregation. Furthermore, increased cAMP can reduce the production of TXA2 within platelets, reduce the release of ADP and 5HT, and thus reduce the amount of secondary platelet aggregation. This dual effect achieves a better inhibition of platelet aggregation. These compounds can be used to better inhibit platelet aggregation, thereby preventing thrombosis and reducing the risk of cardiovascular and cerebrovascular diseases and other vascular thrombotic and embolic diseases. They can be used for extracorporeal circulation to protect platelets, post-arterial angiography treatment, or post-vascular revascularization treatment to prevent postoperative thrombosis.

[0022] The seventh aspect of the present invention is to provide a method for preparing the diphenylpyrazine compound represented by the above formula (I), comprising the following steps:

[0023] S1, 4,4'-dimethylbenzil (Compound 1A) undergoes cyclization reaction with 2-aminoacetamide hydrochloride to obtain Compound 1B;

[0024] S2, compound 1B undergoes chlorination reaction with phosphorus oxychloride to obtain compound 1C;

[0025] S3, compound 1C undergoes substitution reaction with 4-(isopropylamino)butanol to obtain compound 1D;

[0026] S4, compound 1D is oxidized with Dess-Martin reagent to obtain compound 1E;

[0027] S5, compound 1E undergoes nucleophilic addition reaction with triethyl phosphoacetate to obtain compound 1F;

[0028] S6. Compound 1F is hydrolyzed in the presence of sodium hydroxide to obtain a diphenylpyrazine compound represented by formula (I) (ie, compound 1).

[0029] The synthetic route of the preparation method is as follows.

[0030]

[0031] The preparation method of the diphenylpyrazine compound represented by the above formula (I) specifically comprises the following steps:

[0032] S1, mixing 4,4'-dimethylbenzil, 2-aminoacetamide hydrochloride and the first reaction solvent, heating to reflux, adding a strong base solution, keeping the temperature for reaction, cooling to 20-25°C, adjusting the pH to neutral, solid-liquid separation, washing, and drying to obtain compound 1B;

[0033] S2, mixing the compound 1B and phosphorus oxychloride, raising the temperature to 100-105° C., maintaining the temperature for reaction, removing the phosphorus oxychloride, and obtaining a reactant; recrystallizing the reactant, performing solid-liquid separation, and drying to obtain compound 1C;

[0034] S3, mixing the compound 1C and 4-(isopropylamino)butanol, raising the temperature to 160-170° C., maintaining the temperature for reaction, and then cooling to obtain a reaction solution; extracting the reaction solution, separating the organic phase, washing, drying, and purifying to obtain compound 1D;

[0035] S4, mixing the compound 1D with a second reaction solvent, cooling the mixture to 0-10° C., adding a Dess-Martin reagent, and incubating the mixture to react to obtain a reaction solution; washing the reaction solution, drying it, and purifying it to obtain compound 1E;

[0036] S5, triethyl phosphoacetate and the third reaction solvent are mixed, cooled to 0-10°C, sodium hydride is added, and stirred for 0.5-2.0 h. Compound 1E is added at 0-10°C, and the temperature is raised to 20-25°C. The mixture is kept warm for reaction to obtain a reaction solution, the reaction is quenched, extraction is performed, and the organic phase is separated, washed, dried, and purified to obtain compound 1F;

[0037] S6, mixing compound 1F, the fourth reaction solvent and sodium hydroxide, raising the temperature to reflux for reaction to obtain a reaction solution, adjusting the pH to weak acidity, extracting, separating the organic phase, drying, and purifying to obtain a diphenylpyrazine compound represented by formula (I).

[0038] Furthermore, in step S1, the first reaction solvent is methanol.

[0039] Furthermore, in step S1, the strong alkaline solution is a sodium hydroxide aqueous solution.

[0040] Furthermore, in step S1, the pH is adjusted to 6-7.

[0041] Furthermore, in step S1, the washing is to rinse the solid phase obtained after solid-liquid separation with methanol.

[0042] Furthermore, in step S1, the equivalent ratio of 2-aminoacetamide hydrochloride to 4,4'-dimethylbenzil is ≥1.

[0043] Furthermore, in step S1, the mass volume ratio of the 4,4'-dimethylbenzil to the first reaction solvent is 3 g: (15-25) mL.

[0044] Furthermore, in step S1, the equivalent ratio of the 4,4'-dimethylbenzil to the strong base is 1:1-3.

[0045] Furthermore, in step S1, the concentration of the strong alkaline solution is 0.4-0.6 g / mL.

[0046] Furthermore, in step S1, the strong base solution is added dropwise.

[0047] Furthermore, in step S1, the insulation reaction time is 4 to 5 hours.

[0048] Furthermore, in step S1, the insulation reaction is carried out under stirring conditions.

[0049] Furthermore, in step S1, the pH is adjusted to neutral by using hydrochloric acid.

[0050] Furthermore, in step S1, the solid-liquid separation method can be conventional methods such as filtration or centrifugation.

[0051] Furthermore, in step S1, the amount of methanol solvent used in the washing accounts for 10%-20% of the amount of methanol solvent in the reaction.

[0052] Furthermore, in step S1, the drying method may be conventional methods such as vacuum drying.

[0053] Furthermore, in step S2, the method for removing phosphorus oxychloride is to remove residual phosphorus oxychloride by concentrating under reduced pressure; or to remove residual phosphorus oxychloride by concentrating under reduced pressure, then adding toluene, and continuing to concentrate to remove residual phosphorus oxychloride.

[0054] Furthermore, in step S2, the reactant is recrystallized by adding dichloromethane to the reactant, stirring and dissolving to obtain a reaction solution, and adding the reaction solution dropwise to isopropanol. After the addition is completed, stirring is carried out at 0-5°C for 1-3 hours.

[0055] Furthermore, in step S2, the equivalent ratio of phosphorus oxychloride to compound 1B is ≥1, more preferably ≥3.

[0056] Furthermore, in step S2, the mass volume ratio of the compound 1B to toluene is 1 g: (1.5-3.0) mL.

[0057] Furthermore, in step S2, the mass volume ratio of the compound 1B to dichloromethane is 1 g: (0.5-1.5) mL.

[0058] Furthermore, in step S2, the mass volume ratio of the compound 1B to isopropyl alcohol is 1 g: (8-15) mL.

[0059] Furthermore, in step S2, the insulation reaction time is 3 to 5 hours.

[0060] Furthermore, in step S2, the insulation reaction is carried out under stirring conditions.

[0061] Furthermore, in step S2, the solid-liquid separation method is a conventional method such as filtration or centrifugation.

[0062] Furthermore, in step S2, the drying method is a conventional method such as vacuum drying.

[0063] Furthermore, in step S3, the extraction is performed by pouring the reaction solution into water and adding ethyl acetate for extraction.

[0064] Furthermore, in step S3, the washing, drying and purification steps are to wash the separated organic phase with a saturated aqueous ammonium chloride solution, add anhydrous sodium sulfate to dryness, and concentrate to dryness to obtain a concentrate which is then purified by silica gel column chromatography.

[0065] Furthermore, in step S3, the equivalent ratio of 4-(isopropylamino)butanol to compound 1C is ≥1, and more preferably the equivalent ratio is ≥4.

[0066] Furthermore, in step S3, the mass volume ratio of the compound 1C to water is 1 g: (8-12) mL.

[0067] Furthermore, in step S3, the mass volume ratio of the compound 1C to ethyl acetate is 1 g: (8-12) mL.

[0068] Furthermore, in step S3, the mass volume ratio of the compound 1C to the saturated ammonium chloride aqueous solution is 1 g: (8-12) mL.

[0069] Furthermore, in step S3, the insulation reaction time is 20 to 30 hours.

[0070] Furthermore, in step S3, the cooling temperature is 60°C to 80°C.

[0071] Furthermore, in step S4, the second reaction solvent is dichloromethane.

[0072] Furthermore, in step S4, the reaction solution washing, drying and purification steps are washing the reaction solution with a saturated sodium bicarbonate solution, washing with a saturated sodium chloride aqueous solution, adding anhydrous sodium sulfate to dryness, and concentrating the concentrate to dryness to obtain a concentrate which is purified by silica gel column chromatography.

[0073] Furthermore, in step S4, the equivalent ratio of the Dess-Martin reagent to compound 1D is ≥1.

[0074] Furthermore, in step S4, the mass volume ratio of the compound 1D to the second reaction solvent is 1 g: (8-12) mL.

[0075] Furthermore, in step S4, the mass volume ratio of the compound 1D to the saturated sodium bicarbonate solution is 1 g: (8-12) mL.

[0076] Furthermore, in step S4, the mass volume ratio of the compound 1D to the saturated sodium chloride aqueous solution is 1 g: (8-12) mL.

[0077] Furthermore, in step S4, the insulation reaction time is 3 to 5 hours.

[0078] Furthermore, in step S4, the temperature may be lowered to 0-10°C by conventional methods such as ice bath.

[0079] Furthermore, in step S4, the Dess-Martin reagent is added in 3-5 batches.

[0080] Furthermore, in step S5, the third reaction solvent is tetrahydrofuran.

[0081] Furthermore, in step S5, the quenching reaction and extraction steps are to add water dropwise to the reaction solution for quenching, and then add ethyl acetate for extraction.

[0082] Furthermore, in step S5, the washing, drying and purification steps are to wash the separated organic phase with a saturated sodium chloride aqueous solution, add anhydrous sodium sulfate to dryness, and concentrate to dryness to obtain a concentrate which is then purified by silica gel column chromatography.

[0083] Furthermore, in step S5, the equivalent ratio of triethyl phosphoacetate to compound 1E is ≥1.

[0084] Furthermore, in step S5, the mass volume ratio of the compound 1E to the third reaction solvent is 13 g: (80-100) mL.

[0085] Furthermore, in step S5, the equivalent ratio of sodium hydride to compound 1E is 1 to 1.5:1.

[0086] Furthermore, in step S5, the mass volume ratio of the compound 1E to water is 13 g: (40-60) mL.

[0087] Furthermore, in step S5, the mass volume ratio of the compound 1E to ethyl acetate is 13 g: (160-200) mL.

[0088] Furthermore, in step S5, the mass volume ratio of the compound 1E to the saturated sodium chloride aqueous solution is 13 g: (80-100) mL.

[0089] Furthermore, in step S5, the insulation reaction time is 2 to 3 hours.

[0090] Furthermore, in step S5, the temperature may be lowered to 0-10°C by conventional methods such as ice bath.

[0091] Furthermore, in step S5, the sodium hydride needs to be added in 3-5 batches.

[0092] Furthermore, in step S5, the compound 1E needs to be added in 3-5 batches.

[0093] Furthermore, in step S6, the fourth reaction solvent is tetrahydrofuran and purified water.

[0094] Furthermore, in step S6, the pH adjustment and extraction steps are to cool the reaction solution to room temperature, add water, adjust the pH to 5-6, and add ethyl acetate for extraction.

[0095] Furthermore, in step S6, the drying and purification step is to dry the separated organic phase with anhydrous sodium sulfate, concentrate it to dryness, and purify the concentrate by silica gel column chromatography.

[0096] Furthermore, in step S6, the equivalent ratio of sodium hydroxide to compound 1F is 1.5 to 5:1.

[0097] Furthermore, in step S6, the mass volume ratio of the compound 1F to the fourth reaction solvent is 1 g: (8.5-13.5) mL.

[0098] Furthermore, in step S6, the mass volume ratio of the compound 1F to tetrahydrofuran is 1 g: (8-12) mL.

[0099] Furthermore, in step S6, the mass volume ratio of the compound 1F to purified water is 1 g: (0.5-1.5) mL.

[0100] Furthermore, in step S6, the mass volume ratio of the compound 1F to the amount of water added after cooling to room temperature is 1 g: (8-12) mL.

[0101] Furthermore, in step S6, the mass volume ratio of the compound 1F to ethyl acetate is 1 g: (18-22) mL.

[0102] Furthermore, in step S6, the temperature is raised to reflux reaction time is 12 to 15 hours.

[0103] Furthermore, in step S6, the pH is adjusted using hydrochloric acid.

[0104] The positive and progressive effects of the present invention are: the mechanism of action of the diphenylpyrazine compound shown in formula (I) is to inhibit platelet aggregation by increasing the content of cAMP in platelets. The diphenylpyrazine compound shown in formula (I) shows extremely strong inhibitory effects on both AA-induced platelet aggregation and ADP-induced platelet aggregation. The inhibitory effect is significantly better than that of clopidogrel, aspirin and selexipag in the positive control group at the same dose, and is dose-dependent. After the dose concentration is reduced by 10 times, the inhibitory effect is still better than that of clopidogrel, aspirin and selexipag in the positive control group, and compound 1 has a stronger efficacy. In the acute toxicity test, the oral non-lethal dose of selexipag in rats is about 250 mg / kg, and the non-lethal dose of compound 1 is about 2000 mg / kg, which is significantly less toxic than selexipag. In short, compound 1 has a stronger inhibitory effect on platelet aggregation and lower toxicity, with outstanding clinical advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 is the compound 1 in Example 1 of the present invention 1 H-NMR spectrum;

[0106] Figure 2 is the compound 1 in Example 1 of the present invention 13 C-NMR spectrum;

[0107] Figure 3 is the MS spectrum of compound 1 in Example 1 of the present invention; DETAILED DESCRIPTION

[0108] The present invention is further described in detail below through specific implementation methods, but it is only used to help understand the present invention so that professionals in the field can implement or use the present invention, and does not constitute any limitation to the present invention.

[0109] the term

[0110] In the preparation methods of the compounds of the present application, "eq" stands for equivalent, which means equivalent in chemistry. 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.

[0111] 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.

[0112] 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.

[0113] The compounds of the present invention may exist as optical isomers. Depending on the configuration of substituents around the chiral carbon atom, these optical isomers are in the "R" or "S" configuration. Optical isomers include enantiomers and diastereomers. Methods for preparing and separating optical isomers are known in the art.

[0114] 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.

[0115] "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, such as, 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. Isotopically labeled compounds of the present invention 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 isotopically labeled reagents in place of non-isotopically labeled reagents. Such compounds have various potential uses, for example, as standards and reagents in assays for biological activity. In the case of stable isotopes, such compounds have the potential to advantageously alter biological, pharmacological, or pharmacokinetic properties.

[0116] "Pharmaceutically acceptable salts" or "pharmaceutically acceptable salts" refer to salts prepared 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 invention contain one or more acidic or basic groups, the present invention also includes their corresponding pharmaceutically acceptable salts. Thus, compounds of the present invention containing acidic groups may exist in salt form and may be used according to the present invention, for example, as alkali metal salts, alkaline earth metal salts or as ammonium salts, illustrative of which include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines, such as ethylamine, ethanolamine, triethanolamine or amino acids. Compounds of the present invention containing basic groups may exist in salt form and may be used according to the present invention in the form of addition salts with inorganic or organic acids. The example of suitable acid comprises hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic 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 compound of the present invention contains acidic and basic group simultaneously in molecule, the present invention also comprises inner salt or betaine except mentioned salt form.Each salt can be obtained by conventional methods well known by persons skilled in the art, for example, by making these and organic or inorganic acid or base contact or by with other salt anion exchange or cation exchange in solvent or dispersant.

[0117] A "pharmaceutical composition" refers to a composition containing the diphenylpyrazine compound of Formula (I) described herein, or its pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives, isomers, and mixtures thereof. The composition may also include other components (e.g., other active ingredients or pharmaceutically acceptable carriers or excipients). The purpose of a pharmaceutical composition is to facilitate administration to an organism, promote absorption of the active ingredient and thereby exert its biological activity, and promote disease treatment.

[0118] "Pharmaceutically acceptable excipients" refer to additives in pharmaceutical preparations other than the main drug, also known as excipients. These include, but are not limited to, binders, fillers, disintegrants, and lubricants in tablets; alcohol, vinegar, and medicinal juices in traditional Chinese medicine pills; the base component of semisolid ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.

[0119] "Pharmaceutically acceptable carriers" refer to substances that can alter the way a drug enters the human body and its distribution within the body, control the rate of drug release, and deliver the drug to the targeted organ system. These include, but are not limited to, microcapsules, microspheres, nanoparticles, liposomes, microemulsions, nanocapsules, nanospheres, and exosomes.

[0120] As used herein, the term "room temperature" or "RT" refers to an ambient temperature of 20 to 25°C (68 to 77°F).

[0121] The solutions of the present invention are described below through specific embodiments.

[0122] Example 1

[0123] Prepare 6-{N-[5,6-di(4-methylphenyl)pyrazine]-2-yl-N-isopropylamino}-2-hexenoic acid, denoted as compound 1, with the corresponding structural formula as follows.

[0124]

[0125] The preparation route is as follows:

[0126]

[0127] S1: Compound 1A (300 g, 1 eq of 4,4'-dimethylbenzil), 2-aminoacetamide hydrochloride (209 g, 1.5 eq), and methanol (2.0 L) were added to the reaction flask in sequence. The temperature was raised to reflux, and an aqueous sodium hydroxide solution (101 g, 2 eq of sodium hydroxide, 200 mL of purified water) was added dropwise. The mixture was stirred for 4 to 5 hours, cooled to room temperature, and the pH was adjusted to 6 to 7 with hydrochloric acid. The mixture was filtered, and the filter cake was rinsed with 300 mL of methanol and dried in vacuo to obtain 320 g of compound 1B.

[0128] S2: Compound 1B (100 g, 1 eq) and phosphorus oxychloride (277.4 g, 5 eq) were added to a reaction flask in sequence. The temperature was raised to 100-105°C and stirred for 3-5 h. The mixture was concentrated under reduced pressure to remove residual phosphorus oxychloride. 200 mL of toluene was added and the mixture was further concentrated to remove residual phosphorus oxychloride. 100 mL of dichloromethane was added to the reaction mixture and stirred to dissolve. The reaction solution was added dropwise to 1000 mL of isopropanol. After addition, the mixture was stirred at 0-5°C for 1-3 h, filtered, and dried under vacuum to obtain 90.7 g of compound 1C.

[0129] S3: Compound 1C (100 g, 1 eq) and 4-(isopropylamino)butanol (245 g, 5.5 eq) were added to the reaction flask in sequence, the temperature was raised to 160-170°C, the reaction was kept warm for 20-30 h, the temperature was lowered to 60-80°C, the reaction solution was poured into 1.0 L of water, and 1.0 L of ethyl acetate was added for extraction. The organic phase was washed with 1.0 L of saturated ammonium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to dryness. The concentrate was purified by silica gel column chromatography (silica gel column packing: 100-200 mesh silica gel; eluent: n-hexane: ethyl acetate = 10:1→5:1→1:1) to obtain 74 g of compound 1D.

[0130] S4: Compound 1D (70 g, 1 eq) and dichloromethane (700 mL) were added to the reaction flask in sequence. The temperature was cooled to 5-8°C in an ice bath. Dess-Martin reagent (152 g, 2 eq) was added in four batches. The reaction was kept warm for 3-5 h. The reaction solution was washed with 700 mL of saturated sodium bicarbonate solution and 700 mL of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to dryness. The concentrate was purified by silica gel column chromatography (silica gel column packing: 100-200 mesh silica gel; eluent: n-hexane: ethyl acetate = 10:1→5:1→1:1) to obtain 21.5 g of compound 1E.

[0131] S5: Triethyl phosphoacetate (9 g, 1.2 eq) and tetrahydrofuran (90 mL) were added to the reaction flask in sequence. The mixture was cooled to 5-8°C in an ice bath, and sodium hydride (1.34 g, 1 eq) was added in 4 batches. The mixture was stirred for 1 h. Compound 1E (13 g, 1 eq) was added in 4 batches at 0-5°C, and the mixture was heated to room temperature for 2-3 h. 50 mL of water was added dropwise to the reaction solution to quench the mixture. 180 mL of ethyl acetate was added to extract the mixture. The organic phase was washed with 90 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to dryness. The concentrate was purified by silica gel column chromatography (silica gel column packing: 100-200 mesh silica gel; eluent: n-hexane: ethyl acetate = 10:1→5:1→1:1) to obtain 13 g of compound 1F.

[0132] S6: Compound 1F (3 g, 1 eq), tetrahydrofuran (30 mL), and 3 mL of purified water were added to the reaction flask in sequence, followed by sodium hydroxide (1.05 g, 4 eq). The temperature was raised to reflux and the reaction was carried out for 12 to 15 h. The mixture was cooled to room temperature, 30 mL of water was added, and the pH was adjusted to 5 to 6 with hydrochloric acid. 60 mL of ethyl acetate was added for extraction. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified by silica gel column chromatography (silica gel column packing: 100 to 200 mesh silica gel; eluent: dichloromethane: methanol = 30:1 → 20:1 → 10:1) to obtain 1.3 g of compound 1.

[0133] The obtained compound 1 was 1 H-NMR,13 C-NMR and MS detection, the corresponding spectra are as follows Figure 1 、 Figure 2 and Figure 3 The test results are as follows.

[0134] 1 H NMR (500MHz, CDCl3): δ: 8.019 (s, 1H), 7.366~7.350 (m, 2H), 7.282 (s, 2H), 7.158~7.057 (m, 5H), 5.925~5.894 (d, 1 H), 4.714~4.688(m,1H), 3.460~3.429(m,2H), 2.371~2.332(m,8H), 1.918~1.857(m, 2H), 1.291~1.277(m, 6H)ppm.

[0135] 13 C NMR (500MHz, CDCl3): δ: 170.57, 151.64,, 150.50, 138.11, 136.92, 136.59, 129.68, 129.22, 128.84, 128.70, 121.41, 46.59, 42.06, 30.08, 27.65, 21.31, 21.25, 20.40.

[0136] High-resolution mass spectrometry (MS): [M+1] + Measured value (m / z): 430.4.

[0137] The elemental analysis results are shown in Table 1:

[0138] Table 1 Elemental analysis results

[0139]

[0140] The above structural characterization results indicate that the compound has a structural formula as shown in Formula I.

[0141]

[0142] Example 2

[0143] Compound 1 in Example 1 was prepared using the following preparation route:

[0144]

[0145] S1: Compound 1A (300 g, 1 eq of 4,4'-dimethylbenzil), 2-aminoacetamide hydrochloride (279 g, 2.0 eq), and methanol (1.5 L) were added to the reaction flask in sequence. The temperature was raised to reflux, and an aqueous sodium hydroxide solution (50.5 g, 1 eq of sodium hydroxide, 80 mL of purified water) was added dropwise. The mixture was stirred for 4 to 5 hours, cooled to room temperature, and the pH was adjusted to 6 to 7 with hydrochloric acid. The mixture was filtered, and the filter cake was rinsed with 300 mL of methanol and dried in vacuo to obtain 320 g of compound 1B.

[0146] S2: Compound 1B (100 g, 1 eq) and phosphorus oxychloride (221.9 g, 4 eq) were added to a reaction flask in sequence. The temperature was raised to 100-105°C and stirred for 3-5 h. The residual phosphorus oxychloride was removed by concentration under reduced pressure. 150 mL of toluene was added and the reaction mixture was further concentrated to remove the residual phosphorus oxychloride. 50 mL of dichloromethane was added to the reaction mixture and stirred to dissolve. The reaction solution was added dropwise to 800 mL of isopropanol. After addition, the mixture was stirred at 0-5°C for 1-3 h, filtered, and dried under vacuum to obtain 90.7 g of compound 1C.

[0147] S3: Compound 1C (100 g, 1 eq) and 4-(isopropylamino)butanol (178 g, 4.0 eq) were added to the reaction flask in sequence, the temperature was raised to 160-170°C, the reaction was kept warm for 20-30 h, the temperature was lowered to 60-80°C, the reaction solution was poured into 0.8 L of water, and 0.8 L of ethyl acetate was added for extraction. The organic phase was washed with 0.8 L of saturated ammonium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to dryness. The concentrate was purified by silica gel column chromatography (silica gel column packing: 100-200 mesh silica gel; eluent: n-hexane: ethyl acetate = 10:1→5:1→1:1) to obtain 74 g of compound 1D.

[0148] S4: Compound 1D (70 g, 1 eq) and dichloromethane (560 mL) were added to the reaction flask in sequence. The temperature was cooled to 5-8°C in an ice bath. Dess-Martin reagent (228 g, 3 eq) was added in four batches. The reaction was kept warm for 3-5 h. The reaction solution was washed with 560 mL of saturated sodium bicarbonate solution and 560 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to dryness. The concentrate was purified by silica gel column chromatography (silica gel column packing: 100-200 mesh silica gel; eluent: n-hexane: ethyl acetate = 10:1→5:1→1:1) to obtain 21.5 g of compound 1E.

[0149] S5: Triethyl phosphinoacetate (11.25 g, 1.5 eq) and tetrahydrofuran (80 mL) were added to the reaction flask in sequence. The mixture was cooled to 5-8°C in an ice bath, and sodium hydride (1.61 g, 1.2 eq) was added in four batches. The mixture was stirred for 1 h. Compound 1E (13 g, 1 eq) was added in four batches at 0-5°C, and the mixture was heated to room temperature for 2-3 h. 40 mL of water was added dropwise to the reaction solution to quench the mixture. 160 mL of ethyl acetate was added to the reaction solution for extraction. The organic phase was washed with 80 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to dryness. The concentrate was purified by silica gel column chromatography (silica gel column packing: 100-200 mesh silica gel; eluent: n-hexane: ethyl acetate = 10:1 → 5:1 → 1:1) to obtain 13 g of compound 1F.

[0150] S6: Compound 1F (3 g, 1 eq), tetrahydrofuran (24 mL), and 1.5 mL of purified water were added to the reaction flask in sequence, and sodium hydroxide (0.39 g, 1.5 eq) was added. The temperature was raised to reflux and the reaction was carried out for 12 to 15 h. The mixture was cooled to room temperature, 24 mL of water was added, and the pH was adjusted to 5 to 6 with hydrochloric acid. 54 mL of ethyl acetate was added for extraction. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified by silica gel column chromatography (silica gel column packing: 100 to 200 mesh silica gel; eluent: dichloromethane: methanol = 30:1 → 20:1 → 10:1) to obtain 1.3 g of compound 1.

[0151] The obtained compound 1 was 1 H-NMR, 13 C-NMR and MS detection results were consistent with those in Example 1.

[0152] Example 3

[0153] Compound 1 in Example 1 was prepared using the following preparation route:

[0154]

[0155] S1: Compound 1A (300 g, 1 eq of 4,4'-dimethylbenzil), 2-aminoacetamide hydrochloride (251 g, 1.8 eq), and methanol (2.5 L) were added to the reaction flask in sequence. The temperature was raised to reflux, and an aqueous sodium hydroxide solution (151.5 g, 3 eq of sodium hydroxide, 350 mL of purified water) was added dropwise. The mixture was stirred for 4 to 5 hours, cooled to room temperature, and the pH was adjusted to 6 to 7 with hydrochloric acid. The mixture was filtered, and the filter cake was rinsed with 300 mL of methanol and dried in vacuo to obtain 320 g of compound 1B.

[0156] S2: Compound 1B (100 g, 1 eq) and phosphorus oxychloride (332.9 g, 6 eq) were added to a reaction flask in sequence. The temperature was raised to 100-105°C and stirred for 3-5 h. The mixture was concentrated under reduced pressure to remove residual phosphorus oxychloride. 300 mL of toluene was added and the mixture was further concentrated to remove residual phosphorus oxychloride. 150 mL of dichloromethane was added to the reaction mixture and stirred to dissolve. The reaction solution was then added dropwise to 1500 mL of isopropanol. After addition, the mixture was stirred at 0-5°C for 1-3 h, filtered, and dried under vacuum to obtain 90.7 g of compound 1C.

[0157] S3: Compound 1C (100 g, 1 eq) and 4-(isopropylamino)butanol (312 g, 7.0 eq) were added to the reaction flask in sequence, the temperature was raised to 160-170°C, the reaction was kept warm for 20-30 h, the temperature was lowered to 60-80°C, the reaction solution was poured into 1.2 L of water, and 1.2 L of ethyl acetate was added for extraction. The organic phase was washed with 1.2 L of saturated ammonium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated to dryness. The concentrate was purified by silica gel column chromatography (silica gel column packing: 100-200 mesh silica gel; eluent: n-hexane: ethyl acetate = 10:1→5:1→1:1) to obtain 74 g of compound 1D.

[0158] S4: Compound 1D (70 g, 1 eq) and dichloromethane (840 mL) were added to the reaction flask in sequence. The mixture was cooled to 5-8°C in an ice bath, and Dess-Martin reagent (304 g, 4 eq) was added in four batches. The mixture was kept warm for 3-5 h. The reaction solution was washed with 840 mL of saturated sodium bicarbonate solution and 840 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to dryness. The concentrate was purified by silica gel column chromatography (silica gel column packing: 100-200 mesh silica gel; eluent: n-hexane: ethyl acetate = 10:1→5:1→1:1) to obtain 21.5 g of compound 1E.

[0159] S5: Triethyl phosphinoacetate (15 g, 2.0 eq) and tetrahydrofuran (100 mL) were added to the reaction flask in sequence. The mixture was cooled to 5-8°C in an ice bath, and sodium hydride (2.01 g, 1.5 eq) was added in four batches. The mixture was stirred for 1 h. Compound 1E (13 g, 1 eq) was added in four batches at 0-5°C, and the mixture was heated to room temperature for 2-3 h. 60 mL of water was added dropwise to the reaction solution to quench the mixture. 200 mL of ethyl acetate was added to extract the mixture. The organic phase was washed with 100 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to dryness. The concentrate was purified by silica gel column chromatography (silica gel column packing: 100-200 mesh silica gel; eluent: n-hexane: ethyl acetate = 10:1 → 5:1 → 1:1) to obtain 13 g of compound 1F.

[0160] S6: Compound 1F (3 g, 1 eq), tetrahydrofuran (36 mL), and 4.5 mL of purified water were added to the reaction flask in sequence, and sodium hydroxide (1.31 g, 5 eq) was added. The temperature was raised to reflux and the reaction was carried out for 12 to 15 h. The mixture was cooled to room temperature, 36 mL of water was added, and the pH was adjusted to 5 to 6 with hydrochloric acid. 66 mL of ethyl acetate was added for extraction. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified by silica gel column chromatography (silica gel column packing: 100 to 200 mesh silica gel; eluent: dichloromethane: methanol = 30:1 → 20:1 → 10:1) to obtain 1.3 g of compound 1.

[0161] The obtained compound 1 was 1 H-NMR, 13 C-NMR and MS detection results were consistent with those in Example 1.

[0162] Comparative Example 1

[0163] Prepare 6-[N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino]-2-hexenoic acid, denoted as compound 2, with the corresponding structural formula as follows.

[0164]

[0165] The preparation route is as follows:

[0166]

[0167] The preparation method of 6-[N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino]-2-hexenoic acid was the same as the preparation method of 6-{N-[5,6-di(4-methylphenyl)pyrazin-2-yl-N-isopropylamino}-2-hexenoic acid (Compound 1) in Example 1, except that the starting material was benzil. The H NMR spectrum data of Compound 2 are as follows:

[0168] 1 H NMR (500MHz, CDCl3): δ: 8.050 (s, 1H), 7.449 ~ 7.430 (m, 2H), 7.366 ~ 7.347 (m, 2H), 7.285 ~ 7.247 (m, 6H), 7.232 ~ 7.211 (m, 1H), 5.9 20~5.889(m,1H), 4.741~4.715(m,1H), 3.454~3.422(m,2H), 2.354~2.340(m,2H), 1.898~1.867(m,2H), 1.285~1.272(m,6H)ppm.

[0169] Comparative Example 2

[0170] 3-Ethoxy-6-{N-[5,6-di(4-methylphenyl)pyrazine]-2-yl-N-isopropylamino}hexanoic acid was prepared and recorded as compound 3. The corresponding structural formula is as follows.

[0171]

[0172] The preparation route is as follows:

[0173]

[0174] Compound 1F, named 6-{N-[5,6-di(4-methylphenyl)pyrazin-2-yl-N-isopropylamino}-2-hexenoic acid ethyl ester, (the preparation method of this compound is the same as "Compound 1F" in Example 1) (3 g, 1 eq), anhydrous ethanol (30 mL), cooled in an ice bath, sodium hydride (0.79 g, 3 eq) was added, and the reaction was refluxed for 12 to 15 hours, then cooled to room temperature, and the pH was adjusted to 5 to 6 with hydrochloric acid. 30 mL of water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified by silica gel column chromatography to obtain 1.2 g of compound 3. The H NMR spectrum data of compound 3 are as follows:

[0175] 1 H NMR (500MHz, CDCl3): δ: 8.013 (s, 1H), 7.378~7.366 (m, 2H), 7.350 (s, 1H) ,7.090~7.065(m,5H), 4.803~4.776(m,1H), 3.814~3.792(m,1H), 3.594~ 3.536(m,2H), 3.449~3.398(m,2H), 2.628~2.461(m,2H), 2.348~2.337(m ,6H), 1.802~1.642(m,4H), 1.281~1.268(m,6H), 1.212~1.185(t,3H)ppm.

[0176] Comparative Example 3

[0177] Prepare 3-ethoxy-6-[N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino]hexanoic acid, denoted as compound 4, with the corresponding structural formula as follows.

[0178]

[0179] The preparation route is as follows:

[0180]

[0181] Compound 8F (3 g, 1 eq, compound 8F in Comparative Example 1 of the preparation method) and anhydrous ethanol (30 mL) were added to the reaction flask in sequence, cooled in an ice bath, sodium hydride (0.84 g, 3 eq) was added, and the temperature was raised to reflux for 12 to 15 hours, then cooled to room temperature, and the pH was adjusted to 5 to 6 with hydrochloric acid. 100 mL of water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified by silica gel column chromatography to obtain 1.2 g of compound 4. The H NMR spectrum data of compound 4 are as follows:

[0182] 1 H NMR (500MHz, CDCl3): δ: 8.037 (s, 1H), 7.443~7.226 (m, 10H), 4.785 (m, 1H), 3.790 (m, 1H), 3.545~ 3.414(m,4H), 2.585~2.507(m,2H), 1.773~1.661(m,4H), 1.278~1.265(m,6H), 1.175(t,3H)ppm.

[0183] Experimental Example 1: Adenosine diphosphate (ADP)-induced human platelet aggregation inhibitory effect test

[0184] The sample groups of Compound 1 to Compound 4 prepared in Example 1 and Comparative Examples 1 to 3 were subjected to comparative experimental studies on the inhibitory effects of adenosine diphosphate (ADP)-induced human platelet aggregation with the positive control drug clopidogrel group, the positive control drug selexipag group and the blank control group.

[0185] Test method:

[0186] Each time, 20-30 mL of venous blood was drawn from the volunteers and anticoagulated with a citrate dextrose solution at 1 / 6 of the venous blood volume. The blood was centrifuged twice at 800 rpm for 10 minutes each time to prepare platelet-rich plasma (PRP). The remaining plasma was centrifuged at 3000 rpm for 15 minutes to prepare platelet-poor plasma (PPP). The concentration of PRP was adjusted to 3.0 × 10 8 -4.0×10 8 Between 100 and 100 mL.

[0187] Preparation of the test solution: Weigh 10 mg of each of the compound 1-compound 4 samples, clopidogrel sample, and selexipag sample prepared in Example 1 and Comparative Examples 1-3, add an appropriate amount of DMSO to fully dissolve them, and then dilute them with physiological saline to the required concentration (the amount of DMSO is controlled within one thousandth) to prepare the test solution.

[0188] According to the optical principle, the test was performed using the Chrono-log platelet aggregation instrument. The platelet aggregation instrument was turned on and preheated for 30 minutes. 200 μL of PPP and PRP were taken in turbidimetric tubes and placed in the preheated well. For the blank control group, 50 μL of normal saline was added to the PRP and PPP tubes, and for the selexipag, clopidogrel, and compound 1-compound 4 drug groups, 50 μL of the corresponding test solution was added to the PRP and PPP tubes, respectively, with a final concentration of 3.5 μM. After incubation at 37°C for 5 minutes, platelet aggregation was measured. Then, 10 μL of ADP (final concentration 2×10 -4 mol / L) to induce platelet aggregation, record the graph changes within 5 min, and read the maximum aggregation rate of each group.

[0189] Two parallel groups of platelet aggregation assays at lower concentrations of compound 1 were performed: 5 μL of compound 1 test solution was added to the PRP and PPP tubes, with a final concentration of 0.35 μM; 0.5 μL of compound 1 test solution was added to the PRP and PPP tubes, with a final concentration of 0.035 μM. After incubation at 37°C for 5 min, platelet aggregation assay was performed. Then, 10 μL of ADP (final concentration 2×10 -4 mol / L) to induce platelet aggregation, record the graph changes within 5 min, and read the maximum aggregation rate of each group.

[0190] The platelet aggregation inhibition rate was calculated as follows:

[0191] Inhibition rate (%) = (maximum aggregation rate of blank control group - maximum aggregation rate of drug-treated group) / maximum aggregation rate of blank control group × 100%

[0192] The experimental results are shown in Table 1.

[0193] Table 1 Determination results of ADP-induced platelet aggregation inhibition rate in each test group (x±SD, %)

[0194]

[0195]

[0196] As shown in Table 1, in each test group, compound 1 had a significant inhibitory effect on ADP-induced platelet aggregation and showed a dose-response effect. When the concentration of compound 1 reached 3.5μM and 0.35μM, the inhibitory effect on ADP-induced platelet aggregation was 100% and 81.77%, respectively. The effect was significantly better than that of the positive control drugs clopidogrel and selexipag groups at the same concentration of 3.5μM or 10 times higher concentration of 3.5μM. Compound 1 has a very strong inhibitory effect on ADP-induced platelet aggregation. However, the inhibitory effects of compounds 2, 3, and 4 were not ideal and were inferior to those of the clopidogrel and selexipag groups.

[0197] Experimental Example 2: Experiment on the inhibitory effect of arachidonic acid (AA) on human platelet aggregation

[0198] The compound 1-compound 4 sample groups prepared in Examples 1-4 were compared with the positive control drug aspirin group, the positive control drug selexipag group and the blank control group for a comparative study on the inhibitory effect of arachidonic acid (AA)-induced human platelet aggregation.

[0199] Test method:

[0200] Each time, 20-30 mL of venous blood was drawn from the volunteers and anticoagulated with a citrate-dextrose solution at 1 / 6 the volume of the venous blood. The blood was centrifuged twice at 800 rpm for 10 minutes each time to prepare PRP. The remaining plasma was centrifuged at 3000 rpm for 15 minutes to prepare PPP. The concentration of PRP was adjusted to 3.0 × 10 8 -4.0×10 8 Between 100 and 100 mL.

[0201] Preparation of the test solution: Weigh 10 mg of the compound 1-compound 4 samples, aspirin sample and selexipag sample prepared in Examples 1-4, add an appropriate amount of DMSO to fully dissolve them, and then dilute them with physiological saline to the required concentration (the amount of DMSO is controlled within 1 / 1000) to prepare the test solution.

[0202] According to the optical principle, the test was performed using the Chrono-log platelet aggregation instrument. The platelet aggregation instrument was turned on and preheated for 30 minutes. 200 μL of PPP and PRP were taken in turbidimetric tubes and placed in the preheated well. For the blank control group, 50 μL of normal saline was added to the PRP and PPP tubes, and for the selexipag, aspirin, and compound 1-compound 4 drug groups, 50 μL of the corresponding test solution was added to the PRP and PPP tubes, respectively, with a final concentration of 3.5 μM. After incubation at 37°C for 5 minutes, platelet aggregation was measured. Then, 5 μL of AA (final concentration 2×10 -4mol / L) to induce platelet aggregation, record the graph changes within 5 min, and read the maximum aggregation rate of each group.

[0203] Two parallel groups of platelet aggregation assays at lower concentrations of compound 1 were performed: 5 μL of compound 1 test solution was added to the PRP and PPP tubes, with a final concentration of 0.35 μM; 0.5 μL of compound 1 test solution was added to the PRP and PPP tubes, with a final concentration of 0.035 μM. After incubation at 37°C for 5 min, platelet aggregation assay was performed. Then, 10 μL of AA (final concentration 2×10 -4 mol / L) to induce platelet aggregation, record the graph changes within 5 min, and read the maximum aggregation rate of each group.

[0204] The platelet aggregation inhibition rate was calculated as follows:

[0205] Inhibition rate (%) = (maximum aggregation rate of blank control group - maximum aggregation rate of drug-treated group) / maximum aggregation rate of blank control group × 100%

[0206] The experimental results are shown in Table 2.

[0207] Table 2 Determination results of AA-induced platelet aggregation inhibition rate in each test group (x±SD, %)

[0208]

[0209]

[0210] As shown in Table 2, compound 1 exhibited a significant inhibitory effect on AA-induced platelet aggregation in each test group, and the effect was dose-dependent. When the concentration of compound 1 reached 3.5 μM and 0.35 μM, the inhibitory effects on AA-induced platelet aggregation were 100% and 71.67%, respectively. The effect was significantly better than that of the positive control drugs aspirin and selexipag groups at the same concentration of 3.5 μM or 10 times higher concentration of 3.5 μM. Compound 1 also had a strong inhibitory effect on AA-induced platelet aggregation. However, the inhibitory effects of compounds 2, 3, and 4 were not ideal, and were inferior to those of the aspirin and selexipag groups.

[0211] Experimental Example 3: Acute toxicity test

[0212] The acute toxicity comparison test of the compound 1 sample group prepared in Example 1 and the positive control drug selexipag group was conducted.

[0213] Test method:

[0214] Rats were randomly divided into 7 groups according to body weight, 5 rats in each group. Groups 1-3 were given a single oral administration of 250 mg / kg, 500 mg / kg, and 1000 mg / kg of selexipag, respectively. Groups 4-7 were given a single oral administration of 1000 mg / kg, 2000 mg / kg, 3000 mg / kg, and 4000 mg / kg of compound 1, respectively. The administration volume was 10 ml / kg. The rats were observed for 7 days after administration.

[0215] The experimental results are shown in Table 3.

[0216] Table 3 Comparative results of acute toxicity test of compound 1 and selexipag

[0217]

[0218] The results showed that the oral lethal dose of selexipag in rats was approximately 500 mg / kg, and the non-lethal dose was approximately 250 mg / kg. No mortality was observed at 2000 mg / kg of compound 1, indicating that compound 1 is significantly less toxic than selexipag.

[0219] In summary, compound 1 exhibits extremely strong inhibitory effects on both AA-induced platelet aggregation and ADP-induced platelet aggregation, with low toxicity and outstanding clinical advantages.

[0220] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A diphenylpyrazine compound or a pharmaceutically acceptable salt, isomer or mixture thereof, characterized in that: The structural formula of the diphenylpyrazine compound is shown in formula (I): 。 2. A pharmaceutical composition, characterized in that A therapeutically effective dose of the diphenylpyrazine compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt, isomer or mixture thereof.

3. The pharmaceutical composition according to claim 2, wherein Also included are pharmaceutically acceptable carriers or excipients.

4. Use of the diphenylpyrazine compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt, isomer or mixture thereof, or the pharmaceutical composition according to claim 2 or 3, in the preparation of a drug for inhibiting platelet aggregation.

5. The use according to claim 4, characterized in that The inhibition of platelet aggregation is inhibition of AA-induced platelet aggregation or inhibition of ADP-induced platelet aggregation.

6. Use of the diphenylpyrazine compound of formula (I) according to claim 1 or its pharmaceutically acceptable salt, isomer or mixture thereof, or the pharmaceutical composition according to claim 2 or 3 in the preparation of an anticoagulant drug.

7. Use of the diphenylpyrazine compound of formula (I) according to claim 1, or its pharmaceutically acceptable salt, isomer or mixture thereof, or the pharmaceutical composition according to claim 2 or 3, in the preparation of a medicament for preventing or treating cardiovascular and cerebrovascular diseases associated with platelet aggregation, or other vascular thrombosis or embolic diseases.

8. The use according to claim 7, characterized in that The platelet aggregation-related cardiovascular and cerebrovascular diseases, other vascular thrombosis or embolic diseases are cerebral thrombosis, cerebral infarction, myocardial infarction, heart failure, pulmonary embolism, thromboangiitis, chronic arterial occlusive disease or central retinal vein thrombosis.

9. Use of the diphenylpyrazine compound of formula (I) according to claim 1, or its pharmaceutically acceptable salt, isomer or mixture thereof, or the pharmaceutical composition according to claim 2 or 3, in the preparation of a medicament for extracorporeal circulation platelet protection, post-arterial angiography treatment, or post-vascular reconstructive treatment.

10. The method for preparing the diphenylpyrazine compound represented by formula (I) according to claim 1, characterized in that: The steps include: S1, 4,4'-dimethylbenzil and 2-aminoacetamide hydrochloride undergo cyclization reaction to obtain compound 1B; S2, compound 1B undergoes chlorination reaction with phosphorus oxychloride to obtain compound 1C; S3, compound 1C undergoes substitution reaction with 4-(isopropylamino)butanol to obtain compound 1D; S4, compound 1D is oxidized with Dess-Martin reagent to obtain compound 1E; S5, compound 1E undergoes nucleophilic addition reaction with triethyl phosphoacetate to obtain compound 1F; S6, compound 1F is hydrolyzed in the presence of sodium hydroxide to obtain a diphenylpyrazine compound represented by formula (I).

11. The method for preparing the diphenylpyrazine compound represented by formula (I) according to claim 10, characterized in that: The specific steps include: S1, 4,4'-dimethylbenzil, 2-aminoacetamide hydrochloride and the first reaction solvent are mixed, the temperature is raised to reflux, a strong base solution is added, the temperature is kept for reaction, the temperature is lowered to 20-25°C, the pH is adjusted to neutral, the solid-liquid separation is performed, the mixture is washed, and the mixture is dried to obtain compound 1B; S2, mixing the compound 1B and phosphorus oxychloride, raising the temperature to 100-105° C., maintaining the temperature for reaction, removing the phosphorus oxychloride, and obtaining a reactant; recrystallizing the reactant, performing solid-liquid separation, and drying to obtain compound 1C; S3, mixing the compound 1C and 4-(isopropylamino)butanol, raising the temperature to 160-170° C., maintaining the temperature for reaction, and then cooling to obtain a reaction solution; extracting the reaction solution, separating the organic phase, washing, drying, and purifying to obtain compound 1D; S4, mixing the compound 1D with a second reaction solvent, cooling the mixture to 0-10° C., adding a Dess-Martin reagent, and incubating the mixture to react to obtain a reaction solution; washing the reaction solution, drying it, and purifying it to obtain compound 1E; S5, triethyl phosphoacetate and the third reaction solvent are mixed, cooled to 0-10°C, sodium hydride is added, and stirred for 0.5-2.0 h. Compound 1E is added at 0-10°C, and the temperature is raised to 20-25°C. The mixture is kept warm for reaction to obtain a reaction solution, the reaction is quenched, extraction is performed, and the organic phase is separated, washed, dried, and purified to obtain compound 1F; S6, mixing compound 1F, the fourth reaction solvent, and sodium hydroxide, raising the temperature to reflux for reaction to obtain a reaction solution, adjusting the pH to a weakly acidic state, extracting, separating the organic phase, drying, and purifying to obtain a diphenylpyrazine compound represented by formula (I); The first reaction solvent is methanol, the second reaction solvent is dichloromethane, and the third reaction solvent is tetrahydrofuran.

12. The method for preparing the diphenylpyrazine compound represented by formula (I) according to claim 11, characterized in that: In S1, the first reaction solvent is methanol; and / or In S1, the strong alkaline solution is a sodium hydroxide aqueous solution; and / or In S1, the neutral refers to a pH of 6 to 7; and / or In S1, the equivalent ratio of 2-aminoacetamide hydrochloride to 4,4'-dimethylbenzil is ≥1; and / or In S1, the mass volume ratio of the 4,4'-dimethylbenzil to the first reaction solvent is 3g:15mL~25mL; and / or In S1, the equivalent ratio of the 4,4'-dimethylbenzil to the strong base is 1:1-3; and / or In S1, the concentration of the strong base solution is 0.4-0.6 g / mL; and / or In S1, the insulation reaction time is 4 to 5 hours; and / or In S2, the equivalent ratio of phosphorus oxychloride to compound 1B is ≥1; and / or In S2, the insulation reaction time is 3 to 5 hours; and / or In S3, the equivalent ratio of 4-(isopropylamino)butanol to compound 1C is ≥1; and / or In S3, the insulation reaction time is 20 to 30 hours; and / or In S3, the cooling temperature is 60°C to 80°C; and / or In S4, the second reaction solvent is dichloromethane; and / or In S4, the equivalent ratio of the Dess-Martin reagent to compound 1D is ≥ 1; and / or In S4, the mass volume ratio of the compound 1D to the second reaction solvent is 1 g: 8 mL to 12 mL; and / or In S4, the insulation reaction time is 3 to 5 hours; and / or In S5, the third reaction solvent is tetrahydrofuran; and / or In S5, the equivalent ratio of triethyl phosphoacetate to compound 1E is ≥1; and / or In S5, the mass volume ratio of the compound 1E to the third reaction solvent is 13 g: 80 mL to 100 mL; and / or In S5, the equivalent ratio of sodium hydride to compound 1E is 1-1.5:1; and / or In S5, the insulation reaction time is 2 to 3 hours; and / or In S6, the fourth reaction solvent is tetrahydrofuran and purified water; and / or In S6, the equivalent ratio of sodium hydroxide to compound 1F is 1.5 to 5:1; and / or In S6, the mass volume ratio of the compound 1F to the fourth reaction solvent is 1 g: 8.5 mL to 13.5 mL; and / or In S6, the time for heating to reflux reaction is 12 to 15 hours.

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