Diphenylpyrazine compound, composition thereof, uses thereof, and preparation method therefor

Abstract

The present application belongs to the technical field of medical uses of small-molecular compounds, and relates to a diphenylpyrazine compound, a composition thereof, the uses thereof, and a preparation method therefor. The present application mainly relates to the use of the diphenylpyrazine compound and the composition thereof in preparing drugs for inhibiting platelet aggregation, and further particularly relates to the use of the diphenylpyrazine compound and the composition thereof in preparing drugs for treating platelet aggregation related cardiovascular and cerebrovascular diseases and other vascular thrombotic and embolic diseases. By means of an in-vitro human platelet aggregation experiment, the present application proves that the diphenylpyrazine compound exhibits an extremely strong inhibition effect on both AA-induced platelet aggregation and ADP-induced platelet aggregation and also has lower toxicity and outstanding clinical advantages.

Description

Diphenylpyrazine compounds and compositions, applications and preparation methods thereof

[0001] This patent application claims priority to Chinese Patent Application No. CN202311824286.0 filed on December 27, 2023 and Chinese Patent Application No. CN202410952745.1 filed on July 16, 2024. The disclosures of the prior applications are incorporated herein by reference in their entirety. Technical Field

[0002] The present application belongs to the field of medical technology, and specifically relates to diphenylpyrazine compounds and their compositions, applications and preparation methods, and mainly relates to their application in the preparation of drugs for inhibiting platelet aggregation. Background Art

[0003] 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.

[0004] 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; in most patients, 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.

[0005] 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. Technical issues

[0006] The technical problem to be solved by the present application 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. Technical Solutions

[0007] To solve the above technical problems, this application adopts the following technical solutions:

[0008] The first aspect of the present application provides a diphenylpyrazine compound represented by formula (I) or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer and mixture thereof.

[0009] The second aspect of the present application 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.

[0010] Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0011] The third aspect of the present application 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.

[0012] Furthermore, the inhibition of platelet aggregation is inhibition of AA-induced platelet aggregation.

[0013] Furthermore, the inhibition of platelet aggregation is inhibition of ADP-induced platelet aggregation.

[0014] The fourth aspect of the present application 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.

[0015] The fifth aspect of the present application is to provide the diphenylpyrazine compounds represented by the above-mentioned formula (I) or their pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers and mixtures thereof, as well as the use of the above-mentioned 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.

[0016] 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.

[0017] The sixth aspect of the present application 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.

[0018] 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.

[0019] 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 cAMP levels in platelets, convert more free calcium into calcium storage granules, render platelets inert, and thus inhibit primary platelet aggregation. Furthermore, increased cAMP levels can reduce platelet TXA2 production and ADP and 5HT release, thereby reducing secondary platelet aggregation. This dual effect achieves a more effective platelet aggregation inhibition effect. These compounds can be used to effectively 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 platelet protection during extracorporeal circulation, post-arterial angiography treatment, or post-vascular revascularization treatment to prevent postoperative thrombosis.

[0020] The seventh aspect of the present application is to provide a method for preparing the diphenylpyrazine compound represented by the above formula (I), comprising the following steps:

[0021] S1, 4,4'-dimethylbenzil (Compound 1A) undergoes cyclization reaction with 2-aminoacetamide hydrochloride to obtain Compound 1B;

[0022] S2, compound 1B undergoes chlorination reaction with phosphorus oxychloride to obtain compound 1C;

[0023] S3, compound 1C undergoes substitution reaction with 4-(isopropylamino)butanol to obtain compound 1D;

[0024] S4, compound 1D is oxidized with Dess-Martin reagent to obtain compound 1E;

[0025] S5, compound 1E undergoes nucleophilic addition reaction with triethyl phosphoacetate to obtain compound 1F;

[0026] S6. Compound 1F is hydrolyzed in the presence of sodium hydroxide to obtain a diphenylpyrazine compound represented by formula (I) (ie, compound 1).

[0027] The synthetic route of the preparation method is as follows.

[0028] The preparation method of the diphenylpyrazine compound represented by the above formula (I) specifically comprises the following steps:

[0029] 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;

[0030] 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;

[0031] 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 first reaction solution; extracting the first reaction solution, separating the organic phase, washing, drying, and purifying to obtain compound 1D;

[0032] S4, mixing the compound 1D and 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 second reaction solution; washing the second reaction solution, drying it, and purifying it to obtain compound 1E;

[0033] 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 third reaction solution. The reaction is quenched, extracted, and the organic phase is separated. The organic phase is washed, dried, and purified to obtain Compound 1F.

[0034] S6, mixing compound 1F, the fourth reaction solvent and sodium hydroxide, raising the temperature to reflux for reaction to obtain a fourth reaction solution, adjusting the pH to weak acidity, extracting, separating the organic phase, drying, and purifying to obtain the diphenylpyrazine compound represented by formula (I).

[0035] Furthermore, in step S1, the first reaction solvent is methanol.

[0036] Furthermore, in step S1, the strong alkaline solution is a sodium hydroxide aqueous solution.

[0037] Furthermore, in step S1, the pH is adjusted to 6-7.

[0038] Furthermore, in step S1, the washing is to rinse the solid phase obtained after solid-liquid separation with methanol.

[0039] Furthermore, in step S1, the equivalent ratio of 2-aminoacetamide hydrochloride to 4,4'-dimethylbenzil is ≥1.

[0040] Furthermore, in step S1, the mass volume ratio of the 4,4'-dimethylbenzil to the first reaction solvent is 3 g: (15-25) mL.

[0041] Furthermore, in step S1, the equivalent ratio of the 4,4'-dimethylbenzil to the strong base is 1:1-3.

[0042] Furthermore, in step S1, the concentration of the strong alkaline solution is 0.4-0.6 g / mL.

[0043] Furthermore, in step S1, the strong base solution is added dropwise.

[0044] Furthermore, in step S1, the insulation reaction time is 4 to 5 hours.

[0045] Furthermore, in step S1, the insulation reaction is carried out under stirring conditions.

[0046] Furthermore, in step S1, the pH is adjusted to neutral by using hydrochloric acid.

[0047] Furthermore, in step S1, the solid-liquid separation method can be conventional methods such as filtration or centrifugation.

[0048] Furthermore, in step S1, the amount of methanol solvent used in the washing accounts for 10%-20% of the first reaction solvent.

[0049] Furthermore, in step S1, the drying method may be conventional methods such as vacuum drying.

[0050] 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.

[0051] Furthermore, in step S2, the reactant is recrystallized by adding dichloromethane to the reactant, stirring and dissolving to obtain a fifth reaction liquid, and the fifth reaction liquid is added dropwise to isopropanol, and after the addition is completed, stirred at 0-5°C for 1-3h.

[0052] Furthermore, in step S2, the equivalent ratio of phosphorus oxychloride to compound 1B is ≥1, more preferably ≥3.

[0053] Furthermore, in step S2, the mass volume ratio of the compound 1B to toluene is 1 g: (1.5-3.0) mL.

[0054] Furthermore, in step S2, the mass volume ratio of the compound 1B to dichloromethane is 1 g: (0.5-1.5) mL.

[0055] Furthermore, in step S2, the mass volume ratio of the compound 1B to isopropyl alcohol is 1 g: (8-15) mL.

[0056] Furthermore, in step S2, the insulation reaction time is 3 to 5 hours.

[0057] Furthermore, in step S2, the insulation reaction is carried out under stirring conditions.

[0058] Furthermore, in step S2, the solid-liquid separation method is a conventional method such as filtration or centrifugation.

[0059] Furthermore, in step S2, the drying method is a conventional method such as vacuum drying.

[0060] Furthermore, in step S3, the extraction is performed by pouring the first reaction liquid into water and adding ethyl acetate for extraction.

[0061] Furthermore, in step S3, the washing, drying and purification steps are to wash the separated organic phase with a saturated aqueous ammonium chloride solution, dry it with anhydrous sodium sulfate, concentrate it to dryness to obtain a concentrate, and purify it by silica gel column chromatography.

[0062] Furthermore, in step S3, the equivalent ratio of the 4-(isopropylamino)butanol to compound 1C is ≥1, and more preferably the equivalent ratio is ≥4.

[0063] Furthermore, in step S3, the mass volume ratio of the compound 1C to water is 1 g: (8-12) mL.

[0064] Furthermore, in step S3, the mass volume ratio of the compound 1C to ethyl acetate is 1 g: (8-12) mL.

[0065] 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.

[0066] Furthermore, in step S3, the insulation reaction time is 20 to 30 hours.

[0067] Furthermore, in step S3, the cooling temperature is 60°C to 80°C.

[0068] Furthermore, in step S4, the second reaction solvent is dichloromethane.

[0069] Furthermore, in step S4, the second reaction liquid washing, drying and purification step is to wash the second reaction liquid with a saturated sodium bicarbonate solution, a saturated sodium chloride aqueous solution, dry it with anhydrous sodium sulfate, concentrate it to dryness to obtain a concentrate, and purify it by silica gel column chromatography.

[0070] Furthermore, in step S4, the equivalent ratio of the Dess-Martin reagent to compound 1D is ≥1.

[0071] Furthermore, in step S4, the mass volume ratio of the compound 1D to the second reaction solvent is 1 g: (8-12) mL.

[0072] Furthermore, in step S4, the mass volume ratio of the compound 1D to the saturated sodium bicarbonate solution is 1 g: (8-12) mL.

[0073] 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.

[0074] Furthermore, in step S4, the insulation reaction time is 3 to 5 hours.

[0075] Furthermore, in step S4, the temperature may be lowered to 0-10°C by conventional methods such as ice bath.

[0076] Furthermore, in step S4, the Dess-Martin reagent is added in 3-5 batches.

[0077] Furthermore, in step S5, the third reaction solvent is tetrahydrofuran.

[0078] Furthermore, in step S5, the quenching reaction and extraction step is to add water dropwise to the third reaction liquid for quenching, and add ethyl acetate for extraction.

[0079] Furthermore, in step S5, the washing, drying and purification steps are to wash the separated organic phase with a saturated sodium chloride aqueous solution, dry it with anhydrous sodium sulfate, concentrate it to dryness to obtain a concentrate, and purify it by silica gel column chromatography.

[0080] Furthermore, in step S5, the equivalent ratio of triethyl phosphoacetate to compound 1E is ≥1.

[0081] Furthermore, in step S5, the mass volume ratio of the compound 1E to the third reaction solvent is 13 g: (80-100) mL.

[0082] Furthermore, in step S5, the equivalent ratio of sodium hydride to compound 1E is 1 to 1.5:1.

[0083] Furthermore, in step S5, the mass volume ratio of the compound 1E to water is 13 g: (40-60) mL.

[0084] Furthermore, in step S5, the mass volume ratio of the compound 1E to ethyl acetate is 13 g: (160-200) mL.

[0085] 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.

[0086] Furthermore, in step S5, the insulation reaction time is 2 to 3 hours.

[0087] Furthermore, in step S5, the temperature may be lowered to 0-10°C by conventional methods such as ice bath.

[0088] Furthermore, in step S5, the sodium hydride needs to be added in 3-5 batches.

[0089] Furthermore, in step S5, the compound 1E needs to be added in 3-5 batches.

[0090] Furthermore, in step S6, the fourth reaction solvent is tetrahydrofuran and purified water.

[0091] Furthermore, in step S6, the pH adjustment and extraction steps are to cool the fourth reaction liquid to room temperature, add water, adjust the pH to 5-6, and add ethyl acetate for extraction.

[0092] Furthermore, in step S6, the drying and purification step is to dry the separated organic phase with anhydrous sodium sulfate, concentrate to dryness to obtain a concentrate, and purify it by silica gel column chromatography.

[0093] Furthermore, in step S6, the equivalent ratio of sodium hydroxide to compound 1F is 1.5 to 5:1.

[0094] 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.

[0095] Furthermore, in step S6, the mass volume ratio of the compound 1F to tetrahydrofuran is 1 g: (8-12) mL.

[0096] Furthermore, in step S6, the mass volume ratio of the compound 1F to purified water is 1 g: (0.5-1.5) mL.

[0097] 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.

[0098] Furthermore, in step S6, the mass volume ratio of the compound 1F to ethyl acetate is 1 g: (18-22) mL.

[0099] Furthermore, in step S6, the temperature is raised to reflux reaction time is 12 to 15 hours.

[0100] Furthermore, in step S6, the pH is adjusted using hydrochloric acid. Beneficial effects

[0101] The positive progress of the present application is that 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 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 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

[0102] FIG1 is a diagram of compound 1 in Example 1 of the present application. 1 H-NMR spectrum;

[0103] Figure 2 is a diagram of compound 1 in Example 1 of the present application. 13 C-NMR spectrum;

[0104] Figure 3 is the MS spectrum of compound 1 in Example 1 of the present application. Modes for Carrying Out the Invention

[0105] The present application is further described in detail below through specific implementation methods, but this is only intended to help understand the present application so that professionals in the field can implement or use the present application, and does not constitute any limitation to the present application.

[0106] the term

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] "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.

[0113] "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 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 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 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.

[0114] 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.

[0115] "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.

[0116] "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.

[0117] As used herein, the term "room temperature" or "RT" refers to an ambient temperature of 20 to 25°C (68 to 77°F).

[0118] The solution of this application is described below through specific embodiments.

[0119] Example 1

[0120] 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.

[0121] The preparation route is as follows:

[0122] 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.

[0123] 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.

[0124] 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 and the reaction was kept at this temperature for 20-30 h. The temperature was then lowered to 60-80°C. The reaction solution was poured into 1.0 L of water and extracted with 1.0 L of ethyl acetate. The organic phase was washed with 1.0 L of saturated aqueous ammonium 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 (volume ratio) = 10:1 → 5:1 → 1:1) to obtain 74 g of compound 1D.

[0125] 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 and 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 (volume ratio) = 10:1→5:1→1:1) to obtain 21.5 g of compound 1E.

[0126] 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 for extraction. 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 (volume ratio) = 10:1→5:1→1:1) to obtain 13 g of compound 1F.

[0127] 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 (volume ratio) = 30:1→20:1→10:1) to obtain 1.3 g of compound 1.

[0128] The obtained compound 1 was 1 H-NMR,13 C-NMR and MS detections, the corresponding spectra are shown in Figures 1, 2, and 3, respectively, and the detection results are as follows.

[0129] 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.

[0130] 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.

[0131] High-resolution mass spectrometry (MS): [M+1] + Measured value (m / z): 430.4.

[0132] The elemental analysis results are shown in Table 1:

[0133] Table 1 Elemental analysis results

[0134] The above structural characterization results indicate that the compound has a structural formula as shown in Formula I.

[0135] Example 2

[0136] Compound 1 in Example 1 was prepared using the following preparation route:

[0137] 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.

[0138] 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.

[0139] 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 (volume ratio) = 10:1→5:1→1:1) to obtain 74 g of compound 1D.

[0140] S4: Compound 1D (70 g, 1 eq) and dichloromethane (560 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 (228 g, 3 eq) was added in four batches. The mixture 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 (volume ratio) = 10:1→5:1→1:1) to obtain 21.5 g of compound 1E.

[0141] 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 extract the mixture. The organic phase was washed with 80 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 (volume ratio) = 10:1→5:1→1:1) to obtain 13 g of compound 1F.

[0142] 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 (volume ratio) = 30:1→20:1→10:1) to obtain 1.3 g of compound 1.

[0143] The obtained compound 1 was 1 H-NMR, 13 C-NMR and MS detection results were consistent with those in Example 1.

[0144] Example 3

[0145] Compound 1 in Example 1 was prepared using the following preparation route:

[0146] 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.

[0147] 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.

[0148] 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 (volume ratio) = 10:1→5:1→1:1) to obtain 74 g of compound 1D.

[0149] 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 (volume ratio) = 10:1→5:1→1:1) to obtain 21.5 g of compound 1E.

[0150] S5: Triethyl phosphoacetate (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 (volume ratio) = 10:1→5:1→1:1) to obtain 13 g of compound 1F.

[0151] 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 (volume ratio) = 30:1→20:1→10:1) to obtain 1.3 g of compound 1.

[0152] The obtained compound 1 was 1H-NMR, 13 C-NMR and MS detection results were consistent with those in Example 1.

[0153] Comparative Example 1

[0154] Prepare 6-[N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino]-2-hexenoic acid, denoted as compound 2, with the corresponding structural formula as follows.

[0155] The preparation route is as follows:

[0156] 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:

[0157] 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.

[0158] Comparative Example 2

[0159] 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.

[0160] The preparation route is as follows:

[0161] Compound 1F, named 6-{N-[5,6-di(4-methylphenyl)pyrazine]-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, the temperature was raised to reflux, the reaction was carried out for 12 to 15 hours, the temperature was lowered to room temperature, 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:

[0162] 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.

[0163] Comparative Example 3

[0164] 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.

[0165] The preparation route is as follows:

[0166] 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, the temperature was raised to reflux, and the reaction was carried out for 12 to 15 hours. The temperature was then lowered to room temperature, 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:

[0167] 1H 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.

[0168] Experimental Example 1: Adenosine diphosphate (ADP)-induced human platelet aggregation inhibitory effect test

[0169] 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.

[0170] Test method:

[0171] 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.

[0172] 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 DMSO content is controlled within 1 / 1000) to prepare the test solution.

[0173] 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.

[0174] 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.

[0175] The platelet aggregation inhibition rate was calculated as follows: Inhibition rate (%) = (maximum aggregation rate of blank control group - maximum aggregation rate of drug-treated group) / maximum aggregation rate of blank control group × 100%

[0176] The experimental results are shown in Table 1.

[0177] Table 1 Determination results of ADP-induced platelet aggregation inhibition rate in each test group (x±SD, %)

[0178] 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.

[0179] Experimental Example 2: Experiment on the inhibitory effect of arachidonic acid (AA) on human platelet aggregation

[0180] 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.

[0181] Test method:

[0182] 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 × 108 -4.0×10 8 Between 100 and 100 mL.

[0183] 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 appropriate amount of DMSO to fully dissolve them, and then dilute them with physiological saline to the required concentration (the DMSO content is controlled within 1 / 1000) to prepare the test solution.

[0184] 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 -4 mol / L) to induce platelet aggregation, record the graph changes within 5 min, and read the maximum aggregation rate of each group.

[0185] 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.

[0186] The platelet aggregation inhibition rate was calculated as follows: Inhibition rate (%) = (maximum aggregation rate of blank control group - maximum aggregation rate of drug-treated group) / maximum aggregation rate of blank control group × 100%

[0187] The experimental results are shown in Table 2.

[0188] Table 2 Determination results of AA-induced platelet aggregation inhibition rate in each test group (x±SD, %)

[0189] 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.

[0190] Experimental Example 3: Acute toxicity test

[0191] The acute toxicity comparison test of the compound 1 sample group prepared in Example 1 and the positive control drug selexipag group was conducted.

[0192] Test method:

[0193] Rats were randomly divided into 7 groups according to body weight, 5 rats in each group. The animals in groups 1-3 were given a single oral administration of 250 mg / kg, 500 mg / kg, and 1000 mg / kg of selexipag, respectively. The animals in 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 animals were observed for 7 days after administration.

[0194] The experimental results are shown in Table 3.

[0195] Table 3 Comparative results of acute toxicity test of compound 1 and selexipag

[0196] 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.

[0197] 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.

[0198] 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 diphenylpyrazine compound or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer thereof, and mixtures thereof, characterized in that, The structural formula of the diphenylpyrazine compound is shown as formula (I):

2. A pharmaceutical composition, characterized in that, Comprising a therapeutically effective dose of a diphenylpyrazine compound represented by formula (I) or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer thereof, and mixtures thereof.

3. The pharmaceutical composition according to claim 2, wherein, Also comprising a pharmaceutically acceptable carrier or excipient.

4. Use of the diphenylpyrazine compound represented by formula (I) or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer thereof, and mixtures thereof according to claim 1, and the pharmaceutical composition according to claim 2 or 3 in the preparation of a drug for inhibiting platelet aggregation.

5. The application according to claim 4, wherein The inhibition of platelet aggregation is the inhibition of AA-induced platelet aggregation or the inhibition of ADP-induced platelet aggregation.

6. Use of the diphenylpyrazine compound represented by formula (I) or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer thereof, and mixtures thereof according to claim 1, and the pharmaceutical composition according to claim 2 or 3 in the preparation of an anticoagulant drug.

7. Use of the diphenylpyrazine compound represented by formula (I) or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer thereof, and mixtures thereof according to claim 1, and the pharmaceutical composition according to claim 2 or 3 in the preparation of a drug for preventing or treating cardiovascular and cerebrovascular diseases, other vascular thrombi or embolism diseases related to platelet aggregation.

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

9. Use of the diphenylpyrazine compound represented by formula (I) or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer thereof, and mixtures thereof according to claim 1, and the pharmaceutical composition according to claim 2 or 3 in the preparation of a drug for protecting platelets during extracorporeal circulation, treating after arteriography or treating after vascular reconstruction.

10. A method for preparing the diphenylpyrazine compound represented by formula (I), characterized in that, Comprising the following steps: S1, Cyclization reaction of 4,4'-dimethylbenzoyl with 2-aminoacetamide hydrochloride to obtain compound 1B; S2, Chlorination reaction of compound 1B with phosphorus oxychloride to obtain compound 1C; S3, Substitution reaction of compound 1C with 4-(isopropylamino)butanol to obtain compound 1D; S4, Oxidation reaction of compound 1D with Dess-Martin reagent to obtain compound 1E; S5, Nucleophilic addition reaction of compound 1E with triethyl phosphonoacetate to obtain compound 1F; S6. The compound 1F undergoes a hydrolysis reaction under the condition of sodium hydroxide to obtain a diphenylpyrazine compound shown in formula (I); 11. The preparation method of the diphenylpyrazine compound shown in formula (I) according to claim 10, characterized in that, Specifically comprising the following steps: S1, Mix 4,4'-dimethylbenzoyl, 2-aminoacetamide hydrochloride and a first reaction solvent, heat up to reflux, add a strong base solution, keep the temperature for reaction, cool down to 20-25 °C, adjust the pH to neutral, perform solid-liquid separation, washing, drying to obtain compound 1B; S2, Mix the compound 1B and phosphorus oxychloride, heat up to 100-105 °C, keep the temperature for reaction, remove phosphorus oxychloride to obtain a reaction product; perform recrystallization on the reaction product, perform solid-liquid separation, drying to obtain compound 1C; S3, Mix the compound 1C and 4-(isopropylamino)butanol, heat up to 160-170 °C, keep the temperature for reaction and then cool down to obtain a first reaction solution; Extract the first reaction solution to separate the organic phase, wash, dry, and purify to obtain Compound 1D; S4. Mix Compound 1D with a second reaction solvent, cool down to 0 - 10 °C, add Dess-Martin reagent, and carry out a heat preservation reaction to obtain a second reaction solution; wash, dry, and purify the second reaction solution to obtain Compound 1E; S5. Mix triethyl phosphonoacetate with a third reaction solvent, cool down to 0 - 10 °C, add sodium hydride, stir for 0.5 - 2.0 h, add Compound 1E at 0 - 10 °C, heat up to 20 - 25 °C, carry out a heat preservation reaction to obtain a third reaction solution, quench the reaction, extract to separate the organic phase, wash, dry, and purify to obtain Compound 1F; S6. Mix Compound 1F, a fourth reaction solvent, and sodium hydroxide, heat up to reflux for reaction to obtain a fourth reaction solution, adjust the pH to weakly acidic, extract to separate the organic phase, dry, and purify to obtain the diphenylpyrazine compound shown in formula (I).

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 base solution is an aqueous sodium hydroxide solution; and / or In S1, the neutral means the pH is 6 - 7; and / or In S1, the equivalent ratio of 2-aminoacetamide hydrochloride to 4,4'-dimethylbenzil ≥ 1; and / or In S1, the mass-volume ratio of 4,4'-dimethylbenzil to the first reaction solvent is 3 g : (15 - 25) mL; and / or In S1, the equivalent ratio of 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 time for the heat preservation reaction is 4 - 5 h; and / or In S2, the equivalent ratio of phosphorus oxychloride to Compound 1B ≥ 1; and / or In S2, the time for the heat preservation reaction is 3 - 5 h; and / or In S3, the equivalent ratio of 4-(isopropylamino)butanol to Compound 1C ≥ 1; and / or In S3, the time for the heat preservation reaction is 20 - 30 h; and / or In S3, the temperature for cooling is 60 °C - 80 °C; and / or In S4, the second reaction solvent is dichloromethane; and / or In S4, the equivalent ratio of Dess-Martin reagent to Compound 1D ≥ 1; and / or In S4, the mass-volume ratio of Compound 1D to the second reaction solvent is 1 g : (8 - 12) mL; and / or In S4, the time for the heat preservation reaction is 3 - 5 h; and / or In S5, the third reaction solvent is tetrahydrofuran; and / or In S5, the equivalent ratio of triethyl phosphonoacetate to Compound 1E ≥ 1; and / or In S5, the mass-volume ratio of Compound 1E to the third reaction solvent is 13 g : (80 - 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 time for the heat preservation reaction is 2 - 3 h; 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 - 5 : 1; and / or In S6, the mass-volume ratio of the compound 1F to the fourth reaction solvent is 1 g : (8.5 - 13.5) mL; and / or In S6, the time for heating to reflux reaction is 12 - 15 h.

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