A cis 4-(5,6-diphenylpyrazin-2-ylamino)cyclohexyloxyacetic acid compound, its preparation method and application

Abstract

The present application relates to a kind of cis 4-(5,6-diphenylpyrazin-2-aminyl) cyclohexyloxy acetic acid compound and its synthesis method and application, belong to the technical field of drug synthesis.The cis 4-(5,6-diphenylpyrazin-2-aminyl) cyclohexyloxy acetic acid compound has simple molecular structure, simple synthesis path and low production cost.Through in vitro platelet aggregation inhibition activity test, it is found that the synthesized compound shows excellent anti-platelet aggregation effect.In addition, in rat pharmacokinetic study, it is found that the compound shows excellent pharmacokinetic characteristics, has high bioavailability and longer half-life, can effectively improve its activity as antithrombotic drug, and shows excellent prevention and treatment effect in antithrombotic, vasodilator and anti-pulmonary arterial hypertension etc..

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical synthesis technology, specifically relating to a cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound, its preparation method, and its application. Background Technology

[0002] Prostaglandin I2 (PGI2) is a member of the eicosanoid family of lipids and an antagonist of thromboxanes; reduced synthesis of PGI2 promotes thrombus formation. Activation of the PGI2 receptor (IP receptor) not only inhibits platelet-mediated aggregation but also has a strong vasodilatory effect. IP receptor agonists can treat diseases including pulmonary hypertension (PAH), arteriosclerosis obliterans, coronary artery disease, myocardial infarction, transient ischemic attack, angina pectoris, stroke, ischemia-reperfusion injury, restenosis, atrial fibrillation, intermittent claudication, Raynaud's phenomenon, varicose veins, thrombosis, diabetes, diabetic nephropathy, hypertension, hyperlipidemia, cerebral infarction, rheumatoid arthritis, and chronic obstructive pulmonary disease (COPD).

[0003] Currently marketed PGI2-type drugs generally suffer from problems such as short half-life, poor chemical stability, and significant side effects, resulting in poor efficacy. Therefore, there is an urgent need to develop a non-endogenous PGI2-type IP receptor agonist with a long half-life, good chemical stability, higher selectivity, and fewer side effects. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound, its preparation method and application, so as to obtain an antithrombotic drug with good chemical stability, higher selectivity, fewer side effects and long half-life.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is to provide a cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound, the structural formula of which is shown in Formula I:

[0006]

[0007] Wherein, R is an alkyl, deuterated alkyl, or fluoroalkyl group.

[0008] Based on the above technical solution, the present invention can be further improved as follows.

[0009] Furthermore, R can be methyl, deuterated methyl, fluoromethyl, fluoroethyl, fluoropropyl, ethyl, propyl, or isobutyl.

[0010] Furthermore, the 4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound is one of the compounds having the following structural formula:

[0011]

[0012] This invention also discloses a method for preparing the above-mentioned cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound, comprising the following steps:

[0013] S1: 5-Chloro-2,3-diphenylpyrazine, the compound shown in Formula II, and a base are co-dissolved in an organic solvent and reacted at 100–200 °C for 8–48 h to obtain the compound shown in Formula III.

[0014]

[0015] S2: The compound shown in Formula III, tert-butyl bromoacetate, and the catalyst were co-dissolved in a mixed solution under ice bath conditions. The mixture was kept warm and stirred for 30–45 min, then naturally heated to room temperature and the reaction was continued for 0.5–3 h to obtain the compound shown in Formula IV. The mixed solution was prepared by mixing equal volumes of toluene and a 40 wt% KOH solution.

[0016]

[0017] S3: Dissolve the compound shown in Formula IV in a solvent under ice bath conditions, add NaH, keep warm and stir for 30-45 min, then add the iodinated compound RI, naturally warm to room temperature, and continue the reaction for 8-24 h to obtain the compound shown in Formula V; the solvent is tetrahydrofuran, N,N-dimethylformamide or diethyl ether.

[0018]

[0019] Wherein, R is an alkyl, deuterated alkyl, or fluoroalkyl group;

[0020] S4: Dissolve the compound shown in Formula V with LiOH or NaOH in an organic solvent under ice bath conditions, keep the reaction at the temperature for 2-10 hours, then acidify and extract to obtain the product.

[0021] The above-mentioned cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound can also be prepared by the following steps:

[0022] S1: 5-Chloro-2,3-diphenylpyrazine, the compound shown in Formula VI, and a base are co-dissolved in an organic solvent and reacted at 100–180 °C for 12–48 h to obtain the compound shown in Formula VII;

[0023]

[0024] Wherein, R is an alkyl, deuterated alkyl, or fluoroalkyl group;

[0025] S2: The compound shown in Formula VII, tert-butyl bromoacetate, and the catalyst were co-dissolved in a mixed solution under ice bath conditions. The mixture was kept warm and stirred for 30–45 min, then naturally heated to room temperature and the reaction was continued for 0.5–3 h to obtain the compound shown in Formula V. The mixed solution was prepared by mixing equal volumes of toluene and a 40 wt% KOH solution.

[0026]

[0027] S3: Dissolve the compound shown in Formula V with LiOH or NaOH in an organic solvent under ice bath conditions, keep the reaction at the temperature for 2-12 hours, then acidify and extract to obtain the product.

[0028] Furthermore, 5-chloro-2,3-diphenylpyrazine is prepared via the following steps:

[0029] (1) Under an inert atmosphere, biphenyl amide, aminoacetamide and base are dissolved in an organic solvent in a molar ratio of 1:1 to 1.5:2 to 2.5 and heated under reflux for 3 to 5 hours to obtain an intermediate as shown in Formula VIII;

[0030]

[0031] (2) Under an inert atmosphere, the intermediate shown in Formula VIII is mixed with phosphorus oxychloride at a ratio of 1g:0.5-5mL, and heated under reflux until the raw materials react completely to obtain the product.

[0032] Furthermore, the base is potassium carbonate; the catalyst is tetrabutylammonium hydrogen sulfate.

[0033] Furthermore, in the compound shown in Formula VI, R is a methyl group, which is prepared by the following steps:

[0034] Under ice bath conditions, tert-butyl cis-(4-hydroxycyclohexyl)aminocarbamate and LiAlH4 were added to an organic solvent, and then the reaction system was heated to 55-65°C and refluxed for 5-8 hours to obtain the product.

[0035] Alternatively, in the compound shown in Formula VI, R is an alkyl group with 2 or more carbon atoms or a substituted alkyl group with 2 or more carbon atoms, which is prepared by the following steps:

[0036] Under ice bath conditions, cis-4-aminocyclohexanol hydrochloride, aldehyde, and reducing agent are co-dissolved in an organic solvent and reacted overnight to obtain the product; the reducing agent is NaBCNH3, NaB(OAc)3H, or NaBH4; the aldehyde is an aldehyde with 2 or more carbon atoms or a substituted aldehyde with 2 or more carbon atoms.

[0037] The present invention also discloses the use of the above-mentioned cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound in the preparation of IP receptor agonists with high bioavailability and long half-life.

[0038] Furthermore, IP receptor agonists serve as active ingredients in thrombodily vasodilators or anti-pulmonary hypertension drugs.

[0039] The beneficial effects of this invention are as follows: the cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound of this invention has a simple structure, a short synthetic route, and low synthetic cost. Furthermore, in vitro antiplatelet aggregation activity assays revealed that the prepared cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid exhibits excellent antiplatelet aggregation activity, enabling it to exert superior thrombotic prevention and treatment effects as an active ingredient in antithrombotic, vasodilatory, and anti-pulmonary hypertension drugs. Rat pharmacokinetic experiments showed that the cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound possesses excellent pharmacokinetic parameters, high bioavailability, and a long half-life. Attached Figure Description

[0040] Figure 1 The drug concentration-time curve in plasma of SD rats after a single intravenous administration of the test drug. Detailed Implementation

[0041] The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Any equivalent substitutions made in the art based on the content disclosed in the present invention shall fall within the protection scope of the present invention.

[0042] The structure of the compound was determined by nuclear magnetic resonance (NMR). 1 It can be determined by ¹H NMR or liquid chromatography-mass spectrometry (LC-MS).

[0043] LC-MS; Nuclear Magnetic Resonance Spectrometer 1 HNMR) was performed using a Bruker AVANCE-600, nuclear magnetic resonance (NMR) 1 ¹H NMR shifts (δ) are given in parts per million (ppm), the solvents used are CDCl₃ and d⁶-DMSO, the internal standard is tetramethylsilane (TMS), and the chemical shifts are expressed in 10⁻¹⁰ ppm. -6 (ppm) is given as the unit.

[0044] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0045] Example 1

[0046] A cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound, the structural formula of which is as follows:

[0047]

[0048] The compound is cis-2-((4-(5,6-diphenylpyrazin-2-yl)methylaminocyclohexyl)oxy)acetic acid, which is prepared by the following method.

[0049] Method 1:

[0050] Step 1: Preparation of 5,6-diphenyl-2-hydroxypyrazine, the reaction equation is as follows:

[0051]

[0052] Under nitrogen protection, bibenzoyl (21.0 g, 0.10 mol), aminoacetamide (8.9 g, 0.12 mol), and NaOH (9.6 g, 0.24 mol) were added sequentially to 1 L of methanol solvent. The mixture was heated under reflux for 3 h, and the reaction was monitored by TLC. After the reactants had reacted completely, the reaction solution was cooled to 0 °C, and then 12.5 mL of 12N HCl solution was added dropwise. The reaction solution was stirred at room temperature for 30 min, and then 10 g of sodium bicarbonate and 130 mL of water were added. The reaction solution was filtered, and the solid was washed successively with water and methanol, and then dried under vacuum to obtain 22.0 g of white solid, namely 5,6-diphenyl-2-hydroxypyrazine, with a yield of 88.7%.

[0053] Step 2: Preparation of 5-chloro-2,3-diphenylpyrazine, the reaction equation is as follows:

[0054]

[0055] Under nitrogen protection, 19.8 g (0.08 mol) of 5,6-diphenyl-2-hydroxypyrazine was added to 20 mL of phosphorus oxychloride solvent. A tail gas absorption device was added, and the mixture was heated to reflux. The reaction was monitored by TLC until the reactants were completely reacted. The reaction mixture was cooled to room temperature and rotary evaporated under reduced pressure. It was then extracted with ethyl acetate and washed successively with cooled sodium bicarbonate solution, water, and saturated brine. After drying with sodium sulfate, the mixture was filtered and rotary evaporated to obtain a gray solid (crude 5-chloro-2,3-diphenylpyrazine). The solid was then slurried with ethyl acetate, filtered, and dried under vacuum to obtain 18.6 g of white solid, namely 5-chloro-2,3-diphenylpyrazine, with a yield of 87.4%.

[0056] Step 3: Preparation of cis-4-((5,6-diphenylpyrazin-2-yl)amino)cyclohexane-1-ol, the reaction equation is as follows:

[0057]

[0058] Under nitrogen protection, 5-chloro-2,3-diphenylpyrazine (533 mg, 2.00 mmol), potassium carbonate (276 mg, 2.00 mmol), and cis-4-aminocyclohexanol hydrochloride (303 mg, 2.00 mmol) were added to 10 mL of N-methylpyrrolidone (NMP). The mixture was heated to 120 °C and reacted for 48 h. The reaction was monitored by LC-MS until the reactants were completely reacted. The reaction mixture was cooled and ice water was added. The mixture was extracted with ethyl acetate, and the organic mixed phase was washed successively with water and saturated brine. The mixture was dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The mixture was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to give 561 mg of a yellow solid, namely cis-4-((5,6-diphenylpyrazin-2-yl)amino)cyclohexyl-1-ol, yield: 81.3%, ESI-MS: m / z = 346.2 (M+H). + .

[0059] Step 4: Preparation of cis-2-((4-(5,6-diphenylpyrazin-2-yl)aminocyclohexyl)oxy)tert-butyl acetate, the reaction equation is as follows:

[0060]

[0061] Under ice bath conditions, cis-4-((5,6-diphenylpyrazin-2-yl)aminocyclohexyl)-1-ol (519 mg, 1.50 mmol), tetrabutylammonium bisulfate (510 mg, 1.50 mmol), and tert-butyl bromoacetate (438 mg, 2.25 mmol) were added sequentially to a mixture of 2.5 mL toluene and 2.5 mL 40% KOH. The mixture was stirred vigorously for 30 min, allowed to warm naturally to room temperature, and reacted for 2 h. Ice water was then added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic mixture was washed successively with water and saturated brine, dried over MgSO4, filtered, and the solvent removed under reduced pressure. The mixture was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to obtain 471 mg of a yellow oily substance, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)aminocyclohexyl)oxy)tert-butyl acetate, yield: 68.3%, ESI-MS: m / z = 460.3 (M+H). + .

[0062] Step 5: Preparation of cis-2-((4-(5,6-diphenylpyrazin-2-yl)methylaminocyclohexyl)oxy)acetic acid, the reaction equation is as follows:

[0063]

[0064] Under ice bath conditions, cis-2-((4-((5,6-diphenylpyrazin-2-yl)amino)cyclohexyl)oxy)tert-butyl acetate (230 mg, 0.5 mmol) and NaH (200 mg, 5 mmol) were added sequentially to 2 mL of DMF solution. The mixture was stirred vigorously for 45 min, then iodomethane (213 mg, 1.5 mmol) was added and the mixture was allowed to warm naturally to room temperature. The reaction was continued with stirring for 8 h. The reaction was quenched by adding saturated NH4Cl under ice bath conditions. Then, ice water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic mixed phase was washed with water and saturated brine, dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The mixture was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to obtain a yellow oily substance, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)methylaminocyclohexyl)oxy)tert-butyl acetate.

[0065] Under ice bath conditions, 2 mL of cis-2-((4-(5,6-diphenylpyrazin-2-yl)methylaminocyclohexyl)oxy)acetic acid tert-butyl ester (237 mg, 0.50 mmol) and LiOH (48.0 mg, 2.00 mmol) were added sequentially to 2 mL of MeOH solution, and the reaction was allowed to proceed for 10 h. Then, methanol was removed under reduced pressure, and 5 mL of ice water and 5 mL of ethyl acetate were added to the reaction solution. The pH was adjusted to 6 with 1 N HCl, and 5 mL of ice water and 5 mL of ethyl acetate were added again. The mixture was extracted with ethyl acetate, and the organic mixed phase was washed sequentially with water and saturated brine. The mixture was dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The mixture was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to obtain 175 mg of a pale yellow oily liquid, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)methylaminocyclohexyl)oxy)acetic acid, with a yield of 83.9%. 1 H NMR(600MHz,DMSO-d6)δ8.08(s,1H),7.33-7.17(m,10H),4.31-4.28(m,1H),3.62(s,2H), 3.60-3.56(m,1H),2.96(s,3H),1.99-1.96(m,2H),1.87-1.83(m,2H),1.70-1.40(m,4H).

[0066] Method 2:

[0067] Step 1: Preparation of cis-4-methylamino-1-cyclohexanol, the reaction equation is as follows:

[0068]

[0069] Under nitrogen protection and an ice bath, tert-butyl cis-(4-hydroxycyclohexyl)aminocarbamate (2 g, 9.24 mmol) and LiAlH4 (7.4 mL, 18.58 mmol) were added sequentially to 46 mL of THF solution. The mixture was heated to 60 °C and refluxed for 6 h. The reaction was then quenched by cyclically adding NaOH solution and water. After drying with MgSO4, the mixture was filtered through a diatomaceous earth filter cake. The filter cake was washed with ethyl acetate, and the solvent was removed under reduced pressure to give 1.1 g of a white liquid product, cis-4-methylamino-1-cyclohexanol, in 92% yield. ESI-MS: m / z = 129.2 (M+H). + .

[0070] Step 2: Preparation of cis-4-((5,6-diphenylpyrazin-2-yl)methylamino)cyclohexyl-1-ol, the reaction equation is as follows:

[0071]

[0072] Under nitrogen protection, 5-chloro-2,3-diphenylpyrazine (532 mg, 2.00 mmol), potassium carbonate (414 mg, 3.00 mmol), and cis-4-methylamino-1-cyclohexanol (388 mg, 3.00 mmol) synthesized in Method 1 were added to 10 mL of N-methylpyrrolidone (NMP). The mixture was heated to 120 °C and reacted for 48 h. The reaction was monitored by TLC. After the reactants had reacted completely, the reaction mixture was cooled and ice water was added. The mixture was extracted with ethyl acetate, washed with water and saturated brine, dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The mixture was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to give 592 mg of a yellow solid, namely cis-4-((5,6-diphenylpyrazin-2-yl)methylamino)cyclohexyl-1-ol, yield: 82.3%, ESI-MS: m / z = 360.2 (M+H). + .

[0073] Step 3: Preparation of cis-2-((4-(5,6-diphenylpyrazin-2-yl)methylaminocyclohexyl)oxy)tert-butyl acetate, the reaction equation is as follows:

[0074]

[0075] Under ice bath conditions, add 2.5 mL of toluene and 2.5 mL of... To a mixture of 40% KOH, cis-4-((5,6-diphenylpyrazin-2-yl)methylamino)cyclohexyl-1-ol (539 mg, 1.50 mmol), tetrabutylammonium bisulfate (510 mg, 1.50 mmol), and tert-butyl bromoacetate (438 mg, 2.25 mmol) were added sequentially. The mixture was stirred vigorously for 30 min, allowed to warm naturally to room temperature, and then stirred for another 0.5 h. Ice water was then added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic mixture was washed with water and saturated brine, dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The mixture was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to obtain 478 mg of a yellow oily substance, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)methylaminocyclohexyl)oxy)tert-butyl acetate, yield: 67.4%, ESI-MS: m / z = 474.3 (M+H). + .

[0076] Step 4: Preparation of cis-2-((4-(5,6-diphenylpyrazin-2-yl)methylaminocyclohexyl)oxy)acetic acid, the reaction equation is as follows:

[0077]

[0078] Under ice bath conditions, 2 mL of cis-2-((4-(5,6-diphenylpyrazin-2-yl)methylaminocyclohexyl)oxy)acetic acid tert-butyl ester (237 mg, 0.50 mmol) and LiOH (48.0 mg, 2.00 mmol) were added sequentially to 2 mL of MeOH solution, and the reaction was allowed to proceed for 12 h. Methanol was then removed under reduced pressure. 5 mL of ice water and 5 mL of ethyl acetate were added to the reaction solution, and the pH was adjusted to 5 with 2N HCl. The mixture was extracted with ethyl acetate, washed with water and saturated brine, dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The mixture was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to obtain 181 mg of a pale yellow oily liquid, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)methylaminocyclohexyl)oxy)acetic acid, yield: 86.5%, ESI-MS: m / z = 418.2 (M+H). + ; 1 H NMR(600MHz,DMSO-d6)δ8.08(s,1H),7.33-7.17(m,10H),4.31-4.28(m,1H),3.62(s,2H), 3.60-3.56(m,1H),2.96(s,3H),1.99-1.96(m,2H),1.87-1.83(m,2H),1.70-1.40(m,4H).

[0079] Example 2

[0080] A cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound, the structural formula of which is as follows:

[0081]

[0082] The compound is cis-2-((4-(5,6-diphenylpyrazin-2-yl)ethylaminocyclohexyl)oxy)acetic acid, which is prepared by the following method.

[0083] Method 1:

[0084] Step 1: Preparation of 5,6-diphenyl-2-hydroxypyrazine, the reaction equation is as follows:

[0085]

[0086] Under nitrogen protection, bibenzoyl (21.0 g, 0.10 mol), aminoacetamide (8.9 g, 0.12 mol), and NaOH (9.6 g, 0.24 mol) were added sequentially to 1 L of methanol solvent. The mixture was heated under reflux for 5 h, and the reaction was monitored by TLC. After the reactants had reacted completely, the reaction solution was cooled to 0 °C, and then 12.5 mL of 12N HCl solution was added dropwise. The reaction solution was stirred at room temperature for 30 min, and then 10 g of sodium bicarbonate and 130 mL of water were added. The reaction solution was filtered, and the solid was washed successively with water and methanol. The solid was dried under vacuum to obtain 23.0 g of white solid, namely 5,6-diphenyl-2-hydroxypyrazine, with a yield of 92.7%.

[0087] Step 2: Preparation of 5-chloro-2,3-diphenylpyrazine, the reaction equation is as follows:

[0088]

[0089] Under nitrogen protection, 19.8 g (0.08 mol) of 5,6-diphenyl-2-hydroxypyrazine was added to 100 mL of phosphorus oxychloride solvent. A tail gas absorption device was added, and the mixture was heated to reflux. The reaction was monitored by TLC until the reactants were completely reacted. The reaction mixture was cooled to room temperature and rotary evaporated under reduced pressure. The mixture was then extracted with ethyl acetate and washed successively with cooled sodium bicarbonate solution, water, and saturated brine. After drying with sodium sulfate, the mixture was filtered and rotary evaporated to obtain crude 5-chloro-2,3-diphenylpyrazine as a gray solid. The crude solid was then slurried with ethyl acetate, filtered, and dried under vacuum to obtain 16.4 g of white solid, namely 5-chloro-2,3-diphenylpyrazine, with a yield of 77.1%.

[0090] Step 3: Preparation of cis-4-((5,6-diphenylpyrazin-2-yl)amino)cyclohexane-1-ol, the reaction equation is as follows:

[0091]

[0092] Under nitrogen protection, 5-chloro-2,3-diphenylpyrazine (533 mg, 2.00 mmol), potassium carbonate (828 mg, 6.00 mmol), and cis-4-aminocyclohexanol hydrochloride (910 mg, 6.00 mmol) were added to 10 mL of N-methylpyrrolidone (NMP). The mixture was heated to 120 °C and reacted for 48 h. The reaction was monitored by LC-MS until the reactants were completely reacted. The reaction mixture was cooled and ice water was added. The mixture was extracted with ethyl acetate, and the organic mixed phase was washed with water and saturated brine. The mixture was dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The mixture was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to give 525 mg of a yellow solid, namely cis-4-((5,6-diphenylpyrazin-2-yl)amino)cyclohexyl-1-ol, yield: 76.1%, ESI-MS: m / z = 346.2 (M+H). + .

[0093] Step 4: Preparation of cis-2-((4-(5,6-diphenylpyrazin-2-yl)aminocyclohexyl)oxy)tert-butyl acetate, the reaction equation is as follows:

[0094]

[0095] Under ice bath conditions, cis-4-((5,6-diphenylpyrazin-2-yl)aminocyclohexyl)-1-ol (519 mg, 1.50 mmol), tetrabutylammonium bisulfate (255 mg, 0.75 mmol), and tert-butyl bromoacetate (292 mg, 1.50 mmol) were added sequentially to a mixture of 2.5 mL toluene and 2.5 mL 40% KOH. The mixture was stirred vigorously for 45 min, allowed to warm naturally to room temperature, and reacted for 3 h. Ice water was then added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic mixture was washed with water and saturated brine, dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The mixture was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to obtain 586 mg of a yellow oily substance, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)aminocyclohexyl)oxy)tert-butyl acetate, yield: 63.8%, ESI-MS: m / z = 460.3 (M+H). + .

[0096] Step 5: Preparation of cis-2-((4-(5,6-diphenylpyrazin-2-yl)ethylaminocyclohexyl)oxy)acetic acid, the reaction equation is as follows:

[0097]

[0098] Under ice bath conditions, cis-2-((4-((5,6-diphenylpyrazin-2-yl)amino)cyclohexyl)oxy)tert-butyl acetate (230 mg, 0.5 mmol) and NaH (200 mg, 5 mmol) were added sequentially to a 2 mL LMF solution. The mixture was stirred vigorously for 30 min, followed by the addition of iodoethane (234 mg, 1.5 mmol) and natural warming to room temperature. The reaction was then stirred for another 8 h. The reaction was quenched by adding saturated NH4Cl under ice bath conditions. Ice water was then added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic mixed phase was washed with water and saturated brine, dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The mixture was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to obtain a yellow oily substance, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)ethylaminocyclohexyl)oxy)tert-butyl acetate.

[0099] Under ice bath conditions, 243 mg (0.50 mmol) of cis-2-((4-(5,6-diphenylpyrazin-2-yl)ethylaminocyclohexyl)oxy)acetic acid tert-butyl ester and 80.0 mg (2.00 mmol) of NaOH were added sequentially to 2 mL of MeOH solution, and the reaction was allowed to proceed for 2 h. Then, methanol was removed under reduced pressure. 5 mL of ice water and 5 mL of ethyl acetate were added to the reaction solution, and the pH was adjusted to 6 with 1 N HCl. The solution was extracted with ethyl acetate, and the organic mixed phase was washed with water and saturated brine. The mixture was dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The solution was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to obtain 165 mg of a pale yellow powder, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)ethylaminocyclohexyl)oxy)acetic acid, yield: 76.6%. 1 H NMR (600MHz, CDCl3) δ7.90 (s, 1H), 7.41-7.38 (m, 2H), 7.32-7.30 (m, 2H), 7.25-7.17 (m, 6H), 4.22 (q, J = 7.1Hz, 2H), 4.10 (s, 2H), 3.91 -3.88(m,1H),3.62-3.59(m,1H),1.95-1.92(m,2H),1.90-1.84(m,2H),1.80-1.74(m,2H),1.71-1.68(m,2H),1.29(t,J=7.1Hz,3H).

[0100] Method 2:

[0101] Step 1: Preparation of cis-4-ethylamino-1-cyclohexanol, the reaction equation is as follows:

[0102]

[0103] Under ice bath conditions, cis-4-aminocyclohexanol hydrochloride (249 mg, 1.65 mmol), acetaldehyde (0.13 mL, 2.31 mmol), and NaBH3CN (176 mg, 2.8 mmol) were added sequentially to 8 mL of MeOH solution. The reaction was allowed to proceed overnight, methanol was removed under reduced pressure, 5 mL of tetrahydrofuran was added, the mixture was dried over MgSO4, filtered, solvent was removed under reduced pressure, and dried under vacuum to obtain 180 mg of crude cis-2-((4-(5,6-diphenylpyrazin-2-yl)methylaminocyclohexyl)oxy)acetic acid, which could be used directly in the next step without further purification.

[0104] Step 2: Preparation of cis-4-((5,6-diphenylpyrazin-2-yl)ethylamino)cyclohexyl-1-ol, the reaction equation is as follows:

[0105]

[0106] Under nitrogen protection, 5-chloro-2,3-diphenylpyrazine (532 mg, 2.00 mmol), potassium carbonate (414 mg, 3.00 mmol), and cis-4-ethylamino-1-cyclohexanol (429 mg, 3.00 mmol) synthesized in Method 1 were added to 10 mL of N-methylpyrrolidone (NMP). The mixture was heated to 180 °C and reacted for 12 h. The reaction was monitored by TLC. After the reactants had reacted completely, the reaction mixture was cooled and ice water was added. The mixture was extracted with ethyl acetate, washed with water and saturated brine, dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The mixture was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to give 550 mg of a yellow solid, namely cis-4-((5,6-diphenylpyrazin-2-yl)ethylamino)cyclohexyl-1-ol, yield: 73.7%, ESI-MS: m / z = 374.2 (M+H). + .

[0107] Step 3: Preparation of cis-2-((4-(5,6-diphenylpyrazin-2-yl)ethylaminocyclohexyl)oxy)tert-butyl acetate, the reaction equation is as follows:

[0108]

[0109] Under ice bath conditions, add 2.5 mL of toluene and 2.5 mL of... To a mixture of 40% KOH, cis-4-((5,6-diphenylpyrazin-2-yl)ethylamino)cyclohexyl-1-ol (559.5 mg, 1.50 mmol), tetrabutylammonium bisulfate (1.02 g, 3.00 mmol), and tert-butyl bromoacetate (876 mg, 4.50 mmol) were added sequentially. The mixture was stirred vigorously for 30 min, allowed to warm naturally to room temperature, and then stirred for another 3 h. Ice water was then added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic mixture was washed with water and saturated brine, dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The mixture was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to obtain 488 mg of a yellow oily substance, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)ethylaminocyclohexyl)oxy)tert-butyl acetate, yield: 66.8%, ESI-MS: m / z = 488.2 (M+H). + .

[0110] Step 4: Preparation of cis-2-((4-(5,6-diphenylpyrazin-2-yl)ethylaminocyclohexyl)oxy)acetic acid, the reaction equation is as follows:

[0111]

[0112] Under ice bath conditions, 243 mg (0.50 mmol) of cis-2-((4-(5,6-diphenylpyrazin-2-yl)ethylaminocyclohexyl)oxy)acetic acid tert-butyl ester and 80.0 mg (2.00 mmol) of NaOH were added sequentially to 2 mL of MeOH solution, and the reaction was allowed to proceed for 2 h. Methanol was then removed under reduced pressure. 5 mL of ice water and 5 mL of ethyl acetate were added to the reaction solution, and the pH was adjusted to 5 with 2N HCl. The mixture was extracted with ethyl acetate, washed with water and saturated brine, dried over MgSO4, filtered, and the solvent was removed under reduced pressure. The mixture was then purified by silica gel column chromatography, collected under reduced pressure, and dried under vacuum to obtain 191 mg of a pale yellow powder product, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)ethylaminocyclohexyl)oxy)acetic acid, yield: 88.6%. 1 H NMR (600MHz, CDCl3) δ7.90 (s, 1H), 7.41-7.38 (m, 2H), 7.32-7.30 (m, 2H), 7.25-7.17 (m, 6H), 4.22 (q, J = 7.1Hz, 2H), 4.10 (s, 2H), 3.91 -3.88(m,1H),3.62-3.59(m,1H),1.95-1.92(m,2H),1.90-1.84(m,2H),1.80-1.74(m,2H),1.71-1.68(m,2H),1.29(t,J=7.1Hz,3H).

[0113] Example 3

[0114] A cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound, the structural formula of which is as follows:

[0115]

[0116] The compound is cis-2-((4-(5,6-diphenylpyrazin-2-yl)propylaminocyclohexyl)oxy)acetic acid, which is prepared by the following method.

[0117] Method 1:

[0118] The synthesis steps are the same as steps 1 to 5 in Method 1 of Example 1, except that iodomethane in step 5 is replaced with iodopropane, ultimately yielding a pale yellow oily substance, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)propylaminocyclohexyl)oxy)acetic acid. 1 HNMR(600MHz, CDCl3)δ8.01(s,1H),7.45-7.43(m,2H),7.37-7.34(m,2H),7 .30-7.27(m,2H),7.26-7.22(m,4H),4.50-4.44(m,1H),4.16(s,2H),3.45-3 .40(m,1H),3.32(t,J=7.7Hz,2H),2.23-2.18(m,2H),1.94-1.91(m,2H),1.7 0-1.66(m,2H),1.65-1.60(m,2H),1.57-1.52(m,2H),0.98(t,J=7.4Hz,3H).

[0119] Method 2:

[0120] The synthesis steps are the same as steps 1 to 4 in Method 2 of Example 2, except that acetaldehyde in step 1 is replaced with propionaldehyde, ultimately yielding a pale yellow oily substance, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)propylaminocyclohexyl)oxy)acetic acid. 1HNMR(600MHz, CDCl3)δ8.01(s,1H),7.45-7.43(m,2H),7.37-7.34(m,2H),7 .30-7.27(m,2H),7.26-7.22(m,4H),4.50-4.44(m,1H),4.16(s,2H),3.45-3 .40(m,1H),3.32(t,J=7.7Hz,2H),2.23-2.18(m,2H),1.94-1.91(m,2H),1.7 0-1.66(m,2H),1.65-1.60(m,2H),1.57-1.52(m,2H),0.98(t,J=7.4Hz,3H).

[0121] Example 4

[0122] A cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound, the structural formula of which is as follows:

[0123]

[0124] The compound is cis-2-((-4-(5,6-diphenylpyrazin-2-yl)isobutylamino)cyclohexyl)oxy)acetic acid, which is prepared by the following method.

[0125] Method 1:

[0126] The synthesis steps are the same as steps 1 to 5 in Method 1 of Example 1, except that iodomethane in step 5 is replaced with isoisobutane, ultimately yielding a brownish-yellow oily substance, namely cis-2-((-4-(5,6-diphenylpyrazin-2-yl)isobutylaminocyclohexyl)oxy)acetic acid. 1 H NMR (600MHz, CDCl3) δ7.92(s,1H),7.43-7.40(m,2H),7.35-7.31(m,2H),7.29-7.26(m,3H),7.25-7.21(m,3H),4.13(s,2H),3.95(d,J=6.7Hz, 2H),3.92-3.87(m,1H),3.63-3.60(m,1H),1.99-1.92(m,4H),1.91-1. 85(m,2H),1.81-1.75(m,3H),1.73-1.66(m,2H),0.95(d,J=6.8Hz,6H).

[0127] Method 2:

[0128] The synthesis steps are the same as steps 1 to 4 in Method 2 of Example 2, except that acetaldehyde in step 1 is replaced with isobutyraldehyde, ultimately yielding a brownish-yellow oily compound, cis-2-((-4-(5,6-diphenylpyrazin-2-yl)isobutylaminocyclohexyl)oxy)acetic acid. 1 HNMR(600MHz, CDCl3)δ7.92(s,1H),7.43-7.40(m,2H),7.35-7.31(m,2H),7.29-7.26(m,3H),7.25-7.21(m,3H),4.13(s,2H),3.95(d,J=6.7Hz ,2H),3.92-3.87(m,1H),3.63-3.60(m,1H),1.99-1.92(m,4H),1.91-1. 85(m,2H),1.81-1.75(m,3H),1.73-1.66(m,2H),0.95(d,J=6.8Hz,6H).

[0129] Example 5

[0130] A cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound, the structural formula of which is as follows:

[0131]

[0132] The compound is cis-2-((-4-(5,6-diphenylpyrazin-2-yl)2-fluoroethylamino)cyclohexyl)oxy)acetic acid, which is prepared by the following method.

[0133] The synthesis steps are the same as steps 1 to 5 in Method 1 of Example 1, except that iodomethane in step 5 is replaced with iodo-2-fluoroethane, ultimately yielding a pale yellow powder, namely cis-2-((-4-(5,6-diphenylpyrazin-2-yl)2-fluoroethylamino)cyclohexyl)oxy)acetic acid. 1 H NMR(600MHz, CDCl3)δ7.91(s,1H),7.42-7.39(m,2H),7.35-7.31(m,2H),7.30-7.19(m,6H),4.68-4.65(m,1H),4.60-4.57(m,1H),4.44-4.42(m, 1H),4.40-4.37(m,1H),4.18(s,2H),3.90(s,1H),3.64-3.59(m,1H),1.9 7-1.90(m,2H),1.89-1.84(m,2H),1.80-1.74(m,2H),1.71-1.66(m,2H).

[0134] Example 6

[0135] A cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound, the structural formula of which is as follows:

[0136]

[0137] The compound is cis-2-((4-(5,6-diphenylpyrazin-2-yl)(2,2-difluoroethyl)aminocyclohexyl)oxy)acetic acid, which is prepared by the following method.

[0138] The synthesis steps are the same as steps 1 to 5 in Method 1 of Example 1, except that iodomethane in step 5 is replaced with iodo-2,2-difluoroethane, ultimately yielding a brownish-yellow oily substance, cis-2-((4-(5,6-diphenylpyrazin-2-yl)(2,2-difluoroethyl)aminocyclohexyl)oxy)acetic acid. 1 H NMR (600MHz, CDCl3) δ7.91(s,1H),7.43-7.40(m,2H),7.34-7.32(m,2H),7.29-7.27(m,2H),7.26-7.19(m,4H),6.07-5.87(m,1H),4.38-4. 33(m,2H),4.20(s,2H),3.94-3.88(m,1H),3.63-3.60(m,1H),1.96-1 .90(m,2H),1.88-1.85(m,2H),1.81-1.73(m,2H),1.69-1.66(m,2H).

[0139] Example 7

[0140] A cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound, the structural formula of which is as follows:

[0141]

[0142] The compound is cis-2-((4-(5,6-diphenylpyrazin-2-yl)(3,3,3-trifluoropropyl)aminocyclohexyl)oxy)acetic acid, which is prepared by the following method.

[0143] Method 1:

[0144] The synthesis steps are the same as steps 1 to 5 in Method 1 of Example 1, except that iodomethane in step 5 is replaced with iodo-3,3,3-trifluoropropane, ultimately yielding a white oily substance, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)(3,3,3-trifluoropropyl)aminocyclohexyl)oxy)acetic acid. 1H NMR (600MHz, CDCl3) δ7.93(s,1H),7.44-7.39(m,2H),7.34-7.33(m,2H),7.30-7.26(m,3H),7.25-7.22(m,3H),4.39(t,J=6.3Hz,2H),4.14( s,2H),3.92-3.87(m,1H),3.62-3.60(m,1H),2.55-2.47(m,2H),1.96- 1.90(m,2H),1.90-1.85(m,2H),1.82-1.74(m,2H),1.71-1.67(m,2H).

[0145] Method 2:

[0146] The synthesis steps are the same as steps 1 to 4 in Method 2 of Example 2, except that acetaldehyde in step 1 is replaced with 3,3,3-trifluoropropaldehyde, ultimately yielding a white oily substance, namely cis-2-((4-(5,6-diphenylpyrazin-2-yl)(3,3,3-trifluoropropyl)aminocyclohexyl)oxy)acetic acid. 1 H NMR (600MHz, CDCl3) δ7.93(s,1H),7.44-7.39(m,2H),7.34-7.33(m,2H),7.30-7.26(m,3H),7.25-7.22(m,3H),4.39(t,J=6.3Hz,2H),4.14( s,2H),3.92-3.87(m,1H),3.62-3.60(m,1H),2.55-2.47(m,2H),1.96- 1.90(m,2H),1.90-1.85(m,2H),1.82-1.74(m,2H),1.71-1.67(m,2H).

[0147] Example 8

[0148] A cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound, the structural formula of which is as follows:

[0149]

[0150] The compound is cis-2-((4((5,6-diphenylpyrazin-2-yl)deuterated methylaminocyclohexyl)oxy)acetic acid, which is prepared by the following method.

[0151] The synthesis steps are the same as steps 1 to 5 in Method 1 of Example 1, except that iodomethane in step 5 is replaced with deuterated iodomethane, ultimately yielding a pale yellow powder of cis-2-((4((5,6-diphenylpyrazin-2-yl)deuterated methylaminocyclohexyl)oxy)acetic acid. 1H NMR(600MHz,DMSO-d6)δ8.21(s,1H),7.37-7.35(m,2H),7.31-7.26(m,5H),7.26-7.21(m,3H),4.39-4.35(m,1H) ),4.01(s,2H),3.63-3.62(m,1H),2.03-1.98(m,2H),1.90-1.85(m,2H),1.56-1.51(m,2H),1.50-1.46(m,2H).

[0152] Comparative Example 1

[0153] 2-(4-(N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino)butoxy)acetic acid (MRE-269) has the following structural formula:

[0154]

[0155] The preparation was carried out according to the method disclosed in Example 42 of patent CN1516690A, yielding a yellow oily 2-(4-(N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino)butoxy)acetic acid (MRE-269), ESI-MS: m / z = 420.2 (M+H). + ; 1 H NMR(400MHz,d6-DMSO)δ12.59(s,1H),8.14(s,1H),7.38-7.21(m,10H),4.82-4.74 (m,1H),4.00(s,2H),3.53-3.33(m,4H),1.69-1.61(m,4H),1.22(d,J=6.8Hz,6H).

[0156] Comparative Example 2

[0157] trans-2-((4-(5,6-diphenylpyrazin-2-yl)methylaminocyclohexyl)oxy)acetic acid, its structural formula is as follows:

[0158]

[0159] The synthesis steps are the same as steps 1 to 4 in Method 2 of Example 1, except that cis-(4-hydroxycyclohexyl)aminocarbamate tert-butyl ester in step 1 is replaced with trans-(4-hydroxycyclohexyl)aminocarbamate tert-butyl ester, ultimately yielding a pale yellow powder of trans-2-((4-(5,6-diphenylpyrazin-2-yl)methylaminocyclohexyl)oxy)acetic acid, ESI-MS: m / z = 418.2 (M+H). + .

[0160] Experimental Example 1: In vitro platelet aggregation test

[0161] 1. Experimental Objective

[0162] The antiplatelet aggregation activity of the compounds in the embodiments and comparative examples of this invention was evaluated by studying their inhibitory effect on ADP-induced platelet aggregation in vitro.

[0163] 2. Test materials

[0164] 2.1 Test Materials

[0165] Fully automated platelet aggregation analyzer (Talitaikang AG800); ADP (Sigma); DMSO, 0.9% sodium chloride injection, sodium hydroxide, blood collection tubes, etc.

[0166] 2.2 Laboratory Animals

[0167] Domestic rabbit, male, 2.1±0.2kg.

[0168] 2.3 Test Drug

[0169] The compounds in Examples 1-8 and Comparative Examples 1-2.

[0170] 3. Test methods

[0171] 3.1 Preparation of the test solution

[0172] Weigh each of the above test drugs and prepare a stock solution with a concentration of 200 mM using DMSO and an equimolar 1N NaOH aqueous solution. After fully dissolving and mixing, take the stock solution and add 0.9% sodium chloride injection to prepare a series of test solutions of different concentrations (0.15 μM-50 μM).

[0173] 3.2 Preparation of PRP and PPP

[0174] Rabbit blood was collected in plastic centrifuge tubes and anticoagulated with 3.2% sodium citrate (the ratio of anticoagulant to whole blood was 1:9). The blood was centrifuged at 140g for 10 minutes, and the supernatant was carefully aspirated to obtain PRP. The remaining plasma was centrifuged at 2000g for 10 minutes, and the supernatant was obtained to obtain PPP.

[0175] 3.3 Preparation of ADP solution

[0176] Weigh out ADP and dissolve it in 0.9% sodium chloride injection to prepare ADP stock solution. Aliquot the stock solution into centrifuge tubes and store at -20°C. Reconstitute before use and dilute to 300 μM with 0.9% sodium chloride injection.

[0177] 3.4 Maximum Aggregation Rate Detection

[0178] After powering on the platelet aggregation analyzer, preheat for 30 minutes until the temperature reaches 37°C before starting the test. Place 270 μL of PRP and 30 μL of the test solution into the PRP cup of the double-cup combination, and place 300 μL of PPP into the PPP cup of the double-cup combination. The instrument then begins the test. The maximum platelet aggregation rate induced by ADP at different test drug concentrations was statistically analyzed. The results are shown in Table 1.

[0179] Table 1. Maximum platelet aggregation rate of the compounds in the examples and comparative examples.

[0180]

[0181] As shown in Table 1, the compound of Comparative Example 1 exhibited a maximum platelet aggregation rate of 12.3% at a concentration of 12.5 μM, demonstrating antiplatelet aggregation activity. However, the compound of Comparative Example 2 showed a maximum platelet aggregation rate of 38.2% at the same concentration, exceeding 30%, indicating no antiplatelet aggregation activity. The compound of Comparative Example 2 was in the trans configuration; the corresponding cis isomer, i.e., the compound of Example 1, exhibited significant antiplatelet aggregation activity.

[0182] The cis-configured compounds of this invention (Examples 1-8) all exhibited greater inhibitory effects on platelet aggregation than the compound of Preparation Example 1. Among them, the compounds of Examples 1, 2, 5, and 8 showed very strong inhibitory effects on platelet aggregation. In particular, the compounds of Examples 1 and 8 still exhibited anti-platelet aggregation activity at a concentration of 1.25 μM, which was about 10 times higher than that of the compound of Comparative Example 1.

[0183] Experimental Example 2: In vivo rat pharmacokinetics experiment

[0184] 1. Experimental Objective

[0185] After administering the compounds of Comparative Example 1, Example 1 and Example 8 of the present invention to three groups of male SD rats via a single intravenous administration, the drug concentrations of the compounds of Comparative Example 1, Example 1 and Example 8 in plasma samples were measured, and the pharmacokinetic parameters of the drugs were calculated.

[0186] 2. Test materials

[0187] 2.1 Test Materials

[0188] DMSO, Solutol, sodium hydroxide, EDTA-K2, methanol, and acetonitrile, etc.

[0189] 2.2 Laboratory Animals

[0190] SD rats (Beijing Vital River Laboratory Animal Technology Co., Ltd., Laboratory Animal Production License No.: SCXK(Beijing) 2021-0011), male, weighing 220±10 g, 6-8 weeks old at the time of dosing.

[0191] 2.3 Tested drugs

[0192] The sodium salts of the compounds in Comparative Example 1, Example 1 and Example 8.

[0193] 3. Test methods

[0194] 3.1 Preparation of test sample solutions

[0195] Weigh 1.525 mg of the compound in Comparative Example 1, 1.588 mg of the compound in Example 1, and 1.557 mg of the compound in Example 8 in the form of sodium salts respectively, and place them in three 3-mL glass bottles. Add 0.361 mL of DMSO to each bottle and ultrasonically treat for 40 minutes to dissolve, obtaining three colorless solutions. Then add 0.722 mL of Solutol to each solution and ultrasonically treat for 20 minutes to form colorless solutions. Transfer these three solutions to three 10-mL centrifuge tubes, rinse the glass bottles with a small amount of water and transfer the washing liquid to the centrifuge tubes, and add water to make the total volume reach 6.132 mL, finally obtaining three colorless clear solutions with a concentration of 0.2 mg / mL for each tested drug.

[0196] 3.2 Animal experiments

[0197] For each rat experiment with a tested drug: Select 3 qualified adult healthy male rats for intravenous administration. Collect about 0.25 mL of whole blood from the orbital venous plexus of the rats at 0 h before dosing, 0.033, 0.167, 0.5, 1, 2, 4, 6, 8, and 24 hours after dosing, and place it in a centrifuge tube containing EDTA-K2 anticoagulant (previously refrigerated in a 2-8°C refrigerator and placed in crushed ice when in use). After collection, place it in crushed ice. Centrifuge at 2000 g for 10 min within 0.5 h, aliquot all the plasma and place it in another clean centrifuge tube (previously refrigerated in a 2-8°C refrigerator), and place it in a -20°C refrigerator for testing.

[0198] 3.3 Biological sample processing

[0199] Precisely pipette 50 μL of rat blank plasma or unknown sample into a 2.0-mL centrifuge tube, add 10 μL of the corresponding working solution (standard curve, quality control) or working solution diluent (single blank, double blank, and unknown sample) of each concentration, and then add 150 μL of acetonitrile (containing diphenhydramine, concentration 1 ng·mL -1Alternatively, use pure acetonitrile (double blank sample), vortex at speed 5 for 1 min on a multi-tube vortex mixer, centrifuge at 13000g, 2-8℃ for 10 min, take 10 μL of the supernatant into a 0.65 mL injection tube, add 200 μL of acetonitrile-water (1:1, v / v), mix well, and take 10 μL for LC-MS / MS analysis.

[0200] 3.4 Biological Sample Analysis

[0201] Liquid chromatography conditions: Agilent 1200 high-performance liquid chromatograph (G1312A binary pump and G1322A vacuum degasser)

[0202] Mobile phase: acetonitrile-formic acid (100:0.1, v / v); Washing conditions: acetonitrile-water-formic acid (10:90:0.1, v / v / v); Injection volume: 10 μL; Flow rate: 0.40 mL / min; Internal standard: diphenhydramine.

[0203] Column: Inertsustain Swift C18 (2.1 × 50 mm, 5.0 μm) with in-line filter.

[0204] Mass spectrometry conditions: AB API4000 tandem mass spectrometer

[0205] Polarity: Cation mode; Source-related parameters: GS1 (Gas 1): 50; GS2 (Gas 2): 50; TEM (Temperature): 550; CUR (Curtain Gas): 30; IS (Spray Voltage): 5500; CAD (Collision Gas): 6; Ihe (Interface Heater): on.

[0206] 4. Data acquisition, processing, and pharmacokinetic parameter calculation

[0207] Analyst 1.6.2 software was used for chromatographic peak acquisition, integration, calculation, and processing; the ratio of analyte peak area to internal standard peak area was plotted on the ordinate (y) and concentration on the abscissa (x), and the weighted least squares method (W = 1 / x) was used. 2 Regression calculations were performed, and the regression equation was: y = ax + b. When analyzing drug concentrations in samples, a standard curve was established for each analytical batch (Run) to calculate the concentration of the analyte in the batch's quality control and unknown samples. The ratio of the peak area of ​​the analyte in the sample to the peak area of ​​the internal standard was substituted into the corresponding standard curve to calculate the concentration of each analyte. The average of the three blood drug concentrations at the same time point for each test drug was taken to obtain the concentration-time curve of the test drug, as shown in the figure. Figure 1 As shown.

[0208] Pharmacokinetic parameters were calculated using a non-compartmental model based on WinNonlin 6.3, and the main pharmacokinetic parameters, such as AUC, C0, and t, were analyzed. 1 / 2 T max V z Statistical analysis was performed on CL et al., and the results are shown in Table 2.

[0209] Table 2. Pharmacokinetic parameters of the test drug in plasma of SD rats after a single intravenous administration (IV, 1 mg / kg, N=3)

[0210] Pharmacokinetic parameters Comparative Example 1 Example 1 Example 8 No_points_lambda_z 5.00 7.66 8.00 <![CDATA[t 1 / 2 (h)]]> 3.339 11.547 11.999 <![CDATA[T max (h)]]> 0.033 0.033 0.033 <![CDATA[C0(ng / mL)]]> 8117.8 6662.4 6196.5 <![CDATA[AUC 0～t (mg / mL)]]> 2441.87 19772.41 26688.06 <![CDATA[AUC 0～∞obs (mg / mL)]]> 2448.28 31238.15 35458.27 AUC_%Extrap_obs(%) 0.26 36.90 24.90 <![CDATA[AUC 0～t_D_obs (h*kg*ng / mL / mg)]]> 2441.87 19772.41 26688.06 Vz_obs(L / kg) 1.974 0.532 0.492 Cl_obs(mL / min / kg) 6.8147 0.5347 0.4727 <![CDATA[MRT 0～t (h)]]> 1.901 6.764 8.363

[0211] As can be seen from Table 2, at the same dosage, the half-life of Comparative Example 1 is 3.3 h, while the half-lives of Examples 1 and 8 are both extended to approximately 12 h; the area under the curve (AUC) of Comparative Example 1... 0～t The value was 2441 h*ng / mL, while the area under the curve (AUC) of Examples 1 and 8 was... 0～t The half-lives were 19772 h*ng / mL and 26688 h*ng / mL, respectively. This indicates that Examples 1 and 8 have longer half-lives and significantly higher bioavailability.

[0212] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. A cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound, characterized in that, The structural formula of the compound is shown in Formula I: ； Wherein, R is methyl, deuterated methyl, fluoromethyl, fluoroethyl, fluoropropyl, ethyl, propyl or isobutyl.

2. The cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound according to claim 1, characterized in that: The 4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound is one of the compounds having the following structural formula: 、 、 、 、 、 、 、 。 3. The method for preparing the cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound according to any one of claims 1 to 2, characterized in that, Includes the following steps: S1: 5-Chloro-2,3-diphenylpyrazine, the compound shown in Formula II, and a base are co-dissolved in an organic solvent and reacted at 100–200 °C for 8–48 h to obtain the compound shown in Formula III. 、 ； S2: The compound shown in Formula III, tert-butyl bromoacetate, and the catalyst are co-dissolved in a mixed solution under ice bath conditions. The mixture is kept warm and stirred for 30-45 min, then naturally heated to room temperature and the reaction continues for 0.5-3 h to obtain the compound shown in Formula IV. The mixed solution is prepared by mixing equal volumes of toluene and a 40 wt% KOH solution. ； S3: Dissolve the compound shown in Formula IV in a solvent under ice bath conditions, add NaH, keep warm and stir for 30-45 min, then add the iodinated compound RI, naturally warm to room temperature, and continue the reaction for 8-24 h to obtain the compound shown in Formula V; the solvent is tetrahydrofuran, N,N-dimethylformamide or diethyl ether. ； Wherein, R is methyl, deuterated methyl, fluoromethyl, fluoroethyl, fluoropropyl, ethyl, propyl or isobutyl; S4: Dissolve the compound shown in Formula V with LiOH or NaOH in an organic solvent under ice bath conditions, keep the reaction at the temperature for 2-10 hours, then acidify and extract to obtain the product.

4. The method for preparing the cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound according to any one of claims 1 to 2, characterized in that, Includes the following steps: S1: 5-Chloro-2,3-diphenylpyrazine, the compound shown in Formula VI, and a base are co-dissolved in an organic solvent and reacted at 100-180°C for 12-48 h to obtain the compound shown in Formula VII; 、 ； Wherein, R is methyl, deuterated methyl, fluoromethyl, fluoroethyl, fluoropropyl, ethyl, propyl or isobutyl; S2: The compound shown in Formula VII, tert-butyl bromoacetate, and the catalyst are co-dissolved in a mixed solution under ice bath conditions. The mixture is kept warm and stirred for 30-45 min, then naturally heated to room temperature and the reaction continues for 0.5-3 h to obtain the compound shown in Formula V. The mixed solution is prepared by mixing equal volumes of toluene and a 40 wt% KOH solution. ； S3: Dissolve the compound shown in Formula V with LiOH or NaOH in an organic solvent under ice bath conditions, keep the reaction at the temperature for 2-12 hours, then acidify and extract to obtain the product.

5. The preparation method according to claim 3 or 4, characterized in that, The 5-chloro-2,3-diphenylpyrazine was prepared by the following steps: (1) Under an inert atmosphere, biphenyl amide, aminoacetamide and base are dissolved in an organic solvent in a molar ratio of 1:1~1.5:2~2.5 and heated under reflux for 3~5 h to obtain the intermediate shown in Formula VIII; ； (2) Under an inert atmosphere, the intermediate shown in Formula VIII is mixed with phosphorus oxychloride at a ratio of 1g:0.5~5mL, and heated under reflux until the raw materials react completely to obtain the product.

6. The preparation method according to claim 3 or 4, characterized in that: The alkali is potassium carbonate; the catalyst is tetrabutylammonium bisulfate.

7. The use of the cis-4-(5,6-diphenylpyrazine-2-amino)cyclohexyloxyacetic acid compound according to any one of claims 1 to 2 in the preparation of antithrombotic drugs.

8. The application according to claim 7, characterized in that: The antithrombotic drug is a thrombodily vasodilator or an antipulmonary hypertension drug.

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