Par-1 inhibitor, chiral synthesis method therefor, preparation method for salt crystal form thereof, and use thereof

Abstract

The present invention belongs to the technical field of pharmaceutical and chemical engineering and particularly relates to a PAR-1 inhibitor, a chiral synthesis method therefor, a preparation method for a salt crystal form thereof, and a use thereof. The PAR-1 inhibitor of the present invention has a structural formula as shown in formula (I) and is prepared by using five steps. The PAR-1 inhibitor is then dissolved in a solvent, an acid solution is added, and the solution is cooled for crystallization to obtain a salt crystal form of the PAR-1 inhibitor. The PAR-1 inhibitor provided by the present invention has the characteristics of a definite target, a clear mechanism of action, a novel structure, and high safety and activity. The chiral synthesis method therefor yields a compound having an inverted configuration. The present invention further provides a method for preparing a sulfate crystal form of the PAR-1 inhibitor. The prepared crystal form is stable. The PAR-1 inhibitor prepared by the present invention is applied to the treatment of thrombotic diseases and shows high value.

Description

PAR-1 inhibitor, chiral synthesis method thereof, preparation method and use of salt crystal form thereof Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemicals, and in particular relates to a PAR-1 inhibitor, a chiral synthesis method thereof, and a preparation method and use of a salt crystal thereof. Background Art

[0002] Thrombosis refers to the formation of localized blood clots. Arterial thrombosis can lead to conditions such as myocardial infarction, stroke, acute coronary syndrome, and peripheral arterial disease, while venous thrombosis can cause pulmonary embolism. Arterial and venous thrombosis is the leading cause of morbidity and mortality from cardiovascular disease and is also one of the leading causes of death in cancer patients. Antithrombotic drugs include anticoagulants, antiplatelet aggregation drugs, and thrombolytic drugs. However, of these three different types of drugs, only anticoagulants and antiplatelet aggregation drugs are reasonably effective. The shortcomings of existing anticoagulants and the strong market opportunities will drive their development. New anticoagulant drugs that are at least as effective as existing anticoagulants, have improved safety (especially reduced bleeding risk), and are convenient to use (orally, especially for long-term use) are urgently needed.

[0003] Thrombin receptors (PARs) are a type of G protein-coupled receptor (GPCR). Although current PAR-1 inhibitory drugs have good anticoagulant activity, they have caused varying degrees of bleeding side effects in patients during long-term clinical use. The drug has an effective half-life of 3-4 days and a terminal elimination half-life of 8 days. Short-term drug withdrawal is ineffective in treating bleeding, and the effect persists for at least 4 weeks after discontinuation. Currently, there is no suitable treatment to counteract the antiplatelet effect of this drug.

[0004] CN105732595A and CN115043820A both disclose a PAR-1 inhibitor, the general structural formula of which is:

[0005] Compound (II) in CN105732595A is prepared by reacting andrographolide as a starting material with aluminum oxide to undergo a dehydration rearrangement reaction to obtain compound (IV), which is then epoxidized with m-CPBA, then acetalized with 2,2-dimethoxypropane, and finally reacted with ozone to obtain compound (II), and then compound (II) is reacted with Compound (III) is reacted to obtain the above PAR-1 inhibitor. CN115043820A also uses andrographolide as raw material and further optimizes the structure of Shuxinpasha. CN110627710A and CN112759548A disclose a new PAR-1 inhibitor, the structural formula of which is Compound (I) is prepared using sclareolide as the starting material.

[0006] Although PAR-1 inhibitors have been prepared in the above methods, their drug binding to the target is not tight, their efficacy is poor, their pharmacokinetic parameters are poor, and their drugability is poor. Therefore, they still have the problem of rapid metabolism in the body, which is not conducive to drugability.

[0007] Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a PAR-1 inhibitor with the characteristics of clear target, clear mechanism of action, novel structure, high safety and high activity. The chiral synthesis method thereof can obtain a configuration-flipped compound; the present invention also provides a method for preparing a salt crystal form of the PAR-1 inhibitor, and the prepared crystal form is stable. The PAR-1 inhibitor prepared by the present invention is used in the treatment of thrombotic diseases and has high value.

[0009] The PAR-1 inhibitor of the present invention has the following structural formula:

[0010] The preparation method of the PAR-1 inhibitor comprises the following steps:

[0011] (1) Using andrographolide as the starting material, intermediate II is prepared through reaction. The structural formula of intermediate II is shown below:

[0012] (2) The carbonyl group of intermediate II is subjected to chiral selective reductive amination, and the configuration of the methyl group at the ortho position is reconstructed to prepare compound III. The structural formula of compound III is shown below:

[0013] (3) The amino group in compound III was modified with ethyl chloroformate to obtain compound IV. The structural formula of compound IV is shown below:

[0014] (4) Compound IV is hydrolyzed and transesterified under acid catalysis to obtain Compound V. The structural formula of Compound V is shown below:

[0015] (5) Compound V and Compound VI were subjected to a Wittig reaction under the action of lithium diisopropylamide to obtain a PAR-1 inhibitor; the structural formula of Compound VI is shown below:

[0016] The catalyst used for the chiral selective reductive amination in step (2) is one of Ru((R)-BINAP)(OAc)2, Ru((S)-BINAP)(OAc)2, and Ru((S)-Segphos)(OAc)2, preferably Ru((S)-Segphos)(OAc)2.

[0017] The method for preparing the PAR-1 inhibitor salt crystal form comprises the following steps: dissolving the PAR-1 inhibitor in a solvent, adding an acid solution, stirring evenly, cooling to crystallize, filtering or centrifuging, and drying the obtained solid to obtain the PAR-1 inhibitor salt crystal form.

[0018] The acid solution is one of hydrochloric acid, sulfuric acid, phosphoric acid, fumaric acid and tartaric acid, preferably sulfuric acid.

[0019] Cool to -20°C-20°C for crystallization, preferably 0°C-10°C.

[0020] The solvent is one of ethanol, methanol and acetonitrile, and its added amount is 5-100 times the volume of the PAR-1 inhibitor, preferably 10-20 times the volume.

[0021] The PAR-1 inhibitor salt crystal form prepared by the preparation method of the PAR-1 inhibitor salt crystal form is used as a drug for treating thrombotic diseases and can be taken orally.

[0022] Specifically, the method for preparing the PAR-1 inhibitor salt crystal form comprises the following steps:

[0023] (1) Using andrographolide as the starting material, intermediate II is prepared through multi-step reactions. The structural formula of intermediate II is shown below:

[0024] (2) Add intermediate II to a reaction flask, add trifluoroethanol (TFE) to dissolve it, add ammonium acetate (NH4OAc) and a chiral catalyst in sequence, connect the hydrogen cylinder, ventilate three times, stir at 80°C for 10 hours, and after the reaction is complete, remove the catalyst powder in the reaction system by filtration, wash with ethyl acetate, and remove the solvent by reduced pressure distillation at 35°C and ≤-0.08 MPa to obtain a crude compound III, the structural formula of which is shown below:

[0025] (3) The crude product of compound III was dissolved in dichloromethane and transferred to a round-bottom flask. Ethyl chloroformate was added and triethylamine (TEA) was slowly added under stirring at 0°C. After the addition was completed, the mixture was stirred at room temperature for 2 hours. Water was added and stirred for 5 minutes. After the stirring was completed, the mixture was transferred to a separatory funnel and allowed to stand for stratification. The lower organic phase was discharged and the upper aqueous phase was extracted with dichloromethane. After the extraction was completed, the aqueous phase was discarded and the organic phases were combined. The organic phases were washed with water and transferred to a conical flask. The organic phase was dried over anhydrous sodium sulfate. After drying was completed, the mixture was filtered and distilled under reduced pressure. The crude product was separated and purified by column chromatography to obtain a white solid compound, namely compound IV. The structural formula of compound IV is shown below:

[0026] (4) Compound IV was added to a reaction flask, p-toluenesulfonic acid was added, and then acetone and water were added. The mixture was heated and stirred at 80°C for 4 hours. After the reaction was completed, saturated sodium bicarbonate solution was added and stirred for 5 minutes. The mixture was transferred to a separatory funnel, ethyl acetate was added and shaken, and the mixture was allowed to stand for stratification. The upper organic phase was discharged and dried over anhydrous sodium sulfate. After drying, the mixture was filtered and distilled under reduced pressure. The crude product was separated and purified by column chromatography to obtain a white solid compound, namely, compound V. The structural formula of compound V is shown below:

[0027] (5) Compound V was added to a reaction flask, dissolved in tetrahydrofuran, stirred at -20°C for 5 min, and lithium diisopropylamide was slowly added dropwise. After the addition was complete, the reaction was allowed to proceed for 1 h. Compound VI was added and stirred at 0°C for 2 h. After the reaction was complete, a saturated aqueous solution of ammonium chloride was added and stirred for 5 min. The mixture was transferred to a separatory funnel, shaken, allowed to stand and separated, and the upper organic phase was discharged. The lower aqueous phase was extracted with ethyl acrylate (EA). After the extraction was complete, the aqueous phase was discarded, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure. The crude product was purified with anhydrous ethanol to obtain a white solid compound, i.e., a PAR-1 inhibitor. The reaction process is shown below:

[0028] (6) Dissolve the PAR-1 inhibitor in 5-100 times the volume of solvent, add acid solution, stir evenly, cool to -20°C-20°C for crystallization, filter or centrifuge, and dry the obtained solid to obtain the α-crystalline form of the PAR-1 inhibitor salt.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The α-crystalline form of the PAR-1 inhibitor salt prepared by the present invention has good stability and is suitable for long-term storage, effectively avoiding crystal transformation during drug storage and development, thereby avoiding changes in bioavailability and efficacy.

[0031] (2) The method for preparing the PAR-1 inhibitor salt crystal of the present invention has simple process, low cost, good reproducibility, and the solvent used is non-toxic, which is of great value in the application and development of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the NMR spectrum of compound IV prepared in Example 1.

[0033] Figure 2 is the mass spectrum of compound IV prepared in Example 1.

[0034] Figure 3 is the NMR spectrum of compound V prepared in Example 1.

[0035] FIG4 is a mass spectrum of compound V prepared in Example 1.

[0036] FIG5 is the NMR spectrum of the PAR-1 compound prepared in Example 1.

[0037] FIG6 is a mass spectrum of the PAR-1 compound prepared in Example 1.

[0038] FIG7 is an XRPD pattern of the crystalline form of the PAR-1 inhibitor salt prepared in Example 2.

[0039] FIG8 is a single crystal structure diagram of the PAR-1 inhibitor salt crystal prepared in Example 2.

[0040] FIG9 is a graph showing the relationship between the average blood concentration of the PAR-1 inhibitor in SD rats and time when the PAR-1 inhibitor salt prepared in Example 2 was tested on samples. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to specific embodiments.

[0042] The raw materials used in the following examples and comparative examples are commercially available products.

[0043] Example 1

[0044] The preparation method of the PAR-1 inhibitor comprises the following steps:

[0045] (1) Using andrographolide as the starting material, intermediate II is prepared by reaction. The specific preparation steps are referred to the specification section [0095-0114] in Example 1 of Chinese patent CN115043820A published on September 13, 2022.

[0046] (2) 10 g of intermediate II was added to a reaction flask, and 50 mL of trifluoroethanol (TFE) was added to dissolve it. 4.78 g of ammonium acetate (NH4OAc) and 2.49 g of chiral catalyst Ru(OAc)2[(R)-binap] were added in sequence. The mixture was connected to a hydrogen cylinder, ventilated three times, and stirred at 80°C for 10 h. After the reaction was completed, the catalyst powder in the reaction system was removed by filtration and washed with 40 mL of ethyl acetate. The solvent was removed by reduced pressure distillation at 35°C and ≤-0.08 MPa to obtain a crude compound III.

[0047] (3) The crude product of compound III was dissolved in 100 mL of dichloromethane and transferred to a round-bottom flask. 16.8 g of ethyl chloroformate was added and 31.4 g of triethylamine (TEA) was slowly added under stirring at 0°C. After the addition was completed, the mixture was stirred at room temperature for 2 h. 100 mL of water was added and stirred for 5 min. After the stirring was completed, the system was transferred to a separatory funnel and allowed to stand for stratification. The lower organic phase was discharged and the upper aqueous phase was extracted with 100 mL of dichloromethane. After the extraction was completed, the aqueous phase was discarded and the organic phases were combined. The organic phases were washed with 100 mL of water and transferred to a conical flask. The organic phase was dried over anhydrous sodium sulfate. After drying was completed, the organic phase was filtered and distilled under reduced pressure. The crude product was separated and purified by column chromatography to obtain 7.9 g of a white solid compound, namely compound IV, with a yield of 64.44%. The NMR spectrum of compound IV is shown in Figure 1 and the mass spectrum is shown in Figure 2.

[0048] (4) 7.00 g of compound IV was added to a reaction flask, 4.0 g of p-toluenesulfonic acid was added, and then 20 mL of acetone and 2 mL of water were added. The mixture was heated and stirred at 80°C for 4 h. After the reaction was completed, 20 mL of saturated sodium bicarbonate solution was added and stirred for 5 min. The mixture was transferred to a separatory funnel, 20 mL of ethyl acetate was added and shaken, and the mixture was allowed to stand for stratification. The upper organic phase was discharged and dried over anhydrous sodium sulfate. After drying, the mixture was filtered and distilled under reduced pressure. The crude product was separated and purified by column chromatography to obtain 4.85 g of a white solid compound, namely compound V, with a yield of 81.65%. The NMR spectrum of compound V is shown in Figure 3, and the mass spectrum is shown in Figure 4.

[0049] (5) 4.00 g of compound V was added to a 50 mL reaction flask, dissolved in 10 mL of tetrahydrofuran, and stirred at -20°C for 5 min. 2.4 mL of lithium diisopropylamide was slowly added dropwise. After the addition was complete, the reaction was allowed to proceed for 1 h. 4.60 g of compound VI was added and stirred at 0°C for 2 h. After the reaction was complete, 15 mL of saturated aqueous ammonium chloride solution was added and stirred for 5 min. The mixture was transferred to a separatory funnel, shaken, and allowed to stand for stratification. The upper organic phase was discharged, and the lower aqueous phase was extracted with 15 mL of ethyl acrylate (EA). After the extraction was complete, the aqueous phase was discarded, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure. The crude product was purified with anhydrous ethanol to obtain 5.02 g of a white solid compound, i.e., a PAR-1 inhibitor, with a yield of 83.67%. The NMR spectrum of the PAR-1 compound is shown in Figure 5, and the mass spectrum is shown in Figure 6.

[0050] Example 2

[0051] The method for preparing the PAR-1 inhibitor salt crystal form comprises the following steps:

[0052] 4.00 g of the PAR-1 inhibitor prepared in Example 1 was dissolved in 40 mL of ethanol. 1.00 g of concentrated sulfuric acid was dissolved in 20 mL of ethanol and added to the ethanol solution of the PAR-1 inhibitor. The mixture was stirred evenly, cooled to 5° C. for crystallization, stirred for 5 h, filtered, and the resulting solid was dried to obtain an α-crystalline form of the PAR-1 inhibitor sulfate.

[0053] The α-crystalline form of the PAR-1 inhibitor salt prepared in Example 2 above was subjected to XRPD (X-ray powder diffraction) analysis, as shown in FIG7 . A Bruker D8 FOCUS X-ray powder diffractometer was used. As shown in FIG7 , characteristic peaks were found at 2θ values ​​of 4.5°±0.2°, 9.0°±0.2°, 14.5°±0.2°, 14.8°±0.2°, 15.4°±0.2°, 15.6°±0.2°, 18.2°±0.2°, 18.4°±0.2°, and 19.8°±0.2°.

[0054] The single crystal structure of Example 2 was analyzed, and its single crystal structure diagram is shown in Figure 8. The instrument brand used is: German Bruker, model: D8VENTURE, detection method: X-ray operating voltage 50kV, operating current 50mA, maximum 2θ value 68.395°, detection temperature 248K, using a Bruker APEX-IICCD area detector to collect diffraction intensity data, CuKα radiation, total diffraction points are 5948, independent diffraction points are 5644 (Rint=0.0510), and data integrity is 99.1%. The crystal is colorless, transparent, and blocky, belongs to the orthorhombic system, space group is P212121, and unit cell parameters are: α=90°,β=90°,γ=90°,unit cell volume The number of molecules in the unit cell is Z = 4, and there is 1 molecule in an independent area of ​​the unit cell.

[0055] Example 3

[0056] The preparation method of the PAR-1 inhibitor comprises the following steps:

[0057] (1) Using andrographolide as the starting material, intermediate II is prepared by reaction. The specific preparation steps are referred to the specification section [0095-0114] in Example 1 of Chinese patent CN115043820A published on September 13, 2022.

[0058] (2) 10 g of intermediate II was added to a reaction flask, and 50 mL of trifluoroethanol (TFE) was added to dissolve it. 4.78 g of ammonium acetate (NH4OAc) and 2.50 g of chiral catalyst Ru((S)-BINAP)(OAc)2 were added in sequence. The mixture was connected to a hydrogen cylinder, ventilated three times, and stirred at 80°C for 10 h. After the reaction was completed, the catalyst powder in the reaction system was removed by filtration and washed with 40 mL of ethyl acetate. The solvent was removed by reduced pressure distillation at 35°C and ≤-0.08 MPa to obtain a crude compound III.

[0059] (3) The crude product of compound III was dissolved in 100 mL of dichloromethane and transferred to a round-bottom flask. 16.8 g of ethyl chloroformate was added and 31.4 g of triethylamine (TEA) was slowly added under stirring at 0°C. After the addition was completed, the mixture was stirred at room temperature for 2 h. 100 mL of water was added and stirred for 5 min. After the stirring was completed, the system was transferred to a separatory funnel and allowed to stand for stratification. The lower organic phase was discharged and the upper aqueous phase was extracted with 100 mL of dichloromethane. After the extraction was completed, the aqueous phase was discarded and the organic phases were combined. The organic phases were washed with 100 mL of water and transferred to a conical flask. The organic phase was dried over anhydrous sodium sulfate. After drying was completed, the organic phase was filtered and distilled under reduced pressure. The crude product was separated and purified by column chromatography to obtain 5.3 g of a white solid compound, namely compound IV, with a yield of 43.23%.

[0060] (4) 7.00 g of compound IV was added to a reaction flask, 4.0 g of p-toluenesulfonic acid was added, and then 20 mL of acetone and 2 mL of water were added. The mixture was heated and stirred at 80°C for 4 h. After the reaction was completed, 20 mL of saturated sodium bicarbonate solution was added and stirred for 5 min. The mixture was transferred to a separatory funnel, 20 mL of ethyl acetate was added and shaken, and the mixture was allowed to stand for stratification. The upper organic phase was discharged and dried over anhydrous sodium sulfate. After drying, the mixture was filtered and distilled under reduced pressure. The crude product was separated and purified by column chromatography to obtain 4.85 g of a white solid compound, namely compound V, with a yield of 81.02%.

[0061] (5) 4.00 g of compound V was added to a 50 mL reaction bottle, and 10 mL of tetrahydrofuran was added to dissolve it. The mixture was stirred at -20 °C for 5 min, and 2.4 mL of lithium diisopropylamide was slowly added dropwise. After the addition was completed, the reaction was allowed to proceed for 1 h. 4.60 g of compound VI was added and stirred at 0 °C for 2 h. After the reaction was completed, 15 mL of saturated aqueous ammonium chloride solution was added and stirred for 5 min. The mixture was transferred to a separatory funnel, shaken, and allowed to stand for stratification. The upper organic phase was discharged, and the lower aqueous phase was extracted with 15 mL of ethyl acrylate (EA). After the extraction was completed, the aqueous phase was discarded, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure. The crude product was purified with anhydrous ethanol to obtain 5.02 g of a white solid compound, i.e., a PAR-1 inhibitor, with a yield of 82.85%.

[0062] Example 4

[0063] The preparation method of the PAR-1 inhibitor comprises the following steps:

[0064] (1) Using andrographolide as the starting material, intermediate II is prepared by reaction. The specific preparation steps are referred to the specification section [0095-0114] in Example 1 of Chinese patent CN115043820A published on September 13, 2022.

[0065] (2) 10 g of intermediate II was added to a reaction flask, and 50 mL of trifluoroethanol (TFE) was added to dissolve it. 4.78 g of ammonium acetate (NH4OAc) and 2.50 g of chiral catalyst Ru((R)-BINAP)(OAc)2 were added in sequence. The mixture was connected to a hydrogen cylinder, ventilated three times, and stirred at 80°C for 10 h. After the reaction was completed, the catalyst powder in the reaction system was removed by filtration and washed with 40 mL of ethyl acetate. The solvent was removed by reduced pressure distillation at 35°C and ≤-0.08 MPa to obtain a crude compound III.

[0066] (3) The crude product of compound III was dissolved in 100 mL of dichloromethane and transferred to a round-bottom flask. 16.8 g of ethyl chloroformate was added and 31.4 g of triethylamine (TEA) was slowly added under stirring at 0°C. After the addition was completed, the mixture was stirred at room temperature for 2 h. 100 mL of water was added and stirred for 5 min. After the stirring was completed, the system was transferred to a separatory funnel and allowed to stand for stratification. The lower organic phase was discharged and the upper aqueous phase was extracted with 100 mL of dichloromethane. After the extraction was completed, the aqueous phase was discarded and the organic phases were combined. The organic phases were washed with 100 mL of water and transferred to a conical flask. The organic phase was dried over anhydrous sodium sulfate. After drying was completed, the organic phase was filtered and distilled under reduced pressure. The crude product was separated and purified by column chromatography to obtain 4.2 g of a white solid compound, namely compound IV, with a yield of 34.26%.

[0067] (4) 7.00 g of compound IV was added to a reaction flask, 4.0 g of p-toluenesulfonic acid was added, and then 20 mL of acetone and 2 mL of water were added. The mixture was heated and stirred at 80°C for 4 h. After the reaction was completed, 20 mL of saturated sodium bicarbonate solution was added and stirred for 5 min. The mixture was transferred to a separatory funnel, 20 mL of ethyl acetate was added and shaken, and the mixture was allowed to stand for stratification. The upper organic phase was discharged and dried over anhydrous sodium sulfate. After drying, the mixture was filtered and evaporated under reduced pressure. The crude product was separated and purified by column chromatography to obtain 4.85 g of a white solid compound, namely compound V, with a yield of 80.01%.

[0068] (5) 4.00 g of compound V was added to a 50 mL reaction bottle, and 10 mL of tetrahydrofuran was added to dissolve it. The mixture was stirred at -20 °C for 5 min, and 2.4 mL of lithium diisopropylamide was slowly added dropwise. After the addition was completed, the reaction was allowed to proceed for 1 h. 4.60 g of compound VI was added and stirred at 0 °C for 2 h. After the reaction was completed, 15 mL of saturated aqueous ammonium chloride solution was added and stirred for 5 min. The mixture was transferred to a separatory funnel, shaken, and allowed to stand for stratification. The upper organic phase was discharged, and the lower aqueous phase was extracted with 15 mL of ethyl acrylate (EA). After the extraction was completed, the aqueous phase was discarded, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure. The crude product was purified with anhydrous ethanol to obtain 5.02 g of a white solid compound, i.e., a PAR-1 inhibitor, with a yield of 81.62%.

[0069] Example 5

[0070] The method for preparing the PAR-1 inhibitor salt crystal form comprises the following steps:

[0071] 1.00 g of the PAR-1 inhibitor prepared in Example 1 was dissolved in 100 mL of acetonitrile, 0.50 g of sulfuric acid was weighed and dissolved in 20 mL of acetonitrile, and then added to the acetonitrile solution of the PAR-1 inhibitor. The mixture was stirred evenly, cooled to 20° C. for crystallization, stirred for 5 h, filtered, and the resulting solid was dried to obtain an α-crystalline form of the PAR-1 inhibitor sulfate.

[0072] Example 6

[0073] The method for preparing the PAR-1 inhibitor salt crystal form comprises the following steps:

[0074] 1.00 g of the PAR-1 inhibitor prepared in Example 1 was dissolved in 50 mL of methanol, 0.50 g of sulfuric acid was weighed and dissolved in 20 mL of methanol, and then added to the methanol solution of the PAR-1 inhibitor, stirred evenly, cooled to -20°C for crystallization, stirred for crystallization for 5 hours, filtered, and the obtained solid was dried to obtain the α-crystalline form of the PAR-1 inhibitor sulfate.

[0075] The PAR-1 inhibitor prepared in Example 1 above was subjected to bioavailability assay.

[0076] (1) Biological sample collection

[0077] Eight SD rats (♂) were randomly divided into two groups of four. All animals were fasted for 12 hours, weighed, and administered with a PAR-1 inhibitor. Blood was collected by jugular vein puncture before and at 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration. Approximately 0.2 mL of whole blood was collected per rat per time point. The blood was placed in an ice-cold heparinized centrifuge tube and centrifuged within 1 hour to separate plasma (centrifugation conditions: 2-8°C, 8000 rpm, 10 minutes). The collected plasma was stored at -80°C until analysis.

[0078] (2) Sample testing

[0079] Chromatographic conditions: Chromatographic column: Accucore C18 (2.1×50 mm, 2.6 μm), mobile phase: 0.1 wt.% formic acid aqueous solution (A): 0.2 wt.% formic acid acetonitrile (B), gradient elution, column temperature: 40°C, injection volume: 5 μL, as shown in Table 1.

[0080] Table 1 Gradient elution conditions

[0081] Mass spectrometry conditions: AB SCIEX 5500 triple quadrupole tandem mass spectrometry system, ion source: ESI source, CUR: 35 psi, CAD: 8 psi, IS: 5500 psi, TEM: 550°C, Gas1: 55 psi, Gas2: 50 psi, MRM mode. Detection conditions are shown in Table 2.

[0082] Table 2 Test conditions

[0083] Sample processing: 40 μL of plasma sample was placed in a 1.5 mL centrifuge tube, 160 μL of acetonitrile solution (TBTM 100 ng / mL) was added, vortexed for 2 min, centrifuged at 13000 rpm (8°C) for 6 min, and 5 μL of the supernatant was injected for detection.

[0084] Sample analysis: blank SD rat plasma was taken and PAR-1 inhibitor standard series solutions were added in sequence to prepare standard samples. The concentration of the analyte was taken as the horizontal axis and the peak area ratio of the analyte to the internal standard was taken as the vertical axis. The weighted least square method (weight 1 / x 2 ) is the linear regression equation obtained by regression operation, which is the standard curve. The standard curve is Y = 0.00277X + 0.0000757, and the regression coefficient r = 0.9949. According to the standard curve, the sample concentration is analyzed, and accompanying quality control samples are prepared and evenly distributed among the samples to be tested. The deviation of the quality control sample measurement results should be less than 15%. At most, 1 / 3 of the quality control sample results are allowed to exceed the limit, but they cannot appear at the same concentration.

[0085] (3) Statistical analysis

[0086] According to the blood drug concentration data, the non-compartmental model of Phoenix WinNonlin 8.1 software was used to calculate the t 1 / 2 , Vd, CL, AUC, C max 、T max , MRT and other pharmacokinetic parameters. At the same time, according to AUC (0-t) The bioavailability of PAR-1 inhibitors via oral route was calculated as F = AUC(ig) / (10 x AUC(iv)) x 100%.

[0087] (4) Blood drug concentration-time data

[0088] The results of the measurement of the PAR-1 inhibitor concentrations in the plasma of SD rats at different times after oral and intravenous administration of the PAR-1 inhibitor are shown in Table 3. The relationship between the average blood concentration of the PAR-1 inhibitor in SD rats and time is shown in Figure 9, where A represents the relationship between the average blood concentration of the PAR-1 inhibitor in SD rats at a drug concentration of 1 mg / kg and time, and B represents the relationship between the average blood concentration of the PAR-1 inhibitor in SD rats at a drug concentration of 10 mg / kg and time.

[0089] Table 3 Relationship between the average blood concentration of PAR-1 inhibitors and time in SD rats

[0090] (5) Pharmacokinetic parameters and bioavailability

[0091] After oral and intravenous administration of PAR-1 inhibitors to SD rats, the pharmacokinetic parameters were calculated using the non-compartmental model using Phoenix WinNonlin 8.1 software. The average pharmacokinetic parameters are shown in Table 4, where the column for the dose concentration (10 mg / kg) shows the data for SD rats after oral administration of the PAR-1 inhibitors, and the column for the dose concentration (1 mg / kg) shows the data for SD rats after intravenous administration of the PAR-1 inhibitors.

[0092] Table 4 PK parameters of PAR-1 inhibitors in SD rats

[0093] (6) Conclusion

[0094] As shown in Table 4, after intravenous administration of PAR-1 inhibitor 1 mg / kg to rats, the Cmax of PAR-1 inhibitor in plasma was 829.58±48.70 ng / mL, and the AUC (0-t)It was 1716.23±224.74 ng / (mL×h), and AUC(0-∞) was 1755.40±246.10 ng / (mL×h) (n=4).

[0095] As shown in Table 4, after rats were orally administered with 10 mg / kg of PAR-1 inhibitor, the Cmax of PAR-1 inhibitor in plasma was 1127.57±133.59 ng / mL, and the AUC (0-t) The AUC(0-∞) was 9631.51±1629.03 ng / (mL×h), and the AUC(0-∞) was 10692.83±2608.94 ng / (mL×h) (n=4). (0-t) The bioavailability of the PAR-1 inhibitor after oral administration was calculated to be 56.12%.

[0096] The commercially available drug Vorapaxar, which targets the same drug, has an oral bioavailability of 33% in rats (Discovery of a Novel, Orally Active Himbacine-Based Thrombin Receptor Antagonist (SCH 530348) with Potent Antiplatelet Activity. J. Med. Chem. 2008, 51, 3061-3064.), while the PAR-1 inhibitor prepared in the present invention has a bioavailability of 56.12%, showing a significant advantage.

Claims

1. A PAR-1 inhibitor, characterized in that: The structural formula is as follows:

2. A method for preparing the PAR-1 inhibitor according to claim 1, characterized in that: The following steps are involved: (1) Using andrographolide as the starting material, intermediate II is obtained through reaction. The structural formula of intermediate II is shown below: (2) The carbonyl group of intermediate II is subjected to chiral selective reductive amination, and the configuration of the methyl group at the ortho position is reconstructed to obtain compound III. The structural formula of compound III is shown below: (3) The amino group in compound III is modified with ethyl chloroformate to obtain compound IV. The structural formula of compound IV is shown below: (4) Compound IV is hydrolyzed and transesterified under the action of acid catalysis to obtain Compound V. The structural formula of Compound V is shown below: (5) Under the action of lithium diisopropylamide, compound V and compound VI are subjected to a Wittig reaction to obtain a PAR-1 inhibitor; the structural formula of compound VI is shown below:

3. The method for preparing the PAR-1 inhibitor according to claim 2, characterized in that: The catalyst used for chiral selective reductive amination in step (2) is one of Ru((R)-BINAP)(OAc)2, Ru((S)-BINAP)(OAc)2, and Ru((S)-Segphos)(OAc)2.

4. A method for preparing the PAR-1 inhibitor salt crystal form according to claim 1, characterized in that: The method comprises the following steps: dissolving a PAR-1 inhibitor in a solvent, adding an acid solution, stirring evenly, cooling and crystallizing, filtering or centrifuging, and drying the obtained solid to obtain a PAR-1 inhibitor salt crystal form.

5. The method for preparing the PAR-1 inhibitor salt crystal form according to claim 4, characterized in that: The acid solution is one of hydrochloric acid, sulfuric acid, phosphoric acid, fumaric acid and tartaric acid.

6. The method for preparing a PAR-1 inhibitor salt crystal according to claim 4, characterized in that: Cool down to -20℃-20℃ for crystallization.

7. The method for preparing a PAR-1 inhibitor salt crystal according to claim 4, characterized in that: The solvent is one of ethanol, methanol and acetonitrile, and the added amount thereof is 5-100 times the volume of the PAR-1 inhibitor.

8. Use of a PAR-1 inhibitor salt crystal form obtained by the method for preparing a PAR-1 inhibitor salt crystal form according to any one of claims 4 to 7, characterized in that: Medicines used to treat thrombotic disorders.

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