A novel carbamate compound and its use

Abstract

The present invention relates to an antiplatelet aggregation drug and its use. The drug is a compound of formula I structure, or a pharmaceutically acceptable salt, solvate or deuterated product thereof; the compound of the present invention has a significant antiplatelet aggregation effect, suitable water solubility and good stability, and is expected to become a new type of injectable antiplatelet aggregation drug.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and specifically relates to a carbamate compound and use of the compound in preparing an anti-platelet aggregation drug. Background Art

[0002] It is well known in the art that platelet aggregation can cause a series of cardiovascular, cerebrovascular and other arterial circulatory disorders, including (1) acute coronary syndrome (ACS), such as unstable angina (UA), acute ST-segment elevation myocardial infarction (STEMI) and acute non-ST-segment elevation myocardial infarction (NSTEMI); (2) atherosclerotic diseases, such as myocardial infarction, ischemic stroke, peripheral arterial disease; (3) and thrombotic complications. Among the current antiplatelet aggregation drugs, clopidogrel has attracted much attention due to its low risk of bleeding, but the "clopidogrel resistance" phenomenon has greatly limited the drug's onset time and application scenarios, such as acute thrombosis. Although the existing technology also attempts to prepare clopidogrel injections in order to achieve rapid onset and overcome the defects of slow onset in acute treatment environments, such as Ascendia's ASD-002 nanoemulsion, CyDex's MDCO-157 (cyclodextrin inclusion) injection, and Jina Pharmaceuticals' JIN-2013 nanoliposome injection; but so far, due to the solubility of clopidogrel and the stability of water, light, and heat, they have all ended in failure. In addition, 2-oxo-clopidogrel, a clopidogrel metabolic intermediate developed by Chengdu Shibeikang Biopharmaceutical Technology Co., Ltd., has overcome the adverse reactions of "clopidogrel resistance" and has the advantages of rapid onset and higher bioavailability; but it was found in the study that 2-oxo-clopidogrel still has the defect of instability in water, which poses great challenges and limitations for the development of injections. Therefore, the subsequent development of derivatives of the intermediate metabolite 2-oxo-clopidogrel has attracted widespread attention from all walks of life.

[0003] Currently, among the prodrug molecules based on 2-oxoclopidogrel, only Vicagrelor has entered the clinic, and it is in the form of tablets. However, in comparative studies, it was found that Vicagrelor still has many limitations, such as thermal instability, which will also greatly limit its dosage form and clinical application scenarios.

[0004] In summary, developing a prodrug molecule that can release the active metabolite component (H4) of clopidogrel in vivo for a long time and has good water solubility and stability, while solving the problem that clopidogrel is not injectable, is a difficult problem that needs to be solved urgently in clinical practice. Summary of the invention

[0005] One of the purposes of the present invention is to provide a 2-oxo-clopidogrel prodrug molecule compound with good stability and excellent solubility in water.

[0006] The second object of the present invention is to provide a method for preventing and treating heart, brain and other arterial circulation disorders caused by platelet aggregation.

[0007] The technical solution of the present invention is as follows:

[0008] The present invention provides a compound represented by formula I, or a pharmaceutically acceptable salt, solvate or deuterated substance thereof

[0009] in:

[0010] X is selected from C or N;

[0011] R 1 is selected from a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted N-containing heterocyclic group.

[0012] Furthermore, in the compound represented by the above formula I, or a pharmaceutically acceptable salt, solvate or deuterated substance thereof,

[0013] X is selected from C or N;

[0014] R 1 Selected from substituted or unsubstituted C 1 ~C 6 Alkyl, substituted or unsubstituted C 3 ~C 6 Cycloalkyl, or substituted or unsubstituted C 2 ~C 6 Contains N heterocyclic group.

[0015] Furthermore, in the compound of formula I, or a pharmaceutically acceptable salt, solvate or deuterated substance thereof,

[0016] X is selected from N;

[0017] R 1 Selected from substituted or unsubstituted C 1 ~C 6 Alkyl, substituted or unsubstituted C 3 ~C 6 Cycloalkyl; preferably, R 1 is selected from methyl, ethyl, propyl, cyclopropyl, cyclopentyl or cyclohexyl.

[0018] Furthermore, in the compound of formula I, or a pharmaceutically acceptable salt, solvate or deuterated substance thereof,

[0019] X is selected from C;

[0020] R 1 Selected from substituted or unsubstituted C 2~C 6 N-containing heterocyclic group; preferably, R 1 is selected from piperazinyl, piperidinyl or pyrrolyl.

[0021] Furthermore, in the compound of the above formula I, or a pharmaceutically acceptable salt, solvate or deuterated substance thereof, the compound comprises:

[0022]

[0023] Furthermore, in the above-mentioned compounds, or pharmaceutically acceptable salts, solvates or deuterated substances thereof, the pharmaceutically acceptable salts include but are not limited to fumarate, acetate, ascorbate, benzoate, benzenesulfonate, citrate, hydrochloride, hydrobromide, maleate, methanesulfonate, sulfate, bisulfate, nitrate, oxalate, phosphate or succinate.

[0024] Furthermore, the above compound, or a pharmaceutically acceptable salt, solvate or deuterated substance thereof, is selected from:

[0025] or

[0026] .

[0027] The present invention also provides a method for preparing any of the above-mentioned compounds, or a pharmaceutically acceptable salt, solvate or deuterated product thereof, comprising the following steps:

[0028]

[0029] wherein R1 is defined as in any of the above items, and X is a halogen.

[0030] The present invention also provides the use of the above compound, or its pharmaceutically acceptable salt, solvate or deuterated product in the preparation of drugs for preventing and / or treating heart, brain and other arterial circulation disorders caused by platelet aggregation.

[0031] Furthermore, the above-mentioned heart, brain and other arterial circulatory disorders caused by platelet aggregation include but are not limited to acute coronary syndrome, atherosclerotic disease, or thrombotic complications.

[0032] Furthermore, the above-mentioned acute coronary syndrome includes but is not limited to angina pectoris or myocardial infarction.

[0033] Furthermore, the above-mentioned acute coronary syndrome includes but is not limited to unstable angina (UA), acute ST-segment elevation myocardial infarction (STEMI) or acute non-ST-segment elevation myocardial infarction (NSTEMI).

[0034] Furthermore, the above-mentioned atherosclerotic diseases include but are not limited to myocardial infarction, ischemic stroke or peripheral arterial disease.

[0035] Furthermore, the above-mentioned ischemic stroke includes but is not limited to cerebral stroke.

[0036] Furthermore, the above-mentioned thrombotic complications include but are not limited to pulmonary infarction.

[0037] The term "halogen" as used herein refers to fluorine, chlorine, bromine and iodine.

[0038] The term "C 1 ~C 6 "Alkyl" refers to a linear or branched monovalent alkane group having 1 to 6 carbon atoms.

[0039] The beneficial effects of the present invention are as follows:

[0040] The compounds of the present invention have better in vitro efficacy and better pharmacokinetic characteristics. Specifically, the compounds of the present invention have a strong antiplatelet aggregation effect, a fast onset of action, a high blood concentration and bioavailability of the in vivo pharmacokinetic, a long half-life, and better efficacy. In addition, the compounds of the present invention have good in vitro solubility, high stability, low hemolysis risk, no cardiotoxicity, low vascular irritation risk, high safety, and are more conducive to being made into injections to make up for clinical deficiencies. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The concentration-effect curve of compound 2 on hERG channel current is shown;

[0042] Figure 2 A schematic diagram showing the results of the hemolysis test of the low concentration group (0.5 mg / ml) of compound 2;

[0043] Figure 3 A schematic diagram showing the results of the hemolysis test of the high concentration group (2.5 mg / ml) of compound 2;

[0044] Figure 4 A schematic diagram showing the results of a low concentration (0.5 mg / ml) vascular irritation test of compound 2;

[0045] Figure 5 A schematic diagram showing the results of a high concentration (1.5 mg / ml) vascular irritation test of compound 2;

[0046] Figure 6The results of the property comparison of Comparative Example 2 after 0 days and after being placed at high temperature (60)°C for two days are shown. DETAILED DESCRIPTION

[0047] The present invention will be further described in detail below in conjunction with embodiments and test examples. The embodiments and test examples of the present invention are only used to illustrate the technical scheme of the present invention, and are not intended to limit the present invention. Any equivalent substitutions in the art made in accordance with the contents disclosed in the present invention shall fall within the protection scope of the present invention.

[0048] The compounds of the present invention, their stereoisomers or pharmaceutically acceptable salts can be prepared by selecting the synthetic routes of the embodiments, and the conventional conditions of the reaction raw materials and the reaction solvent can be adjusted according to the needs of the substituents or the salt formation, which can be realized by those skilled in the art based on the disclosure of the present invention. In addition, the column chromatography of the present invention refers to silica gel column chromatography unless otherwise specified, and the elution solvent can be determined as a single or mixed elution solvent in combination with the reaction solvent and the common knowledge or common means of those skilled in the art unless otherwise specified.

[0049] The structures of the compounds were determined by NMR ( 1 H NMR) or liquid chromatography-mass spectrometry (LC-MS).

[0050] The liquid chromatography-mass spectrometer (LC-MS) was Agilent G6120B (used with Agilent 1260 liquid phase); the nuclear magnetic resonance instrument ( 1 HNMR) was Bruker AVANCE-400 or Bruker AVANCE-800, NMR ( 1 H NMR) shift ( δ ) are given in parts per million (ppm) and the solvent used for the determination is DMSO-d 6 or CDCl 3 The internal standard is tetramethylsilane (TMS), and the chemical shift is 10 -6 (ppm) is given as the unit.

[0051] The term "room temperature" of the present invention refers to a temperature between 10 and 25°C.

[0052] Example 1: Preparation of (S)-5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl 4-methylpiperazine-1-carboxylate (Compound 1)

[0053]

[0054] Add (2S)-2-(2-oxo-7,7a-dihydrothieno[3,2-c]pyridine-5(2H,4H,6H)-yl)-2-(2-chlorophenyl)-acetic acid methyl ester (500mg, 1.48mmol) and 4-methylpiperazine-1-carbonyl chloride hydrochloride (442mg, 2.22mmol) and DMF (10ml) into a 50ml three-necked flask, stir and cool to 0℃, and add DBU (685g, 4.5mmol) dropwise. After the addition is complete, react at room temperature for 2h. Add EA, wash twice with water and once with brine. The organic phase is washed with anhydrous Na 2 SO 4 The concentrate was separated and purified by chromatography (MeOH:DCM=2:100), and the product was collected and concentrated to dryness to obtain 580 mg of the title product as a yellow oil with a yield of 72.8% and a purity of 96.19%.

[0055] ESI-MS: m / z = 464.1(M+H) + .

[0056] 1 HNMR (400 MHz, DMSO-d6) δ: 7.60~7.54 (m, 1H), 7.51~7.46 (m, 1H), 7.46~ 7.35 (m, 2H), 6.39 (s, 1H), 4.86 (s, 1H), 3.67 (s, 3H), 3.57 (s, 2H), 3.40 (s, 2H), 2.80 (qt, 2H), 2.63 (d, 2H), 2.48~2.39 (m, 6H), 2.34 (s, 3H).

[0057] Example 2: Preparation of (S)-5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl 4-methylpiperazine-1-carboxylate fumarate (Compound 2)

[0058] Add (S)-5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl 4-methylpiperazine-1-carboxylate (220 mg, 0.47 mmol) to a 25 ml flask, add EA (2.5 ml) to dissolve, add fumaric acid (110 mg, 0.95 mmol) and stir, a large amount of solid precipitates. Stir for 3 h, filter, and dry to obtain 192 mg of the title compound. The yield is 58.7% and the purity is 98.39%.

[0059] ESI-MS: m / z = 464.1(M+H) + .

[0060] 1 HNMR (400 MHz, DMSO-d6) δ: 13.82~9.71 (br, 4H), 7.64~7.56 (m, 1H), 7.56~7.47 (m, 1H), 7.46~7.35 (m, 2H), 6.62 (s, 4H), 6.39 (s, 1H), 4.86 (s,1H), 3.67 (s, 3H), 3.57 (s, 2H), 3.46 (s, 2H), 2.82 (qt, 2H), 2.69 (d, 2H), 2.48~2.39 (m, 6H), 2.30 (s, 3H).

[0061] Example 3: Preparation of (S)-5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-4-propylpiperazine-1-carboxylate (Compound 3)

[0062]

[0063] The preparation method is the same as that of Example 1, except that 4-methylpiperazine-1-carbonyl chloride hydrochloride is replaced by an equal molar amount of 4-propylpiperazine-1-carbonyl chloride hydrochloride to obtain the title compound with a yield of 76.3% and a purity of 97.10%.

[0064] ESI-MS: m / z = 492.2(M+H) + .

[0065] 1 HNMR (400 MHz, DMSO-d6) δ: 7.58~7.44 (m, 1H), 7.45~7.36 (m, 1H), 7.31~ 7.22 (m, 2H), 6.29 (s, 1H), 4.75 (d, 1H), 3.90 (d, 2H), 3.71 (s, 3H), 3.40 (m, 4H), 3.15~3.08 (m, 1H), 3.01 (d, 1H), 2.91 (t, 2H), 2.48~2.39 (m, 6H), 1.57 (qt, 2H), 0.88 (t, 3H).

[0066] Example 4: Preparation of (S)-5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl 4-cyclopropylpiperazine-1-carboxylate (Compound 4)

[0067]

[0068] The preparation method is the same as that of Example 1, except that 4-methylpiperazine-1-carbonyl chloride hydrochloride is replaced by an equal molar amount of 4-cyclopropylpiperazine-1-carbonyl chloride hydrochloride to obtain the title compound with a yield of 81.3% and a purity of 97.31%.

[0069] ESI-MS: m / z = 490.1(M+H) + .

[0070] 1 HNMR (400 MHz, DMSO-d6) δ: 7.52~7.43 (m, 1H), 7.43~7.34 (m, 1H), 7.32~ 7.21 (m, 2H), 6.29 (s, 1H), 4.75 (d, 1H), 3.90 (d, 2H), 3.71 (s, 2H),3.45 (dd, 2H), 3.35 (dd, 2H), 3.15~3.08 (m, 1H), 3.02 (t, 1H), 2.91 (t, 2H),2.74 (m, 2H), 2.61 (m, 2H), 2.53~2.45 (m, 1H), 0.77~0.50 (m, 4H).

[0071] Example 5: Preparation of (S)-5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl 4-cyclopentylpiperazine-1-carboxylate (Compound 5)

[0072]

[0073] The preparation method is the same as that of Example 1, except that 4-methylpiperazine-1-carbonyl chloride hydrochloride is replaced by an equal molar amount of 4-cyclopentylpiperazine-1-carbonyl chloride hydrochloride to obtain the title compound with a yield of 84.3% and a purity of 98.11%.

[0074] ESI-MS: m / z = 518.2(M+H) + .

[0075] 1HNMR (400 MHz, DMSO-d6) δ: 7.66~7.51 (m, 1H), 7.46~7.38(m, 1H), 7.33~7.22 (m, 2H), 6.29 (s, 1H), 4.75 (d, 1H), 3.90 (d, 2H), 3.71 (s, 3H), 3.45 (dd, 2H), 3.35 (dd, 2H), 3.15~3.08 (m, 1H), 3.02 (t, 1H), 2.91 (t, 2H), 2.73 (m, 2H), 2.67~2.58 (m, 2H), 2.50 (pent, 1H), 1.88~1.74 (m, 4H), 1.64~1.45 (m, 4H).

[0076] Example 6: Preparation of (S)-5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl 4-cyclohexylpiperazine-1-carboxylate (Compound 6)

[0077]

[0078] The preparation method is the same as that of Example 1, except that 4-methylpiperazine-1-carbonyl chloride hydrochloride is replaced by an equal molar amount of 4-cyclohexylpiperazine-1-carbonyl chloride hydrochloride to obtain the title compound with a yield of 71.4% and a purity of 98.23%.

[0079] ESI-MS: m / z = 532.2(M+H) + .

[0080] 1 HNMR (400 MHz, DMSO-d6) δ: 7.62~7.50 (m, 1H), 7.46~7.35 (m, 1H), 7.30~ 7.18 (m, 2H), 6.29 (s, 1H), 4.75 (d, 1H), 3.88 (d, 2H), 3.71 (s, 3H), 3.45 (dd, 2H), 3.38 (dd, 2H), 3.18~3.08 (m, 1H), 3.02 (t, 1H), 2.91 (t, 2H), 2.73 (m, 2H), 2.67~2.58 (m, 2H), 2.34 (pent, 1H), 1.73~1.58 (m, 4H), 1.62~1.49 (m, 2H), 1.53~1.39 (m, 1H), 1.42~1.32 (m, 1H).

[0081] Example 7: Preparation of (S)-5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl[1,4'-bipiperidine]-1'-carboxylate (Compound 7)

[0082]

[0083] The preparation method is the same as that of Example 1, except that 4-methylpiperazine-1-carbonyl chloride hydrochloride is replaced with an equal molar amount of 4-piperidinylpiperidinylcarbonyl chloride hydrochloride to obtain the title compound with a yield of 88.7% and a purity of 98.45%.

[0084] ESI-MS: m / z = 532.2(M+H) + .

[0085] 1 HNMR (400 MHz, DMSO-d6) δ: 7.52~7.43 (m, 1H), 7.43~7.34 (m, 1H), 7.32~ 7.21 (m, 2H), 6.29 (s, 1H), 4.75 (d, 1H), 3.90 (d, 2H), 3.71 (s, 3H),3.69~3.59 (m, 2H), 3.39 (m, 2H), 3.15~3.08 (m, 1H), 3.02 (t, 1H), 2.91 (t,2H), 2.68 (m, 2H), 2.39 (q, 1H), 2.31 (m, 2H), 1.74~1.37 (m, 10H).

[0086] Example 8: Preparation of (S)-5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl[1,4'-bipiperidine]-1'-carboxylate hydrochloride (Compound 8)

[0087]

[0088] The preparation method is the same as the preparation method of Example 2, except that (S)-5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl 4-methylpiperazine-1-carboxylate is replaced with an equal mole of (S)-5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl[1,4'-bipiperidine]-1'-carboxylate, and fumaric acid is replaced with an equal mole of HCl (ethyl acetate solution) to obtain the title compound with a yield of 85.9% and a purity of 98.69%.

[0089] ESI-MS: m / z = 532.2(M+H) + .

[0090] 1 HNMR (400 MHz, DMSO-d6) δ: 7.58~7.46 (m, 1H), 7.46~7.34 (m, 1H), 7.39~ 7.24 (m, 2H), 6.29 (s, 1H), 4.75 (d, 1H), 3.90 (d, 2H), 3.71 (s, 3H),3.71~3.60 (m, 2H), 3.35 (m, 2H), 3.15~3.08 (m, 1H), 3.02 (t, 1H), 2.91 (t,2H), 2.68 (m, 2H), 2.39 (q, 1H), 2.31 (m, 2H), 1.74~1.37 (m, 10H).

[0091] Example 9: Preparation of 5-((S)-1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-4-(1-methylpyrrolidin-3-yl)piperidine-1-carboxylate (Compound 9)

[0092]

[0093] The preparation method is the same as that of Example 1, except that 4-methylpiperazine-1-carbonyl chloride hydrochloride is replaced with an equal molar amount of 4-(1-methylpyrrolidin-3-yl)piperidinyl chloride hydrochloride to obtain the title compound with a yield of 74.7% and a purity of 97.79%.

[0094] ESI-MS: m / z = 532.2(M+H) + .

[0095] 1HNMR (400 MHz, DMSO-d6) δ: 7.82~7.65 (m, 1H), 7.55~7.44 (m, 1H), 7.38~ 7.25 (m, 2H), 6.29 (s, 1H), 4.75 (d, 1H), 3.90 (d, 2H), 3.73~3.59 (m,5H), 3.34~3.22 (m, 2H), 3.15~3.08 (m, 1H), 3.02 (t, 1H), 2.91 (t, 2H), 2.74~2.52 (m, 4H), 2.23 (s, 3H), 2.13 (m, 1H), 1.86~1.75 (m, 2H), 1.79~1.70(m, 2H), 1.73~1.63 (m, 1H), 1.67~1.60 (m, 1H), 1.63~1.55 (m, 1H), 1.58~1.47 (m, 1H).

[0096] Example 10: Preparation of (S)-5-(1-(2-chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl-4-(4-methylpiperazin-1-yl)piperidine-1-carboxylate (Compound 10)

[0097]

[0098] The preparation method is the same as that of Example 1, except that 4-methylpiperazine-1-carbonyl chloride hydrochloride is replaced with an equal molar amount of 4-(4-methylpiperazine-1-yl)piperidinyl chloride hydrochloride to obtain the title compound in a yield of 68.5% and a purity of 98.08%.

[0099] ESI-MS: m / z = 547.2(M+H) + .

[0100] 1HNMR (400 MHz, DMSO-d6) δ: 7.88~7.66 (m, 1H), 7.53~7.45 (m, 1H), 7.36~ 7.23 (m, 2H), 6.33(s, 1H), 4.75 (d, 1H), 3.90 (d, 2H), 3.71 (s, 3H), 3.65 (m, 2H), 3.39 (m, 2H), 3.15~3.08 (m, 1H), 3.02 (t, 1H), 2.91 (t, 2H), 2.68~2.58 (m, 1H), 2.62~2.54 (m, 3H), 2.57~2.50 (m, 2H), 2.54~2.45 (m,3H), 2.40 (pent, 1H), 2.33 (s, 3H), 1.74~1.56 (m, 4H).

[0101] Comparative Example 1: 2-oxo-clopidogrel (a metabolic intermediate in the activation process of clopidogrel)

[0102]

[0103] Prepared by Chengdu Shibeikang Biotechnology Co., Ltd., ee=98.8%.

[0104] Comparative Example 2: Vicagrelor (in clinical research)

[0105]

[0106] Prepared according to CN201010624329.7, purity 98.22%.

[0107] Test Example 1: Study on Antiplatelet Aggregation Effect

[0108] 1. Purpose of the experiment: To evaluate and compare the therapeutic effects of each compound on antiplatelet aggregation after administration at the same molar dosage.

[0109] 2. Test methods

[0110] (1) Grouping

[0111] 70 rats were randomly and evenly divided into 7 groups: solvent control group, comparative example 1 (2-oxoclopidogrel) group, comparative example 2 (vicagrelor) group, compound 1 group, compound 2 group, compound 7 group, and compound 8 group.

[0112] (2) Anesthesia induction

[0113] Rats were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital.

[0114] (3) Fixed

[0115] After anesthesia, the animal was transferred to the operating table, and the eyelid reflex and pain response of the rat were observed. The operation could be started only after the eyelid reflex response and the pain response of the limbs and tail disappeared.

[0116] (4) Blood collection from the abdominal aorta of rats

[0117] After the injection of anesthetic, the rat can be fixed on the operating table in a supine position until the body becomes soft. After routine disinfection, use surgical scissors to cut the abdominal cavity along the midline of the abdomen, use small tweezers to gently pull apart the fat around the blood vessels, and then use cotton balls to wipe off the excess fat covering the blood vessels until the blood vessels are clearly visible (the abdominal aorta is located above the spine; the abdominal venous vessels are thicker and darker than the abdominal aorta). First fix the blood vessels to avoid vascular displacement as much as possible. The thumb and index finger of the left hand fix the fat on both sides of the blood vessels and other organs. The ring finger presses the upper end of the blood vessel insertion point to lower blood pressure and avoid blood spray. Hold the puncture needle with the right hand, with the needle tip beveled downward, the needle insertion angle is about 30 degrees, and the needle is inserted toward the heart end. The depth is about 5mm. When the needle end returns blood, insert the other end of the blood collection needle into the vacuum tube. After the needle is inserted, the needle can be clamped with a hemostatic clamp to avoid the blood vessels being punctured by the needle due to insufficient anesthesia and struggle.

[0118] (5) Platelet preparation

[0119] Three hours after administration, the rats were anesthetized with sodium pentobarbital, and then blood was collected from the abdominal aorta, anticoagulated with 3.8% sodium citrate at a ratio of 1:9, mixed and centrifuged at 200 g for 10 min, and the supernatant was taken as platelet-rich plasma (PRP); the remaining plasma was centrifuged at 1600 g for 15 min, and the supernatant was taken as platelet-poor plasma (PPP).

[0120] (6) ADP-induced platelet aggregation

[0121] The platelet aggregation rate was measured using a platelet aggregometer (Helena, USA, model: Agg RAM): first, the PPP corresponding to the PRP to be tested in each channel was taken for transmittance correction, and then the PPP was taken out after correction, and then a cuvette with 225 μl of the PRP to be tested was placed in each channel, a stirring bar was added, and then ADP (final concentration was 20 μM) was added to immediately start the platelet aggregation rate test.

[0122] 3. Test results

[0123] After the same molar administration, the platelet aggregation rate data of each group are shown in Table 1.

[0124] The results showed that: (1) the platelet aggregation rate (%) of the solvent control group was 76.06±3.29; the platelet aggregation rates (%) of the comparative example 1 group, comparative example 2 group, compound 1 group, compound 2 group, compound 7 group, and compound 8 group were 31.32±6.07, 39.32±12.38, 24.08±6.00, 21.18±8.51, 27.59±6.99, and 27.13±6.50, respectively. Compared with the solvent control group, each drug-treated group had a significant inhibitory effect on ADP-induced rat platelet aggregation (all P<0.01).

[0125] (2) Compared with control group 2, compound group 1, compound group 2, compound group 7, and compound group 8 had significant advantages in inhibiting ADP-induced platelet aggregation in rats (compound group 1, compound group 2: P < 0.01; compound group 7, compound group 8: P < 0.05), which was statistically significant.

[0126] (3) It can be seen from the test results that after a single intravenous injection at the same dose, the inhibitory effects of Compound 1, Compound 2, Compound 7 and Compound 8 of the present invention on ADP-induced platelet aggregation in rats are significantly better than those of the compound in Comparative Example 2 and the compound in Comparative Example 1.

[0127]

[0128] Experimental Example 2: Effects of Compounds on Stably Overexpressed hERG Channel Currents

[0129] 1. Test samples

[0130] Compounds 1 to 10, and control compound Cisapride.

[0131] 2. Test methods

[0132] Manual patch clamp technique (the gold standard for hERG safety assessment) was used to study the inhibitory effects of test compounds 1 to 10 on hERG potassium channels and to evaluate their risk of inducing ventricular repolarization toxicity.

[0133] 3. Test results

[0134] This study used manual patch clamp technology to detect the concentration-effect relationship of the blocking effect of test compounds 1 to 10 on hERG channels, so as to evaluate the risk of the test compounds inhibiting cardiac hERG potassium channels. The current concentration-effect curve of compound 2 is shown in Figure 1 The test results are shown in Table 2.

[0135]

[0136] The above test results show that the IC50 value of the positive drug is less than 0.1 μM, which is cardiotoxic, proving that the modeling of this test is successful. The IC50 values ​​of the compounds of the present invention are all greater than 10 μM, proving that the compounds of the present invention have a weak inhibitory effect or no inhibitory effect on hERG, that is, the compounds of the present invention have a low risk of inducing ventricular repolarization toxicity and are highly safe.

[0137] The above test results show that the compound of the present invention has a low risk of inducing ventricular repolarization toxicity and is highly safe.

[0138] Test Example 3: Pharmacokinetic Study

[0139] 1. Purpose of the study

[0140] The pharmacokinetic characteristics of each compound after a single oral administration to rats at the same molar dose were investigated, and the main pharmacokinetic parameters were compared.

[0141] 2. Materials and Methods

[0142] (1) Test substance

[0143] Comparative Example 1 (2-oxoclopidogrel), Compound 1, Compound 2, Compound 7, and Compound 8.

[0144] (2) Preparation of drug delivery preparations

[0145] Accurately weigh the test compounds into clean dosing containers, add appropriate amount of Solutol to dissolve, vortex and shake, add pure water, sonicate, and vortex and shake until the compounds are completely dissolved; the dosing preparations are freshly prepared on the day of dosing.

[0146] (3) Trial groups and drug administration

[0147] Thirty healthy adult SD rats were gavaged and given the drug. The specific scheme is shown in Table 3.

[0148]

[0149] (4) Test methods

[0150] Grouping and fasting: SD rats were randomly divided into groups, 6 rats in each group, and the corresponding compounds were administered by gavage according to Table 3.

[0151] Sample collection and processing: 0.2 ml of blood was collected at different time points before administration (0h) and 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h after administration. After anticoagulation with EDTA-K2, the blood was centrifuged at 4°C for 5 min, and the plasma was separated and stored at -80°C for testing.

[0152] Detection: The blood concentration of 2-oxo-clopidogrel in all PK plasma samples was detected by LC / MS / MS method, and the pharmacokinetic parameters of each compound were calculated using WinNonlin7.2 software.

[0153] 3. Test results

[0154]

[0155] As can be seen from the table above, compared with the compound in comparative example 1, the T1 / 2, Cmax and AUC0-last of compound 1, compound 2, compound 7 and compound 8 were significantly prolonged and improved, with statistical significance; among which P<0.01 for compound 1 and compound 2, and P<0.05 for compound 7 and compound 8. This indicates that the absorption of compound 1, compound 2, compound 7 and compound 8 in rats is significantly better than that of the compound in comparative example 1, and has better pharmacokinetic characteristics.

[0156] In summary, the compounds of the present invention are better absorbed, have high bioavailability, and are expected to fully exert their efficacy.

[0157] Test Example 4: In vitro hemolysis test

[0158] 1. Test samples

[0159] Compound 2.

[0160] 2. Experimental Animals

[0161] Adult large white-eared rabbit, male, 1 piece.

[0162] 3. Preparation of red blood cell suspension

[0163] (1) After the rabbit is anesthetized with sodium pentobarbital (about 30 mg / kg, injected into the ear vein), about 20 ml of blood is collected from the heart and placed in an appropriate container;

[0164] (2) Place a stirring bar (or glass beads) in the container and stir slowly for several minutes to remove the fibrinogen in the blood, making it defibrillated blood;

[0165] (3) Add about 10 times the amount of physiological saline, shake well and dispense into centrifuge tubes, centrifuge at 1000-2000 rpm for 15 minutes (due to the different sizes of centrifuge tubes, the speed and time of the centrifuge can be adjusted according to the situation after centrifugation), and remove the supernatant;

[0166] (4) The precipitated red blood cells are then washed with saline until the supernatant no longer appears red;

[0167] (5) Prepare the obtained red blood cells into a 2% suspension with physiological saline.

[0168] 4. In vitro hemolysis test ratio table

[0169]

[0170] Note: The negative control group a The test substance was composed of 2% red blood cell suspension and 0.9% sodium chloride injection 2.5ml each; positive control group b The test substance is composed of a mixture of 2% red blood cell suspension and 2.5 ml of sterile water for injection.

[0171] 5. Result judgment criteria

[0172]

[0173] 6. Test results

[0174] The experimental results are shown in Figure 2 and Figure 3 According to the test results, the low concentration of 0.5 mg / ml and the high concentration of 2.5 mg / ml of compound 2 injection did not cause hemolysis of rabbit blood cells.

[0175] Test Example 5: Vascular stimulation test

[0176] 1. Test samples

[0177] Compound 2.

[0178] 2. Experimental Animals

[0179] 4 Japanese large-eared white rabbits (about 2kg), 2♀ / 2♂.

[0180] 3. Experimental plan

[0181] The left and right sides of the same body were controlled by the same method. Low-dose and high-dose groups of compound 2 injection were set up, with 2 animals in each group, half male and half female. Compound 2 injection was injected into the right ear vein of the low-dose group and the high-dose group, with the dosage of 2.5 and 7.5 mg / kg, respectively, and the administration volume was 5 ml / kg, and the corresponding administration concentrations were 0.5 and 1.5 mg / ml, respectively. The left ear vein of the low-dose group and the high-dose group of compound 2 was injected with the same administration volume of sodium chloride injection as a negative control. The drug was administered by intravenous infusion for 30 min. After administration, the general condition, body weight, food intake, and reactions of the administration site and its surroundings were observed, including edema, congestion, degeneration, necrosis, etc. The drug was administered once a day for 7 consecutive days.

[0182] 72 h after the last administration and after 14 days of recovery, half of the animals (1♀1♂) were killed, and ear tissues were cut from the injection site and the proximal end of the ear vein. The irritation reaction at the injection site was observed with the naked eye, and the animals were fixed with 10% neutral formalin and routine tissue sections were used for histopathological examination.

[0183] Note: The specific administration time should be adjusted according to the situation after administration.

[0184] 4. Dosage regimen

[0185]

[0186] 5. Test results

[0187] The test results are shown in the following table: Figure 4 and Figure 5 As shown, at low concentration (0.5 mg / ml), compound 2 had no vascular stimulating effect on the marginal ear vein of rabbits by naked eye observation; at high concentration (1.5 mg / ml), the marginal ear vein of the female animals on the administration side was congested by naked eye observation at 2 days in the recovery period, but the recovery period was short. Therefore, it was judged that high concentration (1.5 mg / ml) of compound 2 had no vascular stimulating effect on the marginal ear vein of rabbits.

[0188]

[0189] Test Example 6: Solubility Study

[0190] Accurately weigh the samples of Example Compound 2 and Comparative Example 2 (vicagrelor), take 1 ml each of physiological saline and pH 1.2 buffer at 25-30°C, and then add 10 mg of each compound respectively. The solubility data are shown in the table below: Compound 2 of the present invention has excellent solubility and can meet the clinical requirements for preparation of injections with effective concentrations, while the solubility of Comparative Example 2 is extremely low and does not meet the requirements of injections. If other co-solvents are added, it may bring potential safety risks.

[0191]

[0192] Test example 7: Experimental study on influencing factors

[0193] Test method: Weigh the samples of Compound 2 and Comparative Example 2 (Vicagrelor) respectively and place them in weighing bottles, and place them under high temperature (60°C), high humidity (RH80%), and light (5000Lux) conditions respectively.

[0194] The test results of compound 2 are shown in the following table: After 10 days under high temperature, high humidity and light conditions, the content of compound 2 changed slightly, the appearance and shape did not change significantly, and the stability was good.

[0195]

[0196] However, after being placed at high temperature (60°C) for two days, the properties of Comparative Example 2 changed significantly, from a white solid to a yellow-brown oil, indicating that Comparative Example 2 has poor stability under high temperature conditions. Figure 6 .

[0197] In summary, the compound of the present invention has temperature, humidity and light stability, and is superior to Comparative Example 2.

[0198] A person skilled in the art may make various modifications and changes to the compounds, compositions and methods of the present invention without departing from the spirit of the present invention, all of which belong to the same or equivalent scope as the present invention.

Claims

1. A compound represented by the following formula or a pharmaceutically acceptable salt thereof: 。 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The pharmaceutically acceptable salts include fumarate, acetate, ascorbate, benzoate, benzenesulfonate, citrate, hydrochloride, hydrobromide, maleate, methanesulfonate, sulfate, bisulfate, nitrate, oxalate, phosphate or succinate.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound or its pharmaceutically acceptable salt is selected from: 。 4. A method for preparing a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: , wherein R1 is methyl and X is halogen.

5. Use of the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating heart, brain and other arterial circulatory disorders caused by platelet aggregation.

6. The use according to claim 5, characterized in that The heart, brain and other arterial circulatory disorders caused by platelet aggregation are selected from acute coronary syndrome, atherosclerotic disease, or thrombotic complications.

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