An oxypyridazine compound, its preparation method and use

Abstract

The present invention discloses a compound represented by formula (I), its stereoisomers, or a pharmaceutically acceptable salt. The present invention also provides the use of the compound, its stereoisomers or a pharmaceutically acceptable salt in the preparation of a medicament for treating and / or preventing diseases related to the FXIa receptor, especially in the preparation of a medicament for treating and / or preventing cerebrovascular arterial diseases and / or peripheral arterial diseases, with better efficacy and higher safety.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and particularly to oxopyridine compounds or their salts, isomers, and their preparation methods, as well as their use in the preparation of drugs for the treatment and / or prevention of diseases related to the FXIa receptor, especially their use in the preparation of drugs for the treatment of cerebrovascular arterial diseases and / or peripheral arterial diseases, etc. Background Art

[0002] Thromboembolic diseases are diseases caused by abnormal blood clots formed in blood vessels during the survival of humans and animals. Clinically, they may manifest as myocardial infarction, stroke, deep vein thrombosis (DVT), pulmonary embolism, atrial fibrillation, and cerebral infarction, etc., claiming the lives of tens of millions of people globally every year. Coagulation factor XI (FXI) is a plasma serine protease zymogen necessary to maintain the intrinsic pathway. After activation, it generates activated coagulation factor XIa (FXIa), which plays a key role in the amplification process of the coagulation cascade reaction. Therefore, drugs targeting the FXIa target can block the intrinsic pathway and inhibit the amplification of the coagulation cascade reaction, thus having an antithrombotic effect.

[0003] The reported FXIa inhibitors mainly include monoclonal antibodies, antisense oligonucleotides, small chemical molecules, polypeptides or proteins, and polypeptide mimetics, etc. Currently, milvexian jointly developed by BMS and Johnson & Johnson has completed clinical phase II trials, and the results show a relatively low bleeding risk. The clinical phase I trial of BMS's intravenous small molecule FXIa inhibitor BMS-962122 has been completed, and the R & D has been suspended. The small molecule oral FXIa inhibitor ONO-7684 developed by Ono Pharmaceutical Co., Ltd. in Japan has entered clinical phase I research. BAY-2433334 developed by Bayer has completed clinical phase II trials and has become the most promising small molecule FXIa inhibitor currently. Monoclonal antibodies and antisense oligonucleotides need to be administered by injection, and have the disadvantages of high price, slow onset, and possible difficulty in control. Small chemical molecules have the advantages of relatively good oral bioavailability and better patient compliance.

[0004] Therefore, the research and development of new small molecule FXIa inhibitors with high safety, effectiveness, good specificity, and strong activity may make up for the deficiencies of current clinical anticoagulant and antithrombotic drugs that are prone to bleeding complications and meet the unmet clinical needs. Summary of the Invention

[0005] The compounds of the present invention are a novel type of oxopyridine compounds. The example compounds of the present invention exhibit good anticoagulant effects, in vitro affinity for FXIa, and in vivo pharmacokinetics.

[0006] On the one hand, the present invention provides a compound represented by formula (I), its stereoisomers, or a pharmaceutically acceptable salt:

[0007]

[0008] Furthermore, the above-mentioned pharmaceutically acceptable salts are metal salts.

[0009] Furthermore, the above-mentioned metal salts are selected from sodium salts, potassium salts, calcium salts, lithium salts, and magnesium salts.

[0010] On the other hand, the present invention provides a pharmaceutical composition of the above-mentioned compound, its stereoisomer or pharmaceutically acceptable salt, and the composition further contains a pharmaceutically acceptable carrier and / or excipient.

[0011] In the third aspect, the present invention provides a preparation method of the above-mentioned compound, its stereoisomer or pharmaceutically acceptable salt, including the following route:

[0012] .

[0013] In the fourth aspect, the present invention further provides the use of any one of the above-mentioned compounds, its stereoisomers or pharmaceutically acceptable salts or compositions in the preparation of a drug for treating and / or preventing diseases related to the FⅪa receptor.

[0014] Furthermore, the diseases related to the FⅪa receptor are selected from thrombosis or thromboembolism-related disorders.

[0015] Furthermore, the diseases related to the FⅪa receptor are selected from cerebrovascular arterial diseases and / or peripheral arterial diseases.

[0016] Furthermore, the above-mentioned cerebrovascular arterial diseases include but are not limited to transient ischemic attack (TIA), ischemic stroke, or events originating from thrombosis and / or thromboembolism that lead to stroke or TIA; the above-mentioned peripheral arterial diseases include but are not limited to peripheral arterial occlusion, acute limb ischemia, amputation, re-occlusion and restenosis after intervention (such as angioplasty, stent implantation, or surgery and bypass), and / or stent thrombosis.

[0017] Furthermore, the above-mentioned ischemic stroke includes but is not limited to cardiogenic stroke, non-cardiogenic stroke, stroke caused by large artery or small artery diseases, stroke caused by undetermined causes, cryptogenic stroke, embolic stroke, or embolic stroke of undetermined origin.

[0018] Furthermore, the above-mentioned cardiogenic stroke includes but is not limited to stroke caused by atrial fibrillation; the above-mentioned non-cardiogenic stroke includes but is not limited to lacunar stroke.

[0019] Beneficial effects: Compared with the prior art, the present invention has good FXIa inhibitory effect and anti-human plasma coagulation effect. In addition, the pharmacokinetics in rats shows that it has higher AUC and Cmax, and faster metabolism, which can avoid the risk of increased bleeding due to long-term action. Detailed implementation manners

[0020] The present invention will be further described in detail below with reference to examples and experimental examples. The examples and experimental examples of the present invention are only used to illustrate the technical solutions of the present invention, and do not limit the present invention. Any equivalent substitution in the art made in accordance with the content disclosed in the present invention belongs to the protection scope of the present invention.

[0021] The structure of the compound was determined by nuclear magnetic resonance ( 1 H NMR) or liquid chromatography-mass spectrometry (LC-MS).

[0022] The liquid chromatography-mass spectrometer (LC-MS) was Agilent G6120B (used in conjunction with the liquid phase Agilent 1260); the nuclear magnetic resonance spectrometer ( 1 HNMR) was Bruker AVANCE-400 or Bruker AVANCE-800. The nuclear magnetic resonance ( 1 H NMR) chemical shift ( δ ) was given in parts per million (ppm) units, with the internal standard being tetramethylsilane (TMS), and the chemical shift was given in units of 10 -6 (ppm).

[0023] The term "room temperature" in the present invention refers to a temperature between 10 and 30 °C.

[0024] Example 1: Preparation of (S)-4-(2-(4-(5-chloro-2-(1H-tetrazol-1-yl)phenyl)-3-(methoxy-d3)-6-oxopyridazin-1(6H)-yl)butanamido)-2-fluoro-N-(methyl-d3)benzamide (Compound 1):

[0025]

[0026] Step 1: Preparation of intermediate b

[0027] Take 6-methoxypyridazin-3-ol (2 g, 15.8 mmol), dissolve it in 40 ml of DMF, add cesium carbonate (10.3 g, 31.6 mmol), cool to 0 °C, and dropwise add methyl iodide-d (3.5 g, 24.1 mmol). It takes about 30 minutes to add it all. After adding, stir the reaction at room temperature for 4 hours. Add EA and water, extract, wash with water, wash with saturated brine, dry over anhydrous sodium sulfate, filter, evaporate the solvent, and purify by column chromatography to obtain 2 g of intermediate b. Yield: 88.42%, HPLC purity: 98.81%.

[0028] ESI-MS: m / z = 144.1 (M+H) + 。

[0029] Step 2: Preparation of intermediate c

[0030] Take diisopropylamine (1.7 g, 16.8 mmol) and dissolve it in 20 ml of THF. Cool to below -60 °C, and dropwise add 6.4 ml of 2.5 M n-butyllithium in n-hexane solution. It takes about 1 hour to add it all. After adding, stir the reaction at -60 °C for 15 minutes. Dropwise add a 5 ml THF solution of compound b (2 g, 14.0 mmol), which takes about 1 hour. After adding, stir the reaction at -60 °C for 2 hours. Dropwise add triisopropyl borate (2.9 g, 15.4 mmol), which takes 30 minutes. After adding, slowly warm up to room temperature (20 °C) and stir the reaction for 30 minutes. Dropwise add a mixture of 3 g of acetic acid and 15 g of water to terminate the reaction. After adding, stir at room temperature for 30 minutes. Evaporate the organic solvent, add a little water, stir at room temperature for 15 minutes, filter, wash the filter cake with water, and dry it under vacuum at 70 °C to obtain 2.1 g of solid. Yield 80.22%, HPLC purity: 98.12%.

[0031] ESI-MS: m / z = 188.1 (M+H) + 。

[0032] Step 3: Preparation of intermediate e

[0033] Take compound d (2.59 g, 10.0 mmol) and Pd(amphos)Cl2 (107.5 mg, 0.15 mmol) and suspend them in 25 ml of tert-amyl alcohol. Heat to 85 °C, and dropwise add a mixed solution of compound c (2.22 g, 11.9 mmol), sodium carbonate (3.2 g, 30.2 mmol), and 25 ml of water. It takes about 1 hour. After adding, react at 85 °C for 1 hour. Cool to room temperature, add EA / water for extraction treatment, separate the aqueous layer, wash the organic layer successively with water and saturated brine, dry over anhydrous sodium sulfate, filter, and evaporate the solvent. Purify by column chromatography to obtain 2.4 g of intermediate e. Yield 74.59%, HPLC purity: 98.63%.

[0034] ESI-MS: m / z = 322.1 (M+H) + 。

[0035] Step 4: Preparation of Intermediate f

[0036] Take compound e (1.87 g, 5.80 mmol), anhydrous lithium chloride (1.3 g, 30.7 mmol), p-toluenesulfonic acid monohydrate (2.2 g, 11.6 mmol) and 20 ml of isopropanol, mix them, and heat under reflux for 16 hours. Cool to room temperature, distill off half of the solvent, add 30 ml of water, stir at room temperature for 15 minutes, filter, wash the filter cake with water, and dry it under vacuum at 70 °C to obtain 1.61 g of solid. Yield: 90.21%, HPLC purity: 96.43%.

[0037] ESI-MS: m / z = 308.1 (M+H) + 。

[0038] Step 5: Preparation of Intermediate h

[0039] Add compound f (523 mg, 1.70 mmol), tetramethylguanidine (681 mg, 5.91 mmol), 6 ml of isopropanol, and 1.5 ml of acetone to a 25 ml reaction flask, stir at room temperature for 15 minutes, add compound g (684 mg, 1.9 mmol), and stir at room temperature overnight. The next day, add water to terminate the reaction, extract with EA, separate the aqueous layer, wash the organic layer successively with saturated ammonium chloride, water, saturated brine, dry over anhydrous sodium sulfate, filter, evaporate the solvent, purify by column chromatography, collect the product, and obtain 832 mg of pure product. Yield 83.37%, purity 98.32%.

[0040] ESI-MS: m / z = 587.1(M+H) + 。

[0041] Step 6: Preparation of Intermediate j

[0042] Add compound h (587 mg, 1 mmol) and 8 ml of methanol to a 25 ml reaction flask, stir to dissolve, and cool to 0 °C. Weigh lithium hydroxide monohydrate (84 mg, 2 mmol) and dissolve it in 4 ml of water, add it dropwise to the reaction flask. After addition, react at room temperature for 2 h. Add water to terminate the reaction, adjust the pH to weakly acidic with 5% citric acid, extract with EA, separate the aqueous layer, wash the organic layer successively with water, saturated NaCl, dry over anhydrous sodium sulfate, filter, evaporate the solvent, perform column chromatography, and collect the target product to obtain 432 mg of intermediate j, with a yield of 81.36% and a purity of 98.30%.

[0043] ESI-MS: m / z = 531.1 (M+H) + 。

[0044] Step 7: Preparation of (S)-4-(2-(4-(5-chloro-2-(1H-tetrazol-1-yl)phenyl)-3-(methoxy-d3)-6-oxo-pyridazin-1(6H)-yl)butanamido)-2-fluoro-N-(methyl-d3)benzamide (Compound 1)

[0045] Add intermediate j (0.3 g, 0.57 mmol), 3 ml of DMF, deuterated methylamine hydrochloride (48.2 mg, 0.68 mmol), EDCI (218.7 mg, 1.14 mmol), and HOBT (154 mg, 1.14 mmol) to a 50 ml reaction flask. Cool the mixture to 0 °C and add DIPEA (295 mg, 2.28 mmol) dropwise. React at room temperature for 12 h until the raw materials are completely reacted. Add the reaction solution to cold water, extract twice with ethyl acetate, wash twice with water, wash with saturated brine, dry over anhydrous sodium sulfate, evaporate the solvent, and purify by silica gel column chromatography to obtain 210 mg of Compound 1, with a yield of 67.35% and a purity of 98.80%.

[0046] ESI-MS: m / z = 547.2 (M+H) + 。

[0047] 1 H NMR (400 MHz, DMSO-d6) δ: 10.76 (s, 1H), 9.12 (s, 1H), 8.10 (s, 1H), 7.90 – 7.76 (m, 3H), 7.71 – 7.60 (m, 2H), 7.37 (dd, J = 8.6, 2.0 Hz, 1H), 7.13 (s, 1H), 5.52 (s, 1H), 2.10 (d, J = 7.1 Hz, 2H), 0.79 (t, J = 7.2 Hz, 3H).

[0048] Comparative Example 1: Preparation of (S)-4-(2-(4-(5-chloro-2-(4-(trifluoromethyl)-1H-1,2,3-triazol-1-yl)phenyl)-5-methoxy-2-oxo-pyridin-1(2H)-yl)butanamido)-2-fluorobenzamide

[0049]

[0050] Synthesized according to the method described in Patent CN108026072B, purity: 98.5%.

[0051] ESI-MS: m / z = 593.1 (M+H) + 。

[0052] 1 1H NMR (400 MHz, DMSO-d6) δ: 10.78 (s, 1H), 9.14 (s, 1H), 7.88 – 7.77(m, 3H), 7.72 – 7.61 (m, 2H), 7.55 (d, 2H), 7.37 (dd, 1H), 7.13 (s, 1H), 6.54(s, 1H), 5.52 (dd, 1H), 3.25 (s, 3H), 2.18 – 2.00 (m, 2H), 0.78 (t, 3H).

[0053] Test Example 1: Inhibitory Effect on Coagulation Factor FXIa

[0054] 1. Test Samples

[0055] Compound 1 of the Example and Comparative Example 1.

[0056] 2. Test Procedures

[0057] 1) Prepare the experimental buffer (50 mM HEPES, 5 mM KCl, 145 mM NaCl, 1 mg / ml PEG8000, pH 7.4) and equilibrate to room temperature.

[0058] 2) Prepare a 10X compound working solution.

[0059] 3) Prepare a 0.8 nM Human FXIa working solution (2X) and mix well for later use.

[0060] 4) Add 20 μL of the FXIa working solution in step 3) to all experimental wells of a 384-well plate (Coring, 3702), centrifuge at 200 g, RT for 10 s.

[0061] 5) Add 4 μL of the compound working solution in step 2) to the corresponding experimental wells of the 384-well plate, centrifuge at 200 g, RT for 10 s, and then incubate the working plate at 25 °C for 20 min.

[0062] 6) Prepare a 750 μM S-2366 working solution (2.5X) and mix well for later use.

[0063] 7) Add 16 μL of the S-2366 working solution in step 6) to all experimental wells of the 384-well plate, centrifuge at 200 g, RT for 10 s, and then incubate the working plate at 37 °C for 45 min.

[0064] 8) After incubation, use EnVision to read the absorbance at OD405 nm and collect the data.

[0065] Set 5 concentrations, namely: 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, and detect the IC 50 value.

[0066] 3. Data analysis

[0067] 1) Z’ factor = 1 - 3*(SD Max +SD Min ) / (Mean Max -Mean Min );

[0068] 2) CV Max = (SD Max / Mean Max )*100%;

[0069] 3) CVMin = (SD Min / Mean Min )*100%;

[0070] 4) S / B = Singal / Background;

[0071] 5) Blank control: 0.1% DMSO; Positive control: Comparative Example 1;

[0072] 6) IC 50 calculation formula: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC 50 -X)*HillSlope)).

[0073] X: Compound concentration log value; Y: Inhibition%.

[0074] 4. Test results

[0075] The test results are shown in the following table. The results show that under the condition of the same molar concentration, the compound of the present invention has equivalent in vitro inhibitory activity against FXIa to the compound of Comparative Example 1.

[0076] 。

[0077] Test Example 2: Determination of anticoagulant effect in human plasma in vitro

[0078] 1. Test samples

[0079] Compound 1 of the Example and Comparative Example 1.

[0080] 2. Test method

[0081] Use a sodium citrate (1:9) anticoagulant tube. After collecting the blood of healthy individuals, immediately mix it well with the anticoagulant, and centrifuge it at 4000 r / min at room temperature for 15 minutes. After centrifugation, use a pipette to aspirate the plasma sample and store it frozen (-80°C) for later use.

[0082] Weigh an appropriate amount of the compound and prepare a 10 - 100 mM stock solution with 100% DMSO (the specific concentration of the stock solution is determined according to the properties of the compound); then prepare working solutions with different concentrations using healthy human plasma as the solvent (specifically: 0, 0.3, 1, 3, 10, 30, 60, 100, 300 μM), mix well; incubate at 37°C for 3 minutes and then load onto the machine (model CS - 2000I) for the determination of APTT.

[0083] 3. Data processing

[0084] Use GraphPad Prism software for curve fitting and calculate the EC150 value, that is, the effective concentration of the compound corresponding to a 50% prolongation of APTT relative to the blank control group.

[0085] 4. Test results

[0086] The results are shown in the following table. The APTT EC150 of the tested compound 1 is superior to that of the compound in Comparative Example 1.

[0087]

[0088] Experimental Example 3: Pharmacokinetics study in rats

[0089] 1. Test samples

[0090] Compound 1 of the example and Compound 1 of the comparative example.

[0091] 2. Preparation method of the test substance and environmental requirements

[0092] The preparation of the test substance is carried out on a conventional workbench in the preparation room.

[0093] Preparation of the stock solution of the established method: Prepare according to the dispensing procedure, and use methanol as the solvent to prepare a 1.00 mg / mL stock solution of Compound 1 of the example and Compound 1 of the comparative example respectively.

[0094] Preparation of the rat administration solution: Use 0.5% CMC - Na as the solvent for preparation. The oral administration concentration is 30 mg / mL.

[0095] 3. Test operation

[0096] (1)Administration and sample collection

[0097] The rats were fasted for 12 h before drug administration and had free access to water. A total of 12 Sprague-Dawley (SD) rats, half male and half female, were used in the experiment and divided into 2 groups of 6 rats each.

[0098] Blank blood samples were collected before drug administration, and blood samples were collected at predetermined time points after drug administration: 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 5 h, 7 h, 24 h, Approximately 0.5 mL of blood was collected and placed in an EDTA-K2 tube. The plasma was separated by centrifugation and stored at -80 °C.

[0099] (2)Animal Disposal

[0100] After the experiment, all animals were euthanized in accordance with the institutional SOP.

[0101] (3)Instruments

[0102] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis system, including the Shimadzu LC-20AD series binary pump, SIL-20AC autosampler, and AB API-4000 Q-Trap mass detector (including ESI ion source). Chromatographic column: ODS-C18 (4.6×50 mm, 3 µm).

[0103] (4)Sample Preparation

[0104] Standard curve sample preparation: A series of working solutions containing different concentrations of Comparative Example 1 and Compound 1 were prepared. 20 μL of the working solution was taken and added to 100 μL of blank plasma sample. After vortex mixing, 300 μL of acetonitrile solution containing 40 ng / mL propranolol internal standard was added, and vortex mixing was carried out again. The mixture was centrifuged at 4 °C and 14000 g for half an hour, and the supernatant was taken for LC-MS / MS detection.

[0105] Rat plasma sample preparation: 20 μL of acetonitrile was added to 100 μL of plasma sample. After vortex mixing, 300 μL of acetonitrile solution containing 40 ng / mL propranolol internal standard was added, and vortex mixing was carried out again. The mixture was centrifuged at 4 °C and 14000 g for half an hour, and the supernatant was taken for detection.

[0106] (5)Pharmacokinetic Analysis

[0107] Based on the plasma concentration data of the drug, the pharmacokinetic parameters were calculated using DAS 2.0 software.

[0108] 4. Results

[0109] The experimental results of the pharmacokinetic study of oral administration of the drug in rats are shown in the following table. Compared with Comparative Example 1, Compound 1 of the example had better absorption, significantly increased AUC (0-t) and Cmax, and a shorter half-life, showing pharmacokinetic characteristics of rapid elimination and a lower safety risk of potential bleeding.

[0110]

[0111] The above embodiments are only one of the preferred embodiments of the present invention and should not be used to limit the protection scope of the present invention. Any meaningless modifications or polishings made on the main design concept and spirit of the present invention, as long as the technical problems solved are still consistent with those of the present invention, shall be included in the protection scope of the present invention.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: 。 2. The compound or pharmaceutically acceptable salt according to claim 1, wherein, the salt is a metal salt.

3. The compound or pharmaceutically acceptable salt according to claim 2, wherein, the metal salt is selected from sodium salt, potassium salt, calcium salt, lithium salt or magnesium salt.

4. A pharmaceutical composition comprising the compound or pharmaceutically acceptable salt according to any one of claims 1 to 3, wherein, the composition further comprises a pharmaceutically acceptable carrier and / or excipient.

5. A method for preparing the compound or pharmaceutically acceptable salt according to any one of claims 1 to 3, wherein, the method comprises the following route: 。 6. Use of the compound or pharmaceutically acceptable salt according to any one of claims 1 to 3 or the composition according to claim 4 in the preparation of a medicament for treating and / or preventing a disease associated with the FⅪa receptor.

7. The use according to claim 6, wherein, the disease associated with the FⅪa receptor is selected from thrombosis or thromboembolism-related disorders.

8. The use according to claim 6, wherein, the disease associated with the FⅪa receptor is selected from cerebrovascular arterial diseases and / or peripheral arterial diseases.

9. The use according to claim 6, wherein, the disease associated with the FⅪa receptor is selected from transient ischemic attack, ischemic stroke, peripheral arterial occlusion or stent thrombosis.

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