WO2022083707A1 - FXIa抑制剂化合物或其盐的医药用途 - Google Patents

FXIa抑制剂化合物或其盐的医药用途 Download PDF

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WO2022083707A1
WO2022083707A1 PCT/CN2021/125450 CN2021125450W WO2022083707A1 WO 2022083707 A1 WO2022083707 A1 WO 2022083707A1 CN 2021125450 W CN2021125450 W CN 2021125450W WO 2022083707 A1 WO2022083707 A1 WO 2022083707A1
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salt
thrombosis
compound
use according
mmol
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PCT/CN2021/125450
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English (en)
French (fr)
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肖瑛
吴俊军
邢伟
王汝欢
黄伟鑫
陆银锁
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深圳信立泰药业股份有限公司
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Priority to CN202180030744.6A priority Critical patent/CN115427043B/zh
Publication of WO2022083707A1 publication Critical patent/WO2022083707A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/02—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings
    • C07D237/06—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D237/10—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D237/14—Oxygen atoms
    • C07D237/16—Two oxygen atoms

Definitions

  • the invention belongs to the technical field of medicine, and provides a series of medicinal uses of FXIa inhibitor compounds or salts thereof, in particular to their use in preparing medicines for preventing and/or treating arterial and venous thrombosis.
  • cardiovascular and cerebrovascular diseases such as cerebrovascular disease, cerebral infarction, myocardial infarction, coronary heart disease and arteriosclerosis kill nearly 12 million people in the world, which is close to 1/4 of the total number of deaths in the world, and has become the number one enemy of human health. More than 2.6 million people die of cardiovascular disease in China every year, and 75% of the surviving patients are disabled, of which more than 40% are severely disabled. The thrombosis caused by cardiovascular and cerebrovascular diseases and diabetes and its complications has become an urgent problem to be solved today.
  • the human blood coagulation process consists of intrinsic pathway, extrinsic pathway and common pathway (Annu.Rev.Med.2011.62:41–57), which is activated by the sequential activation of various zymogens. A chain reaction in which the process is continuously strengthened and amplified.
  • the coagulation cascade is initiated by the endogenous pathway (also known as the contact activation pathway) and the exogenous pathway (also known as the tissue factor pathway) to generate FXa, and then through the common pathway to generate thrombin (FIIa), and finally form fibrin.
  • the intrinsic pathway refers to the process in which factor XII is activated to form XIa-VIIIa-Ca 2+ -PL complex and activate factor X, while the extrinsic coagulation pathway is released from tissue factor (TF) to TF-VIIa-
  • the process by which Ca 2+ complexes form and activate factor X refers to the process of combining the two pathways into one after the formation of factor Xa, activating prothrombin and finally generating fibrin, in which FXI is necessary to maintain the endogenous pathway, and it is also involved in the amplification of the coagulation cascade. play a key role.
  • thrombin In the coagulation cascade reaction, thrombin can activate FXI feedback, and the activated FXI (FXIa) promotes the production of thrombin in a large amount, thereby amplifying the coagulation cascade reaction. Therefore, antagonists of FXI have been widely developed for the treatment of various thrombi.
  • FXIa is currently an emerging target for inhibiting thrombosis
  • patent applications for compounds with FXIa inhibitory activity are disclosed in WO9630396, WO9941276, WO2013093484, WO2004002405, WO2013056060, WO2017005725, WO2017/023992, WO2018041122, etc.
  • Bayer's antisense oligonucleotide BAY-2306001 has entered the Phase II clinical study.
  • the compounds of the present invention have higher activity.
  • the compound of the present invention exhibits excellent anticoagulant effect on human blood, and has good pharmacokinetic activity, and can be used for effective treatment and/or prevention of cardiovascular and cerebrovascular diseases and thrombosis symptoms.
  • the present invention provides a series of oxopyridazinamide derivatives used in medicine.
  • the pharmaceutical use of the FXIa inhibitor compound or its salt including the use of the FXIa inhibitor compound or its salt in the preparation of a drug for preventing and/or treating arterial thrombosis, venous thrombosis, and arteriovenous bypass thrombosis
  • the compound structural formula is as follows:
  • the salt structural formula of the FXIa inhibitor compound is as follows:
  • n 0.5-3;
  • M forms a salt with a carboxyl group, and the salt is selected from at least one of lithium salt, sodium salt, potassium salt, calcium salt, magnesium salt, aluminum salt, iron salt, zinc salt or ammonium salt; or the salt is selected from methyl Amine salt, dimethylamine salt, trimethylamine salt, ethylamine salt, diethylamine salt, triethylamine salt, isopropylamine salt, 2-ethylaminoethanol salt, pyridine salt, picoline salt, ethanolamine salt, diethanolamine salt, ammonium salt, tetramethylammonium salt, tetraethylammonium salt, triethanolamine salt, piperidine salt, piperazine salt, morpholine salt, lysine salt, arginine salt, L-arginine salt, Histidine, L-histidine, meglumine, dimethylglucamine, ethylglucamine, dicyclohexylamine, 1,6-hexanediamine, glucos
  • n is 0.5, 1, 1.5, 2, 2.5 or 3.
  • the salt is selected from the group consisting of sodium salt, potassium salt, meglumine salt, calcium salt, magnesium salt, and choline salt.
  • the compound or its salt is in crystalline form, or amorphous form, or a mixture thereof.
  • more than one hydrogen atom of the compound or its salt is substituted with the isotope deuterium.
  • the compound or its salt, and one or more pharmaceutically acceptable carriers form a pharmaceutical composition.
  • the arterial thrombus causes arterial embolic diseases, including coronary heart disease, myocardial infarction, ischemic stroke, peripheral arterial disease, atrial fibrillation and valvular heart disease;
  • the venous thrombus causes venous thrombosis Embolic disease, including deep vein thrombosis after joint replacement, pulmonary embolism, deep vein thrombosis; and arteriovenous thrombosis after dialysis.
  • deep venous thrombosis after joint replacement includes venous thrombosis after total knee replacement, venous thrombosis after hip replacement, and the like.
  • Atrial fibrillation is a common arrhythmia, which can aggravate myocardial ischemia, worsen cardiac function, and cause thrombosis. It has been identified as a risk factor for stroke and systemic embolic disease.
  • the mechanism of thrombosis in patients with atrial fibrillation is complex.
  • the existing evidence indicates that thrombosis in atrial fibrillation is related to underlying pathological changes: 1. Left atrial blood flow stasis 2. Damage to the integrity of the vessel wall 3. Abnormal blood components (coagulation factors and platelets) activation, etc.), these changes are fully consistent with Virchow's triad (three elements of venous thrombosis). Therefore, patients with atrial fibrillation are often accompanied by various types of thrombosis, such as deep vein thrombosis, arteriovenous bypass thrombosis, etc.
  • Human arteriovenous fistula has the advantages of sufficient blood flow, few complications, and long-term repeated use. It is the most commonly used vascular channel for hemodialysis patients. With the improvement of dialysis quality, the survival time of maintenance hemodialysis patients is prolonged, but repeated puncture damage to the vascular intima, coupled with improper compression and hemostasis, massive dehydration caused hypotension, slow blood flow, decreased blood flow in internal fistula and blood viscosity Increased thrombosis can promote thrombosis, so thrombosis diseases such as arteriovenous fistula thrombosis are common in dialysis patients.
  • animal experiments of the present invention show that the compounds of the present invention have a good preventive/therapeutic effect on venous thrombosis and arteriovenous bypass thrombosis in rabbits. Therefore, the compounds of the present invention are used for the treatment of arteriovenous thrombosis including: Thrombotic disease in patients with atrial fibrillation and renal dialysis.
  • the present invention further provides a pharmaceutical composition for preventing and/or treating arterial and venous thrombosis, comprising a compound of the following formula or a salt thereof, and one or more pharmaceutically acceptable carriers,
  • the compound of the present invention or a salt thereof, or a pharmaceutical composition containing the compound or a salt thereof has good activity on FXIa and high selectivity to other thrombin; has a significant effect of prolonging APTT, and has the strongest effect on human plasma; In the rabbit model of venous thrombosis, the efficacy level of rivaroxaban can be reached, and there is no risk of bleeding.
  • Salts of the compounds of the present invention are referred to as "pharmaceutically acceptable salts", which are prepared from compounds with specific substituents discovered in the present invention and a pharmaceutically acceptable acid or base.
  • Salts of certain compounds of the present invention may exist in unsolvated as well as solvated forms, including hydrated forms. In general, solvated and unsolvated forms are equivalent and are intended to be included within the scope of the present invention.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which belong to within the scope of the present invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • Optically active (R)- and (S)-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
  • a diastereomeric salt is formed with an appropriate optically active acid or base, followed by conventional methods known in the art
  • the diastereoisomers were resolved and the pure enantiomers recovered.
  • separation of enantiomers and diastereomers is usually accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (eg, from amines to amino groups) formate).
  • the atoms of the molecules of the compounds of the present invention are isotopes, and the isotope derivatization can usually prolong the half-life, reduce the clearance rate, stabilize the metabolism and improve the activity in vivo. Also, an embodiment is included in which at least one atom is replaced by an atom having the same atomic number (number of protons) and a different mass number (sum of protons and neutrons).
  • isotopes included in the compounds of the present invention include hydrogen atom, carbon atom, nitrogen atom, oxygen atom, phosphorus atom, sulfur atom, fluorine atom, chlorine atom, which respectively include 2 H, 3 H, 13 C, 14 C, 15 N, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl.
  • radioisotopes that emit radiation as they decay such as 3 H or 14 C, are useful in the topological examination of pharmaceutical formulations or compounds in vivo. Stable isotopes neither decay or change with their amount nor are they radioactive, so they are safe to use.
  • the isotopes can be converted according to general methods by substituting the reagents used in the synthesis with reagents containing the corresponding isotopes.
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound.
  • compounds can be labeled with radioisotopes, such as deuterium ( 2 H), iodine-125 ( 125 I) or C-14 ( 14 C). All transformations of the isotopic composition of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.
  • one or more hydrogen atoms of the compounds of the present invention are substituted by the isotope deuterium ( 2 H).
  • the compounds of the present invention After deuteration, the compounds of the present invention have the effects of prolonging half-life, reducing clearance rate, stabilizing metabolism and improving in vivo activity.
  • the preparation methods of the isotopic derivatives generally include: a phase transfer catalysis method.
  • a preferred deuteration method employs a phase transfer catalyst (eg, tetraalkylammonium salts, NBu4HSO4 ) .
  • the methylene protons of diphenylmethane compounds are exchanged using a phase transfer catalyst, resulting in higher ratios than with deuterated silanes (eg, triethyldeuterated silane) or with Lewis acids such as trichloro in the presence of an acid (eg, methanesulfonic acid) Aluminium is reduced with sodium deuteroborate to introduce higher deuterium.
  • pharmaceutically acceptable carrier refers to any formulation carrier or medium capable of delivering an effective amount of the active substance of the present invention, without interfering with the biological activity of the active substance, and without toxic side effects to the host or patient
  • representative carriers include water, oil , vegetables and minerals, cream base, lotion base, ointment base, etc. These bases include suspending agents, tackifiers, penetration enhancers, and the like.
  • Their formulations are well known to those skilled in the cosmetic or topical pharmaceutical field. For additional information on carriers, reference can be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.
  • excipient generally refers to the carrier, diluent and/or medium required to formulate an effective pharmaceutical composition.
  • an "effective amount” or “therapeutically effective amount” with respect to a drug or pharmacologically active agent refers to a nontoxic but sufficient amount of the drug or agent to achieve the desired effect.
  • an "effective amount” of one active substance in a composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition.
  • the determination of the effective amount varies from person to person, depends on the age and general condition of the recipient, and also depends on the specific active substance, and the appropriate effective amount in individual cases can be determined by those skilled in the art based on routine experiments.
  • treatment refers to a chemical entity that is effective in treating the target disorder, disease or condition.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by their combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalent to alternatives, preferred embodiments include, but are not limited to, the embodiments of the present invention.
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • the MS was measured using an ISQ EC mass spectrometer (manufacturer: Thermo, model: ISQ EC).
  • HPLC High performance liquid chromatography
  • CombiFlash rapid preparation instrument uses CombiFlash Rf+LUMEN (TELEDYNE ISCO).
  • the thin layer chromatography silica gel plate uses Yantai Yinlong HSGF254 or GF254 silica gel plate, the size of the silica gel plate used for thin layer chromatography (TLC) is 0.17mm ⁇ 0.23mm, and the size of the TLC separation and purification products is 0.4mm ⁇ 0.5mm.
  • Silica gel column chromatography generally uses Rushan Shangbang silica gel 100-200 mesh silica gel as the carrier.
  • Step B Synthesis of 5-bromo-6-methoxy-2-(4-methoxybenzyl)pyridazin-3(2H)-one
  • 2-bromo-4-chloroacetophenone (5.00 g, 21.41 mmol), pinacol biboronate (8.16 g, 32.12 mmol) and potassium acetate (4.20 g, 42.82 mmol) were added to three In the neck flask, nitrogen was replaced, 1,4-dioxane (60.0 mL) was added, nitrogen was replaced, and 1,1'-bisdiphenylphosphinoferrocene palladium dichloride (1.75 g, 2.14 mmol) was added. , replaced nitrogen, and heated to 80 °C for 3 hours.
  • Step D Synthesis of 5-(2-Acetyl-5-chlorophenyl)-6-methoxy-2-(4-methoxybenzyl)pyridazin-3(2H)-one
  • Step F Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- 3-Phenylpropionamido) tert-butyl benzoate
  • Step G Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- 3-Phenylpropionamido)benzoic acid
  • 2-Bromo-4-chloroaniline (3.1 g, 14.5 mmol) was added to 2-bromo-4-chloroaniline (3.0 g, 15.0 mmol), 4,4,5,5-tetramethyl-2-( Tetramethyl-1,3,2-dioxaborol-2-yl)-1,3,2-dioxaborolane (38 g, 150.0 mmol), potassium acetate ( 2.9 g, 30.0 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (1.1 g, 1.5 mmol) was dissolved in dimethyl sulfoxide (75 ml).
  • Step C Synthesis of 4- ⁇ 5-Chloro-2-[4-(trimethylsilyl)-1H-1,2,3-triazol-1-yl]-phenyl ⁇ -6-methoxy -Pyrimidine
  • Step D Synthesis of 4-[5-Chloro-2-(4-chloro-1H-1,2,3-triazol-1-yl)phenyl]-6-methoxypyrimidine
  • Step E Synthesis of 6-[5-Chloro-2-(4-chloro-1H-1,2,3-triazol-1-yl)phenyl]pyrimidin-4-ol
  • Step F Synthesis of (S)-4-(2-(4-(5-chloro-2-(4-chloro-1H-1,2,3-triazol-1-yl)phenyl)-6-oxo tert-Butyl pyrimidin-1(6H)-yl)-3-phenylpropionamido)benzoate
  • Step F Synthesis of (S)-4-(2-(4-(5-chloro-2-(4-chloro-1H-1,2,3-triazol-1-yl)phenyl)-6-oxo Pyrimidine-1(6H)-yl)-3-phenylpropionamido)benzoic acid
  • Step B Synthesis of 1-(4-Chloro-2-(2,5-dimethoxypyridin-4-yl)phenyl)ethan-1-one
  • Step D Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-5-methoxy-2-oxopyridinium-1(2H)-yl)-3 -Phenylpropionamido) tert-butyl benzoate
  • Step E Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-5-methoxy-2-oxopyridinium-1(2H)-yl)-3 -Phenylpropionamido)benzoic acid
  • Example 8 Detection of the biological activity of the compounds of the present invention on the inhibition of human coagulation factor XIa by absorptiometry
  • Enzyme Human Factor XIa (ENZYME RESEARCH, Cat. No. HFXIa 1111a)
  • Buffer 145mM NaCl, 5mM KCl, 1mg/mL PEG 8000, 30mM HEPES, pH7.4
  • 10 mM test compound in 100% DMSO was diluted with 100% DMSO to 1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.00256, 0.00128 ⁇ M; /mL) of FXIa enzyme solution, the blank wells were replaced by 98 ⁇ L of buffer, and then 2 ⁇ L of compounds of different concentrations were added. The blank and control wells were replaced by DMSO, mixed with a shaker, and incubated at 37°C for 20 min.
  • Example 9 Determination of the anticoagulant effect of the compounds of the present invention on human plasma in vitro
  • Plasma Human blood was collected in a vacuum blood collection tube containing 3.2% sodium citrate (volume ratio 1:9), centrifuged at 3000 rpm for 10 min at room temperature, plasma was collected, packaged in EP tubes, and stored at -80°C.
  • APTT assay kit activated partial thromboplastin time assay kit, mindray
  • calcium chloride solution calcium chloride solution
  • SD rats male, 180-250 g, purchased from Guangdong Provincial Medical Laboratory Animal Center, Beijing Weitonglihua.
  • Cynomolgus monkey male, 4-6kg, purchased from Guangzhou Chunsheng Biological Research Institute Co., Ltd.
  • Beagle male, 8-12kg, developed in Kanglong Chemical (Ningbo) New Drug Technology Co., Ltd.
  • DMSO dimethyl sulfoxide
  • PEG-400 polyethylene glycol 400
  • physiological saline physiological saline
  • heparin acetonitrile
  • formic acid formic acid
  • propranolol internal standard
  • the compound was weighed and dissolved in DMSO-PEG-400-physiological saline (5:60:35, v/v/v) system, after intravenous or intragastric administration to rats/monkeys, 5min (gastrically not collected), After 15min, 30min, 1h, 2h, 4h, 6h, 8h, and 24h, 200 ⁇ L of venous blood was collected in a heparinized EP tube, centrifuged at 12000 rpm for 2 min, and the plasma was frozen at -80°C for testing. Precisely weigh a certain amount of the test sample and dissolve it in DMSO to 1 mg/mL as a stock solution.
  • Chromatographic column Thermo Scientific HYPERSIL GOLD C-18 UPLC column, 100*2.1mm, 1.9 ⁇ m.
  • WinNonlin 6.1 software was used to calculate pharmacokinetic parameters by non-compartmental model method. The results are shown in Tables 3, 4 and 5.
  • the compounds of the present invention have certain absorption in rats and monkeys orally.
  • the oral absorption in dogs is good, and the clearance rate in vivo is moderately slow.
  • Most of the compounds have a long oral half-life and have good pharmacokinetic characteristics.
  • Test method The rabbits were fasted for 24 hours, and they were divided into model control group, compound 30 mg/kg/h (calculated as free acid) dose group of Example 4, and positive drug 1 Rivaroxaban dose of 3 mg/kg/h according to their body weight.
  • Group and positive drug 2 Enoxaparin sodium 20mg/kg/h dose group were administered by intravenous infusion once, at a rate of 1.4ml/kg/h for 2 hours, and the model control group was given an equal volume of vehicle.
  • the plastic tubes are placed parallel to the blood vessels and ligated together. After ligation, the plastic tubes are slowly drawn out), and each branch vein at this interval is ligated.
  • a needle electrode was placed on the endothelium of the inferior vena cava in the ligated segment, and a current of 1.5 mA was used to stimulate it for 30 minutes.
  • the artery clip was used to clamp the stenosis and block the vein. After the electrical stimulation was over, the arterial clips were released, and the rabbits were placed for 30 min after the electrical stimulation stopped.
  • the thrombus inhibition rate of the compound of Example 4 at 30 mg/kg/h was 54.3%; rivaroxaban at 3 mg/kg /h thrombus inhibition rate was 59.8%; enoxaparin 20mg/kg/h thrombus inhibition rate was 54.4%, the test results are shown in Table 6 below.
  • Example 4 compound rabbit inferior vena cava thrombosis results
  • Test method The rabbits were fasted for 24 hours and were divided into model control group, positive drug clopidogrel 3mg/kg/h and 10mg/kg/h dose groups, Example 2 compound 10mg/kg/h (calculated as free acid) ) dose group, 3 rabbits in each group.
  • the rabbits were weighed before administration, 40% urethane 1.5mL/kg was anesthetized in the marginal ear vein, fixed, and the right femoral artery, vein and carotid artery were surgically separated. After administration for 2h, the infusion volume was 1.4mL/kg/h, and the model control group was given an equal volume of vehicle. After intravenous infusion for 1 hour, start modeling: add 20 ⁇ L of 50% FeCl3 solution on 10mm*10mm filter paper, apply it around the carotid artery for 5 minutes, remove the filter paper, let it stand for 55 minutes, cut the carotid artery to separate the thrombus and weigh it. .
  • Test results The clopidogrel single-agent 3 mg/kg/h dose group had a thrombus inhibition rate of about 49.1%, and the renal bleeding time had no significant change. When the thrombus inhibition rate reached 72.7% in the 10 mg/kg/h dose group, the renal bleeding time was significantly prolonged. . The thrombus inhibition rate in the 10 mg/kg/h dose group of the compound of Example 2 was about 47.9%, and the renal bleeding time did not change significantly. The test results are shown in Table 7.
  • Clopidogrel has a good effect on carotid artery thrombosis in rabbits, and there is an obvious dose-effect relationship. With the increase of the dose of clopidogrel, the renal bleeding time will be significantly longer. When the compound of Example 2 was administered at 10 mg/kg/h, it showed a certain effect of preventing thrombosis, while reducing the risk of bleeding.
  • Test method The rabbits were divided into a model control group and a 30 mg/kg/h dose group of the compound of Example 2 according to their body weight.
  • the arteriovenous bypass device was connected, the device was filled with normal saline, and the two ends were connected to the right carotid artery and the left jugular vein, and left for 15 minutes.
  • the rabbits were anesthetized with 40 mg/kg sodium pentobarbital injection into the ear vein, supine and fixed, the neck skin was prepared and disinfected, the neck skin was cut with surgical scissors, the right carotid artery and left jugular vein were separated, and firstly ligated At the distal end of the right carotid artery, the proximal end was clamped with a vascular clip, the blood vessel was cut close to the inner side of the distal end ligature, the arteriovenous bypass device filled with normal saline was connected, and two silk non-absorbable sutures were ligated and fixed.
  • the vascular clip is not opened temporarily; the left jugular vein is connected to the arteriovenous bypass device in the same way, ligated and fixed with two silk non-absorbable sutures, and the vascular clip is not opened temporarily.
  • the vascular clip is not opened temporarily.
  • After the end of the infusion time first open the venous vascular clip of the arteriovenous bypass device, and then open the arterial vascular clip for 15 minutes.
  • the arteriovenous bypass device was removed, and the weight of the thrombus was calculated by the decrement method.
  • the renal bleeding time was detected. Using a 1ml disposable syringe needle, pierce a needle hole with a depth of 3mm on the surface of the kidney from which the renal capsule was peeled off, and measure the bleeding time.
  • Test results The compound of Example 2 at a dose of 30 mg/kg/h can significantly reduce the weight of thrombus in rabbit arteriovenous bypass, and the thrombus inhibition rate is 61.9%.
  • the test results are shown in Table 8.
  • the compound of Example 2 has a good effect of preventing thrombosis in the rabbit arteriovenous bypass thrombosis model, and the compound of Example 2 still does not significantly increase the renal bleeding time, indicating that the compound of Example 2 can achieve better efficacy. There is less bleeding risk at the level.

Abstract

提供了一种FXIa抑制剂化合物或其盐的医药用途，具体涉及其在制备预防和/或治疗动脉和静脉血栓药物中的用途。

Description

FXIa抑制剂化合物或其盐的医药用途 技术领域
本发明属于药物技术领域，提供了一系列的FXIa抑制剂化合物或其盐的医药用途，具体涉及其在制备预防和/或治疗动脉和静脉血栓药物中的用途。
背景技术
全球每年脑血管、脑梗塞、心肌梗塞、冠心病、动脉硬化等心脑血管疾病夺走近1200万人的生命，接近世界总死亡人数的1/4，成为人类健康的头号大敌。中国每年死于心血管疾病的人数达到260万人以上，存活的患者75％致残，其中40％以上重残。由心脑血管疾病和糖尿病及其并发症引起的血栓问题，成为当今要解决的刻不容缓的问题。
人体血液凝固过程由内源性途径(intrinsic pathway)、外源性途径(extrinsic pathway)和共同通路组成(Annu.Rev.Med.2011.62:41–57)，是通过多种酶原被顺序激活而过程不断得到加强和放大的一种连锁反应。凝血级联反应由内源性途径(又称接触激活途径)及外源性途径(又称组织因子途径)启动生成FXa，再经共同途径生成凝血酶(FIIa)，最终形成纤维蛋白。
内源性途径是指由XII因子被激活形XIa-VIIIa-Ca 2+-P L复合物、并激活X因子的过程,外源性凝血途径则是从组织因子(TF)释放到TF-VIIa-Ca 2+复合物形成并激活因子Ⅹ的过程。共同通路是指因子Xa形成后,两条途径合二为一,激活凝血酶原并最终生成纤维蛋白的过程，其中FXI是维持内源性途径所必需的，而且在凝血级联反应放大过程中发挥关键作用。在凝血级联反应中，凝血酶可反馈激活FXI，活化的FXI(FXIa)又促使凝血酶的大量产生，从而使凝血级联反应放大。因此，FXI的拮抗剂被广泛开发，用于各种血栓的治疗。
传统的抗凝药物，如华法林、肝素、低分子量肝素(LMWH)，以及近年上市的新药，如FXa抑制剂(利伐沙班、阿哌沙班等)和凝血酶抑制剂(达比加群酯、水蛭素等)，对减少血栓形成均具有较好效果，以其显著有效性占据广大心脑血管市场，然而其副作用也越来越显著，其中“出血风险(bleeding risk)”是首当其冲最为严峻的问题之一(N Engl J Med 1991；325:153-8、Blood.2003；101:4783-4788)。
研究发现，在血栓模型中，抑制FXIa因子可以有效抑制血栓的形成，但在更为严重的血栓情况下，FXIa的作用微乎其微(Blood.2010；116(19):3981-3989)。临床统计显示，提高FXIa的量会增加VTE的患病率(Blood 2009；114:2878-2883)，而FXIa严重不足者其患有DVT的风险性减少(Thromb Haemost 2011；105:269–273)。
FXIa作为目前抑制血栓的新兴靶点，公开具有FXIa抑制活性的化合物的专利申请有WO9630396、WO9941276、WO2013093484、WO2004002405、WO2013056060、WO2017005725、WO2017/023992、WO2018041122等。其中，目前仅拜耳公司的反义寡核苷酸BAY-2306001进入了临床二期研究。
本发明化合物具有更高的活性。特别是本发明化合物表现出优异的对人血液的抗凝血作用，并具有良好的药代活性，可用于有效治疗和/或预防心脑血管疾病及血栓症状。
发明内容
本发明提供了一系列的氧代哒嗪酰胺类衍生物在医药上的应用。
具体而言，FXIa抑制剂化合物或其盐的医药用途，所述用途包括所述在FXIa抑制剂化合物或其盐制备预防和/或治疗动脉血栓、静脉血栓、动静脉旁路血栓药物中的用途药物中的用途，所述化合物结构式如下：
Figure PCTCN2021125450-appb-000001
作为本发明的一种实施方案，所述FXIa抑制剂化合物的盐结构式如下：
Figure PCTCN2021125450-appb-000002
其中：
n为0.5-3；
M与羧基成盐，所述盐选自锂盐、钠盐、钾盐、钙盐、镁盐、铝盐、铁盐、锌盐或铵盐中的至少一种；或所述盐选自甲胺盐、二甲胺盐、三甲胺盐、乙胺盐、二乙胺盐、三乙胺盐、异丙胺盐、2-乙氨基乙醇盐、吡啶盐、甲基吡啶盐、乙醇胺盐、二乙醇胺盐、铵盐、四甲基铵盐、四乙基铵盐、三乙醇胺盐、哌啶盐、哌嗪盐、吗啉盐、赖氨酸盐、精氨酸盐、L-精氨酸盐、组氨酸盐、L-组氨酸盐、葡甲胺盐、二甲基葡糖胺盐、乙基葡糖胺盐、二环己基胺盐、1,6-己二胺盐、葡糖胺盐、肌氨酸盐、丝氨醇盐、三羟基甲基氨基甲烷盐、氨基丙二醇盐、1-氨基-2,3,4-丁三醇盐、L-赖氨酸盐、鸟氨酸盐或胆碱盐中的至少一种。
作为本发明的一种实施方案，n为0.5、1、1.5、2、2.5或3。
作为本发明的一种实施方案，所述的盐选自钠盐、钾盐、葡甲胺盐、钙盐、镁盐、胆碱盐。
作为本发明的一种实施方案，所述的盐选自钠盐、n＝1；钾盐、n＝1；胆碱盐、n＝1；葡甲胺盐、n＝1；钙盐、n＝0.5；镁盐、n＝0.5。
作为本发明的一种实施方案，所述化合物或其盐为晶型、或者无定型，或其混合物。
作为本发明的一种实施方案，所述化合物或其盐的一个以上的氢原子上被同位素氘取代。
作为本发明的一种实施方案，所述化合物或其盐，和一种以上药学上可接受的载体组成药物组合物。
作为本发明的一种实施方案，所述动脉血栓导致动脉栓塞性疾病，包括冠心病、心肌梗死、缺血性脑卒中、外周动脉疾病、房颤和心瓣膜病；所述静脉血栓导致静脉血栓栓塞性疾病，包括关节置换术后深静脉血栓，肺栓塞，深静脉血栓形成；以及透析后的动静脉血栓。
作为本发明的一种实施方案，关节置换术后深静脉血栓包括全膝关节置换术后静脉血栓，髋关节置换术后静脉血栓等。
心房颤动为一种较常见的心律失常,可加重心肌缺血及恶化心功能,并引起血栓形成,已确定为脑卒中及全身性栓塞性疾病的危险因素。房颤患者中血栓形成机制较为复杂，现有证据表明房颤中血栓形成和潜在病理改变相关：1.左心房血流瘀滞2.血管壁完整性破坏3.血液成分异常(凝血因子和血小板激活等)，这些改变完全符合Virchow’s三联征(静脉血栓形成三要素)。所以，房颤患者常伴随有多种类型的血栓，如深静脉血栓、动静脉旁路血栓等。
人体动静脉内瘘具有血流量充足、并发症少、能长期反复使用的优点，是血液透析患者最常用的血管通道。随着透析质量的提高，维持性血液透析患者的生存时间延长，但反复穿刺损伤血管内膜，再加上压迫止血不当、大量脱水致低血压、血流缓慢、内瘘血流量下降及血液黏度增加等可促进血栓形成，所以，透析患者常见动静脉内瘘血栓等血栓疾病。
作为本发明的一种实施方案，本发明动物实验表明本发明化合物对家兔静脉血栓和动静脉旁路血栓均有很好预防/治疗效果，所以，本发明化合物用于动静脉血栓的治疗包括房颤患者和肾透析患者的血栓疾病。
本发明进一步提供了一种预防和/或治疗动脉、静脉血栓的药物组合物，含有下式化合物或其盐，和一种以上药学上可接受的载体，
Figure PCTCN2021125450-appb-000003
本发明化合物或其盐、或含有所述化合物或其盐的药物组合物，对FXIa活性较好，对其他凝血酶选择性高；具有显著的延长APTT的效果，对人血浆的作用最强；对家兔静脉血栓模型可达到利伐沙班药效水平，且无出血风险。
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
本发明化合物的盐是指“药学上可接受的盐”，由本发明发现的具有特定取代基的化合物与药学上可接受的酸或碱制备。
本发明的某些化合物的盐可以以非溶剂化形式或者溶剂化形式存在，包括水合物形式。一般而言，溶剂化形式与非溶剂化的形式相当，都包含在本发明的范围之内。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体，以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本发明化合物分子的原子是同位素，通过同位素衍生化通常可以延长半衰期、降低清除率、代谢稳定和提高体内活性等效果。并且，包括一个实施方案，其中至少一个原子被具有相同原子数(质子数)和不同质量数(质子和中子和)的原子取代。本发明化合物中包括的同位素的实例包括氢原子、碳原子、氮原子、氧原子、磷原子、硫原子、氟原子、氯原子，其分别包括 2H、 3H、 13C、 14C、 15N、 17O、 18O、 31P、 32P、 35S、 18F、 36Cl。特别的是，随其衰退而发射辐射的放射性同位素例如 3H或 14C可用于药物制剂或者体内化合物的局部解剖学检验。稳定的同位素既不随其量衰减或变化，也不具有放射性，因此其可以安全使用。当构成本发明化合物分子的原子是同位素时，通过用包含相应同位素的试剂替代合成中所用的试剂，可以根据通用方法转化同位素。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氘( 2H)，碘-125( 125I)或C-14( 14C)。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
进一步地，本发明的化合物一个或多个氢原子上被同位素氘( 2H)取代，本发明化合物氘代后，具有延长半衰期、降低清除率、代谢稳定和提高体内活性等效果。
所述同位素衍生物的制备方法通常包括：相转移催化方法。例如，优选的氘化方法采用相转移催化剂(例如，四烷基铵盐，NBu 4HSO 4)。使用相转移催化剂交换二苯基甲烷化合物的亚甲基质子，导致比在酸(例如，甲磺酸)存在下用氘化硅烷(例如三乙基氘化甲硅烷)或用路易斯酸如三氯化铝采用氘化硼酸钠还原而引入较高的氘。
术语“药学上可接受的载体”是指能够递送本发明有效量活性物质、不干扰活性物质的生物活性并且对宿主或者患者无毒副作用的任何制剂载体或介质，代表性的载体包括水、油、蔬菜和矿物质、膏基、洗剂基质、软膏基质等。这些基质包括悬浮剂、增粘剂、透皮促进剂等。它们的制剂为化妆品领域或局部药物领域的技术人员所周知。关于载体的其他信息，可以参考Remington:The Science and Practice of Pharmacy,21st Ed.,Lippincott,Williams&Wilkins(2005)，该文献的内容通过引用的方式并入本文。
术语“赋形剂”通常是指配制有效的药物组合物所需要载体、稀释剂和/或介质。
针对药物或药理学活性剂而言，术语“有效量”或“治疗有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。对于本发明中的口服剂型，组合物中一种活性物质的“有效量”是指与该组合物中另一种活性物质联用时为了达到预期效果所需要的用量。有效量的确定因人而异，取决于受体的年龄和一般情况，也取决于具体的活性物质，个案中合适的有效量可以由本领域技术人员根据常规试验确定。
术语“治疗”是指一种化学实体，它可以有效地治疗目标紊乱、疾病或病症。
“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
具体实施方式
下面结合实施例对本发明作进一步详细的描述，但发明的实施方式不限于此。
化合物的结构是通过核磁共振(NMR)或质谱(MS)来确定的。NMR位移(δ)以10-6(ppm)的单位给出。NMR的测定是用Bruker AVANCE-III核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d6)，氘代氯仿(CDCl3)，内标为四甲基硅烷(TMS)。
MS的测定用ISQ EC质谱仪(生产商:Thermo,型号:ISQ EC)。
高效液相色谱法(HPLC)分析使用Thermo U3000 HPLC DAD高效液相色谱仪。
CombiFlash快速制备仪使用CombiFlash Rf+LUMEN(TELEDYNE ISCO)。
薄层层析硅胶板使用烟台银龙HSGF254或GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.17mm～0.23mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm。
硅胶柱色谱法一般使用乳山上邦硅胶100～200目硅胶为载体。
实施例1
合成(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸
Figure PCTCN2021125450-appb-000004
具体合成路线如下：
步骤A：合成5-溴-6-羟基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮
Figure PCTCN2021125450-appb-000005
室温下，将溴马来酸酐(2.00克，11.3毫摩尔)和4-甲氧基苄基肼盐酸盐(2..13克，11.3毫摩尔)加入冰醋酸(50.0毫升)中，100℃反应3小时。
反应结束，冷却至室温，将反应液倒入水中，析出大量固体，搅拌一段时间后抽滤，滤饼用水洗，滤饼烘干得1.50克淡黄色固体5-溴-6-羟基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮，无需纯化，直接用于下步反应。LCMS:RT＝3.44min,[M+H] +＝311.03。
步骤B：合成5-溴-6-甲氧基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮
Figure PCTCN2021125450-appb-000006
室温下，将5-溴-6-羟基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮(1.50克，4.82毫摩尔)和碳酸钾(2.66克，19.29毫摩尔) 加入N,N-二甲基甲酰胺(15.0毫升)中，80℃搅拌15分钟，在该温度下，加入碘甲烷(1.2毫升)，继续反应30分钟。
反应结束，加水淬灭，混合液用乙酸乙酯(50毫升×3次)萃取，合并有机相，有机相先用饱和食盐水(50毫升×2次)，然后用无水硫酸钠干燥，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/正己烷＝1/3)。得到1.10克白色固体5-溴-6-甲氧基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮(收率：70.3％)。LCMS:RT＝3.87min,[M+H] +＝325.01。
步骤C：合成6-乙酰基-3-氯苯硼酸频哪醇酯
Figure PCTCN2021125450-appb-000007
室温下，将2-溴-4-氯苯乙酮(5.00克，21.41毫摩尔)、联硼酸频哪醇酯(8.16克，32.12毫摩尔)和醋酸钾(4.20克，42.82毫摩尔)加入三颈瓶中，置换氮气，加入1,4-二氧六环(60.0毫升)，置换氮气，加入1,1'-双二苯基膦二茂铁二氯化钯(1.75克，2.14毫摩尔)，置换氮气，升温至80℃反应3小时。
反应结束，加水淬灭，垫硅藻土抽滤，乙酸乙酯洗涤滤饼，滤液用乙酸乙酯(80毫升×3次)萃取，合并有机相，有机相先用饱和食盐水(50毫升×2次)，然后用无水硫酸钠干燥，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/正己烷＝1/50)。得到2.1克黄色固体6-乙酰基-3-氯苯硼酸频哪醇酯(收率：35.0％)。LCMS:RT＝4.26min,[M-H] -＝279.08。
步骤D：合成5-(2-乙酰基-5-氯苯基)-6-甲氧基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮
Figure PCTCN2021125450-appb-000008
室温下，将5-溴-6-甲氧基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮(1.10克，3.39毫摩尔)、6-乙酰基-3-氯苯硼酸频哪醇酯(949毫克，3.39毫摩尔)和碳酸钠(718毫克，6.78毫摩尔)加入三颈瓶中，置换氮气，加入混合溶剂(10毫升，1,2-二甲氧基乙烷:乙醇:水＝8:1:1)，置换氮气，加入1,1'-双二苯基膦二茂铁二氯化钯(249毫克，0.34毫摩尔)，置换氮气，升温至90℃反应1小时。
反应结束，加水淬灭，混合液用乙酸乙酯(50毫升×3次)萃取，合并有机相，有机相先用饱和食盐水(50毫升×2次)，然后用无水硫酸钠干燥，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/正己烷＝1/2)。得到676毫克黄色固体5-(2-乙酰基-5-氯苯基)-6-甲氧基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮(收率：50.2％)。LCMS:RT＝3.99min,[M+H] +＝399.07。
步骤E：合成5-(2-乙酰基-5-氯苯基)-6-甲氧基哒嗪-3(2H)-酮
Figure PCTCN2021125450-appb-000009
0℃下，将5-(2-乙酰基-5-氯苯基)-6-甲氧基-2-(4-甲氧基苄基)哒嗪-3(2H)-酮(676毫克，1.70毫摩尔)加入混合溶剂(4毫升，乙腈:水＝3:1)中，再缓慢加入硝酸铈铵(7.46克，13.60毫摩尔)，加毕，室温下反应30分钟。
反应结束，加水淬灭，混合液用乙酸乙酯(30毫升×3次)萃取，合并有机相，有机相先用饱和食盐水(30毫升×2次)，然后用无水硫酸钠干燥，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/正己烷＝1/1)。得到238毫克黄色固体5-(2-乙酰基-5-氯苯基)-6-甲氧基哒嗪-3(2H)-酮(收率：50.0％)。LCMS:RT＝3.23min,[M+H] +＝279.08。
步骤F：合成(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸叔丁酯
Figure PCTCN2021125450-appb-000010
室温下，将5-(2-乙酰基-5-氯苯基)-6-甲氧基哒嗪-3(2H)-酮(50毫克，0.18毫摩尔)、(R)-4-(2-(((4-硝基苯基)磺酰基)氧基)-3-苯基丙酰胺基)苯甲酸叔丁酯(113毫克，0.22毫摩尔)和碳酸钾(50毫克，0.36毫摩尔)加入N,N-二甲基甲酰胺(2.0毫升)中，室温反应过夜。
反应结束，加水淬灭，混合液用乙酸乙酯(10毫升×3次)萃取，合并有机相，有机相先用饱和食盐水(10毫升×2次)，然后用无水硫酸钠干燥，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/正己烷＝1/2)。得到75毫克淡黄色固体(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸叔丁酯(收率：66.7％)。LCMS:RT＝4.53min,[M+H] +＝602.13。
步骤G：合成(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸
Figure PCTCN2021125450-appb-000011
室温下，将(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸叔丁酯(75毫克，0.12毫摩尔)加入二氯甲烷(2.0毫升)中，滴加三氟乙酸(0.25毫升)，室温反应3小时。
反应结束，蒸干二氯甲烷并用油泵抽干三氟乙酸，所得残余物用溶于二氯甲烷(1.0毫升)中，将其滴加入正己烷(10.0毫升)中，析出白色固体，抽滤，滤饼用正己烷洗涤，干燥得到50毫克白色固体(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸(收率：76.5％)。LCMS:RT＝3.98min,[M-H] -＝544.10。 1H NMR(500MHz,DMSO)δ12.79(s,1H),10.52(s,1H),7.99(d,J＝8.4Hz,1H),7.91(d,J＝8.7Hz,2H),7.72(d,J＝8.7Hz,2H),7.69(dd,J＝8.3,2.1Hz,1H),7.50(d,J＝2.1Hz,1H),7.37–7.23(m,4H),7.19(t,J＝7.1Hz,1H),6.91(s,1H),5.74(dd,J＝10.2,4.9Hz,1H),3.67(s,3H),3.52(dd,J＝14.1,10.3Hz,1H),3.41(dd,J＝14.1,4.7Hz,1H),2.53(s,3H)。
实施列2
(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸钠盐
Figure PCTCN2021125450-appb-000012
零摄氏度下，向含有(S)-4-(2-(4-(2-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧并哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸(150.0毫克，0.28毫摩尔)的甲醇(10.0毫升)中，滴加氢氧化钠水溶液(氢氧化钠；6.72毫克，0.28毫摩尔；水：2.0毫升)，保持该温度反应5小时。
反应结束，蒸除甲醇，所得水溶液低温冻干得到155.0毫克Form A白色固体(S)-4-(2-(4-(2-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧并哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸钠Form A(收率：97.5％)。LCMS:RT＝2.00min,[M+H]+＝546.31。 1H NMR(400MHz,DMSO)δ10.37(s,1H),7.99(d,J＝8.4Hz,1H),7.86(d,J＝8.6Hz,2H),7.68(dd,J＝8.3,2.2Hz,1H),7.59(d,J＝8.6Hz,2H),7.50(d,J＝2.1Hz,1H),7.36–7.24(m,4H),7.18(t,J＝7.1Hz,1H),6.90(s,1H),5.75(dd,J＝10.2,4.8Hz,1H),3.68(s,3H),3.47–3.37(m,2H),2.53(s,3H)。
取40毫克Form A样品加入到1毫升丙酮中，加热至50摄氏度，加入20微升水，再加入320毫克Form A样品，固体完全溶解，50摄氏度搅拌24小时后有固体析出，离心得化合物A钠盐结晶性固体Type A。
实施例3
(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸钾盐
Figure PCTCN2021125450-appb-000013
零摄氏度下，向含有(S)-4-(2-(4-(2-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧并哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸(100.0毫克，0.18毫摩尔)的甲醇(10.0毫升)中，滴加氢氧化钾水溶液(氢氧化钾；10.3毫克，0.18毫摩尔；水：2.0毫升)，保持该温度反应5小时。
反应结束，蒸除甲醇，所得水溶液低温冻干得到98.0毫克白色固体(S)-4-(2-(4-(2-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧并哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸钾盐(收率：93.4％)。LCMS:RT＝2.00min,[M+H] +＝546.22。
1H NMR(400MHz,DMSO)δ10.23(s,1H),7.98(d,J＝8.4Hz,1H),7.77(d,J＝8.6Hz,2H),7.68(dd,J＝8.3,2.2Hz,1H),7.50(d,J＝2.1Hz,1H),7.46(d,J＝8.5Hz,2H),7.38–7.24(m,4H),7.18(t,J＝7.1Hz,1H),6.89(s,1H),5.75(dd,J＝10.3,4.7Hz,1H),3.68(s,3H),3.56–3.41(m,2H),2.52(s,3H)。
实施列4
(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸葡甲胺盐
Figure PCTCN2021125450-appb-000014
将(S)-4-(2-(4-(2-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧并哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸(1.0克)和葡甲胺(358毫克)以1：1当量比加入20毫升丙酮中制成混悬液，在温度循环下(1000rpm，50℃～5℃，0.1℃/min，2个循环)混悬搅拌3天，得到(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸葡甲胺盐晶型A。
1H NMR(400MHz,DMSO)δ10.42(s,1H),8.00(d,J＝8.4Hz,1H),7.88(d,J＝8.7Hz,2H),7.69(dd,J＝8.3,2.2Hz,1H),7.66(d,J＝8.7Hz,2H),7.51(d,J＝2.1Hz,1H),7.34–7.25(m,4H),7.22-7.18(m,1H),6.91(s,1H),5.75(dd,J＝10.2,4.9Hz,1H),3.79-3.74(m,1H),3.68(s,3H),3.67–3.65(m,1H),3.60(dd,J＝10.8,3.5Hz,1H),3.56–3.46(m,2H),3.43-3.33(m,3H),2.80–2.66(m,1H),2.55(s,1H),2.53(s,3H),2.39(s,3H)。
实施例5
(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸镁盐
Figure PCTCN2021125450-appb-000015
零摄氏度下，向含有(S)-4-(2-(4-(2-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧并哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲钠(100.0毫克，0.18毫摩尔)的甲醇(10.0毫升)中，滴加氯化镁水溶液(氯化镁；16.8毫克，0.18毫摩尔；水：2.0毫升)，保持该温度反应5小时。
反应结束，蒸除甲醇，析出白色固体，抽滤，干燥得到62.0毫克白色固体(S)-4-(2-(4-(2-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧并哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸镁盐(收率：30.9％)。LCMS:RT＝2.00min,[M+H] +＝546.20。 1H NMR(500MHz,DMSO)δ10.33(s,1H),7.98(d,J＝8.4Hz,1H),7.93(s,2H),7.67(dd,J＝8.3,2.1Hz,1H),7.59(d,J＝8.2Hz,2H),7.49(d,J＝1.9Hz,1H),7.36–7.22(m,4H),7.17(t,J＝7.2Hz,1H),6.88(s,1H),5.73(dd,J＝10.2,4.8Hz,1H),3.66(s,3H),3.41(dd,J＝14.3,4.7Hz,2H),2.51(s,3H)。
实施例6
(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸钙盐
Figure PCTCN2021125450-appb-000016
零摄氏度下，向含有(S)-4-(2-(4-(2-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧并哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲钠(100.0毫克，0.18毫摩尔)的甲醇(10.0毫升)中，滴加氯化钙水溶液(氯化钙；20.0毫克，0.18毫摩尔；水：2.0毫升)，保持该温度反应5小时。
反应结束，蒸除甲醇，析出白色固体，抽滤，水洗，干燥得到58.0毫克白色固体(S)-4-(2-(4-(2-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧并哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸钙盐(收率：28.5％)。LCMS:RT＝2.00min,[M+H] +＝546.17。
实施例7
(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸胆碱盐
Figure PCTCN2021125450-appb-000017
将(S)-4-(2-(4-(2-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧并哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸和胆碱以1:1当量比加入丙酮中，在温度循环下(50℃～5℃,0.1℃/min,2循环)搅拌3天得到胶状样品，胶状样品在室温下真空干燥8小时 得固体粉末(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-甲氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸胆碱盐晶型A。
对比实施例1化合物A1
合成(S)-4-(2-(4-(5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基)-6-氧嘧啶-1(6H)-基)-3-苯基丙酰胺基)苯甲酸
Figure PCTCN2021125450-appb-000018
具体合成路线如下：
步骤A：合成4-氯-2-(四甲基-1,3,2-二氧杂硼杂环戊烷-2-基)苯胺
Figure PCTCN2021125450-appb-000019
将2-溴-4-氯苯胺(3.1克，14.5毫摩尔)加入2-溴-4-氯苯胺(3.0克，15.0毫摩尔)，4,4,5,5-四甲基-2-(四甲基-1,3,2-二氧杂硼杂环戊烷-2-基)-1,3,2-二氧杂硼杂环戊烷(38克，150.0毫摩尔)，醋酸钾(2.9克，30.0毫摩尔)，[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(1.1克，1.5毫摩尔)溶解于二甲基亚砜(75毫升)。氮气保护后,在80℃加热5小时。将反应冷却至室温。加入水溶解盐,然后过滤反应。将剩余的固体悬浮于二氯甲烷中并过滤不溶固体。浓缩滤液,然后通过硅胶柱层析纯化,得到5.2克白色固体4-氯-2-(四甲基-1,3,2-二氧杂硼杂环戊烷-2-基)苯胺(收率：100％)。LCMS：RT＝4.40min,[M+H] +＝254.10。
步骤B：合成4-氯-2-(6-甲氧基嘧啶-4-基)苯胺
Figure PCTCN2021125450-appb-000020
将4-氯-6-甲氧基嘧啶(3.9克,15.4毫摩尔)碳酸钠(3.2克,30.8毫摩尔),乙二醇二甲醚(16毫升),乙醇(2毫升)和水(2毫升)置于三口瓶中。氮气保护后,加入[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(1.3克,1.5毫摩尔)。将4-氯-2-(四甲基-1,3,2-二氧杂硼杂环戊烷-2-基)苯胺(3.31克,23.1毫摩尔)的乙二醇二甲醚(8毫升),将反应在90℃加热2小时。LCMS监测,反应完全后,冷却至室温,垫硅藻土过滤,滤饼用乙酸乙酯(30毫升)洗涤3次,合并滤液及洗涤液,水洗一次,饱和氯化铵洗涤两次,有机相经无水硫酸钠干燥,过滤,旋干,残渣经硅胶柱层析纯化得到1.0克黄色固体4-氯-2-(6-甲氧基嘧啶-4-基)苯胺(收率：28％)。LCMS：RT＝3.95min,[M+H] +＝236.04。
步骤C：合成4-{5-氯-2-[4-(三甲基甲硅烷基)-1H-1,2,3-三唑-1-基]-苯基}-6-甲氧基-嘧啶
Figure PCTCN2021125450-appb-000021
将4-氯-2-(6-甲氧基嘧啶-4-基)苯胺(0.9克,3.8毫摩尔)溶于乙腈(60毫升)中,在0℃下加入3-甲基丁基亚硝酸酯(0.6毫升,5.8毫摩尔),然后滴加叠氮基三甲基硅烷(0.6毫升,5.8毫摩尔)。观察到气体产生。10分钟后,除去冰浴,让反应温热至室温。1小时后,加入乙炔基三甲基甲硅烷(1.8毫升,11.4毫摩尔)和氧化亚铜(0.06g,0.36毫摩尔),将反应再搅拌1小时。向反应液中加乙酸乙酯和饱和氯化铵水溶液分层。有机相用盐水洗涤,经无水硫酸钠干燥,过滤并浓缩。经硅胶柱层析进一步纯化得到730毫克黄色固体4-{5-氯-2-[4-(三甲基-甲硅烷基)-1H-1,2,3-三唑-1-基]苯基}-6-甲氧基嘧啶(收率：45％)。LCMS：RT＝2.04min,[M+H] +＝360.10。
步骤D：合成4-[5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基]-6-甲氧基嘧啶
Figure PCTCN2021125450-appb-000022
将4-{5-氯-2-[4-(三甲基甲硅烷基)-1H-1,2,3-三唑-1-基]苯基}-6-甲氧基嘧啶(700毫克,1.94毫摩尔)溶于乙腈(20毫升)，溶液中加入N-氯代丁二酰亚胺(0.9克,7.2毫摩尔)和硅胶(2.9克,50.44毫摩尔)。反应在80℃搅拌1小时。然后将反应过滤以除去硅胶,将收集的硅胶用乙酸乙酯洗涤。滤液用水洗,盐水洗涤,浓缩。残渣经硅胶柱层析进一步纯化得到450毫克黄色固体4-[5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基]-6-甲氧基嘧啶(收率：72％)。LCMS：RT＝2.00min,[M+H] +＝322.05。
步骤E：合成6-[5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基]嘧啶-4-醇
Figure PCTCN2021125450-appb-000023
向4-[5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基]-6-甲氧基嘧啶(450毫克,1.4毫摩尔)在醋酸(3毫升)中的溶液中加入48％氢溴酸水溶液(1.5毫升,13.3毫摩尔)。混合物在95℃搅拌1小时。将反应浓缩至干,然后用乙酸乙酯和饱和碳酸氢钠溶液分液。有机相浓缩,残渣经硅胶柱层析纯化得到190豪克黄色固体6-[5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基]嘧啶-4-醇(收率：44％)。LCMS：RT＝1.74min,[M-H] -＝305.97。
步骤F：合成(S)-4-(2-(4-(5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基)-6-氧嘧啶-1(6H)-基)-3-苯基丙酰胺基)苯甲酸叔丁酯
Figure PCTCN2021125450-appb-000024
室温下,将6-[5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基]嘧啶-4-醇(45毫克,0.15毫摩尔)和(R)-4-(2-(((4-硝基苯基)磺酰基)氧基)-3-苯基丙酰胺基)苯甲酸叔丁酯(93毫克,0.18毫摩尔)以及碳酸钾(40毫克,0.3毫摩尔)加入N,N-二甲基甲酰胺(3.0毫升)中,室温反应过夜。向反应液中加水淬灭,混合液用乙酸乙酯(40毫升×3次)萃取,合并有机相,有机相先用饱和食盐水(30毫升×2次),然后用无水硫酸钠干燥,最后减压浓缩。所得残余物用硅胶柱层析纯化得到150毫克黄色液体(S)-4-(2-(4-(5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基)-6-氧嘧啶-1(6H)-基)-3-苯基丙酰胺基)苯甲酸叔丁酯(收率：59％)。LCMS：RT＝2.00min,[M+H] +＝631.18。
步骤F：合成(S)-4-(2-(4-(5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基)-6-氧嘧啶-1(6H)-基)-3-苯基丙酰胺基)苯甲酸
Figure PCTCN2021125450-appb-000025
将(S)-4-(2-(4-(5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基)-6-氧嘧啶-1(6H)-基)-3-苯基丙酰胺基)苯甲酸叔丁酯(150毫克,0.25毫摩尔)溶于二氯甲烷(2.0毫升)中。随后,向上述溶液中加入三氟乙酸(0.5毫升),在室温下搅拌1小时。将反应液空气浴中减压浓缩。将所得残余物经制备纯化得到70毫克白色固体(S)-4-(2-(4-(5-氯-2-(4-氯-1H-1,2,3-三唑-1-基)苯基)-6-氧嘧啶-1(6H)-基)-3-苯基丙酰胺基)苯甲酸(收率：59％)。
LCMS：RT＝2.00min,[M+H] +＝573.16。 1H NMR(400MHz,CD 3OD)δ10.36(s,1H),8.36(s,1H),8.18(s,1H),7.87(dd,J＝12.0,5.1Hz,2H),7.72(d,J＝2.3Hz,1H),7.66–7.47(m,4H),7.28–7.07(m,5H),6.22(d,J＝0.8Hz,1H),5.74(dd,J＝10.5,6.2Hz,1H),3.49(dd,J＝14.1,6.3Hz,1H),3.34–3.24(m,1H)。
对比实施例2化合物B
合成(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-5-甲氧基-2-氧吡啶鎓-1(2H)-基)-3-苯基丙酰胺基)苯甲酸
Figure PCTCN2021125450-appb-000026
具体合成路线如下：
步骤A：合成(2,5-二甲氧基吡啶-4-基)硼酸
Figure PCTCN2021125450-appb-000027
将2,5-二甲氧基吡啶(10.0克,71.9豪摩尔)溶于干燥四氢呋喃(40毫升)中,置于干燥三口烧瓶中,氮气保护后,于干冰/乙醇浴中搅拌15分钟后,将二异丙基氨基锂(20毫升,2.0M in THF)缓慢滴加到反应液中,30分钟后滴加完毕,干冰/乙醇浴中搅拌3h后,将硼酸三异丙酯(33.0毫升,143.8豪摩尔)加入混合液中,然后自然升温到室温并恒温搅拌18小时.LCMS监测,反应完全后,向反应液中加稀盐酸调节pH至3～4,搅拌15分钟后,旋蒸除去溶剂,残渣加乙腈打浆得到10.6克白色固体(2,5-二甲氧基吡啶-4-基)硼酸(收率：80％)。LCMS：RT＝1.73min,[M+H] +＝184.08。
步骤B：合成1-(4-氯-2-(2,5-二甲氧基吡啶-4-基)苯基)乙-1-酮
Figure PCTCN2021125450-appb-000028
将2-溴-4-氯苯乙酮(14.8克,63.6豪摩尔)和(2,5-二甲氧基吡啶-4-基)硼酸(9.7克,53.0豪摩尔)溶于1,4-二氧六环(40毫升),碳酸钾(14.6克,106豪摩尔)溶于水(10毫升),置于干燥三口烧瓶中,氮气保护后,将[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(3.87克,5.3豪摩尔)加入反应液中,氮气保护后,升温到100℃并恒温搅拌18小时.LCMS监测,反应完全后,冷却至室温,垫硅藻土过滤,滤饼用EA(30毫升)洗涤3次,合并滤液及洗涤液,水洗一次,饱和氯化铵洗涤两次,有机相经无水硫酸钠干燥,过滤,旋干,残渣经硅胶柱层析纯化得到8.2克黄色固体1-(4-氯-2-(2,5-二甲氧基吡啶-4-基)苯基)乙-1-酮(收率：53％)。LCMS：RT＝4.03min,[M+H] +＝292.03。
步骤C：合成4-(2-乙酰基-5-氯苯基)-5-甲氧基吡啶-2(1H)-酮
Figure PCTCN2021125450-appb-000029
将1-(4-氯-2-(2,5-二甲氧基吡啶-4-基)苯基)乙-1-酮(8.2克,28豪摩尔),吡啶氢溴酸盐(22g,140豪摩尔)溶于N,N-二甲基甲酰胺(20毫升),置于干燥烧瓶中,氮气保护后,升温到110℃并恒温搅拌4h.LCMS监测,反应完全后,冷却至室温,将反应液滴加到100毫升水中,加5％碳酸钠调节pH至10～11,DCM(40毫升×4)萃取四次,合并有机相,有机相经无水硫酸钠干燥,过滤,旋干,残渣用DCM(10毫升)溶解,然后滴加到正己烷(120毫升)中,析出大量固体,过滤,收集滤饼,即产物粗品,经硅胶柱层析进一步纯化得到6.4g黄色固体4-(2-乙酰基-5-氯苯基)-5-甲氧基吡啶-2(1H)-酮(收率：82％)。LCMS：RT＝3.81min,[M-H] -＝277.04。
步骤D：合成(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-5-甲氧基-2-氧吡啶鎓-1(2H)-基)-3-苯基丙酰胺基)苯甲酸叔丁酯
Figure PCTCN2021125450-appb-000030
室温下,将4-(2-乙酰基-5-氯苯基)-5-甲氧基吡啶-2(1H)-酮(1.5克,5.4毫摩尔)和(R)-4-(2-(((4-硝基苯基)磺酰基)氧基)-3-苯基丙酰胺基)苯甲酸叔丁酯(4.0克,7.6毫摩尔)以及碳酸钾(1.5克,10.8毫摩尔)加入N,N-二甲基甲酰胺(20.0毫升)中,室温反应过夜。向反应液中加水淬灭,混合液用乙酸乙酯(40毫升×3次)萃取,合并有机相,有机相先用饱和食盐水(30毫升×2次),然后用无水硫酸钠干燥,最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/正己烷＝1/2)得到1.9克黄色固体(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-5-甲氧基-2-氧吡啶鎓-1(2H)-基)-3-苯基丙酰胺基)苯甲酸叔丁酯(收率：59％)。LCMS：RT＝4.42min,[M+H] +＝601.18。
步骤E：合成(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-5-甲氧基-2-氧吡啶鎓-1(2H)-基)-3-苯基丙酰胺基)苯甲酸
Figure PCTCN2021125450-appb-000031
将(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-3-乙氧基-6-氧代哒嗪-1(6H)-基)-3-苯基丙酰胺基)苯甲酸叔丁酯(1.9克,3.2毫摩尔)溶于二氯甲烷(12.0毫升)中。随后,向上述溶液中加入三氟乙酸(3毫升),在室温下搅拌1小时。将反应液空气浴中减压浓缩。将所得残余物用甲醇打浆纯化,得到1.0克黄色固体(S)-4-(2-(4-(2-乙酰基-5-氯苯基)-5-甲氧基-2-氧吡啶鎓-1(2H)-基)-3-苯基丙酰胺基)苯甲酸(收率：59％)。LCMS：RT＝3.88min,[M-H] -＝543.06。 1H NMR(400MHz,DMSO)δ10.82(s,1H),7.92(d,J＝8.8Hz,2H),7.82(d,J＝8.3Hz,1H),7.76(d,J＝8.8Hz,2H),7.61(dd,J＝8.4,2.3Hz,2H),7.42(s,1H),7.38(s,1H)，7.33–7.23(m,4H),7.22–7.14(m,1H),6.30(s,1H),6.02(dd,J＝9.5,6.6Hz,1H),3.53(s,3H),3.49-3.44(m,2H),2.36(s,3H)。
对比实施例3 CN201680058331实施例143化合物
参照CN201680058331实施例143的制备方法获得相应目标化合物。
实施例8：吸收光法检测本发明化合物对人凝血因子XIa抑制的生物活性
1、实验材料
酶：Human Factor XIa(ENZYME RESEARCH，货号HFXIa 1111a)
底物：S-2366 TM：(CHROMOGENIX，货号82109039)
缓冲液：145mM NaCl，5mM KCl，1mg/mL PEG 8000,，30mM HEPES，pH7.4
2、实验步骤
将溶于100％DMSO的10mM受试化合物用100％DMSO稀释至1000、200、40、8、1.6、0.32、0.064、0.0128、0.00256、0.00128μM；在96孔板中每孔加入98μL(77.7ng/mL)的FXIa酶溶液，空白孔加入98μL缓冲液代替，再加入2μL不同浓度的化合物，空白和对照孔用DMSO代替，用振荡器混匀，37℃孵育20min。
最后每孔加入800μM的底物100μL，在405nm处测其吸光度。
3、数据处理
用GraphPad Prism软件进行曲线拟合，计算IC 50值，见表一。
表一.本发明化合物对人FXIa抑制的IC 50
实施例 hFXIa IC 50(nM)
1 7.61
结论：本发明化合物对人FXIa具有明显的抑制活性。
实施例9：本发明化合物对人血浆体外抗凝血作用的测定
1、实验材料
血浆：人血收集于含3.2％柠檬酸钠(体积比1:9)的真空采血管中，室温3000rpm离心10min，收集血浆，分装在EP管中，-80℃保存。
试剂：APTT测定试剂盒(活化部分凝血活酶时间检测定剂盒，mindray)、氯化钙溶液。
仪器：凝血仪(mindray，C2000-A)
2、实验方法
取分装的冻存人血浆室温融化后，混合均匀。将溶于100％DMSO的10mM受试化合物用100％DMSO稀释至1500、750、375、187.5、93.75、46.88、23.44、11.72μM；在1.5mL EP管中加入98μL人血浆，再加入2μL不同浓度的化合物，空白组加入2μL 100％DMSO，37℃水浴孵育10min，将样品放入凝血仪中对应的位置，进行化合物的APTT测定。
3、数据处理
用GraphPad Prism软件进行曲线拟合，分别计算EC1.5×和EC2×值，即1.5倍和2倍空白对照组的APTT所对应的化合物的浓度，结果见表二。
表二.本发明化合物对人血浆体外抗凝血作用
Figure PCTCN2021125450-appb-000032
结论：从表二中可以看出本发明化合物对人血浆具有明显的抗凝血作用。
实施例10:本发明化合物的药代动力学特征考察
1、实验材料
SD大鼠：雄性，180-250g，购于广东省医学实验动物中心、北京维通利华。食蟹猴：雄性，4-6kg，购于广州春盛生物研究院有限公司。比格犬：雄性，8-12kg，在康龙化成(宁波)新药技术股份有限公司开展。
试剂：DMSO(二甲亚砜)，PEG-400(聚乙二醇400)，生理盐水，肝素，乙腈，甲酸，普萘洛尔(内标)均为市售可得。
仪器：赛默飞LC-MS(U300 UPLC，TSQ QUANTUMN ULTRA三重四级杆质谱)。
2、实验方法
称取化合物溶于DMSO-PEG-400-生理盐水(5：60：35，v/v/v)体系中，大鼠/猴静脉或灌胃给药后，于5min(灌胃不采)、15min、30min、1h、2h、4h、6h、8h、24h采集静脉血200μL于肝素化EP管中，12000rpm离心2min，取血浆-80℃冻存待测。精密称取一定量供试品用DMSO溶解至1mg/mL，作为储备液。准确吸取适量的化合物储备液，加入乙腈稀释制成标准系列溶液。准确吸取上述标准系列溶液各20μL，加入空白血浆180μL，涡旋混匀，配制成相当于血浆浓度为1、3、10、30、100、300、1000、3000和5000ng/mL的血浆样品，每一浓度进行双样本分析，建立标准曲线。取20μL血浆，加入内标普萘洛尔(5ng/mL)的乙腈溶液200μL，涡旋混匀后4000rpm离心5min，取上清LC-MS分析。LC-MS检测条件如下：
色谱柱：赛默飞HYPERSIL GOLD C-18 UPLC柱，100*2.1mm，1.9μm。
流动相：水(0.1％甲酸)-乙腈按下表进行梯度洗脱
时间(min) 水(含0.1％甲酸) 乙腈
0 90％ 10％
0.6 90％ 10％
1 10％ 90％
2.6 10％ 90％
2.61 90％ 10％
4 90％ 10％
3、数据处理
LC-MS检测血药浓度后，采用WinNonlin 6.1软件，非房室模型法计算药动学参数。结果见表三、四、五。
表三.本发明化合物的大鼠药代动力学参数
Figure PCTCN2021125450-appb-000033
表四.本发明化合物的食蟹猴药代动力学参数
Figure PCTCN2021125450-appb-000034
表五.本发明化合物的比格犬药代动力学参数
Figure PCTCN2021125450-appb-000035
结论：本发明化合物在大鼠和猴口服均有一定的吸收，犬口服吸收较好，体内清除速率中等偏慢，多数化合物口服半衰期较长，具有良好的药代动力学特征。
实施例11：本发明化合物的体内药效学征考察
本实施例参考新药(西药)临床前研究指导原则汇编(药学药理学毒理学)要求进行以下试验：
11.1实施例4化合物对电刺激诱导的家兔下腔静脉血栓药效考察
试验方法：家兔禁食不禁水24h，按体重分为模型对照组、实施例4化合物30mg/kg/h(以游离酸计)剂量组、阳性药1利伐沙班3mg/kg/h剂量组、阳性药2依诺肝素钠20mg/kg/h剂量组，均静脉输注给药一次，以1.4ml/kg/h的速率持续2h，模型对照组给予等体积溶媒。
于给药后1h进行电刺激，电刺激前15min，家兔40mg/kg戊巴比妥钠耳缘静脉注射麻醉，仰卧固定，腹部备皮、消毒，经腹中线、腹白线开腹，盐水纱布保护肠管并推向一侧，剪开后腹膜，游离下腔静脉(范围自左肾静脉下至髂腰静脉开口以上)，结扎近心端，间隔2cm处狭窄远心端(使用直径2mm的塑料管平行血管放置，一同结扎，结扎后缓慢抽出塑料管)，结扎此段间隔处的各分支静脉。将针状电极放置于结扎段下腔静脉内皮上，使用1.5mA的电流刺激30min，电刺激时动脉夹夹闭狭窄部位阻断静脉。电刺激结束后松开动脉夹，家兔放置至电刺激停止后30min。
电刺激结束后，使用手术刀在肾脏做长度2mm，深度1mm的切口，测定出血时间。试验结束后，取刺激部位血栓，称量血栓重量，计算血栓形成抑制率。试验结果：实施例4化合物和阳性对照药静脉滴注给药能显著降低家兔下腔静脉血栓重量，实施例4化合物30mg/kg/h血栓抑制率为54.3％；利伐沙班3mg/kg/h血栓抑制率为59.8％；依诺肝素20mg/kg/h血栓抑制率为54.4％，试验结果见下表六。
表六.实施例4化合物家兔下腔静脉血栓药效结果
Figure PCTCN2021125450-appb-000036
***P<0.001vs模型对照
试验结论：实施例4化合物达到和利伐沙班及依诺肝素相当的药效水平时，出血风险明显降低。
11.2实施例2化合物对三氯化铁诱导的家兔颈动脉血栓药效考察
试验方法：家兔禁食不禁水24h，分为模型对照组、阳性药氯吡格雷3mg/kg/h和10mg/kg/h剂量组，实施例2化合物10mg/kg/h(以游离酸计)剂量组，每组3只家兔。
给药前先对家兔称重，耳缘静脉40％乌拉坦1.5mL/kg麻醉，固定，手术分离右股动、静脉和颈动脉，右股静脉给药，右股动脉采血，均静滴给药2h，滴注体积为1.4mL/kg/h，模型对照组给予等体积溶媒。静滴1h后开始造模：用 10mm*10mm滤纸上加入20μL 50％FeCl3溶液，敷在颈动脉周围5min，去除滤纸片，静置55min，剪取颈动脉分离血栓称重。。
兔子血栓实验结束后，剪开腹部皮肤、腹肌，然后剥离肾包膜，将肾放入37℃预热的生理盐水中，用一次性1ml注射器刺破肾组织，保证5mm的深度，引起肾出血，在生理盐水中观察肾出血时间。
试验结果：氯吡格雷单药3mg/kg/h剂量组血栓抑制率约49.1％，肾出血时间无明显变化，10mg/kg/h剂量组血栓抑制率达到72.7％时，肾出血时间有明显延长。实施例2化合物10mg/kg/h剂量组血栓抑制率约47.9％，肾出血时间无明显变化。试验结果见表七。
表七.实施例2化合物家兔颈动脉血栓药效结果
Figure PCTCN2021125450-appb-000037
**P<0.01,***P<0.001vs模型对照
试验结论：氯吡格雷对家兔颈动脉血栓有较好的效果，且有明显的量效关系，氯吡格雷随着给药剂量增加肾出血时间会明显变长。实施例2化合物10mg/kg/h给药时表现出一定的预防血栓形成效果，同时降低了出血风险。
11.3实施例2化合物对三氯化铁诱导的家兔动静脉旁路血栓药效考察
试验方法：家兔按体重分为模型对照组、实施例2化合物30mg/kg/h剂量组，手术分离右颈动脉和左颈静脉，右股动、静脉。右颈动脉和左颈静脉用于连接动静脉旁路装置用于动静脉血栓模型制作；右股静脉插管用于静脉输注给药。均采用静脉输注的方式给药，输注容积1.4ml/kg/h，模型对照组给予等体积溶媒。微量输液泵匀速输注60min后，接通动静脉旁路装置，装置内充满生理盐水，两端连接右颈动脉和左颈静脉，静置15min。
造模前，家兔40mg/kg戊巴比妥钠耳缘静脉注射麻醉，仰卧固定，颈部备皮、消毒，用手术剪刀剪开颈部皮肤，分离右颈动脉和左颈静脉，首先结扎右颈动脉远心端，用血管夹夹闭近心端，靠近远心端结扎线内侧剪开血管，将充满生理盐水的动静脉旁路装置接入，用两道真丝非吸收缝线结扎固定，暂不打开血管夹；左颈静脉用同样的方法接入动静脉旁路装置，用两道真丝非吸收缝线结扎固定，暂不打开血管夹。输注时间结束后首先打开动静脉旁路装置静脉端血管夹，再打开动脉端血管夹，计时15min。取下动静脉旁路装置，利用减量法计算血栓重量。同时检测肾出血时间。使用1ml一次性注射器针头，在剥离肾包膜的肾脏表面，扎一深度3mm的针孔，测定出血时间。
试验结果：实施例2化合物给药剂量30mg/kg/h能明显减少家兔动静脉旁路血栓重量，血栓抑制率为61.9％，试验结果见表八。
表八.实施例2化合物家兔动静脉旁路血栓药效结果
Figure PCTCN2021125450-appb-000038
***P<0.001vs模型对照
试验结论：实施例2化合物在家兔动静脉旁路血栓模型中均有较好的预防血栓形成效果，实施例2化合物肾出血时间仍无明显增加，表明实施例2化合物在达到较好药效水平时出血风险较小。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (11)

  1. FXIa抑制剂化合物或其盐的医药用途，其特征在于，所述用途包括所述在FXIa抑制剂化合物或其盐制备预防和/或治疗动脉血栓、静脉血栓、动静脉旁路血栓药物中的用途，所述化合物结构式如下：
    Figure PCTCN2021125450-appb-100001
  2. 根据权利要求1所述的医药用途，其特征在于，所述FXIa抑制剂化合物的盐结构式如下：
    Figure PCTCN2021125450-appb-100002
    其中：
    n为0.5-3；
    M与羧基成盐，所述盐选自锂盐、钠盐、钾盐、钙盐、镁盐、铝盐、铁盐、锌盐或铵盐中的至少一种；或所述盐选自甲胺盐、二甲胺盐、三甲胺盐、乙胺盐、二乙胺盐、三乙胺盐、异丙胺盐、2-乙氨基乙醇盐、吡啶盐、甲基吡啶盐、乙醇胺盐、二乙醇胺盐、铵盐、四甲基铵盐、四乙基铵盐、三乙醇胺盐、哌啶盐、哌嗪盐、吗啉盐、赖氨酸盐、精氨酸盐、L-精氨酸盐、组氨酸盐、L-组氨酸盐、葡甲胺盐、二甲基葡糖胺盐、乙基葡糖胺盐、二环己基胺盐、1,6-己二胺盐、葡糖胺盐、肌氨酸盐、丝氨醇盐、三羟基甲基氨基甲烷盐、氨基丙二醇盐、1-氨基-2,3,4-丁三醇盐、L-赖氨酸盐、鸟氨酸盐或胆碱盐中的至少一种。
  3. 根据权利要求2所述的医药用途，其特征在于，n为0.5、1、1.5、2、2.5或3。
  4. 根据权利要求2所述的医药用途，其特征在于，所述的盐选自钠盐、钾盐、葡甲胺盐、钙盐、镁盐、胆碱盐。
  5. 根据权利要求2所述的医药用途，其特征在于，其特征在于，所述的盐选自钠盐、n＝1；钾盐、n＝1；胆碱盐、n＝1；葡甲胺盐、n＝1；钙盐、n＝0.5；镁盐、n＝0.5。
  6. 根据权利要求1-5任一项所述的医药用途，其特征在于，所述化合物或其盐为晶型、或者无定型，或其混合物。
  7. 根据权利要求1-5任一项所述的医药用途，其特征在于：所述化合物或其盐的一个以上的氢原子上被同位素氘取代。
  8. 根据权利要求1-5任一项所述的医药用途，其特征在于：所述化合物或其盐，和一种以上药学上可接受的载体组成药物组合物。
  9. 根据权利要求1-5任一项所述的医药用途，其特征在于：动脉血栓导致动脉栓塞性疾病，包括冠心病、心肌梗死、缺血性脑卒中、外周动脉疾病、房颤和心瓣膜病；所述静脉血栓导致静脉血栓栓塞性疾病，包括关节置换术后深静脉血栓，肺栓塞，深静脉血栓形成；以及透析后的动静脉血栓。
  10. 根据权利要求9所述医药用途，其特征在于：关节置换术后深静脉血栓包括全膝关节置换术后静脉血栓，髋关节置换术后静脉血栓等。
  11. 一种预防和/或治疗动脉、静脉血栓的药物组合物，其特征在于，含有下式化合物或其盐，和一种以上药学上可接受的载体，
    Figure PCTCN2021125450-appb-100003
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