TW202227079A - Pharmaceutical use of FXIa inhibitor compound or salt thereof - Google Patents

Abstract

Provided is a pharmaceutical use of an FXIa inhibitor compound or a salt thereof. Specifically, provided is a use of the FXIa inhibitor compound or the salt thereof in preparation of drugs for prevention and/or treatment of arterial and venous thrombosis.

Description

Medicinal use of FXIa inhibitor compound or salt thereof and pharmaceutical composition containing the same

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.

Every year, 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 is composed of intrinsic pathway, extrinsic pathway and common pathway (Annu.Rev.Med.2011.62:41–57), which is initiated by multiple zymogens in sequence. A chain reaction that is continuously strengthened and amplified. The coagulation cascade is initiated by the endogenous pathway (also known as the contact initiation 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 initiate factor X, while the extrinsic coagulation pathway is released from tissue factor (TF) to TF-VIIa- The Ca ²⁺ complex forms and initiates the factor X process. The common pathway means that after the formation of factor Xa, the two pathways are combined into one, starting the process of prothrombin and finally generating fibrin, of which FXI is necessary to maintain the endogenous pathway, and in the process of coagulation cascade amplification play a key role. In the coagulation cascade reaction, thrombin can feed back to initiate FXI, and the activated FXI (FXIa) promotes the massive production of thrombin, thereby amplifying the coagulation cascade reaction. Therefore, antagonists of FXI have been widely developed for the treatment of various thrombi.

Traditional anticoagulant drugs, such as warfarin, heparin, low molecular weight heparin (LMWH), and new drugs launched in recent years, such as FXa inhibitors (rivaroxaban, araguaxaban, etc.) and thrombin inhibitors (Darby Gatran etexilate, hirudin, etc.), all have good effects on reducing thrombosis, and occupy the vast cardiovascular and cerebrovascular market with their significant effectiveness. However, their side effects are becoming more and more significant. Among them, the "bleeding risk" is Bear the brunt of one of the most serious problems (N Engl J Med 1991; 325: 153-8, Blood. 2003; 101: 4783-4788).

Studies have found that in the thrombosis model, inhibition of FXIa factor can effectively inhibit the formation of thrombus, but in more severe thrombosis, the effect of FXIa is minimal (Blood. 2010; 116(19): 3981-3989). Clinical statistics show that increasing the amount of FXIa increases the prevalence of VTE (Blood 2009;114:2878-2883), while those with severe FXIa deficiency have a reduced risk of DVT (Thromb Haemost 2011;105:269–273) .

FXIa is currently an emerging target for inhibiting thrombosis, and patent applications for compounds with FXIa inhibitory activity are disclosed in WO9630396, WO9941276, WO2013093484, WO2004002405, WO2013056060, WO2017005725, WO2017/023992, WO2018041122, etc. Among them, only Bayer's antisense oligonucleotide BAY-2306001 has entered the Phase II clinical study.

The compounds of the present invention have higher activity. In particular, 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.

。 Specifically, 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 Use in medicine, the compound structural formula is as follows:

.

其中： n為0.5-3； M與羧基成鹽，所述鹽選自鋰鹽、鈉鹽、鉀鹽、鈣鹽、鎂鹽、鋁鹽、鐵鹽、鋅鹽或銨鹽中的至少一種；或所述鹽選自甲胺鹽、二甲胺鹽、三甲胺鹽、乙胺鹽、二乙胺鹽、三乙胺鹽、異丙胺鹽、2-乙氨基乙醇鹽、吡啶鹽、甲基吡啶鹽、乙醇胺鹽、二乙醇胺鹽、銨鹽、四甲基銨鹽、四乙基銨鹽、三乙醇胺鹽、呱啶鹽、呱嗪鹽、嗎啉鹽、賴氨酸鹽、精氨酸鹽、L-精氨酸鹽、組氨酸鹽、L-組氨酸鹽、葡甲胺鹽、二甲基葡糖胺鹽、乙基葡糖胺鹽、二環己基胺鹽、1,6-己二胺鹽、葡糖胺鹽、肌氨酸鹽、絲氨醇鹽、三羥基甲基氨基甲烷鹽、氨基丙二醇鹽、1-氨基-2,3,4-丁三醇鹽、L-賴氨酸鹽、鳥氨酸鹽或膽鹼鹽中的至少一種。 As an embodiment of the present invention, the salt structural formula of the FXIa inhibitor compound is as follows:

Wherein: n is 0.5-3; M forms a salt with a carboxyl group, and the salt is selected from at least one of lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, aluminum salts, iron salts, zinc salts or ammonium salts; Or the salt is selected from methylamine salt, dimethylamine salt, trimethylamine salt, ethylamine salt, diethylamine salt, triethylamine salt, isopropylamine salt, 2-ethylaminoethanol salt, pyridine salt, picoline salts, ethanolamine salts, diethanolamine salts, ammonium salts, tetramethylammonium salts, tetraethylammonium salts, triethanolamine salts, oxidine salts, oxazine salts, morpholine salts, lysine salts, arginine salts, L-arginine salt, histidine salt, L-histidine salt, meglumine salt, dimethylglucamine salt, ethyl glucamine salt, dicyclohexylamine salt, 1,6-hexylamine salt Diamine salt, glucosamine salt, sarcosinate, serine alcohol salt, trihydroxymethyl aminomethane salt, amino propanediol salt, 1-amino-2,3,4-butanetriol salt, L-lysine at least one of acid salts, ornithine salts or choline salts.

As an embodiment of the present invention, n is 0.5, 1, 1.5, 2, 2.5 or 3.

As an embodiment of the present invention, the salt is selected from the group consisting of sodium salt, potassium salt, meglumine salt, calcium salt, magnesium salt, and choline salt.

As an embodiment of the present invention, the salt is selected from sodium salt, n=1; potassium salt, n=1; choline salt, n=1; meglumine salt, n=1; calcium salt, n=1 0.5; magnesium salt, n=0.5.

As an embodiment of the present invention, the compound or its salt is in crystalline form, or amorphous form, or a mixture thereof.

As an embodiment of the present invention, more than one hydrogen atom of the compound or its salt is substituted with the isotope deuterium.

As an embodiment of the present invention, the compound or its salt, and one or more pharmaceutically acceptable carriers form a pharmaceutical composition.

As an embodiment of the present invention, 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 thromboembolism Sexual diseases, including deep vein thrombosis after joint replacement, pulmonary embolism, deep vein thrombosis; and arteriovenous thrombosis after dialysis.

As an embodiment of the present invention, 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 shows 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 platelet 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 damages the vascular intima, coupled with improper compression and hemostasis, massive dehydration resulting in hypotension, slow blood flow, decreased blood flow in the internal fistula and blood viscosity Increased thrombosis can promote thrombosis, so thrombosis diseases such as arteriovenous fistula thrombosis are common in dialysis patients.

As an embodiment of the present invention, animal experiments of the present invention show that the compounds of the present invention have good preventive/therapeutic effects 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 atrial Thrombotic disease in patients with 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; The rabbit model of venous thrombosis can reach the level of rivaroxaban's efficacy without the risk of bleeding.

Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear without specific definitions, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commercial product or its active ingredient. As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue , without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.

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, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If an 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 Enantiomers are required. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), 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. In addition, 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). Examples of 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. In particular, 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. When the atoms making up the molecules of the compounds of the present invention are isotopes, 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. For example, 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.

Further, one or more hydrogen atoms of the compounds of the present invention are substituted by the isotope deuterium ( ² H). 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. For example, preferred methods of deuteration employ phase transfer catalysts (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 triethyl deuterated silane) or with Lewis acids such as trisulfuric acid in the presence of an acid (eg, methanesulfonic acid) Aluminum chloride is reduced with sodium deuteroborate to introduce higher deuterium.

The term "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, and representative carriers include water, oil, Vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, tackifiers, skin 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.

The term "excipient" generally refers to the carrier, diluent and/or medium required to formulate an effective pharmaceutical composition.

The term "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. For oral dosage forms of the present invention, 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.

The term "treatment" refers to a chemical entity that is effective in treating the target disorder, disease, or condition.

"Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.

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 equivalents known to those skilled in the art Alternatively, preferred embodiments include, but are not limited to, the embodiments of the present invention.

The present invention will be further described in detail below with reference to the examples, but the embodiments of the invention are not limited thereto.

The structures of compounds were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR shifts ([delta]) are given in units of 10-6 (ppm). NMR was measured by Bruker AVANCE-III nuclear magnetic instrument, and the solvent was deuterated dimethylsulfoxide (DMSO-d6), deuterated chloroform (CDCl ₃ ), and the internal standard was tetramethylsilane (TMS).

The MS was measured using an ISQ EC mass spectrometer (manufacturer: Thermo, model: ISQ EC).

High performance liquid chromatography (HPLC) analysis was performed using a Thermo U3000 HPLC DAD high performance liquid chromatograph.

The CombiFlash Rapid Preparation System uses the 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.

Example 1

Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3 -Phenylpropionamido)benzoic acid

The specific synthetic route is as follows.

Step A: Synthesis of 5-bromo-6-hydroxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one

Bromomaleic anhydride (2.00 g, 11.3 mmol) and 4-methoxybenzylhydrazine hydrochloride (2..13 g, 11.3 mmol) were added to glacial acetic acid (50.0 mL) at room temperature , 100 ℃ reaction for 3 hours.

The reaction was completed, cooled to room temperature, the reaction solution was poured into water, and a large amount of solid was precipitated. After stirring for a period of time, suction filtration, the filter cake was washed with water, and the filter cake was dried to obtain 1.50 grams of light yellow solid 5-bromo-6-hydroxy-2 -(4-Methoxybenzyl)pyridazin-3( 2H )-one was used in the next step without purification. LCMS: RT = 3.44 min, [M+H] ⁺ = 311.03.

Step B: Synthesis of 5-bromo-6-methoxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one

At room temperature, 5-bromo-6-hydroxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one (1.50 g, 4.82 mmol) and potassium carbonate (2.66 g, 19.29 mmol) was added to N,N -dimethylformamide (15.0 mL), and the mixture was stirred at 80°C for 15 minutes. At this temperature, methyl iodide (1.2 mL) was added and the reaction was continued for 30 minutes.

The reaction was completed, quenched by adding water, the mixture was extracted with ethyl acetate (50 ml × 3 times), and the organic phases were combined. The organic phase was first washed with saturated brine (50 ml × 2 times), then dried with anhydrous sodium sulfate, and finally reduced pressure concentrate. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/n-hexane=1/3). 1.10 g of white solid 5-bromo-6-methoxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one was obtained (yield: 70.3%). LCMS: RT = 3.87 min, [M+H] ⁺ = 325.01.

Step C: Synthesis of 6-Acetyl-3-chlorophenylboronic acid pinacol ester

At room temperature, combine 2-bromo-4-chloroacetophenone (5.00 g, 21.41 mmol), pinacol diboronate (8.16 g, 32.12 mmol) and potassium acetate (4.20 g, 42.82 mmol) ear) into a three-necked flask, replacing nitrogen, adding 1,4-dioxane (60.0 ml), replacing nitrogen, adding 1,1'-bisdiphenylphosphinoferrocene palladium dichloride (1.75 g, 2.14 mmol), nitrogen was replaced, and the temperature was raised to 80 °C to react for 3 hours.

The reaction was completed, quenched by adding water, suction filtration through celite, washing the filter cake with ethyl acetate, extracting the filtrate with ethyl acetate (80 ml × 3 times), combining the organic phases, first adding saturated brine (50 ml × 3 times) to the organic phase. 2 times), then dried over anhydrous sodium sulfate, and finally concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/n-hexane=1/50). 2.1 g of 6-acetyl-3-chlorophenylboronic acid pinacol ester was obtained as a yellow solid (yield: 35.0%). LCMS: RT = 4.26 min, [MH] ^- = 279.08.

Step D: Synthesis of 5-(2-Acetyl-5-chlorophenyl)-6-methoxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one

At room temperature, 5-bromo-6-methoxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one (1.10 g, 3.39 mmol), 6-acetyl Alkyl-3-chlorophenylboronic acid pinacol ester (949 mg, 3.39 mmol) and sodium carbonate (718 mg, 6.78 mmol) were added to the three-necked flask, nitrogen was replaced, and mixed solvent (10 mL, 1, 2-dimethoxyethane:ethanol:water=8:1:1), replace nitrogen, add 1,1'-bisdiphenylphosphinoferrocene palladium dichloride (249 mg, 0.34 mmol) , replaced nitrogen gas, heated to 90 °C and reacted for 1 hour.

The reaction was completed, quenched by adding water, the mixture was extracted with ethyl acetate (50 ml × 3 times), and the organic phases were combined. The organic phase was first washed with saturated brine (50 ml × 2 times), then dried with anhydrous sodium sulfate, and finally reduced pressure concentrate. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/n-hexane=1/2). 676 mg of yellow solid 5-(2-acetyl-5-chlorophenyl)-6-methoxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one (acquired rate: 50.2%). LCMS: RT = 3.99 min, [M+H] ⁺ = 399.07.

Step E: Synthesis of 5-(2-Acetyl-5-chlorophenyl)-6-methoxypyridazin-3( 2H )-one

5-(2-Acetyl-5-chlorophenyl)-6-methoxy-2-(4-methoxybenzyl)pyridazin-3( 2H )-one (676 mg, 1.70 mmol) was added to the mixed solvent (4 ml, acetonitrile: water = 3:1), and then slowly added ceric ammonium nitrate (7.46 g, 13.60 mmol), the addition was completed, and the reaction was carried out at room temperature for 30 minutes.

The reaction was completed, quenched by adding water, the mixture was extracted with ethyl acetate (30 ml × 3 times), the organic phases were combined, and the organic phase was first washed with saturated brine (30 ml × 2 times), then dried with anhydrous sodium sulfate, and finally reduced pressure concentrate. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/n-hexane=1/1). 238 mg of 5-(2-acetyl-5-chlorophenyl)-6-methoxypyridazin-3( 2H )-one were obtained as a yellow solid (yield: 50.0%). LCMS: RT = 3.23 min, [M+H] ⁺ = 279.08.

Step F: Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1( 6H )-yl )-3-Phenylpropionamido) tert-butyl benzoate

5-(2-Acetyl-5-chlorophenyl)-6-methoxypyridazin-3( 2H )-one (50 mg, 0.18 mmol), (R)- tert-butyl 4-(2-(((4-nitrophenyl)sulfonyl)oxy)-3-phenylpropionamido)benzoate (113 mg, 0.22 mmol) and potassium carbonate ( 50 mg, 0.36 mmol) was added to N,N -dimethylformamide (2.0 mL) and reacted overnight at room temperature.

The reaction was completed, quenched by adding water, the mixture was extracted with ethyl acetate (10 ml × 3 times), the organic phases were combined, the organic phase was first washed with saturated brine (10 ml × 2 times), then dried with anhydrous sodium sulfate, and finally reduced pressure concentrate. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/n-hexane=1/2). 75 mg of pale yellow solid (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazine -1(6H) was obtained -yl)-3-phenylpropionamido) tert-butyl benzoate (yield: 66.7%). LCMS: RT = 4.53 min, [M+H] ⁺ = 602.13.

化合物A Step G: Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1( 6H )-yl )-3-Phenylpropionamido)benzoic acid

Compound A

At room temperature, (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazine -1(6H)- tert-butyl)-3-phenylpropionamido)benzoate (75 mg, 0.12 mmol) was added to dichloromethane (2.0 mL), trifluoroacetic acid (0.25 mL) was added dropwise, and the reaction was carried out at room temperature for 3 Hour.

After the reaction was completed, dichloromethane was evaporated to dryness and trifluoroacetic acid was dried with an oil pump. The obtained residue was dissolved in dichloromethane (1.0 mL) and added dropwise to n-hexane (10.0 mL) to precipitate a white solid, which was filtered off with suction. The filter cake was washed with n-hexane and dried to give 50 mg of white solid (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridine) oxazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid (yield: 76.5%). LCMS: RT = 3.98 min, [MH] ^- = 544.10. ¹ H NMR (500 MHz, DMSO) δ 12.79 (s, 1H), 10.52 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.72 ( d, J = 8.7 Hz, 2H), 7.69 (dd, J = 8.3, 2.1 Hz, 1H), 7.50 (d, J = 2.1 Hz, 1H), 7.37–7.23 (m, 4H), 7.19 (t, J = 7.1 Hz, 1H), 6.91 (s, 1H), 5.74 (dd, J = 10.2, 4.9 Hz, 1H), 3.67 (s, 3H), 3.52 (dd, J = 14.1, 10.3 Hz, 1H), 3.41 (dd, J = 14.1, 4.7 Hz, 1H), 2.53 (s, 3H).

Implement column 2

化合物A鈉鹽 (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3- Phenylpropionamido)benzoic acid sodium salt

Compound A sodium salt

At zero degrees Celsius, to a compound containing (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazine-1( 6H)-yl)-3-phenylpropionamido)benzoic acid (150.0 mg, 0.28 mmol) in methanol (10.0 mL) was added dropwise aqueous sodium hydroxide (sodium hydroxide; 6.72 mg, 0.28 mmol) Molar; water: 2.0 ml), and the reaction was maintained at this temperature for 5 hours.

The reaction was completed, methanol was evaporated, and the obtained aqueous solution was freeze-dried at low temperature to obtain 155.0 mg of Form A white solid (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3- Sodium Methoxy-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoate Form A (Yield: 97.5%). LCMS: RT = 2.00 min, [M+ H]+ = 546.31. ¹ H NMR (400 MHz, DMSO) δ 10.37 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.6 Hz, 2H), 7.68 (dd , J = 8.3, 2.2 Hz, 1H), 7.59 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 2.1 Hz, 1H), 7.36–7.24 (m, 4H), 7.18 (t, J = 7.1 Hz, 1H), 6.90 (s, 1H), 5.75 (dd, J = 10.2, 4.8 Hz, 1H), 3.68 (s, 3H), 3.47–3.37 (m, 2H), 2.53 (s, 3H).

Take 40 mg of Form A sample and add it to 1 ml of acetone, heat it to 50 degrees Celsius, add 20 microliters of water, and then add 320 mg of Form A sample, the solid is completely dissolved, and after 24 hours of stirring at 50 degrees Celsius, a solid precipitates out, and centrifugation to obtain Compound A sodium Salt crystalline solid Type A.

Example 3

化合物A鉀鹽 (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3- Phenylpropionamido)benzoic acid potassium salt

Compound A Potassium Salt

At zero degrees Celsius, to a compound containing (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazine-1( 6H)-yl)-3-phenylpropionamido)benzoic acid (100.0 mg, 0.18 mmol) in methanol (10.0 mL) was added dropwise aqueous potassium hydroxide (potassium hydroxide; 10.3 mg, 0.18 mmol; water: 2.0 ml), and the reaction was maintained at this temperature for 5 hours.

After the reaction was completed, methanol was evaporated, and the obtained aqueous solution was freeze-dried at low temperature to obtain 98.0 mg of white solid (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3-methoxy) yl-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid potassium salt (yield: 93.4%). LCMS: RT = 2.00 min, [M+H ] ⁺ = 546.22.

¹ H NMR (400 MHz, DMSO) δ 10.23 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.6 Hz, 2H), 7.68 (dd, J = 8.3, 2.2 Hz, 1H), 7.50 (d, J = 2.1 Hz, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.38–7.24 (m, 4H), 7.18 (t, J = 7.1 Hz, 1H), 6.89 (s, 1H), 5.75 (dd, J = 10.3, 4.7 Hz, 1H), 3.68 (s, 3H), 3.56–3.41 (m, 2H), 2.52 (s, 3H).

Implement column 4

化合物A葡甲胺鹽 (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3- Phenylpropionamido) benzoic acid meglumine salt

Compound A Meglumine Salt

(S)-4-(2-(4-(2-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl )-3-Phenylpropionamido)benzoic acid (1.0 g) and meglumine (358 mg) were added to 20 mL of acetone in a 1:1 equiv. ratio to make a suspension, under temperature cycling (1000 rpm , 50 ºC~5 ºC, 0.1 ºC/min, 2 cycles) suspension and stirring for 3 days to obtain (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)- 3-Methoxy-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid meglumine salt form A.

¹ H NMR (400 MHz, DMSO) δ 10.42 (s, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H), 7.69 (dd, J = 8.3, 2.2 Hz, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 2.1 Hz, 1H), 7.34–7.25 (m, 4H), 7.22-7.18 (m, 1H), 6.91 (s , 1H), 5.75 (dd, J = 10.2, 4.9 Hz, 1H), 3.79-3.74 (m, 1H), 3.68 (s, 3H), 3.67–3.65 (m, 1H), 3.60 (dd, J = 10.8 , 3.5 Hz, 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).

Example 5

化合物A鎂鹽 (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3- Phenylpropionamido) benzoic acid magnesium salt

Compound A magnesium salt

At zero degrees Celsius, to a compound containing (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazine-1( To sodium 6H )-yl)-3-phenylpropionamido)benzyl (100.0 mg, 0.18 mmol) in methanol (10.0 mL) was added aqueous magnesium chloride (MgCl; 16.8 mg, 0.18 mmol) dropwise ; water: 2.0 ml), and kept the temperature for 5 hours.

After the reaction was completed, methanol was evaporated to precipitate a white solid, which was filtered off with suction and dried to obtain 62.0 mg of a white solid (S)-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3) -Methoxy-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid magnesium salt (yield: 30.9%). LCMS: RT = 2.00 min, [ M+H] ⁺ = 546.20. ¹ H NMR (500 MHz, DMSO) δ 10.33 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.93 (s, 2H), 7.67 (dd, J = 8.3, 2.1 Hz, 1H), 7.59 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 1.9 Hz, 1H), 7.36–7.22 (m, 4H), 7.17 (t, J = 7.2 Hz, 1H), 6.88 (s, 1H), 5.73 (dd, J = 10.2, 4.8 Hz, 1H), 3.66 (s, 3H), 3.41 (dd, J = 14.3, 4.7 Hz, 2H), 2.51 (s, 3H) ).

Example 6

化合物A鈣鹽 (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3- Phenylpropionamido)benzoic acid calcium salt

Compound A calcium salt

At zero degrees Celsius, to a compound containing (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazine-1( Aqueous calcium chloride (calcium chloride; 20.0 mg, 0.18 mmol; water: 2.0 mL), and the reaction was maintained at this temperature for 5 hours.

After the reaction was completed, methanol was evaporated to precipitate a white solid, which was filtered off with suction, washed with water, and dried to obtain 58.0 mg of white solid (S)-4-(2-(4-(2-(2-acetyl-5-chlorophenyl)) -3-Methoxy-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid calcium salt (yield: 28.5%). LCMS: RT = 2.00 min , [M+H] ⁺ = 546.17.

Example 7

化合物A膽鹼鹽 (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl)-3- Phenylpropionamido)benzoic acid choline salt

Compound A Choline Salt

(S)-4-(2-(4-(2-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl )-3-Phenylpropionamido)benzoic acid and choline were added to acetone in an equivalent ratio of 1:1, under the temperature cycle (50 ^o C ~ 5 ^o C, 0.1 ^o C /min, 2 loops) Stir for 3 days to obtain a colloidal sample, and the colloidal sample is vacuum-dried at room temperature for 8 hours to obtain a solid powder (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-3- Methoxy-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid choline salt form A.

Comparative Example 1 Compound A1

化合物A1 Synthesis of (S)-4-(2-(4-(5-chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl)-6-oxopyrimidine- 1(6H)-yl)-3-phenylpropionamido)benzoic acid

Compound A1

The specific synthetic route is as follows:

Step A: Synthesis of 4-chloro-2-(tetramethyl-1,3,2-dioxaborol-2-yl)aniline

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 sulfite (75 mL). After nitrogen protection, it was heated at 80 °C for 5 hours. The reaction was cooled to room temperature. Water was added to dissolve the salt, and the reaction was filtered. The remaining solids were suspended in dichloromethane and the insoluble solids were filtered. The filtrate was concentrated and then purified by silica gel column chromatography to give 5.2 g of white solid 4-chloro-2-(tetramethyl-1,3,2-dioxaborol-2-yl)aniline (yield : 100%). LCMS: RT = 4.40 min, [M+H] ⁺ = 254.10.

Step B: Synthesis of 4-chloro-2-(6-methoxypyrimidin-4-yl)aniline

Combine 4-chloro-6-methoxypyrimidine (3.9 g, 15.4 mmol), sodium carbonate (3.2 g, 30.8 mmol), ethylene glycol dimethyl ether (16 mL), ethanol (2 mL) and water (2 ml) in a three-necked bottle. After a nitrogen blanket, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (1.3 g, 1.5 mmol) was added. 4-Chloro-2-(tetramethyl-1,3,2-dioxaborol-2-yl)aniline (3.31 g, 23.1 mmol) in ethylene glycol dimethyl ether (8 mL), the reaction was heated at 90 °C for 2 h. LCMS monitoring, after the reaction was completed, cooled to room temperature, filtered through celite, the filter cake was washed three times with ethyl acetate (30 ml), the filtrate and washings were combined, washed once with water, washed twice with saturated ammonium chloride, organic The phase was dried over anhydrous sodium sulfate, filtered, and spin-dried. The residue was purified by silica gel column chromatography to obtain 1.0 g of yellow solid 4-chloro-2-(6-methoxypyrimidin-4-yl)aniline (yield: 28%) . LCMS: RT = 3.95 min, [M+H] ⁺ = 236.04.

Step C : Synthesis of 4-{5-Chloro-2-[4-(trimethylsilyl)-1H-1,2,3-triazol-1-yl]-phenyl}-6-methoxy base-pyrimidine

4-Chloro-2-(6-methoxypyrimidin-4-yl)aniline (0.9 g, 3.8 mmol) was dissolved in acetonitrile (60 mL) and 3-methylbutylidene was added at 0 °C Nitrate (0.6 mL, 5.8 mmol), followed by azidotrimethylsilane (0.6 mL, 5.8 mmol) dropwise. Gas evolution was observed. After 10 minutes, the ice bath was removed and the reaction was allowed to warm to room temperature. After 1 hour, ethynyltrimethylsilane (1.8 mL, 11.4 mmol) and cuprous oxide (0.06 g, 0.36 mmol) were added and the reaction was stirred for an additional 1 hour. Ethyl acetate and saturated aqueous ammonium chloride were added to the reaction solution to separate the layers. The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. Further purification by silica gel column chromatography gave 730 mg of yellow solid 4-{5-chloro-2-[4-(trimethyl-silyl)-1H- 1,2,3 -triazol-1-yl] Phenyl}-6-methoxypyrimidine (yield: 45%). LCMS: RT = 2.04 min, [M+H] ⁺ = 360.10.

Step D: Synthesis of 4-[5-Chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl]-6-methoxypyrimidine

4-{5-Chloro-2-[4-(trimethylsilyl)-1H- 1,2,3 -triazol-1-yl]phenyl}-6-methoxypyrimidine (700 mg, 1.94 mmol) was dissolved in acetonitrile (20 mL), and N -chlorobutanediimide (0.9 g, 7.2 mmol) and silica gel (2.9 g, 50.44 mmol) were added to the solution. The reaction was stirred at 80°C for 1 hour. The reaction was then filtered to remove the silica gel and the collected silica gel was washed with ethyl acetate. The filtrate was washed with water, brine and concentrated. The residue was further purified by silica gel column chromatography to obtain 450 mg of yellow solid 4-[5-chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl]-6-methane oxypyrimidine (yield: 72%). LCMS: RT = 2.00 min, [M+H] ⁺ = 322.05.

Step E: Synthesis of 6-[5-Chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl]pyrimidin-4-ol

To 4-[5-chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl]-6-methoxypyrimidine (450 mg, 1.4 mmol) To a solution in acetic acid (3 mL) was added 48% aqueous hydrobromic acid (1.5 mL, 13.3 mmol). The mixture was stirred at 95°C for 1 hour. The reaction was concentrated to dryness, then partitioned with ethyl acetate and saturated sodium bicarbonate solution. The organic phase was concentrated, and the residue was purified by silica gel column chromatography to obtain 190 mg of yellow solid 6-[5-chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl]pyrimidine -4-ol (yield: 44%). LCMS: RT = 1.74 min, [MH] ^- = 305.97.

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

At room temperature, 6-[5-chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl]pyrimidin-4-ol (45 mg, 0.15 mM ear) and (R)-tert-butyl 4-(2-(((4-nitrophenyl)sulfonyl)oxy)-3-phenylpropionamido)benzoate (93 mg, 0.18 mg) mol) and potassium carbonate (40 mg, 0.3 mmol) were added to N,N-dimethylformamide (3.0 mL) and reacted overnight at room temperature. Water was added to the reaction solution to quench, the mixture was extracted with ethyl acetate (40 mL × 3 times), the organic phases were combined, and the organic phases were first washed with saturated brine (30 mL × 2 times), then dried over anhydrous sodium sulfate, and finally Concentrate under reduced pressure. The obtained residue was purified by silica gel column chromatography to obtain 150 mg of yellow liquid (S)-4-(2-(4-(5-chloro-2-(4-chloro- 1H -1,2,3-triazole-) 1-yl)phenyl)-6- oxopyrimidine -1(6H)-yl)-3-phenylpropionamido)benzoic acid tert-butyl ester (yield: 59%). LCMS: RT =2.00min, [M+H] ⁺ =631.18.

Step F: Synthesis of (S)-4-(2-(4-(5-chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl)-6- Oxopyrimidine -1(6H)-yl)-3-phenylpropionamido)benzoic acid

(S)-4-(2-(4-(5-Chloro-2-(4-chloro- 1H -1,2,3-triazol-1-yl)phenyl)-6-oxopyrimidine- tert-Butyl 1( 6H )-yl)-3-phenylpropionamido)benzoate (150 mg, 0.25 mmol) was dissolved in dichloromethane (2.0 mL). Subsequently, trifluoroacetic acid (0.5 mL) was added to the above solution, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure in an air bath. The resulting residue was preparatively purified to give 70 mg of white solid (S)-4-(2-(4-(5-chloro-2-(4-chloro- 1H -1,2,3-triazole-1-) ( yield : 59%).

LCMS: RT = 2.00 min, [M+H] ⁺ = 573.16. ¹ H NMR (400 MHz, CD ₃ OD) δ 10.36 (s, 1H), 8.36 (s, 1H), 8.18 (s, 1H), 7.87 (dd, J = 12.0, 5.1 Hz, 2H), 7.72 (d , J = 2.3 Hz, 1H), 7.66–7.47 (m, 4H), 7.28–7.07 (m, 5H), 6.22 (d, J = 0.8 Hz, 1H), 5.74 (dd, J = 10.5, 6.2 Hz, 1H), 3.49 (dd, J = 14.1, 6.3 Hz, 1H), 3.34–3.24 (m, 1H).

Comparative Example 2 Compound B

化合物B Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-5-methoxy-2-oxopyridinium-1( 2H )-yl)-3- Phenylpropionamido)benzoic acid

Compound B

The specific synthetic route is as follows:

Step A: Synthesis of (2,5-dimethoxypyridin-4-yl)boronic acid

2,5-Dimethoxypyridine (10.0 g, 71.9 mmol) was dissolved in dry tetrahydrofuran (40 mL), placed in a dry three-necked flask, under nitrogen protection, and stirred in a dry ice/ethanol bath for 15 minutes. , lithium diisopropylamide (20 mL, 2.0 M in THF) was slowly added dropwise to the reaction solution, the dropwise addition was completed after 30 minutes, and after stirring in a dry ice/ethanol bath for 3 h, triisopropyl borate (33.0 milliliter, 143.8 mmol) was added in the mixed solution, then naturally warmed up to room temperature and stirred at constant temperature for 18 hours. LCMS monitoring, after the reaction was complete, add dilute hydrochloric acid to the reaction solution to adjust pH to 3 ~ 4, after stirring for 15 minutes, The solvent was removed by rotary evaporation, and the residue was slurried with acetonitrile to obtain 10.6 g of white solid (2,5-dimethoxypyridin-4-yl)boronic acid (yield: 80%). LCMS: RT = 1.73 min, [M+H] ⁺ = 184.08.

Step B: Synthesis of 1-(4-Chloro-2-(2,5-dimethoxypyridin-4-yl)phenyl)ethan-1-one

2-Bromo-4-chloroacetophenone (14.8 g, 63.6 mmol) and (2,5-dimethoxypyridin-4-yl)boronic acid (9.7 g, 53.0 mmol) were dissolved in 1, 4-Dioxane (40 mL), potassium carbonate (14.6 g, 106 mmol) were dissolved in water (10 mL), placed in a dry three-necked flask, under nitrogen protection, [1,1'-bis( Diphenylphosphine) ferrocene] dichloropalladium dichloromethane complex (3.87 g, 5.3 mmol) was added to the reaction solution, after nitrogen protection, the temperature was raised to 100 ° C and stirred at constant temperature for 18 hours. LCMS monitoring, After the reaction was completed, it was cooled to room temperature, filtered through a pad of celite, the filter cake was washed three times with EA (30 mL), the filtrate and washings were combined, washed once with water, and washed twice with saturated ammonium chloride, and the organic phase was washed with anhydrous sodium sulfate. Dry, filter, spin dry, and the residue was purified by silica gel column chromatography to obtain 8.2 g of yellow solid 1-(4-chloro-2-(2,5-dimethoxypyridin-4-yl)phenyl)ethyl-1- Ketone (yield: 53%). LCMS: RT = 4.03 min, [M+H] ⁺ = 292.03.

Step C: Synthesis of 4-(2-Acetyl-5-chlorophenyl)-5-methoxypyridin-2( 1H )-one

1-(4-Chloro-2-(2,5-dimethoxypyridin-4-yl)phenyl)ethan-1-one (8.2 g, 28 mmol), pyridine hydrobromide (22 g, 140 mmol) was dissolved in N,N-dimethylformamide (20 mL), placed in a dry flask, under nitrogen protection, heated to 110 °C and stirred at constant temperature for 4 h. LCMS monitoring, after the reaction was complete, Cool to room temperature, drop the reaction into 100 ml of water, add 5% sodium carbonate to adjust the pH to 10-11, extract four times with DCM (40 ml × 4), combine the organic phases, and dry the organic phases over anhydrous sodium sulfate, Filtration, spin-dried, the residue was dissolved in DCM (10 mL), then added dropwise to n-hexane (120 mL), a large amount of solid was precipitated, filtered, and the filter cake was collected, that is, the crude product, which was further purified by silica gel column chromatography to obtain 6.4 g 4-(2-Acetyl-5-chlorophenyl)-5-methoxypyridin-2( 1H )-one (yield: 82%) as a yellow solid. LCMS: RT = 3.81 min, [MH] ^- = 277.04.

Step D: Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-5-methoxy-2-oxopyridinium-1( 2H )-yl) -3-Phenylpropionamido) tert-butyl benzoate

At room temperature, 4-(2-acetyl-5-chlorophenyl)-5-methoxypyridin-2( 1H )-one (1.5 g, 5.4 mmol) and (R)-4 tert-butyl-(2-(((4-nitrophenyl)sulfonyl)oxy)-3-phenylpropionamido)benzoate (4.0 g, 7.6 mmol) and potassium carbonate (1.5 g, 10.8 mmol) was added to N,N-dimethylformamide (20.0 mL) and reacted at room temperature overnight. Water was added to the reaction solution to quench, the mixture was extracted with ethyl acetate (40 mL×3 times), and the organic phases were combined. The organic phase was first washed with saturated brine (30 mL×2 times), then dried over anhydrous sodium sulfate, and finally Concentrate under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: ethyl acetate/n-hexane = 1/2) to obtain 1.9 g of yellow solid (S)-4-(2-(4-(2-acetyl-5) -Chlorophenyl)-5-methoxy-2-oxopyridinium-1( 2H )-yl)-3-phenylpropionamido)benzoic acid tert-butyl ester (yield: 59%). LCMS: RT = 4.42 min, [M+H] ⁺ = 601.18.

Step E: Synthesis of (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-5-methoxy-2-oxopyridinium-1( 2H )-yl) -3-Phenylpropionamido)benzoic acid

(S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-ethoxy-6- oxopyridazin -1(6H)-yl)-3 - tert-butyl phenylpropionamido)benzoate (1.9 g, 3.2 mmol) was dissolved in dichloromethane (12.0 mL). Subsequently, trifluoroacetic acid (3 mL) was added to the above solution, followed by stirring at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure in an air bath. The resulting residue was purified by beating with methanol to give 1.0 g of yellow solid (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-5-methoxy-2-oxopyridine) Onium-1( 2H )-yl)-3-phenylpropionamido)benzoic acid (yield: 59%). LCMS: RT = 3.88 min, [MH] ^- = 543.06. ¹ H NMR (400 MHz, DMSO) δ 10.82 (s, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 8.3 Hz, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.61 (dd, J = 8.4, 2.3 Hz, 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, 6.6 Hz, 1H), 3.53 (s, 3H), 3.49-3.44 (m, 2H), 2.36 (s, 3H).

Comparative Example 3 CN201680058331 Example 143 Compound

The corresponding target compound was obtained with reference to the preparation method of Example 143 of CN201680058331.

Example 8: Detection of the biological activity of the compounds of the present invention on the inhibition of human coagulation factor XIa by absorptiometry

1. Experimental materials

Enzyme: Human Factor XIa (ENZYME RESEARCH, Cat. No. HFXIa 1111a)

Substrate: S- ^2366TM : (CHROMOGENIX, Cat. No. 82109039)

Buffer: 145 mM NaCl, 5 mM KCl, 1 mg/mL PEG 8000, 30 mM HEPES, pH 7.4

2. Experimental steps

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 μM; /mL) of FXIa enzyme solution, the blank wells were replaced by 98 μL of buffer solution, and then 2 μL 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.

Finally, 100 μL of 800 μM substrate was added to each well, and its absorbance was measured at 405 nm.

3. Data processing

Curve fitting was performed with GraphPad Prism software, and IC ₅₀ values were calculated, as shown in Table 1. [Table 1] IC ₅₀ of the compounds of the present invention inhibiting human FXIa Example hFXIa _IC50 (nM) 1 7.61

Conclusion: The compounds of the present invention have obvious inhibitory activity on human FXIa.

Example 9: Determination of the anticoagulant effect of the compounds of the present invention on human plasma in vitro

1. Experimental materials

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, and the plasma was collected, divided into EP tubes, and stored at -80 °C.

Reagents: APTT assay kit (activated partial thromboplastin time assay kit, mindray), calcium chloride solution.

Instrument: coagulation meter (mindray, C2000-A)

2. Experimental method

Thaw the frozen human plasma in aliquots at room temperature and mix well. 10mM test compound dissolved in 100% DMSO was diluted with 100% DMSO to 1500, 750, 375, 187.5, 93.75, 46.88, 23.44, 11.72 μM; 98 μL of human plasma was added to a 1.5 mL EP tube, followed by 2 μL of different concentrations 2 μL of 100% DMSO was added to the blank group, incubated in a water bath at 37°C for 10 min, and the samples were placed in the corresponding position in the coagulation instrument to perform APTT determination of the compounds.

3. Data processing

Curve fitting was performed with GraphPad Prism software, and EC1.5× and EC2× values were calculated respectively, that is, 1.5 times and 2 times the concentrations of the compounds corresponding to the APTT of the blank control group. The results are shown in Table 2. [Table 2] The anticoagulant effect of the compounds of the present invention on human plasma in vitro Example aPTT EC1.5×(μM) aPTT EC2×(μM) 1 0.641 2.817

Conclusion: It can be seen from Table 2 that the compound of the present invention has obvious anticoagulant effect on human plasma.

Example 10: Investigation of the pharmacokinetic characteristics of the compounds of the present invention

1. Experimental materials

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.

Reagents: DMSO (dimethyl sulfoxide), PEG-400 (polyethylene glycol 400), normal saline, heparin, acetonitrile, formic acid, and propranolol (internal standard) are commercially available.

Instrumentation: Thermo Fisher Scientific LC-MS (U300 UPLC, TSQ QUANTUMN ULTRA triple quadrupole mass spectrometer).

2. Experimental method

The compound was weighed and dissolved in DMSO-PEG-400-physiological saline (5:60:35, v/v/v) system, and after intravenous or intragastric administration to rats/monkeys, 5 min (gastrically not taken), After 15min, 30min, 1h, 2h, 4h, 6h, 8h, and 24h, 200 μL 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. Accurately draw an appropriate amount of compound stock solution, add acetonitrile and dilute to make standard series solutions. Accurately aspirate 20 μL of each of the above standard series solutions, add 180 μL of blank plasma, vortex and mix to prepare plasma samples with plasma concentrations of 1, 3, 10, 30, 100, 300, 1000, 3000 and 5000 ng/mL. , a double-sample analysis was performed for each concentration, and a standard curve was established. Take 20 μL of plasma, add 200 μL of internal standard propranolol (5 ng/mL) in acetonitrile solution, vortex and mix, centrifuge at 4000 rpm for 5 min, and take the supernatant for LC-MS analysis. LC-MS detection conditions are as follows:

Chromatographic column: Thermo Scientific HYPERSIL GOLD C-18 UPLC column, 100*2.1mm, 1.9μm.

Mobile phase: water (0.1% formic acid)-acetonitrile for gradient elution according to the table below time (min) Water (with 0.1% formic acid) Acetonitrile 0 90% 10% 0.6 90% 10% 1 10% 90% 2.6 10% 90% 2.61 90% 10% 4 90% 10%

3. Data processing

After LC-MS detection of blood drug concentration, using WinNonlin 6.1 software, non-compartmental model method to calculate pharmacokinetic parameters. The results are shown in Tables 3, 4 and 5. [Table 3] Rat pharmacokinetic parameters of the compounds of the present invention Example Mode of administration/dose (mg/kg) Tmax (h) Cmax (ng/mL) AUC (h*ng/mL) T _1/2 (h) CL (mL/min/kg) Vss (L/kg) F (%) 1 iv/1 0.083 2500 1020 0.181 16.4 0.269 / ig/10 1.25 768 2510 2.52 / / 24.6 Compound A1 iv/1 0.083 4600 1410 0.589 11.9 0.124 / ig/5 0.5 180 576 1.26 / / 8.2 Embodiment 143 (CN201680058331) iv/1 0.083 4900 2780 2.4 6.0 0.341 / ig/5 2.0 18.1 105 7.88 / / 0.8 [Table 4] Cynomolgus monkey pharmacokinetic parameters of the compounds of the present invention Example Mode of administration/dose (mg/kg) Tmax (h) Cmax (ng/mL) AUC (h*ng/mL) T _1/2 (h) CL (mL/min/kg) Vss (L/kg) F (%) 1 iv/1 0.083 2690 1430 2.83 12 0.65 / ig/10 2.5 198 2480 7.07 / / 17.3 Compound B iv/1 0.083 8759 4220 1.2 4.1 0.2 / ig/10 2.00 108 1486 8.0 / / 4.1 [Table 5] Beagle dog pharmacokinetic parameters of the compounds of the present invention Example dosing Tmax Cmax AUC T _1/2 CL Vss F Mode/Dose (mg/kg) (h) (ng/mL) (h*ng/mL) (h) (mL/min/kg) (L/kg) (%) 1 iv/1 0.083 2579.7 1405.1 4.2 11.8 0.8 / ig/10 1.25 2320 9232.2 3.6 / / 65.5

CONCLUSION: 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.

Example 11: Investigation of in vivo pharmacodynamics of the compounds of the present invention

This example refers to the compilation of preclinical research guidelines for new drugs (western medicines) (Pharmaceutics, Pharmacology, Toxicology) and requires the following tests:

11.1 Investigating the efficacy of the compound of Example 4 on electrical stimulation-induced thrombus in the inferior vena cava in rabbits

Test method: The rabbits were fasted for 24 hours and could not help but water. According to their body weight, they were divided into a model control group, the compound of Example 4 at a dose of 30 mg/kg/h (calculated as free acid), and the positive drug 1 Rivaroxaban at a dose of 3 mg/kg. /h dose group and positive drug 2 enoxaparin sodium 20 mg/kg/h dose group were administered by intravenous infusion once, at a rate of 1.4 ml/kg/h for 2 hours, and the model control group was given an equal volume of vehicle.

Electrical stimulation was performed 1 hour after administration, and 15 minutes before electrical stimulation, the rabbits were anesthetized by injection of 40 mg/kg sodium pentobarbital into the ear marginal vein, supine immobilized, skin preparation and disinfection of the abdomen, laparotomy through the midline of the abdomen and linea alba, and saline. The gauze protects the bowel and pushes it to one side, cut the retroperitoneum, dissociate the inferior vena cava (ranging from the left renal vein below the opening of the iliolumbar vein), ligate the proximal end, and narrow the distal end at an interval of 2 cm (use a diameter of 2 mm). The plastic tube is placed parallel to the blood vessels, ligated together, and the plastic tube is slowly drawn out after ligation), 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 min. During electrical stimulation, the stenotic site was clamped with an arterial clip and the vein was blocked. After the electrical stimulation was over, the arterial clips were released, and the rabbits were placed until 30 min after the electrical stimulation stopped.

After the electrical stimulation, a scalpel was used to make an incision of 2 mm in length and 1 mm in depth in the kidney, and the bleeding time was measured. After the test, the thrombus at the stimulation site was taken, the weight of the thrombus was weighed, and the inhibition rate of thrombus formation was calculated. Test results: Intravenous infusion of the compound of Example 4 and the positive control drug can significantly reduce the weight of thrombus in the inferior vena cava in rabbits. 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. [Table 6] The results of drug efficacy of the compound of Example 4 in rabbit inferior vena cava thrombosis test substance Example 4 compound Rivaroxaban enoxaparin Dosage (mg/kg/h) 30 3 20 Thrombus inhibition rate (%) 54.3*** 59.8*** 54.4*** Renal bleeding time multiplier 1.47 5.17*** 3.87*** ***P < 0.001 vs model control

Test conclusion: When the compound of Example 4 reaches the same efficacy level as rivaroxaban and enoxaparin, the bleeding risk is significantly reduced.

11.2 Investigation of the efficacy of the compound of Example 2 on ferric chloride-induced carotid artery thrombosis in rabbits

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 2 h, the instillation volume was 1.4 mL/kg/h, and the model control group was given an equal volume of vehicle. After intravenous infusion for 1 hour, start modeling: add 20 μL 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. .

After the rabbit thrombus experiment, cut the abdominal skin and abdominal muscle, then peel off the renal capsule, put the kidney into pre-warmed normal saline at 37°C, and use a disposable 1ml syringe to puncture the kidney tissue to ensure a depth of 5mm to cause renal failure. Bleeding, renal bleeding time was observed in normal saline.

Test results: The clopidogrel single-agent 3mg/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 10mg/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. [Table 7] The results of the drug effect of the compound of Example 2 on carotid artery thrombosis in rabbits drug name dose Thrombus inhibition rate Renal bleeding time (mg/kg/h) (%) multiplier Clopidogrel (clo) 3 49.1%** 1.09 10 72.7%*** 1.83** Example 2 Compound 10 47.9%** 1.04 **P < 0.01, ***P < 0.001 vs model control

Conclusion: 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, and at the same time reduced the risk of bleeding.

11.3 Investigation of the efficacy of the compound of Example 2 on ferric chloride-induced arteriovenous bypass thrombosis in rabbits

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 right carotid artery and left jugular vein, and the right femoral artery and vein were surgically separated. The right carotid artery and left jugular vein were used to connect the arteriovenous bypass device for arteriovenous thrombosis modeling; the right femoral vein was cannulated for intravenous infusion. All patients were administered by intravenous infusion, with an infusion volume of 1.4 ml/kg/h, and the model control group was given an equal volume of vehicle. After the microinfusion pump was infused at a constant rate for 60 minutes, 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.

Before modeling, the rabbits were anesthetized by injection of 40 mg/kg sodium pentobarbital 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 the right carotid artery and left jugular vein were separated first. At the distal end of the carotid artery, clamp the proximal end with a vascular clip, cut the blood vessel near the inner side of the ligation line at the distal end, connect the arteriovenous bypass device filled with normal saline, and ligate and fix it with two silk non-absorbable sutures. Open the vascular clip; the left jugular vein is connected to the arteriovenous bypass device in the same way, and is ligated and fixed with two silk non-absorbable sutures, and 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. At the same time, the renal bleeding time was detected. Using a 1 ml disposable syringe needle, a needle hole with a depth of 3 mm was pierced on the surface of the kidney from which the renal capsule was peeled off, and the bleeding time was measured.

Test results: The compound of Example 2 at a dose of 30 mg/kg/h can significantly reduce the weight of rabbit arteriovenous bypass thrombus, and the thrombus inhibition rate is 61.9%. The test results are shown in Table 8. [Table 8] The results of the drug effect of the compound of Example 2 on arteriovenous bypass thrombosis in rabbits group Pharmacodynamic parameters result Compound of Example 2 (30mg/kg/h) Thrombus inhibition rate (%) 61.9*** Renal bleeding time multiplier 1.1 ***P<0.001 vs model control

Test conclusion: 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.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

none.

none.

none.

Claims (11)

Use of an FXIa inhibitor compound or a salt thereof in the preparation of a medicament for preventing and/or treating arterial thrombosis, venous thrombosis, and arteriovenous bypass thrombosis, wherein the compound structural formula is as follows,

. The use according to claim 1, wherein the salt structure of the FXIa inhibitor compound is as follows,

Wherein, n is 0.5-3; M forms a salt with a carboxyl group, and the salt is selected from at least one of lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, aluminum salts, iron salts, zinc salts or ammonium salts; Or the salt is selected from methylamine salt, dimethylamine salt, trimethylamine salt, ethylamine salt, diethylamine salt, triethylamine salt, isopropylamine salt, 2-ethylaminoethanol salt, pyridine salt, picoline salts, ethanolamine salts, diethanolamine salts, ammonium salts, tetramethylammonium salts, tetraethylammonium salts, triethanolamine salts, oxidine salts, oxazine salts, morpholine salts, lysine salts, arginine salts, L-arginine salt, histidine salt, L-histidine salt, meglumine salt, dimethylglucamine salt, ethyl glucamine salt, dicyclohexylamine salt, 1,6-hexylamine salt Diamine salt, glucosamine salt, sarcosinate, serine alcohol salt, trishydroxymethyl aminomethane salt, amino propanediol salt, 1-amino-2,3,4-butanetriol salt, L-lysine at least one of acid salts, ornithine salts or choline salts. The use according to claim 2, wherein n is 0.5, 1, 1.5, 2, 2.5 or 3. The use according to claim 2, wherein the salt is selected from the group consisting of sodium salt, potassium salt, meglumine salt, calcium salt, magnesium salt and choline salt. The use according to claim 2, wherein the salt is selected from sodium salt, n=1; potassium salt, n=1; choline salt, n=1; meglumine salt, n=1; calcium salt, n=0.5; magnesium salt, n=0.5. The use according to any one of claims 1 to 5, wherein the compound or a salt thereof is in a crystalline form, or an amorphous form, or a mixture thereof. The use according to any one of claims 1 to 5, wherein one or more hydrogen atoms of the compound or its salt are substituted with the isotope deuterium. The use according to any one of claims 1 to 5, wherein the compound or its salt and one or more pharmaceutically acceptable carriers form a pharmaceutical composition. The use according to any one of claims 1 to 5, wherein the arterial thrombus causes arterial embolic diseases, including coronary heart disease, myocardial infarction, ischemic stroke, peripheral arterial disease, atrial fibrillation and valvular heart disease ; Said venous thrombosis leads to venous thromboembolic diseases, including deep vein thrombosis after joint replacement, pulmonary embolism, deep vein thrombosis and arteriovenous thrombosis after dialysis. The use according to claim 9, wherein the deep venous thrombosis after joint replacement includes venous thrombosis after total knee replacement and venous thrombosis after hip replacement. 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,

.