TW202222315A - Salts of FXIa inhibitor compounds, preparation method therefor, and pharmaceutical use thereof - Google Patents

Abstract

Provided are a series of salts of FXIa inhibitor compounds, pharmaceutical compositions containing said compounds, and a use of said compounds in drugs for the treatment of diseases such as thromboembolism.

Description

Salts of FXIa inhibitor compounds, pharmaceutical compositions including the same, and uses thereof

The invention belongs to the technical field of chemical medicine, and provides a series of salts of FXIa inhibitor compounds. The present invention also relates to pharmaceutical compositions comprising the salts of these compounds and their use in medicines for treating diseases such as thromboembolism.

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.

。 In the previous application PCT/CN2020/117257, the applicant applied for a series of FXIa inhibitor compounds including compound A represented by the following formula:

.

The present invention provides a series of salts of oxopyridazinamide derivatives, their preparation method and their application in medicine.

如圖20所示，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。 Specifically, the present invention provides salts of FXIa inhibitor compounds represented by formula (I),

As shown in Figure 20, wherein: n is 0.5-3; M forms a salt with a carboxyl group, and the salt is selected from lithium salt, sodium salt, potassium salt, calcium salt, magnesium salt, aluminum salt, iron salt, zinc salt or ammonium salt At least one of the 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 salts, picoline salts, ethanolamine salts, diethanolamine salts, ammonium salts, tetramethylammonium salts, tetraethylammonium salts, triethanolamine salts, pyridinium salts, oxazine salts, morpholine salts, lysine salts , arginine, L-arginine, histidine, L-histidine, meglumine, dimethylglucamine, ethylglucamine, dicyclohexylamine , 1,6-hexanediamine salt, glucosamine salt, sarcosinate, serine alcoholate, trihydroxymethylaminomethane salt, aminopropanediol salt, 1-amino-2,3,4-butanetriol at least one of salts, L-lysine salts, ornithine salts or choline salts.

As a preferred technical solution of the present invention, n is 0.5, 1, 1.5, 2, 2.5 or 3, particularly preferably n=1 or 0.5.

As a preferred technical solution of the present invention, the salt is selected from sodium salt, potassium salt, meglumine salt, calcium salt, magnesium salt and choline salt.

As a preferred technical solution of the present invention, the salt is selected from sodium salt, n=1; potassium salt, n=1; meglumine salt, n=1; choline salt, n=1; calcium salt, n=0.5; magnesium salt, n=0.5.

As a preferred technical solution of the present invention, the salt is in crystalline form, or amorphous form, or a mixture thereof.

As a preferred technical solution of the present invention, the salt is a sodium salt, n=1, the salt is a crystal form, and the crystal form is represented by 2θ angle at 9.32°, There are characteristic peaks at 15.34°, 16.24°, 18.41°, 19.48°, and 24.05°, and the error is ±0.2°; more preferably, 5.59°, 7.81°, 9.83°, 10.50°, 11.24°, 13.52°, 14.60° , 14.87°, 16.93°, 20.39°, 21.02°, 21.77°, 23.53°, 24.99°, 25.90°, 26.62°, 27.22° have characteristic peaks, the error is ±0.2°; the better X-ray diffraction pattern is shown in the figure 6 or as shown in Figure 9.

As a preferred technical solution of the present invention, the DSC spectrum of the crystal form has a maximum absorption peak at 70.01°C ± 2°C; the preferred DSC spectrum is shown in Figure 7; the preferred TG spectrum of the crystal form is shown in the figure 8 shown.

As a preferred technical solution of the present invention, the salt is a sodium salt, n=1, the salt is amorphous, and the amorphous X-ray diffraction diagram has no obvious characteristic peaks; preferably X The ray diffraction pattern is shown in Figure 5.

As a preferred technical solution of the present invention, the salt is meglumine salt, n=1, the salt is a crystal form, and the crystal form is represented at 9.33 by 2θ angle in the X-ray diffraction diagram. There are characteristic peaks at and 18.79°, and the error is ±0.2°; further preferably, there are characteristic peaks at 10.32°, 13.74°, 16.18°, 24.74°, and the error is ±0.2°; the better X-ray diffraction pattern is shown in the figure 10 or as shown in Figure 12.

As a preferred technical solution of the present invention, the DSC spectrum of the crystal form The DSC of the crystal form has a maximum absorption peak at 122.7°C±2°C; the preferred DSC spectrum is shown in FIG. 11 .

As a preferred technical solution of the present invention, the salt is meglumine salt, n=1, the salt is amorphous, and there is no obvious characteristic peak in the X-ray diffraction diagram of the amorphous; The best X-ray diffraction pattern is shown in Figure 13 or 16.

As a preferred technical solution of the present invention, the amorphous DSC spectrum has a maximum absorption peak at 80.1 °C ± 2 °C; the preferred DSC spectrum is shown in Figure 14; the preferred amorphous TG spectrum is shown in Figure 15 shown.

As a preferred technical solution of the present invention, more than one hydrogen atom of the compound is substituted with the isotope deuterium.

The present invention further provides a pharmaceutical composition comprising the aforementioned salt, and one or more pharmaceutically acceptable carriers.

The present invention further provides the use of the salt in the preparation of a medicament for the treatment of FXIa-related diseases, preferably a thrombus-related disease.

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 that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no 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 described in further detail below with reference to the embodiments and the accompanying drawings, 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-d ₆ ), deuterated chloroform (CDCl3), 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.

Unless otherwise stated, the crystal form and amorphous form of the present invention are detected by the following equipment and conditions: X-ray powder diffraction (XRPD), the XRPD pattern is collected on the X-ray powder diffraction analyzer produced by PANalytacal, and the scanning parameters are as follows: parameter Instrument 1 model Empyrean Cu, Kα X-ray Kα1(Å): 1.540598 Kα2(Å): 1.544426 Kα2/Kα1 intensity ratio: 0.50 X-ray tube settings 45kV, 40mA Divergence slit Automatic scan mode Continuous Scanning range (º2Theta) 5-45 Scan time per step (s) 17.4 Scan step size (º2Theta) 0.019 testing time ~5min 30s

Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), TGA and DSC graphs were collected on TA Q5000/5500 thermogravimetric analyzer and TA2500 differential scanning calorimeter, respectively. The test parameters are as follows: parameter TGA DSC method Linear heating Linear heating sample tray Aluminium pan, open Aluminium pan, with/without gland temperature range Room temperature - set the accent temperature 25ºC - set the key temperature Scanning speed (ºC/min) 10 10 Protective gas nitrogen nitrogen

Dynamic moisture sorption (DVS) curves were collected on DVS Intrinsic of SMS (Surface Measurement Systems). The relative humidity at 25 ^o C was corrected for the deliquescence points of LiCl, Mg(NO ₃ ) ₂ and KCl. The list of DVS test parameters is as follows: parameter set value temperature 25ºC Sample size 10-20mg Shielding gas and flow N ₂ , 200mL/min dm/dt 0.002%/min Minimum dm/dt equilibration time 10min maximum equilibration time 180min RH range 0%RH-95%RH RH gradient 10% (0%RH-90%RH, 90%RH-0%RH) 5% (90%RH-95%RH, 95%RH-90%RH)

Ion chromatography (IC), instrument and analysis conditions are as follows: Ion Chromatograph Thermo Fisher ICS-1100 Column IonPac AS18 Analytical Column, 250*4mm mobile phase 25mM methanesulfonic acid Injection volume 25µL flow rate 1.0mL/min temperature 35ºC column temperature 35ºC current 80mA execution time Sodium/potassium ion is 7.0min

Example 1

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

Compound A

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), 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, and 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.

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

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) oxazine-1(6H)-yl)-3-phenylpropionamido)benzoic acid (yield: 76.5%), obtained by testing (S)-4-(2-(4-(2-acetyl) The compound is crystalline form A. 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).

The X-Ray data of the crystal form A are shown in the following table, and the X-Ray is shown in FIG. 1 . Pos. [°2θ] Rel. Int. [%] 8.81 100 9.14 19.83 10.17 8.77 11.59 34.72 12.79 13.83 15.22 15.11 15.65 47.01 17.43 9.08 18.37 16.6 18.62 7.29 18.88 6.66 19.14 7.94 20.40 8.37 22.44 7.86 23.18 17.54 23.78 14.02 24.13 13.25 25.41 10.91 25.75 5.81 8.81 100 9.14 19.83 10.17 8.77 11.59 34.72 12.79 13.83 15.22 15.11 15.65 47.01 17.43 9.08 18.37 16.6 18.62 7.29 18.88 6.66 19.14 7.94 20.40 8.37

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

Example 2.1

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). Among them, the XRPD diagram, TGA diagram and DSC diagram of Form A are shown in Figures 2, 3 and 4, respectively.

Example 2.2

(S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)-3- Phenylpropionamido)benzoic acid (7.5 g, 13.7 mmol) was added to purified water (75.0 mL) and stirring was turned on. At zero degrees Celsius, slowly add a pre-prepared 5% sodium hydroxide solution (sodium hydroxide, 0.55 g, 13.7 mmol; purified water, 10.0 ml) dropwise for about 30 minutes.

After the dropwise addition, continue to add 5% sodium hydroxide solution to adjust the pH of the aqueous solution to 8~9. Warm up to room temperature and stir for 30-60 minutes to ensure (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazine) -1(6H)-yl)-3-phenylpropionamido)benzoic acid was completely dissolved. Filtered, the aqueous solution was freeze-dried at low temperature to obtain 7.5 g of (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazine-1( Amorphous sample of sodium 6H)-yl)-3-phenylpropionamido)benzoate (yield: 96%; purity: 99.46%).

The (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)-3 The amorphous XRPD spectrum of -phenylpropionamido) sodium benzoate is shown in Figure 5.

Example 2.3

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 stirring at 50 degrees Celsius for 24 hours, a solid is precipitated, and centrifuged to obtain a crystalline solid Type A.

Among them, the X-Ray data of Type A are shown in the following table; the better XRPD, TGA and DSC images are shown in Figures 6, 7 and 8, respectively. Pos. [°2θ] Rel. Int. [%] 5.62 12 7.84 9.21 9.34 100 9.84 16.1 10.54 15.99 11.27 12.92 13.58 16.54 14.67 16.25 14.91 13.94 15.37 22.95 16.20 22.73 16.92 13.36 17.62 10.11 18.49 42.42 19.52 27.2 19.81 16.73 20.48 13.3 21.05 13.14 21.77 12.99 23.57 20.5 24.09 27.84 24.89 18.84 25.83 12.93 26.68 11.88 27.17 14.23

Example 2.4

A 200 mg sample of Form A was taken, added to 1 ml of acetone and 0.02 ml of water, heated to 50 degrees Celsius, suspended at room temperature for 4 days, and centrifuged to obtain a crystalline solid Type A.

Among them, the X-Ray data of Type A are shown in the following table; the better XRPD diagram is shown in Figure 9. Pos. [°2θ] Rel. Int. [%] 5.56 9.72 7.79 7.23 9.30 100 9.82 11.97 10.46 12.53 11.21 15.89 13.46 19.39 14.53 23.37 14.83 28.83 15.31 29.6 16.29 29.47 16.93 23.28 18.33 39.74 19.44 33.53 20.30 15.78 20.99 18.35 21.76 21.64 23.48 16.39 24.01 20.9 25.10 13.54 25.97 12.42 26.56 8.93 27.27 11.47

Comprehensive examples 2.3 and 2.4, according to the average value, the 2θ angle of sodium salt Type A indicates that there are strong characteristic peaks at 9.32, 15.34, 16.24, 18.41, 19.48, 24.05°, and the error is ±0.2°; There are characteristic peaks at 5.59, 7.81, 9.83, 10.50, 11.24, 13.52, 14.60, 14.87, 16.93, 20.39, 21.02, 21.77, 23.53, 24.99, 25.90, 26.62, 27.22° with an error of ±0.2°; better X-ray The diffraction pattern is shown in Figure 6 or Figure 9.

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) Potassium-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoate (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

Example 4.1

(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 stirred 1:1 equiv in ethanol at room temperature for 3 days to give (S)-4-(2-( 4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)-3-phenylpropionamino)benzoic acid meglumine Amine 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).

Among them, the X-Ray data of the meglumine salt crystal form A is preferably as shown in the following table; the XRPD pattern of the meglumine salt crystal form A is more preferably as shown in FIG. 10 , and the TGA/DSC chart is shown in FIG. 11 . Pos. [°2θ] Rel. Int. [%] 9.30 100 10.27 27.47 13.78 14.56 16.19 21.89 18.82 66.78 24.60 29.47

Example 4.2

(S)-4-(2-(4-(2-(2-Acetyl-5-chlorophenyl)-3-methoxy-6- oxopyridazin -1(6H)-yl )-3-Phenylpropionamido)benzoic acid (5.0 g) and meglumine (1786.8 mg) were added to 90 ml of acetone in an equivalent ratio of 1:1 to make a suspension, and 20 ml of meglumine salt crystals were added. The Form A suspension was used as a seed crystal, and was suspended and stirred (300 rpm) at room temperature for about 23 hours, vacuum filtered at room temperature, and the solid was vacuum dried at room temperature for 3 days to obtain 7.17 grams of meglumine salt crystal form A sample (Yield: 89%).

The X-Ray data of the obtained meglumine salt crystal form A is preferably as shown in the following table; the XRPD pattern of the better meglumine salt crystal form A is shown in Figure 12: Pos. [°2θ] Rel. Int. [%] 9.36 70.51 10.37 35.78 13.70 11.69 16.17 25.68 18.76 100 24.88 50.42

Comprehensive examples 4.1 and 4.2, according to the average value, the 2θ angle of meglumine salt crystal form A indicates that there are strong characteristic peaks at 9.33 and 18.79°, and the error is ±0.2°; There are characteristic peaks at 16.18 and 24.74°, with an error of ±0.2°; a better X-ray diffraction pattern is shown in Figure 10 or Figure 12.

Example 4.3

(S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl )-3-phenylpropionamido)benzoic acid (60 g, 109.89 mmol) and meglumine (21.5 g, 109.89 mmol) were added to a mixed solution of acetone and purified water (acetone, 1000 mL). ; purified water, 100.0 ml), stir to dissolve. Stir at room temperature for more than 24 hours.

After the reaction, the acetone was removed by concentration under reduced pressure, and the obtained aqueous solution was freeze-dried at low temperature to obtain 80.0 g of (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-3-methoxyl group) -6-oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid meglumine salt amorphous sample (yield: 98%; purity, 99.9%).

Among them, the XRPD pattern of the amorphous meglumine salt is shown in FIG. 13 , and the DSC and TGA patterns of the amorphous meglumine salt are shown in FIGS. 14 and 15 .

The instruments and specific measurement conditions are as follows: Thermogravimetric analyzer Model: Q500 U.S. TA Chinese Pharmacopoeia 2015 _Edition Four General Principles 0661 Thermal analysis method, N atmosphere (balance chamber flow rate 50ml/min, sample chamber flow rate 100ml/min), detection temperature range: 40 ° C -800 ° C, heating rate 20 ° C / min Differential Scanning Calorimeter Model: Q20 American TA GB/T 28742-2012 Determination of melting point of solid organic chemicals, differential scanning calorimetry, _N2 atmosphere (flow rate 50ml/min), detection temperature range: 20°C -200°C, heating rate 10°C /min Polycrystalline X-ray Derivatizer Model: D8 Advance Bruker D8 Chinese Pharmacopoeia 2015 Edition Four General Principles 0451 The second method of X-ray diffraction, Ni radiation, filter 0.02mm, Sora slit 2.5°, divergence slit 0.6mm, X-ray tube voltage 40kV, X-ray tube current 40mA, scanning range 4-60°(2θ), step size 0.01°, scanning time 0.2s.

Example 4.4

(S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl )-3-phenylpropionamido)benzoic acid (4.30 kg, 7.87 mol) and meglumine (1.62 kg, 8.30 mol) were added to a mixed solution of acetone and purified water (acetone, 65.6 kg; purified water, 8.2 kg), stir to dissolve. Stir at room temperature for more than 48 hours.

After the reaction, the acetone was removed by concentration under reduced pressure, and the obtained aqueous solution was freeze-dried at low temperature to obtain 5.80 kg of (S)-4-(2-(4-(2-acetyl-5-chlorophenyl)-3-methoxyl group) -6-Oxopyridazin-1(6H)-yl)-3-phenylpropionamido)benzoic acid meglumine salt amorphous sample (yield: 98%; purity, 99.78%). Among them, the amorphous XRPD pattern of the meglumine salt is shown in FIG. 16 .

Instrument and specific measurement conditions:

The X-ray powder diffraction (XRD) spectrum was detected by a PANalytical Empyrean X-ray diffractometer. Detection conditions: Cu-Kα radiation, wavelength 1.542 Å, divergence slit 1/4°, anti-diffraction The scattering slit is 1°, the X-ray tube voltage is 45kV, the X-ray tube current is 40mA, the scanning range is 3-40° (2θ), the step size is 0.026°, and the scanning time per step is 30.09 °/s.

Combining Examples 4.3 and 4.4, the amorphous meglumine salt has no obvious characteristic peaks in the X-ray diffraction pattern; further preferred X-ray diffraction patterns are shown in Figure 13 or 16.

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.

Among them, the X-ray data of choline salt crystal form A has strong characteristic peaks at 9.01, 15.90, 16.42 and 22.42°, with an error of ±0.2°; the further preferred X-Ray data are shown in the following table; the better XRPD is shown in the figure 17 and TGA/DSC in Fig. 18. Pos. [°2θ] Rel. Int. [%] 5.47 9.41 7.64 13.26 8.35 36.67 9.01 100.00 10.42 14.53 10.94 14.07 14.39 17.34 15.90 64.08 16.42 72.04 16.79 36.14 18.15 47.87 18.91 29.25 20.45 32.45 22.42 63.10 22.79 43.37 25.23 19.85 27.39 10.00

Example 8: Investigation of the solubility of the compounds of the present invention

8.1 Investigation of Static Solubility

Weigh about 0.5 mg of the sample to be tested in a centrifuge tube, first add an appropriate amount of DMSO to dissolve the sample completely, and then add methanol to make the volume to 1 ml; respectively, weigh about 1.0 mg of each of the two samples to be tested in two centrifuge tubes. Add 1ml of buffer solution of PBS=2.0 and 7.4 respectively; put the prepared control solution and test solution into a 37ºC water bath and heat for 1h, take out and cool to room temperature after 1h, filter with 0.22um filter membrane Calculate the concentration of the sample in the test solution according to C=(A*(m _s /v _s ))/A _S. Among them, m _s , v _s , and A _S are the weight, volume and peak area of the sample in the control solution, respectively, and A is the peak area of the test solution. [Table 1] Investigation of the solubility of the compound of the present invention Example medium Concentration (µg/mL) Molar concentration (µM) 1 pH=7.4 144.0 218.2 2.2 pH=7.4 799.1 1407.0 3 pH=7.4 773.6 1324.5 5 pH=7.4 40.8 73.2 6 pH=7.4 50.2 88.8

From the above results, it can be seen that the sodium and potassium salts provided by the present invention have greatly improved solubility compared with calcium salts, magnesium salts, and compound free acids, which is beneficial to improve the druggability of medicines.

8.2 Dynamic Solubility Investigation

The comparative test methods and results of the dynamic solubility of meglumine salt and sodium salt and free acid are as follows:

The dynamic solubility of each sample in four solvent systems of water, SGF, FaSSIF and FeSSIF was determined at a concentration of 5 mg/mL (15 mg of material into 3 mL of solvent) at 37 ºC by rotary mixing (25 rpm). (1, 4 and 24 hours). The samples at each time point were filtered (0.45 μm PTFE filter) by centrifugation (8000 rpm, 2 min), the HPLC concentration and pH of the filtrate were determined, and the solid samples after centrifugation were tested for XRPD. The solubility test results are summarized in the table below and the solubility curve is shown in Figure 19 below. raw material solvent 1 hour 2 hours 24 hours S pH crystal form change S pH crystal form change S pH crystal form change Example 4.1 H ₂ O 2.800 8.4 Yes 2.700 8.3 Yes 2.700 8.3 Yes SGF 0.013 2.3 Yes 0.020 2.1 Yes 0.005 2.1 Yes FaSSIF 2.200 6.8 Yes 2.000 6.8 Yes 0.160 6.8 Yes FeSSIF 0.098 5.1 Yes 0.190 5.0 Yes 0.670 5.0 Yes Example 2.3 H ₂ O 1.800 8.7 Yes 1.900 8.7 Yes 1.700 8.5 Yes SGF 0.006 2.0 Yes 0.009 2.0 Yes 0.005 2.1 Yes FaSSIF 0.120 6.7 Yes 0.100 6.7 Yes 0.091 6.8 Yes FeSSIF 0.029 4.9 Yes 0.033 5.0 Yes 0.079 5.0 Yes Example 1 H ₂ O 0.040 7.6 no 0.044 7.5 no 0.065 7.7 no SGF 0.007 2.0 no 0.006 1.9 no 0.006 1.9 no FaSSIF 0.065 6.4 no 0.065 6.5 no 0.077 6.3 no FeSSIF 0.012 4.9 no 0.012 4.9 no 0.014 5.0 no

It can be seen from the above results that the sodium salt and meglumine salt of the present invention have a good dynamic dissolution effect, which is better than that of the free acid.

Example 9: Stability investigation of the compound of the present invention

The experimental conditions and experimental results are as follows: starting sample condition time point Purity (area %) Purity/initial purity (%) crystal form change Example 2.3 Starting at 25ºC/60%RH 40ºC/75%RH - 1 week 1 week 100.0 100.0 100.0 - 100.0 100.0 - no no Example 4.1 Starting at 25ºC/60%RH 40ºC/75%RH - 1 week 1 week 100.0 100.0 100.0 - 100.0 100.0 no no

From the above results, it can be seen that the sodium salt and meglumine salt of the present invention are very stable under various humidity conditions.

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 nitrogen blanketing, [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 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- tert-butyl oxopyrimidine -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 mmol 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), 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 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, 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 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 heated 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 completion of the reaction , cooled to room temperature, the reaction was added dropwise to 100 mL of water, 5% sodium carbonate was added to adjust the pH to 10-11, extracted with DCM (40 mL × 4) for four times, the organic phases were combined, and the organic phases were dried over anhydrous sodium sulfate , filtered, spin-dried, the residue was dissolved in DCM (10 mL), and 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 yellow solid 4-(2-acetyl-5-chlorophenyl)-5-methoxypyridin-2( 1H )-one (yield: 82%). 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 10: Detection of the biological activity of the compounds of the present invention on the inhibition of human coagulation factor XIa by absorptiometry

Experimental Materials

Enzyme: Human Factor XIa (ENZYME RESEARCH, Cat. No. HFXIa 1111a) Substrate: S-2366 ^TM : (CHROMOGENIX, Cat. No. 82109039)

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

Experimental procedure

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, 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.

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

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)

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.

1. 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 12: Investigation of the selectivity of the compounds of the present invention to coagulation factors

2. Experimental materials

Enzyme: hFXa: Human Factor Xa: 71nkat. hFIIa: HT5146L. hFVIIa: Human Factor VIIa: hFVIIa 4591L. kallikrein: LOT180223.

Substrate: S-2222 ^™ : CHROMOGENIX, NO864682. S-2238 ^™ : CHROMOGENIX, NO770996. S-2288 ^™ : CHROMOGENIX, NO378902. ADG302.

Buffer: hFXa buffer: 100 mM NaCl, 5 mM CaCl2, 33% ethylene glycol, 50 mM Tris (pH 7.5). hFIIa buffer: 0.145M NaCl, 0.005M KCl, 1 mg/ml PEG-8000, 0.030M HEPES (pH 7.4). hFVIIa buffer: 0.145M NaCl, 0.005M KCl, 1 mg/ml PEG-8000, 0.030M HEPES (pH 7.4). kallikrein buffer: 50 mM Tris, 50 mM Mimidazole and 150 mM NaCl (pH 8.2).

3. Experimental steps

10mM test compound dissolved in 100% DMSO was diluted with 100% DMSO to 1000, 200, 40, 8, 1.6 μM; 98 μL of enzyme solution was added to each well in a 96-well plate, and 98 μL of buffer was added to blank wells, and then 2 μL of compounds at different concentrations were added, the blank and control wells were replaced with DMSO, mixed with a shaker, and incubated at 37° C. for 20 min.

The concentrations of hFXa and S-2222 ^TM were FXa (1:28) and 800 μmol/L, respectively. The concentrations of hFIIa and S-2238 ^TM were hFIIa (0.06 U/ml) and 500 μmol/L, respectively. The concentrations of hFVIIa and S-2288 ^TM were hFVIIa (80 nM) and 1600 μmol/L, respectively. The concentrations of kallikrein and substrate were kallikrein (20 nM) and 1600 μmol/L, respectively.

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

4. Data processing

Curve fitting was performed with GraphPad Prism software, and IC ₅₀ values were calculated, as shown in Table 3. [Table 3] Investigation on the selectivity of the compounds of the present invention to coagulation factors Example hFXa IC50(μM) hFIIa IC50(μM) hFVIIa IC50(μM) hKallikrein IC50(nM) 1 >100 >100 >100 523.9±60.2

Conclusion: The compounds of the present invention have good selectivity to other coagulation factors.

Example 13: Investigation of the pharmacokinetic characteristics of the compounds of the present invention

1. Experimental materials

SD rats: male, 180-250 g, purchased from Guangdong Medical Laboratory Animal Center. 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, WinNonlin 6.1 software was used to calculate pharmacokinetic parameters by non-compartmental model method. The results are shown in Tables 4, 5 and 6. [Table 4] 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 5] 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 6] 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 14: Investigation of the data of the compound caco-2 of the present invention

Experimental Materials:

Medium: DMEM (Corning), FBS (Corning), double antibody (Solarbio), 96-well HTS transwell plate (Corning), Caco-2 cells.

Experimental method: After Caco-2 cells were cultured on 96-well HTS transwell plate for 14-18 days, the TEER value of each well was detected to ensure that the cells in each well formed a complete monolayer, and the drug was cultured for 2h, and the drug concentrations of A-B and B-A were detected.

Data processing: Calculate PappA-B and PappB-A values, Papp = (VA×[drug]acceptor)/(Area×Time×[drug]initial,donor), calculate Efflux Ratio, Efflux Ratio=Papp(BA)/Papp (AB). [Table 7] Data of the compound caco-2 of the present invention Example Papp (AB) (10 ^-6 , cm/s) Papp (BA) (10 ^-6 , cm/s) Efflux Ratio 1 1.54 25.15 16.31 Compound B 1.55 13.56 8.75

Conclusion: The membrane permeability of the compound of the present invention is good.

Example 15: Investigation of CYP enzyme inhibition by the compounds of the present invention

Experimental Materials:

Liver microsomes (150-donor, Corning, Cat. 452117; Lot. 38292), NADPH.

experimental method:

First prepare a 0.2 mg/mL microsome system, add each test substance and substrate, the final concentration of the test substance is 50 μM, after pre-incubating for 8 minutes, add 10 mM NADPH to start the reaction, the final concentration of NADPH is 1 mM, after a period of incubation, add methanol to terminate the reaction. The amount of substrate metabolites generated in each reaction well was detected.

Data processing: Taking the metabolite generation amount of blank well as 100%, calculate the reduction amount of metabolite generation in each test substance well, and calculate the inhibition rate. [Table 8] CYP enzyme inhibition data of the compounds of the present invention Example CYP Inhibition IC50 (μM) CYP1A2 CYP2B6 CYP2C8 CYP2C9 CYP2C19 CYP2D6 CYP3A4 CYP3A5 1 >50 >50 ~50 >50 >50 >50 >50 >50

Conclusion: The compounds of the present invention have no inhibition on major CYP enzymes, and the risk of DDI is small.

Example 16: Investigation on hERG of the compound of the present invention

Experimental Materials:

HEK293-hERG stably transfected cell line (invitrogen). DMEM medium (Gibco), HEPES (invitrogen), Blasticidin (invitrogen)

experimental method:

HEK293-hERG stably transfected cells were used for experiments when they were cultured to a degree of polymerization of 40%-80%. First, the blank vehicle was applied to the cells to establish a baseline. Compounds were tested once the hERG current was found to be stable for 5 minutes. In the presence of the test compound, hERG currents were recorded for approximately 5 minutes to reach steady state, and then 5 frequency sweeps were captured. To ensure good performance of cultured cells and manipulations, the same batch of cells was also tested using the positive control dofetilidone.

data processing

Peak current inhibition = (1-Peak tail current compound/Peak tail current vehicle)*100 [Table 9] hERG experimental data of the compound of the present invention Example hERG IC50 [μM] Comment 1 > 10 1.17% inhibition at 10 μM

Conclusion: The compounds of the present invention have higher IC50 for hERG current and better cardiac safety.

Example 17: Pharmacokinetic study of the compound capsules of the present invention in rats

1. Experimental materials

SD rats: male, 180-250 g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

Reagents: physiological saline, heparin, acetonitrile, formic acid, propranolol (internal standard) are commercially available.

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

2. Experimental method

Weigh the solid powder of each compound (converted to free acid weight of about 3.5 mg), fill it in No. 9 ToRPAC capsules and take it orally, collect 200 μL of venous blood at 15min, 30min, 1h, 2h, 5h, 7h, 24h for heparinization. In EP tube, centrifuge at 12000rpm for 2min, and take the plasma and store it at -80℃ for testing. Precisely weigh a certain amount of the test sample and dissolve it in DMSO to 2 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, and mix by vortexing to prepare plasma equivalent to plasma concentrations of 0.3, 1, 3, 10, 30, 100, 300, 1000, and 3000 ng/mL. Two-sample analysis was performed for each concentration, and a standard curve was established. Take 30 μL of plasma, add 200 μL of internal standard propranolol (50 ng/mL) in acetonitrile solution, vortex to mix, add 100 μL of purified water, vortex again to mix, centrifuge at 4000 rpm for 5 min, take the supernatant LC-MS/ MS analysis. LC-MS/MS detection conditions are as follows.

Chromatographic column: Thermo Scientific HYPERSIL GOLD C-18 UPLC column, 100*2.1mm, 1.7μm.

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/MS detection of blood drug concentration, the pharmacokinetic parameters were calculated using WinNonlin 6.1 software and non-compartmental model method. The results are shown in Table 10 below. [Table 10] Pharmacokinetic results of the salts and free acids of the compounds of the present invention on rats Example Dosage (mg) Route of Administration _Cmax (ng/ml) AUC _last (h*ng/ml) T _1/2 (h) 1 3.5, po 32.2 259 8.62 2.3 3.5, po 97.1 541 5.66 4.1 3.5, po 2130 2900 5.08 2.2 3.5, po 423 717 2.97

It can be seen from the above results that the in vivo exposure of compound A meglumine salt and sodium salt under the same oral disintegration conditions is significantly better than that of compound A free acid, indicating that compared with compound A free acid, it has better absorption .

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.

Figure 1, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- 3-Phenylpropionamido)benzoic acid (Compound A) crystal form XRPD pattern; Figure 2, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- The XRPD pattern of Form A of 3-phenylpropionamido)benzoic acid sodium salt (compound A sodium salt); Figure 3, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- The TGA diagram of Form A of 3-phenylpropionamido)benzoic acid sodium salt (compound A sodium salt); Figure 4, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- The DSC chart of Form A of 3-phenylpropionamido)benzoic acid sodium salt (compound A sodium salt); Figure 5, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- The amorphous XRPD pattern of 3-phenylpropionamido)benzoic acid sodium salt (compound A sodium salt); Figure 6, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- The XRPD pattern of Type A of 3-phenylpropionamido)benzoic acid sodium salt (compound A sodium salt); Figure 7, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- TGA diagram of Type A of 3-phenylpropionamido)benzoic acid sodium salt (compound A sodium salt); Figure 8, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- DSC chart of Type A of 3-phenylpropionamido)benzoic acid sodium salt (compound A sodium salt); Figure 9, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- XRPD pattern of Type A of 3-phenylpropionamido)benzoic acid sodium salt (compound A sodium salt - another example); Figure 10, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- The XRPD pattern of crystal form A of 3-phenylpropionamido) benzoic acid meglumine salt (compound A meglumine salt); Figure 11, (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) crystal form A TGA/DSC chart; Figure 12, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- The XRPD pattern of crystal form A of 3-phenylpropionamido) benzoic acid meglumine salt (compound A meglumine salt - another embodiment); Figure 13, (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) amorphous XRPD pattern; Figure 14, (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) amorphous DSC chart; Figure 15, (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) amorphous TGA diagram; Figure 16, (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 - another example) amorphous XRPD pattern; Figure 17, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- The XRPD pattern of the crystalline form A of 3-phenylpropionamido) benzoic acid choline salt (Compound A choline salt); Figure 18, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- TGA/DSC pattern of 3-phenylpropionamido)benzoic acid choline salt (Compound A choline salt) Form A. Figure 19, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- Schematic diagram of dynamic dissolution of various crystal forms of 3-phenylpropionamido)benzoic acid and its salts. Figure 20, (S)-4-(2-(4-(2-Acetyl-5-chlorophenyl)-3-methoxy-6-oxopyridazin-1(6H)-yl)- Schematic representation of the structural formula of the salt of 3-phenylpropionamido)benzoic acid.

Claims (17)

A salt of an FXIa inhibitor compound represented by the following formula (I),

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. The salt of the FXIa inhibitor compound according to claim 1, wherein n is 0.5, 1, 1.5, 2, 2.5 or 3. The salt of the FXIa inhibitor compound according to claim 1, wherein the salt is selected from the group consisting of sodium salt, potassium salt, meglumine salt, calcium salt, magnesium salt and choline salt. The salt of the FXIa inhibitor compound according to claim 1, 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 salt of the FXIa inhibitor compound according to any one of claims 1 to 4, wherein the salt is a crystalline form, or an amorphous form, or a mixture thereof. The salt of the FXIa inhibitor compound according to claim 1, wherein the salt is a sodium salt, n=1, the salt is a crystal form, and the crystal form is represented by a 2θ angle in an X-ray diffraction diagram There are characteristic peaks at 9.32°, 15.34°, 16.24°, 18.41°, 19.48°, and 24.05°, with an error of ±0.2°; more preferably at 5.59°, 7.81°, 9.83°, 10.50°, 11.24°, 13.52° There are characteristic peaks at °, 14.60°, 14.87°, 16.93°, 20.39°, 21.02°, 21.77°, 23.53°, 24.99°, 25.90°, 26.62°, 27.22°, and the error is ±0.2°; The shot map is shown in Figure 6 or Figure 9. The salt of the FXIa inhibitor compound according to claim 6, wherein the DSC spectrum of the crystal form has a maximum absorption peak at 70.01°C±2°C; a preferred DSC spectrum is shown in FIG. 7 ; The TG spectrum of the type is shown in Figure 8. The salt of the FXIa inhibitor compound according to claim 1, wherein the salt is a sodium salt, n=1, the salt is amorphous, and there is no obvious characteristic peak in the X-ray diffraction pattern of the amorphous ; The preferred X-ray diffraction pattern is shown in Figure 5. The salt of the FXIa inhibitor compound according to claim 1, wherein the salt is a meglumine salt, n=1, and the salt is a crystal form, and the crystal form is represented by 2θ in an X-ray diffraction diagram The angle indicates that there are characteristic peaks at 9.33° and 18.79°, with an error of ±0.2°; further preferably, there are characteristic peaks at 10.32°, 13.74°, 16.18°, and 24.74°, with an error of ±0.2°; more preferably X-ray The diffraction pattern is shown in Figure 10 or Figure 12. The salt of the FXIa inhibitor compound according to claim 9, wherein the DSC pattern of the crystal form has a maximum absorption peak at 122.7°C±2°C; a preferred DSC pattern is shown in FIG. 11 . The salt of the FXIa inhibitor compound according to claim 1, wherein the salt is meglumine salt, n=1, the salt is amorphous, and the amorphous X-ray diffraction pattern has no obvious Characteristic peaks; the preferred X-ray diffraction pattern is shown in Figure 13 or Figure 16. The salt of the FXIa inhibitor compound according to claim 11, wherein the amorphous DSC spectrum has a maximum absorption peak at 80.1°C±2°C; a preferred DSC spectrum is shown in FIG. 14 . The salt of the FXIa inhibitor compound according to claim 11, wherein the DSC spectrum of the amorphous form is shown in FIG. 14 . The salt of the FXIa inhibitor compound according to claim 11, wherein the TG spectrum of the amorphous form is shown in FIG. 15 . The salt of the FXIa inhibitor compound according to any one of claims 1 to 14, wherein one or more hydrogen atoms of the compound are substituted with the isotope deuterium. A pharmaceutical composition comprising a salt of the FXIa inhibitor compound as claimed in any one of claims 1 to 14; and one or more pharmaceutically acceptable carriers. A use of the salt of the FXIa inhibitor compound according to any one of claims 1 to 14 in the preparation of a medicament for the treatment of FXIa-related diseases, preferably in the preparation of a medicament for the treatment of thrombosis-related diseases.

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