WO2020094008A1 - 2-oxo-1,2-dihydroquinoline derivatives, preparation method therefor, and pharmaceutical applications thereof - Google Patents

Abstract

Provided are a 2-oxo-1,2-dihydroquinoline compound represented by formula (I), a preparation method therefor, and use thereof in drugs for preventing and/or treating thromboembolic diseases and/or thromboembolic complications, wherein R 1 is selected from (II); R 2 is selected from C 1-6 alkyl groups; n is selected from 0, 1, and 2. The compound has high activity, high solubility and better pharmacokinetic properties. (I)

Description

2-oxo-1,2-dihydroquinoline derivatives, preparation method and application in medicine Technical field

The invention relates to the field of medicine, in particular to a 2-oxo-1,2-dihydroquinoline compound, and a preparation method and application thereof.

Background technique

Thromboembolic diseases not only have a high morbidity, but also have a high mortality and disability rate. For example, myocardial infarction, cerebral infarction, and pulmonary infarction caused by thromboembolism rank first among various causes of death. The prevention and treatment drugs for thrombotic diseases mainly include anticoagulant, antiplatelet and thrombolytic drugs. Hemorrhage is currently the most important and common complication of clinical antithrombotic drugs. Traditional anticoagulant drugs, such as warfarin, heparin, low molecular weight heparin (LMWH), and new drugs that have been on the market in recent years, such as FXa inhibitors (rivaroxaban, apixaban, etc.) and thrombin inhibitors (Dabi Plus group ester, hirudin, etc.) have a good effect on reducing thrombosis, but they also face a common deficiency-may cause bleeding complications. Therefore, it is of great value to develop new antithrombotic drugs with less bleeding side effects.

Coagulation factor Ⅺ (FⅪ), as a member of the coagulation contact activation pathway, was first recognized by everyone, and it is activated in the body to form coagulation factor Ⅺa (FⅪa). However, with the deepening of research, people have revised the traditional waterfall coagulation theory. The revised theory believes that FⅪ can be activated by thrombin to form FⅪa, and plays a role in the continuous generation of thrombin and the inhibition of fibrinolysis . In recent years, clinical data on human FⅪ defects or elevated levels of FⅪ, and animal anti-thrombotic experimental studies of FⅪ defects or suppression have shown that FⅪ and FⅪa are new targets for antithrombotic prevention and treatment with a small risk of bleeding. The anti-thrombotic drugs at the point have little bleeding side effects. Therefore, the development of FⅪa inhibitor drugs is expected to overcome the common shortcomings of traditional anticoagulant drugs: bleeding complications, with important clinical needs and broad market prospects.

Ola

The article entitled "Creating Novel Activated Factor XI Inhibitors through Fragment Based Lead Generation and Structure Aided Drug Design" published on PLOS ONE | DOI: 10.1371 / journal.pone.0113705 on January 28, 2015 A series of active 2-oxo-1,2-dihydroquinoline FⅪa inhibitors. However, the physicochemical properties of known FⅪa inhibitors have many defects in the pharmacokinetic properties in vivo. Therefore, there is an urgent need in the art for new FⅪa inhibitors with better physical and chemical properties, good activity and good pharmacological properties.

Summary of the invention

An object of the present invention is to provide 2-oxo-1,2-dihydroquinoline compounds that have a strong inhibitory effect on FXIa and have higher solubility and better pharmacokinetic properties.

Another object of the present invention is to provide a method for preparing the compound of the present invention.

Another object of the present invention is to provide the use of the compound of the present invention.

The object of the present invention is achieved by the following technical solutions.

In one aspect, the present invention provides a compound represented by formula (I),

among them:

R ^{1 is} selected from

R ^{2 is} selected from C _1-6 alkyl;

n is selected from 0, 1, and 2.

In certain preferred embodiments of the present invention, in the compound represented by formula (I):

R ^{1 is} selected from

R ^{2 is} selected from C _1-3 alkyl;

n is selected from 0, 1, and 2.

In certain preferred embodiments of the present invention, in the compound represented by formula (I):

R ^{1 is} selected from

R ^{2 is} selected from methyl;

n is selected from 0, 1, and 2.

In certain preferred embodiments of the present invention, the compound represented by formula (I) is selected from the compounds in the following table:

In another aspect, the present invention provides a method for preparing a compound represented by formula (I), including the following steps:

(1) Compound A is reacted with compound C to obtain compound B;

(2) The compound B is reacted with the compound D to obtain the compound represented by the formula (I);

Wherein for formula (I) R is as defined in compound ₁ shown above defined R _1.

In certain embodiments of the present invention, the compound A, compound B, compound C and compound D can be prepared or purchased by conventional synthetic methods.

In still another aspect, the present invention provides a pharmaceutical composition comprising the compound of formula (I) of the present invention and a pharmaceutically acceptable excipient.

In the present invention, the pharmaceutically acceptable excipients refer to conventional carriers and / or excipients that do not affect the compounds of formula (I) of the present invention to exert their pharmaceutical effects.

In yet another aspect, the present invention provides the use of the compound of formula (I) of the present invention in the preparation of a medicament for preventing and / or treating thromboembolic diseases and / or thromboembolic complications.

In still another aspect, the present invention provides a method for preventing and / or treating thromboembolic diseases and / or thromboembolic complications, the method comprising administering to a patient a compound of formula (I) according to the present invention, or administering The patient contains a pharmaceutical composition of the compound of formula (I) according to the present invention.

Within the scope of the present invention, "thromboembolic diseases" especially include diseases such as myocardial infarction with ST segment elevation (STEMI) and without ST segment elevation (without STEMI), stable / unstable angina, coronary artery intervention For example, re-occlusion and restenosis after angioplasty or aortic coronary artery bypass surgery, peripheral vascular occlusive disease, pulmonary embolism, deep venous thrombosis and renal venous thrombosis, transient ischemic attack, and thrombosis type and thrombosis Embolism stroke.

The thromboembolic diseases also include cardiac thromboembolism, such as stroke, cerebral ischemia, systemic thromboembolism and ischemia, and also acute, intermittent or persistent cardiac arrhythmia, cardioversion, heart valve disease .

The thromboembolic diseases also include atherosclerotic vascular diseases and inflammatory diseases (such as rheumatic diseases of the sports system), as well as other diseases (such as diabetes, tumor diseases, especially major surgical interventions or radiotherapy / chemotherapy) Patients) caused thromboembolism.

The thromboembolic diseases also include diffuse osmotic coagulation (DIC).

The thromboembolic complications include microvascular hemolytic anemia, such as complications occurring during extracorporeal blood circulation in hemodialysis and heart valve repair.

Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

When the invention relates to substitution with multiple substituents, each substituent may be the same or different.

The elements carbon, hydrogen, oxygen, sulfur, nitrogen or halogen involved in the groups and compounds of the present invention include their isotopes, and the elements carbon, hydrogen, oxygen involved in the groups and compounds of the present invention , Sulfur or nitrogen is optionally further replaced by one or more of their corresponding isotopes, wherein carbon isotopes include ¹² C, ¹³ C, ¹⁴ C, hydrogen isotopes include protium (H), deuterium (D, also known as heavy hydrogen ), Tritium (T, also known as super heavy hydrogen), oxygen isotopes include ¹⁶ O, ¹⁷ O and ¹⁸ O, sulfur isotopes include ³² S, ³³ S, ³⁴ S and ³⁶ S, nitrogen isotopes include ¹⁴ N and ¹⁵ N , Fluorine isotope ¹⁹ F, chlorine isotopes include ³⁵ Cl and ³⁷ Cl, bromine isotopes include ⁷⁹ Br and ⁸¹ Br.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight and branched chain groups of 1 to 20 carbon atoms. Preferred alkyl groups containing 1 to 10 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, n- Nonyl, and its various branched isomers, etc .; more preferred are lower alkyl groups containing 1 to 4 carbon atoms, non-limiting examples include methyl, ethyl, propyl, isopropyl, n- Butyl, isobutyl or tert-butyl, etc. The alkyl group may be substituted or unsubstituted. When substituted, the substituent is preferably 1 to 5, independently selected from F, Cl, Br, I, = 0, alkyl, alkenyl, alkynyl, alkyl Oxygen, alkylthio, alkylamino, mercapto, hydroxyl, nitro, cyano, amino, alkylacylamino, cycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, Cycloalkylmercapto group, hydroxyalkyl group, carboxyl group, carboxylate group, heterocyclic alkylmercapto group.

"Alkoxy" refers to -O-alkyl, where alkyl is as defined herein above. The alkoxy group may be substituted or unsubstituted, and non-limiting examples thereof include methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentoxy or The hexyloxy group preferably has a 1 to 12-membered alkoxy group. When substituted, the substituents are preferably 1 to 5, independently selected from F, Cl, Br, I, = O, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, Mercapto, hydroxyl, nitro, cyano, amino, alkylacylamino, cycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylmercapto, hydroxyalkyl, carboxy, Carboxylate group or heterocycloalkyl mercapto group.

"Optional", "optional" or "optionally" means that the subsequently described event or environment may but need not occur, including where the event or environment occurs or does not occur. For example, "aryl is optionally substituted with alkyl" means that alkyl may but need not be present. The description includes cases where aryl is substituted with alkyl and cases where aryl is not substituted with alkyl.

"Substituted or unsubstituted" refers to the situation where the group can be substituted or unsubstituted. If no indication is made in the present invention that the group can be substituted, it means that the group is unsubstituted.

"Substitution" refers to the situation where one or more hydrogen atoms in a group are replaced by other groups. If the group is replaced by a hydrogen atom, the formed group is the same as the group substituted by a hydrogen atom. When the group is substituted, for example, amino, C _1-4 alkyl, C _1-4 alkoxy, C _3-6 carbocyclic ring, 3 to 6 membered heterocyclic ring is optionally further substituted by 0, 1, 2, 3 or 4 substituents selected from H, F, Cl, Br, I, hydroxyl, cyano, amino, C _1-4 alkyl or C _1-4 alkoxy, the formed groups include but are not limited to Group, chloromethyl, trichloromethyl, hydroxymethyl, -CH ₂ OCH ₃ , -CH ₂ SH, -CH ₂ CH ₂ CN, -CH ₂ NH ₂ , -NHOH, -NHCH ₃ , -OCH ₂ Cl , -OCH ₂ OCH ₂ CH ₃ , -OCH ₂ CH ₂ NH ₂ , -OCH ₂ CH ₂ SH, -OCH ₂ CH ₂ OH, 1-hydroxycyclopropyl, 2-hydroxycyclopropyl, 2-aminocyclopropyl Group, 4-methylfuranyl, 2-hydroxyphenyl, 4-aminophenyl, phenyl.

The compound represented by the formula (I) of the present invention has a strong inhibitory effect on FXIa, and the animal has an obvious anticoagulant effect after intravenous injection of the compound of the present invention. At the same time, the inventor unexpectedly found that the solubility of the compound represented by formula (I) of the present invention at pH = 8.0 is much higher than the solubility of the FⅪa inhibitor reported in the literature (PLOS ONE | DOI: 10.1371 / journal.pone.0113705) , Which is conducive to the development of pharmaceutical preparations and better administration. At the same time, the inventors also unexpectedly found that the pharmacokinetic indexes AUC and Cmax of the compound of formula (I) of the present invention after injection administration far exceeded the literature (PLOSONE | DOI: 10.1371 / journal.pone.0113705) Reported FⅪa inhibitor. In addition, since FⅪa belongs to the extracellular blood target, the smaller apparent distribution volume at the same dose makes the compound of the present invention easier to distribute in the blood, which is conducive to the distribution of the drug to the target site. The high activity, high solubility, and better pharmacokinetic properties of the compounds of the present invention make the compounds of the present invention have very significant advantages over the compounds of the prior art.

detailed description

The technical solutions of the present invention will be described in detail below with reference to examples, but the protection scope of the present invention includes but is not limited thereto.

The structure of the compound is determined by nuclear magnetic resonance hydrogen spectroscopy ( ¹ H NMR) and / or mass spectrometry (MS). The NMR shift (δ) is given in units of 10 ^-6 (ppm). The measurement of NMR was performed using Bruker AV400 nuclear magnetic resonance spectrometer, and the solvent was determined to be deuterated dimethyl sulfoxide (DMSO-d6).

The measurement of MS was carried out using Thermo Scientific (ESI) mass spectrometer.

The thin-layer chromatography silica gel plate uses Yantai Huanghai GF254 silica gel plate. The specification of the thin-layer chromatography (TLC) silica gel plate is 0.15mm ～ 0.20mm, and the thin-layer chromatography separation purification product uses the specification of 0.4mm ～ 0.5mm .

Column chromatography generally uses Yantai Yellow Sea silica gel 200-300 mesh silica gel as a carrier.

The known starting materials of the present invention can be synthesized using or according to methods known in the art, or can be purchased from Bellingway Technology, Aladdin Technology and other companies.

Nitrogen atmosphere refers to a nitrogen balloon with a volume of about 1L connected to the reaction bottle.

The hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1L.

Unless otherwise specified in the examples, the reaction is carried out under a nitrogen atmosphere.

Unless otherwise specified in the examples, the solution refers to an aqueous solution.

Unless otherwise specified in the examples, the reaction temperature is room temperature.

Room temperature is the most suitable reaction temperature, which is 20-30 ° C.

Intermediate 1: Synthesis of (S) -3- (4-nitrophenyl) -2- (tert-butoxycarbonylamino) propionic acid methyl ester

Step 1: Synthesis of (S) -3- (4-aminophenyl) -2- (tert-butoxycarbonylamino) propionic acid methyl ester (1b)

To (S) -3- (4-nitrophenyl) -2-aminopropionic acid methyl ester hydrochloride (1a) (2.50 g, 9.59 mmol) was added 135 mL of dichloromethane, and the temperature was lowered to 0 ° C. At this temperature, triethylamine (2.43, 24.0 mmol) and di-tert-butyl dicarbonate (2.50 g, 9.59 mmol) were slowly added. The reaction solution was reacted at room temperature overnight. It was suction filtered, and the filtrate was washed successively with 1N hydrochloric acid (45 mL) and water (100 mL × 2). Concentrate under reduced pressure to give the title compound 1b (3.25g, white solid).

¹ H NMR (400 MHz, DMSO-d6): δ 8.15 (d, 2H), 7.52 (d, 2H), 7.39 (d, 1H), 4.26 (td, 1H), 3.62 (s, 3H), 3.15 ( dd, 1H), 2.97 (dd, 1H), 1.45 (s, 2H), 1.29 (s, 9H).

MS / m = z = 324.61 [M + H].

Step 2: Synthesis of (S) -3- (4-nitrophenyl) -2- (tert-butoxycarbonylamino) propionic acid methyl ester (intermediate 1)

To (S) -3- (4-aminophenyl) -2- (tert-butoxycarbonylamino) propionic acid methyl ester (1b) (2.00 g, 6.17 mmol), add methanol (28 mL) and water (14 mL) . With stirring, zinc powder (4.03 g, 61.7 mmol) and ammonium chloride (4.95 g, 92.5 mmol) were added. After the addition, the reaction was carried out at 45 ° C for 0.5h, and then transferred to room temperature and stirred for 3h. Filter and concentrate the filtrate to precipitate a solid. Filtration with suction and drying over anhydrous sodium sulfate gave Intermediate 1 (1.70 g, light yellow solid).

¹ H NMR (400 MHz, DMSO-d6): δ 7.16 (d, 1H), 6.84 (d, 2H), 6.45 (d, 2H), 4.93 (s, 2H), 4.08-3.95 (m, 1H), 3.57 (s, 3H), 2.76 (dd, 1H), 2.64 (dd, 1H), 1.29 (d, 9H).

MS m / z = 294.73 [M + H] ⁺ .

Intermediate 2: Synthesis of 6-chloro-4-((methylamino) methyl) quinoline-2 (1H) -one

Step 1: Synthesis of N- (4-chlorophenyl) -3-oxobutyramide (2b)

4-Chloroaniline (2a) (12.0 g, 94.1 mmol), DMAP (11.5 g, 94.1) and pyridine (48 mL) were added to xylene (240 mL) and heated to reflux. After 8h, the reaction was completed. The reaction solution was cooled to room temperature, poured into a mixed solution of 2N hydrochloric acid (240 mL) and ethyl acetate (240 mL), and stirred for 20 min. The layers were separated, and the aqueous layer was washed with ethyl acetate (120 mL × 2). The organic layers were combined, concentrated to dryness under reduced pressure, and subjected to column chromatography to obtain the title compound 2b (10.7 g, yield 53.7%).

¹ H NMR (400MHz, DMSO-d6): δ 10.2 (s, 1H), 7.57-7.60 (m, 2H), 7.33-7.36 (m, 2H), 3.54 (s, 2H), 2.19 (s, 3H ).

MS m / z = 211.99 [M + H] ⁺ .

Step 2: Synthesis of 4-bromo-N- (4-chlorophenyl) -3-oxobutanamide (2c)

To a solution of N- (4-chlorophenyl) -3-oxobutyramide (2b) (1.0g, 4.72mmol) in glacial acetic acid (10mL) at room temperature, add iodine (1mg) and slowly add bromine dropwise (0.8 g, 4.96 mmol) in glacial acetic acid (15 mL). After the addition, the reaction was carried out at room temperature for 18h. The reaction solution was poured into 250 mL of ice water, stirred, and a white solid precipitated, which was filtered with suction, washed with water, and dried at room temperature to obtain the title compound 4-bromo-N- (4-chlorophenyl) -3-oxobutanamide (2c) ( 1.1g, yield 80.3%).

¹ H NMR (400MHz, DMSO-d6): δ 10.2 (s, 1H), 7.56-7.63 (m, 2H), 7.35-7.40 (m, 2H), 4.47 (s, 2H), 3.73 (s, 2H ).

MS m / z = 289.96 [M + H] ⁺ .

Step 3: Synthesis of 4- (bromomethyl) 6-chloroquinoline-2 (1H) -one (2d)

At room temperature, 4-bromo-N- (4-chlorophenyl) -3-oxobutanamide (2c) (100 mg, 0.38 mmol) was added concentrated sulfuric acid (0.5 mL). After the addition, the reaction was carried out at 80 ° C for 3h. The reaction solution was poured into ice water (15 mL), stirred, and a white solid precipitated, filtered off with suction, washed with water, and dried at room temperature for 12 h to obtain the title compound 4- (bromomethyl) 6-chloroquinolin-2 (1H) -one (2d ) (70 mg, yield 67.8%).

MS m / z = 272.06 [M + H] ⁺ .

Step 4: Synthesis of 6-chloro-4-((methylamino) methyl) quinolin-2 (1H) -one (intermediate 2)

To a suspension of 4- (bromomethyl) 6-chloroquinoline-2 (1H) -one (2d) (110 mg, 0.40 mmol) in tetrahydrofuran (1 mL) and dioxane (1 mL), add 40 drops % Aqueous solution of methylamine (1 mL). After the addition, the reaction was carried out at 30 ℃ for 12h. Water (10 mL) was added, and a white solid precipitated, which was filtered with suction and dried at 50 ° C for 3 h to obtain the title compound Intermediate 2 (35 mg, yield 38.9%).

¹ H NMR (400MHz, DMSO-d6): δ 11.7 (s, 1H), 7.83 (d, 1H), 7.70 (d, 1H), 7.37 (d, 1H), 6.54 (s, 1H), 3.82 ( s, 2H), 2.33 (s, 3H).

MS m / z = 223.00 [M + H] ⁺ .

Example 1: 5-(((2S) -1-(((6-chloro-2-oxo-1,2-dihydroquinolin-4-yl) methyl) (methyl) amino) -3 -(4- (2-Methylcyclopropane-1-carboxamido) phenyl) -1-oxopropane-2-yl) carbamoyl) thiophene-2-carboxylic acid (Compound 1)

The first step: (2S) -2-((tert-butoxycarbonyl) amino) -3- (4- (2-methylcyclopropyl-1-carboxamido) phenyl) propionic acid methyl ester (3b) Synthesis

2-Methylcyclopropanecarboxylic acid (3a) (2.98g, 29.8mmol) and (S) -3- (4-nitrophenyl) -2- (tert-butoxycarbonylamino) propionic acid methyl ester (intermediate 1) (7.30 g, 24.8 mmol) was dissolved in DMF (80 mL), PyBOP (15.5 g, 27.7 mmol), DIEA (6.41 g, 49.6 mmol) and DMAP (0.61 g, 4.96 mmol) were added, and the reaction was stirred at room temperature. After 16h, water (800 mL) was added to the reaction solution to precipitate a solid. It was filtered with suction, washed with water (100 mL), dried at 50 ° C for 3h, and then purified by column chromatography to obtain the title compound 3b (7.40g, yield 79.3%).

¹ H NMR (400MHz, DMSO-d6): δ 10.02 (s, 1H), 7.46 (d, 2H), 7.23 (d, 1H), 7.11 (d, 2H), 4.11 (dd, 1H), 3.59 ( d, 3H), 2.90 (dd, 1H), 2.77 (dd, 1H), 1.48 (td, 1H), 1.29 (d, 9H), 1.23–1.13 (m, 1H), 1.08 (d, 3H), 1.02 –0.93 (m, 1H), 0.65–0.54 (m, 1H).

MS m / z = 377.10 [M + H] ⁺ .

Step 2: Synthesis of (2S) -2- (tert-butoxycarbonylamino) -3- (4- (2-methylcyclopropyl-1-carboxamido) phenyl) propionic acid (3c)

(2S) -2-((tert-butoxycarbonyl) amino) -3- (4- (2-methylcyclopropyl-1-carboxamido) phenyl) propionic acid methyl ester (3b) (3.00g , 7.97mmol) was dissolved in a mixed solution of methanol (15mL) and tetrahydrofuran (15mL). A solution of lithium hydroxide monohydrate (3.50 g, 83.4 mmol) in water (30 mL) was added, and the reaction was stirred at room temperature. After 2.5h, the reaction system was cooled to 0 ° C and adjusted to pH = 3 with 1N hydrochloric acid. The obtained solid was filtered with suction, washed with water (50 mL), and dried at 50 ° C for 3 h to obtain the title compound 3c (2.10 g, yield 72.7%).

¹ H NMR (400MHz, DMSO-d6): δ 13.08–11.98 (s, 1H), 10.08 (s, 1H), 7.46 (d, 2H), 7.12 (d, 2H), 7.01 (d, 1H), 4.03 (m, 1H), 2.93 (m, 1H), 2.74 (m, 1H), 1.51 (m, 1H), 1.31 (s, 9H), 1.22-1.15 (m, 1H), 1.08 (t, 3H) , 1.00–0.93 (m, 1H), 0.59 (m, 1H).

MS m / z = 362.69 [M + H] ⁺ .

The third step: ((2S) -1-(((6-chloro-2-oxo-1,2-dihydroquinolin-4-yl) methyl) (methyl) amino) -3- (4 -(2-Methylcyclopropane-1-carboxamido) phenyl) -1-oxopropane-2-yl) carbamic acid tert-butyl ester (3d)

(2S) -2- (tert-butoxycarbonylamino) -3- (4- (2-methylcyclopropyl-1-carboxamido) phenyl) propionic acid (3c) (1000 mg, 2.76 mmol), Intermediate 2 (614 mg, 2.76 mmol), HOBT (746 mg, 5.52 mmol), EDCI (1060 mg, 5.25 mmol) and DIEA (1070 mg, 8.28 mmol) were dissolved in DMF (20 mL), and the reaction was stirred at room temperature. After TLC showed that the reaction was over, water (200 mL) was added to precipitate a solid. Beating and washing, suction filtration to obtain the title compound 3d (1250mg, yield 79.9%).

¹ H NMR (400MHz, DMSO-d6): δ 11.76 (s, 1H), 10.02 (s, 1H), 7.79 (s, 1H), 7.54 (d, 1H), 7.46 (d, 2H), 7.32 ( d, 1H), 7.18 (d, 2H), 7.03 (d, 1H), 6.32 (s, 1H), 4.72 (s, 1H), 4.61 (s, 2H), 3.00--2.72 (m, 5H), 1.48 (dd, 1H), 1.32 (s, 9H), 1.18 (d, 1H), 1.08 (d, 3H), 0.97 (d, 1H), 0.60 (t, 1H).

MS m / z = 567.04 [M + H] ⁺ .

The fourth step: N- (4-((S) -2-amino-3-(((6-chloro-2-oxo-1,2-dihydroquinolin-4-yl) methyl) (A )) Amino) -3-oxopropyl) phenyl) -2-methylcyclopropylformamide hydrochloride (3e)

To ((2S) -1-(((6-chloro-2-oxo-1,2-dihydroquinolin-4-yl) methyl) (methyl) amino) -3- (4- (2 -Methylcyclopropane-1-carboxamido) phenyl) -1-oxopropane-2-yl) carbamic acid tert-butyl ester (3d) (1.25g, 2.20mmol), ethyl acetate (25mL) was added . With stirring, 1M hydrogen chloride in ethyl acetate (25 mL) was slowly added and reacted at room temperature. After the reaction, suction filtration. It was washed with ethyl acetate and dried at 50 ° C for 3h to obtain the title compound 3e (1.04g, yield 93.7%).

¹ H NMR (400MHz, DMSO-d6): δ 11.86 (s, 1H), 10.26 (s, 1H), 8.34 (s, 3H), 7.81 (s, 1H), 7.57 (dd, 1H), 7.53 ( d, 2H), 7.37 (d, 1H), 7.13 (d, 2H), 6.38 (s, 1H), 4.81 (d, 1H), 4.69–4.46 (m, 2H), 3.07 (dt, 1H), 2.95 (dd, 1H), 2.79–2.64 (m, 3H), 1.55 (dt, 1H), 1.17 (dd, 1H), 1.07 (d, 3H), 1.00-0.92 (m, 1H), 0.61 (s, 1H ).

MS m / z = 467.13 [M + H] ⁺ .

The fifth step: 5-(((2S) -1-(((6-chloro-2-oxo-1,2-dihydroquinolin-4-yl) methyl) (methyl) amino) -3 -(4- (2-Methylcyclopropane-1-carboxamido) phenyl) -1-oxopropane-2-yl) carbamoyl) thiophene-2-carboxylic acid methyl ester (3f)

N- (4-((S) -2-amino-3-(((6-chloro-2-oxo-1,2-dihydroquinolin-4-yl) methyl) (methyl) amino ) -3-oxopropyl) phenyl) -2-methylcyclopropylformamide hydrochloride (3e) (300mg, 0.60mmol), 5- (methoxycarbonyl) thiophene-2-carboxylic acid (111mg , 0.60mmol), HOBT (161mg, 1.19mmol), EDCI (228mg, 1.19mmol) and DIEA (231mg, 1.79mmol) were dissolved in DMF (6mL), the reaction was stirred at room temperature, TLC monitoring (V _DCM : V _MeOH = 15 :1). After the reaction was completed, water (60 mL) was added to precipitate a solid. Beating and washing, suction filtration and drying at 50 ° C for 3h to obtain the title compound 3f (300mg, yield 79.3%).

¹ H NMR (400MHz, DMSO-d6): δ 11.82 (s, 1H), 10.01 (s, 1H), 9.24 (s, 1H), 7.94 (d, 1H), 7.80 (d, 1H), 7.77 ( s, 1H), 7.63 (d, 1H), 7.44 (d, 2H), 7.32 (d, 1H), 7.24 (d, 2H), 6.33 (s, 1H), 5.06 (dd, 1H), 4.71 (d , 2H), 3.83 (s, 3H), 3.04-2.98 (m, 4H), 2.94 (s, 1H), 1.45 (m, 1H), 1.17 (dd, 1H), 1.11-1.02 (m, 3H), 0.94 (m, 1H), 0.59 (t, 1H).

MS m / z = 635.07 [M + H] ⁺ .

The sixth step: 5-(((2S) -1-(((6-chloro-2-oxo-1,2-quinolin-4-yl) methyl) (methyl) amino) -3- ( Synthesis of 4- (2-methylcyclopropane-1-carboxamido) phenyl) -1-oxopropane-2-yl) carbamoyl) thiophene-2-carboxylic acid (Compound 1)

5-(((2S) -1-(((6-chloro-2-oxo-1,2-dihydroquinolin-4-yl) methyl) (methyl) amino) -3- (4 -(2-methylcyclopropane-1-carboxamido) phenyl) -1-oxopropane-2-yl) carbamoyl) thiophene-2-carboxylic acid methyl ester (3f) (300mg, 0.47mmol) dissolved In a mixed solution of methanol (3 mL) and tetrahydrofuran (3 mL). A solution of lithium hydroxide monohydrate (90 mg, 2.14 mmol) in water (6 mL) was added, and the reaction was stirred at room temperature. After the reaction was completed, the system was cooled to 0 ° C and adjusted to pH = 3 with 1N hydrochloric acid. The resulting solid was suction filtered, washed with water (30 mL) and dried at 50 ° C for 3h. After separation by preparative thin layer chromatography, the title compound 1 (57 mg, yield 19.0%) was obtained.

¹ H NMR (400MHz, DMSO-d6): δ 11.85 (s, 1H), 10.08 (d, 1H), 9.15 (d, 1H), 7.89 (d, 1H), 7.77 (s, 1H), 7.64 ( t, 1H), 7.56–7.51 (m, 1H), 7.46 (d, 2H), 7.33 (d, 1H), 7.24 (d, 2H), 6.33 (s, 1H), 5.05 (q, 1H), 4.71 (s, 2H), 3.04–2.97 (m, 4H), 2.94 (s, 1H), 1.48 (m, 1H), 1.15 (t, 1H), 1.06 (d, 3H), 1.00–0.83 (m, 1H ), 0.59 (d, 1H).

MS m / z = 620.99 [M + H] ⁺ .

Example 2: (R) -5-((1-(((6-chloro-2-oxo-1,2-dihydroquinolin-4-yl) methyl) (methyl) amino) -3 -(4- (Cyclopropylcarboxamido) phenyl) -1-oxopropane-2-yl) carbamoyl) thiophene-2-carboxylic acid (Compound 2)

In the first step of this example, cyclopropylcarboxylic acid (4a) was used instead of 2-methylcyclopropanecarboxylic acid (3a) in the first step of Example 1, and the title compound was obtained in a similar manner to Example 1.

¹ H NMR (400MHz, DMSO-d6): δ 13.36 (s, 1H), 11.84 (s, 1H), 10.10 (s, 1H), 9.19 (d, 1H), 7.92 (d, 1H), 7.79 ( d, 1H), 7.70 (d, 1H), 7.59–7.50 (m, 1H), 7.47 (d, 2H), 7.34 (d, 1H), 7.27 (d, 2H), 6.34 (s, 1H), 5.06 (dt, 1H), 4.88–4.65 (m, 2H), 3.05 (d, 4H), 2.98–2.88 (m, 1H), 1.83–1.65 (m, 1H), 0.76 (dd, 4H).

MS m / z = 606.69 [M + H] ⁺ .

Example 3: 5-(((2S) -1-(((6-chloro-2-oxo-1,2-dihydroquinolin-4-yl) methyl) (methyl) amino) -3 -(4- (2,2-Dimethylcyclopropyl-1-carboxamido) phenyl) -1-oxopropane-2-yl) carbamoyl) thiophene-2-carboxylic acid (Compound 3)

In the first step of this example, 2,2-dimethylcyclopropanecarboxylic acid (5a) was used instead of 2-methylcyclopropanecarboxylic acid (3a) in the first step of Example 1, and a method similar to Example 1 was used The title compound is obtained.

¹ H NMR (400MHz, DMSO-d6): δ 13.60–12.87 (m, 1H), 11.82 (s, 1H), 9.97 (s, 1H), 9.17 (d, 1H), 7.91 (d, 1H), 7.78 (d, 1H), 7.68 (d, 1H), 7.52 (d, 1H), 7.46 (d, 2H), 7.32 (d, 1H), 7.24 (d, 2H), 6.33 (s, 1H), 5.05 (d, 1H), 4.72 (s, 2H), 3.06–2.98 (m, 4H), 2.94 (s, 1H), 1.67–1.53 (m, 1H), 1.12 (dd, 6H), 0.92 (d, 1H ), 0.73 (s, 1H).

MS m / z = 634.85 [M + H] ⁺ .

Example 4: (S) -5-((1-((((6-chloro-2-oxo-1,2-dihydroquinolin-4-yl) methyl) (methyl) amino) -3 -(4- (Cyclobutylcarboxamido) phenyl) -1-oxopropane-2-yl) carbamoyl) thiophene-2-carboxylic acid (Compound 4)

In the first step of this example, cyclobutylcarboxylic acid (6a) was used instead of 2-methylcyclopropanecarboxylic acid (3a) in the first step of Example 1, and the title compound was obtained in a similar manner to Example 1.

¹ H NMR (400 MHz, DMSO-d6): δ 13.31 (s, 1H), 11.83 (s, 1H), 9.63 (s, 1H), 9.19 (d, 1H), 7.92 (d, 1H), 7.79 ( d, 1H), 7.69 (d, 1H), 7.53 (dd, 1H), 7.48 (d, 2H), 7.33 (d, 1H), 7.26 (d, 2H), 6.33 (s, 1H), 5.07 (dd , 1H), 4.73 (s, 2H), 3.17 (dt, 1H), 3.03 (s, 4H), 2.95 (s, 1H), 2.19 (dd, 2H), 2.07 (dd, 2H), 1.99--1.84 ( m, 1H), 1.79 (dd, 1H).

MS m / z = 620.78 [M + H] ⁺ .

Example 5: (S) -5-((1-(((6-chloro-2-oxo-1,2-dihydroquinolin-4-yl) methyl) (methyl) amino) -3 -(4- (Cyclohexylcarboxamido) phenyl) -1-oxopropane-2-yl) carbamoyl) thiophene-2-carboxylic acid (Compound 5)

In the first step of this example, cyclohexylcarboxylic acid (7a) was used instead of 2-methylcyclopropanecarboxylic acid (3a) in the first step of Example 1, and the title compound was obtained in a similar manner to Example 1.

¹ H NMR (400 MHz, DMSO-d6): δ 13.40 (s, 1H), 11.82 (s, 1H), 9.69 (s, 1H), 9.17 (d, 1H), 7.90 (d, 1H), 7.77 ( s, 1H), 7.69 (d, 1H), 7.52 (s, 1H), 7.47 (d, 2H), 7.32 (d, 1H), 7.24 (d, 2H), 6.32 (s, 1H), 5.06 (dd , 1H), 4.72 (s, 2H), 3.03 (s, 3H), 3.02–2.97 (m, 1H), 2.94 (s, 1H), 2.33–2.18 (m, 1H), 1.74 (t, 4H), 1.63 (d, 1H), 1.45–1.28 (m, 2H), 1.27–1.09 (m, 3H).

MS m / z = 648.80 [M + H] ⁺ .

Example 6: (S) -5-((1-((((6-chloro-2-oxo-1,2-dihydroquinolin-4-yl) methyl) (methyl) amino) -3 -(4- (Cyclopentylcarboxamido) phenyl) -1-oxopropane-2-yl) carbamoyl) thiophene-2-carboxylic acid (Compound 6)

In the first step of this example, cyclopentylcarboxylic acid (8a) was used instead of 2-methylcyclopropanecarboxylic acid (3a) in the first step of Example 1, and the title compound was obtained in a similar manner to Example 1.

¹ H NMR (400MHz DMSO-d6): δ 13.72-12.88 (m, 1H), 11.82 (s, 1H), 9.75 (s, 1H), 9.18 (d, 1H), 7.91 (d, 1H), 7.78 (d, 1H), 7.69 (d, 1H), 7.52 (t, 1H), 7.47 (d, 2H), 7.32 (d, 1H), 7.25 (d, 2H), 6.32 (s, 1H), 5.06 ( dd, 1H), 4.72 (s, 2H), 3.05–2.98 (m, 4H), 2.94 (s, 1H), 2.71 (td, 1H), 1.87–1.73 (m, 2H), 1.73–1.57 (m, 4H), 1.56–1.46 (m, 2H).

MS m / z = 634.82 [M + H] ⁺ .

Comparative Example 1: Synthesis of Control Compound 1 (PLOS ONE | DOI: 10.1371 / journal.pone.0113705 compound 13 in the literature)

Control compound 1 was synthesized according to the method of this document.

¹ H NMR (400MHz, DMSO-d6): δ 11.78 (s, 1H), 11.36 (s, 1H), 8.77 (d, 1H), 8.67 (t, 1H), 8.34 (d, 1H), 7.93 ( dd, 1H), 7.83 (d, 1H), 7.54 (dd, 1H), 7.33 (d, 3H), 7.22 (dd, 3H), 7.13 (t, 1H), 6.46 (s, 1H), 5.76 (s , 1H), 4.78 (td, 1H), 4.51 (t, 2H), 3.11 (dd, 2H).

MS m / z = 543.00 [M + H] ⁺ .

Test case:

Test Example 1. Compound inhibits FⅪa in vitro enzyme activity

The following method can be used to determine the inhibitory effect of compounds on human FXIa activity in vitro, expressed as IC ₅₀ .

FⅪa enzyme was purchased from: Haematologic Technologies.

BIOPHEN CS-21 (66) activated protein C substrate was purchased from: HYPHEN BioMed.

Solution preparation: reaction buffer: 0.05M Tris, 0.3M NaCl, pH = 7.4, Tris 606mg, NaCl 1753mg, add ddH ₂ O 80mL, adjusted with HCl pH = 7.4, the volume to 100mL.

BIOPHEN CS-21 (66) activated protein C substrate stock solution (10mM): a BIOPHEN CS-21 (66) activated protein C substrate (25mg) dissolved in 5mL sterile deionized water, stored at 4 ℃, protected from light .

BIOPHEN CS-21 (66) Activated Protein C Substrate Working Solution: Use 4 times diluted with reaction buffer before use.

FⅪa working solution: the concentration of the stock solution is 50μg / ml, frozen at -20 ℃, and diluted 1000 times before use.

Method: Add 15 μL of test sample solution (15 μL of DMSO to the control group), 15 μL of FXIa working solution and 100 μL of buffer to a 96-well plate, mix well, incubate at 37 ° C for 5 minutes, add CS-21 (66) substrate to work Start the reaction with 30 μL of solution, measure the absorbance at 405 nm, and measure it every 5 minutes. During the linear reaction period, do a linear regression analysis of the signal value-time, and calculate the slope as the reaction rate. Activity inhibition rate calculated according to the following formula, the value of IC ₅₀ was calculated for each test sample by spass16.0.

Inhibition rate% = (V ₀ -V _i ) / V ₀ x100%

In the formula: V ₀ is the reaction rate of the control well (the test compound is not added and replaced by the same volume of DMSO), and V _i is the reaction rate of the test compound.

The results are shown in Table 1.

Table 1. In vitro FⅪa enzyme activity test results

Chemical compound	FⅪa IC 50 (nM)
Compound 1	14.96
Compound 2	17.69
Compound 3	12.58
Compound 4	21.06
Compound 5	20.06
Compound 6	19.46
Control Compound 1	10.69

It can be seen from Table 1 that the compound of the present invention has a significant FⅪa inhibitory effect, and its IC ₅₀ value for the inhibition of FⅪa enzyme activity is basically equivalent to the compound with the best activity reported in the literature (control compound 1), and the maximum IC of the compound of the present invention _{The 50} value does not exceed twice the IC ₅₀ value of Control Compound 1. In contrast, the literature PLOS ONE | DOI: 10.1371 / journal.pone.0113705 most of the other compounds reported in Table 2. IC ₅₀ values of inhibition of FXIa than the active compound is preferably several orders of magnitude, although some compound The electronic and steric effects of the substituents at the corresponding positions of the control compound 1 are very similar, for example, compounds 8, 18, and 26 in Table 2 of this document. It can be seen that changing the substituent in Control Compound 1 usually significantly affects the activity of the compound. However, the inventors unexpectedly found that although the steric hindrance and electronic effects of some of the substituents in the compound of the present invention are very different from those in the control compound 1, the activity of the compound is different from that of the control compound. The activity of 1 is basically equivalent.

Test Example 2. Effect of compound on coagulation in vitro

Reagents: aPTT reagents were purchased from Virtue Pacific (Tianjin) Biotechnology Co., Ltd.

Coagulation pathways include exogenous coagulation pathway and endogenous coagulation pathway. The parameter related to the exogenous coagulation pathway is prothrombin time, expressed as PT (prothrombin time); the parameter related to the endogenous coagulation pathway is activated partial thromboplastin time, expressed as aPTT (activated partial thromboplatin time).

aPTT (activated partial thromboplastin time) detection method:

After anticoagulation of rabbit blood, centrifuge the plasma and divide into equal portions, add the test compound to make the final concentration of the test compound 0.83-13.32μM, mix and incubate at 37 ℃, then put the sample into the coagulation analyzer Perform aPTT detection. An equal volume of solvent DMSO was added to the blank plasma, and the aPTT values of the sample and blank plasma were recorded. APTT value was used to perform linear regression on the corresponding concentration of the sample, and the regression equation was used to calculate the time for the test sample to double the aPTT relative to the blank sample. The results are shown in Table 2.

Table 2. Effect of Compound 1 on rabbit aPTT

Chemical compound	aPTT ED 2x
Compound 1	6.80μM

Note: aPTT ED _2x : double the concentration of aPTT relative to the blank sample

As can be seen from Table 2, compared with the blank plasma to which no test compound was added, aPTT was doubled after adding 6.8 μM of Compound 1 of Example 1 of the present invention, indicating that the compound of the present invention achieved anti-endogenous coagulation by inhibiting FⅪa effect.

Test Example 3. Effect of compound on blood coagulation in vivo

Rabbits were anesthetized. Compound 1 and control compound 1 were administered intravenously at 1 mg / kg and 3 mg / kg after arterial intubation. Blood samples were taken before (0 min) and 1 min, 5 min, and 15 min after dosing and centrifuged. , Separate the plasma, measure the PT and aPTT with a coagulation analyzer, and calculate the extension multiple of the plasma coagulation time compared with before administration, the extension multiple = aPTT _t / aPTT ₀ , where aPTT _t is the aPTT of the sample at different times, aPTT ₀ is the administration APTT before.

PT and APTT were purchased from Virtue Pacific (Tianjin) Biotechnology Co., Ltd., and the measurement method is described in the instruction manual.

Table 3 Effect of intravenous administration of compound 1 and control compound 1 on rabbit APTT

Table 4 Effect of intravenous administration of compound 1 and control compound 1 on rabbit PT

It can be seen from Tables 3 and 4 that compared with the blank animals not given the test compound, after intravenous injection of Compound 1 of Example 1 of the present invention, aPTT at 1 min and 5 min was significantly prolonged, and the prolongation factor reached 1.17 to 2.01 times. There is no effect, which indicates that the compound of the present invention achieves an anti-endogenous coagulation effect by inhibiting FXIa. In addition, at the same dose, intravenous compound 1 had a higher multiple of aPTT than intravenous control compound 1.

Test Example 4. Solubility test

Take an excessive amount of the test compound into a conical flask with a stopper, add 20mL of pH 8.0 buffer solution respectively, put it into a constant temperature shaker, keep the temperature at (37 ± 1) ° C, and shake at 100 rpm for 24 hours. Then, the solution was taken out and allowed to stand, the supernatant was filtered with a microporous filter membrane, and the filtrate was taken for injection measurement. The test results are shown in Table 5.

Table 5 Compound solubility in pH 8.0 buffer

Chemical compound	Solubility (μg / mL)
Compound 1	17.30
Control Compound 1	0.10

It can be seen from Table 5 that the compound of the present invention has good solubility in a buffer solution of pH 8.0. It is 173 times the solubility of Control Compound 1. The higher solubility of the present invention will facilitate the development of pharmaceutical preparations and better administration.

Test Example 5. Pharmacokinetic properties test

Using SD rats as test animals, the plasma drug concentrations of rats at different times after administration of compound 1 and control compound 1 were measured by LC-MS / MS. The pharmacokinetic behavior of the compound of the present invention in rats was studied, and its pharmacokinetic characteristics were evaluated.

Test drug: Compound 1 of Example 1 and Control Compound 1 of Comparative Example 1

Test animal: Healthy adult SD rats, male, divided into two groups on average, purchased from Sbeef (Beijing) Biotechnology Co., Ltd. Animal production license number: SCXK (Beijing) 2019-0010.

Drug preparation: Weigh a certain amount of drug and prepare a 1.6 mg / mL colorless solution with physiological saline.

Administration: SD rats are given intravenously by sublingual injection. The dosage is 3 mg / kg and the administration volume is 2 mL / kg.

Method: Rats were given compound 1 and control compound 1 by intravenous injection before and after administration (0 min) and 1, 5, 15, 30, 60, 120, 240, and 360 min after administration respectively Blood was collected, heparin anticoagulated, centrifuged at 3500 rpm for 10 minutes, plasma samples were separated, and stored at -20 ° C until measurement.

After the extraction of plasma samples, the internal standard method was used for LC-MS / MS analysis.

Chromatographic mass spectrometry conditions (LC-MS / MS):

1. Liquid chromatography conditions:

LC-MS system: Waters Xevo TQD-ACQUITY UPLC H-Class liquid-mass spectrometer, equipped with infusion pump, automatic sampling system, electrospray ionization source and Masslynx 4.1 data processing workstation.

Column: ACQUITY UPLCTM BEH C18 (2.1mm × 50mm, 1.7μm)

Column temperature: 40 ℃ Sample compartment temperature: 15 ℃

Elution conditions:

Mobile phase A: 10 mM ammonium acetate + 0.05% formic acid in water (V / V)

Mobile phase B: Acetonitrile

Gradient elution:

2. Mass spectrometry conditions:

Capillary voltage: 3.5KV, cone voltage: 50V, solvent removal temperature: 400 ° C, air flow: 800L / h. MRM scanning mode ES ⁺ mode monitors ion pairs.

PKSolver pharmacokinetic and pharmacodynamic data processing software V2.0 was used, and pharmacokinetic non-compartmental model analysis was used to calculate pharmacokinetic parameters. The test results are shown in Table 6. Among them, t _{1/2 in the} table represents the plasma elimination half-life; AUC represents the area under the plasma concentration-time curve; Cl represents the overall clearance; V _dss represents the apparent volume of distribution at steady state; T _max represents the time to peak; C _max Indicates peak concentration.

Table 6 Pharmacokinetic data of intravenous administration in rats

It can be seen from Table 6 that after intravenous administration of 3 mg / kg of compound 1 and control compound 1, the plasma AUC of compound 1 in rats is 3.25 times that of control compound 1, and the C _max is 2.76 times that of control compound 1; in rats In vivo, the pharmacokinetic properties of compound 1 were better than the control compound 1. At the same dose, the apparent distribution volume of Compound 1 is smaller, which is 0.11 times that of Control Compound 1. Therefore, Compound 1 is more likely to be distributed into the blood where the target is located than Control Compound 1.

Claims (4)

1. (1) Compound A is reacted with compound C to obtain compound B;

   (2) The compound B is reacted with the compound D to obtain the compound represented by the formula (I);

   

   Wherein, for R ₁ as defined in claim 1 to 4 for the same requirements as defined for R _1.
2. A pharmaceutical composition comprising a compound represented by formula (I) according to any one of claims 1 to 4 and a pharmaceutically acceptable auxiliary material.
3. Use of the compound represented by formula (I) according to any one of claims 1 to 4 in the preparation of a medicament for preventing and / or treating thromboembolic diseases and / or thromboembolic complications.
4. A method for preventing and / or treating thromboembolic diseases and / or complications of thromboembolism, comprising administering a compound represented by formula (I) according to any one of claims 1 to 4 to a patient, or giving the patient a claim 6. The pharmaceutical composition according to 6.