CN115772196A - Compound with plasmin inhibitory activity, preparation method and application thereof - Google Patents

Abstract

The invention provides a compound with inhibitory activity of plasmin, its preparation method and application, and relates to the field of medicinal chemistry. Specifically, the present invention provides the compound represented by formula I and its application in the field of pharmacy. In formula I, X is CH or N; R ₁ , R ₂ , and R ₃ are each independently selected from H, F, Cl, Br, and I. The compound provided by the invention has excellent blood coagulation and hemostatic activity, long half-life in vivo, high blood drug concentration and exposure, good safety and good clinical application prospect.

Description

A compound with plasmin inhibitory activity, its preparation method and application

technical field

The invention relates to the field of medicinal chemistry, in particular to a compound with inhibitory activity of plasmin, its preparation method and its application in the field of pharmacy.

Background technique

M,Berntorp E.Thromb Res.2015 Feb；135(2):231-42)。Plasmin is a proteolytic enzyme that degrades fibrin. When a blood vessel ruptures due to tissue damage, the hemostatic mechanism is triggered: the blood vessel constricts, a platelet plug forms, and the coagulation process begins, leading to the formation of stable fibrin. At the same time, due to the deposition of fibrin, the fibrinolytic system is activated, which maintains a balance between the formation and cleavage of fibrin, and plays a role in maintaining the smoothness of blood vessels and remodeling the damaged blood vessel walls in the process of repairing damaged blood vessel walls. The role of damaged tissue (Tengborn L,

M, Berntorp E. Thromb Res. 2015 Feb;135(2):231-42).

M.Anaesthesiol Intensive Ther. 2015；47(4):339-50)。1950年初，人们发现赖氨酸氨基酸抑制了纤溶酶原的活化，但是作用太弱，无法用于治疗纤维蛋白溶解性出血病。1953年，Shosuke Okamoto等研究显示几种巯基和氨基碳酸具有抗血浆蛋白的作用，并发现赖氨酸的合成衍生物ε-氨基己酸(EACA)对纤溶酶原具有很强的抑制作用。EACA已在临床上被广泛使用，但是除了轻微的胃肠道副作用如恶心之外，还需要较大剂量。1962年，4-氨基-甲基- 环己烷-碳酸(AMCHA)被发现，该化合物包含两个立体异构体，进一步研究表明其反式形式(反式-4- 氨基甲基环己烷甲酸，即氨甲环酸，TXA)具有抗纤维蛋白溶解能力，活性是EACA的10倍左右，并且被证明具有更强的耐受性(Tengborn L,

M,BerntorpE.Thromb Res.2015 Feb；135(2):231-42)。The fibrinolytic system includes plasminogen, tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Plasminogen binds to lysine residues on the surface of fibrin and is converted to plasmin by an activator (ie, tPA) released from endothelial cells. Fibrinolysis inhibition can be used to treat bleeding. The use of antifibrinolytics can reduce blood loss in cardiac surgery, trauma, orthopedic surgery, solid organ transplantation, obstetrics and gynecology, neurosurgery and non-surgical diseases (Ng W, JerathA,

M. Anaesthesiol Intensive Ther. 2015;47(4):339-50). In the early 1950s, the amino acid lysine was found to inhibit the activation of plasminogen, but the effect was too weak to be useful in the treatment of fibrinolytic haemorrhages. In 1953, Shosuke Okamoto and others showed that several sulfhydryl and aminocarbonic acids had anti-plasma protein effects, and found that ε-aminocaproic acid (EACA), a synthetic derivative of lysine, had a strong inhibitory effect on plasminogen. EACA has been widely used clinically, but in addition to mild gastrointestinal side effects such as nausea, a relatively large dose is required. In 1962, 4-amino-methyl-cyclohexane-carbonic acid (AMCHA) was discovered. This compound contains two stereoisomers, and further research showed that its trans form (trans-4-aminomethylcyclohexane Formic acid, ie, tranexamic acid, TXA) has antifibrinolytic ability, is about 10 times more active than EACA, and has been shown to be more tolerated (Tengborn L,

M, Berntorp E. Thromb Res. 2015 Feb;135(2):231-42).

Tranexamic acid is a synthetic lysine derivative and antifibrinolytic that forms a reversible complex with plasminogen. By combining with plasminogen, blocking the interaction between plasminogen and plasmin heavy chain and fibrin lysine residues, thereby preventing the combination of plasminogen and fibrin surface, thereby delaying fibrinolysis. Tranexamic acid has been approved for the treatment of severe menstrual bleeding and various surgical bleeding disorders, and is currently the most commonly used hemostatic drug in clinical practice. However, a large number of literature reports show that tranexamic acid is prone to gastrointestinal adverse reactions after oral administration, such as nausea, vomiting, diarrhea and indigestion, and its dosage is relatively large, which may cause complications such as epilepsy in patients.

Other similar hemostatic drugs, such as aminocaproic acid, have problems such as rapid excretion in the human body, weak hemostatic effect, short duration of action, and many toxic reactions. It is used in patients with a history of thrombotic vascular disease and patients with renal insufficiency. The mechanism of aminotoluic acid is the same as that of aminocaproic acid, and its effect is 4 to 5 times stronger than that of aminocaproic acid. It has a significant effect on general chronic bleeding, but has no hemostatic effect on traumatic bleeding and cancer bleeding. In addition, excessive dosage can also promote thrombosis. The hemostatic drug aprotinin, which is commonly used in heart bypass surgery, was also withdrawn from the market by the FDA in 2008 because it could induce renal failure, myocardial infarction, heart failure and other reasons.

Hemostatic drugs with other mechanisms, such as carbacola, which acts on blood vessels, can induce epilepsy after repeated use; the hemostatic drug thrombin, which promotes the blood coagulation process, can only be used for gastrointestinal bleeding or local bleeding.

In view of the limited hemostatic drugs that can be selected clinically, there are more or less defects in the dosage and clinical indications, and the existing drugs of the same type have large dosages, many adverse reactions, and are prone to complications such as epilepsy and other problems, it is necessary to develop a new hemostatic drug to better meet the clinical needs.

Contents of the invention

One of the objects of the present invention is to provide a compound represented by formula I, its pharmaceutically acceptable salt, hydrate, isomer, prodrug and their mixture:

Wherein, X is CH or N;

R ₁ , R ₂ , R ₃ are each independently selected from hydrogen or halogen; specifically, R ₁ , R ₂ , R ₃ may each be independently selected from H, F, Cl, Br, I.

In certain specific embodiments, X is CH.

In certain specific embodiments, X is N.

In some specific embodiments, R ₁ , R ₂ , R ₃ are each independently selected from H or F.

In some specific embodiments, the present invention provides the following compounds, their pharmaceutically acceptable salts, hydrates, isomers, prodrugs and mixtures thereof:

Another object of the present invention is to provide a pharmaceutical composition, comprising at least one of the aforementioned compounds, or pharmaceutically acceptable salts, hydrates, isomers, prodrugs and mixtures thereof, and at least one pharmaceutically acceptable Accepted excipients.

Another object of the present invention is to provide the aforementioned compound or its pharmaceutically acceptable salts, hydrates, isomers, prodrugs and mixtures, or pharmaceutical compositions for the preparation of medicines. The medicine has the therapeutic activity of blood coagulation and hemostasis, and can be used for abnormal bleeding caused by hyperfibrinolysis, surgical operation and postoperative bleeding, and the like.

Another object of the present invention is to provide a method for treating and/or alleviating bleeding diseases or conditions, especially treating and/or alleviating abnormal bleeding caused by hyperfibrinolysis, bleeding diseases such as surgery and postoperative bleeding, including One or more of the aforementioned pharmaceutical compositions or compounds of formula I or pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof are administered to a patient in need thereof.

The "pharmaceutically acceptable salt" in the present invention refers to the salt of the compound of the present invention, which is prepared from the compound with specific substituent found in the present invention and a relatively non-toxic acid or base. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base, either neat solution or in a suitable inert solvent. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent, such as the present Invention formula I compound hydrochloride.

The term "pharmaceutically acceptable" as used herein means suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction or other problems or complications, and reasonable benefits / risk ratio is commensurate.

Unless otherwise stated, terms and phrases used herein are to be understood according to their ordinary meanings and should not be regarded as indeterminate or unclear. When a trade name appears herein, it is intended to refer to its corresponding trade name or its active ingredient.

Detailed ways

The synthesis method of the compounds and intermediates of the present invention is illustrated below by way of example, and the following examples are only used as examples of the present invention, and should not be used as limitations to the scope of the present invention. Unless otherwise specified, the raw materials and reagents involved in the present invention can be obtained through commercial channels, and the specific channel source does not affect the implementation of the technical solution of the present invention.

Embodiment 1: the preparation of (4-(aminomethyl) phenyl) phosphonic acid hydrochloride

Step 1: Preparation of tert-butyl (4-(diethoxyphosphoryl)benzyl)carbamate

Weigh tert-butyl (4-iodobenzyl)carbamate (100mg) and dissolve it in toluene (2mL), add tris(dibenzylideneacetone)dipalladium (28mg), 1,1'-bis(diphenyl Phosphine) ferrocene (34 mg), triethylamine (84 μL), diethyl phosphite (83 mg), argon replacement 3 times, reacted at 100 ° C for 4 h, quenched with water, extracted 3 times with ethyl acetate, no Dry over sodium sulfate and purify by Prep-TLC to give the title compound (90mg).

MS (ESI) m/z 344.1 (M+H) ⁺ .

Step 2: Preparation of (4-(aminomethyl)phenyl)phosphonate hydrochloride

Weigh tert-butyl (4-(diethoxyphosphoryl)benzyl)carbamate (90mg) and dissolve it in 12M aqueous hydrochloric acid (6mL), react at 110°C for 6h, concentrate under reduced pressure, and purify by pre-HPLC The title compound (10 mg) was obtained.

MS (ESI) m/z 188.1 (M+H)+.

¹ H NMR (400MHz, Deuterium Oxide) δ7.67 (dd, J=12.7, 7.6Hz, 2H), 7.41 (dd, J=7.6, 2.9Hz, 2H), 4.10 (s, 2H).

Embodiment 2: Preparation of (5-(aminomethyl)pyridin-2-yl)phosphonate hydrochloride

Step 1: Preparation of tert-butyl ((6-chloropyridin-3-yl)methyl)carbamate

Weigh (6-chloropyridin-3-yl)methanamine (1.0g) and dissolve in dichloromethane (20mL), add triethylamine (1.9mL) and di-tert-butyl dicarbonate (1.8g) successively, room temperature After reacting for 3h, TLC showed that the starting material was completely consumed. Concentration under reduced pressure and separation by column chromatography gave the title compound (1.6g).

MS (ESI) m/z 243.1 (M+H) ⁺ .

Step 2: Preparation of tert-butyl ((6-(diethoxyphosphoryl)pyridin-3-yl)methyl)carbamate

Weigh ((6-chloropyridin-3-yl) methyl) tert-butyl carbamate (200 mg) and dissolve it in toluene (5 mL), add tris(dibenzylideneacetone) dipalladium (76 mg) successively, 1, 1'-bis(diphenylphosphino)ferrocene (92 mg), triethylamine (345 μL), diethyl phosphite (230 mg), replaced with argon three times, reacted at 100 ° C for 4 h, quenched with water, acetic acid It was extracted three times with ethyl ester, dried over anhydrous sodium sulfate, and purified by Prep-TLC to obtain the title compound (186 mg).

MS (ESI) m/z 345.2 (M+H) ⁺ .

Step 3: Preparation of (5-(aminomethyl)pyridin-2-yl)phosphonate hydrochloride

Weigh ((6-(diethoxyphosphoryl)pyridin-3-yl)methyl)carbamate tert-butyl ester (186mg) dissolved in 12M aqueous hydrochloric acid (10mL), react at 110°C for 6h, reduce pressure Concentrate and purify by pre-HPLC to give the title compound (30 mg).

MS (ESI) m/z 189.0 (M+H) ⁺ .

¹ H NMR (400MHz, Deuterium Oxide) δ8.52(s, 2H), 8.12(s, 1H), 4.24(s, 2H).

Example 3: Preparation of (6-(aminomethyl)pyridin-3-yl)phosphonate hydrochloride

According to the above synthetic route and referring to the operation method of Example 2, the title compound (6-(aminomethyl)pyridin-3-yl)phosphonate hydrochloride was obtained.

MS (ESI) m/z 188.9 (M+H) ⁺ .

¹ H NMR (400MHz, Deuterium Oxide) δ8.52(s, 2H), 8.12(s, 1H), 4.54(s, 2H).

Example 4: Preparation of (5-(aminomethyl)-3-fluoropyridin-2-yl)phosphonate hydrochloride

Step 1: Preparation of (E)-N-((6-chloro-5fluoropyridin-3-yl)methylene)-2-methylpropane-2-sulfinamide

Weigh 6-bromopyridine-2-carbaldehyde (300 mg), tert-butylsulfinamide (345 mg), and tetraethyl titanate (1 mL) in 2-methyltetrahydrofuran and react at 0° C. for 4 h. TLC showed that the starting material was consumed completely. The reaction solution was diluted with ethyl acetate, added with water, filtered through celite, concentrated under reduced pressure, and separated by column chromatography to obtain the title compound (530 mg).

MS(ESI)m/z 263.0(M+H) ⁺

Step 2: Preparation of N-((6-chloro-5fluoropyridin-3-yl)methyl)-2-methylpropane-2-sulfinamide

Weigh (E)-N-((6-chloro-5fluoropyridin-3-yl)methylene)-2-methylpropane-2-sulfinamide (200mg) and dissolve in tetrahydrofuran (10mL), cool At 0°C, lithium tri-sec-butylborohydride (1.0M solution in tetrahydrofuran, 2.5 mL) was added, and the mixture was raised to room temperature for 1 hour. TLC showed that the starting material was completely consumed. It was quenched by adding water, extracted twice with ethyl acetate, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain the title compound (133 mg).

MS (ESI) m/z 265.0 (M+H) ⁺ .

Step 3: Preparation of tert-butyl ((6-(diethoxyphosphoryl)-5-fluoropyridin-3-yl)methyl)carbamate

Weigh N-((6-chloro-5-fluoropyridin-3-yl)methyl)-2-methylpropane-2-sulfinamide (133mg) and dissolve it in toluene (2mL), add tris(disulfite) successively Benzylacetone) dipalladium (46 mg), 1,1'-bis(diphenylphosphino)ferrocene (55 mg), triethylamine (208 μL), diethyl phosphite (138 mg), argon replacement 3 times , Reaction at 100°C for 4h. TLC showed that the starting material was completely consumed, quenched by adding water, extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and purified by Prep-TLC to obtain the title compound (61 mg).

MS (ESI) m/z 367.1 (M+H) ⁺ .

Step 4: Preparation of (5-(aminomethyl)-3-fluoropyridin-2-yl)phosphonate hydrochloride

Dissolve tert-butyl ((6-(diethoxyphosphoryl)-5-fluoropyridin-3-yl)methyl)carbamate (61mg) in 12M aqueous hydrochloric acid (6mL) and react at 110°C 6h. LCMS showed that the starting material was completely consumed. Concentration under reduced pressure and purification by pre-HPLC gave the title compound (30 mg).

MS(ESI)m/z 207.0(M+H) ⁺

¹ H NMR (400MHz, Deuterium Oxide) δ8.68 (s, 1H), 8.42–8.25 (m, 1H), 4.37 (s, 2H).

Example 5: Preparation of (4-(aminomethyl)-3-fluorophenyl)phosphonate hydrochloride

According to the above synthetic route and referring to the operation method of Example 4, the title compound (4-(aminomethyl)-3-fluorophenyl)phosphonium hydrochloride was obtained. MS (ESI) m/z 206.0 (M+H) ⁺ .

¹ H NMR (400MHz, Deuterium Oxide) δ7.54–7.39 (m, 3H), 4.21 (s, 2H).

Example 6: Preparation of (4-(aminomethyl)-2-fluorophenyl)phosphonate hydrochloride

According to the above synthetic route and referring to the operation method of Example 4, the title compound (4-(aminomethyl)-3-fluorophenyl)phosphonium hydrochloride was obtained.

MS (ESI) m/z 205.9 (M+H) ⁺ .

¹ H NMR (400MHz, Deuterium Oxide) δ7.57–7.36 (m, 3H), 4.21 (s, 2H).

Example 7: Preparation of (6-(aminomethyl)pyridin-2-yl)phosphonate hydrochloride

Step 1: Preparation of (E)-N-((6-bromopyridin-2-yl)methylene)-2-methylpropane-2-sulfinamide

Weigh 6-bromopyridine-2-carbaldehyde (3g), tert-butylsulfinamide (2.34g), and anhydrous copper sulfate (7.7g) in dichloromethane (30mL) and react at room temperature for 12 hours. TLC showed that the starting material was completely consumed, and the mixture was filtered through celite and concentrated under reduced pressure to obtain the title compound (5.2 g).

MS (ESI) m/z 288.8 (M+H) ⁺ .

Step 2: Preparation of N-((6-bromopyridin-2-yl)methyl)-2-methylpropane-2-sulfinamide

Dissolve (E)-N-((6-bromopyridin-2-yl)methylene)-2-methylpropane-2-sulfinamide (5.2g) in tetrahydrofuran (30mL) and cool to 0 At °C, sodium borohydride (2.0 g) was added in portions, and the reaction was continued for 2 hours. TLC showed that the starting material was completely consumed. It was quenched by adding water, extracted twice with ethyl acetate, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain the title compound (4.8 g).

MS (ESI) m/z 290.9 (M+H) ⁺ .

Step 3: Preparation of di-tert-butyl (6-(((tert-butylsulfinyl)amino)methyl)pyridin-2-yl)phosphonate

Weigh N-((6-bromopyridin-2-yl)methyl)-2-methylpropane-2-sulfinamide (150mg) and dissolve it in toluene (2mL), add tris(dibenzylideneacetone) successively ) dipalladium (95 mg), 1,1'-bis(diphenylphosphino)ferrocene (115 mg), triethylamine (144 μL), di-tert-butyl phosphite (202 μL), argon replacement 3 times, React overnight at 100°C. TLC showed that the starting material was completely consumed, quenched by adding water, extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and purified by Prep-HPLC to obtain the title compound (125 mg).

MS (ESI) m/z 404.9 (M+H) ⁺ .

Step 4: Preparation of (6-(aminomethyl)pyridin-2-yl)phosphonate hydrochloride

Weigh (6-(((tert-butylsulfinyl)amino)methyl)pyridin-2-yl)di-tert-butyl phosphonate (66mg) dissolved in 6M aqueous hydrochloric acid (6mL) and react at room temperature overnight. LCMS showed that the starting material was completely consumed. Concentration under reduced pressure and purification by pre-HPLC gave the title compound (64.13 mg).

MS (ESI) m/z 188.9 (M+H) ⁺ .

¹ H NMR (400MHz, Deuterium Oxide) δ8.09–7.97(m,1H),7.83(q,J=6.8Hz,1H),7.63–7.53(m,1H),4.36 (d,J=3.3Hz, 2H).

The preparation method exemplified in the foregoing examples is an illustration of the preparation process of the compound of the present invention. Those skilled in the art can refer to the above specific method and combine with the general knowledge in the field to prepare other compounds within the scope of the present invention. is easily achievable. At the same time, the salt of the prepared compound (as the hydrochloride prepared in the foregoing examples) can be further converted into a free state compound by using conventional experimental means in the art, for example, the following method is used: take the hydrochloride of the compound and dissolve it in water Completely, slowly add 20% sodium bicarbonate solution until a large amount of solids are precipitated, and the obtained solids are filtered and dried to obtain the free compound. According to the common knowledge in the field and the experimental research of the inventors, it is believed that the activity of the compounds of the present invention will not be affected by the salt or free state of the compounds.

biological test

Test Example 1: Plasma Clot Degradation Experiment

1. Purpose of the experiment

The inhibitory effect of the compounds of the invention on the degradation of human plasma clots was determined.

2. Experimental materials and instruments

3. Experimental steps

3.1 Collect fresh healthy human blood, use 0.109M trisodium citrate as an anticoagulant, mix with 1 part of anticoagulant + 9 parts of blood, centrifuge at 2000x g at room temperature for 20 minutes, collect the supernatant (that is, plasma), and pack Store at -80°C for later use.

3.2 On the day of the experiment, the plasma was thawed in a water bath at 37°C, and all reagents except tPA were preheated at 37°C.

3.3 Add 12.5 μL of 80 mM CaCl ₂ (HEPES buffer, pH 7.4) to the 96-well plate, and then add 25 μL of different concentrations of the test compound diluted with normal saline, and add an equal volume of normal saline to the negative control well.

3.4 Mix 50 μL of preheated plasma with 12.5 μL of 4 nM tPA (HEPES buffer, pH 7.4), immediately add to a 96-well plate, detect the absorbance at 405 nm, read every 2 minutes, and measure continuously for 15 hours.

3.5 The absorption value changes with time, rising first and then decreasing. The time corresponding to the median absorption value in the descending segment - the corresponding time to the median absorbing value in the ascending segment is the plasma clot degradation time (Clot lysis time). Taking the plasma clot degradation time of the negative control well as a reference, calculate the plasma clot degradation time relative to the wells with different concentrations of compounds, and obtain the inhibition rate:

Inhibition rate%=(1- _{Clot analysis time} of negative control well/ _{Clot analysis time} of compound well)×100%

3.6 Fitting the dose-effect curve

Taking the log value of the compound concentration as the X-axis and the percentage inhibition rate as the Y-axis, the log (inhibitor) vs. response-variable slope (Variable slope) of the analysis software GraphPadPrism 5 is used to fit the dose-effect curve to obtain the _IC50 values for cell viability.

Calculation formula: Y=min+(max-min)/(1+10^((LogIC ₅₀ -X)×Hillslope)).

The inhibitory effect of the compound of the present invention on plasma clot degradation was determined by the above test, and the IC _{50 ratio} value of the compound of the present invention (IC _{50 ratio} =IC _{50 test compound} /IC _{50 aminomethylbenzoic acid} ) was obtained by calculation, as shown in Table 1.

Table 1 Results of determination of plasma clot-degrading activity of compounds

Note: "/" in Table 1 does not calculate relevant values.

The experimental data in Table 1 shows that the compounds provided by the present invention can effectively inhibit the degradation of plasma clots, have excellent coagulation and hemostatic activities, and thus have excellent drug prospects.

Test Example 2: Rat PK Test

1. Purpose of the experiment

The pharmacokinetic properties of the compound of the present invention in rats were studied by measuring the plasma drug concentration after intravenous and intragastric administration in rats.

2. Experimental animals

SD rats, SFP grade, male, N=3, source: Shanghai Sipro-Bikay Laboratory Animal Co., Ltd.

3. Drug preparation and administration

The compound was weighed and added to physiological saline to dissolve, the drug solution was prepared and then filtered with a filter membrane, and the drug solution before and after filtration was analyzed for preparation.

Preparation: 0.2mg/mL solution for intravenous administration.

The day before the experiment, the rats were fasted overnight and fed 4 hours after administration.

On the day of the experiment, the administration was administered according to the scheme shown in Table 2. At each time point after the administration, approximately 200 μL of blood was collected from the jugular vein and placed in a sodium heparin anticoagulant tube. Blood samples were placed on ice after collection, and the plasma was centrifuged within 1 hour (centrifugation conditions: 6800g, 6 minutes, 2-8°C). Plasma samples were stored in a -80°C freezer prior to analysis.

Table 2 Dosing regimen

4. Bioanalysis

Instruments and equipment: LC-MS/MS-19 (TQ5500, AB SCIEX, USA).

Internal standard: warfarin.

Chromatographic column: ACQUITY UPLC BEH C18, model 1.7um 2.1*50mm, purchased from Shenzhen Nuoyadi Chemical Technology Co., Ltd.;

Flow rate: 0.60 ml/min.

Column temperature: 40°C.

Mobile phase A: 0.1% formic acid in water.

Mobile phase B: 0.1% formic acid in acetonitrile.

The elution gradient is shown in Table 3.

Table 3 Elution gradient

time (min) Mobile phase A(%) Mobile phase B(%) 0 98 2 0.60 12 88 1.10 12 88 1.11 98 2 1.40 98 2

MS detection conditions: electrospray ionization source (ESI), positive ion mode, MRM scan.

Take 30 μL of the plasma sample prepared under item “3” of this example, and use 300 μL of MeOH for protein precipitation, which contains an internal standard of 100 ng/mL. The mixture was vortexed for 1 minute and centrifuged at 18000 g for 7 minutes. Transfer the supernatant to a 96-well plate. Inject 4 µL of the supernatant into LC-MS/MS for analysis.

The above-mentioned LC-MS/MS analysis method was used to determine the concentration of the compound in rat plasma, and the pharmacokinetic parameters were calculated by using Phoenix WinNonlin7.0 based on the blood concentration data at different time points.

The compounds of the present invention were determined through the above experiments, and the measured pharmacokinetic parameters in rats are shown in Table 4.

Table 4 Compound pharmacokinetic parameters

compound T_1/2(h) C_max(ng/mL) AUC_0-t(h*ng/mL) AUC_0-∞(h*ng/mL) CL(mL/h/kg) Aminomethylbenzoic acid 0.27 1811.23 584.3 612.82 1640.65 Example 1 0.65 9174.61 8019.99 8141.32 125.05 Example 2 2.21 4055.76 5908.63 6283.47 159.79 Example 4 1.66 6057.22 14112.35 14571.82 68.67

It can be seen from Table 4 that, compared with aminomethylbenzoic acid, the compound provided by the present invention has a longer half-life in vivo, higher blood concentration and exposure, better safety, and has a good clinical application prospect.

Claims (8)

1. A compound of formula I, a pharmaceutically acceptable salt, hydrate, isomer, prodrug or a mixture thereof,

wherein, X is CH or N;

R ₁ 、R ₂ 、R ₃ each independently selected from H, F, cl, br, I.

2. The compound, pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof of claim 1, wherein X is CH.

3. The compound, pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof of claim 1, wherein X is N.

4. The compound, pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof of any of claims 1-3, wherein R is ₁ 、R ₂ 、R ₃ Each independently selected from H or F.

5. The following compounds, pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof,

6. a pharmaceutical composition comprising a compound of any one of claims 1-5, a pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, and a pharmaceutically acceptable excipient.

7. Use of a compound of any one of claims 1-6, a pharmaceutically acceptable salt, hydrate, isomer, prodrug, or a mixture thereof, or a pharmaceutical composition for the manufacture of a medicament.

8. Use according to claim 7, wherein the medicament has hemostatic, blood clotting activity.