CN110407819B - A Thrombin Receptor Antagonist as Prevention of Surgical Complications - Google Patents

Abstract

Provides a Vorapaxar derivative and a preparation method thereof, the structure of the compound is shown as formula I, wherein R is₁Selected from hydrogen, halogen, C1-5 alkyl, fluoro C1-5 alkyl; the compound has excellent PAR-1 inhibitory activity, is particularly suitable for preventing the occurrence of complications in surgical operations, and is expected to be a potential new medicine for treating myocardial infarction and coronary artery diseases instead of Vorapaxar.

Description

A Thrombin Receptor Antagonist as Prevention of Surgical Complications

technical field

The present invention relates to a thrombin receptor antagonist for preventing surgical complications, in particular, the present invention relates to a vorapasa derivative, in addition, the present invention also relates to a preparation method of the compound and the Its use for the preparation of medicaments.

Background technique

Surgical bleeding is one of the main problems for clinicians in surgical operations. Rational use of hemostatic drugs can reduce surgical bleeding and other complications involving the heart. Platelet activators play a key role in controlling bleeding and hemostasis. Platelet activators include thrombin, adenosine diphosphate (DP), thromboxane A2 (TxA2), epinephrine and collagen, etc. Activated platelets deform, secrete granular components, and express activated protein IIb/IIIa receptors. , binds to fibrinogen in circulating blood, leading to thrombosis. Thrombin, as an important activator, activates the G protein-coupled receptor on the platelet surface, the protease activated receptor (PAR). Vorapaxa sulfate (XII) is a first-in-class protease-activated receptor 1 (PAR-1) antagonist developed by Merck & Co. It is used in patients with a history of myocardial infarction (MI) or peripheral arterial disease (PAD) to reduce the incidence of thrombotic cardiovascular events in reducing the tendency of platelets to aggregate and inhibit the formation of blood clots. The drug was approved by the FDA for marketing in the United States on May 8, 2014, and was approved for marketing in the European Union by the EMA on January 19, 2015. The further optimization of the structure of vorapasa drugs and the research and development of their derivatives have gradually become a research hotspot. Researchers hope to obtain more active vorapasa derivatives by optimizing the structure.

Through long-term and unremitting research, the applicant surprisingly found that a vorapasa derivative with tert-butyl urea has better thrombin receptor antagonistic activity, which is especially suitable for preventing complications in surgical operations, and can also Instead of vorapaxa, it becomes a potential new drug for the treatment of myocardial infarction and coronary artery disease.

SUMMARY OF THE INVENTION

One of the objects of the present invention is to provide a novel vorapasa derivative, which has very excellent thrombin receptor antagonistic activity.

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

Yet another object of the present invention is to provide a pharmaceutical composition comprising the compound.

Another object of the present invention is to provide the application of the compound in the preparation of a thrombin receptor antagonist medicine.

To this end, the present invention provides a compound as shown in formula I:

and pharmaceutically acceptable salts thereof;

Wherein, the substituent R ₁ is selected from hydrogen, halogen, C1-5 alkyl, and fluoro-C1-5 alkyl.

Detailed ways

While a preferred embodiment of the present invention has been shown and described in this application, it will be obvious to those skilled in the art that this embodiment is provided by way of example only. Numerous modifications, permutations, and permutations will occur to those skilled in the art, which are within the scope of the invention. It should be understood that in practicing the invention, various alternatives to the embodiments of the invention described herein may be employed. It is intended that the appended claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned in this application are incorporated herein by reference.

compound

The present invention provides a compound shown in formula I:

and pharmaceutically acceptable salts thereof;

Wherein, the substituent R ₁ is selected from hydrogen, halogen, C1-5 alkyl, and fluoro-C1-5 alkyl.

Preferably, R is selected from hydrogen, fluorine, chlorine, methyl, ethyl, trifluoromethyl.

Preferably, the pharmaceutically acceptable salts of the compound of formula I may include, but are not limited to, vorapasa sulfate, vorapasa phosphate, vorapasa hydrochloride, and vorapasa fumarate.

Preparation

The invention provides a kind of preparation method as shown in formula I and pharmaceutically acceptable salt thereof, and its synthetic route is as follows:

Wherein, R ₂ and R ₃ are independently selected from hydrogen, C1-5 alkyl, aryl C1-5 alkyl, C3-10 cycloalkyl.

The method specifically includes the following steps:

Step (1): adding di-tert-butyl dicarbonate (Boc ₂ O) to the solution formed by the compound of formula II, triethylamine and solvent, and reacting at a temperature of 50° C. to 80° C. for 3 to 8 hours to obtain formula Compound III, wherein the solvent is an alcohol solvent or DMF;

Step (2): the compound of formula III is hydrolyzed in the presence of a base to obtain the compound of formula IV;

Step (3): at a temperature of -80°C to -10°C, the compound of formula IV and the compound of formula V are subjected to a condensation reaction in the presence of an organolithium reagent and a solvent to obtain compound VI;

Step (4): in the solution of compound VI and organic solvent, add dilute acid solution to hydrolyze the Boc protecting group to obtain the compound of formula VII;

Step (5): The compound of formula I is prepared by reacting the compound of formula VII with tert-butylcarbamoyl chloride.

Further, the molar ratio of the compound of formula II, Boc ₂ O and triethylamine in the step (1) is 1:1-2:1-1.5.

Preferably, the base in the step (2) is an organic base or an inorganic base, preferably triethylamine, potassium hydroxide, sodium hydroxide, potassium carbonate, or sodium carbonate.

Further, it is characterized in that the organolithium reagent in the step (3) is selected from n-butyllithium or tert-butyllithium; the solvent is selected from THF, toluene, DMF or dioxane; the formula IV The molar ratio of the compound, the compound of formula V and the organolithium reagent is 1:1-2:0.1-0.5;

Preferably, in the step (4), the solution is selected from dichloromethane, dichloroethane, chloroform, and THF; the dilute acid solution is selected from dilute hydrochloric acid, dilute sulfuric acid, or TFA solution.

combination

Another aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned compound of formula (I) or a pharmaceutically acceptable salt thereof, or a compound prepared by the above-mentioned method, and a pharmaceutically acceptable carrier or excipient.

In pharmaceutical compositions for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration, alone or with other active ingredients The active ingredient can be mixed with conventional pharmaceutical carriers and administered to animals or humans in dosage unit form. Suitable unit dosage forms for administration include oral forms, preferably tablets, capsules, pills, powders, granules and the like.

A preferred preparation method of Vorapaxa preparation provided by the present invention includes: step 1, adding a binder after dissolving Vorapaxa to prepare a granulation solution for later use; Then, the granulation solution described in step 1 is sprayed into the fluidized mixture for granulation and drying, and finally, a lubricant is added to mix evenly, tablet compression, and coating to obtain a Vorapaxa preparation.

Examples of such binders include hypromellose, hypromellose or povidone. Materials that can be used as fillers include lactose, mannitol, starch, microcrystalline cellulose, or a combination of two or more thereof; preferably a combination of lactose and microcrystalline cellulose or a combination of mannitol and microcrystalline cellulose. For the combination of multiple fillers, the amount of each filler is not limited. When choosing two preferred options for fillers, the weight ratio between lactose and microcrystalline cellulose or the weight ratio between mannitol and microcrystalline cellulose can be 1 :1, 4:5 or 11:7.

Examples of the disintegrant specifically include croscarmellose sodium, low-substituted hydroxypropyl cellulose, or sodium carboxymethyl starch.

The lubricant may be selected from magnesium stearate, sodium stearyl fumarate or glyceryl tristearate.

For this type of pharmaceutical composition, the compound of formula I, binder, filler, disintegrant and lubricant are formulated in a suitable weight ratio; for example, in parts by weight, the parts by weight are: compound of formula I 1- 20 parts, binder 1-10 parts, disintegrant 0.5-5 parts, filler 5-75 parts, lubricant 1-5 parts. More preferably, the parts by weight are:

In the pharmaceutical compositions of the present invention, the compounds of formula I are generally formulated in unit dosage form. For one or several daily administrations, each unit dose is 0.5 to 500 mg, advantageously 5 to 200 mg, preferably 5 to 100 mg.

These dosages are examples of average situations, and there may be special circumstances where higher or lower dosages are appropriate and such dosages are also part of the invention. The appropriate dosage for each patient will be determined by the physician according to customary experience, based on the mode of administration, age, weight and response to the patient.

use

The present invention also provides a compound of formula I and a pharmaceutically acceptable salt thereof, or the use of the compound prepared by the above method to prepare a thrombin receptor antagonist; the thrombin receptor antagonist can be used for prevention Surgical complications.

The Vorapaxa derivative with the structure of formula I provided by the present invention has excellent PAR-1 inhibitory activity, and the experimental results show that the shown compound of formula I exhibits better pharmaceutical activity than Vorapaxa, and is expected to replace Vorapax Sand has become a potential new drug for the treatment of myocardial infarction and coronary artery disease, especially for preventing complications in surgical operations.

Example

Example 1: Preparation of Compound I-a:

The reaction route is as follows:

Step (1): Weigh 147 mg (0.5 mmol) of the compound of formula II-a and 60.6 mg (0.6 mmol) of triethylamine and dissolve it in 50 mL of ethanol, and add 163.5 mg (0.75 mmol) of dicarbonate to the resulting solution. The tert-butyl ester (Boc ₂ O) was heated under reflux for 6 hours, cooled, added with 50 mL of water and 30 mL of dichloromethane, separated, and the organic phase was distilled under reduced pressure to obtain 163.5 mg of the compound of formula III-a with a yield of 163.5 mg. 83%.

Step (2): Weigh 118 mg (0.3 mmol) of the compound of formula III-a and dissolve it in 35 mL of methanol, add 5 mL of 20% aqueous sodium hydroxide solution, heat to 50 ° C for hydrolysis, add 30 mL of ethyl acetate, extract, 157 mg of the compound of formula IV were obtained in 96% yield.

Step (3): at -20°C, under nitrogen protection, 136mg (0.25mmol) of formula IV compound and 97mg (0.3mmol) of formula V-a compound in 3.2mg (0.05mmol) of n-butyllithium reagent and 40mL of toluene In the presence of , the condensation reaction was carried out for 4 hours, filtered, and the solvent was recovered by distillation under reduced pressure. The silica gel column chromatography was used for separation and purification (eluent was dichloromethane:methanol=5:1) to obtain 81 mg of compound VI-a with a yield of 62%.

Step (4): Dissolve 78 mg (0.15 mmol) of compound VI-a in 20 mL of THF, add 20% dilute hydrochloric acid solution to the obtained solution, and hydrolyze the Boc protecting group to obtain 62 mg of the compound of formula VII-a with a yield of 62 mg. is 98%.

Step (5): dissolve (0.1 mmol) the compound of formula VII-a in 25 mL of dichloromethane solution, add (0.12 mmol) tert-butylcarbamoyl chloride, heat under reflux for 6 hours, cool, and recover the solvent by distillation under reduced pressure, It was separated and purified by silica gel column chromatography (eluent: dichloromethane:methanol=8:1) to prepare 46.2 mg of the compound of formula I-a with a yield of 89%.

¹ H-NMR (CDCl ₃ , 400MHz): 7.62(s, 1H) 7.54(d, 1H), 7.51(s, 1H), 7.36-7.45(m, 2H), 7.24-7.31(m, 2H), 6.21 (d, 1H), 5.89 (m, 1H), 5.39 (d, 1H), 4.83 (m, 1H), 4.01 (m, 2H), 3.37 (m, 1H), 2.01-2.52 (m, 6H), 1.92 (d, 1H), 1.77 (s, 1H), 1.71 (m, 1H), 1.52 (m, 2H), 1.47 (d, 3H), 1.21 (s, 9H). LC-MS: m/z=520 (M+H ⁺ ), HPLC purity 99.13%.

Example 2: Preparation of Compound I-b

Implement step (3) to step (5) according to the method of embodiment 1;

Wherein, in step (3), 150mg (0.28mmol) compound of formula IV and 125mg (0.35mmol) compound of formula Vb (R ₁ is 2-Cl) in the presence of 5.1mg (0.08mmol) n-butyllithium reagent and 50mL DMF The dilute acid solution used in step (4) was 10% TFA solution, and the eluent for column chromatography in step (5) was dichloromethane:methanol=6:1; 79mg of compound Ib was obtained. The total yield of steps (3) to (5) was 51%.

¹ H-NMR (CDCl ₃ , 400MHz): 7.73(s, 1H) 7.61(d, 2H), 7.54(s, 1H), 7.33-7.42(m, 2H), 6.22(d, 1H), 5.91(m , 1H), 5.37(d, 1H), 4.85(m, 1H), 4.07(m, 2H), 3.41(m, 1H), 2.08-2.55(m, 6H), 1.87(d, 1H), 1.75( s, 1H), 1.68 (m, 1H), 1.55 (m, 2H), 1.49 (d, 3H), 1.23 (s, 9H). LC-MS: m/z=555 (M+H ⁺ ), HPLC purity 99.53%.

Example 3: Preparation of Compound I-c

Implement step (3) to step (5) according to the method of embodiment 1;

Wherein, in step (3), 112 mg (0.21 mmol) of formula IV compound and 98 mg (0.25 mmol) of formula Vb compound (R ₁ is 2-CF ₃ ) in 5.1 mg (0.08 mmol) of n-butyllithium reagent and 35 mL of dioxygen Carry out in the presence of hexacyclic ring; the dilute acid solution used in step (4) is 20% sulfuric acid solution, and the eluent of column chromatography in step (5) is dichloromethane:methanol=4:1, to obtain 53mg of compound Ic. The total yield of steps (3) to (5) was 43%.

¹ H-NMR (CDCl ₃ , 400MHz): 7.75(s, 1H) 7.64(d, 2H), 7.52(s, 1H), 7.36-7.42(m, 2H), 6.24(d, 1H), 5.95(m , 1H), 5.35(d, 1H), 4.76(m, 1H), 3.93(m, 2H), 3.31(m, 1H), 2.10-2.43(m, 6H), 1.84(d, 1H), 1.72( s, 1H), 1.67 (m, 1H), 1.52 (m, 2H), 1.41 (d, 3H), 1.23 (s, 9H). LC-MS: m/z=588 (M+H ⁺ ), HPLC purity 98.78%.

Comparative Example: Preparation of Comparative Compounds 1 and 2:

Steps (1) to (5) were carried out according to the method of Example 1 to prepare Comparative Compound 1 and Compound 2, wherein in step (5), the reactants ethylcarbamoyl chloride and benzylcarbamoyl chloride were respectively used to replace Example 1 tert-butylcarbamoyl chloride in . The NMR and Pristine data of Comparative Compound 1 and Comparative Compound 2 are as follows:

Comparative compound 1: ¹ H-NMR (CDCl ₃ , 400MHz): 7.57(s, 1H) 7.50(d, 1H), 7.47(s, 1H), 7.29-7.42(m, 2H), 7.21-7.25(m, 2H), 6.12(d, 1H), 5.95(m, 1H), 5.40(d, 1H), 4.82(m, 1H), 4.11(m, 2H), 4.02(m, 1H), 1.89-2.45(m , 6H), 1.95(d, 1H), 1.75(s, 1H), 1.68(m, 1H), 1.53(m, 2H), 1.49(d, 3H), 1.21(m, 2H), 1.08(t, 3H). LC-MS: m/z=492 (M+H+, HPLC purity 98.62%).

Comparative compound 2: ¹ H-NMR (CDCl ₃ , 400 MHz): 7.60 (s, 1H) 7.57 (d, 1H), 7.50 (s, 1H), 7.35-7.41 (m, 2H), 7.25-7.32 (m, 5H), 7.20-7.32(m, 2H), 6.22(d, 1H), 5.87(m, 1H), 5.42(d, 1H), 4.81(m, 1H), 4.12(m, 2H), 3.74(m , 1H), 2.26-2.41(m, 6H), 2.01(m, 2H), 1.87(d, 1H), 1.75(s, 1H), 1.67(m, 1H), 1.50(m, 2H), 1.45( d, 3H). LC-MS: m/z=554 (M+H ⁺ ), HPLC purity 99.37%.

Example 5: Determination of Biological Activity (PAR-1 Inhibitory Activity) of Compounds of the Invention

1. Cell Culture

1.1 Cell recovery

The HEK293-Gα15-PAR1 cell line (HD Biosciences stably transfected cell line) was quickly taken out from the liquid nitrogen tank, and kept shaking in a water bath at 37°C until it was completely melted. The cell suspension was quickly added to pre-warmed medium (90% DMEM+10% FBS+1X Pen/Strep), placed in a centrifuge, 1000 rpm, and centrifuged for 10 minutes. Take out the centrifuge tube, discard the supernatant, add fresh pre-warmed medium to the centrifuge tube, resuspend the cells, add the cell suspension to the culture dish, and incubate at 37°C, 5% CO ₂ . 1.2 Passaging

When the cells reach 80-90% of the culture dish, gently wash the cells with 0.05% trypsin-EDTA, remove part of the digestion solution, incubate the cells for 2-3 min, use a new medium to stop the digestion, gently pipette the cells with a pipette tip Cells were resuspended and, typically, passaged 1:4 to 1:8 every 2-3 days.

2. Calcium ion influx experiment

2.1 Cell plate coating

One day before the experiment, 1×Matrigel (Brand: BD, Cat#: 356230) was added to a clean 384-well cell plate, incubated at 37°C for 30 minutes, and then the coating solution was removed by inverting centrifugation at 500 rpm for 30 seconds.

2.2 Plank

The cell pellet was collected by digestion, resuspended to 3×10 ⁵ cells/mL with medium, 50 μL per well was added to the coated cell plate, and then incubated overnight at 37°C, 5% CO ₂ .

2.3 Buffer preparation

On the day of the experiment, fresh experimental buffer and 0.5×Calcium 4 (Brand: Molecular Devices, Cat#: R8141) loading buffer were prepared.

2.4 Compound formulation

The 30 mM DMSO stock solution was first diluted with DMSO to 10 mM, and then 4-fold dilution was started from 10 mM, with a total of 10 concentrations. 10 DMSO concentration gradients of the compound were added to the experimental buffer at a ratio of 1:20 to prepare a working solution of the compound (5 times the final reaction concentration). Afterwards, the working solution of the compound was transferred to a 384-well compound plate for use.

Positive control: 40 mM DMSO stock solution of reference compound SCH79797 was diluted to 2 mM;

Negative Control: 5% DMSO in Assay Buffer

2.5 Formulation of PAR-1 agonist haTRAP

The 10 mM DMSO stock solution of the agonist haTRAP was diluted to 18 μM with experimental buffer (6 times the final reaction concentration of 3 μM), and then at least 25 μl/well was transferred to a 384-well compound plate for use.

2.6 Dye Incubation

Take out the cell plate incubated overnight, remove the cell culture medium by inverted centrifugation at 300 rpm for 30 seconds, add 20 μL of freshly prepared 0.5×Calcium 4 loading buffer to each well, and incubate at 37°C, 5% CO ₂ for 1 hour.

2.7 Adding compounds

Transfer 5 μL/well of compound working solution from the compound plate to the cell plate according to the layout, and then incubate again at 37°C, 5% CO ₂ for 15 minutes.

2.8 Add agonist to read fluorescence signal

Transfer 5 μl/well of agonist from a 384-well compound plate (FLIPR) to a cell plate according to the FLIPR setup program while reading the fluorescent signal in each well of the cell plate.

3. Data Analysis

The inhibition rate (%) of the compounds in each well of each cell plate was calculated based on the fluorescence signal values of the positive and negative controls on the cell plate. The positive control contained a high concentration of the reference compound (400 μM of SCH79797), which was a 100% inhibition control; the negative control did not contain any compound, only DMSO (1% DMSO) as the compound solvent, which was a 0% inhibition control. The calculated inhibition rate and the corresponding compound concentration were imported into the relevant software for graphing, and the IC ₅₀ value of the compound was calculated according to the 4-PL dose-effect formula. The _IC50 result of the reference compound is also one of the criteria to check the quality of each experiment.

The activity screening results of some compounds are shown in Table 1.

Table 1 Compound dose-response results

The results of activity screening showed that compounds Ia, Ib, and Ic all had significant anti-PAR-1 activity in vitro, and their IC ₅₀ values were between 0.87-2.6 μM, and their in vitro PAR-1 inhibitory activity was significantly higher than that of varapasa. , and much higher than the structurally similar comparative compounds 1 (11.20 μM) and 2 (10.83 μM).

Claims (10)

1. A compound of formula I:

and pharmaceutically acceptable salts thereof;

wherein, the substituent R₁Selected from hydrogen, halogen, C1-5 alkyl, fluoro C1-5 alkyl.

2. The compound of formula I as claimed in claim 1, wherein R is a pharmaceutically acceptable salt thereof₁Selected from hydrogen, fluorine, chlorine, methyl, ethyl, trifluoromethyl.

3. A process for the preparation of compounds of formula (I) and pharmaceutically acceptable salts thereof as claimed in claim 1 or 2, which is synthesized as follows:

wherein R is₂And R₃Independently selected from hydrogen, C1-5 alkyl, aryl C1-5 alkyl, C3-10 cycloalkyl.

4. The preparation method according to claim 3, comprising the following steps:

step (1): adding di-tert-butyl dicarbonate (Boc) to a solution of the compound of formula II, triethylamine and a solvent₂O), reacting for 3-8 hours at the temperature of 50-80 ℃ to obtain a compound shown in the formula III, wherein the solvent is an alcohol solvent or DMF;

step (2): hydrolyzing the compound of formula III in the presence of a base to obtain a compound of formula IV;

and (3): carrying out condensation reaction on a compound shown in a formula IV and a compound shown in a formula V in the presence of an organic lithium reagent and a solvent at a temperature of between 80 ℃ below zero and 10 ℃ below zero to obtain a compound VI;

and (4): adding a dilute acid solution into a solution of the compound VI and an organic solvent to hydrolyze and remove a Boc protecting group to obtain a compound of a formula VII;

and (5): reacting the compound of the formula VII with tert-butylamine formyl chloride to obtain the compound of the formula I.

5. The method of claim 4, wherein the compound of formula II, Boc in step (1)₂The molar ratio of O to triethylamine is 1: 1-2: 1-1.5.

6. The production method according to claim 4 or 5, wherein the base in the step (2) is an organic base or an inorganic base.

7. The process according to claim 4 or 5, wherein the organolithium reagent in step (3) is selected from n-butyllithium or t-butyllithium; the solvent is selected from THF, toluene, DMF or dioxane; the molar ratio of the compound shown in the formula IV to the compound shown in the formula V to the organic lithium reagent is 1: 1-2: 0.1-0.5.

8. The method according to claim 4 or 5, wherein the organic solvent in the step (4) is selected from the group consisting of dichloromethane, dichloroethane, chloroform, THF; the dilute acid solution is selected from dilute hydrochloric acid, dilute sulfuric acid, or TFA solution.

9. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 and a pharmaceutically acceptable carrier or excipient.

10. Use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-2, or a pharmaceutical composition as claimed in claim 9, for the preparation of a thrombin receptor antagonist.