CN1052481C - Platelet activating factor antagonist and preparation thereof - Google Patents

Abstract

A 2-(3,4,5-trimethoxyphenyl)-6-(substituted phenyl)tetrahydropyran compound, which is prepared by using ethyl 3,4,5-trimethoxybenzoylacetate and β -Bromosubstituted propiophenone is condensed to form 2-(3,4,5-trimethoxybenzoyl)-5-oxo-(substituted phenyl) ethyl pentanoate, which is formed by removing ethoxyformyl -(3,4,5-trimethoxyphenyl)-5-(substituted phenyl)-1,5-pentanedione is formed by reduction and cyclization, and can be used as a platelet activating factor antagonist. After testing, trans 2-(3,4,5-trimethoxyphenyl)-6-(2,4-difluorophenyl)tetrahydropyran has the highest antagonistic activity.

Description

A novel platelet activating factor antagonist and its preparation method

The invention relates to the field of organic chemistry, in particular to a novel 2-(3,4,5-trimethoxyphenyl)-6-(substituted phenyl)tetrahydropyran compound antagonizing platelet activating factor and its preparation method.

Platelet-Activating Factor (PAF) has been proven to be an important mediator of the human body, which is related to various pathological and physiological processes of the human body. important role in the process. In the past ten years, a lot of research work has been carried out around this aspect at home and abroad, focusing on finding effective PAF antagonists for the treatment of various diseases caused by PAF. Synthetic PAF antagonists that have been discovered include PAF analogs, kelp ketone analogs, benzodiazepine derivatives, substituted tetrahydrofuran compounds, 1,4-diacylpiperazine compounds and 1,3- Disubstituted aryl-α-substituted cyclopentane derivatives, etc., but so far no 2-(3,4,5-trimethoxyphenyl)-6-(substituted phenyl)tetrahydropyran compounds Reported as a PAF antagonist.

The object of the present invention is to provide a novel PAF antagonist: 2-(3,4,5-trimethoxyphenyl)-6-(substituted phenyl)tetrahydropyran compounds and a preparation method thereof.

其中R₁可为：F，    CH₃，HThe general structural formula of the compound is as follows:

Where R ₁ can be: F, CH ₃ , H

R ₂ can be: OCH ₃ , H

R ₃ can be: F, Cl, CH ₃ , OCH ₃ ,

The compound of the present invention is formed by condensing 3,4,5-trimethoxyethyl benzoylacetate as a raw material with β-bromosubstituted phenylacetone, removing ethoxyformyl, reducing, and cyclizing. The synthesis process route is as follows:

1. Condensation of ethyl 3,4,5-trimethoxybenzoylacetate and β-bromosubstituted phenylacetone in alcohol solution containing sodium alcohol to generate intermediate 2-(3,4,5-trimethoxybenzyl Acyl)-5-oxo-(substituted phenyl)pentanoic acid ethyl ester (I) [the following molecular formula is replaced by (1) abbreviated, and the following (II), (III), (IV), (V), etc. are also the same] ;

2. (I) stirred in 40 ℃ 2.5% potassium hydroxide alcohol water (1: 1) liquid for 48 hours to generate 1-(3,4,5-trimethoxyphenyl)-5-(substituted phenyl )-1,5-pentanedione (II);

3. The methanol solution of (II) generates intermediate 1-(3,4,5-trimethoxyphenyl)-5-(substituted phenyl)-1,5-pentanediol (III) through sodium borohydride reduction );

4. The dichloromethane solution of (III) is treated with methanesulfonyl chloride and triethylamine, and cyclized to generate 2-(3,4,5-trimethoxyphenyl)-6-(substituted phenyl)tetrahydropyridine The furan compound was separated by chromatography to obtain cis and trans compounds (IV, V).

Figure 1 is a histogram of the inhibitory effect of drugs on PAF-induced platelet aggregation in rabbits.

Example 1: Preparation of 2-(3,4,5-trimethoxyphenyl)-6-(4-chlorophenyl)tetrahydropyran

A. Preparation of 1-(3,4,5-trimethoxyphenyl)-5-(4-chlorophenyl)-1,5-pentanedione

Add 80ml of absolute ethanol and 0.4g of sodium metal into a 250ml three-necked flask, stir to dissolve, let cool to room temperature, add 4.0g of ethyl 3,4,5-trimethoxybenzoylacetate to it, and stir until the solid is completely After dissolving, 1.0 g of 4-chloro-β-bromopropiophenone ground into powder was added thereto, and after stirring for 10 minutes, 2.5 g of 4-chloro-β-bromopropiophenone ground into powder was added thereto, at room temperature After stirring, a large amount of flocculent precipitates precipitated after about 30 minutes, continued to stir at room temperature for 8 hours, filtered, and the filter cake was washed with a small amount of 95% ethanol to obtain a white solid with a melting point (mp) of 123-125 °C, which was 2-(3,4,5 -ethyl trimethoxybenzoyl)-5-oxo-(4-chlorophenyl)pentanoate.

Proton NMR spectrum ( ¹ HNMR CDCl ₃ , TMS, δppm) 1, 1-1, 3 (t, 3H, -OCH ₂ CH ₃ ), 2, 3-2.5 (m, 2H, 3-CH ₂ -), 3.0-3.2(m, 2H, 4-CH ₂ -) 3.95(s, 3H, 4'-Ar-OCH ₃ ) 3.97(s, 6H, 3'5'-Ar-OCH ₃ ), 4.1-4.3(m , 2H, -OCH ₂ ), 4.5-4.6 (q, 1H, 2-CH-), 7.4-8.0 (m, 6H, Ar-H)

Add 2.3g 2-(3,4,5-trimethoxybenzoyl)-5-oxo-(4-chlorophenyl)ethyl valerate, 25ml ethanol and 1.2g hydrogen to a 250ml three-necked flask Potassium oxide in 25ml of water, stirred and reacted in a water bath at 40°C for 48 hours, filtered, and the filter cake was washed with 50% ethanol until neutral to obtain a white solid 1-(3,4,5-trimethoxyphenyl)-5-( 4-Chlorophenyl)-1,5-pentanedione, mp 86-88°C. ¹ HNMR (CDCl ₃ , TMS, δppm) 2.1-2.3 (m, 2H, 3-CH ₂ ), 3.0-3.2 (m, 4H, 2.4-2×CH ₂ ) 3.92 (s, 3H, 4'-OCH ₃ ) 3.94 (s, 6H, 3', 5'-2×OCH ₃ ) 7.27 (s, 2H, 2', 6'-2×ArH) 7.4-8.0 (dd, 4H, 2", 3", 5" , 6”-4×ArH)

B. Preparation of 2-(3,4,5-trimethoxyphenyl)-6-(4-chlorophenyl)tetrahydropyran

Add 1.25g 1-(3,4,5-trimethoxyphenyl)-5-(4-chlorophenyl)-1,5-pentanedione and 40ml methanol to a 100ml round bottom flask, stir at room temperature, Add 0.3 g of sodium borohydride, react violently to obtain a clear liquid, continue to stir and react at room temperature for 6 hours, evaporate methanol under reduced pressure, add 100 ml of 1N hydrochloric acid to it, and precipitate a viscous solid, extract twice with 100 ml of dichloromethane, extract Wash twice with 50ml of water, 100ml of 2.5% sodium hydroxide solution and twice with 100ml of water, dry, and evaporate the solvent to obtain 1-(3,4,5-trimethoxyphenyl)-5-(4-chlorobenzene base)-1,5-pentanediol thick liquid.

The above-mentioned thick liquid was dissolved in 70ml of dichloromethane, 1.6ml of triethylamine and 0.8ml of methanesulfonyl chloride were added thereto, stirred and reacted at room temperature under nitrogen for 24 hours, and left overnight. 100ml of 2.5% sodium hydroxide was washed twice and 100ml of water twice, dried, evaporated to remove the solvent, and the concentrate was separated by silica gel column chromatography (silica gel G, eluent ethyl acetate:cyclohexane 3:7) to obtain cis Formula and trans 2-(3,4,5-trimethoxyphenyl)-6-(4-chlorophenyl)tetrahydropyran. ¹ HNMR (CDCl ₃ , TMS, δppm) cis 1.5-2.1 (m, 6H, 3×CH ₂ ), 3.84 (s, 3H, OCH ₃ ), 3.88 (s, 6H, 2×OCH ₃ ) 4.4-4.6 (t, 2H, 2.6-H), 6.64(s, 2H, 2×ArH), 7.2-7.4(m, 4H, Ar-H)

Trans 1.8-2.4(m, 6H, 3×CH ₂ ), 3.85(s, 3H, Ar-OCH ₃ ), 3.90(s, 6H, 2×Ar-OCH ₃ ), 5.0(d, 1H, CH- ), 5.60-5.75(m, 1H, CH-), 6.60(s, 2H, 2', 6'Ar-H), 6.9-7.2(dd, 4H, Ar-H)

Example 2: Preparation of 2-(3,4,5-trimethoxyphenyl)-6-(2,4-xylyl)tetrahydropyran

A. Preparation of 1-(3,4,5-trimethoxyphenyl)-5-(2,4-xylyl)-1,5-pentanedione

Add 100ml of absolute ethanol and 0.6g of sodium metal into a 250ml three-necked flask, stir to dissolve, let cool to room temperature, add 5.7g of ethyl 3,4,5-trimethoxybenzoylacetate to it, and stir until the solid is completely After dissolving, 1.0 g of 2,4-dimethyl-β-bromopropiophenone ground into powder was added thereto, and after stirring for 10 minutes, 2,4-dimethyl-β-bromopropiophenone ground into powder was added thereto. Bromopropiophenone 3.9g, stirred at room temperature for 10.5 hours, acidified to pH3, concentrated and cooled to crystallize, filtered, and the filter cake was washed with a small amount of 60% ethanol to obtain a white solid, mp79-81°C, 2-(3,4,5 -Ethyl trimethoxybenzoyl)-5-oxo-(2,4-dimethylphenyl)pentanoate.

¹ HNMR (CDCl ₃ , TMS, δ ppm) 1.1-1.3 (t, 3H, -OCH ₂ CH ₃ ), 2.36 (s, 3H, Ar-CH ₃ ), 2.50 (s, 3H, Ar-CH ₃ ) 2.2 -2.5(m, 2H, 3-CH ₂ -), 3.0-3.2(m, 2H, 4-CH ₂ ), 3.92(s, 3H, 4'-Ar-OCH ₃ ), 3.97(s, 6H, 3 ', 5'-Ar-OCH ₃ ), 4.1-4.3m, 2H, -OCH ₂ ), 4.5-4.6(q, 1H, 2-CH), 7.0-7.7(m, 5H, Ar-H)

Add 2.5g 2-(3,4,5-trimethoxybenzoyl)-5-oxo-(2,4-dimethylphenyl) ethyl valerate, 25ml ethanol and Containing 1.2g of potassium hydroxide in 25ml of water, stirred and reacted in a water bath at 40°C for 48 hours, filtered, and the filter cake was washed with 50% ethanol until neutral to obtain a white solid 1-(3,4,5-trimethoxyphenyl) -5-(2,4-Dimethylphenyl)-1,5-pentanedione, mp 73-75°C. ¹ HNMR (CDCl ₃ , TMS, δppm) 2.1-2.2 (m, 2H, 3-CH ₂ ), 2.35 (s, 3H, Ar-CH ₃ ), 2.50 (S, 3H, Ar-CH ₃ ), 3.0- 3.1(m, 4H, 2.4-2×CH ₂ ), 3.91(s, 3H, 4’-CH ₃ ), 3.93(s, 6H, 3’, 5’-2×OCH ₃ ), 7.0-7.7(m , 5H, Ar-H)

B. Preparation of 2-(3,4,5-trimethoxyphenyl)-6-(2,4-xylyl)tetrahydropyran

In the 100ml round bottom flask, add 1.1g 1-(3,4,5-trimethoxyphenyl)-5-(2,4-dimethylphenyl)-1,5′-pentanedione, 40ml methanol, Stir at room temperature, add 0.3g sodium borohydride to it, react violently to obtain a clear liquid, continue to stir and react at room temperature for 6 hours, distill methanol off under reduced pressure, add 100ml 1N hydrochloric acid to it, precipitate a viscous solid, divide 100ml dichloromethane into two For secondary extraction, the extract was washed twice with 50ml of water, 100ml of 2.5% sodium hydroxide and twice with 100ml of water, dried, and evaporated to obtain 1-(3,4,5-trimethoxyphenyl)-5- (2,4-Dimethylphenyl)-1,5-pentanediol is a viscous liquid.

The above-mentioned thick liquid was dissolved in 70ml of dichloromethane, 1.6ml of triethylamine and 0.8ml of methanesulfonyl chloride were added thereto, stirred and reacted under nitrogen gas at room temperature for 24 hours, and left overnight, the reaction solution was divided into two times with 100ml of 1N hydrochloric acid, 50ml of water, 100ml of 2.5% sodium hydroxide was washed twice with 100ml of water twice, dried, evaporated to remove the solvent, and the concentrate was separated by silica gel column chromatography (silica gel G, eluent: ethyl acetate:cyclohexane 3:7) to obtain the purified product. Formula 2-(3,4,5-trimethoxyphenyl)-6-(2,4-dimethylphenyl)tetrahydropyran.

¹ HNMR (CDCl ₃ , TMS, δppm) 1.6-2.1 (m, 6H, 3, 4, 5-3×CH ₂ ), 2.306 (s, 3H, 2'-CH ₃ ), 2.337 (s, 3H, 4 '-CH ₃ ) 3.818 (s, 3H, 4', -OCH ₃ ), 3.873 (s, 6H, 3'5' 2×OCH ₃ ), 4.49-4.54 (dd, 1H, 2-H), 4.66- 4.71(dd, 1H, 6-H), 6.66(s, 2H, 2', 6'-2×ArH), 6.90-7.50(m, 3H, 3”, 5”, 6”-3×ArH)

Example 3: Preparation of 2-(3,4,5-trimethoxyphenyl)-6-(2,4-difluorophenyl)tetrahydropyran

A. Preparation of 1-(3,4,5-trimethoxyphenyl)-5-(2,4-difluorophenyl)-1,5-pentanedione

Add 80ml of absolute ethanol and 0.4g of sodium metal into a 250ml three-necked flask, stir to dissolve, let cool to room temperature, add 4.0g of ethyl 3,4,5-trimethoxybenzoylacetate to it, and stir until the solid is completely After dissolving, 1.0 g of 2,4-difluoro-β-bromopropiophenone ground into powder was added thereto, and after stirring for 10 minutes, 2.6 g of 2,4-difluoro-β-bromopropiophenone powder was added thereto. g, after stirring at room temperature for 8 hours, adjust the pH to be close to neutral, evaporate part of the solvent and place it in the refrigerator to precipitate needle-like crystals, mp88-89°C, which is 2-(3,4,5-trimethoxybenzoyl)- Ethyl 5-oxo-(2,4-difluorophenyl)pentanoate. ¹ HNMR (CDCl ₃ , TMS, δ ppm) 1.1-1.3 (t, 3H, -OCH ₂ CH ₃ ), 2.3-2.5 (m, 2H, 3-CH ₂ -), 3.1-3.2 (m, 2H, 4 -CH ₂ ) 3.93 (s, 3H, 4'-Ar-OCH ₃ ) 3.95 (s, 6H, 3', 5'-Ar-OCH ₃ ), 4.1-4.2 (m, 2H, -OCH ₂ ), 4.5 -4.6(q, 1H, -2-CH-), 6.8-7.0(m, 2H, Ar-H), 7 43(s, 2H, 2'6'-Ar-H), 7.9-8.0(m, 2H, Ar-H)

Add 2.3g 2-(3,4,5-trimethoxybenzoyl)-5-oxo-(2,4-difluorophenyl) ethyl valerate, 25ml 95% ethanol in a 250ml three-necked bottle and 25ml of water containing 1.2g of potassium hydroxide, stirred and reacted at 40°C for 48 hours, allowed to cool, filtered, and the filter cake was washed with 50% alcohol until neutral to obtain a white solid 1-(3,4,5-trimethoxy Phenyl)-5-(2,4-difluorophenyl)-1,5-pentanedione, mp96-98°C. ¹ HNMR (CDCl ₃ , TMS, δppm) 2.1-2.3 (m, 2H, 3-CH ₂ ), 3.0-3.2 (m, 4H, 2, 4-2×CH ₂ ), 3.92 (s, 3H, 4' -OCH ₃ ), 3.94(s, 6H, 3', 5'2×OCH ₃ )6.8-7.0(m, 2H, Ar-H), 7.28(s, 2H, 2'6'2×Ar-H) , 7.9-8.0 (m, 1H, Ar-H)

B. Preparation of 2-(3,4,5-trimethoxyphenyl)-6-(2,4-difluorophenyl)tetrahydropyran

In a 100ml round bottom flask, add 1.1g 1-(3,4,5-trimethoxyphenyl)-5-(2,4-difluorophenyl)-1,5-pentanedione, 0.3g sodium boron Hydrogen and 40ml of methanol, stirred at room temperature for 8 hours, stopped the reaction, distilled off the methanol under reduced pressure and added 100ml of 1N hydrochloric acid to it, a thick substance was produced, extracted twice with 100ml of dichloromethane, and the extract was mixed with 50ml of water, 100ml of 2.5% sodium hydroxide solution was divided into two times, washed twice with 100ml of water, dried over anhydrous sodium sulfate, and evaporated to obtain 1-(3,4,5-trimethoxyphenyl)-5-(2,4 -Difluorophenyl)-1,5-pentanediol thick liquid.

The above thick matter was dissolved in 80ml of dichloromethane, 0.8ml of methanesulfonyl chloride and 1.6ml of triethylamine were added thereto, stirred and reacted for 24 hours under nitrogen gas, and the reaction was stopped. 2.5% sodium hydroxide solution was divided into two times, 100ml of water was washed twice, dried over anhydrous sodium sulfate, the solvent was evaporated, and the concentrate was subjected to silica gel column chromatography (silica gel G, the eluent was ethyl acetate:cyclohexane 3: 7) Separate cis and trans 2-(3,4,5-trimethoxyphenyl)-6-(2,4-difluorophenyl)tetrahydropyran. ¹ HNMR (CDCl ₃ , TMS δppm) trans 1.5-2.2 (m, 6H, 3×CH ₂ ) 3.83 (s, 3H, Ar-OCH ₃ ), 3.87 (s, 6H, 2×ArOCH ₃ ), 4.5- 4.6(m, 1H, -CH-), 5.1-5.2(m, 1H, CH-), 6.54(s, 2H, 2', 6'-Ar-H), 6.5-7.5(m, 3H, 3× Ar-H)

cis 1.5-2.1(m, 6H, 3×CH ₂ ), 3.82(s, 3H, Ar-OCH ₃ ), 3.89(s, 6H, 2×Ar-OCH ₃ ), 4.4-4.6(d, 1H, CH-), 4.8-4.9 (d, 1H, CH-) 6.58 (s, 2H, 2', 6'-Ar-H), 6.6-7.6 (m, 3H, Ar-H)

Example 4. Preparation of 2-(3,4,5-trimethoxyphenyl)-6-(3,4-dimethoxyphenyl)tetrahydropyran

A. Preparation of 1-(3,4,5-trimethoxyphenyl)-5-(3,4-dimethoxyphenyl)-1,5-pentanedione

Add 80ml of absolute ethanol and 0.4g of sodium metal into a 250ml three-necked flask, stir to dissolve, let cool to room temperature, add 4.0g of ethyl 3,4,5-trimethoxybenzoylacetate to it, and stir until the solid is completely After dissolving, 1.0 g of 3,4-dimethoxy-β-bromopropiophenone ground into powder was added thereto, and after stirring for 10 minutes, 3,4-dimethoxy-β-bromopropiophenone was added thereto Propiophenone powder 2.9g, stirred at room temperature for 8 hours, filtered to obtain a white solid, mp111-113°C, 2-(3,4,5-trimethoxybenzoyl)-5-oxo-(3,4-di Methoxyphenyl) ethyl valerate. ¹ HNMR (CDCl ₃ , TMS, δppm) 1.1-1.3 (t, 3H, -OCH ₂ CH ₃ ), 2.3-2.5 (m, 2H, 3-CH ₂ ), 3.0-3.2 (m, 2H, 4-CH ₂ ), 3.8-4.0(m, 15H, 5)×OCH ₃ ), 4.1-4.3(m, 2H, -OCH ₂ ), 4.5-4.6(q, 1H, 2-CH) 6.8-6.9(d, 1H , ArH), 7.435 (s, 2H, 2', 6'-Ar-H) 7.5-7.7 (m, 2H, Ar-H)

Add 2.3g 2-(3,4,5-trimethoxybenzoyl)-5-oxo-(3,4-dimethoxyphenyl) ethyl valerate in 250ml three-necked bottle, 25ml 95 % ethanol and 25ml aqueous solution containing 1.2g potassium hydroxide, stirred at 40°C for 48 hours, allowed to cool, filtered, and the filter cake was washed twice with 50% alcohol to obtain white solid 1-(3,4,5-trimethoxy Phenyl)-5-(3,4-dimethoxyphenyl)-1,5-pentanedione, mp 105-107°C.

¹ HNMR (CDCl ₃ , TMS, δppm) 2.1-2.3 (m, 2H, 3-CH ₂ -), 3.0-3.2 (m, 4H, 2.4-2×CH ₂ ), 3.8-4.0 (m, 15H, 5 ×OCH ₃ ), 6.8-7.7 (m, 5H, Ar-H)

B. Preparation of 2-(3,4,5-trimethoxyphenyl)-6-(3,4-dimethoxyphenyl)tetrahydropyran

In a 100ml round bottom flask, add 1.5g 1-(3,4,5-trimethoxyphenyl)-5-(3,4-dimethoxyphenyl)-1,5-pentanedione, 0.5g Sodium borohydride and 40ml methanol, reflux reaction in oil bath for 3 hours, let cool to obtain clear liquid, add 100ml 1N hydrochloric acid to it after evaporating methanol under reduced pressure, there is a thick product, extract twice with 100ml dichloromethane, extract solution was washed twice with 50ml water, 100ml 2.5% sodium hydroxide solution, 100ml water twice, dried over anhydrous sodium sulfate, and evaporated to obtain 1-(3,4,5-trimethoxyphenyl)-5 -(3,4-dimethoxyphenyl)-1,5-pentanediol viscous liquid.

The above thick matter was dissolved in 80ml of dichloromethane, 0.8ml of methanesulfonyl chloride and 1.6ml of triethylamine were added thereto, stirred and reacted for 24 hours under nitrogen gas, and the reaction was stopped. 2.5% sodium hydroxide solution was divided into two times, 100ml of water was washed twice, dried over anhydrous sodium sulfate, the solvent was evaporated, and the concentrate was subjected to silica gel column chromatography (silica gel G, the eluent was ethyl acetate:cyclohexane 3: 7) Isolated trans 2-(3,4,5-trimethoxyphenyl)-6-(3,4-dimethoxyphenyl)tetrahydropyran. ¹ HNMR (CDCl ₃ , TMS δppm) 1.6-2.1 (m, 6H, 3×CH ₂ ), 3.8-3.9 (3s, 15H, 5×OCH ₃ ), 4.49-4.6 (2s, 2H, 2.6-H), 6.67(s, 2H, 2', 6'-ArH), 6.8-7.0(m, 3H, ArH)

The 2-(3,4,5-trimethoxyphenyl)-6-(substituted phenyl)tetrahydropyran compound has the effect of antagonizing platelet activating factor (PAF), and can be used as a drug for antagonizing PAF. The results of the pharmacological experiments are described below.

(1) Experimental method

The compounds represented by the following abbreviations are:

SZ-1: trans 2,6-bis(3,4-dimethoxyphenyl)tetrahydropyran

DFTM1: cis 2-(3,4,5-trimethoxyphenyl)-6-(2,4-difluorophenyl)tetrahydropyran

DFTM3: trans 2-(3,4,5-trimethoxyphenyl)-6-(2,4-difluorophenyl)tetrahydropyran

M2T: trans 2-(3,4,5-trimethoxyphenyl)-6-(2,4-xylyl)tetrahydropyran

D2T3: trans 2-(3,4,5-trimethoxyphenyl)-6-(3,4-dimethoxy)tetrahydropyran

1. Antagonism of drugs on PAF-induced platelet aggregation

(1) Preparation of rabbit washed platelets

Healthy New Zealand rabbits (male or female) were awake to collect blood from the heart, put it in a plastic centrifuge tube and mix it with blood maintenance (ACD) solution (6:1). After centrifuging at 500rpm for 10 minutes at room temperature, take the upper layer of platelet-rich plasma (PRP), then centrifuge the PRP at 3500rpm for 15 minutes, pour off the plasma, and wash the precipitated platelets twice with gelatin-containing calcium-free Tyrode's solution (TG-NOCa ⁺⁺ ) , and centrifuged to remove the supernatant. Finally, the platelets were suspended in Tyrode's solution [containing 0.25% phosphate buffer (BSA)], and the platelet count was adjusted to 3×10 ⁸ /ml.

(2) Rabbit washed platelet aggregation test

The platelet aggregation test was carried out with a domestic SPA-3 automatic balance platelet aggregation instrument at 37°C under constant temperature and stirring. Divide platelet suspension into turbidimetric cups (200μl/cup), mix 1μl of different concentrations of SZ-1, DFTM1, DFTM3, M2T, D2T3 and solution control 1μl ethanol with 200μl platelet suspension, and incubate at 37°C After 10 minutes, add a threshold dose of inducer under stirring, observe the effects of different concentrations of drugs on the maximum aggregation rate of platelets induced by PAF within 3 minutes, and calculate the inhibition percentage by the following formula:

Aggregation inhibition percentage=(control aggregation percentage-drug aggregation percentage)/control aggregation percentage×100%

2. Antagonism of drugs on PAF-induced smooth muscle cell proliferation and DNA synthesis

Crystal violet staining method to measure the proliferation rate

(1) The effect of PAF on the proliferation of brain microvascular smooth muscle cells (BCSMC):

Take the BCSMCs that have grown into a dense monolayer, digest them with 0.1% trypsin, dilute the cells to 1×10 ⁵ /ml with 10% serum-containing medium, and inoculate them in 96-well cell culture plates at 100.0 μl per well ( 1×10 ⁴ cells/well), incubated in a 37°C incubator for 12 hours, and added different doses of PAF (final concentrations were 10 ^-16 , 10 ^-13 , 10 ^-10 , 10 ^-8 , 10 ^-7 mol/L ) Add 10% serum-containing medium to each well to 200.0 μl and continue to incubate for different times (24, 48, 72 hours), take it out, dip in cell fixation staining solution for 20 minutes, then rinse with deionized water, soak for 15 minutes, After drying naturally, add 100.0 μl crystal violet extract to each well, and measure the absorbance value at 594 nm with a 511-type enzyme label analyzer.

(2) Effects of drugs on PAF-induced BCSMC proliferation:

Take the BCSMCs that have grown into a dense monolayer, digest them with 0.1% trypsin, dilute the cells to 1×10 ⁵ /ml with 10% serum-containing medium, and inoculate them in 96-well cell culture plates at 100.0 μl per well ( 1×10 ⁴ cells/well), after incubation for 24 hours, add test drug (final concentration is 10 ^-5 , 10 ^-7 , 10 ^-9 mol/L), add 10 ^-10 mol/L concentration of PAF, set at 37 After incubating in a carbon dioxide incubator at ℃ for 48 hours, take it out, and after the cells are fixed and stained, the steps are the same as above.

(3) [ ³ H]-TdR incorporation method to measure cell proliferation

Effect of PAF on DNA synthesis of BCSMC:

The plating method is the same as (1). After incubating in the carbon dioxide incubator for 3 days, replace with 0.4% medium and incubate for 3 days to synchronize the cells. Add [ ³ H]-TdR 1 μCi, add 10% serum-containing medium and After incubating with different concentrations of PAF for 24 hours, wash with D-Hank's to stop the binding of [ ³ H]-TdR, digest with 0.25% trypsin for 3 minutes, collect cells by filtering with 49-type glass fiber filter membrane, and wait for the filter membrane to dry naturally Finally, add 0.2ml of scintillation fluid and count with SN-6904 scintillation counter in the scintillation vial.

(4) Antagonism of drugs:

The planking method is the same as above. After incubating in the carbon dioxide incubator for 3 days, change to 0.4% medium for synchronization for 3 days, add 10% serum-containing medium, different concentrations of drugs, [ ³ H]-TdR, 10 ^-8 mol/L PAF was taken out for testing after 24 hours of incubation, and the following steps were the same as above.

(2) Experimental results

1. The inhibitory effect of drugs on PAF-induced platelet aggregation in rabbits:

SZ-1, DFTM1, DFTM3, M2T and D2T3 could all inhibit the threshold dose of PAF-induced platelet aggregation in a dose-dependent manner within the final concentration range of 10 ^-8 to 10 ^-6 mol/L.

2. Effect of drugs on PAF-induced BCSMC proliferation:

We chose the stimulation concentration of PAF as 10 ^-10 mol/L and the stimulation time as 48 hours to investigate the effects of SZ-1 and DFTM3 on PAF-induced BCSMC proliferation. The results are shown in Table 1.

It can be seen from Table 1 that SZ-1 and DFTM3 significantly inhibited PAF-induced BCSMC proliferation at different concentrations, and there was a good dose-effect relationship.

Table 1 The inhibitory effect of drugs on PAF-induced BCSMC proliferation

X±S n＝6 ^* P＜0.05 ^** P＜0.01 vs control Drug Absorption of drug at different concentrations (mol/L) (inhibition of proliferation percentage%) SZ-1DFTM3 00.351±0.0640.351±0.064 10 ^-9 0.260±0.071 ^** (25.87)0.243±0.056 ^* (30.71) 10 ^-7 0.235±0.063 ^* (33.05)0.235±0.032 ^** (33.11) 10 ^-5 0.237±0.035 ^* (32.56)0.201±0.073 ^* (42.42)

3. The protective effect of the drug on the increase of DNA synthesis of BCSMC induced by PAF:

When the concentration of PAF was 10 ^-8 mol/L, the protective effect of SZ-1 and DFTM3 on DNA synthesis induced by PAF was investigated. The results are shown in Table 2.

It can be seen from Table 2 that different concentrations of SZ-1 and DFTM3 have significant protective effects on BCSMC. Table 2 The protective effect of drugs on the DNA synthesis of PAF-induced BCSMC X±S n=6 ^* P<0.05 ^** P<0.01 vs control Drugs(μmol/L) [ ³ H]TdR, dpm Inhibition rate % controlSZ-10.0010.110DFTM30.0010.110 1617±1481147±156 ^* 1091±172 ^* 830±129 ^** 1222±171 ^* 1013±152 ^* 943±143 ^** 29.0735.8748.6724.4337.3541.87

Claims (3)

1. A 2-(3,4,5-trimethoxyphenyl)-6-(substituted phenyl) tetrahydropyran compound, characterized in that the structure of the compound is: Where R ₁ , R ₂ , R ₃ take different groups and the corresponding compounds are as follows: R ₁ , R ₂ , R ₃ tetrahydropyran compounds H H Cl 2-(3,4,5-trimethoxyphenyl)-6-(4-chlorophenyl)tetrahydropyran CH ₃ H CH ₃ 2-(3,4,5-trimethoxyphenyl)-6-(2,4-dimethylphenyl)tetrahydropyran F H F 2-(3,4,5-trimethoxyphenyl)-6-(2,4-difluorophenyl)tetrahydropyran H OCH ₃ OCH ₃ 2-(3,4,5-trimethoxyphenyl)-6-(3,4-dimethoxyphenyl)tetrahydropyran. 2. The preparation method of the compound described in claim 1, comprising the steps of: (1) Condensation of ethyl 3,4,5-trimethoxybenzoyl acetate and β-bromosubstituted propiophenone in ethanol solution of sodium ethoxide to generate 2-(3,4,5-trimethoxybenzoyl )-5-oxo-(substituted phenyl) pentanoic acid ethyl ester; (2) 2-(3,4,5-trimethoxybenzoyl)-5-oxo-(substituted phenyl) ethyl pentanoate in 2.5% potassium hydroxide alcoholic water (alcohol: water = 1:1) Stirring and reacting at 40°C for 48 hours to generate 1-(3,4,5-trimethoxyphenyl)-5-(substituted phenyl)-1,5-pentanedione; (3) The alcohol solution of 1-(3,4,5-trimethoxyphenyl)-5-(substituted phenyl)-1,5-pentanedione generates 1-(3,4 , 5-trimethoxyphenyl)-5-(substituted phenyl)-1,5-pentanediol; (4) 1-(3,4,5-trimethoxyphenyl)-5-(substituted phenyl)-1,5-pentanediol in dichloromethane solution in the presence of nitrogen, through methanesulfonic Acid chloride and triethylamine ring closure reaction to generate 2-(3,4,5-trimethoxyphenyl)-6-(substituted phenyl)tetrahydropyran. 3. The use of the compound of claim 1 for the preparation of drugs that antagonize platelet activating factor.

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