CN114349746A - A kind of pyridine compound containing 4-fluorophenyl fragment, preparation method and application thereof, and pharmaceutical composition - Google Patents

Abstract

The invention provides a pyridine compound containing a 4-fluorophenyl fragment, and a preparation method and application thereof, and belongs to the technical field of medical chemistry. The invention introduces fluorine atoms (elements with the strongest electronegativity) to the para position of the benzene ring of JNJ4796 to seal the metabolic sites of the JNJ4796, and compared with JNJ4796, the synthesized pyridine compound containing the 4-fluorophenyl fragment has the advantages of remarkably reduced cardiac toxicity and better in-vivo pharmacokinetic property on the basis of maintaining the excellent in-vitro anti-influenza virus activity of the JNJ4796 (R-type), so that the pyridine compound has good drug forming property.

Description

A kind of pyridine compound containing 4-fluorophenyl fragment and its preparation method and application, pharmaceutical composition

This application is a divisional application of the Chinese patent application with the application number CN202011300875.5 and the invention name "a pyridine compound and its preparation method and application, pharmaceutical composition" filed with the China Patent Office on November 19, 2020, The entire contents of which are incorporated herein by reference.

technical field

The invention relates to the technical field of medicinal chemistry, in particular to a pyridine compound containing a 4-fluorophenyl fragment, a preparation method, application and pharmaceutical composition thereof.

Background technique

Seasonal influenza is a major public health problem caused by influenza virus. Influenza virus belongs to the Orthomyxoviridae family (Orthomyxoviridae) negative helix single-stranded RNA virus, according to its nucleo protein (NP) and matrix protein (MP) antigenic determinants are divided into A (A) type, B (B), C (C) and D (D) 4 types. Among them, influenza A virus has a wide host range and can infect humans, pigs, dogs, poultry and wild birds, etc., with high morbidity and mortality, and is likely to cause large-scale outbreaks.

At present, the two main strategies to deal with influenza virus are vaccination and drug treatment. Getting the flu vaccine provides good protection, but mutations in the flu virus reduce the vaccine's effectiveness. At present, the anti-influenza virus drugs used clinically are mainly M2 ion channel inhibitor amantadine-rimantadine; neuraminidase inhibitors oseltamivir, zanamivir, etc., and hemagglutinin inhibitor abirate Dole, the newly launched cap-dependent endonuclease inhibitor Xofluza. However, with the long-term use of anti-influenza virus drugs, the problem of virus resistance has become increasingly serious. Therefore, there is an urgent need to develop anti-influenza drugs with novel mechanisms of action.

In 2018, Jassen Company disclosed the synthesis and in vitro anti-influenza virus activity of pyridine compounds with the following general formula [W] (WO 2018/141854), which has a brand-new target, namely Hemagglutinin (HA) .

In 2019, Jassen Company further disclosed JNJ4796 (R-type), a representative of this class of compounds, and its anti-influenza virus activity (Maria J.P. van Dongen et al., Science, 2019, 363, 1056).

However, although JNJ4796 (R-type) exhibits excellent in vitro anti-influenza virus activity, it has potential cardiotoxicity (53.8% inhibition of hERG K+ channel at 10 μM).

SUMMARY OF THE INVENTION

The object of the present invention is to provide a pyridine compound containing a 4-fluorophenyl fragment, its preparation method, application, and pharmaceutical composition, and the pyridine compound containing a 4-fluorophenyl fragment has excellent in vitro anti-influenza virus Active and low cardiotoxicity.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a pyridine compound containing a 4-fluorophenyl fragment, which has the structure shown in formula I:

In formula I, R is a C1-C7 alkyl group, a cycloalkyl group or an alkenyl group.

Preferably, the pyridine compounds include

The present invention provides the preparation method of the pyridine compound containing 4-fluorophenyl fragments described in the above technical scheme, comprising the following steps:

The compound of the structure shown in formula II, the compound of the structure shown in formula III, a condensing agent, a catalyst and a dipolar solvent are mixed, and a condensation reaction is carried out to obtain a pyridine compound containing a 4-fluorophenyl fragment of the structure described in formula I;

In formula II, R is a C1-C7 alkyl group, a cycloalkyl group or an alkenyl group.

Preferably, the molar ratio of the compound of the structure represented by the formula II and the compound of the structure represented by the formula III is 1:1.

Preferably, the temperature of the condensation reaction is -5～40°C, and the time is 5～20h.

Preferably, the dipolar solvent includes dimethylformamide, acetone, dimethyl sulfoxide or acetonitrile; the condensing agent includes 1-ethyl-3(3-dimethylpropylamine)carbodiimide; the The catalyst includes 4-dimethylaminopyridine.

The present invention provides the pyridine compounds containing 4-fluorophenyl fragments according to the above technical scheme or the pyridine compounds containing 4-fluorophenyl fragments prepared by the preparation method according to the above technical scheme in the preparation of anti-influenza virus infection drugs The application of the pyridine containing 4-fluorophenyl fragment is applied in the form of a compound shown in formula I or a pharmaceutically acceptable salt thereof; the pharmaceutically acceptable salt is the compound shown in formula I and an inorganic acid or an organic acid. Salt.

Preferably, the influenza virus in the anti-influenza virus infection drug includes influenza A, influenza B, influenza C or influenza D.

Preferably, the influenza A includes H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3 or H10N7.

The present invention provides a pharmaceutical composition, comprising a pyridine compound containing a 4-fluorophenyl fragment described in the above technical solution or a pharmaceutically acceptable salt thereof; the pharmaceutically acceptable salt is a compound represented by formula I and an inorganic acid or an organic acid salt formed.

The present application provides a pyridine compound containing a 4-fluorophenyl fragment. The present invention introduces a fluorine atom (the element with the strongest electronegativity) into the para-position of the benzene ring of JNJ4796 to block its metabolic site. On the basis of maintaining the excellent in vitro anti-influenza virus activity of JNJ4796 (R-type), the pyridine compounds containing 4-fluorophenyl fragments have significantly lower cardiotoxicity and better in vivo pharmacokinetic properties than JNJ4796. Therefore, it has good medicinal properties.

Detailed ways

The application provides a pyridine compound containing a 4-fluorophenyl fragment, having the structure shown in formula I:

In formula I, R is a C1-C7 alkyl group, a cycloalkyl group or an alkenyl group.

In the present invention, the C1-C7 alkyl group is preferably methyl, isopropyl, butyl, 1-ethylpropyl, and isopentyl; the cycloalkyl group is preferably cyclopentyl or cyclohexyl; The alkenyl group is preferably a vinyl group.

In the present invention, the pyridine compounds preferably include

The present invention provides the preparation method of the pyridine compound containing 4-fluorophenyl fragments described in the above technical scheme, comprising the following steps:

The compound of the structure shown in formula II, the compound of the structure shown in formula III, a condensing agent, a catalyst and a dipolar solvent are mixed, and a condensation reaction is carried out to obtain a pyridine compound containing a 4-fluorophenyl fragment of the structure described in formula I;

In formula II, R is a C1-C7 alkyl group, a cycloalkyl group or an alkenyl group.

In the present invention, the compound of the formula II is preferably prepared according to the preparation method of compound 9 in the original application (CN202011300875.5, the title of the invention is "a pyridine compound and its preparation method and application, pharmaceutical composition"). Preparation can be done; in the embodiment of the present invention, the compound of the structure shown in the formula II is prepared according to the reaction formula 1:

In formula 1, the iodo compound is preferably the iodo compound corresponding to the R substituent; in the embodiment of the present invention, the iodo compound is specifically iodomethane, 2-iodopropane, iodobutane, 3- iodopentane, 1-iodo-3-methylbutane, 1-fluoro-2-iodoethane, iodocyclopentane or iodocyclohexane.

In an embodiment of the present invention, the specific preparation process of the formula 1 is:

1) Under the protection of argon at room temperature, to a solution of compound 1 (1.38 g, 4.47 mmol) in anhydrous toluene (20 mL) was added carbonyl bis(triphenylphosphine) iridium chloride (351 mg, 0.45 mmol) and 1,1, 3,3-Tetramethyldisiloxane (1.20 g, 8.94 mmol) was stirred at the same temperature for 1 hour, trimethylcyanosilane (886 mg, 8.94 mmol) was added to the reaction solution, stirred at room temperature overnight, and hydroxide was added. Quenched with sodium (1N, 10 mL) aqueous solution, extracted with ethyl acetate (30 mL×3); the organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and obtained by silica gel column chromatography (petroleum ether:ethyl acetate=6:1) White solid compound 2 (1.14 g, yield 80%), MS-ESI (m/z): 320 (M+H)+.

2) Under the protection of argon at room temperature, to a solution of compound 2 (0.92 g, 2.89 mmol) in anhydrous toluene (20 mL) was added tributyltin oxide (1.76 mL, 3.46 mmol), trimethylsilane azide (1.81 mL, 13.8 mmol), stirred at the same temperature for 3 hours, refluxed overnight, quenched by adding aqueous sodium hydroxide (1N, 40 mL), separated the aqueous phase, washed with petroleum ether (30 mL), and adjusted pH with aqueous hydrochloric acid (6N) to 1-2, extracted with ethyl acetate (30 mL×3). The organic layer was dried over anhydrous magnesium sulfate, filtered, concentrated, and subjected to silica gel column chromatography (dichloromethane:methanol=10:1) to obtain a yellow-brown solid compound 3 (0.82 g, yield 78%), MS-ESI (m/ z): 363(M+H)+.

3) RI (319 μL, 5.14 mmol) and potassium carbonate (1.06 g, 7.71 mmol) were added to a solution of compound 3 (0.93 g, 2.57 mmol) in anhydrous acetonitrile (20 mL) at room temperature, stirred at the same temperature for 12 hours, and filtered. , concentrated, and silica gel column chromatography (PE:EA=2:1) gave compound 4a (347 mg, yield 36%), MS-ESI (m/z): 377 (M+H)+.

4) Under an ice bath, trifluoroacetic acid (1 mL) was added to a solution of compound 4a (300 mg, 0.80 mmol) in dichloromethane (5 mL), stirred at the same temperature for 1 hour, and concentrated to obtain an oily compound (II)-a (221 mg, 100%), MS-ESI (m/z): 277 (M+H)+.

In the present invention, the compound of formula III is preferably prepared according to the preparation method of compound 15 in the above-mentioned original application or commercially available products well known in the art.

In the present invention, the molar ratio of the compound of the structure represented by the formula II and the compound of the structure represented by the formula III is preferably 1:1.

In the present invention, the dipolar solvent preferably includes dimethylformamide (DMF), acetone, dimethylsulfoxide (DMSO) or acetonitrile; the condensing agent preferably includes 1-ethyl-3(3-dimethylformamide) carbodiimide (EDCI); the catalyst preferably comprises 4-dimethylaminopyridine (DMAP).

The present invention has no special limitation on the process of mixing the compound of the structure represented by the formula II, the compound of the structure represented by the formula III, the condensing agent, the catalyst and the dipolar solvent, and the materials can be uniformly mixed according to the process well known in the art.

In the present invention, the temperature of the condensation reaction is preferably -5～40°C, and the time is preferably 5～20h; the condensation reaction is preferably carried out under stirring conditions; the present invention has no special limitation on the stirring rate, It is enough to ensure that the reaction proceeds smoothly.

In the present invention, the reaction formula of the condensation reaction is

After completing the condensation reaction, the present invention preferably adds water to the obtained product to dilute, and then sequentially performs extraction, washing, drying, filtration, concentration and column chromatography separation to obtain a pyridine compound containing a 4-fluorophenyl fragment; the The reagent used for extraction is preferably dichloromethane; the washing preferably includes washing with citric acid with a mass concentration of 10% and washing with sodium bicarbonate in sequence; the drying is preferably drying over anhydrous magnesium sulfate; The process is not particularly limited, and can be performed according to a process well known in the art. In the present invention, the separation method of the column chromatography is preferably silica gel column chromatography, the eluent used in the silica gel column chromatography is preferably dichloromethane and methanol, and the volume ratio of the dichloromethane and methanol is preferably 40:1.

The present invention provides the pyridine compounds containing 4-fluorophenyl fragments according to the above technical scheme or the pyridine compounds containing 4-fluorophenyl fragments prepared by the preparation method according to the above technical scheme in the preparation of anti-influenza virus infection drugs The application of the pyridine containing 4-fluorophenyl fragment is applied in the form of a compound shown in formula I or a pharmaceutically acceptable salt thereof; the pharmaceutically acceptable salt is the compound shown in formula I and an inorganic acid or an organic acid. Salt. In the present invention, the inorganic acid is preferably hydrochloric acid or sulfuric acid; the organic acid is preferably acetic acid, trifluoroacetic acid, citric acid, maleic acid, oxalic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, and mandelic acid , ascorbic acid, malic acid, methanesulfonic acid or p-toluenesulfonic acid. The present invention has no particular limitation on the process of forming the salt of the compound represented by the formula I with an inorganic acid or an organic acid, and the process can be carried out according to a well-known process in the art. The method of the application is not particularly limited in the present invention, and it can be applied according to methods well known in the art.

In the present invention, the influenza virus in the anti-influenza virus infection drug preferably includes influenza A, influenza B, influenza C or influenza D; the influenza A preferably includes H1N1, H2N2, H3N2, H5N1, H7N7 , H1N2, H9N2, H7N2, H7N3 or H10N7.

The present invention provides a pharmaceutical composition, comprising a pyridine compound containing a 4-fluorophenyl fragment described in the above technical solution or a pharmaceutically acceptable salt thereof; the pharmaceutically acceptable salt is a compound represented by formula I and an inorganic acid or an organic acid salt formed. The present invention does not have a special limitation on the pharmaceutical composition, and it suffices to include the pyridine compound or a pharmaceutically acceptable salt thereof.

The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1

Under argon protection at room temperature, to a solution of compound 1 (1.38 g, 4.47 mmol) in anhydrous toluene (20 mL) was added carbonylbis(triphenylphosphine)iridium chloride (351 mg, 0.45 mmol) and 1,1,3, 3-Tetramethyldisiloxane (1.20 g, 8.94 mmol) was stirred at the same temperature for 1 hour, trimethylcyanosilane (886 mg, 8.94 mmol) was added to the reaction solution, stirred at room temperature overnight, and sodium hydroxide ( 1N, 10 mL) aqueous solution was quenched, extracted with ethyl acetate (30 mL×3), the organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography (petroleum ether:ethyl acetate=6:1) to obtain a white solid Compound 2 (1.14 g, 80% yield), MS-ESI (m/z): 320 (M+H) ⁺ ;

Under the protection of argon at room temperature, to a solution of compound 2 (0.92 g, 2.89 mmol) in anhydrous toluene (20 mL) was added tributyltin oxide (1.76 mL, 3.46 mmol), trimethylsilane azide (1.81 mL, 13.8 mmol), stirred at the same temperature for 3 hours, refluxed for overnight reaction, quenched by adding aqueous sodium hydroxide (1N, 40 mL), and the aqueous phase was separated. The aqueous phase was washed with petroleum ether (30 mL), adjusted to pH 1-2 with aqueous hydrochloric acid (6N), and extracted with ethyl acetate (30 mL×3). The organic layer was dried over anhydrous magnesium sulfate, filtered, concentrated, and subjected to silica gel column chromatography (dichloromethane:methanol=10:1) to obtain a yellow-brown solid compound 3 (0.82 g, yield 78%), MS-ESI (m/ z): 363(M+H) ⁺ ;

At room temperature, to a solution of compound 3 (0.93 g, 2.57 mmol) in anhydrous acetonitrile (20 mL) was added iodomethane (319 μL, 5.14 mmol), potassium carbonate (1.06 g, 7.71 mmol), stirred at the same temperature for 12 hours, filtered, Concentration, silica gel column chromatography (PE:EA=2:1) to obtain compound 4a (347 mg, yield 36%), MS-ESI (m/z): 377 (M+H) ⁺ ;

Under an ice bath, trifluoroacetic acid (1 mL) was added to a solution of compound 4a (300 mg, 0.80 mmol) in dichloromethane (5 mL), stirred at the same temperature for 1 hour, and concentrated to obtain an oily compound (II)-a (221 mg, 100% ) was directly used in the next reaction without purification, MS-ESI (m/z): 277 (M+H) ⁺ ;

To a solution of compound III (98 mg, 0.33 mmol) in DMF (15 mL) at room temperature was added compound (II)-a (100 mg, 0.33 mmol), EDCI (76 mg, 0.40 mmol) and DMA P (20 mg, 0.16 mmol), The mixture was stirred at the same temperature for 12 hours, diluted with water (100 mL), extracted with dichloromethane (30 mL×3), washed with 10% citric acid, washed with saturated sodium bicarbonate, dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was filtered through a silica gel column (dichloromethane:methanol=40:1) to obtain the target compound LK01 (119 mg, yield 65%), ¹ HNMR (600 MHz, CDCl ₃ ) δ 8.62-8.61 (m, 1H), 8.24 (brs, 1H) ), 8.09-8.07(m, 1H), 7.92(s, 1H), 7.82-7.80(m, 1H), 7.43-7.41(m, 2H), 7.32-7.28(m, 2H), 6.97(t, J ＝8.40Hz, 2H), 4.92(s, 1H), 4.27(s, 3H), 3.82-3.77(m, 2H), 3.54(brs, 2H), 2.60-2.58(m, 1H), 2.49-2.45( m, 2H), 2.33-2.30 (m, 1H), 2.09 (s, 3H); MS-ESI (m/z): 556.20 (M+H) ⁺ .

At room temperature, a solution of compound LKO1 (100 mg, 0.18 mmol) in dichloromethane (10 mL) was passed hydrochloric acid gas, stirred at the same temperature for 1 hour, filtered to obtain LKO1 hydrochloride (74 mg, yield 70%), ¹ HNMR ( 600MHz, DMSO-d ₆ )δ9.04(brs,1H),8.55-8.52(m,1H),8.14(brs,1H),8.08-8.06(m,1H),7.90(s,1H),7.75- 7.73(m,1H),7.43-7.41(m,2H),7.32-7.28(m,2H),6.97(t,J=8.40Hz,2H),4.92(s,1H),4.27(s,3H) ,3.82-3.77(m,2H),3.54(brs,2H),2.60-2.58(m,1H),2.49-2.45(m,2H),2.33-2.30(m,1H),2.09(s,3H) ; MS-ESI (m/z): 556.20 (M+H) ⁺ .

Example 2

For the synthesis of this compound, refer to Example 1, with the only difference that the methyl iodide in Example 1 was replaced with 2-iodopropane, and the nuclear magnetic data of the obtained compound compound LK02 was: ¹ HNMR (500MHz, CDCl ₃ )δ8.70(d , J＝5.10Hz, 1H), 8.21(s, 1H), 8.15(s, 1H), 8.00(d, J＝4.90Hz, 1H), 7.89(s, 1H), 7.52(t, J＝5.50Hz ,2H),7.39-7.36(m,2H),7.04(t,J=8.30Hz,2H),5.05-5.01(m,1H),5.00(s,1H),3.88(s,2H),3.60( s, 2H), 2.68-2.66(m, 1H), 2.56-2.49(m, 2H), 2.39-2.37(m, 1H), 2.16(s, 3H), 1.66(t, J=4.60Hz, 6H) ; MS-ESI (m/z): 584.20 (M+H) ⁺ .

Example 3

For the synthesis of this compound, refer to Example 1, with the only difference that the iodomethane in Example 1 was replaced with iodobutane, and the nuclear magnetic data of the obtained compound LK03 was: ¹ HNMR (400MHz, CDCl ₃ )δ8.72(d, J =4.30Hz,1H),8.26(s,1H),8.05(d,J=4.90Hz,1H),7.96(s,1H),7.76(s,1H),7.52-7.47(m,4H),7.05 (t,J=8.70Hz,2H),4.99(s,1H),4.62(t,J=7.20Hz,2H),3.87(brs,2H),3.62(brs,2H),2.64(brs,1H) ,2.53(brs,2H),2.40-2.34(m,1H),2.20(s,3H),2.01-1.97(m,2H),1.36-1.34(m,2H),0.97(t,J＝7.50Hz , 3H); MS-ESI (m/z): 598.30 (M+H) ⁺ .

Example 4

The synthesis of this compound refers to Example 1, the only difference is that the iodomethane in Example 1 is replaced with 3-iodopentane, and the nuclear magnetic data of the obtained compound LK04 is: ¹ HNMR (400MHz, CDCl ₃ )δ8.70( d, J＝5.10Hz, 1H), 8.61(s, 1H), 8.10(s, 1H), 7.98(dd, J＝1.20Hz, J＝5.20Hz, 1H), 7.88(d, J＝1.60Hz, 1H), 7.51-7.48(m, 2H), 7.38-7.34(m, 2H), 7.05(t, J=8.60Hz, 2H), 5.02(s, 1H), 4.68-4.61(m, 1H), 3.89 (brs,2H),3.61-3.59(m,2H),2.69-2.67(m,1H),2.57-2.56(m,2H),2.39-2.36(m,1H),2.14(s,3H),2.09 -1.92 (m, 4H), 0.79-0.74 (m, 6H); MS-ESI (m/z): 612.4 (M+H) ⁺ .

Example 5

For the synthesis of this compound, refer to Example 1, with the only difference that: the methyl iodide in Example 1 was replaced with 1-iodo-3-methylbutane, and the nuclear magnetic data of the obtained compound LK05 were: ¹ HNMR (400MHz, CDCl ₃ ) δ8.68(d,J＝5.40Hz,1H),8.48(s,1H),8.10(s,1H),7.97(dd,J＝1.30Hz,J＝5.00Hz,1H),7.85(s,1H) ),7.50-7.46(m,2H),7.37-7.31(m,2H),7.03(t,J=8.60Hz,2H),4.98(s,1H),4.62(t,J=7.70Hz,2H) ,3.87-3.86(m,2H),3.59(brs,2H),2.66-2.63(m,1H),2.55-2.49(m,2H),2.38-2.35(m,1H),2.13(s,3H) ,1.91(q,J=7.30Hz,2H),1.58-1.51(m,1H),0.94(d,J=6.70Hz,6H); MS-ESI(m/z):612.30(M+H) ⁺ .

Example 6

For the synthesis of this compound, refer to Example 1, with the only difference that: the methyl iodide in Example 1 is replaced by 1-fluoro-2-iodoethane, and the nuclear magnetic data of the obtained compound LK06 is: ¹ HNMR (400MHz, CDCl ₃ )δ8 .68(d,J＝4.90Hz,1H),8.59(s,1H),8.09(s,1H),7.96(dd,J＝1.30Hz,J＝5.20Hz,1H),7.84(d,J＝ 1.60Hz, 1H), 7.52-7.44(m, 3H), 7.36-7.29(m, 2H), 7.04(t, J=8.40Hz, 2H), 6.22(dd, J=1.40Hz, J=15.00Hz, 1H), 5.40(dd, J=1.40Hz, J=8.60Hz, 1H), 5.01(s, 1H), 3.88-3.87(m, 2H), 3.59(brs, 2H), 2.69-2.66(m, 1H) ), 2.57-2.54 (m, 2H), 2.41-2.37 (m, 1H), 2.12 (s, 3H); MS-ESI (m/z): 568.20 (M+H) ⁺ .

Example 7

For the synthesis of this compound, refer to Example 1, with the only difference that the iodomethane in Example 1 was replaced with iodocyclopentane, and the nuclear magnetic data of the obtained compound LK07 was: ¹ HNMR (400MHz, CDCl ₃ )δ8.67(d , J＝5.3Hz, 1H), 8.61(s, 1H), 8.00(s, 1H), 7.96(dd, J＝1.30Hz, J＝5.30Hz, 1H), 7.85(d, J＝1.60Hz, 1H) ), 7.50-7.47(m, 2H), 7.36-7.29(m, 2H), 7.03(t, J=8.60Hz, 2H), 5.21-5.15(m, 1H), 4.97(s, 1H), 3.86( brs,2H),3.58-3.56(m,2H),2.66-2.63(m,1H),2.55-2.47(m,2H),2.37-2.34(m,1H),2.22-2.19(m,4H), 2.12 (s, 3H), 1.92-1.88 (m, 2H), 1.75-1.71 (m, 2H); MS-ESI (m/z): 610.20 (M+H) ⁺ .

Example 8

For the synthesis of this compound, refer to Example 1, with the only difference that the iodomethane in Example 1 was replaced with iodocyclohexane, and the nuclear magnetic data of the obtained compound LK08 was: ¹ HNMR (400MHz, CDCl ₃ )δ8.69 (brs ,1H),8.16(s,2H),8.00(brs,1H),7.89(s,1H),7.51-7.48(m,2H),7.42-7.37(m,2H),7.03(t,J=8.50 Hz,2H),4.99(s,1H),4.70-4.64(m,1H),3.87(s,2H),3.60(s,2H),2.64(brs,1H),2.51(brs,2H),2.38 -2.36(m,1H),2.23-2.21(m,2H),2.16(s,3H),1.95-1.90(m,4H),1.75-1.72(m,2H),1.49-1.40(m,2H) ; MS-ESI (m/z): 624.30 (M+H) ⁺ .

Biological Test Example 1

In vitro anti-influenza virus (IAVH1N1) activity of the pyridine compounds prepared in Examples 1-8:

1) Determination of in vitro anti-influenza virus (IAVH1N1) activity of the pyridine compounds prepared in Examples 1-8 by cytopathic (CPE) method

The MDCK cells were seeded into a 96-well plate at a density of 2.5×10 ⁴ /well, and after culturing for 24 hours, the medium was discarded, and 100 μL of the pyridine compound solutions prepared in Examples 1 to 8 with different concentration gradients were added to the MDCK cells. Cell control and blank control, continue to culture. After 2 days of administration, the cultured cells were placed upside down under a microscope to detect the toxicity of pyridine compounds to MDCK cells (CPE method), and the Reed-Muench method was used to calculate the half toxic concentration TC ₅₀ , the calculation formula was as follows (1), the results See Table 1:

In formula (1), A represents the drug concentration with cumulative inhibition rate < 50%, B represents the inhibition rate with cumulative inhibition rate > 50%, C represents the inhibition rate with cumulative inhibition rate < 50%, and D represents the log dilution factor.

2) Determination of the anti-influenza efficacy of the pyridine compounds prepared in Examples 1-8 by CPE

The MDCK cells were seeded into a 96-well plate at a density of 2.5×10 ⁴ /well, and after culturing for 24 hours, the MDCK cells were washed once with PBS, and treated with 100 times the tissue culture infective doses (50% tissue culture infective doses, TCID ₅₀ ). Influenza virus infection solution (serum-free MEM medium) was used to infect MDCK cells, and at the same time, virus maintenance solution (MEM medium supplemented with 2 μg·ml ^-1 TPCK-treated pancreas) containing drugs (pyridine compounds prepared in Examples 1 to 8) was added. Enzyme and 0.08% BSA), placed in a 37°C incubator for about 2 days, and when the cytopathic effect (CPE) of the virus control group was 75-100%, the CPE was observed under an inverted microscope. The half effective inhibitory concentration (50% inhibitoroty concentration, IC ₅₀ ) of the drug was calculated by the Reed & Muench method according to the following formula (2), and the results are shown in Table 1;

In formula (2), A represents the drug concentration with cumulative inhibition rate < 50%, B represents the inhibition rate with cumulative inhibition rate > 50%, C represents the inhibition rate with cumulative inhibition rate < 50%, and D represents the log dilution factor.

Table 1 In vitro antiviral (IAV H1N1) activity of the pyridine compounds prepared in Examples 1 to 8

It can be seen from Table 1 that the in vitro activities of the compounds prepared in Examples 1 to 8 of the present invention on IAV H1N1 (IC50: 0.27-0.48 μM) are comparable to those of the control compound (±)-JNJ4796, and the cytotoxicity (CC50: >50 μM) is very high. Low.

Biological Test Example 2

AD-H(250mm×10mm,5μm,PN:19335)；进样量:50μL；流速:4.8-5.2mL/min；检测器：UVat254nm.然后，检测所得(R)-型化合物的体外抗流感病毒(IAV H1N1)活性，活性测试方法同生物测试例1，所得结果见表2。The pyridine compounds prepared in Examples 1-6 and (±)-JNJ4796 were subjected to chiral resolution to obtain the corresponding (R)-type example compounds and JNJ4796. Resolution conditions: instrument Thermo UltiMate 3000 HPLC-UV system; Mobile phase: A:B=65:35, solvent A=10% hexane in IPA, solvent B=MeOH);

AD-H (250mm×10mm, 5μm, PN: 19335); injection volume: 50μL; flow rate: 4.8-5.2mL/min; detector: UVat254nm. Then, the in vitro anti-influenza virus of the obtained (R)-type compound was detected (IAV H1N1) activity, the activity test method is the same as that of biological test example 1, and the obtained results are shown in Table 2.

Table 2 In vitro antiviral (IAV H1N1) activity of (R)-type pyridine compounds

It can be seen from Table 2 that the in vitro activities of some (R)-type example compounds and the reference compound JNJ4796 in the compounds of formula I of the present invention to IAVH1N1 are significantly stronger than the corresponding racemates (Table 1), wherein, (R) The activity (IC50: <0046 μM) of the -type example compounds was better than that of the control compound JNJ4796 (IC50: 0046 μM), while the cytotoxicity (CC50: >50 μM) was very low.

Biological Test Example 3

In vivo pharmacokinetic testing

1. Sample pretreatment method

Take 10.0 μL of plasma sample (take out the sample from the refrigerator at -80°C, vortex for 30 seconds after natural melting at room temperature) into a 1.5 mL centrifuge tube, and add 100 μL of internal standard solution (dexamethasone (60.0ng·mL ^-1 ).) , vortexed for 60 seconds and then centrifuged for 3 minutes (centrifugal force 12000rpm); take 75 μL of supernatant and transfer it to a 96-well sample plate with an equal volume of water. After shaking and mixing, LC-MS/MS sample injection analysis. 10 μL.

2. Standard curve and quality control sample preparation

Internal standard: Precisely weigh an appropriate amount of dexamethasone standard, dissolve and dilute it with dimethyl sulfoxide (DMSO), shake well, prepare a stock solution with a mass concentration of 5000 μg/mL, and dilute it into an internal standard with a concentration of 60 ng/mL. liquid.

Stock solution: Precisely weigh an appropriate amount of compound and prepare a 5.0 mg/mL stock solution with DMSO.

Working solution: The stock solution is diluted according to gradient to obtain standard working solution and low-medium-high quality control working solution with corresponding gradient concentration. Add 5.00 μL of reference standard curve working solution to 45.0 μL blank Balb/c mouse plasma. It is then processed as described in the "Sample processing method" section.

2. Animal experiments

2.1 Experimental Design

Balb/c mice (provided by Suzhou Zhaoyan Experimental Animal Co., Ltd.), the dosage is 25mg/kg, the dosage is 2.5mg/mL, the dosage volume is 20mL/kg, the collected sample is plasma, and the administration The way is oral, the vehicle is 5% DMSO + 95% (0.5% MC).

2.2 Sample collection: 0.030 mL of blood was taken from each animal through the orbit each time, EDTAK ₂ anticoagulation, the collection time point was PO group: 15min, 30min, 1h, 2h, 4h, 6h, 8h, 24h after administration of the test substance. Blood samples were placed on ice after collection, and centrifuged within 30 minutes to separate plasma (centrifugation conditions: 5000 rpm, 10 minutes, 4°C). Store at -80°C until analysis.

data processing

The software of data acquisition and control system is Analyst 1.5.1 software (Applied Biosystem). The integration method of the sample peak in the spectrum is automatic integration; the ratio of the peak area of the sample and the peak area of the internal standard is used as an indicator, and the concentration of the sample is regressed. Regression method: linear regression, the weight coefficient is 1/X ² . Pharmacokinetic parameters were analyzed with WinNonlin Professional v6.3 (Pharsight, USA) using a non-compartmental model. _Cmax is the measured maximum plasma concentration, the area under the plasma concentration-time curve AUC _(0→t) is calculated by the trapezoidal method, and _Tmax is the peak time of the plasma concentration after administration. The experimental data are expressed as "mean±standard deviation" (Mean±ICR, n≥3) or "mean" (Mean, n=2). The results are shown in Table 3.

Biological Test Example 4

Whole-cell current recordings were performed using the manual patch clamp system Multiclamp 700A.

Preparation of test drugs: all were prepared with DMSO, and the concentration of the mother solution was 10 mM. Freeze for later use, and dilute to the required concentration when using.

The detection concentration was 10 μM; an 8-channel perfusion drug delivery system was used, and the drug was administered by perfusion through the drug delivery electrode.

Experimental procedure: HERG-CHO cells were subcultured. Under the microscope, single cells were selected for electrophysiological recording. After the whole-cell mode was formed in the voltage clamp state, the cell membrane was clamped at -80mV to observe the effect of the compound on HERG current. The cells were clamped at -80mV, depolarized to +40mV for 1.5-3s, and then returned to -40mV for 1.5 seconds. ~3s. After the current was stable, the compound to be tested was perfused, and pulse stimulation was given every 10-15s.

Data acquisition and analysis: PClamp 10 software was used for data acquisition. The results are shown in Table 3.

Table 3. Pharmacokinetic properties and hERG K ⁺ inhibitory activity of the (R)-type example compounds

^a Balb/c mice, single oral administration of 25 mg/kg (n=3); ^b Inhibition rate of hERGK+ channel by compound (10 uM)

It can be seen from Table 3 that the pharmacokinetics of some (R)-type example compounds in the compounds of formula (I) of the present application are better than that of the control compound JNJ4796, wherein the drug exposure of the (R)-type example compounds ( C _max , AUC _0-inf ) was significantly greater than that of the control compound JNJ4796, indicating that these (R)-form example compounds have stronger in vivo activity. On the other hand, these (R)-form example compounds (10 uM) exhibited lower inhibition rates of hERGK+ channels than the control compound JNJ4796, indicating lower potential cardiotoxicity.

Tables 1-3 above only list the in vitro activity, cytotoxicity, in vivo pharmacokinetic properties and cardiotoxicity of some compounds of the present invention. Other compounds of the present invention are similar in structure and have the same or similar druggability as the above-mentioned compounds. No more enumerating.

The above description is only to illustrate the present invention, and it should be understood that the present invention is not limited to the above embodiments, and various modifications conforming to the idea of the present invention are all within the protection scope of the present invention.

Claims (10)

1. A pyridine compound containing a 4-fluorophenyl fragment, having the structure shown in formula I:

in the formula I, R is C1-C7 alkyl, cycloalkyl or alkenyl.

2. The pyridine compound according to claim 1, comprising

3. A process for the preparation of a pyridine compound containing a 4-fluorophenyl moiety according to claim 1 or 2, comprising the steps of:

mixing a compound with a structure shown in a formula II, a compound with a structure shown in a formula III, a condensing agent, a catalyst and a dipolar solvent, and carrying out condensation reaction to obtain a pyridine compound containing a 4-fluorophenyl fragment with the structure shown in the formula I;

in the formula II, R is C1-C7 alkyl, cycloalkyl or alkenyl.

4. The method according to claim 3, wherein the molar ratio of the compound having the structure represented by formula II to the compound having the structure represented by formula III is 1: 1.

5. The preparation method according to claim 3, wherein the condensation reaction is carried out at a temperature of-5 to 40 ℃ for 5 to 20 hours.

6. The method of claim 3, wherein the dipolar solvent comprises dimethylformamide, acetone, dimethylsulfoxide, or acetonitrile; the condensing agent comprises 1-ethyl-3 (3-dimethylpropylamine) carbodiimide; the catalyst comprises 4-dimethylaminopyridine.

7. Use of a pyridine compound containing a 4-fluorophenyl fragment according to claim 1 or 2 or a pyridine compound containing a 4-fluorophenyl fragment prepared by the preparation method according to any one of claims 3 to 6 in the preparation of a medicament for resisting influenza virus infection, wherein the pyridine compound containing a 4-fluorophenyl fragment is used in the form of a compound shown as a formula I or a pharmaceutically acceptable salt thereof; the medicinal salt is formed by the compound shown in the formula I and inorganic acid or organic acid.

8. The use of claim 7, wherein the influenza virus in the anti-influenza virus infection medicament comprises influenza a, influenza b, influenza c, or influenza d.

9. Use according to claim 8, wherein the influenza A comprises H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3 or H10N 7.

10. A pharmaceutical composition comprising a pyridine compound containing a 4-fluorophenyl fragment according to claim 1 or 2, or a pharmaceutically acceptable salt thereof; the medicinal salt is formed by the compound shown in the formula I and inorganic acid or organic acid.