Nicotinic acid or isonicotinic acid compound and use thereof

Abstract

Disclosed are a nicotinic acid or isonicotinic acid compound and a use thereof. The compound is the compound shown in Formula I, or a pharmaceutically acceptable salt, ester or solvate thereof. Efficacy tests demonstrate that the nicotinic acid compound can inhibit botulinum toxin endopeptidase activity in vitro, and has a significant protective effect on mice poisoned with botulinum toxin. On this basis, the compound may be used to prepare a drug preventing and/or treating botulinum toxin exposure and/or poisoning. ##STR00001##

Claims

1. A compound of Formula I, or a pharmaceutically acceptable salt or solvate thereof, ##STR00012## in the Formula I, R.sub.1 and R.sub.2 independently represent any one of the groups selecting from the group consisting of hydrogen and —(CH.sub.2).sub.mCOOH; R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 independently represent any one of the groups selecting from the group consisting of hydrogen, hydroxyl, amino, halogen, lower alkyl, lower alkoxy, and lower cycloalkyl; the lower alkyl refers to a linear or branched saturated aliphatic group having 1 to 6 carbon atoms; the lower alkoxy refers to a linear or branched alkoxy group having 1 to 6 carbon atoms; the lower cycloalkyl refers to a ring group having 3 to 7 carbon atoms; X independently represents 0 or —S═O; m is 0, or 1, or 2, or 3.

2. The compound of Formula I, or a pharmaceutically acceptable salt or solvate thereof according to claim 1, characterized in that the compound of Formula I is any one selected from the group consisting of: 2-((2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethyl)sulfonyl)isonicotinic acid; 2-((2-((5-chloro-2-phenoxyphenyl)amino)-2-oxoethyl)sulfonyl)nicotinic acid; 2-((2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethyl)sulfinyl)nicotinic acid; 2-(2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethoxy)nicotinic acid; 2-(2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethoxy)isonicotinic acid.

3. A pharmaceutical preparation comprising the compound of Formula I, or a pharmaceutically acceptable salt or solvate thereof according to claim 1, or a mixture thereof.

4. The pharmaceutical preparation according to claim 3, wherein the botulinum toxin comprises botulinum toxin type A and botulinum toxin type B.

Description

DETAILED DESCRIPTION

(1) Hereinafter, the present disclosure is further illustrated in conjunction with the specific examples, but the present disclosure is not limited to the examples. The methods mentioned are conventional methods unless otherwise specified. The raw materials mentioned can be commercially available unless otherwise specified.

EXAMPLE 1

Synthesis of 2-((2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethyl)sulfonyl)isonicotinic acid

(2) 30 mg of 2-((2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethyl)thio)isonicotinic acid was weighed and dissolved in 3 mL of anhydrous acetic acid, and 0.2 mL of hydrogen peroxide was slowly added thereto. The reaction was carried out at room temperature for 2 h, and a white solid was obtained by column purification with a yield of 42%.

(3) Structural confirmation results of the product were as follows:

(4) .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ: 9.86 (s, 1H), 8.97 (d, J=4.9 MHz, 1H), 8.29 (s, 1H), 8.15 (dd, J=1.2, 4.9 MHz, 1H), 7.92 (d, J=2.4 MHz, 1H), 7.04-7.16 (m, 2H), 4.92 (s, 2H), 3.84 (s, 3H); ESI-MS (m/z): 383.06 (M−H.sup.+), 766.87 (2M−H.sup.+). As can be seen from the above, the compound had the correct structure, and the compound of interest was numbered as 2a.

(5) ##STR00007##

EXAMPLE 2

Synthesis of 2-((2-((5-chloro-2-phenoxyphenyl)amino)-2-oxoethyl)sulfonyl)nicotinic acid

(6) The same procedures as above were followed, except that 2-((2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethyl)thio)isonicotinic acid was replaced with 2-((2-((5-chloro-2-phenoxyphenyl)amino)-2-oxoethyl)thio)nicotinic acid, to obtain 2-((2-((5-chloro-2-phenoxyphenyl)amino)-2-oxoethyl)sulfonyl)nicotinic acid. The product was a white solid with a yield of 53%.

(7) Structural confirmation results of the product were as follows:

(8) .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ: 10.03 (s, 1H), 8.42 (d, J=7.4 MHz, 1H), 8.21 (s, 1H), 7.99 (s, 1H), 7.51 (d, J=7.5 MHz, 2H), 7.32-7.44 (m, 3H), 7.11 (s, 2H), 6.75 (t, 1H), 5.29 (s, 2H), 5.13 (s, 2H); ESI-MS (m/z): 459.01 (M−H.sup.+). As can be seen from the above, the compound had the correct structure, and the compound of interest was numbered as 2b.

(9) ##STR00008##

EXAMPLE 3

Synthesis of 2-((2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethyl)sulfinyl)nicotinic acid

(10) The same procedures as above were followed, except that 2-((2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethyl)thio)isonicotinic acid was replaced with 2-((2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethyl)thio)nicotinic acid, to obtain 2-((2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethyl)sulfinyl)nicotinic acid. The product was a white solid with a yield of 48%.

(11) Structural confirmation results of the product were as follows:

(12) .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ: 7.97 (s, 1H), 8.68 (dd, J=1.6, 4.8 MHz, 1H), 8.28 (dd, J=1.6, 7.7 MHz, 1H), 8.18 (d, J=2.2 MHz, 1H), 7.35 (q, 1H), 6.98-7.09 (m, 2H), 4.00 (s, 2H), 3.78 (s, 3H); ESI-MS (m/z): 383.17 (M−H.sup.+), 767.01 (2M−H.sup.+). As can be seen from the above, the compound had the correct structure, and the compound of interest was numbered as 2c.

(13) ##STR00009##

EXAMPLE 4

Synthesis of 2-(2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethoxy)nicotinic acid

(14) 0.5 g of 2-methoxy-5-chloroaniline was weighed and dissolved in 10 mL of anhydrous dichloromethane, to which 1 mL triethylamine was added, and the mixture was placed in a 50 mL eggplant bottle. 0.25 mL of chloroacetyl chloride was accurately weighed and diluted in 2 mL of anhydrous dichloromethane. The mixture was slowly added dropwise to the reaction flask, and the reaction was carried out at room temperature for 4 h. The reaction was quenched with water, and the product was extracted with dichloromethane 3 times. The organic phases were combined and concentrated, and purified by column chromatography, to obtain 2-chloro-N-(5-chloro-2-methoxyphenyl)acetamide with a yield of 78%.

(15) .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ: 9.68 (s, 1H), 8.12 (d, J=2.5 MHz, 1H), 7.19 (dd, J=2.6, 8.8 MHz, 1H), 7.12 (d, J=8.8 MHz, 1H), 4.43 (s, 2H), 3.89 (s, 3H); ESI-MS (m/z): 272 (M+K.sup.+).

(16) 0.1 g of 2-chloro-N-(5-chloro-2-methoxyphenyl)acetylamine, 0.56 g of 2-hydroxynicotinic acid, 0.19 g of potassium carbonate were weighed and placed in a 20 mL eggplant bottle, to which 5 mL of N, N-dimethylformamide was added, and the etherification was carried out overnight at room temperature. The product was extracted with a large amount of water and ethyl acetate. The organic phases were combined and concentrated, and purified by column chromatography, to obtain 2-(2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethoxy)nicotinic acid as a white solid with a yield of 57%.

(17) Structural confirmation results of the product were as follows:

(18) .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ: 10.02 (s, 1H), 8.42 (d, J=7.2 MHz, 1H), 8.22 (d, J=6.7 MHz, 1H), 8.04 (s, 1H), 7.10 (m, 2H), 6.75 (t, 1H), 5.11 (s, 2H), 3.87 (s, 3 H); ESI-MS (m/z): 335.31 (M−H.sup.+). As can be seen from the above, the compound had the correct structure, and the compound of interest was numbered as 6a.

(19) ##STR00010##

EXAMPLE 5

Synthesis of 2-(2-((5-chloro-2-methoxyphenyl)amino)-2-oxoethoxy)isonicotinic acid

(20) The same procedures as above were followed, except that 2-hydroxynicotinic acid was replaced with 2-hydroxyisonicotinic acid, to obtain a white solid with a yield of 62%.

(21) Structural confirmation results of the product were as follows:

(22) .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ: 9.82 (s, 1H), 8.08 (d, J=2.0 MHz, 1H), 7.63 (d, J=6.8 MHz, 1H), 7.33 (d, J=6.7 MHz, 1H), 7.09-7.12 (m, 1H), 6.80 (d, J=9.8 MHz, 1H), 6.54 (dd, J=1.5, 6.8 MHz, 1H), 4.85 (s, 2H), 3.87 (s, 3H); ESI-MS (m/z): 335.15 (M−H.sup.+). As can be seen from the above, the compound had the correct structure, and the compound of interest was numbered as 6b.

(23) ##STR00011##

EXAMPLE 6

Inhibitory Effects of Novel Thionicotinic Acid-Based Compounds on Endopeptidase Activity of Botulinum Toxin Type A In Vitro

(24) The activities of the compounds were detected by substrate cleavage method. Reference was made to the literature (L. Li, B. R. Singh, High-Level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain, Protein Expr Purif. 1999, 17: 339-344) for expression and purification of Botulinum toxin type A endopeptidase (BoNT/A-LC). Reference was made to the literature (D. R. Ruge, F. M. Dunning, T. M. Piazza, B. E. Molles, M. Adler, F. N. Zeytin, W. C. Tucker, Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters, J. Anal. Biochem. 2011, 411:200-209) for construction, expression and purification of CYA, a substrate of Botulinum toxin type A. To 50 μl of reaction solution (50 mM Hepes-NaOH pH 7.4, 10 mM NaCl, 0.1% Tween 20, 5 mM dithiothreitol, 10 μM ZnCl.sub.2), BoNT/A-LC with a final concentration of 10 nM, and 100 μg of the compounds were added, which were incubated together for 15 min at 37° C., and then the substrate CYA (for Botulinum toxin type A) with a final concentration of 4 μM was added thereto, and the cleavage was carried out in vitro for 30 min SDS-PAGE detection and analysis were performed by a gel imaging system (Bio-Rad Company, Molecular Imager chemiDoc™ XRS Imaging System), and gel image capture was performed by a gel acquisition and image analysis system (ChampGel 5000 Plus Company, SAGECREATION). The data was analyzed and processed by a software Gel-Pro analyzer, and protein fragment gray scale analysis was performed. The inhibition rates of the compounds were calculated in comparison with the control group.

(25) Results of the inhibitory activities of the compounds in vitro detected by substrate cleavage method were shown in Table 1. The compounds had different inhibitory effects on the endopeptidase activity of Botulinum toxin type A, with an inhibition rate in the range from 0 to 60.0%. Among them, the compound as shown in 2c obtained in Example 1 had a remarkable inhibitory effect and the inhibition rate could reach 60.0%.

(26) TABLE-US-00001 TABLE 1 Inhibitory effects of compounds on Botulinum toxin type A detected by substrate cleavage method Compounds dissolved in aqueous 20% by volume of 1,3-propanediol solution No. 2a 2b 2c 6a 6b Inhibition 10 5 60 0 0 rate %

EXAMPLE 7

Inhibitory Effects of Novel Thionicotinic Acid-Based Compounds on Endopeptidase Activity of Botulinum Toxin Type B In Vitro

(27) The activities of the compounds were detected by substrate cleavage method. Reference was made to the literature (J. Gilsdorf, N. Gul, L. A. Smith, Expression, purification, and characterization of Clostridium botulinum type B light chain, Protein Expr Purif 46 (2006) 256-267) for expression and purification of Botulinum toxin type B endopeptidase (BoNT/B-LC). Reference was made to the literature (D. R. Ruge, F. M. Dunning, T. M. Piazza, B. E. Molles, M. Adler, F. N. Zeytin, W. C. Tucker, Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters, J. Anal. Biochem. 2011, 411:200-209) for construction, expression and purification of CYB, a substrate of Botulinum toxin type B. To 50 μl of reaction solution (50 mM Hepes-NaOH pH 7.4, 10 mM NaCl, 0.1% Tween 20, 5 mM dithiothreitol, 10 μM ZnCl.sub.2), BoNT/B-LC with a final concentration of 10 nM, and 100 μg of the compounds were added, which were incubated together for 15 min at 37° C., and then the substrate CYB (for Botulinum toxin type B) with a final concentration of 4 μM was added thereto, and the cleavage was carried out in vitro for 30 min SDS-PAGE detection and analysis were performed by a gel imaging system (Bio-Rad Company, Molecular Imager chemiDoc™ XRS Imaging System), and gel image capture was performed by a gel acquisition and image analysis system (ChampGel 5000 Plus Company, SAGECREATION). The data was analyzed and processed by a software Gel-Pro analyzer, and protein fragment gray scale analysis was performed. The inhibition rates of the compounds were calculated in comparison with the control group.

(28) Results of the inhibitory activities of the compounds in vitro detected by substrate cleavage method were shown in Table 1. The compounds had different inhibitory effects on the endopeptidase activity of Botulinum toxin type B, with an inhibition rate in the range from 0 to 85.0%. Among them, the compound as shown in 6b obtained in Example 2 had a remarkable inhibitory effect and the inhibition rate could reach 85.0%, followed by the compound as shown in 6a obtained in Example 2 which had an inhibition rate of 80%.

(29) TABLE-US-00002 TABLE 2 Inhibitory effects of compounds on Botulinum toxin type B detected by substrate cleavage method Compounds dissolved in aqueous 20% by volume of 1,3-propanediol solution No. 2a 2b 2c 6a 6b Inhibition 0 0 35 80 85 rate %

EXAMPLE 8

Protection Effects of Novel Thionicotinic Acid-Based Compounds against Botulinum Toxin Type A Poisoning in Animals

(30) Reference was made to the literature (C. J. Malizio, M. C. Goodnough, E. A. Johnson, Purification of Clostridium botulinum type A neurotoxin, Methods Mol Biol. 2000, 145:27-39) for extraction and identification of Botulinum toxin type A (BoNT/A), and Balb/c mice of 16-18 g were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences. Reference was made to the method for mouse botulism model described in the literature (C. H. Hatheway, J. D. Snyder, J. E. Seals, T. A. Edell, G. E. Lewis, Jr. Antitoxin levels in botulism patients treated with trivalent equine botulism antitoxin to toxin types A, B, and E. Infect Dis 1984, 150: 145-151) for protection experiments against botulinum toxin poisoning. The method was briefly as follows: Balb/C mice were used as test animals, which were randomly divided into groups with 10 mice of each group, and 1 mg of various compounds were used as samples to be tested. The mice were given Botulinum toxin type A in an amount of 5-fold of the median lethal dose (5LD.sub.50) by injection via the tail vein, and then the mice were given 1 mg of the compounds in a solution (dissolved in 20% of 1,3-propanediol) by injection via the tail vein. A blank group and a control group were set at the same time. The observation was continued for more than 5 days, to observe the symptoms of botulism in mice (such as the occurrence of wasp waist, horripilation, weak breathing, limb paralysis until death), record the survival time of mice, and calculate the survival rate.

(31) The anti-toxic effects of the compounds in the mouse botulism model were shown in Table 36. The compounds could inhibit the lethal effect of Botulinum toxin type A in the test animals to different degrees. Among them, the compound as shown in 2c obtained in Example 1 showed the highest protection rate of 100%, followed by the compound as shown in 6a obtained in Example 2 which showed a protection rate of 60%.

(32) TABLE-US-00003 TABLE 3 Anti-toxic effects of compounds in mouse model poisoned by Botulinum toxin type A Compound Group Antitoxin Placebo 2a 2b 2c 6a 6b Survival 100 0 0 0 100 60 0 rate % Note: the placebo in Table 3 was a solvent (aqueous 20% by volume of 1,3-propanediol solution); the antitoxin was a type A horse serum antitoxin (purchased from National Institutes for Food and Drug Control).

EXAMPLE 9

Protection Effects of Thionicotinic Acid-Based Compounds against Botulinum Toxin Type B Poisoning in Animals

(33) Reference was made to the Literature (H. Arimitsu, K. Inoue, Y. Sakaguchi, J. Lee, Y. Fujinaga, T. Watanabe, T. Ohyama, R. Hirst, K. Oguma, Purification of fully activated Clostridium botulinum serotype B toxin for treatment of patients with dystonia, Infect Immun. 71(2003)1599-1603) for extraction and identification of Botulinum toxin type B (BoNT/B), and Balb/c mice of 14-16 g were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences.

(34) Reference was made to the method for mouse botulism model described in the literature (C. H. Hatheway, J. D. Snyder, J. E. Seals, T. A. Edell, G. E. Lewis, Jr. Antitoxin levels in botulism patients treated with trivalent equine botulism antitoxin to toxin types A, B, and E. Infect Dis 1984, 150: 145-151) for protection experiments against botulinum toxin poisoning. The method was briefly as follows: Balb/C mice were used as test animals, which were randomly divided into groups with 10 mice of each group, and 1 mg of various compounds were used as samples to be tested. The mice were given Botulinum toxin type B in an amount of 3-fold of the median lethal dose (3LD.sub.50) by injection via the tail vein, and then the mice were given 1 mg of the compounds in a solution (dissolved in 20% of 1,3-propanediol) by injection via the tail vein. A blank group and a control group were set at the same time. The observation was continued for more than 5 days, to observe the symptoms of botulism in mice (such as the occurrence of wasp waist, horripilation, weak breathing, limb paralysis until death), record the survival time of mice, and calculate the survival rate.

(35) The anti-toxic effects of the compounds in the mouse botulism model were shown in Table 4. The compounds could inhibit the lethal effect of Botulinum toxin type B in the test animals to different degrees. Among them, the compound as shown in 6b obtained in Example 2 showed the highest protection rate of 80%, followed by the compound as shown in 2c obtained in Example 1 and the compound as shown in 6a obtained in Example 2, both of which showed a protection rate of 20%.

(36) TABLE-US-00004 TABLE 4 Anti-toxic effects of compounds in mouse model poisoned by Botulinum toxin type B Compound Group Antitoxin Placebo 2a 2b 2c 6a 6b Survival 100 0 0 0 20 20 80 rate % Note: the placebo in Table 4 was a solvent (aqueous 20% by volume of 1,3-propanediol solution); the antitoxin was a type B horse serum antitoxin (purchased from National Institutes for Food and Drug Control).

INDUSTRIAL APPLICABILITY

(37) The pharmacodynamic experiments have demonstrated that the thionicotinic acid-based compounds provided by the present disclosure can inhibit the endopeptidase activity of botulinum toxins in vitro, and also have evident protection effects in mice poisoned by botulinum toxins.