RU2396262C2 - N-(2-benzothiazolyl)amide 2-hydroxy-4-oxo-4-(4-chlorophenyl)-2-butenoic acid having antimicrobial and anti-inflammatory activity - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to N-(2-benzothiazolyl)amide 2-hydroxy-4-oxo-4-(4-chlorophenyl)-2-butenoic acid of formula:

, which has antimicrobial and anti-inflammatory activity along with low toxicity.

EFFECT: obtaining a compound which can be used to treat diseases associated with pathogenic microorganisms and inflammation.

1 cl, 2 tbl, 1 ex

Description

The invention relates to the field of organic chemistry, to new biologically active substances of the class of N-heterylamides of 4-aryl-2,4-dioxobutanoic acids, namely to the N- (2-benzothiazolyl) amide 2-hydroxy-4-oxo-4- (4 -chlorophenyl) -2-butenoic acid (1) of the formula

possessing antimicrobial and anti-inflammatory activity, which suggests its use in medicine as a medicinal antimicrobial and anti-inflammatory agent. The closest analogue in structure and action to the claimed compound is N- (2-thiazolyl) amide 2-hydroxy-4-oxo-4- (4-chlorophenyl) -2-butenoic acid (2) [Andreichikov Yu.S., Milyutin A. V., Krylova I.V. et al. Synthesis and biological activity of heteroylamides of aroylpyruvic and 5-arylpyrazole-3-carboxylic acids // Chem. journal - 1990. - No. 7. - S.33-35]

Structural analogue 2 does not have anti-inflammatory activity. There are no data on the antimicrobial activity of structural analogue 2 in the literature.

The following standards were selected as reference standards:

1) the antimicrobial drug chlorhexidine [1,6-di- (4-chlorophenylguanido) hexane] (3), which is widely used in medical practice and is analogous in action [Mashkovsky M.D. Medicines - 15th ed., Rev., Rev. and add. - M .: LLC "Publishing House New Wave", 2005. - S.935-936];

2) the anti-inflammatory drug diclofenac (sodium salt of 2 - [(2,6-dichlorophenyl) amino] phenylacetic acid) (4), which is widely used in medical practice and is an analogue in action [Mashkovsky M.D. Medicines - 15th ed., Rev., Rev. and add. - M .: New Wave Publishing House LLC, 2005. - P.170].

The objective of the invention is to search for a series of N-heterylamides of 4-aryl-2,4-dioxobutanoic acids substances with a pronounced antimicrobial and anti-inflammatory effect and low toxicity.

The problem is achieved by obtaining N- (2-benzothiazolyl) amide 2-hydroxy-4-oxo-4- (4-chlorophenyl) -2-butenoic acid (1), which has antimicrobial and anti-inflammatory activity.

The claimed compound 1 is synthesized by the interaction of 5- (4-chlorophenyl) -2,3-dihydrofuran-2,3-dione with 2-aminobenzothiazole at an equimolar ratio of reactants in anhydrous chloroform at room temperature, followed by isolation of the target product by known methods according to the scheme

Preparation Example 1 of Compound 1. To a solution of 2.09 g (0.01 mol) of 5- (4-chlorophenyl) -2,3-dihydrofuran-2,3-dione in 30 ml of anhydrous chloroform, a solution of 1.50 g (0 , 01 mol) of 2-aminobenzothiazole in 20 ml of anhydrous chloroform, maintained at room temperature for 24 hours. The reaction mixture was cooled to 0 ° C, the precipitate formed was filtered off, recrystallized from toluene and dried. Yield 3.4 g (95%). Mp 210-212 ° C. Found,%: C 57.08; H 3.12; N, 7.92. C ₁₇ H ₁₁ ClN ₂ O ₃ S. Calculated,%: C 56.91; H 3.09; N, 7.81.

IR spectrum (Specord M80, liquid paraffin, v, cm ^-1 ): 3280 (NH), 1710 (CONH), 1640, 1610 (C ⁴ = O, C = C, C = N). ¹ H NMR spectrum (Bruker DRX 500, DMSO-d ₆ , HMDS, δ, ppm): 7.22 s (1H, СН), 7.33-7.99 m (10Н, С ₆ Н ₅ , С ₆ Н ₄ ) NH )

The inventive compound 1 is a yellow crystalline substance soluble in toluene, dioxane, dimethyl sulfoxide, insoluble in water and alkanes.

The determination of bacteriostatic activity was carried out by the method of two serial dilutions in a liquid nutrient medium [Guide to the experimental (preclinical) study of new pharmacological substances / Fisenko V.P. (ed.). - M .: IIA Remedium, 2000. - S.264-273]. For all tested compounds, MICs were determined with respect to pharmacopoeial strains: S.aureus ATCC 6538-P and E.coli ATCC 25922. The test results are shown in Table 1. Inoculations were made in meat and peptone broth, pH 7.0, with different concentrations of the tested compounds. The cultures were grown in test tubes on a beveled agar medium (meat peptone agar). An 18-20 hour culture was used to determine antimicrobial activity. To prepare a working suspension of microbes, the grown culture was washed off with an isotonic sodium chloride solution and the microbial suspension density was set according to the turbidity standard of 5 units. Next, from the obtained microbial suspension (500 million mt / ml), a working solution of bacteria with a concentration of 5 million mt / ml was prepared. This suspension of microbes was introduced in an amount of 0.1 ml into test tubes with serial dilutions of the studied drug. Thus, the microbial load in the determination of PMA was 250,000 mt / ml. The studied compound in an amount of 0.05 g was dissolved in 5 ml of dimethylformamide; 1 ml of the resulting dilution 1: 100 was combined with 4 ml of meat and peptone broth (1: 500). Next, a series of serial dilutions of the compound with a doubling concentration was prepared. The results were taken into account after 18-20 hours of exposure of control and experimental tubes in a thermostat at a temperature of 37 ° C. The minimum inhibitory concentration (MIC) was determined by the absence of signs of growth on a nutrient medium: the last tube with growth retardation (clear broth) corresponds to the MIC of the drug for this strain. The bacteriostatic effect of the studied compounds was compared with the action of chlorhexidine.

Anti-inflammatory activity was studied in experiments on 30 white outbred rats of both sexes weighing 220-260 g. Compound 1 was administered orally at a dose of 50 mg / kg as a suspension in 2% starch solution an hour before modeling acute carrageenin inflammation. Carrageenin edema was caused by subplant implantation of 0.1 ml of a 1% phlogogen solution in the hind paw of a rat. Anti-inflammatory activity was judged by the change in the severity of inflammation in the dynamics, which was recorded oncometrically 1, 3 and 5 hours after modeling inflammation [Methodological recommendations for the experimental study of non-steroidal anti-inflammatory substances / Pharmacological Committee of the Ministry of Health of the USSR, protocol No. 22 of November 11, 1982. - Moscow, 1982. - 47 p.]. Equivolume amount of 2% starch solution was administered to control animals. The comparison drug was diclofenac at a dose of 10 mg / kg. The results were statistically processed using Excel 2003 and are presented in table 2.

Acute toxicity (LD ₅₀ ) of the claimed compound 1 was determined on 24 white mongrel mice of both sexes weighing 18-22 g with an oral route of administration in the form of a suspension in a 2% starch solution according to the express method of VB Prozorovsky [Prozorovsky VB, Prozorovskaya M.P., Demchenko V.M. Express method for determining the average effective dose and its errors. // Pharmacology and toxicology. - 1978. - No. 4. - S.497-502]. The LD _{50 of} compound 1 was found to be 5800 mg / kg. According to the classification of toxicity of drugs, compound 1 belongs to the class of practically non-toxic substances [Izmerov I.F., Sanotsky I.V., Sidorov K.K. Toxicometry parameters of industrial poisons. - M .: Medicine, 1977. - S.196-197]. It is 1.45 times less toxic than chlorhexidine, and 7.44 times less toxic than diclofenac.

Table 1 Antimicrobial activity and acute toxicity of compound 1 Compound LD ₅₀ , mg / kg MIC, μg / ml IPC / LD ₅₀ St.aureus E.coli St.aureus E.coli one 5800 2.0 3.9 0,0003 0,0007 Chlorhexidine 4000 125 125 0,0313 0,0313

As can be seen from table 1, compound 1 exceeds the antimicrobial activity of chlorhexidine in relation to Staphylococcus aureus 62.5 times and Escherichia coli - 32 times. The ratio of MPC (μg / ml) to the toxicity of LD ₅₀ (mg / kg) for compound 1 is 0.0003 for staphylococcus and 0.0007 for E. coli, while these indicators are significantly lower for chlorhexidine.

table 2 Anti-inflammatory activity and acute toxicity of compound 1 Compound Dose mg / kg LD ₅₀ , mg / kg % inhibition of carrageenin edema 1 hour 3 hours 5 o'clock The control - - 0,0 ± 0,0 0,0 ± 0,0 0,0 ± 0,0 one fifty 5800 39.7 ± 2.1 ^{** 1} 64.2 ± 3.2 ^{*** 1} 56.4 ± 3.3 ^{*** 1 * 2 * 2} Diclofenac 10 780 40.2 ± 4.8 65.6 ± 4.3 ^{*** 1} 60.1 ± 4.7 ^{*** 1} * = p <0.05, ^** = p <0.01, ^*** = p <0.001. 1 - compared to control; 2 - compared with diclofenac.

As can be seen from table 2, compound 1 showed a pronounced anti-inflammatory activity throughout the observation period, and at the peak of inflammation, it is practically not inferior to diclofenac. It should be noted that the effective dose of diclofenac is 0.013 from its LD ₅₀ , and the claimed compound has 0.008 from its LD ₅₀ , which indicates certain advantages of compound 1 over diclofenac in terms of its safety and effectiveness.

Thus, 2- hydroxy-4-oxo-4- (4-chlorophenyl) -2-butenoic acid N- (2-benzothiazolyl) amide (1) exhibits a higher antimicrobial and anti-inflammatory activity compared to the structural analogue and reference standards, and is practically non-toxic, which makes it possible to use it to create new drugs with antimicrobial and anti-inflammatory activity.

Literature

1. Andreichikov Yu.S., Milyutin A.V., Krylova I.V. et al. Synthesis and biological activity of heteroylamides of aroylpyruvic and 5-arylpyrazole-3-carboxylic acids // Chem. journal - 1990. - No. 7. - S.33-35.

2. Mashkovsky M.D. Medicines. - 15th ed., Revised., Rev. and add. - M .: New Wave Publishing House LLC, 2005. - P.170, 935-936.

3. Guidance on the experimental (preclinical) study of new pharmacological substances / Fisenko V.P. (ed.). - M .: IIA Remedium. - 2000. - S.264-273.

4. Guidelines for the experimental study of non-steroidal anti-inflammatory substances / Pharmacological Committee of the Ministry of Health of the USSR, protocol No. 22 of November 11, 1982. - Moscow, 1982. - 47 p.

5. Prozorovsky V. B., Prozorovskaya M. P., Demchenko V. M. Express method for determining the average effective dose and its errors. // Pharmacology and toxicology. - 1978. - No. 4. - S.497-502.

6. Izmerov I.F., Sanotsky I.V., Sidorov K.K. Toxicometry parameters of industrial poisons. - M .: Medicine, 1977. - S.196-197.

Claims (1)

N- (2-Benzothiazolyl) amide 2-hydroxy-4-oxo-4- (4-chlorophenyl) -2-butenoic acid of the formula

possessing antimicrobial and anti-inflammatory activity.