RU2582236C1 - 4,4,4-trichlor-1-(4-chlorophenyl)butane-1,3-dione possessing analgesic and antimicrobial activities - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to a new compound - 4,4,4-trichlor-1-(4-chlorophenyl)butane-1,3-dione of formula

(1).

EFFECT: compound possesses antimicrobial and analgesic activities.

2 tbl, 4 ex

Description

The invention relates to the field of organic chemistry, to new biologically active substances of the class 1-substituted 4,4,4-trichlorobutane-1,3-diones, namely to 4,4,4-trichloro-1- (4-chlorophenyl) butane- 1,3-dione (1), the formula:

possessing antimicrobial and analgesic activities, which suggests its use in medicine as a medicine.

An analogue in structure of the claimed compound is 4,4,4-trichloro-1- (2-thienoyl) -butane-1,3-dione (2), exhibiting fungicidal activity [US Patent US 3636214 A, 19720118 (1972)] of the formula:

Sodium metamizole was taken as ethanol for the comparison of analgesic activity [M.D. Mashkovsky, Medicines. - M.: New Wave, 2010 - p. 164]. Phenyl salicylate and nystatin were taken as ethanol for comparison of antimicrobial activity [M.D. Mashkovsky, Medicines. - M.: New Wave, 2010 - p. 950, 913].

The objective of the invention is to search for a series of 1-substituted 4,4,4-trichlorobutane-1,3-dione substances with a pronounced analgesic effect, low toxicity and antimicrobial activity

The problem is achieved by obtaining 4,4,4-trichloro-1- (4-chlorophenyl) butane-1,3-dione, which has analgesic and antimicrobial activities.

The claimed compound (1) is synthesized by the interaction of 1,1-dimethoxy-1- (4-chlorophenyl) propanas with trichloroacetic acid chloride in absolute chloroform, followed by isolation of the target product by known methods according to the scheme.

The technical result is the expansion of the arsenal of means of influence on a living organism.

The invention is illustrated by the following examples:

Example 1. Obtaining compound (1). To a solution of 0.13 mol of 1,1-dimethoxy-1- (4-chlorophenyl) propane and 0.26 mol of pyridine in 40 ml of absolute chloroform cooled to 0 ° C, a solution of 0.26 mol of trichloroacetic acid chloride in 30 ml is added with stirring absolute chloroform. The reaction mixture was left under stirring for 10-12 hours. After time, 2M sulfuric acid was added and stirred for 2 hours at 80 ° C. The resulting mixture was separated on a separatory funnel, the organic layer was dried with sodium sulfate. After removal of solvent, the precipitated product was recrystallized from hexane. Yield (86%). T. pl. 69-71 ° C. (IR spectrum (FSM 1202, liquid paraffin, ν, cm ^-1 ): 1646, 1595, 1583, 1558 (O = CC = C). ¹ H NMR spectrum, (Varian Mercury 300 (300 MHz), DMSO-d ₆ , HMDS, δ, ppm): 6.76 s (1H, = CH), 7.45 d (2H, Ar), 7.86 d (2H, Ar), 14.19 br s (1H, OH). Found,%: C 40.10, H 2.00. C ₁₀ H ₆ Cl ₄ O _2. Calculated,%: C 82.04, H 2.02.

The resulting compound (1) is a pale yellow crystalline substance, soluble in chloroform, acetone, insoluble in water.

Example 2. Acute toxicity of the compound (1). Acute toxicity (LD ₅₀ , mg / ml) of compound (1) was studied on white mice (females) weighing 16-18 g with a single intraperitoneal injection. The animals were monitored for 10 days, recording the behavior, intensity and nature of motor activity, the presence and nature of seizures, motor coordination, muscle tone, reactions to tactile, sound and light stimuli, the frequency and depth of respiratory movements, heart rate, state hair and skin, the color of visible mucous membranes, water and food intake, weight change.

Acute toxicity was calculated, following the recommendations of the state pharmacological committee for the study of the general toxic effects of biologically active substances [Methodological recommendations for the study of the general toxic effects of pharmacological agents. Approved 25.12.97 / Vestn. Pharmacopoeia. committee. - 1998. - No. 1. - C. 27-32.]

Example 3. Analgesic activity of the compound (1). The analgesic activity of compound (1) was studied on outbred mice weighing 18-22 g using the "hot plate" thermal stimulation method [Radell Z.O., Selitto J.J. A method for measurement of analgesic activity on inflamed tissue. // Arch. Intermat. Pharmacodun Etther. 1957. - Vol. 11. - No. 4 - S. 409-419].

The test compound was administered intraperitoneally in the form of 2% starch mucus at a dose of 50 mg / kg 0.5 h before the animals were placed on a metal plate heated to 53.5 ° C. In intact animals, the latent period of the defensive reflex does not exceed 10 seconds. An indicator of pain sensitivity was the duration of the animal’s stay on the hot plate until the hind legs were licked, measured in seconds. Metamizole sodium (analgin) was used as a standard preparation [M.D. Mashkovsky, Medicines - M .: New Wave, 2010 - p. 164]. The results of the experiment are presented in table 1

As can be seen from the table, the claimed compound increases the time of the defensive reflex by 21.67 ± 1.57 s, which is higher than that of the comparison drug metamizole sodium (16.33 ± 3.02 s). It should be borne in mind that the test dose of metamizole sodium (93 mg / kg) is almost 2 times higher than the dose of compound (I) (50 mg / kg). According to the literature data, metamizole sodium is less toxic compared to the proposed compound (LD ₅₀ = 3300 mg / ml). Compound (1) has a value of> 1500 mg / ml for this value.

According to the classification of K.K. Sidorova, compound (1) is of low toxicity [Izmerov N.F., Sanotsky IV, Sidorov K.K. Toxicometry parameters of industrial poisons. - M.Meditsina, 1977 .-- p. 196], which makes it promising as a drug.

Example 4. Antimicrobial activity of the compound (1).

The determination of bacteriostatic, bactericidal and fungicidal activity was carried out by the method of double 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, MIC (minimal inhibitory concentrations) and MBC (minimal bactericidal concentration) were determined. The antimicrobial properties of the claimed compound (1) were studied on 9 museum opportunistic strains of microorganisms: Staphylococcus (St.) aureus, 906, St. epidermidis 52186, St. saprophyticusATCC 15305, Streptococcusfaecalis, 29212, Bacillussubtilis, 6633, Bacilluscereusvaranthracoides, 96, Escherichiacoli, 1257, Pseudomonasaeruginosa, ATCC27853, Salmonellaenteriditis, Medical Research Institute of the Russian Federation microorganisms: St. aureus, No. 154, St. haemolyticus, No. 156, St. cohnii, No. 160. The antifungal effect was studied on the museum strain of Candida (C.) albicans, ATCC 24433, obtained at the Scientific Center for Expertise of Medical Applied FGBI of the Ministry of Health and Social Development of Russia and 4 clinical yeast fungi - C. albicans, No. 23, C. parapsilosis, No. 19 and C. krusei, No. 16.

Bacteria were seeded in fish-peptone broth (pH 7.2-7.4), yeast inoculations were sown in Saburo broth with different concentrations of test compounds. The cultures were grown in test tubes on a beveled agar medium (meat peptone agar and Saburo). 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. Then, 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 2.5 × 10 ⁵ mt / ml. The studied compound in an amount of 0.05 g was dissolved in 5 ml of dimethyl sulfoxide; 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 of control and experimental tubes were taken into account after 18-20 hours (inhibitory effect) and 7 days (bactericidal action) of incubation at a temperature of 37 ± 1 ° C for bacteria, for a Candida yeast - 48 hours culture and 7 days. The current dose was taken as the minimum inhibitory concentration (MIC) of the drug in μg / ml, which inhibits the growth of bacterial and yeast cultures. In the case of opacity of the studied solutions of the compounds, compulsory seeding was carried out on a solid, beveled nutrient medium. The maximum tested concentration corresponded to 1000 μg / ml. Antimicrobial (inhibitory, bactericidal, fungicidal) activity was evaluated by the minimum effective concentration.

The reference standard was phenyl salicylate, an antimicrobial agent and nystatin antifungal agent known in medical practice.

The research results on the claimed compound (1) are set forth in table 2.

Studies of the claimed compounds were studied on 4 groups of microorganisms: gram-negative bacteria, gram-positive bacteria, gram-positive bacteria forming endospores, and yeast fungi (table 1).

Gram-positive bacteria

The effect of the claimed compounds on 4 types of museum strains and 3 hospital cultures was investigated.

Analysis of the obtained data showed that the proposed compound (1) exhibits a pronounced inhibitory and bactericidal effect on both museum and hospital strains.

The claimed compound (1) inhibits the growth of museum strains of Streptococcusfaecalis, St. aureus and hospital culture aureus, 154 at a concentration of 7.8 μg / ml. Bactericidal properties in relation to the above microorganisms were detected at a concentration of 15.6 μg / ml, which is 96 times (inhibitory properties) and 128 times (bactericidal properties) more active than the standard for the pharmacological action of phenyl salicylate.

The impact of the claimed compounds (1) on museum strains of St. epidermidis and St. saprophyticus was observed at a concentration of 3.9 μg / ml and 7.8 μg / ml, respectively, i.e. 256 and 128 times more active than the analogue in pharmacological action. Bactericidal effect on St. epidermidis achieved at a concentration of 15.6 μg / ml, the death of St. saprophyticus was detected at a concentration of 31.2 μg / ml. Compared with the standard, the proposed compound exceeds the bactericidal effect by 128 and 64 times, respectively.

The inventive compound (1) has an inhibitory effect at a concentration of 3.9 μg / ml in relation to the clinical strain St. cohnii, the bactericidal effect comes from exposure to a concentration of 7.8 μg / ml. In relation to the hospital strain of St. Haemolyticus inhibitory effect occurs at a concentration of 7.8 μg / ml, a concentration of 62.5 μg / ml has a detrimental effect on the culture. The analogue in pharmacological action does not possess antimicrobial properties in relation to these microorganisms.

Gram-positive bacteria forming endospores

The effect of the claimed compounds on the 2nd museum culture was investigated. From the obtained data it follows that the compound has inhibitory properties that cause inhibition of culture growth at a concentration of 7.8 μg / ml against Bacillussubtilis and at a concentration of 31.2 μg / ml against Bacilluscereus. The bactericidal effect was achieved at a concentration of 250.0 μg / ml in relation to both cultures. The comparison drug phenyl salicylate does not show antimicrobial activity against Bacillussubtilis and Bacilluscereus.

Gram-negative bacteria

As can be seen from table 1, the effect of the claimed compounds on museum strains is less active. Antimicrobial properties against Escherichiacoli were manifested in the following concentrations: inhibitory effect at a concentration of 250.0 μg / ml, bactericidal - 500.0 μg / ml. The growth of Salmonellaenteriditis culture is inhibited at a concentration of 125.0 μg / ml, death occurs from a concentration of a substance of 250.0 μg / ml. The proposed compound in tested concentrations does not possess antimicrobial activity against Pseudomonasaeruginosa. The reference pharmacological reference phenyl salicylate is not active against gram-negative bacteria.

Yeast

The data in table 2 indicate that the claimed compound has high activity against the museum strain of C. albicans. Inhibitory activity was detected at a concentration of 1.5 μg / ml, the bactericidal concentration is 2.0 μg / ml. Compared with the antifungal agent nystatin, the claimed compound is more active 166 (inhibitory effect) and 250 (fungicidal effect) times, respectively. The prototype in the structure of formula (2) does not possess these properties.

An advantage of the proposed compound is its activity against clinical yeast fungi (C. albicans, 23, C. parapsilosisn C. krusei). The concentration of 3.9 μg / ml of the claimed compound has an inhibitory effect on the culture of C. parapsilosis, the death of the fungus was noted when exposed to a concentration of 7.8 μg / ml.

Growth inhibition of C. albicans, 23 and C. krusei occurs at a concentration of 15.6 μg / ml, and the fungicidal effect was achieved at a concentration of 31.2 μg / ml in relation to both cultures. The antifungal effects of nystatin comparisons have not been studied in clinical cultures.

Due to the fact that the compound of formula (1) has pronounced antimicrobial and antifungal properties against a wide range of both museum and hospital strains of microorganisms, they can find application in practical medicine.

Claims (1)

4,4,4-trichloro-1- (4-chlorophenyl) butane-1,3-dione (1), of the formula:

characterized in that it has antimicrobial and analgesic activities.