RU2638926C2 - Antileprotic of bacteriostatic and bactericide activity relating to m. lufu, representing o-benzoylaminobenzoic acid - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention is an o-benzoylaminobenzoic acid derivative of the following formula

,

which has a bacteriostatic and bactericidal activity against M. lufu.

EFFECT: drug is low-toxic and has the ability to suppress the reproduction of mycobacteria.

1 ex, 5 tbl

Description

The invention relates to the field of the chemical pharmaceutical industry and medicine, more specifically to new derivatives of o-benzoylaminobenzoic acid having antileprosy activity, and relates to compounds that can be used to create drugs that are active against Mycobacterium lufu. A method for the synthesis of a compound of formula I having an antileprosy effect is proposed.

State of the art

It is known that dapsone, ethionamide, as well as antibiotics rifampicin, clofazimine, ofloxacin, monocycline are used to treat leprosy [http://apps.who.int/medicinedocs/en/d/Jh2988e/].

WHO recommended treatment regimens for leprosy include the preparation of 4,4'-diaminodiphenylsulfone (Dapson), which we have chosen as a prototype. However, this drug has significant side effects, including methemoglobinemia, hemolytic anemia, fatal hypoplastic anemia. Long-term use of 4,4'-diaminodiphenyl sulfone leads to the appearance of resistant strains, which requires the search for highly effective and low-toxic drugs. Accordingly, the main disadvantages of this drug include high toxicity and the duration of the course of therapy.

Derivatives of dichlorormaleic acid are known to have anti-leprosy activity, however, the synthesis of these compounds is economically and preparatively inferior to the preparation of the claimed substance [A.S. 1131179 USSR, MKI A61K 31/505. Publ. 03/10/83].

As a result of this, the urgent task is to search for new compounds with pronounced anti-leprosy activity in combination with low toxicity.

Among the derivatives of anthranilic acid amides, many compounds have been discovered that have a wide spectrum of pharmacological activity and low toxicity. These include derivatives of o-benzoylaminobenzoic acid, which exhibit anti-inflammatory, actoprotective, antihypoxic activity and affect the central nervous system [Sochnev B.C., Kodonidi I.P., Oganesyan E.T., Kuleshova S.A. et al. Synthesis and anti-inflammatory activity of anthranilic acid amides with fragments of sulfanilamides and dapsone PMFI - a branch of the SBEI HPE Volgograd State Medical University of the Ministry of Health of Russia. - Pyatigorsk, 2013. - Issue. 68. - S. 339-341; Kodonidi I.P., Oganesyan E.T., Zhoglo E.N., Sochnev B.C., Ivchenko A.V., Manvelyan E.A., Sysa V.Yu. Purposeful synthesis of o-benzoylaminobenzoic acid amides as precursors of quinazolinones-4, having an effect on the central nervous system [New directions in the chemistry of heterocyclic compounds: proc. doc. 3 international scientific conf. - Stavropol, 2013 .-- S. 280-281; E.A. Manvelyan, V.Yu. Sysa., I.P. Codonidi, E.T. Hovhannisyan / Effect of amides of ortho-benzoylaminobenzoic acid, derivatives of quinazolinone-4 precursors, on the behavior of male rats in an “open field” / Science. Innovation Technologies. - Stavropol: Publishing house of SKFU, 2014. - No. 1. - S. 212-220].

The closest in structure to the claimed object are 2-benzoylamino-N- (4-sulfamoylphenyl) benzamides, which have a pronounced effect on the central nervous system.

These data indicate the promise of the search for new highly effective and low-toxic anti-leprosy substances in the series of derivatives of o-benzoylaminobenzoic acid amides.

The aim of the invention is the search for a low-toxic compound that has the ability to suppress the reproduction of mycobacteria, the synthesis of which is technologically simple, cost-effective and environmentally friendly. In this regard, we synthesized a compound related to amides of o-benzoylaminobenzoic acid, obtained by modifying the molecule of 4,4'-diaminodiphenylsulfone.

The method of obtaining compound I is based on the interaction of 2-phenylbenzoxazin-4-one with 4,4'-diaminodiphenylsulfone while boiling in a mixture of glacial acetic acid and dimethyl sulfoxide (Scheme 1).

SYNTHESIS EXAMPLE

N- [4- (4-aminobenzenesulfanyl) phenyl] -2-benzoylaminobenzamide (I)

A mixture of 2.23 g (0.01 mol) of 2-phenylbenzo [d] [1,3] oxazin-4-one and 2.48 g (0.01 mol) of 4,4'-diaminodiphenyl sulfone is boiled for 1 hour in 9 ml glacial acetic acid and 0.5 ml of dimethylformamide. It is cooled, the precipitate is filtered off, washed with diethyl ether. 3.90 g of N- [4- (4-aminobenzenesulfanyl) phenyl] -2-benzoylaminobenzamide are obtained, which is recrystallized from ethanol. Yield 83%. The substance is a white crystalline powder, odorless, insoluble in water, sparingly soluble in ethanol, insoluble in ether. T. pl. 249-250 ° C (from ethanol).

Compound I was identified using IR and ¹ H NMR spectroscopy.

Spectrum ¹ H NMR (300 MHz), δ, ppm, DMSO-d ₆ : 6.11 (s, 2H, NH ₂ ); 6.59-6.62 (d, 2H, ArH); 7.27-7.32 (t, 1H, ArH); 7.45-7.67 (m, 6H, ArH); 7.79-7.93 (m, 7H, ArH); 8.33-8.36 (t, 1H, ArH); 10.80 (s, 1H, NH); 11.33 (s, 1H, NH).

Molecular mass: 471.5. Gross formula: C ₂₆ H ₂₁ N ₃ O ₄ S

Elemental composition:

Found,%: C 66.29; H 4.45; N, 8.91; About 13.61; S 6.74.

Calculated,%: C 66.23; H 4.49; N, 8.94; O 13.56; S 6.78.

Assessment of pharmacological activity

The study of the ability of N- [4- (4-aminobenzenesulfanyl) phenyl] -2-benzoylaminobenzamide to inhibit the growth of mycobacteria was carried out in two stages.

The first stage was carried out in vitro against M.lufu [Irtuganova O.A., Urlyapova N.G. The use of M.lufu for the initial selection of anti-leprosy drugs. - In the book: Actual questions of leprology. Astrakhan, 1984, p. 147-150; SEYDEL, J.K., WEMPE, E.G. Bacterial Growth Kinetics of "M. lufu" in the Presence and Absence of Various Drugs Alone and in Combination. A Model for the Development of Combined Chemotherapy Against M. leprae? // Int. J. Lepr. - 1982. - V. 50, No. 1. - P. 20-30] by the method of serial dilutions [Navashin S. M., Fomin I. P. Handbook of antibiotics. - M .: Medicine, 1974. P. 54] on the environment of Shkolnikova.

The essence of the method of serial dilutions is to create sequential dilutions of a substance in a nutrient medium (in the order of geometric or arithmetic progression). In our experiments, the concentration of the studied compound in the series of serial dilutions decreased exponentially with a coefficient of 2: 128 μg / ml, 64 μg / ml, 32 μg / ml, 16 μg / ml, 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml. The control was tubes containing Shkolnikova’s medium without an inhibitor, as well as a series of serial dilutions of the comparison drug, dapsone.

A weighed portion of the compound (preparation) in 4 mg was dissolved in 0.5 ml of dimexide, then 4.5 ml of physiological saline was added there - a working solution was obtained, from which a series of test tubes with predetermined concentrations were formed by dilution.

For suspension of mycobacteria, we used a two-week culture of M.lufu, synchronized with cold (+ 4 ° C) for 72 hours. A suspension of mycobacteria of a certain density corresponding to the turbidity standard 5 according to McFarland, 0.2 ml was added to each tube of a number of consecutive dilutions of the studied substance, including control. Crops were incubated for 10 days at a temperature of + 31 ° C. After this period, the presence of growth in each of the tubes was visually assessed: “-” - complete transparency of the medium; "+" - weak growth; "++" - moderate growth; "+++" - intensive growth.

The first lowest concentration of the substance at which the turbidity of the medium was not visually determined, i.e. there was no bacterial growth, was considered the minimum inhibitory concentration (MIC).

Next, the contents of the tubes were centrifuged at 1500 rpm for 10 minutes. The supernatant was removed. From each tube, 0.05 ml of sediment was plated on Levenshtein-Jensen medium to determine the viability of M.lufu. After seeding for viability, smears were made from the remaining sediment, which were stained: according to the method of Murohashi (1959) [Small Medical Encyclopedia. - M .: Medicine, 1991-1996] to determine the ratio of “living” and “dead” bacteria, and according to Zil-Nielsen [Small Medical Encyclopedia. - M .: Medicine, 1991-1996] to determine the presence of acid-resistant (KUM) and non-acid-resistant forms (NKUM). Crops on Levenshtein-Jensen medium were incubated for 10 days at a temperature of + 31 ° C. Colonies grown on a school of dense medium were counted. The minimum bactericidal concentration (MBC) of the compound was considered such its concentration, after incubation with which no growth of the test culture colonies was observed on Levenshtein-Jensen medium.

The second stage of research was carried out in vivo on an experimental model of leprosy, proposed by Shepard S.S. [Shepard S.S. The experimental disease that follows the infection of human leprosy bacilli info footpads of mice // J. Exp. Med., 1960, V. 112. P. 445-458], which involves the implementation of intraplanar infection of mice with a suspension of mycobacteria prepared from leprosy or autopsy tissue of untreated patient with leprosy or from tissues of experimental mice previously infected with M. leprae from untreated patients, t .e. "From foot to foot."

Dapsone and Compound I were administered to animals with food. To ensure a constant supply of the drug to the body of the mice, a feed-drug mixture was prepared by mixing compound feed with the test substance. A special rotomixer “Rotomixer” (England) was used to prepare the feed-drug mixture. 100 mg of substance was added per 1 kg of dry feed, thus obtaining a mixture containing 0.01% of compound I (or dapsone).

The introduction of the drug with food is considered the most optimal, since this achieves a constant concentration in the blood of animals. The method allows to achieve adequate results during the study. Given that the mouse eats about 5 g of dry food per day, and this depends on its weight, which varies with age, this method of introducing the substance allows for strict control of the dose entering the body [Batrak G.E., Kudrin A.N. . Dosing of drugs to experimental animals. M.: Medicine, 1979, 167 p.]. The use of feed mixtures is recommended by WHO as a standard method of administering drugs tested for antileprosy activity. In this case, the maximum effective dose of 25 mg / kg (MED) was used [Levi L. Activity of derivatives and analogs of dapsone against Micobacterium leprae // Antimicrobial Agente Chemother., 1978, V. 14. P. 791-793], comparable in exposure with the dose used in the treatment of people with leprosy (100 mg per day).

The animals were infected with M. leprae VIII passage from the 6th M. (class No. 1693). 0.03 ml of suspension containing 10 ⁴ microbial bodies was injected intra-implantally into each mouse. Mice were divided into three groups: 1 - control (infected, not receiving specific treatment), 2 - comparison group (infected, treated with dapsone at a dose of 25 mg / kg), 3 - experimental (infected, treated with compound I at a dose of 25 mg / kg ) Dapsone and compound I began to be administered the day after infection. After 4, 9, and 11 months from the start of the experiment, 10 animals from each group were killed with chloroform anesthesia, and the right hind paw was cut off.

To count mycobacteria, a suspension was prepared. To do this, carefully cut the tissues of the pads of the cut off legs, crushed with scissors. The crushed tissues were ground in a porcelain mortar, adding 2.0 ml of distilled water. The resulting suspension was centrifuged for 3 min at 1000 rpm to precipitate coarse tissue particles, a smear was prepared from the supernatant to count mycobacteria. For the preparation of the smear, special slides were used with 1 cm ² circles deposited on them. In the middle of each circle was placed 10 μl of a 0.1% solution of albumin in distilled water, 10 μl of the test suspension of M. leprae was added. The fluids were mixed and evenly distributed on the surface of the circle so that the outer edge of the smear touched the inner edge of the circle circumference. The smear was dried in air and fixed over the flame of the burner, stained with Ziehl-Nielson.

The count of the number of mycobacteria was carried out according to the method of Shepard C.S., McRae D.H. [Shepard S.S. Drags against M. leprae in the mouse and in man // Int. J. Lepr., 1968, V. 36. P. 650].

A visual assessment of crops on Shkolnikova’s medium showed that, under the action of substance I, in a concentration range of 128–8 μg / ml, the medium remains transparent, which implies the practical absence of bacterial growth. With a decrease in its content in test tubes, the presence of turbidity was noted until complete loss of transparency of the medium (table 1), which was comparable with the effect of dapsone, the main anti-leprosy drug.

Primary (visual assessment) made it possible to determine the IPC of the studied compound. For substance I, it was 8 μg / ml, which was comparable with the performance of the comparison drug.

Analysis of smears stained by Ziehl-Nielsen showed that acid-resistant forms were absent in the dilution series of compound I in the concentration range 128–16 μg / ml, which was identical when analyzing the corresponding samples with dapsone. With a decrease in the concentration of active substances, the KUM content in the samples increased, but even with the largest dilution it did not exceed 40%.

The study of smears stained by Murohashi revealed a decrease in the content of living forms of mycobacteria. The fraction of non-viable bacteria decreased with increasing dilution both under the action of compound I and dapsone and at minimal concentrations did not exceed 50%, while in the control (without exposure) it did not exceed 8-10%.

Assessment of the presence and intensity of M. lufu growth on Levenshtein-Jensen medium showed its complete absence in test tubes after preliminary incubation of mycobacteria on Shkolnikova’s medium with substance I in the concentration range 128–8 μg / ml (table 2), i.e. MBC was 8 μg / ml. At lower concentrations, the growth of atypical colonies was observed (from weak to medium intensity, respectively). MBS of dapsone was significantly higher (Table 2): lack of growth was observed after incubation of M. lufu with dapsone in the range of 128-64 μg / ml, then single atypical colonies were noted, and with a decrease in concentration to 2 μg / ml, their intensive growth.

Thus, the patented derivative of o-benzoylaminobenzoic acid exhibits both bacteriostatic and bactericidal properties against M. lufu, a test culture for evaluating the anti-leprosy activity of substances. In terms of activity in in vitro studies, it is either comparable to dapsone (IPC), or somewhat exceeds it [Luzhnova SA, Gabitova NM, Voronkov AV, Kodonidi IP, Lovyagina S.A. Sochnev BC Evaluation of the antimycobacterial activity of new diazinone derivatives // Fundamental Research. - 2015. - No. 2. Part 11. - S. 2377-2380].

In vivo studies on an experimental Shepard leprosy model showed the following results (Table 3).

As can be seen from table 3, in control mice that did not receive treatment, the average number of mycobacteria in the paws after 4 months from the start of the experiment was almost three orders of magnitude higher than with the introduction. In the groups receiving dapsone and compound I, the inhibition of the mycobacterial reproduction process was observed: more than 15 times. The introduction of dapsone and compound I for nine months also contributed to the inhibition of the process of bacterial reproduction. A pronounced bacteriostatic effect was observed when substance I was used for 11 months: the “yield” of mycobacteria in the paws was 70 times lower than in the control and statistically significantly more effective than in the dapsone group (more than twice).

Thus, compound I in vivo has a pronounced bacteriostatic effect, not only comparable with the action of dapsone, but also with a longer course exceeding it.

Chronic toxicity

Given that dapsone has a number of negative effects that lead to a change in blood counts, a comparative study of the effect of the course of administration of compound I and dapsone on the hemogram and platelet counts was carried out.

The study was performed on 60 Wistar rats of both sexes, weighing 190-205 g (at the beginning of the experiment). Animals were kept on a standard diet with free access to food and water. After two-week quarantine, rats were divided into 6 groups of 10 animals each. Groups 1 (males) and 2 (females) were control and received an equivalent volume of distilled water at the rate of 2 ml per 200 g of animal weight. Animals 3 (males) and 4 (females) groups were administered dapsone, 5 (males) and 6 (females) - compound I. Substances were administered through a tube into the stomach 1 time per day in the morning for 30 days at a dose of 25 mg / kg (Corresponds to the effectiveness of 100 mg / kg per day for humans) [Luzhnova S.A., Abdresheva R.Zh. Correction by alpha-tocopherol of the structural and functional status of erythrocytes in old rats with dapsone administration // Fundamental Research. - 2013. - No. 10. - S. 344-348].

Blood sampling in rats was carried out from the hyoid vein, which was stabilized with heparin (50 IU / ml). Blood counts were determined using a system of veterinary automatic hematological analysis of BC 2800vet (Mindray).

All manipulations were performed according to the requirements for the humane treatment of animals [Guidelines for preclinical studies of drugs. Part One / Ed. A.N. Mironova. - M .: Grif and K, 2012].

Statistical processing of the results was carried out using the BIOSTAT 2009 software. The significance of differences was determined by t - student's criterion.

The results of the study showed that the administration of substance I for 30 days in male rats does not cause a change in the number of red blood cells and the level of hemoglobin of peripheral blood, while when using the comparison drug (dapsone), these indicators are statistically significantly reduced (table 4). The average hemoglobin content in the erythrocyte, the coefficient of variation remain identical to the control, the hematocrit level is comparable to the number of red blood cells and the same indicator in rats not exposed. In animals treated with dapsone, an increase in the average hemoglobin content is observed, which is consistent with a significant decrease in the number of red blood cells in the blood. The coefficient of variation of red blood cells is statistically significantly increased, which indicates a negative load on the blood system [M.S. Gavilanes, A.L. Palacio, P.R. Chellini, [et al]. Dapsone hypersensitivity syndrome in a lepromatous leprosy patient - A Case Report // Lepr.Rev. - 2015. - Vol. 86, No. 2. P. 186-190].

As you know, morphological and functional characteristics that ensure the integrity of red blood cells can change under the influence of a number of factors, including under the influence of drugs, which can lead to a decrease in their resistance. The breakdown of red blood cells is preceded by their spherulation and swelling, which characterizes such an indicator of blood as the average volume of the red blood cell. Under the action of compound I, after a 30-day course in male rats, a slight (3%), but statistically significant increase in the average volume of red blood cells is observed, apparently compensated, which is confirmed by a tendency to increase their number. In comparison, the same course of the drug dapsone increases the average volume of red blood cells by 35% (table 4) with a decrease in their number by 25%, which indicates the irreversibility of the hemolytic effect of dapsone [M.S. Gavilanes, A.L. Palacio, P.R. Chellini, [et al]. Dapsone hypersensitivity syndrome in a lepromatous leprosy patient - A Case Report // Lepr.Rev. - 2015. - Vol. 86, No. 2. P. 186-190].

In female rats after a course of administration of substance I, no significant changes in the parameters of red blood were revealed (table 4). Under the conditions of using dapsone in animals, a tendency toward a decrease in the number of red blood cells and hemoglobin was observed, and the average volume and coefficient of variation of red blood cells were statistically significantly higher than in the control group of females.

Along with erythrocytes, platelets are an important cellular element of internal hemostasis, which are not only components of the blood coagulation system, but also have an angiotrophic effect and participate, highlighting growth factors, in the regeneration of damaged tissues. It is known that some drugs can affect the quantitative and qualitative characteristics of platelets [Kozinets G.I., Vysotsky V.V., Pogorelov V.M., Erovichenkov A.A. Malov V.A. Blood and infection. - M .: Triad-farm, 2001. - S. 456]. A comparative analysis of the effect of the course of the introduction of substance I and dapsone on some platelet counts was carried out.

The introduction of dapsone for 30 days led to a change in the quantitative and qualitative characteristics of platelets in both males and females (table 5). In particular, in comparison with the control, rats of both sexes observed a statistically significant decrease in their number and a decrease in thrombocrit. At the same time, the average platelet volume and the breadth of their volume distribution (coefficient of variation) increased, which indicates a violation of the megakaryocytopoiesis process under the influence of dapsone.

Course introduction of the compound of substance I did not cause significant changes in platelet counts (table 5).

Thus, compound I has a specific antimycobacterial activity and, in contrast to the main anti-leprosy drug dapsone, does not have a toxic effect on the morphofunctional characteristics of red blood cells and megakaryocytopoiesis, which makes it promising for further development. [Evaluation of the effect of a new derivative of diazinon Pyatd1 on some indicators of peripheral blood of rats of both sexes / A.V. Voronkov, S.A. Luzhnova, S.A. Osychenko, N.M. Gabitova // Bulletin of the Volgograd State Medical University. - 2015. - No. 4 (56). - S. 124-126.]

Notes: “-” - full transparency of the environment; “+ -” - incomplete transparency of the environment; "+" - weak growth; "++" - moderate growth; "+++" - intensive growth.

Notes: “-” - absence of colonies; “+” - single colonies; “++” - <= 50%, “+++” - <= 75%; “++++” - <= 100% of the population of the jamb area; AK are atypical colonies.

Note: * - P _to <0.05; ** - P _to <0.01; *** - P _to <0.001 relative to the control; # - P _d <0.05 relative to dapsone.

Note: * - P _to <0.05; ** - P _to <0.02; ** - P _to <0.01; **** - P _to <0.001 relative to the control. #### - P _d <0.001 relative to the "dapsone".

Note: * - P _to <0.05; ** - P _to <0.02; ** - P _to <0.01; **** - P _to <0.001 relative to the control.

Claims (3)

An anti-leprosy agent, which is a derivative of o-benzoylaminobenzoic acid of the general formula I,

possessing bacteriostatic and bactericidal activity against M. lufu.