RU2136668C1 - N,n'-(sulfonyldi-1,4-phenylene)-bis-(n'',n''-dimethylform- -amidine)-1,2,3,4-tetrahydro-6-methyl-2,4-dioxo-5-pyrimidine sulfonate stimulating cellular metabolism and showing immunotropic and antibacterial activity - Google Patents

Abstract

FIELD: organic chemistry. SUBSTANCE: invention relates to new biologically active chemical compounds - derivatives of diaminodiphenyl-sulfone of the general formula (I)

Description

 The invention relates to a new biologically active chemical compound - 1,2,3,4-tetrahydro-6-methyl-2,4-dioxo-5-pyrimidine sulfonate N, N '- (sulfonyldi-1,4-phenylene) -bis- ( N '', N '' - dimethylformamidine, which belongs to the group of sulfonopyrimidines, among which there are known drugs such as diutsifon and dimotsipon (see M.A. Mashkovsky. Medicines. Moscow: Medicine, 1988, part 11, p. 387; N.M. Goloshchapov et al. Invention and rationalization in medicine. Republican Bulletin of Scientific Works. Moscow, 1979, pp. 129-130; N.M. Goloshchapov et al. The use of the drug Dimotsifon in the treatment of patients with leprosy, allergic dermatoses and herpetiform dermatitis Dühring. Methodical recommendations. Moscow, 1978).

и проявляют стимулирующее клеточный метаболизм (обмен клетки) действие, а также высокую иммунотропную и антимикобактериальную активность.The compounds according to the invention correspond to formula I

and exhibit a stimulating cellular metabolism (cell exchange) effect, as well as high immunotropic and antimycobacterial activity.

 The well-known structural analogues - diutsifon hydrate and diutsifon (see USSR author's certificate N 322325) have anti-leprosy activity (see USSR author's certificate N 459228).

 However, they do not have antituberculous activity. In addition, these substances are poorly or completely insoluble in water, and this reduces their bioavailability and, therefore, biological activity. In this regard, therapeutic doses of these drugs should be quite high. Poor solubility and low bioavailability of diuciphons does not allow them to flow through the cell membrane, where, as a rule, the causative agents of mycobacteriosis are located, and to participate in their life.

 The aim of the invention is to obtain a substance that is readily soluble in water and therefore not having the above disadvantages.

 The obtained goal is achieved by the compound of formula I, which is easily soluble in hot water and 0.25 - 0.5% novocaine solution and is able to influence the function of the cell, increasing the content of DNA, RNA and protein in it; it has a more pronounced anti-leprosy and immunotropic activity, and is also active against mycobacteria of leprosy, tuberculosis and atypical mycobacteria, as evidenced by a partial bactericidal action against tuberculosis mycobacteria at a concentration of 4 mg / ml and 128 μg / ml against M.lufu, atypical mycobacteria.

 The advantages of the claimed object in comparison with ducifon are illustrated in table 1 (table. 1 - 12 see at the end of the description).

 From the table it follows that the claimed object has advantages over ducifon: it is less toxic, active against mycobacteria leprosy, tuberculosis and conditionally pathogenic atypical mycobacteria, has a high immunotropic effect on microorganisms (stimulation index is 3 times higher than that of duciphone). In addition, he takes part in the metabolism of the cell, stimulates its function 1.5 - 2 times better than diutsifon. The last conclusion can be made on the basis of an increase in the weight of the organ, since it is known that its hypertrophy contributes to increased function. Given the high effect of leprosy, tuberculosis and other pathogens located intracellularly on mycobacteria, as well as successful clinical trials on HIV carriers (see the attached report), we can allow the proposed substance to participate in the metabolism of various pathogens inside the cell and inaccessible to other drugs whose penetration into the cell is prevented by the cell membrane.

 Compound I is prepared by heating equimolecular amounts of diaminodiphenyl sulfone and 6-methyl-5-uracil sulfochloride in dimethylformamide. Under these conditions, as might be expected, a sulfonamide derivative of DDS is not formed, but primarily diaminodiphenyl sulfone and dimethylformamide react [Can J. Chem., 1965, v. 43, N 9, p. 2640, US patent 3133078]. The resulting dimethylformamidine further forms a salt with 6-methyluracil-5-sulfonic acid.

 The toxicity of compound I was determined on outbred white mice weighing 18 to 20 g in an acute experiment using the Litchfield-Wilcoxon method.

 The invention is illustrated by the following examples.

 Example 1. N, N '- (Sulfonyldi-1,4-phenylene) -bis- (N``, N' '- dimethylformamidine) - 1,2,3,4-tetrahydro-6-methyl-2,4- dioxo-5-pyrimidine sulfonate.

In a flask containing 24.8 g (0.1 mol) of 4,4'-diaminodiphenylsulfone in 300 ml of dry dimethylformamide, 47.3 g (0.2 mol) of 6-methyluracil are sprinkled with stirring and at a temperature of 25-30 ^° C. -5-sulfonyl chloride. Stirred for 1 hour at a temperature of 50 - 53 ^o C, cooled and the precipitate was filtered off. The crude product is stirred for 2 hours at room temperature in 200 ml of chloroform, filtered in air. The yellow powder is boiled for one hour in 250 ml of alcohol, filtered hot, washed with 20 ml of alcohol on the filter. Dried in the air. Amorphous powder of greenish-yellow color, so pl. 245 - 247 ^o C. Easily soluble in hot water, soluble in DMF, DMSO, insoluble in ordinary organic solvents. UV spectrum (in water): λ _max 265 nm, log ε 3.72. 1 H-NMR spectrum in DMSO (ppm): (3H, uracil) 2.46 (s); (6H, NCH ₃ ) 3.35 (s); (6H, NCH ₃ ) 3.42 (s); (8H, phenyl) 7.4 (d), 7.95 (d); (2H, = CH) 8.3 (s).

 Example 2. The study of the effect of drug I on animal cell metabolism.

 Tests of the proposed drug for the exchange of animal cells were carried out in comparison with 6-methyluracil, diuciphon and diuciphon hydrate.

 Found,%: C 48.5; H 5.3; S 11.0; N, 14.5.

C ₂₃ H ₂₈ N ₆ S ₂ O ₇
Calculated,%: C 48.9; H 5.0; S 11.4; N, 14.9.

 Animals (white male rats) were divided into 4 groups of 12 rats each. The preparations were dissolved in distilled water and administered intraperitoneally at 100 mg / kg daily for 6 days. Animals were weighed at the beginning and at the end of the experiment. On the seventh day they were killed by decapitation, organs (kidneys and adrenal glands, spleen and liver) were isolated, weighed and pieces of tissue were taken for examination. RNA and DNA were determined according to the method of R.G. Tsanev and G.G. Markov (1960), protein - according to the method of Loury (1981).

 Animals and isolated organs were weighed before and after the experiment. The results of the studies are presented in tables 2 - 6.

 In the analysis of the data obtained, it is clearly seen that the test drug significantly contributes to an increase in the weight of the liver, kidneys, adrenal glands and spleen as compared with the control, 6-methyluracil and ducifon.

 At the same time, during histological tests of these organs, there are no pathological (cell edema, protein, fatty degeneration, etc.) changes in each organ.

 Thus, the function of the organ under the action of the studied drug increased on average one and a half times or more.

 When analyzing this table, an increase in the amount of protein in each organ is clearly observed when taking the study drug on average by more than 30%.

 The amount of DNA and RNA in the cells of these organs was investigated, since its activity and cell function (phagocytosis, the possibility of cell transformation when the need arises in the body, etc.) depends on their number in the cell.

 When analyzing the table, an increase in DNA is clearly traced when taking various drugs, but most of all when prescribing the studied drug, that is, drug D.

 From the analysis of this table it follows that the amount of RNA with the administration of the studied drug increased by more than 50%, which contributed to a sharp increase in the functional activity of the cell, which was confirmed by us in the following experiment.

 From experimental data, it follows that under the action of drug D, the amount of RNA in the cell increased by more than 50%, which naturally increased the functional activity of the cell, and indicates that diuciphon hydrochloride is actively involved in the exchange of lymphocytes. The antimycobacterial activity of drug D was studied on 3 types of mycobacteria: the causative agent of leprosy Micobacterium leproe, the causative agent of tuberculosis Micobacterium tuberculosis, the atypical mycobacterium, Micobacterium lufu, which is potentially pathogenic.

 Example 3. Determination of anti-leprosy activity

The anti-leprosy activity of drug D compared with duciphone and duciphone hydrate was studied in mice with CBA x C ₅₇ BI hybrids infected with laboratory strain K-1 of mycobacterium leprosy, taken in the amount of 5000 microbial bodies and introduced intraplantally according to the Shepard method.

 Under observation were 4 groups of mice, all were kept under identical conditions of vivarium, 20 mice per group.

 The first group is control: for 6 months, mice of this group received 0.5 ml of starch suspension through a probe 5 times a week. Experimental groups of animals received during the experiment the studied drugs at a dose of 50 μg in 0.5 ml of starch suspension 5 times a week. After 6 months, the animals were clogged with cervical displacement of the spine, organs were subjected to bacterioscopic examination, no pathological abnormalities characteristic of any group were found. During bacterioscopic examination of the site of infection, the paw was triturated in 2 ml of a 0.1% albumin solution, a smear was made from 0.01 ml of the suspension, stained with Ziehl-Nielsen, the number of mycobacteria of the leprosy was counted in 60 fields of view, and the number of mycobacteria per mouse in each group was determined . The research results are presented in table 7.

 As follows from the above data, drug D has a high bacteriostatic activity against leprosy mycobacteria, superior to the antimycobacterial activity of both diuciphon and diuciphon hydrate. In addition, it should be noted that in 50% of the animals in the group that received treatment with drug D, mycobacteria were not observed, which indicates its bactericidal properties.

 Example 4. Determination of anti-tuberculosis activity.

The anti-tuberculosis activity of the drug D in comparison with metacil, diuciphon and diuciphon hydrate was studied on mycobacteria of the Academia strain. Mycobacterium tuberculosis was cultured in Shkolnikova's liquid medium with 10% human plasma. In each tube containing 2 ml of medium with the test substances in different concentrations, 0.2 ml of suspension of tuberculosis mycobacteria prepared according to the bacterial standard of turbidity N 5 and diluted 10 times were inoculated. Incubated for 10 days at 37 ^o C. The minimum inhibitory concentration was determined by the absence of colonies of mycobacterium tuberculosis in a test tube with the lowest content of the test substance. For this purpose, they made a smear, stained it according to Tsil-Nielsen, looked at 7x8,100 magnification fields of view, determined morphological structures (“braids”), indicating the growth of mycobacterium tuberculosis, which was marked with a + sign. The results obtained are presented in table 8.

 Analysis of the data in the table shows that drug D has anti-tuberculosis activity, expressed to a greater extent than the activity of diuciphon hydrate.

 Example 5. The study of the effect of drug D on atypical mycobacteria.

The antimycobacterial activity of the studied drugs was studied on mycobacteria Micobacterium lufu. M.lufu grown on Levenshtein-Jensen medium is ground in a porcelain mortar and a suspension is prepared that meets the X turbidity standard. The suspension is diluted 1000 times and 0.2 ml is added to test tubes with 2 ml solutions of the studied preparations grown in concentrations from 128 μg / ml to 0.25 μg / ml. Crops are kept at 34 ^o C for 10 days, then centrifuged for 5 minutes at 1500 rpm. The supernatant merges over the edge: the precipitate is applied to the glass, evenly smeared, fixed, painted according to Murakhashi.

 The glasses are viewed at a magnification of 7 x 8. The result is expressed in the MIC (minimum inhibitory concentration) of the preparation, in which the number of green (live) colonies is 2 times less than red (dead) colonies than in the control.

 The MIC of the studied drug D is less than the MIC of the comparison drugs, and in the highest studied concentration, the drug D exhibits a bactericidal effect against M.lufu.

 Thus, we can conclude that the proposed drug D has an antimycobacterial effect, both on pathogenic mycobacteria that are causative agents of socially dangerous diseases such as leprosy and tuberculosis, and on atypical mycobacteria, which often under the influence of environmental factors transform into conditionally pathogenic and pathogenic strains.

 Example 6. Determination of immunotropic activity.

a) The effect on humoral immunity was studied using the method of local hemolysis in the gel (reaction of Erne and Nordin). Hybrid mice (CBAxC ₅₇ BI) were immunized with ram erythrocytes at a dose of 5 × 10 ⁶ and immediately after that the studied drugs were administered orally or intraperitoneally. On the 5th day, the number of antibody-forming cells (AOKs) in the spleen of mice was counted and the stimulation index was determined relative to the number of AOKs in the control group. The data are presented in tables 10 and 11.

 Conclusion: pure diuciphone has a weak stimulating effect, and drug D increases the stimulation index to 3.8 and 4.8 with various methods of administration, and diuciphon hydrate is almost 2 times less effective and its immunostimulating activity is 2.2 and 2.6.

 b) The influence of the test drugs on the phagocytic activity of macrophages was studied by carcass clearance from blood taken from the retroorbital sinus in mice that received the drugs intraperitoneally (ip) and orally (p / o). The results were evaluated by the phagocytic index (table 12).

 Conclusion: the phagocytic activity of macrophages under the action of drug D grows by more than 1.5 times, and when taking diuciphon hydrate by 12 - 17% and almost does not change with the introduction of pure diuciphon. Perhaps this is due to the almost complete insolubility of pure diuciphone in water.

 c) The effect of diuciphon, diuciphon hydrate and drug D on cellular immunity was studied in the reaction of blast transformation of lymphocytes under the influence of T-cell mitogens.

The method is based on the fact that cultivating an animal’s in vitro lymphocytes in the presence of mitogens - phytohemagglutinin (PHA) and concanavalin (K _ohm A), they can be transformed into blasts and division. The more blasts are formed during the counting, the more immunomodulatory activity the drug has. The stimulation index is calculated by the ratio of the number of blasts in the experimental group to the number of blasts in the control group.

 As a result, it was concluded that all the studied drugs stimulate the proliferation of lymphocytes (blast formation), but the highest stimulation index for the preparation D. IS = 23.5, then for the diuciphon hydrate, IS = 20.2 and the lowest for diuciphone.

Claims (1)

N, N '- (Sulfonyldi-1,4-phenylene) bis (N``, N''- dimethylformamidine) 1,2,3,4-tetrahydro-6-methyl-2,4-dioxo-5-pyrimidinesulfonate

stimulating cellular metabolism and having immunotropic and antimycobacterial activity.

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