Derivatives of 5h-pyrano[2,3-d:6,5-d']dipyrimidine have antibacterial, antiviral and immunomodulatory effects

 

(57) Abstract:

The invention relates to new derivatives of 5H-pyrano[2,3-d:6,5-d']dipyrimidine General formula I possess anti-microbial, antiviral and immunomodulatory effects. In the compounds of General formula I the radicals have the following meanings: R1selected from the group of the hydroxy-group, mercaptopropyl, halogen, R2selected from the group of the hydroxy-group, the lowest alkoxygroup, halogen, R3selected from the group of hydrogen, phenyl or phenyl substituted by halogen or nitro-group. The preferred connection, where1=R2=OH, R3=4-O2NC6H4(I); R1= R2=Cl, R3=C6H5(II); R1=R2=HE, R3=4-IC6H4(III); R1=R2=HE, R3=N (IV); R1=OH, R2=CO3, R3=4-O2NC6H4(V); R1=R2=OH, R3=4-Cl6H4(VI); R1=R2=OH, R3=4-BrC6H4(VII); R1=SH, R2=OH, R3=4-ClC6H4(VIII); R1=SH, R2=OH, R3=4-O2NC6H4(IX). 9 C.p. f-crystals, 11 PL.

I

The invention relates to the field of organic chemistry and medicine, specifically to barbituric acids and their derivatives and prenuim action.

The level of technology.

Many pyrimidine derivatives are biologically active substances. Derivatives of pyrimidine are nucleic bases (uracil, thymine, cytosine), vitamins (thiamin, fosfotiamina), coenzymes (kokarboksilaza), their use as pharmaceutical drugs with hypnotic, anticonvulsant (barbiturates, primidone, benzonal), diuretic (Mercosul), anti-inflammatory (pentoksil, methyluracil), antithyroid (thiuragyl) action, they are synthetic analogues of vitamins (benfotiamine), anabolic (orotic acid), anti-inflammatory and antibacterial (sulfazin, sulfadimezin, sulfamonometoksin sulfadimetoksin, bactrim, drug), antimalarial (hloridin), cancer (Doan, fastened, etimicin, fluorouracil, Ftorafur, cytarabine) means [Pharmaceutical microbiology, Ed. by W. B. Hugo and A. D. Russel Blackwell Scientific Publications, Oxford, 1987, 511 p.; Mashkovsky M. D. Medicines. The textbook of pharmacotherapy for physicians. Vilnius, Gamma, 1993, 1 o'clock, 544 S.].

In recent decades, considerable interest in systems in which the pyrimidine cycle condensed with other heterocyclic compounds. Often such heterocera in the composition of natural and synthetic biologically active substances: nucleic acids (adenine, guanine), ATP, drugs that excite the Central nervous system, acting on the cardiovascular system (caffeine, theobromine, theophylline, regexen, diprophylline, ksantinola nicotinate) used for suppression of tissue incompatibility with organ transplants (azathioprine) (cytostatic and immunosuppressive effect), anabolic (inosine), antileykemichesky (mercaptopurine); pyrazolo[3,4-d] pyrimidines used for the treatment of diseases associated with hyperuricemia (allopurinol); pteridine used as diuretics (triamterene), vitamins (Riboflavin, folic acid), cancer (methotrexate) drugs; pyrimido [5,4-d]pyrimidines with vasodilator (dipyridamole) [Pharmaceutical microbiology, Ed. by W. B. Hugo and A. D. Russel Blackwell Scientific Publications, Oxford, 1987, 511 p.; Mashkovsky M. D. Medicines. The textbook of pharmacotherapy for physicians. Vilnius, Gamma, 1993, 1 o'clock, 544 S.].

Data on the biological activity of a variety of derivatives of 5-hildenborough acids are summarized in the review [Sans R. G., Chosas M. G. // Pharmazie, 1988, 43 Bd, N 12, p. 827-829] where noted anticonvulsant, antimicrobial, antispasmodic, antipyretic, antitumor activity of these substances. Also received on the 22, N 8, P. 418-422; Singh, S., Gupta G. P., K. Shanker // Indian J. Chem, 1985, Vol. 24 B, N 10, P 1094-1097; Singh, A., Misra V. S. //Pharmacol. Res., 1989, Vol. 21, N 1, P. 59-64; Kumar P., Nath, S., Agarwal J. C., Bhargava, K. P., K. Shanker // Indian J. Chem., 1983, Vol. 22B, N 9, p. 955-958; Japan patent, M. class. a 61 K 31/505, No 05213755 declared 07.02.1992 (92/56671), published 24.08.1993; Kumar A., Singh, S., Saxena A. K., Shanker, K. // Indian J. Chem., Sect. C., 1988, Vol. 27, N 5, P. 443-447; Hirota K., Fukazawa T., Isobe Y., Morita H., EPO application No. 546661, m CL 07 D 239/62 declared 09.10.1991 (91/290538), published 16.06.1993; Bars N. And., Zeev H. L., Ismailov, A., Biktimirov L., A. Ismailov, I., Urazmetova, K. // Chemistry of natural compounds. 1988, 5, S. 647-650], 5-aminomethyltransferase acid [A. Kreutzberger, E. Kreutzberger // Arch. Pharm., 1983, Bd 316, N. 1, S. 6-9], the products of the interaction of barbituric acids with isocyanates, isothiocyanates 5-arylcarbamoyl [Minatelli J. A., Brewer, A. D. , application Germany, m C 07 D 471/04, No 3446371 declared 19.12.1983 published 27.06.1985; Brewer A. D., U.S. Patent No 4920126, m CL a 61 K 31/505, NCI 514-274 declared 10.05.1988 published 24.04.1990; Kratt G., Salbeck G., W. Bonin, Duewel D., application Germany, m CL 07 D 239/62, No. 3903404 declared 06.02.1989 published 09.08.1990; De Sousa Century, Muntwyler R. , Schmidt W. , Ciba-Geigy A.-G., patent Switzerland 653840, 1986 (Chemical Abstracts, Vol. 106, 98105t)], pyrazolo[3,4-a]pyrimidine, obtained by condensation of 6-hydrazinolysis with ISO(thio)cyanate [Naka T., Nagaoka , A., Furukawa Y., EPO application No. 237289 (1987)], 5-deathflower// J. Heterocycl. Chem., 1992, Vol. 29, N 4, p. 763-765; J. Jiang B, D. Isaacson, U.S. patent, M. C 07 D 471/04, NCI 544-250, No. 4656274 declared 12.02.1985 published 07.04.1987], the condensation products of 6-aminouracil with nitrosobenzene - 10-alkyl(halogenarenes)-3-methylflavone [Cowden, W. B. , Clark I. A., Hunt, N. H. // J. Med. Chem., 1988, Vol. 31, N 4, p. 799-801; Cowden, W. B., Clark I. A., PCT application WO 88 04658, m CL C 07 D 474/14 declared 17.12.1986 (86/9548), published 30.06.1988] derived pyrrolo[2,3-d]pyrimidine [Quijano, M. L, M. Nogueras, Melguizo, M., Alvarez de Cienfuegos G. , M. Melgarejo, A. Sanches // Nucleosides &Nucleotides, 1989, Vol. 8, N 8, p 1519-1528], 7-methyl-5-hexyl-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H, 7H)-dione, obtained by the action of the reagent Vilsmeier to the corresponding 6-hydrazinolysis [Gauri K. K., Erbler N., Eltze M, the application of EPO, M C 07 D 487/04, No. 61381, stated 13. 06.1984 (81/2621), published 27.10.1982], pyrano[2,3-d]pyrimidine [Ahluwalia V. K., Batla R., Khurana, A., Kumar R. // Indian J. Chem., 1990, Vol. 29 B, N 12, P 1141-1142; Joshi K. C. , Jain R., Sharma K., Bhattacharya S. K., Goel R. K. // J. Indian Chem. Soc., 1988, Vol. 65, N 3, P. 202-204; Bars N. And, Kamaev F. G., Paisieva R. H., A. Ismailov, I. // Uzbek chemical journal. 1989, No. 3, S. 41-43], 5-(3-Nitrophenyl)-4-oxo-2-thioxo-1,3,7-triphenyl-1,2,3,4-tetrahydropyrido[2,3-d] pyrimidine [Das , A., Sahu, K., Mishra B. K., G. B. Behera// Indian J. Chem., 1985, Vol. 24 B, N 3, p. 310-311; K. Cooper, PCT application, WO 9012015, M C 07 D 519/00, declared 01.04.1989 published 18.10.1990, the priority of great Britain; Jiang J. B., U.S. patent, No. 4596805 (1986).28, N 7, P 1651-1655; Wood, H. C. S, R. Wrigglesworth Yeowell D. A., Gurney F. W., B. Hurlbert S. // J. Chem. Soc. Perkin Trans. 1. 1974, No. 11, P. 1225-1230], pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6N)-diones [Nagamatsu T. , Yamasaki N., Hirota T., Yamato, M., Kido Y., Shibata M., Yoneda F // Chem. Pharm. Bull., 1993, Vol. 41, N 2, p. 363-368], 5-dialkylaminomethyl [Motawia M. S., Joergensen, P. T., Larnkjaer A., Pedersen, E. B., Nielsen, S. // Monatsh. Chem., 1993, Bd 124, H. 1, S. 55-64; Ahluwalia, V. K. , Batla R. Khurana, A., Kumar R. // Indian J. Chem., 1990, Vol. 29 B, N 12, p 1141-1142], pyrimido[4,5-C]pyridazines [Billings C. K., Wagner J. A., Cook, F. D. , Castle, R. N. // J. Heterocycl. Chem., 1975, Vol. 12, N 6, P 1221-1224] . These compounds possess pesticidal, antitumor, antimicrobial, immunosuppressive, nootropic, antihypertension and antiallergic effect.

Known only to a few examples of education pyrano[2,3-d:6,5-d']dipyrimidine system: in the reaction of barbituric acid with 3-allegramente [F. Eiden, Fenner H. // Chemische Berichte, 1968, Bd 101, No. 8, S. 2894-2898; F. Eiden, Schikorr W. // Arch. Pharm., 1972, Bd 305, N 3, P. 187-193]. On their biological activity no data available. At the same time, the available information on the biological activity of 5-substituted barbituric acids and condensed systems containing a fragment of pyrimidinedione, as was shown above, have different biological activity. However, the effectiveness of many of the studied substances is not high enough, magiera is developed resistance to existing drugs, what makes them ineffective [K. M. Stone, Wittington W. L., Treatment of genital gerpes, Rev. of Infect. Dis., 1990, 12, Supl. 6, p. 610-619; R. Saltzman , R. Jurewicz, B. Boon, Safety of famciclovir in patients with herpes zoster and genital gerpes, Antimic. Agents and Chemother., 1994, 38, 10, R. 2454-2457; Gentry G. A., Lawrency N., Lushbaugh N. Isolation and differentiation of Herpis simplex virus and Trichomonas vaginalis in cell culture. J. of Clinical Microbiology, 1985, 22, 2, R. 199-204; Judson B. A., Lambert, P. P. Improved Syva Micro Trac Clamydia trachomatis direct test method. Journal of Clinical Microbiology, 1988, vol. 26, No. 12, p. 2657-2658; Fenelon L. E. , Mumtaz G. , Ridgway, G. L. The in vitro susceptibility of Chlamydia pneumoniae, Journal of Antimicrobial Chemotherapy, 1990, 26, p. 763-767; Boyd, M. R. The Future of new drug development. Current therapy in oncology 1992, 11-22; Grever, M. R., S. A. Schepartz, Chabner, B. A., The National Cancer Institute: cancer drug discovery and development program, Seminars in oncology, 1992, 19, 6, R. 622-638]. The above material shows the viability of scientific research in the field of synthesis of new effective biologically active substances by condensation of barbituric acids with carbonyl compounds, which leads, in particular, to derive pyrano[2,3-d:6,5-d']dipyrimidine.

The prototype of the invention, i.e. the substance, the closest to the chemical nature of the claimed agent selected 1,3,7,9-tetramethyl-5-(3-chromanol)-5H-pyrano[2,3-d: 6,5-d'] dipyrimidine-2,4,6,8(1H, 3H, 7H, N)-tetraen [see F. Eiden, Fenner H. // Chemische Berichte, 1968, Bd 101, No. 8, S. 2894-2898]. It is obtained by heating 1,3-dimethylbarbituric Asa sulfur. As was mentioned previously, the available sources of information information about its biological activity is not present.

The objective of the invention.

The objective of the invention is the creation of new substances with antimicrobial, antiviral and immunomodulatory effects.

The essence of the invention.

The problem is solved by the synthesis of new substances derived pyrano[2,3-d:6,5-d']dipyrimidine General formula

< / BR>
where R1selected from the group of the hydroxy-group, mercaptopropyl, halogen,

R2selected from the group of the hydroxy-group, the lowest alkoxygroup, halogen,

R3selected from the group of hydrogen, phenyl or phenyl substituted by halogen or nitro-group.

The best variants of the claimed substances in

R1=R2=HE, R3=4-O2N6H4(I)

R1=R2=Cl, R3=C6H5(II)

R1=R2=OH, R3=4-IC6H4(III)

R1=R2=OH, R3=N (IV)

R1=OH, R2=CO3, R3=4-O2N6H4(V)

R1=R2=OH, R3-4-ClC6H4(VI)

R1=R2=OH, R3-4-BrC6H4(VII)

R1=SH, R2=OH, R3=the duty to regulate immediately be noted, the use of other representatives of alkoxygroup, mercaptopropyl, halogen and arrow have no fundamental differences in the processes of synthesis and biological properties of the obtained compounds, i.e., are essential not specific elements of the radicals R1, R2and R3and their belonging to the mentioned General formula groups.

The best options are presented in table.1.

The General formula of the claimed compounds and all of the above options specific compounds are new, from the available information sources they are not known to us. Moreover, their synthesis and the presence of appreciable biological activity is not obvious from the current level of technology, i.e. for specialist obvious.

Disclosure of the invention.

The invention is illustrated below are five common examples of the synthesis of the claimed substances, three of the best known authors on the date of filing variants of synthesis, three PivotTables chemical-physical characteristics of the claimed substances, which are the outputs of the melting temperature, and the results of the eight series of experiments to determine the comparative biomedical with the HT synthesis of 5-aryl-2,4,6,8-tetrahydroxy-5H-pyrano[2,3-d: 6,5-d'] dipyrimidine (I, III, IV, VI, VII) and 5-aryl-4,6-dihydroxy-2,8-dimercapto-5H-pyrano[2,3-d:6,5-d']dipyrimidine (VIII, IX).

Example 2 is a variant of the synthesis of 5-aryl-2,4,6,8-tetrahydroxy-5H-pyrano-[2,3-d: 6,5-d']dipyrimidine (I, III, IV, VI, VII) and 5-aryl-4,6-dihydroxy-2,8-dimercapto-5H-pyrano[2,3-d:6,5-d']dipyrimidine (VIII, IX).

Example 3 - a variant of the synthesis of 2,4,6,8-tetrahydroxy-5-(p-nitrophenyl)-5H-pyrano[2,3-d:6,5-d']dipyrimidine (I).

Example 4 - a variant of the synthesis of 5H-phenyl-2,4,6,8-tetrachloro-5H-pyrano[2,3-d: 6,5-d']dipyrimidine (II).

Example 5 - a variant of the synthesis of 2,8-dihydroxy-4,6-dimethoxy-5-(4-nitrophenyl)-5H-pyrano[2,3-d:6,5-d']dipyrimidine (V).

Example 6 - best known authors variant of the synthesis of 4,6-dihydroxy-2,8-dimercapto-5-(p-nitrophenyl)-5H-pyrano[2,3-d:6,5-d']dipyrimidine (IX).

Example 7 - best known authors variant of the synthesis of 5-phenyl-2,4,6,8-tetrachloro-5H-pyrano[2,3-d:6,5-d']dipyrimidine (II).

Example 8 - best known authors variant of the synthesis of 2,8-dihydroxy-4,6-dimethoxy-5-(4-nitrophenyl)-5H-pyrano[2,3-d:6,5-d']dipyrimidine (V).

Table 2 - PMR spectra of solutions of the stated substances 5H-pyrano [2,3-d: 6,5-d']dipyrimidine - in DMSO-d6(, memorial plaques).

Table 3 - NMR spectra13With solutions of the stated substances 5H-pyrano[2,3-d: 6,5-d']dipyrimidine in DMSO-dIran[2,3-d:6,5-d']dipyrimidine.

Experiment 1 (table.5) - determining steps of the claimed substances on the herpes virus.

Experiment 2 (table.6) the determination of the interferon-inducing activity of the claimed compounds.

Experiment 3 (table.7) - define the steps of the claimed 25 substances on C. trachomatis.

Experiment 4 (table. 8) - determination of acute toxicity of the claimed substances.

Experiment 5 - determining steps of the claimed substances in gram-positive bacteria.

Experiment 6 - determining steps of the claimed substances on mycobacteria.

Experiment 7 (table.9 and 10) determination of antiviral activity of the claimed substances against influenza viruses a and B.

Experiment 8 (table.11) - determination of antiviral activity of the claimed substances for respiratory viruses.

Example 1 - variant of the synthesis of 5-aryl-2,4,6,8-tetrahydroxy-5H-pyrano[2,3-d: 6,5-d'] dipyrimidine (I, III, IV, VI, VII) and 5-aryl-4,6-dihydroxy-2,8 - dimercapto-5H-pyrano[2,3-d:6,5-d'] dipyrimidine (VIII, IX).

A mixture of 3 mmol of pyridinium salt of 5,5-arylidene(2-thio)barbituric acid and 55 mmol l3heated with stirring to boiling. After dissolution of sediment (~1 h) to the solution add 3 which constitute at night. The precipitation is filtered off, washed with ethanol or acetone, recrystallized from water. The output of 20-30%.

Example 2 is a variant of the synthesis of 5-aryl-2,4,6,8-tetrahydroxy-5H-pyrano[2,3-d: 6,5-d'] dipyrimidine (I, III, IV, VI, VII) and 5-aryl-4,6-dihydroxy-2,8-dimercapto-5H-pyrano[2,3-d:6,5-d']dipyrimidine (VIII, IX).

A mixture of 0.04 mol pyridinium salt of 5,5'-arylidene(2-thio)barbituric acid, 0.035 mol l3, 0.02 mol P2O5and 7-10 ml of chloroform is stirred at boiling for 4 hours Then the reaction mixture is poured into 100 ml of cold water and leave overnight. Isolation and purification of the obtained compounds is conducted as described above. Output 23-86%.

Example 3 - a variant of the synthesis of 2,4,6,8-tetrahydroxy-5-(p-nitrophenyl)-5H-pyrano[2,3-d:6,5-d']dipyrimidine (I).

To a mixture of 3.4 mmol 2,8-dihydroxy-4,6-dimethoxy-5-(p-nitrophenyl)-5H-pyrano[2,3-d: 6,5-d'] dipyrimidine, 10 ml of acetic acid and 10 ml acetic anhydride add 8-12 drops of concentrated sulfuric acid. The mixture is stirred at boiling for 8-10 h, and then poured into 150 ml of cold water and leave overnight. Isolation and purification of substances carried out as described above. The output of 25-30%.

Example 4 - a variant of the synthesis of 5H-phenyl-2,4,6,8-tetrachloro-5H-pyrano[2,3-d: 6,5-d'] dipyrimidine (II). the live with stirring to a boil. After dissolution of sediment (~1 h) to the solution was added 3.5 mmol P2O5. The boiling mixture is stirred for 4 h, and then poured into 150-200ml of cold water and leave overnight. The precipitation is filtered off, washed with ethanol or acetone. Output 10%.

Example 5 - a variant of the synthesis of 2,8-dihydroxy-4,6-dimethoxy-5-(4-nitrophenyl)-5H-pyrano[2,3-d:6,5-d']dipyrimidine (V).

A mixture of 14 mmol of 6-methoxymethyl, 7 mmol of p-nitrobenzaldehyde in 10 ml of acetic acid and 10 ml of acetic anhydride was stirred at 110-115oC for 3-5 hours the precipitation is filtered off, washed with ethanol and dried.

Examples of the best known authors variants of the synthesis of the inventive compounds of the formula (I).

Example 6 - the best synthesis of 4,6-dihydroxy-2,8-dimercapto-5-(p-nitrophenyl)-5H-pyrano[2,3-d:6,5-d']dipyrimidine (IX).

A mixture of 0.04 mol pyridinium salt of 5,5'-(p-nitrobenzylidene)bis(2-thiobarbiturate) acid, 0.035 mol RO3, 0.02 mol P2O5and 7-10 ml of chloroform is stirred at boiling for 4 hours Then the reaction mixture is poured into 100 ml of cold water and leave overnight. Isolation and purification of the obtained compounds is conducted as described above. Yield 86%.

Example 7 is the best option centreuniversity acid and 55 mmol l3heated with stirring to boiling. After dissolution of sediment (~1 h) to the solution was added 3.5 mmol P2O5. The boiling mixture is stirred for 4 h, and then poured into 150-200ml of cold water and leave overnight. The precipitation is filtered off, washed with ethanol or acetone. Output 10%.

Example 8 is the best variant of the synthesis of 2,8-dihydroxy-4,6-dimethoxy-5-(4-nitrophenyl)-5H-pyrano[2,3-d:6,5-d']dipyrimidine (V).

A mixture of 14 mmol of 6-methoxymethyl, 7 mmol of p-nitrobenzaldehyde in 10 ml of acetic acid and 10 ml of acetic anhydride was stirred at 110-115oC for 3-5 hours the precipitation is filtered off, washed with ethanol and dried. Yield 46%.

The experimental part.

Experiment 1 - determination of the validity of the claimed substances on the herpes virus.

Antiviral activity was studied in relation to herpes virus type I (HSV-I/Leningrad/248/88) by the conventional method [R. Saltzman, R. Jurewicz, Boon Century , Safety of famciclovir in patients with herpes zoster and genital gerpes, Antimic. Agents and Chemother, 1994, 38, 10, P. 2454-2457; Gentry G. A. , Lawrency N. , Lushbaugh N. Isolation and differentiation of Herpis simplex virus and Trichomonas vaginalis in cell culture. J. of. Of Clinical Microbiology, 1985, 22, 2, R. 199-204]. Viruses were grown on transplantable culture of Vero cells obtained from the cell Bank kulturminnen in the medium RPMI-1640 with 10% calf serum and placed in wells of 96-well plateau, added virus at a final concentration of 10 particles/ml and the claimed substances dissolved in DMSO, final concentration of 100, 10 and 1 mg/l For each tested concentration of the substance used 5 independent holes. Plateau were incubated for 60 min at 38oWith CO2-incubator. After incubation, the virus was removed and again made fresh medium containing the claimed substances used in concentrations. The results were evaluated according to the presence cytopathogenic action of the virus on the cells after 36 hours incubation at 38oWith CO2the incubator.

In the experiment we used the following controls:

1. The control cell culture (ability to normal growth).

2. The virus control (ability to reproduce).

3. Control antiviral activity the antiviral drug acyclovir.

4. Control substances (toxic compounds).

5. The control solvent (DMSO) toxicity.

To assess the cytopathic effect of the virus was calculated the number of unchanged cells in 100 fields formed a special grid eyepiece inverted microscope. The results in table.5.

The results obtained are shown in table.5, on the th of the standard drug acyclovir. The rest of the claimed compounds had less pronounced activity in the process of suppressing the reproduction of the herpes virus in the selected experimental conditions.

Experiment 2 - determination of the interferon-inducing activity of the claimed compounds.

The induction of the synthesis of interferons stated substances was carried out on primary cultures of human lymphocytes (data cells in the human body are the main producers of interferon). To obtain a culture of lymphocytes used fresh (12 hours after collection) blood of healthy donors (not the second group). For isolation of lymphocytes of heparinized blood obtained from a healthy donor were subjected to centrifugation in a density gradient ficoll-urografin 1,71 g/cm3to allocate fractions of immunocompetent cells. This fraction was taken and was diluted nutrient medium RPMI-1640 containing 5% fetal bovine serum, 0.3 mg/ml L-glutamine, 100 units /ml penicillin, 50 mg/ml streptomycin. The concentration of lymphocytes was considered after methylene blue staining and counting the number of cells in the cell Goryaeva. The original solutions of the inventive substances were dissolved nutrient medium RPMI-164 is itov. The final concentration of lymphocytes in the induction mixture was 3106cells/ml.

In parallel with experimental samples were stamped with the following controls:

1) control of spontaneous production of interferons (IFN) lymphocytes;

2) control of the process when exposed to a standardized inducer of IFN N-methyl-N-(a,D-glyukopiranozil)ammonium-10-methylene-carboxylate of acridone (cycloferon);

3) control of the process when exposed to a standardized inducer of IFN - Neovia with relevant content of DMSO in the test sample;

4) control of spontaneous production of interferon in the presence of DMSO in the test samples.

Control and test samples were incubated 24 hours at 37oC. After incubation, the samples were tsentrifugirovanie at 2000 g for deposition of cellular elements and samples were selected IFN-containing supernatant, which were analyzed on a quantitative content of IFN. Sediment cells resuspendable in the same volume of nutrient medium were stained with the vital dye Trifanova blue and counted the number of cells in the cell Goriaev (as described above) to determine the cytotoxic effects of substances.

Cooperantes test systems for IFN-a production LLP Protein loop" ProCon IF2 plus. To determine the amount of interferon in the sample used solid-phase immunofermentnyi method using horseradish peroxidase as indicator enzyme. The bound peroxidase activity was measured using an automatic microplate photometer for microprocessor at a wavelength of 450 nm.

For calculation of the parallel activities were determined IFN standard solutions of IFN containing a known amount of the substance. On the basis of the results obtained calibration curve was constructed, allowing the use of microprocessor automatic photometer to obtain data expressed in International Units of activity (ME). The results of the analysis are expressed in ME activity of IFN per ml in this induction system containing 3106lymphocytes/ml

Each experimental and control point was investigated in 4 Parallels.

Controls enzyme reactions:

1. Control DMSO with a nutrient medium.

2. Control system components (according to instructions). All results were taken into account only in accordance controls the passport system.

The obtained results were subjected to statistical analysis is lennyg experiments.

As a result of the research showed that all of the claimed substance is able to induce the synthesis DETAILS, quite comparable with the known interferon-inducing means, indicating their antiviral and antitumor efficacy (table.6).

Experiment 3 - determining steps of the inventive compounds on Chlamydia trachomatis.

Antimicrobial activity of the claimed compounds was studied against C. trachomatis D323 - standard strains from the collection of Department of Microbiology at Saint-Petersburg State University. Acad. I. P. Pavlova. This strain was isolated from a patient with chlamydial urethritis is the morphology and physiological activity typical representatives of this species, sensitive to the action of drugs used for the treatment of chlamydial infection.

Used in the work cell culture Msso and L929 obtained from the Institute of Cytology of the Russian Academy of Sciences.

Scheme of production experience.

Cells were grown in bottles of neutral glass in medium RPMI-1640 with the addition of 10% fetal calf serum. The experience was put in a glass (without toxicity) flat-bottomed vials with the RCTs were made standard infecting dose chlamydia stored frozen at -70oC. Simultaneously to the cells was added compound at a final concentration of 100 mg/L. the Sample was centrifuged at 2400 g for 60 minutes at room temperature and incubated at 37oC for 2 hours. After that changed nutrient medium for a new one, containing 5% fetal calf serum and cycloheximide (2 μg/ml) with re-making the claimed compounds at the same concentration. In parallel, duplicate samples, using a medium without cycloheximide to prevent its influence on the investigated substance. Samples were incubated for 48 hours in CO2the incubator.

Controls included: control of cell cultures, the control action of the solvents, the control actions of chlamydia in the absence of any drugs, the control sensitivity of chlamydia to standard antimicrobial ciprofloxacin and abaktal [Grever, M. R., S. A. Schepartz, Chabner, B. A., The National Cancer Institute: cancer drug discovery and development program, Seminars in oncology, 1992, 19, 6, R. 622-638], the control of the tested compounds on toxicity to the cell cultures.

Evaluation of the results was performed by identifying chlamydial cytoplasmic inclusions using the method of immunologic Road London NW1 7SX) (46). The effect of the drug was determined by analyzing the state of the monolayer and the number of cells with CAI compared with the control (culture of cells infected with C. trachomatis D323), took into account the number of unchanged cells in 100 fields of view obtained using a special grid eyepiece of the microscope. The results of the control samples that meet the requirements of the experiment: control culture of cells - morphology of cells and the state of the monolayer correspond to a given cell type, growth control of chlamydia in cell cultures is the presence of a CAI in the monolayer, the control action standard antimicrobial drug reduction in CAI in the monolayer compared with the previous control, the toxicity of the inventive compounds - toxicity is absent, the control action of solvents toxic effect on the cells is missing. The results of the tests are presented in table.7.

The data obtained indicate that the claimed compounds can be used for the treatment of diseases caused by chlamydia.

Experiment 4 - determination of acute toxicity.

Determination of acute toxicity carried out on outbred white mice weighing 18-20 g of the Emulsion of some declare the unity used in 5 animals. The drug was administered once daily by mouth or intraperitoneally. The observation period was 14 days. 1, 8 and 15 day weighing of animals in each group. For control used animals that were injected emulsion prepared without the tested compounds.

For macroscopic study of the internal organs were opening all animals that died during the experience and survived to the end of the experiment.

In the course of the research was not observed: weight loss, changes in behavior and appearance, as well as death. By necropsy macroscopic pathology of the internal organs of animals in experimental and control groups were found (PL.8).

The results obtained indicate that when taken through the mouth or intraperitoneally some of the claimed compounds at a concentration of 1500 mg/kg not acutely toxic to mice.

Experiment 5 - determining steps of the claimed compounds on gram-positive bacteria.

To assess the validity of the claimed compounds on gram-positive bacteria was determined by minimal inhibitory concentration (MIC) using the method of serial is emesto IX at a concentration of 100 μg/ml completely inhibits growth of the test microbes. Other compounds are less effective against gram-positive bacteria and have a large amount of the IPC.

Experiment 6 - determining steps of the claimed compounds in bacilli.

To assess antimycobacterial steps were used standard strains of Mycobacterium tuberculosis H37RW and Micobacterium smegmatis ATCC607. Minimal inhibitory concentration (MIC) was determined by serial dilution method on environment Shkolnikova, enriched inactivated serum. Substance IX has activity in relation to the used strains of Mycobacterium and inhibits their growth. For Mycobacterium tuberculosis H37RW IPC is 100 μg/ml, and for strain Micobacterium smegmatis ATCC607 - 50 µg/ml.

Other compounds are less effective against mycobacteria and have a large amount of the IPC.

Experiment 7 - determination of the antiviral activity of the claimed compounds against influenza viruses a and B.

Antiviral activity of substances to determine their ability to inhibit the reproduction of influenza viruses on the model experiencing fragments chorioallantoic membrane of chicken embryo (HAO) method Fazekas de St. Growth, modified to determine the inhibitory effect of imipramines (72 holes) by using a special composition. Fragments HAO was prepared from 10-11-day-old chick embryos after processing and removal of the embryo with the yolk SAC. Pieces HAO size 20-25 mm2were placed in the wells of polystyrene panels and covered with glass.

First, determine the toxic effect of the compounds for culture HAO. For wells with different dilutions of the substances on the environment contributed pieces HAO and incubated in a thermostat at 37oC, after 48 and 72 hours assesses the condition of the shell.

Maximum tolerated dose (MTD) believed half the concentration of a substance which has no cytotoxic effect, i.e. does not cause visual changes fragments HAO: violations of drawing blood vessel (losing vessels), the disappearance of the luster of the shell and its complete destruction.

Cited reference strains of influenza virus (A/Hong Kong/1/68(H3N2)) and type b (B/Hong Kong/5/72).

In every experience include positive control drug, rimantadine (Dupont) or mirasol (Virazole,; ICN Pharmaceuticals Inc).

The effectiveness of the substances was evaluated by the reduction of infectious virus activity in experience compared to control - the neutralization index (Ig ID50). Substance sitemapwill with chicken erythrocytes, which was added to the wells of the panel, after removing from them the fragments HAO.

Other compounds were less effective against the flu and had a lower index of neutralization.

Other compounds were less effective against the influenza virus In and had a lower index of neutralization.

Thus, the claimed substances protect cells from infection by influenza viruses a and B. the Most active compound VIII.

Experiment 8 - determination of antiviral activity of the claimed substances for respiratory viruses.

Used and respiratory syncytial virus (strain long). The virus is supported by passages in transplantable cells ner-2 (sowing dose 3105cells/ml). To test the toxicity of substances through 6 days of incubation in an incubator at 37oTo check the status of the cell monolayer and determine the maximum tolerated dose of the substance. Assessment of the condition of the cells was performed microscopic examination for the degree of nonspecific degeneration compared with control: the appearance of graininess in the cytoplasm and the thickening of the cell wall, altering the shape of the cells, the complete destruction of the monolayer. Protivovirusny the specific syncytium, characteristic for reproduction respiratory syncytial virus in heteroploid cultures, reaching maximum size at day 5-7. Antiviral activity of compounds was determined by the index of neutralization. Each experiment included a positive control drug virazole.

Thus, the substance VIII protects cells from infection with respiratory syncytial virus.

Other claimed compounds were less effective and had less neutralization index.

Industrial applicability.

Examples 1-8 practical synthesis and chemical-physical analysis of the claimed compounds are given in table.2-4, confirm the possibility of laboratory and industrial synthesis of all nine of the claimed compounds means mastered the modern pharmaceutical industry, as well as their clear identification of common methods of control.

A series of experiments to determine the biological characteristics in eight presents the experiments showed that the claimed compounds possess biological activity against various microorganisms, including chlamydia, herpes simplex virus, gram-positive bacteria, m is updated. The latter indicates the possibility of their use in the treatment of herpes, and other viral and some cancers.

Provides information to prove the achievement of the objectives of the invention: synthesized a new class of heterocyclic compounds with high biological activity, in particular antimicrobial, antiviral and immunomodulatory.

1. Derivatives of 5H-pyrano[2,3-d:6,5-d']dipyrimidine General formula

< / BR>
where R1selected from the group of the hydroxy-group, mercaptopropyl, halogen;

R2selected from the group of the hydroxy-group, the lowest alkoxygroup, halogen;

R3selected from the group of hydrogen, phenyl or phenyl substituted by halogen or nitro-group,

possessing antimicrobial, antiviral, and immunomodulatory actions.

2. Substance under item 1, wherein R1=R2=OH, R3=4-O2NC6H4(I).

3. Substance under item 1, wherein R1=R2=Cl, R3=C6H5(II).

4. Substance under item 1, wherein R1=R2=HE, R3=4-IC6H4(III).

5. Substance under item 1, wherein R1=R2=HE, R3=H (IVUB>H4(V).

7. Substance under item 1, wherein R1=R2=OH, R3=4-Cl6H4(VI).

8. Substance under item 1, wherein R1=R2=OH, R3=4-BrC6H4(VII).

9. Substance under item 1, wherein R1=SH, R2=HE, R3=4-ClC6H4(VIII).

10. Substance under item 1, wherein R1=SH, R2=HE, R3=4-O2NC6H4(IX).

 

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< / BR>
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< / BR>
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< / BR>
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