DE2136233A1 - Virustatic tetrahydro-benzthiazoles - active against both rna and dna viruses - Google Patents

Abstract

Pharmaceutical prepns with virustatic activity contain as active substance a title cpd of formula (I): (where R is -NH2, -NH-CX-NH-R1 or -NR2-CX-NH-R3 in which X is O or S, R1 is not >4 C alkyl or alkenyl or chlorophenyl, R2 is not >4C alkyl and R3 is not 4C alkyl, phenyl or chlorophenyl). The specification gives results of cell culture tests and animal tests (influenza in mice, parainfluenza in hamsters) demonstrating the activity of (I) against various RNA and DNA viruses.

Description

Pharmaceutical Preparations Containing Tetrahydrobenzthiazole Derivatives The invention relates to pharmaceutical preparations, in particular virustatics, characterized in that they are a tetrahydrobenzthiazole derivative of the general formula wherein R is NH2, NH-CX-NH-R1 or and X is oxygen or sulfur R1 is alkyl or alkenyl with up to 4 carbon atoms or chlorophenyl R2 is alkyl with up to 4 carbon atoms and R3 is alkyl with up to 4 carbon atoms1 phenyl or chloropheyl, contain as active ingredient.

The virustatics according to the invention are distinguished by their effectiveness known antiviral substances are particularly advantageous in that they both act against both RNA and DNA viruses. The substances known as virustatics 5-iodine-2'-deoxyuridine and N-methyl-isatin-ßthiosemicarbazone only work against DNA viruses, the well-known aminoadamantane only on RNA viruses. The broad spectrum effect of the invention Virustatica against RNA and DNA viruses of different virus groups is therefore surprising and was not foreseen.

That these preparations are antiviral substances acts with virustatic effectiveness, results from the proven effectiveness in vitro and in vivo.

The investigation and evaluation of the virustatic effect took place according to the following methods: 1. Cell culture tests In cell cultures, the virustatic Effectiveness of the substances in comparative infection titer determinations in the test tube, tested in plaque inhibition and plaque reduction tests. The evaluation of the tests was carried out in comparison to virus-free and substance-free cell controls and substance-free Virus Control 4 For the evaluation of the tube tests, the Tables based on the Kärber method (Kärber, G .: Naunyn-Schmiedeberg's Arch. exp. Path. Pharmac. 162, 480, 1931, calculated by R.J. Lorenz, Federal Research Institute for virus diseases in animals, Tübbingen 1960) used during the plaque test results according to Lorenz were calculated (Lorenz, R.J .: To the statistics of the plaque test. Arch. Virusforschg. 12, 108-137, 1963).

The probability of error is less than 5.

a) Comparative infection titer determinations in the test tube Each Test tube contains 1 ml maintenance medium with 100 pg test substance. After a two hour Exposure time is added 1 ml of virus agitation. Each virus dilution level is filled with 6 test tubes, the test is read after two days of incubation at 360 ° C.

The mean infection dose (dim) of the substance series is calculated as well as the substance-free control.

b) Plaque inhibition test After 24 hours, adult cell cultures in Plastic dishes are inoculated with 0.5 ml of an appropriate viral dilution and after an incubation time of 2 hours at 360C in a CO2 incubator with a Ion agar layer covered. After the agar has solidified, a test disc with a diameter of 0.6 cm and a test substance content of 33 mg. The test plates are after a two-day incubation at 360C with a second layer of agar, which is used for Staining of the still intact cells neutral red or containing tetrazolium chloride is overlaid. After another overnight incubation, the plates are evaluated. The virus effectiveness of a substance shows up as a ring-shaped plaque inhibition around the substance-containing one Test papers.

c) Plaque Reduction Test Cell cultures grown out after 24 hours in plastic dishes are inoculated with 0.5 ml of a suitable virus dilution and after a two-hour incubation period at 360C in a CO2 incubator with 5 ml of ion agar covered, which contains between 100 and 300 g 'test substance per ml.

After a further two days of incubation, 4 ml of ion agar is overlaid, which contains neutral red or tetrazolium chloride to stain intact cells. After another overnight incubation, the plaques formed by virus become counted. The viral effectiveness of a substance is shown in its ability to To lower the number of plaques compared to the substance-free virus control.

2. Animal experiments All influenza tests were carried out in mice weighing 13-15 g of the NMRI strain carried out the parainfluenza-3 tests in Syrian hamsters with a weight between 40 and 50 g.

a) Influenzater search in the mouse Each mouse receives at least 4 consecutive days in a single dose of 5 mg test substance (# 350 mg / kg / Day) per os. Two hours after the first administration of the substance, the mouse becomes lighter Ethereal anesthesia infected with # 5 dim intranasally. The death rates are recorded within 10 days.

Each substance group consists of 10 mice, the virus control consists of 30 mice.

b) Parainfluenza-3-Veruche in Hamster Each hamster receives 4 consecutive Days in a single dose of 15 mg test substance (# 300 mg / kg / day) orally. One hour After the first administration of the substance, the hamster is placed under light ether anesthesia with 0.1 ml infected intranasally with a suitable dilution of the virus.

On the 5th day of the experiment, the liamsters are bled after ether anesthesia, the lungs are removed and the virus content of the lungs is checked in a plaque test. Effectiveness of the TE substance indicates a significantly reduced Virmegontin the hamster lung.

The results of the investigations are compiled in the table below. In the table, a + symbol means that the tested preparations are in the tested Dose are effective.

The following test viruses were used: I - Influenza A Mannheim / 57; RNA type 2 II - parainfluenza 3; RNA Type III - Vesicular Stomatitis Indiana; RNA type IV - herpes simplex - CAM 13; DNA type V - Vaccinia - Ankara; DNA type VI - Influenza A2 - Japan / 305/57; RNA type VII - Influenza A2 - Hong Kong / 50/68; RNA type VIII - parainfluenza 3; RNA type table Cell culture tests on animals Virus preparation: I II III IV V virus: VI VII VIII Cell primary permanent primary chick animal: mouse mouse hamster culture: chicken human embryonic cell embryonic cell strain (HEZ) cell (HEZ) (HeLa) N-allyl-N'- [2- (4,5,6,7- tetrahydro) - + + + + + + benzthiazolyl] - urea N-methyl-N'- methyl-N '- [2- (4,5,6,7-tet- + + + + rahydro) -benz- thiazolyl] - urea N-methyl-N'- n-butyl-N'- [2- (4,5,6,7- + + + + + tetrahydro) - benzthiazolyl] - urea Cell culture tests on animals Virus preparation: I II III IV V virus: VI VII VIII Cell primary permanent primary chick animal: mouse mouse hamster culture: chicken human embryonic cell embryonic cell strain (HEZ) cell (HEZ) (HeLa) N-methyl-N'- [2- (4,5,6,7- tetrahydro) - + + + + + benzthiazolyl] - thiourea 2-amino 4,5,6,7-tet- + + + + rahydro-benz- thiazole N-methyl-N'- [2- (4,5,6,7- tetrahydro) - + + + + benzthiazolyl] - urea N-butyl-N '- [2- (4,5,6,7-tetra- hydro) -benz- + + + + thiazolyl] - urea N-3-chlorphe- nyl-N '- [2- (4,5,6,7-tet- ++ rahydro) -benz- thiazolyl] - urea Cell culture tests on animals Virus preparation: I II III IV V virus: VI VII VIII Cell primary permanent primary chick animal: mouse mouse hamster culture: chicken human embryonic cell embryonic cell strain (HEZ) cell (HEZ) (HeLa) Nn-butyl-N'- methyl-N '- [2- (4,5,6,7- + tetrahydro) - benzthiazolyl] - urea N-3-chlorphe- nyl-N'-methyl- N '- [2- (4,5,6,7- ++ tetrahydro) - benzthiazolyl] - urea N-4-chloro-phe- nyl-N'-methyl- N '- [2- (4,5,6,7- + + + + tetrahydro) - benzthiazolyl] - urea N-phenyl-N'- methyl-N '- [2- (4,5,6,7-tetra- +++ hydro) -benz- thiazolyl] - urea Cell culture tests on animals Virus preparation: I II III IV V virus: VI VII VIII Cell primary permanent primary chick animal: mouse mouse hamster culture: chicken human embryonic cell embryonic cell strain (HEZ) cell (HEZ) (HeLa) N-phenyl-N'- n-butyl-N'- [2- (4,5,6,7- + tetrahydro) - benzthiazolyl] - urea N-methyl-N'- methyl-N '- [2- (4,5,6,7-tet- rahydro) -benz- + + thiazolyl] - thiourea N-buthyl-N'- [2- (4,5,6,7- tetrahydro) - benzthyiazolyl] - +++ thiourea N-butyl-N'- methyl-N '- [2- (4,5,6,7-tet- + + + + rahydro) -benz- thiazolyl] - thiourea The preparations according to the invention containing tetrahydrobenzothiazole derivatives can be administered orally and parenterally in liquid or solid form. The injection medium used is preferably water, which contains the additives customary in injection solutions, such as stabilizers, solubilizers and / or buffers. Such additives are, for example, tartrate or borate buffers, ethanol, dimethyl sulfoxide, complexing agents (such as ethylenediaminetetraacetic acid), high molecular weight polymers (such as liquid polyethylene oxide 3 for viscosity regulation. Solid carriers are, for example, starch, lactose, mannitol, methyl cellulose, talc, highly dispersed silica Molecular fatty acids (such as stearic acid), gelatin, agar-agar, calcium phosphate, magnesium stearate, animal and vegetable fats, solid high molecular weight polymers (such as polyethylene glycols). Preparations suitable for oral administration can, if desired, contain flavorings and sweeteners. For external treatment are according to the invention, the tetrahydrobenzothiazole derivatives are used in the form of ointments with customary ointment bases or as tinctures.

The Tetrahydrobenzthiazolderivate can in the invention Preparations are in the form of pharmacologically acceptable salts. Pharmacologically acceptable Salts are in particular those with harmless inorganic or organic Acids such as the hydrochlorides, hydrobromides, sulfates, phosphates, tartrates, citrates, Acetates or oxalates.

The invention is explained in more detail in the following examples.

Example 1 1000 g of N-allyl-N'- 9 - (4,5,6,7-tetrahydro) -benzthiazolylurea are made in 60 liters of demineralized water at 300C in a batch kettle acid-proof steel dissolved with stirring. Then dissolve 20 g of a commercially available Preservative (p-hydroxybenzoic acid butyl ester = nipabutyl) in 10 liters Water at 800C and cools down to 300C. The two solutions are then combined and one after the other after complete dissolution with> 2 g dye (amaranth), 35 kg sucrose DAB 7 and 200 g aroma (raspberry) added. The approach is with a 25% aqueous citric acid solution adjusted to pH 3.5 - 4.3 and with demineralized Water made up to 100 liters. Finally stir the solution thoroughly, filtered and bottled. In this way a tasty virustatic is obtained in juice form.

The N-allyl-N '[2- (4,5,6,7-tetrahydro) benzothiazolyl] urea used as active ingredient can be prepared as follows: In a 500 ml three-necked flask equipped with a stirrer, Is equipped with a reflux condenser and dropping funnel, 61.7 g of 2-amino-tetrahydro-benzothiazole dissolved in 300 ml of anhydrous benzene and after adding a few drops of pyridine 50 0C 41.5 g of allylocyanate are added dropwise. One warms up 5 hours at 60 ° C., the mother liquor is then concentrated and the crystalline solution crystallizes Residue from methanol. 73 g of N-allyl-N-s2- (4,5,6,7-tetrahydro) -benzthiazolylS-urea are obtained in this way from m.p. 153-154 C.

Example 2 500 g of N-methyl-N'-methyl-N'-s2- (4,5,6,7-tetrahydro) -benzthiazolylj-urea are micronized to an average grain size of 1-5, i and in 10 liters one Solution of 500 g of a commercially available non-ionized emulsifier heated to 300C (Polyoxyethylene stearate = Myrj 52) and 500 g starch syrup using an intensive stirrer finely suspended. The active ingredient suspension is in 60 liters of one according to the example 1 prepared solution of p-hydroxybenzoic acid butyl ester, dye (tartrazine), Sugar and flavor (vanillin) mixed in homogeneously and by adding water and starch syrup brought to a viscosity of about 500 cp to prevent segregation. In this way, a tasty pharmaceutical preparation with a virustatic effect is obtained.

The N-methyl-N'-methyl-N'-t2- (4,5,6,7-tetrahydro) -benzthiazolylj-urea used as active ingredient can be made as follows: In a 500 ml three-necked flask, which is provided with a stirrer, reflux condenser and dropping funnel, 33.6 g of 2-methylamino-tetrahydro-benzothiazole are dissolved in 200 ml of anhydrous benzene and after adding a few drops of pyridine 30 ° C. mixed with 14.3 g of methyl isocyanate. The mixture is heated to 50 ° C. for 3 hours and crystallized the product obtained from ligroin as a residue when the benzene solution is concentrated. This gives 25 g of N-methyl-N'-methyl-N1-t2- (4,5,6,7-tetrahydro) benzthiazolyl urea of m.p. 84-850C.

The 2-methylamino-4,5,6,7-tetrahydro-benzothiazole used as the starting material in the above procedure is obtained as follows: 360 g of N-methylthiourea and 530 g of M-chlorocyclohexanone are refluxed in 1.2 l of ethanol for 4 hours.

After standing overnight a crystalline precipitate forms, which is removed by filtration. When the mother liquor is concentrated, one obtains after Addition of acetone a further amount of the crystalline product of m.p. 184-1900C.

This product is dissolved in 600 ml of water, filtered and concentrated with Ammonia solution made alkaline. The resulting crystalline precipitate is dried and recrystallized from ligroin. 179 g of 2-methylamino-4,5,6,7-tetrahydro-benzothiazole are obtained in this way of m.p. 113-114 C. Example 3 50 g of N-butyl-N'-methyl-N'- £ 2- (4,5,6,7-tetrahydro) -benzthiazolyl-thiourea are mixed with 40 g lactose and 40 g corn starch and with the addition of 10% Granulated corn starch paste. The finished granulate is passed through a sieve with the Mesh size 0.8 mm sieved. Then 10 g of the granules obtained in this way Methyl cellulose, 10 g talc and 2 g magnesium stearate mixed in.

The mixture is then homogenized and made into tablets with a diameter of 9 mm with a total weight of 202 mg.

Antiviral tablets of the same galenic are obtained, if the active ingredient is instead of N-butyl-N'-methyl-N-C2- (4,5,6,7-tetrahydro) -benzthiazolyl7-thiourea the N-4-chlorophenyl-N'-methyl-N'-z2- (4,5,6,7-tetrahydro) -benzthiazolylj-urea used and otherwise proceed in the specified manner.

The N-4-chlorophenyl-N'-methyl-N'-j2- (4,5,6'7-tetrahydro) used as active ingredient in paragraph 2 of this example benzthiazolyl2-urea can be prepared as follows: In a 500 ml three-necked flask, which is provided with a stirrer, reflux condenser and dropping funnel, 27 g of methylamino-tetrahydro-benzothiazole dissolved in 200 ml of anhydrous benzene and after adding a few drops of pyridine with 30.7 g 4-chlorophenyl isocyanate, dissolved in 50 ml of anhydrous benzene, added drop by drop. The mixture is heated to 80 ° C. for 3 hours and the solvent is removed in vacuo and the residue recrystallizes from benzene. 32 g of N-4-chlorophenyl-N-t-methyl-Nt-s2- (4,5,6,7-tetrahydro) -benzthiazolylS-urea are obtained in this way from m.p. 144-145.

The N-butyl-N'-methyl-N '[2- (4,5,6,7-tetrahydro) -benzthiazolyl] used as active ingredient in paragraph 1 of this example thiourea can be prepared as follows: 33.6 g methylamino-tetrahydro-benzothiazole are dissolved in 100 ml of ethanol and after adding 29 g of n-butyl isothiocyanate 4 Hours under reflux. Crystallized after removal of the solvent the residue from standing for a long time. This is obtained after recrystallization Ethanol 19 g of N-butyl-N'-methyl-Nt-S2- (4,5,6,7-tetrahydro) -benzthiazolyl-thi'ourea of m.p. 65.5-66.5 C.

Example 4 30 g of N-methyl-N'-n-butyl-N'-z2- (4,5,6,7-tetrahydro) -benzthiazolyl-urea are made with 25 g lactose, 25 g corn starch, 5 g methyl cellulose, 5 g talc and 1 g of magnesium stearate is processed into a base, which is then made into curved tablet cores be compressed with a total weight of 110 mg. The cores thus obtained are then in known way with a sugar coating coated and then waxed.

The N-methyl-N'-n-butyl-N'-g2- (4,5> 6,7-tetrahydro) -benzthiazolyl2-urea used as active ingredient can be prepared as follows: In a 500 ml three-necked flask equipped with a stirrer, A reflux cooler and a dropping funnel are provided, 42 g of n-butylamino-tetrahydro-benzthiazole dissolved in 200 ml of anhydrous benzene and after adding a few drops of pyridine with 14.3 g Meshyl isocyanate are added dropwise. The mixture is heated to 50 ° C. for 4 hours, removed then the solvent in vacuo and the residue recrystallized from ligroin. This gives 20 g of N-methyl-N'-n-butyl-N'-z2- (4,5,6,7-tetrahydro) -benzthiazolyl7-urea of m.p. 75-760C.

The n-butylamino-tetrahydro-benzothiazole used as the starting product in the preceding paragraph can be obtained as follows: 400 g of N-n-butylthiourea and 400 g of d; chlorocyclohexanone are refluxed in 1 liter of ethanol for 7 hours.

After removing the solvent in vacuo, the residue is with an acetone-ether mixture added, whereby a crystalline product is obtained, the is separated off by filtration. When the mother liquor is concentrated, a Further Amount of this crystalline product. This product is dissolved in water and with concentrated ammonia solution made alkaline. The precipitating crystalline Precipitate is separated off and recrystallized from ligroin.

432 g of 2-n-butylamino-4,5,6,7-tetrahydrobenzothiazole are thus obtained M.p. 68-700C.

In a manner analogous to that described in the preceding examples, the following tetrahydrobenzothiazole derivatives can be prepared, with the characterization based on the melting points: preparation melting point N-methyl-N '- [2- (4,5,6,7-tetrahydro) -benzthiazolyl] - 221-223 ° C thiourea N-methyl-N '- [2- (4,5,6,7- 1st melting point: 203 ° C tertrahydro) - = benzthiazolyl] - 2nd melting point: 325-330 ° C urea N-butyl-N '- [2- (4,5,6,7- - 1st melting point : 185 - 187 ° C tetrahydro) -benzthiazolyl- 2. Melting point: 290 C urea N-3-chlorophenyl-N '- [2- 318 ° C (4,5,6,7-tetrahydro) -benzthiazolyl-2-urea Preparation melting point N-n-Butyl-N'-methyl-N'-g2- (4,5,6,7-tetrahydro) -benz-37,5-39 C thiazolylj-urea N-3-Chlorophenyl-N'-methyl-N'-2- (4,5,6 7-tetrahydro) -benz-139-1410C thiazolyl) -urea N-Phenyl-N'-methyl-N '- [2- (4,5,6,7-tetrahydro) -benz-113.5-114.5 ° C thiazolyl7-urea N-Phenyl-N'-n -butyl-N '- [2- (4,5,6,7-tetrahydro) -benz-85-86C thiazolyl7-urea N-methyl-N'-methyl-N '- [2- @ (4,5,6,7-tetrahydro) -benz-90.5-91.5 C thiazolyl7-thiourea N-Butyl-N '- [2- (4,5,6,7-tetrahydro) -benzthiazolyl] - 167-169 ° C thiourea

Claims (2)

Patent claims 1. Pharmaceutical preparations with antiviral activity, characterized in that they are a tetrahydrobenzothiazole derivative of the general formula wherein R -NH2, -NH-CX-NH-R1 or and X is oxygen or sulfur R1 is alkyl or alkenyl with up to 4 carbon atoms or chlorophenyl R2 is alkyl with up to 4 carbon atoms and R3 is alkyl with up to 4 carbon atoms, phenyl or chlorophenyl, contain as active ingredient. 2. Use of tetrahydrobenzothiazole derivatives of claim 1 given general formula for the production of pharmaceutical preparations with virustatic effectiveness.