DE2151229A1 - Thiazolid-4-one derivs - prepd from thiocarbonylcyano acetic acid ester derivs and brominated lower fatty acids, virustatics - Google Patents

Abstract

Cpds. of formula (I): (where R1 is opt. substd. alkyl, aryl or aralkyl; R2 is H, Br or opt. substd. alkyl; R3 is lower alkyl) are prepd. e.g. by refluxing butylamino-thiocarbonylcyano acetic acid ethyl ester-butylammonium salt with Dl-alpha-bromo-n-caproic acid in ethanol. (I) have antiviral activity against RNS- as well as DNS-virus. They are therefore used as virustatics in liquid- or solid form for oral or parenteral administration.

Description

Virustatics containing thiazolid-4-one derivatives The invention relates to virustatics, characterized in that they are a thiazolid-4-one derivative of the general formula in which Rj is an optionally further substituted alkyl, aryl or aralkyl radical, R2 is a hydrogen atom, an optionally further substituted alkyl radical or a bromine atom and R3 is a lower alkyl radical, contain as active ingredient.

The radical R1 is preferably by dialkylamino, especially diethylamino, and the radical R2 in the meaning of alkyl is further substituted by a carboxyl group.

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-2t-deoxyuridine and N-methyl-isatin-B-thiosemicarbazone are only effective against DNA viruses, the well-known aminoadamantane only on RNA viruses. The riding band effect of the invention Virustatica against RNA and DNA viruses of different virus groups is therefore surprising and could not be 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 information 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 rolls and substance-free Virus controls.

For the evaluation of the tube tests, the tables after Procedure by Kärber (Kärber, G .: Naunyn-Schmiedeberg's Areh. Exp. Path. Pharmak. 162, 480, 1931, calculated by R.J. Lorenz, Federal Research Institute for Virus Diseases der Tiere, Ttibingen 1960), while the plaque test results according to Lorenz (Lorenz, R.J .: Zur Statistik des Plaque-Testes. Arch. ges. Virusforschg. 12, 108-137, 1963).

a) Comparative infection titer determinations in the test tube Each The test tube contains 1 ml of maintenance medium with 100 μg of test substance. After a two hour 1 ml virus dilution is added to the reaction time. 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 and 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 virus dilution and after an incubation time of 2 hours at 360 C 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' become 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 a substance shows up as a ring-shaped plaque inhibition around the substance-containing test disc c) Plaque reduction test After 24 hours, fully grown cell cultures in plastic dishes are inoculated with 0.5 ml of a suitable virus dilution and after a two-hour period Incubation time at 360C in a C02-3 incubator covered with 5 ml of ion agar containing per ml contains between 100 and 300 Aig of test substance.

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) Influenza experiments 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 is put under light ether anesthesia infected with @ 3 dli intranasally. The mortality rates are recorded within of 10 days.

Each substance group consists of 10, the virus control.

from 30 mice.

b) Parainfluenza 3 experiments in hamsters Each hamster receives 4 in a row Days in a single dose 13 ig test additive (~ 300 mg / kg / day) per 01. 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 hamsters 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 test substance means significantly reduced virus content in 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 A2 Mannheim / 57; RNA type II - parainfluenza 3; RNA type III - herpes simplex - CAM 13; DNA type IV - Vaccinia Ankara; DNA type V - Influenza A2 - Japan / 305/57; RNA Type VI - Influenza A2 - Hongkon £ / 50/68; RNA type VII Y parainfluenza 3; RNA type table Cell culture tests on animals Virus preparation: II III IV virus: V VI VII Cell permanent primary chicken animal: mouse mouse hamster culture: the human embryonic cell cell stem (HEZ) (HeLa) 2- [ethoxycarbonyl- cyanomethylene] -3- ++ [2-dimethylamino- ethyl] thiazoli- don- (4) -hydro- chloride 2- [ethoxycarbonyl- cyanomethylene] - + + + + + + 3-butyl-5-butyl- thiazolidone- (4) 2- [ethoxycarbonyl- cyanomethylene] -3- + phenyl-5-bromo thiazolidone- (4) 2- [ethoxycarbonyl- cyanomethylene] -3- + + + + ß-naphthyl-5- ethyl thiazoli- don- (4) Cell culture tests Virus preparation: I II III IV Cell- primary chick- permanent primary chick- embryonic- culture: embryonic cell human cell stem cell (HEZ). (HEZ) (HeLa) 2- [ethoxycarbonyl- cyanomethylene] -3- +++ methyl thiazoli- don- (4) 2- [ethoxycarbonyl- cyanomethylene] -3- ++ methyl-5-hydroxy carbonyl-methyl- thiazolidone- (4) 2- [ethoxycarbonyl- cyanomethylene] -3- + benzyl-5-hexadecyl- thiazolidone- (4) 2- [methoxycarbonyl- cyanomethylene] -3- ++ benzyl-5-carboxy- methyl thiazoli- don- (4) The preparations according to the invention containing thiazolid-4-one 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) for viscosity regulation. Solid carriers are, for example, starch, lactose, mannitol, methyl cellulose, talc, highly disperse silica, higher molecular fatty acids (such as stearic acid), gelatine, 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, the thiazolid-4-one derivatives are used according to the invention in the form of ointments with customary ointment bases or as tinctures.

The thiazolid-4-one derivatives can be used in the preparations according to the invention present as pharmacologically acceptable salts.

Pharmacologically acceptable salts are in particular those with harmless ones inorganic or organic acids such as the hydrochlorides, hydrobromides, Siilfate, phosphate, tartrate, citrate, acetate or oxalate.

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

Example 1 1000 g of 2- [ethoxycarbonyl-cyanomethylene] -3-butyl-5-butylthiazolidone- (4) are made in 60 liters of demineralized water at 30 ° C 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 of dye (amaranth), 35 kg sucrose DAB 7 and 200 g aroma (raspberry) added. The approach is with a own 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 2- [ethoxycarbonyl-cyanomethylene] -3-butyl-5-butyl-thiazolidon- (k) used as active ingredient can be prepared as follows: 85 g of 3,5-bis- [1-cyano-1-ethoxycarbonylmethylene] -1,2,4-trihiacyclopentane are in chloroform with 2.5 g of n-butylamine to give the butylaminothiocarbonyl cyanoacetic acid ethyl ester butylammonium salt converted from mp 155 ° C.

28 g of butylaminothiocarbonyl cyanoacetic acid ethyl ester butylammonium salt and 16.4 g of DL-oL-bromo-n-caproic acid are refluxed in 300 ml of ethanol for 30 minutes heated, mixed with 200 ml of water and the crystals which precipitated out on cooling sucked off. 20 g of 2- [ethoxycarbouyl-cyanomethylene] -3-butyl-5-butyl-thiazolidone- (4) are obtained of m.p. 39 0C.

Example 2 50 g of 2- [ethoxycarbonyl-cyanomethylene] -3-phenyl-5-bromothiazolidone- (44) 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 mix will then homogenized and into tablets with a diameter of 9 mm with a total weight of 202 mg pressed.

The 2-CAthoxycarbonyl-cyanomethylenel-3-phenyl-5-bromo-thiazolidone- (4) used as an active ingredient can be prepared as follows: 14.5 g of 2-ethoxyearbonyl-cyanomethylene-3-phenyl-thiazolidone- (4) are refluxed with 8 g of bromine in 150 ml of glacial acetic acid until no hydrogen bromide more escapes. The hot solution is filtered and poured into 1 liter of water. The precipitated crystals are suctioned off and made from isopropanol recrystallized. 12 g of 2-ethoxy-carbonyl-cyanomethylenej-3-phenyl-5-bromothiazolidone- (4) are obtained of m.p. = 1560C.

Example 3 30 g of 2- [ethoxycarbonyl-cyanomethylene] -3- [2-dimethylaminoethyl] thiazolidone (4) hydrochloride 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 coated in a known manner with a sugar coating and then waxed.

Example 4: 500 g of 2-ethoxycarbonyl-cyanomethylene-3-ß-naphthyl-5-ethyl thiazolidone- (4) are micronized to an average grain size of i-5 / u and in OK liters of a solution of 500 g of a commercially available solution heated to 3000 not ionized emulsifier (polyoxyethylene stearate = Myrj 52) and 500 g starch syrup means an intensive stirrer finely suspended.

The active ingredient suspension is prepared in 60 liters of one according to Example 1 Solution of butyl p-hydroxybenzoate, color (tartrazine), sugar and flavor (Vanillin) stirred in homogeneously and by adding water and starch syrup to one Brought a viscosity of about 500 cp to prevent segregation. You get such a tasty drug preparation with a virustatic effect.

Claims (2)

Claims i. Virustatica, characterized in that it is a thiazolid-4-one derivative of the general formula in which R1 is an optionally further substituted alkyl, aryl or aralkyl radical, R2 is a hydrogen atom, an optionally further substituted alkyl radical or a bromine atom and R3 is a lower alkyl radical, contain as active ingredient. 2. Use of thiazolid-4-one derivatives specified in claim 1 general formula for the production of virustatics.