HU207218B - Process for producing antiviral pharmaceutical composition containing thiadiazol derivatives as active component - Google Patents

Abstract

The present invention relates to a thiadiazole derivative represented by the general formula (I) wherein R1 is hydrogen, R2 is -C / = S / -NH-R3, where R3 is hydrogen or -COOR4 in which R4 is hydrogen. C 1 -C 8 alkyl, C 2-6 alkenyl or phenyl, or pharmaceutical compositions containing their pharmaceutically acceptable salts. S. R N-R1 (I) EN 207 218 B Scope of the description: 6 pages (including J sheet)

Description

Scope of the description: 6 pages (including 1 page figure)

EN 207 218

The number of viral diseases is increasing alarmingly. Over the past few years, viral and blood transfusive virus infections have been at the center of intensive research. Less serious diseases caused by viruses include, for example, common cold.

Only few effective antiviral agents have been developed to date and in many cases the only possible treatment is the administration of anti-bacterial agents; not to overcome the virus, but rather to prevent bacterial infections of the weakened biological system. Research continues to experiment with effective antiviral agents that are suitable for the treatment and control of simple diseases such as influenza and rhinitis, or diseases caused by different strains of herpes virus, as well as much more serious viral infections, e.g. to fight AIDS (acquired immune deficiency syndrome).

The present invention relates to the recognition that certain 2-amino-1,3,4-thiadiazole derivatives are effective in the treatment of viral infections. Thiadiazoles are generally well known in the art and some have been used in medicine. For example, Naik et al. Describe the antimicrobial activity of a number of thiadiazolylthiazolidinones (J. Ind. Chem. Soc., Vol. LX, July 1983, pp. 674-678), Grant et al. (J. Med. Chem., Vol. 15, No. 10, 1972, pp. 1082-1084) states that certain amine-substituted derivatives of 2-amino-1,3,4-thiadiazole are effective antihypertensive agents. According to U.S. Patent No. 3,723,166, the N-acylated derivatives of 2-amino-1,3,4-thiadiazoles are effective as fungicides. Russo et al. (Farmaco. Ed. Sci., Vol. 30, No. 12, 1975, pp. 1031-1038) describe thiourea derivatives of 1,3,4-thiadiazoles and their cyclization products and determine the antibacterial efficacy of these compounds. Malinoski et al. (Virology, 110: 281-291 (1981)) describe some antiviral activity of 2-amino-1,3,4-thiadiazoles.

The antiviral pharmaceutical compositions of the present invention comprise the compounds of formula (I) wherein R ¹ is hydrogen;

R ² is -C / = S / NH-R ³ ;

R ³ is hydrogen or -COOR ⁴ ;

R ⁴ is C 1 -C 8 alkyl, C 2-6 alkenyl or phenyl.

The compounds of formula (I) can be used as active ingredients in pharmaceutical compositions for treating people and animals suffering from or susceptible to viral infection.

The invention also includes the use of pharmaceutically acceptable salts of said compounds.

Many thiadiazoles used in the antiviral compositions of the present invention are known in the art, but their antiviral activity has not been described so far. Each compound can be prepared by known chemical processes such as those described by Naik et al., Or Russo et al.

Russo et al. (Supra) described the antibacterial activity of the compound of formula (III), but no mention was made of its antiviral activity.

In formula (I), R _{2 is} -C / = S / -NH-R ₃ , where R ₃ may represent an acyl group of the formula -COOR ₄ , and this definition of R ₂ extends the invention to the N-acyl group. thiourea type compounds, many of which are preferred for treatment. These thioureas can be represented by formula (II) wherein R ⁴ is as defined above. Several thiadiazole thioureas of this type are known from the literature, for example, from Japanese Patent Application No. 71/35 262, which is directed to fungicides and bacteria, as well as herbicides, and from 74/7218, which also relates to fungicides and bacteria.

According to the definition of said compound, R ⁴ may be C 1-8 alkyl, where the carbon chain may be straight or branched, such as ethyl, n-hexyl or isooctyl. R ⁴ may also represent C 2-6 alkenyl such as allyl or 4-hexenyl and the like, or phenyl. The most preferred R ⁴ substituents are C 1-6 alkyl and C 2-6 alkenyl.

The active compounds of the present invention have organic and inorganic bases such as sodium acetate, calcium carbonate, sodium hydroxide and the like. are prepared to form pharmaceutically acceptable salts.

The compounds of the invention may exist in different tautomeric forms, all of which are encompassed by the invention.

The following examples illustrate the preparation of certain thiadiazoles used as antiviral agents in the present invention.

/. preparation

Ethyl N- ((3,4-thiadiazol-2-ylamino) thioxotyl) carbamic acid

To a solution of 19.2 g (190 mM) of 2-amino-1,3,4-thiadiazole in 200 ml of acetonitrile with stirring, 25 g (190 mM) of ethoxycarbonyl isothiocyanate are added in one portion and the reaction mixture is further heated at 24 ° C for 16 h. stir for an hour. The precipitate was collected by filtration, washed three times with ethyl acetate, dried to give 35 g (80% yield) of the title compound.

Analysis for C ₆ H _g N ₄ O ₂ S ₂ requires: C: 31.02, H: 3.47, N: 24.12%;

Found: C, 31.32; H, 3.27; N, 24.40.

Preparation 2

N- (], 3,4-Thiadiazol-2-yl) thiourea

10 g of N [(1,3,4-thiadiazol-2-ylamino) thioxomethyl] -carbamic acid ethyl ester prepared as described in Preparation 1 were refluxed for 90 minutes in 150 ml of sodium hydroxide and the solution was cooled. and evaporated by vacuum distillation. Excellent precipitation

It is filtered and dissolved in 20 ml of water. The aqueous solution was acidified with 200 ml of 1N hydrochloric acid. The product was filtered, recrystallized from dimet-dimethylformamide to give the title compound (5 g). 253 ° C.

Analysis calculated for C ₃ H ₄ N ₄ S ₂ : C, 22.49; H, 2.52; N, 34.97; Found: C, 22.73; H, 2.33; N, 34.74; S, 40.26.

Preparation 3

N - [(I, 3,4-thiadiazol-2-yl-amino) thioxomethyl] -carbamic acid phenyl ester

A solution of 3.8 g (50 mM) of ammonium thiocyanate in 100 ml of acetonitrile containing 7.8 g (50 mM) of phenyl chloroformate was stirred at 24 ° C for one hour and then the reaction mixture was stirred at 6.88 ° C. It was filtered to a suspension of g (50 mM) 2-amino-1,3,4-thiazole-HCl in 75 ml of acetonitrile and 25 ml of propylene oxide. The reaction mixture was stirred at 24 ° C for a further two hours, and then the solid precipitate, which was 6.42 g of the title compound, was filtered off.

FD Mass Spec: M ⁺ 280.

4-6. preparation

Following the general procedure for preparation of Formulation 3, the following products were prepared:

N - [(l, 3,4-thiadiazol-2-ylamino) thioxomethyl] -karb-.

amine-acid n-hexyl ester.

Analysis for C ₁₀ H ₁₆ N ₄ O ₂ S ₂ requires: C: 41.65, H: 5.59, N: 19.43, S: 22.24%; Found: C, 41.64; H, 5.31; N, 19.28; S, 22.46.

N - [(l, 3,4-thiadiazol-2-ylamino) thioxomethyl] carbamic acid methyl ester.

FD Mass Spec: M ⁺ 218.

N - [(l, 3,4-thiadiazol-2-ylamino) thioxomethyl] carbamic acid, vinyl ester.

FD Mass Spec: M ⁺ 230.

The thiadiazoles described above exhibit antiviral activity in standard test systems and are therefore useful in the treatment and prevention of diseases caused by various viruses. Thiadiazoles are, for example, active against the following typical virus strains: all tested A and B chain influenza viruses, including A-Ann Arbor, A-Hong Kong, B-Great Lakes, B-Taiwan, B-Singapore, A-Brazil, A-Texas, A-Fukushina, B-Maryland and other similar virus strains. Other viruses that can be controlled by the invention include parainfluenza respiratory syncytial viruses, various Herpes I and II strains, Echo and Vaccinia virus measles "Semlili Sickle", and AIDS-causing viruses.

The thiadiazole-type antiviral agents of the present invention exert their effects in both in vitro and in vivo conditions. For example, the in-vivo activity of agents that counteract the influenza virus A-Ann Arbor is particularly well characterized.

In the in vivo test experiments, groups of 18 CD-1 mice were tested after infection with a predetermined amount of A-Ann Arbor influenza culture. Thiadiazole was also given in a specific dose to the animals, and the control group was the vehicle alone.

The test lasted 10 days. In each group, we counted the number of animals killed on each day. An animal at the end of day 10 was considered a survivor. We used the survival index method to analyze the data as follows:

From the control group data, a "survival index" (TI _xnap ) value was assigned to each day (X) from the day the first death occurred in the control group (usually on day X = 4) until day 10. On the day of the first death, the "survival index value" is by definition equal to zero. Values for other days can be calculated using the following formula:

TI _xnap = (Xl)

Number of control animals killed on day X-1 total number of control animals

For example, if the first day on which a control animal dies, it is X = 4. day 4, and 4 out of 18 control animals die on day 5, since TI ₄ . _nap = zero,

Survivors' T. Survivors I index is also calculated based on the control group data using the following formula:

ΤΙ ₅ " _βρ = (5-1) ^ + 0 = yj = 0.66" _{T in} Number of control _animals died on day 10

T. I · mins = 10x-J; - + TI ^l 10th day

Total number of control animals

The indexes thus calculated can then be used to determine the average survival values for both the control group and the groups of animals tested using the following procedure: The mean survival index of the control group, TI _control | can be calculated by multiplying each TI _day by the number of control animals died on that day, and multiplying the 60 TI survival by the number of survivors, if any, by summing up and multiplying the total number of control animals. For the treated group, an average survival value ("TI _hand | _t ") can be calculated by multiplying each TI _Xnap value by the number of treated animals died that day, and multiplying the "TI, ι-, ΐϋ, ίΐς" by Survivor treated

These values are summed and divided by the total number of animals treated.

Based on the following formula where P is the maximum%, a comparison with the results of the control group can be calculated

TT _TT p _ ^{1 1} ^ control «™ - 1UU ¹ ^ survivors 1 control

If, on the basis of this formula, the average survival value of the treated animals is less than or equal to the mean survival value of the control animals, then P = 0%. If the average survival value for treated animals is equal to the survival index of survivors, then P = 100%.

This P value is then a so-called. relative activity index (RA) is assigned as follows:

P RA 20% or less = 1 (threshold) 20-40%; = 2 (slightly active) 40-60% = 3 (medium active) 60-80% = 4 (good activity) 80-100% = 5 (very active)

Table I shows the representative compounds of the invention.

shows RA values for: /. table Compounds of Formula II * 4 RA ch ₂ ch ₃ 4.5 ch ₃ 5 Ph 5 n-hexyl 5 vinyl 4

In addition, the compounds also showed good activity in mice against various influenza cultures. The II. Table 2 shows the activity of Formulation 2 in a wide range of doses, mixed with the diet of mice infected with different influenza A and B strains.

II. table

flu Strain control Dose (mg / kg / day) 15 30 60 120 B-Great Laké 3/36 11/18 16/18 17/18 18/18 B-Taiwan 1/36 4/18 15/18 18/18 18/18 B-Maryland 13/50 - - 19/20 20/20 B Hong Kong 4/50 - - 10/20 16/20 A-Ann Arbor 0/50 - - 20/20 20/20 A Tcxas 8/36 5/18 6/18 12/18 16/18 A Brazilian 10/28 5/15 5/16 12/18 14/18

Note: (-) means: This dose level has not been evaluated.

Methods for quantifying viral multiplication in tissue cell culture systems in vitro are plaque-reducing assays.

In this test, the sensitized HOCK cells were grown in 25 cm ³ Falcon vessels at 37 ° C in Medium 199 in the presence of 5% inactivated fetal oxide serum (FBS), penicillin (300 units / ml) and streptomycin (300 pg / ml). . After the formation of a coherent single-layer coating, the medium was removed and 0.3 ml of properly diluted virus was added to each bottle. After adsorption at room temperature for 1 hour, the infected cell layer was covered in equal portions with 1% agarose and 2x Medium 199, 2.5% FBS, penicillin, and stereostomycin, with different concentrations of the tested compounds embedded in the agar coating.

Compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10,000 pg / mL, and an aliquot was diluted at the desired concentration using agar. The bottles were incubated until then. until the control vessels showed an optimal plaque size (2-10 mm). A solution of 10% formate and 2% sodium acetate was added to each bottle to inactivate the virus and bind the cell layer to the plastic surface. Plaques were counted after staining the surrounding cell areas with crystal violet. At each concentration, the results obtained for the duplicate bottles were averaged and compared to the control bottle. Inhibition of plaque formation was estimated at 50% (I ₅₀ ) by plotting the results for 10-90% inhibition. The results of three influenza strains are presented in Figure III. table.

III. table

In vitro activity against influenza viruses I ₅₀ [NCG / ML]

Vcgyíilct NWS-A A-Ann Arber B-Great Laké Preparation 1 43 > 50 Preparation 2 3.5 3.0

The preferred use of the compounds can be in vivo when the compounds are administered parenterally, topically, orally or by inhalation into the body of a mammal suffering from or susceptible to the viral infection. For parenteral administration, e.g. for intraperitoneal administration, the chemical is dissolved or suspended in 2% surfactant, in particular emufort (polyhydroxy fatty acid) water. Of course, oral administration is most preferred. In this case, the compound as defined herein may be formulated with one or more standard pharmaceutically acceptable substances such as starch, sucrose, lactose. calcium carbonate, etc. stirring and filling the mixture into empty, reciprocating gelatin capsules so as to be present in each capsule in an amount; compound to effectively suppress influenza or other viruses, whether prospective or present

EN 207 218 B. In other words, these compounds are both prophylactic (prophylactic) and healing. Likewise, the active ingredient can be mixed with any carrier (e.g., starch, lubricants, wetting agents, etc.) such as stearic acid, magnesium stearate, and the like, and the tablet can be compressed, each of which is required to stop and suppress an influenza or other viral attack. included. Dosage of these tablets is suitable for children in half or quarter of a dose, but may be in the form of a solution or suspension.

Ideally, the compounds may be formulated and administered orally, topically or parenterally, for prophylactic or therapeutic purposes, in combination with suitable pharmaceutically acceptable carriers. The compositions may contain from 1 to 95% by weight of the active thiadiazole antiviral agent.

For oral (oral) administration, the compounds may be combined with conventional diluents and excipients (such as sucrose, starch, microcrystalline cellulose, gum arabic, etc.), tablets or pills may be formulated, filled into gelatin capsules, or a solution, elixir, pastilles or the like may be prepared. For topical application, the compositions are prepared by mixing the thiadiazole antiviral agent with a variety of excipients, such as beeswax, lanolin, oil, and so on for ointments, medications, creams, tinctures, and cosmetic solutions.

Intravenous or intramuscular dosing is more appropriate for more serious viral infections. These compositions contain 1-50% of the active ingredient. Compounds are dissolved in common diluents for intravenous infusion, such as isotonic saline or destrose solution; in the case of intravenous or intramuscular injections, the active ingredient is dissolved in polyhydroxy aliphatic alcohols, such as propylene glycol or polyethylene glycol.

Of the acidic or basic compounds of the above formula, pharmaceutically acceptable salts can be prepared by reaction with common organic or inorganic acids or bases such as hydrochloric acid, succinic acid or sodium hydroxide. Pharmaceutically acceptable salts generally have better solubility than the starting compounds and are therefore more suitable for liquid or emulsion formulations.

The thiadiazole antiviral agents described above are effective over a wide dosage range. Although each dose to be administered is the type and severity of the virus infection to be treated or treated, the route of administration and other circumstances are prescribed by the physician, the usual dosages range from 0.1 to 100 mg / kg and most often from 0.5 to 25 mg / kg. fall.

In a preferred application of the treatment, thiadiazole-type compounds have been administered to mammals such as horses, mice, pigs and humans susceptible to influenza virus infection. In the case of humans, we have been treating prophylactic treatment especially for the elderly, young children, nurses, doctors and other hospital and healthcare personnel where a threatening influenza pandemic is expected. The compounds can be administered to anyone who is at risk of being infected with an influenza. A particular advantage of the healing effect described in the present invention is that the compounds can be administered for prophylactic or curative purposes without having previously determined that the influenza virus belongs to strain A or B, since these compounds are effective against both strains.

The following example describes a typical composition comprising a compound of formula (I).

The example

Making pills

The compound of Formulation 1 is 100 mg

Lactose, 200 mg

Corn starch 300 mg

Corn starch chicken 50 mg

Calcium stearate 5 mg

Dicalcium phosphate 45 mg

The active ingredient is uniformly mixed with starch, lactose and dicalcium phosphate. Corn starch chicken, a 10% water paste, is also uniformly mixed into the mixture, then calcium stearate is added and then tablets are compressed.

Claims (1)

Patent Claim Point A process for the preparation of thiadiazole derivatives of the formula I wherein R ¹ is hydrogen, R ² is -C (= S) -NH-R ³ wherein R ³ is hydrogen or -COOR ^{4 in} which R ⁴ is a C 1 -C 8 alkyl group, a C 2 -C 6 alkenyl group, or a pharmaceutically acceptable salt thereof, characterized in that a compound of formula (I), wherein R ¹ , R ² , R ³ and R ⁴ are as defined in the preamble - or a pharmaceutically acceptable salt thereof - with pharmaceutically acceptable carriers and / or excipients to be converted into an antiviral pharmaceutical composition.