KR890004197B1 - Thiadiazole antiviral agents - Google Patents

Abstract

A 2-amino-1,3,4-thiadiazole deriv. of formular (I) is prepd. In (I), R1 is H; R2 is cyano or -C(=S)-NH-R3; R1 and R2 together are =C(NH2)2 or =C(R5)(NHR3'); R3 and R3' are H or -COOR4; R4 is (substd.) C1-10 alkyl, (substd.) C2-10 alkenyl or (sbustd.) phenyl; R5 is H or -S-R6; R6 is C1-4 alkyl, cyano C1-4 alkyl or pyridyl C1-4 alkyl. (I) and its salt are useful as an antiviral agent.

Description

Thiadiazole antiviral agent

The present invention is directed to certain 2-amino-1,3,4-thiadiazole derivatives useful for the treatment of viral infections.

Diseases caused by one or more viruses are surprisingly widespread. Viral infections transmitted by sexual intercourse and viral infections transmitted by blood transfusions have been the subject of intensive research efforts over the past few years. Less serious diseases caused by viruses include colds.

Certainly effective antiviral agents have been rarely developed, and in most cases only useful for treatment, antimicrobial agents have been used that do not rescue viruses but only prevent bacteria from penetrating into a weakened physiological system. Common diseases such as influenza and colds, as well as the treatment and control of diseases caused by various strains of herpes viruses, as well as antiviral agents effective in combating more serious viral infections such as AIDS. Research is ongoing.

The present invention is made by the finding that certain 2-amino-1,3,4-thiadiazole derivatives are useful for the treatment of viral infections. Thiadiazoles are generally known in the art. Some of them are used in the medical field. For example, Naik et al. Describe the antimicrobial activity of many thiadiazolylthiazolidinones in J. Indian Chem. Soc., Vol. LX, July 1983, pp 674-678. have. Grant et al., Disclose certain amine substituted derivatives of 2-amino-1,3,4-thiadiazole in J. Med. Chem., Vol. 15, No. 10, 1972, pp 1082 to 1084. Has been described as useful for hypertension. US Pat. No. 3,772,316 describes a group of N-acylated 2-amino-1,3,4-thiadiazoles known to be useful as fungicides. Russo et al. Describe a series of thiourea derivatives of 1,3,4-thiadiazole and ring closure products thereof in Farmaco. Ed, Sci., Vol. 30, No. 12, 1975, pp 1031 to 1038. It describes. These compounds are known to be antibacterial agents. Malinoski et al. Describe the specific antiviral activity of 2-amino-1,3,4-thiadiazole in Virology, 110, 281-291 (1981).

In one embodiment, the present invention provides a method for preventing, treating and controlling non-rheumatic infections. More particularly, in one aspect the present invention provides an in vitro antiviral method for protecting mammalian cells in a culture, comprising adding an antivirus amount of the following general formula (I) compound to the culture:

In which R is^OneIs hydrogen, R²Is cyano, or -C (= S) -NH-R³Or; R^OneAnd R²Is equal to = C (NH₂)₂Or = C (R⁵) (NHR^3´)ego ; R³And R^3´Is hydrogen or -COOR⁴And; R⁴C_One-C₁₀Alkyl, substituted C_One-C₁₀Alkyl, C₂-C₁₀Alkenyl, substituted C₂-C₁₀Alkenyl, phenyl, or substituted phenyl; R⁵Is hydrogen or -S-R⁶Where R⁶C_One-C₄Alkyl, Cyano-C_One-C₄Alkyl or pyridyl-C_One-C₄Alkyl).

Compounds of general formula (I) are also useful for the treatment of animals suffering from or being susceptible to non-infectious infections. For this purpose, preferred compounds are those of the following general formulas (II), (III) and (IV):

Wherein R ³ is hydrogen or —COOR ⁴ , R ⁵ is hydrogen or SR ⁶ and R ⁶ is C ₁ -C ₄ alkyl.

The present invention also provides a series of preferred novel compounds defined by the general formulas (V), (VI) and (iii):

Compounds of formulas (V) and (VI) are particularly useful as antiviral agents. Compounds of general formula are very active in vitro. The present invention also encompasses the use of pharmaceutically acceptable salts of these compounds.

Various thiadiazoles used in the antiviral method of the present invention are known in the art. All compounds may be prepared by any of the available chemical methods, for example the methods described by Naik et al. And Russo et al. Mentioned above.

Russo et al. Describe in this document 2-thioureido-1,3,4-thiadiazoles, ie compounds of general formula (III), as antimicrobial agents. However, there is no mention of antiviral activity.

In the general formula (I), R ² includes an acyl group defined by -C (= S) -NH-R ³ , wherein R ³ may be another acyl residue -COOR ⁴ . . This definition of R ² provides the N-acyl thioureas of the present invention, most of which are included in the preferred method of treatment. Such thiourea is represented by the following general formula (II):

Wherein R ⁴ is the same as defined above.

Most of these thiadiazole thioureas are known in the art, for example, in Japanese Patent No. 71/35262 for fungicides, fungicides and herbicides, and Japanese patents for fungicides and fungicides. No. 74/7218.

In the definition of the above compound, R ⁴ includes “C ₁ -C ₁₀ alkyl” and “substituted C ₁ -C ₁₀ alkyl”. These terms refer to straight and branched chain alkyl such as ethyl, n-hexyl, isodecyl and 6-ethylheptyl and optionally substituted straight and branched chain alkyl such as haloalkyl, hydroxyalkyl, phenyl alkyl and the like. "Halo" includes fluoro, chloro, bromo and iodine. R ⁴ is also C ₂ -C ₁₀ alkenyl or substituted alkenyl such as allyl, 4-hexenyl, 3-chloro-5-heptenyl, 2-hydroxy-4-isoheptenyl, and the like, as well as chlorophenyl, Substituted phenyl such as hydroxyphenyl, methylphenyl, cyanophenyl and the like. Preferred R ⁴ groups are C ₁ -C ₆ alkyl and C ₂ -C ₆ alkenyl.

Most of the compounds are carbamate is already named Dottie Osan derivative, which is a 1,3,4-thiadiazol-2-yl thiourea of the general formula R ⁶ X (here, R ⁶ are as defined herein above X is a leaving group). Suitable leaving groups include C1, Br, I, and sulfone ester groups such as tosylate and mesylate. Therefore, suitable alkylating agents include, for example, alkyl halides such as methyl bromide, or substituted alkyl halides such as cyanomethyl bromide or 2-pyridylmethyl iodide. The reaction is usually carried out by mixing approximately equal molar amounts of 2-thiadiazolyl thiourea and an alkylating agent in the presence of a base such as sodium carbonate in an unreactive organic solvent such as dimethyl formamide or acetonitrile. The reaction is usually complete within about 16 hours when performed at about 20 ° C to about 50 ° C. The reaction solvent can be removed by evaporation under reduced pressure if necessary. The product is readily purified by conventional methods including crystallization from a solvent such as ethanol, ethyl acetate or hexane, or chromatography on a solid support such as silica or the like.

Particularly preferred compound phrases provided by the present invention include 1,3,4-thiadiazole-2-cyanamide and pharmaceutically acceptable salts thereof. This compound can be prepared by reacting carbamimido thioacid ester with an oxidizing agent such as meta-chloroperbezoic acid, peracetic acid, sodium peroxide, hydrogen peroxide, ozone, chlorine and the like.

According to another aspect of the invention, 1,3,4-thiadiazole-2-cyanamide is also a protected 2-amino-thiadiazole derivative, i.e. 4-imino-3-phenylmethyl-1,3 After reacting, 4-thiadiazole with cyanogen bromide, the 3-position protecting group can be prepared by debenzylation removal with Lewis acid (e.g. aluminum chloride). The debenzylation reaction is carried out in an organic solvent such as methylene chloride, toluene or benzene. Preferably at least 2 equivalents of Lewis acid are used, more preferably 4 to 8 equivalents. The temperature is not important. The tanbenzylation reaction can be carried out, for example, at 0 ° C to 90 ° C. Preferably room temperature is used.

As described in Example 8, when carbamimido thio acid ester is reacted with ammonia, cyanamide is also produced.

Cyanamide reacts with organic and inorganic bases such as sodium acetate, calcium carbonate, sodium hydroxide and the like to readily form pharmaceutically acceptable salts.

Compounds of the invention may exist in a variety of tautomers, all of which are included within the scope of the invention. For example, the preferred compound 1,3,4-thiadiazole-2-cyanamide may be present as follows:

Examples of tautomers of another compound of the invention are represented by the following structural formula:

All possible tautomers of the compounds of the invention are included in the invention.

The following detailed examples illustrate the method for the synthesis of thiadiazole used in the antiviral method of the present invention.

Preparation Example 1

[(1,3,4-thiadiazol-2-ylamino) thioxomethyl] carbamic acid, ethyl ester

To a stirred solution of 19.2 g (190 mmol) of 2-amino-1,3,4-thiadiazole in 200 ml of acetonitrile was added 25 g (190 mmol) of ethoxycarbonyl isothiocyanate at one time. The reaction mixture is stirred at 24 ° C. for 16 h. The precipitates were collected by filtration, washed three times with ethyl acetate and dried to give 35 g (80% yield) of [(1,3,4-thiadiazol-2-ylamino) thioxomethyl] carbamic acid, ethyl ester. .

Elemental Analysis of C ₆ H ₈ N ₄ O ₂ S ₂ :

Theoretic value: C; 31.02, H; 3.47, N; 24.12

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

Production Example 2

1,3,4-thiadiazole-2-ylthiourea

A solution of 150 ml of 1 N sodium hydroxide [(1,3,4-thiadiazol-2-ylamino) thioxomethyl] carbamic acid, 10 g, ethyl ester (obtained from Preparation Example 1) was added at reflux for 90 minutes. Heat. The solution is cooled and concentrated by evaporation under reduced pressure. The precipitated solid is collected by filtration and dissolved in 20 ml of water. The aqueous solution is acidified by adding 200 ml of 1N hydrochloric acid. The product was collected by filtration and recrystallized from N, N-dimethyl-formamide to give 5 g of 1,3,4-thiadiazol-2-ylthiourea (melting point: 253 ° C).

Elemental Analysis of C ₃ H ₄ N ₄ S ₂ :

Theoretic value: C; 22.49, H; 2.52, N; 34.97, S; 40.02

Found: C; 22.73, H; 2.33, N; 34.74, S; 40.26

Example 1

N´-1,3,4-thiadiazol-2-ylcarbamimidothioic acid, methyl ester

A mixture of 4.8 g of 1,3,4-thiadiazol-2-ylthiourea (prepared from Preparation Example 2) of 45 ml of 1N sodium hydroxide, 15 ml of ethanol and 4 ml of methyl iodide is heated at 40 ° C. for 10 minutes. . The mixture is acidified by adding 50 ml of 1N hydrochloric acid. The reaction mixture is concentrated by evaporation under reduced pressure. The precipitated solid was collected by filtration to give 3.02 g of N ¹ -1,3,4-thiadiazol-2-ylcarbamimido thio acid and methyl ester (melting point: 116 to 117 ° C).

Elemental Analysis of C ₄ H ₆ N ₄ S ₂ :

Theoretic value: C; 27.57, H; 3.47, N; 32.15, S; 36.80

Found: C; 27.78, H; 3.55, N; 31.92, S; 36.54

2.42(s,3H,S-CH₃), 8.90(브로드 s,2H,NH₂) 9.08(s,1H,환H). ¹ H nmr (300MHz) (D ₆ DMSO / Me ₄ Si)

2.42 (s, 3H, S-CH ₃ ), 8.90 (broad s, 2H, NH ₂ ) 9.08 (s, 1H, cyclic H).

FD mass spectrum; Parent Ion 174

Example 2

N´-1,3,4-thiadiazol-2-ylcarbamimidothioic acid, ethyl ester

3.2 g (20 mmol) of 1,3,4-thiadiazol-2-ylthiourea in 100 ml of N, N-dimethylformamide containing 1.6 ml (20 mmol) of ethyl iodide and 2.12 g (20 mmol) of sodium carbonate ) Solution is stirred at 24 ° C. for 24 h. The reaction mixture is filtered and the filtrate is concentrated to an oily substance by evaporation of the solvent under reduced pressure. The oil obtained is chromatographed on a column (Waters prep 500 column), eluting with 75% ethyl acetate / hexane (v / v). The appropriate fractions are combined and concentrated to dryness to give a solid, which is crystallized from diethyl ether and hexanes to identify as 2.08 g of N ¹ -1,3,4-thiadiazol-2-ylcarbamididothioic acid.

Elemental Analysis of C ₅ H ₈ N ₄ S ₂ :

Theoretic value: C; 31.90, H; 4.28, N; 29.76, S; 34.06

Found: C; 32.18, H; 4.18, N; 29.59, S; 34.08

1.06(t,3H,CH₃), 3.02(g,2H,CH₂),8.88(브로드 s,2H,NH₂), 9.08(s,1H,환H). ¹ H nmr (300MHz) (D ₆ DMSO / Me ₄ Si)

1.06 (t, 3H, CH ₃ ), 3.02 (g, 2H, CH ₂ ), 8.88 (broad s, 2H, NH ₂ ), 9.08 (s, 1H, ring H).

ir (KBr) cm ^-1 : 3259.9, 3089.2, 1624.2, 1510.4, 1416.8, 1377.3, 1347.4, 1247.1, 1209.5, 781.5

FD Mass Spectrum: 188

[Examples 3 to 5]

The following thiadiazolyl carbamididothio acid esters are prepared by reacting 1,3,4-thiadiazol-2-ylthiourea with alkyl halides according to the processes of Examples 1 and 2.

N ¹ -1,3,4-thiadiazol-2-ylcarbamimidothioic acid, n-butyl ester; Yield 1.68 g; Melting point 40 ° C.

0.90(t,3H,CH₃), 1.38(6중선,2H,-CH₂-), 1.60(5중선,2H,-CH₂-), 3.04(3중선,2H,-CH₂), 8.90(브로드 s,2H,NH₂), 9.09(S,1H,환H). ¹ H nmr (300MHz) (D ₆ DMSO / Me ₄ Si)

0.90 (t, 3H, CH ₃ ), 1.38 (hexadecimal wire, 2H, -CH _2- ), 1.60 (pentine wire, 2H, -CH _2- ), 3.04 (triple wire, 2H, -CH ₂ ), 8.90 ( Broad s, 2H, NH ₂ ), 9.09 (S, 1H, cyclic H).

FD mass spectrum: 216

N ¹ -1,3,4-thiadiazol-2-ylcarbamimidothioic acid, 2-pyridinylmethyl ester, hydrochloride; Yield 3.72 g

4.64(s,2H,CH₂), 7.82(t,1H,피리딘H), 7.98 (d,1H,피리딘H), 8.40(t,1H,피리딘H), 8.79(d,1H,피리딘H), 9.11(s,1H,환H). ¹ H nmr (300MHz) (D ₆ DMSO / Me ₄ Si)

4.64 (s, 2H, CH ₂ ), 7.82 (t, 1H, pyridine H), 7.98 (d, 1H, pyridine H), 8.40 (t, 1H, pyridine H), 8.79 (d, 1H, pyridine H), 9.11 (s, 1 H, cyclic H).

FD Mass Spectrum: 251

N ¹ -1,3,4-thiadiazol-2-ylcarbamimidothio acid and cyanomethyl ester; Yield 1.04 g

4.41(s,2H,CH₂), 9.09(브로드 s,2H,NH₂), 9.15(s,1H,환H). ¹ H nmr (300MHz) (D ₆ DMSO / Me ₄ Si)

4.41 (s, 2H, CH ₂ ), 9.09 (broad s, 2H, NH ₂ ), 9.15 (s, 1H, ring H).

FD Mass Spectrum: 199

Preparation Example 3

[(1,3,4-thiadiazol-2-ylamino) thioxomethyl] carbamic acid, phenyl ester

A solution of 3.8 g (50 mmol) of ammonium thiocyanate in 100 ml of acetonitrile containing 7.8 g (50 mmol) of phenyl chloroformate is stirred at 24 ° C. for 1 hour. The reaction mixture is introduced into a suspension of 6.88 g (50 mmol) of 2-amino-1,3,4-thiadiazole hydrochloride in 75 ml of acetonitrile and 25 ml of propylene oxide. The reaction mixture was stirred at 24 ° C. for 2 hours and then filtered to yield a solid precipitate identified as 6.42 g of [(1,3,4-thiadiazol-2-ylamino) thioxomethyl] carbamic acid, phenyl ester. do.

FD mass spectrum: 280

[Production Examples 4 to 6]

According to the general procedure of Preparation Example 3, the following product was prepared.

[(1,3,4-thiadiazol-2-ylamino) thioxomethyl] carbamic acid, n-hexyl ester

Elemental Analysis of C ₁₀ H ₁₆ N ₄ O ₂ S ₂ :

Theoretic value: 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

[(1,3,4-thiadiazol-2-ylamino) thioxomethyl] carbamic acid, methyl ester

FD mass spectrum: 218

[(1,3,4-thiadiazol-2-ylamino) thioxomethyl] carbamic acid, vinyl ester

FD mass spectrum: 230

Production Example 7

[(Methylthio) (1,3,4-thiadiazol-2-ylamino) methyl] carbamic acid, ethyl ester

[(1,3,4-thiadiazol-2-ylamino) thioxomethyl] carbamic acid, ethyl ester in 45 ml of 1N sodium hydroxide containing 9.0 g (4 ml) of methyl iodide and 15 ml of methanol (Preparation Example 6.96 g (30 mmol) of the mixture as prepared in 1) is heated at 40 ° C. for 10 minutes. The reaction mixture is cooled to 50 ° C. and filtered. The filter cake was washed with water and dried in air to give 4.72 g of [(methylthio) (1,3,4-thiadiazol-2-ylamino) methyl] carbamic acid, ethyl ester.

Elemental Analysis of C ₇ H ₁₀ N ₄ O ₂ S ₂ :

Theoretic value: C; 34.13, H; 4.09, N; 22.75, S; 26.04

Found: C; 34.41, H; 3.84, N; 22.91, S; 25.94

Preparation Example 8

[(Ethylthio) (1,3,4-thiadiazol-2-ylamino) methyl] carbamic acid, ethyl ester

According to the procedure of Preparation Example 7, [(1,3,4-thiadiazol-2-ylamino) -thioxomethyl] carbamic acid (6.96 g) was reacted with 4 ml of ethyl iodide [(ethylthio ) 1.83 g of (1,3,4-thiadiazol-2-ylamino) -methyl] carbamic acid, ethyl ester, are obtained.

FD mass spectrum: 260

Example 6

1,3,4-thiadiazole-2-cyanamide

N ¹ -1,3,4-thiadiazol-2-yl carbamididothioic acid, methyl ester in 200 ml of dichloromethane containing 4 g of meta-chloroperbenzoic acid (20 mmol equivalent based on 85% purity) 3.48 g (20 mmol) of the solution, prepared as described in Example 1, is stirred at 24 ° C. for 2 hours. The mixture is filtered and then the precipitate is stirred with 40 ml of water for 2 hours. The solids were collected by filtration and dried to yield 1.7 g of 1,3,4-thiadiazole-2-cyanamide.

FD mass spectrum: 126

Elemental Analysis of C ₃ H ₂ N ₄ S:

Theoretic value: C; 28.57, H; 1.60, N; 44.42, S; 25.42

Found: C; 28.95, H; 1.80, N; 43.60, S; 24.87

Example 7

1,3,4-thiadiazole-2-cyanamide sodium salt

100 ml of ethanol is added to a solution of 1.38 g of 1,3,4-thiadiazole-2-cyanamide in 10.5 ml of 1N sodium hydroxide. The mixture is filtered and the solvent is removed from the filtrate to give an oil. The oil is evaporated under reduced pressure to slowly remove methanol to crystallize from 20 ml of methanol and 100 ml of isopropyl alcohol to give 980 mg of 1,3,4-thiadiazole-2-cyanamide sodium salt.

¹ H nmr (300MHz) (D ₆ DMSO / Me ₄ Si) δ 8.6 (s, 1H, ring H)

FD mass spectrum: 126

Elemental Analysis of C ₃ HN ₄ SN _a :

Theoretic value: C; 24.33, H; 0.68, N; 37.83, S; 21.65

Found: C; 24.59, H; 0.87, N; 38.06, S; 21.90

Example 8

1,3,4-thiadiazole-2-guanidine

A mixture of methyl ester (prepared from Example 1) of 5.0 g of N ¹ -1,3,4-thiadiazol-2-ylcarbamididothioic acid in 50 ml of ammonia and 100 ml of ethanol was prepared in a bomb at 140 ° C. Heat for hours. TLC (66% CHCI ₃ , 26% MeOH, 8% HOAC) on a silica gel plate reveals two new spots, one of which is identical to 1,3,4-thiadiazole-2-cyanamide (Example 6) It is deployed. The solvent is removed by evaporation and the crude is stirred with 200 ml of 1N HCI and filtered to remove insoluble cyanamide. The HCI solution was evaporated to dryness and the residue was purified by reverse phase HPLC eluting with 90% MeOH / H ₂ O to afford 1.5 g of 1,3,4-thiadiazole-2-guanidine.

¹ H nmr (300 MHz) (D ₆ DMSO / Me ₄ Si) δ 8.37 (Broad s, 4H, NH ₂ ), 9.27 (s, 1H, Ring H).

FD mass spectrum: 144

Example 9

1,3,4-thiadiazole-2-cyanamide

A. Benzylation of 2-amino-1,3,4-thiadiazole

A mixture of 101 g 2-amino-1,3,4-thiadiazole, n-propanol, 650 ml, and 140 ml benzyl bromide is heated in 21 flasks. When the temperature of the reaction mixture reaches 88 ° C., all of the 2-amino-1,3,4-thiadiazoles are in solution. The solution is refluxed at 98 ° C. and the flask is separated from the heating mantle and placed in a water bath to control reflux. An additional 250 ml of n-propanol is added to maintain the fluidity of the mixture, optionally heating and stirring the mixture for 2 hours while maintaining the temperature at 86 ° C. The mixture is then cooled to 30 ° C. The product 4-amino-3 (phenylmethyl) -1,3,4-thiadiazole hydrobromide is collected, washed with n-propanol and dried in air.

Yield: 190 g (70%), melting point: 200 to 202 캜

B. Reaction with Cyanogenbromide

To a stirred mixture of 4-amino-3-phenylmethyl-1,3,4-thiadiazole hydrobromide, water 11, and ethyl acetate 11 was added 44 ml of a 50% by weight solution of sodium hydroxide in water to thiadiazole. Dissolve the starting material. The aqueous layer is then separated off. To the remaining ethyl acetate solution, 800 ml of water containing 84 g of sodium bicarbonate are added. To this solution is added dropwise a solution of 56.3 g of cyanogen bromide in 150 ml of ethyl acetate over 25 minutes. The mixture is stirred for an additional 30 minutes. Thereafter, the aqueous layer is separated and discarded. The remaining ethyl acetate solution is washed with 200 ml of water. A saturated salt solution is added to expedite the separation of the layers, after which the aqueous layer is separated off. The ethyl acetate solution is concentrated to one third of the original volume and then 100 ml of toluene is added. The mixture is concentrated to one third of the original volume, and 100 ml of toluene is also added. This concentration was repeated several times to give the desired (3-phenylmethyl-1,3,4-thiadiazole-2 (3H) -ylidene) cyanamide as a precipitate, which was isolated by filtration and toluene. Wash and dry. Yield: 57.7 g (50.6%)

C. Debenzylation Reaction

6 equivalents (7.4 g) of finely ground aluminum chloride are added to a mixture of 2.0 g of (3-phenylmethyl-1,3,4-thiadiazole-2 (3H) -ylidene) cyanamide in 50 ml of methylene chloride. . The mixture is stirred for 2 hours and 50 ml of THF are added. The mixture is then poured into 500 ml of cold water, stirred for 10 minutes and then filtered to remove the water insoluble matter. Then about 5 g of sodium chloride is added to the mixture to facilitate separation of the layers and to separate the THF layer. Further 2 g of salt is added to the aqueous layer and extracted twice with 25 ml of THF. The THF layers are combined, dried over magnesium sulfate and concentrated to give a solid. 25 ml of methylene chloride was added to the solids, and the solids were collected and dried to yield 0.91 g of 1,3,4-thiadiazole-2-cyanamide (yield 78%), which showed a purity of 96.7% by HPLC. [Melting point: 155 ° C. (decomposition)].

Accordingly, the present invention also provides a) 1,3,4-thiadiazol-2-ylthiourea of the general formula R ⁶ X, wherein R ⁶ is as defined below and X is a leaving group. Reacting with an alkylating agent to yield a compound of formula (I) wherein R ¹ and R ² combine to form C (NH ₂ ) (SR ⁶ ) wherein R ⁶ is defined below, or b) R ¹ and R ² combine to form = C (NH ₂ ) (SR ⁶ ), wherein R ⁶ is defined below, with a oxidizing agent to react with R ¹ Is hydrogen and R ² is —CN, to obtain a compound of formula (I), or c) R ¹ and R ² are bonded to = C (NH ₂ ) (SR ⁶ ), where R ⁶ is And a compound of formula (I) wherein R ¹ is hydrogen and R ² is -CN and R ¹ and R ² are bonded to C (NH ₂₎ compounds of formula (ⅰ) to form a _second Obtained to separate the two compounds, or d) debenzylated (3-phenylmethyl-1,3,4-thiadiazole-2 (3H) -ylidene) cyanamide to yield 1,3,4 -Thiadiazole-2-cyanamide, or e) reacting a compound of formula (I) wherein R ¹ is hydrogen and R ² is -CN with an appropriate organic or inorganic base to obtain a pharmaceutically acceptable salt Characterized in that the present invention provides a method for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In which R ¹ is hydrogen; R ² is -CN; R ¹ and R ² are joined to = C (NH ₂ ) (SR ⁶ ) or C (NH ₂ ) ₂ , wherein R ⁶ is C ₁ -C ₆ alkyl, cyano-C ₁ -C ₄ alkyl, or Pyridyl-C ₁ -C ₄ alkyl).

The thiadiazoles defined above have demonstrated antiviral activity in standard test methods and can therefore be used for the treatment or prevention of diseases commonly caused by a wide range of viruses. Representative viruses in which thiadiazole is active include A-Ann Arbor, A-Hong Kong, B-Great Lakes, B-Taiwan, B-Singapore, A-Brazil, A-Texas, A-Fukushina, and B-Maryland. Both strains A and B of influenza tested, including influenza strains, are included. Other viruses that can be controlled in accordance with the invention include Para-influenza, Respiratory Syncytial Virus, various Herpes I and II strains, Echo and Vaccinia viruses. , Margin viruses, Semliki Forest viruses, and viruses causing AIDS.

Thiadiazole antiviral agents contemplated by the present invention exhibit antiviral activity both in vitro and in vivo. Their in vivo activity against influenza A Ann Arbor strains is particularly well documented.

In a series of in vivo tests, a group of 18 CD-1 species mice were infected with a given amount of influenza A Ann Arbor strain. In addition, one group to which a predetermined amount of thiadiazole was administered and the vehicle to which the vehicle was administered serves as a control group.

This test lasts 10 days. Record the number of animals that die in each group each day. After 10 days, live animals are considered survivors. The data is then analyzed using the "Survival index" method. The analysis is performed as follows.

From the data for the control, survival index values (SI _{DAY X} ) for each _day ( _X ) are determined starting from the day the first death occurred in the control (typically X = 4 days) and continuing until day 10. The survival index for the first day of death is set to zero. The value for the remaining days until 10 days is calculated using the following formula.

이다.For example, if the first day the control animal died on day 4 and four of the 18 control animals died on day 5, SI _{DAY 4} is 0,

to be.

The survival index value "S-I-survivor" for survivors is also calculated from the data for control animals using the following formula.

The resulting set of calculated index values is then used to determine the average survival for both the control group and each group of animals to be tested using the following test methods. Average survival for the control group, "SI control", multiplies each SI _{DAY X} value by the number of control animals that died on the corresponding day (X), and multiplies the SI survivor, if any, by the number of survivors, multiplied by this The sum is divided by the total number of control animals to calculate. Average survival for treatment group, "SI treatment group", multiplies each SI _{DAY X} value by the number of treated animals that died on the corresponding day (X), multiplies the SI survivors by the number of treated animals that survived, and multiplies these multiplied values The sum is divided by the total number of treated animals and calculated.

The comparison with the control is then calculated by the following equation, where P is the maximum percentage:

Using this equation, P is 0% when the average survival for the treated animals is only the same as the average survival for the control. If the mean survival for treated animals is equal to the survival index for survivors, P is 100%.

This maximum percentage is designated RA by the relative activity index as follows.

P RA

Less than 20% = 1 (boundary)

20-40% = 2 (slightly active)

40-60% = 3 (moderate activity)

60-80% = 4 (good activity)

80-100% = 5 (very active)

Table I below shows the RA values for representative compounds of the present invention.

TABLE I

The compounds also show good activity in vivo against various influenza strains in mouse influenza testing. Table II shows the activity of mice infected with various influenza A and B strains when administered orally with the compound of Preparation Example 2 over a wide range of doses. The table shows the number of animals that survived after 10 days in each test group.

TABLE II

Note: (-) means not evaluated at that dosage level.

A plaque-reduction study provides a quantitative assessment of virus proliferation inhibitors in an in vitro tissue culture cell system.

In this test, sensitive MDCK cells were embryonated at 37 ° C with 5% inactivated bovine fetal calf serum (FBS), penicillin (300 units / ml) and streptomycin (300 μg / ml) in medium 199 in a 25 cm ² Falcon flask. do. Once the adherent monolayer is formed, the growth medium is removed and 0.3 ml of the appropriate dilution of the virus is added to each flask. After adsorbing at room temperature for one hour, the infected cell sheets are covered with an equivalent amount of 1% agarose and 2 × medium 199, 2.5% FBS, penicillin, and stereptomycin, with varying concentrations of test compound incorporated into the agar coating. The compound is dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10,000 μg / ml, and the aliquots are diluted to the desired concentration using an agar medium mixture. Incubate the flask until the optimal spot size (2-10 mm) is obtained in the control flask. A solution containing 10% formalin and 2% sodium acetate is added to each flask to inactivate the virus and fix the cellsheet to the plastic surface. Stain the spots after staining the area around the cells using crystal violet. The results for each concentration obtained from the two flasks are averaged and compared with the control flask. The inhibition of speckle production of 50% (I ₅₀ ) plots and predicts all results obtained from 10 to 90% inhibition. The results for the three influenza strains are reported in Table III.

TABLE III

In vitro Activity IC ₅₀ (MCG / ML) for Influenza Virus

When used in vitro, compounds can be added to tissue cultures to inhibit viral growth in the cultures.

Preferred methods of using the compound are in vivo, in which case the compound is parenterally, topically or orally administered, inhaled or parenteral to a mammal suffering from a virus or susceptible to the virus. Administration via the furnace. For parenteral administration, such as intraperitoneal administration, the compound may be dissolved or suspended in water containing 2% of a surfactant, in particular an emulsion (polyhydroxylated fatty acid). Oral administration is, of course, preferred. When used as such, the compounds defined herein are mixed with one or more standard pharmaceutically acceptable bulking media, such as starch, sucrose, lactose, calcium carbonate, and the like, and the mixture is combined with each capsule expected or present. Is filled into a gelatin cocapsule that is embedded to contain an amount of a compound effective to inhibit the growth of influenza or other viruses. In other words, the compound can be used as a prophylactic or therapeutic agent. The agent can also be mixed with various excipients, including starch, lubricants, wetting agents, and the like, such as stearic acid, magnesium stearate, etc., and tableting the mixture, where each tablet prevents the infection of influenza or other viruses or It contains an effective amount of a medicament for treatment. Such tablets may be segmented to provide a half or quarter dose when the drug is administered to a child. The compounds may also be administered in solution or suspension.

In order to carry out the antiviral method of the present invention, all that is needed is to add the antivirus amount of the thiadiazole antiviral agent to the tissue culture to be protected or to an animal suffering from or susceptible to a viral infection. Preferably, the compound may be formulated with a pharmaceutically acceptable diluent, for example for convenient oral, topical or parenteral administration, and may be used for therapeutic as well as prophylactic purposes. The formulations usually contain from about 1 to 95% by weight of active thiadiazole antiviral agent.

For oral administration, the compounds are formulated using conventional diluents and excipients, such as sucrose, starch, microcrystalline cellulose, acacia, etc., molded into tablets or pills, capsuled with gelatin capsules, or It may be formulated in solution, elixirs, lozenges and the like. Topical preparations include mixing thiadiazole antiviral agents with excipients such as beeswax, lanolin, oils, etc. to prepare such preparations as ointments, plasters, creams, tinctures, furnaces, patches, and the like. Include.

For severe viral infections, the antiviral thiadiazole may be formulated into a formulation for intravenous or intramuscular administration. Such formulations contain about 1 to 50 agents. The compound can be dissolved in conventional diluents such as isotonic saline or dextrose solution for intravenous infusion and in polyhydric aliphatic alcohols such as propylene glycol or polyethylene glycol for easy intravenous or intramuscular injection. .

Pharmaceutically acceptable salts can be prepared by reacting compounds of the general formula which are sufficiently acidic or basic with conventional organic and inorganic acids and bases such as hydrochloric acid, succinic acid, sodium hydroxide and the like. In general, pharmaceutically acceptable salts have elevated solubility properties compared to the compounds from which they are derived and are therefore often easier to formulate in liquids or emulsions.

The thiadiazole antiviral agents described above are active over a wide range of dosage levels. The specific dose to be administered is determined by the exact virus infection to be treated or protected and its severity, route of administration and the relevant circumstances to be determined by the attending physician, but conventional doses range from about 0.1 to 100 mg / kg, and more conventional About 0.5 to 25 mg / kg.

In a preferred method of treatment, the thiadiazole compound is administered to mammals, including horses, mice, pigs and humans, susceptible to influenza viruses. Among people, this compound is administered for the purpose of prophylaxis, in particular to the elderly, children, nurses, doctors and other hospitals or public health centers, for example in the case of a sudden infectious "pandemic". This compound may also be administered to a person who has been in contact with a person infected with a "pandemic cold." A particular advantage of the therapeutic methods of the present invention is that the compound is effective against both influenza virus A strain or B strain, so that the compound is prophylactically treated in patients without the need to determine in advance whether the virus is influenza virus A strain or B strain. Or therapeutically.

The following examples illustrate some representative formulations using compounds of formula (I).

Example 10

Manufacture of tablets

100 mg of the compound of Preparation Example 1

Lactose 200mg

Corn Starch 300mg

Corn Starch Paste 50mg

Calcium Stearate 5mg

Calcium Phosphate 45mg

The active ingredient, corn starch, lactose and calcium phosphate are mixed uniformly. Corn starch paste is made of 10% aqueous paste and mixed uniformly in the mixture. The mixture is mixed with calcium stearate and then compressed into tablets.

Example 11

Preparation of suppositories

500 mg of the compound of Example 1

Deobromo Oil 1500mg

The components are mixed uniformly at a temperature of about 60 ° C. and then cooled in a tapered mold.

Example 12

Preparation of Oral Suspension

500 mg of the compound of Example 8

Sorbitol solution (70% N.F.) 40 mg

Sodium Benzoate 150mg

Lactose 10mg

Cherry Flavor 50mg

Ethanol 100mg

The ingredients are mixed to contain 5 mg of active ingredient per ml of syrup. About 5-20 ml of syrup can be administered daily to protect humans from virus infections such as influenza.

Example 13

Intranasal formulations

% By weight

Compound 1.0 of Example 8

Antarox (nonionic polyoxy ethylation from GAF Corp.

Non-volatile oils) 38.5

Ethanol 10.0

Freon 11 (trichloromonofluoromethane) 25.0

Freon 12 (dichlorodifluoromethane) 25.0

Menthol 0.5

The active ingredient is added to Antarox at about 70-80 ° C. and the mixture is stirred until a solution is formed. Cool the solution and dilute with methanol mixture in ethanol. The resulting solution is placed in an aerosol vessel and cooled to 0 ° C., then a freon propellant is added and the aerosol vessel is sealed with a valve.

Claims (12)

A compound of formula (I) or a pharmaceutically acceptable salt thereof.

Wherein R ¹ is hydrogen and R ² is —CN or R ¹ and R ² together form = C (NH ₂ ) ₂ or = C (NH ₂ ) (SR ⁶ ) and R ⁶ is C ₁ -C ₆ alkyl, cyano-C ₁ -C ₄ alkyl, or pyridyl-C ₁ -C ₄ alkyl. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ¹ is hydrogen and R ² is —CN. The compound of formula (I) according to claim 1, wherein R ¹ and R ² together form = C (NH ₂ ) ₂ . The compound of formula (I) according to claim 1, wherein R ¹ and R ² together form = C (NH ₂ ) (SR ⁶ ). 1,3,4-thiadiazole-2-cyanamide, characterized by debenzylating (3-phenylmethyl-1,3,4-thiadiazole-2 (3H) -ylidene) cyanamide How to prepare. 6. The method of claim 5, wherein the 1,3,4-thiadiazole is debenzylated with Lewis acid. The method of claim 6, wherein the Lewis acid is aluminum chloride. Characterized by reacting 1,3,4-thiadiazol-2-ylthiourea with an alkylating agent of the general formula R ⁶ X wherein R ⁶ is as defined below and X is a leaving group To prepare a compound of formula (I) or a pharmaceutically acceptable salt thereof.

Wherein R ¹ and R ² together form = C (NH ₂ ) (SR ⁶ ), wherein R ⁶ is C ₁ -C ₆ alkyl, cyano-C ₁ -C ₄ alkyl, or pyridyl-C ₁ -C ₄ alkyl. A compound of formula (I), characterized by reacting a compound of formula (I) according to claim 1 with R ¹ and R ² together to form = C (NH ₂ ) (SR ⁶ ) with an oxidizing agent. Or a pharmaceutically acceptable salt thereof.

Wherein R ¹ is hydrogen and R ² is —CN. A compound of formula (I), characterized by reacting a compound of formula (I) according to claim 1 with R ¹ and R ² together to form = C (NH ₂ ) (SR ⁶ ) with ammonia. How to prepare.

Wherein R ¹ is hydrogen and R ² is —CN or R ¹ and R ² together form ═C (NH ₂ ) ₂ . The method of claim 10 further comprising the step of separating the two compounds produced. A process for preparing a pharmaceutically acceptable salt thereof, characterized by reacting a compound of formula (I) with an organic or inorganic base.

Wherein R ¹ is hydrogen and R ² is —CN.