THIADIAZOLE ANTIVIRAL AGENTS

Abstract: Compounds of formula (I) wherein: R¹ is hydrogen, and R² is cyano, or -C(=S)-NH-R³; or R¹ and R² taken together are =C(NH₂)₂ or =C(R⁵)(NHR³′); R³ and R³′ are hydrogen or -COOR⁴; R⁴ is C₁-C₁₀ alkyl, substituted C₁ -C₁₀ alkyl, C₂-C₁₀ alkenyl, substituted C₂-C₁₀ alkenyl, phenyl, or substituted phenyl; and R⁵ is hydrogen or -S-R⁶ where R⁶ is C₁-C₄ alkyl, cyano-C₁-C₄ alkyl or pyridyl-C₁-C₄ alkyl, and salts thereof, are useful antiviral agents.

Description: Diseases caused by one or more viruses are becoming alarmingly prevalent. Sexually transmitted viral infections and those transmitted by blood trans­ fusion have been the focus of intensive research efforts over the past several years. Less severe diseases caused by viruses include the common cold. Few truly effective antiviral agents have been developed, and in many cases the only treatment avail­ able is antibacterial agents, not to combat the virus but rather to ward off invasion of a weakened biological system by bacteria. The search continues for effective antiviral agents to treat and control common ailments such as influenza and colds, diseases caused by the various strains of herpes virus as well as to combat more severe viral infections such as acquired immune deficiency syndrome (AIDS). This invention concerns the discovery that certain 2-amino-1,3,4-thiadiazole derivatives are useful in treating viral infections. Thiadiazoles are in general well known in the art. Some have been employed in the medical field. For example, Naik et al. describe the antimicrobial activities of a number of thiadiazolylthiazolidinones; J. Indian Chem. Soc. , Vol. LX, July 1983, pp 674-678. Grant et al., in J. Med. Chem. , Vol. 15, No. 10, 1972, pp 1082-1084, disclose that certain amine substituted derivatives of 2-amino-1, 3,4-thiadiazole are useful hypotensive agents. U.S. Patent No. 3,772,316 describes a class of N-acylated 2-amino-1,3,4-thiadiazoles which are said to be useful as fungicides. Russo et al., in Farmaco. Ed. Sci. , Vol. 30, No. 12, 1975, pp 1031-1038, describe a series of thiourea derivatives of 1,3,4-thiadiazole and their cyclization products. The compounds are said to be antibacterial agents. Malinoski et al., in Virology , 110 , 281-291 (1981) describes certain antiviral activity of 2-amino-1,3,4-thiadiazole. In one embodiment, this invention provides a method for preventing, treating and controlling viral infections. More particularly one embodiment provides an in vitro antiviral method for protecting mammalian cells in culture which comprises adding to the culture an antiviral amount of a compound of formula (I): wherein: R¹ is hydrogen, and R² is cyano, or -C(=S)-NH-R³; or R¹ and R² taken together are =C(NH₂)₂ or =C(R⁵)(NHR³′); R³ and R³′ are hydrogen or -COOR⁴; R⁴ is C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, substituted C₂-C₁₀ alkenyl, phenyl, or substituted phenyl; and R⁵ is hydrogen or -S-R⁶ where R⁶ is C₁-C₄ alkyl, cyano-C₁-C₄ alkyl or pyridyl-C₁-C₄ alkyl. The compounds of formula (I) are also useful in treatment of animals suffering from a viral infection or susceptible thereto. For this purpose, prefered compounds are those of the formulas (II), (III) and (IV): where R³ is hydrogen or -COOR⁴, R⁵ is hydrogen or SR⁶, and R⁶ is C₁-C₄ alkyl. The invention also provides a series of pre­ ferred new compounds defined by the formulas (V), (VI), and (VII): The compounds of formulas (V) and (VI) are particularly useful as antivirals. The compound of formula (VII) is extremely active in vitro . The invention additionally embraces the use of pharmaceutically acceptable salts of the foregoing compounds. Several of the thiadiazoles employed in the antiviral method of this invention are known in the art. All of the compounds can be prepared by chemical processes available, for example by the processes described by Naik et al. and Russo et al. supra . Russo et al. , supra. , discloses 2-thioureido-­ 1,3,4-thiadiazole, i.e. the compound of formula (III), as an antibacterial agent. No mention of antiviral activity is made. R² in the above formula (I) includes an acyl group defined by -C(=S)-NH-R³, where R³ can be another acyl moiety -COOR⁴. This definition of R² provides the N-acyl thioureas of the invention, many of which are included in a preferred method of treatment. Such thioureas have the formula (II): where R⁴ has the above-defined meaning. Many of these thiadiazole thioureas are known in the art, for example as described in Japanese Patent Nos. 71/35262, which is directed to mycocides, bacteriocides and herbicides, and 74/7218, which is directed to fungicides and bacteriocides. In defining the foregoing compounds, R⁴ in­ cludes "C₁-C₁₀ alkyl" and "substituted C₁-C₁₀ alkyl". These terms refer to straight and branched chain alkyl such as ethyl, n-hexyl, isodecyl, 6-ethylheptyl, and optionally substituted alkyl such as haloalkyl, hydroxy­ alkyl, phenylalkyl and the like. "Halo" includes fluoro, chloro, bromo and iodo. R⁴ can additionally be C₂-C₁₀ alkenyl or substituted alkenyl such as allyl, 4-hexenyl, 3-chloro-5-heptenyl, 2-hydroxy-4-isoheptenyl and the like, as well as substituted phenyl such as chlorophenyl, hydroxyphenyl, methylphenyl, cyanophenyl and the like. Preferred R⁴ groups are C₁-C₆ alkyl and C₂-C₆ alkenyl. Most of the foregoing compounds are named herein as carbamimidothioic acid derivatives and can be prepared by reacting a 1,3,4-thiadiazol-2-ylthuourea with an alkylating agent of the formula R⁶X where R⁶ is as defined above and X is a leaving group. Suitable leaving groups include Cl, Br, I, and sulfonic ester groups like tosylate and mesylate. Thus, suitable alkylating agents include, for example, an alkyl halide such as methyl bromide, or a substituted alkyl halide such as cyanomethyl bromide or 2-pyridylmethyl iodide. The reactions are typically accomplished by mixing approx­ imately equimolar quantities of a 2-thiadiazolyl thio­ urea and an alkylating agent in the presence of a base such as sodium carbonate and in an unreactive organic solvent such as dimethylformamide or acetonitrile. The reaction is normally complete within about sixteen hours when carried out at about 20°C to about 50°C. The reaction solvent can be removed by evaporation under reduced pressure if desired. The product is readily purified by routine methods, including crystallization from solvents such as ethanol, ethyl acetate, hexane; or chromatography over solid supports such as silica and the like. A particularly preferred group of compounds provided by this invention includes 1,3,4-thiadiazol-­ 2-cyanamide and its pharmaceutically acceptable salts. The compound can be prepared by reacting a carbamimido­ thioic acid ester with an oxidizing agent such as meta ­ chloroperbenzoic acid, peracetic acid, sodium peroxide, hydrogen peroxide, ozone, chlorine or the like. In accordance with another aspect of the invention, 1,3,4-Thiadiazole-2-cyanamide can also be prepared by reacting a protected 2-amino-thiadiazole derivative, namely 4-imino-3-phenylmethyl-1,3,4-thiadia­ zole with cyanogen bromide and then removing the protect­ ing group at the 3-position by debenzylation with a Lewis acid, e.g. aluminum chloride. The debenzylation is carried out in an organic solvent such as methylene chloride, toluene or benzene. Preferably at least 2 equivalents of Lewis acid are used, and more preferably 4 to 8 equivalents are used. The temperature is not critical. The debenzylation can be carried out, for example, at from 0°C to 90°C. Room temperature is preferred. The cyanamide is also produced when a carbamimi­ diothioic acid ester is reacted with ammonia, as illust­ rated in Example 8. The cyanamide readily forms pharmaceu-tically acceptable salts by reaction with organic and inorganic bases such as sodium acetate, calcium carbonate, sodium hydroxide and the like. The compounds of this invention can exist in several tautomeric forms, all of which are included within the scope of the invention. For example, a preferred compound, 1,3,4-thiadiazol-2-cyanamide, can exist as follows: Another example of tautomeric forms of an invention compound is represented by the following formulas: All possible tautomeric forms of the invention com­ pounds are embraced herein. The following detailed examples illustrate the synthesis of thiadiazoles to be employed in the antiviral method of this invention. Preparation 1 [(1,3,4-Thiadiazol-2-ylamino)thioxomethyl]-­ carbamic acid, ethyl ester To a stirred solution of 19.2 g (190 mM) of 2-amino-1,3,4-thiadiazole in 200 ml of acetonitrile were added in one portion 25 g (190 mM) of ethoxycar­ bonyl isothiocyanate. The reaction mixture was stirred at 24°C for sixteen hours. The precipitate was col­ lected by filtration, washed three times with ethyl acetate and dried to provide 35 g (80% yield) of [(1,3,4-thiadiazol-2-ylamino)thioxomethyl]carbamic acid, ethyl ester. Analysis calc. for C₆H₈N₄O₂S₂ Theory: C, 31.02; H, 3.47; N, 24.12. Found: C, 31.32; H, 3.27; N, 24.40. Preparation 2 1,3,4-Thiadiazol-2-ylthiourea A solution of 10 g of [(1,3,4-thiadiazol-­ 2-ylamino)thioxomethyl]carbamic acid, ethyl ester (from Example 1) in 150 ml of 1N sodium hydroxide was heated at reflux for ninety minutes. The solu­ tion was cooled and concentrated by evaporation under reduced pressure. The precipitated solid was col­ lected by filtration and dissolved in 20 ml of water. The aqueous solution was acidified by addition of 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. m.p. 253°C. Analysis calc. for C₃H₄N₄S₂ Theory: 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 N¹-1,3,4-thiadiazol-2-ylcarbamimidothioic acid, methyl ester A mixture of 4.8 g of 1,3,4-thiadiazol-2-­ ylthiourea (from Preparation 2) in 45 ml of 1N sodium hydroxide, 15 ml of ethanol and 4 ml of methyl iodide was heated at 40°C for ten minutes. The mixture was acidified by adding 50 ml of 1N hydrochloric acid. The reaction mixture was concentrated by evaporation under reduced pressure. The precipitated solid was collected by filtration and dried to give 3.02 g of N¹-1,3,4-thiadiazol-2-ylcarbamimidothioic acid, methyl ester. m.p. 116-117°C. Analysis calc. for C₄H₆N₄S₂ Theory: 27.57; H, 3.47; N, 32.15; S, 36.80. Found: 27.78; H, 3.55; N, 31.92; S, 36.54. ¹Hnmr (300 MHz) (D₆ DMSO/Me₄ Si) δ 2.42 (s, 3H, S-CH₃), 8.90 (broad s, 2H, NH₂) 9.08 (s, 1H, ring H) FD mass spec., parent ion 174. Example 2 N¹-1,3,4-thiadiazol-2-ylcarbamimidothioic acid, ethyl ester A solution of 3.2 g (20 mM) of 1,3,4-thia­ diazol-2-ylthiourea in 100 ml of N,N-dimethylformamide containing 1.6 ml (20 mM) of ethyl iodide and 2.12 g (20 mM) of sodium carbonate was stirred at 24°C for twenty-four hours. The reaction mixture was filtered and the filtrate was concentrated to an oil by evap­ oration of solvents under reduced pressure. The oil was chromatographed over a Waters prep. 500 column, eluting with 75% ethyl acetate/hexane (v/v). The appropriate fractions were collected, concentrated to dryness to leave a solid which upon crystallization from diethyl ether and hexane was identified as 2.08 g of N¹-1,3,4-thiadiazol-2-ylcarbamimidothioic acid. Analysis calc. for C₅H₈N₄S₂ Theory: 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. ¹Hnmr (300 MHz) (D₆DMSO/Me₄Si) δ 1.06 (t, 3H, CH₃), 3.02 (q, 2H, CH₂), 8.88 (broad s, 2H, NH₂), 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, 718.5 FD mass spec.: 188. Examples 3-5 The following thiadiazolylcarbamimidothioic acid esters were prepared by reacting 1,3,4-thiadiazol-­ 2-ylthiourea with an alkyl halide according to the procedures of Examples 1 and 2. N¹-1,3,4-thiadiazol-2-ylcarbamimidothioic acid, n-butyl ester; yield 1.68 g; mp 40°C. ¹Hnmr (300 MHz) (D₆DMSO/Me₄Si) δ 0.90 (t, 3H, CH₃), 1.38 (sextet, 2H, -CH₂-), 1.60 (pentet, 2H, -CH₂-), 3.04 (triplet, 2H, -CH₂), 8.90 (broad s, 2H, NH₂), 9.09 (s, 1H, ring H). FD mass spec. 216. N¹-1,3,4-thiadiazol-2-ylcarbamimidothioic acid, 2-pyridinylmethyl ester, hydrochloride; yield 3.72 g. ¹Hnmr (300 MHz) (D₆DMSO/Me₄Si) δ 4.64 (s, 2H, CH₂), 7.82 (t, 1H, pyridine H), 7.98 (d, 1H, pyridine H), 8.40 (t, H, pyridine H), 8.79 (d, 1H, pyridine H), 9.11 (s, 1H, ring H). FD mass spec. 251. N¹ -1,3,4-thiadiazol-2-ylcarbamimidothioic acid, cyanomethyl ester; yield 1.04 g. ¹Hnmr (300 MHz) (D₆DMSO/Me₄Si) δ 4.14 (S, 2H, CH₂), 9.09 (broad s, 2H, NH₂), 9.15 (s, 1H, ring H). FD mass spec. 199. Preparation 3 [(1,3,4-Thiadiazol-2-ylamino)thioxomethyl]-­ carbamic acid, phenyl ester A solution of 3.8 g (50 mM) of ammonium thio­ cyanate in 100 ml of acetonitrile containing 7.8 g (50 mM) of phenyl chloroformate was stirred at 24°C for one hour. The reaction mixture was filtered into a suspension of 6.88 g (50 mM) of 2-amino-1,3,4-thia­ diazole hydrochloride in 75 ml of acetonitrile and 25 ml of propylene oxide. The reaction mixture was stirred for two hours at 24°C and then filtered to provide a solid precipitate identified as 6.42 g of [(1,3,4-­ thiadiazol-2-ylamino)thioxomethyl carbamic acid, phenyl ester. FD mass spec. 280. Preparation 4-6 By following the general procedure of Preparation 3, the following products were made. [(1,3,4-Thiadiazol-2-ylamino)thioxomethyl]carbamic acid, n-hexyl ester Analysis calc. for C₁₀H₁₆N₄O₂S₂ Theory: 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 spec. 218 [(1,3,4-thiadiazol-2-ylamino)thioxomethyl]-­ carbamic acid, vinyl ester FD mass spec. 230. Preparation 7 [(Methylthio)(1,3,4-thiadiazol-2-ylamino)-­ methyl]carbamic acid, ethyl ester A mixture of 6.96 g (30 mM) of [(1,3,4-thia­ diazol-2-ylamino)thioxomethyl]carbamic acid, ethyl ester (prepared as described in Preparation 1) in 45 ml of 1N sodium hydroxide containing 9.0 g (4 ml) of methyl iodide and 15 ml of methanol was heated at 40°C for ten minutes. The reaction mixture was cooled to 5°C and filtered. The filter cake was washed with water and air dried to provide 4.72 g of [(methylthio)(1,3,4-­ thiadiazol-2-ylamino)methyl]carbamic acid, ethyl ester. Analysis calc. for C₇H₁₀N₄O₂S₂ Theory: 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 8 [(Ethylthio)(1,3,4-thiadiazol-2-ylamino) methyl] carbamic acid, ethyl ester [(1,3,4-Thiadiazol-2-ylamino)thioxomethyl]-­ carbamic acid (6.96 g) was reacted with 4 ml of ethyl iodide according to the procedure of Preparation 7 to give 1.83 g of [(ethylthio) (1,3,4-thiadiazol-2-ylamino)-­ methyl]carbamic acid, ethyl ester. FD mass spec. 260. Example 6 1,3,4-Thiadiazole-2-cyanamide A solution of 3.48 g (20 mM) of N¹-1,3,4-­ thiadiazol-2-ylcarbamimidothioic acid, methyl ester (prepared as described in Example 1) in 200 ml of dichloromethane containing 4 g (20 mM equivalents based on 85% purity) of meta -chloroperbenzoic acid was stirred at 24°C for two hours. The mixture was filtered and the precipitate was then stirred with 40 ml of water for two hours. The solid was collected by filtration and dried to give 1.7 g of 1,3,4-thiadiazole-2-cyanamide. FD mass spec. 126 Analysis calc. for C₃H₂N₄S Theory: 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 To a solution of 1.38 g of 1,3,4-thiadiazole-­ 2-cyanamide in 10.5 ml of 1N sodium hydroxide were added 100 ml of ethanol. The mixture was filtered and the solvent was removed from the filtrate to provide an oil. The oil was crystallized from 20 ml of methanol and 100 ml of isopropyl alcohol by slowly removing the methanol by evaporation under reduced pressure to produce 980 mg of the sodium salt of 1,3,4-thiadia­ zole-2-cyanamide. Analysis calc. for C₃HN₄SNa Theory: 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 5.0 g of N′-1,3,4-thiadiazol-­ 2-ylcarbamidothioic acid, methl ester (from Example 1) in 50 ml of ammonia and 100 ml of ethanol was heated in a bomb at 140°C for 14 hours. TLC on silica gel plates (66% CHCl₃, 26% MeOH, 8% HOAc) showed 2 new spots, one of which ran identically with 1,3,4-thiadiazole-2-­ cyanamide (Example 6). Solvent was removed by evaporation and the crude material was stirred with 200 ml of IN HCl and filtered to remove the insoluble cyanamide. The HCl solution was evaporated to dryness and the residue purfied by reverse phase HPLC, eluting with 90% MeOH/H₂O to give 1.5 g of 1,3,4-thiadiazole-­ 2-guanidine. ¹Hnmr (300 MHz) (D₆DMSO/Me₄ Si) δ 8.37 (broad s, 4H, NH₂), 9.27 (s, 1H, ring H). FD mass spec.: 144. Example 9 1,3,4-Thiadiazole-2-cyanamide A. Benzylation of 2-amino-1,3,4-thiadiazole. A mixture of 101 g of 2-amino-1,3,4-thiadia­ zole, 650 ml of n-propanol, and 140 ml of benzyl bromide was heated in a 2 L flask. When the temperature of the reaction mixture reached at 88°C, all of the 2-amino-­ 1,3,4-thiadiazole was in solution. At 98°C the solution began refluxing and the flask was removed from the heating mantel and placed in a water bath to control the refluxing. A 250 ml additional portion of n-propanol was added to maintain fluidity of the mixture, and the mixture was stirred for 2 hours, heating when necessary to maintain a temperature of 86°C. The mixture was then cooled to 30°C. The product, 4-imino-3-(phenyl­ methyl)-1,3,4-thiadiazole hydrobromide was collected, rinsed with n-propanol, and air dried. Yield 190 g (70%), m.p. 200-202°C. B. Reaction with cyanogen bromide. To a stirred mixture of 146 g of 4-imino-3-­ phenylmethyl-1,3,4-thiadiazole hydrobromide, 1 L of water, and 1 L of ethyl acetate, 44 ml of a 50% by weight solution of sodium hydroxide in water was added to dissolve the thiadiazole starting material. The aqueous layer was then separated and discarded. To the remaining ethyl acetate solution, 800 ml of water containing 84 g of sodium bicarbonate was added. To this solution, a solution of 56.3 g of cyanogen bromide in 150 ml of ethyl acetate was added dropwise over 25 minutes. The mixture was stirred a further 30 minutes. Then, the aqueous layer was separated and discarded. The remaining ethyl acetate solution was washed with 200 ml of water. A saturated salt solution was added to speed separation of the layers, and the aqueous layer was then separated and discarded. The ethyl acetate solution was concentrated down to one third of its original volume, and then 100 ml of toluene was added. This mixture was concentrated down to about the third its original volume, and another 100 ml of toluene was added. This concentration procedure was repeated several times, producing the desired (3-phenyl­ methyl-1,3,4-thiadiazole-2(3H)-ylidene) cyanamide as a precipitate, which was isolated by filtration, washed with toluene and dried, yield 57.7g (50.6%.) C. Debenzylation. Six equivalents (7.4 g) of finely ground aluminum chloride was 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 was stirred for 2 hours, and 50 ml of THF was added. The mixture was then poured into 50 ml of cold water, stirred for ten minutes, and filtered to remove insolubles. Then about 5 g of sodium chloride was added to the mixture to aid separation of the layers and the THF layer was separated. An additional 2 g of salt was added to the aqueous layer, and two extractions with 25 ml portions of THF were carried out. The THF layers were combined, dried with magnesium sulfate, and concentrated to a solid. A 25 ml portion of methylene chloride was added to the solid, then the solid was collected and dried, providing 0.91 g of 1,3,4-thiadia­ zole-2-cyanamide (yield 78%), which was shown to be 96.7% pure by HPLC (m.p. 155d). Accordingly, the invention also provides a process for preparing a compound of the formula (I) wherein R¹ is hydrogen and R² is -CN, or R¹ and R² combine to form =C(NH₂)(SR⁶) or =C(NH₂)₂ where R⁶ is C₁-C₆ alkyl, cyano-C₁-C₆ alkyl, or pyridyl-C₁-C₄ alkyl, or a pharmaceutically acceptable salt thereof, which comprises a) reacting 1,3,4-thiadiazol-2-ylthiourea with an alkylating agent of the formula R⁶X, where R⁶ is as defined above and X is a leaving group to produce a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)(SR⁶); where R⁶ is as defined above; or b) reacting a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)(SR⁶), where R⁶ is as defined above, with an oxidizing agent to produce a compound of formula (I) wherein R¹ is hydrogen and R² is -CN; or c) reacting a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)(SR⁶), where R⁶ is as defined above, with ammonia to produce a compound of formula (I) wherein R¹ hydrogen and R² is -CN and a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)₂ and separating the two compounds; or d) debenzylating (3-phenylmethyl-1,3,4-thia­ diazole-2(3H)-ylidene) cyanamide to provide 1,3,4-­ thiadiazole-2-cyanamide; or e) reacting a compound of formula (I) wherein R¹ is hydrogen and R² is -CN with an appropriate organic or inorganic base to provide a pharmaceutically acceptable salt. The thiadiazoles defined above have demon­ strated antiviral activity in standard tests and thus can be employed to treat or prevent diseases commonly caused by a wide range of viruses. Typical viruses against which the thiadiazoles are active include all A and B strains of influenza tested, including influenza strains such as A-Ann Arbor, A-Hong Kong, B-Great Lakes, B-Taiwan, B-Singapore, A-Brazil, A-Texas, A-Fukushina, B-Maryland and the like. Other viruses that can be controlled according to this invention include Para-­ influenza, Respiratory Syncytial Viruses, the various Herpes I and II strains, Echo and Vaccinia viruses, measles, Semliki Forest, as well as those responsible for causing acquired immune deficiency syndrome. The thiadiazole antiviral agents contemplated by this invention have exhibited their antiviral activity both in vitro and in vivo . Their in vivo activity against influenza A Ann Arbor strain is particularly well-documented. In an in vivo test series, groups of eighteen CD-1 mice were challenged with predetermined dose of influenza A Ann Arbor strain. A predetermined dose of the thiadiazole was also administered, and one group that was given vehicle alone served as a control. The test continued for 10 days. The number of animals dying in each group on each day was recorded. The animals living at the end of 10 days were considered survivors. The "survival index" method was then used to analyze the data. This analysis is carried out as follows. From the data for the control group, a survival index value (SI DayX ) is assigned to each day (X), starting with the day on which a death first occurs in the control group (typically on Day X=4), and continuing through day 10. The survival index value for the day on which a death first occurs is defined to be zero. The values for the remaining days through day 10 are calculated using the formula For example, if the first day on which a control animal dies is day 4, and 4 of 18 control animals die on day 5, then SI Day 4 = Zero, and SI Day 5 = (5-1) + 0 = = 0.66 A survival index value for survivors, "S.I. survivors" is also calculated from data for the control animals, using the formula The resulting set of calculated index values is then used to assign average survival values both to the control group and to each group of tested animals using the following procedure. The average survival value for the control group, "SI controls ", is calculated by multiplying each SI DayX value by the number of control animals dying on the corresponding day (X), multiplying the SI survivors value by the number of survivors, if any, summing these products, and dividing the sum by the total number of control animals. The average survival value for a treated group, "SI treated ," is calculated by multiplying each SI DayX value by the number of treated animals dying on the corresponding day (X), multiplying SI surviviors by the number of surviving treated animals, summing these products, and dividing the sum by the total number of treated animals. Next a comparison with the control group is calculated by the following formula where P = percent of maximum. Using this formula, if the average survival value for the treated animals is only equal to the average survival value for the controls, then P=0%. If the average survival value for the treated animals is equal to the survival index value for survivors, then P=100%. This percent of maximum is then allocated to a relative activity index RA as follows: The following Table I gives RA values for representative compounds of this invention. The compounds have additionally demonstrated good in vivo activity against a wide variety of influ­ enza strains in the mouse influenza assay. Table II shows the activity of the compound of Preparation 2 over a wide dose range when administered orally in the diet to mice infected with various influenza A and B strains. The table shows the number of animals surviving after ten days of each test group. Plaque-reduction studies provide a quantita­ tive evaluation of inhibitors of virus multiplication in a tissue culture cell in vitro system. In this test, susceptible MDCK cells, were grown in 25 cm² Falcon flasks at 37°C in Medium 199 with 5 percent inactivated fetal bovine serum (FBS), penicil­ lin (300 units/ml) and streptomycin (300 µg/ml). When confluent monolayers were formed, growth medium was removed and 0.3 ml of an appropriate dilution of virus was added to each flask. After adsorption for one hour at room temperature, the infected cell sheet was over­ laid with equal parts of 1 percent Agarose and 2 X Medium 199, 2.5 percent FBS, penicillin, and streptomy­ cin, varying concentrations of tested compound were incorporated in agar overlay. The compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentra­ tion of 10,000 µg/ml and then an aliquot was diluted to the desired concentration with the agar medium mixture. Flasks were incubated until control flasks indicated optimum plaque size (2-10 mm). A solution containing 10 percent formalin and 2 percent sodium acetate was added to each flask to inactivate the virus and fix the cell sheet to the plastic surface. The plaques were counted after staining the surrounding cell areas with crystal violet. Results from duplicate flasks at each concen­ tration were averaged and compared with control flasks. The inhibition of plaque formation by 50 percent (I₅₀) was estimated by plotting all results from 10 to 90 percent inhibition. Results for three influenza strains are reported in Table III. TABLE III In Vitro Activity Against Influenza Viruses For in vitro use, the compounds can be added to tissue culture to suppress viral growth therein. The preferred manner of using the compounds is for in vivo use, whereby the compounds can be admin­ istered either parenterally, topically, orally, or by inhalation or the intranasal route to a mammal suffering from a viral infection or susceptible thereto. For parenteral administration, as by the intraperitoneal route, the compound may be dissolved or suspended in water containing 2% of a surface active agent, partic­ ularly an emulphor (a polyhydroxylated fatty acid). Oral administration is, of course, preferred. For such use, a compound as defined herein is mixed with one or more standard pharmaceutically-acceptable extending media such as starch, sucrose, lactose, calcium car­ bonate etc. and the mixture loaded into empty tele­ scoping gelatin capsules, such that each capsule con­ tains an amount of a compound effective to suppress the growth of influenza or other virus, either prospective or present. In other words, the compounds can be used prophylactically or as curative agents. Alternatively, the drug can be mixed with various excipients including starch, lubricating agents, wetting agents, etc., such as stearic acid, magnesium stearate and the like, and the mixture pressed into tablets, each tablet containing an amount of the drug effective to abort or cure an attack of influenza or other virus. Such tablets can be scored so as to provide half or quarter dosages where the drug is to be administered to children. The com­ pounds can also be administered in solution or sus­ pension. To practice the antiviral method of this invention, all that is required is that an antiviral amount of a thiadiazole antiviral agent be applied to the surface to be treated or be administered to an animal suffering from or susceptible to a viral infec­ tion. The compounds will ideally be formulated with pharmaceutically acceptable diluents for convenient administration, for example orally, topically or parenterally, and can be employed for prophylatic as well as therapeutic treatment. The formulations will normally contain from about 1 to about 95 percent by weight of active thiadiazole antiviral agent. For oral administration, the compounds will be formulated with common diluents and excipients such as sucrose, starch, microcrystalline cellulose, acacia and the like, and molded into tablets or pills or encapsulated into gelatin capsules, or formulated as solutions, exlixirs, lozenges or the like. Topical formulations will include mixing the thiadiazole anti­ viral agent with excipients such as bees wax, lanolin, oil and the like for ready formulation as ointments, salves, creams, tinctures, lotions, patches and the like. For severe viral infections, the antiviral thiadiazoles will be formulated for intravenous or intramuscular administration. Such formulations will contain from about 1 to about 50 percent active agent. The compounds will be dissolved in common diluents such as isotonic saline or dextrose solutions, for intravenous infusion, and can be dissolved in poly­ hydric aliphatic alcohols such as propylene glycol or polyethylene glycol for easy intravenous or intra­ muscular injection. Pharmaceutically acceptable salts can be prepared from those compounds of the above formula sufficiently acidic or basic to react with common organic and inorganic acids and bases such as hydro­ chloric acid, succinic acid, sodium hydroxide, and the like. The pharmaceutically acceptable salts generally have enhanced solubility characteristics compared to the compound from which they are derived, and thus are often more amenable to formulation as liquids or emulsions. The thiadiazole antiviral agents described above are active over a wide range of dose levels. While the particular dose to be administered will be determined by the precise viral infection to be treated or guarded against and its severity, the route of administration, and related circumstances that will be determined by attending medical practitioners, the normal dose will range from about .1 to about 100 mg/kg, and more typically about .5 to about 25 mg/kg. In a preferred method of treatment, the thiadiazole compounds are administered to mammals susceptible to infection with influenza virus including horses, mice, pigs and humans. Among humans, the compounds are administered prophylactically particularly to the elderly, young children, nurses, doctors, and other hospital or public health personnel, for example when there is evidence of an imminent "flu" epidemic. The compounds can also be given to anyone having a known exposure to a person with "flu". It is a particular advantage of the therapeutic processes of this invention that the compounds may be administered either prophylac­ tically or therapeutically to patients without a pre­ liminary determination that the virus is influenza virus A strain or B strain, since the compounds are effective against both strains. The following Examples illustrate some typical formulations using compounds of Formula I. Example 10 Preparation of Tablets The compound of Preparation 1 100 mg. Lactose 200 mg. Corn Starch 300 mg. Corn Starch Paste 50 mg. Calcium Stearate 5 mg. Dicalcium Phosphate 45 mg. The active ingredient corn starch, lactose and dicalcium phosphate are uniformly blended. The corn starch paste is prepared as a 10 percent aqueous paste and is blended into the mixture to uniformity. The mixture is blended with the calcium stearate and then compressed into a tablet. Example 11 Preparation for Suppositories The compound of Example 1 500 mg. Theobromo oil 1500 mg. The above ingredients are blended to uniformity at a temperature of about 60°C. and then cooled in a tapered mold. Example 12 Preparation for oral suspension The compound of Example 8 500 mg. Sorbitol solution (70% N.F.) 40 mg. Sodium benzoate 150 mg. Lactose 10 mg. Cherry flavor 50 mg. Ethanol 100 ml. The above ingredients are combined such that each ml. of syrup contains 5 mg. of active ingredient. Administration of about 5 to about 20 ml. of the syrup each day will protect a human subject from viral infections such as influenza. Example 13 Intransal Formulation The active ingredient is added to the Antarox at about 70-80°C. and the mixture is stirred until a solution is formed. The solution is cooled and diluted with a mixture of the menthol in the ethanol. The resulting solution is placed in a aerosol container and chilled to 0°C., and the Freon propellants are added and the aerosol container is sealed with a valve.

Claims: 1. An invitro antiviral method for protecting mammalian cells in culture which comprises adding to the culture an antiviral amount of a compound of formula (I): wherein: R¹ is hydrogen, and R² is cyano or -C(=S)-NH-R³; or R¹ and R² taken together are =C(NH₂)₂ or =C(R⁵)(NHR³′), wherein: R³ is -COOR⁴; R³′ is hydrogen or -COOR⁴; R⁴ is C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, substituted C₂-C₁₀ alkenyl, phenyl, or substituted phenyl; and R₅ is hydrogen or -S-R⁶ where R₆ is C₁-C₄ alkyl, cyano-C₁-C₄ alkyl or pyridyl-C₁-C₄ alkyl. 2. An invitro antiviral method for protecting mammalian cells in culture which comprises adding to the culture an antiviral amount of a compound of formula (I), as set forth in claim 1, wherein R¹ is hydrogen, and R² is cyano or -C(=S)-NH-R³; or R¹ and R² taken together are =C(R⁵)(NHR³′) wherein: R³ and R³′ are hydrogen or -COOR⁴; R⁴ is C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, substituted C₂-C₁₀ alkenyl, phenyl, or substituted phenyl; and R⁵ is hydrogen or -S-R⁶ where R⁶ is C₁-C₄ alkyl, cyano-C₁-C₄ alkyl or pyridyl-C₁-C₄ alkyl. 3. Use of a compound of formula (I), as set forth in claim 1; wherein: R¹ is hydrogen, and R² is cyano or -C(=S)-NH-R³; or R¹ and R² taken together are =C(R⁵)(NHR³′) wherein: R³ and R³′ are hydrogen or -COOR⁴ R⁴ is C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, substituted C₂-C₁₀ alkenyl, phenyl, or substituted phenyl; and R⁵ is hydrogen or -S-R⁶ where R⁶ is C₁-C₄ alkyl, cyano-C₁-C₄ alkyl or pyridyl-C₁-C₄ alkyl, for the manufacture of a medicament for prevention or treatment of viral infection. 4. A compound of the formula (I), as set forth in claim 1, wherein R¹ is hydrogen and R² is -CN, or R¹ and R² combine to form =C(NH₂)(SR⁶) or =C(NH₂)₂, where R⁶ is C₁-C₆ alkyl, cyano-C₁-C₄ alkyl, or pyridyl-C₁-C₄ alkyl, or a pharmaceutically acceptable salt thereof. 5. A compound of claim 4 wherein R¹ is hydrogen and R² is -CN, or a pharmaceutically acceptable salt thereof. 6. A compound of claim 4 wherein R¹ and R² combine to form =C(NH₂)₂. 7. A compound of claim 4 wherein R¹ is hydrogen and R² is -CN, or R¹ and R² combine to form =C(NH₂)(SR⁶), where R⁶ is as defined in claim 4, for use as an antiviral pharmaceutical. 8. A pharmaceutical formulation comprising as active ingredient a compound of claim 4 wherein R¹ is hydrogen and R² is -CN, or R¹ and R² combine to form =C(NH₂)(SR⁶), where R⁶ is as defined in claim 4. 9. A process for preparing a compound of the formula (I), as set forth in claim 1, wherein R¹ is hydrogen and R² is -CN, or R¹ and R² combine to form =C(NH₂)(SR⁶) or =C(NH₂)₂, where R⁶ is C₁ -C₁ alkyl, cyano-C₁-C₄ alkyl, or pyridyl-C₁-C₄ alkyl, or a pharmaceutically acceptable salt thereof, which comprises a) reacting 1,3,4-thiadiazol-2-ylthiourea with an alkylating agent of the formula R⁶X, where R⁶ is as defined above and X is a leaving group, to produce a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)(SR⁶), where R⁶ is as defined above; or b) reacting a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)(SR⁶), where R⁶ is as defined above, with an oxidizing agent to produce a compound of formula (I) wherein R¹ is hydrogen and R² is -CN; or c) reacting a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)(SR⁶), where R⁶ is as defined above, with ammonia to produce a compound of formula (I) wherein R¹ is hydrogen and R² is -CN and a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)₂, and separating the two compounds; or d) reacting a compound of formula (I) wherein R¹ is hydrogen and R₂ is -CN with an appropriate organic or inorganic base to provide a pharmaceutically acceptable salt. 10. A process for preparing 1,3,4-thiadiazole-­ 2-cyanamide which comprises debenzylating (3-phenylmethyl-­ 1,3,4-thiadiazole-2(3H)-ylidene)cyanamide. 11. The process of claim 10 wherein 1,3,4-­ thiadiazole is debenzylated using a Lewis acid. 12. The process of claim 11 wherein the Lewis acid is aluminum chloride. Claims for the following contracting states :AT, GR; and ES 1. An invitro antiviral method for protecting mammalian cells in culture which comprises adding to the culture an antiviral amount of a compound of formula (I): wherein R¹ is hydrogen, and R² is cyano or -C(=S)-NH-R³; or R¹ and R² taken together are =C(NH₂)₂ or =C(R⁵)(NHR³′), wherein: R³ is -COOR⁴; R³′ is hydrogen or -COOR⁴; R⁴ is C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, substituted C₂-C₁₀ alkenyl, phenyl, or substituted phenyl; and R⁵ is hydrogen or -S-R⁶ where R⁶ is C₁-C₄ alkyl, cyano-C₁-C₄ alkyl or pyridyl-C₁-C₄ alkyl. 2. An invitro antiviral method for protecting mammalian cells in culture which comprises adding to the culture an antiviral amount of a compound of formula (I), as set forth in claim 1, wherein R¹ is hydrogen, and R₂ is cyano or -C(=S)-NH-R³; or R¹ and R² taken together are =C(R⁵)(NHR³′) wherein: R³ and R³′ are hydrogen or -COOR⁴; R₄ is C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, substituted C₂-C₁₀ alkenyl, phenyl, or substituted phenyl; and R⁵ is hydrogen or -S-R⁶ where R⁶ is C₁-C₄ alkyl, cyano-C₁-C₄ alkyl or pyridyl-C₁-C₄ alkyl. 3. A process for preparing a compound of the formula (I), as set forth in claim 1, wherein R¹ is hydrogen and R² is -CN, or R¹ and R² combine to form =C(NH₂)(SR⁶) or =C(NH₂)₂, where R⁶ is C₁-C₆ alkyl, cyano-C₁-C₄ alkyl, or pyridyl-C₁-C₄ alkyl, or a pharmaceutically acceptable salt thereof, which comprises. a) reacting 1,3,4-thiadiazol-2-ylthiourea with an alkylating agent of the formula R⁶X, where R⁶ is as defined above and X is a leaving group, to produce a compound of formula (I) wherein R¹ and R² combine to form=C(NH₂)(SR⁶), where R⁶ is as defined above; or b) reacting a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)(SR⁶), where R⁶ is as defined above, with an oxidizing agent to produce a compound of formula (I) wherein R¹ is hydrogen and R² is -CN; or c) reacting a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)(SR⁶), where R⁶ is as defined above, with ammonia to produce a compound of formula (I) wherein R¹ is hydrogen and R² is -CN and a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)₂, and separating the two compounds; or d) reacting a compound of formula (I) wherein R¹ is hydrogen and R₂ is -CN with an appropriate organic or inorganic base to provide a pharmaceutically acceptable salt. 4. The process of claim 3 wherein a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)(SR⁶), and R⁶ is as defined in claim 3, is reacted with an oxidizing agent to produce 1,3,4-thiadiazole-2-cyanamide. 5. The process of claim 3 wherein 1,3,4-thiadiazole-2-cyanamide is reacted with an appropriate organic or inorganic base to provide a pharmaceutically acceptable salt. 6. The process of claim 3 wherein a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)(SR⁶), where R⁶ is as defined in claim 3, is reacted with ammonia to produce 1,3,4-thiadiazole-2-guanidine. 7. The process of claim 3 wherein 1,3,4-thiadiazole-2-ylthiourea is reacted with an alkylating agent of the formula R⁶X, where R⁶ is as defined in claim 3, and X is a leaving group, to produce a compound of formula (I) wherein R¹ and R² combine to form =C(NH₂)(SR⁶). 8. A compound of formula (I) as defined in claim 3 whenever prepared by the process of claim 3. 9. A process for preparing 1,3,4-thiadiazole-­ 2-cyanamide which comprises debenzylating (3-phenylmethyl-­ 1,3,4-thiadiazole-2(3H)-ylidene)cyanamide. 10. The process of claim 9 wherein 1,3,4-­ thiadiazole is debenzylated using a Lewis acid. 11. The process of claim 10 wherein the Lewis acid is aluminum chloride.

Inventors: BLANCHARD, WILLIAM BEVAN, PAGET, CHARLES JOHNSON, JR., RIEDER, BRENT JEFFREY, SPITZER, WAYNE ALFRED, WU, CHYUN-YEH EARNEST
Applicants: ELI LILLY AND COMPANY
Application year: 1986
Main classification: C12N5
IPC codes: C07D417, A61K31, C07D285, A61L2, A01N47, A61P31