The present invention relates to compounds and compositions for preventing and treating virus infections and the diseases associated therewith, particularly those infections and associated diseases caused by viruses in the Flaviviridae family, especially the pestiviruses.
The Flaviviridae family of viruses is composed of three genera: pestivirus, flavivirus and hepacivirus (hepatitis C virus).
The pestivirus genus consists of the prototypic member bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV, also called hog cholera virus) and border disease virus (BDV) of sheep. Human pestiviruses have not been as extensively characterized as the animal pestiviruses. However, serological surveys indicate considerable pestivirus exposure in humans. Pestivirus infections in man have been implicated in several diseases including, but not likely limited to, congenital brain injury, infantile gastroenteritis and chronic diarrhea in human immunodeficiency virus (HIV) positive patients. M. Giangaspero et al., Arch. Virol. Suppl., 7: 53-62 (1993); M. Giangaspero et al., Int. J. STD. AIDS, 4(5): 300-302 (1993); R. Yolken et al., Lancet, 1(8637): 517-20 (1989); C. R. Wilks et al., Lancet, 1(8629): 107 (1989); M. Giangaspero et al., Lancet, 2: 110 (1988); B. J. Potts et al., Lancet, 1(8539): 972-973 (1987).
Pestivirus infections of domesticated livestock (cattle, pigs, and sheep) cause significant economic losses worldwide. BVDV is ubiquitous and causes a range of clinical manifestations including abortion, teratogenesis, respiratory problems, chronic wasting disease, immune system dysfunction and predisposition to secondary viral and bacterial infections. Certain BVDV strains are capable of causing an acute fatal disease, with mortality rates of 17% to 32%. BVDV is also able to establish persistent infections in fetuses infected before 150 days of gestation. When born, these persistently infected (PI) animals are immunotolerant to the infecting BVDV strain and remain viremic throughout life. PI animals constitute 1% to 2% of the cattle population in the United States and serve as virus reservoirs and continuous sources for virus spread in herds. PI animals may also succumb to fatal mucosal disease upon superinfection with closely related, but distinct, BVDV virus strains. CSFV, while eradicated from the United States and Canada, causes widespread disease in Europe and elsewhere in the world.
Flaviviruses and hepaciviruses represent important pathogens of man and are also prevalent throughout the world. There are 38 flaviviruses associated with human disease, including the dengue fever viruses, yellow fever virus and Japanese encephalitis virus. Flaviviruses cause a range of acute febrile illnesses and encephalitic and hemorrhagic diseases. Hepaciviruses currently infect approximately 1 of the world population and cause persistent infections leading to chronic liver disease, cirrhosis, hepatocellular carcinoma and liver failure.
Currently, there are no antiviral pharmaceutical drugs to prevent or treat pestivirus or flavivirus infections. For hepacivirus (hepatitis C virus)infections, interferon alfa (IFN) is currently the only approved drug in the United States. While IFN treatment has been reported to improve symptoms in 20% to 40% of patients, the remainder do not respond favorably to IFN treatment. For patients who do respond, a sustained improvement of liver function reportedly is seen in only 10% to 20% of patients; the majority of patients relapse upon cessation of IFN treatment. Thus, while IFN has been shown to have some utility in treating hepatitis C, its effectiveness is limited and its cure rate is low.
In accordance with one aspect, the present invention provides a compound and composition for preventing and treating pestivirus infection and for preventing and treating diseases associated with pestivirus infection in mammalian hosts. The compounds of the invention have the following structure: 
in which A represents a substituent selected from the group consisting of:
(a) NR1R2 wherein R1 and R2 are radicals independently selected from the group consisting of H, straight or branched chain alkyl groups (C1-C6), substituted or unsubstituted aryl groups, substituted or unsubstituted aralkyl groups in which the alkyl group is C1-C6, alkoxy groups (C1-C6), acyl groups (C1-C7), substituted or unsubstituted carbalkoxy groups (C1-C8 alkoxy), or R1 and R2, together with the nitrogen atom to which they are attached, represent a substituted or unsubstituted heterocyclic ring selected from the group consisting of benzopyridazine, indole, benzotriazole, hexamethyleneimine, imidazole, isoxazole, morpholine, phthalimide, piperidine, piperazine and pyrrolidine;
(b) a substituted or unsubstituted heterocyclic group selected from the group consisting of pyridine, benzimidazole, benzodioxane, benzofurazan, indole, benzothiophene, coumarin, furan, hexamethyleneimine, isoxazole, oxadiazole, piperazine, piperidine, pyridine, pyrimidine, pyrrolidine, quinoline, quinuclidene, tetrahydropyran and thiazole;
(c) a substituted or unsubstituted phenyl group; and
(d) OR3, wherein R3 represents a radical selected from the group consisting of H, a straight or branched chain alkyl (C1-C6) group, a substituted or unsubstituted phenyl group a substituted or unsubstituted phenylalkyl group wherein the alkyl group is C1-C6, or a substituted or unsubstituted tetrahydropyran; Q represents a linking moiety selected from the group consisting of xe2x80x94[(Axe2x80x2)nxe2x80x94(CO)]pxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94(SO)xe2x80x94, xe2x80x94(SO2)xe2x80x94 or a valence bond, Axe2x80x2 being xe2x80x94NRaxe2x80x94 or xe2x80x94Oxe2x80x94 and Ra being H or alkyl (C1-C6); Rb, Rc and Rd independently represent H, alkyl (C1-C4), substituted or unsubstituted phenyl or COOR, R being hydrogen or alkyl (C1-C6); m is an integer from 0 to 6; n and p independently represent 0 or 1; and q and r are independently integers from 0 to 4; said phenyl, aryl, aralkyl, carbalkoxy and heterocyclic substituents and the W, X, Y and Z substituents being selected from the group consisting of H, alkyl (C1-C6), substituted or unsubstituted aryl (C6-C15), substituted or unsubstituted aralkyl (C7-C15), halogen, CF3, CN, O-alkyl (C1-C6), acyloxy (C1-C6 acyl), S-alkyl (C1-C6), SO-alkyl (C1-C6), SO2-alkyl (C1-C6), NH2SO2, NO2, NH2, NHRxe2x80x2, NRxe2x80x2Rxe2x80x3, alkyl COORxe2x80x2, COORxe2x80x2, alkyl CONRxe2x80x2Rxe2x80x3, CONRxe2x80x2Rxe2x80x3, 
Rxe2x80x2 and Rxe2x80x3 being independently selected from the group consisting of hydrogen or alkyl (C1-C6), and the isomers and pharmaceutically acceptable salts of said compound.
The compounds of the invention can be conveniently prepared from known starting materials according to the general synthetic scheme illustrated below. Specific embodiments of anti-pestivirus compounds within the scope of the invention are exemplified below.
In the general synthetic scheme, thiosemicarbazide is reacted with isatin to form 5H-1,2,4-triazino[5,6-b]indole-3(2H)-thione. This product is alkylated with an appropriate halo-derivative to provide the sulfur-substituent desired in the final product. 
Rvar signifies any of the various sulfur substituents mentioned above with reference to the compounds of Formula I.
In vitro studies have been performed demonstrating the usefulness of compounds described herein as antiviral agents against pestiviruses. Antiviral activity was measured on the basis of activity against bovine viral diarrhea virus (BVDV) in a cell culture assay. The specificity of antiviral activity toward pestiviruses was also demonstrated in cell culture. Animal safety and bioavailability studies were also performed. The biological studies of the antiviral activity of the compounds of the invention are also described in the examples that follow.
Compounds with particular utility, including isomeric forms, have the formula: 
in which R1 and R2 are the same or different straight or branched chain alkyl groups (C1-C6), or R1 and R2, together with the nitrogen atom to which they are attached form a substituted or unsubstituted heterocyclic ring having from 5 to 9 ring atoms, with nitrogen being the only heteroatom in the ring, the heterocyclic ring substituents being selected from the group consisting of hydrogen, alkyl (C1-C6), halogen, CF3, CN and O-alkyl (C1-C6), m is an integer from 1-6, and Rb, Rc, W, X, Y, Z, b, c, and q are as previously defined with reference to Formula I, and the pharmaceutically acceptable salts of such compounds.
Particularly preferred are the following compounds:
3-[(2-pyridylmethyl)thio]-5H-1,2,4-triazino[5,6-b]indole, 3-[2-(4-morpholino)ethylthio]-5H-1,2,4-triazino[5,6-b]indole, 3-[4-((6,7-dimethoxy-2-oxo-2H-1-benzopyranyl)methyl)thio]-5H-1,2,4-triazino[5,6-b]indole, 3-[(2-(4-(1-ethylpiperidinyl)ethyl)thio]-5H-1,2,4-triazino[5,6-b]indole, 3-[(2-((4-benzodioxanyl))methyl)thio]-5H-1,2,4-triazino[5,6-b]indole, 3-[(4-(2,6-dichloropyridyl)methyl)thio]-5H-1,2,4-triazino[5,6-b]indole, 3-[4-(((2-tetrahydro-2H-pyranyl)oxy)butyl)thio]-5H-1,2,4-triazino[5,6-b]indole, and the isomers and pharmaceutically acceptable salts of those compounds.
As previously noted, the compounds of formula I above and their pharmaceutically acceptable salts exhibit antiviral activity against pestivirus. The compounds of the invention are particularly useful in treating and preventing pestivirus infections (and diseases) in the livestock industry, and may be used to treat cattle, swine and sheep, or other animals susceptible to pestivirus infection.
Compounds of the invention are similarly useful in treating and preventing pestivirus infections in humans. In addition, the compounds of the invention may further have application in the treatment and prevention of infections and diseases of mammalian species by viruses related to the pestiviruses, in particular, the flaviviruses and hepaciviruses.
Compounds described herein are also useful for the treatment of biological material that is susceptible to infection by, or infected or contaminated with a pestivirus. These compounds are especially useful for preventing or resolving pestiviral infections in cell cultures, tissue cultures and other in vitro applications. For example, inclusion of compounds of the invention as a supplement in cell or tissue culture growth media and cell or tissue culture components will prevent pestiviral infections of cultures not previously infected with pestiviruses. Compounds described above may also be used to eliminate pestiviruses from cultures or other materials infected with pestiviruses (e.g., blood), after a suitable treatment period, under any number of treatment conditions as determined by the skilled artisan. This aspect of the invention may be used to advantage in treating cell cultures comprising bovine serum or other mammalian serum. For example, bovine serum can be contaminated with pestiviruses, including BVDV, classical swine fever virus (hog cholera virus) and border disease virus, and the compounds and compositions of the invention can be used to inhibit the growth of those viruses in applications wherein bovine serum is used.
The utility of such in vitro applications of the compounds of the invention will be broad in scope and will include, but not be limited to, use of the compounds of the invention in research and diagnostic laboratories and use in the manufacture of diagnostic, vaccine and therapeutic products for veterinary and human use.
The compounds of the invention can form useful salts with inorganic and organic acids, including, for example, hydrochloric acid, hydrobromic acid and methane sulfonic acid.
The pharmaceutically acceptable salts of the compounds of formula I are prepared following procedures which are familiar to those skilled in the art.
The antiviral pharmaceutical compositions of the present invention comprise one or more of the compounds of formula I, above, as the active ingredient in combination with a pharmaceutically acceptable carrier medium or auxiliary agent.
The composition may be prepared in various forms for administration, including tablets, caplets, pills or dragees, or can be filled in suitable containers, such as capsules, or, in the case of suspensions, filled into bottles. As used herein, xe2x80x9cpharmaceutically acceptable carrier mediumxe2x80x9d includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington""s Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1975) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the antiviral compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.
In the pharmaceutical compositions of the invention, the active agent may be present in an amount of at least 0.5% and generally not more than 90% by weight, based on the total weight of the composition, including carrier medium and/or auxiliary agent(s), if any. Preferably, the proportion of active agent varies between 5-50% by weight of the composition.
Pharmaceutical organic or inorganic solid or liquid carrier media suitable for enteral or parenteral administration can be used to make up the composition. Gelatine, lactose, starch, magnesium stearate, talc, vegetable and animal fats and oils, gum, polyalkylene glycol, or other known carriers for medicaments may all be suitable as carrier media.
The compounds of the invention may be administered using any amount and any route of administration effective for attenuating infectivity of the pestivirus. Thus, the expression xe2x80x9camount effective to attenuate infectivity of pestivirusxe2x80x9d, as used herein, refers to a nontoxic but sufficient amount of the antiviral agent to provide the desired treatment of viral infection. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular antiviral agent and its mode of administration, and the like.
The anti-pestivirus compounds are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to a physically discrete unit of antiviral agent appropriate for the patient to be treated. Each dosage should contain the quantity of active material calculated to produce the desired therapeutic effect either as such, or in association with the selected pharmaceutical carrier medium. Typically, the antiviral compounds of the invention will be administered in dosage units containing from about 0.1 mg to about 50 mg of the antiviral agent, with a range of about 1 mg to about 25 mg being preferred.
The compounds of the invention may be administered orally, parenterally, such as by intramuscular injection, intraperitoneal injection, aerosol, intravenous infusion or the like, depending on the nature and severity of the infection being treated. The compounds of the invention may be administered orally or parenterally at dosage levels of about 0.01 to about 1000 mg/kg, and preferably from about 1 to about 100 mg/kg of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
Although the triazinoindole derivatives described herein can be administered to any host which is susceptible to pestivirus infection, the compounds are intended for the treatment of mammalian hosts, and especially livestock and humans.
The compounds of the invention will typically be administered from 1 to 4 times a day so as to deliver the above-mentioned daily dosage. However, the exact regimen for administration of the compounds and compositions described herein will necessarily be dependent on the needs of the individual host being treated, the type of treatment administered and the judgment of the attending veterinarian.
In view of the inhibitory effect on pestivirus replication in cell culture produced by the compounds used in the method of the invention, it is anticipated that these compounds will be useful not only for therapeutic treatment of pestivirus infection, but for pestivirus prophylaxis, as well.
The following examples are provided to describe the invention in further detail. These examples, which set forth a preferred mode presently contemplated for carrying out the invention, are intended to illustrate and not to limit the invention.