Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/445—Non condensed piperidines, e.g. piperocaine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4402—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 2, e.g. pheniramine, bisacodyl
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
  • A61P31/22—Antivirals for DNA viruses for herpes viruses

Definitions

• the present invention relates to the fields of virology and cellular signaling. More specifically, the invention provides compositions and methods useful for the treatment of viral infections, particularly those caused by herpes viruses.
• HHV-8 is a recently identified virus that has been associated with Kaposi's sarcoma (KS) and possibly with a type of cancer called body cavity lymphoma (a tumor that arises from the lymph tissue) (1).
• KSHV Kaposi's sarcoma
• Kaposi's sarcoma is an unusual skin tumor that is seen primarily in HIV-infected men.
• HHV-8 has also been isolated in the semen of HIV infected individuals. Because of these factors, it is believed that HHV- 8 may cause a sexually transmitted infection.
• herpesvirus saimiri herpesvirus saimiri
• MHV68 murine gammaherpesvirus 68
• HHV-4 Epstein-Barr Virus
• the -165 kb genome contains over 80 open reading frames arranged in a long unique region flanked by multiple 801bp terminal repeat units of high G+C content.
• the long unique region contains blocks of conserved genes found in most herpesviruses, interspersed with blocks of non-homologous genes that are specific for HHV-8 and related viruses.
• KSHV encodes a G-protein coupled receptor (GPCR) that acts as an oncogene, the expression of which causes malignant transformation of rodent fibroblast cells, and produces tumors in nude mice.
• GPCR G-protein coupled receptor
• Transgenic mice expressing the KSHV-GPCR develop highly vascular endothelial tumors (4-6).
• Such tumorigenicity relates to the ability of the KSHV-GPCR to constitutively activate the extracellular signal-regulated ldnase (ERK) signal-transduction cascade (7), (8).
• ERK extracellular signal-regulated ldnase
• One of the main activators of the ERK cascade is the Ras-Raf-MEKl/2-ERK signaling axis (9).
• GPCRs are known to signal through Ras (10-12). Considering that a number of viruses utilize this signaling axis to establish infection, agents which disrupt this pathway should prove efficacious against viruses which include, for example, Epstein Bar Virus and Hepatitis B virus.
• compositions and methods useful for treating viral infections including KSHV.
• the present application provides such compositions and methods.
• a method for the treatment of viral infection in a patient in need thereof is provided.
• An exemplary method entails administration to a patient of a therapeutically effective amount of a compound having the formula
• U is (CH 2 ) n , CO, SO 2 or CONH; n is 0, 1, 2, 3, 4 or 5; X is CH 2 , CO, SO 2 or CONH; Y is CH 2 , CO, SO 2 or CONH;
• Rl is an optionally substituted aryl, aralkyl, heteroaryl or heteroarylalkyl
• R2 is an optionally substituted heteroalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, heterocycloalkyl or heteroalkylcycloalkyl and
• R3 is an optionally substituted alkyl, alkenyl, alkinyl, heteroalkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, heteroarylalkyl or aralkyl;or a pharmacologically acceptable salt, solvate, hydrate or formulation thereof.
• a preferred embodiment of the invention comprises administration of the compound disclosed above in combination with a MAPK pathway inhibitor and/or an antiproliferative agent. Suitable inhibitors and agents are provided in Table I hereinbelow.
• the methods of the invention should prove efficacious in the treatment of viral infection caused by HHV-8, HHV-4 and hepatitis B virus.
• a pharmaceutical composition for treating or inhibiting viral infection comprises the above-mentioned compound and at least one agent or inhibitor selected from the group provided in Table I in a pharmaceutically acceptable carrier.
• Another embodiment of the invention comprises a method for prophylaxis of viral infection in a host susceptible to said infection.
• One such method comprises administration of a therapeutically effective amount of the above mentioned compound and optionally at least one MAPK pathway inhibitor and/or an antiproliferative agent.
• FIG. 1 Inhibition of anchorage-dependent growth of NIH3T3 cells transformed with KSHV-GPCR oncogene by MCP compounds. IC50 values in a WST-I proliferation assay were established in KSHV-GPCR transformed NEH3T3 cells treated with MCPl, MCPIlO or control compounds BAY43-9006 and U0126.
• FIG. 1 Inhibition of anchorage-independent growth of NIH3T3 cells transformed with KSHV-GPCR oncogene by MCP compounds. IC50 values in a colony formation assay were established in KSHV-GPCR transformed NIH3T3 cells treated with MCPl, MCPIlO or control compounds BAY43-9006 and U0126.
• Figure 3 Inhibition of anchorage-independent growth: combination effect of MCPIlO at 10 ⁇ M with BAY43-9006, U0126 and staurosporine.
• A. NIH3T3 cells transformed with K-Ras(G12V) were treated with the DMSO control, 10 ⁇ M of MCPIlO, compounds at concentrations indicated, or combinations of MCPIlO and compounds, and grown in soft agar for 21 days, then colonies scored.
• B HLR-Elkl cells were incubated with DMSO control, 5 ⁇ M of MCPIlO, compounds at concentrations indicated, or combinations of MCPl 10 and compounds for 6 hours (see Materials and Methods).
• IC50 values for taxol were established in Raf22W, K-Ras(V12) and KSHV-vGPCR-transformed NIH3T3 cells, for growth in soft agar, or by WST-I proliferation assay.
• B. IC50 values for taxol inhibition of NIH3T3-K- Ras(V12) cell growth in soft agar were re-assessed in the presence of MCPl, MCPl 10, BAY43-9006, or U0126 at the concentrations as indicated.
• IC50 values for taxol inhibition of NIH3T3-K-Ras(V12) cell proliferation were re-assessed in the presence of MCPIlO, BAY43-9006, or U0126 at the concentrations as indicated.
• D. IC50 values for taxol inhibition of NIH3T3 -KSHV-GPCR cell growth in soft agar were re-assessed in the presence of MCPl, MCPl 10, B AY43-9006, or U0126 at the concentrations as indicated.
• E. IC50 values for taxol inhibition of EC-vGPCR cell growth in WST-I proliferation assay were determined in the presence of MCPl 10, BAY43-9006, or U0126 at the concentrations as indicated.
• FIG. 1 Cell cycle profile induced by MCPIlO and taxol in K-Ras(V12) versus cRaf22W NIH3T3 transformed cells. Cells were treated with compounds at the concentrations shown for 72 hours then fixed, stained with propidium iodide and analyzed by FACS. The software program CellQuest was used to establish the frequency of Gl versus G2/M cells.
• FIG. 6 Induction of cell death by MCPIlO and taxol in K-Ras(V12) versus cRaf22W NIH3T3 transformed cells.
• Cells were treated with compounds at the concentrations as indicated for 72 hours, then fixed and stained with annexin V and analyzed by FACS. The frequency of annexin V - positive cells is shown.
• Figure 7 Schematic of the signaling cascade that leads to cellular transformation associated with KSHV infection.
• Figure 8 Schematic of the signaling cascade that leads to cellular transformation associated with HHV-4/Epstein Barr virus infection.
• Figure 9. Schematic of the signaling cascade that leads to cellular transformation associated with Hepatitis B infection.
• the compounds described herein block the interaction between Ras and Raf and thus should have demonstrable efficacy as antiviral agents.
• viruses include without limitation, HHV-8, HHV-4 and hepatitis B virus.
• the compounds used in the practice of this invention can be combined with other known anti-viral or antiproliferative agents in methods of treating or preventing viral infection.
• U is (CH 2 ) n , CO, SO 2 or CONH; n is 0, 1, 2, 3, 4 or 5;
• X is CH 2 , CO, SO 2 or CONH
• Y is CH 2 , CO, SO 2 or CONH
• Rl is an optionally substituted aryl, aralkyl, heteroaryl or heteroarylalkyl
• R2 is an optionally substituted heteroalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, heterocycloalkyl or heteroalkylcycloalkyl and
• R3 is an optionally substituted alkyl, alkenyl, alkinyl, heteroalkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, heteroarylalkyl or aralkyl; or a pharmacologically acceptable salt, solvate, hydrate or formulation thereof.
• alkyl refers to a saturated or unsaturated (i. e. alkenyl and alkinyl) straight or branched chain alkyl group, containing from one or two to ten carbon atoms, preferably from one or two to six carbon atoms, e.g.
• 1 or 2 to 6 carbon atoms for example methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert.-butyl, n-pentyl, n-hexyl, 2,2-dimethylbutyl, n-octyl, ethenyl (vinyl), propenyl, iso-propenyl, butenyl, isoprenyl or hexa-2-enyl; ethinyl, propinyl or butinyl groups.
• alkenyl and alkinyl refer to unsaturated straight or branched chain alkyl groups, containing from two to ten carbon atoms, preferably from two to six carbon atoms, e.g. 2 to 6 carbon atoms, for example ethenyl (vinyl), propenyl, iso-propenyl, butenyl, isoprenyl or hexa-2-enyl; ethinyl, propinyl or butinyl groups.
• heteroalkyl refers to an alkyl, alkenyl or allcinyl group as defined herein where one or more carbon atoms are replaced by an oxygen, nitrogen, phosphorous or sulphur atom, for example an alkoxy group containing from one to ten carbon atoms, preferably from one to six carbon atoms, e.g.
• heteroalkyl furthermore refers to a group derived from a carboxylic acid or carboxylic acid amide containing from one to ten carbon atoms, preferably from one to six carbon atoms, e.g.
• 1 to 4 carbon atoms may, for example, be acyl containing from one to ten carbon atoms, preferably from one to six carbon atoms, e.g. 1 to 4 carbon atoms, such as acetyl, propionyl, butyryl or pivaloyl; acyloxy containing from one to ten carbon atoms, preferably from one to six carbon atoms, e.g. 1 to 4 carbon atoms such as acetyloxy, propionyloxy, butyrlyoxy or pivaloyloxy; carboxyalkyl containing from one to ten carbon atoms, preferably from one to six carbon atoms, e.g.
• 1 to 4 carbon atoms such as carboxymethyl, carboxyethyl, carboxypropyl, carboxybutyl, carboxyalkyl ester containing from one to ten carbon atoms, preferably from one to six carbon atoms, e.g.
• 1 to 4 carbon atoms such as carboxyalkyl methyl ester, carboxyalkyl ethyl ester, carboxyalkyl propyl ester, carboxyalkyl isopropyl ester, carboxyalkyl butyl ester or carboxyalkyl tert.-butyl ester, carboxyalklyl aminde or alkylcarbamoyl such as N-(I- 4C)alkylcarbamoyl or N, N'-(l-4C)dialkylcarbamoyl) containing from one to ten carbon atoms, preferably from one to six carbon atoms, e.g.
• 1 to 4 carbon atoms such as N- methylcarbamoyl, N-ethylcarbamoyl, N-propylcarbamoyl, N, N'-dimethylcarbamoyl, N- ethyl-N-methylcarbamoyl or N, N'-dipropylcarbamoyl, alkoxycarbonyl containing from one to ten carbon atoms, preferably from one to six carbon atoms, e. g.
• 1 to 4 carbon atoms such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxy- or tert.-butoxycarbonyl or alkoxycarbonyloxy containing from one to ten carbon atoms, preferably from one to six carbon atoms, e.g. 1 to 4 carbon atoms such as methoxycarbonyloxy, ethoxycarbonyloxy, propoxycarbonyloxy, isopropoxycarbonyloxy, butoxycarbonyloxy, tert.-butoxycarbonyloxy.
• cycloalkyl refers to a saturated or partially unsaturated cyclic group, having one or more rings, formed by a skeleton that contains from three to 14 carbon atoms, preferably from three, four, five or six to nine or ten carbon atoms, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, tetralin, cyclopentenyl or cyclohex-2- enyl groups.
• heterocycloalkyl refers to a cycloalkyl group as defined herein where one or more carbon atoms are replaced by one or more oxygen, nitrogen, phosphorous or sulphur atoms.
• Specific examples for heterocyclalkyl are piperidino, morpholino, N- methyl-piperazino or N-phenyl-piperazino groups.
• aryl refers to an aromatic cyclic group, having one or more rings, formed by a skeleton that contains from five to 14 carbon atoms preferably from five or six to nine or ten carbon atoms, for example phenyl, inden or naphthyl groups.
• heteroaryl refers to an aryl group as defined herein where one or more carbon atoms are replaced by an oxygen, nitrogen, phosphorous or sulphur atom, for example 4- pyridyl, 2-imidazolyl, 3-pyrazolyl, quinolinyl, isoquinolinyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1,2, 3-triazolyl, 1,2, 4-triazolyl, oxadiazolyl, thiadia- zolyl, indolyl, indazolyl, tetrazolyl, pyrazinyl, pyridinyl, pyrimidinyl and pyridazinyl groups.
• aralkyl and heteroarylalkyl refer to groups that comprise both aryl or, respectively, heteroaryl as well as alkyl, alkenyl, alkinyl and/or heteroalkyl (for example alkoxy groups in case of aralkyloxy) and/or cycloalkyl and/or heterocycloalkyl ring systems as defined herein.
• alkylcycloalkyl and heteroalkylcycloalkyl refer to groups that comprise both cycloalkyl or, respectively, heterocycloalkyl as well as alkyl, alkenyl, alkinyl and/or heteroalkyl (for example alkoxy groups in case of aralkyloxy) groups as defined herein.
• Examples of such groups are alkylcycloalkyl, alkenylcycloalkyl, alkinylcycloalkyl, al- kylheterocycloalkyl, alkenylheterocycloalkyl, alldnylheterocycloalkyl, heteroalkylcycloalkyl, heteroalkenylcycloalkyl, heteroallcinylcycloalkyl, heteroalkylheterocycloalkyl, heteroalkenylheterocylcloalkyl, heteroalkinylheterocycloalkyl, which cyclic groups can be saturated or once, twice or three-times unsaturated.
• alkyl, alkenyl, alkinyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl aralkyl or heteroarylalkyl groups as defined herein may be substituted with one or more halogen atoms, NH 2 , SH, NO 2 or OH groups or unsubstituted alkyl, heteroalkyl, aryl, aralkyl, aralkyloxy, heteroaryl, cycloalkyl or heterocycloalkyl groups as defined herein.
• optionally substituted refers to groups wherein one or more hydrogen atoms may be replaced by a halogen atom, a NH 2 , SH, NO 2 or OH group or by an unsubstituted alkyl, heteroalkyl, aryl, aralkyl, aralkyloxy, heteroaryl, cycloalkyl or heterocycloalkyl group as defined herein.
• R4 is H, alkyloxy or aralkyloxy (more preferred H, methoxy or benzyloxy) and R5 is F, Cl, alkyl, heteroalkyl, aryl, aralkyl, heteroaryl or heteroarylalkyl (preferred Cl, methoxy or benzyloxy; more preferred benzyloxy).
• R3 is a Cj-C 6 alkyl group (especially an isopropyl group).
• Het is a pyridyl group; n is 0,1 or 2; X is CH 2 ; Y is CO or SO 2 ; R3 is aryl or aralkyl ; R4 is H, alkyloxy or aralkyloxy (more preferred H, methoxy or benzyloxy) and R5 is F, Cl, alkyl, heteroalkyl, aryl, aralkyl, heteroaryl or heteroarylalkyl (preferred Cl, methoxy or benzyloxy; more preferred benzyloxy).
• Het is a pyridyl group; n is 0,1 or 2; X is CO or SO 2 ; Y is CH 2 ; R3 is aryl or aralkyl ; R4 is H, alkyloxy or aralkyloxy (preferred H, methoxy or benzyloxy) and R5 is F, Cl, alkyl, heteroalkyl, aryl, aralkyl, heteroaryl or heteroarylalkyl (preferred Cl, methoxy or benzyloxy; more preferred benzyloxy).
• Het is a piperidyl group; n is 0,1 or 2; X is CH 2 ; Y is CO or SO 2 ; R3 is aryl or aralkyl ; R4 is H, alkyloxy or aralkyloxy (more preferred H, methoxy or benzyloxy) and R5 is F, Cl, alkyl, heteroalkyl, aryl, aralkyl, heteroaryl or heteroarylalkyl (preferred Cl, methoxy or benzyloxy; more preferred benzyloxy).
• Het is a piperidyl group; n is 0, 1 or 2; X is CO or SO 2 ; Y is CH 2 ; R3 is aryl or aralkyl ; R4 is H, alkyloxy or aralkyloxy (preferred H, methoxy or benzyloxy) and R5 is F, Cl, alkyl, heteroalkyl, aryl, aralkyl, heteroaryl or heteroarylalkyl (preferred Cl, methoxy or benzyloxy; more preferred benzyloxy).
• R3 is a group of the Formula (CH 2 ) m Ph wherein m is 0,1, 2,3, 4 or 5 (more preferred m is 2,3 or 4) and wherein the phenyl group may be optionally substituted.
• the compounds of Formula (I) or (II) do not include N-(4-Benzyloxy-3-methoxy- benzyl)-iV-(2-pyridin-2-yl-ethyl)-2-chloro-benzamide. Especially preferred are the following compounds:
• certain compounds of Formula (I) or (II) may have tautomeric forms from which only one might be specifically mentioned or depicted in the following description, different geometrical isomers (which are usually denoted as cis/trans isomers or more generally as (E) and (Z) isomers) or different optical isomers as a result of one or more asymmetric or chiral carbon atoms (which are usually nomenclatured under the Cahn-Ingold-Prelog or R/S system). Further, some compounds may display polymorphism. Unless expressly excluded, all these tautomeric forms, geometrical or optical isomers (as well as racemates and diastereomers) and polymorphous forms are included in the invention.
• the present invention also relates to pharmacologically acceptable salts, or solvates and hydrates, respectively, and to compositions and formulations of compounds of Formula (1) or (II).
• the pharmaceutical compositions according to the present invention contain at least one compound of Formula (I) or (II) as the active agent and optionally carriers and/or diluents and/or adjuvants.
• Examples of such pharmacologically acceptable salts of sufficiently basic compounds of Formula (I) or (II) are salts of physiologically acceptable mineral acids like hydrochloric, hydrobromic, sulfuric and phosphoric acid; or salts of organic acids like methanesulfonic, p-toluenesulfonic, lactic, acetic, trifluoroacetic, citric, succinic, fumaric, maleinic and salicylic acid.
• a sufficiently acid compound of Formula (I) or (II) may form alkali or earth alkaline metal salts, for example sodium, potassium, lithium, calcium or magnesium salts; ammonium salts; or organic base salts, for example methylamine, dimethylamine, trimethylamine, triethylamine, ethylenediamine, ethanolamine, choline hydroxide, N-methyl-D- aminomethane (meglumin), piperidine, morpholine, tris-(2-hydroxyethyl) amine, lysine or arginine salts.
• Compounds of Formula (I) or (II) may be solvated, especially hydrated.
• the hydration can occur during the process of production or as a consequence of the hygroscopic nature of the initially water free compounds of Formula (1) or (II).
• the compounds of Formula (I) or (II) contain asymmetric C-atoms and may be present either as achiral compounds, mixtures of diastereomers, mixtures of enantiomers or as optically pure compounds.
• the present invention also relates to pro-drugs which are composed of a compound of Formula (I) or (II) and at least one pharmacologically acceptable protective group which will be cleaved off under physiological conditions, such as an alkoxy-, aralkyloxy-, acyl- or acyloxy group as defined herein, e. g. ethoxy, benzyloxy, acetyl or acetyloxy.
• therapeutically useful agents that contain compounds of Formula (I) or (II), their solvates, salts and formulations are also comprised in the scope of the present invention.
• compounds of Formula (I) or (II) will be administered by using the known and acceptable modes known in the art, either alone or in combination with any other therapeutic agent.
• Such therapeutically useful agents can be administered by one of the following routes: oral, e. g. as tablets, dragees, coated tablets, pills, semisolids, soft or hard capsules, for example soft and hard gelatine capsules, aqueous or oily solutions, emulsions, suspensions or syrups, parenteral including intravenous, intramuscular and subcutaneous injection, e. g.
• transdermal for example via an transdermal delivery system (TDS) such as a plaster containing the active ingredient or intranasal.
• TDS transdermal delivery system
• the therapeutically useful product may be mixed with pharmaceutically inert, inorganic or organic excipients e.
• excipients e. g., vegetable, petroleum, animal or synthetic oils, wax, fat, polyols.
• excipients e. g., water, alcohols, aqueous saline, aqueous dextrose, polyols, glycerin, vegetable, petroleum, animal or synthetic oils.
• excipients e.g., vegetable, petroleum, animal or synthetic oils, wax, fat and polyols.
• compressed gases suitable for this purpose e.g., oxygen, nitrogen and carbon dioxide.
• the pharmaceutically useful agents may also contain additives for conservation, stabilisation, e.g., UV stabilizers, emulsifiers, sweetener, aromatisers, salts to change the osmotic pressure, buffers, coating additives and antioxidants.
• Combinations with other therapeutic agents may include other therapeutically useful agents, e.g., those used to prevent or treat cancer (see Table L).
• other therapeutically useful agents e.g., those used to prevent or treat cancer (see Table L).
• NIH3T3 cells stably transfected with K-Ras(G12V), N-terminally truncated, constitutively active Raf-1 (Raf22w) (13), (kindly provided by Drs. Channing Der and Janiel M. Shields), or KSHV-GPCR (7), EC-vGPCR endothelial cells (80) (kindly provided by Dr.
• Bay43-9006 iV-(4-chloro-3-(txifluoromethyl)phenyl)-iV'-(4-(2- (iV-methylcarbamoyl))-4-pyridyloxy)phenyl)urea and UOJ 26 l,4-Diamino-2,3-dicyano- l,4-bis(2-aminophenylthio)-butadiene were purchased from Calbiochem and Promega, respectively. Taxol and staurosporine were obtained from Biomol.
• Proliferation assays 5000 cells were plated in each well of 96-well flat bottom plates, and incubated overnight at 37 0 C in 5% CO 2 . The growth of plated cells was determined by adding 7.5 ⁇ of a prepared WST-I reagent (Roche Applied Sciences, Germany) to 3 control wells and measuring OD 650 and OD 450 absorbances with a SpectraMax250 plate reader. If the OD 65 o-OD 450 values were above 0.5, the remainder of the plate was used for incubation with the above mentioned amine derivatives, other pharmacological agents or solvent control for 48 hours. After this incubation, WST-I reagent was added to the wells and OD 650 -OD 45 o values were calculated as before. Triplicate wells were assayed for each conditions and standard deviation was determined: all experiments were performed independently at least three times.
• Anchorage-independent growth assays were performed as described in (15) with minor modifications. Cells were resuspended in DMEM media supplemented with 10% calf serum and antibiotics, and mixed with 0.5% agar to a final concentration of 6,000 to 25,000 cells/ml in 0.33% agar. 0.3 ml of this cell suspension was layered over a 0.5% agar base in 24 well plates and incubated for 2 to 4 hours at 37 0 C. After solidification of the top agar layer, compounds in DMSO, or DMSO diluted to 1% final concentration, were added, followed by incubation for 12 - 21 days.
• HLR-Elkl cells were plated (30,000 cells per well) in a 96 well plate. After 20 hours medium was replaced with 90 ⁇ per well of starvation medium containing no serum. After 72 hours, compound or DMSO solvent was added. After one more hour, the cells were induced with 10 ng/ml EGF. As a negative control (minimal reporter assay), reference cells were not induced with EGF. After an additional five hours of incubation, the cells were lysed and lucif erase activity was measured by using the Bright-Glo Luciferase Assay kit (Promega Co., Madison, WI).
• Cell cycle analysis Cell cycle compartmentalization was examined by flow cytometry. Cells were trypsinized, collected by centrifugation, washed twice and resuspended in sterile PBS, and fixed with 70% ethanol at -20 0 C for 2-12 hours. Cells were again centrifuged, washed twice with cold PBS and resuspended in 100 ⁇ l of PBS. Cell suspensions were mixed with 0.5 ml of 30 mM sodium citrate, 20 ⁇ .g/ml propidium iodide, and 1 ⁇ of 10 mg/ml DNase-free RNase A. The cells were incubated for 1 hour at 37 0 C in the dark, then DNA content from 10,000 cells was determined using the
• Apoptosis assays To estimate apoptosis of stably transfected cell lines treated with the above mentioned amine derivatives and/or taxol, 500,000 cells were seeded in six-well plates. Cells were treated for 72h with various concentrations of the amine derivatives and/or taxol and collected for flow cytometry or used to prepare lysates. Cells were collected by trypsinization and, labeled with annexin V-FITC and propidium iodide according to the manufacturer's recommendations (Clontech) for fluorescence-activated cell sorting analysis (Becton Dickinson). RESULTS AND DISCUSSION
• MCPl and MCPIlO strongly inhibit proliferation and soft-agar colony formation in KSHV-GPCR-transformed NIH3T3 fibroblasts.
• MCPl and MCPl 10 were tested for their ability to inhibit anchorage dependent and independent growth of the NEH3T3 murine fibroblast cell line stably transformed with the KSHV-GPCR.
• Both MCPl and MCPIlO efficiently inhibited growth of NIH3T3-KSHV-GPCR cells in a WST-I assay of cell proliferation, ' with IC50 of 24 and 28 ⁇ M, respectively ( Figure 1).
• the MCPl and MCPl 10 compounds also effectively inhibited soft-agar colony formation by the same cells, with IC50 values of 16 and 14 ⁇ M, respectively ( Figure 2).
• KSHV-GPCR relays signals through Ras/Raf/MEK/ERK signaling module
• BAY43-9006 the Raf kinase catalytic inhibitor
• U0126 the Mek kinase inhibitor
• Taxol paclitaxel
• Taxol is widely used in the treatment of lung, ovarian, and breast carcinomas (17).
• the FDA approved taxol as a second line treatment of Kaposi's sarcoma in AIDS patients (18).
• a primary mode of action of taxol is as a cytotoxic agent that disrupts microtubule dynamics, and taxol does not directly affect signaling through the MAPK pathway, taxol has also been implicated as a compound that both modulates Raf-1 activation, and has differing activity dependent on Raf-1 status (19- 25), making it of interest to analyze the interaction of MCP compounds with taxol.
• IC50 for inhibition of anchorage dependent or independent-growth by taxol in NIH3T3 cells transformed with K-Ras(V12), cRaf22W (constitutively activated Raf, independent of interaction with Ras), and KSHV-GPCR cells.
• the IC50 value of taxol was determined as 0.9 ⁇ M in K-Ras(V12) transformed cells, 0.2 ⁇ M in Raf22W transformed cells, and 0.75 ⁇ M in KSHV-GPCR transformed cells ( Figure 4A).
• the IC50 for taxol was established at 49.7, 8.7 and 63.3 nM, in K-Ras(V12), Raf22W and KSHV-GPCR, respectively ( Figure 4A).
• MCPIlO induces the G2/M arrest and apoptosis of taxol-treated NIH3T3-K- Ras(V12) cells. Since a strong dose dependent combination effect was established for the above-mentioned amine derivatives and taxol in K-Ras(V12) and KSHV-GPCR, the possible mechanism of the observed synergism was investigated. Previously, we have shown that MCPl 10 used at its IC50 concentration promotes cell cycle arrest in Gl (16). Dependent on the DNA damage and spindle checkpoint status of cells, taxol causes mitotic arrest leading to cell death (25, 26).
• the taxol combination results together with the demonstrated ability of amine derivatives described herein, to inhibit proliferation and anchorage-independent growth properties of KSHV-GPCR transformed NIH3T3 cells, provide logical support for the use of the MCP compound class as anti-KSHV agents.
• the results suggest that the combination of the above-mentioned amine derivatives (or Raf-catalytic inhibitors such as BAY43-9006) with taxol, or combination of MCP compounds with B AY43-9006, may be a particularly potent means of inhibiting KSHV- or Ras- transformed cells.
• Table I lists a variety of anti-proliferative and cytotoxic agents that, when used in combination with the amine derivatives described herein, should have therapeutic efficacy for the treatment of viral infection.
• HHV-8 co-opt cellular signaling pathways to promote their replication, and in some cases may also induce tumorigenesis.
• viruses e.g., HHV-8, HHV-4 and hepatitis B virus
• Kaposi's sarcoma caused by HHV-8, is a multifocal angioproliferative neoplasm induced following long-term infection with Kaposi's sarcoma herpesvirus/human herpesvisrus 8 (KSHV/HHV-8). Development of this neoplasm strictly depends upon the availability of multiple angiogenic growth factors and cytokines, which act in combination from virally encoded oncogenic signals provided by such proteins as the KSHV-encoded viral G-protein coupled receptor (vGPCR).
• vGPCR the KSHV-encoded viral G-protein coupled receptor
• vGPCR induction of transformation of KSHV-infected cells involves direct and indirect autocrine/paracrine mechanisms, which requires enhanced expression and secretion of number of angiogenic factors and cytoldnes. These factors include VEGF, IL-8, IL-6 , Gro ⁇ (42-44), and potentiate vGPCR signaling by enhancing vGPCR direct transformation effect in autocrine fashion (45,46).
• VEGF VEGF
• IL-8 interleukin-6
• Gro ⁇ (42-44) IL-6
• potentiate vGPCR signaling by enhancing vGPCR direct transformation effect in autocrine fashion 45,46.
• the KSHV-vGPCR was implicated in immortalization of human endothelial HUVEC cells via activation of their VEGF receptor-2/KDR protein (47).
• vGPCR induces expression of the cytoldnes and growth factors by activation of key transcription factors, including AP-I, NF-KB and NF-AT (48,7), through activation of p21-activated kinase- 1 (Pakl) that forwards the signaling on Raf-1 and IKK kinases (7).
• Pakl p21-activated kinase- 1
• HHV-4/Epstein-Barr virus belongs to the same group of gammaherpes viruses as KSHV.
• Epstein Barr virus is associated with a number of malignancies of lymphoid and epithelial origin, including endemic Burldtt's lymphoma, T-cell lymphomas, Hodgkin's disease, undifferentiated nasopharyngeal carcinoma and several other carcinomas (53-56).
• the EBV genome is frequently found in the lymphomas and lymphoproliferative disorders of immunocompromised transplant patients and individuals with AIDS (57,58).
• LMPl an integral membrane protein expressed by EBV during type 2 and type 3 latent infections, is the only EBV protein that produces a classic oncogenic effect in Rat-1 and NIH3T3 cells, and in B cells (56, 59).
• An animal model for LMPl exists, as LMPl transgenic mice develop lymphomas at increased frequency (60).
• LMPl produces its effect through activation of two major signaling cascades, the NF-icB pathway and the Ras-MAPK signaling pathway (61-63) (see Figure 7). The induction of these signaling cascades occurs through interaction of cytoplasmic domain of LMPl with TRAF and TRADD proteins (64,65) which subsequently signal through the NIK and IKK kinases towards IKB repressor (66).
• Raf kinase inhibitor protein has been found to also function as a negative regulator of the NFKB pathway (67), and may provide a physical bridge between the two complexes: in any case, this and other data from non-viral systems (68) indicate that the MAPK and NFKB signaling cascades are engaged in significant cross-talk that may be important in specifying the course of EBV infection.
• Hepatitis B (HBV) virus is small hepatotropic pararetrovirus that causes persistent liver infection and cirrhosis, and is strongly associated with development of primary liver cancer (hepatocellular carcinoma, HCC) (69). HCC is one of the most prevalent forms of human cancer worldwide, with extremely limited treatment options (70). In this regard, many studies have focused on identification of potential viral oncogenes. HBV encodes two transcriptional activators, HBx (71) and the pre-S2/S region of LHB/MHB proteins (72), which activate cellular targets and promote the transformation of infected cells(73). See Figure 9. The HBx protein of HBV is essential for infection and is also thought to be an essential cofactor in HCC development. The mechanism mediating HBx-dependent activator function includes the Ras-dependent activation of c-Raf-l/MEK/Erlc2,
• HBx protein activates Ras-Raf-MAPK signaling pathway, most likely through activation of the Pyk2 (76), c-Src and Fyn kinases (77).
• HBx protein activates Ras-Raf-MAPK signaling pathway, most likely through activation of the Pyk2 (76), c-Src and Fyn kinases (77).
• the activity and cellular localization of HBx protein are tightly regulated through phosphorylation by ERK1/2 kinase, causing the HBx protein to shuttle to the nucleus, where it induces the transcription of genes critical for HBV replication and cell transformation.
• the phosphorylation of HBx protein creates a possible feedback regulatory circuit between HBX and MAPK signaling pathway (78), indicating that the MAPK pathway may be a suitable target for developing novel anti-HB V therapeutic agents.
• Combination effect of MCPIlO with taxol synergy in inhibition of anchorage- dependent growth in EC-vGPCR endothelial cells.
• VEGF vascular endothelial growth factor
• MCPIlO was as effective in these cells as it was in NIH3T3- KSHV-GPCR cells in inhibiting proliferation. The proliferation was measured by WST-I assay with IC50 value of 22 ⁇ M. Similar to NIH-3T3 -KSHV-GPCR cells, MCPIlO produced a strong synergistic effect with taxol in the endothelial cells.
• Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway. Cancer Res, 56:
• Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes. J Biol Chem, 270: 26802-26806, 1995.
• MAPK mitogen-activated protein kinase
• Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstain-Barr virus transforming protein. Proc Natl Acad Sci U S A, 93: 11085- 11090, 1996.

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