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WO1998033501A1 - 2-benzoylamino-3-phenylpropenamide derivatives and methods of using the same - Google Patents

2-benzoylamino-3-phenylpropenamide derivatives and methods of using the same Download PDF

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Publication number
WO1998033501A1
WO1998033501A1 PCT/US1998/000986 US9800986W WO9833501A1 WO 1998033501 A1 WO1998033501 A1 WO 1998033501A1 US 9800986 W US9800986 W US 9800986W WO 9833501 A1 WO9833501 A1 WO 9833501A1
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Prior art keywords
piperidinyl
alkyl
nitrogen atom
compound
attached form
Prior art date
Application number
PCT/US1998/000986
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French (fr)
Inventor
Robert B. Perni
Samuel C. Conway
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Avid Therapeutics Inc.
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Application filed by Avid Therapeutics Inc. filed Critical Avid Therapeutics Inc.
Priority to AU59239/98A priority Critical patent/AU5923998A/en
Publication of WO1998033501A1 publication Critical patent/WO1998033501A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
    • C07D295/182—Radicals derived from carboxylic acids
    • C07D295/185—Radicals derived from carboxylic acids from aliphatic carboxylic acids
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/30—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D263/34—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D263/36—One oxygen atom
    • C07D263/42—One oxygen atom attached in position 5
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to certain 2-benzoylamino- 3-phenylpropenamide derivatives which are useful as antiviral agents.
  • the present invention also relates to methods of treating of viral infections, in particular hepatitis.
  • the present invention further relates to pharmaceutical compositions useful for treating viral infections, in particular hepatitis.
  • Viral diseases include such diseases as AIDS, yellow fever, rabies, and poliomyelitis.
  • Viral infections include some of the most common of all human ailments, including the common acute respiratory and gastrointestinal infections, as well as such important chronic infections as hepatitis and genital herpes.
  • HAV Natural hepatitis A virus
  • HAV infection is usually caused by ingestion of feces-contaminated material.
  • the course of HAV infection is extremely variable, with the onset of jaundice sometimes preceded by a prodromal state of several days to more than a week characterized by fever.
  • Human hepatitis B virus (HBV) is a member of the hepadnavirus family which is characterized by a circular,
  • HBV induced disease that of the approximately 300 million individuals worldwide who are chronic carriers of HBV, one million die annually from HBV induced disease (Lau, J.Y.N., et al, Lancet, vol. 342, pp.
  • Hepatitis C virus has recently been identified as the principal agent for transfusion-associated non-A, non-B hepatitis.
  • the mean incubation period for acute HCV infection is intermediate between those of hepatitis A and hepatitis B and the clinical course for acute HCV is similar to those of hepatitis A and hepatitis B.
  • treatment with ⁇ - interferon leads to some improvement, as indicated by reduced alanine amino transferase (ALT) activity, a significant number of patients suffer relapse upon termination of the treatment.
  • ALT alanine amino transferase
  • Hepatitis Delta virus has been identified as the causative agent of delta or type D hepatitis. As in the cases of HBV and HCV, interferon treatment has been found to give only transient, beneficial results.
  • Human papillomaviruses HPV have been implicated in skin warts, epidermodysplasia verruciformis , infections of the genital tract, and anogenital warts, as well as cervical intraepithelial neoplasia and cancer of the cervix. HPV infections of the respiratory tract and oral cavity have also been reported. Although most warts regress spontaneously, there is no treatment which will cure all warts.
  • R 1 and R 2 are C ⁇ C., alkyl, or together with the nitrogen atom to which they are attached form a ring containing 5-6 atoms, including C and/or o;
  • R 3 -R 12 are hydrogen, halogen, C,-C 4 alkyl, C,-C 4 alkoxyl, substituted C, . -C 4 alkoxyl, nitro, cyano, or trifluoromethyl;
  • R J3 is hydrogen, C,-C 4 alkyl, arylalkyl, or C 2 -C- acyl; and X is halogen, Cj-C + alkyl, or substituted 0,-C,, alkyl, or a pharmaceutically acceptable salt thereof.
  • the present invention provides novel methods for the treatment of viral infections, by administering an effective amount of a compound of formula (I) .
  • the viral infections which may be treated by the present method include hepatitis (including hepatitis A, B, C, and Delta virus) and papillomavirus .
  • the present method involves treatment of hepatitis, in particular hepatitis B.
  • Examples of the C,-C 4 alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert- butyl.
  • Examples of the 5-6 membered rings which may be formed by R and R z together with the nitrogen atom to which they are attached include 1-piperidinyl, N-morpholinyl, and 1- pyrrolidinyl.
  • Examples of halogen include F, Cl, and Br.
  • Examples of C -C. alkoxyl include methoxy1, ethoxyl, n-propoxyl, iso-propoxyl, n-butoxyl, iso-butoxyl, sec-butoxyl, and tert-butoxyl.
  • Examples of substituted Cj-C,, alkoxyl include fl ⁇ orinated C,-C 4 alkoxyl such as trifluoromethoxyl and other perfluorinated C z -C 4 alkoxyl groups.
  • Examples of arylalkyl include benzyl, C,-C 4 alkyl substituted benzyl, and nitro substituted benzyl.
  • C 2 -C- acyl examples include acetyl, benzoyl, and nitro substituted benzoyl.
  • substituted C,-C 4 alkyl examples include trifluoromethyl and perfluorinated 0,-C,, alkyl groups.
  • a preferred set of compounds to be used in the present method are those in which R 1 and R 2 together with the nitrogen atom to which they are attached form a 1-piperidinyl group; and one to three of R -R ,z are halogen, N0 2 , or C,-C 4 alkoxyl, with the remainder of R 3 -R 12 being hydrogen; R 13 is hydrogen; and X is Cl or Br.
  • Another preferred set of compounds for use in the present method includes those compounds in which all of R 3 -R 12 are hydrogen; R l and R 2 together with the nitrogen atom to which they are attached form a 1-piperidinyl , 1-pyrrolidinyl, or N- morpholinyl group; X is Cl or Br; and R *J is hydrogen.
  • Another preferred set of compounds for use in the present method includes those compounds in which one of R 8 -R 12 is fluoro, methyl, methoxyl, chloro, or nitro, with the remainder of R -R )2 being hydrogen; all of R 3 -R are hydrogen; R 13 is hydrogen; X is chloro or bromo; and R 1 and R2 together with the nitrogen atom to which they are attached form a 1-piperidinyl group .
  • R ⁇ -R 12 are hydrogen; one or two or R 3 -R are chloro, fluoro, methoxyl, methyl, bromo, nitro, or cyano, with the remainder of R 3 -R being hydrogen; R 13 is hydrogen; X is chloro or bromo; and R 1 and R 2 together with the nitrogen atom to which they are attached form a 1-piperidinyl group.
  • Another preferred set of compounds for use in the present method includes those compounds in which one or two (preferably one) R ⁇ -R 12 of are fluoro, methyl, methoxyl, chloro, or nitro, with the remainder of R s -R 32 being hydrogen; one of R 2 -R is nitro, cyano, bromo, or methyl, with the remainder of R 3 -R 7 being hydrogen; R 23 is hydrogen; X is bromo; and R 1 and R 2 together with the nitrogen atom to which they are attached form a 1-piperidinyl group.
  • Another preferred set of compounds for use in the present method includes those compounds given in Table 1 in the Examples below.
  • R 10 is not methoxyl, chloro, or methyl. In the case of treating hepatitis B, it is also preferred that, when X is chloro, R 10 is hydrogen or fluoro.
  • an antiviral effective amount of a compound according to formula (I) is administered to a subject in need thereof.
  • the compound of formula (I) may be administered orally, parenterally, subcutaneously, intraveneously , topically, or via a suppository or an aerosol spray.
  • the compound of formula (I) is preferably administered orally.
  • a suitable dosage for an adult is 20 mg to 4 g per day, preferably 100 mg to 1500 mg per day, more preferably 250 mg to 750 mg per day.
  • the daily dosage may be given once per day or in small portions.
  • the present method is carried out by administering a compound of formula (I) which is effective for the treatment of the viral infection.
  • the compound is effective for the treatment of the viral infection at a dosage which can be tolerated by the subject being treated.
  • the compound is preferably effective at a dose which is not toxic to the subject.
  • the relative efficacy and toxicity may be determined by routine experimentation. For example in the case of hepatitis B, the relative efficacy and toxicity may be determined using the assays given in the Examples below.
  • the compounds in Table 1 are all effective at doses (EC i0 and/or EC 90 values) which are significantly less than the doses which result in significant cell death (TC 50 values) or significant inhibition of cell replication (IC 50 values) .
  • the present method may also be carried out by coad inistering the compound of formula (I) with one or more Known antiviral agents, such as interferons, ⁇ -thymosin, 3TC, FTC, ganiclovir, (S) -9- (3-hydroxy-2- phosphonylmethoxypropyl) adenine (HPMPA) , and penciclovir. Good results have been achieved with the combined use of 3TC and compound AT-61 in Table 1.
  • Known antiviral agents such as interferons, ⁇ -thymosin, 3TC, FTC, ganiclovir, (S) -9- (3-hydroxy-2- phosphonylmethoxypropyl) adenine (HPMPA)
  • the subject being treated by the present method may be any animal suffering from a viral infection, including dogs, cats, cows, horses, pigs, chickens, turkeys, apes, monkeys or humans .
  • the present invention provides novel compounds.
  • the novel compounds of the present invention are those having formula (II)
  • R 1 and R 2 are C,-C 4 alkyl, or together with the nitrogen atom to which they are attached form a ring containing 5-6 atoms, including C and/or 0;
  • R 3 -R 12 are hydrogen, halogen, C t -C 4 alkyl, C,-C 4 alkoxyl, substituted C,-C 4 alkoxyl, nitro, cyano, or trifluoromethyl;
  • R 13 is hydrogen, C,-C 4 alkyl, arylalkyl, or C-C- acyl
  • X is halogen, C,-c 4 alkyl, or substituted C,-C + alkyl, or a pharmaceutically acceptable salt thereof; with the proviso that, when R 1 and R z together with the nitrogen atom to which they are attached form an N-morpholinyl group, at least one of R 3 -R 12 is not hydrogen.
  • Step one in Scheme A is a slight modification of the procedure described by Buck and Ide in Organic Synthesis , vol.
  • the present invention provides novel pharmaceutical compositions which comprise at least one compound of formulae (I) or (II) and a pharmaceutically acceptable carrier.
  • the present compositions may be in any form which is suitable for the intended route of administration.
  • the present composition may be a sterile solution or suspension suitable for injection; a lotion cream or ointment suitable for topical application; a tablet, pill, capsule, or elixer suitable for ingestion; or a solution or suspension suitable for inhalation.
  • the pharmaceutical composition may also be in the form of a patch for transdermal administration, encapsulated for subcutaneous implantation, or in the form of a suppository.
  • the pharmaceutical composition will be formulated so as to deliver 5 mg to 4 g, preferably 10 mg to 1500 mg, more preferably 50 mg to 750 mg, of the compound of formulae (I) or (II) per unit dosage.
  • the present pharmaceutical compositions may also contain one or more known antiviral agents such as interferons, c_-thymosin, 3TC, FTC, ganciclovir, HPMPA, and penciclovir .
  • hippuric acid derivatives can be prepared according to one or both of the preceding two procedures.
  • Hep AD38 cells were maintained in Dulbecco's Modified Eagle 's/F-12 Medium (DMEM/F-12, Gibco BRL/Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) , 50 ⁇ g/ml penicillin, 50 ⁇ g/ml streptomycin, 100 ⁇ g/ml kanamycin (PSK) at 37°C and 5% carbon dioxide.
  • FBS fetal bovine serum
  • PSK kanamycin
  • Hep AD38 cells were grown in the presence of 0.3 ⁇ g/ml tetracycline and 400 ⁇ g/ml G418 (Gibco BRL/Life Technologies, Gaithersburg, MD) .
  • the 2.2.15 cells (Acs, G. , et al., Proc . Natl . Acad . Sci . USA , vol. 84, pp.
  • Dr. George Acs were maintained in RPMI 1640 medium with 10% FBS, PSK and 400 ⁇ g/ml G418.
  • Hep G2 cells (4 x 10 " cells/35 mm plate) were co- transfected with 0.3 ⁇ g of pUHD 15-lneo (Gossen, M. , et al., Proc . Natl . Acad . Sci . USA , vol. 89, pp. 5547-5551 (1992)) and
  • ptetHBV ptetHBV using a liposome transfection kit as directed by the manufacturer (Transfectace, GIBCO BRL/Life Technologies, Gaithersburg, MD) .
  • Transfected cells were selected in DMEM/F-12 medium containing 10% FBS, 1 ⁇ g/ml of tetracycline and 400 ⁇ g/ml of G418 until individual clones could be identified by visual inspection of the plates (approximately one month) .
  • Individual clones were isolated and maintained in DMEM/F-12 medium supplemented with 10% FBS, PSK, 0.3 ⁇ g/ml tetracycline, and 400 ⁇ g/ml G418.
  • B Plas id constructs
  • Plasmid ptetHBV was created by replacing the cytomegalovirus immediate early (CMV-IE) promoter in pCMVhbv (Fallows, D. A., et al. J . Virol . , vol. 69(5), pp. 3067-73
  • pCMVhbv was created by the fusion of HBV sequences (subtype ayw, Christman, J. K. , et al., Proc .
  • Polyadenylated RNA was isolated from Hep AD38 cells with a modified "fast track" method (In Vitrogen, San Diego, CA) . Briefly, cells were washed twice with PBS and lysed with 10 ml of lysis buffer (0.2 MM Tris HCl pH 7.5, 0.2 M NaCl, 2% SDS , 25 mM EDTA and 0.2 mg/ml proteinase K) /T162 flask. The cell lysate was sheared by repeated (10 times) passage through an 18 gauge needle and incubated at 50°C for 1 hour.
  • lysis buffer 0.2 MM Tris HCl pH 7.5, 0.2 M NaCl, 2% SDS , 25 mM EDTA and 0.2 mg/ml proteinase K
  • oligo (dT) cellulose (Stratagene, La Jolla, CA) in 1 ml of binding buffer (0.5 M NaCl, 10 mM Tris HCl pH 7.5 and 0.1 mM EDTA) on a shaking platform for 1 hour at room temperature.
  • binding buffer 0.5 M NaCl, 10 mM Tris HCl pH 7.5 and 0.1 mM EDTA
  • the oligo (dT) cellulose was pelleted and washed four times with 10 ml of binding buffer, resuspended in 0.5 ml of binding buffer and transferred to a spin column.
  • RNA were eluted with 0.4 ml of elution buffer (10 mM Tris HCl pH 7.5 , 1 mM EDTA) at 65°C, precipitated with ethanol and resuspended in 0.05 ml of DEPC- treated water.
  • elution buffer 10 mM Tris HCl pH 7.5 , 1 mM EDTA
  • One microgram of poly-A selected RNA was electrophoresed through a 1% formaldehyde agarose gel, transferred to a nylon membrane (Hybond-N, Amersham, Arlington Heights, IL) and hybridized with a :: P-labeled HBV RNA probe.
  • ccc covalently closed circular DNA
  • the cells were lysed in 1 ml of lysis buffer without pronase.
  • protein-detergent complexes such as the DNA intermediates of HBV replication
  • 0.25 ml of 2.5 M KCl was added to the lysate.
  • the supernatant containing the ccc DNA was extracted with phenol and ethanol precipitated.
  • the centrifuged pellets were resuspended in 80 ⁇ l of TE and analyzed as described above.
  • the supernatants of Hep AD38 and 2.2.15 cells were clarified of cellular debris of centrifugation (Sorval RT-6000D centrifuge 2000 RMP's for 10 minutes). Virus particles were precipitated from the cleared supernatants with PEG and DNA isolated as described above for viral DNA.
  • HepAD38 cells were plated into 96-well microtiter plates (6 x 10" cells/well) and grown for three days in the presence of 0.3 ⁇ g/ml of tetracycline. On day zero, the cells were washed several times with PBS and treated with tetracycline- free medium that contained either test or control compounds. Each test compound was screened at two, three or six concentrations in quadruplicate. On day three, the medium was removed and replaced with fresh medium containing compound.
  • Radioactivity was quantified with a Bio-Rad GS-363 phospho ⁇ mager .
  • concentration of compound that inhibited HBV replication by 50 or 90% was determined by linear regression.
  • the cell monolayer was washed with PBS and tested for cell viability using a cell proliferation assay kit as directed by the manufacturer (CellTiter 96 Non-Radioactive Cell Proliferation Assay, Promega, Madison, I) .
  • the concentrations of compound that inhibited cell viability by 50 or 90% were determined by linear regression.
  • DPBS fPulbeccos's phosphate buffered saline 0.2 g/1 KCl, 8 g/L NaCl, 0.2 g/L KH 2 P0 4 , 1.15 g/L Na 2 HP0 4 , 133 mg/L CaCL j -2H 2 0, 100 mg/L MgCl z -6H 2 0); bring up to volume with RT deionized water, adjust pH to 7.35 using 1M HCl or 1M NaOH if necessary. Pre-made solution is also acceptable.
  • MTS reagent available from Promega in the CellTiter 96 Aqueous Non-radioactive Cell Proliferation Assay; 2mg/ml in DPBS, filter sterilized with 0.2 ⁇ m pore filter. Store at -20° in a light-protected container. MTS reagent powder must be stored at 4 ; C in a light- protected container. The results of the assays are shown in Tables 1 and 2. Table 1
  • EC ?0 concentration of drug which inhibits synthesis of viral DNA by 50%.
  • EC 90 concentration of drug which inhibits synthesis of viral DNA by 90%.
  • TC 50 concentration of drug which reduces the number -*• of viable cells by 50%.
  • d IC 5e concentration of drug which inhibits the replication of cells by 50%.
  • EG f0 concentration of drug which inhibits synthesis of viral DNA by 50%.

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Abstract

Novel 2-Benzoylamino-3-phenylpropenamides are disclosed as antiviral agents. Compositions containing the 2-benzoylamino-3-phenylpropenamides and methods for treating viral diseases, such as hepatitis, with 2-benzoylamino-3-phenylpropenamides are also disclosed.

Description

TITLE OF THE INVENTION
2-BENZOYLAMINO-3-PHENYLPROPENA IDE DERIVATIVES AND METHODS OF USING THE SAME
BACKGROUND OF THE INVENTION Field of the Invention:
The present invention relates to certain 2-benzoylamino- 3-phenylpropenamide derivatives which are useful as antiviral agents. The present invention also relates to methods of treating of viral infections, in particular hepatitis. The present invention further relates to pharmaceutical compositions useful for treating viral infections, in particular hepatitis.
Discussion of the Background:
Viral diseases include such diseases as AIDS, yellow fever, rabies, and poliomyelitis. Viral infections include some of the most common of all human ailments, including the common acute respiratory and gastrointestinal infections, as well as such important chronic infections as hepatitis and genital herpes.
There are at least four hepatitis viruses: hepatitis A, B, C and Delta. Natural hepatitis A virus (HAV) infection is usually caused by ingestion of feces-contaminated material. The course of HAV infection is extremely variable, with the onset of jaundice sometimes preceded by a prodromal state of several days to more than a week characterized by fever. There is no specific treatment for HAV infection. Human hepatitis B virus (HBV) is a member of the hepadnavirus family which is characterized by a circular,
partially double-stranded DNA genome of approximately 3,000 base pairs in length, an enveloped capsid, and the ability to infect liver cells (Ganem, D. , et al, Ann . Rev . of Biochem . , vol. 56, pp. 651-693 (1987)). After the primary infection of the host, the virus may establish a chronic infection in the liver, which, in turn, can lead to cirrhosis and hepatocellular carcinoma (Beasley, R.P., et al in Viral
Hepatitis and Liver Disease , G.N. Vynas, et al, Eds., Grune
and Stratton, New York, pp.209-224 (1984); and Popper, H. , et al, Hepatology, vol. 7, pp. 764-772 (1987)). It is estimated
that of the approximately 300 million individuals worldwide who are chronic carriers of HBV, one million die annually from HBV induced disease (Lau, J.Y.N., et al, Lancet, vol. 342, pp.
1335-1340 (1993)).
Currently, the only available treatment for chronic hepatitis in the United States is α-interferon. Its efficacy, however, is partial and of limited duration. In clinical studies, approximately 30% of chronic carriers treated with interferon responded to treatment, but virus reappeared in greater than 50% of these patients within two to three months after cessation of treatment (Fattovich, G., et al, Hepatology , vol. 8, pp. 1651-1654 (1988); and Thomas, H.C., in
Viral Hepatitis and Liver Disease , A.J. Zuckerman, Ed., Alan.
R. Liss, Inc., New York, pp. 817-822 (1988)). As an alternative approach, several nucleoside analogs have been investigated as potential inhibitors of HBV replication (see review in Fur an, P. . , et al, Antiviral Chem . Chemother . , vol. 6(6), pp. 345-355 (1995); and Nair, V., et al, Antimicrob . Agents and Chemother. , vol. 39(5), 1017-1029
(1995)). In early clinical trials, both lamivudine and ganciclovir have proven to be effective in decreasing the levels of HBV DNA in the serum of chronically infected patients (Deinstag, J.I., et al, N . E .J . M . , vol. 333(25), 1657-
1661 (1995); Honkoop, P., et al, Lancet, vol. 346, 1156-1157
(1995); and Gish, R.G., et al, Hepatology, vol. 23(1), 1-7
(1996)). However, most patients relapsed shortly after therapy was discontinued, suggesting that virus persisted in the liver during the entire trial period.
Hepatitis C virus (HCV) has recently been identified as the principal agent for transfusion-associated non-A, non-B hepatitis. The mean incubation period for acute HCV infection is intermediate between those of hepatitis A and hepatitis B and the clinical course for acute HCV is similar to those of hepatitis A and hepatitis B. Although treatment with α- interferon leads to some improvement, as indicated by reduced alanine amino transferase (ALT) activity, a significant number of patients suffer relapse upon termination of the treatment.
Hepatitis Delta virus (HDV) has been identified as the causative agent of delta or type D hepatitis. As in the cases of HBV and HCV, interferon treatment has been found to give only transient, beneficial results. Human papillomaviruses (HPV) have been implicated in skin warts, epidermodysplasia verruciformis , infections of the genital tract, and anogenital warts, as well as cervical intraepithelial neoplasia and cancer of the cervix. HPV infections of the respiratory tract and oral cavity have also been reported. Although most warts regress spontaneously, there is no treatment which will cure all warts. Parenteral administration of α-interferon or β-interferon has been shown to induce complete remission of genital warts in a majority of patients, but interferon therapy has been less successful in the treatment of recurrent respiratory papillomatosis.
Thus, there remains a need for methods, compounds, and compositions useful for treating viral infections, such as hepatitis and papillomavirus infections.
SUMMARY OF THE INVENTION
Accordingly, it is one object of the present invention to provide novel methods for treating viral infections.
It is another object of the present invention to provide novel methods for treating hepatitis.
It is another object of the present invention to provide novel methods for treating hepatitis A virus infections.
It is another object of the present invention to provide novel methods for treating hepatitis B virus infections.
It is another object of the present invention to provide novel methods for treating hepatitis C virus infections.
It is another object of the present invention to provide novel methods for treating hepatitis Delta virus infections. It is another object of the present invention to provide novel compounds which are useful for treating viral infections.
It is another object of the present invention to provide novel compounds which are useful for treating hepatitis.
It is another object of the present invention to provide novel compounds which are useful for treating hepatitis A virus infections.
It is another object of the present invention to provide novel compounds which are useful for treating hepatitis B virus infections.
It is another object of the present invention to provide novel compounds which are useful for treating hepatitis C virus infections.
It is another object of the present invention to provide novel compounds which are useful for treating hepatitis Delta virus infections.
It is another object of the present invention to provide novel pharmaceutical compositions which are useful for treating viral infections.
It is another object of the present invention to provide novel pharmaceutical compositions which are useful for treating hepatitis.
It is another object of the present invention to provide novel pharmaceutical compositions which are useful for treating hepatitis A virus infections. It is another object of the present invention to provide novel pharmaceutical compositions which are useful for treating hepatitis B virus infections.
It is another object of the present invention to provide novel pharmaceutical compositions which are useful for treating hepatitis C virus infections.
It is another object of the present invention to provide novel pharmaceutical compositions which are useful for treating hepatitis Delta virus infections.
These and other objects, which will become apparent during the following detailed description, have been achieved by the inventors' discovery that compounds of the formula (I) are useful for the treatment of viral infections.
Figure imgf000008_0001
wherein:
R1 and R2, each independently of the other, are C^C., alkyl, or together with the nitrogen atom to which they are attached form a ring containing 5-6 atoms, including C and/or o; R3-R12, each independently of the other, are hydrogen, halogen, C,-C4 alkyl, C,-C4 alkoxyl, substituted C,.-C4 alkoxyl, nitro, cyano, or trifluoromethyl;
RJ3 is hydrogen, C,-C4 alkyl, arylalkyl, or C2-C- acyl; and X is halogen, Cj-C+ alkyl, or substituted 0,-C,, alkyl, or a pharmaceutically acceptable salt thereof.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Thus, in a first embodiment, the present invention provides novel methods for the treatment of viral infections, by administering an effective amount of a compound of formula (I) . Examples of the viral infections which may be treated by the present method include hepatitis (including hepatitis A, B, C, and Delta virus) and papillomavirus . In a preferred embodiment, the present method involves treatment of hepatitis, in particular hepatitis B.
Examples of the C,-C4 alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert- butyl. Examples of the 5-6 membered rings which may be formed by R and Rz together with the nitrogen atom to which they are attached include 1-piperidinyl, N-morpholinyl, and 1- pyrrolidinyl.
Examples of halogen include F, Cl, and Br. Examples of C -C. alkoxyl include methoxy1, ethoxyl, n-propoxyl, iso-propoxyl, n-butoxyl, iso-butoxyl, sec-butoxyl, and tert-butoxyl. Examples of substituted Cj-C,, alkoxyl include flαorinated C,-C4 alkoxyl such as trifluoromethoxyl and other perfluorinated Cz-C4 alkoxyl groups. Examples of arylalkyl include benzyl, C,-C4 alkyl substituted benzyl, and nitro substituted benzyl. Examples of C2-C- acyl include acetyl, benzoyl, and nitro substituted benzoyl. Examples of substituted C,-C4 alkyl include trifluoromethyl and perfluorinated 0,-C,, alkyl groups.
A preferred set of compounds to be used in the present method are those in which R1 and R2 together with the nitrogen atom to which they are attached form a 1-piperidinyl group; and one to three of R -R,z are halogen, N02, or C,-C4 alkoxyl, with the remainder of R3-R12 being hydrogen; R13 is hydrogen; and X is Cl or Br.
Another preferred set of compounds for use in the present method includes those compounds in which all of R3-R12 are hydrogen; Rl and R2 together with the nitrogen atom to which they are attached form a 1-piperidinyl , 1-pyrrolidinyl, or N- morpholinyl group; X is Cl or Br; and R*J is hydrogen.
Another preferred set of compounds for use in the present method includes those compounds in which one of R8-R12 is fluoro, methyl, methoxyl, chloro, or nitro, with the remainder of R -R)2 being hydrogen; all of R3-R are hydrogen; R13 is hydrogen; X is chloro or bromo; and R1 and R2 together with the nitrogen atom to which they are attached form a 1-piperidinyl group .
Another preferred set of compounds for use in the present method includes those compounds in which all of Rβ-R12 are hydrogen; one or two or R3-R are chloro, fluoro, methoxyl, methyl, bromo, nitro, or cyano, with the remainder of R3-R being hydrogen; R13 is hydrogen; X is chloro or bromo; and R1 and R2 together with the nitrogen atom to which they are attached form a 1-piperidinyl group.
Another preferred set of compounds for use in the present method includes those compounds in which one or two (preferably one) Rβ-R12 of are fluoro, methyl, methoxyl, chloro, or nitro, with the remainder of Rs-R32 being hydrogen; one of R2-R is nitro, cyano, bromo, or methyl, with the remainder of R3-R7 being hydrogen; R23 is hydrogen; X is bromo; and R1 and R2 together with the nitrogen atom to which they are attached form a 1-piperidinyl group.
Another preferred set of compounds for use in the present method includes those compounds given in Table 1 in the Examples below.
In the case of treating hepatitis B, it is preferred that, when X is chloro, R10 is not methoxyl, chloro, or methyl. In the case of treating hepatitis B, it is also preferred that, when X is chloro, R10 is hydrogen or fluoro.
In the present method, an antiviral effective amount of a compound according to formula (I) is administered to a subject in need thereof. The compound of formula (I) may be administered orally, parenterally, subcutaneously, intraveneously , topically, or via a suppository or an aerosol spray. In the case of hepatitis B virus, the compound of formula (I) is preferably administered orally.
Although the exact dosage of the compound of formula (I) will vary with the size and condition of the subject, the exact compound being administered and the viral infection being treated, a suitable dosage for an adult is 20 mg to 4 g per day, preferably 100 mg to 1500 mg per day, more preferably 250 mg to 750 mg per day. The daily dosage may be given once per day or in small portions.
The present method is carried out by administering a compound of formula (I) which is effective for the treatment of the viral infection. Preferably, the compound is effective for the treatment of the viral infection at a dosage which can be tolerated by the subject being treated. In other words, the compound is preferably effective at a dose which is not toxic to the subject. For any given viral infection, the relative efficacy and toxicity may be determined by routine experimentation. For example in the case of hepatitis B, the relative efficacy and toxicity may be determined using the assays given in the Examples below. The compounds in Table 1 are all effective at doses (ECi0 and/or EC90 values) which are significantly less than the doses which result in significant cell death (TC50 values) or significant inhibition of cell replication (IC50 values) . In contrast, the compounds in Table 2, may be effective at doses (EC= values) which are equal to or greater than those which cause significant inhibition of cell replication (IC50 values) .
An in vitro assay for screening compounds for efficacy
against hepatitis A is described in Emerson, S.U. , et al, "The Molecular Basis of Virulence and Growth of Hepatitis A in Cell Culture," Vaccine, vol. 10, supp. 1, pp. S36-S39 (1992), which is incorporated herein by reference in its entirety.
The present method may also be carried out by coad inistering the compound of formula (I) with one or more Known antiviral agents, such as interferons, α-thymosin, 3TC, FTC, ganiclovir, (S) -9- (3-hydroxy-2- phosphonylmethoxypropyl) adenine (HPMPA) , and penciclovir. Good results have been achieved with the combined use of 3TC and compound AT-61 in Table 1.
The subject being treated by the present method may be any animal suffering from a viral infection, including dogs, cats, cows, horses, pigs, chickens, turkeys, apes, monkeys or humans .
A. Lawson et al, J. Chem. Soc. C, pp. 598-603 (1971) disclose a compound having the following formula:
Figure imgf000013_0001
B = N-morpholinyl.
However, the remaining compounds useful in the present method are believed to be novel. Accordingly, in a second embodiment, the present invention provides novel compounds. In particular, the novel compounds of the present invention are those having formula (II)
Figure imgf000014_0001
(II) wherein:
R1 and R2, each independently of the other, are C,-C4 alkyl, or together with the nitrogen atom to which they are attached form a ring containing 5-6 atoms, including C and/or 0;
R3-R12, each independently of the other, are hydrogen, halogen, Ct-C4 alkyl, C,-C4 alkoxyl, substituted C,-C4 alkoxyl, nitro, cyano, or trifluoromethyl;
R13 is hydrogen, C,-C4 alkyl, arylalkyl, or C-C- acyl; and
X is halogen, C,-c4 alkyl, or substituted C,-C+ alkyl, or a pharmaceutically acceptable salt thereof; with the proviso that, when R1 and Rz together with the nitrogen atom to which they are attached form an N-morpholinyl group, at least one of R3-R12 is not hydrogen.
The examples given for the various substituents in the compound of formula (I) are also suitable examples for the analogous substituents in the compound of formula (II) . In addition, the same sets of preferred compounds for use in the present method are also preferred sets of the compounds of formula (II) .
The compounds of formulae (I) and (II) may be synthesized as shown in Scheme A.
Scheme A
Figure imgf000016_0001
NaOAc
Figure imgf000016_0002
1) R,R2NH. CHC13
2) X,. CaCO.
Figure imgf000016_0003
Syntheses of the novel compounds used in the present method may be carried out using the following published methods. Step one in Scheme A is a slight modification of the procedure described by Buck and Ide in Organic Synthesis , vol.
13, pp. 8-9 (1933). The ring opening reaction (Scheme A, Step two, part 1) is described by Barnes and Shriner, J. Am. Chem
SOC , vol. 70, pp. 1769-1772 (1948).
Compound 2 in Scheme A may be prepared as shown in Scheme B.
Scheme B
Figure imgf000018_0001
Figure imgf000018_0002
In another embodiment, the present invention provides novel pharmaceutical compositions which comprise at least one compound of formulae (I) or (II) and a pharmaceutically acceptable carrier. The present compositions may be in any form which is suitable for the intended route of administration. Thus, the present composition may be a sterile solution or suspension suitable for injection; a lotion cream or ointment suitable for topical application; a tablet, pill, capsule, or elixer suitable for ingestion; or a solution or suspension suitable for inhalation. The pharmaceutical composition may also be in the form of a patch for transdermal administration, encapsulated for subcutaneous implantation, or in the form of a suppository.
In a preferred embodiment, the pharmaceutical composition will be formulated so as to deliver 5 mg to 4 g, preferably 10 mg to 1500 mg, more preferably 50 mg to 750 mg, of the compound of formulae (I) or (II) per unit dosage. In addition, the present pharmaceutical compositions may also contain one or more known antiviral agents such as interferons, c_-thymosin, 3TC, FTC, ganciclovir, HPMPA, and penciclovir .
Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.
EXAMPLES I . Synthetic Examples 1. 2-Phenyl-4- (Z) -phenylmethylene-5-oxo-oxazole (1). Hippuric acid (28.8 g, 161 mmol), benzaldehyde (15.3 g,
144 mmol), sodium acetate (12.0 g, 146 mmol) and acetic anhydride (42 mL) were combined in an Erlenmeyer flask and heated on a hot plate until the mixture just began to boil. It was then transferred to a steam bath and heated for 1 hour with occasional manual stirring. Hot ethanol (60 mL) was added; the mixture was stirred until homogenous, and then cooled to room temperature. The resulting solid was collected by suction filtration and washed with a minimum quantity of cold ethanol, then with boiling water, and then dried in
vacuo . Recrystallization from benzene yielded 16.5 g (46%) of
fine yellow needles.
All of the oxazolone derivatives are prepared according to the preceding procedure.
2. l-[ (E) -l-oxo-2- (benzoylamino) -3-phenyl-3- bromopropenyl]piperidine (2) .
To a solution of (1) (10.0 g, 40.1 mmol) in chloroform (200 L) at 03C was added dropwise a solution of piperidine (4.2 g, 49 mmol) in chloroform (60 mL) . The yellow solution was stirred at 0°C for 1 hour. Solid calcium carbonate (6.0 g, 60 mmol) was added, followed by dropwise addition of bromine (6.4 g, 40 mmol) in chloroform (60 mL) . The suspension was filtered to remove calcium salts, and the supernatant liquid was evaporated to dryness. The resulting orange oil was crystallized from ethanol, and further purified by recrystallization from ethanol/water . 8.18 g (49%) of pale green crystals were obtained, p 158.2-158.5.
All of the products in which X=Br are prepared according to the preceding procedure.
3. l-[ (E) -l-oxo-2-(benzoylamino) -3-phenyl-3- chloropropenyl] piperidine (3) .
To a solution of (1) (10.0 g, 40.1 mmol) in chloroform (200 L) at 0DC was added dropwise a solution of piperidine (4.2 g, 49 mmol) in chloroform (60 mL) . the yellow solution was stirred at O^C for lh. Chlorine was introduced through a Pasteur pipet at a rate of approximately 5 bubbles per second until TLC showed no further traces of starting material (approximately 2.5 min per mmol of starting material; an excessive amount of chlorine must be avoided, as it leads to the formation of undesirable side products) . The solvent was removed in vacuo, yielding a yellow oil which was purified by chromatography (silica gel, 2% CH30H/CHC1:.) . The product was further purified by recrystallization from ethanol/water, yielding 7.67 g (52%) of white crystals, mp 175.8-176.6=C.
All of the products in which X=C1 are prepared according to the preceding procedure.
4. N-[ (4-tert-Butyl) benzoyl ]glycine.
Glycine (0.78 g, 1.0 mmol) was dissolved in 1.0-N aqueous NaOH (10 mL) at room temperature. 4-tert-Butylbenzoyl chloride was added, and the biphasic mixture was heated to 60CC and vigorously stirred overnight. A heavy white precipitate gradually formed; it was collected by suction filtration and washed with ether to remove 4-tert-butylbenzoic acid. The product, a white powder, weighed 0.70 g (30%) and was used without further purification.
5. N-[ (4-Carbomethoxy) benzoyl] glycine. Carbonyldiimidazole (1.94 g, 12.0 mmol) was added to a suspension of mono-methylterephthalate (2.16 g, 12.0 mmol) in
CH Cl (50 mL) . A clear solution resulted after approximately 5 min. A solution of sodium hydroxide (0.60 g, 15.0 mmol) and glycine (1.13 g, 15.1 mmol) in water (5 mL) was added, and the mixture was stirred vigorously. A semisolid mass formed and gradually dissolved until a clear biphasic mixture was formed. The reaction mixture was acidified to pH 5 with concentrated HCl. CH:C1 was removed in vacuo , and the solid residue, shown
by TLC (1:2:97 HOAc/CH.OH/CHCl ) to be starting material, was removed by filtration. The supernatant liquid was further acidified and cooled on ice to yield fine, colorless crystals. A second crop was collected after reduction of the volume of the mother liquor. The total yield was 0.54 g (19%) of colorless crystals, which were used without further purification.
All of the hippuric acid derivatives can be prepared according to one or both of the preceding two procedures.
II. Assay Experiments
MATERIALS AND METHODS A. Cell lines and culture conditions Hep G2 cells (Knowles, B. B., et al., Science , vol. 209,
pp. 497-499 (1980)) and Hep AD38 cells were maintained in Dulbecco's Modified Eagle 's/F-12 Medium (DMEM/F-12, Gibco BRL/Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) , 50 μg/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml kanamycin (PSK) at 37°C and 5% carbon dioxide. In addition, Hep AD38 cells were grown in the presence of 0.3 μg/ml tetracycline and 400 μg/ml G418 (Gibco BRL/Life Technologies, Gaithersburg, MD) . The 2.2.15 cells (Acs, G. , et al., Proc . Natl . Acad . Sci . USA , vol. 84, pp.
4641-4644 (1987); Sells, M. A., et al., Proc . Natl . Acad . Sci .
USA , vol. 84, pp. 1005-1009 (1987); and Sells, M.A. , et al.,
J. Virol . , vol. 62(8), pp. 2836-2844 (1988)), a kind gift from
Dr. George Acs, were maintained in RPMI 1640 medium with 10% FBS, PSK and 400 μg/ml G418.
Hep G2 cells (4 x 10" cells/35 mm plate) were co- transfected with 0.3 μg of pUHD 15-lneo (Gossen, M. , et al., Proc . Natl . Acad . Sci . USA , vol. 89, pp. 5547-5551 (1992)) and
2.7 μg of ptetHBV using a liposome transfection kit as directed by the manufacturer (Transfectace, GIBCO BRL/Life Technologies, Gaithersburg, MD) . Transfected cells were selected in DMEM/F-12 medium containing 10% FBS, 1 μg/ml of tetracycline and 400 μg/ml of G418 until individual clones could be identified by visual inspection of the plates (approximately one month) . Individual clones were isolated and maintained in DMEM/F-12 medium supplemented with 10% FBS, PSK, 0.3 μg/ml tetracycline, and 400 μg/ml G418. B . Plas id constructs
Plasmid ptetHBV was created by replacing the cytomegalovirus immediate early (CMV-IE) promoter in pCMVhbv (Fallows, D. A., et al. J . Virol . , vol. 69(5), pp. 3067-73
(1995)) with the tetracycline responsive promoter from pUHD 10-3 (Gossen, M. , et al., Proc . Natl . Acad . Sci . USA , vol. 89,
pp. 5547-5551 (1992)). pCMVhbv was created by the fusion of HBV sequences (subtype ayw, Christman, J. K. , et al., Proc .
Natl . Acad . Sci . USA , vol. 79, pp. 1815-1819 (1982))
corresponding to the cDNA of pg RNA to the CMV-IE promoter (Cullen, B.R., Cell , vol. 46, pp. 973-982 (1986); and Fallows,
D. A., et al, J . Virol , vol. 69(5), pp. 3067-73 (1995)). The nucleotide sequence of ptetHBV is available upon request.
C. Isolation and analysis of polyadenylated RNA
Polyadenylated RNA was isolated from Hep AD38 cells with a modified "fast track" method (In Vitrogen, San Diego, CA) . Briefly, cells were washed twice with PBS and lysed with 10 ml of lysis buffer (0.2 MM Tris HCl pH 7.5, 0.2 M NaCl, 2% SDS , 25 mM EDTA and 0.2 mg/ml proteinase K) /T162 flask. The cell lysate was sheared by repeated (10 times) passage through an 18 gauge needle and incubated at 50°C for 1 hour. Following addition of NaCl to the lysate to a final concentration of 0.5 M, the lysate was incubated with 60 mg of oligo (dT) cellulose (Stratagene, La Jolla, CA) in 1 ml of binding buffer (0.5 M NaCl, 10 mM Tris HCl pH 7.5 and 0.1 mM EDTA) on a shaking platform for 1 hour at room temperature. The oligo (dT) cellulose was pelleted and washed four times with 10 ml of binding buffer, resuspended in 0.5 ml of binding buffer and transferred to a spin column. RNA were eluted with 0.4 ml of elution buffer (10 mM Tris HCl pH 7.5 , 1 mM EDTA) at 65°C, precipitated with ethanol and resuspended in 0.05 ml of DEPC- treated water. One microgram of poly-A selected RNA was electrophoresed through a 1% formaldehyde agarose gel, transferred to a nylon membrane (Hybond-N, Amersham, Arlington Heights, IL) and hybridized with a ::P-labeled HBV RNA probe.
D. Isolation and characterization of viral and chromosomal
January 31. 1997DNA
For the isolation of viral DNA a method described by Summers et al (Summers, J., et al., J . Virol . , vol. 64(6), pp.
2819-24 (1990)) was followed. Briefly, cells from a 60 mm plate were lysed in 1 ml of lysis buffer (50 mm Tris HCl pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% sodium dodecyl sulfate, 0.5 mg of pronase per ml) at 37°C for 60 minutes followed by a phenol extraction and ethanol precipitation. The pellets containing nucleic acids were resuspended in 80 μl of TE (10 mM Tris HCl pH 8.0, 1 mM EDTA). One quarter of each sample was electrophoresed through a 1.5% agarose gel, transferred to a nylon membrane and hybridized with a radioactive RNA probe.
For the isolation of covalently closed circular (ccc) DNA, the cells were lysed in 1 ml of lysis buffer without pronase. To precipitate protein-detergent complexes, such as the DNA intermediates of HBV replication, 0.25 ml of 2.5 M KCl was added to the lysate. Following centrifugation of the precipitate, the supernatant containing the ccc DNA was extracted with phenol and ethanol precipitated. The centrifuged pellets were resuspended in 80 μl of TE and analyzed as described above.
For the isolation of DNA in secreted virus particles, the supernatants of Hep AD38 and 2.2.15 cells were clarified of cellular debris of centrifugation (Sorval RT-6000D centrifuge 2000 RMP's for 10 minutes). Virus particles were precipitated from the cleared supernatants with PEG and DNA isolated as described above for viral DNA.
E. Chemicals
Human o(-interferon, tetracycline and β-2',31- dideoxycytidine, (ddC) were purchased from Sigma Chemical Corp. (St. Louis, MO). (±) -2-amino-l , 9-dihydro-9- [ ( la, 3β , 4 ) - 3-hydroxy-4- (hydroxymethyl) cyclopentyl] -6H-purine-6-one, (2 '- CDG) was received from Dr. Jack Secrist (Southern Research Institute; Birmingham, AL) . 2 ' -deoxy-3 ' -thiacytidine, (3TC) and 2 ', 3 ' -dideoxy-5-fluoro-3 ' -thiacytidine, (FTC) were received from Dr. Raymond Schinazi (Emory University; Atlanta, GA) .
F. Antiviral and cytotoxicity assay
HepAD38 cells were plated into 96-well microtiter plates (6 x 10" cells/well) and grown for three days in the presence of 0.3 μg/ml of tetracycline. On day zero, the cells were washed several times with PBS and treated with tetracycline- free medium that contained either test or control compounds. Each test compound was screened at two, three or six concentrations in quadruplicate. On day three, the medium was removed and replaced with fresh medium containing compound. Twenty four hours later the medium was collected, clarified by centrifugation (Sorval RT-6000D centrifuge, 1000 RPM's, 5 minutes) and assayed for HBV DNA by dot-blot analysis as described previously with the exception that 1% NP-40 was added to the denaturing solution (Korba, B.E., et al., Antiviral Res . , vol. 19, pp. 55-70 (1992); Korba, B.E., et
al., Antiviral Res . , vol. 15, pp. 217-228 (1991)).
Radioactivity was quantified with a Bio-Rad GS-363 phosphoπmager . The concentration of compound that inhibited HBV replication by 50 or 90% (the EC,0 or EC90, respectively) was determined by linear regression.
To determine the cytotoxic effects of test compounds, the cell monolayer was washed with PBS and tested for cell viability using a cell proliferation assay kit as directed by the manufacturer (CellTiter 96 Non-Radioactive Cell Proliferation Assay, Promega, Madison, I) . The concentrations of compound that inhibited cell viability by 50 or 90% (the TC,0 or TC90, respectively) were determined by linear regression.
G . AD38 CELL PROLIFERATION ASSAY/CYTOSTATICITY ASSAY fIC.,1
1. Day -1:
(a) 96-well plates are seeded with 1 x 10" Hep AD38 cells/well in 100 μl of F-12/DMEM medium containing O 98/33501
10% fetal bovine serum (FBS), tetracycline (0.3 μg/ml) , G418 (400 μg/ml) , penicillin, streptomycin, and either kanamycin or genatamicin. (b) Incubate plates at 37°C in a humidified, 5% C02 incubator.
2. Day 0:
(a) The positive controls [actinomycine-D (ActD) , cycloheximide (CHx, and colchecine (colch) ] are added to two wells each per plate, at 50 μg/ml. The concentration of these initial doses in 100 μg/ml prior to the addition to the plates.
(b) Serial dilutions of 30 μg/ml, 10 μg/ml, 3 μg/ml, 1 μg/ml, 0.3 μg/ml, and 0.1 μg/ml are attained by gently pipetting up and down the media in the well containing the initial dose (yielding a concentration of 30 μg/ml) . 50 μl of each well in the initial dosage row is carried to the next well and again mixed gently. This process is carried out four more times until the final dilution (0.1 μg/ml) has been attained. At this point, the 50 μl that has been removed from the final dilution well is discarded.
(c) Incubate plate at 37°C in a humidified, 5% CO; incubator.
3. Dav 3 :
(a) Remove medium from each well.
(b) To each well, add 100 μl of warm D-PBS. (c) To each well add 20 μl of a mixture of 2ml MTS + 100 μl PMS. It is necessary to mix the MTS and PMS immediately prior to their addition.
(d) Plate is incubated at 37°C in a humidified, 5% C0: incubator for one hour.
(e) Plate is removed from incubator and placed in a Molecular Devices Co. Vt4ΛX Kinetic Microplate Reader in order to measure the light absorption per well at 490 nm. The data are printed out via the software program SOFTmas, version 2.34 by Dale Quantz , Molecular Devices Company.
DPBS fPulbeccos's phosphate buffered saline): 0.2 g/1 KCl, 8 g/L NaCl, 0.2 g/L KH2P04, 1.15 g/L Na2HP04, 133 mg/L CaCLj-2H20, 100 mg/L MgClz-6H20); bring up to volume with RT deionized water, adjust pH to 7.35 using 1M HCl or 1M NaOH if necessary. Pre-made solution is also acceptable.
PMS fPhenazine methosulfate) : 0.92 mg/ l in DPBS; filter sterilize through a 0.2 μm pore filter into a sterile, light-protected container. Store at -20°C.
MTS . Owen's reagent): available from Promega in the CellTiter 96 Aqueous Non-radioactive Cell Proliferation Assay; 2mg/ml in DPBS, filter sterilized with 0.2 μm pore filter. Store at -20° in a light-protected container. MTS reagent powder must be stored at 4;C in a light- protected container. The results of the assays are shown in Tables 1 and 2. Table 1
Figure imgf000030_0001
Designation EC SO EC90 τcsc IC 50
AT- 61 Cl 1-piperidinyl C6H5 C6H5 1.2 13 >81 >271
AT- 65 Cl 1-piperidinyl 4- -F-C6H4 C6HS 3.1 28 >75
CGX-67438 Cl 1-morpholinyl C6H5 C6HS 7.3 >81 >270
AT-56 Cl 1-pyr- c.n, C6Hg 5.6 54 >75 rolidinyl
AT- 58 CH3 1-piperidinyl C6HS C6HS 21.2 >86
AT-64 Br 1-piperidinyl C6Hς C6H5 1.3 21 >72
AT-68 Br 1-morpholinyl C6H? C6Hβ 10.8 48 >72
AT- 71 Br 1-piperidinyl 4- -F-C6H4 CβH5 3.9 >70
AT-75 Br 1-piperidinyl 2- -F-CeH4 C6H5 1.5 70
Designation EC, a.fe ECa TC •so -so
AT-81 Br 1-piperidinyl 1-F-C6H4 C6Hβ 4.4 >69
AT-111 Br 1-piperidinyl 4- -CHS-C6H4 C6H5 2.8 >70
AT-112 Br 1-piperidinyl 4- CH30-C6H4 C*H6 13.1 >67
AT-113 Br 1-piperidinyl 4 -Cl-C6H< C«HS 11.4 55
AT-127 Br 1-piperidinyl 2- -CH3-CβHΔ 6HS 0.89 >70
AT-129 Br 1-piperidinyl 2- CH30-C6H4 c6a. 2.1 >70
AT-131 Br 1-piperidinyl 2 -C1-C6HΛ CfiH5 3.3 >67
AT-133 Br 1-piperidinyl 3 -Cl-CβH4 CsHf 11.2 >22
AT-135 Br 1-piperidinyl 2- -NOj-C^ C6Hy 12.0 >21
AT-83 Cl 1-piperidinyl CeH, 3,4-Cl2-C6H3 22.8 68
AT-101 Br 1-piperidinyl C6HS 3,4-Cl2-C6H, 33.0 >62
AT-85 Cl 1 -piper id inyl CβH5 4-F-C6H4 1.3 5. .5 >67
AT-96 Cl 1-piperidinyl C*H5 2,4-F2-CβH3 7.2 >74
AT-93 Br 1-piperidinyl C6HS 4-F-C6H4 0.86 4. ,4 >69
AT-86 Cl 1-piperidinyl C6H5 4-CHa0-C6H4 5.2 >75
AT-97 Cl 1-piperidinyl C6HS 3-CH3-C,H4 4.2 18 >78
AT-88 Cl 1 -pi pe id inyl C6H5 4-Cl-C6H4 0.52 3. ,5 >74 >248
AT-99 Br 1-piperidinyl C6H5 4-Cl-C6H4 0.54 3. 1 >67 >223
AT-104 Br 1-piperidinyl C6H? 3-CH3-C,H4 2.2 11. 0 >70
AT-124 Br 1-piperidinyl C6H? 4-CH3-C6H, 2.1 12. 3 >70
Designation EC 50 '90 TC S.a τc50*
AT-105 Br 1-piperidinyl C6Hς 2 , 4— Fj— CgH4 3.3 >66
AT-106 Br 1 -piper id inyl CeH_j 4-CF3-C6H4 5.0 34 >62
AT-107 Br 1-piperidinyl C6H, 4-CH30-CfiH4 1.9 19 >67
AT-108 Br 1-piperidinyl C6H? 4-Br-C6H4 0.53 3.7 >61
AT-109 Br 1-piperidinyl C6H5 4-N0^-CfiH4 0.35 3.0 >65
AT-137 Br 1-piperidinyl C«Hς 2-N02-C6H„ 5.9 >21
AT-141 Br 1-piperidinyl CέH? 3-N0,-C H4 7.6 >21
AT-114 Br 1-piperidinyl C6H5 2-Cl-C6H4 1.2 8.9 >67
AT-125 Br 1-piperidinyl C6H5 4-CN-C6H4 2.3 26 >68
AT-148 Br 1-piperidinyl CβHs 2-Br-CβH4 2.2 10.2 >20
AT-118 Br 1-piperidinyl 2-F-C6H4 4-NOz-C6H4 0.31 1.8 42
AT-140 Br 1-piperidinyl 3-F-C6H4 4-N02-C6H4 2.0 11.6 >21
AT-119 Br 1-piperidinyl 4-F-C6H4 4-N02-C6H4 0.72 11.0 >63
AT-120 Br 1-piperidinyl 4-CH3-C6H4 4-N02-CβH4 1.4 >63
AT-121 Br 1-piperidinyl 4-CH30-C6H4 4-N02-C6H4 2.7 >61
AT-122 Br 1-piperidinyl 4-Cl-CβH4 4-N02-C6H4 1.6 >60
AT-128 Br 1-piperidinyl 2-CH3-C6H4 4-N02-CέH4 0.60 4.6 >63
AT-130 Br 1-piperidinyl 2-CHjO-C6H4 4-N02-CβH« 0.13 0.92 >61
AT-146 Br 1-piperidinyl 2,6-(CH?0) -C6H3 4-N02-C6H4 0.17 1.2 >19
AT-132 Br 1 -piper id inyl 2-Cl-C6H4 4-N02-CέH4 0.90 8.2 >60
Figure imgf000033_0001
AT-134 Br 1-piperidinyl 3-Cl-CέH4 4-mz-cj 1.4 14.7 >60
AT-136 Br 1-piperidinyl 2 -NO -C I^ 4-NOt-C*H4 21.8 >60
AT-142 Br 1-piperidinyl 2-F-CέH4 3-N02-CfaHΛ 11.1 >21
AT-126 Br 1-piperidinyl 2-F-CβH4 4-CN-C6H4 5.0 30 >65
AT-147 Br 1-piperidinyl 2-F-C6H4 4-Br-C6H4 0.37 2.5 >19
AT- 90 Br 1-piperidinyl 2-F-C6H4 4-CH3-C6H4 0.72 4.0 >67
EC?0=concentration of drug which inhibits synthesis of viral DNA by 50%. ' EC90=concentration of drug which inhibits synthesis of viral DNA by 90%. TC50=concentration of drug which reduces the number -*• of viable cells by 50%. dIC5e) concentration of drug which inhibits the replication of cells by 50%.
Figure imgf000033_0002
Table 2
Figure imgf000034_0001
esignation X A Y Z EC^fl IC 5t>
AT-62 Cl 1-Piperidinyl 4-CH30-C6H4 C6HS >75 >75
AT-66 Cl 1 -Piper id inyl 4-Cl-C6H4 C6H5 >45 45
AT-67 Cl 1-Piperidinyl 4-CH3-C6H4 C«HS >75 >75
AT-102 Br 1-Piperidinyl 3 , 4-dichloro C6H5 >44 44
AT-110 Br 1-Piperidinyl 4-CH3C02-C6H<, C6HS >63 >63
AT-72 Br 1-Piperidinyl 1-Naphthyl C6HS >50 50
AT-73 Br 1-Piperidinyl 2-Naphthyl C6HS >65 >65
AT-78 Br 1-Piperidinyl 3-Pyridinyl C6H5 >70 >70
AT-79 Br 1-Piperidinyl 2-f uranyl C6HS >74 >74
CGX-67440 Cl N(CH CH() C6HS C6HS >75 >75
AT-63 Cl 1-hexamethylenimine C6H? C6H5 >75 >75
Designation X A Y Z EC5β s ιc?0 b
AT-57 Cl 1-] .leptamethylenimine C6H? CέHs >75 >75
Figure imgf000035_0001
CGX-67441 Cl NHCH C&H5 CέUζ C6H-r >75 >75
CGX-67442 Cl 2-Thiazolyl C6H? C«H5 >75 >75
AT-82 Br N(CH2CH^)2NCH3 C6H5 CέHε >70 >70
( -methylpiperazine)
AT-84 Br NHCeHs C6H? CfeH5 >75 >75
AT-94 Cl l-(3,5- -dimethyl) -piperidinyl C6H5 C^H5 >75 75
AT-59 CH3 N-Morpholinyl C6H5 C6HS >85 >85
AT- 60 CH3 N(CI^CH2)2NCH3 C6H5 6H5 >82 >82
(N- -methylpiperazine)
AT-69 Cl 1-Piperidinyl CβH? CH3 >97 >97
AT-89 Cl 1-Piperidinyl C6Hf 4 -CHj-C^ >78 <78
AT-87 Cl 1-Piperidinyl C6H5 2 -CHj-C^H4 >21 21
AT-95 Cl 1-Piperidinyl C6H5 4 -CF-j-CβH4 >69 >69
AT-100 Cl 1-Piperidinyl C6HS C6F5 >33 33
AT-138 Br 1-Piperidinyl C6Hf 3, 5- -(N02)2-CβH3 >20 >20
AT-115 Br 1-Piperidinyl C6H5 4- -CeHs-CβH4 >61 >61
AT-116 Br 1-Piperidinyl C6Hf 4- tert-C4H9 >63 >63
AT-117 Br 1-Piperidinyl C6H5 2- Naphthyl >64 >64
3EGf0=concentration of drug which inhibits synthesis of viral DNA by 50%. IC5o concentration of drug which inhibits the replication of cells by 50%.
Figure imgf000036_0001
O -»
Figure imgf000036_0002
Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims

O 98/33501CLAIMS :
1. A method for treating a viral infection, comprising administering, to an animal in need thereof, an effective amount of a compound of formula (I) :
Figure imgf000038_0001
wherein:
RJ and Rz, each independently of the other, are C^C^ alkyl, or together with the nitrogen atom to which they are attached form a ring containing 5-6 atoms, including C and/or 0;
R3-R12, each independently of the other, are hydrogen, halogen, CL -C4 alkyl, C.-C. alkoxyl, substituted C_-C alkoxyl, nitro, cyano, or trifluoromethyl;
R13 is hydrogen, C,-C4 alkyl, C2-C- acyl, or arylalkyl; and
X is halogen, 0,-04 alkyl, or substituted C,-C4 alkyl, or a pharmaceutically acceptable salt thereof.
2. The method of Claim 1, wherein said viral infection is hepatitis infection.
3. The method of Claim 1, wherein said viral infection is hepatitis B virus infection.
4. The method of Claim 1, wherein R1 and R1 together with the nitrogen atom to which they are attached form a 1- piperidinyl group.
5. The method of Claim 1, wherein R1 and R2 together with the nitrogen atom to which they are attached form a 1- piperidinyl group, Ri3 is hydrogen, and X is chloro, bromo, methyl or trifluoromethyl .
6. The method of Claim 1, wherein R1 and R2, together with the nitrogen atom to which they are attached form a group selected from the group consisting of 1-piperidinyl , N- morpholinyl, and 1-pyrrolidinyl .
7. The method of Claim 1, wherein said compound of formula (I) is administered intravenously, orally, parenterally , topically, or via a suppository or an aerosol spray.
8. The method of Claim 1, wherein said compound of formula (I) is administered in an amount of 20 mg to 4 g per day.
9. The method of Claim 1, wherein said compound of formula (I) is administered in an amount of 100 mg to 1500 mg per day.
10. The method of Claim 1, wherein said compound of formula (I) is administered in an amount of 250 mg to 750 mg per day.
11 . A compound of f ormula ( Ii ;
Figure imgf000040_0001
wherein:
R1 and R2, each independently of the other, are C,-C4 alkyl, or together with the nitrogen atom to which they are attached form a ring containing 5-6 atoms, including C and/or O;
R3-R/z, each independently of the other, are hydrogen, halogen, CL -C alkyl, Cj-C4 alkoxyl, substituted Cx-C4 alkoxyl, nitro, cyano, or trifluoromethyl ;
R13 is hydrogen, Ct-C4 alkyl, C2-C- acyl, or arylalkyl; and
X is halogen, C,-C4 alkyl, or substituted Ct-C4 alkyl, or a pharmaceutically acceptable salt thereof; with the proviso that, when R1 and R2 together with the nitrogen atom to which they are attached form an N-morpholinyl group, at least one of R3-R12 is not hydrogen.
12. The compound of Claim 11, wherein R1 and R together with the nitrogen atom to which they are attached form a group selected from the group consisting of 1-piperidinyl, N- morpholinyl, and 1-pyrrolidinyl .
13. The compound of Claim 11, wherein R1 and R2 together with the nitrogen atom to which they are attached form a 1- piperidinyl group.
14. The compound of Claim 11, wherein R1 and R2 together with the nitrogen atom to which they are attached form a 1- piperidinyl group, R13 is hydrogen, and X is chloro, bromo, methyl, or trifluoromethyl.
15. A pharmaceutical composition, comprising: (a) a compound of formula (II)
Figure imgf000041_0001
wherein:
RJ and Rz , each independently of the other, are Ct-C4 alkyl, or together with the nitrogen atom to which they are attached form a ring containing 5-6 atoms, including C and/or O; R3-R1Z, each independently of the other, are hydrogen, halogen, Cj-C4 alkyl, C,-C4 alkoxyl, substituted C,-C4 alkoxyl, nitro, cyano, or trifluoro ethy1;
Ru is hydrogen, C(-C4 alkyl, C2-C- acyl, or arylalkyl; and X is halogen, Ct-C4 alkyl, or substituted Ct-C4 alkyl, or a pharmaceutically acceptable salt thereof; with the proviso that, when R1 and R2 together with the nitrogen atom to which they are attached form an N-morpholinyl group, at least one of R3-R12 is not hydrogen; and (b) a pharmaceutically acceptable carrier.
16. The composition of Claim 15, wherein R- and R: together with the nitrogen atom to which they are attached form a group selected from the group consisting of 1- piperidinyl, N-morpholinyl, and 1-pyrrolidinyl.
17. The composition of Claim 15, wherein R1 and R2 together with the nitrogen atom to which they are attached form a 1-piperidinyl group.
18. The composition of Claim 15, wherein R1 and R2 together with the nitrogen atom to which they are attached form a 1-piperidinyl group, R13 is hydrogen, and X is chloro, bromo, methyl, or trifluoromethyl.
19. The composition of Claim 15, which is in the form of a unit dosage which comprises 5 mg to 4 g of said compound of formula (II) .
20. The composition of Claim 15, which is in the form of a unit dosage which comprises 10 mg to 1500 mg of said compound of formula (II) .
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WO2004050613A3 (en) * 2002-12-02 2004-09-23 Gilead Sciences Inc 2-substituted-3-propenamide derivatives and methods of using the same
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US7041690B2 (en) 2002-07-01 2006-05-09 Pharmacia & Upjohn Company, Llc Inhibitors of HCV NS5B polymerase
JP2008528600A (en) * 2005-01-26 2008-07-31 アラーガン、インコーポレイテッド 3-Aryl-3-hydroxy-2-amino-propionic acid amide, 3-heteroaryl-3-hydroxy-2-amino-propionic acid amide and related compounds having analgesic activity and / or immunostimulatory activity
EP2514750A1 (en) 2007-06-18 2012-10-24 Sunshine Lake Pharma Co., Ltd Bromo-phenyl substituted thiazolyl dihydropyrimidines
CN107011206A (en) * 2017-03-06 2017-08-04 徐州医科大学 A kind of acrylamides, its preparation method and medical usage
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WO2000024392A1 (en) * 1998-10-26 2000-05-04 Sumitomo Pharmaceuticals Company, Limited β-AMYLOID FORMATION INHIBITORS
WO2001045712A1 (en) * 1999-12-22 2001-06-28 Bayer Aktiengesellschaft Combinations of medicaments for treating viral diseases
US6930106B2 (en) 2002-07-01 2005-08-16 Pharmacia & Upjohn Company Inhibitors of HCV NS5B polymerase
US7041690B2 (en) 2002-07-01 2006-05-09 Pharmacia & Upjohn Company, Llc Inhibitors of HCV NS5B polymerase
WO2004050613A3 (en) * 2002-12-02 2004-09-23 Gilead Sciences Inc 2-substituted-3-propenamide derivatives and methods of using the same
US7026339B2 (en) 2003-11-07 2006-04-11 Fan Yang Inhibitors of HCV NS5B polymerase
US8927589B2 (en) 2005-01-26 2015-01-06 Allergan, Inc. 3-aryl-3-hydroxy-2-amino-propionic acid amides, 3-heteroaryl-3-hydroxy-2-aminopropionic acid amides and related compounds having analgesic and/or immuno stimulant activity
JP2008528600A (en) * 2005-01-26 2008-07-31 アラーガン、インコーポレイテッド 3-Aryl-3-hydroxy-2-amino-propionic acid amide, 3-heteroaryl-3-hydroxy-2-amino-propionic acid amide and related compounds having analgesic activity and / or immunostimulatory activity
US8288556B2 (en) 2005-01-26 2012-10-16 Allergan, Inc. 3-aryl-3-hydroxy-2-amino-propionic acid amides, 3-heteroaryl-3-hydroxy-2-amino-propionic acid amides and related compounds having analgesic and/or immuno stimulant activity
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WO2018085619A1 (en) 2016-11-07 2018-05-11 Arbutus Biopharma, Inc. Substituted pyridinone-containing tricyclic compounds, and methods using same
CN107011206A (en) * 2017-03-06 2017-08-04 徐州医科大学 A kind of acrylamides, its preparation method and medical usage
CN107011206B (en) * 2017-03-06 2020-01-03 徐州医科大学 Acrylamide compound, preparation method and medical application thereof
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