Background of the Invention Hepatitis is an inflammation of the liver that is most often caused by infection with one of five known viruses, hepatitis A, B, C, D or E. In many cases of infection, particularly in cases of hepatitis B and hepatitis C infection,"chronic hepatitis"may result. Chronic hepatitis occurs when the body is unable to completely clear the virus even though the symptoms may not persist.

Continued presence of the virus over a number of years can lead to cirrhosis (scarring of the liver) or hepatoceliular carcinoma (liver cancer).

Hepatitis B virus (HBV) is transmitted through sexual contact, vertical transmission (mother to child at birth) or by coming into contact with contaminated blood. It is estimated that over 2 billion people worldwide have been infected with hepatitis B virus. Of these 2 billion people, approximately 350 million people have developed chronic HBV infection, putting them at high risk of developing cirrhosis and liver cancer (World Health Organization, fact sheet on Hepatitis B, November, 1998). In the United States alone, 140,000 to 320,000 HBV infections are reported every year, and an estimated 1 to 1.25 million Americans are chronically infected with HBV. The estimated costs of medical treatment and work loss is over 700 million dollars per year.

Chronic carriers of the HBV have been defined as those who are HBV surface antigen positive for greater-than 6 months. Approximately 5-10% of those people who are infected with the virus will become carriers, an estimated 5-10% of those people infected each year will progress to chronic liver disease, cirrhosis and possibly liver cancer. About 5,000 people die in the United States each year from causes related to HBV, and 1,000 die of HBV-related liver cancer.

The incubation period for HBV usually lasts from 2 to 4 months, although it may be very short (10 days) or extremly long (9 months). Before the outbreak of the acute disease, hepatitis B surface antigen (HBs-Ag) and"enigma"antigen (HBe- Ag) are detectable in the patient's serum.

The onset of acute hepatitis is characterized by the appearance of antibodies to hepatitis B core antigen (anti-HBc-Ag Abs), which at first exclusively belong to the IgM class. From this point on in the course of infection, anti-HBc antibodies will be detectable in the patient's serum for the rest of his life, no matter whether there is an acute hepatitis B, a form of persisting viral infection, or some naturally acquired immunity to HBV.

The most significant event indicating a chronic course of hepatitis B is the absence of the HBsAg/anti-HBs seroconversion. If this phenomenon has not occurred within 6 months after the onset of the disease, persistence of the HBV infection and the related clinical pictures (asymptomatic HBsAg carrier, chronic hepatitis, cirrhosis, or hepatoma) have to be reckoned with.

While there are effective vaccines for prevention of HBV infection, the treatments available to those who become infected are not satisfactory. Current treatments focusing on compounds like Epivir-HBV (lamivudine, 3TC), or cytokines, e. g., Intron A (alpha interferon, IFN-cc) show some benefits for chronic hepatitis B, but there is clearly a need for additional therapies. It has now been discovered that a class of guanyl hydrazones have the unexpected activity of inhibiting HBV replication, and these compounds are useful for the treatment of HBV infections, particularly chronic HBV infections.

Summary of the Invention A first aspect of the present invention relates to compounds having the general formula (11) : wherein: Xi represents-CHGhy or-C (CH3) Ghy; X2 represents-H,-OCH3,-CHGhy or-C (CH3) Ghy; Ghy represents a guanidino group: =N-NH-C (NH) NH2; Z is-A- (CH2) n-CH=CH- (CH2) p-A'-B, -A-(CH20n-C6H4- (CH2) p-A'-B, -A- (CH2) n-C5H3N- (CH2) p-A'-B,-A- (CH2) n-CR'R"- (CH2) p-A'-B, -A- (CH2) n-CH=CH- (CH2) p-B,-A- (CH2) n-C6H4-(CH2)p-B, -A- (CH2) n-C5H3N- (CH2) p-B,- (CH2) nB,-A- (CH2) n-CR'R"- (CH2) p-B, -A- (CH2) n-B or-A- (CH2) n-A'-B ; R', R"are independently of each other-OH,-SH,-NH2, methyl, ethyl or propyl ; A and A'are independently of each other-NH (CO)-, -NH-, -(CO)NH-, -NH (CO) NH- or-O- ; B represents X'1 is independently of X1 -CHGhy or-C (CH3) Ghy; X'2 is independently of X2-H,-OCH3,-CHGhy or-C (CH3) Ghy; n and p are independently of each other an integer of 0 to 10;

under the proviso that A # A' ; and pharmaceutical acceptable salts thereof.

A second aspect of the present invention relates to the use of compounds of the general formula (11) : wherein: Xi represents-CHGhy or-C (CH3) Ghy; X2 and X'2 represents-H,-OCHs,-CHGhy or-C (CH3) Ghy; Ghy represents a guanidino group: =N-NH-C (NH) NH2; Z is-A- (CH2) n-CH=CH- (CH2) p-A'-B,-A- (CH2) n-C6H4- (CH2) p-A'-B, -A- (CH2) n-C5H3N- (CH2) p-A'-B, -A-(CH2)n-CR'R"-(CH2)p-A'-B, -A- (CH2) n-CH=CH- (CH2) p-B,-A- (CH2) n-C6H4- (CH2) p-B, -A- (CH2) n-C5H3N- (CH2) p-B,- (CH2) nB,-A- (CH2) n-CR'R"- (CH2) p-B, -A- (CH2) n-B or-A- (CH2) n-A'-B ; R', R"are independently of each other-OH,-SH,-NH2, methyl, ethyl or propyl ; A and A'are independently of each other-NH (CO}-,-NH-,- (CO) NH-, -NH (CO) NH- or-0- ; B represents X'i is independently of X1 -CHGhy or-C (CH3) Ghy; X'2 is independently of X2-H,-OCH3,-CHGhy or-C (CH3) Ghy ; n and p are independently of each other an integer of 0 to 10;

under the proviso that A ; A' ; and pharmaceutical acceptable salts thereof as pharmaceutically active agents.

A third aspect of the present invention relates to the use of compounds of the general formula (II) : wherein: Xi represents-CHGhy or-C (CH3) Ghy; X2 represents-H,-OCH3,-CHGhy or-C (CH3) Ghy; Ghy represents a guanidino group: =N-NH-C (NH) NH2 ; Z is-H,-NH (CO) NHB,-C6H4B,-NHC (NH) NHC (NH) NH2,-C5NH3B, -C (CH3) Ghy,-CHGhy,-NH (CO)-Ph,-CO-NHPh,-CH=CH-COOH, -A- (CH2) n-CH=CH- (CH2) p-A'-B, -A- (CH2) n-C6H4-(CH2)p-A'-B, -A- (CH2) n-C5H3N- C5H3N- (CH2) n-CR'R"- (CH2) p-A'-B, -NH (CO) B, -NHB, -(CO)NHB, -COB, -SB, -OB, -CO-OB, -O-COB, -NH (CO) OB,-A- (CH2) n-CH=CH- (CH2) p-B,-A- (CH2) n-C6H4- (CH2) p-B, -A- (CH2) n-C5H3N- (CH2) p-B,- (CH2) nB,-A- (CH2) n-CR'R"- (CH2) p-B, -A- (CH2) n-B, -A- (CH2) n-A'-B,-NH2,-O (CO) NHB, R', R"are independently of each other-OH,-SH,-NH2, methyl, ethyl or propyl ; A and A'are independently of each other-NH (CO)-, -NH-, -(CO)NH-, -NH (CO) -NH- or -O-; B represents

X'1 is independently of Xi-CHGhy or-C (CH3) Ghy; X'2 is independently of X2-H,-OCH3,-CHGhy or-C (CHs) Ghy; n and p are independently of each other integer of 0 to 10; and pharmaceutical acceptable salts thereof as pharmaceutical active agents for prophylaxis and/or treatment of Hepatitis B virus infections and associated diseases, including opportunistic infections.

The guanylhydrazone compounds of the invention are basic and form pharmaceutical acceptable salts with organic and inorganic acids. Examples of suitable acids for such acid addition salt formation are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, p-aminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, sulfonic acid, phosphonic acid, perchloric acid, nitric acid, formic acid, propionic acid, gluconic acid, lactic acid, tartaric acid, hydroxymaleic acid, pyruvic acid, phenylacetic acid, benzoic acid, p-aminobenzoic acid, p-hydroxybenzoic acid, methanesulfonic acid, ethanesulfonic acid, nitrous acid, hydroxyethanesulfonic acid, ethylenesulfonic acid, p-toluenesulfonic acid, naphthylsulfonic acid, sulfanilic acid, camphersulfonic acid, china acid, mandelic acid, o-methylmandelic acid, hydrogen-benzenesulfonic acid, picric acid, adipic acid, d-o-tolyltartaric acid, tartronic acid, cc-toluic acid, (o, m, p)-toluic acid, naphthylamine sulfonic acid, and other mineral or carboxylic acids well known to those skilled in the art. The salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce a salt in the conventional manner.

The free base forms may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous sodium hydroxide, potassium carbonate, ammonia and sodium bicarbonate. The free base forms differ from their corresponding salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the salts are otherwise

equivalent to their corresponding free base forms for purposes of this invention.

Preferred is the use of a compound of formula (II) wherein the guanylhydrazone substituents characterized as, Xi and X2, or within the residue B as X'1 and X'2, are in meta or para position. In the case that X2 (X'2) is not hydrogen the meta position of Xi and X2 (X', and X'2) to Z is most preferred, because of steric reasons.

Furthermore, the use of compounds of the general formula (II) is preferred wherein Z represents -A-(CH2) n-A'-B or-A- (CH2) n-B and n is an integer of 1 to 10 or these compounds wherein Z represents -A-(CH2)n-CH=CH-(CH2)p-A'- B,-A- (CH2) n-CH=CH- (CH2) p-B, -A- (CH2) n-C6H4- (CH2) p-A'-B,-A- (CH2) n-C6H4- (CH2) p-B,-A- (CH2) n- CR'R"- (CH2) p-A'-B,-A- (CH2) n-CR'R"- (CH2) p-B,-A- (CH2) n-C5H3N- (CH2) p-A'-B, or-A- (CH2) n-C5H3N- (CH2) p-B and n and p are independently of each other integer of 1 to 5. A, A', and B represent the residues as mentioned above.

More preferred is the use of the inventive guanylhydrazone compounds wherein Z is-NH (CO)- (CH2) n- (CO) NHB and n is an integer of 3 to 10 and these compounds wherein Z stands for-NH (CO}- (CH2) n-C6H4-(CH2)p-(CO) NHB, -NH (CO)- (CH2) n-CH=CH- (CH2) p- (CO) NHB, -NH (CO)- (CH2) n-CR'R-(GH2) p-(CO) N HB, or -NH (CO)- (CH2) n-C5H3N-(CH2)p-(CO) NHB, and n and p are independently of each other integer of 1 to 5. B represents another benzene substituted with one or two guanylhydrazone residues as shown above.

Most preferred is the use of the inventive compounds selected from the group comprising : N-(4-acetylphenyl)-N'-(3,5-diacetylphenyl)urea tris (amidinohydrazone), N, N'-bis (3-acetylphenyl) pentane diamide bis (amidinohydrazone), N, N'-bis (3,5-diacetylphenyl) pentane diamide tetrakis (amidinohydrazone), N, N'-bis (3,5-diacetylphenyl) decane diamide tetrakis (amidinohydrazone), N, N'-bis (3,5-diacetylphenyl) butane diamide tetrakis (amidinohydrazone),

N, N'-bis (3,5-diacetylphenyl) hexane diamide tetrakis (amidinohydrazone), N, N'-bis (3,5-diacetylphenyl) heptane diamide tetrakis (amidinohydrazone), N, N'-bis (3,5-diacetylphenyl) isophthalic acid diamide tetrakis (amidino- hydrazone) or a salt of one of these compounds.

The compounds of the general formula (I), as defined below, and/or (II) and/or pharmaceutically acceptable salts thereof can be used to manufacture pharmaceutical formulations useful for prophylaxis and/or treatment of Hepatitis B virus infections, including opportunistic infections, and associated diseases.

The inventive compounds are especially useful for prophylaxis and/or treatment of acute and/or chronic Hepatitis B virus infections. Furthermore, said inventive guanylhydrazone compounds can be used to treat diseases induced by HBV infection or diseases associates with HBV infection. Said diseases comprise chronic liver disease, cirrhosis or hepatocellular carcinoma induced by HBV infection.

The compounds of the general formula (I), as defined below, and (II) and their pharmaceutical active salts are especially useful for treating drug resistant Hepatitis B virus strains. Said drug resistant hepatitis B virus strains are especially Lamivudine resistant strains.

The compounds of the general formula (I) and/or (II) and/or pharmaceutical acceptable salts are also useful for regulating the production and/or replication of Hepatitis B viruses in an individual and/or in cells.

As used herein the term"individual"refers to mammals, especially humans.

The compounds of the general formula (I) and/or (11) and/or pharmaceutical acceptable salts are administered in a dosage corresponding to an effective concentration in the range of 0.01-100, M, preferably in the range of 0.1-100 , M, more preferably in the range of 1-10 uM, and most preferably in the range of 1-5, Jet.

As used herein, a"pharmaceutical effective concentration"of an inhibitor is an amount effective to achieve the desired-physiological result, either in cells treated in vitro or in a subject treated in vivo. Specifically, a pharmaceutical effective amount is an amount sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the viral infection. The effective amount may vary depending on the specific inhibitor selected, and is also dependent on a variety of factors and conditions related to the subject to be treated and the severity of the infection. For example, if the inhibitor is to be administered in vivo, factors such as the age, weight and health of the patient as well as dose response curves and toxicity data obtained in pre- clinical animal work would be among those considered. If the inhibitor is to be contacted with the cells in vitro, one would also design a variety of pre-clinical in vitro studies to assess such parameters as uptake, half-life, dose, toxicity, etc.

The determination of a pharmaceutical effective concentration for a given agent is well known within the ability of those skilled in the art.

Another preferred embodiment of the present invention describes the use of at least one compound of the general formula (I) and/or (II) and/or pharmaceutically active salts thereof in combination with further therapeutic compounds. Said further therapeutic compounds are selected from the group comprising lamivudine, Zeffixe, Heptovir@, 3TC@, Epivir-HBV, Combivir@, Trizivir° by GlaxoSmithKline), alpha interferon (e. g., Intron As by Schering-Plough), FTC (e. g., Coviracil'by Triangle Pharmaceuticals), DAPD (DXG, by Triangle), L- FMAU (e. g., Clevudineo by Triangle), Adefovir dipivoxil (by Gilead Sciences), tenofovir, epavudine, epcitabine, lobucavir, Penciclovir (GlaxoSmithKline), Famvir, Entecavir/BMS-200475 (by Bristol-Myers Squibb), Racivir (by Pharmasset), L-ddA prodrug, HDP-P-acyclovir, ara-AMP prodrugs, thymosin alpha-1 (e. g., Zadaxino by SciClone), (-)-Carbovir, hammerhead ribozymes, HBV DNA vaccine such as Genevaxo (by Wyeth-Lederle Vaccine), PreS1/S2 vaccine (e. g., Hepagenee by Medeva PLC), HBV immunoglobulin (e. g., Nabi-HBV by Nabi), glycosidase inhibitors such as Nonyl-DNJ (by Synergy), human monoclonal antibodies directed against HBV (e. g., by XTL Biopharmaceuticals), therapeutic vaccine strategies including live, attenuated and replication incompetent virus; killed, inactivated virus; envelope subunit protein; core subunit

protein; peptides; nucleic acids of the HBV; antiviral gene-therapy approaches, including-antisense or dominant-negative HBV core mutants, and any'other antiviral composition in use or in development for use to treat hepatitis B infection, and any other antiviral composition in use or in development for use to treat hepatitis B infection.

A further aspect of the present invention is directed to a method for preventing and/or treating Hepatitis B virus infections, including opportunistic infections, and associated diseases in a mammal, including a human, which comprises administering to the mammal an amount of at least one compound of the general formula (II) and/or pharmaceutical acceptable salts thereof effective to prevent and/or treat Hepatitis B virus infections, including opportunistic infections, and associated diseases.

Furthermore, a method for regulating the production and/or replication of Hepatitis B viruses in an individual is disclosed in the present invention, that method comprises administering to the individual an amount of at least one compound of the general formula (II) and/or pharmaceutical acceptable salts thereof effective to at least partially inhibit the production and/or replication of Hepatitis B virus. A similar aspect of the present invention describes a method for regulating the production and/or replication of Hepatitis B viruses in cells, which comprises administering to the cells an amount of at least one compound of the general formula (II) and/or pharmaceutical acceptable salts thereof effective to at least partially inhibit the production and/or replication of Hepatitis B virus.

The methods disclosed herein are especially useful for treating drug resistant Hepatitis B virus strains. Another aspect of the present invention relates to the use of a compound having the formula (I) :

wherein: X2 is GhyCH-, GhyCCH3-or H-, wherein Ghy being a guanidino group {(H2N) (HN) C-NH-N=} ; Xi, X'i and X'2 are, independently, GhyCH-or GhyCCH3-; Z is-NH (CO) NH-,- (C6H4)-,- (C5NH3)-, or-A- (CH2) n-A-, where n is an integer from 2-10, and monounsaturated or diunsaturated derivatives thereof, and wherein Z may be unsubstituted or may be methyl-, dimethyl-or trimethyl-substituted at any carbon atom, or may be substituted with a heteroatom (e. g., N, O, S); and A is independently-NH (CO)-,- (CO) NH-,-NH (CO) NH-,-NH-or-O-, and salts thereof, as an inhibitor of Hepatitis B Virus (HBV). Preferably, when X2 is GhyCH-or GhyCCH3-, X2 is meta or para to Xi and X'2 is meta or para to X'1.

More preferably, Z is-NH (CO)-Y- (CO) NH- and Y is (CH2) n, wherein n is an integer from 6-10 and wherein up to 3 CH2 groups may be substituted by heteroatom groups such as NH, O or S. Most preferably, the compound is N, N'-bis (3,5- diacetylphenyl) decanediamide-tetrakis (amidino) hydrazone (Fig. 1) or a salt thereof, particularly a pharmaceutical acceptable salt such as the tetrahydrochloride.

A still further aspect of the present invention relates to the preparation of medicines for treatment of HBV infected individuals. Pharmaceutical compositions including a compound according to formula (I) and/or (II) and/ortheir pharmaceutically active salts in a pharmaceutical acceptable carrier are contemplated. Such pharmaceutical compositions include inserts instructive as to the treatment of HBV infection using the compound of formula (I) and/or (II) and/or pharmaceutical active salts thereof, and may also advantageously include additional active ingredients either separately or in admixture with the compound

of formula (I) and/or (II) and/or pharmaceutically active salts thereof, such additional active ingredient can be selected form-the group comprising lamivudine, Zeffixo, Heptovir@, 3TC@, Epivir-HBV, Combiviro, Trizivirs by GlaxoSmithKline), alpha interferon (e. g., Intron Ao by Schering-Plough), FTC (e. g., Coviracil'by Triangle Pharmaceuticals), DAPD (DXG, by Triangle), L-FMAU (e. g., Clevudinee by Triangle), Adefovir dipivoxil (by Gilead Sciences), tenofovir, epavudine, epcitabine, lobucavir, Penciclovir (GlaxoSmithKline), Famvir, Entecavir/BMS- 200475 (by Bristol-Myers Squibb), Racivir (by Pharmasset), L-ddA prodrug, HDP-P-acyclovir, ara-AMP prodrugs, thymosin alpha-1 (e. g., Zadaxino by SciClone), (-)-Carbovir, hammerhead ribozymes, HBV DNA vaccine such as Genevaxo (by Wyeth-Lederle Vaccine), PreS1/S2 vaccine (e. g., Hepageneo by Medeva PLC), HBV immunoglobulin (e. g., Nabi-HBV by Nabi), glycosidase inhibitors such as Nonyl-DNJ (by Synergy), human monoclonal antibodies directed against HBV (e. g., by XTL Biopharmaceuticals), therapeutic vaccine strategies including live, attenuated and replication incompetent virus; killed, inactivated virus; envelope subunit protein; core subunit protein; peptides; nucleic acids of the HBV; antiviral gene-therapy approaches, including antisense or dominant-negative HBV core mutants, and any other antiviral composition in use or in development for use to treat hepatitis B infection, and any other antiviral composition in use or in development for use to treat hepatitis B infection.

Brief Description of the Drawings Fig. 1 shows the structures of: Compound 1: N- (4-acetylphenyl)-N'- (3, 5-diacetylphenyl) urea tris (amidinohydrazone), Compound 2: N, N'-bis (3-acetylphenyl) pentane diamide bis (amidinohydrazone), Compound 3: N, N'-bis (3,5-diacetylphenyl) pentane diamide tetrakis (amidinohydrazone), Compound 4: N, N'-bis (3,5-diacetylphenyl) decane diamide tetrakis (amidinohydrazone), Compound 5: N, N'-bis (3,5-diacetylphenyl) butane diamide tetrakis (amidinohydrazone),

Compound 6: N, N'-bis (3,5-diacetylphenyl) hexane diamide tetrakis (amidinohydrazone), Compound 7: N, N'-bis (3,5-diacetylphenyl) heptane diamide tetrakis (amidinohydrazone), and Compound 8: N, N'-bis (3,5-diacetylphenyl) isophthalic acid diamide tetrakis (amidino-hydrazone).

The compounds are provided in the form of their corresponding bis-, tris-, or tetrahydrochloride salts.

Fig. 2 shows a plot of the effect of various concentrations of a guanyl hydrazone compound (Compound 4, see Fig. 1) on hepatitis B virus expressing HepG2- 2.2.15 human hepatoma cells.

Fig. 3 shows a plot of the effect of various concentrations of a guanyl hydrazone compound (Compound 1, see Fig. 1) on Hepatitis B virus expressing HepG2- 2.2.15 human hepatoma cells.

Fig. 4 shows a plot of the effect of various concentrations of a guanyl hydrazone compound (Compound 4, see Fig. 1) on 3TC-resistant HBV production in transiently transfected human hepatoma cells.

Fig. 5 shows data and synergy plot of combination anti-HBV activity of a guanyl hydrazone compound (Compound 4, see Fig. 1) with 3TC in Hepatitis B virus expressing HepG2-2.2.15 human hepatoma cells.

Fig. 6 shows the effect of various concentrations of a guanyl hydrazone compound (Compound 4, see Fig. 1) on encapsidated plus strand viral nucleic acid in Hepatitis B virus expressing HepG2-2.2.15 human hepatoma cells.

Detailed Description The present invention relates to the use of certain guanyl hydrazone compounds for inhibiting HBV replication. The guanyl hydrazone compounds disclosed herein are suitable for use in treating HBV infection and are particularly useful for treating chronic HBV infection. The compounds of formula (I) and (II), above, are potent inhibitors of HBV replication in the low micromolar range and exhibit low cellular toxicity at effective antiviral dosages. Thus, the compounds described

herein may be used as the active ingredient in medicines for the treatment of HBV- infection and in methods for treating patients suffering from hepatitis B.

Compounds having the formula (II), wherein A = A', or (I) are known from U. S.

Patent 5,599,984 (incorporated herein by reference) to be inhibitors of the uptake of arginine by macrophages and/or its conversion to urea. Further, the suitability of these compounds in preventing the generation of nitric oxide (NO) by cells and so to prevent NO-mediated inflammation and other responses is disclosed.

International patent publication WO 98/20868 discloses a method for treating diseases and disorders involving T-cell activation and HIV infection using the p38 mitogen activated protein kinase (MAPK) signaling pathway as target for the intervention. It is suggested that inventive guanylhydrazone compounds according to formula (I) or (II) act as MAPK inhibitors. However, there has been no disclosure of the compounds of formula (I) or (II) as having activity to inhibit HBV.

Unlike HIV and other retroviruses that belong to the group of RNA viruses, HBV belongs to the DNA virus group of hepadnaviruses, which multiply by an entirely different mechanism and show an entirely different multiplication cycle.

Specifically for HBV, no viral encoded protein is known that acts on nucleo- cytoplasmic translocation of viral RNA, unlike the Rev-type mechanism of HIV, which has been shown to be the relevant mechanism that is inhibited by the compounds of formula (I) and (II) and/or pharmaceutical active salts thereof with respect to that virus.

Surprisingly, it has now been found that the compounds of formula (1) and (II) are potent inhibitors of HBV replication in the micromolar range by screening in a relevant HBV cellular assay. The compounds of Fig. 1 were observed to reduce virus production in HepG2-2.2.15 human hepatoma cells to very low levels at concentrations between about 10 and 20 M, while having little cytotoxic effect on control cells. These results indicated that the compounds of Fig. 1 are potent inhibitors of HBV replication in the low micromolar range with an therapeutic index TI >6.

These findings indicate that the class of aromatic guanylhydrazones disclosed herein may advantageously be used in the treatment of HBV infection, particularly chronic HBV infection.

While the specific mode of action of these inhibitors is not certain, it is believed that they may act as inhibitors of the biological function of certain cellular proteins involved in the translocation of essential viral mRNAs from the nucleus of an infected cell to the cytoplasm, notably elF-5A (Bevec et al., 1996, Science, 271, p 1858-1860), TAP (human homologue of yeast Mex67p ;-Gruter et al., 1998, Mol.

Cell, 1, p 649-659; Segref et al., 1997, EMBO J., 16, p 3256-3271), p15 (human homologue of Mtr2p ;- Katahira et al., 1999, EMBO J., 18, p 2593-2609; Santos- Rosa et al., 1998, Mol. Cell. Biol., 18, p 6826-6838), L5, TFIIIA (Rosorius et al., 2000, J. Biol. Chem., 275, p 12061-12068), and/or Dbp5/Rat8p (DEAD box type of RNA helicases ;- Tseng et al., 1998, EMBO J., 17, p 2651-2662; Snay-Hodge et al., 1998, EMBO J., 17, p 2663-2676) proteins.

The compounds of formula (I) and (II) may be formulated in suitable pharmaceutical carriers and may be administed by any appropriate means, including but not limited to injection (intravenous, intraperitoneal, intramuscular, subcutaneous) by absorption through epithelial or mucocutaneous linings (oral mucosa, rectal and vaginal epithelial linings, nasopharyngial mucosa, intestinal mucosa); orally, rectally, transdermally, topically, intradermally, intragastrally, intracutaneously, intravaginally, intranasally, intrabuccally, percutaneously, sublingually, or any other means available within the pharmaceutical arts. The compound may be administered to a subject in need thereof, e. g., a human patient, by itself or in pharmaceutical compositions where they are mixed with suitable carriers or excipients at doses which are sufficient for an at least partial reduction of virus production. Therapeutically effective doses may be administered alone or as adjunctive therapy in combination with further therapeutic compounds, particularly antiviral agents and more particularly antiviral agents suitable for treatment of HBV infection, e. g., alpha interferon and/or reverse transcriptase inhibitors. The compounds of formula (I) and/or (II) may be specifically combined with compounds such as lamivudine (e. g., Epivir-HBV),

alpha interferon (e. g., Intron A), Coviracil/FTC, DAPD, L-FMAU, adefovir dipivoxil, tenofovir, epavudine, epcitabine, lobucavir, Famvir/penciclovir, entecavir/BMS- 200475, racivir, DXG, L-ddA prodrug, HDP-P-acyclovir, LM-019c, CS-1091, PS- 019, PS-018, ara-AMP prodrugs, thymosin, (-)-Carbovir, hammerhead ribozymes, glycosidase inhibitors, and any other antiviral composition in use or in development for use to treat hepatitis B infection; with therapeutic vaccine strategies including live, attenuated and replication incompetent virus; killed, inactivated virus; envelope subunit protein; core subunit protein; peptides; nucleic acids of the HBV; with antiviral gene-therapy approaches like antisense or dominant-negative HBV core mutants. This invention also relates to the combination of the compounds of formula (I) and/or (II) with at least one of the above mentioned drugs. The compounds described herein can be used as inhibitors of HBV as well as for treating diseases associated with HBV, especially where treatment with other drugs, particularly NRTIs or NNRT ! s, has caused viral resistance against conventional antiviral drugs.

Techniques for the formulation and administration of the compounds of the present invention may be found in Remington's Pharmaceutical Sciences, 18th ed., Alfonso R. Gennaro, ed. (Mack Publishing Co., Easton, PA 1990). A suitable composition comprising the compound of the invention may be a solution of the compound in a suitable liquid pharmaceutical carrier or any other formulation such as tablets, pills, dragees, capsules, gels, syrups, slurries, suspensions and the like.

A therapeutical effective dosage of the compound of formula (I) or (II) refers to that amount of the compound that results in an at least partial inhibition of virus production in the patient, which may be measured in several ways, e. g., reduction in viral DNA in a patient hepatocyte sample, increase in cell viability, and/or reduction of HBV"enigma"antigen (HBe-Ag) and/or HBs-Ag in a patient's serum, and consequently results in a desired pharmaceutical effect. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical, pharmacological, and toxicological procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the

population). The dose ratio between toxic and therapeutic--effect is the therapeutic-index and can be expressed as the ratio between LD50 and ED50.

The dosage of the compound lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. More preferably, the dosage of the compound corresponds to an effective concentration in the range of 0.1-100, M, most preferably 1-10 pLM. The actual amount of the composition administered will be dependent on the subject being treated, on the subject's weight, the severity of the HBV infection, the manner of administration, and the judgement of the prescribing physician.

The present invention will be further illustrated by the following non-limiting examples.

Examples Example 1: Effect of N, N'-bis (3,5-diacetylphenyl) decanediamide-tetrakis- (amidino) hydrazone on HBV production in the HepG2-2.2.15 cell culture system.

HepG2-2.2.15 human hepatoma cells, obtainable according to the procedure described in Sells et al., 1987, Proc. Natl. Acad. Sci. USA, Vol. 84, pp. 1005- 1009, which is fully incorporated by reference, were plated in 96-well microtiter plates in DMEM medium supplemented with fetal bovine serum. After incubation at 37°C in a humidified, 5% C02 environment for 16-24 hours, the monolayer of HepG2-2.2.15 cells was washed and the medium was replaced with complete medium containing various concentrations of test compound. Every three days, the culture medium was replaced with fresh medium containing the appropriately diluted test compound. Six days following the initial administration of the test compound, the cell culture supernatant was collected and clarified by centrifugation. The cell viability was evaluated by a dye uptake procedure (Cell Titer Aqueous One Solution, by Promega). Virion-associated HBV DNA present in the tissue culture supernatant was amplified employing PCR technology using primer pairs derived from the HBV strain ayw. Subsequently, the PCR-amplified HBV DNA was detected in real time by monitoring increases in fluorescence signals that result from exonucleolytic degradation of a quenched fluorescent probe molecule following hybridization of the probe to the amplified HBV DNA

(TaqMan ; Perkin-Etmer). A standard curve using a known copy number of HBV genome was generated and the copy number of HBV genome in each sample was then derived from the curve. The O. D number obtained with CeIlTiter 96° AQueous Cell Proliferation Assay and the HBV DNA copy number obtained with Real Time PCR were analyzed using a computer program which calculates the percentage of DNA copy number, IC50, the percentage of cell viability, TC so, and TI (therapeutic index) and displays the graphic results.

The results of the effect of Compound 4 (see Fig. 1) on virus production in HepG2-2.2.15 cells is shown below in Table 1 and 2.

Table 1: Effect of compound 4 on viral DNA (copy number per 3 pI) conc of 20 10 5 2. 5 1.25 0.75 0 comp 4(pM) sample1 12333.7 55581.4 235252.8 360110.3 721040.6 721220.3 687817.7 sample2 9345.3 53845.6 207368.7 385169.0 524674.6 711419. 3 667661.5 sample3 10162.6 35977.9 170819.2 421169.9 646427.3 731017.2 670182.8 Mean 10613. 9 48468.3 204480.2 388816.4 630714.2 721218.9 675220.7 viral DNA 1.6 7.2 30.3 57.6 93.4 106.8 100 (% of control) Table 2: Effect of compound 4on toxicity values (O. D. at 450/650 nm) conc of 20 10 5 2.5 1.25 0.75 0 comp 4 (1jM) sample1 1.103 1.158 1.295 1.108 1.291 1.426 1.353 sample2 1.106 1.169 1.285 1.198 1. 097 1.538 1.316 sample3 1.095 1.021 1.217 1.350 1.401 1. 458 1.303 Mean 1.101 1.095 1.251 1. 274 1.249 1.498 1.310 cell 84.0 83.6 95.5 97.3 95.4 114.4 100 viability (% of control)

IC50 (, uM) = 3.1945

TC50 (µM)=>20 TC50/IC50 = therapeutic index (TI) > 6.26 A plot of the data above appears in Fig. 2, demonstrating the efficacy of Compound 4 as a potent inhibitor of HBV replication at a low molar range with a therapeutic index (TI) greater than about 6, without apparent cellular toxic effects.

From the results observed with N, N'-bis (3,5-diacetylphenyl) decanediamide- tetrakis (amidino) hydrazone, the class of guanyl hydrazones of formula (I) is indicated to provide potent inhibitors of hepatitis B viral replication with low toxicity.

Example 2: Effect of N- (4-acetylphenyl)-N'- (3, 5-diacetylphenyl) urea tris (amidinohydrazone) on HBV production in the HepG2-2.2.15 cell culture system.

The assay was performed as described above in example 1. Briefly, the toxicity of the compound was evaluated by CeIITiter 96@ AQueous One Solution Cell Proliferation Assay (Promega) and the activity of the compound against HBV by quantification of virion-associated DNA in the cell culture supernatants with Real Time PCR.

The results of the effect of Compound 1 (see Fig. 1) on virus production in HepG2-2.2.15 cells is shown below in Table 3 and 4: Table 3: Effect of compound 1 on viral DNA (copy number/3pt) conc of 100 31.6 10 3.16 1 0.31 0 comp 1 (, uM) sample1 0 80038 216039 337312 400579 418796 419066 sample2 0 83853 188278 320470 371832 393452 342701 sample3 0 89824 270451 327438 431797 390429 365386 Mean 0 84571 224922 328407 401403 400892 375717 viral 0.0 22.5 59.9 87.4 106. 8 106.7 100 DNA (% control) Table 4: Effect of compound 1 on toxicity (XTT-OD at 450/650 nm) conc of 100 31.6 10 3. 16 1 0.31 0 comp 1 (µM) sample1 0.086 0.581 0.666 0.767 0.773 0.802 0.694 sample2 0.101 0.615 0.671 0.686 0.729 0. 778 0. 726 sample3 0. 089 0.597 0. 659 0. 666 0. 781 0.753 0. 773 Mean 0. 092 0.598 0.665 0.706 0.761 0.778 0. 731 cell 12. 6 81. 8 91.0 96.6 104. 1 106. 4 100. 0 viability (% of control)

! C50 (uM) =15. 7 TC 50 uM) = 63. 0 TC50/ ! C50 = therapeutic index (TI) = 4 A plot of the data above appears in Fig. 3, demonstrating the efficacy of Compound 1 as a potent inhibitor of HBV replication at a low molar range with a therapeutic index (TI) of 4, without apparent cellular toxic effects.

From the results observed with N- (4-acetylphenyl)-N'- (3, 5-diacetylphenyl) urea tris (amidinohydrazone), the class of guanyl hydrazones of formula (1) is indicated to provide potent inhibitors of hepatitis B viral replication with low toxicity.

Example 3: Effect of Compound 4 on 3TC-resistant (M552V) HBV production.

HepG2 cells were plated in 24-well plates and transfected with either wild type or 3TC resistant (M552V) HBV genome containing plasmid DNA. After an incubation of 16 hours, Compound 4 was added at five half-log dilutions in duplicates. The culture medium was replaced one day later with fresh medium containing the appropriately diluted drug. Six days post transfection, the cells were used for testing toxicity with the CelITiter 96pro AQueous Cell Proliferation Assay (Promega) and the cell supernatants were harvested. Following toxicity testing, the cells were lysed. The culture supernates or cell lysates were subjected to treatment with DNase after a low speed centrifugation. Virus DNA

present in the tissue culture supernatants or cell lysates was PCR amplified using primers derived from HBV strain ayw. Subsequently, the PCR-amplified HBV#DNA was detected in real-time by monitoring increases in fluorescence signals that result from exonucleolytic degradation of a quenched fluorescent probe molecule following hybridization of the probe to the amplified HBV DNA.

The O. D number obtained with CeIlTiter 9600 AQueous Cell Proliferation Assay and the HBV DNA copy number obtained with Real Time PCR were analyzed using a computer program which calculates the percentage of DNA copy number, (Cso, the percentage of cell viability, TC so and TI (therapeutic index) and displays the graphic results.

The results of the effect of Compound 4 (see Fig. 1) on 3TC resistant mutant HBV production in transiently transfected HepG2cells is shown below and in Fig. 4a:

Table 5: Effect of compound 4 on 3TC resistant HBV (copy number/3µl) conc of 50 15. 8 5 1.5 0. 5 0.15 0 comp 4 (, uM) sample1 0.0 1392. 3 73920.3 288300.2 377075.1 371567.1 419066. 0 sample2 0.0 559.4 65338.1 209569.0 338293.0 364952.8 342700.6 sample3 0.0 1855. 7 56756.0 277361.1 431797.0 438868.0 365385.8 Mean 0. 0 1269. 1 65338.1 258410.1 382388.4 391796.0 375717.5 viral 0.0 0.3 17. 4 68.8 101. 8 104. 3 100. 0 DNA (% control) Table 6: Effect of compound 4 on toxicity (XTT-OD at 450/650 nm) conc of comp 4 50 15. 8 5 1.5 0.5 0.15 0 (UM) sample1 0.160 0.493 0.629 0.797 0.849 0.859 0.694 sample2 0.160 0.458 0.549 0.747 0.794 0.727 0.726 sample3 0.154 0. 444 0.662 0.763 0.797 0.822 0.773 Mean 0.157 0.451 0.606 0.755 0.796 0.775 0.750 cell viability (% 20.9 60.2 80.8 100. 7 106.1 103.3 100 ofcontrol) IC 50 (pM) = 2.78

TC 50 (pM) = 24.67 TC50/IC50-therapeutic index (TI) = 8.73 As a control for the resistance of the transfected HBV genome against 3TC (lamivudine), data on the effect of 3TC on mutant virus production are provided below and graphically displayed in Fig. 4b.

Table 7: Effect of 3TC on 3TC resistant HBV (copy number/3µl) conc of 10 3 1 0.3 0.1 0 3TC(IJM) sample1 13987 12549 6354 10467 17020 18201 sample2 21296 22025 14527 18188 6216 10513 Mean 17642 17287 10441 14328 11618 14357 viral 122.9 120.4 72.7 99.8 80.9 100. 0 DNA (% control) Table 8: Toxicity of 3TC (XTT OD at 450/650nm) conc of 10 3 1 0.3 0.1 0 3TC (µM) sample1 0.565 0. 810 0.708 0.671 0.682 0.811 sample2 0.940 0. 573 0.649 0.726 0.725 0.576 Mean 0. 753 0. 692 0. 679 0. 699 0. 704 0. 694 viability 108. 5 99.7 97.8 100. 7 101. 4 100. 0 (% of control)

The IC50 of 3TC against the 3TC resistant mutant HBV was > 10, uM, while it is around 0.05uM against HBV ayw (data not shown).

A plot of the data above appears in Fig. 4, demonstrating the efficacy of Compound 4 as a potent inhibitor of 3TC (lamivudine) resistant HBV replication at a low molar range with a therapeutic index (TI) of approx. 8, without significant cellular toxic effects.

From the results observed with N, N'-bis (3,5-diacetylphenyl) decanediamide-

tetrakis (amidino) hydrazone7 the class of guanyl hydrazones of formula (I) is indicated to provide potent inhibitors of 3TC-resistant hepatitis B viral replication with low toxicity.

Example 4: Combination of Compound 4 with 3TC (lamivudine).

This in vitro combination assay was designed to define the antiviral interaction of the two compounds and to determine if their interaction is additive, synergistic or antagonistic. In addition, the data are evaluated for indications of enhanced toxicity when the compounds are used in combination with each other.

HepG2-2.2.15 cells were plated in 96-well microtiter plates in DMEM medium supplemented with fetal bovine serum. After incubation at 37°C in a humidified, 5% C02 environment for 16-24 hours, the monolayer of HepG2-2.2.15 cells was washed and the medium replaced with complete medium containing five concentrations of the test compound in all possible combinations with five concentrations of 3TC. Every three days, the culture medium was replaced with fresh medium containing the appropriately diluted drug. Six days following the initial administration of test compound, the cell culture supernate was collected and clarified by centrifugation.

The cell viability was evaluated by Cet ! Titer 96@ AQueous One Solution Cell Proliferation Assay. This assay is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays. The reagent contains a novel tetrazolium compound, MTS, and an electron coupling agent, PES, which, when combined, form a stable solution. The MTS tetrazolium compound is bioreduced into a formazan product by NADPH or NADH produced by dehydrogenase in metabolically active cells. Therefore, the quantity of formazan product measured is directly proportional to the number of living cells in culture.

Virion-associated HBV DNA present in the tissue culture supernate was PCR amplified using primers derived from HBV strain ayw. Subsequently, the PCR- amplified HBV DNA was detected in real-time by monitoring increases in

fluorescence signals that result from exonucleolytic degradation of a quenched fluorescent probe molecule following hybridization of the probe to the amplified HBV DNA.

Evaluation of Combination Anti-HBV Data : Analysis and statistical evaluation of drug combination assays was performed according to the method of Prichard and Shipman (Antiviral Research 14: 181-206 [1990]). Combination antiviral assays were performed utilizing the HepG2.2.15 cell-line and a quantitative TaqMan HBV assay to measure virus-associated DNA levels at the experimental endpoint. Five concentrations of 3TC were tested in all possible combinations with five concentrations of Compound 4. Effects of the drug combinations were calculated based on the activity of each compound when tested alone. The expected additive antiviral protection was subtracted from the experimentally determined antiviral activity at each combination concentration resulting in a positive value (synergy), a negative value (antagonism), or zero (additivity). Data were analyzed by the most stringent statistical means by assuming the compounds inhibited HBV replication by action at the same site (mutually exclusive). The results of the combination assays are presented three dimensionally at each combination concentration, yielding a surface of activity extending above (synergy) or below (antagonism) the plane of additivity. The volume of the surface is calculated and expressed as a synergy volume (µM2%) calculated at the 95% confidence interval. For these studies, synergy is defined as drug combinations yielding synergy volumes greater than 50 pM2%. Slightly synergistic activity and highly synergistic activity have been defined as yielding synergy volumes of 50-100 pM2% and >100 pM2%, respectively. Additive drug interactions have synergy volumes in the range of-50 M2% to 50, M2%, while synergy volumes less than-50 µM2% are considered slightly antagonistic or antagonistic (<-100fi, M2%).

The results are presented in Fig 5a, 5b and 5c, demonstrating a mostly additive interaction of Compound 4 in combination with 3TC (lamivudine).

From the results observed with N, N'-bis (3,5-diacetylphenyl) decanediamide- tetrakis (amidino) hydrazone, the class of guanyl hydrazones of formula (I) is

indicated to provide potent inhibitors of hepatitis B virus replication in combination with other anti-HBV drugs such as 3TC.

Example 5 : Compound 4 mechanism of action.

HepG2-2.2.15 cells were plated in 24-well microtiter plates in DMEM medium supplemented with fetal bovine serum. After incubation at 37°C in a humidified, 5% C02 environment for 16-24 hours, the monolayer of HepG2-2.2.15 cells was washed and the medium replaced with complete medium containing five concentrations of Compound 4 or 3TC. Each concentration was tested in duplicates. Every three days the culture medium was replaced with fresh medium containing the appropriately diluted drug. Six days following the initial administration of test compound, cell viability was evaluated by a dye (Ce ! ! Titer 96@ AQueous) uptake procedure. Following the cell viability testing, the cells were lysed and the nuclei removed. The capsids present in the cell lysates were resolved via agarose/sodium phosphate electrophoresis. Following transfer onto the nylon membrane, the viral positive strand nucleic acids associated with the viral capsids were detected with a P32-labeled negative strand riboprobe prepared in vitro and visualized by exposing the membrane onto X-ray film.

The results from the capsid gels are shown in Fig. 6. In capsids from untreated cells (0 uM compound 4 or 0 uM 3TC), encapsidated pregenomic HBV RNA as well as plus strand viral DNA are responsible for the hybridization signal seen with the minus strand riboprobe. As predicted, the signal for plus strand nucleic acid was significantly higher in capsids from cells treated with the reverse transcriptase inhibitor 3TC, since the pregenomic RNA is not transcribed into DNA and cytoplasmic capsids containing RNA presumably accumulate in the cytoplasm in the absence of virus secretion. In contrast, treatment of HepG2 2.2.15 cells with compound 4 led to a reduction in encapsidated plus strand viral nucleic acid, indicating that the reduction of viral DNA observed in the cell supernatants (examples 1,3 and 4) is not due to inhibition of reverse transcription, but rather to an inhibition of viral RNA synthesis or transport, or protein synthesis, or pregenomic RNA encapsidation or capsid assembly.

From the results observed with N, N'-bis (3,5-diacetylphenyl) decanediamide-

tetrakis (amidino) hydrazone, the class of guanyl hydrazones of formula (I) is indicated to inhibit hepatitis B viral replication by a mechanism different from the mode of action of reverse transcriptase inhibitors like 3TC, and is therefore indicated to provide potent inhibitors in combination with such drugs. In addition, resistance development during drug combination treatments is less likely.

From the foregoing discussion and examples, additional embodiments of the present invention will be apparent to those skilled in this art. All such obvious additional embodiments are included in the scope of the invention as contemplated and described herein and in the claims that follow. The publications cited above are incorporated herein by reference.