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WO2002000613A2 - Inhibitors of hepatitis b virus infection - Google Patents

Inhibitors of hepatitis b virus infection Download PDF

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Publication number
WO2002000613A2
WO2002000613A2 PCT/EP2001/007260 EP0107260W WO0200613A2 WO 2002000613 A2 WO2002000613 A2 WO 2002000613A2 EP 0107260 W EP0107260 W EP 0107260W WO 0200613 A2 WO0200613 A2 WO 0200613A2
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Prior art keywords
hbv
compound
independently
ghy
hepatitis
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PCT/EP2001/007260
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French (fr)
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WO2002000613A3 (en
Inventor
Dorian Bevec
Sabine Obert
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Axxima Pharmaceuticals AG
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Axxima Pharmaceuticals AG
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Priority to AU2001269092A priority Critical patent/AU2001269092A1/en
Publication of WO2002000613A2 publication Critical patent/WO2002000613A2/en
Publication of WO2002000613A3 publication Critical patent/WO2002000613A3/en
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
    • C07C281/16—Compounds containing any of the groups, e.g. aminoguanidine
    • C07C281/18—Compounds containing any of the groups, e.g. aminoguanidine the other nitrogen atom being further doubly-bound to a carbon atom, e.g. guanylhydrazones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • the present invention relates to a class of guanyl hydrazones discovered to have activity as inhibitors of Hepatitis B Virus (HBV) replication.
  • the inhibitor compounds are suitable as agents for the treatment of HBV infection, including opportunistic infections, in particular chronic HBV infection and diseases caused by HBV infections.
  • the present invention discloses pharmaceutical compositions useful for prophylaxis and/or treatment of HBV infections, including opportunistic infections, and diseases caused by HBV infections.
  • 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 hepatocellular carcinoma (liver cancer).
  • Hepatitis B virus 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.
  • 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).
  • hepatitis B surface antigen HBs-Ag
  • HBe- Ag HBe- Ag
  • anti-HBc-Ag Abs antibodies to hepatitis B core antigen
  • IgM class antibodies to hepatitis B core antigen
  • 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.
  • a first aspect of the present invention relates to compounds having the general formula (II):
  • Xi represents -CHGhy or -C(CH 3 )Ghy
  • X 2 represents -H, -OCH 3 , -CHGhy or -C(CH 3 )Ghy;
  • R', R" are independently of each other -OH, -SH, -NH 2 , methyl, ethyl or propyl;
  • a and A are independently of each other -NH(CO)-, -NH-, -(CO)NH-, -NH(CO)NH- or -O-;
  • X'1 is independently of X1 -CHGhy or -C(CH 3 )Ghy;
  • X' 2 is independently of X 2 -H, -OCH 3 , -CHGhy or -C(CH 3 )Ghy;
  • n and p are independently of each other an integer of 0 to 10; under the proviso that A ⁇ A'; and pharmaceutically acceptable salts thereof.
  • a second aspect of the present invention relates to the use of compounds of the general formula (II):
  • Xi represents -CHGhy or -C(CH 3 )Ghy
  • X 2 and X' 2 represents -H, -OCH 3 , -CHGhy or -C(CH 3 )Ghy;
  • R', R" are independently of each other -OH, -SH, -NH 2 , methyl, ethyl
  • a and A are independently of each other -NH(CO)-, -NH- -(CO)NH-, -NH(CO)NH- or -O-;
  • X' ⁇ is independently of Xi -CHGhy or -C(CH 3 )Ghy;
  • X' 2 is independently of X 2 -H, -OCH 3 , -CHGhy or -C(CH 3 )Ghy;
  • n and p are independently of each other an integer of 0 to 10; under the proviso that A ⁇ A'; and pharmaceutically 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):
  • Xi represents -CHGhy or -C(CH 3 )Ghy
  • X 2 represents -H, -OCH 3 , -CHGhy or -C(CH 3 )Ghy;
  • X' ⁇ is independently of Xi -CHGhy or -C(CH 3 )Ghy;
  • X' 2 is independently of X 2 -H, -OCH 3 , -CHGhy or -C(CH 3 )Ghy;
  • n and p are independently of each other integer of 0 to 10; and pharmaceutically acceptable salts thereof as pharmaceutically 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 pharmaceutically acceptable salts with organic and inorganic acids.
  • 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,
  • 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.
  • 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.
  • X 2 (X' 2 ) is not hydrogen the meta position of Xi and X 2 (X'1 and X' 2 ) to Z is most preferred, because of steric reasons.
  • A, A', and B represent the residues as mentioned above.
  • inventive guanylhydrazone compounds wherein Z is -NH(COHCH 2 ) n -(CO)NHB and n is an integer of 3 to 10 and these compounds wherein Z stands for -NH(COHCH 2 ) n -C 6 H 4 -(CH 2 ) p -(CO)NHB,
  • B represents another benzene substituted with one or two guanylhydrazone residues as shown above.
  • inventive compounds selected from the group comprising: N-(4-acetylphenyl)-N'-(3,5-diacetylphenyl)urea tris (amidinohydrazone),
  • N,N'-bis (3,5-diacetylphenyl) hexane diamide tetrakis (amidinohydrazone)
  • N,N'-bis (3,5-diacetylphenyl) heptane diamide tetrakis (amidinohydrazone)
  • 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.
  • inventive compounds are especially useful for prophylaxis and/or treatment of acute and/or chronic Hepatitis B virus infections.
  • 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 pharmaceutically acceptable salts are also useful for regulating the production and/or replication of Hepatitis B viruses in an individual and/or in cells.
  • mammals As used herein the term "individual” refers to mammals, especially humans.
  • the compounds of the general formula (I) and/or (II) and/or pharmaceutically 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 ⁇ M, and most preferably in the range of 1 - 5 ⁇ M.
  • 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.
  • a pharmaceutically 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 pharmaceutically 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, Zeffix ® , Heptovir ® , 3TC ® , Epivir-HBV, Combivir ® , Trizivir ® by GlaxoSmithKline), alpha interferon (e.g., Intron A ® by Schering-Plough), FTC (e.g., Coviracil ® by Triangle Pharmaceuticals), DAPD (DXG, by Triangle), L- FMAU (e.g., Clevudine ® by Triangle), Adefovir dipivoxil (by Gilead Sciences), tenofovir, epavudine, epcitabine, lobucavir, Penciclovir (GlaxoSmithKline), Famvir, Entecavir
  • 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 pharmaceutically acceptable salts thereof effective to prevent and/or treat Hepatitis B virus infections, including opportunistic infections, and associated diseases.
  • a method for regulating the production and/or replication of Hepatitis B viruses in an individual comprises administering to the individual an amount of at least one compound of the general formula (II) and/or pharmaceutically 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 pharmaceutically acceptable salts thereof effective to at least partially inhibit the production and/or replication of Hepatitis B virus.
  • Another aspect of the present invention relates to the use of a compound having the formula (I):
  • Xi, X' ⁇ and X' 2 are, independently, GhyCH- or GhyCCH 3 -;
  • Z is -NH(CO)NH-, -(C 6 H 4 )-, -(C 5 NH 3 )-, or -A-(CH 2 ) 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 -0-, and salts thereof, as an inhibitor of Hepatitis B Virus (HBV).
  • HBV Hepatitis B Virus
  • X 2 is GhyCH- or GhyCCH 3 -
  • X 2 is meta or para to Xi
  • X' 2 is meta or para to XV
  • Z is -NH(CO)-Y-(CO)NH- and Y is (CH 2 ) n , wherein n is an integer from 6-10 and wherein up to 3 CH 2 groups may be substituted by heteroatom groups such as NH, O or S.
  • the compound is N,N'-bis(3,5- diacetylphenyl) decanediamide-tetrakis(amidino)hydrazone (Fig. 1) or a salt thereof, particularly a pharmaceutically 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/or their pharmaceutically active salts in a pharmaceutically 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 pharmaceutically 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 he group comprising lamivudine, Zeffix ® , Heptovir ® , 3TC ® , Epivir-HBV, Combivir ® , Trizivir ® by GlaxoSmithKline), alpha interferon (e.g., Intron A ® by Schering-Plough), FTC (e.g., Coviracil ® by Triangle Pharmaceuticals), DAPD (DXG, by Triangle), L-FMAU (e.g., Clevudine ® by Triangle), Adefovir dipivoxil (by Gilead Sciences), tenofovir, epa
  • Fig. 1 shows the structures of:
  • 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-
  • 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-
  • 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
  • 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.
  • 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.
  • HBV belongs to the DNA virus group of hepadnaviruses, which multiply by an entirely different mechanism and show an entirely different multiplication cycle.
  • 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 pharmaceutically active salts thereof with respect to that virus.
  • the compounds of formula (I) 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.
  • inhibitors 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; - Gr ⁇ ter et al., 1998 ,Mol. Cell ,1 , p 649-659; Segref et al., 1997, EMBO J.
  • elF-5A Bevec et al., 1996, Science, 271 , p 1858-1860
  • TAP human homologue of yeast Mex67p; - Gr ⁇ ter et al., 1998 ,Mol. Cell ,1 , p 649-659; Segref et al., 1997, EMBO J.
  • 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.
  • injection intravenous, intraperitoneal, intramuscular, subcutaneous
  • epithelial or mucocutaneous linings oral mucosa, rectal and vaginal epithelial linings, nasopharyngial mucosa, intestinal mucosa
  • transdermally topically, intradermally,
  • 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-acydovir, 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,
  • 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 NNRTIs, has caused viral resistance against conventional antiviral drugs.
  • 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 therapeutically 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 ⁇ M.
  • 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.
  • 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, Prnc Natl. Acart. Sci. USA t 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% C0 2 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.
  • 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-Elmer).
  • 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 CellTiter 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 supematants with Real Time PCR.
  • 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 CellTiter 96® AQueous Cell Proliferation Assay (Promega) and the cell supernatants were harvested. Following toxicity testing, the cells were lysed. The culture supemates or cell lysates were subjected to treatment with DNase after a low speed centrifugation.
  • M552V 3TC resistant
  • Virus DNA present in the tissue culture supernatants or cell lysates was PCR amplified using primers derived from HBV strain ayw. Subsequently, the PCR-ampIified 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 CellTiter 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 50 and Tl (therapeutic index) and displays the graphic results.
  • the IC50 of 3TC against the 3TC resistant mutant HBV was > 10 ⁇ M, while it is around 0.05 ⁇ M against HBV ayw (data not shown).
  • 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% C0 2 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 CellTiter 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.
  • MTS novel tetrazolium compound
  • PES electron coupling agent
  • 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.
  • 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).
  • synergy is defined as drug combinations yielding synergy volumes greater than 50 ⁇ M 2 %.
  • synergistic activity and highly synergistic activity have been defined as yielding synergy volumes of 50-100 ⁇ M 2 % and >100 ⁇ M 2 %, respectively.
  • Additive drug interactions have synergy volumes in the range of -50 ⁇ M 2 % to 50 ⁇ M 2 %, while synergy volumes less than -50 ⁇ M 2 % are considered slightly antagonistic or antagonistic ( ⁇ -100 ⁇ M 2 %).
  • 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% C0 2 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 (CellTiter 96® AQueous) uptake procedure. Following the cell viability testing, the cells were lysed and the nuclei removed.
  • CellTiter 96® AQueous a dye 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 P -labeled negative strand riboprobe prepared in vitro and visualized by exposing the membrane onto X-ray film.
  • capsid gels The results from the capsid gels are shown in Fig. 6.
  • capsids from untreated cells (0 ⁇ M compound 4 or 0 ⁇ M 3TC)
  • pregenomic HBV RNA as well as plus strand viral DNA are responsible for the hybridization signal seen with the minus strand riboprobe.
  • 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.

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Abstract

The present invention relates to inhibitors of Hepatitis B Virus (HBV). A class of guanyl hydrazones are disclosed to be powerful inhibitors of HBV infection. Such compounds are useful for the treatment of HBV infection, in particular chronic HBV infection and for the treatment of opportunistic infections and diseases caused by HBV infections. Furthermore, the present invention discloses pharmaceutical compositions useful for prophylaxis and/or treatment of HBV infections, including opportunistic infections, and diseases caused by HBV infections.

Description

Inhibitors of Hepatitis B Virus Infection
Specification
Field of the Invention
The present invention relates to a class of guanyl hydrazones discovered to have activity as inhibitors of Hepatitis B Virus (HBV) replication. The inhibitor compounds are suitable as agents for the treatment of HBV infection, including opportunistic infections, in particular chronic HBV infection and diseases caused by HBV infections. Furthermore, the present invention discloses pharmaceutical compositions useful for prophylaxis and/or treatment of HBV infections, including opportunistic infections, and diseases caused by HBV infections.
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 hepatocellular 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-α) 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 (II):
Figure imgf000004_0001
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-(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 -O-;
B represents
Figure imgf000004_0002
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 pharmaceutically acceptable salts thereof.
A second aspect of the present invention relates to the use of compounds of the general formula (II):
Figure imgf000005_0001
wherein:
Xi represents -CHGhy or -C(CH3)Ghy;
X2 and X'2 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-(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-C6H -(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
Figure imgf000005_0002
X'ι is independently of Xi -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 pharmaceutically 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):
Figure imgf000006_0001
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-(CH2)p-A'-B, _A-(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-C6H -(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,
or — N
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
Figure imgf000007_0001
X'ι is independently of Xi -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 integer of 0 to 10; and pharmaceutically acceptable salts thereof as pharmaceutically 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 pharmaceutically 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, α-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'1 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-C6H -(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(COHCH2)n-(CO)NHB and n is an integer of 3 to 10 and these compounds wherein Z stands for -NH(COHCH2)n-C6H4-(CH2)p-(CO)NHB,
-NH(COMCH2)n-CH=CH-(CH2)p-(CO)NHB,
-NH(COHCH2)n-CR'R"-(CH2)P-(CO)NHB, or
-NH(COHCH2)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 (amidinohydrazone) 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 pharmaceutically 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 (II) and/or pharmaceutically acceptable salts are administered in a dosage corresponding to an effective concentration in the range of 0.01 - 100 μM, preferably in the range of 0.1 - 100 μM, more preferably in the range of 1 - 10 μM, and most preferably in the range of 1 - 5 μM. 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 pharmaceutically 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 pharmaceutically 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, Zeffix®, Heptovir®, 3TC®, Epivir-HBV, Combivir®, Trizivir® by GlaxoSmithKline), alpha interferon (e.g., Intron A® by Schering-Plough), FTC (e.g., Coviracil® by Triangle Pharmaceuticals), DAPD (DXG, by Triangle), L- FMAU (e.g., Clevudine® 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., Zadaxin® by SciClone), (-)-Carbovir, hammerhead ribozymes, HBV DNA vaccine such as Genevax® (by Wyeth-Lederle Vaccine), PreS1/S2 vaccine (e.g., Hepagene® 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 pharmaceutically 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 pharmaceutically 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 pharmaceutically 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):
Figure imgf000012_0001
wherein: X2 is GhyCH-, GhyCCH3- or H-, wherein Ghy being a guanidino group {(H2N)(HN)C-NH-N=};
Xi, X'ι 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 -0-, 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 XV 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 pharmaceutically 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/or their pharmaceutically active salts in a pharmaceutically 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 pharmaceutically 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 he group comprising lamivudine, Zeffix®, Heptovir®, 3TC®, Epivir-HBV, Combivir®, Trizivir® by GlaxoSmithKline), alpha interferon (e.g., Intron A® by Schering-Plough), FTC (e.g., Coviracil® by Triangle Pharmaceuticals), DAPD (DXG, by Triangle), L-FMAU (e.g., Clevudine® 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., Zadaxin® by SciClone), (-)-Carbovir, hammerhead ribozymes, HBV DNA vaccine such as Genevax® (by Wyeth-Lederle Vaccine), PreS1/S2 vaccine (e.g., Hepagene® 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 pharmaceutically active salts thereof with respect to that virus.
Surprisingly, it has now been found that the compounds of formula (I) 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 μM, 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 Tl >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; - Grϋter 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-acydovir, 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 NNRTIs, 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 therapeutically 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 μM, most preferably 1 - 10 μM. 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, Prnc Natl. Acart. Sci. USAt 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-Elmer). A standard curve using a known copy number of HBV- genoηne was generated and the copy number of HBV genome in each sample was then derived from the curve. The O.D number obtained with CellTiter 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 50, and Tl (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.
Figure imgf000019_0001
Figure imgf000019_0002
!C50 (μM) = 3.1945 TC50 (μM) = >20
TC50/IC50 = therapeutic index (Tl) > 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 (Tl) 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 CellTiter 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 supematants 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:
Figure imgf000020_0001
Figure imgf000021_0001
IC 50 (μM) = 15.7 TC 50 (μM) = 63.0
TC50/IC50 = therapeutic index (Tl) = 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 (Tl) 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 (I) 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 CellTiter 96® AQueous Cell Proliferation Assay (Promega) and the cell supernatants were harvested. Following toxicity testing, the cells were lysed. The culture supemates 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-ampIified 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 CellTiter 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 50 and Tl (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:
Figure imgf000022_0001
Figure imgf000022_0002
IC 50 (μM) = 2.78 TC 50 (μM) = 24.67
TC50/IC50 = therapeutic index (Tl) = 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.
Figure imgf000023_0001
The IC50 of 3TC against the 3TC resistant mutant HBV was > 10μM, while it is around 0.05μM 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 (Tl) of approx. 8, without significant cellular toxic effects.
From the results observed with N,N'-bis(3,5-diacetylphenyl) decanediamide- tetrakis(amidino)hydrazone, ..the class of guanyl hydrazones of formutø (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 CellTiter 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.
Fvaluation 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 (μM %) calculated at the 95% confidence interval. For these studies, synergy is defined as drug combinations yielding synergy volumes greater than 50 μM2%. Slightly synergistic activity and highly synergistic activity have been defined as yielding synergy volumes of 50-100 μM2% and >100 μM2%, respectively. Additive drug interactions have synergy volumes in the range of -50 μM2% to 50 μM2%, while synergy volumes less than -50 μM2% are considered slightly antagonistic or antagonistic (< -100μM2%).
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 (CellTiter 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 P -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 μM compound 4 or 0 μM 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 supematants (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.

Claims

Claims
1. Compounds having the general formula (II):
Figure imgf000028_0001
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-(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
Figure imgf000028_0002
X'ι is independently of Xi -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 pharmaceutically acceptable salts thereof.
Use of a compound having the general formula (II):
Figure imgf000029_0001
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-(CH2)n-C6H4-(CH2)p-A'-B, -A-(CH2)π-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
Figure imgf000029_0002
X'ι is independently of Xi -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 pharmaceutically acceptable salts thereof as pharmaceutically active agents.
Use of a compound having the general formula (II):
Figure imgf000030_0001
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'-BJ
-A-(CH2)n-C5H3N-(CH2)p-A'-B, -A-(CH2)n-CR'R"-(CH2)P-A,-BI
-NH(CO)B, -NHB, -(CO)NHB, -COB, -SB, -OB, -CO-OB, -O-COB, -NH(CO)OB, -0(CO)NHB, -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,
Figure imgf000030_0002
R', R" are independently of each other -OH, -SH, -NH2, methyl, ethyl or propyl;
A and A' are independently of each other -NH(CO)-l -NH-, -(CO)NH-, -NH(CO)NH- or -0-;
Figure imgf000030_0003
B represents
X'ι is independently of Xi -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; and pharmaceutically acceptable salts thereof as pharmaceutically active agents for prophylaxis and/or treatment of Hepatitis B virus infections and associated diseases, including opportunistic infections.
4. Use of a compound of formula (I) according to claims 2 or 3 wherein, when X2 is not hydrogen, Xi and X2 are meta to Z and when X2 is hydrogen, Xi is meta or para to Z.
5. Use of a compound of any one of claims 2 - 4 wherein Z is -A-(CH2)n-A'-B or -A-(CH2)n-B and wherein A, A', and B represent the residues mentioned above and wherein n is an integer of 1 to 10.
6. Use of a compound of any one of claims 3 - 5 wherein Z is -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 wherein A, A', and B represent the residues mentioned above and wherein n and p are independently of each other integer of 1 to 5.
7. Use of a compound according to claim 5 wherein Z is -NH(CO)-(CH2)n-(CO)NHB and B represents the residue shown above and wherein n is an integer of 3 to 10.
8. Use of a compound according to claim 6 wherein Z is -NH(CO)-(CH2)n-CH=CH-(CH2)p-(CO)NHB, -NH(COHCH2)n-C6H4-(CH2)p-(CO)NHB, -NH(CO)-(CH2)n-CR'R"-(CH2)p-(CO)NHB, or -NH(COHCH2)n-C5H3N-(CH2)p-(CO)NHB, and B represents the residue shown above and wherein n and p are independently of each other integer of 1 to 5.
9. Use of a compound of formula (II) according to claim 3 wherein the compound is 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) and/or a salt thereof.
10. Use of at least one compound of the general formula (II) and/or pharmaceutically acceptable salts thereof for the manufacture of a pharmaceutical formulation for prophylaxis and/or treatment of Hepatitis B virus infections, including opportunistic infections, and associated diseases.
11. Use according to claims 2 - 10 wherein the Hepatitis B virus is a drug resistant virus strain.
12. Use of a compound having the general formula (II) and pharmaceutically acceptable salts thereof according to any one of claims 2 - 11 for regulating the production and/or replication of Hepatitis B viruses in an individual and/or in cells.
13. Use according to claims 2 - 12 wherein the compound of the general formula (II) or pharmaceutically acceptable salts thereof is administered in a dosage corresponding to an effective concentration in the range of 0.01 - 100 μM.
14. Use according to claim 13 wherein the compound of the general formula (II) or pharmaceutically acceptable salts thereof is administered in a dosage corresponding to an effective concentration in the range of 0.1 - 100 μM.
15. Use according to claim 14 wherein the compound of the general formula (II) or pharmaceutically acceptable salts thereof is administered in a dosage corresponding to an effective concentration in the range of 1 - 10 μM.
16. Use according to claims 2 - 15 wherein at least one compound of the general formula (II) and/or pharmaceutically active salts thereof is administered in combination with further therapeutic compounds.
17. Use according to claim 16 wherein the further therapeutic compounds are selected from the group comprising hepatitis B virus drugs or vaccines comprising lamivudine, Zeffix®, Heptovir®, 3TC®, Epivir-HBV, Combivir®,
Trizivir®, alpha interferon, Intron A®, FTC, Coviracil®, DAPD, DXG, L- FMAU, Clevudine®, Adefovir dipivoxil, tenofovir, epavudine, epcitabine, lobucavir, Penciclovir, Famvir, Entecavir/BMS-200475, Racivir, L-ddA prodrug, HDP-P-acyclovir, ara-AMP prodrugs, thymosin alpha-1 , Zadaxin®, (-)-Carbovir, hammerhead ribozymes, HBV DNA vaccine,
Genevax®, PreS1/S2 vaccine, Hepagene®, HBV immunoglobulin, Nabi- HBV®, glycosidase inhibitors, Nonyl-DNJ, human monoclonal antibodies directed against HBV, 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.
18. 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):
Figure imgf000034_0001
Xi and X'ι are independently of each other -CHGhy or -C(CH3)Ghy;
X2 and X'2 are independently of each other -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, -NHfCO -Ph, -CO-NHPh, -CH=CH-COOH, -A-(CH2)n-CH=CH-(CH2)p-A,-Bl -A-(CH2)n-C6H4-(CH2)p-A'-B1
-A-(CH2)n-C5H3N-(CH2)P-A'-B, -A-(CH2)n-CR'R"-(CH2)p-A'-B, -NH(CO)B, -NHB, -(CO)NHB, -COB, -SB, -OB, -CO-OB,
-O-COB, -NH(CO)OB, -0(CO)NHB,
Figure imgf000034_0002
-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,
Figure imgf000034_0003
R', R" are independently of each other -OH, -SH, -NH2, methyl, ethyl or propyl;
A and A' are independently of each other -NHfCO)-, -NH-, -(CO)NH-, -NH(CO)NH- or -0-;
B represents
Figure imgf000034_0004
n and p are independently of each other integer of 0 to 10; and/or pharmaceutically acceptable salts thereof effective to prevent and/or treat Hepatitis B virus infections, including opportunistic infections, and associated diseases.
19. Method according to claim 18 wherein, when X2 is not hydrogen, Xi and X2 are meta to Z and when X2 is hydrogen, Xi is meta or para to Z.
20. Method according to claim 18 or 19 wherein Z represents -A-(CH2)n-A'-B or -A-(CH2)n-B and wherein A, A', and B represent the residues mentioned above and wherein n is an integer of 1 to 10.
21. Method according to any one of claims 18 - 20 wherein Z is -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 wherein A, A', and B represent the residues mentioned above and wherein n and p are independently of each other integer of 1 to 5.
22. Method according to claim 20 wherein Z is -NH(COHCH2)n-(CO)NHB and B represents the residue shown above and wherein n is an integer of 3 to 10.
23. Method according to claim 21 wherein Z is -NH(COHCH2)n-CH=CH-(CH2)p-(CO)NHB, -NH(COHCH2)n-C6H4-(CH2)p-(CO)NHB, -NH(COHCH2)n-CR'R"-(CH2)p-(CO)NHB, or -NH(COHCH2)n-C5H3N-(CH2)P-(CO)NHB, and B represents the residue shown above and wherein n and p are independently of each other integer of 1 to 5.
24. Method according to claim 18 wherein the compound is selected from: 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 (amidinohydrazone) and/or a salt thereof.
25. Method according to any one of claims 18 - 24 wherein the Hepatitis B virus is a drug resistant virus strain.
26. Method for regulating the production and/or replication of Hepatitis B viruses in an individual, which comprises administering to the individual an amount of at least one compound of the general formula (II) and/or pharmaceutically acceptable salts thereof effective to at least partially inhibit the production and/or replication of Hepatitis B virus.
27. 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 pharmaceutically acceptable salts thereof effective to at least partially inhibit the production and/or replication of Hepatitis B virus.
28. A method for inhibiting Hepatitis B Virus (HBV) production comprising administering to cells infected with HBV a compound having the formula (I):
Figure imgf000036_0001
wherein:
X2 is GhyCH-, GhyCCH3- or H- wherein Ghy is a guanidino group,
(H2N)(HN)C-NH-N=;
Xi, X'ι and X'2 are, independently, GhyCH- or GhyCCH3-; Z is -NH(CO)NH-, -(C6H )-, -(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 -0-, and salts thereof.
29. The method of claim 28, wherein:
X2 is GhyCH- or GhyCCH3- and is meta or para with respect to Xi; and wherein X'1 is meta or para with respect to X'2.
30. The method of Claim 29, wherein:
Z is -NH(CO)-Y-(CO)NH- and Y is (CH2)n, wherein n is an integer from 6 to 10, and Y may be unsubstituted or substituted at up to 3 carbon atoms with NH. O, or S.
31. The method of Claim 30, wherein the compound is N,N'-bis(3,5- diacetylphenyl) decanediamide-tetrakis(amidino)hydrazone or a salt thereof.
32. The method of Claim 28, wherein the cells are human cells.
33. The method of Claim 32, wherein the compound is administered in vivo to treat a subject infected with HBV.
34. Method according to any one of claims 18 - 33 wherein the compound of the general formula (I) and/or pharmaceutically acceptable salts thereof is administered in a dosage corresponding to an effective concentration in the range of 0.01 - 100 μM.
35. Method according to claim 34 wherein the compound of the general formula (I) and/or pharmaceutically acceptable salts thereof is administered in a dosage corresponding to an effective concentration in the range of 0.1 - 100 μM.
36. Method according to claim 35 wherein the compound of the general formula (I) or pharmaceutically acceptable salts thereof is administered in a dosage corresponding to an effective concentration in the range of 1 - 10 μM.
37. Method according to any one of claims 18 - 36 wherein at least one compound of the general formula (I) and/or pharmaceutically active salts thereof is administered in combination with another therapeutic agent.
38. Method according to claim 37, wherein the additional therapeutic agent is selected from the group comprising lamivudine, Zeffix®, Heptovir®, 3TC®, Epivir-HBV, Combivir®, Trizivir®, alpha interferon, Intron A®, FTC, Coviracil®, DAPD, DXG, L-FMAU, Clevudine®, Adefovir dipivoxil, tenofovir, epavudine, epcitabine, lobucavir, Penciclovir, Famvir,
Entecavir/BMS-200475, Racivir, L-ddA prodrug, HDP-P-acyclovir, ara- AMP prodrugs, thymosin alpha-1 , Zadaxin®, (-)-Carbovir, hammerhead ribozymes, HBV DNA vaccine, Genevax®, PreS1/S2 vaccine, Hepagene®, HBV immunoglobulin, Nabi-HBV®, glycosidase inhibitors, Nonyl-DNJ, human monoclonal antibodies directed against HBV, 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.
39. Pharmaceutical composition comprising at least one compound of the general formula (I) and/or (II) and/or pharmaceutically acceptable salts thereof as an active ingredient and a pharmaceutically acceptable carrier, excipient, adjuvant, and/or diluents.
40. Pharmaceutical composition according to claim 39 further comprising an additional therapeutic agent selected from the group comprising lamivudine, Zeffix®, Heptovir®, 3TC®, Epivir-HBV, Combivir®, Trizivir®, alpha interferon, Intron A®, FTC, Coviracil®, DAPD, DXG, L-FMAU, Clevudine®, Adefovir dipivoxil, tenofovir, epavudine, epcitabine, lobucavir, Penciclovir, Famvir, Entecavir/BMS-200475, Racivir, L-ddA prodrug, HDP-P-acyclovir, ara-AMP prodrugs, thymosin alpha-1 , Zadaxin®, (-)-
Carbovir, hammerhead ribozymes, HBV DNA vaccine, Genevax®, PreS1/S2 vaccine, Hepagene®, HBV immunoglobulin, Nabi-HBV®, glycosidase inhibitors, Nonyl-DNJ, human monoclonal antibodies directed against HBV, 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.
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003006426A1 (en) * 2001-07-13 2003-01-23 Axxima Pharmaceuticals Ag Aromatic guanylhydrazones as effective compounds against neurodiseases
US7001924B2 (en) 2001-09-21 2006-02-21 Message Pharmaceuticals Inhibitors of RNase P proteins as antibacterial compounds
WO2006130697A1 (en) * 2005-06-01 2006-12-07 Cytokine Pharmasciences, Inc. Hmgb1 expression and protective role of semapimod in nec
EP1572095A4 (en) * 2002-09-13 2008-11-05 Novartis Ag S-l-2 -deoxynucleosides for the treatment of resistant hbv strains and combination therapies
US7863334B2 (en) 2004-08-17 2011-01-04 Cytokine Pharmasciences, Inc. Guanylhydrazone compounds, compositions, methods of making and using
US8034840B2 (en) 2004-06-25 2011-10-11 Cytokine Pharmasciences, Inc. Guanylhydrazone salts, compositions, processes of making, and methods of using
US8642752B2 (en) 2011-04-21 2014-02-04 Isis Pharmaceuticals, Inc. Modulation of Hepatitis B virus (HBV) expression
US8765802B2 (en) 2007-06-12 2014-07-01 Provid Pharmaceuticals, Inc. Kinase inhibitors, compositions thereof, and methods of use therewith
AU2013355220B2 (en) * 2012-12-06 2018-08-02 Baruch S. Blumberg Institute Functionalized benzamide derivatives as antiviral agents against HBV infection
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Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB923398A (en) * 1960-08-09 1963-04-10 Vismara Francesco Spa Diphenyl mono-and bis-formyl guanylhydrazones
CH474486A (en) * 1965-11-19 1969-06-30 Ciba Geigy Process for the production of new diphenylureas
US5455863A (en) * 1993-06-29 1995-10-03 Motorola, Inc. Method and apparatus for efficient real-time authentication and encryption in a communication system
US5599984A (en) * 1994-01-21 1997-02-04 The Picower Institute For Medical Research Guanylhydrazones and their use to treat inflammatory conditions
AU746647B2 (en) * 1996-11-15 2002-05-02 Ferring B.V. Guanylhydrazones useful for treating diseases associated with T cell activation
US6319894B1 (en) * 1997-01-08 2001-11-20 The Picower Institute For Medical Research Complexes and combinations of fetuin with therapeutic agents
WO2001025192A1 (en) * 1999-10-06 2001-04-12 Melacure Therapeutics Ab Guanidine derivatives and their use in the production of a medicament for blocking xanthine oxidase/dehydrogenase
AU2001240578A1 (en) * 2000-02-02 2001-08-14 Axxima Pharmaceuticals Ag Pharmaceutically active aromatic guanylhydrazones
GB0019357D0 (en) * 2000-08-07 2000-09-27 Melacure Therapeutics Ab Novel phenyl guanidines

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US7001924B2 (en) 2001-09-21 2006-02-21 Message Pharmaceuticals Inhibitors of RNase P proteins as antibacterial compounds
US8158606B2 (en) 2002-09-13 2012-04-17 Novartis, Ag β-L-2′-deoxynucleosides for the treatment of resistant HBV strains and combination therapies
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US7928086B2 (en) 2002-09-13 2011-04-19 Novartis Ag β-L-2′-deoxynucleosides for the treatment of resistant HBV strains and combination therapies
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EP2540703A1 (en) * 2004-06-25 2013-01-02 Cytokine Pharmasciences, Inc. Guanylhydrazone salts, compositions, processes of making and methods of using
US8034840B2 (en) 2004-06-25 2011-10-11 Cytokine Pharmasciences, Inc. Guanylhydrazone salts, compositions, processes of making, and methods of using
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US8247455B2 (en) 2004-08-17 2012-08-21 Ferring B.V. Guanylhydrazone compounds, compositions, methods of making and using
US7863334B2 (en) 2004-08-17 2011-01-04 Cytokine Pharmasciences, Inc. Guanylhydrazone compounds, compositions, methods of making and using
US7795314B2 (en) 2005-06-01 2010-09-14 Cytokine Pharmasciences, Inc. Protective role of semapimod in necrotizing enterocolitis
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US9677076B2 (en) 2011-04-21 2017-06-13 Ionis Pharmaceuticals, Inc. Modulation of hepatitis B virus (HBV) expression
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