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WO1994021658A1 - Derives antiviraux de nucleoside imidazolinone - Google Patents

Derives antiviraux de nucleoside imidazolinone Download PDF

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Publication number
WO1994021658A1
WO1994021658A1 PCT/US1993/002472 US9302472W WO9421658A1 WO 1994021658 A1 WO1994021658 A1 WO 1994021658A1 US 9302472 W US9302472 W US 9302472W WO 9421658 A1 WO9421658 A1 WO 9421658A1
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Prior art keywords
compound
virus
hydrogen
hydroxy
reverse transcriptase
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PCT/US1993/002472
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English (en)
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Thomas I. Kalman
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Kalman Thomas I
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Priority to PCT/US1993/002472 priority Critical patent/WO1994021658A1/fr
Publication of WO1994021658A1 publication Critical patent/WO1994021658A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D233/70—One oxygen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/052—Imidazole radicals

Definitions

  • the present invention relates to novel nucleoside analogs having a 4-acetylimidazolinone ring in place of the pyrimidine or purine rings present in most natural nucleosides and nucleotides.
  • nucleoside analogs can inhibit viral DNA polymerases, particularly viral reverse transcriptases, as well as nucleotide biosynthetic enzymes. Such nucleoside analogs can also inhibit replication of retroviruses and hepatitis B virus.
  • the present invention also provides a means for inhibit viral DNA polymerases, particularly viral reverse transcriptases, as well as nucleotide biosynthetic enzymes. Such nucleoside analogs can also inhibit replication of retroviruses and hepatitis B virus.
  • compositions and methods for treating diseases caused by retroviruses and RNA viruses e.g., acguired immunodeficiency syndrome (AIDS), hepatitis B and T-lymphocytic leukemias and the like.
  • AIDS acguired immunodeficiency syndrome
  • hepatitis B and T-lymphocytic leukemias e.g., hepatitis B and T-lymphocytic leukemias and the like.
  • viruses including all retroviruses and the hepatitis B virus rely upon an RNA-dependent DNA- polymerase, or reverse transcriptase, for replication.
  • RNA-dependent DNA- polymerase or reverse transcriptase
  • this DNA genome is transcribed into full length RNA which is then packaged into an immature core containing an RNA "pre-genome” and hepatitis B reverse transcriptase.
  • the reverse transcriptase then simultaneously copies and degrades the RNA "pre-genome” to produce a hepatitis B DNA.
  • reverse transcriptase Unlike mammalian DNA polymerases, e.g., nuclear DNA polymerase ⁇ or mitochondrial DNA polymerase ⁇ , reverse transcriptase is notoriously error-prone and permits a high degree of mispairing in the production of a new DNA strand. Moreover, unlike mammalian (nuclear) DNA polymerase, reverse transcriptase cannot edit and thus does not repair mismatched bases as DNA synthesis proceeds. Various nucleoside analogs have been designed to inhibit viral DNA synthesis, without adversely affecting normal cellular DNA synthesis.
  • Nucleoside analogs are structurally related, but not identical, to the nucleosides normally used by cells and microorganisms to synthesize DNA.
  • the degree of structural relatedness between a nucleoside analog and the corresponding normal nucleoside is thought to control the extent of incorporation of the analog by DNA polymerases into DNA; the more structurally similar the analog the greater likelihood of its incorporation into DNA.
  • replication mediated e.g., by reverse transcriptase
  • a nucleoside analog must retain sufficient structural similarity to a normal nucleoside to be recognized and used by the enzyme or such an analog will not be useful for treating diseases caused by retroviruses or hepatitis B.
  • Nucleoside analogs can inhibit viral DNA synthesis in several ways.
  • a nucleoside analog can be a DNA chain terminator if the 3'-OH normally present on a nucleoside either is not present or has been replaced with a substituent which cannot form a phosphodiester bond to the next nucleotide in the growing DNA chain.
  • Non-chain terminating analogs which are incorporated into viral DNA, can lead to viral chromosomal mutation as the analogs are misread in subsequent rounds of DNA synthesis.
  • nucleoside analogs can also competitively inhibit reverse transcriptase activity, thereby reducing the amount of viral replication. Nucleoside analogs are therefore useful both as viral mutators and as
  • cells are unaffected by treatment with nucleoside analogs designed to inhibit viral replication because cellular DNA replication is more precise than replication by reverse transcriptase.
  • nucleoside analogs designed to inhibit viral replication because cellular DNA replication is more precise than replication by reverse transcriptase.
  • replication not only discriminate between nucleosides to a greater degree than reverse transcriptase, but also proofread the incorporated nucleoside for correct base pairing.
  • nucleoside analogs which are readily incorporated into chromosomal and mitochondrial DNA are of limited value in the treatment of viral infections. In practice, a
  • nucleoside analog can also inhibit the conversion of normal nucleosides into the nucleoside triphosphate used to synthesize DNA. While inhibition of triphosphate formation can slow the rate of viral replication, inhibition of normal nucleoside triphosphate formation can also have detrimental effects upon the cell. While a number of nucleoside analogs are known to inhibit viral replication, these analogs cause side effects such as anemia, neutropenia, neuropathy, pancreatitis and other problems (see Saunders et al. 1992 DN&P 5:153-169 for a review).
  • AZT 3'-azido-3'-deoxythymidine
  • AZT AZT
  • AZT can penetrate the blood- brain barrier and therefore has efficacy against HIV caused dementia.
  • serum half-life of AZT is only about 1.1 hours and its major metabolite is an inactive 5'-glucuronide (Yarchoan et al. 1989 N. Eng. J. Med. 321: 726-738).
  • dd dideoxycytidine
  • dideoxyadenine (ddA) and dideoxyinosine (ddl) (Mitsuya et al. 1986 Proc. Natl. Acad. Sci. USA 83 : 1911-1915).
  • ddC The ddC analog was the first, after AZT, to be evaluated clinically. This analog is not readily metabolized to an inactive form and is quite stable in plasma. In clinical trials ddC has provided evidence of activity against HIV (Yarchoan et al. 1988 Lancet i:76- 81; and Merigan et al. 1989 Ann. Intern. Med. 110: 189- 194). Moreover, ddC has good bioavailability after oral administration. However, ddC causes peripheral
  • the ddT analog has only weak activity against HIV and has not been further developed as an anti- retroviral agent.
  • ddA and ddl are both converted to an active ddATP species.
  • ddATP is less potent than the AZT triphosphate (AZTTP) or ddCTP
  • the intracellular half-life of ddATP is 12 hr, at least 4-fold longer than AZTTP and ddCTP.
  • both ddA and ddl are highly susceptible to solvolysis of the glycosidic linkage which liberates the free purine base.
  • the free base of ddl, hypoxanthine is less toxic than the free base of ddA, adenine, which has been shown to cause renal damage (Lindbald et al. 1973 Acta Pharmacol. Toxicol. 32 : 246- 256). Accordingly, ddl has been pursued in clinical trials over ddA as a therapeutic agent. While ddl does not cause the severe anemia cause by AZT, ddl does have its own side effects: neuropathy and pancrea
  • Nucleoside analogs have also been developed which have a variety of 3'-substituents, other than the azide on AZT, and in place of the 3'-OH present on naturally occurring nucleosides.
  • a 3'- fluoro analog of thymidine (FDT) has been developed which has potent in vitro activity against HIV; however, initial studies indicate that this analog can be toxic and therefore would have no advantage over AZT (Mansuri et al. 1990 Antimicrob. Agents Chemother. 34: 637-641).
  • Uridine and cytidine analogs of AZT, 3'- azidodideoxyuridine (AZU) and 3'-azidodideoxycytidine have also been developed (Eriksson et al. 1989
  • d4 compounds 2',3'-dideoxy-2',3'- didehydronucleoside analogs, commonly referred to as d4 compounds, have been made and preliminarily tested.
  • the most notable d4 analogs are d4C and d4T (Mansuri et al. 1990; Balzarini et al. 1986 Biochem. Biophys. Res.
  • the present invention is directed to novel nucleoside analogs having a 4-acetylimidazolinone ring, as well as to methods of using such nucleoside analogs for inhibiting reverse transcriptase, viral replication and diseases caused by retroviruses and hepatitis B.
  • Imidazolinone nucleosides have been synthesized (Otter et al. 1969 J. Org. Chem. 34: 2636-2642). However these synthetic procedures did not yield the specific
  • the present analogs do not exhibit many of the toxicity problems associated with known nucleoside analogs.
  • the present invention provides novel nucleoside or nucleotide analogs having a 4- acetylimidazolin-2-one base.
  • One embodiment the present invention provides a compound of the following formula: wherein R is hydrogen or
  • R 1 is hydroxy, monophosphate, diphosphate, triphosphate, phosphonate,
  • R 4 is hydrogen, cation, lower alkyl or acyloxymethyl
  • X and Y each are independently -CH-, -O-, -S-,
  • R 2 is hydrogen, lower alkoxy or hydroxy
  • R 3 is hydrogen, lower alkoxy, hydroxy, halo, azido
  • n and q are independently 0 or 1;
  • the present invention further provides a method of inhibiting DNA synthesis catalyzed by reverse transcriptase which includes contacting the reverse transcriptase with a reverse transcriptase inhibiting amount of at least one nucleoside or a nucleotide analog having a 4-acetylimidazolin-2-one base.
  • the present invention is still further directed to a method of inhibiting retroviral
  • replication which includes contacting a retrovirus with at least one retrovirus replication-inhibiting amount of a nucleoside or a nucleotide analog having a 4- acetylimidazolin-2-one base.
  • the present invention also provides a method of treating or preventing animal retroviral infection which includes administering to an animal an anti- retroviral effective amount of at least one nucleoside analog or a nucleotide analog having a 4- acetylimidazolin-2-one base.
  • the present invention further provides a method of treating or preventing human hepatitis B infection which includes administering to a patient an anti-hepatitis B effective amount of at least one nucleoside or a nucleotide analog having a 4- acetylimidazolin-2-one base.
  • HIV human immunodeficiency virus
  • nucleotide analog having a 4-acetylimidazolin-2-one base.
  • Fig. 1 depicts the structures of
  • deoxythymidine and 1-( ⁇ -D-2-deoxyribofuranosyl)-4- acetylimidazolin-2-one (abbreviated deoxyimidine or dlmd).
  • deoxyimidine 4- methoxycarbonyl derivative i.e., 1-( ⁇ -D-2- deoxyribofuranosyl)-4-methoxycarbonylimidazolin-2-one indicates that the carbonyl oxygen of the imidine base is correctly oriented to permit internucleotide hydrogen bonding (base pairing).
  • Fig. 2 illustrates the close structural similarity between the energy minimized structures of thymidine (dThd) and 1-( ⁇ -D-2-deoxyribofuranosyl)-4- acetylimidazolin-2-one (dlmd).
  • Fig. 3 provides a comparison of the distances and angles of NH-O and N-HN hydrogen bonds in an
  • Fig. 4 provides a graph comparing the percent inhibition of human immunodeficiency virus reverse transcriptase (HIV-RT, filled circles) with the percent inhibition of a nuclear human DNA polymerase ⁇ from MOLT-4 human lymphocytes (MOLT-4 POLY ⁇ , filled
  • dImdTP 1-( ⁇ -D-2- deoxyribofuranosyl)-4-acetylimidazolin-2-one 5'- triphosphate
  • IC 50 molar concentration of dImdTP required for 50% inhibition (IC 50 ) of both HIV-RT (38 nM) and MOLT-4 POLY ⁇ (17 uM) is also provided. As illustrated, about 500-fold less dImdTP is required to inhibit HIV-RT than MOLT-4 POLY ⁇ .
  • Fig. 5 depicts the percent inhibition of HIV reverse transcriptase during synthesis of a poly(dG) strand on a poly(dC) template when varying
  • dImdTP 1-( ⁇ -D-2-deoxyribofuranosyl)-4- acetylimidazolin-2-one 5'-triphosphate
  • dImdTP a much more effective competitive inhibitor of HIV reverse transcriptase than is dTTP (filled diamonds).
  • Fig. 6 depicts the percent protection afforded to HIV infected cells by varying concentrations of 1-( ⁇ - D-2-deoxyribofuranosyl)-4-acetylimidazolin-2-one (dlmd).
  • the percent protection (solid line -------) was defined as the percent viable HIV-infected cells relative to uninfected dlmd-treated cells. The cytopathic effect of HIV on untreated cells is provided for comparison
  • Fig. 6 also depicts the cytoxicity of dlmd (broken line -------), defined as the percent viable non-infected cells treated with dlmd. Little or no cytoxicity was observed for dlmd concentrations up to
  • a 50% protection reference line is also provided (-•-•-•-•).
  • the present invention relates to novel nucleoside or nucleotide analogs having a 4- acetylimidazolin-2-one ring in place of the pyrimidine rings found in naturally occurring nucleosides. As described herein these analogs have utility for
  • R is hydrogen or
  • R 1 is hydroxy, monophosphate, diphosphate, triphosphate, phosphonate,
  • R 4 is hydrogen, cation, lower alkyl or
  • X and Y each are independently -CH-, -O-, -S-,
  • R 2 is hydrogen, lower alkoxy or hydroxy
  • R 3 is hydrogen, lower alkoxy, hydroxy, halo or azido
  • n and q are independently 0 or 1;
  • a 4-acetyl-2-imidazolinone nucleoside having a deoxyribose sugar is referred to as imidine or dlmd; the 4-acetyl-2-imidazolinone base is abbreviated as Im.
  • a lower alkyl contains up to six carbon atoms in the main chain and a total of 10 carbon atoms if the alkyl is branched.
  • Lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, t- butyl, sec-butyl, isobutyl, amyl, isoamyl, pentyl, isopentyl, hexyl and the like.
  • Methyl and ethyl groups may be abbreviated herein as Me and Et, respectively.
  • the preferred lower alkyl groups contain one to four carbon atoms.
  • a lower alkoxy substituent is a lower alkyl covalently attached via an oxygen atom, i.e., -O-lower alkyl.
  • a lower alkanoyl substituent is a lower alkyl containing a carbonyl group.
  • an acyloxymethyl group is a lower alkyl group covalently attached to a -CO-O-CH 2 - group, i.e., a lower alkyl-CO-O-CH 2 -.
  • An azido group is an -N 3 group.
  • Halogen or halo groups include fluoro (-F), chloro (-Cl), bromo (-Br) and iodo (-l). Fluoro is a preferred halo oroup.
  • aryl refers to an aromatic moiety containing 6-10 ring carbon atoms and includes phenyl, ⁇ -naphthyl, ⁇ -naphthyl, and the like.
  • An aryl-lower alkyl refers to an aryl group with one or more lower alkyl substituents.
  • a sulfonate ester is a -OSO 2 - group; and a sulfinate ester is an -SO-O- group.
  • a lower alkyl sulfonate ester is a -OSO 2 -lower alkyl and a lower alkyl sulfinate ester is a -SO-O-lower alkyl.
  • An arysulfonate ester is a -OSO 2 -aryl and an arylsulfinate ester is a - SO-O-aryl wherein the aryl may be substituted with 1-3 lower alkyl groups, 1-2 halogens or 1-2 nitro group.
  • n and q define the number of R 3 and R 2 groups, respectively, wherein n and q are independently 0 or 1.
  • n is 0 when X is -O- or -S-. Therefore, R 3 is not present when X is -O- or -S- and can only be present when X is >CH-.
  • q is 0 when Y is -O- or -S- and there are no R 2
  • R 2 can only be present when Y is >CH-.
  • R 3 can be hydrogen, lower alkoxy, hydroxy, halo or azido.
  • a preferred halo group for R 3 is fluoro.
  • R 3 is hydrogen, hydroxy, fluoro or azido.
  • Analogs of the present invention where R 3 is hydrogen, lower alkoxy, halo or azide can terminate a growing DNA strand, i.e., can cause chain termination, because such substituents cannot form a bond to the 5'-phosphate of another nucleotide.
  • analogs of the present invention where R 2 is hydroxy are non-chain terminating since such a hydroxy group can bond with a 5'-phosphate of another nucleotide.
  • R 2 can be hydrogen, lower alkyl, hydroxy, lower alkoxy.
  • R 2 is hydrogen, i.e., the present nucleoside analogs preferably contain 2 'deoxyribose or a 2'-deoxyribose with one of the present X or R 3 substituents.
  • R 1 is hydroxy, monophosphate, diphosphate, triphosphate, phosphonate, phosphorylphosphonate, pyrophosphorylphosphonate and the like.
  • R 1 moieties can have an R 4 substituent attached to a phosphate oxygen or a phosphonate oxygen.
  • the R 4 group can be hydrogen, cation, lower alkyl,
  • acyloxymethyl and the like.
  • Such cations include Na+, K + , Li + , Ca ++ , Mg ++ , Ba ++ , NH 4 + , monoethanolammonium, tri-cyclohexylammonium, and the like.
  • X is -O- and Y is -CH-. Therefore, the present compounds are preferably of the formula:
  • n and q are both 1 and Y, R 1 , R 2 and R 3 are as defined hereinabove.
  • Each of the present analogs can have a 5'- hydroxy, a 5'-monophosphate, a 5'-diphosphate or a 5'- triphosphate or a derivatized mono-, di- or triphosphate.
  • Preferred nucleoside analogs have a 5'- hydroxy, a 5'-monophosphate, a 5'-phosphonate or a 5'- triphosphate, i.e., R 1 is preferably -OH, -OPO 3 -, -CH 2 -PO 3 -, -PO 3 - or -O-PO 2 --OPO 2 --OPO 3 + .
  • These preferred phosphate groups can also have a proton or cation associated with one or more phosphate oxygens; when such a cation is present a pharmaceutically acceptable salt can form.
  • nucleoside analogs of the present invention are depicted below.
  • novel compounds of the present invention were designed with consideration for the size and geometry of the 4-acetylimidazolinone base as relating to normal nucleoside bases, e.g. thymine, and the base pairing properties thereof. Careful molecular modeling indicated that 4-acetylimidazolinone deoxyriboside
  • dlmd was an excellent structural match for thymidine (dT) and could mimic the base pairing of thymine (T) and, to a lesser degree, cytosine (C).
  • dlmd 1-( ⁇ -D-2- deoxyribofuranosyl)-4-acetylimidazolin-2-one
  • the methyl within the present 4- acetyl group corresponds to the 5-methyl on thymidine.
  • the structures of the present novel nucleotide analogs are similar enough to normal nucleotides, e.g., thymidine 5'-triphosphate, that they are readily
  • discriminating than reverse transcriptase can detect the structural differences between these analogs and natural nucleotides, and do not as readily recognize or bind these analogs.
  • the present analogs are recognized and bound by reverse transcriptase, these analogs are also sufficiently different from normal nucleotides to act as competitive inhibitors of the enzyme.
  • the present analogs can be bound within the active site of reverse transcriptase but are only slowly released and replaced by an incoming normal nucleotide.
  • dImdTP 1-(2-Deoxy- ⁇ -D-ribofuranosyl)-4- acetylimidazolin-2-one 5'-triphosphate
  • dImdTP 1-(2-Deoxy- ⁇ -D-ribofuranosyl)-4- acetylimidazolin-2-one 5'-triphosphate
  • dImdTP does not inhibit DNA synthesis by chain termination; rather dImdTP acts as a competitive inhibitor of reverse transcriptase.
  • thymidine incorporation into DNA synthesized by a human nuclear DNA polymerase is not detectably inhibited by dImdTP. Inhibition of a mammalian nuclear DNA polymerase required at least a 500-fold greater concentration of dImdTP than was required for inhibition of reverse transcriptase (Fig. 4).
  • the angle of hydrogen bonding in the A:Im base pair differs from that in the A:T base pair, by about 15° to 20° (Fig. 3). This difference in bonding angles can weaken the base pairing between adenine and imidine resulting in recognition and
  • transcriptase which can cripple or kill the virus, e.g., a retrovirus or hepatitis B virus.
  • the present analogs can be chain terminators when the 3'-OH is replaced with a hydrogen, an azido, a halo and the like. Therefore, in addition to causing mutations in the viral genome, the present analogs can actually inhibit DNA replication catalyzed by reverse transcriptase in at least two different ways, i.e., by competitive inhibition and by chain termination. This three-fold effect of the present analogs upon retroviruses and hepatitis B, i.e., mutation of viral genomes, competitive inhibition of viral DNA synthesis and chain termination of newly replicated viral DNA, makes these analogs extremely effective anti-viral agents.
  • the present 4-acetylimidazolinone analogs are more selective for reverse transcriptase than known nucleoside analogs which contain normal pyrimidine or purine bases, e.g. thymidine analogs AZT, d4T, ddT, DFT and the like. This higher selectivity for reverse transcriptase, over mammalian DNA polymerases, makes the present 4-acetylimidazolinone analogs less toxic and therefore, more efficacious than known
  • the present invention thus contemplates a method of inhibiting DNA synthesis catalyzed by reverse transcriptase which includes contacting the reverse transcriptase with a reverse transcriptase-inhibiting amount of at least one nucleoside or nucleotide analog of the present invention.
  • the present invention also provides a method of inhibiting viral replication catalyzed by reverse transcriptase which includes contacting a virus with a reverse transcriptase-inhibiting amount of at least one nucleoside or a nucleotide analog of the present
  • the virus can be contacted with a viral-replication inhibiting amount of at least one of the present analogs.
  • the present invention is directed to a method of treating or preventing retroviral infection in an animal which includes
  • an anti-retroviral effective amount of a compound of the present invention can be an amount sufficient to inhibit retroviral replication.
  • the present nucleoside analogs can be used alone or in combination with other therapeutic agents to inhibit DNA synthesis catalyzed by reverse transcriptase, to inhibit viral replication catalyzed by reverse transcriptase and to treat or prevent retroviral or hepatitis B infection.
  • treating retroviral infections means to slow the progress of the disease, to ameliorate symptoms of such infections which are already visible and to preclude or diminish the onset of new symptoms.
  • Preventing animal retroviral infections refers to delaying or preventing the onset of initial symptoms of the infection.
  • Retroviral infections which can be treated or prevented by administration of the nucleoside analogs of the present invention include infections caused by a lentivirus, oncovirus C, oncovirus A, oncovirus B, cisternavirus, Spumavirus F and the like.
  • the present compounds have efficacy against
  • HIV-1 human immunodeficiency virus-1
  • human immunodeficiency virus-1 human immunodeficiency virus-1 (HIV-1)
  • human immunodeficiency virus-1 human immunodeficiency virus-1 (HIV-1)
  • HIV-2 immunodeficiency virus-2
  • human intracisternal retrovirus human T cell leukemia/lymphoma virus type I (HTLV-I), human T cell leukemia/lymphoma virus type II (HTLV-II), Spumavirus F foamy virus
  • mouse mammary tumor virus-S MMTV-S or Bittner's virus
  • mouse mammary tumor virus-P MMTV-P or GR virus
  • mouse mammary tumor virus- L MMTV-L
  • Rous sarcoma virus RSV
  • Rous-associated viruses Rous-associated viruses
  • related chicken sarcoma viruses avian leukosis viruses (ALV), reticuloendotheliosis viruses, pheasant viruses
  • murine sarcoma viruses MSV
  • murine leukosis virus G Gross or AKR virus
  • murine leukosis virus-Friend MLV-F
  • murine leukosis virus-Moloney MLV-M
  • endogenous virus RD114
  • hamster leukosis virus porcine leukosis virus
  • bovine leukosis virus bovine leukosis virus
  • simian immunodeficiency virus primate sarcoma viruses (woolly monkey; gibbon ape), primate sarcoma-associated virus, primate endogenous viruses including baboon endogenous virus (BaEV), stumptail monkey virus (MAC-1), owl monkey virus (OMC-1), viper virus, mason-pfizer monkey virus (MPMV), langur virus, squirrel monkey virus, visna virus of sheep, caprine arthritis-encephalitis virus, equine infectious anemia, and the like.
  • nucleoside analogs of the present invention are used to prevent infections caused by a lentivirus, an oncovirus C and the like, e.g., human immunodeficiency virus-1 (HIV-l), human immunodeficiency virus-2 (HIV-2), human immunodeficiency virus-2 (HIV-2), human immunodeficiency virus-1 (HIV-l), human immunodeficiency virus-2 (HIV-2), human immunodeficiency virus-1 (HIV-l), human immunodeficiency virus-2 (HIV-2), human
  • HTLV-I leukemia/lymphoma virus type I
  • HTLV-II human T cell leukemia/lymphoma virus type II
  • feline immunodeficiency virus simian immunodeficiency virus
  • visna virus of sheep caprine arthritis-encephalitis virus
  • equine infectious anemia and the like.
  • analogs of the present invention can be used to prevent or treat a human immunodeficiency virus infection, i.e., infections caused by HIV-1, HIV-2 and the like.
  • the present methods can be used in a method of treating or preventing hepatitis B infection in a patient which includes administering to a patient an anti-hepatitis B effective amount of a compound of the present invention. While hepatitis B is not
  • the present compounds have utility for inhibiting hepatitis B virus replication and for treating and preventing hepatitis B infections.
  • Reverse transcriptases which can be inhibited by the present methods also include any reverse transcriptases
  • transcriptase of the foregoing retroviral and hepatitis B viral species are preferably employed to inhibit the reverse transcriptases of HIV-1, HIV-2, HTLV-I, HTLV-II, hepatitis B and the like.
  • an amount of the novel nucleoside analogs of the present invention which is sufficient to inhibit reverse transcriptase is an amount which detectably inhibits the synthesis of DNA by reverse transcriptase.
  • the amount of DNA synthesized by reverse transcriptase in the presence of varying amounts of the present compounds can be observed by any combination of the novel nucleoside analogs of the present invention.
  • Such methods can involve the use of a detectable reporter molecule which is covalently attached to a nucleotide.
  • This labeled nucleotide is provided to the reverse transcriptase under the conditions where inhibition is to be effected, e.g., either in vivo or in vitro. After permitting DNA synthesis to occur for the desired length of time, the DNA synthesized by reverse transcriptase can be separated from the unincorporated labeled
  • nucleotide and the amount of reporter molecule present in such DNA is measured.
  • reporter molecule is a molecule which, by its chemical nature, provides an analytically identifiable signal allowing detection of an incorporated nucleotide. Detection is preferably quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclides covalently linked to nucleotides which are incorporated into DNA synthesized by reverse
  • transcriptase or by mammalian DNA polymerases include horseradish peroxidase, alkaline phosphatase, glucose oxidase and ⁇ -galactosidase, among others.
  • the substrates used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change.
  • p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for horseradish peroxidase, 1,2-phenylenediamine, 5- aminosalicyclic acid or tolidine are commonly used.
  • a virus replication-inhibiting amount of the present compounds is an amount sufficient to detectably reduce the rate of virus replication catalyzed by reverse transcriptase. Such an amount can also inhibit reproduction of a retrovirus or hepatitis B virus.
  • the rate of viral reproduction can be determined by observing the number of viruses or the amount of viral antigen (e.g., p24 of HIV), the amount of reverse transcriptase activity or the amount of viral nucleic acid present over time.
  • the detection of antibodies in animal body fluids e.g., serum, urine and the like
  • animal body fluids e.g., serum, urine and the like
  • An anti-retroviral effective amount is an amount of at least one of the present analogs which detectably reduces the number of infective retroviruses, the retroviral infectivity, the symptoms or progression of a retroviral infection.
  • Procedures for determining the number or infectivity of retroviruses are known and readily available to the skilled artisan as described hereinabove.
  • the symptoms associated with retroviral disease are well documented and can be used to assess the progression of the disease (e.g., see Wilson et al. 1991 Harrison's Principles of Internal Medicine, twelfth edition, McGraw-Hill, Inc., New York; Centers for Disease Control 1986 Morb. Mort. Week Rep. 35:334; Centers for Disease Control 1987 Morb. Mort. Week Rep. 36 :1S; Centers for Disease Control 1989 Morb. Mort. Week Rep. 38:S-6).
  • An anti-HIV effective amount is an amount of at least one of the present analogs sufficient to inhibit or reduce the replication of HIV DNA, the amount of HIV antigen, the number of HIV-induced syncytia, the number of infective HIV virions, the HIV infectivity or the progression of a HIV infection.
  • the amount of DNA replicated by HIV can be measured in vivo or in vitro. Measurements of the amount of HIV DNA replicated include enzymatic assays, e.g., as described in Eriksson et al. (1989), cell culture measurements, e.g., as described in Weislow et al. and the like.
  • HIV antigen can be routinely detected by the skilled artisan in patient body fluids, e.g., blood (serum), urine and the like.
  • patient body fluids e.g., blood (serum), urine and the like.
  • Commonly available procedures for HIV antigen detection include enzyme-linked immunosorbant assays (ELISA), Western analyses, immunofluorescence assays, radioimmunoprecipitation assays and the like.
  • the number of infective HIV virions can be assayed e.g., as described in Balzarini et al. (1991 AIDS 5 : 21-28) and Balzarini et al. (1988 Biochem. Pharmacol. 37 : 2847- 2856).
  • the progression of HIV infection in humans is well documented (e.g., Wilson et al. 1991; Centers for Disease Control 1986; Centers for Disease Control 1987; Centers for Disease Control 1989).
  • An anti-hepatitis B effective amount is an amount of the present analogs which detectably reduces the amount of hepatitis B antigens observed, the anti- hepatitis B antibody titer in a host serum sample, the number of infective hepatitis B viruses, the hepatitis B infectivity or the progression of a hepatitis B
  • Hepatitis B antigens which can be detected by routine procedures available to the skilled artisan include hepatitis B surface antigen, hepatitis B core antigen, hepatitis B e antigen, and the like.
  • the number and infectivity of hepatitis B virions can be detected by cell culture assay and by observing the number of Dane particles or the number of large 42 nm spherical intact hepatitis B virions in a given volume, e.g., the number of intact virions in a blood sample.
  • a reverse transcriptase inhibiting amount does not substantially inhibit DNA synthesis catalyzed by nuclear or mitochondrial DNA polymerase. Moreover such amounts preferably inhibit DNA synthesis mediated by reverse transcriptase by at least about 50% to at least about 80%, and more preferably by at least 90%. Preferred dosages for compositions comprising the present
  • the compounds of the present invention can be prepared by art recognized techniques using protecting groups, leaving groups, activating groups and the like as needed. Starting compounds can be chosen which have X, Y, R 2 and R 3 groups in the desired positions.
  • a leaving group may be used in the desired R 2 or R 3 position, and the appropriate R 2 or R 3 group may replace the leaving group in a later synthetic step.
  • Another alternative is to employ a protecting group on a reactive group which may be present on starting materials, e.g., an amine, amide, carboxylate, hydroxy or similar reactive group on the chosen starting material. The use of leaving or protecting groups prevents undesirable side reactions from occurring, while permitting desired reactions to take place.
  • a leaving group is defined as a group which is readily broken away from its union with a carbon atom. These groups are readily recognizable by one skilled in the art. Suitable leaving groups are generally electron attracting groups, either because of their electronegativity or because they have an inductive effect, and may include groups such as halides, N 3 , HO-Aryl, or HSO 3 -Aryl groups, and the like.
  • a protecting group is covalently bound to a reactive group to render the reactive group unreactive while allowing desired reactions to take place.
  • a protecting group must in addition be easily removed without chemically altering the remainder of the molecule, and must regenerate the correct structure of the reactive group.
  • Examples of protecting groups effective with, for example, primary and secondary amino groups include acetyl, carbobenzoxy (cleaved by
  • Alcohols may be
  • an activating group is a group which, when bound to an oxygen, facilitates cleavage and removal of the oxygen from the present nucleoside analogs.
  • Activating groups contemplated by the present invention include lower alkyl sulfonate, arylsulfonate, trifluoromethylene, cyano, fluoroalkylsulfonate, aryloxy and the like.
  • Such a lower alkyl sulfonate can be a methyl sulfonate (i.e., mesylate), ethyl sulfonate, ammonio-alkylsulfonate (i.e., betylate) and the like.
  • An arylsulfonate can be a tolylsulfonate (i.e.,
  • lower fluoroalkylsulfonate includes a trifluoromethylsulfonate (i.e., lower alkyl-OS0 2 CF 3 or triflate), a nonafluorobutyl-sulfonate (i.e., lower alkyl-OSO 2 -C 4 F 9 or nonaflate), a 2,2,2- trifluoroethylsulfonate (i.e., lower alkyl-OSO 2 -CH 2 - CH 2 CF 3 or tresylate) and the like.
  • Preferred activating groups are lower alkyl sulfonic esters. More preferred activating groups are mesylates.
  • an activator Prior to attachment of the activating group, a leaving group can present on the activating group at the position which will be attached to the nucleoside.
  • an activator is an activating group with an attached leaving group.
  • activation can occur intermolecularly, intramolecularly or by changing the stereoisomeric configuration of a specific carbon to facilitate attachment of a substituent.
  • Intermolecular activation occurs when an activating group is attached to a oxygen on a precursor for one of the present nucleoside analogs by reacting such a precursor with an activator.
  • a nucleoside oxygen atom which is to be activated is bound to a reactive atom present within the nucleoside.
  • a ribose ring atom can be activated by linkage to a reactive imidazolinone oxygen.
  • the present compounds are prepared from readily available starting materials.
  • Two generalized synthetic strategies can lead to the present nucleoside analogs. See, for example, Ueda, 1988 in Chemistry of Nucleosides and Nucleotides, Townsend, ed. Vol. 1, pp 1-112.
  • a first, “total synthesis”, strategy involves condensing the present 4-acetylimidazolin-2-one base with the desired ribose sugar derivative.
  • a second strategy involves chemical modification of both the heterocyclic base and the sugar moiety of an available nucleoside derivative.
  • a free 4-acetylimidazolin-2-one base can be prepared by carbamylation of
  • aminoacetaldehyde dialkylacetal wherein e.g., the alkyl can be ethyl, using a salt of HOCN, such as KOCN in the presence of strong acid (e.g., 5N HCl) at low
  • Such a reaction forms NH 2 -CO-NH-CH 2 -CH(O-alkyl) 2 which can be cyclized to an imidazolinone ring by reaction with acid (e.g., H 2 SO 4 ).
  • acid e.g., H 2 SO 4
  • An acetyl group can be added to the 4-position of the imidazolinone ring by known procedures, e.g. by using an acetylating agent such as CH 3 COCl in the presence of a Lewis acid (e.g. AlCl 3 ).
  • a Lewis acid e.g. AlCl 3
  • the 4-acetylimidazolin-2-one base can be
  • silylated e.g., with trimethylsilyl, t-butyldimethyl- silyl, t-butyldiphenylsilylchloride, trimethyl-t- butyldimethyl-t-butyldiphenylsilylchloride or the like
  • 1-halo e.g. chloro or bromo
  • 1-acetyl ribose derivative in the absence or presence of a catalyst (e.g. SnCl 2 , TiCl 4 and the like).
  • a catalyst e.g. SnCl 2 , TiCl 4 and the like.
  • the present imidazolinone nucleoside analogs can be conveniently prepared from commercially available reagents, e.g., uridine or, preferably, derivatives of uridine.
  • Particularly useful derivatives of uridine have a free 5' -OH (R 1 is hydroxy) on the ribose and another
  • substituent at the 5-position of the pyrimidine ring e.g., a 5-halo, 5-hydroxy and the like.
  • 5-bromo-2'-deoxyuridine After formation of the 4-acetylimidazolinone ring by ring contraction the 2'-deoxyribose ring of this starting material can be modified as described hereinbelow.
  • 5- bromo-2'-deoxyuridine can be prepared by techniques known to the skilled artisan, e.g., by dissolving a small molar excess of bromine in an aqueous solution of uridine followed by neutralization and deionization.
  • a uridine derivative (VIII) having a modified ribose ring and a substituent at the 5-position of the pyrimidine ring is reacted with base to promote ring contraction and formation of a imidazolinone intermediate (IX), as depicted below.
  • the R 5 group is halo or hydroxy and X, Y, R 2 , R 3 , n and q are as defined hereinabove.
  • the carboxylate group at the 4-position of imidazolinone of IX can be protected by addition of a protecting group, e.g., by esterification.
  • Esterification can be performed by known procedures as described in Greene, e.g., by addition of an alcohol such as methanol, ethanol or the like in the presence of acid. This procedure permits higher yields of sugar protected IX after hydrolysis of the ester.
  • the 4- carboxylate-protected derivative of IX has the following structure (X).
  • the R 6 group is a protecting group and X, Y, R 2 , R 3 , n and q are as defined hereinabove.
  • R 7 and R 6 are separate protecting groups and X, Y, R 2 , R 3 , n and q are as defined hereinabove.
  • R 2 or R 3 is a hydroxy
  • compound XI will have a R 7 -O- substituent in place of the R 2 or R 3 hydroxy.
  • the carboxylate protecting group (R 6 ) is first removed. For example if the carboxylate
  • protecting group is an alkyl
  • compound XI can be treated with base in an aqueous lower alkanol solvent ( e . g . , aqueous alcohol ) . Removal of this protecting group yields compound XII.
  • R 7 , X, Y, R 2 , R 3 , n and q are as defined hereinabove.
  • Conversion of the -COOH group of compound XII to -CO-CH 3 using methyllithium provides the 4-acetyl imidazolinone nucleoside (XIII).
  • the reaction requires protection of the NH group, preferably by acetylation.
  • Such acetylation can be formed by reacting XII with an acetylating agent, e.g., acetic anhydride, acetyl halide and the like, in nonaqueous solvent.
  • R 7 , X, Y, R 2 , R 3 , n and q are as defined hereinabove.
  • R 7 hydroxy protecting groups can be removed by known procedures, e.g., when R 7 is silyl, compound XIII can be treated with acid in aqueous solution or with fluoride (e.g., Bu 4 NF) to yield a compound of formula I as described hereinabove.
  • fluoride e.g., Bu 4 NF
  • the desired X, Y, R 2 and R 3 substituents can be added to the ribose ring after synthesis of the 4-acetylimidazolinone ring by art- recognized procedures. Replacement of a ribose hydroxy with a R 2 or
  • R 3 substituent can require activation of a 2' or 3' hydroxy oxygen. As described above, such activation can occur intermolecularly, intramolecularly or by changing the stereoisomeric orientation of the hydroxy oxygen.
  • a 3'-OH or a 2'-OH can be
  • XIV 4-acetylimidazolinone nucleoside
  • R 7 is a primary hydroxyl protecting group, preferably trityl, monomethoxytrityl and the like.
  • the XIV starting material can have a 3'-OH if a R 3 group is to replace such a 3'-OH, or the XIV starting material can have a 2'-OH which is replaced with an R 2 group.
  • starting material XIV having a 3'-OH can be activated via XV to form the anhydronucleoside XVI.
  • Intermolecular activation of XIV can provide intermediate XV.
  • R 8 is an activating group and R 7 , Y and R 2 are as defined hereinabove.
  • Intramolecular activation requires formation of a bond between the atom to be activated and another atom in the nucleoside, e.g., to intramolecularly activate a ribose oxygen, a covalent linkage can be formed between a 3'-position and the oxygen atom of the 2-carbonyl in the imidazolinone ring.
  • a covalent linkage can be formed between a 3'-position and the oxygen atom of the 2-carbonyl in the imidazolinone ring.
  • the stereoisomeric configuration of the 3'-OH must be reversed from the ribo-configuration to the xylo-configuration.
  • a 5'-protected 4- acetyl imidazolinone nucleoside XV having an activating group i.e. R 8 wherein R 6 is preferably mesyl or the like
  • R 8 an activating group
  • XVI intramolecularly activated intermediate
  • R 8 is an activating group and R 7 , Y and R 2 are as defined hereinabove.
  • Activation can also occur by changing the stereoisomeric configuration of the atom to be
  • a ribose oxygen can be activated for later removal by forming a 3'-OH with the xylo-configuration.
  • the activated intermediate XV can be reacted with sodium acetate, followed by mild base-catalyzed hydrolysis to produce such a xylose derivative (XVII).
  • R 7 , Y and R 2 are as defined hereinabove.
  • the xylose 3'-OH of XVII can be activated by procedures similar to those described above for the ribose 3'-OH, to produce XVIII.
  • Preferred activating groups are mesyl, trifyl and the like.
  • R 8 , R 7 , Y and R 2 are as defined hereinabove. Any of the foregoing activated intermediates can be used to replace a 3'-OH present on the selected starting compound with a desired R 3 substituent.
  • An azido group can be placed on the 3'- position of the ribose ring of the present 4- acetylimidazolinone compounds by known procedures, e.g., as described in Chu et al. (1989 J. Med. Chem. 32 : 612- 617). Alternatively, the foregoing activated
  • intermediates can be utilized to synthesize a 3'-azido- 4-imidazolinone compound (III) of the present invention.
  • an azido group can be placed on any of the 3'-activated intermediates (XV, XVI and XVIII) by heating one these intermediates with sodium azide or lithium azide in a non-aqueous solvent, e.g., dimethyl formamide.
  • a reaction temperature of about 75°C to 150°C can be used.
  • compounds XV and XVI may require a somewhat higher reaction temperature than XVIII.
  • a preferred reaction temperature of about 130°C can be used for intermediates XV and XVI, while a reaction temperature of about 100°C or lower temperature is preferred for intermediate XVIII.
  • Such reactions yield a 5'-protected (3-azido-2-deoxy- ⁇ -D- ribofuranosyl)-4-acetylimidazoline-2-one (XIX).
  • R 7 , Y, R 2 are as defined hereinabove.
  • protecting group can be removed by procedures described hereinabove, e.g. a preferred 5'-trityl protecting group is removed by treatment of XIX with 80% acetic acid.
  • a fluoro group can be placed on the 3'- position of the ribose ring by known procedures, e.g Herdewijn et al. (1987 J. Med. Chem. 30: 1270-1278) and Herdewijn et al. (1989 Nucleoside Nucleotides 8 : 65-96).
  • a 3'-fluoro compound of the present invention e.g. compound IV
  • a fluorinating agent e.g., HF (or KHF 2 ) or diethylamino- sulfur trifluoride (DAST), respectively.
  • HF or KHF 2
  • DAST diethylamino- sulfur trifluoride
  • R 7 , Y, R 2 are as defined hereinabove.
  • the 5'-R 7 protecting group can then be removed by procedures described hereinabove, e.g. a preferred 5'-trityl protecting group is removed by treatment of XX with 80% acetic acid.
  • a 2',3'-dideoxyribose analog of the present invention can be made e.g., by the method of Prisbe et al. (1985 Synthetic Commun. 15: 401-409) or Robins et al. (1983 J. Am. Chem. Soc. 105: 4059-4065).
  • a thionocarbonate compound (XXI, wherein R 9 is methyl, phenyl and the like) can be formed by reaction of XIV with phenyl chlorothionocarbonate or methyl chlorothionocarbonate.
  • Thionocarbonate compound XXI can be reduced, e.g. by tin hydride in the presence of azobisisobutyronitrile (AIBN).
  • AIBN azobisisobutyronitrile
  • the compound formed from this reaction is a 5'-protected 2',3'-dideoxyribo- furanosyl-4-acetylimidazolinone (
  • the 5'-R 7 protecting group can then be removed by procedures described hereinabove, e.g. a preferred 5'- trityl protecting group is removed by treatment of XXII with 80% acetic acid or HCl in chloroform.
  • a 2',3'-unsaturation can also be created within a ribose ring present on the subject compounds, e.g., as in Horwitz et al. (1966 J. Org. Chem. 29 : 205).
  • a 2'deoxy-derivative of intermediate XVII can be used for synthesizing such a 2',3'-unsaturated compound.
  • intermediate XVIII can be reacted with a strong base, e.g., tetrabutylammonium fluoride (TBAF),
  • a dioxolane-4-acetylimidazolinone compound (VII) of the present invention (wherein X is O) can be made by procedures available to the skilled artisan, e.g., by the method of (Choi et al. 1991 J. Amer. Chem. Soc. 113: 9377-9379).
  • a modified mono-, di- or tri-phosphate can be placed on the nucleoside analogs by any method available to the skilled artisan. For example, Uhlmann et al.
  • corresponding preferred 5'-triphosphates can be obtained by pyrophosphorylation of the 5'-monophosphate (e.g. as in Kovacs et al. 1988 Tetrahedron Lett. 29: 4525).
  • the preferred 5'-phosphonates and the corresponding 5'- triphosphates can be prepared as described by Freeman et al. (1992 J. Med. Chem. 35.: 3192-3196).
  • the present compounds which contain one or more phosphates can form salts with cations. All such cationic salts are contemplated by the invention, but preferred salts are formed with pharmaceutically
  • acceptable cations such as sodium, potassium, lithium, calcium, magnesium, barium, ammonium,
  • the present invention provides a pharmaceutical composition containing a pharmaceutically effective amount of at least one of the present compounds.
  • such a pharmaceutically effective amount is an anti-viral effective amount, a reverse transcriptase-inhibiting amount, a retrovirus replication-inhibiting amount, a hepatitis B
  • the pharmaceutically effective amount is chosen as one that does not substantially inhibit mammalian DNA replication mediated by cellular DNA polymerases, including nuclear and mitochondrial DNA polymerases.
  • the present compounds generally inhibit reverse transcriptase at about 500- fold lower concentrations than required for inhibition of mammalian DNA polymerases involved in cell
  • the compounds of the present invention are generally administered to birds and mammals, including but not limited to humans.
  • the mg/kg/day dosage required for humans is less than that required for small warm-blooded animals, e.g. mice.
  • a pharmaceutically effective amount of the present compounds is about 0.001 mg/kg/day to about 500 mg/kg/day as needed to attain beneficial therapeutic effects. In a preferred embodiment such a
  • mg/kg/day For example, about 1 mg to about 500 mg of the present compounds can be administered approximately every 4-12 hr.
  • Specific dosage amounts can be readily determined by one of ordinary skill in the art taking into account factors which generally tend to modify drug action, e.g. age, weight, sex, diet, disease state, times and methods of administration, and the like.
  • a dosage unit can include a single compound of the present invention or a mixture of the present compounds; a dosage unit can further include other therapeutic agents beneficial for the treatment of diseases caused by retroviruses or hepatitis B viruses. Such combinations of the present compounds with other therapeutic agents can be administered either
  • the compounds of the present invention can be administered to an animal in a variety of forms adapted to the chosen route of administration, e.g., oral, topical, intradermal, intravenous, intramuscular, intraperitoneal or subcutaneous routes.
  • the subject compounds can also be administered parenterally by osmotic pump to permit continuous infusion of the active compound, for example, as described in Rataiczak et al. (1992 Proc. Natl. Acad. Sci. USA 89: 11823-11827).
  • osmotic pumps are commercially available, e.g., from Alzet, Inc (Palo Alto, CA).
  • the present nucleoside analogs can be suitably protected, e.g., by enclosure in hard or soft shell gelatin capsules.
  • the present nucleoside analogs can be suitably protected, e.g., by enclosure in hard or soft shell gelatin capsules.
  • the active compound may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, incorporated directly with the food of the diet and the like.
  • the subject compounds can be incorporated into a cream, solution or suspension for topical administration.
  • the active compounds may be incorporated into liposomes or liposomes modified with polyethylene glycol for
  • parenteral administration Incorporation of additional substances into the liposome, for example, antibodies reactive against membrane proteins found on specific target cells, can help target the present compounds to specific cell types.
  • the percentage of such additives and stabilizers can be varied as needed, however the amount of active compound is at least 0.1%. More conveniently the active compound can constitute about 2% to about 60% of the weight of the unit. The amount of active
  • compositions according to the present invention are prepared in unit dosage form so that an oral dosage unit form contains an amount ranging from about 0.01 mg to about 1 g of active compound. Preferred dosage ranges from about 0.01 mg to about 500 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid or the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, fructose, lactose or saccharin; or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • an excipient such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid or the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, fructose, lactose or saccharin
  • any material used in preparing any dosage unit form should be pharmaceutically pure and
  • the active compound may also be administered parenterally or intraperitoneally. Solutions of the active compound as a free base, acid or
  • pharmacologically acceptable salt can be prepared in water. Such solutions can be mixed with a surfactant such as hydroxypropylcellulose or a dispersing agent such as glycerol, a liquid polyethylene glycol, an oil and a mixture thereof. Under ordinary conditions of storage and use these preparations contain a surfactant such as hydroxypropylcellulose or a dispersing agent such as glycerol, a liquid polyethylene glycol, an oil and a mixture thereof. Under ordinary conditions of storage and use these preparations contain a surfactant such as hydroxypropylcellulose or a dispersing agent such as glycerol, a liquid polyethylene glycol, an oil and a mixture thereof. Under ordinary conditions of storage and use these preparations contain a surfactant such as hydroxypropylcellulose or a dispersing agent such as glycerol, a liquid polyethylene glycol, an oil and a mixture thereof. Under ordinary conditions of storage and use these preparations contain a surfactant such as hydroxypropylcellulose or
  • injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or
  • microorganisms such as bacteria and fungi.
  • pharmaceutical forms for injection must be fluid to the extent that easy syringability exists.
  • pharmaceutical composition is stable under the
  • a pharmaceutical carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, polyethylene glycol and the like), vegetable oil and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of
  • microorganisms can be brought about by various means
  • antibacterial and antifungal agents for example, parabens, chlorobutanol, phenol, sorbic acid,
  • thimerosal and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • solvents dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is
  • compositions incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • the reaction mixture was passed through a column of ion exchange resin (Dowex 50W X8, 100-200 mesh, H + form) to convert the sodium salt of the product into the free acid.
  • the solution was concentrated under reduced pressure. The black residue obtained was dissolved in
  • the thionocarbonate (8) was then reduced with tri-n-butyltinhydride (Bu 3 SnH) in the presence of azobisisobutyronitrile (AIBN) at 75°C to 80°C, using toluene as solvent.
  • This reaction produced a 5'-trityl- 1-(2,3-dideoxy- ⁇ -D-ribofuranosyl)-4-acetylimidazolin-2- one compound 9.
  • the 5'-trityl protecting group was removed by treatment of compound 9 with 80% acetic acid in chloroform to provide 1-(2,3-dideoxy- ⁇ -D-ribofuranosyl)-4- acetylimidazolin-2-one, i.e. compound V.
  • dlmd 1-(2-Deoxy- ⁇ -D-ribofuranosyl)-4- acetylimidazolin-2-one (dlmd) was synthesized as described in Example 1. This compound had a 2'-deox ⁇ , a 3' -OH and a 5'-OH. dlmd was treated with trityl chloride in pyridine to place a trityl (Tr) protecting group on the 5'-OH and thereby generate compound 7, as described in Example 2.
  • the POM protecting group was removed from compound 12 using concentrated ammonium hydroxide in methanol to generate compound 13.
  • the 5'-trityl protecting group was removed by treatment of compound 13 with 80% acetic acid in chloroform to provide the 2',3'-unsaturated-1- ( ⁇ -D-ribofuranosyl)-4-acetylimidazolin-2-one, i.e.
  • transcriptase was determined to be about 500-fold less than the amount of dImdTP required to similarly inhibit the activity of human nuclear DNA polymerase ⁇ .
  • dlmd having a 2'-deoxy, a 3'-OH and a 5'-OH was prepared as described in Example 1.
  • the 5'- triphosphate derivative of dlmd was prepared by direct phosphorylation of the 5'-OH using phosphorous- oxychloride (POCl 3 ) to form the 5'-monophosphodi- chloridate, followed by pyrophosphorylation according to the method of Kovacs et al. (1988 Tetrahedron Lett. 29: 4525).
  • HIV reverse transcriptase was obtained from the National Institutes of Health AIDS Reagent.
  • DNA polymerase ⁇ was isolated from MOLT-4 human lymphocytes according to the procedure of Ho et al. (1985 Cancer Biochem. Biophys. 8: 85-94).
  • the template for reverse transcription was poly-rA (Pharmacia) using an oligo dT primer
  • RT HIV reverse transcriptase
  • a control reaction contained no nucleoside analog
  • the concentration of analog required for 50% inhibition of the control activity is referred to as the IC 50 .
  • the assay for HIV reverse transcriptase was performed essentially as described by Eriksson et al. (1989 Antimicrobial Agents and Chemotherapy 33 : 663- 669).
  • the standard reaction mixture contained, in a total volume of 100 ⁇ L, 100 mM Tris-HCl buffer (pH 8.0), 50 mM KCl, 2 mM MgCl 2 , 5 mM dithiothreitol, 9 ⁇ g/mL bovine serum albumin, 0.001 O.D. unit (0.06 ⁇ g) of poly(rA) n • (dT) 12-18 , 0.13 ⁇ M 3 H-dTTP (specific activity 47 Ci/mmol) and 10 ⁇ L HIV reverse transcriptase (0.16 unit). After incubation for 1 hour at 37°C, the reaction was terminated by addition of 1 mL 10%
  • TCA trichloroacetic acid
  • nucleotide substrates were provided at 0.1 mM (i.e., dATP, dCTP and dGTP).
  • 3 H-dTTP (0.13 ⁇ M with specific activity 47 Ci/mmol) was used as described above.
  • Fig. 4 provides a graph of the percent
  • dImdTP 1-(2-Deoxy- ⁇ -D-ribofuranosyl)-4- acetylimidazolin-2-one 5'-triphosphate (dImdTP) analog inhibitor.
  • concentration of analog inhibitor required for 50% inhibition (IC 50 ) of human immunodeficiency virus reverse transcriptase was found to be 38 nM.
  • IC 50 for normal human DNA polymerase ⁇ was determined to be about 500-fold higher, i.e., 17 ⁇ M.
  • the IC 50 value determined for HIV reverse transcriptase was 3.8 X 10 -8 molar, i.e., 38 nM.
  • IC 50 value determined for MOLT 4 human DNA polymerase ⁇ was 1.7 X 10 -5 molar, i.e., 17 ⁇ M.
  • HIV-1 was originally obtained from the culture supernatant of a persistently HIV-infected H9 cell line, H9-HTLV-III B , described in Popovic et al. (1984 Science 224: 497-500).
  • HIV-l stocks were prepared from the supernatants of HIV- 1 infected MOLT-4 human T-lymphocytes.
  • HIV-2 stocks were prepared from the supernatants of HIV-2 infected MOLT-4 human T-lymphocytes.
  • MOLT-4 and CEM cells (5 x 10 5 cells ml) were suspended in fresh culture medium and infected with either HIV-l or HIV-2 at 100% to 50% cell culture infective doses (CCID) per ml cell suspension (CCID 50 is the dose required for infection of about 50% of the cultured cells).
  • CCID 50 is the dose required for infection of about 50% of the cultured cells.
  • 100 ⁇ l infected cell suspension were transferred to microtiter plate wells, mixed with 100 ⁇ l of the appropriate dilutions of dlmd and further incubated at 37°C.
  • Cells were then pelleted, suspended in fresh RPMI-1640 culture medium containing 13% fetal calf serum (FCS), 11% interleukin-2 (vol/vol), 50 ⁇ mol/l ⁇ -mercaptoethanol, 4 mmol/l L-glutamine, 50 units/ml penicillin and 50 ⁇ g/ml streptomycin, and infected with 2 x 10 3 HIV virions/cell for 60-90 min at 37°C. After infection, cells were reconstituted in culture medium and seeded in culture tubes at 2 ml per tube in the presence or absence of the test compound.
  • FCS fetal calf serum
  • vol/vol interleukin-2
  • 50 ⁇ mol/l ⁇ -mercaptoethanol 4 mmol/l L-glutamine
  • penicillin and 50 ⁇ g/ml streptomycin 50 units/ml penicillin and 50 ⁇ g/ml streptomycin
  • the 50% effective dose was defined as the concentration of compound required to reduce the non-viability of infected cells by 50%.
  • the 50% cytotoxic dose (CD 50 ) was defined as the concentration of compound required to reduce by 50% the number of viable cells in mock- infected cell cultures.
  • a chain terminating analog of the present invention therefore would likely be an even more effective anti-HIV agent than a non-chain terminating compound of the present invention.
  • dlmd 1-(2-deoxy- ⁇ -D- ribofuranosyl)-4-acetylimidazolin-2-one
  • the standardized assay performed was a
  • dlmd was dissolved in dimethyl sulfoxide and diluted 1:100 in cell culture medium before serial half- logio dilutions of dlmd were prepared.
  • T4 lymphocytes CEM cell line
  • Uninfected cells were treated with the compound to serve as a toxicity control, while infected and uninfected cells cultured without dlmd served as basic controls. Cultures were incubated at 37° in a 5% carbon dioxide atmosphere for 6 days.
  • the tetrazolium salt, XTT was added to all wells, and cultures were incubated to allow formazan color development by viable cells. Individual wells were analyzed spectrophotometrically to quantitate formazan production and viewed
  • Drug-treated virus-infected cells were compared with drug-treated noninfected cells and with other appropriate controls (untreated infected and untreated noninfected cells, drug-containing wells without cells, etc) on the same plate. Data were reviewed in comparison with other tests done at the same time and a determination of activity was made.
  • a chain-terminating analog of the present invention can have greater efficacy against HIV than a non-chain terminating compound of the present invention.
  • dlmd 1-(2-Deoxy- ⁇ -D- ribofuranosyl)-4-acetylimidazolin-2-one
  • the assay was performed as described by
  • Mitogen-stimulated human PBMC were infected with HIV-1 (strain LAV), Schinazi et al. (1988
  • RT reverse transcriptase
  • the virus concentration used for infection was about 63,000 dpm of reverse transcriptase (RT) activity per 10 7 cells per 10 ml of medium. Analog dlmd was added about 45 min. after infection. Cultures were maintained in a humidified 5% CO 2 -95% air incubator at 37°C for 6 days after infection, at which point all cultures were sampled for supernatant RT activity. Previous studies by Schinazi and coworkers had indicated that maximum RT levels were obtained at that time, Chu et al. (1989 J. Med. Chem. 32: 612-617); Chu et al. (1988 Biochem.
  • dlmd was also evaluated for toxic effects on uninfected phytohemagglutinin-stimulated human PBMC using a radioactive thymidine uptake method. Briefly, cells in a 96-well plate were grown in the presence of drug for 24 h, and then 1 ⁇ Ci of [ 3 H]thymidine (specific activity, 69 Ci/mmol) was added to each well. After 24 h, the cells were harvested on glass fibers, washed, and dried, and the amount of radioactivity associated with the cells was determined. Cycloheximide was included as a control for toxicity in every assay.
  • dlmd for 50% inhibition of HIV-l reverse transcriptase (EC 50 ) by viable virus particles was 8.4 ⁇ M. In contrast no cytotoxicity was observed for cultured PBMC, CEM and Vero cells treated with up to 100 ⁇ M dlmd. Therefore, dlmd, even with a free 3'-OH, is a highly selective inhibitor of HIV replication.
  • a chain-terminating analog of the present invention would be expected to have greater efficacy against HIV than a non-chain terminating compound of the present invention.

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Abstract

Cette invention concerne de nouveaux analogues nucléosidiques ou nucléotidiques comprenant une base 4-acétylimidazolin-2-one, ainsi que des procédés d'inhibition des transcriptases inverses codées par virus, d'inhibition de la réplication virale des virus qui utilisent la transcriptase inverse pour la réplication, et de traitement ou de prévention des maladies provoquées par des virus dont le cycle de vie nécessite une transcriptase inverse, tels que les virus du déficit immunitaire humain, le virus de l'hépatite B, les virus humains de leucémie/lymphome des cellules T et autres.
PCT/US1993/002472 1993-03-15 1993-03-15 Derives antiviraux de nucleoside imidazolinone WO1994021658A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1027359A2 (fr) * 1996-10-16 2000-08-16 ICN Pharmaceuticals, Inc. L nucleosides monocycliques, analogues et leurs utilisations
WO2001016150A3 (fr) * 1999-08-31 2001-11-15 Nycomed Amersham Plc Analogues de nucleoside
EP1302474A1 (fr) * 1996-10-16 2003-04-16 Ribapharm, Inc. L-nucléosides monocycliques, analogues et leurs utilisations
US7524831B2 (en) 2005-03-02 2009-04-28 Schering Corporation Treatments for Flaviviridae virus infection
US20210363169A1 (en) * 2020-05-21 2021-11-25 Thomas I. Kalman Broad-spectrum antiviral nucleoside derivatives

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US2441933A (en) * 1945-07-30 1948-05-18 Hoffmann La Roche Substituted imidazolones and process of making them
EP0079049A2 (fr) * 1981-11-04 1983-05-18 Merrell Dow Pharmaceuticals Inc. Dérivés substitués d'alcanoylimidazol-2-one
JPS60109595A (ja) * 1983-11-17 1985-06-15 Yamasa Shoyu Co Ltd イミダゾ−ルヌクレオシド誘導体およびその製造法
JPS60109594A (ja) * 1983-11-17 1985-06-15 Yamasa Shoyu Co Ltd ３−デアザグアノシンの製造法
WO1992006102A1 (fr) * 1990-10-02 1992-04-16 Medivir Ab Derives de nucleoside

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US2441933A (en) * 1945-07-30 1948-05-18 Hoffmann La Roche Substituted imidazolones and process of making them
EP0079049A2 (fr) * 1981-11-04 1983-05-18 Merrell Dow Pharmaceuticals Inc. Dérivés substitués d'alcanoylimidazol-2-one
JPS60109595A (ja) * 1983-11-17 1985-06-15 Yamasa Shoyu Co Ltd イミダゾ−ルヌクレオシド誘導体およびその製造法
JPS60109594A (ja) * 1983-11-17 1985-06-15 Yamasa Shoyu Co Ltd ３−デアザグアノシンの製造法
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CHEMICAL ABSTRACTS, vol. 103, no. 25, 23 December 1985, Columbus, Ohio, US; abstract no. 215737b, "3-Deazaguanosine derivatives" page 939; column 1; *
J. L. LAMATTINA ET AL: "The reaction of 5-acetyl-2-aminooxazole with amines: an approach to 1H-5-acetyl-2-aminoimidazoles", TETRAHEDRON LETTERS, vol. 25, no. 28, 10 August 1984 (1984-08-10), OXFORD, UK, pages 2957 - 2960 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1027359A2 (fr) * 1996-10-16 2000-08-16 ICN Pharmaceuticals, Inc. L nucleosides monocycliques, analogues et leurs utilisations
EP1027359A4 (fr) * 1996-10-16 2001-07-25 Icn Pharmaceuticals L nucleosides monocycliques, analogues et leurs utilisations
EP1302474A1 (fr) * 1996-10-16 2003-04-16 Ribapharm, Inc. L-nucléosides monocycliques, analogues et leurs utilisations
WO2001016150A3 (fr) * 1999-08-31 2001-11-15 Nycomed Amersham Plc Analogues de nucleoside
US7524831B2 (en) 2005-03-02 2009-04-28 Schering Corporation Treatments for Flaviviridae virus infection
US7816339B2 (en) 2005-03-02 2010-10-19 Schering Corporation Treatments for Flaviviridae virus infection
US20210363169A1 (en) * 2020-05-21 2021-11-25 Thomas I. Kalman Broad-spectrum antiviral nucleoside derivatives
US11618765B2 (en) 2020-05-21 2023-04-04 Thomas I. Kalman Broad-spectrum antiviral nucleoside derivatives

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