WO2008052722A2 - Use of ribavirin-conjugates as an anti-viral drug - Google Patents

Use of ribavirin-conjugates as an anti-viral drug Download PDF

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WO2008052722A2
WO2008052722A2 PCT/EP2007/009354 EP2007009354W WO2008052722A2 WO 2008052722 A2 WO2008052722 A2 WO 2008052722A2 EP 2007009354 W EP2007009354 W EP 2007009354W WO 2008052722 A2 WO2008052722 A2 WO 2008052722A2
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ribavirin
conjugate
formula
crc
nucleotide
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PCT/EP2007/009354
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French (fr)
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WO2008052722A3 (en
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Hans-Georg Opitz
Christoph Müller
Joris Braspenning
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Heidelberg Pharma Ag
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    • 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
    • C07H19/056—Triazole or tetrazole radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7024—Esters of saccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds

Definitions

  • Objective of this invention is the use of ribavirin conjugates as antiviral drugs, which selectively protect human red blood cells from ribavirin induced haemolysis while not inhibiting the pharmacological activity of ribavirin in other cells.
  • Ribavirin is a nucleoside analogue, which has been shown to be a broad spectrum antiviral agent.
  • An approved medical application is its use in the treatment of chronic hepatitis C in combination with interferon (IFN)- ⁇ .
  • IFN interferon
  • the addition of ribavirin has increased the probability of sustained response to over 50 % if combined with pegylated IFN (Friend, MW et al. Peginterferonalfa-2b plus ribavirin for chronic hepatitis C virus infection. N. Engl.J Med 2002;347:97555-982).
  • ribavirin improves the liver function in patients suffering from HCV caused chronic hepatitis, it does not show an anti HCV viral activity in monotherapy.
  • Various modes of action of ribavirin have been proposed. A conclusive proof of the mode of action of ribavirin in HCV caused chronic hepatitis is still missing.
  • Ribavirin is responsible for the haemolysis of erythrocytes, the cause for anaemia. It acts directly on mature erythrocytes in the blood system. In order to exert its deleterious effects on erythrocytes ribavirin must be within the cell. Ribavirin actively enters cells by way of the equilibrative nucleoside transporter (Jarvis SM et al. Ribavirin uptake by human erythrocytes and the involvement of nitrobenzyl- thioinosine-sensitive (es) nucleoside transporters. Br J Pharmacol 1998; 123: 1587- 1592).
  • ATP adenosyl triphosphate
  • Glue P The pharmacology of ribavirin. Semin Liver Dis 1999;19:17-24. It is supposed to act on the ATP and redox metabolism of erythrocytes, causing an intracellular oxidative stress which is supposed to lead to fragility of the cell membrane finally resulting in haemolysis.
  • phosphorylation of ribavirin is irreversible in the anucleate erythrocytes. Phosphorylated ribavirin accumulates because it cannot be exported by the nucleoside carrier.
  • the concentration of ribavirin in erythrocytes is 50- to 70-fold higher than the therapeutic plasma concentration of 9 -15 ⁇ M ( Page T, Connor JD. The metabolism of ribavirin in erythrocytes and nucleated cells, lnt J Biochem 1990;22:379-383).
  • the high concentration of ribavirin in erythrocytes is responsible for the haemolysis occurring during ribavirin therapy. Based on these data a selective prevention of uptake of ribavirin by erythrocytes should lead to the prevention of haemolysis.
  • ribavirin-nucleotide conjugate Surprisingly by using a ribavirin-nucleotide conjugate a selective lack of ribavirin uptake by red blood cells has been found, whereas uptake of ribavirin by other cells is not affected.
  • the ribavirin-nucleotide conjugate itself is stable and pharmacologically inactive in the blood plasma. It is not able to enter cells. The conjugate is readily fixed on the outer side of the cell membranes. In order to become active ribavirin has to be released from the conjugate.
  • NPP nucleotide pyrophophatase/ phosphodiesterase
  • Ribavirin-nucleotide conjugate will be cleaved into the nucleotide and the conjugated moiety only by cells which express the extra cellular membrane bound enzyme NPP. Immediately thereafter an alkaline phosphatase will release ribavirin from ribavirin nucleotide. Finally the nucleoside ribavirin will be taken up into the cell by the equilibrative nucleoside transporter. It mediates the facilitative diffusion (equilibrative) transport of nucleosides. The release and uptake of ribavirin occurs in close vicinity of both enzymes and the involved transporter, resulting in a highly efficient uptake of ribavirin by the cell. Consequently free ribavirin has been found only in very low concentration in the plasma of animals.
  • red blood cells do not express NPP on their cell surface. Therefore they are not capable to cleave the ribavirin-nucleotide conjugate and as a consequence erythrocytes are not exposed to free ribavirin. Uptake of ribavirin by red blood cells does not occur because free ribavirin is available only in negligible concentrations in the blood plasma when a ribavirin- nucleotide conjugate is applied.
  • target cells for ribavirin like lymphocytes or hepatocytes, express NPP on their cell surface and are therefore able to generate and to incorporate ribavirin.
  • Ribavirin-nucleotide conjugates delivering ribavirin to sensitive cells without effecting red blood cells must fulfill the criteria of a substrate for NPP.
  • NPPs hydrolyze both pyrophosphate bonds (in, e.g. ATP) and phosphodiester bonds (in, e.g. oligonucleotides) and thereby produce nucleoside ⁇ '-monophosphates.
  • Substrates of NPPs are all ribavirin conjugates composed of ribavirin-mono-/diphos- phate and a chemical moiety R linked by a second ester bond to the phosphate group according formula I.
  • the nucleotide phosphodiester is the essential configuration of the conjugate to be recognized by NPP as a substrate.
  • the Ribavirin conjugated moiety R has no influence on the specific cleavage of the conjugates.
  • Substrates for NPPs can be ribavirinmono/diphosphates according to formula I: O
  • n 1 or 2
  • n 1 or 2
  • Sugar moieties S of formula I are preferably sugars like ribose, glucose or galactose.
  • Aminoacids AA of formula I are preferably amino acid residues of natural amino acids like tyrosin, serin or threonin.
  • Lipid residues are preferably residues of glycerol derivatives.
  • Preferred phospholipid derivatives of formula I are compounds of formula Il
  • R 1 is a straight-chain or branched, saturated or unsaturated alkyl residue having 1-
  • R 2 is hydrogen, a straight-chain or branched, saturated or unsaturated alkyl chain having 1-20 carbon atoms, optionally mono- or polysubstituted by halogen, Ci-C 6 alkoxy, CrC ⁇ alkylmercapto, C 1 -Ce alkoxycarbonyl Or C 1 -Ce alkylsulfonyl groups, preferably a C 8 -C 12 alkyl group,
  • X represents an oxygen, a sulfur, a sulfinyl or sulfonyl group
  • Y is an oxygen atom, their stereoisomeric forms and their physiologically acceptable salts of inorganic and organic acids and bases.
  • the invention also provides medicaments containing these compounds as active ingredients.
  • Preferred phospholipid derivatives of ribavirin are shown in formula III, Ilia, INb, and INc.
  • Embodiments of the invention also encompass salts of the compounds of the general formula I to III including alkali, alkaline earth and ammonium salts of the phosphate group.
  • alkali salts include lithium, sodium and potassium salts.
  • Alkaline earth salts include magnesium and calcium.
  • Ammonium salts are understood to be those containing the ammonium ion, which may be substituted up to four times by alkyl residues having 1-4 carbon atoms, and/or aryl residues such as benzyl residues. In such cases, the substituents may be the same or different.
  • the compounds of general formula I to III may contain basic groups, particularly amino groups, which may be converted to acid addition salts by suitable inorganic or organic acids.
  • the acids are, in particular: hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, fumaric acid, succinic acid, tartaric acid, citric acid, lactic acid, maleic acid or methanesulfonic acid.
  • R 1 preferably represents a straight-chain C 8 -Ci 6 alkyl residue which may be further substituted by a Ci-C 6 alkoxy or a CrC 6 alkylmercapto group. More specifically, R 1 represents a nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl or pentadecyl residue, preferably dodecyl. Preferably, methoxy, ethoxy, butoxy, pentyloxy and hexyloxy groups are possible as substituents of R 1 residue.
  • R 1 is substituted by a Ci-C 6 alkylmercapto residue, this is understood to be the methylmercapto, ethylmercapto, propylmercapto, butylmercapto and hexyl- mercapto residue, in particular.
  • R 2 represents a straight-chain C ⁇ -Cis alkyl group which may be further substituted by a Ci-C 6 alkoxy or a Ci-C 6 alkylmercapto group. More specifically, R 2 represents an octyl, nonyl, decyl, undecyl, dodecyl, tridecyl or tetradecyl group preferably decyl. Methoxy, ethoxy, propoxy, butoxy, pentyloxy and hexyloxy groups are preferable as the CrC 6 alkoxy substituents of R 2 .
  • R 2 is substituted by a CrC 6 alkylmercapto residue
  • this is understood to be the methylmercapto, ethylmercapto, propylmercapto, butylmercapto, pentylmercapto and hexyl- mercapto residue, in particular.
  • X is sulfur, sufinyl or sulfonyl
  • Y is oxygen
  • X is oxygen and Y is oxygen.
  • R 2 is C 10H21
  • X is S , SO or SO 2 and
  • Nucleoside conjugates are generally disclosed in WO 1996/015234, however the disclosed compounds do not mention the specific use of ribavirin conjugates.
  • the compounds of example 1 to 4 of the present invention are not specifically disclosed and therefore new.
  • the compounds of the general formula I to III may be prepared by
  • R 3 and R 4 are hydrogen or suitable protective groups, e.g. esters, ethers silylethers or acetals,
  • an activating acid chloride such as 2,4,6-triisopropylbenzene- sulfonyl chloride
  • a tertiary nitrogen base e.g., pyridine or lutidine
  • an inert solvent such as toluene, or immediately in anhydrous pyridine, and optionally, subsequent to hydrolysis, removing the oxygen protecting groups according to procedures conventional in nucleoside chemistry, or
  • L has the meaning as indicated, with a compound of formula Vl in the presence of phospholipase D from Streptomyces in an inert solvent, such as chloroform, in the presence of a suitable buffer, and optionally, subsequent to reaction, removing the oxygen protecting groups according to procedures conventional in nucleoside chemistry.
  • Salts of compounds of general formula I are prepared by reacting the free acid with alkali or alkaline earth hydroxides, alcoholates or acetates.
  • the enantiomers in the lipid part of the compounds of formula I may be prepared by separation via diastereomeric salts or by enantioselective synthesis of the lipid residues starting with optically active C 3 -precursors.
  • the drugs containing compounds of formula I for the treatment of antiviral infections may be administered in liquid or solid form on the oral or parenteral route. Common application forms are possible, such as tablets, capsules, coated tablets, syrups, solutions, or suspensions.
  • water is used as the injection medium, containing additives such as stabilizers, solubilizers and buffers as are common with injection solutions.
  • additives are, e.g., tartrate and citrate buffers, ethanol, complexing agents such as ethylenediaminetetraacetic acid and its non-toxic salts, high-molecular polymers such as liquid polyethylene oxide for viscosity control.
  • Liquid vehicles for injection solution need to be sterile and are filled in ampoules, preferably.
  • Solid carriers are, for example, starch, lactose, mannitol, methylcellulose, talc, highly dispersed silicic acids, higher-molecular fatty acids such as stearic acid, gelatine, agar-agar, calcium phosphate, magnesium stearate, animal and plant fats, solid high-molecular polymers such as polyethylene glycol, etc.
  • formulations suitable for oral application may include flavorings or sweeteners.
  • the dosage may depend on various factors such as mode of application, species, age, or individual condition.
  • the compounds according to the invention may suitably be administered orally or intravenously (i.v.) in amounts in the range of 0.1 - 100 mg, preferably in the range of 0.2 - 80 mg per kg of body weight and per day.
  • the daily dose is divided into 2-5 applications, with tablets having an active ingredient content in the range of 0.5 - 500 mg being administered with each application.
  • the tablets may have sustained release, reducing the number of applications, e.g., to 1-3 per day.
  • the active ingredient content of sustained-release tablets may be in the range of 2-1000 mg.
  • the active ingredient may also be administered by i.v. bolus injection or continuous infusion, where amounts in the range of 5-1000 mg per day are normally sufficient.
  • the compound according to the invention may be administered orally or intravenously in combination with interferon ⁇ a for the treatment of viral infections like hepatitis C.
  • the crude material is suspended in 100 ml tert.-butyl methyl ether and filtrated, and the solvent removed by evaporation.
  • the residue is dissolved in 120 ml Acetone and 20 ml Toluene and 6.0 g Calcium acetate is added.
  • the precipitated product calcium salt is filtrated, dried and vigorously stirred with 20 ml 2 N hydrochloric acid and 100 ml tert.-butylmethyl ether.
  • the organic phase is separated and evaporated, and the residue is purified by preparative HPLC on Lichroprep RP-18 with methanol / water 87:13 as the eluent.
  • the product containing fractions are com- bined and the product is precipitated by addition of 5.0 g Calcium acetate.
  • the solid is filtrated and distributed between tert.-butylmethyl ether and 2 N hydrochloric acid.
  • the organic layer is washed twice with water. After evaporation the residue is dissolved in 25 ml toluene and the pH is adjusted to pH 7 by addition of sodium methanolate (30 % in methanol). The sodium salt is then separated by addition to 100 ml acetone, filtration and drying in vacuum.
  • the yield is 7.4 g (62 %) white powder.
  • R f 0.27 (silicagel; mobile phase: isopropanol / n-butyl acetate / water / cone, ammonia 50/30/15/5)
  • the phosphoric acid-(3-dodecylmercapto-2-decyloxy)propyl ester is prepared as described in WO 92/03462.
  • the crude material is suspended in 250 ml tert.-butylmethyl ether and filtrated, and the solvent removed by evaporation.
  • the residue is purified by preparative HPLC on LiChrosphere RP-select B with methanol / sodium acetate (40 mM, pH 6) 85:15 as eluent.
  • the product containing fractions are combined, the solvent is reduced to 1/3 of the volume by evaporation and the product is precipitated by addition of 1.14 g calcium acetate.
  • the solid is filtrated and distributed between tert.-butylmethyl ether and 2 N hydrochloric acid.
  • the organic layer is evaporated, the residue is dissolved in 20 ml methanol and the pH is adjusted to pH 7 by addition of sodium methanolate (30 % in methanol).
  • the sodium salt is then separated by addition of 200 ml acetone, filtration and drying in vacuum.
  • the yield is 4.33 g (71 %) white powder.
  • Rf 0.27 (silicagel; mobile phase: isopropanol / n-butyl acetate / water / cone, ammonia 3/5/1/1 ).
  • the synthesis can be performed according to the method described in example 1 starting from phosphoric acid-(2,3-didodecyloxy)propyl ester
  • the synthesis can be performed according to the method described in example 1 starting from phosphoric acid-(2,3-didecyloxy)propyl ester
  • phosphoric acid-(2,3-didodecyloxy)propyl ester and phosphoric acid-(2,3- didecyloxy)propyl ester are analogously prepared as described in WO 96/06620, whereas the corresponding lipid alcohols are synthesized by the method disclosed in EP 315973.
  • Murine and human erythrocytes were isolated out of blood by ficoll density gradient centrifugation.
  • a cell homogenate of the purified erythrocytes and lymphocytes has been prepared by treatment with ultrasound.
  • the membrane fraction containing NPP was isolated by ultra centrifugation.
  • the enzymatic cleavage activity of the cell membrane preparation has been determined with 14 C radiolabeled ribavirin-conjugate by determining the amounts of free 14 C ribavirin and 14 C ribavirin-conjugate at different times.
  • the specific enzymatic activity (pmol / min / mg protein / assay) was calculated dependent on the protein content of the membrane preparation. Values are the result of three determinations.

Abstract

The invention refers to ribavirin -nucleotide conjugates which are a substrate for nucleotide pyrophosphatases/phosphodiesterases for the treatment of viral diseases, preventing specifically ribavirin induced haemolysis of erythrocytes. These conjugates have the following structure (I). Ribavirin nucleotide conjugates of formula I which are substrates for nucleotide-pyrophosphatases/phosphodiesterases for a use as a medicament (structure (I)) wherein n is 1 to 2 and R is chosen among another ribavirin molecule, a sugar rest S, selected from the groups consisting of ribose, glucose or galactose, an amino acid residue AA, selected from the group consisting of residues of natural amino acids, and a lipid residue L, selected from the groups consisting of glycerol derivatives, as well as their stereoisomeric forms and pharmacological acceptable salts, for the preparation of a medicament for the treatment of viral infections preventing ribavirin induced haemolysis of erythrocytes.

Description

Use of ribavirin-conjuqates as an anti-viral drug
Objective of this invention is the use of ribavirin conjugates as antiviral drugs, which selectively protect human red blood cells from ribavirin induced haemolysis while not inhibiting the pharmacological activity of ribavirin in other cells.
Ribavirin is a nucleoside analogue, which has been shown to be a broad spectrum antiviral agent. An approved medical application is its use in the treatment of chronic hepatitis C in combination with interferon (IFN)-α. The addition of ribavirin has increased the probability of sustained response to over 50 % if combined with pegylated IFN (Friend, MW et al. Peginterferonalfa-2b plus ribavirin for chronic hepatitis C virus infection. N. Engl.J Med 2002;347:97555-982). Whereas ribavirin improves the liver function in patients suffering from HCV caused chronic hepatitis, it does not show an anti HCV viral activity in monotherapy. Various modes of action of ribavirin have been proposed. A conclusive proof of the mode of action of ribavirin in HCV caused chronic hepatitis is still missing.
Both drugs in combination cause severe adverse effects. The primary toxicity associated with oral ribavirin therapy is a dose related anaemia. This side effect often requires an adaptation of the dosage or even a discontinuation of the therapy. A reduction of haemoglobin is observed in nearly all patients. Some of them must be treated with transfusion of blood or erythropoietin. This adverse effect prevents the use of higher doses of ribavirin and probably a higher cure rate.
Ribavirin is responsible for the haemolysis of erythrocytes, the cause for anaemia. It acts directly on mature erythrocytes in the blood system. In order to exert its deleterious effects on erythrocytes ribavirin must be within the cell. Ribavirin actively enters cells by way of the equilibrative nucleoside transporter (Jarvis SM et al. Ribavirin uptake by human erythrocytes and the involvement of nitrobenzyl- thioinosine-sensitive (es) nucleoside transporters. Br J Pharmacol 1998; 123: 1587- 1592). Subsequently it undergoes phosphorylation at the expense of adenosyl triphosphate (ATP) (Glue P. The pharmacology of ribavirin. Semin Liver Dis 1999;19:17-24). It is supposed to act on the ATP and redox metabolism of erythrocytes, causing an intracellular oxidative stress which is supposed to lead to fragility of the cell membrane finally resulting in haemolysis. In contrast to nucleated cells such as hepatocytes, phosphorylation of ribavirin is irreversible in the anucleate erythrocytes. Phosphorylated ribavirin accumulates because it cannot be exported by the nucleoside carrier. Therefore, the concentration of ribavirin in erythrocytes is 50- to 70-fold higher than the therapeutic plasma concentration of 9 -15 μM ( Page T, Connor JD. The metabolism of ribavirin in erythrocytes and nucleated cells, lnt J Biochem 1990;22:379-383). The high concentration of ribavirin in erythrocytes is responsible for the haemolysis occurring during ribavirin therapy. Based on these data a selective prevention of uptake of ribavirin by erythrocytes should lead to the prevention of haemolysis.
Surprisingly by using a ribavirin-nucleotide conjugate a selective lack of ribavirin uptake by red blood cells has been found, whereas uptake of ribavirin by other cells is not affected. The ribavirin-nucleotide conjugate itself is stable and pharmacologically inactive in the blood plasma. It is not able to enter cells. The conjugate is readily fixed on the outer side of the cell membranes. In order to become active ribavirin has to be released from the conjugate. Investigations into the selective mode of action of the ribavirin-nucleotide conjugate revealed that release of ribavirin is dependent on the presence of nucleotide pyrophophatase/ phosphodiesterase (NPP) (Bollen M et al. Nucleotide pyrophosphatases/ phosphodiesterases on the move. Critical reviews in biochemistry and molecular biology 2000,35:393-432). NPPs release nucleoside 5'-monophosphates from their di- and triphosphates and phosphodiester derivatives. The best known NPPs are the mammalian ecto-enzymes NPP1 (PC-1 ), NPP2 (autotaxin) and NPP3 (B10). Ribavirin-nucleotide conjugate will be cleaved into the nucleotide and the conjugated moiety only by cells which express the extra cellular membrane bound enzyme NPP. Immediately thereafter an alkaline phosphatase will release ribavirin from ribavirin nucleotide. Finally the nucleoside ribavirin will be taken up into the cell by the equilibrative nucleoside transporter. It mediates the facilitative diffusion (equilibrative) transport of nucleosides. The release and uptake of ribavirin occurs in close vicinity of both enzymes and the involved transporter, resulting in a highly efficient uptake of ribavirin by the cell. Consequently free ribavirin has been found only in very low concentration in the plasma of animals.
Surprisingly it has been found that human red blood cells do not express NPP on their cell surface. Therefore they are not capable to cleave the ribavirin-nucleotide conjugate and as a consequence erythrocytes are not exposed to free ribavirin. Uptake of ribavirin by red blood cells does not occur because free ribavirin is available only in negligible concentrations in the blood plasma when a ribavirin- nucleotide conjugate is applied. In contrast target cells for ribavirin, like lymphocytes or hepatocytes, express NPP on their cell surface and are therefore able to generate and to incorporate ribavirin.
Ribavirin-nucleotide conjugates delivering ribavirin to sensitive cells without effecting red blood cells must fulfill the criteria of a substrate for NPP. NPPs hydrolyze both pyrophosphate bonds (in, e.g. ATP) and phosphodiester bonds (in, e.g. oligonucleotides) and thereby produce nucleoside δ'-monophosphates. Substrates of NPPs are all ribavirin conjugates composed of ribavirin-mono-/diphos- phate and a chemical moiety R linked by a second ester bond to the phosphate group according formula I.
The nucleotide phosphodiester is the essential configuration of the conjugate to be recognized by NPP as a substrate. The Ribavirin conjugated moiety R has no influence on the specific cleavage of the conjugates. Substrates for NPPs can be ribavirinmono/diphosphates according to formula I: O
R- -O- P- -O — Ribavirin (I) OH
wherein R is
• another ribavirin molecule
• a sugar rest S
• an amino acid residue AA or
• a lipid residue L, n is 1 or 2, as well as their stereoisomeric forms and their pharmacologically acceptable salts.
Sugar moieties S of formula I are preferably sugars like ribose, glucose or galactose. Aminoacids AA of formula I are preferably amino acid residues of natural amino acids like tyrosin, serin or threonin. Lipid residues are preferably residues of glycerol derivatives.
Preferred phospholipid derivatives of formula I are compounds of formula Il
O
Il
L- - -O-P— -O — Ribavirin
(II)
OH
wherein n is 1 or 2 and L is the group
Figure imgf000005_0001
wherein
R1 is a straight-chain or branched, saturated or unsaturated alkyl residue having 1-
20 carbon atoms, optionally mono- or polysubstituted by halogen, C1-C6 alkoxy, C-1-C6 alkylmercapto, C1-C-6 alkoxycarbonyl, C1-Ce alkylsulfinyl or Ci-Cβ alkylsul- fonyl groups, preferably a C8-C12 alkyl group, and
R2 is hydrogen, a straight-chain or branched, saturated or unsaturated alkyl chain having 1-20 carbon atoms, optionally mono- or polysubstituted by halogen, Ci-C6 alkoxy, CrCβ alkylmercapto, C1-Ce alkoxycarbonyl Or C1-Ce alkylsulfonyl groups, preferably a C8-C12 alkyl group,
X represents an oxygen, a sulfur, a sulfinyl or sulfonyl group, and
Y is an oxygen atom, their stereoisomeric forms and their physiologically acceptable salts of inorganic and organic acids and bases.
The invention also provides medicaments containing these compounds as active ingredients.
Since the compounds of the general formula Il contain asymmetric carbon atoms, all stereoisometric forms and racemic mixtures of these compounds are also the subject of the present invention.
Preferred phospholipid derivatives of ribavirin are shown in formula III, Ilia, INb, and INc.
Figure imgf000006_0001
Figure imgf000007_0001
or their tautomers, their stereoisomeric forms, their physiological acceptable salts of inorganic or organic acids.
Embodiments of the invention also encompass salts of the compounds of the general formula I to III including alkali, alkaline earth and ammonium salts of the phosphate group. Examples of the alkali salts include lithium, sodium and potassium salts. Alkaline earth salts include magnesium and calcium. Ammonium salts are understood to be those containing the ammonium ion, which may be substituted up to four times by alkyl residues having 1-4 carbon atoms, and/or aryl residues such as benzyl residues. In such cases, the substituents may be the same or different. The compounds of general formula I to III may contain basic groups, particularly amino groups, which may be converted to acid addition salts by suitable inorganic or organic acids. To this end, possible as the acids are, in particular: hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, fumaric acid, succinic acid, tartaric acid, citric acid, lactic acid, maleic acid or methanesulfonic acid.
In general formula II, R1 preferably represents a straight-chain C8-Ci6 alkyl residue which may be further substituted by a Ci-C6 alkoxy or a CrC6 alkylmercapto group. More specifically, R1 represents a nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl or pentadecyl residue, preferably dodecyl. Preferably, methoxy, ethoxy, butoxy, pentyloxy and hexyloxy groups are possible as substituents of R1 residue. In case R1 is substituted by a Ci-C6 alkylmercapto residue, this is understood to be the methylmercapto, ethylmercapto, propylmercapto, butylmercapto and hexyl- mercapto residue, in particular.
Preferably, R2 represents a straight-chain Cβ-Cis alkyl group which may be further substituted by a Ci-C6 alkoxy or a Ci-C6 alkylmercapto group. More specifically, R2 represents an octyl, nonyl, decyl, undecyl, dodecyl, tridecyl or tetradecyl group preferably decyl. Methoxy, ethoxy, propoxy, butoxy, pentyloxy and hexyloxy groups are preferable as the CrC6 alkoxy substituents of R2. In case R2 is substituted by a CrC6 alkylmercapto residue, this is understood to be the methylmercapto, ethylmercapto, propylmercapto, butylmercapto, pentylmercapto and hexyl- mercapto residue, in particular.
In some embodiments X is sulfur, sufinyl or sulfonyl, and Y is oxygen.
In some embodiments X is oxygen and Y is oxygen.
An example of a preferred moiety L is the group
Figure imgf000009_0001
wherein
Figure imgf000009_0002
R2 is C 10H21
X is S , SO or SO2 and
Y is O
Nucleoside conjugates are generally disclosed in WO 1996/015234, however the disclosed compounds do not mention the specific use of ribavirin conjugates. The compounds of example 1 to 4 of the present invention are not specifically disclosed and therefore new.
The compounds of the general formula I to III may be prepared by
1. reacting a compound of general formula V
Figure imgf000009_0003
wherein R and n have the meaning as indicated above, with a compound of general formula Vl
Figure imgf000009_0004
wherein R3 and R4 are hydrogen or suitable protective groups, e.g. esters, ethers silylethers or acetals,
in the presence of an activating acid chloride, such as 2,4,6-triisopropylbenzene- sulfonyl chloride, and a tertiary nitrogen base, e.g., pyridine or lutidine, in an inert solvent, such as toluene, or immediately in anhydrous pyridine, and optionally, subsequent to hydrolysis, removing the oxygen protecting groups according to procedures conventional in nucleoside chemistry, or
2. reacting a compound of general formula VII
Figure imgf000010_0001
wherein L has the meaning as indicated, with a compound of formula Vl in the presence of phospholipase D from Streptomyces in an inert solvent, such as chloroform, in the presence of a suitable buffer, and optionally, subsequent to reaction, removing the oxygen protecting groups according to procedures conventional in nucleoside chemistry.
The preparation of the compounds of the general formula V and VII is performed in analogy to Lipids 22, 947 (1987) and J. Med. Chem. 34, 1377 (1991). The compound of formula Vl (ribavirin) is prepared in analogy to DE 2220246.
Salts of compounds of general formula I are prepared by reacting the free acid with alkali or alkaline earth hydroxides, alcoholates or acetates.
The enantiomers in the lipid part of the compounds of formula I may be prepared by separation via diastereomeric salts or by enantioselective synthesis of the lipid residues starting with optically active C3 -precursors. The drugs containing compounds of formula I for the treatment of antiviral infections may be administered in liquid or solid form on the oral or parenteral route. Common application forms are possible, such as tablets, capsules, coated tablets, syrups, solutions, or suspensions.
Preferably, water is used as the injection medium, containing additives such as stabilizers, solubilizers and buffers as are common with injection solutions. Such additives are, e.g., tartrate and citrate buffers, ethanol, complexing agents such as ethylenediaminetetraacetic acid and its non-toxic salts, high-molecular polymers such as liquid polyethylene oxide for viscosity control. Liquid vehicles for injection solution need to be sterile and are filled in ampoules, preferably.
Solid carriers are, for example, starch, lactose, mannitol, methylcellulose, talc, highly dispersed silicic acids, higher-molecular fatty acids such as stearic acid, gelatine, agar-agar, calcium phosphate, magnesium stearate, animal and plant fats, solid high-molecular polymers such as polyethylene glycol, etc. If desired, formulations suitable for oral application may include flavorings or sweeteners.
The dosage may depend on various factors such as mode of application, species, age, or individual condition.
The compounds according to the invention may suitably be administered orally or intravenously (i.v.) in amounts in the range of 0.1 - 100 mg, preferably in the range of 0.2 - 80 mg per kg of body weight and per day. In some dosage regimens, the daily dose is divided into 2-5 applications, with tablets having an active ingredient content in the range of 0.5 - 500 mg being administered with each application. Similarly, the tablets may have sustained release, reducing the number of applications, e.g., to 1-3 per day. The active ingredient content of sustained-release tablets may be in the range of 2-1000 mg. The active ingredient may also be administered by i.v. bolus injection or continuous infusion, where amounts in the range of 5-1000 mg per day are normally sufficient.
The compound according to the invention may be administered orally or intravenously in combination with interferon α a for the treatment of viral infections like hepatitis C.
EXAMPLES
Example 1
Synthesis of (1-β-D-Ribofuranosyl-[1 ,2,4]triazole-3-carboxamide), 5'-phosphoric acid (3-dodecylmercapto-2-decyloxy)propyl ester (formula III).
9.5 g phosphoric acid-(3-dodecylmercapto-2-decyloxy)propyl ester are treated twice with 50 ml anhydrous pyridine and concentrated by evaporation. The residue is dissolved in 60 ml anhydrous pyridine at room temperature, treated with 9.65 g 2,4,6-triisopropyl-benzenesulfonic chloride under nitrogen and stirred at 25 0C for 4 hours. Then 3.89 g ribavirin are added at once, and the reaction is stirred under nitrogen for 14 hours. Hydrolysis is performed by adding 20 ml water. The mixture is evaporated from the solvent and stripped twice using 30 ml of toluene. The crude material is suspended in 100 ml tert.-butyl methyl ether and filtrated, and the solvent removed by evaporation. The residue is dissolved in 120 ml Acetone and 20 ml Toluene and 6.0 g Calcium acetate is added. The precipitated product calcium salt is filtrated, dried and vigorously stirred with 20 ml 2 N hydrochloric acid and 100 ml tert.-butylmethyl ether. The organic phase is separated and evaporated, and the residue is purified by preparative HPLC on Lichroprep RP-18 with methanol / water 87:13 as the eluent. The product containing fractions are com- bined and the product is precipitated by addition of 5.0 g Calcium acetate. The solid is filtrated and distributed between tert.-butylmethyl ether and 2 N hydrochloric acid. The organic layer is washed twice with water. After evaporation the residue is dissolved in 25 ml toluene and the pH is adjusted to pH 7 by addition of sodium methanolate (30 % in methanol). The sodium salt is then separated by addition to 100 ml acetone, filtration and drying in vacuum.
The yield is 7.4 g (62 %) white powder.
Rf = 0.27 (silicagel; mobile phase: isopropanol / n-butyl acetate / water / cone, ammonia 50/30/15/5)
1H-NMR (500 MHz, D6-DMSO, 25 0C): 8.78 (s, 1 H, -triazole-H), 7.79 (s, 1 H, -CONH), 7.55 (S1 1 H, -CONH), 5.82 (s, 1 H, 11 H), 4.32 (t, 1 H, 2' Oder 31 H), 4.18 (t, 1 H, 2' Oder 31 H), 4.05 (m, 2 H, 41 H und S-C-C-CH2-O), 3.92 (m, 1 H, S-C-C- CH2-O), 3.82 (m, 2H, 0-CH2-C9H19), 3.46 (m, 2H, 5' H und S-C-CH-C-O), 3.36 (m, 1 H, 5' H), 2.57 (m, 4 H, -CH2-S-CH2-), 1.41 (m, 4 H, S-C-CH2-Alkyl; O-C-CH2- Alkyl), 1.20 (m, 32 H, S-C-C-C9H18-CH3, 0-C-C-C7Hu-CH3), 0.81 (t, 6 H, -CH3).
The phosphoric acid-(3-dodecylmercapto-2-decyloxy)propyl ester is prepared as described in WO 92/03462.
Example 2
Synthesis of (1-β-D-Ribofuranosyl-[1 ,2,4]triazole-3-carboxamide), 5'-phosphoric acid (3-dodecyloxy-2-decyloxy)propyl ester (formula Ilia)
4.02 g phosphoric acid-(3-dodecyloxy-2-decyloxy)propyl ester are treated with 100 ml anhydrous pyridine and concentrated by evaporation. The residue is dissolved in 100 ml anhydrous pyridine at room temperature, treated with 4.56 g 2,4,6-triisopropyI-benzenesulfonic chloride under nitrogen and stirred at 25 0C for 2 hours. Then 3.09 g ribavirin-2',3'-acetonicle are added at once, and the reaction is stirred under nitrogen for 3 hours. Hydrolysis is performed by adding 30 ml water. The mixture is evaporated from the solvent and coevaporated twice using 100 ml of toluene. The crude material is suspended in 250 ml tert.-butylmethyl ether and filtrated, and the solvent removed by evaporation. The residue is purified by preparative HPLC on LiChrosphere RP-select B with methanol / sodium acetate (40 mM, pH 6) 85:15 as eluent. The product containing fractions are combined, the solvent is reduced to 1/3 of the volume by evaporation and the product is precipitated by addition of 1.14 g calcium acetate. The solid is filtrated and distributed between tert.-butylmethyl ether and 2 N hydrochloric acid. The organic layer is evaporated, the residue is dissolved in 20 ml methanol and the pH is adjusted to pH 7 by addition of sodium methanolate (30 % in methanol). The sodium salt is then separated by addition of 200 ml acetone, filtration and drying in vacuum.
The yield is 4.33 g (71 %) white powder.
Rf = 0.27 (silicagel; mobile phase: isopropanol / n-butyl acetate / water / cone, ammonia 3/5/1/1 ).
1H-NMR (300 MHz, D6-DMSO, 25 0C): 8.94 (s, 1 H, triazole-H), 8.02 (s, 1 H, -CONH), 7.71 (s, 1 H, -CONH), 6.00 (d, 1 H, 2"-OH or 3'-OH), 5.82 (d, 1 H, 11 H), 5.78 (d, 1 H, 2'-OH or 3'-OH), 4.44 (m, 1 H, 2' or 31 H), 4.23 (t, 1 H, 2' or 3' H), 4.09 (m, 1 H, 41 H), 3.88-3.24 (m, 11 H, CH-O-CH2-C9H19, CH2-O-CH2-C11H23, CH2-O- PO3, 51 H), 1.42 (m, 4 H, 2x O-C-CH2-Alkyl), 1.22 (m, 32 H, 0-C-C-C9H18-CH3, O- C-C-C7H14-CH3), 0.84 (t, 6 H, -CH3).
The phosphoric acid-(3-dodecyloxy-2-decyloxy)propyl ester is prepared as described in WO 96/06620. Example 3
Synthesis of (1-β-D-Ribofuranosyl-[1 ,2,4]triazole-3-carboxamide), 5'-phosphoric acid (2,3-didodecyloxy)propyl ester (formula Mb)
The synthesis can be performed according to the method described in example 1 starting from phosphoric acid-(2,3-didodecyloxy)propyl ester
Example 4
Synthesis of (1-β-D-Ribofuranosyl-[1 ,2,4]triazole-3-carboxamide), 5'-phosphoric acid (2,3-didecyloxy)propyl ester (formula IHc)
The synthesis can be performed according to the method described in example 1 starting from phosphoric acid-(2,3-didecyloxy)propyl ester
The phosphoric acid-(2,3-didodecyloxy)propyl ester and phosphoric acid-(2,3- didecyloxy)propyl ester are analogously prepared as described in WO 96/06620, whereas the corresponding lipid alcohols are synthesized by the method disclosed in EP 315973.
Example 5
Comparison of Nucleotide pyrophosphatase/phosphodiesterase mediated cleavage of ribavirin lipid-conjugate by cell membrane-preparations from human and mouse erythrocytes and lymphocytes.
Murine and human erythrocytes were isolated out of blood by ficoll density gradient centrifugation. A cell homogenate of the purified erythrocytes and lymphocytes has been prepared by treatment with ultrasound. The membrane fraction containing NPP was isolated by ultra centrifugation. The enzymatic cleavage activity of the cell membrane preparation has been determined with 14C radiolabeled ribavirin-conjugate by determining the amounts of free 14C ribavirin and 14C ribavirin-conjugate at different times. The specific enzymatic activity (pmol / min / mg protein / assay) was calculated dependent on the protein content of the membrane preparation. Values are the result of three determinations.
Specific enzymatic ribavirin - conjugate cleaving activity:
Figure imgf000016_0001
In contrast to mouse and human lymphocytes no free ribavirin could be detected in the mouse and human membrane fraction of erythrocytes.
Example 6
Comparison of nucleotide pyrophosphatase/phosphodiesterase mediated cleavage of ribavirin lipid-conjugate by human lymphocytes, T-cell leukemia cells and erythrocytes.
Human erythrocytes, human peripheral blood cells treated with or without phythaemagglutinine (PHA), and the human T-cell Leukemia CEM-SS were incubated with 14C-nucleoside labeled ribavirin-lipid-conjugate for 72 h. After incubation period, cells were harvested and washed twice. Radioactive compounds/ metabolites were extracted with ice cold ethanol 60% and separated on thin layer chromatography. Visualisation of radioactivity was performed using phosphor screen and Cyclone™ (Packard, USA). The result is show in figure 1. No intracellular nucleotides could be detected in human erythrocytes. Low levels have been found in resting human PBLs. In contrast, significant levels of nucleotide monophosphate and triphosphate (-MP, -TP) were detected in human PBLs, stimulated with PHA (human PBLs +PHA) and in CEM-SS.

Claims

1. Ribavirin nucleotide conjugates of formula I which are substrates for nucleotide-pyrophosphatases/phosphodiesterases for a use as a medicament
R- -O- P- -O— Ribavirin (I) I OH
wherein n is 1 to 2 and
R is chosen among another ribavirin molecule, a sugar rest S, selected from the groups consisting of ribose, glucose or galactose, an amino acid residue AA, selected from the group consisting of residues of natural amino acids, and a lipid residue L, selected from the groups consisting of glycerol derivatives, as well as their stereoisomer^ forms and pharmacological acceptable salts, for the preparation of a medicament for the treatment of viral infections preventing ribavirin induced haemolysis of erythrocytes.
2. Conjugates for use according to claim 1 wherein R is a compound of formula Il
O Il
L- - -O-P- -O — Ribavirin
(II)
OH
wherein n is 1 or 2 and L is a group
Figure imgf000019_0001
wherein
R1 is a straight-chain or branched, saturated or unsaturated alkyl residue having 1-20 carbon atoms, optionally mono- or polysubstituted by halogen,
CrC6 alkoxy, Ci-C6 alkyl mercapto, CrC6 alkoxycarbonyl, CrC6 alkylsulfinyl or CrC6 alkylsulfonyl groups,
R2 is hydrogen, a straight-chain or branched, saturated or unsaturated alkyl chain having 1 -20 carbon atoms, optionally mono- or polysubstituted by halogen, CrC6 alkoxy, CrC6 alkylmercapto, CrC6 alkoxycarbonyl or CrC6 alkylsulfonyl groups,
X represents an oxygen, a sulfur, a sulfinyl or sulfonyl group, and
Y is an oxygen atom.
3. Conjugate for use according to claim 1 or 2 wherein the compound is as of formula III
Figure imgf000019_0002
its tautomers, stereoisomeric forms or its pharmacologically acceptable salts.
4. Conjugate for use according to claim 1 or 2 wherein the compound is as of formula Ilia
Figure imgf000020_0001
its tautomers, stereoisomeric forms or its pharmacologically acceptable salts.
5. Conjugate for use according to claim 1 or 2 wherein the compound is as of formula IMb
Figure imgf000020_0002
its tautomers, stereoisomeric forms or its pharmacologically acceptable salts.
6. Conjugate for use according to claim 1 or 2 wherein the compound is as of formula NIc
Figure imgf000020_0003
its tautomers, stereoisomeric forms or its pharmacologically acceptable salts.
7. A ribavirin nucleotide conjugate according to formula I, II, or III of claim 1 to 6 for use as a substrate for nucleotide pyrophosphatases/phosphodiesterases type 1 and /or type 3 (NPP1/NPP3).
8. Use of a conjugate of formula I, II, III, as respectively mentioned in claims 1 to 6 for the preparation of medicaments for treating viral infections without inducing haemolysis of erythrocytes.
9. Composition comprising a conjugate of formula I, II, III as respectively mentioned in claim 1 to 6 and interferon α.
10. Method of treating viral infections without inducing haemolysis of erythrocytes, wherein a conjugate according to at least one of claims 1 to 6 is administered to a patient in need thereof in an effective amount for an effective period of time.
11. Method according to claim 10, wherein the viral desease is chronic hepatitis C.
12. Method according to claims 10 or 11 , wherein the conjugate is mixed with suitable carriers and optionally usual additives and/or one or more additional active ingredient.
13. Method according at least one of claims 10 to 12, wherein the conjugate is administered together with interferon α.
PCT/EP2007/009354 2006-11-02 2007-10-29 Use of ribavirin-conjugates as an anti-viral drug WO2008052722A2 (en)

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