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WO1994005687A1 - Antiviral pyrimidine nucleosides - Google Patents

Antiviral pyrimidine nucleosides Download PDF

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Publication number
WO1994005687A1
WO1994005687A1 PCT/GB1993/001858 GB9301858W WO9405687A1 WO 1994005687 A1 WO1994005687 A1 WO 1994005687A1 GB 9301858 W GB9301858 W GB 9301858W WO 9405687 A1 WO9405687 A1 WO 9405687A1
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WO
WIPO (PCT)
Prior art keywords
formula
compound
thio
deoxy
group
Prior art date
Application number
PCT/GB1993/001858
Other languages
French (fr)
Inventor
John Allen Miller
Robert John Young
Saad George Rahim
David Lawrence Selwood
Richard Walker
Original Assignee
University Of Birmingham
The Wellcome Foundation Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Birmingham, The Wellcome Foundation Limited filed Critical University Of Birmingham
Priority to JP6506988A priority Critical patent/JPH08504753A/en
Priority to EP94908867A priority patent/EP0658166A1/en
Priority to AU49733/93A priority patent/AU4973393A/en
Publication of WO1994005687A1 publication Critical patent/WO1994005687A1/en

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Classifications

    • 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
    • C07H19/06—Pyrimidine radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals

Definitions

  • the compounds according to the invention may be administered to mammals including humans by any route appropriate to the condition to be treated, suitable routes including oral, rectal, nasal, topical (including buccal and sublingual) , vaginal and parenteral -(including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) . It will be appreciated that the preferred route may vary with, for example, the condition of the recipient. For each of the above-indicated utilities and indications the amount required of the individual active ingredients will depend upon a number of factors including the severity of the condition to be treated and the identity of the recipient and will ultimately be at the discretion of the attendant physician.
  • An edible foam or whip formulation as described above may be prepared in a conventional manner, for example by mixing the edible oil, surfactant (s) and any other soluble ingredients, adding the viscosity modifier(s) and milling the mixture to form a uniform dispersion and suspension. The active ingredient is blended into the milled mixture until evenly dispersed. Finally, a metered quantity of propellant is incorporated to the mixture after said mixture has been measured into a suitable dispensing container.
  • Pharmaceutical formulations for topical administration according to the present invention may be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil.
  • the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, 0.075 to 20% w/w, preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w.
  • the active ingredients may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredients may be formulated in a cream with an oil-in- water cream base.
  • the aqueous phase of the cream base may include, for example, at least 30% w/w of a polyhydric alcohol, i.e.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • the formulations of this invention may include other agents conventional in the art having regard to the type of formulation ir. question, for example those suitable for oral administration may include flavoring agents.
  • the compounds according to the invention may be employed alone or in combination with other therapeutic agents for the treatment of the above infections or conditions.
  • Combination therapies according to the present invention comprise the administration of at least one compound of the formula (I) or a physiologically functional derivative thereof and at least one other pharmaceutically active ingredient.
  • the active ingredient (s) and pharmacologically active agents may be administered together or separately and, when administered separately this may occur simultaneously or sequentially in any order.
  • the amounts of the active ingredient (s) and pharmacologically active agent (s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.
  • the combination therapy involves the administration of one compound of the formula (I) or a physiologically functional derivative thereof and one of the agents mentioned herein below.
  • the combination therapy involves the administration of one of the above-mentioned agents and a compound within one of the preferred or more-preferred sub-groups within formula (I) as described above.
  • the combination therapy involves the joint use of one of the above named agents together with one of the compounds of formula (I) specifically named herein.
  • the compounds of formula (I) may be produced by various methods known in the art of organic chemistry in general and nucleoside synthesis in particular. Starting materials are either known and readily available from commercial sources or may themselves be produced by known and conventional techniques.
  • the present invention further provides a process for producing a compound of formula (I) as hereinbefore defined which process comprises: A) reacting a compound of formula (II)
  • R 3a either forms a carbon-carbon bond with R 2 or when R 2 is H, R 3a is hydrogen, hydroxy or a group OZ 3 where Z 3 is a hydroxyl protecting group;
  • the groups R 3a and Z 5 are preferably hydroxyl protecting groups, particularly benzyl, toluoyl or p-nitrotoluoyl groups.
  • the reaction may be performed using standard methods including the use of a Lewis Acid catalyst such as mercuric chloride or bromide or stannic chloride or trimethylsilyltrifluoromethane-sulphonate in solvents such as acetonitrile, 1-2 dichloroethane, dichloromethane, chloroform or toluene at reduced, ambient or elevated temperature such as from -78°C to reflux; or b) reaction of the compound of formula (III) , or a protected form thereof, with a compound of formula (VI)
  • a Lewis Acid catalyst such as mercuric chloride or bromide or stannic chloride or trimethylsilyltrifluoromethane-sulphonate in solvents such as acetonitrile, 1-2 dichloroethane, dichloromethane, chloroform or
  • Acid catalyst such as trimethylsilyltrifluoromethane sulphotonate in a solvent such as acetonitrile.
  • Py is preferably uracil or thymine.
  • silylation 25 by silylation.
  • Suitable silylating agents include bis- (trimethylsilyl) acetamide.
  • Silylation is conducted in a suitable solvent, for example acetonitrile. The reaction may be conducted at from about 20°C to 100°C and is preferably preformed at an elevated temperature, e.g. about 50° to 100°C, e.g. about
  • the compound of formula (V) is reacted with the protected base in the presence of a Lewis acid catalyst such as those mentioned above and, as a co-catalyst a N-halosuccinimide, eg. N- iodosuccinimide or N-bromosuccinimide.
  • a Lewis acid catalyst such as those mentioned above and, as a co-catalyst a N-halosuccinimide, eg. N- iodosuccinimide or N-bromosuccinimide.
  • the Lewis acid is
  • the solvent may be any suitable solvent including chlorinated solvents such as chloroform, dichloromethane, 1-2-dichloromethane but is preferably acetonitrile.
  • the Lewis acid and N-halosuccinimide are preferably used in equimolar proportions, although a range of from 1:5 to 5:1 molar ratio may be used. Desirably, the ratio of Lewis acid to compound of formula (V) is 1:1.
  • nucleoside of formula (VI) When the nucleoside of formula (VI) is protected, it may be deprotected using standard de-esterification reactions, eg by reaction with a base (organic or inorganic) in a suitable solvent such as alcohol (eg. methanol, ethanol or propanol) .
  • a base organic or inorganic
  • suitable solvent such as alcohol (eg. methanol, ethanol or propanol)
  • alcohol eg. methanol, ethanol or propanol
  • 5-Halopyrimidines are commercially available and may be coupled to the 4-thiosugar compound by conventional techniques, for instance by reacting a protected 5-halopyrimidine with a protected 4-thio sugar compound having a leaving group in the 1- position.
  • the leaving group on the 4-thio sugar compound may be a halogen, benzylthio or preferably acetate group.
  • Reaction with iodine and nitric acid also introduces the 5-iodo substituent.
  • Reaction with bromine and acetic acid introduces a 5-bromo substituent to the unprotected nucleoside. Deprotection where necessary is by conventional techniques and is performed as the final step.
  • X is C 2 6 alkenyl
  • 5-Alkenyl compounds may be produced by partial hydrogenation of the corresponding 5-alkynyl pyrimidine of formula (III) or of the nucleoside of formula (II) for instance using Lindlar catalyst poisoned with quinoline, and subsequently, in the case of the pyrimidine, coupling with a 4-thio sugar compound as described above.
  • a 5-iodo nucleoside of formula (II) may be reacted with an appropriate alkenylating agent for example a 2- alkenoic acid ester (for instance the methyl ester) in the presence of palladium (II) acetate and triphenylphosphine to form the 5- (2-methoxycarbonyl alkenyl) derivative.
  • an appropriate alkenylating agent for example a 2- alkenoic acid ester (for instance the methyl ester) in the presence of palladium (II) acetate and triphenylphosphine to form the 5- (2-methoxycarbonyl alkeny
  • the 5- (2-carboxyvinyl) compound may also be produced by treating an unprotected 5- (hydroxymethyl)pyrimidine of formula (III) with an oxidising agent such as persulphate or manganese dioxide to form the corresponding aldehyde and followed by treatment of the aldehyde with malonic acid.
  • an oxidising agent such as persulphate or manganese dioxide
  • the 5- (2-haloalkenyl) base may alternatively be made by a novel route starting with a 2,4-dimethoxy protected 5- bromopyri idine.
  • This may be converted to the corresponding 5- lithium derivative by treatment with an organolithium reagent, preferably n-butyllithium at reduced temperature such as -70°C in an ethereal solvent such as diethylether.
  • Reaction of the lithio derivative j-n situ with an appropriate ester of formic acid, such as ethyl formate at reduced temperature such as -70°C gives rise to the corresponding 5-formyl compound.
  • Treatment of the formyl compound with malonic acid as described above gives rise to the 5- (2-carboxyvinyl) derivative.
  • Similar halogenation gives rise to the required 5- (2-haloalkenyl) compound which is in the 2,4- dimethoxy protected from. Deprotection can then be carried out by conventional techniques.
  • X is C ?f alkyl
  • alkyl eg. 5-ethyl substituted nucleosides may be produced by hydrogenation of the corresponding 5-alkynyl or 5- alkenyl pyrimidine base followed by coupling to the 4-thio sugar compound. Conventional hydrogenation conditions, such as hydrogen over palladium/charcoal catalysts, may be adopted.
  • 5-Fluoroalkyl substituents may be generated from the corresponding 5-hydroxyalkyl substituents, preferably starting from nucleosides having protected sugar hydroxyl groups on the 4- thio sugar moiety.
  • Suitable protecting groups include tert-butyl diphenylsilyloxy groups which may be introduced using tert- butyldiphenylsilylchloride.
  • the protected 5-hydroxyalkyl nucleoside is treated with a fluorinating agent such as diethylaminosulphurtrifluoride followed by deprotection of the hydroxyl groups using tetra-n-butylammonium fluoride to give the monofluoroalkyl derivative.
  • the above 5-hydroxyalkyl nucleoside starting materials where the alkyl group is a methylene are obtained from the corresponding 5-methyl-nucleosides by protection (for instance using tert-butyldiphenylsilylchloride) of the hydroxyl groups of the 4-thio sugar moiety, photolytic bromination (for instance, using bromine, N-bromosuccinimide in carbon tetrachloride) and hydrolysis of the bromoalkyl side chain using sodium bicarbonate.
  • X is nitro or optionally substituted amino
  • Nitro-substituents are introduced at the 5-position of the 5-unsubstituted 4'thio-nucleosides by reaction with a nitrating agent for example nitronium tetrafluoroborate (N0 2 BF 4 ) , and these may be reduced using hydrogen over palladium/charcoal or tin (II) chloride to provide the corresponding amino substituent.
  • a nitrating agent for example nitronium tetrafluoroborate (N0 2 BF 4 )
  • II palladium/charcoal or tin
  • 5-Alkylamino and 5-dialkylamino substituents may be introduced by reacting a suitably protected 5-iodo-nucleoside with a corresponding alkylamine or dialkylamine. Protection is preferably by acylation for example by acetylation using acetic anhydride in pyridine.
  • X is alkoxy
  • alkoxy substituents may be introduced by treatment of the corresponding 5-iodo-4' -thionucleoside with an alkoxylating agent such as sodium alkoxide in an appropriate solvent such as methanol or dimethylformamide or the corresponding alkanol.
  • an alkoxylating agent such as sodium alkoxide in an appropriate solvent such as methanol or dimethylformamide or the corresponding alkanol.
  • Cyano substituents are introduced at the 5-position by reaction of the corresponding 5-iodo 4' -thio-nucleoside with potassium cyanide in the presence of potassium acetate in a suitable solvent such as dimethylformamide, preferably at elevated temperature, for example 80°C-120°C, preferably 100°C
  • X is thiocyanate. alkylthio, mercapto
  • 5-hydroxy-4' -thio-nucleosides may be prepared by the method described above in connection with the preparation of alkoxy compounds.
  • the starting compound, 5-unsubstituted 4'-thio- nucleoside may conveniently be prepared by condensation of the appropriate sugar moiety with commercially available uracil.
  • X is hvdroxy-C 13 alkyl. These compounds may be prepared as described above in connection with the preparation of haloalkyl compounds. Hydroxymethyl uracil itself is commercially available.
  • X is C, ⁇ alkoxyC, 7 alkyl or C /.alkylthiomethyl.
  • X is alkoxymethyl or alkylthiomethyl
  • bases in which the group X is of the formula -CH 2 0H may be made starting from bases in which the group X is of the formula -CH 2 0H.
  • the alkoxymethyl compounds may be made by reacting this starting material with an appropriate alkanol group in the presence of an acid catalyst or an acidic ion exchange resin.
  • the alkylthiomethyl compounds may be made in a similar way but using the appropriate alkylmercaptan group or an appropriate metal salt thereof.
  • the resulting base may be condensed with the desired 4-thio sugar as described herein.
  • alkoxyethyl compounds may be made in an analogous manner starting from the appropriate base in which the group X is hydroxyethyl.
  • These starting bases are either commercially available or may be made as described above in connection with the preparation of haloalkyl compounds.
  • these alkoxyalkyl and alkylthiomethyl compounds may be made from nucleosides of the formula I or a protected derivate thereof in which the group X is -CH 2 L where L is a leaving group, eg halo such as bromo, or alkyl or arylsulphonyloxy such as trifluoromethanesulphonyl or p- toluenesulphonyl or a secondary acyclic or cyclic amino group, such as dimethylamino or pyrrolidinyl.
  • L is a leaving group, eg halo such as bromo, or alkyl or arylsulphonyloxy such as trifluoromethanesulphonyl or p- toluenesulphonyl or a secondary acyclic or cyclic amino group, such as dimethylamino or pyrrolidinyl.
  • the reaction is carried out by treatment of one of these with a suitable nucleophilic
  • the procedure may be performed using methods analogous to those described by Barwolff and Langen, Nucleic Acid Chemistry - Improved and New Synthetic Procedures, Methods and Techniques, Part 1, Eds. L.B. Townsend and R.S. Tipson, p359.
  • the above reference also describes the procedures which may be utilised to make compounds in which L is OH.
  • Such compounds may be used to make compound where L is O-alkyl or S-alkyl using the procedures described above.
  • the compounds where L is dimethylamino or pyrrolidinyl may be prepared by methods analogous to those described by Badman and Reese in J. Chem. Soc. Commun. 1987, 1732-1734 and by Jones et al, Synthesis 1982, 259-260.
  • the 4-thio-sugar compound may be produced by conventional methods prior to coupling with the base or derived by modification of another sugar moiety which is already part of a nucleoside.
  • the process may be carried out using the following procedures to prepare compounds of formula (I) in which R 2 and R 3 have the following meanings include:-
  • R 2 is hydrogen and R 3 is hydroxy.
  • Such compounds may be prepared from a corresponding 3'5' , - anhydro compound for example by treatment with a strong base eg. potassium ter -butoxide.
  • a strong base eg. potassium ter -butoxide.
  • Such 3' ,5' -anhydro compounds may be prepared by treating the corresponding 3 ' , 5' -methanesulphonate diester with a base.
  • the 3' 5' -methanesulphonate diester may be obtained by esterification of the 2-deoxy-L-ribose sugar which may be synthesised by analogous methods to those of Smejkal and Sor , (1964) , Nucleic Acids. Components and their Analogues, part Lii, volume 29, 809.; Genu-Dellac e_t al.
  • acetylated uracil nucleoside (produced for instance by reactions as described above and acetylated using acetic anhydride in pyridine) is treated with p-chlorophenyl- phosphorodichloridate, 1,2,4-triazole and pyridine to produce the 4- (1,2,4-triazol-l-yl) derivative which is then treated with ammonia in dioxane (which also removes the 4-thio sugar protecting group(s) ) to form the corresponding unprotected cytosine 4' -thionucleoside.
  • esters may be prepared by treating a compound of formula (I) with an appropriate esterifying agent, for example, an acyl halide or anhydride.
  • Salts may be prepared by treating a compound of formula (I) with an appropriate base, for example an alkali metal, alkaline earth metal or ammonium hydroxide, or where necessary, an appropirate acid, such as hydrochloric acid or an acetate, eg. sodium acetate.
  • compounds of the formula (V) in which in which R 2 is hydrogen, R 3a is OZ 3 and W is a group -S-CH 2 -Ar as defined above may be made by ring closure of a compound of the formula (VII)
  • Z 3 and Z 5 are hydroxyl protecting groups as defined above, for example optionally substituted benzyl or acyl groups as defined above.
  • the groups Z 3 and Z 5 are acyl groups.
  • the group A is a leaving group, for example an organosulphonyl group such as an optionally substituted alkyl- or aryl-sulphonyl group, for instance methanesulphonyl, a haloalkylsulphonyl group (eg. trifluoromethylsulphonyl) and optionally substituted phenyl- sulphonyl (eg. toluylsulphonyl or bromobenzenesulphonyl) , and Ar is as defined above.
  • A is preferably a methanesulphonyl group.
  • the ring closure may be performed under appropriate basic conditions. Suitable conditions include those described by J. Harness and N.A. Hughes (Chem. Comm. 1971, 811) , which includes the use of sodium iodide and barium carbonate.
  • the compound of the formula (VII) may be made from a compound of formula (VIII) Ar-CH 2 -S.. S-CH 2 -Ar
  • the group M is a group of the formula Ar ** -CO- where Ar 1 is a phenyl group which may be optionally substituted by any of the substituents described above for the group Ar.
  • Removal of this group M may be performed under standard conditions, for example with a base such as an alkali metal alkoxide, for instance sodium methoxide in methanol.
  • a base such as an alkali metal alkoxide, for instance sodium methoxide in methanol.
  • the compounds of formula (IX) may be obtained by the concomitant inversion and derivatization of the 4-hydroxy group of a compound of formula (X) :
  • the inversion and derivatization may be effected by reacting the compound of formula (X) with a derivative of the group M, such as an acid of the formula Ar'-COOH, for example benzoic acid (or a reactive derivative thereof) where Ar 1 is as defined above.
  • a derivative of the group M such as an acid of the formula Ar'-COOH, for example benzoic acid (or a reactive derivative thereof) where Ar 1 is as defined above.
  • the reaction is performed typically at room temperature and under neutral conditions in a suitable polar solvent, for instance tetrahydrofuran.
  • a suitable polar solvent for instance tetrahydrofuran.
  • the Mitsunobu reaction is used for the inversion and derivatization; diethyl azodicarboxylate (DEAD) and triphenylphosphine are used as coreactants together with the acid Ar'COOH.
  • the compound of formula (X) may be made from a glycoside compound of formula (XI)
  • R is a defined above.
  • the hydroxyl groups of the compound of formula (XII) are protected under conventional conditions with the reactive derivative of the groups Z 3 and Z 5 .
  • the bromo derivative may be used.
  • Z 3 and Z 5 are benzyl groups
  • benzyl bromide may be used.
  • the reaction may be performed in an organic solvent such as tetrahydrofuran in the presence of a suitable base such as sodium hydride and a phase transfer catalyst such as tetrabutylammonium iodide.
  • Compounds of the formula (XII) may be made by standard techniques from 2-Deoxy-L-ribose, which can be made by methods described in .J. Robins, T.A.Khwaja, R.K. Robins, J. Org. Chem., 1970, 35(3) 636.
  • 2-Deoxy-L-ribose may be reacted with an alcohol of formula R-OH (where R is as defined above) in the presence of an acid. Hydrochloric acid is suitable.
  • R-OH When R is a methyl group, the alcohol R-OH will be methanol.
  • the compound of the formula (VIII) may also be made directly from the compound of formula (X) using a Mitsunobu reaction under conditions analogous to those described by D.R. Williams et al, JACS (1990) 112, 4552.
  • R, Z 3 and Z ⁇ are as defined for a compound of formula (XI) ; with a compound of the formula Ar-CH 2 -SH where Ar is defined above.
  • the reaction may be conducted using similar conditions to those described above for the preparation of the compound of the formula (X) .
  • the reaction is performed in the presence of an acid, for example an inorganic acid such as HCl or a Lewis acid such as TiCl 4 . TiCl 4 is preferred.
  • the groups Z 3 and Z 5 are preferably acyl groups, in particular p-nitrobenzoyl groups.
  • a compound of the formula Ar-CH 2 -SH which is preferred include p-methoxybenzyl mercaptan.
  • Z n OH is reacted with a compound or compounds of formula Z n OH where Z n is Z 3 and/or Z 5 .
  • Z 3 and Z 5 will be the same and a single compound Z n 0H may be used. If different values of Z 3 and Z 5 are required, then the required mixture of compounds of ZOH may be used, and the desired reaction products separated from the resulting reaction mixture.
  • Preferred compounds of the formula Z n OH are those where Z n is an acyl group as defined above.
  • Z n 0H is p-nitro- benzoic acid, although other benzoic acids may also be used.
  • the reaction is performed in the presence of an azido- carboxylate such as diethylazidodicarboxylate or preferably diisopropylazidodicarboxylate.
  • the solvent for the reaction may be DMF, tetrahydrofuran, dichloromethane or toluene. Toluene is preferred.
  • the reaction may be performed at room temperature.
  • Z 3 and Z 5 are both p-nitrobenzyl.
  • Compounds of the formula (XIV) may be made from 2- deoxyribose using techniques known in the art, for example as described above in connection with the production of compounds of formula (XII) .
  • Compounds of the formula (I) may also be made by reaction of a compound of formula (III) with a compound of formula (XV)
  • L is a leaving group, for example, an acyloxy group such as C alkanoyloxy, for instance, acetoxy
  • P 1 is a protecting group or hydrogen
  • Z is a directing group.
  • Suitable groups P 1 include groups such that P'O is an ether group, e.g. a silyl ether group (such as tertbutyldiphenylsilyl ether or tertbutyldimethylsilyl ether) , a straight or branch chain alkyl ether group, a cyclic ether group (such as tetrahydropyran-2-yl ether) or an optionally substituted aryl ether group (such as benzyl ether, trityl ether or benzhydryl ether) .
  • the group P'O- can also be an ester group e.g.
  • the reaction of the compound of formula (XV) with the base of formula (III) may be carried out for example in the presence of nonafluorobutane sulphonic acid or a Lewis acid catalyst, e.g. tin (IV) chloride, a mercury (II) salt or trimethylsilyl triflate.
  • a Lewis acid catalyst e.g. tin (IV) chloride, a mercury (II) salt or trimethylsilyl triflate.
  • the reaction can be carried out, for example, at a temperature of from 0°C to room temperature in a suitable solvent such as acetonitrile or a chloroalkane.
  • Z is phenylselenyl it may be eliminated under oxidising conditions which are capable of oxidising selenium without oxidising sulphur, e.g. by treatment with m- chloroperbenzoic acid in dichloromethane at -20°C (Toru et al , Tetrahedron Letters, 1986, 22; 1583) .
  • Compounds of the formula (I) in which R 2 and R 3 are both hydrogen may be made by elimination of the group Z (from the reaction product of (XV) and (III) ) under reducing conditions, e.g. using tributyltin. ' hydride and triethyl borane (see Nozaki et al , Tetrahedron Letters-,; 1988, 23; 6125) .
  • EP-A-514 036 describes the production of D-thionucleosides from, inter-alia , the isomer of the compound of formula (XV) which has the D-configuration.
  • the reactions described in EP-A-514 036, the contents of which are incorporated herein by reference, may be applied to the production of compounds of formula (I) .
  • Compounds of the formula (XV) may be made by acylation under standard conditions (eg. acetic anhydride with pyridine) of a compound of formula (XVI) :
  • the compound of formula (XVI) may be made from a compound of formula (XVII) ,
  • E-5- (2-bromovinyl) -2' -deoxy-4' -thio- ⁇ -L-uridine is dissolved in a solution of sodium methoxide in methanol (7.5ml, 0.1m) and the mixture is allowed to stand at 22°C for 24 hours.
  • the solution is neutralised by careful addition of Dowex 50 ion exchange resin (H + form) to pH6. The resin is filtered off and washed with methanol and the filtrate and washings evaporated to a white solid.
  • MeOH-CH 2 Cl 2 (1:1, 18 ml) is added slowly and the mixture allowed to warm to ambient temperature then evaporated. The residue is re-evaporated from MeOH (3x) , taken up in MeOH-CHCl 3 (1:1, 15ml) and the solid collected by filtration, yielding the desired ⁇ -anomer of the product.
  • Benzyl 3,5-di-0-benzyl-2-deoxy-1 , 4-dithio-D-erythro- pentofuranoside (5.6 mmol) is dissolved in CC1 4 (30 ml) and bromine (6.2 mmol) in CC1 4 (30 ml) is added. After stirring for 55 min. at ambient temperature the solvent is evaporated and the residue re-evaporated from CC1 4 (10 ml) to remove excess bromine.
  • This compound is prepared by a method similar to the iodo compound above with the following modifications:
  • the total solvent (MeCN) volume for the reaction is 3 ml.
  • 5-ethynyluracil this compound is prepared in a similar manner to that described in Example 5.
  • 5-ethynyluracil may be prepared from 5-ioduracil using the methodology analogous to that described by M.J. Robins et al (ibid) .
  • E-5- (2-Bromovinyl) -2,4-dimethoxypyrimidine To a solution of E-5- (2-carboxyvinyl) -2,4-dimethoxypyrimidine (0.300 g; 1.43 mmol) in dry DMF (5 ml) was added K 2 C0 3 (0.45 g: 5.25 mmol) . After stirring at ambient temperature for 15 min. a solution of N.-bromosuccinimide (0.258 g; 1.45 mmol) in dry DMF (4 ml) was added dropwise over 10 min. The suspension was immediately filtered, the solid washed with DMF and the filtrate evaporated in high vacuum.
  • the carboxymethyllactone was dissolved in 75 ml of dimethylsulphoxide, to which were added 20 drops of brine. After 2 h at 170°C the reaction was complete. After cooling, the reaction solution was directly transferred onto a pre-packed silica column, and the lactone eluted off with 30% ether in petrol, to give a colourless oil on evaporation.
  • the lactone (7.5 mmol) was reduced by diisobutylaluminiumhydride (7.9 mmol) in 100 ml of dry toluene at -78°C over 3 h, after which the reaction was quenched with 100 ml of saturated ammonium chloride and vigorously stirred for 1 h. After filtering through a hyflo pad, the organic layer was separated and washed twice with brine, dried and evaporated.
  • the crude lactol was dissolved in 200 ml of dichloromethane and treated with 4-dimethylamino pyridine (1.0 g, 8.25 mmol) and acetic anhydride (0.84 g, 8.25 mmol) . Reaction was complete in 2 h at room temperature, then the solution was washed sequentially with water, copper sulphate, water then brine, dried and evaporated. Purification was achieved by on a short flash column.
  • the 2'-selenyl nucleoside was dissolved in dry dichloromethane and cooled to -20°C under nitrogen. To this was added an equivalent of metachloroperoxvbenzoic acid in one portion, and the temperature maintained during the course of the reaction (45 0 min) . 5 eq of pyridine were then added and the solution allowed to warm to room temperature over 1 hour. After dilution with dichloromethane the solution was washed successively with water, copper sulphate (x2) , sodium bicarbonate (x2) , water, and brine, before drying and evaporation. Purification was achieved on a 5 flash column, eluted with methanol/chloroform/ammonia (7:92:1) .
  • silyl ether was stirred in a thf solution with tetraethylammonium fluoride (1.1 eq) for 1 hour at room
  • aqueous layer was further extracted with dichloromethane (3 x 50 ml) , the combined organic layers dried (Na 2 S0 4 ) and evaporated to dryness. Residual pyridine was co-evaporated with ethanol (2 x 50 ml) to give the title compound.
  • N-iodosuccinimide (6 mg, 0.027 mmol) was added and after stirring overnight the reaction mixture was diluted with dichloromethane

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Abstract

Antiviral nucleosides of formula (I), wherein: Y is hydroxy or amino; X is hydrogen, hydroxy, mercapto, halo, trifluoromethyl, methyl, C2-6alkyl, C1-6haloalkyl, hydroxyC1-3alkyl, formyl, C2-6alkenyl, C2-6haloalkenyl, C2-6alkynyl, C1-6alkoxy, C1-6alkylthio, C1-6alkoxyC1-2alkyl, C1-6alkylthiomethyl, amino, monoC1-6alkylamino, diC1-6alkylamino, cyano, thiocyanate or nitro; R2 is hydrogen and R3 is hydroxy or hydrogen, or together R?2 and R3¿ form a carbon-carbon bond; and physiologically functional derivatives thereof. Compositions containing the compounds, their use in treatment and therapy of viral disease, and processes for the production of the compounds are also provided.

Description

ANTIVIRAL PYRIMIDINE NUCLEOSIDES The present invention relates to pyrimidine nucleosides and their use in medical therapy particularly for the treatment or prophylaxis of virus infections.
Of the DNA viruses, those of the herpes group are the sources of the most common viral illnesses in man. The group includes herpes simplex virus types 1 and 2 (HSV) , varicella zoster virus (VZV) , cytomegalovirus (C V) ; Epstein-Barr virus (EBV) ; human herpes virus type 6 (HHV-β) and human herpes virus type 7 (HHV-7) . HSV 1 and HSV 2 are some of the most common infectious agents of man. Most of these viruses are able to persist in the host's neural cells; once infected, individuals are at risk of recurrent clinical manifestations of infection which can be both physically and psychologically distressing.
HSV infection is often characterised by extensive and debilitating lesions of the skin, mouth and/or genitals. Primary infections may be subclinical although tend to be more severe than infections in individuals previously exposed to the virus. Ocular infection by HSV can lead to keratitis or cataracts thereby endangering the host's sight. Infection in the newborn, in immunocompromised patients or penetration of the infection into the central nervous system can prove fatal.
Transmission of the virus is by direct physical contact between a host and a recipient; the spread of HSV infection is therefore considered a very significant social problem, particularly as no effective vaccine is yet available.
Varicella zoster (VZV) is a herpes virus which causes chickenpox and shingles. Chickenpox is the primary disease produced in a host without immunity and in young children is usually a mild illness characterised by a vesicular rash and 0 fever. Shingles or zoster is the recurrent form of the disease which occurs in adults who were previously infected with varicella-zoster virus. The clinical manifestions of shingles are characterised by neuralgia and a vesicular skin rash that is unilateral and dermatomal in distribution. Spread of 5 inflammation may lead to paralysis or convulsions. Coma can occur if the meninges become affected. In immunodeficient
■ patients VZV may disseminate causing serious or even fatal illness. VZV is of serious concern in patients receiving immunosuppressive drugs for transplant purposes or for treatment of malignant neoplasia and is a serious complication of AIDS patients due to their impaired immune system. In common with other herpes viruses, infection with CMV leads to a lifelong association of virus and host and, following a primary infection, virus may be shed for a number of years. Congenital infection following infection of the mother during pregnancy may give rise to clinical effects such as death or gross disease (microcephaly, hepatosplenomegaly, jaundice, mental retardation) , retinitis leading to blindness or, in less severe forms, failure to thrive, and susceptibility to chest and ear infections. CMV infection in patients who are immunocompromised for example as a result of malignancy, treatment with immuno- suppressive drugs following transplantation or infection with Human Immunodeficiency Virus may give rise to retinitis, pneumonitis, gastrointestinal disorders and neurological diseases. CMV infection in AIDS patients is a predominant cause of morbidity as, in 50-80% of the adult population, it is present in a latent form and can be re-activated in immunocompromised patients.
Epstein-Barr virus (EBV) , a member of the herpes virus family, was discovered in 1964 and is now believed to be carried by 90% of the world's population. In the West, the main disease caused by EBV is acute or chronic infectious mononucleosis (glandular fever) . Examples of other EBV or EBV associated diseases include lymphoproliferative disease which frequently occurs in persons with congenital or acquired cellular immune deficiency, X-linked lymphoproliferative disease which occurs namely in young boys, EBV-associated B-cell tumours, Hodgkin's disease, nasopharyngeal carcinoma, Burkitt lymphoma, non-Hodgkin /3-cell lymphoma, i munoblastic lymphoma, thymomas and oral hairy leukoplakia. EBV infections have also been found in association with a variety of epithelial-cell-derived tumours of the upper and lower respiratory tracts including the lung.
HHV-6 has been shown to be a causative agent of infantum subitum in children and of kidney rejection and interstitial pneumonia in kidney and bone marrow transplant patients respectively and may be associated with other diseases. There is also evidence of repression of stem cell counts in bone marrow transplant patients. HHV-7 is of undetermined disease etiology. HBV is a viral pathogen of world-wide major importance. The virus is aetiologically associated with primary hepatocellular carcinoma and is thought to cause 80% of the world's liver cancer. In the United States more than ten thousand people are hospitalised for HBV illness each year, and an average of 250 die with fulminant disease. The United States currently contains an estimated pool of 500,000 to 1-million infectious carriers. Chronic active hepatitis generally develops in over 25% of carriers, and often progresses to cirrhosis. Clinical effects of infection with HBV range from headache, fever, malaise, nausea, vomiting, anorexia and abdominal pains. Replication of the virus is usually controlled by the immune response, with a course of recovery lasting weeks or months in humans, but infection may be more severe leading to persistent chronic liver disease outlined above.
One group of viruses which has assumed a particular importance is the retroviruses. Retroviruses form a sub-group of RNA viruses which, in order to replicate, must first 'reverse transcribe' the RNA of their genome into DNA ('transcription' conventionally describes the synthesis of RNA from DNA) . Once in the form of DNA, the viral genome may be incorporated into the host cell genome, allowing it to take advantage of the host cell's transcription/ translation machinery for the purposes of replication. Once incorporated, the viral DNA is virtually indistinguishable from the host's DNA and, in this state, the virus may persist for the life of the cell. A species of retrovirus, the lentivirus Human Immuno¬ deficiency Virus (HIV) , has been reproducibly isolated from humans with Acquired Immune Deficiency Syndrome (AIDS) or with the symptoms that frequently precede AIDS. AIDS is an immunosuppressive or immunodestructive disease that predisposes subjects to fatal opportunistic infections. Characteristically, AIDS is associated with a progressive depletion of T-cells, especially the helper-inducer subset bearing the OKT4 surface marker. HIV is cytopathic and appears to preferentially infect and destroy T-cells bearing the OKT4 marker and it is now generally recognised that HIV is the aetiological agent of AIDS. Since the discovery that HIV is the aetiological agent of AIDS, numerous proposals have been made for the anti-HIV chemotherapeutic agents that may be effective in treating AIDS. Thus, for example, European Patent Specification No. 196 185 describes 3' -azido-3' -deoxythymidine (which has the approved name zidovudine) , its pharmaceutically acceptable derivatives and their use in the treatment of human retrovirus infections including AIDS and associated clinical conditions.
Examples of retroviral infections include human retroviral infections such as Human Immunodeficiency Virus (HIV) , for example, HIV-1 or HIV-2, and Human T-cell Lymphotropic Virus (HTLV) , for example, HTLV-I or HTLV-II, infections. AIDS and related clinical conditions such as AIDS-related complex (ARC) , progressive generalized lymphadenopathy (PGL) , Karposi's sarcoma, thrombocytopenic purpura, AIDS-related neurological conditions, such as multiple sclerosis or tropical paraparesis, and also anti-HIV antibody-positive and HIV-positive conditions, including such conditions in asymptomatic patients, are also conditions which may be treated by appropriate anti-viral therapy.
Another RNA virus which has been recognised as the causative agent of an increasingly serious international health problem is the non-A, non-B hepatitis virus. At least 80% of cases of chronic post-transfusional non-A, non-B hepatitis have been shown to be due to the virus now identified as hepatitis C and this virus probably accounts for virtually all cases of post- transfusional hepatitis in clinical settings where blood products are screened for hepatitis B. Whereas approximately half of the cases of acute hepatitis C infection resolve spontaneously over a period of months, the remainder become chronic and in many if not all such cases chronic active hepatitis ensues with the potential for cirrhosis and hepatocellular carcinoma. The structure of the hepatitis C virus genome has recently been elucidated and the virus has been characterised as a single stranded RNA virus with similarities to flaviviruses.
Coxsackie viruses belong to the enterovirus genus. They have a single stranded RNA genome contained in an icosahedral nucleocapsid. Coxsackie virus infection is increasingly recognised as a cause of primary myocardial disease in adults and children.
Coxsackie infection is also associated with meningitis, pleurodynia, herpangia, hand-feet and mouth disease, respiratory disease, eye disease, diabetes and post-viral fatigue syndrome. In the latter case viral RNA has been detected in the muscle and in monocytes.
We have now found that certain pyrimidine L-4' -thionucleosides have activity against viruses. Such compounds include pyrimidine L-4' -thionucleosides of the following general formula (I) :
Figure imgf000007_0001
wherein: Y is hydroxy or amino;
X is hydrogen, hydroxy, mercapto, halo, trifluoromethyl, methyl,
C2^alkyl, C^haloalkyl, hydroxyC..3alkyl, formyl,
Figure imgf000007_0002
C^alkylthio, C^alkoxyC-^alkyl, C^alkylthiomethyl, amino, monoCj. 6alkylamino, diC^alkylamino, cyano, thiocyanate or nitro; R2 is hydrogen and R3 is hydroxy or hydrogen, or together R2 and R3 form a carbon-carbon bond; and physiologically functional derivatives thereof.
It will be appreciated that by virtue of the definition of the group Y the compounds of formula (I) are derivatives either of uracil or of cytosine.
It will also be appreciated that the compounds of formula (I) may exist in various tautomeric forms.
The compounds of formula (I) may exist as a- or β-anomers; β-anomers are preferred. It will be appreciated that the compounds of formula (I) may exist in various isomeric forms and as mixtures thereof in any proportions. The present invention includes within its scope the use of such isomeric forms or mixtures thereof, including the individual a- and 0-isomers of the compound of formula (I) as well as mixtures of such isomers in any proportion. A preferred group of compounds of the formula (I) are those in which Y is hydroxy or amino and X is hydrogen, hydroxy, mercapto, halo, trifluoromethyl, methyl, C^alkyl, C^haloalkyl, hydroxyC^alkyl, formyl, C2^alkenyl, C2^ haloalkenyl, C2.6 alkynyl, C,^alkoxy, C,.6alkoxyC,.2alkyl, amino, monoC-.6alkylamino, diCu 6alkylamino, cyano or nitro; R2 is hydrogen and R3 is hydroxy or hydrogen, or together R2 and R3 form a carbon-carbon bond; and physiologically functional derivatives thereof.
A particularly preferred group of compounds of formula (I) are those in which Y is hydroxy or amino, X is hydrogen, halo, methyl, C2-6alkyl, C^haloalkyl, C2-6alkenyl, C2-6 haloalkenyl, C2^ alkynyl, cyano or nitro; R2 is hydrogen and R3 is hydroxy or hydrogen, or together R2 and R3 form a carbon-carbon bond; and physiologically functional derivatives thereof.
In the definition of formula (I) , references to alkyl groups include groups which, . when they contain at least three carbon atoms may be branched or cyclic but which are preferably straight (particular alkyl groups include methyl and ethyl) ; references to alkenyl groups include groups which may be in the E- or Z- form or a mixture thereof and which, when they contain at least three carbon atoms, may be branched but are preferably straight; and references to alkynyl groups include groups which, when they contain at least four carbon atoms may be branched but which are preferably straight; particular alkenyl groups include vinyl and E- (1-propenyl) and particular alkynyl groups include ethynyl and prop-1-ynyl. References to halo-substituted groups include chloro, bromo, iodo and fluoro substituted groups and groups substituted with two or more halogens which may be the same or different, for example perhalo substituted groups. In the case where the alkoxy group is part of the group alkoxyC,.
2alkyl, the alkoxy substituent may be attached to either the C or C2-carbon atom of the group.
Preferred compounds of the formula (I) include those in which the group X is methyl, C2. alkyl or haloalkenyl preferably
C2.3 alkyl, 3 i alkenyl or alkynyl, or halovinyl. Preferred haloalkenyl groups are straight chain haloalkenyl groups having a single halogen group on the terminal carbon. Also preferred are haloalkenyl groups having a double bond in the 1-position. Of such compounds, those having a 2-halovinyl group which is in the E configuration are preferred. Particular halovinyl groups include E- (2-bromovinyl) .
Particularly preferred compounds of formula (I) are those: (i) wherein Y is amino; (ii) wherein R2 and R3 form a carbon-carbon bond;
(iii) wherein Y is amino, X is hydrogen or halo and R2 and
R3 form a carbon-carbon bond.
Particular compounds of the invention are compounds of formula (I) and physiologically acceptable derivatives thereof wherein the pyrimidine base is selected from:
Uracil;
Cytosine;
Thymine;
5-Iodouracil; 5-Bromouracil;
5-Chlorouracil;
5-Fluorouracil;
5-Iodocytosine;
5-Bromocytosine; 5-Chlorocytosine;
5-Fluorocytosine;
5-Ethynyluracil;
5-Prop-1-ynyluracil;
5-Vinyluracil; E-5- (2-Bromovinyl)uracil;
E-5- (1-Propenyl)uracil;
5-Ethyluracil;
5-Trifluoromethyluracil; E-5- (2-Bromovinyl) cytosine; or
5-Propyluracil.
Compounds of formula (I) having the beta configuration which are of special interest as antiviral agents are: 2' -deoxy-4' -thio-L-uridine,
2' -deoxy-4' -thio-L-cytidine,
2' -deoxy-5-fluoro-4' -thio-L-cytidine
2' -deoxy-5-methyl-4' -thio-L-uridine,
5- (2-chloroethyl) -2' -deoxy-4' -thiouridine; 5-nitro-2' -deoxy-4' -thiouridine;
5-amino-2' -deoxy-4' -thiouridine;
5-methylamino-2' -deoxy-4' -thiouridine;
E-5- (2-bromovinyl) -2' -deoxy-4' -thio-L-uridine,
2' -deoxy-5-iodo-4' -thio-L-uridine, 5-bromo-2' -deoxy-4' -thio-L-uridine,
5-chloro-2' -deoxy-4' -thio-L-uridine,
2' -deoxy-5-ethyl-4' -thio-L-uridine,
2' -deoxy-5-prop-l-ynyl-4' -thio-L-uridine,
2' -deoxy-5-fluoro-4' -thio-L-uridine, 2' -deoxy-5-trifluoromethyl-4' -thio-L-uridine,
2' -deoxy-5-ethynyl-4' -thio-L-uridine,
2' -deoxy-5-E- (2-bromovinyl) -4' -thio-L-cytidine,
2' -deoxy-5-propyl-4' -thio-L-uridine,
E-2 ' -deoxy- 5 - (propen-1-yl) -4 ' -thio-L-uridine , 1 - ( 2 , 3 - didehydro - 2 , 3 - dideoxy- 4 - thio - L - ribof uranosyl ) - 5 - methyluracil ,
1- (2 , 3 -dideoxy-4-thio-L-ribofuranosyl) -5-methyluracil ,
5 -bromo- 2 ' 3 ' -didehydro-2 ' , 3 ' -dideoxy-4 ' -thio-β-L-cytidine ,
5-chloro-2' ,3' -didehydro-2' ,3' -dideoxy-4' -thio-/3-L-cytidine, and 2' ,3'-didehydro-2' ,3' -dideoxy-5-iodo-4' -thio-3-L-cytidine. Especially preferred compounds of the invention are:
2' ,3' -didehydro-2' ,3' -dideoxy-4' -thio-0-L-cytidine, and
2' ,3' -didehydro-2' ,3' -dideoxy-5-fluoro-4' -thio-0-L-cytidine.
These compounds have particularly potent activity against HIV and HBV.
The above-mentioned derivatives include the pharmaceutically acceptable salts; esters and salts of esters, or any other compound which, upon administration to a human subject, is capable of providing (directly or indirectly) the antivirally active metabolite or residue thereof.
Preferred mono- and di-esters according to the invention include carboxylic acid esters in which the non-carbonyl moiety of the ester grouping is selected from straight or branched chain alkyl, (methyl, n-propyl,, n-butyl or t-butyl) ; cyclic alkyl (e.g. cyclohexyl) ; alkoxyalkyl (e.g. methoxymethyl) , carboxyalkyl (e.g. carboxyethyl) , aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl) , aryl (e.g. phenyl optionally substituted by halogen, CM alkyl or CM alkoxy or amino) ; sulphonate esters such as alkyl- or aralkyl- sulphonyl (e.g. methanesulphonyl) ; mono-, di- or tri-phosphate esters which may or may not be blocked, amino acids esters (e.g. L-valyl or L-isoleucyl esters) and nitrate esters. With regard to the above-described esters, unless otherwise specified, any alkyl moieties present in such esters advantageously contain 1 to 18 carbon atoms, particularly 1 to 4 carbon atoms, in the case of straight chain alkyl groups, or 3 to 7 carbon atoms in the case of branched or cyclic alkyl groups. Any aryl moiety present in such esters advantageously comprises a phenyl group. Any reference to any of the above compounds also includes a reference to a physiologically acceptable salt thereof.
Salts according to the invention which may be conveniently used in therapy include physiologically acceptable base salts, eg derived from an appropriate base, such as alkali metal (e.g. sodium) , alkaline earth metal- (e.g. magnesium) salts, ammonium and NR4 (wherein R is C alkyl) salts. When Y represents an amino group, salts include physiologically acceptable acid addition salts, including the hydrochloride and acetate salts. Derivatives of the compounds of the formula I include the corresponding sulphones and sulphoxides.
Such nucleosides and their derivatives will be hereinafter referred to as the compounds according to the invention. The term "active ingredient" as used hereafter, unless the context requires otherwise, refers to a compound according to the invention.
The present invention further includes: a) compounds according to the invention for use in a method of treatment or therapy of the human or animal body . b) compounds according to the invention for use in the treatment or prophylaxis of viral infections, eg. HBV, HIV, Hepatitis C and herpes virus infections such as those mentioned above and more particularly
HSV, HHV-6, VZV, EBV or CMV infections. c) a method for the treatment or prophylaxis of a virus infection such as those mentioned above in a mammal including man, eg. HBV, HIV, Hepatitis C and herpes virus infections such as those mentioned above and more particularly HSV, HHV-6, VZV, EBV or CMV infections, which comprises treating a subject with an effective non-toxic amount of a compound according to the invention. d) use of a compound according to the invention in the manufacture of a medicament for use in the treatment or prophylaxis of a virus infection, such as those mentioned above, eg. HBV, HIV, Hepatitis C and herpes virus infections such as those mentioned above and more particularly HSV, HHV-6, VZV, EBV or CMV infections.
Examples of clinical conditions which may be treated in accordance with the invention include those which have been discussed in the introduction hereinbefore and in particular those caused by infections of-HIV, HBV, Hepatitis C, HSV 1 and
2, HHV-6, VZV, EBV or CMV.
The compounds according to the invention are particularly useful for the treatment of humans diagnosed as HIV positive or anti-HIV antibody-positive and especially useful for the treatment of patients who are immunocompromised, for example, as a result of malignancy, treatment with immunosuppressive drugs following transplantation or humans suffering from AIDS, AIDS related complex (ARC) or progressive generalised lymphadenopathy
(PGL) . The compounds according to the invention may be administered to mammals including humans by any route appropriate to the condition to be treated, suitable routes including oral, rectal, nasal, topical (including buccal and sublingual) , vaginal and parenteral -(including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) . It will be appreciated that the preferred route may vary with, for example, the condition of the recipient. For each of the above-indicated utilities and indications the amount required of the individual active ingredients will depend upon a number of factors including the severity of the condition to be treated and the identity of the recipient and will ultimately be at the discretion of the attendant physician. In general, however, for each of these utilities and indications, a suitable, effective dose will be in the range 0.05 to 250 mg per kilogram body weight of recipient per day, preferably in the range 0.1 to 100 mg per kilogram body weight per day and most preferably in the range 0.5 to 20 mg per kilogram body weight per day; an optimum dose is about 2 to 5 mg per kilogram body weight per day (unless otherwise indicated all weights of active ingredient are calculated as the parent compound; for salts and esters thereof the figures would be increased proportionately.) The desired dose may if desired be presented as two, three, four or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing 10 to 1000 mg, preferably 20 to 500 mg and most preferably 100 to 400 mg of active ingredient per unit dosage form. While it is possible for the compounds to be administered alone it is preferable to present them as pharmaceutical formulations. The formulations of the present invention comprise at least one active ingredient, as above defined, together with one or more acceptable carriers thereof and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipients thereof.
The formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual) , vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintergrant (e.g. sodium starch glycolate, cross- linked povidone, cross-linked sodium carboxymethyl cellulose) , surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored an may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxpropyl ethylcellulose in varying proportions to provide desired release profile.
Formulations suitable for oral use may also include buffering agents designed to neutralise stomach acidity. Such buffers may be chosen from a variety of organic or inorganic agents such as weak acids or bases admixed with their conjugated salts.
A capsule may be made by filling a loose or compressed powder on an appropriate filling machine, optionally with one or more additives. Examples of suitable additives include binders such as povidone; gelatin, lubricants, inert diluents and disintegrants as for tablets. Capsules may also be formulated to contain pellets or discrete sub-units to provide slow or controlled release of the active ingredient. This can be achieved by extruding and spheronising a wet mixture of the drug plus an extrusion aid (for example microcrystalline cellulose) plus a diluent such as lactose. The spheroids thus produced can be coated with a semi-permeable membrane (for example ethyl cellulose, Eudragit WE30D) to produce sustained release properties.
An edible foam or whip formulation ideally comprises; 50- 70% of an edible oil, particularly a vegetable oil, including corn oil, peanut oil, sunflower oil, olive oil and soybean oil; 2-10% of one or more surfactants particularly lecithin, polyols, polyol polymer esters including glyceryl fatty acid esters, polyglyceryl fatty and acid esters (e.g. decaglycerol tetraoleate) , or sorbitan fatty acid esters (e.g. sorbitan monostearate) ; 1-4% of a propellant which is suitable for ingestion, notably a compressed gas propellant especially nitrogen, nitrous oxide or carbon dioxide, or a gaseous hydrocarbon especially propane, butane or isobutane; 0.5-30% of one or more viscosity modifiers of particle size in the range 10-50 microns in diameter, particularly powdered sugars or colloidal silicon dioxide; and optionally 0.5-1% of one or more suitable, non-toxic colourings, flavourings or sweetners. The active ingredient is preferably present in such formulations in a concentration of 10-46%, advantageously 30%. An edible foam or whip formulation as described above may be prepared in a conventional manner, for example by mixing the edible oil, surfactant (s) and any other soluble ingredients, adding the viscosity modifier(s) and milling the mixture to form a uniform dispersion and suspension. The active ingredient is blended into the milled mixture until evenly dispersed. Finally, a metered quantity of propellant is incorporated to the mixture after said mixture has been measured into a suitable dispensing container. Pharmaceutical formulations for topical administration according to the present invention may be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil. Alternatively, a formulation may comprise a dressing such as a bandage or adhesive plaster impregnated with active ingredients and optionally one or more excipients or dilue'nts. Compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such patches suitably contain the active compound 1) in an optionally buffered, aqueous solution or 2) dissolved in an adhesive or 3) dispersed in a polymer. A suitable concentration of the active compound is about 1% to 35%, prefrably about 3% to 15%. As one particular possibility, the active compound may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6), 318 (1986) .
For infections of the eye or other external tissues, e.g., mouth and skin, the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, 0.075 to 20% w/w, preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in- water cream base. If desired, the aqueous phase of the cream base may include, for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulphoxide and related analogues. The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While this phase may comprise merely an emulsifier (otherwise known as an emulgent) , it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulphate.
The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2- ethylhexyl palmitats or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or. in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oil can be used.
Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient. The active ingredient is preferably present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10% particularly about 1.5% w/w. Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.
Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as for example a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Injection solutions and suspensions may be prepared extemporaneously from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.
It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation ir. question, for example those suitable for oral administration may include flavoring agents. The compounds according to the invention may be employed alone or in combination with other therapeutic agents for the treatment of the above infections or conditions. Combination therapies according to the present invention comprise the administration of at least one compound of the formula (I) or a physiologically functional derivative thereof and at least one other pharmaceutically active ingredient. The active ingredient (s) and pharmacologically active agents may be administered together or separately and, when administered separately this may occur simultaneously or sequentially in any order. The amounts of the active ingredient (s) and pharmacologically active agent (s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect. Preferably the combination therapy involves the administration of one compound of the formula (I) or a physiologically functional derivative thereof and one of the agents mentioned herein below.
Examples of such further therapeutic agents include agents that are effective for the treatment of HIV infections or associated conditions such as 3' -azido-3' -deoxythymidine (zidovudine) , other 2' ,3' -dideoxynucleosides such as 2',3'- dideoxycytidine, 2' ,3' -dideoxyadenosineand2' ,3' -dideoxyinosine, carbovir, pentoxifylline, N-acetylcysteine, procysteine, a- trichosanthin, acyclic nucleosides (for example, acyclovir) , 2' ,3' -didehydrothymidine, protease inhibitors such as N-tert- butyl-dechydro-2- [ 2 (R) -hydroxy-4 -phenyl-3- (S) - [ [N- (2 - quinolycarbonyl) -L-asparginyl] -butyl] - (4aS, 8aS) -isoquinoline-3-
(S) -carboxamide (RO 31-8959) , oxathiolan nucleoside analogues such as cis-1- (2-hydroxymethyl) -1,3-oxathiolan-5-yl)cytosine
(BCH-189) or cis-1- (2- (hydroxymethyl) -1,3-oxathiolan-5-yl) -5- fluoro-cytosine, 3' -deoxy-3' -fluorothymidine, 2' ,3' -dideoxy-5- ethynyl-3' -fluorouridine, 5-chloro-2'3-dideoxy-3' -fluorouridine, Ribavirin, 9- [4-hydroxy-2- (hydroxymethyl)but-1-yl]guanine(H2G) , TAT inhibitors such as 7-chloro-5- (2-pyrryl) -3H-1,4- benzodiazapin-2 (H) -one (R05-3335) or 7-chloro-l,3-dihydro-5- (1H- pyrrol-2-yl) -3H-1, 4-benzodiazapin-2-amine (R024-7429) , interferons such as α-interferon, renal excretion inhibitors such as probenecid, nucleoside transport inhibitors such as dipyridamole, phosphonoformic acid, as well as immunodulators such as interleukin II, granulocyte macrophage colony stimulating factors, erythropoetin, soluble CD4 and genetically engineered derivatives thereof. Examples of such further therapeutic agents which are effective for the treatment of HBV infections include carbovir, oxathiolan nucleoside analogues such as cis-1- (2- hydroxymethyl) -1,3-oxathiolan-5-yl) -cytosine (BCH-189) or cis-1- (2- (hydroxymethyl) -1,3-oxathiolan-5-yl-5-fluoro-cytosine, 2' ,3' - dideoxy-5-ethynyl-3' -fluorouridine, 5-chloro-2' ,3' -dideoxy-3' - fluorouridine, 1- (/3-D-arabinofuranosyl) -5-propynyluracil, acyclovir and interferons, such as α-interferon. Examples of further therapeutic agents which are effective for the treatment of herpes virus infections are acyclovir, 9- [4-hydroxy-2- (hydroxymethyl) butyl] guanine (H2G) , 9- [4-hydroxy-3- (hydroxymethyl) -but-l-yl)guanine (penciclovir) , famciclovir, the 6-deoxy-diacetate ester of penciclovir, BVaraU, 1- (3-D- arabinofuranosyl-5-propynyluracil, 2- [ (2-amino-l,6-dihydro-6-oxo- 9H-purin-9-yl)methoxy]ethyl L-valinate, phosphonoformic acid and phosphonoacetic acid, Ganciclovir, (S) -1- (3-hydroxy-2- phosphonylmethoxypropyl) -cytosine (HPMPC) , Oxetanocin G, 2'- deoxy-5-iodouridine, E-5-2-bromovinyl-2' -deoxy-uridine (BVDU) and 9- (3-hydroxypropoxy)guanine. Further compounds include those disclosed in EP-A-0 409 575 and EP-A-0 427 777.
More preferably the combination therapy involves the administration of one of the above-mentioned agents and a compound within one of the preferred or more-preferred sub-groups within formula (I) as described above. Most preferably the combination therapy involves the joint use of one of the above named agents together with one of the compounds of formula (I) specifically named herein. The compounds of formula (I) may be produced by various methods known in the art of organic chemistry in general and nucleoside synthesis in particular. Starting materials are either known and readily available from commercial sources or may themselves be produced by known and conventional techniques.
The present invention further provides a process for producing a compound of formula (I) as hereinbefore defined which process comprises: A) reacting a compound of formula (II)
Figure imgf000021_0001
wherein X1 is a precursor for the group X as defined in relation to formula (I) ; Y and R2 are as defined in relation to formula (I) ;
R3a either forms a carbon-carbon bond with R2 or when R2 is H, R3a is hydrogen, hydroxy or a group OZ3 where Z3 is a hydroxyl protecting group; and
Z5 is hydrogen or a hydroxyl-protecting group, with a reagent or reagents serving to convert the group X1 to the desired group X;
B) reacting a compound of formula (III)
Figure imgf000021_0002
wherein X and Y are as defined in relation to formula (I) or a protected form thereof with a 4-thio sugar compound serving to introduce the 4-thio sugar moiety, or a protected form thereof, at the 1-poεition of the compound of formula (III) ; or C) reacting a compound of formula (IV)
Figure imgf000022_0001
wherein X and Y are as defined in relation to formula (I) , Z5 is a hydroxyl protecting group or hydrogen; R2 and R3a are as defined above wherein at least one of R3a and Z5 represents a precursor group for the group (s) R3 and/or R5 in formula (I) under conditions or with a reagent serving to convert the groups R3a and/or Z5 into the respective groups R3 and/or H.
Where necessary or desired, one or more of the following further steps may be additionally performed in any desired or necessary order: a) removing each of the protecting groups, b) converting a compound of formula (I) or a protected form thereof into a further compound of formula (I) or a protected form thereof, c) converting the compound of formula (I) or a protected form thereof into a physiologically acceptable derivative of the compound of formula (I) or a protected form thereof, d) converting a physiologically acceptable derivative of the compound of formula (I) or a protected form thereof into the compound of formula (I) or a protected form, thereof, e) converting a physiologically acceptable derivative of the compound of formula (I) or a protected form thereof into another physiologically acceptable derivative of the compound of formula (I) or a protected form thereof, f) performing an anomerisation reaction in order to convert an α-anomer of a compound of formula (I) into a 3-amoner or to convert a 3-anomer of a compound of formula (I) into an α- anomer, and g) where necessary, separating the a and β anomers of the compound of formula I or a protected derivative thereof or of a physiologically acceptable derivative of a compound of formula (I) or a derivative thereof.
The term "4-thio sugar compound" is used herein to denote a compound containing the 4-thio-L-ribofuranose ring wherein the 5-hydroxyl group thereof is optionally protected, the l-position is optionally substituted by a leaving group and the 2- and 3- positions thereof are either the groups R2 and R3 or a derivative thereof, the derivatives of R3 including protected hydroxyl groups. With regard to the protecting groups referred to herein, including the groups Z3 and Z5, it will be appreciated that the particular nature of such groups will be dependent on the identity and nature of the particular group(s) to be protected and will therefore be selected in accordance with conventional techniques. Examples of protecting groups that may be generally used include acyl groups. Acyl groups include for example Cw alkanoyl groups, e.g. acetyl, or aroyl groups, e.g. benzoyl optionally substituted with one or more CMalkyl (particularly methyl), halo, nitro or amino groups. Preferred substituted aroyl groups include toluoyl and p-nitrobenzoyl groups. Other protecting groups include ether groups such as tri-C^alkylsilyl (e.g. trimethylsilyl) or tert-butyl diphenylsilyl; or arylmethyl groups such as a triphenylmethyl group or a benzyl group optionally substituted on the phenyl ring by one or more halogen atoms, C1 alkyl eg. methyl, CM haloalkyl, CM alkoxy, nitro or amino groups.
Protection of hydroxy groups with trialkylsilyl, eg. trimethylsilyl, groups on the pyrimidine ring is conveniently achieved by reaction with (a) chlorotrimethylsilane together with triethylamine or with (b) hexamethyldisilazane, optionally together with chlorotrimethylsilane and/or ammonium sulphate.
The above groups may be removed in conventional manner, for example the acyl groups being removed advantageously under basic conditions (e.g. using sodium methoxide) , the silyl ether groups being removed advantageously under aqueous or acidic conditions (e.g. using aqueous methanol to remove trimethylsilyl groups) and the arylmethyl groups being removed advantageously under reducing conditions.
Process A may be effected by, for example, the means described below.
Process B may be effected, for example, by a) reaction of the compound of formula (III) , or a protected form thereof, with a 4-thio sugar compound of formula (V) :
Figure imgf000024_0001
wherein R2, R3a and Z5 are as defined above and W is a leaving group, for example halogen, e.g. chloro, acyloxy (e.g. Cw alkanoyloxy such as acetoxy) , or an optionally substituted S- benzyl group of the formula -S-CH2-Ar where Ar is an optionally substituted aryl group, for example optionally substituted phenyl or toluyl. Optional substituents of the aryl groups include one or more halogen atoms, CM alkyl eg. methyl, CM haloalkyl, CM alkoxy, nitro or amino groups. Ar is preferably a 4- methoxyphenyl group. In formula (V) , the groups R3a and Z5 are preferably hydroxyl protecting groups, particularly benzyl, toluoyl or p-nitrotoluoyl groups. The reaction may be performed using standard methods including the use of a Lewis Acid catalyst such as mercuric chloride or bromide or stannic chloride or trimethylsilyltrifluoromethane-sulphonate in solvents such as acetonitrile, 1-2 dichloroethane, dichloromethane, chloroform or toluene at reduced, ambient or elevated temperature such as from -78°C to reflux; or b) reaction of the compound of formula (III) , or a protected form thereof, with a compound of formula (VI)
Figure imgf000025_0001
wherein R2, R3a and Z5 are as defined above and Py represents a 0 pyrimidine base in the presence of a silylating agent such as
N,0-bis- (trimethylsilyl) acetamide and in the presence of a Lewis
Acid catalyst such as trimethylsilyltrifluoromethane sulphotonate in a solvent such as acetonitrile. In the compound of formula
(VI) , Py is preferably uracil or thymine.
15 The 4-thio-sugar compound of formula (V) may be produced by conventional methods prior to coupling with the base or derived by modification of another sugar moiety which is already part of a nucleoside. Particular methods are as described in the Examples.
20 The compound of formula (VI) may also be produced by coupling a compound of formula (V) with an appropriate base. Particular methods are as described in the Examples. For the preparation of compounds of the formula (I) the base (III) is preferably modified to protect the 2-carbonyl and 4-amine groups
25 by silylation. Suitable silylating agents include bis- (trimethylsilyl) acetamide. Silylation is conducted in a suitable solvent, for example acetonitrile. The reaction may be conducted at from about 20°C to 100°C and is preferably preformed at an elevated temperature, e.g. about 50° to 100°C, e.g. about
3080°C.
The compound of formula (V) is reacted with the protected base in the presence of a Lewis acid catalyst such as those mentioned above and, as a co-catalyst a N-halosuccinimide, eg. N- iodosuccinimide or N-bromosuccinimide. The Lewis acid is
35 preferably trimethylsilyltrifluoromethanesulphonate. The solvent may be any suitable solvent including chlorinated solvents such as chloroform, dichloromethane, 1-2-dichloromethane but is preferably acetonitrile.
The Lewis acid and N-halosuccinimide are preferably used in equimolar proportions, although a range of from 1:5 to 5:1 molar ratio may be used. Desirably, the ratio of Lewis acid to compound of formula (V) is 1:1.
When the nucleoside of formula (VI) is protected, it may be deprotected using standard de-esterification reactions, eg by reaction with a base (organic or inorganic) in a suitable solvent such as alcohol (eg. methanol, ethanol or propanol) . The final product will be an anomeric mixture which may be separated by standard techniques such as fractional crystallisation or chromatography.
Particular methods for producing the compounds of formula
(I) in accordance with the above processes will be described below and these may be combined in order to produce further compounds within formula (I) , in accordance with Process A above. Reference may be made to the following texts:
Synthetic Procedures in Nucleic Acid Chemistry, Eds. W.W. Zorbach R.S. Tipson, Vol. 1, Interscience, 1973;
Nucleic Acid Chemistry - Improved and New Synthetic Procedures, Methods and Techniques, Eds. L.B. Townsend and R.S.Tipson, Parts 1 and 2, Wiley-Interscience, 1978 and Part 3, Wiley-Interscience, 1986;
Nucleoside Analogues-Chemistry, Biology and Medical Applications Eds R.T Walker, E. De Clercq &. F. Eckstein, NATO Advanced Study Institutes Series, Plenum Press, 1979; Basic Principles in Nucleic Acid Chemistry, Eds. P.O.P Ts'O, Academic Press, 1974.
The following techniques are particularly convenient: X is haloσen
5-Halopyrimidines are commercially available and may be coupled to the 4-thiosugar compound by conventional techniques, for instance by reacting a protected 5-halopyrimidine with a protected 4-thio sugar compound having a leaving group in the 1- position. The leaving group on the 4-thio sugar compound may be a halogen, benzylthio or preferably acetate group. Reaction of the protected 4-thio sugar compound with the protected 5-halopyrimidine is conducted under conventional conditions using Lewis Acid catalysis such as by treatment with mercuric chloride or mercuric dibromide with cadmium carbonate or with stannic chloride, or preferably trimethylsilyl¬ trifluoromethane sulphonate in toluene, acetonitrile, dichloromethane or 1,2-dichloroethane, as solvent followed by treatment as necessary with aqueous methanol (which also serves to remove the protecting groups from any hydroxyls on the pyrimidine ring) .
Protecting groups may be removed by conventional techniques, for instance trimethylsilyl groups may be removed from hydroxyl groups on the pyrimidine ring by treatment with aqueous methanol, benzyl groups are removed from the hydroxyl groups on the 4-thio sugar compound by treatment with boron trichloride in dichloromethane at -78°C, and p-toluyl groups are removed from the hydroxyl groups on the sugar by treatment with sodium methoxide in methanol at room temperature. Alternatively the 5-halo substituent may be introduced into the pre-formed 5-unsubstituted 4' -thio-pyrimidine nucleosides having protected or unprotected hydroxyl groups. When the hydroxyl groups on the 4-thio sugar compound are protected (for instance with ethers such as silyl ethers or esters such as acetate, benzoate or p-toluate esters) , reaction with N- chlorosuccinimide in glacial acetic acid or with chlorine and iodobenzene and glacial acetic acid will introduce a 5-chloro substituent and reaction with iodine monochloride in dichloromethane will introduce a 5-iodo substituent, while reacting the unprotected 4' -thio sugar pyrimidine nucleoside with chlorine in carbon tetrachloride and acetic acid also introduces the 5-chloro substituent. Reaction with iodine and nitric acid also introduces the 5-iodo substituent. Reaction with bromine and acetic acid introduces a 5-bromo substituent to the unprotected nucleoside. Deprotection where necessary is by conventional techniques and is performed as the final step.
The 5-unsubstituted 4' -thionucleoside starting material of formula (II) (in which X1 = H) may be produced as described above by coupling a 5-unsubstituted pyrimidine to a 4-thio sugar compound. Protection of the hydroxy groups of the 4-thio sugar moiety may be effected at any convenient stage.
X is C-,6 alkvnyl 5-Alkynyl compounds may be produced by reacting a 5-iodo nucleoside of formula (II) wherein the hydroxyl groups of the 4- thio sugar are optionally protected (for instance by reaction of the unprotected nucleoside with p-toluoylchloride in pyridine to introduce p-toluoyl ester groups on the hydroxyl groups of the 4- thio sugar) with an appropriate alkynylating agent, for example trimethylsilyl acetylene or a terminal alkyne in the presence of a palladium catalyst such as bis (triphenylphosphine) palladium dichloride, and a copper catalyst such as cuprous iodide and triethylamine and, where necessary, removal of the protecting groups using sodium methoxide in methanol [c.f. M.J. Robins e_t al; Can. J. Chem.. £0:554 (1982)] .
Alternatively the 5-alkynyl group may be introduced by reacting a 5-iodo pyrimidine with trimethylsilyl-acetylene or a terminal alkyne in the presence of bis(triphenyl¬ phosphine)palladium dichloride, cuprous iodide, triethylamine and dimethylformamide followed, where necessary, by removal of the protecting groups and reacting the 5-alkynyl pyrimidine of formula (III) in suitably protected form (for instance the trimethylsilyl-protected form) with a protected 4-thio sugar compound as previously described followed by deprotection of the pyrimidine and sugar moieties as required.
X is C2 6 alkenyl
5-Alkenyl compounds may be produced by partial hydrogenation of the corresponding 5-alkynyl pyrimidine of formula (III) or of the nucleoside of formula (II) for instance using Lindlar catalyst poisoned with quinoline, and subsequently, in the case of the pyrimidine, coupling with a 4-thio sugar compound as described above. Alternatively a 5-iodo nucleoside of formula (II) may be reacted with an appropriate alkenylating agent for example a 2- alkenoic acid ester (for instance the methyl ester) in the presence of palladium (II) acetate and triphenylphosphine to form the 5- (2-methoxycarbonyl alkenyl) derivative. The ester group is then removed by hydrolysis using sodium hydroxide forming the 2- carboxy alkenyl compound which itself is subjected to treatment with triethylamine in dimethylformamide at 100°C to give the 5- vinyl analogue [c.f. S.G. Rahim et. al.. , Nucleic Acids Research. 10 (17) :5285(1982)1.
Yet another method for producing the 5-alkenyl compounds involves coupling the terminal alkene with a 5-iodo or 5- chloromercuri nucleoside of formula (II) (formed by for example reaction of the 5-unsubstituted nucleoside with mercury (II) acetate and sodium chloride) , in the presence of a palladium catalyst such as palladium (II) acetate and a copper salt such as copper (I) chloride, or preferably a palladium catalyst such as dilithiu palladium tetrachloride. Reaction of a 5-iodo- or 5- chloromercuri-nucleoside of formula (II) with allyl halides such as chloride or bromide in the presence of dilithium palladium tetrachloride leads to the formation of the corresponding 5- (alk- 2-enyl) derivative which can be rearranged to form the 5- (alk-1- e n y 1 ) derivat ive s by t reatment wi t h tris (triphenylphosphine)rhodium chloride. This process may also be applied to the free pyrimidine base, which is subsequently condensed with the 4-thio sugar compound. The above processes are exemplified by J.L. Ruth S. D.E. Bergstrom, J. Orσ. Chem, 43 (14) : 2870 (1978), J. Goodchild et al. , J. Med. Chem. 26: (1983), D.E. Bergstrom & J.L. Ruth, J. Am. Chem. Soc.. 98: 1587 (1976) and D.E. Bergstrom & M.K. Ogawa, J. Am. Chem. Soc.. 100: 8106 (1978).
X is Cϊ6 haloalkenyl 5- (Haloalkenyl) substituents may be introduced into a nucleoside of formula (II) by conventional methods. For example, in order to prepare 5- (2-halovinyl) compounds the corresponding 5- (2-carboxyvinyl) nucleoside is treated with an appropriate halogenating agent, for example N-halosuccinimide in aqueous potassium acetate, or with potassium carbonate in dimethylformamide when the halogen is bromo or iodo. A 5- (2- chlorovinyl) nucleoside may also be made from the corresponding 5- (2-carboxyvinyl) nucleoside using chlorine gas in, for example, dimethylformamide (DMF) . Alternatively the 5-haloalkenyl group may be introduced into the appropriate free pyrimidine base to form a compound of formula (III) which is subsequently coupled with a 4-thio compound as described above; this may be achieved for example by treating a 2,4-dimethoxy-protected 5-iodo-pyrimidine with an 2- alkenoic acid ester in the presence of palladium (II) acetate, triphenylphosphine and dioxane followed by removal of the methoxy protecting groups, hydrolysis of the ester with sodium hydroxide and reaction of the resulting 5- (2-carboxyviny1) derivative with N-halosuccinimide (where halo is bromo or iodo) or chlorine gas (where halo is chloro) in the presence of a base such as sodium hydrogen carbonate in dimethylformamide. The 5- (2-carboxyvinyl) compound may also be produced by treating an unprotected 5- (hydroxymethyl)pyrimidine of formula (III) with an oxidising agent such as persulphate or manganese dioxide to form the corresponding aldehyde and followed by treatment of the aldehyde with malonic acid. The above processes are exemplifed by A.S. Jones et al, Tetrahedron Letts, 45; 4415 (1979) and P.J. Barr et al. J. Chem. Soc. Perkin Trans 1, 1981, 1665.
The 5- (2-haloalkenyl) base may alternatively be made by a novel route starting with a 2,4-dimethoxy protected 5- bromopyri idine. This may be converted to the corresponding 5- lithium derivative by treatment with an organolithium reagent, preferably n-butyllithium at reduced temperature such as -70°C in an ethereal solvent such as diethylether. Reaction of the lithio derivative j-n situ with an appropriate ester of formic acid, such as ethyl formate at reduced temperature such as -70°C gives rise to the corresponding 5-formyl compound. Treatment of the formyl compound with malonic acid as described above gives rise to the 5- (2-carboxyvinyl) derivative. Similar halogenation gives rise to the required 5- (2-haloalkenyl) compound which is in the 2,4- dimethoxy protected from. Deprotection can then be carried out by conventional techniques.
5-Halovinyl compounds having more than one halogen substituent may be produced from a 5-halo-substitued 2,4- dimethoxy protected pyrimidine of formula (III) by reaction with a strong base such as butyl lithium and the resulting lithio derivative treated with the appropriate haloalkene followed by removal of the protecting groups and coupling to the 4-thio sugar compound as described above [c.f. P.L. Coe et. al. , J. Med. Chem. 2^:1329 (1982) ] . Alternatively, the halogen atoms may be introduced sequentially into a 5-substituent of the pyrimidine base. Thus, for example treatment of 5-acetyl uracil with a chlorinating agent such as phosphorus oxychloride provides the 5- (1- chlorovinyl) group with simultaneous chlorination of the hydroxyl groups of the pyrimidine base. Treatment with potassium ethoxide then hydrogen chloride and finally bromine leads to bromination of the 5-unsaturated side chain of the pyrimidine base with simultaneous conversion of the 2,4-dichloro groups on the pyrimidine ring to form the corresponding uracil derivative. The resulting pyrimidine base can then be coupled to the 4-thio sugar compound as described above [c.f. P.J. Barr et, al. Nucleic Acids Res. 3_: 2845 (1976) and P.J. Barr et al. , J. Chem. Soc. Perkin Trans 1. 1981: 1665] .
X is C?f alkyl
5-C2.6 Alkyl eg. 5-ethyl substituted nucleosides may be produced by hydrogenation of the corresponding 5-alkynyl or 5- alkenyl pyrimidine base followed by coupling to the 4-thio sugar compound. Conventional hydrogenation conditions, such as hydrogen over palladium/charcoal catalysts, may be adopted.
X is trifluoromethyl
5-Trifluoromethyl uracil is commercially available and this may be condensed with a 4-thio sugar compound in accordance with process B described above. The 5-trifluoromethyl cytosine analogue may be made from the uracil compound/using an analogous procedure to that described by Sung as mentioned below.
X is 5-haloalkyl
5-Fluoroalkyl substituents may be generated from the corresponding 5-hydroxyalkyl substituents, preferably starting from nucleosides having protected sugar hydroxyl groups on the 4- thio sugar moiety. Suitable protecting groups include tert-butyl diphenylsilyloxy groups which may be introduced using tert- butyldiphenylsilylchloride. The protected 5-hydroxyalkyl nucleoside is treated with a fluorinating agent such as diethylaminosulphurtrifluoride followed by deprotection of the hydroxyl groups using tetra-n-butylammonium fluoride to give the monofluoroalkyl derivative. Alternatively treatment of the 4'- thio sugar- protected 5-hydroxyalkyl nucleoside with manganese dioxide or pyridinium dichromate produces the corresponding aldehyde which may be treated with diethylamino- sulphur trifluoride. Treatment with tetra-n-butylammonium fluoride removes the protecting groups and liberates the 5-difluoroalkyl derivative.
Other 5-haloalkyl, for example haloethyl, substituents may also be generated using the corresponding 5-hydroxyalkyl substituents. The 5-hydroxyalkyl compound, in the form of either a base or a nucleoside is reacted with carbon tetrachloride and triphenylphosphate to introduce a chloro substituent, or with N- bromosuccinimide and triphenylphosphate to introduce a bromo substituent, or with N-bromosuccinimide, triphenylphosphine and tetrabutyl-ammonium iodide to introduce an iodo substituent [c.f. J.D. Fissekis & F. Sweet, J. Org. Chem. 1973, 3_8, 264, and WO84/00759] .
The above 5-hydroxyalkyl nucleoside starting materials where the alkyl group is a methylene are obtained from the corresponding 5-methyl-nucleosides by protection (for instance using tert-butyldiphenylsilylchloride) of the hydroxyl groups of the 4-thio sugar moiety, photolytic bromination (for instance, using bromine, N-bromosuccinimide in carbon tetrachloride) and hydrolysis of the bromoalkyl side chain using sodium bicarbonate.
X is nitro or optionally substituted amino
Nitro-substituents are introduced at the 5-position of the 5-unsubstituted 4'thio-nucleosides by reaction with a nitrating agent for example nitronium tetrafluoroborate (N02BF4) , and these may be reduced using hydrogen over palladium/charcoal or tin (II) chloride to provide the corresponding amino substituent. [c.f. G- F. Huang and P.F. Torrence, J. Org. Chem. , 42 : 3821 (1977)] . 5- Nitro-substituted pyrimidines are readily available and may be coupled with 4-thio sugar compounds as described above. 5-Alkylamino and 5-dialkylamino substituents may be introduced by reacting a suitably protected 5-iodo-nucleoside with a corresponding alkylamine or dialkylamine. Protection is preferably by acylation for example by acetylation using acetic anhydride in pyridine.
X is alkoxy
Alkoxy substituents are introduced at the 5-position by reaction of the corresponding 5-hydroxy-4' -thio-nucleoside with a base, for example sodium hydroxide followed by alkylation with an appropriate alkyl halide in a suitable solvent such as methanol. The starting 5-hydroxy 4' -thio-nucleoside may be obtained from the 5-unsubstituted 4' -thio-nucleoside by treatment with bromine in an aqueous solvent such as aqueous tetrahydrofuran followed by treatment with base such as trimethylamine.
Alternatively, alkoxy substituents may be introduced by treatment of the corresponding 5-iodo-4' -thionucleoside with an alkoxylating agent such as sodium alkoxide in an appropriate solvent such as methanol or dimethylformamide or the corresponding alkanol.
X is cyano
Cyano substituents are introduced at the 5-position by reaction of the corresponding 5-iodo 4' -thio-nucleoside with potassium cyanide in the presence of potassium acetate in a suitable solvent such as dimethylformamide, preferably at elevated temperature, for example 80°C-120°C, preferably 100°C
[c.f. P.F. Torrence & B. Bhoosham J. Med. Chem.. 20, 974 (1977)].
X is thiocyanate. alkylthio, mercapto
Compounds of the formula (I) with these substituents may be prepared from a suitably protected 5-halogen-nucleoside (for example bromo or iodo) by methods analogous to those described in Lin et al. J Med Chem, 3JL, 336-340 1988. Methods for the synthesis of a 5-halogen-nucleoside are described in the above mentioned specifications.
X is hvdroxy
5-hydroxy-4' -thio-nucleosides may be prepared by the method described above in connection with the preparation of alkoxy compounds. The starting compound, 5-unsubstituted 4'-thio- nucleoside may conveniently be prepared by condensation of the appropriate sugar moiety with commercially available uracil.
X is hvdroxy-C13alkyl. These compounds may be prepared as described above in connection with the preparation of haloalkyl compounds. Hydroxymethyl uracil itself is commercially available.
X is C,<alkoxyC,7 alkyl or C /.alkylthiomethyl.
Compounds in which X is alkoxymethyl or alkylthiomethyl may be made starting from bases in which the group X is of the formula -CH20H. The alkoxymethyl compounds may be made by reacting this starting material with an appropriate alkanol group in the presence of an acid catalyst or an acidic ion exchange resin. The alkylthiomethyl compounds may be made in a similar way but using the appropriate alkylmercaptan group or an appropriate metal salt thereof. The resulting base may be condensed with the desired 4-thio sugar as described herein.
The corresponding alkoxyethyl compounds may be made in an analogous manner starting from the appropriate base in which the group X is hydroxyethyl. These starting bases are either commercially available or may be made as described above in connection with the preparation of haloalkyl compounds.
Alternatively these alkoxyalkyl and alkylthiomethyl compounds may be made from nucleosides of the formula I or a protected derivate thereof in which the group X is -CH2L where L is a leaving group, eg halo such as bromo, or alkyl or arylsulphonyloxy such as trifluoromethanesulphonyl or p- toluenesulphonyl or a secondary acyclic or cyclic amino group, such as dimethylamino or pyrrolidinyl. The reaction is carried out by treatment of one of these with a suitable nucleophilic reagent serving to introduce the O-alkyl or S-alkyl group, eg. alkyl-OH, alkyl-SH or alkyl-SM where M is a metal ion such as sodium. The procedure may be performed using methods analogous to those described by Barwolff and Langen, Nucleic Acid Chemistry - Improved and New Synthetic Procedures, Methods and Techniques, Part 1, Eds. L.B. Townsend and R.S. Tipson, p359. The above reference also describes the procedures which may be utilised to make compounds in which L is OH. Such compounds may be used to make compound where L is O-alkyl or S-alkyl using the procedures described above. The compounds where L is dimethylamino or pyrrolidinyl may be prepared by methods analogous to those described by Badman and Reese in J. Chem. Soc. Commun. 1987, 1732-1734 and by Jones et al, Synthesis 1982, 259-260.
X is formyl Compounds of formula I wherein X is formyl are typically prepared from the corresponding compound wherein X is CH2L, and L is Br. The latter compound may be prepared by methods analogous to those described by Barwolff and Langen, Nucleic Acid Chemistry - Improved and New Synthetic Procedures, Methods and Techniques, Part 1, Eds. L.B. Townsend and R.S. Tipson, p359.
Sugar moieties
The 4-thio-sugar compound may be produced by conventional methods prior to coupling with the base or derived by modification of another sugar moiety which is already part of a nucleoside. When the compounds of formula I are produced in accordance with process (C) , the process may be carried out using the following procedures to prepare compounds of formula (I) in which R2 and R3 have the following meanings include:-
a) R2 is hydrogen and R3 is hydroxy.
The synthesis of the 2' -deoxy sugar moiety may be conducted in accordance with the methods disclosed in the examples which follow. Reference may also be made to the methods described in M.J. Robins, T.A.Khwaja, R.K. Robins, J. Org. Chem., 1970, 35(3) 636.
b) R2 and R3 together form a carbon-carbon bond.
Such compounds may be prepared from a corresponding 3'5' , - anhydro compound for example by treatment with a strong base eg. potassium ter -butoxide. Such 3' ,5' -anhydro compounds may be prepared by treating the corresponding 3 ' , 5' -methanesulphonate diester with a base. The 3' 5' -methanesulphonate diester may be obtained by esterification of the 2-deoxy-L-ribose sugar which may be synthesised by analogous methods to those of Smejkal and Sor , (1964) , Nucleic Acids. Components and their Analogues, part Lii, volume 29, 809.; Genu-Dellac e_t al. , Nucleosides and Nucleotides, J-0. (6) , 1345-1376, (1991) ; Robins et al. J. Org. Chem. 3_5 (3) , 636-639 (1970) , ; and Schimmel &. Bevill, Analytical Biochemistry 3_7, 385-394, (1970) .
c) R2 and R3 are both hydrogen.
The 2,3-dideoxy-4-thio-L-ribonucleosides may be obtained by a method analogous to that disclosed by E.J. Prisbe and J.C. Martin (Synth. Commun. (1985), 15.(4) , 401-409) , which describes the synthesis of D-dideoxy nucleosides from 2-deoxy nucleosides, or by a method analogous to J.A. Secrist et. al (J. Med. Chem. (1992) 3.5,533-539) which discloses the synthesis of D-dideoxy-4- thionucleosides from L-glutamic acid. The use of D-glutamic acid (Aldrich Chemical Company Ltd) will result in the synthesis of dideoxy-4-thio-L-nucleosides. The above reactions are all suitable for producing uracil nucleosides; most of such reactions may also be used to form cytosine nucleosides. When this is not convenient or possible, cytosine analogues can be prepared most conveniently from the uracil compounds using an analogous procedure to that described by W.L. Sung, J. Chem. Soc. Chem. Commum. , 1981, 1089] : for example the acetylated uracil nucleoside (produced for instance by reactions as described above and acetylated using acetic anhydride in pyridine) is treated with p-chlorophenyl- phosphorodichloridate, 1,2,4-triazole and pyridine to produce the 4- (1,2,4-triazol-l-yl) derivative which is then treated with ammonia in dioxane (which also removes the 4-thio sugar protecting group(s) ) to form the corresponding unprotected cytosine 4' -thionucleoside.
The derivatives of the compounds of formula (I) may be prepared in conventional manner. For example, esters may be prepared by treating a compound of formula (I) with an appropriate esterifying agent, for example, an acyl halide or anhydride. Salts may be prepared by treating a compound of formula (I) with an appropriate base, for example an alkali metal, alkaline earth metal or ammonium hydroxide, or where necessary, an appropirate acid, such as hydrochloric acid or an acetate, eg. sodium acetate.
The anomers of compounds of the formula (I) may be separated by conventional means, for example by chromatography or fractional crystallisation.
In a further aspect of the invention, compounds of the formula (V) in which in which R2 is hydrogen, R3a is OZ3 and W is a group -S-CH2-Ar as defined above may be made by ring closure of a compound of the formula (VII)
Ar-CH2-S. ^S-CH,-Ar CH
CH2 (VII)
Z30-CH I
HC-O-A
CH2OZ5 where Z3 and Z5 are hydroxyl protecting groups as defined above, for example optionally substituted benzyl or acyl groups as defined above. Preferably the groups Z3 and Z5 are acyl groups. The group A is a leaving group, for example an organosulphonyl group such as an optionally substituted alkyl- or aryl-sulphonyl group, for instance methanesulphonyl, a haloalkylsulphonyl group (eg. trifluoromethylsulphonyl) and optionally substituted phenyl- sulphonyl (eg. toluylsulphonyl or bromobenzenesulphonyl) , and Ar is as defined above. A is preferably a methanesulphonyl group.
The ring closure may be performed under appropriate basic conditions. Suitable conditions include those described by J. Harness and N.A. Hughes (Chem. Comm. 1971, 811) , which includes the use of sodium iodide and barium carbonate.
Preferably the reaction is carried out in a solvent such as acetone or dimethylformamide (DMF) . DMF is preferred. The use of sodium iodide to debenzylate the intermediate arylthio cation is also preferred. Triethylamine may also be used as an alternative base.
The compound of the formula (V) in which R2 is hydrogen, R3a is OZ3 and W is -S-CH2-Ar may be converted to a compound of formula (V) where W represents other leaving groups by techniques known in the art, for example those disclosed in EP-A-0 409 575. For example, the compounds may be reacted with an appropriate acylating agent such as acetic anhydride (optionally in the presence of acetic acid) , in the presence of a mineral acid such as sulphuric acid. This will provide compounds in which the group W is acyloxy, which is a suitable leaving group for the reactions described herein. The acyloxy group may be converted to other leaving groups, eg. halo groups, using known methods.
The compound of the formula (VII) may be made from a compound of formula (VIII) Ar-CH2-S.. S-CH2-Ar
CH
I (VIII)
CH2 Z30-CH
I
HC-O-H CH2OZ5 where Ar, Z3 and Zs are as defined above. Preferably the groups Z3 and Z5 are acyl groups. Conversion of a compound of formula (VIII) to a compound of formula (VII) is carried out according to standard procedures such as treatment with an appropriate optionally substituted alkyl- or aryl-sulphonyl halide, eg. methanesulphonylchloride in a basic solvent such as pyridine. Other suitable alkyl or aryl sulphonyl halides include trifluoro- methanesulphonyl chloride and p-toluene-sulphonyl chloride. The compound of formula (VIII) may be made from a compound of formula (IX) : r-CHj S-CH2-Ar
(IX)
Figure imgf000039_0001
CH2OZ5 where Ar, Z3 and Z5 are as defined above and M is a hydroxyl protecting group which may be removed under conditions which leave the -S-CH2-Ar groups and the groups Z3 and Z5 in place.
Preferably, the group M is a group of the formula Ar**-CO- where Ar1 is a phenyl group which may be optionally substituted by any of the substituents described above for the group Ar.
Removal of this group M may be performed under standard conditions, for example with a base such as an alkali metal alkoxide, for instance sodium methoxide in methanol.
The compounds of formula (IX) may be obtained by the concomitant inversion and derivatization of the 4-hydroxy group of a compound of formula (X) :
(X)
Figure imgf000039_0002
wherein Ar, Z3 and Z5 are as defined above. The inversion and derivatization may be effected by reacting the compound of formula (X) with a derivative of the group M, such as an acid of the formula Ar'-COOH, for example benzoic acid (or a reactive derivative thereof) where Ar1 is as defined above. The reaction is performed typically at room temperature and under neutral conditions in a suitable polar solvent, for instance tetrahydrofuran. Preferably the Mitsunobu reaction is used for the inversion and derivatization; diethyl azodicarboxylate (DEAD) and triphenylphosphine are used as coreactants together with the acid Ar'COOH.
The compound of formula (X) may be made from a glycoside compound of formula (XI)
Figure imgf000040_0001
where Z3 and Z5 are as defined above and R is a hydrocarbyl group such as a CM hydrocarbyl group, eg. a CM alkyl group, preferably methyl. The compound of the formula (X) is reacted under acid conditions at an elevated temperature with a compound of formula Ar-CH2-SH, where Ar is as defined above. Suitably the reaction is' performed in the presence of hydrochloric acid which may be in aqueous or anhydrous form. Preferably the elevated temperature is from 30°C to 60°C, for example 40°C. When Ar is a phenyl group, the compound Ar-CH2-SH will be benzyl thiol .
Compounds of the formula (XI) may be made from a compound of formula (XII)
Figure imgf000040_0002
where R is a defined above. The hydroxyl groups of the compound of formula (XII) are protected under conventional conditions with the reactive derivative of the groups Z3 and Z5. Suitably, the bromo derivative may be used. Thus when Z3 and Z5 are benzyl groups, benzyl bromide may be used. The reaction may be performed in an organic solvent such as tetrahydrofuran in the presence of a suitable base such as sodium hydride and a phase transfer catalyst such as tetrabutylammonium iodide.
Compounds of the formula (XII) may be made by standard techniques from 2-Deoxy-L-ribose, which can be made by methods described in .J. Robins, T.A.Khwaja, R.K. Robins, J. Org. Chem., 1970, 35(3) 636. 2-Deoxy-L-ribose may be reacted with an alcohol of formula R-OH (where R is as defined above) in the presence of an acid. Hydrochloric acid is suitable. When R is a methyl group, the alcohol R-OH will be methanol.
The conversion of 2-deoxy-L-ribose to a compound of formula (XII) will also produce a small proportion of the corresponding pyranoside compound, substituted at the l-position by the group - OR. This may remain in the reaction mixture during the converions of (XI) to (X) , (X) to (IX) and the subsequent reactions described above and it will undergo analogous reactions. These by-products may be separated at any convenient step by conventional means, eg. chromatography.
The compound of the formula (VIII) may also be made directly from the compound of formula (X) using a Mitsunobu reaction under conditions analogous to those described by D.R. Williams et al, JACS (1990) 112, 4552.
Compounds of the formula (VIII) may also be made by reaction of a compound of the formula (XIII)
Figure imgf000041_0001
where R, Z3 and Z~ are as defined for a compound of formula (XI) ; with a compound of the formula Ar-CH2-SH where Ar is defined above. The reaction may be conducted using similar conditions to those described above for the preparation of the compound of the formula (X) . The reaction is performed in the presence of an acid, for example an inorganic acid such as HCl or a Lewis acid such as TiCl4. TiCl4 is preferred.
The groups Z3 and Z5 are preferably acyl groups, in particular p-nitrobenzoyl groups. A compound of the formula Ar-CH2-SH which is preferred include p-methoxybenzyl mercaptan. When compounds of the formula (I) are made using a synthetic route which includes the production of compounds of formula (VIII) from compounds of formula (XIII) , this substituent is preferred, desirably in conjunction with the groups Z3 and Z5 being p-nitrobenzoyl.
Compounds of the formula (XIII) may be prepared from compound of the formula (XIV)
Figure imgf000042_0001
where R is as defined above, using a Mitsunobu reaction to protect the 3' and 5' hydroxy groups and to invert the 3-hydroxyl of the ribo-sugar ring at the same time. The compound of formula
(XIV) is reacted with a compound or compounds of formula ZnOH where Zn is Z3 and/or Z5. Desirably Z3 and Z5 will be the same and a single compound Zn0H may be used. If different values of Z3 and Z5 are required, then the required mixture of compounds of ZOH may be used, and the desired reaction products separated from the resulting reaction mixture. Preferred compounds of the formula ZnOH are those where Zn is an acyl group as defined above. Preferably, Zn0H is p-nitro- benzoic acid, although other benzoic acids may also be used.
The reaction is performed in the presence of an azido- carboxylate such as diethylazidodicarboxylate or preferably diisopropylazidodicarboxylate.' The solvent for the reaction may be DMF, tetrahydrofuran, dichloromethane or toluene. Toluene is preferred. The reaction may be performed at room temperature.
When compounds of the formula (I) are made starting from compounds of the formula (XIV) , it is preferred that Z3 and Z5 are both p-nitrobenzyl.
Compounds of the formula (XIV) may be made from 2- deoxyribose using techniques known in the art, for example as described above in connection with the production of compounds of formula (XII) . Compounds of the formula (I) may also be made by reaction of a compound of formula (III) with a compound of formula (XV)
Figure imgf000043_0001
where L is a leaving group, for example, an acyloxy group such as C alkanoyloxy, for instance, acetoxy; P1 is a protecting group or hydrogen, and Z is a directing group.
Suitable groups P1 include groups such that P'O is an ether group, e.g. a silyl ether group (such as tertbutyldiphenylsilyl ether or tertbutyldimethylsilyl ether) , a straight or branch chain alkyl ether group, a cyclic ether group (such as tetrahydropyran-2-yl ether) or an optionally substituted aryl ether group (such as benzyl ether, trityl ether or benzhydryl ether) . The group P'O- can also be an ester group e.g. wherein P1 is QC=0 where Q is alkyl (such as methyl) , cycloalkyl or an optionally substituted aryl . Suitable groups Z include sulphenyl groups and selenyl groups. The preferred group Z is phenylse1enyl .
The reaction of the compound of formula (XV) with the base of formula (III) may be carried out for example in the presence of nonafluorobutane sulphonic acid or a Lewis acid catalyst, e.g. tin (IV) chloride, a mercury (II) salt or trimethylsilyl triflate. The reaction can be carried out, for example, at a temperature of from 0°C to room temperature in a suitable solvent such as acetonitrile or a chloroalkane.
Following reaction of a compound of formula (XV) with a compound of formula (III) , the group Z may be eliminated to provide a compound of formula (I) in which R2 and R3 together form a carbon-carbon bond. The protecting group P1 may be removed either before or after elimination of Z.
For example when Z is phenylselenyl it may be eliminated under oxidising conditions which are capable of oxidising selenium without oxidising sulphur, e.g. by treatment with m- chloroperbenzoic acid in dichloromethane at -20°C (Toru et al , Tetrahedron Letters, 1986, 22; 1583) . Compounds of the formula (I) in which R2 and R3 are both hydrogen may be made by elimination of the group Z (from the reaction product of (XV) and (III) ) under reducing conditions, e.g. using tributyltin.'hydride and triethyl borane (see Nozaki et al , Tetrahedron Letters-,; 1988, 23; 6125) .
Reference may also be made to EP-A-514 036 which describes the production of D-thionucleosides from, inter-alia , the isomer of the compound of formula (XV) which has the D-configuration. The reactions described in EP-A-514 036, the contents of which are incorporated herein by reference, may be applied to the production of compounds of formula (I) .
Compounds of the formula (XV) may be made by acylation under standard conditions (eg. acetic anhydride with pyridine) of a compound of formula (XVI) :
Figure imgf000044_0001
in which P1 and Z are as defined above.
The compound of formula (XVI) may be made from a compound of formula (XVII) ,
Figure imgf000044_0002
in which P1 and 2 are as defined above, by reduction of the carbonyl group of (XV) , for example using conventional reagents such as diisobutylaluminium hydride.
Compounds of the formula (XVII) may be prepared by methods analogous to those described in EP-A-514 036 but starting from R- (+) -glycidol instead of the (S) - (-) -enatiomer described in this reference. Example 13 also describes the preparation of 4-(R)-
(tert-butyl-diphenylsiloxy-methyl) -2- (S) -phenylselenyl-4-thio- butanoluctone, and those of skill in the art may refer to Example
13 for the preparation, by analogous methods, of other compounds of the formula (XVII) . The anomerisation of a- or 0-anomers of compounds of the formula (I) into β- or α-anomers respectively may be perfomed using known techniques, see for example Yamaguchi, T. and Saneyoshi, M. , Chem. Pharm. Bull. __ 2 , 1441-1450 (1984) vol. 4. The process may also be conducted by the use of an anhydride in the presence of a strong acid, as disclosed in British Patent application 9218737.6, in the name of the University of Birmingham, filed on 4th September 1992.
The invention is illustrated by the following Examples.
Example A
Preparation of Methyl 3 ,5-di-0-benzyl-2-deoxy-L-ervthro- pentoside
To a solution of 2-deoxy-L-ribose (50g, 373 mmol) in dry methanol
(900ml) is added a 1% solution of dry hydrogen chloride in methanol (100ml) . The mixture is kept in a stoppered flask for 30 minutes after which the reaction is stopped by adding, with vigorous stirring, silver carbonate (lOg) . The mixture is filtered by gravity and the colourless filtrate evaporated to a syrup using a dry rotary evaporator. Residual methanol is then removed by repeated evaporation with dry THF. The syrup is then dissolved in dry THF (470ml) . Under an atmosphere of dry nitrogen, at 0°C, with stirring sodium hydride in a 50% oil- dispersion (39.4g, 821 mmol) is slowly added to the THF mixture. Next, dry tetrabutylammonium iodide (30.3g, 82.1 mmol) is added followed by benzyl bromide (140g, 821 mmol) , which is added over 1 hour. The THF is removed in vacuo, the residue dissolved in dichloromethane and then poured into ice/water. The dichloromethane solution is extracted from this mixture and then dried over magnesium sulphate. The dichloromethane is evaporated under reduced pressure and the resulting residue applied to a silica gel column eluted with hexane-ethyl acetate (4:1). Combination of the appropriate fractions gives the a and β isomers of the title product as a syrup.
Preparation of 3,5-di-0-benzyl-2-deoxy-L-ervthro-pentose dibenzyl dithioacetal
Concentrated hydrochloric acid (150ml) is added dropwise to a stirred mixture of methyl 3,5-di-.0-benzyl-2-deoxy L-erythro- pentoside (77.5g, 236 mmol) and benzyl thiol (147g, 1.19 mol) at room temperature. The temperature is then raised to 40°C and the mixture stirred for 18 hours. The mixture is dissolved in chloroform, poured into ice/water, neutralised with sodium hydrogen carbonate and extracted with chloroform. The chloroform extracts are dried over magnesium sulphate and the chloroform evaporated under reduced pressure. The residue is applied to a silica gel column and eluted with hexane-ethyl acetate (4:1) to give the title product.
Preparation of 4-0-benzoyl-3 ,5-di-0-benzyl-2-deoχy-D-threo- pentose dibenzyl dithioacetal To a solution of 3,5-di-0-benzvl-2-deoxy-L-ervthro- pentose dibenzyl dithioacetal (54.lg,99.3 mmol) ,triphenyl- phosphine (39.lg, 149 mmol) and benzoic acid (18.2g, 149 mmol) in dry THF (800ml) is added a solution of DEAD (26.Og, 149 mmol) in dry THF (200ml) dropwise, with stirring, at room temperature for 18 hours. The THF is removed in vacuo and the residue applied to a silica gel column eluted with hexane-ethyl acetate (85:15). Combination of the appropriate fractions gives the title product.
Preparation of 3.5-di-0-benzyl-2-deoxy-D-threo-pentose -dibenzyl dithioacetal
To a solution of 4-0.-benzoyl-3,5-di-0.-benzyl-
2-deoxy-D-threo-pentose dibenzyl dithioacetal (88.8g, 137 mmol) in dichloromethane (500ml) is added a solution of sodium methoxide (11.lg, 206 mmol) in methanol (205ml) dropwise, with stirring, at 0°C. The reaction mixture is allowed to warm to room temperature over a period of 3 hours. The mixture is then poured into a 5% solution of NaH2P04 and extracted with dichloromethane. The dichloromethane extracts are then washed with a 5% solution of sodium hydrogen carbonate and water, dried
(magnesium sulphate) and evaporated. The crude title product is applied to a silica gel column eluted with hexane-ethyl acetate (4:1) . Combination of the appropriate fractions gives the title product.
Preparation of 3 , 5-di-0-benzyl-2-deoxy-4-0-methane-sulphonyl-D- threo-pentose dibenzyl dithioacetal
To a solution of 3,5-di-.0-benzyl-2-deoxy-D-threo-pentose dibenzyl dithioacetal (61.4g, 113 mmol) in dry pyridine (700ml) is added methanesulphonyl chloride (19.4g, 169 mmol) in dry pyridine (200ml) dropwise, with stirring, at 0°C. The temperature of the mixture is raised to room temperature and stirring continues for 18 hours. The pyridine is then removed in vacuo and the residue dissolved in dichloromethane. The dichloromethane extracts are then successively washed with 2M hydrochloric acid, 1M sodium carbonate and water, dried (magnesium sulphate) and evaporated to give the title product.
Preparation of benzyl 3,5-di-0-benzyl-2-deoxy-l.4-dithio- L-ervthro-pentofuranoside
A suspension of 3 , 5-di-0.-benzyl-2-deoxy-4-0- methanesulphonyl D-threo-pentose dibenzyl dithioacetal (29.4g, 47.4 mmol), sodium iodide (74.Og, 494 mmol), barium carbonate
(148g, 750 mmol) and dry acetone (1L) is boiled under reflux for
42 hours. At the end of this time the suspension is filtered and the solids washed with chloroform. The filtrate is sequentially washed with water, sodium thiosulphate solution (5%) and water, dried (magnesium sulphate) and evaporated. The resultant residue is applied to a silica gel column, eluted with hexane-ethyl acetate (9:1). Combination of the appropriate fractions gives the title product.
Preparation of 3' .5' -di-0-benzyl-4' -thio-L-thvmidine and its o.- anomer
A suspension of benzyl 3,5-di-.0-benzyl-2-deoxy-l,4-dithio-
L-ervthro-pentofuranoside (22.5g, 51.6 mmol), bis TMS-thymine
(46g, 170 mmol), mercuric bromide (20.5g, 56.7 mmol), cadmium carbonate (29.3g, 170 mmol) and dry toluene (1L) is boiled under reflux, with stiring, for 24 hours. The hot mixture is then filtered and the solids are washed with toluene. The filtrate is successively washed with potassium iodide solution (30%) and water and then evaporated. The residue is taken up in 4:1 methanol-water, stirred for 30 minutes, the suspension filtered and the filtrate evaporated. The residue is applied to a silica gel column (hexane-ethyl acetate (1:1)) and combination of the appropriate fractions gives the title product.
Preparation of β-4' -thio-L-thymidine To a 2M boron trichloride solution in dry dichloromethane
(55ml) cooled to -78°C, is added a solution of β-3' ,5' -di-O- benzyl-4' -thio-L-thymidine (1.6g, 3.7mmol) in dry dichloromethane
(30ml) . Stirring is continued for 5 hours at -78°C. This is followed by the dropwise addition of a 1:1 methanol- dichloromethane solution (200ml) over 40 minutes. The reaction mixture is allowed to warm to room temperature over 1 hour and the solvent removed in vacuo and coevaporated with dry methanol
(3 x 30 ml) . The residue is applied to a silica gel column eluted with chloroform-methanol (85:15) to give the title product.
Preparation of benzyl 2-deoxy-l,4-dithio-3,5-di-O-p-toluoyl-L- ervthro-pentofuranoside
To a 2M boron trichloride solution in dry dichloromethane
(150ml) cooled to -78°C, is added a solution of benzyl 3,5-di-O- benzyl-2-deoxy-1.4-dithio-L-ervthro -pentofuranoside (4.2g, lOmmol) in dry dichlormethane (100ml), dropwise, over 30 minutes.
Stirring is continued for 5 hours at -78°C. This is followed by the dropwise addition of a 1:1 methanol-dichloromethane solution
(200ml) over 40 minutes. The reaction mixture is allowed to warm to room temperature over 1 hour and the solvent removed in vacuo and coevaporated with dry methanol (3x30ml) . The crude residue is dissolved in dry pyridine (25ml) , cooled to 0°C, and a solution of p-toluoyl chloride (4.6g, 30 mmol) in dry pyridine
(25ml) added, dropwise, with stirring. The pyridine is removed in vacuo, the residue extracted with chloroform, and the extract successively washed with 2M hydrochloric acid, 1M sodium carbonate and water, dried (magnesium sulphate) and evaporated. The residue is applied to a silica gel column eluted with hexane- ethyl acetate (9:1) to give the title product.
Preparation of E-5 (2-bromovinyl-2' -deoxy-4' -thio-3' ,5'di-O- p-toluoyl-L-uridine and its α-anomer
To a solution of benzyl 2-deoxy-1,4-dithio-3,5-di-O- p-toluoyl-L-erythro-pentofuranoside (1.4g,2.8 mmol) in carbon tetrachloride (15ml) is added a solution of bromine (0.49g, 3.1mmol) in carbon tetrachloride (15ml) with stirring at room temperature. After 5 minutes the mixture is concentrated under diminished pressure and then carbon tetrachloride (5ml) added and the mixture evaporated to remove the excess bromine. The evaporation procedure is repeated four times. The resulting syrupy bromide is used directly in the next step. To a solution of the bromide in carbon tetrachloride (10ml) is added the bis TMS-derivative of E-5- (2-bromovinyl)uracil (1.7g, 4.7mmol) in carbon tetrachloride (10ml). The mixture is stirred until homogenous, evaporated and the residue heated for 1 hour at 90-100°C. The cooled, dark residue is dissolved in 4:1 methanol-water (30ml) , the solution boiled for 15 minutes under reflux and then evaporated. The residue is triturated with chloroform (40ml) and the solid 5- (2-bromovinyl) uracil that separates filtered off. The filtrate is successively washed with aqueous sodium hydrogen carbonate and water, dried (sodium sulphate) and evaporated. The residue is applied to a silica gel column eluted with hexane-ethyl acetate (3:2). Combination of the apporpirate fractions gives the title product.
EXAMPLE 1
Preparation of E-5- (2-bromovinyl) -2' -deoxy-4' -thio-β-L-uridine. E-5- (2-bromovinyl) -2' -deoxy-4' -thio-3, '5'-di-0-p_-toluoyl-/3- uridine (200mg, 0.34mmol) is dissolved in a solution of sodium methoxide in methanol (7.5ml, 0.1m) and the mixture is allowed to stand at 22°C for 24 hours. The solution is neutralised by careful addition of Dowex 50 ion exchange resin (H+form) to pH6. The resin is filtered off and washed with methanol and the filtrate and washings evaporated to a white solid. This is applied to a silica gel column eluted with chloroform-methanol (9:1) . Combination of the appropriate fractions gives E-5- (2- bromovinyl) -2' -deoxy-4 ' thio-/3-L-uridine which is crystallised from methanol-water.
EXAMPLE B
3 ' , 5' -Di-O-benzyl-2' -deoxy-5-iodo-4 ' -thio-L-uridine
Mercuric bromide (370 mg; 1.03 mmol) and cadmium carbonate (480 mg; 2.8 mmol) are added to a stirred solution, protected from moisture, of benzyl 3 , 5-di-0-benzyl-2-deoxy-l,4-dithio-L- erythro-pentofuranoside (436 mg; 1.0 mmol) in dry MeCN (3 ml) .
A solution of 5-iodo-bis-O-trimethylsilyluracil (3 mmol) in MeCN
(12 ml) is added via syringe. The progress of the reaction is monitored by analytical HPLC while the mixture is heated under reflux for 1 h. When cooled to ambient temperature, water (200 μl) is added and after stirring for 30 min. the suspension is filtered. The filtrate is evaporated and redissolved in dry MeCN, the precipitated 5-iodouracil is removed by filtration. The filtrate is purified by preparative HPLC on a 2.5 cm (1 in.) Zorbax C8 reverse phase column eluted at 20 ml min"1 with a gradient [0 - 95% MeCN-water containing a constant 0.2% trifluoroacetic acid] over 20 min.; half-minute fractions are collected. Fractions containing pure product are pooled.
EXAMPLE 2
2' -Deoxy-5-iodo-4' -thio-L-uridine The above product (240 mg; 0.44 mmol) dissolved in dry CH2C12 (10 ml + 2 ml rinse) is added over 30min to a stirred 1M solution of
BC13 in CH2C12 (18 ml, 18 mmol) at
-78°C under N2. The reaction is followed by analytical HPLC.
After 6h at -78°C, MeOH-CH2Cl2 (1:1, 18 ml) is added slowly and the mixture allowed to warm to ambient temperature then evaporated. The residue is re-evaporated from MeOH (3x) , taken up in MeOH-CHCl3 (1:1, 15ml) and the solid collected by filtration, yielding the desired β-anomer of the product.
EXAMPLE 3 2' -Deoxγ-5-ethyl-4'thio-L-uridine
Benzyl 3,5-di-0-benzyl-2-deoxy-1 , 4-dithio-D-erythro- pentofuranoside (5.6 mmol) is dissolved in CC14 (30 ml) and bromine (6.2 mmol) in CC14 (30 ml) is added. After stirring for 55 min. at ambient temperature the solvent is evaporated and the residue re-evaporated from CC14 (10 ml) to remove excess bromine. To a solution of this crude 1-bromo-thiosugar in CC14 (15 ml) is added bis-O-trimethylsilyl-5-ethyl uracil (16.6 mmol) [prepared by refluxing 5-ethyluracil (16.6 mmol) in a mixture of 0 hexamethyldisilazane (50 ml) and chlorotrimethylsilane (5 ml) for 2h., and evaporation of the solvents], HgBr2 (1.99 g; 5.5 mmol) and CdC03 (2.36 g; 16.6 mmol) . The solvent is evaporated and the residue heated at 100°C for lh. The residue is worked up as for the thymidine analogue and the product purified by column
15 chromatography. The benzyl ether protecting groups are removed by treatment with BC13 as described for the 5-iodo analogue. The a,β anomers are separeted by preparative reverse phase HPLC using MeCN.H20 as eluant.
EXAMPLE C 203'5' -Di-0-benzyl-5-bromo-2'deoxy'β-4' -thio-L-uridine.
This compound is prepared by a method similar to the iodo compound above with the following modifications:
1. The total solvent (MeCN) volume for the reaction is 3 ml.
2. The CdC03 is omitted.
253. The excess of 5-bromo-bis-O-trimethylsilyuracil is reduced to 1.5 mole equivalents.
EXAMPLE 4
5-Bromo-2' -deoxy-4' -thio-L-uridine
The BC13 deprotection of the compound of Example C is conducted 30 as for the iodo-compound.
EXAMPLE D l-Acetoxy-3.5-di-p-toluoyl-2-deoxy-4-thio-L-ervthro- pen ofuranoside
A solution of benzyl 3 , 5-di-0-benzyl-2-deoxy-l-4-dithio-L- 35 erythro-pentofuranoside (3.68 g; 8.76 mmol) in dry CH2CH2 (20ml) is added dropwise to a stirred 1M solution of BC13 in CH2C12 (125 ml; 0.125 ml; 0.125 mol) at 78°C under N2. The mixture is stirred at -78°C for 4.5h, then a mixture of MeOH-CH2Cl2(1 :1,v/v) is added slowly. After warming to room temperature the solvents are evaporated to give the crude O-debenzylated thiosugar. The gum is dissolved in dry pyridine at 0°C under N2 and a solution of p- toluoyl chloride (3.47 ml; 26.3 mmol) added slowly. The mixture is stirred at 0°C for 3 hours then the solvents are evaporated. The residue is dissolved in CH2C12, washed with 2M HCl, 1M Na2C03 and water, dried over MgS04 and evaporated. The residue is purified by flash chromatography on Si02 eluted with EtOAc-hexane
(1:9, v/v) to give the bis-toluoylthiosugar derivative. The product is dissolved in acetic anhydride (16 ml) and stirred at
0°C. Cone. H2S04 (8 μl) is added followed after 10 min. by a second aliquot (8 μl) ; the reaction is monitored by TLC. After a further 55 min. stirring NaHC03 (100 mg.) is added and after 20 min. the mixture is cautiously poured into ice-water containing NaHC03. The product is extracted into CH2C12, dried and evaporated. The residue is purified by flash chromatography on Si02 eluted with 20-25% EtOAc-hexane.
EXAMPLE 5
2' -Deoxy-5-prop-l-ynyl-4' -thio-L-uridine
5-Prop-l-ynyluracil (0.112 g; 0.75 mmol) is heated in hexamethyl¬ disilazane (3 ml) containing trimethylsilyl chloride (1 ml) until the solid dissolves (4h) . The solvents are evaporated and the residue dissolved in dry MeCN (6 ml) . The solution is added, under N2, to a stirred solution of the above thiosugar ester (0.2 g; 0.5 mmol) in MeCN (10 ml) at 0°C. Trimethylsilyl triflate
(0.096 ml; 0.5 mmol) is added and the mixture stirred for 15 min. The mixture is diluted with CH2C12 (20 ml) , poured into saturated aqueous NaHC03 and the organic layer separated. The aqueous layer is further extracted with CH2C12 and the combined organics dried and evaporated. Flash chromatography on Si02 eluted with EtOAc- hexane (3.2, v/v) gives the protected thionucleoside as a mixture of anomers contaminated with a little propynyluracil . This material (0.206 g; 0.397 mmol) is dissolved in MeOH (15 ml) containing NaOMe (0.021 g; 0.397 mmol) and the mixture kept at ambient temperature overnight. The solution is neutralised with Dowex 50 (H+) ion-exchange resin, filtered and the filtrate evaporated to dryness. The solid is washed with ether (3 x 4 ml) and digested with hot acetone to give the required product. Methanol is added to the mixture and the solid filtered off to give the β-anomer.
5-Prop-l-ynyluracil may be obtained from 5-iodouracil using the methodology analogous to that described by M.J. Robins et. al (ibid) .
EXAMPLE 6
2' -Deoxy-5-chloro-4' -thio-L-uridine.
Starting with 5-chlorouracil, this compound is prepared in a similar manner to that described in Example 5. 5-chlorouracil is commercially available. The compound is purified by HPLC as described above.
EXAMPLE 7
2' -Deoxy-5-trifluoromethyl-4' -thio-L-uridine:
Starting with 5-trifluoromethyluracil, this compound is prepared in a similar manner to that described in Example 5. 5- trifluoromethyluracil is commercially available. A sample of this compound of is obtained by trituration of the crude deprotected nucleoside mixture with acetone, filtration and evaporation.
EXAMPLE 8
2' -Deoxy-5-ethynyl-4' -thio-L-uridine:
Starting with 5-ethynyluracil, this compound is prepared in a similar manner to that described in Example 5. 5-ethynyluracil may be prepared from 5-ioduracil using the methodology analogous to that described by M.J. Robins et al (ibid) .
A sample of the pure β-anomer of this compound is obtained by boiling the crude anomer mixture with MeOH and filtering off the product. EXAMPLE 9
2' -Deoxy-5-E- (2-bromovinyl) -4' -thio-L-cytidine.
To a solution of benzyl 3,5-di-0-benzyl-2-deoxy-l,4-dithio-L- erythro-pentofuranose (4 g; 9.5 mmol) in acetic acid (50 ml) and acetic anhydride (50 ml) is added cone, sulphuric acid (50 μl) and the mixture stirred at ambient temperature for 30 min. The mixture is poured into excess sodium bicarbonate Na2HC03, extracted with CHC13, the extracts dried over MgS04 and evaporated. The residue is purified by column chromatography on Si02 in the TLC solvent to give the 1' -acetoxy-di-O- benzylthiosugar derivative which is used directly below. To the above derivative (0.33 g; 0.89 mmol) in dry CH2C12 (3 ml) at 0°C is added SnCl4 (0.33 g; 0.89 mmol) in dry CH2C12 (3 ml) and E-5- (2-bromovinyl) -2,4-dimethoxypyrimidine (0.218 g; 0.89 mmol) in dry CH2C12 (3 ml) . The stirred mixture is allowed to warm to ambient temperature and stirred for a further 4 h. The mixture is poured onto water, washed with saturated NaHC03 and dried over MgS04. After evaporation, the residue is chromatographed on Si02 in toluene-acetone (9:1, v/v) to give the β-anomer of the protected thionucleoside, which is crystallised from MeOH. The above methoxy derivative of the protected thionucleoside is converted to the cytidine analogue by dissolution in NH3/MeOH at ambient temperature for 2d. The product is isolated by column chromatography on Si02 eluted with CHCl3-MeOH (9:1, v/v), then deprotected directly with BC13. as described above.
EXAMPLE 10
2' -Deoxy-5-propyl-4'thio-L-uridine:
2' -Deoxy-5-propynyl-4'thio-L-uridine, β-anomer, (26 mg) and 5% Pd/C (40 mg) in MeOH (80 ml) is stirred in an atmosphere of hydrogen for 45 min. The mixture is filtered and evaporated to give a gum. Trituration with ether-hexane gives the product as a solid.
EXAMPLE 11
E-2' -Deoxy-5- (propen-1-γl) -4' -thio-L-uridine (a) 5-Allyluracil.
Uracil (1 g; 9 mmol) is dissolved in water (200 ml) at 70°C and Hg(0Ac)2 (2.9 g; 9.1 mmol) added. The mixture is stirred at 70°C for 1 week. After cooling to ambient temperature NaCl (1.5 g) is added and the mixture stirred for 4h. The resulting thick suspension of 5-chloromercury-uracil is filtered, the solid washed with 0.1 M NaCl solution and dried in vacuo at 85°C for 2 days. To the crude solid (1 g; 2.9 mmol) in MeCN (25 ml) is added Li2PdCl4 (0.76 g) and allyl chloride (2.9 ml) and the mixture stirred at ambient temperature for 1 week. The suspension is filtered and the filtrate evaporated to dryness. The residue is dissolved in MeOH (75 ml) and treated with H2S gas; a black precipitate of HgS is removed by filtration and the filtrate is evaporated to leave a white solid. The desired product is isolated by flash chromatography on Si02 eluted with 8% MeOH-CH2Cl2 (v/v) .
(b) 5- (E-propen-1-yl) uracil
To a solution of 5-allyluracil (80 mg; 0.5 mmol) in 95% aq. EtOH (50 ml) is added (Ph3P)3RhCl (90 mg; 0.1 mmol) and the mixture heated under reflux for 3 days. The solvent is evaporated and the product isolated by flash chromatography on Si02 eluted with 5% MeOH-CH2Cl2.
(c) E-2' -Deoxy-5- (propen-1-yl) -4' -thio-L-uridine 5- (E-propen-1-yl)uracil (110 mg; 0.78 mmol) is converted to the bis-TMS-ether, coupled with the protected thiosugar and deprotected with methoxide as described for the 5-propynyl analogue. The crude product is purified by chromatography on Si02 eluted with 5% MeOH-CH2Cl2.
EXAMPLE 11
5- (2-Chloroethyl) -2' -deoxy-4'thio-L-uridine
5- (2-chloroethyl)uracil (prepared by the method J.D. Fissekis & F. Sweet, J-Org. Chem., 1973, .28., 264(0.122 g, 0.7 mmol) is added to hexamethyldisilazane (3 ml) and chloromethylsilane (0.1 ml) and the mixture is heated at reflux for 2 hours. The mixture is evaporated to dryness and to the residue is added a solution of l-acetoxy-3 , 5-di-0-p-toluoyl-2-deoxy-4-thio-L- erythropentofuranoside (See Example D) (0.2 g, 0.46 mmol) in dry dichloromethane (10 ml) , the mixture is cooled to 0°C with stirring and trimethylsilyltrifluoromethanesulphonate (0.1ml) is added. After stirring at 0°C for 2 hours, dichloromethane (30 ml) is added and the reaction is quenched with a saturated solution of sodium bicarbonate (20 ml) . The aqueous phase is extracted with dichloromethane (2 x 25 ml) and the combined organic phases dried (Na2S04) and evaporated to dryness to give the crude product in the p-toluoyl protected form. This intermediate (0.23 g) is added to a solution of sodium methoxide (0.9 mmol) in dry methanol (20 ml) and the mixture stirred at room temperature overnight. The solution is neutralised with Dowex 50 (H+) resin, the resin filtered and washed with methanol. The combined filtrate and washings are evaporated to dryness, the residue partitioned between ether and water, the organic layer re-extracted with water and the combined aqueous phases evaporated to dryness. The residue is chromatographed on silica gel eluting with 7% methanol/dichloromethane, the product freeze dried from water to give the title compound.
EXAMPLE 12
2' -Deoxy-5-nitro-4' -thio-L-urdine
A solution of 5-nitrouracil-2,4-bis-trimethylsilyl ether [from 5- nitrouracil (118 mg, 0.75 mmol) and hexamethyldisilazide (3 ml)- chlorotrimethylsilane (3 drops) , reflux 1 h.] in dry CH2C12 (6 ml) is added to a solution of l-acetoxy-3,5-di-p-toluoyl-2-deoxy-4- thio-L-erythro-pentofuranoside (200 mg, 0.5 mmol) in dry CH2C12 (10 ml) . The stirred mixture is cooled in an ice-bath and freshly distilled trimethylsilyl trifluoromethanesulphonate (96 μl) added. After 30 min. at 0°C the mixture is poured into saturated NaHC03 (50 ml) , the layers separated and the aqueous phase extracted with CH2C12. The combined CH2C12 phase is dried over MgS04, then evaporated and the residue triturated with ether to leave the crude protected nucleoside. Deprotection is accomplished by adding a solution of sodium (20 mg) in MeOH (1 ml) to a suspension of the product (140 mg) in MeOH (10 ml) . After 2 hours the solution is neutralised with Dowex 50X8 H+, filtered and evaporated. The residue is triturated with ether and the residue purified by HPLC on Zorbax C8 reversed phase eluted with MeCN-H20 (1:9, v/v) .
EXAMPLE E
Preparation of E-5- (2-bromovinyl) -Uracil-5-Bromo-2.4- dimethoxypyrimidine
A solution of 5-bromo-2,4-dichloropyrimidine (16 g: 70.2 mmol) [D.M. Mulvey et al. J. Het. Chem., 1973,p79] in dry MeOH (55 ml) was added slowly to a stirred solution of sodium (3.23 g: 140.4 mmol) in MeOH (55 ml) at 0°C over 30 min. The ice-bath was removed and the reaction mixture stirred at ambient temperature for 18 h. The precipitated salt was removed by filtration and the filtrate evaporated to give an oil. To this was added an aqueous solution of NaOH (30 ml; 30% w/v) ; the product separated as an upper layer and was extracted into Et20. The organic extracts were dried over MgS04 and evaporated. The residue was crystallised from thanol to give the product as colourless plates, yield 14.3 g, 93%, mp 62-63°C. Mass spectrum, elm/z 219 (M+, 11%). Analysis, found: C,33.20, H,3.26, Br 36.90, N, 12.7%; C6H7BrN202 requires: C,32.90, H,3.33, Br 36.50, N, 12.80%. 5-Formyl-2.4-dimethoxypryrimidine
A solution of 1.6 M n-Buli in hexane (48 ml, 73.6 mmol) was added over 5 min. to a stirred suspension of 5-bromo-2,4- dimethoxypyrimidine (16 g; 72.9 mmol) in dry Et20 (240 ml) at - 70°C under an atmosphere of dry N2. Dry ethyl formate (28 g: 377 mmol) was added and the orange solution stirred at -70°C for 1 h then allowed to warm slowly to ambient temperature. Water (400 ml) was added and the aqueous layer separated and extracted with Et20 (3 x 200 ml) . The ether layer was combined with the extracts and dried over MgS04, filtered and evaporated. The residue was purified by column chromatography by preloading in Si02 and eluting with EtOAc-hexane (3:7, v/v). Product fractions were combined and evaporated to give fine white needles, yield 6.89 g, (56%). Mass spectrum m/z 169 (M+H) + Analysis, found: C, 50:l;H,4.5;N,16.9%;C7H8N2O3 requires C,50.00; H,4.79; N, 16.66% E-5- (2-carboxyvinyl) -2,4-dimethoxypyrimidine
Malonic acid (13.03 g; 126.2 mmol) and redistilled piperidine (
2ml) were added to a solution of 5-formyl-2,4,dimethoxypyrimidine
(10.52 g; 6.2.6 mmol) in dry pyridine (60 ml). The mixture was heated on a steam bath for 10 h then the solvent was removed by distillation under reduced pressure. The residual oil was re- evaporated from water (3 x 25 ml) and the solid thus obtained recrystallised firstly from water and then from dry methanol to give the product as white needles, yield 6.45 g; a second crop was obtained from the filtrate (1.08 g) . Total yield 7.53g
(57%) . Mass spectrum: (El) m/z 210 (M+) . Analysis, found: C,52.1;H,4.8;N, 13.1%: CgH^O,, requires: C, 52.43; H, 4.79; N,
13.33%.
E-5- (2-Bromovinyl) -2,4-dimethoxypyrimidine To a solution of E-5- (2-carboxyvinyl) -2,4-dimethoxypyrimidine (0.300 g; 1.43 mmol) in dry DMF (5 ml) was added K2C03 (0.45 g: 5.25 mmol) . After stirring at ambient temperature for 15 min. a solution of N.-bromosuccinimide (0.258 g; 1.45 mmol) in dry DMF (4 ml) was added dropwise over 10 min. The suspension was immediately filtered, the solid washed with DMF and the filtrate evaporated in high vacuum. The solid residue was purified by column chromatography by preloading on Si02 and eluting with EtOAc-hexane (7:3, v/v) . Product fractions were pooled and evaporated to give fine white crystals, yield 0.561 g (45%) . FAB mass spectrum: m/z245 and 247 (M+H)+. Analysis, found: C,39.9; H, 3.6; N, 11.5% C8H9BrN202 requires C, 40.20; H, 3.70; N. 11.43%.
E-5- (2-Bromovinyl)uracil
To a solution of E-5- (2-bromovinyl) -2,4-dimethoxypyrimidine (2.45 g; 10 mmol) in AcOH (10 ml) was added Nal (3.3 g; 2.2 eq. ; 22 mmol) and the solution heated under reflux for 3h. The hot mixture was filtered and diluted with water (15 ml) . After cooling, the precipitated product was filtered off, washed with acetone (50 ml) and ether (20 ml) and dried to give a pale yellow powder (1.40 g, 65%) . Mp >320°C; 60 MHz ]H-NMR, DMSO-6d, δ: 7.60 (s, IH, H-6) ; 7.30 (d, IH, J=13 Hz, vinyl H) ; 6.80 (d, IH, J=13 Hz, vinyl H) . EXAMPLE 13 .
(A) General Experimental Procedures
Reagents (including R- (+)glycidol, tert-butylchlorodiphenyl silane, phenylselenyl bromide, cytosine and 5-fluorocytosine) were purchased from the Aldrich Chemical Company, Gillingham, Dorset, U.K., except 4-dimethylaminopyridine and dried tetrahydrofuran (thf) which were purchased from Fluka, Glossop, U.K. Dimethylmalonate was dried and distilled over calcium chloride, acetonitrile was similarly treated over calcium hydride; all other solvents were stored over molecular sieves when appropriate. Petrol refers to the .distillate collected between 40 and 60°C. Organic solutions were dried over anhydrous magnesium sulphate.
(R) -rert-butyl-diphenylsilyl glycidol To a solution of tert-butylchlorodiphenyl silane (19.5 g, 70.9 mmol), imidazole (4.82 g, 70.9 mmol), and 4-dimethylamino pyridine (0.41 g, 3.3 mmol) in dichloromethane (150 ml), cooled to 0°C under nitrogen, was added the R- (+) -glycidol (5.0 g, 67.5 mmol) in a dropwise manner in 40 ml of dichloromethane. Reaction was complete after 2.5 h. The solution was washed with water, a back extraction combined, then washed with iced IN HCl (x2) , water and brine. After drying, the solution was evaporated and treated directly with the next reagent. Η NMR spectrum (CDCl3)δH, 1.05 (9H, s, CMe3) , 2.60 and 2.75 (ea IH, dd, H-3) , 3.10 (IH, m, H-2) , 3.70 and 3.85 (ea IH, dd, H-l) , 7.40 to 7.70 (10H, m, Ar-H) .
(S) - (rert-butyl-diphenylsilyloxy-methyl) -thiirane The crude (R) -silyl glycidol (67.5 mmol) was dissolved in 300 ml of methanol to which was added thiourea (5.13 g, 67.5 mmol) in one portion. The solution was stirred for 14 h, after which the solvent was evaporated. The residue was taken up in ether and washed with water (x2) and brine, then dried and evaporated. The residue was purified on a flash column eluted with neat petrol, to give the pure thiirane as a mobile oil. -H NMR spectrum (CDCl3)δH, 1.05 (9H, s, CMe3) , 2.10 and 2.45 (ea IH, dd, H-3) , 3.05 (IH, m, H-2) , 3.55 and 3.95 (ea IH, dd, H-l) , 7.40 to 7.70 (10H, m, Ar-H) . CD Spectrum (hexane) 261 (+6.9)nm.
2- (R/S) -Carboxymethyl-4- (R) - ( tert-butyl-diphenylsilyloxy-methyl) - 4-thio-butanolactone
Dimethyl malonate (6.6 g. 50.1 mmol) in 75 ml of dry thf, was added dropwise to a solution of sodium bis (trimethylsilyl) amide (50.1 mmol) in 250 ml of thf at room temperature. On completion of the addition, the (S) -thiirane (13.7 g, 41.8 mmol) was added in one portion in 400 ml of thf, and the solution was refluxed for 80 h. On cooling, the solution volume was reduced and the residue partitioned between ether and saturated ammonium chloride. A second extraction was combined and washed with water then brine before drying and evaporation. The residue was columned on silica, eluted with a gradient of 10-30% ether/petrol, which gave the required carboxymethyllactone as a colourless oil.
*H NMR spectrum (CDC13) δH, 1.05 (9H, s, CMe3) , 2.30 and 2.70 (3H, m, H-2/3) , 3.60 to 4.10 (3H, m, H-4/5) , 3.75 (3H, s, OMe) , 7.40 to 7.70 (10H, m, Ar-H) . Mass Spectrum (m/z) (FAB+) 429 (M+H+, 25%) .
4- (R) - (Tert-butyl-diphenylsilyloxy-methyl) -4-thio-butanolactone
The carboxymethyllactone was dissolved in 75 ml of dimethylsulphoxide, to which were added 20 drops of brine. After 2 h at 170°C the reaction was complete. After cooling, the reaction solution was directly transferred onto a pre-packed silica column, and the lactone eluted off with 30% ether in petrol, to give a colourless oil on evaporation.
Η NMR spectrum (CDC13) δH, 1.05 (9H, s, CMe3) , 2.05 to2.55 (4H, m, H-2/3) , 3.80 (2H, m, H-5) , 4.05 (IH, m, H-4) , 7.40 to 7.70 (10H, , Ar-H) . Mass Spectrum (m/z) (FAB+) 371 (M+H+, 5%), 313 (M+H-CMe3, 80) . CD Spectrum (EtOH) 231 (-1.2) nm.
Microanalysis, found: C, 67.73; H, 6.94; C21H2602SSi requires C, 68.06; H, 7.07%.
4- (R) - (Tert-butyl-diphenylsilyloxy-methyl) -2- (S) -phenylselenyl-4- thio-butanolactone
The lactone (5.25 g, 14.2 mmol) in 45 ml of dry thf was added dropwise to a 1M solution of lithium bis(trimethylsilyl)amide
(15.6 ml) in thf at -78°C under nitrogen (this temperature was maintained throughout the reaction) . This solution was stirred for 1 h on completion of the addition, then a single portion of trimethylsilylchloride (1.69 g, 15.6 mmol) was added and stirred for 2 h. At this time, phenylselenyl bromide (3.68 g, 15.6 mmol) was added slowly in 60 ml of thf, and the reaction stirred for a further 1 h before it was allowed to warm to room temperature. The mixture was poured into water, which was extracted with three portions of ether. These combined fractions were washed twice with brine, dried and evaporated. The resulting syrup was purified by elution of a flash column with a 0-10% ether/petrol gradient, which gave the desired 2- (S) -phenylselenyl lactone, free of the small quantities of the 2-(R)-epimer which was also formed during the reaction.
'H NMR spectrum (CDClj) δH, 1.05 (9H, s, CMe3) , 2.35 (2H, m, H-3), 3.75-3.85 (4H, m, H-2/4/5) , 7.30 to 7.65 (15H, m, Ar-H). Mass Spectrum (m/z) (FAB+) 526 (M+H+, 10%) .
1-0-Acetoxy-5-0- tert-butyl-diphenylsilyloxy-2 , 3-dideoxy-2- phenylselenyl-4-thio-o./ι8-L-ribofuranose
The lactone (7.5 mmol) was reduced by diisobutylaluminiumhydride (7.9 mmol) in 100 ml of dry toluene at -78°C over 3 h, after which the reaction was quenched with 100 ml of saturated ammonium chloride and vigorously stirred for 1 h. After filtering through a hyflo pad, the organic layer was separated and washed twice with brine, dried and evaporated. The crude lactol was dissolved in 200 ml of dichloromethane and treated with 4-dimethylamino pyridine (1.0 g, 8.25 mmol) and acetic anhydride (0.84 g, 8.25 mmol) . Reaction was complete in 2 h at room temperature, then the solution was washed sequentially with water, copper sulphate, water then brine, dried and evaporated. Purification was achieved by on a short flash column.
-E NMR spectrum (CDCl3)δH, 1.00 and 1.05 (9H, s, CMe3) , 1.95 and 2.10 (3H, 2s, CH3CO) , 2.30 to 2.70 (2H, m, H-3), 3.45 to 3.95 (4H, m, H-2/4/5) , 6.10 and 6.15 (IH, 2d, H-l) , 7.40 to 7.70 (15H, m, Ar-H) .
(B) General procedure for glvcosylation of cvtosines with l'-O- acetoxy-2'phenylselenyl-4'thio-riboside
The 1' -0-acetoxy-2' -selenyl-4'thio-riboside (2.0 mmol) was dissolved in 20 ml of acetonitrile under nitrogen. To this was added the cytosine (3.0 mmol) , then potassium nonafluorobutane-1- sulphonate (6.3 mmol), hexamethyldisilazane (2.0 mmol) and trimethylsilylchloride (9.0 mmol), each in one portion. This suspension was then vigorously stirred at room temperature for 14 h, then poured into a saturated aqueous solution of sodium bicarbonate, and the mixture extracted with 3 x 50 ml portions of dichloromethane. The combined organic fractions were washed twice with brine, dried and evaporated, prior to purification on a flash silica column eluted with methanol/chloroform/ammonia
(7:92.1). This purification gave the single required diastereomer as a white foam, free of small quantities of the other diastereomer formed in the reaction.
5' -0- (Tert-butyl-diphenylsilyl) -2.3'-dideoxy-2' -phenylselenyl-4' - thio-ff-L-cvtidine
-R NMR spectrum (CDCl3)δH. 1.05 (9H, s, CMe3) , 2.15 and 2.35 (ea IH, m, H-3'), 3.80 (4H, m, H-2'/4'/5'), 5.30 (IH, d, H-5) , 6.40
(IH, d, A-H-l') 7.25 to 7.75 (lδH, m, Ar-H), 7.85 (IH, d, H-6).
5 ' - 0 - ( Ter t -butyl - diphenyl silyl ) - 2 , 3 ' -dideoxy- 5 - f luoro - 2 ' - phenylselenyl -4 ' -thio-β-L-cvtidine Η NMR spectrum (CDC13) δH, 1.05 (9H, s, CMe3) , 2.15 and 2.35 (ea IH, m, H-3') , 3.70 (4H, m, H-2'/4'/5') , 6.35 (IH, dd, H-l') , 7.25 to 7.75 (16H, m, Ar-H, H-6) .
(C) General procedure for oxidative removal, by elimination, of 5 phenylselenyl functionality
The 2'-selenyl nucleoside was dissolved in dry dichloromethane and cooled to -20°C under nitrogen. To this was added an equivalent of metachloroperoxvbenzoic acid in one portion, and the temperature maintained during the course of the reaction (45 0 min) . 5 eq of pyridine were then added and the solution allowed to warm to room temperature over 1 hour. After dilution with dichloromethane the solution was washed successively with water, copper sulphate (x2) , sodium bicarbonate (x2) , water, and brine, before drying and evaporation. Purification was achieved on a 5 flash column, eluted with methanol/chloroform/ammonia (7:92:1) .
5' -0- ( Ter -butyl-diphenylsilyl) -2' ,3' -didehvdro-2' ,3' -dideoxy-4' - thio-<3-L-cvtidine
'H NMR spectrum (CDC13) δH, 1.05 (9H, s, CMe3) , 3.85 (2H, m, H-5') , 4.40 (IH, m, H-4'), 5.30 (IH, d, H-5), 5.80 and 6.20 (each IH, m, 20 H-2 3'), 7.20 (IH, dd, H-l') , 7.35 to 7.70 (10H, m, Ar-H) , 7.50 (1-H, d, H-6) .
5' -0- (Tert-butyl-diphenylsilyl) -2' .3' -didehydro-2' .3' -dideoxy-5- Fluoro-4' -thio-/3-L-cvtidine
Η NMR spectrum (CDCl3)δH, 1.05 (9H, s, CMe3) , 3.80 (2H, m, H-5') , 254.40 (IH, m, H-4'), 5.75 and 6.25 (each IH, m, H-2'/3') , 7.15 (IH, dd, H-l') , 7.35 to 7.70 (11H, m, Ar-H, H-6) .
(D) General procedure for desilylation of nucleosides
The silyl ether was stirred in a thf solution with tetraethylammonium fluoride (1.1 eq) for 1 hour at room
30 temperature when completion was evident by tic
[methanol/chloroform/ammonia (7:92:1)] . Silica gel was added to this mixture, the solvent evaporated, and the pre-absorbed mixture placed onto a short silica column. The column was eluted with a gradient based on the tic solvent system; the required product was either crystallised from ethanol or lyophilised.
(i) 2' , 3 ' -Didehvdro-2' , 3 ' -dideoxy-4' -thio-<3-L-cytidine 'H NMR spectrum (DMS0-d6) δH, 3.60 (2H, m, H-5') , 4.30 (IH, m, H- 4') , 5.15 (lH,brt, OH) , 5.75 (IH, d, H-5) , 5.85 and 6.30 (each IH, dt, H-2'/3') , 6.85 (IH, dd, H-l') , 7.30 (2H, brd, NH2) , 7.65 (IH, d, H-6) .
Infra red spectrum v^ (KBr disc) 3 344, 3 199, 1 649, 1 607, 1 526, 1 499 cm'1.
Mass spectrum (m/z) (FAB+) 226 (M+H+, 20%) .
Microanalysis, found: C, 45.42, H, 4.70; N, 17.33;
C9Hn02N3SF.0.14CHCl3 requires C, 45.41; H, 4.64, N,17.39%.
CD Spectrum (H20) 275 (+1.70) , 231 (-6.41) , 213 (+4.20)nm.
(ii) 2' , 3 ' -Didehvdro-2' ,3' -dideoxy-5-Fluoro-4' -thio-β-L-cvtidine
'H NMR spectrum (DMSO-d6) δH, 3.65 (2H, m, H-5') , 4.35 (IH, m, H- 4' ) , 5.20 (IH, t, OH) , 5.85 and 6.25 (each IH, dt, H-2'/3' ) , 6.85 (IH, m, H-l') , 7.65 (2H, brd, NH2) , 7.95 (IH, d, H-6) . Infra red spectrum v^ (KBr disc) 3 351, 3 186, 1 682, 1 647, 1 607, 1 508 cm"1.
Mass spectrum (m/z) (FAB+) 243 ' (M+, 65%) .
Microanalysis, found: C, 43.01, H, 4.26; N, 16.34;
CJHJOO^S.O.SSHJO requires C, 42.79; H, 4.41, N,16.63%.
CD Spectrum (EtOH) 288 (+7.62), 239 (-3.86) , 214 (+5.85)nm.
EXAMPLE 14.
Methyl 2-Deoxy-3.5-di-O- (p-nitrobenzoyl) -D-threo-pentoside
To a cold (0°C) stirred solution of methyl 2-deoxy-D-erythro- pentoside (1.2g, 8.1 L) and triphenylphosphine (8.5 g, 32.4 mmol) in dry toluene (160 ml) was added a solution of p-nitrobenzoic acid in dry toluene (30 ml) , followed by diisopropyl azodicarboxylate (6.4 ml, 32.4 mmol) under N2. The reaction mixture was allowed to warm up to room temperature and after 6 hrs of stirring the solid was filtered, the filtrate evaporated, the residue resuspended in toluene (100 ml) , the precipitate filtered and filtrate evaporated to dryness the residue was 5 purified by column chromatography eluting sequentially with ethyl-acetone hexane (3:7, 2.3 and 1:1) , ethyl-acetate/toluene (3:7) and methanol/dichloro methane (1:99) to give the title compound.
NHR: CH) δ (CDC13) : 8.4-8.1 (8H, m, aromatic) , 5.85 (IH, m, H-3) , 105.3 (0.64H, dd, H-l, α-anomer) , 5.2 (0.36H, dd, H-l, β anomer) , 4.75-4.55 (3H, m, H-4, H-5) , 3.48 (3H, s, OMe) , 3.43 (3H, s, OMe), 2.65-2.25 (2H, m, H-2) .
MASS SPECTRUM
FAB 447 (H+l) ; NBA Matrix 15
2-Deoxy-3.5-di-0- (p-nitrobenzoyl) -D-threopentose di- (p-methoxybenzyl) dithioacetal
To a stirred solution of methyl 2-deoxy-3, 5-di-O- (p- nitrobenzyol) -D-threopentoside (218 mg, 0.488 mmol) in dry
20 toluene (40 ml) was added p-methoxybenzylmercaptan (218 μl; 2.44 mmol) followed by titanium tetrachloride (54 μl, 0.488 mmol) at room temperature under N2. After 20 mins. the reaction mixture was quenched with NaHC03 (100 ml) and extracted with ether (3 x 40 ml) . The combined organic layers were dried (Na2S04) ,
25 evaporated to dryness and the residue was purified by column chromatography eluting with ethyl-acetate/hexane (1:4-2:3) to give the title compound.
NMR Spectrum
CH) δ (CDC13) : 8.32-7.9 (8H, m, aromatic) , 7.2-7 (4H, 30 dd, aromatic) , 6.8-6.55 (4H, dd, aromatic) , 5.52 (IH, m, H-3) , 4.35 (2H, m, H-5) , 3.95 (IH, m, H-4) , 3.85-3.6 (10H, m, PhCH? S,OMe) , 3.52 (IH, m, H-l) , 2.5-2.1 (2H, m, H-2) .
MASS SPECTRUM FAB - 723 (M+l) NBA - Matrix
2-Deoxy-3,5-di-O- (p-nitrobenzoyl) -4-O-MethaneBulphonyl-D-threo- Pentose-Di- (p-methoxybenzyl) dithioacetal
To a cold (0°C) stirred solution of 2-deoxy-3,5-di-O- (p- nitrobenzoyl) -D-threopentose-di- (p-methoxybenzyl) dithioacetal (1.45g, 2 mmol) in dry pyridine (20 ml) was added methanesulphonyl chloride (188 μl, 2.44 mmol) under N2. After stirring at room temperature for 5 hrs. the reaction mixture was quenched with water, the solvent evaporated to dryness under high vacuum and the residue was partitioned between dichloromethane/water (100 ml / 75 ml) . The aqueous layer was further extracted with dichloromethane (3 x 50 ml) , the combined organic layers dried (Na2S04) and evaporated to dryness. Residual pyridine was co-evaporated with ethanol (2 x 50 ml) to give the title compound.
NMR SPECTRUM
H) δ (CDCl3) : 8.4-7.9 (8H, m, aromatic), 7.3-7 (4H, dd, aromatic), 6.8-6.5 (4H, dd, aromatic), 5.3 (IH, m, H-3), 5.0 (IH, m, H-4), 4.65-4.5 (IH, m, H-5) , 4.45-4.3 (IH, m, H-5) , 3.9-3.6 (10H, m, PHCH2 S, OMe), 3.55 (IH, dd, H-l), 3.0 (3H, s, OMs) , 2.5-2 (2H, m, H-2) .
p-Methoxybenzyl-2-deoxy-3.5-di-O- (p-Nitrobenzoyl) -1.4-dithio-L- ervthro-pentofuranose
To a stirred solution of 2-deoxy-3,5-di-O- (p-nitrobenzoyl) -4-0- methanesulphonyl-D-threo-pentose-di- (p-methoxybenzyl) - dithioacetal (1.46 g, 1.82 mmol) in dry dimethylformamide (40 ml) was added triethylamine (381 μl, 2.73 mmol) followed by sodium iodide (2.2 g, 14.6 mmol) at room temperature under N2. After 20 hrs. of stirring at 100°C the reaction mixture was quenched with water, the solvent evaporated to dryness under high vacuum and the residue was partitioned between dichloromethane and water. The organic layer was washed with water (2 x 30 ml) dried (Na2S04) , evaporated to dryness and the residue was purified by column chromatography eluting with ethyl-acetate/hexane (3:7) to give the title compound (5)
NMR SPECTRUM
(H) δ (CDC13) : 8.3-8.1 (8H, , aromatic), 7.35-7.2 (2H, d, aromatic), 6.9-6.8 (2H, d, aromatic), 5.8 (IH, m, H-3), 4.65-4.4 (3H, m, H-l, H-5) , 4-3.75 (6H, , H-4, PhCH2S, OMe 2.7-2.3 (2H, m, H-2) .
2 ' -Ωeoxy-3 ' . 5 ' -di-O- (p-nitrobenzoyl) -5-fluoro-4/ -thio-L-lfB- cvtidine
A mixture of 5-fluorocytosine (42 mg, 0.324 mmol) and bis (trimethylilyl) -acetamide (133 μl, 0.43 mmol) in dry acetonitrile (15 ml) was stirred at 80°C under N2. After 1 hr. the solution was cooled to room temperature and to it was added dropwise a solution of p-methoxybenzyl-2-deoxy-3,5-di-O- (p- nitrobenzoyl) -1,4-dithio-L-erythro-pentofuranose (158 mg, 0.27 mmol) in dry acetonitrile (10 ml) , followed by N-iodosuccinimide (61 mg 0.27 mmol) in dry acetonitrile (5 ml) and trimethyl¬ silyltrifluoromethane sulphonate (52 μl, 0.27 mmol). After stirring for 2 hrs. at room temperature TLC (ethyl-acetate/hexane
3.7) showed presence of starting material. A further amount of
N-iodosuccinimide (6 mg, 0.027 mmol) was added and after stirring overnight the reaction mixture was diluted with dichloromethane
(30 ml) , quenched with 10% NaHC03 (30 ml) and to the mixture was added a solution of sodiumthiosulphate (30m m) . The organic layer was separated and the aqueous layer was further extracted with dichloromethane (2 x 30 ml) and the combined organic layers were washed with water (3 x 30 ml) , dried (Na2S04) and evaporated to dryness. The resulting residue was purified by column chromatography eluting with methanol/dichloromethane (1:9) to give title compound (6) as an anomeric mixture α:/3 = 1.8:1.
NMR SPECTRUM CH) δ (deDMSO) :8.45-8 (9H, m, aromatic, H-6) , 7.9-7.5 (2H, m, NH2) , 6.45 (0.35H,t, H-l', β ano er) , 6.25 (0.65H, m, H-l', a anomer) , 5.9-5.65 (IH, m, H-3') , 4.8 -4.5 (2H m, H-5) , 3.65 (IH, m, H-4') , 3-2.5 (2H, m, H-2') .
2 ' -deoxy-5-fluoro-4' -thio-L-ot,β-cvtidine
To a stirred solution of 2' -deoxy-3' , 5' -di-O- (p-nitrobenzoyl) -5- fluoro-4' -thio-L-α,B-cytidine (55.6 mg, 0.01 mmol) was added a 30% w/v solution of sodium methoxide in methanol (360 μl 0.198 mmol) at room temperature under N2. After 3 hrs. TLC in methanol/dichlromethane (3:17) showed presence of product and starting material . The reaction mixture was neutralised with "DOWEX" 50W-X8 (H) to pH 7, filtered washed with methanol and evaporated to dryness. The residue was purified by column chromatography, eluting with methanol/dichloromethane (3:17 and 1:4) to give the first batch of product. The recovered starting material was deprotected with 30% w/v solution of sodium methoxide in methanol (180μl; 0.09 mmol) and worked up as above. The combined products were purified by HPLC (ZORBAX C8, using a 0-35% 20 minute gradient of 10% H20/ACN in 0.IM NH4OAc pH4.0, to give the title compound (7) as an anomeric mixture o.:3=1.2.
NMR - SPECTRUM
C4) δ (d4-MeOH) 8.5 (0.35H, d, H-6, a -anomer) , 8.4 (0.65H, d, H- 6, β -anomer) , 6.35 (0.65H, m, H-l', β anomer), 6.25 (0.35H, m, H- 1', a -anomer) , 4.6-4.35 (IH, m, H-3'), 3.8-3.4 (3H, m, H-4', H- 5') , 2.6-2.0 (2H, m, H-2') .
MASS SPECTRUM
E170 MI=261 BIOLOGICAL DATA
a) Anti-HSV Activity
Herpes Simplex Virus types 1 (HSV 1) and 2 (HSV2) were assayed in monolayers of Vero cells in multiwell trays. The virus strains used were SC16 and 186 for HSV-1 and HSV-2 respectively. Activity of compounds was determined in the plaque reduction assay, in which a cell monolayer was infected with a suspension of the appropirate HSV, and then overlaid with nutrient agarose in the form of a gel to ensure that there was no spread of virus throughout the culture. A range of concentrations of compound of known molarity was incorporated in the nutrient agarose overlay. Plaque numbers at each concentration were expressed as percentages of the control and a dose-response curve was drawn. b) Anti-CMV Activity
Human cytomogalovirus (HCMV) was assayed in monolayers of either MRC5 cells (human embryonic lung) in multiwell trays. The standard CMV strain AD 169 was used. Activity of compounds is determined in the plaque reduction assay, in which a cell monolayer is infected with a suspension of HCMV, and then overlaid with nutrient agarose in the form of a gel to ensure that there is no spread of virus throughout the culture. A range of concentrations of compound of known molarity was incorporated in the nutrient agarose overlay. Plaque numbers at each concentration of drug are expressed as percentage of the control and a dose-response curve is drawn. c)Anti-VZV Activity
Clinical isolates of varicella zoster virus (VZV) were assayed in monolayers of MRC-5 cells. MRC-5 cells are derived from human embyonic lung tissue. A plaque reduction assay was used in which a suspension of the virus stock was used to infect monolayers of the cells in multiwell trays. a range of concentrations of the compound under test of known molarity was added to the wells. Plaque numbers at each concnetration were expressed as percentages of the control and a dose response curve was constructed. From these curves the 50% inhibitory concentration of each drug was determined. d) HBV Assay (Method 1) Materials Virus/Cells
The cell line used was derived from a hepatoblastoma cell line, Hep G2, which had been transfected with a plasmid containing four 5' -3' tandem copies of the hepatitis B virus genome, subtype ayw, to produce the cell line designated 2:2:15.
(Sells et al PNAS 84 1005-1009, 1987) . These cells carry the Hep
B DNA both as chromosomally integrated sequences and episomally. The cells constitutively produce small amounts of virus particles. A higher virus producing clone P5A, was obtained from the 2.2.15 cells for use in the assay. Media
Cells were grown in RPMI 1640 containing 0.5% penicillin and streptomycin, 2mML-glutamine and 10% foetal calf serum. Methods
Assays were performed in 24 well plates which were seeded with approximately 2.5xl04 cells/well and grown for 5 days at 37°C in 5% C02, the monolayers were then incubated with RPMI 1640, 0.5% penicillin and streptomycin, 2mM L-glutamine and 2% FCS containing the test compounds at the required concentrations. Medium was replaced every 48 hours with fresh medium containing the test compound. The plates were incubated for 10 days, the medium was removed and the cells scraped from the wells in 0.5ml of PBS, the cells were pelleted at 5000 rpm for 5 minutes the supernatant discarded and the cells frozen at -20°C. The cells were thawed and resuspended in 500μl of lysis buffer (150 mM NaCl, 20mM Tris/HCl pH7.4, lOmM EDTA and 0.6% SDS) and 50 μL of proteinase K (20mg/ml) added and the samples incubated at 37°C for 2 hours. DNA was extracted on an Autogen 540 DNA extractor and dissolved in a final volume of 50μl of water. DNA was digested with the restriction enzyme Hind III at 37°C for 16 hours and the DNA fragments separated on 1% agarose gel. The separated DNA was transferred by capillary blotting to hybond N+ nylon membrane (Amersham International) and, after prehybridisation, hybridized with a 32P labelled positive strand RNA transcript of the core region of the hepatitis B genome, subtype ayw, at 42°C overnight in the presence of 50% formamide. After extensive washing the blot was exposed to X-ray film and the intensity of the hybridization to the replicative intermediate DNA analysed by a Milli Pore 610 imager. Results were compared to a control sample containing no test compound.
e) HBV Assay (Method 2)
Anti-HBV activity of compounds of formula (I) was determined with a high capacity assay for assessing efficacy. Supernatants from growing HBV-producing cells (HepG2 2.2.15, P5A cell line) in 96-well plates are applied to microtiter plate wells which have been coated with a specific monoclonal antibody to HBV surface antigen (HBsAg) . Virus particles present in the supernatants bind to the antibody and remain immobilized while other debris is removed by washing. These virus particles are then denatured to release HBV DNA strands which are subsequently amplified by the polymerase chain reaction and detected with a colorimetric hybrid-capture assay. Quantitation is achieved through fitting of a standard curve to dilutions of a cell supernatant with known HBV DNA content. By comparing HBV DNA levels of untreated control cell supernatants with supernatants containing a compound of formula (I) , a measure of anti-HBV effectiveness is obtained.
Immunoaffinitv Capture of HBV
HBV producer cells, 2500 cells/well, were seeded in 96-well culture dishes in RPMI/10% fetal bovine serum/2mM glutamine (RPMI/10/2) . Media were replenished on days 1, 3, 5 and 7 with dilutions of a compound of formula (I) in RPMI/10/2 to a final volume of 150 μl. Fifty μL of mouse monoclonal anti-HBsAG antibody (lOμg/mL in PBS) were added to each well of a round- bottom microtiter plate. After incubation overnight at 4°C, the solutions were aspirated and replaced with 100 μL of 0.1% BSA in PBS. Samples were incubated for 2 hours at 37°C and washed three times with PBS/0.01% Tween-20 (PBS/T) using a Nunc Washer. Ten μL of 0.035% Tween 20 in PBS were then added to all wells by Pro/Pette. Cell supernatants (25 μL) containing extracellular virion DNA were transferred into wells by Pro-Pette; the final Tween concentration is 0.01%. Twenty-five μL HBV standard media dilutions in RPMI/10/2 were added to 2 rows of wells to serve as an internal standard curve for quantitation, and the plates were sealed and incubated at 4°C overnight. Samples were washed 5 times with PBS/T and 2 times with PBS, aspirating the last wash. Next, 25 μL of 0.09N NaOH/0.01% NP40 were added to each well by Pro/Pette, and the sample wells were sealed and incubated at 37°C for 60 minutes. Samples were then neutralized with 25 μL of 0.09N HCI/100 mM tris (pH 8.3) .
Polymerase Chain reaction (PCR) Polymerase chain reaction (Saiki, R.K. et al, Science, 239 (4839) 487-91 (1988)) was carried out on 5μL samples, using a Perkin Elmer PCR kit. PCR is performed in "MicroAmp tubes" in a final volume of 25 μL. Primers were chosen from conserved regions in the HBV genome, as determined by alignment of several sequences. One primer is biotinylated at the 5-prime end to facilitate hybrid-capture detection of the PCR products. All primers were purchased from Synthecell Corp, Rockville, MD 20850.
Hybrid-Capture Detection of PCR Products
PCR products were detected with horse radish peroxidase- labelled oligonucleotide probes (Synthecell Corp, Rockville, MD
20850) , which hybridize to biotinylated strands of denatured PCR products directly in streptavidin-coated microtiter plate wells, using essentially the method of Holodiniy, M et al, Bio Techniques, 12 (1) 37-39 (1992) . Modifications included the use of 25k PCR reaction volumes and sodium hydroxide denaturation instead of heat. Simultaneous binding of the biotin moiety to the plate-bound streptavidin during the hybridization serves to "capture" the hybrids. Unbound labelled probes were washed away before colorimetric determination of the bound (hybridized) horse radish peroxidase. Quantities of HBV DNA present in the original samples were calculated by comparison with standards. These values were then compared to those from untreated cell cultures to determine the extent of anti-HBV activity.
IC50 (the median inhibitory concentration) is the amount of compound which produces a 50 percent decrease in HBV DNA. f) HeLa-CD4^ cell assay for evaluating susceptibility of HIV to antiviral compounds
Susceptibility of HIV to inhibitors was determined by infection of HT4-6C cell monolayers as described by Larder, B.A. Chesebro, B & Richman, D.D. Antimicrob. Agents Chemother. 1990 34. 436-441. Briefly cells were seeded in 24-well multiwells at 5 x 104 cells per well and incubated overnight at 37°C in growth medium (DMEM10) . Monolayers were infected with 100-200pfu of cell-free virus in 0.2ml of DMEM containing 5% fetal bovine serum plus antibiotics (DMEM5) and incubated for 1 hour at 37°C to allow virus adsorption. Following this time 0.3m, of DMEM5 (with or without inhibitor) was added to each well and cultures were incubated at 37°C for 2-3 days. Monolayers were fixed with 10% formaldehyde solution in PBS and stained with 0.25% crystal violet in order to visualize virus plaques. Individual foci of multinucleated gian cells (plaques) were apparent using this staining procedure. ID50 values were derived from plots of percent plaque reduction versus inhibitor concentration.
g) MT4/MTT dye uptake assay lOOμl of RPMI growth medium, with or without inhibitor, was added to each well of a 96-well microplate. MT4 cells, either mock-infected or infected with HIV at a m.o.i. of 0.01 for 1 hour at 37°C then washed three times, were added to the plate at a concentration of 40,000 cells per well, and the cultures incubated at 37°C for 5 days. 20μl of a 5mg/ml solution of 3-
(4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) was added to each well and the panels incubated at 37°C for 2 hours. Acidified isopropanol (AIP) was then added (170μl/well) and the plates maintained at room temperature overnight prior to reading spectrophotometrically at 590 nm. Viable cells are able to reduce the yellow MTT to its purple formozan product, which is solubilized by the AIP, while wells containing killed cells remain yellow. The concentration of drug (IC50) required to protect 50% of the cells from viral killing was determined from regression analysis of percentage cell death against drug concentration. h) Cell Toxicity
Cell toxicity is assessed in cell growth inhibition assay. Subconfluent cultures of Vero cells grown on 96-well microtiter dishes are exposed to different dilutions of drug, and cell viability determined daily on replicate cultures using uptake of a tetrazolium day (MTT) . The concentration required for 50% inhibition of cell viability at 96 hours is termed CCID50.
Biological test results.
The compounds 2' ,3' -didehydro-2' ,3' -dideoxy-4' -thio-jS-L- cytidine and 2' ,3' -didehydro-2' ,3' -dideoxy-5-fluoro-4' -thio-/3-L- cytidine were tested for activity against HBV using both assay methods described above, and against HIV using the assay described above. The results of the tests, together with toxicity data (derived as described in (g) above are shown in Table 1.
TABLE 1
Figure imgf000075_0001
EXAMPLES
The following examples illustrate pharmaceutical formulations according to the invention in which the active ingredient is a compound of formula (I) .
Formulation Example A Tablet
Active ingredient 100 mg Lactose 200 mg Starch 50 mg
Polyvinylpyrrolidone 5 mg Magnesium stearate 4 mg
359 mg Tablets are prepared from the foregoing ingredients by wet granulation followed by compression.
Formulation Example B Qpthalmic Solution Active ingredient 0.5 g
Sodium chloride, analytical grade 0.9 g
Thiomersal 0.001 g
Purified water to: 100 ml pH adjusted to: 7.5
Formulation Example C; Tablet Formulations
The following formulations a and b are prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression.
Tablet Formulation a
(a) Active ingredient
(b) Lactose B.P.
(c) Povidone B.P
(d) Sodium Starch Glycolate (e) Magnesium Stearate
Figure imgf000076_0001
Tablet Formulation b
(a) Active ingredient
(b) Lactose (c) Avicel PH 101
(d) Povidone B.P.
(e) Sodium Starch Glycollate
(f) Magnesium Stearate
Figure imgf000077_0001
500 300
Tablet Formulation c mg/tablet
Active ingredient 100 Lactose 200 Starch 50 Povidone 5
Magnesium stearate 4
359
The following formulations, D and E, are prepared by direct compression of the admixed ingredients. The lactose used in formulation E is of the direct compression type.
Tablet Formulation d mg/capsule
Active Ingredient 250 Pregelatinised Starch NF15 150 400
Ta t Form lation e
Figure imgf000077_0002
500
Tablet Formulation £ (Controlled Release Formulation)
The formulation is prepared by wet granulation of the ingredients (below) with a solution of povidone followed by the addition of magnesium stearate and compression. g/tablet (a) Active Ingredient 500 (b) Hydroxpropylmethylcellulose 112 (Methocel K4M Premium)
(c) Lactose B.P. 53
(d) Povidone B.P.C. 28
(e) Magnesium Stearate 7 700
Drug release takes place over a period of about 6-8 hours and was complete after 12 hours.
Formulation Example D; Capsule Formulations Capsule Formulation a A capsule formulation is prepared by admixing the ingredients of Formulation D in Example C above and filling into a two-part hard gelatin capsule. Formulation B (infra) is prepared in a similar manner. Capsule Formulation b mσ/capsule
(a) Active ingredient 250
(b) Lactose B.P. 143
(c) Sodium Starch Glycollate 25
(d) Magnesium Stearate 2 420
Capsule Formulation c mσ/capsule
(a) Active ingredient 250
(b) Macrogol 4000 BP 3_5£ 600
Capsules are prepared by melting the Macrogol 4000 BP, dispersing the active ingredient in the melt and filling the melt into a two-part hard gelatin capsule. Capsule Formulation d
Figure imgf000079_0001
Capsules are prepared by dispersing the active ingredient in the lecithin and arachis oil and filling the dispersion into soft, elastic gelatin capsules.
Capsule Formulation e (Controlled Release Capsule)
The following controlled release capsule formulation is prepared by extruding ingredients a, b, and c using an extruder, followed by spheronisation of the extrudate and drying. The dried pellets are then coated with release- controlling membrane (d) and filled into a two-piece, hard gelatin capsule. mσ/capsule
(a) Active Ingredient 250
(b) Microcrystalline Cellulose 125
(c) Lactose BP 125 (d) Ethyl Cellulose 13
513
Formulation Example E; Injectable Formulation
Active ingredient 0.200 g
Sterile, pyrogen free phosphate buffer, pH 7.0 to 10 ml
The active ingredient is dissolved in most of the phosphate buffer (35-40°C) , then made up to volume and filtered through a sterile micropore filter into a sterile 10ml amber glass vial (type 1) and sealed with sterile closures and overseals. Formulation Example F: Intramuscular in-jection
Active Ingredient 0.20 g
Benzyl Alcohol 0.10 g
Glucofurol 75 1.45 g Water for Injection q.s. to 3.00 ml
The active ingredient is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml.
The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1) . Formulation Example G; Syrup Suspension
Active ingredient 0.2500 g
Sorbitol Solution 1.5000 g
Glycerol 2.0000 g
Dispersible Cellulose 0.0750 g Sodium Benzoate 0.0050 g
Flavour, Peach 17.42.3169 0.0125 ml
Purified Water q.s. to 5.0000 ml
The sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added. The active ingredient is added and dispersed. In the glycerol is dispersed the thickener
(dispersible cellulose) . The two dispersions are mixed and made up to the required volume with the purified water. Further thickening is achieved as required by extra shearing of the suspension. Formulation Example H; Suppository mg/suppositorv Active Ingredient (63μm)* 250
Hard Fat, BP (Witepsol H15 - Dynamit NoBel) 1770 2020
* The active ingredient is used as a powder wherein at least 90% of the particles are of 63μm diameter or less.
One-fifth of the Witepsol H15 is melted in a steam- jacketed pan at 45°C maximum. The active ingredient is sifted through a 200μm sieve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45°C, the remaining Witepsol H15 is added to the suspension and stirred to ensure a homogenous mix. The entire suspension is passed through a 250μm stainless steel screen and, with continuous stirring, is allowed to cool to 40°C. At a temperature of 38°C to 40°C 2.02g of the mixture is filled into suitable plastic moulds. The suppositories are allowed to cool to room temperature.
Formulation Example I: Pessaries mg/pessarv Active ingredient 63μm 250 Anydrate Dextrose 380 Potato Starch 363
Magnesium Stearate 7
1000
The above ingredients are mixed directly and pessaries prepared by direct compression of the resulting mixture.

Claims

1. A pyrimidine 4' -thio-L-nucleoside of the general formula (I) :
Figure imgf000082_0001
wherein: Y is hydroxy or amino;
X is hydrogen, hydroxy, mercapto, halo, trifluoromethyl, methyl,
C^alkyl, Cj^haloalkyl, hydroxyCj.3alkyl, formyl,
Cwalkenyl, C2^ haloalkenyl, C2^ alkynyl, C^alkoxy, C^alkylthio, C,^alkoxyC..2alkyl, C^alkylthiomethyl, amino, monoC,. 6alkylamino, diC^alkylamino, cyano, thiocyanate or nitro; R2 is hydrogen and R3 is hydroxy or hydrogen, or together R2 and R3 form a carbon-carbon bond; and physiologically functional derivatives thereof.
2. A compound according to claim 1 in which X is hydrogen, hydroxy, mercapto, halo, trifluoromethyl, methyl, C2- 6alkyl, C^haloalkyl, hydroxyC^alkyl, formyl, C2-6 alkenyl, Cw haloalkenyl, C2^ alkynyl, C^alkoxy, CwalkoxyCι.2alkyl, amino, monoCi^alkylamino, diC^alkylamino, cyano or nitro.
3. A compound according to claim 1 or 2 in which X is hydrogen, halo, methyl, C^alkyl, C^aloalkyl, C^alkenyl, Cw haloalkenyl, Cw alkynyl, cyano or nitro.
4. A compound according to any one of claims 1 to 3 wherein X is fluoro, C2.3 alkyl, CM alkenyl, halovinyl or C3-4 alkynyl.
5. A compound according to any one of claims 1 to 4 wherein Y is amino.
6. A compound according to any one of claims 1 to 5 wherein R2 and R3 form a carbon-carbon bond.
7. A compound according to claim 1 wherein the pyrimidine 4' -thio-L-nucleoside is:
2' -deoxy-4' -thio-L-uridine,
2' -deoxy-4' -thio-L-cytidine,
2' -deoxy-5-fluoro-4' -thio-L-cytidine 2' -deoxy-5-methyl-4' -thio-L-uridine,
5- (2-chloroethyl) -2' -deoxy-4' -thiouridine;
5-nitro-2' -deoxy-4' -thiouridine;
5-amino-2' -deoxy-4' -thiouridine;
5-methylamino-2' -deoxy-4' -thiouridine; E-5- (2-bromovinyl) -2' -deoxy-4' -thio-L-uridine,
2' -deoxy-5-iodo-4' -thio-L-uridine,
5-bromo-2' -deoxy-4' -thio-L-uridine,
5-chloro-2' -deoxy-4' -thio-L-uridine,
2' -deoxy-5-ethyl-4' -thio-L-uridine, 2' -deoxy-5-prop-l-ynyl-4' -thio-L-uridine,
2' -deoxy-5-fluoro-4' -thio-L-uridine,
2' -deoxy-5-trifluoromethyl-4' -thio-L-uridine,
2' -deoxy-5-ethynyl-4' -thio-L-uridine,
2' -deoxy-5-E- (2-bromovinyl) -4' -thio-L-cytidine, 2' -deoxy-5-propyl-4' -thio-L-uridine,
E-2' -deoxy-5- (propen-1-yl) -4' -thio-L-uridine,
1- (2 , 3 -didehydro-2 , 3 -dideoxy-4 -thio-L-ribof uranosyl ) -5- methyluracil ,
1- (2, 3 -dideoxy-4-thio-L-ribof uranosyl) -5-methyluracil, 2' ,3' -didehydro-2' ,3' -dideoxy-4' -thio-3-L-cytidine,
2' ,3' -didehydro-2' ,3' -dideoxy-5-f luoro-4' -thio-3-L-cytidine,
5-bromo-2'3' -didehydro-2' ,3' -dideoxy-4' -thio-/3-L-cytidine,
5-chloro-2' , 3' -didehydro-2' , 3' -dideoxy-4 ' -thio-|3-L-cytidine, or
2' ,3' -didehydro-2' ,3' -dideoxy-5-iodo-4' -thio-3-L-cytidine.
8. A compound according to any one of claims 1 to 7 wherein the pyrimidine 4' -thio-L-nucleoside is the β-anomer.
9. A physiologically functional derivative of a pyrimidine nucleoside of Formula (I) according to any one of claims 1 to 8 .
10. A derivative according to claim 9 which is an alkali metal, alkali earth metal, ammonium, tetra (CM alkyl) ammonium, hydrochloride or acetate salt, or a mono- or di-carboxylic acid ester or an alkali metal, alkali earth metal, ammonium or tetra (CM) alkyl ammonium salt.
11. A composition comprising a compound according to any one of claims 1 to 10 in association with a pharmaceutically acceptable carrier or diluent.
12. A compound according to any one of claims 1 to 10 or a composition according to claim 11 for use in a method of treatment or prophylaxis of virus infections.
13. Use of a compound according to any one of claims 1 to 10 or a composition according to claim 11 for the manufacture of a medicament for use in the treatment or prophylaxis of virus infections.
14. A process for the production of a pyrimidine 4'-thio- L-nucleoside of the formula (I) :
Figure imgf000084_0001
wherein Y is hydroxy or amino;
X is hydrogen, hydroxy, mercapto, halo, trifluoromethyl, methyl, Chalky1, C^haloalkyl, hydroxyC,.3alkyl, formyl, CM alkenyl, C2^ haloalkenyl, C^ alkynyl, C^alkoxy, C^alkylthio, C^alkoxyC^alkyl, C-^alkylthiomethyl, amino, monoC1_6alkylamino, diC-^alkylamino, cyano, thiocyanate or nitro; R2 is hydrogen and R3 is hydroxy or hydrogen, or together R2 and R3 form a carbon-carbon bond; and physiologically functional derivatives thereof, which process comprises:
A) reacting a compound of formula (II)
Figure imgf000085_0001
wherein X1 is a precursor for the group X as defined in relation to formula (I) ;
Y and R2 are as defined in relation to formula (I) ;
R3a either forms a carbon-carbon double bond with R2 or when R2 is
H, R3a is hydrogen, hydroxy or a group OZ3 where Z3 is a hydroxyl protecting group; and
Z5 is hydrogen or a hydroxyl-protecting group, with a reagent or reagents serving to convert the group X1 to the desired group X; or
B) reacting a compound of formula (III)
Figure imgf000085_0002
wherein X and Y are as defined in relation to formula (I) or a protected form thereof with a 4-thio sugar compound serving to introduce the 4-thio sugar moiety, or a protected form thereof, at the l-position of the compound of formula (III) ; or C) reacting a compound of formula (IV)
Figure imgf000086_0001
wherein X and Y are as defined in relation to formula (II) , Z5 is a hydroxy protecting group or hydrogen; R2 and R3a are as defined above wherein at least one of R3a and Z5 represents a precursor group for the group (s) R3 and/or R5 in formula (I) under conditions or with a reagent serving to convert the groups R3a and/or Z5 into the respective groups R3 and/or H; and, where necessary or desired, thereafter optionally effecting one or more of the following further steps in any desired or necessary order: a) removing each of the protecting groups, b) converting a compound of formula (I) or a protected form thereof into a further compound of formula (I) or a protected form thereof, c) converting the compound of formula (I) or a protected form thereof into a physiologically acceptable derivative of the compound of formula (I) or a protected form thereof, d) converting a physiologically acceptable derivative of the compound of formula (I) or a protected form thereof into the compound of formula (I) or a protected form, thereof, e) converting a physiologically acceptable derivative of the compound of formula (I) or a protected form thereof into another physiologically acceptable derivative of the compound of formula (I) or a protected form thereof, f) performing an anomerisation reaction in order to convert an α-anomer of a compound of formula (I) into a /3-amoner or to convert a 0-anomer of a compound of formula (I) into an α-anomer, and g) where necessary, separating the ct and β anomers of the compound of formula I or a protected derivative thereof or of a physiologically acceptable derivative of a compound of formula (I) or a derivative thereof.
15. A process according to claim 14 for the production of a compound according to any one of claims 2 to 10. 16. A process according to claim 14 or claim 15 wherein the 4-thio sugar derivative is a compound of formula (V)
Figure imgf000087_0001
wherein R2, R3a and Z5 are as defined in claim 14 and W is a leaving group. 0 17. A process according to any one of claims 14 to 16 wherein the 4-thio sugar derivative is a l-acetoxy-4-thio sugar derivative.
18. A compound of the formula (V)
Figure imgf000087_0002
0 wherein R2 is hydrogen, R3 is a group OZ3 where Z3 is a hydroxyl protecting group, Z5 is a hydroxyl protecting group and and W is - S-CH2-Ar where Ar is an optionally substituted aryl group.
19. A compound according to claim 18 wherein Z3 and Z5 are benzyl and Ar is optionally substituted phenyl . 5
20. A compound according to claim 18 wherein Z3 and Z5 are • toluyl and Ar is optionally substituted phenyl .
21. A compound according to claim 19 or 20 in which Ar is phenyl.
22. A method of treatment of virus infections which comprises administration to a recipient in need of treatment an effective amount of a compound according to claim 1.
23. A process for the preparation of a compound of the formula (XIII)
Figure imgf000088_0001
where R is a hydrocarbyl group and Z3 and Z5, which may be the same or different, are acyl groups, which comprises the reaction of a compound of the formula (XIV)
Figure imgf000088_0002
where R is as defined above, with a compound or compounds of formula ZnOH where Zn is Z3 and/or Z5.
24. A compound of the formula (XIII)
Figure imgf000088_0003
where R is a hydrocarbyl group and Z3 and Z5, which may be the same or different, are acyl groups.
25. A compound of the formula (VII)
Ar-CH2-S. ,S-CH2-Ar CH
Figure imgf000089_0001
CH2OZ5 where Z3 and Z5 are acyl groups, A is a leaving group and Ar is an optionally substituted aryl group.
26. A compound according to claim 25 wherein A is a methanesulphonyl group.
27. A compound of formula (VIII) Ar-CH2-S N*- fS-CHj-Ar
(VIII)
Figure imgf000089_0002
CH2OZ5 where Z3 and Z5 are acyl groups and Ar is an optionally substituted aryl group.
28. A compound according to any one of claims 25 to 27 wherein Ar is a phenyl or toluyl group optionally substituted by one or more halogen atoms, C alkyl eg. methyl, C haloalkyl, CM alkoxy, nitro or amino groups.
29. A compound according to any one of claims 24 to 28, or a process according to claim 23 wherein the groups Z3 and Z5 are p-nitrobenzoyl.
PCT/GB1993/001858 1992-09-04 1993-09-03 Antiviral pyrimidine nucleosides WO1994005687A1 (en)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990001036A1 (en) * 1988-07-20 1990-02-08 Medivir Ab Nucleoside derivatives
EP0409575A1 (en) * 1989-07-17 1991-01-23 The University Of Birmingham Antiviral pyrimidine nucleosides
WO1991004033A1 (en) * 1989-09-15 1991-04-04 Southern Research Institute 2'-deoxy-4'-thioribonucleosides as antiviral and anticancer agents
EP0421777A1 (en) * 1989-10-04 1991-04-10 The University Of Birmingham Further antiviral pyrimidine nucleosides
WO1991016333A1 (en) * 1990-04-20 1991-10-31 Southern Research Institute 2',3'-dideoxy-4'-thioribonucleosides as antiviral agents
WO1992006102A1 (en) * 1990-10-02 1992-04-16 Medivir Ab Nucleoside derivatives
WO1992006993A1 (en) * 1990-10-22 1992-04-30 Pasteur Merieux Serums Et Vaccins Novel ribonucleoside compounds, process for their preparation and drugs containing said compounds
WO1992008727A1 (en) * 1990-11-13 1992-05-29 Consiglio Nazionale Delle Ricerche L-2'-desoxyuridines and pharmaceutical compositions containing them

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990001036A1 (en) * 1988-07-20 1990-02-08 Medivir Ab Nucleoside derivatives
EP0409575A1 (en) * 1989-07-17 1991-01-23 The University Of Birmingham Antiviral pyrimidine nucleosides
WO1991004033A1 (en) * 1989-09-15 1991-04-04 Southern Research Institute 2'-deoxy-4'-thioribonucleosides as antiviral and anticancer agents
EP0421777A1 (en) * 1989-10-04 1991-04-10 The University Of Birmingham Further antiviral pyrimidine nucleosides
WO1991016333A1 (en) * 1990-04-20 1991-10-31 Southern Research Institute 2',3'-dideoxy-4'-thioribonucleosides as antiviral agents
WO1992006102A1 (en) * 1990-10-02 1992-04-16 Medivir Ab Nucleoside derivatives
WO1992006993A1 (en) * 1990-10-22 1992-04-30 Pasteur Merieux Serums Et Vaccins Novel ribonucleoside compounds, process for their preparation and drugs containing said compounds
WO1992008727A1 (en) * 1990-11-13 1992-05-29 Consiglio Nazionale Delle Ricerche L-2'-desoxyuridines and pharmaceutical compositions containing them

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EA007491B1 (en) * 2001-01-22 2006-10-27 Мерк Энд Ко., Инк. Nucleoside derivatives as inhibitors of rna-dependent rna viral polymerase
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US8481712B2 (en) 2001-01-22 2013-07-09 Merck Sharp & Dohme Corp. Nucleoside derivatives as inhibitors of RNA-dependent RNA viral polymerase
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US7125907B2 (en) 2003-03-03 2006-10-24 Guilford Pharmaceuticals Inc. Thiolactones
US7553866B2 (en) 2003-03-03 2009-06-30 Eisai Corporation Of North America Thiolactones
US7968593B2 (en) 2003-03-03 2011-06-28 Eisai Inc. Thiolactones
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