IL144465A - 9-[4,4-bis(etherifiedhydroxy)-2-(hydroxymethyl)butyl]guanine derivatives - Google Patents

Abstract

A compound of the formula 2766 י" ב בתשרי התשס" ה - September 27, 2004 wherein R4 and R7 are lower alkyl or benzyl or R4 and R7 taken together are -CH2CH2- or -CH2CH2CH2- or -CH2CH2CH2CH2- and R9 is H or an alcohol protecting group, or the tautomeric form thereof wherein OR9 is =O.

Description

GUANINE DERIVATIVES The present invention is a divisional patent application from Israeli Patent Application 124760 parent The parent application relates to diesters of process for their preparation and pharmaceutical compositions containing The present invention relates to compounds of formula Π protecting grou The compounds of the invention are useful intermediates in the preparation of compounds of tile parent 1a Technical Field This invention relates to the field of antivirais and in particular to derivatives of acyclic nucleosides useful against herpes and retroviral Tne invention provides novel pharmaceutical compositions comprising these methods for the or prophylaxis of viral infections employing methods for their manufacture and novel Background to the invention The practical utility of many acyclic nucleosides is limited by their relatively modest A number of prodrug approaches been explored an effort to improve the bioavailability of acyclic nucleosides in One of these approaches involves the preparation of ester particulariy aliphatic of one or more of the hydroxy groups on the acyclic side European patent EP165 to describes the promising antiherpes agent otherwise known as European patent EP 640 to 1L discloses European patent EP 343 discloses that these particularly the are additionally active against retroviral infections such as Various derivatives of such as aliphatic esters the diacetate and the and ethers of the hydroxy groups on the acyclic side chain are EP 343 This patent also discloses methods for the preparation of these derivatives comprising the condensation of the acyclic side chain to the position of a typically purine moiety the imidazole ring closure of a pyrimidine or pyrimidine moiety or the pyrimidine ring closure of an imidazoie where the acyciic side chain is already present in the precursor pyrimidine or imidazole In the broadest description of of these methods the acyclic side chain is but individual examples aiso show a diacylation of K2G with acetic or proprionic anhydride and et 173S investigated a number of short chain aliphatic esters of the acyclic nucleoside otherwise known as and its a marketed antiviral is the ciiacetyt derivative of et 4 120 discloses short chain aliphatic esters of otherwise as The dipropionate ester is disclosed to be the preferred et discloses in Agents 33 diacetate and dipropionate derivatives of H2G and monoacetate and diacetate derivatives of The diacetate and dipropionate derivatives of H2G are reported to result in only modest improvements in bioavailability relative to International patent application published October discloses aliphatic ester prodrugs of the analog of including the and stearoyl International patent application published April 1993 and International patent application published October both disclose derivatives of nucleoside analogs derived from unsaturated C18 or C20 fatty Patent issued June 1 also discloses long chain fatty acid derivatives of nucleoside A second approach to providing prodrugs of acyclic nucleosides involves the preparation of amino acid esters of one or more of the hydroxy groups on the side European patent EP 99 493 discloses generally amino acid esters of acyclovir and European patent application EP 308 published March discloses the valine isoleucine esters of European patent application EP 375 published June discloses acid ester derivatives of including the glycine and ester International patent application published April discloses amino acid ester derivatives of including the and ester DE published February 1 1996 and Patent issued August disclose achiral amino acid esters of European parent application EP 694 published January discloses the ester of ganciclovir and its preparation from European patent application EP 654 published May discloses various bis amino acid ester derivatives of International patent application published August discloses aliphatic amino acid and mixed esters of the acyclic nucleoside This reference discloses that bioavailability is reduced when one of the valine esters of the trivaline ester derivative is replaced with an acetate Brief Description of the Invention We have found that diester derivatives of H2G bearing specific combinations of an amino acid ester and a fatty acid ester are able to provide significantly improved oral bioavailability relative to the parent compound In accordance with a first aspect of the invention there is thus provided novel compounds of the formula I 4 where is or and is saturated or aliphatic which is optionally substituted with up to 5 similar or different substituents independently selected from the group consisting of mercapro and nitro or is saturated or 1997 optionally substituted aliphatic group as defined and is or and is H or OH includes the tautomeric group and pharmaceutically acceptable salts 15 The advantageous effect on oral bioavailability of the mixed fatty acid and amino acid esters of the invention is particularly unexpected in comparison to the oral bioavailability of the corresponding fatty acid Based on the results using a 1997 urinary recovery assay 1 or a plasma assay of H2G from neither the mono or acid esters of H2G provide any improvement in 20 oral bioavailability relative to the parent compound Indeed the derivative provided significantly lower bioavailability than the parent indicating a stearate ester may be detrimental for improving oral bioavailability of Converting one or both of the hydroxyls in certain other acyclic nucleoside analogues to the corresponding valine or valine ester has been reported to 25 improve Conversion of H2G to the coresponding ester derivatives produced similar improvement in bioavailability relative to the parent Given fatty acid derivatives of H2G are shown to be detrimental for improving it was unexpected that a amino acid acid diester derivative of would provide improved comparable oral btoavailabilicy to of the valine diester derivative of based recovery plasma drug Table see Biological Exam le 2 below for details The invention also provides pharmaceutical compositions comprising the compounds of Formula I and their pharmaceutically acceptable salts in conjunction with a pharmaceutically acceptable carrier or diluent Further aspects of the invention include the compounds of Formula I and their pharmaceutically acceptable salts for use in and the use of these compounds and salts in the preparation of a medicament for the treatment or prophylaxis of viral infection in humans or 6 The compounds of the invention potent especially herpes such as caused by Varicella zoster Herpes simplex virus rypes 1 Herpes type 6 and type 8 The compounds are particularly useful against Varicella zoster virus infections such as shingles in the elderly including post herpetic neuralgia or chicken pox in the young where the duration and severity of the disease can reduced by several Epstein Barr virus infections amenable to treatment with the compounds include infectious fever which has previously not been treatable but which can cause many months of scholastic incapacity amongst The compounds of the invention are also active against certain retroviral notably and and against infections where a transacrivating virus is Accordingly a further aspect of the invention provides a pharmaceutical composition for the prophylaxis or treatment of a viral infection in humans or animals by the administration of an effective amount of a compound of Formula I or its acceptable salt to the human or Advantageously group is hydroxy or its tautomer so that the base portion of the compounds of the invention is the naturally occuring for instance in the event that the side chain is cleaved in may be hydrogen thus defining the generally more soluble derivative which can be oxidised in vivo by xanthine to the guanine The compound of formula I may be present in that is a mixture of the 2R and 25 the compound of formula I has at least preferably at least R for example greater than Most preferably the compound of formula 1 is enantiomerically pure R Preferably the amino acid of group is derived from an 7 Preferably the fatty acid of group has in total an even number of carbon in decanoyl lauryl myristoyl stearoyl or eicosanoyl Other useful groups include octanoyl or behenoyl Further useful RJ groups include those 5 derived from erucic or briissidic Monounsaturated fatty acid esters typically have the double bond in the trans preferably in the or dependent upon their Preferably the R2 group is derived from a fatty acid which comprises a to aliphatic The saturated or unsaturated fatty acid or may optionally be substituted with up to five similar or different substituents independently selected from the group 1997 consisting of such as allcoxy mercapto and Most preferred compounds of the formula I are those where is or and is saturated 20 The term as used herein refers to straight or branched chain alkyl radicals containing from 1 to 7 carbon atoms but not limited and 25 the The term or as used herein refers to those groups intended to protect the of an amino acid or peptide or to protect an amino group against undesirable reactions during synthetic 30 Commonly used groups are disclosed in Groups in Organic Wiley New which is hereby incorporated by groups include acyl groups such as 8 and the groups such as and the carbamate forming groups such as 3 1 methoxycarbon and the substituted methyl such as and the and groups such as trimethylsilyl and the Favoured groups include and benzyloxycarbonyl The term ester as used herein refers to acid such as acid and activated esters but not limited formic acetic acid derived anhydrides anhydrides derived from halides such as isobutyloxycarbonylchloride and the derived derived derived derived derived esters and the 9 Preferred compounds of formula I 1 1997 1 10 5 1 1 or and their pharmaceutically preferred compounds 10 1 15 20 25 1 J 30 11 5 10 or and their pharmaceutically acceptable Other preferred compounds of formula I include I 20 30 12 1 J or and their pharmaceutically acceptable The compounds of formula I can form salts which form an additional aspect of the Appropriate pharmaceutically acceptable salts of the compounds of formula 1 include salts of organic especially carboxylic including 5 not limited to undecanoate and 0 organic sulphonic acids such as and and inorganic acids such as phosphoric and sulphonic Hydrochloric acid salts are convenient The compounds of Formula I may be isolated as the The compounds of the invention may be isolated in crystal preferably thus an additional aspect of the invention provides the compounds of Formula I in substantially pure crystalline comprising preferably homogeneous crystalline material for example homogeneous crystalline The compounds of the invention are particularly suited to oral but may also be administered transdermally or for instance intravenously or The compounds may be administered for instance in a but will generally be in conjunction with a pharmaceutically acceptable carrier or The invention extends to methods for preparin a pharmaceutical composition comprising bringing axompound of Formula I or its pharmaceutically acceptable salt in conjunction or association with a pharmaceutically acceptable carrier or Oral formulations are conveniently prepared unit dosage such as capsules or employing conventional carriers or binders such as magnesium gum or Liposomes or synthetic or natural polymers such as HPMC or PVP may be used to afford a sustained release Alternatively the formulation may be presented as a nasal or eye gel or cream comprising a or preparation in conventional vehicles such as vegetable oil or optionally with flavourant preservative The compounds of the invention may be administered at a daily dose generally in the range to 200 to 100 more preferably 10 to such as to 25 A typical dosage rate for a normal adult will be around 50 to 500 for example 300 once or twice per day for herpes infections and 2 to times this dosage for As is prudent in antiviral the compounds of the invention can be administered in combination with other antiviral such as ganciclovir and its foscamet and the like for herpes indications and Venex VX Agouron AG 1343 and the like for retroviral The compounds of the invention can be prepared de novo or by esterification of the H2G parent compound which is for by the synthesis methodology disclosed in European Patent EP 343 which is incorporated herein by A typical reaction scheme preparation of H2G is depicted H2G The condensation in step 1 is typically carried out with a base catalyst such as NaOH or in a solvent such as Step 2 involves a reduction which can be performed with in a solvent such as The substitution in step 3 of the chlorine with an amino group can be performed under pressure with Step 4 employs adenosine deaminase which can be conveniently n a solid support Cooling the reaction mixture allows unreacted isomeric precursor to remain in solution thereby enhancing Starting materials for compounds of the invention in which is hydrogen may be prepared as shown in European Patent EP the contents of which are incorporated herein by These starting materials may be acylated as described for H2G optionally after protecting the purine amino group with a conventional group as defined especially BOC Z or The compounds of the invention may be prepared from H2G as described below in Schemes A and Direct acylation method Scheme A Scheme A depicts the preparation of compounds in which Ri is derived from the amino acid and is derived from the fatty but the converse scheme is applicable to where is derived from the fatty acid and is derived from the amino acid In the variant specifically depicted in scheme A G is guanine or PG is an optional protecting group or is the valine or isoleucine side chain and is the fatty is depicted above as a starting material but this of course may be optionally protected at or the 2 position of the purine with conventional groups The H2G reacts in the first step with an activated Ri acid as further described in a solvent such as dimethylformamide or to give a monoacylated The acid may be suitably with or or the Under controlled the first acylation can be made to predominantly take place at the side chain group on the side chain of These controlled conditions can be for by manipulating the reagent concentrations orrate of 17 especially of the acylating by lowering the temperature or by the choice of The reaction can be followed by TLC to monitor the controlled After the monoacylated compounds are further acylated on the side chain group with the appropriate activated fatty acid derivative to give diacylated products using similar procedures as for the first esterification The diester products are subsequendy subjected to a conventional deprotection treatment using for example or hydro genation in the presence of catalyst to give the desired compound of Formula The compound may be in salt form depending on the deprotection The activated acid derivative used in the various acylations may comprise the acid acid activated acid ester or the acid in the presence of coupling for example where in each case represents the corresponding amino acid or the fatty Representative activated acid derivatives include the acid formic and acetic acid derived mixed anhydrides derived from alkoxycarbonyl as isobutyloxycarbonylchloride and the derived derived derived derived esters and the 13 Via protection of the side chain Scheme B deprotectiorv Formula 1 wherein are as described for scheme Scheme B has been exemplified with reference to the preparation of a compound where is derived from an amino acid and is derived from the fatty acid but a converse scheme will be applicable to compounds where is derived from the amino acid and is derived from the fatty This scheme relies on regioselective protection of the H2G side chain group with a bulky protecting In scheme B above this is depicted as but other regioselective protecting groups such as l may also be Tne resulting product is at the side chain 19 group using analogous and procedures as described in A but wherein activated acid derivative is the fatty for rn acid chloride and the The thus compounds are subjected to appropriate deprotection 5 treatment to remove the chain protecting group which be done in a highly selective manner with such on the rcgioselective protecting as and the and manipulation of the reaction viz reagent speed of and solvent as elaborated The then side chain 10 group is with the activaied acid in a way as described in A Additional techniques for introducing the group of for instance in schemes C or D herein include the method described in international patent application WO 94 Additional techniques for introducing the fatty acid ester of for instance in schemes C or D herein include the enzymatic routs in 20 Preparative Biotransformations S J Wiley and 1 with a lipase such as SP 435 immobilized Candida antarcticus porcine pancreatic lipase or Candida rugosa Enzymatic is especially convenient where it is desired to avoid and deprotection steps on the other acyl group or the purine An alternative route to compounds of Formula I in which is hydrogen is to the guanine compound of Formula I the amino acid ester moiety of is optionally protected with conventional gronps such as with an activating group such as The thus acti ated is subsequently reduced to for instance with a palladium catalyst and to the desired 20 A further aspect of the thus provides a method for the preparation of the compounds of formula I comprising optionally the purine 2 6 positions of a compound formula I wherein and are each regioselectively acylating the of 1 at the side chain group with either an optionally valine or isoleucine an optionally saturated or monounsaturated or a regioselective protecting acylating at the side chain group with an optionally valine or isoleucine or an optionally saturated or monounsaturated C2tCOOH replacing the regioselective protecting group at if with an optionally valine isoleucine or an optionally saturated or monounsaturated C21COOH and the resulting compound as Schemes A and B above employ selective acyiation to stepwise add the amino acid and fatty acid An alternative process the preparation of the compounds of formula I starts with a diacylated H2G wherein both the groups are the and selective removal of one of the acyl groups to obtain a monoacyl intermediate which is then acylated with the acyl group in the same manner as Schemes A and B Accordingly a further aspect of the invention provides a method for the preparation of a compound of the formula as defined which method comprises the monodeacylation of a diacylated compound corresponding to formula 1 wherein are both a or isoleucyl ester is optionally or and are both saturated or optionally substituted aliphatic and 1997 acylating the thus liberated side chain or side chain group with the corresponding or saturated or optionally substituted aliphatic and as This alternative process has the advantage that the preparation of the diacylated H2G derivative is facile and requires little or no purification Selective removal of one only of the acyl groups of a diacylated H2G derivative can be achieved by manipulating the reaction in particular the rate of reactant addition and choice of Compounds amenable to this alternative synthesis route are thus of the 15 ly or a 97 stituted aliphatic and is 20 For ease of synthesis in this alternative it is preferred that and are both initially identical and are most preferably the same amino acid Such a acid ester will generally be during its preparation and may be used directly in this condition in the selective such an H2G derivative may be deprotected optionally 25 as The unprotected H2G derivative thus comprises one of the following 22 These unprotected H2G diacylated derivatives can be directly subject to selective deacylation of one of the acyl groups the side chain followed by enzymatic acylation of the liberated as described the unprotected H2G diacylated derivative can be and then subjected to the selective followed in turn by conventional acylation with the fatty acid as described in Schemes A and such a reprotection step is done with a different having properties appropriate to the subsequent For it is convenient to employ a lipophilic such as Fmoc when preparing a acid H2G as the lipophilic nature of the protecting group assists with separation of the acylated On the other the lipophilic nature of Fmoc is of less utility when conducting an acylation with a fatty and thus it is convenient to reprotect a diacylated H2G with an alternative group such as It also be apparent that the preparation of the compounds of formula I can commence with the novel monoacylated intermediates of step b or in the above defined first method aspect the These compounds are thus of the 23 a saturated or optionally subsotuted aliphatic or a regioselective protecting the other of and is and Useful compounds thus 24 and Regioselectively sidechain intermediates step of the above described first method aspect of the invention are also novel Useful compounds thus An alternative process for the preparation of compounds of invention of the formula I 1997 wherein is is shown in Scheme 26 Referring to Scheme and R5 lower alkyi or benzyl or he is alkylated by with from about to about equivalents of 2 5 and R7 are lower or benzyl and tbe like or and taken together or or is a leaving group Br or L or a sulfonate such as benzenesulfonate and tbe in the presence of from about to about molar equivalents of a base potassium or sodium ethoxide or NaH or KH and the an inert sol vent D or THF or dioxane or dioxolane or and the at a temperature of from about to about to provide alkylated malonate Reduction of 3 with about to about molar equivalents of an ester to alcohol Feb 14 reducing agent or or or and the in an 1997 inert solvent THF or methyl ether or and the at a temperature of from about to about provides diol Enzymatic of 4 by reaction with from about to about molar equivalents of a vinyl ester 5 is saturated or optionally substituted in the presence of 20 lipase Lipase or Lipase PPL or Lipase CCL and the or a example phospholipase D and the provides the desired stereoisomer of ester This reaction can be carried out in the absence of solvent or in the presence of an inert solvent methyl ether or toluene or and the Tie reaction is carried at a of from about to about 25 The alcohol subsatuent of 6 is converted to a leaving group a halogen or a by reaction with a halogenating agent example or or or NCS in acetone and an inert solvent methylene chloride or toluene or and the or by reaction with from about 30 molar equivalent to about molar equivalents of a haiide toluenesuifonylchioride or methane suifcnylchioride and the 27 in the presence of from about to about molar equivalents of a base triethyiamine or potassium carbonate or pyridine dimethylarninbpyridine or amine and the an inert solvent example methylene chloride or toluene or ethylacetate or pyridine or methyl ether and the at a temperature of 5 from about to about to provide ester is a halogen or sulfonate leaving Reaction of 7 with from about to about molar equivalents of chloropurine 8 in the presence of from about to about molar equivalents of a base 10 potassium carbonate or aH or H or or or lithium and the in an inert solvent DMF or or or or ethanol and the at a of from about to about provides substituted purine 15 Alternatively Mitsunobu coupling example of 14 1997 alcohol 6 with 8 provides Reaction of 9 with from about to about 20 molar equivalents of an alcohol R9OH is an alcohol protecting such as benzyl and the in the presence of from about 20 to about molar equivalents of a base potassium or potassium carbonate or NaH or KH lithium and the in an inert solvent THF or DMF and the at a of from about to about provides alcohol Removal of the alcohol protecting group of JO by catalytic hydrogenation in an inert solvent such as benzyl alcohol or methanol or THF and the like in the presence of hydrogenation catalyst such as Pd C or and the provides substitucd guanine 30 Estcrincaiion of reacdon with about to about molar equivalents of and a coupling agent example DCC and the like in an inert solvent example THF or DMF and the or from about to about molar 29 shown Maionate and are or benzyl and the is alkylated with about to about molar equivalents of ether 15 wherein is a leaving example Br or or sulfonate such as methane benzsnesulfonate and the and is or and the like the presence of from about to about equivalents of a base example potassium or sodium ethoxide or or and the in an inert solvent example or THF or dioxane or dioxolane pyrrolidinone and the at a temperature of from about to about to provide alkylated maionate Reduction of with from about to about molar equivalents of an ester to alcohol reducing agent or or or and the in an inert solvent example THF or meth l or and the at a temperature of from about to about provides dioi Enzymatic of 17 by reaction with from about to about molar equivalents of a vinyl ester 5 is saturated or optionally substituted in the presence of a lipase lipase or lipase PPL or Lipase CCL and or a phosphalipase example phospholipase D and the provides the desired stereoisomer of ester The reaction can be carried in the absence of solvent or in the presence of an inert solvent example methyl ether or toluene or hexane or the The reaction is carried out at a temperamre of about to about The alcohol substituent of is convened to a leaving group example halogen or by reaction with a halogenating agent example NCS or in acetone and the in an inert solvent example methylene chloride or toluene or ethyiacstate and the or by reaction with from about molar equivalent to about molar equivalents of a suifonyl halide example or methane suifonylchloride and the in the presence of from about to about molar equivalents of a base example triethvlamine or potassium carbonate or pyridine or methyl ether and the at a of from about to about to provide ester is a halogen or sulfonate leaving Reaction of 19 with from abour to about molar equivalents of 8 in presence of rom about to about molar equivalents of a base potassium carbonate or NaH or or aOH or KOH or lithium 5 diisopropylamide and the in an inert solvent or THF or acetonitrile or or ethanol and the at a temperature of from about to about provides substituted purine Mitsunobu coupling of 10 alcohol with 8 provides Reaction of 20 with from about to about molar equivalents of an alcohol RoOH is a alcohol protecting group such as benzyl and the in the presence of from about to about molar equivalents of a base potassium or 14 15 potassium carbonate or NaH or KH or lithium and the like in an inert 1997 solvent THF or DMF and the at a temperature of from about to about provides alcohol Removal of the alcohol protecting group of example by catalytic hydrogenation 20 in an inert solvent such as ethanol or benzyl alcohol or methanol or THF and the like in the presence of an hydrogenation catalyst such as C or and the provides substituted guanine The ether of 23 is deprotected by reaction with a reducing agent HC02H and and the wherein is or or a desilyiating agent example and the wherein is and the like to provide Alcohol can be converted to as outlined in scheme 30 An additional alternative involves enzymatic esterification of alcohol 4 or 7 with the Yinyl ester in Schemes C and to directly incorporate 12 or Detailed Description of the Invention The invention will now be illustrated by way of example only with reference to the following comparative examples and the accompanying in which Figure 1 depicts plasma K2G levels a function of time in cynomdgus monkeys administered with compound of the invention or with an alternative prodrug derivative of as further explained in Biological Example and Figure 2 depicts survival as a function of time for Herpes simplex infected mice administered wi h various doses of a compound of the invention or a prior arc as further explained in Biological Example Passages of description which are not within the scope of claims do not constitute part of the 33 EXAMPLE 1 This example the application of preparation scheme was dissolved in DMF under heating and was cooled to room temperature before addition of DMAP and DCC S2 The mixture was stirred at room temperature for 24 h and was then filtered The product was chromatographed on silica gel and eluted with MeOH to give g of the desired intermediate δ The product from step was dissolved in pyridine the solution was cooled in an ice bath and stearoyl chloride was The solution was kept in the ice bath for 2 then at room temperature for 1 It was then evaporated and chromatographed on silica It was eluted with dichloromethane to give mg of the desired intermediate The product from step was cooled in an ice bath and trifluoroacetic acid was The solution was kept in the ice bath for 45 was then evaporated to give an to 1 was added and evaporated The residue was once more dissolved in water filtered and to give 148 mg of the desired product as the 34 MR 28 EXAMPLE 2 guanine The titled compound was obtained as the bistrifluoracetate salt in a manner analogous to Example 1 using myristoyl chloride instead of stearoyl chloride in step NM δ EXAMPLE 3 guanine The titled compound was obtained as the bistrifluoroacetyl salt in a manner analogous to Example 1 using oleoyl chloride instead of stearoyl chloride in step NMR 1 EXAMPLE 4 DCC 10 was dissolved in dichloromethane 10 and butyric acid was After 4 hours at room temperature the mixture 35 was filtered and filtrate was The residue was dissolved in pyridine and guanine step was The mixture was stirred for 120 hours at room According to TLC the reaction was incomplete and more anhydride was made using the procedure This anhydride was added and the mixture was stirred for an additional 20 The reaction mixture was evaporated and chromatographed first on silica gel and then on aluminium in both cases eluted with dichioromethane methanol to give 79 mg of the intermediate The intermediate product of step a was deprotected in a manner analogous to Example step c to give 84 mg of the desired product as the bistrifluoracetate NMR δ EXAMPLE 5 The titled comp was o 1btained as the bistrifluoroacetate salt in a manner analogous to Example 1 using decanoyl chloride instead of stearoyl chloride in step NMR S 36 EXAMPLE 6 janine The titled compound was obtained as the bistrifluoroacetate salt in a manner analogous to Example 1 but using in step b the DMAP DCC conditions of Example step in conjunction with docosanoic acid in place of the stearoyl chloride and a mixture of and dichloromethane as NMR δ 36 EXAMPLE 7 This example illustrates the application of preparative scheme butyl H2G 8 was coevaporated with dry DMF two times and was then suspended in dry DMF pyridine To the suspension was added dropwise in dichloromethane at 0 over a period of 30 The reaction mixture became a clear solution at the completion of the dropwise The reaction continued at 0 for two hours and was then kept at 4 Methanol was added to the After 20 min at room the reaction mixture was evaporated to a small poured into aqueous sodium hydrogen carbonate solution and extracted with dichloromethane two The organic phase was dried over sodium sulphate and evaporated in The product was isolated by silica gel column chromatography using a system with a stepwise increasing MeOH concentration The product was eluted with in to yield 37 5 was coevaporated with dry pyridine twice and dissolved in pyridine To the solution was slowly added dropwise stearoyl chloride technical in dichloromethane at The reaction was kept at the same temperature for 1 hr and then at 5 for 2 The reaction was monitored by Additional stearoyl chloride at C was added due to incompletion of After 30 min at 5 methanol was added and the reaction mixture stirred for 20 It was then poured into aqueous sodium hydrogen carbonate and extracted with The organic phase was dried and the product purified by silica ge column chromatography with eluting with in was dissolved in dry and hydrogen added to the The reaction was kept at overnight and monitored by The reaction reached about 80 Additional pyridine was added After 4 TLC showed that the staninf material had The reaction mixture was concentrated in vacuo without raising the temperature and more pyridine was added and evaporated The product was isolated by silica gel column guanine and were coevaporated with dry DMF twice and dissolved in the same solvent To the solution was added and After reaction for the reaction mixture was filtered through Celite and worked up in a conventional product was 3S isolated by silica gel coiumn eluting at 5 MeOH in from step was treated with trifluoroacetic acid at for 20 The solution was evaporated in The residue was coevaporated with toluene twice and kept under vacuum for several The residue was dissolved in MeOH and evaporated to give the triflnoracetate salt as a product 191 NMR δ EXAMPLE 8 The title compound was obtained as the bistrifluoroacetate salt in a manner analogous to Example 7 using decanoyl chloride instead of stearoyi chloride in step NMR δ 1 2H 1H 122 12H 39 EXAMPLE 9 r The title compound was obtained as the bistrifluoroacetate salt in a manner analogous to Example 1 using instead of and myristoyl chloride instead of stearayl chloride in step δ br 148 EXAMPLE 10 15 The tided compound was obtained as the bistrifluoroacetate salt in a manner analogous to Example 1 but using in DMAP conditions of Example step in conjunction with acid instead of stearayl 6H 20 EXAMPLE 1 1 25 The compound was obtained as the salt in a manner analogous to Example 1 using dodecanoyl chloride instead of chloride in step 40 EXAMPLE The tided compound was obtained as the bistriflouroacetate salt in a manner analogous to Example 1 using palmitoyl chloride instead of stearoyl chloride in step 250 δ 2H EXAMPLE 13 This example shows the deoxygenation of group To a solution of from step b of Example I in acetpnitrile were added tetramemylammonium chloride dieth and phosphorus oxychloride The reaction was kept under reflux and the progression monitored by After 3 hours the reaction mixture was evaporated in vacuo and residue was dissolved in then poured into cold sodium hydrogen carbonate aqueous The organic phase was evaporated and purified by silica gel column δ 1 2H To the solution of in acetate were added ammonium formate and palladium on carbon The reaction was kept under reflux for 1 hour and recharged with ammonium formate After one hour more the TLC showed completion of the reaction and the mixture was filtered through Celite and washed extensively with The filtrate was evaporated and purified by silica gel was treated with trifluoroacetic acid at for 40 It was then evaporated in vacuo and coevaporated successively with toluene and The residue was overnight to give 195 mg of the desired product EXAMPLE 14 Preparation of ethyl Potassium was dissolved dry D F 1 Diethyl was added 5 Bromoacetaidehyde was added over 5 Tne mixture was heated to C stirred at C for 5 The mixture was allowed cool to room poured wacer and extracted with ether 3 x 600 Tne organic solution was dried ove disdlled to yield the desired diestsr as a colorless Ή 5 Preparation of 2M in and the product of Example 14 step g in 5 mL of were combined and to C and stirred at C for 4 Tne reaction mixture was allowed to cool to room temperature and the reaction vessel was placed in a cool water Then I was added at such a rate that the of the reacdon mixture was maintained between Brine was added at a rate such that gas evolution was controiled and the mixture was stirred for 45 minutes at room The layers the organic layer was washed with brine The brine washes were extracted with MTBE 3 x 20 The combined extracts were evaporated the residue was dissolved in and washed with brine The brine layer was with MTBE x 25 The combined organic extracts were dried over and concentrated to yield the desired as a coioriess Ή NMR δ 6H Preparation of a 10 ml 1 neck round bottom flask was charged the product of Example 14 step 20 followed by addition of vinyl acetate 30 and Lipase PS 30 urchased from The mixture was stirred at ambient temperature for Progress of the was monitored by TLC stained with charred on hot of diol is rnonoacetaic is bis acetate is The reaction mixture was diluted with and through a 5 micron The filter was washed with additional The filtrate was then concentrated in vacuo to afford the desired Preparation of toiuenesuifonate 45 300 MHz Ή NMR δ 1 Preparation of a 100 mL 1 round boccom flask was charged the crude produc of Example 14 step g of che benzyl alcohol containing of the product step dissolved in absolute To this was 14 added g of slurried in 5 mL absolute The reaction flask was evacuated and charged with three times with a balioon of The reaction was pressurized with 1 and the reaction mixture was sdrred The reaction mixture was filtered through a pad of earth to remove 15 The were removed in The residue was mixed with 25 of isopropyl acetate and then concentrated in The residue was diluted with seeded with the desired heated to reflux and MTBE The mixture was for 30 The precipitate was filtered and dried to a constant weight of 600 mg of the desired 20 300 MHz lH NMR δ 1 1 2 MS 41 6 47 of Into a 25 mL 1 neck round bottom flask was charged the product of Example step pyridine and DMAP The mixture was cooled to and stearoyl chloride dissolved in was added over 5 The resulting mixture was stirred hours at Absolute EtOH was added and the was stirred an additional 1 The reaction mixture was concentrated in Toluene was added to the residue and then the mixture was concentrated in toluene was added to the and then the mixture was concentrated in To the residue was added 1 and this mixture was extracted with The organic layer was separated and was dried filtered and concentrated in vacuo to a constant weight of The crude product was chromatographed on 40 g of eluting with affording 367 mg of the desired product 300 MHz N R δ 30 1 1 1 Preparation of Into a 25 mL 1 neck round bottom flask was charged the product of step dissolved in 1 7 To this solution was 14 1997 49 McOH 5 to obtain of the desired EXAMPLE 15 Alternative preparation of butyll K2G di ethylfonnamide were charged into a Bucchi evaporator and the mixture warmed to dissolve the The solution was concentrated to dryness under at no more The resulting powder was transferred to a 22 litre with addition funnel and and temperature 1 was added followed by pyridine The resulting suspension was cooled to under nitrogen and stirred at as was added resulting mixture was stirred at until the reaction was complete monitored by TLC 1 methylene chioride and x 250 mm Zorbax at m UV detecdon at 254 Water was added and the mixture was stirred for 30 minutes to precipitate the then the mixture was cooled to 30 The solid was isolated by filtration and the product cake was washed cold water and sucked dry with air to provide the crude product as an The crude solid was taken up in pydridine and concentrated under vacuum at to remove The dry solid residue was slurried with methanol at for hours and filtered while Tne filtrate was concentrated under vacuum and the soiid residue was with isopropyi acetate for 30 The mixture was cooled to and The filter cake was dried under vacuum at to provide the title compound as a white soiid The product of Example step was charged to a 50 litre Buchi Pyridine was added dropwise to dissolve the solid and the mixture was distilled to dryness under at The residue was taken up in fresh pyridine and transferred to a 22 litre flask with addition and temperature The solution was cooled under A solution of chloride in methylene chloride was 5 added so as to maintain a temperature below 15 was added and the mixture was stirred at for hours until conversion svas complete monitored by TLC 1 methylene and HPLC x 250 mm Zorbax detection at 254 At the 10 end of the was added and the mixture was stirred for not less than minutes to precipitate the The slurry was cooled to for 2 hours and the solid isolated by filtration the filter cake washed with acetonitrile The desired product was obtained as a white solid 14 1997 15 A solution of the product of Example step in tetrahydrofuran was prepared in a A solution of fluoride in tetrahydrofuran kg of 1 M was added and the resulting clear soluuon was stirred at 20 for 4 20 Water was added and the resulting slurry was stirred for 1 hour and then cooled to for 30 The precipitate was isolated by filtration the filter cake was washed successively with water and acetonitrile After drying under vacuum at 702 g of crude product was The crude product was dissolved in refiuxing THF and water 160 then cooled to 25 and created with methylene chloride The mixture was allowed to cool for 1 then it was cooled to for 1 hour to complete Tne slightly powder was isolated by filtration and dried under vacuum at to yield the desired product A solution of in dry THF was prepared in a 2 litre flask with mechanical thermometer and addition A solution of in was added over 5 minutes and the resulting slurry was stirred at for 2 The siurry filtered and the filter cake was washed with TKF Tne filtrate and wash were charged to a 3 litre flask with stirrer and The product of Example step was added as a with a of THF was added and the white slurry stirred at the solids were all dissolved and the reaction was complete within 1 hour determined fay x 250 mm Zorbax at 1 UV detection at 254 starting material elutes at and product elutes at The reaction was quenched by addition of water and the was concentrated under vacuum to leave a light yellow This was taken up in methanol and warmed to reflux for 30 The solution was cooled to and the precipitate was removed by The filtrate was concentrated under vacuum to leave a pale yellow was added and white suspension was stirred at 20 for 90 The crude solid product was isolated by washed with x 10Q and overnight to provide the desired product as a sticky solid This was further purified by crystallization from ethyl acetate and drying under vacuum at to provide the desired product as a waxy solid 104 A soludon of the product of Example step in warm ethanol was charged to an hydrogenation reactor with 5 The mixture was agitated at under 40 psi hydrogen for 4 evacuated and hydrogenated for an additional The catalyst was removed by filtration and the filtrate was concentrated under vacuum to provide a white This was stirred with ethanol at for 1 cooled to and The filter cake was dried with then under vacuum at to yield the title compound as a white powder 52 EXAMPLE was dissolved in dry with pyridine and Stearoyl chloride was added and the mixture kept overnight room Most of the solvent was evaporated in the residue stirred with 70 ml ethyl acetate and 70 ml and the solid filtered washed with ethyl acetate and water and dried to yield 680 mg of crude Column chromatography on silica gel gave pure title compound as a white NMR NMR 1 A mixture of and carbodiimide in dichloromethane was stirred overnight at room dicyclohexylurea filtered off and extracted with a small volume of and the filtrate evaporated in vacuo to a small mg and dry N were added and the mixture was stirred for 4h at under The solvent was evaporated vacuo to a small Column chromatography on silica 53 then on aluminum oxide water 1 as gave 185 pure title compound as a white NMR 1 2 NMR Val Chilled trifluoroacetic acid was added to and the solution kept at room temperature for evaporated to a small and lyophilized repe tedly with dioxane until a white amorphous powder was The yield of title obtained as the trifluoracetate was NMR NMR Val 54 1 1 l J EXAMPLE 18 Alternative preparation of H2G 30 was heated to solution in dry solution was filtered to remove solid cooled to C and stirred at that temperature during addition of pyridine 114 dimethylaminopyridine and stearoyl chloride 66 Stirring was continued at room temperature Most of the solvent was then evaporated off in the residue stirred with 200 ml ethyl acetate and 200 and the solid filtered washed with ethyl acetate and water and dried to yield crude As an alternative to the crude product was briefly heated to almost boiling with 100 ml of ethyl water 1 and the suspension slowly cooled to C and filtered to leave most of the isomer in solution isomer would crystallize at lower The was repeated once more to after drying g of almost isomer free EXAMPLE 19 Preparation of crystalline The product of Example step was dissolved in absolute ethanol with and further diluted with ethanol To this solution was added water and the mixture was allowed to cool to After the mixture was water was added at a constant rate over hours with efficient After all the water was stirring was continued for 4 hours at room The resulting precipitate was filtered through paper and dried under vacuum at room 55 temperature to obtain the title compound as a free flowing crystalline powder m pt EXAMPLE 20 guanine To a solution of in DMF were added 225 pyridine and DCC After 16 the reaction was recharged with and DCC and was kepi an additional 5 The reaction mixture was filtered through Celite and poured into sodium hydrogen carbonate aqueous and then it was extracted with The organic phase was evaporated and purified by silica gel column giving 950 mg of the monoamino acyl The above intermediate was dissolved in To the solution was added hydrogen fluoride in pyridine I 30 After two the solution was evaporated coevaporated with Purification by silica gel column chromatography gave mg of the protected monoamino acyl R δ The product of step was treated with a mixture of trifluoroacetic acid and dichloromethane for 1 The solution was evaporated and give mg of the unprotected monoaminoacyl product 56 δ EXAMPLE 21 guanine To a solution of 10 in DMF were added 12 methylarninopyridine and DCC After reaction for 16 hr at and DCG were and the reaction was kept overnight at room The reaction mixture was filtered through Celite and the filtrate was evaporated and purified by silica gel column giving g of the monoamino acyl δ 5 The intermediate from step was treated with trifluoroacetic acid and for 30 min at The solution was evaporated and give the titled unprotected product in quantitative 57 EXAMPLE 22 The product of step was deprotected with trifluoro acid in the same manner as Example step δ 2H EXAMPLE 23 Application of the technique described in Example step but using and eqs of and resuhed in the title NMR The compound was obtained as the acetate salt from the intermediate of Example 23 step by deprotection in a manner analogous to Example 1 step NMR 1 EXAMPLE 24 guanine The titled compound is prepared according to steps to of Example 5S 1 EXAMPLE 25 The titled compound is prepared by the procedure of Example step H NMR 3H 334 2H EXAMPLE 26 Alternative preparation of Dry H2G 1 and ester were dissolved in dry dimethyl formamide After stirring at for 30 the organic solvent was removed and the residue carefully chromatographed 2 to afford the desired product as a white solid A solution of stearoyl chloride in dry methylene chloride was added slowly dropwise under nitrogen to a solution of the product of step and in dry pyridine at The reaction mixture was stirred at that temperature for Methanol 59 was added and the reaction stirred for 1 After removal of the the residue was triturated with acetonitrile and chromato to afford the desired product The product of step was dissolved in methanol and A balloon filled with hydrogen was placed on top of the reaction After hours at TLC showed the absence of starting The reaction mixture was filtered through a micron nylon membrane to remove the catalyst and the solvent was removed to afford the desired product as a white solid which was identical and analytical to Example EXAMPLE 27 Alternative preparation of fP from Example 23 step was dissolved in N NaOH aqueous solution at room At an aliquot was taken and neutralized with N trifluoroacetic The aliquots were evaporated and analyzed by HPLC to monitor the progress of the After 4 N J 1 trifluoroacetic acid solution was added to the solution and the reacdon mixture was The desired product was purified by 50 x gradient TFA TFA in in 20 UV detection at 254 δ 60 EXAMPLE 28 Alternative preparation of HPLC separation of the reaction solution from Example 27 gave the titled compound in 1 br l EXAMPLE 29 monohvdrochloride The product of Example step was dissolved in a mixture of methanol 10 and ethyl acetate To the solution was added Pd C and IN HC1 micro The reaction mixture was stirred at room temperature for 2 hours under 1 The reaction mixture was filtered and the solvent evaporated from the filtrate to provide the desired product as a crystalline solid FORMULATION EXAMPLE A Tablet formulation The following ingredients are screened through a mm sieve and ed 10 g 40 g lactose 49 g crystalline cellulose g magnesium stearate A tabletiing machine is used to mixture to tablets containing 250 mg of active FORMULATION EXAMPLE B 6 Enteric coated tablet The tablets of Formulation Example A are spray coated in a tablet coater with a solution comprising 120 g ethyl cellulose 30 g propylene glycol sorbitan monooleate ad 1 000 ml FORMULATION EXAMPLE C Controlled release formulation 50 g 12 g hydroxypropylmethylcellulose g lactose are and granulated with an aqueous paste of Magnesium stearate is added and the mixture compressed in a tabletting machine to mm diameter tablets containing 500 active FORMULATION EXAMPLE D Soft capsules 250 g 100 g lecithin 100 g arachis oil The compound of the invention is dispersed in the lecithin and arachis oil and filled into soft gelatin 62 BIOLOGY EXAMPLE 1 Bioavailab3itv testine in The bioavailability of compounds of the invention were compared to the parent compound H2G and other H2G derivatives in a rat Compounds of the invention and comparative compounds were per oral catheter into the to multiples of three individuall weighed animals to kg of the dissolved prodrug in an aqueous Comparative example 1 peanut oil examples or propylene glycol 1 6 Comparative example vehicle dependent on the solubility of the test compound The animals were fasted from 5 hours before to approximately hours after administration and were maintained in metabolic Urine was collected for the 24 hours following administration and frozen until IDG was analysed in the urine using the assay of Antimicrob Agents 36 No modified as samples upon thawing are diluted in aq dist and filtered through an Amicon filter with centrifugarion at 3000 rpm for Duplicate 30 samples are on HPLC Zorbax 75 x 3 Mobile hase 3 4 pH 33 254 retention time for H2G at MeOH and pH Bioavailability is calculated as the measured H2G recovery from each animal averaged over at least three animals and expressed as a percentage of the averaged 24 hour urinary H2G recovery from a group of 4 individually weighed rats respectively injected with H2G in a buffer vehicle and analysed as Comparative example 1 was from the same batch as used for preparation of Examples 1 to The preparation of Comparative example 2 and 63 3 are shown in Examples 20 and Comparative example 4 was prepared by of unprotected H2G in comparable conditions to step b of Example Comparative examples 5 8 Ac prepared analogously to Example 4 using acetic anhydride with relevant monovaJine Comparative example 6 stearoyl was prepared analogously to Example 6 using in step Comparative example 7 was prepared analogously to Example 5 but using the step a intermediate made with The preparation of Comparative examples 9 and is shown in Examples 24 and 25 The results appear on Table 2 64 TABLE 2 Comparison of the bioavailabilities of the compounds of the invention with the comparative examples indicates that the particular combination of Ihe fatty acids at with the amino acids at produces bioavailabilities significantly greater 65 than the diamino acid ester or difarty acid For in this the compound of Example 1 displays 55 better bioavailability than the corresponding valine ester of Comparative example The compound of Example 4 displays 25 better availability than the corresponding divaline It is also for instance from Comparative examples 6 and 7 that only the specified fatty acids of this invention in combination with the specified amino acids produce these dramatic and unexpected increases in pharmacokinetic BIOLOGY EXAMPLE 2 Plasma concentrations in rats A plasma concentration assay was done in Sprague Dawley derived The animals were fasted overnight prior to dosing but were free access 15 Each of the compounds evaluated was prepared as a in propylene glycol at a concentration corresponding to 10 mg H2G and shaken at room temperature for eight Groups of rats least 4 rats in each received a 10 ml dose of each of the the dose was administered by At selected time points after dosing 0 and 24 hours after hepaiinized blood samples ml were obtained from a tail vein of each The blood samples were immediately chilled in an ice Within two hours of the plasma was separated from the red cells by centrifugation and frozen till The components of interest were separated from the plasma proteins using acetonitrile Following and the plasma concentrations were determined by reverse phase HPLC with fluorescence The oral uptake of H2G and other test compounds was determined by comparison of the H2G area under the curve derived from the oral dose compared to that obtained from a 10 intravenous dose of administered to a separate group of The results are depicted to Table BIOLOGY EXAMPLE 3 66 Bioavailability in The compounds of Example 1 and Comparative example 3 Biology Example 1 were administered by gavage to cynomolgus The solutions Example 1 dissolved in ml propylene corresponding to 25 or Comparative 164 mg dissolved in ml corresponding to Example 3 or mmol samples were taken at 30 and 24 Plasma was separated by centrifugation at 2500 rpm and the samples were inactivated at for 20 minutes before being frozen pending Plasma H2G levels were monitored by the assay of Example 30 Figure 1 depicts the plasma H2G recovery as a function of Although it is possible to draw statistically significant conclusions from single animal it appears that the animal receiving the compound of the invention experienced a somewhat more rapid and somewhat greater exposure to H2G than the animal which received an alternative prodrug of BIOLOGY EXAMPLE 4 Antiviral activity Herpes simplex 1 infected mouse serves as an animal model to determine the efficacy of antiviral agents in Mice inoculated intraperitoneally with at 1000 times the were administered either with a formulation comprising the currently marketed agent acyclovir and 83 in a propylene glycol in sterile water three times or the compound of Example 29 and 83 in a propylene glycol in sterile 67 water three times for 5 consecutive days beginning 5 hours after Tnc daily for The results are displayed in Figure 2 which charts the survival rate against In the the compound of the invention is denoted and acyclovir is denoted The of mice surviving the 1 infection was significantly greater following a given dose of the compound of the invention relative to an equivalent dose of The foregoing is merely illustrative of the invention and is not intended to limit the invention to the disclosures made Variations and changes which are obvious to one skilled in the art to be within the scope and nature of the invention as defined in the appended Material which is outside the scope of the claims does not constitute a part of the claimed 14446573 68 insufficientOCRQuality

Claims (1)

1. CLAIMS group, LUZZATTO & LUZZATTO