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Title:
THERAPEUTIC NUCLEOSIDES
Document Type and Number:
WIPO Patent Application WO/1993/006112
Kind Code:
A1
Abstract:
The present invention relates to an antiviral phosphonate derivate of an acyclic purine nucleoside analog, to pharmaceutically acceptable derivatives thereof and to their use in medical therapy, particularly in the treatment of cytomegalovirus infections. Methods for preparing the R-enantiomer of the compound of the invention, substantially free from the corresponding S-enantiomer are disclosed.

Inventors:
Miller
Wayne
Howard, Chamberlain
Stanley
Dawes, Biron
Karen
Kathleen
Application Number:
PCT/GB1992/001748
Publication Date:
April 01, 1993
Filing Date:
September 23, 1992
Export Citation:
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Assignee:
THE WELLCOME FOUNDATION LIMITED MILLER
Wayne
Howard, Chamberlain
Stanley
Dawes, Biron
Karen
Kathleen
International Classes:
C07C69/78; C07D473/00; C07F7/18; C07F9/6561; C07H9/04; (IPC1-7): A61K31/675; C07C69/78; C07D473/00; C07D473/18; C07F7/18; C07F9/6561; C07H9/04
Domestic Patent References:
WO1988005438A11988-07-28
Foreign References:
EP0130126A11985-01-02
EP0452935A11991-10-23
Other References:
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY vol. 110, no. 14, 1988, WASHINGTON, DC US pages 4672 - 4685 K.C.NICOLAOU 'Total Synthesis of Amphoteronolide B and Amphoterivin B. 1.Strategy and Stereocontrolled Construction of Key Building Blocks'
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Claims:
Claims :
1. The compound (R)3 [ (2aminol,6dihydro6oxo9Hpurin9yl) methoxy] 4hydroxybutylphosphonic acid substantially free from the corresponding Senantiomer.
2. The compound of claim 1 in admixture with less than 5% w/w of the Senantiomer.
3. The compound of claim 1 in admixture with less than 2% w/w of the Senantiomer.
4. A pharmaceutically acceptable salt, ester, or ester salt of (R)3 [ (2aminol,6dihydro6oxo9Hpurin9yl)methoxy] 4hydr oxybutylphosphonic acid substantially free from the corresponding salt, ester, or ester salt of the Senantiomer.
5. A pharmaceutically acceptable salt, ester, or ester salt of claim 4 in admixture with less than 5% w/w of the corresponding salt, ester, or ester salt of the Senantiomer.
6. A pharmaceutically acceptable salt, ester, or ester salt of claim 4 in admixture with less than 2% w/w the corresponding salt, ester, or ester salt of the Senantiomer.
7. An ester according to claim 4 which is a mono or diphosphate.
8. An ester according to claim 4 which is (R)ethyl hydrogen 3[(2 aminol,6dihydro6oxo9Hpurin9yl)methoxy] 4hydroxybutylpho sphonate.
9. An ester according to claim 4 which is (R)dibenzyl 3[(2amino 1,6dihydro6oxo9Hpurin9yl)methoxy] 4hydroxybutylphosphon ate.
10. A process for the preparation of (R)3 [(2aminol,6dihydro6 oxo9Hpurin9yl)methoxyJ4hydroxybutrylph.ospb.onIc acid substan¬ tially free from the corresponding Senantiomer which comprises dephosphorylating (R)3[(2aminol,6dihydro6oxo9Hpurin9yl) methoxy]4hydroxybutylphosphonic acid monophosphate using a suitable phosphatase enzyme.
11. A process according to claim 10 wherein the enzyme used is alkaline phosphatase.
12. A process according to claim 10 or 11 wherein the monophosphate is obtained by: (a) phosphorylating a racemic mixture of 3[(2ammol,6dihydro 6oxo9Hρurm9yl)methoxy]4hydroxybutylphosphonIc acid with an enzyme which preferentially phosphorylates the Renantiomer to give the corresponding monophosphate; and (b) separating the monophosphate from from the unphosphorylated Senantiomer by suitable means.
13. A process according to claim 12 wherein the enzyme used in step (a) is GMP kinase.
14. A process for the preparation of (R)3[(2aminol,6dihydro6 oxo9Hpurin9yl)methoxy]4hydroxybutylphosphonic acid substan¬ tially free of the corresponding Senantiomer which comprises deesterifying (R)ethyl hydrogen 3[(2aminol,6dihydro6oxo 9Hpurin9yl)methoxy]4hydroxybutylphosphonate.
15. A process according to claim 14 wherein the deesterisation is carried out using a suitable enzyme.
16. A process according to claim 15 wherein the enzyme is snake venom phosphodiesterase.
17. A process for the preparation of (R)3 [ (2aminol,6dihydro6 oxo9Hpurin9yl)methoxy] 4hydroxybutylphosphonic acid substan¬ tially free of the corresponding Senantiomer which comprises deesterifying (R) dibenzyl3 [ (2aminol,6dihydro6oxo9H purin9yl)methoxy] 4hydroxybutylphosphonate.
18. A process according to claim 17 wherein the deesterification is carried out by chemical hydrolysis.
19. A process according to claim 18 wherein the chemical hydrolysis comprises treatment with Pd(0H)_ on carbon.
20. An ester according to any of claims 4 to 9 when in a protected form.
21. A compound according to claim 20 selected from (R)diethyl3 [ (2 acetamido1,6dihydro6oxo9Hpurin9yl)methoxy] 4benzoyloxy butylphosphonate and (R)Dibenzyl3 [(2acetamidol,6dihydro 6oxo9Hpurin9yl)methoxy] 4benzoyloxybutylphosphonate.
22. A compound of formula (Ilia), (Illb) or a mixture thereof in any proportions (Ula) (Illb) wherein L is a leaving group, OQ is a protected hydroxyl group, Q 1 is an aminoprotecting group and OQ2 is a protected hydroxyl group.
23. A compound according to claim 22 selected from (R)2[(2Acet amido1,6dihydro6oxo7Hpurin7yl)methoxy] 4iodobutyl benzoate and (R)2[(2Acetamidol, 6dihydro6oxo9Hpurln 9yl)methoxy]4Iodobutyl benzoate, or a mixture thereof in any proportions.
24. A compound of formula (Va) , (Vb) or a mixture thereof in any proportions (Va) (Vb) 1 2 wherein Q is an aminoprotecting group, OQ is a protected hydroxyl group and L is a precursor of L, wherein L is a leaving group.
25. A compound according to claim 24 which is (R)2 [(2Acetamidol, 6dihydro6oxo7Hpurin7yl)methoxy]4chlorobutyl benzoate.
26. A compound of formula (VI) 2 2 wherein L is an acid labile group or a leaving group, OQ is a protected hydroxyl group and L is as defined in claim 24.
27. A compound according to claim 26 selected from (R)4Chloro2(methoxymethoxy) butyl benzoate and (R)1 [ (tert butyldiphenylsilyl)oxy)4chloro2(methoxymethoxy)butane.
28. (R)10(tertButyldiphenylsilyl)4chlorol,2butanediol.
29. (R)10(tertButyldiphenylsilyl)1,2,4butanetriol.
30. 50(tertButyldiphenylsilyl)3deoxyl,20isopropylidene/3L threopentofuranose.
31. A pharmaceutical composition comprising (R)3 [ (2aminol,6dihy dro6oxo9Hpurin9yl)methoxy] 4hydroxybutylphosphonic acid substantially free from the corresponding Senantiomer, or a pharmaceutically acceptable salt, ester, or ester salt thereof and a pharmaceutically acceptable carrier therefor.
32. The pharmaceutical composition of claim 31 wherein the phosphonic acid is in admixture with less than 5% w/w of the Senantiomer.
33. The pharmaceutical composition of claim 31 wherein the phosphonic acid is in admixture with less than 2% w/w of the Senantiomer.
34. Use of (R)3[(2aminol,6dihydro6oxo9Hpurin9yl)methoxy] 4hydroxybutylphosphonic acid substantially free from the corresponding Senantiomer in the manufacture of a medicament for the treatment of a herpes virus Infection.
35. Use according to claim 34 wherein the viral infection is selected from cytomegalovirus, EpsteinBarr virus, human herpes virus 6 and VZV infections.
36. (R)3[(2AmInol,6dihydro6oxo9Hpurin9yl)methoxy] 4hydr oxybutylphosphonic acid substantially free from the corresponding Senantiomer for use in medical therapy.
37. A compound according to claim 36 for use in the treatment or pro¬ phylaxis of a herpes virus infection.
38. A compound according to claim 36 for use in the treatment or pro¬ phylaxis of a herpes virus infection selected from cytomegalo¬ virus, EpsteinBarr virus, human herpes virus 6 and VZV infect¬ ions.
39. A method of treating a herpes virus infection in a mammal compri¬ sing administration to said mammal of an effective antiherpes virus treatment amount of (R)3[(2aminol,6dihydro6oxo9H purin9yl)methoxy]4hydroxybutylphosphonIc acid substantially free from the corresponding Senantiomer.
40. The method of claim 39 wherein the herpes virus infection is a cytomegalovirus infection.
41. The method of claim 39 wherein the herpes virus infection is an EpsteinBarr virus infection.
42. The method of claim 39 wherein the herpes virus infection is a human herpes virus 6 infection.
43. The method of claim 39 wherein the herpes virus infection is a VZV infection.
Description:
THERAPEUTIC NUCLEOSIDES

The present invention relates to an antiviral phosphonate derivative of an acyclic purine nucleoside analog and pharmaceutically acceptable derivatives thereof, to compositions containing them, to their use in medical therapy, particularly for the treatment of certain viral infections, and to methods for preparing the R-enantiomer of the phosphonate derivative substantially free from the corresponding S-enantiomer.

Of the DNA viruses, the herpes group is the source of many common viral illnesses in man. The group includes cytomegalovirus (CMV) , Epstein-Barr virus (EBV) , varicella zoster virus (VZV) , herpes simplex virus (HSV) and human herpes virus 6 (HHV6) .

In common with other herpes viruses, infection with CMV leads to a life-long association of virus and host and, following a primary infection, virus may be shed for a number of years. Clinical effects range from death and gross disease (microcephaly, hepatosplenomegaly, jaundice, mental retardation) through failure to thrive, susceptibility to chest and ear infections to a lack of any obvious ill effect. CMV infection in AIDS patients is a predominant cause of morbidity as, in 40 to 80% of the adult population, it is present in a latent form and can be reactivated in immunocompromised patients.

EBV causes infectious mononucleosis and is also suggested as the causative agent of nasopharyngeal cancer, immunoblastic lymphoma, Burkitt's lymphoma and hairy leukoplakia.

VZV causes chicken pox and shingles. Chicken pox is the primary disease produced in a host without immunity. In young children, it is usually a mild illness characterized by a vesicular rash and fever. Shingles is the recurrent form of the disease which occurs in adults who were previously infected with varicella. The clinical manifestations of shingles include neuralgia and a vesicular skin rash

that is unilateral and dermatomal in distribution. Spread of inflammation may lead to paralysis or convulsions and coma can occur if the meninges become affected. In immunodeficient patients, VZV may disseminate causing serious or even fatal illness.

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 manifestation of infection which can be both physically and psychologically distressing. HSV infection is often characterized by extensive lesions of the skin, mouth and/or genitals. Primary infections may be subclinical although they tend to be more severe than infections in individuals previously exposed to the virus.

Ocular infections by HSV can lead to keratitis or cataracts.

Infection in the newborn, in immunocompromised patients or penetration of infection into the central nervous system can prove fatal. HHV6 is the causative agent of roseola infan um (exanthum subi urn) in children which is characterized by fever and the appearance of a rash after the fever has declined. HHV6 has also been implicated in syndromes of fever and/or rash and pneumonia or hepatitis in immunocompromised patients.

Acyclic nucleoside phosphonates have been previously proposed for the treatment of herpes virus infections. For example, EPO 0 173 624 describes 9-(3-phosρhono-l-propoxymethyl)guanine. Duke, A.E. et. al., _ntiviral Res. (1986) £.299-308; Prisbe, E.J. et. al. , J. Med. Chem. (1986) 29:671-675 and PCT Patent Application WO 88/05438 describe racemic 9-(3-phosphono-l-hydroxymethyl-l-propoxymethyl)gua- nine (ie. ganciclovir phosphonate) and related compounds.

We have now discovered that the R-enantiomer of ganciclovir phosphonate, having the formula (la)

R-€naπtiαrπer

(la) (lb)

and pharmaceutically acceptable salts, esters and salts of esters thereof have an especially advantageous combination of therapeutic properties in terms of their antiviral activity and/or other properties relevant to their use as antivirals that render these enantiomeric compounds of exceptional benefit as therapeutic agents, in particular as anti-herpes virus agents, more particularly as anti-CMV, .-EBV, VZV or -HHV6 agents, especially in comparison with the above-mentioned racemic ganciclovir phosphonate and also ganciclovir.

Furthermore, we have discovered methods of preparing the compound of formula (la) substantially free of the compound of formula (lb). Prior to this discovery, there was no known method of separating enantiomers (la) and (lb) or of preparing the individual enantiomers.

The R-enantiomer of ganciclovir phosphonate, the compound of formula (la), can also be named (R)-3- [(2-amino-l,6-dihydro-6-oxo-9H-purin-9- yl)methoxy] -4-hydroxybutylphosphonic acid.

It will be appreciated that the compounds of formula (la) and (lb) may exist in tautomeric forms, since the 6-oxo substituent may also be a hydroxy group.

According to one feature of the present invention, we provide the R-enantiomer of ganciclovir phosphonate substantially free from the S-enantiomer thereof, and salts, esters or salts of esters of said R-enantiomer.

The R-enantiomer of ganciclovir phosphonate is substantially free from the corresponding S-enantiomer to the extent that it is generally in admixture with less than 10% w/w, preferably less than 5% w/w and most preferably less than 2% w/w of S-enantiomer, based on the total weight of the mixture. In particular, the above R-enantiomer, according to the invention, advantageously contains less than 1% w/w of the corresponding S-enantiomer.

By "pharmaceutically acceptable salts, esters or salts of esters," is meant any salt, ester or salt of an ester which upon administration to the recipient, is capable of providing (directly or indirectly) the compound of formula (la) or an active metabolite or residue thereof.

Preferred esters, .in accordance with the invention, include carboxylic acid esters of the hydroxymethyl group in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl e.g. , n-propyl, t-butyl, n-butyl, alkoxyalkyl (e.g., methoxymethyl) , alkenyl (e.g., 2-butenyl) , arylalkyl (e.g., benzyl), arylox alkyl (e.g., phenoxymethyl) , aryl (e.g., phenyl optionally substituted by halogen, C. .alkyl or C- .alkoxy) ; sulfonate esters such as alkyl- or alkylarylsulfonyl (e.g., methanesulfonyl) ; amino acid esters (e.g., L-valyl or L-isoleucyl); and mono- di- or tri-phosphate esters. The phosphate esters may be further esterified by, for example, a C- __ alcohol or reactive derivative thereof, or by a 2,3-di(C fi „ )acyl glycerol. Additional preferred esters include mono or diesters of the phosphonic acid group in which the phosphonic acid group is mono or dis bstituted with substituents selected from straight or branched chain alkyl e.g., n-propyl, t-butyl, n-butyl, alkoxyalkyl (e.g., methoxymethyl), acyloxyalkyl (e.g., acetoxymethyl) , alkenyl (e.g., 2-bu enyl) , arylalkyl (e.g., benzyl), aryloxyalkyl (e.g., phenoxymethyl), aryl (e.g., phenyl optionally substituted by halogen, C. .alkyl or C_ .alkoxy) . The two substituents on the phosphonic acid group may be the same or different. Preferred esters may also include

combinations of carboxylic acid esters and mono or diesters of phosphonic acid.

With regard to the above-described esters, unless otherwise specified, any alkyl moiety present advantageously contains 1 to 18 carbon atoms, particularly 1 to 4 carbon atoms. 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 pharmaceutically acceptable salt thereof.

Examples of pharmaceutically acceptable salts of the compound of formula (la) include base salts, e.g., derived from an appropriate base, such as alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium) salts, ammonium and NX, , (wherein X is C. , alkyl). Pharmaceutically acceptable salts further include acid salts, e.g., derived from an appropriate organic carboxylic acid, such as acetic, fumaric, citric, lactic, tartaric, maleic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, hydrobromic, sulfuric, phosphoric and sulfamic acids.

With regard to the especially advantageous therapeutic properties of the compound of formula (la), referred to above, its increased anti-CMV activity is an improvement over racemic ganciclovir phosphonate.

The present invention further includes:

(a) A method for the treatment of a viral infection, more preferably a herpes virus infection, in particular a CMV, EBV, VZV or HHV6 infection, in a mammal, including a human, which comprises treating said mammal with an effective anti-herpes virus amount of the R-enantiomer which is substantially free of the

S-enantiomer of ganciclovir phosphonate or a pharmaceutically acceptable salt, ester or salt of such ester thereof.

(b) Use of an amount of the R-enantiomer of ganciclovir phosphonate or a pharmaceutically acceptable salt, ester or salt of such ester which is substantially free of the S-enantiomer in the preparation of a medicament for the treatment of a viral infection, preferably a herpes virus infection, most preferably a CMV, EBV, VZV or HHV6 infection.

(c) A pharmaceutical formulation comprising the R-enantiomer of ganciclovir phosphonate substantially free of the S-enantiomer or a pharmaceutically acceptable salt, ester or salt of such ester thereof together with a pharmaceutically acceptable carrier therefor.

(d) The R- enantiomer of ganciclovir phosphonate or a pharmaceutically acceptable salt, ester or salt of such ester which is substantially free of the S- enantiomer for use in medical therapy particularly for the treatment or prophylaxis of a herpes virus infection, preferably a CMV, EBV, VZV or HHV6 infection.

(e) A process as described below for the preparation of compounds according to the invention.

In an additional feature of the present invention, there is provided any and all novel intermediates disclosed herein.

Examples of clinical conditions caused by herpes viruses such as CMV, EBV, VZV and HHV6 infections, which may be treated in accordance with the invention, include those referred to above. It will be recognized that while a racemic mixture of the compounds of formulae (la) and (lb) can also be used for treating the aforementioned conditions, for

example those caused by HHV6 infection, use of the compound of formula (la) is preferred.

The compound of formula (la) may be employed in combination with other therapeutic agents for the treatment of the above infections or conditions. Examples of such further therapeutic agents include agents that are effective for the treatment of herpes virus infections such as l-3-D-arabinofuranosyl-5- (l-propynyl)uracil, foscarnet, acyclovir or ganciclovir, interferons, for example, α-interferon, or benzimidazole nucleosides such as those disclosed in WO 92/07867. The component compounds of such combination therapy may be administered simultaneously, in either separate or combined formulations, or at different times, e.g., sequentially, such that a combined effect is achieved.

The compound of formula (la) , also referred to herein as the active ingredient, may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, buccal and sublingual) , vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient and the nature of the infection.

In general, a suitable dose for each of the above named viral infections, e.g., CMV, EBV, VZV and HHV6 infections, is in the range of 0.1 to 250 mg per kilogram body weight of the recipient per day, preferably in the range of 1 to 100 mg per kilogram body weight per day and most preferably in the range 5 to 20 mg per kilogram body weight per day. (Unless otherwise indicated, all weights of active ingredient are calculated as the parent compound of formula (la) ; for salts or esters thereof, the weights would be increased proportionately.) The desired dose may be presented weekly or daily as one, two, three, four, five, six or more doses administered at appropriate intervals throughout the week or the day. These 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.

Ideally, the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.25 to about 100 μK, preferably about 0.5 to 70 μK, most preferably about 1 to about 50 μK. This may be achieved, for example, by the intravenous injection of a 0.1 to 5% solution of the active ingredient, optionally in saline, or orally administered, for example, as a tablet, capsule or syrup containing about 0.1 to about 250 mg per kilogram of the active ingredient. Desirable blood levels may be maintained by a continuous infusion to provide about 0.01 to about 5.0 mg/kg/hour or by intermittent infusions containing about 0.4 to about 15 mg per kilogram of the active ingredient.

While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation comprising at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic agents. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

Formulations include those suitable for oral, rectal, nasal, topical

(including transder al, buccal and sublingual) , vaginal, parenteral

(including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any 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 suspension in an aqueous or 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, disintegrant (e.g., sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxmethyl 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 and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

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 mouthwashes comprising the active ingredient in a suitable liquid carrier.

Pharmaceutical compositions 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 patch or a dressing such as a bandage or adhesive plaster impregnated with active ingredients and optionally one or more excipients or diluents.

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 25% w/w and most preferably 0.5 to 10% w/w. hen formulated in an ointment, the active ingredient 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-l,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 palmitate 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 oils 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 for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

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 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 parenteral administration Include aqueous and non-aqueous isotonic sterile injections 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 sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, or example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those suitable for administration either once daily or once or twice weekly 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 in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavouring agents.

The compound of formula (la) may also be presented for the use in the form of veterinary formulations, which may be prepared, for example, by methods that are conventional in the art.

Ganciclovir phosphonate, in racemic form, may be prepared by methods that are known in the art of nucleoside synthetic chemistry for the preparation of the same or similar compounds, for example, PCT Patent Application WO 88/05438 and Prisbe, E.J. ' , et. al., J. Med. Chem. (1986) 29: 671-675.

Resolution of R- and S-enantiomers can then be achieved by adding an enzyme, such as GMP kinase, which preferentially phosphorylates the R-enantiomer of racemic ganciclovir phosphonate. The phosphorylated R-enantiomer may be separated from the S-enantiomer chromatographically and then dephosphorylated with a phosphatase enzyme, such as alkaline phosphatase. Hog brain GMP kinase is commercially available from Sigma Chemical Company, St. Louis, MO 63178 and calf intestine alkaline phosphatase may be obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN 46250.

Alternatively, R-ganciclovir phosphonate (la) may be prepared by deesterification of a corresponding phosphono diester of formula (II)

(ID

wherein both R groups are independently selected from straight or branched chain alkyl (eg., n-propyl, t-butyl, n-butyl), alkoxyalkyl (eg. , methoxymethyl) , acyloxyalkyl (eg. , acetoxymethyl) , alkenyl (eg. , 2-butenyl) , arylalkyl (eg., benzyl), aryloxyalkyl (eg., phenoxymethyl), aryl (eg..phenyl optionally substituted by halogen, C- , alkyl or C- , alkoxy) .

The above deesterification process may be effected either in one step using conventional methods or stepwise via the corresponding mono-ester using any combination of standard deesterification methods such as chemical hydrolysis, for example, treatment with base, or enzymic hydrolysis eg., treatment with an esterase enzyme, for instance snake venom phosphodiesterase.

Compounds of formula (II) may be obtained by deprotection of the product formed by reaction of a mixture of compounds of formula (Ilia) and (Illb)

(Ilia) (Illb)

wherein L is a suitable leaving group such as a halo group, for example, iodo or bromo, or an organosulphonyloxy group, for example, methanesulphonyloxy or 4-toluenesulphonyloxy;

-OQ is a protected hydroxyl group such as an alkyl-, aryl-, or arylalkyl ether, for example, benzyl ether or a silylether group, for example, a trimethylsilyloxy-, or t-butyldiphenylsilyloxy group, or an

ester, for example, nonanoyl or benzoyl ester, or a carbamate, for example, N,N-diphenyl carbamate.

Q is an amine protecting group, such as an acyl group, for example C- . alkylcarbonyl, for instance acetyl, or a dialkylaminoalkylene group, for example, diC- ,alkylaminoC- .alkylene, for instance, N,N-dimethylaminomethylene, or an alkoxycarbonyl group, for example, tert-butoxycarbonyl; and

2 -0Q is a protected hydroxyl group, such as an ether, for example, a silyl ether (for instance tert-butyldiphenylsilyl ether, triphenylsilyl ether or tert-butyldimethylsilyl ether) , a straight or branched chain alkyl ether, a tetrahydropyranyl ether or an optionally substituted aryl ether (for instance benzylether, trityl ether or

4-methoxybenzyl ether), or as an ester, for example, an alkyl ester, cycloalkyl ester or optionally substituted aryl ester, or as a carbonate, for example, an alkyl or arylalkyl carbonate (for instance, methyl,isobutyl or benzyl carbonate);

with the anion of the appropriate disubstituted phosphite of formula (IV)

0

RO P0MΘ

(IV)

OR

wherein both R groups are as hereinbefore defined for formula(II) and M is a suitable counterion, such as an alkali metal ion, for example, Na or K . This reaction may be carried out in a suitable solvent, typically a polar aprotic solvent, for example, tetrahydrofuran or acetonitrile.

A mixture of compounds of formula (Ilia) and (Illb) may be obtained by treating a compound of formula (Va) , (Vb) or a mixture thereof

1 2 wherein Q and -0Q are as defined for formulae (Ilia) and (Illb) ; and

L is a precursor of L, such as an alternative leaving group, for example, chloro-, or an organosulphonyloxy group, such as methanesulphonyloxy or 4- toluenesulphonyloxy;

so as to convert L into L and/or to effect the migration of the side chain from N-7 to N-9. This may be effected by heating a compound of formula (Va) , (Vb) or a mixture thereof in polar solvent, such as, methyl ethyl ketone or acetone in the presence of an appropriate reagent to introduce L, for example, when L is iodo, an Iodide salt may be used, for instance sodium iodide; or by heating a compound of formula (Va) , (Vb) or a mixture thereof in a non-polar solvent, such as benzene or dichloromethane in the presence of a silylating agent, such as N,0-bis(trimethylsilyl) acetamide, and a Lewis acid, such as trimethylsilyltriflate or tin tetrachloride.

Compounds of formula (Va) , (Vb) or a mixture thereof may be prepared by reaction of a compound of formula (VI)

(VI)

2 1 2 wherein -0Q and L are as defined for formulae (Va) and (Vb) and L is an acid labile substituent such as an alkyl ether, for example C. alkoxy (eg. methoxy) , or an acyloxy group, for example, acetoxy.

2 2 AAlltteerrnnaattiivveellyy,, LL mmaayy bbee a leaving group, such as a halo group, for example, chloro or bromo;

with a suitably protected and/or activated guanine derivative. The guanine derivative may be prepared from commercially available starting materials using conventional techniques well known to those skilled in the art.

2 Compounds of formula (VI) in which L is an acyloxy group may be synthesised directly form the corresponding compound of formula (VI)

2 2 in which L is an alkoxy group. Compounds of formula (VI) in which L is a halo group may be prepared by coupling a compound of formula

(VII) as defined below, with formaldehyde and HCl. Compounds of

2 formula (VI) in which L is alkoxy may be prepared by coupling a compound of formula (VII)

1 2 wherein L and -OQ are as defined for formulae (Va) and (Vb) , or an activated form thereof; with a compound of formula (VIII)

18 -

2 3 wherein L is an alkyl ether, typically methoxy, and L is either the

2 same as L or is a leaving group, such as a halo group, typically chloro-, using conventional techniques well known to those skilled in the art.

Compounds of formula (VIII) are commercially available or may be prepared from readily available starting materials using methods well known to those skilled in the art.

Compounds of formula (VII) may be prepared from compounds of formula (IX)

2 wherein -OQ is as defined for formulae (Va) and (Vb) ; or an activated and/or protected form thereof,

by treatment with an agent so as to convert the primary hydroxyl group into a leaving group L as defined for formulae (Va) and (Vb) . For example, when L is chloro, this reaction may be effected by treatment with carbon tetrachloride in the presence of triphenylphosphine with a polar aprotic solvent, such as acetonitrile. Alternatively, methane sulphonylchloride in N,N-dimethylformamide (DMF) or triphenylphosphine and N-Chlorosuccinimide in DMF may be used to effect the substitution.

Compounds of formula (IX) may conveniently be prepared from a compound of formula (X)

2 wherein -OQ is as defined for formulae (Va) and (Vb) ;

3 4 and Q and Q may be the same or different and are hydroxyl protecting groups such as those described above, or together form a diol protecting group such as an acetonide, methylene acetal or benzylidine acetal. The above conversion may be effected by initial treatment with aqueous acid, such as hydrochloric, acetic, tosic or trifluoroacetic acid, followed by oxidative cleavage of the resulting diol with sodium periodate in water or an alcoholic solvent, such as methanol or ethanol, or with lead tetraacetate in an organic solvent.

Reduction of the resulting aldehyde may be effected by a variety of techniques well known to those skilled in the art, including use of sodium borohydride in an alcoholic solvent such as methanol or ethanol.

Compounds of formula (X) may be prepared form a compound of formula (XI)

2 3 4 wherein -OQ , Q and Q are as defined for formula (X) ; by reductive elimination of the 3'-hydroxyl group using any one of a number of methods well known to those skilled in the art, including conversion of the 3'-hydroxy group to an aryloxythiocarbonyloxy group (eg. phenoxythiocarbonyloxy) and treatment with tributyltinhydride and a radical initiator such as bis azoisobutyrylnitrile (AIBN) .

Compounds of formula (XI) may be prepared from L-arabinose (Aldrich Chemical Co.) by the method of Dahlman et. al, Acta Chem. Scand. B 40 (1986) 15-20.

For a better understanding of the invention, the following examples are given by way of illustration.

Example 1

Preparation of CR)-3-T(2-Amino-l.6-dihydro-6-oxo-9H-τ>urin-9-yl)- methoxyl-4-hvdroxybutylphosphonic Acid and its Monoethyl Ester

A. 5-0-(tert-Butyldinhenylsilyl)-L-arabinofuranose

L-Arabinose was dried under vacuum in the presence of P.-0,. for 72 h. A total of 25 g (166 mmol) of L-arabinose was weighed into a flame dried round bottom flask fitted with a stirring bar and gas inlet adapter. Imidazole (25 g, 367 mmol) was added followed by anhydrous N,N-diraethylformamide ( DMF) (100 mL) . This solution was stirred at ambient temperature until the solids had dissolved. tert-Butylchlorodiphenylsilane (50 g, 182 mmol) (Aldrich Chemical Company, Milwaukee, WI 53233) was added directly to this mixture. The reaction was heated at 60°C, under nitrogen, for two hours. The DMF was then removed under vacuum at 60°C over a period of two hours. The resulting thick yellow oil was dried on the vacuum pump.

The crude product was purified on a silica gel column which was eluted with ethyl acetate : hexanes (1:1). Pure product was obtained as a clear oil 24.3 g (57.3 mmol):

MS (CI): m/z 371 (C^.O^i) , 353 C^H^Si) , 311 (C-^H^- Si), 293 (C 15 H 21 0 4 Si) , 275 (C^H^Si) , 251 (C^.O j Si) , 233 C 13 H 17 0 2 Si) , 221 (C 12 H 17 0 2 Si) , 215 C^H^OSi) , 173 C^O^i , 161 C 10 H 13 Si), 133 C 5 H 9 0 4 );

1 H-NMR (DMSO) 8 , 7.12-1.61 (m, 4H, Ar-H) , 7.44-7.40 (m, 6H, Ar-H), 6.23 (d, IH, /3-anomer, J=5.5 Hz, l-OH) , 6.10 (d, IH, α-anomer, l-OH, J-8.2), 5.22 (d, IH, 0-anomer, OH, J=4.1Hz),

5.14-5.12 (m, 2H, α and /3-anomers , OH), 4.97-4.94 (m,2H, α and 3-anomers, H-l) , 4.72 (d, IH, a anomer, OH, J=6.6Hz), 3.90-3.60 (m,5H, a and /3-anomers, H-2,3,4 and 5), 0.97 (s, 9H, C(CH 3 ) 3 ):

Analysis Calculated for C.-H-gO Si 0.4 C H~0 : Calcd: C, 64.05; H, 7.42. Found: C, 63.95; H, 7.36.

B. 5-0-(tert-Butyldrphenylsilyl)-l,2-0-isopropylidene-jg-L- ar ab ino f ur ano s e

A 24 g (62 mmol) portion of 5-0-(tert-butyldiphenylsilyl)-L- arabinofuranose was weighed into a dried round bottom flask fitted with, a stir bar and gas inlet adapter. Anhydrous cupric sulfate 27 g (170 mol) was added followed by a solution of concentrated sulfuric acid (1.2 mL, 18 M ) in acetone (200 mL) . The suspension was flushed with nitrogen and stirred overnight at room temperature. After 18 h the suspension was filtered and the filtrate was neutralized with Ca(0H)„ (14.4 g) and concentrated. The resulting thick yellow oil was dried on a vacuum pump and purified on a silica gel column eluted with ethyl acetate : hexanes (1:5). Pure product was obtained as a gold oil 17.0 g (40 mmol) :

MS (CI): m/z 427 (C^H^Si) , 413 (C^H^Si) , 371 (C^H^- Si), 353 (C 21 H 25 0 3 Si), 351 (C^H^Si) , 294 (C-^H-^Si) , 293 (C 15 H 21 0 4 Si) , 275 (C 15 H 19 0 3 Si , 251 (C^H^Si) , 221 (C 12 H 17 0 2 Si) , 215 (C 13 H 15 0Si), 173 (C^O^i) , 161 (C^H^Si ; ■

Sϊ-NMR (CDC1 ) δ, 7.68-7.65 (m, 4H, Ar-H), 7.43-7.36 (m, 6H, Ar-H), 5.88 (d,lH, H-l, J=4.1Hz), 4.54 (d, IH, H-2, J=4.0Hz), 4.45-4.42 (m, IH, H-3) , 4.08-4.00 (m, IH, H-4) , 3.83-3.80 (m, 2H, H-5), 2.12 (d, IH, 3-0H, J=4.3Hz), 1.32 (s, 3H, C(CH 3 ) 2 ), 1.29 (s, 3H, C(CH 3 ) 2 ), 1.06 (s, 9H, C(CH 3 ) 3 );

Analysis Calculated for C„,H 3 „0-.Si: Calcd: C, 67.26; H, 7.53. Found: C, 66.88; H. 7.80.

C. 5-0-ftert-Butrldiphenylsilyl)-1.2-0-Isonropylidene-3-0- f henoxythiocarbon 1)- β -L-arabinofuraπose

A 16 g (37 mmol) portion of 5-0-(tert-butyldiphenylsilyl)-l,2-0- isopropylidene-3-L-arablnofuranose was weighed into a dried round bottom flask fitted with a stir bar and gas inlet adapter. N,N-DimethylamInopyridine (8.2 g, 67 mmol) was added followed by anhydrous acetonitrile (150 mL) . Phenyl chlorothionoformate was added (6.2 g, 45 mmol) and the solution was flushed with nitrogen and stirred at ambient temperature overnight. After 18 h the reaction mixture was treated with ethyl acetate:hexanes (1:1) (200 mL) and the precipitate was filtered off. The precipitate was washed with ethyl acetate. The washings and filtrate were concentrated to a thick yellow oil which was purified on a silica gel column eluted with ethyl acetate:hexanes (1:10). Pure product was obtained as a clear oil 14 g (25 mmol) :

MS (CI): m/z 565 SSi), 429 C 2] H 21 0 6 SSi) , 411 (C^H^O^i) , 353 (C^H^OgSSi) , 275 (C 15 H lg 0 3 Si) , 269 (C 17 H 21 OSi), 179 , 155 C 7 H-,0 2 S), 137 (C^OS) ;

ϊ-NMR (CDC1 3 ) δ , 7.70-7.64 ( , 4H, Ar-H), 7.48-7.30 (m, 9H,

Ar-H), 7.14-7.09 (m, 2H, Ar-H), 5.96 (d.lH, H-l, J-4.0 Hz), 5.80

- (d, IH, H-3, J=1.3 Hz), 4.76 (d, IH, H-2, J=4.0 Hz), 4.46 (t, IH,

H-4, J=7.0 Hz), 3.90-3.83 (m, 2H, H-5) , 1.32 (s, 3H, C(CH ) ) , 1.29 (s, 3H, C(CH 3 ) 2 ), 1.05 (s, 9H, C(CH 3 ) 3 );

Analysis Calculated for C,_H_ fi 0,SSI: Calcd: C, 65.93; H, 6.42.

Found: C , 66 . 18 ; H , 6 . 62.

5-0-(tert-Butyldiphenylsilyl)-3-deoxy-l.2-0-isopropyliden e-θ-L- threo-pentofuranose

A 13 g (23 mmol) portion of 5-0-(tert-butyldiphenylsilyl)-1,2-0- isopropylidene-3-0-(phenoxythiocarbonyl)-y9-L-arabinofuranos e was weighed into a dried round bottom flask fitted with a stir bar and gas inlet adapter. 2,2' -Azobis- [2-methylpropionitrile] (AIBN) 0.30 g (1.8 mmol) was added followed by anhydrous toluene (200 mL) . This solution was degassed under reduced pressure for 15 min. Tributyltin hydride (7 mL, 26 mmol) was added by syringe and needle.

The reaction mixture was stirred under nitrogen at 90°C for six hours, then concentrated and dried on the vacuum pump. The resulting thick yellow oil was purified on a silica gel column eluted with ethyl acetate:hexanes (1:20). Pure product was obtained as a clear oil 9.3 g (22 mmol):

MS (El): m/z 397 (C^H^Si) , 355 (C^H^Si) , 337

(C 21 H 25°2 Si) ' 335 (C 18 H 25°3 Si) ' 297 (C 18 H 21°2 Si) ' 277 (C 14 H 15 0) ' 241 (C 14 H 13 0 2 Si), .221 (C^H^Si) , 199 (C^H-^OSi) , 163

(C 10 H 15 Si), 135 (C 7 H ? 0Si);

"Ή-NMR (CDC1 3 ) δ , 7.68-7.64 (m, 4H, Ar-H), 7.41-7.32 (m, 6H,

ArOH), 5.77 (d,lH, H-l, J=3.9 Hz), 4.74-4.69 (m, IH, H-2),

4.30-4.22 (m, IH, H-4) , 3.81-3.76 (m, IH, H-5) , 2.25-2.10 (m, 2H,

H-3), 1.31 (s, 3H, C(CH 3 ) 2 ), 1.26 (s, 3H, C(CH 3 ) 2 ), 1.04 (s, 9H, C(CH 3 ) 3 );

Analysis Calculated for C H 3 -0 Si 0.10 C H g 0 2 : Calcd: C, 69.54; H, 7.84. Found: C, 69.18; H, 7.67.

(R)-1-0-(tert-Butyldiphenylsilyl)-1.2.4-butanetriol

A total of 33.5 g (81.2 mmol) of 5-0-tert-butyldiphenylsilyl-3- deoxy-l,2-0-Isopropylidene-beta-L-arabinofuranose was weighed Into a round bottom flask fitted with a stir bar and treated with 80% acetic acid (300 mL) . The solution was stirred for 30 minutes at 90°C, then concentrated on a rotoevaporator to a thick oil and treated with ethanol and rotoevaporated (3 x 50 mL) . The residue was dissolved in methanol (450 mL) and treated with sodium periodate (34.9 g, 163 mmol) dissolved in water (300 mL) . This solution was stirred at room temperature for 1.5 hours, filtered, cooled in a ice bath and treated with excess sodium borohydride (13.1 g, 346 mmol). After fifty minutes, the reaction was neutralized with concentrated HCl (20 mL) to pH 7.5. The neutral solution was filtered and the solids were washed with methanol. The solution was concentrated to about 500 mL and extracted with ethyl acetate : hexanes (2:1) (3 x 300 mL) . The combined organics were washed with 5% sodium bisulfite (300 mL) , filtered, dried (MgSO.) and concentrated to a clear yellow oil. This material was purified on a silica gel column (230-400 mesh, 1300 mL of silica) eluted with ethyl acetate : hexanes (1:2) (3L) then with ethyl acetate : hexanes (1:1) (3L) providing 18.0 g (52.2 mmoL) of a yellow oil after drying on the vacuum pump:

MS (El): m/z 327 (C^H^Si) , 309 C^H^OSi) , 267 C-^H^Si), 249 C 14 H 2] 0 2 Si) , 209 C-^H-^Si , 199 (C-^H-^OSi) , 189 C g H 17 0 3 Si , 181 (C 12 H g Si), 161 (C^H^Si) , 131 (C^O^i) , 105 C 4 H 9 0 3 ) ;

Sl-NMR (DMSO) δ , 7.67-7.64 (m, 4H, Ar-H), 7.45-7.36 (m, 6H, Ar-H), 4.01-3.94 (m,lH, H-2) , 3.81 (dt 2H, H-4, J- 5.4, J=5.5Hz), 3.64 (dd, IH, H-l, J=3.8Hz, J-lO.lHz), 3.54 (dd, IH, H-l', J=7.3Hz, J=10.1Hz), 2.80 (d, IH, 2-0H, J=3.2Hz), 2.44 (t, IH, 4-0H, J=5.4Hz), 1.68-1.63 (m, 2H, H-3), 1.06 (s, 9H, C(CH ) ) ;

Analysis Calculated for C n H o 0_Si:

ΔU Δ O J

Calcd: C, 69.72; H, 8.19 Found: C, 69.49; H, 8.15.

F. (R)-1-0-(tert-Butyldiphenylsilyl)-4-chloro-l.2-butanediol

A total of 1.5 g (4.3 mmol) of (R)-1-0- (tert-butyldiphenylsilyl)- 1,2,4-butanetriol was dried by co-evaporation with toluene, then taken up in anhydrous acetonitrile (50 mL) . Carbon tetrachloride (4.2 mL, 43.5 mmol) was added. Portions of a solution of triphenylphosphine (2.3 g, 8.7 mmol) dissolved in anhydrous acetonitrile (50 mL) were added so that all the starting material was used up and no triphenyl phosphine was left. The reaction mixture was concentrated to 60 mL and applied directly to a silica gel column packed in ethyl acetate:hexanes (1:10). The column was eluted with the same solvent and provided product as a clear oil: 1.25 g (3.44 mmol):

4. NMR (CDC1 3 ) δ , 7.68-7.64 (m, 4H, Ar-H), 7.46-7.40 (m, 6H, Ar-H), 4.00-3.90 (m, IH, H-2) , 3.72-3.66 (m, 3H, H-l and H-4) , 3.56-3.50 (dd, IH, H-l', J=3.9 Hz, J=10.1 Hz), 2.46 (d, IH, 2-0H, J=5.0 Hz), 1.98-1.75 (m, 2H, H-3) , 1.08 (s, 9H, C(CH ) ).

G. (R)-l-((tert-Butyldiphenylsilyl)oxy)-4-chloro-2- (methoxymethoxy) butane

Phosphorous pentoxide 7.0 g (49.3 mmol) was weighed into a dry round bottom flask and treated with chloroform pre-dried with phosporous pentoxide. Dimethoxyme hane was added and the solution was stirred at ambient temperature for 15 minutes. A total of 1.38 g (3.8 mmol) of (R)-1-0-(tert-butyldiphenylsilyl)- 4-chloro-l,2-butanediol was dissolved in dry chloroform and added to the reaction mixture. This solution was stirred for 24 hours at ambient temperature. The reaction mixture was decanted from the phosporous pentaoxide and the solids were washed with

chloroform (2 x 50 mL) . The combined organics were dried (MgS0 4 ) filtered and concentrated to an oil which was purified on a silica gel column (2.5 x 22 cm) with 1:15 ethyl acetate:hexanes. Eroduct was obtained as a clear oil (1.13 g, 2.8 mmol) after drying:

MS(CI): m/z 371 (M+H-Cl);

4. NMR (CDC1-) δ , 7.69-7.65 (m, 4H, Ar-H), 7.44-7.36 (m, 6H, Ar-H), 4.71 (d, IH, OC^O, J=6.8 Hz), 4.62 (d, IH, OCH 2 0, J=6.7 Hz), 3.88-3.84 (m, IH, H-2) , 3.73-3.61 (m, 4H, H-l and H-4), 3.34 (s, 3H, OCH..) , 2.06-1.99 (m, 2H, H-3) , 1.06 (s, 9H, C(CH 3 ) 3 );

13 C NMR (CDC1 3 ) δ , 135.6 (Ar) , 133.3 (Ar) , 129.8 (Ar) , 127.7 (Ar), 96.3 (CH 2 ), 74.9 (CH) , 65.8 (O^) , 55.7 (CH 3 ), 41.5 CU^ , 35.1 (CH 2 ), 26.8 (CH 3 ) , 19.2 (C) ;

Analysis Calculated for C -H-.O-ClSi 0.15 C H. : Calcd: C, 65.50; H, 7.94. Found: C, 65.58; H, 7.74.

H. (R)-4-Chloro-2-(methoxymethoxy) butyl benzoate

(R)-1-((tert-Butyldiphenylsilyl)oxy)-4-chloro-2-(methoxym ethoxy) butane 0.50 g (1.23 mmol) and benzoic anhydride 1.1 g (4.9 mmol) were weighed into a dry flask fitted with a constant addition funnel. Anhydrous acetonitrile (20 mL) was added and the constant addition funnel was charged with a 1 M solution of tetrabutyl ammonium fluoride in tetrahydrofuran (2.5 mL) . The tetrabutyl ammonium fluoride was added in two portions over two hours. The reaction was stirred at ambient temperature for 20 hours. An additional 1.0 mL (1 mmol) of tetrabutyl ammonium fluoride was added and the reaction was continued for 20 hours more.

The reaction was passed through a silica gel filter pad (5 x 5 cm) with 1:10 ethyl acetate:hexanes. The UV absorbing fractions were concentrated and purified on a silica gel column (2.5 x 18 cm) with 1:10 ethyl acetate:hexanes. Product was obtained as a clear oil (0.24 g, 0.88 mmol) after drying:

1 H NMR (CDC1 3 ) δ , 8.05 (d, 2H, Ar-H, J-7.1 Hz), 7.58 (t, IH, Ar-H, J-6.7 Hz), 7.45 (t, 2H, ArH, J-7.5 Hz), 4.82 (d, IH, OCH 0, J=6.9 Hz), 4.73 (d, IH, OCHgO, J-6.9 Hz), 4.46 (dd, IH, H-l, J=4.4 Hz, J=11.7 Hz), 4.36 (dd, IH, H-l', J=5.0 Hz, J=11.7 Hz), 4.20-4.12 (m, IH, H-2), ' 3.74-3.69 (m, 2H, H-4) , 3.40 (s, 3H, 0CH 3 ), 2.17-2.04 (m, 2H, H-3) ;

13 C NMR (CDC1 3 ) δ , 166.3 (C=0) , 133.2 (Ar) , 129.8 (Ar) , 129.6 (Ar), 128.4 (Ar) , 96.5 (CH 2 ) , 72.5 (CH) , 66.3 (CH 2 ) , 55.8 (CH 3 ) , 41.0 (CH 2 ), 35.3 (CH 2 ).

Analysis Calculated for C, _H-..0.Cl 0.15 C-H..: J 13 17 4 6 14

Calcd: C, 58.45; H, 6.74. Found: C, 58.61; H, 6.35.

(R)-2- r (2-Acetamido-l.6-dihydro-6-oxo-7H-nurin-7-yl)methoxy1 -4- chlorobutyl benzoate

Diacetylguanine 0.35g (1.4 mmol) (U.S. 4,355,032) was treated with N,0-bistrimethylsilylacetamide 1.8 mL (7.3 mmol) in 1,2- dichloroethane (10 mL) . The solution was stirred for 1.5 hours until all the solids had dissolved. The 1,2-dichloroethane was removed under vacuum, and the residue was taken up in anhydrous acetonitrile. This solution was treated with trimethylsilyl triflate 2.0 mL (10 mmol), stirred for 15 minutes then treated with a solution of (R)-4-chloro-2-(methox methoxy) butyl benzoate 0.27g (1.0 mmol) in anhydrous acetonitrile (5 mL) . After 30 minutes at ambient temperature, the reaction was diluted with ethyl acetate (150 mL) and hexanes (50 mL) and extracted with H„0

(2x50mL) . The organics were washed with 5% NaHCO, (25 mL) and then with H„0 (25 mL) followed by drying (MgS0 ) and concentration to a clear oil (0.48g). The crude product was purified by silica gel chromatography with methanoldichloro¬ methane (1:10) providing 0.37 g (0.84 mmol) of the N-7 regioisomer:

1 H-NMR (Me o S0-d,) δ , 12.1 (s, IH, NH) , 11.6 (s.lH, NH) , 8.40 λ b

(s,lH, H-8), 7.90 (d, 2H, Ar-H, J-6.9Hz), 7.66 (t, IH, Ar-H, J=7.4Hz), 7.51(t, 2H, ArH, J-7.6Hz), 5.82 (d, IH, 0CH 2 0, J=10.9Hz), 5.73 (d, IH, 0CH 2 0, J-10.9Hz), 4.53 (dd.lH, H-l, J=3.0Hz, J=11.7Hz), 4.23 (dd.lH, H-l', J=5.6Hz, J=11.7Hz), 4.18-4.14 (m,H-2, CH) , 3.64 - 3.60 (m, IH, H-4) , 3.48 - 3.45 (m, IH, H-4'), 3.34 (s, 3H, 0CH 3 ) , 2.18 (s, 3H, CH 3 C(0)NH-), 2.02 - 1.96 ( , 2H, H-3) ;

13 C NMR (Me„S0-d.) δ , 173.3 (NC-0) , 165.4 (PhC-0) , 157.5 (Pur), 2. o

152.4 (Pur), 147.1 (Pur), 145.1 (Pur), 133.3 (Ar) , 129.2 (Ar) , 129.0 (Ar), 128.6 (Ar) , 111.0 (Pur), 73.6 (CH 2 ) , 72.0 (CH) , 65.2 (CH 2 ), 41.2 (CH 2 ), 33.9 (CH.-,) , 23.6 (CH 3 ) .

Analysis Calculated for C j^ H..N-0 Cl 0.20 CH,00.05 CH 2 C1 : Calcd: C, 52.02; H, 4.74; N, 15.76. Found: C, 51.93; H, 4.80; N, 15.87.

J. (R)-2-r(2-Acetamido-1.6-dihvdro-6-oxo-7H-purin-7-yl)methoxy1 -4- iodobutyl benzoate and (R)-2- \(2-Acetamido-1.6-dihydro-6-oxo-9H- purin-9-yl)methoxy1-4-iodobutyl benzoate

Freshly distilled 2-butanone (25 mL) was added to (R)-2-[(2- acetamido-1,6-dihydro-6-oxo-7H-purin-7-yl)methoxy]-4-chlorob utyl benzoate 0.52g (1.2 mmol) along with anhydrous sodium Iodide 0.36 g (2.4 mmol). This solution was stirred under nitrogen at 85°C. After three hours the reaction mixture was diluted with dichloromethane and extracted with H_0 (2x50 mL) . The aqueous

layers were back-extracted with dichloromethane (50 mL) then the combined organics were washed with H„0 (50 mL) , dried (MgSO, ) and concentrated. The residue was purified on a silica gel column (2.5x16 cm) with methanol:dichloromethane (1:10). Product was obtained as a yellow glass after drying 0.32 g (0.60 mmol). H-NMR data showed that the product was a mixture of N-9 (30%) and N-7 (70%) regioisomers:

1 H-NMR (Me.SO-d^) δ , 12.10 (s, IH, NH, N-7), 12.00 (s, IH, NH, o

N-9), 11.71 (s, IH, NH, N-9), 11.61 (s, IH, NH, N-7), 8.43 (s, IH, H-8, N-7), 8.40 (s,lH, H-8, N-9) , 8.20 - 7.82 (m, Ar-H, N-7 and 9), 7.70 - 7.64 (m, Ar-H, N-7 and 9), 7.54 - 7.48 (m, Ar-H, N-7 and 9), 5.82 (d, IH, 0CH..0, J-10.9Hz, N-7), 5.73 (d, IH, 0CH 2 0, J=10.9Hz, N-7), 5.62 (d, IH, OC^O, J=11.4Hz, N-9) , 5.56 (d, IH, OCH 0, J=11.4Hz, N-9) , 4.58 - 4.47 (m, H-l, N-7 and 9), 4.28 - 4.21 (m, H-l', N-7 and 9), 4.00 - 3.93 (m, H-2, N-7 and 9), 3.35 - 3.20 (m, H-4, N-7 and 9), 3.15 - 3.00 (m, H-4' , N-7 and 9), 2.16 (s, 3H, CH 3 C(0)N, N-9), 2.09 (s, 3H, CH.-C(0)N, N-7), 2.20 - 2.00 (m, H-3, N-7 and 9);

MS (Cl) 526 (M+l).

Analysis Calculated for C H N 0 I 0.15 C-H-O: Calcd: C, 43.91; H, 3.99; N, 13.06 Found: C, 44.12; H, 4.05; N, 13.26.

K. (R)-Diethyl-3-r (2-acetamido-l.6-dihydro-6-oxo-9H-purin-9-yl) methoxyl -4-benzoyloxybutylphosphonate

The mixture of (R)-2- [(2-acetamido-l,6-dihydro-6-oxo-7H-purin-7- yl)methoxy] -4-iodobutyl benzoate and (R)-2- [ (2-acetamido-l,6-di- hydro-6-oxo-9H-purin-9-yl)methoxy] -4-iodobutyl benzoate (0.31 g, 0.60 mmol) was treated with toluene (4mL) and N,0-bistrimethyl- silylacetamide (0.3 mL, 1.2 mmol) and heated to 80°C in an oil bath. After two hours the solution was cooled to ambient

temperature. In a separate flask a 60% dispersion of sodium hydride In mineral oil (0.12 g, 3.0 mmol) was treated with anhydrous tetrahydrofuran (20 mL) and cooled to -78°C. A solution of diethylhydrogen phosphite dissolved in tetrahydrofuran was added and the mixture was stirred at -78°C for 30 minutes then at 0°C for 45 minutes. The two solutions were combined and stirred at 0°C for five hours then diluted with dichloromethane (200 mL) and H_0 (50 mL) . The aqueous phase was neutralized with IN HCl, then the two phases were shaken and separated. The aqueous phase was further extracted with dichloromethane (150 mL) and the combined organics were dried (MgSO,), filtered and concentrated to a yellow oil. This residue was purified on a silica gel column (230-400 mesh, 2.5x18 cm) using methanol:dichoromethane (1:10) to give 0.13 g (0.25 mmol) of title compound as a white foam:

UV λ (e): pH=7.00: 259.6 nm (13,400), 236.4 nm (14,500); 0.1 N max

HCl: 262.3 (14,100), 236.2 (14,400); 0.1 N NaOH: 263.2 (12,100);

1 H-NMR (Me„S0-d ) 8 , 11.9 (s, IH, NH) , 11.7 (s,lH, NH) , 8.19 .. fa

(s,lH, H-8), 7.79 (d, 2H, Ar-H, J-7.3Hz), 7.66 (t, IH, Ar-H, J-7.6Hz), 7.51 (t, 2H, ArH, J-7.7Hz), 5.62 (d, IH, OCE Q, J-11.4HZ), 5.56 (d, IH, OC^O, J-11.4Hz) , 4.37 (dd,lH, H-4, J-3.3HZ, J-=11.9Hz), 4.22 (dd.lH, H-4, J-6.3Hz, J-11.8Hz), 3.99 - 3.88 (m,5H, H-3 and OCJ^CH.-, 2.16 (s, 3H, CH 3 C(0)NH-), 1.75 - 1.50 (m, 4H, H-l and H-2) , 1.20 (t, 6H, OCH^H... J-7.0 Hz);

13 C NMR (Me 2 S0-dg) δ , 173.8 (NC-0) , 165.7 (PhC-0) , 155.1 (Pur), 149.0 (Pur), 148.3 (Pur), 140.4 (Pur), 133.7 (Ar) , 129.5 (Ar) , 129.2 (Ar), 128.9 (Ar) , 120.2 (Pur), 75.6 (d, CH, J_ -,=17.5 Hz), 71.8 (0CH 2 0), 65.3 (CH 2 0C-0) , 61.2 (d, 0CH 2 CH 3 , J c -6.3 Hz), 24.2 (d, CH 2 , J c p= 4.1 Hz), 23.9 (NC(-0)CH 3> 20.3 (d, CH 2> J c p -141 Hz), 16.7 * (d, 0CH 2 CH 3 , J c p -5.7 Hz);

P NMR (Me„SO-d, ) δ , 32 .43 ; o

MS (CI) 536 (M+l) .

Analysis Calculated for C 2;- H 30 N 5 O g P 0.10 CH 0 0.15 CH 2 C1 : Calcd: C, 50.64; H, 5.61; N, 12.70 Found: C, 50.53; H, 5.71; N, 12.81.

(R -3- T(2-Amino-l.6-dihvdro-6-oxo-9H-purin-9-yl methoxy1 -4- hydroxybutylphosphonic acid:

The protected product (R)-diethyl-3- [ (2-acetamido-l,6-dihydro-6- oxo-9H-purin-9-yl)methoxy]-4-benzoyloxybutylphosphonate (0.10 g, 0.19 mmol) was treated with methanol (100 mL) saturated with methylamine at 0°C. This solution was kept at 0-5°C for 18 hours. The solution was concentrated then redissolved in 0.5 N NaOH (50 mL) and stirred at ambient temperature for 24 hours. The solution was neutralized with slow addition of dry ice to pH 7.5, then extracted with dichloromethane (3x50 mL) . The aqueous phase was concentrated while maintaining a neutral pH. The white solid residue weighed 0.26 g after drying. This material was taken up in 10 mL of 50 mM NaHC0_ buffer (pH 9) and 100 units of snake venom phosphodiesterase I (Pharmacia LKB Biotechnology, Uppsala, Sweden) were added. This solution was incubated at 37°C for five days. HPLC on a SAX column with isocratic 10 mM ammonium phosphate showed the reaction was 50% complete. An additional 100 units of enzyme were added and the reaction was kept at 37°C for 41 hours more. HPLC showed the reaction was 65% complete. The reaction mixture was diluted with 40 mL of deionized water, adjusted to pH 9, and purified on a Sephadex DEAE A-25 ion exchange column prepared in 50 mM ammonium bicarbonate. (R)-Ethyl hydrogen 3- [ (2-amino-l,6-dihydro-6-oxo- 9H-purin-9-yl)methoxy]-4-hydroxybutylphosphonate was isolated from the column with 50 mM ammonium bicarbonate and was

repeatedly concentrated from deionized water to give 0.023 g (0.06 mmol) of a white solid:

^I-NMR (D 2 0) δ , 7.80 (s.LH, H-8) , 5.49 (d, IH, NCH 2 0, J=11.4Hz), 5.38 (d, IH, NCH 2 0, J=11.4Hz), 3.58 - 3.50 (m, 4H, 0CH 2 CH 3 and H-3 andH-4), 3.39 (dd, IH, H-4' , J-6.9 Hz, J-13.0Hz), 1.50 - 1.00 (m, 4H, H-l and H-2) , 0.99 (t, 3H, 0CH 2 CH 3 , J-7.1 Hz). A linear gradient was started with 500 mM ammonium bicarbonate as the high concentration buffer. The title compound phosphonic acid eluted off early In the gradient and was repeatedly concentrated from deionized water. (R)-3-[(2-Amino-l,6-dihydro- 6-oxo-9H-purin-9-yl)methoxy]-4-hydroxybutylphosphonic acid was obtained as a white solid, which weighed 0.034 g (0.09 mmol), and was shown to be the mono-ammonium salt by the spectrophotometric assay of Nathans, G.R., et. al. , Vol. LI, Methods in Enzymology, Article 66, p.497:

UV λ (e): pH=7.00: 252.0 nm (12,100), 270 nm (9,500); 0.1 N HCl: 255.0 (10,900), 275 (8,300); O.l NaOH: 265.0 (10,000);

hl-NMR (D 2 0) δ , 7.78 (s.lH, H-8) , 5.40 (br.s, 2H, CH 2 0 , 3.55 - 3.32 (m, 4H, H-3 and H-4) , 1.56-1.46 (m, 2H, H-2) , 1.32 - 1.13 ( , 2H, H-l);

13 C NMR (Me 2 S0-dg) δ , 158.8 (Pur), 153.9 (Pur), 151.5 (Pur), 139.8 (Pur), 115.9 (Pur), 79.9 (d, C-3, J 17.6 Hz), 71.7

(0CH 2 0), 62.7 (C-4), 24.7 (d, C-2, J c __«3.1 Hz), 23.5 (d, C-l, J c p -134 Hz);

31 P NMR (Me„S0-d ) δ , 24.51. L fa

20 Optical Rotation at 5.23 mg/mL in water: [α] 589 - -1.5°;

20 20 20

[a] 578 - -1.5°; [a] Δ 546 « -1.8°; [a]^ 436 = -3.3°;

20 [α] 365 - -5.5°.

M. (R)-Dibenzyl-3- r ( " 2-acetamido-l.6-dihvdro-6-oxo-9H-purin-9-yl) methoxyl -4-benzoyloxybutylphosphonate

The mixture of (R)-2- [ (2-acetamido-l,6-dihydro-6-oxo-7H-purin-7- yl)methoxy] -4-iodobutyl benzoate and (R)-2- [ (2-acetamido-l,6- dihydro-6-oxo-9H-purin-9-yl)methoxy] -4-iodobutyl benzoate (from part J) (1.25g, 2.38 mmol) was treated with benzene (15mL) and N,0-bistrimethyisilylacetamide (1.8mL, 7.1 mmol)) and heated to reflux in an oil bath. After two hours the solution was cooled to ambient temperature and the benzene was removed under vacuum. The residue was dissolved in anhydrous tetrahydrofuran (20mL) . In a separate flask potassium tert-butoxide (l.lg, 9.5mmol) was dissolved in anhydrous tetrahydrofuran (20mL) and cooled to 0-5 C. Dibenzylhydrogen phosphite (2.2mL, 9.8mmol) was added and the mixture was sturred at 0-5 C for one hour. The two solutions were combined and stirred at 0 C for three hours then quenched by pouring into 10% aqueous NH,Cl(lOOmL) . This solution was extracted with chloroform (2x400mL) . The combined organics were dried (MgSO,), filtered and concentrated to a yellow oil. This residue was purified on a silica gel column (230-400 mesh, 2.5x18cm) using first neat dichloromethane (2L) then methanol: dichloromethane (1:20) to give 1.5g of a mixture of N-9 and N-7 regio isomers which was further purified on a chromatotron fitted with a 2mm rotor and using methanol:dichloromethane (1:25). The chromatotron provided 0.77g (1.2mmol) of pure title product;

UV λ (e): pH=7.00: 238nm(10,000) , 261nm(10,000) , 280 sh (6700);

JΩcLX

0.1 N HCl: 238 (11,000), 263(11,300); 277 sh (9,400); 0.1 N NaOH; 263 (12,300);

"Ή-NMR (Me 2 S0-dg) δ : 11.9 (s.lH. H), 11.7 (s.lH, NH) , 8.17 (s,lH,H-8), 7.74 (d, 2H, Ar-H, J-7.1Hz), 7.63 (t.lH.Ar-H, J-7.5Hz), 7.45(t,2H,ArH,J-7.6Hz), 7.38-7.29 (m.lOH, Ar-H), 5.54(d,lH,NCH 2 0, J=11.5Hz), 5.49(d, lH.NCHgO, J-11.4Hz) , 4.95 (quintet, 4H, J-7.5Hz, 0CH,-Ph) , 4.32 (dd,lH,H-4\ J=3.3Hz ,

J-11.9HZ) , 4.17 (dd, lH,H4" ,J=6 .3Hz ,J=11.9Hz) , 3.97-3.94

(m, lH,H-3 ' ) , 2.12 (s , 3H, CH 3 C(0)NH- ) , 1.85-1.70 (m, 4H,H-l' and H-2' ) ;

13 C NMR (Me„SO-d,.)S: 173.4 (NC-O) , 165.3 (PhC=0) , 154.8 (Pur), 2. fa

148.7 (Pur) , 148.0 (Pur) , 140.0 (Pur) , 136. 6 (Ar,J _ =6 .1Hz) , > , a

133.4 (Ar), 129.2 (Ar) , 129.0 (Ar) , 128.9 (Ar) , 128.7 (Ar) , 128.6 (Ar), 128.5 (Ar), 128.4 (Ar) , 128.3 (Ar) , 128.2 (Ar) , 127.7 (Ar) , 120.4 (Pur), 75.2 (d,CH,J L_i, -17.6Hz), 71.5 (NCH Z..O) , 66.3

(d,P0CH 2 Ph,J c p -5.0Hz), , 24.0 (d,CH 2 , J c p -3.5Hz) 139Hz) ;

31.. NMR (Me„S0-d /- )5:33.7; fa

MS (CI) m/z (rel. intensity) 335 (100), 263 (13.9), 227 (11.5), 181 (61.8) 137 (44.6).

Analysis Calculated for C 33 H_,N g O P. 0.75H,_0; Calcd: C, 58.88; H, 5.32; N, 10.40 Found: C, 59.18; H, 5.35; N, 10.03.

20 Optical Rotation at 1.51 mg/mL in ethanol (95%): [a] 589 -

-4.64°,

N. (R)-Dibenzyl 3-F (2 -amino-1.6-dihvdro-6-oxo-9H-purin-9-vDmethoxy1 -4-hvdroxybutylphosphonate

The protected product from part M, (R)-dibenzyl-3-[(2-acetamido- l,6-dihydro-6-oxo-9H-purin-9-yl)methoxy]-4-benzoyloxybutylph osph- onate (0.36g, 0.55mmol) was treated with 95% ethanol (lOOmL) saturated with methylamine at 0-5 C. This solution was kept at 0-5 C for five days. The solution was concentrated then triturated with dlethylether to give 0.17g of a white powder. This material was further purified on a silica gel flash column

(2.5x12 cm) with ethanol: dichloromethane (1:2) to give 0.063g (0.12mmol) of a white solid;

4. -NMR (Me 2 S0-dg) δ : 10.63 (s.lH.NH), 7.80 (s,lH,H-8), 7.39-7.30 (m,10H,ArH), 6.50 (s,2H,NH,_), 5.39 (d ( lH,NCH 2 0, J=11.4Hz), 5.36 (d,lH,NCH 2 0, J=11.4Hz), 4.97-4.84 (m,4H,0CH 2 Ph) , 4.76

(t,lH,CH 2 0H, J-5.4Hz), 3.55-3.52 (m,lH, H-3'), 3.36-3.30 (m,2H,CH 0H), 1.70-1.45 (m,4H,H-l' and H-2');

MS (CI) m/z (rel. intensity): 514 (0.90, M+H) , 363 (46.8, M-guanine) .

Analysis Calculated for O;

Calcd: C, 54.98; H, 5.61; N, 13.36 Found: C, 54.88; H, 5.43; N, 13.54.

0. (R -3- T (2-amino-l.6-dihvdro-6-oxo-9H-purin-9-yl)methoxy1 -4-hydr- oxybutylphosphonic acid

(R)-Dibenzyl 3-[ (2-amino-l,6-dihydro-6-oxo-9H-purin-9-yl)methoxy] -4-hydroxybutylphosphonate, from part N, 1.5g (2.9mmol) was combined with 20% Pd(0H)~ on carbon (1.5g), cyclohexene (24mL) , deionized water (45mL) and absolute ethanol (45mL) . The solution was refluxed at an oil bath temperature of 100 C for three hours. The reaction mixture was cooled and filtered through a celite filter with water (200mL) and the filtrate was extracted with dichloromethane (2x200mL) . The aqueous layer was concentrated to an off white solid (0.75g). The residue was dissolved in 120mL of deionized water, adjusted to pH 9, and purified on a Sephadex DEAE A-25 ion exchange column prepared in 50 mM ammonium bicarbonate. A linear gradient was started with 500mM ammonium bicarbonate as the high concentration buffer. The free phosphonic acid eluted off early in the gradient and was repeatedly concentrated from deionized water. A white solid was obtained which weighed 0.356g (0.94mmol);

UV λ (e): pH=7.00; 252.Onm (10,000), 270nm (6,900); 0.1 N max

NaOH: 265.0 (8,000);

^I-NMR D 2 0) 8 : 7.78 (s,lH,H-8), 5.40 (br.s,2H, NCH 2 0 , 3.55 -3.32(m,3H,H-3'and H-4'), 1.56-1.46 (m,2H, H-2) , 1.32-1.13 (m,2H,H-l);

31 P NMR (Me 2 SO-dg) 8 : 25.9 ppm;

MS (CI) m/z (rel.Intensity): 335 (2.73, M+2H) , 183 (16.07, M-guanine), 151 (100, Guanine+ H)

Optical Rotation at 5.0 mg/mL In water: [α] 589 - -4.6 ; [ ] 578 - -4.8°; [ex] 20 546 - -5.8°; [α] 20 436 - -10.6°; [a] 20 365 - -17.2°.

Analysis Calculated for C-QH g 5 0gP.1.5 H 2 0.1 NH " Calcd: C, 31.83, H, 5.88, N, 22.27 Found: C, 31.88, H, 5.75, N, 22.23.

Analyzed as the mono-ammonium salt in a spectrophotometric assay by method of Nathans, G.R. et. al. , Volume LI, Methods in Enzymology, Article 66, pg 497.

Example 2

Preparation of Racemic 3-f(2-Amino-1.6-dihydro-6-oxo-9H-purin-9-yl methoxyl-4-hydroxybutylρhosphonic Acid

A. 2-T(Benzyloxy)methylloxirane

A two phase system consisting of 240 mL of 50% aqueous sodium hydroxide, 4.89 g (0.014 M)tetra-n-butylammonium hydro ensulfate and 150 mL (1.92 M) of epichlorohydrin was stirred at room temperature for ten min. Anhydrous benzyl alcohol, 36 mL (0.36

M) , was then added and the resulting mixture stirred at room temperature for 4 h (after 3.5 h, a temperature increase to 50°C was noted) .

The mixture was poured into 700 mL of ice and water and the organic product extracted with ether (2 x 200 mL) . The combined ethereal extracts were washed with saturated aqueous sodium bicarbonate (2 x 200 mL) and dried over magnesium sulfate. Filtration and evaporation in vacuo at 30°C, gave an orange oil which was distilled at 0.3 mm. The desired product, 2- [(benzyl- oxy)methyl]oxirane, 47.2 g (80%) distilled at 100-117"C.

X H NMR (CDC1 3 ): δ , 7.35 (m, 511, phenyl), 4.59 (q, J-12, 2H, phenyl C^O) , 3.75 (q, ^ He . Hd -ll^. J He -Hc" 3 -°' 1H ' ' 3 Λ6 (q '

J Hd-Hc= 5 - 5 ' 1H ' V- 3'2 < m> 1H ' ' 2 ' 8 <* ■ J Hc-Hb= J Ha-Hb- 4 - 6 ' IH, H, ) , 2.63 (q, J -2.6, IH, Ha); a trace amount of benzyl alcohol impurity was also present as noted from small extra multiplets, 7.3 (aromatic) and 4.7 (CΗ O) .

13 C NMR (CDC1 3 ): δ , 137.87, 128.43, 127.76, (phenyl); 73.32 (phenyl CH 2 0) , 70.08 (CH j O) , 50.87 (CH) , 44.30 (oxirane ring CH 2 ).

B. Diethyl 4-benzyloxy-3-hydroxybutylphosphonate

A solution of diethylmethyl phosphonate (7.7 mL) in THF (500 mL) was chilled to -75°C under a nitrogen atmosphere. A solution of 1.6 M nBuLi (34 L) in hexanes was then added dropwise over the

course of 20 min to the chilled solution by an addition funnel. Boron trifluoride etherate (7.0 mL) was added dropwise by syringe over the course of 5 min. The resulting solution was then treated with a solution of benzyloxymethyloxirane (7 g) In THF (50 mL) over the course of 20 min. The reaction mixture was then sealed and the reaction flask was packed in a styrofoam container packed with dry ice and allowed to stand for 20 h (at -66°C). The reaction mixture was quenched with saturated NaHC0_ solution (100 mL) and the resulting suspension was allowed to warm to room temperature. The THF was removed in vacuo to afford a concentrate which was extracted with CH^Cl- (2 x 350 mL) . The combined organic layers were filtered through a pad of celite, dried over MgS0 and then filtered again through a pad of celite. The filtrate was concentrated In vacuo to an opaque oil which was dissolved in a minimal amount of EtOAc and applied to a column (9 x 22 cm) packed with silica gel (300 g) in EtOAc (neat) . The column. was eluted with EtOAc (neat) , then EtOAc:isopropano1/97:3, and finally 95:5, collecting 175 mL fractions. The fractions containing a product with an R..-0.29 were pooled and concentrated in vacuo to an oil which was co-evaporated with CH„C1„ (3 x 50 mL) . The colorless oil was further dried in vacuo (25°C, oil pump) for 30 min to provide 9.88 g of the title compound.

NMR (200 MHz. CDC1 3 ): δ , 7.35 (s, 5H, Ph H's), 4.55 (s, 2H, PhCH 2 ), 4.08 (q, 4H, OCH 2 CH 3 , 3.83 (m, IH, CHOH) , 2.92 (m, 2H, BnOCH ), 2-5 (br s, IH, OH), 2.0-1.6 (m, 4H, CJ^CfLP), 1.31 (t, 6H, CH,). A small doublet centered at 1.46 which corresponds to a small amount of contaminating diethylmethylphosphonate was visible in the spectrum.

13 C NMR (50 MHz, CDC1 3 ) : δ , 138.4 (Ph C) , 129.0 (Ph C) , 128.3 (Ph C), 74.4 (PhCH 2 0), 73.8 (CH^Bn) , 70.6 (CHO) , 62.0 (OCH^H , 26.7 (CH 2 CH 2 P), 22.2 (d, J -142 Hz, CH 2 CH 2 P , 16.8 CH 3 ) .

Small doublet at 11.6 corresponding to diethyl methyl phosphonate.

31 P NMR (121 MHz, CDC1 3 ) : δ , 33.14.

Diethyl 4-benzyloxy-3-chloromethoxy-3-butylphosphonate

A suspension of diethyl 4-benzyloxy-3-hydroxybutylphosphonate (9.56 g) (200 mL) and paraformaldehyde (95%, 2.58 g) in CH Cl 2 was cooled to 0°C by an external ice bath. HCl (g) was introduced into the chilled reaction mixture by a bubble tube over the course of 3.5 h. The bubble tube was removed and the reaction was kept at 0°C for an additional 16 h. The cold reaction mixture was then dried over CaCl„ pellets and filtered. The filtrate was concentrated in vacuo to an opaque oil. The oil was co-evaporated with CH-C1„ (2 x 50 mL) and dried further in vacuo (25°C, oil pump) for 40 min to furnish 11.81 g of the title compound.

. NMR (200 Hz, CDC1 3 ) : δ , 7.35 (m, 5H, Ph H) , 5.65-5.55 (m, 2H, 0CH 2 C1), 4.53 (s, 2H, Cg 2 Ph) , 4.15-3.90 (m, 5H, OCH CH 3 , CHO), 3.54 (m, 2H, CH 2 0Bn) , 1.95-1.70 (m, 4H, CH 2 CH 2 P) , 1.32 (m, 6H, OCH-CH.) . There are peaks corresponding to minor impurities at 5.30 and from 5.06-4.80 ppm.

13 C NMR (75 MHz, CDC1 3 ): δ , 137.7 (Ph C) , 128.4 (Ph C) , 127.7 (Ph C), 82.2 (0CH 2 C1), 77.7 (CH) , 73.4 (PhCH ) , 71.8 (CH OBn) , 61.7 (CH 2 CH 3 ), 24.3 (CH 2 CH 2 P), 21.4 (d, J p . c -1 3 Hz), 16.3 (CH 3 ) .

31 P NMR (300 MHz, CDC .): δ , 32.37.

2-Amino-6-chloro-9-(3-diethylphosphono-1-benzyloxymethyl- propoxymethyl) urine

A suspension of 2-amino-6-chloropurine (5.02 g) and cesium carbonate (9.64 g) in DMF (60 mL) was stirred at room temperature for 1 h and then a solution of diethyl. 4-benzyloxy-3-chlorometh- oxy-3-butylphosphonate (13.5 g) In DMF (20 mL) was added in one portion. The resulting suspension was stirred at room temperature for 16 h and then it was filtered. The filtrate was concentrated in vacuo (oil pump) to an oil which was partitioned between EtOAc (300 mL) and H_0. The H.O layer was re-extracted with additional EtOAc (1 x 200 mL) . The combined organic layers were dried over Na„S0, and then concentrated in vacuo to an oil. The oil was dissolved in a minimal amount of EtOAc and applied to a column (13.5 x 16 cm sintered glass funnel) packed with silica gel (340 g) in EtOAc (neat) . The column was eluted with EtOAc (neat) and then EtOAc:isopropano1/9:1, collecting fractions. The fractions containing a product with an R..-0.23 (EtOAc: isopropanol/9:l) were pooled and concentrated in vacuo to a white gum, 6.00 g. A portion of this gum (5.40 g) was suspended in CH„C1„ (15 mL) , filtered, and applied to a lobar column (5 x 35 cm) packed with silica gel in CH_C1 (neat) . The column was eluted with a gradient of CH-C1_:MeOH/10:0 to 9:1, collecting fractions. The product-containing fractions were pooled and concentrated in vacuo to furnish the title compound as a gum (3.71 g) .

4ϊ NMR (CDCL-, 300 MHz): 8 , 7.91 (s, IH, H-8) , 7.35-7.26 (m, 5H, Ph H's), 5.61 (s, 2H, OCH^N) , 4.49 (s, 2H, PhCH^O) , 4.08 (m, 4H, 0CH 2 CH 3 ), 3.83 ( , IH, CHOH) , 3.47 (m, 2H, CH 2 OBn) , 1.91-1.60 (m, 4H, CH CH 2 P), 1.28 (m, 6H, OCH 2 CH 3 ) . Small peaks corresponding to minor impurities were evident at 7.85, 5.65-5.49, 4.80, and 3.5 ppm.

13 C NMR (75 MHz, CDC1 3 ) : 8 , 159.5 (C-6), 153.9 (C-2) , 151.3 (C-4). 142.4 (C-8), 137.6 (Ph C) , 128.5 (Ph C) , 127.7 (Ph C), 124.9 (C-5), 77.7 (CHO), 73.5 NCH 2 0) , 73.4 (CH OBn) , 72.8 (0CH 2 Ph), 61.7 (OCH 2 CH 3 ), 24.3 (CH 2 CH 2 P) , 21.5 (d, J p _ c -142 Hz,

CH CH-P) , 16.4 (CH,). Small peaks corresponding to minor impurities are evident at 24.9, 22.7 and 20.8 ppm.

31

P NMR (121 MHz, CDC1 ) : 8 , 32.42. Small peaks corresponding to minor impurities are evident at 33.36 and 32.84 ppm.

Ethyl Hydrogen 3- f (2-Amino-l.6-dihvdro-6-oxo-9H-purin-9-yl) methoxyl -4-benzyloxybutylphosphonate

A suspension of 2-amino-6-chloro-9-(3-diethylphosphono-l- benzyloxymethylpropyloxymethyl)purine (304 mg) , DABCO (14 mg) , 1 N NaOH (10 mL) and EtOH (1 mL) was heated at 55°C for 1 h. Another portion of EtOH (6 mL) was added and the reaction mixture immediately became a solution. The solution was diluted to 120 mL with H.0 and then sufficient 1 N HCl was added to effect a pH-9. The solution was applied to a lobar column packed with DEAE (HC0 ~ form) Sephadex. The column was eluted with H„0 and then a linear gradient from 0 to 0.2 N NH,HC0_ , collecting fractions. The product-containing fractions were pooled and concentrated in vacuo. The concentrate was dissolved in H 0 (3 mL) and applied to a Regis C-18 HPLC column (21 x 250 mm). The column was eluted at 10 mL/min with 0.1 M ammonium acetate buffer (pH-5.8) :acetonitrile/88:12, collecting fractions. The product-containing fractions were pooled and concentrated in vacuo to a white amorphous solid. This solid was dissolved in H«0 and lyophilized to provide 74 mg of the title compound as a white fluffy solid.

4i NMR (300 MHz, D 2 0) : 8 , 8.23 (br s, IH, H-8), 7.33-7.10 (m, 5H, Ph H's), 5.59 (d, IH, J 2 -11.7 Hz, NCH^O) , 5.49 (d, J 2 -11.7 Hz, NCH 2 0), 4.33 (s, 2H, 0CH 2 Ph) , 3.83 (m, 3H, OCH 2 CH 3 , CHO), 3.47 (m, 2H, CH 2 0Bn), 1.72-1.42 (m, 4H, CH^P) , 1.19 (t, 3H, 0CH 2 CH 3 ).

13 C NMR (75 MHz, D £ 0) : 8 , 157.0 (purine C) , 139.8 (C-8), 131.1 (Ph C), 130.8 (Ph C), 130.4 (Ph C) , 82.0 (CHO), 75.9 (NCH 2 0) , 75.5 (CH OBn), 74.2 (OCH £ Ph) , 63.5 (OCH 2 CH 3 ) , 27.6 (CH 2 CH P) , 24.4 (d, J c _ p -136 Hz, CH 2 CH 2 P) , 18.6 (CH 3 CH 2 ) .

31 P NMR (121 MHz, D 2 0) : 8 , 28.27.

F. 3-f2-Amino-1.6-dihvdro-6-oxo-9 H -purin-9-yl)methoxy1-4- hydroxybutylphosphonic acid

A solution of previous title compound (2E) (120 mg) in DMF (4 mL) was treated with bromotrimethylsilane (0.4 mL) dropwise over the course of 3 min while being chilled by an external dry ice- isopropanol bath. After the addition was complete the ice bath was removed and the reaction mixture was allowed to stir at room temperature for 20 h. The reaction mixture was recooled by a dry ice-isαpropanol bath and then another portion of bromotrimethyl¬ silane (0.2 mL) was added. The resulting mixture was allowed to stir at room temperature for another 2 days and then the reaction mixture was quenched with EtOH (4 mL) . Sufficient 15% aq. NH OH was then added to the solution to effect a pH=8. The resulting mixture was concentrated in vacuo to a sticky white solid.

This solid was taken up in EtOH (5 mL) and H„0 (5 mL) . Cyclohexene (0.5 mL) and 20%Pd(OH) 2 on carbon (97 mg) was added and the resulting mixture was heated at reflux for 4 h. Another portion of cyclohexene (1 mL) was added and the heating was resumed overnight. The reaction mixture was allowed to cool to room temperature and then filtered through a pad of celite. The filtrate was concentrated in vacuo to a white solid. The solid was again taken up in EtOH:H„0/1:1, and more 20% Pd(0H) on carbon (115 mg) and cyclohexene (1.5 mL) was added. The resulting mixture was heated at reflux for another 20 h and then filtered through a thick pad of celite. The filtrate was concentrated in vacuo to a sticky white solid. The solid was

recrystallized from H-0 to afford a solid whose HPLC tracing (Supelco C-18 analytical column, 100% H_0, rate 2 mL/min) revealed the presence of a more nonpolar impurity. The solid and mother liquor were combined and dissolved in H„0 (60 mL) and the pH of the resulting solution was adjusted to 9 with concentrated NH.OH. The solution was applied to a lobar column (2 x 8.5 cm) packed with DEAE Sephadex (HCO,- form). The column was eluted with H_0 and then with a linear gradient from 0 to 0.4 N NH HC0_, collecting fractions. The product-containing fractions were pooled and concentrated in vacuo to a white solid. An HPLC analysis of this solid (using the same conditions described above) again revealed the presence of a more nonpolar impurity. The solid was dissolved in H„0 (4 mL) and loaded onto a Regis C-18 prep column (21 x 250 mm) . The column was . eluted at 10 mL/min with H„0, collecting fractions. The fractions were analyzed by HPLC (Supelco C-18, 2 mL/min, 100% H„0) . The fractions containing the desired product were homogeneous, containing a single component which eluted at 48 seconds. These fractions were pooled and concentrated in vacuo and then lyophilized to furnish 28 mg of the title compound as a white fluff.

The 300 MHz H NMR (DMSO-d ) spectrum of the title compound revealed the presence of 2 singlet peaks at 8.1 and 7.8 ppm corresponding to the H-8 of the N-7 and N-9 isomers, respectively. Peaks at 5.59 (br s) and 5.38 ppm (dd) corresponding to the NCH„0 protons of the N-7 and N-9 isomers, respectively, were also evident in a ratio of 1 to 9 (N-7:N-9).

31 P NMR (121 MHz, D 2 0) : δ , 25.62.

Example 3

Resolution of 3- [ (2-Amino-l,6-dihydro-6-oxo-9H-purin-9-yl)-methoxyl - 4-hvdroxy-butylphosphonic Acid Into Its Enantiomers

GMP kinase was used to resolve the compound of Example 2F into its R- and S-enantiomers and separate the N-7 isomers. The enzyme preferentially phosphorylates only one of the enantiomers of the N-9- isomers and will not phosphorylate the N-7-isomers. The monophosphate of the R-phosphonic acid is obtained as a product of GMP kinase. It is separated from unreacted materials and then converted back to the R-phosphonic acid using alkaline phosphatase.

A. CS)-3-T(2-Amino-l.6-dihvdro-6-oxo-9H-purin-9-yl)methoxy1-4- hydroxybutylphosphonic acid

A reaction mixture, final volume 22 ml, contained 0.11 mmol racemic phosphonate of Example 2F, 1.2 mmol adenosine 5'- triphosphate, 1 mmol magnesium chloride and 14 units of hog brain GMP kinase (Sigma Chemical Company, St. Louis, MO 63178). After about 4 h at 37° C, the reaction mixture was frozen. Separation of the reaction products was performed using anion exchange chromatography on DE4AE Sephadex A-25: the reaction mixture was thawed, diluted to 70 ml with water, and applied to a chromatography column containing about 50 ml of DEAE Sephadex A-25 which had been previously equilibrated in 50 mM ammonium bicarbonate. The column was washed with one liter of 50 mM ammonium bicarbonate, and compounds were eluted with 1.2 liters of a linear gradient of 50 - 500 mM ammonium bicarbonate. The unreacted S-enantiomer (fraction 1) eluted first, followed by the monophosphate of the R-phosphonic acid (fraction 2) , which was overlapped by the later-eluting adenosine 5' -diphosphate and adenosine 5' -triphosphate. Fractions containing each enantiomer were pooled and dried in vacuo to remove water and ammonium bicarbonate. Fraction 1 was determined to be a mixture of the S-enantiomer of the N-9-isomer (title compound) and a racemic mixture of the R- and S- N-7-isomers (0.052 mmol).

20

Optical Rotation at 3.3 mg/ml in water: [ ] 589 +0.91°;

[a] 578 - +0.30° [α] 20 546 +0.61 c [α] 2 °436 +0.91°;

20 [α] 365 +2.1°.

The presence of N-7 isomers may have contributed to the variations in optical rotation from the expected equal and opposite values to those of Example 3B.

31

P NMR (D 2 0) : δ , 25.2 (s)

H-NMR (D 2 0): 5, 8.03 (s, 0.2H, H-8, N-7-isomer), 7.79 (s.O.βH, H-8, N-9 isomer), 5.57 (m, 0.4H, NCH 2 0, N-7 isomer), 5.41 (m,

1.6H, NCH 0, N-9 isomer), 3.61 3.35 (m, 3H, CH and CH 2 0H) ,

1.52 (m, 2H, CH 2 > , 1.10 - 1.40 (m, 2H, CH 2 ) . Peaks at 5.57 and 5.41 and at 8.03 and 7.79 indicate a ratio of N-7 and N-9 isomers of 1:4.

The title compound was obtained in pure form by preparative HPLC on a 0.9x50cm Whatman Partisil 10 SAX column, eluting with 4ml/min lOmM ammonium phosphate (pH 2.4) containing 5% methanol. (R,S)-3- [ (2-Amino-l,6-dihydro-6-oxo-7H-purin-7-yl)methoxy] -4-hy- droxybutylphosphonic acid eluted at 16 min followed by (S)-3-[(2- A ino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy] -4-hydroxybutylphos phonic acid at 31 min. The latter compound was separated from ammonium phosphate by chromatography on DEAE Sephadex as described above to yield the desired product (13 μmol) .

UV (10 mM ammonium phosphate, pH 5.5) λ 253 nm, shoulder

267 nm, λ . 221 nm. mm

(10 mM ammonium phosphate, pH 2.4) λ 254 nm, shoulder r max

269 nm, λ . 222 nm. mm

H-NMR (D 2 0): δ , 7.79 (s, IH, H-8), 5.41 (m, 2H, NCH..0) , 3.58-3.32 (m, 3H, CH and CH,_0H) , 1.50 (m, 2H, CH 2 ) , 1.40-1.10 (m, 2H, CH 2 ).

B. (R)-3-rC2-Amlno-l.6-dihvdro-6-oxo-9H-Purin-9-yl)methoxyl-4- hydroxybutylphosphonic acid

The monophosphate of the R-phosphonic acid (fraction 2 of Example 3A) (0.05 mmol), was treated with 5,600 units alkaline phosphatase from calf intestine (Boehringer Mannheim Biochemicals, Indianapolis, IN 46250) for one hour at 37° C. The adenosine 5'-diphosphate and adenosine 5'-triphosphate were converted to adenosine, and the monophosphate of the R-phosphonic acid was converted to the title compound. This mixture was chromatographed using DEAE Sephadex, as in Example 3A. Fractions containing the title compound were pooled and dried in vacuo to remove water and ammonium bicarbonate (0.047 mmol).

20 Optical Rotation at 3.3 mg/ml in water: [ ] 589 - -0.61°;

20 20 20 20

[a] 578 - -0.61°; [ ] 546 - -0.91°; [α] 436 - -2.4°; [α] 365

- -4.8°.

On an ion exchange HPLC column (Whatman Partisil 10 SAX, gradient of ammonium phosphate, pH 5.5, 10 mM-800 mM monitored with Perkin Elmer LC480 diode array UV detector) , the title compound R-enantiomer eluted with an identical retention time to the racemic mixture of Example 2F, and to the R-enantiomer of Example IL. All of these compounds had UV spectra indicating the presence of a guanine base, with λ at 255 nm and a shoulder at 270 nm.

31 P NMR (D 2 0): δ , 25.1 (s)

" " H-NMR (D 2 0): δ , 7.84 (s,lH, H-8), 5.41 (m, 2H, NCH 2 0 , 3.52 - 3.36 (m, 3H, CH and Cg 2 0H) , 1.49 ( , 2H, CH 2 ) , 1.10 - 1.29 (m, 2H, CH 2 ).

C. fR.S)-3-r(Amino-1.6-dihvdro-6-oxo-7H-purin-7-yl)methoxyl -4- hydroxybutylphosphonic acid

The purified N-7 isomers from Example 3A were separated from ammonium phosphate by chromatography on DEAE Sephadex as described in Example 3A to yield the title product (1.8 μmol) .

UV (10 mM ammonium phosphate, pH 5.5) λ 243 nm, 286 nm, λ max mm

235 nm, 261 nm. ( (1100 mmMM aammmmoonniiuuπm p r hosp r hate,» prH 2.4) / λ m 2 75 nm, λ m a a χ 48 nm, 2 x mm 232 nm, 264 nm.

H-NMR (D 2 0) δ , 8.05 (s, IH, H-8), 5.58 (m, 2H, NCH,,0) ,

3.58-3.33 (m, 3H, CH and CH 2 0H) , 1.48 (m, 2H, CH 2 ) , 1.38-1.05 (m, 2H, CH 2 ).

Example 4

Preparation of phosphate esters

A. (R)-3- f(2-Amino-1.6-dihydro-6-oxo-9H-purin-9-yl)methoxyl -4- hydroxybutylphosphonic acid, diphosphate

Fraction 2 of Example 3A (39 μmol) was incubated for 24 hours at 37 C with creatine phosphate (5.6 mM) , magnesium acetate (61 mM) and creatine kinase, (Boehringer Mannheim, rabbit muscle), 1,350 IU/ml (57 mM) . The resulting mixture was purified on DEAE Sephadex by the method described in Example 3A to afford the title compound (25 μmol) .

UV (0.5 M ammonium phosphate, pH 5.5) λ 253 nm, shoulder

270 nm, λmm. 222 nm.

B. (S)-3-r(2-Amino-l,6-dihvdro-6-oxo-9H-purin-9-yl)methoxy1-4- hvdroxybutylphosphonic acid, diphosphate

The title compound of Example 3A (9.6 μmol) was dried from triethylammonlum bicarbonate then co-evaporated twice with acetonitrile. The compound was dissolved in 0.2 ml 1,3-dimethyl- 3,4,5,6, -tetrahydro-2(IH)-pyrlmidinone (Aldrich) then 0.012 g 1,1'-carbonyldiimldazole (Aldrich) was added. This mixture was stirred overnight at room temperature. Methanol (0.01 ml) was added and stirring continued for a further 30 min. Trlbutylammonium pyrophosphate (Sigma, 0.046 g) was added, stirred 90 min at 44 C, then 1 ml water and 0.1 ml 1M sodium bicarbonate was added. This mixture was chromatographed on DEAE Sephadex as described in Example 3A. The fractions containing the title compound were combined and dried in vacuo to remove water and ammonium bicarbonate affording the desired product (3.2 μmol) .

UV (0.5 M ammonium phosphate, pH 5.5) λ 252 nm, shoulder r r r max

271 nm, λ . 222 nm. mm

Pharmaceutical formulations

In the following formulation examples, the "Active Ingredient" may be a compound of formula (la) or a pharmaceutically acceptable salt, ester or salt of such ester thereof.

Example 5

Tablet Formulations

The following formulations A, B and C are prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression.

- 49

Formulation A

(a) Active ingredient

(b) Lactose B.P.

(c) Povidone B.P.

(d) Sodium Starch Glycollate

(e) Magnesium Stearate

Formulation B

(a) Active Ingredient

(b) Lactose

(c) Avicel PH 101

(d) Povidone B.P.

(e) Sodium Starch Glycollate

(f) Magnesium Stearate

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 in formulation E is of the direct compression type (Dairy Crest - "Zeparox").

Formulation D mg/tablet

Active Ingredient 250

Pregelatinized Starch NF15 150

400

Formulation E mg/tablet

Active Ingredient 250

Lactose 150

Avicel 100

500

Formulation F (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.

mg/tablet

(a) Active Ingredient 500

(b) Hydroxypropylmethylcellulose 112 (Methocel K4M Premium)

(c) Lactose B.P. 53

(d) Povidone B.P. 28

(e) Magnesium Stearate 7

700

Drug release takes place over a period of about 6-8 hours and is complete after 12 hours.

Example 6

Capsule Formulations

Formulation A

A capsule formulation is prepared by admixing the ingredients of Formulation D in Example 5 above and filling into a two-part hard gelatin capsule. Formulation B (infra) is prepared in a similar manner.

Formulation B mg/capsule

(a) Active Ingredient 250

(b) Lactose B.P. 143

(c) Sodium Starch Glycollate 25

(d) Magnesium Stearate 2

420

Formulation C mg/capsule

(a) Active Ingredient 250

(b) Macrogol 4000 B.P. 350 600

Formulation D mg/capsule

Active Ingredient 250 Lecithin 100 Arachis Oil 100 450

Capsules of Formulation D are prepared by dispersing the active Ingredient in the lecithin and arachis oil and filling the dispersion into soft, elastic gelatin capsules.

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 spheronization 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. mg/capsule

(a) Active Ingredient 250

(b) Microcrystalline Cellulose 125

(c) Lactose B.P. 125

(d) Ethyl Cellulose 13

513

Example 7

Iniectable Formulation

Formulation A

Active Ingredient 0.200 g

Hydrochloric acid solution, 0.1M, or

Sodium hydroxide solution, 0.1M q.s. to pH 4.0 to 7.0

Sterile water q.s. to 10 mL

The active ingredient is dissolved in most of the water (35°C-40°C) and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate. The batch is then made up to volume with the water and filtered through a sterile micropore filter

into a sterile 10 mL amber glass via (type 1) and sealed with sterile closures and overseals.

Formulation B

Active Ingredient 0.125

Sterile, Pyrogen-free, pH 7 Phosphate Buffer q.s. to 25 mL

Example 8

Intramuscular Injection

Active Ingredient 0.20 g

Benzyl Alcohol 0.10 g

Glycofurol 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 amber glass vials (type 1) .

Example 9

Syrup

Active Ingredient 0.25 g

Sorbitol Solution 1.50 g

Glycerol 2.00 g

Sodium Benzoate 0.005 g

Flavor, Peach 0.0125 mL

Purified Water q.s. to 5.00 mL

The active ingredient is dissolved in a mixture of the glycerol and most of the purified water. An aqueous solution of the sodium

benzoate is then added to the solution, followed by addition of the sorbitol solution and finally the flavour. The volume is made up with purified water and mixed well.

Formulation B

Active Ingredient 0.125 g

Sterile, pyrogen-free, pH 7 phosphate buffer q.s. to 25 mL

Example 10

Suppository mg/suppository

Active Ingredient 250

Hard Fat, B.P. (Witepsol H15-Dynamit NoBel) 1770

2020

One-fifth of the Witepsol H15 is melted in a stem- acketed pan at 45 C maximum. The active ingredient is sifted through a 200 μm sieve and added to the molten base with mixing, using a Silverson fitted with a cutting head, until smooth dispersion is achieved. Maintaining the mixture at 45 C r the remaining Witepsol H15 is added to the suspension and stirred to ensure a homogenous mix. The entire suspension is passed through a 250 μM stainless steel screen and, with continuous stirring, is allowed to cool to 40 C. At a temperature of 38 C to 40 C, 2.02 g of the mixture is filled into suitable, 2 mL plastic moulds. The suppositories are allowed to cool to room temperature.

Example 11

Pessaries mg/pessary

Active Ingredient 250 Anhydrate 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.

Example 12

Antiviral Activity

The antiviral activities of the compounds of formulae (la) and (lb) and of racemic ganciclovir phosphonate were assessed against human cytomegalovirus (HCMV) , laboratory strain AD169. The assay was conducted, in triplicate, in MCR-5 lung fibroblasts (Whittaker Bioproducts, Inc., Walkersville, MD) using the DNA hybridization assay developed by Diagnostic Hybrids, Inc., Athens, OH (Dankner, W.M. , et.al, 1990, J. Virol. Methods, 28:293-298) with ganciclovir as a control.

Compound ED 50 (μM)t

R-enantiomer (Ex. 1L) 1.7 ± 0.1; 3.0 + 0.1 R-enantiomer (Ex. 3B) 2.8 ± 1.6 S-enantiomer (Ex. 3A) 57 ± 35 Ganciclovir Phosphonate * 5.6 ± 0.2 Ganciclovir 1.7 + 0.1; 1.9 + 0.3

j- 50% effective antiviral dose

* Ganciclovir phosphonate was obtained from E.J. Reist, SRI International, Menlo Park, CA 94025.

The data indicates that the R-enantiomer of ganciclovir phosphonate is unexpectedly more potent than the S-enantiomer. The R-enantiomer of ganciclovir phosphonate has twice the activity of racemic ganciclovir phosphonate.

Example 13

Toxicity

The compound of formula (la) was tested for toxicity in human bone marrow progenitor cells, in vitro, by the methods of Dornsife, R.E. et.al., 1991, Antimicrob. Agents. Chemother., 3_5:322-328. Three separate assays were performed using marrow from three different donors.

Compound CFU-GM BFU-E

R-enantiomer (Ex. 1L) 100 + 10 120 ± 10

t 50% Inhibition of bone marrow progenitor cells.