Phosphoramidate, and mono-, di-, and tri-phosphate esters of (1R, cis)-4-(6-amino-9H-purin-9-yl)-2-cyclopentene-1-methanol as antiviral agents

The present invention relates to phosphoramidate, and phosphate esters of (1R,cis)-4-(6-amino-9H-purin-9-yl)-2-cyclopentene-1nethanol, processes for their preparation, and their use in treating viral infections.

FIELD OF THE INVENTION

The present invention relates to certain analogs of (1R,cis)-4-(6-amino-9H-purin-9-yl)-2-cyclopentene-1-methanol and their use in medical therapy.

BACKGROUND OF THE INVENTION

Retroviruses form a sub-group of RNA viruses which, in order to replicate, must first “reverse transcribe” the RNA of their genome into DNA (“transcription” conventionally describes the synthesis of RNA from DNA). Once in the form of DNA, the viral genome may be incorporated into the host cell genome, allowing it to take advantage of the host cell's transcription/translation machinery for the purposes of replication. Once incorporated, the viral DNA is virtually indistinguishable from the host's DNA and, in this state, the virus may persist for the life of the cell.

A species of retrovirus, the Human immunodeficiency virus (HIV) has been reproducibly isolated from patients with AIDS (acquired immunodeficiency syndrome) or with the symptoms that frequently precede AIDS. AIDS is an immunosuppressive or immunodestructive disease that predisposes subjects to fatal opportunistic infections. Characteristically, AIDS is associated with a progressive depletion of T-cells, especially the helper-inducer subset bearing the CD4 surface marker. HIV is cytopathic and appears to preferentially infect and destroy T-cells bearing the CD4 marker, and it is now generally recognized that HIV is the etiological agent of AIDS. Clinical conditions such as AIDS-related complex (ARC), progressive generalized lymphadenopathy (PGL), Kaposi's sarcoma, thrombocytopenic purpura, AIDS-related neurological conditions, such as AIDS dementia complex, multiple sclerosis or tropical paraparesis, and also anti-HIV antibody-positive and HIV-positive conditions, including such conditions in asymptomatic patients, are also conditions which may be treated by appropriate anti-viral therapy.

Another RNA virus which has been recognized as the causative agent of an increasingly serious international health problem is the non-A, non-B hepatitis virus. At least 80% of cases of chronic post-transfusional non-A, non-B hepatitis have been shown to be due to the virus now identified as hepatitis C and this virus probably accounts for virtually all cases of post-transfusional hepatitis in clinical settings where blood products are screened for hepatitis B. Whereas approximately half of the cases of acute hepatitis C infection resolve spontaneously over a period of months, the remainder become chronic and in many if not all such cases chronic active hepatitis ensues with the potential for cirrhosis and hepatocellular carcinoma. The structure of the hepatitis C virus genome has been elucidated and the virus has been characterized as a single stranded RNA virus with similarities to flaviviruses.

Hepatitis B virus (HBV) is a small DNA containing virus which infects humans. It is a member of the class of closely related viruses known as the hepadnaviruses, each member of which selectively infects either mammalian or avian hosts, such as the woodchuck and the duck. Recent insights into the mechanism of replication of the hepadnavirus genome indicate the importance of reverse transcription of an RNA intermediate, suggesting that the reverse transcriptase is a logical chemotherapeutic target. HBV is a viral pathogen of major worldwide importance. The virus is etiologically associated with primary hepatocellular carcinoma and is thought to cause 80% of the world's liver cancer. Clinical effects of infection with HBV range from headache, fever, malaise, nausea, vomiting, anorexia and abdominal pains. Replication of the virus is usually controlled by the immune response, with a course of recovery lasting weeks or months in humans, but infection may be more severe leading to persistent chronic liver disease outlined above.

U.S. Pat. No. 4,916,224 discloses 2′,3′-dideoxy-2′,3′-didehydro-carbocyclic nucleosides and their use in the treatment of HIV. WO 96/29336 discloses masked monophosphate nucleoside analogues for the treatment of HIV. Wang et al. (Bioorganic Et Medicinal Chemistry Letters8, pp. 1585–1588, 1998) disclose the synthesis of L-carbocyclic 2′,3′-didehydro-2′,3′-dideoxyadensosine and its use in HIV infections.

It has now been discovered that certain phosphoramidates of (1R,cis)-4-(6-amino-9H-purin-9-yl)-2-cyclopentene-1-methanol are useful for the treatment of viral infections, particularly hepatitis B and retroviral infections, especially HIV. Compounds of the present invention have pharmacokinetic properties which render them advantageous as therapeutic agents.

SUMMARY OF THE INVENTION

The present invention relates to compounds of formula (I)

wherein:
R1is hydrogen; C6-14aryl; or heteroaryl, optionally substituted with one or more substituents selected from the group consisting of C1-6alkoxy, nitro, halogen, amino, hydroxy, carboxylate and esters thereof, carboxyalkyl, —CONHR6, and —CONR6R7, wherein R6and R7, which may be the same or different, are independently selected from C1-8alkyl, C1-8alkylaryl or C6-14aryl;
R2and R3are independently selected from hydrogen or C1-8alkyl, C3-8cycloalkyl, C2-8alkenyl, C5-8cycloalkenyl, C6-14aryl, or aralkyl wherein each C1-8alkyl, C3-8cycloalkyl, C2-8alkenyl, C5-8cycloalkenyl, C6-14aryl or aralkyl may be optionally substituted with one or more substituents selected from the group consisting of C1-8alkyl, halo, hydroxy, alkoxy, amino, aminoalkyl, aminodialkyl, —SH, thioalkyl, heterocycle, carboxylate and esters thereof, carboxyalkyl, —CONHR6, and —CONR6R7, wherein R6and R7, which may be the same or different, are independently selected from C1-8alkyl, C1-8alkylaryl or C6-14aryl; or R2and R3can together form a 3 to 8-membered ring;
R4is —OR8, —NR8R9or —SR8, where R8and R9, which may be the same or different, are independently selected from hydrogen; or C1-8alkyl, C3-8cycloalkyl, C2-8alkenyl, C5-8cycloalkenyl, aralkyl, heteroaryl, or C6-14aryl wherein each C1-8alkyl, C3-8cycloalkyl, C2-8alkenyl, C5-8cycloalkenyl, aralkyl, heteroaryl, or C6-14aryl may be optionally substituted with one or more substituents selected from the group consisting of halo, hydroxy, alkoxy, amino, aminoalkyl, aminodialkyl, —SH, thioalkyl, carboxylate and esters thereof, carboxyalkyl, —CONHR6, and —CONR6R7, wherein R6and R7, which may be the same or different, are independently selected from C1-8alkyl, C1-8alkylaryl or C6-14aryl;
R5is hydrogen; C1-8alkyl; or C6-14aryl; or R2and R5may together form a 5- or 6-membered ring or R3and R5may together form a 5- or 6-membered ring;
or a pharmaceutically acceptable derivative thereof, and their use in the treatment of viral infections.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features compounds of formula (I)

wherein:
R1is hydrogen; C6-14aryl; or heteroaryl, optionally substituted with one or more substituents selected from the group consisting of C1-6alkoxy, nitro, halogen, amino, hydroxy, carboxylate and esters thereof, carboxyalkyl, —CONHR6, and —CONR6R7, wherein R6and R7, which may be the same or different, are independently selected from C1-8alkyl, C1-8alkylaryl or C6-14aryl;
R2and R3are independently selected from hydrogen or C1-8alkyl, C3-8cycloalkyl, C2-8alkenyl, C5-8cycloalkenyl, C6-14aryl, or aralkyl wherein each C1-8alkyl, C3-8cycloalkyl, C2-8alkenyl, C5-8cycloalkenyl, C6-14aryl or aralkyl may be optionally substituted with one or more substituents selected from the group consisting of C1-8alkyl, halo, hydroxy, alkoxy, amino, aminoalkyl, aminodialkyl, —SH, thioalkyl, heterocycle, carboxylate and esters thereof, carboxyalkyl, —CONHR6, and —CONR6R7, wherein R6and R7, which may be the same or different, are independently selected from C1-8alkyl, C1-8alkylaryl or C6-14aryl; or R2and R3can together form a 3 to 8-membered ring;
R4is —OR8, —NR8R9or —SR8, where R8and R9, which may be the same or different, are independently selected from hydrogen; or C1-8alkyl, C3-8cycloalkyl, C2-8alkenyl, C2-8cycloalkenyl, aralkyl, heteroaryl, or C6-14aryl wherein each C1-8alkyl, C3-8cycloalkyl, C2-8alkenyl, C5-8cycloalkenyl, aralkyl, heteroaryl, or C6-14aryl may be optionally substituted with one or more substituents selected from the group consisting of halo, hydroxy, alkoxy, amino, aminoalkyl, aminodialkyl, —SH, thioalkyl, carboxylate and esters thereof, carboxyalkyl, —CONHR6, and —CONR6R7, wherein R6and R7, which may be the same or different, are independently selected from C1-8alkyl, C1-8alkylaryl or C6-14aryl;
R5is hydrogen; C1-8alkyl; or C6-14aryl; or R2and R5may together form a 5- or 6-membered ring or R3and R5may together form a 5- or 6-membered ring;
or a pharmaceutically acceptable derivative thereof, and their use in the treatment of viral infections.

An embodiment of the present invention features compounds of formula (II)

or a pharmaceutically acceptable derivative thereof, and their use in the treatment of viral infections.

A further aspect of the present invention features a compound of formula (III)

wherein n is 0, 1, or 2, and wherein R12is optionally substituted by C6-14aryl.

The compounds of the present invention include diastereomers differing in the absolute configuration at phosphorus. Diastereomers may be present as a single isomer or as mixtures of diastereomers.

The term “alkyl” refers to a straight-chain or branched-chain saturated aliphatic hydrocarbon radical containing the specified number of carbon atoms, or where no number is specified, preferably from 1 to about 10, more preferably from 1 to about 8 carbon atoms. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, n-hexyl and the like.

The term “alkenyl,” alone or in combination with any other term, refers to a straight-chain or branched-chain mono- or poly-unsaturated aliphatic hydrocarbon radical containing the specified number of carbon atoms, or where no number is specified, preferably from 2–10 carbon atoms and more preferably, from 2–6 carbon atoms. Examples of alkenyl radicals include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, isobutyenyl, pentenyl, hexenyl, hexadienyl and the like.

The term “alkoxy” refers to an alkyl ether radical, wherein the term “alkyl” is defined above. Examples of suitable alkyl ether radicals include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy and the like, with methoxy being preferred.

The term “halo” or “halogen” refers to a radical of fluorine, chlorine, bromine or iodine.

The term “aryl” refers to a carbocyclic aromatic radical (such as phenyl or naphthyl) containing the specified number of carbon atoms, preferably from 6–14 carbon atoms, and

more preferably from 6–10 carbon atoms, optionally substituted with one or more substituents selected from C1-6alkoxy (for example, methoxy), nitro, halogen (for example chloro), amino, carboxylate and hydroxy. Examples of aryl radicals include, but are not limited to phenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl, anthracenyl and the like.

The term “heterocycle”, alone or in combination with another term, refers to a stable 3–7 membered monocyclic heterocyclic ring or 8–11 membered bicyclic heterocyclic ring which is either saturated or unsaturated, and which may be optionally benzofused if monocyclic. Each heterocycle consists of one or more carbon atoms and from one to four heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. As used herein, the terms “nitrogen and sulfur heteroatoms” include any oxidized form of nitrogen and sulfur, and the quaternized form of any basic nitrogen. A heterocyclyl radical may be attached at any endocyclic carbon or heteroatom which results in the creation of a stable structure. Preferred heterocycles include 5–7 membered monocyclic heterocycles and 8–10 membered bicyclic heterocycles. Examples of such groups include imidazolyl, imidazolinoyl, imidazolidinyl, quinolyl, isoqinolyl, indolyl, indazolyl, indazolinolyl, perhydropyridazyl, pyridazyl, pyridyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazinyl, quinoxolyl, piperidinyl, pyranyl, pyrazolinyl, piperazinyl, pyrimidinyl, pyridazinyl, morpholinyl, thiamorpholinyl, furyl, thienyl, triazolyl, thiazolyl, carbolinyl, tetrazolyl, thiazolidinyl, benzofuranoyl, thiamorpholinyl sulfone, oxazolyl, benzoxazolyl, oxopiperidinyl, oxopyrrolidinyl, oxoazepinyl, azepinyl, isoxozolyl, isothiazolyl, furazanyl, tetrahydropyranyl, tetrahydrofuranyl, thiazolyl, thiadiazoyl, dioxolyl, dioxinyl, oxathiolyl, benzodioxolyl, dithiolyl, thiophenyl, tetrahydrothiophenyl, sulfolanyl, dioxanyl, dioxolanyl, tetahydrofurodihydrofuranyl, tetrahydropyranodihydrofuranyl, dihydropyranyl, tetradyrofurofuranyl and tetrahydropyranofuranyl.

The term “pharmaceutically acceptable derivative”, as used herein, means any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention or an inhibitorily active metabolite or residue thereof. Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.

Compounds of formula (I), (II), and (III) and their pharmaceutically acceptable derivatives may hereinafter be referred to as compounds according to the invention.

Preferred compounds of formulae (I) and (II) include the compounds listed in Table 1.

A further aspect of the present invention features a compound of formula (II) wherein R1is H or C6-14aryl, R2is C1-6alkyl or aralkyl, R3is hydrogen, C1-6alkyl or aralkyl and R4is OR10wherein R10is C1-6alkyl or C3-8cycloalkyl.

A preferred aspect of the present invention features a compound of formula (II) wherein R1is C6-14aryl, R2is methyl, R3is hydrogen, and R4is OR10where R10is methyl or ethyl. More preferably, R1is phenyl.

In another aspect of the present invention there is provided compounds of formula (II) wherein R1is hydrogen.

In another aspect of the present invention there is provided compounds of formula (I) and (II) wherein R2and R3are not both hydrogen.

When R2and R3are different, the L-configuration of naturally occurring amino acids is preferred.

Pharmaceutically acceptable salts of the compounds of the present invention include salts of a basic or acidic portion of the molecule. Salts of a basic moiety may be formed by organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic, and succinic acids, organic sulphonic acids, such as methanesulphonic, ethanesulphonic, benzenesulphonic and p-toluenesulphonic acids and inorganic acids, such as hydrochloric, sulphuric, phosphoric and sulphamic acids. Salts of an acidic moiety may be formed by an appropriate base, such as an alkali metal (for example, sodium), an alkaline earth (for example, magnesium, calcium), ammonium and ammonium salts.

In such esters, unless otherwise specified, any alkyl moiety present advantageously contains from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more particularly from 1 to 4 carbon atoms, Any cycloalkyl moiety present in such esters advantageously contains from 3 to 6 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group.

Esters of carboxylate may include alkyl, cycoalkyl, aralkyl, and aryl esters.

Compounds of formula (I) and (II) may be made by modifications of the procedures described in Biochem. Biophys. Res. Commun. 225:363–369, 1997.

The present invention further includes a process for the preparation of a compound of formula (I) which comprises reaction of a compound of formula (IV)

with a compound of formula (VI)

wherein R1–R5are as hereinbefore defined for formula (I).

The reaction may be carried out in pyridine, pyridine-tetrahydrofuran or acetonitrile in the presence of t-butyl magnesium chloride (Balzarini et al.,Biochem. Biophys. Res. Comm.225:363–369 (1996). The phosphochloridate intermediates, compounds of formula (VI), may be prepared according to WO 96/29336, incorporated herein by reference hereto; McGuigan et al, J. Med. Chem., 1996, 39, 1748–1753; and McGuigan et al, Antiviral Res., 1997, 35, 195–204.

Compounds of formula (IV) may be made according to Example 1 or by any method known in the art.

The present invention further includes a process for the preparation of a compound of formula (II) which comprises reaction of a compound of formula (IV)

with a compound of formula (V)

wherein R1–R4are as hereinbefore defined for formula (II).

The reaction may be carried out in pyridine, pyridine-tetrahydrofuran or acetonitrile in the presence of t-butyl magnesium chloride (Balzarini et al.,Biochem. Biophys. Res. Comm.225:363–369 (1996). The phosphochloridate intermediates, compounds of formula (V), may be prepared according to WO 96/29336, incorporated herein by reference hereto; McGuigan et al, J. Med. Chem., 1996, 39, 1748–1753; and McGuigan et al, Antiviral Res., 1997, 35, 195–204.

Compounds of formula (III) may also be prepared by any method known in the art.

Separation of isomers may be accomplished by methods known in the art, for example, by high-pressure liquid chromatography with chiral columns, particularly using liquid carbon dioxide as the mobile phase, or by crystallization of salts with chiral acids or bases.

Phosphate isomers may be separated with Supercritical Fluid Chromatography using a Chiralpak AS column, 25% methanol in carbon dioxide as the eluent, flow rate 2 mL/min, temperature 40° C., and pressure 3000 psi.

One aspect of the invention features the compounds according to the invention for use in medical therapy, particularly for the treatment or prophylaxis of retroviral infections and hepatitis B virus infections.

A further aspect of the invention features the compounds according to the invention for use in the manufacture of a medicament for the treatment or prophylaxis of viral infections, particularly for the treatment of retroviral infections, for example HIV infections, and hepatitis B virus infections.

In a further aspect of the present invention there is provided a method for the treatment of viral infections, for example, retroviral infections, particularly HIV infections, and hepatitis B virus infections in a host comprising administering to said host a therapeutically effective amount of a compound according to the invention.

Examples of retroviral infections which may be treated or prevented in accordance with the invention include human retroviral infections such as human immunodeficiency virus (HIV), HIV-1, HIV-2 and human T-cell lymphotropic virus (HTLV), for example, HTLV-I or HTLV-II infections. The compounds according to the invention are especially useful for the treatment of AIDS and related clinical conditions such as AIDS-related complex (ARC), progressive generalized lymphadenopathy (PGL), Kaposi's sarcoma, AIDS-related neurological conditions, such as multiple sclerosis, tropical paraparesis, and AIDS dementia, anti-HIV antibody-positive and HIV-positive conditions and thrombocytopenic purpura.

The compounds according to the invention are particularly applicable for the treatment of asymptomatic infections or diseases in humans caused by or associated with human retroviruses.

The compounds according to the invention may be employed in combination with other therapeutic agents for the treatment of the above infections or conditions. Other therapeutic agents may include agents that are effective for the treatment of viral infections or associated conditions such as reverse transcriptase inhibitors, for example, zidovudine or abacavir; (1 alpha, 2 beta, 3 alpha)-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine [(−)BHCG, SQ-34514]; oxetanocin-G (3,4-bis-(hydroxymethyl)-2-oxetanosyl]guanine); acyclic nucleosides (e.g. acyclovir, valaciclovir, famciclovir, ganciclovir, penciclovir); acyclic nucleoside phosphonates (e.g. (S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine (HPMPC) or PMEA or PMPA; ribonucleotide reductase inhibitors such as hydroxyurea, 2-acetylpyridine 5-[(2-chloroanilino)thiocarbonyl) thiocarbonohydrazone; other 2′,3′-dideoxynucleosides such as 2′,3′-dideoxycytidine, 2′,3′-dideoxyadenosine, 2′,3′-dideoxyinosine, 3′-deoxy-2′,3′-didehydrothymidine (d4T); protease inhibitors such as saquinavir, indinavir, ritonavir, nelfinavir, amprenavir; oxathiolane nucleoside analogues such as lamivudine,cis-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-5-fluorocytosine (FTC); 3′-deoxy-3′-fluorothymidine, 5-chloro-2′,3′-dideoxy-3′-fluorouridine, ribavirin, 9-[4-hydroxy-2-(hydroxymethyl)but-1-yl]-guanine (H2G); tat inhibitors such as 7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2-(H)one (Ro5-3335), 7-chloro-1,3-dihydro-5-(1H-pyrrol-2yl)-3H-1,4-benzodiazepin-2-amine (Ro24-7429); interferons such as α-interferon; renal excretion inhibitors such as probenecid; nucleoside transport inhibitors such as dipyridamole; pentoxifylline, N-acetylcysteine (NAC), Procysteine, α-trichosanthin, phosphonoformic acid, as well as immunomodulators such as interieukin II or thymosin, granulocyte macrophage colony stimulating factors, erythropoetin, soluble CD4and genetically engineered derivatives thereof; or non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as nevirapine (BI-RG-587), loviride (α-APA) and delavuridine (BHAP), and phosphonoformic acid, and 1,4-dihydro-2H-3,1-benzoxazin-2-ones NNRTIs such as (−)-6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one (L-743,726 or DMP-266), and quinoxaline NNRTIs such as isopropyl (2S)-7-fluoro-3,4-dihydro-2-ethyl-3-oxo-1(2H)-quinoxalinecarboxylate (HBY1293). The component compounds of such combination therapy may be administered simultaneously, in either separate or combined formulations, or at different times, for example, sequentially such that a combined effect is achieved.

Another aspect of the present invention features a method of delivering a compound of formula (III), wherein R12and n are defined as above, into cells by treating said cells with a compound of formula (I) or (II) as defined above. The cells to be treated may be within a human or ex vivo, for example, in culture.

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

The amounts required of the active ingredient will depend upon a number of factors including the severity of the condition to be treated and the identity of the recipient and will ultimately be at the discretion of the attendant physician or veterinarian. In general however, for each of these utilities and indications, a suitable effective dose of a compound of formula (I) will be in the range of 0.01 to 200 mg per kilogram body weight of recipient per day, advantageously in the range of 1 to 100 mg per kilogram body weight per day.

The desired dose is preferably presented as one, two, three or four or more subdoses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing about 0.5 to 2000 mg, preferably about 5, 25, 50, 150, 200, or 250 mg of active ingredient per unit dose form.

A further aspect of the present invention features a patient pack comprising at least one active ingredient selected from a compound of formula (I), (II), and (III) and an information insert containing directions on the use of the compound.

While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical composition. A further aspect of the present invention features pharmaceutical compositions comprising a compound of formula (I), (II) or (III) or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier therefor.

The compositions of the present invention comprise at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers thereof and optionally other therapeutic agents. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. Compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.

The compositions may conveniently be presented in unit dosage form prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing in to association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, sachets of granules or tablets (such as a swallowable, dispersible or chewable tablet) each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multidose sealed containers, for example, ampoules and vial, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

The active ingredient may also be presented in a composition comprising micrometer- or nanometer-size particles of active ingredient.

Preferred unit dosage compositions are those containing a daily dose or unit daily sub-dose (as herein above recited) or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the composition 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 flavoring agents or taste masking agents.

A further aspect of the invention relates to kits to be used in the treatment of patients suffering from viral infections. These kits include one or more oral dosages of a compound of formula (I), (II), or (III) and may include one or more additional therapeutic agents. By way of illustration, a kit of the invention may include one or more tablets, capsules, caplets, gelcaps or liquid formulations containing a compound of formula (I) and one or more tablets, capsules, caplets, gelcaps or liquid formulations containing a compound of formula (I) in dosage amounts within the ranges described above. The kits may include as an insert printed dosing information for the co-administration of the agents.

The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way.

(1R,cis)-4-(6-Amino-9H-purin-9-yl)-2-cyclopentene-1-methanol (part f of example 1,925 mg, 4.00 mmol) was stirred in dry pyridine (100 ml) and tert-butyl magnesium chloride (Aldrich, 1 M in tetrahydrofuran, 4.3 ml) was added. After 15 minutes, a solution of phenyl(methoxy-L-alaninyl)phosphorochloridate (prepared as described by McGuigan, C. et al.,J. Med. Chem.1993, 36: 1048–1052) (2.22 g, 8.00 mmol) in tetrahydrofuran (10 ml) was added. After 24 hours, additional tert-butyl magnesium chloride (4.4 ml) and phenyl(methoxy-L-alaninyl)phosphorochloridate (2.22 g) were added and stirring continued for an additional 24 hours. Volatiles were removed and the residual gummy solid was partitioned between chloroform (200 ml) and water (50 ml). The chloroform layer was dried (sodium sulfate) and concentrated to a colorless glass. The glass was chromatographed on silica gel. Title compound was eluted with 5% methanol-chloroform.

In the same manner as Example 2, (1R,cis)-4-(6-amino-9H-purin-9-yl)-2-cyclopentene-1-methanol (part f of example 1, 200 mg, 0.860 mmol) was reacted with phenyl(methoxy-D-alaninyl)phosphorochloridate (prepared as described by McGuigan, C. et al.,J. Med. Chem.1993, 36: 1048–1052). Title compound was eluted with 5% methanol-chloroform.

In the same manner as Example 2, (1R,cis)-4-(6-amino-9H-purin-9-yl)-2-cyclopentene-1-methanol (part f of example 1, 500 mg, 2.16 mmol) was reacted with phenyl(methoxy-L-phenylalaninyl)phosphorochloridate (prepared as described by McGuigan, C. et al.,J. Med. Chem.1993, 36: 1048–1052). Title compound was eluted with 5% methanol-chloroform.

The following general procedures were used in the preparation of compounds of Examples 10–41.

Standard Procedure for Phosphorochloridate Preparation

Dry triethylamine (2.0 mol equiv.) in dry dichloromethane (40 ml) was added dropwise to a stirred solution of phenyl dichlorophosphate (1.0 mol equiv.) and the appropriate amino acid ester salt (1.0 mol equiv.) in dry dichloromethane (40 ml), at −78° C. under nitrogen. Following the addition, the reaction mixture was allowed to warm slowly to room temperature and stirred overnight. The solvent was removed under reduced pressure and the crude residue was resuspended in dry diethyl ether or THF, and filtered under nitrogen. The solvent was removed under reduced pressure to leave the crude product as an oil.

All crude phosphorochloridates were used as solutions in dry THF or dry acetonitrile in subsequent coupling reactions.

Standard Procedure 1 for Phosphoramidate Preparation

To (1R,cis)-4-(6-Amino-9H-purin-9-yl)-2-cyclopentene-1-methanol (200 mg, 0.87 mmol) suspended in anhydrous acetonitrile (15 ml) under a nitrogen atmosphere, was added 1M t-butyl magnesium chloride dissolved in tetrahydrofuran (1.73 ml, 1.74 mmol). After 15 minutes, phosphorochloridate (2.61 mmol) dissolved in acetonitrile (15 ml) was added dropwise over 1 minute and the reaction mixture allowed to stir for a further 4 hours. Following the removal of volatiles in vacuo, the product was purified by column chromatography (silica) eluting with 4–5% MeOH in chloroform or dichloromethane.

Standard Procedure 2 for Phosphoramidate Preparation

To (1R,cis)-4-(6-Amino-9H-purin-9-yl)-2-cyclopentene-1-methanol (200 mg, 0.87 mmol) suspended in anhydrous tetrahydrofuran (15 ml) under a nitrogen atmosphere, was added 1M t-butyl magnesium chloride dissolved in tetrahydrofuran (1.73 ml, 1.74 mmol). After 15 minutes, phosphorochloridate (2.61 mmol) dissolved in acetonitrile (15 ml) was added dropwise over 1 minute and the reaction mixture allowed to stir for a further 4 hours. Following the removal of volatiles in vacuo, the product was purified by column chromatography (silica) eluting with 4–5% MeOH in chloroform or dichloromethane.

Standard Procedure 3 for Phosphoramidate Preparation

To (1R,cis)-4-(6-Amino-9H-purin-9-yl)-2-cyclopentene-1-methanol (200 mg, 0.87 mmol) suspended in anhydrous pyridine (15 ml) under a nitrogen atmosphere, was added 1M t-butyl magnesium chloride dissolved in tetrahydrofuran (1.73 ml, 1.74 mmol). After 15 minutes, phosphorochloridate (2.61 mmol) dissolved in acetonitrile (15 ml) was added dropwise over 1 minute and the reaction mixture allowed to stir for a further 4 hours. Following the removal of volatiles in vacuo, the product was purified by column chromatography (silica) eluting with 4–5% MeOH in chloroform or dichloromethane.

(1R,cis)-4-(6-Amino-9H-purin-9-yl)-2-cyclopentene-1-methanol (Example 1, 200 mg, 0.865 mmol) was dissolved in 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (2 mL). Phosphoryl chloride (0.24 mL, 0.26 mmol) was added to the stirred, cooled (0° C.) solution. After 1 minute, 1.0 M sodium hydrogen carbonate (3.3 mL, 3.3 mmol) was added and stirring was continued at 0° C. for 30 minutes and then at 25° C. for 1 hour. The reaction solution was diluted to 125 mL with deionized water and applied to a 1.1×7.0 cm DEAE Sepahadex A25 (Aldrich) ion exchange chromatography column which had been washed with 1.0 M ammonium bicarbonate buffer and then equilibrated with deionized water. The title compund was eluted with a 0 to 0.5 M gradient (2 L) of ammonium bicarbonate. The appropriate fractions were combined and the volatiles removed by evaporation in vacuo. The residue was redissolved in deionized water (20 mL) and evaporated in vacuo three times. Lyophylization of the residue from water gave the ammonium salt of the title compound (270 mg, 90% as mono ammonium salt, monohydrate);1H-NMR (D2O) δ 8.10 (S, 2H), 6.14–6.20 (m, 1H), 5.84–5.90 (m, 1H), 5.48–5.58 (m, 1H), 3.71–3.86 (m, 2H), 3.02–3.14 (br m, 1H), 2.66–2.80 (m, 1H), 1.56–1.66 (m, 1H);31P-NMR (D2O) 0.62. Mass spectrum (ES−) m/e 310 (M−H).

Compounds according to the invention were tested for anti-HIV activity in MT4cells according to the method described by Averett, D. R.,J. Virol. Methods,23, 1989, 263–276. Activity of the compounds was in the range of IC500.009–2.1 μM.

Anti-Hepatitis B Virus Activity

Compounds were tested for anti-Hepatitis B Virus activity according to the method described by Jansen, R. et al.,Antimicrobial Agents and Chemotherapy, Vol.37, No. 3, pp. 441–447, 1993, and results are shown in the table below. IC50values of the compounds according to the invention demonstrated improved activity by as much as 500-fold over that of the corresponding nucleoside analog, (1R,cis)-4-(6-amino-9H-purin-9-yl)-2-cyclopentene-1-methanol.

Tablet Formulation

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.

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”).

The formulation is prepared by wet granulation of the ingredients with a solution of povidone followed by the addition of magnesium stearate and compression.

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

Capsule Formulations

Formulation A

A capsule formulation is prepared by admixing the ingredients of formulation D in Example 49 above and filling into a two-part hard gelatin capsule. Formulation B (infra) is prepared in a similar manner.

Capsules of formulation C are prepared by melting the Macrogel 4000 B.P., dispersing the active ingredient in the melt and filling the melt into a two-part hard gelatin capsule.

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.

Four (4) kilograms (kg) of Vitamin E TPGS (obtained from Eastman Chemical Co.) was heated at 50° C. until liquefied. To the liquefied Vitamin E TPGS, 2.005 kg of polyethylene glycol 400 (PEG400) (low aldehyde, <10 ppm, obtained from Union Carbide or Dow Chemical Co.) heated to 50° C. was added and mixed until a homogeneous solution was formed. The resultant solution was heated to 65° C. 1.5 kg of active ingredient was dissolved in the liquefied solution of Vitamin E TPGS and PEG 400. 0.395 kg of propylene glycol at room temperature was added and mixed until a homogenous solution was formed. The solution was cooled to 28–35° C. The solution was then de-gassed. The mixture was preferably encapsulated at 28–35° C. at a fill weight equivalent to 150 mg of volatiles-free compound, into Size 12 oblong, white opaque soft gelatin capsules using a capsule filling machine. The capsule shells were dried to a constant fill moisture of 3–6% water and a shell hardness of 7–10 Newtons, and placed in a suitable container.

Formulation F (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.

Injectable Formulation

The active ingredient is dissolved in most of the water (35°–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 water and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals.

Intramuscular Injection

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).

Syrup

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 flavor. The volume is made up with purified water and mixed well.

One-fifth of the Witepsol H15 is melted in a steam-jacketed 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 a smooth dispersion is achieved. Maintaining the mixture at 45° C., the remaining Witepsol H15 is added to the suspension and stirred to ensure a homogenous mix. The entire suspension is passed through a 250 μm stainless steel screen and, with continuous stirring, is allowed to cool to 45° C. At a temperature of 38° C. to 40° C., 2.02 g of the mixture is filled into suitable, 2 ml plastic molds. The suppositories are allowed to cool to room temperature.

The above ingredients are mixed directly.

Acid Stability

Compounds according to the invention were tested for their stability towards acid-mediated hydrolytic decomposition employing a test designed to simulate stomach conditions. Each compound was incubated at an initial concentration of 0.3 mg/ml in dilute hydrochloric acid at pH 1 at 37° C. HPLC was run immediately for t=0 and at intervals up to approximately 24 hours. The half-life of title compound from Example 7 was 76 hours under these conditions. Comparative phosphoramidates of 2′,3′-dideoxy-adenosine (Compound 1093) and 2′,3′-didehydro-2′,3′-dideoxy-adenosine (Compound 1001), described in PCT/GB96/00580, were significantly less stable at pH1. Compound 1001 was completely decomposed in <1 minute at pH 1 (25° C.). Compound 1093 was completely decomposed after 13 hours at pH 1 (25° C.).

Biological Stability

Title compound of Example 7 and phosphoramidates of 2′,3′-dideoxy-adenosine (Compound 1093) and 2′,3′-didehydro-2′,3′-dideoxy-adenosine (Compound 1001), described in PCT/GB96/00580, were tested for their stability towards biological decomposition. Each compound was incubated in normal heparized human plasma at 37° C. At selected time points duplicate samples were removed and deproteinated by acetonitrile extraction. Drug concentrations were then determined by LC/MS/MS analysis using standard methods. Half-lives were calculated and are shown in the table below.

The half-life in human plasma of compound of the present invention is more than 10-fold greater than those of compounds 1001 and 1093.