NL8900122A - DIDEOXYNUCLEOSIDES ANTI-VIRUSES AND PREPARATIONS CONTAINING THEM. - Google Patents

Description

- 1-

Anti-virus dideoxynucleosides and preparations containing them.

This invention relates to dideoxyribose nueleoside analogues. More specifically, these are carbocyclic Z ^ S-dideoxy-Z, 3 * -didehydropurin nucleoside analogues and their use in therapy, especially against viruses.

Given the similarities between the cell functions of viruses and their hosts, it is difficult to selectively attack a virus and leave the host cell intact. As a result, there are relatively few anti-virus agents per se, and it is difficult to find anti-virus agents with an acceptable therapeutic index, ie substances with significant anti-virus activity at a dose in which the agent has an acceptable toxicity or side effect profile. .

A group of viruses that have become very important in recent times are the retroviruses that are responsible for acquired immunodeficiency syndrome (AIDS).

Such viruses have previously been referred to by various names, but are now commonly referred to as "human immunodeficiency viruses (HIVs)"; two of them, HIV-I and HIV-II, have been reproducibly isolated from patients suffering from AIDS or related conditions such as AIDS-related complex (ARC) or from persistent general lymphadenopathy.

Although a number of nucleosides have been reported to be useful in the treatment of conditions associated with HIV infections, only zidovudine (ΔΖΤ, Retrovir) has regulated consent to treat such conditions.

Acquired. But zidovudine is known to have serious side effects, it suppresses the bone marrow leading to a decrease in white blood cell count, resulting in marked anemia, and there is a need for effective agents that are less cytotoxic.

A new class of nucleoside analogues with anti-virus activity has now been found. A first aspect of the invention is a compound of formula 1 wherein X is hydrogen, NRRj, SR, OR or halogen and Z is hydrogen, OR2 or NRR ^ 35, wherein R, R ^ and R ^ are the same or different and are water- 'emuu - 2 -' '> *' * can be substance, alkyl and aryl, as well as the pharmaceutically acceptable derivatives thereof.

Those skilled in the art will recognize that the compounds of formula 1 are cis compounds, and further that the cyclopentene-3 ring has two chiral centers (indicated by * in the formula) and thus in the form of two optical isomers (dz enantiomers) and mixtures of which may exist, including racemic mixtures. All of those isomers and mixtures thereof, including the racemic mixtures, are within the scope of the invention. Thus, 10 in compounds of formula 1 or the chiral center to which the base is attached has the R-configuration and the chiral center to which the group CH 2 OH is attached has the S-configuration (hereinafter referred to as the D-isomer), or the chiral center to which the base is attached is the S-configuration and the group to which the group C 1-20 is attached is the R-configuration (hereinafter referred to as the L-isomer). Suitable are the compounds in the form of a racemic mixture or in those of essentially pure D isomer. The D isomers can be represented by formula la, wherein X and Z have the previously given definitions. Hereinafter, reference to compounds of formula 1 includes reference to compounds of formula 1a.

One of ordinary skill in the art will also appreciate that certain compounds of Formula 1 may exist in a number of tautomeric forms, and all of these tautomers are also within the scope of the invention.

Here, the term "halogen" includes fluorine, chlorine, bromine and iodine; if X is halogen, it is preferably chlorine.

Here, "C 1-6 alkyl" includes branched and unbranched alkyl groups, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl and t-butyl. alkyl is suitably methyl.

Here, "aryl" includes all mono- and polycyclic aromatic debris, including substituted or unsubstituted aryl (such as phenyl, tolyl, xylyl, and anisyl) and substituted or unsubstituted aralkyl having 1 to 4 ° C in the alkyl portion, such as phenyl (C 1-4) alkyl, for example benzyl and phenylene.

In the compounds of formula 1, Z is preferably amino.

θ> 9 0 0 12.2 - 3 - *

In a preferred class of compounds of formula 1, X is OR, especially OH.

In another preferred class of compounds of formula 1, X is NRR 4, especially NH 4 or hydrogen.

Particularly preferred compounds of formula 1 are those wherein Z is M2 and X Η, M2 or (and especially) OH.

These compounds have attractive therapeutic indices in particular against viruses.

By "pharmaceutically acceptable derivative" is meant any pharmaceutically acceptable salt, ester or salt of an ester of such a compound of formula 1, or any other compounds which, when administered, directly or indirectly contain a compound of formula 1 or an anti-virus metabolite or residue can give.

Preferred esters of the compounds of formula 1 include the carboxylic acid esters whose non-carbonyl portion is hydrogen, branched or unbranched alkyl (eg, methyl, ethyl, n-propyl, t-butyl, n-butyl), alkoxyalkyl (e.g. methoxymethyl, aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl) or aryl (e.g. optionally substituted by halogen, ^ -alkyl or ^ -alkoxy), sulfonates such as the esters of alkane and arylalkanesulfonic acids ( e.g. methanesulfonic acid), the amino acid esters (e.g. those of L-valine and L-isoleucine) and the mono-, di- and triphosphoric acid esters.

As to the esters described above, unless otherwise indicated, any alkyl moiety present advantageously has 1 to 18 and especially 1 to 4 carbon atoms.

Any aryl moiety present in such esters is advantageously a phenyl group.

The pharmaceutically acceptable salts of the compounds of formula 1 include those derived from acceptable inorganic and organic acids and bases. Examples of such acids are hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, p-toluenesulfonic, tartaric, acetic, citric, methanesulfonic, formic, benzoic, benzoic acid 2-sulfonic acid and benzenesulfonic acid. Other acids such as oxalic acid, while not pharmaceutically acceptable in themselves, may be 8900 122.

V '-4.- useful in the preparation of salts which serve as intermediates. Salts derived from appropriate bases include the alkali metal (eg, sodium), alkaline earth metal (eg, magnesium) and ammonium salts (eg, of NR ^ where R is C 1 -alkyl 5).

Hereinafter, mentioning a compound of the invention also includes mentioning the pharmaceutically acceptable derivatives thereof.

Specific compounds of formula 1 are: 10 (1a, 4a) -4- (6-Chloro-9H-puriny-1-9) -2-cyclopentenylcarbinol; (Ια, 4a) -4- (6-hydroxy-9H-purinyl-9) -2-cyclopentenylcarbinol; (1a, 4a) -4- (6-amino-9H-purinyl-9) -2-cyclopentenylcarbinol; (1a, 4a) -4- (6-mercapto-9H-purinyl-9) -2-cyclopentenylcarbinol; (1a, 4a) -4- (2-amino-6-chloro-9H-purinyl-9) -2-cyclopentenyl-15 carbinol; (Ια, 4a) -4- (2-amino-6-hydroxy-9H-purinyl-9) -2-cyclopentenylcarbinol; ...

(1a, 4a) -4- (2,6-diamino-9H-purinyl-9) -2-cyclopentenylcarbinol; in the form of racemic mixtures or individual enantiomers. Either the compounds of the invention themselves act against viruses and / or they are metabolizable to such compounds. In particular, these compounds are effective in inhibiting the proliferation of retroviruses, including human retroviruses such as the human immunodeficiency viruses (HIVs) that cause AIDS.

Some compounds of the invention, especially those in which "Z is hydrogen, have anti-cancer activity.

Thus, another aspect of this invention relates to a compound of formula 1 or a pharmaceutically acceptable derivative thereof for use as a therapeutically active agent, in particular against viruses, for example in the treatment of retrovirus infections, or as an anti-cancer agent.

Another aspect of the invention pertains to a method of treating viral infections, especially infections caused by a retrovirus such as HIV, wherein an effective amount of anti-virus compound of formula 1 or pharmaceutically acceptable its derivative to a mammal, including a human.

Another or additional aspect is the use of a compound of formula 1 or a pharmaceutically acceptable derivative thereof in the manufacture of a medicament for the treatment of a virus infection or against cancer.

The compounds of the invention have activity against viruses, and are also useful in the treatment of AIDS-related disorders such as the AIDS-related complex (ARC), progressive general lymphadenopathy (PGL), AIDS-related neurological disorders (such as dementia or tropical paraparesis), states of being positive in antibodies to HIV and / or in HIV, Kaposi's sarcoma and thrombocytopenia purpurea.

The anti-virus compounds of the invention are also useful in preventing and spreading clinical diseases in individuals who have antibodies to HIV or are HIV antigen positive, and in prophylaxis after exposure to HIV.

The anti-virus compounds of formula 1 and the pharmaceutically acceptable derivatives thereof can also be used to prevent virus contamination of physiological fluids such as blood and semen in vitro.

Some compounds of formula 1 are also useful as intermediates in the preparation of other compounds of the invention.

Those skilled in the art will recognize that "treatment" here also includes "prophylaxis" and treating already established infections or symptoms.

It will further be appreciated that the amount of compound required for treatment according to the invention will vary not only with the choice of the compound, but also with the mode of administration, the nature of the condition treated and the age and condition of the patient, and that will ultimately be at the discretion of the doctor or veterinarian on duty.

In general, however, a suitable dose will be between about I and 750 mg / kg per day, e.g. between about 10 and 750, 35 mg / kg per day, such as 3 to 120 mg per kg of body weight per day, preferably between 6 and 90 mg / kg.day and most preferably between 15 and 60 mg / kg.day.

The desired dose may conveniently be administered in a single portion, or in multiple doses with appropriate 8900 122.

* / - 6 - intervals, for example over two, three or four or more. portions per day.

The compound is suitably administered in unit doses containing, for example, 10 to 1500 mg, suitably 20 to 1000 mg, and even more suitably 50 to 700 mg, of active ingredient per dose.

Ideally, so much of the active agent is administered that the plasma concentration thereof exhibits a peak between about 1 and 75 µM, preferably between 2 and 50 µM, and even more preferably between 3 and 30 µM. This can be achieved, for example, by intravenous injection of a 0.1 to 5% solution of the active substance, suitably in physiological saline, or with an oral administration of a bolus containing approximately 1 to 100 mg per kg body weight of active substance. stand. Desirable blood levels can be maintained by a continuous infusion delivering about 0.01 to 5.0 mg / kg per hour, or with infusion pulses providing about 0.4 to 15 mg / kg of active substance.

While it is possible for a compound of the invention to be administered as such in therapeutic use, it is preferable to present the active ingredient as a pharmaceutical composition.

Thus, the invention further relates to pharmaceutical preparations containing a compound of formula 1 or a pharmaceutically acceptable derivative thereof, together with one or more pharmaceutically acceptable carriers therefor, and optionally other therapeutic and / or prophylactic ingredients.

The carrier (s) must be "acceptable" in the sense that they are compatible with the other components of the composition and are not harmful to the recipient.

Pharmaceutical preparations include those suitable for oral, rectal, nasal, topical (including in the mouth and under the tongue), vaginal or parenteral (including intramuscular and intravenous) administration and which are in a form suitable for inhalation and inhalation. Those formulations, where convenient, may conveniently be in the form of individual units and may be prepared by any method known in pharmacy. All methods include the step of combining the active substance with liquid carriers or fine-azeism.

Divided solid supports (or both) and then, if necessary, shaping the product into the desired formulation.

Pharmaceutical preparations suitable for oral administration can conveniently be presented in separate units, such as capsules, cachets and tablets, each containing a certain amount of active ingredient, either as powder or as granules or as a solution or as a suspension or as an emulsion. The active ingredient can also be offered in a bolus, in a lick pot or as a paste. Tablets and capsules for oral administration may contain the usual excipients, such as binders, fillers, lubricants, disintegrants and wetting agents. The tablet may be coated by methods known in the art. Oral liquid preparations may, for example, take the form of suspensions in water or oil, of solutions, emulsions, syrups and elixirs, or may be presented as a dry product to be prepared for use with water or other suitable carrier. Such liquid preparations may contain the usual additives such as suspending agents, emulsifying agents, non-aqueous carriers (including edible oils) and preservatives.

The compounds of the invention may also be formulated for parenteral administration (eg, by injection, as boluses or by continuous infusion) and may be presented as unit doses in ampoules, pre-filled syringes or in multidose packs containing added preservative. The formulations can take forms such as suspensions, solutions and emulsions in oil or water, and they can contain formulation aids such as stabilizers and suspension and / or dispersing agents. However, the active substance may also be in powder form, obtained by aseptically isolating the sterile solid or by freeze-drying a solution which is prepared for use with a suitable carrier (eg sterile, pyrogen-free water).

For topical application to the epidermis, the compounds of the invention may be formulated as ointments, creams or lotions or as a healing patch. For example, ointments and creams can be formulated on a water or oil basis, with the addition of suitable thickening and / or gelling agents. Lotions can be formulated at 8900122 ..

ft.

- based on water or oil and will generally also contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickeners and / or dyes.

Suitable preparations for topical administration in the mouth include the lozenges, with the active ingredient in a flavorful base, usually sucrose and gum arabic or tragacanth, the pastilles with the active ingredient in an inert base such as gelatin with glyceol or sucrose with gum arabic, and the mouthwashes with the active ingredient in a suitable liquid carrier.

Pharmaceutical preparations, the carrier of which is a solid, suitable for rectal administration are preferably provided as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in this art, and the suppositories may be suitably formed by mixing the active agent with the softened or melted carrier followed by cooling and molding.

Suitable preparations for vaginal administration may be provided as diaphragms, tampons, creams, gels, pastes, foams and sprays, which in addition to the active ingredient contain carriers known in the art as suitable therefor.

For intranasal administration, the compounds of the invention can be used as a liquid spray or in the form of drops. Drops can be formulated on an aqueous or non-aqueous basis and may also contain one or more dispersants, stabilizers and suspending agents. Liquid sprays are conveniently administered from aerosol cans.

For inhalation administration, the compounds of the invention are conveniently provided in an inserter, nebulizer or aerosol or any other suitable aerosol administration means. Aerosol cans may contain a suitable propellant gas such as CC 2 F 2, C 2 F 2 F, C 2 Cl 2 F 2, CO 2, or any other suitable gas. In the case of an aerosol in an aerosol, the dosage is determined by the valve that delivers a certain amount.

Alternatively, the compounds of the invention may be of form 89 DO 121 for administration by sniffing or inhalation.

- 9 - S 'adopt a dry powder, for example a mixture of the compound and a suitable base such as lactose or starch. The powder preparation can be dispensed in unit doses, for example in capsules or cartridges of, for example, gelatin, which powder is then taken by means of a breathing device.

If desired, the formulations described above can be adapted to a sustained release of the active agent.

The pharmaceutical preparations according to the invention may also contain other active ingredients, for example anti-microbial agents and preservatives.

The compounds of the invention can also be used in combination with other therapeutics, for example other anti-infection agents. In particular, the compounds according to the invention can be used together with known anti-virus substances.

Thus, in another aspect, the invention provides a combination of a compound of formula 1 or a physiologically acceptable derivative thereof with another therapeutically active agent, in particular an anti-virus agent.

The above combinations may suitably be in the form of a pharmaceutical composition for use, and thus pharmaceutical compositions comprising in combination as defined above and a pharmaceutically acceptable carrier therefor comprise yet another aspect of the invention.

Suitable therapeutics for such combinations include acyclic nucleosides such as aciclovir, interferons such as α-interferon, renal secretion inhibitors such as probenicid, nucleoside transport inhibitors such as dipyridamole, 21,3'-dideoxynucleosides such as 2 ', 3 * -dideoxy-cytidine, 2 ', 3'-dideoxyadenosine, 2', 3'-dideoxyinosine, 2 *, 3f-dideoxythymidine and 2 ', 31-dideoxy-21,31-didehydrothymidine, and immunomodulators such as interleukin II (IL2 ) and granulocyte macrophage colony stimulating factor (GM-CSF), arythropoetin 35 and ampligen.

The individual components of such combinations can be administered consecutively or simultaneously in separate or combined preparations.

If the compound of formula 1 or an M00.12Z.

- 10 - Pharmaceutically acceptable derivative thereof used in combination with a second therapeutic acting against the same virus, the dose of both substances may be different than when used alone. Those skilled in the art will readily come to mind with suitable doses.

The compounds of formula 1 and their pharmaceutically acceptable derivatives can be prepared by any method known in the art for preparing compounds of analogous structure.

Suitable methods for preparing compounds of formula 1 and their pharmaceutically acceptable derivatives are described below; unless otherwise indicated, groups X and Z have the previously given definitions. It will be appreciated that the following reactions require or require starting materials that have protected functional groups, and deprotection may be required as an intermediate or final step to arrive at the desired compound.

Protection and deprotection of functional groups can be done by conventional means. For example, affino-20 groups can be protected, for example, with aralkyl (e.g. benzyl), acyl and aryl (e.g. 2,4-dinitrophenyl) groups; subsequent removal of the protecting group is optionally effected by hydrolysis or hydrogenolysis under standard conditions. Hydroxy groups can be protected with the groups commonly used therefor, for example as described in "Protective Groups in Organic Chemistry", ed. J.F.W. McOlie (Plenum Press, 1973) and in "Protective Groups in Organic Synthesis" by Theodora W. Greene (John Wiley & Sons, 1981). Examples of suitable hydroxy protecting groups are alkyl, (eg, methyl, t-butyl and methoxymethyl), aralkyl (eg, benzyl, diphenylmethyl and triphenylmethyl), heterocyclic groups such as tetrahydropyranyl, acyl (eg, acetyl and benzoyl) ) and silyl groups such as trialkylsilyl (e.g. t-butyldimethylsilyl). The hydroxy protecting groups can be removed by conventional techniques. For example, alkyl, silyl, acyl and heterocyclic groups can be removed by solvolysis, e.g. by hydrolysis under acidic or basic conditions. Aralkyl groups such as triphenylmethyl can also pass through. solvolysis, e.g. by hydrolysis $ 9 ¢ 0122.

-Light under acidic conditions. Aralkyl groups such as benzyl can also be removed by hydrogenolysis in the presence of a noble metal catalyst such as palladium-on-carbon. Silyl groups can conveniently be removed with a source of fluoro-ride ions such as tetra-n-butylammonium fluoride.

In a first method (A), compounds of formula 1 and the pharmaceutically acceptable derivatives thereof can be prepared by reacting a compound of formula 2, wherein X and Z have the meanings given in formula 1, or reacting a protected form thereof with formic acid or a reactive derivative thereof, followed if necessary by removing undesired groups introduced with that reagent and / or removing any protecting groups present, Examples of suitable derivatives of formic acid which may be used in method (A) are the orthoformates (e.g. triethyl orthoformate), the dialkoxymethyl acetates (e.g. diethoxymethyl acetate), dithiomic acid, form amide, s-triazine and formamidinium acetate. Undesirable groups introduced with formic acid or a reactive derivative thereof can conveniently be removed by milder hydrolysis, for example with an inorganic acid such as aqueous hydrochloric acid.

If a trialkyl orthoformate such as triethyl orthoformate is used, it suitably also serves as a solvent. Other useful solvents are the amides (e.g., dimethylformamide or dimethylacetamide), the chlorohydrocarbons (e.g., dichloromethane), the ethers (e.g., tetrahydrofuran), and the nitriles (e.g., acetonitrile).

In some cases (eg when a trialkyl ortho-formate such as triethyl orthoformate is used), the reaction preferably occurs in the presence of a catalyst such as a strong acid (eg concentrated hydrochloric, nitric or sulfuric acid). The reaction can take place at a temperature between -25 ° and + 150 ° C, eg between 0 ° and 100 ° C and suitably at room temperature.

In another method (B), a compound of formula 1 or a protected form or pharmaceutically acceptable derivative thereof undergoes an exchange reaction in which the initial substituent X present by another 8S00U2.

-12 - α substituent X is replaced and / or the initially present group Z is replaced by another group Z, if necessary followed by removal of any protective groups present.

In an embodiment of method (B), a compound of formula 1, wherein X represents a group RR ^ (where R and R ^ have the previously given definitions) can be prepared by amination of the corresponding compound of formula 1 wherein X is a halogen atom (e.g. chlorine).

The amination can be done by reaction with a substance HNRR ^ 10 (in which R and R ^ have the previously given definitions), suitably in a solvent such as methanol. The reaction can be carried out at any suitable temperature and suitably at an elevated temperature such as under reflux, or, when using liquid ammonia, in a sealed tube at 50 ° to 80 ° C. Suitable conditions for converting halides to secondary or tertiary amines have also been described by I.T. Harrison et al. In the Compendium of Organic Synthetic Methods, (Wiley-Interscience, New York, 1971) at biz. 250-252 ..

In another embodiment of method (B), a compound of formula 1, wherein X represents a group OR (wherein R has the previously defined definition) is prepared by replacing the halogen atom with an appropriate anion R0. When R represents a hydrogen atom, the displacement is a hydrolysis carried out in water or in a mixture of water and a miscible solvent such as methanol, ethanol, dioxane, tetrahydrofuran, acetone, dimethylformamide or dimethyl sulfoxide, suitably in the presence of an acid or a base. Suitable acids include t-toluenesulfonic acid and hydrochloric, nitric and sulfuric acids. Suitable bases include the alkali metal hydroxides and carbonates (e.g. KOH,

NaOH and Na 2 CO 2). The aqueous acid or base can also serve as a solvent. The hydrolysis can conveniently be carried out at a temperature between -10 ° and + 150 ° C, eg under reflux. When R represents an alkyl or aryl group, the anion RO is conveniently formed from the corresponding alcohol RÖH with an inorganic base such as an alkali metal (e.g. sodium) or an alkali metal hydride (e.g. sodium hydride).

The reaction with the anion formed in situ can be effected at room temperature.

In another embodiment of method (B), a compound of formula 1, wherein X represents a group SH, is prepared by reacting the halogen compound of formula 1 with thiourea in a suitable solvent such as an alcohol (e.g. n propanol) at elevated temperature (e.g., at the boiling point), followed by alkaline hydrolysis. Suitable bases include the alkali metal hydroxides (e.g., NaOH).

The reaction is conveniently performed by the method of 10 G.G. Urquart et al. (Org. Syn. Coll. Vol. 3 (1953) 363), boiling the intermediate with, for example, 15 to 5 hours with aqueous NaOH.

In yet another embodiment of method (B), a compound of formula 1 wherein X represents a hydrogen atom is prepared by reduction of the halogen compound of formula 1 with a reduction system that does not affect the rest of the molecule. A suitable reducing agent for this dehalogenation is the combination of zinc metal with water, as described by J. R, Marshall et al. In J. Chem. Soc. 1004, 20 (1951). But the reaction can also be carried out by photolysis in a suitable solvent such as tetrahydrofuran containing 10% triethyl amine, and suitably in a photohemic reactor according to Rayonet, According to the method of V. Nair et al. (J. Org. Chem. 52 (1987) 1344).

In yet another embodiment of method (B), a compound of formula 1, wherein X represents a halogen atom from another halogen compound of formula 1, is prepared by the methods usual for halogen exchange. But when X is chlorine, this substituent can be replaced by a different halogen atom by known methods using the various p-halobenzeniazonium chlorides.

Compounds of formula 1 wherein X represents a group SR, wherein R represents a C 1-6 alkyl or aryl group, may be prepared from the corresponding thiols by the standard alkylation or arylation methods, for example as described in U.S. U.S. Patent 4,383,114.

Compounds of formula 1 wherein Z represents a hydroxy group can suitably be reacted with saline 6900122.

Petrigic acid can be prepared from the corresponding compound in which Z represents N 11, for example as described by J. Davoll in J. Am. Chem. Soc. 73 (1951) 3174.

Many of the reactions described above have been extensively reported in the context of the synthesis of purine nucleosides, for example, in Nucleoside Analogs - Chemistry, Biology and Medical Applications edited by RTWalker et al. (Plenum Press, New York, 1979) at biz . 193-223.

Pharmaceutically acceptable salts of the compounds of the invention can be prepared as described in U.S. Patent 4,383,114. For example, the product of any of the above processes can be converted into a salt by treating the resulting free base in the usual manner with the appropriate acid. Pharmaceutically acceptable acid addition salts can be prepared by reacting the free base with the appropriate acid, optionally in the presence of a suitable solvent such as an ester (e.g. ethyl acetate), or an alcohol (e.g. methanol, ethanol or isopropanol). Inorganic base salts can be prepared by reacting the free compound with an alkoxide (eg sodium methylate), optionally in the presence of a solvent such as methanol. Pharmaceutically acceptable salts can also be prepared from other salts by conventional methods, including other pharmaceutically acceptable salts.

A compound of formula 1 can be converted into a pharmaceutically acceptable phosphate or other ester by reaction with a phosphorylating agent such as POCl 4 or another suitable esterification agent such as an acid halide or anhydride, as it suits. An ester or a salt of a compound of the formula 1 can be converted back into the parent compound, for example by hydrolysis.

The compounds of formula 5 and the salts thereof are new compounds and thus form another axis of this invention.

The compounds of formula 5 wherein Z represents hydrogen or hydroxy can be prepared directly from the compound of formula 3 by reaction with an excess of pyrimidine of formula 4, wherein Y is a halogen atom (e.g.

89 0 0 1 2.2 ..

- chlorine) and Z is hydrogen or hydroxy, in the presence of a base such as triethylamine and in an alcoholic solvent (e.g. n-butanol), suitable at boiling temperature.

Compounds of formula 5 wherein Z 5 represents NH 2 can be prepared by reacting the compound of formula 3 under similar conditions with a pyrimidine of formula 6, wherein Y has the definition given in formula 4 and wherein X represents hydrogen or hydroxy, which a compound of formula 7 which can be reacted in a solvent such as water, an organic acid such as acetic acid or a mixture thereof, suitably at room temperature, with a diazonium salt ArN ^ E (wherein Ar is an aromatic group , e.g. p-chlorophenyl and E represents an anion such as chloride), which gives a diazo compound of formula 8 (wherein Ar has the definition just given) which can be converted into the desired compound of formula 5 by reduction with a metal such as zinc in the presence of an acid, e.g. acetic acid. It will be appreciated that the choice of the reducing agent will depend on the nature of the group X.

The compound of formula 3 can be prepared from the versatile precursor loKacetylamino) -3a- (acetoxymethyl) -cyclopentene-2 (formula 2) by hydrolysis with a mild base such as an alkaline earth metal hydroxide.

A particularly suitable synthesis of compounds of formula 1 from 6-chloro compounds of formula 2 is shown in reaction scheme A.

The compounds of formulas 3, 7 and 8 are new intermediates and thus form another aspect of this invention. The compound of formula 2 is a known compound described in U.S. Patent 4,138,562.

If a single isomer of the compound of formula 1 is obtained, this can be obtained by splitting up the end product or by stereospecific synthesis from isomers of pure starting materials or between products. It. cleavage of the final product or an intermediate or a precursor can be done by any method known in the art, see, for example, "Stereochemistry of Carbon Compounds" from E.L. Eliel (McGraw Hill, 1962) and & 90 0 121.

- 16 - "Tables of Resolving Agents" by S.H. Wanting.

A suitable method for obtaining chirally pure compounds of formula 1 is the enzymatic conversion of a racemic mixture of that compound 5 or precursor thereof. With such a method, both (+) and (-) compounds of formula 1 can be obtained in optically pure form. Suitable enzymes are the deaminases such as adenosine deaminase.

The invention is now further illustrated by the following detailed examples. The elemental analyzes were performed by the M-H-W-Laboratories of Phoenix, AZ. Melting points were performed on the Mel-Temp device and corrected. NMR spectra were recorded with Jeol FX 90QFT or Nicollet NT300 spectrometers and in DMSO-Dg. The chemical shifts are given in ppm downstream from that for

Me ^ Si. IR spectra were recorded in KBr pellets with a Nicollet 50XC FT-IR spectrophotometer and the UV spectra were recorded in a Beckmann DU-8 spectrophotometer. Mass spectra were recorded with an AEI Scientific Apparatus Limited 20 MS-30 mass spectrometer. Thin layer chromatography (TLC) was performed on 0.25 mm thick layers of Merck silica (37-62 yam). All chemicals and solvents were of reagent quality unless otherwise specified. The term "active ingredient" used in the examples refers to a compound of formula 1 or a pharmaceutically acceptable derivative thereof.

Example I

(+) - (1 ^, 43.) - 4- ^ (5-amino-6-chloro-4-pyrimidinyl) amino 7-2-cyclopentenylcarbinol (5a)

A mixture of 3.0 g (15 mmol) of loc-acetylamino-30- (acetoxymethyl) cyclopenten-2 and 300 ml of 0.5 N barium hydroxide solution was refluxed overnight.

After cooling, it was neutralized with solid carbon dioxide. The precipitate was filtered off and the aqueous filtrate was evaporated to dryness. The residue was extracted with absolute ethanol and concentrated again to give 1.6 g (14 mmol) of the compound of formula 3, a colorless syrup.

To this syrup were added 4.59 g (28 mmol) of 5-amino-4,6-dichloropyrimidine, 4.2 g (42 mmol) of triethylamine and 50 ml of n-butanol and the mixture was returned to 8900 122 for 24 hours. .

- 17 - boiled fluently. The volatile solvents were removed and the residue adsorbed on 7 g of silica gel contained in a 4.0 x 12 cm column; this was eluted with 5 Z methanol in chloroform to give 2.69 g (74%) of the compound of formula 5a 5; mp. 130-132 ° C. An analytical sample was obtained by recrystallization from ethyl acetate (EtOAc), mp. 134-135 ° C; MS (30 ev, 200 ° C) m / e = 240 and 242 (M + and M ++ 2), 209 (M + ~ 31), 144 (B +); IR: 3600-2600 (OH), 1620, 1580 (C = C, C = N);

Example II

10 (i) - (1, 4¾) -h- (2-amino-6-chloro-4-pyrimidinyl) amino J-2-cyclopentenylcarbinol (formula 6a)

To 14 mmol of crude product of Example I were added 3.74 g (22.8 mmol) of 2-amino-4,6-dichloropyrimidine, 15 ml of triethylamine and 75 ml of n-butanol and that mixture was refluxed for 48 hours. cooked. The volatile solvents were removed and the residue extracted with methanol; the insoluble by-product (the double pyrimidine nucleoside) was discarded. The methanol solution was adsorbed on 8 g of silica gel packed into a 4.0 x 14 cm column; this was eluted with 2% methanol in chloroform to give 1.52 g (42%) of crude product of formula 6a. This was recrystallized from ethyl acetate and the melting point was 132-134 ° G; MS (30 ev, 200 ° C): m / e 240 240 and 242 (M + and M ++ 2), 209, (M + “31) and 144 (B +); IR: 3600-3000 (NH2, OH), 1620, 1580 (C-C, C = N);

Example III

(±) - (1k, 4 ».) - 4- (C (2-amino-6-chloro-5- (4-chlorophenyl) azo J-4-pyrimidinylamino) -2-cyclopentenylcarbinol (Formula 7a)

A cold diazonium salt solution was made from 1.47 g (11.5 mmol) p-chloroaniline in 25 ml 3N HCl and 870 mg (12.5 mmol) sodium nitrite in 10 ml water. This solution was added to a mixture of 2.40 g (10 mmol) of the product of Example II, 50 ml of glacial acetic acid, 20 g of sodium acetate trihydrate and 50 ml of water. The reaction mixture was stirred at room temperature overnight. The yellow precipitate was filtered and washed with cold water to neutral, then it was air dried under the hood to give 3.60 g (94 Z) of the compound of formula 7a; mp. 229 ° C (dec.). The analytical sample was obtained from acetone / methanol 1: 2; mp. 241-243 ° C (dec.).

MS (30 ev, 260 ° C): m / e 378 and 380 (M + and M + + 2), 282 (B +); IR: 3600-3000 (NH2, OH), 1620, 1580 (C = C, C = N).

8900 122 ..

- 18 -

Example IV

(±) - (Ibv, 4οΟ-4- ^ (2,5-diamino-6-chloro-4-pyrimidinyl) amino J-2-cyclopentenylcarbinol (formula 8a)

A mixture of 379 mg (1 mmol) of compound vol-5 of formula 7a, 0.65 g (10 mmol) of zinc powder, 0.32 ml of glacial acetic acid, 15 ml of water and 15 ml of ethanol was refluxed for 3 hours. The zinc was removed and the solvents evaporated. The residue was adsorbed on 2 g of silica gel packed into a 2.0 x 18 cm column; this was eluted with 6% methanol in chloroform. A pink syrup was obtained. * Further purification from methanol and ether gave 170 mg (66%) of the compound of formula 8a as pink crystals; mp. 168-170 ° C; MS (30 ff, 220 ° C): m / e 255 and 257 (M + and M + + 2), 224 (M + _31) and 159 (B +); IR: 3600-3000 (NH 2> OH) 1620, 1580 (C = C, C = N).

Example V

(t) - (lt (, 4oQ -4- (6-chloro-9H-purinyl-9) -2-cyclopentenyl-carbinol (formula 2a, Z = H)

A mixture of 1.30 g (5.4 mmol) of the compound of formula 5a, 30 ml of triethyl orthoformate and 0.50 ml of 12N hydrochloric acid was stirred at room temperature overnight. The solvent was evaporated under vacuum at 35 ° C. To the residue, 30 ml of 0.5 N hydrochloric acid was added and that mixture was stirred for 1 hour and then neutralized with 1N NaOH to pH = 7-8 and adsorbed on 8 g of silica gel which was added to a column of 4.0 x 8 cm 25 packs; this was eluted with 5% methanol in chloroform to give 1.12 g (82%) of white crystals of the compound of formula 1, X = Cl and X = H. The crude product was recrystallized from ethyl acetate, now m.p. = 108-110 ° C, MS (30 ff, 200 ° C): m / e 250 and 252 (M + and M + + 2), 219 (M + ”31), 154 (B +); IR: 3600-2800 (OH), 1600 (C = C, C-N).

Example VI

(±) - (1 «, 4cQ -4- (6-hydroxy-9H-purinyl-9) -2-cyclopentenyl-carbinol

A mixture of 251 mg (1 mmol) of product of Example V and 10 ml of 0.2 N NaQH was refluxed for 3 hours. After cooling, the pH was adjusted to 5-6 with acetic acid. The reaction mixture was adsorbed on 2 g of silica gel packed into a 2.0 x 11 cm column. Elution with 9% methanol in chloroform gave 105 mg (45%) of the compound of formula 1.89D0122.

- 19 - X = OH and Z = H. The crude white product was recrystallized from water / methanol 3: 1, now it was m.p. 248-250 ° C (dec.), Ms (30 ff, 300 ° C): m / e 232 (M +), 214 (M + ~ 18), 136 (B +); IR: 3600-2600 (OH), 1680, 1600 (C = 0, C = C, C = N).

Example VII

(t) - (1 $ c, 4oQ -4- (6-amino-9H-purinyl-9) -2-cyclopentenylcarbinol Liquid ammonia was charged into a bomb containing a solution of 250 mg (1 mmol) cooled to -80 ° C ) product of Example V in 5 ml of methanol The bomb was closed for 10 hours and heated at 60 ° C for 24 hours. Ammonia and methanol were evaporated and the residue recrystallized from water, yielding off-white crystals of the compound of formula I, X 2 NH 2. and Z = H, mp 198-200 ° C MS (30 ev, 210 ° C): m / e 231 (M +), 213 (M + _18), 135 (B +); IR: 3600-2600 ( NH 2> OH), 1700, 1600 (C = C, C = N).

Example VIII

(±) - (let, 4 te) -4- (6-mercapto-9H-purinyl-9) -2-cyclopentenyl-carbinol

A mixture of 125 mg (0.5 mmol) of product of Example V, 40 mg (0.64 mmol) of thiourea and 5 ml of n-propanol was refluxed for 2 hours. After cooling, the precipitate was filtered off, washed with n-propanol and dissolved in 5 ml 1N NaOH. The pH was adjusted to 5 with acetic acid.

90 mg (73 Z) of the crude compound of formula 1 precipitated with 25 X = SH and Z = H; 260-262 ° C (dec.). This was recrystallized from dimethylformamide, now it was m.p. 263-265 ° C (dec.). MS (30 ev, 290 ° C): m / e 248 (M +), 230 (M + ~ 18), 152 (B +); IR: 3600-3200 (OH), 3100, 2400 (SH), 1600 (OC, C = N).

Example IX

30 (±) - (1a, 4cQ -4- (2-amino-6-chloro-9H-puriny1-9) -2-cyclopentenyl carbinol

A mixture of 1.41 g of 85.5 mmol) of the compound of formula 8a, 30 ml of triethyl orthoformate and 1.40 ml of 12N hydrochloric acid was stirred overnight. The suspension was evaporated to dryness under vacuum and 40 ml of 0.5 N hydrochloric acid were added to the residue and the mixture was allowed to react for 1 hour at room temperature. After adjusting the pH with 1N NaOH to 8, the mixture was adsorbed on 7.5 g of silica gel packed into a 4.0 x 10 cm column. Elution with 5% methanol in chloroform gave 1.18 g of 8900122.

20 (80%) off-white crystals of the compound of formula 1 with X = Cl and Z = NH2. The crude product was recrystallized from ethanol, m.p. 145-147 ° C. MS (30 ff, 220 ° C): m / e 265 and 267 (M + and M + + 2), 235 (M + "30), 5 169 (B +); IR 3600-2600 (NH2> OH), 1620-1580 (C = C, C = N).

Example X.

(±) - (lft, 4cQ -4- (2-amino-6-hydroxy-9H-purinyl-2-cyclopentenyl-carbinol)

A mixture of 266 mg (1 mmol) product of Example IX with 0.33 N NaOH was refluxed for 5 hours

boils. The residue was adsorbed on 2 g of silica gel, which was in a 2.0 x 7.5 cm column, which was eluted with CHCl 4 / MeOH 5: 1. The crude product was recrystallized from methanol / water 1: 4 to give 152 mg (61%) of white crystals of compound of formula 1 with X = OH and X = NH 2; mp. 254-256 ° C

(dec.). MS (30 ff, 200 ° C): m / e 247 (M +), 217 (M + "30), 151 (B +); IR: 3600-2600 (NH2, OH), 1700, 1600 (C = 0, C = C, C = N) Example XI

(i) - (lot, 4¾) -4- (2,6-diamino-9H-purinyl-9) -2-cyclopentenyl-20 carbinol

Liquid ammonia was placed in a bomb containing a solution of 265 mg (1 mmol) of the product of Example IX cooled to -80 ° C in 10 ml of methanol. The bomb was sealed and heated at 75 ° C for 48 hours. Ammonia and methanol 25 were evaporated. The residue was adsorbed on 2 g of silica gel which was packed into a 2.0 x 10 cm column and eluted with CHCl 3 / MeOH 15: 1. The crude product was recrystallized from ethanol to give 196 mg (80%) of the compound of formula 1 with X = Z = NH 2; mp. 152-155 ° C. MS (30 ev, 200 ° C): m / e 246 (M +), 229 (M + "17), 216 (M +" 30), 150 (B +); IR: 3600-3000 (NH2, OH), 1700, 1650, 1600 (C = 0, C = C, C = N).

Example XII

(IS, 4R) -4- (2,6-diamino-9H-purinyl-9) -2-cyclopentenylcarbinol / (1S, 4R) -4- (2,6-diamino-9H-purinyl-9) -2- cyclopentene-methanol 7 35 (a) Intermediate 1: (1R, 2S, 3R, 5R) -3- / 6-amino-9H-purinyl-9-7-5- / ((1,1-dimethylethyl) dimethylsilyloxy) methyl 7-1, 2-cyclopanediol. A mixture of 12.505 g (-) - Aristeromycin, 7.8 g of tert-butyldimethylsilyl chloride and 12.96 g of imidazole in 85 ml of dry dimethylformamide was stirred at room temperature for 2 hours at 83 ° C. Soc. 105 81983) 4049-55-21 - stirred. The resulting solution was diluted with 500 ml of ethyl acetate and then washed with 3 x 100 ml of water and with 50 ml of brine before a white substance crystallized. It was collected by filtration, washed with ethyl acetate and dried under vacuum; this gave 3.92 g of the title product; ^ H-NMR: Peaks at 8.15 (1H), 8.09 (1H), 7.19 (2H), 5.00 (1H), 4.72 (1H), 4.69 (1H), 4 .36 (1H), 3.85 (1H), 3.67 (2H), 2.23 (1H), 2.09 (1H), 1.79 (1H), 0.89 (9H), 0, 07 (6H).

(b) Intermediate 2: (4R, 3aS, 6R, 6aR) -4- / 6-amino-9H-purinyl-9 Ζ-10 6- / ((1-dimethylethyl) dimethylsilyloxy) methyl-3a, 5.6 6a-tetrahydro-4H-cyclopenta-1,3-dioxole-2-thione

To a suspension of 3.45 g of intermediate 1 in 56 ml of dry dimethylformamide, 3.3 g of 1.1T-thiocarbonyldimidazole was added to give a yellow solution. After 15 | at room temperature for 1 hour, this solution was combined with that of an earlier test (on a 6% scale) and the solvent was removed by evaporation. The residual oil was diluted with 100 ml ethyl acetate and then washed with 2 x 20 ml water and 2 x 20 ml brine, dried over MgSO 4 and evaporated to a yellow substance. This was stirred with 25 ml diethyl ether, collected by filtration, washed another 25 ml of ether and dried under vacuum to give 3.61 g of the title product as a pale yellow solid; λ (in ethanol) 240.0 nm (eJ% = 459); XH NMR 8.27 (1H), 8.13 max 1.cm (1H), 7.33 (2H), 5.81 (1H), 5.37 (1H), 5.28 (1H), 3, 78 (2H), 2.60 (1H), 2.28 (2H), 0.90 (9H), 0.09 (6H).

(c) Intermediate 3: (1, 4'S) -6-amino-9- / 4 - ((t-butylddimethylsilyloxy) methyl) -2-cyclopentenyl-1 / -9H-purine

To a solution of 3.57 g of intermediate 2 in 25 ml of dry tetrahydrofuran was added a solution of 4.94 g of 30 l, 3-dimethyl-2-phenyl-1,2,2-diazaphospholidine in 10 ml of dry tetra hydrofuran, after which 8 | stirred at room temperature for an hour. The solvent was removed by evaporation. The residual oil was combined with that of an earlier test (on a 40% scale) and then chromatographed on a column of 200 g of silica (Merck 7734), first with chloroform and then with chloroform / ethanol mixtures to give a white gave dust.

This substance was stirred with 25 ml diethyl ether and then collected by filtration, washed with an additional 10 ml ether and dried under vacuum. This gave 1.47 g of the title product; 8900122 ..

22 - 17 λ (in ethanol) 261.4 nm (E, ° = 443); max 1cm -1 H NMR: 8.14 (1H), 8.00 (1H), 7.20 (2H), 6.12 (1H), 5.95 (1H), 5.60 (1H) , 3.66 (2H), 2.96 (1H), 2.69 (1H), 1.65 (1H), 0.74 (9H), 0.02 (6H).

5 (d) Intermediate 4: (1, R, 4, S) -6 "amino-9- / 4 - ((t-butyl-dimethylsilyloxy) methyl) -2-cyclopentenyl-1 / -9H-purine-1- oxide 1.29 g of 80-90% m-chloroperoxybenzoic acid was added to a solution of 1.37 g of intermediate 3 in 30 ml of chloroform, followed by stirring at room temperature for 3 hours. The solvent was removed by evaporation and the leftover gum was dissolved in 10 ml of ethyl acetate. A white matter crystallized out. This substance and material recovered by evaporation of the filtrate were taken up in 100 ml of chloroform and then washed with 3 x 10 ml of saturated NaHCO 2 solution and with 2 x 10 ml of brine. The washings were back-extracted with 50 ml of chloroform. The combined organic solutions were dried over MgSO 4 and then evaporated to a solid. This solid was stirred with 25 ml diethyl ether and collected by filtration, washed with an additional 10 ml ether and then dried under vacuum to give 1.16 g of the title product; λ (in ethanol) 235.4 nm (eJ% = 1324), 263.2 nm (E * Z = 248), 300.2 nm (eJ £ = 75); n NMR (in CDCl 3): 8.72 (1H), 8.02 (1H), 7.16 (2H), 6.21 (1H), 5.87 (1H), 5.72 (1H), 3.68 (2H), 3.04 (1H), 2.82 (1H), 1.74 (1H), 0.89 (9H), 0.06 (6H).

(E) Intermediate 5: (1TR, 4TS) -7 / 4 - ((t-butyldimethyl-silyloxy) methyl) -2-cyclopentenyl-9 7-2-cyclopentenyl-1 7-2-imino-1,2 -dihydro / 1,2,4 7oxadiazolo / 3,2-7purine hydrobromide To an ice-cooled suspension of 1.08 g of intermediate 4 in 20 ml of methanol, 0.34 g of cyanogen bromide in 20 ml of methanol was stirred over 5 minutes. added. After 15 minutes, the suspension was allowed to warm to room temperature to give a solution. After 90 minutes, the solvent was removed by evaporation. The residue was stirred in 25 ml diethyl ether, collected by filtration and washed with an additional 25 ml ether and then dried under vacuum to give 1.37 g of the title product; λ (in ethanol): 228.2 nm (E, 1% = 530), 285.2 nm (eJ% = 445); .max Icm Icm H-NMR (in CDCl3): 10.20 (1H), 10.02 (1H), 8.37 (1H), 6.25 (1H), 6.01 (1H), 5.90 (1H), 3.69 (2H), 3.05 (1H), 2.86 (1H), 1.73 (1H), 0.86 (9H), 0.03 (6H).

8900 (22 ..

- 23 - (f) Intermediate 6: (1'R »4TS) -9— / 4 - ((t-butyldimethyl-silyloxy) methyl) -2-cyclopentenyl-1-7-6-cyanoimino-1,6-dihydro-1 -me thoxy-9H-purine

To a solution of 1.36 g of intermediate 5 in 10 ml of dimethylformamide was added triethylamine with stirring at room temperature, and 40 minutes later 0.54 ml of iodomethane to give a yellow solution. After 45 minutes, the solvent was removed by evaporation. The residue was diluted over 100 ml of ethyl acetate and 20 ml of water. The organic solution was then washed with a further 2 x 20 ml of water and 20 ml of brine, dried over MgSO 4 and evaporated to a solid.

This substance was stirred in 25 ml diethyl ether, collected by filtration and washed with an additional 10 ml ether and dried under vacuum. This gave 0.865 g of the title product; 15 λ (in ethanol) 227.2 nm (E *% = 449), 287.0 nm - 544); ax ax Icm in combination with tt-NMR: 8.23 (1H), 7.96 (1H), 6.24 (1H), 5.85 (1H), 5.65 (1H), 4.21 (3H), 3.66 (2H), 3.04 (1H), 2.77 (1H), 1.68 (1H), 0.88 (9H), 0.05 (6H).

(g) Intermediate 7ï (1, R, 4, S) -2-amino-9- / 4 - ((t-butyl-20-dimethylsilyloxy) methyl) -2-cyclopentenyl-1- / -6-methoxyamino-9H-purine

A solution of 802 mg of intermediate 6 and 0.45 ml of 1,8-diazabicyclo / 5,4-0 / undecene-7 in 80 ml of ethanol was stirred and refluxed for 9 hours. Thereafter, heating was stopped and the solution was left overnight.

The solvent was removed by evaporation. The residual oil was chromatographed together with that of an earlier test (on a 4% scale) over a column of 40 g of silica (Merck 9385), first with chloroform and then with chloroform / ethanol-30 mixtures to give a foam. This foam was stirred in 10 ml of diethyl ether, the resulting solid collected by filtration and washed with an additional 5 ml of ether and dried under vacuum to give 594 mg of the title product; 11 1 λ ^ in ethano1) 282'2 ^ (Eicm = 409) * H NMR: 9.76 (1H), 7.32 (1H), 6.53 (2H), 6.08 (1H), 5.88 (1H), 5.26 (1H), 3.72 (3H), 3.61 (2H), 2.90 (1H), 2.50 (1H), 1.52 (1H), 0 .83 (9H), 0.02 (6H).

(h) Intermediate 8: (1S, 4R) -4- / 2-amino-6-methoxyamino-9H-purinyl-9 7-2-cyclopentenemethanol 89 0 0 1 22 ..

f - 24 -

To a solution of 356 mg of intermediate 7 in 35 ml of tetrahydrofuran, 1.4 ml of 1.0 M tetrabutylammonium fluoride in tetrahydrofuran was added with stirring at room temperature. After stirring for 90 minutes, the reaction was quenched with 1 ml of water and the solvents were removed by evaporation.

The remaining oil was chromatographed on a column of 20 g of silica (Merck 7734), first with chloroform and then with chloroform / ethanol mixtures; this gave 243 mg of the title product, a solid; 10 λ (in buffer with pH = 6): 280.2 nm (eJ% = 534); 1 H-NMR: max in combination with 9.75 (1H), 7.39 (1H), 6.52 (2H), 6.10 (1H), 5.84 (1H), 5.27 (1H), 4.73 ( 1H), 3.40 (2H), 2.83 (1H), 2.55 (1H), 1.52 (1H), (1S, 4R) -4-2.6-diamino-9H-purinyl- 9 J-2-cyclopentene carbinol

Aluminum amalgam (from 237 mg aluminum and 0.5% aqueous mercuric chloride solution) was added to the ice-cooled solution of 210 mg of intermediate 8 in 10 ml of water and 50 ml of tetrahydrofuran, which was added in small amounts over 15 minutes. After 40 minutes, the mixture was allowed to warm to room temperature with stirring. After 15 hours, the insoluble material was removed by filtration over kieselguhr; this was washed 1: 5 with 60 ml water / tetrahydrofuran. The filtrates were evaporated to dryness and the residue was chromatographed on a 10 g column of silica (Merck 9385) with chloroform / ethanol mixtures. This gave 159 mg of the title product, a foam; / α 7 ^ -81 ° (c = 1.04 in methanol); λ (in buffer with pH = 6): 255.0 nm (E? - ^ = 302), 280.8 nm max. X cm (E: / o = 381); H-NMR: 7.61 (1H), 6.66 (2H), 6.10 (1H), 5.87 (1H), in combination with 5.76 (2H), 5.38 (1H), 4.76 (1H) ), 3.45 (2H), 2.87 (1H), 2.60 (1H), 1.60 (1H).

Example XIII

(1S, 4R) -4- (2-amino-6-hydroxy-9H-purinyl-9) -2-cyclopentenylcarbinol (11R, 4'S) -2-amino-1,9-dihydro-9- / 4- hydroxymethyl-2-cyclopenyl-1-7-6-ketopurin 35 A cloudy solution of 144 mg of the title compound

thing of Example XII in 10 ml 0.1 M buffer with pH = 6 (28.4 g Na 2 HPO 2 in 2 liters of water, pH adjusted with H 2 PO 2) was treated with adenosine deaminase (778 units in 0.5 ml of liquid which was 50% glycerol and 50% 0.01N

890012.2 ..

- 25 - phosphate buffer with pH = 6.0). After everything was put together with stirring, the mixture was heated to 37 ° C. After 18 hours, the resulting suspension was cooled. The precipitated solid was recrystallized from water to give 86 mg of the title product, a white matter; / a -49 ° (c 0.5 in dimethylsulfoxide); λ (in buffer with pH = 6) ï 252.6 nm (E ^ = 531), ^ H-NMR: max r lcm 10.60 (1H), 7.60 (1H), 6.47 (2H), 6.10 (1H), 5.86 (1H), 5.33 (1H), 4.72 (1H), 3.45 (2H), 2.59 (1H), 1.58 (1H).

Example XIV

Preparation of the enantiomers of (lot, 4 ») - 4- (2-amino-6-hydroxy-9H-purinyl-9) cyclopentenyl-2-carbinol (a) (IS, 4R) -4- (2-amino -6-hydroxy-9H-puriny1-9) -cyclopentenyl-2-carbinol

100 mg of the diamino analog of Example XI was dissolved in 3 ml of 0.05 M K 2 PO 2 buffer (pH 7.4) under heating to 50 ° C. The solution was cooled to room temperature and 40 units of adenosine deaminase (type VI, from calf intestinal mucus, from Sigma) were added. After 3 days of incubation at room temperature, a precipitate was formed which was separated by filtration, yield 18.2 mg. The filtrate was concentrated to 1.5 ml and kept in the refrigerator for 2 days. Even more solid was obtained by filtration, yield 26.8 mg. The two fractions were recrystallized from water together, pure title compound with m.p.

-24 ° C gave 269-272 ° C, / α7 * = -62.1 (c = 0.3 in MeOH).

(b) (IR, 4R) -4- (2-amino-6-hydroxy-9H-purinyl-9) cyclopentenyl-2-carbinol

The filtrates from the preparation of the 1S, 4R isomer (Example XIVa) were evaporated to dryness. The starting material, the unchanged diamino compound, was separated on a column of silica gel by chromatography with 10% methanol in chloroform. This diamino compound was dissolved in 15 ml of 0.05 M 10 P 10 buffer (pH 7.4) and 800 units of adenosine deaminase were added. The solution was incubated at 37 ° C for 96 hours. TLC showed that some unchanged starting material was left over. The solution was heated in boiling water for 3 minutes and filtered to remove denatured protein. Another 800 units of adenosine deaminase were added and the treatment was repeated. The protein-liberated solution 8800 122. " 26 was evaporated to dryness and the product was crystallized from water. The title compound was obtained as a white solid, m.p. 265-270 ° C, / ct_7p4 = + 61.1 ° (c = 0.3 in MeOH).

Example XV

5 (i) - (loc, 4ft) -4- (2-amino-6-hydroxy-9H-purinyl-9) cyclopentenyl-2-acetoxycarbinol

To a suspension of 130 mg (0.50 mmol) of product of Example X and 5 mg (0.04 mmol) of 4-dimethylaminopyridine in a mixture of 6 ml of acetonitrile and 0.09 ml (0.66 mmol) of 10 triethylamine, 0 0.06 ml (0.6 mmol) acetic anhydride. The mixture was stirred at room temperature for 3 hours. 1 ml of methanol was added to quench the reaction. The solution was evaporated to dryness and adsorbed on 1.5 g of silica which was packed into a 2.0 x 12 cm column. This was eluted with 15 CHCl 3 / MeOH 20: 1. The product fractions were concentrated to dryness to a white matter. This was washed with MeOH / AcOEt; yield 123 mg (85%). Further purification from methanol gave the title compound as needle-shaped crystals, m.p. 237-239 ° C.

Example XVI

(1S, 4R) -4-jf 2-amino-9H-purinyl-9-cyclopentenyl-2-carbinol

To an ice-cooled solution of 1.202 g (IS, 4R) -4- / 2-amino-6-methoxyamino-9H-purinyl-9 / cyclopentenyl-2-methanol (intermediate 8 of Example XII) in 250 ml of tetra-25 hydrofuran and 50 ml of water were added over 1 hour 47 minutes in small pieces of aluminum amalgam from 1.761 g of aluminum and 0.5% aqueous mercuric chloride solution. After stirring for 35 minutes, the mixture was allowed to warm to room temperature.

After 16 hours and 50 minutes, some aluminum-30 amalgam (from 235 mg aluminum) was added over 14 minutes. After an additional 4 hours and 10 minutes, the mixture was filtered through kieselguhr, which was washed with tetrahydrofuran / water 5: 1. The filtrates were evaporated to dryness, leaving a yellow foam. This was chromatographed on a column prepared in chloroform from 33.8 g of silica (Merck no. 7734); elution with chloroform / ethanol mixtures gave multiple fractions (578 mg, 420 mg and 40 mg). The two larger fractions were crystallized separately from isopropanol. The filtrates were combined with the smallest fraction and underwent preparative thin layer chromatography (on a Merck No. 5717 plate), developing three times with chloroform / methanol 10: 1. The plates were washed with ethyl acetate and with ethyl acetate / ethanol eluted 1: 1 to give 45 mg of brown matter, which was chromatographed on a column prepared in 2.7 g of silica (Merck no. 7734) in chloroform, eluting with chloroform / methanol / triethyl amine mixture gave 17 mg of gum After an unsuccessful crystallization from isopropanol and treatment with carbon in methanol, an aqueous solution of the conserved material was freeze-dried to give 15 mg of the title compound.

1 H NMR (in DMSO-dg): 1.62 (1H), 2.63 (1H), 2.89 (1H), 3.45 (2H), 4.73 (1H), 5.48 (1H) ), 5.91 (1H>, 6.14 (1H), 6.50 (2H), 7.98 (1H), 8.57 (1H).

Mass spectrum: / MH 7 * "232.

Example XVII

Tablet formulations A. The following formulation was prepared by wet granulation, drying and sieving the ingredients with a provider of providon in water, followed by addition of magnesium stearate and pressing.

mg / tablet (a) active substance 250 (b) lactose (B.Ph.) 210 (c) Povidone (B.Ph.) 15 25 (d) sodium starch glycollate 20 (e) magnesium stearate 5 500 B. De the following formulation was prepared by direct compression; the lactose was of the appropriate type.

mg / tablet

Active substance 250

Lactose 145

Avicel 100 35 Magnesium Stearate 5 500 C. (Delayed Release Formulation). This was prepared by wet granulation of the ingredients listed below with a solution of providon in water, drying and sieving, followed by addition of magnesium stearate and compression.

& 900 122.'- 28 - mg / tablet (a) Active substance 500 (b) Hydroxypropylmethylcellulose 112 (Methocel K4M Premium) 5 (c) Lactose (B.Ph.) 53 (d) Povidone (B.Ph.) 28 ( e) Magnesium stearate 7 700

Example XVIII

10 Capsule formulation

A capsule formulation was prepared by mixing the ingredients listed below and putting them into two-piece hard gelatin capsules.

mg / capsule 15 Active substance 125

Lactose 72.5

Avicel 50

Magnesium stearate 2.5 250

20 Example XIX

Injection formulation

Per ampoule 0.200 g of active substance, enough 0.1 M NaOH solution for a pH of about 11, and sterile water to 10 ml.

The active substance was suspended in part of the water (which should have been warmed up) and the pH was adjusted to 11 with the NaOH solution. The portion was then made to volume and filtered through a sterilizing membrane filter into 10 ml sterile ampoules, which were sterile sealed and given protective caps.

Example XX Suppository mg per suppository

Active substance (63 µm) 250 35 Hard fat (BPh.) 1770 2020

About a fifth of the hard fat was melted in a steam jacketed pan at a maximum of 45 ° C. The active substance was passed through a 200 µm sieve and charged with 8800122.

With the aid of a high-speed stirrer, mixed with the melted base until a smooth dispersion was obtained. While the mixture was kept at 45 ° C, the rest of the hard fat was added to the slurry and stirred until a homogeneous mixture was obtained. The entire suspension was passed through a stainless steel sieve with 250 µm holes and allowed to cool to 40 ° C with continuous stirring. At a temperature of 38 ° to 40 ° C, 2.02 g portions of the mixture were placed in suitable 2 ml plastic molds. The suppositories were allowed to cool to room temperature.

Example XXI Anti-virus effect

(A) Determination of activity against HIV

Compounds of formula 1 were screened for HIV activity at the National Cancer Institute (Frederick Cancer Research Facility) in Frederick, Maryland (FCRF). The following is the usual method of screening at the FCRF. The protocol includes three areas: (I) preparation of the infected cells and distribution thereof on the test plates, (II) preparation of the dilution plates and division thereof on the test plates, and (III) the XTT test. See D.A. Scudiero et al. In "A New Simplified Tetrazolium Assay for Cell Groth and

Drug Sensitivity in Culture ", in Cancer Res., 48 (1988) 4827.

I. Infection and distribution of ATBH cells across microtiter plates. Cells to infect (a normal lymphoblastoid cell line expressing CD4) were placed in 50 ml conical centrifuge tubes and 1 h at 37 ° C with 1-2 µl. Ig / ml polybrene treated. The cells were then sedimented at 1200 rpm for 8 minutes. Now HIV virus, diluted 1:10 with medium (RMPI-1640, 10% human serum or 15% fetal calf serum (FCS), with IL-2 and antibiotics) was added to an MOI of 0.001. The virus-free blank cells received only -4 medium. Assuming that the infectivity of the virus was 10, an MOI of 0.001 means eight infectious virus particles per 10,000 cells. About 500,000 cells were exposed to 400 µl of diluted virus per centrifuge tube. The resulting mixture was incubated with CO2 for 1 hour at 37 ° C under air. The cells infected or not L-4 were diluted to 1 x 10 (with human serum) or 2 x 10 (with fetal calf serum) cells per 89 0 P122.

- 30 - 100 µl.

100 µL aliquots of infected or uninfected cells were placed in the appropriate wells of a 96 U-well microtiter plate. Each dilution was tested in duplicate with infected cells, and singly on the uninfected cells for sensitivity to the test substance. Test cells free blank cells, infected and uninfected, ran in triplicate. Wells B2 through G2 served as reagent blanks and received medium only. The plates were incubated with CO 2 at 37 ° C under air until the test substance was added.

II. Dilute the test substance and add

Dilution plates (flat microtiter plates with 96 flat bottom wells) were overnight with phosphate buffered saline (PBS) or with medium containing at least 1% FCS

or 1% human serum (depending on the medium used in the trial) starting one day before the trial. This "blocking" served to limit adsorption of the test substance to the microtiter plate during the actual treatment.

The wells were completely filled with blocking solution and left to stand at room temperature in a humid space under a hood.

The dilution procedure started with diluting the test substance 1:20. Blocked dilution plates were prepared by shaking the blocking solution from the wells and patting them dry with sterile gauze. All wells of each plate were then filled with 225 µl of the appropriate medium using a Cetus liquid handling system. From each diluted 1:20 compound, 25 µl was then manually placed in row A of a "blocked" and filled dilution plate. Four compounds were administered per dilution plate, enough to provide two test plates. The four compounds were then diluted tenfold using Cetus's liquid handling system from row A to row H. At this time, the initial dilution of each compound in row A was 1: 200. The dilution plates were kept on ice until needed.

Using a six microtip multiple pipette, 100 µl of each test dilution was mixed on the test plate.

- 31 - which already contained 100 µl medium plus cells. The final dilution starts on the test plate with 1: 400 (wells B4 to G4). This dilution (up to 0.25% DMSO) prevents the carrier from interfering with DMS0 in cell growth. Test-free, infected and uninfected cells (wells B3 to G3) and reagent blanks (B2 to G2) received medium only. The last two compounds were then transferred from wells H7 to H12 to a second test plate using the same procedure. The test plates were then incubated with CO 2 at 37 ° G under air for 7-14 days or until virus blanks were visibly lysed macroscopically.

III. Quantitative determination of the cytopathogenicity of the viruses and the activity of the test substances A. Materials 15 1. A solution of 2,3-bis / 2-methoxy-4-nitro-5-sulfophenyl 7-5- / (phenylamino) carbonyl_7-2H- tetrazolium hydroxide. (XTT) - 1 mg / ml in medium without FCS. Store at 4 ° C.

Prepare fresh every week.

2. Phenazine methosulfonate (PMS). A stock solution can be prepared and kept frozen at -20 ° C until needed. This must be made in PBS to a concentration of 15.3 mg / ml.

B. The microculture assay with tetrazolium (MTA) 1. Preparation of the XTT-PMS solution - The XTT-PMS solution is prepared just before it is placed in the wells of the culture plate. The stock solution of the PMS is diluted 1: 100 (to 0.153 mg / ml). Diluted PMS is added to each ml of XTT to a final PMS concentration of 0.02 mM. Each appropriate well receives a 50 µl aliquot of the XTT-PMS mixture, and the plate is incubated at 37 ° C for 4 hours. The plate covers are then removed and replaced by adhesive plate seals (Dynatech cat. No. 001-010-3501). The sealed plate is shaken in a microculture plate mixer and the absorbance at 450 nm is taken up.

35 IV. Outcomes

Figure 1 is a graph of the percentage of test cells relative to the uninfected cells in both infected and non-infected cells, against the increasing compound concentration of Example X.

69001ZZ.

- 32 -

The figures plotted in Figure 1 can be calculated as follows: the effective concentration (EC ^) for infected cells is about 0.15 µg / ml and normal cells are inhibited by about 100 µg / ml (IC ^ q), which a thera-5 peutical index (Th.I) of about 667.

In a previous trial conducted at the Southern Research Institute, a Th.I. of about 200 when MT-2 cells were grown with H9 / HTLV-IIIB.

The inhibitory concentrations against HIV determined in the manner described for the compounds of Examples VII, IX, X, XI and XIV (b) are shown in Table 1.

Table 1

Verb. van Cellijn ED,.,. ID ..-. Th.I.

No. example 15 9a VII MT-2 2.3 50 21.4 13a IX MT-2 0.41 6.97 17.3 14a X MT-2 0.15 100 667 15a XI MT-2 2.9 > 125> 42.7 (-) 14a XIV (b) CEM 0.66 189 284 20

In these tests, the compounds of Examples V and VIII also exhibited anti-virus activity.

B. Action against the cat leukemia virus

Screening for activity against the cat virus 25 FeLV-FAIDS was done on 96 well Corning microtiter plates containing 81 C indicator cells in the Dulbecco's Iscove modified medium supplemented with 10% heat inactivated fetal bovine serum (FBS). 20 hours prior to the 3 run, plates containing 8lC cells were seeded to 5 x 10 cells per well. On the day of the experiment, the cells were pretreated for 30 minutes at 37 ° C with 25 µg / ml DEAE-dextran in 0.1 ml Hanks balanced salt solution. That was removed and 0.1 ml culture medium containing 32 TCID ^ q of FeLV-FAIDS or 0.1 ml culture medium alone was placed in each well. The virus was allowed to adsorb for 1 hour and then 0.1 ml of test substance or positive blank (the 2 ', 3'-dideoxycytidine, DDC) in growth medium or only that medium was added. The plates were incubated at 37 ° C. the

On the 4 day after infection, the cells received fresh growth medium containing the intended substance. On the 7 day after infection, the culture medium was completely changed and replaced with fresh medium containing the substance. On the 10 day after infection, the cells were fixed with formalin, stained with 0.1% Coomassie Brilliant Blue R-25Q, and examined microscopically for CPE and cytotoxicity of the test substance.

The compound of Example X had an ED 2 -q of 1.9 µg / ml.

(C) Action against murine AIDS

Tissue culture plates from 6 well Falcon were seeded with 1.75 x 10 cells per well in a total volume of 2.5 ml EMEM containing 5% heat inactivated FBS. 20 hours after the cells were seeded, the medium was decanted and 2.5 ml of phosphate buffered saline containing 25 µg / ml DEAE-dextran was placed in each well.

Then it was incubated for 1 hour at 37 ° C, after which the DEAE-dextran-15 solution was decanted and the cell layers rinsed once with 2.5 ml PBS. The normal cell blanks again received 2.5 ml medium (without virus or test substance). Test-control cultures received 2.5 ml of medium containing test substance but no virus.

The virus infected blank cultures received 0.5 ml of the appropriate dilution of the CAS-BR-M solution (such that the number of plaques could be counted) plus 2.0 ml of medium. The test samples received 0.5 ml of the appropriate virus dilution plus 2.0 ml medium or test substance dilution. Six concentrations of each test substance were tested which differed by a factor of 25 VTÖ. Three concentrations of the positive blank, DDC, were used. Each concentration of the test substance was tested in triplicate, and in each series there were six virus and six blank cell cultures. On the 3 day after infection, the toxicity of the test substance to the SC-1 cells was verified in duplicate by microscopic examination after fixing and staining. The remaining test and blank cultures were irradiated with an ultraviolet lamp for 20 seconds and XC cells were added to each culture (5 x 10 4 cells per well in 2.5 ml EMEM containing 10% heat-inactivated FBS). On the 35th day after UV irradiation, the cultures were fixed with formalin and stained with crystal violet. The plaques were counted under a dissection microscope.

The anti-virus activity, evidenced by the reduction in the number of CAS-BR-M plaques, was based on 89 00 12.2.

i - 34 virus infected cultures, whether or not treated with test substance. The compound of Example X had an ED ^ q of 1.1 µg / ml.

(D) Action against retrovirus SAIDS in the monkey (SRV-2)

Screening for activity against SAIDS virus 5 (D / Washington) was done with a syncytia inhibition test on

Raji cells. The test substance was diluted with Iscove complete medium and then 100 µl of each dilution was placed in the appropriate wells of a 96-point plate. In each well 3, 5 x 10 actively growing Raji cells were placed in 50 µl complete medium of Iscove. This was followed by adding 50 µl of clarified supernatant from a co-culture of SRV-2 and Raji cells. As a positive blank, DDC was included in this test. The plates were incubated under a humid atmosphere with 5% CO2 at 37 ° C. The occurrence of syncytia was counted on the day after infection.

Test substance toxicity was seen when comparing the counts of viable cells in an uninfected test substance treated sample and an uninfected sample without test substance. The compound of Example X had a 20 U: D50 Van µg / ml.

(E) Action against the Visna Maedi virus

The anti-virus activity against the Visna Maedi virus (VMV), strain WLC-1, was determined by monitoring the reduction of the virus specific immunohistochemical staining. Sheep monolayers of choroid plexus cells were infected with VMV and coated with serial dilutions of the test substances. After 5 days of incubation, the monolayers were further incubated with virus specific antisera conjugated to radish asoxidase (HRP). Subsequent incubation of the monolayers with a chromogenic substrate of the HRP stained areas where the virus could multiply. The individual colored points were counted and the concentration of the test substance needed to reduce the number of points by 50% relative to the untreated blanks was calculated.

The compound of Example XIII had an ED 2 of 0.2 µg / ml.

Example XXII Cytotoxic activity

The compounds according to examples V, VII and 8900122 / - 35 -

VIII showed cytotoxicity when tested on cell cultures of murine leukemia P388, as described by R.G. Alonquist and R. Vince described in J. Med. Chem. JJ3 (1973) 1396. The EDr values obtained were 5U

Substance of Example V 12 µg / ml

Substance of Example VII 40 µg / ml

Substance of Example VIII 3 µg / ml 10 8900 ni.

Claims (18)

A compound according to formula 1, wherein X is hydrogen, NRR ^, SR, OR or halogen and Z is hydrogen, OR ^ or NRR ^ 5, where R, R ^ and R £ are the same or different and hydrogen, 0χ ^ -alkyl and aryl, as well as the pharmaceutically acceptable derivatives thereof. A compound of the formula 1 according to claim 1 and the pharmaceutically acceptable salts thereof. A compound according to claim 1 or 2, wherein Z is hydrogen, hydroxy or amino. A compound according to any preceding claim, wherein Z is NHL. 5. A compound according to any one of the preceding claims, wherein X is hydrogen, chlorine, N 2, SH or OH. A compound according to claim 5 wherein X is OH. A compound according to claim 5, wherein X is H or. 8. One of the following compounds: 20 (10i, 4A) -4- (6-chloro-9H-purinyl-9) cyclopentenyl-2-carbinol; (1 «t» 4 «.) -4- (6-hydroxy-9H-purinyl-9) cyclopentenyl-2-carbinol; (ltf, 4a) -4- (6-amino-9H-purinyl-9) cyclopentenyl-2-carbinol; (loi 4 × 4) -4- (6-mercapto-9H-purinyl-9) cyclopentenyl-2-carbinol; (lol,, 4 a) -4- (2,6-diamino-9H-purinyl-9) cyclopentenyl-2-carbinol; 25 (lcC> 4 R.) -4- (2-amino-6-chloro-9H-purinyl-9) cyclopentenyl-2-carbinol, and (10t4oO-4- (2-amino-9H-purinyl-9) cyclopentenyl -2-carbinol. 9. (li, 4ot) -4- (2-amino-6-hydroxy-9H-purinyl-9) -cyclopent enyl-2-carb ino1. A compound according to any one of claims 1 to 9 in the form of a substantially racemic mixture. A compound according to any one of claims 1 to 9 substantially in the form of an optically pure isomer. 12. A compound according to any one of claims 1 to 9 consisting essentially in the form of the D isomer. A compound of formula 1 as defined in any one of claims 1 to 12 or a pharmaceutically acceptable derivative thereof for use as a medicament. 14. A compound of the formula 1 as defined in any of claims 1 to 12 or a pharmaceutically acceptable derivative thereof for use in the manufacture of a medicament for the treatment of a viral infection. A pharmaceutical composition containing a compound 5 of formula 1 as defined in any one of claims 1 to 12 or a pharmaceutically acceptable derivative thereof together with a pharmaceutically acceptable carrier therefor. A pharmaceutical composition containing a compound of formula 1 as defined in any one of claims 10 to 12 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier therefor. Pharmaceutical composition according to claim 14, which additionally contains yet another therapeutic agent. 18. A compound of formula 2, wherein X is hydrogen, NRR 1, SR, OR, halogen or a protected form thereof, wherein Y is hydroxy or a protected form thereof and Z is hydrogen, OR 1, NRR 1, or a protected form form thereof, wherein R, R 1 and are the same or different and may be hydrogen, C 1-6 alkyl and aryl, as well as the pharmaceutically acceptable derivatives thereof. -o-o-o-o-o- 8900122 ..