PL163814B1 - Method of obtaining novel analogous compounds of dideoxy-didehydrocarbocyclic nucleosides - Google Patents

Abstract

Compounds of the formula <IMAGE> in which X = H, NRR<1>, SR, OR or halogen; Z = H, OR<1> or NRR<1>; R, R<1> and R<2> are H, C1-4-alkyl or aryl and pharmaceutically acceptable derivatives thereof are described. The use of the compounds as antiviral and antitumour agents is likewise described as well as pharmaceutical formulations and methods for the preparation of the compounds and their intermediates.

Description

The subject of the invention is a method for the preparation of new analogs of didaoxydidehydrocarbocyclic nucleosides, namely 2 ',: 3 * -dideox ^' ^! 2 '/ 3 * -didahydro-purine nucleoside analogs, which are suitable for use in medicine, especially as antiviral agents.

Due to the similarity between the cellular functions of viruses and the host, it is difficult to selectively attack the virus while leaving the host cell intact. There are relatively few agents that are effective against viruses per se, and it is difficult to obtain antimiral agents with an acceptable therapeutic index, i.e. agents that have a significant effect. antiviral in a dose which shows acceptable toxicity or. acceptable side effect.

A group of viruses that have gained prominence recently are the retroviruses responsible for human immunodeficiency syndrome / AIDS /. Such viruses have previously been referred to by different names, but now they are commonly referred to as human immunodeficiency viruses / HIV / j such two viruses, HIV- I and HIV-II, repeatedly isolated in patients suffering from AIDS and related conditions such as AIDS-related complex / ARC / and chronic systemic lymphadenopathy

Although a number of nucleosides are considered to be suitable for the treatment of conditions associated with HIV infection, only Jewovudine (AZT, Retrovir) is approved for treatment, however, Jewovudine is known to have serious side effects and to induce bone marrow suppression leading to a decrease in white blood cell counts. blood and, consequently, to pronounced anemia. There is therefore a need for effective agents that are less cytoxic.

The method of the invention produces a new class of nucleoside analogues which have antiviral activity as defined by formula 1 in the figure, wherein X

21 is hydrogen, NRR, SR or ORj Z is hydrogen, OR or NRRj

2.

R, R and R may be the same or different and are selected from substituents consisting of hydrogen, C ^ _ ₄ alkyl and aryl, and pharmaceutically acceptable derivatives thereof.

The preparation of nucleoside analogues according to the invention consists in reacting a compound of the formula 10, wherein X * is a halogen atom and Z is as defined above, optionally in a functionally protected form, with a compound of the formula ONRr, wherein R and r! have the meanings as defined above, with RO ion-donating reagent "in which R is as defined above, with thiourea or with a reducing agent, then optionally a racemic mixture of a compound of formula 1 is converted to substantially a single optical isomer of the same or a different compound of formula" 1 by enzymatic conversion or a compound of formula 1 in which X is SH group is alkylated or arylated to compounds of formula 1 in which X is SR or from a protected derivative of a compound of formula 9 in which X and Z are as defined above and Y is a hydroxyl protecting group, the protecting group is removed, and optionally a compound of formula 1 is converted to a pharmaceutically acceptable derivative by reaction with an acid, base, phosphorylating or esterifying agent.

It is obvious to those skilled in the art that the compounds of formula I are cis compounds and that the cyclopentene ring in these compounds contains two chiral centers (which are indicated by x in formula Ia), thus these compounds may exist as optical isomers / i.e. enantiomers) and mixtures thereof, including racemic mixtures. The scope of the present invention includes the preparation of all such isomers and mixtures thereof, including racemic mixtures. Thus, in the compounds of formula (I) either the chiral center to which the base is attached has the R configuration and the chiral center to which the CH1OH group is attached has the S configuration (O-isomer), or the chiral center with the base attached has the configuration

S, a enntuum with which ruppa CHoOH aa confiurracee R (lzmear) is attached. Preferably the compounds are in the form of a mixture of racamfclaaC or substantially pure isomer 0. The D isomer can be represented by formula Ib where X and Z have the lanaraafn defined for formula 1.

When further reference is made to compounds of formula 1 in this specification, it is understood that this also includes compounds of formula 1b.

It is also apparent to those skilled in the art that some of the compounds of formula I may be present

163,814 in several teutomeric forms, and all such tautomers are also within the scope of the invention.

The term "halogen as used herein refers to fluorine, chlorine, bromine and iodine, X as halogen being preferably chlorine.

The term _Ci-4 alkyl refers to an alkyl group of straight or branched chain group. For example methyl, ethyl, n-propyl, 1-propyl, n-butyl, sec-butyl and tert-butyl. Preferably, the C 1-4 alkyl group is a methyl group.

Aryl refers to any mono- or polycyclic aromatic moiety and includes unsubstituted and substituted aryl / mash as phenyl, tolyl, xylyl, anizylowa / and unsubstituted and substituted aralkyl containing the group C ^^ alkyl group such as a phenyl-C ₄ alkyl, e.g. benzyl or phenethyl.

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

A preferred group of compounds of formula I are those in which X is OR, especially OH.

Another preferred group of compounds consists of those compounds of formula I wherein X is NRr, especially NH ', or hydrogen.

Particularly preferred are those compounds of formula I wherein Z is NH2 and X is hydrogen, NH2 or, especially, OH. Such compounds have particularly desirable therapeutic indices as antiviral agents.

By pharmaceutically acceptable derivatives is meant pharmaceutically acceptable salts, esters or salts of such esters or compounds of Formula 1 or other compounds which, when administered to a patient, are capable of providing an antiviral active metabolite or the remainder thereof.

Preferred esters of compounds of formula I include esters of carboxylic acids in which the non-carbonyl group of the ester is selected from hydrogen, straight and branched chain alkyl groups (e.g. methyl, n-propyl, tert-butyl, n-butyl), alkoxyalkyl / e.g. methoxymethyl / aralkyl / e.g. benzyl /, aryloxyalkyl / e.g. phenoxyrnetyl /, aryl / e.g. phenyl optionally substituted by halogen, C ^^ alkyl or alkoxy Cj_ ₄ / sulphonate esters such as alkyl- or aralkylsulphonyl / example. methanesulfonyl / amino acid esters / e.g. L-valyl or L-isoleucyl / and mono-, di- or tri-phosphate esters.

The alkyl group present in the above esters, unless otherwise stated, preferably has 1 to 18 carbon atoms, in particular 1 to 18 carbon atoms. The aryl group in these esters preferably contains the group pheoyylone.

The pharmaceutically acceptable salts of the compounds of formula I include the salts of pharmaceutically acceptable inorganic and organic bases. Examples of suitable acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene p-sulfonic, tartaric, acetic, citric, methanesulfonic, formic, benzoic, malonic, naphthalene. -Z-Sufon and Benzenesulfon. Other acids, such as oxalic, although not pharmaceutically acceptable, may be useful in the preparation of salt intermediates for the preparation of compounds of Formula I and their pharmaceutically acceptable acid addition salts.

Salts with bases include alkali metal (e.g. sodium), alkaline earth metal salts / e.g. megnesium /, ammonium and NR4 + / R is a ^^ alkyl group /

When reference is made to the compounds according to the invention, this applies both to the compounds of the formula I and to their pharmaceutically acceptable derivatives.

Particular compounds of formula I include i / o C, 4 oC, -4- (6-choozn-9H-purin-9-yl) -2-cyclopeπtenyl-oatannl, and oC, 406 (-4- / 6). -hydroxy-9H-puzy-9-y0o / -2-cyclopentanyo-methanol, (loC, 4oC) -4- (6-aminn-9H-puzy-9-yo) / -2-cyclopentanyo-oethanol, / and oC, 4 oC / -4- / 6-mercapto-9H-pazin-9-yln / -2-cyclopantenyl-methanol, / lo, 4 oC /-4-/2- aoyyn-6-chl.oro- 9H-purin-9-yOn / -2-cyclopentayyl-oatano / 1oC 14oN -4- / 2-amino-6-hydroxy-9H-purin-9-yl / -2-cyclopentenyl-methanol, / 1 cC, 4 ° C / -4- /

163 814 (2,6-diamino-9H-purin-9-yl) -2-cyclopentenyl-methanol, either as a racemic mixture or as a single enantiomer.

The compounds of the invention either have antiviral activity themselves and / or metabolize into such compounds. In particular, these compounds are effective in inhibiting the replication of retroviruses, including human retroviruses such as human immunodeficiency viruses (HIV), which are the causative agents of AlOS.

Some of the sppsobbe-prepared compounds of the invention have anti-cancer activity, especially those compounds of formula 1 where Z is hydrogen.

The compounds of the present invention having anti-lipus activity are also suitable for the treatment of AlOS-related conditions such as the AIDS-related complex (ARC), general progressive disease (PGL), AIDS-related neurological conditions (such as dementia or tropical paraper), positive reactions for anti-HIV antibodies and positive for HIV, Kaposi's muscle and hemorrhagic purpura.

The compounds of the present invention are also useful for preventing the progression of clinical disease in individuals who are positive for anti-H ^ antibodies or for the HIV antigen, and for prophylaxis after exposure to HIV.

The pzzeclywlrus compounds of formula 1 or their pharmaceutically acceptable derivatives can also be used to prevent viral contamination of physiological fluids such as blood or semen.

Some of the compounds of the formula I are also suitable as intermediates for other compounds of the formula I.

Compounds with V-1 are suitable for the prophylaxis as well as the treatment of existing infections or symptoms.

The amount of the compound used in treatment will vary not only with the particular compound employed but also with the mode of administration, the nature of the condition being treated, the age and condition of the patient, and should ultimately be decided by the attending physician or veterinarian. In general, suitably, however, the dosage ranges from about 1 to about 750 mg / kg, e.g., from about 10 to about 750 mg / kg of body weight per day, such as 3 to about 120 mg per kilogram of recipient body weight, daily, preferably in the range of 6 to 90 mg / kg / day, and most preferably in the range of 15 to 60 mg / kg / day.

The desired amount may conveniently be presented in single or divided doses at appropriate intervals, e.g., 2, 3, 4 or more sub-doses per day.

The compound is conveniently administered in unit dosage form, e.g., containing 10 to 1500 mg, suitably 20 to 100O mg, and especially 50 to 700 mg of active ingredient per unit dosage form. Ideally, the active ingredient should be administered in such an amount that maximum plasma concentrations of the active ingredient ranging from about 1 to about 75%, preferably about 2 to 50 and most preferably about 3% ox, 30 mM, are obtained, e.g. by intravenous injection of 0.1% of a 9% solution of the active ingredient, optionally in saline, or by oral administration in the form of large pills, containing 100 mg / kg of active ingredient. Undesirable blood circulation of the active ingredient can be maintained by continuous infusion, which will provide about 0.10 to about 5.0 mg / kg / hr. or by periodic infusions containing about 0.4 to about 15 mg / kg of active ingredient.

For therapeutic purposes, a compound according to the invention can be administered as a raw compound, but preferably the active ingredient should be in the form of a pharmaceutical.

The pharmaceutical composition comprises a compound of formula 1 or a pharmaceutically acceptable derivative thereof together with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic and / or prophylactic ingredients. The carrier or carriers must be acceptable in the sense that they must be compatible with the other ingredients of the medium and not deleterious to the recipient.

Pharmaceuticals include agents suitable for oral, rectal, intravenous administration, localized application (including buccal or sublingual), intravaginal or parenteral administration / including gastritis and intravenous administration / or in the form of

163 814 suitable for inhalation or insufflation. the agents, if desired, may be in the form of divided unit doses and may be prepared by any known methods. All methods include the step of bringing the active compound into contact with liquid carriers or finely divided solid carriers or both, and, optionally, shaping the product into the desired form.

the pharmaceutical compositions for oral administration may conveniently be in the form of a unit dose such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, powder or granule form, solution, suspension or emulsion. The active ingredient may also be in the form of a large pill, electuary or mush. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrating agents, or wetting agents. The tablets can be coated by methods known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles / which may include edible oils / or preservatives.

The compounds of the invention may also be formulated for parenteral administration (e.g. by injection or continuous infusion / and may be presented in unit dose form in ampoules, pre-filled syringes or in multi-dose containers with added preservatives. the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization of a solution which is constituent with a suitable vehicle, e.g. sterile pyrogen-free water, before use.

For topical application to the epidermis, the compounds according to the invention may be formulated as ointments, creams or lotions, or as transdermal patches. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and / or gelling agents. Liquid powders (lotions) may be formulated with an aqueous or oily base and will usually also contain one or more emulsifying, stabilizing, dispersing, suspending, thickening or coloring agents.

agents suitable for topical application in the mouth include diamond-shaped lozenges containing the active ingredient in a flavored base, usually sucrose with acacia or tragacanth, lozenges containing the active ingredient in a neutral base such as gelatin and glycerin or sucrose and acacia, and mouthwashes containing the active ingredient in a suitable liquid carrier.

pharmaceutical compositions for rectal administration which use a solid carrier, most preferably, are in the form of unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used, and suppositories can be prepared by mixing the active compound with a softened or molten carrier or carriers, and then cooling and shaping into a mold.

agents suitable for vaginal administration may take the form of pessaries, tampons, creams, gels, pastes, foams or spray agents containing, in addition to the active ingredient, carriers known in the pharmaceutical art and suitable for the application in question.

For nasal administration, the compounds of the invention may be in the form of a liquid spray or in the form of drops.

The drops may be formulated with an aqueous or non-aqueous base also containing one or more dispersing, pulping or suspending agents. The sputtering liquids are conveniently supplied from pressurized packs.

For inhalation, the compounds of the present invention are conveniently administered from an insufflation device, a nebulizer or pressurized canister, or in any other convenient manner that produces a nebulized aerosol. The pressure packs may also contain a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethene, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized nebulized aerosols, the unit dose can be determined by using a valve which produces the amount indicated.

Alternatively, for administration by inhalation or insufflation, the compounds of the invention may also be in the form of a dry powder composition, e.g. a powder mix of the compound and a suitable powder base such as lactose or starch. The powder mixture can be in unit dose form, for example, in capsules or cartridges, for example, of gelatin, or in blister packs, from which the powder may be administered via an inhaler or insufflator.

If desired, the above-described formulations can be adapted to give a prolonged release form.

Pharmaceutical compositions containing the compounds of the present invention may also contain other active substances, such as antimicrobials or preservatives.

The compounds of the invention may also be used in combination with other therapeutic agents, e.g. other anti-infective agents. In particular, these compounds can be used together with known antiviral agents.

The above-mentioned combinations may conveniently be used in the form of a pharmaceutical which contains such combination together with a pharmaceutically acceptable carrier.

Suitable therapeutic agents that are suitable for use in such combinations include acyclic nucleosides such as acyclovir, interferons such as α-interferon, renal excretion inhibitors such as probenecid, nucleoside transport inhibitors such as dipyridamole, 2 *, 3'-dideoxynucleosides such as 2 ', 3'-dideoxycytidine, 2', 3 * -dideoxyadenosine, 2 ', 3'-dideoxyinosine, 2 *, 3 * -didaoxytimidine, and 2', 3'-dideoxy-2 ', 3 * -didehydrothymidine and immunomodulators such as interleukin II / IL2 / and granulocyte macrophage colony stimulating factor / GM-CSF /, erythropoietin and ampligen.

The individual components of such combinations may be administered either sequentially or simultaneously, in separate or combined pharmaceutical agents.

When a compound of Formula 1 or a pharmaceutically acceptable derivative thereof is used in combination with another therapeutic agent active against the same virus, the dose of each compound may differ from that when each compound is used alone. Appropriate dosages can be readily determined by one skilled in the art.

In the process according to the invention, it may be necessary or convenient to use starting compounds which have protected functional groups, and then deprotection of these groups may be an intermediate or final step in the synthesis of the desired compound.

The introduction of protecting groups and their removal can be accomplished by conventional methods. Thus, e.g., amino groups may be protected with a group selected from aralkyl groups (e.g. benzyl /, acyl or aryl / e.g. 2,4-dinitrophenyl), and these protecting groups can be removed by hydrolysis or hydrogenolysis under standard conditions if desired. Hydroxy groups can be protected using conventional protecting groups for hydroxyl groups, e.g., as described in Protective Groups in Organic CheιlasSΓy, Ed. 0. F. W. McOmie (Plenum Press, 1973) or in "Protective Groups in Organic Synthesis Theodora W. Greene (Oohn Wiley and Sons, 1981). Examples of protecting groups for hydroxyl groups may include groups selected from alkyl groups (e.g. methyl, tert-butyl or methoxymethyl / aralkyl / e.g. benzyl, diphenylmethyl or triphenylmethyl /, heterocyclic groups such as tetrahydropyranyl, acyl / e.g. acetyl or benzoyl / and silyl such as trialkylsilyl / e.g. tert-butyldimethylsilyl /. The protecting groups for hydroxyl groups can be removed by conventional methods. Thus, e.g. alkyl, silyl, acyl groups and heterocyclic groups can be removed by solvolysis, e.g. by hydrolysis under acidic or basic conditions.

163 814 data. Aralkyl groups such as triphenylmethyl groups may similarly be removed by solvolysis, e.g. by acid hydrolysis. Aralkyl groups such as benzyl can be cleaved by hydrogenolysis in the presence of a noble metal catalyst such as palladium on charcoal. Silyl groups can also be conveniently removed using a fluoride ion source such as tetra-n-butylammonium fluoride.

The starting materials for the preparation of the compound of formula 1 as defined above and their pharmaceutically acceptable derivatives can be prepared by reacting a compound of formula 2 wherein X is halogen, Z is as defined in formula 1 and Y is OH, optionally protected, with acid. formic acid or a reactive derivative thereof and then, if desired, removal of undesirable groups introduced by formic acid or a derivative thereof and / or removal of protecting groups.

Examples of suitable formic acid derivatives which can be used in the above method include the orthoformates / e.g. triethyl orthoformate /, dialkoxymethyl acetates / e.g. diethoxymethyl acetate /, dithioformic acid, formamide, s-triazine or formamidine acetate.

Unwanted groups introduced by formic acid or a reactive derivative thereof can typically be removed by mild hydrolysis, e.g., using an inorganic acid such as aqueous hydrochloric acid.

When a trialkyl orthoformate such as triethyl orthoformate is used, it is also a solvent. Other solvents that can be used include amides / e.g. dimethylformamide or dimethylacetamide /, chlorinated hydrocarbons / e.g. dichloromethane /, ethers / e.g. tetrahydrofuran / or nitriles / e.g. acetonitrile /.

In some cases / e.g. when a trialkyl orthoformate such as triethyl orthoformate is used, the reactions may preferably be carried out in the presence of a catalyst such as a strong acid / e.g. concentrated hydrochloric, nitric or sulfuric acid /. The reaction may be carried out at a temperature in the range of -25 ° to + 15O ° C, e.g. 0 ° to 100 ° C, and conveniently at room temperature.

In the process according to the invention, the compound of formula 10 in which x * represents a halogen atom, optionally in the form of protected functional groups, is replaced with other substituents and, if necessary, the protecting groups are removed.

Compounds of formula I in which X is NRR *, wherein R and R 1 are as defined above are prepared by amination of a corresponding compound of formula 10 in which X is halogen / e.g. chlorine /. The amination can be performed by reaction with an HNRR * reagent wherein R and R * are as defined above, preferably in a solvent such as an alcohol / e.g. methanol /. The reactions can be carried out at any suitable temperature, preferably at elevated temperatures such as reflux or, when liquid ammonia is used, in a closed tube at about 50 to 80 ° C. Suitable conditions for the conversion of halides to secondary and tertiary amines are described in 1.1. Harrison and, inter alia, Compendium of Organic Synthetic tiethods, Wiley-Interaciewcience. New York / 1971 / pp. 250-252.

According to the invention, compounds of formula I in which X is OR / R is as defined above / are prepared by replacing a halogen with the appropriate anionene RO ~. When R is hydrogen, the replacement reactions can be carried out by hydrolysis in water or in a mixture of water with a water-miscible solvent such as an alcohol / e.g. methanol or ethanol /, ether / e.g. dioxane or tetrahydrofuran / ketone / e.g. acetone /, amide / e.g. dimethylformamide / or sulfoxide / e.g. dimethylsulfoxide /, preferably in the presence of an acid or a base. Suitable acids include organic acids such as p-toluenesulfonic acid, and inorganic acids such as hydrochloric, nitric or sulfuric acid. Suitable bases include inorganic bases such as alkali metal hydroxides or carbonates / e.g. sodium or potassium hydroxide or charcoal /. Aqueous solutions of acids or bases can also be used as reaction solvents. The hydrolysis can preferably be carried out at a temperature in the range -10 ° to + 15O ° C, e.g. at reflux temperature. When R is a C 1-4 alkyl group or an aryl group, the RO anion is preferably formed from the corresponding ROH alcohol using an inorganic base such as an alkali metal (sodium metal) or an alkali metal hydride (e.g. sodium hydride /. The reaction with the in situ anion formed is preferably carried out at ambient temperature.

163 814

For the preparation of compounds of formula I in which X is SH, the compound of formula 10 in which X 'is halogen is reacted with a thiourea in a suitable solvent such as an alcohol / e.g. n-propanol / at elevated temperature / e.g. at boiling point / followed by alkaline hydrolysis. Suitable bases that can be used include alkali metal hydroxides / e.g. sodium hydroxide/. The reaction can preferably be carried out as described by G.G. Urguarta et al. in Org.Syn.Coli. Vol. 3, 363 (1953), e.g., by refluxing the intermediate with an aqueous NaOH solution for about 0.25 to about 5 hours.

For the preparation of compounds of Formula 1 where X is hydrogen, X 'is halogen is reduced using a reducing system that does not affect the rest of the bottle. Suitable reducing agents that can be used to effect the desired dechlorinated reaction include zinc metal and water according to the method described by 3.R. Marshall et al., 3. Chem. Soc., 1004 (1951). Alternatively, the reaction can be carried out through ^ ^ ^ ^ ^ in a suitable solvent such as tetrahydroonurane. in the method of V.Nair et al., □ .Org. 01-1601., 52, 1344 (1987), and preferably in the pho-amphase of Raakkorla Ryneta (25374).

Compounds of Entry 1 wherein X is an SR group in which R Olaαcα is a C1-4alkyl or aryl group, are prepared from the corresponding thiols by standard alkylation or arylating methods, for example as described in US Pat. No. 4,383,114.

Many of the more extinct parts of the world have been found synonymous with purine nuclosides, for example in Nucleoside Analkgs-Chemfskry. Biology and Nadical Applfcatfons. R .T. Walker 1 et al., Ed. Planum Plass, Nowy Oork / 1979 / sSó. 193-223.

The pharmaceutically acceptable salts of the compounds of the present invention can be prepared as described in US Pat. Thus, the free base product of any of the above-described processes is reacted in the form of the free base with the appropriate acid by conventional methods to form an acid addition salt. This reaction may be carried out in the presence of a suitable solvent such as aster / e.g. ethyl acetate / or alcohol / e.g. methanol, ethanol or IIkZtopanol /. A salt with an inorganic base can be prepared by reacting the free base with a suitable base such as an alkoxide / e.g. sodium methane / optionally in the presence of a solvent such as alcohol / e.g. mJkankl / · A pharmaceutically acceptable salt can also be prepared from other salts, including other pharmaceutically acceptable salts, compounds of application 1 using conventional methods.

A compound of formula 1 can be converted to a pharmaceutically acceptable phosphate or other ester by reaction with a phosphating agent such as POCl 4 or with a suitable esterifying agent such as an acid halide or anhydride, acidic acid. The ester or salt of a compound of subsume 1 can be converted to the parent compound e.g. by hrdtklflą.

Compounds of formula 2 in which z is a hydrogen αkk or a hydroxyl group and Y olnaala with an OH group can be prepared directly after a compound of formula 3 by reaction with an excess of pyrimidine of formula 4 where Y is a halogen atom, e.g. chlorine, and Z represents a hydrogen atom or a hydroxyl group in the presence of an amphacMjj base such as trfacrylate 1 in an alcoholic solvent, e.g. n-butanol, preferably at kJmetor boiling point.

Compounds of formula 2 in which Z is NO2 and Y olnaala is OH can be prepared from compounds of formula 3 by reaction with an excess of pyrimidine of formula 5 in which Y has the zaazeapha as defined for vloro 4 above under similar conditions to those described above in the method of preparing compounds of formula 2 in which Z is hydrogen or a hydroxyl group and dosing the resulting compound of formula 6 with the salt dfαzonicwjC ArN ₂ ⁺ F in which Aromacm with an aromatic group, e.g. and F-kasease anion, e.g. a halide such as chloride, in a solvent such as water, an organic acid such as acetic acid or a mixture thereof, preferably at ambient temperature, to produce a chlorine 7 compound where Ar is as defined above, which can be convert to the desired compound of formula 2 after reduction, using, for example, a reducing metal such as

Zinc in the presence of an acid, e.g. acetic. Of course, the choice of the reducing agent will depend on the nature of the X group.

The compound of formula 3 can be prepared from the universal precursor 1-acetylamino-3 O-acetoxy-methylcyclopent-2-ene of formula 8 by hydrolysis in the presence of a mild base such as an alkaline earth metal hydroxide.

A particularly preferred synthesis of a compound of formula 1 by the 6-chloro compound of formula 2 is shown in the figure in the scheme. The compound of formula 8 is a known compound described in the US patent specification. No. 4,138,562. A compound of Formula 1 as a single isomer can be obtained either by cleavage of the final product or by stereospecific synthesis from the isomerically pure starting compound or any suitable intermediate.

The cleavage of the final product or preparation of a suitable intermediate or starting material can be carried out by any suitable method known in the art, see, e.g., Stereocnemistry of Carbon Compounds E.I. Eliel / McGraw Hill, 1962 / and Tables of Resolving Agent's S.H. Wilan.

One convenient method of making chirally pure compounds of Formula 1 is the enzymatic conversion of a racemic mixture of the compound or a precursor thereof. Both the / + / and / - / compounds in optically pure form can be obtained by this method. Suitable enzymes include deaminases such as adenosine deaminase. Tests confirming the antiviral activity of the compounds according to the invention are presented below.

Antiviral activity / A / Assay for anti-HIy activity

Compounds of formula I were screened for anti-HIV activity at the National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland (FCRF). The following generally accepted practical screening procedures were used in FCRF. The assay consists of 3 steps: / I / preparation of infected cells and their spreading on test plates, / II / preparation of drug dissection plates and spreading on test plates, and / III / XTT test procedure, see D.A. Scudiero et al. "A New Simplnfiad Tetrazolium Assay for Cell Growth and Drug Sensntvity in Culture Cancer Res., 48, 4827/1988 /.

I Infection and spreading of ATH8 cells in microtiter trays

Cells to be infected / normal CD4-expressing lymphoblastoid cell line / are placed in 50 ml conical centrifuge tubes and within

1 hour is treated with 1-2 µg / ml of polyrene at 37 ° C. The cells are then harvested for min at 1200 revolutions / minute. HIV diluted 1:10 in medium (RMPl-1640, 10% human serum or 15% fetal calf serum (FCS) with IL-2 and antibiotics is added to provide 0.001 MOI. Medium itself is added to virus-free control cells. Assuming the infecting virus titer is 10-a, the MOI of 0.001 is 8 viral contamination particles per 10,000 cells. About 500,000 cells / tube are exposed to 400 µL of virus. The resulting mixture is incubated for 1 hour. at 37 ° C in an air-COg atmosphere. Infected and uninfected cells are diluted to obtain 1 x 10 / ⁴ / ₂ human serum / or 2 x 10 ~4 / with fetal calf serum / cells / 100, ul.

Infected or uninfected cells (100 µl) are plated into the appropriate wells in a U-bottomed 96-well microtiter plate. Each compound dilution is tested in duplicate with infected cells. Uninfected cells are tested for drug per well for each compound dilution. Triplicate studies are performed for drug-free control cells, infected and uninfected cells. Wells B2 through G2 served as controls and contained only substrate. The plates are incubated at 37 ° C in an air-COg atmosphere until drug addition.

II Drug dilution and addition

Dilution plates / 96-well multiplication plates with flat bottom / Treats

Overnight in phosphate buffered saline or medium containing at least 10 FCS or 1% human serum (depending on the vehicle used in the test), starting the day before the test. This "blocking procedure" is used to minimize drug adsorption through the microtiter tray during the dilution process. The wells are completely filled with blocking solution and allowed to stand at room temperature in a fume cupboard humidifier.

The dilution process begins with the first 1:20 dilution of the test compound. Blocked Dilution Plates are prepared by removing the blocking solution and drying the block in sterile gauze. Then all wells in each plate are filled with 225 µl of the appropriate medium using the manipulation system and the filled dilution plate is manually added with 25 µl of each compound at a 1-20 dilution. Four compounds sufficient for two assay plates are added to one dilution plate. The four compounds are then serially diluted 10 times from row A to row H using the Cetus liquid handling system. The starting dilution of each compound in the row is now 1: 200. The dilution plates are kept on ice until use. Using a multichannel pipette filler with 6 microtips, 100 µl of each drug dilution is transferred to the assay plate, which already contains 100 µl of medium + cells. The final dilution, on the assay plate, starts at 1: 400 (wells B4 to G4). This dilution (to 0.25% of OMSO) prevents the OMSO carrier from interfering with cell growth of drug-free, infected or uninfected cells (wells B3 through G3) and controls (BB through G2) will be obtained from the same maeiuu. I transfer the final axis ppzzenss from H7 to Hl2 onto a second test plate using this deer procedure. The test plates are kept at 37 ° C in a poeιatzka-CO 2 atmosphere for 7-14 Iii or until lysis of the in vitro viruses, take makzosOnDnwo.

III. Quantitative determination of ^^ viral pathogenicity and drug activity

A. Materials

1. Hydroxide solution /, 3-bιs [/ - meto0ay-4-nltzo-5-aulfnfanmnw7-5- / ^- (fenmloauιloBcarbonmlo7 “2H-tatzazonl. / XTTB-equiv. 1 mg / ml in FCS-free medium. Stored at 4 ° C C. Prepared every week.

2. Fenakynm metosulfnliate stock solution (PMS). It can be prepared and kept frozen until use at -2O ° C. It should be prepared in PBS and have a concentration of 15.3 mg / ml.

B. Attempting tatraznllnea z mikroculture / MTA /

1. Preparation of the XTT-PMS solution. It is prepared just before adding it to the wells of the culture vessel. The PMS stock solution is diluted 1: 100 (0.153 mg / ml). The diluted PMS is added to each .ιΙΙΙιϊ ^ XTT needed to obtain a coCcoee PMS concentration of 0.02 mM. An aliquot of 50 µl of the XTT-PMS mixture is added to each of the other wells and the plate is washed for 4 hours at 37 ° C. The lids of the plates are removed and the BDmnatech cat 001-010-3501 / adhesive closure plate is replaced. The closed plate is shaken in a microculture plate mixer and determined for abaorbance at 450 nm.

IV. Results

The data obtained are plotted as the percentage of test cells converted to nlesa-aola cells for both infected and mea-infected cells with functions of growing at the same time of the test compound. Such graphs allow the calculation of the effective concentration / ECg / for llezaOzone cells, the inhibitory concentration / ICgg / for z0zwwmch cells and the therapeutic index / Tlgg /

The inhibitory concentrations in µgBml against HIV determined as described above for the compounds of Examples VII, IX, X, XI and XIVBbB are shown in Table 1.

163 814

Table 1 Relationship Example AND 1 1 1 1 Cell line [ ^ED 50 1 1 1 1 ^ID S0 and TI ₅ O I 9a VII 1 ! MT-2 ' 2.3 and ! 50 ! 21.4 13a IX 1 l | MT-2 1 0.41 1 | 6.97 '1 17.3 l4a 1 X 1 AND MT-2 j 0.15 1 1 100 [667 15a 1 ! XI vol ! MT-2! 2.9 1 ! > 125 '> 42.7 • / - / 148 ! XIV / b / 1 1 1 CEM J > 0.66 1 1 1 189 J 284 1

The compounds of Examples 5 and 8 also showed anti-viral activity in this screening.

An earlier trial with the compound of Example 10 at the Southern Research Institute had a TI50 value of about 200 when MT-2 cells were cultured with H9 / HILV-IIIB.

/ B / Activity against feline leukemia virus

Antiviral selection for activity against FeLV-FAIDS was performed in 96-well plates (Corning) using 81C indicator cells in Dulbecco's modified medium

Iscove supplemented with 10% heat-dactivated fetal bovine serum (FBS). For 24 hours 3 before assay, 81C cells were plated at 5x106 cells / well. On the day of the test, cells were pretreated for 30 minutes at 37 ° C with DEAE-dextran (25 µg / ml) in 0.1 ml saline equilibrated in Hank's solution. Usuniąto it and added to each well nastąpnie 0.1 ml of growth medium containing _32/0 TCIDg FeLV-FAIOS or 0.1 ml of the same medium. After being left for 1 hour to adsorb the virus, 0.1 ml of test compound or positive control compound (2 ', 3 * -dxdeoxycytidine, ddc / or growth medium was added). The plates were incubated at 37 ° C. Cells were supplied with fresh growth medium containing compound, on day 4 after infection, on day 7 after infection, the culture medium was completely changed and replaced with fresh? On day 10 after infection, cells were formalin-fixed, stained with 0.1% Coomassia brilliant blue R -25O and observed macroscopically on CPE and drug cytotoxicity.

The compound of Example 10 had an ED value of 1.9 g / ml / C / Activity against murine AIDS

1.75χ1ϋ5 cells per well were seeded in 6-well Falcon tissue culture plates in a total volume of 2.5 ml EMEN containing 5% heat-inactivated FBS. 24 hours after inoculating the cells, the medium was decanted and 2.5 ml of DEAE-dextran (25 µg / ml in phosphate buffered saline) was added to each well. The cultures were incubated at 37 ° C for 1 hour after which the DEAE-dextren solution was decanted and the cell layers were washed once with 2.5 ml PBS. 2.5 ml of medium alone (no virus and no drug) was added to normal control cells. Drug control cultures received 2.5 ml of medium containing drug but no virus. Virus-infected control cultures received 0.5 ml of the appropriate CAS-BR-M stock dilution to produce countable plaques and 2.0 ml of medium. The test samples received 0.5 ml of the appropriate virus dilution and 2.0 ml of vehicle with the drug dilution. Six concentrations of the test compound diluted over a series of half-log µg dilutions were tested. Three concentrations of the positive control drug, ddC, were tested. 3 wells for each concentration of the test compound and 6 viruses and 6 cultures of control cells were used in each trial. On the 3rd day after virus inoculation, drug toxicity to 5C-1 cells was determined by microscopic double examination of staining cells and drug control cultures.

The remaining control cultures and test cultures were irradiated with an ultraviolet lamp for 20 c seconds and XC cells (5x10 cells / well in 2.5 ml EMEM containing 10% heat-reactivated FBS) were added to each culture. On day 3 after UV irradiation, the cultures were fixed in formalin and stained with crystal violet. The plaques were counted using a slide microscope.

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Antimicrobial activity manifested by reduction in the number of plaques CAS-BR-M was expressed as the mean number of plaques counted in drug-treated virus-infected cultures, compared to the mean number of plaques counted in untreated virus-infected cultures (% control).

The compound of Example 10 had an ED 50 value = 1.1 µg / ml.

/ D / Activity against Simian SA10S / SRV-2 retrovirus /

Antiviral testing against SAIOS virus (D-Washington) was performed in an anastomosis-inhibition assay on Raji cells. Drug was diluted in complete Iscoye® medium and 100 µl of each dilution was added to the appropriate wells of a 96-well plate. Then 50 µl of clavnwneas sepnanatauau was added and in common Oowiilk mornerek VV / 2 / bnji, DDC was used as a positive control drug in this case. The tiles are primed at 37 ° C in a humid atmosphere containing 5% CO2. Salt syndrome on the 7th day after infection. Drug efficacy was assessed by comparing the number of viable cells in a drug-treated laud sample with the viability of a neet-infected control sample. The compound of Example X had a ED50 value of 2.8 ^m l.

/ E / Activity against the virus of wisny naeOi sheep

The viral viral activity of sheep maedi virus (VMV), strain WCL-1, was determined by measuring the reduction of virus specific inmunahnsnacnemicunega staining. The sheep choroid plexus cells were infected with VMV and covered with a series of dilutions of the test compounds. After incubation for 5 days, manowα prnwy nnwas further bought with antisera specific for the virus, prestressed with horseradish seraxidase / HRP /. Subsequent incubation of the monovarils with the cnpine-genic medium of HRP distorts the viral replication regions. These isolated foci were counted and the concentration of the test compound was calculated, which is needed to unnnajruaniα the number of foci to 50% of their number in the lacquer-treated controls.

The compound of Example 13 had an ED value of 0.2 µg / ml

The following examples illustrate the process of the invention. The elemental analysis shown in the example was performed at M-H-W Laboratories, Pnoennx, AZ. Melting dams were determined on a Mel-Temp apparatus and corrected values are reported. Nuclear magnetic resonance spectra were obtained on Jsol FX 90QFT or Nicol ^ t NT3OO spectrometers and determined in OMSO-Dg. Chemical shifts are expressed in ppm relative to Me ^ Si. Infrared spectra were obtained by examining KBr tablets with the memory of the N4 ollat 5OXC FT-IR spectrometer, and ultraviolet spectra were determined using a Beckmann DU-b spectrophotometer. Mass spectra were obtained using an AFI kcnettnfnc Apsαrαtur Lnnitad MS-3O mass spectrometer. Thin layer chromatography was carried out on 0.25 mm layers of Merck silica gel (23O-4OO ιοο). All compounds and solvents used are analytical reagents, unless otherwise stated. By the term active ingredient, as used in the Examples, it is meant the compound of Formula 1 or a pharmaceutically acceptable derivative thereof.

Example I. / + / - / 1 O, 4 oC / ^ - // b-amno-6-cnlarO4Snpymn0ynylaαnnno (2-cyclapetethylmethanol / formula 2a). .

A mixture of 3.0 g (15 mmol) of 1 06 -acetyl-acyclapent-2-ene (formula 8) and 0.5 N aqueous barium hydroxide (3OO ml) was heated to reflux overnight. . After cooling with dry ice, the mixture was neutralized. The resulting precipitate was filtered off and the aqueous solution was concentrated to dryness. The residue was extracted with absolute ethanol and concentrated again to give the compound of Formula 3 as a colorless syrup at 1.6 g / 14 mmol / L.

To this syrup was added 4.59 g (28 mmol) of 5-amita-4,6-0chloPosiryndyπy, 4.2 g (42 mol) of thienylamine and 50 ml of n-butanol and the mixture was heated to reflux for 24 hours. Volatile solvents were removed and the residue absorbed onto 7 g of silica gel in a flash chromatography column (4.20 x 12 cm) and eluted with CHCl2-MeCH (2Osl) to yield 2.69 g (74%) of the compound of formula 2a and melting point 13 ° -132 ° C. An analytical sample was obtained by recrystallization from ethyl acetate (EtOAc), m.p.

163 814

134-135 ° C, MS / 3O ev, 2OO ° C / IU / e 240 and 242 / M ⁺ 1 M ⁺ + 2 /, 209 / M * 31 /, 144 / B ⁺ / l

IR: 36OO-26OO (OH), 1620, 1580 (CC, C = a / j Analysis (C _in H ₁₁ ClN ₄ O) (C, H, N.

Example II. / + / - / 1 oC, 4 oC B-4 - / "/ 2-auino-6-chloro-4-plasmidinyl / -amlno J -2-cyOloDaltan mlouatanol / formula 6a /

Oo 14 14ζ .. n and a slushy head on / decoupled from SkzyrkmO- d ZoZaoo ^ / 4 7 /2/.2, ι8ζ ../ l- / mino-4.6-dlozoplrimldine, 15 ml of triethylamine and 75 ml of n-butanol and the mixture was heated to reflux for 48 hours. Volatile solvents were removed, the residue was treated with methanol for the purpose of a double nulliprimidine side effect. The methanol solution was absorbed onto silica gel (8 g) in a column (4.20 x 14 cm) and enuivaled in the CHCl -MeOH system (40: 1) to give 1.52 g of the crude compound of formula 6a (42%). The product was extracted from ethyl acetate to give the compound of formula 6a, m.p. 132-134 ° MS (3O ev, 200 ° CB) m / e 240 1-242 (M + and M + + 2), 209 (M + -31).

144 (B + / 1 IR: 36OO-3OOO (NH 4 OH), 1620, 1580 / C "C, C = Nj Analysis / Cχc ^h 13 4 / C, H, N.

Example III. / - / - / l OĆ, 4oB / 4- / aU / l-amino-Wzlhl-r4-5- / Ozohelrnfθnylo / -azo J -4-pιryuι0ynylo-amιno / -2-cyclopθntθnylouetanol / pattern 7a /

Cold solution of the diazonium salt was prepared from 1.47 g (11.5 mmol) of p-chlorwallllly in 3N HCl (25 ml) and 870 mg (12.5 mmol) of sodium nitrite in 10 ml of water. This solution was added to a mixture of 2.40 g (10 mmol) of the compound of formula VIa, 50 ml of acetic acid, 50 ml of water and 20 g of sodium acetate trihydrate. The reaction mixture was stirred overnight at room temperature. The yellow precipitate was filtered off and washed with cold water until neutralized until neutralized, then air dried in a fume hood to give 3.60 g (94%) of wwwzza 7a with a melting point of 229 ° C. An analytical sample was obtained from the acetone-methanol system (1: 2), m.p. 241-243 ° C, composition B. MS / 30 ev, 26O [deg.] C / µm / e 378 and 380 [mu] ^{M +} i

M + ♦ 2 /, 282 / B + / t IR: 36OO-3OOO / NH ^ OH /, 1620, 1580 / C = C, C = N / L Analysis / C ₁₆ Hj ^ Cl2NgO /

C, Η, N.

Example IV. d oU | n0-6 ”/ 2,0zW-4mlιrmmιomlmlOB - plrymldynyio / -amlno_7-2-cmOInpentelmloueralol / pattern 2b /

With a mixture of 379 mg of the compound of formula 7a (I uuoI), 0.65 g (100%) of zinc dust,

0.32 mL of acetic acid, 15 mL of water and 15 mL of ethanol was heated to reflux under nitrogen for 3 hours. The zinc was removed, and the solvents were evaporated. The posoethase was absorbed onto 2 g of krcamlonowwago gel in a column (2.20 x 18 cm) and I had 1 1 Oem CHCl2 -MeOH / 15: 1 /. A pink syrup was obtained. After further purification from the methanol-atar system, the compound of formula 2b was obtained as pink crystals at 170 mg (66%), m.p. 168-170 ° C, MS (3Oev, (2O ° C), m / e 255 and 257 / M + and M + + 2 /, 224 / M + "31 /, 159 / B + / I IR: 36OO-3OOO / NH2, OH / 1620,1580 / C = C, C = N / and Analysis / CjoH ^ ClNg / C, H, N.

Example V. / - / - / I oC, 4 in £ / -4-B6-chloro-9H-DJZyn-9-mln / - / - cyclDaltenmlnuatanol

A mixture of 1.30 g (5.4 mmol) of a 2a ecose compound, 30 ml of methyl ortho-mutate and 0.50 ml of 12N hydrochloric acid was stirred overnight at the lower temperature. The solvent was evaporated under vacuum at 35 ° C. 30 ml of 0.5 N hydrochloric acid solution was added and mixed for 1 hour, then it was mixed to pH 7-8 with IN EVO and absorbed on 8 g of gel in the column (4, O x 8 cm), followed by aluoeal in Jk-aOem CHCl6-MeOH // OιlB yielding 1.12 g (82%) of white crystals of the title compound. The crude product was crystallized from ethyl acetate to give the pure compound, m.p. 108-110 ° C, MS (3O ev, MO 2 (1m) 250 and 252 (M + and M + + 2), 219 (M + +, "31), 154 (B +) (and IR? 3600-2800 (OH), 1600 (C = C, C ° N) Anallza / CllHą CLN _{1 4} 0 / C, H, N.

Example VI. / - / - / I oC, 4 «C B-4-B6-hy0rwkay-9H-Durmn-9-yloB-2“ Cy0lwDentalmlvuethanwl

A mixture of 251 mg / 1 mmol / of the compound with prkmk-aOu V 1 10 ml of a 0.2 N aqueous solution of sodium indoxole was heated to reflux for 3 hours. After degoldenl with a mixture of water additive to pH 5-6 with hydrochloric acid. The reaction mixture was absorbed onto a 2 g OrwaulolOowago gel in a column (2.0 x 11 cm) and a small amount of CHCl 3 -MeOH (10: 1) to give 105 mg (4S) of the title compound. The crude white product was crystallized from water-uatal in 1B 3: 1B and obtained

Title compound with decomposition melting point 248-25O ° C, MS / 3O ev, 3OO ° C / j, m / e 232 / M * /, 214 / M * 18 /, 136 / B * / j IR: 36OO-26OO / OH /, 1680, 1600 / C »O, C = C, CN / j Analysis: / c ₁₁ h ₁₂ n ₄ ° ₂ / C * _H * N.

Example VII. / - / - /! oC .4 oć / -4t / 6-amfnot90tZurrat9-ylo / -2-crklkpankanylomat anol

A solution of 250 mg / L of the title compound from Example 5 in 5 ml of methanol was passed through the lαwiJtα bomb at -8O ° C. Liquid zmoniαk · The bomb was sealed and heated at 6O ° C for 24 hours. The ammonia and methanol were evaporated and the total was recrystallized from water to give white cloudy crystals of the title compound, kampJrnkurla 198-2OO ° C, in an amount of 187 mg (81%).

MS (30 av, 21O [deg.] C (µm) and 231 (M *). 213 / M * "18 /, 135 / B * / j IR: 36OO-26OO (NH ₂ , OH), 1700, 1600 / C = C, C = N / j Analysis / CnH ^ N ^ / C, H, N.

Example VIII. / - / - / 1 zC, 4 o // - 4- / b-merkBpt / -OH-ptιryn-9-ylo / -2-cyclopenten yioyekαnol

A mixture of 125 mg / 0.15 µl / the title compound from Example 5, 40 mg / 0.14 mmol / siomkclaik and 5 ml n-propanol was heated to reflux for 2 hours. After kzfabienfu, the precipitate formed was separated by filtration, washed with n-propanol and dissolved in 5 ml of 1N sodium hydroxide. The solution was adjusted to pH 5 with acetic acid. The crude title compound at 90 mg (73%) was re-isolated (mp 26O-262 ° C with decomposition) and converted with N, N (dfmatrlknkrmαmfdu) to give a compound with mp 263-265 ° C with decomposition. MS / 3O ev, 29O ° C / j u / j 248 / M * /, 230 / M * 18 /, 152 / B * / j IR: 36OO-32OO / OH /, 3100,

2400 / SH /, 16OO / C = C, C = N / j Analysis / C ^ C ^ I ^ OS / C, H, N.

Example IX. / ”/ - / 1 cCC.4 oC // 4- / 2 / amino-6-aalkrk / 9H / zurrn-ylk // 2-cyclopantJ / aylomasankl

The mfaelαaphine of the title compound of Example 4 was stirred overnight in an amount of 1.41 H (5.5 ιιτι), 30 ml krsk / formate and 1.40 ml 12N hydrochloric acid. The slurry was dried in vacuo. 40 ml of 0.5 N dilute hydrochloric acid were added and the mixture was added to ZtlarJzgkwαnfα at room temperature for 1 hour. The mixture was neutralized to pH 8 with 1 N sodium hydroxide and absorbed onto 7.5 g of silica gel in a column (4.0 x x 10 cm), then eluted with CHClg-MeDH (2O: 1) to give off-white crystals of the title compound in the amount of 1 , 18 g (SO%). The crude product was crystallized from ethanol to give the compound, m.p. 145-147 ° C. MS (30 ev, 22O [deg.] C ([mu] m) 265 and 267 (M * and M *>), 235 (M * 30), 169 (B *); IR: 36OO-26OO (NH ₂ , OH), 1620-1580 (C = C, C = N) Analysis (C, O, I, OC, H 2 O / C, H, N.

Example X. / - / - / I oR, 4 C // 4- / 2 / αmino / 6-hrdtkksr-9H / purrn-9-rlo / -2-cycloper: tany lornet ano 1

A mixture of 266 mg / I mmol / of the title compound from Example 9 and 0.33 N aqueous sodium hydroxide solution was heated to reflux for 5 hours, then absorbed onto 2 g of silica gel in a column (2.0 x 7.5 cm) and alucMano CHClH "MaOH / 5: 1 /. The crude product was crystallized from methanol-water (1: 4) to give 152 mg (61%) of white crystals of the title compound, mp 254-256 ° C (decomposition). MS (30 ev, 200 ° C) m / j 247 (M *). 217 / M * ^_ 30 /, 151 / B * / and IR: 36OO-26OO (NH2 »OH), 1700, 16OO, / C = O, C = C, C = N /; Analysis / CuH N 3/4 HgO / C, H, N.

Example XI. / - / - / i C, 4C //4-/2.6-dizmfno-9H/puryn-9-rlk/-2/crklopenseny/ lomeSanol

Liquid ammonia was introduced into a solution of 265 mg / I mmol / of the title compound of Example 9 in 10 ml of methanol at -8O ° C in a bomb. The bomb was sealed and heated at 75 ° C for 48 hours. Ammonia and methanol were evaporated. The residue was absorbed onto 2 g of silica gel in a column (2.0 x 10 cm) and CHClH "MaOH eluent / 15: 1). The crude product was recrystallized from ethanol to give 196 mg (80%) of the title compound, mp 152 "155 ° C. MS / 3O ev, 2OO ° C / sm / e 246 / M * /, 229 / M * ~ 17 /, 216 / M * ~ 3O /, 150 / B * / t IR: 36OO-3OOO / NH ₂ , OH /, 1700, 1650, 1600 / C = O, C = C, C = N / | Analysis / C 1 H 2 N 6 O / C, H, N.

Example XII. /1S,4R/-4//2.6/dfαyino-9H-zuryn [9-ylo//2/cyklkZentenyl/ methanol 1b3 814 [~ / 1S, 4R / -4- / 2,6-diamino-9H-purine- 9-yl (-2-cyclopentenomethanol) 7 / a) Intermediate 1j (1H, 2S, 3R, 5R) -3 - "6-amino-9H-purin-9-yl-7-5 - (1,1-dimethylethyl) - dimethylsilyloxy (methyl 7-1,2-cyclopanthanediol)

12.505 g / - / aristeromycin / Journal ot the American Chemical Society 1983, vol. 105, 4049-4055 /, 7.8 g of tert-butyldimethylalyl chloride and 12.96 g of imidazole in 85 ml of dry dimethylformamide were stirred for 2.5 hours in ambient temperature. The resulting solution was diluted with 5OO mL of ethyl acetate, then washed with 3 x 100 mL of water and 50 mL of brine. The crystallized white crystals were collected by filtration, washed with ethyl acetate and then dried in vacuo to give 3.92 g of the title compound; * H NMR (DMSO-d6) (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 (2HH).

2.23 (1H), 2.09 (1.79) (1H), 0.89 (9H). O0D7 / 6HH.

/ b / Intermediate 2: / 4R, 3aS, 6R, 6aR / -4- ~ 6-amino-9H-purin-9-yl-7-6 - / 1,1-dimethylethyl) -dimethylsilylk9y / methyl_7-3a, 5 , 6,6a-tet rahydro-4H-cyclopenta-1,3-dioxole-2-thion

3.3 g of 2I, C * -teocarbonyldllmldazmCu were added to 15 ml of eimβtelttormamldu. After 15.5 hours at ambient temperature, the resulting solution was combined with the solution from the previous experiment (S weight) and the solvent was removed by evaporation. The residual oil was diluted with 100 mL of ethyl acetate then washed with 2 x 20 mL of water and 2 x 20 mL of brine, dried over MgSO4 and evaporated to a yellow solid. It was washed with 25 ml of dimethyl ether, then mellted, and washed with 25 ml of ether and dried in vacuo to give 3.61 g of the title compound as a pale black solid.

λ max / ethanol / 240.0 nm / eH ⁴ 59 / ί ^{1r NM} ^ / DtOO-d ^ / 8.27 / 1/1, .1, (CH). Ρ, 3, / CH /,

5.81 (H), 5.37 (1H), 5.28 (1), 3.78 (2H), 1.6, (1H), 2.28 (iZ), 0.90 (9H) , 0.09 (6H).

/ c / Intermediate 3: / 1 * R, 4 * S / -9 - / "4 - /// 1,1, -dimethylatyl / dimethylsilyloxy / methyl / -2-cyclopenten-1-yl _7-9H-purine- 6- a.m. on

A solution of 3.57 g of Intermediate 2 in 25 mL of dry tetrahydrofuran was mixed with the solution

4.94 g of C, 3-UeuetyCo-H-phenyCo-e, 3, H-deazofoafoCedin in 10 ml of dry tetrahydrofuran, followed by stirring at ambient temperature for 8 3/4 hours. The solvent was removed by evaporation. The residual oil was combined with that obtained from the previous experiment (40% by weight), and then subjected to a silica column (200 g, Merck 7734), and chloroform-ethanol mixture, followed by a white solid, washed with 25 ml of Ueethyl ether, then collected by filtration, further washed with ether, then dried in vacuo to give 1.47 g of the title compound;

(ethanol / 2bl, 4 nm / Εχ ,,,, 443); H NMR (DMSO-d6) (8.14 21/2.8.00 2 HH), 7.20 µTH (f b, 12 (1H),

5.95 6, (H, J, H), 66 H, (2, 26, 96,), 1, H, H, 04 7 (H / H), 02 H / H).

/ d / Intermediate 4: 2l * R »4 * S / -2- / ~ 4-222e, C-UimetyCoetyeo / UimetyCmaelilokay / metyCm2-2-cycCm ^ nten ^ -yl 0-9H-purinm-b-yuenej l- oxide

A solution of 1.37 g of Intermediate 3 at 30 ° C of chloroform was treated with 10-910ι; m-chlormnabenzoic acid (1.29 g) and then stirred at ambient temperature for 3 hours. The solvent was removed by evaporation and it remained? the resin was dissolved in 10 ml of ethyl acetate. A white solid was crystallized, which, together with the substance obtained by evaporating the filtrate, was dissolved in 100 ml of chloroform, then washed with 3 x 10 ml of a saturated aqueous solution of sodium carbonate and 2 x 10 µC of brine. The aqueous washes were extracted with 50 mL of chloroform. The combined organic solutions were dried over MgSO 4 then evaporated to a solid. The solid was washed with 25 ml of Ueethylmwegm ether then filtered off. The white solid was further washed with 10 ml of ether and then dried in vacuo to yield 1.1H g of the title compound: λ max (ethanol) 235.4 nm (eH, 1324), 263.2 nm (Eg, 248), 300 , 2 nm (eUc 75);

1 H NMR (CDCl ₃ (8.72 (1H2, 8.02 (1H)), 7.16 (1H), 6.21 (W, 5.87 (1H), 5.72 21H), 3.68 (HH), 3.04 (21H), 2.82 (21H), 1.74 (1H), 0.89 29H2. O.06 (HH).

/ e / Intermediate 5: Brmmohydmrek / 1 * R, 4'S / -0 - "- 2 // c0, ldelyttcmyttcl2 / dlmθtylo8ilyloxyZmethyl / -2-cyclopintin-C-ylm _7-2-emeno-1.2-d1 / ydro- ₍ ^, 2,4_7okaadiazolm [/. 2, -e_7-2H-pwryn

An ice-cooled suspension of 1.08 g of Intermediate 4 in 20 mL of methanol, while stirring, was charged with a solution of 0.34 g of cyanogen bromide in 20 mL of methanol which was fed over 5 minutes. After allowing the slurry to warm to ambient temperature for 15 minutes, a solution was obtained. After 9O minutes, the solvent was removed by evaporation. The residue was washed with 25 ml of diethyl ether then collected by filtration. The collected solid was washed with a further 25 ° C of eerTu, with the amount of increased difference and the reduction of 1.7Z in the taper bond: λ / ethanol / 228.2 nm / E1% J _m 530 /, 285.2 nm / Ε ^ 445 (1 H NMR (CDCl 3) (10.20 (1H), 10.02 (1H), 8.37 (1H), 6.25 (1H), 6.01 (1H), 3.69 (2H), 3 .05 (lH), 2.86 (lH), 1.73 (lH), 0.86 (9H), 0.03 / 6H / / f / Intermediate 6: / l'R, 4'S / -9- [ "4 - /// 1,1-dimethylethyl / dimethylsilyloxy / methyl / -2-cyclopenten-1-yl) - 5-cyαnolmino-1,6-dlhydzo-O-matoxy-9H-pszyyα

A solution of Intermediate 5 (1.36 g / in 10 L of dimethylformamide) was stirred at ambient temperature then treated with 1.2 ol of triethyl3.8 g. After 40 minutes, 0.54 ol iodine methyl was added to give a yellow solution. After 3 3/4 hours the solvent was removed by evaporation. The residue was partitioned between 100 ol ethyl acetate and 20 ol water. The organic solution was further washed with 2 x 20 ml of water and 20 ml of brine, dried over MgSO ₄ and evaporated to a solid. The solid was washed with 25 ml of diethyl ether and filtered off. They were washed further with 10 ols of ether and then dried under vacuum to give 0.865 g of the title compound: -Amaw / ethanol / 227.2 nm, c '10/1 max / E1co ⁴⁴ 9 /, ²⁸ 7.0 nm / ^Ε 1 £ ο ⁵⁴⁴ (ίH 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 7: / 1'R, 4'S / -9- ^ ~ 4 - /// 1,1-diinethylethyl / dimethylsilylk8y / methyl / -2-cyclopenten-1-yl [7-6-methoxyamino-9H-purine -2-amine

A solution of 802 mg of Intermediate 6 and 0.45 ml of 1,8-diazabicyclo (5.4.0) undecetene in 80 ml of ethanol was stirred and heated to reflux. After 9 hours, heating was stopped and the solution was allowed to stand overnight at ambient temperature. The solvent was removed by evaporation. The residual oil was combined with that obtained in the previous experiment (4% by weight), then column chromatographed on 40 g of silica (Merck 9385), eluted with chloroform and then with a mixture of chloroform and ethanol to give a foam. The foam was triturated with 10 ol of diethyl ether and the resulting solid was collected by filtration. They were washed with 5 ml of ether and then dried in vacuo to give 594 mc of the title compound: na / ethanol / 282.2 nm / ^E 1 (1c 4 ° 9) .NMR / DMSO-d 6 (9.76 / 1H), 7.7. 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 a: / lS, 4R / -4 - ["2-amino-6-methoxyamino-9H-chin-9-yln / - 2-cyclopentenemethanol

A solution of 356 mg of intermediate 7 in 35 ml of tetzahydrofurays was stirred at room temperature and then quenched with 1.4 ol of 1.0 M tetrabstylamine fluoride solution in tetrαhydrate. After 90 minutes the reaction was quenched suddenly by adding 1 ml of water and then the solvents were removed by evaporation. The remaining oil was subjected to a column of 20 g of silica (Merck 7734), eluted with chloroform, followed by a mixture of chloroform and ethanol and the title as a solid in the amount of 243 mg JA oax / pH 6 buffer / «• C / τ n» ex

280.2nm (Ej ^ ³⁴ ); NMR (DMSO-d6 (S, 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-diemino-9H-purin-9-yl) - 2-cyclopentenemethanol

Aluminum amalgam (from 237 g of aluminum and 0.5% aqueous mercury chloride solution) was added in small pieces for 15 minutes to an ice-cooled solution of 210 g of intermediate in 10 ml of water and 50 ml of tetzahydrofshan with stirring. After 40 minutes, the stirred miaucayin was allowed to warm to ambient temperature. After 15 hours, the resulting mixture was filtered through diatomaceous earth to remove insoluble matter. It was washed with a mixture of water: tetzahydroOuran / 1: 5, 60 ol /. The combined filtrates were evaporated. The residue was subjected to silica column chromatography (10 g, Merck 9385) and eluted with chloroform-ethanol mixtures to give the title compound as a foam, 159 mg.

-81 ° / cO, 04, methanol / j / L oax ^{/ p} H 6 buffer / 255.9 nm / ε | * _η 302 /, 280.8 nm, / eOco 381 /. * H NMR? DMSO-d 6 (7.61 (1H), 6.66 (2H), 6.10 (1H), 5.87 (1H), 5.76 (2H), 5.38 (1H), 4.76 (1H), 3.45 (2H), 2.87 (1H), 2.60 (1H), 1.60 (1H).

lb

163 814

Example XIII. (IS, 4R) -4- (2-amino-6-hydroxy-9H-pupyn-9-yl) -2-cyclopenWenylmethanol / 1 'R, 4 ^, S / -2-amino-S, 9-dlHydrs-9 - / ^_ 4-hcdrsksymewyls-2-eyklspenten-l-ylo_7-6H-pu rin-6-one

A cloudy solution of 144 mg of the title compound from Example 12 in 10 ml of 0.1 M buffer at pH 6 / with 28.4 g of disodium phosphate in 2 liters of water, saturated with orthophosphoric acid / treated with 0.5 ml of adenosine dβaminarc solution / 778 units / in a mixture of 50% glycerin - 0.11 M potassium phosphate, pH 6.0, followed by stirring and heating to 37 ° C. Ps 18.5 hours yypyopzsną znwiepnyę frozen. Zsbrαyz the solid was crystallized from water to give the title compound as a white solid in the amount of 86 mg, / ο £ _ / θ-49 ° / £ 0.5, dimethylxulfctilizek7ί Λ max / ^pH 6 buffer / 252.6 nm / Ecm 531 /, ^X H NMR (DMSO-d6) (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. Production of enαnejsmzróy / I oG, 4 oC / -4- / 2-amino-6-hydroxy-9Hpousin ^ -yl / -2-ecklspθntenyOwetanol / a / / 1S, 4R / -4- / 2-8mlno-6-hydroxy- 9H-pιJryn-9-clo7-2-ccklopznWzny binoculars ans 1

100 mg of the predominant analog (Example 11) was dissolved in 3 ml of 0.12 M buffer (pH 7.4) by heating at 50 ° C. The solution was cooled to room temperature and 40 units of adenosine deαminarc / Sigma, Type VI, calf intestinal mucosa / · Ps were added. After 3 days of incubation at room temperature, a precipitate formed which was removed by filtration to yield 18.2 mg. The filtrate was concentrated to ds 1.5 mL and frozen for 2 days. A further 26.8 mg of solid was obtained by filtration. Both solid fractions were crystallized from water to afford pure title compound, m.p. 269-272 ° C, "62.1 (c 0.3 MeOH).

/ b / / IR, 4S7-4- / 2-amino-6-hydroxy-9H-pupyy-9-clo / -2-ccklopozyy binornet ano 1

The filtrates obtained during the preparation of the 1S, 4R isomer / Example 14a were combined and evaporated to d dry. Unchanged predominant input compound was separated on silica gel in a flash column using 10% methanol / chlorophyll. The dominant compound was dissolved in 15 ml of 0.05 M 1 4 PO 4 buffer and 800 units of adenosine deamiyαrc were added. The solution was incubated for 96 hours at 37 ° C. TLC showed unreacted compound remained. The solution was heated in hot water for 3 minutes and filtered to remove any depleted protein. Another 800 units of adenosine actin was added and the process was repeated. The deproteinized solution was evaporated to dryness and the solution was taken up in water. After filtering the water, the title compound was collected as a white solid, m.p. 265-27 ° C. Cc = jqA + 61.1 (c 0.3 MzOH).

Example XV. / i / / I oC, 4 cC / -4-72-amlyo-6-hedrskse-9H-puryy-9-yl7-2-cyclopentenylacetoxymetayol

To a suspension of 130 mg / 0.10 mmol / of the product from translation X and 5 mg / 0.24 mmol / 4-dimzwelaminspyridenes in a mixture of 6 ml of aeetsniwpel and 0.09 ml (0.66 mmol) in methylamine, 0.06 ml was added / 0.6 mmol / acetic anhydride. The mixture was stirred at room temperature for 3 hours. 1 ml of ethanol was added to quench the reaction quickly. The solution was concentrated and absorbed onto 1.5 g of silica gel in a column (2.0 x 12 cm), eluted with CHCl6-MeOH 72O- 17 mixture. The product fractions were collected and denoted to give a white solid. The diluent solid was washed with MeOH-AcOEW, 123 mg of the product 785% 7 was obtained. Further purification from methanol gave the starting compound as needle-shaped crystals, mp 237-239 ° C. Analysis / ^c i3 ^H i5 ^N 5 ⁰ ; j / C, H, N.

Example XVI. 7lS, 4R7-4 - "'' - αalys - 9Hppwcyn9-ey0o772-ecyk0ρpnwzycy0mmenaso1

To an ice-cooled solution of 1.202 g of 7lS, 4R / -4- / ~ 2-αmino-6-mevoxseamins-9H-purey-9els_7-2-ceclopzn-zanol / intermediate 8 of Example 12 / in 250 ml of intrahydrofuran and 50 ml of water were added with stirring aluminum amalgam (1.761 g of aluminum and 0.5% aqueous solution of mercury chloride) in small pieces for 1 hour and 47 minutes. After stirring for 35 minutes, the mixture was allowed to warm to ambient temperature. After 16 hours and 50 minutes, aluminum amalgam (with 235 mg aluminum) was added over a further 14 minutes. After a further 4 hours and 10 minutes, the resulting mixture was filtered through diatomaceous earth

To remove insolubles which were washed with netpahydrofuran: water (5: 1, 300 ml). The combined filtrates were evaporated to a yellow foam which was column chromatographed on 33.8 g of silica (Merck 7734) in chloroform and eluted with chloroform-ethanol mnate to give several fractions (578 mg, 420 mg and 40 mg). The two fractions of SPzekpystαlnawα are separate from isaspopanol The filtrates were combined with the smallest fraction from the column and subjected to thin layer chromatography / Merck 5717 / developed three times in a 10: 1 cnlapafopn system: matanal · ePuowαta plates with ethyl acetate / 1 ethyl acetate and ethanol mixture : 1 / and 45 mg of a spray solid are obtained. They were subjected to column chromatography on 2.7 g of silica (Merck 7734) prepared in chloroform and eluted with chloroform-enenylnenyl-methyl mixtures to obtain 17 mg of gum substance. After recrystallization from iuospopanol and treatment with charcoal in methanol, an aqueous solution of the recovered material was freeze-dried to give 15 mg of the title compound. ^X H NMR (OMSO-On (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) + 232.

163 814

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163 814

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163 814

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Publishing Department of the UP RP. Circulation of 90 copies. Price PLN 1.00.

Claims (12)

Patent claims 1. A method for the preparation of new analogs of dideoxydidahydrocarbocyclic nucleosides of formula I, wherein X is hydrogen, NRR1, SR or OR) Z is hydrogen, OR 'or NRR) R, R and R may be the same or different and are selected from hydrogen, C-alkyl and aryl groups and pharmaceutically acceptable derivatives thereof, characterized in that X * is halogen and Z is as defined above, optionally in protected form functions, is reacted with a compound of formula HNRr 1 where R and R 6 are as defined above, with a RO- ion yielding reagent in which R is as defined above, with a thiourea or with a reducing agent, or a compound of formula I, where X is SH group is alkylated or arylated to compounds of formula 1 in which X is SR and optionally the compound of formula 1 is converted to a pharmaceutically acceptable derivative by reaction with an acid, with a base, with a phosphorylating or esterifying agent. 2. The method according to p. A compound according to claim 1, characterized in that the starting compound is a compound of formula 10 in which X 'is a halogen atom and Z is H, OH or NHg. 3. The method according to p. A compound according to claim 2, characterized in that the starting compound is a compound of formula 10 in which X * is a halogen atom and Z is a NHg group. 4. The method according to p. A compound according to claim 1, characterized in that the compound of formula 10 in which X * is a halogen atom is reacted with a reducing agent, with NH 6, OH ~ or thiourea. 5. The method according to p. A compound according to claim 4, characterized in that the compound of formula 10 in which X * is a halogen atom and Z is as defined above, is reacted with OH. 6. The method according to p. The process of claim 4, wherein X * is halogen is reacted with a reducing agent or NHg. 7. The method according to p. A process as claimed in claim 1, characterized in that in the preparation of (I cC, 4-4) (22-amino-y-hydroxy-9H-purin-9-yl) -2-cyclopentenylmethanol), AcC), S-chloro-1H-purin-1-yl) -cyclopentanylmethanol is reacted with the reagent OH-. 8. The method according to p. The process of claim 1, wherein the starting material is the compound of formula 10 in the form of a substantially racemic mixture. 9. The method according to p. The process of claim 1, wherein the starting material is a compound of formula 10 which is essentially an optical isomer. 10. The method according to p. The process of claim 9, wherein the starting compound of formula 10 is essentially an O-isomer. 11. The method according to p. 2. The process of claim 1, wherein the racemic mixture of the compound of formula I is converted to a substantially single optical isomer of the same or a different compound of formula I by enzymatic conversion. 12. The method according to p. The method of claim 1, wherein the protected derivative of the compound of formula 9, wherein X and Z are as defined above, and Y is a hydroxyl protecting group, is removed with the protecting group. 163 814