AP196A - Therapeutic nucleosides - Google Patents

Abstract

The present invention relates to 6-substitued purine carbocyclic nucleosides and their use in medical therapy particularly in the treatment of HIV and HBV infections. The invention also relates to pharmaceutical formulations and processes for the preparation of compounds according to the invention.

Description

Therapeutic Nuc^os^des ’he present invention relates to purine nucleoside analogues containing an unsaturated carbocyclic ring in place of the sugar residue, pharmaceutically acceptable derivatives thereof, and their use in medical therapy, particularly for the treatment of certain viral infections.

AIDS (acquired immunodeficiency syndrome) is an immunosuppressive or immunodestructive disease that predisposes subjects to fatal opportunistic infections. Characteristically, AIDS is associated with a progressive depletion of T-cells, especially the helper-inducer 4 subset bearing the OKT surface marker.

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Human immunodeficiency virus (HIV) has Been reproducibly isolated from patients with AIDS or with the symptoms that frequently precede AIDS. HIV is cytopathic and appears to preferentially infect and destroy T-cells bearing the OKT marker, and it is now generally recognized that HIV is the etiological agent of AIDS.

Since the discovery that HIV is the etiological agent of AIDS, numerous proposals have been made for anti-HIV chemotherapeutic agents that may be effective in treating AIDS sufferers. Thus, for example, U.S. Patent 4,724,232 describes 3'-azido-3'-deoxythymidine (which has the approved name zidovudine), its pharmaceutically acceotable derivatives and their use in the treatment of human retrovirus infections including AIDS and associated clinical conditions. Vince et al., Antiviral Research. 9 (1/2), 120 (1988) describes certain carbocyclic nucleoside analogs and their use against HIV. At the Second International Conference on Antiviral Research, Williamsburg, VA, April 10-14, 1988, (±)-9-(cis-4-(hydroxymethyl)-2-cyclopentenyI) guanine (NSC-614846), also known as carbovir, was disclosed.

AP 0 0 0 1 9 6

Worldwide, hepatitis B virus (H8V) is another viral pathogen of major consequence. It is most common in Asian countries, and prevalent in

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MG.MF.21st November 1990

I

PSI 193

5uc-uaⁿaran Africa. The virus is etό 1 og^; cal¹y assoc-ated «’*.n orrnary neoacoce ’ !u lar carcinoma.

ihe united States currently contains an estimated pool of 500,300-1 million infectious carriers. Chronic active hepatitis «ill deve'op in over 25½ of carriers, and often progresses to cirrhosis. It ’S estimated that 5000 people die from HBV-related cirrhosis eac.n year in the USA, and that perhaos 1000 die from HBV-related ’i.e’cancer. Even «hen a universal HBV vaccine is in place, the need ^for effective anti-HBV compounds will continue. The large reservoir of persistently infected carriers, estimated at 220 million worldwide, «i1' receive no benefit from vaccination and «ill continue at high risk for HBV-induced liver disease. This carrier population serves as the source of infection of susceptible individuals perpetuating the incidence of disease particularly in endemic areas or high risk groups such as IV drug abusers and homosexuals. Thus, there is a great need for effective antiviral agents, both to control the chronic infecfon and reduce progression to hepatocellular carcinoma.

Clinical effects of infection with the HBV virus range from headache, fever, malaise, nausea, vomiting, anorexia and abdominal pains. Replication of the virus is usually controlled by the iimnune response, with a course of recovery lasting weeks or months in humans, but infection may be more severe leading to persistent chronic ' ’/er disease as outlined above.

European Patent Specification No. 349242 discloses certain 5substituted purine carbocyclic nucleosides and their use in mec’ca’ therapy particularly in the treatment of HIV and HBV infect’:'·;. Among such nucleosides are the compounds (i)-c_ts-4-[2-amino-0-i:..c^,o propy ιamino)-9H-pur i n-9-y 1 ]-2-cyclopentene-l-methanol and (J-C'5-4[2-amino-6-(cyclopropylmethylami no)-9H-purin-9-yl]-2-cyclopentene-1 methanol, i.e. each in the form of a racemic mixture of their re’e-ant enantiomers.

— bad ORIGINAL

MG.MF.21st November 1990 *e have new ’ound that the ¹-;· /' tua 1 ’cclatsc enantiomers of tne :.·»ο oomccuncs ment^;cned above an; the·· pharmaceutica1 cen^;/atives have ad/antagecus antiviral acti/’ty, particularly against H'7 and 487 mrecfons, coupled with low cytotoxicity and/or are useful as intermediates for the preparation of compounds having such activity.

According to one feature of the present invention there are provided enantiomeric compounds of the general formula

(I) (wherein R represents a cyclopropyl amino or N-cyclopropyl-N-methyl amino group and A represents the 2-cyc1opentene-l-methano1-4-y1 group in either the (IS,4R) or (1R,4S) configuration) and their derivatives (for example, esters, salts and salts of esters), the said compounds and their derivatives each being in the form of an enantiomer substantially free (for example to the extent of less than 10% w/w, preferably less than 5%w/w) of the corresponding enantiomer.

It will be appreciated that the compounds of formula (I) comprise the compounds having the following configurations:AP 0 0 0 1 9 6

MG.MF.21st November 1990 BAD

Μ

Α)

λ-5'-3··' R is as defined above).

The enantiomeric compounds of formula (I), i.e. substantially free of the corresponding enantiomer, thus comprise:1) (IS,AR)-cj_s-A-[2-amino-6-(cyclopropy1 amino)-9H-purin-9-y11-2cyclopentene-1-methanol

2) (IR. AS) -cis-Α-Γ 2-amino-6-fcvclopropy!ami no)-9H-purin-9-vl]-?eye 1epentene-1-methanol

3) (IS, 4R)-cis.-4-[ 2-ami no-6-(N-cyc lopropy 1-N-methy lami no)-9H-pur in -9-y1]-2-cyc1opentene-l-methanol

A) (IR, AS)-cis-4-Γ2-ami no-6-(N-cyclopropy1-N-methylamino)-SH-our in

-9-y!J-2-cyclopentene-L-methanol

Compounds 1) and 3) above, hereinafter referred to as the (IS,AR) e.naoiome’·’C compounds of formula (I), with a negative (-) optical rotation, nave been found to have especially potent activity against :-ίC / and HBV infections, and these compounds and their pharmaceutical ly acceptable derivatives represent preferred embodiments of the present invention. ^The compounds have the further advantage that, upon aom-pistrat ^;on, they are capable of penetrating the blood-brain

MG.MF.21st November 1990

BAD ORIGINAL .ζ3ΑΑ*9 barrier to provide high levels of tne compounds or active metabo’^;tes thereof in tne central .nervous system where man 1festat'ons of Ό n-'ect’ons re particularly debilitating. Compound 1) above <s especially preferred in view of its exceptionally potent activity against HIV and HBV infections. The compound has also been found to have significantly lower toxicity against bone marrow progenitor cells than 3'-azido-3'-deoxythymidine (zidovudine) referred to above.

«#»«»/·

We have further found that phosphate derivatives of compounds 2) and

4) above, hereinafter referred to as the (1R.4S) enantiomeric compounds of formula (I), with a positive (*) optical rotation, have potent activity against viral infections such as those referred to above. These phosphate derivatives thus represent a further preferred embodiment of the present invention.

S* _ i s> uv ·Ο3· Jii O’-.iieji - ¹

Yhe reference herein to “phosphate derivatives of the (1R.4S) enantiomeric compounds of formula (I) denotes derivatives in which a phosphate grouping is attached to the 1-methanol group of formula (I) and includes mono-, di- and tri-phosphates.

The parent (1R,4S) enantiomeric compounds of formula (I) and non-phosphate derivatives thereof are useful as intermediates for the preparation of the said phosphate derivatives.

AP o 0 019 6

The above (1S.4R) enantiomeric compounds of formula (I) and their pharmaceutically acceptable derivatives, and the phosphate derivatives of the (1R,4S) enantiomeric compounds of formula (I), are hereinafter referred to as the antiviral compounds according to the invention.

According to further features of the present invention we provide:a) antiviral compounds according to the invention for use in medical therapy particularly for the treatment or prophylaxis of a retroviral infection or a hepatitis B viral infection;

MG.MF.21st November 1990 c; a neThccl for the treatment tr orocn,’ax^;s of ret-?,·-:' ·> e⁽_ t or 5 ano hepatites 3 'nfecfons m a subject, e.g. a mamma' such as a human, which comprises treating the subject «'th a therapeutically effective amount of an antiviral compound according to the invention; and '

c) use of an antiviral compound according to the invention -n tne manufacture of a medicament for the treatment or prophylaxis of any of the above-mentioned infections or conditions.

examples of retroviral infections which may be treated or prevented in accordance with the invention include human retroviral infections such as numan immunodeficiency virus (HIV), HIV-1, HIV-2 and human T-cell lymphotropic virus (HITV), e.g. HTLV-I or HTLV-II infections. The antiviral compounds according to the invention are especially useful for the treatment of AIDS and related clinical conditions such as AIDS-related complex (ARC), progressive generalised lymphadenocatny (PGL), AIDS-related neurological conditions, such as mult'ple sclerosis or tropical paraparesis, and anti-HIV antibody-positive and HIV-positive conditions for example in asymptomatic patients, and thrombocytopenic purpura. The compounds may also be used in the treatment or prevention of psoriasis.

By 'a pharmaceutically acceptable derivative¹' in relation to the (1S,4R) enantiomeric compounds of formula (I) is meant any pharmaceutical ly acceptable salt, ester or salt of such ester, of 'a (1S,AR) enantiomeric compounds of formula (I), or any other comccund which, upon administration to the recipient, is capable of pre, ding (directly or indirectly) such an enantiomeric compound, c an ant'/irally active metabolite or residue thereof.

^referred esters of the (1S.4R) enantiomeric compounds of formu'a (I) include carboxylic acid esters in which the non-carbonyl moiety c* tne ester grouping is selected from straight or branched chain alky', e.g. n-propyl, t-butyl, n-butyl, alkoxyalkyl (e.g. methoxymethyl), aralkyl

MG.MF.21st November 1990 bad ORIGINAL

It····

' V - ’ «top*·**·**» (e.g. oenzy'), aryloxyalky! (e.g. phenoxymethy11, ary^: 'e.g. ere·.' opfonal’y substituted by nalogen. C_1U alky: or C, ₄ a'koxy or amino:; sulfonate esters such as alkyl- or aralkylsulfony1 (e.g. met.nanesuifonyl); ammo acid esters (e.g. L-valyl or L-isoleucy1); and mono-, di- or tri-phosphate esters.

The phosphate esters of compounds of formula (I), may be further esterified by, for example, a C^q alcohol or react!/e derivative thereof, or by a 2,3-di(C_g_₂₄)acy 1 glycerol, for example

2,3-bis-(hexanoyloxy)propyl hydrogen phosphate and 2,3-bis-(hexadecan yloxy)propyl hydrogen phosphate derivatives. In addition to such further esterified phosphate derivatives of the compounds of formula (I), the present invention further includes such derivatives of the racemic compounds of formula (I).

With regard to the above-described esters, unless otherwise specified, any alkyl moiety present advantageously contains I to 18 carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group.

Pharmaceutically acceptable acid addition salts of the (IS,4R) enantiomeric compounds of formula (I) include mono- or di- basic salts with the appropriate acid for example organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesuIfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulphate methanesulohonate sulfamic acids. The hydrochloric acid salts (ie. the mono- and dihydrochlorides) are particularly preferred.

AP 0 0 o19 6

The above antiviral compounds according to the invention may be employed in combination with other therapeutic agents for the treatment of the above infections or conditions. Examples of such further therapeutic agents include agents that are effective for the treatment of viral infections or associated conditions such as

MG.MF.21st November 1990

BAD ori«^nM?31:9ό

-az’do-3' -deoxythymidine (zidovudine), 2' , 3'-dideoxynuc’eos' des 3-:~ ²⁵ 2 ,3’-didecxycytidine, 2',3¹-d’ceoxyadenosine a 3 ; -1 · deoxy ’ nos i ne, acycl'c nucleosides (e.g. acyclovir;, sucn as a- nter^feron, renal excretion innibitors sucn as probenicid, nucleoside transport inhibitors such as dipyridamo’e, di azeo, mio-, lido- or soluflazine, or hexobendine, immunomodu1ators sucn as interleukin II and granulocyte macrophage colony stimulat’ing factors, soluble CD^ or genetically engineered derivatives thereof, and phosphonoformic acid. The component compounds of such combination therapy may be administered simultaneously, in either separate or combined formulations, or at different times, e.g. sequentially such tnat a combined effect is achieved.

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

In general a suitable dose for each of the above-mentioned conditions (e.g. AIDS) will be in the range of 3.0 to 120 mg per kilogram body weight of the recipient (e.g. a human) per day, preferably in the range of 6 to 90 mg per kilogram body weight per day ano mdst preferably in the range 15 to 60 mg per kilogram body weight per d£y. ^The desired dose is preferably presented as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage ^orms, for example, containing 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably 50 to 700 mg of active ingredient per unit dosage form.

BAD ORIGINAL

MG.MF.21st November 1990

'.cea'ly, ’.he active ^;ngredient snould he acm’n·stewed to achieve teak c'asjia roncentrations of the active compound p- from about I to about '5 _M, preferably aDcut 2 to 50 uM, most preferably about 3 to 30 J4. 'his may be achieved, for example, by the intravenous injection of a 0.1 to 5¾ solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1 to about ICO mg/kg of the active ingredient. Oesirable blood levels may be maintained by a continuous infusion to provide about 0.01 to about 5.0 mg/kg/hour or by intermittent infusions containing about 0.4 to about 15 mg/kg of the active ingredient.

While it is possible for the active ingredient to be administered alone it is preferable to present it as a pharmaceutical formulation.

The formulations of the present invention comprise at least one active ingredient_t. Hr. feS¹ v4mu·pharmaceutically acceptable carrier or excipient. Formulations include those adapted for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Formulations of the present invention adapted for oral administration may be presented as discrete units such as capsules or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

AP 0 0 0 1 9 6

MG.MF.21st November 1990 bad 0W®’^nM·

A taP’et nay be nade cy bcmc^res5 όπ or mou’d^;t. bDticna’¹'/ w’b.n -nore accessory 'ngrecients, Concressed tab’ets nay be prepared :, ccnoress;ng m a suitat’e machine the active ’ngredsent in a freer’j'/v'ng ^forn such as a powder or granules, optionally nixed wit.n a binder (e.g. povidone, gelatin, hydroxypropyImethyl cellulose), 'ubr-cant, inert diluent, preservative, disintegrant (e.g. sod'un starch glycol late, cross-linked povidone, cross-linked sodium

Λ carboxymethyl cellulose) surface-active or dispensing agent.

Moulded tablets nay be nade by noulding in a suitable nachine a nixture of the powdered compound moistened with an inert l-'quid driuent. The tablets nay optionally be coated or scored and nay be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethy1 cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach. may—beparticuiarly—advantageous—fee—the—purine—nucleoside derivatives according to the invention, as such compounds arc often susceptible to acid hydrolysis·;—

Formulations adapted for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in' a suitable liquid carrier.

Formulations adapted for rectal administration nay be presented as a suppository with a suitable base comprising for example cocoa butter or a sal icy late.

Formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations

MG.MF.21st November 1990

BAD ORIGINAL

- il ccnti’O’ng ’n addition to t^he act’/® ingredient such car^r'e^r: as a^e <ncwn ·η the art to he appropriate.

or-Tiu'at'ons adapted for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain ant·-oxidants, buffers, bacteriostats and solutes which render :-e formuat ion isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may incite suspending agents and thickening agents. The formulations may be presented in unit-dose or multidose sealed containers, for example, ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

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

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents.

The present invention further includes the following process *:r the preparation of enantiomeric compounds of formula (I) atc-e and derivatives thereof. The enantiomeric starting materia’s and precursors for such materials which are employed as described be’ow in relation to the process are each in the form of an enantiomer substantially free (e.g. to the extent referred to above in '•e'aticn

AP 0 0 0 1 9 6

MG.MF.21st November 1990 bad “Tu j ' u ''“«O'* a n 3 <*» * : Ome*3.

a:::rr-; ;; ;^e i^enc-cr comer ; an enant:omeric compound of formula (I i) :-

^nere^?n A is as hereinbefore defined and Z represents a precursor group for the said R group as defined in formula (·)) or a derivative thereof with an agent or under conditions serving to convert the precursor Z group to the desired R group; or

B) reacting an enantiomeric compound of formula (III):-

A (wherein A and R are as hereinbefore defined, R reoresents hydrogen or a formyl group and R^ represents an amino protecting group, e.g. an acyl group such as a alkanoyl group, e.g.

formyl, acetyl or isobutyryl) or a derivative thereof wth an agent serving to effect formation of the imidazole ring ·? tne desired compound of formula (I) followed by removal of tne R amino protecting group; or bad original

MG.MF.21st November 1990 '•eact^g an enant •erne’· : comccunc 3* fermu'a

(wherein A and R are as hereinbefore defined and R^ is an am-no arctecting group, e.g. as described above in relation to formula (III)) or a derivative thereof with an agent serving to effect removal of the R^ amino protecting group, ano optionally effecting one or both of the following conversions in any desired order:i) where a compound of formula (I) is formed, converting the said compound to a derivative thereof; or ii) where a derivative of a compound of formula (I) is formed, converting the said derivative to the parent compound of formula (I) or to a further such derivative.

Process A) may be carried out in conventional manner, for example, by treatment of a compound of formula (II) in which Z represents a leaving group (e.g. a halo such as a chloro grouo) with an approDriate amine, i.e. cyclopropylamine or N-cyclopropyl-N-methyiam’ne, preferaoly in an excess to introduce the amino R group as defined above, advantageously at reflux or at a temperature greater than 50³C, pre*'e^raoly in the presence of an organic solvent, for example methanol or ethano1.

AP 0 0 0 1 9 6

Process B) may be carried out, for example, by reacting a compound of formula (III) with formic acid or a reactive formic acid derivative (e.g. triethylortnoformate cr diethoxymethyl acetate) optionally with

MG.MF.21 st November 1990 «.,muAL

a -e-t such as a	:·me: .' ·: e:	amfe or time:
,3 — , 2 “ □ * 'O’mrqrj» ’·*α		a: '5-lC:I.
.er'er:’, e**ected b	y one acf	:-on o* s 1 'gnt
ecu ·.a'ent of a strong	an.nycrous ac	id, e.g. with
ethanesuIconic acid per	equiva’ent	of compound of

which case lower temperatures (e.g. 25’C) are used.

. '•ormam· ce :: '5 r e a c:; n y tic re t r a .1 eouivalents formula (III), rs of i n

Process C) may be carried out, for example, by reacting an enartt’omeric compound of formula (IV) with an acidic agen:, -or example, dilute aqueous hydrochloric acid.

''.e oompcunos of 'ormu’a (II) employed as starflng mater-'als process A) may be prepared for example, in an analogous manner process 3), i.e. by reacting a corresponding enantiomeric compound formula (V) ·» w>

of

A (wherein A, Z, R and R are as hereinbefore defined) or a derivative thereof with an agent serving to effect formation of the imidazole ring in the desired compound cf formula (II) and to effect removal of tne R^ amino protecting group. The reaction may be carried out using those agents and conditions described above for process 8).

'he compounds of formula (III) employed as starting materials in orocess 3) may be prepared for example by treating an enantiomeric compound of formula (V) above with an agent or conditions serving to convert the precursor group Z to the desired R group, in an analogous manner to that described for process A).

MG.MF.21st November 1990

BAD ORIGINAL

(,.*re^rein A, 2 and R^ are hereinbefore defined) with an agent cr j.ncer oond'tions serving to convert the precursor group Z to the des'>eo R group, i.e. in an analogous manner to that described for process .A).

The compounds of formula (VI) above may be prepared for example by reacting an enantiomeric compound of formula (V) above with an agent serving to effect formation of the imidazole ring in the desired compound of formula (VI), for example by treatment with formic ac^;d or a reactive formic acid derivative, as described above in relation to process 3).

Enantiomeric compounds of formulae (II), (III), (IV), (V) and (VI) above represent further features of the present invention, especially

3 those in which R represents a formyl group and/or R represents a C._« alkanoyl group, particularly acetyl or isobutyryl, and/or Z represents a halo such as a chloro group.

Particularly preferred intermediates for the preparation of 1S, AR'-c i s-A-^r2-ami nc-6-cyclooroov 'amino)-SH-purin-9-y 1 j-Z-cyc'ooentene -'.-methanol, i.e. the preferred compound i) above, include:a) (IR,4S)-cis-N-rb-(cyclopropyl ami no )-9-(4-(hydroxymethylj-Zcyc 1 open ten-1-y) )-9H-purin-2-yl)isobutyramide;

AP 0 0 0 1 9 6

BAD ORIGINAL

MG.MF.21st November 1990

R .1S -' ~ 5 -N-d - on o^o- 5-' 3nmam· to-ό- ' -· crox ,met . ’ -2: .³, -5, - T ·; - Ί -(d -- n^?orc-;--;mam ^- :c-5-( ,d - nydro^mecnyribeye .open tene-1-yl) ami no)-2-pyrimi di ny: ] acetamide;

d(15, -R} -c i s-( 2-amino-5-chloro-9H-purin-9-y1' -2-eye 'opentene -L-methanol.

e) (IR, 45)-cis-N-T5-chloro-9-(4-(hydroxymethyl)-2-cyclopentene-lyl )-3H-purin-2-yI ) isobutyramide.

The enant^;cmer'c compounds of formula (V) employed as starting materials as described above may be prepared for example by reacting a compound of formula (VII)

(VII)

3 4 (wherein Z, R and R are as hereinbefore defined and R leaving group, e.g. a halo such as a chloro group) or thereof with an enantiomeric compound of formula (VIIIA) represents a a denivat've or ( Η 1131

Ot

MG.MF.21st November 1990

O' (v 1118)

BAD ORIGINAL

3r 3 ter · . at ·ve tner=e ast-ment^;oned -eact^n Is advantageous’y * ΟΓ * O j · *» “ « ft τ r j r □ ;* a case sucn as a tertiary amine for examole tmetny'amine or trimet.ny lamine advantageously in an organic solvent such as dimethoxyethane or ethanol.

or (VIIIS) having the aporocnate be octaineo by comp'exing tne i.e. (2)-4-ami no-2-eye 1epentene-1’ne compounds of formula (VIIIA) enanfomeric configuration can corresoonding racemic compound, metnanol with an optically d^;de^pcoy'-O-tartaric ac’dl and resu’t^;ng ciasteneomeric salts, may be employed as described for and J.Med.Chem., 1985, 28, 1385.

act ive then carboxyl ic fractional acid (*'or exame'e crystal 1 isatiort cf l

y· ·

Alternatively, enzymatic resolution example in J.Med.Chem., L9S7, 30, 746

The enantiomeric compounds of formula (VIIIA) or (VIIIS) and the’r derivatives, particularly salts thereof with optically active carbox^ytlic acids such as dibenzoyl-Q-tartaric acid, for example (lS,4R)-4-amino-2- cyclopentene-l-methanol and its dibenzoyl-D-tartrate represent a further feature of the present invention.

6 KJOO cWy

The compounds of formula (VII) employed as starting materials above may be prepared in a conventional manner for example by reducing a compound of formula (IX)

N»C?

/-, 4» * fix'

4 («herein Σ, R and R are as hereinbefore defined) to e''ect conversion of the N0₂ group to an NH₂ group and optionally converfng

MG.MF.21st November 1990

BAD ORIGINAL ;~e -esj't'ng crcuo co a *crmamido p^cuc, ^;cr e<ic'e :.

t **? i t t ·*/ * t C * 3 ^r *7 ’ * } r ’ 5C 5t ’ C 3 - Γ > C ** ' ^Λ 6 ?6 z^mccuncs o* ’srjiutd (;X) mey Oe prepared in conventions’ nanner. ’hose compounds in which Z represents a halo, for example chloro group may be prepared for example by halogenating, for examc'e using phosphorus oxych’cride, a corresponding compound of formula '<;

*

(X)

4 (wherein R and R are as hereinbefore defined).

The compounds of formula (X) may also be prepared in conventional manner, for example by reaction of a compound of formula (XI)

(wherein R⁴ is as hereinbefore defined) with an appropriate aceⁿt serving to introduce the amino protecting group, for examole byreaction with an appropriate carboxylic acid or a funcfcraMG.MF.21st November 1990

BAD ORIGINAL ec-' < i ent tnerect. e-j. ’scout/r-c annydnoe. The comocund :* -:-01/3 '</ may oe O'-ecared cy oit-a/on :- 3 tor-ssccnc^;ng oomccuod * :-ou '3 .' </ ‘

(wherein R⁴ is as hereinbefore defined).

The compounds of formulae (VII), (IX), (X) and (XI) represent further features of the present invention particularly those in which I represents a halo such as a chloro group, and/or R³ represents a C^galkanoyl group, especially acetyl or isobutyryl, and/or R⁴ represents a halo such as a chloro group.

Particularly preferred compounds of formulae (VII), (IX) and (X) according to the invention include:N-(A,5-dichloro-5-formamido-2-pyrimidinyl)isobutyramide;

N-(A,5-dichloro-5-nitro-2-pyrimidiny1)isobutyramide; and

N-(A-chloro-1,6-di hydro-5-nitro-6-oxo-2-pyri midinyl)isobutyramide.

A comcound of formula (I) may generally be converted into an ester thereof by reaction with an appropriate esterifying agent, e.g. an

6 I 0 0 0 dV

MG.MF.21st November 1990

BAD ORIGINAL ac · i '¹a’^-de cr ann>cr-:e. 'he comocund of *;rmu'a 1', inc'u:‘g es*. e^s tre^eo’’, nay ce corye^rted sats .ai'sor ’n convene·;'^' ma-’re'·, ’or examole, o_? treatment witn an aoc^r:cr-ats acid. An esteo- sa't of a compound of formula J) may be converted into the parent compound, for example by hydrolysis.

Thus the O-monoDhosphate of a comoound of formula (I) may be prepared by treating the parent with an appropriate phosphorylating agent, e.g. , phcsohorus oxychloride as in M.Yoshikawa, T.Kato and T.Takenisni, /i Bulletin Chem. Soc. Japan, 1969, 42, 3505. The correspondirig 0-di- ' and 3- tripnosphates may be prepared by methods described in 'Nuc'eotide Analogs’ by K.H.Sheit, John Wiley and Sons, New fork 1,980, pp. 211-215, and in O.E.Hoard and D.G.Ott, J.Amer.Chem.Soc. 1365, 37,

1785, e.g. by making the imidizolate derivative of the relevant O-monophosphate and by subsequent reaction of this derivative with phosphate to give O-diphosphate or with pyrophosphate to give Q-triphosphate. For the preparation of esterified phospnate derivatives referred to above, the parent compound of formula (I) may be treated with an appropriate di-alkanoyl phosphatidyl choline derivative in the presence of an appropriate phospholipase for example phospholipase 0, as described in S.Shuto et al, Nucleic Acid Research,

1988, 20, page 35 or by reaction of a compound of formula (I) with an appropriate phosphorylating agent such as phosphorus oxychloride followed by work-up with an appropriate alcohol as described in A.Rosowsky & S.Kim, Nucleic Acid Chemistry, Part 3, 1.3.Townsend & R.S.Tipson (Editors), John Wiley & Sons, New York, 1986, 255.

The enantiomers of the compounds of formula (I) may be resolved or isolated in conventional manner, e.g. by chromatographic separation of diastereomeric esters prepared by acylation of the hydroxyl on the eye¹ocenteny1 moiety with appropriate optically active carboxylic acid derivatives as, e.g., with naproxen (J. Qrq. Chem. 1986, 51., 1287).

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MG.MF.21st November 1990 ';.. .-c

The fc’lowing Examples are intended for il!ustrat^;on only and are •ncenoed to limit the scope of the invention in any way. In t.ne

Examcles the optica! rotations were assigned with respect to tne sodium 0 line (5S9nm) at 20°C. The term ‘active ingredient¹ as used in Examples A to G means an antiviral compound according to the invention, especially compound 1) above.

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Example 1 . -. -c.'S-A-; ; no-d-,-.η loro-iS-ovr -m·' b · t/ ' am^;no 1-2-0 vc¹ opente^re’. -metnano i :i s-4-Acetamidocyclocent-2-enemethyl acetate fU.S. Patent 4,253.572] (’„4.38 g, 0.073 mo’) and barium hydrox-de octahydrate g,

0.146 mol) were refluxed in water (300 ml) under nitrogen -‘or 13 hours. The resulting solution was neutralized with carbon d’ox-de. ’he orecipitate was washed with water, then ethanol. The comb-ned fi’trate-wasn was evaporated to a syrup (11.15 g) which was condensed with 2-amino-4,6-dichloropyrimidine (23.91 g, 0.146 mol) and tniethylamine (30.5 mL, 0.219 mol) in refluxing 1-butanol (100 mL) tor

1.5 hours. After addition of 1 N NaOH (73 mL), the resulting mixture was evaporated to dryness and the residual solid slurried in CHCl^ (200 mL). Unreacted 2-amino-4,6-dichloropyrimidine was filtered off and washed with chloroform (100 mL). The chloroform fi 1 trate-wasn was concentrated and chromatographed on a silica gel column. Adoitonal pyrimidine starting material was eluted with 2.5¾ methanol-chIcro^'orm. The title compound was eluted with 3.5¾ methanol-chloroform as an off-white solid foam (15.90 g, 91%).

^-NMR: (Me₂S0-d^) s 1.15-1.28 and 2.26-2.41 (2m, 2, CH₂);

2.60-2.71 (m, 1, l'-H); 3.4 (m overlapping H₂0, CH₂OH); 4.525 (t,

2=5.3, 1, CH₂0H); 4.95 (br s, 1. CH-N); 5.57-5.87 (m, 2, 2- = 2-',; 5.38 (br s, 1, NH₂); 7.12 (br s, 1, NH); MS (CI) M+l, 241, 243.

Anal. Calcd. for C^qH,^N^OClΌ.2 H₂0: C, 48.99; H, 5.55; N, 22.35; 21, 14.45.

-ound: C, 49.10; H, 5.57; N, 22.81; Cl, 14.40.

Exaapl· 2 ( =)-cls-4-rr2-Amino-6-chloro-5-i(4-chlorophenyl)azo]-4-pyrimid^;nv' 'ami no 1-2-cyclopentene-1-methanol

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-51193 ; 2..' ‘ ?-4-Γ :2--mino-4-chIoro-6-pyr’'midinyl)amino]-2-cyc'ooe^r'tenel-me:?ano! *rcm Example I (11.53 g, 43.1 mmol) and scd'um acetate tnhydrate (97 g) were dissolved in glacial acetic acid (225 mL) and water (225 mL). A cold solution (0-5’C) of 4-chlorobenzenediazon’urn chloride was prepared from 4-chloroani1ine (6.74 g, 52.3 mol), concentrated hydrochloric acid (14.7 mL) water (52 mL), and sodium n-f’te (4.01 g, 53.2 mmol in 47 mL of water). This cold so^;ut^;cn was added dropwise over 5 minutes to the first solution. The resu’f'ng yellow precipitate was filtered after 18 hours, washed with water, and extracted with ethanol to give title compound as dark yellow powder (12.56 g, 69%), m.p. 218-220’C dec.

‘H-NMR: (Me^O-d^) 5 10.25 (d, 1, NH); 7.69 and 7.54 (both, d, J=8.9, C_gH₄) overlapping 7.6 (br, 6, NH₂); 5.80-5.95 (m, 2, CH=CH); 5.24 (m, 1, CHN); 4.75 (t, 1, CH₂0H); 3.41 (t, 2, CH₂0H); 2.75 (m, 1, CH); 2.41 (m, 1, CH); 1.44-1.53 (m, 1, CH).

Anal. Calcd. for C^H^NgCljO: C, 50.67; H, 4.25; N, 22.16; Cl, 18.70.

Found: C, 50.59; H, 4.29; N, 22.10; Cl, 18.66.

Ex*·»!· 3 (=)-cis-4-r(2.5-0iamino-4-chloro-6-pyrimidinyl)-amino1-2cvclopentene-1-methanol

The title compound of Example 2 (11.67 g) was suspended in ethanol (235 mL), glacial acetic acid (30 mL), and water 235 mL). The mixture was heated to reflux under nitrogen. Zinc dust (13.5 g) was added in small portions over 30 minutes during which time the compound dissolved. The reaction was heated an additional 20 minutes, and then the excess zinc was filtered of from the hot solution, and it was washed with ethanol. The filtrates were evaporated, and the residue was purified on a silica gel column eluting with chloroform (1 L) and

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MG.MF.21st November 1990

BAD ORIGINAL ?3U ?o on oroform:metnarc’,4:i ('..3 _ i. the ^act’cns contain^;oo te O'Oduct were comb’ned, ano the solvent «as removed under reduced pressure to j’ve tne title compound as a red-orange oil (LI.2 g, > LGO% vie'di^{1 * * * * *}. 4 pure sample was obtained during another small scale reaction to obtain the product as a light yellow solid in a 76¾ yield.

VwR·· (Me₂S0-d₆) J 1.29 and 2.39 (m, 2. CH₂); 2.69 (t, L, l'-H); 3.37 (d, 2, CH₂OH); 3.91 (br, 2, NH₂); 4.60 (br, 1, CH₂OH); 5.02‘⁷m, 1, CHNH); 5.56 (br s, 2, NH?); 5.74 (m, i, = CH); 5.86 (m, 1, = CH); 6.36 (d, 1, CHNH).

Exaaple 4 (;)-cis-4-(2-Amino-6-chloro-9H-purin-9-vl)-2-cvclopentene-l-methanol

The title compound of Example 3 (about 9.7 g) was dissolved in diethoxymethyl acetate (100 g), and refluxed for two days. The solvent was removed under high vacuum at 50°C, and dioxane (40 mL) and 0.5 N HC1 (60 mL) was added. The reaction was stirred at room temperature for 1.25 hours, and then chilled. The reaction was neutralized to pH 7 with cold 5 N sodium hydroxide, and then it was extracted with chloroform:methanol/3:1 several times. The organic layers were dried with magnesium sulphate, filtered, and evaporated. The residue was purified by chromatography on a silica gel column, eluting with 2¾ MeOH-CHCl^ to give 3.7 g (46¾ yield) of the title compound, m.p. 138-139°C.

¹H-NMR: (Me₂S0-d^) a 1.53 and 2.61 (m, 2, CH₂); 2.87 (m, 1, l'-H);

3.44 (d, 2, CH₂0H); 5.44 (m, 1, CH-N); 5.39 (m, 1, = CH); 6.14 (m, 1, = CH); 6.82 (br s, 2, NH₂); 3.02 (s, 1, 8-H); (CH₂OH not seen - under

H₂0 peak). UV: pH 1 Amax 315 (ξ 7370); 218 (26200); a sh

239.5 (5650). pH 7.4 \max 307 (ξ 8000); 245.5 (4500); 223 (26400).

MS (El) 265,267 (m) (CI) 266,268 (m * 1).

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Ar.a’. Ca'cd.

:r C,,H,.N-C’0.2H_?0: C, 43.'9 _ j . -.

Found: c, 43.57; H, 5.29; N, 23.05; CL, 11.70.

Exawl· 5 ( a )-c i S-4-Γ2-Ami no-6-(eyelopropv1 am ino)-9H-pur^;n-9-y11-2-cve ¹ opentene-l-methanol

The title compound of Example 4 (0.50 g) was dissolved in ethanol (40 mL), and cyclopropylamine (0.55 mL, 5 equivalent) was added. ’he reaction was refluxed under nitrogen for 6 hours. An additional 0.55 mL of cyclopropylamine was added, and the reaction refluxed for an additional 5.5 hours. The solvents were evaporated, and chloroform (25 ml) and saturated sodium bicarbonate solution (5 mL) was added. The aqueous layer was extracted several times with CHC1 to obtain the crude product. This was purified on a silica gel column eluting with 3% methanol-ethyl acetate to give 0.43 g (80¾) of (t-cls-4-f2-amino-6-(cvclopropv1 ami no)-9H-purin-9-vH-2-cyc1opentene-l-methanol. This was recrystal 1ized from acetonitrile to give 0.30 g of white powder; m.p. collapses at 93-130°C; melts at 165°C.

(Me₂S0-d^) & 0.56 and 0.63 (2m, 4, 2-cyclopropyl CH₂); 1.56 and 2.50 (2m, 2, cyclopentenyl-CH₂); 2.85 (m, 1, Γ-Η); 3.02 (m, 1, cyclopropy! CH-NH); 3.43 (m, 2, CH₂CH); 4.71 (t, 1, CH₂0H); 5.40 (m, 1, 4’-H); 5.77 (s, 2, NH₂), overlapping 5.84 (m, 1, = CH₂); 6.09 (m, 1, =CH); 7.23 (d, 1, NH-CH); 7.58 (s, 1, purine-8-H); ms (CI)

287 (m*l). UV: pH 1: „max 296 (ξ 14000), 255 (10700); pH 7.0: \max 234 (L59OG); 259 (9200); pH 13 \max 284 (15800), 259 (9100).

Anal. Calcd. for C^H^NgO.O.25 H₂0: C, 57.82; H, 6.41; N, 28.90.

Found: C, 57.84; H, 6.45; N, 28.86.

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Exawle 6 ς , · s-4 -; 2-Um·:πο-6- (cvc looropy Imethyanr no ?- 9H-our 1 n-9-v 1 i - 7eye'opentene-I-methanol (:)-c¹ s -4-(2-Amino-6-chloro-9H-purin-9-y1)-2-eye 1opentene-1-methano1 (0.53 g, 2 mmol) from Example 4, N-methyl-N-cyclopropylamine (Karl Industries, Aurora, OH; 0.3477 g, 12 mmol) and methanol (20 mL) ^ere placed in a Parr bomb and heated to 62°C for 5 hours. The solution was concentrated and then diluted with ethanol before being brought to pH 12 oy the addition of 1.0 N NaOH. This solution was concentrated and the residue was purified by elution from a silica gel column A^;th 3% methano1-chloroform (0.547 g, 91.2%). Crysta11ization of such a sample from water-ethanol yielded a white powder, m.p. 130-131’C.

IhjimR: (Me₂S0-d₆) S 7.51 (s, 1H, purine H-8), 6.10 (m, 1H, CH=),

5.34 (m, 1H, CH=), 5.7 (br s, 2H, NH₂), 5.40 (m, 1H, CHN), 4.70 (.br t, 1H, OH), 3.43 (m, 2H, CH₂OH) 3.24 (br s, 4H, 0Η_ρ NCH cycloprooyl), 2.85 (m, IK, CH), 2.66-2.57 and 1.61-1.51 (m, 2, cyclopenteny1 CH~),

0.90-0.65 (m, 4H, 2CH₂ of cyclopropyl).

Anal. Calcd. C^H^NgO’0.5 H₂0: C, 58.24; H, 6.84; N, 27.16.

Found: C, 58.15; H, 6.86; N, 27.14.

Exaapl· 7 (- i-c^s-4-f2-Amino-6-(eye iopropvlamiho)-9H-pur in-9-yl 1-2-cyc'ooentene-l-methanol

The title compound of Example 5 (0.600 g, 2.00 mmol) was disso'/ed in i,3-dimethyl-3,4,5,6-tetrahydro-2-(lH)-pyrimidinone (Aldrich, 12 mL).

Phosphoryl chloride (0.76 mL, 8.0 mmol) was added to the stirred, cooled (-10°C) solution. After 3 minutes, cold water (100 mL) was added and the resulting solution neutralized with 3 M ammonium

MG.MF.21st November 1990

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PS nyO'-ox’de. The neutralized solution was di’uted to 1 liter w’t.h .vats'* and aocl-ed to a 2.5 x 20 cm column of CE.AE Seohadex A25 (?harmac*a;

λί·;ί nad been preeou ’¹ ’.prated with 50 mM ammonium bicarbonate. the column was first washed with 4 liters of 50 mM ammonium bicarbonate.

'he Q-monophosphate of (=)-cis-4-[2-amino-6-(cyclopropylamino)-9Hcurin-9-yl]-2-cyclopentene-l-methanol was then eluted with a 2-1’ter grad-lent of 50 to 300 mM ammonium bicarbonate. The fractions containing nucleotide (i.e. the above O-monophosphate) were evaporated to a white powder to remove ammonium bicarbonate; 71% calculated by

UV absorbance; one peak by HPLC (see below). Snake venom

5¹-nucleotidase (EC 3.1.3.5) from Crotalus atrox (1000 IU, Sigma) was added to 1.4 mmoles of the nucleotide dissolved in water (20 ml), the solution was incubated at 37°C for 22 hours, at which time additional enzyme (1000 III) was added. Incubation was continued for another days. HPLC analysis (0.4 x 10 cm Whatman Partisi 1 10 strong anion exchange column; elution with a gradient of 20 mM to 1 M ammonium phosphate, pH 5.5, containing 5% methanol; UV detection at 234 nM, at this point showed that 50% of the starting nucleotide had been dephosphorylated to the parent nucleoside. This mixture was again applied to a OEAE Sephadex column of the type described above.

Elution with 4 liters of 50 mM ammonium bicarbonate gave fractions containing the title compound. Evaporation of the water left white powder. This material was further purified by chromatography on silica gel with MeOH:CHC11:9 to give colorless glass. The glass was solidified in acetonitrile to give (-)-ci_s-4-[2-anrino-6-(cyclcpr*cpyl amino)-9H-purin-9-yl ]-2-cyclopentene-l-methanol as white gummy solid which was dried to a solid foam at 0.5 mm Hg at 68’C (260 mg, 86-¾ *'rom racemate); ^H-NMR in DMS0-dr and mass spectrum identical with these of on the racemate (title compound of Example 5); [a] θ -59.7°,

Ζ;²°436 ‘127.8°, M²⁰365 ‘218.1°, (c = 0.15, methanol).

Ana¹. Calcd. for C^H^NgO-O.3 H₂0: C.55.91; H, 6.57; N, 27.94.

Pound: C, 56.05; H, 6.65; N, 27.88.

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MG.MF.21st November 1990 :;^Λί'υβ3 e'ution or the 'ast-mentioned Seohaaex column witn a 2-' -ter :--30-ent o' ;C to JCG mM ammonium picarponate gave the u-moncprcicⁿate .0' the enantiomer correspond·ng to the title compcunc) wh'on *as staple to o -nucleotidase; the preparation of this monophosphate is described in more detail in Example 9.

Exawl· 8 (-1 ' s-4-l~ 2-Ami no-6-( eye ‘cpropvI ami no )-9H-pur in-9-vl 1-2-cyc lopentere

-l-metnanol Q-monophosphate

The title compound of Example 7 (0.35 g, 1.2 mmol) was dissolved in

1,3-dimethyl-3,4,5,6-tetrahydro-2-(lii)«pyrimidinone (Aldrich, 5 mL).

Phosphoryl chloride (Aldrich, 0.43 mL, 4.6 mmol) was added to the stirred, cooled (-10°C) solution. After 3 minutes, cold water (20 mL) was added and the resulting solution neutralized with 3 M ammonium hydroxide. Ion exchange chromatography as described in Example 7 gave the nucleotide as the diammonium salt after evaporation of water, white powder (95¾ yield, quantitated by UV); HPLC analysis as in

Example 7 shows one peak; UV Amax nM (0.1 M HC1 ):254, 297; (pH 7 phosphate buffer):259, 284; (0.1 M NaOH):259, 284. The base/ phosphate ratio was 1.0/1.3 as determined by the method of B. Ames ,20 -_{cn n9} r i20 (Methods in Enzymoloqy 8:115, 1966). [a] & '69.9°, [a]‘

73.0°,

578 ,20 ₅₄₆ 34.0° (c = 0.52, MeOH: H₂0/4:1)

Exawl· 9

-1 - ς,5-4-12-Amino-6-(cvc loo^opvlamino) -9H-ourln-9-yl1-2-cvclopentene

-1-methanol Q-monophosphate

Elution of the DEAE Sephadex column described in Example 7 after 5'-nucleotidase incubation with a 2-1 iter gradient of 50 to 300 mM

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- £9 ammcrrum bicarbonate gave nuc’eot ^;qe-con:aining ’racccrs wP-cn. a-'f e,accretion of water, gave tne title ccmoound as the diammonium salt; «η-te ocwder (55¾ from title compound of Example 5); HPLC analysis as m Example 7 shows one peak; UV phosphate buffer): 259, 284;

< ^nM (0-1 ^M HCl): 254, 297;

ΓΠα a (0.1 M base/phosphate ratio was 1.0/0.98. [al ,20

NaOH): 62-0’,

259, 284.

(pH hhe ,20

173 '55.2°, [a]

546 '75.O’, (c = 0.54, MeCH:H₂0/4:

Ex awl· 10 ί-)-cis-4-[2-Amino-6-(eye lopropylamino)-9H-purin-9-yl1-2-cyclopentene-l-methanol

The title compound of Example 9 (0.67 mmole) was dissolved in water (20 mL) and alkaline phosphatase (EC 3.1.3.1) from calf intestine (3000 IU, Boehringer Mannheim) was added. The solution was incubated at 37’C for 19 hours, at which point HPLC analysis as described in Example 7 showed that all of the nucleotide had been dephosphorylated. The solution was evaporated to dryness and the residual solids extracted with refluxing ethanol (100 ml). The ethanol-soluble material was adsorbed on silica gel and applied to a silica gel column. Title compound was eluted with methanol:chloroform/l:9. Evaporation of an acetonitrile-ethanol solution gave white solid foam (154 mg, 79%); ^H-NMR in DMSO-dL and mass spectrum identical with those of the racemate (title compound of Example 5); [a] _Q *53.7°, [aj²⁰436 *126.2°, Laj²°₃₆₅ *217.5°, (c = 0.10, methanol).

Arab Calcd. for C^H^NgO-0.60 H₂0-.15 EtOH: C, 56.49; H, 6.66;

N, 27.64.

Found: C, 56.60; H, 6.63; N, 27.55.

6 I· 0 0 0 dV

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ExampIt 11

4-: 2-Am-no-6-'loyc'oorco7¹methv'amino)-?H-ourⁱ’-i-9-v¹ ;-2-cvc’oceotene-’.-metnanol ’he t^;tle compound of Exampie 5, (2.00 g, 5.50 mmol) *as dissolved :n

1,3-dimethy1-3,4,5,6-tetrahydro-2(lH)-pyr-midinone (Aldr-ch, EC mL,:. Phosphoryl chloride (2.28 ml, 24.0 mmol) was added to the stirrec, cooled (-10°C) solution. After 3 minutes, cold water (80 mL) was added. The solution was extracted with chloroform (3 x 80 mL). The aqueous layer was diluted with ethanol (400 mL) and the pH adjusted to 5 with saturated aqueous NaOH. The precipitated inorganic salts were -'•'Itered off. The filtrate was further diluted with ethanol to a volume of 1 liter and the pH adjusted to 8 with additional NaOH. The resulting precipitate was filtered and dried to give the O-monophosphate of (± )-cis -4-(2-amino-6-(cyclopropylmethy 1 amino)-9Hpurin-9-yl]-2-cyclopentene-l-methanol as white powder (4.0 mmoles, 62¾ quantitated by UV absorbance); HPLC analysis as in Example 7 shows one peak. This racemic O-monophosphate was dissolved in water (200 mL) and snake venom 5'-nucleotidase (EC 3.1.3.5) from Crotalus atrox (5,000 IU, Sigma) was added. After incubation at 37 ^aC for 10 days, HPLC analysis as described in Example 7 showed that 50% of the starting nucleotide had been dephosphorylated to the nucleoside. These were separated on a 5 x 14 cm column of OEAE Sephadex A25 (Pharmacia) which had been preequilibrated with 50 mM ammonium bicarbonate. The title compound was eluted with 2 liters of 50 mM ammonium bicarbonate. Evaporation of water gave white powder whicn was dissolved in methanol, adsorbed on silica gel, and applied to a silica gel column. Title compound was eluted with metnanol:chloroform/!:9 as a colorless glass. An acetonitrile solution was evaporated to give white solid foam, dried at 0.3 mm Hg over ^Ρ2θ5' (72¾ from racemate); ^H-NMR in DMSO-d^ and mass soectrum identical with those of the racemate (title compound of

methano1).

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?6,' H , 5.^2’ Ί , 2 3. > ά

Continued elution of the Sephadex column with 2 liters of 100 mM ammonium bicarbonate and then with 2 liters of 200 mM ammon'um bicarbonate gave O-monophcsphate of tne (-) enantiomer cor>-esoc'ⁱc^;nc to the title compound, which *as stac’e to 5'-nuc’eotidase.

Example 12 (-1-cl 5-4-f2-Am100-6-(0vcloproov'methvlamino9H-our in-9-yl1-2-cyclopentene-l-methanol

The fractions containing O-monophosphate of the (+) enantiomer eluted from the Sephadex column of Example 11 were combined and a^]ka’ine phospnatase (EC 3.1.3.1) from calf intestine (4800 ID, Boen'-nger Mannheim) was added. The solution was incubated at 25°C for 18 hours, at which point HPLC analysis showed that all of the nucleotide nas been dephosphorylated. The solution was evaporated to dryness arc the residual solids extracted with refluxing ethanol (100 mL). The ethanol-soluble material was adsorbed on silica gel and applied to a silica gel column. Title compound was eluted with methanol: chloroform/l:9 as a colorless glass. An acetonitrile solution was evaporated to give white solid foam, dried at 0.3 mm Hg over Ρ-,Ο-; 639 mg (73% from racemate); ^H-NMR in OMSC-d^ and mass icectrum identical with those of the racemate (title comoound of Examo’e 6);

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Ana¹. Calcd. for C₁₅H_2QN₆0-0.1 CH-jCN: C

59.95; H, 6.72; N, 23.:6.

Found: C, 59.92; H, 6.80; N, 27.96.

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Example 13

4m 1 no - 2-eye ¹ open te^e - L - me faro ' d i benzoyl - D-tar fate (±)-c^js-4-AcetamldocvcIopent-2-enemethyl acetate ['JS Patent 4,268,572] ^14.38^, 0.073mo’' and barium hydroxide octahydrate (J-G.iGg, 3.146mc’; we^re refluxed ή water (3C0m1) under nitrogen for 18 hours. *he resulting solution was neutralised with carbon d’oxide. 'he precipitate was washed with water, then ethanol. The comoined filtrate wash was evaporated to a syrup, (acetic acid salt of (χ)-4-amiπο-2-cyclopentene-1-methanol) which was converted to ^iree amine by stirring with an excess of Amcerlite IRA-400 (CH‘) ^res^;n ^;n water. The resin was filtered off, washed with water, and the filtrate-wash evaporated to a pale yellow syrup which was dried by evaporation of portions of ethanol. Such a sample of amine (2.26 g, 20.0 mmol) and dibenzoyl-D-tartaric acid (Aldrich, 3.62 g, 10.0 mmol as 99%) were dissolved in het absolute ethanol (35 mL). Ref’uxing acetonitrile (ca. 150 mL) was added to the cloud point and tne solution was allowed to cool slowly to room temperature. The white needles which formed were recrystallized three times from the same solvent combination to give title compound as white plates (1.07 g,

(c = 0.28, methanol). X-ray crystallography of this salt allowed the absolute configuration of the cation to be fixed by the known configuration of the D-dibenzoyl tartaric acid dianion. This salt crystallized in the space group C2 with one C^H^NO cafcn and one-ha If C, g^gOg di an ion as the asymmetric unit.

round: C, 61.56; H, 6.24; N, 4.74.

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Example 14

IP A>'-4-Am’ro-2-C7Criceⁿ*erg-'·-met nano’ d^; benzov’-L-ate

This salt was formed and crystallized as described in Example 13, except that dibenzoyl-L-tartaric acid was used. Three crystallizations from ethano1-acetonitrile gave the title compound as white plates (l.OOg, 34%); m.p. 150-162°; [aj²⁰Q -58.2°, [aj²⁰J36 -169°, [aj²⁰365 -333°, (c = 0.24, methanol).

Anal. Calcd. for CgH^NO-1/2 (C^H^Og): C, 51.63; H, 6.21; N, 4.79. Fount: C, 61.59; Η, Y2I; N, 4.75*

Example IS ( = 1 -c^’s-N-TA-chloro-5-formamido-5-rr4-(hydroxymethyl )-2-cyclopentene1-yllamino1-2-pyrimidinyl1acetamide

N-(5-Amino-4,6-dichloropyrimidin-2-yl)acetamide (J. Orq. Chem. 1975, 40, 3141) was formylated by addition of 96% formic acid (20 ml) to a solution of (0.75 g, 3.4 mmoles) dissolved in acetic anhydride (20 ml). The resulting solution was stirred at 25°C for one hour and then evaporated to give N-(4,6-dichloro-5-formamido-2-pyrimidiny1)acetamide as tan powder (0.77 g, 91%); structure confirmed by ¹H-NMR and mass spectrum. This tan powder (840 mg, 3.37 .mmol), (=)-cis-4-amino-2-cyclopentene-l-methanol (940 mg, 8.2 mmol), and triethylamine (0.80 g, 8.0 mmol) were warmed in ethanol (50 mL) in an oil bath (70-80°C) under nitrogen for 50 minutes and evaporated to a dark oil which was chromatographed on silica gel. Title compound was eluted with 5% methanol-chloroform as a peach-colored solid foam (340 mg). Crystallization from methanol gave white granules (575 mg, 52%); m.p. 139-193°; ^!H-NMR (OMSO-d^) 5 10.23 (br, 1.0, NHAc),

9.3 (or, 1.0, NHCHO), 8.15 and 7.90 (both s, total 1.0, HC=O from two conformers, peaks coalesce at 60°C), 7.42 and 7.22 (both d, 0=8.3, bad ORIGINAL

6 I· 0 0 0 dV

MG.MF.21st November 1990

:. jc :n τι, 2. 2-u2Hi, .,0 „ r! J · ‘-NH ^from two cpnformers, peaks coalesce at oC’C«, 5.? arc 2.2, 2H=CH;, 5.25 .'m, 1, CH-Nj, 4.2 3 (m, 1, CH), 3.59 ,71, '2 (71,

CH'

:.40 (m,

1/2 CH-), 2.36 τι,

Anal. Calcd. for C^H^N-O-jCb C, 47.93; H, 4.95; N, 21.50; Cl, 10.38.

Found: C, 47.99; H, 4.96; N, 21.42; Cl. 10.96.

Exaapl· 16 (=)-c1s-4-Γ2-Ami no-6-(cyclopropyl ami no)-9H-purin-9-yl1-2-cyclopentene-l-methanol

The title compound of Example 15 (0.91 g, 2.79 mmol) was dissolved in dry DMF (1 mL). Triethylorthoformate (10 mL) and ethane sulfonic acid (0.29 mL, 3.4 mmol) were added and the solution heated at 65°C for 24 hours. The solution was evaporated to a syrup. The syrup was dissolved in IN HCl (15 mL) and stirred for three hours. The pH was adjusted to 7 with 5N sodium hydroxide and the resulting mixture (oil formed) was extracted with i-propanol:chloroform/l:3 (3 x 100 mL). The combined organic layers were dried (MgSO^) and evaporated to a red glass (0.93 g). A solution of this glass in methanol (20 mL) was heated with cyclopropylamine (2 mL) in a Parr bomb at 70°C for 18 hours. The resulting solution was evaporated to a dark glass which was adsorbed on silica gel. Elution with 7¾ methanol-ethylacetate gave title compound (148 mg, 19¾) as white powder, after trituration with acetonitrile; ^LH-NMR (DMSO-d^) identical with that of the title compound of Example 5.

MG.MF.21st November 1990

BAD ORIGINAL ?311?3

Exawle 17 (-¹ -' L9,43- c^; s-N - Γ4-Ch oro-5--ormam^;da-5--/4-(hydroxymethyl) 2-: vc1 ooen tene-1 - y 1 lami no'--2-pvr imi d^; ny 11 acetamide (-i,4R)-4-Ami no-2-eye 1 opentene-1-methano 1 dibenzoy1-0-tartrate prepared as described in Example 13 (2.75 g, 9.02 mmol) was dissolved in water (20 mL) and applied to a column of 65 mL of Amberlite lA-J-CC (QH'r'orm) anion exchange resin. The column was washed with water. Basic fractions were combined and evaporated to a residual oil which was dried by evaporation of absolute ethanol and then at 0.5 mm to give (lS.,4R)-4-amino-2-cyclopentene-l-methanol (1.2 g) as a pa'e yellow oil (darkens rapidly in air) which was used immediately. This oil was dissolved in ethanol (5 ml) and added to a solution of N-(4,6-dichloro-5-formamido-2-pyrimidinyl)acetamide (2.07 g, 8.31 mmol), prepared as described in Example 15, and triethylamine (2.50 g, 24.8 mmol). The resulting dark solution was heated (oi¹ batn 75-80°C) under nitrogen for 50 minutes. The solution was evaporated to a syrup which was applied to a silica gel column. Title compound was eluted with 3 to 5¾ methanol-chloroform as a pale yellow solid foam (1.59 g, 54%); ^H-NMR identical with that of crystallized sample. Such a sample was crystallized from ethanol to give white granules, m.p. 194-195°C; ^H-NMR (DMSO-d^) identical with that of the title compound of Example 15; [=»]²°0 *2.7°, M²°57g *3.6°, Η^2θ546 ‘2.9°, La]²⁰436 -2.5°, M²°₃₆₅ -41.2^s (c=0.238, methanol).

AP 0 0 0 1 9 6

Exawle 18 (-)-(lS,4R)-cis-(2-Amino-6-chloro-9H-purin-9-yl)-2-cyc1 opentene1-methanol

The title compound of Example 17 (1.15 g, 3.53 mmol) was gently refluxed in diethoxyImethyl acetate (45 mL) under nitrogen for

3.5 hours. The resulting pale yellow solution was concentrated at

BAD ORIGINAL

MG.MF.21st November 1990

3.5 mm -g to a yellow syrup. *ⁿe syrup was stirred in ’n hC’ '53 'S’· ... our. ,his sciutic.n was neutralized with sodium bicarbonate Arc a.acc^ated to dryness. The residual sol’ds were extracted w’ch metranol and the methanol-soluble material applied to a silica gel co^;umn. Elution of the column with 10¾ methano1-ethy1 acetate gave ft'e compound as a pale yellow solid foam ( 730 mg), 73¾); ‘H-WR (SMSO-d/: identical w’th that of racemate (title compound c*

Example 4); [_aJ^dUQ -114.9° (c = 0.26, MeOH).

Exawl· 19 (-)-(IS, 4R)-cis-4-Γ 2-Ami no-6 - (cycIopropylamino)-9H-purin-9-yn2-cyclopentene-1-methanol

The title compound of Example 18 (560 mg, 2.11 mmol) in metrarol (12 ml) was heated with cyclopropylamine (2.4 mL) in a Parr bomb at 78°C for 17 hours. The solvent was evaporated and the residue chromatographed on silica gel. Title compound was eluted with 5-7¾ methanol-ethyl acetate as a colorless solid foam (367 mg, 59¾); ^XH-NMR (CMSO-d^) identical with that of Example 7; -59.0° (c = 0.28,

MeOH) confirms the absolute configuration of the title compound of Example 7.

Exawl· 20 (1S, 4R)-4-Ami no-2-cyclopentene-1-methano1 dibenzov1-D-tartrate

2-Azabicyclo[2.2.1 ]hept-5-en-3-one [Daluge and Vince, J. Qrq. 3^hem. 1973, 43, 2311 and U.S. Patent 4,268,672] (44.0 g, 0.400 mole) was stirred in 2N HC1 in methanol (0.5 L) at 25 °C for 1.5 hours. Volatiles were evaporated to leave (±)-cis-methyl-4-amino-2-eyelopentene-l-carboxylate hydrochloride as an off-white powder (71.1 g).

MG.MF.21st November 1990 non ³31Iii

Tn'Cura τι. o . 32

2Μ^ς 2-1 *> · 1

J . - b •on of such a samp'e «if diethylether gave a «nite bCAde·, :-95’C i'J. Qrq. Cnem. 1982, 46, 3271; m.p. 32-33’Cj; f-WR ; 3.25 (br s, 3, NH,’., 5.1 and 5.9 (both m, 2, CH=CH), o,e<apping 3.75-3.5 (m, total 4, OMe and CM), 2.55-2.35 and

2.25-1.35 (both m, 2, CH^).

Ana¹. Ca’cd for C-H^NC^-HCl: Found: C, 47.41; H, 6.84; N,

C, 47.33; H, 6.31; N, 7.85; Cl, 19.89.

89; Cl, 19.95.

(= )-cis-Methy1-4-amino-2-cvc’opentene-1-carboxy late hydroch londe (17.7 g, 0.100 mole) and di isobutyl-aluminum hydride (0.500 mole as a 1 M so’ution in hexane) «ere refluxed in hexane (200 mL) for 6 hours. The resulting solution «as cooled and 10 ml of 1 M aqueous ammonium chloride and then methanol (200 mL) «ere added. This mixture «as refluxed for 30 minutes and MgSO^ (10 g) added. Solids were filtered off and «ashed with additional methanol. The filtrate-wash was evaoorated to a dark oil (15.5 g); ^H-NMR (DMSO-d^) identical to that of (=)-4-amino-2-cyclopentene-l-methanol prepared as described in Example 13. Such a sample, after purification by chromatography on silica gel (EtQH:CHC1:NH^OH/10:90:1) was crystallized with dibenzoyl-O-tartaric acid to form the title compound.

AP 0 0 0 1 9 6

Example 21

Γ cf-3-ί 2-Ami no-6-(cvc loproov lamino)-9H-pur in-9-vl)-2-cycl open ten1-yllmethvl R-2,3-bis-(hexadecanoyloxy)propyl hydrogen phosphate

A solution of L-a-dipalmitoyl phosphatidyl choline (150 mg, 0.2 mmol, Sigma) in 6 mL of chloroform was added to a flask containing ( = )-cis-4-(2-amino-6-(cyclopropyl amino)-9H-purin-9-yl)-2cyciopentene-l-methanol (300 mg, 1.03 mmol) .phospholipase D, Type VII (from Streptomyces, 1.0 mg, specific activity 185 units/mg, Sigma) and pH 4.5 buffer (1.5 ml, 250 mM in CaCl₂, 200 mM in NaOAc adjusted to bad ORIGINAL

MG.MF.21st November 1990 '3 . . .-3

ο.¹- addition of 3.1 N HC 1). 'he resulting biphase was sc^-ec at 4;·; ο-t batn,j *or 1 hour. ^rhe ’ayers were separated ana tne apjeCuS layer extracted with chloroform (3x5 mL). 'he cornered organ·: layers were washed with 1 N. HC 1, dried and concentrated. Such a sample was purified by elution from 2 silica gel columns with 12% methanol-chloroform to yield the title compound, 120 mg (47%). 'his mater’al was solidified using ethylacetate-acetonitrile to produce a lignt yellow powder m.p. 155-157°C; ^H-NMR (CO-jCD-CDC 1₃) S 7.73 fs, overlapping solvent, purine H-8), 6.12 and 5.88 (m, 2,. HC=CH),

5.53 (m, 1, CHN cyclopentene), 5.22 (m, 1, CO₂CH), 4.37 (dd, J=3, 12; 1, 0.5 POCH^ glycerol), 4.12 (m, 1, 0.5 P0CH.₂ glycerol), 3.42 (m, 4,

OCH_? glycerol, OCH₂), 3.11 (br m, 1, CH), 2.90 (m, 1, NCW), 2.78 (m, 1, 0.5 CH₂ cyclopentene), 2.27 (m, 4, 2CH₂C0₂), 1.70 (m, 1, 0.5 CH₂ cyclopentene), 1.56 (br m, 4, 2CH₂CH₂C0₂), 1.27 (br m, 38, 24 CH?),

O. 88 (m, 6, 2CHg), 0.83 (m, 2, CH₂ cyclopropyl), 0.60 (a, 2, CH₂ cyclopropyl).

Anal. Calcd. for C_4gHg₅N₆0gP-2.4 H₂0: C, 61.28; H, 9.42; N, 8.75;

P, 3.22.

Found: C, 60.97; H, 9.12; N, 8.78; P, 2.96.

ΕχμρΊ· 22

Tcis-4-(2-Ami no-6-(eyelopropy!amino)-9H-purin-9-yl)-2-cyclopenten1-vllmethvl R-2,3-bis-(hexanoyloxy)propyl hydrogen phosphate

A solution of L-a-dicaproyl phosphatidylcholine (300 mg, 0.66 mmol, S’lgma) in 15 mL of CHC1₃ was added to a flask containing (χ)-c^;s-4(2-ami no-6-(eyelopropylamino)-9H-purin-9-yl)-2-eye 1 opentene-1-metnano! (373 mg, 1.32 mmol), phospholipase 0, Type VII (from Streptomvces, 1.34 mg, specific activity 185 units/mg, Sigma), pH 4.5 buffer (4.5 mL, 250 nfd in CaCl₂, 200 mM in NaOAc adjusted to pH 4.5 with HC1) and CHCl^ (3 mL). The resulting biphase was stirred at 45’C (oil

MG.MF.21st November 1990 ** bad ORIGINAL

-:)- 3.. ;C

d a t n '	or 4	hours.	he	layers	were seoarated and the	organ·'* '	a:,er
*asned	,v': n	1 N HCI	(2 x	A ml; .	Τπθ comDined aqueous ’	avers «ere	bac'x
wasred	w- th	cn loroform	(10 mL),	’he combined organ-	c layers	*ere
cr · ad	(Mg SC	>4) and	concentrated. The residue was	placed on	' a
silica	ge 1	column	and	the	title compound was	eluted	with

16% methanol-chloroform and concentrated to yield a fine yellow powder. This material was dissolved in ethanol and concentrated (3 x 50 mL) before drying under high vacuum to yield 103 mg (21% yield) of a light yellow powder, m.p. 182-185°C.

*H-NMR:

6.09 (m, CH=CH, NH (DMSO-d^)

1,

7.61 (s, 1, purine H8), 7.22 (br s, 1, NH), 0.5 CH=CH), 5.89 (m, overlapping br s at 5.83, 3, 0.5

1, CHN), 5.09 (br m, 1, CO₂CH), 4.30 (dd;

₂), 5.41 (br in,

J=2.7, 12; 1, 0.5 POCH₂ glycerol),4.08 (m, 1, 0.5 P0CH₂ glycerol), 3.80 (br m overlapping br m at 3.75, 4, 0CH₂ glycerol, OCH₂), 3.02 (br m, 2, CH, NCH cyclopropropyl), 2.65 (m, 1, 0.5 CH₂ eyelopentene), 2.23 ( + , J=7.5, 4, 2 CH₂C0₂), 1.48 (br m, 5, 2 CH₂CH₂C0₂, 0.5 CH₂ cyclopentene), 1.23 (br m, 8, 2 (CH₂)₂), 0.84 (m, 6, 2 CH^), 0.67 and 0.58 (m, 4, 2 CH₂ cyclopropyl).

Anal. Calcd. for 0₂₉Η₄₅Ν₆0θΡ-3.9 H₂0, 0.2 CHC1₃, 0.05 EtOH: C, 48.00; H, 7.33; N, 11.46; Cl, 2.9.

Found: C, 48.65; H, 6.61; N, 10.81; Cl, 2.5.

The preceding example is an adaptation of the procedure by Satoshi Shuto et ak Tetrahedron Letters, Vol. 28, No. 2, pp. 199-202, 1987.

Example 23

N-'A-Chloro-1,6-dlhydro-5-nitro-6-oxo-2-pyrImid iny1)isobutyramide

6-Chlcro-5-nitroisocytosine (J.Chem.Soc. 1960, 5041; J.Org.Chem, 1975, 40, 3141) was protected by heating the yellow solid (14.88g, 78.09 mmol) to 100°C for one hour in isobutyric anhydride (250ml) and

MG.MF.21st November 1990

BAD ORIGINAL

RSI 1Η concentrated sulphu-’c acid «3-4 drops). The resulting solution was treated with anhydrous metnanol ;LCOml), stirred at 50°C for half an noun, concentrated to a th'ro of one orig-nal <o’ume, and toe tit’e compound (I4.97g, 74%) was collected by filtration as pale yellow crystals; m.p. 196-199°C (dec); Ή-NMR (0M$Q-d6) 5 1.12 (d, J=6.9, Hz, 5H, (CH₃)₂CH), 2.75(m, J=5.9, Hz, 1H, (CH^CH), 12.41 (Pr s, Ή).

Ana¹. Calcd for C_gH_gN₄0₄C1: C, 36.87; H, 3.48; N, 21.50; *1, 13.50. Found. C, 36.94; H, 3.48; N, 21.44; Cl, 13.53.

Exawl· 24

N-(4.6-Dichloro-5-nitro-2-pyrinridinvl)isobutvramide

The title compound of Example 23 (10.Og, 38.37 mmol) was heated to reflux in phosphorus oxychloride (200ml) and Ν,Ν-diethylani1ine (3-4 drops) for 5 hours under nitrogen. The solution was then cooled to room temperature, concentrated to dryness, and the syrup was dissolved in cold (~-10°C) methylene chloride (200ml). The organic layer was treated with saturated aqueous sodium bicarbonate (100ml) with vigorous stirring,· and the temperature was kept below 5°C as solid sodium bicarbonate was added portionwise to elevate the pH to between 5 and 7. The layers were separated and the aqueous phase was extracted with methylene chloride. The combined organic layers were ^f;'tered over phase-seoarator paper, concentrated and dried under vacuum to give the title compound (7.71g, 72%) as a yellow-white sol’d sufficiently pure to employ in the next step. Recrystallisat-on of the solid from hexane/methylene chloride provided an analytical sample, m.p. 166-169°C; Ή-NMR (0MS0-d6) & 1.09 (d, J=6.9Hz, 6H, (CH₃)₂CH), 2.79 (m, J=6.9Hz, 1H, (CH^CH), 11.51 (s, 1H).

Anal. Calcd. for CgHgN^C^: C, 34.43; H, 2.89; N, 20.08; CI, 25.41. Found: C, 34.53; H, 2.89; N, 20.02; Cl, 25.40.

MG.MF.21st November 1990

BAD ORIGINAL ?32 2 A3

Example 25

N- 4 3-Q*cn'orc-5-formanr Co - 2-pvr^;d^;ny1)isobufyramide

The title compound of Example 24 (6.77g, 24.26 mmol) was placed in a

Parr bottle containing 220ml absolute EtOH and 10.Og (wet) Raney nickel catalyst that had been previously shaken under hydrogen (4Cpsi) for 10 minutes. The mixture was shaken under hydrogen (40psi) for an hour, filtered over celite, and the filtrate was concentrated to a yellow-white solid that was dried under vacuum overnight. This so’id was stirred in 1,2-dichloroethane (250ml) at 0°C. Acetic anhydride (30ml) was added, followed by formic acid (30ml), dropwise under nitrogen. The resulting mixture was stirred at room temperature for 2 hours, concentrated to half the original vlume, and azeotroped with toluene to remove residual formic/acetic acid. The crude solid was triturated with methanol to give the title compound (4.92g, 73%) as an off-white solid; m.p. 206-209°C (dec); Ή-NMR (DMS0-d6) 5 1.08 (d,

J-6.8HZ, 6.0 (CH₃)₂CH), 2.74 (m, J=6.8Hz, 1.0 (CH₃)₂CH), 8.13 (d,

J=L0.3Hz) and 10.26 (br s) [total 1.0, NHCHO from two conformers], 11.17 (br :, 1.0).

Anal. Calcd. for CgH^N^C^: C, 39.01; H, 3.64; N, 20.22; Cl, 25.59. Found: C, 39.13; H, 3.68; N, 20.12; Cl, 25.67.

AP 0 0 0 1 9 6

Example 26 f - M IR,4$)-cis-N-r4-Chloro-5-formamido-6-'iΓ4-(hydroxymethyl '- 2-~.: 'a peⁿtene-l-vllaminoT-2-pyrimid invllisobutyramide.

(1S..4R)-4-Amino-2-cyclopentene-l-methanol dibenzoy1-D-tartrate (2.44g, 3.15mmol) prepared as described in Example 13, was dissolved in 90¾ ethanol (20ml) and the solution added to a column of Amberlite ISA-400 (OH*) resin (30ml) which had been prewashed with the same so^rv%nt.

bad origin*¹.,MG.MF,21st November 1990 with 90¾ ethanol gave oasic fractions wnich on concentrafcn arc evaporat^;on of portions of to’uene-ethano1, ie’t i L S_, -if-4-ami no-2-cyc looentene-1 - methanol as pa'e yellow o·' i'L.Ag wr·;·' ,vas condensed immediately with N-(4,5-d;chloro-5-formamido-2pynm'dinyl isobutyramide (2.26g, 3.15mmol) prepared as described an Example 25, in 1,2-dimethoxyethane (ICCml) with triethylamine (2.3ml, L6.3mmol) at 95-110^C for 1.5 hours. The resulting solution *as evaporated to a dark yellow syrup which was chromatographed on silica gel. Elution of column with 5-7.5¾ methanol-chloroform gave the title compound as pale yellow solid (2.45g, 84¾). Crystallisation of such a sample from acetonitrile gave the title compound as fine white crystals, mp. 194.5-195.5°C.

Ή-NMR (DMSQ-dg) 5 10.21 (s, 1, NHCOCHMep, 9.29 (s, 1, NHCHO), 8.12 (s, 1, CHO), 7.18 (d, J=7.9, 1, CHNH), 5.8 and.,^ (both ra, 2, CH=CH), 5.08 (m, 1, CHN), 4.71 (t, 0=5.06, 1, OH), 3.37 (m, 2, CH.₂OH), 2.9-2.6 (m,2, CHMe. and CH), 2.40 (m, 1, 0.5CH₂), 1.33 (m, 1, 0.5CH₂); 4.4°, 20.7° (c=0.237, MeOH).

Anal.Calcd. for C^H^NgClO^

Calcd : C, 50.92; H, 5.70; N, 19.79; Cl, 10.02.

Found : C, 50.67; H, 5.78; N, 19.62; Cl, 9.92.

ExmpI· 27 (-)-(IR,4S)-cis-N-f6-(cyclopropvlamino)-9-(4-(hydroxymethyl )-2cvclooentene-l-yl)-9H-purin-2-yl1isobutyramide (-)-(IR,4S)-cis-N-f4-chloro-5-formamido-6-K4-(hydroxymethyl )-2cyclopentene-l-yl]ami no}-2-pyrimidinyl]i sobutyramide (1.949g,

5.44mmol) prepared as described in Example 26, was stirred with triethylorthoformate (30ml) in an ice-water bath while concentrated hydrochloric acid (2.0ml) was added dropwise over two minutes. The resulting clear solution was stirred at ambient temperature overnight.

BAD ORIGINAL

MG.MF.21st November 1990

A3

PSL196

The /o’ati’es were removed under vacuum and the residual Sv-uc /cnta’n’ng a (IP , AS)-cs-N-f5-ch loro-3-f A-hydroxymethy1)-2-cyc ’o pencere-l-yt )-9H-pur^;π-2-y1 ] ’soDutyramide orthoester conjugate; refluxed ’n ethanol (30ml) with cyclopropylamine (lOg) for 2.5 hours. Evaporation left a syrup which was dissolved in 10% isopropanolchlorofcrm (20Cml). This solution was stirred vigorously with saturated aqueous sodium bicarbonate (25ml). The organic layer was separated and the aqueous layer washed with additional 10% isopropanoI-chloroform. The combined organic layers were dried (MqSOj). Evaporation left a pale yellow glass (2.4g) which was chromatographed on silica gel. The title compound was eluted with 2-3% methanol-ethyl acetate as a white solid (1.02g, 53%);

recrystal 1isation of such a sample from methanol-acetonitrile gave the title compound as white needles; mp. 197.5-198.5°.

Ή-NMR (OMSO-dg) 5 9.75 (s, 1, NHCO), 7.93 (s, 1, purine H-8), 7.82 (br s, 1, NH-cyclopropyl), 6.12 and 5.92 (both m, 2, CH=CH), 5.50 (m,

1, CH-N), 4.73 (t, J»5.3, 1, OH), 3.46 (m, 2, CH₂-O), 3.25-3.00 (m, 2, CHMe₂ and CH), 2.91 (m, 1, CH), 2.75-2.6 (m,l, 0.5CH₂), 1.7-1.6 (m, 1, 0.5CH₂), 1.07 (d, J=6.8, 6, CHMe₂), 0.75-0.6 (m, 4, 2 cyclopropyl,

CH₂); [_q]2° -70.7°, Μ4θ₆- 159.0° (c»1.02, MeOH).

Anal Calcd. for C|gH₂₄NgO₂:

Ca’cd : C, 60.66; H, 6.79; N, 23.58

Found : C, 60.62; H, 6.83; N, 23.51.

Continued elution of the column with 5% methanol-ethyl acetate gave additional title compound contaminated by ca. 10% of f-)-(l$.4R)-cisA-j2-ami no-5-(cyclopropyl ami no)-9H-purin-9-y1]-2-cyclopentene-1metnanol as a pale yellow solid foam (928mg).

6l 0 0 0 dV _bM) owew*L

MG.MF,21st November 1990

-g::

Exawl· 28 ’,3,4R;-;is-4-f2-Amino-6-(cvciopropv1amino)-9H-purin-9-yl eye 'opentene-1-methanol (- )-( IR ,4S)-cis.-N-[ 6-(eye 1 opropyl ami no )-9-(4-(hydroxymethyl )-2cyc ’opentene-L-y1)-9H-purin-2-y1Jisobutyramide (1.33g, 3.73mmol) prepared as described in Example 27, was stirred with IN hydrochloric acid (4QmL) for 2 days at ambient temperature. The pH was adjusted to 7.0 with sodium hydroxide and the mixture evaporated to dryness. ’he residual solids were triturated with hot EtOH (3x25ml). The ethanol was evaporated to leave yellow glass which was chromatographed on silica gel. The title compound was eluted with 3% methanol-ethyl acetate as a colourless solid foam (857mg, 80%), Ή-NMR and [α]θ°, identical with that of the title compound of Example 19.

Exawl· 29 (-MlS^Rl-cis-A-i^-Amino-e-icyclopropylaminol-gH-purin-g-vll-Zcvc1 opentene-1-methanol hydrochloride.

(-)-(1$,4R)-cis-4-Γ2-Amino-6-(cyclopropylamino)-9H-purin-9-yl ]-2cyclopentene-l-methanol (1.90g, ca. 6.3mmol by Ή-NMR) was dissolved in IN hydrochloric acid (7.0mL) and ethanol. The solution was evaporated to dryness and the residue redissolved in ethanol (15ml). Ethyl acetate was added slowly, to a total volume of 80ml. The offwhite powder which formed was filtered off and dried under vacuum to give the title compound (2.07g, 97%); mp. collapses at 125-130°, dec. above 138°C, -27.1°, [₃]2θ₆ -52.3° (c=0.199, MeOH).

Anal. Calcd. for C^H^gNgO.HCl .0.8H₂0:

Calcd : C, 49.87; H, 6.15; N, 24.92;

Found : C, 49.91; H, 6.16; N, 24.96;

MG.MF.21st November 1990

Cl, 10.51.

Cl, 10.52.

BAD ORIGINAL

- 45 P3;:;a

Exawl· 30 .-- 15 . 4R ,i^-c * 3-4-^2-Ami ηο-ό - (eye lopropy¹ aml^o) - ?H-our i π-? - y i ~-2e->e lcoentene-l-methanol Oinydrochloride.

( -)-(IS, 4R)-cis-4-Γ2-Amino-6-(eyelopropylamino)-9H-purin-q-y1]-?cyclooentene-l-methanol (857mg, 3.00mmol) was dissolved in ethanolethyl acetate and IN ethereal hydrochloric acid (12ml) was added. The fine white precipitate was washed with ethyl acetate and dried - ar vacuum to give the title compound (642mg, 75%); mp. 176-180° dec.

Ana’. Calcd. for C^H^NgO.ZHCl :

Calcd. : C, 46.81; H, 5.61; N, 23.39; Cl, 19.74,

Pound. : C, 46.82; H, 5.64; N, 23.33; Cl, 19.67.

Exawl· 31 (IR ,4S)-4-( 2-Amino-6-(eyelopropylamino)-9H-purin-9-yl)-2-cyclo pentene-1-methanol Q-diphosphate (*)-( 1R,4S)-4-(2-Amino-6-(cyclopropyl amino)-9H-purin-9-y1)-2cyclopentene-l-methanol O-monophosphate, prepared as described in Example 9, was converted to the triethyl ammonium salt by taking a solution containing 0.5 mmol of the monophosphate as the ammonium salt, combining it with 10 ml of 0.5 M triethylammonium bicarbonate and drying in vacuo, followed by another addition of 10 ml of 0.5M triethylammonium bicarbonate, then drying. Then, three times, 10 ml of acetonitrile were added and dried in vacuo. This was dissolved in 10 ml of 1,3-dimethy1-3,4,5,6- tetrahydro-2(lH)- pyrimidinone (Aldrich) then 0.43 g of 1,1'-carbonyl diimidazole (Aldrich, 2.6 mmol) was added and stirred for 2 hours at room temperature. Methanol (0.18 ml, 4.5 mmol) was added and stirred for 30 minutes. Tri butylammonium pyrophosphate (Sigma, 1.2 g, 2.6 mmol) was added,

BAO ORIGNAL

6 l 0 0 0 dV

MG.MF.21st November 1990

- 4ό PS..ίό stirred for 13 hours at 'oom temperature, then 1 5 of add'hO-a’ t-•rit.,^: ammon :um pyrophosp^ha te .2.2 mmc '' was added and ifr^eo 3 ^ηουΓ5 at 4Q u, then oO ml of water was added. 9oth O-oioncspnate aⁿd 0-tr-pnospnate were formed since the tributyl ammonium pyropnospnate contained orthophosphate impurity.

The reaction products were separated by OEAE Sephadex ion exchange chromatography in a 2.5 x 18 cm column of OEAE Sephadex 425 (Pharmacia) which had been equilibrated with 50 mM of ammonium bicarbonate (ABC). The column was washed with 1 1 of 50 mM A8C then with a 2 1 linear gradient of 50 to 800 mM ABC to elute the title comccund followed by triphosphate, as described in more detail in Example 32. The fractions containing diphosphate were combined, dried jn. vacuo, redissolved in water then dried again to yield the ammonium salt of the title compound (0.077 mmol, 15 % yield). UV scan: in 0.1 M HCl λ max = 254 and 298 nm; at pH 7 x max - 259 and 284 nm: in 0.1 M NaOH x max = 259 and 284 nm.

An aliquot of diphosphate was treated with alkaline phosphatase (calf intestine, Boehringer Mannheim), sampled at various times and developed on thin layer chromatography (PEI- cellulose, Brinkman, 1M LiCl/lM formic acid 1:1). A sequential conversion of diphosphate to monophosphate to nucleoside was observed. The final amount of phosphate released was determined by the method of Bencini (Bencini, D.A., Wild, J.R., and O'Oonovan, G.A. Analytical Biochemistry 132:254-258 (1983)) and the base/phosphate ratio was determined to be 1.0/11.5, indicating the presence of inorganic phosphate. UV purity was 99.8% on analytical HPLC (strong anion exchange column eluted with a gradient of lOmM to 1M ammonium phosphate, pH 5.5).

Exawl· 32

M-( IR ,4S)-4-(2-Aroino-6-(cyclopropylamino)-9H-purin-9-yl )-2-cvc^vd pentene-1-methanol 0-triphosphate

BAD ORIGINAL

MG.MF.21st November 1990

- a; ?s::33

Cont-nued e'ution of the to'umn desorbed in Sxamp'e 31 gave, on e/apona:;on, the ammonium salt of the title compound, his salt «as converted to the sodium sa't by passage through a Ocwex AG 5Cl*-<3 (3ic-Rad) resin column (sodium form, 20 ml). The fractions containing nucleotide «ere concentrated in vacuo to yield 0.31 mmol (51 %). iJV scan: in 0.1 M HC1 a max = 254 and 299 nm; at pH 7 a max = 259 and 234 nm: in 0.1 M NaOH \ max = 259 and 284 nm. Optical rotation in «ater at 3.83 g/ 100 ml was [a] 20 = *43.2° at 589nm. UV purity «as 39.1¾ on analytical HPLC (strong anion exchange column eluted with a gradient of lOmM to 1M ammonium phosphate, pH 5.5) «ith 0.9 % diphospnate present. An aliquot of triphosphate «as treated «ith alkaline phosphatase (calf intestine, Soehringer Mannheim), sampled at various times and developed on thin layer chromatography (PEIcellulose, 8rinkman, 1M LiCl/lM formic acid 1:1). A sequential conversion of triphosphate to diphosphate to monophosphate to nucleoside was observed. The final amount of phosphate released was determined by the method of Sencini (Sencini, D.A., Wild, J.R., and O'Donovan, G.A. Analytical Biochemistry 132:254-258 (1983)) and the base/phosphate ratio was determined to be 1.0/2.7.

AP 0 0 0 1 9 6

Exawlo 33 (lS,4R)-4-(2-Amino-6-(cyclopropy1anrino)-9H-Durin-9-yl)-2cvclopentene-1-methanol Q-diphosphate

The (-)-(IS,4R)-4-(2-Amino-δ-(cyclopropylami no)-9H-purin-9-yl)-2-cyclo pentene-l-methanol O-monophospnate, prepared as described in Example 8, was converted to the triethylammonium salt by taking a solution containing 0.49 mmol of the monophosphate as the ammonium salt, combining with 5 ml of 0.5 M tri ethyl amnionium bicarbonate and drying in vacuo, followed by another 5 ml of 0.5M triethylammonium bicarbonate then repeating twice. Then, three times, 5 ml of acetonitrile were added and dried j_n vacuo. This was dissolved in 7 ml of 1,3-dimethyl-3, 4,5,6-tetra hydro-2(lH)-pyrimidinone (Aldrich) bad ORIGINAL

MG.MF.21st November 1990

- 48 ps:;98

2.4 mmc^: Methano1 Tri butyl ammonium of tetrasodium wd S (0.15 tnen 3.39 5 of 1,1’- carbonyl diimiqazole (Aidrioh aoded and stirred for 30 minutes at room temperature, mi, 4.3 mmo') was added and stirred for 30 minutes, pyropnoscnate (made by exchanging the salt pyrophosphate for hydrogen on an ion-exchange resin, then neutralizing with tr 1'butylamine and drying, 2.4 mmol) was added, stirred ^for 18 hours at room temperature, then 50 ml of water was added. 3otn

O-diphosphate and O-triphosphate were formed since the tributylammonium pyrophosphate contained orthophosphate impurity.

The reaction products were separated by DEAE Sephadex ion exchange chromatography in a 2.5 x 18 cm column of DEAE Sephadex 425 (Pharmacia) which had been equilibrated with 50 mM ammonium bicarbonate (ABC). The column was washed with 1 L of 100 mM ABC then with a 2 L linear gradient of 100 to 800 mM ABC to elute the to elute the title compound followed by the triphosphate as described in more detail in Example 34. The fractions containing diphosphate were combined, dried in vacuo, redissolved in water then repeated twice to yield the ammonium salt of the title compound (0.032 mmol, 6 % yield). UV scan: in 0.1 M HC1 λ max = 254 and 298 nm; at pH 7 λ max = 259 and 284 nm: in 0.1 M NaOH λ max = 258 and 284 nm.

An aliquot of diphosphate was treated with alkaline phosphatase (calf intestine, 8oehringer Mannheim), sampled at various times and developed on thin layer chromatography (PEI- cellulose, Brinkman, 1M LiCl/lM formic acid 1:1). A sequential conversion of diphosphate to monophosphate to nucleoside was observed. The final amount of phosphate released was determined by the method of Bencini (Bencinf,

3.A., Wild, J.R., and Q'Oonovan, G.A. Analytical Biochemistry 132:254-258 (1983)) and the base/phosphate ratio was determined to be 1.0/4.7, ’ndicating the presence of inorganic phosphate. UV purity was 97% on analytical HPLC (strong anion exchange column eluted with a gradient of lOmM to 1M ammonium phosphate, pH 5.5).

bad original

MG.MF.21st November 1990

P81198

Example 34 ·, 13.4Ρ '· - 4- ί 2-aml πο-6 - (c vc looropy 2 am* no ; - 9H-pur I π - 9-y > '· - 2-cvc lo pentene-1-metnanol Q-triphosphate

Continued elution of the column described in Example 33 gave, on evaporation, the ammonium salt of the title compound. This salt was converted to the sodium salt by passage through a Dowex AG 50W-X3 (8io-Rad) resin column (sodium form, 20 ml). The fractions containing nucleotide were concentrated in vacuo to yield 0.4 mmol (81 %). UV scan: in 0.1 M HCl a max = 254 and 299 nm; at pH 7 a max = 259 and 284 nm: in 0.1 M NaOH a max = 259 and 284 nm. Optical rotation in water at 6.14 g/ 100 ml was [a]20 = -47.1° at 589nm. UV purity was 99.5 % on analytical HPLC (strong anion exchange column eluted with a gradient of 10mM to 1M ammonium phosphate, pH 5.5) with 0.5 % diphosphate present. An aliquot of triphosphate was treated with alkaline phosphatase (calf intestine, Boehringer Mannheim), sampled at various times and developed on thin layer chromatography (PEIcellulose, Brinkman, 1M LiCl/lM formic acid 1:1). A sequential conversion of triphosphate to diphosphate to monophosphate to nucleoside was observed. The final amount of phosphate released was determined by the method of Bencini (Bencini, O.A., Wild, J.R., and O'Donovan, G.A. Analytical Biochemistry 132:254-258 (1983)) and the base/phosphate ratio was determined to be 1.0/2.8.

Ex aw 1b 35 (l$,4R)-4-f2-Amino-6-(cvclopropvlmethvlamino)-9H-purin-9-vl1-2cvclopentene-1-methanol (IS,4R)-4(2-Amino-6-ch1oro-9H-purin-9-y1)-2-cyclopentene-1-methanol (274mg, 1.00 mmol), N-cydopropyl-N-methylamine (0.71g, lOmmol), and absolute ethanol (6 mL). The residue was chromatographed on silica

6 I 0 0 0 dV _BAO OWQ’NMMG.MF.21st November 1990

- 50 PS1198 gel. The Title compound was eluted with 10% methanol-chlorc*orm as a solcr'ess g'ass. Evaporation of an ethanol solution and drying with pnoscnorus oentoxide at 0.2 mm Hg gave the title compound as a white solid foam (293 mg, 98%); ^LH-NMR and [a]²⁰589 identical with those of the title compound of Example 11.

Example A

Taoiet Formulations

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

MG.MF.21st November 1990

BAD ORIGINAL

PS::98

-ormu’it ion A
	mq/’tao'et	mq/tab !et
(a) Active ingredient	250	250
(b) Lactose 8.P.	210	26
(c) Povidone 3.P.	15	9
(d) Sodium Starch Glycollate	20	12
(e) Magnesium Stearate	5	3
	500	300
-onmu’ation 9
	mq/tab’et	mq/tablet
(a) Active ingredient	250	250
(b) Lactose	150	-
(c) Avicel PH 101	60	26
(d) Povidone B.P.	15	9
(e) Sodium Starch Glycollate	20	12
(f) Magnesium Stearate	5	3
	500	300

AP 0 0 0 1 9 6

Formulation C	mq/tablet
Active ingredient	100
lactose	200
Starch	50
?ovi done	5
Magnesium stearate	4 359
The following formulations,	D and E, are prepared

compression of the admixed ingredients. The lactose in formulation £ is of the direct compression type (Dairy Crest - Zeparox).

MG.MF.21st November 1990 bad original

- 52 ?s::;s at-on g mg/tab 'et

Active ingredient 250

P^rege'atinised Starch NF 15 150

400

Formulation E mg/tablet

Active ingredient

Lactose

Avicel

250

150

10£

500

Formulation F (Controlled Release Formulation)

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

(a) (b)	Active ingredient Hydroxypropy1 methy Ice 1lulose (Methocel K4M Premium)
(c)	Lactose B.P.
(b)	Povidone B.P.
! a '< \ w V	Magnesium Stearate

Drug release takes place over a complete after 12 hours.

mg/tablet

500

112

700 period of about 6-8 hours and is

BAD ORIGINAL

MG.MF.21st November 1990

- OJ

P8L193

Examp'e 3

ZiOSu'e -ormu1at^;ons

Formulation A

A capsule formulation is prepared by admixing the ingredients of Formulation D in Example A above and filling into a two-part hard gelatin capsule. Formulation 8 (infra) is prepared in a similar manner.

Formu’ation 3 mg/capsule (a) Active ingredient 250 (b) Lactose B.P. 143 (c) Sodium Starch Glycol late 25 (d) Magnesium Stearate 2

420

Formulation C mg/capsule (a) Active ingredient 250 (b) Macrogol 4000 B.P. 350

600

AP 0 0 0 1 9 6

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

Formulation D

Active ingredient Lecithin Aracnis Oil mg/capsule

250

100

100

450

BAD ORIGINAL

MG.MF.21st November 1990 λ****»''¹

ΡβΙΙϊβ

Capsules or formulation 0 are prepared by dispersing the acc-e •oredien: in the lecithin and arachis oi! and filling the discers-on ’to soft, elastic gelatin capsules.

Formulation E (Controlled Release Capsule)

The following controlled release capsule formulation is prepared by extruding ingredients a, b and c using an extruder, followed by spheronisation of the extrudate and drying. The dried pellets are then coated with release-controlling membrane (d) and filled into a two-piece, hard gelatin capsule.

mq/capsule

(a)	Active ingredient	250
(b)	Microcrystalline Cellulose	125
(c)	Lactose B.P.	125
(d)	Ethyl Cellulose	13 513

Example C

Injectable Formulation

Formulation A.

Active ingredient

Hydrochloric acid solution, 0.1M, or Sodium hydroxide solution, 0.1M q.s. to pH Sterile water q.s. to

0.200 g

4.0 to 7.0 10 ml

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

MG.MF.21st November 1990 bad ORIGINAL

A3:

into a sterile 10 ml amber glass vial (type 1) and sealed with ster^’e ;'osjres and oversea 1s.

,-or-nu^:at^;on 3.

Active ingredient Sterile, pyrogen-free, pH 7 phosphate buffer q.s. to	0.125 g 25 ml
Examole D
Intramuscular injection
Active ingredient	0.20 g
Benzyl Alcohol	0.10 g
Glycofurol 75	1.45 g
Water for Injection q.s. to	3.00 ml
The active ingredient is dissolved in the glycofurol. The benzyl
alcohol is then added and dissolved, and water added to 3 ml. The
mixture is then filtered through a	sterile micropore filter and sealed
in sterile 3 ml amber glass vials	(type 1).
Example E
S vruD
Active ingredient	0.25 g
Sorbitol Solution	1.50 g
Glycerol	2.00 g
Sodium Benzoate	0.005 g
Flavor, Peach 17.42.3169	0.0125 ml
Purified Water q.s. to	5.00 ml

AP 0 0 0 1 9 6

BADOfMG'N*¹MG.MF.21st November 1990

- 5ό P81L98 ιhe active ingredient is dissolved in a mixture of tne qlycerol arc most of the purified water. An aqueous solution of tne socum oenzoate is then added to t.ne solution, followed by addition of tne sorbitol solution and finally the flavor. The volume is made up with purified water and mixed well.

Example F

Suppository mq/supoos I tory

Active ingredient HJUmV’ , 250 ·' Hard Fat, 8P (Witepso1 H15 - Oynamit -WeSel) 1770 \ 2020

-7 / »£h_e active ingredient -is-usad-as a powder wherein- at--least 90% o-f *he particles-are of 634m· diameter· or less.

z

One-fifth of the Witepsol H15 is melted in a steam-jacketed pan at

45°C maximum. The active ingredient is sifted through a 200 S’eve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45°C, the remaining Witepsol H15 is added to the suspension and stirred to ensure a homogenous mix. The entire suspension is passed through a 250 Mm stainless steel screen and, «'th continuous stirring, is allowed to cool to 40°C. At a temperature of 38°C to 40°C, 2.02 g of the mixture is filled into suitable, 2 ml plastic molds. The suppositories are allowed to cool to 'oom temperature.

BAD ORIGINAL

MG.MF.21st November 1990

PSI158

Examp'e G

Pessaries

Active ingredient Annydrate Dextrose Potato Starch Magnesium Stearate mq/pessary

250 380 363 7

1000

The above ingredients are mixed directly and pessaries prepared by direct compression of the resulting mixture.

. Antiviral Activity

a) Anti-HIV Activity (IS,4R)-cj_s-4-[2-amino-6-(cyclopropyl ami no)-9H-purin-9-yl]-2cyclopentene-l-methanol was tested for anti-HIV activity in MT^ cells according to the method described by Averett, 0. R., J. Virol, Methods. 23 1989, 263-276 and was found to have an

ΙΟςθ value of 4.0 * 1.4 μΜ (average of 10 determinations).

b) Antl-HBV Activity

The human HBV producer cell line of HepG2,2.2.15, described and characterised by Sells et al.. PNAS 84: 1005, 1987 and J.V:rol. 62: 2836, 1988 has been shown to share many characteristics of che HBV chronically infected hepatocyte. It is infectious as demonstrated by the ability to cause disease in chimpanzees.

To test compounds for anti-HBV activity, monolayer cultures were treated with the test compound: 50-200μΜ for ten days.

MG.MF.21st November 1990 BAD OWQ'^nM9 6 l 0 0 0 dV

- 53 PB1198

Supernatant media containing extrace 1lular virion ONA 'Dane part’t’es) were harvested on days three, six and ten, treated w'tn proteinase K (1 mg/ml) and sodium dodecyl sulfate (1%), and incubated at 50°C for one hour. DNA was extracted with equal volumes of phenol followed by chloroform and then precipitated by ammonium acetate and propanol. The DNA precipitate was dissolved and collected on nitrocellulose using the procedure of Schleicher and Schuell (S4S, 10 Optical Ave., Keene, NH 03431, Publication #700, 1987), and treated as described by Southern, J.Mol.Biol., 98, 503, 1975. Cells were harvested, and the ’htracellular DNA was obtained after cell lysis with guanidine -sothiocyanate. The int-acellular DNA was handled in the same manner as the extracellular ONA. After precipitation by ammonium acetate and propanol, the intracellular DNA precipitate was dissolved, cut by restriction endonuclease, Hind III, applied to agarose gel and then treated as described by Southern to determine the quantity of replicative intermediate forms. The antiviral effect of the drug was determined by measuring at least a 100-fold reduction of the amount of Oane particles extruded into the culture medium and a similar decrease in the intracellular replicative intermediates.

(IS, 4R)-cis.-4-[ 2-Ami no-6-(N-cyclopropyl-N-methy lamino )-9H-purin9-y1]-2-cyc1 opentene-1-methano1 was tested by the above procedure and found to have potent anti-HBV activity at 100μΜ.

Claims (29)

1. Enantiomeric compounds of the general formula (I) (wherein R represents a cyclopropyl ami no or N-cyclopropyl-Nmethyl amino group and A represents the 2-cyclopentene-1-methanol-4-yl group in either the (1S.4R) or (1R,4S) configuration) and their derivatives, the said compounds and their derivatives each being in the form of an enantiomer substantially free of the corresponding enantiomer.
2. 2. (1S.4R)-cis-4-r2-Amino-6-(cvclopropylamino)-9H-ourin-9-vl1-2cyclopentene-l-methanol substantially free of the corresponding (1R,4S) enantiomer.
3. 3. (lS,4R)-cis-4-[2-Amino-6-(N-cyclopropyl-N-methylamino)-9H-purin9-yl]-2-cyclopentene-l-methanol substantially free of the corresponding (1R,4S) enantiomer.
4. 4. Pharmaceutically acceptable salts, esters and salts of esters of the (1S,4R) enantiomeric compounds of formula (I) claimed in any of the preceding claims.
5. 5. Phosphate derivatives of the (1R.4S) enantiomeric compounds of formula (I) claimed in claim 1.

   AP 0 0 0 1 9
6. 6

   MG.MF.21st November 1990

   BAD ORIGINAL

   PB1198CC

   - 60 6. Pharmaceutical formulations comprising a (1S,4R) enantiomeric compound of formula (I) (as defined in claim 1), a pharmaceutically acceptable derivative thereof or a phosphate derivative of a (1R,4S) enantiomeric compound of formula (I) (as defined in claim 1),together with at least one pharmaceutically acceptable carrier or excipient.
7. 7. Pharmaceutical formulations as claimed in claim 6 in the form of tablets or capsules.
8. 8. Pharmaceutical formulations as claimed in claim 6 or claim 7 wherein the said (1S,4R) enantiomeric compound of formula (I) is (1S. 4R)-cis-4-f2-aniino-6-(cyclopropyl amino)-9H-purin-9-vl1-2cyclopentene-l-methanol.
9. 9. A (1S,4R) enantiomeric compound of formula (I) (as defined in claim 1), a pharmaceutically acceptable derivative thereof or a phosphate derivative of a (1R,4S) enantiomeric compound of formula (I) (as defined in claim 1) for use in medical therapy.
10. 10. Use of a (1S,4R) enantiomeric compound of formula (I) (as defined in claimj) a pharmaceutically acceptable derivative thereof or a phosphate derivative of a (1R.4S) enantiomeric compound of formula (I) (as defined in claim 1) in the manufacture of a medicament for the treatment or prophylaxis of a retroviral infection or a hepatitis 8 virus infection.
11. 11. Use as claimed in claim 10 of (1$,4R)-cis-4-f2-amino-6-(eyele propylamino)-9H-purin-9-yl ]-2-cyclopentene-l-methanol.
12. 12. Use as claimed in claim 10 or claim 11 for the manufacture of a medicament for the treatment or prophylaxis of a human immuno deficiency virus (HIV) infection.

    MG.MF.21st November 1990 OR'G^AL

    PB1198CC
13. 13.

    A)

    13.

    A)

    A process for the preparation of compounds of formula (I) (as defined in claim 1) and their derivatives which comprises:treating an enantiomeric compound of formula (II):Z

    B)

    Β) (wherein A is as defined in claim 1 and Z represents a precursor group for the said R group as defined in formula (I)) or a derivative thereof with an agent or under conditions serving to convert the precursor Z group to the desired R group; or reacting an enantiomeric compound of formula (III):AP 0 0 0 1 9 6 (wherein A and R are as defined in claim 1, R represents hydrogen or a formyl group and R represents an amino protecting group) or a derivative thereof with an agent serving to effect formation of the imidazole ring in the desired compound of

    MG.MF.21st November 1990

    - 62 PB1198CC formula (I) and to effect removal of the R^ amino protecting group; or

    C) reacting an enantiomeric compound of formula (IV):- (wherein A and R are as defined in claim 1 and R^ is an amino protecting) or a derivative thereof with an agent serving to effect removal of the R^ amino protecting group, and optionally effddting one or both of the following conversions In any desired order: i) where a compound of formula (I) is formed, converting the said compound to a derivative thereof; or ii) where a derivative of a compound of formula (I) is formed, converting the said derivative to the parent compound of formula (I) or to a further such derivative.
14. 14. Enantiomeric compounds of formula (II):-

    A bad original

    MG.MF.21st November 1990

    - 63 PB1198CC (wherein A is as defined in claim 1 and Z represents a halo group) and derivatives thereof.
15. 15. (lS.4R)-cis-(2-Amino-6-chloro-9H-purin-9-yl)-2-cyclopentene

    -1-methanol substantially free of the corresponding (1R,4S) enantiomer.

    Enantiomeric compounds of formula (III):

    (HI) (wherein A and R are as defined in claim 1, R represent hydrogen or a formyl group and R represents a alkanoyl group) and derivatives thereof.
16. 17. Enantiomeric compounds of formula (IV):-

    MG.MF.21st November 1990

    BAD ORIGINAL $

    PB1198CC

    - 64 (wherein A and R are as defined in claim 1 and represents a alkanoyl group) and derivatives thereof.
17. 18. (IR.4Sl-cis-N-i6-(Cyclopropylamino )-9-(4-(hydroxymethyl )-2cyclopenten-l-yl)-9H-purin-2-y1]isobutyramide substantially free of the corresponding (1S,4R) enantiomer.
18. 19. Enantiomeric compounds of formula (V):- (wherein A is as defined in claim 1, Z represents a halo group,

    2 3

    R represents hydrogen or a formyl group and R represents a C|_g alkanoyl group) and derivatives thereof.
19. 20. (IR.4$)-cis-N-r4-Chloro-5-formamido-6-((4-(hydroxymethyl )-2cyclopentene-l-yl)amino)-2-pyrimidinyl]isobutyramide substantially free of the corresponding (1S,4R) enantiomer.

    Enantiomeric compounds of formula (VI):- (VI)

    MG.MF.
20. 21st November 1990

    BAD ORIGINAL

    - 65 P81198CC (wherein A is as defined in claim 1, Z represents a halo group, and represents a g alkanoyl group) and derivatives thereof.
21. 22. (IS.4R)-cis-N-r6-Ch1oro-9-(4-(hydroxymethyl)-2-cvclopentene-ly1)-9H-purin-2-yl]isobutyramide substantially free of the corresponding (1R,4S) enantiomer.
22. 23. Enantiomeric compounds of formula (VIIIA) or (VIIIB):- (VIIIA) (VIIIB) and derivatives thereof.

    AP 0 0 0 1 9 6
23. 24. (lS,4R)-4-Amino-2-cyclopentene-l-methanol and the corresponding dibenzoyl-D-tartrate, substantially free of the corresponding (1R,4S) enantiomer.
24. 25. (lR,4S)-4-Amino-2-cyclopentene-l-methanol and the corresponding dibenzoyl-L-tartrate substantially free of the corresponding (1S,4R) enantiomer.
25. 26. Compounds of formula (VII):MG.MF.21st November 1990

    BAD ORIGINAL

    - 66 PB1198CC n AR¹ (VII) (wherein Z represents a halo group, R represents a hydrogen or a

    Ο Λ formyl group, R^J represents a Cj_g alkanoyl group and R⁴ represents a halo group) and derivatives thereof.
26. 27. N-(4,6-Dichloro-5-formamido-2-pyrimidinyl)isobutyramide.

    Compounds of formula (IX):- (IX) (wherein Z, and R⁴ are as defined in claim 26) and derivatives thereof.
27. 29. N-(4,6-0ichloro-5-nitro-2-pyrimidinyl)isobutyramide.
28. 30. Compounds of formula (X):-

    BAD OFUG^,NAL $ .

    MG.MF.21st November 1990

    PB1198CC (wherein and R^ are as defined in claim 26) and derivatives.
29. 31. N-(4-Chloro-l, 6-dihydro-5-nitro-6-oxo-2-pyrimidinyl) isobutyramide.