AP73A - Therapeautic nucleosides. - Google Patents

Abstract

The present invention relates to

Description

The [upspnl invention rnlalps Co 6 - subs C i. tuted 2' ,3' -didcoxynuc1eosides , pharmaceutically acceptable derivatives thereof, and their use in therapy, particularly for the treatment or prophylaxis of certain viral infections.

AIDS is an immunosuppressive or Lminunodestructive disease that predisposes subjects to fatal opportunistic infections. Characteristically, AIDS is associated with a progressive depletion of T-cells, especially the In· 1 pi’ r - i nthice i: subset hearing the OKT surface marker.

Human linmunodef Ic i ency Virus (HIV) has been reproducibly isolated from paLients with AIDS or with the symptoms that frequently precede AIDS. HIV is cytopathic and appears to preferentially infect and destroy T-cells bear i ng the Oi<T * marker , and it is now generally recognized that HIV Is tire 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 “Ifeetive in treating AIDS sufferers. Thus, for example, European Patent Specification No, 196185 describes 3azido - 3’-deoxy thymidine (which has the approved name zidovudine·), its pharmaceutically acceptable derivatives and their use in the treatment of human retrovirus infections including Ait’S and associated clinical conditions.

European Patent Publication No. 0206497 relates generally to purine nucleosides for use in the treatment of HIV infections and ijd.iud conditions. In particular this publication discloses 2,6-diaminopurint?-¹)/! · D - 2 ' , 3didcoxyr ibofuranos ide for the treatment of HIV infections.

U’e have now discovered that certain 6- subs ti tuted 2' , 3 ' -dideoxynucle.osides . as referred to below, are useful for the treatment or prophylaxis of viral infections, particularly retroviral infections and especially ALDS.

NJB/KT/AC/9th March 1988 λ

Β50'?

Cerf./1 Ln 6 - subs Li Luted purine nucleosides have previously been described, and in par [.icu I a r 6 - me thy 1 aininopur ine - 9 - β - D - 2 ' - , 3 ' - d i deoxy r ibo fur a nos ide , described hereinafter lor its use in the treatment of HIV infections and related conditions, has been disclosed in Bioorg Khim 9(1) 52-59 (1983).

in a first aspect of the present invention, there are provided novel 6-substituted 2',3'- dideoxynucleosides having the following general formula (1)

AP 0 0 0 0 7 3 wherein R^ represents hydrogen or amino; and R^ represents halogen (e.g. chlorine), G^ _f alkoxy (e.g. propyloxy or isopropoxy), optionaiiv substituted for example by G^ cycloalkyl (e. g. cyclopropylincthoxy) ; G., „ eye loalkyloxy (e.g. cyclobutyioxy or cyclopentyloxy); aryloxy (e.g. pbenyloxy), aralkyl (e.g. benzyl) or aralkyloxy (e.g. benzyloxy) in which the aryl may optionally be substituted with lower alkyl, hydroxy or halogen; cycloalkylthio; alkylthio; arylthio, or aralkylthio in which the aryl may optionally be substituted with lower alkyl, hydroxy, or halogen; or R.^ represents a heterocyclic group containing an oxygen atom or one nr two nitrogen atoms, and 3-7 carbon atoms with optional double bonds in the ring (e.g. piperidino, pyrroLidino or furfuryl) optionally containing a sulphur and/or oxygen heteroatom and optionally substituted on the ring by one or more lower alkyl, hydroxy or halogen groups, θ cycloalkylthio, aralkylthio In which the aryl may be substituted with lower alkyl, hydroxy or halogen; or R^ represents an imidazolylthio group in which the iinidazolyl moiety may be substituted with lower alkyl and/or C-substituted with nitro;

NJB/KT/AC/9th March 1968

SAD ORIGINAL Ά

3» ο

7» ο

&

fc* in· , ι j, ι ' ί η s an ,υ,ι ι ι j; ι ο' ιρ ',·;!) i ch i s mono- or d j. - subs l i l ιι ι ed b·.· ι a)kvi < < . g. methyl, or -u.iv.l.), h alkoxy (e.g. methoxy), hvdroxv C alkvl if·./.. Iivdroxye Lliyl·) ,τιυΐ/ον eycloalkyl (c.£. cyclopropyi. or cy.· loppotiyl) . aryl· (e.g. phenyl·), aralkyl· (e.g. benzyl) in which the aryl may optionally be substituted with lower alkyl, hydroxy or halogen, aLlyl optionally substituted with mono- or di-alkyl or alkoxy groups (e.g. diinethylallyl) ; and represents hydrogen or aiaino, and pharmaceutically acceptable derivatives thereof other than the compounds of formula (I) in which R^ and R^ represent hydrogen and represents a methoxy, inethylthio or methylainino. Examples of substituted amino groups represented by K, in formula (1) include elbylainino, ethylmethylamino, cyclopropyl amino and i. sop ropy I am i no

The above references to lower alkyl denote groups containing I to 6 carbon atoms preferably methyl, or ethyl. The references to halogen include chlorine, bromine, iodine and fluorine, chlorine and iodine being particularly preferred.

Preferred classes of the compounds of formula (1) include those in which E and R^ represent hydrogen and R^ represents a substituted amino group, for example a ιηοηο-C^ eye ioalky lamino group, a mono- or di- C alkvlami.no group or a heterocyclic group such as piperi.di.no or pyrrolidino.

The following compounds are preferred compounds of the present invention:1. 6 -N-Pipe ridinopurine- 9-ft-D-2' ,3'- dideoxyribofuranos ide . 6 - Chloro pur ine - 9 -/3- D - 2 ' , 3 ' - dideoxyrib of uranos ide . 6 - Etby lamino pur ine -') -(3 - D - 2 ' , 3' - dideoxyribofuranos ide . 6 - Ethyl me thy lamino - 9 -il - ϋ - 2 ' , 3' - dideoxyr ibofuranos ide . 6-1 o do pur ine - 9 - /3 - D - 2 ' , 3 ’ - dideoxyr ibofuranos ide . 6 - Cyc lop ropy Lme thy Lam Lnopur ine - 9 - /) - D - 2 ' , 3'- d ideoxyr ibofuranos ide

7. 6 -1 sopropylaminopurine - 9 - /3 - D - 2 ',3'-dideoxyribofuranos ide

8. Thiamiprine- 9-β-D-2' , 3'-dideoxyribofuranoside

NJB/KT/AC/9th March 1988

13G'' in i no - Ij - ii p ropQx ·.·(· ir i u<·· - ’' -/>' - 1' ' - ci ideoxyr i fun f iminos i ib

Hi i I

- (, t li v 1 th i op’iv i m· -9 - /3 -1) - 2 ' • 1 i deoxyr ibofuranos i cle

1.6

Lz

9

2D λ 1 ? 2 ,’3

1(1

S3 a 2 p

3?

6(.) .

/. I

- Am ί ιιυ - G - benzy L t h i opur i up. -9 - p -1) - 2 ' , 3 ' - d ideoxyr ibo Curanos ide

- E ( 1P '.·: ypu r i ne - 9 - /3 - D - 2 ' , 3 ' - <1 i deoxy r ibo [uranos ide

- Dime thy 1 ami no pur inc -') -β - D - 2 ’ , 3 ' - dideoxyr ibofuranos ide

- Hydroxy e thy laininopu fine - 9 - /3-D- 2 ' , 3' - dideoxyr ibofuranos ide

- Cyc Lop ropy laininopu r ine- 9 -β- D- 2' , 3' - dideoxyr ibo furanoside

- Cyc iopenty laminopur ine - 9 -/3- D- 2 ' , 3' -dideoxyribofuranos ide

2-Amino - 6-methoxypurine- 9 - /3 - D-2 ',3'-dideoxyribofuranoside

- n - Propoxy pur ine - 9 - /3 - D - 2 ’ ,3'-d ideoxyr ibofuranos ide

- n - F.i 1Coxypur i no - 9 - β - D - 2 ' , 3 ' - dideoxyr ibofuranos ide

- (:-r_: | op ropy 1 me thoxypur i ne - 9 - β - D - 2 ' . 3 ' - dideoxyr ibo furano s ide

- Cyc Lope n tyloxypur ine - 9 - /3 - D - 2 ’ , 3 ‘ - dideoxyr ibofuranos ide

- Cvc 1 oliexy loxypur ine -/3 -1) - 2 ' . 3 ' - dideoxyribofuranos ide

- Cyc Lob u ty Lam i no pur inc - 9 -/3 - D- 2 ' , 3 ’ - dideoxyr ibofuranos ide

-1) to thyLaminopur ine-9-β-D-2' ,3'-dideoxyribofuranos ide · Pyr fo 1. id i.nopur inc - 9 - /3 - D - 2 ' , 3 ' - dideoxyr ibofuranos ide

- ilo r pho J j nopur i no - 9 - /3 - 0 - 2 ' . 3 ' - d ideoxyr ibo f uranos ide

- y , y - L) ime 11 iv La ily laminopur ine - 9 - /3 - D - 2 ' , 3 ' - dideoxyr ibofuranos ide

- hir fur v Laminopur i.ne - 9 - /3 - D - 2 ' , 3 ' - dideoxyr ibofuranos ide

- bonny Line reap to pur ine - 9 -/3 - 0 - 2 ' , 3' - dideoxyr ibofuranos ide

- An i 1 ί i io pur ine - 9 - /j - D - 2 ' , 3 ' - dideoxyr ibo f uranos ide

- Am i. no - 6 - e thoxvpi i r ine - 9 -/3 -1) - 2 ’ , 3 ' - dideoxyr ibofuranos ide

2,6.5 - Tr i a ini no pur ine - 9 -/3 - D- 2 ' , 3 ’ -dideoxyribofuranos ide

- Amino - 6 - benzyl ami nopur ine - 9 - /3 - D - 2 ' , 3 ' - dideoxyr ibofuranos ide

- Amino-6 -cyclopropy Laminopurine-9-/J-D-2' ,3'-dideoxyribofuranos ide

- Am i no - 6 - ine thy la ini no pur ine - 9 - /3 - D - 2 ' , 3 ' - dideoxyribofuranos Lde

- Amino - 6 - n- propoxy pur ine - 9 -/3-D- 2 ' ,3' - dideoxyr ibo furanoside

- Benzy laminopur ine - 9 - /3 - D - 2 ' , 3 ' - dideoxyr ibofuranos ide

-1 sopropoxypurine - 9 - /3 - D - 2' , 3 ‘ - dideoxyr ibofuranos ide

- Propylaminopurine - 9 - /3- D - 2' , 3' - dideoxyr ibofuranos ide

- Cyc lobexy lamino - 9 - /3 - D - 2 ' , 3 ' - dideoxyribofuranos ide

- He thy laminopur ine - 9- /3-D-2' ,3' - dideoxyribofuranos ide

AP 0 0 0 0 7 3

HJB/KT/AC/9th March 1988

BAD ORIGINAL j

; ;', ί ) ’ i comp. ” mils )., 6, L3,13,16. 2 3 and 61 above are pa r t i eu lar Ly p re ί > r red _O1) ,-. cc,,' ii it <>f thⁿir surprisingly hii;ii anti-iliV activity.

liic compounds of formuLa (1) above and their pharmaceuticaliy acceptable Jevivatives, also Including the compound of formula (I) in which R^ is hvdrogen and R? is mcthylamino, referred to in the above Bioorg. Khin, reference, are hereinafter referred to as the compounds according to the invention.

In one aspect of the invention there are provided the compounds according to the invention for use in medical therapy particularly for the treatment or prophylaxis of retroviral infections.

Examples of retroviral infections which inav be treated or prevented in accordance with the invention include human retroviral infections such as Human Immunodeficiency Virus (HIV), HIV-2 and Human T-cell Lymphotropic Virus (HLTV) e.g. HTLV-I or HTLV-IV infections. The compounds according, to the invention are especially viseful for the treatment or prophylaxis of AIDS and related ciincial conditions such as AIDS-related complex (ARC), progressive generalised ivmphadenopathy (PGL), AIDS-related neurological conditions, such as multiple sclerosis or tropical paraparesis, antr-i ί IV antibody-positive and li .1 V - pos i t ive conditions, Kaposi’s sarcoma and th roml.iocy topen i a purpura. The compounds may also be us^'l in the ' re.,; me n; or prevention of psoriasis.

Ln a further aspect of the present invention there is included : A method for the treatment or prophylaxis of retroviral infer which comprises treating the subject with a Lherapcu; i effective amount of a compound according to the invention.

ions a 1 1 ·;

bi Use. of a compound according to the invention in the manufacture of a medicament for the treatment or prophylaxis of an;· of Live above-mentioned infections or conditions.

N.IB/KT/AU/Vth March 1988

BAD ORIGINAL ft rp tab 1 S-iH:, ester •eej.' ah 1 e ilerivaLive is meant any pharmaceutical 1 ; ΊΓ sail· uC such ester, of a compound according l·’ [’·.<> i nv°n t i nil or anv oilier compound which, upon administration to the C' ·i p i ·- n i, is capable of providing (directly or indirectly) a compound according. to the invention, or an anti.viraLly active metabolite or residue ( hereof .

Preferred esters of the compounds of the Invention include carboxylic acid esters in which the non-carbonyl inoiety of the ester grouping is selected from straight or branched chain alkyl e.g. n-propyl, t-butyl, n-butyl, alitor.·,-a l.kyl (e.g. inethoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g,.

APO 00 0 7 3 (e.g. luethanesulphonyl); amino acid esters (e.g. L-valyL or L-iso leucyl); and mono-, di - or tri - phosphate esters.

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

Any reference to any of the above compounds also includes a reference to a pharmaceutically acceptable salt thereof.

Examples of pharmaceutically acceptable salts of tlie compounds according in the invention and pharmaceutically acceptable derivatives thereof include lease salts, eg derived from an appropriate base, such as alkali moral (e.g,. sodium), alkaline earth metal (e.g. magnesium) salts, ammonium and lix' (wherein X is alkyl). Physiologically acceptable salts of a hydrogen aioni or an amino group include salts of organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesu1fonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, suLfuric, phosphoric and sulfamic acids. Phys i o log, i.ea 1 1 / acceptable salts of a compound with a hydroxy group include tin· anion of

III I’./il /AC/'H I. tlarch 1988

BAD ORIGINAL o5d‘J .·ι ϊ <Ί compound in combination with a suitable cation such as I .'a . Nil * . and ολ (wherein X is a C, , alkvl r.coun'i .

1. - n

Specific examples of pharmaceutically acceptable derivatives of the compound of formula (I) that may be used in accordance with the present invention include the monosodium salt and the following 5' esters: monophosphate; disodium monophosphate; diphosphate; triphosphate; acetate;

- methyl-butyrate; octanoate; palmitate; 3-chloro benzoate; benzoate; A-methyL benzoate; hydrogen succinate; pivalate; propionate; valerate and mn·.· late.

The above compounds according to the invention and their pharmaceutically acc-optable derivatives may he employed in combination with other therapeutic agents for the treatment or prophylaxis of the above infections or conditions. Examples of such further therapeutic agents include agents that are effective for the treatment or prophylaxis of HIV infections or associated conditions such as 3 ' - az i do - 3 ’ - deoxy tliym i d ine (zidovudine), other 2 ' , 3 ' - d ideoxynucleosides such as 2 ' , 3dideoxycytidine, 2 ' , 3' - did^oxy •Tihivsinc and 2 ' , Jd i.deoxy inos ine , acyclic nucleosides (eg acyclovir), interferons such as o-interferon, renal excretion inhibitors such as proben i c i d. nucleoside transport inhibitors such as dipyridamole, as veil ,is i ininui lomodu I a t ο r s such as interleukin ii and granulocyte macrophage colon·, stimulating factors. The component compounds of such combination therapy may be administered simultaneously, in either separate or combined formulations, or at different times, e.g. sequentially such that a combined effect is achieved.

the compounds according l.o the invention, also referred to herein as ί he active ingredient, may be administered for therapy by any suitable unit:·’ 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 varv with the condition and age of the recipient, the nature of the infection ami the chosen active ingredient.

N.J B/KT/AC/9 th March 19811

BAD ORIGINAL β 30‘?

hi i',·' ii rn 1. a si t i Labi c dose will lie Lu the range of. 3.0 io 120 mg per I. i I i Ί-υ I,.» ly v.>j.gh! ο I i.l )·? recipient per day, preferably in L.he range of 6 n. °0 in-,-. per I: i Ingram body weight per day and must; preferably in tiie vanr.e 14 lo tai in'·, per kilogram body weight per day. The desired dose is preCerabLy 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 forms, 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.

Ideally, the. active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 1 to about 75 /iii, preferably about to 50 /iii, most preferably about 3 to about 30 /ill. This iiipv lie achieved, for example, by the intravenous injection of a 0.1 to 5¹. solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1 to about 100 mg/kg of the active ingredient. Desirable blood levels may be maintained by a continuous infusion to provide about 0.01 to about 5.0 mg/kg/hour or by intermittent infusions containing about 0.4 to about 15 mg/kg of the active ingredient..

AP o n 0 0 7 3 rhi I.e il is possible for the active ingredient to be administered ,i!mm ii is p i. c f. e r.-ib i.e to present it as a pharmaceutical formulation. Tii·· formulations of the present invention comprise at Least one active ingredient, as defined above, together with one or more acceptable carriers I hereof and optionally other therapeutic agents. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. formulations incind,· those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral, (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations in.r,· conveniently be presented in unit dosage form and may be prepared by ,ur; methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitute-s one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the fUb/KT/AC/ytb March 1988

BAD ORIGINAL Cyp “V, *·

Ο

Ο cs net ]-·<' i i';-, i vj L’ · ri L with ί ί«ρ.ιΐ«1 tnrciers of finely divided solid cnriiers or both. and : hen it nr <- ⁿ··/> 1’ ·.· shap int; I.he produe I..

l-'eruHil nt i on;; ο Γ the present invention suitable Cot' oral administration may lie presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or 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 act ive ingredient, may also be presented as a bolus, electuary or paste.

Λ tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropyImethyL ceLlulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, c.ross-linked sodium carboxymethyl cellulose) surface - act ive or dispersing agent.. Moulded tablets may be made by moulding in a suitable machine a mixture of the. powdered compound moistened with an inert liquid diluent. The tablets 'nay optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein nsing, for example, hydroxypropy line thy I cellulose in varying proporlimis to provide the desired release profile. Tablets may optional. 1.7 !>·'· provided with an enteric coating,, to provide release in parts of the gut other than (he stomach. This is particularly advantageous for purine nucieositle. derivatives as such compounds are susceptible to acid hydrolysis.

formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usual!·.· sucrose and acacia or tragacanth; pastilles comprising tiie 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.

I'ormu I a t i ons for rectal, administration may be presented as a suppos i t or·.· with a suitable base comprising for example cocoa butter or a salicylate.

N.l P./KT/AC/')t h Ma rch I ^r)RR

BAD ORIGINAL Cj

Lu -

. ’ r nr I I 1 i i a	suitable	i'o i: ·.·a,-, iaia L administration ma y	be	present··'!
(. ; s 1' i e ; ,	tampons, ci.	i' ;iuis ,	('•.els, pastes, foams or	;pray	lormulati o
on l a i n inr.	i 11 add ί f i ο n	Ln llir	active ingredient, such car	r ί e r s	as are kno
η 1.1 ie art	in be appropi	i a ί r .

Γ'οπιιυ lat i ons sui table for parenteral administration include aqueous and non.iqueous isotonic sterile injection .solutions which may coni a i n anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-close or multi-dose sealed containers, for example, ampoules and vials, and may bo ct.ored 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 desc r i.bed .

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

ad ft n ft 0 7 3

Ihe compounds according to the. invention may aLso be presented lor use in the form of veterinary formulations, which may be prepared, for example, bv methods that are conventional in the art.

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

The present invention further includes a process for the preparation oi a compound according to the invention and pharmaceutically acceptable derivatives thereof which comprises either:

N.f B/KT/AC/9 th March 198,.

BAD ORIGINAL

Γ O ft ft fl A (wherein Rj , ^anc* ^3 are ^as hereinbefore defined and A represents a precursor group for the hydroxy group, or for a pharmaceutically acceptable derivative group thereof) with an agent or under conditions serving to cmiverL the said precursor group into the corresponding desired group; or reacting a • urine lja.se ot formula (HI) (wherein B is the required purine moiety of a compound according to the i ΙΙ'.’,'ΙΐΙ i Ol) ) .

or a functional, equivalent thereof, with a compound serving to introduce the desired rihofuranosvl ring at the 9- position of the purine base of formula dll);

and 1.hereafter, or s i mo 1 ta neons Ly therewith, effecting one or more ol :.1,-f e i 1 ov.· log optional conversions: (ii when a compound of formula (1) is formed, converting it into a pharmaceutLeal1v acceptable derivative thereof, (, i i) when a pharmaceutical Ly acceptable derivative of a compound of formula

(I) i.s formed, converting	the said	derive t ive	into a	compound ο 1
formula (1), or a different	de r iva t ive	thereof .
in the above - described process	according	to the invention,	it	will lx
appreciated that the precursor	compounds	of formula	(I) as wt	Ί1	as 111 r

above-mentioned agents and conditions, will he selected from those ihat ire known in the art of nucleoside synthetic chemistry. Examples of such i!JB/KT/AC/9th March 1988

BAD ORIGINAL

ΒΙΟ·’ c'ii’.· I 'umi pi ncriiiirr-.s .31(-- dose r i lied lie re ina f to r feu' git Ldance and it will ho iiikI.'isi (ιού i h -r ί hey can be modified in conveiii ional manner depending on the i!«sii’p'l compound of formula (I). In particular. where a conversion is ib-sc r ibe.d which would otherwise result in the undesired reaction of labile groups then such groups may be protected in conventional manner, with subsequent removal of the protecting groups after completion of the conversion.

With regard to process (.Λ), Λ may represent a protected hydroxy group e.g. an ester grouping of the type referred to above in relation to formula (I) particularly acetoxy, or an ether group such as a trialkylsilyloxy group, e.g. t- but vid lrne thvls i. Ivloxy or an aralkoxy group e.g. t r ipheny Ime thoxy. Such groups may be converted for example by hydrolysis to the desired hydroxy group or, by transesterification, to an alternative ester group.

AP 0 0 0 0 7 3

With regard to process (B), this may be effected for example by treating an appropriate purine base of formula (III) or a salt or protected derivative thereof, with Jdeoxy thymidine for example in the presence of the appropriate pentosyl transferring enzyme.

Λ compound of formula (I) may he converted into a pharmaceut i c ai 1v acceptable phosphate or oilier ester by reaction with respectively a phosphor?lating agent, e.g. I’OCL^ or an appropriate esterifying agent, e.g,. an acid halide or anhydride. The compound of formula (I), including esters thereof, may be converted into pharmaceutically acceptable salts thereof in conventional manner, e.g. by treatment with an appropriate base. An ester or salt of a compound of formula (I) may be converted into the parent compound, e.g. by hydrolysis.

lhe following Examples are intended for illustration only and are not intended to limit the scope of the invention in any way. The term 'active ingredient' as used In the Examples means a compound of formula (.1) or a pharmaceutically acceptable derivative thereof.

Ex ample_1

NJB/KT/AC/9th March 1958

BAD ORIGINAL ι_ιΗι //1/17331.1/ i' - H i’i.per

’))' 11''t in i.’.vl. mmo L , 0.5g, Sigma Chemicals, St. Lot;

HO) was

After cooling to room temperature 3'-deoxyi.hyini dine (3 . 62minol, 0.82g)(Howitz,J .P. et a I. J· Org. them. j_l., 205 ( 1966)) was added along with 30ml of lOtitM potassium phosphate buffer with «a pH of 6.8 containing 0.04% potassium azide.

dissolved in iOml of d ime thy is· 11. Co:·; ide with hen

'•<*r
	Puri f ied	thymidine pho
-Tz	phosphory	lase (20,000 1..
	1/7 3 1 and	US i’a tent A . )3 1
*··*. ·*·««·	wo i e add'	•vl, .and the ,.u
j3	re,-ic t. i on	was filtered, ;
P*		......

itirred at 35^LC.

After 8 hours the columns . i lie initial roiiumi contained L ΛΖ hyuroxi.de resin (7.5 x lOemt while the second column was filled wi th Ainberlite XAD-2 resin (2.5 ;·; 20cm) . After sample application, the columns were washed with a large volume of water and the product was eluted with inethanol. After removal of the solvent, and redissolving in chi orofonn: methanol (9:1, v/v) , additional chromatography was performed on a column containing silica gel (5 X 20cm). 'L'lie mobile pha.se was c'n 1 o rof orm: me lhano 1 (9:1, v/v). Product containing tractions wore combined, redissoLved in ethanol, and filtered through a 0.2? μ filter. The ethanol was evaporated, and the product was red i ssm 1 ve.l in water. After lyophilization, the 6-N - pipe r i di nopur iue - o - b-7.' . 3 ’ ί hydrate coutainiuii 0. i

d i do ,.v ·,· r ibo 1. u r a nos i. do	1.0.	265g)	analyzed as a
e thanoI .
Anal. Calcd. for C j II	N 21 5	0? 0.3	W:
Cal cd.: C, 58.74; H,	7 . 2 /	; o’, 2	1.96
Found: C , 58.86; H, 7	• 14;	N, 21	.82
Hilli: Λ 8.36 (s, 1 11,	Η,,ι,	3 . 15»	( s , l Η , H ,) , 6
11, J - 5.54, 011 ) , 6 j	. 1 2	(m , 5	Π, H. , , C1L) , 3 '4 Z
1! , 11 , ) , 2.04 (in, 2 II	' 3	,). 1.	61 (b. 2 H, CH.,) z

Examp_le_,2.

- Chloropur inc - 9 - <3-1) - 2 ' , 3 ' - di deoxyr ibofuranos ide

HIR/KT/AC/91h March 198»

BAD ORIGINAL

i. i 11 - s ·,·η rl i ,·,-. i s of 6 - ch loropur ine - 9-/1-0-2 ’ , 3 ' - d ideoxyr ibofuranos ide v.is P'· i i; - imp rl us ,' n ; < ·: · · · i a, I in Example 1 except. that Che 6 - chloropur inc (3 i gma bhemicals, St. . L.oui s Ho) was dissolved in 5 m I each of d ime thy iforinam i de and i.l i me thox ye ! i in i k- .

After filtering off the solids, the filtrate was reduced to —5ml under vacuum then dissolved in 100ml water. This material was chromatographed on a 2.5 X 20 cm column containing XAD-2 resin. After washing this column with 500ml of water, the product was eluted with methanol. Product containing I tactions were combined and 20ml of dry silica gel added. All solvent was einoved under vacuum. The dry silica gel was applied to the top of a silica el column equiLibrated with chloroform: methanol ('J : i , v/v). Product onl.aining fractions free of deoxythymidine were combined, and after removal, of the solvent under vacuum, the residue was dissolved in ethanol, filtered, then dissolved in water and lyophilized. This material was further purified bv chromatography on a methanol;water (8:2, v/v).

AP 0 0 0 0 7 3 column containing Polygosii resin in

Alter removal of the solvent in vacuo, the product was dissolved in water and lyophilized yielding 0. Γ-'Gg u( b - eh 1 oropur ine - 9-/3-D-2 ' , 3' -dideoxyr ibofuranoside (mp - 100°C).

Anal, baled, lor C, 11,,bid,0,:

1011 A 2 baled.: b, A/. 16; ii, 4. 33; 11, 22.00; Cl, 13.92 i'nund: I,, A/. 1.0; !i, A.’,? Id, 2 1.. 9A ; 01. 13.86

Exampie 3

- Ethyl aniinopur ine - 9 - d - 0 - 2 ' , 3' - dideoxyr ibofuranos ide b E I. by 1 am inopur ί ne (prepared by uueLeophi1ie displacement of the chlorine gioup ou f> - ch I oropur i uc (Sigma Chemicals, St. Louis Mo) by the amino group oL ethvlamine) (2.69mmol, 0.5g) and 3'-deoxythymidine (Ilorwitz, J.P. e 1: nJ, 1 . Οι g. Ghent. , 3.1 205 ( 1966)) (3.33mmol, 0.755g) were combined along with 30ml of l.Oim’l potassium phosphate buffer with a pH of 6.8, containing 0.04? potassium azide. Purified thymidine phosphorylase (400 1. U.) and purine nucleoside phosphorylase (/00 1. U.) were added and the suspension was

NJB/KT/AC/9th March 1988

BAD ORIGINAL

t.) Γ) J Ο 9

ΓΙ' ic I ' ' '.' i ‘ I !

ad:!-', ,ut(

V. Λ(ί ρ! ms ρΙ m ι ·,' 1 .-· ·:. 1,1 ic r·'· ;tc : ί ί ή

ί. no'trs an adml. ι οηηΐ Zt.u.) units of purine 'u'H units of. tp.'/midinu p! mspl ic ι ·: 1 a se wpi·,· 0

H. i at 3/ G. Five days later the reaction

..•as filtered, and tit (i Itralc was applietl to a column containing AG - 1. X?

hydroxide resin 1 2 . x idem). The product was eluted with a water wash and chromatographed on AmherLite ΧΛ0-2 resin (2.5 x 20cm), After sample application, this column was washed with a large volume of water. The product was eluted with methanol. After removal of the solvent, the product was ipilissolvp.d in water and acetone then lyophilized yielding 0.299g of

- e Lhv lam inopu r Lne - 9-/?-D - 2 ' , 3 ’-d ideoxyr ibofuranos ide that analyzed for 0.2 water and 0.L acetone (mp - < 30°C, [o]20°C ”-29.95°C (0.5, LJHF)).

AnaL. Calcd. for 0,.,11,.11,0., 0.2 11,,0 0.1 C,H.O:

12J/52 2 36

Calcd.: C, 59.17; Π. 6.69; N, 25.68

Found: C , 59.13; I I, 6.69; ί 1, 25.75

Example 9

- E thy line t hy laminopur ine - 9 - Z) - D - 2 ' . 3 ' - d ideoxyr ibofuranos ide

The procedure for the synthesis of 6 - e thylme thy laminopur ine - 9-/3 - D-2 ’ , 3 'd i.deoxyrihofuranoside was identical to Example 2. The reaction was lilt· red and the fi 1.1 rate applied to a Dowex-1-hydroxide column (2.5 x 10cm) . The product was e Luted with 90^-. methano 1/water (v/v) and chromatographed on Amberlite XAD-2 resin (2.5 x 20cm) after removal of the solvent to -3ml and redissolving in water (100ml). After sample application, the column was washed with a iarge volume of water, and the product was eluted with 9 5’. ethnnoί/water (v/v). Product containing fractions were combined and 20ml of dry silica gel added. All· solvent was removed under vacuum. The dried si I ica gnl was applied to the top of a silica gel column (9.8 X 20cm) equilibrated with chloro form: methanol (98:2, v/v). Product containing fractions were combined arid after removal of the solvent under vacuum, were dissolved first in ethanol and filtered. After removal of the solvent and redissolving in water, the solution was lyophilized yielding 0.3g of. 6-e thyline thy lamino - 9-/?-D-2 ' , 3 '-dideoxyribofuranosyl pur ine that analyzed for a 0.05 hydrate (mp < 30°C).

MJB/KT/AC/9tli March 1.988

BAD ORIGINAL

- ί ϋ i’.3U9

Ana!. >	f a 1 c d. for d	1 .’	Π, 7.0? Π.0:
A a i < d . :	f . 3 A . | y ;	II .	6 *!, 2 5.1. /
! 'r' rnd :	i f , 5 6.12; ί1	.	ύ . 9 * ; 0', 2 5 . 1.
Nil?: 5	8.36 <s, 1	11	, IL; . 8.19 (s. 1 H, IL·) , 6.23 o 2	(dd.	1 H. 0,,7.	5.05	(t.
111, .1	- A. 58, (III ' J	),	, 4 .ιη tin , 1. , 11, 11^, ) , 4.08 ς in.	2 H,	CH ), 3.51	(m . 2	11,
IL ’ ) , )	5.33 (s. 3 11	•	CH ), 2.41 (tn, 2 H, H2,), 2.03	(m, 2	H, H3,), 1.	.14 (t	, 3
H. J -	7.01, CH3).

Example 5

- [odopui ine - 9 - Z?- D- 2 ' , 3 ’ - d ideoxyr lb o Cur ano s ide.

-1 odopur Lne (0.626g 2.54mmol, Sigma Chemicals, St. Louis MO) and 3' - deoxythymidine (O./lg, 3.13minol) (llorwitz, J.P. e t al J . Org. Cbein . , 31.,205 (1966)) wore combined with 700inl 10 mt-I potassium phosphate buffer with a pH of 6.8, containing 0.04% potassium azide. Purified thymidine phosphorylase (2,000 1.0.) and purine nucleoside phosphorylase (7,000 I. U.) (Frenilsl:v Ϊ.Λ., pt aj... , Biochemistry. 20, 3615, 1981 and US Patent 4,381,464) were added and the suspension was stirred at 35°C. “ After 48 hours the rear! ion

AP000073

•.π·· I'iLtere	d , a oil the 1 i 1 1 ra t.e was dried	mule r	vacuum	'['be result:	. ί n r
residue was	di.s.solved in 95? e thanol/wa te r	(v/v),	and a	fter addinr, -2	n,n 1
s i 1 i.c a ge 1 ,	the solvent was removed under	vacuum .	The	dried silica	V.’ -1 s
app1 i ml to	t'iie top of a ·- i. 1 i.ra gel column	(2.8 ;	K 50cm)	and the prod	’.i<·;

rioted with ehloroform/rnethanol (95:5, v/v). Fractions containing only product were combined, and the solvent was removed under vacuum. The residue was redissolved in ethanol and filtered through a 0.22/i filter. After removing most of the ethanol and adding ~25ml of water, the material was ivophilized yielding O.O88g of 6 - iodopur ine - 9-/3-D-2 ' , 3' dideoxyrlbofuranoside that analyzed as a 0.2 hydrate (mp « 151-153°C).

Anal. Calcd. for C, ,31,,0.0., 0.2 IL0:

1.1 Ί λ 7.

baled.: C, 35.15; H, 3.46; N, 15.77

Found: C, 3 5.31.: 11, 3.31 ii, 15.83

NJf’./KT/AC/9th March 1988

BAD ORIGINAL Λ i j I'.-c 1 opiupy line t.hy 1. iin i. nopu r i nc- 9 - A - b - 2 ’ . 3' - Ί idco::y r L no furanos ide

6-Cyclopropylinethylaininopurine was prepared by nucleophilic displacement of the chlorine group on 6 - chioropurine (Sigma Chemicals. St. Louis MO) by the amino group on cyclopropylmethylamine (Sigma Chemicals, St. Louis MO).

- Cvc l.opropy 1 inc thy 1 am inopu r ine (2.64 mmol 0.50 g) was dissolved in 5 ml of dimethy1 formamido . After cooling to room temperature 3'- deoxythymidine < i.'tf’· mmol, 0.4() μ) (Jlo rw i t z., J.P. e_t a_l . , J_. (..¹ r f.. Chem. 31. 205 ( L⁰At'>) ) was added along with 30 ml n| l.o ipit potassium phosphate buffer with a pH of 6.3 containing 0.04?: potassium azide. Purified thymidine phosphorylase (LG.OOO

l.U) and purine nucleoside phosphorylase (20,000 I.U) (Krenitsky Ϊ.Λ. e t nj_. , Biochemistry 20. 3615, 1981 and US Patent 4,381,444) absorbed onto 10 ml DEAR cellulose (Whatman) were added, and the suspension was stirred at (1

1.3 <L After 8 hours the reaction was filtered, and the filtrate was nppl i«-<l to a series of coupled columns. Tire initial column contained AG1-X2 resin (( ill - form) , 2..5 x 10 cm, while the second column contained Ainberlite ΧΛΡ-7 resin, 2.5 x 20 cm. After sample application, the columns were washed with 5 DO ml water and die product was eluted with methanol. The product was then Clash chromatographed on a silica gel column, 5 x 20 cin, with a mixinr»· of ch 1 o ro C ortn: me thano 1 (9:1, v/v) . Solvent was removed in vacuo and I he product gum was transferred in acetone to a vinL. Lyophilisation yielded 0.588 g of 6 - eye lopropy line thy lnminopur ine - 9-/3-D-2 ' , 3 ’-d ideoxy r i bo furanos i do that analysed for 0.15 water and 0.15 acetone.

Anal. Calcd. for C, ,11.,d!_r0_o 0-15 ILO 0.15 C.,H_fO:

I 4 I 9 .> z 2 3 6 (tiled. ; G. 5 7. /1; ii, 6. 77; II, 23.29

Found: C, 57./3; H, 6.94, H, 23.39

Example 7

- 1 sop ropy lain! nopur ine - 9 - /1 - D - 2 ' . 3 * - di deoxy r ibo furanoside

MJB/KT/AC/9th March 1980

BAD ORIGINAL &

IS iW .·;·,·ηί hes i.s <> f 6 - i sopropy I.am i.noptir i.ne - 9 - β - i) - 2 ' , i' - d idcoxvr ibo Cur,-, nos i de ρι> r ( o vine d as (ίπ3<?) ibod in f.xamp 1 c i except that 6 - Isopropyl.-nn inopLU'i ne ( [ it pal'ed tom

- eh 1 οι :opi i v inc (Si.fy.na Clieinic.a Ls . Si.. Louis ! to I ί so prop 7 1 am i. lie) w.is d Line thy l.su L foxide .

dissolved in 5ml each of dimethy1formaraide ar.u and

After lyophilization, the 6-isopropylaminopurine-9-β-ΰ-2’,3'dideoxyribofuranoside (O.5O2g) analyzed for 0.2 hydrate (inp « 55-57°C)

Anal, baled, for C, ., II. _nN_r 0., 0.2 11.,0:

1J 1 9 5 ί I baled.: C, 55.38; H. 6.96; N, 24.93 I'm 11 h 1 : f, 3 5.56; 11 , 6 . ^!) 4 ; ΐί, 2.3.0 1

AP 0 0 0 0 7 3

1> xa.inpLe_3

Th l a in i p tine - 9 - /3-D - 2 ' , 3' - d idcoxyribofuranoside

Th i am i pi i up (Burroughs Wellcome Co.. Research Triangle Park Nil) (0.5g) was dissolved in 2.5ml diinethylsLiifoxide and 15ml dimethoxyethane and combined with 3'-tleoxy thymidine (O.Sg) (Horwitz J.P. e t al J.Org. Chern, 31. 203, ( 1^61.1 ) in 30inL potassium phosphate pll 6.3. Purified thymidine phosphor/1 asc (1600 1.11. ) and purine nucleoside phosphoryLase (70,000 1.1-.5

3615, 198 L and IP . 0

Pa ten I (hr cn it.sky T.A., et aL . , B i 0 eh e in is try . 20 .

4,381,444) were added and the suspension was stirred at 35'Tj. After 96 hours the reaction was filtered and the volume reduced in vacuo to a syrup. Water (25ml) was added and the solution stored overnight at 3°C. The precipitate was collected by filtration, suspended in 5inl dime tlivi form..mi de and filtered. To the filtrate was added 15ml methanol, and the solution was stored at d i. s 5 ο ί ved ,O

-20'C.

ί. n 6 5 % hydroxide resin.

After 5 days the solids were collected by filtration, methanol/watcr (v/v) and chromatograhed on a AG-I X2 The product was eluted with 65% methanol/water (v/v).

After removal of the solvent in vacuo, the solids were dissolved in 20ml ch Loroform/inethanol (9:1) and chromatographed on a bed of silica gel <3 x 511cm) equilibrated with chloro forin/ine thano 1 (9;1, v/v). Product containing, fract. ions were combined and 11κ· solvent removed under vacuum. The residual

HJB/KT/AC/9th March 1938

BAD ORIGINAL

HI

- i 1 i ·' .-1 p.c i	'•71': ]’<	'· ,ΊΙΟ '.ΊΊ 1	1 .-am ihc pl-	< ic t; by <.l L	/solving in 95%	e t liano 1 /wa
t v/v ’ .uid 1	i h I’l i Ilf. 1 h ;	ough a 0.22/	i i’ 1 lLov .	ihe et. Hanoi was	evapotai cd
and - 7 ‘ Όιιι 1	•..al iU		aUUc.i The resu 1 h in	,g suspension v.	is I vopli i I i
; i U · 1 i n r. 1 1		i| li.'lllii	.) 1 it· -0./1-1)- ,	’ ' ,3' di.,1 co:-:	:y r Lbo i u ra no ?: 1 de	that ana 1y
. i s a n. 4 by	Urate	cant a i	11 i Itg 0./ equ	ivaleuLs of	me tlianol (mp ~	1 30* C , part
melt, j ng at	110°C)
Anal. Calcd	. for	C14HL6	SN„0.0.4 H„0 8 4 2	0.7 CH.O: 4
Calcd.: C ,	41.84;	H, 4.	68; S, 7.60;	N, 26.55
foi md : C . 4	1.. 9 3 ;	11, 4.4	3; S. 7.48;	», 26.34

ExamirL_e

Am i no - 6 -n-propoxypur in»- 9 -fl-b- 2¹ , 3 * - d ideoxy r iho furanos itle

2-Amino-6-n-propoxypurine (prepared by nucleophilic displacement of the chlorine group on 2-amino-6-chloropurine (Aldrich Chemical Co., Milwaukee UI) by the alkoxy anion formed between sodium hydride and propanoL) (D.21g) and 3'-deoxythymidine (0.29g) (Horwitz J.P. et a 1. J.Org. Chem. 3_1 , 205 (1946)) were combined in LOOmL potassium phosphate, pH 6.8, with 0. Oa '·. potassium azide. Purified thymidine phosphorylase (1200 I.U.) and purine nucleoside phosphorylase (8600 I.U.) (Krenitsky T. Λ . , e t al. . Bioclieini.s try. i0. 3615, L 9 81. and US Patent 9,381,664) were added and the suspension was stirred at 35°C. After 48 hours the reaction was filtered, and the filtrate was chromatographed on a column containing AG-1 X2 hydroxide resin (2 X 5cm). The product was eluted with 90% methanol/water (v/v) . The solvent was removed under vacuum, and the residue was dissolved in methanol. lOmls of dry silica geL were added, and the methanol was removed under vacuum. The dried silica gel was applied to a silica gel column (2.5 X 30cm) equilibrated in chloroform/me tlianol (9:1, v/v). This was also the eluting, solvent. fractions containing only product were combined and t:he solvent was removed under vacuum. The residual silica gel was removed, and the product was dried as described in Example 8. This yielded O.132g of

2-amino-6-n-propoxypurine-9-J-D-2' ,3'-dideoxyribofuranoside that analyzed as a 0.2 hydrate (mp - 70°C).

N.l L’,/KT/AC/9 th March 1988

BAD ORIGINAL

'i id: (..-, 5 / . j 2. , i I, 4 . 4 2 ; N , 2 3.49

Example 10;

- Et.l iv 1 th Iopur i nc -*>-/>-1) · 2 ' . 3 ’ - d i deoxy r ibo furanos tde

- E i.l i y 1 l h i opu r i no (5.5 mmoles, lg) obtained from Sigina Chemical Co., Si. I.oni.·: i!o and I ’- deoxythymidine (4.47 mmoles) (liorwitz, J . 1’. e. t a_l . , J. gyrg. Client. , 31., 205 (1.946 ;) were suspended in 50 ml. of a 15 mil potassium phosphate solution with a pH of /.2. Purified thymidine phosphorylase (7890 I. II.) and purine nucleoside, phosphorylase (i960 I, U.) (Krenitsky Τ.Λ., et nl. , Bip_chemis_Ljry, 20 3615, 1981 and US Patent 4,381,444) were added and the suspension si irred at 3s‘’(;. After 147» hours the reaction was filtered and

Q the filtrate stored at -20 C. After thawing, the. filtrate was adjusted to pH 10.7 with ammonium hydroxide and chromatographed on a column containing Dowstx - 1. - Co rma te resin (2.5 x 8 cin) . This column was eluted wiili 30¹n-propane1/water (v/v;. Fractions containing product were combined and lb” solvent removed under varmiin. The residue was dissolved in it» n - ρ ropa no 1./wa I.e r <v/v) and chromatographed on a column containing Biol’.id P-2 (4 x 90 cm). The product was eluLed from the column with u-propanol/watev (v/v). Product containing fractions were combined and the solvent removed under vacuum yielding 0.427g of 6 - e thyl th iopur i lie - 9-ft - 02 ' . 3 ’ - d i.deoxy r ibofuranos i de that analyzed as a 0.5 hydrate. Anal. ruled.

AP000073

Cm C, ,H. _rSN.0.,0.5 11.,0:

I 2 L6 a 2 z (hied: C, 49.31; 11, 5.92; N, 19.36; s, 11.47»

BAD original

NJB/KT/AC/9th March 1988

Iii J

1, L

2_w\m) no - 4 - Ρ'-ήχ·,' 1 Oh iopur ine - '.* -;! - D - 2 ' , 3' - <1 [deoxy f Lb ο [ u ra no:; ide

2-Amino-6-benzylthiopurine (1.9 mmoles, 0,5 g) obtained from Sigma Chemical Co., St. Louis, MO and 3deoxy thymidine (2.0 mmoles, 0.543 g) (Horwitz,

J.P. et cil., J. Org. Chem. 31 205 (1966)) were dissolved in 20 ml of 10 tnM potassium phosphate buffer, pH 7, containing 0.04% potassium azide. Purified thymidine phosphorylase (2,000 I.U.) and purine nucleoside phosphorylase (2,900 I.U.) (Krenitsky T.A., e t a 1. . Biochemistry, 20. 3615. 1981 .uni US Patent 4,181,444) were added and the suspension was stirred at 15 C. Alter llirve day::. 8i) ml ol. 1.0 mM potassium phosphate bufier, pii i , were added. One day later the reaction was filtered. The cake was dissolved in 90% methanol/water (v/v), filtered, and the filtrate was chromatographed on a 2.5 x L0 cm column containing Dowex-1-hydroxide . The product was eluted from the column with 90% methanol/water (v/v). Product containing fractions were combined and after lyophilization yLelded 0.0!’()g of 2 - amino-6-benzyl thiopur ine- 9-β· D-2' ,3'- dideoxyribofuranoside.

Ana 1 Γοιιη	. Cal cd . ,.1: C ,	for Cl7ll1() 57.02; H,	SIR <>.,: 5 . 39 ;	ι.ι u,	, 9' 19 .	'.13; 5 i;	H, 5.36; S, 8.89	N, 1	9.59; S 8.9 7
EUR	data; 6	8 . 18	( s , 1	li, lln 8	),	7 . 3	(m ,	5 H, S3) ,	6.6	(s , 211, NH ?) , 6.08 (. d«i.
1 l	,), 4.93	t t),	1 H, 5	' OH) ,		.45	(b ,	2H, CH ),	4.08	(in, 1H, H ' ) , 3.4 3 - 3 . f<? 4
(m,	2 li, 5’	Cil2),	2.35	(m, 2	H ,	2'	cup	• , 2.0 (m,	2 H,	3' CH2).

Ex a m pie 12

-11 tlioxypur ί tin - 9 - (1 - D - 2 ' , 3 ' - d i deoxy r ibo fur a nos ide

- Lthoxypur ine (3.0 minoies, 0.5g: Sigma Chemicals Co., St. Louis Mm and 3' - deoxy thyroidine (3.3 mmoles, O.75g) (Horwitz, J.P., e t al. . J. Org. Chem. 31, 205, (1966)) were suspended in 25ml of 10 inM potassium phosphate buffer pH 6.8 and containing 0.04% potassium azide. Purified thymidine phosphorylase (800 1. U.) and purine nucleoside phosphorylase ( 1.,200 1. U.) (Krenitsky Τ.Λ. e t a_l.. Biochemistry. 20. 3615, 1981 and US Patent

NJB/KT/AC/9Ih March 1988

BAD ORIGINAL ί , ’-‘ί I , ύ 'ι ύ} .···)-'’ added ·ΐϋ·Ι ί He suspension was stirred at 35°C. Alter hours , ' ^r>mi of Jo uu‘1 ρ·>;-ass i.um phosphate buffer pH 6.8, were added and tie· ι·«·ι<;ΐ'ίοιι stirred for an additional five days at 33°C. The reaction precipitate was removed by filtration and the fiLtrate chromatographed on a 2.5 x 10 cm column containing Dowex-1-hydroxide . The product was eluted with 90% methanol/water (v/v) and the product containing fractions combined. After removing the solvent by vacuum, the material was dissolved in 30% n-propanol/water (v/v) and chromatographed on a 5 x 90 cm column containing BioRad P-2 resin. Product containing fractions were pooled and after lyophilization yielded <>.225g of 6-e thoxypurine - 9 - β - 1) - 2 ' . 3' - dideoxy riboCuranoside that analyzed as a 0.15 hydrate.

Anal. Calcd. for 111111,.0 0.1511 0: C, 53.98; 11, 6.15; N, 20.98 hound: C, 5^Zi.(J5; II, 6.15; N, 20.88

NMR data: 68.6 (s, 1 11, H_g) , 8.5 (s, 111, 1^), 6.3 (dd, I 11, 11 ) , 4.97 (t. 1

II, 5' Oil), 4.6 (m, 2 II, -Cl -), 4.1 (m 1 Η, H ), 3,53 (m, 2 II, 5' Cl J , 2.41 (in, 2 II, 2' Clip, 2.03 (m, 2 II, 3' Clip, 1.4 (t, 3 H, J-O.O35 11::, Cl .).

LCamp 1e___13

- p jine thv Lam i ιν.ιρ-η· i up - 9 - /1 - 0 - 2 * 3 ' - dideoxyribo fur a no s i de

-1) Line thy lam i nopurine (6.13 mmoles, lg, Sigma Chemical Co., St. Louis, MO) and 3' - doo.xy thymidine (4.44 mmoles, lg) (Horwitz, J. P. e t a L . . J_._ 0g.

Chem· i ϋ· 205 (1966) were suspended in 50ml of a 10 mil potassium phosphate solution pll 7.0 and containing 0.04% potassium azide. Purified thvmidine phosphorylase (.2000 I. 0) and purine nucleoside phosphorylase (3000 1 . 0.) (Krenitsky T. A . ct aL. , B1ochemis try. 20. 3615, (1981) and US Latent

4,381,444) were added and the suspension stirred at 35^tC. After L20 hours the reaction was filtered and the filtrate chromatographed on a column containing Dowex -1.-hydroxide resin (2.5 x 8 cm) with 90% inethanol and wa t o r (v/v) ns the eluent. Iractions containing product were combined and the solvent, removed under vacuum. The residue was dissolved in 25 ml 30'· ii-propanol/water (v/v) and chromatographed on a column containing BioRad i’-2 (5 x 90cm). The product was eluted with 30% n-propanol/water (v/v).

APtl η ft 0 7 3

NJB/KT/AC/9th March 1968 bad ORIGINAL ft ί'. J i)) • ’Λ ©

•5 >

Product con: .1 i ;i i ng 1 rac ri <iis were combined and Lbe solvent removed under '.’.emuin. The residue sus dissolved i.n 3 Om I. de-Ionized water ami chiomatogtaphed on a cnlu.’im containing Sephadex C-LO resin (3 x 90cm; . The eluent was water. Appropriate fractions were combined and after

I. yopli LIizat ion yielded 6 -di me thy i aminopurine -9-/J-D-2' , 3' -dideoxyribofuranosi.de that analyzed as a 0.3 hydrate and (inp = 162°C) .

Anal. Calcd. for CUN 00.31^0 C, 53.64; H, 6.60; N 26.06

Found: C, 53.63; II, 6.63; N, 25.8

Exampd.e__14

C'nflyjdrjjxy-e tlc,jlajn_i nonur ine - 9 - B- D - 2 ' 3 ' -dideoxyr ibofuranos ide

- HydroxyethylaminopurIne (2.8 mmoles, 0.5g, Sigina Chemical Co. St. Louis, MO) and 3'-deoxythymidine (3.30 mmoles, 0.76 g) (Horwitz J.P. et al, J.Org. Chem., 31 205, (1966)) were suspended in 75 ml of a 10 mH potassium buffer, pll of 6.8 and containing 0.04+, potassium azide. Purified thymidine phosphorylase (400 l.M.) and purine nucleoside phosphorylase (700 i.U.) (Krenilsky Τ.Λ.

iochemistr ·, _20 361.5, 198 1 and U.S. Patent

4,3 3 L , 4 44) were added and the suspension was stirred at 35°C. After P. d.ay.s, 600 i.U. thymidine phosphorylase and 1050 J.U. purine 11ucleosi.de phosphorylase were added. After an additional day, the reaction was filtered and the filtrate was applied to a 2.5 x 10cm column contained Dowex-1-hydroxide . The product was eluted with methanol. Product containing fractions were combined and evaporated under vacuum. The residue was then applied and eluted from a 2.5 x 50 cm silica geL column under pressure with a mixture of (8:2) chloroform: inethanol. Product containing fractions were combined and after lyophilization yielded

6-hydroxyethyLaminopurine-9-β-D-2’,3’-dideoxyribofuranoside that analyzed as a 0.63 hydrate and (inp - 153°C) .

Anal Calcd. for C, ,ΙΙ,.,Ν of). 6511.,0:

1 / 5 3 2

Calcd: C, 49.33; II, 6.34; N, 24.07 Found: C, 49.85; H. 6.07; N, 23.70.

>·! IK/rV/AC/’>t h March l8B

BAD ORIGINAL &

t'.Ui'.l aic’ ip J a

6-_b.yo [ 'c t'-'i¹ 1. am inopur i n - - .1? - 0 - , 1' - d ideoxy r ibo futanos ide

6-f.yc 1 opropy l.aminopuri ne (prepared from 6-chloropurine (Sigma Chemicals, St. Louis MO) and cyclopropy lainine) (2.86 mmoles, 0.5 g) and 3deoxythymidine (6.30 mmoles, 1 g) (Horwitz J.P. ct al J.Org.Chem., 31. 205 (1966)) were dissolved in 10 ml of a 1:1 dimethylsulfoxide: N',N'- dime thyIfovmnmido mixture and further diluted with 30 ml of a 10 mM potassium phosphate buffer pH 6.8 and containing 0.04% potassium azide. Purified thymidine phosphorylase (10,000 I . U.) and purine nucleoside phosphorylase (20,000 i .0. ) (liven i . Csky Τ.Λ., e t a 1. . Biochemistry, 20. 3615, 1981 and US Patent

4.381,464) absorbed onto 10 ml of DEAE resin (Whatman) were added and the suspension was stirred at 35°C. After 8 hours the reaction was filtered and the filtrate was applied to a series of coupled columns. The initial column contained Dowex-1-hydroxide (2.5 x 10 cm) while the second column was filled with AmberIito XAD-2 resin (2.5 x 20 cm). After sample application, the Cidmiins were washed with a large volume of water and the product was eluted with methanol. Product containing fractions were combined and after 1 vopli i I. i za I ion yielded 0.54 g ;

- c vc 1 op ropy lam ί no pur i nc - 9 - /) - D - 2 ' . 3' - d ideoxyr ibofu ratios ide that anal ed a s a H.4S lu'ilmO·. .and (mp -- 63-65°C).

AP000073

Anal. Cal	cd. for	(J i.	'd. , M - 0 ,0.5 3 11., 0 3 1 / i 2 2
Ca led : C,	56. / 5 ;	11,	6,60; N, 26.55
iouml: C ,	46.6 / ;	H ,	6.63; U, 24.5 7

Exampic 16

- Cvc lopcntv lainitiopur ine -d - D- 2 * . 3' - d ideoxyr ibofuranos ide

6-UycJopentylaminopurine (prepared from 6 - chloropurine (Sigma Chemicals, St. Louis MO) and cyclopenty]amine) (2.49 mmoles, 0.506 g) was dissolved in 5 ml N, M - tl i me thy I f nrmamide and 5 ml. dimethylsulfoxide. 3'-deoxythymidi iv' (3.'>/i mmoles, 0.894 g) (Horwitz, J . 1’. , et al J . Org. Chem. , 31, 205 (I966))wa.s added fiJB/KT/AC/9th March 1988 bad original &

i i Ong, ·. i : h 30 in I of I.¹'» mil potassium phosphate buffer, [ill 6 . 8 and ΰ.ό'Π, ·,ό L a ss i i mi azide. The pH was adjusted to 6.8 with acetic acid. ί’ιι r i f i < d

11 yin i. i I i. 11 phosphorylase (10,000 I.U.) and purine nucleoside phosphorvlase (,’<1,1)00 I.II.) (Krenitsky !’. A . . e_L _ajL, Biochemis try . 20 . 3615, 1981 and OS

Patent 4,381,944) bound to DEAE-cellulose (Whatman) was added to the reaction mixture. The suspension was stirred at 35°C for 8 hours, filtered, and Che filtrate stored overnight at -20°C. Upon thawing, the filtrate was applied to a 2.5 x 10 era column containing Dowex-1-hydroxide resin. The product was eluted with water. Product containing fractions were combined and chvomaLographed on a column containing XAD-2 resin (2.5 x 20 cm). This product was eluted with 350 ml of water followed by methanol. Product containing fraction.'; ••ere combined and the methanol removed under vacuum. The residue was dissolved in water and after lyophilization, yielded 0.459 g of 6 - cyclopentyLaini nopur i.ne-β-1)-2 ' , 3 '-dideoxyribofuranoside that analyzed as a 0.05 hydrate and (mp - 88°C).

Anal. CaLcd. for C, .11,,, N_r0-0.05 ILO a 21. j 2 2 baled; C, 59.21 11, 6.99; N, 2 3.02 Found; C, 59.24; li, 7.05; N.22.95

Fxnmpic 1/ .--.rA'll i no - 6 - in'' Ih_<ixvpur i i;c - 9 - <9 - D - 2 * , 3' -dideoxyr Lbo furanns 1dc .

- Amino-6 - methoxypurinc (3.0 inmoles, 0.5 g, prepared from 2 - amino - 6ch loropurine (Aldrich Chemical Co., Milwaukee Wl) and methanol) ,ni«i 3'-deoxy thymidine (4.50 mmoles, 1 g) (llorwitz J.P. c t a 1. J . Or g. CIkmii . , 31, 205 ( 1966)) were dissolved in 10 ml of a 1:1 dimethylsui lox i .Ip . M' , 11d ime thy 1 f ormainide mixture and further diluted with 30 ml of a 10 mM potassium phosphate buffer with a pH of 6.8 and containing 0.04* potassium azide. Purified thymidine phosphorylase (10,000 I.U) and purine nucleoside phosphorylase (20,000 I.U.) (Krenitsky, e t al.. Biochemistry. 20. 3615. 1'53 I.

and US Patent 4.381,444) adsorbed onto 10 ml of DEAE resin were added and the suspension was stirred at 35°C. After 8 hours the reaction was filtered

NJB/KT/AC/9th March 1988

APO 0 0 0 7 3 bad original

- i /list the ί i 1 ί' i: p

I a - ! - hydroside.

.-ί vn 1 ί ΐιΐι·'-· o L ’0 in I s .

i t h Λint'f’ ill! c ΧΛΙ) as applied Lo a 2.5 x 10 cm column containin'; 'a <· t j a ms containing product were pooled and r-educed to 'i n i a sample was applied to a 2.5 x 20 cm column fill.-H I'esin. The roluimi was washed with a large voIiiiiic of water and (lie product, was eluted with methanol. Product contaiui fractions were combined and after lyophilization yielded 2 - amino - 6 me( boxypurine - 9-p-D-2' , 3' -dideoxyribofuranoside.

*3

Anal. Calcd. for 6,,11,,.11,.0., : C, 69.81; 11, 5.70; N, 26.60 1.1 IJ > 3

Foimd : o , 6^ . 10 ; 11, 5 . / ; N , 26.36 .

ExaiiiiJ

- n -1' rope:: ypu r ? ne - 9 - /? - D - 2 ’ , 3 ' - d ideoxy r ihofuranos ide

6-n-Pmpoxypui'j ne (0.5g, 2.8 mmoles, Sigma Chemicals, St. Louis, M0) and 3 ' - deoxy thym i d i no (<1.96g, 6.2 mmoIes)(Horwitz, J.I’., ct a I .1 . Org . Client. , 31,

()’> ( 1996)) were d i. me t h y .1. f o riiiam ide. containing O.U-'T¹.

dissolved in 5ml. dimethyl. sulfoxide and 3ml h . ii 30ini of lOmtl potassium phosphate buffer, pH ',.3. potassium azide and purified purine nucleoside phosphorylase (20,000 1.U.) and thymidine phosphorylase (10,000 ί .1’. ' (Kmnitsky, Τ.Λ., e.t ni . , Biochemistry. 20, 361 3, 1981. and l;S Patent 6, 18 1 ,666) absori.'ed onto 10ml of DEAE - c e i lulose resin were added and i.h·· react;! on was stirred at 33°C for 7 hours. The resin was removed bv cent, r i fuga t Lon anti the supernatant applied to a column of AG1-K2 (OH ioniu, coupled to a column of NAD-2, 2.5 x 20cm. The coliraws wc is waslipd with jOOinl of water and the product was eluted with methanol. Tiie product was flash chromatographed on a silica gei column, 3 x 3D cm, with

10cm , chi o rof o tin me thanoi (9:1 v/v)

Lyophilization afforded 0.55‘tg

- n - propoxy pur i no - 9 -/1-D - 2 ’ , 3 ' - d ideoxy r ibo furanos ide that; analyzed as a 0.

hydrate .

Analysis Calculated for C<11 . 3!l?0

Calculated : C, 55.06; 11, 6.61; N, 19.75

NJB/KT/AC/9th March 1988

BAD ORIGINAL ft ij JI I ’) ί'ιιυ,ικΙ ; C , 11 , i· . h 1.; i , ί V . /7

G7HHrLo__]/2

- ii - Butoxypur inc - 9 - ft - D - 2' , 3' - dideoxyL ibofuranos ide

6-n-Butoxypurine (0.5g, 2.6 mmoles, Sigma Chemicals, St. Louis, MO) and ' - deoxythymidine (0.70g, 3.L mmoles) (Horwitz J.P. et al. J .Org.Chem., 31, 203 (1966)) were suspended in 100ml of lOmM potassium phosphate buffer, pi! 6.8, containing 0.04?, potassium azide. Purified purine nucleoside phosphorylase (3,500 i.U.) and thymidine phosphorylase (800 I.U.) (Krenitsky, Τ.Λ., cl .((1.., i‘ iochemistry. 20, 3615, 1981 and US Patent

4,381,444) were addeil and the solution was stirred at 32°C. After 7 days the reaction was filtered and the filtrate applied to a column containing ACL-X2 (OH- form), 2.5 x 10cm. Product was eluted with 90% aqueous methanol. Solvent was removed in vacuo from the product and the residue was flash chromatographed on a silica gel column, 2.5 x 80 cm, with chloroform ; methanol (8:2, v/v). The product was dissolved in water and applied to a column containing XAl)-2, 2.5 x 20cm. The column was washed with 500ml ol water and then developed with methanol. Lyophilization yie.lded 0.276g of 6 ii - bi i¹ ovypu,-1 ne - 9 - β - h - 2 ' , I' . d j deoxyr i i io furanos i.dⁿ (mp 51 °C ) .

Analysis Calculated for C . , il ,,,ίί, ΟΙ a 70 4 3

Caiculated : C. 57.52; II, 6.90; N, 19.17 found : C, 57.86; H, 7.29; H, 18.83

Exarnp. 1 e_2 0

- Cyc Lopvopylmc thoxypur ino - 9-D-2' , 3' -dideoxyr ibofuranos ide

6-Cyclopropylinetboxypurine was prepared by nucleophilic displacement of the chlorine group on 6 - chloropurine (Sigma Chemical Co., St. Louis M0) by the alkoxy anion formed between sodium hydride and cyclopropy1 methyl alcohol.

AP000073

M.IB/V l/AC/9tb March L988

BAD ORIGINAL

- I /.Ό ί.' > 1' 1' (? μ ·: 1 Ιίΐ- ί. 11 , *'ί

-'T

J3 >

! ι νιιι ί d ί ί ie (' ι.>. ’Ό 3 j;, AG.· // > L “ .<; , (Ho vi; i Lz o_L P k · J · t’l'g . Ghent. , 11. .’Go ; ;i ···«> cn ie-icl'p<l and eh roma r.i ρ!ι e d on AG1-X2 (OH form; and XAD-2 as desc r i bed in Example L8. Product nmi ai.niur, fractions were flash chromatographed on a silica gel column, 1 30 cm, with acetonitrile. : water (98:2, v/ν'» .

Lyophilization yielded 0.6'.?6g of 6-cyclopropylmethoxypurine-9-β-D-2 ’ , 3 ' dideoxyribofuranoside.

Analysis Calculated for C Calculated : C, 57.92; H, round : C. 57.9?; h./g i¹¹! 8^N/*°3

6.25; H, 19.30 M, 19.2 7

E.x a mqj. e _2..L · Cyclopenty J.oxypur ine - 9 - 0 - 0 - 2 * . 3 ' - dideoxyr ibofuranos ide

6-Cyc1opentyloxypurine was prepared by nucleophilic displacement of the chlorine group on 6 - ch l.o iopur ine (Si.gma Chemical Co., St. Louis, 51, /06 (1966)) by the alkoxy anion formed between sodium hydride and cyclopenl ano 1.

- Cyc lopenty Loxypur ine (O.5U6g, 2.68 mmoles) and 3'-deoxythymidLae (0. l.o.,-., 1. 78 iiunol.es) i.llorwitz .1 . P . , al, d.Org.Chein, 3 1, /03 (1966); wit.· ι·.·,ίι·: ··.!

and chromatographed on AC1-X2 (UH form; and XAH-2 as deseriheil in 1./.,:: p i

13. Solvent was removed in vacuo from product fractions and the tesiduo was f Lash chromatographed on a silica gel column, 3 x 50cm, with chloroform methanol (95.5, v/v). Lyophilization yielded 0.385,; .,/

- c vc.1 open l; y 1 oxypur i no - 9 -/> -1; - λ ' , 3 ' - d j deoxy r ibo f u ratios i de that ana i-.-,-./-0 as a 0. 1 5 hydrate .

Analysis Calculated for G. _rll. .11.0. 0.1511 0 i,) /.0 3 L

Calculated : C, 58.68; II, 6.66; N, 18.25 Found : C, 58.61; H, 6.53; N, 18.25

Example 22

HJB/KT/AC/9(h March 1983

BAD ORIGINAL

830''

puv	in·' - o - n’ -11 -’ , 3 ' - d id'-OX’i	rribofuranoside
pu r	ine was prepared by	nucleophilic	di sp1acement	of
Oil	6 - eli 1 oropur i up (S j.gma	ChemicaL Co . ,	St. Louis MO)	bv

alkoxy anion formed between sodium hydride and cyclohexanol.

6-Cyclohexyloxypurine (0.50g, 2.29 mmoles) and 3'-deoxythymidine (0.776g,

3.42 mmoles) (Horwitz J.P. el al J.Org.Chem., 31, 205 (1966)) chromatographed on AGl-X2 (OH form) and XAD-2 as described in Example 18 with the exception that lOinl glyine in addition to the 5ml dimethyl sulfoxide and 'm| N , N-di me thyl formaini de , and a total of 70 nil of ΙΟιηΜ potassium phosphate buffer, pH 6.8, containing 0.04% potassium azide were used. Lyophilization yielded 0.102g of 6 - eye lohexyloxypur ine - 9 -/3 - D - 2 ' , 3 ' dideoxyribofuranoside (inp L05°C) that analyzed as a 0.2 hydrate.

Analysis Calculated for C.,11, ,Ν,Ο, 0.211-0 J. 6 ./ 4 3 2

Calculated : G, 59.69; H, 7.01; N, 17.40

I'ound ; C, 39.1/9; II, 6.91; N, 1././/

Ex a inp i_e_ 23.

· i.iyc 1 ol.'utv 1 am inopur i tv - 9 - ii -I) - 2' , 3 ' · d ideoxy r ibofuranos ide

6-CycJ ol.iutyl aini nopuririe was prepared by nucleophilic displacement, of the chlorine group on 6 - chLoropurine (Sigma Chemical Co., St. Louis M0) by the amino group on cyclobutyl amine .

b-Cyi. 1 ohut y I ami uopuriiie (O.blOg, 2.62 mmoles) and 3' - deoxy Lhymid inv (<). 8 ’> 6 g, 3.'96 mmoles) (Horwi.tz .1.1', et al J. Org. Chem. , 31, 205 ( 1.966)) were reacted and chromatographed on AGJ-X2 (Oil form) and XAD-2 as described in Example 18. Solvent was removed from product containing fractions and the residue was fLash chromatographed on a silica gel column, 3 x 50cm, with chloroform : methanol. (9:1, v/v) . Lyophilization yielded 0,524g of 6-cycl.obuty1 ain inopur ine - 9 - β-D 2 ' , 3 '-dideoxyribofuranoside (mp 96-98°C) .

£ L 0 0 0 0 dV

NJB/KT/AC/9th March 1988

SAD ORIGINAL ft

JO ·,· s Ia ί c 111 a t.e d •noted <,, 58 I.<·

Id : C. -dd 1 '7; II,

I.O]' I.

Example 2d

FS

- Diethvlaminopurine- 9 - - D - 2¹ 3'-dideoxyribofuranoside

Λ ⁶ -Diethylaminopuvinc was prepared by nucleophilic displacement of the chlorine group on 6-chl.nropurine (Sigma Chemical Co., St. Louis MO) by the amino group on diethylain inc .

- Die thy 1 aminopurine (0.266g 1.28 mmoles) and 3'-deoxythymidine (0.663g,

2.0d mmoles) (Horwi.tz J . I’. c_l a 1 J . Or g. Client. , 31, 205 ( 1966)) were, reacted and chromatographed on AC1-X2 (Oli form) and XAD-2 as described in Example 18. Solvent was removed in vacuo from product containing fractions and the residue was flash chromatographed on a silica gel column, 5 x 20cm with chloroform : methanol (9:1, v/v). Solvent was removed in vacuo from product containing fractions and the residue was flash chromatographed on a second silica gel column, 2.5 x 50cm, with ethyl acetate. The product gum was i r,i ns f“ r red in aceloiv* ^: a vial and lyophilization yielded O.Hdgg of (,.

d i e f hv i am i nopur i.no - 9-/1 - 0 - 2.' , t' - d ideoxyr i.bofuranos ide that analyzed io,· u . ; > water and 0.20 acetone.

Analysis Calculated for C Η N O 0.2C_H,0 0.2511,,0 1.6 2 1 5 2 3b 2

Calculated : C, 5/.Ο3; H, 7.66; W. 22.78 Eound : C , 57.02; 11. / . 39 ; b . 22 . 72

Example 25 (Λ Γγ 1ΊΟ1 id i nopur inc - 9 - /1 - i> - 2 ' , 3 ' -di deoxyr i ij o furanos ide

- I’yr ro I idinopur ine was prepared by nucleophilic displacement of the chlorine group on 6 - chloropurine by the amino group on pyrrolidine.

HIB/KT/AC/9th March 1988

BAD ORIGINAL Ά

- 8 1 3 3( J^!1

- i’v1 ί-· I i.d it ιορ’.ι r i ικ· id. 3d0r., /..bb mmoles) and ileoxv thyin idine (0.7)1./. I.OS Mind I ·· -,) (Hot wit.z I.)’. fj: ,i I, Org. Chein., 31, 205 ( 1966)) were dissolved in 3 ml dimethyl, sulfoxide and 3 ml. ί!, N-dimethylformamlde . Thirty ml of 10 ini I potassium phosphate buffer, pH 6.8 containing 0.04% potassium azide and purified purine nucleoside phosphorylase (20,000 i.U) and thymidine phosphorylase (L0.000 I.U) (Krenitsky, T.A. e t al. . Biochemistry. 20. 3615, 1981 and US Patent 4,381,444) adsorbed onto 10 ml of DEAE-cellulose resin were added and the reaction was stirred at 35°C for 7 hours. The resin was removed by centrifugation and the supernatant applied to a column of AGL-X2 (OH-form). 2.5 x 10 cm, coupLed to a column of XAD-2, 2.5 x 20 cm. The

........... were washer! with 500 ml of water and the product was eluti’d with methanol. lyophilization yielded 0.385/ of 6-pyrrolidincipurine-9-d-D2 ' , 3 ' - d i rieox y r i ho fu ratios ί de that analyzed as a 0.05 hydrate (mp 1 58 -1 59°ii.

Analysis Calculated for C, , Η, _N,0,. 0.051L0 1.4 19 j z 2

Calculated : C, 57.94; H, 6.63; N. 24.13 Pound : C, 57.92; H, 6.67; N, 24.11

Exanip 1 e_ 2_6

- Ho rpito 1 inotnirine - 9 - d - D - 2 ' , 3 ' - d ideoxyribofuranos ide

6-Morpholίnopurine was prepared by nucleophilic displacement of the ci,’urine group on 6 - ch Loropur ine (Sigma Chemical. Co., St. Louis MO) bv the am i no group on morpholine.

6-MorpholInopurine (0.50lg, 2.44 nmoles) and 3'-deoxy thymidine (0.842/, 3. minoles ) (llorwj. tz J.P. e_t al J . Org. Client. , 31, 205, (L966)) were reactm! and chromatographed on AGL-X2 (OH form) and XAD-2 as described In Example 13. Lyophi lization yielded 0.292g of 6-morpholinopurine-9-/3-D-2 ' ,3'-dideoxyribofuranoside that analyzed as a 0.2 hydrate (mp 97°C).

Analysis Calculated for C,,ll,_nN_rO_n 0.2011,0 14 19 5 3 2

Calculated : C, 54.43; II, 6.33; N, 22.67

Found : C, 54.48; H, 6.28; N, 22.51

NJB/KT/AC/9th March 1988 ,

BAD ORIGINAL

AP 0 0 0 0 7 3 due M ryi.L_i lie -'.' -/1 - D - 2 ' . 3 ' - dideuxyr Lbofurnnos icl

Ml »

:z>

.3» >

- v , y - 0 i.me tbv la 1 J.y lam i nopur i no (0.500g, 2.46 mmoles , Sigina Chemicals, Sc.

Louis, HO) and 3deoxy thyinid ine (0 . 7 5 2 g , 3.32 mmoles) (Horwitz J.P. e t a 1.

J.Org.Chem., 31, 205 (1966)) were reacted and chromatographed on AG1-X2 (OH form) as described in Example 18. Solvent was removed in vacuo from product containing fractions and the residue was Clash chromatographed on a silica gel. column, 3 x 50 cm, with chloroform : methanol (95:5, v/v). Product, 'on ta i n i ng fractious were then appLied Lo an XAD-2. column, 2.5 x 20 cm, arid •Luted with methanol. The product gum was transferred in acetone Co a vial and l.yophi 1 i za I. ion yielded 0.645g of 6-y,y-dimethylaliylaminopi irine9-l> - 0 - 2 ' , 3 ' - ill deoxy r j.ho Cura nos Lde that analyzed for 0.45 water and 0.20 ace tone .

Analysis Calculated for C_irH H 0„ 0.451LO 0.2C.H.0 152152 2 36

Calculated : C, 5 7.99; 11, 7.21; ti, 21.68

Found : C, 57.77; 11, 6.91; H, 21.41

E x a_i η pie 23

-Fu r fury 1 am i input inc - 9 -/-02' , 3 ' -di deoxy r i ho furanos ide

- Fur furyLaminopurLne (O.5O2g, 2.33 mmoles, Sigma Chemicals, St. Louis, HO) and 3'-deoxy thymidine (O./54g. 3.33 nnnoles) (Horwitz J.P. e t a L J . Org.Chem. , 3 1, 20 5 , ( 1966 )) were reacted and chromatographed on AG1.-X2 (Oil farm) and

ΧΛ0-2 as described In Example 18. Solvent was removed i.n vacuo from product containing fractions and the residue was flash chromatographed on a silica gel column, 5 x 50 cm. with chloroform ; methanol (9:1, v/v). Lyophilization yielded O.3O3g of 6 - fur furylaminopur ine - 9-/?-D-2 ' . 3 ' - d 1 deo;·:-,-ribofuranoside that analysed as a 0.2 hydrate.

Analysis Calculated for C,-11, ,N_r0., 0.211.0 1.6 1 7 5 3 2

Calculated : C. 56.49; ||, 5.50, N, 21.96

NJB/KT/AC/9th (larch 1988

BAD ORIGINAL

6_-J*<Ί11^:-V Lme l~caρ Lopui: i nc - 9 -/3 - D - 2' , 3 ’ ·dideoxvribofuranos ide

6-Benzylmercaptopurine (O.SOlg, 2.07 inmoles, Sigma Chemicals, St. Louis, HO) mid 3’deoxythymidine (0.704g, 3.11 mmoles) (Horwitz J.P. et al J,Org.Chem., 31, 205, (1.966)) wore reacted and chromatographed on AG1-X2 (OH form) ns described in Example 1.8 except that 1.0ml glyme was used to dissolve the purine base. Solvent was removed In vacuo from product containing fractions and the residue was flash chromatographed on a silica gei column, 3 x 50 cm, with chloroform : methanol (95:5, v/v). The product was transferred in ethanol io a vial, and lyophilization yielded 0.304g of 6-benzyl inc reap t,>pur i.ne - 9-/3 -1' - 2 ' , 3 '- d ideoxvr ibo furanoside that analyzed for 0.05 water and 0.05 ethanol (nip 81-83°C).

Analysis Calculate,! for C 11 M (1 8 0 0511 Ω Ω 05(1 II ()

Pound : 0., 59.49; 11, 5.38: ii. 16.32; S, 9.30 i ::ainp fe___)J.J

6_-_Aiii.! Lit')¹ ILl1-1)-2¹ .3’ -dideoxvribofuranosidc

6-Anil i nopurine (0.500g, 2.37 mmoles, Sigma Chemicals, St. Louis, lit') and ' - deoxy thymidine (O./5'2g, 3.32 mmoles) (Horwitz J.P. e t al J .Org.Chem. . 3i. 205 (1966)) were reacted and chromatographed on AG1-X2 (Oil form) as described in Example 18. Solvent was removed in vacuo from product containing fractions and the residue was flash chromatographed on a silica gel. column, 2.5 x 50 cm, with chloroform : methanol (95:5, Lyophilization yielded 0.4/0g of 6-anil inopur ine - 9-/?-D-2 ' , 3' - d i deo:: v libofuranoside that analyzed as a 0.05 hydrate (mp 17O-172°C).

Analysts Calculated for C_lrH,,N_rO„ 0.051LO 161/52 2

.G/ranjii»,

MJB/KT/AC/9th March 1988

A. 2 2.43 ί .y.aii'g i e _j_i.

•*1

- Amino - 6 - e ihoxvpurinc -9-/1- 0 - ?.' , 3 ' -dideoxyrlbofuranoside (0.5g, 2.8 mmoles prepared by nucleophilic ne group on 2 - amino - 6-chloropurine , (Aldrich

2-Amino - 6 -e tboxypurine

- w*	di splacement	of the cb1
	Ahemical	Co .	, Milwaukee
	by dr i. de	and	ethanol) and
	A . 1’. e_t	_al 3	.Org. Ahem . , 3

205 (1966)) were reacted and chromatographed mi ΛΑ1-Χ2 (OH Torni) and XAb-2 as described in Example 18. Solvent was remove,i in vacuo from product containing fractions and the residue was Clash chromatographed on a silica gel column, 5 x 20 cm, with chloroform v/v). Lyophilization yielded 0.443g of 2-ainino thanol (9:1 ,

- e tboxypur i ne - 9-/)-D-2 ' , 3 ' - dideoxy r ibofuranos ide that analyzed as a 0. hydrate (mp 150°A, partial, inelt at 65°C).

Analysis Calculated for A II ^A_r 0 0.311.,0

Calculated : A, 60.6), Ii, Ay); fi , 24.60 I’ound : A , 5(.). 1 7 ; 11, 6.21; A , 24.6 3

Lxnmt) I. e 3 2.

2. 6 . S - Tr iaminopur inc - 9 - /3 - D - 2 ' , 3 ' - tl tdeoxyribofuranos ide

2,6,8-Triaminopurine (0.500g, 3.0 nunoles) (0avi.es, R. , e t al. , iM oh ijn.

1’ i ophys , Ac ta . . 564(3), 668, 1979) and 3'-dideoxythymidine (1.()2,-,, 4 . AC· mmoles) (llorwitz J . 1’. e_t ai J.Org.Ahem. 31, 205 ( 1966)) were reacted and chromatographed on AG1-X2 (OH form) and XAD-2 as described in Example if). Lvophi i izat ion yielded 0.148g of 2,6,8 -1r iaminopur i ne - 9 -/3 - D - 2 ' , 3 ' dideoxyrlbofuranoside that analyzed for 0.7 methanol (mp 154°C).

Analysis calculated for C. ,.,11. _rN,0, 0.7CH.O - 10 1j 7 2 4

N IB/KT/AC/”t Ii March

1988

BAD ORIGINAL ft ί ί i c.11 ί a : _ e (I : G . 5 . ’ 6 ; ί 1, 6 .2 5 ; Μ , 39.08

I'oui id : G, 99.51; II, 5.9’; Μ. 3 5./8

Ε gamp L'\

- Am ί no - 6 - benzy 1aminopur ine- 9-g-D- 2’ , 3 * - dideoxyr ibofuranos ide

2-Amino-6-benzylaminopurine (0.2g, 0,8 mmoLes prepared by nucleophilic displacement of the chlorine group on 2-amino-6-chloropurine (Aldrich Chemical Co. Mi Lwauk.ee Wi) by benzylamine) and 3' -deoxythymidine (O.282g, 1. 2 11111)010:1) (llorwitz .1.1’. ct al J .Org.Chem. , 31, 205 (1966)) were reacted and chromatographed on AG I.-X2 (UH form) and XAD-2 as described in Example 18 except smaller amounts of purine nucleoside phosphorylase (10,000 l.U.) and thymidine phosphorylase (3,000 l.U.) were used. Lyophilization yielded

O.L82g of 2 - amino - 6 - benzy l amLnopur ine-9-/3-U - 2 ’ ,3'-dideoxy- r ibofuranos ide that analyzed for 0.60 inethanol (mp 92-99°C).

Analysis Calculated Cor 0 II M .0,, 0.60CI1.0 1/ /0 6 2 9

Calculated : C, 58.28; II, 6.28; M, 23.37 found : C. 58.60; 11, 6.06; M, 2 3.93

E.xarug le_3_9

- Amino - 6 - eye, lopropy lam i nopur inc - 9 - /3- D - 2 , * 3 ' - didcoxy r Ibofuranos ide

2-Amino-6-cyclopropylaminopurine (0.995g, 2.1 mmoles prepared by nucleophilic displacement of the chlorine group on 2 - amino - 6-chlovopurino (AJdt'ich Chemical Co. Milwaukee Wi) by cyclopropylami no) and 3'-deoxy thymidine (O.73g, 3.2 mmoles) (Horwitz J.I’, e t al J.Org. Chem., 31, 205 (1966)) were, reacted and chromatographed on AG1-X2 (Oil form) ami XAD-2 as described in Example 18. Lyophilization yielded 0.9l9g of

- ainino - 6 - cyclop ropy lamino - purine - 9 - /3-D - 2'-d ideoxyr ibofuranos ide that analyzed as a 0.3 hydrate (mp 82-89°C).

APO 0 0 0 7 3

Analysis Calculated for C.,IL_oN.0„ 0.311.,0 13 io 6 2 2

MJ B/KT/AC/9 th March 1.988

bad ORIGINAL

4-,1.,-,1 .ΐίοΗ : 52.80; Π, 4.34; II. 2.8.7»?.

ί-’οιιiv 1 ; , ' 2.8 3; 1 ί . 6.33; Ν . 2 8 . h

Exam ι > 1 e_ 3_ 5

- Amino - 6 - ine thy I aininopur ine - 9 - - D - 2' .3 -dideoxyr ibofuranos ide

2-Amino - 6-ine tliylaminopur ine (0.5 g, 3.0 mmoles prepared by nucleophilic displacement of the chlorine group on 2 - amino - 6 - chloropurine (Aldrich Chemical Co. Milwaukee WI) by methyLamine) and 3'-deoxythymLdine (0.893 g, 3.9 imno les ) (1 Io rw i 1z , J . i’. e t a 1_., J . Ore. . Chem., 3 1, 205 ( 19 46 ) ) wp re suspended in .1.00 ml of id mil potassium phosphate buffer, pH 6.8, cmit:iini.n<; 0.04». potassium azide. Purified purine nucleoside phosphorylase )2,880 I 0) and thymidine phosphorylase (1.200 I.U.) (Krenitsky, Τ.Λ. e t; a 1 . .

Biochemis try, _2_0, 3615, 1981 and US Patent 4,381,447+) were added and the reaction was stirred at 33°C for 72 hours. The reaction was applied to a column of ACI-X2 (OH-form) 2.5 x 10 cm, and the product eluted with 9<H aip.ieou.·: inel Hanoi . Solvent was removed in vacuo and the residue was riash chromatographed on a silica go i column, 2.5 x 30 cm, with chloroform: me (.Hanoi (9/.3, v/v). Lyophilization yielded 0.3 g. o!

- am i no - 6 - me t hy lain (no pur i. up 9-/3-1)-2 ' , 3-dideoxyri.bofurano·-,ide that analysed as a 0.4 hydrate i.m.p. 3c partial me It at / 5 C)

Analysis Calculated for C^ll^N^O? 0.411^0 Calculated: C, 48.66; H, 6.24; N, 30.95 Pound: C, 48.57; II, 6.27: H, 30./7

Example 36

7jj\m i no - 6 - n- propoxy pur i ne - 9 - ft - 0 - 2 ' . 3 ' - dideoxy r i.bo fu ranos j. do

2-Amino - 6 - n-propoxypur ine. (0.21 g, 1.1 mmoles prepared by nucleophilic displacement of the chlorine group on 2 - amino - 6 - chloirpur ine (Aldrich Chemical Co. Milwaukee WI) by the alkoxy anion formed bei.ween sodium hydride

NJB/KT/AC/9th March 1988

BADORIQINAL $ [Odi'j

-3/•u>d ι: - ( rnp •·_υη| > .ίιιΊ i' - (1 o (> x v^: J i yin id i no (U.29J /,. 1.3 mmoles) (liorwi; , J.P. /_t at, J, mrg. Cljem. . ', 205, ('1966)) were suspended in 100 ml of 10 peiassium piiosphat'' bull e r , pll /.0 containing 0.04% potassium azide, fur i f i nJ purine nucleoside phosphorylase (2,880 I.U) and thymidine phosphorylase (1200 10) (Krenitsky, T.A, e t. al. . Biochemist ry. 20. 3615, 1981 and OS Patent 4,381,446) were added and the reaction was stirred at 33°C for 48 hours. The reaction was applied to a column of AG1-X2 (OH form) 2.5 x 5 cm, and eluted with 90% aqueous methanol. Solvent was removed in vacuo and the residue was flash chromatographed on a silica gel column 2.5 x 30 cm, with chloroform: methanol (9:1 v/v) . Lyophilization yielded 0.132 g, of 2 - amino - 6 - n - propoxvpur ine - 9 - /3 - D - 2 ' , 3 ' di.di.oxyrLbniui.aiiosi.de : hat analysed as a 0.2 hydrate (m.p. 70°C)

Analysis Calculated for C, H^N^O^ 0.21^0

Calculated: C, 52.59; 11, 6.59; N, 23.59

Pound: C, 52.52; H, 6.62; M, 24.49

h.TdbPjT-.JlZ

- Benzy lam i nopur ine - 9 - /3 - 0 - 2 ' . 3 ' - d ideoxyr ibofuranos ide

6-Benzylaminopurine (1.0 g , 4.44 mmoles, Sigina Chemicals, St. Louis, 00/ and. 3'-deoxy thymidine (L.O g. 4,6 inmoles) (Uorwitz, J.P. c t a 1. . J. t)rg. Chem. 31, 205, ( 1.966)) were suspended in 50 ml of 15 mM potassium phosphate buffer, pH 7.2. Purified purine nucleoside phosphorylase (2,000 I.U.) and hvmidine phosphorylase (/,900 I.U.) (Krenitsky, Τ.Λ., et. a 1 . . B i g/itc mi s_: ry 21), 36L5, 1981 and US Patent 4,381,464) were added and the reaction was stirred at 25^JC. After L hour, 6ml of diglyme were added and the reaction was stirred at 37°C for 6 days. The reaction filtrate was adjusted to pH LU. 5 with ammonium hydroxide, applied to a column of- AC1-X2 (formate form/, x 6 cin, and the product eluted with 30% aqueous propanol. The product was then chromatographed on a P-2 column, 2.5 x 90 cm, eluted with 30% aqueous propanol and lyophilization yielded 0.063 g of 6-benzy laminopur ine - 9-/3 - 02 ' . 3'-dideoxyribofuranosjde that analysed as a 0.5 hydrate (m.p. 65°C).

APO00073

NJB/KT/AC/9fch March 1988

BAD ORIGINAL

Β ίι i' Ι' '' · ι¹ ί 11··· οχ -.· r ί >ι ι Γη ran os i d ο that analysed as a 0 . / hvd ra te .

Vb ϋ Ο Ο Ο λ

Analysis ‘ a ί ι' u 1 a I ml (or A i i . N. 0 A . /11 0 ί ι ι ° 3 λ / ι,,' 11 u I a ι erl: b, (.<3.83; II, /.09; ii. 2.4.15 found: C, 3 3.93; II, /.08; il, 24.13

Example 40

- Cyclohexy lain i nopur i ne - 9 - Zl - D - 2 ' , 3 ' -dldeoxyribofuranos ide '· -bye I ohⁿxyl ain Lnopur i ne was prepared by nucleophi 1 ic displacement of the chlorine group of 6 - eh 1.oropur ine, by cyclohexy laiaine .

- byelohexylamLnopurLne (1.0 g, 5 mmoles) and 3deoxythymidine (2.07 g, 9.1 imnoLes) (Horwitz, J.P. et a_l. , J. Org . Chern. 31, 205, (1966)) were dissolved in 23 ini 2 - ine thoxye thy 1 ether and 500 ml of 10 mM potassium phosphate buffer, pH /.2. Purified purine nucleoside phosphorylase (5,000 l.U) anti thymidine phosphorylase ( 3350, 1.1).) (Krenitsky, Τ.Λ. e t. ad_., Bi.oc.hemi stry.

20. 361.5, 1931. anti US Patent 4,381,444) were added and the reaction was stirred at 3/°C for 7 days. The reaction mixture was applied to a column of λΛ0-2 and washed extent ive Ly with water. Product was eluted with W.

aqueous methanol	UV	absorbing
co 1 ι min o f At' 1 - .'12	(Oil- fe	nan) . 2x12
aqueous me thano 1. .	The	product was
2.5 x 90 cm, and	a 0-10	column, 2.5
3(5% aqueous	propanol . Ly
6 - c vc lohexy 1aminopur i.n>~	- ’’ - d - 1) - 2 ' , 3 ’

0.09 3

Analysis Calculated for b Π N U , ι 4 / 3 j 2 calculated: b, 60.53; II, /.30; ti. 22.0/

Found: b. 60.37; H, 7.39; ii, 21.94

Exampic 41

- He thy laininopur ine - 9 - Z? - 0 - 2 ' . 3 ' - di deoxyribofuranos ide

NJB/KT/AC/9th March 1988

BAD ORIGINAL

- '.0 ; 3 η'¹ η - E'S ’ · ί ·>ιι> i nojii i r i no t/' · ’ 1 mmoles, lg) ,) 1 > (..i i 11 e <1 from Sigma Chemical Co.. 5;. i/'ui.s. !’>(' ami i' -deoxy thymidine (4.40 mmoles, Lg) (Horwitz J . 1'. ·- g a 1 ; .1 . !V<·,. eiicm. Si., 20 j (, 1.91·4 ) ) wore suspended in 5<'ml ol LOrnll potassium phosphate buffer, pH i , and 0.04% potassium azide. Purified thymidine phosphorylase (2,000 I.U.) and purine nucleoside phosphorylase (2,400 I.U.) (Krenitsky

Τ.Λ. , ct a 1. , Biochemistry 2_0, 3615, 1981 and US Patent 4,381,444) were added and the suspension was stirred at 35°C. After three days, the reaction was stored at -20°C. Upon thawing, the reaction was filtered and the filtrate applied to a 2.5 x 10 cm column containing Dowex-1-hydroxide. The product was eluted from the column with 90% methanol/water (v/v). Product containing fractions were combined and the solvent removed under vacuum. This material was chromatographed twice on a 5 x 90 cm column containing BioRad P-2 resin with 30% n-propanoL/water (v/v). Product containing fractious were pooled, and after lyophilization yielded 0.191;', ot 6 - me thylaminopurine-9-ft-D-2',3' - didcoxyribofuranoside that analysed as a 0.1 hydra te.

Ana I.. Calcd. for C, Jlj _r>N_r0_?0.111 0: C, 52.62; H, 6.10; N, 27.89

Round; C. 52.75; 11. 6.16; H, 28.01

AP 0 0 0 0 7 3

UilR data: 6 8. .)7. (s, I. II 4 . 8.12 (s , 1 H, II.,). /.72 (b. 1 II, Nil) 6.?3 (<!-!, Ϊ ' ’ Z.

Η. H.'.i, 3.06 ft, I 11, 3' OH), 4.10 (in, 1 Η. 11,,) 3.58-3.69 (,Μ. 1 11 5’ CH , i L 4 ' 2

3.45-3.35 (in. 1 11, 5' Clip , 2.95 (b, 3H, CH ) , 2.40 (in, 211. 2' CH J and 2.07 (m. 2 H, 3' CH„ ) ·

E x a iup i e 4 2

Tubjet Formulations

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

H H'./KT/A<;/9i h Hard. 1.988

BAD ORIGINAL

'ι i ί· j .·> rin_u 1, .i_l. J. υ η_Λ

(a)	Act ive ingredient
(b)	Lactose B.P.
(c )	Povidone 11. P.
(d)	Sodium S La veil Given
(e)	Magnesium Stearate

mr / ta1.) re t	t.iί i ’ ? L p _
250	2 50
210	26
15	9
20	12
_5	_3
500	300

1tormu 1 a t ion 11
(a)	Active ingredient
(b)	I .a c Lose
(c)	Av reel PH 101
( d ?	Povidone B.P.
'/ ° )	Sodium Starch Give
ti.)	Magnesium Stearate
Lo run	11.a L ion C

Active ingredient

Lac to.se

S ta tcli

I’ov [done

M a gi io s i uni s t e a r ate

mg/table t	ing/tablet
2 50	2 50
150	-
60	26
15	9
20	12
_3	_3
500	300
mg/tab 1 c t
100
200
50
J
_4
359

Ran nomiMAL

M.l Β/ΚΤ/Λ0/9Ι 11 March I'MU’t i;?< i' ‘

I'l·:- ί c i i nr. I nini'i i a t i<»ns , !» and E. are prepared by direct compression ο I. ; ii- admixed i η ί; i ci 1 i e nI~ · . The lactose in formulation E is of the di reel (mp i ei mi <J).;ii cv Crest - Zeparox).

ij \t mp i.. >J-.iojL_b ing/tablet

Active ingredient 250

Pregelatinised Starch ίϋ·Ί3 150

400

Io rintila t ion _E ιιΐΕ,/tablet

Ac live ingredient 250

Lactose 150

Av ic-'I 100

500 foriy 11 a t i m i 1'_(Controlled Release formulation)

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

AP000073

NJB/KT/AC/9th March 1988

•s	, a )	Ac t i ”c i ngreil Len I	?00
	51’>	Hydtoxypropy line thyl ce I lul ose	L12
		(Kethocel K4M Premium)
4+	(c)	Lac tose 15. P .	53
	(<»)	Povidone B.P.	28
.0	(p)	Magnesium Stearate	_7
			700

Iirug ri'le.-isr takes plac·· ver a period of about G-S hours and is comp i e ;.<? af ter I 2 hours .

Example 43

C a psule Fori ik i_L a t ions

Formulation Λ

Λ capsule formulation is prepared by admixing the ingredients of Fe rmu ! ,11 i, ,n 1' in Ex-amp Le 19 above and fi ll inp; into a two-part hard gelatin capsu i ··. Formulation 15 CLLLC fa) i^s prepared in a similar manner.

Formulation 15 mg/capsule

i1	Active ingredient	2 50
(.!>)	Lac tose 8.P.	14 3
ic)	Sodium St.arch Glycol late	23
id)	Magnesium Stearate	Ί

420

NJB/K’f/AC/9th March 1988

BAD ORIGINAL

- 44 B509

Form11 LaC ion C	mg/capsule
(a) Active ingredient	250
(b) Macrogol 4000 B.P.	350
	600
Capsules of formulation C are prepared by melting the Macrogol 4000 BP,
dispersing the active ingredient two-part hard gelatin capsule.	in the melt and filling the melt Into a
Formulation 0
	mg/capsule
Active ingredient	250
Lecithin	100
Arachis Oil	100
	450

AP 0 0 0 0 7 3

Capsules of formulation D are prepared by dispersing the active ingredient in the lecithin and arachis oil and filling the dispersion into soft, plastic 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 bv 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.

NJB/KT/AC/9th March 1988 bad ORIGINAL

509 mg/capsu re

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

Λ' ts·;

Example 46

Injectable Formulation

Formulation A.

Active ingredient

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

0.200g

6.0 to 7.0 lOinl

The active ingredient is dissoLved in most of the water (35°-60°C) and the pH adjusted to between 6.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate. The batch is then made up to volume with the. water and filtered through a sterile micropore filter into a sterile 10ml amber glass vial (type 1) and sealed with sterile closures and overseals.

NJH/KT/AC/9th March 1988

BAD ORIGINAL

B5O9

Formulation B .

Active ingredient

Sterile, pyrogen-free, pH / phosphate buffer , q.s. to

Example 45 n t ramiiscu lar injection

Active ingredient

Benzyl Alcohol

Glycofurol 75

Water for Injection q.s. to

The active ingredient is dissolved in then added and dissolved, and water filtered through a sterile inicropore glass vials (type 1).

Example 46

Syrup

Active ingredient

Sorbitol Solution

Glycerol

Sodium Benzoate

Flavour, Peach 17,42.3169

Purified Water q.s. to

The active ingredient the purified water, added to the solution

0.125 g ml

0.20 g

0,10 g

1.45 g

3.00 ml the glycofurol. The benzyl alcohol is added to 3 ml. The mixture is then filter and sealed in sterile 3 ml amber

0.25 g· 1.50 g

2.00 g

0.005 g 0.0125 ml 5.00 ml is dissolved In a mixture of the glycerol and most oi An aqueous solution of the sodium benzoate is then , followed by addition of the sorbitol solution and

AP 0 0 0 0 7 3

NJB/KT/AC/9th

March 1988

bad ORIGINAL

B5O9 tiii.il ly : he flavour. Th;· volume is made lip w i th purified water and mixed we 11.

Example 47

Suppos i torv

J3

Active ingredient (63pm)* Hard Fat, BP (Witepsol H15

	ma/supDositorv
	250
Dynamic NoBel)	1770
	2020

*The active ingredient is used as a powder wherein at least 90% of the particles arc of 63pm diameter or less.

One-fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45°C maximum. The active ingredient is sifted through a 200pm sieve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45°C, the remaining Witepsol 1115 is added to t:he suspension and stirred to ensure a homogenous mix. The entire suspension is passed through a 250pm stainless steel screen and, with continuous stirring, is allowed to cool to 40°C. At a temperature of 33°C tc? 40°C, 2.02g of the mixture is filled into suitable, 2 ml plastic moulds. The suppositories are. allowed to cool to room temperature .

NJB/KT/AC/9th March 1988

BAD ORIGINAL

B5O9

Exitnple 98 i’cssar Les mg/pessary

Active ingredient (63/im)	250
Anhydrate Dextrose	380
Potato Starch	363
Magnesium Stearate	7

1000

AP 0 0 0 0 7 3

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

Antiviral Activity

-Cyclopropy Laminopur ine- 9-p-D-2’ , 3'-dideoxyribofuranos ide and 6-methylaminopurine-9-β-0-2' ,3'-dideoxyribofuranoside, were tested for activity against HIV generally in accordance with the method described bv Mitsuya e t a L. Proc. Nat. Acad. Sci, USA Vol 82, pp 7096-7100, Oct. 1985 and found to have activity against HIV at concentrations of ΙμΜ.

Claims (8)

1. Λ compound of formula (I)

   J3 (I) wherein R^ represents hydrogen or amino; and represents halogen, alkoxy optionally substituted by C.

   cycloalkyl; C.

   1-6 —·/ ------ ~₃₆ __y-------..., „₃_₈ eyeloalkyloxy; aryloxy, aralkyl or aralkyloxy in which the aryl tnay optionally be substituted with lower alkyl, hydroxy or halogen; ₍ cycloalkylthio; alkylthio; arylthio or aralkylthio in which the.

   aryl may optionally be substituted with lower alkyl, hydroxy, or halogen; or R^ represents a heterocyclic group containing an oxygen atom or one or two nitrogen atoms, and 3-7 carbon atoms with optional double bonds in the ring optionally containing a sulphur and/or oxygen hcteroatoiu and optionally substituted on the ring by one or more Lowei; alkyl, hydroxy or halogen groups, & cycloalkylthio, aralkylLhio in which the aryl may be substituted with lower alkyl, hydroxy or halogen; or R^ represents an imidazolylthio group in which the\^ imidazolyl moiety may be substituted with lower

   C-substituted with nitro; or R„ represents an. amino mono- or di - substituted by θ alkyl, ,, *...*./*.* .., cycloalkyl, aryl, aralkyl in which the aryl may optionally he substituted with lower alkyl, hydroxy or halogen, allyl optionally substituted with mono- or di-alkyl or alkoxy groups and R^ represents hydrogen or ainino; and pharmaceutically acceptable derivatives thereof, other than the compounds of formula (I) in which R, and R„ i j

   NJB/KT/9th March 1988

   BAD ORIGINAL fi

   -/<Q B309CC represent hvdrogen and R^ represents me thy I. ami no group, a methoxy, methylthio or /3 compound of formula (I) according to claim 1 wherein R^ and each represent hydrogen.
2. 3. Λ compound of formula (I) according to claims 1 or 2 wherein R, represents a mono- or di- substituted amino group.

   APO00073
3. 4. Λ compound of formula (I) according to claim 3 wherein the amino group is mono- or di-substituted by alkyl or cycloalkyl.
4. 5. Λ compound of formula (I) according to claims 1 or 2 wherein represents a heterocyclic group containing a nitrogen atom and 3-7 carbon atoms.
5. 6, A compound of formula (I) according to claim 1 selected from the following;

   a) 6-N-Piperidinopurine-9-/3-D-2' ,3' -dideoxyribofuranoside

   b) 6 - Cyclopropylmethylaminopurine- 9 - /J-D-2',3'-dideoxyribofuranoside

   c) 6 -Dimethylaminopurine- 9-β-D-2' ,3'-dideoxyribofuranos ide

   d) 6-Cyclopropylaininopurine-9-/3-D-2' , 3' - dideoxyribofuranoside

   e) 6 - Cyc 1 ope n ty lam i nopur ine - 9 -/3-D - 2' , 3 ' - dideoxyr ibo furanos ide

   f) 6-Pyrrolidinopurine-9-/3-D-2' , 3' -dideoxyribofuranoside
6. 7. A compound of formula (1)A

   NJB/KT/9th March 1988

   BAD ORIGINAL .,ηB509CC wherein R represents hydrogen or ainino; and R, represents halogen, ilkoxy optional·!.·/ substituted by C cycioalkyi; C, „

   1 -6 c ycLoa Ikyloxy;

   aryloxy, aralkyl or aralkyloxy in which Che aryl may optionally be substituted with lower alkyl, hydroxy or halogen; rye 1 onLkyIchio, C alkyl.Lhio; arylthio or araLkylthio in which Liic aryl may optionally be substituted with lower alkyl, hydroxy, or halogen; or R^ represents a heterocyclic group containing an oxygen atom or one or two nitrogen atoms, and 3-7 carbon atoms with optional double bonds in the ring optionally containing a sulphur and/or oxygen heteroatom and optionally substituted on the ring by one or more lower alkyl, hydroxy or haLogen groups, cycloalky1 thio, aralkylthio in which the aryl may be substituted with lower alkyl, hydroxy or halogen; , or R£ represents an imidazolylthio group in which the imidazolyl moiety may be substituted with Lower alkyl and/or C-substituted with nitro; or R, represents an amino group which is mono- or di-substituted by alkyl, alkoxy ,/anu/or &

   cycioalkyi, aryL, aralkyl in which the aryl may optionally be substituted with lower alkyl, hydroxy or halogen, allyl optionally substituted with mono- or di-alkyl or alkoxy groups and R^ represents hydrogen or ainino; and pharmaceutically acceptable derivatives thereof, other than the compounds of formula (I) in which R^ and represent hydrogen and R^ represents methoxy or inethylthio, for use

   in medical therapy. 8. A compound according to claim 7 for use in the treatment or prophylaxis of a human retrovirus infection. 9 . A compound according to claim 8 for use in the treatment O 1' prophylaxis of a Human Immunodeficiency Virus (HIV) infection. 10. A compound according to claim 7 for use in the treatment or prophylaxis of Acquired Immune Deficiency Syndr ome (AIDS).
7. 11. Λ process for the preparation of a compound of formula (I) according to claim 1 comprising:

   NJB/KT/9th March 1988

   BAD ORIGINAL

   )Α·

   B509CC (II) (a) reacting a compound of formula (II)

   AP000073 (wherein R., , R_ and R, are as defined in claim 1) and A represents a precursor group for the hydroxy group, with an agent or under conditions to convert .said precursor group into the desired group; or (b) reacting a purine base of formula (III)

   B-H (III) wherein B is a purine base according to claim 1 or a functional equivalent thereof, with a compound serving to introduce the desired dideoxyribofuranosyl ring at the 9-position of the purine base of formula (III);

   and thereafter, or simultaneously therewith, effecting one or more of the following optional conversions:(i) when a compound of formula (I) is formed, converting it into a pharmaceutically acceptable derivative thereof.

   (ii) when a pharmaceutically acceptable derivative of a compound of formula (I) is formed, converting the said derivative into a compound of formula (£), or a different derivative thereof.
8. 12. A pharmaceutical formulation comprising as active ingredient a compound of formula (I)A (as defined in claim 7) or a pharmaceutically acceptable derivative thereof, together with a pharmaceutically acceptable carrier therefor.