AP11A - Antiviral nucleosides. - Google Patents

Abstract

3'-Azido-3'-deoxythymidine and its pharmaceutically acceptable derivatives have been found to possess patent activity against human retrovirus infections particularly HTLV-1 and HTLV-111 infections.

Description

2445 -i-

Antiviral Nucleosides

The present invention relates to 3’-azido-3'-deoxythymidine, its pharmaceuticallyacceptable derivatives and their use in the treatment or prophylaxis o' humanretroviral inactions.

Retroviruses *orm a sub-group of RNA viruses which, in order to replicate, must*irst 'reverse transcribe’ the RNA of their genome into DNA (’transcription’conventionally describes the synthesis of RNA from DNA). Once in the form ofDNA, the viral genome is incorporated into the host cell genome, allowing it totake 'ull advantage o* the host cell's transcription/translation machinery 'or thepurposes of replication. Once incorporated, the viral DNA is virtuallyindistinguishable *rom the host's DNA and, in this state, the virus may persist'or a3 long as the cell lives. As it is virtually invulnerable to attack in this form,any treatment must be directed at another state of the li'e cycle and will, o*necessity, have to be continued until all virus-carrying cells have died. HTLV-I and HTLV-II are both retroviruses and are known to be causative agentsof leukaemia in man. HTLV-I Infections are especially widespread and areresponsible 'or many deaths world-wide each year. A species of retrovirus has now been reproducibly isolated from patients withAIDS. While it has been extensively characterised, there is, as yet, no agreed e name' 'or the virus, and it is currently known either as human T-celllymphotropic virus ΙΠ (HTLV ΠΙ), AIDS-associated retrovirus (ARV), orlymphadenopathy associated virus (LAV). It is anticipated that the name to beagreed on internationally is aquired immune deficiency virus (AIDV). This virus(re'erred to herein as AIDV) has been shown preferentially to infect and destroyT-cell3 bearing the OKT sur'ace marker and is now generally accepted as theaetiologic agent of AIDS. The patient progressively loses this set of T-cells,upsetting the overall balance o* the immune system, reducing his ability tocombat other infections, and predisposing him to opportunistic infections which'requently prove fatal. Thus, the usual cause o' death in AIDS victims is byopportunistic infection, such as pneumonia or viraliy induced cancers, and not asa direct result of AIDV infection. APOOOO 1 1

Recently, AIDV has also been recovered *rom other tissue types, including S-cells expressing the T4 marker, macrophages and non-blood associated tissue inthe central nervous system (CNS). This latter In'ection has been discovered In

BAD ORIGINAL

HDL/L\D,'26th February 1926 Q443 · ts expr-jsi'·. ; classical ^iDS symp toms and is associated with progressivecar i. elinaiion, .ending to casting and such symptoms as encepna; pathy,ongressive dysarthria, ataxia and disurientation. c; ’here are at least 'our clinical mar:'estations o* AIDV in'ection. In the initial'carrier' state, the only indication of in'ection is the presence o* arti-AiDVantibodies in the blood-stream. It is believed that such 'carriers' are capable o'passing on the in'ection, e.g. by blood trans'usion, sexual intercourse or usedsyringe needles. The carrier state may often never progress to the second stagecharacterised by persistent generalised lymphadenopathy (PGL). It is currentlyestimated that about 20% o* PGL patients progress to a more advanced conditionknown as 'AIDS related complex' (ARC). Physical symptoms associated withARC may include general malaise, increased temperature and chronicin'ections. This condition usually progresses to the 'inal, fatal AIDS condition,when the patient comoletely loses the ability to fight infection.

The existence o' these human retroviruses and others has only recently beenrecognised and, as the diseases with which they are linked are of a li'e-tt-; eaten I ng nature, there exists an urgent need to de· -lop ways to combat theseviruses.

Various drugs have now been proposed as ’cures1 for AIDS. These includeantimonictungstate, su amin, ribavirin and isoprinosine, wl !ch are cithersomewhat toxic or have shown no marked anti-retroviral activity. As the AIDVrename is incorporated into the host cell DNA after infection and is virtuallyinvulnerable to attack in this state, it will persist as long as the host ceilsurvives, causing new infection in the meantime. Thus, any treatment of AIDSwould have to be 'or an extended period, possibly life, requiring substances withan acceptable toxicity.

Reports have described the testing c‘ compounds against various retroviruses,'or example, Friend Leukaemia Virus (FLV), a murine retrovirus. For instanceKrieg e^al. (Fxp. Cell Res., 116 (1978) 21-29) found J'-azido-J’-deoxythymidineto be active against FLV in in vitro experiments, and Ostertag et al. (Proc. Nat.Acad. Sci. (1974) 71, 4980-85) stated that, on the basis o* antiviral activityrelated to FLV and a lack of cellular toxicity, 3'-azido-3'-deoxythymidine "might'avourably replace bromodeoxyuridine for medical treatment o* diseases causedby DNA viruses”. However, De Clerq et al. (Biochem. Pharm. (1980 29, 1949-1851) established, six years later, that 3'-azido-3-deoxythymidir; ad no

BAD ORIGINAL -3- !_)ΰ ύ 5 appreciab'e activity against any viruses used in their tests, including v .cciaia,Η.>νΊ and varicella zoster virus <VZV). Giinski et al. (J. Urg. ' 'hem. (1975’, 39»429/-Λ305) discloses certain derivatives o* 3'-azido-3'-ceoxythy[nidine (tn'r'A andtheir ability to block mammalian exuribenu^lease activity.

We have now discovered that J'-azido-i’-deoxythymidine ha; a surprisinglypotent activity against human retroviruses, with a particularly high activityagainst A1DV as demonstrated by the experimental data re'erred to below. Suchactivity renders the compound useful in the therapy o' human retroviralin'ections.

Thus, in a 'irst aspect o' the present invention, there is provided a compound o*

(I) (i.e. 3'-azido-3’-deoxythymidine) or a pharmaceutically acceptable derivative,thereo', for use in the treatment or prophylaxis of human retrovirus infections.Th,? cnrnpnu; d of 'orc ;’.a (I,’ and its pharmaceutically acceptable derivative- areoerea'ter re'erred to as the compounds according to the invention.

Activity o' 3,-azido-3,-deoxythy.-Tiid»ne against human retroviruses has beenestablished in various in vitro assay systems. For example, in'ection o' the H9human lymphoblastoid ceil-line by AIDV is effectively prevented byconcentrations o' 3'-arido-3'-deoxy thymidine as low as 0.D13 mcg/ml up tohours a'ter in'ection. AIDV in'ection o' U93 7 human lymphoblastoid cells, PHA-3tim.Jated white blood cells and cultured peripheral blood lymphocytes is alsoprevented at similarly low concentrations. In addition, 10-day challenge•xperiments using up to 3(100 AIDV virions per cell and cloned T4, tetanus-speci'ic, T-helper lymphocytes, showed no decrease in cells treated with 3’-azido-3'-deoxythymidine, while untreated cells had decreased 3-'old. Cytopathice"c-ts were also completely blocked in the same cell-line trans'ormed byHII V-I and super-in'ected with AIDV.

Other studies using peri'ied AIDV reverse transcriptase have shown that the «activity o' this enzyme is blocked by the triphosphate o' 3'-azido-3'-ieov, thymidine by a competitive inhibition mechanism. ΑΡ0 0 0 0 1 1 J»

BAD ORIGINAL 43 Ρί?3ί» 1 clinical tri ala have r'.:* shown tr.ot 3'-azido-3’-deoxythy midine is -ocaclerrossina the binej/brain be", er :.. clinically e^cti.e quantities. .h'3 rrccerty is both unusual and valuable *or the treatment ,r J prophyli' is ·>*infections caused by b;TOn retroviruses.

The ability o' T’-azxdo^’-.-'-ioxythymidine to modify the course o* retrov · s-in-Jueed malignancy has ber-n demonstrated in a mouse modul, \ herebyadministration o' J'-azido-T'-deoxychymidine prevented splenomegaly causes u*intravenously administered Ra seher Murine Leukaemia Virus, the murineequi.-a'ent of HTLV-l. in *urther experiments, 3'-azido-3'-Ceoxythymidine hasbeen shown to inhibit the jnj/itjm replication of HTLV-I at concentrations as lowas 0.8 mcg/ml.

Thus, in a *urther, preferred aspect of the present invention, there is providedtne use of a comcound accord:.-,a to the invention in the manufacture o' a —, medicament for the treatment or prophylaxis of human retrovirus infections. -·' The present invention further provides a method for the treatment or proohylaxis of AIDS in a hu nan subject which comprises administering to the said humansubject an elective amount of a compound according to the invention. >

The present invention also includes a method for the trestme.' t or prop, lax:? cfinfections caused by retroviruses in a human subject, which compriseradministering to the said human subject an effective antiviral amount o* acompound according to the invention.

The present invention 'urther provides a compound according to the invention 'oruse in the treatment or orophylaxis o' AIDS or a virus infection as ic-:ntifiedabove.

Examples o* human retrovirus infections which may be treated or prevented inaccordance with the present invention include T-cell lymphotropic retroviruses(HTLV), especially HTLV-I, HTLV-II and AIDV (HTLV-ΠΙ). Clinical conditionsthat may be treated or prevented in accordance with the invention include AIDS,AIDS-related complex and HTLV-l positive leukaemia and lymphoma. Suitablepatients *or treatment also include those having antibodies to AIDV, AIDV CNSinfections, PGL and ARC.

By "a pharmaceutically acceptable derivative" is meant any pharmaceutically

BAD_ORIGINAL -5- 8445 εcceur-:tle suit, est-i, or salt o* such ester, or any othe.- compound which, up-nadministration to a human subject, is capable o‘ providing (directly ori. Erectly) P-azido-l'-deux/thymidine or an anti-retrov^rall/ active metaboliteor resiov · thereo*. An example of a non-ester compound is the derivativewherein the > -C- end 2-C-atoms are linked by an oxycrn atom to ‘arm ananhyoro group.

Preferred esters o* the compound of *ormula (I) include carboxylic acid esters inwhich the non-carbonyl moiety of the ester grouping is selected *rom straight orbranched chain alkyl, alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl),aryloxyd'kyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionally substituted byhalogen, alkyl or C,_^ alkoxy); sulphonate eaters such as alk7i- or a-aik/lsulphonyl (e.g. methanesulphonyl); and mono-, di- or tri-phesphate esters.With regard to the acove-described esters, unless otherwise speci*ied, eny alkylmoieties present in such esters advantageously contain 1 to 18 carbon atoms,particularly 1 to 4 carbon atoms. Any aryl moiety present in such estersadvantageously comprises a phenyl group. Any reference to any of the abovecompounds also inc!.ides a reference to a pharmaceutically acceptable saltthereo*. t xperiments have shown that 3’-azido-3'-deoxythymidine is converted, in vivo, bythe action of cellul· r enzymes into the 5*-monophosphate. The monophosphate isthen ‘urther phoschorylated by other enzymes to form the triphosphate via thediphosphate, and other studies have demonstrated that it is the triphosphaterorm o* 3'-azido-3'-deoxythymidine which is believed to be the elective chainterminator in the reverse transcription of A1DV, as evidenced by its e**ect onavian myeloblastosis virus and Moloney murine leukaemia virus. This *orm abonhibits AIDV reverse transcriptase in vitro whilst having a negligible e*'ect onhuman ONA polymerase activity. AP 0 0 0 0 1 1

Examples of pharmaceutically acceptable salts of the compound o* *ormula (I)and its pharmaceutically acceptable derivatives include base salts, eg derivedfrom an appropriate base, such as alkali metal (e.g. sodium), alkaline earthmetaKe.g. magnesium) salts, ammonium and NX* (wherein X is alkyl).

The present invention thus further provides the novel pharmaceuticallyacceptable derivatives o* the compound a* formuie (I), other than the 'ohowmg5' derivatives, namely monophosphate, di sodium monophosphate, 2-cysnoetny1monophosphate, triphosphate, p-toluene sulphonate, acetate, triphenylmeth/ί andmethanesulphonate derivatives or where the 5' C is linked to a *urtheg^gp|5pfgj^Lor nucleoside derivative. BAD original -6- 5ceci*ic examples o' charmaceutically acceptable derivatives of the compound-i* ‘ormuia (1) that r,?v b«s used if: accordance with the -resent invention .nc·1 ‘~the -nonosodium salt and tie 'c'.Uwing 5' esters: monophosphate; di-jd.ummorophosohate; diphoschate; trichoscnate; acetate; J-m^’.’.yi-bLl/raCi::nctancate; palmitate; 3-chioro tenroate; benzoate; '.-methyl benzoate; hydrogensuccinate; pivaiate; and mesy.aie. I’-Azido-J'-deoxythymidine, or = pharmaceutically acceptable deriv^riv^ tncren*(hereafter re*erreu r.o as the active muradient), may be administered to humans*or prophylaxis or treatment o* retroviral infections by any suitableincluding oral. rect_i, nasal, topical (.including buccal and subungual). ·. ay;nai c· iparenteral (incluring subcutor ec :s. intramuscular» intravenous and -,--01..It will be apprecictao that the preferred route will var> with the con j>ion cr-rage of the recipient, the nature of the infection and the chosen act --incredient. er ο

X > !rs general a sc table dose will be in the range of 3.0 to 12C mg per kilogram bodyweight of the - itient per cay, preferably in the range of 6 to 90 mg pet kilogramcody weight per day and most preferably in the range 15 to 60 mg per kilogrambody weight per day. The desired dose is preferably presented as two, three,four, f;ve, six or or more sub-doses administered at appropriate : terveisthroughout the day. These sub-doses may be administered in unit dosaqe *urrr,s,'or example, containing 10 to 1500 mg, preferably 20 to 1000 mg, and mostpreferably 50 to 70C mg of active ingredient per unit dosage *orm,

Fxoeriments with 3'-az;do-3'-decxythymidine suggest that a dose should beadministered to achieve peak -jiaama concentrations o‘ the active compound z*‘rom about 1 to about 75 pM, pre'erably about 2 to 50 pM, most preferablyat nut 3 to ao out 30 yM. This mg/ be achieved, *or example, by the intravenousInjection o~ a 0.1 to 5'c solution cr the active ingredient, optionally in saline, ororally administered as a bolus containing about 1 to about 100 mg/kg a* theactive ingredient. Desirable blood levels may be maintained by a continuousirr'usion to provide aoout 0.01 tc about 5.0 mg/kg/hour or by intermittentin'usions 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 pre*erable to present it as a pharmaceutical *ormulation. The *ormulations of bad original -/- 5443 the present invention comprise at least one active ingredient, as above dr'ined,together with one or more acceptable carriers thereo' and optionally othertherapeutic agents. Fach carrier must be "acceptable" in the sense o' beingcomoatible with the other ingredients o' the 'ormulat’on and rot injurious to ·,'?oatient. Formulations inckde these suitable 'or oral, rec’ul, nasal, topical'including buccal and sublingual), vaginai or parenteral (including subcutaneous,intramuscular, intravenous and intradermal) administration. The 'ormuiationsmay conveniently be presented in unit dosage 'orm and may be prepared by anymethods well known in the art of pharmacy. Such methods include the step o'bringing into association the active ingredient with the carrier which constitutesone or more accessory ingredients. In general, the formulations are prepared byuni'ormly and intimately bringing into association the active ingredient withliquid carriers cr 'inely divided solid carriers or both, and then i* necessaryshaping the product.

Formulations, of the present invention suitable 'or oral administration may bepresented as discrete units such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient; as a powder or grp^ules; as asolution or a suspension in an aqueous or non-aqueous liquid; or as'an oil-in-waterliquid emulsion or a water-in-oil liquid emulsion. The active ingredient may alaobe presented as a bolus, electuary or paste. Oral formulations may *urther includeother agents conventional in the art, such as sweeteners, flavouring agents andthickeners. A tablet may be made by compression or moulding, optionally with one or moreaccessory ingredients. Compresstd tablets may be prepared by compressing In asuitable machine the active ingredient in a 'ree-flowing 'orm. such as a . wderor granules, optionally mixed .-. ith a binder (e.g. . ovicane, :e· .tin, hydroxypropylmethyl cellulose), lubricant, inert uiiuent, prese-vati/0.disintegrant fe.g. sodium starch glycollate, cross-linked pc.ioone. cr·- ?:-!inkedsodium carboxymethyl cellulose), sur'ace-active or dispersing ager.t. Mouldedtablets may be made by moulding in a suitable machiie a mixture 0' thepowaered compound moistened w?h an inert liquid diluent. The tablets mayoptionally be coated or scored and may be 'ormulated so as to provide slow orcontrolled release 0' the active ingredient therein using, 'or examole,hydroxypropylmethyicellulose in varying proportions to provide the desiredrelease pro'ile.

II 0 0 0 OdV

BAD ORIGINAL j· 3 - ·?? nulations s^d-ibic ‘c: '. •dmi.-'^i-ation in the mouth ;r.< . ~i· .:urr.r,rising tha acuve .ngrciicnt .:t a fiovoured basis, jj'iui»,. sucivie -.’’v.>? tranacanth; ; eet!’irs . :.ncrl.' ''j vhe s-u.ve ingred’u.·· ‘ . ·. un ·.··<?:; ., :ugei.rin and 0^,00::-1, s;-cruse and acacia; and mo· tn#a<·'^ comp;>.-4 ·-.*active ingredient in a suitable lie·.id carrier. formulations *oi rectal administration may be presented as a suppository with asuitacie base comprising ‘or example cocoa butter or a salicylate.

Formulations suitable *or vaginal administration may be presented as pessaries,tampons, creams, gels, pastes, foarns or spray formulations containing in auctionto the active ingredient such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants,buyers, bacteriostats and solutes which render the formulation isotonic with theblood of the intended recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending .egents and thickening agents. The formulationsmay be presented in unit-dose or multi-dose sealed containers, *or exampie,ampoules and vials, and may be stored i.i a freeze-dried (lyophilized) conditionreauiring oniy the addition of the sterile liquid carrier, for exampjewater fori.lections, immediately p.-'or rj use. Fxtemporaneous inject’jn solution,. andsuspensions may be prepared *.-om sterile powders, granules and tablets o* thekind previously described.

Fre'erred unit dosage formulations are those containing a daily dose or unit,daily sub-dose, as herein above recited, or an appropriate faction thereo*, o* anactive ingredient.

The administered ingredients may also be used in therapy in conjunction withother medicaments such as 9-l(2-hydroxy-l-(hydroxy- r.ethyl)ethoxy]methyl]guanine, 9-(2-hydroxyethoxymethyl)guanine (ncyclovir), 2-amino-9-(2-hydroxyethoxymethyl)purine, interferon, e.g., a-inter*eron,interleukin II, and phosphono'ormate (Foscarnet) or in conjunction with otherimmune modulating theraoy including bone marrow or lymphocyte transplants ormedications such as levamisol or thymosin which serve to increase lymphocytenumbers and/or function as is appropriate.

It should be understood that in addition to the ingredients particularly mentioned

BAD ORIGINAL -9- B--3 above the formulations o' this invention may include other agents con\entionalin the art o* formulation.

The compound o* 'ormula (1) 3nd its pharmaceutically acceptable derivati- es--3/ be prerared in ccnve .tiona. manner. *or example as described in thefollowing references, or by mett'?ds analogous thsreto:J.R. Horwi“c m a1., 2.Crg. Chem. 29, July 1964) 2076-76; M. Imazawa et al.. 3. Crg. Chem, 43 15)(1978) 3044-3043; Κ.Ά. Watanab-· et al., J. Org. Chem., 45, 3274 (19.3), andR..P. Glinski et al., J. Chem. Sac. Chem. Commun., >15 (1970), as ·,. eii as theprocesses described ir the F-.an pies.

The present invention furthe- mctudes a process *or the preparation o' acompound of 'or-ula (I) and pharmaceutically acceptable derivatives h.ereo*which comprises (A) reacting a compound o* 'ormula:

(II) AP o 0 0 0 1 1 (wherein M rep-esents s pr* tursor group 'or the 3-azido group) or a derivative''e.g. an ester or salt) thereof with an agent or under conditions serving toconvert the said precursor group into the desired azido, group; or (b) :eactir a compound o* formula:

(III) pharmaceutically acceptable derivative group thereo') with an agent or undereruditions serving to convert the said precursor group into the correspondingasired group; or

BAD ORIGINAL -10- !•-'acting 3 ur ..·3; A ·-<’ ''or^jn

HN □,· a 'unctions! equivalent thereo', with a compound serving to ir.troducs th?

desired ribo'uranosvl ring at the 1-position o* the compound o' 'ormula (IV). orC (j) reacting a comoound of formula

(wherein R^ is hydroxy or R as defined above), with an agent or under condl;serving to convert the said compound into a cot mound according toinvention; -, ns the and thereafter, or simultaneously h erewith, ejecting one or more o* the (. 'rnlowing optional conversions:- ’ «» « Χ-» (i) when o'-azico-J'-deoxythymidins, is *ormed, convert;. 7 it into cpharmaceutically acceptable derivative thereo*, when a pharmaceutically acceptable derivative o* 3’-azido-3’-deoxythymicine is 'armed, converting the said derivative into thecompound o' 'ormula (I), or a di'*erent derivative thereof.

In the above-described process according to the invention, it will be appreciatedthat the precursor compounds o* 'ormula? (II) and (ill), as well as the above-mentioned agents and conditions, will be selected 'rom those that are known .inthe art o* nucleoside synthetic chemistry. Examples o* such conversionprocedures are described hereinafter *or guidance and it will be understood thetthey can be modi'ied in conventional manner depending on the desired compoundo' 'ormula (I). In particular, where a conversion is described which .'ouldotherwise result in the undecired reaction o* labile groups then such groups maybe protected in conventional manner, with subsequent removal o' the protect’nggroups a'ter completion o* the conversion. Thus, in process (Aj, the group N < BAD original -11- tbe compound -~>e 'ormuia (It) may represent, *or example, a '-aluj?^ . · q.chlorine;. m. proxy or organosulpr.oiixio·./ e.g. tri'luoromethyipulpbuir..meth.-.nesulph ••avl,·* y or p-tulu.· ie sulphonylixy rac.cal.

Fjr -/·? prepacstiw o* the compound o' 'oimula (P, a -ompcund j* *or; ui,· '11:in which the group M la a halogen (eg chloro) group in the three co-'i'igim.ot.on (; 1«bich the b'-hydroxy is advantageously protected, eg with a trityl group) m v; betreated 'or example with lithium or sodium azide. The 3’- ibrec-halogon (egchlorine) starting material may be obtained *or example by reaction a‘ thecorresponding y-erythro-hydrox/ compound with 'or example triphenylpho mhireand carbor. tetrachloride, or alternatively by treatment with organosulohonylhe:Ida (eg tri'iuoromethanesulpbooyi chloride) to *orm a correspondingc: ^‘':~n-c~ganosu.Lnony'~\y cnrrtuund which is then halogenated. Alternativelya ^'-th'mj-hydroxy ccmpounu o* 'otcuia (II) may be treated, 'or example withtripber-ylphasphine, carbon tetrabromidc and lithium azide to 'orm thecorresponding 3-eryphro azido compound. Removal o' any protecting group maythen subsequent.y be e"eci.ed, e.g. as ioove.

With regard to process (B), R mav represent a protected hydroxy g, jup e.g. anester grouning o' the type referred to above in relation to formula GOparticularly acetoxy, or an ether group such as a trialkylsilyloxy group* e.g. Λ-butyldimethylsliyloxy or an aralkoxy group e.g. triphenylmethoxy. Such groupsmay be converted 'or example by hydrolysis to the desired hydroxy group or, bytransesteri'icatio.n, be converted to an alternative ester group o' 3'-az.do-3'-deoxy thymidine.

With regard to precess (C), this may be e"ected 'or example by treating theancropriate pyrimidine o' 'ormuia (IV) or a salt or protected derivative "hereo',^.ith a compound o' 'ormuia ΑΡ0 0 0 0 1 1

(wherein A represents a leaving group, e.g. an acetoxy or benzoyloxy or halo, eg chloro group and B represents an optionally protected hydroxy group eg a p- toljenesulphonytoxy group), and subsequently removing any protecting groups. BAD ORIGINAL ft j • \.. ,r ji·. ; . , - i ’ i - >'*·()['I· .··ι i! .1 |iiri i.; ,·;· .,γίι.;ι us . ’ ‘ < ·' *.· · .i.i 11 5'-·'hym-diiK· ·η.·|. then, ' ; . ···., : · .v.tn · alkali inolal , e.g. lithium adv.ti’la .cl, ,n ,m uropriate solvent such a;, moist ΩΜΓ 'olinv.ed ’>.ly.'i: ’· hyorolym a Jv.onta msly under mild conditions. ΠΓ e* I': - .d or ;'-,\zidn-3’-,;eo'-thyn ..line may bo converted into a pharmaceutic all,acccptaolc pbos. bate or other ester by reaction with respectively aphosphorylatrng agent, e.g. POCl^ or an appropriate esteri'ying agent, e.g. anacid halide or an.b.dride. The compound o* *ormula (I), including esters tbereo',may be converted into pharmaceutically acceptable salts thereo* in conventionalmanner, e.g. by treatment with ar. appropriate base. An ester or salt of U'-azido-3'-deo».yth,mi dine may be converted into the parent compound, e.g. byhydrolysis. c;> o

The 'ollowing Examples are intenoed 'or illustration only and are not intended tolimit the scope □ * the invention in any way. The term ’active ingredient1 as usedin the Fsamples means a compound o* *ormula (I) or a pharmaceuticallyacceptable derivative thereo*.

Example 1: Tablet Formulations

The *ollowing *ormulations A and B were prepared by wet granulation o* theingredients with a solution o* povidone, *ollowed by addition o* magnesiumstearate and compression. mq/tablet mg/tabtet

Formulation A (a) Active ingredient 250 250 (b) Lactose B.P. 210 26 (c) Povidone B.P. 15 9 (d) bodium Starch Glycollate 20 12 (e) Magnesium btearate _5__ _3 500 300 d/.tr, rnbr^ar, 1906

BAD ORIGINAL

A ‘'ΐ” '- *1 ·Η ‘ - ;\cti·/'; <' S 't 2 SO ’ , '· actose : so - ._·; A·,led !-· Ί i ’Ji 60 26 (ci) i'ovidcne Β.Γ. 15 O e) Sodium Starch Glycoltate 20 12 (‘j Magnesium Stearate _5 _5 500 TOO Γ muiationC. icq/tablet Active ingrecient 100 l_ nctose 200 S t -1cn 50 A (J Ldc.ne 5 Magnesium stec*ace 4 359 The following formulations, D and r , were prepared by direct compression o* the admixed ingrr'"’onts. The lactose used in *ormulaticompression type (Dairy Crest - "Zeparox"). on F was of the direct Form^iaticc, D mq/capsule Active Ingredient 250 A.-egelatinised Starch NF15 150 600 r "rrnulation F mq/capsule Active ingredient 250 Lactose 150 A vied 100 500 Formulation F (Controlled Release Formulation) ΑΡ0 0 0 0 1 1

The 'ormulation was prepared by wet granulation o* the ingredients (below) witha solution o* povidone 'ollowed by the addition o* magnesium stearate and.<>( npression.

.BAD ORIGINAL it**’ '.Wii 3 .'li ve : i jrer’:i. >t '1 ) i; H.,τορ* in'-'thyleeliclose 112 ?/ U-: ··' »< ;’·' Pre.nium) setose B.P. 53 C, p ?v . ' ;;|i L.P.C. ~ ·> Λ1' Magnc^.uin otearc.te 7 700

Drug release took place over a period o' about 6-8 hours and was complete a'ter12 hours. r v3rr.pie 2: Capsule Formulations

Formuiat'.on A A capsule 'ormulaticn was prepared by 3dmixing the ingredients o* FormulationD in Example 1 above and filling into a two-part hard gelatin capsule.Formulation B (in'ra) was prepared in a similar manner.

Formulation B W mq/caoscle (a) Active ingredient 250 (b) Lactose 3.P. 143 (c) Sodium Starch Glycollate 25 (d; Magnesium Stearate _2 420

Γcnnulat'.on C mg/capsule 250 350 600 (a) Active ingredient

(b) Marrcgo! 2000 BP

Capsules were prepared by melting the Maerogol 6000 BP, dispersing the activeingredient in the melt and 'illing the melt into a two-part hard gelatin capsule. H·'.·! /1 '/.J/2pthFepru- : v 1'C’u

BAD ORIGINAL * . .Ή .· Γ I Τ’.3 ( . • * e »

Capsule Acre ureeared by dispersing the active ingredient if: 're lecithin anuarachis ml and 'lliin ; the dis,···.-.-;cn into so*t, elastic qr tatin cap Ces.

Fe-mulation F (CcHrc led Release Cjpsule)

The follr-Am; controlled release capsule formulation was prepared by extrudingir. /ei'.ientj a, a and c using an extruder, *ollowed by sphero lisotion c* theextrudate and dr ' j. The dried pellets were then coated with re'.ease-controllirg membrane (d< and 'ilitd into a two-piece, hard gelatin capsule. mq/capsub ·,<») Active lr r Client (o' Microcrystalline Cellulose ,q'> Lactose »F •d) Fthyi Cellulose ?;o

12S 125 Π 515

.ample 3: li /.c.-ihie Forme'"’nn I . muA. A :live inc: 1client 0.200g

Hydrochloric acid solution, 0.1MSodium hydroxide solution, 0.1MSterile wat-.r q.s. to pHq.s. to pHq.s. to 4.0 to 7.04.0 to 7.013ml

The active ingredient was dissolved in most o* the water (55*-40*C) and the pHadjusted to : etween 4.0 and 7.0 with the hydrochloric acid or tlx? sodium hydroxide asappropriate. The batch was then made up to volume with the water and Alteredthrough mnlc miernpore Citer into a sterile 10ml amber glass vial (type 1) andsealed with sterile e'osures and eve: seals.

BAD ORIGINAL -V;· Ge ingrrUc'i it 0.12b ij ••t-τιίπ. c .γ·'··?·ι,-rr ·. i · ! phosphate bu"er, q.s. to ?b t' !

Fxample u: Intramuscular injection z Active Ingredient 0.20 g Benzyl Alcohol 0.10 g Glyco'urol 75 1.45 g Water 'or Injection q.s. to 3.00 ml The active ingredient was dissolved in the glycofurol. The benzyl alcohol was then added and dissolved, and water added to 3 ml. The mixture was then Altered through asterile rr.icropore 'ilter and sealed in sterile 3 ml amber glass vials (type 1).

Fxample 3; Ingredients • s·, '· ?*' ·

Active ingredient 0.2500 g

Sorbitol Solution 1.5000 g

Glycer-H 2.0000 g

Sodium Benzoate 0.0050 g i L·

Flavour, Peach 17.42.3169 0.0125 ml

Purified Water q.s. to 5.0000 ml

The active ingredient was dissolved in a mixture o' the glycerol and most o' thepuri'ied water. An aqueous solution o* the sodium benzoate wae then added to thesolution, 'ollowed by addition o' the sorbitol solution and finally the 'lavour. Thevolume was made up with puri'ied water and mixed well.

Fxample 6: Sup.posl l ory me/ .uppository

Active Ingreu.ent (63 pm)·

Hard Fat, BP (Wit jpsol H15 - Dynarnit NoBel) 250 1 770?020

BAD ORIGINAL ..t:i it

It s i

Capsules were pre; .->r?d by dispr sing the active ingredient in '.he teeithln i.-irfor a.-his oil and *;li:ng the dispersum into so't, elastic gelatin cap··;, Hu

Formulation f (Cei-.trolled Re'etse Capsule)

The 'allowing contr'’ied release capsule 'ormulatior was prepared py βχΰ.--υ:ί.igingredierts a, b and c using an extruder, 'ollowed by spherunisation o' theextrudate and drying. The dried pellets were then coated with release-..y Telling membrane (d) and 'illed into a two-piece, hard gelatin capsule. rnq/eapsule •a) Active Ir rrdient (b) Microcrystalline Cellulose V' ) I octose BP Ftn/ΐ Cellulose xarrnh· \ Ιη.ττ; !p Formulation 250 125 12; 13 513 : 3ti

Active ingredient

Hydrochloric acid solution, C.1MSodium hydroxide solution, 0.1MSterile water q.s. to pHq.s. to pHq.s. to 0.200g4.0 to 7.04.0 to 7.010ml

The active ingredient was dissolved in most o* the water (35*-4G*C) and the pHadjusted to between '».0 and 7.0 with the hydrochloric acid or the sodium hydroxide asap; ropriate. The batch was then made up to volume with the water and filteredtbr jugh a sterile micrupore 'liter into a sterile 10ml amber glass vial (type 1) andseab-d with steric .res and o-erseals.

BAD ORIGINAL

): .lot:· ·

Act!-e u gr-d.cnt 0.125 g yterile. ;a- -gen-'rr·»·. ;>H 7 phosp* ate bu"er, ().3. t) " 25 ml

Fxample 4: Intrarm.scular injection

Active Ingredient q ^o g

Benzyl Λ’cohol 0.10 g

Glyco'urol 75 1.45 g

Water 'or Injection q.s. to 3.00 ml

The active ingredient was dissolved in the glyco'urol. The benzyl alcohol was thenadded and dissolved, and water added to 3 ml. The mixture was then Altered through asterile micropore 'liter and sealed in sterile 3 ml amber glass vials (type 1).

Pxample 5: Ingredients

O

JQ 5>

Active ingredient

Sorbitol SolutionGlycerol

Sodium BenzoateFlavour, Peach 17.42.3169Puri'ied Water q.s. to ·ϋ&amp;- , 0.2500 g1.5000 g2.0000 g0.0050 g0.0125 ml5.0000 mt

The active ingredient was dissolved in a mixture o' the glycerol and most o' thepuri'ied v/ater. An aqueous solution c' the sodium benzoate was then added to thesolution, 'allowed by addition o' the sorbitol solution and 'inally the 'favour. Thevolume was made up with puri'ied water and mixed well.

Fxampte 6: Suppository mq/sup; - -tory

Active Ingredient lidym)*

Hard Fat, BP (V/Persol H15 - Dynamit Nof’el) 2ri?n 250 1770 BAD ORIGINAL ft 4 if. . I ,Ί·: , il: ,»'rΐ’ Μμπι < ' • Γ w h > · 1’ ι; ι ,'ι . t ·· ,.ηπυ ne-Vth ·.' the 'Λ'ite;.s ' Η15 ννίϊΐ melt,*.] id a ste.un-ja. -etc·! pan at .;'>*C ιη«».:·πι. T fie .ictive ingr-u’ 'd w.’· r.i'ti*·· through a dOOum ::ive .an! added to c molten b i,,with rni'ing, using u silver ·.·>:, 'it t ed w ith a rutting bead, until a s.noofp dispersion wasachieved. Maintains g the mixture at 45 *C, the remaining '.Vitepsol HIS was added tothe suspension and stired to ensure a homogenous mix. The entire suspension waspassed through a 2Sdum stainless steel screen and, with continuous stirring, wasallowed to cool to 4U*C. At a temperature of 38*C to 40*C 2.02g o* the mixture was'illed into suitable plastic moulds. The suppositories were allowed to cool to roomtemperature.

Fxamole 7: Pessaries mq/pessary

Active ingredient 6)yrn 2Su

Anhydrate Dextrose 5θθ

Potato Starch «* 563 .Magnesium Stearate 7 1000

The above ingredients were mixed directly and pessaries prepar·, a bv dire ’compression o' the resulting mixture.

Fxnmple 3: 3'-Αζίΰ 3'-dpoxy-5'-(.i-octanoyl thy midjf.e io a solution o‘ 3'-azido-~ -deo*y thymidine in pyridine (0*C), octanoyl chloride G.2equivalents) was ached, ’he reaction was allowed to warm to room temperature,.'/hen tic (DHCl^:MeOH;2U: 1, on silica get) indicated complete reaction, the solutionwas poured onto ice water. The aqueous phase was decanted. The resulting oil waschromatographed on silica gel eluted with CHCl^iMeOH. The title compound wasobtained as an oil by evaporation o' the solvent 'rom the appropriate 'ractions.CHNnal. C-54.95 H-6.92 N-17.80 'nd. C-54.82 H-6.96 N-17.6&amp; 67.46(d,lH,J5 6=1Hz,6H), 06.13(t,lH,l‘H), 64.5-4.2(m,3H,3'H and 5’CH2)64.0-3.B(rn,lH,4’H), 62.3-2.l(m,4H,2'H and (CH^l o' octanoyl)), 6 1.81(d,3H,J5 5=l-OHz, SCHj), 61.5-0.6(m, 1 3H,5' octanoyl (C'r^^CHj)

BAD ORIGINAL

APO 000 1 1 ti'.-: *· ,., .tier ; .T'tO ice water with

Γ., S' . ‘ «fj·?- Ϊ’-ilc· •./thymidine '’ZOn 'll pyf.di.·.·'; '5.. .P t“ ’ '· !.*r t’c.-f. ϊ ·. et .· i chloride C. I -usi vaients) was add? i. ίί : r, •wa h-mrs .im! kc-.-.t u to i‘C *or 20 hours. It was -,. i?· s".irr,μ). Tc.c ague >cs ph.-v-e v.as dec-anted. The oily .prnduc’ was dissolved in a^itsnd extracted with water (5 times), 0.5 N hydrochlor.c acid, water (2x), and dried everrria-:nesium sulphate. The solution was Pitered and evaporated in vacuo. The , ---.idu. 1oil was u:~-c!veJ in chlorc 'ίγιγ , applied to a silica cel column, and 'lashchromatographed using 2% me:Hanoi in chloroform. Fractions with product weree·.apor_.t“d and the oii was ch: cmaiograpned again using ethyl acetate: '*z f£:iiv). r ractior.s .-.-is''» product were evaporated in vacu^- to a white solid.

m.p. 9-.-95 * C •Ml. ·. 46.6'? H-4.89 N-22.65<nc. C-46.67 H-4.96 N-22-59 f x~mcle Id

Tne '-'Lv.nc ·-ι,.ιτ.pounds ware prepared according to the procedure o* Example δ or 9as appropriate 'rorr, the appropiiate acid halide or anhydride. 3' -Azido-9'-0-benzoyl-3’-deoxythymidine cal. C-53.68 H-4.77 N-18.41 'nd. C-53.81 H-4.7? N-18.46m.p. 54-59 · £ 5‘-Azidj-3’-deox;-5'-0-pivaloylthyinidine cal. £-51.27 1-:-6.03 Γ.-19.93 'nd. C-51.07 H-6.95 N-19.83m.p. 99-100 T. 5'-Azido-3'-deoxy-5'-0-(3-methylbutyryl)thymidine cal. £-50.24 1-.-6.13 Ν-Ϊ9.53 'nd. C-50.27 H-6.ll N-19.49 67.46(-.1,1.-0,J, -l.2Hz,6H), 66.l3(t,lH,l’H),

X I O 64.55-4.15(n,3H,3'H and 5'CH2), 6 3.8-4.1Xm,lH,4’H), 62.4-1.78.1m,3Η.2Ή and 5'methine). 61.80(d,3H,J^ 6=1.2H7,5CHj), 60.9(d,6i l,J-6.4Hz, methyls on 5’butyryl) BAD ORIGINAL fi -ϊ'>- ".Λ.·?!' ?-('.··ι .-ilinit.·. πυ Hr·'* · 1. ' -< .> 7 ·'5 7 Ν. 1 ;.jp, 'n<i. ! . -t, [.h'f ) ·-(?.' · ϊ J-1 <. 7 6 7.4 5(,), ι Μ, J- ϊ · k j f~ i ζ. ti H J, ό η. I 2 t, 1 ( (, 1 Ή), 6.».5-4.'!5<m,3H, "i · an·) 64.U- 3.8(m. 1Η.4Ή)

L 62.3Ϊ--· 11'm,4H,2Ή aid (CH9)' .j‘ palmitoyl), 6 1.8 d, 3H, j. - l.ul iz,5Cl ,) 6 I. jCHj) 3'-Azido-5'-deoxy-5'-U-tolLylthymidine Cal. C-56.10; H-4.97; N-10.17

Fnd. C-55.88; H-5.00; N-18.09m.p. 73‘C NMR taken in CMOO-d^ NMR: 67.95-7.25 (m,5H; bH,cH,6H), 66.16 (ΐ,ΙΗ,ΙΉ), 64.6-4.4 (ηο,ίΗ,ί'Η^Ή), 64.2-4.0 (m,lH,4'H), 62.39 (s,3H,dCH3), 61.63 (s,3H,5CH.j) 3'-Azido-3'-deoxytbymidine 5'-Q-(hydrcgen succinate) Cal. C-44.98; H-4.83; N-17.96Fnd. C-44.90; H-4.77; N-17.85 NMR taken in DM5O-d o NMR: 67.46 (s,lH,6H), 66.13 (m.lH.l'H), 64.48-4.40 (γπ,1Η,3Ή), 64.34-4.20 (m,2H,5'H), 63.99-3.94 (γγ,ΙΗ,ΑΉ), 61.73 (s,3H,5CH.j) 3,-Azido-3i-deoxy-?'-incsy:thyrridine Cal. C-38.25; H-4.37; N-20.23; 5-9.23

Fnd. C-38.I5; H-4.38; ' m.p. 253 “C (dec.) AP 0 0 0 0 1 1 NMR: 67.49 (d,lH,J , -- i.O Hz, 6H), 66.15 (t,lH,J,, 7, = ,..6 Hz, 1Ή). 64.54-4.41 (m,3H:3’H,5'H), 64.14-4.02 (m,lH,4'H), 63.24 (s,3H,5'-mesyl CH,i, 61.79 (d, 3H,J, ί = ’.0 Hz, 5CH,) 3’-Azido-5'-0-(3-cb!<jrobenzoyl)-3'-deoxythymidine Cal. C-50.31; H-3.97; N-17.26; C1-0M4Fnd. C-50.16; H-4.03; N-17.13; Cl-8.66 NMR taken in C'MSO-d, 6 NMR: 611.37 (s,lH,3-NH), 67.98-7.43 (m,5H;5’-phenyl,6H), 66.17 (dd.lH;

Jp 2g, = 6.1 Hz, Jp 2 = 7.2 Hz, 1Ή), 64.68-4.48 (m,3H;3'H,5’H), 64.14-4.il (ιη,1Η,4'Η), 62.48-2.41 (m,2H,2'H), 61.64 (d,3H,J, r χ 1.2 Hz, 5CH-)J,o i » -Cl./; M.'/?6th Fp· .- irv 1 °36

BAD ORIGINAL

J - I ZI <: - )’ - ..) V y t f> » it ·(<}';:.. · ’ mu. !U:, U.u mMH, 111,-..1 : b. fa· a '.uitinu 0' ninth.irwsukdnnyl cmor.de (2.7 ml ) to n solution 0' the S' or t cv i : i ·' ill 1 )r . j . 1 j t (i <- (1 * 1 .. I'he r· . ‘ι··η .·/;·, Olowd to proceed at 5 " 'GT Of,.; ’Π . . !' ,-pfj onto iue w iter. T he precipitate was collected by 'iltration. The product l’5'---,z:da-7 me-;,1 thymidine) was reacted witn potassium carbonate (0.7b g, 5.6 mMot) in DMF (75ml.). The reactants were heated in an 80°C oil bath ‘or six hours, then poured into icewater. The product was extracted 'rom the water with ethyl acetate. The solventwas removed in vacuo and the resultant oil was 'lash chromatographed on silica gel byelution with CHCl^MeOH (9:1 v/v). The title compound was obtained as a solid a'terevaporation o' the. solvent 'rom the approprate 'ractions.

m.c. = 184 - 186°C

Ci <c

Vb F xample 12: 31-Az i do-3’-deoxy thy mi dine a) 2,3'-AnhydrG thymidine

Thymidine (85.4 g: 0.353 mol) was dissolved in 500 ml dry DMF and added to N-(2-chloro-l,i,2-trifluoroethyl)diethylamine (100.3 gj 0.529 moi) (preparedaccording to the method o' D.F. Ayer, 3. Med. Chem. 6, 608 (196^))^ Thissoiutmn wu heated at 70*0 'or 30 minutes then poured into 950 ml ethanol(FtOH) with vigorous stirring. The product precipitated 'rom this solution andwas 'iltered. The FtOH supernatant was refrigerated then 'iltered tc yield metitle compound, mp. = 226 -270*0. b) .’-Azido-?'-dPO mthyrrrdlnn 2,3'-0-Anhydrof bymidine (25 g: 0.1115 mol) and Γ' iN^ (2? g, 0.446 n.-a!) w;··suspended in 0 mixture o‘ 250 mi DMF and 38 ml water. The reactionmixture was refluxed 'or 5 hours at which time it was poured into 1 iit»-r 0'water. The aqueous solution was extracted with FtOAc (3 x 700 rnl). TheFtOAc extracts were dried over Na.^SO^, 'iltered and the FtOAc wasremoved in vacuo to yield a viscous oil. This oil was stirred with 200 n!water providing the title compound as a solid which was collected by'iltration. mp = 116-118*0 bad original . ... 5

f.S r ' ’ :ί ·: · · ' '-·'!/. ·. t1 .)·, - .μ -.ι”) in ;·· : ι; / :. ; - J .·, it.· . . i · «· ... t ά .>3 adju t«'J t j ,2 .·. ,tl lΓ » ‘....j; J. Λ,.,.roxim.itel. h.al' ’ · ' w is Tie'!. iH»j tit!·.· compound was obtain»’.' ns a ; .j; raised fowd·:.'.

Anal;, sis calculated 'or C^HpN^NaO^ 6/10 N7Ocat: C-40.03; H-4.43; Ν-23.Ϊ-; Na-7.66x.-,d: C-39.83; H-4.34; N-23.23; Na-7.90

Example 14: Preparation o' ^'-Monophosphate o* 3l-Azido-3’-deoxythymidine 3'-Azido-3’-deoxythymidine (0.5 g, 1.87 mmol) was dissolved in 5 ml. o' triethylphosphate and the mixture was cooled to -5*C. Phosphorus oxychloride (0.685ml., 7 mmol) was added in cne portion to the rapidly stirred solution which wasthen maintained at -10 *C for 22 hours. An aliquot was removed and added toconcentrated ammonium hsdroxide. Analysis of this sample on TLC (cellulose,n-PrOKE-^O, 7:3 v/v) showed no remaining starting material and a singlefluorescent spot with lower mobility than the nucleoside. The reaction mixturewas poured onto 20ml_ o* ice and water. This was placed in an ice bath and thepH o' tee solution was adjusted to a value of 7.5 by the addition of 7N NaOH.The basic mixture was extracted once with chloroform and once withe : sr. Theaqueous layer was again adjusted to give a pH of 7,5 and concentrated in vacuoto remove residual organic solvent. The material was stored at -10*C untilpuri'ied as 'ollows: ΑΡ0 0 0 0 1 1

Deactivated charcoal was prepared by washing coconut charcoal (50-200 mesh, 100 g) with 500 ml. o' 1 N HC1, 3 I. o' water, 35 ml. o' 3% toluene in 95%ethanol, 600 ml. o' 95% ethanol and 'inally extensively with water. Deactivatedcharcoal (12 mL of settled wet charcoal) was added with stirring to themonophosphate solution (0.72 g, 1.8 mmol, 30 ml.). The supernatant wasdecanted and the charcoal was washed with 150 ml. o' water. The nucleotidewas eluted 'rem the charcoal by washing with 120 ml. o* 1.5 M ammoniumhydroxide in 50% ethanol. This solution was Altered through a 0.22 micron•'liter, concentrated in vacuo to 10 ml., 'iltered through a Amicon Centri'lo CF-25 membrane, and lyophilized to yield the diammonium azido-3'- <1»Oxythymidine-5'-monophosphate as a solid. This compound was characteris»'das a nucleoside 5'-monophosphate by the ability o' 5'-nucleotidase to degrade itto the nucleoside.

•Eh 1/ V2uth February 1956 BAD ORIGINAL

•W -- tc . t η vr’i .· ::r H ·

• it

VbC i us ‘.tri-^·: · l.i-’p; " mphosuhate A c.jlu. - - o' DUW 5(J dDnwex’ ion exchange r--;in - DOW Cncmim-ilI aboratorl«’u; fy. idiniurn resin was prepared by pouring r.3 m! o' resin intoa 23 cm diameter column and washed with water until no more coloureluted. Pyrophosphate decahydrate (1.12 g, 2.51 mM) was dissolved in 30ml. o' water and applied to the column. The column was eluted withwr-tor. A 125 ml. 'raction o' the eluant which contained UV adsorbingmaterial was collected. The volume was reduced to 10 ml. in vacuo andtri-n-butylamire (1.2 ml.) was added. The volume was reduced in vacuaand the residue was dried by coevaporation with pyridine 'our cimms. Thepraduct was stored in a 'reezer (-5*0. (b) Hydrogen Form o* 3l-Azido-5'-monophosphate-3l-deoxythyrridine

The hydrogen form o' the monophosphate was prepared by passing theammonium salt obtained in Example 14 (0.1 g, 0.283 mMol) dissolved in 6mL of water, through a 1.5 ml. (lOeq.) column of DOW 50 H+. (c) Phosphoromorpholidate Derivative o' 3’-Azido~3l-deoxythymidine

In 9 mL o* water was dissolved 0.283 mMol o' the hydrogen fqiyp o* themonophosphate obtained in stage b). Morpholine (99 yl., 1.13 mMol, 4 eq.)was added and the solution heated to re'lux. Dicyclohexyl carbodiimide(9.234 g, 1.13 mMol, 4 eg.) dissolved in ^-butanol (5 ml.) was added over athree hour period The reaction was re'luxed overnight. The reaction wascooled to ro .io. temperate.e, 'iltered, and the solvents removed in vacuo.Ethanol was ,-.dded and r.aparatod in vacuo *our times. The r^si^ue wasdissolved in methanol and the phosphoromorpholidate precipitated by theaddition o' etne-r. The precipitate was triturated with ether 'our time' <,r.cdried on a rotary evaporator. The title compound was obtained. (u) 3’- A.-ldo-3*-deo xy thymidine-^'-triphosphate

The phosphoromorpholidate derivative obtained in stage c), was dricci by aremoval o' pyridine in vac o 'our times. The bis (n-Qu)jt J pyi uphoonhalL 1 j.M}/26th February 1986

BAD ORIGINAL ϊ< Η ’ ' · ., 'i in si i < .·.·.·)3 J s,j . .-i-no'/a! <>* pyfi D ·· in var ,·ι. - .'•sphorun,':: ph.nit'jte was dissolved in ;·, ridinn, ’’ ml., »nd addc 1 M* t.»· contaJ,:" ] the p/rophusph ,te reagent.. The rean'ion wr·-. nl: »·.% : to continue overnight at room tern, ..rati.re. The .//ridine was removed m. acu·?. Water was added to the residue and removed in varco three times.Tre residue w..·· 'razen.

The residue was thawed and dissolved in 50 ml. o' water. The solution wasapplied to a column (1 x 10 cm) of DFAF Saphadex A-25 which had beenequilibrated with 50 mM ammonium bicarbonate. The phosphates wereeluted with a 300 mL linear gradient o' 50-000 mM ammonium bicarbonate.The factions containing the diphosphate nucleotide were pooled as werethose containing the triphosphate nucleotide. The pooled diphosphate andtriphosphate factions were each dried in vacuo, redissolved in water, drieda;ain, redissolved in water and lyop..ilized.

Fxample Ιά: Enzymatic Sy ithesis o* 3,-Azido-5l-triphosphate-3l-deoxythymidire

The 5'-triphosphate was synthesized from the 5'-diphosphate using pyruvatekinase and nucleoside diphosphate kinase. The reaction mixture contained: 6mM3'-azido TOP, 12 mM adenosine triphosphate, 40 mM MgC^. f'O piM potassiumpiperazine-N,N’-bis(2 -etivanesulphonic acid) PIPES bu"er (r l 6.0: 30 rnM phosphcenolpyruvate, 40 lu/ml nucleoside oiphosphate kinase and ICO U/mlpyr.vate kinase in a 'ir.al volume c* 5 ml.· The reaction mixture -.vac incubaU dat 37 *C 'or 5 days. The reaction mixture was applied to a cclumn - · ;0 cm)o* DFAF Sephaaex A-25 which had been equilibrated w’’-' bicarbonate. The nucleotides were eluted with a gradient a' luO -ICJO mMan.manii.n. bicarbonate. Fractions containii.j the triphosphate were .moled, .n.:evaporated to dryness in vacuo. The compound was 'urther puri'ied using 3...-operative HP1.C column (Whatman, Inc., Magnum 9 SAX) eluted with .,gradient 0* 10 - 100 mM potassium phosphate, pH 3.5. The resulting compoundwas 'urther puri'ied using a DFAF Sephadex A-25 column as above. The'ractions containing the tetraammonium 3'-az: jo-3'-deoxythyrnidine-5'-triphosphate were pooled, dried in vacuo, redissolved in water and lyophilized to-mid the title compound. APO 00 0 1 1

Muth

BAD ORIGINAL A 5Ϊ...

Vb υ ’.'-Azidj-’’-dc'.'V; -h/mldine --.15 -. 1,· i·u„·tored to 'emale P-Ai Q/c mice in'etieawith F'u o' the RVb' strn.n o' P.au.w'nct Murine ! -m,-r. virus.

Treatment was started 4 hours a*ter in'eetion at dusa., ‘ :-0 .m./kqintrnp^rit0 -eaily every 8 hours or 0.9 or 1.0 n.g/ml nmlly in <t-i.-.L:ng water,i uch feat:, e-ot was 'ound to prevent in'eetion 0’ spleen cells and iubse ,-.er«t-sei.clap oen: a* splenomegaly end also suppressed viraemia. • 1:'' ~~!Ρν':ί TM-11 cells (T-reil clor 2 susceptible to HTLV-i in'eetion) were co-cultivatedwitn irradiated, HTLV-I producer MJ-tumour cells as 'ollows: a) b) c) d)

0 I

On day 13, ·; 1 e*ate aano u-i.ing radio-labelled lamucha Ml-2. a standard probe recognising the Dam. Hl ‘rajm.i ,;rHTLV-I.

No hybridisation was obser.ed 'or a), indicating a lack 0' virus in the un'n'ecfccontrol. Λ strong signal was seen 'or b), the untreated, infected control. Λ wea-signal was observed with c), indicating incomplete eradication o* the virus, and nohybridisation -was noted in il) or e) indicating complete extermination 0' the virus.

Fach culture was also probed with a probe 'or T-cell receptor β chain, with a strong- signal being generated 'or all cultures, showing the continued presence o' JM-ll 'orthe ration 0' tin: experiment. TM-11 cells only; TM-11 cells and MJ-tumour cells TV-].' cells , MJ-tumour cells and J’-azido-J'-deoxythymidine fTiiM); TM-11 cells, MJ-tumour cells and J’-azido-J’-deorythymidine (9yM); M-ll cells, MJ-turrour cells and 3’-azido-3’-deoxythymidine >,27yM). total DNA was extracted 're η each culture and digested with Barn I L tr.fi ο ,cent 0' the HTLV-1 genome, independent 0* any host 'lacking -equon:-·3 standard molecular weight o* 3.3 kD. The digest was then or-.0.--.-1 w.t

BAD ORIGINAL A Λ I i. R- V-A7/J.--5'-·rte-3'-ceoγ»‘hrmicrne was tosted m '· >rr? against A'DVs '<· tcf,·· -'.AIL?'/ RT). AIDV KT was : uri'ied 'ro.n pelleted and extracted AIOV by elution throughOf A" and pf ispt'ocel!ulose columns. The enzyme activity was linear through 60minutes and sialic 'or at least 2 months when stored in 60% glycerol and 1 mgco vine sercn albumin per ml. Using rA-odT (12-18) 83 template-primer, AIOV RT had a pH optimum of 7.0 to 7.3, a MnCl^ opt.mum c* 0.3 mNi and ah*c,Cl7 optimum q* 5 mM, The activity in the presence 5 mid WgC^ '-*3S 10-'old greater than the activity in the presence o' 0.3 mM MnCl.^. Maximalenzyme activity was also 'cund in 80 to 140 mM KO and 6C to 100 m.M N-;C1.Incorporation c* pH) dTTP vas linear with respect to enzyme concentration.

When testc-j. 3’ azido-5'- .riphosphate-3'-deoxythymid>na was 'ound te be gcompetitive inh.h.tor of AIDV RT, giving a Ki o' 0.04 μΜ when using rA-nm , 18} as ti e tempia? e-primer. The enzyme had a Km *cr nTTP o* 2.3)n A.suggesting that 3,-azido-‘>’-triphosphate-3'-deoxythymidin binds tighter to *heenzyme than does dTTP. further experiments wit the RT’s o' avionmyeloblastosis virus, Moloney murine leukemia virus and AIO7, showed 3' c. ldo-y-triphosphat^.-o'-deaxyihymiJir.e to be a terminator o' OiAA c-'-ain elongat? v (ii,- In Vitro Anti-APDV Activity 3'-Azido-3'-d._· xyth^n.idiuc was tested and 'ound to possess activity in a ..-nbet‘ in vitro assay systems. Drug et,e<.f< were measured by assaying reversetranscriptase RT) activity in the supernates 'rom injected, unin'ected, and drugtreated ceils. 3‘-Azido-3,-d.;ox7thymidine effectively blocked the infection byή.AV a* the H9 and U937 human lymphoblastoid celt lines at concentrations 'rom 2.7 to 1).0013 m.; ;,'ml. Similarly, in'ection of normal FHA stimulated whiteMood cells and cultured peripheral blood lymphocytes was inhibited at drugconcent: «limns os low as 0.013 mcg/ml. Drug addition and subtractionexperinrents in H9 cells revealed that 3'-azido-3'-deoxythymidine was mosteffective v.' a present at the time o' virus in'ection o' susceptible cells, cutstill retained most o' its aniivual activity even when added as late as 20 hoursa'ter initial AIOV in'ection. Inhibition o' viral replication was also evident whenf “ drug /->'. - r-sent in th·· a .-01,1 only during ; he 2iJ h<u.r period e' virus BAD ORIGINAL 05 AP o 0 0 0 1 1 -/(,-

. I

oqA -.•«sorption.Sytily. .· ! . 'ons. ώι.ιo* virions *ro; it Ί.Π and . .tJI J t-ij/rnt. !·» «"‘.uited ;\o direct anti-Hl activity a jains* pi. ‘'itiriy, t'«e had little or no e“e t on the production an,i the chronically ir/ected 1 19 AIDV cell line. .1,) Preventing Insertion by AID'.’

.MOV lease

The ability o* 3'-azido-3'-deoxythymidine to block infection o' cells by AIDV wasdetermined as 'ollows.

Cloned T4 positive tetanus speci'ic T helper lymphocytes were injected with apool o* AIDV isolates [at challenge doses o* up to 5000 virions/csll] and cellsurvival a'ter infection was monitored. A'ter 10 days in culture no viralcytopathic ejects were seen in injected T cells treated with 8.8 and 1.3 mcg/ml3'-azido-3'-deoxythymidine, while untreated, injected cells were 5-*olddecreased. Cell survival was also evaluated in an HTLV-I trans*ormed, AIDVsuperinfected cell line derived from the cells above. 3'-azido-3'-deoxythymidineat concentrations of 2.7, 0.27 and 0.13 mcg/ml totally blocked cytopathic effectsat 7 days. Protective ejects were seen in infections induced by both cell freevirions and cell associated virus. J'-Azido-J'-deoxythymidina^lt 0.27 mcg/mlconcentraion also e*fectively prevented cytopathic effect induction by a lessrelated Haitian isolate of AIDV.

Fxample 18: Toxicity Assay 3'-Azido-3'-deoxythymidine was administered to both mice and rats,value was in excess o' 750 mg/kg in both species.

The I.D 50

BAD ORIGINAL

Claims (11)

1. :: ,: .. ,'h.< c. . ‘ ' r. rh.tru c* ·.: rte-i in treat the lcMv·-· i* .-re h :: * '-^id;- 3'/-. -./rid ini, or a pnarmaeouticaily .ic . ba. Leden.vi.ve thereof, is capable cf use in the rreatment v:prcthy? -xis of --.-.in retrovirus infections . Λ f.'r.r.uiaticn according to Claim 1 woF-rem the carrier isother than water.
2. 3. A formulation according to Claim 1 or Claim 2 which is
3. 4. A foruiruLation according to Claim 3 contained in a sealedvial.
4. 5. A rcr~ulation aocordirc tc either cf Claims 2 or 3 whereinthe carrier is sterile water.
5. 6 . A formulation according to Claims 1, 2 or 3 providing sustainedrelease of the ret ve ingredient after oral administration. A formulation according to Claim 6 further carprising afla veuring agent.
6. 8. A formulation accoroing to any one of Claims ' to ' cunprisir-ja unit dose of the active ingredient, or a mu’.tick thereof.
7. 9. A formulario:: accordm. -o Claim 8 wherein tu ? nit ---ose istit/i i and laCOng of tne active ingredient. iv. A formula*·.ion according to Cliim. 8 wherein die utit d. >a. a isbet; »>a-i * ' and IhOPmg of the active ingredient. ' 1. A t·* wd -••-ion .accordinc to Claim 8 wherein the un: t dose isbetw-.· t . ? ird T'd'-g of ·he active ingredient.
8. 12. A fcrtxlation according to any ore of Claims 1 to π whereinthe active ingredient is 3 '-a2ido-3 1 mloo;c/doymddir.e. '2. A process for the preparation of a formulation according toany of Cluuis 1 to 12 which corpriscs bringing 3'-azido-3'-deextnhymidine cr a pharmaceutically acceptable derivativethereof into association with the said pharmaceuticallyacceptable carrier. AP 0 0 0 0 1 1 BAD ORIGINAL 1 28
9. 14. Γη unnc·-. .ically .-i.wpiable iti'.nhvcs of j'-azido- 21 -u·cxythymidine which, upon administration to a human^ubkot, are capable of providing (directly or indirectly) 3 '-azrdo-3'-deox'/thimidir.e, or an anti-rotrovirally activemetabolite or residue thereof, other titan the following5'-derivatives, namely the monophosphate, disodiummonophosphate 2-cyanoethyl monophosphate, triphosphate,p-toluene sulphonate, acetate, metha: asulphonate andtriphenylmethyl derivatives and where the 5'-C of2'-azido-3'-deoxythymidine is linked to a further nucleotideor nucleoside derivative.
10. 15. Pharmaceutically acceptable derivatives as claimed inClaim 14 in the form of pharmaceutically acceptable salts,esters or salts of such esters of 3'-azido-3'-deoxythymidine.
11. 16. A derivative as claimed in Claim 14 selected from the groupconsisting of the 5'-diphosphate, 5'-(3-methyl-butyrate) , 5'-octanoate, 5'-palmitate, 5'-(3'-chlorobcnzoate^. 5' -benze ate, 5' -hydrogen succinate, and 5' -pivalate estersof 3'-azido-3'-deoxythymidine. BAD ORIGINAL Λ ", :r th · '>:·; ' Γ υ..7' Ο* < claims Η t.j ’.C ccmpriti'·; either jf t!' C.'CT; June·, ' ο

    GO (wherein M represents a precursor group for the 3'-azldo group) or a derivative(e.g. an ester or salt) thereof, with an agent or under conditions serving toconvert the sai<' precursor group into the desired azido group; or (0) reacting a compound of formula:

    (Ill) ΑΡ0 00 0 1 1 ('••/herein R repr«s-'its a precursor croup for ti-e n.drcxy gr.-n-p.pharmaceutical!» ac.···.:.-'hie derivative group thereo') with :o ; je-er· ditions serv.nj to o··'.'.-;rt the said precursor group into th·- ·.in-sired group: or (C) reacting a compound of formula CH. nder

    (IV) BAD ORIGINAL fu-,<'tio ·\·ι1 cjnivalent thereof, with a compound serving to i--stroduce thej<3 r.jjofuiHi :s/I ring at ’he l-josiLion cf the compound jf formula (IV?; or

    £> (wherein R^ is hydroxy or R as defined above), with an agent under conditionsserving to convert the said compound into 3’-azido-3'-deoxythymidine or apharmaceutically acceptable derivative thereof, and (i) when V-azido-3’-deoxythymidine is formed, converting the said compoundinto a pharmaceutically acceptable deriative thereofiW (ii) when a pharmaceutically acceptable derivative of 3’-ezido-3'-deoxythymidine is formed, optionally converting the said derivativesimultaneously or thereafter into 3'-azido-3'-deoxythymdine or a differentderivative thereof. BAD ORIGINAL ft ’’· V-Azldj-J’-cie? - f-.yir'.· !.· m ,i f * .;· . · ί ', t. 1 > .,c·; nta>:? :· f · •..hereof, ' ' use in t.L·: \ :,·.:?.·.*,· :f 1 jmr: : >··.«: .nfectio· 3. 1'’. ’*· Azido-J'-deoxythymidine f sr ose in rhe trc.'itr··: i ~j·. prophy'.; ,3 .,f human retr :v.fis infs ’ tic-im. 2.·. }‘-Azido-3‘-JcjX; thymic'iui for use in Lie treatment or rem· >. i-. xi.-» '( .-. DS. 22. i’-Azido-i’-deoxythymidir.i, for use in the treatment or pr->; v Ian's e.nA iDV in faction. 22,· 3*-Azido-3‘ deo\ythym: i'.re for use .r the treatment or picobyhx.s o* tn HTLV-I or HTLV-Π infection. AP 0 0 0 0 1 1 BAD ORIGINAL

    » · ' “ " ‘ - t CL·· O 2-1 25 phtr r j·::.-iral .y acC.1 -;- -orivat b.-e the. <f, -.zd · in. th·.. .'.os 1 t< i?, f: r \ ··. , s desci.’tel in toy of the Cl?.~s 1S to ^2. A f nxt'ulat; :n as dc'cnhod in any of the Claims 1 to1 ' ·.·’ - rein the active ingredient is a ccrpc-^td as deter Lbedin any ot the Clairs 14 to 15, A formulation as described in any of Claims 1 to 12 wherein the attrve ingredient is a compound prepared by the processof Claim 17. A fcrr-’.d.ation, or said fcmJr.tien for use according to anyof Ci?.inis 23 to 25, wrien said formulation is prepared to thecroce;s of Claim 13. Any novel ccrrcund prepared according to any of 'Examples4 to 11.

    Γ -17CL : j * h γ;λ Λ 0- ‘t.rch ' / /986 / •T .ΥΊΙ27Γ '’P I’rlh BAD ORIGINAL