PH26859A - 3-azido-2'3'-dideoxy pyrimidine nucleosides - Google Patents

Description

Ca ' oo AN { - 7 [ i; w-1- DX/85 5 J ? | RE 26859 : d ’ . “a » A Co

Therapeutic Compounds 5) Jbl la a Aru yy Soli erdenn vd ore ert jor / . . — Ce Co

Senipd Ip. BE poled ad Send - N, IT8E. 1

The present invention relates to 2 3 -dideoxy-3 -azido-nucleosides and their use for the treatment or prophylaxis of certain viral and bacterial infections.

Acquired immunodeficiency syndrome (AIDS) is an immunosuppressive or : immunodestructive disease that predisposes subjects to ‘atal opportunistic infections. Characteristically, AIDS is associated with a progressive depletion of

T-cells, especially the helper-inducer subset bearing the oKT4 surface marker.

Human T-cell lymphotropic retrovirus HTLV-II has been reproducibly isolated from patients with AIDS or with signs and symptoms that frequently precede AIDS. .

HTLV-II, unlike HTLV-I or HTLV-II, is cytopathic and appears to preferentially infect and destroy OKT*-bearing T-cells. It is now believed that HTLV-II is the etiologic agent of AIDS. :

We have now discovered that a broad class o* 2',3'-dideoxy-furanosyl nucleosides 1 characterised by the presence of an azido group in the 3 -position, and particularly those referred to below are useful for the treatment or prophylaxis of AIDS as well as other viral and bacterial infections.

Examples of such 2',3'-dideoxy-3'-azido-nucleosides include those having the following formula:- 3

R

2 4

RG > R \ \

R! N om

Cy

Ns

HDL. /LLMJ/17th September 1985

: -2- D be wherein Rl is hydroxy, mercapto, amino, alkylthio, aralkoxy, alkoxy, cyanog o 1 alkylamino; 3

R2 is hydrogen, acyl, alkyl or sulphonate;

R> is hydroxy, mercapto, amino, alkylamino, arylalkoxy, alkoxy or alkylthio; \ £

Rs alkyl, halo, perhalomethy], hydroxy, alkylthio, cyano, nitro, alkenyl, halo- L substituted alkenyl, alkynyl or hydrogen; and '

R° is hydroxy or mercapto; and pharmaceutically acceptable derivatives thereo* ‘ including esters and salts and other compounds that are bioprecursors of the compounds of formula (I) or are capable of hydrolysis thereto, as well as the derivatives of the compounds of formula (I) in which the 5-C and 2-C atoms are linked to form an anhydro group. \

In the above general formula (I) the dotted lines in the 2- to 6- positions are intended to indicate single or double bonds inthe positions in the pyrimidine ring, the relative positions of the single and double bonds being determined by whether the substituents rl and RZ are groups capable of e.g. keto-enol tautomerism.

Also within the scope of the Bresent invention are the compounds of “armula (II)

R

N

"ON (1D PY

R N N

CY

Ns wherein R’ is as defined above; and RO and R7 may be the same or different and are selected from amino, hydrogen, hydroxy, mercapto, alkylthio, alkoxy, aralkoxy,

Cyano or alkylamino; and pharmaceutically acceptable derivatives ag referred to above.

Preferred esters of the compounds of ‘ormula (I), particularly when X represents an

Oxygen atom, include acyl esters such as straight or branched chain alkyl (e.g. Ci. A

BAD ORIGINAL &

HDL /LLMJ/17th September 1985

! Oy, ’ iil -3- DX/85 ; 3 EL 8 alkyl), aralkyl (e.g. phenylacetyl), aryl (e.g. benzoyl optionally substituted by

J halogen, C;_, alkyl and/or Cy _, alkoxy), organosulphonyl (e.g. methanesulphonyl) of and mono-, di- or tri-phosphate esters as well as the corresponding thio esters including dithiocarbamoy! e.g. dialkyl (e.g. methyl) carbamoyl esters.

Examples of pharmaceutically acceptable salts of the compounds of formula (Im : include base salts, e.g. derived from an appropriate base, such as alkali metal (e.g. - sodium) or alkaline earth metal salts.

Compounds of the present invention have been found to be active against the following diseases: virus infections caused by animal and human retroviruses including T-cell lymphotropic retroviruses (HTLV), especialy HTLV-II and other

AlIDS-associated viruses including lymphadenopathy-associated virus (LAV), feline leukaemia virus, equine infectious anaemia virus and other lentiviruses, and other human viruses such as non-A, non-B hepatitis virus, hepatitis B virus, Epstein-Barr virus (EBV) and cytomegalovirus (CMV); and certain bacterial infections caused by . such clinically significant gram-negative bacteria as Escherichia coli, Salmonella dublin, Salmonella typhosa, Salmonella typhimurium, Shigella flexneri, Citrobacter freundii, Klebsiella pneumoniae, Vibrio cholerae, Vibrio anquillarum, Enterobacter aerogenes, Pasteurella multocida, Haemophilus influenzae, Yersinia enterocolitica,

Pasteurella haemolytica, Proteus mirabilis and Proteus vulgaris, the causative organisms of such ailments: as travellers diarrhoea, urinary tract infections, shigellosis, typhoid fever and cholera in humans, as well as animal diseases such as calf neonatal enteritis, pig post-weaning enteritis and chicken colisepticaemia. '

The compounds according to the invention are hereinafter referred to broadly as 3 - azido-2,3 -dideoxy nucleosides, which term is used herein to include purine or pyrimidine nucleoside analogues and derivatives (including salts and esters) which are essentially structurally based on a purine or pyrimidine ring linked at the 9- position or 1-position respectively to a furanosyl ring.

Thus, in a ‘irst aspect of the present invention is provided pharmaceutically acceptable 3'-azido-2 ,3 -dideoxy nucleosides, for use in therapy.

In a second aspect is provided pharmaceutically acceptable 3'-azido-2,3 dideoxy nucleosides for use in the treatment or prophylaxis of diseases as described above.

In a further aspect of the present invention is provided the use of pharmaceutically acceptable 3'_azido-2 ,3 -dideoxy nucleosides, particularly where the said {

HDL/LMJ/17th September 1985 BAD ORIGINAL 9 | -

Lo oo | 4- A nucleoside is a compound of formula (D, in the manufacture of a medication fg The treatment of any of the above-mentioned diseases.

Preferred classes of pyrimidine nucleoside according to the invention are cytiding * derivatives, e.g. compounds of formula (I) wherein R> ig amino or alkylamino, wherein the 3' azido group is in the erythro configuration ("down azido"), thymidine derivatives (e.q. compounds of formula (I) wherein R> is as defined other than amino or alkylaming and RY is as defined not including H) with the 3' azido in either the erythro or threo ("up azido") configuration, and nucleosides unsaturated between the 5C and 6C positions. \

Preferred classes of purine nucleoside according to the invention are adenine derivatives, e.q. compounds of formula (11) wherein RS ig amino or substituted amino, and guanine derivatives, €.9. compounds of ‘ormula (I) wherein RS is as defined, other than amino or substituted amino, and R’ is amino or substituted : amino.

With regard to the Compounds of formulae (ID) and (11) above, the above-mentioned alkyl groups advantageously contain 1 to 8 carbon atoms, particularly 1 to 4 carbon atoms, e.g. methyl or ethyl groups. The above-mentioned aryl groups Including the aryl moieties of such groups as aralkoxy are preferably phenyl groups optionally substituted by one or more appropriate substituents. The above-mentioned alkenyl and alkynyl] groups advantageously contain 2 to 8, particularly 2 to 4, carbon atoms, e.g. ethenyl or ethynyl.

Further classes of preferred Compounds of formula (I) include those wherein one or more of RL RZ, R> and RY are as de‘ined below, namely

Rl is hydroxy, mercapto, C, 4 alkoxy or amino;

RZ is hydrogen, methyl or acyl;

R> is hydroxy or mercapto when a thymidine derivative (as defined above) or amino when a cytidine derivative (as defined above); and

R4 is hydrogen when a cytidine derivative or halogen or perhalomethy]l when a 1 thymidine derivative; and 5 derivatives of such compounds including straight or

BAL wisn NAL 9 —

HDL/LMJ/17th September 1985

¢ se DX/85 brdéiched chain alkyl esters optionally substituted with carboxy groups, (e.g. ¢, u £cinate), Cig thio esters, optionally substituted aryl esters, mesylate,

Jrylucuronide or mono-, di- or tri-phosphates.

Fd § Further classes of preferred compounds o* formula (II) include those wherein ’ ; R® is amino, Cia alkylamino, mercapto, hydroxy or Cia alkoxy; and/or / rR’ is amino, Cia alkylamino or hydrogen; / and 5 derivatives of such compounds including straight or branched chain alkyl esters optionally substituted with carboxy groups (e.g. succinate), C,_g thio esters, optionally substituted aryl esters, mesylate, glucuronide or mono-, di- or tri- phosphates.

The 3'-azido-2',3'-dideoxy nucleosides according ta the invention may be prepared : in conventional manner using techniques that are well known In the art, e.g. as described in Synthetic Procedures in Nucleic Acid Chemistry, 1, 321 (1968), T.A.

Krenitsky et al, J. Med. Chem, 26, 981 (1983), and Nucleic Acid Chemistry,

Improved and New Synthetic Processes, Methods and Techniques, Parts 1 and 2, Ed.

L. D. Townsend, R.S. Tipson, (J.Wiley) 1978, which disclosures are herein incorporated by reference.

The present invention further includes a process for the preparation of compounds of formulae (I) and (II) and pharmaceutically acceptable derivatives thereof which comprises (A) reacting a compound of formula: 3 7 2 4 N

R R

~~ NT N* O \ / \ RQ / 1 ! 6,” (IIIa) R o (I111b)

R o ~~

M ne (wherein RL, RZ, rR, RY, rR, R® and rR’ are as defined above and M represents a 1 precursor group for the 3 -azido group) or a derivative (e.g. an ester or salt) a0 ORIGINAI A

HDL /LMJ/17th September 1985 0 BERL a

. -6- ORE: thereof, with an agent or under conditions serving to convert the said prec sor group inte the desired azide group; | \ (B) reacting a compound of formula % 7, 3

Ra N %

R%a 7 “4 N N 2 ™W- \ E 6 NN $i.

PR : [3 N N : 1 N R O :

Ra UN : 1° % ~ |!

N- : 3 . (wherein RL, RZ, RZ, RS, RZ, R$ and R4 respectively represent the groups RL, RZ, rR’ RY, rR, R® and R’ or precursor groups therefor, providing that at least one of

RL RZ, rR RS and R3 in formula (IV a) or at least one of the groups R3, RS and

RY in formula (IV b) represents a precursor group) with an agent or under conditions serving to convert the said precursor group(s) into the corresponding desired groups; (C) reacting a compound of ‘ormula rR’

RS N

RS rR > . Rh LO )

N f \

Jo (V a) or r® N—" UN (Vb)

R N Fl.

H

. 1 52 3 _4 _6 7 . . (wherein R*, R y R% RY R” and R’ are as defined above) or a ‘functional equivalent thereof, with a compound serving to introduce the desired ribofuranosyl ring at the l-position of the compound of “ormula (IVa) or the 9-position of formula (Ivb); or (D) or the preparation of compounds of formula (II) reacting a purine of formula (V b) with a pyrimidine nucleus of formula r— a

BAD ORtamAr ben,

HDL/LLMJ/17th September 1985 i gE 7 : Py -7- DX/85 ay R Oo

B J

79 (v1)

Ff N

A wherein Py represents a 1-pyrimidinyl group); and thereafter, or simultaneously

Co 7 therewith, effecting one or more of the #ollowing optional conversions:-

E (i) when a compound of formula (I) or (II) is formed, converting the said compound into a pharmaceutically acceptable salt or ester thereof, / / (ii) when a pharmaceutically acceptable salt or ester of formula (I) or (II) is / ¢ormed, converting the said salt or ester into the parent compound of formula (0) or (ID.

In the above-described process according to the invention, it will be appreciated that the choice of the precursor compounds in processes (A) to (D) will be dictated

Jargely by the particular compound of formula (I) or (I) that it is desired to prepare, the above-mentioned agents and conditions being selected accordingly : from those that are known in the art of nucleoside synthetic chemistry. Examples of such conversion procedures are descirbed hereinafter for guidance and it will be understood that they can be modified in conventional manner depending on the desired compound of formula (I) or (I). In particular, for example, where a conversion is described 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.

Thus, for example, with regard to process (A) the group M in the compound of formula (Illa) or (IIIb) may represent, for example, a halogen (e.g. chlorine) hydroxy or anganosulphonyloxy (e.q. trifluoromethylsulphonyloxy, methanesulphonyloxy or p-toluene sulphonyloxy) radical.

For the preparation of compounds of formulae (I) and (I) in which the 3'-azido group is in the threo configuration, a compound of formula (Ila) or (IIb) (in which the group M is a hydroxy group in the erythro configuration in which the 5'-hydroxy group is advantageously protected e.g. with a trityl group) may be treated for example with triphenylphosphine, carbon tetrabromide and lithium azide.

Alternatively M may represent an organosulphonyloxy leaving-group in the threo

BAD ORIGINAL 9d

HDL/LMJ/17th September 1985

-8- D configuration which may be converted into an azido group in the threo configuration by treatment, for example, with lithium or sodium azide, \. hydroxy group being similarly protected as described above, Removal of the §'— . trityl protecting group may be subsequently effected, e.q. by treatment under mild acidic conditions or zine bromide. i

For the preparation of Compounds of formulae (I) and (I) in which the 3'-azido \ group is in the erythro configuration, a compound of formula (Illa) or (IIb) in which the group M is a halogen (e.q. chloro) group in the threo configuration (in which the : >'-hydroxy is advantageously protected, e.g. with a trityl group) may be treated for example with lithium or sodium azide. The 3'-threo-halogen (e.g. chlorine) starting material may be obtained, for example, by reaction of the corresponding 3'- erythro-hydroxy compound with, for example, triphenylphosphine and carbon tetrachloride, or alternatively by treatment with organosulphonyl halide (e.g. . triflucromethanesulphony! chloride) to “orm a corresponding 3'-erythro- organosulphonyloxy Compound which is then halogenated e.g. as described above.

Alternatively a 3'-threo-hydroxy or organosulphonyloxy compound of formula (Illa) or (IIIb) may be treated, for example with triphenylphosphine, carbon tetrabromide and lithium azide to form the corresponding 3'-erythro azido compound.

With regard to process (B) the following represent examples o¢ various procedures by which the precursor groups in formula (Iva) may be converted into the desired

RL RZ, R> and RY groups:- a) When RZ represents an alkoxy (e.g. methoxy or ethoxy) group, such compounds may be prepared from corresponding compounds of formula (IVa) in which R} represents a hydroxy, e.g. by treatment with an appropriate alkylating agent, e.g. an alkanol, conveniently in the presence of potassium carbonate; b) When Rl Fepresents a mercapto group, such compounds may be prepared from corresponding compounds of formula (IVa) in which R1 represents an alkoxy (e.g. ethoxy) group, e.g. by treatment with hydrogen sulphide; c) When R? represents an alkyl group, such Compounds may be prepared from corresponding compounds of formula (IVa) in which RZ represents a hydrogen atom, e.g. by treatment with an alkylating agent, e.g. N,N- dimethylformamide dimethylacetal;

BAD ORIGINAL ol

HDL/LMJ/17th September 1985

/ -9- DX/85 it 3 £ 7 When R’ represents a mercapto group, such compounds may be prepared from

FS , . ; * corresponding compounds of formula (IVa) in which R represents an i appropriate leaving group, e.g. 1,2,4-triazolyl, by treatment ‘or example a with an alkali metal (e.g. sodium) mercaptan; 4 e) When R> represents an amino group, such compounds may be prepared from ! corresponding compounds of formula (Iva) in which rR represents a hydroxy / group by treating with an aminating agent, e.g. ammonium sulphate, in a bomb; £) When RY represents a halo (e.g. chloro) radical, such compounds may be prepared from corresponding compounds of formula (IVa) in which R% represents a hydrogen atom by treatment with a halogenating agent e.g. m- chloroperbenzoic acid.

Similar procedures may be used to e“fect conversion of the precursor group in : formula (IVb) into the desired R® and rR’ group, as well as procedures described for example in the above-mentioned references.

With regard to process (C), this may be effected for example by treating the appropriate pyrimidine or purine of formula (Va) or (Vb) or a salt or protected derivative thereof, with a compound of formula

B

A

Ko 3 (wherein A represents a leaving group, e.g. an acetoxy or benzoyloxy or halo, e.g. chloro group and B represents an optionally protected hydroxy group e.g. a p- toluenesulphonyloxy group), and subsequently removing any protecting groups.

With regard to process (D) the reaction of the compounds of formulae (Vb) and (VI) is conviently effected in the presence of a phosphorylating enzyme, and if desired, separation of the 3'-azido anomers in conventional manner.

HDL /LMJ/17th September 1985

\ -10- oy

The compounds of formula (1 wherein rR” is hydroxy and the azido group is in chee r configuration may also be Prepared jn conventiong] Manner for example as : described jn the following references op by methods analogous thereto, JK.

Horwitz et al., J, Org. Chem. 29, (July 1964) 2076-78, and M.Imazawg et al,, a

Org. Chem, 43 (15) (197g) 3044-3043; K.A. Watanabe et al., J. Org. Chem., 45, 3274 (1980); or R.P. Hlinski et al., J, Chem., soc. Chem. Commun, 215 (1970). ,

Where a Compound of formyj, Mor (I) is formed, such 4 Compound may be :

Converted into 4 Pharmaceuticalyy acceptable phosphate gp other ester by reacting : the compound of formula (1) op (II) with respectively phosphorylating agent, e.g.

POCl, Or an appropriate esterifying agent , e.q. an acid halide ofp anhydride, The

Compounds of formula (p) including esters thereof may be converted into pharmaceutical]y acceptable gat thereof jn Conventiong] Manner, e.g. by vo treatment with an appropriate base, b

Where an ester or salt of a compound of formula (I) or (I) is formed, such a

Compound may be Converted intg the parent Compound e.g. by hydrolysis.

Where an estep or salt of 4 compound of formula (1) op Mm) is formed, sych g compound May be converteq into the parent Compound, e.g. by hydrolysis, ' ! 7 A pharmaceutically acceptable 3 -azido-2,3 ~dideoxy nucleoside (hereafter referred to as the active ingredient) may be administered by any Suitable route Including oral, rectal, nasal, topical (including buccal ang sublingual), vaginal ang Parenterg] (including subcutaneous, intramuscy]ar, intravenoys and intradermay), It will pe appreciated that the preferred route may vary with, for Example, the condition ang age of the recipient, :

In general, for each of the above-mentioneq viral infections, a suitable effective dose wil] be in the range 1.0 to 250 Mg per kilogram body weight of recipient per day, preferably in the range of } tq 100 mg per kilogram body weight per day and most preferably in the range 5 to 40 mg Per kilogram body weight per day. The desired doge is preferably Presented gag two, three, four Or more Sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, Containing 5 tq 500 mg, preferably to 200 Mg and mogt preferably 20 to 100 mg of active ingredient per unit dosage } form. r P)]

L 0 .

HOL/LMI/17¢eh September 198%

3 -11- DX/85

A Jreferred dose is administered to achieve peak plasma concentrations of the said nucleoside of from about 1 to about 100 uM, preferably about 2 to 80 uM, most reforadly about 3 to about 50 uM. This may be achieved, for example, by the / intravenous injection of a 0.1 to 5% solution of the active ingredient in saline as a } " bolus containing about 1 to about 40 mg/kg of the active ingredient. Desirable blood levels may be maintained by a continuous infusion to provide about 0.01 to about 0.4 ma/ kg/hour or by intermittent infusions containing about 0.4 to about 10 ¥ mg/kg of the active ingredient. / 7 While it is possible for the active ingredient to be administered alone it is preferable to present it as pharmaceutical formulations. The formulations of the present invention comprise at least one active ingredient, as above defined, together with one or more acceptable carriers thereof and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the . : recipient thereof.

The formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual, vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.

Such methods’ include the step of bringing into association the active ingredient with the carrier which constitutes one Or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers of finely divided solid carriers or both, and then if necessary shaping the product.

Formulations of the present invention suitable for oral administration may be 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 liquid or a non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or molding, 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, lubricant, inert diluent, preservative,

CL eac BAD ORIGINAL - a surface-active or dispersing agent. Molded tablets may be made by molding int suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.’ The tablets may optionally be Coated or scored and may oe ; formulated so as to provide slow Or controlled release of the active ingredient L therein. \

Formulations suitable for topical administration in the mouth include lozenges *

Comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles Comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier, §

Formulations for rectal administration may be presented as ga Suppository with a ’ suitable base comprising for example cocoa butter or a salicylate. \

Formulations suitable for nasa] administration wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range 20 to 500 microns which is administered in the manner in which snuff ig taken, i.e. by rapid inhalation through the nasa] Passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as for example a nasa] Spray or as nasa) drops, include aqueous or oily solutions of the active ingredient,

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams op spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Formulations suitable for Parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, bu*fers, 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-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, ‘or example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. r ~

BAD ORIGINAL 0)

HDL /LMJ/17th September 1985

1 -13- DX/85 & erred unit dosage formulations are those containing 2 daily dose or unit, fily sub-dose, as herein above recited, or an appropriate fraction thereof, of an 4 tive ingredient. 7 1t should be understood that in addition to the ingredients particularly mentioned ’ above the £grmulations of this invention may include other agents conventional - in the art having regard to the type of formulation in question, for example, © J those suitable for oral administration may include flavouring agents. 3 ' [IR . / It should be appreciated that the said 3 -azido-2,3 _dideoxynucleosides may be / administered singly or in conjunction with other therapeutic agents. For . example, one possible opportunistic infection frequently observed in suffers of . AIDS is herpetic meningitis. The nucleoside according to the invention may then / be administered for the treatment of AIDS in conjunction with, for example, 9- / (2-hydroxyethoxymethy)) guanine (acyclovir) or 2-amino-9-(2- hydroxyethoxymethyl) guanine for the treatment of meningitis. ‘ - - - - * '

Thus, in 2a further aspect of the present invention 18 provided 3 -azido- t 1 2,3 dideoxy furanosyl nucleosides in conjunction with another therapeutic agent in therapy. 1 Tt

The following £xamples illustrate pharmaceutical formulations and 3 -azido-2,3 - dideoxy-furanosyl nucleosides according to the invention.

Example 1 Tablet

Active ingredient 100 mg }

Lactose 200 mg

Starch 50 mg

Polyvinylpyrrolidone 5 mg

Magnesium stearate 4 mg

A————————————. 359 mg a ——————————

Tablets were prepared from the foregoing ingredients by wet granulation followed by compression. \ —

BAD ORIGINAL 9 eet oc amtamber 1985 | i

Example 2

Injectable t ,

Active ingredient 0.125 ¥

Sterile, pyrogen-free, pH 7 phosphate buffer, q.s. to 25 ml t

Example 3 %

Preparation of 3'-Azido-3'-Deoxy-5'-0-Octanoyl Thymidine \

To a solution of 3'-azido-3'-deoxy thymidine in pyridine at 0°C, 1.2 eq. of octanoylchloride was added. The reaction was allowed to warm to room . temperature. When tlc (CHC14:MeOH;20:1, on silica gel) indicated complete reaction, the solution was poured onto ice water. The aqueous phase was decanted. 7 The resulting oil was chromatographed on silica gel eluted with

CHC14:MeOH. The compound was obtained as an oil.

CHN: cal. C-54.95 H-6.92 N-17.80 fnd. C-54.82 H-6.96 N-17.66

Example 4 2'-Acetyl-3'-Azidothymidine 5'-Acetyl-3'-azidothymidine was prepared by the method of M. Imazawa and F.

Eckstein, J. Org. Chem, 43, 3044 (1978). T-S.Lin, Y-S Gas and W.R. Mancini, J.

Med. Chem., 26, 1691 (1983).

Example 5

Compounds

The following compounds were prepared according to the procedure of Example 3 from the appropriate acid halide or anhydride. 3 -Azido-5'-0-benzoyl-3'-deoxythymidine cal. C-53.68 H-4.77 N-18.41 ‘nd. C-53.81 H-4.72 N-18.46

HDL/LLMJ/17th September 1985

Ota. . - ——

J ff -15- DX/85 ~ ! : 3. /\zido-3'-deoxy-5'-Q-pivaloylthymidine cal. C-51.27 H-6.03 N-19.93 7 fnd. C-51.07 H-6.05 N-19.83 #-Azido-3'-deoxy-5'-0-(3-methylbutyrylthymidine cal. C-50.24 H-6.13 N-19.53 #nd. C-50.27 H-6.11 N-19.49 7 31A2ido-3'-Deoxy-5'-0-succinylthymidine 31_Azido-3'-Deoxy-5'-0-mesylthymidine 31 Azido-5'-0-(4-methylbenzoyl)-3'-deoxythmidine / / 51-0-Acetyl-3'-azido-3'-deoxythymidine cal. C-46.60 H-4.89 N-22.65 fnd. C-46.67 H-4.94 N-22.59 31Azido-5'-0-(3-chlorobenzoyl)-3'-deoxythymidine cal. C-50.31 H-3.97 N-17.26 Cl1-8.74 #nd. C-50.16 H-4.03 N-17.13 C1-8.66 31_Azido-3'-Deoxy-5'-0-palmitoylthymidine cal. C-61.67 H-8.57 N-13.85 nd. C-61.85 H-8.59 N-13.75

Example 6 :

Preparation of 1-(3-Azido-2,3-Dideoxy-8-D-Threo -

Pentofuranosyl)Thymine5'-Monophosphate Disodium Salt 200mg (0.75 mmoles) of 1-(3-azido-2,3"-dideoxy- B-D-threo-pentofuranosyljthymine 2.

Org.Chem. (1980) 43, 3274-3278) were dissolved in 5ml of triethylphosphate and chilled to -8°C. 274ul (3mmoles, 4eq.) of phosphorus oxychloride was added all at once. TLC (n-PrOH:H,O:conc. NH, 0H,7:1:3 on cellulose) indicated product formation but incomplete reaction after 3 hours. The reaction was stored in a -5°C freezer overnight. The next morning the tlc indicated 80-90% complete reaction. The reaction was poured onto 15-20 ml of ice water and the pH was adjusted to 7. The aqueous solution was extracted with chloroform (2x) and ether (2x). The traces of organic solvents were removed by gentle stripping on a rota-vap.

Freshly prepared deactivated charcoal was added to the aqueous solution until UV assay indicated 90% of the nucleotide had been absorbed. The mixture was filtered and washed with water until no UV absorption was found in the filtrate. The compound ! N

HDL/LMJ/17th September 1985 DAD ORIGIN! A

- -16- DX/85

Cl ve was washed off the charcoal by slurrying in 50% aqueous ethanol containing 2p’ ¢O»C -

NH, 0H and filtering. The wash was repeated a second time. The ethanol and NHLOH were removed by evaporation. The pH was adjusted to 7. The solution was passes} . through 10ml of DOW 50 (Na™) to obtain the compound as the disodium salt. The solution was freeze dried. The title compound (104 mg) was obtained as a solid why af, was one peak by HPLC. NMR H 87.77 (d,36,5:1.2Hz &¢/) §6.18(dd,J1',2a'=3.0Hz,J1",2b'=7.7Hz 1'H) 64.64-4.45(m,3'H) 84.45-4.28(m,4'H) 54.28 — 4.08(m,5'H) 63.06-2.70(m,2a'H) §2.50-~2.24(m,2b'H) §1.95(d,J5,6=1.2Hz,5CH3) NMR 31p. 82.33 (JPH=6.3) \

Example 7 v

Sodium Salt of 3~Azidothymidine €

Approximately one gram of 3 -azidothymidine as prepared by the method of Example 6 was dissolved in 50mL of distilled water. The pH was adjusted to 12 with IN NaOH.

Approximately half of the solution was freeze dried. A white powder, 0.415 g, resulted. Elemental analysis indicated the formation of a full sodium salt.

Example 8 S~Phosphate of 3-Azidothymidine t 1

The 5 -phosphate of 3 -azidothymidine was prepared by the method of R.P. Glinski,

M.S. Khan, R.L. Kalamas and C.L. Stevens, J. Chem.Soc. Chem. Commun., 915 (1970).

Example 9 1-(3~Azido-2,3~Dideoxy-8-D-Threo-Pentofuranosyl)Thymine t 1 1-(3 -azido-2 ,3 -dideoxy-B8-D-threo-pentofuranosylthymine was prepared by the method of A.Matsuda, K.A. Watanabe and J.J. Fox, J. Org. Chem., 45, 3274 (1980).

Example 10 . (a) 2,5-Anh dro-3~Azido-3~Deoxythymidine

I 1 1 1 2,5 -Anhydro-3 -azido-3 deoxythmidine was prepared from 3 -azidothymidine by a two step reaction sequence.

BAD ORIGINAL 9

/ -17- DX/85 / es -nydroxyl of 5 azidothymidine (3.09, 11-2 Mol) was mesylated by the addition of snethanesulphonyl chloride (2.7 mL) to a solution of the starting material in dry pyridine (20 mL). The reaction was allowed to proceed at 5eC for one hour, then poured onto ice water. The precipitate was collected by filtration. The desired : product was obtained by reacting the 3" azido-5 -mesylthymidine obtained from the first step with potassium carbonate (0.789, 5.6 mMol) in DMF (75 mL). The reaction was heated in an g0 °C oil bath for six hours, then poured into ice water. The product was extracted from the water with ethyl acetate. The solvent was removed in vacuo and the resultant oil was flash chromatographed on silica gel by elution with : CHC1,:MeOH (9:1 v/v). The product was obtained in low yield. / mp = 184-186°C (b) igo spideon SOs Dimetnnesrban IRS

The sodium salt of dimethyldithiocarbamic acid dihydrate (0.642 g, 3-58 mMol) and 3.58 mL of a solution o® IN tetrabutylammonium hydroxide in MeOH was added to 25 mL of DMF. The solution was boiled to remove water and MeOH. After cooling, 5',2- anhydro-3 -azidothymidine (0.85 g, 3.4 mMol) dissolved in 15 mL of DMF was added.

The reaction was heated in a 55°C oil bath overnight. The reaction was poured onto ice water and 3 precipitate was removed by filtration. The product was extracted from the filtrate with ethyl acetate. The ethyl acetate was removed in vacuo and the resulting oil was purified by flash chromatography on silica gel by elution with

CHC1,:MeOH (95:5 v/v). Chromatography was required a second time on silica gel.

The second elution Was with CHC15:MeOH (98.2 v/v). Final purification was accomplished by reverse phase chromatography on Cia eluted with water:methanol (3:7). The yield was 2.5%.

Example 11 51_Azido-5'-0-Acetyl-4-Thiothymiding 1 Azido-5"-O-acetyl-i-(1,2;-triszoleXthy icine (Lin, et al, ,J. Med. Chem. 26, 1691 (1983)) (1.419; 3.9 mMol) was dissolved in 100 mL acetone and 30 mL Ho 0s then treated with 0.39 9 NaSH.xH,0 (Sung, J. Chem. Soc. Chem. Comm. 522, (1982)) . The mixture was stirred for 30 min, the volume reduced by 1/2 and extracted with 200 mL CHCl. r Cr anI17eh Ceptember 1985

The CHCl, was washed with 100 mi H,0 and dried over Na,SO,. The solv E+ Way removed in vacuo and the resultant oil placed on a silica gel pad (6.5 x 3 cm) fa lowed] by elution with 750 mL. CHC1,. The solvent was removed in vacuo to yield a ye fou oil which was recrystallized ‘rom 1-PrOH to yield 0.64g (1.9 mMol; 48.7%).

Lo mp = 75-78 *C. t

Example 12 E: 3'-Azido-4-Thiothymidine %. 3'-Azido-5'-0-acetyl-4-thiothymidine (0.25 g; 0.76 mMol, Example 11) was dissolved in { a mixture of 5 ml of dioxane and 5 mL of conc. NH, OH and stirred for 18 hrs. The solvent was removed in vacuo and the residue applied to a silica gel column followed - by elution with CHC13/EtOAc (3:1 v/v). The appropriate fractions were combined and the solvent removed in vacuo to yield a yellow oil which was dissolved in Et,0, forming crystals upon concentration: 0.16g (0.56 mMol; 74%). m.p. = 116-118°C,

Example 13 3-N-Methyl-3'-Azidothymidine 3'-Azidothymidine (0.5 g; 1.9 mMol) and N,N-dimethylformamide dimethylacetal (Zemlicka, Coll. Czech. Chem. Comm. 35, 3572 (1972) (0.9 mL; 7.5 mMol) were refluxed to 20 mL CHCl, for 48 hours. The solvent was removed in vacuo and the material placed on a silica solumn. Elution with EtOAc/CHC1, (1:1 v/v) resulted in pure material as a viscous oil: 0.26 g (0.9 mMol, 47%).

Example 14 3'-Azido-2-Thiothymidine

The synthesis of 3'-Azido-2-thiothymidine was accomplished by a five step reaction sequence starting from 3',2-0-anhydro-5'-tritylthymidine (3.3. Fox, J. Org. Chem, 28, 936, (1963).

Fr

BAD ORIGINAL »

HDL /l M1/17+R Cate be — Ye0e

/ -19- DX/85 3/.0-Anhydro-5'-tritylthymidin® (10.5 gq, 22.4 mMol) was added to 3 solution of spdium (0.52.9, 22.4 mMol) in dry ethanol (1.2L) and the reaction was refluxed £or six fours. The reaction was cooled and neutralized with IN HCl. The solvent was removed in_vacug and the resultant oil purified by flash chromatography on silica gel by elution with CHC15:MeOH (96:4 v/v). A 30% yield of 1-(2'-deoxy-5'-trityl-D- lyxofuransyl)-2-ethoxythymine was obtained. The 2-ethoxythymidine derivative (3.5g, 16.8 mMol) was dissolved in 35 mL of DMF containing 2.2 mb of triethylamine. : The cold solution was saturated with H,S. The reaction was placed in a steel bomb and heated at gseC. After twenty seven hours TLC indicated no starting material remained. The reaction was purged with N, for several hours and poured onto ice water. The product was collected by ¢j]tration and purified by s]ash chromatography on silica gel by elution with CHC15:MeOH (97:3 viv). A 37% yield of 1-(2'-deoxy-5'- trityl- B-D-lyxofuranosyl)-2-thiothymine was obtained. The UV max at pH1 of 277 nm and at pH11 of 241 nm indicated the formation of a 2-thiothymidine. The 3'-hydroxyl of the thiothymidine derivative was mesylated as follows: methanesulfonyl chloride (665 ul, 3.5 eq.) was added in four portions over six hours to a solution o¢ 1-(2'-deoxy- : &1_trityl- -D-lyxofuranosyl)-2-thiothymidin® (1.25 g) in dry pyridine (15 mL) at 5°C.

The reaction was maintained at 5eC overnight. The reacton was poured onto ice water and the product collected by filtration. Purification was accomplished by flash chromatography on silica gel by elution with ethyl acetateshexane (1:1 v/v). The yield was 50%. Lithium azide (0.3 g, 6 mMol) was dissolved in 20 mL of dry DMF and 1-(2'- deoxy-3'-mesyl-5'-trityl- 8-D-lyxofuranosyl)-2-thiothymine 0.72 9, 1.2 mMol) was added. The DMF solution was heated at g5°C for 2.5 hours. The reaction was poured onto ice water and the product collected by filtration. Purification was accomplished by flash chromatography on silica gel by elution with CHC15:MeOH (98:2 viv). The yield was 78%. A band in the IR at 2100 cmt indicated the presence of an alkyl azide. The UV confirmed the presence of a 2-thiothymidine. The final product was prepared by deblocking the 5t_hydroxyl of 31_azido-2-thio-5'-tritylthymidine 0.1 @) in 80% acetic acid (5 mL) on a steambath for fortyfive minutes. 3-Azido-2- thiothymidine (0.021 g) was obtained by chromatography on silica gel by elution with

CHC14:MeOH (96:4 v/v) in 37% yield.

Example 15 31_Azido-2-E thoxythymidine : : 3'_Azido-2-ethoxythymidine was prepared by refluxing 3'_azido-5 -mesylthymidine (2.6 g, 7.5 mMol) in dry ethanol (25 mL) with two equivalents of potassium carbonate (1.08 nt g, 7.2 mMol) for five hours. The solution was neutralized and taken to an oil in vacuo.

BAD ORIGINAL A

Co 1 sat /17+h Segtember 1985 ER =

-20- DXx/85

The oil was purified by flash chromatography on silica gel by elution wi : elley acetate:methanol. The desired product was isolated in 39% yield. 3 mp = 98-100°C EO

Example 16 % % 3~Azido-2-Methoxythymidine Y 3'-Azido-2-methoxythymidine was prepared from 3'-azido-5 -mesylthymidine (1.6q, 4.6 i mMol) by the procedure of Example 15. The yield was 42%. mp = 47-51°C

Example 17 ’ 3-Azido-2~Deoxy-5-Methylisocytidine 2,5 -Anhydro-3 -azido-3 -deoxythymidine (0.35 g; 1.4 mMol) was dissolved in 15 mL of

MeOH presaturated with ammonia and placed in a bomb at 77 *C (oil bath) for 48 hours (Skaric and Matulic-Adamic, Helv. Chim. Acta. 63, 2179 (1980). By TLC (6:1 v/v

CHCl;/MeOH) the reaction was incomplete. The solvent was removed in vacuo and the resultant oil placed on a silica gel column followed by elution with CHCl ;/MeOH (6:1 v/v). The appropriate fractions were combined to yield 0.14 g (0.53 mMol; 38%). mp = 107 - 108°C

Example 18 5-Chloro-3'-Azido-2'-Deoxyuridine 3'-Azido-2'-deoxyuridine (0.25g; 1 mMol) was dissolved in 2 mL dry dimethylacetamide (DMAC), cooled to 0°C and 2 mL of 0.5 m HCl in DMAC was added. m-

Chloroperbenzoic acid (0.277 g; 1.6 mMol) was added in two portions over ten minutes and the mixture was allowed to come to ambient temperature. After two hours, 4 mL

H,0 was added and the solution filtered. The aqueous DMAC solution was extracted with Et,0 (3 x 3 mL) and the Et,0 was evaporated in vacuo to an oil which was applied to a silica gel column. Elution with CHC13/MeOH (15:1 V/Vjsgqmbination of

BAD ORIGINAL A

HDL /LMJ/17th September 1985 L—.

/ | _21- DX/85 aggeopriate fractions and evaporation in vacuo yielded an oil which was crystallized hem Et,0- vield 58.5 mg (0.2mMol , 20%). mp = 169-170°C- ! Example 19 4 Azido-5-Bromo-2-Deoxyuriding 3 Agzido-5-bromo-2-deoxyuriding was prepared from the known 51_gzido-2'- deoxyuridine (T.A. Krenitsky, et al., J. Med. Chem., 26; 891, (1983) (0.827 9» 3.3 mMol) by first acetylating the 51-hydroxyl with acetic anhydride (15 mL) then by brominating the 5 position bY the addition of acetic acid (0.5 mL) and bromine (0.566 g). The red-brown solution was stirred at room temperature £or two hours. The reaction was taken to an oil in vacuo and triturated with ethyl ether. The oil was dissolved in methanol ammonia to remove the acetyl group. The desired product was isolated by chromatography on silica gel by elution with CHC14:MeOH (95:5 v/v). The yield was 32%. mp = 148-149°C

Example 20 1 Agido-2-Deoxy-5:lodouridine 1+ Azido-2-deoxy-5-iodouridin® was prepared from 21_deoxy-5-iodouridine (10 g, 28 mmol) by 8 four step reaction sequence described in the literature (T.A. Krenitsky, et ale, J Med. Chem. 26, 891, (1983): mp = 126-130°C

Example 21 + Agido2-Deoxy:5-TrifluprometIISCE + Agido-2-deory-5-trifluorometYIUFIETE was prepared by a four step reaction sequence. \ pr —— +11 7+k Ceptember 1985 BAD ORIGINAL 9

-22- Dx/es

The 5'-hydroxyl of 2'-deoxy-5-trifluoromethyluridine (5.0 g, 16.9 mMol) was t th fof by the addition of triphenylmethyl choride (5.65 g, 20.3 mMoal) to the starting mofercaf in a suspension of dichloromethane (1.4 L ml), pyridine (70 mL), and 3 A molticeiLof— sieves (55 g). The reaction was stirred at room temperature for four days, Affe, — filtration and evaporation in vacuo, the oily product was chromatographed on silicaige f by elution with CH, C1,:Me0OH (95:5 v/v). The product fractions were combined ayf the solvent removed in Yacuo. The resulting oil was triturated with water and the solid that formed was collected by filtration. The 3'-hydroxyl was chlorinated by dissolving the 5'-protected uridine (3.0 g) in dimethylacetamide (30 mL) containing’ triphenylphosphine (3.27 g) and adding carbon tetrachloride (51 mL). The reaction was stirred at room temperature overnight. One millilitre of methanol was added. The reaction was taken to an oil and chromatographed on silica gel by elution with

CH, Cl1,:Et0AC (9:1 v/v). The desired product was collected as an oil. The oil, 1-(3- chloro-2 -deoxy-5 trityl-threo-8-D-ribouranosyl)-5-trifluoromethyluracil, was ‘ deblocked by dissolving in nitromethane (80 ml.) and adding zinc bromide (4.45 g) dissolved in nitromethane (80 mL) by the method of Vv. Kohli, et_al., Tetrahedron

Letters, 21, p 2683, 1980. The reaction was stirred at room temperature overnight.

More zinc bromide (3.0 9) was added the next day and the reaction was allowed to go overnight. A “inal addition of zinc bromide (3.7 g) with gentle heating pushed the reaction to a stopping point. The reaction was poured into 1M ammonium acetate.

The product was extracted into dichloromethane. The dichloromethane was removed in _vacuo and the resulting oil was chromatographed on silica gel by elution with

CH,C1,:MeOH (95:5 v/v). The *inal product was obtained by treating 1-(3-chloro-2 - deoxy-8-D-ribofuranosyl)-5-trifluoromethyluracil (0.48 g, 1.53 mMol) with lithium azide (0.19 g, 3.8 mMol) in dimethylacetamide (4.8 mL). The reaction was heated at 90°C for four hours. The reaction was taken to an oil and chromatographed on silica gel by elution with CHC15:MeOH (95:5 v/v). Chromatography was required a second time. Elution on silica gel with CH,C1,:MeOH (97:3 v/v) resulted in a nearly pure product. Crystallization from toluene produced the pure product in 10% yield.

Example 22 3~Azido-2-Deoxyeytidine 3'-Azido-2'-deoxycytidine ‘was prepared from 3'-azido-2 deoxyuridine (2.2q, 7.9mMol) as the HCI salt by the procedure of T.A. Krenitsky, et _al., J. Med. Chem., 26, 891, (1983). The yield was 40% mp = 174.5-176.5*C Pr,

Bap ORIGINAL

HDL/LLMJ/17th September 1985 sv -23- DX/85 \ 4 fe dfxample 23 ¢ f 3" Azido-2-Deoxy-5-Methylcytidine . oF ¢ = 3 3 -Azido-2 _deoxy-5-methylcytidine was prepared from 3'azidothymidine (0.89,

J 3.0mMol) by the procedure of Example 22. The yield was 19%. 7 { Example 24 j Threo-3'-Azido-2'-Deoxycytidine

The synthesis of threo-3'-azido-2'-deaxyctidine was accomplished from 2'- deoxyuridine in four steps.

The S5'-hydroxyl of 2'-deoxyuridine was tritylated by the method described in

Synthetic Procedures in Nucleic Acid Chemistry, 1, 321, (1968). py, Threo-3'-Azido-2'-deoxy-5'-trityluridine was prepared by reacting 2'-deoxy-3'- trityluridine (5.0 G 10.6 mMol) with triphenylphosphine (3.07 g, 11.7 mMoles, 1.1 eq.) and carbon tetrabromide (3.88 g, 11.7 mMol, 1.1 eq.) and lithium azide (5.21g, 106 mMol, 10 gq.) in DMF (80 mL). The carbon tetrabromide was added last. The reaction was allowed to go at room temperature overnight. Methanol (5 mL) was added. The solution was taken to an oil in vacuo and flash chromatographed on silica gel by elution with ethyl acetate. Deblocking the 5'- hydroxyl was accomplished by heating in 80% acetic acid on 8 steambath for twenty minutes. Upon cooling, the tritylcarbinol precipitated and was filtered off. The filtrate was taken to dryness and slurried in ethyl ether. The product, threo-3'-azido-2'-deoxyuridine, was carried on without yrther purification. The final product, threo-3'-azido-2'-deoxycytidine as the HC1 salt, was prepared from the uridine analogue by exactly the same procedure as used for the preparation of the erythro isomer (T.A. Krenitsky, et al, J. Med. Chem., 26, 891, (1983)). The yield was 0.021 g, 7%. / 84p - &

-24- A

Example 25 \

Preparation of 2-Azido-3'-Deoxythymidine % a) 2,3'-Anhydrothymidine % % -*

Thymidine (85.4 g: 0.353 mol) was dissolved in 500 ml dry DMF and added to N- vo (2-chloro-1,1,2-trifluorcethyl) diethylamine (100.3 g: 0.529 mol) (prepared % according to the method of D.E. Ayer, J. Med. Chem. 6, 608 (1963). This t solution was heated at 70°C for 30 minutes them poured into 950 m] ethanol t (EtOH) with vigorous stirring. The product precipitated from this solution was ¥ filtered. The EtOH supernatant was refrigerated then “iltered to yield a tota] o* Y 47.75 g (0.213 mol. 60.3%) mp = 228 - 230°C. y b) 3'-Azido-3'-Deoxythymidine : 2,3'-Anhydrothymidine (25 0: 0.1115 mol) and NaN; (29 g, 0.446 mol) was ° suspended in a mixture of 250 ml DMF and 38 m] H,0. The reaction was refluxed for 5 hours at which time it was poured into 1 liter of H,0. The aqueous solution was extracted with EtOAc (3 x 700 ml). The EtOAc was dried over Na,S0,, filtered and the EtOAoac was removed in vacuo to yield a viscous oil. This oil was stirred with 200 ml water resulting in a solid 9.15g (0.0342 mol, 30.7%). mp = 116-118°C

Example 26 -

Triphosphate of 3'-Azido-3'-Deoxthymidine

The triphosphate of 3'-azido-3'-deoxythymidine was prepared from the §5'- monophosphate of 3'-azido-3'-deoxythymidine in four stages: (a) Bis (nBu)sN Pyrophosphate

A column of DOW 50 pyridinium resin was prepared by pouring 40 mL of resin into a 25 cm diameter column and washed with water until no more colour eluted. Pyrophosphate decahydrate (1.12 g, 2.51 mM) was dissolved in 30 mL o* water and applied to the column. The column was eluted with water. A 125 mL fraction o* the eluant which contained UV absorbing material was collected. The

HDL /LMJ/17th September 1985 f -25- DX/85 © volume was reduced to 10 mL in vacuo and tri-n-butyl amine (1.2 ml) was added.

The volume was reduced in vacuo and the residue was dried by coevaporation with pyridine four times. The product was stored in a freezer (-5°C). - ! * 3 * (b) Hydrogen Form of the Monophosphate of 31_Azido-3~Deoxythymidine

The hydrogen form of the monophosphate was prepared by passing the ammonium salt (0.1 g, 0.283 mMol) dissolved in 6 mL of water, through a 1.5 mL (10 eq.) column of DOW 50 H'. (e) Phosphormorpholidate Derivative of 31_Azido-3'-Deoxythymidine

In 9 mL of water was dissolved 0.283 mMol of the hydrogen form of the monophosphate obtained in stage b). Morpholine (99 ul, 1.13 mMol, 4 eq.) was added and the solution heated to reflux. Dicyclohexyl carbodiimide (0.234 a, 1.13 mMol, 4 eq.) dissolved in t-butanol (5 mL) was added over a three hour ~ period. The reaction was refluxed overnight. The reaction was cooled to room temperature, filtered, and the solvents removed in vacuo. Ethanol was added and evaporated in vacuo four times. The residue was dissolved in methanol and the phosphormorpholidate precipitated by the addition of ether. The precipitate was triturated with ether four times and dried on a rotary evaporator. A weight yield of 97 mg, 50%, was noted. (d) 31. Azido-3'-Deoxythymidine-5'- Triphosphate

The phosphormorpholidate derivative obtained in stage c), was dried by a removal of pyridine in vacuo four times. The bis (n-Bu)sN pyrophosphate obtained in stage a) was also dried by removal of pyridine in vacuo. The phosphormarpholidate was dissolved in pyridine, 5 mL, and added to the vessel containing the pyrophosphate reagent. The reaction was allowed to continue overnight at room temperature. The pyridine was removed in vacuo. Water was added to the residue and removed in vacuo three times. The residue was frozen. vill

La Mh M1/17¢th September 1985

-26- i ple 27 v

Exam le. oo ”

Preparation of 2 AZId0- 105 Dideoxy-aD-Ribouranosyadenine : 5-0 -Azida-2.5' dideoxy a-0-riboturanos aden. Was prepared in two steps. ' :, ‘rom N,-octanoyladenine (2.0q, 7.7mMoal) and 3 -azido-3 ~deoxythymidine (1.13q, ¥ 4.2mMo)) by the procedure described by M. Imazawa and F. Eckstein, J. Org.

Chem., 43, 3044 (1978). i mp = 120-122 eC -

Uv pH 1 max 258nm min 230 nm \ pH 13. max 260nm min 229 nm }

CHN calculated ‘or CG oH; 2Ng0, | )

Calculated: C-43.48; H-4.38; N-40.5¢

Found: C-43.28; H-4.45; N40-.38

Example 28 . £ ! . ' ! - . .

Preparation 9-(3 -Azido-2 3 =Dideoxy- 8-D-Ribofuranosy) Adenine 2-5 -A2ido-2, dideoxy 8-D-riboruranasy igen. Was prepared in two steps f _27- DX/85

Framete 29

J . * Anti-HTLVIL Activity of 3 Azido-3~Deoxypyrimidine Nucleosides f

Compounds of formula (I) were tested in vitro against HTLVII and were found to have °°

EDgq values as shown in table 1.

Table

Compound EDg(uM) 3'_Azido-2,3 ~dideoxythymidine 0.005 1 1 3 -Azido-2,3 _gideoxythymidine-5 -phosphate 50 [I 1-(3'-Azido-2 0 ideoxy- 8-D-threo-pentofuranosylthymine 0.5 1 1 3' _Azido-2,3 _dideoxy-5-bromothymidine 0.5 1 1 5'_Azido-2,3 -dideoxycytidine 0.5

Example 30

Toxicity Assay t v1 3 -Azido-2,3 _dideoxythymidine was administered to both mice and rats. The LDgq value was found to be in excess Of 750mg/ kg in both.

Vi av J17+h September 1985

EXANPLE 3 Te - 3'-Azido-5-Chloro-2',3' -Dideoxyuridine . 3'-Azido-2' -deoxyuridine (0.25 g; 1 mMol) was dissolved in 2 mL dry \ . dimethylacetamide (DMAC), cooled to 0° and 2 mL of 0.5 M HCl in DMAC was \ ) added. m-Chloroperbenzoic acid (0.277 8; 1.6 mMol) was added in two 3 portions over ten minutes and the mixture was allowed to come to ambient % temperature. After two hours, 4 mL H20 was added and the solution filtered. 7

The aqueous DMAC solution was extracted with Et20 (3 x 3 mL) and the Et,0 Y

Was evaporated in vacuo to an oil which was applied to a Silica gel column, }

Elution with CHC13/MeOH (15:1 v/v), combination of appropriate fractions and evaporation in vacuo yielded an oi} which was crystallized from Eto0 to give . 58.5 mg (0.2 mMol, 20%): mp = 169-170°C; UV (nm): at PH 1 xpay = 276 (e = 7400), Amin = 23 (e = 500); at pH 13 ‘max = 274 (e = 6400), apy, . 219 (e = 3800); H! NMR (DMSO-dg) ¢ 8.29 (s,1H,H6), 6.04 (t,1H,H1', J=5.5 Hg), 5.49-5.29 (m, 1H,5'-0H), 4.44.4 29 (m, 1H,H3'), 3.88-3.71 (m, 1H,HY'), 3.71-3.53 (m,2H,H5"), 2.63-2.31 (m,2H,H2') ; analysis calculated for

CgH1oN504C1: C 37.58, H 3.50, N, 24.35, Cl 12.32

Found: ¢ 37.67, H 3.54, N 24.39, C1 12.40

PAT 2/11/AF/sp/1

~ ) WG

Enent] % . - DX/85 + _acetyl-3'-Azido-3-Benzoyl-3' -Deoxythymidine

Yockj1-3" -azido-3' ~deoxythymidine (0.75 8, 2.4 mMol) was dissolved in pyridine (5 mL) and benzoyl chloride (1.4% mL, 12 mMol, 5 eq.) was added at room fenperature. The reaction was stirred overnight then poured onto ice water (1.50 mL). The pH of the aqueous solution was adjusted to 1. The product as extracted with chloroform. The organic phase was washed with water, dried with MgSOy and filtered. The chloroform was removed and the oily product was flash chromatographed on silica gel eluted with chloroform. The product was collected as an oil.

NMR was taken in DMS0-dg \MR: 68.04-7.50 (m,6H;3N-benzoyl and 6H), 66.12 (dd, 1H, oF

Jiv,2a' * 5.6 Hz, Ji1',2b' = 6.7 Hz, 1'H), §4.55-3.96 (m,

UH; 3'H,4'H,5H"), §2.62-2.38 (m,2H,2'H), §2.07 (s,34,5' acetyl CH3), 51.90 (d, 3H, J5,6 © 1.0 Hz, S5CH3)

CHN calculated for C1gH19N506

Calculated: C-55.20; H-4.63; N-16.94

Found: c-55.29; H-U4.64; N-16.93

PAT 2/11/AF/sp/2

Toeen B

Preparation of 5'-Esters of threo-3'-Azido-3' -Deoxythymidine G: threo-3'-Azido-3'-deoxy-5'-0-phenoxyacetyl thymidine . “ threo-3'-Azido-3'-deoxythymidine (1.0 g, 3.7 mMol) was dissolved in pyridine % (10 mL). water was removed by boiling the solution until the temperature of - the vapor coming off reached 115°C. The reaction was chilled to Q°c, The t phenoxyacetyl chloride (1 mL, 7.4 mMol, 2 eq.) was added all at once. The tv reaction was allowed to continue for three hours. After Pouring onto ice Y water (350 mL), the product oiled out of solution. The aqueous phase was \ decanted and the oi] dissolved in ethyl acetate. After drying with MgS0y . and filtering, the solvent was removed. The resultant oil was flash chromatographed on Silica gel eluted with chloroform:methanol (40:1, vrv). i

The product was collected in 15% yield.

NMR taken in DMS0-dg

NMR: 411.3 (s,1H,3-NH), §7.50 (d, 1H,Jg 5 = 1.2 Hz, 6H), 8§7.41-6.86 (m, 5H,5'-phenoxy), 56.10 (dd, 1H; J1r 230 = 4.5 Hz, J1' 2p = 7.5 Hz, 1'H), 64.86 (s,2H,CHy of 5'-acetyl), §4.57-4 21 (m, 4H; 3'H,4'H,5'H), §2.75-2.21 (m, 2H,2'H), §1.79 (d, 3H, J5,6 = 1.0 Hz, 5CH3)

CHN Calculated for C18H19N506

Calculated: C-53.86; H-4.77; N-17.45

Found: C-53.80; H-4.82; N-17.35

PAT 2/AF/sp/3

E oo . oe TI eee EE i RRNA yor

+ - 31

Gan f * _ DX/85 preparation of 1 ceo-3' -Azido-5-Bromo-2' 3! -Dideoxyuridine £ \con3' -Azido-5-bromo=2' 3 ~dideoxyuriaine was prepared from 2' deoxyuridine by a five step reaction sequence. . protection of the 5'-hydroxyl of 21 deoxyuridine with a triphenylmethyl group was accomplished in the usual manner. (1) The 3'-hydroxyl was mesylated in the usual fashion. (2) An azide was introduced into the 3' position with the correct stereochemistry by adding 51 _deoxy-3' -mesyl-5' ~trityluridine (22 g, 40 mMol) to a solution of sodium azide (7.84 g, 120 mMol, 3 eq.) in dimethylformamide (380 mL) at 80°C. The reaction was continued for 35 hours.

The solution was poured onto ice water (2 L) and the precipitate collected . by filtration. The product was isolated by chromatography on silica gel } eluted with chloroform:methanol (1:1, v/v) in 514 yield. The 5'-hydroxyl was deblocked with 80% acetic acid on a steambath for 25 minutes. after cooling, the tritylcarbinol was filtered off. The filtrate was reduced to a thick oil in vacuo. The product was isolated by flash chromatography on silica gel eluted with chloroform:methanol (85:15 V/V). The 5 position of chreo-3' -azido-2' ,3' -dideoxyuridine was brominated py exactly the same procedure as outlined for the bromination of erythro-3'-azido-2',3"- dideoxyuridine.

UV pH 1 Apax 280, © = 9400, Amin 244, € F 2600 pH 13 Amax 276 ¢ = 6700, ‘min 251 ¢ = 3700

NMR taken in pDMSO-dg

PAT 2/11/AF/sp/Y

BAD ORIGINAL 9 mn pn ST

NMR: 611.86 (s,1H,3-NH), 68.02 (s,1H,6H), 65.99 (dd, 1H, Jy» par = 3.0 %

Ji, 2p! = 7.5 Hz, 1'H), 65.1 (t, MH, J51CH,,5' OH = 5.4 Hz, 5'0H), 84.49 \ (m,1H,3'H), 84.05 (m,1H,4'H), 63.71 (m,2H,5'H), 62.72 (m,1H,2b'), 62.18 §& (m, 1H,2a") x

CHN calculated for CqH1gBrN5Oy-0.25 H30-0.1 CoHyOo y

Calculated: (C-32.25; H-3.21; N-20.44; Br-23.32

Found: C-32.17; H-3.21; N-20.33; Br-23.19

References: (1) Zorbach, W. Syn. Proc. in Nucleic Acid Chem., 1968, 1, 321 . (2) Michelson, A. J. Chem. Soec., 1955, 816

PAT 2/11/AF/sp/5

, ~33-

Frade / | ~ Dx/85 ; ‘Preparation of -(3-Azido-2, 3-dideoxy-B-D-erythro-pentofuranosyl)- '»_(Benzyloxo)-5-Methyl-4-( 1H) -Pyrimidinone

Sodium (0.4 g, 17.4 mMol, 2.6 eq.) was allowed to react with dry benzyl alcohol (10 mL) for one hour at room temperature. 2,5" -Anhydro-3'-azido-3'- . deoxythymidine (1.65 g, 6.6 mMol) was added. The reaction was allowed to continue for one hour. After pouring onto ice water (250 mL), the pH was adjusted to 7 and the aqueous phase was extracted with ethyl acetate. The organic phase was extracted with water (4 times). After drying with MgSOy the ethyl acetate was removed in vacuo. The resultant oil was chromatographed on silica gel eluted first with ethyl acetate then with ethyl acetate:methanol (9:1 v/v). ‘The product containing fractions were collected and the solvents : removed in vacuo yielding an oil. The oil crystallized after covering with } ethyl ether. m.p. = 125-126.5°C uv pH 1 unstable pH 13 Amax 256, ¢ = 10700, Amin 240, € = 8900

NMR taken in DMSO-d

NMR: 87.8 (d,1H,J§,5 = 1.2 Hz, 6H), §7.49-7.38 (m,5H,2-phenyl), 56.08 (dad, 1H,J11 2a' * 5.0 Hz; J1,2b' = 7.0 Hz,1'H), 65.37 (s, 2H,2CH2), 85.25 (t,'H,J5'CH,,5'OH = 5.4 Hz, 5'0H) 64.36-4.32 (m,1H,3'H), 63.85-3.81 (m, 1H,4'H), §3.7-3.58(m,2H,5'H), §2.53-2.34 (m,2H,2'H), 61.82 (d,3H,J5,6 = 1.0 Hz, SCH3)

PAT 2/11/AF/sp/6 : ce vel OFCINAL 9 es rn rr ST —

- 34 - Ke. o

CAN calculated for C17H1gN50y :

Calculated: C-57.14; H-5.36; N-19.60 Ee

Found: C-57.02; H-5.43; N-19.53 t :

PAT 2/11/AF/sp/7 Fm ™

BAD ORIGINAL €; bos

Ae te ett 4 ras Were mm. CME a SB ig om, Wher egy, ET as sr HA i aes Pole abe. as ome FF

0 - 35 -

Cased! 36 / } } pX/85 5'-Esters of 31 _Azido-3' -Deoxy thymidine /

J . fro ronnie 5'-esters of 31 -Azido-3' -deoxythymiding were prepared in the . fysual way . b) J+ _Azido-3' ~deoxy-5' -Q-boLUYLERYIIALAS / NMR taken in DMSQ-dg / NMR: 87.95-7.29 (m,SH; bi, cH, 6H), 86.16 (t,1H,1'H), o4.6-4.8 (m,3H,3'H,5'H), sl.2-4.0 (m, 1H,4'H), §2.39 (s,3H,dCH3), 61.63 (s,3H,5CH3) f CHN calculated for c1gH1gNs05

Calculated: Cc-56.103 H-4.97; N-18.17

J Found: c-55.88; H-5.00; N-18.09

A b) 31 -Azido-3' -deoxythymidine 5'-0- (Hydrogen succinate)

NMR taken in DMSO-dg

NMR: §7.46 (s,1H,6H), 66.13 (m, 1H, 1'H), gl. 48-u.40 (m,1H,3'H), sl. 34-4.20 (m,2H,5'H), §3.99-3.94 (m, 1H,4'H), 51.78 (s,3H,5CH3)

CHN calculated for c1yH17N507 calculated: c-hy.98; H-4.835 ’

Found: c-44.903 H-4.77; N f

PAT 2/AF/sp/B BAD ORIGINAL 0) i \ ore

- 36 - po rE : : by Sh sa ¢) 3'-Azido-3" -Deoxy-5'_Mesy thymidine TE - bg

NMR taken in DMSO-d, % ¥ Te

Con

NMR: 47.49 (d,1H,06 5 = 1.9 Hz, 6H), 56.15 (6, 1H,010 50 = 6.6 Hz, 1'H), bm $504.41 (m,30;30, 50g) 4.02 (m, 10, 40h 43 4 (5:34,5" -mesy1 cu), Yo 61.79 (d,3H,05 ¢ = 1.0 Hz, 5CH3) eo

CHN Calculated for C11H15N504s Kilo

Calculateq: C-38.25; H-4.37, N-20.28; S-9.28 . vo

Found; €-38.15; H-4.38; _20, q. $-9.30 b. d) eS 0 Chlorobenzost) 3: eosyenymnay 2

NMR taken jp DMS0-qg

NMR: 5171.37 (s, 1H,3-NH) 67.98-7.43 (m,5H;5" -pheny1, 6) 66.17 (dd, 14;

J1v 2a = 6.1 Hz, Ji opr = 7.2 Hz, 11H), 64.68-4 4g (m,3H;3'H,51y), sh. 14-y qq (m, 1H, 4h), 82.48-2. 41 (m,2H,2'H) | 41 ¢y (d,34,05 6 = 1.2 Hz, 5CH3) :

CHN calculateq fop C17H16C1NS05 i

Calculated: c-50, 3, H-3.97; N-17.26; c.g. ]

Found; €-50.16; H-4.03, N-17.13; c1-8.66 EE

AT 2/8F/sp/q -

Example 27 lo, | ~ DX/85 : 1-(3-Azido-2, 3-deoxy-8-D-threo-pentofuranosyl)-2-Ethoxy-5-Nethyl- 4-( 1H) -Pyrimidinone ; threo-3'-Azido-5'-0-mesylthymidine (1.08 g, 3.13 mMol) [prepared according : to a standard method (1) from threo-3'-azidothymidine] was dissolved in © © 100 mL EtOH and treated with NaHCO3 (0.26 g, 3.13 mMol) at reflux for 18 nours. The reaction was cooled and filtered. The solvents were removed in vacuo and the residue placed on a silica gel column followed by elution with 9:1 (v/v) CHC13/MeOH. Combination of appropriate fractions and removal of solvents in vacuo yielded 0.7 g (2.4 mMol, 75.7%). mp = 120-122°C; UV (nm): at pH 1 Agax = 260 (e = 9300), Amin = 237 (e = 5500), A shoulder = 221 . (e = 7500); at pH 13 Agax = 256 (e = 10000), Amin = 240 (e = 7700); H! NMR (DMSO-dg) 67.58 (s,1H,H6), 6.0 (dd,1H,H1", J = 2.9, 4.56 Hz), 5.06 (t, H,5'0H, J = 4.91 Hz), 4.51-4.47 (m,1H,H3'), 4.34 (q,2H,-0CH2-,

J = 7.14 Hz), 4.10-4.05 (m,1H,H4'), 3.73 (t,2H,H5', J = 5.62 Hz), 2.82-2.73 (m, 1H,H2'b), 2.21-2.14 (m,1H,H2'a), 1.82 (s,3H,5CH3), 1.31 (t,3H,-CHp-CHg,

J = 6.65 Hz). Analysis calculated for CqoH17N50y: C 48.81, H 5.80, N 23.72

Found: c 48.59, H 5.86, N 23.64. 1) J. Horwitz et al. J. org. Chem. (1964), 29, 2076.

PAT 2/11/AE/sp/10 ire0-3"-421do-3" “Deoxy-4-Thiothymiq ine Eo SH ree n3 ha do-S 0 brityl he (1,2, bt az01e) thymine (1.25 g, \ 2.2 mMol) (1) was dissolved in 109 ml acetone ang 30 mL Hy0 then treated with 0.22 g NaSH. xH,0, The mixture was stirred 3 hours. The volume was Lo reduced by half ang extracted with 309 mL CHCl13. The CHC13 was washed with \ - 50 mL H20, dried over Napsoy, then removed in vacuo to yield an oj], The \ 5'-0-trityl group was removed by dissolving this oil in 100 mL 80% HOAc. \

The solution Was heated on a Steam bath for 2 hours then cooled, diluted \

With 100 mL H,0 and filtered. The solvents Were removed in vacuo ang the ye oil placed on a silica gel column. Elution with 20:1 (v/v) CHC13/MeOH, Lo collection of appropriate fractions followed by removal of Solvents in vacuo Vv yielded 0.18 g (0.62 mMol; 28%). mp = 65-67°C;: yy (nm): at pH 1 Amax = 337 L (e = 20700), api, = 280 (c - 1200), A shoulder = 238 (¢ - 3400); at pH 13 \

Amax = 320 (e¢ = 18400), Amin = 257 (¢ = 1700); H' NMR (DMSO-dg) 87.63 \ (s,1H,H6), 5.97 (dd, 1H, H1", J - 2.93, 4.89 Hz), 5.06 (s,H,5'0H), 4.50-4_ 46 ] (m, 1H,H3"), 4.10-4. 04 (m, 1H,HY4") 3.73 (d,2H,Hs5" J = 5,61 Hz), 2.78-2.68 (m, 1H,H2'b), 2.20-2.14 (m, 1H,H2'a), 2.00 (s,3H,5CH3), Analysis calculated 4 for C10H13N503s 0.1 CoHgO 0.25 Hp0: C 41.90, H 4.86, N 23.95, s 10.97 a

Found: 41.99, 4 4.73, N 23.88, 5 10.91. i 1) W. Sung Nucleic Acid$ Research (1981), 9, 6139. i b

PAT 2/11/48F/sp/11 . Pe !

ree tt t——————— ry

Ty 1. A’ compound of the formula: : - . R® 2 . 4 : oo R™. ’ R

SN TY :

Pt ;

N ! (ID)

RY N ~~ : pl

HO — O J > | wid

N2 wherein rR! is mercapto or C;_, alkoxy; Co]

R? is hydrogen, C;_4 alkyl, C,.4 alkanoyl, benzoyl, acetyl or sulphonate; : : | :

EET mercapto or amino; Co rR is hydrogen, Cia alkyl, Cia alkoxy, halo or : Co . trifluoromethyl; : oo oT a pharmaceutically acceptable salt or ester thereof.