GB2459779A - Process for the preparation of 2'-cyano-2'-deoxy-N4-palmitoyl-1-beta-D-arabinofuranosylcytosine - Google Patents

Abstract

The present invention relates to a process for preparing a compound of formula 682-3', said process comprising the steps of converting a compound of formula 682-1 into a compound of formula 682-2'; and converting said compound of formula 682-2' into a compound of formula 682-3'. 682-2' may be prepared by reaction of triol 682-1 with 1,3-dichloro-1,1,4,4-tetraisopropyldisiloxane (CIPS) in pyridine. Amide 682-3' may then be prepared using palmitic anhydride in DMF. Further aspects of the invention relate to the use of the above process in the preparation of 2'-cyano-2'-deoxy-N 4 -palmitoyl-l-beta-D-arabinofuranosylcytosine (682), a pyrimidine nucleoside which is therapeutically useful in the treatment and/or prevention of cancer. In particular, the synthesis of (682) may proceed via TEMPO oxidation of the 2'-OH of 682-3', followed by treatment with TMSCN and AlCl 3 or acetone cyanohydrins and triethylamine to give cyanoalcohol (682-5'), which may then be treated with ethyl dithiochloroformate in the presence of triethylamine and dimethylaminopyridine (DMAP) to afford dithiocarbonate ester (682-6"). Treatment of (682-6") with lauroyl peroxide and collidine followed by deprotection results in the formation of (682). 682-6" is also claimed per se.

Description

PROCESS

The present invention relates to the preparation of intermediates useful in the synthesis of T-cyano-T-deoxy-N4-palmitoyl-1 -J-D-arabinofuranosylcytosine, a s pyrimidine nucleoside therapeutically useful in the treatment and/or prevention of cancer. Specifically, the invention provides an improved process for the preparation of 2'-cyano-2'-deoxy-N4-palmitoyl-1 --D-arabinofuranosylcytosine.

BACKGROUND TO THE INVENTION

The therapeutic use of pyrimidine nucleosides in the treatment of proliferative disorders has been well documented in the art. By way of example, commercially available antitumor agents of the pyrimidine series include 5-fluorouracil (Duschinsky, R., et a!., J. Am. Chem. Soc., 79, 4559 (1957)), Tegafur (Hiller, SA., et a!., Doki.

Akad. Nauk USSR, 176, 332 (1967)), UFT (Fujii, S., et al., Gann, 69, 763 (1978)), Carmofur (Hoshi, A., et al., Gann, 67, 725 (1976)), Doxyfluridine (Cook, A. F., et a!., J. Med. Chem., 22, 1330 (1979)), Cytarabine (Evance, J. S., eta!., Proc. Soc. Exp. Bio. Med., 106. 350 (1961)), Ancytabine (Hoshi, A., et a!., Gann, 63, 353, (1972)) and Enocytabine (Aoshima, M., et a!., Cancer Res., 36, 2726 (1976)).

EP 536936 (Sankyo Company Limited) discloses various 2'-cyano-2'-deoxy-derivatives of 1 -3-D-arabinofuranosylcytosine which have been shown to exhibit valuable anti-tumour activity. One particular compound disclosed in EP 536936 is 2'-cyano-2'-deoxy-N4-palmitoyl-1 -3-D-arabinofuranosylcytosine (referred to hereinafter as "682" or "CYC682"); this compound is currently under further investigation.

CYC682, also known as 1 -(2-C-cyano-2-dioxy-13-D-arabino-pentofuranosyl)-N4-palmitoyl cytosine, (Hanaoka, K., et a!, mt. J. Cancer, 1999:82:226-236; Donehower R, et a!, Proc Am Soc Gun Oncol, 2000: abstract 764; Burch, PA, et a!, Proc Am Soc Gun Oncol, 2001: abstract 364), is an orally administered novel 2'-deoxycytidine antimetabolite prodrug of the nucleoside CNDAC, 1 -(2-C-Cyano-2-deoxy-3-D-arabino-pentafuranosyl)-cytosine. Fl2

C,5H3, H[''" CYC682 CNDAC CYC682 has a unique mode of action over other nucleoside metabolites such as gemcitabine in that it has a spontaneous DNA strand breaking action, resulting in potent anti-tumour activity in a variety of cell lines, xenograft and metastatic cancer model.

CYC682 has been the focus of a number of studies in view of its oral bioavailability and its improved activity over gemcitabine (the leading marketed nucleoside analogue) and 5-FU (a widely-used antimetabolite drug) based on preclinical data in solid tumours. Recently, investigators reported that CYC682 exhibited strong anticancer activity in a model of colon cancer. In the same model, CYC682 was found to be superior to either gemcitabine or 5-FU in terms of increasing survival and also preventing the spread of colon cancer metastases to the liver (Wu M, et al, Cancer Research, 2003:63:2477-2482). To date, phase I data from patients with a variety of cancers suggest that CYC682 is well tolerated in humans, with myelosuppression as the dose limiting toxicity.

More recent studies have focussed on different crystalline forms of CYC682 (see for example, WO 02/064609 in the name of Sankyo Company Limited) and optimised formulations containing CYC682 which exhibit improved stability and which allow easier processing (see for example, WO 07/072061 in the name of Cyclacel Limited).

The preparation of CYC682 described in EP 536936 (see Scheme 1 below) involves reacting cytidine [1] with palmitic anhydride in DMF to form N4-palmitoylcytidine [2] and subsequently protecting with 1,3-dichloro-1,1,4,4-tetraisopropyldisiloxane (CIPS) to form intermediate [3]. Oxidation of [3] with pyridinium dichromate/acetic anhydride in dichioromethane produces intermediate ketone [4], which is then reacted with sodium cyanide and sodium dihydrogen phosphate dihydrate in ethyl acetate to S form the cyanohydrin [5]. Intermediate [5] is then reacted with N,N-dimethylaminopyridine, phenoxythiocarbonyl chloride and triethylamine to form intermediate [6], which is subsequently reacted with AIBN and tributyltin hydride in toluene to give intermediate [7]. Deprotection of [7] with acetic acid and tetrabutylammonium fluoride in THF yields the desired product, CYC682.

:de LN2PaIm HO OH 0 DMF HO OH 0 Py OH Ac,O [1] (2] [3] __2(H,O) 0rm CS)CI EtOAc DCM [4] [5] [6) Bu,SnH IBAF Toluene THF Hd \

TN 7 N

[7] 682 Scheme 1: Preparation of CYC682 as described in EP 536936 Further modifications to the above described route have been disclosed in JP 07053586 (Sankyo Company Limited). In particular, JP 07053586 discloses that the oxidation step can be achieved using 2,2,6,6-tetramethyl piperidinyloxy free radical (TEMPO), NaClO and an alkali metal halide (see conversion of [3a] to [4a] in Scheme 2 below). Furthermore, conversion of ketone [4a] to cyanohydrin intermediate [5a] can be achieved by treating [4a] with acetone cyanohydrin instead of NaCN. The resulting cyanohydrin [5a] can then be treated with 2-naphthylchlorothioformate to give intermediate [6a].

cyanohydnn .srO OH NaOCI sr0 0 0S1 HO "N I DCM I / DCM [3a] [4a] (5a1

H

Si \-.1 "17 ° NTFCI.

EMAAP 16a]

Scheme 2: Alternative conditions described in JP 07053586 However, in spite of these modifications, the above described routes are associated with relatively poor yields andlor a high level of variability, thereby highlighting the need for improved synthetic strategies.

The present invention thus seeks to provide an improved process for preparing CYC682. More specifically, the invention seeks to provide a synthetic route which gives rise to improved yields and/or easier purification of CYC682 and precursors thereof and/or which is suitable for the large scale preparation of this compound.

STATEMENT OF INVENTION

A first aspect of the invention relates to a process for preparing a compound of formula 682-3', said process comprising the steps of °:? bH N

HO OH

682-1 682-2' 682-3' (i) converting a compound of formula 682-1 into a compound of formula 682-2'; and (ii) converting said compound of formula 682-2' into a compound of formula 682-3'.

A second aspect of the invention relates to a process for preparing a compound of formula 682, 3Palm sr00H Nd 682-3' 682 said process comprising converting a compound of formula 682-3' to a compound of formula 682.

is Advantageously, incorporating the palmitoyl group in step (ii), i.e. at an early state in the process, leads to a reduction in the total number of steps in the synthesis, thereby leading to improved yields overall.

A third aspect of the invention relates to a compound of the formula: I 0Si0 \ ) 682-6" and use thereof as an intermediate in the preparation of 682-9 or 682.

Another aspect of the invention relates to a process for preparing a compound of formula 682, said process comprising converting a compound of 682-6" to a compound of formula 682.

DETAILED DESCRIPTION

As mentioned above, a first aspect of the invention relates to a process for preparing a compound of formula 682-3', said process comprising the steps of 10dbH0

HOOH

682-1 682-2' 682-3' (i) converting a compound of formula 682-1 into a compound of formula 682-2'; and (ii) converting said compound of formula 682-2' into a compound of formula 682-3.

In one preferred embodiment, step (i) comprises treating said compound of formula 682-1 with I,3-dichloro-1,1,4,4-tetraisopropyldisiloxane (CIPS) in pyridine. Further details of this reaction are reported in Org. Process Dev., 4, 172 (2000); US Pat 6,531,584 Bi (2003); Org. Let!., , 55 (2006).

Advantageously, incorporating the CIPS protecting group first in step (i) yields a solid product, 682-2', which can be more easily purified (for example, by washing) to remove unwanted by-products and any excess of the CIPS protecting group reagent.

Once purified, the solid 682-2' intermediate so produced is then reacted with palmitic anhydride to give intermediate 683-3', which is then oxidised to give intermediate 682-4'. The ability to purify 682-2' in solid form leads to better quality material for use in the subsequent steps of the process, leading to higher yields and improved reproducibility. More particularly, the above route leads to better quality ketone 682- 4' (which is the substrate for the subsequent cyanohydrin reaction in the synthesis of CYC682), and avoids the need for purification by chromatography.

In one preferred embodiment, step (ii) comprises treating said compound of formula 682-2' with palmitic anyhydride in DMF. Further details of this reaction are described in EP 536936 (Sankyo Company Limited). Other conditions for this conversion will be familiar to the skilled artisan.

Another aspect of the invention relates to a process for preparing a compound of to formula 682, Palm Palm TSOoH Hd 682-3' 682 said process comprising converting a compound of formula 682-3' to a compound of formula 682.

As mentioned above, incorporating the palmitoyl group in step (ii), i.e. at an early state in the process, leads to a reduction in the overall number of steps in the synthesis.

Preferably, said process comprises the steps of: (A) preparing a compound of formula 682-3' as described above; and (B) converting said compound of formula 682-3' to a compound of formula 682.

In one preferred embodiment, step (B) comprises the steps of: O(Ncb1m flPaIm (B2) (B3) S 682-4' 682-5' 682-6"

H

(B4) SI \\ 1N 682-7' (Bi) converting said compound of formula 682-3' to a compound of formula 682-4'; (B2) converting said compound of formula 682-4' to a compound of formula 682-5'; (B3) converting said compound of formula 682-5' to a compound of formula 682- (B4) converting said compound of formula 682-6" to a compound of formula 682-7'; and (B5) converting said compound of formula 682-7' to a compound of formula 682.

Oxidising agents for converting compound 682-3' to compound 682-4' in step (Bi) will be familiar to the skilled artisan. By way of example, the conversion can be achieved by Dess-Martin periodinane oxidation [analogous to methods described in Helv. Chim. Acta, 85, 224 (2002) & I Org. Chem., 55, 5186 (1990)], Swern oxidation [Org. Process Res. Dev., 4, 172 (2000) & J. Med. Chem., 48, 5504 (2005)], oxidation with pyridinium dichromate or with 2,2,6,6-tetramethyl piperidinyloxy free radical (TEMPO) and NaOC1.

In one particularly preferred embodiment, step (Bi) comprises treating said compound of formula 682-3' with 2,2,6,6-tetramethyl piperidinyloxy free radical (TEMPO) and NaOC1 in the presence of an alkali metal halide. Further details of this reaction are described in JP 07053586 (Sankyo Company Limited).

In one preferred embodiment, step (B2) comprises treating said compound of formula 682-4' with TMSCN and Aid3. Further details of this reaction are described in Tet, , 9197 (2004).

In another preferred embodiment, step (B2) comprises treating said compound of formula 682-4' with acetone cyanohydrin and NEt3 in heptane. Advantageously, the use of acetone cyanohydrin and NEt3 in heptane leads to the improved yield and easier purification of intermediate 682-5 compared to reaction conditions previously known in the art.

In one preferred embodiment, step (B3) comprises treating said compound of formula 682-5' with ethyl dithiochloroformate in the presence of NEt3 and dimethylaminopyridine. Ethyl dithiochioroformate may be prepared by the method disclosed mi. Org. Chem., 26, 4047 (1961).

Advantageously, the use of ethyl dithiochloroformate in step (B3) leads to improved yields of intermediate 682-6". Prior art procedures for this step have typically involved the use of bulky aryl agents such as 2-naphthylchlorothioformate or phenoxythiocarbonyl chloride which react with only one out of the two cyanohydrin isomers, thereby leading to only modest yields of the desired product. Without wishing to be bound by theory, it is believed that the less bulky dithiocarbonylation agent ethyl dithiochloroformate is capable of reacting with both cyanohydrin isomers, thereby leading to an increased yield of the dithiocarbonylated product (in this case intermediate 682-6").

Another advantage of the claimed process is that the 682-6" so formed can be used directly in the next step (B4) without the need for further costly and time consuming purification, for example, by chromatography.

In one preferred embodiment, step (B4) comprises treating said compound of formula 682-6" with lauroyl peroxide and collidine. Further details of this reaction may be found in Tett. Left, 37, 5877 (1996) and Tett. Left, 39, 9435 (1998). The use of lauroyl peroxide and collidine is particularly favoured for economic reasons in view of its low cost. Advantageously, this conversion gives rise to 682-7' in the form of a colourless oil which solidifies on standing and requires no further purification, for example, by chromatography. Accordingly, this renders the process ideal for the large scale preparation of 682 and intermediates thereof Alternatively, the conversion of 682-6" to 682-7 may be achieved by treating said compound of formula 682-6 with tris(trimethylsilyl)silane (TTMSS) and azobisisobutyronitrile (AIBN) in toluene.

In one preferred embodiment, step (B5) comprises treating said compound of formula 682-7' with tetrabutyl ammonium fluoride (TBAF) and acetic acid. Further details of this reaction are described in I Med. Chem., 34, 2917 (1991), Ibid, 36, 4183 (1993).

The resulting 682 is preferably purified by recrystallization using solvent systems and methods familiar to the skilled person. Preferably, the 682 is recrystallized from a mixture of ethyl cyclohexane, 1 -4-dioxane and methanol. More preferably, the 682 is recrystallized from a mixture of ethyl cyclohexane, 1 -4-dioxane and methanol in a ratio of 15:2:1.

A third aspect of the invention relates to a compound of the formula: _,oNçr-1m °Si° \O 25) 682-6" and use thereof as an intermediate in the preparation of 682-9 or 682.

Another aspect of the invention relates to a process for preparing a compound of formula 682, said process comprising converting a compound of 682-6" to a compound of formula 682.

Preferably, said process comprises converting said compound of 682-6" to a compound of formula 682-7', and then converting said compound of formula 682-7' to a compound of formula 682.

The present invention is further described by way of non-limiting examples, and with reference to the following figures, wherein; Figure 1 shows synthesis of CYC682 via Route 1, a modification of the prior art procedure.

Figure 2 shows synthesis of CYC682 via Route 3, in accordance with a preferred embodiment of the invention.

EXAMPLES

CYC682-2'---* CYC682-3'

PALM

NH2 HN O(PALM)2 TIPDS\f 91% TIPDS/ 3,5 -O-( 1,1,3,3-Tetraisopropyl-1,3-disiloxanediyl)cytidine (240mg, 0.494mmo1) was dissolved in dry DMF (2m1) and palmitic anhydride (269mg, 1.leq) added. The mixture was heated (oil bath 100°C) for 6h. LCMS showed product with only trace of starting material remaining. The mixture was cooled to room temperature and the DMF evaporated on RV to give a clear oil which solidified on cooling. The crude material was dissolved in petroleum ether (lOOm! -containing 2% NEt3) and filtered through a Si02 (lOg) plug. The silica was washed successively with 1:1 petroleum ether-EtOAc (lOOmi) and EtOAc (lOOmi). TLC showed the product was present in the 1:1 petroleum ether-EtOAc fraction. Evaporation of the solvent gave the desired product as a colourless oil (91% yield). See NMRILCMS 579-42-2 (MW -MT-I 724).

CYC682-3' -* CYC682-4'

HNAL HNALM

TI

O OH o 0 682-3' (564mg, O.78mmol) was dissolved in DCM (5m1) and cooled to 5°C in an ice-bath. Dess-Martin periodinane (677mg, 2.O5eq.) was added in small portions. The resulting cloudy solution was stirred at room temperature overnight. The mixture was diluted with DCM (20m1) and washed with aq NaHCO3 in which Na2S2O3.5H20 (23.4g) had been dissolved. The aqueous phase was extracted with DCM (70m1). The combined organics were washed (sat NaHCO3, followed by brine), dried (MgSO4) filtered and evaporated to give a colourless oil (84% yield). See NMRILCMS 579-44- 1 (MW -M1-I 722).

CYC682-4' -* CYC682-5' HN HN'

N NCOH N

TIPDS \ / q* TIPDS Q 0 OOH 682-4' (250mg, 0.35mmol) was partially dissolved in heptane (5m1) to give a hazy solution. Acetone cyanohydrin (63u1, 2eq) was added in a steady stream, followed by addition of triethylamine (5ii, lOmol%) and the mixture stirred at room temperature.

LCMS after lh showed some starting material remained. Another portion of acetone cyanohydrin (63ul, 2eq) was added and the mixture stirred for a further lh. LCMS showed no starting material. The solvent was evaporated to give a colourless semi- solid (quantitative crude yield). See LCMS 5 79-45 (MW -MH 749) & NMR 579-70cropA.

CYC682-5' -* CYC682-6" HNLM s HNLM N CISEt TI! °° OH s SEt 682-5' (crude, 0.3Smmol) and DMAP (9mg, 0.2eq.) were dissolved in DCM (Sm!) in a dry flask and the vessel purged with argon. The yellowish solution was cooled in an ice-bath. Ethyl dithiochloroformate 1 (54mg, 1.1 eq.) was dissolved in DCM (1 ml) and triethylamine (6lul, l.25eq) added. The reaction mixture darkened to a brown

F

solution on addition of triethylamine. The mixture was stirred lh with cooling, allowed to warm to ii, then stirred for a further 2h. Water (lOml) was added and the organic phase separated. The organic phase was extracted with DCM (1 Omi). The combined organics were washed (brine), dried (MgSO4), filtered and evaporated to a yellow oil. The resulting oil was used in the next step without further purification. See LCMS 579-47a1 (MW -MH 853) & NMR 579-69Et.

CYC682-6" -� CYC682-T

HN HN

[j Lauroyl ("& ° TIPDS \ \/ 68% TIPDS

S SEt

682-6" (229mg, 0.269mmol) was suspended in IPA and 2,4,6-collidine (49mg/54u1, 1.5eq) added. The mixture was heated to 100°C. Lauroyl peroxide (80mg, 0.75eq.) was added when the heating bath was at 7O°C. The mixture was heated for lh.

Another portion of lauroyl peroxide (80mg, 0.75eq.) was added and the mixture heated for 1.5h. On cooling the solvent was evaporated and the crude product dissolved in petroleum ether (lOOmi -containing 2% NEt3) and filtered through a Si02 (lOg) plug. The silica was washed successively with 1:1 petroleum ether-EtOAc (lOOml) and EtOAc (lOOmi). TLC showed the product was present in the 1:1 petroleum ether-EtOAc fraction. Evaporation of the organic solvent gave the desired product as a colourless oil which solidified on standing (62% yield from CYC682-5').

See NMRJLCMS 579-48-3 (MW -MH 733).

CYC682-7' -* CYC682 HN HN/AM 0 TBAF HO ( 0 AcOH N

TIPDS 8O/

OH

CYC682-7' (25mg, 0.O34mmol) was dissolved in dry-THF (2m1) and AcOH (2mg, leq.) added. A THF solution of TBAF (1M, O.068m1, 2eq.) was added and the mixture stirred at room temperature for 1 5mm. The solvent was removed in vacuo and the crude product suspended in MeOH (2m1) and placed in a refrigerator overnight.

The resulting precipitate was collected by filtration and was characterised as the desired product (85% yield). See NMRILCMS 579-51-1 (MW MH 491). NMR identical to that of CYC682 provided by Sankyo (batch AM7O1). A sample of this material was recrystallised from ethylcyclohexane, 1,4-dioxane, MeOH (15:2:1).

CYC682 is initially isolated as Form K which is a methanol solvate. Form K is converted to Form B which is a hemihydrate by a suspension form change reaction.

Form K or Form B can be further purified by recrystallisation. The recrystallisation yields Form K which is then converted, or reconverted to Form B. (i) 682: Form K Palmitic anhydride (3.53kg/kg 682-9) is added to a mixture of 682-9 in 1,4-dioxane (20L/kg 682-9) and low pyrogen water (1.OL/kg 682-9) and the reaction mixture is heated to 800 to 90°C (target range 80° to 85°C). The reaction is monitored by HPLC and continued until the 682-9 content is 52.0%. At the completion of the reaction, the mixture is hot filtered and the filter washed with I,4-dioxane (1 OL/kg 682-9) at 70° to 90°C. The resultant combined filtrate is concentrated to less than 30% of its original volume (7.3L/kg 682-9) at or below 60°C (target internal temperature 45°C to 5 5°C, or less). The water content is checked by Karl Fischer titration. If the water content is <2%, additional dioxane is added and the distillation repeated. If required, 1,4-dioxane is added to dilute the mixture to 30% of the original volume.

Ethylcyclohexane (48.3L1/kg 682-9) and 1,4-dioxane (3.66L/kg 682-9) are added and the temperature adjusted into the range 430 to 47°C. Methanol (3.23L/kg 682-9) is added at 40° to 45°C over at least 5 minutes.

In a separate reactor CYC682 seed crystals (Form B) (lOg/kg 682-9) are added to a mixture of ethylcyclohexane (1 333mL/kg 682-9), 1,4-dioxane (1 77mL/kg 682-9) and methanol (89mLIkg 682-9) (15:2:1 v/v/v). The resultant mixture is stirred at 20° to 25°C for at least 1 hour, then added to the crude reaction solution at 40° to 45°C.

After crystallisation of the Form K occurs, the reaction mixture is stirred at 40° to 45°C for at least a further 30 minutes. The reaction mixture is cooled to 20° to 23°C over at least 120 minutes, and held in the range 20° to 23°C for at least 1 hour. The resultant solid is isolated by centrifugation in up to two loads and each load washed with a mixture of ethylcyclohexane (7.5L/kg 682-9), 1,4-dioxane (1.OL/kg 682-9) and methanol (0.5Ukg 682-9) at 0° to 5°C. The product is dried under vacuum at 35° to 40°C, to constant weight to yield CYC682 (Form K).

(ii) 682: Form B CYC682 (Form K) is suspended in methyl acetate (8.9L/kg CYC682) containing approximately 1.5 to 2% low pyrogen water (1 69.3mUkg CYC682). The suspension is stirred at 20° to 25°C (target 22° to 24°C) for 1.5 hours and undergoes form conversion. The product is isolated by Nutsehe filtration and washed with a mixture of methyl acetate (2.2L/kg CYC682) and low pyrogen water (42.3mUkg CYC682) 20° to 25°C. The product is dried under vacuum at or below 40°C, to constant weight, to yield CYC682 (Form B).

Recrystallisation of CYC682 (Form K or B) CYC682 (Form K or B) is suspended in a mixture of 1,4-dioxane (3.33L/kg CYC6S2) and ethylcyclohexane (25L/kg CYC682) and the mixture adjusted into the range 43° to 47°C. Methanol (l.66L/kg CYC682) is added at 40° to 50°C over at least 5 minutes to achieve dissolution. Additional heating up to 60°C may be required to achieve dissolution of CYC682 Form B. In a separate reactor CYC682 seed crystals (4 to 15g/kg CYC682) are added to a mixture of ethylcyclohexane, 1,4-dioxane and methanol (15:2:1 v/v/v) as in Section 9 above. The resultant mixture is stirred at 200 to 25°C for at least 1 hour, then added to the crude reaction solution at 40° to 45°C. After crystallisation of the Form K occurs, the reaction mixture is stirred at 40° to 45°C for at least a further 30 minutes. The reaction mixture is cooled to 20° to 23°C over at least 120 minutes, and held in the range 20° to 23°C for at least 1 hour. The resultant solid is isolated by centrifugation in up to two loads and each load washed with a mixture of ethylcyclohexane (3.852L/kg CYC682), 1,4-dioxane (0.514L/kg CYC682) and methanol (257mL/kg CYC682) at 0° to 5°C. The product is dried under vacuum at 35° to 40°C, to constant weight to yield CYC682 (Form K).

Comparative Studies Studies by the Applicant have shown that the process steps as presently claimed lead to improved yields over methodology previously used in the art. By way of example, Table 1 below compares the yields for each step in Route 1 (see Figure 1; prior art methodology) and Route 3 (see Figure 2; in accordance with the invention).

TabLe 1: Comparison of yields for Route I and Route 3 -.4 -* -+ -* -� -� -.4 -3. -5 -� Total 2 3 4 5 6 7 8 9 K B ________ _____ ____ _____ ____ _____ ____ _____ ____ form form ______ Route 1 98 38 90 91 85 97 89 89 19.9 Route 3 86 88 90 100 77 68 90 32.1 Table 1 shows that incorporating the CIPS protecting group and then the palmitoyl group, followed by cyanation using TMSCN/A1C13 (Route 3) gives rise to intermediate 682-5 in high yield. By way of comparison, protecting the cytidine -NH2 with an acyl group prior to incorporating the CIPS protecting group (Route 1), and using standard cyanation conditions (e.g. NaCN, NaHCO3 in H2OIEtOAC) leads to a significantly lower yield of intermediate 682-5. Moreover, introducing the palmitoyl group early on in the synthesis, rather than in the final step, reduces the overall number of steps by two, which is of considerable economic value. The reduction in steps also has a significant impact on the final yield; overall, a comparison of the two routes gives 19.9% CYC682 for Route 1, compared to 32.1 /o CYC682 for Route 3.

Various modifications and variations of the described aspects of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims (15)

1. CLAIMS1. A process for preparing a compound of formula 682-3', said process comprising the steps of Lr oçj4Q2 682-1 682-2' 682-3' (i) converting a compound of formula 682-1 into a compound of formula 682-2'; and (ii) converting said compound of formula 682-2' into a compound of formula 682-3'.
2. 2. A process according to claim 1 wherein step (i) comprises treating said compound of formula 682-1 with 1,3-dichloro-1,1,4,4-tetraisopropyldisiloxane (CIPS) in pyridine.
3. 3. A process according to claim 1 or claim 2 wherein step (ii) comprises treating said compound of formula 6 82-2 with palmitic anyhydride in DMF.
4. 4. A process for preparing a compound of formula 682, Ny Palm Si0H Hd 682-3' 682 said process comprising converting a compound of formula 682-3' to a compound of formula 682.
5. 5. A process according to claim 4 which comprises the steps of: (A) preparing a compound of formula 682-3' according to any one of claims 1 to 3; and (B) converting said compound of formula 682-3' to a compound of formula 682.
6. 6. A process according to claim 5 wherein step (B) comprises the steps of: 0o: rPam (B2) (B3) S 682-4' 682-5' 682-6"R682-7' (Bi) converting said compound of formula 682-3' to a compound of formula 682-4'; (B2) converting said compound of formula 682-4' to a compound of formula 682-5'; (B3) converting said compound of formula 682-5' to a compound of formula 682- (B4) converting said compound of formula 682-6" to a compound of formula 682-7'; and (B5) converting said compound of formula 682-7' to a compound of formula 682.
7. 7. A process according to claim 6 wherein step (Bi) comprises treating said compound of formula 682-3' with 2,2,6,6-tetramethyl piperidinyloxy free radical (TEMPO) and NaOCI.
8. 8. A process according to claim 6 or claim 7 wherein step (B2) comprises treating said compound of formula 682-4' with TMSCN and Aid3.
9. 9. A process according to claim 6 or claim 7 wherein step (B2) comprises treating said compound of formula 682-4' with acetone cyanohydrin and NEt3 in heptane.
10. 10. A process according to any one of claims 6 to 9 wherein step (B3) comprises treating said compound of formula 682-5' with ethyl dithiochioroformate in the presence of NEt3 and dimethylaminopyridine.
11. 11. A process according to any one of claims 6 to 10 wherein step (B4) comprises treating said compound of formula 682-6" with lauroyl peroxide and collidine.
12. 12. A process according to any one of claims 6 to 11 wherein step (B5) comprises treating said compound of formula 682-7' with tetrabutyl ammonium fluoride (TBAF) and acetic acid.
13. 13. A compound of the formula: o ) 682-6"
14. 14. Use of a compound as defined in claim 13 as an intermediate in the preparation of 682-9 or 682 HO 7'_C15H31 HO(Lç NN N682-9 682
15. 15. A process for preparing a compound of formula 682, said process comprising converting a compound of 682-6" to a compound of formula 682.