AU659008B2 - Stereoselective anion glycosylation process - Google Patents

Description

X-7773 -1- STEREOSELECTIVE ANION GLYCOSYLATION PROCESS The invention relates to a stereoselective anion glycosylation process for preparing 2'-deoxyfluoro- P -nucleosides.

The continued interest in the synthesis of 2'deoxyfluoronucleosides and their analogues is predicated on their successful use as therapeutic agents for treating viral and cancerous diseases. A compound of particular interest is gemcitabine; see European Patent Specification No. 211354 and U.S. Patent No. 4,526,988. Since these compounds are nucleosides, there is a need to provide such compounds in high yield.

A critical step in the synthesis of 2'deoxyfluoronucleosides is the condensation or glycosylation of the nucleobase and carbohydrate to form a N-glycoside bond. However, processes'for synthesis of 2'deoxynucleosides are typically non-stereoselective forming mixtures of a and p nucleosides. For instance, U.S. Patent .20 4,526,988 did not stereoselectively produce 2-deoxy-2,2difluoro-P -nucleosides but instead produced a 4:1 a to P anomer ratio of 2-deoxy-2,2-difluoronucleoside. Even optimizing the protecting groups could not increase the a to ratio beyond 1:1; see U.S. Patent No. 4,965,374 which utilized benzoyl protecting groups.

According to the present invention there is provided a stereoselective anion glycosylation process for preparing a P anomer enriched nucleoside of the formula HO T X-7773 -2wherein T is selected from fluoro, and hydrogen and R is a nucleobase selected from the group consisting of OH OH N KRI

N

2 HNi N

NH

2

NH

2

N

N RIN O N N N NH2

OH

NCH=CHR3 N CH=CHR 3 I II N 0 N

NH

2

N

0 N HO NN X-7773 R2 0 N O

R

6 R2 N N R4 N

Q

N N

R

2 NS R,

N^J

O N R7 N and

N

I

C

I

wherein R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl and halo; R 2 is selected from the group consisting of hydroxy, halo, cyano, azido, primary amino and secondary amino; R 3 is selected from the group consisting of hydrogen, alkyl, substituted alkyl and halo;

R

4

R

5 and Rg are independently selected from the group consisting of hydrogen, -OH, -NH2, N(alkyl), halo, cyano, .0 azido, alkoxy and thioalkyl; R 7 is selected from the group consisting of hydrogen, halo, cyano, alkyl, alkoxy, alkoxycarbonyl, thioalkyl, thiocarboxamide and carboxamide; Q is selected from the group consisting of CH, CR 8 and N; wherein R 8 is halo, carboxamide, thiocarboxamide, .5 alkoxycarbonyl and nitrile; comprising reacting an a anomer enriched fluorocarbohydrate of the formula X o H

F

H

(II);

XO T X-7 773 -4wherein T is as defined above; X is a hydroxy protecting group; and Y is selected from iodo and bromo; with at least a molar equivalent of a nucleobase salt selected from the group consisting of Oz oz

R

1

N'N

N WHNN

N

NHW NHW N N jRi N N- N NH-W OZ N: CH=CH- N CH=CHR, S.**NHW

OZ

NO

0ZO N N

SD

X-7773

R

2

R,

NR N RI RR6 R4 A-IR6 and Rs N N wherein R 1 through R 7 and Q are as defined above; Z is a hydroxy protecting group; W is an amino protecting group; and M is a cation; in an inert solvent; and deblocking to form a compound of formula Throughout this document, all temperatures are in degrees Celsius, all proportions, percentages and the like, are in weight units and all mixtures are in volume units, except where otherwise indicated. Anomeric mixtures are .expressed as a weight/weight ratio or as a perrent.

The term "xylenes" alone or in combination refers to all isomers of xylene and mixtures thereof. The term "lactol" alone or in combination refers to a 2-deoxy-2,2-difluoro-D- 15 ribofuranose or 2-deoxy-2-fluoro-D-ribofuranose. The term "carbohydrate" alone or in combination refers to a lactol wherein the hydroxy group at the C-l position has been replaced by a desirable leaving group. The term "halo" alone or in combination refers to chloro, iodo, fluoro and bromo.

The term "alkyl" alone or in combination refers to straight, cyclic and branched chain aliphatic hydrocarbon groups which contain up to 7 carbon atoms and more preferably contain up to 4 carbon atoms such as, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, n-hexyl, 3- X-7773 -6methylpentyl groups and the like or substituted straight and branched chain aliphatic hydrocarbons such as chloroethyl, 1,2-dichloroethyl and the like. The term "alkoxy" alone or in combination refers to the general formula AO; wherein A is alkyl. The term "aryl" alone or in combination refers to carbocyclic or heterocyclic groups such as phenyl, naphthyl, thienyl and substituted derivatives thereof. The term "thioalkyl" alone or in combination refers to the general formula BS; wherein B is alkyl or hydrogen. The term "ester" alone or in combination refers to the general formula EOOC; wherein E is alkyl or aryl. The term "aromatic" alone or in combination refers to benzene like structures containing (4n+2) t delocalized electrons. The terms "sulfonate" or "sulfonyloxy" alone or in combination refer to the general formula GSO 3 wherein G is alkyl, substituted alkyl, aryl or substituted aryl. The term "substituted" alone or in combination refers to substitution by at least one or more of the groups selected from cyano, halo, carboalkoxy, toluoyl, nitro, alkoxy, hydroxy and dialkylamino. The phrase "anomer enriched" alone or in combination refers to an anomeric Smixture wherein the ratio of a specified anomer is greater than 1:1 and includes a substantially pure anomer.

S" According to the present anion glycosylation Sprocess, b anomer enriched 2'-deoxy-2',2'-difluoronucleosides 25 and 2'-deoxy-2'-fluoronucleosides of formula are prepared by reacting an a anomer enriched carbohydrate of formula (II) with at least a molar equivalent of a nucleobase salt, in an Sinert solvent as shown by the following reaction scheme:

HO

o0 H 0 R R Solvent Deblock H 1 .H R' IN 30 H XO T HO T (II)

(I)

X-7773 -7wherein X, T, Y, R' and R are as defined above.

The glycosylation reaction proceeds via SN2 displacement. Therefore, 3 anomer enriched nucleosides of the present invention are derived from a anomer enriched carbohydrates.

The lactol starting materials suitable for use in the present anion glycosylation process are commonly known in the art and can be readily synthesized by standard procedures commonly employed by those of ordinary skill in the art. For example, U.S. Patent 4,526,988 teaches the synthesis of 2,2difluoro-2-deoxy-D-ribofuranoses having the formula

XO

OH

H F(III); XO F wherein X is a hydroxy protecting group. In addition, Reichman, et al., Carbohvdr. Res., 42, 233 (1975) teaches the synthesis of 2-deoxy-2-fluoro-D-ribofuranoses of the formula

X•

X,

I 0

OH

F f" (VI); 0*

*H

X• H S wherein X is a hydroxy protecting group. A preferred embodiment of the present invention employs 2-deoxy-2,2difluoro-D-ribofuranose-3,5-dibenzoate as the lactol starting *5 material.

Glycosylation reactions typically require protecting the hydroxy groups of the lactol of formulas (III) X-7773 -8and (IV) to prevent the hydroxy groups from reacting with the nucleobase derivative, or being decomposed in some manner.

Hydroxy protecting groups suitable for use in the present glycosylation process may be chosen from known protecting groups used in synthetic organic chemistry. The hydroxy protecting group selected is preferably capable of being efficiently placed on the lactol and easily removed therefrom once the glycosylation reaction is completed. Hydroxy protecting groups known in the art are described in Chapter 3 of Protective Groups in Organic Chemistry, McOmie Ed., Plenum Press, New York (1973), and Chapter 2 of Protective Groups in Organic Synthesis, Green, John, J. Wiley and Sons, New York (1981); preferred are ester forming groups such as formyl, acetyl, substituted acetyl, propionyl, butanoyl, pivalamido, 2-chloroacetyl, benzoyl, substituted benzoyl, phenoxycarbonyl, methoxyacetyl; carbonate derivatives such as phenoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, vinyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl and benzyloxycarbonyl; alkyl ether forming groups such as benzyl, diphenylmethyl, triphenylmethyl, t-butyl, methoxymethyl, tetrahydropyranyl, allyl, tetrahydrothienyl, 2-methoxyethoxy methyl; and silyl ether forming groups such as trialkylsilyl, trimethylsilyl, isopropyldialkylsilyl, alkyldiisopropylsilyl, triisopropylsilyl, t-butyldialkylsilyl and 1,1,3,3tetraisopropyldisloxanyl; carbamates such as Nphenylcarbamate and N-imidazoylcarbamate; however more preferred are benzoyl, mono-substituted benzoyl and disubstituted benzoyl, acetyl, pivaloyl, triphenylmethyl ethers, and silyl ether forming groups, especially tbutyldimethylsilyl; while most preferred is benzoyl.

In attaching the hydroxy protecting groups to the lactol, typical reaction conditions are employed and depend on the nature of the protecting group chosen. Suitable Sreaction conditions are discussed in U.S. Patent 4,526,988, incorporated herein by reference.

To obtain an efficient reaction of the nucleobase X-7773 salt and carbohydrate, an appropriate leaving group is stereoselectively attached to the lactol at the C-1 position to activate the lactol and generate the a anomer enriched carbohydrate of formula Suitable leaving groups (Y) are selected from iodo and bromo; more preferred is iodo.

The preparation of the a anomer enriched carbohydrates of formula (II) is described in USSN 07/902,306 and requires contacting a hydroxy protected 2-deoxy-2,2difluro-D-ribofuranoysl-l-P-sulfonate with a halide source in an inert solvent to form a anomer enriched 2-deoxy-2,2difluoro-D-1-a-halo-ribofuranosyl.

The nucleobases employed herein are commonly known to organic chemist and no discussion of their synthesis is necessary. However, in order to be useful in the present glycosylation process the nucleobases or their tautomeric equivalents bearing amino or hydroxy groups preferably contain protecting groups such as amino protecting groups (W) and/or hydroxy protecting groups depending on the nature of the nucleobase derivative selected. The protecting group prevents the hydroxy or amino groups from providing a competing reaction site for the a anomer enriched carbohydrate of formula The protecting groups are attached to the nucleobase before it is reacted with the a anomer enriched carbuydrate of formula (II) and are removable subsequent thereto. A procedure for protecting nucleobases is described in U.S. Patent 4,526,988.

Preferred amino protecting'groups for Spyrimidine nucleobases are selected from the group consisting of silyl ether forming groups such as trialkylsilyl, tbutyldialkylsilyl and t-butyldiarylsilyl; carbamates such as t-butoxycarbonyl, benzyloxycarbonyl, 4methoxybenzyloxycarbonyl, and 4-nitrobenzyloxycarbonyl; Sformyl, acetyl, benzoyl and pivalamido; ether forming groups Ssuch as methoxymethyl, t-butyl, benzyl, allyl and tetrahydropyranyl; more preferred is trimethylsilyl.

Preferred amino protecting groups for purine nucleobases X-7773 are selected from the group consisting of alkylcarboxamides, haloalkylcarboxamides and arylcarboxamides such as 2trialkylsilylethoxymethyl, 4-methoxybenzyl, 3,4dimethoxybenzyl, t-butyl, phthalamido, tetrahydropyranyl, tetrahydrofuranyl, methoxymethyl ether, methoxythiomethyl, trityl, pivalamido, t-butyldimethylsilyl, thexyldimethylsilyl, triisopropylsilyl, trichloroethoxycarbonyl, trifluoroacetyl, naphthoyl, formyl, acetyl; sulfonamides such.as alkylsulfonamido and arylsulfonamido, and more preferred is pivalamido. Besides serving as an amino protecting group, the pi',,lamido protecting group increases the solubility of notoriously insoluble purine nucleobase derivatives and directs the Nglycosidic coupling of the purine base to the 9 regioisomer as opposed to the 7 regioisomer.

Preferred hydroxy protecting groups for pyrimidine nucleobases are selected from silyl ether forming groups such as trialkylsilyl; carbamates such as tbutoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl and 4-nitrobenzyloxycarbonyl; carbocyclic esters such as formyl, acetyl, and pivalamido; preferred is trimethylsilyl.

Preferred hydroxy protecting groups for purine ncleobases are selected from the group consisting of ether forming groups such as benzyl, t-butyl, trityl, tetrahydropyranyl, tetrahydrofuranyl, methoxymethyl, trityl; esters such as formyl, acetylpropionyl, pivalamido, benzoyl, substituted benzoyl; carbonates such as carbobenzoxy, tbutoxycarbonyl, carbethoxy, vinyloxycarbonyl; carbamates, such as N,N-dialkylcarbamoyl; trialkylsilyl ethers such as tbutyltrimethylsilyl, t-hexyldimethylsilyl, triisopropylsilyl; more preferred is pivalamido.

In providing protecting groups to the nucleobases of the present process, the protecting group itself m.-y be protected.

In addition, it is often advisable to convert any keto oxygen atoms on the nucleobases to a protected enol X-7773 -11form. This makes the nucleobases more nucleophilic and enhances the reactivity of the nucleobase with the a anomer enriched carbohydrate of formula It is most convenient to enolize the keto oxygens and provide silyl protecting groups for them.

The nucleobases employed in the present process are converted to anions (salts) to further enhance their reactivity with the aanomer enriched carbohydrate of formula The formation of the nucleobase anions involve adding a base to the nucleobase in a solvent. The base may be selected from the group consisting of sodium t-butoxide, potassium hydroxide, potassium-t-butoxide, potassium ethoxide, potassium methoxide, sodium ethoxide, sodium methoxide, sodium hydride, lithium hydride and potassium hydride. Alternatively the base may be selected from trialkylamine or tetraalkylammonium. The solvent may be selected from the group consisting of acetonitrile, dimethylformamide, dimethylacetamide, 1,3-dimethyl-2imidazolidinone, N-methylpyrrolidinone, sulfolane, dimethylsulfoxide, and mixtures thereof. The solvent used to prepare the nucleobase may be removed prior to the glycosylation reaction or admixed with the reaction solvent, provided the admixture is inert to the glycosylation reaction.

The reaction solvents suitable for use in the presen: glycosylation process must be inert to the glycosylation reaction conditions. Preferred reaction solvents are selected from the group consisting of dichloromethane, 1,2-dichloroethane, dichlorofluoromethane, acetone, toluene, anisole, chlorobenzene, dimethylformamide, ~acetonitrile, N,N-dimethylacetamide, methanol, tetrahydrofuran, ethyl acetate, dimethoxymethane, 1,2dimethoxyethane, dimethylsulfoxide, and mixtures thereof.

In accordance with the present process, at least an equimolar amount of nucleobase salt is employed, relative to the total amount of carbohydrate employed. However, more X-7773 -12preferably an excess of nucleobase salt is used in an amount from greater than 1 equivalent to about 10 equivalents and more preferably from about 2 equivalents to about 4 equivalents.

The glycosylation reaction temperature employed in the present process is from about 23 0 C to about 170 0 C; more preferably from about 23 0 C to about 130 0 C, and most preferably about 23 0 C to about.50 0 C. The glycosylation reaction is preferably carried out under atmospheric conditions and is substantially complete in about 5 minutes to about 6 hours.

Although not critical, it is advisiable that the reaction between the a anomer enriched carbohydrate of formula (II) and the nucleobase salt be carried out in a dry atmosphere, e.g. in the presence of dry air, nitrogen, or argon. This is because ce:rtain nucleobase salts are moisture sensitive.

The progress of the present glycosylation process may be followed by procedures well known to one of ordinary skill in the art such as high pressure liquid chromatography (HPLC) and thin layer chromatography (TLC) which can be used to detect the presence of nucleoside product.

In accordance with the present glycosylation process, the p anomer enriched nucleosides are prepared in a to a anomer ratio of greater than 1:1 to about 10:1.

The final phase of the reaction sequence is the removal of the protecting groups X, Z and/or W from the blocked nucleoside of formula The same anomeric ratio of unprotected nucleoside is obtained by removal of the protecting groups.

Most silyl and silyl-amino protecting groups are easily cleaved by use of a protic solvent, such as water or an alcohol. The acyl protecting groups, such as benzoyl and the acyl-amino protecting groups, are removed by hydrolysis with a strong base at a temperature from about 0°C to about 100 0 C. Strong or moderately strong bases X-7773 -13suitable for use in this reaction are bases which have a pKa (at 25 0 C) of about 8.5 to about 20.0. Such bases include alkali metal hydroxides such as sodium or potassium hydroxide; alkali metal alkoxides such as sodium methoxide or potassium t-butoxide; alkali metal amides; amines such as diethylamine, hydroxylamine, ammonia and the like; and other common bases such as hydrazine and the like. At least one equivalent of base is needed for each protecting group.

The acyl protecting groups can also be removed with acid catalysts, such as methanesulfonic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, or with acidic ion exchange resins. It is preferred to carry out such hydrolysis at relatively high temperature, such as the reflux temperature of the mixture, but temperatures as low as ambient may be used when particularly strong acids are used.

The removal of ether protecting groups is carried out by known methods, for example, with ethanethiol and aluminum chloride.

The t-butyldimethylsilyl protecting group requires acid conditions, such as contact with gaseous 'hydrogen halide, for its removal.

Removal of the protecting groups may be conveniently carried out in alcoholic solvents, especially aqueous alkanols such as methanol. However, the deblocking reaction may also be carried out in any convenient solvent, such as polyols including ethylene glycol, ethers such as tetrahydrofuran, ketones such as acetone and methyl ethyl ketone, or dimethylsulfoxide.

In a preferred embodiment, the deblocking reaction employs ammonia to remove a benzoyl hydroxyprotecting group at a temperature of about 100C. It is preferable, however, to use an excess of base in this reaction, although the amount of excess base used is not crucial.

X-7773 -14- The 3 anomer enriched nucleosides of the present invention may be extracted and/or isolated from the reaction mixture by the procedure described in U.S. Patent 4,965,374, Chou, which is incorporated herein by reference, or by conventional methods known in the art such as extraction, crystallization, etc.

The following examples illustrate specific aspects of the present invention and are not intended to limit the scope thereof in any respect and should not be so construed.

Example 1 Preparation of beta-anomer enriched l-(2'-deoxy- 2',2'-difluoro-3 ,5'-di-O-benzoyl-D-ribofuranosyl)-4-(Npivalamido)aminopyrimid-2-ohe in acetonitrile N-pivaloylcytosine (0.098 g, 0.5 mmol) was suspended in acetonitrile (1.5 ml) and treated with potassium t-butoxide (0.062 g, 0.55 mmol) and stirred under a nitrogen atmosphere at 25 0 C to form the potassium salt of Npivaloylcytosine.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5dibenzoyl-l-a-iodo (0.244 g, 0.5 mmol), in acetonitrile 25 ml), was added to the above salt and the entire mixture was reacted for 24 hours at 60°C to form a blocked nucleoside.

HPLC analysis confirmed completion of the reaction and indicated a beta to alpha anomeric ratio of 1.13:1.

Example 2 oPreparation of beta-anomer enriched l-(2'-deoxy- '2',2'-difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)-l,2,4triazole-3-carbonitrile in acetonitrile X-7773 1,2,4-triazole-3-carbonitrile (0.101 g, 1.03 mmol) was suspended in acetonitrile (10 ml) and treated with sodium hydride (0.0445 g, 1.12 mmol) and stirred under a nitrogen atmosphere at 25 0 C to form the corresponding sodium salt of the triazole. 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5dibenzoyl-l-a-bromo (0.451 g, 1.02 mmol), in acetonitrile ml), was added to the above salt and the entire mixture was reacted for 78 hours at 82 0 C to form a blocked nucleoside.

HPLC analysis confirmed completion of the reaction and indicated a beta to alpha anomeric ratio of 1.2:1.

To isolate the nucleoside product, the reaction mixture was evaporated to from an oily solid, diluted with ethyl acetate, washed with sodium bicarbonate and dried over magnesium sulfate and concentrated. The residue crystallized from ethanol to give 30 mg of a titled product at a yield of 6 percent; m.p. 225 0 C-226 0 C. MS(FD) M/Z 455 Elemental Analysis for C 22

H

1 6

F

2

N

4 0 5 (Theoretical) C, 58.15; H, 3.55; N, 12.33; (Empirical) C, 58.36; H, 3.79; N, 12.10.

20 ExamDle 3 Preparation of beta-anomer enriched l-(2'-deoxy- 2',2'-difluoro-3 ',5.'-di-O-benzoyl-D-ribofuranosyl)-1,2,4triazole-3-carbonitrile in acetonitrile 1,2,4-triazole-3-carbonitrile (0.272 g, 2.89 nsnol) was suspended in acetonitrile (20 ml), treated with sodium hydride (0.094 g, 2.7 mmol) and stirred under a nitrogen atmosphere at 25 0 C to form the sodium salt of the triazole.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5dibenzoyl-l-a-iodo (0.941 g, 1.9 mmol), in acetonitrile ml), was added to the above salt and the entire mixture was S. reacted for 48 hours at 82 0 C to form a blocked nucleoside.

HPLC analysis confirmed completion of the reaction and indicated a beta to alpha anomeric ratio.of 3.5:1.

To isolate the nucleoside product, the reaction X-7773 -16mixture was evaporated to.from an oily solid, diluted with ethyl acetate, washed with sodium bicarbonate, dried over magnesium sulfate and concentrated. The residue crystallized from ethanol to give 0.421 g of the titled product; m.p.

225 0 C-226C at a yield of 48 percent. MS(FD) M/Z 455 (M+1) Elemental Analysis for C 2 2

H

1 6

F

2

N

4 0 5 (Theoretical) C, 58.15; H, 3.55; N, 12.33; (Empirical) C, 58.35; H, 3.65; N, 12.33.

Example 4 Preparation of (9)regioisomer-beta-anomer enriched 1-(2'-deoxy-2',2'-difluoro-3',5'-di-0-benzoyl-Dribofuranosyl)-6-cyanopurine in N,N-dimethylacetamide 6-cyanopurine (0.92 g, 6.35 mmol) was suspended in N,N-dimethylacetamide (12 ml) and treated with sodium hydride S(0.396 g, 8.25 mmol) and stirred under a nitrogen atmosphere at 25 0 C to form the sodium salt of 6-cyanopurine.

2-deoxy-2, 2-difluoro-D-ribofuranosyl-3,5dibenzoyl-l-a-iodo (3.09 g, 6.35 mmol), in N,N- Sdimethylacetamide (4 ml), was added to the above salt and the Sentire mixture was reacted for 5 hours at 70 0 C to form a blocked nucleoside. HPLC analysis confirmed completion of the reaction and indicated a beta to alpha anomeric ratio of 1.2:1.

To isolate the nucleoside product, the reaction mixture was cooled, the solvent removed under vacuum, the Sresidue was dissolved in ethyl acetate, washed with a 0.2 M lithium chloride solution, dried over magnesium sulfate and concentrated. Column chromatography (silica gel, toluene/ethyl acetate 9:1) gave 0.21 g of the titled product at a yield of 6.5 percent. MS(FD) 506 Elemental Analysis for C 25

H

17

F

2

N

5 0 5 (Theoretical) C, 59.41; H, 3.39; N, 13.86; (Empirical) C, 59.85; H, 3.49; N, 13.48.

X-7773 -17- Examnle Preparation of (9)regioisomer-beta-anomer enriched 1-(2'-deoxy-2',2'-difluoro-3',5'-di-0-benzoyl-Dribofuranosyl)-2,6-(dipivalamido)diaminopurine in acetonitrile 2,6-(dipivalamido)diaminopurine (0.159 g, 0.5 mmol) was suspended in acetonitrile (1.5 ml) and treated with potassium t-butoxide (0.062 g, 0.55 mmol) and stirred under a nitrogen atmosphere at 25 0 C to form the potassium salt of 2,6-(dipivalamido)diaminopurine.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5dibenzoyl-l-a-iodo (0.244 g, 0.5 mmol), in acetonitrile 15 ml), was added to the above salt and the entire mixture was reacted for 16 hours at 60 0 C to form a blocked nucleoside.

HPLC analysis confirmed completion of the reaction and indicated a beta to alpha anomeric ratio of 2.2:1.

To isolate the nucleoside product, the reaction mixture was diluted with ethyl acetate, the organic layer was washed with sodium bicarbonate, dried over magnesium sulfate S" separated and concentrated to an oil. Column chromatography (silica gel, toluene/ethyl acetate 1:1) followed by recrystallization gave 0.085 g of the titled product at a yield of 25 percent. MS(FD) 679 Example 6 Preparation of beta-anomer enriched l-(2'-deoxy- 2',2'-difluoro-3',5'-di-O-benzoyl-D-ribofuranosyl)-4- (benzylamino)pyrimid-2-one in N,N-dimethylacetamide N-benzylcytosine (0.099 g, 0.493 mmol) was suspended in N,N-dimethylacetamide (2.0 ml) and treated with sodium hydride (0.0256 g, 0.534 mmol) and stirred under a X-7773 -18nitrogen atmosphere at 25 0 C to form the sodium salt of Nbenzylcytosine.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5dibenzoyl-l-a-iodo (0.201 g, 0.411 mmol), in N,Ndimethylacetamide (1.5 ml), was added to the above salt and the entire mixture was reacted for 5 hours at 23 0 C to form a blocked nucleoside. HPLC analysis confirmed completion of the reaction and indicated a beta to alpha anomeric ratio of 1.9:1.

The reaction solvents were removed under vacuum and the residue was dissolved in ethyl acetate, washed with sodium bicarbonate, dried over magnesium sulfate and concentrated to an oil. Column chromatography (silica gel, toluene/ethyl acetate 9:1) gave 0.015 mg of the titled 1' 5 product at a yield of 6.5 percent. MS(FD) 562 Example 7 Preparation of beta-anomer enriched ethyl deoxy-2',2'-difluoro-3',5'-di-0-benzoyl-D-ribofuranosyl)- 1,2,4-triazole-3-carboxylate in N,N-dimethylacetamide Ethyl 1,2,4-triazole-3-carboxylate (0.723 g, 5.13 mmol) was suspended in N,N-dimethylacetamide (2.5 ml), '"025 treated with sodium hydride (0.123 g, 5.13 mmol) and stirred under a nitrogen atmosphere at 25 0 C to form the sodium salt of the triazole.

2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5dibenzoyl-1-a-iodo (2.0 g, 4.11 mmol), in N,Ndimethylacetamide (2.5 ml), was added to the above salt and the entire mixture was reacted for 24 hours at 23 0 C to form a blocked nucleoside. HPLC analysis confirmed completion of the reaction and indicated a beta to alpha anomeric ratio of 3:1.

The crude reaction mixture was purified by removing the solvent under reduced pressure and employing column X-7 773 -9 -19chromatography (silica gel, toluene/ethyl acetate 9:1) The combined theoretical yield of alpha and beta regiolsomers (A and B below) of blocked nucleosides was 67 percent.

A. Ethyl l-(2',-deoxy-2' ,2'-difluoro-3',5'-dibenzoyl-j3-D--ribofuranosyl) 4-triazole-3-carboxylate (436 mg, 21.2 percent yield).

C(,H~C F iCO Recrystallization of "All from ethyl acetateisooctane provided 267 mg of the pure V3anomer in 13% yield.

B. Ethyl l-(2'-deoxy-2' ,2'-difluoro-3' benzoyl-p-D-ribofuranosyl)-1,2,4-triazole-5-carboxylate (855 mg, 41.5 percent yield).

X-7773 Example 8 Preparation of beta-anomer enriched 2-deoxy-2,2difluoro-D-ribofuranosyl-1--(2-amino-6-chloropurine) in dimethylacetamide To a suspension of 2-amino-6-chloropurine (82.6 mmol, 14.0 g) in dimethylacetamide (900 ml) at 0°C under nitrogen was added powdered potassium hydroxide (99.12 mmol, 5.55 The mixture was stirred for 30 minutes to form a solution. 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5dibenzoyl-l-a -iodo (82.6 mmol, 40.31 g) in dimethylacetamide (450 ml) was added. The reaction was allowed to warm to room temperature and stirred under nitrogen overnight.

The product was extracted by adding ethyl acetate and brine. The organic layer was washed successively with IN HC1, saturated sodium bicarbonate solution, H 2 0, and brine.

The orga' layer was then dried over sodium sulfate and evaporate- in vacuo.

The crude product was purified with silica gel chromatography to yield a 3:1 beta to alpha anomer ratio of 2-deoxy-2,2-difluoro-D-ribofuranosyl-3,5-dibenzoyl-l-(2amino-6-chloropurine) 1 H NMR (300 MHz, CD 3 0D), 5 4.68(m, 2H, 4.90(m, 1H, 6.02(m, 1H, 6.29 25 1H, 7.53(m, 6H, Bz), 7.92(s, 1H, 8.05(m, 4H, Bz).

The dibenzoyl intermediate (.49 mmol, 260 mg) was Sdeprotected by suspending it in methanol at 0 C and saturating the mixture with anhydrous ammonia. The resulting solution was warmed to room temperature and stirred overnight. The solution was then purged with nitrogen and evaporated. The titled product was then purified by washing with a non-polar solvent such as methylene chloride to remove the benzoate by products. The beta anomer was separated by reversed phase HPLC.

X-7773 -21- 1H NMR (300MHz, CD3OD), D3.90 (in, 3H, 4.58 (n 1H, 6.27 (dd, IH, 8.31 IH, 8-H).

Claims (7)

1. A stereoselective anion glycosylation for preparing a Panomer enriched nucleoside of the HO 0 R R HO T .10 wherein T is selected from fluoro or hydrogen and R is a nucleobase selected from the group consisting of S S S S OH N N 2HN N NHN N NN X-7773 (OUS) -23- NH 2 OH I CH=CHR3 N CH=CHR 3 0 N O N NH 2 N N O N OH .N pN HO N r r ~I o s R 2 N 0 R 2 N R4 N N R2 N RI 0 N R^ N and N I wherein R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl and halo; R 2 is selected from the group consisting of hydroxy, halo, azido, primary amino and secondary amino; R 3 is selected from the group consisting of hydrogen, alkyl and halo; R 4 R 5 and R 6 are independently selected from the group consisting of hydrogen, -OH, -NH2, N(alkyl), halo, alkoxy and thioalkyl; R 7 is selected from the group consisting of hydrogen, halo, X-7773 (OUS) -24- cyano, alkyl, alkoxy, alkoxycarbonyl, thioalkyl, thiocarboxamide and carboxamide; Q is selected from the group consisting of CH, CRg and N; wherein R 8 is selected from the group consisting of halo, carboxamide, tniocarboxamide, alkoxycarbonyl and nitrile; comprising reacting an a anomer enriched fluorocarbohydrate of the formula XO xo H (II); XO T wherein T is as defined above; and X is a hydroxy protecting group Y is selected from iodo and bromo; with at least a molar equivalent of a nucleobase salt selected from the group consisting of OZ oz N NHW NHW N RI N N r: 6 X-7773 (OUS) NHW OZ MN ^CH=CHR 3 N CH=CHR3 NHW OZ N N N 0 ZO N G R2 R2 R N :R2" N C. I R 4 R 7 N R6 and N M R 5 wherein R 1 through R 7 and Q are as defined above; Z is a hydroxy protecting group; W is an amino protecting group and M is a cation; in an inert solvent; and deblocking to form a compound of formula

2. The process of Claim 1 wherein M is sodium or potassium metal cation.

3. The process of Claims 1 or 2 wherein Y is iodo. V 26

4. The process of Claims 1 or 2 wherein Y is bromo.

The process of any one of the preceeding claims wherein the reaction temperature is from about 23'C to about 130'C.

6. A stereoselective anion glycosylation process for preparing a P-anomer enriched nucleoside substantially as hereinbefore described with reference to any one of the Examples.

7. A fP-anomer enriched nucleoside whenever prepared by the process of any one of claims 1 to 6. D!ted 7 June, 1993 Eli Lilly and Company Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON ILIbMfjO578:GSA 2o 2 of 4 2-1 Stereoselective Glycosylation Process Abstract This invention relates to a stereoselective glycosylation process for preparing 2'- deoxyfluoro-f3-nucleosi~des. According to the present invention there is provided a stereoselective anion glyc-osylation process for preparing a P3 anomer enriched .,ucleoside of the formula HO F H H HO T OH 0 X1N a wherein T is H or F and R NW2 ON NW is RI a nucleobase selected from NW2 N N N N 0 ,CH=C HR 3 .CH=CHR 3 OH R HO Nc N NR2R I 0 6-e N> 0 N R 5 N NN 1 and wherein R, is H, alkyl, substituted alkyl ance halo; R 2 is OH, halo, azido, priiaary amino and secondary amino; R 3 is H, alkyl, and halo; R 4 R 5 and R 6 are H, OH, NH 2 N(alkyl), halo, alkoxy and thioalkyl; R 7 is H, halo, CN, alkyl, alkoxy, alkoxycarbonyl, thioalkyl, thiocarboxamide and carboxamide; Q is CH, CR 8 and N; wherin R 8 is halo, carboxamide, thiocarboxamide, alkoxycarbonyl, and nitrile comnprising reacting an (x anomer enriched fluorocarbohydrate of the formula [L~bFI00696:JOC Io I of 2 wherein T is as defined above; and X is a hydroxy protecting group Y is I or Br with at least a oz 0 N. mlarld eLjuiv JenL Ut d 11ucleouade oz NHW N N R WHN N 0 N 0 ON6 MeM NHW N N N N Ne Me NHW CH=CHR 3 Me oz N-11 CH==CHR 3 0 Ne NHW N-3 R2 M R2 R4 N1 Q N N 0 aO R 6 I 0 Ne Nt N NN Me Me and MeI wherein R, through R 7 and Q are as defined above; Z is a hydroxy protecting group; W is an amino protecting group; and M+ is a cation; in an inert solvent; and deblocking to form a compqyind of formula ILbMh00566:JOC