AU777502B2 - Intermediates in the synthesis of appetite suppresant steroid glycosides - Google Patents

Description

S&F Ref: 476761D2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

o Name and Address of Applicant Actual Inventor(s): Address for Service:

CSIR

Corporate Building Scientia Pretoria 0001 Republic of South Africa Fanie Retief Van Heerden, Robert Vleggaar, Roelof Marthinus Horak, Robin Alec Learmonth, Vinesh Maharaj, Rory Desmond Whittal Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Intermediates in the Synthesis of Appetite Suppresant Steroid Glycosides Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me/us:- 8 MAR 2002 5845c Intermediates in the Synthesis of Appetite Suppressant Steroid Glycosides This invention relates to certain carbohydrates and their use in synthesis.

This application is divided out of our co-pending Australian patent application No.

70613/98 which describes and claims a process for preparing an extract of a plant of the genus Trichocaulon or of the genus Hoodia, the extract comprising an appetite suppressant agent, the process including the steps of treating collected plant material with a solvent to extract a fraction having appetite suppressant activity, separating the extraction solution from the rest of the plant material, removing the solvent from the extraction solution and recovering the extract. The extract so recovered may be further purified, eg by way of suitable solvent extraction procedures.

S

The compounds and synthetic methods in accordance with the present invention S' find application in preparing an active appetite suppressant agent which is found in the c extract defined above.

S

To give a better appreciation of the applications of the compounds of the present o:o, invention, some further information about the plant extracts and processes described in S:'o AU 70613/98 is included herein.

o

S

The extract may be prepared from plant material such as the stems and roots of said 25 plants of the genus [R:\LIBA06570.doc:NSS Trichocaulon or of the genus Hoodia. The genus Trichocaulon and the genus Hoodia include succulent plants growing in arid regions such as are found in Southern Africa. In one application of the invention, the active appetite suppressant extract is obtained from the species Tric.hocaulon piliferum. The species Trichocaulon officinale may also be used to provide an active appetite suppressant extract. In another application of the invention, the active appetite suppressant extract may be obtained from the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii. Bioassays conducted by the Applicant on rats have indicated that certain of the extracts possess appetite suppressant activity.

The plant material may be homogenised in the presence of a suitable solvent, for example, a methanol/methylene chloride solvent, by means of a device such as a Waring blender. The extraction solution may then be separated from the residual plant material by an appropriate separation procedure such as, for example, filtration or 20 centrifugation. The solvent may be removed by means of the rotary evaporator, preferably in a water bath at a temperature of 60 0 C. The separated crude extract may then be further extracted with methylene chloride and water before being separated into a methylene chloride extract S" 25 and a water extract. The methylene chloride extract may have the solvent removed preferably by means of evaporation on a rotary evaporator and the resultant extract may be further purified by way of a methanol/hexane extraction.

The methanol/hexane extraction product may then be separated to yield a methanol extract and a hexane extract.

The methanol extract may be evaporated to remove the solvent in order to yield a partially purified active extract.

The partially purified active extract may be dissolved in methanol, and may be further fractionated by column chromatography, employing silica gel as an adsorption medium and a chloroform/30% methanol mixture as an eluent.

A plurality of different fractions may be obtained, and each may be evaluated, by suitable bioassaying procedures, to determine the appetite suppressant activity thereof.

A fraction having appetite suppressant activity may preferably be further fractionated such as by column chromatography using silica gel as an adsorption medium and a 9:1 chloroform:methanol solvent, and the resultant subfractions bioassayed for their appetite suppressant activity. A sub-fraction displaying appetite suppressant activity may, if desired, be further fractionated and purified, conveniently using a column chromatographic procedure with silica gel as the adsorption medium and a 9:1 ethylacetate:hexane solvent. The resultant purified fractions may again be evaluated by suitable bioassay procedures for their appetite suppressant activity.

The Applicant has found that at least one such purified fraction has good appetite suppressant activity, and the active principle in the fraction was identified by 20 conventional chemical techniques including nuclear magnetic resonance, and was found to be a compound of the structural formula 0 l o *2 1121 20 oCCH3 Me 6 6 e Me M e lOH HO--X-O n O

OH

OMe OMe C B A (1) in accordance with S.I. nomenclature, the active principle is the compound 3-0- C-j-D-thevetopyranosyl- -o-D-cymaropyranosyl- (1-4)-o-D-cymaropyranosyl] -l23tilyoy1-yrx-4-rg--n2-n

(C

4 7

YH

7 4 0 1 M*878) this structure and certain derivatives and analogues thereof are described and claimed in a second co-pending patent application No. 26126/02 which, like the present application, is divided out of AU 70613/98.

The present invention provides compounds having the structural formulae set out hereinafter as formulae (37), (50A), (5OB), (54), (57) and (58).

Compound may be synthesized f rom, a first carbohydrate intermediate in the form of an activated monosaccharide cyruarose moiety, which can be prepared f rom a compound having the formula Compound (36) can be prepared by a process which includes the steps treating methyl-a-D-glucose having the formula

HO;O

HO' WOe

OH

(31) with benza ldehyde and zinc chloride to produce- a compound methyl -4,6-0 -benzylidene -a-D-glucopyr-anoside of the-formula 0 .O01 (32) (ii) treating the 0 (3A it oychrieAn~d pyridine at OOC, to produce a compound methyl-4,6-0of the formula .0 Ph

OH

00 0Me 0 S (33) (iii) treating the-compound. with NaOI~e at iOOGC to produce a compound methyl 4, 6-O-benzylidene-3-Omethyl-a-.D-altropyranoside of the formula 0 PhHO 0 .OMe OWe (34) (iv) treating the compound (34) with N-bromosucci-namide (NBS) to produce a compound methyl 6-bromo-4-Ol--0-methyl- 6 -deoxy-a-D-altrpyanside of the B~r 0' PhO UOye OMe (3S) and treating the compound (35) with NaBH, and NiJC1 2 to produce a compound methyl 4-O-benzoy"L-3-0methyl-6-deoxy-a-D-altropyraloside cf. the f ormula 0 0 HO

II

PhCO OMe OMe (36) in which Ph is phenyl A first aspect of the present invention provides a process for the preparation of a carbohydrate intermediate in the form of an activated monosaccharide cymarose moiety which includes the steps of treating the compound (36) with PhSSiMe 3 and trimethylsilylfluoromethane o0 sulphonate to produce a compound 4-0-benzoyl-3-0-methyl-6-deoxy-ap-Dphenylthioaltroside of the formula

O

0 HO SPh PhCO OMe (37) 15 in which Ph phenyl; (ii) optionally treating the compound (37) with diethylaminosulphur trifluoride (DAST), e.g. at 0°C, to produce a compound 4-0-benzoyl-3-0-methyl-2-phenylthio-2,6dideoxy-cp3-D-fluorocymaropyranoside having the formula 0 o0 PhCO rPh I SPh 2 OMe (38) in which Ph phenyl; or [R:\L1BAj06570.doc:NSS 8 (iii) optionally, treating the compound (37) with t-butyldimethylsilylchloride and imidazole in a solvent, e.g. pyridine, to produce 4-0-benzoyl-3-0-methyl-2-0-tbutyldimethylsilyl-cp-D-phenylthioaltroside having the formula 0 0 ZO SPh PhCO OMe (39) in which Ph phenyl, Z TBDMS t-butyldimethylsilyl; and (iv) treating the compound (39) with a base, e.g. sodium methoxide, to produce 3- 0-methyl-2-0-t-butyldimethylsilyl-cp3-D-phenylthioaltroside having the formula ZO SPh .HO OMe in which Ph phenyl, Z TBDMS t-butyldimethylsilyl.

15 A related aspect of the present invention provides a carbohydrate intermediate of formula (40) when produced according to the process of the first aspect of the present o invention defined above.

Reaction Scheme C shows the procedure for the synthesis of the activated monosaccharide cymarose moiety (40) from compound (36) according to the invention (and includes the preparation of compound (36) from compound (31) for illustrative purposes).

[R:\LIBA]06570.doc:NSS Reaction Scheme C

HO-

HO

(31) 0* Ph HO0 0 OlMe OMe* 'TOMe

OH

OH

OTs *Ph 9 9* 9 .9 A*99 9SS 9 (34Y.

PhCO 0 PhCO W~e OMe L36)~ OMe ONke 0 U1 PhCO.

SPh OM e 37 (381 0 z SPh HO0 OMe (40) SPh 0 1: PhCO OMe (39 Z =t-butyldimethysily! The synthesis of compound may also involve a second carbohydrate *intermediate in the form of an activated monosaccharide thevetose moiety, which. can be Prepared from a compound having the *formula Compound can be by a process -which includes the steps of treating a-D-glucose having the formula 64 0 6 6 9 990 6 *6 6 66 4.

0

S

4066*.

*6

S

44 0 *00c.

46 6 *4 94 4 0 966.

609* 9046 0 6.S* a.

S

6 066*

I.

49 969 0 66* 660 0

HO

0

(OH)

HO fOH

CH

(41) with acetone and sulphuric acid.-to produce a compound 1,2 :5,6-di-O-isopropylidene-a-D-glucofuranose of the formula (42) (ii) treating the compound (42) with Nal and Mel to produce a compound 1, 2 G-Di-Q-isopropylidene-3 -0methyl-a-D-glucofuranose of the formula 0 OMe 0 (43) ii)treating the compound (43) with acetic acid to.

produce a compound 3 -O-methyl-a-D-glucopyranose of the formula

HO

HO

(44) (iv) treating the compound with -methanol and hydrochloric acid to produce a compound methyl methyl-a-D-glucopyranoside having the formula HO-1_ OMe OMe

HO

12 treating the compound (45) with benzaldehyde and zinc chloride to produce a compound methyl 4,6-0-benzylidene-3-0-methyl-ap-glucopyranoside having the formula OMe (46) (vi) treating the compound (46) with N-bromosuccinamide, nickel chloride and sodium borohydride to produce a compound methyl 4-0-benzoyl-3-0-methyl-6-deoxy-apglucopyranoside having the formula OMe 1o

OH

(47) The invention extends in a second aspect to a process for the preparation of an activated monosaccharide thevetose moiety which includes the steps of treating the compound (47) with phenylthiotrimethylsilane and trimethylsilyltrifluoromethanesulphonate to produce a compound 4-0-benzoyl-3-0methyl-l-phenylthio-6-deoxy-ap-glucopyranoside having the formula 0 O OMe SPh

II

PhCO

OH

(48) in which Ph phenyl; [R:\LIBA]06570.doc:NSS 13 (ii) treating the compound (48) with pivaloyl chloride and a solvent, e.g. pyridine, to produce a compound 4-0-benzoyl-3-0-methyl-2-0-pivaloyl-l-phenylthio-6-deoxy-capglucopyranoside having the formula 0 0 OMe SPh

II

PhCO OPv (49) in which Ph phenyl and Pv pivaloyl; and (iii) treating the compound (49) with a brominating bromosuccinimide, and diethylaminosulphur trifluoride to produce benzoyl-3-0-methyl-2-0-pivaloyl- 1-fluoro-6-deoxy-P-glucopyranoside stereoisomers having the formula agent, e.g.

a compound occurring

N-

as O F O OMe

II

PhCO OPv

A)

O

O OMe

II

PhCO F OPv (50 B) in which Ph phenyl, Pv pivaloyl.

A related aspect of the present invention provides a carbohydrate intermediate of formula (50 A) or (50 B) when produced according to the process of the second aspect of the present invention defined above.

[R:\LIBA]06570.doc:NSS 14 Reaction Scheme D shows the procedure for the synthesis of the activated monosaccharide thevetose moiety (50 A) and (50 B) from compound (48) according to the invention (and includes the preparation of compound (47) from compound (41) for illustrative purposes).

[R:\LIBA06570.doc:NSS Reaction Scheme D

HO

ZOH0 OH HO OH

OH

(41)

OH-

0t (43)

HO

1OMe

HO

0e 0 *0 0 0 0 *0 00 0**0 00.0 0 0 0*0b *0 0 0000 p *000* (44)' 1T(45r 0 e Me PhCO e

OH

147) 0 OMe SPh Ph CO

OH

(489) 0 OMe

'OH

(46).

OMe' S Fh PhCQ OPv (49) 0 11 PhCO O F PhCOb Op&D'

B)

Certain of the compounds of this invention may be utilised in a process of synthetically producing a compound of the formula and analogues and derivatives thereof; the process involves the steps of synthesising a suitable steroid intermediate or precursor and coupling the required number of suitable monosaccharides with the steroid intermediate.

One such process involves coupling a monosaccharide cymarose of this invention with a steroid intermediate, which includes the steps of reacting a cymarose moiety (38) with a steroid intermediate e.g. at -15 0 C, and in the presence of tin chloride, in a solvent, e.g. ether, to produce *a compound 3-0-[4-0-benzoyl-2-phenylthio-8-D- 15 cymaropyranosyll-12,14-d-dihydroxy-pregn-5-ene-20-one of the formula 0 B* z Ph We e.g. NaOMe, to produce a compound phenylthio-g-D-cymaropyranosyll-128-tigloyloxy- 14-hydroxy-14-pregn-5-ene-20-one of the formula and (ii) treating the compound (51) with tiglic acid chloride in pyridine and thereafter with a base, e.g. NaOMe, to produce a compound 3-0-1-2phenylthio-8-D-cymaropyranosyl] -lS-tigloyloxy- 14-hydroxy-14B-pregn-5-ene-20-one of the formula 0

HO

1SPh OMe (52) A similar process includes coupling a monosaccharide cymarose moiety of this invention to a monosaccharide thevetose moiety of this invention and coupling the resultant disaccharide with the combined steroid product 5 (52) to form compound An example of such a process of coupling the monosaccharide cymarose moiety to the monosaccharide thevetose moiety and coupling the resultant disaccharide to the combined steroid product (52) may Sinclude the steps of 0 coupling a selectively protected cymarose moiety and a selectively protected thevetose moiety (50 A) using tin chloride (SnCl 2 and silver trifluoromethanesulphonate, e.g. at -15°C, to produce a compound of the formula Ome.

0 OMe O~v (53) in which Z =TBDMS =t-butyldimethylsilyl treating compound (53) w it h tetrabutylammoniumfluoride to produce a compound of the formula 00 PhCO OE'v (54) (iii) treating compound (54) with diethylaminosuiphur trifluoride, e.g. at OOW, to produce a compound of the formuia 0 II FhCO (iv) reacting compound (55) with compound (52) to produce a compound of the formula

OH

SPh OPv (56) and treating compound (56) in a Raney-Nickel reaction and thereafter with a base, e.g. NaOMe, to produce compound as described above.

Reaction Scheme E shows the procedure for the synthesis of intermediates (52) and (55) and coupling them to form compound (56).

Reaction Scheme E (38) 0 0**0 0 0 00 0@ 9 0 0 00 0000 0000 0.

000 0 000000 0

OH

(51) SzO !*SPh OMe'

I.

QA~>~2OH (52) I SPh OMe 0 Me -Sfh PhCO HO OPv OMe 0- HO SPh 0o S~ PhOO_0 eP~ OMe 0 '0 h 0 4..L54) (53) a. 0

F

0 0. SPh 449r 0 OMe/ OMe 4 0 PhC 0 0 HO (52) SPh OMe (56) Z-t-butyldimethylsilyI .OPv Certain compounds of this invention may find application in an alternative synthetic process which includes coupling cymarose and thevetose moieties of this invention to form a trisaccharide and coupling the trisaccharide onto a steroid derivative to form a compound of the formula An example of such a process of forming the trisaccharide and coupling. the resultant trisaccharide to a steroid derivative may include the steps of 0 coupling a selectively protected cymarose moiety and compound (45) using tin (II) chloride, AgOTf, Cp 2 ZrCl 2 to produce a compound of the formula S SPh O ZO S OMe 0 SPh O OMe OMe PhCO OPv (57) in which Z TBDMS t-butyldimethylsilyl treating compound (57) with tetrabutylammoniumfluoride and diethylaminosulphur tri fl tirid e +o-r rmvr\ ra ,A m Tpi having the having the formula I SPh OMe (58) and (iii) coupling the trisaccharide (58) with a steroid intermediate of the formula (59) using tin (II) chloride, AgOTf, Cp 2 ZrCI 2 to produce compound 10 The steroid intermediate (59) may be produced by treating steroid (15) with tiglic acid chloride.

Reaction Scheme F shows the procedure for the synthesis of the trisaccharide (58) and the synthesis of compound by coupling the trisaccharide (58) with the steroid intermediate (59).

[R:\LIBA]06570.doc:NSS Reaction Scheme F 0

F

O OMe0 PhC /4 SPh PhCO OMe ONv 0 ZO SPh

HO

OMe

S.

S

S

S S

S..

0 zO SPh o0 OMe 0 0 O OMe SPh PhCO OMe OPv (57) 0 11 PhCO 7 1 SPh OMe ONv (58) (59) Z t butyldimethylsilyl [R:\LIBA]06570.doc:NSS EXAMPLE 1 Methvl-4,6-0-benzvlidene-a-D-glucopvranoside (32) A mixture of methyl-a-D-glucopyranoside (30 g, 0,15 mol), benzaldehyde (70 ml) and zinc chloride (20 g) is stirred at room temperature for 24 hours. The reaction product is poured into ice water and stirring continued for 15 min. The white precipitate is filtered and washed with diethyl ether. The solid material is stirred with a solution of sodium metabisulphite (10% soln), for 15 min, filtered and washed with water. The solid material is crystallized from chloroform and ether to yield the benzylidene product (32) (31 g, 72%).

EXAMPLE 2 Methyl-4,6-0-benzvlidene-2-0-tosyl-a-D-glucopyranoside (33) p-Toluene sulfonyl chloride (25 g, 1,2 eq) in pyridine (100 ml) is added dropwise to a solution of the benzylidene glucose (32) (31 g, 0.12 mol) in pyridine (100 ml) at 0°C.

15 The reaction is stirred at room temperature for 48 hours. Ice is added to the reaction mixture. The resulting white solid material is washed with water and recrystallized from hot ethanol to yield the tosylated glucose (33) (28 g, EXAMPLE 3 20 Methyl-4,6-0-benzylidene-3-0-methvl-a-D-altropvranoside (34) The tosylate (33) (28 g, 64 mmol) in a solution of sodium (7 g) in methanol (150 ml) is heated at 110 0 C for 48 hour in an autoclave. The reaction vessel is cooled and solid carbon dioxide is added to the reaction mixture. After filtration, the methanol is evaporated and the solid material is then taken up in water. The aqueous layer is extracted with chloroform (X The chloroform is dried (MgSO 4 filtered and evaporated. The crude mixture is purified by silica gel column chromatography eluting with chloroform:acetone to yield the altroside (34) (10 g, 52%) EXAMPLE 4 Methvl-6-bromo-4-0-benzovi-3-0-methyv-6-deoxyv--D-aliropyranoside The benzylidene altroside (34) (10 g, 33 mmol) is added to a solution of Nbromosuccinimide (7.6 g) and barium carbonate (20 g) in carbon tetrachloride and the reaction mixture is refluxed at 75 0 C for 3 hours. The reaction mixture is filtered and the [R:\LIBA]06570.doc:NSS carbon tetrachloride layer is washed with water. The organic layer is dried (MgSO 4 filtered and evaporated to yield 6-bromo-altroside (9 g, 69%).

EXAMPLE Methyl-4-0-benzovl-3-0-methyl-6-deoxv-ct-D-altrolvranoside (36) Sodium borohydride (18 g) in water (30 ml) is added dropwise to a solution of the bromoaltroside (35) (9 g, 23 mmol) and nickel chloride (18 g) in ethanol (300 ml) at 0' C.

The reaction mixture is refluxed at 750 C for 1 hour and then it is filtered. The ethanol is evaporated and the remaining aqueous layer is extracted with chloroform (X The chloroform is dried (MgSO 4 filtered and evaporated, to yield the 6-deoxy-altroside (36) g, 72%) EXAMPLE 6 4-0-Benzoyl-3 -0-methyl-6-deoxy-cd3-D-phenvlthioaltropyranoside (37) *Phenylthiotrimethylsilane (5 ml) and trimethylsilyltrifluoromethane sulphonate (2 i ml) are added at 0 0 C to a solution of the 6-deoxy-altroside (36) (5 g, 17 mmol) in dichloromethane (200 ml). The reaction mixture is stirred at room temperature for 6 :hours. Saturated sodium bicarbonate is added to the reaction mixture. The dichloromethane layer is dried (MgSO 4 filtered and evaporated. The crude mixture is purified by silica gel colum chromatography eluting with chloroform: acetone to yield the cto-phenylthioaltroside (37) (4 g, 63%) EXAMPLE 7 4-0-Benzoyl-3 -0-methyl-2-phenlthio-2,6-dideox-c3-D-fluorocymaropvranoside (38) Di ethylaminosulphurtri fluoride (0,65 g) is added rapidly to a solution of the OLDphenylthioaltroside (37) (0,5 g, 1,33 mmol) in dichioromethane at 0 0 C. The reaction is stirred for 0,5 h at 0 0 C and then saturated sodium bicarbonate is added. The dichloromethane is separated from the aqueous layer, dried (MgSO 4 filtered and evaporated to yield the ccp-fluorocymnarose (38) (450 mg, EXAMPLE 8 4-0-Benzoyl-3 -O-methyl-2-0-t-butyldimethvlsilyl-c43-D-phenlthio-altroside (39) [R:\LIBAj06570.doc:NSS 27 The 6-deoxy altroside (37) (5 g) is silylated using t-butyldimethylsilylchloride (3 g) and imidazole (3 g) in pyridine (50 ml). The reaction is worked-up by extracting with ethyl acetate, washing the ethyl acetate with hydrochloric acid (6 then with sodium bicarbonate, and finally with water. The ethyl acetate layer is dried (MgSO 4 filtered and evaporated to yield the silylated benzoyl phenylthioaltroside (39) EXAMPLE 9 3-0-methl-2-0-t-butmethtb ethvlsilvl--D-phenylthioaltroside The silylated benzoyl phenylthioaltroside (39) (6 g) is treated with sodium 0o methoxide (100 ml) for 4 hours. The methanol is evaporated and water is added to the reaction. The water layer is acidified (pH 5, ACOH) and extracted with ethyl acetate.

The ethyl acetate is washed with water, dried (MgSO 4 filtered and evaporated to yield silylated methyl phenylthioaltroside (40) Examples 10 to 18 illustrate the procedures synthetic whereby the intermediate 15 compounds may be prepared to form the second monosaccharide EXAMPLE 1,2: 5,6-Di-0-isopropvlidene-a-D-glucofuranose (42) Sulfuric acid (40 ml) is added dropwise to a solution of a-D-glucose (41) (50 g, 20 0,28 mol) in acetone (1 f) at 0°C. The reaction mixture is stirred for 24 h and then it is neutralized using sodium hydroxide (6 The acetone is evaporated and the aqueous layer is extracted with chloroform The chloroform is dried (MgSO 4 filtered and 0 .'evaporated. Crystallization from cyclohexane yielded the di-isopropylidene glucose (42) (41 g, 57%).

EXAMPLE 11 1,2: 5,6-Di-0-isopropvlidene-3-0-methyl-a-D-glucofuranose (43) The a-D-glucofuranose (42) (41 g, 0,16 mol) in tetrahydrofuran (300 ml) is added dropwise to a suspension of sodium hydride (5 g) in tetrahydrofuran (200 ml). After methyl iodide (25 g) in tetrahydrofuran (100 ml) is added dropwise to the reaction mixture which is then stirred for 24 h. Water is added to the reaction mixture which is then extracted with ether The ether layer is dried (MgSO 4 filtered and evaporated to yield the methyl protected glucose (43) (38g, 83%).

[R:\LIBA]06570doc:NSS EXAMPLE 12 (44) The methyl diisopropylidene compound (43) (38 g, 0,14 mol) is dissolved in acetic acid 700 ml) and the solution refluxed for 18 h. After cooling the acetic acid is evaporated. The crude product is purified by column chromatography eluting with chloroform: methanol: acetone: water (70:27:2:1) to yield 3-0-methyl-a3glucopyranoside (44) (13 g, EXAMPLE 13 Methyl 3-0-methyl-cp3-D-glucovranoside The 3-0-methyl-ap-glucopyranoside (44) (10 g) is dissolved in methanol (50 ml) and HCI (conc.) (1 ml) and refluxed overnight. Solid NaHCO 3 is added and the reaction is filtered. The methanol is evaporated to give 1,3-di-0-methyl-ap-D-glucopyranoside 15 EXAMPLE 14 Methyl 4,6-0-benzylidene-3-0-methyl-ac-glucopyranoside (46) The glucopyranoside (45) (8 g) is stirred at room temperature in a solution of benzalaldehyde (20 ml) and zinc chloride (5 After 24 hours, ice is added and the S 20 aqueous layer is extracted with chloroform. The chloroform layer is dried (MgSO 4 Sfiltered and evaporated. The benzalaldehyde is removed by vacuum distillation and the product is purified by silica gel column chromatography eluting with acetone:chloroform to yield benzylidene-cp-glucopyranoside (46) EXAMPLE Methyl 4-0-benzoyl-0-methyl-6-deoxv-ap-glucopvranoside (47) The benzylidene compound (46) (5 g) is refluxed at 80 0 C in a mixture of Nbromosuccinimide (3,7 g) and barium carbonate (4 g) in carbon tetrachloride. After 4 hours. the reaction is filtered and the carbon tetrachloride is washed with water, dried (MgSO4), filtered and evaporated to give the bromo compound The bromo compound (4,3 g) is dissolved in a solution of ethanol (300 ml) and nickel chloride (8,6 g) at o0C. To this solution, sodium borohydride (8,6 g) in water ml) is added dropwise over a period of 15 minutes. The reaction mixture is refluxed at [R:\LIBA]06570.do:NSS 100 0 C for 45 minutes, cooled, filtered and evaporated. Chloroform is added, and the chloroform layer is washed with water, dried (MgSO 4 filtered and evaporated to give the 6-deoxy sugar (47) SEXAMPLE 16 4-0-Benzovl-3-0-methyl-l-phenvlthio-6-deoxy-aB-glucolranoside (48) The 6-deoxy glucopyranoside (47) (3 g) is dissolved in dichloromethane (50 ml).

To this solution, phenylthiotrimethylsilane (2g) and trimethylsilyltrifluoromethanesulphonate (0,2 ml) are added. The solution is stirred at room temperature overnight, after which saturated sodium bicarbonate is added. The dichloromethane layer is dried (MgSO 4 filtered and evaporated. The product is purified by silica gel column chromatography eluting with ethyl acetate:hexane to give the compound (48)

*S

15 EXAMPLE 17 4-0-Benzoyl-3-0-methyl-2-0-pivalovl-l-Phenylthio-6-deoxy-a -glucopyranoside (49) To a solution of the glucopyranoside (48) (2 g) in pyridine (20 ml), pivaloyl -chloride (2 ml) is added. The solution is stirred at room temperature overnight after which water is added. The aqueous layer is extracted with ethyl acetate, and the organic 20 layer is washed with HCI (6 The organic layer is dried (MgSO 4 filtered and evaporated to give the pivaloyl ester (49) 8 EXAMPLE 18 4-0-Benzoyl-3-0-methvl-2-0-Pivalovl-l-fluoro-6-deoxy-p-glucopranoside N-Bromosuccinimide (1,2 g) and diethylaminosulphur trifluoride (1,2 g) are added to a solution of the pivaloyl ester (49) (2 g) in dichloromethane (100 ml) at 0°C. After 1 hour, saturated sodium bicarbonate is added. The dichloromethane layer is dried (MgSO 4 filtered and evaporated. The p-fluoropyranoside (50) is purified by silica gel column chromatography eluting with ethyl acetate:hexane (yield Examples 19 to 21 illustrate the synthetic procedures whereby the cymarose and thevetose moieties may be coupled.

[R:\LIBA]06570.doc:NSS EXAMPLE 19 Thevetose-cvmarose dissaccharide (53) A solution of thevetose (50 A) (1,5 cymarose (40) (1,3 and molecular sieves 4A in dichloromethane is stirred at room temperature for 1 hour. The reaction mixture is cooled to -15 0 C, and tin (II) chloride (0,8 g) and silver trifluoromethanesulphonate (1,1 g) are added. The mixture is stirred at -15 0 C for 16 hours, after which triethylamine (0,5 ml) is added. The reaction product is filtered and the dichloromethane is evaporated. The dissaccharide (53) is purified by silica gel column chromatography eluting with ethyl acetate:hexane yield EXAMPLE Thevetose-cvmarose dissaccharide (54) To a solution of the dissaccharide (53) (200 mg) in tetrahydrofuran (20 ml), tetrabutylammonium fluoride (0,4 ml) is added. The mixture is stirred at room S 15 temperature for 1 hour, after which saturated sodium bicarbonate is added. The reaction mixture is extracted with ethyl acetate and the ethyl acetate layer is dried (MgSO 4 filtered and evaporated. The dissaccharide (54) is purified by silica gel column Schromatography (acetone:chloroform, 0,5:9,5) yield 20 EXAMPLE 21 4 Thevetose-cymarose dissaccharide To a solution of the dissaccharide (54) (80 mg) in dichloromethane (10 ml), diethylamino sulphur trifluoride (801l) is added at 0°C After stirring at 0°C for 0,5 hour, saturated sodium bicarbonate and more dichloromethane are added. The dichloromethane is dried (MgSO 4 filtered and evaporated. Purification by silica gel column chromatography (ethyl acetate:hexane gives the dissaccharide (55) in a 65% yield.

[R:\LIBA06570.doc:NSS

Claims (21)

1. A process for the preparation of a carbohydrate intermediate in the form of a monosaccharide cymarose moiety which includes the steps of treating the compound of the formula 0 O HO PhCO OMe OMe (36) in which Ph is phenyl with PhSSiMe 3 and trimethylsilylfluoromethane sulphonate to produce a compound benzoyl-3-0-methyl-6-deoxy-ap-D-phenylthioaltroside of the formula 0 0 HO SPh PhCO OMe (37) in which Ph phenyl; (ii) optionally treating the compound (37) with diethylaminosulphur trifluoride (DAST) to produce a compound 4-0-benzoyl-3-0-methyl-2-phenylthio-2,6-dideoxy-cp- S 15 D-fluorocymaropyranoside having the formula O 0 F PhCO SPh OMe (38) in which Ph phenyl; or (iii) optionally, treating the compound (37) with t-butyldimethylsilylchloride and imidazole in a solvent to produce 4-0-benzoyl-3-0-methyl-2-0-t-butyldimethylsilyi-pe-D- phenylthioaltroside having the formula fR:\LIBA]06570.doc:NSS ZO SPh OMe (39) in which Ph phenyl, Z TBDMS t-butyldimethylsilyl; and (iv) treating the compound (39) with a base to produce the monosaccharide methyl-2-0-t-butyldimethylsilyl-c43-D-phenylthioaltroside having the formula 0 ZO SPh HO OMe in which Ph phenyl, Z TBDMS t-butyldimethylsilyl. A process for the preparation of a carbohydrate intermediate in the form of an activated thevetose moiety which includes the steps of treating the compound 0 OMe OMe PhCO OH (47) in which Ph =phenyl, with phenyithiotrimethylsi lane and trimethylsilyltrifluoromethanesulphonate to produce a compound 4-0-benzoyl-3-0-methyl-lI-phenylthio-6-deoxy-c43-glucopyranoside having the formula J-O O OMe SPh 11I X PhCO OH (48) in which Ph phenyl; [R:\LIBA]06570.doc:NSS (ii) treating the compound (48) with pivaloyl chloride and a solvent to produce a compound 4-0-benzoyl-3-0-methyl-2-0-pivaloyl- 1 -phenylthio-6-deoxy-a3- glucopyranoside having the formula 0 0 OMe SPh II PhCO OPv (49) in which Ph phenyl and Pv pivaloyl; and (iii) treating the compound (49) with a brominating agent and diethylaminosulphur trifluoride to produce the monosaccharide compound 4-0-benzoyl-3-0-methyl-2-0- pivaloyl-l-fluoro-6-deoxy-P-glucopyranoside occurring as stereoisomers having the 0o formula F 0 0 /OMe 0 OMe 1 II II PhCO PhCO F O iOPv OPv (50 A) (50 B) in which Ph phenyl, Pv pivaloyl.

3. A carbohydrate intermediate of formula (40) when produced by a process as 5 claimed in claim 1.

4. A carbohydrate intermediate of formula (50 A) or formula (50 B) when produced by a process as claimed in claim 2.

5. A compound having the structural formula *0 0 HO SPh II PhCO OMe (37) in which Ph phenyl.

6. A compound having the structural formula [R:\LIBA]06570.doc:NSS PhCOI F 4R/Ph OMe (38) in which Ph phenyl.

7. A compound having the structural formula 0 o ZO SPh PhCOII Z OMe (39) in which Ph phenyl, Z TBDMS t-butyldimethylsilyl. A compound having the structural formula 0 HOZO SPh OMe in which Ph =phenyl, Z =TBDMS t-butyldimethylsilyl. A compound having the structural formula 0 0 OMe SPh PhCO OH (48) in which Ph =phenyl. A compound having the structural formula 0 O OMe SPh PhCOI/ OPv (49) [R:\LIBA106570.doc:NSS in which Ph phenyl and Pv pivaloyl.

11. A compound occurring as stereoisomers having the structural formnula O F 0 0 /AOMe 0 OMe PhCOPhCO F A) in which Ph phenyl, Pv pivaloyl.

12. A compound having the structural formula 0 ZO SPh 0 0 O OMe Oe 11II1 PhCO (53) 9in which Ph =phenyl, Pv =pivaloyl, Z TBDMS t-butyldimethylsilyl. 0 13. A compound having the structural formula 0 HO SPh 0 0 0 OM e(I~ PhCO ONv (54) in which Ph =phenyl, Pv =pivaloyl.

14. A compound having the structural formnula F 0 1o OOt~' e PhCOO e OPv in which Ph phenyl, Pv pivaloyl. [R:ULBA]06570.doc:NSS 36 A compound having the structural formula SPh OMe 0 11 PhCO ONv (57) wherein Ph phenyl, Pv pivaloyl, Z TBDMS t-butyldimethylsilyl.

16. A compound having the structural formula I S~h OMe OPv (58) in which Ph phenyl, Pv pivaloyl.

17. A process for the preparation of a carbohydrate intermediate in the form of a 10 monosaccharide cymarose moiety which includes the steps of treating the compound of the formnula 0 O HO I I PhCO OMe OMe (36) in which Ph is phenyl. with PhSSiMe-a and tnmethylsilyltluoromethane suiphonate to produce a compound benzoyl-3-O-methyl-6-deoxy-c43-D-phenylthioaltroside of the formula [R:ULBA]06570.doc:NSS 37 0 O HO SPh PhCO OMe (37) in which Ph phenyl; (ii) optionally treating the compound (37) with diethylaminosuiphur trifluoride (DAST) to produce a compound 4-O-benzoyl-3-O-methyl-2-phenylthio-2,6-dideoxy-Lp- D-fluorocymaropyranoside having the formula 0 O F I I PhCO SPh OMe *(38) in which Ph =phenyl; (8 10 or (iii) optionally, treating the compound (37) with t-butyldimethylsilylchloride and imidazole in a solvent to produce 4-O-benzoyl-3-O-methy1-2-O-t-butyldimethylsilyl-c4p-D- phenylthioaltroside having the formula 0 O ZO SPh 0 PhCO OMe (39) in which Ph =phenyl, Z =TBDMS t-butyldimethylsilyl; and (iv) treating the compound (39) with a base to produce the monosaccharide methvl-2-0-t-butyldimethylsilyl-43-D-phenylthioaltroside having the formula 0 HO OMe [R:\LIBA]06570.doc:NSS in which Ph phenyl, Z TBDMS t-butyldimethylsilyl, substantially as hereinbefore described with reference to any one of Examples 1 to 9.

18. A process for the preparation of a carbohydrate intermediate in the form of an activated thevetose moiety which includes the steps of treating the compound 0 O /OMe OMe 11 RiCO OH (47) in which Ph phenyl, with phenyithiotrimethyl si lane and trimethylsilyltrifluoromethanesulphonate to produce a o0 compound 4-0-benzoyl-3 -0-methyl-i -phenylthio-6-deoxy-c43-glucopyranoside having the formnula 0 0 OMe S~h PhCO O SOH (48) in which Ph =phenyl; (ii) treating the compound (48) with pivaloyl chloride and a solvent to produce a :compound 4-O-benzoyl-3-0-methyl-2-0-pivaloyl-l1-phenylthio-6-deoxy-c43- glucopyranoside having the formula 0 0 OMe SPh RiCO OPv (49) in which Ph =phenyl and Pv =pivaloyl; and (iii) treating the compound (49) with a brominating agent and diethylaminosuiphur trifluoride to produce the monosaccharide compound 4-0-benzoyl-3-0-methyl-2-O- pivaloyl-l1-fluoro-6-deoxy-o3-glucopyranoside occurring as stereoisomers having the formnula [R:\LIBA]06570.doc:NSS SF 0 0 OMe 0 OMe I I I I PhCO PhCO F OPv OPv A) (50 B) in which Ph phenyl, Pv pivaloyl, substantially as hereinbefore described with reference to any one of Examples 10 to 18.

19. A carbohydrate intermediate of formula (40) when produced by a process as claimed in claim 17. A carbohydrate intermediate of formula (50 A) or formula (50 B) when produced by a process as claimed in claim 18.

21. A compound having the structural formula 0 .O HO SPh PhCO S 10 OMe (37) in which Ph phenyl, substantially as hereinbefore described with reference to Example 6.

22. A compound having the structural formula PhCO II .CO OMSPhP (38) in which Ph phenyl, substantially as hereinbefore described with reference to Example 7.

23. A compound having the structural formula I-- 0 Z SPh PhCO \r V OMe (39) [R:\LIBA]06570.doc:NSS in which Ph phenyl, Z TBDMS t-butyldimethylsilyl, substantially as hereinbefore described with reference to Example 8.

24. A compound having the structural formula 0 HO SPh HO OMe in which Ph phenyl, Z TBDMS t-butyldimethylsilyl, substantially as hereinbefore described with reference to Example 9. A compound having the structural formula 0 0 OMe SPh II PhCO OH 0 (48) in which Ph phenyl, substantially as hereinbefore described with reference to Example 16.

26. A compound having the structural formula OMe SPh PhCO 0 OPv 15 (49) *0g in which Ph phenyl and Pv pivaloyl, substantially as hereinbefore described with reference to Example 17.

27. A compound occurring as stereoisomers having the structural formula SF 0 0 OMeY 0 OMe PhCO PhCO \F OPv OPv (50 A) (50 B) in which Ph phenyl, Pv pivaloyl, substantially as hereinbefore described with reference to Example 18. [R:\LIBA]06570.doc:NSS 41

28. A compound having the structural formula 0 ZO SPh OO 0 OMe OMe PhCO OPv (53) in which Ph phenyl, Pv pivaloyl, Z TBDMS t-butyldimethylsilyl, substantially as hereinbefore described with reference to Example 19.

29. A compound having the structural formula 0 HO SPh 00 0 OMe OMe PhCO OPv (54) in which Ph phenyl, Pv pivaloyl, substantially as hereinbefore described with reference to Example A compound having the structural formula O O0 0 OMe SPh PhCO OMe OPv in which Ph phenyl, Pv pivaloyl, substantially as hereinbefore described with reference to Example 21. Dated 16 August, 2004 CSIR Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON (R:LIBA]06570.doc:NSS