AU2612602A - Appetite suppresant steroidal glycosides - Google Patents

Description

S&F Ref: 476761D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

*r Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title:

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) Appetite Suppresant Steroidal Glycosides The following statement is a full description of this invention, including the best method of performing it known to me/us:- IIP ~Ch8~~l ri Documents recev? r, wx Raftha Q B &J f G^HW> B..8nAK ti^ 5845c Appetite Suppressant Steroidal Glycosides THIS INVENTION relates to steroidal glycosides, to compositions containing such steroidal glycosides and to a new use for these steroidal glycosides and the compositions containing them. The invention further relates to a method of extracting and isolating these steroidal glycosides from plant material, to a method of synthetically producing these steroidal glycosides, and to the products of such an 10 extraction and such a synthesis process.

In a particular application, the invention relates to an appetite suppressant agent, to a process for synthetically producing the appetite suppressant agent, to a process for extracting the appetite suppressant agent 15 from plant material, to an appetite suppressant composition containing the appetite suppressant agent, and to a method of suppressing an appetite.

According to the invention, there is provided 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.

The invention also provides a plant extract made of plants of the group comprising the genus Trichocaulon and the genus Hoodia and having appetite suppressant activity.

The extract may be prepared from plant material such as the stems and roots of said plants of the genus 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 Trichocaulon 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 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 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 10 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 conventional chemical techniques including nuclear magnetic resonance, and was found to be a compound of the structural formula 0 o 4 0 aoC CH 3 I D Me Me M e

OH

HO' Oo 4J..sjo OMe OMe C B A 4 In accordance with S.I. nomenclature, the active principle is the compound 3-0-(-0-D-thevetopyranosyl- -ft-D-cymaropyranosyl- -fl-D-cymaropyranosyl] -12f- 0tigloyloxy- 14 -hydroxy- 14fl-pregn- 50-en- 20-one (C 47

H

74 0 1 M+878).

According to another aspect of the invention, there is provided 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 S 10 including the steps of pressing collected plant material to "separate sap from solid plant material and recovering the sap free of the solid plant material to form the extract.

The extract may be dried to remove moisture, e.g. by spray-drying, freeze-drying or vacuum drying, to form a free-flowing powder.

The invention extends to a composition having appetite suppressant activity comprising an extract as described above.

The composition may be admixed with a pharmaceutical excipient, diluent or carrier and optionally it. is prepared in unit dosage form.

The invention also extends to the use of an extract as described above in the manufacture of a medicament having appetite suppressant activity, to an extract as described above for use as a medicament having appetite suppressant activity, and to a method of suppressing an appetite by administering to a human or animal an effective dosage of a composition as described above.

Compound is a novel compound and the invention extends to compound and certain analogues or derivatives of this steroidal trisaccharide having appetite suppressant properties. The molecules chosen as the analogues or derivatives are intended to affect the properties of the steroidal trisaccharide with the aim of increasing the activity of the active ingredient. The following effects were taken into consideration when the analogues were chosen: Hydrophobic interactions and lipophilicity Functional group modifications of the active molecule is intended to change the hydrophobicity and lipophilicity of the molecule. Increased 10 lipophilicity has been shown to correlate with increased biological activity, poorer aqueous solubility, increased detergency/cell lysis, increased storage in tissues, more rapid metabolism and elimination, increased plasma protein binding and S 15 faster rate of onset of action.

(ii) Electronic properties and ionization constants Functional group modification of the molecule is also intended to change the acidity and basicity which would have a major role in controlling the transport of the compound to its site of action and the binding at this target site.

(iii) Hydrogen bonding Functional group modifications of carboxyl and carbonyl groups in the active molecule are intended to change the interactions between the proteins in biological systems and the chemically modified functional groups.

(iv) Steric parameters The purpose of changing the steric features of the molecule is to increase binding to its receptor and 6 thus increase its biological activity.

The following chemical modifications to the molecule are intended to affect the hydrophobicity and lipophilicity electronic properties, hydrogen bonding and steric parameters on the molecule: a) Chemical modification of the C-12 group and ester functionality; b) Chemical modification of the 5,6-double bond, e.g.

hydrogenation and migration; 10 c) Chemical modification of the C-20 carbonyl and C-17 acetyl group; d) Chemical modification of the ring of the steroid or aglycone ring; e) Modification of the carbohydrates of the trisaccharide 15 moiety.

Accordingly, the invention provides a compound having the general structural formula

OH

R^0 (2) in which R alkyl; Ri H, alkyl, tigloyl, benzoyl, or any other organic ester group; R2 H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4-C5 or C5-C6.

7 The invention also provides a compound as described above wherein there is a further bond between C5 C6, R methyl, R 1 tigloyl, R 2 3-0- 3 -D-thevetopyranosyl- 1-D-cymaropyranosyl-(1-4) D-cymaropyranosyl] and having the structural formula.

0 0.

V..

I

0 0 0 0 Further active analogues or derivatives of the appetite suppressant compound in accordance with the invention are compounds having the following structural formulae: 0O OMe 0 O OMe OMe Ho

OH

in which R alkyl; and

S

8 Rl= H, or benzoyl, or. tigloyl, Or any other organic ester group 00OH OMe 0 OMe 0 OM.

HoOH (4) in. which R =alkyl; and

R

1 H, or tigloyl, or benzoyl, or any other organic ester group

O~OH

00 00

OH

in which R =alkyl; and =l H, or tigloyl, or benzoyl, or any other organic ester group

OH

OMo 0* e* 0* 0 in which R =alkyl; and =l H, or tigJloyl, or benzoyl, or any other organic ester group 014f in which R =alkyl; R H, or tigloyl, or benzoyl, or any other organic ester group.

OH

C

C

in which R alkyl; and R H, alkyl, tigloyl, benzoyl, or any other organic ester group; R2 H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4 C5 or C5 C6.

R H OR, OH

C

(9) in which R alkyl; and

R

I H, alkyl, tigloyl, benzoyl,. or any other organic ester group;

R

2 H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose 11 molecules, or combinations thereof; and in which the broken lines indicate the presence of a further bond between C4 C5 or C6.

0 0 5 in which R alkyl; and

R

1 H, alkyl, tigloyl, benzoyl, or any other organic ester group; R2 H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4 or C5 C6.

R)

(11) in which R alkyl; and Ri H, alkyl, tigloyl, benzoyl, or any other organic ester group;

R

2 H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4 C5 C6 or C14 00 *S e to 00 *0 a 0 00

I

(12) in which R alkyl; and

R

1 H, alkyl, tigloyl, benzoyl, any other organic ester group;

R

2 H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4 C5 C6 or C14 (13) @0 0e 0 *600 0 0@ 0 @000 0@ 0@0@ in which

M

R alkyl; and

R

1 H, alkyl, tigloyl, benzoyl, any other organic ester group; R2 H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4 C5, C5 C6 or C14 C15; and

R

3 H, alkyl, aryl, acyl, or glucoxy.

00

S.

Me

OH

Me Me OMe OR OMe (14) in which R H, alkyl, aryl or any steroid possessing a C14 beta hydroxy group, or a C12 beta hydroxy functionality, or a C17 acyl group, or a C5 C6 olefin, or combinations thereof.

The invention still further extends to a process for synthetically producing a compound having appetite suppressant activity.

The process uses a steroid as. a starting material (or intermediate or precursor), the steroid having the chemical formula

OH

OH

HO

The steroid (15) can be prepared from a compound having the formula (22) by a process which includes the steps of treating progesterone having the formula (16) with the micro-organism Calonectria decora to produce a compound 12S, 15a- dihydroxy progesterone of the formula

OH

(17) 0 0 *0.

*.o (ii) treating compound (17) with tosyl chloride and pyridine to produce a compound 129-hydroxy-l1r- (ptoluene sulf onyl) -progesterone of the f ormula 0OTS (18) (iii) treating the compound (18) with collidine at 150 0 C to produce a compound 129-hydroxy- 5 4 progesterone, of the formula

OH-

(19) (iv) treating the compound (19) with acetyl chloride and acetic anhydride at 1200C, to produce a compound 3,l2I9-diacetoxyoregna-3,5, 14-trien-20-one Of the formula 5 treating the compound (20) with ethylene glycol and a catalytic amount of p-toluene suiphonic acid, to produce a compound 3,129-diacetoxy-20,20ethylenedioxypregna-3, 5, 14-triene of the formula (21) (vi) treating the compound (21) with NaBH 4 to produce a compound 3S~, 121-dihydroxy-20, 5,14-diene-12-acetate of the formula 01 0

HO

(22) In a first alternative procedure, a process for the preparation of steroid (15) according to the invention includes the steps of treating compound (22) with a reducing agent, e.g.

LiA1H 4 to produce a compound 313, 1218-dihydlroxy-2O,2ethylenedioxypregna-5,14-diene of the formula (23) treating compound (23) with N-bromoacetamide (NBA) and a base, e.g. pyridine, to produce a compound 3I3,12I9dihydroxy- 14, 15 -epoxy- 20, 2 0-ethylenedioxypregn- 5- ene of the formula 0 01 (24) treating compound (24) with a reducing agent, e.g.

LiAIH 4 e.g. with refluxing, to produce a compound 38, 12B, 148-trihydroxy-20,20-ethylenedioxypregn-5-ene of the formula OH 0] ¥i r

OH

and treating compound (25) with an acid, e.g. acetic acid, and water to produce the steroid intermediate compound 121, 14i-trihydroxy- Reaction Scheme A depicts the procedure for the preparation of steroid intermediate (15) from compound (22) according to "the first alternative procedure" of the idvention (and includes the preparation of compound (22) from compound (16) for illustrative purposes).

19 Reaction Scheme A 0

OH

00 (16) (7 ~OH 0

OH

a. 0 o(18) OAc 0 QAc AcO '-.NAcO (21) 0]

HO,

(23) (22) 0 0]

OH

HO'

t241

OH

115) Ini a second alternative procedure, a process f or the preparation of steroid (15) according to the invention i ncludes the steps of treating compound (22) -(30l, 120-dihydroxy.-2,2o..

ethylenedioxypregna-..s14-diene- 12-acetate) with ptoluenesulfonyl chloride and a base, e.g. pyridine, to produce a compound 30, 12a-dihydroxy-20,2..

ethylenedioxypregna.s, 4 -diene-3-tosyl-12-acetate of the formula

OAO]

(26) 10 treating compound (26) with potassium acetate in a solvent, e.g. acetone, to produce a compound 6ft, 12/3- 20-ethylenedioxy-3, Sa-cyclopregnan14ene- 12-acetate of the formula

OH

(27) 0 0* 0 0 treating the compound (27) with a reducing agent, e.g.

LiA1H 4 and e.g. tetrahydrofuran, to produce a compound 6J3, 120-dihydroxy-2O,2O-ethylenedioxy-3,sacyclopregnan-14-ene of the formula (28) 5 treating the compound (28) with N-bromoacetamide, optionally acetic acid, and a base, e.g. pyridine, to produce a compound 6f9, 120-dihydroxy-2O,20ethylenedioxy- 14, 15-epoxy-3, 5a-cyclopregnane of the formula (29) treating the compound (29) with a reducing agent,,.e.g.

LiAi 4 and e.g. tetrahydrof uran, to produce a compound 6t3, 1203, 140-trihydroxy-2O, 20-ethylenedioxy-3. cyclopregnane of the formula

OH

OH

OH

S.

'S S

S

0S

C

*5 S S @555 *5 S 5* I. 6* 55.

S

*55* Ge S 0 5* S S

S

*55 0 and treating compound (30) with an acid, e.g.

hydrochloric acid, and a solvent e.g. acetone, to produce compound Reaction Scheme B shows the procedure for the preparation of steroid intermediate (15) from compound (22) according to "the second alternative procedure" of the invention.

Reaction-Scheme R OAc 00] QAc ~0]

T

5 0 1 (26) (22)

S

5.55

S

S

S.

S

5555 55

S

S

S

S.

0 0] QAc 0 .01 OH 27) 0 O H (29) (151 Wixture atepimers (152) C- 17a3 aUeyl (I Sbl C. I7daeyI Compound may be synthesized from a first carbohydrate intermediate in the form of an activated monosaccharide cymarose moiety,-which can be prepared from 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

0. HO Oe

OH

(31) *.:with benzaldehyde and zinc chloride to produce a comound ethy-4, 6-O-benzylidene-cr-D-glucopyranoside of the formula .0 *Ph O H CM e

OH

(32) (ii) treating the compound (32) with tosyl chloride and pyridine at O*W, to produce a compound methyl-4,6-O- ~benzylidene-2-O-tosyl-a-D-glucopyranoside of the formula Ph -4 OMe OTs (33) (iii) treating the compound (33) with NaOMe at 100C to produce a compound methyl 4,6 -O-benzylidene-3 -0methyl-a-D-a-tropyrafloside of the formula 00 Ph HO 0 OMe (34) (iv) treating the compound (34) with N-bromosuccinamide (NBS) to produce a compound methyl 6-bromo-4-Qbezy---ehl6dox---lrprnsd of the formula Br 0 PhCOOe OMe and treating the compound (35) with NaBH 4 and NiCl 2 to produce a compound methyl. 4-0-benzoyl-3-0methyl-6-deoxy-a-D-atropyranoside of the formula 9/ 1Y P hCO Ome OMe (36) The invention extends to a process for the preparation of a carbohydrate intermediate in the form of an activated :~.'monosaccharide cymarose moiety which includes the steps of Wi treating the compound (36) with PhSSiMe 3 Zn1 2 and SBu4'I- to produce a compound 4-O-benzoyl-3-O-methyl-6deoxy-aig-D-phenylthioaltroside of the formula 0 H O S~h ph0 *(37) (ii) optionally treating the compound (37) with diethylaminosuiphur trifluoride (DAST), e.g.'at 0 0

C,

to produce a compound 4-O-benzoyl-3-0-methyl-2phenylthio 6-dideoxy-aIZ-D-fluorocymaropyranoside having the formula 0 PhCO (38) 28 or (iii) optionally, treating the compound (37) with tbutyldimethylsilylchloride and imidazole in a solvent, e.g. pyridine, to produce 4-0-benzoyl-3phenylthioaltroside having the formula *0 0 o- SPh PhcZO t~ OMe (39) in which Z TBDMS t-butyldimethylsilyl and (iv) treating the compound (39) with a base, e-.g.

sodium methoxide, to produce 3-0-methyl-2-0-tbutyl dime thyl s ilyl ao-D -phenyl thioal troside having the formula OMe in which Z TBDMS t-butyldimethylsilyl.

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).

Reaction Scheme C

HO

0

OH

HO

OH

OH

(32) OMe 0- Ph PhCO 0 PhCO lOMe OTs (33) 0 11 1, PhCO OMe OMe (351 IOMe OMe IL36) SPh CM e (37) (38) Z SPh HO0 OMe (401 SPh 0 PhC OMe (9 Z but yId imet hy Isily I 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 (47) can be prepared by a process which includes the steps of treating a-D-glucose having the formula

HO

OH

HO- H

OH

C

(41) with acetone and sulphuric acid to produce a compound 1,2 5,6-di-0-isopropylidene-a-D-glucofuranose of the formula

O.

0 0

XO

OR

(42) (ii) treating the compound (42) with NaH and Mel to produce a compound 1,2 5,6-Di-0-isopropylidene-3-0methyl-a-D-glucofuranose of the formula 0 OMe 0 0 (43) (iii) treating the compound (43) with acetic acid to produce a compound 3-O-methyl-aIB-D-glucopyranose of the formula

HO

'0

HOAO

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

HO

0 H OMerOMe treating the compound (45) with benzaldehyde and zinc chloride to produce a compound methyl 4,6-0benzylidene-3 -0-methyl -aIg-glucopyranoside having the f ormula .0 0 OMe OMe

OH

(46) 5 (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-atglucopyranoside having the formula O~le OMe Ph CO OH (47) The invention extends to a process for the preparation 10 of an activated monosaccharide thevetose moiety which includes the steps of Wi treating the compound (47) w it h phenyithiotrimethylsilane and trimethylsilyltrifluoromethanesuiphonate to produce a compound 4-0-benzoyl-3-0-methyl-1-phenylthio-6deoxy-aO-glucopyranoside having the formula O~e SPh Ph CO

OH

(48) treating the compound (48) with pivaloyl chloride and a solvent, e.g. pyridine, to produce a compound benzoyl-3-0-methyl-2-0-pivaloyl--phenylthio-6-deoxyaft-glucopyranoside having the formula OMe Sh Ph CO

O*

(49) and (iii) treating the compound (49) with a brominating agen t, e.g. N-bromosuccinimide, and diethylaminosuiphur trifluoride to produce a compound 4-0-benzoyl-3-O-methyl-2-O-pivaloyl-1fluoro- 6-deoxy-/3-glucopyranoside occurring as stereo-isomers having the formula 34

OF

0 OM 0 OMe PhCO PhCO F OPv OPv A) (50 B) Reaction Scheme D shows the procedure for the synthesis of the activated monosaccharide thevetose moiety (50 and 50 from compound (48) according to the invention (and includes the preparation of compound (47) from compound 5 (41) for illustrative purposes) o* o Reaction Schenme D

HO

OH0 HO OH

OH

(41) 0 0- (43) OMe

HO

(44) (451 1 0 OMe OMe Ph CO/ O

OH

t47) OMe (46).

O OMe SPh Ph CO

OH

(48) O OMe 11 PhCO OF OMe S Ph PhCO/4oe OPv (49) O ON* Ph CO ONv (5 0A) B) According to a still further aspect of the invention there is provided a process of synthetically producing a compound of the formula and analogues and derivatives thereof which includes the steps of synthesising a suitable steroid intermediate or precursor and coupling the required number of suitable monosaccharides with the steroid intermediate.

The invention also provides a process of coupling a monosaccharide cymarose with the steroid intermediate, 10 which includes the steps of reacting a cymarose moiety (38) with a steroid intermediate e.g. at -15°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-E-D- 15 cymaropyranosyl] -12,14-S-dihydroxy-pregn-5-ene-20-one o* of the formula

*OH

H

BzO SPh OMe (51) and (ii) treating the compound (51) with tiglic acid chloride in pyridine and thereafter with a base, e.g. NaOMe, to produce a compound phenylthio- -D-cymaropyranosyll -128-tigloyloxy- 14-hydroxy-149-pregn-5-ene-20-one of the formula 0 Ha. o

OH

SPh The invention extends to a process which includes coupling a monosaccharide cymarose moiety to a disaccharide with the combined steroid product (52) to form compound The process of coupling the monosaccharide cymarose (52) may include the steps of The invention extends to a process which includes 0 coupling a monoselectively prtctedharide cymarose moiety to and a selectively protectedosaccharide thevetose moiety and coupling the resultant disaccharide with the combined steroid product (52) to fand silver trifluoromethanesulphonate, e.g. at -15 0 C, to produce Scompound of the formula The process of coupling the monosaccharide cymarose 0 moiety to the monosaccharide thevetose moiety and coupling 0 l the resultant disaccharide to the combined steroid product (52) may include the steps of coupling a selectively protected cymarose moiety and a selectively protected thevetose moiety (50 A) using tin chloride (SnCl2) and silver trifluoromethanesulphonate, e.g. at -15*C, to produce a compound of the formula 38 0 o Oe 0 OmeM OFv (53) :in which Z TEDMS =t-butyldirnethylsilyl t r. ei tr at in g compound (53) w it h tetrabutylammoniumfluoride to produce a compound of the formula HO S?* HO SO OPv (54) (iii) treating compound (54) with diethylaminosuiphur trifluoride, e.g. at 0 0 OC, to produce a compound of the formula I SPh OMe so a 0. 0 060S 0 0. 0 00 0 a 0 so 0 0 0 04 0 0 0 (iv) reacting compound (55) with compound (52) to produce a compound of the formula OMe (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 coupli ng them to f orm compound (56) Reaction SchemeE 0

BZO

SPh OMer

*OH

*OH

*A51 H* 0 *Ph **h *e (52) 0 0 0 0 OMe( +o ZO PhCO- HOSh 1 OPv OMe **HO SPh ZO S 0 Z__Oe OMe OMe 0 Q OMe e PhCO PhCO *.L54) (53)

F

0 SPht 0 OMe O Ie 0 PhCO 0 p 0OHt HO (52) I SPh OMe (56) Zut-butyidimethylsilyl opv 42 According to the invention, an alternative process is provided which includes coupling cymarose and thevetose moieties to form a trisaccharide and coupling the trisaccharide onto a steroid derivative to form a compound of the formula The process of forming the trisaccharide and coupling the resultant trisaccharide to a steroid derivative may include the steps of coupling a selectively protected cymarose moiety .0 and compound (45) using tin (II) chloride, AgOTf, Cp 2 ZrClz to produce a compound of the formula 'SPh OMe iPh 0

II

PhC OPv (57) in which Z TBDMS t-butyldimethylsilyl (ii) treating compound 57 with tetrabutylammoniumfluoride and diethylaminosulphur trifluoride to produce a trisaccharide compound having the formula OMe 7. (58) and (iii) coupling the trisaccharide (58) with a steroid intermediate of the formula (59) using tin (II) chloride, AgOTf, Cp 2 ZrC12 to produce compound 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 (1) by coupling the trisaccharide (58) with the steroid intermediate (59).

Reaction Scheme F 0 0 OMe PhCO ONv 0 MNF 0O SPh

HO

)h OMe Zo SPh 0 0 OMe 0 mo/ SPh 11O~ OMe PhCO Opv (57) OMe OMe (58) +H o (59) Z ut -butyidimethylsilyl The intermediates (29), (53), (57) and (58) described above are novel compounds and the invention extends to these compounds as such.

Compound 3-0- (-f-D-thevetopyranosyl- -0-Dcymaropyranosyl- (1-44) D-cymaropyranosyl]-120-0tigloyloxy-14-hydroxy-140-pregn-5-en-20-one, and various analogues and derivatives thereof have been found to have appetite suppressing activity.

•The invention extends also to a composition or formulation having appetite suppressant activity, in which the active ingredient is an extract obtained from a plant of the genus Trichocaulon or the genus Hoodia.

The active ingredient may be a compound of the formula extracted from a plant of the genus Trichocaulon or Hoodia or a derivative thereof. The plant may be of the species Trichocaulon officinale or Trichocaulon piliferum, or the species Hoodia currorii, Hoodia gordonii or Hoodia 20 lugardii.

The invention extends also to a composition or formulation having appetite suppressant activity, in which the active ingredient is a synthetically produced compound of the formula or a derivative or analogue thereof, as hereinbefore set out with reference to compounds to (14).

According to another aspect of the invention there is provided a method of suppressing an appetite by administering to a human or animal a suitable dosage of an appetite suppressant agent comprising an extract of a plant of the genus Trichocaulon or Hoodia. The extract may be incorporated in a composition or formulation including also pharmaceutically acceptable other ingredients.

46 The appetite suppressant agent may be an isolated natural chemical or a synthetic chemical compound of the f ormula: 0o Me Me Me0H (1) :or derivatives or analogues thereof, as set out before.

The appetite suppressant composition or formulation may consist of the appetite suppressant agent admixed with a *pharmaceutical excipient, diluent or carrier. other suitable additives, including a stabilizer and such other ingredients as may be desired may be added.

The invention extends to the use of compound or its derivatives or analogues in the manufacture of a medicament having appetite suppressant activity.

The invention further extends to compound or its derivatives or analogues as set out before, for use as a medicament having appetite suppressant activity.

A method of suppressing an appetite by administering to a human or animal an effective dosage of a composition as described above is also provided.

A method has been described herein for extracting a steroidal glycoside having appetite suppressant activity from plant material obtained from a plant of the Trichocaulon or Hoodia genus. The invention thus extends to an extract obtained from plant material of the Trichocaulon or Hoodia genus and containing a substantially pure steroidal glycoside of formula The invention extends also to a foodstuff or a beverage containing an effective quantity of the steroidal glycoside of the formula or its derivatives or analogues as set out before, to have an appetite suppressant effect when ingested.

10 Molecular genetic studies have led to a considerable S* increase in the understanding of the regulation of appetite, satiety and bodyweight. These studies have revealed numerous central regulatory pathways, mediated by a number of neuropeptides. The maintenance of a normal 15 body weight is achieved by an intricate balance between energy intake, food consumption, and energy expenditure.

Energy homeostasis is subject to a wide range of Sinfluences, ultimately controlled by the brain. The different signals include such things as sense of smell and 20 taste and gastro-intestinal signals such as distension of the gastro-intestinal tract, chemical signals to the gastric mucosa and blood-borne metabolites such as fatty acids and glucose.

Centrally, neuropeptide (NPY) which is negatively regulated by leptin, has been established as one of the positive regulators of feeding behaviour. Expression of the endogenous antagonist for melanocortin receptors has also been shown to be the basis for obesity in a particular model (the ob/ob mouse). Indeed deficiency at the MC4 melanocortin receptor completely replicates the obesity syndrome. Other mediators which have been shown to have roles in the energy balance include bombesin, galonin and glucagon-like peptide-1.

48 Without being bound by theory, the Applicant believes that compound and its analogues as described above act as an agonist of the melanocortin 4 receptor. The effect of this is to regulate NPY but also to increase cholecystokinin. The effect of cholecystokinin amongst other things is to inhibit gastric emptying.

Accordingly, the invention extends to a composition having appetite suppressant activity comprising a melanocortin 4 receptor agonist.

The agonist may be an extract or compound as previously S. described, in particular the compound of formula The composition may be admixed with a pharmaceutical excipient, diluent or carrier and is optionally prepared in unit dosage form.

15 The invention still further extends to the use of a melanocortin 4 receptor agonist in the manufacture of a medicament having appetite suppressant activity, to a melanocortin 4 receptor agonist for use as a medicament having appetite suppressant activity, to a method of 20 suppressing an appetite by administering to a human or animal an effective dosage of a composition comprising a melanocortin 4 agonist as described above, and to the use of a melanocortin 4 receptor agonist to suppress the appetite of and/or to combat obesity in a human or animal.

The invention and its efficacy will now be further described, without limitation of the scope of the invention, with reference to the following examples and drawings.

In the'drawings, Figure 1 shows a flow diagram of the general method of extracting a first crude appetite suppressant extract and a purified appetite suppressant extract from plant material of the genus Trichocaulon or Hoodia; 49 Figure 2 shows a graphical representation of a bioassay carried out on rats using a partially purified methanol extract of Trichocaulon piliferum; Figures 3 and 4 together show a schematic representation of a preferred embodiment of the process of the invention for producing an extract of plant material of the genus Trichocaulon or Hoodia; and Figures 5 and 6 show a graphical representation of the percentage change of body mass of rats for different groups for days -7 to 7 and days 0 to 7 respectively in a repeat dose study using a sap extract and a spray-dried sap S. extract of plant material of the species Hoodia gordonii.

EXAMPLE 1 The general method of extracting a first crude appetite suppressant extract and a purified appetite suppressant extract from plant material of the genus Trichocaulon or of the genus Hoodia is illustrated by way of the flow diagram of Figure 1.

EXAMPLE 2 Bioassays carried out on rats using a partially purified methanol extract obtained in the manner illustrated in Example 1, indicated that the extract does in fact exhibit appetite suppressant activity. The appetite suppressant activity of the active extract can be illustrated by way of a typical example of the effect of the methanol extract of Trichocaulon piliferum on rats, by way of the graphic representation in Figure 2.

It will be evident from Figure 2 that the test group of rats dosed with the extract on day 5 displayed a substantially diminished food intake over the next two days, while a control group did not disclose a comparable reduced food intake. The food intake of the test group returned to normal, and in fact increased, from day 8 onwards.

EXAMPLE 3 A preferred embodiment of a process in accordance with the invention for producing an extract having appetite suppressant activity is illustrated schematically by way of example in Figures 3 and 4, which two Figures together illustrate the comprehensive process. However, various 10 other procedures may be used, as will be understood by persons skilled in the art.

Referring to Figure 3, plant material of the genus Trichocaulon or the genus Hoodia is fed into a blender 3, eg a Waring blender, by way of feedline 1, with a solvent in the form of a methylene chloride/methanol solution introduced via feedline 2. The homogenised product is fed via line 4 into a separation stage 5, eg in the form of a filter or centrifuge, and the residual plant material is removed via line 27.

The solvent/extract mixture is fed via line 6 into an evaporation stage 7, where the solvent is removed, for example by means of a rotor evaporator. The dried crude extract is fed via line 8 into a further extraction stage 9 with the addition of a methylene chloride/water solution introduced via feedline 29 for further extraction, and then to a separation stage 13 by way of line 11, where the water fraction is removed via line 31. The dissolved extract fraction is fed via line 15 into a drier stage 17 where the solvent is evaporated, for example by a rotor evaporator.

Referring to Figure 4, the -dried extract is fed via line 10 into an extraction stage 12. A methanol/hexane solution is also fed via line 14 into the extraction stage 12 for further purification and extraction of the dried extract. The extract/methanol/hexane mixture is fed via line 16 into a separation stage 18, the hexane fraction is removed via line 20, and the methanol/extract mixture is then fed via line 22 into a drying stage 24. In the drying stage 24, the solvent is removed, eg by evaporation on a rotor evaporator.

The dried, partially purified active extract is fed via line 26 and with the addition of methanol via line 28 into a solution stage 30, and the dissolved fraction is fed via line 36 to a chromatography column 38.

In the column 38 the methanol soluble fraction is further fractionated, using silica gel and a methanol solvent, into different fractions schematically indicated as fractions I to V. According to an actual fractionation procedure carried out by the Applicant, the fractionation procedure yielded the following fraction weights 1(3.9 11(2.6 111(2.1 IV(1.1 g) and These fractions are individually evaluated by a suitable bioassaying procedure (in a step not shown) and those fractions identified as fractions I and II, displaying marked appetite suppressant activity, are fed by feedlines 40 and 42 into columns 44 and 46 respectively where they are further fractionated and purified by column chromatography, again by using silica gel and a 9:1 chloroform:methanol system.

The sub-fractions II(A) obtained from column 44 do not, when assayed, display a noteworthy appetite suppressant activity, and may be recycled for further chromatography.

The sub-fractions I(A) obtained from column 46 are also evaluated (by an assaying step not shown), and the sub-fraction I(C) is found to have marked appetite suppressant activity.

The sub-fraction I(C) is fed-via line 48 into column for a further fractionation and purification, using silica gel and a 9:1 ethyl acetate:hexane eluent. Of the resultant purified fractions, fraction I(C) (ii) is found, after assaying, to possess marked appetite suppressant activity.

The purified product is identified by nuclear magnetic resonance spectroscopy (as indicated in Tables 1 and 2 below), to be compound 0o Table 1: 1 H (300.13 MHz) n.m.r. data for compounid f c e 3 Compound (1) Hydrogen Atom J(HH)/Hz SH/p.p.M.

Aglycone-3 -3.522 m 6 5.381 m 12 11.5,4.1 4.607 dd 17 9.3, 9.3 3.157 dd 18 1.029 s 19 0.951 s 21 -2.164 s 3*7.1, 1.5 6.888 qq 4*7.1, 1.2 1.806 dq 1.6, 1.2 1.853 dq Cym-1' 9.4, 2.1 4.816 dd 2 'aq 13.8, 3.7, 2.1 2.055 ddd 3'a 13.8, 9.4, 2.6 1.552 ddd 9.4, 2.9 3.179 dd 6.3, 9.4 3.821 dd 6.3 1.279 da 3'-OMe 3.408 sd ill 9.4, 2.1 4.730 dd 13.8, 3.7. 2.1 2.108 ddd 2.q13.8, 9.4, 2.6 1.601 ddd 3-x3.7, 2.9, 2.6 3.755 ddd 4" 9.4, 2.9 3.239 dd 6.3, 9.4 3.898 dd 6"v 6.3 1.243 db 3"-OMe -3.392 se Thev-i"1 7.7 4.273 d 7.7, 8.0 3.469 dd 3' 8.0, 2.9 3.099 dd 9.3, 2.9 3.179 dd 6.3, 9.3 3.351 dd 6.3 1. 183 d'c 3"'-OMe -3.622 s a,b,c in each column may be interchangeable. d,e in each column may be interchangeable, R~efes to the tigloate group atoms Table 2: Relevant 1 3 r (75.25 MHz) n.mn.r. data for CompDound in CDCE 3 Aglycone moiety Sugar moiety Carbon 6cIp.p.m. Carbon J6IP.P.M.

1 37.04 T cym- 1' 95.84 D 2 29." T 2' 35.57 T 3 77.24 D 3' 77.05 D 4 38.62 T 4' 82.57 D 138.95 S 5' 68.48 D 6 131.900D 6' 18.14 Q 7 27.30 T 3'-OMe 57.93 Q 35.30 D 1"t 9954 D 9 43.040 2"T 35.17 T 37.22 S 3" 76.99 D 11 26.04 T 4" 82.520D :12 75.88 D 5" 68.30 D 13 53.71 S 6" 18.36 Q .:14 85.69 S 3"-OMe 57.09 Q 34.36 T They- 104.280D 16 24.31 T 74.62 D 17 57.18 D 85.300D 18 9.850Q 74.62 D 19 19.27 Q 71.620D 216.85 S 17.750Q *21 33.01 Q 3"'-OMe 60.60 Q 1 167.60S 2* 128.69 D 3 137.66 D 4* 14.41 Q 12.080Q Refers to the tigloate group atoms Compound (1) IR data: 3440 cm- 1 (OH) 2910 ctfl 1 1700 cmn 1

(C=O)

]~)205839 12,670 CHC1 3 m.p. 147 0 C 152 0

C

Examples 4 to 13 illustrate the synthetic procedures whereby the intermediate compounds and steroid (15) may be prepared according to "the first alternative procedure".

EXAMPLE 4 12. 15a-Dihydroxy progesterone (17) Cultures of Calonectria decora (ATCC 14767) are prepared by the inoculation of a culture medium comprised of sucrose (900 K 2

HPO

4 (30 Czapek concentrate (300 ml), corn steep liquor (300 ml) and distilled water (30 f) (150 X 500 ml flasks). After 5 days of shaking at 26°C, Sprogesterone (16) (150 g) in a suspension of Tween 80 (0,1 S% soln., 1,5 2) is added to the flasks. The cultures are incubated for a further 5 days and then worked-up by centrifugation, decantation, extraction of the medium with 15 chloroform, and then evaporation to yield the dihydroxy progesterone (17) (75 g, 45 1 H NMR (CDC13) 5,71 (1H, s, 4,12-4,22 (1H, m, 4,43 (1H, br, s, OH); 3,46-3,53 (1H, dd, J 4,6Hz, 20 H-12); 2,16 Hz (3H, s, H-21); 1,18 (3H, s, H-19); 0,74 (3H, s, H-18) EXAMPLE 12S-Hydroxv-15- (p-toluene sulfonvl)-progesterone (18) The dihydroxy progesterone (17) (75 g, 0.22 mol) is dissolved in dry pyridine (300 ml) and cooled to 0°C. p- Toluene sulfonyl chloride (46 g, 0,24 mol) in dry pyridine (200 ml) is added dropwise to the reaction mixture at 0°C.

The reaction is stirred overnight at 0 0 c, and quenched by the addition of H20 (500 ml). The water layer is extracted with ethyl acetate (1 and the organic extract washed with hydrochloric acid (6M, 3 X 1 aqueous saturated sodium bicarbonate (500 ml), aqueous saturated sodium chloride (500 ml), and water (500 ml). The organic layer is dried (MgSO 4 I filtered and evaporated to yield p-toluene sulfonated progesterone (18) (98 g, 92 U) as a viscous dark yellow-oil.

1H NM (CDCl 3 7, 7 (2H, d, J =14Hz, 2, 6) 7,34 (2H, d, J 8, 4Hz, H- 3,5) 5, 67 (1K, s, H-4) 4,86-4,93 (1K, mR, H-15) 3,45-3,50 (1H, dd, J 4,6Hz, H-12); 2,44 (3H, s, H-4Me) 2,15 (3HI, s, H- 21) 1,13 (3H, s, H-19); 0,74 (3H, s, H -18 EXAMPLE 6 1219-Hydroxy-A 1 4 _rrocesterone (19) A solution of the tosylated progesterone (18) (98 g, 0,19 mol) in 2,4,6-trimethyl collidine (500 ml) is refluxed *at 1500C for 3 h. The reaction mixture is cooled and poured into water (500 ml) The water layer is extracted with ethyl acetate (1 after which the organic layer is washed with hydrochloric acid (SM, 3 X 1 aqueous saturated sodium bicarbonate (500 ml) aqueous saturated sodium chloride (500 ml) and water (500 ml) After drying (MgSO 4 and filtering, the ethyl acetate is evaporated and the crude mixture is purified by silica gel chromatography, eluting with acetone: chloroform (1:10) to afford A 14 -progesterone (50 g, 78 k) as a dark red oil.- 1 H NMR (CDCl 3 5,73 (1H, s, 5,28 (1H, dd, J 2,2Hz, H-iS), 4,41 (1H, br, s, OH), 3,49-3,52 (1H, dd, J 4,3Hz, H4-12), 2,80-2,84 (1K, dd, J 9, 2Hz, H-17), 2,14 (3H, H- 21), 1,19 M.1, s, H4-19), 0.89 (3M, s, H-18).

EXAMPLE 7 3,12S-Diacetoxvpreana-3,5.14-trien-20-one A solution of a 14 -progesterone (19) (50 g, 0,15 mol) in acetyl chloride (1,5 f) and acetic anhydride (750 ml) is refluxed for 2 hours. The reaction mixture is poured into cold ethyl acetate (1 f) and aqueous saturated sodium bicarbonate is added with stirring until the effervescence ceases. The ethyl acetate layer is separated from the sodium bicarbonate layer and washed with further portions 10 of aqueous sodium bicarbonate (3 X 700 ml), thereafter with aqueous saturated sodium chloride (700 ml) and finally with water (700 ml) The organic layer is dried (MgSO 4 filtered and evaporated to afford the 3,12idiacetoxypregna-3,5,14-trien-20-one (20) (60 g, 93 as an orange oil.

1 H NMR(CDC1 3 5,68 (1H, s, 5,44 (1H, m, 5,31 (1H, dd, J 2,2Hz, H-15), 4,82-4,86 (1H, dd, J= 4,5Hz, H- 12), 3,10-3,18 (1H, t, J 9,5Hz, H-17), 2,18 (3H, s, 3- Ac), 2,11 (3H, s, 12-Ac), 2,08 (3H, s, H-21), 1,02 (3H, s, H-19), 1,01 (3H, s, H-18) EXAMPLE 8 3,126-Diacetoxy-220 ,20-ethvlenedioxvprerna-3,5,14-triene (21) The diacetoxy compound (20) (60 g, 0,14 mol) is dissolved in benzene (1 f) and ethylene glycol (60 ml) and p-toluene sulfonic acid (1 g) are added. (The benzene is previously refluxed with a Dean-Stark trap). The mixture is refluxed with stirring and azeotropic removal of water for 16 hours. Aqueous saturated sodium bicarbonate solution (500 ml) is added to the.cooled solution. This is then washed with brine (500 ml), and with water (500 ml), and dried (MgSO 4 The solvent is evaporated and the crude mixture purified by silica gel column chromatography, eluting with ethyl acetate: hexane to yield the -ethylenedioxypregna-3,5,14-triene (21) (35 g, 53 1H1 NMR '(cDCl 3 5,68 (111, s, 5,45 (111, m, H-6), 5,31 (1H1, dd, J 2,2Hz, H-15), 4,73-4,85 (11, dd, j 4,4Hz, H-12), 3,78-3,98 (4H, m, ethylenedioxy), 2,16 (311, s, 3-Ac), 2,04 O3H, s, 12-Ac), 1,29 (31, s, H1-21), 1,12 (3H, s, H-19), 1,02 (31, s, H1-18).

EXAMPLE 9 3 9- 129- D ihvdroxy -2 0, 2 0 -ethylenedioxvoregna 5,14 -diene -12 10 acetate (22) The dienolacetate (21) (35g, 0,077 mol) is suspended in ethanol (500 ml) and sodium borohydride (2,8g, 0.074 mol) is added at Q 0 C. The mixture is allowed to warm to *:room temperature and stirred overnight. Most of the 15 solvent is removed in vacuo and the mixture is diluted with water (500 ml) and extracted with ethyl acetate (500 ml) Work-up followed by chromatography on silica gel with acetone /chloroform (1:10) yields the 319-alcohol (22) (25 g, 80 NMR (CDCl 3 41 mn, H 5, 28 (11, dd, J= 2,2Hz, H-15), 4,72-4,81 (lH, *dd, J 4,4Hz, 11-12)., 3,82- 4,02 (411, mi, ethylene dioxy), 3,45-3,59 (1H1, mi, 2,03 O3H, s, 12-Ac) 1,28 (311, s, H-21), l,1Q (3H, s, H-19), 1,01 (31, s, H-18).

EXAMPLE 3g. 12g-Dihydroxv-20,20- ethyl enedioxvt~recrn 14 -diene (23) The 3g.-alcohol (22) (25 g, 6 0. 2 mol) in dry tetrahydrofuran (300 nil) is added dropwise- to a suspension of lithium aluminium hydride (2,7 g, 72,2 mnmol) in dry tetrahydrofuran (500 The reaction mixture is stirred at room temperature f or 24 hours *af ter which water (2,7 ml) is carefully added and stirred for a further 10 min.

Sodium hydroxide (15 soln, 2,7 Ml) is then added and the suspension stirred. After 10 min, water (8,1 ml-) is added and the suspension stirred for 10 minutes, filtered, dried (MgSO 4 and the solvent evaporated to afford the 3i9, 1219 dihydroxypregna-diene (23) (20 g, 90 -01.

1H1 MIR (CDC1 3 5,36 (1H, m, H1-6), 5,23 (1H, dd, j= 2,2Hz, H-15), 3,94-4,06 (4H1, mi, ethylene dioxy), 3,41-3,52 (1H1, m, 3,32-3,36 (111, dd, J 4,3Hz, H-12), 1,31.

(3H1, s, H) 1, 01 (3H1, s, H1-19) 0, 96 (3H1, s, H1-18).

13C NMR (CDC1 3 152,4 140,2 121,1 119,7 (c6,111,1 79,8 71,6 63, 7 and 63, 6 (ethylene dioxy) 58, 8 19,0o 15 11,9 (C-18).

39g. 12f9-DihvdroxV-14. 15-eToxv-20. 20-ethylenedioxvprecrn-5ene; 3191219-Dih droxy- 5. 6 -epoxy-20, 2 20-ethylenedioxvmrecn- 14 -ene N-Bromoacetamide (211 mg, 1, 5 mmol) is added to a stirred solution of the 5,14-diene (23) (500 mg, 1.34 mmol) in acetone (100 ml), acetic acid (2,5 ml), and water (5 ml) at 0 0 C. After 15 min sodium sulphite (S soin, 50 ml) is added to the reaction mixture. The acetone is evaporated, and the aqueous layer extracted with dichioromethane (3 X 50 ml) The organic layer is dried (MgSO 4 filtered and evaporated. Pyridine (U ml) is added to the product, and stirred for 0,5 h. Dichloromethane (100 ml) is then added to the reaction mixture, and the dichioromethane is washed with citric acid (5 11 soln, 3 X 100 ml), saturated sodi um bicarbonate (50 ml), and water (50 ml) The organic layer is dried (MgSO 4 )1 filtered and evaporated to give the mixture of 14,15- and 5-,6-epoxides (360 mg, 69 as a white foam. The mixture of epoxides could not be separated by silica gel column chromatography.

EXAMPLE 11.

313, 1213-Dihvdroxv-14. 15-e~oXV-20,20-ethyenedo1cvpreC....

ene (24) The mixture of 14,15- and 5,6- epoxides (14,4 g, 37,0 mmol) in dry tetrahydrofuran. (200 ml) is added to a suspension of lithium aluminium hydride (1,69 g, 44,4 mmol) in dry tetrahydrofuran (300 ml). The reaction mixture is stirred at room temperature for 24 hours, after which it is worked up as described earlier by the addition of water (1,69 ml), and sodium hydroxide (15 soln, 1,69 ml).

After filtration and evaporation of the solvent, the crude product is purified by silica gel column chromatography using methanol /chloroform as solvent to give the unreacted 14,15 epoxy- 20, 20-ethylenedioxypregn-S-ene (24) (300 mg, 2,1 1 H NMR (CDCl 3 5,31 (1H, mn, 3,82-3,98 (4H, m, ethylene dioxy) 3,43-3,52 (lH, m, 3,41 (1H, s, H- 3,31-3,35 (1H, dd, LJ=4,3 Hz, H-12), 1,29 (3H, s, H- 21), 1,17 M3, s, H-19), 1,02 O3H, s, H-18).

20 13 C NMR (CDCl 3 1 139,8 ,120,8 112,1 77,2 75,4 61,0 22,3 (C- 21), 19,2 9,5 (C-18).

EXAMPLE 12 313. 1213. 1413-Trihydroxy- 20, 2 0-ethyl enedioxy~rec 5 ne The 14,15-epoxide (24) (300 mg, 0,77 mmol) in dry tetrahydrofuran (10 ml) is added to a suspension of lithium aluminium hydride (300 mg, 7,89 mmol) in tetrahydrofuran and the reaction ref luxed for 48 h. After the addition of water 3 ml), sodium hydroxide (15 soln, 0,3 ml) and filtration as described earlier, the mixture is purified by silica gel column chromatography using methanol: chloroform as solvent to give the trihydroxy pregnene (25) (250 mg, 83 1 H NMR (CDCl 3 :5,38 (1H, m, 3,98 (4H, m, ethylene dioxy), 3,43-.3,53 (1H, m, 3,25-3,32 (1H, dd, J 4,1Hz, H-12), 1,32 (3H, s, H-21), 14,01 (3H, s, H-19) 0, 98 (3H, s, H-18) 1 3 C NMR

CDC.

3 S. a a.

a 139,1 122,1 112,2 85,1 75,1 (C-12), 71,6 23,4 19,4 (C- 19), 8,9 (C-18) EXAMPLE 13 319, 129, 149-Trihvdroxv-porean-5-ene The ethylenedioxypregnene (25) (250 mug, 0,64 rumol) is dissolved in acetic acid (13,4 ml) and water which after freeze drying affords the trihydroxy steroid (15) (200 rmg, 89 2281 0 -235 0 C (lit 225 0 -235 0 M+ 348, tUD' 350 (lit (aD]2 2.90).

1 H NmR (CDC1 3 5,39 (1H, m, 3,56-3,62 (11H, t, J =8,1 Hz, H-17), 3,42-3,51 (1H, mH-3) 3,28-3,39 (1H, dd, J 4,3Hz, H-12), 2,23 (3H, s, H- 21), 1,03. (3H, s, H-19), 0,90 O3H, s, H-18) 217,7 (C-20) 138,9 122,2 85,5 73,6 71,6 57,0 55,1. 43,6 42,1. 37,3 36,8 35,9 34,5 32,9 (C-21) 31, 5 (C-16) 30,1 27,4 24,4 19,4 (C-19), 13 C NMR (CDCl 3 8, 3 (C-18).

Examples 14 to 19 illustrate the synthetic procedures whereby the intermediate compounds and steroid (15) may be prepared according to "the second alternative procedure-.

EXAMPLE 14 20-Ethylenedioxy-30 toluefe-p-sulphonyloxy-pregqn-5,.14diene-32f-ol acetate (26) A solution of ptoluenesuiphonyl chloride (650 mg, 3.4 mmol) in pyridine ml) was added dropwise to a mixture of the 20,20- Ethyl enedioxcypregna 14 -diee 30, 12P -diol 12-acetate (22) (1.3 g, 3.1 mmol) in pyridine (15 ml) at DOC. The reaction so.. mixture was left stirring at room tempe rature for 24 hours after which water was added to the reaction mixture. The solution was extracted with ethyl acetate (2x50 ml) the 15 ethyl acetate layer was washed citric acid (5x50 ml) saturated sodium bicarbonate solution (100 ml) saturated sodium chloride solution (100 ml) and water (100 ml). The 9 ethyl acetate was dried (MgSO 4 filtered, and evaporated and purified by flash column chromatography using hexaneethyl acetate (8:2 v/v) as the eluant to give the 0-0-tosyl steroid (1.5 g, 84ks), as a yellow oil, (Found M 570.271, C 3 2

H

4 2 OIS requires:. M 570.273).

6H 1. 021 s, 19-H) 1. 131 (3M, s, 18-H), 1.282 (3H, s, 21-H) 2.021 (acetateOcH 3 12.431 (3H, s, Ar-CM 3 3.883 (4H, m, 0CM 2

CH

2 4.750 (1H, dd, 3 J 10.8 Hz, 5.2 Hz, 12-H) 4.890 (1H, m, 30H), 5.281 (1H, dd j 4.2 Hz, 2.1 Hz, 15-H), 5.388 (1H, m, 7.341 (2H, d' J 8.2 Hz, ArH) 7.746 (2H, d, 3 J 8.2 Hz, ArH) ac 13. 493Q 19.002Q 21.612Q .(Armethyl)*, 21.671Q 24.1750 (acetate methyl), 63 .401T (ethylenedioxy) 63 .498T (ethylenedioxy), 71-531S 80.912D 82.531D 111.363S 120.881D (C-15) 121.461D 123.715- 133.917 (Aromatic), 139,903S 151,722S 170.819S (ester carbonyl).

may be interchanged EXAMPLE 2 0, 20 -Ethyl enedi oxy-3a, 5- Cyc1o-5a -pregn -14 -ene- 69,29s diol -22-acetate (27) A solution of 3S9-toluene-psulphonyloxy-pregn-5,14-diene (26) (1.2 g, 2.1 mmol) and potassium acetate (2.2 g, 22.4 nunol) in water (250 ml) and acetone (500 ml) was refluxed at 60 0 C for 16 hours. The 10 acetone was evaporated and the water was extracted with .:ethyl acetate (200 ml) The ethyl acetate was dried (MgSO 4 filtered, and evaporated. Flash chromatographic separation of the mixture using chloroform- acetone (9:1 v/v) as the eluant gave the 3a,5-cyclo derivative (27), is (530 mg, as a yellow oil, (Found M4 416.262, C25 1 1 36 0 requires M 416-263).

6H0.288 (lH, dd, 3 J 8.l Hz, 4. 9 Hz, 4 -Ha) 0. 477 (iH, dd, J 4.4 Hz, 4.4 Hz, 4 -Hb) 1.025 (3H, s, 19-H), 1.121 (3H, s, 18-H) 1.256 (3H, s, 21 1. 9 89 (3H, s, acetate-OH 3 3.302, (1H, ad, 3 J 2.8 Hz 2.8 Hz, 6- H) 3.784-3.947 (4H, m, OCH 2 CH4 2 4.721 L1,dd Hz, 5.6 Hz, 12-H), 5.232 (1H, dd, 3 J3.9 Hz, 1.9 Hz, 23.623Q(C-21), 24.153Q (acetate methyl) 63.700T (ethylenedioxy) 63.788T (ethylenedioxy) 73.591D 80.551D 111.126S 118.778D 152.959S 170.991S (ester carbonyl).

EXAMPLE 16 2 0,20 -Ethyl enedioxy-3, 5 cyclo-5a -pregn-24 -ene- 6S~,22B -dio01 (28) A solution of the 3a,5-cyclo derivative (500 mg, 1.2 mmol) in tetrahydrofuran (20 ml) was added dropwise to a suspension of lithium aluminium hydride (50 mg, 1.3 mmol) in tetrahydrofuran (10 ml) The reaction mixture was stirred for 4 hours and quenched by the addition of water pl) After 30 minutes, sodium hydroxide was added solution, 50 yl) and stirring continued for a further minutes. Water (150 p1 was added and the reaction mixture was filtered. The tetrahydrofuran was dried (MgSo 4 filtered and evaporated and flash chromatographic purification using chlorof orm- acetone (8:2 v/v) as the 10 eluant to give the diol (370 mg, 83110 as an oil, :(Found M 374.250, C 2 3

H

3 4 0 4 requires: M 374.252) 6 H 0.298 (1H, dd, J 8.1 Hz, 4. 9 Hz, 4-12), 0.510 (1H, dd, 3 J 4. 4 Hz, 4.4 Hz,4-Hb) 0. 985 (3H, s, 19-H) 1.055 (3H, s, 18-H), 1.325 (3H1, s, 21-H), 3.318 (1H1, dd, 3 LJ3. 0 Hz, 3. 0 Hz, 6 3.3 63 (111, dd 3 J 11.4 Hz, 4.2 Hz, 12-H) 4.019 (4H1, m, OCH Ch 0) 4.622 (111, s, OH) S. 255 (11, dd, 3 J 3.9 Hz, 1. 9 Hz, 23.604Q(C-21), 63.620T (ethylenedioxy), 63.733T 20 (ethylenedioxy), 73.569D 77.478D (C-12), 111.125S 118.702D 152.912S (C-14).

EXAMPLE 17 2O0-Ethyl enedioxy-1.4, 15B- epoxy- 3 5- cycl 148 pregnane-63,1213-diol (29) N-bromoacetamide (150 mg, 1.1 mmol) was added to a solution of the 20,20-ethylenedioxy- 3a,5-cyclo-5a-pregn-14-ene-69,12Z-diol (28) (340 mg, 0.91 mmol) in acetone (20 ml), water (0.25 ml) and acetic acid (0.25 ml) at OOC. After 15 min., sodium sulphite (51; solution, 20 ml) was added to the reaction mixture. The acetone was evaporated under reduced pressure and the remaining solution was extracted with dichioromethane (3x30 ml). The dichloromethane layer was dried (MgSO 4 filtered and evaporated to a concentrated volume (50 ml). Pyridine ml) was added to the mixture and stirred for a further 1 hour after which the dichloromethane layer was washed with a citric acid solution 3x30 ml), saturated sodium bicarbonate solution (30 ml) and water (30 ml) The dichloromethane layer was dried (MgSO) 4 filtered and evaporated and purified by flash column chromatography using chloroform-methanol (9.5:0.5 v/v) as the eluant to give the epoxide (29) (180 mg, 51% as a foam, (Found M 390.245, C 2 3

H

3 4 0 2 requires: M 390.247).

6 H 0.287 (1H, dd, 3 J8.1 Hz, 4.9 Hz, 4 0.501 (1H, dd, 3 J 4.4 Hz, 4.4 Hz,4-Hb), 0.978 (3H, s, 19-H), 1.048 (3H, s, 18-H), 1.321 (3H, s, 21-H), 3.318 (1H, "dd, 3 J 3.1 Hz, 3.1 Hz, 3.355 (1H, dd, 3 J 11.2 Hz, 4.1 Hz, 12-H), 3.491 (1H, s, 15-H), 4.001 (4H, Sm, OCH2Ch20) 4.901 (1H, s, OH).

15 6 c 11.668T(C-4), 11.973Q(C-18), 19.515Q (C-19), 23.519Q(C-21), 59.910D 63.601T (ethylenedioxy), 63.713T (ethylenedioxy), 72.501S 73.571D 77.471D 111.085S EXAMPLE 18 S 20 20, 20-Ethylenedioxy-6B, 12B, 14-trihydroxy-3a,5-cyclo-5a,143pregnane (30) .A solution of the epoxide (29) (170 mg,.

0-.44 mmol) in tetrahydrofuran (10 ml) was added to a suspension of lithium aluminium hydride (20 mg, 0.53 mmol) in tetrahydrofuran (5 ml) The reaction mixture was refluxed for 2 hours after which water (20 ti) was added and stirring continued for 05 hour. Sodium hydroxide solution 20Al) was added and stirring continued for a further 0.5 hour. A further quantity of water was added Al) and the suspension was stirred for 1 hour. After filtration, the suspension was dried (MgSO 4 filtered, and the tetrahydrofuran was evaporated. Flash chromatographic separation of the resulting mixture eluting with chloroform-methanol (9:1 v/v) gave the required triol mg, 53%) as a clear oil, (Found M 392.261, C23H 3 8 requires: M 392.263).

6H 0.287 (1H, dd, 3 J 8.1 Hz, 4.9 Hz, 4-H 2 0.510 (1H, dd, 3 J 4.4 Hz, 4.4 Hz,4-Hb), 0.971 (3H, s, 19-H), 1.042 (3H, s, 18-H), 1.319 (3H, s, 21-H), 3.321 (1H, dd, 3 J 3.0 Hz, 3.0 Hz, 3.321 (1H, dd, 3 j 11.1 Hz, 3.9 Hz, 12-H), 3.561 (1H, s, OH), 4.084 (4h, m,

OCH

2 Ch20O) 4.671 (1H, s, OH).

6c 11.668T(C-4), 11.971Q(C-18), 19.511Q 23.520Q 1: 0 63.612T (ethylenedioxy), 63.711T (ethylenedioxy), 73.483D 76.051D 84.307S 111.099S EXAMPLE 19 12S, 14-Trihydroxy-14B-pregn-5-en-20-one (15) A mixture of the triol (30) (80 mg, 0.20 mmol) in acetone (20 ml) and hydrochloric acid (IM, 10 ml) was refluxed at 60 0 C for 2 hours. The reaction mixture was cooled and saturated sodium bicarbonate solution (20 ml) was added. The acetone was evaporated and the aqueous layer extracted with chloroform (3 x 20 ml), the chloroform layer was dried (MgSO 4 filtered and evaporated to give the epimeric trihydroxy steroids (15a, 15b) (42 mg, Separation of the epimeric mixture (15a, 15b) (15 mg) was achieved, by flash chromatographic separation using chloroform methanol (9:1 v/v) as the eluant to give the pure 176epimer (15a), (10 mg), m.p. 224-229 0 C (acetone), (lit. 226- 2230), (Found M 348.234, C; 72.32, H 9.21% C 2 1

H

32 0 4 requires: C, 72.38; H 9.26%, M 348.236), and the 17aepimer (15B) (3 mg), m.p. 183-191°C (acetone), (lit 184- 1960).

3&,12, 14-Trihydroxy-14S-pregn-5-en-20-one 6 H 0.963 (1H, s, 19-H), 1.192 (3H, s, 18-H), 2.236 (3H, s 21-H), 3.325 (1H, dd, 3 J 11.2 Hz, 3.9 Hz, 12-H), 67 3.464 (1H, s, OH), 3.5140 (1H, Mn, 3.598 (1H1, dd, 3J 9.6 Hz, .9.6 Hz, 17-H), 4 .255 (1H, s, OH) 5.383 (1H, m, 6C 8.275Q 19.414Q AC-19), 24.400T (C-11) 24.581T 27.443T 30.062T 32 .972Q (C-21) 34.543T 35.864D 36.975S 37.337T 42.144T 43.565D 55.101.S 57.038D 71.597D 73.558D (C-12), 85.566S 122.223D 138.932S 217.011S 3fB,122.14-Trihydroxy-14S-pr-egn-5 -en-2O -one 6 H 0. 996 (1H, s, 19-H) 1.144 (3H, s, 18-H) 2.221 (3H, s 21-H) 3.339 (1H, dd, 3 J 9.4 Hz, 9.4 Hz, 17-H) 153.492 (1H, m, 3.629 (1H, ad, J 11i.1 Hz, 3.9 Hz, 12 3. 712 (lH, s, OH) 4. 32 5 (1H, s, OH) 5. 3 83 (IH, mn, Examples 20 to 28 illustrate the procedures whereby the intermediate compounds may be prepared to form the first monosaccharide EXAMPLE Methyl-4,6-0-benzvlidene-a-D-cglucopvranoside (32) A mixture of methyl-ct-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 min. The white precipitate is filtered and washed with diethyl ether. The solid material is stirred with a solution of sodium metabisuiphite (10 k soin) 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 21 Methyl-4,6-0-benzvlidene-2-0-tosyl-a-D -lucopyranoside(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.

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, 10 EXAMPLE 22 Methyl-4, 6-0-benzylidene-3-0-methvl-a-D-altropyranoside (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) g, 52 EXAMPLE 23 Methyl-6-bromo-4 -0-benzoyl-3-0-methyl-6-deoxy-ce-Daltropyranoside The benzylidene altroside (34) (10 g, 33 mmol) is added to a solution of N-bromosuccinimide (7.6 g) and barium carbonate (20 g) in carbon tetrachloride and the reaction 69 mixture is refluxed at 75 0 C for 3 hours. The reaction mixture is filtered and the carbon tetrachloride layer is washed with water. The organic layer is dried (MgSO4), filtered and evaporated to yield 6-bromo-altroside (9 g, 69 EXAMPLE 24 Methyl-4-0-benzovl-3-0-methyl-6-deox-a-D-altropyranoside S(36) Sodium borohydride (18 g) in water (30 ml) is added 10 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 75 0 C for 1 hour and then it is filtered. The ethanol is evaporated and the remaining aqueous layer is extracted with chloroform (X 3).

The chloroform is dried (MgSO 4 filtered and evaporated, to yield the 6-deoxy-altroside (36) (5 g, 72 EXAMPLE 4-0-Benzovl-3-0-methvl -6 -deoxy- -Dphenvlthioaltropyranoside (37) Phenylthiotrimethylsilane (5 ml) and trimethylsilyltrifluoromethane sulphonate (2 ml) are added at 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 column chromatography eluting with chloroform acetone to yield the a-phenylthioaltroside (37) (4 g, 63 EXAMPLE 26 4 -0-Benzovl-3-0-methyl-2-Rhenlvlthio-2. 6-dideoxv-ag-Dfluorocvrnaropvranoside (38) Diethylaminosulphurtrifluoride (0,65 g) is added rapidly to a solution of the a1g-phenylthioaltroside (37) g, 1,33 mmol) in dichioromethane at OOC. The reaction is stirred for 0,5 h at 0OC and then saturated sodium bicarbonate is added. The dichioromethane is separated .:from the aqueous layer, dried (MgSO 4 f iltered and evaporated to yield the (x-fluorocymarose (38) (450 EXAMPLE 27- 4-0-Benzoyl-3-0-methvl-2 -0-t-butvldimethvlsilvl-a3-Dphenylthio-altroside (39) is1 The 6-deoxy altroside (37) (S g) is silylated using tbutyldimethylsilylchloride (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 f inally with water.- The ethyl acetate layer is dried (MgSO 4 I f iltered and evaporated to yield the silylated benzoyl phenylthioaltroside (39) (80 Is) EXAMPLE 28 3 y1-2-0-t-bu Phenvlthioaltroside The silylated benzoyl phenylthioaltroside (39) (6 g) is treated with sodium methoxide (100 ml) for 4 hours. The methanol is evaporated and water is added to the reaction.

The water layer is acidified (pH 5, ACQE) and extracted with ethyl acetate. The ethyl acetate is washed with water, dried (MgS04), filtered and evaporated to yield silylated methyl phenylthioaltroside (40) Examples 29 to 37 illustrate the procedures synthetic whereby the intermediate compounds may be prepared to form the second monosaccharide EXAMPLE 29 1,2 5,6-Di-O-isopropylidene-a-D-Qlucofuranose (42) o Sulfuric acid (40 ml) is added dropwise to a solution of a-D-glucose (41) (50 g, 0,28 mol) in acetone (1 2) at 10 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 S. chloroform The chloroform is dried (MgSO 4 filtered and evaporated. Crystallization from cyclohexane yielded the di-isopropylidene glucose (42) (41 g, 57 EXAMPLE 1,2 5.6-Di-0-isopropylidene-3-0-methyl-a-D-qlucofuranose (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 h, 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 (X The ether layer is dried (MgS04), filtered and evaporated to yield the methyl protected glucose (43) (38 g, 83 EXAMPLE 31 (44) The methyl diisopropylidene compound (43) (38 g, 0,14 mol) is dissolved in acetic acid (50 li, 700 ml) and the solution ref luxed 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-at1- ;glucopyranoside (44) (13 g, 50 k) EXAMPLE 32 *Methyl 3-0-methvl-aB--D-QlucoDovrafloside The 3-0-methyl-af3-glucopyranoside (44) (10 g) is dissolved in methanol (50 ml) and HCl (conc.) (1 ml) and ref luxed overnight.- Solid NaHCO 3 is added and the reaction *i filtered. The methanol is evaporated to give 1,3-dimethyl-afl-D-glucopyranoside EXAMPLE 33 Methyl 4. 6-0-benzvlidene-3-0-methl-a-crlucoDraloside, (46) The glucopyranoside (45) (8 is stirred at room temperature in a solution of benzalaldehyde (20 ml) and zinc chloride (5 After 24 hours, ice is added and the aqueous layer is extracted with chloroform. The chloroform layer is dried (MgSO 4 )1 filtered 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-ap3-glucopyranoside (46) 73 EXAMPLE 34 Methyl 4-0-benzovl-0-methyl-6-deoxv- qlucopyranoside (47) The benzylidene compound (46) (5 g) is refluxed at 80 0

C

in a mixture of N-bromosuccinimide (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 (MgSO 4 filtered and evaporated to give the bromo compound SeThe bromo compound (4,3 g) is dissolved in a solution 10 of ethanol (300 ml) and nickel chloride (8,6 g) at 0°C. To this solution, sodium borohydride (8,6 g) in water (50 ml) is added dropwise over a period of 15 minutes. The reaction mixture is refluxed at 100°C for 45 minutes, cooled, filtered and evaporated. Chloroform is added, and the chloroform layer is washed with water, dried (MgS0 4 filtered and evaporated to give the 6-deoxy sugar (47) EXAMPLE 4-0-Benzovl-3 0-methyl-l -henvlthio-6-deoxy-a~t- Tlucopvranoside (48) The 6-deoxy glucopyranoside (47) (3 g) is dissolved in dichloromethane (50 ml) To this solution, phenylthiotrimethylsilane (2 g) 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) EXAMPLE 36 4 0-Benzovl -3 0-methyl -2 -O-pivaloyl- -phenylthio-6-deox-y- 0d-alucopyranoside (49) To a solution of the glucopyrarloside (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 layer is washed with HCl (6 The organic layer is dried (MgS0 4 filtered and 10 evaporated to give the pivaloyl ester (49) (80-1).

EXAMPLE 37 4-0-BenzoVl-3-0-methvl-2-0-rivalol-l-f luoro-6-deox-3- K: I iuconyranoside 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 OOC. After 1 hour, saturated sodium bicarbonate is added. The dichioromethane layer is dried (MgSO 4 filtered and evaporated. The fl-fluoropyranoside (50) is purified by silica gel column chromatography eluting with ethyl acetate:hexane (yield Example 38 illustrates the synthetic procedure whereby the compound 3-0- (4-0-benzoyl-2-phenylthio-g-Dcymaropyranosyll -12, l4I-dihydroxy-pregnan-5-ene-20-one(5l) may be prepared.

EXAMPLE 38 f4-0-benzovl-2-phenylthio--D-cYTmaropralosylI -12. 149dihvdroxv-Preqn-5-en-20-ofle (51) Tin chloride (190 mg, 1 mmol) is added to a solution of 3,12,14 19-trihydroxy pregnan-5-ene-20-one (15) (100 mg, 0,28 mmol) and the fluorocymaropyranoside (38) (210 mg, 0,56 mmol), in dry diethyl ether and 4A molecular sieves at 0 C. The reaction mixture is maintained at -15 0 C for 3 days. Saturated sodium bicarbonate is added to the reaction mixture. The ether layer is dried (MgSO 4 filtered and evaporated. The product is purified by silica gel column chromatography eluting with chloroform methanol 5:0,5) to yield the glycoside (51) (30 mg, 1: 0 Examples 39 to 41 illustrate the synthetic procedures whereby the cymarose and thevetose moieties may be coupled.

EXAMPLE 39 Thevetose-cvmarose dissaccharide (53) A solution of thevetose (50 A) (1,5 cymarose (1,3 and molecular sieves 4A in dichloromethane is stirred at. room temperature for 1 hour. The reaction mixture is cooled to -15CC, and tin (II) chloride (0,8 g) and silver trifluoromethanesulphonate (1,1 g) are added.

The mixture is stirred at -15°C for 16 hours, after which triethylamine 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-cymarose 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 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 chromatography (acetone:chloroform, 0,5:9,5) yield EXAMPLE 41 Thevetose-cvmarose dissaccharide To a solution of the dissaccharide (54) (80 mg) in ;dichloromethane (10 ml), diethylamino sulphur trifluoride 10 (80 pl) is added at 0°C. After stirring at 0°C for 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 15 dissaccharide (55) in a 65% yield.

EXAMPLE 42 The results of the following three bioassays on the appetite suppressant are set out below, viz.

a) Irwin Test; b) Acute Toxicity Test; and c) Oral Dose Anorectic Test.

a) Irwin Test The purpose of this test was to evaluate the appetite suppressant of the invention produced from a plant extract as hereinbefore described, according to the reduced animal Irwin test for tranquillising and sedative action.

Experimental Procedure The appetite suppressant was extracted from plant material by the Applicant by the method as hereinbefore described and administered to two of four groups of three animals each: one group receiving no treatment, one group receiving the solvent dimethylsulfoxide (DMSO), one group receiving the test sample at 50 mg/kg, and one group receiving the test sample at 300 mg/kg. Treatment took 999*** o place by intraperitoneal injection, and observations were 10 made at specific intervals up to five hours post treatment.

Only symptoms other than those observed in the DMSO-treated animals were used in the interpretation of the results.

Results It was clear that the solvent, DMSO, had a marked 9 effect on the animals, especially on the heat regulating mechanism. Body temperatures of all the animals treated with the solvent, alone or together with the test sample, showed a marked drop.

Animals in the low dose group showed decreased dispersion in the cage and decreased locomotor activity, as in all the other groups, including the control group.

Apathy was seen in the same degree as in the DMSO-treated group. Decreased respiration was observed 15-60 minutes after treatment. Ptosis (closing of the eyelids) was also observed to a larger degree than in the DMSO group. A 0O CI S 6 0 4 C.

0000 pinna (ear) response was seen as well as a positive finger response, indicating fearfulness. Body temperature dropped to 32,7 0 C after treatment.

Animals in the high dose group showed as in the other groups an initial decreased dispersion in the cage and decreased locomotor activity, but showed increased dispersion and locomotor activity before death, which occurred approximately 1 hour after treatment. Severe clonic symmetrical convulsions occurred 30 minutes after 10 treatment. Respiration decreased initially, but increased before death. A pinna (ear) response was delayed and a positive finger response was observed, indicating fearfulness, both as observed in animals in the low dose group. Body temperature dropped to 30,7 0 C after treatment.

15 Increased positional passivity was observed as well as decreased body tone. Abnormal limb rotation was observed, the grip strength decreased, no pain response was present and loss of righting reflex occurred.

Discussion When compared with the control and DMSO-treated animals, animals receiving the low dose (50 mg/kg) only showed decreased respiration and an increased degree of ptosis. Animals receiving the high dose (300 mg/kg) of the test sample reacted very intensely by showing 'convulsions and death. All other observations made in these animals can be ascribed to the animals being in convulsions and 0* o 79 dying. Signs suggestive of tranquillising and sedative actions such as marked decreased dispersion in the cages, decreased locomotor activity and apathy in the test groups that could be ascribed to the test sample were not seen.

It can therefore be concluded that the test sample is lethal to mice at 300 mg/kg and has respiratory suppressive effects on mice at 50 mg/kg, when given intraperitoneally with DMSO as solvent.

b) Acute Toxicity Test S 10 The purpose of this test was to gain information on the .toxicity of the test sample.

Experimental Procedure A plant extract prepared in accordance with the invention as hereinbefore described, and having appetite suppressive action was purified and one test sample was tested at increasing doses by oral treatment in mice. Two animals were used per dose group, except in the highest dose group where only one animal was treated. Animals were examined for good health and their body masses determined on the day of treatment.

Doses ranged from 100 mg/kg up to 3 028,5 mg/kg. The dose was calculated and mixed into prepared potato starch, so that each animal received a total dose of 0,2 ml.

Animal 13 received 0,25 ml. Potato starch was prepared by mixing 20 g starch into a small volume of cold water, and adding it to boiling water, to make up a volume of 1 litre.

The suspension was allowed to cool to room temperature before dosing.

Animals in groups 1 and 2 were treated on the same day.

They were observed for 24 hours and if no signs of toxicity developed, the next group was treated. The same approach was followed until all the animals were treated. This 10 schedule was followed to ensure that animals were not unnecessarily treated when an acute toxic dose had been reached in the previous group.

Animals were observed for clinical signs of toxicity immediately (1-2 hours) after treatment and daily S15 thereafter. Body mass was determined once a week and total food and water intakes of each animal were measured.

Surviving animals were euthanased by intraperitoneal injection of pentobarbitone sodium (commercially available under the trade name Euthanaze, Centaur

R

on day 14 of the experiment. A post-mortem examination was performed on these animals, as well as on the one animal which died during the experiment. Samples for histopathology were collected.

81 Results Group 1 (Control Group) No clinical signs of toxicity were observed during the 14-day observation period. Food and water intakes were within the normal parameters. Changes in body mass were also within normal parameters. No histopathological changes were recorded in the liver samples.

Group 2 (100 mq/kq) SNo clinical signs of toxicity were observed during the 10 observation period. Food and water intakes were normal and changes in body mass over the observation period were also normal. No macroscopical pathology was observed and no histopathological or morphological changes were recorded in the liver samples.

Group 3 (200 ma/kc) Animals in this group showed no clinical symptoms of toxicity during the experiment. Food and water intakes were normal, as was the change in body mass. No macroscopic pathology was observed, but the livers showed histopathological changes on examination. Cloudy swelling of the hepatocytes was mild in animal 6, but moderate in animal 5. Moderate hydropic degeneration also occurred in the hepatocytes of animal Group 4 (400 mc/kr) No clinical signs of toxicity were observed during the observation period, and no macroscopic pathology was observed during the post-mortem examination. Moderate cloudy swelling and mild hydropic changes of the hepatocytes were observed on histology.

Water and food intakes and the increase in body mass in animal 7 were normal. Animal 8 consumed almost double the total food intake of animal 7 (144,6 g and 73,9 g 10 respectively), but the increase in body mass was only 0,81 g compared to 2,7 g.

Group 5 (800 mc/kq) One animal (animal 10) died three hours after dosing without showing any specific signs. The other animal (animal 9) survived the entire observation period without any signs of toxicity. Water intake in the surviving animal was normal (42,42 ml), while food intake was high (134,2 The body mass increased by 2,85 g which was the highest of all animals in the experiment.

At the post-mortem examination of animal 10, which died shortly after oral dosing, the lungs were congested. No foreign body reaction which would have indicated inhalation of test material was present. No macroscopic pathology was observed in animal 9. Mild cytoplasmic vacuolisation 83 (hydropic degeneration) was present in animal 10, but moderate in animal 9. The glandular cytoplasmic appearance of the liver was classified as moderate in both animals.

Group 6 (1 600 mq/kr) None of the animals presented any clinical signs of toxicity during the duration of the experiment. No macroscopic pathology was observed at post-mortem examination, but moderate degenerative changes in the liver of animal 11 were observed at histopathological 10 examination. Animal 12 showed moderate cloudy swelling and mild hydropic changes of the hepatocytes. Food and water intakes were normal, as was the increase in body mass over the experimental period.

SGroup 7 (3 028,5 mc/kr) Only one animal was treated at this dose. This animal showed no signs of toxicity during the observation period, and no macroscopic pathology was observed. At histopathological examination, moderate cloudy swelling and hydropic degeneration of the hepatocytes was observed. The animal showed a loss of body mass over the observation period (-0,82 but food and water intakes were normal.

84 Discussion Since a very small number of animals were used in each dose group, it is difficult to make any conclusions. The fact that only one animal died at a low dose rate, without showing any symptoms, might indicate that death was not related to the test sample, but due to stress during and/or after treatment. No animals in higher dose groups died or showed any signs of toxicity, which further supports this assumption.

1 0 The increased food intake observed in animal 8 could possibly be ascribed to excessive spillage of food as was reflected in the small increase in body mass. It should be kept in mind that all the animals in this experiment were *"*only treated once, and that it is unlikely that an appetite suppressor will have a marked influence on either the food or water intakes, or body mass over a 14 day period, as was the case in this experiment.

From the histopathological examination of the liver samples, it was clear that the pathological changes were dose related, with animals receiving higher doses showing the extensive changes. The pathology observed was not metabolic of nature, but possibly test sample-induced. The changes were only degenerative and therefore reversible.

No signs of irreversible hepatocellular changes were observed.

It can, therefore, be concluded that only one animal died at a lower dose (800 mg/kg), but that the death was possibly not test sample related. None of the other animals in any of the dose groups showed any signs of toxicity during the 14 day observation period after treatment, or died as result of the treatment. A single oral dose of the test sample induced reversible doserelated hepatocellular changes.

"o c) Oral Dose Anorectic Test 10 The purpose of this test was to determine the activity of a plant extract prepared in accordance with the invention, and the minimum effective dose, and at the same time investigate any possible side-effects such as respiratory suppression, as experienced in the Irwin Test (referred to above).

Experimental Procedure Animals were allocated to treatment groups using randomisation tables. Each treatment group consisted of three animals, with 6 animals in the control group. The test sample was dosed to young female rats with body weight 100-150 g at acclimatisation, for three consecutive days.

Animals were identified by means of metallic ear tags and KMnO 4 skin markings for easy identification. Animals were housed individually in standard rodent polycarbonate cages, and water and powdered commercial rodent pellets were 86 available ad libitum. Water and food intakes were measured and calculated for each day. In order to find the minimum effective dose of the test sample, five doses were tested.

Treatment was by oral gavage, with the test sample suspended in potato starch.

The test substance was compound a white granular powder prepared from an extract from plant material in accordance with the invention, and the measured quantity of the test sample was mixed with prepared potato starch and 10 dosed. Mixing with potato starch took place immediately before dosing on each day. Before withdrawal of the dosing volume for each animal, the suspensions were mixed thoroughly using a Vortex.

A range of five doses was tested, with a control group 15 receiving only the carrier substance. Doses were chosen on the basis of the effects observed in the aforedescribed Irwin Test and were: Group 1: 0,00 mg/kg (Control Group) Group 2: 6,25 mg/kg Group 3: 12,50 mg/kg Group 4: 25,00 mg/kg Group 5: 37,50 mg/kg Group 6: 50,00 mg/kg 87 Results Treatment did not affect the health of the animals during the study period. Animals treated with the test sample in all dose groups, showed a significantly reduced S mean body mass gain over the total study period, and animals in three of the five treatment groups actually lost body mass.

Mean food intakes for all the treatment groups were reduced over the study period. Animals in the higher dose 1 0 groups showed an increased water consumption.

e•Respiratory rate in none of the animals in any dose group was significantly effected.

Animals in all dose groups presented with friable livers at post-mortem examination, but no macroscopic pathology was observed.

Discussion Data collected during the acclimatisation period confirmed that all animals included in the experiment were healthy and body mass gain was comparable between the animals.

The reduction, and in some animals even a loss, in body mass gain, in combination with the reduced food intake is 88 strongly indicative of suppression of the appetite centre.

Reduced food intake and reduced body mass gain was experienced even with the lowest dose group (6,25 mg/kg).

Actual loss in body mass was experienced in the 12,50 mg/kg group.

It is important to note that the treatment groups all had an increased water consumption when feed consumption decreased (Figure This could be due to a diuretic effect of the test sample, or to stimulation of the thirst S 10 centre in the brain.

The fact that no respiratory suppression occurred as had been observed in the acute toxicity test referred to above, with the intraperitoneal route, is seen as a positive aspect. This could be due to reduced absorption 15 from the gastrointestinal tract, with consequent reduced bioavailability. The bioavailability at the oral doses tested was, however, sufficient for the test sample to be effective. The slight reduction in respiratory rate 1 hour post treatment in most groups could be ascribed to filling of the stomach with the dose volume and consequent passivity of the animals.

The friable livers observed in the treatment groups could be due to a change in the energy metabolism secondary to the reduced food intake, causing increased fat metabolism and overload on the liver. If this was indeed 89 the case, these changes could possibly be regarded these changes as transitory which might recover with time after a steady state had been reached, or after withdrawal of the test sample. The possible effect on the liver also needs further investigation.

Since this study was intended primarily as a screening test, small groups of test animals were used. This makes statistical interpretation of the data difficult, especially where individual animals react totally oeeoo 1 0 differently. However, the data indicates that the test sample has appetite suppressive action, even at the lowest dose tested (6,25 mg/kg). No clinical signs of respiratory ""suppression occurred at the doses tested.

.EXAMPLE 43 Harvested Hoodia plants received either from the natural environment or through a cultivation programme are first stored at 4 0 C 'for a maximum of 48 hours. The plants are washed in tap water and thereafter sliced into 1 cm slices. The sliced pieces are all combined and then pressed through a hydraulic press at 300 bar pressure for a minimum of 0.5 hour per pressing. During the pressing the sap of.the plant is collected separately. The sap is stored at -18 0 C until further processing is required.

The sap is spray-dried under suitable conditions to obtain a free flowing powder. The moisture content in the powder is preferably less than 5% after spray drying and, if necessary, it is further dried in a vacuum oven or using a fluid bed drier.

Both the sap and the spray-dried material have been shown effective as an appetite suppressant in biological assays in rats.

Experimental S. 50 kg of Hoodia gordonii plants were washed with tap water and thereafter sliced into 1 cm slices. The sliced plants were then pressed through a hydraulic press at 300 bar for g a minimum of 0.5 hour per batch. The sap was collected and the mass was found to be 10 kg when Hoodia gordonii plants from the environment were used, and 20 kg when Hoodia gordonii plants from the cultivation programme was used.

15 The sap (500 g) was spray-dried using the following conditions: Flow rate 2.85 ml/min Inlet temperature 110-C Outlet temperature 70 0

C

Chamber temperature 78 0

C

The spray-dried powder obtained was a free flowing powder (22 g) with a moisture content of 6.9%.

The spray dried powder was analysed for active ingredient concentration using HPLC techniques. The concentration of the active was determined to be 13 g/kg of spray dried powder.

HPLC Analysis Method Eluant Acetonitrile: water isocratic Column Reverse phase C-18 UV absorbance 225 nm Flow rate 1 ml/min Injection volume 101 Method Spray-dried powder (10 mg) was dissolved in water (0.5 ml) 10 and acetonitrile (0.5 ml) 10Al of this solution was injected into the HPLC and the concentration of the active compound was determined using a standard curve which S*was prepared from the pure compound (1) SEXAMPLE 44 15 The results of a study designed to assess the possible anorectic effects of compound in the rat are presented below. In the following, the samples tested are pure sap (Sample spray-dried sap (Sample 2) and active moiety (Sample Samples 1 and 2 are the sap and the spray- 20 dried sap respectively, as described in Example 43 above.

Sample 3 is solvent-extracted compound of 295% purity.

Sample 1 to 3 were each administered as a single oral dose to male Wistar rats. Two additional control groups received vehicle (distilled water or DMSO). Orally administered fenfluramine (7.5 mg/kg) was included as a reference standard.

Sample 1 (pure sap) administered orally, produced dosedependent reductions in food consumption which were statistically significant at doses of 1600 mg/kg and above when compared with vehicle-treated controls. Concomitant reductions in bodyweight (or growth rate) were also recorded. On the day of dosing, statistically significant increases in water consumption were recorded at 3 hours post-dose (6400 and 10000 mg/kg) and 6 hours post-dose (10000 mg/kg). Between 24 and 48 hours post-dose, statistically significant reductions in water consumption were recorded at doses of 3200 mg/kg and above.

Sample 2 (spray-dried sap) administered orally at 76 mg/kg also produced statistically significant reductions in food consumption and bodyweight when compared with vehicletreated animals. No statistically significant effects on S. 10 water consumption were recorded.

Sample 3 (active moiety) produced statistically significant reductions in food consumption at an oral dose of mg/kg. No statistically significant effects on bodyweights were produced by the active moiety although examination of the data revealed a slight delay in growth when compared with vehicle-treated control animals. No statistically significant effects on water consumption were recorded.

SThe reference standard, fenfluramine (7.5 mg/kg), produced statistically significant reductions in food consumption at 20 6 and 24 hours post-dose when compared with the relevant vehicle-treated control group. No statistically significant effects on water consumption or bodyweight were recorded.

No treatment-related effects on the livers were recorded.

0%.

Identity sample 1 (pure sap) Appearance Brown liquid Storage'conditions -20 0 C in the dark TEST SUBSTANCE Sample 2 (spray-dried sap) Powder Room temperature in the dark Pure spray-dried sap Distilled water Sample 3 (active moiety) White powder 4 0 C in the dark Purity Vehicle Pure sap.

Distilled water 2t95% Dimethyl.suiphoxide

(DMSO)

Experimental Procedure Fifty-five male Wistar rats were used for the study.

Bodyweights, food consumption (food hopper weight) and water consumption (bottle weight) were recorded daily at the same time each day from the day of arrival until the termination of the study.

On Day 1, the rats received a single oral (gavage) dose according to the following table: S.Group n Oral treatment Dose (mg/kg) 1. 10 1 5 Vehicle (distilled water) 2 4 Sample 1 (pure sap) 800 3 5 Sample 1 (pure sap) 1600 4 5 Sample 1 (pure sap) 3200 5 5 Sample 1 (pure sap) 6400 6 5 Sample 1 (pure sap) 10000 7 5 Sample 2 spray-dried sap 38 8 5 Sample 2 spray-dried sap 76 9 5 Sample 3 (active moiety) 10 5 Sample 3 (active moiety) 11 3 Fenfluramine 12 3 Vehicle (DMSO) Groups 1 8 were dosed using a constant dose volume of ml/kg and groups 9 12 were dosed using a dose volume of 1 ml/kg.

Food and water consumption were also measured at 1,3 and 6 hours after dosing on Day 1.

Following the measurements of Day 8, the animals were killed by carbon dioxide asphyxiation, and the livers excised and placed in 10% buffered formalin, prior to histology.

Paraffin wax sections of each liver were taken at 4 5 Am and stained with haematoxylin and eosin. Additional sections were cut on a cryostat at 12 gm and stained for fat with Oil Red 0 (ORO).

Data Analysis The post-dose food and water consumption measurements and bodyweights at each time-point for the P57-treated animals were compared with those for the relevant, similarlytreated vehicle control group using analysis of variance 10 followed by Williams' test for comparisons with controls.

0 000 The data for the fenfluramine-treated animals was compared with that for the vehicle-treated control group using Student's t test.

Results The results are summarised in the tables.

Sample 1 (pure sap) administered orally produced marked, 0.dose-related reductions in daily food consumption. The duration and amplitude of these reductions in food consumption were dose-dependent. At 24 hours post-dose, Sample 1 (pure sap) produced statistically significant reductions in food consumption at doses of 1600 mg/kg and above when compared with vehicle-treated controls. The highest dose of Sample 1 (sap) (10000 mg/kg) produced statistically significant reductions in food consumption on a daily basis up to 5 days post-dose.

Sample 2 (spray-dried sap) and Sample 3 (active moiety) produced marked and statistically significant reductions in food consumption at oral doses of 76 and 5.0 mg/kg respectively. In both cases the effects lasted 48 hours post-dose.

The reference standard, fenfluramine (7.5 mg/kg, p.o.) produced statistically significant reductions in food consumption at 6 and 24 hours post-dose when compared with the relevant vehicle-treated control group (Group 12).

Sample 2 (spray-dried sap) and Sample 3 (active moiety) produced no marked, dose-related effects on water consumption. On the day of dosing, the pure sap produced statistically significant increases in water consumption at 3 hours post-dose (6400 and 10000 mg/kg) and 6 hours postdose (10000 mg/kg). Two days after dosing however, statistically significant decreases in water consumption were recorded in animals receiving Sample 1 (sap) at 3200, 6400 and 10000 mg/kg. These reductions however, were not clearly dose-related and only occurred between 1 and 2 days S 15 post-dose. The biological significance of these effects therefore remains unclear.

Sample 1 (pure sap) produced dose-related, statistically significant effects on bodyweights when compared with the vehicle-treated control group (Group When administered orally at doses of 3200 mg/kg and above, Sample 1 (pure sap) produced statistically significant reductions in bodyweight or decreased growth rates when compared with vehicle-treated animals. These effects were statistically significant from 48 hours post-dose until the end of the study.

Sample 2 (spray-dried sap) administered orally at 76 mg/kg also produced statistically significant reductions in growth of the animals when compared with the vehicletreated control group (Group These effects were statistically significant between Days 3 (48 hours postdose) and 5 inclusive.

Although Sample 3 (active moiety) appeared to delay. the growth of the animals at the highest dose (5.0 mg/kg) when compared with the relevant vehicle-treated control group 97 (Group 12), this effect was not statistically significant.

Fenfluramine, (7.5 mg/kg) produced no marked or statistically significant effects on water consumption or bodyweights when compared with the vehicle-treated control group (Group 12).

No treatment-related effects on the livers were recorded.

*o ooo eo 0ooo S.0 TABLE 1la Effects of oral administration on food consumption In the rat (daily pre-dose data) Group Oral treatment Dose (mg/kg) Group mean food consumption (q sd) between Days: -2- 5 -4 -3 -2 -1 1 Vehicle (water) 27.8 24.2 27.6 28.3 29.4 1.54 *1.83 ±3.67 ±3.50 ±2.66 2 Sample 1 gap 800 28.3 24.9 27.7 28.4 30.1 ±0.82 ±0.76 ±1.51 ±0.27 3 Sample 1 sap 1600 29.0 25.0 27.4 28.8 29.5 ±1.39 ±2.16 ±1.96 ±0.61 ±1.55 4 Sample 1 sap 3200 27.2 25.1 26.0 28.5 27.6 ±2.33 2.46 ±2.52 ±2.29 '±1.15 Sample 1 sap 6400 28.7 25.3 27.3 29.2 30.3 1.64 ±1.73 ±1.45 *1.09 *0.90 6 Sample 1 sap 10000 28.5 .23.7 26.0 27.0 28.7 2.38 ±2.73 ±2.31 ±3.50 ±2.26 7 Sample 2 spray-dried 38 28.1 23.9 24.5 27.6 28.5 ±1.79 ±2.30 ±1.61 ±1.87 8 Sample 2 spray-dried 7 6 28.7 26.5 27.1 28.7 28.9 1.55 ±1.01 ±1.99 ±1.37 9 Sample 3 active moiety 2.5 28.8 26.4 29.0 29.4 29.5 ±3.12 ±1.99 ±1.76 ±2.81 Sample 3 active moiety 5.0 28.3 25.8 28.1 28.0 28.5 ±2.1 ±1.86 ±2.65 ±2.65 ±3.03 11 Fenfluramine 7.5 29.1 25.3 27.0 30.8 29.7 0.66 *4.03 ±1.53 ±0.54 2.84 .12 Vehicle (DMS0) 27.9 26.7 28.7 28.1 30.5 ±2.11 ±1.99 *4.06 sd Standard deviation 0 0 9* 0 TABLE lb administration on food consumption In the rat (daily post-dose data) Effects of oral Group Oral treatment Dose (mg/kg) G roup mean food consumption (a sd) between Days.

12 12-3 3-4 4-5 5-6 6-7 7-8 1 Vehicle (water) 29.5 29.8 30.6 31.8 30.7 31.7 32.9 3.15 ±t2.84 ±3.49 3.21 2.24 ±3.03 3.18 2 Sample 1 sap 800 26.1 29.3 30.7 30.9 33.3 32.7 40.1 ±1.49 ±1.15 ±0.60 ±1.69 ±0.80 I± 13.40 3 Sample 1 sap 1600 22.6 28.9 30.9 30.9 34.1 33.7 33.8 ±2.06 ±2.54 ±1.22 ±1.38 ±1.69 ±1.61 4 Sample 1 sap 3200 20.1 19.0 22.8 28.0 31.4 32.3 33.0 1.88 ±1.77 ±3.14 2.02 ±2.91 ±3.01 Sample 1 sap 6400 18.2** 14.80 18.4* 22.4'" 26.9 31.0 32.0 ±4.18 ±1.75 ±0.97 ±3.01 ±2.81 ±2.31 ±2.34 6 Sample 1 sap 10000 16.1* 12.4 16.0~ 19.7 22.6* 30.1 32.6 ±2.61 ±3.15 ±4.31 ±5.70 ±4.79 ±5.90 7 Sample 2 spray-dried 38 256 27.3 30.3 31.0 31.8 31.1 31.8 ±0.95 ±2.06 ±2.13 ±1.63 ±1.94 2.45 8 Sample 2 spray-dried. 76 24.2 25.2* 29.9 30.2 31.2 32.3 33.1 ±3.25 ±3.24 ±1.85 ±2.28 2.26 ±t1.44 ±0.61 9 Sample 3 active moiety 2.5 26.8 29.1 31.7 34.0 34.4 33.1 34.8 ±3.43 ±3.08 ±2.95 ±4.32 ±4.11 ±3.71 Sample 3 active moiety 5. 0 22.1 t 2 l.Ott 27.6 30.5 33.0 32.4 33.0 ±2.19 ±3.07 ±5.26 ±3.33 3.16 3.25 ±3.84 11 Fenfluramlno 7.5 22.t~ 31.9 32.7 33.0 30.4 32.7 32.4 ±0.84 ±2.50 2.55 ±0.23 ±1.90 ±1.60 12 Vehicle (DMSO) 29.9 30.6 30.1 32.4 31.8 32.8 33.3 +4.43 ±4.17 ±5.2 ±3.08 ±3.98 ±3.76 ad Standard deviation 0 Groups 2 8 were compared with vehicle Group 1: W-005, At<0.01 Groups 9 11 were compared with vehicle Group 12: lp<O.05, IIp<0.01 '.0 ~0 6 6 ~6 6 *OB 6* C C C C a. C. ~g C CC C C *C C C C C C p C CC* *CS

C

TABLE 2a Effects of oral administration on water consumption In the rat (daily pre-dose data) Group Oral treatment Dose (mg/kg) .Group mean water consumption (q rd) between Days: .2- .4 -3 -2 -1 1 Vehicle (water) 40.9 34.8 37.6 33.5 32.2 *4.61 £4.15 £5.63 £7.42 ±6.32.

2 Sample 1 sap. 800 38.6 37.1 36.4 28.1 30.4 1.96 ±9.74 £4.81 £1.83 £4.75 3 Sample 1 sap 1600 43.4 35.9 38.4 31.1 36.5 £3.84 £4.56 ±4.47 ±5.39 4 Sample 1 sap 3200 40.1 33.3 37.3 31.3 31.7 5.58 3.01 £4.46 £3.48 3.18 Sampla 1 sap 6400 43.8 36.3 35.4 34.0 35.1 £9.02 8.18 £6.62 5.72 6 Sample 1 sap 10000 37.4 32.7 33.2 29.0 32.2- ±3.35 *±4.86 5.11 ±3.27 7 Sample 2 spray-dried 38 40.0 35.8 34.7 30.2 31.4 £4.92 ±3.20 ±1.88 ±2.98 8 Sample 2 spray-dried 76 38.6 37.0 48.8 31.6 39.0 1.98 £1.96 £21.6 £4.56 ±17.27 9 Sample 3 active moiety 2.5 42.0 37.0 34.1 28.0 31.6 6.70 £5.05 £3.16 ±2.58 £3.12 Sample 3 active moiety 5.0 40.9 34.2 32.7 28.2 33.1 £3.00 £1.26 ±1.65 ±4.82 11 Fenfluramine 7.5 47.0 35.5 34.7 30.9 31.6 5.3 £7.4 9 £3.73 ±2.12 £2.80 12 Vehicle (DMSO) 43.3 34.5 35.2 28. 31.4 5.67 £4.9j7 t£4.34 ±4.64 ±6.44sd Standard deviation

C

C C

C..

TABLE 2b

C.

C C. C C C C Effects of oral administration on water consumption In the rat (daily post-dose data) Group Oral treatment Dose (m g/kg) Gopmean water con umtion (II±s)be e as 1-2 2-3 3-4 4-5 5-6 6-7 7-8 1 Vehicle (water) 34.9 36.9 38.0 37.2 37.7 35.3 36.5 ±6.06 ±7.59 ±6.18 ±5.54 ±2.86 .±5.85 2 Sample 1 sap 800 30.9 34.4 38.2 35.9 39.5 28.B 31.8 ±8.12 ±13.71 ±13.51 ±11.20 ±1.22 3 Sample 1 Sap 1600 29.2 31.7 41.3 34.6 48.1 37.8 36.9 ±5.35 ±11.21 ±4.10 ±12.27 ±7.28 ±9.28 4 Sample 1 sep 3200 35.9 26.2 30.5 34.1 45.8 51.0 42.6 ±2.66 ±2.44 ±4.80 ±18.54 ±35.21 ±13.88 Sample 1 sap 6400 33.4 27.4* 32.6 35.4 46.2 36.2 35.9 ±8.13 ±10.67 ±10.78 ±8.72 ±6.72 ±9.58 6 Sample 1 sap 10000 31.7 28.r, 32.4 36.6 40.7 38.0 37.5 ±8.85 ±8.87 ±6.50 ±11.51 ±6.66 ±6.21 7 Sample 2 spray-dried 38 36.0 34.6 38.2 39.6 42.7 45.6 46.1 ±1.79 ±7.16 ±7.09 ±9.74 ±17.15 ±9.49 8 Sample 2 spray-dried 76 45.0 39.1 46.9 35.9 41.9 36.9 38.1 ±16.59 ±18.34 ±3.40 ±12.37 ±8.47 8.93 9 Sample 3 active moiety 2.5 32.2 36.1 38.3 41.5 34.7 33.0 35.3 ±12.42 ±11.71 ±16.60 ±7.57 ±4.20 ±8.70 Sample 3 active moiety 5.0 33.9 31.5 35.1 37.7 39.5 37.4 37.8 2.40 ±8.12 ±3.82 ±5.99 7.78 ±11.07 ±6.42 1 Fenfluramine 7.5 34.1 37.2 36.7 33.8 33.7 32.1 33.6 ±1.48 ±3.92 ±2.89 ±5.43 ±1.93 ±2.50 12 Vehicle (DMSO) 40.7 3. 329 52 3.8 32.3 32.0 937 ±7.07 ±11.491 9.82 1±7.44 ±7.22 sd Standard deviation Groups 2 8 were compared with vehicle Group 1: p <0.05 Groups 9 11 were compared with vehicle Group 12 (no significances) TABLE 3a Effects of oral administration on bodyweight in the rat (daily pre-dose data) Group Oral treatment Dose (mglkg) Group mean bodyweight (q sd on Day: -4 -3 -2 -1 1 Vehicle (water) 130.9 150.7 157.3 168.1 177.5 ±5.50 t5.3 7 5.29 ±h6.20 ±E6.70 2 Sample 1 sap 800 131.6 150.1 158.5 1.69.6 177.7 k 4.34 ±4.84 ±4.35 ±4.99 ±4.10 3 Sample 1 sap 1600 130.1 148.6 156.7 167.5 176.6 ±6.59 ±6.38 ±6.04 ±6.37 4 Sample 1 sap 3200 130.8 147.7 154.4 165.2 175.8 ±7.56 ±8.06 ±8.43 ±9.10 Sample 1 sap 6400 132.6 151.3 158.4 169.0 178.1 ±7.01 ±7.23 ±8.50 ±8.79 7.75.

6 Sample 1 sap 10000 132.3 151.8 157.3 167.1 175.4 ±8.75 ±9.08 ±9.37 ±10.41 ±10.90 7 Sample 2 spray-dried 38 131.7 149.0 156.2 166.7 175.6 :E8.28 ±L5.85 ±L5.81 :t:6.54 *8.42 8 Sample 2 spray-dried 76 130.0 146.1 155.9 166.0 175.1 ±6.00 ±13.59 ±6.87 ±6.55 9 Sample 3 active moiety 2.5 132.6 148.9 1567.3 169.8 179.4 ±7.63 ±8.61 :E8.91 ±8.96 ±8.71 Sample 3 active moiety 5.0 133.5 150.5 168.8 171.0 179.0 ±6.45 ±9.55 ±8.48 ±7.72 ±9.20 11 Fenfluramrlne 7.5 133.2 152.7 160.0 170.0 182.8 ±9.09 ±9.82 ±9.15 ±10.21 12 Vehicle (DMS0) 129.1 147.3 155.0 166.0 174.8 ±4.37 6.29.. ±5.91 ±8.26 6d Standard deviation 0 0 0** TABLE 3b 0 Effects of oral administration on bodyweight In the rat (daily post-dose data) Group Oral treatment Dose Imglkg) Group mean bodyweight (n ad) onDay: 2 3 4 5 6 7 8 1 Vehicle 185.4 192.6 202.0 211.2 220.2 227.2 235.0 242.8 water) :0.77 ±7.16 ±10.17 ±7.98 ±10.35 ±10.28 *11.82 :h11.97 2 Sample 1 Sap 800 186.0 187.0 198.5 206.8 214.8 222.0 231.5 240.0 ±4.66 ±4.20 ±5.01 ±4.65 ±4.99 3.70 ±L3.05 3 Sample 1 sap 1600 185.0 180.0 193.2 204.0 212.4 223.0 232.6 240.4 ±8.20 ±6.42 1±6.40 ±5.81 ±6.33 ±7.70 ±6.00 4 Sample 1 sap 3200 181.8 184.8 186.2' 189.8'' 199.2"' 210.6"* 219.0' 228.4' *8U.00 ±0.67 ±9.99 *±9.34 10.21 11.29 ±t12,10a Semple 1 Sep 6400 106.6 185.0. 103.8" 1852" 191.2 201.0 213.0 222.0' 7.98 ±6.39 ±L6.07 ±t9.10 ±7.89 ±6.89 0.90 7.94 6 Sample 1 sap 10000 182.8 181.4 179.8 1 180.6, 195.6"0 192.2"a 203.4 212.4"' ±12.22 ±14.06 ±15.85 ±13.85 ±11.28 *10.99 11.00 ±11.35 7 Sample 2 spray-drlid 38 183.4 .185.8 195.8 205.6 214.4 222.6 231.4 239.0 ±0.11 9.23 ±7.79 ±L9.79 ±9.61 ±9.34 ±-10.02 :L11.4 0 a Sample 2 spray-dtied 76 100.6 103.4 188.6 0 190.2 0 200.0' 214.0 222.0 232.2 ±7.57 ±6.73 ±8.50 ±9.43 ±9.51 :L-9.4 9 :L9.60 9 Sample 3 active moiety 2.5 100.2 191.2 200.0 209.6 219.6 229.4 2311.4 247.0 *11.15 ±11.25 ±12.28 12.95 13.69 14.50 ±14.35 Sample 3 active moiety 5.0 188.4 192.0 192.4 201.0 209.4 219.8 220.2 236.0 9.93 9.04 ±11.27 ±12.70 11.00 12.20 ±13.95 11 Fonfluramine 7.5 190.3 190.3 197.7 207.7 217.7 224.3 234.3 243.3 9.71 *10.97 7.37 7.23 10.69 10.12 +±12.70 9.24 12 Vehicl~e 183.3 190.3 199.0 207.7 215.7 222.3 230.7 239.0 (DMSOI ±8.33 *10.20 *10.B2 *12 i .66 ±14.05 ±14.04. ±15.95 *17.35 sd Standard deviation Groups 2 0 were compared with vehicle Group 1: p<0.05, P<0.01 Groups 9 11 wore compared with vohicle Group 12 (no significances) 104 Histonatholov Report Histological examination was restricted to the liver. No treatment-related changes were detected for Sample 1 (liquid) Sample 2 (spray-dried sap) Sample 3 (active moiety), fenfluramine or the DMSO control group.

The findings recorded were of a similar incidence in control and treated groups.

TABLE

Microscopic pathology incidence summary Group 1 Group 2 Group 3 Group 4 Group S. Group 6 0 G00 1600 3200 6400 10000 Sex: Males mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Males on study 5 4 5 5 Animals completed S 4 5 5 5 Liver Examined 54 55 5 0 No abnormalities detected 0 0 1 2 3 3 Parenchymal inflammatory cell foci (Total) 0 1 0 0 0 3 Minimal 0 1 0 0 0 1 iepatocyte. hypertrophy centrilobular (Total) 0 0 0 0 0 1 Extramedullary haemopoiesis (Total200000 Minimal 2 0 0 0 0 0 llepatocyte necrosis focal (Total) 1 0 0 0 0 0 Minimal 1 0 0 0 0 0 Portal lymphoid infiltration (Total) 3 4 4 3 2 2 Minimal 3 4 4 3 2 2.

Eosinophilic hepatocytes focal (Total) 1 0 0 0 0 0 Minimal 1 0 0 0 00 Portal fibrosis (Total) 0 0 1 0 0 0 Minimal 0 0 1 0 0 0 Liver (ORO stain) Examined 5 4 555 No abnormalities detected 2 3 2 4 3 3 Hepatocyte fat -centrilobular (Total) 3 1 2 1 2 2 Minimal 3 1 2 1 2 2 flepatocyte fat -periportal (Total) 0 0 1 0 0 0 Minimal 1 o 0 1 0 0 .0

TABLE

(continued) i I I Group 7 Group B Group 9 Group 10 Group 11 Sex: Males Males on study Group 7 38 mg/kg Group a 76 mg/kg Group 9 2.5 mg/kg 5 30 76 2roup 10.

Anml 5 S 5 3e 4 II __5 Liver Examined No abnormalities detected Parenchymal. inflammatory cell foci (Total) Minimal Hepatocyte, necrosis focal (Trotal) Minimal Portal lymphoid. infiltration (Total) Minimal Portal leucocytes (Total) Minimal Liver (ORO staln) Examined No abnormalities detected Hepatocyte fat centrilobular (Total) Minimal

S

2 0 0 0 0 3 3 0 0 5 5 0 mg/kg mg/k 33 33 0 01 0 3 00 00 31 3.

0 33 Group 12 3 00* 2 1 a 121 0 107 EXAMPLE A further bioassay, which employed the same test samples as described in Example 44, is described below. Animals in this study received a restricted diet i.e. animals only received food between 12:00 and 3:00pm daily. This is different from all other biological assays conducted thus far, whereby food was available to the rats at lib.

Animals were acclimatised over a seven day period (days -7 to dosing took place from day 0 to day 6 at 9:00am by 10 oral gavage. The recovery period was from days 7 to day 13. Dosage groups are described in Table 1 below. It should be noted that the actual control group is labelled Group 09. Group 5 is a controlled group which received a diet equivalent to that of Group 4. The purpose of this group was to evaluate the effect a restricted diet has on the lives of the animals.

Results The results generated during the study showed that the acclimatization period was too short. Rats feed mainly during the night and the sudden change to a restricted access to feed for 3 hours during day-time, resulted in low daily intakes. The daily intake of feed was still increasing in most groups at the end of the acclimatization period when dosing with the test items started. As a result of this, the effect of the test materials did not significantly affect the food intake of the rats during the period of dosing.

The mean body masses for the different groups for day -7 to -1 and days 0 to 6 are shown in the Table Dl and Table D2.

The effect of the different dosages of the sap and spray-dried sap is shown in the accompanying graphs as change in body mass day 0 to 7 (Figure and change in body mass day -7 to 7 (Figure The loss in body mass is clearly dose-related especially with the higher dosages.

108 The histopathological examination of the livers did not show any significant pathology in the groups receiving the test items.

Food Food consumption was measured daily, during acclimatization and during the study. Food was available for a 3 hour feeding period daily, starting at 12:00 and ending at 15:00. The animals were fasted for the remainder of the S time. Animals in Group 5 received a measured quantity food S 10 on Day 1, equivalent to the average food consumption of Group 4 on Day 0. This controlled feeding pattern for S"Group 5, as determined from the average food consumption of Group 4 from the previous day, was followed for Days 1 7.

Water 15 Water was provided in standard containers. Water (Magalies Water Board Tap Water, suitable for human consumption) was available ad libitum. Water consumption was measured once daily, at the same time each day, after food consumption *"determination.

oooeo Acclimatization The animals were acclimatized for seven days before the start of the study, during which time food and water consumption were determined as described above. The body masses were determined on a daily basis during this time.

Study Design and Procedures TABLE 1 STUDY DESIGN GROUP TEST NUMBERS DOSE TEST ITEM 01 66 001 006 100 mg/kg Frozen sap 02 66 007 012 400 mg/kg Frozen sap 03 6d 013 018 1 600 mg/kg Frozen sap 04 66 019 024 3 200 mg/kg Frozen sap 05 66 025 030 CONTROL Elga Option 4 Purified Water 06 66 031 036 2.2 mg/kg Spray-dried sap 07 6d 037 042 8.8 mg/kg Spray-dried sap 08 66 043 048 35 mg/kg Spray-dried sap 09 66 049 -054 CONTROL Elga Option 4 Purified Water Route of Administration The test items were administered on a daily basis for seven days, using an intra-gastric needle. Animals were fasted for 18 hours prior to the item administration (starting at 09:00).

Duration of Treatment Animals were treated for seven consecutive days (from Day 0 Day Three animals of each group were sacrificed 24 hours after the last dosing (Day The remaining three animals were sacrificed 7 days after the last treatment (Day 13). This procedure was followed for all the groups except for Group 5 where three animals were sacrificed 24 hours after the last controlled feeding (Day the remaining three animals were sacrificed 7 days after the last treatment (Day 13).

110 Body Masses Body masses were determined daily, at approximately the same time each day for the duration of the study, including during the acclimatization period.

Euthanasia Three animals of each group were sacrificed 24 hours after the last dosing (Day 7).

The remaining three animals were sacrificed 7 days after the last treatment. This procedure was followed for all 10 the groups except for Group 5 where three animals were sacrificed 24 hours after the last controlled feeding (Day the remaining three animals were sacrificed 7 Days after the last treatment (Day 13). The animals were euthanased at the end of the study period with CO 2 gas.

S 15 Ophthalmoscopic Examinations Ophthalmoscopic examinations, using an ophthalmoscope, were

V

done prior to the first adminstration of the test item and at termination, in all animals in all groups.

Macroscopic Pathology A full post mortem examination was performed on every animal which was euthanased at the end of the study period.

Histopatholoqy Histopathological examination was performed on the liver of each of the animals.

S S S S S 5.5 5..

TABLE D.1 MEAN BODY MASSES GROUP WEEK Group Oral treatment Dose Mean body masses Standard deviation (mg/kg) Day -7 Day -6 Day -5 Day -4 Day -3 Day -2 Day -1 01. Sample 1 (Sap). 100 203.38 197.13 192.75 188.62 184.95 182.48 182.25 ±95.39 ±90.63 ±89.49 ±86.76 ±84.80 ±83.47 ±82.57 02 Sample 1 (Sap) 400 192.53 183.92 178.25 173.17 170.82 168.25 169.37 ±65.60 ±61.20 ±59.37 ±58.10 ±57.42 ±68.40 ±59.25 03 Sample 1 (Sap) 1 600 149.25 142.87 136.85 132.37 131.50 129.67 131.12 ±54.80 ±51.89 ±52.17 ±49.64 ±49.60 ±48.89 ±48.22 04 Sample 1 (Sap) 3200 224.15 214.45 207.10 201.82 198.25 194.83 196.77 i_80.70 ±77.25 ±76.38 ±75.42 ±74.82 ±75.34 ±74.56 Elga Option 4 214.55 204.85 198.57 193.48 192.40 190.87 190.15 purified water (control) ±74.90 ±72.41 ±71.79 ±68.49 ±67.48 ±67.39 ±65.24 06 Sample 2 (Spray-dried sap) 2.2 208.65 199.37 193.18 188.25 186.22 184.55 185.97 ±65.74 ±62.49 ±61.18 ±60.89 ±59.98 ±58.86 +58.76 07 Sample 2 (Spray-dried sap) 8.8 256.95 246.02 237.47 232.62 229.78 228.07 228.45 ±77.65 ±73.67 ±73.53 ±71.73 ±71.76 ±69.88 ±68.81 08 Sample 2 (Spray-dried sap) 35 194.37 185.83 177.53 172.05 170.10 167.25 168.00 ±43.74 ±42.70 ±41.10 ±40.13 ±39.49 ±37.61 ±38.83 09 Elga Option 4 171.52 162.67 154.95 151.38 149.63 148.30 149.07 purified water (control) ±69.81 ±62.68 ±61.83 ±59.48 ±57.66 ±57.12 ±56.01 S S S 5 S S S S S TABLE D.2 MEAN BODY MASSES GROUP WEEK (CONTINUED) Group Oral treatment Dose Mean body masses Standard deviation (mg/kg) Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 01 Sample 1 (Sap) 100 183.87 175.83 175.72 175.48 175.53 177.95 178.43 ±83.33 ±81.82 ±79.05 ±77.54 ±76.20 ±73.99 ±72.68 02 Sample 1 (Sap). 400 173.45 164.58 164.75 166.22 166.55 169.93 171.77 ±60.73 ±58.62 ±58.37 ±57.69 ±57.79 ±57.47 ±57.29 03 Sample (Sap) 1 600 134.38 129.20 127.53 127.20 126.70 128.00 128.07 ±46.01 ±44.74 ±43.20 ±41.36 ±39.19 ±39.22 ±38.66 04 Sample (Sap) 3200 199.60 196.38 192.20 189.05 186.57 186.05 185.68 ±75.16 ±73.96 ±71.20 ±69.11 ±66.29 ±67.45 ±65.73 Elga Option 4 194.27 187.93 181.97 177.53 174.73 172.85 171.45 purified water (control) ±67.46 ±65.48 ±65.01 ±64.73 ±61.08 ±58.63 ±56.79 06 Sample 2 (Spray-dried sap) 2.2 189.07 181.52 181.48 184.42 185.75 189.35 189.68 ±60.15 ±58.99 ±57.79 ±55.64 ±55.29 ±54.66 ±53.70 07 Sample 2 (Spray-dried sap) 8.8 230.28 221.55 220.17 221.80 222.82 224.82 224.90 ±69.32 ±68.02 ±66.63 ±63.88 ±63.56 ±62.38 ±62.05 OB Sample 2 (Spray-dried sap) 35 169.10 164.42 162.50 162.75 162.52 164.30 164.22 ±38.40 ±38.03 ±36.81 ±36.36 ±36.93 ±37.69 ±37.18 09 Elga Option 4 151.02 146.55 148.10 149.70 152.58 155.82 157.85 purified water (control) ±55.45 ±53.77 ±52.67 ±52.05 ±50.37 ±49.91 ±49.70 .00* 4 TABLE D.3 MEAN BODY MASSES GROUP WEEK (CONTINUED) Group Oral treatment Dose Mean body masses Standard deviation (gk) Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 01' Sample 1 (Sap) 100 185.38 234.73 236.73 234.07 236.33 239.07 238.43 (GHA I 35A) ±72.64 ±62.44 ±62.39 ±62.09 ±62.31 ±60.24 ±59.85 02 Sample 1 (Sap) 400 178.83 225.63 277.13 227.10 229.43 234.93 236.20 (GHA I 35A) ±58.24 ±13.05 ±14.18 14.03 ±16.97 ±18.35 15.97 03 Samp le 1 (Sap) 1 600 132.22 133.80 135.23 134.63 138.30 139.30 142.80 (GHA I 35A) ±55.17 ±455.74 ±54.96 53.03 51.10 ±49.51 04 Sample 1 (Sap) 3 200 188.57 199.63 198.90 198.70 194.73 194.93 197.93 (GHA 9 35A) ±66.14 ±61.07 ±57.48 ±54.55 ±52.78 0.78 ±51.57 Elga Option 4 173.97 172.98 157.80 158.87 160.80 163.40 167.80 purified water (control) ±54.29 ±52.06 ±58.62 ±57.76 ±567.67 56.27 ±58.49 06 Sample 2 (Spray-dried sap) 2.2 196.00 190.27 190.27 192.60 194.73 196.97 19B.60 (GH-A 1 59) ±t5 3.09 ±27.78 29.54 29.09 29.68 ±29.04 ±30.18 07 Sample 2 (Spray-dried sap) 8.8 231.30 177.27 178.17 180.67 182.03 186.10 189.73 (GHA 1 59) ±61.91 24.48 23.79 ±25.04 25.31 24.60 ±23.58 08 Spray-dried sap' 35 167.48 164.90 166.63 168.43 171.67 174.90 178.57 (GHA 1 59) ±36.75 22.54 23.08 22.66 ±24.42 ±25.70 _±23.58 09 Elga Option 4 165.50 193.73 196.87 198.07 199.83 204.93 207.13 purified water (control) ±49.27 22.37 ±21.86 ±21.02 ±20.21 ±E18.65 1±18.22 114 TABLE 1: HISTOLOGICAL EVALUATION OF LIVER SECTIONS FROM MALE RATS Sample 1 p. p

S

*5*S p. *5 S. *5 5".

S

555.

S

S GROUP 1: 100 mg/kg Sample 1 GROUP 2:.400 mng/kg Sample 1 Animal no Hepatic lesions Animal no Hepatic lesions Day 7 01 NPL 07 FHSl 02 NPL 08 NPL C1 03 NPL C1 09 NPL Day 13 04 NPL MLC Day 13 10 DHS1.g+ FHS1+ 11 NPL 06 NPL __12 DHS1 GROUP 3: 1 600 mg/kg Sample 1 GROUP 4: 3 200 mg/kg Sample 1 Animal no Hepatic lesions Animal no Hepatic lesions Day 7 13 NPL 19 NPL 14 NPL 20 NPL 15 NPL 21 NPL Day 13 116 NPL Day 13 22 DHS1 17 DHS1+ 23 FHS1+ 18 NPL 24 NPL GROUP 5: CONTROL: ELGA OPTION 4 PURIFIED WATER: RESTRICTED FOOD INTAKE Legend: GROUP 5: Control: Elga option 4 purified water Animal no Hepatic lesions Day 7 25 NPL MLC 26 NPL 27 NPL Day 13 28 DHS1 29 DHS1

NPL

C Congestion

DHS

FHS

NPL

MLC

1 2+ =Diffuse hydropic cell swelling =Focal hydropic cell swelling =No parenchymal lesions =Minimal lymphocytic cuffing =mild =moderate 3+ severe 11is TABLE 2: HISTOLOGICAL EVALUATION OF LIVER SECTIONS FROM MALE RATS Sample 2 GROUP 6:.2.2 mg/kg Sample 2 GROUP 7: 8.8 mg/kg Sample 2 Animal no Hepatic lesions Animal no Hepatic lesions Day 7 31 NPL 37 NPL 32 NPL MLC 38 NPL 33 FHS1 39 NPL C1 Day 13 34 NPL Day 13 40 DHS1 35 DHS1 41 NPL.

36 NPL IL 142 MLC FHS1 0e 9 9. 9 9.

*9999* 9 .r 9 S. 9 S 9 4O 9 9.

9 9* 9S*S 5 GROUP 8: 35 mg/kg Sample 2 Animal no Hepatic lesions Day 7 43 NPL 44 NPL

NPL

Day 13 46 NPL 47 NPL C1+ 4 8 MLC FH-S1 0 .0 GROUP 9: CONTROL: ELGA OPTION 4 PURIFIED WATER GROUP 9: Control: Elga option 4 purified water Animal n6 Hepatic lesions Day 7 49 NPL

NPL

51 FHS1 Day 13 52 DHS1 53 NPL 54 FHS1 Legend: C =Congestion DHS =Diffuse hydropic cell swelling FHS =Focal hydropic cell swelling NPL =No parenchymal lesions MLC =Minimal lymphocytic cuffing 1 =mild 2+ =moderate 3+ severe 116 No specific lesions were recorded in the liver sections from the experimental rats which received the frozen sap as well as the spray-dried sap that could be attributed to the oral adminstration of the abovementioned chemicals. The hydropic cell swelling recorded in both control and experimental rats may indicate normal metabolic cell swelling and anoxic changes. Minimal foci of lymphocytic perivascular cuffing were found in some animals and is most likely an incidental observation. In a few rats congestion of mild degree is present in the hepatic sinusoids and should be regarded as an incidental observation.

An important feature of the invention shown by the results of this study is that no tolerance to any of the samples developed over the test period. This may provide S**i considerable benefit, particularly in relation to the use of the compounds and compositions of the invention in the treatment of obesity.

While the compounds and compositions of the invention have primarily been described in relation to their properties as appetite suppressants, it should be noted that this expression "appetite suppressant" is used herein to denote activity which tends to limit appetite and/or increase the sense of satiety, and thus tends to reduce total calorific intake; this in turn tends to counteract obesity. Accordingly, this invention extends to a method of treating, preventing or combating obesity in a human or non-human animal which comprises administering to said human or non-human animal an obesity treating, preventing or combating amount of a compound of formula A preferred embodiment of this aspect of the invention utilises a composition or extract containing a compound of formula The term "animal" as used herein extends to, but is riot restricted to, companion animals, e.g. household pets and domesticated animals; non-limiting examples of such animals include cattle, sheep, ferrets, swine, camels, horses, poultry, fish, rabbits, goats, dogs and cats.

As an anorectic agent or in the treatment or prevention of obesity in a human, a compound of formula preferably of formula or the composition defined in any one of claims 9 and 25-31 hereafter, is advantageously administered to said human in a dosage amount of from about 0.01 mg/kg/day to about 10 mg/kg/day.

A preferred dosage range is 0.05 mg/kg/day to 0.5 mg/kg/day. When using the spray dried powder form of the extract of this invention, a preferred dosage range is 0.1 mg/kg/day to 20 mg/kg/day; especially preferred is 0.5 mg/kg/day to mg/kg/day.

Claims (51)

1. 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.

2. A process as claimed in claim 1 wherein the plant of the genus Trichocaulon is selected from the species Trichocaulon piliferum and Trichocaulon officinale and the plant of the genus Hoodia is selected from the 15 species Hoodia currorii, Hoodia gordonii and Hoodia lugardii.

3. A process as claimed in claim 1 or claim 2 which includes the step of concentrating the active agent in the extracted material by further extraction with a solvent.

4. A process as claimed in claim 1, claim 2, or claim 3, wherein the solvent in the solvent extraction step or steps is one or more of methylene chloride, water, methanol, hexane, ethyl acetate or mixtures thereof.

5. A process as claimed in any one of claims 1 to 4 inclusive which includes the step of concentrating the active agent in the extracted material by chromatographic separation.

6. A process as claimed in claim 5 wherein the chromatographic separation employs one or more of chloroform, methanol, ethyl acetate, hexane or mixtures thereof as an eluant. 118

7. A process as claimed in claim 5 or claim 6 which includes carrying out the chromatographic separation on a column, collecting the eluate in fractions from the column, evaluating the fractions to determine their appetite suppressant activity, and selecting the at least one fraction containing the appetite suppressant agent.

8. A process as claimed in any of the preceding claims wherein the extract is processed to form a free- flowing powder.

9. An extract comprising an appetite suppressant agent when produced by a process as claimed in any of the preceding claims. A composition having appetite suppressant activity comprising the extract claimed in claim 9.

11. A composition as claimed in claim 10 when admixed with a pharmaceutical excipient, diluent or carrier.

12. A composition as claimed in claim 10 or claim 11 which is prepared in unit dosage form.

13. The use of an extract as claimed in claim 9 -in the manufacture of a medicament having appetite suppressant activity.

14. An extract as claimed in claim 9 for use as a medicament having appetite suppressant activity.

15. A method of suppressing an appetite by administering to a human or animal an effective dosage of a composition as claimed in claim 10, claim 11, or claim 12. 119

16. 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 pressing collected plant material to separate sap from solid plant material and recovering the sap free of the solid plant material to form the extract.

17. A process as claimed in claim 16 wherein the extract is dried to form a free-flowing powder. "0 18. An extract comprising an appetite suppressant agent when produced by a process as claimed in claim 16 or e. claim 17.

19. A composition having appetite suppressant activity comprising the extract as claimed in claim 18.

20. A composition as claimed in claim 19 when admixed with a pharmaceutical excipient, diluent or carrier.

21. A composition as claimed in claim 19 or claim 20 which is prepared in unit dosage form.

22. The use of an extract as claimed in claim 19 in the manufacture of a medicament having appetite suppressant activity.

23. An extract as claimed in claim 18 for use as a medicament having appetite suppressant activity.

24. A method of suppressing an appetite by administering to a human or animal an effective dosage of a composition as claimed in claim 19, claim 20, or claim 21. 120 An extract obtainable from a plant of the genus Trichocaulon or of the genus Hoodia which comprises an appetite suppressant agent having the formula 0 o 0/O CCH 3 iM O Me Me Me OH OMe OMe (1)

26. An extract as claimed in claim 25 wherein the plant of 5 the genus Trichocaulon is selected from the species Trichocaulon piliferum and Trichocaulon officinale and the plant of the genus Hoodia is selected from the species Hoodia currorii, Hoodia gordonii and Hoodia lugardii.

27. An extract as claimed in claim 26 wherein substantially all the non-active impurities have been removed.

28. An extract as claimed in any one of claims 25 to 27 inclusive which has been processed to a free-flowing powder.

29. A composition having appetite suppressant activity comprising the extract as claimed in any one of claims to 28 inclusive. A composition as claimed in claim 29 when admixed with a pharmaceutical excipient, diluent or carrier. 121

31. A composition as claimed in. claim 29 or claims which is prepared in unit dosage form.

32. The use of an extract as claimed in any one of claims to 28 inclusive in the manufacture of a medicament having appetite suppressant activity.

33. An extract as claimed in any one of claims 25 to 28 inclusive for use as a medicament having appetite suppressant activity.

34. A method of suppressing an appetite by administering to a human or animal an effective dosage of a composition as claimed in claim 29, claim 30 or claim 31. A compound having the general structural formula RO R R0 F O2^ r in which R alkyl; R 1 H, alkyl, tigloyl, benzoyl, or any other organic ester group; R 2 H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between or C5-C6. 122

36. A compound as claimed in claim 35 wherein there is a bond between C5- C6, R =methyl, RI tigloyl, R 2 =3- 0- [-#-D-thevetopyranosyl- -fl-D-cymaropyranosyl- -#-D-cymaropyranosy1] the compound having the structural formula 0 I1 CCH 3 4 Me Me Me 0 0 00 OH OMe OMe

37. A compound having the general structural formula OH Ome HO0 (3) in which R =alkyl; and Ri H, alkyl, tigloyl, benzoyl, or any other organic ester group. 123

38. A compound having the general structural formula OMtO 5 (4) in which R =alkyl; and R H, alkyl, tigloyl, benzoyl, or any other organic ester group.

39. A compound having the general structural formula OMe in which R =alkyl; and R 1 H, alkyl, tigloyl, benzoyl, or any 124 other organic ester group. A compound having the general structural formula OH S. 5 OMO 0 Me in which R alkyl; and RI H, alkyl, tigloyl, benzoyl, or any other organic ester group.

41. A compound having -the. general str-uctural formula in which R =alkyl; R 1 H, alkyl, tigloyl, benzoyl, or any other organic ester group. 125

42. A compound having the general structural formula OR in which R alkyl; and R 1 H, alkyl, tigloyl, benzoyl, or any other organic ester group; R 2 H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4 C5 or C5 C6.

43. A compound having the general structural formula R H OR, yOH in which R alkyl; and R 1 H, alkyl, tigloyl, benzoyl, or any 126 other organic ester group; R 2 H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose molecules, or combinations thereof; and in which the broken lines indicate the presence of a further bond between C4 or C5 C6.

44. A compound having the general structural formula R OR, -OH ioJoIOH in which R alkyl; and R, H, alkyl, tigloyl, benzoyl, or any other organic ester group; R 2 H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4 C5 or C5 C6. 127 A compound having the general structural formula S. S (11) in which S 5 S S 2. R alkyl; and Ri H, alkyl, tigloyl, benzoyl, or any other organic ester group; R 2 H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4 C5, C5 C6 or C14

46. A compound having the general structural formula R on. O (12) in which R alkyl; and 128 S SOS. S R 1 H, alkyl, tigloyl, benzoyl, any other organic ester group; R 2 H, or one or more 6-deoxy carbohydrates, or one or more 2 ,6-dideoxy carbohydrates, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4 C5, CS C6 or C14

47. A compound having the general structural formula R OR, O UOR3 R2 S S. S (13) in which R alkyl; and R H, alkyl, tigloyl, benzoyl, any other organic ester group; R H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxy carbohydrates, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4 C5, C5 C6 or C14 C15; and R 3 H, alkyl, aryl, acyl, or glucoxy. 129

48. A compound having the general structural formula Me Me Me HO~ 0 OR .Mc I -1- OH OMe OMe (14) in which R H, alkyl, aryl, or any steroid possessing a C14 beta hydroxy group, a C12 beta hydroxy functionality, a C17 acyl group, a C5 C6 olef in, or combinations thereof.

49. A process for the preparation of a steroid intermediate of the formula 0 a OH- OH which includes the steps of treating the compound OAc HO. (22) with a reducing agent to produce a compound 39, 12I9- 20-ethylenedioxypregna-5, 14'-diene of the formula 130 E0 0 HO 0.0 (23) treating compound (23) with N-bromoacetamide (NBA) and a base to produce a compound 39,129~- dihydroxy- 14, 15 -epoxy- 20, 2 0 -ethylenedioxypregn-5 ene of the formula 0HJ~] (24) treating compound (24) with a reducing agent to produce a compound 39, 1219, 14I9-trihydroxy-2O,2O- ethyl enedioxypregn- -ele of the formula OH~4 OH 131 and. (d) treating compound (25) with an acid and water to produce compound SO. A process for the preparation of compound (15) which includes the steps of treating the compound (22) with p-toluenesulfonyl chloride and a base to produce a compound 39, 12#-dihydroxy-20, 20-ethylenedioxypregna-5,14- diene-3-tosyl-12-acetate of the formula 10 01] TsO' (26) treating the compound (26) with potassium acetate in a solvent to produce a compound 6fl, 120- 20-ethylenedioxy-3, Sce-cyclopregnan- 14 -ene-12 -acetate of the formula Q~0 OH (27) 132 treating the compound (27) with a reducing agent to produce a compound 60, 12fl-dihydroxy-20,20- ethylenedioxy-3,SoL-cyclopregnan-l4-ene of the formula OH. C C OH (28) treating the compound (28) with N-bromoacetamide, and a base to produce a compound 6fl, 12fl- dihydroxy- 20, 2 0 -ethylenedioxy- 14, 15 -epoxy- 3, cyclopregnane of the formula O H 0 OH (29) treating the compound (29) with a reducing agent to produce a compound 6fl, 1.20, 140-trihydroXy- 20,20-ethylenedioxy-3,5a-cyclopregnane of the 133 formula OH OH n S 5 and treating compound (30) with an acid and a solvent to produce compound

51. 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 PhCO OMe (36) with PhSSiMe 3 ZnI 2 and Bu4 4 I to produce a compound 4- 0-benzoyl-3-0-methyl-6-deoxy-aB-D-phenylthioaltroside of the formula 134 S~h PhCQ "M e S (37) (ii) optionally treating the compound (37) with diethylaminosuiphur trif luoride (DAST) to produce a compound 4-O-benzoyl-3 -O-methyl-2-phenylthio- 2,6 -dideoxy-ag-D- fluorocymaropyranoside having the formula -0 (38) or (iii) optionally, treating the compound (37) with t- butyldimethylsilylchloride and imidazole in a solvent to produce 4-O-benzoyl-3-O-methyl-2-O- t-butyldimethylsilyl -co-D-phenylthioaltroside having -the formula SPh 0 PhQO OMe (39) in which Z TBDMS t-butyldimethylsilyl; 135 and (iv) treating the compound with a base to p~roduce the monosaccharide 3-Q-methyl-2 -0-t butyldimethyl silyl -cfl-D-phenylthioaltroside having the formula z SPh HO Ome S. in which Z TBDMS t-butyldimethylsilyl.

52. 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 OMe OWe Ph CO OH (47) w it h phenyithiotrimethyls ilane and t rime thyls ilyl tri flIuoromethalesulphoflate to produce a compound 4-O-benzoyl-3-O-methyl-l-phenylthio--6-deoxy- agi-glucopyranoside having the formula 136 0 OMe SPh Ph CO k OH (48) (ii) treating the compound (48) with pivaloyl chloride and a solvent to produce a compound 4-O-benzoyl- 3 -o-methyl-2-O -pivaloyl-l-phenylthio- 6-deoxy-o3- glucopyranoside having the formula a t SPh Ph CO (49) and (i ii) treating the compound (49) with a brominating agent and diethylaminosulphur trifluoride. to produce the monosaccharide compound benzoyl-3-O-rnethyl-2-0-pivaloyl-l-fluoro-6- deoxy-f3-glucopyranoside occurring as stereo- isomers having the formula O F O OWe If PhCO W OPv O Me a PhCOO F ONv 0 A) (0 A)(5 0 B) 137

53. A steroid intermediate of formula (15) when produced by a process as claimed in claim 49 or claim

54. A carbohydrate intermediate of formula (40) when produced by a process as claimed in claim 51.

55. A carbohydrate intermediate of formula (50 A) or formula (50 B) when produced by a process as claimed in claim 52.

56. A process of coupling a monosaccharide cymarose to a steroid intermediate, which includes the steps of i) reacting a cymarose moiety of formula (38) with "a steroid intermediate of formula (15) as claimed in claim 53 in the presence of tin chloride in a solvent to produce a compound 3-0- [4-0-benzoyl-2- phenylthio-B-D-cymaropyranosyl]-12,14-6- dihydroxy-pregnan-5-ene-20-one of the formula OH OH BzO SPh OMe (51) and (ii) treating the compound (51) with tiglic acid chloride in pyridine and thereafter with a base to produce a compound 3-0-[4-0-benzyol-2- phenylthio-f-D-cymaropyranosyl]-129-tigloyl-149- hydroxy-pregnan-5-ene-20-one of the formula 138 0 HO I SPh OMe 4 (52)

57. A compound of formula as claimed in claim 56. 52) when produced by a process 4*

444.4 58. A process of coupling a monosaccharide cymarose moiety to a monosaccharide thevetose moiety and coupling the resultant disaccharide to the compound of formula (52) as claimed in claim 57 which includes the steps of i) coupling a selectively protected cymarose moiety of formula (40) as claimed in claim 54 and a monosaccharide thevetose moiety of formula (50 A) as claimed in claim 55 using tin chloride (SnCl 2 and silver trifluoromethanesulphonate to produce a compound of the formula (53) in which Z TBDMS t-butyldimethylsilyl jl 139 ii) treating compound (53) with tetrabutylammoniumf luoride to produce .a compound of the formula *5 S. S Ph (54) iii) treating compound (54) with diethylaminosuiphur trifluoride to produce a compound of the formula S S 5S5S55 0 It RiCO iv) reacting compound (55) with compound (52) as claimed in claim 57 to produce a compound of the f ormula 140 SPh OMe C) hCOSPh PCO e OMe OPv (56) and treating compound (56) in a Raney-Nickel reaction and thereafter with a base to produce compound as claimed in claim 36. 59. A process of forming a trisaccharide and coupling the resultant trisaccharide to a steroid intermediate, which includes the steps of i) coupling a selectively protected cymarose moiety of formula (40) as claimed in claim 55 and compound (45) using tin (II) chloride, AgOTf, Cp 2 ZrCl 2 to produce a compound of the formula OMe 0 o SPh 0 OMe OMe PhCO OPv (57) in which Z TBDMS t-butyldimethylsilyl; 141 ii) treating compound (57) with tetrabutylammonium fluoride and diethylaminosulphur trifluoride to produce a trisaccharide compound having the formula 0 F 0 OBe OMe Ph 0 OPv (58) and iii) coupling the trisaccharide of formula (58) with a steroid intermediate of the formula i C 0 (59) using tin (II) chloride, AgOTf, Cp 2 ZrCl 2 to produce compound as claimed in claim 36. A composition having appetite suppressant activity comprising a compound as claimed in any one of claims to 48 inclusive. 61. A composition as claimed in claim 60 wherein .the compound is the compound of formula as claimed in claim 36. 142 62. A composition as claimed in claim 60 or claim 61 when admixed with a pharmaceutical excipient, diluent or carrier. 63. A composition as claimed in claim 60, claim 61, or claim 62, which is prepared in unit dosage form. 64. The use of a compound as claimed in any one of claims to 48 inclusive in the manufacture of a medicament having appetite suppressant activity. 65. The use as claimed in claim 64 of a compound of 010 formula as claimed in claim 36. 66. A compound as claimed in any one of claims 35 to 48 inclusive for use as a medicament having appetite suppressant activity. 67. A compound as claimed in claim 66 which is the :15 compound of formula as claimed in claim 36. 68. A method of suppressing an appetite by administering to a human or animal an effective dosage of a composition as claimed in any one of claims 60 to 63 inclusive. 69. A foodstuff or beverage comprising an effective quantity of a compound as claimed in any one of claims to 48 inclusive to have an appetite suppressant effect when ingested. A foodstuff or beverage as claimed in claim 69 wherein the compound is the compound of formula as claimed in claim 36. 71. Use of a compound of formula as claimed in claim 36 isolated from a plant of the genus Trichocaulon or of the genus Hoodia for the manufacture of a 143 medicament having appetite suppressant activity. 72. Use according to claim 71 wherein the compound is isolated from the species Trichocaulon piliferum or Trichocaulon officinale or from the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii. 73. A composition having appetite suppressant activity comprising a compound of formula isolated from a plant of the genus Trichocaulon or of the genus Hoodia. 74. A composition as claimed in claim 73 wherein the compound is isolated and/or purified from a plant of the species Trichocaulon piliferum or Trichocaulon officinale or from of the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii. 75. A composition as claimed in claim 73 wherein the compound is isolated and/or purified from an extract derived from a plant of the species Trichocaulon piliferum, Trichocaulon officinale or from a plant of the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii. 76. A composition as claimed in claim 73, claim 74, or claim 75, when admixed with a pharmaceutical excipient, diluent or carrier. 77. A composition as claimed in claim 76 which is prepared in unit dosage form. 78. A compound of formula as claimed in claim isolated from a plant of the genus Trichocaulon or from the genus Hoodia for use as a medicament having appetite suppressant activity. 144 79. A compound as claimed in claim 78 wherein the compound is isolated from a plant of the species Trichocaulon piliferum or Trichocaulon officinale of from Hoodia currorii, Hoodia gordonii or Hoodia lugardii. 80. A method of suppressing an appetite by adminstering to a human or animal an effective dosage of a composition as claimed in any one of claims 73 to 77 inclusive. 81. A composition having appetite suppressant activity comprising a melanocortin 4 receptor agonist. .010 82. A composition as claimed in claim 81 wherein the agonist is an extract as claimed in claim 9, claim 18 or claim 25, or a compound as claimed in any one of claims 35 to 48 inclusive. 83. A composition as claimed in claim 81 or claim 82 wherein the compound is the compound of formula as claimed in claim 36. 84. A composition as claimed in claim 81, claim 82, or claim 83 when admixed with a pharmaceutical excipient, diluent or carrier. 85. A composition as claimed in any one of Claims 81 to 84 inclusive, which is prepared in unit dosage form. 86. The use of a melanocortin 4 receptor agonist in the manufacture of a medicament having appetite suppressant activity. 87. The use as claimed in claim 86 of an extract as claimed in claim 9, claim 18 or claim 25, or a compound as claimed in any one of claims 35 to 48 inclusive. 88. The use as claimed in claim 87 of a compound of 145 formula as claimed in claim 36. 89. A melanocortin 4 receptor agonist for use as a medicament having appetite suppressant activity. A melanocortin 4 receptor agonist as claimed in claim 89 which is an extract as claimed in claim 9, claim 18 or claim 25, or a compound as claimed in any one of claims 35 to 48 inclusive. 91. A melanocortin 4 receptor as claimed in claim 90 which is the compound of formula as claimed in claim 36. 92. A method of suppressing an appetite by administering to a human or animal an effective dosage of a composition as claimed in any one of claims 81 to inclusive. 93. The use of a melanocortin 4 receptor agonist to suppress the appetite of and/or to combat obesity in a human or animal. 94. The use as claimed in claim 93 of an extract as claimed in claim 9, claim 18 or claim 25, or a compound as claimed in any one of claims 35 to 48 inclusive. The use as claimed in claim 94 of a compound of formula as claimed in claim 36. 146 96. A compound having the structural formula OH E 0 0 S. S 59 S S (23) 97. A compound having the structural formula 0HfO0 (24) 98. A compound having the structural formula OH 147 99. A compound having the structural formula QAc 0 0 0:0. of *9* (27) 100. A compound having the structural formula oHJJ (28) 101. A compound having the-structural formula 0H0 OH (29) S. 0O S S 0e 0 S. S 0 0@0* 0@ S 0* S. S. S S. 0 S. 0@ 00 S S S. *0 148 102. A compound having the structural formula 103. A compound having the structural formula HO S~h PhCO Oe (37) 104. A compound having the structural formula 0 0 F It PhCO OSPh (38) 149 105. A compound having the structural formula SPh 0 PhCO OMe (39) 106. A compound having the structural formula 0 z -nSPh H 0 OMe 107. A compound having the structural formula 0 OMe SF'h Ph CO OK (48) ISO 108. A compound having the structural formula 0 OMe SPh ph CO e OH (49) 109. A compound occurring as stereo-isomers having the structural formula O F o ON 0 PhCO PhC' OpY 0 A) 110. A compound having the structural formula Me 001F ONv (5 0 B) BZO I SPh W~e (51) 151 111. A compound having the structural formula 0 HO I SPh Ome C (52) 112. A compound having the structural formula (53) in which Z TBDMS =t-butyldimethylsilyl. 113. A compound having the structural formula 0 SI'h 0 oe OMe PhCO OPV (54) IS2 114. A compound having the structural formula 0 It RICO 115. A compound having the structural formula 0 V OMe ONv (56) 153 .16. A compound having the structural formula SPh 0 W~e o 1 OMe ON~e It PhCO CON (57) 117. A compound having the structural formula MNe (58) 154 118. A method of combating obesity in a human or animal comprising administering to said human or animal an obesity combating amount of an extract as claimed in any one of claims 9, 25, 26, 27 or 28. 119. A method of combating obesity in a human or animal comprising administering to said human or animal an obesity combating amount of a composition as claimed in claim 120. A method of combating obesity in a human or animal .0 comprising administering to said human or animal an obesity combating amount of a compound as claimed in claim 35 or 36. 121. A structure of the formula 3-0-P-D-theverosyl-( 1-4)-3- D-cymaropyranosyl-(1-4)-p-D-cymaropyranoside-12p-0- 15 tigloyl-14p-hydroxy-pregnane-5-ene-20-one. o* 155 1. An extract obtainable from a plant of the genus Trichocaulon or of the genus Hoodia which comprises an appetite suppressant agent having the formula 0 0 0 0 CCH3 Me Me Me J OH HO OH r S: OMe OMe 15 (1) 2. An extract as claimed in claim 1 wherein the plant of the genus Trichocaulon is selected from the species Trichocaulon piliferum and Trichocaulon officinale and the plant of the genus Hoodia is selected from the species Hoodia currorii, Hoodia gordonii and Hoodia lugardii. 3. An extract as claimed in claim 2 wherein substantially all the non-active impurities have been removed. 4. An extract as claimed in any one of claims 1, 2 or 3 which has been processed to a free-flowing powder. A composition having appetite suppressant activity comprising the extract as claimed in any one of claims 1 to 4 inclusive. 6. A composition as claimed in claim 5 when admixed with a pharmaceutical excipient, diluent or carrier. 156 7. A composition as claimed in claim 5 or claim 6 which is prepared in unit dosage form. 8. The use of an extract as claimed in any one of claims 1 to 4 inclusive in the manufacture of a medicament having appetite suppressant activity. 9. An extract as claimed in any one of claims 1 to 4 inclusive for use as a medicament having appetite suppressant activity. 10. A compound having the general structural formula R,0R O R OH R2J in which R alkyl; R, H, alkyl, tigloyl, benzoyl, or any other organic ester group; R2 H, or one or more 6-deoxy mono-, di- or trisaccharides, or one or more 2,6-dideoxy mono-, di- or trisaccharides, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4-C5 or C5-C6, except. where R=CH 3 R.=H or 157 benzoy.l; R 2 and there is a double bond at C. 5 -C 6 and (b) where R=C.. 4 alkyl; R 1 R 2 and the C. 5 -Cg bond is saturated. 11. A compound as claimed in claim 10 wherein there is a double bond between C5-C6, R methyl, R, tigloyl, R 2 3-0- [-O-D-tbevetopyraflosyl- -1-D- cymaropyranosyl- -1-D-cymaropyraflosyll, the compound having the structural formula 4* C C C 4* C C C C. C. C C C CC C 0 ft CCH 3 Me Me OH We. 12. A compound having the general structural formula OH- HO0 158 in which R= alkyl; and R= H, alkyl, tig'loyl, benzoyl, or any other organic ester group. 13. A compound having the general structural formula OH 4e *0 a Ome in which R alkyl; and R, H, alkyl, tigloyl, benzoyl, or any other organic ester group. 14. A compound having the general structural formula HO 159 in which R alkyl; and R= H, alkyl, tigloyl, benzoyl, or any other organic ester group. S 15. A compound having the general structural formula OH .c V 966@ 6O be V 06396 C 9 69 69 V **96 6@ C 96 6* 66 6 9 6O 9 *6 9~* 6 69b6 OW e in which R alkyl; and R= H, alkyl, tigloyl, benzoyl, or any other organic ester group. 16. A compound having the general structural formula *6 6 6 6V 066e6@ S Nq O14 NO' in which R =alkyl; R, H, alkyl, tigloyl, benzoyl, or any other organic ester rroup, except where PR CH 3 R, H or benzoyl; and there is a double bond at CB-C 6 and (b) R=C 1 4 alkyl; R 1 and the C. 5 -C 6 bond 160 is saturated. 17. A compound having the general structural formula R qR[ 0 9* 0* in which R alkyl; and R= H, alkyl, tigloyl, benzoyl, or any other organic ester group; R= H, or one or more 6-deoxy mono-, di- or trisaccharides, or one or more 2,6-dideoxy mono-, di- or trisaccharides, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a furt her bond between C4 CS or CS C6, except where R=Cfl 3 R 1 =H or benzoyl; there is a double bond at C 5 -C 6 and R 2 H. 161 18. A compound having the general structural formula .in which R =alkyl; and R, H, alkyl, tigloyl, benzoyl, or any other organic es-ter group; R= HI or one or more 6-deoxy mono-, di- or trisaccharides, or one or more 2,6-dideoxy mono-, di- or trisaccharides, or glucose molecules, or combinations thereof; and in which the broken lines indicate the presence of a further bond between C4 or C5 C6, except where R=CH 3 R 1 =H; R 2 =digitopyranosyl; and there is a double bond at C 5 -C 6 R=C2H 3 R 1 benzoyl, tigloyl or angeloyl, Rz=H; and there is a double bond at C 5 -C 6 R=CH 3 R 1 =H or benzoyl; R 2 and the C,-C 6 bond is saturated; R CH3, R, benzoyl, R 2 6- deoxy-3-O-methkrl-I3-D-allopyranosyl- -1- D -cymaropyranosyl -13-D- cyiaropyranosyl; and where R CH 3 R, benzoyl, R 2 6- deoxy-3-O-methyl-O-D-allopyranosyl- -13- D-oleandropyranosyl-(l-4)-p-D- cymaropyranosyl. 162 19. A compound having the general structural f ormula in which R alkyl; R, H, alkyl, tigloyl, benzoyl or any other organic ester group; R 2 H, or one or more :6-deoxy mono-,. di- or trisaccb~arides or one or more 2,6-dideoxy mono-, di- or trisaccharides, or glucose molecules, or combinations thereof; and in which the ~.:broken lines indicate the optional presence of a further bond between C4 CS or CS C6, except where R=CH 3 R 1 R 2 =digitopyranosyl; and there is a double bond at C. 5 -Cs; R=CE 3 R 1 =H, benzoyl, tigloyl or angeloyl, R 2 and there is a double bond at Cs-C, 6 R=CH 3 R,=H or benzoyl; R 2 =H; and the C. 5 -C 6 bond is saturated; R CE 3 R 1 R 2 6-deoxy-3-O-methyl-p-D-allopyralosyl- -13-D-cyraropyraflosyl- -0-D-cymaropyra1osyl; and where R CH 3 R, benzoyl, R 2 6-deoxy-3-O- methyl- p-D-allopyranly- p-D-oleandropyraflosyl- (1-4)-pI-D-cymaropyraInosyl. 20. A compound having the general structural formula (11) 163 in which R alkyl; and RI H, alkyl, tigloyl, benzoyl, or any other organic ester group; R2 H, or one or more 6-deoxy mono-, di- or trisaccharides, or one or more 2,6- dideoxy mono-, di- or trisaccharides, or glucose molecules, or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4-C5, C5-C6 or C14-C15. 21. A compound having the general structural formula (12) in which R alkyl; and R, H, alkyl, tigloyl, benzoyl, any other organic ester group; R2 H, or one or more 6-deoxy mono-, di- or trisaccharides, or one or more 2,6- dideoxy mono-, di- or trisaccharides, or glucose molecules, or 164 combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4-C5, C5-C6 to C14-C15, except where R=CH 3 RI=H; R 2 and the C 5 C 6 bond is saturated. 22. A compound having the general structural formula R OR, OR O R2 e.(13) in which R alkyl; .and RI H, alkyl, tigloyl, benzoyl, any other organic ester group; R, H, or one or more 6-deoxy mono-, di- or trisaccharides, or one or more 2,6- dideoxy mono-, di- or trisaccharides, or glucose molecules or combinations thereof; and in which the broken lines indicate the optional presence of a further bond between C4-C5, C5-C6 or C14-C15; and R 3 H, alkyl, aryl, acyl, or glucoxy. 165 23. A process for the preparation of a steroid intermediate of the formula OH OH HO 0 0-0 O 0 0 which includes the steps of treating the compound OAc (22) with a reducing agent to produce a compound 33, 12(3- dihydroxy-20,20-ethylenedioxypregna-5,14-diene of the formula 01 O 1 OH (23) 166 treating compound (23) with N-bromoacetamide (NBA) and a base to produce a compound 3P3, 1203-dihydroxy- 14 1 5-epoxy-2O,2Q-ethylefledioxypregn-5-ene of the formula 0 OH HO' (24) a. 9* a a. a a a treating compound (24) with a reducing agent to produce a compound 3f3, '12P3, l4p-trihydroxy-2O,2O- ethylenedoxypregn-5-ene of the formula OH and treating compound (25) with an acid and water to produce compound 24. A process for the preparation of compound (15) which includes the steps of treating the compound (22) as shown in claim 23 with p-toluenesulfonl chloride and a base to produce a compound 3P3, 12r-dihydroxy-20,20- ethylenedioxyprega5,4diene3tosyl1 2 acetate of the formula 167 OAc TsO (26) 0. *l 00 0 0:* 0 treating the compound (26) with potassium acetate in a solvent to produce a compound 603, 12I3-dihydroxy-20,2O-ethylelediozy-3, a cyclopregnan-14-ene-12-acetate of the formula 0Ac~o OH (27) -treating the compound (27) with a reducing agent to produce a compound 6 3, 2Q,20-ethylenedioxy-3,5cX-cyclopregfa 4 -ene of the formula 168 a. a a a. a. a a a (28) treating the compound (28) with N- bromoacetamide, and a base to produce a compound 6P3, 1213-dihydroxy-20, 14,15-epoxy-3, Sa-cyclopregnane of the formula OH OH (29) .treating the compound (29) with a reducing agent to produce a compound 6f3, 1213, 1413- trihydroxy-20,20-ethylefledioxy-3, cyclopregnane of the formula 169 OOH OH OH OH and treating compound (30) with an acid and a solvent to produce compound e* 15 25. A steroid intermediate of formula (15) when produced by a process as claimed in claim 23 or claim 24. 26. A process of coupling a monosaccharide cymarose to a steroid intermediate, which includes the steps of 1) reacting acymarose moiety of formula (38) 0 O F phCO OMeSPh OMe (38) with a steroid intermediate of formula (15) as shown in claim 23 in the presence of tin chloride in a solvent to produce a compound 3- 0-[4-0-benzoyl-2-phenylthio- -D- 170 cymaropyranosyl] -12 ,14- 3-dihydroxy-pregnal-5 of the formula BZO I SPjh OMe S S S *5 S S 555554 S (51) and (ii) treating the compound (51) with tiglic acid chloride in pyridine and thereafter with a base to- produce a compound 3-0- [2-phenylthio-p-D- cymaropyranosyl] -12D3-tigloyl-14f3-hydroxy- pregnan-5-efle-20-one of the formula 0 <Kr OH HO I SPh Ome (52) 27. A compound of formula (52) when produced by a process as claimed in claim 26. 171 28. A process of coupling a monosaccharide cymarose moiety to a monosaccharide thevetose moiety and coupling the resultant disaccharide to the compound of formula (52) as claimed in claim 28 which includes the steps of i) coupling a selectively protected cymarose moiety of formula (40) below and a monosaccharide thevetose moiety of formula below: 0 Z -SPh HO OMe OF 0 OMe PhCO OPv A) using tin chloride (SnCl 2 trifluoromethanesulphonate to compound of the formula and silver produce a 0 0 OVO OPv (53) in which Z TBDMS t-butyldimethylsilyl 172 ii) treating compound tetrabutylammoniumf luoride compound of the formula (53) with to produce a 0* I I. I I. I I I I. I I 0e II I I I I I I. I I I 0 11 F'hCI opy (54) iii) treating compound (54) with diethylaminosuiphur trifluoride to produce a compound of the f ormula 173 iv) reacting compound (55) with compound (52) as claimed in claim 28 to produce a compound of the formula @0 00 0600 0@ 0 0 g S.

0900.0 0 00 *i 0 OS.. 55 0 40 £0 00 0 t *S 0@ 0500 0 005w OMe I SPh OMe OPv 0@ S 00 0C0550 (56) and (v) treating compound (56) in a Raney-Nickel reaction and thereafter with a base to produce compound as claimed in claim 11. 29. A process of forming a trisaccharide and coupling the resultant trisaccharide to a steroid intermediate, which includes the steps of i) coupling a selectively protected cymarose moiety of formula (40) and compound (45) using tin (II) chloride, AgOTf, CP 2 ZrCl 2 to produce a compound of the formula 174 OMe PhC 0 ONv (57) in which Z TBDMS t-butyldimethylsilyl; ii) treating compound (57) with tetrabutylammolium~ fluoride and di ethyl aminosuiphur trif luoride to produce a trisaccharide compound having the formula Ph 0 PtI& (58) and (i ii) coupling the trisaccharide of formula (58) with a steroid intermediate of the formula 175 HO (59) using tin (II) chloride, AgOTf, CpZrC 2 1 to produce compound as claimed in claim 11. 30. A composition having appetite suppressant activity 15 comprising a compound as claimed in any one of claims 10 to 22 inclusive. 31. A composition as claimed in claim 30 wherein the compound is the compound of formula as claimed in claim 11. 32. A composition as claimed in claim 30 or claim 31 when admixed with a pharmaceutical excipient, Sdiluent or carrier. 33. A composition as claimed in claim 30, 31 or 32, which is prepared in unit dosage form. 34. The use of a compound as claimed in any one of claims 10 to 22 inclusive in the manufacture of a medicament having appetite suppressant activity. The use as claimed in claim 34 of a compound of formula as claimed in claim 11. 176 36. A compound as claimed in any one of claims 10 to 22 inclusive for use as a medicament having appetite suppressant activity. 37. A compound as claimed in claim 36 which is the compound of formula as claimed in claim 11. 38. A foodstuff or beverage comprising an effective quantity of a compound as claimed in any one of claims 10 to 22 inclusive to have an appetite suppressant effect when ingested. 39. A foodstuff or beverage as claimed in claim 38 15":wherein the compound is the compound of formula (1) 15 as claimed in claim 11. Use of a compound of formula as claimed in claim 11 isolated from a plant of the genus Trichocaulon or the genus Hoodia for the manufacture of a medicament having appetite suppressant activity. 41. Use according to claim 40 wherein the compound is isolated from the species Trichocaulon piliferum or Trichocaulon officinale or from the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii. 42. A composition having appetite suppressant activity comprising a compound of formula isolated from a plant of the genus Trichocaulon or of the genus Hoodia. 43. A composition as claimed in claim 42 wherein the compound is isolated and/or purified from a plant of the species Trichocaulon piliferum or Trichocaulon officinale or from the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii. 177 44. A composition as claimed in claim 42 wherein the compound is isolated and/or purified from an extract derived from a plant of the species Trichocaulon piliferum or Trichocaulon officinale or from the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii. A composition as claimed in claim 42, 43 or 44, when admixed with a pharmaceutical excipient, diluent or carrier. 46. A composition as claimed in claim 45 which is -prepared in unit dosage form. 15 47. A compound of formula as claimed in claim isolated from a plant of the genus Trichocaulon or from the genus Hoodia for use as a medicament having appetite suppressant activity. 20 48. A compound as claimed in claim 47 wherein the compound is isolated from a plant of the species Trichocaulon piliferum or Trichocaulon officinale of from Hoodia currorii, Hoodia gordonii or Hoodia lugardii. 49. A compound having the structural formula OH E 0 HO (23) 178 A compound having the structural formula 0 0r OH HO, (24) 51. A compound having the structural formula OH 179 52. A compound having the structural formula OH (27) 53. A compound having the structural formula OH. OH (28) 180 54. A compound having the structural formula OH (29) A compound having the structural formula OH OH- OH 56. A compound having the structural formula OH BzO I SPh Ome (51) 181 57. A compound having the structural formula 0 OH HO I SPh OM (52) 58. A compound having the structural formula OH I SPh Me OPv (56) Dated 13 March, 2002 CSIR Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON