AU780886B2 - Appetite suppresant steroidal glycosides - Google Patents

Description

S&FRef: 476761D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

9

C

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:- 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 synthetically producing these steroidal glycosides, and to the products of 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 an appetite suppressant composition containing the appetite suppressant agent, and to a method of ~0o suppressing an appetite.

The invention also relates to a plant extract obtainable from plants of the group comprising the genus Trichocaulon and the genus Hoodia and having appetite suppressant activity.

Our co-pending Australian patent application No. 199870613 from which the is present application is divided describes and claims a process for preparing an extract of a e plant of the genus Trichocaulon or of the genus Hoodia, the extract comprising an i 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 extract may be prepared from plant material such as the stems and roots of said plants of the genus [RA\LIBA106800 docNSS 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 S 20 centrifugation. The solvent may be removed by means of the rotary evaporator, preferably in a water bath at a temperature of 60°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 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 I5 fractions may again be evaluated by suitable bioassay procedures for their appetite suppressant activity.

The Applicant has found that at least one such purified fraction has good appetite suppressant activity, and the active principle in the fraction was identified by 20 conventional chemical techniques including nuclear magnetic resonance, and was found to be a compound of the structural formula 0 o 2oCCH 3 6 It Me e Me

DD

MeHO O O 0 UO sO OMe OMe C B A 4 In accordance with S.I. nomenclature, the active principle is the compound [-P-D-thevetopyranosyl-(1- 4)-P-D-cymaropyranosyl-(1- 4)-P-D-cymaropyranosyl]- 12p-0-tigloyloxy-14-hydroxy-14p-pregn-5-ene-20-one (C 47

H

74 0 1 5 M+878); this structure constitutes one aspect of the present invention.

Accordingly, a first aspect of the present invention provides 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 I I 0 CCH 3 Me Me Me OH a 0 S. MeOP OH r OMe OMe (1) The invention extends in a second aspect to a composition having appetite to suppressant activity comprising an extract in accordance with the first aspect of the g* present invention 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 in a third aspect to the use of an extract in accordance with the present invention 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 present invention extends to compound and certain analogues or derivatives of this steroidal trisaccharide having appetite suppressant properties. The molecules chosen as the [R \LIBA]06800 doc:NSS a 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 1 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 faster rate of onset of action.

o (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 20 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 tc increase binding to its receptor and 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; 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; 10 e) Modification of the carbohydrates of the trisaccharide moiety.

Accordingly, the invention provides in a fourth aspect a compound having the general structural formula

O*

O

:R2 (2) Sin which R alkyl; RI H, alkyl, or an 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; (R.\LIBA06800 doc:NSS 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 Ri=H or benzoyl; R 2 and there is a double bond at Cs-C 6 and where R=CI-4 alkyl; RI=H; R2=H; and the C 5

-C

6 bond is saturated.

Compounds having the same ring structure as shown in formula above may be synthesised using general procedures known in the art. For example, suitable synthetic routes are disclosed in: EP 0 101 383A at page 3, line 22 et seq. Zeitschrift fur Naturforschunq, Teil B, 40B(5), 659, column 1, paragraph 1; Chem. Pharm. Bull.

1 13(11), 1965, Charts 1 and 2 at pages 1336-1337; and 15 Steroids 483-493. These known synthetic procedures may be modified using standard procedures known to synthetic chemists in order to achieve incorporation of the substituent groups as defined in formula above.

o* *oo o o*oo* *e 0 0.00 *0 0 0* *0 0 00 0 0 *00 0 *0*0 0 0 000* 0e 00 0 0*0* 0 0 0.00 0*0000 0 0@ 0 8 The invention also provides a compound as described above wherein there is a further bond between C5 C6, R methyl, R, tigloyl, R 2 thevetopyranosyl-(- 4)-p-D-cymaropyranosyl-(- 4)-f3-D-cymaropyranosyl], and having the structural formula: 0 0 I~zz 0 CCH 3 Me Me Me ZOH HO0--,1 0 0 00 MeO 0 0

OH

OMe OMe Further active analogues or derivatives of the appetite suppressant compound in accordance with the fifth to fifteenth aspect of the present invention are compounds having the following structural formulae:

OR,

O~OH

0/00 HO( OMe OH (3) in which R =alkyl; and [R\LIBAjO6800 doc:NSS 9 H, alkyl or an organic ester group 0@ 4 4 4. 4 04 4.

6 4

S

*9 4949 S. S 4* *4

S

54 S 4 S S 454 5 *S4S

S

4 @4 5 4.

9* 4 4~45 4

S

444* 4 *4 4 44 4*

S

in which R= alkyl; and H, alkyl or an organic ester group R 1

R

0 0 0 O~e

OH

in which R= alkyl; and [R \LIBAJO6800.doc:NSS R, H, alkyl or an organic ester group ~0 00 0 0 A S. S

'S

0.

0 ~00O0.

0

S

0 0 00t0 *0 0 .0 4.

0 *0 0 0 5 0 050 0 oSot 0 .0 0 0~ 0S 0 **00 4

S

@000 000004 0 .0 00

S

(6) in which R alkyl; and H, alkyl or an organic ester group.

in which R alkyl; R= H, alkyl, or an organic ester group, and the broken lines indicate the optional presence of a further bond between C 4

-C

5 or C-6 [R \LIBA]06800 dccNSS 11 except where: R CH 3 Ri H or benzoyl; and there is a double bond at

C

5

-C

6 and R Ci-4 alkyl; Ri H; and the C 5

-C

6 bond is saturated.

OR, R\O

OH

R2" (8) in which R alkyl; and RI H, alkyl or an 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, except where R CH 3 Ri H or benzoyl; there is a double bond at C 5

-C

6 and R 2

H.

R

H

ORi R OH

OH

0f R2 (9) [R \LIBA]06800 doc:NSS in which R= alkyl; and R, H, alkyl, or an 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 presence of a further bond between C4-C5 or C5-C6, except where R CH 3 R, H; R 2 digitopyranosyl or H; and there is a double bond at C 5

-C

6 R CH 3 R, H, benzoyl, tigloyl or angeloyl, R 2 H; and there is a to double bond at C -C 6 R CH 3 R, H or benzoyl; R 2 H; and the C 5

-C

6 bond is saturated; R CH 3 R, benzoyl, R 2 6-deoxy-3-O-methyI-p-D-allopyranosyl- 4)-j3-D-cymaropyranosyl-( 1- 4)-J3-D-cymaropyranosyl; and where R CH 3 R, benzoyl, R 2 6-deoxy-3-O-methyl-3-Dallopyranosyl-( I 4)-f3-D-oleandropyranosyl-( 4)-j3-D-cymaropyranosyl.

*H

R

OR OH R2 in which R =alkyl; and H, alkyl, or an 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; [R \LiBAIO68G0.docNSS 13 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; R 2 =digitopyranosyl or H; and there is a double bond at C 5

-C

6 R CR 3 R, H, benzoyl, tigloyl or angeloyl, R 2 H; and there is a double bond at C 5

-C

6 R CR 3 R, H or benzoyl; R 2 H; and the C 5

-C

6 bond is saturated; R CR 3 R, benzoyl, R 2 6-deoxy- 3-O-methyl-f3-D-allopyranosyl-( 4)-f3-D-cymaropyranosyl-( 4)-f3-Dcymnaropyranosyl; and where R CH 3 R, benzoyl, R 2 6-deoxy-3-Omethyl-f3-D-allopyranosyl-( 4)-j3-D-oleandropyranosyl-(1- 4)-J3-Dcymnaropyranosyl.

S S

S

(11) in which R alkyl; and R, H, alkyl, or an 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.

RALIBA]06800doc:NSS (12) in which 10 R= alkyl; and RI H, alkyl, or an 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, except where R CH 3

R

1 H; R 2 H; and the C 5

-C

6 bond is saturated.

[R\LIBA]06800.doc NSS OR1 R

O

OR3 R2 (13) in which R alkyl; and RI H, alkyl, or an 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 o R3 H, alkyl, aryl, acyl, or glucoxy.

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

(R:\LIBA]06800doc:NSS The process uses a steroid as a starting material (or internediate or precursor) the steroid having the chemical f O rmu -a

OH

0 *000 *0 0 0* 0* *0 0 0 0 0 0*00 0 0 0* 0

S

0 0 0 0 0 0* 0 0*

OH

HO

A.e steroid can be prepared from a compound having Sthe formula (22) by a process which includes the steps of treating progesterone having the formula (16) with the micro-oraanism Caloneccz-ia decora to produce a compound !29, !5a- di4hvdroxy progesterone of the formula

~OH

0. 0 0 00 0 0. 0 0 00 0 0*0000 0 0 0 0 0*00 0* @0 0* *0 @0 0 0 0@0 0 0 0 0* 0* 00 0 0000 0 0 (1 7) treating compound (17) with tosyl chloride and pyridine to produce a compound 129-hydroxy-15a- (ptoluene sulfonyl) -progesterone of the formula 0OTS (18) 00 0 (iii) treating the compound (18) with collidine at to produce a compound 129-hydroxy-A 14 progesterone of the formula O H (19) (iv) treating the compound (19) with acetyl chloride and acetic anhydride at 120 0 C, to produce a compound 3,12g-diacetoxypregna-3,5,14-triel-2O-one ofL the formula AcO 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 AcO (21) treating the compound (21) wizh NaBH 4 to produce a compDound 3S, 12S-dihydroxy-20, 5,14-diene-12-acetate of the formula (22).

S

S

S S

S.

S

S

*.SS

*5 S

S.

S

*5 S S S S

S

S

5*5* S. S S S *5

S

5*55

S

S

S. 55 In a sixteenth aspect of the present invention there is provided a process for the preparation of steroid intermediate (15) which is termed "the first alternative procedure" and includes the steps of: treating compound (22) with a reducing agent, e.g. LiAlH 4 to produce a 10 compound 3P3, 12f-dihydroxy-20, 20-ethylenedioxypregna-5, 14-diene of the formula (23) treating compound (23) with N-bromoacetamide (NBA) and a base, e.g.

pyridine, to produce a compound 3P~, 12f-dihydroxy- 14,15-epoxy-20, ethyl enedioxypregn- 5-ene of the formula I R.L1BAO6800.doc NSS 0 01

HO

S S

S

S

S.

*5

S

S

S. S S S (24) treating compound (24) with a reducing agent, e.g.

LiA1E 4 ,1 e.g. with refluxing, to produce a compound 3B, 12BG, 14B.-tr--ihydr-oxy-2O,20-ethylenedioxypregl-5-ene of the formula

OH

and treating compound (25) with an acid, e.g. acetic acid, and water to produce the steroid intermediate compound 3i9, 1213, 1413-trihydroxypregn-S-ene Reaction Scheme A depicts the procedure for the preparation cf st eroid intermediate (15) from compound (22) according to "the firs-, alternative procedure", of the ifivention (and includes the preparation of compound (22) from compound !116) for illustrative purposes).

Reaction Scheme A (16) (17) es S S S 5e50 SS S S S

S.

S

**SSSS

S

S S 5 0 .5

SS

S 55 0S S S

S

0

OSOS

S

S

55 *5 00

S

S

S

S

550555

S

S. S 5* OTs (19) (18) .0] OAc AcO AcO I(21)

OH

'S

S 0 0 0**S

S

SO

06 0 0 0 4 00 S 00 .0

S

*0 a S 0 S 0@S 0040 a S 0 0005 00 0 00 00 0 .650 j 0 0000 so.

0 45 0 oc

OS

S

(23) (22) 0- 0] (24) 23 In a second alternative procedure, there is provided a process for the preparation of steroid (15) according to a seventeenth aspect of the invention which includes the steps of treating compound (22) 12/-dihydroxy-20, 20-ethylenedioxypregna-5,14diene-12-acetate) with p-toluenesulfonyl chloride and a base, e.g. pyridine, to produce a compound 3P/, 12fl-dihydroxy-20,20-ethylenedioxypregna-5,14-diene-3-tosyl-12-acetate of the formula OAc

OO

o0- (26) treating the compound (26) with potassium acetate in a solvent, e.g. acetone, to produce a compound 6P, 12p-dihydroxy-20,20-ethylenedioxy-3, 50x-cyclopregnan-14ene-12-acetate of the formula OAci o• (26) (27) treating the compound (26) with potassium acetate in a solvent, e.g. acetone, to produce a compound 613, 1213-dihydroxy-20,20-ethylenedioxy-3, 5a-cyclopregnan-14ene-12-acetate of the formula OAc ,y0"

OH

(27) [R\LI BA]06800.doc:NSS treating the compound (27) with a reducing agent, e.g.

LiAlH 4 and e.g. tetrahydrofuran, to produce a compound 121-dihydroxy-20,20-ethylenedioxy-3,Sacyclopregnan-14-ene of the formula (28) treating the compound (28) with N-brornoacetamide, optionally acetic acid, and a base, e.g. pyridine, to produce a comp~ound 60, 120-di4hydroxy-2O,2Oethylenedioxy- 14, 15-epoxy-3, 5a-cycloprecnane of the formula 0

OH

(29) treating the compound (29) with a reducing agent, e.g.

LiA.1H 4 and e.g. tetrahydrofuran, to produce a compound 603, 1213, 141-trihydroxy-20,20-ethylenedioxy-3,crcyclopregnane of the formula

OH

OH

4* 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) acccrding to rthe second alternative procedure" of the invention.

QAc Reaction Scheme B 01 Tso QAc 0]

HO

(22) (26)

S

S*

S

S S 555 5 S S 55

S

55.5 S S

S

0 0]

OR.

OH L.28) 00]

OH>

0 01 '0 OH (29)

OH

(tS) Mixture flevimers (153) C-17a acetyl (I Sb) C. l7aacetyl S 9 S S

S.

S

S

S S *5S5 9* S

S.

S

55 S S

S

*5 S *5

S

S

5*5* *9*555 S S

S

Compound may be synthesized from a first carbohydrate intermediate in the form of an activated monosaccharide cymarose moiety, which can be prepared from a comp~ound having the formula Compound (36) can be prepared by a process which includes the steps W'treating methyl -a-D-glucose having the formula

HO

SOH

HO 0POMe

OH

(31) with benzaldehyde and zinc chloride to produce a compound methyl G-O-benzylidene-ea-D-glucopyrafloside of the formula 0 0 Ph

OH

OM e

OH

(32) Ui4) treating the compound (32) with tosyl chloride and pyridine at 0 0 C, to produce a compound methyl-4,6-0benzylidene-2-O-tosy1-a-D-g1ucopyraloside of the formu.L Ph IlOme OTs 0 0 *0*00* 0 *000 *0 0 0* 00 0 0* 0 0 0 0*00 0 0 0*00 0* 0 0* *0 *000 0000 *0 0 (33) 4i treating the compound (33) with NaOMe at 1001 0 C to produce a compound methyl 4,6-O-benzylidene-3-Omethyl-a-D-altropyranoside of the formula 00 0 0 Ph

HO

0 OMe (34) (iv) treating the compound (34) with N-bromosuccinamide (NBS) to =roduce a compound methyl 6-bromo-4-Obenzoyl-3-O-mrezhyl-6-deoxy-a-D-altropy-anoside of the formula Br- PhO OMe OMe and treating the compound (35) with NaBH 4 and NiC1 2 0 to produce a compound methyl 4-O-benzoyl-3-Orethyl-6-deoxy-a-D-altropyranoside of the formula 0

H

PhCo OMe OMe (36) Our co-pending Australian patent application No. 200226125, which is also divided from 199870613, describes and claims a process for the preparation of a carbohydrate inten-nediate in the forrm of an activated monosaccharide cyrnarose moiety which includes the steps of i treating the compound (36) with PhSSiMe3 and trimethylsilylfluoromethane sulphonate to produce a compound 4-0-benzoyl-3 -0-methyl-6-deoxy-cP -Dt phenylthioaltroside of the formula PhCO, OMe i (37) (ii) optionally treating the compound (37) with diethylaminosulphur trifluoride (DAST), e.g. at OOC, to produce a compound 4-0-benzoyl-3-0-methyl-2-phenylthio-2,6dideoxy-(xp-D-fluorocymaropyranoside having the formula 0 0

IF

PhCO, qIh SPh OMe (38) I R \L1 BA106800 doc: NSS or (iii) optionally, treating the compound (37) with tbutyldimethylsilylchloride and inidazole in a solvent, e.g. pyridine, to produce 4-O-benzoyl-3phenylthioaltroside having the formula 0 O SPh 0 if PhCQ 'S(39) in which Z =TBDMS =t-butyldimethylsilyl and (iv) treating the compound (39) with a base, e*.g.

sodium methoxide, to produce 3-0-methyJ.-2-0-tbutyldimethylsilyl -ao-D-phenylthioaltroside 10having the formula 0 z NSPh

HOO

OMe in which Z =TBDMS t-butyldimethylsilyl.

Reaction Scheme C shows the procedure for the synthesis of the activated monosaccharide cvmarose moiety (40) from compound iand includes the preparation of compound (36) from czmound (31) fEo r illustrative purposes).

Rgartion nchamp r Reaction Scheme

C

HO

0

OH

HOu OM e

OH

0 Ph- OH0 OMe

OH

(32)1 .0 0 6

S

*0 0~ *S

U

6.0600 0 4 *000 *0 0 0b 0 *0 0 0 0 *0.6 0 V VS.

.0 0 *0 0 @00*

JO.

060 bt U 40 V. 0.

4 0 -0 Ph HO 0 OlMe OMe (34)1I F3r f 4 ~Ph

Z.

W~e (33) 0 11 1 PhCO 0to PhCO OMe OMe (35) IOWe W~e I 36) F 0 11 PhCO SPh W~e i37) (38) I-X 0 Z SPh

HO

OWe (40) SPh 0 PhCO OMe (9 Z -butyldimethylsilyl Reaction Scheme D sb~ws the procedi~re for the synthesis of the activated .monosaccb.aride thevetose moiety (50 and 3s Reaction Schemne D

HO

0 OM e 0 (43)

OH

.(4D)

HO

HO

O~e

HO

(44)* 0 OMe rOMe Ph CO

O

t47)* 0 -4 1OMe (46).

0 OMe SPh Ph

CO

OH

(48) 0 OMe SPh PhCO OPv (491 0 OMe PhE II PhCO F Opv

B)

0 OW/ If Ph CO OPv (50 A) 34 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 in a seventeenth aspect, a process of coupling a monosaccharide cymarose with the steroid intermediate, which includes the steps of reacting a cymarose moiety of formula (38) with a steroid intermediate of formula e.g. at -15 0 C, and in the presence of tin chloride, in a solvent, e.g. ether, to 10 produce a compound 3-0-[4-0-benzoyl-2-phenylthio--D-cymaropyranosyl]-12,14-pdihydroxy-pregn-5-ene-20-one of the formula O*

OH

BzO SPh

O

OMe in which Ph phenyl and Bz= benzoyl and (ii) treating the compound (51) with tiglic acid chloride in pyridine and thereafter with a base, e.g. NaOMe, to produce a compound 3-0-[2-phenylthio-3-Dcymaropyranosyl]- 1 20-tigloyloxy- 14-hydroxy- 140 -pregn-5-ene-20-one of the formula [R:\LIBA06800 doc:NSS (52) 5 in which Ph phenyl.

9 The invention extends in an eighteenth aspect to a process which includes coupling a monosaccharide cymarose moiety to a monosaccharide thevetose moiety and coupling the resultant disaccharide with the combined steroid product (52) to form compound i 10 The process of the eighteenth aspect of the invention, namely coupling the monosaccharide cymarose moiety to the monosaccharide thevetose moiety and coupling the resultant disaccharide to the combined steroid product (52) may include the steps of coupling a selectively protected cymarose moiety (40) and a selectively protected thevetose moiety (50 A) using tin chloride (SnCl 2 and silver trifluoromethanesulphonate, e.g. at -15 0 C, to produce a compound of the formula (R\LIBA]06800 doc:NSS 0 0 0 ZO Sh 0. Ome.

0 o oMe (53) in which Z =TBDMS =t-butyldimethylsilyl treating compound w it h tetrabutylammoniUmfluoride to produce a compound of the formula O~ie 0 PhC( (54).

(iii) treating compound with diethylamilosuJPhu= trifluoride, e.g. at OOC, to produce a compound of the formula 1.

0 It FfiC'

PV'

S S

S

SSSS

S. S S S

S.

S

*5

S

S S 5 *55*

S

S

S

*5

S

*SS*

S

S. S

S

*5

S

(iv) reacting compound (55) with compound to produce a compound of the formula V

OH

0 .11 AiC' OMe SPh le ONv and treating compound (56) in a Raney-Nickel. reaction and thereafter with a base, NaOMe, to produce compound as described above.

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

Reaction Scheme E 0 o +OH.

F

BZO

sph, Omer-

OH

(38) *0 0H 00 0 *0 0 00 Sh *~We *(52 0 9OMe +zo Sfh P Co HO OPv OMe 0- 9 H S~ Z SPh 0 0 0 OMe 0 O~ Me 0 OMe.

PhCO opy ONv L 54) (53) -S PP h 0s 00 0 OO~v 00 If eoSSh F'hCO OWe (56) Z-t-butyidimethylsilyl op" According to a nineteenth aspect of 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 includes the steps of coupling a selectively protected cymarose moiety (40) and compound (55) using tin (II) chloride, AgOTf, CP 2 ZrCl 2 to produce a compound of the formula (57) in which Ph phenyl, Pv pivaloyl and Z TBDMS t-butyldimethylsilyl; (ii) treating compound (57) with tetrabutylammoniumfluoride and diethylaminosulphur trifluoride to produce a trisaccharide compound having the formula [R.\LIBA]06800.doc:NSS 41 0 SSPh OV OMe

O

11 OMe SPh PhCO OMe OPv (58) in which Ph phenyl, Pv pivaloyl and (iii) coupling the trisaccharide of formula (58) with a steroid intermediate of the formula HO (59) using tin (II) chloride, AgOTf, CP2ZrCI2 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 by coupling the trisaccharide (58) with the steroid s15 intermediate (59).

intermediate (59).

[R:\LIBA]06800 doc NSS Reaction Scheme F F 0 OM e0 II SPh PhCO OMe P OPv 'SPh 0 ZO SPh 0 0 OMe D0 0 OMe SPh 11 PhC01 OMe OPv (57) (59) (58) Z t-butyldimethylsilyl [R\LIBA16800 doc NSS 43 The intermediates (48), (57) and (58) described above are novel compounds; the present invention extends to compounds (29), s (52) and (56) as such.

Compound 3-0-[-p-D-thevetopyranosyl-(1- 4)-p-D-cymaropyranosyl-(l- 4)- P-D-cymaropyranosyl]-12p-0-tigloyloxy-14-hydroxy-14P-pregn-5-ene-20-one, and various analogues and derivatives thereof have been found to have appetite suppressing activity.

10 The invention extends also in a twentieth aspect to a composition or formulation having appetite suppressant activity comprising a compound of formula isolated 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 lugardii.

i 15 The invention extends also in a twenty first aspect 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 namely the fourth to fifteenth aspects of the invention described above.

A composition or extract of this invention may be used to suppress appetite or to treat, prevent or combat obesity in a human or animal. The composition or formulation may also include pharmaceutically acceptable other ingredients.

[R\LIBA]06800 docNSS 44 The appetite suppressant agent may be an isolated natural chemical or a synthetic chemical compound of the formula: 0 0

II

0 CCH 3 Me Me Me OH HO 0 0 0 MeO 0 t 0

OH

OMe OMe (1) or derivatives or analogues thereof, as set out before.

The appetite suppressant composition or formulation may consist of the appetite I 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 o" io be added.

The invention extends in a twenty second aspect to the use of compound or its .derivatives or analogues described in the fourth to fifteenth aspects of the present invention above, disregarding the disclaimers therein, in the manufacture of a medicament having appetite suppressant activity or for use in treating, preventing or combating ••oo s15 obesity.

S"The invention further extends in a twenty third aspect to compound or its o* *derivatives or analogues as set out before, disregarding the disclaimers therein, for use as a medicament having appetite suppressant activity or for treating, preventing or combating obesity.

A method of suppressing an appetite or for treating, preventing or combating obesity 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 (R \LIBA]06800.doc:NSS 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 in a twenty fourth aspect 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 above in the fourth to fifteenth aspects of the present invention, disregarding the disclaimers therein, for use as an appetite suppressant or for use in treating, preventing or combating obesity.

The invention extends in its twenty fifth aspect to thirty fourth aspects, to 1o compounds having the structural formula (52) or (56) as defined above.

invention also provides in its twenty sixth aspect a method of suppressing appetite comprising administering to a human or an animal an effective amount of extract S•of the first aspect of the present invention or a composition of the second aspect of the 15 invention as defined above.

The invention also provides in its twenty seventh aspect a method of suppressing appetite comprising administering to a human or an animal an effective amount of a compound as defined in any one of the fourth to fifteenth aspects of the present invention above, disregarding the disclaimers therein, or of a composition of the second aspect of o1* 20 the present invention as described above, disregarding the disclaimers therein; or of a foodstuff or beverage as defined in the twenty fourth aspect above.

A twenty eighth aspect of the present invention provides a method of treating, preventing or combating obesity, comprising administering to a human or an animal an effective amount of a compound as defined in any one of the fourth to fifteenth aspects of the present invention above, disregarding the disclaimers therein, or of a composition of the second aspect of the present invention as described above, disregarding the disclaimers therein; or of a foodstuff or beverage as defined in the twenty fourth aspect above.

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 (as described and claimed in our co-pending patent application AU 199870613) of extracting a first crude appetite [R-\LIBA]06800 doc:NSS suppressant extract and a purified appetite suppressant extract from plant material of the genus Trichocaulon or Hoodia; 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 AU 199870613 for producing an extract of plant material of the genus Trichocaulon or Hoodia; and

S

S

*S

o•i o•i [R \LIBA]06800.doc:NSS 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 extract of plant material of the species Hoodia gordonii.

EXAMPLE 1 10 0 0

S

0

S

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 twz 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 for producing an extract having appetite suppressant activity in accordance with GB9919797.2 (from which the present application is divided) is illustrated schematically by way of example in Figures 3 and 4, which two Figures together illustrate the comprehensive process. However, various other procedures may be used, as will be understood by persons skilled in the art.

0 00 10 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 i, with a solvent in the form of a methylene chloride/methanol solution introduced via feedline 2. The homogenised product is fed 15 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.

oo The solvent/extract mixture is fed via line 6 into an evaporation stage 7, where the solvent is removed, for 20 example by means of a rotor evaporator. The dried 6rude 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 V(2.0 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 20 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 *o **oo* *oo o: Table 1: 'IH (300.13 MHz) n.m.r. data for compound CDCP 3 Compound (1) J(HH)/Hz Hydrogen Atom Aglycone-3 6 12.

17 18 19 21 3' 4' 5* CYM-1' 3- 4, 5, 6' 3'-OMe 2"a 3-a 4,' 6" 3"-OMe They-i"'.

2' 501.

3"'-OMe abc in each column interchangeable, 3.522 m 5.381 m 11.5, 4.1 4.607 dd 9.3, 9.3 3.157 dd 1.029 s 0.951 s 2.164 s 7.1,1.56.888 qq 7.1,1.2 1.806 dq 1.6,1.2 1.853 dq 9.4, 2.1 4.816 dd 13.8, 3.7, 2.1 2.055 ddd 13.8, 9.4, 2.6 1.552 ddd 3.7, 2.9, 2.6 3.776 ddd 9.4,2.9 3.179 dd 6.3, 9.4 3.821 dd 6.3 1.279 d a 3.408 Sd 9.4, 2.1 4.730 dd 13.8, 3.7, 2.1 2.108 ddd 13.8, 9.4, 2.6 1.601 ddd 3.7, 2.9, 2.6 3.755 ddd 9.4, 2.9 3.239 dd 6.3, 9.4 3.898 dd 6.3 1.243d b ~3.392 s* 7.7 4.273 d 7.7, 8.0 3.469 dd 2.9 3.099 dd 9.3, 2.9 3.179 dd 6.3, 9.3 3.351 dd 6.3 1.183dc 3.622 s may be interchangeable. dein each column may be Refers to the igloate group atoms Table 2: Relevant 3 C (75.25 in CDC 3 Carbon 1 2 3 4 6 7 8 9 11 12 13 14 15 16 17 18 19 21 1* 3.

4.

Aglycone moiety 6.P..

37.04 T 29.44 T 77.24 D 38.62 T 138.95 S 131.90 D 27.30 T 35.30 D 43.04 D 37.22 S 26.04 T 75.88 D 53.71 S 85.69 S 34.36 T 24.31 T 57.18 D 9.85 Q 19.270Q 216.85 S 33.01 Q 167.60 S 128.69 D 137.66 D 14.41 Q 12.08 Q Carbon cym- 1V 21 3, 4' 5, 6' 3'-OMe it, 2" 3" 4" 51, 6" 3"-OMe They- 3V' 4"'I 5.

6# 3"'-OMe MHz) n.m.r. data for Compound Sugar moiety 64-p..

95.84 D 35.57 T 77.05 D 82.57 D 68.48 D 18.14 Q 57.93 Q 99.54 0 35.17 T 76.99 D 82.52 0 68.30 0 18.36 Q 57.09 0 104.28 D 74.62 0 85.30 0 74.62 D 71.62 D 17.75 Q 60.60 Q Refers to the tigloate group atoms Compound (1) IR data: 3440 cm1 (OH) 2910 cm'1 (CH) 1700 cmf' (C=0) Carj]2 5 89 12, 670 CHCl 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-Dihvdroxy progesterone (17) 10 o 15 5 *0 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 P) (150 X 500 ml flasks). After 5 days of shaking at 26 0

C,

progesterone (16) (150 g) in a suspension of Tween 80 (0,1 soln., 1,5 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 chloroform, and then evaporation to yield the dihydroxy progesterone (17) (75 g, 45 'H NMR (CDC1 3 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, H-12); 2,16 Hz (3H, s, H-21); 1,18 (3H, s, H-19); 0,74 (3H, s, H-18) EXAMPLE 129-Hvdroxv-15a-(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 c, and quenched by the addition of H 2 0 (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 0 chloride (500 ml), and water (500 ml). The organic layer is dried (MgSO 4 filtered and evaporated to yield p-toluene sulfonated progesterone (18) (98 g, 92 as a viscous dark yellow oil.

'H NMR (CDC1 3 7,7 (2H, d, J 14Hz, 7,34 0

S

S S( S S S. S

S

S.

S

S

S

(2H, d, J 8,4Hz, 5,67 (1H, s, 4,86-4,93 (1H, m, 3,45-3,50 (1H, dd, J 4,6Hz, H-12); 2,44 (3H, s, H-4Me); 2,15 (3H, s, H- 21) 1,13 (3H, s, H-19); 0,74 (3H, s, H-18).

EXAMPLE 6 12-Hydroxv-a' 4 -progesterone (19) A solution of the tosylated progesterone (18) (98 g, 0,19 mol) in 2,4,6-trimethyl collidine (500 ml) is refluxed at 150 0 C 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 (6M, 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"-progesterone (19) (50 g, 78 as a dark red oil.

c oC C 0C S 10 5H NMR (CDC1 3 5,73 (1H, s, 5,28 (1H, dd, J 2,2Hz, c C C, H-15), 4,41 (1H, br, s, OH), 3,49-3,52 (1H, dd, J 4,3Hz, Co c oC'o H-12), 2,80-2,84 (1H, dd, J 9,2Hz, H-17), 2,14 (3H, s, H- C CI 21), 1,19 (3H, s, H-19), 0.89 (3H, s, H-18).

C c C C C EXAMPLE 7 3,129-Diacetoxyprenca-3,5,14-trien-20-one A solution of a"-progesterone (19) (50 g, 0,15 mol) in acetyl chloride (1,5 4) and acetic anhydride (750 ml) is refluxed for 2 hours. The reaction mixture is poured into cold ethyl acetate (1 4) 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 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,), filtered and evaporated to afford the 3,12Sdiacetoxypregna-3,5,14-trien-20-one (20) (60 g, 93 as an orange oil.

'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, 2,18 (3H, s, 3o@ 10 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 o.

3.12 -Diacetoxy-20,20-ethylenedioxypreana-3,5,14-triene (21) 15 The diacetoxy compound (20) (60 g, 0,14 mol) is dissolved in benzene (1 9) 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 ethyl enedioxypregna 5,14 -triene (21) (35 g, 5 3 !k) 'H NMR (CDCl 3 5,68 (1H, s, 5,45 (1H1, mf, H-6), 5,31 (1H, dd, J 2,2Hz, H1-15), 4,73-4,85 (1H, dd, J 4,4Hz, H-12), 3,78-3,98 (4H, mn, ethylenedioxy), 2,16 (3H, s, 3-Ac), 2,04 (3H, s, 12-Ac), 1,29 (3H, s, H-21), 1,12 (3H, s, H-19), 1,02 (3H, s, H-18).

EXAMPLE 9 3Ig.1fiDivdox-20,20-ethylenedioxprana..S14-diefle-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 0 0 C. The mixture is allowed to warm to :room temperature and stirred overnight. Most of the 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 3I9-alcohol (22) (25 g, 'H NMR (CDC'l 3 5,41 (1H1, m, H1-6), 5,28 (1H1, dd, J 2,2Hz, H-15), 4,72-4,81 (1H1, dd, J 4,4Hz, H-12), 3,82- 4,02 (411, m, ethylene dioxy), 3,45-3,59 (1IH, m, H1-3), 2,03 (31, s, 12 -Ac) 1, 28 (3H, s, H-21), 1,10 (3H, s, H1-19), 1,01 O3H, s, H-18).

EXAMPLE 3f9, 12f9,Dihvdrox-2020-ethlenedioxvmrecn-5,14-diene (23) The 39-alcohol (22) (25 g, 60.2 mmol) in dry tetrahydrofuran (300 ml) is added dropwise to a suspension of lithium aluminium hydride (2,7 g, 72,2 mmol) in dry tetrahydrofuran (500 ml). The reaction mixture is stirred at room temperature for 24 hours after 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 3I, 129 dihydroxypregna-diene (23) (20 g, 90 'H NMR (CDC 3 5,36 (1H, m, 5,23 (1H, dd, J 2,2Hz, H-15), 3,94-4,06 (4H, m, ethylene dioxy), 3,41-3,52 (1H, m, 3,32-3,36 (1H, dd, J 4,3Hz, H-12), 1,31 (3H, s, H) 1,01 (3H, s, H-19), 0,96 (3H, s, H-18).

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

3. 12S-Dihydroxy-14.15-epoxv-20.20-ethylenedioxyprecm-5ene: 39, 121-Dihydroxy-5,6-epoxy-20,20-ethvlenedioxvprecn-14-ene go 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°C. After 15 min sodium sulphite (5 soln, 50 ml) is added to the reaction mixture. The acetone is evaporated, and the aqueous layer extracted with dichloromethane (3 X 50 ml). The organic layer is dried (MgSO 4 filtered and evaporated. Pyridine (1 ml) is added to the product, and stirred for 0,5 h. Dichloromethane (100 ml) is then added to the reaction mixture, and the dichloromethane is washed with citric acid (5 soln, 3 X 100 ml), saturated sodium bicarbonate (50 ml), and water (50 ml). The organic layer is dried (MgSO) 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 39, 121-Dihydroxv-14,15-epoxy-20.20-ethvlenedioxyprecn-5ene (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 10 (1,69 ml), and sodium hydroxide (15 soln, 1,69 ml).

After filtration and evaporation of the solvent, the crude Sproduct is purified by silica gel column chromatography using methanol/chloroform as solvent to give the e* unreacted 14,15 epoxy- 20,20-ethylenedioxypregn-5-ene (24) (300 mg, 2,1 H NMR (CDC1 3 5,31 (1H, m, 3,82-3,98 (4H, m, ethylene dioxy), 3,43-3,52 (1H, m, 3,41 (1H, s, H- 15), 3,31-3,35 (1H, dd, J=4,3 Hz, H-12), 1,29 (3H, s, H- 21), 1,17 (3H, s, H-19), 1,02 (3H, s, H-18).

20 "C NMR (CDC1 3 139,8 120,8 112,1 (C- 77,2 75,4 61,0 22,3 (C-21 19,2 9,5 (C-18).

EXAMPLE 12 39, 129. 146-Trihydroxv-20.20-ethylenedioxyprecn-5-ene (2E 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 refluxed for 48 h. After the addition of water (0,3 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 4*

S

S

*5

S

S*S..

S

S.

S

SSSS

S. S S C *S

S

S.

S

(A S *5 S

S

55

S

S

S

S. S

S

0* as solvent to give the trihydroxy pregnene (25) (250 ing, 8 3 'i) 'H NIVR (CDCl,) :5,38 (1H, mn, 3,98 (4H1, mn, ethylene dioxy) 3,43-3,53 (1H1, mn, 3,25-3,32 (1H, dd, J 4,1Hz, H-12), 1,32 (31, s, H-21), 1,01 (311, s, 11-19), 0,98 (3H, s, H-18) NMR CDC1 3 :139,1 122,1 112,2 85,1 75,1 71,6 23,4 19,4 8,9 (C-18) EXAMPLE 13 31g. 129. 141g-Trihydroxv-precmn-5-ene The ethylenedioxypregnene (25) (250 mg, 0,64 iniol) is dissolved in acetic acid (13,4 ml) and water which after freeze drying affords the trihydroxy steroid (15) (200 ing, 89 1) m.p. 228 0 -235 0 C (lit 225 0 -235 0 C) M+ 348, (aD] 0 350 (lit CaD 20 2.90) 'H NMR (CDC1,) 1 3 C NMR (CDC13) 5,39 (1H1, m, H1-6), 3,56-3,62 (1H, t, J 8,1 Hz, H1-17), 3,42-3,51 (111, mn, H- 3,28-3,39 (1H1, dd, J 4,3Hz, H- 12), 2,23 (311, s, 11-21), 1,01 (3H, s, 1-19), 0,90 (3H, s, H1-18) 217,7 138,9 122,2 85,5 73,6 71,6 57,0 55,1 43,6 42,1 37,3 36,8 35,9 34,5 32,9 31,5 30,1 27,4 24,.4 19,4 (C-19), 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 0-ty ndoy36 oun pslhnlx-rg 51 -e 0 *0.S 0 0@ S

C

*0 0

S

0 9.

S S S @550 S. 0

S

t C.

S

00 0 0 S. 0 000 0 0

S

S. *i 0O

S

01*5

J

COO.

20

S

a. 0 00 0.

S

diene-1213-ol acetate (26) A solution of ptoluenesulphonyl chloride (650 mg, 3.4 mmol) in pyridine ml) was added dropwise to a mixture of the 20,20- Ethylenedioxpregna-5,14-diene-3,2-diol 12-acetate (22) (1.3 g, 3.1 mmol) in pyridine (15 ml) at 0 0 C. The reaction mixture was left stirring at room temperature for 24 hours after which water was added to the reaction mixture. The solution was extracted with ethyl acetate (2x50 ml), the 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 ethyl acetate was dried (MgSO4), filtered, and evaporated and purified by flash column chromatography using hexaneethyl acetate (8:2 v/v) as the eluant to give the 0-O-tosyl steroid (1.5 g, as a yellow oil, (Found M 570.271, C 3 2

H

4 2 0 7 S requires: M 570.273).

6 H 1.021 s, 19-H), 1.131 (3H, s, 18-H), 1.282 (3H, s, 21-H), 2.021 (acetateOCH 3 2.431 (3H1, s, A-r-CH 3 3.883 (4H, m, OCH 9

CH

2 4.750 (1H, dd, J 10O.8 Hz, 5.2 Hz, 12-H), 4.890 (1H, m, 30H), 5.281 (1H1, dd, 3 J 4.2 Hz, 2.1 Hz, 15-H) 5.388 (1H1, m, 6-H) 7.341 (2H, d, J 8.2 Hz, ArH) 7.746 (211, d, J 8.2 Hz, ArH).

13.493Q 19.002Q 21.612Q (Armethyl)*, 21.671Q 24.175Q (acetate methyl) 63 .401T (ethylenedioxy), 63 .498T (ethylenedioxy), 71.531S 80.912D 82.531D 111.363S 120-881D 121.461D 123.715- 133 .917 (Aromatic) 139, 903S (C-14) 151, 722S 61 170.819S (ester carbonyl).

may be interchanged EXAMPLE 20-Ethyl enedioxy- 3ai,5 -cyclo-5a- -pregn- 4- ele 6,2213diol-22-acetate (27) A solution of 3I9-toluene-psulphonyloxy-preg'n-5,14-diene (26) (1.2 g, 2.1 mmol) and potassium acetate (2.2 g, 22.4 mmol) in -water (250 ml) and acetone (500 ml) was ref luxed at 60 'C for 16 hours. The acetone was evaporated and the water was extracted with 10 ethyl acetate (200 ml) The ethyl acetate was dried (MgSOj filtered, and evaporated. Flash chromatographic separation of the mixture using chlorof orm- acetone (9:1 v/v) as the eluant gave the 3c,5-cyclo derivative (27), (530 mg, 6110) as a yellow oil, (Found M 416.262, C 2 5

H

3 6 0 15 requires M 416.263).

61 0.288 (1H, dd 3 J 8.1 Hz, 4.9 Hz, 0.477 (1H, dd 3 J 4.4 Hz, 4.4 Hz, 1. 025 (3H, s, 19-H) 1.121 O3H, s, 18-H), 1.256 (3H, s, 21-H), 1.989 (3H, s, acetate-CH 3 3.302 (lH, dd 3 J 2.8 Hz 2.8 Hz, 6- H) 3. 784 947 (4H, m, OCH 2

CH

2 O) 4.721 (1H, dd 3 J Hz, 5.6 Hz, 12-H), 5.232 (1H, dd, J 3.9 Hz, 1.9 Hz, 23.623Q(C-21) 24.153Q .(acetate methyl), 63.700T (ethylenedioxy), 63.788T (ethylenedioxy), 73.591~D (C- 80.551D 111.126S 118.778D 152.959S 170.991S (ester carbonyl).

EXAMPLE 16 20 -Ethyl enedioxy- 3az, 5-cyclo-5c-pregn-14 -ene-69, 129-diol (28) A solution of the 3cr,5-cyclo derivative (27), (500 mg, 1.2 rnmol) in tetrahydrofuran (20 ml) was added dropwise to a suspension of lithium aluminium hydride mg, 1.3 mmol) in tetrahydrofuran (10 ml). The reaction mixture was stirred for 4 hours and quenched by the addition of water (50 After 30 minutes, sodium hydroxide was added (15k solution, 50 Aul) and stirring continued for a further 30 minutes. Water (150 p1l was added and the reaction mixture was filtered. The tetrahydrofuran was dried (MgSo,) filtered and evaporated and flash chromatographic purification using chloroformacetone (8:2 v/v) as the eluant to give the diol (370 mg, 83k) as an oil, (Found M 374.250, C 23

H

34 0 4 requires:1' 374 .252) 6,1 0.298 (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), 0.985 (3H, s, 19-H), 1.055 (3H, s, 18-H), 1.325 (3H, s, 21-H) 3.318 (1H1, dd, J J3.0 Hz, 3.0 Hz, 3.363 (1H1, dd, J J11.4 Hz, 4.2 Hz, 12-H), 4.019 (411, m, OCH 2 Ch 2 O) 4.622 (111, s, OH) 5.255 (1H1, dd 3 J 3.9 Hz, 1.9 Hz, 6C~ 11.681T(C-4), 12. 243Q (C-18) 19.844Q (C-19), 23.604Q(C-21) 63.620T (ethylenedioxy), 63.733T (ethylenedioxy), 73.569D 77.478D (C-12), 111.125S 118.702D 152.912S (C-14).

EXAMPLE 17 20, 20-Ethyl enedioxy-14, 15j-epoxy-3, 5-cyclo-5(, 4,6pregnane-69,12fB-diol (29) N-bromoacetamide (150 mg, 1.1 mmol) was added to a solution of the 20,20-ethylenedioxy- 3a,5-cyclo-5ca-pregn-14-ene-613,129-diol (28) (340 mg, 0.91 mmol) in acetone (20 ml), water (0.25 ml) and acetic acid (0.25 ml) at 0 0 C. After 15 min., sodium sulphite (501 solution, 20 ml) was added to the reaction mixture. The acetone was evaporated under reduced pressure and the remaining solution was extracted with dichloromethane (3x30 ml). The dichloromethane layer was dried (MgSO,), 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) 0.287 (1H, dd, 3 J 8.1 Hz, 4.9 Hz, 0.501 (1H, dd, 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, 15 dd, 3 J 3.1 Hz, 3.1 Hz, 3.355 (1H, dd, J *g 11.2 Hz, 4.1 Hz, 12-H), 3.491 (1H, s, 15-H), 4.001 (4H, m, OCH 2 ChO), 4.901 (1H, s, OH) .c 11.668T(C-4), 11.973Q(C-18), 19.515Q (C-19), S" :23.519Q(C-21), 59.910D 63.601T (ethylenedioxy), 20 63.713T (ethylenedioxy), 72.501S 73.571D 77.471D 111.085S EXAMPLE 18 20,20-Ethylenedioxy-6,12B, 14-trihydroxy-3a, 5-cyclo-5a, 148pregnane (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 pl) 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 l) and the suspension was stirred for 1 hour. After 4_& filtration, the suspension was dried (MgSO 4 filtered, and the tetrahydrof uran was evaporated. Flash chromatographic separation7 of the resulting mixture eluting with chloroform-methanol (9:1 v/v) gave the required triol (90 mg, 5311) as a clear oil, (Found M 392.261, C 23

H

38 0 requires: M 392.263).

511 0.287 (1H, dd 3' J 8.1 Hz, 4.9 Hz, 4-H 2 0.510 (1H, dd 3J 4.4 Hz, 4.4 Hz, 4-Hb),1 0. 971 (3H, s, 19-H) 1.042 s, 18-H) 1.319 (3H, s, 21-H), 3.321 (1H, dd 3 LT3. 0 Hz, 3. 0 Hz, 6 3A32 1 (1H, dd, 13J11. 1 Hz, 3.9 Hz, 12-H), 3.561 (1H, s, 0OH), 4.084 (4h, mn,

OCH

2 Ch 2 O) 4.671 (1H1, s, OH) 63.612T (ethylenedioxy) 63.711T 15 (ethylenedioxy) 73.483D 76. 051D (C-12) 84 .307S (C1) 11.9S.C2) EXAMPLE 19 3B, 12S, 14 -Trihydroxy-14t9-pregn-5-en -20O-one (15) A mixture of the trial (30) (80 mg, 0.20 mmol) in acetone (20 ml) hydrochloric acid (IM, 10 ml) was ref luxed at 60*C fcr hours. The reaction mixture was cooled and saturate-_ sodium bicarbonate solution (20 ml) was added. The acetcn.' was evaporated and the aqueous layer extracted w::t.

chloroform (3 x 20 ml), the chloroform layer was dr-'e: (MgSO 4 filtered and evaporated to give the epimer:.= trihydroxy steroids (15a, 15b) (42 mg, 6111). Separatio.cf the epimeric mixture (15a, 15b) (15 mng) was achieved bv flash chromnatographic separation using chloroform methanol (9:1 v/v) as the eluant to give the pure 179~epimer (15a), (10 mg), rn.p. 224-229 0 C (acetone), (lit. 226- 2230) (Found M 348.234, C; 72.32, H 9.21-0i C,:H37O 4 requires: C, 72.38; H M 348.236) and the 17cr-epimer (15B) (3 mg) 1 m.p. 183-191 0 C (acetone), (lit 184-1960).

3LB,12B,14 -Trihydroxy- 1 4I-pregn -5-en -20O-one (15a) s 21-H) 3.325 (1H, dd, 3J 11.2 Hz, 3.9 Hz, 12-H) 3.464 (1H1, s, OH) 3.5140 (1H1, mn, 3-H) 3.598 (1H1, dd, 3J 9.6 Hz, 9.6 Hz, 17-H) 4.255 (1H, s, OH) 5.383 (11, m, 5 C 8.275Q 19.414Q 24.400T (C-li) :24.581T (C-16) 27.443T 30.062T 32.972Q 10 34.543T (C-15) 35.864D 36.975S 37.337T 42.144T 43.565D 55-101S 57.038D 71.597D 73.558D (C-12), 85.566S 122.223D 138.932S 217.011S 3I.,12f,14-Trihydroxy-14I8-pregn-5-en-2O-one :6H 0. 996 (1H, s, 19-H) 1. 144 (3H1, s, 18-H) 2.221 (3H, S 2-H) 3.339 (1H, dd, 3J 9.4 Hz, 9.4 H,1-H), *3.492 (1H, m, 3-H) 3.629 (1H, dd, 1J11.1 Hz, 3.9 Hz, :12-H) 3.712 (1H, s, OH) 4.325 (1H, s, OH) 5.383 (lE, m, Examples 20 to 28 illustrate the procedures whereby e intermediate compoundg may be prepared to form thefrs monosaccharide EXAMPLE Methv1-4,6-0-benzvlidene-ca-D-glucopvrano ide (32) A mixture of methyl-a-D-glucopyranoside (30 g, 0, 1S ml) benzaldehyde (70 ml) and zinc chloride (20 g) is stirred at room temperature for 24 hours. The reaction product is poured into ice water and stirring continued for 15 min. The white precipitate is filtered and washed with r diethyl ether. The solid material is stirred with a solution of sodium metabisulphite (10 soln), for 15 min, filtered and washed with water. The solid material is crystallized from chloroform and ether to yield the benzylidene product (32) (31 g, 72 EXAMPLE 21 Methyl-4,6-0-benzvlidene-2-0-tosyl-a-D-qlucopyranoside (33) p-Toluene sulfonyl chloride (25 g, 1,2 eq) in pyridine (100 ml) is added dropwise to a solution of the benzylidene :i 10 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, 15 S* EXAMPLE 22 "Methyvl-4,6-0-benzvlidene-3-0-methyl-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-deoxv-a-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 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 69 EXAMPLE 24 Methyl-4-0-benzoyl-3-0-methyl-6-deoxv-a-D-altropyranoside (36) Sodium borohydride (18 g) in water (30 ml) is added dropwise to a solution of the bromoaltroside (35) (9 g, 23 mmol) and nickel chloride (18 g) in ethanol (300 ml) at 0°C.

The reaction mixture is refluxed at 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 filtered and evaporated, to yield the 6-deoxy-altroside (36) (5 g, 72 Examples 25 to 33 illustrate the procedures synthetic 0* 4 *44* *4 4 .4 whereby the intermediate compounds may be prepared to form the second monosaccharide EXAMPLE 1,2 5,6-Di-0-isopropylidene-a-D-glucofuranose (42) Sulfuric acid (40 ml) is added dropwise to a solution of a-D-glucose (41) (50 g, 0,28 mol) in acetone (1 R) at 0 C. The reaction mixture is stirred for 24 h and then it is neutralized using sodium hydroxide (6 The acetone is evaporated and the aqueous layer is extracted with chloroform The chloroform is dried (MgSO 4 filtered and evaporated. Crystallization from cyclohexane yielded the di-isopropylidene glucose (42) (41 g, 57 EXAMPLE 26 1.2 5.6-Di-0-isopropylidene-3-0-methyl-a-D-qlucofuranose (43) 10 S* 10 5*15 4 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 (MgSO 4 filtered and evaporated to yield the methyl protected glucose (XXV) (38 g, 83 EXAMPLE 27 (44) The methyl diisopropylidene compound (43) (38 g, 0,14 mol) is dissolved in acetic acid (50 1, 700 l)adthe 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 glucopyranoside (44) (13 g, 50 e EXAMPLE 28 Mehl.0mehla-..lcprnoie ~Methyl--mehv-c-lucopranoside (45) U M the 460bnie-3-0-methyl-c4-glucopyranoside (44) i The glucopyranoside (45) (8 g) is stirred at room temperature in a solution of benzalaldehyde (20 ml) and zinc chloride (5 After 24 hours, ice is added and the aqueous layer is extracted with chloroform. The chloroform layer is dried (MgSO,), 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-ap-glucopyranoside (46) EXAMPLE Methyl 4-0-benzovl-0-methyl-6-deoxv-aB -clucopvranoside (47) *o The benzylidene compound (46) (5 g) is refluxed at 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,), filtered and evaporated to give the bromo compound The bromo compound (4,3 g) is dissolved in a solution of ethanol (300 ml) and nickel chloride (8,6 g) at 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 0 C for 45 minutes, cooled, filtered and evaporated. Chloroform is added, and the chloroform layer is washed with water, dried (MgSO,), filtered and evaporated to give the 6-deoxy sugar (47) EXAMPLE 31 4-0-Benzoyl-3-0-methyl-l-phenylthio-6-deoxy-aqlucopyranoside (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,), filtered and evaporated. The product is purified by silica gel column chromatography eluting with ethyl acetate:hexane to give the compound (48) EXAMPLE 32 4-0-Benzoyl-3-0-methyl-2-0-pivaloyl-1-phenylthio-6-deoxyca-qlucopvranoside (49) To a solution of the glucopyranoside (48) (2 g) in pyridine (20 ml), pivaloyl chloride (2 ml) 'is added. The solution is stirred at room temperature overnight after which water is added. The aqueous layer is extracted with ethyl acetate, and the organic layer is washed with HCl (6 N) The organic layer is dried (MgSO 4 filtered and evaporated to give the pivaloyl ester (49) -1V- EXAMPLE 33 4-0-Benzovl-3-0-methvl-2-0-D2ivalov1-l-f luoro-6-deoxy-Bglucor~vranoside N-Bromosuccinimide 2 g) and diethylaminosuiphur trif luoride 2 g) are added to a solution of the pivaloyl :ester (49) (2 g) in dichioromethane (100 ml) at 0 0 C. After 1 'hour, saturated sodium bicarbonate is added. The *dichioromethane layer is dried (MgSO 4 filtered and evaporated. The 13-f luoropyranoside (50) is purified by silica gel column chromatography eluting with ethyl acetate:hexane (yield Example 34 illustrates the synthetic procedure whereby the compound 3-0- [4-O-benzoy1-2-phenylthio-g-Dcymaropyranosyl] 12, 141-dihydroxy-pregnan-5 -ene -20 -one (51) may be prepared.

EXAMPLE 34 r4-0-benzovl-2-phenlthio-g-D-cvnaropyranosvl1 -12 .149dihydroxvy-preqn-5-en-20-one (51) Tin chloride (190 mg, 1 mmcl) is added to a solution of 3,12,14 B-trihydroxy pregnan-5-ene-20-one (15) (100 mg, 0,28 mmcl) 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 73 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, EXAMPLE o..

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

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

*o 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 place by intraperitoneal injection, and observations were 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 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 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 00 0 0 0 0 o 0 0o C. 0 cl: 0 0 0 o 0 0 0 o 0 o o o o S oo0 o o r, o 0 o 0 o 0 0 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 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°C after treatment.

0 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 .0 C 15 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 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

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S*

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 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.

5 55 5

S.

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55 5

S.

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.

0 71 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 schedule was followed to ensure that animals were not unnecessarily treated when an acute toxic dose had been reached in the previous group.

9@ 9*

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9

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S. 15 9.

Animals were observed-for clinical signs of toxicity immediately (1-2 hours) after treatment and daily 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, CentaurR) 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.

Results Group 1 (Control Group) No clinical signs of toxicity were observed during the 14-day observation period. Food and water intakes were -Th 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 mc/kq) No clinical signs of toxicity were observed during the observation period. Food and water intakes were normal and changes in body mass over the observation period were also S* normal. No macroscopical pathology was observed and no 0 histopathological or morphological changes were recorded in S 10 the liver samples.

Group 3 (200 mg/kq) 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 i.

animal 5. Moderate hydropic degeneration also occurred in the hepatocytes of animal Group 4 (400 mq/kq) 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 respectively), but the increase in body mass was only 0,81 g compared to 2,7 g.

Groun 5 (800 ma/ka)

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*5 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. Nc foreign body reaction which would have indicated inhalation of test material was present. No macroscopic pathology was observed in animal 9. Mild cytoplasmic vacuolisation (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/kq) 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 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.

10 Group 7 (3 028,5 mq/kg) .0.

.00.* 0000 *0*0 0 00 0 0 00. 0000 0 15 *0 0* 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.

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 0@

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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.

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.

5 0

S

*5 From the histopathological examination of the liver samples, it was clear that the pathological changes were 15 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.

c) Oral Dose Anorectic Test 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 6 (referred to above)

.S

S: 10 Experimental Procedure Animals were allocated to treatment groups using randomisation tables. Each treatment group consisted of t 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 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.

^J

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 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 10 receiving only the carrier substance. Doses were chosen on the basis of the effects observed in the aforedescribed Irwin Test and were: o*o• *o *oo *oo *o *oo o *o Group i: 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 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 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 groups showed an increased water consumption.

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.

9 9 9* 9 *9*9 9 The reduction, and in some animals even a loss, in body mass gain, in combination with the reduced food intake is 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 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 from the gastrointestinal tract, with consequent reduced bioavailability. The bioavailability at the oral doses tested was, however, sufficient for the test sample to be 0 <g-b 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 a a o* g o* o *ooo o*o o*o *o* qj 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, o 0: .especially where individual animals react totally I10 differently. However, the data indicates that the test g sample has appetite suppressive action, even at the lowest 00. dose tested (6,25 mg/kg). No clinical signs of respiratory o suppression occurred at the doses tested.

*.00 EXAMPLE 36 06060: 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 3 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.

S

S

S.

S

S

S

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

Experimental 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 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.

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

C

70 0

C

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.

9 9 0* 9 9. 9* 9 9 9 9**9 9@ 9 5 HPLC Analysis Method Eluant Column UV absorbance Flow rate Injection volume Acetonitrile: isocratic Reverse phase C-18 225 nm 1 ml/min water Method Spray-dried powder (10 mg) was dissolved in water (0.5 ml) 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 was prepared from the pure compound EXAMPLE 37 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 spraydried 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.

10 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 water consumption were recorded.

Sample 3 (active moiety) produced statistically significant reductions in food consumption at an oral dose of mg/kg. Nostatistically 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.

The reference standard, fenfluramine (7.5 mg/kg), produced statistically significant reductions in food consumption at 6 and 24 hours post-dose when compared with the relevant 10 vehicle-treated control group. No statistically o.

significant effects on water consumption or bodyweight were recorded.

t No treatment-related effects on the livers were recorded.

9 9 0@ 9 9 9 9 C 9 9 9 *99* *9 *99*9*9 Identity Sample 1 (pure sap) Appearance Brown liquid storage conditions -20 0 C in the dark Purity Pure sap Vehicle Distilled water TEST SUBSTANCE Sample 2 (spray-dried sap) Powder Room temperature in the dark Pure spray-dried sap Distilled water

I.

Sample 3 (active moiety) White powder 4 0 C in the dark Dimethylsulphoxide

(DMSO)

C

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

*4* 4*e p L. 00 0 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: Group n Oral treatment 04 0

S

4P00 0

S

0*0 48*000 4 S 15 Vehicle (distilled water) Sample 1 (pure sap) Sample 1 (pure sap) Sample 1 (pure sap) Sample 1 (pure sap) Sample 1 (pure sap) Sample 2 spray-dried sap Sample 2 spray-dried sap Sample 3 (active moiety) Sample 3 (active moiety) Fenfluramine Vehicle (DMSO) Dose (mg/kg) 800 1600 3200 6400 10000 38 76 Groups 1 8 were dosed using a constant dose volume of 1C 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

C

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 Am and stained for fat with Oil Red 0 (ORO) 9 10 Data Analysis i. 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, similarly- o treated vehicle control group using analysis of variance followed by Williams' test for comparisons with controls.

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, 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 10 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 eo". 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 post-dose. The biological significance of these effects therefore remains unclear.

Sample 1 (pure sap) produced dose-related, statistically :l: significant effects on bodyweights when compared with the vehicle-treated control group (Group i) When administered Si.l 10 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 15 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 (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.

e 0* 0 09 0900 9 0 9 0 S @9 0 *9 *0 0 00 *0 0* 00 9 09 9@*0 S @909 9 9 0 0 9 9 9 0 0 9*99 99 TABLE la Effects of oral administration on food consumption In the rat (daily pre-dose data)

F-

Group 1 2 3 4 6 7 8 9 I Oral treatment I Dose (mgfkg) Grop manfood cosumption (g±t sd) between Days: L5 Vehicle (water) Sample I sap Sample I sap Sample 1 sap Sample I sap Sample 1 sap Sample 2 spray-dried Sample 2 spray-dried Sample 3 active moiety Sample 3 active moiety 800 1600 3200 6400 10000 38 76 2.5 5.0 27.8 ±0.54 28.3 ±1.43 29.0 ±1.39 27.2 ±2.33 28.7 ±1.64 28.5 ±2.38 28.1 1.24 28.7 ±0.91 28.8 ±1.49 28.3 ±2.1 29.1 ±0.66 27.9 24.2 ±1.83 24.9 ±0.82 25.0 ±2.16 25.1 ±2.46 25.3 ±1.73 23.7 ±2.73 23.9 1.79 26.5 ±1.55 26.4 ±3.12 25.8 ±1.86 25.3 ±4.0 3 26.7 -4- -3 27.6 ±3.67 27.7 ±0.76 27.4 ±1.96 26.0 ±2.52 27.3 ±1.45 26.0 ±2.31 24.5 ±2.30 27.1 ±1.01 29.0 ±1.99 28.1 ±2.65 27.0 ±1.53 28.7 -3- -2 28.3 ±3.50 28.4 ±1.51 28.8 ±0.61 28.5 ±2.29 29.2 ±1.09 27.0 ±3.50 27.6 ±1.61 28.7 ±1.99 29.4 ±1.76 28.0 ±2.65 30.8 ±0.54 28.1 -2- -1 29.4 ±2.66 30.1 ±0.27 29.5 1.55 27.6 ±1.15 30. 3 ±0.90 28.7 _±2.26 28.5 _±1.87 28.9 1.37 29.5 _±2.81 28.5 ±3.03 29.7 ±2.84 30.5

I

11J Fenfluramine -2 Vehicle (DMSO) j ±1.8 ±2.11 ±1.99 ±4.06 ±2.54 sd Standard deviation 0 00 n0 n* 00o 0 00 00 00 .f00l, 00 000 0 0 0 0 0-00 00 0-00!00o 00 TABLE lb ______Effects of oral administration on food consumption In the rat (daily post-dose data) Group Oral treatment IDose (mgfkg) Group mean food consumption (g sd) between Days: I I I 1 2 3 4 6 7 8 9 Vehicle (water) Sample 1 sap Sample 1 sap Sample 1 sap Sample 1 sap Sampl'e I sap SaMmp2sra-re Sample 2 spray-dried Sample 3 active moiety 800 1600 3200 6400 10000 38 76 2.5 1--2 j2-3 3-4 29.5 ±3.15 26.1 ±0.98 22.6- ±3.17 20. 1 ±1.39 18.2- ±4.18 15. 1 ±2.98 25.6 ±2.85 24.2* ±3.25 26.8 29.6 ±2.84 29.3 ±1.49 26.9 ±2.06 19.0** _±1.88 14.8- 75 12.4** ±2.61 27.3 ±0.95 25.2' ±3.24 29.1 ±3.43 21.Ott ±3.07 31.9 ±0.84 30.6 ±4.43 30.6 ±3.49 30.7 ±1.15 30.9 ±2.54 22.8** ±1.77 18.4" ±0.97 16.0** ±3.15 30.3 ±2.06 29.9 ±1.85 31.7 ±3.08 27.6 ±5.26 32.7 ±2.50 30.1 4-5 31.8 ±3.21 30.9 ±0.60 30.9 ±1.22 28.0 ±3.14 22.4- ±3.01 19.7** ±4.31 31.0 ±2.13 30.2 ±2.28 34.0 30.

33.0 ±2.55 32.'4 5-6 6-7 7-8 30.7 31.7 32.9 ±2.24 ±3.03 ±3.18 33.3 32.7 40.1 ±1.69 ±0.80- ±13.40 34.1 ±1.36 31.4 ±2.08 2 26.9 ±2.81 22.6' ±5.70 31.8 ±1.63 31.2 ±2.26 34.4 ±4.32 33.0 ±3.16 30.*4 ±0.23 31.8 33.7 ±1.69 32.3 ±2.91 31.0 ±2.3 1 30.1 ±4.79 31.1 1.94 32.3 ±1.44 33.1 ±4.11 32.4 ±3.25 32.7 ±1.90 32.8 33.8 ±1.61 33.0 ±3.01 32.0 ±2.34 ±5.90: 31.8 ±2.45 33.*1 ±0.61 34.8 ±3.71 33.0 ±3.84 32.4 ±1.60 33.3 Sample 3 active moiety 5.0 I 22 1 Fenfluramine

I-

Vehicle (DMS0) 36-.- I I sd Standard deviation Groups 2 8 were compared with vehicle Group 1: 'p<0.05,-p<0.0j Groups 9 11 were compared with vehicle Group 12:"p<0.05, 11 p<0.01 ±4.17 I ±5.26 I ±3.08 ±3.98 ±3.76 SO *e *ee@ S. S *e S S S 55 *S~iS S S S S S S S 0 S OS S SOS 55 *S*S S. S S. S* S 55 *5*S S S S S S S S S S S SS 55 555 *SSS 55 TABLE 2a Effects of oral administration on water consumption In the rat (daily pre-dose data) I -I I Group Oral treatment Dose (mg/kg) 1 ~Vehicle (water) J_ Sample 1 sap 800 3 Sample 1 sap 1600 4 Sample 1 sap 3200 Sample I sap 6400 6 Sample I sap 10000 7 Sample 2 spray-dried 38 8 Sample 2 spray-dried 76 9 Sample 3 active moiety 2.5 Sample 3 active moiety 5.0 11 Fenfluramine 7.5 12 Vehicle (DMS0)- Group mean water consumption (gt: sd) between Days: -2- -4 -3 -1 40.9 34.8 37.6 33.5 32.2 ±t4.61 ±4.15 ±5.63 ±7.42 ±6.32 38.6 37.1 36.4 28.1 30.4 ±1.96 ±9.74 ±4.81 ±1.83 ±4.75 43.4 35.9 38.4 31.1 36.5 ±1 0.53 ±3.84 ±4.56 ±4.47 ±5.39 40.1 33.3 37.3 31.3 31.7 ±5.58 ±3.01 ±4.46 ±3.48 ±3.18 43.8 36.3 35.4 34.0 35.1 ±8.57 ±9.02 ±8.18 _±6.62 ±5.72 37.4 32.7 33.2 29.0 32.2 ±5.34 ±t3.35 ±4.86 ±5.11 ±3.27 40.0 35.8 34.7 30.2 31.4 ±4.36 ±4.92 ±3.20 ±1.88 ±2.98 38.6 37.0 48.8 31.6 39.0 ±1.98 ±1.96 ±21.5 ±4.56 ±17.27 42.0 37.0 34.1 28.0 31.6 ±6.70 ±5.05 ±3.16 ±2.58 ±3.12 40.9 34.2 32.7 28.2 33.1 ±4.48 ±3.00 ±1.26 ±1.65 ±4.82 47.0 35.5 34.7 30.9 31.6 ±5.3 ±7.49 ±3.73 ±2.12 ±2.80 43.3 34.5 35.2 28.3 31.4 ±5.67 ±4.97 24.34 ±46 6.44 sd Standard deviation S S S S S S S .5 5*55 555 55 5555 *5 S. 55 5 55 S. S 55 S S S S S S S S TABLE 2b Effects of oral administration onwater consumption In the rat (daily post-dose data) 1~ I I I Group Oral treatment Dose (mg/kg) Group mean water consumption (g sd) between Days: 1-2 j 2-3 3-4 1 4-5 5-6 1 6-7 1 -8 1i Vehicle (water) j J 34.9 36.9 38.0 ±606 t7 f~.59r Sample 1 sap 30.9 34.4 38.2 ±3~77 ±812 ±13.71*Y Sample 1 sap 1600 29.2 4 Sample 1 sap 3200 35.9 Sample I sap 6400 33.4 12.04 6 Sample 1 sap 10000 31.7 7 Sample 2 spray-dried 38 36.0 31.7 ±5.35 26.2* ±2.66 27.4' ±8.13 28.5' ±8.85 34.5 ±1.79 39.1 ±16.59 36.1 ±12.42 31.5 ±8.12 41.3 ±11.21 30.5 ±2.44 32.6 ±10.67 32.4 ±8.87 38.2 ±7.16 46.9 ±18.34 38.3 ±11.71 35.1 ±3.82 36.7 ±3.92 37.2 ±6.18 35.9 ±13.51 34.6 ±4.10 34.1 ±4.80 35.4 ±10.78 36.6 ±6.50 39.6 ±7.09 35.9 ±3.40 41.5 ±16.60 37.7 ±5.99 33.8 ±2.89 35.2 37.7 ±5.54 39.5 ±11.20 48.1 ±12.27 45.8 ±18.54 45.2 ±8.72 40.7 ±11.51 42.7 ±9.74 41.9 ±12.37 35.3 ±2.86 28.8 ±1.22 37.8 ±7.28 51.0 ±35.21 36.2 ±6.72 38.0 ±6.66 45.6 ±17.15 36.9 ±8.47 36.5 ±5.85 31.8 ±5.58 36.9 ±9.28 42.6 ±13.88 35.9 ±9.58 37.5 ±6.21 46.1 ±9.49 38.1 ±8.93 Sample 2 spray-dried 76 45.0 1±19.031 9 Sample 3 active moiety Sample 3 active moiety 2.5 32.

34.7 I33.00 I 35.3 ±7.57 4±4.20 ±8.70 I 4 j~±240 Fenfluramine 7.5 34.1 37.2 39.5 ±7.78 33.7 ±5.43 33.8 37.4 ±11.07 32.1 ±1.93 32.3 ±7.44 37.8 ±6.42 33.6 ±2.50 ±360 148R 1 1 Vehicle (DMSO) 40.7 33.8 32.9 I32.0 +q910 4±937 ±&707 1 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) ±11.49 ±9.82 ±7.22 0 0 0 0 so TABLE 3a Effects of oral administration on bodyweight in the rat (daily pre-dose data) Group Oral treatment Dose (mg/kg) Group mean bodyweight (g sd on Day -4 -3 -2 -1 SVehicle (water) 130.9 150.7 157.3 168.1 177.5 ±5.56 ±5.37 ±5.29 ±6.20 ±6.70 2 Sample 1 sap 800 131.6 150.1 158.5 169.6 177.7 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 ±4.3 ±6.59 ±6.38 ±6.04 ±6.37 4 Sample 1 sap 3200 130.8 147.7 154.4 165.2 175.8 ±6.19 ±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 -6.75 ±9.

0 8 ,9.37 10.41 10.90 7 Sample 2 spray-dried 38 131.7 149.0 156.2 166.7 175.6 ±8.28 ±5.85 ±5.81 ±5.54 ±8.42 8 Sample 2 spray-dried 76 130.0 146.1 155.9 166.0 175.1 9 ±6.00 ±6.59 ±6.87 ±6.55 9 Sample 3 active moiety 2.5 132.6 148.9 157.3 169.8 179.4 ±7.63 ±8.51 ±8.91 ±8.96 ±8.71 Sample 3 active moiety 5.0 133.5 150.5 158.8 171.0 179.0 ±6.45 ±9.55 ±8.48 ±7.72 ±9.20 11 Fenfluramine 7.5 133.2 152.7 160.0 170.0 182.8 :±9.21 ±9.09 9.82 ±9.15 ±10.21 12 Vehicle (DMSO) 129.1 147.3 155.0 166.0 174.8 I I ±3.17 ±4.37 ±6.29 ±591 ±8.26 sd Standard deviation *0 1 TAB3LE 3b Effects of oral administration on bodyweight In the rat (daily post-dose data) I Group 2 3 4 6 7 8 Oral treatment Vehicle (water) Sample 1 sap Sample 1 sap Dose (mg/kg) 800 1600

I

Sample 1 sap Sample 1 sap Sample 1 sap Sample 2 spray-dried Sample 2 spray-dried 3200 6400 10000 38 76 Group meanbdwgh(g±s)o Day: 2 3 4 5 6 192.6 202.0 211.2 220.2 227.2 ±7.16 ±10.17 ±7.98 ±10.35 ±10.26 187.0 198.5 206.8 214.8 222.8 ±4.55 ±4.20 ±5.91 ±4.65 ±4.99 186.0 193.2 204.0 212.4 223.0 ±8.28 ±6.42 ±6.40 ±5.81 ±6.33 184.6 186.2' 189.8- 199.2* 210.6** ±8.88 ±8.67 ±9.99 ±9.34 ±10.21 18. 138 185.2- 191.2- 201.0 ±856.3 ±63.87 ±9.18 ±7.89 ±6.89 181.4 179.8- 180.6- 185.6- 192.2- ±14.06 ±15.85: ±13.85 ±11.28 ±10.99 185.8 195.8 205.6 214.4 222.6 ±9.23 ±7.79 ±9.79 ±9.61 ±9.34 183.4 188.6' 198.2* 206.0' 214.0 t7.57 ±t6.73 ±8.50 ±9.43 ±9.51 191.2 200.0 209.6 219.6 229.4 ±11.15 ±11.25 ±t12.28 ±12.95 ±t13.69 192.0 192.4 201.0 209.4 219.8 ±9.93 ±9.84 ±11.27 ±12.70 ±11.86 190.3 197.7 207.7 217.7 224.3 ±10.97 ±7.37 ±7.23 ±10.69 ±10.12 190.3 199.0 207.7 215.7 1222.3 ±10.26 ±1082 ±12.66 ±1405 ±14.84 231.5 ±3.70 232.6 ±7.70 219.0' ±11.29 213.0- ±6.96 203.4 ±11.68 231.4 ±10.62 222.0 ±9A49 238.4 ±14.50 228.2 ±12.28 234.3 ±12.70 230.7 8 242.8 ±11.97 240.0 ±3.65 240.4 ±6.66 228.4' _±12.18 222.0- ±7.94 212.4- ±11. 239.6 ±1 1.46 232.2 ±9.68 247.0 ±14.35 236.0 ±13.95 243.3 _±9.24

.W."I

9 Sample 3 active moiety 2.5 188.2 Sample 3 active moiety 5.0 186.4 0.02 11 Fenfluramine 7.5 190.3 12 Vehicle -183.3 sd Standard deviation Groups 2 8 were compared with vehicle Group 1: 'p<0.05, -p<0.01 Groups 9 11 were compared with vehicle Group 12 (no significance! 239.0

S)

±15.95 ±17.35 Histonathology Report Histological examination was restricted to the liver. No treatment-related changes were detected for sample (liquid), Sample 2 (spray-dried sap), Sample 3 (active moiety) f enf luramine or the DMSO control group.

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

0 0 to* 0@ ofS

TABLE

Microscopic pathology incidence summary Group 0 mg/kg Sex: Males Males on study Animals completed Group 2 800 mg/kg 4 Group 3 1600 mg/kg 5 Liver Exami ned 5 4 5 No abnormalities detected 0 0 1 Parenchymal inflammatory cell foci (Total) 0 1 0 Minimtal1 0 1 0 Hepatocyte hypertrophy centrilobular (Total) 0 0 0 Minimal 0 0 0 Extramedullary haemopoiesis (Total 2 0 0 Minimal 2 0 0 Hepatocyte necrosis focal (Total) 1 0 0 Minimal 1 0 0 Portal lymphoid infiltration (Total) 3 4 4 Minimal 3 4 4 Eosinophilic hepatocytes focal (Total) 1 0 0 Minimal 1 0 0 Portal fibrosis (Total) 0 0 1 Minimal 0 0 1 Liver (ORO stain) Examined 5 4 5 No abnormalities detected 2 3 2 fepatocyte fat centrilobular (Total) 3 1 2 Minimal 3 1 2 Hepatocyte fat peit i j-t t il I 0 0 1 Minimal 0 0 1 Group 4 3200 mg /kg 5 5 2 0 0 0 0 0 0 0 0 3 3 0 0 0 0 5 4 1 0 GroupS 5 Group 6 6400 I 10000 mg/kg mg/kg 5 5 .5 3 0 0 0 0 0 0 0 0 2 2 0 0 0 0 5 3 2 2 0 0 3 3 0 0 0 0 0 0 0 0 3 2 2 0 0 0 0 0

TABLE

(continued) T sex: Males Males on study Animals completed Liver Examined No abnormalities detected Parenchymal inflammatory cell foci (Total) Min imal1 Ilepatocyte necrosis focal (Total) Minimal Portal lymphoid infiltration (Total) Minimal Portal leucocytes (Total) Minimal Liver (ORO stain) E xami ine d No abnormalities detected Ilepatocyte fat centrilobular (Total) Minimal Group 7 38 mg/kg 5 5 5 2 0 0 0 0 3 3 0 0 5 5 0 0 Group 8 76 mg/kg 5 5 5 2 0 0 0 0 3 3 0 0 5 3 2 Group 9 2.5 mg/kg 5 5 5 0 0 0 1 1 5 3 2 Group 10 5 mg/kg 5 5 5 1 0 0 0 0 4 4 0 0 Group 1 7.5 mg/kg 3 3 3 0 0 0 0 0 3 3 0 0 Gr~up 12 0 mg/kg 3 3 3 2 1 1 0 0 1 1 0 5 3 3 3 2 2 2 0

I

I

2 2 EXAMPLE 38 A further bioassay, which employed the same test samples as described in Example 37, 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.

o Animals were acclimatised over a seven day period (days -7 *see.: to dosing took place from day 0 to day 6 at 9:00am by 0 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 15 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

U

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 D1 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.

S. 1 0 The histopathological examination of the livers did not show any significant pathology in the groups receiving the test items.

Food

S

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 time. Animals in Group 5 received a measured quantity food on Day i, equivalent to the average food consumption of Group 4 on Day 0. This controlled feeding pattern for Group 5, as determined from the average food consumption of Group 4 from the previous day, was followed for Days 1 7.

Water 'N /09 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.

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 S 10 masses were determined on a daily basis during this time.

*•g Ito Study npicmnand Procedures TABLE 1 STUDY DESIGN GROUP TEST NUMBERS DOSE TEST ITEM 01 6d 001 006 100 mg/kg Frozen sap 02 6o 007 012 400 mg/kg Frozen sap 03 6c 013 018 1 600 mg/kg Frozen sap 04 6e 019 024 3 200 mg/kg Frozen sap 05 6e 025 030 CONTROL Elga Option 4 Purified Water 06 6d 031 036 2.2 mg/kg Spray-dried sap 07 6" 037 042 8.8 mg/kg Spray-dried sap 08 6o 043 048 35 mg/kg Spray-dried sap 09 6d 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).

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 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 o* euthanased at the end of the study period with CO 2 gas.

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

0. go go 00 go go 0* 00 0 00 *0 *O 0@ 0 00*0 0 0 0 0 0 0 0 0 0 0 *0*0 0* *0 TABLE D.1 MEAN BODY MASSES I GROUP WEEK Group 01 Oral treatment Sample 1 (Sap) Dose (mg/kg) 400 Mean body masses Standard deviation 02 Sample I (Sap) 03 J Sample I (Sap) 1 600 04 Sample I (Sap) 3200 06 Elga Option 4 purified water (control) Sample 2 (Spray-dried sap) Day -7 203.38 ±95.39 192.53 ±65.60 149.25 ±54.80 224.15 ±80.70 214.55 ±74.90 208.65 ±65.74 256.95 ±77.55 194.37 ±43.74 171.52 D2ay -6 197.13 ±90.63 183.92 ±61.20 142.87 ±51.89 214.45 ±77.25 204.85 ±72.41 199.37 ±62.49 246.02 ±73.67 185.83 _±42.70 162.67 Day -5 192.75 ±89.49 178.25 ±59.37 136.85 ±52. 17 207.10 ±76.38 198.57 ±71.79 193.18 ±61.18 237.47 ±73.53 177.53 ±41.10 154.95 Day -4 188.62 ±86.75 173.17 ±58. 10 132.37 ±49.64 201.82 ±75.42 193.48 ±68.49 188.25 ±60.89 232.62 ±71.73 172.05 ±40.13 151.38 _Ray -3 184.95 ±84.80 170.82 ±57.42 131.50 ±49.50 198.25 ±74.82 192.40 ±67.48 186.22 ±59.98 229.78 ±71.76 170.10 ±39.49 149.63 D_2ay -2 182.48 ±83.47 168.25 ±58.40 129.67 ±48.89 194.83 ±75.34 190.87 ±67.39 184.55 t58.86 228.07 ±69. 88 167.25 ±37.61 148.30 Day -1 182.25 ±82.57 131.12 ±48.22 196.77 ±74 .56 190.,15 ±65.24 185.97 ±58.76 228.45 ±68.81 168.00 ±38.83 149.07 2.2 07 Sample 2 (Spray-dried sap) j 8.8 08 Sample 2(Spray-dried sap) 1 35 09 Elga Option 4 Purified water (control) .1 I ±69.81 ±62.68 I I ±61.83 ±59.48 ±57.66 ±57.12 ±56.01

S

to a* q..4 004 o 0. 0 to aS S* 10 00~ 5.e S 00: S 000 .5 S 0 S 0 *0 TABLE D.2 MEAN BODY MASSES GROUP WEEK (CONTINUED) Group Oral treatment Dose (mg/kg) 100 01 Sample 1 (Sap) 02 Sample I (Sap) 03 J Sample (Sap) 11600 04 06 Sample (Sap) Elga Option 4 purified water (control) Sample 2 (Spray-dried sap) 3 200 DayO0 183.87 ±83.33 173.45 ±60.73 134.38 ±46.01 199.60 ±75.16 194.27 ±67.46 189.07 ±60.15 230.28 ±69.32 169.10 ±38.40 151.02 Mean body masses Standard deviation Dayl1 Day 2 Day3 DaY 4 Da 5 :Day 6 175.83 175.72 175.48 175.53 177.95 1'78.43 ±81.82 ±79.05 ±77.54 ±76.20 ±73.99 ±72.68 164.58 164.75 166.22 166.55 169.93 171.77 ±58.52 ±58.37 ±57.69 ±57.79 ±57.47 ±57.29 129.20 127.53 127.20 126.70 128.00 128.07 ±44.74 ±43.20 ±41.36 ±39.19 ±39.22 ±38.66 196.38 192.20 189.05 186.57 186.05 185.68 ±73.96 ±71.20 ±69.11 ±66.29 ±67.45 ±65.73 221.55 220.17 221.80 222.82 224.82 224.90 ±68.02 ±t66.63 ±63.88 ±63.56 ±62.38 ±62.05 164.42 162.50 162.75 162.52 164.30 164.22 ±38.03 ±36.81 ±36.36 ±36.93 ±37.69 ±37.18 146.55 148.10 149.70 152.58 155.82 157.85 2.2 07 Sample 2 (Spray-dried sap) 8.8 08 Sample 2 (Spray-dried sap) I 35 09 1 Elga Option 4 purified water (control)

&I

±55.45 14 X't V. V I 7 0 ±53.77 ±52.67 UC .1

I

6 6 4'.1q4'4 6-00 S 0 .s 55 5 5. *5 4* 4 S 5555 S 5 S 0 S S S

S

0@SS 5505506 50 TABLE D.3 MEAN BODY MASSES GROUP WEEK (CONTINUED) Group Oral treatment Dose (mgfIkg) Day 7 f 4 I Sample 1 (Sap) (GHA I 35A) 185.38 J 4.79 ~A 1 4 L Sample 1 (Sap) (GHA I 35A) 400 178.83 1 ±5 2 Sample 1 (Sap) (GHA I 35A) 1 600 04 06 r .4 1 Sample 1 (Sap) (GHA 9 35A) Elga Option 4 purified water (control) Sample 2 (Spray-dried sap) (GHA 1 59) 3 200 2.2 132.22 ±37.08 188.57 ±66. 14 173.97 ±54.29 196.00 ±53.09 231.30 ±61.91 167.48 ±36.75 [165.50 Mean body masses Standard deviation Day 8 Day 9 Day 10 Day 11 Day 12 Dy1 34.73 236.73 234.07 236.33 239.07 238.43 62.44 ±62.39 ±62.09 ±62.31 ±60.24 ±59.85 25.63 277.13 227.10 229.43 234.93 236.20 13.05 ±14.18 ±14.03 ±16.97 ±18.35 ±15.97 33.80 135.23 134.53 138.30 139.30 142.80 55.17 ±455.74 ±54.96 ±53.03 ±51.10 ±49.51 99.63 198.90 198.70 194.73 194.93 197.93 61.07 ±57.48 ±54.55 ±52.78 ±50.78 ±51.57 72.98 157.80 158.87 160.80 163.40 167.80 52.06 ±58.62 ±57.76 ±57.67 _±56.27 ±58.49 90.27 190.27 192.60 194.73 196.97 198.60 27.78 ±29.54 ±29.09 ±29.68 ±29.04 ±30.18 77.27 178.17 180.67 182.03 185.10 189.73 24.48 ±23.79 ±25.04 ±25.31 ±24.60 ±23.58 64.90 166.63 168.43 171.67 174.90 178.57 :22.54 ±23.08 ±22.66 ±24.42 ±25.70 ±23.58 93.73 196.87 198.07 199.83 204.93 207.13 :22.37 ±21.86 1±21.02 ±20.21 ±18.65 ±18.22 07 Sample 2 (Spray-dried sap) 8.8 (GHAI_59) 08 Spray-dried sap 35 (GHA 1 59) Elga Option 4 purified water (control) 4 i 16 TABLE 1: HISTOLOGICAL EVALUATION OF LIVER SECTIONS FROM MALE RATS Sample I GROUP 1: 100 mg/kg Sample 1 GROUP 2:400 mg/kg Sample 1 Animal no Hepatic lesions IAnimal no Hepatic lesions Day 7 101 NPL 107 FHS1+ 02 NPL 108 NPL C1+ 03 NPL C1+ 1 109 NPL Day 13 104 NPL MLC IDay 13 110 ]DHS1+ FHSI+ ill NPL 06 NPL 1 H1 *~GROUP 3: 1 600 mg/kg Sample I GROUP 4: 3 200 mg/kg Sample I ____Animal no IHepatic lesions Animal no Hepatic lesions Day 7 113 NPL 1 P 14 P192 NPL NPL .121 NPL :Dayl13 116 NPL IDayl13 122 DHS1+ *17 DHS1+ 123

FHSI+

.:18 NPL 124 JNPL GROUP 5: CONTROL: ELGA OPTION 4 PURIFIED WATER. RESTRICTED FOOD INTAKE GROUP 5: Control: Elga option 4 purifiedwae Animal no Hepatic lesions Day 7 25 NPL MLC 26 NPL F__t_2 7 NPL Day 13 128 DHS1+ 29 DHS1+

NPL

Legend: C Congestion

OHS

FHS

NPL

MLC

1+ 2+ 3+ Diffuse hydropic cell swelling Focal hydropic cell swelling No parenchymal lesions Minimal lymphocytic cuffing mild moderate severe i 16 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 Animjal noj Hepatic lesions jj Animal no Hepatic lesionsIi Da 1NPL 37 NPL INPL MLC 138

NPL

I Day13 134 135 36 FHS1+ 1 3 NPL Day 3 14 DHS1+ 4 NPL ±±4 NPL C1+ DHS1+

NPL

MLC FHS1+ II .1 GROUP 8: 35 mg/kg Sample 2 Animal no Hepatic lesions Day 7 143 NPL GR U44

NPL

45 NPL Day 1 3 46 NPL 'N 47 NPL

C+

148 IMLC FHS1+ GROUP 9: CONTROL: ELGA OPTION 4 PURIFIED WATER Legend: GROUP 9: Control: Elga option 4 purified water Animal no Hepatic lesions Day 7 49 NPL

NPL

51 FHS1.

Day 13 52 DHS1+ 53 NPL 54 1FHS1.

C Congestion DHS Diffuse hydropic cell swelling FHS Focal hydropic cell swelling NPL No parenchymal lesions MLC Minimal lymphocytic cuffing mild moderate severe 1' 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 .ooo o of mild degree is present in the hepatic sinusoids and should be regarded as an incidental observation.

go 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 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 118 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 (1) The term "animal" as used herein extends to, but is not 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 1 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 5 mg/kg/day.

Claims (75)

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 Io 0 CCH 3 Me Me Me OH HO 0 0 MeO J_ 0 0 0 OH OMe OMe (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 S* and Hoodia lugardii.

3. An extract as claimed in claim 2, wherein the plant is selected from the genus i10 Hoodia and the species is selected from Hoodia currorrii, Hoodia gordonii and Hoodia lugardii. S* 4. An extract as claimed in claim 2 or 3 wherein substantially all the non-active impurities have been removed.

5. An extract as claimed in any one of claims 1, 2, 3 or 4 which has been 15 processed to a free-flowing powder.

6. A composition having appetite suppressant activity comprising the extract as claimed in any one of claims 1 to 5 inclusive.

7. A composition as claimed in claim 6 when admixed with a pharmaceutical excipient, diluent or carrier.

8. A composition as claimed in claim 6 or claim 7 which is prepared in unit dosage form.

9. The use of an extract as claimed in any one of claims 1 to 5 inclusive in the manufacture of a medicament having appetite suppressant activity. An extract as claimed in any one of claims 1 to 5 inclusive for use as a medicament having appetite suppressant activity.

11. An extract as claimed in any one of claims 1 to 5 for use in treating, preventing or combating obesity. (R\LIBA]06800 doc:NSS 120

12. Use of an extract as claimed in any one of claims 1 to 5 in the manufacture of a medicament for treating, preventing or combating obesity.

13. A compound having the general structural formula RiO 0 R OH R 2 (2) in which R alkyl; RI H, alkyl, or an 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 S 10 between C4-C5 or C5-C6, except where R CH 3 RI H or benzoyl; R2 H and there is a double bond at C 5 -C 6 and where R C 1 -4 alkyl; R 1 H; R 2 H; and the C 5 -C 6 bond is saturated. So** 14. A compound as claimed in claim 13 wherein there is a double bond between C5-C6, R methyl, R, tigloyl, R 2 3-0-[-p-D-thevetopyranosyl-(1-4)-P-D- 15 cymaropyranosyl-(1 4)-p-D-cymaropyranosyl], the compound having the structural formula III cCH 3 Me Me Me OH HO ,O _J p 0 MeO 0 O0 OH I OMe OMe (1) A compound having the general structural formula [R \LIBAj06800.doc:NSS in which R alkyl; and R, H, alkyl or an organic ester group.

16. A compound having the general structural formula 0 0 0* 00 0 000000 0 0 0 0 0 0000 00 0 0* 0 00 0 0 000 0 0000 0@ 0000 0 0000 0 00 0 0@ 0 in which R= alkyl; and R, H, alkyl or an organic ester group.

17. A compound having the general structural formnula in which R= alkyl; and H, alkyl or an organic ester group.

18. A compound having the general structural formula (R:\LIBAO6800 doc:NSS 122 0 o OMe HO OMeOH OMe (6) in which R alkyl; and RI H, alkyl or an organic ester group.

19. A compound having the general structural formula OH RO R OH s HO (7) Sin which R= alkyl; RI H, alkyl, or an organic ester group, and the broken lines indicate the optional presence of a further bond between C 4 -C 5 or C5-C 6 except where R CH 3 R 1 H or benzoyl; and there is a double 10 bond at C 5 -C 6 and R Cl-4 alkyl; RI H; and the C 5 -C 6 bond is saturated. A compound having the general structural formula OR R OH R21 (8) in which R alkyl; and R 1 H, alkyl or an 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, except where R CH 3 RI H or benzoyl; there is a double bond at C 5 -C 6 and R 2 H. [R \LIBA]06800 doc:NSS 123

21. A compound having a general structural formula R2 1-1(9) in which R= alkyl; and R, H, alkyl, or an 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; 9*and in which the broken lines indicate the presence of a further bond between C4-C5 or C5-C6, except where R CH 3 R, H; R 2 digiLtJpyrwinsyl or H;adthere isadouble bodat C5-C6; (b CH13, R, H, benzoyl, tigloyl or angeloyl, R 2 and there is a double bond at C 5 -C 6 R CH 3 R, H or benzoyl; R(2 H; and the C 5 -C 6 bond is saturated; (d) R CH 3 R, benzoyl, R 2 6-deoxy-3-O-methyl-j3-D-allopyranosyl-(1- 4)- j3-D-cymaropyranosyl-(- 4)-f3-D-cymaropyranosyl; and where R CH 3 R, benzoyl, R 2 6-deoxy-3-O-methyl-3-D-allopyranosyl-(- 4)-j3-D- 15 oleandropyranosyl-(1-~ 4)-f3-D-cymaropyranosyl.

22. A compound having the general structural formula .0 ID6: H R2 in which R =alkyl; and H, alkyl, or an 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, except where R CH 3 R, H; R2 digitopyranosyl or H; and there is a double bond at C 5 -C 6 R CH 3 R, [R \LIBA]068oo doc:NSS 124 H, benzoyl, tigloyl or angeloyl, R 2 H; and there is a double bond at C 5 -C 6 R CH3, Ri H or benzoyl; R 2 H; and the C 5 -C 6 bond is saturated; (d) R CH 3 Ri benzoyl, R 2 6-deoxy-3-O-methyl-P-D-allopyranosyl-(1- 4)- P-D-cymaropyranosyl-(1- 4)-P-D-cymaropyranosyl; and where R CH 3 RI benzoyl, R 2 6-deoxy-3-O-methyl--D-allopyranosyl-(1- 4)-P-D- oleandropyranosyl-(1- 4)-P-D-cymaropyranosyl.

23. A compound having the general structural formula R ORi R O R2O (11) 10 in which R alkyl; and RI H, alkyl, or an 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 15 between C4-C5, C5-C6 or C14-C15.

24. A compound having the general structural formula *R 0 O R o R2 (12) in which R= alkyl; and RI H, alkyl, or an 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 to C14-C15, except where R CH 3 RI H; R 2 H; and the C 5 -C 6 bond is saturated. [R:\LIBA106800.docNSS 125 A compound having the general structural formula OR1 R O OR3 R2 (13) in which R alkyl; and RI H, alkyl, or an 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.

26. A compound according to any one of claims 12, 14-24 wherein R 1 is tigloyl or 0o benzoyl.

27. A process for the preparation of a steroid intermediate of the formula OH 0 OH HO which includes the steps of treating the compound OAc HO (22) with a reducing agent to produce a compound 33, 121-dihydroxy-20, 14-diene of the formula [R\LIBA]06800.doc NSS HO'v v (23) treating compound (23) with N-bromoacetamide (NBA) and a base to produce a compound 3p, 12p-dihydroxy-14,15-epoxy-20, 20-ethylenedioxypregn-5-ene of the formula HO (24) treating compound (24) with a reducing agent to produce a compound 33, 123, 14p-trihydroxy-20,20-ethylenedioxypregn-5-ene of the formula OH O HOO and treating compound (25) with an acid and water to produce compound

28. A process for the preparation of compound (15) which includes the steps of treating the compound (22) as shown in claim 27 with p-toluenesulfonyl chloride and a base to produce a compound 31, 12p-dihydroxy-20, 14-diene-3-tosyl-12-acetate of the formula OAc -O 0 TsOC (26) treating the compound (26) with potassium acetate in a solvent to produce a compound 6p, 12p-dihydroxy-20,20-ethylenedioxy-3, 5ac-cyclopregnan-14-ene-12- acetate of the formula fR:\LIBA]06800 doc:NSS OH (27) treating the compound (27) with a reducing agent to produce a compound 6P3, I 21-dihydroxy-20,20-ethylenedioxy-3, 5cc-cyclopregnan- 14-ene of the formula OH (28) treating the compound (28) with N-bromoacetamide, and a base to produce a compound 6P, 1 21-dihydroxy-20,20-ethylenedioxy- 14,1 5-epoxy-3, 5ec-cyclopregnane of the formula OH (29) treating the compound (29) with a reducing agent to produce a compound 6P3, 12P3, 14p-trihydroxy-2O,2O-ethylenedioxy-3, 5a-cyclopregnane of the formula OH 2 0 dyUH0 OH and treating compound (30) with an acid and a solvent to produce compound I R\LI BAO6800.doc:NSS

29. A steroid intermediate of formula (15) when produced by a process as claimed in claim 27 or claim 28. A process of coupling a monosaccharide cymarose to a steroid intermediate, which includes the steps of 1) reacting a cymarose moiety of formula (38) OMe (38) in which Ph=phenyl with a steroid intermediate of formula (15) as shown in claim 27 in the presence of tin chloride in a solvent to produce a compound 3-0-[4-0-benzoyl-2-phenylthio-p-D- 10 cymaropyranosyl]-12,14-p-dihydroxy-pregnan-5-ene-20-one of the formula OH 0 BzO SPh OMe (51) in which Ph phenyl and Bz= benzoyl 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]-123- tigloyloxy-14-hydroxy-14p-pregn-5-ene-20-one of the formula 0 O H HO I SPh OMe (52) in which Ph phenyl. [R \LIBA]06800 doc:NSS 129

31. A compound of formula (52) when produced by a process as claimed in claim

32. 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 shown in claim 30 which includes the steps of i) coupling a selectively protected cymarose moiety of formula (40) below and a monosaccharide thevetose moiety of formula (50A) below: 0 O F ZO SPh 0 OMe HO PhCO OMe (40) OPv (50 A) using tin chloride (SnC12) and silver trifluoromethanesulphonate to produce a compound of the formula *ZO SPh ""0 *o 1I1 OMe OMe PhCO o9 OPv (53) in which Ph phenyl and Pv pivaloyl, Z TBDMS t-butyldimethylsilyl ii) treating compound (53) with tetrabutylammoniumfluoride to produce a compound of the formula HO SPh O•M II OMe OMe PhCO OPv (54) in which Ph phenyl, Pv pivaloyl iii) treating compound (54) with diethylaminosulphur trifluoride to produce a compound of the formula [R.\LIBA]06800 doc.NSS I SPh OMe OPv in which Ph phenyl, Pv pivaloyl iv) reacting compound (55) with compound (52) as shown in claim 29 to produce a compound of the formula 9* 5 OPv (56) in which Ph phenyl and Pv pivaloyl and treating compound (56) in a Raney-Nickel reaction and thereafter with a base to produce compound as claimed in claim 13.

33. 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 shown in claim 32 and compound (55) as shown in claim 32 using tin (II) chloride, AgOTf, Cp 2 ZrCI 2 to produce a compound of the formula 0 zo SPh OMe 11 OMe SPh PhCO OMe OPv (57) is in which Ph phenyl, Pv pivaloyl, Z TBDMS t-butyldimethylsilyl; [R:\LIBA]06800.doc:NSS 131 ii) treating compound (57) with tetrabutylammonium fluoride and diethylaminosulphur trifluoride to produce a trisaccharide compound having the formula F SPh o O OMe O 0 OMe SPh PhCO OMe OPv (58) in which Ph phenyl, Pv pivaloyl and (iii) coupling the trisaccharide of formula (58) with a steroid intermediate of the formula OH :claim 14. o 34. A composition for use as an appetite suppressant comprising a compound as .claimed in any one of claims 13 to 26. using tin (II) chloride, AgOTf, CP 2 ZrCI 2 to produce compound as claimed in claim 14. 10 34. A composition for use as an appetite suppressant comprising a compound as claimed in any one of claims 13 to 26. A composition for use in treating, preventing or combating obesity comprising a compound as claimed in any one of claims 13 to 26.

36. A composition as claimed in claim 34 or 35 wherein the compound is the compound of formula as claimed in claim 14.

37. A composition as claimed in any one of claims 34 to 36 when admixed with a pharmaceutical excipient, diluent or carrier.

38. A composition as claimed in any one of claims 34 to 37, which is prepared in unit dosage form.

39. The use of a compound as claimed in any one of claims 13 to 26 disregarding the disclaimers therein in the manufacture of a medicament having appetite suppressant activity, or for use in treating, preventing or combating obesity. [R:\LIBA]06800.doc:NSS The use of a compound as claimed in claim 39 wherein the compound is the compound of formula as claimed in claim 14.

41. A compound as claimed in any one of claims 13 to 26 disregarding the disclaimers therein for use as a medicament having appetite suppressant activity or for use as a medicament in treating, preventing or combating obesity.

42. A compound as claimed in claim 41 wherein the compound is the compound of formula as claimed in claim 14.

43. A method of manufacturing a composition according to either of claims 37 or 38, comprising admixing a compound as claimed in any one of claims 13 to 26, disregarding the disclaimers therein, with a pharmaceutical excipient, carrier or diluent.

44. A foodstuff or beverage comprising an effective quantity of a compound as claimed in any one of claims 13 to 26 disregarding the disclaimers therein to have an appetite suppressant effect when ingested.

45. A foodstuff or beverage according to claim 44, for use as an appetite 15 suppressant or for use in treating, preventing or combating obesity.

46. A foodstuff or beverage as claimed in claim 44 or 45 wherein the compound is the compound of formula as claimed in claim 14.

47. Use of a compound of formula as claimed in claim 14 isolated from a plant of the genus Trichocaulon or the genus Hoodia for the manufacture of a medicament having appetite suppressant activity or for use in treating, preventing or combating obesity.

48. Use according to claim 47 wherein the compound is isolated from the species Trichocaulon piliferum or Trichocaulon officinale or from the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii.

49. Use according to claim 48, wherein the compound is isolated from the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii. A composition having appetite suppressant activity comprising a compound of formula isolated from a plant of the genus Trichocaulon or of the genus Hoodia.

51. A composition as claimed in claim 50 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.

52. A composition as claimed in claim 51 wherein the compound is isolated and/or purified from a plant of the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii. [R \LIBA106800.doc:NSS 133

53. A composition as claimed in claim 50 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.

54. A composition as claimed in claim 53 wherein the compound is isolated and/or purified from an extract derived from a plant of the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii. A composition as claimed in any one of claims 50 to 54, when admixed with a pharmaceutical excipient, diluent or carrier.

56. A composition as claimed in claim 55 which is prepared in unit dosage form.

57. A compound of formula as claimed in claim 14 isolated from a plant of the genus Trichocaulon or from the genus Hoodia for use as a medicament having appetite suppressant activity or for use in treating, preventing or combating obesity.

58. A compound as claimed in claim 57 wherein the compound is isolated from a 5i plant of the species Trichocaulon piliferum or Trichocaulon officinale or from Hoodia currorii, Hoodia gordonii or Hoodia lugardii.

59. A compound as claimed in claim 58 wherein the compound is isolated from a plant of the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii.

60. Use of a composition according to any one of claims 50 to 56 in the 20 manufacture of a medicament having appetite suppressant activity.

61. Use of a composition according to any one of claims 50 to 56 in the manufacture of a medicament for use in treating, preventing or combating obesity.

62. A compound having the structural formula OH O0""\ OH H (23).

63. A compound having the structural formula [R:\LIBAj06800 doc:NSS 134 (24).

64. A compound having the structural formula

65. A compound having the structural formula (27).

66. A compound having the structural formula (28).

67. A compound having the structural formnula (29).

68. A compound having the structural formula [R \LIBA]06800.doc:NSS

69. A compound having the structural formula (51) wherein Bz= benzoyl and Ph phenyl. A compound having the structural formnula OMe (52) wherein Ph phenyl.

71. A compound having the structural formula [R %LIBAJO6800doc*NSS 136 0 00 0 OH 0 SPh o0 OMe O~e' SPh PhCO OMe OPv (56) wherein Ph phenyl and Pv pivaloyl. *72. A method of suppressing appetite comprising administering to a human or an animal an effective amount of extract as claimed in any one of claims 1 to 5 or of a composition as claimed in any one of claims 6 to 8.

73. A method of treating, preventing or combating obesity, comprising administering to a human or an animal an effective amount of extract as claimed in any one of claims 1 to 5 or of a composition as claimed in any one of claims 6 to 8.

74. A method of suppressing appetite comprising administering to a human or an i o animal an effective amount of a compound as claimed in any one of claims 13 to 26 disregarding the disclaimers therein, or of a composition as claimed in any one of claims o000 34 and 36 to 38, disregarding the disclaimers therein; or of a foodstuff or beverage as S: claimed in any one of claims 44 to 46. A method of treating, preventing or combating obesity, comprising administering to a human or an animal an effective amount of a compound as claimed in any one of claims 13 to 26 disregarding the disclaimers therein, or of a composition as claimed in any one of claims 35 to 38, disregarding the disclaimers therein; or of a foodstuff or beverage as claimed in any one of claims 44 to 46.

76. A method of suppressing appetite comprising administering to a human or an animal an effective amount of a compound of formula as claimed in claim 14 isolated from a plant of the genus Trichocaulon or the genus Hoodia, or of a composition as claimed in any one of claims 50 to 56.

77. A method of treating, preventing or combating obesity, comprising administering to a human or an animal an effective amount of a compound of formula (1) [R \LIBA]06800 doc:NSS 137 as claimed in claim 14 isolated from a plant of the genus Trichocaulon or the genus Hoodia, or of a composition as claimed in any one of claims 50 to 56.

78. 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 I I O CCH 3 Me Me OH MeO OH OMe OMe substantially as hereinbefore described with reference to Example 3.

79. A process for the preparation of a steroid intermediate of the formula OH 0 SOH HO' which includes the steps of treating the compound OAc HO- V (22) with a reducing agent to produce a compound 31, 120-dihydroxy-20, 14-diene of the formula OH HO (23) [R\LIBA]06800 doc:NSS 138 treating compound (23) with N-bromoacetamide (NBA) and a base to produce a compound 33, 12p-dihydroxy-14,15-epoxy-20, 20-ethylenedioxypregn-5-ene of the formula OH "O O 0 HOe (24) treating compound (24) with a reducing agent to produce a compound 3p, 12P, 14p-trihydroxy-20,20-ethylenedioxypregn-5-ene of the formula OH OD O H HO and treating compound (25) with an acid and water to produce compound substantially as hereinbefore described with reference to Examples 10 to 13.

80. A process for the preparation of compound (15) which includes the steps of treating the compound (22) as shown in claim 27 with p-toluenesulfonyl chloride and a base to produce a compound 3p, 12P-dihydroxy-20, 14-diene-3-tosyl-12-acetate of the formula OAc D TsO(26) treating the compound (26) with potassium acetate in a solvent to produce a compound 6p, 12p-dihydroxy-20,20-ethylenedioxy-3, 5a-cyclopregnan-14-ene-12- acetate of the formula [R \LIBA]06800 doc.NSS 139 0 0@ 0@ S S 0* *0000 S *0S~ S. S 0* S S S *0S* S *5 5* 0@ *5 S S OH (27) treating the compound (27) with a reducing agent to produce a compound 6P, 1 2f-dihydroxy-20,20-ethylenedioxy-3, 5cc-cyclopregnan- 14-ene of the formula 0- 0 OH (28) treating the compound (28) with N-bromoacetamide, and a base to produce a compound 6P3, 1 2p-dihydroxy-2O,2-ethylenedioxy- 14,1 5-epoxy-3, 5ax-cyclopregnane of the formula OH (29) treating the compound (29) with a reducing agent to produce a compound 6P, 12P3, 14j-trihydroxy-20,20-ethylenedioxy-3, 5cc-cyclopregnane of the formula OH and treating compound (30) with an acid and a solvent to produce compound substantially as hereinbefore described with reference to Examples 14 to 19. RA\L BA ]06800.doc: NSS 140

81. A process of coupling a monosaccharide cymnarose to a steroid intermediate, which includes the steps of 1) reacting a cymnarose moiety of formula (38) S p 4. p. p 4 p. S P 4*S* *5 6* p. 0 p 5 Op. p 4P** 0* 4* p S *4 I S. OMe (38) in which Ph=phenyl with a steroid intermediate of formula (15) as shown in claim 27 in the presence of tin chloride in a solvent to produce a compound 3-0-[4-0-benzoyl-2-phenylthio-P-D- cymaropyranosyl]- 12,1 4-f3-dihydroxy-pregnan-5-ene-20-one of the formula BzO SPh OMe (51) in which Ph phenyl and Bz= benzoyl and (ii) treating the compound (5 1) with tiglic acid chloride in pyridine and thereafter with a base to produce a compound 3-0-[2-phenylthio-3-D-cymaropyranosyl]-123- tigloyloxy-1I4-hydroxy- 1 41-pregn-5 -ene-20-one of the formnula 0 0, .0= OMe (52) in which Ph phenyl, substantially as hereinbefore described with reference to Example 34.

82. A compound having the structural formnula (R \LIBA]068DO doc:NSS HO' (23), substantially as hereinbefore described with reference to Example

83. A compound having the structural formula HO- (24), substantially as hereinbefore described with reference to Example 11.

84. A compound having the structural formula OH OH HOe substantially as hereinbefore described with reference to Example 12. A compound having the structural formula OAc o OH (27), substantially as hereinbefore described with reference to Example

86. A compound having the structural formula [R:\LIBA106800doc:NSS OH (28), substantially as hereinbefore described with reference to Example 16.

87. A compound having the structural formula OH (29), substantially as hereinbefore described with reference to Example 17.

88. A compound having the structural formula OH substantially as hereinbefore described with reference to Example 18.

89. A compound having the structural formula OH OH BzO SPh OMe (51) wherein Bz= benzoyl and Ph phenyl, substantially as hereinbefore described with reference to Example 34. [R:\LIBA)06800.doc:NSS 143 A method of suppressing appetite comprising administering to a human or an animal an effective amount of extract as claimed in any one of claims 1 to 5 or of a composition as claimed in any one of claims 6 to 8, substantially as hereinbefore described with reference to any one of Examples 35, 37 and 38.

91. A method of treating, preventing or combating obesity, comprising administering to a human or an animal an effective amount of extract as claimed in any one of claims 1 to 5 or of a composition as claimed in any one of claims 6 to 8, substantially as hereinbefore described with reference to any one of Examples 35, 37 and 38.

92. A method of suppressing appetite comprising administering to a human or an animal an effective amount of a compound of formula as defined in claim 14 isolated from a plant of the genus Trichocaulon or the genus Hoodia, or of a composition as claimed in any one of claims 50 to 56, substantially as hereinbefore described with reference to any one of Examples 35, 37 and 38. 15 93. A method of treating, preventing or combating obesity, comprising administering to a human or an animal an effective amount of a compound of formula (1) •as defined in claim 14 isolated from a plant of the genus Trichocaulon or the genus Hoodia, or of a composition as claimed in any one of claims 50 to 56, substantially as hereinbefore described with reference to any one of Examples 35, 37 and 38. 20 Dated 21 February, 2005 CSIR Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [R \LIBA]06800 doc:NSS