CZ359999A3 - Pharmaceutical composition reducing appetite - Google Patents

Abstract

The pharmaceutical composition comprises an extract that can be obtained from a plant of the genus Trichocaulon or Hoodia containing an agent flavor suppressant of Formula 1. Also included a method for obtaining an extract and a process for preparing compound 1 a its analogues. The solution is further extended by the use of extracts Compounds 1 and their analogs for the preparation of medicaments having flavor-reducing activity. The solution also provides new solutions intermediates for the preparation of compound 1.

Description

The present invention relates to steroid glycosides, compositions comprising steroid glycosides and a novel use of these steroid glycosides and compositions comprising them. The invention further relates to a process for the extraction and isolation of these steroid glycosides from plant material, a process for the synthetic preparation of such steroid glycosides and products of such extraction and such synthetic methods.

SUMMARY OF THE INVENTION

In particular, the invention relates to an appetite reducing agent, a method for the synthetic preparation of a appetite reducing agent. for eating, a method for extracting an appetite reducing agent from plant material, an appetite reducing composition comprising an appetite reducing agent, and a method for reducing appetite.

The present invention provides a process for preparing an extract of a plant of the genus Trichocaulon or Hoodia, the extract comprising an appetite reducing agent, the process comprising the steps of treating the collected plant material with a solvent to extract fractions having appetite reducing activity, separating the extraction solution from the remainder of the plant material; from the extraction solution and recovering the extract. The extract thus obtained may be further purified, for example by suitable solvent extraction procedures.

The invention further provides a plant extract prepared from the group comprising the genus Trichocaulon and the genus Hoodia and having appetite reducing activity.

• · • ·

The extract may be prepared from plant material such as the stems or roots of said plants of the genus Trichocaulon or of the genus Hoodia. The genus Trichocaulon and genus Hoodia are succulent plants growing in arid areas such as those found in South Africa. In one application, the appetite reducing extract is obtained from the species Trichocaulon piliferum. Trichocaulon officianale can also be used to obtain a taste-reducing extract. to eat. In another application, the appetite reducing extract can be obtained from Hoodia currorii, Hoodia gordonii or Hoodia lugardii. Bioassays performed by the applicant have shown that some of these extracts exhibit appetite-reducing activity.

The plant material may be homogenized in the presence of a suitable solvent, such as a mixture of methanol and methylene chloride, by a device such as a Waring mission. The extraction solution may then be separated from the remainder of the plant material by a suitable separation technique such as filtration or centrifugation. The solvent may be removed on a rotary evaporator, preferably in a water bath at 60 ° C. The separated crude extract can be further extracted with methylene chloride and water before being separated into methylene chloride extract and water extract.

The methylene chloride extract may have a solvent which can be preferably removed on a rotary evaporator and the resulting extract can be further purified by extraction with a mixture of methanol and hexane. The extraction product of the methanol / hexane extraction can then be separated into a methanol extract and a hexane extract. The methanol extract can be evaporated to remove the solvent to obtain a partially pure active extract.

The partially pure active extract can be dissolved in methanol and further fractionated by column chromatography using silica gel as the adsorption medium and a mixture of chloroform and 30% methanol as eluent.

A large number of different fractions are obtained and each can be evaluated by a suitable bioassay to determine appetite-reducing activity.

The fractions having appetite reducing activity may advantageously be further fractionated by silica gel column chromatography as an adsorption medium and 9: 1 chloroform: methanol solvent, and the resulting sub-fractions are biologically tested for appetite reducing activity. Furthermore, sub-fractions characterized by appetite-reducing activity can be fractionated and purified, if desired, using column chromatography with silica gel as the adsorption medium and a 9: 1 mixture of ethyl acetate and hexane as solvent. The resulting purified fractions can be re-evaluated by appro- priate biological procedures for appetite-reducing activity.

The Applicant has found that at least one such purified fraction has good appetite-reducing activity and the active ingredient in the fraction has been identified by conventional chemical techniques involving nuclear magnetic resonance and has been found to be a compound of the structural formula

• • • •

In accordance with the SI nomenclature, active substance 1 is compound 3-0 - [- β-D-thevetopyranosyl- (1-> 4) -β-D-cymaropyranosyl- (1-> 4) -β-D-cymaropyranosyl] - 12B-O-thigloyloxy-14-hydroxy-14H-pregn-5-en-20-one (C ₄₇ H ₇₄ O ₁₅ M ⁺ 878).

Another aspect of the invention is a method of preparing a plant extract of the genus Trichocaulon or Hoodia, the extract comprising a taste-reducing agent. for eating, a process comprising compressing the collected plant material into separate juices from the solid plant material and obtaining free juice of the solid plant material to form an extract.

The extract may be dried to remove moisture, for example by spray drying, freeze drying or vacuum drying to give a free-flowing powder.

The invention further relates to a composition having an appetite reducing activity comprising an extract as described above.

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

The invention is further extended to the use of an extract as described above for the preparation of a medicament having an appetite reducing activity, to an extract as described above for use as a medicament having an appetite reducing activity, and to a method for reducing appetite by administering to the animal an effective dose of the composition described above.

Compound 1 is a novel compound and the invention extends to compound 1 and certain appetite-reducing steroid trisaccharide analogs or derivatives thereof. Molecules selected as analogs or derivatives are believed to confer on the steroid trisaccharide properties that enhance the activity of the active ingredient. The following effects have been considered when selecting analogs:

(i) Hydrophobic interactions and lipophilicity

Modification of the functional group of the active molecule is accomplished to alter the hydrophobicity and lipophilicity of the molecule.

Increased lipophilicity has been shown to correlate with increased biological activity, less water solubility, increased detergency and cell lysis, increased tissue deposition, faster metabolism and elimination, increased plasma protein binding, and faster onset of action.

(ii) Electronic properties and ionization constants

Modification of the functional group is also carried out in order to achieve a change in acidity and basicity, which will play a major role in regulating the transport of the compound to the sites of action and binding at that target site.

(iii) Binding of hydrogen

Modification of the carboxyl and carbonyl functional groups in the active molecule is carried out in order to alter the interaction between proteins in biological systems and chemically modified functional groups.

(iv) Steric parameters

The purpose of altering the spherical features of a molecule is to increase binding to its receptor and thus increase its biological activity.

The following chemical modifications are made to achieve hydrophobicity and lipophilicity, electronic binding, and

molekuly spherical parameters of the molecule:

a) Chemical modification of the C-12 group and the ester functional group;

b) Chemical modification of the 5,6-double bond, for example hydrogenation and migration;

c) Chemical modification of the C-20 carbonyl group and the C-17 acetyl group;

d) Chemical modification of ring D of a steroid or aglycone ring;

e) Modification of the sugars of the trisaccharide moiety.

The present invention provides a compound of formula

(2) wherein R = alkyl

R @ 1 = H, alkyl, tigloyl, benzoyl, or another organic ester group;

R ₂ = H or one or more 6-deoxy-saccharides or one or more 2,6-dideoxysacharidů, or glucose molecules, or combinations thereof;

and wherein the dashed line represents optionally the presence of an additional bond between C4-C5 or C5-C6.

The present invention also includes a compound as described above, wherein further there is an additional bond between C 5 -C 6, R = methyl, R 1 = tigloyl, R ₂ = 3-0 - [- β-D-thevetopyranosyl- (1-> 4) ) • · i

β-D-cymaropyranosyl- (1-> 4) -β-D-cymaropyranosyl], and wherein the compound has the general formula

Other active analogues of the appetite-reducing compounds of Compound 1 of the invention are compounds having the following structural formula

OH (2) •

wherein R = alkyl; and

R-L = H, alkyl, tigloyl, benzoyl or any other organic ester group

(4)

OH

wherein R = alkyl; and

R 1 = H, alkyl, tigloyl, benzoyl or any other organic ester group

OH (5) wherein R = alkyl; and

R @ 1 = H, alkyl, tigloyl, benzoyl or any other organic ester group

OMe (6) wherein R = alkyl; and

R 1 = H, alkyl, tigloyl, benzoyl or any other organic ester group

wherein R = alkyl; and

R @ 1 = H, alkyl, tigloyl, benzoyl or any other organic ester group.

• ·!

(8) wherein R = alkyl

R @ 1 = H, alkyl, tigloyl, benzoyl, or another organic ester group;

R ₂ = H or one or more 6-deoxy-saccharides or one or more 2,6-dideoxysacharidů, or glucose molecules, or combinations thereof;

and wherein the dashed line represents optionally the presence of an additional bond between C4-C5 or C5-C6.

(9) wherein R = alkyl

R @ 1 = H, alkyl, tigloyl, benzoyl, or another organic ester group;

R ₂ = H or one or more 6-deoxy-saccharides, or a • · ·· ··· "or more 2,6-dideoxysacharidů, or glucose molecules, or combinations thereof;

and wherein the dashed line represents optionally the presence of an additional bond between C4-C5 or C5-C6.

(10) wherein R = alkyl

R-L = H, alkyl, tigloyl, benzoyl, or another organic ester group;

R ₂ = H or one or more 6-deoxy-saccharides or one or more 2,6-dideoxysacharidů, or glucose molecules, or combinations thereof;

and wherein the dashed line represents optionally the presence of an additional bond between C4-C5 or C5-C6.

R

(11) wherein R = alkyl

R @ 1 = H, alkyl, tigloyl, benzoyl, or other organic species;

Fcfc fcfc fc ester group;

R ₂ = H or one or more 6-deoxy-saccharides or one or more 2,6-dideoxysacharidů, or glucose molecules, or combinations thereof;

and wherein the dashed line represents optionally the presence of an additional bond between C4-C5, C5-C6 or C14-C15.

(12) wherein R = alkyl

R @ 1 = H, alkyl, tigloyl, benzoyl, or another organic ester group;

R ₂ = H or one or more 6-deoxy-saccharides or one or more 2,6-dideoxysacharidů, or glucose molecules, or combinations thereof;

and wherein the dashed line represents optionally the presence of an additional bond between C4-C5, C5-C6 or C14-C15.

(13)

9994

9 «» 9

9 9 9 9 • 9 9 9 4

9 9 9 9

9

9999 99 94

94

9 9 9

9 9 9

9

9 4 9

99 wherein R = alkyl

R 1 = H, alkyl, tigloyl, benzoyl, or another organic ester group;

R2 = H or one or more 6-deoxysaccharides or one or more 2,6-dideoxysaccharides, or glucose molecules or combinations thereof;

and wherein the dashed line represents optionally the presence of an additional bond between C 4 -C 5, C 5 -C 6 or C 14 -C 15; and R ₃ = H, alkyl, aryl, acyl or glukoxy.

wherein R = H, alkyl, aryl, or any steroid having a C 14 beta hydroxyl group, a C 12 beta hydroxyl group and a C 17 acyl group, a C 5 -C 6 olefin, or a combination thereof.

The invention is further extended to a process for the synthetic preparation of a compound having appetite reducing activity.

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

(15) • 4 '' frfr '' frfrfr fr frfr fr frfr fr frfr fr frfrfr frfrfr frfrfr frfrfr frfrfr fr frfrfr fr frfrfr fr frfrfr fr frfrfr fr frfrfr frfrfr frfrfr frfrfr frfrfr frfrfr Fr· frfr frfr frfr

The steroid 15 may be prepared from a compound having the formula 22, which comprises the steps of (i) treating a progesterone having the formula

(16) with Calonectrica decora to produce 12S, 15α-dihydroxyprogesterone of formula

(17) (ii) treating compound 17 with tosyl chloride and pyridine to give 12S-hydroxy-15a- (p-toluenesulfonyl) progesterone of the formula

(18) • (iii) treating compound 18 with a collidine at 150 ° C to give the compound 12E-hydroxy- ^14- progesterone of formula

(19) (iv) treating compound 19 with acetyl chloride and acetic anhydride at 120 ° C to give compound 3,12S-diacetoxypregna-3,5,14-trien-20-one of formula

And what

(20) (v) treating compound 20 with ethylene glycol and a catalytic amount of p-toluenesulfonic acid to give compound 3,12S-diacetoxy-20,20-ethylenedioxypregna-3,5,14-triene of formula i

• ·· ·

(21) (vi) treating compound 21 with NaBH ₄ to give compound 3S, 12S-dihydroxy-20,20-ethylenedioxypregna-5,14-diene-12-acetate of formula

In a first alternative process, the process for preparing steroid 15 of the invention comprises the steps of (a) treating compound 22 with a reducing agent such as LiAlH ₄ to give compound 3E, 12S-dihydroxy-20,20-ethylenedioxypregna-5,14-diene of formula

(23) • 9 • 999 ί

17 (b) treating compound 23 with N-bromoacetamide (NBA) and a base such as pyridine to give compound 3S, 12S-dihydroxy-14,15-epoxy-20,20-ethylenedioxypregnen-5-ene of formula

(24) (c) treating compound 24 with a reducing agent to give compound 3S, 12S, 14S-trihydroxy-20,20-ethylenedioxypregn-5-ene of formula

and (d) treating compound 25 with an acid such as acetic acid and water to form the steroid compound 15, 3S, 12S, 14S-trihydroxypregn-5-ene.

Reaction Scheme A illustrates a process for preparing steroid intermediate 15 from compound 22 according to the first alternative process of the invention (and includes preparing compound 22 from compound 16 for illustrative purposes).

• • • •

120) (21)

U5)

AND

In a second alternative process for preparing steroids 15 of the invention, the process comprises the steps of (a) treating compound 22 (3S, 12S-dihydroxy-20,20-ethylenedioxypregna-5,14-diene-12-acetate) with p-toluenesulfonyl chloride and a base, e.g. pyridine to form the compound

3S, 12S-dihydroxy-20,20-ethylenedioxypregna-5,14-diene-3-tosyl-12-acetate of formula

(b) treating compound 26 with potassium acetate in a solvent such as acetone to give compound 6S, 12S-dihydroxy-20,20-ethylenedioxy-3,5α-cyclopregnan-14-en-12-acetate of formula

(27) (c) treating compound 27 with a reducing agent such as LiAlH ₄ and, for example, tetrahydrofuran to give compound 6S, 12S-dihydroxy-20,20-ethylenedioxy-3,5α-cyclopregnan-14-ene of the formula • 4 • 4

(28) (d) treating compound 28 with N-bromoacetamide, optionally acetic acid, and a base such as pyridine to give compound 6β, 12β-dihydroxy-20,20-ethylenedioxy-14,15-epoxy3, β-cyclopregnan of formula

OOOH (29) (e) treating compound 29 with a reducing agent such as LiAlH ₄ and, for example, tetrahydrofuran to give compound 6β, 12β, 14β-trihydroxy-20,20-ethylenedioxy-3,5α-cyclopregnane of the formula

OH (30); and (f) treating compound 30 with an acid such as hydrochloric acid and a solvent such as acetone to form compound 15.

Reaction Scheme B depicts a process for preparing intermediate 15 from compound 22 according to a second alternative process of the invention.

• ·

Reaction Scheme B

(15) A mixture of epimers of (15a) C-17pacety ((15b) C-17aacetyl)

O

OH

HIM • ·

Compound 1 may be prepared by synthesis from the first carbohydrate intermediate in the form of an activated monosaccharide cymarose moiety, which may be prepared from a compound having the formula 36. Compound 36 may be prepared according to a process comprising the steps of (i) processing α-D-methyl- glucose having the formula

with benzaldehyde and zinc chloride to give methyl-4,6-O-benzylidene-α-D-glucopyranoside of formula

(32) (ii) treating compound 32 with tosyl chloride and pyridine at 0 ° C to give methyl-4,6-O-benzylidene-2-O-tosyl-α-D-glucopyranoside compound of formula

(33) · (iii) treating compound 33 with NaOMe at 100 ° C to give methyl 4,6-O-benzylidene-3-O-methyl-α-D-methylpyranoside of formula

(34) (iv) and treating compound 34 with N-bromosuccinimide (NBS) to give methyl 6-bromo-4-O-benzoyl-3-O-methyl-6-deoxy-α-D-altropyranoside of formula

and (v) treating compound 35 with NaBH ₄ and NiCl ₂ to give methyl 4-O-benzoyl-3-O-methyl-6-deoxy-α-D-altropyranoside compound of formula

(35) • ·

The invention is further extended to a process for preparing a cabohydrate intermediate in the form of an activated monosaccharide cymarose moiety, comprising the steps of (i) treating compound 36 with PhSSiMe 2, Zn ₁₂ and Bu 4 ^{+ 1} "to provide 4-O-benzoyl-3-O-methyl -6-deoxy-αS-D-phenylthioaltroside of formula

SPh (37) (ii) optionally treating compound 37 with diethylaminosulfur trifluoride (DAST) at, for example, 0 ° C to give 4-O-benzoyl-3-O-methyl-2-phenylthio-2,6-dideoxy-α-D -fluorocaropyranoside having the formula

or (iii) optionally treating compound 37 with tert-butyldimethylsilyl chloride and imidazole in a solvent such as pyridine to give 4-O-benzoyl-3-O-methyl-2-O-tert-butyldimethylsilyl-cdS-D-phenylthioaltroside having the formula

»99

9

9 9 9 <· ♦

99.

Italy (39)

SPh where Z = TBDMS = t-butyldimethylsilyl;

and (iv) treating compound 39 with a base such as sodium methoxide to give 3-O-methyl-2-O-tert. butyldimethylsilyl-ε-D-phenylaltroside having the formula

(40)

SPh where Z = TBDMS = t-butyldimethylsilyl.

Reaction Scheme C illustrates a process for preparing activated monosaccharide cymarose moiety 40 from compound 36 of the invention (and includes preparing compound 36 from compound 31 for illustrative purposes).

- 28 Reaction Scheme C

Ph

(34);

Br-

Z = t-butyldimelhylsilyl of • ·

The synthesis of Compound 1 may also include a second carbohydrate intermediate in the form of an activated monosaccharide thevetose moiety that can be prepared by a compound of Formula 47. Compound 47 can be prepared by a process comprising the steps of (i) processing α-D-glucose having the formula

(41) with acetone and sulfuric acid to give compound 1,2: 5, β-di-O-isopropylidene-Q-D-glucofuranose of formula

(42) (ii) treating compound 42 with NaH and Mel to give compound 1,2: 5,6-di-O-isopropylidene-3-O-methyl-of-D-glucofuranose of formula

(43)

- (iii) treating Compound 43 with acetic acid to give 3-O-methyl-α-D-glucopyranose of formula

(iv) treating compound 44 with methanol and hydrochloric acid to give the methyl 3-O-methyl-β-D-glucopyranoside compound having the formula

OMe (v) treating compound 45 with benzaldehyde and zinc chloride to give methyl 4,6-O-benzylidene-3-Omethyl-β-glucopyranoside compound having the formula

(Vi) treating compound 46 with N-bromosuccinamide, nickel chloride, and borohydride solution to form methyl

4-O-benzoyl-3-O-methyl-6-deoxy-GI-glucopyranoside having the formula

ί

(47)

The invention is further extended to a process for preparing an activated monosaccharide thevetose moiety, comprising the steps of (i) treating compound 47 with phenylthiotrimethylsilane and trimethylsilyl trifluoromethanesulfonate to provide 4-O-benzoyl-3-O-methyl-1-phenylthio-6-deoxy-o; -glucopyranoside having the formula

SPh (48) (ii) treating compound 48 with pivaloyl chloride and a solvent such as pyridine to give 4-O-benzoyl-3-O-methyl-2-O-pivaloyl-1-phenylthio-6-deoxy-αS-glucopyranoside having the formula

.............. 0 * 0 • · 0 · 0 · ·

00 0 ·

0 0 0 0 0

0 0 0 0 », 0-0,

0 0 »

0 0 0 0 0 0 0

(49) and (iii) treating compound 49 with a brominating agent, for example, N-bromosuccinimide and diethylaminosulfur trioxide to provide 4-O-benzoyl-3-O-methyl-2-O-pivaloyl-1-fluoro-6-deoxy-5-glucopyranoside occurring as stereoisomers having the formula

Reaction Scheme D illustrates a process for preparing activated monosaccharide thevetose moieties 50A and 50B from compound 48 of the invention (and includes preparing compound 47 from compound 41 for illustrative purposes).

· «« Φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ

- 33 Reaction scheme D

149)

• 99 4 4 * 9 * 94-99

4944 9,949,449 4 «9 9 4 9,999« 4,949 9 9 99 49 9

44 499 4949

4499 44 99 9999 99 99

According to a further aspect, the invention provides a process for the synthetic preparation of a compound of formula 1, analogous derivatives thereof, comprising the steps of synthesizing a suitable steroid intermediate or precursor and coupling the desired number of suitable monosaccharides to the steroid intermediate.

The invention also provides a method of coupling a monosaccharide cymarose to a steroid intermediate, comprising the steps of (i) reacting the cymarose moiety of formula 38 with a steroid intermediate of formula 15, for example at -15 ° C in the presence of tin chloride in a solvent such as ether to form 3-0 - [4-O-benzoyl-2-phenylthio-SD-cymaropyranosyl] -12,14-S-dihydroxypregnan-5-en-20-one of formula

BzO

(51) and (ii) treating compound 51 with tiglic acid chloride in pyridine and then with a base such as NaOMe to give compound 3-O- [-2-phenylthio-β-D-cymaropyranosyl] -12S-tigloyloxy-14- hydroxy-14S-pregn-5-en-20-one of the formula. frfr frfrfr fr frfr frfr frfr fr frfr frfr frfr frfr frfr frfr frfr frfr frfr fr

(52)

The invention is further extended to a method which comprises coupling a monosaccharide cymarose moiety to a monosaccharide thevetose moiety and coupling the resulting disaccharide to the combined steroid product 52 to form Compound 1.

Coupling of the monosaccharide cymarose moiety to the monosaccharide thevetose moiety and coupling of the resulting disaccharide to the combined steroid product 52 may include the steps of (i) coupling the selectively protected cymarose moiety of Formula 40 and the selectively protected thevetose moiety 50A using stannous chloride (SnCl ₂ ) and silver trifluoromethanesulfonate. -15 C to give the compound of formula

(53)

wherein Z = TBDMS = tert-butyldimethylsilyl (ii) treating compound 53 with tetrabutylammonium fluoride to form a compound of formula

SPh (54) (iii) treating compound 54 with diethylaminosulfur trifluoride at, for example, 0 ° C to give a compound of formula

(55) (iv) reacting compound 55 with compound 52 to form compound ·· · - · - · ·· ♦ '- ···· ^1' · '9 9 - '99 • 999 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 99

(56) and (v) treating compound 56 with a Raney nickel reaction and then with a base such as NaOMe to form compound 1 as described above.

Reaction Scheme E illustrates the procedure for synthesizing intermediates 52 and 55 and coupling them to give compound 56.

> · · ·

- 38 Reaction Scheme E

BzO

(15)

(52) 9

(50A)

SPh * ·

53ν (53)

ΟΡν

154)

ΟΡν • ·

According to the invention, an alternative method involves coupling the cymarose and the thetose moieties to form a trisaccharide and coupling the trisaccharide to a steroid derivative to form a compound of formula 1.

The process of trisaccharide formation and coupling of the resulting trisaccharide to a steroid derivative may include the steps of (i) coupling a selectively protected cymarose moiety of Formula 40 and Compound 45 using stannous chloride, AgOTf,

Cp ₂ ZrCl ₂ to give a compound of formula

(57) wherein Z = TBDMS = t-butyldimethylsilyl;

(ii) treating compound 57 with tetrabutylammonium fluoride and diethylaminosulfur trifluoride to give a trisaccharide compound having the formula

(58) and (iii) coupling the trisaccharide of formula 58 with the steroid intermediate of formula

(59) using stannous chloride, AgOTf, Cp ₂ ZrCl ₂ to give compound 1.

Steroid intermediate 59 can be prepared by treating steroid 15 with tiglic acid chloride.

Reaction Scheme F shows a procedure for the synthesis of trisaccharide 58 and the synthesis of compound 1 by coupling trisaccharide 58 with the steroid intermediate 59.

· · Φ · • · • · • · • · · · · · · · · · · · · · · ·

49- * ·· φφφ φφφ <

φφφφ φφ φφ φφφφ φφ φφ

Reaction Scheme F

(40)

ΟΡν

SPh

^0Ρν 158)

Β-t-butyl [dimethyl Isilyl

Intermediates 23, 24, 25, 27, 28, 29, 30, 37, 38, 39

40, 48, 49, 50, 51, 53, 54, 55, 56, 57 and 58 described above are novel compounds and the invention extends to these compounds as such.

Compound 1 3-0 - [- β-D-thevetopyranosyl- (1-> 4) -β-Dcymaropyranosyl- (1-> 4) -β-D-cymaropyranosyl] -12 µ-O-tigloyloxy-14-hydroxy-14B -pregn-5-en-20-one and its various analogs and derivatives have a taste-reducing activity. to eat.

The invention is extended to a composition or formulation having an appetite reducing activity, wherein the active ingredient is an extract obtained from plants of the genus Trichocaulon and the genus Hoodia.

The active ingredient may be a compound of Formula 1 extracted from a plant of the genus Trichocaulon or Hoodia or a derivative thereof. The plant may be of the species Trichocaulon officinale or Trichocaulon piliferum or of the species Hoodia currorii, Hoodia gordonii or Hodia lugardii.

The invention is further extended to a composition or formulation having an appetite-reducing activity, wherein the active ingredient is a synthetically prepared compound of Formula 1 or a derivative thereof or an analogue thereof, as described above with reference to compounds 2 to 14.

In another aspect, the invention provides a method of reducing appetite by administering to a human or animal a suitable dose of an appetite reducing agent comprising an extract of a plant of the genus Trichocaulon or Hoodia. The extract may be incorporated into a composition or formulation which also includes pharmaceutically acceptable other ingredients.

The appetite reducing agent may be an isolated natural or synthetic chemical compound of the formula:

Or

derivatives or analogues thereof,

as above.

A taste reducing composition or formulation. for eating may consist of an appetite reducing agent mixed with a pharmaceutically acceptable excipient, diluent or carrier. Other suitable additives, including stabilizer and other components, may also be added.

The invention extends to the use of Compound 1 or derivatives or analogs thereof in the preparation of a medicament having appetite reducing activity.

The invention is further extended to Compound 1 or derivatives or analogs thereof as described above for use as a medicament having appetite reducing activity.

Also included is a method of reducing appetite by administering to a human or animal an effective dose of the composition as described above.

Disclosed herein is a method of extracting a steroid glycoside having appetite reducing activity from plant material obtained from a Trichocaulon or Hoodia genus. Thus, the invention extends to the extract obtained from

plant material of the genus Trichocaulon or Hoodia and containing substantially pure steroid glycoside of formula 1.

The invention is also extended to foods or beverages containing an effective amount of the steroid glycoside of formula 1 or derivatives or analogs thereof as described above, which have an appetite-reducing effect when ingested.

Molecular genetic studies have led to a significant increase in understanding of regulation of taste, supersaturation and body weight. These studies have revealed a number of central regulatory pathways mediated by a number of neuropeptides. Maintaining normal body weight is achieved by a complex balance between energy intake, food intake, and energy expenditure. Energy homeostasis is subject to a wide range of influences, mainly controlled by the brain. Different signals include parameters such as sense, smell and taste.

and gastrointestinal signals such as gastrointestinal extension, chemical signals to gastric mucosa, and blood-borne metabolites such as fatty acids and glucose.

Central neuropeptide Y (NPY), which is negatively regulated by leptin, has been identified as one of the positive regulators of food intake. Expression of an endogenous antagonist to melanocortin receptors has been shown to underlie obesity in a particular model (ob / ob mouse). In fact, lack of MC4 melanocortin receptors completely replicates obesity syndrome. Other agents reported to play a role in energy balance include bombesin, galonin, and glucagon-like peptide-1.

Without being bound by theory, the Applicant believes that Compound 1 and its analogs as described above act as agonists of melanocortin 4 receptors. The effect of this action is to regulate NPY but also to increase it. · • ·· 9 9 9 9 9

9 ·· ·· 9 4 3 4

9999 99 99

- 46 cholecystokinin. The action of cholecystokinin inter alia inhibits gastric emptying.

Accordingly, the invention is extended to a composition having an appetite reducing activity comprising a melanocortin 4 receptor agonist.

The agonist may be an extract or a compound as described above, in particular a compound of formula 1. The composition may be admixed with a pharmaceutical excipient, diluent or carrier and optionally prepared in unit dosage form.

The invention is further extended to the use of melanocortin 4 receptor agonists in the preparation of a medicament having appetite-reducing activity, to melanocortin 4 receptor agonists for use as a medicament having appetite-reducing activity, for a method of reducing appetite by administering to a human or animal an effective dose comprising a melaocortin 4 agonist as described above and for use of melanocortin 4 receptor agonists to reduce appetite and / or combat obesity in humans or animals.

The invention and its efficacy will now be further described by the following examples and drawings, which, however, are for the purpose of illustration only and are not intended to limit the scope of the invention in any way.

In the drawings,

Figure 1 shows a flow chart of a general method of extracting a first raw appetite-lowering extract and purifying an appetite-lowering extract from plant material of the genus Trichocaulon or Hoodia;

Figure 2 shows a graphical representation of a bioassay performed on rats using partially purified

9 «

999 · 9 «methanol extract of Trichocaulon piliferum;

Figures 3 and 4 together show a schematic representation of a preferred embodiment of the process of the invention for preparing an extract of plant material of the genus Trichocaulon or Hoodia; and

Figures 5 and 6 show a graphical representation of the percent change in body weight of rats for the various groups on days -7 to 7 and days 0 to 7 in a repeated dose study using a juice extract and a spray dried juice extract of plant material of Hoodia gordonii.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

The general method for extracting the first raw appetite-lowering extract and purified appetite-lowering extract from plant material of the genus Tricocaulon or Hoodia is illustrated by the method shown in the flow chart of Figure 1.

Example 2

Bioassays conducted in rats using the partially purified methanol extract obtained as illustrated in Example 1 showed that the extract actually exhibited appetite reducing activity. The appetite-reducing activity of the active extract can be illustrated by a typical example of the effect of Trichocaulon piliferum methanol extract on rats by the graphical representation of Figure 2.

From Figure 2, it will be seen that the test group of rats will be tested. Which received a dose of extract on day 5 showed significantly less food consumption for the next two days, while the control the group did not show comparatively lower food intake. Food intake returned to normal in the test group, but actually increased from day 8.

Example 3

A preferred embodiment of the process of the invention for preparing an extract having appetite-reducing activity is illustrated schematically in the example of Figures 3 and 4, wherein the two figures together show the complete process. However, the skilled artisan can use various other procedures.

According to Figure 3, the plant material of the genus Trichocaulon or Hoodia is fed into a mixer, for example a Waring mixer, by means of a conveyor 1, with a solvent in the form of a methylene chloride / methanol solution fed via line 2.

The homogenized product is fed via line 4 to a separation stage 5, which is, for example, a filter or centrifuge, and the remaining plant material is discharged via line 27.

The solvent / extract mixture is fed via line 6 to the evaporation stage where the solvent is removed, for example by a rotary evaporator. The dried crude extract is passed through line 8 to the next extraction stage 9 with the addition of a methylene chloride / water mixture supplied via line 29 for further extraction and then to the separation stage 13 via line 11 where the water fraction is removed through line 31. The dissolved extract fraction is fed. via line 15 to a drying stage where the solvent is evaporated, for example by a rotary evaporator.

Referring to Figure 4, the dried extract is passed through line 10 to the extraction stage 12. The methanol and hexane solution is also passed through line 14 to the extraction stage 12 to the next stage 9. Purification and extraction of the dried extract. The mixture of extract, methanol and hexane is passed through line 16 to separation stage 18, the hexane fraction is removed through line 20 and the mixture of methanol and extract is then passed through line 22 to drying stage 24. In the drying stage 24 the solvent is removed e.g. evaporator.

The dried, partially purified active extract is passed through line 26 and with the addition of methanol through line 28 to the solution stage and the dissolved fraction is passed through line 36 to the chromatography column 38.

In column 38, the methanol soluble fraction was further fractionated using silica gel and chloroform / 30% methanol, to various fractions, schematically labeled I to V. According to the current fractionation procedure carried out by the applicant, the fractionation procedure achieved the following weights of the individual fractions: 9 g); II (2.6 g); III (2.1 g); IV (1.1 g) and V (2.0 g). These fractions are individually evaluated by appropriate bioassays (not reported at the stage) and those fractions identified as fractions I and II showing significant taste-reducing activity. They are fed through columns 40 and 42 to columns 44 and 46, where they are further fractionated and purified by column chromatography, again using silica gel and a 9: 1 chloroform: methanol system.

The subfractions II (A) - (C) obtained from column 44 do not show appreciable appetite-reducing activity in the bioassays and can be recycled for further chromatographic processing.

Subfractions I (A) - (L) obtained from column 46 are also evaluated (assay not shown), and subfraction I (C) has been found to have significant appetite-reducing activity.

Subfraction I (C) is passed through line 48 to column 50 k. Frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr frfr Frfrfr frfrfr frfrfr frfr frfr frfr frfr

50 further fractionation and further purification using silica gel and a 9: 1 ethyl acetate / hexane solvent. Of the resulting purified fractions, a significant appetite-reducing activity was found in fraction (C) (ii) after the bioassay.

The purified product is identified by nuclear magnetic resonance spectroscopy (as shown in Tables 1 and below) and is Compound 1.

····················· · 9

9999 99 99

Table 1: - ^ (300.13 MHz) NMR data for Compound 1 (CDCl3)

Compound 1

Hydrogen atom J (HH) / Hz Aglykon-3 6 - 12 11.5, 4.1 17 9.3, 9.3 18 - 19 Dec - 21 - 3 * 7.1, 1.5 4 * 7,1,1,2 5 * 1.6, 1.2

Cym-1 _H and ppm

3.522 m 5.381 m 4.607 dd 3.157 dd 1.029 s 0.951 s 2.164 s 6.888 qq 1.806 dq 1.853 dq

2 ', aq

3 '

4 '

5 '

6 'ax

3'-OMe

1 ”

2 ”

3 ^ax

5 ”

6 ”

3 ”-OMe

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 s ^d 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 1.243 d ^b - 3,392 s®

Thev-1

3 '

4 '»

5 '

6 '”

3'-OMe

7.7, 8.0 8.0, 2.9

9.3, 2.9

6.3, 9.3

4,273 d 3,469 dd 3,099 dd 3,179 dd 3,351 dd 1,183 d ' ^c 3,622 sa, b, c _{in the} column can be interchangeable ^ ^that in each column can be interchangeable refers to atoms of the tigloate group ·· aa aa ·· aa aa · · Aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

Table 2: compound Relevant ¹³ C (75.25 1 in cdci ₃₎ MHz) n.m.r Aglycone part Sugar part Carbon Jp / p.p.m. Carbon J _C / ppm 1 37,04 T cym- 1 ' 95.84 D 2 29,44 T 2 ' 35,57 T 3 77.24 D 3 ' 77.05 D 4 38,62 T 4 ' 82.57 D 5 138,95 S 5 ' • 68.48 D 6 131.90 D 6 ' 18.14 Q 7 27/30 T 3'-OMe 57.93 Q 8 35.30 D 1 99.54 D 9 43.04 D 2 35,17 T 10 37.22 N 3 76.99 D 11 26,04 T 4 ” 82.52 D 12 75.88 D 5 68.30 D 13 53.71 N 6 18/36 Q 14 85.69 N 3 ”-0Me 57.09 Q 15 Dec 34,36 T Thev- 1 ' 104.28 D 16 24,31 T 2 ' 74.62 D 17 57.18 D 3 ' 85.30 D 18 9.85 Q 4 ' 74.62 D 19 Dec 19.27 Q 5 ' 71.62 D 20 May 216.85 S 6 ' 17.75 Q 21 33.01 Q 3'-ΟΜθ 60.60 Q • 1 167.60 N 2 * 128.69 D 3 ’ 137.66 D ♦ 4 14.41 Q 5 * 12.08 Q

refers to atoms of the tigloate group

Compound 1

IR data: 3440 cm ^-1 (OH), 2910 cm ^-1 (CH), 1700 cm ^-1 (C = O) [α] 589 = 12.67 ^{2θ 0} (c = 3, CHC1 _3), mp 147-152 ° C .

te ·· te · ♦ · «9« «9 9 · 9 * * * * * 9 9 9 · · · · 9 · 9 9 9 9 9 9 9 · «· · · · · · · · · ·

Examples 4-13 illustrate synthetic procedures while intermediate compounds and steroid 15 may be prepared according to the first alternative process.

Example 4

12S, 15α-Dihydroxyprogesterone 17

Cultures of Calonectria decora (ATCC 14767) are prepared by inoculating a culture medium including sucrose (90 g), K ₂ HPO ₄ (30 g) Czapek's concentrate (300 ml), a soaked corn beverage (300 ml) and distilled water (30 L) ( 150 x 500 ml containers). After shaking for 5 days at 26 ° C, progesterone 16 (150 g) in Tween 80 suspension (0.1% solution, 1.5 L) was added to the flasks. The cultures were incubated for an additional 5 days and processed by centrifugation, decanting, extraction of the medium with chloroform and evaporation to give dihydroxyprogesterone 17 (17.5 g, 45%).

¹ H NMR (CDCl ₃ ): 5.71 (1H, S, H-4); 4.12-4.22 (1 H, m, H-15); 4.43 (1 H, br, s, OH); 3.46-3.53 (1H, dd, J = 4.6Hz, H-12); 2.16 Hz (3 H, s, H-21); 1.18 (3H, s, H-19); 0.74 (3H, s, H-18)

Example 5

12S-Hydroxy-15α- (p-toluenesulfonyl) progesterone (18)

Dihydroxyprogesterone 17 (75 g, 0.22 mol) was dissolved in dry pyridine (300 mL) and cooled to 0 ° C. Then, p-tolenesulfonyl chloride (46 g, 0.24 mol) in dry pyridine (200 mL) was added dropwise at 0 ° C. The reaction mixture was stirred overnight at 0 ° C and cooled by addition of H ₂ O (500 mL). The aqueous layer was extracted with ethyl acetate (1 L) and the organic extract was washed with hydrochloric acid (6M, 3 x 1 L), aqueous saturated sodium bicarbonate (500 mL), brine (500 mL) and water (500 mL). Organic

the layer was dried, filtered and evaporated to give p-toluenesulfonated progesterone 18 (98 g, 92%) as a viscous dark yellow oil.

NMR (CDC1 _3): 7.7 (2H, d, J = 14Hz, H-2,6); 7.34 (2H, d, J = 8.4Hz, H-3.5); 5.67 (1 H, s, H-4); 4.86-4.93 (1 H, m, H-15); 3.45-3.50 (1H, dd, J = 4.6Hz, H-12); 2.44 (3H, s, H-4 Me); 2.15 (3H, s, H-21); 1.13 (3H, s, H-19); 0.74 (3H, s, H-18).

Example 6

12S-hydroxy and ¹⁴ ogesteron ^EXAMPLE 19

A solution of tosylated progesterone 18 (98 g, 0.19 mol) in 2,4,6-trimethylcolidine (500 mL) was heated to reflux at 150 ° C for 3 hours. The reaction mixture was cooled and poured into water (500 mL). The aqueous layer was extracted with ethyl acetate (1 L) and the organic layer was washed with hydrochloric acid (6M 3 x 1 L), aqueous saturated sodium bicarbonate (500 mL), aqueous saturated sodium chloride solution (500 mL), and water. After drying (MgSO ₄ ), the ethyl acetate was evaporated and the crude mixture was purified by silica gel chromatography, eluting with acetone / chloroform (1:10) to give ^ ¹⁴ -progesterone 19 (50 g, 78%) as a dark red oil.

¹ H NMR (CDC1 _3): 5.73 (1H, s, H-4); , 28 (1H, dd, J = 2.2Hz, H-15), 4.41 (1 H, br, s, OH), 3.49-3, 52 (1H, dd, J = 4.3Hz, H-12), 2.80-2.84 (1H, dd, J = 9.2Hz, H- 17), 2.14 (3H, S, H-21), 1.19 (3H, S, H-19), 0.89 (3H, WITH, H-18).

Example 7 /

3,12S-Diacetoxypregna-3,5,14-triene-20-one 20

A solution of ¹⁴ -progesterone 19 (50 g, 0.15 mol) in acetyl chloride (1.5 L) and acetic anhydride (750 mL) was heated at reflux for 2 hours. Pour the reaction mixture into dry ethyl acetate (11) and add aqueous saturated sodium bicarbonate until the solution is effervescent. The ethyl acetate layer was separated from the sodium bicarbonate layer and washed with another portion of aqueous sodium bicarbonate (3 x 700 mL) followed by saturated sodium chloride (700 mL) and finally water (700 mL). The organic layer was dried (MgSO 4), filtered and evaporated to give 3,12S-diacetoxypregna-3,5,14-trien-20-one 20 (60 g, 93%) as an orange oil.

¹ H NMR (CDCl ₃ ): 5.68 (1H, s, H-14), 5.44 (1H, m, H-6), 5.31 (1H, dd, J = 2.2Hz, H- 15), 4.82-4.86 (1H, dd, J = 4.5Hz, H-12), 3.10-3.18 (1H, t, J = 9.5Hz, H-17), 2 18 (3H, s)

3-Ac), 2.11 (3H, S, 12-Ac), 2.08 (3H, S, H-21), 1.02 (3H, S, H-19), 1.01 (3H, S, H-21); s, H-18).

Example 8

3,12S-Diacetoxy-20,2 O-ethylenedioxypregna-3,5,14-triene 21

Diacethoxy compound 20 (60 g, 0.14 mol) was dissolved in benzene (11) and ethylene glycol (60 mL) and p-toluenesulfonic acid (1 g) was added. (Benzene was previously heated to reflux in a Dean-Stark trap). The mixture was heated to reflux with stirring and water was removed azeotropically for 16 hours. To the cooled solution was added aqueous saturated sodium bicarbonate solution (500 mL). The solution was washed with brine (500 mL) and water (500 mL) and dried (MgSO ₄ ). The solvent was evaporated and the crude mixture was purified by silica gel column chromatography, eluting with ethyl acetate / hexane (2: 8) to give ethylenedioxypregna-3,5,14-triene 21 (35 g, 53%).

¹ H NMR (CDCl ₃ ): 5.68 (1H, s, H-4), 5.45 (1H, m, H-6), 5.31 (1H, dd, J = 2.2Hz, H- 15), 4.73-4.85 (1H, dd, J = 4.4 Hz, H-12), 3.78-3.98 (4H, m, ethylenedioxy), 2.16 (3H, s, 3 -Ac), 9 · 9 · · · · · · · · · · · · · · · · · ·

56 ”2.04 (3Η, s, 12-Ac), 1.29 (3Η, s, 21-21), 1.12 (3Η, s, Η-19), 1.02 (3Η, s, Η-21) -18).

Example 9

3S, 12S-Dihydroxy-20,20-ethylenedioxypregna-5,14-diene-12-acetate 22

Diene acetate 21 (35 g, 0.077 mol) was suspended in ethanol (500 mL) and sodium borohydride (2.8 g, 0.074 mol) was added at 0 ° C. The mixture was allowed to warm to room temperature and stirred overnight. Most of the solvent was removed in vacuo and the mixture was diluted with water (500 mL) and extracted with ethyl acetate (500 mL). Chromatography on silica gel with acetone / chloroform (1:10) gave 3S-alcohol 22 (25 g, 80%).

¹ H NMR (CDCl ₃ ): 5.41 (1H, m, H-6), 5.28 (1H, dd, J = 2.2Hz, H-15), 4.72-4.81 (1H, dd, J = 4.4 Hz, H-12), 3.82-4.02 (4H, m, ethylenedioxy), 3.45-3.59 (1H, m, H-3), 2.03 (3H) s, 12-Ac), 1.28 (3H, s, H-21), 1.10 (3H, s, H-19), 1.01 (3H, s, H-18).

Example 10

3S, 12S-Dihydroxy-20,20-ethylenedioxypregn-5,14-diene 23

3E-Alcohol 22 (25 g, 60.2 mmol) in dry tetrahydrofuran (300 mL) was added dropwise to a suspension of lithium aluminum hydride (2.7 g, 72.2 mmol) in dry tetrahydrofuran (500 mL). The reaction mixture was stirred at room temperature for 24 hours and water (2.7 mL) was added cautiously and the mixture was stirred for an additional 10 minutes. Sodium hydroxide (15% 2.7 ml) was then added and the suspension was stirred. After 10 minutes, water (8.1 mL) was added and the suspension was stirred for 10 minutes, filtered, dried (MgSO ₄ ) and the solvent was evaporated to give 3S, 12E dihydroxypregnadien 23 (20 g, 90%).

• »ϊ

- 57 ¹ H NMR (CDCl ₃ ): 5.36 (1H, m, H-6), 5.23 (1H, dd, J = 2.2Hz, H-15), 3.94-4.06 ( 4H, m, ethylenedioxy), 3.41-3.52 (1H, m, H-3), 3.32-3.36 (1H, dd, J = 4.3Hz, H-12), 1.31 (3H, s, .delta.), 1.01 (3H, s, H-19), 0.96 (3H, s, H-18).

¹³ C NMR (CDCl ₃ ): 152.4 (C-14), 140.2 (C-5), 121.1 (C-15), 119.7 (C-6), 111.1 (C- 20), 79.8 (C-12), 71.6 (C-3), 63.7 and 63.6 (ethylenedioxy), 58.8 (C-17), 19.0 (C-19), 11.9 (C-18).

35.125-Dihydroxy-14,15-epoxy-20,20-ethylenedioxypregn-5-ene

35.125-Dihydroxy-5,6-epoxy-20,20-ethylenedioxypregn-14-ene

N-Bromoacetamide (211 mg, 1.5 mmol) was added at 0 ° C to a stirred solution of 5,4-diene 23 (500 mg, 1.34 mmol) in acetone (100 mL), acetic acid (2.5 mL). ) and water (5 mL). After 15 minutes, sodium sulfite (5% solution, 50 mL) was added to the reaction mixture. The acetone was evaporated and the aqueous layer was extracted with dichloromethane (3 x 50 mL). The organic layer was dried (MgSO ₄ ), filtered and evaporated. Pyridine (1 mL) was added to the product and the mixture was stirred for 0.5 h. Dichloromethane (100 mL) was then added to the reaction mixture and the dichloromethane was washed with citric acid (5% solution, 3 x 100 mL), saturated sodium bicarbonate solution (50 mL), and water (50 mL). The organic layer was dried (MgSO ₄ ), filtered and evaporated to give a mixture of 14,15- and 5,6-epoxides (360 mg, 69%) as a white foam. The epoxide mixture cannot be separated by column chromatography.

Example 11

3E, 12S-Dihydroxy-14,15-epoxy-20,20-ethylenedioxypregn-5-ene 24

A mixture of 14,15- and 5,6-epoxides (14.4 g, 37.0 mmol) in dry tetrahydrofuran (200 mL) was added to the suspension. Lithium aluminum hydride (1.69 g, 44.4 mmol) in dry tetrahydrofuran (300 mL). The reaction mixture was stirred at room temperature for 24 hours and then treated as described above by adding water (1.69 mL) and sodium hydroxide (15% solution, 1.69 mL). After filtration and evaporation of the solvent, the crude product was purified by silica gel column chromatography using a 1: 9 mixture of methanol and chloroform to give unreacted 14,15-epoxy-20,20-ethylenedioxypregn-5-ene 24 (300 mg, 2, 1%).

¹ H NMR (CDCl ₃ ): 5.31 (1H, m, H-6), 3.82-3.98 (4H, m, ethylenedioxy), 3.43-3.52 (1H, m, H- 3), 3.41 (1H, s, H-15), 3.31-3.35 (1H, dd, J = 4.3Hz, H-12), 1.29 (3H, s, H- 21), 1.17 (3H, s, H-19), 1.02 (3H, s, H-18).

¹³ C NMR (CDCl ₃ ): 139.8 (C-5), 120.8 (C-6), 112.1 (C-20), 77.2 (C-12), 75.4 (C- 14), 61.0 (C-15), 22.3 (C-21), 19.2 (C-19), 9.5 (C-18).

Example 12

3S, 12S, 14S-Trihydroxy-20,20-ethylenedioxypregn-5-ene 25

14,15-epoxide 24 (300 mg, 0.77 mmol) in dry tetrahydrofuran (10 mL) was added to a suspension of lithium aluminum hydride (300 mg, 7.89 mmol) in tetrahydrofuran and the reaction mixture was heated under reflux for 48 hours. After addition of water (3 mL), sodium hydroxide (15% solution, 0.3 mL) and filtration as described above, the mixture was purified by silica gel column chromatography using a 1: 9 mixture of methanol and chloroform as solvent to give trihydroxypregnene. 25 (250 mg, 83%).

^{Χ Η} NMR (CDC1 _3): 5.38 (1H, m, H-6), 3.98 (4H, m, ethylenedioxy), 3.43-3.53 (1H, m, H-3), 3 25-3.32 (1H, dd, J = 4.1Hz, H-12), 1.32 (3H, s, H-21), 1.01 (3H, s, H-19), O, 98 • 9 · ♦ · ♦ · ·

9999

99

I · · *

I · * »· · ·

59 (3H, s, H-18) ¹³ C NMR (CDCl ₃ ): 139.1 (C-5), 122.1 (C-6), 112.2 (C-20), 85.1 ( C-14), 75.1 (C-12), 71.6 (C-3), 23.4 (C-21), 19.4 (C-19),

8.9 (C-18)

Example 13

3S, 12S, 14β-Trihydroxypregn-5-ene 15

Ethylendioxypregnen 25 (250 mg, 0.64 mmol) was dissolved in acetic acid (13.4 ml) and water and freeze-drying afforded trihydroxysteroid 15 (200 mg, 89%), mp 228 ^0-235 ° C, M + 348 , [α] ²⁰ + 35 ° (lit. [of] ²⁰ + 29 °).

^{Τ Η} NMR (CDC1 _3): 5.39 (1H, m, H-6), 3.56-3.62 (1H, t, J = 8.1 Hz, H-17), 3,42-3 51 (1H, m, H-3), 3.28-3.29 (1H, dd, J = 4.3Hz, H-12), 2.23 (3H, S, H-21), 1, Δ (3H, s, H-19), 0.90 (3H, s, H-18) ¹³ C NMR (CDCl ₃ ): 217.7 (C-20), 138.9 (C-5), 122 2 (C-6), 85.5 (C-14), 73.6 (C-12), 71.6 (C-3), 57.0 (C-17), 55.1 (C- 13), 43.6 (C-9), 42.1 (C-4), 37.3 (C-1), 36.8 (C-10), 35.9 (C-8), 34.5 (C-8) C-15), 32.9 (C-21), 31.5 (C-16), 30.1 (C-2), 27.4 (C-7), 24.4 (C-11), 19.4 (C-19), 8.3 (C-18).

Examples 14-19 illustrate synthetic procedures wherein intermediate compounds and steroid 15 may be prepared according to a second alternative procedure.

Example 14

20,20-Ethylenedioxy-3B-toluene-p-sulfonyloxypregn-5,14-trans-12β-ol acetate 26

A mixture of p-toluenesulfonyl chloride (650 mg, 3.4 mmol) in 9% · e · · · e · · · · e »* * * * * * * *

60 pyridine (10 mL) was added dropwise at 0 ° C to a mixture of 20,20-ethylenedioxypregna-5,14-diene-3S, 12S-diol 12-acetate 22 (1.3 g, 3.1 mmol) in pyridine (15 mL). The reaction mixture was allowed to stand at room temperature for 24 hours and then water was added to the reaction mixture. Extract the solution with ethyl acetate (2 x 50 mL), wash the ethyl acetate layer with citric acid (5 x 50 mL), saturated sodium bicarbonate solution (100 mL), saturated sodium chloride solution (100 mL) and water 100 mL). The ethyl acetate layer was dried (MgSO _4), filtered and and after concentration and purification by flash chromatography using hexane: ethyl acetate (8: 2, v: as eluant afforded β-0-tosylsteroid 26 (1.5 g, 84 %) as a yellow oil (found M 570.271, ^C 32 ^H 42 ° 7 ^S requires: M 570.273).

δ _Η 1.021 (3H, 21-H), 2.021 m, OCH ₂ CH ₂ O), (1H, m, 30H), (1H, m, 6-H), s, 19-H), 1.131 (3H, s, 18-H), 1.282 (3H, s, (OCH ₃ acetate), 2.431 (3H, s, Ar-CH ₃ ), 3.883 (4H,

4.750 (1H, dd, ³ J 10.8 Hz, 5.2 Hz, 12-H), 4.890

5.281 (1H, dd ³ J 4.2 Hz, 2.1 Hz, 15-H), 5.388

7.341 (2H, d, ³ J 8.2 Hz, ArH), 7.746 (2H, d, ³

J 8.2 Hz, ArH).

δ _c 13.439Q (C-18), 19.002Q (C-19), 21.612Q (Ar-methyl) *, 21.671Q (C-21) *, 24.175Q (methyl acetate), 63.401T ethylenedioxy), 63.498T (ethylenedioxy), 71.531S (C-13),

80.912D (C-12), 82.531D (C-3), 111.363S (C-20), 120.881D (C-15), 121.461 (C-6), 123.715-133.917 (aromatic), 139.903S (C) -4), 151.722S (C-5), 170.819S (carbonyl ester).

Example 15

20,20-Ethylenedioxy-3α, 5-cyclo-5α-pregn-4-ene-6S, 12S-diol-12-acetate 27

A solution of 3E-toluene-p-sulfonyloxypregn-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). ml) are heated at reflux to 60

- 61 ° C for 16 hours. The acetone was evaporated and the water was extracted with ethyl acetate (200 mL). The ethyl acetate was dried (MgSO _4), filtered and evaporated. Flash chromatography of the mixture using chloroform / acetone (9: 1 v / v) as eluent gave 3α, 5-cycloderivative 27 (530 mg, 61%) as a yellow oil (found M 416.262, C ₂₅ H ₃₆ O) ₅ requires: M 416.263).

Δ _H 0.288 (1H, dd, ³ J 8.1 Hz, 4.9 Hz, 4-H &amp;), 0.477 (1H, dd, ³ J 4.4 Hz, 4.4 Hz, 4-Hb), 1.025 ( 3H, s, 19-H), 1.121 (3H, s, 18-H), 1.256 (3H, s, 21-H), 1.989 (3H, s, acetate-CH ₃ ), 3.02 (1H, dd) ³ J 2.8 Hz, 6-H), 3.784-3.947 (4H, m, OCH ₂ CH ₂ O), 4.721 (1H, dd, ³ J 8.5 Hz, 5.6 Hz, 12-H) 5.232 (1H, dd, ³ J 3.9 Hz, 1.9 Hz, 15-H).

5 _C 11.678T (C-4), 12.298Q (C-18), 19.971Q (C-19), 23.623Q (C-21), 24.153Q (methyl acetate) 63.700T (ethylenedioxy), 63.788T (ethylenedioxy) ), 73.591D (C-6), 80.551D (C-12), 111.126S (C-20), 118.778D (C-15), 152.959S (C-14), 170.991S (carbonyl ester).

Example 16

20,20-Ethylenedioxy-3α, 5-cyclo-5α-pregn-14-en-6S, 12S-diol 28

A solution of 3α, 5-cycloderivative 27 (500 mg, 1.2 mmol) in tetrahydrofuran (20 mL) was added dropwise to a suspension of lithium aluminum hydride (50 mg, 1.3 mmol) in tetrahydrofuran (10 mL). Stir the reaction mixture for 4 hours and then cool by adding water (50 μΐ). After 30 minutes, add sodium hydroxide (15% solution, 50 μΐ) and continue stirring for an additional 30 minutes. Add water (150 μΐ) and filter the reaction mixture. Tetrahydrofuran was dried (MgSO ₄ ), filtered and evaporated and purified by flash chromatography using chloroform and acetone (8: 2 v / v) as eluant to afford diol 28 (370 mg, 83%) as an oil (found M 374.250, C ₂₃ H ₃₄ O ₄ requires M 374.252).

···········································

- 62 δ _Η 0.298 (1Η, dd, ³ J 8.1 Hz, 4.9 Hz, 4-¾), 0.510 (1H, dd, ³ J 4.4 Hz, 4.4Hz, 4-¾), 0.985 (3H, s, 19-H), 1.055 (3H, s, 18-H) 1.355 (3H, s, 21-H), 3.318 (1H, dd ³ J 3.0 Hz, 3.0 Hz, 6- H), 3.333 (1H, dd, ³ J 11.4 Hz, 4.2 Hz, 12-H), 4.019 (4H, m, OCH ₂ CH ₂ O), 4.622 (1H, s, OH), 5.255 ( 1H, dd, 3 J 3.9 Hz, 1.9 Hz, 15-H).

5 _C 11.681T (C-4), 12.243 (C-18), 19 / 844Q (C-19), 23.604Q (C-21), 63.620T (ethylenedioxy), 63.733T (ethylenedioxy),

73.569D (C-6), 77.478D (C-12), 111.125S (C-2), 118.702D (C-15), 152.912S (C-14).

Example 17

20,20_Ethylenedioxy-14,15S-epoxy-3α, 5-cyclo-5cY, 14S-pregnan-6S, 12S-diol 29

N-bromoacetamide (150 mg, 1.1 mmol) was added at 0 ° C to a solution of 20,20-ethylenedioxy-3α, 5-cyclo-5α-prgn-14-en-6S, 12S-diol 28 (340 mg). , 0.91 mmol) in acetone (20 mL), water (0.25 mL). After 15 minutes, sodium sulfite (5% solution, 20 mL) was added to the reaction mixture. The acetone was evaporated under reduced pressure and the remaining solution was extracted with dichloromethane (3 x 30 mL). The dichloromethane layer was dried (MgSO 4), filtered and evaporated to a concentrated volume (50 ml). Pyridine (0.5 mL) was added and the mixture stirred for an additional hour and then the dichloromethane layer was washed with citric acid solution (5%, 3 x 30 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 eluant to afford epoxide 29 (180 mg, 51%) as an eluent. foam (found M 390.245, .delta.3.4-4.2 requires: M 390.247).

δ _Η 0.287 (1H, dd,

J 8.1 Hz, 4.9 Hz,

4-¾), 0.501 (1 H, dd,

-63 ³ J 4.4 Hz, 4.4 Hz, 4-¾), 0.978 (3H, s, 19-H), 1.048 (3H, s, 18-H), 1.321 (3H, s, 21-H) ), 3,318 (1H, dd, ³ J 3.1 Hz, 3.1 Hz, 6-H), 3.355 (1H, dd, ³ J 11.2 Hz, 4.1 Hz, 12-H), 3.491 (1H 1H, s, 15-H), 4.001 (4H, m, OCH ₂ CH ₂ O), 4.901 (1H, S, H).

δ _c 11.668T (C-4), 11.973Q (C-18), 19.515Q (C-19), 23.519Q (C-21), 59.910D (C-15), 63.601T (ethylenedioxy), 63.713T (ethylenedioxy), 72.501S (C-14), 73.571D (C-6), 77.471D (C-12), 111.085S (C-20).

Example 18

20,20-Ethylenedioxy-6E, 12S, 14-trihydroxy-3α, 5-cyclo-5α, 14β-pregnane 30

A solution of epoxide 29 (170 mg, 0.44 mmol) in tetrahydrofuran (10 mL) was added to a suspension of lithium aluminum hydride (20 mg, 0.53 mmol) in tetrahydrofuran (5 mL). The reaction mixture was heated to reflux for 2 hours and water (20 μΐ) was added and stirring continued for 0.5 hour. Add sodium hydroxide solution (15%, 20 μΐ) and continue stirring for an additional 0.5 hours. An additional amount of water (60 μΐ) was added and the suspension was stirred for 1 hour. After filtration, the suspension was dried (MgSO ₄ ), filtered and the tetrahydrofuran was evaporated. Flash chromatography of the resulting mixture eluting with chloroform / methanol (9: 1 v / v) gave the desired triol 30 (90 mg, 53%) as a clear oil. (Found: M 392.261, C ₂₃ H ₃₈ O ₅ requires: M 392.263).

Δ _H 0.287 (1H, dd, ³ J 8.1 Hz, 4.9 Hz, 4-¾) 0.510 (1H, dd ³ J 4.4 Hz, 4.4 Hz, 4-¾), 0.971 (3H, s, 19-H), 1.042 (3H, s, 18-H), 1.329 (3H, s, 21-H), 3.321 (1H, dd, ³ J 3.0 Hz, 3.0 Hz, 6-H) ), 3.321 (1H, dd, ³ J 11.1 Hz, 3.9 Hz, 12-H), 3.561 (1H, S, OH), 4.084 (4H, m, OCH ₂ CH ₂ O) 4.671 (1H, s, OH).

6 _C 11.668T (C-4), 11.971Q (C-18), 19.511Q (C-19), 23.520Q • · • ·

- 64 (C-21), 63.612T (ethylenedioxy), 63.711T (ethylenedioxy), 73.483D (C-6), 76.051D (C-12), 84.307S (C-14), III, 099S (C- 20).

Example 19

3β, 12E, 14-Trihydroxy-14β-pregn-5-en-20-one 15

A mixture of triol 30 (80 mg, 0.20 mmol) in acetone (20 mL) and hydrochloric acid (1M, 10 mL) was heated at reflux at 60 ° C for 2 hours. The reaction mixture was separated and saturated sodium bicarbonate solution (20 mL) was added. The acetone was evaporated and the aqueous layer was extracted with chloroform (3 x 20 mL), the chloroform layer was dried (MgSO ₄ ), filtered and evaporated to give epimeric trihydroxysteroids (15a, 15b) (42 mg, 61%). Separation of epimeric mixture 15a, 15b (15 mg) was accomplished by chromatographic separation using a 9: 1 v / v mixture of chloroform and methanol as eluent to give pure 17β-ερίϊΏΘΓ (15a) (10 mg), m.p. 229 ° C (acetone), (lit. 226-223 ° C), (found M 348.234, C 72.32, H 9.21% ^C 21 ^H 32 ° 4 requires C, 72.38, H 9.26% , M 348,236) and 17α-epimer 15B (3 mg), m.p. 183-191 ° C (acetone), (lit. 184-196 ° C).

3β, 12β, 14-Trihydroxy-14β-pregn-5-en-20-one 15a:

δ _Η 0.963 (1H, s, 19-H), 1.192 (3H, s, 18-H), 2.236 (3H, s, 21-H), 3.325 (1H, dd, ³ J 11.2 Hz, 3, 9 Hz, 12-H), 3.444 (1H, S, OH), 3.5140 (1H, m, 3-H), 3.598 (1H, dd, ³ J, 9.6 Hz, 9.6 Hz, 17- H), 4.255 (1H, s, OH), 5.383 (1H, m, 5-H).

No. _c 8,275Q (C-18), 19.414Q (C-19), 24.400T (C-11), 24.581T (C-16), 27.443 (C-7), 30.062T (C-2), 32.972 Q (C-21), 34.543T (C-15), 35.864D (C-8), 36.975S (C-10), 37.337T (C-1), 42.144T (C-4), 43.565D (C- 9), 55.101S (C-13), 57.038D (C-17), 71.597D (C-3), 73.558D (C-12), 85.566S (C-14), 122.223D (C-6) ,

138.932S (C-5), 217.011S (C-20).

3β, 12β, 14-Trihydroxy-14β-pregn-5-en-20-one 15b:

δ _Η 0.996 (1H, s, 19-H), 1.144 (3H, s, 18-H), 2.221 (3H, S, 21-H), 3.339 (1H, dd, ³ J 9.4 Hz, 9, 4 Hz, 17-H), 3.492 (1H, m, 3-H), 3.629 (1H, dd, ³ J, 11.1 Hz, 3.9 Hz, 12.H), 3.712 (1H, s, OH) 4.325 (1H, s, OH), 5.383 (1H, m, 5-H).

Examples 20-28 illustrate procedures by which intermediate compounds can be prepared to prepare the first monosaccharide 40.

Example 20

Methyl-4,6-O-benzylidene-α-D-glucopyranoside 32

A mixture of methyl-α-D-glucopyranoside (30 g, 0.15 mol), benzaldehyde (70 mL), and zinc chloride (20 g) was stirred at room temperature for 24 hours. The reaction product is poured into water and stirring is continued for 15 minutes. The white precipitate was filtered and washed with diethyl ether. Stir the solid with sodium metabisulphite solution (10% solution) for 15 minutes, filter and wash with water. The solid material was crystallized from chloroform and ether to give benzylidene product 32 (31 g, 72%).

Example 21

Methyl 4,6-O-benzylidene-2-O-tosyl-α-D-glucopyranoside 33 p-Toluenesulfonyl chloride (25 g, 1.2 eq.) In pyridine (100 mL) was added dropwise at 0 ° C to the solution. benzylidenglucose 32 (31 g, 0.12 mol) in pyridine (100 mL). The reaction mixture was stirred at room temperature for 48 hours and then ice was added to the reaction mixture. The resulting white solid was washed with water and recrystallized from hot ethanol to give.

66 tosylated glucose 33 (28 g, 60%).

Example 22

Methyl-4,6-O-benzylidene-3-O-methyl-α-D-altropyranoside 34

Tosylate 33 (28 g, 64 mmol) was heated in a solution of sodium (7 g) in methanol (150 mL) to 110 ° C for 48 hours in an autoclave. The reaction vessel was cooled and solid carbon dioxide was added to the reaction mixture. After filtration, the methanol was evaporated and the solid material was taken up in water. The aqueous layer was extracted with chloroform (3x). The chloroform was dried (MgSO ₄ ), filtered and evaporated. The crude mixture was purified by silica gel column chromatography eluting with chloroform / acetone (9: 1) to give altroside 34 (10 g, 52%).

Example 23

Methyl-6-bromo-4-O-benzoyl-3-O-methyl-6-deoxy-α-D-altropyranoside 35

Benzylidene altroside 34 (10 g, 33 mmol) was added to a solution of N-bromosuccinimide (7.6 g) and barium carbonate (20 g) in carbon tetrachloride, and the reaction mixture was heated to reflux at 75 ° C for 3 hours. The reaction mixture was filtered and the carbon tetrachloride layer was washed with water. The organic layer was dried (MgSO ₄ ), filtered and evaporated to give 6-bromo-altroside 35 (9 g, 69%).

Example 24

Methyl 4-O-benzoyl-3-O-methyl-6-deoxy-α-D-altropyranoside 3 6

Sodium borohydride (18 g) in water (30 mL) was added dropwise at 0 ° C to a solution of bromaltroside 35 (9 g, 23 mmol) and nickel (II) chloride (18 g) in ethanol (300 mL).

• · 9 9 »9 9

The reaction mixture was heated to reflux at 75 ° C for 1 hour and then filtered. The ethanol was evaporated and the remaining aqueous layer was extracted with chloroform (3x).

The chloroform was dried (MgSO ₄ ), filtered and evaporated to give 6-deoxyaltroside 36 (5 g, 72%).

Example 25

4-O-Benzoyl-3-O-methyl-6-deoxy-α-D-phenylthioaltropyranoside 3 7

Phenylthiotrimethylsilane (5 mL) and trimethylsilyl trifluoromethanesulfonate (2 mL) were added at 0 ° C to a solution of 6-deoxyaltroside 36 (5 g, 17 mmol) in dichloromethane (200 mL). The reaction mixture was stirred at room temperature for 6 hours. Saturated sodium bicarbonate was then added to the reaction mixture. The dichloromethane layer was dried (MgSO ₄ ), filtered and evaporated. The crude mixture was purified by silica gel column chromatography eluting with chloroform / acetone (9: 1) to afford α-phenylthioaltroside 37 (4 g, 63%).

Example 26

4-O-Benzoyl-3-O-methyl-2-phenylthio-2,6-dideoxy-.alpha.-D-fluorocymaropyranoside 38

Diethylaminosulfurtrifluoride (0.65 g) was added rapidly at 0 ° C to a solution of α-phenylthioaltroside 37 (0.5 g, 1.33 mmol) in dichloromethane. The reaction mixture was stirred at 0 ° C for 0.5 h and then saturated sodium bicarbonate was added.

Dichloromethane was separated from the aqueous layer, dried (MgSO ₄ ), filtered and evaporated to give α-fluorocymarose 38 (450 mg,

%).

Example 27

4-O-Benzoyl-3-O-methyl-2-O-tert. butyldimethylsilyl-ceS-D · · · · · · · · · · · · · · · · · · · · · · ·

-phenylthioaltrosici 39

6-Deoxyaltroside 37 (5 g) was silylated using tert-butyldimethylsilyl chloride (3 g) and imidazole (3 g) in pyridine (50 mL). The reaction was worked up by extraction with ethyl acetate, washed with ethyl acetate with hydrochloric acid (6 N), then with sodium bicarbonate and finally with water. The ethyl acetate layer was dried (MgSO ₄ ), filtered and evaporated to give silylated benzoylphenylthioaltroside 39 (80%).

Example 28

3-O-methyl-2-O-tert-butyldimethylsilyl-α-D-phenylthioaltroside 40

Silylated benzoyl phenylthioaltroside 39 (6 g) was treated with sodium methoxide (100 mL) for 4 hours. Methanol was evaporated and water was added to the reaction mixture. The aqueous layer was acidified (pH 5, AcOH) and extracted with ethyl acetate.

The ethyl acetate layer was washed with water, dried (MgSO ₄ ), filtered and evaporated to give silylated methylphenylthioaltroside 40 (75%).

Examples 29-37 illustrate procedures by which intermediate compounds can be prepared to prepare the second monosaccharide 50.

Example 29

1,2: 5,6-Di-O-isopropylidene-α-D-glucofuranose 42

Sulfuric acid (40 mL) was added dropwise at 0 ° C to a solution of αD-glucose 41 (50 mg, 0.28 mol) in acetone (1 L). The reaction mixture was stirred for 24 hours and then neutralized using sodium hydroxide (6 M). The acetone was evaporated and the aqueous layer was extracted with chloroform (2x). The chloroform was dried (MgSO ₄ ), filtered and evaporated. Crystallization from cyclohexane gave di-isopropylidene glucose 42 (41 g, 57%).

Example 30

1,2: 5,6-Di-O-isopropylidene-3-O-methyl-α-D-glucofuranose 43 and D-glucofuranose 42 (41 g, 0.16 mol) in tetrahydrofuran (300 mL) were added dropwise to a suspension of sodium hydride (5 g) in tetrahydrofuran (200 mL). After 0.5 h, methyl iodide (25 g) in tetrahydrofuran (100 mL) was added to the reaction mixture and stirred for 24 h. Water was added to the reaction mixture, and the mixture was extracted with ether (3x). The ether layer was dried (MgSO ₄ ), filtered and evaporated to give methyl protected glucose 43 (38 g, 83%).

Example 31

3-O-Methyl-β-D-glucopyranoside 44

Methyldiisopropylidene compound 43 (38 g, 0.14 mol) was dissolved in acetic acid (50%, 700 mL) and the solution was heated under reflux for 18 hours. After cooling, acetic acid was evaporated. The crude product was purified by column chromatography eluting with a mixture of chloroform, methanol, acetone and water (70: 27: 2: 1) to give 3-O-methyl-o; S-glucopyranoside 44 (13 g, 50%).

Example 32

Methyl 3-O-methyl-α-D-glucopyranoside 45

3-O-methyl-β-glucopyranoside 44 (10 g) was dissolved in methanol (50 ml) and concentrated hydrochloric acid (1 ml) was heated at reflux overnight. Adds solid • · ·· · · · · ···· *

NaHCO 3 and the reaction mixture was filtered. Methanol was evaporated to give 1,3-di-O-methyl-αS-D-glucopyranoside 45 (95%).

Example 33

Methyl 4,6-O-benzylidene-3-O-methyl-5-glucopyranoside 46

Glucopyranoside 45 (8 g) was stirred at room temperature in a solution of benzaldehyde (20 ml) and zinc chloride (5 g).

After 24 hours, ice was added and the aqueous layer was extracted with chloroform. The chloroform layer was dried (MgSO ₄ ), filtered and evaporated. The benzalaldehyde was removed by vacuum distillation and the product was purified by silica gel column chromatography eluting with acetone / chloroform (0.5: 9.5) to give benzylidene-α-glucopyranoside 46 (60%).

Example 34

Methyl 4-O-benzoyl-O-methyl-6-deoxy-.alpha.-glucopyranoside 47

The benzylidene compound 46 (5 g) was heated to reflux at 80 ° C in a mixture of N-bromosuccinimide (3.7 g) and barium carbonate (4 g) in carbon tetrachloride. After 4 hours the reaction mixture is filtered and the carbon tetrachloride is washed with water, dried (MgSO _4), filtered and evaporated to give bromo compound (70%).

The bromo compound (4.3 g) was dissolved at 0 ° C in a solution of ethanol (300 ml) and nickel chloride (8.6 g). To this solution was added sodium borohydride (8.6 g) in water (50 mL) dropwise over 15 minutes. The reaction mixture was heated to reflux at 100 ° C for 45 minutes, cooled, filtered and evaporated. Chloroform was added and the chloroform layer was washed with water, dried (MgSO ₄ ), filtered and evaporated to give 6-deoxy-sugar 47 (70%).

··

ί

- 71 Example 35

4-Ο-Benzoyl-3-O-methyl-1-phenylthio-6-deoxy-αS-glucopyranoside 48

6-Deoxyglucopyranoside 47 (3 g) was dissolved in dichloromethane (50 mL). To this solution was added phenylthiotrimethylsilane (2 g) and trimethylsilyl trifluoromethanesulfonate (0.2 mL). The solution was stirred at room temperature overnight and then saturated sodium bicarbonate was added.

The dichloromethane layer was dried (MgSO ₄ ), filtered and evaporated. The product was purified by silica gel column chromatography eluting with ethyl acetate / hexane (2: 8) to give compound 48 (60%).

Example 36

4-O-Benzoyl-3-O-methyl-2-O-pivaloyl-1-phenylthio-6-deoxy-α-glucopyranoside 49

To a solution of glucopyranoside 48 (2 g) in pyridine (20 mL) was added pivaloyl chloride (2 mL). The solution was stirred at room temperature overnight and then water was added. The aqueous layer was extracted with ethyl acetate and the organic layer was washed with HCl (6 N). The organic layer was dried (MgSO ₄ ), filtered and evaporated to give pivaloyl ester 49 (80%).

Example 37

4-O-Benzoyl-3-O-methyl-2-O-pivaloyl-1-fluoro-6-deoxy-E-glucopyranoside 50

N-Bromosuccinimide (1.2 g) and dimethylaminosulfur trifluoride (1.2 g) were added at 0 ° C to a solution of pivaloyl ester 49 (2 g) in dichloromethane (100 mL). After 1 hour, saturated sodium bicarbonate was added. The dichloromethane layer was dried (MgSO ₄ ), filtered and evaporated.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Φ · · · · Β-Fluoropyranoside 50 was purified by silica gel column chromatography eluting with a 2: 8 mixture of ethyl acetate and hexane (45% yield).

Example 38 illustrates a process by which 3-O- [4-O-benzoyl-2-phenylthio-β-D-cymaropyranosyl] -12,14-dihydroxypregnan-5-en-20-one (51) can be prepared.

Example 38

3-O- [4-O-benzoyl-2-phenylthio-β-D-cymaropyranosyl] -12,14β-dihydroxypregn-5-en-20-one 51

Stannous chloride (190 mg, 1 mmol) was added at -15 ° C to a solution of 3,12,14-trihydroxypregnan-5-en-20-one 15 (100 mg, 0.28 mmol) and fluorcymaropyranoside 38 (210 mg). ,

0.56 mmol) in dry diethyl ether and 4A molecular sieves. The reaction mixture is maintained at -15 ° C for 3 days. Saturated sodium bicarbonate was added to the reaction mixture. The ether layer was dried (MgSO ₄ ), filtered and evaporated. The product was purified by silica gel column chromatography eluting with chloroform-methanol (9.5: 0.5) to give glycoside 51 (30 mg, 15%).

Examples 39 to 41 illustrate synthetic procedures by which cymarose and thevetose moieties can be coupled.

Example 39

Thevetosy-cymarose disaccharide 53

A solution of thevetose 50 A (1.5 g), cymarose 40 (1.3 g) and molecular sieves 4A in dichloromethane was stirred at room temperature for 1 hour. The reaction mixture was cooled to -15 ° C and stannous chloride (0.8 g) and silver trifluoromethanesulfonate (1.1 g) were added. The mixture was stirred at -15 ° C for 16 hours and triethylamine (0.5 mL) was added. The reaction product was filtered and the dichloromethane was evaporated. The disaccharide was purified by column chromatography on silica gel, eluting with a mixture of ethyl acetate and hexane (2: 8), yield 15%.

Example 40 β · * «« «« 40 40 40 40 40 40 40 40 40 40 40 40 40 40 · * · · · · · · · · · · · ·

Thevetosy-cymarose disaccharide 54

To a solution of disaccharide 53 (200 mg) in tetrah-yrofuran (20 mL) was added tetrabutylammonium fluoride (0.4 mL). The mixture was stirred at room temperature for 1 hour and saturated sodium bicarbonate was added. The reaction mixture was extracted with ethyl acetate and the ethyl acetate layer was dried (MgSO ₄ ), filtered and evaporated. The disaccharide 54 is purified by column chromatography on silica gel with a mixture of acetone and chloroform (0.5: 9.5), yield 60%.

Example 41

Thevetosy-cymarose disaccharide 55

To a solution of the disaccharide 54 (80 mg) in dichloromethane (10 mL) was added diethylaminosulfur trifluoride (80 μΐ) at 0 ° C. The mixture was stirred at 0 ° C for 0.5 hour and saturated sodium bicarbonate and more dichloromethane were added. The dichloromethane was dried (MgSO _4), filtered and evaporated. Purification by silica gel column chromatography (ethyl acetate: hexane = 1: 9) gave disaccharide 55 in 65% yield.

Example 42

The results of the following appetite reduction bioassays are shown below

(a) Irwin test;

(b) Acute toxicity test; a ·· φ • # # # # #........ ·.

♦ ♦ • 9 9 9 9 9 «« 9 «

c) Anorectic oral dose test.

a) Irwin test

The purpose of this test was to evaluate the appetite reducing agent of the invention obtained from the plant extract as described above according to the Irwin test for tranquilizing and sedative effects.

Experimental procedure

The appetite reducing agent is extracted from the plant material according to the method described above and administered with two of four groups of animals, each group containing three animals: one group is not treated, one group is given dimethylsulfoxide (DMSO), one group is given a test sample at a dose of 50 mg / kg and one test group is administered at a dose of 300 mg / kg. Treatment is performed by intraperitoneal injection and observations are made at specific intervals up to five hours after administration. When interpreting the results, only those symptoms other than those observed in DMSO-treated animals are observed.

Results

It is clear that the solvent, DMSO, has a considerable effect on animals, in particular on the temperature-regulating mechanism. The body temperature of all animals treated with solvent, alone or with the test sample showed a significant decrease.

Animals in the group treated with the lower dose of the test compound exhibited reduced cage dispersion and reduced physical activity, as in all other groups, including the control group. Apathy was observed at the same level as the DMSO treated group.

• ·

- 75 Reduced breathing was observed 15 to 60 minutes after treatment. Ptosis (eyelid drop) was also observed to a greater degree than in the DMSO treated group. The response to the auricle was positive as well as the response to the finger and showed timidity. Body temperature after processing to 32.7 ° C.

Animals in the treatment group treated with a high dose of the compound exhibited an initial reduced cage dispersion and decreased physical activity, but exhibited increased dispersion and increased activity before death at 1 hour post-treatment. Serious clonic symmetric seizures occurred 30 minutes after treatment. Breathing decreased initially but increased before death. Response to auricle was delayed, finger response was positive and showed timidity as observed in low dose treated animals. Body temperature dropped to 30.7 ° C after treatment. Increased positional activity as well as decreased tone were observed. Abnormal limb rotation was observed, the clamping force decreased, no painful response, and the righting reflex was lost.

Discussion

Compared to the control and DMSO treated animals, animals that were only treated with a low dose (50 mg / kg) exhibited reduced breathing and an increased degree of ptosis. Animals receiving a high dose (300 mg / kg) of the test sample responded very intensely, resulting in convulsions and death. All other observations made with these animals are attributed to the convulsive and dying animals. Signals suggesting sedative and sedative effects, such as significantly reduced cage dispersion, decreased mobility and apathy in the test groups that could be attributed to the test sample were not observed.

In conclusion, the test sample is lethal in 300 mg / kg mice and causes reduced respiration in mice at 50 mg / kg when administered intraperitoneally with DMSO as solvent.

(b) Acute toxicity test

- 76 The purpose of this test was to obtain information on the toxicity of the sample.

Experimental procedure

The plant extract prepared according to the invention as described above and causing appetite reduction is purified and one test sample is tested at an increased dose by oral treatment of mice. Two animals per group were used in the test, except that only one animal was treated at the highest dose. The animals were examined for good health and their body weights were determined on the day of treatment.

Doses administered ranged from 100 mg / kg to 3,028.5 mg / kg. Doses were determined and mixed with potato starch such that each animal received a total dose of 0.2 ml. Animal No. 13 received 0.25 ml. Potato starch was prepared by mixing 20 g of starch with a small amount of cold water and boiling water was added to make the volume 1 liter. The suspension was allowed to cool to room temperature before dosing.

Animals in Groups 1 and 2 were treated on the same day. The animals were observed for 24 hours and another group was treated if no signs of toxicity appeared. The same procedure continued until all animals were treated. In this way, the animals were not necessarily treated if the acute toxic dose was reached in the previous group.

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

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

- 77 weight was measured once a week and also the total food and water consumption was measured for each animal.

Living animals were euthanased by intraperitoneal injection of sodium pentobarbitone (commercially available under the tradename Euthanaze, Centaur ^R ) on day 14 of the experiment. A post-mortem examination was then performed on these animals as well as animals that 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 consumption were in normal parameters. Changes in body weight were also within normal parameters. No histopathological changes were detected in the liver sample.

Group 2 (100 mg / kg)

No clinical signs of toxicity were observed during the observation period. Food and water consumption were normal, and changes in body weight were also normal. No macroscopic pathology was observed and no histopathological or macrophological changes were detected in the liver sample.

Group 3 (200 mg / kg)

Animals in this group showed no clinical signs of toxicity during the experiment. Water and food consumption were normal as well as changes in body weight. No macroscopic pathology was observed, but the liver showed histopathological changes in the examination. The turbid swelling of hepatocytes was mild in animal # 6, but mild in animal # 5. Mild hydropic degeneration also occurs in animal hepatocytes # 5.

Group 4 (400 mg / kg)

No clinical signs of toxicity were observed during the observation and no macroscopic pathology was found in the post-mortem examination. Histology revealed slight turbid swelling and slight hydropic changes in hepatocytes.

Water and food intake and weight gain in animal # 7 was normal. Animal # 8 consumed nearly twice the total food of animal # 7 (144.6 g respectively 73.9 g), but the increase in body weight was only 0.81 g compared to 2.7 g.

Group 5 (800 mg / kg)

One animal (animal # 10) died three hours after dosing without showing any specific symptoms. Another animal (animal # 9) survived the entire observation period without signs of toxicity. The water consumption of the surviving animal was normal (42.42 ml), while the food consumption was high (134.2 g). Body weight increased by 2.85 g, which was the highest increase of the animals participating in the experiment.

Post-mortem examination of animal # 10, which died shortly after administration, showed blood vessels in the blood. No foreign physical reaction indicating inhalation of the test material was detected. No pathology was observed in animal # 9. Mild cytoplasmic vacuolization (hydropic degeneration) did not occur in animal # 10, but mild in animal # 9. The glandular cytoplasmic appearance of the liver was classified as mild in both animals.

··············································

- 79 Group 6 (1600 mg / kg)

None of the animals present showed any signs of toxicity during the duration of the experiment. No macroscopic pathology was observed in the post-mortem examination, but slight degenerative changes in the liver were observed in animal # 11 in histopathological examination. Animal # 12 showed slight turbid swelling and mild hydropic changes in hepatocytes. food and water consumption were normal as well as weight gain.

Group 7 (3028.5 mg / kg)

Only one animal was treated with this dose. This animal showed no signs of toxicity during the experiment and also no macroscopic pathology was observed.

Histopathological examination revealed slight turbid swelling and hydropathic hepatocyte degeneration. The animals showed weight loss (-0.82 g) during the observation, but food and water consumption were normal.

Discussion

Since only a very small number of animals were used in each group, it is difficult to draw any conclusions. The fact that only one animal at a low dose without any symptoms died in the experiment may indicate that death was not related to the test sample but due to stress during and / or after treatment. No animal in the higher dose group died or showed any signs of toxicity, again supporting this assumption.

The increased food requirement observed in animal 8 could be attributed to excessive spillage of food, as evidenced by a small increase in body weight. It should be taken into account that

all animals in the experiment were treated only once, and that the taste-reducing agent is unlikely. food will have a significant effect on food or water consumption or body weight over a 14 day period, as is the case with this test.

The histopathological examination of the liver sample shows that the pathological changes are dose-dependent, the animals receiving the higher dose show larger changes. The pathology observed was not metabolic in nature but was probably due to the test sample. The changes were only degenerative and therefore reversible. No irreversible hepatocellular changes were observed.

In summary, only one animal died at a lower dose (800 mg / kg), but its death was probably not due to the test sample. No other animal in any dose group showed any symptoms during the 14th day observation after treatment or died as a result of treatment. A single oral dose of the test sample introduced dose-dependent reversible hepatocellular changes.

c) Theoretical test for oral dose

The purpose of this test was to determine the activity of the plant extract prepared according to the invention and the minimum effective dose and to examine at the same time any side effects, such as reduced breathing, as seen in the Irwin test above.

Experimental procedure

The animals were divided into groups at random. Each treatment group consisted of three animals, with 6 animals in the control group. The test sample was dosed to young female rats weighing 100-150 g acclimated for three consecutive days. Animals

was labeled with metal rings for easy identification and the skin was labeled with KMnO ₄ . Animals were housed individually in standard polycarbonate cages and water and powdered commercial animal pellets were available ad libitum. Water and food consumption were measured and calculated daily. Five doses were tested for the minimum effective dose of the test sample. The treatment was oral with a test sample suspended in potato starch.

The test substance was compound 1, in the form of a white granular powder prepared from an extract of plant material according to the invention, and a measured amount of the test sample was mixed with the prepared potato starch and dosed. Mixing with potato starch was performed every day, just prior to dosing. Prior to preparing a dose volume for each animal, the suspension was thoroughly mixed using Vortex.

A range of five doses was tested with a control group receiving only the carrier substance. Doses were selected based on the effects observed in the Irwin test described above and were as follows:

Group 1: 0.00 mg / kg (control group)

Group 2: 6.25 mg / kg

Group 3: 12.50 mg / kg

Group 4: 25.00 mg / kg

Group 5: 37.50 mg / kg

Group 6: 50.00 mg / kg

Results

The treatment did not affect the health of the animals during the study period. The animals treated with the test sample in all dose groups showed a significantly reduced average body weight obtained over the entire study period, and animals in three of the five treatment groups actually lost body weight.

The average food consumption of all treatment groups decreased over the period. Animals in the higher dose groups had increased water consumption.

Respiration rate was not affected by any animal in any dose group.

Animals in all dose groups had a crumbly liver at post-mortem examination, but no macroscopic pathology was observed.

Discussion

The data collected during the acclimatization period confirmed that all animals included in the experiment were healthy and body weight was comparable between animals.

A decrease and, in some cases, even weight loss in combination with reduced food intake is strongly indicative of a decrease in appetite.

Reductions in food intake and observed decreased body weight were found even in the lowest dose group (6.25 mg / kg). Current weight loss was found in the 12.50 mg / kg group.

It is important to note that all treatment groups had increased water consumption and reduced food consumption (Figure 2). This could be due to the diuretic effect of the test sample or the stimulation of the third center in the brain.

A positive aspect is that there was no reduction in breathing as observed in the acute test

toxicity mentioned above with the intraperitoneal route. This could be due to decreased absorption from the gastrointestinal tract, with consequent reduced bioavailability. The oral bioavailability was, however, sufficient for the test sample to be effective. A slight decrease in respiratory rate 1 hour after treatment in most groups could be attributed to gastric filling with dosed volume and subsequent animal passivity.

The crumbly liver observed in the treated groups could be due to metabolic changes depending on decreased food intake, causing increased fat metabolism and liver congestion. If this assumption is true, these changes may be considered transient, which cease with time after a steady state is reached or upon interruption of the experiment. The possible effect on the liver requires further research.

Since this study was primarily designed as a test, small groups of test animals were used. This makes statistical evaluation of the data difficult, especially if individual animals react quite differently. However, the data indicate that test samples act as appetite reducing agents, even at the lowest doses tested (6.25 mg / kg). There were no clinical signs of breathing in the test.

Example 43

Harvested Hoodia plants obtained either from nature or by culture are first stored at 4 ° C for a maximum of 48 hours. The plants are washed under a tap and cut into cuttings of about 1 cm. The cuttings are joined and pressed on a hydraulic press at a pressure of 303 MPa for at least 0.5 hours. During the pressing, the juice from the plants is separately collected. The juice is stored at -18 ° C until further processing.

• 9

9 • 9 «· 9

99 9

9

9900 ·· 99

The juice is spray dried under suitable conditions to give a flowing powder. The moisture content of the powder is preferably less than 5% after spray-drying and, if necessary, further dried in a vacuum oven or using a fluid bed dryer.

Both the juice and the spray dried material are effective as appetite reducing agents in rat bioassays.

The kg kg of Hoodia Gordonii experiment was washed under a tap and cut into cuttings of about 1 cm. The cuttings are joined and pressed on a hydraulic press at a pressure of 303 MPa for at least 0.5 hours. The juice shall be collected and its weight shall be 10 kg for Hoodia Gordonii natural plants and 20 kg for Hoodia Gordonii. The juice (500 g) is spray dried under the following conditions:

Speed of current : 2.85 ml / min Inlet temperature : 110 ° C Outlet temperature : 70 ° C Chamber temperature: : 77 ° C

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

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

HPLC analysis method

Eluant

Column

Absorbance

Flow rate: 1 ml / min Injection volume: 10 μΐ

Acetonitrile: water (7: 3), isocratic Reverse Phase C-18 225 nm

Method

The powder obtained by spray drying was dissolved in water (0.5 ml) and acetonitrile (0.5 ml). Inject 10 μΐ of the solution into HPLC and determine the concentration of active compound using a curve prepared from pure compound 1.

Example 44

The results of the study designed to determine the possible theoretical effects of Compound 1 in rats are presented below. The following are the test samples as pure juice (sample 1), the juice-dried sample (sample 2) and the active portion (sample 3). Samples 1 and 2 are the juice and the spray-dried substance as described in Example 43 above. Sample 3 is a solvent-extracted compound (with 95% purity).

Wistar rats were treated with a single oral dose using samples 1-3. Two additional control groups were given vehicle (distilled water or DMSO). Fenfluramine (7.5 mg / kg) was orally administered as a reference standard.

Sample 1 (pure juice) administered orally produced a dose-dependent reduction in food consumption, which was statistically significant at doses of 1600 mg / kg and above compared to the vehicle dosing control. Concomitant weight loss (or growth rate) was also recorded. On the dosing day, a statistically significant water consumption was recorded 3 hours after dosing (6400 mg / kg ΐ)

Φ · · · φ φ φ · · φ φ φ a a a a a a a a a a a a 1000 a a 1000 a a 1000 1000 1000 a a a a 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000) and 6 hours after dosing. Between 24 and 48 hours post-dose, there was a statistically significant reduction in water consumption at doses of 3200 mg / kg and above.

Sample 2 (a juice spray) administered orally at 76 mg / kg also produced a statistically significant decrease in food consumption and body weight compared to vehicle treated animals. No statistically significant effects on water consumption were observed.

Sample 3 (active portion) produced a statistically significant reduction in food consumption at an oral dose of 5.0 mg / kg. No statistically significant effects on body weight were achieved by the active part, although examination of the data revealed a slight growth delay compared to vehicle-treated control animals. There were also no statistically significant effects on water consumption.

The reference standard, fenfluramine (7.5 mg / kg) produced a statistically significant reduction in food at 6 and 24 hours post-dose compared to vehicle-treated control. No statistically significant effects on water consumption or body weight were noted.

No effects on the liver due to treatment were noted.

·································································· · fcfc ··· · ···· ·· ·· ···· ·· ··

Test substance

Identity

Sample 1 (pure juice) Sample 2 (substance obtained by spray-drying juice)

Sample 3 (active part)

Appearance Brown liquid Storage conditions 20 ° C in the dark Purity Clear juice

Vehicle Distilled water

Powder White powder

Room temperature in the dark 4 ° C in the dark

Juice spray-dried pure substance> 95%

Distilled water Dimethylsulfoxide

Experimental procedure

Fifty-five Wistar rats were used for the study

Body weight, food consumption (food container weight) and water consumption (container weight) are recorded daily at the same time each day from start to end of the study.

On the first day, rats received a single oral dose according to the following table:

Group n Oral treatment Dose (mg / kg) 1 5 Vehicle (distilled water) - 2 4 Sample 1 (pure juice) 800 3 5 Sample 1 (pure juice) 1600 4 5 Sample 1 (pure juice) 3200 5 5 Sample 1 (pure juice) 6400 6 5 Sample 1 (pure juice) 10000 7 5 Sample 2 (substance obtained by spray - drying juice 38 8 5 Sample 2 (substance obtained by spray - drying juice 76 9 5 Sample 3 (active part) 2.5 10 5 Sample 3 (active part) 5.0 11 3 Fenfluramine 7.5

• ·

3 Vehicle (DMSO)

Groups 1 to 8 were dosed using a constant dose volume of 10 ml / kg and groups 9 to 12 were dosed using a dose volume of 1 ml / kg.

On the first day, food and water consumption were also measured at 1, 3 and 6 hours after dosing.

After measurement on day 8, the animals were sacrificed by carbon dioxide asphyxiation and the liver excised and placed in 10% buffer formalin prior to histology. Wax sections embedded in waxes of 4 to 5 μτη were prepared and stained with hematoxylin and eosin. Other sections were cut to 12 μτη on a cryostat and stained for the presence of oily red O (ORO) fats.

Data analysis

Food and water consumption after dosing was measured and the weight at each time point for treated P57 animals was compared to a similarly treated control group using vehicle, using a difference analysis, followed by a Williams test for comparison with the control.

The data for animals treated with fenfluramine were compared with the data for the vehicle treated control group using the Student's test.

Results

The results are summarized in the tables.

Sample 1 (pure juice), administered orally, produced a significant, dose-dependent daily reduction in food. The duration and amplitude of these reductions in food consumption were dose-dependent. 9 9 hours post-dose, Sample 1 (pure juice) produced a statistically significant reduction in food consumption compared to vehicle control at 1600 mg doses. / kg and higher. The highest dose of Sample 1 (juice) (10,000 mg / g) produced a statistically significant reduction in food consumption within 5 days of dosing.

Sample 2 (juice-dried substance) and Sample 3 (active part) produced a significant and statistically significant reduction in food consumption at an oral dose of 76 and 5.0 mg / kg. In both cases the effect lasted 48 hours after the dose.

The reference standard, fenfluramine (7.5 mg / kg p.o.) produced a statistically significant reduction in food, as compared to the vehicle control group, 6 and 24 hours post-dose (group 12).

Sample 2 (the juice-dried substance) and Sample 3 (the active part) did not produce significant dose-dependent effects on water consumption. On the dosing day, pure juice produced a statistically significant increase in water consumption 3 hours post dose (6400 and 10000 mg / kg) and 6 hours post dose (10000 mg / kg). However, two days post-dose, a statistically significant reduction in water consumption was observed in animals sampled 1 (juice) at 3200, 6400, and 10000 mg / kg. These reductions are of course not dose-dependent and occurred between day 1 and day 2 post-dose. The biological nature of these effects is unclear.

Sample 1 (pure juice) produced dose-dependent, statistically significant effects on body weight when compared to the vehicle control group (Group 1). At an oral dose of 3200 mg / kg and above, Sample 1 (pure juice) produced a statistically significant decrease in body weight or a decrease in velocity compared to the control when the vehicle was used to treat the animals.

49

9 9 4

44

4949 44

4 4

4444 growth. These effects were statistically significant from 48 hours post-dose to the end of the study.

Sample 2 (a juice-dried substance) administered orally at 76 mg / kg also produced a significant reduction in the growth of animals compared to the control group (Group 1) when vehicle was used for treatment. These effects were statistically significant between day 3 (48 hours post dose) and day 5 inclusive.

Sample 3 (active portion), although delayed the growth of animals at the highest dose (5.0 mg / kg) compared to the vehicle control group (group 12), this effect was not statistically significant.

Fenfluramine (7.5 mg / kg) did not produce statistically significant effects on reducing water consumption or body weight compared to control (group 12) when vehicle was used for treatment.

No treatment-related effects were found with respect to the effect on the liver.

' ^Uo ^ Oiuibjq

-91 aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa «··

Table 1a

Effects of oral administration on rat food consumption (pre-dose, daily)

tn (0 Q C O O 1 CN τ— 1 what ^ .co X »· * <- <M S + i »- » ^ CM íb ' LO 10 ¹ ¾ c57 cmΉ LO <0 ^ Z CN Ή '8 WHAT CN  OOY CN -H p * -4 ® JJ <M Ή <η <2. ® jj CN + t it" <M Ή WHAT 10¾ C?" ”+ L CN Ή xť to ^ £ 2 5 4-4 O O in- r ~ en cn O Τ '* cn what it <- what 1 - ¹⁰ CO p - <O CD® to 5 * CN® o®. co® - cn r> o® 00® Ό M -H CO CM and 03 * “ S-H -? ** "· ® jj CM +1 c * CM * H enSJ CM +1 CM - ^ what CN Ή CN Ή what * ' ^- * SH 04 * CD ^ “• H °° 00 ” C r * WHAT what CM LO 1- O O LO what cn O _t co® o® . tot O" it" ,_O O" _C0 o® • Q E □ tower 1 1 CN Ή *O CN Ή + i what with) CN Ή CN Ή what <?) CM Ή _t <n CN + i CN Ή CM +1 ® ή CN Ή “44 CN + f (0 C O O WHAT CM what WHAT WHAT what cn LO CM what WHAT IT 1 cn " cn " ot ν-'ΐ, co ^. cn ^ - LO IO 0. CD" . CO ^ O H- C ΙΟ’Φ »» 1 cn Ή χ * * ° CM * H CM · Η CM Ή LOT CM · « CO * ^N cn * CM “Π <0 * 2 CM loV CM Ή CM Ή c5 ^ J CM · Η ca O E Xj WHAT O what xt- 00 xt τ— O what □ 1 cd®. CD ^ O" CM - - ^ CO • P · - it" _ CM IN M, ^ 05 P ; cd'v WHAT T-® cn " at_ O coin 1 ^C H44 CM “Π CN Ή ** CM CN -H CN * “44 CN Ή eoT CN + i * About CO Ύ CM Ή ΤΤ CN · Η co ™ CN Ή CN · Η c? H σ> O) E, what • O O CD O O what in· O O CN WHAT O O TT WHAT O O O O r— 00 what WHAT r> it O? O * » LO it • > 'WHAT O 00 _of - 00 'WHAT Έ what Ό O ra > 'WHAT WHAT > 'WHAT > (/) IN £ O N WHAT > what > WHAT > if c c CD £ 'WHAT > o > Q Έ C 0 w > £ ω > ω O Έ 'WHAT E • 3 Jč SW > </> IN" s> O Her) > <Λ IN" £ O N 'WHAT > ω IN" £ O 'WHAT > w IN" £ O > ω c 3'CO ω> CM θ what CD HE »EN) • 5 ω> CM O * ’S N N C > 2 what what CD > 2 what WHAT 4lC 2 E 2 3 C C <u LL with Q E 3 3 ic ω > O Φ > > > > N > .Ng £ S at- O with O 2 what C CL r- CN CD in it what Γ ca cn O T— CM T-7 of w

sd Standard deviations>

· · R 9 9 • 9 9 9 9 r 9

-92 V V V V V 92 92 92 92

Table 1b

Effects of oral administration on food consumption in the rat (dose, daily)

sd Standard deviation

Group 2-8 was compared to Group 1 vehicle: Pf <0.05, p _f <0.01 Group 9-11 was compared to Group 12 vehicle: p <0.05, p <0.01 · ·· ·· ············

-93Table 2a

Effects of oral administration on food consumption in the rat (dose, daily)

Group average food consumption (g ± sd) between days CN r- CN ν '! cn 'cn-H LO «-Η cn . to® cn + t 31.7 ± 3.18 CN R - * \ LO ^ ω-Η r> . CN * CN® cn Ή 31.4 ± 2.98 »- 39.0 ± 17.27 CN ‘CQ» r7® «* 33.1 ± 4.82 O . CO® cn Ή •E . • what cn +1 COCN 1 CN LO * cTh ro 00 * r * «« CN -H r- 'R- *. W-H CD . cn ^ ~ cn cn -H CN j5 cn +1 T ' . ot CN + 1 O l CN®. cn Ή WHAT “4 cn * About him ¹⁰ · CTI CN Ή it . CN ^ Ο 11 CN Ή CN . 0) ¾ n- . cn " <N Ή »» rtcn t WHAT what" Tn CO Ή CO Ή WHAT <3 CO +1 WHAT . co ^ s ~; what . -e ': t * ® S-h WHAT ®4 O . rs®. cn Ή it * CO 1 " WHAT in-" O* WHAT rs® cn * what. F* P> M> _rcn cn +1 rř . C". S3 1 LO rf ¹ LO co -H xř σΑ tcT® co + » cn $ éó-H CN . ω °. cn + 1 to .CO. rs with CN * ⁰ cn * CN . 00¾ what O® ω * it . o ° cn Ή About CN °. , + N c cn -H cn . it* P *  * 4 ' 1 what the " σ, ζ sť what . co®. s + i what LO . M-o 35 00 * · LO r> it • “* co® , 4. what cn +1 what L o® 5Ϊ 00 • “3 σ> * O o ^. Sl 00 §5 §9 .co® Dose (mg / kg) 1 O O what O O WHAT T— ’ O O CN WHAT O O Those " WHAT O O O O r- 00 WHAT WHAT r * it oT O, in * it r < 1 Oral treatment / • “X ro Ό O >, E = 3 Ξ what > ro > > ro > </) r · SW CD L_ O 2 ro > '<0 > L0 ω O with ro > 'CD > w CD at. O 2 ro > 'WHAT > 00 in 9 O with ro > 'WHAT «Λ IN" 2 O N > c E <u => w 5 3 5 CN ° eats 2a O N Ng Se * 3 = « CN on> (/) 5 8 07 / 'WHAT > Q Έ .> £ ro what CD at. O with 07 / 'CD > o Έ .> 2 ro WHAT 2 O with C E 2 3 ¢ = C CD If. O w 2 Q 's-x' E 3 3 Ξ CD > ro C ’5L □ ω r- CN cn Ν ’ LO WHAT Γ- 00 cn O T ' t- · τ— CN r-

sd Standard Deviations · Standard Deviations · Deviations Standard Deviations

-94Table 2b

Effects of oral administration on food consumption in the rat (dose, daily)

sd Standard deviation

Group 2-8 was compared to Group 1 vehicle: 'p <0.05 Group 9-11 was compared to Group 12 vehicle (insignificant).

-95Table 3a

Effects of oral administration on rat body weight (dose, daily)

sd Standard deviation

Table 3b

Effects of oral administration on rat body weight (dose, daily)

sd Standard deviation

Group 2-8 was compared to Group 1 vehicle: * p _f <0.05, ** p _f <0.01 Group 9-11 was compared to Group 12 vehicle (insignificant).

Histological report

Histological examination was limited to the liver. No treatment-induced changes were found in Sample 1 (liquid), Sample 2 (becoming spray-dried), Sample 3 (active portion), fenfluramine or DMSO control group.

The findings had a similar incidence in the control and treatment groups.

• · · φ φ • * • φ

- 98’Table

Summary of occurrences of microscopic pathological changes

Gender: males Males in the study Total males Group 1 0 mg / kg 5 5 Group 2800 mg / kg 4 4 Group 3 1600 mg / kg 5 5 Group 4 3200 mg / kg 5 5 Group 5 6400 mg / kg 5 5 Group 6 10000 mg / kg 5 5 Liver Examined 5 4 5 5 5 5 No abnormalities 0 0 1 2 3 3 Inflammatory foci of parenchymal cells 0 1 0 0 0 3 (total) 0 1 0 0 0 1 Minimal Hepatocyte hypertrophy-centrilobular 0 0 0 0 0 1 (total) 0 0 0 0 0 0 Minimal 2 0 0 0 0 0 Extramedullary haemopoiesis (general) 2 0 0 0 0 0 Minimal 1 0 0 0 0 0 Hepatocyte necrosis - focal (general) 1 0 0 0 0 0 Minimal 3 4 4 3 2 2 Portal lymphoid infiltration (total) 3 4 4 3 2 2 Minimal 1 0 0 0 0 0 Eosinophilic hepatocytes - focal (total) 1 0 0 0 0 0 Minimal 0 0 1 0 0 0 Portal fibrosis (total) Minimal 0 0 1 0 0 0 Liver (ORO stain) 5 4 5 5 5 5 Examined 2 3 2 4 3 3 No abnormalities 3 1 2 1 2 2 Hepatocyte fat - centrilobular (total) 3 1 2 1 2 2 Minimal 0 0 1 0 0 0 Hepatocidal fat - periportal (total) Minimal 0 0 1 0 0 0

Table (continued)

Gender: males Males in the study Total males Group 7 38 mg / kg 5 5 Group 8 76 mg / kg 4 4 Group 9 2.5 mg / kg 5 5 Group 10 5 mg / kg 5 5 Group 11 7.5 mg / kg 5 5 Group12 0 mg / kg 5 5 Liver Examined 5 5 5 5 3 3 No abnormalities 2 2 0 1 0 2 Inflammatory foci of parenchymal cells 0 0 0 0 0 1 (total) 0 0 0 0 0 1 Minimal Hepatocyte hypertrophy-centrilobular 0 0 1 0 0 0 (total) 0 0 1 0 0 0 Minimal 3 3 5 4 3 1 Portal lymphoid infiltration (total) 3 3 5 4 3 1 Minimal 0 0 1 0 0 0 Portal fibrosis (total) Minimal 0 0 1 0 0 0 Liver (spot OŘO) 5 5 5 5 3 3 Examined 5 3 3 3 2 2 No abnormalities 0 2 2 2 1 1 Hepatocyte fat - centrilobular (total) Minimal 0 2 2 2 1 0

I · · · gg _ · ♦ ··

Example 45

Another bioassay using the same test samples as described in Example 44 is described below. Animals in this study received limited food, i.e., animals were fed between 12 and 15 pm daily. This differs from all bioassays conducted to date where food was available to rats as needed. Animals were acclimatized for a seven-day period (days -7 to -1), dosing was performed 0 to 6 days at 9.00 by oral gavage. The recovery period ran from day 7 to day 13. Dosage groups are described in Table 1 below. It should be noted that the actual control group is Group 9.

Group 5 is a control group that received the same amount of food as group 4. The purpose of this test was to evaluate the effect of restricted food on animal lives.

Results

The results obtained during the study showed that the acclimatization period is very short. Rats were fed mainly during the night and the sudden change to limited access to food during the daytime led to low daily consumption. Daily food intake increased in most groups towards the end of the acclimatization period when dosing with the test compound began. As a result, the effect of the test materials had no significant effect on food intake in rats during dosing.

The mean body mass for the different groups for days -7 to -1 and days 0 to 6 are shown in Table D1 and Table D2. The effect of different dosages of juice and spray-dried substance is shown in the accompanying graphs as% change in body weight 0 to 7 days (Figure 5) and% change in body weight -7 to 7 days (Figure 6). Weight loss is dose dependent, especially at higher doses.

AND

100 ·· ·· ·· ··················

Histopathological examination of the liver did not reveal significant pathology in the groups receiving the test compound.

Food

Food consumption was measured daily during acclimatization and during the study. Food was available for 3 hours a day, the period started at 12.00 and ended at 15.00. The animals were fasted for the remaining time. Animals in group 5 received a measured amount of food on day 1, which was equivalent to the average food consumption of group 4 day 0. This regulated feeding model for group 5, as determined from group 4 average consumption from the previous day, continued 1 to day 7.

Water

Water was provided in a standard container. Water (Magalies Water Board Tap Water, suitable for human needs) was available as needed. Water consumption was measured once a day, at the same time each day, after determining food consumption.

Acclimatization

The animals acclimatized seven days prior to the start of the study, during which time food and water consumption were determined. Body weight was determined on a daily basis during this time.

101

Study design and procedure

TABLE 1 Study design

group exam numbers dose test substance 01 6o 001-006 100 mg / kg frozen juice 02 / 6o 007-012 400 mg / kg frozen juice 03 / 6o 013-018 1600 mg / kg frozen juice 04 / 6o 019-024 3200 mg / kg frozen juice 05 / 6o 025-030 control Elga Option 3 Cleaned water 06 / 6o 031-036 2.2 mg / kg spray drying juice 07 / 6o 037-042 8.8 mg / kg spray drying juice 08 / 6o 043-048 35 mg / kg spray drying juice 09 / 6o 049-054 control Elga Option 3 Cleaned water

Route of administration

The test substance was administered on a daily basis for 7 days, using an intragastric probe. Animals were not fed 18 hours prior to compound administration (start at 9.00).

Duration of treatment

The animals were treated for seven consecutive days (day 0-6). Three animals from each group were sacrificed 24 hours after the last dose (day 7). The remaining three animals were sacrificed on day 7 after the last dose (day 13). This procedure was performed for all groups except group 5 where animals were sacrificed 24 hours after the last controlled feeding (day 8), the remaining three animals were sacrificed 7 days after the last dose (day 13).

Weight

Body weight was determined daily, approximately at the same time each day for the duration of the study, including during •

102 acclimatization periods.

Euthanazie

Three animals of each group were sacrificed 24 hours after the last dose (day 7).

The remaining three animals were sacrificed 7 days after the last treatment. This procedure followed for all groups except group 5, where three animals were sacrificed 24 hours after the last controlled feeding (day 8) and the remaining three animals were sacrificed on the 7th day after the last treatment (day 13). The animals were euthanized at the end of the study period with CO ₂ gas.

Ophthalmoscopic examination

Ophthalmoscopic examination, using an ophthalmoscope, was performed in all animals in all groups before the first administration of the test substance and after treatment.

Macroscopic pathology

Full examination of each dead animal was performed at the end of the study period.

Histopathology

Histopathological examination was performed on the liver of each animal.

9 99

9999

Table D.1

Mean body weight / group / week

-103-

9 9 9 9 9 9 99 9 9 9 99 99

Table D.2

Mean body weight / group / week (continued)

-104-

-105-

Table D.3

Mean body weight / group / week (continued)

99 ·························· ··· ··· ···

Table 1: Histological evaluation of liver in male rats Sample 1

Group 1: 100 mg / kg Sample 1 Group 2: 400 mg / kg Sample 1 animal no. Hepatic lesions animal no. Hepatic lesions 7th day 01 NPL 07 / FHS1 + 02 / NPL 08 / NPL C1 + 03 / NPL C1 + 09 / NPL 13th day 04 / NPL MLC 13th day 10 DHS1 + 05 / FHS1 + 11 NPL 06 / NPL 12 DHS1 +

Group 3: 1600 mg / kg Sample 1 Group 2: 400 mg / kg Sample 1 animal no. Hepatic lesions animal no. Hepatic lesions 7th day 13 NPL 19 Dec NPL 14 NPL 20 May NPL 15 Dec NPL 21 NPL 13th day 15 Dec NPL 13th day 22nd DHS1 + 16 DHS1 + 23 FHS1 + 17 NPL 24 NPL

Group 5: Group 5: Control: Purified Water Elga Optin 4: Limited food consumption

Group 5: Control: Purified Water Elga option 4 animal no. Hepatic lesions 7th day 25 NPL MLC 26 NPL 27 Mar: NPL 13th day 28 DHS1 + 29 DHS1 + 30 NPL

Legend:

C = Blood flow

DHS = Diffuse Hydropic Cell Swelling FHS = Focal Hydropic Cell Swelling NPL = No Parenchymal Lesions MLC = Minimal Lymphocyte Cuff + = Fine + = Mild + = Serious

-107.

· »·· ··

99 99

9 9 · 9 1 • · · · <

• · 9 9 9 I • · · · ·

Table 2: Histological evaluation of liver in male rats Sample 2

Group 6: 2,2 mg / kg Sample 2 Group 7: 8.8 mg / kg Sample 2 animal no. Hepatic lesions animal no. Hepatic lesions 7th day 31 NPL 37 NPL 32 NPL MLC 38 NPL 33 FHS 1+ 39 NPL C1 + 13th day 34 NPL 13th day 40 DHS1 + 35 DHS1 + 41 NPL 36 NPL 42 MLC DHS1 +

Group 8: 35 mg / kg Sample 2 animal 6. Hepatic lesions 7th day 43 NPL 44 NPL 45 NPL 13th day 46 NPL 47 NPL C1 + 48 MLC FHS1 +

Group 9: Control: Purified Water Elga Option 4

Group 9: Control: Purified Water Elga option 4 animal no. Hepatic lesions 7th day 49 NPL 50 NPL 51 FHS1 + 13th day 52 DHS1 + 53 NPL 54 FHS1 +

Legend:

C = congestion

DHS = Diffuse Hydropic Cell Swelling FHS = Focal Hydropic Cell Swelling NPL = No Parenchymal Lesions MLC = Minimal Lymphocyte Cuff + = Mild + = Mild + = Serious 9 9 « 9 9 9 9

98 9 9 9 9 9 9 9

9 9 9 9 9 · 9 9 9 _Λ · ······ 9 9 9 9

108 - ···· ........ ·· ··

No specific lesions were detected on the liver of experimental rats that received frozen juice, as well as a substance obtained by dry spraying the juice that could be attributed to the above chemicals. Hydroponic cell swelling, recorded in both control and experimental rats, may indicate metabolic cell swelling and anoxic changes. In some animals minimal lesions of lymphotic perivascular changes have been found, which is most likely a random observation. Mild congestion in hepatic sinusoids was found in several rats.

An important feature of the invention shown by the results of this study is that no tolerance has developed to any of the samples. This may provide considerable benefit, particularly with respect to the use of the compounds and compositions of the invention for the treatment of obesity.

In particular, the compounds and compositions of the invention have been described with respect to their properties as appetite reducing agents. It should be noted that the term appetite reducing agent is used herein to determine an activity that results in reduced appetite and / or an increase in satiety and thus leads to a reduction in overall caloric food consumption, which counteracts obesity. The invention further extends to a method of treating, preventing or combating obesity in a human or animal comprising administering to a human or animal an amount of a compound of Formula 2 that treats and is effective in preventing or combating obesity. A preferred embodiment of this aspect of the invention employs a composition or extract comprising a compound of Formula 1.

The term animal as used herein includes, but is not limited to, a species of animal, such as domestic domestic animals; non-limiting examples of such animals include cattle, sheep, ferrets, pigs, elephants, horses, poultry, fish, rabbits, goats, dogs and cats.

Compound of formula 2, preferably of formula 1, or composition?

109 as defined in any one of claims 9 and 25 to 31 above, as an theoretic agent, or in the treatment or prevention of obesity in a human, is preferably administered to said human in an amount of about 0.01 mg / kg / day to about 10 mg / kg / day . A preferred dose is in the range of 0.05 mg / kg / day to 0.5 mg / kg / day. When using a spray-drying powder or an extract according to the invention, the preferred dose is in the range of 0.1 mg / kg / day to 20 mg / kg / day, particularly preferred is 0.5 mg / kg / day to 5 mg / day. kg / day.

Claims (95)

A method for preparing an extract of a plant of the genus Trichocaulon or Hoodia, the extract comprising an appetite reducing agent, the process comprising the steps of treating the collected plant material with a solvent to extract fractions having appetite reducing activity, separating the extraction solution from the remainder of the plant removing the solvent from the extraction solution and recovering the extract. A method of preparation as claimed in claim 1, wherein the plant of the genus Trichocaulon is selected from the species Trichocaulon piliferum and Trichocaulon officinale and the plant of the genus Hoodia is selected from the species Hoodia currorii, Hoodia gordonii and Hoodia lugardii. A method as claimed in claim 1 or claim 2, wherein the step comprises concentrating the active agent in the extracted material by further extraction with a solvent. The method as claimed in claim 1, claim 2 or claim 3, wherein the solvent in the solvent extraction step or steps is one or more solvents selected from the group consisting of methylene chloride, water, methanol, hexane, ethyl acetate or a solvent. mixtures thereof. A method as claimed in any one of claims 1 to 4 inclusive, comprising the step of concentrating the active agent in the extracted material by chromatographic separation. 6. A method as claimed in claim 5, wherein the chromatographic separation comprises one or more of the following: 111 more solvents selected from the group consisting of chloroform, methanol, ethyl acetate, hexane or mixtures thereof as eluant. A method as claimed in claim 5 or claim 6, characterized in that chromatographic separation is carried out on the column, collecting the eluate in the fractions from the column, evaluating the fractions to determine the activity of the appetite reducing agent, and selecting at least one fraction containing appetite reducing agent. A method as claimed in any one of the preceding claims, characterized in that the extract is processed to form a free-flowing powder. 9. An extract comprising an appetite reducing agent produced according to the process claimed in any preceding claim. A composition having an appetite reducing activity comprising the extract claimed in claim 9. The composition as claimed in claim 10, wherein the composition is admixed with a pharmaceutical excipient, diluent or carrier. Composition as claimed in claim 10 or 11, characterized in that it is prepared in unit dosage form. Use of an extract as claimed in claim 9 in the preparation of a medicament having appetite reducing activity. 14. The extract as claimed in claim 9, wherein the medicament has an appetite reducing activity. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 15. A method of reducing appetite, comprising administering to a human or animal an effective dose of a composition as claimed in claim 10, claim 11 or claim 12. A method for preparing an extract of a plant of the genus Trichocaulon or of the genus Hooclia, wherein the extract comprises a taste reducing agent. characterized in that the process comprises the steps of compressing the collected plant material to separate the juice from the solid plant material and obtain a juice not containing the solid plant material to form an extract. A method as claimed in claim 16, characterized in that the extract is dried to form a free-flowing powder. An extract comprising an appetite reducing agent, characterized in that it is prepared by a process as claimed in claim 16 or claim 17. A composition having an appetite reducing activity, comprising an extract as claimed in claim 18. A composition as claimed in claim 19, which is admixed with a pharmaceutical excipient, diluent or carrier. A composition as claimed in claim 19 or claim 20, characterized in that it is prepared in unit dosage form. Use of an extract as claimed in claim 19 in the preparation of a medicament having appetite reducing activity. • • • • 113 An extract as claimed in claim 18, characterized in that it is used as a medicament having appetite reducing activity. 24. A method of reducing appetite, comprising administering to a human or animal an effective dose of the composition as claimed in claim 19, claim 20 or claim 21. 25. An extract obtainable from a plant of the genus Trichocaulon or of the genus Hoodia, comprising an appetite reducing agent having the formula: O II About me OMe (i) An extract as claimed in claim 25, wherein the plant of the genus Trichocaulon is selected from the species Trichocaulon piliferum and Trichocaulon officinale and the plant of the genus Hoodia is selected from the species Hoodia currorii, Hoodia gordonii and Hoodia lugardii. An extract as claimed in claim 26, characterized in that all inactive impurities are removed. • • • • • 114 An extract as claimed in any one of claims 25 to 27 inclusive, characterized in that it is processed into a free-flowing powder. A composition having an appetite reducing activity comprising an extract as claimed in any one of claims 25 to 28 inclusive. A composition as claimed in claim 29, which is admixed with a pharmaceutical excipient, diluent or carrier. A composition as claimed in claim 29 or claim 30, characterized in that it is prepared in unit dosage form. Use of an extract as claimed in any one of claims 25 to 28 inclusive for the preparation of a medicament having appetite reducing activity. An extract as claimed in claims 25 to 28 inclusive for use as a medicament having appetite reducing activity. 34. A method of reducing appetite, comprising administering to a human or animal an effective dose of the composition as claimed in claim 29, claim 30 or claim 31. 35. A compound of formula 115 r ₂ (2) where R = alkyl R ₄ = H, alkyl, tigloyl, benzoyl, or another organic ester group; R ₂ = H or one or more 6-deoxy-saccharides or one or more 2,6-dideoxysacharidů, or glucose molecules, or combinations thereof; and wherein the dashed line represents optionally the presence of an additional bond between C4-C5 or C5-C6. A compound as claimed in claim 35, wherein the bond is between C 5 -C 6, R = methyl, R 1 = tigloyl, R ₂ = 3-0 - [- SD-thevetopyranosyl- (1-> 4) -β- D-cymaropyranosyl- (1-> 4) -β-D-cymaropyranosyl], wherein the compound has the general formula (1) 116 37. A compound having the general formula (3) wherein R = alkyl; and R 1 = H, alkyl, tigloyl, benzoyl or any other organic ester group. 38. A compound having the general formula (4) wherein R = alkyl; and R ₁ = H, alkyl, tigloyl, benzoyl or any other organic ester group. 39. A compound having the general formula 9 9 Φ · 9 · · · 9 9 9 Φ ΦΦ »» »· Φ · - 117 - OH (5) wherein R = alkyl; and R @ 1 = H, alkyl, tigloyl, benzoyl or any other organic ester group. 40. A compound having the general formula OMe (6) wherein R = alkyl; and R @ 1 = H, alkyl, tigloyl, benzoyl or any other organic ester group. 41. A compound having the general formula 118 (7) wherein R = alkyl; and = H, alkyl, tigloyl, benzoyl or any other organic ester group. A compound of formula (8) wherein R = alkyl R ₁ = H, alkyl, tigloyl, benzoyl, or another organic ester group; R ₂ = H or one or more 6-deoxy-saccharides or one or more 2,6-dideoxysacharidů, or glucose molecules, or combinations thereof; and wherein the dashed line means optionally the presence of another 9 · ♦ · 9 · 9 9 9 9 9 99 99 9 9999 ♦ ····· 9 9 9 9 9 9 ¢ 9 9 9 9 9 9 9 9 9 9999 99 99 119 bonds between C4-C5 or C5-C6. A compound of the formula wherein R = alkyl R-L = H, alkyl, tigloyl, benzoyl, or another organic ester group; R ₂ = H or one or more 6-deoxy-saccharides or one or more 2,6-dideoxysacharidů, or glucose molecules, or combinations thereof; and wherein the dashed line represents optionally the presence of an additional bond between C4-C5 or C5-C6. A compound of formula (10) • 4 • 4 4 4 120 wherein R = alkyl R ₄ = H, alkyl, tigloyl, benzoyl, or another organic ester group; R2 = H or one or more 6-deoxysaccharides or one or more 2,6-dideoxysaccharides, or glucose molecules or combinations thereof; and wherein the dashed line represents optionally the presence of an additional bond between C4-C5 or C5-C6. 45. A compound of formula (r) (11) wherein R = alkyl R ₄ = H, alkyl, tigloyl, benzoyl, or another organic ester group; R ₂ = H or one or more 6-deoxy-saccharides or one or more 2,6-dideoxysacharidů, or glucose molecules, or combinations thereof; and wherein the dashed line represents optionally the presence of an additional bond between C4-C5, C5-C6 or C14-C15. A compound of formula (12) wherein R = alkyl R 1 = H, alkyl, tigloyl, benzoyl, or another organic ester group; R ₂ = H or one or more 6-deoxy-saccharides or one or more 2,6-dideoxysacharidů, or glucose molecules, or combinations thereof; and wherein the dashed line represents optionally the presence of an additional bond between C4-C5, C5-C6 or C14-C15. A compound of formula (13) wherein R = alkyl R-L = H, alkyl, tigloyl, benzoyl, or another organic ester group; φ φ 122 R ₂ = Η or one or more 6-deoxy-saccharides or one or more 2,6-dideoxysacharidů, or glucose molecules, or combinations thereof; and wherein the dashed line represents optionally the presence of an additional bond between C 4 -C 5, C 5 -C 6 or C 14 -C 15; and R ₃ = H, alkyl, aryl, acyl or glukoxy. A compound of the formula wherein R = H, alkyl, aryl, or any steroid having a C 14 beta hydroxyl group, a C 12 beta hydroxyl group and a C 17 acyl group, a C 5 -C 6 olefin, or a combination thereof. 49. A process for preparing a steroid intermediate of the formula comprising the steps of (a) treating a compound. 123 • · · 9 · ♦ 999 99 99 9 · 9 9 with a reducing agent to give 3S, 12S-dihydroxy-20,20-ethylenedioxypregna-5,14-marrow of the formula OH (23) (b) treating compound 23 with N-bromoacetamide (NBA) and a base to give compound 3S, 12S-dihydroxy-14,15-epoxy-20,20-ethylenedioxypregnen-5-ene of formula (24) Λ · · · Λ · 124 (c) treating compound 24 with a reducing agent to form compound 3S, 12E, 14E-trihydroxy-20,20-ethylenedioxypregn-5-ene of formula and (d) treating compound 25 with acid and water to form compound 15. 50. A process for preparing compound 15, comprising the steps of (a) treating compound 22 with p-toluenesulfonyl chloride and a base to give compound 3E, 12E-dihydroxy-20,20-ethylenedioxy pregna-5,14-diene -3-tosyl-12-acetate of formula (b) treating compound 26 with potassium acetate in a solvent to form compound 6E, 12E-dihydroxy-20,20-ethylenedioxy. 9 9,999 · 9 9 9 9 9 9 125 9 9 9 9 9 9 9 9 9 9999 99 99 -3,5α-cyclopregnan-14-en-12-acetate of formula (27) (c) treating compound 27 with a reducing agent to give 6S, 12B-dihydroxy-20,20-ethylenedioxy-3,5α-cyclopregnan-14- ene of formula (28) (d) treating compound 28 with N-bromoacetamide and a base to give 6S, 12E-dihydroxy-20,20-ethylenedioxy-14,15-epoxy-3,5α-cyclopregnane of formula (29) OH • · • 9 126 ··· 99 9 9 9 99,999 • 9 9 9 (E) treating compound 29 with a reducing agent to give compound 6S, 12S, 14S-trihydroxy-20,20-ethylenedioxy-3,5α-cyclopregnane of formula (30); and (f) treating compound 30 with an acid and solvent to form formation of compound 15. A process for preparing a carbohydrate intermediate in the form of a monosaccharide cymarose moiety, comprising the steps of (i) treating a compound of formula (36) 127 ^ PhSSiMe, _ZnI2 and Bu4 ⁺ I to produce a compound 4-0-benzoyl-3-O-methyl-6-deoxy-D-AS-fenylthioaltrosidu formula SPh (37) (ii) optionally treating compound 37 with diethylaminosulfur trifluoride (DAST) to provide 4-O-benzoyl-3-O-methyl-2-phenylthio-2,6-dideoxy-α-D-fluorocaropyranoside having the formula or (iii) optionally treating compound 37 with tert-butyldimethyl silyl chloride and imidazole in a solvent to give 4-O-benzoyl-3-O-methyl-2-O-tert-butyldimethylsilyl-α-D-phenylthioaltroside having the formula 128 -phenylthioaltroside having the formula 9 9 9 9 9 9 9999 99 99 99 99 SPh (39) wherein Z = TBDMS = t-butyldimethylsilyl; and (iv) treating compound 39 with a base to form the 3-O-methyl-2-O-tert-butyldimethylsilyl-α-D-phenylaltroside monosaccharide having the formula SPh (40) wherein Z = TBDMS = t-butyldimethylsilyl. 52. A process for preparing a saccharide intermediate in the form of an activated thevetose moiety, comprising the steps of (i) treating the compound AND 129 • fc ·· I fcfc < • fcfc fc · «• · 4 ► ··· ·· 99 (9) fcfcfcfcfcfc (47) with phenylthiotrimethylsilane and trimethylsilyltrifluoromethanesulfonate to give 4-0-benzoyl-3-0-methyl-1 -phenylthio-6-deoxy- α -glucopyranoside having the formula (48) (ii) treating compound 48 with pivaloyl chloride and a solvent to give 4-O-benzoyl-3-O-methyl-2-O-pivaloyl- 1-phenylthio-6-deoxy-af-glucopyranoside having the formula SPh (49); and (iii) treating compound 49 with a brominating agent and diethylaminosulfur trioxide to give the monosaccharide compound 4-O-benzoyl-3-O-methyl-2-O-pivaloyl-1-fluoro-600,000. 0 0 0 130 • 0 ·· W »0 0 • 00 0 0 0 0000 0 » 0 0 0 00 00 * 0 0 0 · 0 0 0 0 0 0 · 0 0 0 0 0 0 * 00 -deoxy-S-glucopyranoside occurring having the formula as stereoisomers 53. The steroid intermediate of formula 15, which is prepared by a process as claimed in claim 49 or claim 50. 54. A carbohydrate intermediate is prepared 51. of formula 40, characterized by the process as claimed in the claim A carbohydrate intermediate of formula 50A or formula 50B, characterized in that it is prepared by a process as claimed in claim 52. 56. A method of coupling a monosaccharide cymarose to a steroid intermediate comprising the steps of (i) reacting a cymarose moiety of formula 38 with a steroid intermediate of formula 15 as claimed in claim 53 in the presence of stannous chloride in a solvent to form compound 3-O- [4-O-benzoyl-2-phenylthio-ED-cymaropyranosyl] -12,14-S-dihydroxypregnan-5-en-20-one of formula 131 BzO (51) and (ii) treating compound 51 with tiglic acid chloride in pyridine and then with base to give compound 3-0- [4-0-benzoyl-2-phenylthio-β-β-cymaropyranosyl] -12β-tigloyl- 14? -Hydroxypregnan-5-en-20-one of formula (52) A compound of formula 52, characterized in that it is prepared by a process as claimed in claim 56. 58. A method of coupling a monosaccharide cymarose moiety to a monosaccharide thevetose moiety and coupling the resulting disaccharide to a compound of Formula 52 as claimed in claim 57, comprising the steps of (i) coupling a selectively protected cymarose moiety of Formula 40 as claimed in of claim 54 and the monosaccharide thevetose moieties of formula 50A as claimed in claim 55 using stannous chloride (SnCl ₂ ) and silver trifluoromethanesulfonate to form a compound of formula OPv (53) wherein Z = TBDMS = tert-butyldimethylsilyl (ii) treating compound 53 with tetrabutylammonium fluoride to form a compound of formula (54) (iii) treating compound 54 with diethylaminosulfur trifluoride to form a compound of formula 57 of compound 55 with compound 52 as claimed to form a compound of formula OPv (56) and (v) treating compound 56 with a Raney-nickel reaction and then with a base to form compound 1 as claimed in claim 36. 59. A method for preparing a trisaccharide and coupling the resulting trisaccharide to a steroid intermediate, comprising the steps of: 134 (i) coupling a selectively protected cymarose moiety of formula 40 as claimed in claim 55 and compound 45 using stannous chloride, AgOTf, Cp ₂ ZrCl ₂ to form a compound of formula (57) SPh where Z = TBDMS = t-butyldimethylsilyl; (ii) treating compound 57 with tetrabutylammonium fluoride and diethylaminosulfur trifluoride to give a trisaccharide compound having formula (58); and (iii) coupling the trisaccharide of formula 58 with the steroid intermediate of formula. ··. . ·· .. ·················································································································· · ·· ·· Using tin (II) chloride, AgOTf, Cp ₂ ZrCl ₂ to give compound 1 as claimed in claim 36. 60. A composition having appetite reducing activity comprising a compound as claimed in any one of claims 35 to 48 inclusive. The composition as claimed in claim 60, wherein the compound is a compound of formula 1 as claimed in claim 36. A composition as claimed in claim 60 or claim 61, which is admixed with a pharmaceutical excipient, diluent or carrier. A composition as claimed in claim 60, claim 61 or claim 62, characterized in that it is prepared in unit dosage form. Use of a compound as claimed in any one of claims 35 to 48 inclusive for the preparation of a medicament having appetite reducing activity. Use as claimed in claim 64 of a compound of formula 1 as claimed in claim 36. ·· ·· .. · ♦. • · » - 136 A compound as claimed in any one of claims 35 to 48 inclusive for use as a medicament having appetite reducing activity. A compound as claimed in claim 66 which is a compound of formula 1 as claimed in claim 36. 68. A method of reducing appetite, comprising administering to a human or animal an effective dose of the composition as claimed in any one of claims 60 to 63 inclusive. 69. A food or beverage comprising an effective amount of a compound as claimed in any one of claims 35 to 48 inclusive, which has an appetite-reducing effect when ingested. 70. The food or beverage as claimed in claim 69, wherein the compound is a compound of formula 1 as claimed in claim 36. Use of a compound of formula 1 as claimed in claim 36, isolated from a plant of the genus Trichocaulon or of the genus Hoodia for the preparation of a medicament having appetite reducing activity. Use according to claim 71, characterized in that the compound is isolated from the species Trichocaulon piliferum or Trichocaulon officinale or from the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii. 73. A composition having an appetite reducing activity comprising a compound of Formula 1 isolated from the genus Trichocaulon or the genus Hoodia. Ί4. The composition as claimed in claim 73, wherein the compound is. - 137 isolate and / or purify from plants of Trichocaulon piliferum or Trichocaulon officinale and from Hoodia currorii, Hoodia gordonii or Hoodia lugardii. 75. A composition as claimed in claim 73, wherein the compound is isolated and / or purified from an extract derived from a plant of the species Trichocaulon piliferum or Trichocaulon officinale or from a plant of the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii. 76. A composition as claimed in claim 73, claim 74 or claim 75, wherein the composition is admixed with a pharmaceutical excipient, diluent or carrier. A composition as claimed in claim 76, characterized in that it is prepared in unit dosage form. A compound of formula 1 as claimed in claim 35, isolated from a plant of the genus Trichocaulon or of the genus Hoodia, for use as a medicament having appetite reducing activity. 79. A compound as claimed in claim 78, wherein the compound is isolated from a plant of the species Trichocaulon piliferum or Trichocaulon officinale or from a plant of the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii. 80. A method of reducing appetite, comprising administering to a human or animal an effective dose of the composition as claimed in any one of claims 73 to 77 inclusive. 81. A composition having an appetite reducing activity, 9 9 · 9 '9 9 99 9 9 · 99 999 999 9999 99 99 138 comprising a melanocortin 4 receptor agonist. 82. The composition as claimed in claim 81, wherein the agonist is an extract as claimed in claim 9, claim 18 or claim 25 or a compound as claimed in any one of claims 35 to 48 inclusive. 83. A composition as claimed in claim 81 or claim 82 wherein the compound is a compound of formula 1 as claimed in claim 36. 84. A composition as claimed in claim 81, claim 82 or claim 83, wherein the composition is admixed with a pharmaceutical excipient, diluent or carrier. A composition as claimed in any one of claims 81 to 84 inclusive, characterized in that it is prepared in unit dosage form. 86. The use of melanocortin 4 receptor agonists in the preparation of a medicament having appetite reducing activity. Use as claimed in claim 86 of an extract as claimed in claim 9, claim 18 or claim 25 or a compound as claimed in any one of claims 35 to 48 inclusive. Use as claimed in claim 87 of a compound of formula 1 as claimed in claim 36. 89. Melanocortin 4 receptor agonist for use as a medicament having appetite reducing activity. 90. The melanocortin 4 receptor agonist as claimed in U.S. Pat 139 of claim 89, which is an extract as claimed in claim 9, claim 18 or claim 25, or a compound as claimed in any one of claims 35 to 48 inclusive. A melanocortin 4 receptor agonist as claimed in claim 90 which is a compound of formula 1 as claimed in claim 36. 92. A method of reducing appetite, comprising administering to a human or animal an effective dose of the composition as claimed in any one of claims 81 to 85 inclusive. 93. Use of receptor agonists to reduce appetite and / or combat obesity in a human or animal. Use as claimed in claim 93 of an extract as claimed in claim 9, in claim 18 or claim 25 or a compound as claimed in any one of claims 35 to 48 inclusive. 95. The use as claimed in claim 94 of the compound of formula 1 as claimed in claim 36. 96. A compound having a structural formula