PHARMACEUTICAL COMPOSITIONS HAVING APPETITE SUPPRESSANT ACTIVITY
THIS INVENTION relates to steroidal glycosides, to compositions containing such steroidal glycosides and to a new use for these steroidal glycosides and the compositions containing them. The invention further relates to a method of extracting and isolating these steroidal glycosides from plant material, to a method of synthetically producing these steroidal glycosides, and to the products of such an extraction and such a synthesis process.
In a particular application, the invention relates to an appetite suppressant agent, to a process for synthetically producing the appetite suppressant agent, to a process for extracting the appetite suppressant agent from plant material, to an appetite suppressant composition containing the appetite suppressant agent, and to a method of suppressing an appetite.
According to the invention, there is provided a process for preparing an extract of a plant of the genus Trichocaulon or of the genus Hoodia, the extract comprising an appetite suppressant agent, the process including the steps of treating collected plant material with a solvent to extract a fraction having appetite suppressant activity, separating the extraction solution from the rest of the plant material, removing the solvent from the extraction solution and recovering the extract. The extract so recovered may be further purified, eg by way of suitable solvent extraction procedures.
The invention also provides a plant extract made of plants of the group comprising the genus Trichocaulon and the genus Hoodia and having appetite suppressant activity.
The extract may be prepared from plant material such as the stems and roots of said plants of the genus

Trichocaulon or of the genus Hoodia. The genus
Trichocaulon and the genus Hoodia include succulent plants
growing in arid regions such as are found in Southern
Africa. In one application of the invention, the active
appetite suppressant extract is obtained from the species
Trichocaulon piliferum. The species Trichocaulon
officinale may ajso be used to provide an active appetite suppressant extract. In another application of the invention, the active appetite suppressant extract may be obtained from the species Hoodia currorii, Hoodia gordonii or Hoodia lugardii- Bioassays conducted by the Applicant on rats have indicated that certain of the extracts possess appetite suppressant activity.
The plant material may be homogenised in the presence of a suitable solvent, for example, a methanol/methylene chloride solvent, by means of a device such as a Waring blender. The extraction solution may then be separated from the residual plant material by an appropriate separation procedure such as, for example, filtration or centrifugation. The solvent may be removed by means of the rotary evaporator preferably in a water bath at a temperature of 60°C. The separated crude extract may then be further extracted with methylene chloride and water before being separated into a methylene chloride extract and a water extract. The methylene chloride extract may have the solvent removed preferably by means of evaporation on a rotary evaporator and the resultant extract may be further purified by way of a methanol/hexane extraction. The methanol/hejcane extraction product may then be separated to yield a methanol extract and a hexane extract. The methanol extact may be evaporated to remove the solvent in ordei to yield a partially purified active extract.
The partially purified active extract may be dissolved in methanol, and may be further fractionated by column chromatography, employing silica gel as an adsorption

medium and a chloroform/30% methanol mixture as an eluent. A plurality of different fractions may be obtained, and each may be evaluated, by suitable bioassaying procedures, to determine the appetite suppressant activity thereof.
A fraction having appetite suppressant activity may preferably be further fractionated such as by column chromatography using silica gel as an adsorption medium and a 9:1 chloroform:methanol solvent, and the resultant sub-fractions bioassayed for their appetite suppressant activity. A sub-fraction displaying appetite suppressant activity may, if desired, be further fractionated and purified, conveniently using a column chromatographic procedure with silica gel as the adsorption medium and a 9:1 ethylacetate:hexane solvent. The resultant purified fractions may again be evaluated by suitable bioassay procedures for their appetite suppressant activity.
The Applicant has found that at least one such purified fraction has good appetite suppressant activity, and the active principle in the fraction was identified by conventional chemical techniques including nuclear magnetic resonance, and was found to be a compound of the structural


In accordance with S.I. nomenclature, the active principle (1) is the compound 3-0-[-/3-D-thevetopyranosyl-(14) -/3-D-cymaropyranosyl- (l-»4) -(3-D-cymaropyranosyl] -12/3-0-tigloyloxy-14-hydroxy-14/3-pregn-50-en-20-one (C47H74O15 M"*"878) .
According to another aspect of the invention, there is provided a process for preparing an extract of a plant of the genus Trichocaulon or of the genus Hoodia, the extract comprising an appetite suppressant agent, the process including the steps of pressing collected plant material to separate sap from solid plant material and recovering the sap free of the solid plant material to form the extract.
The extract may be dried to remove moisture, e.g. by spray-drying, freeze-drying or vacuum drying, to form a free-flowing powder.
The Invention extends to a composition having appetite suppressant activity comprising an extract as described above.
The composition may be admixed with a pharmaceutical excipient, diluent or carrier and optionally it is prepared in unit dosage form.
The invention also extends to the use of an extract as described above in the manufacture of a medicament having appetite suppressant activity, to an extract as described above for use as a medicament having appetite suppressant activity, and to a method of suppressing an appetite by administering to a human or animal an effective dosage of a composition as described above.
Compound (1) is a novel compound and the invention extends to compound (1) and certain analogues or derivatives of this steroidal trisaccharide having appetite suppressant properties. The molecules chosen as the

analogues or derivatives are intended to affect the properties of the steroidal trisaccharide with the aim of increasing the activity of the active ingredient, The following effects were taken into consideration when the analogues were chosen:
(i) Hydrophobic interactions and lipophilicity
Functional group modifications of the active molecule is intended to change the hydrophobicity and lipophilicity of the molecule- Increased lipophilicity has been shown to correlate with increased biological activity, poorer aqueous solubility, increased detergency/cell lysis, increased storage in tissues, more rapid metabolism and elimination, increased plasma protein binding and faster rate of onset of action.
(ii) Electronic properties and ionization constants
Functional group modification of the molecule is also intended to change the acidity and basicity which would have a major role in controlling the transport of the compound to its site of action and the binding at this target site.
(iii) Hydrogen bonding
Functional group modifications of carboxyl and carbonyl groups in the active molecule are intended to change the interactions between the proteins in biological systems and the chemically modified functional groups,
(iv) Steric parameters
The purpose of changing the steric features of the molecule is to increase biiiding to its receptor and

thus increase its biological activity.
The following chemical modifications to the molecule are intended to affect the hydrophobicity and lipophilicity electronic properties, hydrogen bonding and steric parameters on the molecule:
a) Chemical modification of the C-12 group and ester
functionality;
b) Chemical modification of the 5,6-double bond, e.g.
hydrogenation and migration;
c) Chemical modification of the C-20 carbonyl and C-17
acetyl group;
d) Chemical modification of the "D" ring of the steroid
or aglycone ring;
e) Modification of the carbohydrates of the trisaccharide
moiety.
Accordingly, the invention provides a compound having the general structural formula

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

The invention.also provides a compound as described above wherein there is a further bond between C5 - C6, R = methyl, R = tigloyl, R2 = 3-0-[-;3-D-thevetopyranosyl-(l-»4}-i3-D-cymaropyranosyl- (l->4) -;S-D-cymaropyranosyl] and having the structural formula.

molecules, or combinations thereof; and in which the broken lines indicate the presence of a further bond between C4 - C5 or C5 - C6.

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


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

in which R = alkyl; and
R- = H, alkyl, tigloyl, benzoyl, any other
organic ester group;
R2 = H, or one or more 6-deoxy carbohydrates, or
one or more 2,6-dideoxy carbohydrates, or glucose
molecules, or combinations thereof;
and in which the broken lines indicate the
optional presence of a further bond between C4 -
C5, C5 - C6 or C14 - Cl5.


organic ester group;
R2 = H, or one or more 6-deoxy carbohydrates, or
one or more 2,6~dideoxy carbohydrates, or glucose
molecules, or combinations thereof;
and in which the broken lines indicate the
optional presence of a further bond between
C4 - C5, C5 - C6 or C14 - C15; and
R3 = H, alkyl, aryl, acyl, or glucoxy.

C14 beta hydroxy group, or a C12 beta hydroxy functionality, or a C17 acyl group, or a C5 - C6 olefin, or combinations thereof.
The invention still further extends to a process for synthetically producing a compound having appetite suppressant activity.

and (d) treating compound (25) with an acid, e.g. acetic acid, and water to produce the steroid intermediate compound 3E, 12E, 14E-trihydroxy-pregn-5-ene (15) .
Reaction Scheme A depicts the procedure for the preparation of steroid intermediate (15} from compound (22) according to "the first alternative procedure" of the invention (and includes the preparation of compound (22) from compound (16) for illustrative purposes).

In a second alternative procedure, a process for the preparation of steroid (15) according to the invention includes the steps of
(a) treating compound (22) (3/3, 12|3-dihydroxy-20,20-
ethylenedioxypregna-5,14-diene-12-acetate) with p-
toluenesulfonyl chloride and a base, e.g. pyridine, to
produce a compound 20, 12/3-dihydroxy-20,20-
ethylenedioxypregna-5,14-diene-3 -tosyl-12-acetate of the formula

Reaction Scheme B shows the procedure for the preparation of steroid intermediate {15) from compound (22) according to "the second alternative procedure" of the invention.

compound (1) may be synthesized from a first carbohydrate intermediate in the form of an activated monosaccharide cymarose moiety, which can be prepajred from a compound having the formula (36). Compound (36) can be prepared by a process which includes the steps

(ii) treating the compound (32) with tosyl chloride and pyridine at OC, to produce a compound methyl-4,6-0-benzylidene-2-0-tosyl-Q'-D-glucopyranoside of the formula


(iii) treating the compound (33} with NaOMe at lOO''C to produce a compound methyl 4,6-0-benzylidene-3-0-methyl-a-D-altropyranoside of the formula

(iv) treating the compound (34) with N-bromosuccinamide (NBS} to produce a compound methyl 6-bromo-4 -0 -benzoyl-3-0-methyl-6-deoxy-a'D-altropyranoside of the formula

and (v) treating the compound (35) with NaBH and NiCl2, to produce a compound methyl 4-0-benzoyl-3-0-methyl-6-deoxy-Q!-D-altropyranoside of the formula


The invention extends to a process for the preparation of a carbohydrate intermediate in the form of an activated monosaccharide cyraarose moiety which includes the steps of
(i) treating the compound {36) with PhSSiMe3, Znl2 and Bu4'''I' to produce a compound 4-0-benzoyl-3-0-methyl-6-deoxy-aS-D-phenylthioaltroside of the formula

(ii) optionally treating the compound (37) with diethylaminosulphur trifluoride (DAST), e.g. at 0°C, to produce a compound 4-0-benzoyl-3-0-raethyl-2-phenylthio-2,6-dideoxy-aE-D-fluorocymaropyranoside having the formula


or (iii) optionally, treating the compound (37) with t-butyldimethylsilylchloride and imidazole in a solvent, e.g. pyridine, to produce 4-0-benzoyl-3-0-methyl-2-0-t-butyldimethylsilyl-a)3-D-phenylthioaltroside having the formula

and (iv) treating the compound (39) with a base, e.g. sodium methoxide, to produce 3-O-methyl-2-O-t-but yldime t hyl silyl-Q!/3-D-phenyl thioalt reside having the formula

Reaction Scheme C shows the procedure for the synthesis of the activated monosaccharide cymarose moiety (40) from compound (36) according to the invention (and includes the preparation of compound (35) from compound (31) for illustrative purposes).

The synthesis of compound (1) may also involve a second carbohydrate intermediate in the form of an activated monosaccharide thevetose moiety, which can be prepared from a compound having the formula (47). Compound (47) can be prepared by a process which includes the steps of

Cii) treating the compound (42) with NaH and Mel to produce a compound 1,2 : 5,6-Di-0-isopropylidene-3-0-methyl-a-D-glucofuranose of the formula

(v) treating the compound (45) with benzaldehyde and zinc chloride to produce a compound methyl 4,6-0-benzylidene-3-0-methyl-o;E-glucopyranoside having the formula

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


(ii) treating the compound (48) with pivaloyl chlotide and a solvent, e.g. pyridine, to produce a compound 4-0-benzoyl-3 - 0-methyl-2 - 0-pivaloyl-1-phenylthio-6-deoxy-a(9-glucopyranoside having the formula

and (iii) treating the compound (49) with a brominating agent, e.g. N-bromosuccinimide, and diethylaminosulphur trifluoride to produce a compound 4 - 0-benzoyl-3 - 0-methyl-2 - 0-pivaloyl-1-f luoro-6-deoxy-j3-glucopyranoside occurring as stereo-isomers having the formula


Reaction Scheme D shows the procedure for the synthesis of the activated monosaccharide thevetose moiety (50(A) and 50(B)) from compound (48) according to the invention (and includes the preparation of compound (47) from compound (41) for illustrative purposes).

According to a still further aspect of the invention
there is provided a process of synthetically producing a
compound of the formula (1) and analogues and derivatives
thereof which includes the steps of synthesising a suitable
steroid intermediate or precursor and coupling the required ;
number of suitable monosaccharides with the steroid intermediate.
The invention also provides a process of coupling a monosaccharide cymarose with the steroid intermediate, which includes the steps of
(i) reacting a cymarose moiety (38) with a steroid intermediate (15), e.g. at -ISC, and in the presence of tin chloride, in a solvent, e.g. ether, to produce a compound 3-0- [4 -0-benzoyl-2-phenylthio-£-D-cymaropyranosyl]-12,14-fi-dihydroxy-pregn-5-ene-20-one of the formula

and (ii) treating the compound (51) with tiglic acid chloride in pyridine and thereafter with a base, e.g. NaOMe, to produce a compound 3-0-[-2-phenylthio-S-D-cymaropyranosyl] -12fi-tigloyloxy-14-hydroxy-14fi-pregn-5-ene-20-one of the formula


The invention extends to a process which includes coupling a monosaccharide cymarose moiety to a monosaccharide thevetose moiety and coupling the resultant disaccharide with the combined steroid product (52) to form compound (1).
The process of coupling the monosaccharide cymarose moiety to the monosaccharide thevetose moiety and coupling the resultant disaccharide to the combined steroid product (52) may include the steps of
(i) coupling a selectively protected cymarose moiety (40) and a selectively protected thevetose moiety {50 A) using tin chloride (SnCl2) and silver trifluoromethanesulphonate, e.g. at -15°C, to produce a compound of the formula

and (v) treating compound (56) in a Raney-Nickel reaction and thereafter with a base, e.g. NaOMe, to produce compound (1) as described above.
Reaction Scheme E shows the procedure for the synthesis of intermediates (52) and (55) and coupling them to form compound (56).

According to the invention, an alternative process is provided which includes coupling cymarose and thevetose moieties to form a trisaccharide and coupling the trisaccharide onto a steroid derivative to form a compound of the formula (1).
The process of forming the trisaccharide and coupling the resultant trisaccharide to a steroid derivative may include the steps of
(i) coupling a selectively protected cymarose moiety (40) and compound (45} using tin (II) chloride, AgOTf, Cp2ZrCl2 to produce a compound of the formula

(ii) treating compound (57) with tetrabutylamraoniumfluoride and diethylaminosulphur trifluoride to produce a trisaccharide compound having the formula


using tin (II) chloride, AgOTf, Cp2ZrCl2 to produce compound (l).
The steroid intermediate (59) may be produced by treating steroid (15) with tiglic acid chloride.
Reaction Scheme F shows the procedure for the synthesis of the trisaccharide (58) and the synthesis of compound (1) by coupling the trisaccharide (53) with the steroid intermediate (59).

The intermediates (23), (24), (25), (27) , (28), (29), (30), (37), (381, (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- [-/3-D-thevetopyranosyl- (l-*4) -(3-D-cymaropyranosyl - (l-»4 ) -/3-D - cymaropyranosyl ] -12(3-0-t igloyloxy-14-hydroxy-14/3-pregn-5-en-20-one, and various analogues and derivatives thereof have been found to have appetite suppressing activity.
The invention extends also Co a composition or formulation having appetite suppressant activity, in which the active ingredient is an extract obtained from a plant of the genus Trichocauion or the genus Hoodia..
The active ingredient may be a compound of the 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 the species Hoodia currorii, Hoodia gordonii or Hoodia Ingardii.
The invention extends also to a composition or formulation having appetite suppressant activity, in which the active ingredient is a synthetically produced compound of the formula (1) or a derivative or analogue thereof, as hereinbefore set out with reference to compounds (2) to (14) .
According to another aspect of the invention there is provided a method of suppressing an appetite by administering to a human or animal a suitable dosage of an appetite suppressant agent comprising an extract of a plant of the genus Trichocaulon or Hoodia. The extract may be incorporated in a composition or formulation including also pharmaceutically acceptable other-ingredients.


The appetite suppressant composition or formulation may consist of the appetite suppressant agent admixed with a pharmaceutical excipient, diluent or carrier. Other suitable additives, including a stabilizer and such other ingredients as may be desired may be added.
The invention extends to the use of compound {1) or its derivatives or analogues in the manufacture of a medicament having appetite suppressant activity.
The invention further extends to compound (1), or its derivatives or analogues as set out before, for use as a medicament having appetite suppressant activity.
A method of suppressing an appetite by administering to a human or animal an effective dosage of a composition as described above is also provided.
A method has been described herein for extracting a steroidal glycoside having appetite suppressant activity from plant material obtained ' ■from a plant of the

Trichocaulon or Hoodia genus. The invention thus extends to an extract obtained from plant material of the Trichocaulon or Hoodia genus and containing a substantially pure steroidal glycoside of formula (1).
The invention extends also to a foodstuff or a beverage containing an effective quantity of the steroidal glycoside of the formula (1), or its derivatives or analogues as set out before, to have an appetite suppressant effect when ingested.
Molecular genetic studies have led to a considerable increase in the understanding of the regulation of appetite, satiety and bodyweight. These studies have revealed numerous central regulatory pathways, mediated by a number of neuropeptides. The maintenance of a normal body weight is achieved by an intricate balance between energy intake, food consumption, and energy expenditure. Energy homeostasis is subject to a wide range of influences, ultimately controlled by the brain. The different signals include such things as sense of smell and taste and gastro-intestinal signals such as distension of the gastro-intestinal tract, chemical signals to the gastric mucosa and blood-borne metabolites such as fatty acids and glucose.
Centrally, neuropeptide "Y" (NPY) which is negatively regulated by leptin, has -been established as one of the positive regulators of feeding behaviour. Expression of the endogenous antagonist for melanocortin receptors has also been shown to be the basis for obesity in a particular model (the ob/ob mouse) . Indeed deficiency at the MC4 melanocortin receptor completely replicates the obesity syndrome. Other mediators which have been shown to have roles in the energy balance include bombesin, galonin and glucagon-like peptide-1.

without being bound by theory, the Applicant believes that compound (1) and its analogues as described above act as an agonist of the raelanocortin 4 receptor. The effect of this is to regulate NPY but also to increase cholecystokinin. The effect of cholecystokinin amongst other things is to inhibit gastric emptying.
Accordingly, the invention extends to a composition having appetite suppressant activity comprising a raelanocortin 4 receptor agonist.
The agonist may be an extract or compound as previously described, in particular the compound of formula (1). The composition may be admixed with a pharmaceutical excipient, diluent or carrier and is optionally prepared in unit dosage form.
The invention still further extends to the use of a raelanocortin 4 receptor agonist in the manufacture of a raedicament having appetite suppressant activity, to a raelanocortin 4 receptor agonist for use as a medicament having appetite suppressant activity, to a method of suppressing an appetite by administering to a human or animal an effective dosage of a composition comprising a raelanocortin 4 agonist as described above, and to the use of a melanocortin 4 receptor agonist to suppress the appetite of and/or to combat obesity in a human or animal.
The invention and its efficacy will now be further described, without limitation of the scope of the invention, with reference to the following examples and drawings.
In the drawings. Figure 1 shows a flow diagram of the general method of extracting a first crude appetite suppressant extract and a_purified appetite suppressant extract from plant material of the genus Trichocaulon or Hoodia;

Figure 2 shows a graphical representation of a bioassay carried out on rats using a partially purified methanol extract of Trichocaulon piliferum;
Figures 3 and 4 together show a schematic representation of a preferred embodiment of the process of the invention for producing an extract of plant material of the genus Trichocaulon or Uoodia.; and
Figures 5 and 6 show a graphical representation of the percentage change of body mass of rats for different groups for days -7 to 7 and days 0 to 7 respectively in a repeat dose study using a sap extract and a spray-dried sap extract of plant material of the species Hoodia. gordonii.
EXAMPLE 1
The general method of extracting a first crude appetite suppressant extract and a purified appetite suppressant extract from plant material of the genus Trichocaulon or of the genus Hoodia is illustrated by way of the flow diagram of Figure 1.
EXAMPLE 2
Bioassays carried out on rats using a partially purified methanol extract obtained in the manner illustrated in Example 1, indicated that the extract does in fact exhibit appetite suppressant activity. The appetite suppressant activity of the active extract can be illustrated by way of a typical example of the effect of the methanol extract of Trichocaulon piliferum on rats, by way of the graphic representation in Figure 2.
It will be evident from Figure 2 that the test group of rats dosed with the extract on day 5 displayed a substantially diminished food intake over the next two days, while a control group did "riot disclose a comparable

reduced food intake. The food intake of the test group returned to normal, and in fact increased, from day 8 onwards.
EXAMPLE 3
A preferred embodiment of a process in accordance with the invention for producing an extract having appetite suppressant activity is illustrated schematically by way of example in Figures 3 and 4, which two Figures together illustrate the comprehensive process. However, various other procedures may be used, as will be understood by persons skilled in the art.
Referring to Figure 3, plant material of the genus Trichocaulon or the genus Hoodia. is fed into a blender 3, eg a Waring blender, by way of feedline 1, with a solvent in the form of a methylene chloride/methanol solution introduced via feedline 2. The homogenised product is fed via line 4 into a separation stage 5, eg in the form of a filter or centrifuge, and the residual plant material is removed via line 27.
The solvent/extract mixture is fed via line 6 into an evaporation stage 7, where the solvent is removed, for example by means of a rotor evaporator. The dried crude extract is fed via line 8 into a further extraction stage 9 with the addition of a methylene chloride/water solution introduced via feedline 29 for further extraction, and then to a separation stage 13 by way of line 11, where the water fraction is removed via line 31. The dissolved extract fraction is fed via line 15 into a drier stage 17 where the solvent is evaporated, for example by a rotor evaporator.
Referring to Figure 4, the dried extract is fed via line 10 into an extraction stage 12. A methanol/hexane solution is also fed via line 14 into the extraction stage 12 for further purification and"'extraction of the dried

extract. The extract/methanol/hexane mixture is fed via line 16 into a separation stage 18, the hexane fraction is removed via line 20, and the methanol/extract mixture is then fed via line 22 into a drying stage 24. In the drying stage 24, the solvent is removed, eg by evaporation on a rotor evaporator.
The dried, partially purified active extract is fed via line 26 and with the addition of methanol via line 28 into a solution stage 30, and the dissolved fraction is fed via line 36 to a chromatography column 38.
In the column 3 8 the methanol soluble fraction is further fractionated, using silica gel and a chloroform/30% methanol solvent, into different fractions schematically indicated as fractions I to V. According to an actual fractionation procedure carried out by the Applicant, the fractionation procedure yielded the following fraction weights : 1(3.9 g) ; II{2.6 g) ; 111(2.1 g) ; IV(1.1 g) and V(2.0 g) . These fractions are individually evaluated by a suitable bioassaying procedure (in a step not shown) and those fractions identified as fractions I and II, displaying marked appetite suppressant activity, are fed by feedlines 40 and 42 into columns 44 and 4 6 respectively where they are further fractionated and purified by column chromatography, again by using silica gel and a 9:1 chloroform:methanol system.
The sub-fractions 11(A) - (C) obtained from column 44 do not, when assayed, display a noteworthy appetite suppressant activity, and may be recycled for further chromatography.
The sub-fractions I(A) - (L) obtained from column 46 are also evaluated (by an assaying step not shown), and the sub-fraction 1(C) is found to have marked appetite suppressant activity.

t— The sub-fraction I(C) is fed via line 48 into column 50 for a further fractionation and purification, using silica gel and a 9:1 ethyl acetate:hexane eluent. Of the resultant purified fractions, fraction 1(C)(ii) is found, after assaying, to possess marked appetite suppressant activity.
The purified product is identified by nuclear magnetic resonance spectroscopy (as indicated in Tables 1 and 2 below}, to be compound (1).

Examples 4 to 13 illustrate the synthetic procedures whereby the intermediate compounds and steroid (15) may be prepared according to "the first alternative procedure".
EXAMPLE 4
12fi, 15a-Dihydroxv progesterone (17)
Cultures of Calonectria decora {ATCC 14767) are prepared by the inoculation of a culture medium comprised of sucrose (900 g) , K2HPO4 (30 g) , Czapek concentrate (300 ml), corn steep liquor (300 ml) and distilled water {30 I) {150 X 500 ml flasks). After 5 days of shaking at 26°C, progesterone (16) {150 g) in a suspension of Tween 80 {0,1 % soln., 1,5 i) is added to the flasks. The cultures are incubated for a further 5 days and then worked-up by centrifugation, decantation, extraction of the medium with chloroform, and then evaporation to yield the dihydroxy progesterone (17) (75 g, 45 %).
=H NMR (CDCI3) : 5,71 (IH, s, H-4); 4,12-4,22 (IH, m, H-15)
4,43 (IH, br, s, OH); 3,46-3,53 (IH, dd, J = 4,6Hz, H-12); 2,16 Hz {3H, s, H-21); 1,18 (3H, s, H-19); 0,74 {3H, s, H-18)
EXAMPLE 5
12E-Hydroxv-15Q'- (p-toluene sulfonyl) -progesterone (18)
The dihydroxy progesterone (17) (75 g, 0.22 mol) is dissolved in dry pyridine (300 ml) and cooled to 0°C, p-Toluene sulfonyl chloride (46 g, 0,24 mol) in dry pyridine (200 ml) is added dropwise to the reaction mixture at 0°C. The reaction is stirred overnight at 0 °c, and quenched by the addition of H2O (500 ml). The water layer is extracted with ethyl acetate (1 £) , and the organic extract washed with hydrochloric acid (6M, 3 X 1 O , aqueous saturated sodium bicarbonate (500 ml), aqueous saturated sodium

chloride {500 ml), and water {500 ml) , The organic layer is dried (MgS04), filtered and evaporated to yield p-toluene sulfonated progesterone (18) (98 g, 92 %) as a viscous dark yellow oil.
H NMR (CDCI3) : 7,7 (2H, d, J = 14Hz, H-2,6); 7,34
(2H, d, J = 8,4Hz, H-3,5); 5,67 (IH, s, H-4); 4,86-4,93 (IH, m, H-15); 3,45-3,50 (IH, dd, J = 4,6H2, H-12); 2,44 (3H, s, H-4Me); 2,15 {3H, s, H-21} 1,13 {3H, s, H-19); 0,74 (3H, s, H-18).
EXAMPLE 6
12E-HydrQxy-A-'--progesterone (19)
A solution of the tosylated progesterone (18) (98 g, 0,19 mol) in 2,4, 6-trimethyl collidine (500 ml) is refluxed at 150°C for 3 h. The reaction mixture is cooled and poured into water {500 ml). The water layer is extracted with ethyl acetate (1 i) , after which the organic layer is washed with hydrochloric acid (6M, 3X1?}, aqueous saturated sodium bicarbonate (500 ml) , aqueous saturated sodium chloride (500 ml}, and water {500 ml). After drying (MgSO4) and filtering, the ethyl acetate is evaporated and the crude mixture is purified by silica gel chromatography, eluting with acetone:
chloroform (1:10) to afford A-progesterone (19) {50 g, 78 %) as a dark red oil.
H NMR (CDCI3): 5,73 (IH, s, H-4), 5,28 (IH, dd, J=2,2Hz, H-15}, 4,41 (IH, br, s, OH), 3,49-3,52 {IH, dd, J = 4,3Hz, H-12}, 2,80-2,84 (iH, dd, J = 9,2Hz, H-17}, 2,14 {3H, s, H-21), 1,19 (3H, s, H-19), 0.89 (3H, s, H-18).

EXAMPLE 7
3 .12]5-DiacetoxYPreqna-3 . 5.14-trien-2Q-one (20)
A solution of A-"--progesterone (19) (50 g, 0,15 mol) in acetyl chloride (1,5 I) and acetic anhydride (750 ml) is refluxed for 2 hours. The reaction mixture is poured into cold ethyl acetate (1 i) and aqueous saturated sodium bicarbonate is added with stirring until the effervescence ceases. The ethyl acetate layer is separated from the sodium bicarbonate layer and washed with further portions of aqueous sodium bicarbonate (3 X 700 ml} , thereafter with aqueous saturated sodium chloride (700 ml) and finally with water (700 ml) . The organic layer is dried (MgS04) , filtered and evaporated to afford the 3,12E-diacetoxypregna-3.5,14-trien-20-one (20} (60 g, 93 %) as an orange oil.
H NMR(CDCl3) : 5,68 (IH, s, H-4}, 5,44 (IH, m, H-6), 5,31 (IH, dd, J = 2,2Hz, H-15), 4,82-4,86 {IH, dd, J = 4,5Hz, H-12), 3,10-3,18 {IH, t, J = 9,5Hz, H-17), 2,18 (3H, s, 3-Ac), 2,11 (3H, S, 12'Ac}, 2,08 (3H, S, H-21), 1,02 {3H, s, H-19), 1,01 (3H, s, H-18)
EXAMPLE 8
3 . 12i5-DiacetDXV-2 0 , 2 0-ethylenedioxvpreqna-3 . 5 , 14-triene {21}
The diacetoxy compound {20} (60 g, 0,14 mol) is dissolved in benzene (1 I) and ethylene glycol (60 ml} and p-toluene sulfonic acid (1 g) are added. (The benzene is previously refluxed with a Dean-Stark trap). The mixture is refluxed with stirring and azeotropic removal of water for 16 hours. Aqueous saturated sodium bicarbonate solution (500 ml) is added to the cooled solution. This is then washed with brine (500 ml), and with water (500 ml), and dried (MgSO4). The solvent is evaporated and the crude mixture purified by silica gel' column chromatography.

/
eluting with ethyl acetate: hexane (2:8) to yield the ethylenedioxypregna-3,5,14-triene (21) (35 g, 53 %).
H NMR (CDCI3): 5,68 (IH, s, H-4), 5,45 (IH, m, H-6), 5,31 (IH, dd, J = 2,2Hz, H-15), 4,73-4,85 (IH, dd, J = 4,4Hz, H-12), 3,78-3,98 (4H, iti, ethylenedioxy) , 2,16 (3H, s, 3-Ac) , 2,04 (3H, s, 12-Ac) , 1,29 (3H, s, H-21) , 1,12 (3H, s, H-19), 1,02 (3H, s, H-18).
EXAMPLE 9
3fi-12E-Dihydroxy-2 0 . 2 0-ethvlenedioxvpreQna-5 ,14-diene-12-acetate (22)
The dienolacetate (21) (35g, 0,077 mol) is suspended in ethanol (500 ml) and sodium borohydride (2, 8g, 0.074 mol) is added at 0°C. The mixture is allowed to warm to room temperature and stirred overnight. Most of the solvent is removed in vacuo and the mixture is diluted with water (500 ml) and extracted with ethyl acetate (500 ml). Work-up followed by chromatography on silica gel with acetone/chloroform (1:10) yields the 3iS-alcohol (22) (25 g, 80 %) .
H NMR (CDCI3): 5,41 (IH, m, H-6), 5,28 (IH, dd, J= 2,2H2, H-15), 4,72-4,81 (IH, dd, J = 4,4HZ, H-12), 3,82-4,02 (4H, m, ethylene dioxy}, 3,45-3,59 (IH, 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
3fi. 12fi-Dihvdroxy-20,2 0-ethylenedioxypreqn-5,14-diene (23)
The 3E-alcohol (22) (25 g, 50.2 mmol) in dry tetrahydrofuran (300 ml) is added dropwise to a suspension of lithium aluminium hydride (2,7 g, 72,2 mmol) in dry tetrahydrofuran (500 ml). The reaction mixture is stirred

at room temperature for 24 hours after which water (2,7 ml) is carefully added and stirred for a further 10 min. Sodium hydroxide (15 % soln, 2,7 ml) is then added and the suspension stirred. After 10 min, water (8,1 ml) is added and the suspension stirred for 10 minutes, filtered, dried (MgSO4) , and the solvent evaporated to afford the 3E, 1215 dihydroxypregna-diene (23) (20 g, 90 %).
% NMR (CDCI3): 5,36 (IH, m, H-6), 5,23 (IH, dd, J = 2,2H2, H-15), 3,94-4,06 (4H, m, ethylene dioxy), 3,41-3,52 (IH, m, H-3), 3,32-3,36 (IH, dd, J = 4,3Hz, H-12), 1,31 (3H, s, H) 1,01 (3H, s, H-19), 0,96 (3H, s, H-18). C NMR (CDCI3): 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 (ethylene dioxy), 58,8 (C-17), 19,0 (C-19), 11,9 (C-18).
3E. 12£-Dihvdroxv-14.15-epoxy-20.20-ethylenedioxvpreqn-5-
ene ,-
3£.12fi-Dihydroxy-5 . 6-epoxy-20 , 20-ethylenedioxyprecfn-14-ene
N-Bromoacetamide (211 mg, 1,5 mmol) is added to a stirred solution of the 5,14-diene (23) (500 mg, 1,34 mmol) in acetone (100 ml), acetic acid (2,5 ml), and water (5 ml) at 0°C. After 15 min sodium sulphite (5 % soln, 50 ml) is added to the reaction mixture. The acetone is evaporated, and the aqueous layer extracted with dichloromethane (3 X 50 ml) . The organic layer is dried (MgSO4) , filtered and evaporated. Pyridine {1 ml) is added to the product, and stirred for 0,5 h. Dichloromethane (100 ml) is then added to the reaction mixture, and the dichloromethane is washed with citric acid (5 % soln, 3 X 100 ml), saturated sodium bicarbonate (50 ml), and water (50 ml). The organic layer is dried (MgSO4) , filtered and evaporated to give the mixture of 14,15- and 5,6-epoxides (360 mg, 69%) as a white foam. The mixture of epoxides could not be separated by silica gel column chromatography.
EXAMPLE 11
3fi. l2fi-Dihvdroxv-14 ,15~e-Doxy-2Q , 20-ethylenedioxyprecrn-5-ene (24)
The mixture of 14,15- and 5,6- epoxides (14,4 g, 37,0 mmol) in dry tetrahydrof uran {200 ml) is added to a suspension of lithium aluminium hydride (1,69 g, 44,4 mmol) in dry tetrahydrofuran (300 ml). The reaction mixture is stirred at room temperature for 24 hours, after which it is worked up as described earlier by the addition of water (1,69 ml), and sodium hydroxide (15 % soln, 1,69 ml). After filtration and evaporation of the solvent, the crude product is purified by silica gel column chromatography using methanol/chlorof orm (1:9) as solvent to give the unreacted 14,15 epoxy- 20,20-ethylenedioxypregn-5-ene (24} (300 mg, 2,1 %).
% NMR {CDCI3): 5,31 (IH, m, H-6), 3,82-3,98 (4H, m,
ethylene dioxy), 3,43-3,52 (IH, m, H-3), 3,41 (IH, s, H-
15), 3,31-3,35 {IH, dd, J=4,3 Hz, H'12} , 1,29 (3H, s, H-
21), 1,17 (3H, s, H-19), 1,02 (3H, s, H-18}.
C NMR (CDCI3) : 139,8 {C-5}, 120,8 (C-6), 112,1 CC-2Q), 77.2 CC-12), 75,4 (C-14), 61,0 (C-15}, 22,3 (C-21), 19,2 (C-19}, 9,5 (C-18).
EXAMPLE 12
3fi. 12s. I4fi-Trihydroxv-20.20-ethvlenedioxypreqn-5-ene {25)
The 14, 15-epoxide (24) (300 mg, 0,77 mmol) in dry tetrahydrofuran (10 ml) is added to a suspension of lithium aluminium hydride (300 mg, 7,89 ravaol) in tetrahydrofuran and the reaction refluxed for 48 h. After the addition of water (0,3 ml), sodium hydroxide (15 % soln, 0,3 ml) and filtration as described earlier, the mixture is purified by silica gel column chromatography using methanol: chloroform

(1:9) as solvent to give the trihydroxy pregnene (25) (250 mg, 83 %) .
H NMR (CDCI3) -.5,38 (IH, m, H-6), 3,98 (4H, m, ethylene dioxy),
3,43-3,53 {IH, m, H-3), 3,25-3,32 ClH, dd, J = 4,lHz, H-12), 1,32 (3H, s, H-21), 1,01 (3H, s, H-19), 0,98 (3H, s, H-18}
C NMR CDCI3): 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-ia)
EXAMPLE 13 3E, 12E. 14fi-Trihydroxy-preqn-5-ene (15)
The ethylenedioxypregnene {25) (250 mg, 0,64 mmol) is dissolved in acetic acid (13,4 ml) and water which after freeze drying affords the trihydroxy steroid (15) (200 mg, 89 %} , m.p.: 22S°-235''C (lit 225'=-235'*C) , M+ 348, [a] + 35° (lit laj]° + 29°) .
H NMR {CDCI3): 5,39 (IH, m, H-6), 3,56-3,62 {IH,
t, J = 8,1 Hz, H-17), 3,42-3,51
(IH, m, H-3), 3,28-3,39 (IH, dd, J = 4,3Hz, H-12), 2,23 (3H, s, H-21) , 1,01 (3H, s, H-19) , 0,90
{3H, s, H-18)
C NMR (CDCI3) : 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-15), 32,9 {C-21), 31,5 CC-16) , 30,1 {C-2) , 27,4 {C-7} , 2-4,4 (C-ll), 19,4 (C-19),

8,3 (C-18}.
Examples 14 to 19 illustrate the synthetic procedures whereby the intermediate compounds and steroid (15) may be prepared according to "the second alternative procedure".
EXAMPLE 14
20, 2O-Ethylenedioxy-30-toluene-p-sulphonyloxy-pregn-5,14-diene-120-ol acetate (26) - A solution of p-toluenesulphonyl chloride (650 mg, 3.4 mmol) in pyridine
(10 ml) was added dropwise to a mixture of the 20,20-Ethylenedioxypregna-5,14-diene-3(3,12j3-diol 12-acetate (22)
(1.3 g, 3.1 mmol) in pyridine (15 ml) at 0°C. The reaction mixture was left stirring at room temperature for 24 hours after which water was added to the reaction mixture. The solution was extracted with ethyl acetate {2x50 ml) , the ethyl acetate layer was washed citric acid (5x50 ml) , saturated sodium bicarbonate solution (lOO ml) , saturated sodium chloride solution (100 ml) and water (100 ml). The ethyl acetate was dried (MgSO) , filtered, and evaporated and purified by flash column chromatography using hexane-ethyl acetate (8:2 v/v) as the eluant to give the (3-0-tosyl steroid (26), (1.5 g, 84%), as a yellow oil, {Found M 570.271, C32H42O7S requires: W 570.273).
6jj 1.021 {3H, s, 19-H) , 1.131 {3H, s, 18-H), 1.282 (3H, s, 21-H), 2.021 (acetateOCHj) , 2.431 (3H, s, Ar-CH3) , 3.883 {4H, m, OCH2CH2O) , 4.750 (IH, dd, J 10.8 Hz, 5.2 Hz, 12-H), 4.890 (IH, m, 30H), 5.281 {IH, dd, J 4.2 Hz, 2.1 Hz, 15-H), 5.388 (IH, m, 6-H), 7.341 {2H, d, 3 J 8.2 HZ, ArH), 7.746 {2H, d, J 8.2 Hz, ArH).
6 13.493Q (C-18), 19.002Q (C-19), 21.612Q (Ar-methyl)*, 21.671Q (C-21)*, 24.175Q (acetate methyl), 63.4 01T {ethylenedioxy), 63.498T (ethylenedioxy), 71.531S (C-13), 80.912D {C-12), 82.531D (C-3), 111.353S (C-20), 120.881D {C-15), 121.461D {C-6), 123.715-

133.917 (Aromatic), 13 9,9033 (C-14), 151,7223 (C-5), 170.819S (ester carbonyl). may be interchanged
EXAMPLE 15
20, 2 0-Ethylenedioxy-3a, 5-cyclo-5a--pregn-14-ene-6S, 12S-diol-12-acetate (27} - A solution of 3S-toluene-p-sulphonyloxy-pregn-5,14-diene (26) (1.2 g, 2.1 ramol) and potassium acetate (2.2 g, 22.4 ramol) in water (250 ml) and acetone (500 ml) was refluxed at 60 °C for 16 hours. The acetone was evaporated and the water was extracted with ethyl acetate (2 00 ml) . The ethyl acetate was dried (MgSO) , filtered, and evaporated. Flash chromatographic separation of the mixture using chloroform-acetone (9:1 v/v) as the eluant gave the 3Q;,5-cyclo derivative (27) , (530 mg, 61%) as a yellow oil, (Found M 416.262, 'zszes requires : W 416.263).
5H 0-288 (IH, dd, J" 8.1 Hz, 4.9 Hz, 4-Hg) , 0.477 (IH, dd, J 4.4: Hz, 4.4 Hz, 4-Hb), 1.025 (3H, s, 19-H), 1.121 (3H, s, 18-H), 1.256 {3H, s, 21-H), 1.989 {3H, s, acetate-CH3) , 3.302 (IH, dd, J 2.8 Hz 2.8 Hz, 6-H) , 3.784-3.947 (4H, m, OCH2CH2O) , 4.721 (IH, dd, j 8.5 Hz, 5.6 Hz, 12-H), 5.232 (IH, dd, J 3.9 Hz, 1.9 Hz, IS-H).
5 11.678T(C-4) , 12.298Q(C-18) , 19.971Q (C-19}, 23.623QCC-21), 24.153Q (acetate methyl), 63.700T (ethylenedioxy), 63.788T (ethylenedioxy), 73.591D (C-6), 8 0.551D (C-12), 111.1263 (C-20), 118.778D (C-15), 152.959S (C-14), 170.991S (ester carbonyl).
EXAMPLE 16
20,20-Ethylenedioxy~3a,5-cyclo-5a-pregn-14-ene-6B,12S-diol
(28) - A solution of the 30,5-07010 derivative (27), (500
mg, 1.2 ramol) in tetrahydrofuran (2Q ml) was added dropwise

to a suspension of lithium aluminium hydride (50 mg, 1.3 mmol) in tetrahydrofuran (10 ml) . The reaction mixture was stirred for 4 hours and quenched by the addition of water (50 1) . After 30 minutes, sodium hydroxide was added (15% solution, 50 1) and stirring continued for a further 30 minutes. Water (150 1 was added and the reaction mixture was filtered. The tetrahydrofuran was dried (MgSO} filtered and evaporated and flash chromatographic purification using chloroform-acetone [8:2 v/v) as the eluant to give the did {28), (370 mg, 83%) as an oil, (Found M 374.250, C23H34O4 requires: M 374.252)
Sjj 0.2 98 (IH, dd, J 8.1 Hz, 4 .9 Hs, i-H) , 0.510 (IH, dd, J 4.4 Hz, 4.4 Hz,4-Hb), 0.985 (3H, s, 19~H) , 1.055 (3H, s, 18-H), 1.325 (3H, s, 21-H), 3-318 (IH. dd, J- 3.0 Hz, 3.0 Hz, 6-H) , ) , 3 .363 (IH, dd, 1/11.4 Hz, 4.2 Hz, 12-H), 4.019 (4H, m, OCH2Ch20) 4.622 (IH, s, OK) , 5.2 55 (IH, dd, j- 3 .9 HZ , 1.9 Hz, 15-H) .
5 11.681T(C-4}, 12.243Q(C-1B), 19.844Q (C-19), 23.604Q(C-21), 63.620T (ethylenedioxy), 63.733T (ethylenedioxy), 73.569D (C-e), 77.478D (C'12), 111.125S (C-20), 118.702D {C-15), 152.912S (C-14).
EXAMPLE 17
20,20-Ethylenedioxy-14,15S-epoxy-3a , 5-cyclo-5a,142-pregnane-6S,l2E-diol (2 9) - K-bromoacetamide (150 rag, 1.1 mmol) was added to a solution of the 20,20-ethylenedioxy-3o;,5-cyclo-5o'-pregn-14-ene-6fi, 12E-diol (28) (340 mg, 0.91 mmol) in acetone (20 ml), water (0.25 ml) and acetic acid
(0.25 ml) at 0°C. After 15 min. , sodium sulphite (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 (3x30 ml) . The dichloromethane layer was dried (MgS04) , filtered and evaporated to a concentrated volume (50 ml). Pyridine
(0.5 ml) was added to the mixture "and stirred for a further

1 hour after which the dichloromethane layer was washed with a citric acid solution (5%, 3x30 ml) , saturated sodium bicarbonate solution (3 0 ml) and water (30 ml) . The dichloromethane layer was dried (MgSO)), filtered and evaporated and purified by flash column chromatography using chloroform-methanol (9.5:0.5 v/v) as the eluant to give the epoxide (29) (180 mg, 51% as a foam, (Found M 390.245, C23H34O2 requires: M 390.247).
6jj 0.287 (IH, dd, J" 8.1 Hz, 4 .9 Hz, 4-Ha) . 0.501 (IH, dd, J 4.4 Hz, 4.4 H2,4-H53), 0.978 (3H, s, 19-H) , 1.04 8 (3H, s, 18-H), 1.321 (3H, s, 21-H), 3.318 (IH, dd, J 3.1 Hz, 3.1 Hz, 6-H}, ), 3.355 (IH, dd, j 11.2Hz, 4.1Hz, 12'H), 3.491 (IH, s, 15-H), 4.001 (4H, ra, 0CH2Ch20) , 4.901 (IH, s, OH).
5 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-6S, 12B, 14 - trihydroxy-3a, 5-cyclo-5a, 14B-pregnane (30) - A solution of the epoxide (29) {170 mg, 0.44 mmol) in tetrahydrofuran (10 ml) was added to a suspension of lithium aluminium hydride (20 mg, 0.53 mmol) in tetrahydrofuran (5 ml) . The reaction mixture was refluxed for 2 hours after which water (20 1) was added and stirring continued for 05 hour. Sodium hydroxide solution (15%, 201) was added and stirring continued for a further 0.5 hour. A further quantity of water was added (60 jil) and the suspension was stirred for 1 hour. After filtration, the suspension was dried (MgS04) filtered, and the tetrahydrofuran was evaporated. Flash chromatographic separation of the resulting mixture eluting with chloroform-methanol (9:1 v/v) gave" the required triol (30),

(90 mg, 53%) as a clear oil, (Found M 392.261, C23H33O5 requires: M 392.263}.
6jj 0.287 (IH, dd, J" 8.1 Hz, 4.9 Hz, 4-H2) , 0.510 (IH, dd, 3 J 4.4 Hz, 4.4 Hz,4-Hi,), 0.971 (3H, s, 19-H) , 1.042 (3H, s, 18-H), 1.319 (3H, s, 21-H), 3.321 (IH, dd, J 3.0 Hz, 3.0 Hz, e-H), 3.321 (IH, dd, J 11.1 Hz, 3.9 Hz, 12-H), 3 .561 (IH, s, OH) , 4.084 (4h, m, OCH2Ch20) 4.671 {IH, s, OH).
5 11.668T(C-4), 11.971Q{C-ia), 19.511Q (C-19), 23.520Q
(C-21}, 63.612T (ethylenedioxy), 63.711T
(ethylenedioxy), 73.483D (C-6), 76.051D (C-12), 84.307S {C-14), 111.099S (C-20}.
EXAMPLE 19
3E,12B,14-Trihydroxy-14S-pregn-5-en-20-one (15) - A mixture of the triol (30) (80 mg, 0.20 mmol) in acetone (20 ml) and hydrochloric acid (IM, 10 ml) was refluxed at 60°C for 2 hours. The reaction mixture was cooled and saturated sodium bicarbonate solution (20 ml) was added. The acetone was evaporated and the aqueous layer extracted with chloroform (3 x 20 ml) , the chloroform layer was dried (MgS04), filtered and evaporated to give the epimeric trihydroxy steroids (15a, 15b) (42 mg, 61%) . Separation of the epimeric mixture (15a, 15b) (15 mg) was achieved by flash chromatographic separation using chloroform : methanol (9:1 v/v) as the eluant to give the pure 1715-epimer (15a), (10 mg), m.p. 224-229°C (acetone), (lit. 226-223°) , (Found M 348.234, C; 72.32, H 9.21% C21H32O4 requires: C, 72.38; H 9.26%, M 348.236), and the 17a-epimer (15B) (3 mg), m.p. 183-191°C (acetone), (lit 184-196°) .
3E, 12IS,14-Trihydroxy-14B-pregn-5-en-20-one (15a) : 60.963 (IH, s, 19-H), 1.192 (3H, s, 18-H), 2.236 (3H, s 21-H), 3.325 (IH, dd, J-11.2 Hz, 3.9 Hz, 12-H),

3.464 (IH, S, OH), 3.5140 (IH, m, 3-H}, 3.598 (IH, dd, J 9.6 Hz, 9.6 Hz, 17-H), 4,255 (IH, s, OH), 5.383 (IH, m, 5-H) .
5 8.275Q (C-18), 19.414Q (C-19}, 24.400T (C-11) 24.581T (C-16), 27.443T (C-7), 30.062T (C-2), 32.972Q (C-21), 34.543T {C-15), 35.864D {C-8), 36.975S (C-IO), 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}, 138.9323 (C-5), 217.011S (C-20).
3S,12S,14-Trihydroxy-14B-pregn-5-en-20-one (15b): 5jj 0.996 (IH, s, 19-H) , 1.144 (3H, s, 18-H) , 2.221 (3H, S 21-H) , 3.339 (IH, dd, J 9.4 Hz, 9.4 Hz, 17-H) , 3.492 (IH, m. 3-H) , 3.629 (IH, dd, J 11.1 Hz, 3.9 Hz, 12-H), 3.712 (IH, s, OH), 4.325 (IH, s, OH), 5.383 (IH, m, 5-H).
Examples 20 to 28 illustrate the procedures whereby the intermediate compounds may be prepared to form the first monosaccharide (40).
EXAMPLE 20
Methyl-4,6-0-benzylidene-a-D-qlucopvranoside (32)
A mixture of methyl-a-D-glucopyranoside (30 g, 0,15 mol} , benzaldehyde (70 ml) and zinc chloride (20 g) is stirred at room temperature for 24 hours. The reaction product is poured into ice water and stirring continued for 15 min. The white precipitate is filtered and washed with diethyl ether. The solid material is stirred with a solution of sodium metabisulphite (10 % soln), for IB min, filtered and washed with water. The solid material is crystallized from chloroform and ether to yield the benzylidene product (32) (31 g, 72 %) .

EXAMPLE 21 Methyl-4.6-Q-benzvlidene-2-0-tosyl-a-D-qlucopyranoside(33 )
p-Toluene sulfonyl chloride (25 g, 1,2 eq) in pyridine (100 ml) is added dropwise to a solution of the benzylidene glucose (32) (31 g, 0.12 mol) in pyridine (100 ml) at 0°C. The reaction is stirred at room temperature for 48 hours. Ice is added to the reaction mixture. The resulting white solid material is washed with water and recrystallized from hot ethanol to yield the tosylated glucose (33) (28 g, 60
EXAMPLE 22
Me thy 1-4 . 6-0 -benzylidene-S-O-methyl-a-D-altropyranoside (34)
The tosylate (33) (28 g, 64 mmol) in a solution of sodium (7 g) in methanol (150 ml) is heated at 110°C for 48 hour in an autoclave. The reaction vessel is cooled and solid carbon dioxide is added to the reaction mixture. After filtration, the methanol is evaporated and the solid material is then taken up in water. The aqueous layer is extracted with chloroform (X 3). The chloroform is dried (MgSO} , filtered and evaporated. The crude mixture is purified by silica gel column chromatography eluting with chloroform : acetone (9:1) to yield the altroside (34) (10 g, 52 %).
EXAMPLE 23
Methyl-6-bromo-4-0-benzoyl-3-0-methyl-6-deoxv-o-D-altropyranoside (35)
The benzylidene altroside (34) (10 g, 33 mmol) is added to a solution of N-bromosuccinimide (7.6 g) and barium carbonate (2 0 g) in carbon tetrachloride and the reaction

mixture is refluxed at 75°C for 3 hours. The reaction mixture is filtered and the carbon tetrachloride layer is washed with water. The organic layer is dried (MgS04), filtered and evaporated to yield 6-bromo-altroside (35) , (9 g, 69 %)-
EXAMPLE 24
Methyl - 4 - 0 -benzoyl -3 - 0 -methyl - 6 -deoxy-a-D-altropyranoside (36)
Sodium borohydride (18 g) in water (30 ml) is added dropwise to a solution of the bromoaltreside (35) (9 g, 23 ramol) and nickel chloride (18 g) in ethanol (300 ml} at 0°C. The reaction mixture is refluxed at 7S°C for 1 hour and then it is filtered. The ethanol is evaporated and the remaining aqueous layer is extracted with chloroform (X 3) . The chloroform is dried (MgSO) , filtered and evaporated, to yield the 6-deoxy-altroside (36) (5 g, 72 %).
EXAMPLE 25
4-0-Benzoyl-3-0-methvl-6-deoxy-aE-D-phenylthioaltropyranoside (37)
Phenylthiotrimethylsilane (5 ml) and trimethylsilyltrifluoromethane sulphonate (2 ml} are added at 0°C to a solution of the 6-deoxy-altroside (36) (5 g, 17 mmol) in dichloromethane (200 ml) . The reaction mixture is stirred at room temperature for 6 hours. Saturated sodium bicarbonate is added to the reaction mixture. The dichloromethane layer is dried {MgS04), filtered and evaporated. The crude mixture is purified by silica gel column chromatography eluting with chloroform : acetone (9:1) to yield the aE-phenylChioaltroside (37) (4 g, 63 %) .

EXAMPLE 7 6
4-Q-Benzovl-3-0-methvl~2-phenylthio-2.6-dideoxY-gfi-D-f luorocymaropyranoside (3 8}
Diethylaminosulphurtrifluoride {0,65 g) is added rapidly to a solution of the aE-phenylthioaltroside (37) (0,5 g, 1,33 mmol) in dichloromethane at 0°C. The reaction is stirred for 0,5 h at 0°C and then saturated sodium bicarbonate is added. The dichloromethane is separated from the aqueous layer, dried (MgSO), filtered and evaporated to yield the aS-fluorocymarose (38) (450 mg, 90 %) .
EXAMPLE 22
4-Q-Benzoyl-3-0-methvl-2-0-t-butYldimethylsilvl-gg-D~ phenvlthio-altreside (3 9)
The 6-deoxy altroside (37) (5 g) is silylated using t-butyldimethylsilylchloride (3 g) and imidazole (3 g) in pyridine (50 ml). The reaction is worked-up by extracting with ethyl acetate, washing the ethyl acetate with hydrochloric acid (6 N), then with sodium bicarbonate, and finally with water. The ethyl acetate layer is dried (MgSO), filtered and evaporated to yield the silylated benzoyl phenylthioaltroside (39) (80 %).
EXAMPLE 2 8
3 -Q-methvl -2- Q-t-butvldimethylsilyl-Qn3-D-phenvlthioaltroside (4 0)
The silylated benzoyl phenylthioaltroside (39) (6 g) is treated with sodium methoxide (100 ml) for 4 hours. The methanol is evaporated and water is added to the reaction. The water layer is acidified (pH 5, ACOH) and extracted with ethyl acetate. The ethyl"acetate is washed with

water, dried (MgSO) , filtered and evaporated to yield silylated methyl phenylthioaltroside (40} (75%).
Examples 29 to 37 illustrate the procedures synthetic whereby the intermediate compounds may be prepared to form the second monosaccharide (50).
EXAMPLE 2 9
1,2 : 5.g-Di-Q-isopropylidene-g-D-qlucofuranose (42}
Sulfuric acid (40 ml) is added dropwise to a solution of a-D-glucose (41) (50 g, 0,28 mol) in acetone (1 £) at 0°C. The reaction mixture is stirred for 24 h and then it is neutralized using sodium hydroxide (6 M) . The acetone is evaporated and the aqueous layer is extracted with chloroform {X2). The chloroform is dried (MgSO) filtered and evaporated. Crystallization from cyclohexane yielded the di-isopropylidene glucose (42) (41 g, 57 %).
EXAMPLE 3 0
1,2 : 5 . 6-Di-0"isopropvlidene-3-0-methvl-a-D-qIuco£uran-ose (43)
The a-D-glucofuranose (42} (41 g, 0,16 mol) in tetrahydrofuran (300 ml) is added dropwise to a suspension of sodium hydride (5 g) in tetrahydrofuran (200 ml) . After 0,5 h, methyl iodide (25 g) in tetrahydrofuran (100 ml) is added dropwise to the reaction mixture which is then stirred for 24 h. Water is added to the reaction mixture which is then extracted with ether (X 3). The ether layer is dried (MgSO), filtered and evaporated to yield the methyl protected glucose (43) (38 g, 83 %).

EXAMPLE 31
3 - Q -Methyl "Q!fi"D"