EP2235036A1

EP2235036A1 - Saccharide lupane derivatives, their use and pharmaceutical compositions containing these derivatives

Info

Publication number: EP2235036A1
Application number: EP09706942A
Authority: EP
Inventors: Zbigniew Andrzej Pakulski; Piotr Cmoch; Jana Oklestkova; Miroslav Strnad
Original assignee: Bio Apex Sro; Universita Palackeho v Olomouci; Palacky University Olomouc; Institute Of Organic Chemistry Polish Academy Of Sciences
Current assignee: Bio Apex Sro; Universita Palackeho v Olomouci; Palacky University Olomouc; Institute Of Organic Chemistry Polish Academy Of Sciences
Priority date: 2008-01-30
Filing date: 2009-01-28
Publication date: 2010-10-06
Also published as: WO2009094958A1; CZ200851A3; ZA201004771B

Abstract

This invention relates to saccharide lupane derivatives of general formula (I), wherein R denotes substituent independently selected from the group comprising hydrogen, hydroxy, amino, mercapto, alkyloxy, alkyl and saccharide group, R´ denotes substituent independently selected from the group comprising hydrogen, hydroxy, alkyl, carboxyl, acyl and saccharide group, wherein at least one of R and R´ contains a saccharide group. A further object relates to these compounds for use as medicaments and growth regulators. This invention further includes pharmaceutical compositions containing said derivatives.

Description

Saccharide lupane derivatives, their use and pharmaceutical compositions containing these derivatives

Field of invention

This invention relates to saccharide lupane derivatives and their use for inhibition of hyperproliferation in mammalian cells and for treating proliferative diseases in mammals, particularly in anticancer therapy. This invention also relates to preparations containing these derivatives.

Background Art

Saponins are a large family of steroid or triterpenoid glycosides, widely occurring in plants and in some marine organisms, in which hydrophilic mono- or oligo-saccharides are attached to a hydrophobic sapogenin backbone (Hostettmann et al., Saponins, Cambridge University Press, 1995). They have received considerable attention because of their diverse, promising biological and pharmaceutical properties, including antitumour, antiviral, antifungal, antiinflamatory and other activities (Ikeda et al., Biol. Pharm. Bull., 23, 2000, 363-364; Takechi et al., Phytochemistry, 30, 1991, 3943-3944; Mimaki et al., Phytochemistry, 38, 1995, 1279-1286). They have recently also been shown to have significant effects on plant growth (Ohara et al., J. Wood Sd., 49, 2003, 59-64).

Lupeol [3β-lup-20(29)-ene] is found in many plant species (Mutai et al., Phytochemistry, 65, 2004, 1159-1164; Krasutsky, Nat. prod. Rep., 23, 2006, 919- 942), betulin (lup-20(29)-ene-3β,28-diol) is a highly abundant component of birch bark, and betulinic acid [3β-hydroxy-lup-20(29)-ene-28-oic acid], which has very interesting and promising biological properties (Sami et al., Eur. J. ^"Pharm. ScL, 29, 2006, 1-13; Dzubak et al., Nat. Prod. Rep., 23, 2006, 394-411), can also be isolated from various plants (Baglin et al., Mini Rev. Med. Chem., 3, 2003, 525-539) or easily prepared from betulin (Kim et al., Synth. Commun., 27, 1997, 1607-1612). Natural saponins based on lupeol (Minocha, Phytochemistry, 20, 1981, 135-137) and betulinic acid (Tapondjou et al., Phytochemistry, 67, 2006, 2126-2132) are rarely found in nature, and reports on their synthesis are also sporadic (Ma et al., Tetrahedron Lett., 45, 2004, 3261-3263; Gauthier et al., Bioorg. Med. Chem., 14, 2006, 6713-6725). Various aspects of the chemistry and biological activity of betulin and its derivatives are discussed in various papers (e.g., Pakulski, Polish J. Chem., 79, 2005, 361-367).

Promisingly for their potential clinical use, these triterpenoids have shown no haemolytic activity at high concentrations (100 mmol/1), and very weak activity even at extremely high concentrations (500 mmol/1) (Yamashita et al., Clinica Chim. Acta, 325, 2002, 91-96). For instance, EC₅₀ values (50% effective concentration) < 6.6xlO^"4 μM and a remarkably high therapeutic index (TI), exceeding 20 000, have been obtained for some derivatives of betulin and betulinic acid, compared to 1.5 μM and 12 000, respectively, for azidothymidine (AZT), and furthermore, some derivatives of betulin and betulinic acid have shown high anti-HIV activity (Sun et al., J. Med. Chem., 41, 1998, 4648^1-657). Some derivatives have also shown a significant cytotoxicity and anti-tumour properties, their concentration leading to 50% inhibition of viability (IC₅o) was approx. 20-80 μM. Although betulin and lupeol themselves are usually inactive, betulinic acid was found to be selectively cytotoxic against several cancer cell lines (Sami et al., Eur. J. Pharm. Sci., 29, 2006, 1-13).

Recently we were interested in the preparation of saponins from several lupane-type triterpenes (lupeol, betulinic acid and betulin). Attaching sugar moieties to sapogenins as a hydrophilic transport-facilitating functional group could both improve their ability to enter the target cells via interactions with mannose receptors and increase their solubility, thus providing a convenient drug delivery strategy. Additionally, due to the presence of the sugar fragment and potential affinity to the dendritic cells, these derivatives may be considered as components of carbohydrate- based anticancer drugs. Hence, they can be used as antimitotic and apoptotic drugs, particularly as anticancer drugs. It is an object of this invention to provide anticancer compounds having high selectivity and therapeutic index, i.e. that they are less toxic and yet more efficacious than their non-saccharide analogues known heretofore.

Disclosure of the Invention

The object of this invention are saccharide lupane derivatives of the general formula I

I and the pharmaceutically acceptable salts thereof with alkali metals, ammonium or amines, in the form of racemates or optically active isomers, as well as their addition salts with acids, wherein

R denotes substituent independently selected from the group comprising hydrogen, hydroxy, amino, mercapto, alkyloxy, alkyl and saccharide group, R' denotes substituent independently selected from the group comprising hydrogen, hydroxy, alkyl, carboxyl, acyl and saccharide group, wherein at least one of R and R' contains saccharide group.

As used herein, the generic groups have the meaning definined herein below: amino denotes the group -NH₂, azido denotes the group -N₃, hydroxy denotes the group -OH, carboxyl denotes the group -COOH, mercapto denotes the group -SH, alkyl denotes branched or unbranched alkyl chain containing 1 to 6 carbon atoms, which is optionally substituted with 1 to 5 substituents selected from the group containing hydroxyl, alkyloxy, amino, carboxyl and acyl group, aryl denotes the aromatic carbocyclic group containing 6 to 18 carbon atoms, which is formed by at least one aromatic ring or multiple condensed rings, from which at least one ring is aromatic, which is optionally substituted with 1 to 7 substituents selected independently from the group containing alkyl, hydroxy, alkyloxy, amino, carboxyl, and acyl group, arylalkyl denotes the group -Rl-Ar, wherein Ar refers to aryl group and Rl is branched or unbranched alkylene chain containing 1 to 6 carbon atoms, the aryl group being optionally substituted with 1 to 5 substituents selected independently from the group containing alkyl, hydroxy, alkyloxy, amino, carboxyl, and acyl group, acyl denotes the group -C(O)R, wherein R is alkyl, aryl, arylalkyl, alkyloxy or a saccharide, alkyloxy denotes the group -OR, wherein R is alkyl, aryl, or arylalkyl, alkylamino denotes the group -NR"R"', wherein R" can be hydrogen, alkyl or aryl, and R" ' can be alkyl or aryl, saccharide group denotes monosaccharides, disaccharides, or trisaccharides selected from the group comprising glycosyl or glycoside derivatives occuring in both enantiomeric forms, which may be either the D-form (e.g. D-glucose or D- galactose) or the L-form (e.g. L-rhamnose or L-fucose), their anomeric linkages can be selected from α or β linkages and the saccharide group can be optionally substituted with azido, amino, alkylamino, acyl, aryl or arylalkyl groups.

In a preferred embodiment the invention relates to saccharide lupane derivatives of the general formula I and the pharmaceutically acceptable salts thereof with alkali metals, ammonium or amines, in the form of racemates or optically active isomers, as well as their addition salts with acids, wherein the saccharide group is monosaccharide, disaccharide, or trisaccharide, wherein the carbohydrate group is glycosyl or glycoside occuring in both enantiomeric forms, which may be either the D-form (e.g. D-glucose or D-galactose) or the L-form (e.g. L-rhamnose or L-fucose), their anomeric linkage is selected from α or β, and the saccharide group is selected from glucosyl, glucoside, mannosyl, mannoside, galactosyl, galactoside, fucosyl, fucoside, rhamnosyl, rhamnoside, idosyl, idoside or their combinations, which can be optionally substituted independently with azide, amino, alkylamino, acyl, aryl or arylalkyl group.

In another preferred embodiment, the compounds of the invention are monosaccharide lupane derivatives of the general formula I wherein at least one of R or R' groups contain at least one glycosyl or glycoside group selected from the group comprising glucosyl, glucoside, mannosyl, mannoside, galactosyl, galactoside, fucosyl, fucoside, rhamnosyl, rhamnoside, idosyl, idoside or their corresponding amino, acyl, or benzyl substituted mono-, di- and trisaccharide derivatives and the salts thereof with alkali metals, ammonium or amines, in the forms of racemates or optically active isomers, as well as their addition salts with acids.

In yet another embodiment, the compounds of the invention are the heterosaccharide lupane derivatives of the general formula I wherein at least one of R or R' contain at least one glycosyl or glycoside group selected from the group comprising glucosyl, glucoside, mannosyl, mannoside, galactosyl, galactoside, fucosyl, fucoside, rhamnosyl, rhamnoside, idosyl, idoside, which can be optionally substituted with one or two glycosyl or glycoside group selected from glucosyl, glucoside, mannosyl, mannoside, galactosyl, galactoside, fucosyl, fucoside, rhamnosyl, rhamnoside, idosyl, idoside or their corresponding amino, acyl, or benzyl derivatives and the salts thereof with alkali metals, ammonium or amines, in the forms of racemates or optically active isomers, as well as their addition salts with acids.

In the preferred embodiment, the saccharide lupane derivatives of the general formula I are: 3β-O-(α-D-mannopyranosyl)-lup-20(29)-ene, 3β-O(α-D- mannopyranosyl)-(l— >3)-[(α-D-mannopyranosyl)-(l— >6)]-α-D-mannopyranosyl-lup- 20(29)-ene, l-O-[3-β-acetoxy-lup-20(29)-ene-28-oyl]-α-D-mannopyranosyl, l-Ο-[3- β-acetoxy-lup-20(29)-en-28-oyl]-(α-D-mannopyranosyl)-(l— >3)-[(α-D- mannopyranosyl)-(l→6)]-α-D-mannopyranosyl, 3β-O-acetyl-28-O-(α-D- mannopyranosyl)-lup-20(29)-ene, 3 β-O-acetyl-28-O-(α-D-mannopyranosyl)-(l ->3)- [(α-D-mannopyranosyl)-(l→6)]-α-D-mamopyranosyl-lup-20(29)-ene, 28-O-acetyl- 3 β-O-(α-D-mannopyranosyl)-lup-20(29)-ene, 28-O-acetyl-3 β-O-(α-D- mannopyranosyl)-(l→-3)-[(α-D-mannopyranosyl)-(l— >6)]-α-D-mannopyranosyl-lup- 20(29)-ene (12), 3β-O-(β-D-glucopyranosyl)-lup-20(29)-ene, 3β-Ο-(α-D- mannopyranosyl)-(l— )-3)-[(α-D-mannopyranosyl)-(l→-6)]-β-D-glucopyranosyl-lup- 20(29)-ene, l-O-[3-β-acetoxy-lup-20(29)-en-28-oyl]-(α-D-mannoρyranosyl)-(l→3)- [(α-D-mannopyranosyl)-(l->6)]-β-D-glucopyranosyl, 3β-O-acetyl-28-O-(β-D- glucopyranosyl)-lup-20(29)-ene, 3 β-O-acetyl-28-O-(α-D-mannopyranosyl)-(l ->3)- [(α-D-mannopyranosyl)-(l ->6)]-β-D-glucopyranosyl-lup-20(29)-ene, 28-O-acetyl-3 β- O-(β-D-glucopyranosyl)-lup-20(29)-ene, 28-O-acetyl-3β-O-(α-D-mannopyranosyl)- (l->3)-[(α-D-mannopyranosyl)-(l→-6)]-β-D-glucopyranosyl-lup-20(29)-ene (12), 3β- O-(β-D-galactopyranosyl)-lup-20(29)-ene, 3 β-O-(α-D-mannopyranosyl)-(l ->3)-[(α- D-mannopyranosyl)-( 1 ->6)] -β-D-galactopyranosyl-lup-20(29)-ene, 1 -O- [3 - β-acetoxy- lup-20(29)-ene-28-oyl]-β-D-galactopyranosyl l-O-[3-β-acetoxy-lup-20(29)-en-28-oyl]-(α-D-mannopyranosyl)-(l→3)-[(α-D- mannopyranosyl)-(l-^6)]-β-D-galactopyranosyl, 3β-O-acetyl-28-O-(β-D- galactopyranosyl)-lup-20(29)-ene, 3 β-O-acetyl-28-O-(α-D-mannopyranosyl)-(l →3)- [(α-D-mannopyranosyl)-(l →6)]-β-D-galactopyranosyl-lup-20(29)-ene, 28-O-acetyl- 3β-O-(β-D-galactopyranosyl)-lup-20(29)-ene, 28-O-acetyl-3β-O-(α-D- mannopyranosyl)-(l-»3)-[(α-D-mannopyranosyl)-(l— >6)]-β-D-galactopyranosyl-lup- 20(29)-ene (12), 3β-0-(L-rhamnoρyranosyl)-lup-20(29)-ene, l-O-[3-β-acetoxy-lup- 20(29)-ene-28-oyl]-L-rhamnopyranosyl, 3β-O-acetyl-28-O-(L-rhamnopyranosyl)-lup- 20(29)-ene, 28-O-acetyl-3β-O-(L-rhamnopyranosyl)-lup-20(29)-ene, 3β,28-di-O-(α- D-mannopyranosyl)-lup-20(29)-ene, 3β,28-di-O-(α-D-mannopyranosyl)-lup-20(29)- ene.

A further object of this invention are the compounds of the invention for use as medicaments.

The next object of this invention is to provide a method for inhibiting cell proliferation and inducing apoptosis by means of a compound of general formula I.

Another object of this invention are the saccharide lupane derivatives of general formula I for use for inhibiting cell proliferation and inducing apoptosis.

This invention further comprises a method for inhibiting cell proliferation in mammals comprising administering to a mammal in need of such treatment an effective amount of the saccharide lupane derivative of general formula I.

Yet another object of this invention are the saccharide lupane derivatives of general formula I for use in the treatment of hyperproliferative diseases. Thus, the present invention also provides a method for treating hyperproliferative diseases in mammals, said method comprising an application of an effective amount of the saccharide lupane derivative of general formula I to a mammal in need of such treatment.

In another embodiment, this invention is a method for inhibiting cell proliferation and for inducing apoptosis in mammals, comprising administration of a therapeutically effective amount of the saccharide lupane derivative of general formula I to a mammal in need of such treatment.

Saccharide lupane derivatives are useful for treating disorders, some of them involving cell proliferation, such as cancer, Alzheimer disease, Huntington disease, steroid-induced osteonecrosis, sexual differentiation disorders, hyperadrenocorticism associated with sex steroid excess, androgen insensitivity syndrome, glucocorticoid insensitive asthma, steroid-induced cataracta, and deficiency of P450 oxidoreductase, osteoporosis, cholesterol metabolism defects.

A further object of this invention are the saccharide lupane derivatives of general formula I for use as growth factors, preferably in animal and human tissue cultures for regulation of proliferation and morphogenesis.

Another object of this invention is a pharmaceutical composition, which comprises at least one saccharide lupane derivative of general formula I and a pharmaceutically acceptable carrier.

In yet another embodiment, this invention relates to the pharmaceutical composition further comprising one or more pharmaceutical excipients.

The invention relates also to the pharmaceutical composition further comprising commonly used cytostatics, such as mitoxantrone, cis-platinum, methotrexate, taxol, or doxorubicin.

The saccharide lupane derivatives of this invention may be used in compositions containing the form of free compounds of the above given general formulae I or as pharmaceutically acceptable salts thereof. The pharmaceutically acceptable salts may be formed with, for example, alkali metals, ammonium, or amines. They may also be in the form of addition salts with acid. The derivatives or their salts may be in the form of a racemic mixture or optically active isomers.

PROCESSES FOR PREPARATION

Saccharide lupane derivatives of the general formula I can be conveniently prepared by various methods used in general carbohydrate synthesis including particularly glycoside bond formation methodologies (e.g. Toshima, K.; Tatsuta, K. Chem. Rev. 1993, 93, 1503-1531; Nicolaou, K. C, Mitchell, H. J. Angew. Chem. Int. Ed. Engl. 2001, 40, 1576-1624) as well as by using "unprotected acceptor" methodologies (e.g. Furneaux, R. H.; Pakulski, Z.; Tyler, P. C, Can, J. Chem. 2002, 80, 964-972).

According to the invention there is further provided a process for the preparation of compound of formula (I),

I wherein R and R' have the above defined meanings.

For example, the compound of the formula I wherein R = OAc and R' = mannosyl may be prepared by the treatment of 3-O-acetyl-lupeol with 2,3,4,6-tetra-O- benzoyl-α-D-mannopyranosyl trichloroacetimide or 2,3,4,6-tetra-O-acetyl-α-D- mannopyranosyl trichloroacetimide in the presence of trimethylsilyl trifluoromethanesulphonate (TMSOTf) and further debenzoylation with potassium carbonate in methanol.

Another method of preparation according to the invention consists in that the compound of the formula I wherein R = OAc and R' = (mannopyranosyl)-(l— »3)- [(mannopyranosyl)-(l→6)]-mannopyranosyl may be prepared by treatment of the above-mentioned product with 2 equivalents of 2,3,4,6-tetra-O-benzoyl-α-D- mannopyranosyl trichloroacetimide and further deprotection.

In yet another method of preparation according to the invention, the compound of the formula I wherein R = OAc and R' = mannosyl may also be prepared by using phenyl 2,3,4,6-tetra-O-benzoyl-l-thio-D-mannopyranoside or phenyl 2,3,4,6-tetra-O- acetyl-1-thio-D-mannopyranoside as the glycosyl donor.

According to another method of preparation according to the invention, the compound of the formula I wherein R = OAc and R' = mannosyl may also be prepared by using of 2,3,4,6-tetra-O-benzoyl-D-mannopyranosyl bromide or 2,3,4,6- tetra-O-acetyl-D-mannopyranosyl bromide as glycosyl donor.

All compounds of the formula I wherein R and R' have the above defined meanings may be obtained by the described methods.

Therapeutic administration

Suitable routes for administration include oral, rectal, topical (including ocular, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravitreous, intravenous, intradermal, intrathecal and epidural) ways. The preferred route of administration will depend upon the condition of the patient, the toxicity of the compound and the type and site of infection, among other considerations known to the clinician.

The therapeutic composition comprise about 1% to about 95% of the active ingredient, single-dose forms of administration preferably comprising about 20% to about 90% of the active ingredient and administration forms, which are not single- dose preferably comprising about 5% to about 20% of the active ingredient. Unit dose forms may be, for example, coated tablets, tablets, ampoules, vials, suppositories or capsules. Other forms of administration are, for example, ointments, creams, pastes, foams, tinctures, lipsticks, drops, sprays, dispersions and the like. Examples are capsules containing from about 0.05 g to about 1.0 g of the active ingredient. The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, coating, dissolving or lyophilizing processes.

Preferably, solutions of the active ingredient, and in addition also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions, are used, if being possible for these to be prepared before use, for example in the case of lyophilised compositions which comprise the active substance by itself or together with a carrier, for example mannitol. The pharmaceutical compositions can be sterilised and/or comprise excipients, for example preservatives, stabilisers, wetting agents and/or emulsifiers, solubilizing agents, salts for regulating the osmotic pressure and/or buffers, and they are prepared in a manner known per se, for example by means of conventional dissolving or lyophilising processes. The solutions or suspensions mentioned can comprise viscosity-increasing substances, such as sodium carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatine. Suspensions in oil comprise, as the oily component, the vegetable, synthetic or semi-synthetic oils customary for injection purposes. Oils which may be mentioned are, in particular, liquid fatty acid esters which contain, as the acid component, a long- chain fatty acid having 8 - 22, in particular 12-22, carbon atoms, for example lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidonic acid, behenic acid or corresponding unsaturated acids, for example oleic acid, elaidic acid, euric acid, brasidic acid or linoleic acid, if appropriate with the addition of antioxidants, for example vitamin E, β-carotene or 3,5-di-fert-butyl-4-hydroxytoluene. The alcohol component of these fatty acid esters has not more than 6 carbon atoms and is mono- or polyhydric, for example mono-, di- or trihydric alcohol, for example methanol, ethanol, propanol, butanol, or pentanol, or isomers thereof, but in particular glycol and glycerol. Fatty acid esters are, for example: ethyl oleate, isopropyl myristate, isopropyl palmitate, "Labrafϊl M 2375" (polyoxyethylene glycerol trioleate from Gattefosee, Paris), "Labrafil M 1944 CS" (unsaturated polyglycolated glycerides prepared by an alcoholysis of apricot kernel oil and made up of glycerides and polyethylene glycol esters; from Gattefosee, Paris), "Labrasol" (saturated polyglycolated glycerides prepared by an alcoholysis of TCM and made up of glycerides and polyethylene glycol esters; from Gattefosee, Paris) and/or "Miglyol 812" (triglyceride of saturated fatty acids of chain length C₈ to C₁₂ from HuIs AG, Germany), and in particular vegetable oils, such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and, in particular, groundnut oil.

The preparation of the injection compositions is carried out in the customary manner under sterile conditions, as are bottling, for example into ampoules or vials, and closing of the containers.

For example, pharmaceutical compositions for oral use can be obtained by combining the active ingredient with one or more solid carriers, if appropriate granulating the resulting mixture, and, if desired, processing the mixture or granules to tablets or coated tablet cores, if appropriate by addition of additional excipients.

Suitable carriers are, in particular, fillers, such as sugars, for example lactose, sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium diphosphate, or calcium hydrogen phosphate, and furthermore binders, such as starches, for example maize, wheat, rice or potato starch, methylcellulose, hydroxypropylmethylcellulose, sodium carboxyniethylcellulose and/or polyvinylpyrrolidine, and/or, if desired, desintegrators, such as the above mentioned starches, and furthermore carboxymethyl-starch, cross-linked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate. Additional excipients are, in particular, flow regulators and lubricants, for example salicylic acid, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol, or derivatives thereof.

Coated tablet cores can be provided with suitable coatings which, if appropriate, are resistant to gastric juice, the coatings used being, inter alia, concentrated sugar solutions, which, if appropriate, comprise gum arabic, talc, polyvinylpyrrolidine, polyethylene glycol and/or titanium dioxide, coating solutions in suitable organic solvents or solvent mixtures or, for the preparation of coatings which are resistant to gastric juice, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments can be admixed to the tablets or coated tablet coatings, for example for identification or characterisation of different doses of active ingredient.

Pharmaceutical compositions, which can be used orally, are also hard capsules of gelatine and soft, closed capsules of gelatine and a plasticiser, such as glycerol or sorbitol. The hard capsules can contain the active ingredient in the form of granules, mixed for example with fillers, such as maize starch, binders and/or lubricants, such as talc or magnesium stearate, and stabilisers if appropriate. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable liquid excipients, such as greasy oils, paraffin oil or liquid polyethylene glycol or fatty acid esters of ethylene glycol or propylene glycol, it being likewise possible to add stabilisers and detergents, for example of the polyethylene sorbitan fatty acid ester type.

Other oral forms of administration are, for example, syrups prepared in the customary manner, which comprise the active ingredient, for example, in suspended form and in a concentration of about 5% to 20%, preferably about 10% or in a similar concentration which results in a suitable individual dose, for example, when 5 or 10 ml are measured out. Other forms are, for example, also pulverulent or liquid concentrates for preparing of shakes, for example in milk. Such concentrates can also be packed in unit dose quantities.

Pharmaceutical compositions, which can be used rectally, are, for example, suppositories that comprise a combination of the active ingredient with a suppository base. Suitable suppository bases are, for example, naturally occurring or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols.

Compositions which are suitable for parental administration are aqueous solutions of an active ingredient in water-soluble form, for example of water-soluble salt, or aqueous injection suspensions, which comprise viscosity-increasing substances, for example sodium carboxymethylcellulose, sorbitol and/or dextran, and, if appropriate, stabilizers. The active ingredient can also be present here in the form of a lyophilisate, if appropriate, together with excipients, and be dissolved before parenteral administration by addition of suitable solvents. Solutions such as are used, for example, for parental administration can also be used as infusion solutions. Preferred preservatives are, for example, antioxidants, such as ascorbic acid, or microbicides, such as sorbic or benzoic acid.

Ointments are oil-in-water emulsions which comprise not more than 70%, preferably 20 - 50% of water or aqueous phase. The fatty phase consists, in particular, hydrocarbons, for example vaseline, paraffin oil or hard paraffins, which preferably comprise suitable hydroxy compounds, such as fatty alcohols or esters thereof, for example cetyl alcohol, or wool wax alcohols, such as wool wax, to improve the water- binding capacity. Emulsifiers are corresponding lipophilic substances, such as sorbitan fatty acid esters (Spans), for example sorbitan oleate and/or sorbitan isostearate. Additives to the aqueous phase are, for example, liumectants, such as polyalcohols, for example glycerol, propylene glycol, sorbitol and/or polyethylene glycol, or preservatives and odoriferous substances. Fatty ointments are anhydrous and comprise, as the base, in particular, hydrocarbons, for example paraffin, vaseline or paraffin oil, and furthermore naturally occurring or semi-synthetic fats, for example hydrogenated coconut-fatty acid triglycerides, or, preferably, hydrogenated oils, for example hydrogenated groundnut or castor oil, and furthermore fatty acid partial esters of glycerol, for example glycerol mono- and/or distearate, and for example, the fatty alcohols. They also contain emulsifiers and/or additives mentioned in connection with the ointments which increase uptake of water.

Creams are oil-in-water emulsions, which comprise more than 50% of water. Oily bases used are, in particular, fatty alcohols, for example lauryl, cetyl or stearyl alcohols, fatty acids, for example palmitic or stearic acid, liquid to solid waxes, for example isopropyl myristate, wool wax or beeswax, and/or hydrocarbons, for example vaseline (petrolatum) or paraffin oil. Emulsifiers are surface-active substances with predominantly hydrophilic properties, such as corresponding non-ionic emulsifiers, for example fatty acid esters of polyalcohols or ethyleneoxy adducts thereof, such as polyglyceric acid fatty acid esters or polyethylene sorbitan fatty esters (T weens), and furthermore polyoxyethylene fatty alcohol ethers or polyoxyethylene fatty acid esters, or corresponding ionic emulsifiers, such as alkali metal salts of fatty alcohol sulphates, for example sodium lauryl sulphate, sodium cetyl sulphate or sodium stearyl sulphate, which are usually used in the presence of fatty alcohols, for example cetyl stearyl alcohol or stearyl alcohol. Additives to the aqueous phase are, inter alia, agents which prevent the creams from drying out, for example polyalcohols, such as glycerol, sorbitol, propylene glycol and/or polyethylene glycols, and furthermore preservatives and odoriferous substances.

Pastes are creams and ointments having secretion-absorbing powder constituents, such as metal oxides, for example titanium oxide or zinc oxide, and furthermore talc and/or aluminium silicates, which have the task of binding the moisture or secretions present. Foams are administered from pressurised containers and they are liquid oil-in- water emulsions present in aerosol foam. As the propellant gases halogenated hydrocarbons, such as polyhalogenated alkanes, for example dichlorofluoromethane and dichlorotetrafluoroethane, or, preferably, non-halogenated gaseous hydrocarbons, air, N₂O or carbon dioxide are used. The oily phases used are, inter alia, those mentioned above for ointments and creams, and the additives mentioned there are likewise used.

Tinctures and solutions usually comprise an aqueous-ethanolic base to which, humectants for reducing evaporation, such as polyalcohols, for example glycerol, glycols and/or polyethylene glycol, and re-oiling substances, such as fatty acid esters with lower polyethylene glycols, i.e. lipophilic substances soluble in the aqueous mixture to substitute the fatty substances removed from the skin with ethanol, and, if necessary, other excipients and additives, are admixed.

The present invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefor. Veterinary carriers are materials for administering the composition and may be solid, liquid or gaseous materials, which are inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route. The invention also relates to a process or method for treatment of the disease states mentioned above. The compounds can be administered prophylactically or therapeutically as such or in the form of pharmaceutical compositions, preferably in an amount, which is effective against the diseases mentioned. With a warm-blooded animal, for example a human, requiring such treatment, the compounds are used, in particular, in the form of pharmaceutical composition. A daily dose of about 0.1 to about 5 g, preferably 0.5 g to about 2 g, of a compound of the present invention is administered here for a body weight of about 70 kg.

Brief Description of Drawings Fig. 1 displays structure of saccharide lupane derivatives of general formula I.

Fig. 2 shows induction of apoptosis by new lupane drivatives. Ultrastructural analysis of treated/untreated CEM cells by electron microscopy. Compounds 4, 12, and 20 were examined at different time points (24h, 48 h, and 72 h). Fig. 3 shows induction of apoptosis in MCF-7 cells by new lupane derivative 4. MCF-7, apoptotic and secondarily necrotic cells (i.e. necrotic following apoptosis): 24 h, 4, 20μM. Hoechst 3342 (green) and ethidium homodimer (red). Fig. 4 shows detection of cell damage by Annexin and Propidium Iodide. A: Detection of apoptotic cells by Annexin (green fluorescence). B: Detection of necrotic cells by PI (red fluorescence) analysed by Olympus image analysis after treatment of MCF-7 cells by new lupine derivative 4. C and D: Fluorescence image of late stage apoptotic and necrotic cells by fluorescence microscopy. D: Fused microscopic image of Annexin labeled apoptotic and PI labeled necrotic MCF-7 cells acquired in fluorescence mode and in transmitted light with phase contrast.

Fig. 5 displays the effect of new lupine derivative 4 and paclitaxel (an internal control) on cell cycle distribution. CEM cells were treated with compound 4 and paclitaxel (IC₅₀) for 48 h and analyzed by flow cytometry. The percentages of cells in sub- Go/Gl cell cycle phase and in apoptosis are indicated. Fig. 6 shows tumor volume analysis of treated mice transplanted with K562 human leukemia. Fig. 7 shows tumor volume analysis of treated mice transplanted with B16F melanoma.

Examples of carrying out the Invention Silica gel HF-254 and silica gel 230-400 mesh (Merck) were used for TLC and column chromatography, respectively. ¹H and ¹³C NMR spectra were recorded at 303K (500 and 125 MHz, respectively) with a Bruker Avance DRX-500 spectrometer. An internal tetramethylsilane (TMS) was used as the ¹H and ¹³C NMR chemical shift standard. Signals of the aromatic groups observed for the typical values were omitted for simplicity. High resolution mass spectra (HR-MS) were acquired with a MARINER mass spectrometer. Optical rotations were measured with a JASCO P- 1020 automatic polarimeter. Configurational assignments were based on NMR measurements including DEPT and two-dimensional techniques, including gradient-selected COSY, ¹H-¹³C gradient selected HSQC (g-Heteronuclear Single Quantum Correlation; C, H correlation via double INEPT transfer in the phase sensitive mode), ¹H-¹³C gradient selected HMBC (g-Heteronuclear Multiple Bond Correlation; long-range correlation), as well as TOCSY experiments. The following examples serve to illustrate the invention without limiting the scope thereof.

EXAMPLE 1 Lupeol [3 β-lup-20(29)-ene] (1). A sample (300 g) of the outer bark of a white birch tree collected in Poraj (South Poland) was air-dried for 7 days, cut into small pieces and extracted with methanol in a Soxhlet apparatus for 8 h. The extract was concentrated to yield 58.0 g of crude betulin, which was purified by acetylation in a refluxed mixture of acetic anhydride (120 ml), acetic acid (50 mL) and pyridine (1 ml) for 2 h. Solvents were evaporated to dryness and the residue was purified by column chromatography (hexane-diethyl ether 40:1 -> 5:1 as eluent). The fastest moving fraction during chromatographic separation of individual components contained lupeol acetate (3). It was further purified by column chromatography (hexane-ethyl acetate 50:1) to yield pure 3 (4.0 g). ¹H NMR spectral data matched that reported. (Hiroya, K.; Takahashi, T.; Miura, N.; Naganuma, A.; Sakamoto, T. Bioorg. Med. Chem. 2002, 10, 3229-3236). ¹³C NMR (CDCl₃) δ: 170.9 (C=O), 150.9 (C-20), 109.3 (C-29), 81.0 (C-3), 55.4, 50.4, 48.3, 48.0, 43.0 (C), 42.8 (C), 40.9 (C), 40.0 (CH₂), 38.4 (CH₂), 38.1, 37.8 (C), 37.1 (C), 35.6 (CH₂), 34.2 (CH₂), 29.8 (CH₂), 27.9, 27.4 (CH₂), 25.1 (CH₂), 23.7 (CH₂), 21.3, 20.9 (CH₂), 19.3, 18.2 (CH₂), 18.0, 16.5, 16.2, 16.0, 14.5. A solution of lupeol acetate (3, 3.93 g, 8.4 mmol) and potassium hydroxide (1.25 g, 22 mmol) in ethanol (30 ml) was refluxed for 5 h, cooled to room temperature and concentrated. The oily residue was suspended in dichloromethane and filtered through a short silica pad (hexane-ethyl acetate 5:1). The filtrate was concentrated and the residue was purified by column chromatography (hexane-ethyl acetate 9:1) to yield lupeol (1, 3.06 g, 85%): mp 209-211 ⁰C; [α]o⁰ +25.0 (c 1.15, CHCl₃). ¹H and ¹³C NMR spectral data matched that reported (Hiroya et al. 2002; Reynolds et al. 1986).

EXAMPLE 2

3β-O-(2, 3, 4, 6-Tetra-O-benzoyl-a-D-mannopyranosyl)-lup-20(29)-ene (5). A solution of 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl trichloroacetimidate (24,

572 mg, 0.77 mmol) and lupeol (1, 320 mg, 0.75 mmol) in CH₂Cl₂ (15 mL) was stirred for 30 min at room temperature over molecular sieves (4 A, 700 mg, finely ground), then cooled to -4O⁰C and TMSOTf (50 μL) was added. After 20 min the reaction was quenched with Et₃N (0.2 mL), and the solvents were evaporated in vacuo. Column chromatography (hexane-ethyl acetate 9:1 -> 5:1) of the residue gave 5 (716 mg, 95%) as a foam. [αβ° -2.4 (c 0.5, CHCl₃). ¹H NMR (CDCl₃) δ: 6.08 (t, IH, J_4,3 = J_4;5 = 10.1 Hz, H-4), 5.92 (dd, IH₅ J_3;2 3.2 Hz, H-3), 5.62 (dd, IH, J_2;1 1.8 Hz, H-2), 5.28 (d, IH, H-I), 4.70 (d, IH, J 2.0 Hz, lupene-H-29), 4.67 (dd, IH, J_6j5 2.3, J_6,6> 11.9 Hz, H-6), 4.56 (m, 2H, H-5, lupene-H-29), 4.48 (dd, IH, J_6,5 5.1 Hz, H- 6), 3.35 (dd, IH, J 4.3, 11.7 Hz, lupene-H-3), 2.38 (td, IH, J 5.7, 11.0 Hz, lupene-H- 19), 1.78-1.98 (m, 2H), 0.66-1.75 (m), 1.70 (s, 3H, CH₃), 1.11 (s, 3H, CH₃), 1.05 (s, 3H, CH₃), 0.95 (s, 3H, CH₃), 0.94(s, 3H, CH₃), 0.89 (s, 3H, CH₃), 0.80 (s, 3H, CH₃). ¹³C NMR (CDCl₃) δ: 166.1 (C=O), 165.6 (C=O), 165.6 (C=O), 165.5 (C=O), 150.9 (lupene-C-20), 109.4 (lupene-C-29), 94.4 (C-I), 84.3 (lupene-C-3), 71.6, 70.3, 69.5, 67.1, 63.1 (CH₂), 55.7, 50.5, 48.3, 48.0, 43.0 (C), 42.9 (C), 40.9 (C), 40.0 (CH₂),38.6 (C), 38.3 (CH₂), 38.1, 37.1 (C), 35.6 (CH₂), 34.3 (CH₂), 29.9 (CH₂), 28.8, 27.5 (CH₂), 25.2 (CH₂), 22.2 (CH₂), 21.0 (CH₂), 19.3, 18.3 (CH₂), 18.0, 16.5, 16.2, 16.0, 14.6. HR-MS (ESI) calc. for C₆₄H₇₆NaOi₀ (M+Na)⁺: 1027.5331. Found: 1027.5388.

EXAMPLE 3

3β-O-(a-D-Mannopyranosyl)-lup-20(29)-ene (6). To a solution of 5 (910 mg, 0.91 mmol) in methanol (20 mL), K₂CO₃ (200 mg) was added, the mixture was stirred overnight and concentrated to dryness. Column chromatography (hexane-ethyl acetate 5:1 then hexane-ethyl acetate-methanol 5:3:1 -> 1:1 :1) of the residue gave 6 (420 mg, 79%) as white crystals. M.p.: 244-245°C; [α]r> ⁰ +77.5 (c 0.5, CHCl₃-methanol 1:1). ¹H NMR (CDCl₃-CD₃OD 1 :1) δ: 4.94 (bs, IH), 4.70 (d, IH, J 2.0 Hz, lupene-H-29), 4.57 (m, IH), 3.72-3.84 (m, 5H), 3.68 (m. IH)₅ 3.35 (m, IH), 3.23 (dd, IH, J4.3, 11.7 Hz, lupene-H-3), 2.40 (td, IH₅ J 5.8, 11.1 Hz, lupene-H-19), 1.92 (m, 2H), 0.66-1.80 (m), 1.69 (s, 3H, CH₃), 1.06 (s, 3H, CH₃), 0.99 (s, 3H, CH₃), 0.96 (s, 3H, CH₃), 0.86 (s, 3H, CH₃), 0.81 (s, 3H₅ CH₃), 0.78 (s, 3H₅ CH₃). ¹³C NMR (CDCl₃-CD₃OD 1 :1) δ: 151.3 (lupene-C-20)₅ 109.7 (lupene-C-29),

97.0 (C-I), 83.1 (lupene-C-3), 73.6, 72.3, 72.1, 67.6, 62.0 (CH₂), 56.2, 51.0, 48.9, 48.6, 43.5 (C)₅ 43.3 (C), 41.4 (C), 40.5 (CH₂),38.9 (C), 38.8 (CH₂), 38.6, 37.6 (C)₅

36.1 (CH₂), 34.8 (CH₂), 30.3 (CH₂), 28.9, 27.9 (CH₂), 25.7 (CH₂), 22.4 (CH₂), 21.5 (CH₂), 19.5, 18.8 (CH₂), 18.3, 16.6, 16.5, 16.4, 14.9. HR-MS (ESI) calc. for C₃₆H₆₀NaO₆ (M+Na)⁺: 611.4282. Found: 611.4311. Anal. Calcd for C₃₆H₆₀O₆ x H₂O: C, 71.25; H, 10.30. Found: C, 71.02; H, 10.38.

EXAMPLE 4 3β-O-(2, 3, 4, 6-Tetra-O-benzoyl-a-D-mannopyranosyl)-(l →3)-[(2, 3, 4, 6-tetra~0- benzoyl-a-D-mannopyranosyl)-(l->6)]-a-D-mannopy?'anosyl-lup-20(29)-ene (7). A solution of 6 (295 mg, 0.5 mmol) in CH₂Cl₂-acetonitrile (1 :1, 20 niL) was stirred at room temperature over molecular sieves (4 A, 500 mg, finely ground) for 30 min, then cooled to -40 °C and TMSOTf (55 μL) was added followed by 2,3,4,6-tetra-O- benzoyl-α-D-mannopyranosyl trichloroacetimidate (24, 815 mg, 1.1 mmol) in CH₂Cl₂ (10 mL) dropwise over 15 min. The solution was stirred for a further 30 min, neutralized with Et₃N (0.2 mL), and concentrated to dryness. Column chromatography (hexane-ethyl acetate 5:1 -» 7:3, then hexane-ethyl acetate-methanol, 5:3:1) afforded 430 mg (37%) of crude 7, which was used in the next reaction without further purification.

EXAMPLE 5

3β-O-(2, 3, 4, 6-Tetra-O-henzoyl-a-D-mannopyranosyl)-(l →3)-[(2, 3, 4, 6-tetra-O- benzoyl-a-D-mannopyranosyl)-(l—>6)]-2,4-di-O-acetyl-a-D-mannopyranosyl-lup- 20(29)-ene (8).

The above crude product (7) was acetylated under standard conditions (Ac₂O₅ Py) and purified by column chromatography (hexane-ethyl acetate 5:1 — » 7:3) to yield 8 (251 mg, 27% after two steps) as an amorphous glass. ¹H NMR (CDCl₃) δ: 6.23 (t, IH, J_4,3

(¹J_C-H 171.3 Hz, C-I), 84.4 (lupene-C-3), 75.2 (C-3), 72.0 (C-2), 70.7 (C-2'), 70.3, 70.2 and 70.2 (C-2",3",5), 69.6 (C-5'), 69.4 (C-3'), 68.7 (C-5"), 68.4 (C-4), 66.9 (C- 6), 66.8 (C-4"), 66.3 (C-4'), 62.9 (C-6"), 62.4 (C-6'), 55.3, 50.1, 48.2, 47.9, 42.9 (C), 42.5 (C), 40.7 (C), 39.9 (CH₂), 38.6 (C)₅ 38.3 (CH₂), 37.9, 37.0 (C), 35.4 (CH₂), 34.1 (CH₂), 29.8 (CH₂), 28.9, 27.2 (CH₂), 24.9 (CH₂), 22.5 (CH₂), 21.1 (CH₃), 20.9 (CH₃), 18.2 (CH₂), 17.9, 16.5, 16.0, 15.9, 14.0. HR-MS (ESI) calc. for C₁₀₈H₁₁₆NaO₂₆ [MH-Na]⁺: 1851.7647. Found: 1851.7730.

EXAMPLE 6 3 β-O-(a-D-mannopyranosyl)-(l →3)-[(a-D-mannopyranosyl)-(l —>6)]-a-D~manno- pyranosyl-lup-20(29)-ene (9).

A suspension of 8 (190 mg, 0.1 mM) and K₂CO₃ (40 mg) in methanol (5 niL) was stirred for 2 h, then neutralized with Amberlyst 15 resin (H⁺ form), filtered through a short silica pad (methanol as eluent) and the filtrate was evaporated to dryness. The residual methyl benzoate was removed by adding water (3 niL) and freeze drying to afford 9 (84 mg, 88%) as white powder. ¹³C NMR (pyridine-<fc) δ: 151.1 (lupene-C- 20), 109.9 (lupene-C-29), 103.8 (C-I), 101.8 (C-I), 98.6 (C-I), 82.9, 81.1, 75.5, 75.1, 74.5, 73.2, 73.1, 72.4, 72.1, 72.1, 69.5, 69.3, 67.2 (CH₂), 67.1, 63.2 (CH₂), 63.0 (CH₂), 55.8, 52.0, 50.5, 48.6, 48.3, 43.2 (C), 43.1 (C), 41.1 (C), 40.3 (CH₂), 38.7 (C), 38.5 (CH₂), 38.3, 37.3 (C), 35.8 (CH₂), 34.7 (CH₂), 30.2 (CH₂), 27.8 (CH₂), 25.6 (CH₂), 22.7 (CH₂), 21.1 (CH₂), 19.5, 18.6 (CH₂), 18.2, 16.7, 16.3, 16.2, 14.7. HR-MS (ESI) calc. for C₄₈H₈₀NaO₁₆ [M+Na]⁺: 935.5339. Found: 935.5343.

EXAMPLE 7 3-β-O-Acetyl-lup-20(29)-en-28-oic acid 2, 3, 4, 6-tetra-O-benzoyl- a-D-mannopyranosyl ester (10).

Betulinic acid acetate (4, 500 mg, 1.0 mM) was converted into the glycoside 10 using 2,3,4,6-tetra-<9-benzoyl-α-D-mannopyranosyl trichloroacetimidate (24) and the procedure described for 5 to yield 10 (985 mg, 91%) as a foam. [α]o⁵ -18.6 (c 0.6, CHCl₃). ¹H NMR (CDCl₃) δ: 6.44 (d, IH, J_lj2 2.0 Hz, H-I), 6.19 (t, IH, J₄,₃ = J₄,₅ = 10.1 Hz, H-4), 5.87 (dd, IH, J_3j2 3.3 Hz, H-3), 5.72 (dd, IH, H-2), 4.80 (bs, IH, lupene-H-29), 4.66 (m, 2H), 4.42-4.52 (m, 3H), 3.05 (ddd, IH, J 5.0, 11.1 Hz), 2.42 (m, IH), 2.31 (m, IH), 2.12 (m, IH), 2.04 (s, 3H, CH₃). ¹³C NMR (CDCl₃) δ: 173.1 (C=O), 171.0 (C=O), 166.1 (C=O), 165.5 (C=O), 165.3 (C=O), 165.1 (C=O), 150.0 (lupene C-20), 110.0 (lupene C-29), 90.3 (C-I), 80.9 (lupene C-3), 71.5, 70.0, 69.3, 66.3, 62.6 (C-6), 57.1 (C), 55.5, 50.5, 49.4, 46.9, 42.5 (C), 40.7 (C), 38.4 (CH₂), 38.2, 37.8 (C), 37.1 (C), 37.0 (CH₂), 34.2 (CH₂), 32.4 (CH₂), 30.5 (CH₂), 29.6 (CH₂), 27.9, 25.5 (CH₂), 23.7 (CH₂), 21.3 (CH₃), 20.8 (CH₂), 19.4, 18.2 (CH₂), 16.4 (CH₃), 16.1 (CH₃), 16.0 (CH₃), 14.7 (CH₃). HR-MS (ESI) calc. for C₆₆H₇₆NaO₁₃ [M+Na]⁺: 1099.5178. Found: 1099.5142.

EXAMPLE 8 3-β-O-Acetyl-lup-20(29)-en-28-oic acid 2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl ester (11).

Betulinic acid acetate (4, 500 mg, 1.0 niM) and 2,3,4,6-tetra-O-acetyl-α-D- mannopyranosyl trichloroacetimidate (25) were converted into the glycoside 11 using the procedure described for 5 to yield 11 (772 mg, 93%) as a foam. [α]o +32.4 (c 0.6, CHCl₃). ¹H NMR (CDCl₃) δ: 6.13 (d, IH, J_lj2 2.0 Hz, H-I), 5.35 (t, IH, J₄,₃ = J₄,₅ = 10.0 Hz, H-4), 5.29 (dd, IH, J_3j2 3.3 Hz, H-3), 5.24 (dd, IH, H-2), 4.74 (d, IH, J 1.8 Hz, lupene H-29), 4.61 (bs, lupene H-29), 4.46 (dd, IH, J 8.0 and 10.3 Hz, lupene H- 17), 4.29 (dd, IH, J₆,₅ 4.9, J_6;6- 12.4 Hz, H-6), 4.06 (dd, IH, J₀ 2.5 Hz, H-6'), 3.98 (m, IH, H-5), 2.94 (m, IH, lupene H-19), 2.18 (s, 3H, CH₃), 2.08 (s, 3H, CH₃), 2.05 (s, 3H, CH₃), 2.03 (s, 3H, CH₃), 1.99 (s, 3H, CH₃), 1.68 (s, 3H, CH₃), 0.97 (s, 3H, CH₃), 0.90 (s, 3H, CH₃), 0.83 (s, 6H, 2 x CH₃), 0.81 (s, 3H, CH₃). ¹³C NMR (CDCl₃) δ: 172.9 (C=O), 171.0 (C=O), 170.6 (C=O), 169.9 (C=O), 169.7 (C-O), 169.5 (C=O), 149.8 (lupene C-20), 110.0 (lupene C-29), 90.1 (C-I), 80.9 (lupene C-3), 71.1, 69.0, 68.3, 65.4, 62.2 (C-6), 56.9 (C), 55.4, 50.5, 49.3, 46.8, 42.4 (C), 40.7 (C), 38.4 (CH₂), 38.1, 37.8 (C), 37.1 (C), 36.8 (CH₂), 34.2 (CH₂), 32.2 (CH₂), 30.3 (CH₂), 29.5 (CH₂), 27.9, 25.4 (CH₂), 23.7 (CH₂), 21.3, 20.8 (CH₂), 20.7, 20.7, 20.6, 20.5, 19.3, 18.1 (CH₂), 16.4, 16.1, 16.0, 14.6. HR-MS (ESI) calc. for C₄₆H₆₈NaO₁₃ [M+Na]⁺: 851.4552. Found: 851.4578

EXAMPLE 9

3-β-O~Acetyl-lup-20(29)-en-28-oic acid a-D-mannopyranosyl ester (12). To a solution of 11 (1.27 g, 1.53 mM) in methanol (15 niL), a solution of freshly prepared sodium methoxide in methanol (0.24 M, 0.2 niL) was added and stirred for 90 min. The solution was neutralized with Amberlyst 15 resin (H⁺ form), filtered through a short silica pad (methanol as eluent) and the filtrate was evaporated to dryness. Column chromatography (hexane-ethyl acetate, 7:3 then hexane-ethyl acetate-methanol, 5:3:1 -» 1:1 :1) of the residue gave 12 (910 mg, 90%) as a foam. [α]o ° +48.4 (c 0.6, CHCl₃-methanol 1:1). ¹H NMR (CDCl₃-CD₃OD 1 :1) δ: 6.04 (d, IH, J_1;2 1.8 Hz, H-I), 2.01 (s, 3H, CH₃), 1.66 (s, 3H, CH₃), 0.96 (s, 3H, CH₃), 0.90 (s, 3H, CH₃), 0.83 (s, 3H, CH₃), 0.81 (s, 6H₅ 2 x CH₃). ¹³C NMR (CDCl₃ / CD₃OD, 1 : 1) δ: 173.7 (C=O), 171.3 (C=O), 149.5 (lupene C-20), 109.1 (lupene C-29), 92.7 (C-I), 80.9 (lupene C-3), 75.0, 70.8, 69.3, 65.7, 60.6 (C-6), 56.3 (C), 54.9, 50.0, 48.7, 46.4, 41.9 (C), 40.1 (C), 37.8 (CH₂), 37.7, 37.1 (C), 36.5 (C), 36.1, 33.7 (CH₂), 31.4 (CH₂), 29.7 (CH₂), 29.0 (CH₂), 27.1 (CH₂), 24.9, 23.0 (CH₂), 20.3 (CH₂), 20.2, 18.3, 17.5 (CH₂), 15.6, 15.3, 15.2, 13.9. HR-MS (ESI) calc. for C₃₈H₆₀NaO₉ [M+Na]: 683.4130. Found: 683.4161. Anal. Calcd for C₃₈H₆₀O₉ x 1.5H₂O: C, 66.35; H, 9.23. Found: C, 66.35; H, 9.14.

EXAMPLE 10

3-β-O-Acetyl-lup-20(29)-en-28-oic acid (2, 3, 4, 6-tetra-O-benzoyl-a-D- mannopyranosyl)-(l ->3)-[(2, 3, 4, 6-tetra-O-benzoyl-a-D-mannopyranosyl)-(l —>6)]-a- D-mannopyranosyl ester (13). Mannoside 12 (330 mg, 0.5 mM) and 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl trichloroacetimidate (24, 815 mg, 1.1 mM) were converted into trimannoside 13 using the procedure described for 7 and used in the next reaction without further purification.

EXAMPLE I l

3-β-O-Acetyl-lup~20(29)-en-28-oic acid (2, 3, 4, 6-tetra-O-benzoyl-a-D- mannopyranosyl)-(l —>3)-[(2, 3, 4, 6-tetra-O-benzoyl-a-D-mannopyranosyl)-(l —>6)]- 2,4-di-O-acetyl-a-D-mannopyranosyl ester (14). The crude product 13 was acetylated under standard conditions (Ac₂O, Py) and purified by column chromatography (hexane-ethyl acetate 7:3 then hexane-ethyl acetate-methanol 5:3:0.2 -» 5:3:0.5) to yield 14 (391 mg, 41% after two steps) as an amorphous glass. [α]o° -13.9 (c 0.5, CHCl₃). ¹H NMR (CDCl₃) δ: 6.27 (t, IH, J_4;3 = J_4;5 = 10.1 Hz, H-4"), 6.25 (d, IH, J_1;2 1.9 Hz, H-I), 6.17 (m, IH, J 9.6, 10.9 Hz, H- 4'), 5.83 (m, 3H, H-2', 3\ 3"), 5.63 (t, IH, J_4,3 = J_4;5 = 9.9 Hz, H-4), 5.56 (dd, IH, J_2;1 2.0, J_2,3 3.1 Hz, H-2"), 5.45 (dd, IH, J₂,₃ 3.3 Hz, H-2), 5.39 (d, IH, H-I"), 5.14 (bs, IH, H-I'), 4.75 (m, 2H₃ H-6\ lupene H-29), 4.44-4.65 (m, 6H, H-5', 5", 6', 6", 6", lupene H-29), 4.38 (m, 2H, H-3, lupene H-3), 4.02 (m, 2H, H-5, 6), 3.76 (dd, IH, J_6;5 2.3, J_6,6' 10.6 Hz, H-6), 2.98 (m, IH, J4.7, 11.1 Hz), 2.38 (s, 3H, CH₃), 2.33 (s, 3H, CH₃), 2.01 (s, 3H, CH₃), 1.69 (s, 3H, CH₃), 0.93 (s, 3H, CH₃), 0.87 (s, 3H, CH₃), 0.66 (s, 3H, CH₃), 0.61 (s, 3H, CH₃), 0.57 (s, 3H, CH₃). ¹³C NMR (CDCl₃) δ: 173.3 (C=O), 171.0 (C=O), 170.4 (C=O), 170.1 (C=O), 166.2 (C=O), 166.0 (C=O), 165.5 (C=O), 165.4 (C=O)₅ 165.4 (C=O), 165.3 (2 x C-O), 165.2 (C=O), 149.8 (lupene C-20), 110.2 (lupene C-29), 99.4 (¹Jc-^ 174.2 Hz, C-I'), 98.0 C Jc-_H 175.5 Hz, C-I"), 90.2 (¹Jc-^ 179.3 Hz, C-I), 80.9 (lupene C-3), 75.5 (C-3), 72.3 (C-5), 70.7 (C-2"), 70.4 (C- 3' or C-3"), 70.1 (C-2'), 69.8 (C-5"), 69.8 (C-2), 69.4 (C-3' or C-3"), 68.9 (C-5')₅ 67.6 (C-4), 67.2 (C-6), 66.6 (C-4'), 66.4 (C-4"), 62.8 (C-6'), 62.4 (C-6"), 57.1 (C), 55.4, 50.4, 49.3, 47.1, 42.5 (C), 40.7 (C), 38.4, 38.2 (CH₂), 37.6 (C), 37.1 (CH₂), 37.1 (C), 36.9 (C), 34.3 (CH₂), 32.4 (CH₂), 30.5 (CH₂), 29.5 (CH₂), 27.8, 25.5 (CH₂), 23.6 (CH₂), 21.3, 21.0, 20.8, 20.8 (CH₂), 19.3, 18.0, 16.3, 16.1, 16.0, 14.6. HR-MS (ESI) calc. for C₁₁₀H₁₁₆NaO₂₉ [M+Na]⁺: 1923.7495. Found: 1924.7516. Anal. Calcd for C₁₁₀H₁₁₆O₂₉ x 2H₂O: C, 68.17; H, 6.24. Found: C, 68.05; H, 6.29.

EXAMPLE 12

3-β-O-Acetyl-lup-20(29)-en-28-oic acid (a-D-mannopyranosyϊ)-(l —>3)-[(a-D- mannopyranosyl)-(l—>6)]-a-D-mannopyranosyl ester (15).

A suspension of 14 (160 mg, 0.084 mM) and K₂CO₃ (40 mg) in methanol (5 niL) was stirred for 2 h, neutralized with Amberlyst 15 resin (H⁺ form), filtered through a short silica pad (methanol as eluent) and the filtrate was evaporated to dryness. The residual methyl benzoate was removed by adding water (3 mL) and freeze drying to afford 15 (82 mg, quant.) as white powder. ¹³C NMR (pyridine- d₅) δ: 174.3 (C=O), 170.7 (C=O), 150.7 (lupene C-20), 110.2 (lupene C-29), 104.1 (C-I), 102.2 (C-I), 95.1 (C- 1), 80.8, 80.5, 76.8, 75.5, 75.2, 73.1, 73.1, 72.3, 72.0, 70.3, 69.5, 69.2, 66.9 (CH₂), 66.5, 63.1 (CH₂), 63.0 (CH₂), 57.2 (C), 55.7, 52.0, 50.8, 49.7, 47.6, 42.8 (C), 41.1 (C), 38.6 (CH₂), 38.0 (C), 37.3 (C), 37.2 (CH₂), 34.6 (CH₂), 32.5 (CH₂), 30.9 (CH₂), 29.9 (CH₂), 28.1, 26.0 (CH₂), 24.1 (CH₂), 21.2 (CH, CH₂), 19.4, 18.5 (CH₂), 16.8, 16.3, 14.9. HR-MS (ESI) calc. for C₅₀H₈₀NaO₁₉ [M+Na]⁺: 1007.5186. Found: 1007.5177. Anal. Calcd for C₅₀H₈₀O₁₉ x H₂O: C, 59.86; H, 8.24. Found: C, 59.77; H, 8.21.

EXAMPLE 13 Testing of in vitro cytotoxicity

The cell lines (T-lymphoblastic leukemia cell line CEM; breast carcinoma cell lines MCF-7, lung carcinoma cell lines A-549, human peripheral blood myeloma cells RPMI 8226, epitheloid carcinoma cell line HeLa, malignant melanoma cell lines G361, and human fibroblast BJ-H-tert) were cultured in DMEM medium (Gibco BRL) supplemented with 10 % fetal calf serum, 4 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, at 37 °C in a fully humidified atmosphere containing 5% CO₂. Suspensions of these lines (ca. 1.25 x 10⁵ cells/ml) were placed in 96-well microtitre plates and after 3 h of stabilization the tested saponins were added in serially diluted concentrations. Saponins were dissolved in dimethylsulfoxide (DMSO) before addition to cultures. Control cultures were treated with DMSO alone. The final concentration of DMSO in the reaction mixtures never exceeded 0.6 %. Four-fold dilutions of the test compound solutions were added at time zero in 20 μl aliquots to the microtiter plate wells. Usually, each test compound was evaluated at six 4-fold dilutions and in routine testing, the highest well concentration was 50 μM, although this varied in a few cases, depending on the test compound. After 72 h of culture, the cells were incubated with Calcein AM solution (Molecular Probes) for 1 h. The fluorescence of viable cells was quantified using a Fluoroscan Ascent instrument (Microsystems). The percentage of surviving cells (IC₅₀) in each well was calculated from the equation IC₅₀ = (ODd_rug exposed well / mean OD_controi wells) x 100%. The IC₅₀ value, the drug concentration lethal to 50% of the tumour cells, was calculated from the obtained dose-response curves.

Table 1. IC₅₀ (μM) values obtained from the Calcein AM assays with the tested cancer cell lines and means ±SD obtained from three independent experiments performed in triplicate. Betulinic acid (2) was used as a positive control.

Structures of the compounds shown in Table 1.

1. R = H, R¹ = CH₃ (lupeol) 2: R = H, R' = CO₂H (betulinic acid) 3: R = Ac, R' = CH₃ 4: R = Ac, R¹ = CO₂H

5: R = Bz 6: R = H EXAMPLE 14: Novel Compounds Induce Apoptosis in Cancer Cells

To analyse the mechanisms of induced cytotoxicity by novel compounds, it is important to distinguish apoptosis from the other major form of cell death, necrosis. First, at the tissue level, apoptosis produces little or no inflammation, since the neighbouring cells, especially macrophages, rather than being released into the extracellular fluid, engulf shrunken portions of the cell. In contrast, in necrosis, cellular contents are released into the extracellular fluid, and thus have an irritant affect on the nearby cells, causing inflammation. Second, at the cellular level, apoptotic cells exhibit shrinkage and bleeding of the cytoplasm, preservation of structure of cellular organelles including the mitochondria, condensation and margination of chromatin, fragmentation of nuclei, and formation of apoptotic bodies, thought not all of these are seen in all cell types. Third, at the molecular level, a number of biochemical processes take an important role in induction of apoptosis. However, majority of them is not well understood, and they result in activation of proteases and nucleases, which finally destruct key biological macromolecules - proteins and DNA. For detection of apoptotic versus necrotic mode of cell death, two independent methods were employed: assessment of morphology by fluorescence microscopy and analysis of DNA fragmentation by flow cytometry using the TUNEL technique. Determination of apoptosis and cell cycle distribution

Microscopy: Nuclear morphology of the cells was analysed with the fluorochromes Hoechst 33342 (λε_x max 346 nm; λ_Em max 460 nm) (Sigma) prepared in phosphate-buffered saline (PBS at 0.1 mg/ml, added to the culture medium at a final concentration of 2 μg/ml and ethidium homodimer (EB) (λβ_X max 540 nm; λE_m max 625 nm) (Sigma) prepared in PBS at 100 μg/ml and added to the culture medium at a final concentration of 2μg/ml (Lizard, 1995). Hundred cells were counted for each sample and percentage of apoptosis was determined. Annexin V-FITC Apoptosis Detection Annexin V-FITC kit allows fluorescent detection of annexin V bound to apoptotic cells and quantitative determination by flow cytometry. The AnnexinV- FITC kit uses annexin V conjugated with fluorescein isothiocyante (FITC) to label phosphatidylserine sites on the membrane surface. The kit includes propidium iodide (PI) to label the cellular DNA in necrotic cells where the cell membrane has been totally compromised. This combination allows the differentiation among early apoptotic cells (annexin V positive, PI negative), necrotic cells (annexin V positive, PI positive), and viable cells (annexin V negative, PI negative). Detection of apoptosis by this kit is shown in. Fig. 4. TdT-Mediated dUTP nick end labeling (TUNEL) assay. For detection of apoptotic cells, the TUNEL assay was used. The cells were seeded in a density 1.4xlθ⁴cells/cm² (MCF-7) or 1.6><10⁴cells/cm² (G-361) using appropriate culture medium to 60-mm culture dishes with coverslips. Cells were grown 24 h and then treated with 4 and 12 (IC₅₀) for 6, 12, and 24 h. After given period of treatment, the cells were washed with phosphate-buffered saline (PBS) and fixed on the slides with cold acetone-methanol (1 :1, v/v) for lO min. Apoptosis-induced nuclear DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) technique according to the protocol (In Situ Cell Death Detection Kit; Roche Diagnostics; Mannheim, Germany). The cells were then washed three times in PBS and incubated with 4'-6-diamidmo-2-phenylindole (DAPI; 50 μg/mL; Sigma) for 10 min in the dark. The coverslips with cells were washed in deionized water and mounted on glass slides, using the hydrophilic medium Mowiol (Calbiochem; Fremont, CA) in glycerol-PBS (1:3, v/v) for fluorescence. Cells were visualized using using a fluorescence microscope (BX50F, Olympus; Japan) and compared with control untreated cells.

Apoptosis and cell cycle analysis. Flow cytometry was used to evaluate the number of cells in the particular phases of the cell cycle, including SUbG₁ peak detection. The cells were seeded in a density 1.4xl0⁴cells/cm² (MCF-7) and 1.6x10⁴cells/cm² (CEM) using culture medium in 60-mm culture dishes. After 24 h, the cells reaching approximately 70-80 % confluence were treated with IC_5O concentrations of novel compounds. DMSO was used as a vehicle for controls. After 24 h treatment, the cells (IxIO^"6) were washed twice with cold PBS (10 mM, pH 7.4), pelleted, and fixed with chilled ethanol (70%; v/v) by low-speeded vortexing. Low molecular weight apoptotic DNA was extracted in citrate buffer and RNA was cleaved by RNAse (50μg/mL). For DNA content measurements, propidium iodide staining was used. The cells were analyzed using a FACSCaliburflow cytometer (BD Biosciences; San Jose, CA). Pro-apoptotic effects of new compounds Fluorescence microsopy analysis of apoptosis and necrosis: Cell cultures treated with different doses of novel compounds were examined microscopically for apoptosis. Apoptotic cells exhibit a very bright Hoechst 33342 fluorescence, while viable cells display a very faint fluorescence. Late apoptotic cells or secondarily necrotic cells display a fragmented nucleus with bright red ethidium bromide fluorescence. Primary necrotic cells show a red fluorescence and do not have fragmented nuclei. An illustration of these different features can be found in Figure 3 (MCF-7-cell line incubated with compound 4). The cells were also labelled with Anexin FITC V (MoI. Probes) and propidium iodide. The results are shown in Figure 4.

Figure 4 shows the result of microscopic examination of MCF7 cells incubated with 4. Viable, apoptotic, necrotic (= primary necrosis, not following apoptosis) and secondarily necrotic cells (= late apoptotis, evolving to necrosis) were scored differentially after 24 hours of exposure to novel compounds. For the product a different apoptosis-inducing pattern could be observed. Apoptosis induction occurs fast after incubation with 4.

Flow cytometric detection of apoptosis and cell cycle analysis: The induction of apoptotic death of MCF-7 cells by the novel compounds was confirmed using the TUNEL reaction technique.

Table 2. Detection of DNA strand breaks in apoptotic nuclei of MCF-7 and G361 cells by TUNEL staining. Cells were treated with Compound 4 or 12 (IC₅₀) for 6/12/24/48 h. Data indicate mean (± SD) percentages of TUNEL-positive cells obtained from three independent experiments. Asterisks (*) denote values that are significantly different from the respective control values at p < 0.05.

Compound 12 , 24 h 1.7 ± 3.8* 38.3 ± 3.0*

Control, 48 h 0 .7 ± 0 .6 0.6 ± 0 .0

Compound 4, 48 h 91 .8 ± 2 .6* 87.3 0 .6*

Compound 12 , 48 h 91 .7 ± 3 .8* 88.9 ± 3 .0*

Initial phase contrast microscopy examinations indicated that the lupane treated MCF- 7 line exhibit typical morphological features of apoptotic cells and this was later confirmed by electron microscopy on CEM cells (Fig. T). Corresponding results were obtained from flow cytometric analysis of the DNA content in CEM cells treated with various lupane derivative 4 (Fig. 5). Extensive apoptosis of tumor cells, measured as a percentage of sub-Go/Gl, was initiated in the treated cells as early as 12 hours after the treatment. The distribution of cells within the cell cycle showed an early disappearance of G2/M and S-phase cells in the treated cells. Further experiments were designed to manipulate the apoptotic process in order to elucidate the mechanisms of cell death.

EXAMPLE 15 Inhibition of tumor growth by new lupane derivatives was performed according to previously described procedures (Bartolazzi et al, 1994, J Exp. Med., 180, 53-66, 1994). In the first experiment, athymic nude mice (BALB/c, nu/nu) were anesthetized with 2,2-dichloro-l,l-difluoroethyl-methyl ether (methoxyflurane) in a semiclosed system and a single osmotic pump 1007D (Alza, Palo Alto, CA), filled with 100 μg lupane derivative 4 or 100 μg CD44HRg in 100 μl PBS, was implanted subcutaneously (s.c.) into the retroscapular region. Ten mice were used in each group. The 1007D pump is designed to deliver a fixed volume of 0.5 μl/hr for a period of 7 days. On the second day, Bl 6F10 melanoma cells were detached with EDTA, washed in PBS resuspended in PBS at a concentration of 125x10³ cells/0.2 ml and inoculated s.c. into the pocket of the pump. In the second experiment, 2x10⁵ Bl 6F10 cells were injected into each mouse 2 days prior to implantation of pumps which were filled with 1 mg CS-A or, 10 mg, 50 mg, 100 mg, and 150 mg/kg lupane derivative 4 in 100 μl PBS. Ten mice were used in each group. Primary endpoint for analysis was reduction of tumor volume (10 mice/group), which was quantified by caliper ation. Analysis of treated animals was preformed in comparison with vehicle treated mice. Body weight of experimental animals was also evaluated in parallel in order to reflect toxicity of the therapy. Comparative analysis of tumor volumes and body weights was performed using non-parametric t-test.

Screening of biological activity of compound 4 was also performed on survival model of K562 leukemia transplanted intraperitoneally with 2.10⁵ cells. CD-I nude mice were used as a host. One day following the leukemia transplantation, treatment with 4 was initiated. The compound 4 was applied as described above (Days 1-45).

Survival analysis of treated animals (10 mice/group) was preformed in comparison with vehicle treated mice using Kaplan-Meier method and the significance was evaluated by the log-rank test. Body weight of experimental animals was evaluated in parallel in order to reflect toxicity and efficacy of therapy. Comparative analysis of body weight was performed using non-parametric t-test.

New lupane derivatives inhibit tumor growth in vivo Alzet osmotic pumps containing 1 mg/ml were inserted into s.c. tissue in the immediate vicinity of the site of injection of Bl 6F10 cells. Pumps containing PBS or 1 mg/ml new lupane derivative 4 were inserted into separate groups of animals. The pumps deliver 0.5 μl/hr over the course of 7 days; therefore, the tumor would be exposed to approximately 0.5 μg/ml lupane derivative 4 of for this period of tumor growth. The tumors were allowed to grow for 14 days whereupon the animals were measured and tumor size was determined. The lupane derivative 4 were found to inhibit tumor growth by 35%. Results of our analysis demonstrated that compound 4 has also good tolerability. There was only slight reduction the body weight of treated animals, which was however significant in one time point (Day 12) only. Tumor volume analysis showed significantly smaller tumors in mice treated with 4 (P=O.149) that has been translated to longer mean survival time (25.9 days for drug treated group versus 20.4 days for vehicle treated group).

Results of our analysis also demonstrated that compound 4 is reducing body weight of treated animals, which is in the model of intraperitoneally transplanted K562 leukemia indicative for both toxicity and efficacy of the treatment (reduction of ascites and intraperitoneal tumor formation). Survival analysis demonstrated significantly better survival of mice treated with compound 4 (P=O.807) that has been translated to longer mean survival time (>50 days for drug treated group versus 34.5 days for vehicle treated group).

EXAMPLE 16 Dry Capsules

5000 capsules, each of which contains 0.25 g of one of the compounds of the general formula I mentioned in the preceding examples as active ingredient, are prepared as follows:

Composition Active ingredient 125O g

Talc 180 g

Wheat starch 120 g

Magnesium stearate 80 g

Lactose 20 g Preparation process: The powdered substances mentioned are pressed through a sieve of mesh width 0.6 mm. Portions of 0.33 g of the mixture are transferred to gelatine capsules with the aid of a capsule-filling machine.

EXAMPLE 17 Soft Capsules

5000 soft gelatine capsules, each of which contain 0.05 g of one of the compounds of the formula I mentioned in the preceding Examples as active ingredient, are prepared as follows: Composition Active ingredient 250 g Lauroglycol 2 litres

Preparation process: The powdered active ingredient is suspended in Lauroglykol^® (propylene glycol laurate, Gattefosse S. A., Saint Priest, France) and ground in a wet- pulveriser to a particle size of about 1 to 3 μm. Portions of in each case 0.419 g of the mixture are then transferred to soft gelatine capsules by means of a capsule-filling machine. EXAMPLE 18

Soft Capsules

5000 soft gelatine capsules, each of which contain 0.05 g of one of the compounds of the formula I, II or III mentioned in the preceding Examples as active ingredient, are prepared as follows:

Composition

Active ingredient 250 g

PEG 400 1 litre

T ween 80 1 litre Preparation process: The powdered active ingredient is suspended in PEG 400

(polyethylene glycol of Mr between 380 and about 420, Sigma, Fluka, Aldrich, USA) and Tween" 80 (polyoxyethylene sorbitan monolaurate, Atlas Chem. Inc., Inc., USA₃ supplied by Sigma, Fluka, Aldrich, USA) and ground in a wet-pulveriser to a particle size of about 1 to 3 mm. Portions of in each case 0.43 g of the mixture are then transferred to soft gelatine capsules by means of a capsule-filling machine.

Claims

1. Saccharide lupane derivatives of the general formula I

2. The saccharide lupane derivatives of claim 1, wherein the saccharide is a monosaccharide, disaccharide, or trisaccharide, wherein the carbohydrate group is glycosyl or glycoside occuring in both enantiomeric forms, their anomeric linkage is α or β, preferably the carbohydrate group is selected from glucosyl, glucoside, mannosyl, mannoside, galactosyl, galactoside, fucosyl, fucoside, rhamnosyl, rhamnoside, idosyl, idoside or their combinations, which can be optionally substituted independently at each occurrence with azido, amino, alkylamino, acetyl, aryl or arylalkyl group.

3. The saccharide lupane derivatives of claim 1, wherein at least one of R or R' contains at least one glycosyl or glycoside group selected from glucosyl, glucoside, mannosyl, mannoside, galactosyl, galactoside, fucosyl, fucoside, rhamnosyl, rhamnoside, idosyl, idoside or their corresponding amino, acyl, and benzyl substituted di- and trisaccharide derivatives.

4. The saccharide lupane derivatives of claim 1, wherein at least one of R or R' contains at least one glycosyl or glycoside group selected from glucosyl, glucoside, mannosyl, mannoside, galactosyl, galactoside, fucosyl, fucoside, rhamnosyl, rhamnoside, idosyl, idoside which are substituted by one or two different glycosyl or glycoside group selected from glucosyl, glucoside, mannosyl, mannoside, galactosyl, galactoside, fucosyl, fucoside, rhamnosyl, rhamnoside, idosyl, idoside or their corresponding amino, acyl, or benzyl derivatives.

5. The saccharide lupane derivatives of claim 1, selected from the group comprising 3β-O-(α-D-mannopyranosyl)-lup-20(29)-ene, 3β-O-(α-D-mannopyranosyl)-(l->3)- [(α-D-mannopyranosyl)-(l→6)]-α-D-mannopyranosyl-lup-20(29)-ene, l-O-[3-β- acetoxy-lup-20(29)-ene-28-oyl]-α-D-mannopyranosyl, l-Ο-[3-β-acetoxy-lup-20(29)- en-28-oyl]-(α-D-mannopyranosyl)-(l->3)-[(α-D-mannopyranosyl)-(l-»6)]-α-D- mannopyranosyl, 3 β-O-acetyl-28-O-(α-D-mannopyranosyl)-lup-20(29)-ene, 3 β-O- acetyl-28-O-(α-D-mannopyranosyl)-(l→-3)-[(α-D-mannopyranosyl)-(l-^-6)]-α-D- mannopyranosyl-lup-20(29)-ene, 28-O-acetyl-3β-O-(α-D-mannopyranosyl)-lup- 20(29)-ene, 28-O-acetyl-3β-O-(α-D-mannopyranosyl)-(l→3)-[(α-D- marmopyranosyl)-(l->6)]-α-D-mannopyranosyl-lup-20(29)-ene (12), 3β-6>-(β-D- glucopyranosyl)-lup-20(29)-ene, 3β-0-(α-D-mannopyranosyi)-(l-»3)-[(α-D- mannopyranosyl)-(l→6)]-β-D-glucopyranosyl-lup-20(29)-ene, l-O-[3-β-acetoxy-lup- 20(29)-en-28-oyl]-(α-D-mannopyranosyl)-(l→-3)-[(α-D-mannopyranosyl)-(l->6)]-β- D-glucopyranosyl, 3β-O-acetyl-28-O-(β-D-glucopyranosyl)-lup-20(29)-ene, 3β-O- acetyl-28-O-(α-D-mannopyranosyl)-(l->3)-[(α-D-mannopyranosyl)-(l-»6)]-β-D- glucopyranosyl-lup-20(29)-ene, 28-O-acetyl-3β-O-(β-D-glucoρyranosyl)-lup-20(29)- ene, 28-O-acetyl-3β-O-(α-D-mannopyranosyl)-(l— >3)-[(α-D-mannopyranosyl)- (l→6)]-β-D-glucopyranosyl-lup-20(29)-ene (12), 3β-O-(β-D-galactopyranosyl)-lup- 20(29)-ene, 3β-O-(α-D-mannopyranosyl)-(l→-3)-[(α-D-mannopyranosyl)-(l->6)]-β- D-galactopyranosyl-lup-20(29)-ene, l-O-[3-β-acetoxy-lup-20(29)-ene-28-oyl]-β-D- galactopyranosyl l-O-[3-β-acetoxy-lup-20(29)-en-28-oyl]-(α-D-mannopyranosyl)-(l→3)-[(α-D- mannopyranosyl)-(l-=>6)]~β-D-galactopyranosyL 3β-O-acetyl-28-O-(β-D- galactopyranosyl)-lup-20(29)-ene, 3β-O-acetyl-28-O-(α-D-mannopyranosyl)-(l->3)- [(α-D-marmopyranosyl)-(l-»6)]-β-D-galactopyranosyl-lup-20(29)-ene, 28-O-acetyl- 3β-O-(β-D-galactopyranosyl)-lup-20(29)-ene, 28-O-acetyl-3β-O-(α-D- marmopyranosyl)-(l->3)-[(α-D-maiinopyranosyl)-(l-»6)]-β-D-galactopyranosyl-lup- 20(29)-ene (12), 3β-O-(L-rhamnopyranosyl)-luρ-20(29)-ene, l-O-[3-β-acetoxy-lup- 20(29)-ene-28-oyl]-L-rhamnopyranosyl, 3β-O-acetyl-28-O-(L-rhamnopyranosyl)-lup- 20(29)-ene, 28-O-acetyl-3 β-O-(L-rhamnopyranosyl)-lup-20(29)-ene, 3 β,28-di-O-(α- D-mannopyranosyl)-lup-20(29)-ene, 3 β,28-di-O-(α-D-mannopyranosyl)-mp-20(29)- ene.

6. The saccharide lupane derivatives of any of claims 1 to 5 for use as medicaments.

7. The saccharide lupane derivatives of any of claims 1 to 5 for use for inhibiting cell proliferation and inducing apoptosis.

8. The saccharide lupane derivatives of any of claims 1 to 5 for use in the treatment of hyperproliferative diseases.

9. The saccharide lupane derivatives of any of claims 1 to 5 for use in the treatment of a disorder selected from the group comprising cancer, osteoporosis, cholesterol metabolism disorders, Alzheimer disease, Huntington disease, steroid-induced osteonecrosis, sexual differentiation disorders, hyperadrenocorticism associated with sex steroid excess, androgen insensitivity syndrome, glucocorticoid insensitive asthma, steroid-induced cataracta, and deficiency of P450 oxidoreductase.

10. The saccharide lupane derivatives of any of claims 1 to 5 for use as growth regulators, preferably in animal and human tissue cultures for regulation of proliferation and morphogenesis.

11. A pharmaceutical composition, which comprises at least one saccharide lupane derivative of any of claims 1 to 5 and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition of claim 11, further comprising one or more pharmaceutical excipients.

13. The pharmaceutical composition of claim 11 or 12, further comprising commonly used cytostatics, preferably mitoxantrone, cis-platinum, methotrexate, taxol, or doxorubicin.