Natural isoflavones, especially genistein (5,7,4\'-trihydroxy-3- phenylchromen-4-on) are characterised by multidirectional biological activity, especially by ability to inhibit protein tyrosine kinases activity-the group of enzymes of significant importance for protooncogens and oncogens expression.

This activity influences potential use of genistein in therapy and prevention of neoplastic diseases, circulatory diseases, osteoporosis and other diseases. The total amount of isoflavones in plant raw materials is small. For example, different soy products contain from 0.1 to 0.3% of various components which in case of Glycine max Merill seeds (soy beans) are ß-D-glucosides : genistin, daidzin, glycitin. The aglycones corresponding to them (genistein, daidzein and glycitein) are practically not present in the raw material and appear in significant amount only after thermal processing or fermentation.

Due to a suggestion of researchers that glycosides of isoflavones, and not their aglycones are responsible for advantageous action of soy, interest in synthetic preparation of glycosides and other genistein derivatives is growing.

Recent reports on genistin (7-0-p-D-glucopyranosylgenistein) preparation (7-0-p-D-glucopyranosylgenistein) indicate, that hydroxy groups of genistein are not equivalent regarding their chemical reactivity and that 7-hydroxyl group shows ca. 100 times greater acidity than 4\'-hydroxyl group. The expected (on the basis of calculation of acidity) sequence of substitution of hydrogen atoms in phenol groups (7-OH » 4\'-OH> 5-OH) is, however, not reflected in known structures of simple

genistein derivatives that are usually a result of exhaustive alkylation or acylation (for example, there are tris-trimethylsilyl, trimethyl, triethyl, triacetyl and tribenzoyl derivatives known).

Recently, in Tetrahedron 56 (2000), 7805 publication, mono-and diesters of genistein with oleinic and stearic acids were described along with regioselective method of their preparation.

The present invention provides a number of genistein derivatives containing selectively introduced certain functional groups which may play important biological roles, especially they alter the pathways by which genistein may be eliminated or transformed as a result of metabolic processes in the organism.

The invention resides provides the new genistein derivatives of formula 1, wherein: Rl and Ra are the same or different and independently represent: - hydrogen atom - alkyl - allyl - aryl - alkyloaryl - alkylcarbonyl - arylcarbonyl, while each of the above mentioned groups may be substituted in a chain or ring by amino, nitro or nitrile groups, -R5 (R6) R7-Si- group, wherein Rs, R6 and R7 are the same or different and denote Cl 6alkyl or aryl, - mono-, di-or oligosaccharide group, whereby at least one hydroxyl group of saccharide group may be substituted by the same or different acyl, alkyl, acyloxyalkyl or aryl groups; R3 denotes hydrogen atom or-COCH3 group; and R4 denotes hydrogen atom, group-S03H, SO3-,-NH2 or-N02 groups.

The term"alkyl"should be understood as a straight or branched hydrocarbon chain containing from 1 to 18 carbon atoms and, eventually, one or more unsaturated bonds excluding chains containing 17 carbon atoms corresponding to residues of oleinic and stearic acids.

The term"aryl"should be understood as cyclic unsaturated group containing at least four carbon atoms and, eventually, one or more heteroatoms chosen out of nitrogen, oxygen and sulfur.

The term"saccharide group"should be understood as a sugar derivative: pentose, hexose or heptose in a chain or cyclic form.

In the first embodiment of the invention, the preferable genistein derivatives are expressed by formula 1, wherein Rl represent alkyl, allyl, aryl, alkyloaryl, alkylcarbonyl, arylcarbonyl, R5 (R6) R7-Si-group, R2 denotes hydrogen atom or group-COCH3, an R3 and R4 are simultaneously hydrogen atom.

In the second embodiment of the invention, the preferable genistein derivatives, according to the invention, are expressed by formula 1, wherein Rland R4 are hydrogen atom, R3 is hydrogen atom or-COCH3 group, and R2 represents alkyl, allyl, aryl, alkyloaryl, alkylcarbonyl, arylcarbonyl or R5 (R6) R7-Si- group.

In another embodiment of the invention, the preferable genistein derivatives, according to the invention, are expressed by formula 1, wherein Rl and R2 are the same or different and denote alkyl, allyl, aryl, alkyloaryl, alkylcarbonyl, arylcarbonyl or R5 (R6) R7-Si-, and R3 is hydrogen atom or-COCH3, and R4 is hydrogen atom.

The preferable group of compounds according to the invention are genistein glycosides expressed by formula 1, wherein Rl, R3 and R4 are hydrogen atom, and R2 is saccharide group.

Especially preferable glycosides are furanosides.

Genistein derivatives according to the invention exhibit a potent and selective antiproliferative and cytotoxic action and some of them, in addition, for example silyl derivatives, may be valuable intermediates for preparation of other derivatives.

Genistein derivatives expressed by formula 1 may be prepared, depending upon a kind and a place of a substituent position, by use of genistein, its synthetic precursor-2,4,6,4\'-tetradeoxybenzoin or genistin (7-O-ß-D- glucopyranosylgenistein) as a substrate.

Genistein with hydroxyl groups in positions 7 and 4\'protected in the form silyl derivatives, expressed by formula 1, wherein Ri and R2 simultaneously represents R5 (R6) R7-Si-, and R3 and R4 are hydrogen atom, is prepared by reaction

of genistein with a molar excess of alkylsilyl chloride in the presence of imidazole, in organic solvent such as dimethylformamide. Disilyl derivatives obtained by this way may be then subjected to the reaction of selective acylation in position 7, by use of proper chloride or acid anhydride in the presence of organic base, such as pyridine. Mono-O-acyl derivatives of genistein of formula 1 are obtained in this way, wherein Rl represents alkylcarbonyl or arylcarbonyl, R2 represents R5 (R6) R7- Si-, and R3 and R4 are hydrogen atom.

Mono-O-acyl derivatives of genistein have conveniently diversified functional groups: base-labile 7-0-acyl group, acid-labile 4-O-alkylsilyl group and non-reactive hydroxyl group in position 5,"protected"by intramolecular hydrogen bond. Such derivatives may serve (after partial deprotection) as substrates for another genistein derivatives preparation, which contain two different substituents in positions 4\'-and 7-.

7-Mono-O-acyl genistein derivatives expressed by formula 1, wherein Rl denotes alkylcarbonyl or arylcarbonyl, and R2, R3 and R4 are hydrogen atom, are obtained from disilyl compounds by removing silyl protective group from position 4\'by reaction with diluted acids or reagents liberating fluoride anion..

Diacyl genistein derivatives expressed by formula 1, wherein Rl and R2 are the same and represent alkylcarbonyl or arylcarbonyl, and R3 and R4 are hydrogen atom, are prepared by reaction of genistein with at least 4-fold molar excess of acylating agent, such as acid chloride or acid anhydride in the presence of a base.

Suitable bases are pyridine, substituted pyridines, trialkylamines and cyclic tertiary amines.

In the reaction of genistein with slight molar excess of acylating agent (1.2-1.8 M for 1 M of genistein), the mixture of 7-0-acetyl and 4\'-O-acetyl derivatives of genistein is obtained, which may be then separated by chromatography method.

Genistein derivatives expressed by formula 1, wherein Ri, R3 and R4 are hydrogen atom, R2 represents alkyl, allyl or arylalkyl, are prepared by known in the art method consisting in the reaction of genistein with a slight molar excess of alkylating agent, such as alkyl halide in the presence of base or by generating genistein phenoxide anion through the reaction with a potent base or metal hydride first, and then by the reaction with alkylating agent.

Next, genistein derivatives containing an alkyl substituent in position 5 are prepared by monoalkylation of tetradeoxybenzoin, which is then subjected to a formylation and cyclization reaction, similarly as described in the Polish Patent application pending No. 343505 for genistein preparation.

Finally, genistein derivatives substituted in position 3\'expressed by the general formula 1, wherein R4 represents SO3H, SO3-,-NH2 or-N02 group, are prepared by typical reactions of sulfonation, nitration or stepwise introduction of an amino group applying genistein as a substrate.

Genistein derivatives expressed by formula 1, wherein Rl denotes hydrogen atom, R2 denotes saccharide group, and R3 and R4 are hydrogen atom, may be prepared by one, known in the chemistry of sugars, method of creating glycoside bond in the reaction of glycosyl donor with glycosyl acceptor in the presence of promoter, preferably in organic solvent, by use of thermal or microwave activation.

In the methods widely known in the art, 1-glycosyl derivatives are applied as glycosyl donors, such as halides, esters, ethers or glycals in which anomeric substituent may be transformed in a good leaving group. Depending upon a type of anomeric substituent, glycosidation reaction may be carried out in acidic conditions in the presence of Lewis\'or Brönsted\'s acids or, in the case of anomeric exchange reaction, in basic conditions. Proper Brönsted\'s acids are, for example, toluenesulfonic or trifluoromethylsulfonic acids, and proper Lewis\'acids are boron trifluoride, tin tetrachloride, aluminium trichloride or titanium tetrachloride.

Genistein glycosides according to the invention are preferably prepared by anomeric exchange reaction known, for example, from publication in Nucleosides, Nucleotides, 15,771 (1996).

As a glycosyl acceptor, genistein or its acyl, alkyl or silyl derivatives are used. Use of selectively functionalized derivatives is preferable due to their better solubility in reaction medium. This reaction exhibits unexpected regioselectivity of glycosidation reaction, allowing for preparation of genistein glycosides substituted in position 4\', as main reaction products.

Glycosidation reaction is carried out in anhydrous conditions, preferably in neutral gas atmosphere, in aprotic solvent or their mixture, at elevated temperature.

As a result of glycosidation, a mixture of anomers of genistein glycosides is obtained which, if needed, may be separated by gel chromatography or crystallization.

The obtained glycoside derivatives of genistein may be further selectively functionalised in position 5"of a pentofuranose or 6"of hexopyranose in the saccharide group by use of acylating, alkylating, silylating or glycosidating reagents. Especially, the hydroxyl group in position 5"may be selectively protected by the method known in chemistry of sugars in the form of esters of mono-or dicarboxylic acids, orthoesters, carbamates, sulfonates, phosphates and ethers, for example: trityl, silyl, alcoxyalkyl o tetrahydropyranyl.

Genistein derivatives according to the invention are characterized by various physicochemical properties while maintaining the desired biological activity of the parent compound.

Selective fwctionalising of genistein allows obtaining compounds of desired properties, such as better solubility in body fluids, affinity to biopolymers, susceptibility to degradation, lipophilicity (affinity to cell membranes) which, in turn, influence pharmacokinetic and pharmacodynamic properties of the compound.

Especially 4\'-furanosides and 7-pyranosides of genistein and their ester and ether derivatives show multidirectional biological activity, both as direct precursors of genistein of high bioavailability and as integral ligands of functionally important endogenous biopolymers, so the range of possible biological activity and medical applications of new derivatives includes all actions of genistein, but is not limited by them.

Genistein derivatives according to the invention were examined from the point of view of biological activity in selected tumour cell lines in various tests of cytotoxicity and cytostatics using statistical cytometry (SC) and flow cytometry (FC) techniques.

Methods Examination of antitumour activity of genistein derivatives was carried out by evaluation of influence of these compounds on survival rate and proliferation in vitro. Leukemic cell lines were used in experiments: Tib-152, Molt-4, Hl-60, L- 1210 which were incubated in the presence of genistein (the reference substance) or

tested substances in concentrations from 1 to 150 pM/l. Incubation time was 12 to 72 hours. Then the cells were stained with a set of fluorescent FDA stains (fluorescein diacetate) and PI (propidium iodid). FDA stains alive cell, while PI- dead cells. (FDA/PI method is routinely used for cytotoxicity examination: Kenneth H., Senft JA., SenftJ.: J. Histochem. And Cytometry 33,77,1985). The stained samples were analysed by flow cytometry. The results were presented in two- dimensional cytograms and analysed taking into consideration the following criterions: a degree of FDA staining, a degree of PI staining, size and granularity of the cells. Each experimental point was repeated twice, and 5000-10000 cells were analysed during cytometric examination. Survival rate in the control culture was higher than 90%.

Results The potency of toxic activity (relative cytotoxicity) of the tested compounds was expressed with regard to genistein, as a ratio of percentage of alive cells in a culture with genistein to percentage of alive cells in a culture with the tested compound.

The influence of the examined compounds on the rate of proliferation (relative cytostatic activity) was expressed with regard to genistein as a ratio of cell culture density in the presence of genistein to cell density in the presence of the tested compound.

The results of the experiments are summerized in Table 1.

Tablel No Substance Relative cytotoxity Relative cytostatic activity (genistein = 1) (genistein = 1) (range of values obtained in (range of values obtained in experiments) experiments) 1 11 1.0-32 1. 0-4.3 2 12a and 12b 1. 0-19 1. 0-3.5 3 19 1. 0-17 1. 1-3.9 4 18 n. act. * 0. 1 5 16 n. act. * 0. 3 6 4 0. 9-23 0. 9-4.6 7 5 0. 9-11 0. 9-5.0 8 6 0. 8-1. 0 0. 4-1.3 9 7 1. 0-12 0. 9-10 10 20 1. 0-85 1. 0-1.8

n. act. *-non-active compound Antyproliferative (cytostatic) and antitumour (cytotoxic) activity of genistein derivatives exhibited in vitro unequivocally indicate the possibility of their potential use in clinical oncology (chemotherapy of neoplasms) and/or chemoprevention of neoplasms.

Moreover, the invention includes the pharmaceutical preparation containing the active substance which is the genistein derivative of formula 1, wherein Rl and R2 are the same or different and independently represent: - hydrogen atom - alkyl - allyl - aryl - alkyloaryl - alkylcarbonyl - arylcarbonyl

while each of the above mentioned groups may be substituted in a chain or ring by amino, nitro or nitrile groups; - Rs (R6) R7-Si- group, wherein R5, R6 and R7 are the same or different and denote alkyl Cl-6 or aryl, - mono-, di-or oligosaccharide group, while at least one hydroxyl group of saccharide group may be substituted by the same or different acyl, alkyl, acyloxyalkyl or aryl groups; R3 denotes hydrogen atom or-COCH3 group, and R4 denotes hydrogen atom,- S03H, SO3-, -NH2 or-NO2 group or its pharmaceutically acceptable salt, together with the usual carriers and/or auxiliaries.

The preparation according to the invention may be in a pharmaceutical dosage form suitable for oral, parenteral, intranasal, sublingual or rectal use or for administration by inhalation or insufflation. Especially the preparation may be in the dosage form of a tablet, pill, capsule, powder, granules, sterile solution or suspension, aerosol, ampoule, liposomal preparation or suppository.

Appropriate pharmaceutical dosage forms of the preparation according to the invention are prepared by the methods known by those skilled in the art.

Solid dosage forms, such as tablets, pills, powders, granules or capsules are prepared by mixing the active substance with the pharmaceutically acceptable carrier, such as corn starch, lactose, saccharose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums and other pharmaceutical diluents, for example water, in order to obtain solid preliminary mixture containing homogenous mixture of the compound according to the invention or its pharmaceutically acceptable salt. The mixture obtained in this way may be then tabletted or drageetted or the capsules may be filled with it. Tablets or granules of the new composition may be coated or processed in another way in order to obtain unit form ensuring preferable prolonged action. A range of various substances may be used for preparation of such protective or coating layers including various polymeric acids and mixtures of polymeric acids with such substances as shellac, cetyl alcohol or cellulose acetate.

Liquid dosage forms appropriate for oral or parenteral administration of the preparations according to the invention include water solutions, syrups, water or oil

suspensions, emulsions with edible oils such as cotton seeds oil, sesame oil, coconut or peanut oil as well as elixirs with similar pharmaceutical carriers. Appropriate dispergating or suspending agents for water suspensions include synthetic or natural gums such as tragacanth, acacia, alginate, dextran, carboxymethyl sodium cellulose, methylcellulose, polyvinyl pyrrolidone or gelatine.

Pharmaceutical preparations according to the invention may be used in chemotherapy and/or chemoprevention of neoplasms.

The invention is further illustrated by the following examples.

Example 1 7-tert-Butyldimethylsilyloxy-5-hydroxy-3- (4\'-hydroxyphenyl) chromen-4-on (2a) and 7-dihydroxy-3- (4\'-t-utyldimethylsilyloxyphenyl) chromen-4-on (2b) Imidazole (1.36g, 20 mmol) and tert-butyldimethylsilyl chloride (1.65 g, 11 mmol) were added to genistein solution 1 (2.7 g, 10 mmol) in anhydrous N, N- dimethylformamide (DMF), the reaction was carried out at room temperature for 24 hours. After that time the reaction solution was diluted by toluene (300 ml) and rinsed with water (2 x 50 ml). Toluene solution was dried with anhydrous MgS04, concentrated on a rotary evaporator and the residue of 0.3 g of yellow oil soldifying at room temperature was purified by column chromatography method (flash) eluting with the mixture of hexan : ethyl acetate (10: 1 v/v). 0.27 g (yield: 7%) of the mixture of products 2a and 2b was obtained in the form of white crystals.

For compounds 2a and 2b : 1H NMR (CDC13, 8 (ppm) ; 0.21 (s, 6H, CH3), 0. 99 (s, 9H, t-Bu), 6.33-638 (m, 2H, H-6, H-8), 6,86-6.92 (m, 2H, H-3\', H-5\'), 7.38 (d, 2H, H-2\'. H-6\', J=8.6 Hz), 7.85 (s, 1H, H-2), 12.92 (s, lH, 5-OH).

HR MS for C2lH240sSi : calculated 384. 1393, found 384. 1382 ; M/z 384 (M+), 327 (100%).

Example 2 7-tert-Butyldimethylsilyloxy-5-hydroxy-3- (4\'-t- butyldimethylsilyloxyphenyl) chromen-4-on (3)

Following the procedure described in Example 1, imidazole (2.72 g, 40 mmol) and tert-butyldimethylsilyl chloride (3.3 g, 22 mmol) were added to genistein solution 1 (2.7 g, 10 mmol) in DMF (30ml). The reaction was carried out at room temperature for 1.5 hours. 5.34 g of yellow precipitate was obtained, it was purified by chromatography with eluting mixture of hexan : ethyl acetate (20: 1 v/v). 4.19 g of the product 3 was obtained (yield: 84%) and 0.26 g (yield: 6%) of the mixture of products 2a and 2b was obtained in the form of white crystals.

For compound 3 : lH NMR (CDC13), 8 (ppm) ; 0.23 (s, 6H, CH3), 0.28 (s, 6H, CH3) 0.99 (s, 18H, t-Bu), 6.31 (d, 1H, H-6, J=2. 2 Hz), 6.35 (d, 1H, h-8, J=2. 2 Hz), 6.91 (d, 2H, H-3\', H-5\', J=8. 6 Hz), 7.39 (d, 2H, H-2\', H-6\', J=8.6 Hz), 7.86 (s, 1H, H-2), 12.82 (s, 1H, 5-OH).

HR MS for C27H3gO5Si2 : calculated 498.2257, found 498.2248; m/z 498 (M+), 441 (100%).

Example 3 7-tert-Butyldimethylsilyloxy-5-acetoxy-3- (4\'-acetoxyphenyl) chromen-4-on (4) 7-t-butyldimethylsilyloxy-5-hydroxy-3- (4\'-acetoxyphenyl) chromen-4-on (5) Acetic anhydride (1. 5ml, 16 mmol) was added to the solution of compound 3 (4g, 8 mmol) (obtained according to the example 2) in anhydrous pyridine (15 ml). The reaction was carried out at room temperature for 3 hours. After that time the mixture was diluted with toluene (100 ml) and rinsed with cold water (3 x 30 ml). Toluene solution was dried with anhydrous MgS04, concentrated under vacuum on the evaporator and additionally evaporated with toluene (2 x 10 ml). The obtained yellow solid (4.03 g) was purified on chromatographic column with use of eluting mixture of hexan : ethyl acetate (15: 1 vol). 2.32g (yield: 62%) of product 4 in the form of white crystals and 1.02 g (yield: 30%) of product 5 in the form of light yellow crystals was obtained.

For compound 4 : 1H NMR (CDC13), 8 (ppm) ; 0.21 (s, 6H, CH3), 0.99 (s, 9H, t-Bu), 2.34 (s, 3H, CH3CO), 6.84 (d, 1H, J=2.4 Hz), 6.87 (d, 2H, H-3\', H-5\', J=8.6 Hz), 7.23 (d, 1H, h-6, J=2.4 Hz), 7.34 (d, 2H, H-2\', H-6\', J=8.6 Hz), 7.85 (s, 1H, H-2).

HR MS for C25H2807Si : calculated 468.1604, found 468.1620; M/z 468 (M+), 426 (54%), 369 (100%), 327 (99%).

For compound 5 : 1H NMR (CDC13), 8 (ppm), 0.23 (s, 6H, CH3), 1.00 (s, 9H, tBu), 2.33 (s, 3H, CH3CO), 6.58 (d, 1H, H-6, J=2.2 Hz), 6.75 (d, 1H, H-8, J=2.2), 6.92 (d, 2H, H-3\', H-5\', J=8.6 Hz), 7.40 (d, 2H, H-2\', H-6\', J=8. 6 Hz), 7.94 (s, 1H, 5-OH)..

HR MS for C23H2606Si : calculated 426.1498, found 426.1506; m/z 426 (M+), 369 (62%), 327 (100%).

Example 4 7-Hydroxy-5-acetoxy-3- (4\'-acetoxyphenyl) chromen-4-on (6) 1M water solution of Bu4NF (0.8 ml, 0.8 mmol) was added to the solution of compound 4 (0.8 g, 1.6 mmol) (obtained according to Example 3) in THF. The solution was stirred at room temperature while controlling the reaction course by TLC method. After several minutes the reaction was ended, the reaction mixture was diluted with methylene chloride (50 ml) and rinsed with water (20 ml).

Methylene chloride solution was dried with anhydrous MgS04, concentrated and the residue in the form of precipitate was purified by column chromatography method using eluting mixture of benzene : ethyl acetate (4: 1/). 0.3 g of product 6 (yield: 53%) was obtained in the form of white crystals.

For compound 6 : 1H NMR (CDC13), & (ppm), 2.28 (s, 3H, CH3CO), 2.29 (s, 3H, CH3CO0, 6.60 (d, 1H, H-6, J=2.38 Hz), 6.80 (d, 1H, H-8, J2.38 Hz), 7.21 (d, 2H, H-3\', H-5\', J=8.6 Hz), 7.50 (d, 2H, H-2\', H-6\', J=8.6 Hz), 8.36 (s, 1H, H-2), 11.19 (s, 1H, 7-OH).

HR MS for C19H1407 : calculated 354.0739, found 354.0722; m/z 354 (M+), 312 (37%), 27- (100%).

Example 5 7-O-Benzoil-4\'-O-tert-butyldimethylsilylgenistein (7) Following the procedure described in Example 3, anhydride of benzoic acid (452 mg, 2 mmol) was added do the solution of 7,4\'-di (O-tert- butyldimethylsilyl) genistein (3) in anhydrous pyridine. The solution was stirred at room temperature for 24 hours. The raw product 7 was purified by chromatography

with use of eluting mixture of hexan: ethyl acetate. 312 mg (yield: 64%) of genistein monobenzoate was obtained. (7).

For compound 7: UV nm (log s) 257 (4.62), 328; IR (KBr) 3073,2955,2929,2858, 1742 and 1670 cm-1 ; MS (LSIMS (+) NBA); m/z 489 (M. +H) +; Properties of the compounds obtained in examples 1-5 are presented in table 2. R4 for all compounds is hydrogen atom. Compound R1 R2 R3 T. t. No (°C) 2a t-Bu (CH3) 2Si-OH OH 2b OH t-Bu (CH3) 2Si- OH- 3 t-Bu (CH3) 2Si-t-Bu (CH3) 2Si- OH 72-74 t-Bu (CH3) 2Si-CH3CO-CH3CO-120-121 5 t-Bu (CH3) 2Si-CH3CO-OH 134-135 6 OH CH3CO-CH3CO-183-190 7 Bz t-Bu (CH3) 2Si- OH 152-154 Example 6 7-O-Genistein palmitate 7,4\'-O-Genistein dipalmitate Following the procedure described in Example 3, palmitoyl chloride (328 mg, 1. 2 mmol) was added to genistein solution (1) (270 mg, 1 mmol) in anhydrous pyridine (5 ml). The solution was stirred at room temperature for 24 hours. The raw mixture of 565 mg of mono-and diacyl derivative of genistein (8 and 9) was separated by chromatography with use of eluting mixture of chloroform : ethanol (98 : 2 v/v). 125 mg of compound 8 (yield 24.6%) and 190 mg of compound 9 (yield : 25.5%) was obtained in the form of white precipitates.

For compound 8 :\'H NMR (CDC13), 8 (ppm), 0.9 (t, 3H, J=6.2 Hz), 1.16-1.36 (m, 24H), 1.7 (m, 2H), 2.58 (t, 2H, J=7.4 Hz), 6.56 (d, 1H, J=2.2 Hz), 6.7 (d, 1H, J=2.2 Hz), 6.85 (dd, 2H, Jl=8. 8 Hz, J2=2. 2 Hz), 7.4 (dd, 2H, Jl=8. 8 Hz, J2=2. 2 Hz), 7.9 (s, 1H), 12.8 (s, 1H); HR MS for C31H4006 : calculated 508. 2825, found 508.2837; M/z 508 (M+), 270 (100%).

For compound 9 : IH NMR (CDC13), 5 (ppm) ; 0.9 (t, 6H, J=6.2 Hz), 1.16-1.36 (m, 48H), 1.7 (m, 4H), 2.58 (t, 4H, J=7.4 Hz), 6.56 (d, 1H, J=2.2 Hz), 6.7 (d, 1H, J=2. 2 Hz), 7.17 (dd, 2H, Jl=8. 8 Hz, J2=2. 2 Hz), 7.54 (dd, 2H, Jl=8. 8 Hz, J2=2.2 Hz), 7.96 (s, 1H), 12.8 (s, 1H); HR MS (LSIMS (+), [M. +H] +: for C47H7107 : calculated 747.5199, found 747.5249; m/z 747 [M. +H] +.

Example 7 Genistein 7-O-palmitate (8) Following the procedure described in Example 6, potassium tert-butanolate (45 mg, 0.4 mmol) was added to the genistein solution (1) (108 mg, 0.4 mmol) in N, N-dimethylformamide (DMF) (15 ml) and the mixture was stirred at room temperature for 1 hour. Then palmityloyl chloride (121 mg, 0.44 mmol) was added to the mixture and stirring was continued at room temperature for 24 hours. After that time the mixture was poured on water with ice (75 g) and extracted with 2 x 30 ml of mixture of diethyl ether: ethyl acetate (1: 1 v/v). Collected extracts were rinsed with saturated solution of NaHCO3, water, dried with anhydrous sodium sulfate, concentrated on the evaporator and the residue was purified by chromatography. 90 mg of 7-O-palmitate derivative of genistein (8) (yield: 44.3%) was obtained in the form of white precipitate for which spectroscopic IH NMR data are consistent with those described for compound 8 obtained according to Example 6.

Example 8 Genistein 4\'-O-palmitate (10) Following the procedure described in Example 7,95 mg (yield: 46.8%) of 4\'-O-palmitate derivative of genistein (10) was obtained in the form of white precipitate in the reaction of genistein (1) (108 mg, 0.4 mmol) with palmityloyl chloride (121 mg, 0.44 mmol) in the presence of potassium tert-butanolate (148 mg, 1.32 mol).

For compound 10 : IH NMR (CDC13), b (ppm), 0.9 (t, 3H, J=6.2 Hz), 1.26-1.35 (m, 24H), 1.7 (m, 2H), 2.58 (t, 2H, J=7.4 Hz), 6.24 (d, lH, J=2.0 Hz), 6.30 (d, 1H, J=2.0 Hz), 7.15 (d, 2H, J=8.4 Hz), 7.5 (d, 2H, J=8.4 Hz), 7.8 (s, 1H), 12.8 (s, 1H) ;

HR MS for C3lH4006 : calculated 508.2825, found 508.2800; m/z 508 (M+), 270 (100%).

Example 9 Genistein 4\'-O-anthranilate (11) Following the procedure described in Example 7, isatin anhydride (0.897 g, 5.5 mmol) and catalytic amount of 4-dimethylaminopyridine (DMAP) (61 mg, 0.5 mmol) were added to genistein solution (1) (1.35 g, 5 mmol). The mixture was stirred at 95-100°C for 6 hours. After cooling to room temperature, the mixture was poured on water with ice (90 g), raw precipitate of product 11 was filtered, rinsed with water, dried and purified by chromatography with use of eluting mixture of chloroform : ethanol (100: 2 v/v). 1.33 g (yield: 68%) of compound 11 was obtained in the form of light-yellow precipitate.

For compound 11 : IHNMR (DMSO-d6), 8 (ppm); 6.25 (d, 1H, J=2.2 Hz), 6.42 (d, 1H, J=2. 2), 6.6 (m., 1H), 6.7 (bs, 2H), 6.84 (dd, 1H, J=8.6 Hz), 7.3 (m., 3H), 7.6 (dd, 2H, Jl=6. 8 Hz, J2=2.0 Hz), 7.9 (dd, 1H, Jl=8. 2 Hz, J2= 1.6 Hz), 8.49 (s, 1H), 10.95 (bs, 1H), 12.88 (s, 1H) ; UV nm (log F) 259 (4.57); IR (KBr) 3458,3352,1689,1652 and 1620 cm-1 ; HR MS for C22H15O6N : calculated 389.0899, found 389. 0882 ; m/z 389 (M+).

Properties of the compounds obtained in Examples 6-9 are presented in Table 3. R4 is hydrogen atom in all compounds.

Table 3 Compoun Ri R2 R3 T. t. d No (°C) 8 ClsH3lCO OH OH 120-121 9 CisHslCO CisH3iC0 OH 97-98 10 OH ClsHsiCO OH 115-116 11 OH 2-(H2N)-Ar-CO OH 241 Example 10 7-O-Pivaloyloxymethylgenistein (12a) and 4\'-O-pivaloyloxymethylgenistein (12b)

Genistein solution (1) (270mg, 1 mmol) in DMF (3 ml) with addition of diisopropylethylamine (DIPEA) (155 mg, 1.2 mmol) was stirred at room temperature for 20 minutes. After that time, pivaloyloxymethyl chloride (180, 1.2 M) and catalytic amount of 4-dimethylaminopyridine (DMAP) were added to homogenous mixture. The mixture was stirred at room temperature for 5 days. Then the mixture was poured on water with ice (50 g) and agitated with methylene chloride (2x 25 ml). Combined extracts were rinsed with water to neutral reaction, dried with anhydrous Na2S04 and concentrated on the evaporator. The oily residue was dissolved in methylene chloride and injected on a column filled with silica gel. Products of genistein alkylation (12a and 12b) were washed out with the mixture of chloroform : ethanol (100: 2 v/v). After concentration of the fraction, 120 mg (yield: 31%) of white-cream precipitate of the mixture of compounds 12a and 12 b was obtained.

For compounds 12a and 12b: UV nm (log s) 260 (4.59); IR (KBr) 3438 2973 1733 1653and 1614 cm~l ; HR MS (LS1MS (+), [M. +H] +): for C21H2107 : calculated 385.1287, found 385.1299; m/z 385 [M. +H] +.

Example 11 7-O-Allylgenistein (13) Following the procedure described in Example 10, the mixture of genistein (1) (270 mg, 1 mmol) with allyl bromide (145 mg, 1.2 mmol) in the presence of diisopropylethylamine (155 mg, 1. 2mmol) in DMF (5 ml) was heated at 60°C for 24 hours. The raw product 13 was purified by chromatography with use of eluting mixture of hexan : ethyl acetate (7: 3 v/v). 150 mg (yield: 48%) of compound 13 was obtained in the form of white-cream precipitate.

For compound 13 : lH NM : R (DMSO-d6), 6 (ppm); 4.7 (d, 2H, J=5.2 Hz), 5.27-5.47 (m, 2H), 5.95-6.14 (m., 1H), 6.43 (d, 1H, J=2.2 Hz), 6.68 (d, 1H, J=2.2 Hz), 6.81 (d, 2H, J=8. 6 Hz), 7.38 (d, 2H, J= 8.6 Hz), 8.41 (s, 1H), 9.62 (bs, lH), 12.95 (s, 1H); UV nm (log s) 262 (4.57); IR (KBr) 3126,1666,1612 1573 and 1517 cm~l.

Example 12 7-O-Allyl-5-acetoxy-3- (4\'-acetoxyphenyl) cliromen-4-on (14) Acetate anhydride (5 ml) was added to the solution of compound (13) (310 mg, 1 mmol) in anhydrous pyridine (5 ml). The mixture was stirred at room temperature for 24 hours. Then the mixture was diluted with ethyl acetate (50 ml), rinsed with water (50 ml), 1M solution of hydrochloric acid, saturated solution of NaHC03 and brine. The extract of ethyl acetate was dried with anhydrous Na2S04, concentrated on the evaporator and the residue was purified by chromatography with use of eluting mixture of hexan : ethyl acetate (7: 3 v/v). 315 mg (yield: 80%) of compound 14 was obtained in the form of white precipitate.

For compound 14 :\'H NMR (CDC13), b (ppm); 2.31 (s, 3H), 2.41 (s, 3H), 4.63 (d, 2H, J=5.3 Hz) 5.33-5.49 (m, 2H), 5.95-6.15 (m, 1H), 6.64 (d, 1H, J=2.4 Hz), 6.79 (d, 1H, J=2.4 Hz), 7.14 (d, 2H, J=8. 6Hz), 7.83 (s, lH) ; UV nm (log s) 251 (4.45); IR (KBr) 3123,3081 1773,1644 1564 and 1507 cm-1.

MS (LSIMS (+), NBA): m/z 395 [M. +H] +.

Example 13 4\'-O- [2- (3-Cyano-5-nitropyridyl)] genistein (15) Following the procedure described in Example 10, the mixture of genistein (1) (540 mg, 2 mmol), 2-chloro-3-cyano-5-nitropyridine (403 mg, 2.2 mmol) in the presence of diisopropylethylamine (400 mg, 3.1 mmol) in DMF (6 ml) was heated at 80°C for 3.5 hours. Then the mixture was chilled to room temperature and poured on water with ice (60g). The precipitate of product 15 was filtered, rinsed with water and dried. 718 mg (yield: 86%) of product 15 was obtained in the form of light-beige precipitate. The analytical sample (100 mg) was purified by chromatography with use of eluting mixture of hexan: acetate (2: 1 v/v).

For compound 15 :\'H NMR (DMSO-d6), 8 (ppm), 6.25 (d, 1H, J=2.2 Hz), 6.43 (d, 1H, J= 2.2 Hz), 7.41 (d, 2H, J=8.7 Hz), 7.70 (d, 2H, J=8.7 Hz), 8.51 (s, 1H), 9.25 (d, 1H, J=2. 7), 9.35 (d, 1H, J=2.7), 9.95 (bs, lH), 12.85 (s, 1H); UV nm (log s) 262 (4.57); IR (KBr), 3382,3082,2248 (CN), 1654,1605 1578 and 1516 cm ;

MS (LSIMS (+), NBA): m/z 418 [M. +H] +.

Example 14 Caesium salt of genistein 4\'-O-propylsulfonate (16) The mixture of caesium genisteinate (100 mg, 0.25 mmol) (obtained in the form of cream precipitate in the reaction of genistein (270 mg, 1 mmol) with caesium carbonate (162 mg, 0.5 mmol) in methanol (10 ml) carried out at room temperature for l/z hour and evaporating methanol to dryness), propano-1, 3-diyl sulfate (42 mg, 0.3 mmol) in DMF (3 ml) was heated at 60°C for 1.5 hours. Then the mixture was evaporated under vacuum and the residue was crystallised from ethanol. 85 mg (yield: 65%) of compound 16 was obtained in the form of crystalline precipitate.

For compound 16 : 1H NMR (DMSO-d6), 8 (ppm) ; 1.87 (t, 2H, J=6.0 Hz), 3.55 (t, 2H, J=6.0 Hz), 4.15 (t, 2H, J=6.0 Hz), 6.39 (d, 1H, J=2.2 Hz), 6.64 (d, 1H, J=2. 2 Hz), 6.82 (d, 2H, j=8.6 Hz), 7.38 (d, 2H, J=8. 6 Hz), 8.39 (s, 1H), 9.59 (bs, 1H), 12.95 (s, 1H); UV nm (log s) 262 (4.38); IR (KBr), 1668,1573 and 1520 cm-1; Properties of the compounds obtained in Examples 10-14 are presented in Table 3. R4 is hydrogen atom for all compounds.

Table 3 Compoun Ri R2 R3 T. t. d No (°C) 12a (CH3) 3C02CH2 OH OH 12b OH (CH3) 3C02CH2 OH 13 CH2=CHCH2 OH OH 146 14 CH2=CHCH2 CH3CO-CH3CO-176-177 15 OH 2- (3-CN-5- OH 266 N02) pyridyl 16 OH CH2CH2CH2SO OH 3

cs+

Example 15 3\'-Nitrogenistein (17) Solution of 65% nitric acid (1 ml) was added to the mixture of genistein (1) (270 mg, 1 mmol) in glacial acetic acid (3 ml) and the mixture was stirred for 2 hours at 0-5°C, and then at room temperature for 4 hours. After that time the mixture was poured to water with ice (40 g). The precipitate of product 17 was filtered, rinsed with water and dried. 242 mg (yield: 77%) of compound 17 was obtained in the form of yellow precipitate. The analytical sample (100 mg) was purified by chromatography with use of the eluting mixture of hexan: acetate (2: 1 v/v).

For compound 17 : 1H NMR (DMSO-d6), 8 (ppm), 6.07 (d, 1H, J=2. 0Hz), 6.25 (d, 1H, J=2.2 Hz), 7.02 (d, lH, J=8.79 Hz), 7.58 (d, 1H, Jl=8. 6Hz, J2=2.2 Hz), 7.97 (d, 1H, J=2.2 Hz), 8. 33 (s, 1H), 10.8 (bs, 1H), 11.05 (bs, 1H), 12.6 (bs, 1H); UV nm (log s) 262 (4.57); IR (KBr), 3515,3449,3279,3087,2656,1663,1633, 1612,1581,1537 and 1495 cm~l ; Example 15 3\'-Sulfogenistein (18) Genistein suspension (1) (270 mg, 1 mmol) in concentrated sulfuric acid (5 ml) was stirred at room temperature for 12 hours. After that time the mixture was poured to water with ice (50 g) and extracted with the mixture of n-butanol : ethyl acetate (8: 2 v/v) 2 x 25 ml. The combined extracts were rinsed with the mixture of brine: water (6: 4 v/v) 3 x 25 ml to pH = 2.5 and dried with anhydrous sodium sulfate. After removal of solvents to dryness, 213 mg (yield: 60%) of compound 18 was obtained in the form of crystalline precipitate.

For compound 18 : 1H NMR (DMSO-d6), 8 (ppm); 6.22 (d, 1H, J=2.2 Hz), 6.34 (d, 1H, J=2.2 Hz), 6.81 (d, 1H, J=8.4 Hz), 7.37 (dd, 1H, J1=8. 4 Hz, J2=2.3 Hz), 7.67 (d, 1H, J=2.2 Hz), 8.4 (s, 1H), 10.6 (bs, 1H), 10.85 (s, 1H), 12.95 (s, 1H); UV nm (log s) 262 (4.49); IR (KBr), 1665,1633,1612,1512,1437 and 1197 cm~l.

Properties of the compounds obtained in Examples 15-16 are presented in Table 5.

Table 5 Compound Ri R2 R3 R4 T. t. No (°C) 17 OH OH OH N02 262-263 18 OH OH OH SO3H Example 17 7-0- (3", 4"-di-O-Acetyl-6-deoxy-glucopyranosyl) genistein (19) 3,4-di-O-Acetyl-6-deoxy-L-glucal (428 mg, 2 mmol) and catalytic amount of triphenylphosphine hydrobromide (PPh3xHBr) (34 mg, 0.1 mmol) were added to genistein solution (1) (270 mg, 1 mmol) in anhydrous THF (5 ml). The mixture was stirred at 50°C for 24 hours. After cooling to room temperature, the mixture was poured water with ice (50 g) with addition of saturated solution of NaHCO3, the neutral mixture was rinsed with ethyl acetate (50 ml) and water. The extract of ethyl acetate was dried with anhydrous Na2S04, concentrated and the residue was purified by chromatography with use of the eluting mixture of chloroform : ethanol (100: 2 v/v). 90 mg (yield: 18%) of compound 19 was obtained in the form of colourless precipitate.

For compound 19 : 1H NMR (CDC13), 8 (ppm), 1.16 (m, 3H), 2.07 (s, 6H), 2.46 (m, 1H), 3.94 (m, 1H), 4.84 (t, 1H), 5.47 (m, 1H), 5.62 (d, lH), 6.36 (d, 1H, J=2.2 Hz), 6.39 (d, 1H, J=2.2 Hz), 7.12 (d, 2H, J=8.6 Hz), 7.42 (d, 2H, J=8. 6 Hz), 7.79 (s, 1H), 9.40 (bs, 1H), 12.78 (s, 1H) ; UV nm (log s) 261 (4.53); IR (KBr) 1745,1654,1512 and 1369 cm-1.

Example 18 7-0- (2", 3", 5"-tri-O-Benzyl-arabinofuranosyl) genistein (20) Catalytic amount of SnCl4 (333mg, 1.28 mmol) was added to the mixture of genistein (1) (270 mg, 1 mmol), 2,3,5-tri-O-benzylarabinofuranose (428 mg, 2 mmol) in anhydrous acetonitrile (5 ml) cooled to °C. Then the mixture was stirred at room temperature for 40 minutes. After that time the mixture was poured on water with ice (30 g) with addition of saturated solution of NaHC03 (20 ml), the neutral mixture was rinsed with ethyl acetate (50 ml) and water. The extract of ethyl acetate was dried with anhydrous Na2S04, concentrated and the residue was purified by chromatography with use of eluting mixture of hexan : ethyl acetate (8: 2 v/v). After evaporation of appropriate fractions, 150 mg (yield: 22%) of compound 20 was obtained in the form of colourless precipitate.

For compound 20 : 1H NMR (CDC13), 8 (ppm); 3. 69 (d, 2H), 3.99 (, 1H), 4.19-4.28 (m, 2H) 4.26 (q, 2H), 4.48 (q, 2H), 5.59 (s, 2H), 6.41 (s, 1H), 6.89 (d, 2H), 7.05 (m, 2H), 7.18-7.35 (m, 14H), 7.39 (d, 2H), 7.83 (s, 1H), 9.79 (s, 1H), 13.21 (s, 1H); HR MS (LSIMS (+), {M. +H] +): for C41H3709 : calculated 673.2437, found 673.2452; m/z 673 [M. +H] +.

Example 19 4\'-O- (2", 3", 5"-tri-O-Acetyl-a, [3-D-ribofuranosil-genistein and 4\'-O- (2", 3", 5"-tri-O-Acetyl-a, P-D-ribofuranosil) genistein (21a, 21b) 4.36 ml of 0.87M solution of SnCl4 in CH2C12 (990 mg, 3.8 mmol) was added at room temperature to anhydrous solution of tetraacetylribose (954.8mg, 3.0 mmol) in 40 ml of methylene chloride. After 20 minutes this solution was added to stirred genistein suspension (540.5 mg, 2.0 mmol) in anhydrous acetonitrile and clear, yellow solution was obtained. After 50 minutes of stirring at room temperature the reaction was ended by adding 40 ml of cold saturated solution of NaHC03 and 80 ml of chloroform. The organic layer was separated, the water layer was rinsed with the mixture of chloroform-acetonitrile 4: 1 (50 ml). The combined

organic layers were dried over Na2S04 and then evaporated to dryness. The residue after evaporation was treated with chloroform and the obtained suspension was filtered. The filtrate was evaporated to oil which was dissolved in 5 ml of the mixture chloroform : ethanol 4: 1 (v/v) and separated on chromatographic column with silica gel 60H (5 x 8 cm) in chloroform-ethanol gradient (from 98: 2 to 9: 1).

After evaporation of the solvent, 550 mg (52%) of the main product was obtained, which was the mixture of a and ß anomers in a ratio of 1: 2 (NMR).

Example 20 4\'-O- (a-D-Ribofuranosil) genistein and 4\'-O- (P-D-Ribofuranosil) genistein (22a i22b).

The raw mixture of a and ß anomers of triacetylribogenistein obtained in the previous example (400 mg, 0.757 mmol) was dissolved in 12 ml of methanol and 4 ml of 25% NH40H was added. After 24 hours at room temperature, a complete unblocking of hydroxyl groups was determined on the base of TLC. The mixture was evaporated to dryness, then it was co-evaporated with water (2 x 50 ml) and with ethanol (2 x 50 ml). The obtained oil was dissolved in ethanol (100 ml) and adsorbed on silica gel (70-230 mesh) by evaporation. The product was separated on a column with silica gel 60H (3.5 x15 cm) in toluene-ethanol system (4: 1).

The following products were obtained: 128.8 mg (42%) of anomer ß in the form of white precipitate which was crystallised from water, then fine needles of slightly pink colour were obtained, t. t. 199.5- 201°C, purity according to HPLC-98. 9%. lH-NMR (DMSO-d6), 8 (ppm) : 12.91 (s, 1H); 10.92 (s, 1H), 8.38 (s, 1H), 7.49 (d, 2H), 7.04 (d, 2H), 6.40 (d, 1H); 6.23 (d, 1H), 5.51 (s, 1H) ; 5.36 (d, 1H), 5.03 (d, 1H), 4.70 (t, lH) ; 4.02 (m, 2H); 3.91 (m, 1H) ; 3.54 (m, 1H) ; 3.36 (m, 1H); 64.4 mg (21.1%) of anomer oc which was crystallised from 30% ethanol to obtain very fine pink needles of t. t. 158-160°C, purity according to HPLC-90.0%. lH-NMR (DMSO-d6), 8 (ppm) : 12.92 (s, 1H) ; 10.93 (bs, 1H); 8. 39 (s, 1H); 7.49 (d, 2H); 7.11 (d, 2H); 6.40 (d, 1H); 6.24 (d, 1H); 5.63 (d, 1H) ; 4.72 (d, 1H); 4.92 (d, 1H); 4.82 (t, 1H) ; 4.07 (m, 1H), 3.96 (m, 2H), 3.49 (m, 2H). Properties of the compounds obtained in Examples 17-20 are presented in Table 6.

R4 is hydrogen atom in all compounds.

Table 6 Compound Ri R2 R3 T. t. No (°C) 19 A OH OH 20 B OH OH 133-134 21 a OH C OH 21b OH D OH - 22a OH E OH 158-160 22b OH F OH 199-201

Structures of substituents in formulae of compounds 19-22 are presented below.

19 : Rl= a: 20 : R1= b: 21a: R2= 21b : Ra= d: 22b: R2= e :

22b: R2= f :