WO2009012910A1

WO2009012910A1 - Biologically-active stilbene derivatives and compositions thereof

Info

Publication number: WO2009012910A1
Application number: PCT/EP2008/005751
Authority: WO
Inventors: Luisella Verotta; Fulvia Orsini; Clarissa Gerhauser; Karin Klimo
Original assignee: Universita'degli Studi Di Milano; Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority date: 2007-07-20
Filing date: 2008-07-15
Publication date: 2009-01-29

Abstract

The invention relates to stilbene derivatives displaying a large spectrum of biological activities, particularly anti-cancer, antioxidant and antiinflammatory activities, pharmaceutical and nutraceutical compositions containing such compounds and their uses as therapeutic agents or nutritional supplements.

Description

BIOLOGICALLY-ACTIVE STILBENE DERIVATIVES AND COMPOSITIONS THEREOF

The present invention relates to stilbene derivatives which are structurally close to the natural compound resveratrol and which possess a large spectrum of biological activities, particularly anti-cancer, antioxidant and antiinflammatory activities. The invention further concerns pharmaceutical and nutraceutical compositions containing such stilbene derivatives and their uses as therapeutic agents or nutritional supplements. Background of the invention

Resveratrol (trans-S^'.δ-trihydroxystilbene), a polyphenols phytoalexin present in grapes, peanuts and mulberry, has attracted incerasing interest of the biomedical researchers due to its relatively simple structure and the number of beneficial physiological effects it produces (Pervaiz et al., 2003) An inverse correlation between red wine assumption and the incidence of cardiovascular diseases gave rise to a number of reseacrh studies on the effects of resveratrol on platelet aggregation (Orsini et al., 1997) potassium channel modulation (Orsini et al., 2004) and lipid peroxidation inhibition (Miller et al., 1995) all mechanisms involved in coronary artery disease.

Resveratrol has been identified as a potent cancer chemopreventive agent in assays representing the three major stages of carcinogenesis (i.e. tumor initiation, promotion and progression) (Jang et al., 1997). Recent investigations in this field have bee directed at understanding the molecular mechanisms that underlie this important physiological effect and found resveratrol to suppress cellular proliferation via inhibition of key steps in signal transduction pathways (Haworth et al., 2001 ; Pozo-Guisado et al., 2002) and cyclin-dependent kinases (Delia Ragione et al., 1998; Kim et al., 2003) scavenging/suppression of intracellular reactive oxygen species (ROS) (Manna et al., 2000) anti-inflammatory activity via inhibition of cyclooxidenase (Cox) and down-regulation of pro-inflammatory cytokines (Wadsworth et al., 1999) androgen receptor function inhibition and estrogenic activity (Hsieh et al., 2000; Lu et al. 1999) and induction of apoptotic cell death (Huang et al., 1999; Roberti et al., 2003). State of the art

US2004/014778 discloses stilbene derivatives having specific antagonistic ligand properties with respect to the Aryl Hydrocarbon Receptor (AhR) and their use for the prevention and treatment of poisoning and pathologies caused by toxic aryl hydrocarbons and other ligands of the AhR.

WO2004/00302 discloses resveratrol analogs which have antioxidant properties and are used in skin desquamation compositions, for reducing the adhesion of microorganisms on the skin and in skin-fortifying compositions. WO2004/041758 discloses stilbene derivatives having specific antagonistic ligand properties with respect to the Aryl Hydrocarbon Receptor

(AhR) and their use for the prevention and treatment of poisoning and pathologies caused by toxic aryl hydrocarbons and other ligands of the AhR.

WO03010121 discloses medicinal compositions for treating hyperpigmentation and cosmetic compositions for whitening skin containing stilbene derivatives as an active ingredients.

CN1566054 discloses a veratrum album alcohol oligomer stilbene compounds and their preparing process, pharmaceutical compositions containing them, and their use as medicaments, especially as medicaments for treating chronic infectious arthritis, asthma and allergic inflammation.

The European Journal of Organic Chemistry (2006), 15:3457-3463, discloses prenylated stilbenes derivatives isolated from the stems of Artocarpus chama, and their cytotoxicity against HepG2 cells. Description of the invention

The invention provides stilbene derivatives resembling the structure of the natural compound resveratrol, which proved particularly effective as chemopreventive, antioxidant, antiestrogenic, antitumor and antinflammatory agents. According to a first embodiment, the invention provides a compound of formula (I):

(I) wherein:

R1 , R2 and R3, independently from one another, represent H or (Ci-C₃)alkyl;

R4 and R5 are identical or different and represent hydrogen, linear or branched (Ci-C5)alkyl, a prenyl group -CH2-CH=C(CH3)2, a geranyl group

-CH2-CH=C(CH₃)(CH2)2CH=C(CH₃)2 or R₄ and R1 , and independently R5 and R2, together with the atoms they are linked to, form one of the following groups:

with the provisos that R4 and R5 are not both hydrogen and that when

R1=R2=R3=H, R4 and R5 are not a prenyl group and hydrogen, respectively.

The compounds of formula (I) wherein R1 , R2 and R3 represent H, R4 and R5 are identical or different and represent hydrogen, a prenyl group -CH₂-CH=C(CHa)₂ or a geranyl group -CH₂-CH=C(CH₃)(CH₂)₂CH=C(CH₃)₂ or R₄ and R1 , and independently R5 and R2, together with the atoms they are linked to, form one of the following groups: subject to the above provisos, are preferred.

In a particularly preferred embodiment, the compounds of formula (I) are selected from the group consisting of and

The compounds of the invention can be prepared according to the general procedures illustrated in the following scheme:

Rl, R2 and R3 = H or C1-C3 alkyl R4, Ri = H or C1-C5 alkyl or (CH3)2C=CHCH2- (prenyl) or

Rl, R2 and R3 = H or C1-C3 alkyl (CH3)2C=CH(CH2)2C(CH3)=CHCH2- R4 =R5 =H (geranyl)

R = H, or (CH3)2C=CHCH2- (prenyl) Rf = H or (CH3)2C=CHCH2- (prenyl) R2 and R3 = H or C1-C3 alkyl

R = H, or (CH3)2C=CHCH2- (prenyl) R3 =H or Cl-C3 alkyl

In a further embodiment, the invention provides a pharmaceutical or nutraceutical composition containing a compound of formula (I) together with physiologically-compatible vehicles and/or excipients. In a preferred embodiment, the compositions are suitable for oral intake. Examples of oral compositions according to the invention include aqueous solutions or dispersions, powders, capsules, tablets and syrups. The liquid formulations can be added with a solubilizer or co-solvent such as an alcohol. The compounds can also be embedded in liposomes or nanoparticles. In alternative, the compounds of the invention can be combined with food stuff or beverages to prepare nutritional supplements.

The antioxidant properties exhibitied by the invention compounds render their use as cell and tissue radical scavengers particularly attractive. The invention compounds showed to possess significant anti-inflammatory, chemopreventive and anti-tumor activities, especially against estrogen-dependent cancer. The estrogen-modulating activity in particular allows for their use in the treatment or prevention of osteoporosis and in substitutive hormone therapy as alternatives to estrogens of animal origin (such as conjugated equine estrogens) or to beta-estradiol. EXPERIMENTAL SECTION

Resveratrol, BuLi and dimethylallylbromide were purchased from

Fluka; dry THF was purchased from Aldrich. Proton and carbon nuclear magnetic resonance (1 H NMR and 13C NMR) spectra were recorded at 400

MHz on Bruker Avance spectrometer. MS spectra were recorded with a

VG7070 E9 spectrometer. Reversed phase chromatography was performed on RP18 (25-40 μm, Merck). Thin-layer chromatographies were performed on silica gel plates (60 F254, Merck): spots were detected visually by ultraviolet irradiation (254 nm) or by spraying with methanol:H2SO4 9:1 , followed by heating at 100⁰C.

Synthesis of compounds ES

A solution of resveratrol (500 mg, 2.2 mmol) in dry THF (10 mL), is stirred at -40⁰C₁ under N2, then n-BuLi is added dropwise (2.5 mL of a solution 1.6 M in n-hexane; 2 eq/mol). After 1 h the reaction mixture is allowed to reach -30⁰C, 3,3-dimethylallylbromide (512 μL, 2 eq/mol) is added dropwise and the temperature is slowly raised to -2O⁰C. After 6 h, a solution of 1M NH4CI is added and the reaction is extracted with EtOAc (3 x 15 mL). The organic phases are pooled and washed with water, then dried (Na2SO4) and evaporated to dryness under vacuum. The crude extract (600 mg) is purified by medium pressure chromatography on RP 8 (25-40 μm; 25 x 2.5 cm) eluted with MeOH-H2O 75:25 (1 L) and MeOH-H2O 8:2 (500 mL). (Fractions (10 mL each) were combined according to their composition to give ES32 (27-36, 90 mg), ES35 (41-45, 80 mg), ES33 (56-70, 80 mg), ES34 (87-100, 85 mg). The column was washed with MeOH (200 ml_) and this fraction was re-cromatographed on RP 8 eluted with MeOH-H2O 9:1 to give ES22 (50 mg). ES32: pale yellow viscous oil. ¹H-NMR (CDCI₃, 400 MHz):δ 7.28 (2H,d,

J=7.8 Hz, H-Z+6'), 7.08 (1 H, d, J= 16.5, H-1α), 6.81 (1H, d, J=16.5 Hz, H- 1 β), 6.81 (2H, d, J= 7.8 Hz, H-3'+5'), 6.60 (1H, bs, H-2), 6.33, 1 H₁ bs, H-4), 5.12 (1 H, bt, J= 7.0 Hz, H-3"), 3.35 (2H₁ bd, J= 7.0 Hz, H-2"), 1.76 (3H, s, H-4"), 1 -67 (3H, s, H-5"). ¹³C (100 MHz) see Table 1. EIMS (70 eV) m/z: 296 [M]⁺

ES35: pale yellow viscous oil. ¹H-NMR (CDCI₃, 400 MHz): δ 7.34 (2H₁(I₁ J=7.8 Hz, H-2'+6'), 7.03 (1H, d, J= 16.5, H-1α), 6.86 (1H, d, J=16.5 Hz, H-1 β), 6.82 (2H, d, J= 7.8 Hz, H-3'+5'), 6.55 (1 H, d, J= 2Hz, H-2), 6.24 (1 H, d, J= 2Hz, H-4), 2.71 (2H, bt, J= 7.2 Hz, H-2"), 1.77 (2H, t, J= 7.2 Hz, H- 3"), 1.30 (6H, s, H-4"+5"). .¹³C (100 MHz) see Table 1. EIMS (70 eV) m/z: 296 [M]⁺

ES33: pale yellow viscous oil. ¹H-NMR (CDCI₃, 400 MHz): δ 7.35 (2H,d, J=7.8 Hz, H^'+δ¹), 6.94 (1 H, d, J= 16.5, H-1α), 6.81 (2H₁ d, J= 7.8 Hz, H-3'+5'), 6.37 (1H, d, J=16.5 Hz, H-1 β), 6.23 (1 H, s, H-4), 5.21 (2H, m, H- 3"+3'"), 3.39 (4H₁ bt, J= 7.0, H-2"+2'"), 1.74 (12H, s, H-4"+4"'+5"+5"'). ¹³C (100 MHz) see Table 1. EIMS (70 eV) m/z: 364 [M]⁺, 296 (100).

ES34: pale yellow viscous oil. ¹H-NMR (CDCI₃, 400 MHz): δ 7.35 (2H,d, J= 8.8 Hz₁ H-2'+6'), 6.87 (1 H, d, J= 16.6, H-1α), 6.83 (2H₁ d, J= 8.8 Hz, H-3'+5'), 6.46 (1H₁ d, J=16.6 Hz₁ H-1β), 6.28 (1 H, s, H-4), 5.23 (1H, bt, J= 7.0 Hz₁ H-3"), 3.40 (2H₁ m, H-2¹"), 2.64 (2H, bt, J= 7.2 Hz, H-2"), 1.81 (2H₁ m, H-3"'), 1.74 (6H, s, H-4"+5"), 1.33 (6H, s, H-4"'+5'"). ¹³C (100 MHz) see Table 1. EIMS (70 eV) m/z: 364 [M]⁺, 296 (100).

ES22: pale yellow viscous oil. ¹H-NMR (CDCI₃, 400 MHz): δ 7.32 (2H,dd, J= 7.5,1.0 Hz, H^'+δ¹), 6.83 (2H, dd, J= 7.5,1.0 Hz, H-3'+5'), 6.79 (1 H, d, J= 15.5, H-1α), 6.51 (1 H, d, J=15.5 Hz, H-1 β), 6.18 (1 H, s, H-4), 2.67 (4H, bt, J= 7.0 Hz, H-2"), 1.71 (4H, bt, J= 7.0 Hz, H-3"¹), 1.28 (12H, s, H- H- 4"+4'"+5"+5"¹). ¹³C (100 MHz) see Table 1. EIMS (70 eV) m/z: 364 (100) [M]⁺, 296, 253.

Table 1. ¹³C (100 MHz) NMR data (δ; multiplicity) for compounds (1-5). The spectra were recorded in CDCb, and the solvent signal (77.0 ppm) was used as reference. The multiplicities of the carbon signals were determined from DEPT experiments or indirectly from HMQC experiments. C ES32 ES35 ES33 ES34 ES22

1 138.2, S 138.0, s 139.0, S 138.3, S 137.2, s

2 117.5, d 111.2, S 117.7, S 112.0, S 111.5, s

3 155.5, S 155.0, s 153.6, S 152.8, S 153.2, s

4 102.4, d 103.3, d 102.6, d 103.3, d 104.2, d 5 155.6, S 155.0, s 153.6, S 153.7, S 153.2, s

6 104.0, d 104.4, d 117.7, S 117.4, S 111.5, s

1¹ 129.4, S 130.0, s 129.0, S 130.2, S 130.1 , s

127.7, d 128.0, d 127.8, d 127.7, d 127.6, s

3' 115.7, d 115.7, d 115.5, d 115.5, d 115.2, d 4' 156.9, S 156.0, s 155.3, S 155.4, S 155.9, s

5¹ 115.7, d 115.7, d 115.5, d 115.5, d 115.2, d

6' 127.7, d 128.0, d 127.8, d 127.7, d 127.6, d

1α 124.0, d 123.3, d 124.6, d 124.2, d 123.7, d

1 β 129.6, d 130.1 , d 134.2, d 134.2, d 133.7, d 1" 24.5 , t 19.9, t 26.9 , t 21.4 , t 21.4, t

2" 124.1 , d 32.9, t 122.9, d 33.2 , t 33.7, t

3" 130.4, S 76.6, s 133.8, S 73.6, S 73.3, s

4" 18.0, q 26.6, q 17.0, q 26.8, q 26.8, q

5" 25.8, q 26.6, q 25.8, q 26.8, q 26.8, q V" 26.9 , t 26.6 , t

2'" 122.9, d 123.3, d

3'" 133.8, S 133.9, , s

4- 17.0, q 17.3, q 5'" 25.8, q 25.4, q

Biological assays. Materials and methods

Chemicals.

All cell culture media and supplements were obtained from Invitrogen (Eggenstein, Germany). Fetal calf serum was provided by PAA Laboratories

(Pasching, Austria). 4-Methylumbelliferyl phosphate (MUP), 7-hydroxy-4- methylcoumarin, 3-cyano-7-ethoxycoumarin (CEC), and 3-cyano-7- hydroxycoumarin (CHC) were purchased from Molecular Probes (Mobitec,

Gδttingen, Germany). Human Cyp19 Supersomes were obtained from Gentest, BD Biosciences (Woburn, USA). The human Ishikawa cell line was obtained from S. Mader, Department of Biochemistry, University of Montreal

(Montreal, Canada). All other chemicals were purchased from Sigma

Chemical Co. (Deisenhofen, Germany).

Animals 19 days old female Sprague Dawley rats were purchased from Charles

River Wiga (Sulzfeld, Germany) and were housed in a climate controlled room with a 12 h light/12 h dark cycle. Rats were fed standard diet (Altromin, Lage, Germany). Animals were given free access to diet and water.

Test systems to detect potential cancer chemopreventive activity Inhibition of cytochrome P450 1A (Cyp1A) enzymatic activity β-Naphthoflavone-induced H4IIE rat hepatoma cell preparations were used as a source of Cyp1A enzyme activity. Cyp1A activity was determined via the time-dependent dealkylation of 3-cyano-7-ethoxycoumarin (CEC) to 3-cyano-7-hydroxycoumarin (CHC) based on the method of Crespi et al. (1997, with modifications). Briefly, H4IIE rat hepatoma cells were cultured in 100 mm tissue culture dishes at a density of 5 x 10⁴ cells/ml in MEME medium containing 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulfate and 250 ng/ml amphotericin B supplemented with 10% 51

11 fetal bovine serum at 37°C in a 5% CO2 atmosphere. After a preincubation period of 24 h, Cyp1A was induced by addition of 10 μM β-naphthoflavone (dissolved in DMSO, 0.1 % final concentration). Cells were harvested after 38 h by scraping in 1 ml/plate buffer P (200 mM potassium phosphate buffer, pH 7.4 containing 10 mM MgC^) and snap frozen in liquid nitrogen. For determination of Cyp1 A activity, test compounds dissolved in 10% DMSO (10 μl, final concentration 0.5%) were pipetted to a 96-well microplate. A freshly prepared reaction mixture (100 μl), consisting of 5.2 μl 50 mm NADP+ in water, 4.4 μl 150 mM glucose-6-phosphate in water, 0.5 U glucose-6- phosphate dehydrogenase, 0.1 μl 10 mM CEC (in DMSO) and 90.25 μl buffer P, was then added to each well. The reaction was started by addition of 90 μl cell lysate, passed once through a 27 gauge needle and diluted with buffer P to a final protein concentration of 50 to 100 μg/ml depending on Cyp1A activity. The rate of dealkylation of CEC to fluorescent CHC was measured for 45 min at 37°C in a Cytofluor 4000 microplate fluorescence reader (PE Applied Biosystems, reading every 2.5 min with prior shaking, excitation 408/20 nm, emission 460/40 nm). Vmax values were computed, and the halfmaximal inhibitory concentration IC50 was generated from the data obtained with 8 serial two-fold dilutions of inhibitors in a final concentration range of 0.004 - 0.5 μM or 0.04 - 5 μM, respectively, tested in duplicates. Enzyme activities were calculated in comparison with a CHC standard curve and normalized to protein content determined by the BCA method (Smith et al., 1985). α-Naphthoflavon as a known Cpy1A inhibitory compound was used as a positive control. Determination of NAD(P)H:quinone reductase (QR) activity in mouse hepatoma cell culture.

For the detection of phase 2 enzyme inducers, QR activity was measured in Hepa 1c1c7 cells as described previously (Prochaska and Santamaria, 1995). QR activity was determined by measuring the NADPH- dependent menadiol-mediated reduction of MTT [3-(4,5-dimethylthiazo-2-yl)- 2,5-diphenyltetrazolium bromide] to a blue formazan. Protein was determined by crystal violet staining of an identical set of test plates. Induction of QR activity was calculated from the ratio of specific enzyme activities of compound-treated cells in comparison with a solvent control, and CD values (concentration required to double the specific enzyme activity in μM) were generated. In addition, IC50 values for the inhibition of cell proliferation were calculated. Only nontoxic inhibitor concentrations resulting in greater than 50% cell viability were considered to calculate inducing potential. Scavenging of diphenyl-picryl-hydrazyl (DPPH) radicals. Radical scavenging potential was determined spectrophotometrically by reaction with 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals in a microplate format at 515 nm (van Amsterdam eta/., 1992, with modifications). Briefly, test compounds dissolved in DMSO were treated with a solution of 100 μM DPPH in ethanol for 30 min at 37°C. Scavenging potential was compared with a solvent control (0% radical scavenging) and ascorbic acid (250 μM final concentration, 100% radical scavenging, used as a blank), and the halfmaximal scavenging concentration SC50 was generated from data obtained with 8 serial two-fold dilutions in a final concentration range of 2 - 250 μM tested in duplicates. Correction for colored samples was achieved by preparing identical dilutions of the sample in ethanol instead of DPPH solution.

Measurement of oxygen radical absorbance capacity (ORAC).

For the determination of peroxyl or hydroxyl radical absorbance capacity of test compounds, the ORAC assay (Ou et a/., 2001 ; Huang et al., 2002) was used in a 96-well plate format. Fluorescein was used as a redox- sensitive fluorescent indicator, and 2,2,-azobis-(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator. Results were expressed as ORAC units, where 1 ORAC unit equals the net protection of fluorescein produced by 1 μM Trolox, a water soluble Vit. E analog used as a reference compound. Briefly, reaction mixtures contained 170 μl fluorescein solution (42.1 μM in 75 mM sodium potassium phosphate buffer, pH 7.0), 10 μl solvent dilution, and 10 μl Trόlox (1 μM final concentration) or 10 μl of the inhibitor solution (1 to 5 μM final concentration) or phosphate buffer as a negative control, respectively. The reaction was initiated by addition of 10 μl 382 mM AAPH in phosphate buffer (19.1 mM final concentration). The decline of fluorrescein fluorescence was measured at 37°C until completion for 100 min using a Cytofluor 4000 fluorescent microplate reader (excitation wavelength Ex 530/25 nm, emission wavelength Em 585/30 nm). Scavenging capacities > 1 ORAC unit were considered as positive.

Scavenging of superoxide anion radicals formed enzymatically by xanthine oxidase (X/XO) Superoxide anion radicals were generated by oxidation of hypoxanthine to uric acid by xanthine oxidase and quantitated by the concomitant reduction of XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)- 2H-tetrazolium-5-carboxanilide] (modified from Ukeda et a/., 1997 and adjusted to a 96-well microplate format). The reaction mixture (170 μl), consisting of 50 mM sodium carbonate buffer, pH 9.4, EDTA (100 μM final concentration), NBT (12.5 μM final concentration) and hypoxanthine (50 μM final concentration), was mixed with test compounds (10 μl, in 100% DMSO, 5% final DMSO concentration) or DMSO (10 μl) as a solvent control. 1 U SOD (in 10 μl 50 mM sodium carbonate buffer, pH 9.4) was used as a positive control. The reaction was started by addition of 3 mil xanthine oxidase (in 10 μl buffer), and the rate of reduction of XTT was monitored for 6 min at 480 nm in a microplate reader (Spectramax 340, Molecular Devices). Vmax values were computed, and the halfmaximal scavenging concentration SC50 was generated from the data obtained with 8 serial two- fold dilutions of inhibitors in a final concentration range of 2 - 250 μM tested in duplicates. To exclude a direct inhibitory effect on xanthine oxidase, formation of uric acid was monitored directly at 290 nm under identical conditions as described above without addition of XTT. In the reaction mixture, 50 μM hypoxanthine was replaced by 100 μM xanthine. 2.6 Inhibition of cyclooxygenase (Cox) activity.

Cox activity was measured at 37°C by monitoring oxygen consumption during conversion of arachidonic acid to prostaglandins in a 1.0 ml incubation cell of an Oxygen Electrode Unit (Hansatech DW, based on a Clark-type O2 electrode) according to Jang and Pezzuto (1997) with modifications. The reaction mixture, containing 0.1 M sodium potassium phosphate buffer, pH 7.4, 1 mM hydroquinone, 0.01 mM hemin and approximately 0.2 U Cox-1 in 100 μl microsome fraction derived from ram seminal vesicles as a crude source of Cox-1 (specific activity 0.2 - 1 U/mg protein), was incubated with 10 μl DMSO (negative control) or inhibitor solution (10 mM in DMSO), respectively, for 90 s. The reaction was started by addition of 2 μl 50 mM arachidonic acid in ethanol (100 μM final concentration), and oxygen consumption was monitored for 20 sec. For calculation, the rate of O2 consumption was compared to a DMSO control (100% activity). Inhibition of LPS-mediated inducible nitric oxide synthase (/^'NOSJ induction in murine macrophages

Inhibition of lipopolysaccharide-mediated iNOS induction in murine Raw 246.7 macrophages was determined via the Griess reaction (Heiss et al., 2001). Briefly, murine macrophages were cultured in DMEM medium containing 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulfate and 250 ng/ml amphotericin B supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 atmosphere. Cells were plated at a density of 1-2 x 105 cells/well in DMEM in 96-well plates. After a preincubation period of 24 h, the medium was replaced by 170 μl serum-free DMEM. Inhibitors (10 μl in 10% DMSO₁ 8 serial 2-fold dilutions, final concentration range 0.8 to 50 μM) were added, and iNOS was induced by addition of 20 μl LPS solution (500 ng/ml in serum-free DMEM). After 24 h, iNOS activity was determined via the quantitation of nitrite levels according to the Griess reaction. Briefly, 100 μl aliquots of cell culture supematants were incubated with an equal volume of Griess reagent (1 % sulphanilamide/0.1% naphthylethylene diamine dihydrochloride/ 2.5% H3P04) at room temperature for 10 min. The absorbance at 550 nm was determined in a microplate reader and compared to a nitrite standard curve. To determine cytotoxic effects of test compounds, residual cell culture medium was removed, cells were fixed at 4°C for 30 min with 50 μl ice-cold 10% trichloroacetic acid solution in water, washed 5 times with tap water and briefly dried. Cell numbers were estimated by sulforhodamin B staining (Skehan et al., 1990). Generally, compounds were tested at non-toxic concentrations (cell staining > 50% of LPS- treated control cells).

Inhibition of aromatase activity (Cyp19)

For the measurement of aromatase inhibition the method of Stresser et al. (2000) was modified. Cofactor buffer (100 μl/well of a 50 mM phosphate buffer, pH 7.4 containing 2.6 mM NADP⁺, 6.6 mM glucose 6-phosphate and 0.8 U/ml glucose-6-phosphate dehydrogenase) was incubated in black 96 well plates at 37°C for 10 minutes. Inhibitor dilutions (10 μl) and 90 μl freshly made, prewarmed enzyme-substrate-mix (containing 0.4 μM dibenzylfluorescein, 0.2 μM final and 4 pmol/ml human recombinant Cyp19, 2 pmol/ml final, in 50 mM phosphate buffer, pH 7.4) were added. 10 μl 10% DMSO was used as negative control. After incubation at 37°C for 30 minutes the reaction was stopped by adding 50 μl 2.2 N NaOH and the mixture was again incubated at 37⁰C for 2 hours. The fluorescent product of DBF, which is formed proportionally to the activity of aromatase, was determined in a Spectramax fluorescent microplate reader (excitation 490 nm; emission 530 nm; cutoff 515 nm). Incubations were corrected for background fluorescence to test compounds by adding 90 μl of phosphate buffer to sample/cofactor dilutions without addition of enzyme and substrate. Halfmaximal inhibitory concentration IC50 was generated from data obtained with 8 serial two-fold dilutions of inhibitors in a final concentration range of 0.8 to 100 μM, respectively, tested in duplicates.

Determination of estrogenic and antiestrogenic capacity in cultured Ishikawa cells Measurement of the enhancement of alkaline phosphatase (ALP) activity in the Ishikawa human endometrial adenocarcinoma line allows the assessment of intrinsic estrogenic activity of test compounds. Antiestrogenic effects are determined by co-treatment with β-estradiol and inhibitors. Cell culture conditions were essentially as described earlier (Littlefield et al., 1990; Markiewicz et al., 1992). Ishikawa cells were routinely maintained in α-MEM medium containing 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulfate and 250 ng/ml amphotericin B supplemented with 10% charcoal-stripped fetal bovine serum at 37°C in a 5% CO2 atmosphere. One day before the start of an experiment, the medium was changed to an estrogen- and phenol red-free D-MEM/F-12 mix (1 :1) (estrogen free mix) containing L-glutamate and pyridoxine HCI (Gibco BRL), supplemented with 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulfate, 250 ng/ml amphotericin B and 5% charcoal-stripped fetal bovine serum. For the determination of estrogenic activity, cells were trypsinized with 0.25% phenol-red free trypsin/EDTA, passed through a 27 gauge injection needle to obtain a single-cell suspension, and plated in 96-well microplates at a density of 2 x 10⁴/well in 200 μl estrogen free mix. After a preincubation period of 24 h, the medium was replaced by 190 μl fresh estrogen free mix. Test compounds (10 μl in 10% DMSO, 8 serial 2- or 5-fold dilutions, final concentration range 10-¹² to 10⁵ M in duplicate) were added to a final volume of 200 μl, and the plates were incubated at 37°C in a humidified 5% CO2 atmosphere for 72 h. Plates were washed three times with PBS (pre-warmed to 37°C), 50 μl/well 0.5% Triton X in PBS were added, and plates were kept at -8O⁰C overnight. To determine ALP activity, plates were thawed at 37°C for 2 min, 100 μl/well 15 μM 4-methyl-umbelliferyl phosphate (MUP) in 1 M diethanolamine buffer, pH 9.8, containing 0.24 mM MgCk were added, and plates were shaken thoroughly for 5 min on a microplate shaker. Dephosphorylation of MUP to fluorescent 4-methyl-7-hydroxy-coumarin (4-methylumbelliferon) was monitored for 45 min at 37°C (excitation 360 nm, emission 460 nm). ALP activity was determined from the rate of product formation (in arbitrary fluorescent units/min). Protein was determined by SRB staining of an identical set of test plates. For calculation of estrogenic activity, results were expressed as a percentage in comparison with a control sample treated with 50 nM β-estradiol (set as 100%) to determine the half- maximal effective concentration.

For the determination of antiestrogenic activity, the medium pre-incubated cells (as described above) was changed to 170 μl estrogen free mix, and cells were treated simultaneously with test samples (10 μl in 10% DMSO) on 10 μl 10% DMSO as a solvent control, respectively, and 20 μl 50 nM β-estradiol in estrogen free mix. Inhibition of ALP induction was calculated in comparison with the result obtained with the β-estradiol control set as 100%. Tamoxifen was used as a known antiestrogenic reference compound and produced an inhibition of >50% at a test concentration of 0.5 μM. Uteroptrophic assay in prepubertal rats

Six groups of prepubertal (19 days old) female Sprague-Dawley rats (n = 6 per treatment) were injected with ES34 at a concentration of 100 μg/kg b.w., 1 mg/kg b.w. or 10 mg/kg b.w. s.c, respectively on three consecutive days (two group for each dose). One of each dose group was injected simultaneously with 1 μg/kg b.w. ethinylestradiol (EE) s.c. every day. As positive and negative controls, two groups were treated with either solvent or EE (1 μg/kg b.w.), respectively. Body weight changes were monitored daily. On day four, rats were sacrificed by CO2 and uteri and ovaries were weighed. The ovaries and uteri weights were calculated in relation to body weight. Normalized uterine and ovaries weights were calculated as a percentage of the untreated control to assess estrogenic effects. To quantify anti-estrogenic activity, uteri weights from untreated animals were deduced from all other groups. The EE-treated group was set as 100%. Normalized uteri weights of groups treated simultaneously with EE and ES34 were calculated as a percentage in relation to the EE group.

Results Three resveratrol derivatives ES32, ES33, ES34 (Figure 1) were tested for potential cancer chemopreventive activities.

The results are summarized in Table 1 and illustrated in the attached Figures 2-5, wherein:

Figure 2 A: Inhibition of Cyp1 A activity Figure 2 B: Induction of QR activity

Figure 3 A: Scavenging of DPPH radicals

Figure 3 B: Scavenging of peroxyl radicals

Figure 4 A: Inhibition of Cox-1 activity

Figure 4 B: Inhibition of iNOS induction Figure 5 A: Inhibition of aromatase activity

Figure 5 B: Inhibition of estrogen mediated alkaline phosphatase activity (antiestrogenic effect)

Figure 5 C: Induction of alkaline phosphatase activity (estrogenic effect) Figure 6 A: Body weight changes in ES 34 treated prepubertal rats Means significantly different from other ES 34 treated groups (p < 0.05, n=6)

Figure 6 B: Influence of ES 34 on uterine weights in prepubertal rats a means significantly different from untreated control (p < 0.05, n=6) b means significantly different from EE-treated control (p < 0.05, n=6) Figure 6 C: Influence of ES 34 on ovarian weights in prepubertal rats Table 2. Potential chemopreventive activities of prenylated Resveratrol derivatives

Fig. ES32 ES33 ES34 Resveratrol

Anti-initiation mechanisms

2 A Inhibition of Cyp 1A activity 0.03 + 0.005 0.09 ± 0.03 0.18 ± 0.08 0.52 ± 0.10 (ICso)^a

2 B Induction of QR activity in 3.2 ± 0.4 0.53 ± 0.07 2.9 ± 0.5 10.0 ± 1.5

Hepa1c1c7 (CD)_b

Inhibition of Hepa1c1c7 cell 21.7 ± 1.6 4.9 ± 0.9 6.1 ± 1.4 22.7 ± 5.8 proliferation (ICso)

3 A DPPH scavenging (SCso)^c 26.0 ± 8.4 28.7 ± 5.8 113.4 ± 10.6 75.2 ± 8.0

3 B ORACROO (units)^d 2.5 ± 0.3 1.8 ± 0.5 4.2 ± 0.3 3.9 ± 1.1

Superoxide anion radical >250 97.6 ± 7 >250 >250 scavenging (SCso) Antitumor promoting mechanisms

4 A Inhibition of Cox-1 activity (ICso) 27.8 36.6 >100 2.9

4 B Inhibition of iNOS induction (ICso) 22.2 ± 0.9 11.5 ± 0.4 20.2 ± 2.0 39.1 ± 2.0

Inhibition of Raw 264.7 cell >50 36.4 ± 2.4 47.6 ± 1.4 >50 proliferation (ICso)

5 A Inhibition of Aromatase activity 7.1 ± 2.9 2.5 ± 0.8 2.3 ± 0.8 >50

(ICso)

5 B Antiestrogenic (Ishikawa) (ICso) 14.8 ± 4.5 6.1 ± 0.4 5.8 ± 2.2 32.5 ± 11.0 Inhibition of Ishikawa cell >50 18.8 ± 8.1 24.4 ± 9.5 >50 proliferation (ICso)

5 C Estrogenic activity (Ishikawa) 0.1 ± 0.09 0.001 ± 0.00006 ± 10.5 ± 0.7 (ECso)^e 0.0005 0.00004 ^a IC50: half-maximal inhibitory concentration (in μM). b CD: concentration required to double the specific activity of QR (in μM). c SC50: half-maximal scavenging concentration (in μM). d 1 ORAC unit equals the net protection of fluorescein produced by

1 μM Trolox. Compounds were tested at a concentration of 1 μM. e Effective concentration for halfmaximal induction of alkaline phosphatase activity (marker of estrogen response) (in μM).

Anti-initiating mechanisms Modulation of xenobiotics metabolism and anti-oxidant activities contribute to the inhibition of carcinogenesis during the initiation phase. Cytochrome P450 1A (Cyp1A), which is e.g. involved in the activation of carcinogens from cigarette smoke or grilled meat, was used as a model phase 1 enzyme, and NAD(P)H:quinone reductase (QR) was tested as a phase 2 enzyme which is induced concomitantly with other phase 2 enzymes such as glutathione S-transferases.

Modulation of xenobiotics metabolism

As indicated in Figure 2 A, all three derivatives were more potent inhibitors of Cyp1A activity as Resveratrol. IC50 values were in the range of 0.03 to 0.52 μM. ES ES32 was identified as the most potent inhibitor.

All three derivatives were also more potent inducers of QR activity, indicative of enhanced potential to detoxify carcinogens (Figure 2B). CD values (concentration required to double the specific activity of QR) were 3-0-fold lower than that of resveratrol. ES33 was the most active derivative. Anti-oxidant capacity

It is well established that excessive production of reactive oxygen species (ROS) caused by immune diseases, chronic inflammation and infections leads to continuous oxidative stress and is causally linked with processes during tumor initiation, promotion and progression.

First, we used 2,2-diphenyl-1-picrylhydrazyl stable radicals to investigate the radical scavenging potential (DPPH assay). All compounds dose-dependently scavenged DPPH radicals, but IC50 values were relatively high, as indicated in Figure 3 A.

Substitution of the stilbenes structure reduced the potential to scavenge peroxyl radicals, measured in the ORACROO assay (Figure 3 B). Only ES 34 was equally active as resveratrol. Only ES 33 was demonstrated weak potential to scavenge superoxide anion radicals (IC50 97.6 μM) whereas the two other derivatives as well as resveratrol were not active up to a final test concentration of 250 μM (data not shown).

Antitumor promoting activities

It is estimated that 18% of all cancer cases are related to chronic inflammatory processes. We therefore tested for potential to inhibit the activity of cyclooxygenase 1 (Cox-1) and the induction of inducible nitric oxide synthase (iNOS). Both enzymes are involved in the generation of inflammatory mediators in the form of prostaglandins and nitric oxide.

Anti-inflammatory mechanisms

Resveratrol is a potent plant derived Cox-1 inhibitor with an IC50 vale of 2.9 μM. Derivatisation with prenyl groups reduced the Cox-1 inhibitory potential, as depicted in Figure 4 A.

In contrast, the derivatives were more potent in inhibiting the induction of iNOS in murine macrophages. IC50 values were about 2-4-fold lower that those obtained with resveratrol (Figure 4B). Antiestrogenic and estrogenic activity

Excessive exposure to estrogen is regarded as a factor contributing to carcinogenesis. During menopause, lack of estrogen however leads to an increase in bone fractures etc. We first assessed the potential to inhibit aromatase activity as an indirect anti-estrogenic mechanism. Aromatase (Cyp19) converts the male hormone testosterone to the female hormone estrogen. Inhibition will reduce circulating estrogen levels. Whereas resveratrol was not able to inactivate aromatase, all three derivatives demonstrated considerable activity in inhibiting the enzyme, with IC50 values of 2.3 to 7.1 μM (Figure 5 A).

Pro- and antiestrogenic properties of resveratrol and the prenylated derivatives derived were further tested in Ishikawa cell culture. This human endometrial cancer cell line responds to estrogens with elevated alkaline phosphatase activity. Concomitant treatment with estrogens and test compounds allows the identification of anti-estrogens.

With respect to anti-estrogenic potential, all derivatives were more potent in inhibiting the estrogen mediated induction of alkaline phosphatase activity (Figure 5 B). In addition, all three derivatives potently induced alkaline phosphatase activity, partly at extremely low concentrations, as indicated in Figure 5 C. ES34 was more effective than β-estradiol in inducing alkaline phosphatase activity. The halfmaximal effective concentration (EC50) was determined as 6O pM. In vivo rat uterotrophy assay

To determine whether the strong estrogenic activity of 34 is also detectable in vivo, the compound was tested in the prepubertal rat uterotropy assay. 34 was applied s.c. at three dose levels (100 μg per kg body weight (b.w.) per day, 1 mg per kg b.w. per day and 10 mg per kg b.w. per day) in the presence and absence of 17-ethinylestradiol (EE, 1 μg per kg b.w. per day, s.c). Body weights of all animals were measured daily. After 3 days, the experiment was terminated, and uterine and ovarian wet weights were determined. As indicated in Figure 6 A, application of 34 at the highest dose level significantly delayed the weight gain of the animals. Since mean body weights of both groups treated with 34 at 10 mg per kg b.w. per day were the lowest of all groups starting from day 0, the experiment has to be repeated to confirm whether 34 has growth-delaying or toxic effects.

Treatment of prepubertal rats with estrogens leads to a rapid increase in uterine weights within 3 days. As shown in Figure 6 B, three daily s.c. injections of 1 μg EE per kg b.w. resulted in a 3.4-fold increase in uterine weights in the EE-treated control group. ES 34 in the absence of EE demonstrated dose-dependent estrogenic effects. Uterine weights (normalized to body weights) increased 1.7, 2.2 and 4.2-fold, respectively, in comparison with the untreated control after injection of ES34 at increasing concentrations (dark blue bars). In the presence of EE (light blue bars), ES34 at the two lower doses inhibited the EE-induced uterine weight gain by 37 % and 50 %, respectively. At the highest concentration, the weight increase was similar to that seen in the absence of EE, indicating that at a concentration of 10 mg per kg b.w. per day, ES 34 functions as a pure estrogen and its effect overrules that of EE given at a 10.000-fold lower dose. Overall, ES34 has dose-dependent anti-estrogenic and estrogenic effects. At low doses, it functions as an estrogen in the absence of EE and as an anti-estrogen in the presence of EE. At a high dose, it is estrogenic independent of the effect of EE.

We also analysed the influence of ES 34 on ovarian weights in perpubertal animals. Similar to the effect of EE, ES 34 did not significantly influence ovarian weights after three days (Figure 6 C). References

Crespi, C. L., Miller, V.P. and Penman, B.W. (1997) Anal. Biochem. 248,

188-90 Delia Ragione, F.; Cucciolla, V.; Borriello, A.; Pietra, V. D.; Racioppi, L.;

Soldati, G.; Manna, C; Galletti, P.; Zappia, V. Resveratrol arrests the cell division cycle at S/G2 phase transitino. Biochem. Biophys. Res. Commun.

1998, 250, 53-58

Haworth, R. S.; Avkiran, M. Inhibition of protein kinase D by resveratrol. Biochem. Pharmacol. 2001, 62, 1647-1651.

Heiss, E., Herhaus, C, Klimo, K., Bartsch, H. and Gerhauser, C. (2001) J.

Biol. Chem. 276, 32008-32015

Hsieh, T. C; Wu, J. M. Grape-derived chemopreventive agent resveratrol decreases prostate-specific antigen (PSA) expression in LNCaP cells by an androgen receptor (AR)-independent mechanism. Anticancer Res. 2000, 20,

225-228;

Huang, C; Ma₁ W.Y.; Goranson, A.; Dong, Z. Resveratrol suppresses cell transformation and induces apoptosis through a p53-dependent pathway.

Carcinogenesis 1999, 20, 237-242 Huang, D., Ou, B., Hampsch-Woodill, M., Flanaganm J.A., Prior, R.L. (2002)

J. Agric. Food Chem. 50, 4437-4444

Jang, M.S. and Pezzuto, J. M. (1997) Methods Cell Sci. 19, 25-31

Kanno, J., Onyon, L., Haseman, J., Fenner-Crisp, P., Ashby, J. and Owens,

W. (2001) Environ. Health Perspect. 109, 785-794 Kim, Y.-A. Rhee, S.-H. Park, K.-Y.; Choi Y. H. Antiproliferative effect of resveratrol in human prostate carcinoma cells. J. Med. Food 2003, 6, 273-

280

Littlefield, B. A., Gurpide, E., Markiewicz, L, McKinley, B. and Hochberg, R. B. (1990) Endocrinology 127, 2757-2762

Lu, R.; Serrero, G. Resveratrol, a natural product derived from grape, exhibits antiestrogenic activity and inhibits the growth of human breast cancer cells. J. Cell Physiol. 1999, 179, 297-304 Markiewicz, L., Hochberg, R. B. and Gurpide, E. (1992) J. Steroid Biochem.

MoI. Biol. 41 , 53-58

Manna, S. K.; Mukhopadhyay, A.; Aggarwal, B. B. Resveratrol suppresses

TNF-induced activation of nuclear transcription factors NF-kappa B, activator-protein 1 , and apoptosis: potential role of reactive oxygen intermediates and lipid peroxidation. J. Immunol. 2000, 164, 6509-6519

Miller, N. J.; Rice-Evans, C. A. Antioxidant activity of resveratrol in red wine.

CHn. Chem. 1995, 41, 1789

Orsini, F.; Pelizzoni, F.; Verotta, L.; Aburjai, T.; Rogers, C. B. Isolation,

Synthesis and Antiplatelet Aggregation Activity of Resveratrol 3-O-β-D- glucopyranoside and Related Compounds. J. Nat. Prod. 1997, 60, 1082-087.

Orsini, F.; Verotta, L.; Lecchi, M.; Restano, R.; Curia, G.; Redaelli, E.;

Wanke, E. Resveratrol analogues as K⁺ channels modulators. J. Nat. Prod.

2004, 67, 421-426

Ou, B., Hampsch-Woodill, M. and Prior, R.L. (2001) J. Agric. Food Chem. 49, 4619-4626

Pervaiz, S. Resveratrol: from grapevines to mammalian biology. FASEB

2003, 17, 1975-1985

Pozo-Guisado, E.; Alvarez-Barrientos, A.; Mulero-Navarro, S.; Santiago-

Josefat, B.; Fernandez-Salguero, P. M. The antiproliferative activity of resveratrol results in apoptosis in MCF-7 but not in MDA-MB-231 human breast cancer cells: cell-specific alteration at the cell cycle. Biochem.

Pharmacol. 2002, 64, 1375-1386

Prochaska, H.J. and Santamaria, A.B. (1988) Anal. Biochem. 169, 328-336 Roberti, M.; Pizzirani, D.; Simoni, D.; Rondanin, R.; Baruchello, R.; Bonora,

C; Buscemi, F.; Grimaudo, S.; Tolomeo, M. Synthesis and biological evaluation of resveratrol and analogues as apoptosis-inducing agents. J.

Med. Chem. 2003, 46, 3546-3554 Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D.,

Warren, J.T., Bokesch, H., Kenney, S. and Boyd, M. R. (1990) J. Natl. Cancer

Inst. 82, 1 107-1112

Stresser, D. M., Turner, S. D., McNamara, J., StocKer, P., Miller, V. P., Crespi,

L. and Patten, CJ. (2000) Anal. Biochem. 284, 427-430 Takeuchi, T., Nakajima, M. and Morimoto, K. (1994) Cancer Res. 54, 5837-

5840

Ukeda, H., Maeda, S., Ishii, T. and Sawamura, M. (1997) Anal. Biochem.

251 , 206-209 van Amsterdam, FT., Roveri, A., Maiorino, M., Ratti, E. and Ursini, F. (1992) Free Radic. Biol. Med. 12, 183-187

Wadsworth, T. L.; Koop, D. R. Effects of the wine polyphenols quercetin and resveratrol on pro-inflammatory cytokine expression in RAW 264.7 macrophages. Biochem. Pharmacol. 1999, 57, 941-949

Claims

1. A compound of formula (I):

(I) wherein:

R1 , R2 and R3, independently from one another, represent H or (Ci-C3)alkyl; R4 and R5 are identical or different and represent hydrogen, linear or branched (Ci-C5)alkyl, a prenyl group -CH2-CH=C(CH3)2, a geranyl group -CH2-CH=C(CH₃)(CH2)2CH=C(CH₃)2 or R₄ and R1 , and independently R5 and R2, together with the atoms they are linked to, form one of the following groups:

with the provisos that R4 and R5 are not both hydrogen and that when R1=R2=R3=H, R4 and R5 are not a prenyl group and hydrogen, respectively.

2. A compound according to claim 1 , wherein R1 , R2 and R3 represent H, R4 and R5 are identical or different and represent a prenyl group -CH₂-CH=C(CH₃)2 or a geranyl group -CH2-CH=C(CH₃)(CH₂)2CH=C(CH3)2 or R4 and R1, and independently R5 and R2, together with the atoms they are linked to, form one of the following groups:

3. A compound according to claim 2, which is selected from the group consisting of:

and

4. A pharmaceutical or nutraceutical composition containing a compound of claims 1-3.

5. The composition of claim 4, which is in a form suitable for oral intake.

6. The use of a compound as defined in any of claims 1 to 3, for the preparation of a composition for preventing or treating the oxidative damage to cells or tissues.

7. The use of a compound as defined in any of claims 1 to 3, for the prevention or treatment of cancer.

8. The use according to claim 7, wherein said cancer is of estrogen- dependent type.

9. The use of a compound as defined in any of claims 1 to 3, for the preparation of a composition for the treatment or prevention of osteoporosis and in substitutive hormone therapy.

10. The use of a compound as defined in any of claims 1 to 3 for the preparation of an anti-inflammatory agent.