WO2003018013A1

WO2003018013A1 - Stilbene derivative having cytochrome p450 1b1 inhibitory activity, pharmaceutically acceptable salt thereof, method for preparing the same, and composition including the same

Info

Publication number: WO2003018013A1
Application number: PCT/KR2002/000977
Authority: WO
Inventors: Sang-Hee Kim; Young-Jin Chun
Original assignee: Promeditech Inc
Current assignee: Promeditech Inc
Priority date: 2001-08-31
Filing date: 2002-05-23
Publication date: 2003-03-06
Anticipated expiration: 2004-02-29
Also published as: KR20030018800A; WO2003018013B1

Abstract

A stilbene derivative having cytochrome P450 1B1 inhibitory activity, pharmaceutically acceptable salt thereof, method for preparing the same, and composition including the same are disclosed. The stilbene derivative is a strong and selective inhibitor of cytochrome P450 1B1, thereby to decrease or prevent chemical carcinogenesis in mammals, in particular the development of a breast cancer, and can be used as a pharmaceutical tool to elucidate the function of cytochrome P450.

Description

STILBENE DERIVATIVE HAVING CYTOCHROME P450 1B1 INHIBITORY

ACTIVITY, PHARMACEUTICALLY ACCEPTABLE SALT THEREOF, METHOD

FOR PREPARING THE SAME, AND COMPOSITION INCLUDING THE SAME

FIELD OF THE INVENTION

The present invention relates to a stilbene derivative having cytochrome P450 1B1 inhibitory activity, pharmaceutically acceptable salt thereof, method for preparing the same, and composition including the same.

BACKGROUNDS OF THE INVENTION

Cytochrome P450 catalyzes phase I oxidative metabolism of a number of xenobiotics such as drugs, carcinogens, or pesticides, and of a number of endogenous compounds such as steroids or eicosanoids (Ortiz de Montellano, P.R. Cytochrome P450; Structure, Mechanism, and Biochemistry 2^nd ed.; Plenum Press: New York, 1995). Cytochrome P450 1 subfamily including 1A1 , 1A2 and

1 B1 , have been studied for a long time due to its relevance to chemical carcinogenesis.

In particular, cytochrome P450 1B1 is expressed preferentially in steroidogenic tissues such as the adrenal, testis and ovary, and in steroid- sensitive tissue such as the breast, prostate, testis and embryonic cells (Savas, U., et al., Carcinogenesis 14: 2013-2018, 1993). Like cytochrome P450 1A1 , cytochrome P450 1 B1 is involved in the metabolic activation of polycyclic aromatic hydrocarbons such as benzo[a]pyrene, dibenzo[a,/]pyrene, 7,12- dimethylbenz[a]anthracene (DMBA) and 5-methylchrysene (Shimada, T., et al., Cancer Res. 56: 2979-2984, 1996). Cytochrome P450 1 B1 is more active than cytochrome P450 1A1 in converting DMBA to the carcinogenic metabolite 3,4- dihydrodiol 1,2-epoxide. The expression of cytochrome P450 1B1 is regulated by Ah receptor-mediated signal pathway, and 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent agonist of Ah receptor, activates a transcription of cytochrome P450 1 B1. Also, cytochrome P450 1 B1 is known as a main metabolic enzyme for 17β- estradiol (E2).

Various evidences for carcinogenicity of estrogen suggest that 4- hydroxyestradiol (4-OHE2) produced by cytochrome P450 1 B1 is involved in the development of breast cancer in humans (Liehr, J. G. and Ricci, M.J., Proc. Natl.

Acad, Sci. USA, 93: 3294-3296, 1996). 4-OHE2 is also known to be a long- acting estrogen (Barnea, E. R., et al., Steroids 41 :643-656, 1993) and the role of 4-OHE2 as a tumor-inducing substance has been explained by Cavalieri (Cavalieri, E.L., et al., Proc. Natl. Acad. Sci. USA 94: 10937-10942, 1997). 4-OHE2 can be oxidized to E2-3,4-semiquinone and E2-3,4-quinone, which binds to N⁷-guanine of

DNA to thereby form depurinating DNA adducts. Therefore, the resulting apurinic sites in DNA can results in mutations in critical gene.

Since it is known that the hydroxylation in 4-position of E2 by cytochrome P450 1B1 is important in the carcinogenic effects of estrogen, a selective inhibitor of cytochrome P450 1B1 may prevent or decrease the formation of tumors in several tissues, especially in mammary glands. Moreover, the inhibitor can be used as a pharmaceutical tool to elucidate the function of cytochrome P450. To find out a selective inhibitor of cytochrome P450 1 enzymes, various compounds have been tested for this purpose (Shimada, T., et al., Chem. Res. Toxicol. 11 : 1048-1056, 1998). However, only a few potently selective inhibitors have been known with respect to specific P450 enzymes.

Recently, a rhapontigenin, a natural hydroxystilbene having a structure of the chemical formula 3, has been known as a selective inhibitor of cytochrome

P450 1A1(Chun, Y. J., et al., Drug Metab. Dispos. 29: 389-393, 2001). It is also suggested that the selectivity and a potency of cytochrome P450 1A1 inhibition by stilbene compounds are sensitive to the substituent on ring structure of the trans- stilbene moiety. [Chemical formula 3]

On the basis of the above studies, we, the present inventors investigated the synthesis and biological evaluation of various trans-stilbene analogues in order to find a potent and selective cytochrome P450 1B1 inhibitor. We designed and prepared a series of compounds in which the left phenyl ring of chemical formula 3 contains dimethoxy groups on the 3-, 4- and/or 5-positions in order to obtain compounds having the potent activity. Several structural changes were also made on the right phenyl ring in order to obtain compounds having the high selectivity.

The compound of the following chemical formula 1 , its salts, a method for preparing the same, and the use of the compound as a whitening agent and a melanin formation inhibitor are disclosed in Korean patent application Nos. 2000- 78490 and 2000-78491. However, the present invention is directed to a new use of the compounds as strong and selective inhibitors of cytochrome P450 1 B1 which functions under a completely different pharmacological mechanism compared with the mechanism of the melanin formation inhibitor. The present invention also directed to a novel compound of chemical formula 2.

SUMMARY OF THE INVENTION The main object of the present invention is to develop a strong and selective inhibitor of cytochrome P450 1 B1 for preventing chemical carcinogenesis in mammals, in particular the occurrence of a breast cancer or an ovarian cancer.

Development of these inhibitors as a pharmaceutical tool for analyzing function of cytochrome P450 is also proposed. For these purposes, the present invention provides a pharmaceutical composition including stilbene derivatives having a chemical formula 1 which has strong and selective inhibitory activity to cytochrome P450 1B1. [Chemical formula 1]

In chemical formula 1 , Ri_, R₂ and R₃ independently represent hydrogen, hydroxy or methoxy group and, and Ri and R₃ cannot be hydroxy groups at the same time.

M represents (i) R -substituted 2-pyridyl, 3-pyridyl or 4-pyridyl; (ii) R₄-substituted 2-furyl, 3-furyl, 2-pyrrole, 3-pyrrole, 2-thiophenyI, or 3- thiophenyl; or

(iii) phenyl ring of the following chemical formula I -1 ;

wherein R₄, R₅, Re, R₇, Re, and Rg independently represent hydrogen, hydroxy, halogen or Cι_-4 alkoxy group, and Ri to Rg substituents cannot be hydrogen at the same time.

The present invention also provides a novel compound of chemical formula 2. [Chemical formula 2]

In chemical formula 2, M' represents R -substituted 2-thiophenyl, or 3- thiophenyl, and R-i, R₂, R₃ and R₄ represents the same functional groups defined in chemical formula 1 , and Ri to R₄ substituents cannot be hydrogen at the same time.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention, and many of the attendant advantages thereof, will be readily apparent as the same becomes better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings, wherein:

Fig. 1 is a graph showing the IC₅₀ values of stilbene derivatives according to the present invention with respect to the activities of cytochrome P450 1A1 , cytochrome P450 1A2, or cytochrome P450 1B1; and

Fig. 2 is a graph showing the ratios of IC₅₀ values(1A1/1B1, 1A2/1B1) of stilbene derivatives according to the present invention with respect to the activities of cytochrome P450 1A1 , cytochrome P450 1A2, or cytochrome P450 1 B1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will become more clearly understood from the following detailed description with reference to the accompanying drawings.

The stilbene derivative of the chemical formula 1 according to the present invention, can be prepared by a method comprising the steps of (1) producing olefin mixture by reacting phosphonate derivative of chemical formula 4 with aromatic aldehyde of chemical formula 5 in the presence of base and catalyst in an organic solvent, (2) purifying the olefin mixture by removing the excess aldehyde, and (3) transforming the olefin mixture into trans isomer by adding iodine to the olefin compound and refluxing the olefin mixture in an organic solvent.

[Chemical formula 4]

[Chemical formula 5]

M-CHO In chemical formulae, R represents hydrogen or lower alkyl, Ri to Rg and M represent the same functional groups defined above.

In detail, an example of the reaction is shown in the following reaction equation 1. [Reaction equation 1]

(4) (6)

Girard's reagent R, γ^^

The preparing method comprises the steps of:

(1) producing olefin mixture(6) by reacting phosphonate(4) and aromatic aldehyde(5) in the presence of base and catalyst in an organic solvent;

(2) purifying the olefin mixture(δ) by removing the excess aldehyde(5) from the olefin mixture(6); (3) transforming the olefin mixture(6) into trans isomer(7) by adding iodine to the olefin mixture(δ) and refluxing the olefin mixture(6) in an organic solvent; and

(4) obtaining the stilbene derivative(7) by washing and vacuum concentrating the trans isomer(7). In more detail, the phosphonate(4) and aromatic aldehyde(5) can react with mole ratio of 1 :1 to 1 :2, preferably with mole ratio of about 1 :1.2 in the presence of a strong base such as KOH and catalyst such as 18-crownether-6 of catalytic amount, and in an organic solvent such as dichloromethane(CH₂Cl2) in step (1). Then, the olefin mixture(6) having Z/E ratio of about 1 :3 to 1 :5 by TLC intensity is obtained through Honer-Wadsworth -Emmons reaction. In this case, the structure of the phenyl ring (M) depends on the kind of the aromatic aldehyde(5). In step (2), the excess aldehyde(δ) is removed from the olefin mixture(6) by, for example, being treated with Girard's reagent T and acetic acid in an organic solvent such as dichloromethane. Then, insoluble substances can be removed from the product by filtration, and the residue solution is dissolved in an organic solvent, and washed with salt water. Then the organic solvent is removed under low pressure to obtain the stilbene derivatives in the form of E- and Z-isomer mixture. The obtained product has the purity of more than 98% according to NMR analysis.

In step (3), the Z/E mixture(6) obtained from step (2) is heated with iodine of catalytic amount while refluxing with an organic solvent such as heptane to be transformed into E-isomer(7).

In step (4), the reaction mixture is diluted with an organic solvent such as diethylether, and washed with saturated aqueous sodium bisulfite and distilled water to remove the iodine from the produced stilbene derivatives. Generally, stilbene moiety can be formed by Wittig reaction between aromatic aldehyde and aromatic phosphonium ylide. However, semi-stabilized ylide such as benzyl ylide produces triphenylphosphine oxide as an inevitable byproduct as well as Z and E-isomer mixture as the target products. In order to overcome these drawbacks and to synthesize trans stilbene more practically and effectively, the present inventors utilized Honer-Wadsworth-Emmons reaction (Kucerovy, A., et al., Org. Process Res. Dev. 1 : 287-293, 1997), which produces the target trans isomer as a main product, and forms water soluble dimethyl phosphoric acid as the byproduct instead of triphenylphosphine oxide. The reaction does not need silica gel chromatography procedure which is a main obstacle in producing trans stilbene derivatives, thus can be preferably used for synthesizing trans stilbene libraries in solvent.

The preferable stilbene derivative according to the present invention includes a compound of chemical formula 6 in which Ri represents hydrogen, hydroxy or methoxy group, and M of chemical formula 1 is an hetero aromatic compound(Ar) such as 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, 2-pyrrole, 3-pyrrole, 2-pyridyl, 3-pyridyl or 4-pyridyl; and a compound of chemical formula 7 in which Ri represents hydrogen, hydroxy or methoxy group, and M of chemical formula 1 is a phenyl ring of chemical formula I -1.

[Chemical formula 6]

In chemical formula 6, Ri represents hydrogen, hydroxy or methoxy group, and Ar represents 2-thiophenyl, 3-thiophenyl, 2-furyl, 3-furyl, 2-pyrrole, 3-pyrrole, 2-pyridyl, 3-pyridyl or 4-pyridyl. [Chemical formula 7]

In chemical formula 7, Ri represents hydrogen, hydroxy or methoxy group, and R₂ and R₃ independently represents hydrogen, hydroxy or C₁-₃ alkoxy group.

The more preferable stilbene derivative according to the present invention includes 2,4-dimethoxyphenyl (chemical formula 8), 3,4,5-trimethoxyphenyl

(chemical formula 9), 3,5-dimethoxyphenyl (chemical formula 10), 3,4- dimethoxyphenyl (chemical formula 11 ), 4-methoxyphenyl (chemical formula 12), 2-hydroxy-4-methoxyphenyl (chemical formula 13), 2-fluoro-4-methoxyphenyl (chemical formula 14), 4- pyridyl (chemical formula 15), 3- furanyl (chemical formula 16) or 2- thiophenyl (chemical formula 17) substituted compounds, and their chemical structures are as follows.

[Chemical formula 8] (E)-1-(2',4'-dimethoxyphenyl)-2-(3,5-dimethoxyphenyl)ethene

[Chemical formula 9]

(E)-1-(3',4^, ₎5^,-trimethoxyphenyl)-2-(3,5- dimethoxyphenyl)ethene

[Chemical formula 10] (E)-1 ,2-di(3,5-dimethoxyphenyl)ethene

[Chemical formula 11]

(E)-1 -(3',4'-dimethoxyphenyl)-2-(3,5- dimethoxyphenyl)ethene

[Chemical formula 12]

(E)-1 -(4'-methoxyphenyl)-2-(3,5- dimethoxyphenyl)ethene

[Chemical formula 13] (E)-1-(2'-hydroxy-4'-methoxyphenyl)-2-(3,5-dimethoxyphenyl)ethene

[Chemical formula 14] (E)-1-(2'-fluoro-4'-methoxyphenyl)-2-(3,5- dimethoxyphenyl)ethene

[Chemical formula 15] (E)-4-[2-(3,5-dimethoxyphenyl)-vinyl]-pyridine

[Chemical formula 16] (E)-3-[2-(3,5-dimethoxyphenyl)-vinyl]-furan

[Chemical formula 17] (E)-2-[2-(3,5-dimethoxyphenyl)-vinyl]-thiophene

The stilbene derivative of chemical formula 1 according to the present invention can be provided in the form of a pharmaceutically acceptable salt or solution composition. As the salt, acid addition salt formed with a pharmaceutically acceptable free acid is preferable. The acid addition salt can be prepared by conventional methods, for example, by dissolving the compound with excess acid aqueous solution, and precipitating the salt using water miscible organic solvent such as methanol, ethanol, acetone and acetonitrile. Alternatively, acid or alcohol (ex. glycolmonomethylether), the active ingredient and water solution can be heated, evaporated and dried, or the precipitated salt can be filtered. As the free acid, organic acid or inorganic acid can be used. Examples of the inorganic acid include hydrochloric acid, phosphoric acid, hydro idodic acid, sulfuric acid, and nitric acid, and examples of the organic acid include methane sulfonic acid, p-toluene sulfonic acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, phenylglycolic acid, propionic acid, lactic acid, glycollic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carboxylic acid, and vanillic acid.

Alternatively, pharmaceutically acceptable metal salt can be prepared with

5 a base. Alkali metal or alkaline earth metal salt can be prepared by conventional methods, for example, by dissolving the compound with excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering insoluble salt, and evaporating or drying the residue solvent. The pharmaceutically preferable metal salts include sodium, potassium and calcium salts. In addition, the corresponding

.0 silver salt is produced by reacting a proper silver salt (ex.: silvernitrate) with alkali metal or alkaline earth metal salt.

In addition, the present invention also provides pharmaceutical composition including the stilbene derivative having inhibitory effects of

L5 cytochrome P450 1 B1 or the pharmaceutically acceptable salt thereof as active ingredient. From the experimental data regarding the inhibition of cytochrome P450 1A1 , 1A2 or 1B1 by stilbene derivative, the stilbene derivative newly synthesized in this present invention shows significantly higher inhibitory activity on cytochrome P450 1 enzymes compared with the conventional_oxyresveratrol, 0 its amine or imine derivatives. From the stilbene derivatives tested, the most selective inhibitor of cytochrome P450 1B1 is 2',3,4\5- tetramethoxystilbene(chemical formula 8), and the strongest inhibitor of cytochrome P450 1B1 is 3,5-dimethoxyphenyl-vinyl-thiophene (chemical formula 17). (See Table 1) The above results suggest that the substitution at the 2-position of phenyl ring of the stilbene moiety plays important role in the selectivity and pharmaceutical activity of the compound with respect to cytochrome P450 1A and cytochrome P450 1B1. In addition, the results propose the relationship between the cytochrome P450 1 inhibitory activity and the chemical structure of the stilbene inhibitor, and it will be possible to produce other stilbene derivatives having higher potential. The active trans stilbene derivatives, 2', 3,4', 5- tetramethoxystilbene(chemical formula 8) of the present invention can be used as a chemical probe to characterize the properties of cytochrome P450 1B1 , and can be used as chemopreventive agent or therapeutic agent for chemical carcinogenesis by protecting the metabolism of 17β -estradiol by cytochrome P450 1 B1.

The compound of the chemical formula 1 can be administrated orally and parenterally for clinical inoculation, and can be used in the form of conventional drug preparations. Namely, the compound of the chemical formula 1 according to the present invention can be administered in various forms of oral or parenteral preparation. In formulation of the oral or parenteral preparation, conventional diluents or vehicles such as filler, expander, bonding agent, wetting agent, releasing agent, and surfactant can be used. The solid preparation for oral administration can include tablet, pilula, pulvis, granulum, and capsule, and can be produced by mixing the compound of the chemical formula 1 with at least one vehicle, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. Besides the vehicles, lubricant such as magnesium, stearate, and talc can be used. The liquid preparation for oral administration can include suspension, liquid drug, emulsion, and syrup, and can include water as a conventional diluent, and various vehicles such as wetting agent, sweetening agent, odorant, preservative and liquid paraffin. The preparation for parenteral administration can include sterilized aqueous solution, non-aqueous solution, suspension, emulsion, lyophilization, and suppository. Examples of solvent for the non-aqueous solution or suspension include propylene glycol, polyethyleneglycol, vegetable oil such as olive oil, injectable ester such as ethylolate. The basement for suppository includes witepsol, macrogol, tween 61 , cacao, laurine, glycerogelatin, etc.

The effective amount of the compounds of the chemical formula 1 according to the present invention is 0.1 - 100 mg/kg, preferably 0.1 - 10 mg/kg, and the compounds of the chemical formula 1 may be administered 1 - 3 times a day. However, the effective amount can be varied according to the individual constitution of the subject, specificity, weight, type and seriousness of the disease, characteristics of preparation, administration method of drug, administration period and interval.

Hereinafter, the preferable examples and manufacturing examples are provided for better understanding of the present invention. However, the present invention should not be restricted to the following Examples.

[Manufacturing Example 1] Preparation of (3,5-dimethoxy-benzyl)- phosphonic acid dimethyl ester (chemical formula 4)

In order to produce (3,5-dimethoxy-benzyl)-phosphonic acid dimethyl ester as a starting material for producing the trans stilbene derivative of the present invention, a mixture of Sjδ-dimethoxy-benzyl bromide (2.31 g, 10 mmol) and trimethylphosphite (3.75 g, 30 mmol) in a sealed tube was heated at 180^°C in an oil bath for 8 hours. After the mixture was cooled, the excess trimethylphosphite was removed in vacuo. Purification of the residue by short flash column chromatography on silica gel (eluent: EtOAc) gave 2.42 g (93%) of (3,5- dimethoxy-benzyl)-phosphonic acid dimethyl ester (chemical formula 4) as an oil.

¹H NMR (CDCI₃, 300MHz) δ 6.45 (t, J = 2.4 Hz, 2H), 6.35 (dd, J = 4.2, 2.1 Hz, 1H), 3.77 (s, 6H), 3.70 (s, 3H), 3.66 (s, 3H), 3.10 (d, J = 21.6 Hz, 2H).

[Example 1] Preparation of (E)-2-[2-(3,5-dimethoxy-phenyl)-vinyl]- thiophene (Chemical formula 17)

To a well-stirred suspension of phosphonate(1.0 mmol, chemical formula 4), fleshly powered KOH (2.0 mmol), and 18-crownether-6 (0.1 mmol) in 2 m£ of CH₂CI₂ was added 2-thiophenylaldehyde(1.1 mmol, chemical formula 5) at room temperature. After the mixture was additionally stirred for 3-6 hours, the mixture was diluted with 15 m£ of CH₂CI , and washed with distilled water(10 ml) and brine (2 x 10 mi). The organic layer was dried over MgS0₄, and concentrated in vacuo. The residue was dissolved in 2 ml of CH₂CI₂. To this solution were added Girard's reagent T((carboxymethyl) trimethylammonium chloride hydrazide, 0.5 mmol) and acetic acid(5 mmol), and the obtained mixture was stirred for 2 hours at room temperature. The insoluble material was filtered off, and the filtrate was concentrated in vacuo, and the residue was dissolved in 15 ml of EtOAc. The solution was washed with brine (3 10 mil), and dried over MgS0₄, and the solvent was removed in vacuo to give the target stilbene derivative (0.95 mmol) as

5 a mixture of E- and Z-isomers.

To the solution of this mixture in heptane (5 ml) was added a catalytic amount of iodine, and heated to reflux for 12 hours. The reaction mixture was diluted with 20 ml of ether, and washed with saturated aqueous sodium bisulfite (10 ml) and brine (2 X 10 mi). The organic layer was dried over MgS04, and

.0 concentrated in vacuo to provide the target compound, (E)-2-[2-(3,5-dimethoxy- phenyl)-vinyl]-thiophene (0.91 mmol, Chemical formula 17).

Yield = 91%; oily state

¹H NMR (300MHz, CDCI₃) δ 7.13 (d, J = 15.9 Hz, 1 H), 7.12 (d, J = 8.1 Hz, L5 1 H), 7.00 (d, J = 3.0 Hz, 1 H), 6.93 (dd, J = 3.3, 5.1 Hz, 1 H), 6.78 (d, J = 16.2 Hz,

1 H), 6.55 (d, J = 2.4 Hz, 2H), 6.31 (t, J = 2.4 Hz, 1 H), 3.75 (s, 6H)

[Example 2] Physicochemical analysis of stilbene derivatives.

Various stilbene derivatives were produced according to methods similar to 0 the method of Example 1 , and the physicochemical analysis of the derivatives were carried out. In those Examples, the physicochemical characteristics of the derivatives were analyzed as follows.

First, the melting point was measured with Buchi melting point B-540 apparatus, and was not corrected. NMR spectrum was obtained with Varian Gemini 2000 spectrophotometer at 300MHz with tetramethylsilane as an internal standard. Electron impact mass spectrum was obtained with HP 5989B mass spectrometer at 70eV with using electrospray(ES) ionizing technology. The elemental analysis was carried out on an EA1110 elemental analyzer. The 5 results were within 0.4% of the calculated values. Reaction was monitored by

TLC analysis using E. Merk silica gel 60 F-254 thin layer plates. Flash chromatography was carried out on E. Merk Kieselgel 60 silica gel.

The results of the physicochemical analysis of the derivatives are as .0 follows.

(EV1-(2'.4'-dimethoxyphenvπ-2-(3.5-dimethoχyphenvπethene (Chemical formula 8)

Yield = 95%; Melting point = 78-79 ^°C; L5 ¹H NMR (300MHz, CDCI₃) δ 7.42 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 16.5 Hz,

1 H), 6.85 (d, J = 16.5 Hz, 1 H), 6.59 (d, J = 2.1 Hz, 2H), 6.42 (m, 2H), 6.28 (t, J = 2.1 Hz, 1 H), 3.79-3.69 (m, 12H)

(E)-1-(3',4',5'-trimethoxyphenyl)-2-(3,5-dimethoxyphenyl)ethene (Chemical 0 formula 9)

Yield= 95%; Melting point = 136-138 ^°C ;

¹H NMR (300MHz, CDCI₃) δ 6.95 (d, J = 16.2 Hz, 1 H), 6.86 (d, J = 16.2 Hz, 1 H), 6.67 (s, 2H), 6.59 (d, J = 2.1 Hz, 2H), 6.33 (dd, J = 2.1 , 2.1 Hz, 1H), 3.85 (s, 6H), 3.80 (s, 3H), 3.77 (s, 6H) (E)-1 ,2-di(3,5-dimethoχyphenv0ethene (Chemical formula 10) Yield = 94%; Melting point = 129-135 ^°C;

¹H NMR (300MHz, CDCI₃) δ 6.93 (s, 2H), 6.58 (d, J = 2.1Hz, 4H), 6.32 (t, J

= 2.1 Hz, 2H), 3.75 (s, 12H)

(E)-1-(3'.4'-dimethoxyphenvπ-2-(3.5-dimethoχyphenvπethene (Chemical formula 11)

Yield = 92%; Melting point = 66-67 ^°C; ¹H NMR (300MHz, CDCI₃) δ 6.99-6.94 (m, 3H), 6.83 (d, J = 16.5 Hz, 1 H),

6.79 (d, J = 8.1 Hz, 1H), 6.59 (d, J = 2.1 Hz, 2H), 6.31 (t, J = 2.1 Hz, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 3.76 (s, 6H)

(E)-1-(4'-methoxyphenvπ-2-(3,5-dimethoxyphenyl)ethene (Chemical formula 12)

Yield = 94%; Melting point = 55-57 ^°C ;

¹H NMR (300MHz, CDCI₃) δ 7.4-7.35 (m, 2H), 6.97 (d, J = 16.5 Hz, 1 H), 6.83 (d, J = 16.2 Hz, 1H), 6.84-6.81 (m, 2H), 6.58 (d, J = 2.1 Hz, 2H), 6.30 (t, J = 2.1 Hz, 1H), 3.76 (s, 9H)

(E)-1-(2'-hvdroxy-4'-methoxyphenyl)-2-(3,5-dimethoxyphenyl)ethene (Chemical formula 13)

Yield = 90%; oily state

¹H NMR (300MHz, CDCI₃) δ 7.34 (d, J = 8.7 Hz, 1 H), 7.20 (d, J = 17.1 Hz, 1H), 6.85(d, J = 16.2 Hz, 1H), 6.58 (d, J = 2.1 Hz, 2H), 6.43 (dd, J = 2.4, 8.4 Hz, 1 H), 6.30-6.29 (m, 2H), 3.74 (s, 6H), 3.69 (s, 3H)

(E)-1-(2'-fluoro-4'-methoxyphenvπ-2-(3.5-dimethoχyphenyl)ethene (Chemical formula 14)

Yield = 90%; Melting point = 51-55^°C ;

¹H NMR (300MHz, CDCI₃) δ 7.43 (t, J = 8.7 Hz, 1 H), 7.10 (d, J = 16.5 Hz, 1 H), 6.91 (d, J = 16.5 Hz, 1 H), 6.64 (dd, J = 2.7, 8.7 Hz, 1 H), 6.59 (d, J = 2.1 Hz, 2H), 6.56 (dd, J = 2.7, 12.6 Hz, 1H), 6.32 (t, J = 2.1 Hz, 1 H), 3.76 (s, 6H), 3.75 (s, 3H)

(E)-4-f2-(3,5-dimethoxyphenyl)-vinvn-Pyridine (Chemical formula 15) Yield = 90%; Melting point = 139-144 ^°C;

¹H NMR (300MHz, CDCI₃) δ 8.51 (d, J = 5.7 Hz, 2H), 7.39-7.33 (m, 2H), 7.19 (d, J = 16.5 Hz, 1 H), 6.93 (d, J = 16.2 Hz, 1 H), 6.63 (d, J = 2.1 Hz, 2H), 6.40

(t, J = 2.1 Hz, 1 H), 3.77 (s, 6H)

(E)-3-r2-(3,5-dimethoxyphenyl)-vinyl1-furan (Chemical formula 16) Yield = 91 %; oily state ¹H NMR (300MHz, CDCI₃) δ 7.53 (s, 1 H), 7.4 (m, 1 H), 6.95 (d, J = 16.2 Hz,

1H), 6.74 (d, J = 16.2 Hz, 1 H), 6.65 (m, 1 H), 6.61 (d, J = 2.1 Hz, 2H), 6.38 (t, J = 2.1 Hz, 1 H), 3.82 (s, 6H)

[Example 3] Biological analysis of stilbene derivatives. In order to investigate the inhibitory activities of the stilbene derivatives obtained in Examples 1 and 2 on cytochrome P450 1 enzymes, ethoxyresorufin O- deethylation reaction was performed to measure the activity of cytochrome P450 1 enzymes. First, bicistronic bacterial membranes of human cytochrome P450 1A1 ,

1A2 or 1 B1 were prepared in accordance with the method of Guengerich et al. (Guengerich, F. P., et al., Methods Enzymol. 272: 35-44, 1996). Bicistronic bacterial membranes containing 5 nM of cytochrome P450 1A1 , 1A2 or 1B1 were added to 0.1 M phosphate buffer containing 2 μ M of ethoxyresorufin and varying concentrations of inhibitors. As the inhibitor, stilbene derivatives obtained in

Examples 1 and 2 (chemical formulas 8-17) according to the present invention, and oxyresveratrol were used.

The reaction mixtures were preincubated at 37 ^°C for 3 minutes, and NADPH-generating systems consisting of 5mM of glucose 6-phosphate, 0.5 mM NADP+ and 0.5 unit glucose 6-phosphate dehydrogenase were added to initiate the reaction. The reaction mixtures were incubated for 10 minutes at 37 ^°C, and 1 ml of methanol was added to terminate the reaction.

The formation of resorufin was determined fluorometrically using Perkin- Elmer LS5 spectrofluorometer at the excitation and emission wavelengths of 550 nm and 585 nm, respectively. The results are shown in Table 1. In addition, IC₅₀ values of stilbene derivatives according to the present invention with respect to the activities of cytochrome P450 1A1, cytochrome P450 1A2, or cytochrome P450 1B1 are depicted in Fig. 1 , and the ratios of IC₅₀ values(1A1/1B1 or 1A2/1B1) of stilbene derivatives according to the present invention with respect to the activities of cytochrome P450 1A1 , cytochrome P450 1A2, or cytochrome P450 1B1 are depicted in Fig. 2.

[Table 1] Effects of the stilbene derivatives on ethoxyresorufin O-deethylation activity of human cytochrome P450 1 A1 , 1 A2, or 1 B1.

As shown in Table 1 , Figs 1 and 2, 2,3',4,5'-tetramethoxystilbene (Chemical formula 8) is found out to be the most potently selective inhibitor of cytochrome P450 1B1 from the stilbene derivatives tested. The compound of chemical formula 8 shows the inhibitory activity on cytochrome P450 1B1 (IC₅₀ = 6 nM), cytochrome P450 1A1 (IC₅₀ = 300 nM) and cytochrome P450 1A2 (IC₅₀ = 3 μ M). The compound of chemical formula 8 is a methylated derivative of naturally occurring oxyresveratrol. The oxyresveratrol inhibits cytochrome P450 1 with IC₅₀ values of 15 μ M, 150 μ M and 34 μ M with respect to 1A1 , 1A2 and 1 B1 , respectively (Chun, Y. J., Ryu, S. Y., Jeong, T. C, Kim, M. Y., Drug Metab.

Dispos., 29:389-393, 2001). These results propose that the substitution of four hydroxy groups of oxyresveratrol to methoxy groups improves the inhibitory potency and selectivity.

From the results of the compounds having chemical formulas 9 - 12, the modification of phenyl ring by addition, deletion or change in the position of methoxy groups results in the reduced efficiency and selectivity. The test results suggest that the position of methoxy group is very important in selectivity. With compared to the compound of chemical formula 8, the compound of chemical formula 12 is structurally different in methoxy group at 2-position. The inhibitory activities of the compound of chemical formula 12 on 1A1 and 1A2 are a little less than those of the compound of chemical formula 8. In contrast, the inhibitory activity of the compound of chemical formula 12 on 1 B1 is 130 times reduced (IC₅₀ = 790 nM) with compared to the inhibitory activity of the compound of chemical formula 8 on 1 B1. It means the existence of methoxy group at 2-position plays important role in binding with a functional group of cytochrome P450 1B1. The compound of chemical formula 13 or 14 in which 2-methoxy group is substituted with F or OH has more selective inhibitory activity on 1 B1 rather than 1A1 and 1A2, and has more improved characteristics than the compound of chemical formula 12. However, the inhibitory activity and selectivity thereof are less than those of the compound of chemical formula 8. However, the compounds of chemical formulas 15 to 17 in which phenyl groups are substituted with 4-pyridyl, 2-thiophenyl, or 3-puranyl ring do not show any selectivity similar to the compound of chemical formula 8. Though the compounds of chemical formulas 15 and 16 have weak inhibitory activities on cytochrome P450 1 , the compound of chemical formula 17 having 2-thiophenyl ring inhibits cytochrome P450 1 with IC₅₀ values of 61 nM, 11 nM, or 2 nM for 1 A1 , 1A2 and 1B1 , respectively. The compound of chemical formula 17 has 30 times improved selectivity on 1 B1 compared to 1A1 , but the selectivity on 1B1 and 1A2 is much less than that of the compound of chemical formula 8 (selectivity ratio = about 5 times : about 500 times).

[Example 4] Acute toxicity test of oral administration in rats. To test in vivo toxicity of the compound of chemical formula 8, the acute toxicity test was carried out with SPF(Specific Pathogen Free) SD rat of 6-weeks old. The stilbene derivative compounds of chemical formulas 8 - 17 were suspended with corn oil, and orally administered to 5 animals per group with the amount of 0.5 g/kg . After administration, the survival of animal, clinical manifestation, and changes in weight were investigated, and hematological and hemato-biological examinations were also carried out. In addition, the abdominal and thoracic organs were examined with naked eye after autopsy.

After the experimental test, all animals administered with the test compounds did not show particular clinical manifestations, and were survived. In addition, the changes due to toxicity were not found from the hematological and hemato-biological examinations, autopsy and change of weight. Therefore, the tested compounds do not induce any change due to toxicity within the administration amount of 0.5 g/kg for rat, and can be considered as safe, and the minimum lethal dose (LD50) for oral administration is more than 0.5 g/kg.

As described above, stilbene derivatives according to the present invention have the inhibitory activity on human cytochrome P450 family, i.e., cytochrome P450 1A1 , 1A2 and 1 B1. In particular, 2,3',4,5'-tetramethoxystilbene compound has the strongest inhibitory activity on and most selective to cytochrome P450 1B1. In addition, stilbene derivatives according to the present invention is useful in evaluating the relation between the structure of stilbene as the inhibitor of cytochrome P450 1 and its activity, and in preparing stilbene derivatives having the strong and selective activity. Thus, 2,3',4,5'-tetramethoxystilbene according to the present invention is useful not only as the strong and selective inhibitor of cytochrome P450 1B1 , but also as a compound for studying the enzymatic properties of cytochrome P450 1 B1. Stilbene derivatives according to the present invention can be used as chemopreventive or therapeutic agent for inhibiting carcinogenesis in mammals, in particular the development of breast cancer.

Claims

1. A pharmaceutical composition comprising stilbene derivative of the following chemical formula 1 for inhibiting the activity of cytochrome P450 1B1

enzyme. [Chemical formula 1]

In chemical formula 1 , R-i_, R₂ and R₃ independently represent hydrogen, hydroxy or methoxy group and, and Ri and R₃ cannot be hydroxy groups at the same time.

M represents (i) R₄-substituted 2-pyridyl, 3-pyridyl or 4-pyridyl;

(ii) R₄-substituted 2-furyl, 3-furyl, 2-pyrrole, 3-pyrrole, 2-thiophenyl, or 3- thiophenyl; or

(iii) phenyl ring of the following chemical formula I -1 ;

wherein R , R₅, Rβ, R7, Re. and Rg independently represent hydrogen, hydroxy, halogen or Cι- alkoxy group, and Ri to Rg substituents cannot be hydrogen at the same time.

2. The pharmaceutical composition according to claim 1 , wherein the stilbene derivative is a compound of chemical formula 6. [Chemical formula 6]

In chemical formula 6, Ri represents hydrogen, hydroxy or methoxy group, and Ar represents 2-thiophenyl, 3-thiophenyl, 2-furyl, 3-furyl, 2-pyrrole, 3-pyrrole, 2-pyridyl, 3-pyridyl or 4-pyridyl.

3. The pharmaceutical composition according to claim 1 , wherein the stilbene derivative is a compound of chemical formula 7. [Chemical formula 7]

In chemical formula 7, Ri represents hydrogen, hydroxy or methoxy group, and R2 and R₃ independently represents hydrogen, hydroxy or C₁-₃ alkoxy group.

4. The pharmaceutical composition according to claim 1 , wherein the stilbene derivative is a compound of chemical formula 8.

[Chemical formula 8]

5. The pharmaceutical composition according to claim 1 , wherein the stilbene derivative is a compound of chemical formula 17. [Chemical formula 17]

6. An anticancer composition comprising the pharmaceutical composition according to one of claims 1 to 5.

7. The anticancer composition according to claim 6, wherein the cancer is a breast cancer.

8. A stilbene derivative of the following chemical formula 2 or pharmaceutically acceptable salt thereof. [Chemical formula 2]

In chemical formula 2, M' represents R₄-substituted 2-thiophenyl, or 3- thiophenyl, and R-i, R₂, R₃ and R₄ represents the same functional groups defined in chemical formula 1 , and Ri to R substituents cannot be hydrogen at the same time.

9. A method of preparing stilbene derivatives of the above chemical formula 1 comprising the steps of:

(1 ) producing olefin mixture by reacting phosphonate derivative of chemical formula 4 and aromatic aldehyde of chemical formula 5 in the presence of base and catalyst in an organic solvent;

(2) purifying the olefin mixture by removing the excess aldehyde; and

(3) transforming the olefin mixture into trans isomer by adding iodine to the olefin compound and refluxing the olefin mixture in an organic solvent. [Chemical formula 4]

[Chemical formula 5]

M-CHO In above chemical formulas, R represents hydrogen or lower alkyl, and M represents the same functional groups defined in chemical formula 1.