PL194989B1 - Method of obtaining genisteine - Google Patents

Abstract

1. The method of obtaining genistein of formula 1, characterized in that the floroglucin of formula 2 is reacted with 4-hydroxybenzyl cyanide 3, followed by isolation of the product intermediate - deoxybenzoin IV is subjected formylation and cyclization reactions in one stage and then purifies the raw genistein out

Description

Description of the invention

The subject of the invention is a method of obtaining genistein, which is a substance with potential therapeutic use, inter alia, in anti-cancer prophylaxis.

Genistein (4 ', 5,7-trihydroxyisoflavone) represented by the formula 1, belonging to the group of flavonoids, has so far been a reagent for biochemical and pharmacological studies, as it has been established that it is a competitive inhibitor of protein tyrosine kinases (PTK), acting as a structural analog of adenosine triphosphate (ATP) ) (TJO'Dell et al., Nature, 353, p. 558 (1991). Studies on the biological role of PTK enzymes in the cascade of chemical signal transmission from cell membrane receptors to nuclear effectors regulating gene expression and transcription are now one among the most promising directions of medical chemistry (PW Groundwater et al., Progr. Med. Chem., 33, p. 233 (1996), and it is expected that selective phosphorylation inhibitors will give rise not only to a new generation of drugs, e.g. anti-cancer drugs, but also compounds that will make it possible to inhibit oncogenesis, and thus prevent cancer.

On the other hand, the results of epidemiological studies show that genistein, which is mainly derived from soybeans, is the cause of the low incidence of breast cancer in Asian women. Studies of healthy volunteers consuming soy-rich foods led to the detection in their urine of significant amounts of a substance that inhibits the growth of endothelial cells of blood vessels, which turned out to be identical to genistein (T. Fotsis et al., J. Nutr., 125, 790S (1995) The discovered ability of genistein to inhibit angiogenesis may be of great importance in the control of solid tumors. Further studies of genistein's anti-tumor activity in in vitro and in vivo models (S. Barnes, J. Nutr., 125, 777S (1995)), despite confirmation of its inhibition. effects on neoplastic cells have not yet led to a clear indication of the biomacromolecular targets of this ligand (Z.-M. Shao et al., Cancer Res., 58, p. 4851 (1998)). Currently, genistein is medically classified as a phytoestrogen and belongs to a new class of biologically active compounds known as selective estrogen receptors (SERM).

Obtaining genistein from natural sources is very difficult and expensive, hence the growing interest in synthesizing the compound.

Among the known methods of obtaining isoflavones, collected by K. Wahal et al. (Proc. Spc. Exp. Biol. Med. 208, 1995 pp. 27-32), an important place is occupied by the oxidative conversion of the intermediate chalcones obtained from acetophenones and aromatic aldehydes.

An alternative method of obtaining isoflavones is to close the polyhydroxydeoxybenzoin ring. This method achieves relatively high yields of the end product only with a substrate hydroxyl group of less than 2. A preferred variant of the method of ring closure of partially unprotected deoxybenzoin derivatives described by R.J. Bass (J. Chem. Soc., Chem. Commun., 1976, p. 78) is based on the formylation and subsequent cyclization of polyhydroxydeoxybenzoins with N, N-dimethylformamide and methanesulfonic chloride in the presence of boron trifluoride etherate. This method produces a 4'-methoxy derivative of genistein (5,7-dihydroxy-4'-methoxyisoflavone) which must be demethylated in the presence of boron tribromide to form genistein.

These methods require the introduction and removal of protecting groups, and a particularly careful choice of reaction conditions due to the high reactivity of polyphenolic substrates, which are readily susceptible to polycondensation or oxidation reactions. In addition, a significant limitation of these processes is the availability of the used starting materials, hydroxyl phenol derivatives and aromatic aldehydes in the synthesis of the intermediate chalcones or deoxybenzoins.

The method of obtaining polyhydroxyisoflavones in a one-step reaction not requiring the protection of hydroxyl groups described by K. Wahal and T.Hase (J. Chem. Soc. Perkin Trans, 1, 1991, p. 3005) is based on the Friedel-Crafts reaction of substituted phenols and acetic acids in boron trifluoride etherate. The reaction can be stopped at the stage of the deoxybenzoin formed or, without isolation of the intermediate, catalyzed cyclization against DMF and methanesulfonic chloride to form isoflavones.

Despite the total efficiency of the reaction of genistein production in the reaction of floroglucin with 4-hydroxyphenylacetic acid in the environment of boron trifluoride etherate of 50%, as declared by the authors, in practice it turned out that a large amount of unreacted substrates remains in the reaction mixture, a large amount of by-products and only traces of deoxybenzoin.

PL 194 989 B1

Surprisingly, it was found that it was possible to obtain genistein with a high degree of purity and high yield by using completely unprotected raw materials in the regioselective C-acylation reaction of the aromatic phenol ring.

The method of obtaining genistein of the formula I consists in reacting the floroglucin of the formula 2 with 4-hydroxybenzyl cyanide of the formula 3, and then the isolated intermediate product - deoxybenzoin of the formula 4 is formylated and cyclized in one step, and then crude genistein is purified.

The reaction of floroglucin with 4-hydroxybenzyl cyanide is carried out in the presence of catalytic amounts of zinc chloride in an ethereal solvent by passing a stream of dry hydrogen chloride at 0-5 ° C through the reaction mixture. The ether solvent used is, for example, diethyl ether, diisopropyl ether, tert-butyl methyl ether. Preferably, an ether of the glim type (ethers of ethylene glycol and its oligomers), especially dimethoxyethane, is used as the solvent.

The cyclization of deoxybenzoin is carried out in N, N-dimethylformamide in the presence of boron trifluoride etherate and methanesulfonium chloride. N, N-dimethylformamide plays a dual role in the reaction - the formylating agent and the solvent. Preference is given to using a five molar excess of boron trifluoride and a three molar excess of methanesulfonium chloride over deoxybenzoin. These reagents are added successively to the reaction mixture at a temperature of 20-45 ° C, while the ether is distilled off from the reaction mixture, which allows the final stage of the synthesis to be carried out at an elevated temperature (65-70 ° C).

The obtained crude genistein is purified by hydrolysis in a water-methanol medium at pH 8-13.5 at room temperature or higher, and then cooled to 0-10 ° C, the clear reaction mixture is acidified with a mineral acid solution to pH 0.5 -3.5 and pure crystalline precipitate is isolated in a known manner.

Two parts by mole of an alkali metal hydroxide solution are added to a suspension of one mole part of crude genistein. After the product suspension has dissolved, stirring is continued at a pH of 8-13.5, preferably at a pH of 11, at room temperature or higher for 2 hours. Then, two parts of a solution of mineral acid, preferably hydrochloric acid, are added to the clear and cooled to 0-10 ° C solution until the pH is 0.5-3.5. The crystalline precipitate is filtered off, washed with water and dried to obtain high purity genistein (ca. 99.8% by HPLC).

The method according to the invention makes it possible to obtain a substance with a potential anti-tumor application - genistein with a high degree of purity and in good yield from readily available raw materials, as a result of several uncomplicated reaction steps.

The following examples illustrate the invention.

P r z k ł a d 1.

4-Hydroxybenzyl cyanide

A. 4-Hydroxybenzyl alcohol

4-hydroxybenzaldehyde (900 g; 7.37 mol) was dissolved in 4 L of methanol at room temperature. After cooling the mixture to approx. + 10 ° C in an ice-water bath, sodium borohydride (174 g; 4.6 mol) was added in portions (approx. 5 g each) over approx. 3 hours, keeping the temperature of the mixture within + 15- 20 ° C. The progress of the reaction was monitored by TLC chromatography on silica gel using chloroform: ethanol: acetic acid (90: 10: 0.01). The mixture was then acidified with acetic acid (270 ml) at 15 ° C to a pH of 5.5. The solvents were evaporated to recover 3.5 L of distillate. The residue was taken up in cold water (1 L) and ethyl acetate (2 L) and stirred for 1 hour. The organic and aqueous layers were separated and the aqueous layer was further extracted with ethyl acetate (3x1 L). The combined organic phases were washed with brine (2 x 1.5 L) and dried anhydrous. sodium sulfate (150 g). After filtration, the ethyl acetate solution was concentrated on an evaporator to recover 4 L of solvent. 2x0.5 l of toluene was added to the wet residue of the crystalline precipitate and the solvents were evaporated on an evaporator at a temperature of about 70 ° C. 870 g of pure 4-hydroxybenzyl alcohol were obtained. 95% yield.

M.p. 114-122 ° C.

IR ⁽ KB11 cm ^-1): 3387 ^, 3111, 16 ^ 1598 ^, 1454 ^, 1370 ^, 1236 ^, 1208 ^, 994 ^, 838 ^, 565 ^, 491.

B. 4-Hydroxybenzyl cyanide

186.2 g (1.5 mol) of 4-hydroxybenzyl alcohol, 91.6 g of sodium cyanide and 1 L of dry dimethylformamide were placed in the flask. The mixture was vigorously stirred and heated under nitrogen at 110-130 ° C in an oil bath for 20 hours. The mixture was then cooled to

At room temperature, 200 ml of cold water, 25 g of sodium hydroxide were added and the mixture was stirred for 1 hour. Solvents were removed from the reaction mixture on a rotary evaporator at a pressure of 0.1-1 mm Hg (0.13-1.33 hPa) with a water bath temperature of 45 ° C. 500 ml of cold water was added to the residue and stirred until the solids dissolved. It was neutralized with 75 ml of acetic acid to pH 5.5. The aqueous solution was extracted with chloroform (2x200 ml and 2x100 ml). The combined organic layers were washed with 200 ml of brine and dried anhydrous. sodium sulfate. The drying agent was filtered off and the chloroform solution was concentrated on a rotary evaporator under reduced pressure. The residue (358.8 g) of dark brown oil was subjected to preliminary vacuum distillation at 18-20 mm Hg (24.0-26.6 hPa) at 70 ° C, yielding 157.1 g of forerunner. The main distillation was carried out at the temperature of 157 ° C under pressure. 0.5 mm Hg (0.66 hPa). 172.5 g of pure 4-hydroxybenzyl cyanide crystallizing in the form of colorless to pale straw crystals at the temperature below 70 ° C was obtained. Yield: 86.4%.

Mp 67 ^- 70 ° C. ¹ H NMR ⁽²⁰⁰ MHz, _CDCl3), d ^{(pp m): 3,} 68 (s, ^{2H); 5,} 19 (s, ^{1H); 6,} 83 (dd.J ^^ Hz, J2 = 6.6 Hz, 2H); 7.18 (dd, J (= 2.2Hz, J2 = 6.6Hz, 2H).

P r z k ł a d 2.

2,4,6,4'-Tetrahydroxydeoxybenzoin.

152.15 g (1 mol) of molten 4-hydroxybenzyl cyanide were placed in the flask. The cooled 4-hydroxybenzyl cyanide was dissolved in 500 ml of anhydrous dimethoxyethyl ether over 15 minutes. Then 126.11 g (1 mol) of floroglucin was added. 68.14 g (0.5 mol) of anhydrous zinc chloride and 500 ml of dimethoxyethyl ether were added to the clear solution. The mixture was vigorously stirred, cooled in an ice-water bath to 0-5 ° C, and a dry stream of hydrogen chloride (generated from 535 g of NH ₄ Cl and 535 ml of conc. H 2 SO 4) was dispensed. Hydrogen chloride saturation was continued for 2 hours and then the reaction mixture was left at below 10 ° C for 24 hours. After this time, a small amount of a yellow crystalline precipitate of ketimine hydrochloride dropped out of the mixture. For the reactants to react completely, the reaction mixture was re-saturated with dry hydrogen chloride in an analogous manner. A large amount of crystalline sediment fell out of the mixture, which was left at the temperature below 10 ° C for 3 days. The course of the Friedel-Crafts reaction was monitored by TLC on silica gel in the system chloroform: ethanol: acetic acid 90: 10: 0.01 (v / v). The resulting yellow product precipitate was filtered off and washed with dimethoxy ether (400 ml). The precipitate was treated with a 2% aqueous solution of hydrochloric acid (1000 ml) and it was heated under reflux for 3.5 hours. After cooling to room temperature, the product was filtered off and washed with water (2 x 500 ml) and then crystallized from a water-methanol mixture (2: 1, 2.4 l). The separated yellow crystals are filtered off, washed with a water-methanol mixture (2: 1.240 ml) and dried at 100-105 ° C. Yield 161 g (61.9%). Mp 270-272 ° C.

IR ⁽ KBl cm ^1): 3490 ^, 3323 ^, 3205 ^, 1646 ^, 1611, 1592 ^, 1540 ^, 15 ^ 1475 ^, 14 ^ 12 ^ 1079 ^, 994 ^, 819, 799, 529.

¹ H ^- NMR ⁽²⁰⁰ MH + acetone-d6), d ^{(pp m): 4,} 30 (s, ^{2H); 5,} 93 (s, ^{2H); 6,} 75 (dd, J ^ = 2.0, J2 = 6.4 Hz, 2H); 7.10 (dd, J (= 2.0 Hz, J2 = 6.4 Hz, 2H); 8.13 (bs, 1H); 11.74 (bs, 1H).

P r z k ł a d 3.

4 ', 5,7-Trihydroxyisoflavone (genistein)

150 g (0.576 mol) of deoxybenzoin and 858 g of dry N, N-dimethylformamide were placed in the flask. The mixture was stirred for 15 minutes at room temperature. 408 g (2.874 mol) of boron trifluoride etherate was added dropwise to the clear solution over 45 minutes, while the temperature of the mixture increased to 40 ° C. Then, diethyl ether was distilled off by heating the mixture in an oil bath to 45 ° C for 1 hour. 100 mL of ether was collected. While stirring at 45 ° C was continued, 197.9 g (1.728) of methanesulfonium chloride were added dropwise over 30 minutes. The temperature of the mixture rose to 56 ° C. The mixture was heated at 65-70 ° C for 4 hours with an oil bath temperature of 80-85 ° C. A yellow precipitate formed after 30 minutes. After cooling to room temperature, the reaction mixture was poured into 4.5 L of cold water and stirred for 12 hours. The separated light cream precipitate was filtered off and washed with water (2 x 3 l) until the pH was ca. 381.3 g of a wet sludge was obtained, of which, after drying at the temperature of 100-105 ° C, 153 g of the product was obtained with the title compound content 97.7% (HPLC). Yield 96.03%.

The crude reaction product (2.7 g) was suspended in 80 ml of methanol and 20 ml of 1M NaOH were added. It was stirred for 2 hours at 24 ° C. To the clear methanol-aqueous solution was added 20 ml of 1M HCl over 5 minutes. After 30 minutes, 40 ml of cold water was added to the mixture containing the crystalline precipitate, it was cooled in an ice-water bath to 5-10 ° C and stirred for 1 hour. The precipitate was filtered off and washed with chilled MeOH: H 2 O (1: 1). After drying

After exposure to air (temperature 30-35 ° C, 12 hours), 2.59 g of purified genistein (99.9%, HPLC) was obtained. Yield 96.07%.

M.p. 300-301 ° C.

IR ^(KBL cm ^-1): 3411 ^{3105, 1652,} 16 ^ ^{1520, 1504, 1310, 1274, 1203,} 1180, 1145, ^1044, 791. ¹ H ^- NMR ⁽²⁰⁰ MH + DMSO ^{- d6)} d ^(pp m ^{) 6,} 20 (d, J = 2,0H ^{z, 1H); 6,} 33 (d, J = 2,0H ^{z, 1H);} 6 ^, 83 (dd,

J1 = 2.0 Hz, J2 = 6.0 Hz, 2H); 7.33 (dd, J1 = 2.0 Hz, J2 = 6.6 Hz, 2H); 8.09 (s. 1H); 9.35 (s, 1H); 10.55 (s, 1H); 12.87 (s, 1H).

Claims (7)

Patent claims 1. The method of extracting moldistin of formula 1, changing the fact that florooluucin of formula 2 is reacted with 4-hydroxybenzyl cyanide of the formula 3, and then the isolated intermediate product - deoxybenzoin of formula 4 is subjected to formylation and cyclization in one step, and then the raw genistein is purified. 2. How to do it in ^ e ^^^ g a ^ r. f 1 is changed by the fact that the florzoluccny fiction with cc ^^^ r ^ n ^ h ^ r ^ 4-hyyrscobenzyl is carried out in the presence of catalytic amounts of zinc chloride in an ethereal solvent by passing a stream of dry hydrogen chloride at 0-5 ° C through the reaction mixture . 3. Follow the steps as shown above. f or 2. with changes that, like a phososocial element, there is a glimu-type ether. 4. Sppsób according to zzstrz. 3, with the change that j j ^^ o fcs ^ F ^ L ^^^ c ^^^ lr ^ ik. I am studying myself; dimethoxyethyl eem. 5. The method according to principles. The process of claim 1, wherein the deoxybenzoin cyclzacation is in N, N-dimethylformamide in the presence of boron trifluoride etherate and methanesulfonium chloride. 6. Sppsób according to zzstrz. f, with the fact that I suck in ζ ^ Ιο ^ ccklizzaci? five-molar excess of boron trifluoride and a tri-molar excess of methanesulfonium chloride in relation to deoxybenzoin. 7. Spłc ^ oit) according to zzstrz. The reaction of the hydrolysis reaction at pH 8-13.5 in the water-methanol medium is acidified to pH 0.5-3.5 and gives off a crystalline precipitate. .