Process for preparing 14-alpha-hydroxy-4-androstene-3,6,17-trione

Abstract: A process for the preparation, in high yield, of 14-α-­ hydroxy-4-androstene-3,6,17-trione which has an aromatase inhibition activity and is useful as an antitumor agent, in which a hydroxyl group at position 6-β of 6-β,14-α-­ dihydroxy-4-androstene-3,17-dione is selectively oxidized with the aid of light energy.

Description: The present invention relates to a process for preparing a kind of steroid, 14-α-hydroxy-4-androstene-3,6,17-trione, and particularly to a process for preparing said compound easily and in high yield by oxidation caused by light. Steroids show various activities in physiology due to their tertial configurations. In particular, it is now evident that such physiological activities are greatly affected by the fact that whether oxygen atoms bound to the steroid skeleton are those of hydroxyl groups or those of double-bonded oxygens. Consequently, various methods for oxidation of the hydroxyl groups bound to the steroid backbones and for reduction of carbonyl groups thereof have been investigated. The present inventors previously found that 6-β,14-α-dihydroxy-4-androstene-3,17-dione was produced by microbial transformation of steroid when 4-androstene-3,17-dione was added as a substrate to a culture medium for Acremonium strictum , and thus applied for a patent as Japanese Patent Application No. 24598/1987 since the compound has a strong aromatase inhibitory activity. Thereafter, as a result of intensive study on 6-β,14-α-dihydroxy-4-androstene-3,17-dione, the present inventors confirmed that 14-α-hydroxy-4-androstene-3,6,17-trione have a stronger aromatase inhibitory activity than 6-β,14-α-dihydroxy-4-androstene-3,17-dione and that 14-α-hydroxy-4-androstene-3,6,17-trione is useful as an antitumor agent, and then applied for a patent as Japanese Patent Application No. 24594/1987. These patent applications by the present inventors have been applied in an integrated form in U.S.S.N. 279,596 or in EP-A-0300062. 14-α-hydroxy-4-androstene-3,6,17-trione is a compound having the following formula. Then, in order to obtain 14-α-hydroxy-4-androstene-3,6,17-trione, the hydroxyl group at the 6-β position of 6-β,14-α-dihydroxy-4-androstene-3,17-dion has to be selectively oxidized as mentioned above using an oxidizing agent, wherein the use of activated manganese dioxide or chromic acid as the agent is required. Steroid compounds in which an oxygen atom is bound to the carbon at position 6, not being limited to the compound above, have prominent physiological activities and hence various investigations have been conducted. However, most of the compounds have been obtained using oxidation with the use of metal catalysts or oxidizing agents such as activated manganese dioxide and chromic acid. However, in the oxidation reaction using such oxidizing agents, in order to remove metal catalysts such as chromium or manganese and to remove excessive oxidizing agents remaining in the reaction system, complicated steps such as extraction of the solvents or fractionation using silica gel chromatography were required. Moreover, the recovery of a desired compound was not necessarily satisfactory. On the other hand, introduction of a hydroxyl group to position 6-β using excitation energy with the aid of light has been reported by R. Gandi et al. (Journal of Organic Chemistry 32, 2647-2649, 1967). However, a process for oxidizing a hydroxyl group at position 6-β of steroid compounds with the aid of light has not been reported and not yet known. In the course of the intensive study on the oxidation of steroids having a hydroxyl group at position 6-β, the present inventors have discovered that 14-α-hydroxy-4-androstene-3,6,17-trione can be obtained in a high yield by dissolving the above-mentioned 6-β,14-α-dihydroxy-4-androstene-3,17-dione in an organic solvent, sealing the reaction system after blowing oxygen therein and selectively oxidizing the hydroxyl group at position 6-β by radiating with light containing visible light; and thus completed the present invention. Hence, according to the method of the present invention, it is possible to obtain 14-α-hydroxy-4-androstene-3,6,17-trione, which is useful as an antitumor agent, in a highly purified form in a simple manner and in a high yield, by selectively oxidizing the hydroxyl group at position 6-β of 6-β,14-α-dihydroxy-4-androstene-3,6,17-trione with the aid of light energy. Furthermore, the use of oxidizing agents, catalysts or the like are not required, so that production at low cost can be achieved industrially. The Figures attached show the characteristics of 14-α-hydroxy-4-androstene-3,6,17-trione. Figure 1 shows the UV absorption spectrum, Figure 2 shows the IR spectrum, Figure 3 shows the Mass spectrum, Figure 4 shows the proton-NMR spectrum and Figure 5 shows the ¹³C-NMR spectrum. An oxidation reaction facilitated in the process according to the present invention is as follows: In order to selectively oxidize a hydroxyl group at position 6-β of a starting material of the formula shown above, according to the present invention, firstly, 6-β,14-α-dihydroxy-4-androstene-3,17-dione as the starting material is dissolved in a solvent of halogenated hydrocarbons. Said starting material may be obtained by the method disclosed in the above-mentioned U.S.S.N. No. 279,596, in which the above-mentioned starting material is produced by the action of Acremonium strictum on a known 4-androstene-3,17-dione as a substrate. In the process according to the present invention, independently of the production method or the degree of purity required, 6-β,14-α-dihydroxy-4-androstene-3,17-dione can be used as a starting material. Examples of halogenated hydrocarbon solvents to dissolve said starting materials include chloroform, dichloromethane, dichloroethane, trichloroethane, ethyl bromide, dibromoethane, ethyl fluoride, propyl bromide and chlorobenzene; chloroform is preferably used. As to the above-mentioned solvent, 6-β,14-α-dihydroxy-4-androstene-3,17-dione is dissolved at a concentration of 5-200g/liter, preferably 10-50g/liter and placed in a reaction vessel. After blowing oxygen gas therein, said reaction vessel is sealed. Oxygen is blown directly into the solvent with a nozzle or the like or into the reaction vessel, so that the reaction vessel is sufficiently filled with oxygen gas. The reaction vessel is sealed immediately after so blowing oxygen gas. As to the reaction vessel, a sealable glass vessel is preferably used; however, a plastic vessel which is solvent resistant may be used. Since the material of the reaction vessel effects the transmissivity and transmissive wavelength of the light in the irradiation step to be followed, it has to be selected depending on the light source to be used. Subsequently, the sealed reaction vessel is irradiated for 4 to 24 hours. Any light sources which emit light containing visible light can be used; for example, the sunlight, incandescent electric light, a white fluorescent lamp, a metal arc such as tungsten lamp, a carbon arc, a mercury arc and a discharge tube can be used. Namely, in order to selectively oxidize a starting material at position 6-β, wavelengths of visible rays (about 7800 to 3800Å) are most effective in respect to light energy. Generally, lights with shorter wavelength having high chemical energy are not suitable, and the lights in which the ultraviolet area is rather small and a portion of visible rays is 50 % or more are preferably used. If the light energy is too high, the oxidation reaction on carbon atoms other than that at position 6-β may occur, or steroid structures may be affected, which interferes with the selective oxidation. Since visible rays for the generation of the above-mentioned selective oxidation have to reach the solvent in which the starting substance is dissolved, the material of the above-mentioned reaction vessel is preferably selected so as not to interfere with transmission of the visible rays. For this, transparent glass is preferably used. Although a composition of the glass may affect the transmitted light, any kinds of glass which do not interfere with the transmission of visible rays can be used. Further, when a light source which contains lights of many different wavelength areas as well as visible rays is used, an appropriate filter or an ultraviolet absorbing agent may be used so as to increase a ratio of visible rays. Intensity of the light for the irradiation of the solvent is selected depending on the amount of the starting material to be treated and the material of the reaction vessel; the intensity to be used ranges over 500 lux, which approximately corresponds to the range from the intensity of irradiation with a 40 W white fluorescent lamp at a distance of 10 cm to that of the sunlight. When the intensity of irradiation light is too strong, selective oxidation reaction as desired is difficult to occur; further when it is too weak, the oxidation reaction does not proceed. During the irradiation, temperature control is not particularly required unless temperature of the solvent increases excessively high; room temperature may be satisfactory for the reaction. Then the solvent is removed, and 14-α-hydroxy-4-androstene-3,6,17-trione can be recovered either by recrystallization from the resultant residue using organic solvents such as methanol, ethanol and acetone or by subjecting the residue to thin-layer chromatography or the like. According to the process of the present invention, it is possible to obtain the above-mentioned compound at the yield of 90% or more; even the yield of almost 100% can be achieved depending on the conditions. The following Examples demonstrate the present invention more in detail. In this regard, the method of identifying 14-α-hydroxy-4-androstene-3,6,17-trione was disclosed in Japanese Patent Application No. 24594/1987 (integrated in U.S.S.N. 279,596) which had been previously applied by the present inventors. Namely, the compound of the present invention is specified to have the following physicochemical characteristics. 1) Appearance: White powder 2) Molecular weight: 316 3) Molecular formula: C₁₉H₂₄O₄ 4) Ultraviolet absorption spectrum: See Fig. 1 attached 5) EI mass spectrum: m/Z = 316, see Fig. 2 6) Infrared absorption spectrum (KBr method): 3520, 3410, 2790, 1735, 1690, 1675, 1605 cm⁻¹, see Fig. 3 7) Rf value (developing solvent, chloroform:methanol = 9:1): 0.43 8) Solubility: Soluble in ethanol, methanol, ethyl acetate, and chloroform; insoluble in water and hexane 9) Melting point: 241-244°C Example 1 6-β,14-α-dihydroxy-4-androstene-3,17-dione used as a starting material in this Example was prepared according to the method disclosed in Japanese Patent Application No. 192798/1988 (also included in N.S.S.N. 279,596). Namely, Acremonium strictum NN 106 (deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, under the deposition number of 9143) was cultured; after culturing, 4-androstene-3,17-dione was added to the culture as a substrate; steroid conversion was carried out by the microorganisms; solvent extraction from the culture was carried out; and finally purified preparation to be used was obtained. 6-β,14-α-dihydroxy-4-androstene-3,17-dione (50 g) thus prepared in the above-mentioned manner was dissolved in 1 liter of chloroform and transferred to a glass sealable vessel; after blowing oxygen therein, the vessel was sealed and allowed to stand for 4 hours under the sunlight. Thereafter, chloroform was removed by evaporation at 40°C. After the evaporation, the resultant residue was analyzed by thin layer chromatography. As a result, a spot for 14-α-hydroxy-4-androstene-3,6,17-trione only was confirmed. According to this method, 49.2 g of 14-α-hydroxy-4-androstene-3,6,17-trione was obtained. The yield was 98.4%. Example 2 6-β,14-α-dihydroxy-4-androstene-3,17-dione (1 gram) obtained in the same manner as described in Example 1 was dissolved in 100 ml of chloroform and transferred to a 300ml glass flask with a screw cap; after blowing oxygen therein, the flask was sealed and then irradiated with the light of a white fluorescent lamp (50 W) at a distance of 10 cm for 12 hours at room temperature. Thereafter, chloroform was evaporated at 40°C under reduced pressure. The resultant residue was dissolved in methanol and then cooled for recrystallization. Thus, 0.9 g of crystallized 14-α-hydroxy-4-androstene-3,6,17-trione was obtained. The UV absorption spectrum, IR absorption spectrum, mass spectrum and proton NMR and ¹³C-NMR measurements for the crystallized compound revealed that the 14-α-hydroxy-4-androstene-3,6,17-trione thus obtained was identical to that previously reported. These absorption spectra are shown in Figs. 1 to 5. Example 3 6-β,14-α-dihydroxy-4-androstene-3,17-dione (1 gram) obtained in the same manner as described in Example 1 was dissolved in 100 ml of chloroform and transferred to a 300ml glass flask with a screw cap; after blowing oxygen therein, the flask was sealed and then irradiated with the light of a tungsten lamp (80 W) at a distance of 10 cm for 12, hours at room temperature. Thereafter, chloroform was evaporated at 40°C under reduced pressure. The resultant residue was dissolved in methanol and then cooled for recrystallization. Thus, 0.95 gram of 14-α-hydroxy-4-androstene-3,6,17-trione was obtained. The UV absorption spectrum, IR absorption spectrum, Mass spectrum and proton NMR and ¹³C-NMR analyses for the crystallized compound revealed that the 14-α-hydroxy-4-androstene-3,6,17-trione thus obtained was identical to that previously reported. These absorption spectra are shown in Figs. 1 to 5. Although the invention has been described with reference to preferred embodiments, it is to be understood that variations and modifications will be apparent to those skilled in the art and are included within the invention. Such variations and modifications are to be considered within the purview and the scope of the claims appended hereto.

Claims: A process for the preparation of 14-α-hydroxy-4-androstene-3,6,17-trione, comprising the steps of dissolving 6-β,14-α-dihydroxy-4-androstene-3,17-dione in a halogenated hydrocarbon solvent; sealing the reaction system after blowing oxygen therein; irradiating the system with light of a wavelength which is at least partly in the visible range (λ = 380-780 nm) to oxidize a hydroxyl group of position 6-β; and recovering the thus-produced oxidized derivative thereof. A process as set forth in claim 1, in which said halogenated hydrocarbon solvent is chloroform. A process as set forth in claim 1 or 2, in which a light source of said irradiation light contains visible radiation at a rate of 50 % or more of the total. A process as set forth in claim 1 or 2, in which the irradiation is carried out with a light intensity of from 500 lux to sunlight illuminance.

Inventors: MIYATA NOBUO, NAKAKOSHI MASAMICHI, TAMURA KOHJI, YOSHIHAMA MAKOTO, MIYATA, NOBUO, NAKAKOSHI, MASAMICHI, TAMURA, KOHJI, YOSHIHAMA, MAKOTO
Applicants: SNOW BRAND MILK PROD CO LTD, SNOW BRAND MILK PRODUCTS CO., LTD.
Application year: 1990
Main classification: C07J1
IPC codes: A61P43, A61K31, C07J1, A61P35, C07J75