PROCESS FOR THE SYNTHESIS OF 6-HYDROXYMETHYL-l,4- ANDROSTADIEN-3J7-DIONE

The invention relates to a new bioconversion process for the synthesis of 6- hydroxymethyl-l,4-androstadiene-3,17-dione (short name: 6-hydroxymethyl-ADD). The invention particularly relates to the microbiological δ'-dehydrogenation of 6- hydroxymethyl-4-androsten-3,17-dione (short name: 6-hydroxymethyl-AD). The obtained compound is an intermediate in the synthesis of 6-methylene-l,4-androstadien- 3,17-dione (short name: 6-methylene-ADD, exemestane), which is an aromatase inhibitor used in the treatment of breast cancer.

Several methods are known from the literature for the synthesis of exemestane, but the majority of these methods is merely chemical synthesis. For example 6- hydroxymethyl-ADD was synthesized by Wojciechowska and co-workers (Polish Journal of Applied Chemistry, 47(3-4), 63-74/2004/) starting from 1,4-androstadien- 3,17-dione (ADD) via several intermediates and the 6-methylene-ADD was obtained by water elimination from 6-hydroxymethyl-ADD.

6-Methylene-4-androsten-3,17-dione was synthesized by Longo and Lombardi (see EP 326,340) starting from 4-androsten-3,17-dione (AD) via enolether, followed by bromination and subsequent hydrogen bromide elimination to yield exemestane.

Shao, Li and You (Zhongguo Yiyao Gongye Zazhi, 32(8), 345-346 /2001/) used 2,3-dichloro-5,6-dicyano-l,4-benzoquinone for the dehydrogenation of 6-methylene- androstendione.

The 6-methylene group of exemestane was formed via a several-step synthesis by Kunnen and co-workers (see WO 2005/070951) starting from l,4-androstadien-3,17- dione.

Zhu and Pan (see Chinese patent No. CN 1,491,957) used 2-iodoxy-benzoic acid for the formation of δ^double bond in the 6-methylene-4-androsten-3,17-dione.

The only process containing a bioconversion step was applied by Krook and

Hewitt (see: WO 2001/004342) in which the enzymatic dehydrogenation of 6- methylene-4-androsten-3,17-dione directly resulted in the exemestane. Because of its poor solubility in water the starting material was dissolved in a solvent (toluene) immiscible with water and the Arthrobacter simplex bacterium cells were added to the system in an aqueous suspension. The dehydrogenation reaction was carried out in the presence of electron acceptor and catalase enzyme, by introducing a mixture of air and nitrogen into the stirred mixture and the product was crystallized from the toluene phase by addition of octane.

Summarizing the disadvantages of the above mentioned processes we can say that large amounts of highly flammable and/or toxic reagents and solvents were used. Moreover the yields of these processes were below the economical level of an industrial manufacturing.

As it can be seen from the above mentioned facts, there is no publication in the literature about the direct bioconversion synthesis of 6-hydroxymethyl-l,4- androstadien-3 , 17-dione.

The aim of the present invention is to elaborate an industrially applicable, economical and environmental friendly bioconversion process for the synthesis of 6- hydroxymethyl- 1 ,4-androstadien-3 , 17-dione.

After investigating the microbiological δ'-dehydrogenation possibility of different intermediates obtained in the synthesis of exemestane starting from 4- androstene-3,17-dione surprisingly it was found, that using the Arthrobacter simplex bacterium cells the 6-hydroxymethyl-4-androsten-3,l 7-dione can be transformed into 6- hydroxymethyl-l,4-androstadien-3,l 7-dione in excellent yield without formation of considerable amount of by-products and this way we found a favourably accomplishable process, through which both the disadvantages resulting from product inhibition in the transformation of 4-androsten-3, 17-dione or from the poor solubility of

6-methylene-4-androsten-3,17-dione in water and the use of hazardous reagents and solvents can be eliminated.

According to this finding the invention relates to a process for the synthesis of 6- hydroxymethyl-l,4-androstadien-3,17-dione comprising dehydrogenating 6- hydroxymethyl-4-androsten-3,17-dione in the presence of a biocatalyst.

The 6-hydroxymethyl-4-androsten-3,17-dione used as substrate was prepared as described in the literature (J. Am. Chem. Soc. 78 _^ 430-436 (1956) and HeIv. Chim. Acta 56(7), Nr. 247., 2396-2404 (1973)) starting from 4-androsten-3,l 7-dione.

According to a preferred embodiment of the invention the dehydrogenation is carried out the following way: the bacterium cells able to produce steroid-δ ¹ - dehydrogenase enzyme, preferably Arthrobacter simplex (ATCC 6946) microorganism, are cultured under aerobic conditions by known method, the biosynthesis of the enzyme is induced by addition of an inducer, the so obtained biocatalyst is interacted with 6- hydroxymethyl-4-androsten-3,l 7-dione used as starting material, optionally stabilizer and electron acceptor are added to the mixture and after completion of the bioconversion the product is isolated by known method.

In the process according to our invention hydrocortisone or 4-androsten-3,17- dione can be used as inducer, 13 -ethyl- 10,l l-dihydroxy-4-gonen-3,l 7-dione or sterols containing C ₈ -C ₁₀ alkyl side-chain in position 17 can be used as stabilizer and 2-methyl- 1 ,4-naphtoquinone or the bisulfite derivative thereof can be used as electron acceptor.

During the realization of the process according to our invention from among the known methods (whole cells, enzyme extracts free of cells, or the combination thereof) whole cell culture of Arthrobacter simplex can be used for the production of the biocatalyst, which is incubated in liquid culture medium containing carbon source, preferably glucose in a concentration of 1-35 g/1, yeast extract in a concentration of 1-10 g/1 and inorganic salts. The culture is incubated at 25-38 ⁰ C, preferably at 35 ⁰ C for 20-

72 hours, then a solution of compound/compounds inducing the production of the bacterial δ'-dehydrogenase (STDH) enzyme is added to the culture.

The induction is triggered by known method: a methanol solution of hydrocortisone - in a concentration of 10-100 mg/1 - is added to the culture. If the induction effect is decreased because of the enzymatic decomposition of hydrocortisone further amount of hydrocortisone can be added in order to maintain the activity needed for the conversion.

According to our discovery a more advantageous solution for reducing the inactivation is the application of - besides hydrocortisone - a slowly degrading, non- inducing steroid compound, for example β-sitosterol or the addition of 13 -ethyl- 10,11- dihydroxy-4-gonen-3,17-dione, which was used successfully in the bioconversion synthesis of finasteride by K. Olasz and coworkers (see US patent No. 6,762,302), in the beginning of the induction in a concentration of 10- 100 mg/1.

If cultivation is continued under the same fermentation condition during the induction the STDH activity reaches 4000-9000 U/dm ³ and the OD value reaches 18-25 in 45-53 hours. This induced culture can be used directly for bioconversion or can be kept at 2-8 ⁰ C for several weeks.

In a preferred embodiment of the process according to our invention the properly induced culture is diluted to 3-15 fold of its original volume with sterilized water, then the solutions of 6-hydroxymethyl-4-androsten-3,17-dione (1-10 g/dm ³ ) used as starting material and 2-methyl-l,4-naphtoquinone (10-100 mg/dm ³ ) used as electron carrier are added in an organic solvent miscible with water, preferably in methanol.

The bioconversion mixture is incubated at 32 ⁰ C under aerobic conditions. The conversion is monitored in every hour by TLC or HPLC, after completion of the transformation (3-12 hours) the obtained 6-hydroxymethyl-l,4-androstadien-3,17-dione is isolated by extraction with an organic solvent immiscible with water.

The invention is illustrated by the following not limiting examples.

Example 1

Step a) Preparation of Arthrobacter simplex culture containing STDH enzyme

The culture of Arthrobacter simplex (ATCC 6946) bacterium is maintained on agar slopes of the following composition:

The inoculated medium was incubated at 32 ⁰ C for 4 days in order to initiate proliferation, then it was kept at +4-10 ⁰ C for further 30 days. Vegetative culture was made by transferring the suspension of the surface culture into 100 cm ³ of sterilized culture medium of the following composition in a 500 cm ³ flask:

The culture was shaken at 32 ⁰ C for 24 hrs with 200 rpm, then 2-2 cm ³ of the obtained inoculum culture was used to inoculate 100-100 dm ³ of culture medium of the following composition in two 500 cm ³ flasks:

The culture was shaken at 32-37 ⁰ C, preferably at 32 ⁰ C for 24 hrs with 200 rpm, then 200 cm ³ of the so obtained inoculum culture was used to inoculate 5 dm ³ of sterilized main phase culture medium of the following composition into a 9 dm ³ jar fermenter:

The culture was sterilized at 121°C for 40 min and the pH was adjusted to 6.3-6.4 with NaOH solution before inoculation.

The culture was agitated at 35 ⁰ C with 300 1/min speed and 60 dm ³ /hr aeration rate. The consumption of glucose is accompanied by lowering of the pH, which could decrease the growth rate. Therefore the pH of the culture was controlled at 6.3-6.5 by the addition of 200 g/dm ³ NaOH solution. The cultivation time was 24-26 hours followed by a further 48-96 hours of induction period.

Before the induction of the formation of the STDH enzyme 0.5 g of glycine and 0.75 g of zinc sulfate was dissolved in 40 cm ³ of water and the mixture was sterilized at 121°C for 30 min. 0.5 g of hydrocortisone was dissolved in 50 cm ³ of methanol which contained 0.35 g of dry calcium chloride. The so obtained solution was mixed with the glycine - zinc sulfate solution and added to the culture. 0.5 g of sitosterol was soaked with 0.5 cm ³ of 1 % Tween-80 solution and 40 cm ³ of water, boiled until foaming was stopped and sterilized at 121°C for 30 min, then it was shaken till cooling to room temperature. The obtained suspension was added to the culture and cultivation was continued under the same fermentation conditions. During the induction the STDH activity reaches 5000-7000 U/dm ³ and the OD value reaches 20-25 in 48-96 hours.

Step b) Synthesis of 6-hydroxymethyl-l,4-androstadien-3,17-dione (shortly: 6-hydroxymethyl- ADD) from 6-hydroxymethyl-4-androsten-3,17-dione (shortly: 6-hydroxymethyl-AD)

The dehydrogenation was carried out by using the aqueous suspension of the biocatalyst obtained in the previous step. The bioconversion medium was prepared by sterilizing pH=7 phosphate buffer: 16.15 g of KH ₂ PO ₄ and 35.65 g Na ₂ HPO ₄ .2H ₂ O was dissolved in 4.8 dm ³ water and solution was sterilized at 121 ⁰ C for 30 min in a 9 dm ³ jar fermenter equipped with probes to measure the pH and the concentration of the dissolved oxygen. The temperature was adjusted to 32 ⁰ C and 330 cm ³ of the induced culture was added to the phosphate buffer under aseptic conditions. Then a solution of 14.5 g of 6-hydroxymethyl-4-androsten-3 , 17-dione in a mixture of 202 cm ³ of methanol and 22 cm ³ of water - dissolved previously by boiling - was added to the bioconversion system and finally a solution of 0.5 g of 2-methyl-l,4-naphtoquinone -sodium bisulfite

(shortly: menadione-bisulfite) - used as electron acceptor - in 10 cm ³ of sterile water was added.

The bioconversion was monitored by TLC or HPLC, along with the STDH activity and the optical density (OD) characteristic for the cell-concentration.

When 90 % of the starting 6-hydroxymethyl-AD was transformed the bioconversion was stopped, 5 g/dm ³ of filtration aid (Perfil) was added and the fermentation broth was filtered. The filtered biomass and the filtration aid were removed for incineration and the filtrate was extracted with 2 dm ³ of ethyl acetate. The aqueous phase was again extracted with 1 dm ³ of ethyl acetate, the combined organic phases were washed with 0.8 dm ³ of NaOH solution (50g/dm ³ ), then 0.2 dm ³ of water was added and the ethyl acetate was distilled off in vacuum at 65-70 ⁰ C in a Rotavapor. The obtained material was poorly crystalline. It was purified the following way: the precipitated material was dissolved in 0.2 dm ³ of warm methanol and 0.15 dm ³ of water and 4 dm ³ of NaOH solution (50 g/dm ³ ) were added. Then the solvent was distilled off, the precipitated homogenous crude crystalline material was filtered after stirring for 3 hours, washed with 10 cm ³ of water and dried at 80 ⁰ C in vacuum oven. The weight of the so obtained crude crystalline material was 10 g, it contained 92 % of 6-hydroxymethyl-ADD.

Further purification: 10 g of crude crystalline material was dissolved in 20 cm ³ of dichloromethane and 100 cm ³ of methyl-cyclohexane was added dropwise to the stirred solution. The product was precipitated, the mixture was stirred for 1 hour, then filtered, washed with 10 cm ³ of methyl-cyclohexane and dried at 80 ⁰ C in vacuum oven. The weight of the so obtained crystalline material was 9.5 g, it contained 93 % of 6- hydroxymethyl-ADD (yield: 60.9 %).

The structure elucidation was performed by NMR analysis. The characteristic chemical shifts are as follows:

¹ H NMR {Varian NMRS-500, DMSO-d ₆ (TMS), δ(ppm)}: 7.17d (C-I); 6.08 dd (C-2); 6.02 d (C-4); 2.7 m (C-6); 1.16 m & 2.01 m (C-7); 1.92 m (C-8); 1.06 m (C-9); 1.59 m

& 1.83 m (C-I l); 1.20 m & 1.69 m (C-12); 1.28 m (C- 14); 1.54 m & 1.88 m (C-15); 2.01 m & 2.42 m (C-16); 0.89 s (C-18); 1.20 s (C-19); 3.57 m & 3.70 m (-CH ₂ (6)); 4.76 1 (-OH)

¹³ C NMR {Varian NMRS-500, DMSO-d ₆ (TMS), δ(ppm)}: 156.8 (C-I); 125.9 (C-2); 184.8 (C-3); 126.3 (C-4); 168.2 (C-5); 48.1 (C-6); 31.9 (C-7); 30.4 (C-8); 50.6 (C-9); 43.0 (C-IO); 21.0 (C-I l); 30.9 (C-12); 46.9 (C-13); 49.7 (C- 14); 21.4 (C-15); 35.1 (C- 16); 219.1 (C-17); 13.5 (C-18); 19.5 (C- 19); 62.8 (-CH2(6))

Example 2

The STDH enzyme was prepared as described in Example 1, but the following modification was applied in the preparation of the main phase culture:

The induction was carried out as described in Example 1, but the formation of δ ¹ - dehydrogenase enzyme was stabilized by addition of a non-inducing compound. Hydrocortisone is the most preferred STDH enzyme inducer, but the high cell density culture could metabolise it, which would lead to the inactivation of the formed STDH. hi order to avoid inactivation in addition to hydrocortisone 13 -ethyl- 10,1 l-dihydroxy-4- gonen-3,17-dione (shortly: 10,11-dihydroxy-levodione) was also added at the beginning of the induction.

The formation of the -δ ¹ -dehydrogenase enzyme was induced by addition of 100 mg/dm ³ of hydrocortisone, simultaneously 75 mg/dm ³ of 13-ethyl-10,l l-dihydroxy-4- gonen-3,17-dione dissolved in methanol was added as enzyme stabilizer. After 48-72 hours induction time the culture was diluted with pH=7 phosphate buffer of 14-fold volume and this was used in the bioconversion step. The latter and the isolation of the product were carried out as described in Example 1. This way 9.8 g crude crystalline product was obtained, which contained 92.2 % of 6-hydroxymethyl-ADD (yield: 62.3

%).

Example 3

The process was carried out as described in Example 2, but the main phase cultivation and the enzyme induction were carried out in a volume of 80 dm culture medium. During the fermentation the culture was agitated at 35 ⁰ C with 200 1/min speed and 960 dm ³ /hr aeration rate. Foaming was blocked by addition of Struktol SB2020 antifoam agent. The culture could be used for the bioconversion step 48-72 hours after starting the enzyme induction.

70 dm ³ of pH=7 sterilized phosphate buffer was added to 5 dm ³ of the so obtained culture. 217.5 g of 6-hydroxymethyl-4-androsten-3,17-dione was dissolved in 3300 cm of methanol at 50 ⁰ C and it was added to the stirred, diluted culture. Then 7.5 g of menadione-bisulfite was dissolved in 150 cm ³ sterile water and added to the

bioconversion mixture. The bioconversion mixture was stirred in a 100 dm ³ fermenter at 32 ⁰ C with 200 1/min speed and 0.1 v/v/min aeration rate. The conversion was monitored in every hour by TLC, after completion of the transformation the obtained 6- hydroxymethyl-l,4-androstadien-3,17-dione was isolated by extraction with ethyl acetate as described in Example 1. The weight of the so obtained crystalline material was 152 g, which contained 91.5 % of 6-hydroxymethyl-ADD (yield: 64 %).