HU201353B - Process for microbiological redcingt prostacyclin intermediates containing 15-keto-group - Google Patents

Abstract

The invention thus relates to a process for the enantioselective reduction of the 15-keto group in bicyclic prostaglandin intermediate production to 15a-hydroxy compounds of the general formula wherein X is oxygen or a CH 2 group, a trans CHCH or CC group, oxygen, residues or or with R 3 in the meaning of a C 1 -C 4 -alkyl group, R 1 is hydrogen, tetrahydropyranyl, benzoyl, tert-butyldimethylgilyl or tert-butyldiphenylgilyl and R 2 is the residue substituent, characterized in that a ketone of the general formula II in which A, B, X, R 1 and R 2 are as above have been indicated, treated with Candida or Pichia-Staemmen and isolated the resulting 15-hydroxy prostaglandin intermediate. Formula (I), (II)

Description

This invention relates to a microbiological process for the enantioselective reduction of 15-keto groups present in bicyclic prostacyclin intermediates to 15 ° C-hydroxy.

The process of the invention is particularly suitable for microbiological reduction of the prostacyclin intermediates of formulas (I) to (4).

The microbiological reduction of the above-described prostacyclin intermediates according to the invention is preferably carried out with the following microorganism strains:

Candida solani (NCYC 41),

Pichia farinosa (NRRL-γ-118) and Pichia farinosa (CBS 185).

Of the above strains, Candida solani is particularly preferred.

European Patent 12710 discloses the chemical reduction of the 15-keto group of prostaglandins and prostaglandin intermediates using, for example, sodium borohydride. These 15-keto compounds can only be converted to the corresponding 15oC-hydroxy-prostaglandins via a mixture of the appropriate 15oC and 15u-hydroxy compounds, and significant yield losses occur due to the required separation. U.S. Patent No. 3,687,811 discloses a number of microorganisms which, in addition to the 11-hydroxyl group already present and formed in the 11-hydroxy-15-oxo-prostaglandins, are the corresponding trans-15-hydroxyl group. into a group. DE-OS 2,357,815 discloses a microbiological process for, for example, converting an 11-hydroxy-15-oxo-prostaglandin into a mixture of HcC, 15oC and 11a, 15u-dihydroxy-prostaglandins. The cis arrangement of the 10C, 15C hydroxy groups corresponds to the configuration present in the biologically active prostaglandins.

DE-OS 2 401 761 also discloses similar processes, which also have the above disadvantages. European Patent No. 12,710 discloses Kloeckera, Saccharomyces, and Hansenula strains which only partially reduce the 15-keto group present in prostaglandin intermediates and thus yield very poor yields of 15oC-hydroxyl-containing prostaglandin intermediates.

Surprisingly, it has been found that the above-mentioned prostaglandin intermediates can be enantioselectively reduced to the corresponding 15 ° C-hydroxy compounds in very good yields using the Candida or Pichia strains for microbiological reduction.

The 15C-hydroxy compounds of the present invention can be used to prepare pharmaceutically effective prostacyclines while maintaining the 15-center asymmetry. For example, in Scheme A, the (1S, 2S, 2R, 5R) -7,7-ethylenedioxy-3-benzoyloxy-2 - [(1E), (4RS) -4-methyl of formula (1) described above Starting from -3-oxooct-1-en-6-yn-yl] bicyclo [3.3.0] octane, the active ingredient Iloprost can be prepared. (see European Patent 11591).

Naturally, depending on the microorganism strains employed, the efficiency of the reduction according to the invention will vary. Good results are obtained with the strains Candida solani (NCYC 41), Pichia farinosa (NRRL-γ-118) and Pichia farinosa (CBS 185), and the reductions with Candida solani (NCYC 41) are particularly good.

The present invention therefore relates to a process for the enantioselective reduction of 15-keto groups present in bicyclic prostaglandin intermediates, which comprises preparing 15 ° C-hydroxy compounds of formula (I):

X is oxygen or -CH2-,

The trans is -CH = CH- or -C = C-,

B is oxygen, a group of formula (a) or (b), wherein

R 1 is benzoyl, and

R2 is a group of formula (e) or (f).

According to the present invention, a ketone of formula II wherein A, B, X, R 1 and R 2 are as defined above, is fermented with Candida solani or Pichia farinosa and the resulting 15-hydroxy-prostaglandin intermediate is obtained. isolated.

Depending on the type of R 1 which is desired in the final product, the protecting groups may be removed by methods known per se.

In the process of the present invention, the aforementioned microorganisms are cultured under culture conditions known per se, on suitable medium and under submerged aeration. The substrate is then added (dissolved in a suitable solvent or preferably in an emulsified form) and the fermentation is carried out until maximum substrate conversion occurs.

Suitable solvents for dissolving the substrate are, for example, methanol, ethanol, glycol monomethyl ether, dimethylformamide or dimethylsulfoxide. For example, the emulsification of a substrate may be accomplished by dissolving it in micronized form or in a water miscible solvent such as methanol, ethanol, acetone, glycol monomethyl ether, dimethylformamide, dimethyl sulfoxide, preferably in deionized water, Contains conventional emulsifiers. Suitable emulsifiers include non-ionogenic emulsifiers, such as ethylene oxide adducts or fatty acid esters of polyglycols. Suitable emulsifiers include, for example, commercially available Tegin, Tagat and Span wetting agents.

Emulsification of substrates often promotes an increase in substrate concentration. Of course, the addition of the substrate

The process of the invention may be enhanced by other methods well known to those skilled in the art of fermentation.

The optimum substrate concentration, the time of substrate addition and the duration of fermentation will always depend on the substrate structure used and the microorganism employed. As is common with biological transformations, it is advisable to carry out preliminary experiments in this case, which are readily accomplished by those skilled in the art.

Canida solani NCYC 41 and

Pichia farinosa CBS 185 strains in the German microorganism collection DSM 3315 and

DSM 3316, Pichia farinosa Y-118 is available from the NRRL.

The following examples illustrate the invention.

Example 1

Into a Erlenmeyer Flask 1, place a nutrient solution containing 1.5% glucose monohydrate, 0.5% yeast extract (Difco), 0.5% corn equivalent and 0.5% ammonium sulfate (for 30 minutes at 120 ° C). sterilized at pH 6.3), then inoculated from an oblique culture of Candida solani (NCYC 41) and shaken on a rotary shaker at 30 ° C for 2.5 days.

250 ml of the above culture was inoculated with 20 L of a pre-fermenter containing 15 L of the above-seeded medium sterilized at 1.1 bar at 121 ° C for 30 minutes at 121 ° C. Silicone SH was used as an antifoam and fermentation was continued at 25 ° C, 0.7 bar overpressure, 15 l / min aeration and 220 rpm with stirring for 36 hours.

Subsequently, 0.9 L of pre-fermentation broth was harvested under sterile conditions and inoculated with 20 L of a fermenter containing 10 L of medium. After a 6-hour growth phase, 4 g of (1S, 2S, 3R, 5R) -7,7-ethylenedioxy-3-benzoyloxy-2 - [(1E), 4 (RS) -4-methyl-3 was added to the fermentor. A sterile solution of -oxooct-1-en-6-yn-yl] -bicyclo [3.3.0] octane (compound 1) in DMF (100 mL) was added and stirring and aeration continued.

after a contact time of 1 hour, the fermentation is completed. The culture broth was extracted three times with methyl isobutyl ketone, the extracts combined and evaporated to dryness in vacuo. The oily residue was taken up in methanol, filtered from silicone oil and evaporated again to dryness. The residual oily crude product was dissolved in methylene chloride and further purified by silica gel column chromatography (5 L hexane / 4 L hexane + 11 acetone solvent). Thus 2.75 g of pure (1S, 2S, 3R, 5R) -7,7-ethylenedioxy-3-benzoyloxy-2 - [(1E), (3S, 4RS) -3-hydroxy-4 methyl oct-1-en-6-yn-yl] -bicyclo [3.3.0] octone is obtained as a colorless oil. Nuclear Magnetic Resonance Spectrum delta (ppm):

0.85, 0.86, 1.73, 1.75, 3.83-3.96, 4.02-4.06, 4.97-5.08, 5.09-5.17,

7.42-7.47, 7.53-7.59, 7.99-8.03.

IR spectrum (cm ^-1 ):

3480, 3060, 3030, 2960, 2930, 2880, 1718, 1601, 1585, 1275, 1115, 1090, 1070, 1027, 972, 713.

Example 2

The procedure described in Example 1 was followed, however, using Pichia farinosa (NRRL-γ-118) for the reduction of the keto group.

Example 3

The procedure of Example 1 is followed, however, for keto reduction, Pichia farinosa (CBS 185) is used.

Example 4

In the same manner as in Example 1, compound (3) was reduced with the strain Candida solani (NCYC 41) to the corresponding 15 ° C hydroxy compound.

Nuclear Magnetic Resonance Spectrum delta (ppm):

0.90, 0.91, 1.78, 3.95-4.12, 4.12-4.28, 4.97, 5.34, 5.56-5.70, 7.31-7, 67, 7.90-8.05.

IR spectrum (cm ^-1 ):

3500 3060, 3030, 2965, 2920, 2875, 2860, 1771 1715, 1601, 1585, 1275, 1114, 1071, 1051,

1027, 972, 714.

Example 5

In the same manner as in Example 1, compound (4) was reduced to the corresponding 15 ° C-hydroxy compound with strain Candida solani (NCYC 41).

Nuclear Magnetic Resonance Spectrum delta (ppm):

0.98, 1.07, 3.00, 3.84-3.96, 4.32, 5.19-5.26, 7.43-7.48, 7.56-7.61,

8.00-8.05.

IR spectrum (cm ^-1 ):

3465, 3065, 3035, 2970, 2939, 2882, 2855, 2233, 1721 1602, 1584, 1275, 1115, 1090, 1071, 1027 714th

Claims (1)

Patent Claim Point A process for the preparation of 15 ° C-hydroxy-prostaglandin intermediates of formula I wherein X is oxygen or --CH2 -, The trans is -CH = CH- or -C = C-, B is oxygen, a group of formula (a) or (b), wherein R 1 is benzoyl; R2 is a group of formula (e) or (f), wherein a ketone of formula (II) wherein A, Β, X, R1 and R2 are fermented with Candida solani or Pichia farinosa strains as defined above and the resulting 15-hydroxy-prostaglandin intermediate is isolated.