CS258141B2 - Method of 15 alpha-hydroxyprostacycline intermediate products' microbiological production - Google Patents

Description

The invention relates to a process for the microbiological production of 15α-hydroxyprostacycline intermediates. The invention is particularly suitable for the microbiological reduction of the following prostacyclin intermediates 1 to 6.

co.c _tf %

O · COiCβHg

The microbiological reduction of the above prostacyclin intermediates is carried out according to the invention using the following strains of microorganisms: Candida solani (NCYC 41) and Pichia farinosa (CBS 185). Candida solani has proven to be particularly suitable for these strains.

EP 12 710 discloses the following prior art: While the chemical reduction of 15-keto groups in prostaglandins and prostaglandin intermediates, for example sodium borohydride, results only in mixtures of the respective 15a and 150-hydroxy compounds and due to subsequent separation only yield losses to the desired 15a-hydroxyprostaglandins , is US Pat. No. 3,687,811 discloses a series of microorganisms which convert the 15-keto group in 1-hydroxy-15-oxoprostaglandins, depending on the 11-hydroxy conformation present, to the corresponding trans-15-hydroxy group. Due to the method of German Offenlegungsschrift C. 23 57 815, the prior art has a microbiological method, for example by converting 11-hydroxy-15-oxoprostaglandin into a mixture of 11α-, 15α and 110-, 150-dihydroxyprostaglandins. The cis-arrangement of 11α-, 15α-hydroxyl groups corresponds to the same situation in biologically active prostaglandins.

The method described in German Patent Publication No. 24 01 761 fails just as all the methods mentioned.

The Kloecker, Saccharomyces and Hansenula strains disclosed in EP 12 710 reduce the 17-keto group in prostacyclin intermediates only partially and the yields of the 15α-hydroxy prostacyclin intermediates are very poor.

It has now been found that the above prostacyclin intermediate products can be reduced in the

258141 5 very good yields enantioselectively to the respective 15α-hydroxy compounds when Candida or Pichia strains are used,

Pharmacologically active prostacyclins can be prepared from the 15α-hydroxy compounds prepared by the process of the invention while maintaining the center of asymmetry at position 15. For example, from (1S, 2S, 5R) -7,7-ethylenedioxy-3-benzoyloxy-2 - [(1E), (4RS) -4-methyl-3-oxooct-1-en-6-yn] bicyclo [3.3.0] octane (1) is obtained by multistage synthesis to the active substance Iloprost (described in EP 11591):

Óthp. ^0Thp

O.CO.C _b H ₅

Of course, the different classes of microorganisms mentioned show differences in the reduction efficiency of the invention. The use of Candida solani (NCYC 41) and Pichia farinosa (CBS 185) yields good yields, while reduction with Candida solani (NCYC 41) yields particularly high yields.

The present invention provides a process for the microbiological production of 15α-hydroxyprostacycline intermediates of formula (I)

(I) in which

X represents an oxygen atom or a methylene group,

A represents a trans-CH = CH- or a —G = C-group,

В represents an oxygen atom, a group

В

O

í ll)

-O or

CM taking

R3 represents a C1-C6 alkyl group,

R 1 represents a hydrogen atom, a tetrahydropyranyl group, a benzoyl group, a tert-butyldimethylsilyl group or a tert-butyldiphenylsilyl group, and

R2 represents a group

c ^ -csc-c ^ or ii

CH-C-CC _from H ₅ wherein

А. V, X, Ri and R'2 are as defined above, which is deposited in the German Collection of Microorganisms in Gottingen under number DSM 3,315, or the Pichia farinosa strain, which is deposited in the German Collection of Microorganisms in Gottingen under number DSM 3,316, For example, methanol, ethanol, glycol monomethyl ether, dimethylformamide or dimethylsulfoxide are used as substrate solvents, or the substrate is used in emulsified form and the resulting 15 ' -hydroxyprostacycline derivative is isolated after fermentation.

C1-C1-allyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl.

The process is particularly preferred when R1 in the compounds of formula II is benzoyl.

Depending on the desired meaning of R.t, the supine protectors can be cleaved in a manner known per se.

They are first grown under the usual conditions for said microorganisms in a suitable culture medium under aeration of a submerged culture. Subsequently, the substrate (dissolved in a suitable solvent or preferably in the form of an emulsion) is added to the cultures and fermentation is performed until the maximum conversion of the substrate is achieved.

Suitable substrate solvents are, for example, methanol, ethanol, glycol monomethyl ether, dimethylformamide or dimethylsulfoxide. The emulsification of the substrate can be carried out, for example, by mixing it in micronized form or dissolved in a water-miscible solvent (such as methanol, ethanol, acetone, glycol monomethyl ether, dimethylformamide, dimethylsulfoxide) and mixing under strong turbulence into water (preferably de-calcium) containing conventional emulsifiers. Suitable emulsifiers include nonionic emulsifiers such napřípříklad ethylene oxide adducts or fatty acid esters of polyglycols. Vhodkterý characterized in that the ketone of formula II APPLICABLE emulsifiers include commercial wetting agents Tegin ^R), Tagat ^(R) and Span ^{(R )} .

Often, the emulsification of the substrate allows for greater throughput and thus an increase in its concentration. However, it is of course also possible to use other methods of increasing the throughput of the substrate in the process according to the invention, as is known to the person skilled in the art of fermentation.

The optimum substrate concentration, substrate addition time and fermentation time depend on the structure of the substrate used and the type of microorganism used. These quantities must be determined, as is usually the case in microbiological transformations, necessary for individual cases by preliminary experiments, as is common to the skilled person.

Candida solani (NCYC 41) and Pichia farimosa strains. (CBS 185) were deposited in the German Collection of Microorganisms under numbers DSM 3,315 and DSM 3,316 on May 21, 1985.

The following examples serve to explain the method according to the invention.

Example 1 _v

Two-liter Erlenmeyer flask containing 500 ml of sterilized medium at 120 ^° C in an autoclave consisting of 1,5% glucose monohydrate, 0,5% yeast extract (Difco), 0,5% corn extract and 0, 5% ammonium sulphate, pH 6.3, is inoculated with a Candida solani (NCYC 41) slant agar culture and shaken for 2.5 days at 30 ° C on a rotary shaker.

Inoculate a 250-liter seed tank with 250 ml of this seed culture and add 15 liters of culture medium sterilized for 30 minutes at 121 ° C and 15 Pa of the same composition as the seed culture. With the addition of Silicone SH as an anti-foaming agent, it is cultivated for 36 hours at 25 ° C and a pressure of 9 Pa under aeration (15 liters / min) and stirring (220 rpm).

Then, 0.9 liters of this inoculum culture is removed under sterile conditions and inoculated into a 201 liter main fermenter tank containing 10 liters of sterilized culture medium of the same composition as the inoculum culture. After 6 hours of growth phase under inoculation culture conditions, 4 g of (1S, 2S, 3R, 5R) -7,7-ethylenedioxy-3-benzoyloxy-3 - [(IE), (4RS) are added with further stirring and aeration. 4-methyl-3-oxo-oct-1-en-6-ynyl] bicyclo (3,3,0) octane dissolved in 100 mL of dimethylformamide.

After 90 hours of contact, the reaction is complete. The culture liquid is extracted three times with methyl isobutyl ketone, the extracts are combined and evaporated to dryness under reduced pressure. The oily residue was taken up in methanol, filtered from silicone oil and evaporated to dryness again. The residual oily crude product was dissolved in methylene chloride and further purified by silica gel column chromatography using a mixture of 5 liters of hexane, 4 liters of hexane, and 4 liters of acetone as eluent. Thus, 2.75 g of pure (1S, 2S, 3R, 5R) -7,7-ethylenedioxy-3-benzoyloxy-2 - [(IE), (38,4RS) -3-hydroxy-4-methyloct-1] are obtained. -en-6-ynylbicyclo [3.3.0] octane as a colorless oil.

Example 2

The procedure is the same as in Example 1, except that the Pichia farinosa strain (CBS 185) is used for the desired reduction of the ketone.

Example 3

Under the conditions of Example 1, a Pichia farinosa strain (CBS 185) was used to obtain the desired ketone reduction.

Example 4 ·

Under the conditions of Example 1, compound 3 was reduced with the appropriate 15α-hydroxy compound by Candida solani strain (NCYC 41).

Example 5

Under the conditions of Example 1, compound 5 is reduced with the Candida solani strain (NCYC 41) to the corresponding 15α-hydroxy compound.

Claims (1)

Process for the microbiological production of 15α-hydroxyprostacycline intermediates of the general formula I (I) wherein X represents an oxygen atom or a N-group, A trans-CH = CH- or —C = C- group, Or an oxygen atom, residues or characterized in that the ketone of the general formula II or Οχ -O COOR where R3 is C1-C4-alkyl, R 1 represents a hydrogen atom, a tetrahydropyranyl or tert-butyldiphenylsilyl group and R2 denotes residues where A, V, X, R 1 and R 2 are as defined above, acting by Candida solani, which is deposited in the German Collection of Microorganisms in Gdttingen under number DSM 3 315 or by Pichia farinosa, which is deposited in the German Collection of Microorganisms in Gottingen under DSM 3,316, wherein methanol, ethanol, glycomonomethyl ether, dimethylformamide or dimethylsulfoxide are used as substrate solvents, or the substrate is used in emulsified form and the resulting 15α-hydroxyprostacycline derivative is isolated after fermentation. Сн-CžC - Сн _а £ 3