DE2038926A1 - Microbiological reduction - Google Patents

Description

PATENT LAWYERS

DR. W. SCHALK DJPL.-ING. P. WlRTH · DIPL.-ING. G. DAN N EN BERG DR. V. SCHMIED-KOWARZIK · DR. P. WE! NHOLD · DR. D. GUDEL

6 FRANKFURT AM MAIN

Ott. ESCHENHEIMER ITRAFSE 39

Syntex Corix ration Apartado Postal 7386 Panama / Panama

ΟΛ / ΟΛ

Microbiological reduction

The present invention relates to the reduction of oxo-containing compounds through microbiological measures. It relates in particular on the selective microbiological reduction of symmetrical dioxocycloalkanes to the corresponding, optically active oxohydroxycycloalkanes.

The use of microorganisms to achieve the reduction of an oxogroup i3t not new. For example, U.S. Patent 3,432,393 is directed on the microbiological reduction of symmetrical cycloalkane-l ^ -diones.

It has now been found that symmetrical uioxocycloalkanes are good and improved Exploitation clay by means of a new and different range of Microorganisms can be selectively reduced, according to the invention the selective reduction of the Uioxocycloalkane by using the species of the genus Schizosaccharomycea to target the corresponding üxohydroxycycloalkana.

109810/2251 bad original

The cultures of the species of genus Scnizosaccharomyoes which can be used in the process according to the invention can be obtained from known sources, such as, for example, American Type Culture Collection (ATCC), Rockville, Maryland; the Northern Utilization Research and Development Branch, US Department of Agriculture (NRKL) ₁ Peoria, 111 .; and Centraalbureau voor Schimmelcultures (CBS) ₁ Baarn, Holland. The following species are typical of the materials available from these sources are representative of those that can be used in the method of the invention.

japonicus ATCC 10660 octosporus ATCC
ATCC 2479
42ü6 pombe ATCC
ATCC
ATCC
ATCC
ATCC 2476
2478
14548
16491
16979 versatilis ATCC 9987

The strains Schizosaccharomyces pombe ATCC 24-6 and are particularly important ATCC 2478.

There are a large number of suitable substrates for the process according to the invention symmetrical 1,3-dioxocycloalkanes such as those described in U.S. Patent 3 432 393, and in particular those of the following formula:

_R l0

0 '

in which
R stands for methylene or ethylene

R means methyl or ethyl?

ο ·

R for an aliphatic group with 3-18 carbon atoms or a carbo-

oyolischo group of the partial formula:

109810/2251 ^BAD

(2)

and derivatives thereof in which the aliphatic group and carbocyclic Group are optionally substituted and unsaturated. These optional In the case of the aliphatic group, substituents are, for example, oxo, carboxy, carboalkyloxy, hydroxy, acyloxy, alkyloxy, alkyl, aryl, cycloalkyl or Halogen, and in the case of the carbocyclic group, oxo, hydroxy, acyloxy, alkyloxy ^ Alkyl or halogen. The optional unsaturation is as one or several double bonds (olefinic unsaturation) present, which in the aliphatic or carbocyclic groups are present.

The method of the present invention can be represented by the following equation will:

1 °

12th
in which R, R and R each have the meaning given above.

The method consists in reducing a 1,3-dioxocycloalkane with a Species of the genus Schizosaccharomyces for obtaining the corresponding 1β-hydroxy-3-oxocycloalkane.

109810/2251

_ Ii- _

The inventive method is particularly suitable for the production of lß-Hydroxy-3-oxocyclopentanes (R = methylene) in a first group, the

contain a 2ß-methyl or -ethyl group (represented by R), and - as

2 second group - those compounds of the first group in which R the

can have the following meaning:

2-C arboalkyloxyethyl,

3-oxo-n-butyl

3-oxo-n-pentyl,

3-Οχο-ό-carboalkyloxy-n-hexyl,

3,7-dioxo-n-octanyl,

3,7-dioxo-n-nonanyl and

in which
3
Ir stands for lower alkyl, cyclopentyl, cyclohexyl or a lower carboxylic hydrocarbon acyl radical and

12 3
Z, Z and Z each represent a carbon-carbon single bond or a

2 signify carbon-carbon double bond, provided that Z and Z

1 1

Single bonds are when Z is a double bond and when Z is one

2 3?

Single bond, äst, Z and Z each stand for a single bond or Z "

3 2

and Z each represent a double bond, or Z represents a double bond and

Z ^ is a simple tie.

BATH ORIGINAL

10981n /? 251

The 13-hydroxy-3-oxocycloalkane products of the process of the invention are valuable intermediate products for the production of well-known and valuable steroid

Rotal compounds according to known / synthetic methods. Because of this sufficiency

the cycloalkane ring is determined as the Hing D of the steroid nucleus, which is why the

12th
substituents represented by K, and in particular by R, can be selected in accordance with the preparation of the valuable basic steroid compounds. The above-mentioned US patent and numerous other patents and publications show the known synthesis sequence in which the products according to the invention are suitable. M.

An important consideration in these synthesis methods is usage of an optically resolved 1-hydroxy-3-oxocycloalkane. The present 3rfindung enables the achievement of particularly expedient optical Isomers required for further elaboration in the synthesis sequence for production of a steroid nucleus with the correct stereochemistry are particularly suitable.

According to the invention, the microbiological reduction takes place by adding a 1,3-dioxocycloalkane in contact with a species of the genus Schizosaccharomyces brings. This reduction takes place at temperatures between about 20-35 C. for a time sufficient to achieve the reduction, preferably between about 2 ** ~ 120 hours. The reduction is expediently carried out in a nutrient medium, which contains a source of carbon, minerals, nitrogen and vitamins. The medium has such a set pH value or an ionic strength, that we are isotonic with the microorganism. The incubation continues under aerobic conditions.

BATH ORIGINAL

10981D / 2251

When carrying out the reduction process, the microorganism and substrate become brought into contact and in each, in raicrobial fermentation processes in the usual order or manner. The respondents are in a suitable nutrient medium within a given temperature range and held in contact for a time sufficient for the reduction to occur. When the reduction is complete, the mixture becomes more customary V / s treated for the isolation and recovery of the product, such as filtration, decanting, extraction, evaporation, chromatography, etc.

Various known and common materials are used as the carbon source that are high in carbohydrates, such as sugars, starches and low in organic hydrocarbons.

Minerals are added by adding inorganic salts. Often they are sufficient Amounts of minerals present in the other constituents constituting the fermentation medium for the process according to the invention.

As nitrogen sources, e.g. peptone, soy flour, corn flour, yeast extract, Use casein, hydrolyzate, etc.

Sources of vitamins are generally those necessary for the intake of the others Sources used of nutrients, with yeast extract being a particularly suitable source of vitamins.

The pH of the medium used is expediently adjusted to or around the neutral value, ie about 7 ». This ensures that the medium is isotonic with the microorganism used. The isotonic system can be adjusted by using salts or buffers, such as ammonium and dihydroorthophosphate, calcium carbonate, alkali metal phosphates, etc. _t .

109810/2251 _{BAD 0RlelNAL}

The inventive method is expediently carried out by first the Cultivates the microorganism in a suitable medium containing sufficient carbon, mineral, nitrogen and vitamin nutrients. The culture medium is expedient under sterile, aerobic conditions and for about 12 - ^ - 8 Hours to about 20-35 ° C. held. During this time, the medium becomes appropriate touched.

After the cultivation period, the culture is transferred, preferably under sterile conditions, for incubation in a medium of the same or different nutrient composition. The culture is preferably about 8-20 hours W in this medium further grown. After this additional time, the substrate can optionally be added and the mixture obtained can be stirred under aerobic conditions within the given temperature and time range. The product can then be isolated by column chromatography with an appropriate eluent or by other customary measures.

Another embodiment is the culture of agar plates on which they are kept was transferred into a prepared nutrient medium of the above definition. The culture is in this medium at about the same temperature and preference ™ grown wisely with stirring. After the cultivation period, the culture is used to inoculate a nutrient medium of the same or different composition used. The culture is preferably in the new medium after inoculation let grow further.

10981Π / 2251

After the culture has reached a satisfactory stage of growth, the substrate is added to the medium, preferably as a solution in an organic solvent, and the incubation is continued. Suitable Solvents include acetone, methyl ethyl ketone, methanol, ethanol, propanol, Diethyl ether, dioxane, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide etc. and mixtures thereof. The incubation can be achieved by using thin-film or paper chromatography aliquot samples can be followed. To separate and purify the product after the reaction has ended, preference is given to ^ column chromatography used wisely.

The following examples illustrate the present invention without it ■ to restrict.

Example 1

A culture of Schizosaccharomyces pombe (ATCG ? Λ7β \ NRMj Y-Io ^) was grown for 5 days on agar plates of the following composition at a temperature

held between 23-28 G.
Maltose ^ Q g

Proteose Peptone No. 3 15 E.

Agar 20 g

least. Water 1000 ecm

. The culture was removed from the agar plates by dilution with dist. water and Scrape into a nutrient broth / De :: tro £ e41edium of the following composition

transferred:

Nutrient broth, Difco 8g

Dextrose (Cerelose) 20 g

least. Water 1000cm

1 C 9 8 1 0/1 2 S 1

The mixture vmrde by 15 minute heating at 121 ⁰ C, under 1.05 'i -' / ⁰¹ * ν sterilized, then the culture was aerobically for 30 hours at 25 ° C. grown in a rotating shaker (250 rev / min 2.5 cm rash) in nutrient broth / dextrose medium. The culture was then removed and used as a vaccine for a separate nutrient broth / dextrose medium of the same composition to provide 10.28 liters of a nutrient mix with 1.028 liters or 10 # of vaccine. After inoculation, the culture was grown aerobically for an additional 16 hours. Thereafter, the medium 3iO8 gl, 3-dioxo-2- (2'-carboraethoxyäthyl) -2-methyl cyclopentane, were dissolved in 30.8 cc of acetone added and the Bebrü-tung was cultured under shaking as above at 22-26 ⁰ C continued.

The incubation was allowed to continue in this manner for from time to time Time aliquots were taken from the inoculated mixture to determine the extent and rate of reduction. In this determination the corresponding aliquot was acidified with glacial acetic acid and the acidified Mixture shaken with chloroform. The chloroform extracts were analyzed by thin layer chromatography on silica using a 80:20 (v / v) - solvent system of chloroform and acetone and the common method of detection using a $ 3 spray material Uori ——— sulfate in 3N sulfuric acid solution and heating to 110 C. separately.

After incubation for 24 hours, the entire mixture was chromatographed on a silica gel column (0.05-0.2 mm particle size), eluting with chloroform / methanol (95% SO, 5 v / v). 2i (- (2 ¹ -carbcmethoxyiithyl) -2i3-methyl-3-oxocyclopentane ale product «

109810/2251

example

* 100 ecm of a broth-dextrose medium of the composition mentioned in Example 1 were prepared and inoculated with Scliizosaccharomyces pomte (ATCC 2478; NRRL y-9). After the culture had grown for 16 to 10 hours, 200 ml of 1,3-dioxo-2- (o'-carbomethoethyl) -2-methylcyclopentane in 2 ecm of dimethylformamide were added. The incubation was continued and after 8 and 2k hours a further 200 mg of substrate in dimethylformamide were added in each case. Incubation was allowed to continue 96 hours after the last addition of Sübstrat. After this time the mixture was chromatographed in Example 1 and gave li3-hydroxy-2 (j (~ (2 '-carbomethoxyethyl) -2ß-methyl-3-oxocyclopentane as the product.

Example 3

Example 2 was carried out using Schizosaccharomyces pombe ATCC 164 ° -1 instead of ATCC 2478 and a nutrient medium of the following composition repeated:

Dextrose 10 g

Corn soft liquid 6 g

NH ^ H ^ PO ^ 3 g

Yeast extract 2.3 g

CaCl ₂ 2.5 g

least. Water to a total of 1000 ecm

The pH was washed with 2N-NADH Lb ¹ solution of 7 _t 0 + 0, set L * was thus obtained LSS-hydroxy-2 (j (- (2 ^l -carbomethoxyäthyl) ^ ß-methyl-S-oxoeyciopentan as Product.

108810/2251

-I-

example

A culture of Schizosaccharomyces versatilis (ATCC 9987; NIlRL Y-3.026) was kept at room temperature on an agar plate for 7 days and then from this transferred into a medium of the following composition:

Nutrient broth, Difco 16 g _

Cerelose * to g

least. Water to a total of 2000 ecm

The culture medium wurdenim with stirring for 30 hours at a temperature between 25-3O ⁰ C. grown _t then removed and, as lO-jäiger vaccine to a medium of the same composition. After the inoculum addition, the culture was allowed to grow for an additional 16 hours. After this time, 50 cc of dimethylformamide was added, the 5 gl _s 3-dioxo-2- (3 ^'-oxo-1 -earbomethoxy 6-n-hexyl) -methylcyclopentan contained, added and continue the incubation at room temperature. During this time, aliquots were occasionally withdrawn and chromatographed on paper using a toluene / propylene glycol solvent system and standard detection method with Zimmermann's reagent followed by heating to determine the extent of the reaction. After 50 hours, the Bebrütungsmischung was _w i _e in Example 1 and chromatographed gave _t

lß-Hydroxy - ^ - iS'-oxo ^ ö'-carbomethoxy-n-hexylJ ^ ß-methyl ^ -oxocyclopentane as a product.

Example ^

360 mg of 1,3-Dioxo-2- (3'-oxo-6 ¹ -carbomathoxy-n-hexyl) -2-methylcyclohexane in 6 ecm of dimethylformamide were mixed with Schizosaccharomyces pombe (ATCC 2476) in 1.2 liters (of 400 each ecm divided into 3 2-1 bottles) of a nutrient broth / dextrose medium of the composition mentioned in Example 1 at room temperature HQ. Hours incubated? In this way, lß-hydroxy-2o (- (3 ^l -oxo-6 ¹ -carbomethoxy-n- · hexyl) -2ß-methyl-3-oxocyclohexane was obtained as the product

109810/2251

Example 6

Example 5 was repeated using 1,3-dioxo-2- (3 ^l -oxo-6'_carbomethoxyn-hexyl) -2-ethylcyclopentane at a concentration of 300 / Ug / ccm for 72 hours} so one obtained β-hydroxy -2e (- (3'-oxo-6 ¹ -carbomethoxyn-hexyl) -2ß-ethylcyclopentane as the product.

example 2

The incubation process of Example 5 was repeated with 500 / Ug substrate per ecm of incubation mixture, 1,3-Dioxo-2- (3 'carbomethoxy-n-hexyl) -2-methylcyelopentane in dimethyl sulfoxide being added as substrate; in this way, lß-hydroxy-2o (- (3 ^l -oxo-6 ¹ -carbomethoxy-n-hexyl) -2ßraethyl-3-oxocyclopentane was obtained as the product.
Example 8_

Example 5 was repeated using a kQO -ecm medium, wherein

200 mg 1,3-dioxo-2- (3 '-oxo-6 ¹ -carbomethoxy-n-hexyl) ^ -methylcyclopentane / were added as substrate at the beginning and 4, 8 and 2k hours after the first addition. Incubation continued for 48 hours after the last addition; at a final concentration of 2 mg of substrate per cm of medium, the product obtained was β-hydroxy-2fl (- (3 ^l -oxo-6 ¹ -carbomethoxy-n-hexyl) -2β-methyl-3-oxocyclopentane.

Example 2

The incubation procedure of Example 5 was carried out in a ΙΛΐ-1 fermentation vessel for Zh hours at 25 ° C. repeated with stirring at a speed of 250 rpm, the vessel 10 l of a medium containing 5 g of 1,3-Di-0x0-2- (3'-oxo-6 ¹ -carbomethoxy-n-hexyl) -2 -methylcyclopentane as a dispersed substrate. In addition, the incubation mixture was aerated during the entire incubation period at an amount of 0.81 per liter of medium per minute; the product obtained was 13-hydroxy-20V- (3'-oxo-6 ¹ -carbomethoxy-n-hexyl) -2j3-methyl-3-oxocyclopentane.

109810/2251

Example 10

Example 5 was repeated with an incubation period of 16 hours at 30 ° C., whereby 1β-hydroxy-20 (- (3 ^l -oxo-6'-carbomethoxy-n-hexyl) -2i3-methyl-3-oxocyclohexane was obtained as the product .

According to the above process, 1,3-dioxo-2- (3'-oxo-n-butyl) -2-methylcyclopentane was obtained by incubating with Schizosaccharomyces pombe

^ (ATCC Ik ¹ ^ Q) in a nutrient medium de ^ following composition the Il3-Hydroxy-

2et- (3'-oxo-n-butyl) -2ß-methyl-3-oxocyclopentane as product:

Corn soft liquor. 6g ™

NH ₂₁₁ H ₂ PO ^ 3g

CaCO ^ 2.5 g

Soybean oil 2.2 g

Yeast extract 2.5 g

Dextrose 10 g

least. Water to a total of 1000 ecm

By incubating 1,3-dioxo-2- (3'-oxo-n-butyl) -2-ethylcyclohexane with Schizosaccharomyces pombe (ATCC 16491) in a nutrient medium of the composition given in Example 1, I3-hydroxy ~ 20 was obtained (~ (3 '-oxo-n-butyl) 2ß-ethyl-3-oxocyclopentane as product. A

Incubation of 1,3-oxo-2- (3'-oxo-6'-carboethoxy-n-hexyl) -2-ethylcyclopentane with Schizosaccharomyces pombe (ATCC 16979) in a nutrient medium of the following composition gave li-i-hydroxy-2 ^ - (3'-oxo-6'-carboethoxy-n-hexyl) ~ 23-ethyl-3-oxocyclopentane as a product

109810/2251

Glucose 3.0 g

KH ₂ PO ^ 0.1 g

Corn soft liquid ² »0 ecm

0.05 g

NaNO ₃ 0.2 g

0.002 g 0.2 g

KCl 0.005 g

least. Water to a total of 1000 ecm

By incubating l ^
Pentane with Schizosaccharomyces japonicus (ATCC 1066O) in a nutrient medium of the composition mentioned in Example 1 gave β-hydroxy-2ot- (3'-chlorobut-2'-enyl) -2Ji-n-propyl-3-oxocyclopentane as the product.

By incubating 3-methoxy-8, l ^ -secoö'stra-l, 3i5 (10), 9 (ll) -tetraen-14-, 17-dione with Schizosaccharomyces octosporus (ATCC 2479), 3-methoxy-8,14-secoestra-1,3,5 (10), 9 (II) -tetraen-17i3-ol-14-one was obtained as a product.

Incubation of 3-methoxy-8,14-secoöstra-l, 3t5i (10) lo ^ - dione 'with Schizosaccharomyces octosporus. In a nutrient medium of the following composition gave 3-methoxy-8,14 ~ secoö'stra-l, 3 _f 5 (10), 6,8-pentaen-17ί3-ο1-1Λ-οη as a product:

Corn soft liquor 6g glucose 35 g Peptone 20 g least. Water on total 1000 ecm

109310/2251

Obtained by incubation of 8,1A-Secoestra-1,3 »5 (10), 8-tetraen-3 ~ ol-14-, 17-dione with Schizosaccharomyces pombe (ATCC 2476) in a nutrient medium of the composition mentioned in Example 1 man 8, lA-Secoöstra-l, 3i5 (10) _f 8-tetraen-3ß | 17ß-diol-lA-one as the product.

By BebrUtung of 3-ethoxy-8, l4-seco-estra-t _t 3 "5 (10) -triene-la, 17-dione with Schizosaccharomyces pombe (ATGC 2 ^ 78) received in a nutrient medium mentioned in Example 1 composition 3-ethoxy-8 _t 14-secoestra-I »3i5 (10) -trien-17β-ol-14-one is the product.

Incubation of 1,3-dioxo-2- (2 ^l -carbopropoxyethyl) -2-ethylcyclohexane with Schizosaccharomyces pombe (ATCC Ih ^ d) in a nutrient medium of the composition mentioned in Example 1 gave lß-hydroxy-2e (- (2 ^l -carbopropoxyethyl) -Zß-ethylcyclohexane as the product.

The following products, among others, can be manufactured according to the invention:

lß-Hydroxy-2c (-. (3 ¹ -oxo-n-butyl) -2ßHmethyl-3-oxocyclopentane; lß-Hydroxy-2c4- (3 ¹ -oxo-n-butyl) -2ß-ethyl-3-oxocyclopentanej lß -Hydroxy ^^ - C 3 '-oxo-n-butyl) -2ß-ethyl-3-oxocyclohexane ^

lß-Hydroxy-2i (- (3 '-oxo-n-pentyl) -2ß-methyl-3-oxocyclopentane; Ä

lß-Hydroxy-2fl (- (3 '-oxo-6 -carboethoxy-n-hexyl) -2ß-methyl-3-oxocyclopentanej' lß-hydroxy-2fl (- (3 ¹ -oxo-6 ¹ -carboethoxy- n-hexyl) -2ß-ethyl-3-oxocyclopentane; lß-hydroxy-2 »(- (3 ^l -oxo-6 ¹ -carboethoxy-n-hexyl) -2ß-methyl-3-oxocyclohexane} lß-hydroxy-2 ((- (carbomethoxyethyl) -2ß-methyl-3-oxocyclopentane {lß-hydroxy-2fl {- (carboethoxyethyl) -2ß-ethyl-3-oxocyclopentane ·, ^ lß-hydroxy ^ pKcarboethoxyethylJ ^ ß-ethyl-S-oxocyclohexane; "lß-Hydroxy-2 (jt- (3 ^l _f 7 ^l -dioxo-n-octanyl) -2ß-metlTyl-3-oxocyclopentanj ''

T09810 / 2251

lß-Hydroxy - ^ (3 ' _t 7' -dioxo-n-octanyl) ^ fS-methyl ^ -oxocyclohexane; lß-Hydroxy-2el- (3 ', 7' -dioxo-n-nonanyl) -2ß-methyl-3-oxocyclopentane; H3-Hydroxy-2ct- (3 ', 7' -dioxo-n-nonanyl) -2ß-methyl-3-oxocyclohexane and iß-hydroxy-2ol- (3 ', 7' -dioxo-n-octanyl) ^ i- ethyl ^ -oxocyclopentane.

1 ο 9 8 1 η /? -y β

Claims (1)

Claims (ll ~ Process for the reduction of a l ^ -dioxocycloalkane with a species of the genus Schizosaccharorayces to form the corresponding liä-hydroxy-3-oxocycloalkane. 2. Process according to claim 1, characterized in that the 1,3-dioxocycloalkane the following formula: 0
and the product li3-hydroxy-3-oxocycloalkane the i'oxtael: has in which R represents methylene or ethylene, R is methyl or ethyl, 2 C § R is an aliphatic group with 3-1B carbon atoms or a carbocyc- ~ ic group of the formula CO is »and derivatives of the same, in which the aliphatic group and carbocyol ohe ur group are optionally eubatituiort and unsaturated. BAD ORIQlNM- 109810/2251 203B926 3.- The method according to claim 2, characterized in that H is methylene 1 2 is, H is methyl or ethyl and R 2-carboalkyloxyethyl,
3-oxo-n-butyl,
3-oxo-n-pentyl, 3-oxo-6-carboalkyloxy-n-hexyl,
3,7-dioxo-n-octanyl
3,7-Üioxo-n-nonanyl or 3
means, where H is a lower alkyl, cyclopentyl, Cydohexyl- or 12th lower carboxylic hydrocarbon acyl group and Z, Z and Z in each case a carbon-carbon bond or a carbon-carbon -Double bond, provided that when Z is a double 2 3 bond, Z and Z each stand for a single bond, and, if 1 2 3 2 Z is a single bond, Z and Z are each a single bond, or Z 3 2 and Z are each a double bond. or Z for a double 3
and Z stands for a single bond. ** .- The method according to claim 1 to 3i, characterized in that the species from the genus Schizosaccharoncyces Schizosaccharomyces poraba is used. 5.- The method according to claim 2 to h _t, characterized in that H is methyl or ethyl. 109810/2251 - 19 -. 6.- The method according to claim 1 to 5 »characterized in that a material from the group of Schizosaccharomyces pombe ATCC 24? 6 _f ATCC 24-78 or ATCC 1 ^ 5 ^ 8 is used as Schizosaccharoiqyces porabe. The patent attorney / lVTpt ^ Xsy ^^^ ~ ^ 10981 (1/2251