HU216874B - Method for microbial production of 11alfa-hydroxi-gonan compounds - Google Patents

Abstract

The present invention is a novel process for 11a-hydroxylation of 13-alkyl-4-ynyne-3,17-diene compounds under aerobic conditions by microbial pathway fermentation, in anhydrous form, as well as Aspergillus awamy (NCAIM F 0030) having sterol-11a-enigenase activity, orPenicilli. albidom (NRRL 2043, ATCC 10 408) with a spawn steamer, or at the National Collection of Field Horticultural and Industrial Microorganisms (University of Horticulture and Food Industry, Bűdapest, Natiő Cőllectiőnőf Agricűltű and Indűstrial Micrőőrganisms, Bűdapest, Hytisgary) NCAIF 001 229 Cylindrőcarpőndydimme richter is provided with microbial microorganism BB 0599, administered as a dermatologic acid and / or charcoal gap. ŕ

Description

The present invention relates to a process for the preparation of 11a-hydroxy derivatives of gonan compounds, in particular 13-alkyl-4-gonene-3,17-dione compounds, by microbiological hydroxylation.

19-Norteroids are very important steroid hormone and drug starting materials, and hydroxylated derivatives are important key intermediates in further syntheses.

As the first hydroxy steroid produced synthetically demanded very high cost and labor, the results of Horváth and Krámli (Natúré 160 639 (1947)) on the biotransformation utilization of microorganisms were of great interest. The first practical results came from the hydroxylation of steroids. An industrial scale process for the la-hydroxylation of progesterone was developed by Peterson and Murray in 1952 (U.S. Patent No. 2,602769). Later, 80% conversion was made to 11-hydroxy-progesterone (U.S. Pat. Nos. 26,86791 and 2686792). In the fifties - it confirmed that all of the carbon atoms in the steroid skeleton - - strain collections systematic screening result, with the exception of C ₄ to C ₁₃ - hydroxylation well. Hydroxylation of C ₃ and C ₁₀ is rarely possible because they are usually joined by other groups.

The hydroxylation of 19-norteroids has been successfully dealt with since the 1950s. The strain Rhizopus nigricans, which has been frequently and successfully used for α-hydroxylation of progesterone 11, has undergone bioconversion on 19norsteroid, namely 19-nortestosterone. In addition to the main 63-hydroxy derivative, the all-α-hydroxy derivative as well as the i-hydroxy derivative [J. Chem. Soc. 78, 1512-1513 (1956)].

Initial results have already provided sufficient information on the basic properties of enzymatic hydroxylation of microorganisms. In the sterane backbone, bioconversion is substrate specific, i.e., molecules with different functional groups may be converted at different positions. Region and stereospecificity are typical. Mild reaction and the possibility of introducing a functional group into a non-activated site by microbiological hydroxylation are definitely advantages over synthetic conversion.

Of course, many researchers were interested in the reaction mechanism of enzymatic steroid hydroxylation. Early experience has shown that hydroxylations at different positions are carried out by separable oxygenase enzymes. It has been found that the formation of Aspergillus ochraceus 63-hydroxylase is induced by 1α-hydroxy progesterone (U.S. Patent Nos. 2,802,775 and 2,905,593); by removing the resulting Ια-hydroxy product from the reaction mixture, it was achieved that the formation of the undesired 6β, 1 1α-dihydroxy derivative was reduced.

Further information on the mechanism of reaction was provided by the recognition that Reichstein-S is also hydroxylated at the 11a and 11b positions of the filamentous fungus Absidia orchidis [Folia Microbiol. 6, 237 (1961); Appl Microbiol. 11,256 (1963)]. The ratio of the two 11hydroxy products could not be changed, although thousands of mutants have been screened. It was concluded that oxygen is supplied to the C _H atom by a single enzyme. The substrate may be bound to the active site at various positions and will determine the orientation of the hydroxyl group formed. The enzyme system that performs hydroxylation works in a membrane-bound, microsomal, intracellular, protease-protected vesicle.

There are relatively few commercially available steroid hydroxylation procedures for hydroxylation of 19-noreroids, especially when a longer alkyl group is attached to the C ₁₃ atom. Conversion of estradiol to Fusarium and Absidia strains yielded a 15α-hydroxy derivative (US Patent 3,332,888). The Ια-hydroxylation of nortestosterone 1 has been described by Sehgal et al., Aspergillus ochraceus strain [Can. J. Microbiol. 14: 529-532 (1968)]. Only many years later, the economical 1α-hydroxylation of 19-nortestosterone with the Rhizopus nigricans strain associated with Petzoldt (US Patent No. 3248434) was successfully solved.

Etβ-hydroxylation of norethisterone has been solved at several research sites with a variety of microorganisms, such as Botryodiplodia malorum (U.S. Pat. No. 3,880,895, U.S. Patent 2,546,062), Acremonium strictum and A. kiliense [Ambrus et al., Steroids 99 (1975)], microorganisms of Calonectria decora (U.S. Patent No. 2,450,106), Mucor spinosus (U.S. Patent No. 2,539,261). Zakelj-Mavric et al. Converted the 19-norethisterone from the 1? -Hydroxylase enzyme complex from Rhizopus nigricans, which has a very good la-hydroxylation property, and surprisingly gave 10β- and 6β-hydroxy derivatives. The same enzyme extract converts etisterone to 7β-1ή (ΐΓοχί-6ύ8ΖΙεΓοη.). The deviation from expected is explained by the fact that the ethinyl group interferes with normal seniosity (FEMS Microbiol. Lett. 33, 117-120 (1986)). -hydroxylation with Penicillium raistrickii (German Patent No. 2546062).

It was only from the 1970s that researchers began to cope with the hydroxylation of 19-nor-13-ethyl steroids. The 13β-εelί11,3,5 (10) -βοηΗϋ · ίέη-3,17β-όίο1 substrate was capable of 16a-hydroxylation with the bacterial strain Bacillus megaterium (U.S. Pat. No. 3,733,254). The strain Botryodiplodia malorum, which also hydroxylates norethisterone at 11 β-positions, is unable to hydroxylate norgestrel (13β-είϊ1-17α-εύηί1-17β-1ιί <ΐΓθχί-4gonen-3-on) on C _n atom, due to the presence of German Patent Specification). Many strains have been found capable of ßα-hydroxylation of various gonan derivatives: Rhizoctonia, Sclerotium, Calonectria, Glomerella (U.S. Patent No. 2450106). The Ια-hydroxylation of norgestrel has been solved by several research groups: Calonectria decora strain (U.S. Patent No. 2,450,106), Aspergillus clavatus strain (U.S. Patent No. 2539261). The 6β-hydroxylation of norgestrel is in Mucor griseocyanus

HU 216 874 Β (German Patent No. 2330159). 9-Hydroxylation of gonan derivatives with Glomeretta fungal strain (U.S. Patent No. 4,397,947) was also performed.

German researchers also dealt with the hydroxylation of 11-phenylgonane derivatives: they were able to create a 6ct-hydroxy derivative with Streptomyces and Nigrospora strains, convert a 7a-hydroxy product with a Streptomyces strain, and a 15P-hydroxy product with a Neurospora strain (No. 3,527,517). patent).

The 15α-hydroxy derivative of levodione (13β-ethyl-4-gonene-3,17-dione) could be produced by two strains: Penicillium raistrickii and Fusarium sp. strains were suitable for the preparation of an important intermediate of gestestene, 15ahydroxy-133-ethyl-4-gonene-3,17-dione (German Patent No. 2,546,068).

Of the many examples of hydroxylation of gonan derivatives, only one description has been found which relates to the hydroxylation of the C1 atom. Petzoldt et al. (U.S. Patent No. 3,248,434) performed two bioconversion reactions in space, but not in time, on 3oxo-4-estrene derivatives and their 18-alkyl homologues. The main disadvantage of their process was that they were able to use low substrate concentration (1 g / l), which was subjected to aerosolization of ring A by addition of 11 α-hydroxylated strains of Aspergillus ochraceus, followed by addition of Arthrobacter simplex or Bacillus sphaericus under aerobic conditions. a total yield of 42% was achieved. Due to the low substrate concentration mentioned above, the process is not suitable for industrial production.

It is an object of the present invention to provide a novel fermentation process which enables safe and economical microbial 11-hydroxylation of 13-alkylgonan derivatives. To this end, microorganisms have been used which safely hydroxylate the 13-alkylgonane derivatives on the Cn carbon atom at appropriate concentrations and with technological parameters suitable for scaling.

Screening for microorganisms with the potential steroid 11-oxygenase enzyme found strains of microorganisms that show good or poor hydroxylation of corticoids and pregnane skeletal steroids; and found strains capable of hydroxylating 4-estrene-3,17-dione and 13-ethyl-4-gonene-3,17-dione at the α-position of the Cn carbon, although such enzyme activity has not been known so far.

In the course of our further research work, we developed a seedling line and medium composition that provides optimal bioconversion conditions and favorable yield parameters. Selection of appropriate media components resulted in well-settled pellet growth (d = 1 -4 mm) of suitable microorganisms, with the higher amount of converted substrate bound to the mycelium and the more water-soluble hydroxy product in the fermentation broth. The original 1.5-fold solubility ratio is modified to 7.5-fold in culture by the end of the bioconversion.

It is often difficult to introduce a substrate with very low water solubility into the fermentation broth. The addition in the commonly used dimethylformamide solution was favorable but did not achieve the desired effect. Therefore, a further solubility enhancer was sought and a sorbimacragole oleate (Tween-80) emulsifier, a polyethylene-polypropylene diblock copolymer based (Struktol J-633) foam and β-cyclodextrin were found to be suitable. Surprisingly, it has been found that adding ascorbic acid to the culture results in a significant increase in bioconversion efficiency. The latter finding is all the more surprising since ascorbate was previously mentioned as an electron donor only in mammalian monoxygenases [Angew. Chem. 84 (15) 689-724 (1972)].

In our experiments, it has also been observed that microbiological hydroxylation can be prolonged, provided that the culture is adequately supplied with a suitable carbon source.

SUMMARY OF THE INVENTION The present invention relates to a process for the la-hydroxylation of 13-alkyl4-gonene-3,17-dione compounds by aerobic microbiological fermentation, which comprises fermentation with Aspergillus awamori (NCAIM F 0030, 1966), or Penicillium albidum (NRRL 2043, ATCC 10408, 1972) or the National Collection of Agricultural and Industrial Microorganisms (University of Horticulture and Food Industry, Budapest; National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary) NCAIM F 001229 deposited under the sign Cylindrocarpon majorimum var. richter SZB 0599, optionally with ascorbic acid and / or carbon source.

For example, the microorganisms Penicillium albidum (NRRL 2043, ATCC 10408) and Aspergillus awamori (NCAIM F 0030) not previously introduced for the hydroxylation of gonan derivatives and strains specifically selected for these properties (3612/94) may be advantageously used. Cylindrocarpon majority var. richter SZB 0599 [filed with the National Collection of Agricultural and Industrial Microorganisms (University of Horticulture and Food Industry, Budapest) under the accession number NCAIM F 001229 on 25 July 1995] a new filamentous fungal strain.

The strain was isolated from a soil sample and was found to form flat, woolly white colonies on potato dextrose agar. Conidium supports are simple, conidia can be of two types: unicellular, ovoid, 1-3 cell long ellipsoid, or cylindrical.

The growth of the microorganisms and the hydroxylation were performed in shake culture (240 rpm, 2 cm flat plate shaker) and in fermentors. The composition of the culture media varies depending on the microorganisms involved in the transformation: soybean meal, yeast extract, malt extract, corn jam are the preferred sources of nitrogen

EN 216 874 Β and various protein hydrolysates; and carbon sources of mono-, oligo- and polysaccharides, such as glucose, sucrose and starch, and vegetable oils and organic acids. Fungi that carry out 1α-hydroxylation are preferably grown at 24-32 ° C.

The pre-culture of the bioconversion microorganism is carried out in the conversion fermenter by adding the substrate in the optimal state of the culture. The optimum state is the end of the decrease in pH indicating the utilization of the carbon source, which occurs at 10-24 hours depending on the microorganism and the medium. Simultaneously with the addition of the substrate, the dissolution and bioconversion enhancers are added to the fermentation broth at a concentration of 0-2 g / l, preferably 1 g / l.

In a preferred embodiment of the invention, the carbon source is added to the culture after the substrate is added. The carbon source can be added either batchwise or continuously. Thus, the hydroxylating activity of the culture can be sustained over an extended period of time, which may provide an opportunity to convert additional substrate amounts.

To monitor the fermentation, the steroid compounds may be isolated from the fermentation broth by extraction with chlorinated hydrocarbons, esters, water-immiscible ketones or alcohols. It is advisable to homogenize the culture prior to extraction to obtain the fullest possible amount of steroids in the fermentation broth.

Quantitation of the individual components after thin layer chromatography is performed by densitometry on pre-fabricated Kieselgel 60 F ₂₅₄ (Merck, N ° 5554). As a developmental system, we have developed a system for the good separation of the compounds in question, consisting of cyclohexane-ethyl acetate-ethanol (35: 56: 8). The measurement was performed on a Shimadzu CS-9000 densitometer at 242 nm.

The compounds of the present invention were purified by preparative liquid chromatography and confirmed by Ή-NMR and ¹³ C-NMR.

The following examples are intended to illustrate the practice of the present invention without limiting it thereto.

Example 1

From a 1-4 week culture of Aspergillus awatnori (NCAIM F 0030) malt slant on agar agar, 30 ml of BK-2 medium sterilized in 100 ml Erlenmeyer flask was inoculated with 3 ml of cell suspension in 10 ml of sterile water as follows:

peptone 30.0 g glucose 10.0 g

1000 ml tap water.

The pH of the medium was adjusted to 7.3 before sterilization. The medium was sterilized at 121 ° C for 20 minutes.

The culture was grown for 2 days on a flat shaker at 28 ° C and 100 ml of BK-A5 medium sterilized in a 500 ml Erlenmeyer flask with 5 ml of culture. The composition of BK-A5 medium is as follows:

glucose 10.0 g diammonium hydrogen phosphate 2.0 g urea 1.0 g potassium chloride 0.2 g

1000 ml tap water.

The medium is adjusted to pH 6.0-6.3 and sterilized at 121 ° C for 20 minutes.

The resulting culture was shaken at 28 ° C for 16 hours on a shaker and 0.1 g of ascorbic acid and 0.15 g of 4-estrene-3,17-dione dissolved in 2 ml of dimethylformamide were added. After another two days of shaking, the culture contained 7.5 mg (5%) of 1α-hydroxy-4-estrene-3, 7-dione by TLC.

Ultraviolet spectrum (methanol): A _max = 245 nm.

Retention time (preparative liquid chromatography, ISCO prep Si 10 µm, 60A, Davisil, 250 x 21.2 mm column; n-hexane-THF 62:38, 9 mL / min eluent): 24.5 min.

Mass Spectrum: 288 1 H NMR δ (ppm): 0.95 (3H, s, 18-Me), 3.92 (1H, m, H-11), 5.87 (1H, t, H-4). ^{, 3} C NMR δ (ppm): 27.4 (Cl), 35.9 (C-2), 200.5 (C-3), 124.8 (C-4), 166.5 (C-). 5), 36.0 (C-6), 30.7 (C-7), 39.3 (C-8), 54.8 (C-9), 43.9 (C-10), 71, Δ (C-11), 42.6 (C-12), 48.2 (C-13), 49.2 (C-14), 21.7 (C-15), 35.7 (C-16) ), 218.6 (C-17), 14.6 (C-18)

Example 2

100 ml BK-2 medium sterilized in a 500 ml Erlenmeyer flask was inoculated from a 1-4 week culture of Penicillium albidum (ATCC 10408) malt slant on agar agar with 10 ml of sterile water. The composition of the medium and the method of preparation are as follows

Example 1 contains. The culture was grown for 2 days on a flat-bed shaker at 26 ° C and then 1 liter of BK-7 medium sterilized in a laboratory glass fermenter was inoculated with 50 ml of the culture:

glucose 1.0 g peptone 5.0 g Dipotassium hydrogen phosphate 1.0 g NaCI 0.5 g magnesium sulfate water (1: 7) 0.5 g iron (II) sulfate water (1: 7) 0.01 g

1000 ml of distilled water.

The medium was adjusted to pH 6.6 before sterilization. The medium was sterilized at 121 ° C for 20 minutes.

The inoculated fermentor was thermostated at 26 ° C and incubated with a flatblade mixer at 600 rpm and a flow rate of 15 liters per hour. After 24 hours pre-culture, 1.5 g of 4-estrene-3,17-dione and 1.0 g of ascorbic acid in 20 ml of dimethylformamide containing 0.2 g of Tween-80 are added.

After 3 days, the culture contained 0.17 g (11.3%) of 11-hydroxy-4-estrene-3,17-dione by TLC.

HU 216 874 Β

Example 3

Cylindrocarpon magnitude var. Richter SZB 0599 (NCA1M F 001229) from a 1-4 week culture of malted slant agar was inoculated with 2 x 100 ml BK-2 medium sterilized in 10-10 ml sterile water in a 500 ml Erlenmeyer flask. The composition and preparation of BK-2 medium is described in Example 1. The culture was grown for 2 days on a flat shaker at 26 ° C and 5 x 5 mL of culture was inoculated into 3 x 100 mL malt medium sterilized in a 500 mL Erlenmeyer flask. The composition of the malt medium is as follows:

malt extract 60 g

1000 ml tap water.

The medium was adjusted to pH 6.4 before sterilization. The medium was sterilized at 121 ° C for 20 minutes.

The resulting culture was shaken on a flat shaker at 26 ° C for 2 days and then inoculated with 250 ml of culture medium in 5 liters of AF-15 sterilized in a laboratory glass fermenter. The composition of AF-15 medium is as follows:

sucrose (sterilized separately) 75 g yeast extract 100 g succinic acid 5 g

Structol J-633 in 0.5 g liter of tap water.

The pH of the medium was adjusted to 6.4 before sterilization. Sterilization was carried out at 121 ° C for 60 minutes.

The inoculated fermentor was thermostated at 26 ° C and incubated with a flatblade mixer at 300 rpm and air flow at 60 L / h. After 18 hours of pre-culture, 22.5 g of 133-ethyl-4-gonene-3,17 dione in 0.5 ml of Tween-80 detergent and 5 ml of Structol J-633 foam inhibitor are added and 100 ml of 50% sucrose solution.

After two days, the culture contained 9.65 g (42.8%) of 11a-hydroxy-13-ethyl-4-gonene-3,17-dione by thin layer chromatography.

Ultraviolet spectrum (methanol): A _max = 242 nm.

Retention time (preparative liquid chromatography, ISCO prep Si 10 μπι, 60A, Davisil, 250 x 21.2 mm column; n-hexane-THF 62: 38.9 mL / min eluent): 20.7 min.

Mass Spec: 302

1 H-NMR δ (ppm): 0.87 (3H, t, 19-Me), 3.83 (1H, m, H-11), 5.84 (1H, t, H-4)

DCMR δ (ppm): 27.3 (Cl), 35.9 (C-2), 200.7 (C-3), 124.6 (C-4), 167.0 (C-5) , 36.0 (C-6), 30.9 (C-7), 39.1 (C-8), 54.7 (C-9), 44.0 (C-10), 71.2 ( Cl 1), 38.3 (C-12), 51.3 (C-13), 50.0 (C-14), 21.1 (C-15), 35.8 (C-16), 217 , 8 (C-17), 18.7 (C-18), 7.6 (C-19).

Example 4

As in Example 3, Cylindrocarpon majority var. 300 ml of a second inoculum prepared using richter SZB 0599 (NCAIM F 001 229) was inoculated with 6 liters of BK-A8 medium in a laboratory fermenter, the composition of which is as follows:

sucrose (sterilized separately) 60 g yeast extract 120 g

Structol J-633 in 0.3 g liter of tap water.

The pH of the medium was adjusted to 6.4 before sterilization.

Sterilization was carried out at 121 ° C for 60 minutes.

The pre-cultivation provides agitation of 300 rpm and air flow of 60 liters / hour. 80 g of β-cyclodextrin in 200 ml of water and 6 g of ascorbic acid were added at 17 hours to the pre-culture, followed immediately by 27 g of N-ethyl-4-gonene-3,7-dione in 120 ml of dimethylformamide. Aeration and agitation are controlled by a microprocessor controller so that the dissolved oxygen content is not less than 30% of the saturation value of the uninoculated medium.

The two-day culture contained 12.1 g (44.8%) of 11α-ύίύΓθχί-13β-ethyl-4-gonene-3,7-dione by TLC.

Example 5

Cylindrocarpon magnitude var. richter SZB 0599 (NCAIM F 001229) In a Blake dish, wash 500 ml BK-2 medium sterilized in a 3000 ml Erlenmeyer flask with 50 ml sterile distilled water from a 1-4 week culture of malt agar. The cultures were incubated at 26 ° C on a flat shaker for 60 hours and then 50 ml of the culture was inoculated with 5 liters of laboratory medium sterilized malt medium. Cultivation is performed at 26 ° C with a flat-blade mixer at 300 rpm and air flow at 60 L / h. After 32 hours of culture, 4 liters of inoculum is inoculated into the main phase AF-15 medium with 80 liters of tap water, the composition and preparation of which is given in Example 3.

The culture was performed at 26 ° C with stirring at 150 rpm and air bubbling at 500 L / h. At 17 hours of the culture, 360 g of 13? -Ethyl-4-gonene-3,17-dione in 80 ml of ascorbic acid and 1600 ml of dimethylformamide containing 8 g of Tween-80 and 80 g of Structol J-633 are added.

Fermentation was continued for a further 2 days when TLC showed 184 g (51.5%) of 11α-1ιί0Γθχί-13β-εΰ1-4 ^ οη ^η3,17-dione.

Claims (1)

PATIENT PERSONALITY 1. A process for the hydroxylation of 13-alkyl-4-gonene-3,17-dione compounds by microbial fermentation under aerobic conditions, characterized in that the fermentation is carried out with Aspergillus awamori [NCAIM F 0030, steroid-1? (1966)], or Penicillium albidum [NRRL 2043, ATCC 10408, (1972)] with filamentous fungi or the National Collection of Agricultural and Industrial Microorganisms (University of Horticulture and Food Industry, Budapest; National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary) Cylindrocarpon size var. Deposited under NCAIM F 001229 identification mark. richter is administered with the microorganism <RTI ID = 0.0> BB </RTI> 0599, optionally with ascorbic acid and / or carbon source.