NO863077L - PROCEDURE FOR MICROBIAL HYDROXYLATION OF PRECOLINE AND ITS DERIVATIVES BY NEUROSPORA CRASSA. - Google Patents

Description

The invention relates to forskolin analogues and derivatives, a method for their preparation by means of micro-

biological transformations by means of Neurospora crassa, and the use of the substances as a pharmaceutical agent, especially as drugs for the treatment of glaucoma, heart failure, hypertension, inflammations, allergies, bronchial diseases and diseases of the immune systems.

Forskolin and its naturally occurring analogues are one group

of labdan terpenoids, which are isolated from the plant Coleus for-skolii (Briqu.). The structural formulas of forskolin and its naturally occurring isolated analogues are shown in formula I

For forskolin, R2 = OH, R' = Ac, as well as R3 to R7 and R" = H.

To date, the following naturally occurring for-choline analogs have been identified:

1,9-dideoxy- R" = Ac, as well as R.^ to R7 and R" = H,

forskolin:

9-deoxy- R1= OH, R<1>= Ac, as well as R2 to R7 and R" = H, forskolin:

1,9-dideoxy-

7-deacetyl-

forskolin: R<1>, R" and R^ to R^= H,

1-deoxy-

forskolin: R2 = OH, R' = Ac, as well as R" and Rx, R^ to R^ = H,

7-deacetyl-

forskolin: R^, R2= OH, R<1>,R", as well as R3 to R7= H.

6- acetyl-7- R, = OH, R" = Ac, as well as R' and R2 to R^ = H, deacetyl-

forskolin:

(Tetrahedron Lett. 19, 1669 (1977), J. Med. Chem. 26, 486 (1983).

Forskolin is one of the important diterpenes of the plant Coleus forskoli. This substance and its derivatives can be used as valuable drugs in the treatment of various diseases such as glaucoma, hypertension, heart failure, inflammation, allergies, bronchoconstriction and, in general, diseases that are produced by disturbances in cAMP production and metabolism (Indian Patent No. 143 875 and corresponding DE-OS 25 57 784, Indian Patent 145 926, Indian Patent No. 147 630 and corresponding DE-OS 26 40 275, Med. Res. Revs. 3, 201-19 (1983)).

A method for isolating the main part of forskolin from Coleus forskohlii has already been described (see Indian Patent Nos. 143,975 and 145,926, DE-OS 25 57 784). By this method, forskolin analogues are also recovered. These analogues can be used as intermediates for the production of forskolin. Methods have already been discussed through which analogues with the same oxidation step as forskolin, e.g. 7-deacetyl-forskolin and 6-acetyl-7-deacetylforskolin can be converted to forskolin (Indian Patent 147,007, J. Chem. Perk. Trans. 1, 767

(1982)).

A method of microbial conversion has already been described, through which the less hydroxylated forskolin analogues are converted into forskolin, 7-deacetyl-forskolin or 6-acetyl-7-deacetylforskolin, or into such derivatives which, by known chemical methods, can easily be converted into forskolin (German search-nad P 35 02 685.5) .

The object of the invention is a microbial method for the hydroxylation of Labdan terpenoids of formula I

in which

Rlog R2 is hydrogen or hydroxy, to Rg means hydrogen, R' means the acetyl group or hydrogen and R" means hydrogen, using Neurospora strains, especially using strains Neurospora crassa ATCC 9279, ATCC 9344, ATCC 10336, ATCC 10870. The method is characterized in that a compound of formula I, in which R^-R^, R<1> and R" have the above meaning, is fermented in a normal nutrient medium of defined composition containing one of the aforementioned microorganism strains. The fermentation takes place for 3-5 days at a temperature of 25-30°C, preferably at 28°C with shaking.

The nutrient medium contains carbon and nitrogen sources, possibly also inorganic nutrient salts and trace elements';

Suitable carbon sources are e.g. glucose, starch, dextrin and glycerol. Suitable nitrogen sources are e.g. soya flour, yeast extract, meat extract, malt extract, maize bran water, peptone and casein. As inorganic nutrient salts, there are e.g. in terms of sodium chloride, magnesium sulphate or calcium carbonate, as trace elements, e.g. iron, magnesium, copper, zinc and cobalt in the form of suitable salts.

Until the end of the reaction, the fungal mycelium is filtered off and the resulting culture filtrate is extracted with an organic solvent such as dichloromethane, chloroform or ethyl acetate. The oak extract is suitably dried over desiccants such as Na2SO^

and concentrated in vacuo. The residue is then purified by separation methods such as chromatography and by crystallization

sering.

The hydroxylation preferably takes place in the 1-, 2- and/or 3-position.

General procedure description:

a) Tribal management and cultivation.

Spore suspensions of the above-mentioned strains are inoculated onto small inclined tubes

with the following nutrient medium and grown for 14 days at 30°C:

Stock culture medium:

For cultivation in liquid cultures, spore suspensions are extracted from the stems by flooding the small inclined tubes with sterile NaCl 0.8%, and this is diluted to a titer of 1 x 10^ spores/ml. 0.5 ml of this suspension serves as an inoculum of 50

ml main culture (in 100 ml Erlenmeyer flask) of the following composition:

Main culture nutrition solution:

After 30-40 hours at 29°C and a shaking frequency of 190 rpm, a dense mycelial growth is achieved.

b) B io t r an s f o rrnation.

For biotransformation, 50 ml of the main culture is transferred into a

sterile 300 ml Erlenmeyer flask and add 16 mg of substrate (dissolved in methanol). The suspensions are then shaken at 28°C and 240 rpm for 3-5 days. The progress of the biotransformation is followed by means of thin-layer chromatography (HPTCL plates, methylene chloride/ethyl acetate = 3/1, anisaldehyde-sulfuric acid as detection reagent). After the end of the reaction, the fungal mycelium is filtered off, the resulting culture filtrate is extracted with the same amount of dichloromethane and evaporated.

c) Transformation products (structures see table 1)

I) 1,9-dideoxyforskolin (1) 1 α-HO-DDF (5)

(DDF) ( = 9-deoxyforskolin)

2 a-HO-DDF (8)

3a-HO-DDF (9)

II) 7-dAc-1,9-didesoxforskolin (11)

7-dAc-9-deoxyforskolin (12)

III) 1-deoxyforskolin (3)->forskolin (2)

IV) forskolin (2) 3a-HO-forskolin (10)

6Ac-7dAc-3a-HO-forskolin (13) .

Turnover examples with N. crassa ATCC 10336

Example 1

Biotransformation of 1,9-dideoxyforskolin (DDF). Implementation according to 1 b) 120 hours

DC analysis of the reaction product:

Example 2

Biotransformation of 1-deoxyforskolin

Implementation according to 1 b) 72 hours,

DC analysis of the reaction product:

Example 3

Biotransformation of forskolin,

execution according to 1 b) 72 hours,

DC analysis of the reaction product:

Example 4

A biotransformation of DDF carried out according to example 1

gave after extraction with ethyl acetate 97.8 mg dry matter.

This quantity was dissolved in dichloromethane, impregnated on 1 g of silica gel 60 (Grace, 31 µm, pH 7.0), and then filled into a pre-column. Above the pre-column, the sample was then chromatographed on the main column (102 ml silica gel, see above) at a flow rate of 2 ml/min with dichloromethane/acetone = ..15/1. After approx. 22 hours, the polarity of the mobile phase was increased to 10/1. The individual fractions (a 14 ml) were analyzed for substance content per DC (CH 2 Cl 2 /acetone = 7/1, detection with anisaldehyde/H 2 SO 4 /acetic acid as discussed above).

Next to unreacted DDF, 2.1 mg of ND1 (Rf 2 0.64) was obtained. 4.2 mg of ND2 (Rf = 0.46) and 7.9 mg of a mixture of ND2 and ND3

(Rf = 0.36). This mixture was resolved by preparative thin layer chromatography to 3.5 mg of ND2 and 4.0 mg of ND3.

Example 5

A biotransformation of forskolin carried out according to example 3 gave, after extraction with ethyl acetate, 57.1 mg of dry matter.

This amount was dissolved in 1.7 ml of CH2Cl2 and chromatographed over a silica gel column (Merck, KG 60, 15-40 ym, 102.5 ml column volume) with the mobile phase CH2Cl2/acetone = 12/1 at 2.5 ml/ my. After 8 hours, the mobile phase was applied with a ratio of 9/1.

According to DC"^, forskolin, 7-desacetyl-forskolin and the new substances NF2, NF3 and NF4 are essentially not converted.

A further purification of the intermediate fractions led to 0.9 mg of NF3 and 0.7 mg of NF4.

1) Silica gel plates Merck, F254 10 x 20 cm

mobile phase: petroleum ether/ethyl acetate = 2/1

Detection: see example 4.

3g- hydroxy-forskolin (NF 3)

N-NMR (270 MH 2 , CDCl 3 , TMS) δ = 5.96 (dd, J = 10 Hz, J<»>=18 Hz 14-H), 5.57 (d, J = 4 Hz, 7 a-H) , 5.30 (d, J = 18 Hz +

homoallyl coupling, 15 t-H), 4.99 (d, J = 10 Hz + homoallyl coupling, 15 c-H), 4.65 (consumed t. J = 4 Hz, 1 a-H), 4.41 (consumed, t, J = 5 Hz, 6 a-H), 3.52 (consumed, t. J = 4Hz, 3 B-H), 2.85 (AB with 6A = 3.20 and 6= 2.50, JAB= 17 Hz, 12-H), 2.63 (d, J=4 Hz, 5 a-H)

2.14 (AB with 6A2 2.34 and 6B 2 1.94, JAB= 16 Hz, added J'= 3 Hz to t, 2-H) , 2.20 (s, OCOCH3) ,1.88-0 .80 (remaining signals therein, 5g, each 3H at 6 = 1.73, 1.43, 1.37, 1.28 and 1.12, CH3).

MS m/z = 427 (18, M<+>+H), 426 (4, M<+>), 408 (78, M<+>H2O)

391 (46), 380 (16), 331 (28), 125 (100), 109 (90%) .

3a- hydroxy- T, 9- dideoxy- forskolin (ND2)

1 un

H-NMR (270 MHz, CDCl 3 , TMS) δ = 5.95 (dd, 10 Hz, J'= 18 Hz,

14-H), 5.27 (d, J = 18 Hz + homoallyl coupling, 15 t-H), 5.14 (d, J= 4Hz + 7 a-H), 5.06 (d, J = 10 Hz + homoallyl coupling, 15 c-H), 4.21 (spent s, 6 a-H), 3.38 (spent.s, 3 B-H), 2.82 (s, 9 a-H), 2.60 (AB with 6A= 2.65 and 6B = 2.55, JAfi= 16 Hz, 12-H), 2.30 - 2.00 (m, 5 H therein 2.21, s, OCOCH3), 1.78 - 0.75

(resid. H, therein 4s, 1.52, 1.43, 1.22, 1.03 for CH3)

MS m/z= 379 (M<+->CH3), 361 (M<+->CH3H2O), 334 (M<+->AcOH),

319 (M<+->CH 3 -AcOH), 251, 222, 167, 151, 95, 71%.

Fig. 1 and 2 show the H-NMR signals of the train connections ND2 and NF3 in the range 5 = 1.80 - 6.50.

Claims (7)

1. Process for microbial hydroxylation or oxidation of labdan terpenoids of formula I wherein and R2 means hydrogen or hydroxy, R^ to Rg means hydrogen, R^ means hydrogen or hydroxy, R' means the acetyl group or hydrogen, and R" means hydrogen, characterized in that on one of the aforementioned compounds of formula I as substrate in a normal nutrient medium, a strain of microorganism Neurospora crassa is allowed to act and the compounds formed are isolated after completion of the fermentation from the culture liquid by extraction with an organic solvent, and subsequent chromatography, and/or crystallization. 2. Method according to claim 1, characterized in that the strain Neurospora crassa ATCC 9279, ATCC 9344, ATCC 10336 or 10780 is used. 3. Method according to claim 2, characterized in that the fermentation is carried out for 3-5 days at 25-30°C with shaking and the culture filtrate is extracted with dichloromethane. 4. Method according to claim 3, characterized in that it is fermented at 28°C. 5. Method according to claims 1-4, characterized in that the hydroxylation takes place in the 1-, 2- and/or 3-position. 6. 3a-Hydroxyforskolin. 7. Medicine, containing 3a-hydroxyforskolin.