1
012
Vol. 53, No. 8
Incubation of 17b-methoxymestranol (4) with Cunning- dispersion in oil), followed by the addition of methyl iodide 0.1 ml
(
1.61 mmol). The mixture was protected from the atmosphere with a drying-
hamella elegans for 12 d has not afforded any transformed
product (Table 1).
tube, and was stirred at room temperature for 2 h. The solvent was evapo-
rated, the residues were distributed between 20 ml of water and 100 ml of
CH Cl , and the CH Cl layer was washed twice with water, dried over
2
2
2
2
Experimental
Na SO and evaporated to obtain compound 1 (413 mg, 82.6%). The single-
2 4
16)
General Experimental Procedure The IR spectra were recorded in crystal X-ray data and synthesis of compound 4 was earlier reported by us.
CHCl3 on an FTIR-8900 spectrophotometer. Melting points were deter-
mined on a Buchi 535 melting point apparatus. Optical rotations were mea-
sured on a Jasco DIP-360 digital polarimeter. UV Spectra were recorded in
Organism and Media Microbial cultures were obtained from the
Northern Regional Research Laboratories (NRRL). Cultures were main-
tained on SDA and stored at 4 °C prior to use. The medium for Cunning-
1
13
CHCl on a Hitachi U-3200 spectrophotometer. The H- and C-NMR spec- hamella elegans (NRRL 1392, kingdom: fungi, Phylum: Zygomycota, Class:
3
tra were recorded in CDCl solutions on Bruker Avance-400 NMR at 400
Zygomycetes, Family: Cunninghamellaceae: Genus: Cunninghamella) was
3
and 100 MHz, respectively. The EI and HR-EI-MS were measured on a Jeol
JMS-600H mass spectrometer. TLC was performed with precoated plates
prepared by mixing the following ingredients into distilled H O (3.0 : 1):
2
glucose (30.0 g), peptone (15.0 g), yeast extract (15.0 g), KH PO (15.0 g),
2
4
(Silica gel 60, PF254, 0.2 mm, E. Merck). Compounds 1 and 4 were synthe- and NaCI (15.0 g).
sized from compound 5, while compound 5 was isolated from Primolut N, a
patent product of Schering AG, Berlin, Germany.
Preparation of Compounds 1 and 4 To 500 mg (1.61 mmol) of com-
pound 5 in 30 ml THF (dried), was added 64.4 mg (1.61 mmol) NaH (60%
General Fermentation and Extraction Protocol The fermentation
media was distributed among 30 flasks of 250 ml capacity (100 ml in each),
which were autoclaved. The fermentation was carried out according to a
standard two-stage protocol. Substrates were dissolved in DMSO and were
evenly distributed into 30 flasks (20 mg/0.5 ml in each flask) containing
17)
13
1
Table 2.
C-NMR Spectral Data of Compounds 1—3, and H-NMR Data 24 h-old stage II cultures. Fermentation was continued for additional 12 d on
of New Metabolite 3 in CDCl3
a rotatory shaker (200 rpm) at 29 °C. During the fermentation period,
aliquots from flasks were drawn daily and analyzed by TLC in order to de-
termine the degree of transformation of the substrate. In all experiments, one
control flask without biomass (for checking substrate stability) and one flask
without substrate (for checking the endogenous metabolite) were used. The
culture media and mycelium were separated by filtration. The mycelium
were washed with CH Cl (1 l) and the filtrate was extracted with CH Cl
1
dC
2
dC
3
Position
dC
dH (J in Hz)
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
0
1
126.1, d
114.2,
126.5, d
114.4,
126.3, d
7.18, d (8.5)
6.74, dd (8.5, 2.8)
—
6.89, d (2.8)
—
4.78, t (2.1)
2.13, m; 1.97, m
1.78, m
2
2
2
2
a)
a)
a)
d
d
d
d
114.4,
d
d
(3ꢃ1.5 l). The combined organic extract was washed with brine and dried
158.2, s
158.0, s
158.3, s
over anhydrous Na SO , evaporated under reduced pressure, and analyzed by
2
4
a)
a)
a)
114.5,
114.2,
114.2,
thin layer chromatography. Control flasks were also harvested and checked
by TLC to detect the bio-transformed products.
137.8, s
29.0, t
27.2, t
39.2, d
42.8, d
131.3, s
27.8, t
33.1, t
44.3, s
47.2, d
22.1, t
39.0, t
79.5, s
12.4, q
87.6, s
74.4, d
55.5, q
139.0, s
67.7, d
34.8, t
32.6, d
42.6, d
131.5, s
27.9, t
34.0, t
44.0, s
47.8, d
22.5, t
38.9, t
79.8, s
12.7, q
87.4, s
74.0, d
55.8, q
139.0, s
68.2, d
34.6, t
32.8, d
42.4, d
131.4, s
38.5, t
74.8, d
48.4, s
47.8, d
22.6, t
38.9, t
79.9, s
Fermentation of Mestranol (1) with Cunninghamella elegans (NRRL
1392) Compound 1 (600 mg) was dissolved in DMSO (15 ml) and distrib-
uted among 30 flasks containing stage II cultures. Fermentation was contin-
ued for 12 d. Culture filtrates were extracted with CH Cl . The resulting or-
2.25, m
2
2
1
1
1
1
1
1
1
1
1
2
2
—
ganic extract was dried to afford a brown gum (3.5 g). The crude extract was
subjected to column chromatography (silica gel). Elution with a gradient of
petroleum ether and ethyl acetate afforded unchanged substrate 1 (121 mg,
petroleum ether–EtOAc, 81 : 19), and metabolites 2 (16.7 mg, petroleum
ether–EtOAc, 76 : 24) and 3 (21.7 mg, petroleum ether–EtOAc, 71 : 29).
6b-Hydroxymestranol (2): Colorless crystalline solid, mp 181—182 °C.
2.48, dt (4.3, 8.9); 2.21 m
4.32, dd (11.2, 4.6)
—
1.69, m
1.81, m; 1.58, m
2.14, m; 1.52, m
—
2
5
ꢁ1
[a] ꢁ107° (cꢂ0.1, MeOH). IR (CHCl ) n : 3421, 2927 and 2869 cm
.
D
3
max
1
UV (CHCl ) l nm (log e): 202 (2.9). H-NMR (CDCl , 400 MHz) d: 7.24
(1H, d, J1,2ꢂ8.5 Hz, H-1), 6.82 (1H, dd, J2,1ꢂ8.5 Hz, J ꢂ2.9 Hz, H-2), 6.89
3
max
3
7.38, q 0.85, s
86.9, s
2,4
—
(1H, d, J4,2ꢂ2.9 Hz, H-4), 4.76 (1H, t, Jꢂ2.2 Hz, H-6), 0.89 (3H, s, Me-18),
1
3
74.4, d
55.3, q
2.58, s
3.43, s
2.58 (1H, s, H-21), 3.79 (3H, s, OMe). C-NMR (CDCl , 100 MHz) d:
3
ꢀ
OCH3
Table 2. MS (EI, 70 eV): m/z (%): 326 (12) [M ], 308 (7), 273 (5), 243 (16),
2
25 (54), 197 (18), 171 (56), 128 (33), 115 (56), 91 (53), 83 (100). HR-EI-
Carbon multiplicities were determined by DEPT experiments; sꢂquaternary, dꢂme-
thine, tꢂmethylene, qꢂmethyl carbons. a) May be exchangeable.
MS m/z: 326.1739 (Calcd for C H O : 326.1766).
2
1
26
3
Chart 1. Metabolism of Compound 1 by Cunninghamella elegans