1
458
Vol. 53, No. 11
Experimental
3), 1.91 (6H, s, AcO).
3b,6a,11a,12b,15b-Pentahydroxypreg-4-en-20-one (4): Crystalline solid
General Experimental Procedures Melting points were determined by
a Buchi 535 melting-point apparatus. Optical rotations were measured on a
Jasco DIP-360 digital polarimeter, and a Hitachi U-3200 spectrophotometer IR (KBr) cm : 3374 (OH), 1693 (CꢀO); EI-MS m/z (rel. int., %): [M
2
5
(4%); [a]D ꢁ6.0° (cꢀ0.3, CHCl ); UV l
(MeOH) nm (log e): 204 (3.8);
3
max
ꢁ1
ꢂ
ꢂ
was used for UV spectrophotometry. The IR spectra were obtained on a FT-
missing], 348 [Mꢁ32] (9), 330 (100), 312 (15), 257 (6), 145 (20), 55 (30);
1
ꢂ
IR 8900 or FT-IR Bruker vector-22 spectrophotometer in CHCl . The H-
HR-FAB-MS m/z: 363.3780 [Mꢁ18ꢂNa] (Calcd for C H O ꢂNa:
3
21 30
5
13
1
13
and C-NMR spectra were recorded on a mixture of CDCl and CD OD on 363.3808); H-NMR (CDCl ꢂCD OD, 400 MHz) for d, see Table 1. C-
3
3
3
3
a Bruker AM 400 NMR spectrometer using the UNIX data system at 400
and 100 MHz, respectively. 2D experiments in CDCl3 were made on the
NMR (CDCl ꢂCD OD, 100 MHz) for d, see Table 1.
3 3
3b,6b,11a-Trihydroxypreg-4-en-20-one (5): Crystalline solid (2.6%);
2
5
Bruker AM 400 NMR spectrometer. The EI-MS and HR-EI-MS [ion source [a] ꢁ1.11° (cꢀ0.36, CHCl ); UV l
energy (70 eV), ion source temperature 250 °C] were recorded on a JEOL (KBr) cm : 3422 (OH), 1697 (CꢀO); EI-MS m/z (rel. int., %): 348 (M )
(MeOH) nm (log e): 203 (3.3); IR
D
3
max
ꢁ1
ꢂ
JMS 600 H mass spectrometer. FAB-MS spectra were obtained on a JEOL
JMS-AX505WA. TLC was carried out on silica gel precoated plates (Merck,
PF254: 20ꢄ20 cm, 0.25 mm). Spots on TLC were detected by spraying ceric
sulphate and vanillin solution, followed by heating. Column chromatography
(21), 332 (94), 315 (11), 279 (18), 251 (5), 149 (83), 105 (21), 81 (26), 57
1
(35); HR-EI-MS m/z: 348.2591 (Calcd for C H O : 348.2664); H-NMR
2
1
32
4
(CDCl ꢂCD OD, 400 MHz) for d, see Table 1.
3
3
Fermentation of Pregnenolone (1) with Gibberella fujikuroi (ATCC
10704) Compound 1 (300 mg) was dissolved in 20 ml acetone and equally
distributed among 40 flasks and kept for fermentation. The media was fil-
(CC) was performed on silica gel (70—230 mesh size). Compounds 1 and 2
were purchased from Fluka.
Fungi and Culture Conditions Cultures of Cunninghamella elegans tered after 12 d, extracted with dichloromethane and evaporated under re-
(
TSY 0865), Rhizopus stolonifer (TSY 0471), and Gibberella fujikuruoi
duced pressure to obtain a thick brown crude (0.92 g). The crude obtained
(0.92 g) was subjected to silica gel CC to obtain 6 (9.1 mg) on elution with
pet. ether–EtOAc (60 : 40), and 7 (7.23 mg) on elution with pet. ether–EtOAc
(50 : 50).
(ATCC 10704) were grown on Sabouraud-4% glucose-agar (Merck) at 25 °C
and stored at 4 °C. C. elegans broth media was prepared by mixing the fol-
lowing ingredients into distilled H O (3.0 l): glucose (30.0 g), glycerol
2
(
30.0 g), peptone (15.0 g), yeast extract (15.0 g), KH PO (15.0 g), and NaCl
Incubation of Pregnenolone Acetate (2) with Cunninghamella elegans
(TSY 0865) Four liters media of Cunninghamella elegans was prepared for
2
4
(15.0 g). R. stolonifer broth media was prepared by adding glucose (100 g),
peptone (25 g), KH PO (25 g) and yeast extract (15 g) to distilled water (4 l) incubation of pregnenolone acetate (2). Compound 2 (500 mg) was dis-
2
4
and pH was maintained at 5.6. G. fujikuroi broth media was prepared by solved in acetone and fed into each flask (15 mg) and flasks were placed on a
adding the following ingredients to distilled H O (3.0 l): glucose (80.0 g), shaker for 8 d. A time-course study was also conducted after 2 d of incuba-
KH PO (5.0 g), MgSO ·2H O (1.0 g), NH NO (0.5 g) and Gibberella trace tion. The resulting mixtures were extracted with dichloromethane and
2
2
4
4
2
4
3
element solution (2 ml). The latter solution was prepared by mixing transformations were detected through TLC. Fermentation was continued
Co(NO ) ·6H O (0.01 g), FeSO ·7H O (0.1 g), CuSO ·5H O (0.1 g),
for further 6 d. After 8 d, all the flasks were filtered and extracted with
dichloromethane. Crude extract thus obtained (0.9 g) was subjected to col-
umn chromatography (silica gel) to afford compound 1 (20 mg) on elution
3
2
2
4
2
4
2
ZnSO ·7H O (0.161 g), MnSO ·4H O (0.01 g) and NH molybdate (0.01 g)
4
2
4
2
4
into distilled water (100 ml).
General Fermentation and Extraction Conditions The fungal media with pet. ether–EtOAc (70 : 30), 10 (11.5 mg) on elution with pet.
were transferred into conical flasks (100 ml each) and autoclaved at 121 °C.
Seed flasks were prepared from three-day old slants and fermentation was
ether–EtOAc (55 : 45) and two biodegraded products 8 (10.2 mg) on elution
with pet. ether–EtOAc (65 : 35) and 9 (10.2 mg) on elution with pet.
allowed for 2 d on a shaker at 25 °C. The remaining flasks were inoculated ether–EtOAc (60 : 40).
from the seed flasks. After 2 d, pregnenolone (1) and pregnenolone acetate Incubation of Pregnenolone Acetate (2) with Rhizopus stolonifer (TSY
2) were dissolved in acetone and transferred into each flask (12 mg/ml) and
(
0471) Three liters media of Rhizopus stolonifer (TSY 0471) was prepared
for incubation of pregnenolone acetate (2). Compound 2 (300 mg) was dis-
solved in acetone and fed into each flask (20 mg) and flasks were placed on a
shaker for 12 d. After 12 d, all the flasks were filtered and extracted with
flasks were placed on a rotatory shaker (128 rpm) at 25 °C for fermentation.
Time course studies were carried out after 2 d and the transformation was
analyzed on TLC. The culture media was filtered and extracted with CH Cl
2
2
and ethyl acetate. The extract was dried over anhydrous Na SO , evaporated dichloromethane and the resulting crude extract was analyzed by TLC.
2
4
under reduced pressure, and the brown gummy crude was analyzed by TLC.
Crude extract (0.9 g) was subjected to column chromatography (silica gel) to
Fermentation of Pregnenolone (1) with Cunninghamella elegans (TSY afford two hydroxylated metabolites, 7 (11.2 mg) on elution with pet.
0
865) Compound 1 (370 mg) was dissolved in 20 ml acetone and placed in
ether–EtOAc (50 : 50) and 11 (12 mg) on elution with pet. ether–EtOAc
(55 : 45).
30 flasks for fermentation. All the media were filtered after 6 d of fermenta-
tion and extracted with dichloromethane. The CH Cl extract was evapo-
In Vitro Cholinesterase Inhibition Assay Electric-eel AChE (EC
3.1.1.7), horse-serum BChE (E.C 3.1.1.8), acetylthiocholine iodide, bu-
tyrylthiocholine chloride, 5,5ꢅ-dithiobis [2-nitrobenzoic acid] (DTNB), eser-
ine and galanthamine were purchased from Sigma Co. (St. Louis, MO,
U.S.A.). Cholinesterase inhibiting activities were measured by the spec-
2
2
rated under reduced pressure and a thick brown crude (1.02 g) was obtained,
which was subjected to silica gel CC to afford compound 3 (104 mg) on elu-
tion with pet. ether–EtOAc (25 : 75), compound 4 (15 mg) on elution with
pet. ether–EtOAc (22 : 78) and compound 5 (8.5 mg) on elution with pet.
ether–EtOAc (30 : 70).
23)
trophotometric method developed by Ellman et al. Acetylthiocholine io-
3
b,7b,11a-Triacetoxypreg-5-en-20-one (3a): Metabolite 3 (20 mg) was dide and butyrylthiocholine chloride were used as substrates to assay AChE
treated with pyridine/Ac O (2 : 1) and stirred (5 h) at r.t. The reaction mix-
ture was diluted with CH Cl (18 ml) and shaken with 2% aqueous NaHCO
and BChE inhibition, respectively. The assay conditions and protocol were
2
23)
the same as described previously.
2
2
3
(
15 ml) and 2% HCl (15 ml). The CH Cl extract was then dried over anhy-
Determination of IC50 Values The concentrations of test compounds
that inhibited hydrolysis of substrates (acetylthiocholine and butyrylthio-
choline) by 50% (IC ) were determined by monitoring the effect of increas-
2
2
drous Na SO4 and evaporated under reduced pressure. The crude extract
2
(17 mg) was then subjected to silica gel CC to afford compound 3a. Crys-
5
0
2
5
talline solid (18%); mp 159—162 °C; [a]D ꢁ8.8° (cꢀ0.18, CHCl ); UV ing concentrations of these compounds on the inhibition values. The IC50
3
ꢁ1
l
(MeOH) nm (log e): 203 (4.3); IR (KBr) cm : 2941 (C–H), 1732 values were then calculated using the EZ-Fit Enzyme Kinetics program (Per-
max
1
(
CꢀO), 1669 (CꢀC); H-NMR (CDCl ꢂCD OD, 400 MHz): d 2.01 (3H, s, rella Scientific Inc., Amherst, U.S.A.).
3 3
Me-21), 1.13 (1H, s, Me-19), 0.70 (3H, s, Me-18), 5.23 (1H, m, H-11), 4.56
(
1H, m, H-7), 5.1 (1H, m, H-3), 2.00 (9H, s, AcO).
b,7b-Diacetoxy-11a-hydroxypreg-5-en-20-one (3b): Metabolite
10 mg) was treated with pyridine/Ac O (2 : 1) and stirred (6 h) at r.t. The re-
References
3
3
1) Choudhary M. I., Musharraf S. G., Khan M. T. H., Abdelrahman D.,
Parvaz M., Shaheen F., Atta-ur-Rahman, Helv. Chim. Acta, 86, 3450—
3460 (2003).
(
2
action mixture was diluted with CH Cl (18 ml) and shaken with 2% aque-
2
2
ous NaHCO3 (15 ml) and 2% HCl (15 ml). The CH Cl2 extract was then
2) Choudhary M. I., Shah S. A. A., Musharraf S. G., Shaheen F., Atta-ur-
Rahman, Nat. Prod. Res., 17, 215—220 (2003).
3) Choudhary M. I., Musharraf S. G., Shaheen F., Atta-ur-Rahman, Nat.
Prod. Lett., 16, 377—382 (2002).
4) Choudhary M. I., Azizuddin, Atta-ur-Rahman, Nat. Prod. Lett., 16,
101—106 (2002).
2
dried over anhydrous Na SO , evaporated under reduced pressures and the
2
4
crude extract was analyzed by TLC. The crude obtained (8 mg) was sub-
jected to silica gel CC to afford compound 3b. Crystalline solid (29.8%); mp
2
5
1
2
65—167 °C; [a] ꢂ2.8° (cꢀ0.14, CHCl ); UV l (MeOH) nm (log e):
D
3
max
ꢁ1
1
03 (4.1); IR (KBr) cm : 3430 (OH), 1728 (CꢀO), 1668 (CꢀC); H-NMR
(
0
CDCl ꢂCD OD, 400 MHz): d: 2.00 (3H, s, Me-21), 1.12 (1H, s, Me-19),
5) Atta-ur-Rahman, Choudhary M. I., Asif F., Farooq A., Yaqoob M.,
Nat. Prod. Lett., 14, 217—224 (2000).
3
3
.69 (3H, s, Me-18), 3.8 (1H, m, H-11), 4.56 (1H, m, H-7), 5.2 (1H, m, H-