Z. Hussain et al. / Steroids 112 (2016) 68–73
71
Illkirch, France). Upon reaching 70% confluency, 2.5 ꢁ 105 cells/mL
were plated in 24-well tissue culture plates, and differentiated into
macrophage mimicking cells with the addition of 20 ng/mL phor-
bol myristate acetate (PMA), (SERVA, Heidelberg, Germany). The
plates were then incubated for 24 h at 37 °C in 5% CO2. The cells
were then stimulated with 50 ng/mL of Escherichia coli lipopolysac-
charide B (DIFCO Laboratories, Michigan, USA), followed by treat-
rated carbonyl functionalities, respectively. The presence of
a
,b-
unsaturated carbonyl was also inferred from the UV–visible spec-
trum (kmax 247 nm). The 1H NMR spectrum showed a downfield
methine singlet at d 3.77 which was assigned to a geminal proton
to a hydroxyl group, whereas in the 13C NMR spectrum, only 20 sig-
nals appeared, clearly indicating the loss of the ester side chain. An
additional downfield quaternary carbon signal at d 218.3 indicated
a ketonic group. The OH group was placed at C-17 (d 87.3) based
on the HMBC experiment (Scheme 2). Key HMBC correlations of H-
17 (d 3.77 s) with C-16 (d 218.3), C-13 (d 44.3), and C-18 (d 12.5) sup-
ported the presence of a ketonic carbonyl at C-16, and an OH at C-17
of ring D. The relative stereochemistry at C-17 was deduced by the
NOESY interactions between H-14 (d 1.46 m, axial), and H-17 (d
3.77 s, axial), suggesting a b orientation of the geminal OH group
(Scheme 3). Thus metabolite 2 was identified as 17b-hydroxy-1-
ment with the test compounds at a concentration of 25
The plates were again left for incubation at 37 °C in 5% CO2 for
duration of 4 h. The level of TNF- in supernatants was analyzed
lg/mL.
a
by performing ELISA using human Duo Set kit (R & D Systems, Min-
neapolis, USA) kit, according to instructions prescribed by the
manufacturer.
2.9. Cytotoxicity assay
methyl-5a-androst-1-ene-3,16-dione.
Metabolite 3 (7 mg, brown gummy solid, TR = 30 min) was
obtained from the reverse phase recycling HPLC, using isocratic
methanol and water system (7:3). The HREI-MS data of 3 sup-
ported the molecular formula C20H30O3 m/z 318.2211 [M+] (calcd.
318.2195) with six degrees of unsaturation. The IR absorptions at
tmax (cmꢀ1) 3374, 1658, and UV absorption at kmax 230 nm, sup-
Cytotoxic activity of the transformed products was evaluated by
using the standard MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphe-
nyl-tetrazolium bromide) colorimetric assay in the 96-well flat-
bottomed microplates [20]. For this assay, the Dulbecco’s Modified
Eagle Medium, supplemented with 5% of fetal bovine serum (FBS),
along with the 100 IU/mL of penicillin, and 100 lg/mL of strepto-
mycin in 75 cm2 flasks, were used to culture the 3T3 (mouse
fibroblast) cells in the incubator at 37 °C in 5% CO2. Cells were har-
vested, and 5 ꢁ 104 cells/mL were plated in the 96-well plates
ported the presence of hydroxyl, and
a,b-unsaturated carbonyl
groups. In the 1H NMR spectrum, an additional methine multiplet
at d 4.08, and a signal at d 70.6 in the 13C NMR spectrum, suggested
hydroxylation. The HMBC (Scheme 2) correlations of H-14 (d 1.24,
m) with C-9 (d 49.3), C-13 (d 43.5), and C-16 (d 38.8), supported the
presence of an OH group at C-15. Besides this, H-17 (d 3.33, t) also
showed HMBC correlations with C-15 (d 70.6), C-12 (d 36.1), C-13
(d 43.5), C-16 (d 38.8), and C-18 (d 12.3). The NOESY (Scheme 3)
spectrum showed cross peaks between H-14 (m 1.24, axial) and
H-15, and H-17. Thus the relative stereochemistry of OH groups
at C-15, and C-17 was deduced as b. These data permitted to estab-
lish the structure of metabolite 3 as 15b, 17b-dihydroxy-1-methyl-
(100 lL/well), and allowed to incubate overnight. After incubation,
the medium was removed, followed by the addition of 200
fresh medium with different concentrations of the test compounds
(1–30 M) in triplicates. The plate was then further incubated for
48 h. It was preceded with the addition of 200 L MTT (0.5 mg/
mL) to each well, and incubated for 4 h. Finally, 100 L of dimethyl
lL of
l
l
l
sulphoxide (DMSO) was added to each well. The microplate reader
(Spectra Max plus, Molecular Devices, CA, USA) was used to calcu-
late the extent of the MTT reduction to formazan within cells by
measuring the absorbance at 540 nm. The cytotoxicity was
recorded as concentration causing 50% growth inhibition (IC50) of
3T3 cells. The percent inhibition was calculated by using the fol-
lowing formula, and the results were processed with the help of
EZ-FitTM enzyme kinetics program (Perellela Scientific, Inc.,
Amherst, Mars, USA).
5a-androstan-1-ene-3-one.
Fraction 4 afforded metabolite 4 (12 mg, TR = 22 min) as a yel-
lowish solid through reverse phase recycling HPLC. The HRFAB-
MS of 4 ([M+H]+, m/z 317.2100) was consistent with the formula
C
20H30O3 (calc. 318.2195). The IR showed absorption at tmax
(cmꢀ1) at 3733, 1734, 1651 for OH, carbonyl and
a,b-unsaturated
carbonyl group and UV spectra showed absorptions kmax (nm)
229. The 1H NMR showed an additional methine proton triplet at
d 3.77 (J12,13 = 2.4 Hz). Broad-band decoupled 13C NMR spectrum
of compound 4 was distinctly similar to that of 3. The HMBC
(Scheme 2) spectrum showed the correlations of H-18 (d 0.83, s)
with C-12 (d 71.0), C-13 (d 44.2), C-14 (d 52.6), and C-17 (d 75.5),
supporting the OH groups at C-12 (d 71.0), and C-17 (d 75.5). In
addition, H-17 (3.22, t, J17,16 = 8.4 Hz) also showed HMBC correla-
tions with C-12 (d 71.0), C-13 (d 44.2), and C-18 (d 14.2), indicating
an OH at C-12 (d 71.0). The geminal H-12 (d 3.77, t) showed NOESY
(Scheme 3) interactions with H-9 (d 1.97, m), H-14 (d 1.15, m), and
H-17 (d 3.22, t), suggesting a b disposition of the OH groups at C-12
(equatorial) (d 71.0), and at C-17. In addition to the NOESY spec-
trum, the key COSY correlations between H-11 and H-12 and H-
16 and H-17, led to the deduction of the structure of 4 as
% Inhibition
100 ꢀ ðOD of test compound ꢀ OD of negative controlÞ
¼
ꢁ 100
ðOD of positive control ꢀ OD of negative controlÞ
3. Results and discussion
First microbial biotransformation of methenolone enanthate (1)
with Aspergillus niger, and Cunninghamella blakesleeana is being
reported here. Fermentation of 1 with A. niger afforded six metabo-
lites 2–7, three of them (i.e., 2–4) were found to be new, whereas C.
blakesleeana yielded a known metabolite 6 (Scheme 1). Structures
of compounds 2–7 were deduced by comparing their spectroscopic
data with the substrate 1, and other related metabolites reported
earlier.
Compound 2 (6 mg) was obtained from fraction 2 as a white crys-
talline material from the reverse phase recycling HPLC with an iso-
cratic elution of MeOH and H2O (75:25). Compounds 7 and 2 were
obtained with retention times (TR) 21, and 27 min, respectively.
The molecular formula for 2 was deduced as C20H28O3, based on its
HRFAB-MS at m/z 315.2006 [MꢀH]+ (calc. 316.2038); 96 amu less
than 1, indicating the hydrolytic removal of the ester side chain
along with the addition of a ketonic moiety in the steroidal skeleton.
12b,17b-dihydroxy-1-methyl-5
Structures of metabolites 5 (7 mg), and 6 (14 mg) were identi-
fied as 1-methyl-5 -androstan-1-ene-3,17-dione, and 17b-
hydroxy-1-methyl-5 -androstan-1-ene-3-one, respectively, based
a-androstan-1-ene-3-one.
a
a
on their HREI-MS, and by comparing their spectral data with the
reported data for metabolites of methenolone acetate in horses
[17], and fungal transformation of mesterolone by Fusarium lini,
and Cephalosporium aphidicola [9]. Metabolite 7 (7 mg) was identi-
fied as 16b, 17b-dihydroxy-1-methyl-5a-androstan-1-ene-3-one
by comparison of its spectral data with the data reported for a
In the IR spectrum, peaks at
cated the presence of hydroxyl group, a carbonyl, and
t
max (cmꢀ1) 3733, 1746, and 1655 indi-
,b-unsatu-
a
metabolite of methenolone acetate in horses [17].