Synthesis antimicrobial and antioxidant studies of new oximes of steroidal chalcones

Article abstract of DOI:10.1016/j.steroids.2013.05.015

A convenient synthesis of oximes of steroidal chalcones (4a-4j) was performed and structural assignment of the products was confirmed on the basis of IR, 1HNMR, 13C NMR, MS and analytical data. The synthesized compounds were screened for in vitro antioxid

Full text of DOI:10.1016/j.steroids.2013.05.015

Steroids 78 (2013) 945–950
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis antimicrobial and antioxidant studies of new oximes
of steroidal chalcones
a
b
c
Imtiyaz H. Lone a,b, , Khaliquz Z. Khan , Bharat I. Fozdar , Fida Hussain
⇑
a Department of Chemistry, University of Kashmir, Srinagar 190009, J&K, India
b Department of Chemistry, IGNOU, New Delhi 110068, India
c Department of Zoology, Bundelkhand University, Jhansi 284128, UP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 April 2013
Received in revised form 4 May 2013
Accepted 20 May 2013
Available online 5 June 2013
A convenient synthesis of oximes of steroidal chalcones (4a–4j) was performed and structural assignment
of the products was confirmed on the basis of IR, 1HNMR, 13C NMR, MS and analytical data. The synthesized
compounds were screened for in vitro antioxidant activity by using DPPH method and in vitro antimicrobial
activity against different bacterial and fungal strains by agar diffusion method. The activity of the tested
compounds against each microbe varied due to structural differences between them. Presence and position
of different substituents on the benzene ring of the chalconyl pendent had a marked effect on the activity of
the compounds. From the results it can be inferred that the compounds 4a–j showed significant antioxidant
activity and antimicrobial activity against all microbial strains used for testing.
Keywords:
Oximes
Pregnenolone
Antimicrobial
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
cancer, osteoarthritis, etc. [7]. Most of the steroid based pharma-
ceuticals are semi-synthetic compounds prepared by connecting
In the last few decades there has been an extensive focus of re-
search towards the rational modification of steroid molecules. This
is due to the fact that such type of compounds are less toxic, less
vulnerable to multi-drug resistance (MDR) and highly bioavailable
because of being capable of penetrating the cell wall. Oxime com-
pounds are used as antidotes for nerve agents. A nerve agent inac-
tivates acetylcholinesterase molecules by phosphorylation of the
molecule. Oxime compounds can reactivate acetylcholinesterate
by attaching to the phosphorus atom and forming an oxime-phos-
phonate which then splits away from the acetylcholinesterase
molecule. The most effective oxime nerve-agent antidotes are pra-
lidoxime (also known as 2-PAM), obidoxime, methoxime, HI-6,
Hlo-7, and TMB-4 [1]. Methyl Ethyl Ketoxime is a skin-preventing
additive in many oil-based paints. Steroidal oximes are different
class of compounds and have well validated biological effects. Ste-
roids and their synthetic congeners including their oxime derva-
tives have extensively been studied during the last decade [2,3].
These molecules have always attracted considerable attention be-
cause of being a fundamental class of biological signaling mole-
cules and their profound biological, scientific and clinical
importance [4]. They can regulate a variety of biological processes
and thus have the potential to be developed as drugs for the treat-
ment of a large number of diseases including cardiovascular [5],
autoimmune diseases [6], brain tumors, breast cancer, prostate
a special functionality to the core structure of a steroid [8]. Struc-
tural modification of steroids would provide a platform to ap-
proach the synthesis of new drugs for tackling important
biological problems. To meet these ends, attention has been de-
voted in the literature to the synthesis of steroidal compounds be-
cause of their potent receptor binding properties and valuable
pharmacological activities [9–11]. Steroid molecules and their
oxime derivatives have been tested against variety of microorgan-
isms for antimicrobial activities, cholestanes, deoxycorticosterone,
progesterone and androsterone are notable ones [12]. The advan-
tage of employing hydrophobic steroid units with oxime group
(@NOH) enhance their ability to interact with cell membranes
and thus pave the way for biological activity of such hybrid mole-
cules. This has been proved by different ring modification studies
of steroidal molecules and their chalcone dervatives involving
the A and D-ring whereby incorporation of heteroatom (N or O)
have been reported to enhance the biological activities of these
molecules. Such systems have been shown to bear a lot of different
biological activities such as anti-microbial, anti-inflammatory,
hypotensive, hypocholesterolemic and diuretic activities [13–17].
Very fewer efforts have been reported related to the synthesis of
oximes of chalcones of steroidal moieties and their biological
screening. Though there are reports for the synthesis of other such
analogs, the same is not true for the oximes of chalconyl
derivatives at the D-ring of pregnenolone. Taking inspiration from
the number of reported biological activities associated with
structurally related analogs, we, in continuation of our efforts to-
wards the synthesis of oximes of chalcones of preglenenolone
⇑
Corresponding author at: Department of Chemistry, IGNOU, New Delhi 110068,
India. Tel.: +91 9796540195; fax: +91 11 29572824.
E-mail address: hussainjamia5@gmail.com (I.H. Lone).
0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.05.015

946
I.H. Lone et al. / Steroids 78 (2013) 945–950
derivatives starting from readily available 20-keto pregnenanes
[18]. We efficiently synthesized new oxime derivatives and studied
their antimicrobial and antioxidant properties which we wish to
report herein.
2.2.1.1. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-
17-yl)-3-p-methylphenylprop-2-en-1-imine (4a). Coloured powder
(78%). M.P: 235–240 °C; IR (KBr) cmꢁ1: 3525, 3385, 2948, 1814,
1617, 1512, 1423, 1051, 609; 1H NMR (CDCl3): d 0.78 (s, 3H),
1.200 (s, 3H), 1.54–2.0 (m, 6H), 2.41–2.48 (m, 3H), 2.42 (t,
J = 8.80, 1H); 3.21 (m, 1H); 5.98 (s, J = 16.00, 1H), 6.89 (m, 3H),
7.05 (m, 3H), 8.68 (s, 1H); 13C NMR (500 MHz, CDCl3): d 14.23,
21.06, 22.07, 24.02, 31.03, 31.48, 32.09, 36.52, 42.11, 45.21,
48.01, 48.75, 49.32, 49.79, 50.84, 57.44, 61.07, 73.12, 123.26,
127.69, 128.89, 131.41, 135.72, 142.15, 158.6, 201.12; ESI-MS:
420 (M+H); Anal. Calcd. for C28H37NO2: C, 80.15; H, 8.89; N, 3.34;
O, 7.63; Found C, 80.10; H, 8.71; N, 3.04; O, 7.52.
2. Experimental
2.1. General methods
Solvents and organic reagents were purchased from Sigma Al-
drich, Merck (Germany) and Loba Chemie (India) and were used
without further purification. Melting points were recorded on Buc-
ci Melting point apparatus D-545; IR spectra (KBr discs) were re-
corded on Bruker Vector 22 instrument. NMR spectra were
recorded on Bruker DPX200 instrument in CDCl3 with TMS as
internal standard for protons and solvent signals as internal stan-
dard for carbon spectra. Chemical shift values are mentioned in d
(ppm) and coupling constants are given in Hz. Mass spectra were
recorded on EIMS (Shimadzu) and ESI-esquire 3000 Bruker Dalton-
ics instrument. The progress of all reactions was monitored by TLC
on 2 ꢀ 5 cm pre-coated silica gel 60 F254 plates of thickness of
0.25 mm (Merck). The chromatograms were visualized under UV
254–366 nm and iodine.
2.2.1.2. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-
17-yl)-3-p-nitrophenylprop-2-en-1-imine
(4b). Yellow
powder
(81%). M.P: 255–260 °C; IR (KBr) cmꢁ1: 3410, 3315, 2941, 1764,
1651, 1323, 1061, 797; 1H NMR (CDCl3): d 0.71 (s, 3H), 1.09 (s,
3H), 1.93–2.0 (m, 6H), 2.26–2.39 (m, 3H), 2.31 (s, 3H), 3.12 (t,
J = 8.43, 1H); 3.82 (m, 1H), 6.12 (s, 1H), 7.01 (d, J = 15.78, 1H),
7.72 (m, 3H), 7.89 (d, J = 6.83, 1H), 8.12 (d, J = 15.78, 1H), 9.31 (s,
1H),; 13C NMR (500 MHz, CDCl3): d 13.94, 19.96, 20.15, 21.11,
22.14, 24.87, 31.07, 32.19, 36.71, 37.37, 37.82, 39.82,42.12, 45.18,
51.23, 57.75, 63.27, 71.30, 71.86, 121.82, 126.57, 128.11, 129.18,
130.58, 131.93, 134.81, 138.87, 139.76, 140.53, 160.3, 200.51;
ESI-MS: 465 (M+H); Anal. Calcd. for C28H36N2O4: C, 72.39; H,
7.81; N, 6.03; O, 13.77; Found: C, 72.09; H, 7.68; N, 5.83; O, 13.57.
2.2. Chemical synthesis
2.2.1. General procedure for the synthesis of oxime derivatives
To a solution of pregnenolone 1 (0.316 g, 1 mmol, 1 eq.) in eth-
anol (10 ml) was added a conc. aq. solution of KOH (2 eq.). Then
aldehyde 2 (1.2 eq.) was charged into the reaction mixture and
the reaction mixture was stirred for 1–2 h at room temperature
to get the corresponding benzylidine derivative 3. The benzylidene
dervatives were isolated and recrystallized from ethylacetate and
were further reacted (0.2 g, 1 mmol), with hydroxylamine hydro-
chloride (2 ml) in 10 ml of ethanol to get the corresponding oxime
4. After completion as revealed by thin layer chromatography (TLC
run in ethylacetate:hexane) in an average span of around 3–4 h
(Scheme 1). The precipitate obtained was filtered, dried and mon-
itored through TLC for the purity. Thin layer chromatography re-
vealed just a single spot which proved the presence of a single
product. For further purification, the product was recrystallized
from ethylacetate to give product as solid powder. In some cases
the products were purified by column chromatography using eth-
ylacetate: hexane (70:30 v/v) as eluent. It is to be mentioned that
when non-aromatic aldehydes were used, the chalcones were
formed in a very minor quantity and that too not stable enough
at ambient conditions. Thus the study was restricted to the use
of aromatic aldehydes only. The spectral data of various oxime der-
vatives are given as under (Most of the peaks due to steroidal skel-
ton were merged and could not be differentiated in the 1H NMR.
Thus d values of only those peaks that distinguish the product
and could easily be differentiated are reported as under).
2.2.1.3. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-
17-yl)-3-(3,3,4-trimethoxyphenyl)prop-2-en-1-imine (4c). Solid yel-
lowish powder (74%). M.P: 230–235 °C; IR (KBr) cmꢁ1: 3407,
3323, 2981, 1627, 1433, 1216, 1029, 778; 1H NMR (CDCl3): d
0.73 (s, 3 H), 1.00 (s, 3H), 1.81–2.10 (m, 6H), 2.30 (m, 3H), 2.55
(s, 3H), 2.88 (t, J = 8.43, 1H); 3.55 (m, 1H), 5.76 (s, 1H), 6.94 (d,
J = 15.98, 1H), 7.32 (d, J = 7.42, 2H), 7.58 (d, J = 7.42, 2H), 7.64 (d,
J = 15.98, 1H), 10.41 (s, 1H); 13C NMR (500 MHz, CDCl3): d 12.82,
18.48, 20.43, 20.88, 22.18, 23.76, 30.72, 30.85, 31.13, 35.64,
36.67, 38.34, 41.57, 44.96, 50.11, 57.21, 58.89, 71.73, 120.24,
125.14, 128.29, 128.95, 131.28, 139.63, 159.9, 199.42; ESI-MS:
510 (M+H); Anal. Calcd. for C31H43NO5: C, 73.05; H, 8.5; N, 2.75;
O, 15.70; Found C, 72.85; H, 8.05; N, 2.25; O, 15.12.
2.2.1.4. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-
17-yl)-3-(4-dimethylamine)prop-2-en-1-imine (4d). Solid powder
(75%). M.P: 231–236 °C; IR (KBr) cmꢁ1: 3415, 3335, 2944, 1723,
1621, 1512, 1413, 1112, 789; 1H NMR (CDCl3): d 0.58 (s, 3H), 1.00
(s, 3H), 1.71–1.80 (m, 6H), 2.10–2.44 (m, 3H), 2.60 (s, 3H), 2.90 (t,
J = 8.73 ,1H); 3.65 (m, 1H), 6.36 (s, 1H), 6.95 (d, J = 15.93, 1H), 7.37
(m, 4H), 7.61 (d, J = 15.93, 1H) 10.10 (s, 1H); 13C NMR (500 MHz,
CDCl3): d 13.36, 20.18, 20.48, 21.78, 24.19, 30.78, 30.99, 31.63,
R
R
HON
O
O
OHC
R
2
NH2OH.HCl
EtOH
EtOH/ KOH
HO
HO
HO
4
1
3
Scheme 1. Synthesis of D-ring substituted oximes of steroidal chalcones.

I.H. Lone et al. / Steroids 78 (2013) 945–950
947
35.64, 36.17, 38.64, 44.26, 45.16, 49.51, 56.61, 61.99, 72.73, 121.54,
124.04, 127.19, 128.05, 132.18, 139.73, 159.54, 198.42; ESI-MS: 462
(M+H); Anal. Calcd. for C30H42N2O2: C, 77.88; H, 9.15; N, 6.05; O,
6.92; Found: C, 77.68; H, 9.03; N, 5.95; O, 6.81.
(70%). M.P: 240–250 °C; IR (KBr) cmꢁ1: 3425, 3300, 2917, 1718,
1611, 1513, 1296, 1024, 763; 1H NMR (CDCl3): d 0.61 (s, 3H),
1.03 (s, 3H), 1.65–1.92 (m, 6H), 2.22–2.54 (m, 3H), 2.76 (t,
J = 9.12, 1H), 3.62 (m, 1H), 5.45 (s, 1H), 6.74 (d, J = 15.31, 1H),
6.96–7.21 (m, 4H), 7.54 (d, J = 15.31, 1H), 9.96 (s, 1H),; 13C NMR
(500 MHz, CDCl3): d 13.13, 19.58, 20.23, 24.04, 25.21, 30.92,
31.66, 35.04, 36.17, 38.44, 43.16, 45.76, 49.21, 57.28, 61.29,
75.03, 121.64, 124.04, 127.19, 129.45, 130.98, 142.13, 160.04,
199.83; ESI-MS: 455 (M+H); Anal. Calcd. for C28H36ClNO2: C,
74.07; H, 7.99; N, 3.08; O, 7.05; Cl, 7.81; Found C, 74.01; H, 7.89;
N, 3.01; O, 6.99; Cl, 7.78.
2.2.1.5. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-
17-yl)-3-(3-fluorophenyl)-prop-2-en-1-imine (4e). Solid powder
(74%). M.P: 240–245 °C; IR (KBr) cmꢁ1: 3407, 3312, 2940, 1773,
1618, 1518, 1232, 1040, 781; 1H NMR (CDCl3): d 0.66 (s, 3H),
1.00 (s, 3H), 1.81–2.00 (m, 6H), 2.40–2.62 (m, 3H), 2.94 (t,
J = 8.92, 1H); 4.12 (m, 1H), 5.36 (s, 1H), 6.90 (d, J = 15.93, 1H),
7.18 (m, 2H), 7.73 (m, 3H) 9.36 (s, 1H); 13C NMR (500 MHz, CDCl3):
d 12.16, 19.18, 20.43, 22.78, 25.90, 30.82, 30.96, 31.13, 35.74,
36.87, 38.24, 43.56, 44.96, 49.71, 56.81, 60.99, 72.73, 122.14,
124.14, 126.79, 129.12, 132.98, 159.73, 196.92; ESI-MS: 438
(M+H); Anal. Calcd. for C28H36FNO2 C, 76.85; H, 8.29; N, 3.02; O,
7.31 F, 4.34; Found: C, 76.76; H, 8.21; N, 2.92; O, 7.11 F, 4.14.
2.2.1.10. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-
17-yl)-3-(4-bromophenyl)-prop-2-en-1-imine (4j). Solid brown
powder (69%). M.P:238–244 °C; IR (KBr) cmꢁ1: 3454, 3305, 2939,
2872, 1605, 1511, 1355, 1200, 754; 1H NMR (CDCl3): d 0.61 (s,
3H), 1.03 (s, 3H), 1.71–1.81 (m, 6H), 2.21–2.35 (m, 3H), 2.79 (t,
J = 8.68, 1H), 3.62 (m, 1H), 5.54 (s, 1H), 6.38 (s, 1H), 6.67–6.89
(dd, J = 3.68, 2H), 7.37 (m,1H), 7.58 (s, 1H), 10.16 (s, 1H); 13C
NMR (500 MHz, CDCl3): d 13.26, 20.18, 21.34, 22.48, 22.78, 24.11,
30.52, 30.96, 32.13, 35.59, 37.85, 38.74, 43.96, 49.12, 56.23,
61.39, 72.53, 121.04, 124.91, 127.09, 121.05, 130.19, 138.95,
160.14, 197.44; ESI-MS: 499 (M+H); Anal. Calcd. for C28H36BrNO2:
C, 67.46; H, 7.28; N, 2.81; O, 6.42; Br, 16.03; Found: C, 67.34; H,
7.21; N, 2.76; O, 6.36; Br, 15.96.
2.2.1.6. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-
17-yl)-3-(4-fluorophenyl)-prop-2-en-1-imine (4f). Solid powder
(71%). M.P:243–250 °C; IR (KBr) cmꢁ1: 3417, 3315, 2946, 1774,
1641, 1518, 1232, 1045, 759; 1H NMR (CDCl3): d 0.67 (s, 3H),
1.09 (s, 3H), 1.71–1.99 (m, 6H), 2.23–2.53 (m, 3H), 2.83 (t,
J = 8.61, 1H), 3.73 (m, 1H), 5.57 (s, 1H), 6.65 (d, J = 15.84, 1H),
7.18 (m, 2H), 7.14–7.22 (m, 2H), 7.52 (d, J = 15.84, 1H) 8.86 (s,
1H); 13C NMR (500 MHz, CDCl3): d 12.13, 19.36, 20.23, 20.58,
21.68, 23.45, 30.42, 31.86, 32.03, 35.34, 36.17, 38.24, 43.16,
44.96, 49.21, 56.01, 61.99, 70.73, 121.52, 125.04, 127.19, 128.35,
132.08, 139.53, 158.14, 196.82; ESI-MS: 438 (M+H); Anal. Calcd.
for C28H36FNO2 C, 76.85; H, 8.29; N, 3.02; O, 7.31 F, 4.34; Found:
C, 76.74; H, 8.22; N, 2.91; O, 7.12 F, 4.14.
3. Biology
3.1. Antimicrobial activity
The bacterial strains used for the analysis were Bacillus subtilis
(MTCC 619), Staphylococcus epidermidis (MTCC 435), Proteus vulga-
ris (MTCC 426) and Pseudomonas aeruginosa (MTCC 424). The fun-
gal strains used were Aspergillus niger (MTCC 1344) and Penicillium
chrysogenum (MTCC 947). All the bacterial and fungal strains were
obtained from The Microbial Type Culture Collection and Gene
Bank (MTCC), Institute of Microbial Technology (IMTECH), Chandi-
garh. Kanamycin and Fluconazole were used as standard antibacte-
rial and antifungal substances respectively, under similar
conditions for comparison. Dimethyl sulphoxide (DMSO) was used
as negative control.
The test organisms were cultured on agar slants, incubated 24 h
at 37 0.5 °C and 24–48 h at 27 0.2 °C for bacteria and fungi
respectively to get the freshly prepared cultures. The steroidal
oxime derivatives were evaluated for antimicrobial activity against
these freshly prepared strains of test organisms by agar diffusion
method [19–21]. Muller Hinton Agar (MHA) and Potato Dextrose
Agar (PDA) were used as nutrient media for bacterial and fungal
strains respectively. The media (MHA &PDA) was prepared using
distilled water and 20 ml of it was transferred into 50 ml test
tubes, the test tubes were tightly plugged with cotton and steril-
ized in autoclave at 15 lb/in2 for 15 min as directed by the manu-
facturer. After sterilization the medium was inoculated with
freshly cultured bacterial strains under sterile condition i.e. under
Laminar Flow. The inoculation was done when the temperature of
the medium reached to 50–40 °C, so that test organism may not die
at higher temperature. The medium inoculated with test microor-
ganisms was transferred into the plates of 90 mm size under sterile
conditions. The medium was allowed to solidify and the wells (4/
2.2.1.7. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-
17-yl)-3-(4-methoxyphenyl)prop-2-en-1-imine (4g). Solid powder
(72%). M.P: 250–255 °C; IR (KBr) cmꢁ1: 3405, 3322, 2916, 2853,
1803, 1637, 1521, 1275, 1036, 769; 1H NMR (CDCl3): d 0.68 (s, 3H),
1.2(s, 3H), 1.7–1.90 (m, 6H), 2.3–2.56 (m, 3H), 2.93 (t, J = 8.89, 1H),
3.13 (m, 1H), 4.17 (s, 3H), 5.46 (s, 1H), 6.55 (d, J = 15.92, 1H), 6.81 (d,
J = 8.72, 2H), 7.61 (d, J = 8.72, 2H), 7.83 (d, J = 15.84, 1H), 9.36 (s, 1H);
13C NMR (500 MHz, CDCl3): d 12.23, 19.31, 21.21, 21.78, 24.23,
31.51, 32.13, 34.42, 36.17, 38.24, 43.56, 44.96, 49.12, 56.31, 61.89,
75.83, 121.54, 124.74, 127.19, 128.45, 132.46, 139.63, 160.14,
199.94; ESI-MS: 450 (M+H); Anal. Calcd. for C29H39NO3: C, 77.47;
H, 8.74; N, 3.12; O, 10.68 Found:: C, 77.38; H, 8.64; N, 3.02; O, 10.56.
2.2.1.8. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-
17-yl)-3-(2-methoxyphenyl)prop-2-en-1-imine (4h). Solid powder
(73%). M.P: 255–260 °C; IR (KBr) cmꢁ1: 3485, 3330, 2950, 1718,
1623, 1528, 1246, 1014, 763; 1H NMR (CDCl3): d 0.70 (s, 3H),
1.70 (s, 3H), 1.9–2.10 (m, 6H), 2.60–2.72 (m, 3H), 2.99 (t, J = 8.85,
1H), 3.52 (m, 1H), 3.98 (s, 3H), 5.57 (s, 1H), 6.84 (d, J = 16.16,
1H), 7.01 (m, 2H), 7.46 (m, 1H), 7.59 (d, J = 6.37, 1H), 7.98 (d,
J = 16.16, 1H), 10.06 (s, 1H),; 13C NMR (500 MHz, CDCl3): d 13.34,
19.14, 20.38, 21.78, 25.11, 30.67, 30.88, 31.03, 35.44, 36.27,
38.44, 43.56, 44.76, 49.01, 55.29, 61.89, 71.13, 118.75, 124.44,
127.09, 128.15, 130.38, 140.93, 160.34, 199.93; ESI-MS: 450
(M+H); Anal. Calcd. for C29H39NO3: C, 77.47; H, 8.74; N, 3.12; O,
10.68 Found:: C, 77.38; H, 8.64; N, 3.02; O, 10.56.
plate) of 6 mm diameter and 50
sterile cork borer. The solution of test compound 1000
prepared in DMSO and the wells bored on the medium were each
filled (50 g) with test compound using micropipette (20–200 l).
Four wells were bored on the plates and each filled with same
l
l volume were bored on it using
lg/ml was
2.2.1.9. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-
17-yl)-3-(4-chlorophenyl)-prop-2-en-1-imine (4i). Brown powder
l
l

948
I.H. Lone et al. / Steroids 78 (2013) 945–950
Table 1
Antibacterial and antifungal screening data of compounds 4a–j.
Compound
(Zone of inhibition in ‘‘mm’’)
Antibacterial activities
Antifungal activities
B. subtilis
S. epidermidis
P. Vulgaris
P. aeruginosa
A. niger
P. chrysogenum
(MTCC 619)
(MTCC 435)
(MTCC 426)
(MTCC 424)
(MTCC1344)
(MTCC 947)
4a
4b
4c
4d
4e
4f
4g
4h
20
18
13
17
14
13
15
16
11
21
–
18
17
-
15
11
15
13
–
14
10
–
24
–
12
10
12
14
–
15
–
11
12
11
–
13
14
15
14
12
14
12
12
15
12
–
11
14
11
14
11
15
11
14
14
12
–
11
13
–
15
17
20
14
12
19
14
–
4i
4j
Control
Kenamycin
Flucanazole
24
–
22
–
17
–
18
–
14
P. vulgaris-Proteus vulgaris, B. subtilis-Bacillus subtilis, S. epidermidis-Staphylococcus epidermidis, P. aeruginosa Pseudomonas aeruginosa. DMSO-Negative control, well
diameter/vol.- 6 mm/50 l, Kanamycin- Standard for antibacterial activity. Fluconazole-Standard for anti fungal activity.
l
compound and two plates for each test compound were taken and
the experiment was repeated thrice. The discs of Kanamycin and
Fluconazole were also incorporated into the medium for compari-
with the help of sterile loop, a loop of organisms were transferred
into the different antibiotic dilutions (4 mg/mL, 8 mg/mL etc.) un-
der laminar flow and incubated for 24 h at 37 0.5 °C. The inocu-
lums after incubation were tested for bacterial growth by
transferring them with the help of sterile loop on the nutrient agar
plates. The nutrient agar plates were incubated for 24 h at
37 0.5 °C to determine the visible growth of microorganism.
0 mg/mL is antibiotic free inoculums, was taken as growth or posi-
tive control for microorganism.
son (10–30 lg). The plates containing test organism and test mate-
rial in contact were incubated at 37 0.5 °C for 24 h. Same
procedure was employed for antifungal activity however, the cul-
ture strains of fungi were maintained on potato dextrose agar
and spores were transferred in the PDA medium and the plates
were incubated at 27 0.2 °C for 24–48 h. Inhibition of growth of
test organisms (bacterial & fungal) in presence of test material
and standard was measured with the help of standard scale and
the mean values of inhibition zones are reported in Table 1.
3.3. Antioxidant activity
3.3.1. General free radical scavenging-DPPH assay
The antioxidant potential of any compound can be determined
on the basis of its capacity to trap the stable 1, 1-diphenyl-2-pieryl
hydrazyl (DPPH) free radical [23,24]. The antioxidant activity by
this method is measured as the decrease in the absorbance of DPPH
at 517 nm resulting from the colour change from purple to yellow.
The decrease in absorbance is because of formation of stable mol-
ecule of DPPH on reaction with an antioxidant through donation of
hydrogen or electron by an antioxidant. The free electron on DPPH
radical is responsible for giving absorbance peak at 517 nm and ap-
pears purple in colour. The antioxidant agent pair up through
donation of electron or release of hydrogen with the free electron
on DPPH radical and form stable molecule of DPPH–H. The change
of colour from purple to yellow is attributed to decrease of molar
absorptivity of DPPH radical when odd electron of DPPH pair up
with the antioxidant agent. The resulting decrease in colour is also
stiochiometric with number of electrons captured. Antiradical
activity of Compounds was performed by DPPH model. Stock solu-
tion of DPPH (1.3 mg/ml) in methanol was prepared. Stock solution
3.2. Minimum inhibitory concentration
Minimum inhibitory concentrations (MICs) are defined as the
lowest concentration of an anti-microbial that will inhibit the vis-
ible growth of a microorganism after overnight incubation and
minimum bactericidal concentrations (MBCs) as the lowest con-
centration of antimicrobial that will prevent the growth of an
organism after subculture onto antibiotic-free media. Minimum
inhibitory concentrations (MICs) are considered the ‘gold standard’
for determining the susceptibility of organisms to antimicrobials
and are therefore used to judge the performance of all other meth-
ods of susceptibility testing [22]. MICs are used in diagnostic labo-
ratories to confirm unusual resistance, to give a definitive answer
when a borderline result is obtained by other methods of testing,
or when disc diffusion methods are not appropriate. The Minimum
Inhibitory Concentration (MIC) was determined in vitro by macro-
dilution method for four bacterial isolates. Antibiotic dilution
ranges were prepared from stock solution having concentration
of DPPH 100 ll was added in 3 ml of methanol and absorbance was
recorded at 517 nm. The various concentrations of Compounds (25,
of 1000 lg/mL as 0, 4, 8, 16, 32, 64, 128, 256, 512 (lg/mL) using
DMSO as solvent with the help of micropipette. The Muller Hinton
Agar medium was prepared using distilled water and 20 mL of it
was transferred into 50 mL test tubes, the test tubes were tightly
plugged with cotton and sterilized in autoclave at 15 lb/in2 for
15 min as directed by the manufacturer. The inoculums was pre-
pared by transferring the bacterial colonies with the help of sterile
loop into the freshly prepared and autoclaved 1 mL of Muller Hin-
ton Broth under laminar flow and were taken for incubation for
24 h at 37 0.5 °C. The visible turbity of these organism suspen-
sions were matched with 0.5 Mc Farland standard (0.5 mL of
0.048 M BaCl2 in 99.5 mL of 0.18 M H2S04) by measuring the absor-
bance of organism suspension and Mc Farland so that suspension
should contain 107–108 CFU/mL. From the bacterial suspensions
50, 75, 100, 125 mg/ml) were prepared. All sample solutions 1 ml
each is diluted to 3 ml and 100 ll of stock solution of DPPH was
added then absorbance were recorded at 517 nm.
% Inhibition ¼ ½Blank ꢁ Testꢂ=Blank ꢀ 100
4. Results and discussion
4.1. Chemistry
Present study was undertaken to synthesize some novel
D-ring oxime derivatives of chalcones of 20-keto pregnenolone to

I.H. Lone et al. / Steroids 78 (2013) 945–950
949
investigate their probable antimicrobial and antioxidant effects.
Target compounds were obtained in two step reaction, in first
step pregnenolone was converted into corresponding chalcone
with different aldehydes. In the second step the chalcones on
reaction with hydroxylamine hydrochloride got converted into
corresponding oximes which were isolated and recrystallized
from ethylacetate. The preparation of the latter involves the fol-
lowing synthetic approach (Scheme 1). Most products were
found to be homogeneous by TLC (ethylacetae:hexane) and 1H
NMR analyses, but when needed, heterogeneous products were
readily purified by silica gel column chromatography using eth-
ylacetate/hexane eluent. The structure of all the compounds was
confirmed using NMR, IR, mass spectrometry, and elemental
analysis.
antimicrobial activity of afore mentioned oxime derivatives were
enhanced against some or all bacterial and fungal strains used
for testing, this can be attributed to the incorporation of oxime
group (@NOH) in the core molecule of steroidal chalcone, thus
the aim of incorporating oxime group in the steroidal chalcone to
enhance its biological properties proved to be successful. Among
the oxime derivatives, the compounds 4a and 4j were found more
potent against Bacillus subtilis, similar behavior were observed in
other oxime derivatives as well, 4a and 4b against Staphylococcus
epidermidis,4f against Proteus vulgaris,4c and 4i against Pseudomo-
nas aeruginosa, 4b, 4d, 4h and 4i against Aspergillus niger. The com-
pounds 4d, 4e, 4f, and 4i showed the highest inhibition against
Penicillium chrysogenum.
SAR studies on these oxime derivatives revealed that the
oxime derivatives of steroids were more potent than its chal-
cones. Both electron donating and electron withdrawing groups
on the aromatic nucleus had no remarkable effect on the antimi-
crobial properties of oximes. Since the compound exist in two
isomeric forms i,e syn- and anti- isomers, However, only one iso-
mer formed predominantly may be due to the bulky nature of
steroid nucleus and the methyl group present at D-ring of the
steroid molecule.
Entry
4a
Nature of R
Entry
4f
Nature of R
NO2
4b
4g
OCH3
OCH3
CH3
N
CH3
OCH3
4c
4h
4i
4.2.2. Minimum inhibitory concentration
F
The MIC of the synthesized compounds was determined against
two gram positive and two gram negative bacteria and two fungal
strains using macrodilution method. Table 2 summarizes the
in vitro susceptibilities of all the types of isolates against all the
synthesized test steroidal oximes. Evaluation of MIC showed that
all the test compounds were active in vitro against all the tested
microorganisms with varying degrees of inhibition except P. vulga-
ris, within the reference range. Our study revealed that all the com-
pounds had stronger antibacterial activity against gram positive
bacteria when compared to gram negative bacteria, which may
be due to the presence of the lipopolysaccharides in the outer
membrane of the gram-negative bacteria, which may hinder the
penetration of these oximes into the cells. As already demon-
strated, our results also showed that the activity of the tested
agents against each microbe varies due to structural differences
between the microorganisms. It was interesting to observe that
the gram negative P. vulgaris bacteria did not show susceptibility
F
4d
OCH3
H3CO
4e
4j
Cl
Br
4.2. Biology
4.2.1. Antimicrobial activity
The antimicrobial activity of the synthesized compounds was
determined against some bacterial and fungal strains. Table 1 sum-
marizes the in vitro activity of both the types of microbial strains
against all the synthesized test chalconyl oximes. Evaluation of
antimicrobial activity showed that all the test compounds were ac-
tive in vitro against all the tested microorganisms with varying de-
grees of inhibition. The oxime derivatives showed enhanced
antimicrobial activity than the reported chalcones of pregneno-
lone. The substituents on aromatic ring in 4a, 4e, 4f, 4g and 4h
were same as chalcones of the reported paper [25], However, the
against any of the steroidal oxime at the concentration 6512 lg/
mL.
4.2.3. Antioxidant activity
The in vitro antioxidant activity and scavenging effects of the
steroidal oximes were evaluated by using different reactive species
assay containing DPPH radical scavenging activity. The free radical
scavenging activity of all the steroidal oximes (4a–j) were
Table 2
MIC values (
l
g/mL) of steroidal oximes against bacterial and fungal strains.
Compound
Bacteria
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
B. subtilis
S. epidermidis
P. vulgaris
664
6128
664
>512
6128
664
6128
6128
>512
6128
664
6128
664
>512
6512
664
6128
6256
>512
6128
6128
664
>512
6256
6512
6128
>512
6512
6128
>512
6256
664
6128
>512
6256
6128
>512
6256
>512
6256
P. aeruginosa
Fungi
A. niger
P. chrysogenum
6128
6128
664
6128
6128
664
6128
6128
6128
6256
–
6256
664
6128
6256
6256
6256
6256
–
6128
Bacillus subtilis (MTCC 619), Staphylococcus epidermidis (MTCC 435), Proteus vulgaris (MTCC 426), Pseudomonas aeruginosa (MTCC 424).A. niger (MTCC 1344),
P.chrysogenum (MTCC 947)
Table gives the MIC data obtained after treating different bacterial and fungal strains against test doses of the different steroidal chalcones and the values are reported in
terms of lg.

950
I.H. Lone et al. / Steroids 78 (2013) 945–950
Table 3
Antioxidant activity (DPPH assay data) of compounds 4a–4j.
Compound
% inhibition
25 g/ml
l
50
lg/ml
75
lg/ml
100
lg/ml
125 lg/ml
4a
4b
4c
4d
4e
4f
4g
4h
6.14 0.02
10.87 0.04
8.99 0.05
9.56 0.01
13.41 0.04
12.56 0.06
11.24 0.01
16.88 0.08
11.15 0.0
12.78 0.01
–
9.43 0.03
13.64 0.01
14.18 0.06
15.75 0.09
21.68 0.01
19.96 0.02
16.81 0.06
22.22 0.07
15.20 0.0
16.43 0.04
–
12.17 0.02
18.43 0.06
28.90 0.06
26.61 0.09
28.17 0.08
24.41 0.04
22.18 0.01
32.28 0.02
22.39 0.0
20.21 0.03
–
19.22. 0.01
23.19 0.02
33.63 0.08
40.89 0.06
38.56 0.03
37.42 0.01
34.76 0.03
41.23 0.08
30.13 0.0
27.61 0.09
–
24.23 0.01
27.51 0.03
40.29 0.06
51.27 0.08
56.59 0.09
48.80 0.04
45.85 0.01
58.24 0.01
38.13 0.04
35.72 0.05
–
4i
4j
Control
Standard
26.73 5
l
g/ml
48.63 10
l
g/ml
56.46 15
l
g/ml
84.10 20
l
g/ml
96.27 25 lg/ml
1. Values represent the mean standard error mean (SEM) of three experiments.
2. (–): No inhibition, standard: ascorbic acid.
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attributed to the field effects or can be associated with the steroid
nucleus. The potencies for the antioxidant activity of the test com-
pounds to the reference drug are shown in Table 3.
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