Rapid microwave assisted synthesis and antimicrobial bioevaluation of novel steroidal chalcones

Article abstract of DOI:10.1016/j.steroids.2012.12.003

A novel class of chalconoyl pregnenolones has been prepared via Claisen-Schmidt condensation under microwave activation and solvent free reaction conditions. The compounds were screened for antimicrobial activity against two bacterial strains Bacillus subtilis and Escherichia coli and two fungal strains Aspergillus niger and Candida albicans. Some of the compounds exhibited significant inhibitory activity against the microbial strains. Presence of the α,β-unsaturated carbonyl moiety in the synthesized compounds was found to be essential for the activity as manipulation of the same through epoxidation of the double bond diminished the activity.

Full text of DOI:10.1016/j.steroids.2012.12.003

Steroids 78 (2013) 321–326
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Rapid microwave assisted synthesis and antimicrobial bioevaluation of novel
steroidal chalcones
b
b
a
a,
Dwipen Kakati a, , Rupak K. Sarma , Ratul Saikia , Nabin C. Barua , Jadab C. Sarma
⇑
⇑
a Natural Products Chemistry Division, CSIR North East Institute of Science & Technology, Jorhat, Assam, India
b Biotechnology Division, CSIR North East Institute of Science & Technology, Jorhat, Assam, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of chalconoyl pregnenolones has been prepared via Claisen–Schmidt condensation under
microwave activation and solvent free reaction conditions. The compounds were screened for antimicro-
bial activity against two bacterial strains Bacillus subtilis and Escherichia coli and two fungal strains Asper-
gillus niger and Candida albicans. Some of the compounds exhibited significant inhibitory activity against
Received 3 September 2012
Received in revised form 30 November 2012
Accepted 16 December 2012
Available online 31 December 2012
the microbial strains. Presence of the
a,b-unsaturated carbonyl moiety in the synthesized compounds
was found to be essential for the activity as manipulation of the same through epoxidation of the double
bond diminished the activity.
Keywords:
Chalcone
Pregnenolone
Antibacterial
Ó 2013 Elsevier Inc. All rights reserved.
Antifungal
Claisen–Schmidt condensation
1. Introduction
Literature studies reveal that, the nature of substituents on the
phenyl rings and their conformation in the core structure play a
The occurrences of life-threatening fungal infections have con-
tinued to rise over the last two decades, particularly within popu-
lations of immune compromised individuals [1]. Bacterial
infections are caused by multidrug resistant Gram-positive and
Gram-negative pathogens which affect millions of people in the
subtropical regions of the world and 20,000 deaths every year
are due to these parasitic bacterial infections [2]. Different antibi-
otics are extensively used for the treatment of these infectious dis-
eases but with commonly increasing multidrug resistant microbial
strains there is a constant need for the development of novel anti-
microbial agents.
Steroids find widespread application as anti-inflammatory,
diuretic, anabolic, contraceptive, antiandrogenic, progestational
and anticancer agents [3]. Steroid based compounds turn out to
be nontoxic, less vulnerable to multi-drug resistance (MDR) and
highly bioavailable because of being capable of penetrating the cell
wall [4].
vital role in determining their activities. In some specific study it
was found that variations leading to conformational changes like
epoxidation or substitution on the double bond result in a
decreased bioactivity [9].
O
H
H
H
HO
O
1
2
Pregnenolone (2) is a naturally occurring neurosteroid that is
synthesized from cholesterol in the adrenal gland and the central
nervous system known as a precursor to other hormones, including
cortisone, oestrogen, testosterone and progesterone [10–11]. It is
used in the synthesis of anti-depressive drugs [12] and its deriva-
tives and analogues were prepared and evaluated for different bio-
logical activities [13].
The coupling of two or more natural products to make hybrids
leads to an almost inexhaustible reservoir of new types of
compounds with diverse structures [14]. The underlying expecta-
tion being that a combination of structural features of two or more
functionally active substances into one molecule or their covalent
Chalcones (1,3-diphenyl-2-propen-1-one) are well known for
their diverse array of bioactivities. In particular, they have been
reported to possess pharmacological activities like anticancer, anti-
malarial, anti-inflammatory [5], anti-tubercular, cytotoxic, gastro-
protective [6] and antimicrobial activities [7–8]. Chalcone
structure (1) consists of three important components, viz. two
phenyl rings and an a,b-unsaturated carbonyl system joining them.
⇑
Corresponding authors. Tel.: +91 9678004037; fax: +91 0376 2370011.
E-mail address: dwipen_kakati@yahoo.co.in (D. Kakati).
0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.12.003

322
D. Kakati et al. / Steroids 78 (2013) 321–326
coupling may either enhance or modulate the desired characteris-
tics of individual components or lead to new types of properties
[15].
wave reactor and irradiated for the stipulated time at 250 W
power. Maximum internal temperature observed was 88–95 °C
and pressure 5.5–5.7 bar. After cooling to room temperature, ethyl
acetate was (15 mL) added to the RM and washed with Na2S2O3
solution (15 mL Â 1) followed by H2O (2 Â 15 mL). The separated
organic layer was dried over anhydrous sodium sulphate and con-
centrated in reduced pressure. Products were purified by column
chromatography over silica gel (100–200 mesh) with ethyl acetate
and hexane as the mobile phase.
Working close to the realm of hybrid molecules, we prepared a
novel class of modified steroids or chalcone analogues or chalco-
noyl pregnenolones using pregnenolone acetate as the steroid pre-
cursor. The 17-acetyl group of pregnenolone acetate was used to
form the enol using our own method of Claisen–Schmidt conden-
sation using I2-Al2O3 [6] to condense with different aldehydes in
order to synthesize a novel class of chalconoyl pregnenolones. Dur-
ing the preparation of this manuscript, Banday et al. [4] reported
the synthesis of 17-chalconoyl derivatives of pregnenolone with
their antimicrobial bioevaluation. We report here the synthesis of
another novel class of steroidal chalcones using pregnenolone ace-
tate with potent antimicrobial activities.
2.2.3.1.
(E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-phenyl-2-
propen-1-one (5). Reaction time: 6 min. Brown solid (86%); mp
171–173 °C; IR (KBr) 1732, 1681, 1631, 1605, 1366, 1248,
1037 cmÀ1 1H NMR (CDCl3, 300 MHz) d 7.61–7.52 (m, 3H), 7.40–
;
7.38 (m, 3H), 6.81 (d, J = 15.9 Hz, 1H), 5.39 (d, J = 4.56 Hz, 1H),
4.65–4.55 (m, 1H), 2.90–2.84 (m, 1H), 2.34 (d, J = 6.9 Hz, 2H),
2.04 (s, 3H), 1.88 (d, J = 11.2 Hz, 2H), 1.62–1.50 (m, 5H), 1.28–
1.25 (m, 6H), 1.01 (s, 3H), 0.63 (s, 3H); 13C NMR (CDCl3, 75 MHz)
d 200.36, 170.58, 141.5, 139.6, 134.8, 130.3, 128.9, 128.3, 126.8,
122.3, 73.8, 62.0, 57.1, 49.9, 44.9, 39.0, 38.0, 36.9, 36.6, 31.9,
27.7, 26.7, 24.6, 22.7, 21.0, 19.3, 13.4. MS (ESI) m/z 447 (M + H)+.
Anal. Calcd. for C30H38O3: C, 80.68; H, 8.58. Found: C, 80.64; H,
8.55.
2. Experimental
2.1. General remarks
All commercially available chemicals and reagents were pur-
chased from Aldrich and used without further purification. Melting
points were determined with a Buchi B 540 apparatus in open cap-
illaries and are uncorrected. IR spectra were recorded on a Perkin-
Elmer 1640 FT-IR instrument. The 1H- and 13C NMR spectra were
recorded on a Bruker DPX-300 NMR machine using CDCl3 as sol-
vent with TMS as the internal standard. Mass spectra were re-
corded with a Trace DSQ GCMS system. Elemental analyses were
carried out using a Perkin-Elmer series II CSNS/O Model 2400
analyser.
2.2.3.2. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(4-methoxy-
phenyl)-2-propen-1-one (6). Reaction time: 7 min. Brown solid
(82%); mp 179 °C. IR (KBr) 2941, 1731, 1677, 1598, 1511, 1251,
1035 cmÀ1 1H NMR (CDCl3, 300 MHz) d 7.55–7.52 (m, 2H), 7.50
;
(d, J = 1.38 Hz, 1H), 6.93–6.89 (m, 2H), 6.69 (d, J = 15.9 Hz, 1H),
5.39 (d, J = 4.62 Hz, 1H), 4.66–4.55 (m, 1H), 3.84 (s, 3H), 2.88–
2.82 (m, 1H), 2.37–2.31 (m, 3H), 2.05–1.98 (m, 5H), 1.88 (d,
J = 9.96 Hz, 2H), 1.75–1.46 (m, 12H), 1.28–1.25 (s, 3H), 0.63 (s,
3H); 13C NMR (CDCl3, 75 MHz) d 200.3, 170.6, 161.4, 141.3,
139.6, 129.9, 127.4, 124.7, 122.4, 114.3, 73.8, 61.9, 57.1, 55.4,
49.9, 44.9, 39.0, 38.0, 36.9, 36.6, 31.9, 31.8, 27.7, 24.6, 22.7, 21.4,
21.0, 19.3, 13.4. MS (ESI) m/z 477 (M + H)+. Anal. Calcd. for
2.2. Chemistry
2.2.1. Preparation of the catalyst
The catalyst was prepared as reported earlier [6].
C31H40O4: C, 78.11; H, 8.46. Found: C, 78.09; H, 8.43.
2.2.2. Pregnenolone acetate (3)
Pregnenolone acetate (3) was prepared by chemoselective cata-
lytic hydrogenation of 16-DPA over Pd-charcoal. 16-DPA (2.0 g,
5.61 mmol) was dissolved with brief warming in a minimum vol-
ume of absolute ethanol (70 mL). 10% Pd-C (0.2 g) was added to
it and subjected to hydrogenation at 45 psi. The reaction was mon-
itored by taking 1H NMR of the reaction mixture at regular inter-
vals. Disappearance of the signal for 16-CH proton at d 6.72
indicated the complete reduction of the 16–17 carbon–carbon dou-
ble bond after 3.5 h. After separating the Pd-C by filtration, ethanol
was distilled off at reduced pressure to obtain 3 in the form of pink
crystals which was used in the synthesis of compounds 5–20 with-
out further purification. Yield: 1.96 g, 98%; mp 132–133 °C. IR (KBr)
2.2.3.3. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(4-chloroph
enyl)-2-propen-1-one (7). Reaction time: 6 min. Brown solid
(83%); mp 126–127 °C; IR (KBr)1732, 1680, 1591, 1511,
1058 cmÀ1 1H NMR (CDCl3, 300 MHz) d 7.52–7.46 (m, 3H), 7.37–
;
7.34 (m, 2H), 6.77 (d, J = 15.96 Hz, 1H), 5.38 (d, J = 4.14 Hz, 1H),
4.65–4.55 (m, 1H), 2.57–2.51 (m, 1H), 2.33 (d, J = 6.94 Hz, 2H),
2.12 (s, 3H), 1.89 (d, J = 10.1 Hz, 2H), 1.69–1.50 (m, 9H), 1.47 (d,
J = 9.06 Hz, 2H), 1.25–1.15 (m, 4H), 1.02 (s, 3H), 0.63 (s, 3H); 13C
NMR (CDCl3, 75 MHz) d 200.0, 170.5, 140.0, 139.6, 136.1, 133.2,
129.4, 127.1, 122.3, 73.8, 63.6, 56.7, 49.8, 43.9, 38.7, 38.0, 36.9,
36.5, 31.9, 31.78, 31.73, 31.5, 27.7, 24.4, 22.7, 21.0, 19.2, 13.2. MS
(ESI) m/z 482 (M + H)+. Anal. Calcd. for C30H37ClO3: C, 74.90; H,
7.75. Found: C, 74.87; H, 7.73.
;
1732, 1698, 1631, 1366, 1248, 1036 cmÀ1 1H NMR (CDCl3,
300 MHz) d 5.38 (d, J = 4.95 Hz, 1H), 4.64–4.55 (m, 1H), 2.57–2.51
(m, 1H), 2.33 (d, J = 7.08 Hz, 2H), 2.12 (s, 3H), 2.03 (s, 3H), 1.89
(d, J = 9.89 Hz, 2H), 1.68–1.53 (m, 11H), 1.2–1.15 (m, 4H), 1.02 (s,
3H), 0.63 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 209.6, 170.5, 139.6,
122.3, 73.8, 63.6, 56.8, 49.8, 43.9, 38.7, 38.0, 36.9, 36.5, 31.7,
31.5, 27.7, 24.4, 22.8, 21.4, 21.0, 19.2, 13.2; MS (ESI): m/z 359
(M + H)+; Anal. Calcd. for C23H34O3: C, 77.05; H, 9.56; Found C,
77.01; H, 9.53.
2.2.3.4. 1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(2-bromophenyl)
-2-propen-1-one (8). Reaction time: 5 min. White solid (86%); mp
64.5 °C; IR (KBr) 1732, 1686, 1631, 1606, 1246, 1026 cmÀ1; 1H NMR
(CDCl3, 300 MHz) d 7.91 (d, J = 16.02 Hz, 1H), 7.63 (d, J = 7.9 Hz, 2H),
7.35–7.30 (m, 1H), 7.23–7.20 (m, 1H), 6.72 (d, J = 15.96 Hz, 1H), 5.39
(d, J = 4.35 Hz, 1H), 4.65–4.59 (m, 1H), 2.98–2.93 (m, 1H), 2.34 (d,
J = 7.23 Hz, 2H), 2.03 (s, 3H), 1.88 (d, J = 10.29 Hz, 2H), 1.74–1.58 (m,
9H), 1.51 (d, J = 9.96 Hz, 2H), 1.29–1.25 (m, 4H), 1.01 (s, 3H), 0.65 (s,
3H); 13C NMR (CDCl3, 75 MHz) d 200.3, 170.6, 140.1, 139.6, 134.8,
133.5, 131.1, 129.8, 127.7, 125.8, 122.3, 73.8, 61.2, 57.0, 49.9, 44.9,
39.2, 38.07, 36.9, 36.6, 35.5, 31.9, 31.8, 27.7, 24.7, 22.9, 21.0, 19.3,
13.3. MS (ESI) m/z 526 (M + H)+. Anal. Calcd. for C30H37BrO3: C,
68.57; H, 7.10. Found: C, 68.55; H, 7.08.
2.2.3. General experimental procedure for the synthesis of compounds
5–20
Pregnenolone acetate (3, 0.2 g, 0.557 mmol), benzaldehyde
(0.557 mmol) and I2-Al2O3 (20 mol% I2 as 10% I2 in Al2O3) were ta-
ken and homogenized in a mortar. Then the reaction mixture was
transferred to a reaction vessel of Anton Paar Synthos 3000 micro-

D. Kakati et al. / Steroids 78 (2013) 321–326
323
2.2.3.5. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(4-fluorophe
nyl)-2-propen-1-one (9). Reaction time: 6 min. Brown solid (84%);
mp 151–152 °C, IR (KBr) 1729, 1682, 1599, 1509, 1234,
62.0, 57.1, 49.9, 44.8, 39.0, 38.8, 36.7, 31.9, 31.8, 27.7, 24.6, 22.7,
21.0, 19.3, 13.4. MS (ESI) m/z 501 (M + H)+.
1159 cmÀ1
;
1H NMR (CDCl3, 300 MHz) d 8.01 (d, J = 15.6 Hz, 1H),
2.2.3.10. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(4-hydroxy
phenyl)-2-propen-1-one (14). Reaction time: 7 min. Thick brown oil
(82%); IR (CHCl3) 3390, 1735, 1686, 1631, 1556, 1366, 1248,
7.78–7.75 (m, 2H), 7.72–7.62 (m, 3H), 5.38 (d, J = 4.57 Hz, 1H),
4.65–4.54 (m, 1H), 2.90–2.84 (m, 1H), 2.33 (d, J = 6.89 Hz, 2H),
2.04 (s, 3H), 1.88 (d, J = 11.1 Hz, 2H), 1.73–1.54 (m, 9H), 1.52 (d,
J = 8.68 Hz, 2H), 1.28–1.25 (m, 4H), 1.01 (s, 3H), 0.63 (s, 3H); 13C
NMR (CDCl3, 75 MHz) d 200.3, 170.5, 141.2, 139.1, 136.7, 132.1,
129.1, 126.6, 122.7, 122.4, 73.8, 62.5, 57.08, 49.9, 45.3, 39.1, 38.0,
36.7, 36.4, 31.8, 31.7, 27.7, 24.6, 22.6, 21.0, 19.3, 13.5. MS (ESI)
m/z 465 (M + H)+. Anal. Calcd. for C30H37FO3: C, 77.55; H, 8.03.
Found: C, 77.53; H, 8.01.
1037 cmÀ1 1H NMR (CDCl3, 300 MHz) d 8.08 (d, J = 7.75 Hz, 2H),
;
7.79 (d, J = 15.5 Hz, 1H), 7.65–7.55 (m, 2H), 6.88 (d, J = 15.9 Hz,
1H), 5.39 (d, J = 4.35 Hz, 1H), 4.65–4.57 (m, 1H), 2.98–2.91 (m,
1H), 2.33 (d, J = 10.1 Hz, 2H), 2.03 (s, 3H), 1.89 (d, J = 10.1 Hz,
2H), 1.73–1.57 (m, 9H), 1.54 (d, J = 9.3 Hz, 2H), 1.28–1.23 (m,
4H), 1.02 (s, 3H), 0.63 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 200.1,
170.5, 156.6, 141.5, 139.6, 132.7, 127.8, 126.3, 122.2, 117.2, 73.7,
62.0, 57.1, 49.9, 44.8, 39.2, 38.0, 36.9, 36.7, 32.0, 31.9, 27.7, 24.6,
22.7, 21.0, 19.3, 13.4. MS (ESI) m/z 463 (M + H)+.
2.2.3.6. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(4-nitrophen
yl)-2-propen-1-one (10). Reaction time: 7 min. Yellow solid (79%);
mp 184–185 °C, IR (CHCl3) 1732, 1704, 1510, 1436, 1326, 1242,
2.2.3.11. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(4-hydroxy
3-methoxyphenyl)-2-propen-1-one (15). Reaction time: 7 min. Yel-
low oil (84%); IR (KBr) 3340, 1731, 1680, 1599, 1511, 1251,
1036 cmÀ1 1H NMR (CDCl3, 300 MHz) d 8.26 (d, J = 8.64 Hz, 2H),
;
7.71 (d, J = 8.55 Hz, 2H), 7.58 (d, J = 15.96 Hz, 1H), 6.89 (d,
J = 15.9 Hz, 1H), 5.40 (d, J = 5.13 Hz, 1H), 4.62–4.58 (m, 1H), 2.66–
2.60 (m, 1H), 2.34 (d, J = 4.11 Hz, 2H), 2.03 (s, 3H), 1.88 (d,
J = 9.96 Hz, 2H), 1.69–1.52 (m, 9H), 1.43–1.38 (m, 2H), 1.28–1.13
(m, 4H), 1.01 (s, 3H), 0.65 (s, 3H); 13C NMR (CDCl3, 75 MHz) d
199.87, 170.6, 141.0, 139.6, 138.3, 130.2, 130.1, 128.8, 124.1,
123.3, 122.3, 73.8, 62.5, 57.0, 49.9, 45.2, 39.1, 38.0, 36.9, 31.9, 31.7,
27.7, 24.6, 22.6, 21.0, 19.3, 13.5. MS (ESI) m/z 492 (M + H)+. Anal.
Calcd. for C30H37NO5: C, 73.29; H, 7.59; N, 2.85. Found: C, 73.26; H,
7.57; N, 2.84.
1042 cmÀ1 1H NMR (CDCl3, 300 MHz) d 7.81 (d, J = 15.7 Hz, 1H),
;
7.32–7.26 (m, 4H), 5.38 (d, J = 4.41 Hz, 1H), 4.62–4.58 (m, 1H),
3.8 (s, 3H), 2.87–2.82 (m, 1H), 2.34 (d, J = 6.46 Hz, 2H), 2.04 (s,
3H), 1.88 (d, J = 9.8 Hz, 2H), 1.75–1.54 (m, 9H), 1.48 (d,
J = 11.0 Hz, 2H), 1.41–1.29 (m, 4H), 1.04 (s, 3H), 0.63 (s, 3H); 13C
NMR (CDCl3, 75 MHz) d 200.1, 170.1, 147.5, 141.2, 140.2, 139.0,
131.4, 127.1, 126.6, 120.9, 117.7, 113.4, 73.8, 62.0, 57.1, 56.0,
49.9, 44.9, 39.0, 38.0, 36.9, 36.6, 31.9, 27.7, 26.7, 24.6, 22.7, 21.0,
19.3, 13.4. MS (ESI) m/z 493 (M + H)+.
2.2.3.12. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(3,4-dihydr
oxyphenyl)-2-propen-1-one (16). Reaction time: 6 min. Brown oil
(81%); IR (KBr) 3410, 1737, 1683, 1636, 1556, 1361, 1248,
2.2.3.7. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(4-bromoph
enyl)-2-propen-1-one (11). Reaction time: 6 min. White solid (85%);
mp 99–100 °C; IR (CHCl3) 1736, 1682, 1630, 1601, 1243, 1029 cmÀ1
;
1037 cmÀ1 1H NMR (CDCl3, 300 MHz) d 8.01–7.95 (m, 2H), 7.81
;
1H NMR (CDCl3, 300 MHz) d 7.57–7.49 (m, 3H), 7.11–7.02 (m, 2H),
6.73 (d, J = 15.9 Hz, 1H), 5.39 (d, J = 4.44 Hz, 1H), 4.62–4.59 (m,
1H), 2.87–2.83 (m, 1H), 2.34 (d, J = 6.45 Hz, 2H), 2.04 (s, 3H), 1.87
(d, J = 9.9 Hz, 2H), 1.73–1.49 (m, 9H), 1.47 (d, J = 11.4 Hz, 2H),
1.39–1.25 (m, 4H), 1.04 (s, 3H), 0.63 (s, 3H); 13C NMR (CDCl3,
75 MHz) d 200.1, 170.5, 164.4, 141.1, 139.5, 134.7, 127.7, 127.6,
122.3, 115.3, 115.1, 73.6, 61.2, 57.1, 49.9, 44.9, 39.1, 38.0, 37.1,
36.8, 35.6, 31.8, 27.8, 24.6, 22.9, 21.0, 19.3, 13.5. MS (ESI) m/z 526
(M + H)+. Anal. Calcd. for C30H37BrO3: C, 68.57; H, 7.10. Found: C,
68.54; H, 7.07.
(d, J = 15.9 Hz, 1H), 7.19–7.14 (m, 2H), 5.39 (d, J = 4.4 Hz, 1H),
4.61–4.58 (m, 1H), 2.88–2.83 (m, 1H), 2.34 (d, J = 6.46 Hz, 2H),
2.04 (s, 3H), 1.87 (d, J = 9.8 Hz, 2H), 1.75–1.53 (m, 9H), 1.49 (d,
J = 10.9 Hz, 2H), 1.40–1.28 (m, 4H), 1.04 (s, 3H), 0.63 (s, 3H); 13C
NMR (CDCl3, 75 MHz) d 200.1, 170.5, 146.5, 145.2, 141.5, 139.1,
138.1, 123.3, 122.3, 119.4, 114.1, 73.8, 61.9, 57.0, 49.9, 44.9, 39.1,
38.0, 36.9, 36.6, 31.9, 31.8, 27.7, 24.6, 22.7, 21.0, 19.3, 13.4. MS
(ESI) m/z 479 (M + H)+.
2.2.3.13. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(2,4-dichlo
rophenyl)-2-propen-1-one (17). Reaction time: 6 min. Brown gum
(83%); IR (CHCl3) 1732, 1681, 1590, 1513, 1062 cmÀ1 1H NMR
;
2.2.3.8. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(2,4-difluoro
phenyl)-2-propen-1-one (12). Reaction time: 6 min. Brown solid
(CDCl3, 300 MHz) d 7.61 (d, J = 15.9 Hz, 1H), 7.54 (d, J = 6.8 Hz,
1H), 6.86–6.81 (m, 2H), 5.39 (d, J = 4.21 Hz, 1H), 4.61–4.57 (m,
1H), 2.84–2.81 (m, 1H), 2.34 (d, J = 6.29 Hz, 2H), 2.03 (s, 3H), 1.89
(d, J = 10.9 Hz, 2H), 1.75–1.70 (m, 9H), 1.64 (d, J = 4.81 Hz, 2H),
1.56–1.51 (m, 4H), 1.01 (s, 3H), 0.63 (s, 3H); 13C NMR (CDCl3,
75 MHz) d 200.7, 170.6, 141.1, 139.0, 136.1, 136.3, 131.4, 129.7,
127.1, 126.3, 122.1, 73.8, 62.0, 57.2, 49.9, 44.9, 39.0, 38.0, 36.9,
31.9, 31.8, 27.8, 24.6, 22.7, 21.0, 19.3, 13.4. MS (ESI) m/z 516
(M + H)+.
(84%); mp 128–129 °C; IR (CHCl3) 1729, 1686, 1611, 1489 cmÀ1
;
1H NMR (CDCl3, 300 MHz) d 7.63–7.59 (m, 2H), 7.09 (s, 1H), 6.85
(d, J = 16.1 Hz, 1H), 5.39 (d, J = 5.01 Hz, 1H), 4.63–4.59 (m, 1H),
2.87–2.84 (m, 1H), 2.34 (d, J = 6.42 Hz, 2H), 2.04 (s, 3H), 1.88 (d,
J = 11.2 Hz, 2H), 1.71–1.54 (m, 9H), 1.53 (d, J = 8.5 Hz, 2H), 1.29–
1.26 (m, 4H), 1.01 (s, 3H), 0.63 (s, 3H); 13C NMR (CDCl3, 75 MHz) d
200.1, 170.1, 164.4, 162.3, 141.4, 139.1, 128.1, 126.4, 122.1, 118.1,
109.9, 73.8, 62.6, 57.08, 49.9, 45.3, 39.1, 38.2, 36.7, 31.9, 31.8, 27.7,
24.6, 22.7, 21.1, 19.3, 13.3. MS (ESI) m/z 483 (M + H)+. Anal. Calcd.
for C30H36F2O3: C, 74.66; H, 7.52. Found: C, 74.63; H, 7.49.
2.2.3.14. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(4-methyl-
phenyl)-2-propen-1-one (18). Reaction time: 6 min. Brown solid
2.2.3.9. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(2,3,5-trifluo
rophenyl)-2-propen -1-one (13). Reaction time: 6 min. Brown liquid
(84%); mp 171–173 °C; IR 3050, 2916, 1655, 1597 cmÀ1 1H NMR
;
(CDCl3, 300 MHz) d 8.02–7.96 (m, 2H), 7.81 (d, J = 15.5 Hz, 1H),
7.60–7.46 (m, 4H), 5.38 (d, J = 4.58 Hz, 1H), 4.64–4.55 (m, 1H),
2.90–2.84 (m, 1H), 2.40 (s, 3H), 2.34 (d, J = 6.9 Hz, 2H), 2.03 (s,
3H), 1.88 (d, J = 11.2 Hz, 2H), 1.71–1.55 (m, 9H), 1.51 (d,
J = 8.67 Hz, 2H), 1.29–1.25 (m, 4H), 1.01 (s, 3H), 0.63 (s, 3H); 13C
NMR (CDCl3, 75 MHz) d 200.1, 170.3, 141.1, 139.4, 135.7, 136.2,
131.1, 130.5, 127.1, 126.9, 122.1, 73.8, 61.9, 57.1, 49.9, 44.9, 39.1,
38.3, 36.9, 36.6, 31.9, 31.7, 27.7, 24.6, 22.7, 21.2, 21.0, 19.3, 13.4.
(84%); IR (CHCl3) 1736, 1688, 1615, 1491, 1145 cmÀ1 1H NMR
;
(CDCl3, 300 MHz) d 7.79 (d, J = 16.0 Hz, 1H), 7.57 (s, 1H), 7.02 (s,
1H), 6.78 (d, J = 16.0 Hz, 1H), 5.39 (d, J = 4.24 Hz, 1H), 4.62–4.59
(m, 1H), 2.85–2.83 (m, 1H), 2.34 (d, J = 6.37 Hz, 2H), 1.74–1.70
(m, 9H), 1.64 (d, J = 4.9 Hz, 2H), 1.57–1.50 (m, 4H), 1.01 (s, 3H),
0.63 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 200.8, 170.6, 158.1,
151.5, 145.2, 141.5, 139.5, 126.3, 122.3, 121.2, 114.3, 103.1, 73.8,

324
D. Kakati et al. / Steroids 78 (2013) 321–326
MS (ESI) m/z 461 (M + H)+. Anal. Calcd. for C31H40O3: C, 80.83; H,
8.75. Found: C, 80.80; H, 8.71.
IR (CHCl3) 1729, 1628, 1601, 1511, 1251, 1230, 1159,
1038 cmÀ1 1H NMR (CDCl3, 300 MHz) d 7.57–7.49 (m, 2H), 7.11–
;
7.02 (m, 2H), 5.39 (d, J = 4.44 Hz, 1H), 4.62–4.59 (m, 1H), 4.23 (d,
J = 1.75 Hz, 1H), 4.19 (d, J = 1.75 Hz, 1H), 2.87–2.83 (m, 1H), 2.34
(d, J = 6.45 Hz, 2H), 2.04 (s, 3H), 1.87 (d, J = 9.9 Hz, 2H), 1.73–1.49
(m, 9H), 1.47 (d, J = 11.4 Hz, 2H), 1.39–1.25 (m, 4H), 1.04 (s, 3H),
0.63 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 200.1, 170.5, 164.4,
139.5, 134.7, 127.7, 122.3, 115.3, 115.1, 73.6, 62.1, 61.2, 59.4,
57.1, 49.9, 44.9, 39.1, 38.0, 37.1, 36.8, 35.6, 31.8, 27.8, 24.6, 22.9,
21.0, 19.3, 13.5. MS (ESI) m/z 481 (M + H)+.
2.2.3.15. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(2,4-dimet
hoxyphenyl)-2-propen-1-one (19). Reaction time: 7 min. Brown so-
lid (83%); mp 188–189 °C; IR (KBr) 3062, 1732, 1699, 1643, 1249,
1044 cmÀ1 1H NMR (CDCl3, 300 MHz) d 8.01 (d, J = 15.7 Hz, 1H),
;
7.79–7.73 (m, 1H), 6.51–6.34 (m, 3H), 5.38 (d, J = 4.56 Hz, 1H),
4.65–4.55 (m, 1H), 3.88 (s, 3H), 3.81 (s, 3H), 2.91–2.85 (m, 1H),
2.34 (d, J = 6.9 Hz, 2H), 2.03 (s, 3H), 1.88 (d, J = 11.1 Hz, 2H), 1.72–
1.54 (m, 9H), 1.52 (d, J = 8.66 Hz, 2H), 1.29–1.26 (m, 4H), 1.01 (s,
3H), 0.63 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 200.4, 170.5, 161.2,
160.8, 141.7, 139.4, 129.3, 126.3, 116.1, 109.4, 101.0, 73.8, 62.0,
57.1, 49.9, 44.8, 39.1, 38.1, 36.9, 36.6, 31.9, 31.8, 27.7, 24.6, 22.7,
21.0, 19.3, 13.4. MS (ESI) m/z 507 (M + H)+. Anal. Calcd. for
2.3. Antimicrobial bioevaluation
2.3.1. Microorganisms and media
The antimicrobial activity of the new chalconoids of pregneno-
lone acetate was studied against fungal and bacterial pathogens.
All the pathogens, Aspergillus niger (MTCC 281), Candida albicans
(MTCC 183), Bacillus subtilis (MTCC 736) and Escherichia coli (MTCC
1692) were obtained from Microbial Type Culture Collection, IM-
TECH, Chandigarh, India.
The bacterial strains were grown in nutrient broth (NB) at
30 2 °C with continuous agitation at 200 rpm for 24 h, whereas
the fungal strains were grown in potato dextrose broth (PDB) at
25 2 °C. The compounds were solubilized in 10% dimethyl sulfox-
ide (DMSO). The bactericidal as well as fungicidal effects were
C32H42O5: C, 75.86; H, 8.36. Found: C, 75.84; H, 8.31.
2.2.3.16. (E)-1-[(3b)-3-(Acetyloxy)-androst-5-en-17-yl]-3-(2-nitrophe
nyl)-2-propen-1-one (20). Reaction time: 6 min. Brown solid (81%);
mp 143–144 °C, IR (CHCl3) 2990, 1732, 1704, 1510, 1436, 1329,
1247, 1039 cmÀ1
; d 8.14 (d,
1H NMR (CDCl3, 300 MHz)
J = 15.7 Hz, 1H), 8.08 (d, J = 7.3 Hz, 1H), 8.03–8.01 (m, 3H), 7.51
(d, J = 15.9 Hz, 1H), 4.65–4.59 (m, 1H), 2.98–2.93 (m, 1H), 2.33 (d,
J = 7.77 Hz, 2H), 2.04 (s, 3H), 1.88 (d, J = 10.27 Hz, 2H), 1.74–1.58
(m, 9H), 1.52 (d, J = 9.97 Hz, 2H), 1.28–1.25 (m, 4H), 1.01 (s, 3H),
0.65 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 200.4, 170.5, 161.2,
160.8, 141.7, 139.4, 129.3, 126.3, 116.1, 109.4, 101.0, 73.8, 62.0,
57.1, 56.1, 49.9, 44.8, 39.1, 38.1, 36.9, 36.6, 31.9, 31.8, 27.7, 24.6,
22.7, 21.0, 19.3, 13.4. MS (ESI) m/z 492 (M + H)+. Anal. Calcd. for
studied with 1000
ent agar (NA) and potato dextrose agar (PDA) medium, respectively
through agar well diffusion assay [17]. 100 L of compound sus-
pension and 60
L of bacterial/fungal cultures (approx. 108 colony
lg/mL solution of the compounds using nutri-
l
l
forming units [CFU]) were applied for each study. The zone of inhi-
bition was recorded after 24 h of incubation at 30 2 °C for bacte-
rial strains and after 72 h at 25 2 °C for fungal strains. Kanamycin
for bacteria and fluconazole for fungus were used as positive con-
trol with same dilution as of the compounds.
C
30H37NO5: C, 73.29; H, 7.59; N, 2.85. Found: C, 73.27; H, 7.56;
N, 2.82.
2.2.4. Epoxidation of compound 9
Compound 9 was converted to its epoxide by reacting with
H2O2 in the presence of K2CO3 (Scheme 2) [16]. Compound 9
(0.4 g, 0.86 mmol) and K2CO3 (0.356 g, 2.58 mmol) were added to
MeOH (30 mL) to get a suspension. Excess aqueous hydrogen per-
oxide (35%, 1 mL, 10 mol equiv.) was then added to the suspension
over 10 min. Then the mixture was stirred at room temperature
and reaction progress was monitored by TLC. Upon completion
(4 h), the MeOH was removed under reduced pressure and the
resulting residue was dissolved in CH2Cl2 (50 mL) and washed with
H2O (2 Â 30 mL). The organic phase was separated, dried (Na2SO4)
and was concentrated under reduced pressure. The resulting crude
oil was purified by column chromatography over silica gel (100–
200 mesh) using EtOAc/hexane as the eluent to yield the corre-
sponding epoxide (0.284 g, 0.591 mmol, 69%) as a brown oil.
Acetic acid 17-[3-(4-fluoro-phenyl)-oxiranecarbonyl]-10,13-di-
methyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-3-yl ester (21).
2.3.2. Determination of minimum inhibitory concentration
Minimum inhibitory concentration (MIC) is the lowest concen-
tration of the applied material that can inhibit the growth of an
organism. MIC was determined through batch cultures containing
different volumes of the compounds (1000
300 g/mL, 250 g/mL, 200 g/mL, 150 g/mL, 100
50 g/mL). 100 L of compound suspensions of different concen-
lg/mL, 500
lg/mL,
l
l
l
l
l
g/mL and
l
l
trations was added to 100 mL of sterile NB and PDB taken in Erlen-
meyer flask (250 mL).The flasks were then inoculated with 1 mL of
24 h old bacterial and 72 h old fungal suspension (approx. 108 CFU)
and incubated at 30 2 °C and 25 2 °C with continuous shaking
at 200 rpm for 18–24 h for bacteria and 72 h for fungus. After the
incubation period, microbial growth was observed by taking opti-
cal density (OD) at 600 nm. Microbial culture with only DMSO was
treated as control for the experiments. Kanamycin for bacteria and
5, R = H
6, R = 4-OMe
R
7, R = 4-Cl
O
O
8, R = 2-Br
9, R = 4-F
10, R = 4-NO2
11, R = 4-Br
12, R = 2,4-F
13, R = 2,3,5-F
14, R = 4-OH
R
I2-Al2O3, MW
5-7 min
H
+
O
AcO
AcO
15, R = 3-OMe, 4-OH
16, R = 3,4-OH
17, R = 2,4-Cl
5-20
4
3
18, R = 4-CH3
19, R = 2,4-OCH3
20, R = 2-NO2
Scheme 1. Synthesis of compounds 5–20.

D. Kakati et al. / Steroids 78 (2013) 321–326
325
Table 1
F
F
Antimicrobial activity of steroidal chalcones.
O
O
O
Entry
Compound
Zone of inhibition (mm)a
K2CO3, MeOH
H2O2, rt, 4h
E. coli
B. subtilis
C. albicans
A. niger
1
2
3
4
5
3
–
–
20
–
18
–
–
–
22
–
–
–
–
–
–
16
–
–
–
–
–
–
–
AcO
AcO
4 (R = 4ÀF)
9
21
5
6
7
8
Scheme 2. Conversion of 9 to its epoxide 21.
6
–
7
8
9
10
26
–
28
–
22
–
19
–
fluconazole for fungus were used as standard drugs. The value of
OD for each concentration of compound and standard was sub-
tracted from the value of control for the calculation of the killing
percentage.
9
11
12
13
14
15
16
17
18
–
–
–
–
10
11
12
13
14
15
16
17
18
19
20
22
24
–
–
–
21
–
–
–
28
–
24
25
–
–
–
–
–
–
–
20
20
–
–
–
18
–
–
–
–
17
18
–
–
–
16
–
–
–
–
3. Results and discussions
19
20
3.1. Chemistry
Kanamycin
Fluconazole
31
–
The condensation between pregnenolone acetate (3) and benz-
aldehyde was tried using I2-Al2O3 as catalyst under microwave
activation and solvent free conditions. The desired product was
isolated in 86% pure yield after 6 min of MW exposure at 250 W
of microwave power in the presence of 20 mol% catalyst. Using 3
as the ketone component sixteen novel steroidal chalcones were
prepared via its condensation with different substituted benzalde-
hyde (Scheme 1). In general, the reactions were clean and no side
products were detected. Products were isolated in good yields
(79–86%); further enhancement of the same was not possible as
increment of microwave power or reaction time or catalyst loading
resulted in the decomposition of the product formed. No signifi-
cant variation in product yield was observed with variations in
the nature of substituents in the benzaldehyde aromatic ring. In
activity screening, compound 9 was found to be the most potent
antimicrobial agent. In order to have a gist of the structural
requirements for the activity, it was converted to its epoxide using
H2O2 in the presence of K2CO3.
24
20
a
Zone of inhibitions less than 10 mm are not shown.
Table 2
MIC of the active compounds.
Entry
Compound
MIC (
l
g/mL)
B. subtilis
E. coli
C. albicans
A. niger
1
2
3
4
5
6
7
8
5
7
9
12
13
17
300
350
150
300
200
300
100
–
300
–
150
200
200
–
–
500
300
350
350
500
–
–
–
300
500
500
350
–
Kanamycin
Fluconazole
100
–
150
150
‘–’ No activity.
Results of antimicrobial activity screening indicate that the
presence of the fluoro group in the para position of the benzalde-
hyde aromatic ring enhances the activity. A comparison of the zone
of inhibition produced by the unsubstituted compound 5 and the
most active compound 9 is shown in Fig. 1. The presence of Cl
group on that position also increased the activity compared to
unsubstituted 5. However, the presence of the other halogen Br
or any other groups whether electron withdrawing or electron
donating, reduces the activity compared to 5. In addition to the
one in para position, another F in the ortho position of benzalde-
hyde or in both of the meta and one ortho position could not en-
hance the activity more than the para substituted compound. It
is also found that compounds 3 and 4 (R = 4ÀF), which are the
starting compounds in the synthesis of 9, possess no activity
against the select microbes (entry 1 and 2, Table 1). Again manip-
fies the necessity of the condensation between 3 and 4 and the
presence of the specific conformation for the bioactivity.
3.2. Biology
The synthesized compound 5, 7, 9, 12, 13 and 17 showed good
antimicrobial activity in general (Table 1). Bacterial strain E. coli
was sensitive to all of these compounds with inhibition zones vary-
ing from 18 to 26 mm. B. subtilis was found to be resistant to 7 and
17, while sensitive to samples 5, 9, 12 and 13 with the zone of inhi-
bitions from 22 to 28 mm. In the case of fungal pathogens C. albi-
cans was found to be resistant to 5 and A. niger to 5 and 7.
Compound 9 was found to be much effective against C. albicans
with the inhibition zone at 22 mm. In the case of A. niger zones
of inhibition were 19, 18, 17 and 16 mm for samples 9, 13, 12
ulation of the
a,b-unsaturation of the most active compound 9 via
its epoxidation (21), resulted in the total loss of activity. This signi-
Fig. 1. Zone of inhibition produced by compounds 5 & 9 in (a) A. niger, (b) C. albicans, (c) B. subtilis and (d) E. coli.

326
D. Kakati et al. / Steroids 78 (2013) 321–326
[2] Khan SA. Synthesis, characterization and in vitro antibacterial activity of new
and 17, respectively. MIC was determined for the active com-
pounds 5, 7, 9, 12, 13, 17 and the results are plotted in Table 2. Ex-
cept compounds 7 and 17, all have the property to inhibit the
growth of both of the bacterial strains-Gram positive B. subtilis as
well as Gram negative E. coli. Among all the compounds analysed,
compound 9 was found to be the most promising having both bac-
steroidal 5-en-3-oxazolo and thiazoloquinoxaline. Eur
2008;43:2040–4.
J
Med Chem
[3] Shan L-H, Liu H-M, Huang K-X, Dai G-F, Cao C, Dong R-J. Synthesis of 3b, 7
a,
11 -trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives and their
a
cytotoxic activities. Bioorg Med Chem Lett 2009;19:6637–9.
[4] Banday AH, Mir BP, Lone IH, Suri KA, Kumar HMS. Studies on novel D-ring
substituted steroidal pyrazolines as potential anticancer agents. Steroids
2010;75:805–9.
[5] Nowakowska Z. A review of anti-infective and anti-inflammatory chalcones.
Eur J Med Chem 2007;42:125–37.
tericidal and fungicidal activities with MIC values 150 and 300
mL, respectively.
lg/
[6] Kakati D, Sarma JC. Microwave assisted solvent free synthesis of 1,3-
diphenylpropenones. Chem Cent J 2011;5:8 [references cited therein].
[7] Cheenpracha S, Karalai C, Ponglimanont C, Subhdhirasakul S, Tewtrakul S. Anti-
HIV-1 protease activity of compounds from Boesenbergia pandurata. Bioorg
Med Chem 2006;14:1710–4.
4. Conclusions
In conclusion, we have synthesized a novel series of chalconoyl
pregnenolones for the first time under convenient reaction condi-
tions in a very short reaction time. These new steroidal compounds
possess potent antimicrobial activities against some common
[8] ElSohly HN, Joshi AS, Nimrod AC, Walker LA, Clark AM. Antifungal chalcones
from Maclura tinctoria. Planta Med 2001;67:87–9.
[9] Bhat BA, Dhar KL, Puri SC, Saxena AK, Shanmugavel M, Qazi GN. Synthesis and
biological evaluation of chalcones and their derived pyrazoles as potential
cytotoxic agents. Bioorg Med Chem Lett 2005;15:3177–80.
[10] Baulieu EE, Robel P. Neurosteroids: a new brain function? J Steroid Biochem
Mol Biol 1990;37:395–403.
[11] Choudhary MI, Alam MS, Rahman A-U, Yousuf S, Wu Y-C, Lin A-S.
Pregnenolone derivatives as potential anticancer agents. Steroids
2011;76:1554–9.
[12] Bianchi M, Baulieu EE. 3b-Methoxy pregnenolone (MAP-4343) as an
innovative therapeutic approach for depressive disorders. Proc Natl Acad Sci
USA 2012;109:1713–8.
[13] Pérez-Ornelas V, Cabeza M, Bratoeff E, Heuze I, Sánchez M, Ramírez E, et al.
New 5alpha-reductase inhibitors: in vitro and in vivo effects. Steroids
2005;70:217–24.
pathogens. Necessity of the presence of the
a,b-unsaturated car-
bonyl system in the compounds was also verified by manipulation
of the conformation via epoxidation. Further studies on structure
activity relationships and on the scope of application of the com-
pounds is going on.
Acknowledgements
The authors thank the Director, CSIR-NEIST for providing neces-
sary facilities, CSIR New Delhi for funding the project (NWP-0037),
Analytical Chemistry Division, CSIR-NEIST for recording the spectra
and the reviewers for their valuable suggestions.
[14] Magyar A, Wölfling J, Kubas M, Seijo JAC, Sevvana M, Herbst-Irmer R, Forgó P,
Schneider G. Synthesis of novel steroid-tetrahydroquinoline hybrid molecules
and
D-homosteroids by intramolecular cyclization reactions. Steroids
2004;69:301–12.
[15] Mehta G, Singh V. Hybrid systems through natural product leads: an approach
towards new molecular entities. Chem Soc Rev 2002;31:324–34.
[16] Pal S, Tak YK, Song JM. Does the antibacterial activity of silver nanoparticles
depend on the shape of the nanoparticle? A study of the Gram-negative
bacterium Escherichia coli. Appl Environ Microbiol 2007;73:1712–20.
[17] Schillinger U, Lücke FK. Antimicrobial activity of Lactobacillus sake isolated
from meat. Appl Environ Microbiol 1989;55:1901–6.
References
[1] Cammarata A, Upadhyay SK, Jursic BS, Neumann DM. Antifungal activity of
2a,3b-functionalized steroids stereoselectively increases with the addition of
oligosaccharides. Bioorg Med Chem Lett 2001;21:7379–86.