Microwave-assisted synthesis and in vitro antibacterial activity of novel steroidal thiosemicarbazone derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.09.083

Herein, we reported the synthesis of 16 novel steroidal thiosemicarbazone derivatives via the condensation of steroidal ketones and substituted thiosemicarbazides under solvent-free conditions using microwave irradiation. The yields obtained are in the ra

Full text of DOI:10.1016/j.bmcl.2012.09.083

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7730–7734
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Microwave-assisted synthesis and in vitro antibacterial activity of novel
steroidal thiosemicarbazone derivatives
⇑
Zhigang Zhao , Zhichuan Shi, Min Liu, Xingli Liu
College of Chemistry and Environmental Protection Engineering, Southwest University for Nationalities, Chengdu 610041, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2012
Revised 18 September 2012
Accepted 24 September 2012
Available online 11 October 2012
Herein, we reported the synthesis of 16 novel steroidal thiosemicarbazone derivatives via the condensa-
tion of steroidal ketones and substituted thiosemicarbazides under solvent-free conditions using micro-
wave irradiation. The yields obtained are in the range of 84–96% using microwave method and 46–62%
using conventional method. All the synthesized compounds (7a–p) have been characterized by 1H NMR,
ESI-MS, IR and elemental analyses. All the series compounds (7a–p) were evaluated for their antibacterial
activity against and the results were compared with the standard drug Amoxicillin. Some of the com-
pounds from the series like 7c, 7o and 7p were equipotent with Amoxicillin against Pseudomonas aeru-
ginosa. Also compound 7h was better than Amoxicillin against Staphylococcus aureus and Bacillus subtilis.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Thiosemicarbazones
Microwave
Antibacterial activity
Steroid
The diverse parasitic bacteria such as Staphylococcus aureus and
Pseudomonas aeruginosa have significant impact on the mucosal
health of humans. P. aeruginosa is one of the leading causes of hos-
pital-acquired pneumonia and urinary tract infections and is the
major cause of death in Cystic Fibrosis patients.1 Hence, the pres-
ent strategy for new drug development is directed towards identi-
fying the essential enzyme systems in the bacterial and developing
molecules to inhibit them. The present work is aimed towards
developing novel molecules with improved potential for treating
bacterial infections and with decreased probability for developing
drug resistance.
The biological activity of Schiff base thiosemicarbazones were
exploited in the 1960s using N-methylisatin-b-thiosemicarbazone,
which was effective as prophylactic agent against smallpox and
vaccinia.2,3 3-Aminpyridine-2-carboxaldehyde thiosemicarbazone
is now being evaluated in clinical trials against several malignan-
cies.4 Currently, compounds with a thiosemicarbazone structure
are known to possess antibacterial, antitumous, antiviral and anti-
malarial properties.5–7
going facile reactions to provide high yields of pure products, thus
eliminating or minimizing the use of organic solvents.9–13
Recently, steroidal building blocks have attracted the interest of
research groups in many branches of science and technology, such
as the medical and pharmacological fields, supramolecular chemis-
try and nanotechnology.14–17 To the best of our knowledge, the re-
search about steroidal (cholesterol) thiosemicarbazone derivatives
screening on bacteria has been rarely reported.18,19 However, above
reported synthetic methods suffer from one or more drawbacks like
prolonged reactions times, the use of environmentally unfavorable
solvents and frequently with low yields. Our research group has
been reported with solid inorganic support (such as: Al2O3) in a
special microwave oven to synthesize the thiosemicarbazone com-
pounds.20,21 Besides, earlier reports on N(4)-substituted thiosemi-
carbazones have concluded that the presence of bulky group at
the N(4) position of the thiosemicarbazone moiety greatly en-
hances biological activity.22 Inspired by these earlier work, herein,
we report the environmentally friendly, highly efficient and selec-
tive method for the synthesis of novel steroidal thiosemicarbazone
derivatives in the solvent-free conditions under microwave irradia-
tion. The synthetic route is depicted in Scheme 1.
The substituted thiosemicarbazides were synthesized by using
the literature procedure.21 All the thiosemicarbazone derivatives
were prepared through the condensation of steroidal ketones
and thiosemicarbazides which mixture were placed in a porcelain
mortar in the presence of a few drops of HCl using neutral alumi-
num oxide as supporter under microwave. In the experiment,
ketones of compounds 4, 6 used 2 equiv of substituted thiosemic-
arbazides for the preparation of disubstituted compounds (7a–h).
Reaction of ketones 5 or 6 with one equivalent of substituted
The application of microwave techniques for organic synthesis
has attracted considerable interest in recent years. Microwave-
assisted organic synthesis has proven to be a valuable technique for
reducing reaction times, giving cleaner reactions, improving yields,
simplifying work-up and designing energy-saving protocols.8
Moreover, with the development of ‘green chemistry’, the focus
has now shifted to less cumbersome solvent-free methods, under-
⇑
Corresponding author. Tel.: +86 28 85522315; fax: +86 28 85524386.
E-mail address: zzg63129@yahoo.com.cn (Z. Zhao).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.083

Z. Zhao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7730–7734
7731
S
C
R1
H
N
H
N
NH2
1. CH3OH, H3PO4
COOH
COOCH3
COOCH3
COOCH3
2. PCC, CH2Cl2
O
OH
MWI Solvent-free
HO
O
4
5
1
2
S
C
R1
H
N
H
N
NH2
1. CH3OH,CH3COCl
2. PCC, CH2Cl2
COOH
COOH
MWI Solvent-free
O
OH
O
HO
HO
S
R1
H
N
H
N
C
NH2
1. CH3OH, p-CH3-Ph-SO3H
MWI Solvent-free
2. PCC, CH2Cl2
O
O
OH
6
3
Disubstituted compounds:
COOCH3
COOCH3
N
NH
N
N
N
NH
NH
C
HN
C
C
S
S
C
S
S
NH
NH
NH
NH
R1
Hyodeoxycholic 7a~d
R1
Chenodeoxycholic 7e~h
R1
R1
COOCH3
Monosubstituted compounds:
COOCH3
O
O
N
N
NH
C
NH
S
S
C
NH
NH
R1
Deoxycholic 7m~p
Chenodeoxycholic 7i~l
R1
Scheme 1. Synthetic protocol of titled compounds 7a–p.
thiosemicarbazides only gave the monosubstituted compounds
(7i–p). It has been known carbonyl group at C-3 position is more
reactive to the nucleophilic addition reaction than that of C-7
position of steroidal scaffold.23
ious supports like silica gel H, neutral Al2O3 and K2CO3 have also
been studied under the catalyst of HCl. Among the results obtained,
the use of HCl in neutral Al2O3 received the best yield (94%) for the
synthesis of compound 7a (Table 1). Finally, it showed a dramatic
improvement that is in the yield the reaction time (2 min), when
we employed a microwave-assisted method. Thus this protocol
effectively worked for the affording new steroidal thiosemicarba-
zones 7a–p: mixture of steroidal ketones (1 mmol), substituted
thiosemicarbazide (1 mmol or 2 mmol) and netural aluminium
oxide (0.8 g) in the presense of a catalytic amount of HCl was
placed in a microwave oven for 1.5–6.0 min at 240–500 W. The
reaction mixture was cooled to room temperature and was dis-
solved in DMSO and filtered. The filtrate was added water and
the product was formed. The crude product was recrystallized by
ethanol to give pure compounds 7a–p.
In our quest to search an optimal reaction condition, we
examined the condensation of steroidal ketones with substituted
thiosemicarbazide, employing conventional and microwave
solvent-free thermal procedures. The reaction was monitored with
or without additives catalyst such as HCl, acetic acid or H2SO4 un-
der refluxing in ethanol, DMF or DMSO. The result showed that the
yields 7a was in the 18–50% range. But it required a longer reaction
time. Among the results obtained, the use of HCl in EtOH received
the yield (50%) for the synthesis of compound 7a. So catalytic prop-
erty of HCl has been studied considering synthesis of compound 7a
in the solvent-free conditions under microwave. Then effect of var-

7732
Z. Zhao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7730–7734
showed another sharp peak at 1697–1710 cmÀ1 attributable ste-
roidal ketones carbonyl (7-C or 12-C). In the 1H NMR spectra, the
peaks between d 8.59 and 9.27 ppm can be assigned to the protons
of the NHN. Peaks due to the other NH proton were observed at d
8.56–9.45 ppm. In addition, the singlet peaks at 0.69 (7a–d), 0.68
(7e–l), 1.05 (7m–p), 0.94 (7a–d), 1.16–1.27 (7e–l), 1.09 (7m–p)
ppm and the doublet at 0.94–0.96 (7a–d), 0.93–0.96 (7e–l), 0.86
(7m–p) ppm were the characteristics of steroidal structure. The
singlet at 3.66–3.68 ppm was assigned to the protons of COOCH3.
The in vitro antibacterial activities of compounds 7a–p were
carried out using the culture of gram-negative bacteria (P. serugin-
osa) and gram-positive bacterium (S. aureus and B. subtilis). Amox-
Table 1
Optimization of the reaction conditions synthesizing compound 7a
Entry
Solvent
Catalyst
Supporter
Time (min)
Yield (%)
1
2
3
4
5
6
7
8
EtOH
EtOH
EtOH
EtOH
DMF
DMSO
—
—
—
—
—
—
HCl
acetic acid
H2SO4
—
—
—
HCl
—
HCl
—
—
—
—
—
—
—
380
380
380
380
380
380
2.0
2.0
2.0
2.0
2.0
18a
50a
42a
37a
31a
33a
37b
48b
23b
94b
14b
39b
Silica gel H
Silica gel H
Neutral Al2O3
Neutral Al2O3
K2CO3
9
10
11
12
icillin (30 lg) was used as the standard drug, where MIC was
—
HCl
K2CO3
2.0
evaluated by the macro-dilution test using standard inoculums of
10À5 CFU mLÀ1. Serial dilutions of the test compounds, previously
dissolved in DMSO were prepared to final concentrations of 128,
a
Conventional method.
Microwave method.
b
64, 32, 16, 8, 4 and 2 lg/mL. To each tube was added 100 lL of
24 h old inoculums. The MIC which inhibits the visible growth after
18 h, was determined visually after incubation for 18 h, at 37 °C.
The lowest concentration, which showed no visible growth, was ta-
ken as an end point for minimum inhibitory concentration (MIC).
The MIC level of the tested compounds against these organisms
is given in Table 2.
The preliminary results of antibacterial activities indicated that
some of the compounds exhibited a moderate to good activity
against gram-negative bacteria (P. seruginosa) and gram-positive
bacterium (S. aureus and B. subtilis). Notably, compounds 7a, 7b,
7c, 7f, 7n, 7o and 7p found active against Gram-negative bacteria
We compared the synthesis of compounds 7a–p between
microwave in the solvent-free conditions and conventional heat-
ing. Compared to conventional thermal heating, microwave irradi-
ation technique decreased the reaction time from 300–480 min to
1.5–6.0 min. It was obvious that yields were increased from 46–
62% to 84–96%. Consequently, the use of microwave technology
in conjunction with solvent-free conditions allows expeditious,
environmentally friendly and efficient procedures in organic meth-
odology. The comparison data are given in Table 2.
The purity of the compounds was checked by the TLC and ele-
mental analyses, and the structures of compounds were identified
by spectral data. Compounds 7a–p analytical data (IR, ESI-MS, 1H
NMR, Elemental analysis) are in good agreement with the compo-
sition of thiosemicarbazones. The elemental analysis results were
within 0.4% of the theoretical values.24 Their mass spectra showed
the expected molecular peaks at high intensity. Selected diagnostic
bands of the IR spectra of the steroidal thiosemicarbazone deriva-
tives (7a–p) showed useful information about the structure of the
compounds. All the compounds exhibited a characteristic strong
absorption at 3270–3370 cmÀ1 due to N–H stretching vibration.
Intense absorption bands in the regions 1166–1200 cmÀ1 and
1505–1546 cmÀ1 were attributed to the v (C@S) and v (C@N)
stretching vibration which also confirms the formation of desired
thiosemicarbazones in all compounds. The sharp peaks at 1717–
1739 cmÀ1 attributable to ester carbonyl. But compounds 7i–p
P. seruginosa with MIC values lying in the 2–4 lg/mL range. Fur-
thermore, compounds 7c, 7o and 7p were found resembled with
a standard Amoxicillin drug against P. seruginosa, whilst three
compounds 7d, 7i, 7j were found weak activity. Rapid glance at
the Table 2, compounds 7a, 7d, 7g and 7h found activity against
Gram-positive bacteria S. aureus with MIC values lying in the 8–
32
resembled with a standard Amoxicillin drug against S. aureus.
Additionally compounds 7g (IC50 = 2.85 0.10 g/mL) and 7h
(IC50 = 1.02 0.05 g/mL) also showed as potent as a standard ref-
erence drug Amoxicillin (IC50 = 2.84 0.05 g/mL) against S. aureus
lg/mL range. Among compounds 7a, 7d and 7g were found
l
l
l
(Table 3). More exceptional result was due to 7h which showed
activity against Gram-positive B. subtilis bacteria with a MIC value
of 8
lg/mL, a more potency then all other reference drugs used in
the study. But majority of compounds showed poor activity against
Table 2
Synthesis comparison of between microwave irradiation and conventional, in vitro antibacterial activity of compounds 7a–p
Compd
R1
Conventional
Microwave
Yield (%)
MIC (lg/mL)
T (min)
Yield (%)
T (min)
S. aureus
B. subtilis
P. seruginosa
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
4-OCH3
4-CH3
4-Br
3-F
380
300
420
450
360
450
400
450
360
420
360
480
360
380
420
450
—
50
61
60
58
52
46
52
49
53
62
52
49
58
57
60
59
—
2.0
2.0
1.5
1.5
5.0
5.5
4.5
5.0
4.0
5.5
4.0
6.0
2.0
2.0
1.5
2.0
—
94
90
96
95
91
84
93
89
94
86
90
87
91
84
94
89
—
32
128
64
32
>128
64
128
128
128
128
128
>128
>128
8
32
128
32
32
64
4
4
2
128
8
H
4-OCH3
4-Br
3-F
4-OCH3
4-F
4
32
8
16
32
128
128
16
16
32
4
128
128
>128
>128
>128
>128
>128
>128
32
4-CH3
NAa
H
4-F
4-Br
4-OCH3
—
>128
>128
>128
16
2
2
2
7p
Standardb
a
Naphthalene.
Amoxicillin.
b

Z. Zhao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7730–7734
17. Savage, P. B. Eur. J. Org. Chem. 2002, 759.
7733
Table 3
18. Salman, A. K.; Mohamad, Y. Eur. J. Med. Chem. 2009, 44, 2270.
19. Salman, A. K. Eur. J. Med. Chem. 2008, 43, 2040.
20. Li, G. H.; Shi, Z. C.; Li, X. R.; Zhao, Z. G. J. Chem. Res. 2011, 35, 278.
21. Liu, L. L.; Yang, J.; Zhao, Z. G.; Shi, P. Y.; Liu, X. L. J. Chem. Res. 2010, 34, 57.
22. Li, Q. H.; Chen, S. H. Chin. J. Org. Chem. 2006, 26, 528 (in Chinese).
Antibacterial activity of compounds 7f–h against S. aureus (IC50
lg/mL)
7f
7g
7h
Amoxicillin
2.84 0.05
S. aureus
3.25 0.12
2.85 0.10
1.02 0.05
23. Richard, P. B. L.; Anthony, P. D. Tetrahedron 1993, 49, 9829.
24. Compound 7a: white solid, mp 208–209 °C. 1H NMR (400 MHz, CDCl3): d 9.09
(d, 2H, J = 8.8 Hz, NH), 8.72–8.68 (m, 2H, NH), 7.53–7.43 (m, 4H, ArH), 6.90 (t,
4H, J = 6.8 Hz, ArH), 3.81 (s, 6H, Ar-OCH3), 3.67 (s, 3H, OCH3), 0.96 (d, 3H,
J = 6.4 Hz, 21-CH3), 0.92 (s, 3H, 19-CH3), 0.69 (s, 3H, 18-CH3). IR (KBr, cmÀ1):
3301, 2941, 1739, 1594, 1525, 1480, 1178, 1034, 827. ESI-MS m/z (%): 761
([M+1]+, 100). Ana1. Calcd for C41H56N6O4S2: C, 64.71; H, 7.42; N, 11.04; found:
C, 64.59; H, 7.40; N, 11.01. Compound 7b: white solid, mp 176–177 °C. 1H NMR
(400 MHz, CDCl3): d 9.17 (d, 2H, J = 9.6 Hz, NH), 8.74–8.65 (m, 2H, NH), 7.54–
7.44 (m, 4H, ArH), 7.17 (d, 4H, J = 4.4 Hz, ArH), 3.67 (s, 3H, OCH3), 2.34 (s, 6H,
Ar-CH3), 0.96 (d, 3H, J = 6.4 Hz, 21-CH3), 0.93 (s, 3H, 19-CH3), 0.69 (s, 3H, 18-
CH3). IR (KBr, cmÀ1): 3295, 2943, 1737, 1591, 1529, 1478, 1175, 1051, 814. ESI-
MS m/z (%): 729 ([M+1]+, 100). Ana1. Calcd for C41H56N6O2S2: C, 67.55; H, 7.74;
N, 11.53; found: C, 67.63; H, 7.69; N, 11.56. Compound 7c: white solid, mp
170–171 °C. 1H NMR (400 MHz, CDCl3): d 9.22 (t, 2H, J = 6.4 Hz, NH), 8.71 (d,
2H, J = 10.8 Hz, NH), 7.63–7.46 (m, 8H, ArH), 3.68 (s, 3H, OCH3), 0.95 (d, 3H,
J = 6.0 Hz, 21-CH3), 0.93 (s, 3H, 19-CH3), 0.69 (s, 3H, 18-CH3). IR (KBr, cmÀ1):
3280, 2942, 1735, 1587, 1534, 1488, 1179, 1066, 1008, 824. ESI-MS m/z (%):
859 ([M+1]+, 100). Ana1. Calcd for C39H50Br2N6O2S2: C, 54.54; H, 5.87; N, 9.79;
found: C, 54.67; H, 5.89; N, 9.81. Compound 7d: white solid, mp 189–190 °C.
1H NMR (400 MHz, CDCl3): d 9.31 (d, 2H, J = 5.6 Hz, NH), 8.76 (s, 2H, NH), 7.75–
7.63 (m, 2H, ArH), 7.41–7.15 (m, 4H, ArH), 7.06–6.83 (m, 2H, ArH), 3.68 (s, 3H,
OCH3), 0.94 (d, 3H, J = 6.8 Hz, 21-CH3), 0.92 (s, 3H, 19-CH3), 0.69 (s, 3H, 18-
CH3). IR (KBr, cmÀ1): 3290, 2945, 1736, 1602, 1539, 1487, 1442, 1198, 1048,
775. ESI-MS m/z (%): 737 ([M+1]+, 100). Anal. Calcd for C39H50F2N6O2S2: C,
63.56; H, 6.84; N, 11.40; found: C, 63.77; H, 6.82; N, 11.45. Compound 7e:
white solid, mp 188–189 °C. 1H NMR (400 MHz, CDCl3): d 9.36–9.25 (m, 2H,
NH), 8.66 (s, 1H, NH), 8.63 (s, 1H, NH), 7.71–7.64 (m, 4H, ArH), 7.43–7.34 (m,
4H, ArH), 7.22 (dd, 2H, J = 9.6, 7.6 Hz, ArH), 3.67 (s, 3H, COOCH3), 1.19 (s, 3H,
19-CH3), 0.96 (d, 3H, J = 6.4 Hz, 21-CH3), 0.73 (s, 3H, 18-CH3). IR (KBr, cmÀ1):
3303, 2942, 1734, 1595, 1536, 1441, 1327, 1266, 1182, 1064, 751. ESI-MS m/z
(%): 701 [(M+1)+, 100]. Anal. Calcd for C39H52N6O2S2: C, 66.82; H, 7.48; N,
11.99; found: C, 66.85; H, 7.47; N, 12.01. Compound 7f: white solid, mp 197–
198 °C. 1H NMR (400 MHz, CDCl3): d 9.15–9.08 (m, 2H, NH), 8.61 (s, 1H, NH),
8.59 (s, 1H, NH), 7.53–7.45 (m, 4H, ArH), 6.96–6.89 (m, 4H, ArH), 3.83 (s, 3H,
Ar-OCH3), 3.81 (s, 3H, Ar-OCH3), 3.66 (s, 3H, COOCH3), 1.16 (s, 3H, 19-CH3),
0.96 (d, 3H, J = 6.4 Hz, 21-CH3), 0.74 (s, 3H, 18-CH3). IR (KBr, cmÀ1): 3301, 2943,
1733, 1595, 1520, 1469, 1244, 1177, 1034, 829. ESI-MS m/z (%): 761 [(M+1)+,
100]. Anal. Calcd for C41H56N6O4S2: C, 64.71; H, 7.42; N, 11.04; found: C, 64.76;
H, 7.40; N, 11.03. Compound 7g: white solid, mp 164–165 °C. 1H NMR
(400 MHz, CDCl3): d 9.32–9.21 (m, 2H, NH), 8.77 (s, 1H, NH), 8.70 (s, 1H, NH),
7.62–7.52 (m, 4H, ArH), 7.50–7.44 (m, 4H, ArH), 3.68 (s, 3H, COOCH3), 1.19 (s,
3H, 19-CH3), 0.96 (d, 3H, J = 6.0 Hz, 21-CH3), 0.68 (s, 3H, 18-CH3). IR (KBr,
cmÀ1): 3291, 2944, 1732, 1585, 1530, 1260, 1179, 1065, 1010, 823. ESI-MS m/z
(%): 859 [(M+1)+, 100]. Anal. Calcd for C39H50Br2N6O2S2: C, 54.54; H, 5.87; N,
9.79; found: C, 54.59; H, 5.85; N, 9.81. Compound 7h: white solid, mp 188–
187 °C. 1H NMR (400 MHz, CDCl3): d 9.45–9.29 (m, 2H, NH), 8.74 (s, 1H, NH),
8.69 (s, 1H, NH), 7.79–7.65 (m, 2H, ArH), 7.39–7.19 (m, 4H, ArH), 6.85–6.94 (m,
2H, ArH), 3.68 (s, 3H, COOCH3), 1.17 (s, 3H, 19-CH3), 0.97 (d, 3H, J = 6.0 Hz, 21-
CH3), 0.73 (s, 3H, 18-CH3). IR (KBr, cmÀ1): 3300, 2944, 1735, 1604, 1539, 1486,
1436, 1272, 1200, 1064, 858, 775. ESI-MS m/z (%): 737 [(M+1)+, 100]. Anal.
Calcd for C39H50F2N6O2S2: C, 63.56; H, 6.84; N, 11.40; found: C, 63.54; H, 6.86;
N, 11.39. Compound 7i: white solid, mp 164–165 °C. 1H NMR (400 MHz,
CDCl3): d 9.07 (s, 1H, NH), 8.58 (d, 1H, J = 7.2 Hz, NH), 7.46 (d, 2H, J = 8.8 Hz,
ArH), 6.91 (d, 2H, J = 7.6 Hz, ArH), 3.81 (s, 3H, Ar-OCH3), 3.67 (s, 3H, COOCH3),
1.27 (s, 3H, 19-CH3), 0.93 (d, 3H, J = 6.4 Hz, 21-CH3), 0.69 (s, 3H, 18-CH3). IR
(KBr, cmÀ1): 3286, 3174, 2951, 2875, 1721, 1594, 1547, 1516, 1244, 1177,
1033, 833. ESI-MS m/z (%): 604 ([M+23]+, 100). Anal. Calcd for C33H47N3O4S: C,
68.12; H, 8.14; N, 7.22; found: C, 68.04; H, 8.12; N, 7.20. Compound 7j: white
solid, mp 115–116 °C. 1H NMR (400 MHz, CDCl3): d 9.15 (s, 1H, NH), 8.75 (d,
1H, J = 7.2 Hz, NH), 7.56 (t, 2H, J = 8.0 Hz, ArH), 7.07 (t, 2H, J = 8.4 Hz, ArH), 3.67
(s, 3H, COOCH3), 1.27 (s, 3H, 19-CH3), 0.93 (d, 3H, J = 6.4 Hz, 21-CH3), 0.69 (s,
3H, 18-CH3); IR (KBr, cmÀ1): 3293, 2950, 2871, 1738, 1710, 1609, 1521, 1258,
1186, 1053, 835. ESI-MS m/z (%): 570 ([M+1]+, 100). Anal. Calcd for
B. subtilis. Among the thiosemicarbazones, substituents with deac-
tivating electron withdrawing groups like fluoric group in the phe-
nyl ring showed excellent antibacterial activity. It was also
observed that the Chenodeoxycholic acid thiosemicarbazone
disubstituted derivatives of biological activity better than mono-
substituted Deoxycholic acid and Hyodeoxycholic. The importance
of such work lies in the possibility that the new compounds might
be more effective against bacteria for which a thorough investiga-
tion regarding the structure–activity relationship, toxicity and the
biological effects which would be helpful in designing more potent
antibacterial agents for therapeutic use is required.
In conclusion, we have demonstrated a rapid, efficient and eco-
friendly method for synthesis of steroidal thiosemicarbazones un-
der solvent-free conditions using microwave irradiation. The reac-
tion was conducted in the presence of neutral aluminum oxide,
without using solvent. The present method has some distinct
advantages comparing to the conventional method, such as shorter
reaction times, good product yields and finally agreement with the
green chemistry protocols. All the synthesized compounds were
tested for in vitro antibacterial activity. Based on the activity data,
many new synthesized compounds are good antibacterial activity.
Among compounds 7a, 7b, 7c, 7f, 7n, 7o and 7p exhibited potency
as close as a standard drug Amoxicillin against Gram-negative bac-
teria. Compound 7h even received more than reference drugs
against Gram-positive bacterium S. aureus and B. subtilis. Com-
pound 7h can serve as an important pharmacophore for the design
of new antibacterial agent. Studies to establish their in vitro effi-
cacy and safety are being planned for their further development.
Acknowledgments
This research was financially supported by the Science and
Technology Department of Si Chuan Province (No. 2011JY0035)
and the Fundamental Research Funds for Central University, South-
west University for Nationalities (No. 11NZYTH05).
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.09.
083.
References and notes
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C32H44FN3O3S: C, 67.46; H, 7.78; N, 7.37; found: C, 67.43; H, 7.75; N, 7.35.
Compound 7k: white solid, mp 151–152 °C. 1H NMR (400 MHz, CDCl3): d 9.15
(s, 1H, NH), 8.59 (d, 1H, J = 7.2 Hz, NH), 7.48 (d, 2H, J = 8.0 Hz, ArH), 7.18 (t, 2H,
J = 8.0 Hz, ArH), 3.67 (s, 3H, COOCH3), 2.35 (s, 3H, Ar-CH3), 1.27 (s, 3H, 19-CH3),
0.93 (d, 3H, J = 6.4 Hz, 21-CH3), 0.69 (s, 3H, 18-CH3). IR (KBr, cmÀ1): 3282, 3220,
2951, 2870, 1737, 1708, 1591, 1546, 1481, 1439, 1269, 1176, 1045, 819. ESI-MS
m/z (%): 1131 ([2 M+1]+, 100). Anal. Calcd for C33H47N3O3S: C, 70.05; H, 8.37; N,
7.43; found: C, 69.99; H, 8.35; N, 7.40. Compound 7l: white solid, yield 87%, mp
118–119 °C. 1H NMR (400 MHz, CDCl3):
d 9.42 (s, 1H, NH), 8.75 (d, 1H,
J = 9.2 Hz, NH), 7.93–7.85 (m, 3H, ArH), 7.82 (d, 1H, J = 8.0 Hz, ArH), 7.55–7.51
(m, 3H, ArH), 3.67 (s, 3H, COOCH3), 1.29 (s, 3H, 19-CH3), 0.94 (d, 3H, J = 6.4 Hz,
21-CH3), 0.70 (s, 3H, 18-CH3). IR (KBr, cmÀ1): 3307, 2946, 2871, 1735, 1708,
1596, 1505, 1470, 1380, 1268, 1199, 1166, 1093, 1019, 775. ESI-MS m/z (%):
1225 ([2M+23]+, 100). Anal. Calcd for C36H47N3O3S: C, 71.84; H, 7.87; N, 6.98;
found: C, 71.82; H, 7.85; N, 6.97. Compound 7m: white solid, mp 175–176 °C.
1H NMR (400 MHz, CDCl3): d 9.27 (s, 1H, NH), 8.57 (d, 1H, J = 10.8 Hz, NH), 7.66

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Z. Zhao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7730–7734
(d, 2H, J = 8.0 Hz, ArH), 7.38 (dd, 2H, J = 8.0, 7.6 Hz, ArH), 7.22 (dd, 1H, J = 7.2,
J = 10.0 Hz, NH), 7.59–7.56 (m, 2H, ArH), 7.48 (dd, 2H, J = 7.2, 1.6 Hz, ArH), 3.67
(s, 3H, COOCH3), 1.09 (s, 3H, 19-CH3), 1.05 (s, 3H, 18-CH3), 0.86 (d, 3H,
J = 6.4 Hz, 21-CH3). IR (KBr, cmÀ1): 3270, 2931, 2867, 1735, 1699, 1582, 1519,
1488, 1383, 1328, 1267, 1175, 1061, 1009, 76. ESI-MS m/z (%): 1261 [(2M+3)+,
100]. Anal. Calcd for C32H44BrN3O3S: C, 60.94; H, 7.03; N, 6.66; found: C, 60.78;
H, 7.01; N, 6.68. Compound 7p: white solid, mp 198–199 °C. 1H NMR
(400 MHz, CDCl3): d 9.09 (s, 1H, NH), 8.56 (d, 1H, J = 11.2 Hz, NH), 7.49–7.46
(m, 2H, ArH), 6.91 (d, 2H, J = 8.4 Hz, ArH), 3.82 (s, 3H, Ar-OCH3), 3.67 (s, 3H,
COOCH3), 1.09 (s, 3H, 19-CH3), 1.05 (s, 3H, 18-CH3), 0.86 (d, 3H, J = 6.4 Hz, 21-
CH3). IR (KBr, cmÀ1): 3285, 2931, 2866, 1734, 1699, 1589, 1522, 1379, 1329,
1238, 1174, 1036, 840. ESI-MS m/z (%): 604 [(M+23)+, 100]. Anal. Calcd for
7.6 Hz, ArH), 3.67 (s, 3H, COOCH3), 1.09 (s, 3H, 19-CH3), 1.05 (s, 3H, 18-CH3),
0.86 (d, 3H, J = 6.4 Hz, 21-CH3). IR (KBr, cmÀ1): 3286, 2937, 2872, 1717, 1697,
1594, 1528, 1490, 1439, 1352, 1265, 1178, 1040, 750. ESI-MS m/z (%): 1103
[(2M+1)+, 100]. Anal. Calcd for C32H45N3O3S: C, 69.65; H, 8.22; N, 7.62; found:
C, 69.57; H, 8.19; N, 7.64. Compound 7n: white solid, mp 198–199 °C. 1H NMR
(400 MHz, CDCl3): d 9.17 (s, 1H, NH), 8.57 (d, 1H, J = 10.4 Hz, NH), 7.59–7.55
(m, 2H, ArH), 7.07 (dd, 2H, J = 8.4, 8.4 Hz, ArH), 3.67 (s, 3H, COOCH3), 1.09 (s,
3H, 19-CH3), 1.05 (s, 3H, 18-CH3), 0.86 (d, 3H, J = 6.4 Hz, 21-CH3). IR (KBr,
cmÀ1): 3271, 2931, 2867, 1738, 1699, 1519, 1380, 1330, 1268, 1197, 1053, 847.
ESI-MS m/z (%): 570 [(M+1)+, 100]. Anal. Calcd for C32H44FN3O3S: C, 67.46; H,
7.78; N, 7.37; found: C, 67.47; H, 7.77; N, 7.39. Compound 7o: white solid, mp
C
33H47N3O4S: C, 68.12; H, 8.14; N, 7.22; found: C, 68.01; H, 8.13; N, 7.24.
196–197 °C. 1H NMR (400 MHz, CDCl3):
d 9.24 (s, 1H, NH), 8.64 (d, 1H,