Solvent-free synthesis of novel unsymmetric chenodeoxycholic acid bis thiocarbazone derivatives promoted by microwave irradiation and evaluation of their antibacterial activity

Article abstract of DOI:10.1016/j.crci.2013.05.009

Eleven new non-symmetric chenodeoxycholic acid bis thiocarbazones were efficiently synthesized via the condensation of steroidal diketones (methyl (5β)-3,7-dioxocholan-24-oate) and substituted benzaldehyde thiocarbohydrazones by means of microwave irradiation. Compared with the conventional heating method, the yields were increased from 31-58% to 80-89% and the reaction times were reduced from 420-520 min to 5.0-7.5 min. The structures of these novel compounds were characterized by spectral data and elemental analysis. Four compounds (7a, 7c, 7f and 7g) possess excellent activity against Bacillus subtilis and Staphylococcus aureus.

Full text of DOI:10.1016/j.crci.2013.05.009

C. R. Chimie 16 (2013) 977–984
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´
Full paper/Memoire
Solvent-free synthesis of novel unsymmetric chenodeoxycholic acid
bis thiocarbazone derivatives promoted by microwave irradiation
and evaluation of their antibacterial activity
*
Zhichuan Shi, Zhigang Zhao , Min Liu, Xiaohong Wang
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:
Eleven new non-symmetric chenodeoxycholic acid bis thiocarbazones were efficiently
Received 6 March 2013
Accepted after revision 17 May 2013
Available online 5 July 2013
synthesized via the condensation of steroidal diketones (methyl (5b)-3,7-dioxocholan-24-
oate) and substituted benzaldehyde thiocarbohydrazones by means of microwave
irradiation. Compared with the conventional heating method, the yields were increased
from 31–58% to 80–89% and the reaction times were reduced from 420–520 min to 5.0–
7.5 min. The structures of these novel compounds were characterized by spectral data and
elemental analysis. Four compounds (7a, 7c, 7f and 7g) possess excellent activity against
Bacillus subtilis and Staphylococcus aureus.
Keywords:
Thiocarhydrazone
Microwave irradiation
Antibacterial activity
Steroid
ß 2013 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
Thiocarbazone
Solvent-free conditions
1. Introduction
their capacity of penetrating the cell wall. Several steroidal
derivatives have been investigated as new curative agents
The steroid system has been a prime focus of research
throughout scientific history and displays a variety of
biological functions [1–4]. Steroidal compounds, widely
used in traditional medicines, exhibit a wide range of
activities, including anticancer and antibacterial [5,6], and
have the potential to be developed as drugs for the
treatment of a large number of diseases. They have also
become the basis of many important discoveries in organic
chemistry. Recently, steroidal building blocks have
attracted interest with a view to modifying steroidal
for cancer, and exhibited diverse biological activities, such
as antimicrobial, anti-inflammatory, anticancer ones [9–
11].
The biological activity of Schiff bases containing thione
(C5S) was exploited in the 1960s using N-methylisatin-b-
thiosemicarbazone, which was effective against smallpox
and vaccinia [12,13]. 3-Aminpyridine-2-carboxaldehyde
thiosemicarbazone is now being evaluated in clinical trials
against several malignancies [14]. Compounds containing
thione (C5S) groups occupy an important position among
organic reagents. Organic compounds with this structure
are known to display a wide range of pharmacological
activity, including anticancer, antibacterial and antifungal
effects [15–18]. A good deal of research has been carried
out on the potential of coordination chemistry of
thiocarbohydrazones, and thiocarbohydrazide attached
to carbonyl compounds having heterocyclic moiety to
give potential drugs [19,20]. Further optimization of this
compound is required to improve its antimicrobial activity.
molecules [7,8]. Their preparation is
a stimulating
challenge to organic chemists, often demanding the
development of new and generally useful reactions. These
compounds are often non-toxic, capable of overcoming
multi-drug resistance, and highly bioavailable because of
*
Corresponding author.
E-mail address: zzg63129@163.com (Z. Zhao).
1631-0748/$ – see front matter ß 2013 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.crci.2013.05.009

978
Z. Shi et al. / C. R. Chimie 16 (2013) 977–984
Although there are many reports based on steroidal
reactions than a carbonyl at C-7 [30]. In our quest to find
optimal reaction conditions, we studied steroidal dike-
tones with substituted benzaldehyde thiocarbohydra-
zones, employing conventional and microwave-mediated
solvent-free thermal procedures. The conventional reac-
tion was conducted with or without catalysts, such as HCl,
acetic acid or H2SO4 by refluxing in ethanol, acetic acid,
methanol, THF, DMF, or DMSO. The result showed that the
yield for 7a was in the range 24–52% (Table 1). But it
required a longer reaction time. So, the catalytic properties
of HCl have been studied considering the synthesis of
compound 7a.
We then studied the effect of various supports, such as
silica gel H, neutral Al2O3 and K2CO3 with or without HCl
catalyst for the synthesis of compound 7a using the
microwave-assisted method. Among the results obtained,
the use of HCl in neutral Al2O3 received the best yield (84%)
(Table 1). Finally, we evidenced a dramatic improvement
in the yield and the reaction time (5.5 min) when
employing a microwave-assisted method. The micro-
wave-assisted procedure was then applied to the synthesis
of compounds 7b–k.
In order to study the advantages of the microwave-
promoted reaction, we compared the synthesis of
compounds 7a–k using conventional heating and the
microwave-assisted method under solvent-free condi-
tions. Compared to the conventional thermal heating,
the microwave irradiation technique decreased the
reaction time from 420–540 min to 5.0–7.5 min and
increased the yields from 31–58% to 80–89% (Table 2).
Consequently, the use of the microwave technology
in conjunction with solvent-free conditions allows
units’ modification, including derivatization, cyclization
[21], there are few reports about steroidal Schiff base.
Steroid is known for its abundance and importance in
biological systems, which is expected to show relatively
low toxicity, augmented biocompatibility [22]. Further-
more, thiourea and its related analogues have become
the focus of interest in recent years on account of their
pharmacological activities. According to the active
principle of superposition. We hoped that thiosemicar-
bazone derivatives containing steroid unit and thiourea
group have potent antibacterial activity. In addition,
microwave synthesis is a well-established technique in
green chemistry and has many advantages compared
with the traditional methods, such as faster reaction
times, higher yields; moreover, it is
a solvent-free
method [23–26]. It was being applied in many organic
synthesis. Accordingly, as a further development of our
green agenda [27–29], herein we report a microwave-
mediated, efficient, high yield and environmentally
friendly synthesis of novel steroidal bis thiocarbazone
derivatives under solvent-free conditions (Scheme 1) as
well as an evaluation of the antibacterial activities of
these compounds against four pathogenic strains,
Escherichia coli (ATCC 35218), Bacillus subtilis (ATCC
6633), Pseudomonas aeruginosa (ATCC 27853), and
Staphyloccocus aureus (ATCC 6538).
2. Result and discussion
It is known that a carbonyl group at C-3 of steroidal
scaffold is more susceptible to nucleophilic addition
CH3OH, p-CH3-Ph-SO3H
COOCH3
COOH
OH
OH
HO
HO
5
4
S
R1
H
N
H
H2N
C
N
N
C
H
PCC, CH2Cl2
COOCH3
MWI Solvent-free
O
O
6
COOCH3
O
N
NH
S
C
NH
N
R1
7a
=
7b
7f
7j
7c
7g
7k 4-NO2
7d
7h
H
4-OCH3
2-OH
4-F
3-OH
2-Cl
CH
7e 2,4-OH
7i 2,4-F
2-Br
R1
7a-k
4-Cl
Scheme 1. Synthetic protocol of titled compounds 7a–k.

Z. Shi et al. / C. R. Chimie 16 (2013) 977–984
979
Table 1
Effect of the reaction conditions on the synthesis of compound 7a.
Entry
Solvent
Catalyst
Support
Time (min)
Yield (%)
1
EtOH
–
–
420
420
420
420
420
420
420
420
420
5.5
24a
52a
43a
39a
40a
42a
48a
32a
36a
53b
61b
32b
84b
12b
40b
2
EtOH
HCl
–
3
EtOH
acetic acid
–
4
EtOH
H2SO4
–
–
5
Acetic acid
–
6
THF
HCl
HCl
–
–
7
MeOH
–
8
DMF
–
9
DMSO
–
–
10
11
12
13
14
15
–
–
–
–
–
–
–
Silica gel H
Silica gel H
Neutral Al2O3
Neutral Al2O3
K2CO3
HCl
–
5.5
5.5
HCl
–
5.5
5.5
HCl
K2CO3
5.5
a
Conventional method.
b
Microwave method.
expeditious, environmentally friendly and efficient
procedures in organic methodology.
The singlet at 3.58 ppm was assigned to the protons of
COOCH3. Mass spectra showed the expected molecular
peaks at high intensity.
Assignments of selected characteristic IR bands of the
steroidal bis thiocarbazone derivatives (7a–k) provided
useful information about the structure of the compounds.
All the compounds exhibited a characteristic strong
The preliminary results of antibacterial assays indi-
cated that some of the compounds exhibited a moderate
to good activity against Gram-negative bacterium
(P. seruginosa) and Gram-positive bacteria (S. aureus
and B. subtilis). As shown in Table 3, compounds 7b, 7i–k
exert no potent inhibition activity against any of the
bacterial strains tested. Notably, compounds 7d, 7e and
7h were effective against the Gram-positive S. aureus and
compounds 7a, 7c, 7f and 7g were significantly more
active against S. aureus than the standard reference drug,
amoxicillin. The minimal inhibitory concentration (MIC)
absorption in the region 3229–3309 cmÀ1 due to the
v
(N–H) stretching vibration. Intense absorption bands in
the regions 1153–1171 cmÀ1 and 1503–1533 cmÀ1 were
attributed to the
v (C5S) and v (C5N) stretching
vibrations, which also confirms the formation of the
desired thiocarbazones in all the compounds. All com-
pounds showed
a ,
sharp peak at 1731–1735 cmÀ1
attributable to the ester carbonyl and another sharp peak
at 1707–1709 cmÀ1, due to the steroidal ketones carbonyl
(C-7), which also confirms the formation of the mono-
substituted compounds. Further evidence of the structure
of steroidal bis thiocarbazones was obtained from the 1H
values were 0.5–2
mg/mL (7a, 7c, 7f and 7g) and 16 mg/
mL (7d, 7e and 7h). The activities of compounds 7a, 7c, 7d,
7f and 7g were found similar to that of amoxicillin against
B. subtilis. Most of the compounds showed poor activity
against the Gram-negative E. coli and P. aeruginosa. A
rapid glance at Table 4 shows that the methoxy derivative
7c was effective at inhibiting Gram-positive S. aureus and
B. subtilis. These results reveal that the electron-donating
substituent group in these compounds plays a vital role in
the inhibitory activity towards Gram-positive bacteria
NMR spectra. The signal between
can be assigned to the protons of the NHN. The signals
due to the other NH protons were observed at 8.56–
d 8.59 and d 9.27 ppm
d
9.45 ppm. In addition, the singlet peaks at 0.64, 1.17–
1.21 ppm and the doublet at 0.82–0.89 ppm were char-
acteristics of the methyl group of the steroidal structure.
Table 2
Synthesis of compounds 7a–k using microwave irradiation or conventional method.
Compound
R1
Conventional heating
Microwave method
TC/TMW
T (min)
Yield (%)
T (min)
Yield (%)
7a
7b
7c
7d
7e
7f
H
420
480
420
540
420
420
480
520
480
520
520
47
49
58
37
41
48
39
34
39
31
35
5.5
6.0
5.0
7.0
5.5
5.0
6.0
7.0
5.0
7.0
7.5
84
86
89
82
85
84
82
80
86
84
85
76
80
84
77
76
84
80
84
96
74
69
2-OH
4-OCH3
3-OH
2,4-OH
4-F
7g
7h
7i
2-Br
2-Cl
2,4-F
4-Cl
7j
7k
4-NO2
TC: conventional method needs time; TMW: microwave method needs time.

980
Z. Shi et al. / C. R. Chimie 16 (2013) 977–984
Table 3
Antibacterial activity of compounds 7a–k (unit, mm).
Compounds
Zone of inhibition (mm)a,b
E. coli
P. aeruginosa
B. subtilis
S. aureus
7a
–
–
20.1 Æ 0.2
25.0 Æ 0.1
–
7b
–
–
–
7c
–
–
25.5 Æ 0.1
25.2 Æ 0.2
20.5 Æ 0.3
20.5 Æ 0.2
25.0 Æ 0.3
25.0 Æ 0.4
20.2 Æ 0.5
–
7d
–
–
20.5 Æ 0.1
7e
–
–
16.1 Æ 0.3
7f
15.2 Æ 0.2
21.1 Æ 0.3
20.1 Æ 0.2
7g
11.9 Æ 0.3
–
20.4 Æ 0.3
7h
11.9 Æ 0.4
–
–
7i
–
–
–
7j
–
–
–
–
7k
–
–
–
–
Amoxicillin
Ciprofloxacin
DMSO
22.9 Æ 0.4
25.3 Æ 0.5
–
23.5 Æ 0.2
31.4 Æ 0.4
–
25.9 Æ 0.4
30.0 Æ 0.3
–
23.5 Æ 0.3
29.6 Æ 0.5
–
a
Zones of inhibition less than 10 mm are not shown in the table.
b
Reference and compounds (40
mg/mL).
strains. Monosubstituted halogen compounds (except 7j)
showed greater antibacterial activity, whereas disubsti-
tuted halogen compounds showed weaker antibacterial
activity. With respect to P. aeruginosa, only compound 7f
showed strong inhibition. On the other hand, only 7f–h
showed moderate inhibitions for E. coli.
the design of more potent antibacterial agents for
therapeutic use.
3. Conclusion
In conclusion,
a microwave-assisted solvent-free
Further, the determination of MIC and 50% inhibition of
bacteria growth (IC50) of the active compounds suggested
that the fluorine-containing derivative 7f showed the
maximum activity against most of the strains tested, with
synthetic procedure in the presence of neutral aluminum
oxide was developed for the preparation of asymmetric
chenodeoxycholic acid bis thiocarbazones. The present
method has many advantages compared to the conven-
tional method, such as shorter reaction times, high yields
and compliance with green chemistry protocols. Com-
pound 7c exhibited promising antibacterial activity
similar to that of the standard drug amoxicillin against
B. subtilis, indicating that this compound is a promising
antibacterial compound for further research. Compounds
7d, 7e and 7h exhibited potency similar to that of
amoxicillin against Gram-positive S. aureus, and com-
pounds 7a, 7c, 7f and 7g were found to be very promising
and almost comparable to amoxicillin, indicating that
they can serve as important pharmacophores for the
design of new leading antibacterial agents. Studies to
MIC of 32
mL for P. aeruginosa, and 1
pounds 7a, 7c, 7f and 7g demonstrated notable activity,
with MIC values ranging from 0.5 to 2 g/mL and IC50 from
0.28 to 1.1 g/mL against S. aureus, showing their
m
g/mL for E. coli, 8
m
g/mL for B. subtilis, 16
mg/
m
g/mL for S. aureus. Com-
m
m
potential to become a drug against the drug-resistant
S. aureus. Compound 7c was as potent as the standard
reference drug amoxicillin against B. subtilis. The impor-
tance of this work lies in the possibility that the new
compounds might be more effective against bacteria. A
thorough investigation regarding the structure–activity
relationship, toxicity and the biological effects may allow
Table 4
Antibacterial activity of compounds 7a–k.
Compounds
B. subtilis
S. aureus
E. coli
P. aeruginosa
MIC
IC50
MIC
IC50
MIC
IC50
MIC
IC50
7a
16
9
1
0.88
> 64
1.1
> 128
> 128
> 128
> 128
> 128
32
> 64
> 64
> 64
> 64
> 64
19.17
70.5
48.5
> 64
> 64
> 64
7.3
> 128
> 128
> 128
> 128
> 128
16
> 64
> 64
> 64
> 64
> 64
8.65
> 64
> 64
> 64
> 64
> 64
2.5
7b
> 128
1
> 64
0.86
8.1
> 128
2
7c
7d
16
16
8.2
7e
64
35.8
4.7
16
8.5
7f
8
1
0.69
0.28
8.3
7g
16
8.2
0.5
128
128
7h
> 128
> 128
> 128
> 128
2
> 64
> 64
> 64
> 64
1
16
64
> 128
> 128
> 128
> 128
4
7i
> 128
> 128
> 128
16
> 64
> 64
> 64
6.4
> 128
> 128
> 128
16
7j
7k
Amoxicillin
Ciprofloxacin
0.128
0.1
3.12
1.82
0.25
0.18
0.5
0.41
MIC: minimal inhibitory concentration; IC50; 50% inhibition of bacteria growth; MIC and IC50 are given in
mg/mL.

Z. Shi et al. / C. R. Chimie 16 (2013) 977–984
981
1
S
C
S
C
R
1
R
H
N
H
N
H
H
N
H O/EtOH
2
H N
2
N
N
C
H
H N
2
NH
2
CHO
+
MWI
3a-k
1
2a-k
Scheme 2. Synthetic protocol of compounds 3a–k.
establish their in vitro efficacy and safety are being
planned for their further development.
benzaldehyde thiocarbohydrazones 3a–k are shown in
Table 5.
4.1.1. Compound 3i
4. Experimental
White crystal. Yield 79%. Mp 169–170 8C (MeOH–H2O).
IR (KBr, cmÀ1): 3409, 3356, 3265, 2930, 2791, 1615, 1564,
1507, 1429, 1283, 1253, 1141, 1077, 1018, 848, 611. 1H
The melting points were measured with a micro-
melting point apparatus and are uncorrected. IR spectra
were recorded on 1700 PerkinElmer FTIR using KBr
disks. 1H NMR spectra were recorded on a Varian INOVA
400 MHz spectrometer using CDCl3 or DMSO-d6 as a
solvent and TMS as an internal standard. Mass spectra
were determined with a Finnigan LCQ-DECA instrument.
Elemental analysis (C, H, and N) was performed with a
Carlo Erba EA1106 auto analyzer and the results
were within Æ 0.4% of the theoretical values. All the
experiments were monitored by thin layer chromatogra-
phy (TLC) on pre-coated silica gel plates. Optical rotation
was measured on a Wzz-2B polarimeter. All the reactions
were performed in a commercial microwave apparatus
(XH-100A, 100–1000 W, Beijing Xianghu Science and
Technology Development Co. Ltd, Beijing, China). All the
solvents were purified before use. Pyridinium chlorochro-
mate (PCC) and methyl (5b)-3,7-dihydroxycholan-24-oate
NMR (DMSO-d6, 400 MHz): d 11.53 (s, 1H, NH), 9.96 (s, 1H,
NH), 8.45–8.39 (m, 1H, ArH), 8.17 (s, 1H, 5CH), 7.33–7.27
(m, 1H, ArH), 7.15 (dd, J = 6.4 and 8.4 Hz, 1H, ArH), 4.88 (s,
2H, NH2). ESI–MS m/z (%): 231 ([M + 1]+, 100). Anal. calcd
for C8H8F2N4S: C, 41.73; H, 3.50; N, 24.33. Found: C, 41.78;
H, 3.48; N, 24.35%.
4.2. Synthesis of methyl (5
[37]
b)-3,7-dioxocholan-24-oate (6)
Pyridinium chlorochromate (PCC) (3.03 mmol) was
added to a solution of methyl (5 )-3,7-dihydroxycholan-
b
24-oate 5 (0.25 g, 0.615 mmol) in dried CH2Cl2 (20 mL) at
room temperature. The reaction was completed in 12 h.
The solvent was removed under reduced pressure, and the
crude product was purified by chromatography on silica
gel using ethyl acetate to give a white crystal. Yield 86%.
Mp 155–156 8C (ethyl acetate) (lit. [38] Mp 154–156 8C),
5
were prepared according to the methods reported
[31,32].
[
a
]
D = À21.5 (c 0.12, CH2Cl2). IR (KBr, cmÀ1): 2953, 2873,
1709, 1403, 1375, 1214, 1174, 1010, 966. 1H NMR (CDCl3,
4.1. Preparation of substituted benzaldehyde
400 MHz):
d 3.67 (s, 3H, COOCH3), 1.31 (s, 3H, 19–CH3),
thiocarbohydrazones (3a–k)
0.93 (d, J = 6.4 Hz, 3H, 21–CH3), 0.69 (s, 3H, 18–CH3). ESI–
MS m/z (%): 827 ([2M + 23]+, 100). Anal. calcd for C25H38O4:
C, 74.48; H, 9.54. Found. C, 74.51; H, 9.53%.
The aromatic aldehyde (10 mmol) in ethanol (40 mL)
was added dropwise to a solution of thiocarbohydrazide
(15 mmol) in H2O (50 mL) and acetic acid (1 mL). The
mixture was refluxed in the microwave oven at 300 W for
15 min. The reaction was monitored by TLC until comple-
tion. The precipitate was collected by filtration and washed
with water (3 Â 10 mL). The resulting solid was recrys-
tallised from ethanol to give the product (Scheme 2). The
analytical data for 3i are given below. The melting points of
4.3. Microwave procedure for the preparation of bis
thiocarbazones (7a–k)
The steroidal diketone methyl (5b)-3,7-dioxocholan-
24-oate (6) (0.1 mmol) and the substituted benzaldehyde
thiocarbohydrazones 3a–k (0.1 mmol) and natural alumi-
num oxide (0.3 g) were placed in a porcelain mortar, and
concentrated HCl (two drops) was added. After grinding,
the mixture was put in a round-bottom flask (25 mL) and
irradiated in a microwave oven for 5.0–7.5 min at 350 W.
The reaction mixture was cooled to room temperature,
dissolved in DMSO and filtered. The product was recrys-
tallised from ethanol in 80–89% yields. The physical and
spectral data of the compounds 7a–k are as follows.
Table 5
The melting points of substituted benzaldehyde thiocarbohydrazones
3a–k.
Product
Formula
R1
Mp (8C)
Lit. Mp (8C)
3a
3b
3c
3d
3e
3f
C8H10N4S
H
197–198
218–219
198–199
201–202
214–215
208–209
200–201
209–210
169–170
211–212
204–206
198 [33]
C8H10N4OS
C9H12N4OS
C8H10N4OS
C8H10N4O2S
C8H9FN4S
2-OH
4-OCH3
3-OH
2,4-OH
4-F
218 [33]
198–200 [34]
200–201[28]
215–216 [35]
208–209 [28]
200–201 [35]
210 [33]
4.3.1. Methyl (5b)-3-[2-
[[(phenylideneamin)amino]thioxomethyl]hydrazinylidene]-
7-oxocholan-24-oate (7a)
3g
3h
3i
C8H9BrN4S
C8H9ClN4S
C8H8F2N4S
C8H9ClN4S
C8H9N5O2S
2-Br
White crystal. Yield 84%. Mp 125–126 8C (EtOH),
2-Cl
D = À108.7 (c 0.10, CH2Cl2). IR (KBr, cmÀ1): 3263,
2,4-F
4-Cl
–
[
a
]
3j
211–212 [28]
204–206 [36]
2945, 2871, 1735, 1708, 1630, 1517, 1380, 1235, 1215,
1169, 1099, 1021, 757. 1H NMR (DMSO-d6, 400 MHz):
3k
4-NO2
d

982
Z. Shi et al. / C. R. Chimie 16 (2013) 977–984
11.72 (s, 1H, NH), 10.60 (s, 1H, NH), 8.10 (s, 1H, 5CH), 7.72
(s, 2H, ArH), 7.43 (t, J = 5.6 Hz, 3H, ArH), 3.58 (s, 3H,
COOCH3), 1.21 (s, 3H, 19–CH3), 0.89 (d, J = 6.4 Hz, 3H, 21–
CH3), 0.64 (s, 3H, 18–CH3). ESI–MS m/z (%): 1157 ([2M + 1]+,
100). Anal. calcd for C33H46N4O3S: C, 68.48; H, 8.01; N,
9.68. Found. C, 68.45; H, 8.04; N, 9.69%.
COOCH3), 1.21 (s, 3H, 19–CH3), 0.89 (d, J = 6.4 Hz, 3H, 21–
CH3), 0.64 (s, 3H, 18–CH3). ESI–MS m/z (%): 611 ([M + 1]+,
100). Anal. calcd for C33H46N4O5S: C, 64.89; H, 7.59; N,
9.17. Found. C, 64.92; H, 7.57; N, 9.15%.
4.3.6. Methyl (5b)-3-[2-[[(4-fluorophenylideneamin)-
amino]thioxomethyl]hydrazinylidene]-7-oxocholan-24-oate
(7f)
4.3.2. Methyl (5b)-3-[2-[[(2-hydroxyphenylideneamin)-
amino]thioxomethyl]hydrazinylidene]-7-oxocholan-24-oate
(7b)
White crystal. Yield 84%. Mp 124–125 8C (EtOH),
[
a]
D = À97.9 (c 0.10, CH2Cl2). IR (KBr, cmÀ1): 3229, 2945,
White crystal. Yield 86%. Mp 173–174 8C (EtOH),
2870, 1735, 1708, 1601, 1503, 1380, 1232, 1156, 1097,
1017, 896, 835. 1H NMR (DMSO-d6, 400 MHz):
d 11.72 (s,
[
a
]
D = À58.9 (c 0.10, CH2Cl2). IR (KBr, cmÀ1): 3265, 3128,
2949, 1735, 1707, 1615, 1533, 1488, 1438, 1379, 1272,
1226, 1199, 1153, 1038, 958. 1H NMR (DMSO-d6,
1H, NH), 10.61 (s, 1H, NH), 8.08 (s, 1H, 5CH), 7.77 (t, J = 12
and 5.2 Hz, 2H, ArH), 7.29 (t, J = 8.8 and 8.8 Hz, 2H, ArH),
3.58 (s, 3H, COOCH3), 1.21 (s, 3H, 19–CH3), 0.89 (d,
J = 6.4 Hz, 3H, 21–CH3), 0.64 (s, 3H, 18–CH3). ESI–MS m/z
(%): 1193 ([2M + 1]+, 100). Anal. calcd for C33H45FN4O3S: C,
66.41; H, 7.60; N, 9.39. Found. C, 66.48; H, 7.58; N, 9.37%.
400 MHz):
d 11.74 (s, 1H, NH), 11.64 (s, 1H, OH), 10.89
(d, J = 9.2 Hz, 1H, NH), 8.69 (s, 1H, 5CH), 7.35 (s, 1H, ArH),
7.29 (t, J = 8.0 Hz, 2H, ArH), 6.91 (t, J = 4.4 Hz, 1H, ArH), 3.58
(s, 3H, COOCH3), 1.21 (s, 3H, 19–CH3), 0.89 (d, J = 6.4 Hz, 3H,
21–CH3), 0.64 (s, 3H, 18–CH3). ESI–MS m/z (%): 1189
([2M + 1]+, 100). Anal. calcd for C33H46N4O4S: calcd. C,
66.64; H, 7.79; N, 9.42. Found. C, 66.59; H, 7.81; N, 9.43%.
4.3.7. Methyl (5b)-3-[2-[[(2-bromophenylideneamin)-
amino]thioxomethyl]hydrazinylidene]-7-oxocholan-24-oate
(7g)
4.3.3. Methyl (5
b
)-3-[2-[[(4-methoxyphenylideneamin)-
White crystal. Yield 82%. Mp 129–130 8C (EtOH),
amino]thioxomethyl]hydrazinylidene]-7-oxocholan-24-oate
(7c)
[
a]
D = À79.4 (c 0.10, CH2Cl2). IR (KBr, cmÀ1): 3236, 2944,
2869, 1734, 1708, 1589, 1516, 1466, 1435, 1378, 1228,
1203, 1168, 1100, 1021, 821, 757. 1H NMR (DMSO-d6,
400 MHz): d 11.80 (s, 1H, NH), 10.68 (s, 1H, NH), 8.47 (s, 1H,
5CH), 8.12 (s, 1H, ArH), 7.67 (d, J = 8.0 Hz, 1H, ArH), 7.47–
7.41 (m, 1H, ArH), 7.37–7.33 (m, 1H, ArH), 3.58 (s, 3H,
COOCH3), 1.21 (s, 3H, 19–CH3), 0.89 (d, J = 6.4 Hz, 3H, 21–
CH3), 0.64 (s, 3H, 18–CH3). ESI–MS m/z (%): 657 ([M + 1]+,
100), 658 ([M + 2]+, 93). Anal. calcd for C33H45BrN4O3S: C,
60.26; H, 6.90; N, 8.52. Found. C, 60.30; H, 6.87; N, 8.50%.
White crystal. Yield 89%. Mp 120–121 8C (EtOH),
[
a
]
D = À154.8 (c 0.10, CH2Cl2). IR (KBr, cmÀ1): 3253,
2944, 1735, 1708, 1605, 1505, 1379, 1301, 1250, 1169,
1103, 1027, 931. 1H NMR (DMSO-d6, 400 MHz):
d 11.63 (s,
1H, NH), 10.51 (s, 1H, NH), 8.05 (s, 1H, 5CH), 7.65 (t,
J = 4.4 Hz, 2H, ArH), 7.01 (d, J = 8.4, 2H, ArH), 3.80 (s, 3H, Ar–
OCH3), 3.58 (s, 3H, COOCH3), 1.21 (s, 3H, 19–CH3), 0.89 (d,
J = 6.4 Hz, 3H, 21–CH3), 0.64 (s, 3H, 18–CH3). ESI–MS m/z
(%): 631 ([M + 23]+, 100). Anal. calcd for C34H48N4O4S: C,
67.07; H, 7.95; N, 9.20. Found. C, 67.00; H, 6.97; N, 9.26%.
4.3.8. Methyl (5b)-3-[2-[[(2-chlorophenylideneamin)-
amino]thioxomethyl]hydrazinylidene]-7-oxocholan-24-oate
(7h)
4.3.4. Methyl (5b)-3-[2-[[(3-hydroxyphenylideneamin)-
amino]thioxomethyl]hydrazinylidene]-7-oxocholan-24-oate
(7d)
White crystal. Yield 80%. Mp 127–128 8C (EtOH),
[
a]
D = À86.7 (c 0.10, CH2Cl2). IR (KBr, cmÀ1): 3239, 2945,
White crystal. Yield 82%. Mp 144–145 8C (EtOH),
2870, 1735, 1708, 1594, 1516, 1468, 1437, 1379, 1228,
1204, 1169, 1049, 893. 1H NMR (DMSO-d6, 400 MHz):
d
[
a
]
D = À100.7 (c 0.10, CH2Cl2). IR (KBr, cmÀ1): 3272,
2946, 2870, 1707, 1581, 1522, 1451, 1380, 1225, 1170,
955, 784. 1H NMR (DMSO-d6, 400 MHz):
11.79 (s, 1H, NH), 10.68 (s, 1H, NH), 8.51 (s, 1H, 5CH), 8.19
(s, 1H, ArH), 7.58–7.48 (m, 1H, ArH), 7.45–7.42 (m, 2H,
ArH), 3.58 (s, 3H, COOCH3), 1.21 (s, 3H, 19–CH3), 0.89 (d,
J = 6.4 Hz, 3H, 21–CH3), 0.64 (s, 3H, 18–CH3). ESI–MS m/z
(%): 1225 ([2M + 1]+, 100), 1226 ([2M + 2]+, 42). Anal. calcd
for C33H45ClN4O3S: C, 64.63; H, 7.40; N, 9.14. Found. C,
64.60; H, 7.41; N, 9.17%.
d 11.36 (s, 1H,
NH), 10.57 (s, 1H, NH), 9.62 (d, J = 5.2 Hz, 1H, OH), 8.02 (s,
1H, 5CH), 7.28–7.18 (m, 1H, ArH), 7.14–7.07 (m, 2H, ArH),
6.82 (d, J = 7.6 Hz, 1H, ArH), 3.58 (s, 3H, COOCH3), 1.21 (s,
3H, 19–CH3), 0.89 (d, J = 6.4 Hz, 3H, 21–CH3), 0.64 (s, 3H,
18–CH3). ESI–MS m/z (%): 1189 ([2M + 1]+, 100). Anal. calcd
for C33H46N4O4S: C, 66.64; H, 7.79; N, 9.42. Found. C, 66.68;
H, 7.75; N, 9.45%.
4.3.9. Methyl (5b)-3-[2-[[(2,4-difluorophenylideneamin)-
amino]thioxomethyl]hydrazinylidene]-7-oxocholan-24-oate
(7i)
4.3.5. Methyl (5b)-3-[2-[[(2,4-dihydroxyphenylideneamin)-
amino]thioxomethyl]hydrazinylidene]-7 -oxocholan-24-oate
(7e)
White crystal. Yield 86%. Mp 126–127 8C (EtOH),
[
a]
D = À102.7 (c 0.10, CH2Cl2). IR (KBr, cmÀ1): 3253,
White crystal. Yield 85%. Mp 222–223 8C (EtOH),
2948, 2872, 1734, 1708, 1614, 1523, 1499, 1431, 1381,
1279, 1230, 1171, 1140, 1100, 965, 850. 1H NMR (DMSO-
d6, 400 MHz): d 11.80 (s, 1H, NH), 10.68 (s, 1H, NH), 8.72 (s,
1H, 5CH), 8.19 (d, J = 10.4 Hz, 1H, ArH), 7.41–7.33 (m, 1H,
ArH), 7.22 (d, J = 8.0 Hz, 1H, ArH), 3.58 (s, 3H, COOCH3),
1.21 (s, 3H, 19–CH3), 0.82 (d, J = 6.0 Hz, 3H, 21–CH3), 0.64
(s, 3H, 18–CH3). ESI–MS m/z (%): 615 ([M + 1]+, 100); Anal.
[
a
]
D = À97.4 (c 0.10, CH2Cl2). IR (KBr, cmÀ1): 3309, 3233,
3149, 2950, 1732, 1709, 1624, 1515, 1378, 1330, 1208,
1171, 1111, 979, 885. 1H NMR (DMSO-d6, 400 MHz):
d
11.76 (s, 1H, NH), 11.55 (s, 1H, OH), 10.75 (s, 1H, NH), 9.92
(s, 1H, OH), 8.56 (s, 1H, 5CH), 7.12 (s, 1H, ArH), 6.34 (d,
J = 7.2 Hz, 1H, ArH), 6.28 (d, J = 2.0 Hz, 1H, ArH), 3.58 (s, 3H,

Z. Shi et al. / C. R. Chimie 16 (2013) 977–984
983
calcd for C33H44F2N4O3S: C, 64.47; H, 7.21; N, 9.11. Found.
C, 64.50; H, 7.20; N, 9.10%.
nutrient agar medium and 20
m
L (40, 20, 10, 5 and 0 mg/
mL) of the sample were added per disc in five replications.
Then, the paper discs impregnated with the solutions of
the tested compounds in DMSO were placed on the surface
of the media inoculated with the microorganism. The
plates were incubated at 37 8C for 16–18 h (Table 3). The
IC50 was computed from the diameter values of growth
inhibition zones.
4.3.10. Methyl (5b)-3-[2-[[(4-chlorophenylideneamin)-
amino]thioxomethyl]hydrazinylidene]-7-oxocholan-24-oate
(7j)
White crystal. Yield 84%. Mp 131–132 8C (EtOH),
[
a
]
D = À84.2 (c 0.10, CH2Cl2). IR (KBr, cmÀ1): 3250, 2948,
2864, 1731, 1708, 1633, 1591, 1521, 1489, 1241, 1209,
1168, 1086, 1016, 828. 1H NMR (DMSO-d6, 400 MHz):
d
4.5.2. Microdilution assay
11.75 (s, 1H, NH), 10.49 (s, 1H, NH), 8.49 (s, 1H, 5CH), 7.71
(dd, J = 10.4 and 8.8 Hz, 2H, ArH), 7.56–7.44 (m, 2H, ArH),
3.58 (s, 3H, COOCH3), 1.17 (s, 3H, 19–CH3), 0.82 (d,
J = 6.0 Hz, 3H, 21–CH3), 0.64 (s, 3H, 18–CH3). ESI–MS m/z
(%): 613 ([M + 1]+, 100), 614 ([M + 2]+, 43). Anal. calcd for
The MIC were determined by microdilution, both
methods [41] using standard inoculums of 2 Â 106 CFU/
mL. Serial dilutions of the test compounds were prepared
to final concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5,
0.25 and 0
mg/mL in DMSO. To each tube was added 1 mL
C
33H45ClN4O3S: C, 64.63; H, 7.40; N, 9.14. Found. C, 64.68;
of a bacterial fluid (0.5 McFarland standard). The MIC that
inhibits the visible growth after 18 h was determined
visually after incubation for 18 h at 37 8C (Table 4).
H, 7.42; N, 9.12%.
4.3.11. Methyl (5b)-3-[2-[[(4-nitrophenylideneamin)-
amino]thioxomethyl]hydrazinylidene]-7-oxocholan-24-oate
(7k)
Acknowledgments
White crystal. Yield 85%. Mp 129–130 8C (EtOH),
This research was financially supported by the Science
and Technology Department of Sichuan Province (No.
2011JY0035), the Fundamental Research Funds of the
Central University, Southwest University for Nationalities
(No. 13NZYQN12) and the Postgraduate Degree Construc-
tion Project of Southwest University for Nationalities of
P.R. China (No. 2013XWD-S0703).
[a
]
D = À135.7 (c 0.11, CH2Cl2). IR (KBr, cmÀ1): 3238,
2947, 2871, 1735, 1708, 1589, 1519, 1484, 1439, 1380,
1241, 1207, 1171, 1009, 822. 1H NMR (DMSO-d6,
400 MHz):
d 11.76 (s, 1H, NH), 10.65 (s, 1H, NH), 8.48 (s,
1H, 5CH), 7.83–7.61 (m, 4H, ArH), 3.58 (s, 3H, COOCH3),
1.17 (s, 3H, 19–CH3), 0.82 (d, J = 6.4 Hz, 3H, 21–CH3), 0.64
(s, 3H, 18–CH3). ESI–MS m/z (%): 1247 ([2M + 1]+, 100).
Anal. calcd for C33H45N5O5S: C, 64.54; H, 7.27; N, 11.23.
Found. C, 64.59; H, 7.25; N, 7.29%.
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