Synthesis and in vitro antibacterial activity of new steroidal thiosemicarbazone derivatives

Article abstract of DOI:10.1016/j.ejmech.2007.12.004

We investigated the antibacterial activity of some new steroidal thiosemicarbazone derivatives, prepared from the reaction of cholest-5-en-7-one with thiosemicarbazides, in ethanol in the presence of a few drops of HCl at 80 °C in high yield. All the compounds have been characterized by means of elemental analyses, IR, ¹H NMR and mass spectroscopic data, to find an effective antibacterial agent. The antibacterial activity was first tested in vitro by the disk diffusion assay against two Gram-positive and two Gram-negative bacteria, and then the minimum inhibitory concentration (MIC) of compounds was determined. The results showed that the steroidal thiosemicarbazones derivatives inhibit growth of both types of the bacteria (Gram-positive and Gram-negative). The acetoxy and chloro derivatives of cyclopentyl and cyclohexyl amine thiosemicarbazones were found to have more antibacterial activity than the other derivatives.

Full text of DOI:10.1016/j.ejmech.2007.12.004

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European Journal of Medicinal Chemistry 43 (2008) 2029e2034
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Short communication
Synthesis and in vitro antibacterial activity of new steroidal
thiosemicarbazone derivatives
*
Salman Ahmad Khan, Praveen Kumar, Rajkumar Joshi, Prince F. Iqbal, Kishwar Saleem
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
Received 19 May 2007; received in revised form 4 December 2007; accepted 6 December 2007
Available online 23 December 2007
Abstract
We investigated the antibacterial activity of some new steroidal thiosemicarbazone derivatives, prepared from the reaction of cholest-5-en-7-
one with thiosemicarbazides, in ethanol in the presence of a few drops of HCl at 80 ꢀC in high yield. All the compounds have been characterized
by means of elemental analyses, IR, 1H NMR and mass spectroscopic data, to find an effective antibacterial agent. The antibacterial activity was
first tested in vitro by the disk diffusion assay against two Gram-positive and two Gram-negative bacteria, and then the minimum inhibitory
concentration (MIC) of compounds was determined. The results showed that the steroidal thiosemicarbazones derivatives inhibit growth of
both types of the bacteria (Gram-positive and Gram-negative). The acetoxy and chloro derivatives of cyclopentyl and cyclohexyl amine thio-
semicarbazones were found to have more antibacterial activity than the other derivatives.
Ó 2008 Published by Elsevier Masson SAS.
Keywords: Thiosemicarbazone; Steroids; Antibacterial activity
1. Introduction
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. Considerable attention has been focused on
substituted thiosemicarbazone derivatives due to their interest-
ing biological activity. Compounds with a thiosemicarbazone
structure are known to possess tranquilizing, muscle relaxing,
psychoanaleptic, hypnotic, ulcerogenic, antidepressant, anti-
bacterial, antifungal, analgesic and anti-inflammatory proper-
ties [3e10]. Steroidal thiosemicarbazones dramatically
increase the diversity of certain biological properties [11e13].
As evident from the literature, it was noted that a lot of research
has been carried out on thiosemicarbazone derivatives but no
work has been done on steroidal (cholesterol) thiosemicarba-
zone derivative screening on bacteria. In this paper the
steroidal thiosemicarbazone derivatives (Fig. 1) have been
synthesized by the condensation of the steroidal ketones with
thiosemicarbazide. The activities of these compounds were
screened in vitro against bacteria S. aureus, S. pyogenes,
S. typhimurium, and E. coli. To the best of our knowledge this
is the first report that thiosemicarbazone analogues having
The diverse parasitic bacteria such as Staphylococcus aureus,
Staphylococcus pyogenes, Salmonella typhimurium, and
Escherichia coli have significant impact on the mucosal health
of humans. Infection with S. aureus, S. pyogenes, S. typhimu-
rium, and E. coli may have resulted in massive destruction of
host tissue and life-threatening diseases. These bacterial para-
sites cause food poisoning, rheumatic fever and diarrhea that
affect millions of individuals in developing countries. More
than 50 million people worldwide are infected and up to
110,000 of these die every year. Amoxicillin, Norfloxacin,
Ciprofloxacin are the most common drugs used for this bacterial
infection but are associated with severe side effects. Toxicity
and resistance to the drugs also play an important role in the
failure of treatment [1,2]. Hence, the present strategy for new
drug development is directed towards identifying the essential
* Corresponding author. Tel.: þ91 11 26981717/3253; fax: þ91 11
26980229/1232.
E-mail address: kishwarsaleem2003@yahoo.co.in (K. Saleem).
0223-5234/$ - see front matter Ó 2008 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2007.12.004

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S.A. Khan et al. / European Journal of Medicinal Chemistry 43 (2008) 2029e2034
C8H17
about the structure of the compounds. All the compounds
C8H17
showed intense bands in the region 1138e1185 cmꢁ1 due to
the n(C]S) stretching of the thiocarboxamide group. In addi-
tion, the absorption bands at 1528e1590 cmꢁ1 were attributed
to the n(C]N) stretching vibration, which also confirms the for-
mation of desired thiosemicarbazones in all the compounds. The
compounds (1e9) showed additional sharp bands in the region
3345e3388 cmꢁ1 due to the n(NeH) stretching.
S
H2N-NH-C-R1
HCl-C2H5OH
O
R
N-NH-CS-R1
R
NH-
NH-
(1) R, OAc , R1,
(2) R , Cl , R1
(3) R ,H R1
,
2.2. Nuclear magnetic resonance spectral studies
NH-
,
Further evidence for the formation of steroidal thiosemicar-
1
bazone compounds was obtained from the H NMR spectra,
(4) R , OAc ,R1 ,
(5) R, Cl , R1 ,
NH-
which proved to be a diagnostic tool for the elucidation of
position of the protons. Assignments of the signals are based
on the chemical shift and intensity pattern. The NeH protons
of all the thiosemicarbazones (1e9) are singlets in the range of
9.50e10.50 ppm. Other spectral studies of thiosemicarbazones
(1e9) are given in Section 4.
NH-
(6) R , H ,R1
,
NH-
NH-
(7) R, OAc ,R1 ,
(8) R , Cl, R1 ,
NH-
NH-
2.3. FAB mass studies
Characteristic peaks were observed in the mass spectra of all
thiosemicarbazone derivatives (1e9), which followed a similar
fragmentation pattern. The spectrum of compounds 1, 4 and 7
(9) R, H, R1,
ꢂ
showed molecular ion peak (Mþ ) at m/z 584, 598 and 626. The
Fig. 1. Showing the number of thiosemicarbazone compounds.
other fragments within the mass spectra of thiosemicarbazones
(1e9) are given in Section 4.
steroidal moiety inhibit the growth of bacteria (Gram-positive
and Gram-negative). The present work deals with the synthesis
of new thiosemicarbazones (Scheme 2). The structures of all
compounds were elucidated by IR, 1H NMR, mass spectra and
elemental analysis. These compounds were tested in vitro
against bacteria such as S. aureus, S. pyogenes, S. typhimurium,
and E. coli.
2.4. In vitro evaluation of antibacterial activity against
Gram-positive and Gram-negative bacteria
The in vitro antibacterial activities of thiosemicarbazone
derivatives (1e9) were carried out using the culture of S.
aureus, S. pyogenes, S. typhimurium, and E. coli by the disk dif-
fusion method [16] and then the minimum inhibitory concentra-
tion (MIC) of all the compounds were determined. Amoxicillin
(30 mg) was used as the standard drug, whereas DMSO poured
disk was used as negative control and then minimum inhibitory
concentration (MIC) was evaluated by the macro-dilution test
using standard inoculums of 10ꢁ5 CFL mLꢁ1. Serial dilutions
of the test compounds, previously dissolved in dimethyl sulfox-
ide (DMSO) were prepared to final concentrations of 512, 256,
128, 64, 32, 16, 8, 4, 2 and 1 mg mLꢁ1. To each tube was added
100 mL of 24 h old inoculums. The MIC, defined as the lowest
concentration of the test compound, which inhibits the visible
growth after 18 h, was determined visually after incubation
for 18 h, at 37 ꢀC, The thiosemicarbazone derivatives 1, 4 and
7 have acetoxy group at 3b position and compounds 2, 5 and
8 have chloro group at 3b position, respectively. The in vitro
study results showed that the compounds chloro and acetoxy
derivatives of cyclopentyl and cyclohexyl thiosemicarbazones
were found to be more active among all the thiosemicarbazone
derivatives. The susceptibility of the bacteria to the test com-
pounds was determined by the formation of an inhibitory
zone after 18 h of incubation at 36 ꢀC. Table 1 reports the inhi-
bition zones (mm) of each compound and the results are
2. Results and discussion
The yields of thiosemicarbazide products were in the range
of 60e82%. The thiosemicarbazide derivatives were synthe-
sized by using the literature procedure [14]. All thiosemicarba-
zone derivatives (1e9) were prepared by the corresponding
ketones (c, d and f) and thiosemicarbazide in dry ethanol in
the presence of a few drops of HCl and gave 62e70% yield
(Scheme 2) [15]. The obtained compounds are stable in the
solid state as well as in the solution state. Analytical data of
these compounds are in good agreement with their composition
(see Section 4). The compounds are insoluble in water but sol-
uble in most of the organic solvents. The structures of all
compounds were established by means of their spectral data
(IR, 1H NMR, FAB mass) and elemental analysis. These com-
pounds were tested in vitro against bacteria such as S. aureus,
S. pyogenes, S. typhimurium, and E. coli.
2.1. IR spectral studies
Selected diagnostic bands of the IR spectra of the steroidal
thiosemicarbazone derivatives (1e9) showed useful information

S.A. Khan et al. / European Journal of Medicinal Chemistry 43 (2008) 2029e2034
2031
Table 1
point apparatus and were uncorrected. Electronic spectra were
recorded in DMSO. IR spectra were recorded in KBr on a Per-
kineElmer model 1620 FTIR spectrophotometer. 1H NMR
spectra were recorded at ambient temperature using a Brucker
spectroscopin DPX-300 MHz spectrophotometer in DMSO.
The following abbreviations were used to indicate the peak
multiplicity: s-singlet, d-doublet, t-triplet and m-multiplet.
FAB mass spectra were recorded on a JEOL SX 102 mass
spectrometer using argon/xenon (6 kV, 10 mB gas). Steroidal
compounds aef were synthesized by the published method
[17e19].
Antibacterial activity of steroidal thiosemicarbazone derivatives, positive con-
trol (Amoxicillin), and negative control (DMSO) measured by the Halo Zone
Test (unit, mm)
Compounds
Corresponding effect on microorganisms
S. aureus
S. pyogenes
S. typhimurium
E. coli
1
12.5 ꢃ 0.5
16.2 ꢃ 0.2
10.4 ꢃ 0.5
14.6 ꢃ 0.2
13.4 ꢃ 1.4
10.2 ꢃ 1.4
12.6 ꢃ 0.6
15.6 ꢃ 0.4
9.9 ꢃ 0.4
21.0 ꢃ 0.5
e
11.8 ꢃ 0.4
13.4 ꢃ 0.5
9.5 ꢃ 0.5
16.8 ꢃ 0.4
12.2 ꢃ 1.6
9.2 ꢃ 0.3
11.4 ꢃ 0.4
14.0 ꢃ 0.4
11.3 ꢃ 0.2
22.2 ꢃ 0.4
e
14.6 ꢃ 0.2
12.5 ꢃ 0.4
9.2 ꢃ 0.5
14.5 ꢃ 0.5
14.4 ꢃ 0.6
10.4 ꢃ 0.8
13.6 ꢃ 0.5
14.6 ꢃ 0.5
10.2 ꢃ 0.4
25.2 ꢃ 0.8
e
12.4 ꢃ 0.4
15.6 ꢃ 0.3
9.4 ꢃ 0.4
13.2 ꢃ 0.2
11.8 ꢃ 1.6
7.8 ꢃ 0.4
13.8 ꢃ 0.4
12.4 ꢃ 0.4
10.0 ꢃ 0.4
20.0 ꢃ 0.2
e
2
3
4
5
6
7
8
4.1. Synthesis of thiosemicarbazides: a general method
9
Amoxicillin
DMSO
All the thioglycolic acids were prepared by the same
method (Scheme 1). Cycloalkyl amino and thio carbonyl hy-
drazines were prepared by refluxing the alkaline solution of
thioglycolic acid with hydrazine hydrate [20].
presented in Table 2. The tests use DMSO and Amoxicillin as
negative and positive controls. The molecular structure of these
active compounds showed enhanced activity. The distinct dif-
ference in the antibacterial property of the thiosemicarbazones
further justifies the purpose of this study. The importance of
such work lies in the possibility that the new compound might
be more effective against bacteria for which a thorough
investigation regarding the structureeactivity relationship,
toxicity and the biological effects which would be helpful in
designing more potent antibacterial agents for therapeutic use
is required.
4.2. Synthesis of thiosemicarbazones: a general method
Steroidal thiosemicarbazones were synthesized (Scheme 2)
by refluxing the solution of cyclopentyl, cyclohexyl and cyclo-
octyl thiosemicarbazide (0.03 mol) in ethanol in the presence
S=C=S
KOH
+
HR1
+
ClCH2COONa
3. Conclusion
This research examined the antibacterial activities of new
steroidal thiosemicarbazones prepared by the reaction of ste-
roidal ketones with thiosemicarbazides substituted by different
amines. The in vitro antibacterial activity of these compounds
revealed that the compounds chloro and acetoxy derivatives of
cyclopentyl and cyclohexyl thiosemicarbazones were found to
be more active among all the thiosemicarbazone compounds.
S
NaOOCCH2-S-C-R1
HCl
S
4. Experimental
HOOCCH2-S-C-R1
Reactions were monitored by TLC analysis using Merck
silica gel 60F-254 thin layer plates. All the chemicals were
purchased from Aldrich chemical company (USA). Elemental
analysis (C, H, N) was carried out by Central Drug Research In-
stitute, Lucknow, India, and the results were within 0.6% of cal-
culated values. Melting points were recorded on a KSW melting
NH2-NH2.H2O
S
NH2-NH-C-R1
Table 2
Minimum inhibitory concentration (MIC) of steroidal thiosemicarbazone
derivatives and positive control (Amoxicillin)
MIC (mg mLꢁ1
Strain
)
Compounds
3
Positive
control
NH-
NH-
R 1 =
NH-
,
,
1
2
4
5
6
7
8
9
S. aureus
S. Pyogenes
32 64 256 64 32 128 128 64 256 32
64 64 128 64 64 256 64 128 128 32
Where R 1 = Cyclopentyl, cyclohexyl and cyclooctyl amine
S. typhimurium 64 128 128 64 128 256 128 128 128 32
E. coli 32 64 256 32 64 128 128 64 256 32
Scheme 1. Schematic diagram showing the synthesis of thiosemicarbazide.

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S.A. Khan et al. / European Journal of Medicinal Chemistry 43 (2008) 2029e2034
HO
pyridine
acetic anhydride
thionyl chloride
CH3COO
Cl
(b)
(a)
t- butyl chromate
amyl alcohol
t- butyl chromate
Na
S
(c)
(d)
O
O
CH3COO
Cl
(e)
H2N-NH-C-R1
S
HCl-C2H5OH
HCl- C2H5OH
t- butyl chromate
H2N-NH-C-R1
S
NNH-C-R1
S
NNH-C-R1
CH3COO
O
(f)
Cl
S
HCl-C2H5OH
H2N-NH-C-R1
S
NNH-C-R1
Scheme 2. Showing the synthesis of compounds 1e9.
of a few drops of HCl and the alcoholic solution of steroidal
ketones (0.03 mol) at 60 ꢀC for 5 h with continuous stirring.
After cooling the compounds were filtered and recrystallized
from the appropriate solvent.
4.2.1. 3b-Acetoxy
thiosemicarbazone (1)
cholest-5-en-7-one
cyclopentyl
Yield: 53.6%; m.p. 172 ꢀC; Anal. calc. for C35H57N3O2S:
C, 72.04; H, 9.77; N, 7.20. Found: C, 72.02; H, 9.75; N,

S.A. Khan et al. / European Journal of Medicinal Chemistry 43 (2008) 2029e2034
2033
7.18%. IR (KBr) nmax cmꢁ1: 3380 (NH), 1725 (OCOCH3),
(M ꢁ Cl), 491 (M ꢁ C6H11), 476 (M ꢁ C6H12N), 432
(M ꢁ C7H12NS), 417 (M ꢁ C7H13N2S), 403 (M ꢁ C7H13N3S).
1
1625 (weak, C]C), 1590 (C]N), 1162 (C]S). H NMR
(DMSO) (d): 10.5 (s, 1H, NH), 7.54 (d, 1H, eNH), 6.08 (s,
1H, C6eH), 4.5 (br m, 1H, J ¼ 17 Hz, C3aeH, axial), 2.50
(s, 3H, OCOCH3), 1.2 (C10eCH3), 0.71 (C13eCH3) 0.97,
4.2.6. Cholest-5-en-7-one cyclohexyl thiosemicarbazone (6)
Yield: 53.6%; m.p. 122 ꢀC; Anal. calc. for C34H57N3S: C,
76.73; H, 10.12; N, 7.46. Found: C, 76.68; H, 10.08; N,
7.42%. IR (KBr) nmax cmꢁ1: 3388 (NH), 1618 (C]C), 1546
(C]N), 1185 (C]S). 1H NMR (DMSO) (d): 9.9 (s, 1H,
NH), 6.2 (d, 1H, NH), 5.4 (s, 1H, C6eH), 1.06 (C10eCH3),
0.68 (C13eCH3), 0.86, 0.80 (other methyl protons). Mass
ꢂ
0.87 (other methyl protons). Mass spectra (Mþ ) at m/z 584,
525 (M ꢁ AcO), 515 (M ꢁ C5H9), 500 (M ꢁ C5H10N), 456
(M ꢁ C6H10NS), 441 (M ꢁ C6H11N2S), 427 (M ꢁ C6H11N3S).
4.2.2. 3b-Chloro cholest-5-en-7-one cyclopentyl
thiosemicarbazone (2)
ꢂ
spectra (Mþ ) at m/z 540, 457 (M ꢁ C6H11), 442
Yield: 53.6%; m.p. 164 ꢀC; Anal. calc. for C33H54N3SCl:
C, 70.52; H, 9.94; N, 7.47. Found: C, 70.48; H, 9.97; N,
7.44%. IR (KBr) nmax cmꢁ1: 3345 (NH), 1620 (C]C), 1585
(M ꢁ C6H12N), 398 (M ꢁ C7H12NS), 383 (M ꢁ C7H13N2S),
369 (M ꢁ C7H13N3S).
1
(C]N), 1145 (C]S), 725 (CeCl). H NMR (DMSO) (d):
4.2.7. 3b-Acetoxy cholest-5-en-7-one cyclooctyl
10.46 (s, 1H, NH), 7.38 (d, 1H), 6.06 (s, 1H, C6eH), 4.05
(br m, 1H, J ¼ 15 Hz, C3aeH, axial), 1.20 (C10eCH3),
0.69 (C13eCH3), 0.94 and 0.84 (remaining methyl protons).
thiosemicarbazone (7)
Yield: 53.6%; m.p. 108 ꢀC; Anal. calc. for C38H63N3O2S:
C, 72.96; H, 10.08; N, 6.72. Found: C, 72.92; H, 10.04; N,
6.70%. IR (KBr) nmax cmꢁ1: 3365 (NH), 1715 (OCOCH3),
1605 (C]C), 1570 (C]N), 1148 (C]S). 1H NMR
(DMSO) (d): 10.3 (s, 1H, NH), 7.4 (d, 1H, NH), 4.2 (br m,
J ¼ 17 Hz, C3aeH, axial), 2.18 (s, 3H, OCOCH3) 4.12 (m,
14H, eCH2), 1.22 (C10eCH3), 0.74 (C13eCH3) 0.98, 0.87
ꢂ
Mass spectra (Mþ ) at m/z 560, 525 (M ꢁ Cl), 491
(M ꢁ C5H9), 476 (M ꢁ C5H10N), 432 (M ꢁ C6H10NS), 417
(M ꢁ C6H11N2S), 403 (M ꢁ C6H11N3S).
4.2.3. Cholest-5-en-7-one cyclopentyl thiosemicarbazone (3)
Yield: 53.6%; m.p. 142 ꢀC; Anal. calc. for C33H55N3S: C,
76.50; H, 10.00; N, 7.65. Found: C, 76.45; H, 9.92; N,
7.62%. IR (KBr) nmax cmꢁ1: 3386 (NH), 1628 (C]C), 1568
ꢂ
(other methyl protons). Mass spectra (Mþ ) at m/z 626, 567
(M ꢁ AcO), 515 (M ꢁ C8H15), 500 (M ꢁ C8H16N), 456
(M ꢁ C9H16NS), 441 (M ꢁ C9H17N2S), 427 (M ꢁ C9H17N3S).
1
(C]N), 1185 (C]S), 2928 (CeH). H NMR (DMSO) (d):
9.7 (s, 1H, NH), 5.9 (d, 1H, NH), 5.22 (s, 1H, C6eH), 0.07
(C10eCH3), 0.66 (C13eCH3), 0.90 and 0.82 (remaining pro-
4.2.8. 3b-Chloro cholest-5-en-7-one cyclooctyl
thiosemicarbazonee (8)
Yield: 53.6%; m.p. 114 ꢀC; Anal. calc. for C36H60N3SCl:
C, 71.5; H, 10.2; N, 6.95. Found: C, 71.2; H, 9.08; N,
6.88%. IR (KBr) nmax cmꢁ1: 3355 (NH), 1618 (C]C), 1554
ꢂ
tons). Mass spectra (Mþ ) at m/z 526, 457 (M ꢁ C5H9), 442
(M ꢁ C5H10N), 398 (M ꢁ C6H10NS), 383 (M ꢁ C6H11N2S),
369 (M ꢁ C6H11N3S).
1
(C]N), 1142 (C]S), 705 (CeCl). H NMR (DMSO) (d):
4.2.4. 3b-Acetoxy cholest-5-en-7-one cyclohexyl
thiosemicarbazone (4)
9.86 (s, 1H, NH), 7.1(d, 1H, NH), 5.53 (s, 1H, C6eH) 4.23
(br m, J ¼ 15 Hz: C3aeH, axial), 3.6 (m, 14H, eCH2), 1.24
(C10eCH3), 0.67 (C13eCH3), 0.91, 0.89 (remaining methyl
Yield: 53.6%; m.p. 146 ꢀC; Anal. calc. for C36H59N3O2S:
C, 72.36; H, 9.88; N, 7.03. Found: C, 72.32; H, 9.81; N,
7.02%. IR (KBr) nmax cmꢁ1: 3375 (NH), 1728 (OCOCH3),
1612 (C]C), 1582 (C]N), 1152 (C]S). 1H NMR
(DMSO) (d): 10.05 (s, 1H, eNH), 7.26 (d, 1H, eNH), 6.04
(s, 1H, C6eH), 4.3 (br m, J ¼ 17 Hz, C3aeH axial), 2.2 (s,
3H, OCOCH3), 4.5 (m, 10H, eCH2), 1.20 (C10eCH3), 0.73
(C13eCH3), 0.94, 0.87 (other methyl protons). Mass spectra
ꢂ
protons). Mass spectra (Mþ ) at m/z 602, 567 (M ꢁ Cl), 491
(M ꢁ C8H15), 476 (M ꢁ C8H16N), 432 (M ꢁ C9H16NS), 417
(M ꢁ C9H17N2S), 403 (M ꢁ C9H17N3S).
4.2.9. Cholest-5-en-7-one cyclooctyl thiosemicarbazone (9)
Yield: 53.6%; m.p. 102 ꢀC; Anal. calc. for C36H61N3S: C,
77.15; H, 10.32; N, 7.10. Found: C, 77.12; H, 10.26; N,
7.06%. IR (KBr) nmax cmꢁ1: 3362 (NH), 1623 (C]C),1518
(C]N), 1138 (C]S). 1H NMR (DMSO) (d): 9.5 (s, 1H,
NH), 5.4 (d, 1H, NH), 4.8 (s, 1H, C6eH), 1.16 (C10eCH3),
0.70 (C13eCH3), 0.92 and 0.86 (other methyl protons).
ꢂ
(Mþ ) at m/z 598, 539 (M ꢁ AcO), 515 (M ꢁ C6H11), 500
(M ꢁ C6H12N), 456 (M ꢁ C7H12NS), 441 (M ꢁ C7H13N2S),
427 (M ꢁ C7H13N3S).
ꢂ
4.2.5. 3b-Chloro cholest-5-en-7-one cyclohexyl
thiosemicarbazone (5)
Mass spectra (Mþ ) at m/z 568, 457 (M ꢁ C8H15), 442
(M ꢁ C8H16N), 398 (M ꢁ C9H16NS), 383 (M ꢁ C9H17N2S),
369 (M ꢁ C9H17N3S).
Yield: 53.6%; m.p. 126 ꢀC; Anal. calc. for C34H56N3SCl:
C, 70.89; H, 10.0; N, 7.29. Found: C, 70.84; H, 9.82; N,
7.25%. IR (KBr) nmax cmꢁ1: 3380 (NH), 1622 (C]C), 1566
4.3. Organism culture and in vitro screening
1
(C]N), 1155 (C]S), 715 (CeCl). H NMR (DMSO) (d):
10.27 (s, 1H, NH), 7.3 (d, 1H, NH), 4.2 (br m, 1H,
J ¼ 15 Hz, C3aeH), 4.28 (m, 10H, eCH2), 5.85 (s, 1H,
C6eH), 1.21 (C10eCH3), 0.68 (C13eCH3), 1.09 and 0.88
Antibacterial activity was done by the disk diffusion method
with minor modifications. S. aureus, S. pyogenes, S. typhimu-
rium, and E. coli were subcultured in BHI medium and incu-
bated for 18 h at 37 ꢀC, and then the bacterial cells were
ꢂ
(other methyl protons). Mass spectra (Mþ ) at m/z 574, 539

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S.A. Khan et al. / European Journal of Medicinal Chemistry 43 (2008) 2029e2034
suspended, according to the McFarland protocol in saline solu-
tion to produce a suspension of about 10ꢁ5 CFU mLꢁ1: 10 mL
of this suspension was mixed with 10 mL of sterile antibiotic
agar at 40 ꢀC and poured onto an agar plate in a laminar flow
cabinet. Five paper disks (6.0 mm diameter) were fixed onto
nutrient agar plate. One milligram of each test compound
was dissolved in 100 ml DMSO to prepare stock solution and
from the stock solution different concentrations 10, 20, 25,
50, and 100 mg/ml of each test compound were prepared. All
these concentrations of the different compounds 1e9 were
poured over disk plate. Amoxicillin (30 mg) was used as a stan-
dard drug (positive control). DMSO poured disk was used as
negative control. The susceptibility of the bacteria to the test
compounds was determined by the formation of an inhibitory
zone after 18 h of incubation at 36 ꢀC. Table 1 reports the
inhibition zones (mm) of each compound and the controls.
The minimum inhibitory concentration (MIC) was evaluated
by the macro-dilution test using standard inoculums of
10ꢁ5 CFL mLꢁ1. Serial dilutions of the test compounds, previ-
ously dissolved in dimethyl sulfoxide (DMSO) were prepared
to final concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2
and 1 mg mLꢁ1. To each tube was added 100 mL of a 24 h old
inoculum. The MIC, defined as the lowest concentration of
the test compound, which inhibits the visible growth after
18 h, was determined visually after incubation for 18 h, at
37 ꢀC, and the results are presented in Table 2. The tests use
DMSO and Amoxicillin as negative and positive controls.
References
[1] S.A. Puertoa, G.J. Fernandeza, L.D.J. Castillob, M. Jose, S. Pinoa,
P.G. Anguloa, Diagn. Microbiol. Infect. Dis. 54 (2006) 135e139.
[2] C.M. Nolan, G.E. Chalhub, G.D. Nash, T. Yamauchi, Antimicrob. Agents
Chemother. (1979) 171e175.
[3] H.I. Hall, Y.S. Cheen, J.B. Barnes, D.X. Wext, Met. Based Drugs 6
(1999) 143.
[4] E. Bermejo, R. Carballo, A. Castineiras, R. Dominguez,
E.A. Liberta, C. Maichle-Mossmer, Z.X.D. Wext, Z. Naturforsch.
54 (1999) 777.
[5] M.J. Perez, I.A. Matesanz, A. Marin-Ambite, P. Navarro, C. Alonso,
P. Souza, J. Inorg. Biochem. 75 (1999) 255.
[6] H.K. Reddy, S.P. Reddy, R.P. Babu, J. Inorg. Biochem. 77 (1999)
169.
[7] P.F. Kelly, A.Z.M. Slawin, A. Soriano-Rama, J. Chem. Soc., Dalton
Trans. 53 (1996) 53e59.
[8] X.D. West, B.S. Pardhye, B.P. Sonawane, Struct. Bonding 76 (1991).
[9] E.A. Liberta, X.D. West, BioMetals 5 (1992) 121.
[10] X.D. West, E.A. Liberta, B.S. Padhye, C.R. Chikate,
B.P. Sonawane, S.A. Kumbhar, G.R. Yerande, Coord. Chem. Rev.
49 (1993) 123.
[11] M. Merlani, S.L. Amiranashvili, G.M. Davitishvili, P.E. Kemertelidze,
K. Papdopoulos, E. Yannakopoulos, Chem. Nat. Compd. 42 (2006)
194e197.
[12] M. Merlani, P.E. Kemertelidze, K. Papadopoulos, N. Men’Shova, Russ. J.
Bioorg. Chem. 30 (2004) 497e501.
[13] A. Omar, E.M. Mohsen, M.S. El-Khawass, B.A. Makar, M.N. Bakry,
T.T. Daabees, Pharmazie 33 (1978) 577e580.
[14] M. Abid, A. Azam, Bioorg. Med. Chem. 13 (2005) 2213e2220.
[15] S. Singh, F. Athar, M.R. Maurya, A. Azam, Eur. J. Med. Chem. 41 (5)
(2006) 592e598.
[16] S.A. Khan, K. Saleem, Z. Khan, Eur. J. Med. Chem. 42 (2007) 103e108.
[17] A.W. Bauer, W.M. Kirby, J.C. Sherris, M. Turck, Am. J. Clin. Pathol.
(1966) 493e496.
Acknowledgment
[18] G.W. Dauben, G.J. Fonken, J. Chem. Soc. 48 (1956) 4736.
[19] A.H. Millurn, E.V. Truter, J. Chem. Soc. (1956) 1736.
[20] N. Bharti, K. Husain, M.T. Gonzalez, D.E. Cruz-Vega, J. Castro-Garza,
B.D. Mata-Cardenas, F. Naqvi, A. Azam, Bioorg. Med. Chem. Lett. 12
(2002) 3475e3478.
Authors are thankful to Dr. Amir Azam, Department of
Chemistry, Jamia Millia Islamia, New Delhi for providing lab-
oratory facilities and chemicals for research.