Synthesis, spectral studies and in vitro antibacterial activity of steroidal thiosemicarbazone and their palladium (Pd (II)) complexes

Article abstract of DOI:10.1016/j.ejmech.2008.06.008

We investigated the antibacterial activity of some new steroidal thiosemicarbazone and their Pd(II) metal complexes. Metal complexes were prepared from the reaction of steroidal thiosemicarbazone with [Pd(DMSO)₂Cl₂]. Coordination via

Full text of DOI:10.1016/j.ejmech.2008.06.008

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European Journal of Medicinal Chemistry 44 (2009) 2270e2274
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, spectral studies and in vitro antibacterial activity of steroidal
thiosemicarbazone and their palladium (Pd (II)) complexes
*
Salman Ahmad Khan , Mohamad Yusuf
Department of Chemistry, Punjabi University, Patiala, Punjab 147002, India
Received 9 December 2007; received in revised form 10 June 2008; accepted 13 June 2008
Available online 21 June 2008
Abstract
We investigated the antibacterial activity of some new steroidal thiosemicarbazone and their Pd(II) metal complexes. Metal complexes were
prepared from the reaction of steroidal thiosemicarbazone with [Pd(DMSO)2Cl2]. Coordination via the thionic sulphur and the azomethine
nitrogen atom of the thiosemicarbazone to the metal ion, the thiosemicarbazone derivatives were obtained by the thiosemicarbazide with
steroidal ketones. All the compounds have been confirmed by spectral data. The antibacterial activity of these compounds 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) was determined. The results showed that steroidal complexes are better inhibit growth as compared to steroidal thiosemicarbazones
of both types of the bacteria (gram-positive and gram-negative); compound Ia is better antibacterial agent as compared to amoxicillin.
Ó 2008 Published by Elsevier Masson SAS.
Keywords: Thiosemicarbazone; Palladium(II); Antibacterial activity
1. Introduction
square planarcomplexes[10]. Thoughtheversatilityofthiosemi-
carbazone ligands for binding to the metal ion has been well
established, there remains an ambiguity in predicting the actual
coordination mode of thiosemicarbazones. The biological activ-
ities of thiosemicarbazones are considered to be due to their abil-
ity to form chelates with heavy metal. Biological activities of
metal complexes differ from those of either ligands or the metal
ions and increased and/or decreased biological activities are
reported for several transition metal complexes, such
as copper(II) and nickel(II). The present work deals with synthe-
sis of new thiosemicarbazones (Fig. 1) and their Pd(II)
complexes (Fig. 2). The structures of all compounds were eluci-
Awide range of biological activities possessed by substituted
thiosemicarbazones and their metal complexes include
cytotoxic, antibacterial, antitumor and antileukemic properties
[1e6]. They are well known chelating ligands coordinating to
the metal ion through the sulphur and one of the hydrazinic nitro-
gen atoms (N2 or N1). Coordination through N2 results in an
unusual four membered chelate ring [7e9] while with N1 hydra-
zinic nitrogen a more stable five membered chelate ring results
[10e13]. However, on incorporation of an additional donor site
into the thiosemicarbazones, their coordination behavior
becomes unpredictable. If there is intramolecular hydrogen
bonding between either N2 nitrogen or N1 nitrogen and phenolic
OH which reduces the availability of lone pair on the imines
nitrogen preventing its coordination which might lead to the for-
mation of either unusual four member rings or more stable five
member rings. The satiric hindrance created by the bulky ligands
in six coordinated complexes is not present in four coordinated
1
dated by IR, H NMR, 13C NMR, Fab mass and elemental
analyses. These compounds were tested in vitro against bacteria
such as Staphylococcus aureus, Streptococcus pyogenes,
Salmonella typhimuurium and Escherichia coli.
2. Results and discussion
Reaction
of
steroidal
thiosemicarbazones
with
[Pd(DMSO)Cl2] gave amorphous solid compounds. All the
compounds were isolated in good yields and were stable
* Tel.: þ91 935 6201007; fax: þ91 175 2283073.
E-mail address: sahmad_phd@yahoo.co.in (S.A. Khan).
0223-5234/$ - see front matter Ó 2008 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2008.06.008

S.A. Khan, M. Yusuf / European Journal of Medicinal Chemistry 44 (2009) 2270e2274
2271
bromine
solution
HO
jones reagent
HO
Br
Br
(a)
Zn
glacialo acetic acid
O
S
Br
O
Br
C2H5OH
(c)
(b)
R-C-NHNH2
S
R-C-NH-N
(1-3)
CH3
CH3
N
N
Where R =
CH3
N
H
H
H
(1)
(2)
(3)
Fig. 1. Synthesis of compounds 1, 2, 3: 1 e cholest-5-en-3-one-o-toludinethiosemicarbazone; 2 e cholest-5-en-3-one-m-toludinethiosemicarbazone; 3 e cholest-5-
en-3-one-p-toludinethiosemicarbazone.
both in the solid and solution state. Analytical data of these
compounds were in good agreement with their composition
(see Section 4). The complexes were insoluble in water,
methanol and ethanol, soluble in DMF and DMSO. The com-
plexes do not undergo any weight loss up to 245 ꢀC, which
suggest their fair thermal stability. The structures of the
ligands and complexes presented in Figs. 1 and 2a,b were es-
tablished by comparing spectral data (IR, 1H NMR, 13C NMR
and Fab mass) with the free ligand along with their thermo
gravimetric analysis.
2.1. IR spectral studies
Assignments of selected characteristic IR band positions
provide significant indication for the formation of steroidal thi-
osemicarbazones and their complexes. The thiosemicarbazones
S
SH
R-C-NH-N
R-C=N-N
Thione form (IA)
Thiol form (IB)
N
Cl
NH
C
Pd
Cl
S
R
Fig. 2. (a) Structure of palladium(II) complexes: (1a) R ¼ eNHC7H7; (2a) R ¼ eNHC7H7; (3a) R ¼ eNHC7H7. (b) Synthesis of complexes 1a, 2b, 3c
(TSCN ¼ thiosemicarbazones).

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S.A. Khan, M. Yusuf / European Journal of Medicinal Chemistry 44 (2009) 2270e2274
can exist as thione and thioltautomeric from IA and IB,
respectively.
product. The total % weight loss corresponds to the loss of
the respective ligand after considering the transfer of one
sulphur atom to the metal ion and residue corresponds to the
metal sulphide.
S
SH
2.4. FAB mass analysis
R-C-NH-N
R-C=N-N
Characteristic peaks were observed in the mass spectra of
ligand and their metal complexes, which followed the similar
fragmentation pattern. The spectrum of compound (1) showed
Thione form (IA)
Thiol form (IB)
However, the existence of a strong in the region
1034e1042 cmꢁ1 due to v (C]S) and absence of any band
in the region 2500e2600 cmꢁ1 due to v (CeSH) suggesting
that all the thiosemicarbazones remain in their thione form.
The negative shift (18e33 cmꢁ1) of v (C]N) band
observed in all complexes indicates the involvement of azome-
thine nitrogen upon complexation. This was further supported
by the upward shift of NeN band of ligand on coordination.
The strong band at 1028e1082 cmꢁ1 ascribed to v (C]S)
of ligands is shifted to lower frequency (12e28 cmꢁ1) indicat-
ing the bonding of metal through thionic sulphur. The broad
band observed in region 3226e3252 cmꢁ1 due to v (NeH)
stretch is only slightly affected on the coordination.
ꢂ
a molecular ion peak (Mþ ) at m/z ¼ 548 and its complex
ꢂ
compound (1a) showed a molecular ion peak (Mþ ) at m/
z ¼ 725. The characteristic peaks observed within the mass
spectra of thiosemicarbazones and their metal complexes are
given in Section 4.
2.5. In vitro evaluation of antibacterial
The in vitro antibacterial activities of thiosemicarbazone
derivatives (1e3) and their metal complexes (1ae3a) were
carried out using the culture of S. aureus, S. pyogenes, S. typhi-
murium, and E. coli by the disk diffusion method [18] and then
the minimum inhibitory concentration (MIC) of all the
compounds were determined. Amoxicillin (30 mg) was used
as the standard drug, where as DMSO poured disk was used
as negative control and then minimum inhibitory concentra-
tion (MIC) was evaluated by the macrodilution test using stan-
dard inoculums of 10ꢁ5 CFL mLꢁ1. Serial dilutions of the test
compounds, previously dissolved in dimethyl sulfoxide
(DMSO) were prepared to final concentrations of 512, 256,
128, 64, 32, 16, 8, 4, 2 and 1 mg/mL. 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 in vitro studies result showed that the
compound o-toludinethiosemicarbazone(I) derivative of
steroidal was found to be more active among the all thiosemi-
carbazon compounds and their complex (Ia) is better antibac-
terial agent as compared to amoxicillin. 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
1
2.2. H NMR spectral analysis
Further evidence for the formation of thiosemicarbazones
1
and their metal complexes was obtained from the H NMR,
which provides diagnostic tools for the positional elucidation
of the protons. Assignments of the signals are based on the
1
chemical shifts and intensity patterns. The H NMR spectra
of thiosemicarbazones (1e3) recorded in DMSO-d6 exhibit
a broad peak at 9.82e10.42 ppm due to eNH proton, which
indicate that even in a polar solvent they remain in the thione
form. The NeH proton signal of the thiosemicarbazones
usually shifts to upfield and appears at 3.32e3.84 ppm in their
respective complexes. This information suggests the adjust-
ment of electronic current upon coordination of pC]S group
to the metal ion.
The 13C NMR spectra of ligand (I) were recorded in DMSO
and the spectral signals are in good agreement with the prob-
able structures. The ligand (I) showed two signals at 187.6 and
156.7 ppm assigned to (C]S) and (C]N), respectively and
their complex (Ia) at 185.6 and 155.4 ppm assigned to
(C]S) and (C]N), respectively. Other carbons in this
complex resonate nearly at the same region as that of free
ligands is given in Section 4.
Table 1
Antibacterial activity of steroidal thiosemicarbazons and their complexes,
positive control A (amoxicillin), and negative control (DMSO) measured by
the Halo Zone Test (unit, mm)
2.3. TGA analysis
Compound
Corresponding effect on microorganisms
S. aureus
S. pyogenes
S. typhimurium
E. coli
The TGA (under nitrogen, 10% min) profiles of complexes
along with the % weight at different temperatures were
recorded. These complexes do not lose weight up to 245 ꢀC.
Further increment of temperature causes decomposition of
the complexes in two steps being 245e315 ꢀC where loss of
mixed fragment was observed. The second step starts immedi-
ately after one and continues until the complete decomposition
of the ligand and formation of MS [M ¼ Pd(II)] as the end
1
14.2 ꢃ 0.4
12.4 ꢃ 0.2
12.6 ꢃ 0.4
18.4 ꢃ 0.2
16.2 ꢃ 0.4
16.8 ꢃ 1.1
21.0 ꢃ 0.5
e
12.8 ꢃ 0.4
10.6 ꢃ 0.2
11.5 ꢃ 0.5
19.8 ꢃ 0.4
14.4 ꢃ 0.8
14.8 ꢃ 0.6
22.2 ꢃ 0.4
e
12.4 ꢃ 0.2
10.6 ꢃ 0.4
10.2 ꢃ 0.5
21.5 ꢃ 0.5
18.2 ꢃ 0.4
18.8 ꢃ 0.4
25.2 ꢃ 0.8
e
14.2 ꢃ 0.5
11.2 ꢃ 0.5
10.2 ꢃ 0.4
23.2 ꢃ 0.2
15.6 ꢃ 0.8
19.2 ꢃ 0.8
20.0 ꢃ 0.2
e
2
3
1a
2a
3a
A
DMSO

S.A. Khan, M. Yusuf / European Journal of Medicinal Chemistry 44 (2009) 2270e2274
2273
Table 2
Minimum inhibition concentration (MIC) of steroidal thiosemicarbazons and
their complexes and positive control (amoxicillin)
4.1. Synthesis of thiosemicarbazides: a general method
Carbon disulphide (50 mmol) was added drop wise, a solu-
tion of amine and potassium hydroxide in water/ethanol (1:3)
mixture. The temperature of the reaction was maintained below
10 ꢀC. Sodium chloroacetate (50 mmol) was added and the re-
action mixture was left overnight at room temperature. Addi-
tion of conc. hydrochloric acid (to pH w 1) precipitated
substituted thioglycolic acid was recrystallized from methanol.
A solution of thioglycolic acid (40 mmol) in water (15 mL)
containing sodium hydroxide (40 mmol) and hydrazine hydrate
(40 mmol) was refluxed for 2 h with continuous stirring. The
compound separated out during the reaction or on cooling at
0 ꢀC for12 h. The product was filtered and crystallized from
methanol [15].
Strain
MIC (mg/mL)
Positive
control
1
2
3
1a
2a
64
3a
64
S. auras
64
64
128
128
128
256
256
256
128
128
32
32
64
64
32
32
32
32
S. pyogenes
S. typhimurium
E. coli
64
64
64
128
64
128
128
128
compound and the result is presented in (Table 2). Tests using
DMSO and amoxicillin as negative and positive controls. The
molecular structure of these active compounds showed
enhanced activity. The distinct difference in the antibacterial
property of the thiosemicarbazones and their metal complexes
further justifies the purpose of this study. The importance of
such work lies in the possibility that the new compound might
be more efficacious drugs against bacteria for which a thorough
investigation regarding the structureeactivity relationship tox-
icity and in their biological effects which could be helpful in
designing more potent antibacterial agents for therapeutic use.
4.2. Synthesis of thiosemicarbazones: a general method
Steroidal thiosemicarbazone was synthesized (Fig. 1) by
refluxing the solution of thiosemicarbazide (0.03 mol) in
methanol and the alcoholic solution of steroidal ketones
(0.03 mol) at 60 ꢀC for 5 h with continuous stirring after cool-
ing the compounds were filtered and recrystallized from
methanol [16].
3. Conclusion
4.2.1. Compound 1
Although the synthesis of thiosemicarbazones and their
metal complexes was reported several years ago, very little
is known about their antibacterial activity. This research exam-
ined the biological activities of the new thiosemicarbazones
from steroids and their Pd(II) complexes. o-Toludinethiosemi-
carbazone (I) derivative of steroidal thiosemicarbazines was
found to be more active among the all thiosemicarbazon
compounds and their complex (Ia) is better antibacterial agent
as compared to amoxicillin.
Yield: 64%; m.p. 210 ꢀC; Anal. Calc. for C35H53N3S: C,
76.78; H, 9.68; N, 7.67. Found: C, 75.65; H, 8.82: N, 7.35.
IR (KBr) nmax cmꢁ1: 3246 (NeH), 1568 (C]N), 1618
1
(C]C), 1124 (CeN), 1034 (C]S). H NMR (DMSO) (d):
10.42 (2H, s, eNH), 1.96 (3H, s, CH3), 7.20e8.04 (6H, m,
aryl protons), 5.36 (1H, s, C6eH), 1.08 (C10eCH3), 0.76
(C13eCH3), 0.88, 0.96 (other methyl protons). 13C NMR
(DMSO) (d): 187.6 (C]S), 156.7 (C]N), 135.5 (CeNH),
21.4 (C10eCH3), 19.6 (C13eCH3), 18.6, 18.4 (remaining
methyl carbon), 16.4 (CH3 of o-toludine). Mass spectra
ꢂ
(Mþ ) at m/z 548, 533 (M ꢁ CH3), 457 (M ꢁ C7H7), 442
(M ꢁ C7H8N), 398 (M ꢁ C8H8NS), 383 (M ꢁ C8H9N2S).
4. Experimental
4.2.2. Compound 2
All melting points were measured with a capillary appara-
tus and are uncorrected. All the compounds were routinely
Yield: 54%; m.p. 218 ꢀC; Anal. Calc. for C35H53N3S: C,
76.78; H, 9.68; N, 7.67. Found: C, 74.85; H, 9.24; N, 7.82.
IR (KBr) nmax cmꢁ1: 3226 (NeH), 1572 (C]N), 1522
1
checked by IR, H NMR mass spectrometry and elemental
analysis. IR spectra were recorded in KBr on a PerkineElmer
1
1
model 1620 FTIR spectrophotometer. H NMR spectra were
(C]C), 1138 (CeN), 1038 (C]S). H NMR (DMSO) (d):
9.62 (2H, s, eNH), 7.08e8.62 (6H, m, aryl protons), 5.48
(1H, s, C6eH), 2.42 (3H, s, CH3), 1.12 (C10eCH3), 0.78
(C13eCH3), 0.92, 0.98 (other methyl protons).
recorded at ambient temperature using a Brucker spectrosco-
pin DPX-300 MHz spectrophotometer in CDCl3 and DMSO.
The following abbreviations were used to indicate the peak
multiplicity s e singlet, d e doublet, t e triplet, m e
multiplet. FAB mass spectra were recorded on a JEOL
SX102 mass spectrometer using argon/xenon (6 kV, 10 mB
gas). Column chromatography was performed on silica gel
(Merck). Thin layer chromatography (TLC) was carried out
on 2.5 ꢄ 7.5 cm plates with a large thickness of 0.25 mm using
the indicator elements. Anhydrous sodium sulfate was used as
a drying agent for the organic phase. Compounds a, b and c
were prepared according to published methods [14].
4.2.3. Compound 3
Yield: 58%; m.p. 232 ꢀC; Anal. Calc. for C35H53N3S: C,
76.78; H, 9.68; N, 7.67. Found: C, 76.55; H, 97.92; N, 6.55.
IR (KBr) nmax cmꢁ1: 3252 (NeH), 1546 (C]N), 1513
1
(C]C), 1148 (CeN), 1042 (C]S). H NMR (DMSO) (d):
9.80 (2H, s, eNH), 6.80e7.92 (6H, m, aryl protons), 5.62
(1H, s, C6eH), 2.64 (3H, s, CH3), 1.18 (C10eCH3), 0.84
(C13eCH3), 0.98, 1.02 (other methyl protons).

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S.A. Khan, M. Yusuf / European Journal of Medicinal Chemistry 44 (2009) 2270e2274
4.3. Preparation of palladium(II) complexes
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 stock solution different concentrations 10, 20, 25, 50, and
100 mg/mL of each test compound were prepared. These
compounds of different concentrations were poured over
disk plate onto it. Amoxicillin (30 mg) was used as standard
drug (positive control). DMSO poured disk was used as nega-
tive 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 results of minimum inhibitory concentration (MIC) are
presented in Table 2. Tests using DMSO and amoxicillin as
negative and positive controls.
All the complexes were prepared by mixing the equimolar
ratio of ligand and [Pd(DMSO)2Cl2] in refluxing methanol.
The solution was kept at 0 ꢀC overnight, the product was
separated by filtration and finally washed with methanol.
Recrystallization was effected in methanol/DMF (6:4) [17].
4.3.1. [Pd(C35H53N3S)Cl2] (1a)
Yield:
72%;
m.p.
248 ꢀC;
Anal.
Calc.
for
[Pd(C35H53N3S)Cl2]: C, 58.01; H, 7.32; N, 5.80. Found: C,
57.02; H, 7.32; N, 5.80. IR (KBr) nmax cmꢁ1: 3446 (NeH),
1535 (C]N), 1522 (C]C), 1148 (CeN), 1022 (C]S), 432
1
(MeN, MeS). H NMR (DMSO) (d): 9.60 (2H, s, eNH),
7.26e8.14 (6H, m, aryl protons), 5.52 (1H, s, C6eH), 3.72
(1H, s, eNH), 2.10 (3H, s, CH3), 1.14 (C10eCH3), 0.84
(C13eCH3), 0.96, 1.04 (other methyl protons). 13C NMR
(DMSO) (d): 185.6 (C]S), 155.4 (C]N), 134.8 (CeNH),
21.2 (C10eCH3), 19.5 (C13eCH3), 18.2, 18.5 (remaining
methyl carbon), 16.2 (CH3 of o-toludine). Mass spectra
Acknowledgment
Authors are thankful to Dr. Kishwar Saleem Department of
Chemistry, Jamia Millia Islamia New Delhi for usefull
discussion.
ꢂ
(Mþ ) at m/z 725, 689 (M ꢁ Cl), 653 (M ꢁ Cl2), 548
(M ꢁ Pd), 533 (M ꢁ CH3), 457 (M ꢁ C7H7), 442
(M ꢁ C7H8N), 398 (M ꢁ C8H8NS), 383 (M ꢁ C8H9N2S).
References
4.3.2. [Pd(C35H53N3S)Cl2](2a)
Yield:
68%;
m.p.
262 ꢀC;
Anal.
Calc.
for
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C.L. Zani, E.M.S. Fagundes, H. Beraldo, Eur. J. Med. Chem. 40
(2005) 467e472.
[Pd(C35H53N3S)Cl2]: C, 58.01; H, 7.32; N, 5.80. Found: C,
56.86; H, 6.82; N, 5.20. IR (KBr) nmax cmꢁ1: 3452 (NeH),
1552 (C]N), 1518 (C]C), 1146 (CeN), 1014 (C]S), 498,
[2] J.G. Tojal, A.G. Orad, J.L. Serra, J.L. Pizarro, L. Lezama, M.I. Arriortua,
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J.L. Serra, M.I. Arriortua, T. Rojo, J. Inorg. Biochem. 86 (2001)
627e633.
1
445 (MeN, MeS). H NMR (DMSO) (d): 9.28 (2H, s, e
NH), 7.40e8.08 (6H, m, aryl protons), 5.32 (1H, s, C6eH),
3.84(1H, s, NH), 2.12 (3H, s, CH3), 1.16 (C10eCH3), 0.76
(C13eCH3), 0.94, 0.96 (other methyl protons).
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4.3.3. [Pd(C35H53N3S)Cl2] (3a)
Yield:
78%;
m.p.
278 ꢀC;
Anal.
Calc.
for
[6] P.F. Kelly, A.M.Z. Slawin, A. S-Rama, J. Chem. Soc., Dalton Trans.
(1996) 53e59.
[Pd(C35H53N3S)Cl2]: C, 58.01; H, 7.32; N, 5.80. Found: C,
56.45; H, 6.20; N, 4.50. IR (KBr) nmax cmꢁ1: 3468 (NeH),
1528 (C]N), 1478 (C]C), 1146 (CeN), 1018 (C]S), 428,
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5645e5647.
[8] F. Basuli, M. Ruf, C.G. Pierpont, S. Bhattacharya, Inorg. Chem. 37
(1998) 6113e6116.
1
447 (MeN, MeS). H NMR (DMSO) (d): 9.48 (2H, s, e
NH), 7.26e8.12 (6H, m, aryl protons), 5.38 (1H, s, C6eH),
3.32 (1H, s, NH), 2.05 (3H, s, CH3), 1.14 (C10eCH3), 0.78
(C13eCH3), 0.96, 0.98 (other methyl protons).
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[10] R. Prabhakaran, R. Karvembu, T. Hashimoto, K. Shimizu, K. Natarajan,
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4.4. Organism culture and in vitro screening
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Antibacterial activity was done by the disk diffusion
method with minor modifications. S. aureus, S. pyogenes, S.
typhimurium, and E. coli were sub cultured in BHI medium
and incubated for 18 h at 37 ꢀC, and then the bacterial cells
were suspended, according to the McFarland protocol in saline
:
10 mL of this suspension was mixed with 10 mL of sterile an-
tibiotic agar at 40 ꢀC and poured onto an agar plate in a laminar
[14] L.F. Fiesser, J. Am. Chem. Soc. 75 (1953) 542C.
[15] D.G. OSullivan, P.W. Sadler, C. Webley, Chemotherapia 7 (1963) 17.
[16] S. Singh, F. Athar, M.R. Maurya, A. Azam, Eur. J. Med. Chem. 41 (2006)
592e598.
solution to produce a suspension of about 10ꢁ5 CFU mLꢁ1
[17] A. Budakoti, A. Abid, A. Azam, Eur. J. Med. Chem. 42 (2007) 544e551.
[18] S.A. Khan, K. Saleem, Z. Khan, Eur. J. Med. Chem. 42 (2007) 103e108.