Novel steroidal (6R)-spiro-1,3,4-thiadiazoline derivatives as anti-bacterial agents

Article abstract of DOI:10.1002/cjoc.201200126

Novel steroidal (6R)-spiro-1,3,4-thiadiazoline derivatives have been synthesized by the cyclization of steroidal thiosemicarbazones. Thiosemicarbazones have been synthesized by the reaction of steroidal ketones with thiosemicarbazide. All the compounds ha

Full text of DOI:10.1002/cjoc.201200126

FULL PAPER
DOI: 10.1002/cjoc.201200126
Novel Steroidal (6R)-Spiro-1,3,4-thiadiazoline Derivatives as
Anti-bacterial Agents
Khan, Salman A.*,a
Asiri, Abdullah M.a,b
a Chemistry Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203,
Jeddah 21589, Saudi Arabia
b Center of Excellence for Advanced Materials Research, King Abdulaziz University,
Jeddah 21589, P.O. Box 80203, Saudi Arabia
Novel steroidal (6R)-spiro-1,3,4-thiadiazoline derivatives have been synthesized by the cyclization of steroidal
thiosemicarbazones. Thiosemicarbazones have been synthesized by the reaction of steroidal ketones with thiosemi-
carbazide. All the compounds have been characterized by IR, 1H NMR, mass and elemental analyses. The antibac-
terial activities of these compounds have been 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) values have
been determined with the reference of standard drug amoxicillin. The results showed that steroidal thiadiazoline de-
rivatives exhibited better antibacterial activity than the steroidal thiosemicarbazone derivatives. Chloro and acetoxy
substituents on the 3β-position of the steroidal thiadiazoline ring increased the anti-bacterial activity. Among all the
compounds, compounds 7 and 8 were found better inhibitors as compared to the respective drug amoxicillin.
Keywords steroidal thiosemicarbazones, steroidal thiadiazolines, cholesterol, amoxicillin, antibacterial activity
Introduction
potential antibacterial activity has been studied in our
laboratory.[10] It is evident from the literature that no
work has been done on steroidal (cholesterol) thiadia-
zoline derivative screening on bacteria. Considering the
facts that nearly all the classes of the cyclic thiosemi-
carbazones are biologically active and as a part of our
continuous efforts towards the development of more
potent antibacterial agents, we herein report the synthe-
sis, characterization and in vitro antibacterial activity of
novel spiro-1,3,4-thiadiazoline derivatives. In continu-
ing our previous works to report steroidal thiosemicar-
bazones as antibacterial agents[11] and heterocyclic
chemistry,[12] herein, we have developed the steroidal
thiosemicarbazone as key starting materials to form
novel heterocyclic compounds, to exhibit antibacterial
activitives of new substituted steroidal compounds and
to synthesize some new steroidal thiadiazolne deriva-
tives for their antibacterial screening.
Food poisoning, rheumatic, salmonellosis and diar-
rhoea are the serious health problem mainly in develop-
ing country. It is caused by the bacterial infection such
as S. aureus, S. pyogenes, S. typhimurium and E. coli.[1]
More than 50 million people worldwide are infected and
up to one hundred fifty thousand die every year due to
these bacterial infections.[2] Amoxicillin norfloxacin,
ciprofloxacin and chloramphenicol are most common
drug for the treatment of these bacterial infection but it
is associated with some side effects.[3] Hence, the pre-
sent work is aimed towards developing novel molecules
with improved potential for treating bacterial infections
and with decreased probability for developing drug re-
sistance. Heterocyclic compounds with reference of S
and N containing are of great importance in treating
biological systems. Compounds such as thiazole, thia-
zolidinone, thiadiazoline are abundant in nature and are
of great significance to life because their structural
subunits exist in many natural products such as vitamins,
hormones, antibiotics etc. Compounds with a thiadia-
zoline structure are known to possess tranquilizing,
muscle relaxing, psychoanaleptic, hypnotic, ulcerogenic,
antidepressant, antibacterial, antifungal, analgesic and
anti-inflammatory properties.[4-9] Recently a number of
thiazolidinone derivatives were synthesized and their
Experimental
All chemicals were purchased from Aldrich Co. and
were used without further purification. Precoated alu-
minium sheets (silica gel 60 F254, Merck) were used for
thin-layer chromatography (TLC) and spots were visu-
alized under UV light. All melting points were meas-
ured with a capillary apparatus and were uncorrected.
*
E-mail: sahmad_phd@yahoo.co.in
Received February 12, 2012; accepted April 6, 2012; published online XXXX, 2012.
Chin. J. Chem. 2012, XX, 1—5
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1

Khan & Asiri
FULL PAPER
1
General procedure for oxidative cyclization of ste-
riodal 6-ketone thiosemicarbazones (4—6) to ster-
oidal 6R-spiro-1',3',4'-thiadiazolines (7—9)
All the compounds were routinely checked by IR, H
NMR and mass spectrometry. IR spectra were recorded
on a Perkin-Elmer model 1620 FTIR spectrophotometer
as KBr discs. 1H NMR spectra were recorded on
Brucker spectroscopin DPX-400 MHz spectrophotome-
ter in CDCl3 and DMSO. The following abbreviations
were used to indicate the peak multiplicity s—singlet,
d—doublet, t—triplet, m—multiple. FAB mass spectra
were recorded on a JEOL SX102 mass spectrometer
using Argon/Xenon (6 kV, 10 mB gas.) Column chro-
matography was performed on silica gel (Merck Co.).
Anhydrous sodium sulfate was used as a drying agent
for the organic phase.
Steroidal thiosemicarbazones 4—6 (1.0 mmol) were
dissolved in chloroform (25 mL) and treated with
freshly distilled acetic anhydride (11.0 mmol) and pyri-
dine (2.5 mmol) and the mixture was stirred for 3—4 h
over an oil bath at 80 ℃. Reaction progress was moni-
tored by TLC. After completion of the reaction, solvent
was removed under reduced pressure and the residue
was purified by column chromatography over silica gel
(petroleum ether∶diethyl ether, 8∶2) to give the re-
spective steroidal (6R)-spiro-1',3',4'-thiadiazolines 7—9.
3β-Acetoxy-5α-cholestan-(6R)-spiro-6,4'-acetyl-2'-
(acetylaminomethylbenzene)-Δ2'-1',3,4'-thiadiazoline
Synthesis of steroidal thiosemicarbazones (4—6): a
general method
1
(7) Yield 78%; m.p. 146 ℃; H NMR (400 MHz,
Steroidal thiosemicarbazones were synthesized by
refluxing the solution of thiosemicarbazide (0.03 mol)
in methanol (10 mL) and the alcoholic solution (10 mL)
of steroidal ketones (0.03 mol) at 60 ℃ for 5 h with
continuous stirring. After cooling the compounds were
filtered and recrystallized from methanol (4—6).
CDCl3) δ: 4.88 (m, W1/2 18 Hz, C3 α-H), 2.28, 2.16 (s,
3H Ac), 7.38—8.42 (m, 4H, aryl protons), 1.96 (s, 3H,
CH3), 0.78 (s, 3H, 13-CH3), 0.92 (s, H, 18-CH3), 1.10 (s,
3H, 10-CH3), 0.80 (s, 3H, 19-CH3); 13C NMR (CDCl3) δ:
171.6, 169.0, 156.5, 135.5, 133.5, 129.2, 51.8, 46.4,
30.3, 26.2, 20.8, 17.9; IR (KBr) νmax: 2968 (CH), 1728,
1698 (amide), 1654 (C＝N), 1738 (OCOCH3), 1168
3β-Acetoxy-5α-cholestan-6-one-m-toluidinethio-
semicarbazone (4) Yield 65%; m.p. 168 ℃; H
1
－
＋•
1
(CN), 646 (CS) cm ; Mass spectra (M ) at m/z 691,
677 (M－CH3), 601 (M－C7H7), 650 (M－CH3CO),
545 (M－C9H10O), 428 (M－C12H13N3SO2), 632 (M－
CH3COO). Anal. calcd for C41H61N3O4S: C 71.20, H
8.82, N 6.07; found C 70.95, H 8.78, N 6.07.
3β-Chloro-5α-cholestan-(6R)-spiro-6,4'-acetyl-2'-
(acetylaminomethylbenzene)-Δ2'-1',3,4'-thiadiazoline
NMR (DMSO) δ: 10.42 (s, H, NH), 7.25—8.38 (m, 4H,
aryl protons), 4.72 (m, W1/2 18 Hz, 1H, C3 α axial),
1.96 (s, 3H, CH3), 1.10 (s, 3H, 10-CH3), 0.98 (s, 3H,
18-CH3), 0.90 (s, 3H, 19-CH3), 0.82 (s, 3H, 13-CH3); IR
(KBr) νmax: 3247 (NH), 1735 (AcO), 1564 (C＝N),
1625 (C＝C), 1118 (CN), 1032 (C＝S) cm ; Mass
spectra (M＋•) at m/z 608, 593 (M－CH3), 517 (M－
C7H7), 502 (M－C7H8N), 458 (M－C8H8NS), 443 (M－
C8H9N2S), 549 (M － AcO). Anal. calcd for
C37H57N3O2S: C 73.14, H 9.39, N 6.91; found C 73.09,
H 9.25, N 6.55.
－
1
1
(8) Yield 65%; greenish solid; m.p. 136 ℃; H NMR
(400 MHz, CDCl3) δ: 4.56 (m, W1/2 18 Hz, C3 α-H),
2.32, 2.18 (s, 3H Ac), 7.42—8.48 (m, 4H, aryl protons),
1.92 (s, 3H, CH3), 0.76 (s, 3H, 13-CH3), 0.98 (s, H,
18-CH3), 1.18 (s, 3H, 10-CH3), 0.86 (s, 3H, 19-CH3);
13C NMR (CDCl3) δ: 175.4, 164.8, 144.4, 138.2, 135.8,
3β-Chloro-5α-cholestan-6-one-m-toluidinethio-
1
semicarbazone (5) Yield 74%; m.p. 152 ℃; H
NMR (DMSO) δ: 10.38 (s, 1H, NH), 7.28—8.30 (m, 4H,
aryl protons), 4.48 (m, br, 1H, W1/2 17 Hz, C3α-H, ax-
ial), 1.92 (s, 3H, CH3), 1.05 (s, 3H, 10-CH3), 0.98 (s, 3H,
18-CH3), 0.85 (s, 3H, 19-CH3), 0.76 (s, 3H, 13-CH3); IR
(KBr) νmax: 3246 (NH), 1572 (C＝N), 1624 (C＝C),
128.6, 55.6, 45.8, 29.3, 24.7, 21.5, 20.8; IR (KBr) νmax
:
2956 (CH), 1736, 1696 (amide), 1648 (C＝N), 1162
－
＋•
1
(CN), 715 (CCl), 652 (CS) cm ; Mass spectra (M ) at
m/z 668, 653 (M－CH3), 577 (M－C7H7), 625 (M－
CH3CO), 520 (M－C9H10NO), 405 (M－C12H13N3SO2),
533 (M－Cl). Anal. calcd for C39H58O2N3SCl: C 70.16,
H 8.69, N 6.29; found C 69.98, H 8.45, N 6.08.
－
1
1118 (CN), 1028 (C＝S), 718 (CCl) cm ; Mass spectra
( M ＋ • ) a t m / z 5 8 4 , 5 6 9 ( M － C H 3 ) , 4 9 3
(M－C7H7), 478 (M－C7H8N), 458 (M－C8H8NS), 419
(M － C8H9N2S), 549 (M － Cl). Anal. calcd for
C35H54N3SCl: C 72.04, H 9.26, N 7.20; found C 71.96,
H 9.16, N 7.18.
5α-Cholestan-(6R)-spiro-6,4'-acetyl-2'-(acetyla-
minomethylbenzene)-Δ2'-1',3,4'-thiadiazoline
(9)
1
Yield 76%; semi-solid; H NMR (400 MHz, CDCl3) δ:
4.58 (m, W1/2h 18 Hz, C3 α-H), 2.28, 2.12 (s, 3H Ac),
7.28—8.42 (m, 4H, aryl protons), 0.78 (s, 3H, 13-CH3),
0.96 (s, H, 18-CH3), 1.14 (s, 3H, 10-CH3), 0.88 (s, 3H,
19-CH3); 13C NMR (CDCl3) δ: 175.4, 164.8, 152.2,
134.8, 135.3, 127.2, 54.2, 43.4, 27.5, 24.5, 21.4, 19.8;
IR (KBr) νmax: 2954 (CH), 1722, 1694 (amide), 1655 (C
5α-Cholestan-6-one-m-toludinethiosemicarbazone
1
(6) Yield 78%; m.p. 218 ℃; H NMR (DMSO) δ:
10.35 (s, 1H, NH), 7.28—8.60 (m, 6H, aryl protons),
1.95 (s, 3H, CH3), 1.08 (s, 3H, 10-CH3), 0.99 (s, 3H,
18-CH3), 0.88 (s, 3H, 19-CH3), 0.78 (s, 3H, 13-CH3); IR
(KBr) νmax: 3256 (NH), 1576 (C＝N), 1636 (C＝C),
－
＋•
1
－
＋•
1
＝N), 1158 (CN), 638 (CS) cm ; Mass spectra (M ) at
m/z 634, 619 (M－CH3), 543 (M－C7H7), 591 (M－
CH3CO), 486 (M－C9H10NO), 371 (M－C12H13N3SO2);
Anal. calcd for C39H59O2N3S: C 73.93, H 9.30, N 6.63;
found C 73.85, H 9.18, N 6.45.
1132 (CN), 1024 (C＝S) cm ; Mass spectra (M ) at
m/z 550, 535 (M－CH3), 459 (M－C7H7), 441 (M－
C7H8N), 400 (M－C8H8NS), 385 (M－C8H9N2S). Anal.
calcd for C35H55N3S: C 76.50, H 10.01, N 7.65; found C
75.52, H 9.96, N 7.62.
2
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Chin. J. Chem. 2012, XX, 1—5

Novel Steroidal (6R)-Spiro-1,3,4-thiadiazoline Derivatives as Anti-bacterial Agents
Organism culture and in vitro screening
(4—6) were prepared by condensing the steroidal ke-
tones with m-toluidinethiosemicarbazide in the presence
of catalytic amount of conc. HCl.[13] 3β-Acetoxycholest-
6-one (1),[14] 3β-chloro-cholest-6-one (2),[15] 5α-cholest-
6-one (3) were prepared according to the published
methods.[16] The 1,3,4-thiadiazoline derivatives were
synthesized according to the literature procedure by the
acetylation of steroidal thiosemicarbazone derivatives as
shown in Scheme 1.[17] All the compounds were soluble
in DMSO and ethanol. The structures of all the com-
pounds were established on the basis of spectral studies
Antibacterial activity was treated by the disk diffu-
sion 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 ℃, and
then the bacterial cells were suspended, according to the
McFarland protocol in saline solution to produce a sus-
－
1
pension of about 10－ CFU•mL : 10 μL of this sus-
pension was mixed with 10 mL of sterile antibiotic agar
at 40 ℃ and poured onto an agar plate in a laminar
flow cabinet. Five paper disks (05 mm diameter) were
fixed onto nutrient agar plate. 1 mg of each test com-
pound was dissolved in 100 μL DMSO to prepare stock
solution and from stock solution different concentra-
tions 10, 20, 25, 50, and 100 μg/μL of each test com-
pound were prepared. These compounds of different
concentration were poured over disk plate on to it.
Amoxicillin (30 μg/disk) was used as standard 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 ℃. Table
1 reports the inhibition zones (mm) of each compound
and the controls. The minimum inhibitory concentration
5
1
such as IR, H NMR, 13C NMR, FAB mass spectra and
the elemental analyses were carried out to check the
purity of the compounds.
Scheme 1 Schematic diagram showing the synthesis of com-
pounds 7, 8 and 9
S
C8H17
H2N
N
N
H
H
MeOH
R
C8H17
O
1 R = OAc
2 R = Cl
(MIC) was evaluated by the macro dilution test using
－
standard inoculums of 10－5 CFL•mL . Serial dilutions
1
3 R = H
C8H17
R
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 μg/mL, and to
each tube was added 100 μL 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
℃, and the results are presented in Table 2. In the tests,
DMSO and amoxicillin were used as negative and posi-
tive controls.
Ac
N
S
Ac2O/Pyridine
N
Ac
N
R
S
N
N
N
H
H
7 R = OAc
8 R = Cl
9 R = H
4 R = OAc
5 R = Cl
6 R = H
Assignments of selected characteristic IR band posi-
tions provide significant indication for the formation of
the cyclized thiadiazoline analogues of thiosemicarba-
zones. All the compounds showed v(C＝N) stretch at
Table 1 Antibacterial activities of steroidal thiosemicarbazones
(4—6) and steroidal thiadiazoline derivatives (7—9)a
1648—1655 cm－ due to the ring closure. In addition,
1
Corresponding effect on microganisms
Compd
the absorption band at 1158—1168 cm－ was attributed
1
S. aureus S. pyogenes S. typhimurium E. coli
to the v(C—N) stretch vibrations. The compounds
showed intense bands at 638—652 cm－1 due to v(C—S)
stretch, which also confirm the formation of thiazole
ring in all the compounds. Further evidence for the for-
mation of thiadiazoline compounds was obtained from
the 1H NMR spectra, which provide diagnostic tools for
the positional elucidation of the protons. Assignments of
the signals are based on the chemical shifts and intensity
patterns. The acetylation proton of thiadiazoline ring of
all the compounds is shown as singlet in the range δ
4
5
6
7
8
9
11.2±0.3 11.2±0.4 10.8±0.5
11.2±0.5 12.5±0.3 11.0±0.4
12.4±0.5
10.6±0.4
9.2±0.4
21.6±0.4
22.5±0.6
14.2±0.4
20.0±0.2
—
8.8±0.5 9.2±0.2
9.6±0.3
17.2±0.4 20.5±0.6 17.4±0.4
20.5±0.4 20.8±0.4 21.9±0.3
12.8±0.5 15.0±0.5 13.8±0.4
Amoxicillin 17.0±0.5 18.2±0.4 17.2±0.8
DMSO
a
—
—
—
13
Positive control amoxicillin and negative control (DMSO)
measured by the Halo Zone Test (mm).
2.12—2.32. C NMR spectra of the compounds (7—9)
were taken in CDCl3 and the signal obtained further
confirmed the proposed structures. All the compounds
showed a signal at δ 144.4—156.5 due to (N＝C—S)
and δ 43.4—55.6 due to N—C(6)—S, indicating the
cyclization of thiocarbamoyl carbon. The characteristic
peaks observed within the 13C NMR spectra of thiadia-
Results and Discussion
Chemistry
The starting material steroidal thiosemicarbazones
Chin. J. Chem. 2012, XX, 1—5
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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3

Khan & Asiri
FULL PAPER
zoline derivatives are given in Experimental section.
Characteristic peaks were observed in the mass spectra
of compounds 7—9, which followed the similar frag-
mentation pattern. The spectrum of compound 7 showed
a molecular ion peak (M•＋) at m/z 691, compound 8
showed a molecular ion peak (M•＋) at m/z 668 and
compound 9 showed a molecular ion peak (M•＋) at m/z
634. Further fragmentation pattern of these compounds
has been given in the experimental section.
compounds (4—9) were tested for their antibacterial
activities by disc-diffusion method using nutrient broth
medium [contained (g/L): beef extract 3; peptone 5; pH
7.0].[19] The Gram-positive bacteria and Gram-negative
bacteria utilized in this study included S. aureus, S. pyo-
genes, S. typhimurium and E. coli. In the disc-diffusion
method, sterile paper discs (5 mm) impregnated with
compound dissolved in dimethylsulfoxide (DMSO) at
concentration 100 μg/mL were used. Then, the paper
discs impregnated with the solution of the compound
tested were placed on the surface of the media inocu-
lated with the microorganism. The plates were incu-
bated at 35 ℃ for 24 h. After incubation, the growth
inhibition zones are shown in Table 1. The thiadiazoline
derivatives were further checked by MIC method. The
results are presented in Table 2. The molecular structure
of these active compounds showed enhanced activity.
The distinct differences in the antibacterial property of
these compounds further justify the purpose of this
study. The importance of such work lies in the possibil-
ity that the new compound might be more efficacious
drugs against bacteria for which a thorough investiga-
tion regarding the structure-activity relationship, toxic-
ity and in their biological effects could be helpful in
designing more potent antibacterial agents for therapeu-
tic use.
Stereochemistry of spiro-1,3,4-thiadiazolines
All the 1,3,4-thiadiazolines derivatives (7—9) have
C(6)—S bond axially and C(6)—NAc bond as equato-
rially oriented. Although sulphur atom is more bulky
than the nitrogen but NAc group becomes more bulky
than the sulphur atom, so during cyclisation (Scheme 1)
the thiadiazoline ring preferably closes at C-6 from the
front (β, axial) side by the attack of sulphur (C＝S) of
the thiosemicarbazone moiety and as the bulky group,
already attached at C-6, is moved toward back (α,
equatorial) side to avoid the 1,3-diaxial interactions
mainly due to C(10)β-Me, and leaving front (β, axial)
side for the attack of incoming group. So, the com-
pounds (7—9) of this reaction have R geometry at C-6
in which C(6)—S and C(6)—NAc bonds are oriented
axially (β) and equatorially (α), respectively. This ar-
rangement (geometry) provides greater stability to the
molecule as they have less 1,3-diaxial interactions.
The mechanism (Scheme 2) for the formation of
1,3,4-thiadiazolines can also be explained on the basis
of the hard soft acid and base principle.[18] The harder
acetylating reagent reacts with the harder nitrogen atom
rather than the softer sulphur atom and this acetylation
favours cyclization of thiosemicarbazones to (6R)-spiro-
1,3,4-thiadiazolines.
Table 2 Minimum inhibition concentration (MIC) of steroidal
thiadiazoline derivatives, positive control (Amoxicillin).
－
MIC/(μg•mL )
1
Strain
7
8
9
Positive control
S. aureus
64
32
64
32
32
32
32
16
128
64
32
32
32
32
S. Pyogenes
S. typhimurium
E. coli
128
64
Scheme 2 Mechanism of steroidal thiadiazoline derivatives
C8H17
C8H17
Conclusions
Ac2O/Pyridine
R
S
This research involves the synthesis of novel ster-
oidal (6R)-spiro-1,3,4-thiadiazoline derivatives by the
cyclization of steroidal thiosemicarbazones. The anti-
bacterial activity of these compounds was carried out by
disc-diffusion method against culture of bacteria. The
biological behavior of these compounds revealed that
chloro and acetoxy substituents on the 3β-position of the
steroidal thiadiazoline ring increased the anti-bacterial
activity. Among all the six compounds, compounds 7
and 8 showed better antibacterial activity than the re-
spective drug Amoxicillin. These results identified that
steroidal (6R)-spiro-1,3,4-thiadiazoline derivatives are
new leads in antibacterial chemotherapy. The study
suggests the beneficial potential of these leads that need
to be further explored in order to discover and develop
better and yet safer therapeutic agents for bacterial in-
fections.
R
S
N
N
N
H
N
N
N
H
H
H
AcO
AcOH
C8H17
R
Ac
N
S
N
N
Ac
Anti-bacterial activity
Disc-diffusion and micro dilution assay The
4
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Chin. J. Chem. 2012, XX, 1—5

Novel Steroidal (6R)-Spiro-1,3,4-thiadiazoline Derivatives as Anti-bacterial Agents
251.
Acknowledgement
[7] Satyanarayana, K.; Rao, M. N. A. J. Pharm. Sci. 1995, 84, 263.
Authors are thankful to Head, Department of Chem-
istry, King Abdulaziz University, for providing the re-
search facilities.
[8] Satyanarayana, K.; Rao, M. N. A. Eur. J. Med. Chem. 1995, 30, 641.
[9] Kavali, J. R.; Badami, B. V. II Farmaco 2000, 55, 406.
[10] Khan, S. A.; Yusuf, M. Eur. J. Med. Chem. 2009, 44, 2597.
[11] Asir, A. M.; Khan, S. A. Molecules 2010, 15, 4784.
[12] Khan, S. A.; Saleem, K.; Khan, Z. Eur. J. Med. Chem. 2007, 42,
103.
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