Synthesis and antituberculosis activity of new thiazolylhydrazone derivatives

Article abstract of DOI:10.1016/j.ejmech.2007.07.001

The increasing clinical importance of drug-resistant mycobacterial pathogens has lent additional urgency to microbiological research and new antimycobacterial compound development. For this purpose, new thiazolylhydrazone derivatives were synthesized and

Full text of DOI:10.1016/j.ejmech.2007.07.001

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 981e985
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antituberculosis activity of new thiazolylhydrazone
derivatives
a
a
Gulhan Turan-Zitouni a, , Ahmet Ozdemir , Zafer Asim Kaplancikli ,
¨
*
¨
Kadriye Benkli a, Pierre Chevallet b, Gulsen Akalin c
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskixsehir, Turkey
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Montpellier University I, LAAP, CNRS-UMR 5810, 34000 Montpellier, France
c Department of Biochemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskixsehir, Turkey
Received 5 April 2007; received in revised form 18 June 2007; accepted 2 July 2007
Available online 15 July 2007
Abstract
The increasing clinical importance of drug-resistant mycobacterial pathogens has lent additional urgency to microbiological research and new
antimycobacterial compound development. For this purpose, new thiazolylhydrazone derivatives were synthesized and evaluated for antituber-
culosis activity. The reaction of thiosemicarbazide with acetophenone derivatives gave 1-(1-arylethylidene)thiosemicarbazide (1). The N-(1-ar-
ylethylidene)-N0-[4-(indan-5-yl)thiazol-2-yl]hydrazone (3) derivatives were synthesized by reacting 1-(1-arylethylidene)thiosemicarbazide with
1
1-(5-indanyl)-2-bromoethanone (2). The chemical structure of the compounds was elucidated by elemental analyses, IR, H NMR, MS-FABþ
spectral data. Antituberculosis activities of the synthesized compounds were determined by broth microdilution assay, the Microplate Alamar
Blue Assay, in BACTEC12B medium and the results were screened in vitro, using BACTEC 460 Radiometric System against Mycobacterium
tuberculosis H37Rv (ATCC 27294) at 6.25 mg/ml and some of the tested compounds showed important inhibition ranging from 92% to 96%. The
compounds were also investigated for their cytotoxic properties on normal mouse fibroblast (NIH/3T3) cell line and the results obtained here
showed that all the compounds used have no significant cytotoxicity at the concentrations under 50 mg/ml.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Thiazolylhydrazone; Antituberculosis activity; Toxicity
1. Introduction
number of deaths, is the emergence of multiple drug resistance
(MDR) [2e4].
According to alarming data from the World Health Organi-
sation, tuberculosis (TB) has spread to every corner of the
globe [1]. As much as one-third of the world’s population is
currently infected and more than 5000 people die from TB ev-
ery day. A large number of the infected people are carriers of
the latent form, which creates a potentially dangerous source
of the illness for the future. The HIV pandemic has led to
the rapid growth of the TB epidemic and increased the likeli-
hood of people dying from TB. Another factor contributing to
the rise in TB infections, and consequently to the increased
There is an urgent need to develop new TB drugs [5]. How-
ever, no new TB drugs have been developed in about 40 years.
Although TB can be cured with the current therapy, the six
months needed to treat the disease is too long, and the treat-
ment often has significant toxicity. These factors make patient
compliance to therapy very difficult and this noncompliance
frequently selects for drug-resistant TB bacteria. The current
TB problem clearly demonstrates the need for a reevaluation
of our knowledge of the current TB drugs and chemotherapy
and the need for new and better drugs that are not only active
against drug-resistant TB but also, more importantly, shorten
the requirement for six months of therapy [6].
To pursue this goal, our research efforts are directed to find
new chemical classes of antimycobacterially active agents.
The methods of investigation of structureeactivity relationships
* Corresponding author. Tel.: þ90 222 335 05 80/37 77; fax: þ90 222 335
07 50.
E-mail address: gturan@anadolu.edu.tr (G. Turan-Zitouni).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.07.001

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G. Turan-Zitouni et al. / European Journal of Medicinal Chemistry 43 (2008) 981e985
(SARs) enabled us to find some new pharmacophores of the
above-mentioned activity. Many studies were carried out on het-
erocyclic systems bearing a hydrazone structure as a pharmaco-
phore [7e13]. Among them especially thiazole residue [14e17]
had taken our interest.
3.2. Toxicity
The level of cellular MTT (Sigma) reduction was quantified
as previously described in the literature with small modifica-
tions [20e22].
Keeping these observations in mind, we decided to under-
take the synthesis of N-(1-arylethylidene)-N0-[4-(indan-5-yl)
thiazol-2-yl]hydrazones carrying a thiazolylhydrazone moiety
and to study their antituberculosis activity and toxicity.
4. Results, discussion and conclusion
In the present work, six new compounds were synthesized.
The formulas of compounds (3aef) were found by elemental
analyses and their structures were determined by IR, 1H NMR
and MS-FABþ spectral data. The IR data were very informa-
tive and provided evidence for the formation of the expected
structures. In the IR spectra, some significant stretching bands
due NeH, C]N and C]C were at 3309e3224 cmꢀ1, 1612e
2. Chemistry
The synthetic route of compounds is outlined in Scheme 1.
For the synthesis of the title compounds, 1-(1-arylethylidene)-
thiosemicarbazide (1) required as starting material was pre-
pared by the reaction of acetophenone derivatives with
thiosemicarbazide [18]. The reaction of equimolar quantities
of thiosemicarbazide (1) with 1-(5-indanyl)-2-bromoethanone
(2) in the presence of isopropyl alcohol resulted in the forma-
tion of the title compounds (3aef) (Table 1).
1
1484 cmꢀ1, respectively. The H NMR spectra data were also
1
consistent with the assigned structures. In the 250 MHz H
NMR spectrum of compounds, protons C2 of indane resonated
as a pentaplet at 1.95e2.10 ppm, protons C1 and C3 of indane
resonated as a quartet at 2.70e3.00 ppm, NH proton was ob-
served as a broad at 5.95e6.90 ppm, and all the other aromatic
and aliphatic protons were observed at expected regions. The
mass spectra (MS(FAB)) of compounds showed [M þ 1]
peaks, in agreement with their molecular formula. All com-
pounds gave satisfactory elemental analysis.
3. Biology
3.1. Antituberculosis activity
In conclusion, a series of novel N-(1-arylethylidene)-N0-
[4-(indan-5-yl)thiazol-2-yl]hydrazone (3) derivatives (3aef)
were synthesized and their antituberculosis activities and tox-
icity have been evaluated. According to the primary screening,
the compounds 3b, 3c, 3e and 3f showed the highest inhibi-
tions with 96%, 96%, 94%, 92%, respectively. SAR observa-
tion showed that a thiazolylhydrazone structure and a
substitution on phenyl affect the activity.
The IC50 values obtained for the six compounds are shown
in Table 3. As can be seen for the six compounds, there were
no significant cytotoxicity at the concentrations under 49 mg/ml
after 24 h incubation. Among the compounds used, compound
3b showed greater toxicity at 49e200 mg/ml range. On the
other hand, 3a and 3c showed no significant toxicity at
the concentrations used. Also compounds 3d, 3e and 3f
All of the compounds were evaluated for in vitro antitu-
berculosis activity against Mycobacterium tuberculosis, as
a part of the TAACF TB screening program under direction
of the US National Institute of Health, the NIAID division.
Rifampicin was used as a reference drug. Primary screening
was conducted at a single concentration, 6.25 mg/ml, against
M. tuberculosis H37Rv (ATTCC 27294), in BACTEC 12B
medium, using the Microplate Alamar Blue Assay
(MABA) [19]. Compounds effecting <90% inhibition in
the primary screening (MIC > 6.25 mg/ml) were not gener-
ally evaluated further. Some of the compounds showed sig-
nificant antituberculosis activity as can be inferred from
Table 2.
R1
R1
S
S
R2
R2
N
NH2
+
CH3
N
H
N
H
NH2
H2N
CH3
O
1
S
CH3
O
R1
R2
N
H
Br
N
N
1
+
2
3a-f
Scheme 1. Synthetic route of the title compounds.

G. Turan-Zitouni et al. / European Journal of Medicinal Chemistry 43 (2008) 981e985
983
Table 1
Some characteristics of the compounds
Table 3
Cytotoxicity of compounds to mouse fibroblast (NIH/3T3) cell line
Comp. R1 R2
M.p. (ꢁC)
Yield (%) Mol. for.
MW
Comp.
Cytotoxicity
IC50 (mg/ml)a
3a
3b
3c
3d
3e
3f
H
H
H
H
H
H
CH3
258e260 dec. 70
248e250 dec. 64
C20H19N3S
C21H21N3S
333
347
363
378
367
3a
3b
3c
3d
3e
3f
a
200 ꢂ 9.1
49 ꢂ 7.6
180 ꢂ 8.5
90 ꢂ 3.3
120 ꢂ 15
110 ꢂ 5.0
OCH3 253e255
NO2
Cl
61
78
C21H21N3OS
C20H18N4O2S
212e214
243e244 dec. 63
237e239 59
C20H18ClN3S
Cl Cl
C20H17Cl2N3S 401
Incubation for 24 h. IC50 is the drug concentration required to inhibit 50%
of the cell growth. The values represent mean ꢂ standard deviation of triplicate
determinations.
showed moderate cytotoxic effects on NIH/3T3 cell line, dem-
onstrating that 3d, 3e and 3f were the cause of 90, 120 and
110 mg/ml IC50 values, respectively.
In comparison of the results of toxicity and antimicrobial
activity tests, it is seen that the antimicrobial activity of the
compounds is not due to their general toxicity effect, however,
their antimicrobial activity can be possibly because of their
selective antimicrobial effect.
refluxing isopropyl alcohol (80 ml) to complete dissolution,
until a white foaming product is formed. The mixture is then
allowed to cool and the solid is filtered, washed with saturated
NaHCO3 water solution and then with cool water, dried, and
crystallised from ethanol.
We concluded from our investigations that 3b, 3c, 3e and 3f
may be considered promising for the development of new an-
tituberculosis agents with their antituberculosis activity and
toxicity screening.
Some characteristics of the synthesized compounds are
1
shown in Table 1. Analytical and spectral data (IR, H NMR,
MS-FABþ) confirmed the structures of the new compounds.
Compounds 3aef: IR (KBr) nmax (cmꢀ1): 3309e3224
(NH), 1612e1484 (C]C and C]N).
5. Experimental
5.1.2.1. N-(1-Phenylethylidene)-N0-[4-(indan-5-yl)thiazol-2-yl]
hydrazone (3a). 1H NMR (250 MHz, DMSO-d6) d (ppm):
1.95e2.15 (2H, pentaplet, protons C2 of indane), 2.85 (3H, s,
CH3), 2.80e3.00 (4H, q, protons C1 and C3 of indane), 5.95
(1H, br, NH), 7.20e7.85 (9H, m, aromatic protons). For
C20H19N3S calculated: 72.04% C, 5.74% H, 12.60% N; found:
72.16% C, 5.82% H, 12.64% N. MS-FABþ: m/z: 334 [M þ 1].
5.1. Chemistry
All melting points (m.p.) were determined in open capillar-
ies on a Gallenkamp apparatus and are uncorrected. The purity
of the compounds was routinely checked by thin layer chro-
matography (TLC) using silica gel 60G (Merck). Spectro-
scopic data were recorded on the following instruments: IR,
Shimadzu 435 IR spectrophotometer; 1H NMR, Bruker
250 MHz NMR spectrometer in DMSO-d6 using TMS as an
internal standard; elemental analyses were performed on a Per-
kin Elmer EAL 240 elemental analyser; MS-FABþ, VG Quat-
tro mass spectrometer.
5.1.2.2. N-(1-(4-Methylphenyl)ethylidene)-N0-[4-(indan-5-yl)
thiazol-2-yl]hydrazone (3b). 1H NMR (250 MHz, DMSO-d6)
d (ppm): 1.95e2.10 (2H, pentaplet, protons C2 of indane),
2.40e2.45 (6H, two s, CH3), 2.75e2.95 (4H, q, protons C1
and C3 of indane), 6.90 (1H, br, NH), 7.10e7.70 (8H, m, aro-
matic protons). For C21H21N3S calculated: 72.59% C, 6.09%
H, 12.09% N; found: 72.56% C, 6.04% H, 12.12% N. MS-
FABþ: m/z: 347 [M], 348 [M þ 1].
5.1.1. Preparation of 1-(1-arylethylidene)thiosemicarbazide
(1)
In a flask equipped with a reflux condenser, a mixture of
thiosemicarbazide (40 mmol) and the appropriate acetophe-
none derivatives (40 mmol) are reacted in 80 ml isopropyl al-
cohol in the presence of a catalytic amount of acetic acid. The
mixture is then refluxed for 1 h and the obtained solid is
filtered and used without further purification.
5.1.2.3. N-(1-(4-Methoxyphenyl)ethylidene)-N0-[4-(indan-5-yl)
thiazol-2-yl]hydrazone (3c). 1H NMR (250 MHz, DMSO-d6)
d (ppm): 1.90e2.10 (2H, pentaplet, protons C2 of indane),
2.30 (3H, s, CH3), 2.70e2.95 (4H, q, protons C1 and C3 of in-
dane), 3.80 (3H, s, OCH3), 5.95 (1H, br, NH), 7.00 (2H, d,
J ¼ 8.89 Hz, phenyl C2 and C6 protons), 7.20 (1H, s, indane
C4 proton), 7.25 (1H, d, J ¼ 7.80 Hz, indane C7 proton),
7.60 (1H, d, J ¼ 7.94 Hz, indane C6 proton), 7.70 (1H, s, thi-
azole C5 proton), 7.75 (2H, d, J ¼ 8.88 Hz, phenyl C3 and C5
protons). For C21H21N3OS calculated: 69.39% C, 5.82% H,
11.56% N; found: 69.38% C, 5.82% H, 11.54% N. MS-
FABþ: m/z: 364 [M þ 1].
5.1.2. Preparation of N-(1-arylethylidene)-N0-[4-(indan-5-yl)
thiazol-2-yl]hydrazones (3aef)
1-(1-Arylethylidene)thiosemicarbazide (1) (20 mmol) and
1-(5-indanyl)-2-bromoethanone (2) (20 mmol) are stirred in
Table 2
Antituberculosis activity of the compounds
Comp.
MIC (mg/ml) >6.25 <6.25 <6.25 >6.25 <6.25 <6.25 0.25
% Inhibition 89 96 96 54 94 92 98
3a
3b
3c
3d
3e
3f
Rifampicin
5.1.2.4. N-(1-(4-Nitrophenyl)ethylidene)-N0-[4-(indan-5-yl)th-
iazol-2-yl]hydrazone (3d). 1H NMR (250 MHz, DMSO-d6)
d (ppm): 1.90e2.00 (2H, br, protons C2 of indane), 2.35

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G. Turan-Zitouni et al. / European Journal of Medicinal Chemistry 43 (2008) 981e985
(3H, s, CH3), 2.70e2.90 (4H, br, protons C1 and C3 of indane),
6.50 (1H, br, NH), 7.40e8.30 (8H, m, aromatic protons). For
C20H18N4O2S calculated: 63.47% C, 4.79% H, 14.80% N;
found: 63.55% C, 4.84% H, 14.91% N. MS-FABþ: m/z: 379
[M þ 1].
5.3. Toxicity
5.3.1. Cell culture and drug treatment
NIH/3T3 cells were obtained from the American Type Cul-
ture Collection (ATCC, USA). The cells were incubated in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal calf serum (Life Technologies, UK), 100 IU/ml
penicillin (Gibco, Paisley, Scotland) and 100 mg/ml streptomy-
cin (Gibco) at 37 ꢁC in a humidified atmosphere of 95% air
and 5% CO2. Exponentially growing cells were plated at
5 ꢃ 104 cells/ml into 96-well microtiter tissue culture plates
(Nunc, Denmark) and incubated for 24 h before the addition
of the drugs (the optimum cell number for cytotoxicity assays
was determined in preliminary experiments). Stock solutions
of compounds were prepared in dimethyl sulphoxide
(DMSO; SigmaeAldrich, Poole, UK) and further dilutions
were made with fresh culture medium (the concentration of
DMSO in the final culture medium was <0.6% which had
no effect on the cell viability).
5.1.2.5. N-(1-(4-Chlorophenyl)ethylidene)-N0-[4-(indan-5-yl)
thiazol-2-yl]hydrazone (3e). 1H NMR (250 MHz, DMSO-d6)
d (ppm): 1.90e2.10 (2H, pentaplet, protons C2 of indane),
2.80 (3H, s, CH3), 2.80e2.95 (4H, q, protons C1 and C3 of
indane), 6.85 (1H, br, NH), 7.25 (1H, s, indane C4 proton),
7.30 (1H, d, J ¼ 7.37 Hz, indane C7 proton), 7.50 (2H, d,
J ¼ 8.63 Hz, phenyl C2 and C6 protons), 7.65 (1H, d,
J ¼ 7.49 Hz, indane C6 proton), 7.75 (1H, s, thiazole C5 pro-
ton), 7.80 (2H, d, J ¼ 8.64 Hz, phenyl C3 and C5 protons).
For C20H18ClN3S calculated: 65.30% C, 4.93% H, 11.42%
N; found: 65.29% C, 4.99% H, 11.54% N. MS-FABþ: m/z:
367 [M], 368 [M þ 1], 369 [M þ 2].
5.1.2.6. N-(1-(3,4-Dichlorophenyl)ethylidene)-N0-[4-(indan-5-
yl)thiazol-2-yl]hydrazone (3f). 1H NMR (250 MHz, DMSO-
d6) d (ppm): 1.95e2.10 (2H, pentaplet, protons C2 of indane),
2.35 (3H, s, CH3), 2.70e2.95 (4H, q, protons C1 and C3 of in-
dane), 6.40 (1H, br, NH), 7.20e8.00 (7H, m, aromatic pro-
tons). For C20H17Cl2N3S calculated: 59.71% C, 4.26% H,
10.44% N; found: 59.89% C, 4.43% H, 10.59% N. MS-
FABþ: m/z: 401 [M], 402 [M þ 1], 403[M þ 2].
5.3.2. Cell culture and drug treatment
It is widely used as a measure of cytotoxicity. After 24 h of
preincubation, the tested compounds were added to give final
concentration in the range of 1.56e200 mg/ml and the cells
were incubated for 24 h. At the end of this period, MTT was
added to a final concentration of 0.5 mg/ml and the cells
were incubated for 4 h at 37 ꢁC. After the medium was re-
moved, the formazan crystals formed by MTT metabolism
were solubilized by addition of 200 ml DMSO to each well
and absorbance was read at 540 nm with a microtitre plate
spectrophotometer (Elx808-IU Bio-Tek plate reader). Every
concentration was repeated in three wells and IC50 values
were defined as the drug concentrations that reduced absor-
bance to 50% of control values.
5.2. Biology
5.2.1. BACTEC radiometric method of susceptibility testing
Inocula for susceptibility testing were either from a posi-
tive BACTEC isolation vial with a growth index (GI) of 500
more, or suspension of organism isolated earlier on conven-
tional medium. The culture was mixed well with a syringe
and 0.1 ml of a positive BACTEC culture was added to
each of the vials containing the test drugs. The drug vials
contained rifampicin (0.25 mg/ml). A control vial was inoc-
ulated with a 1:100 microdilution of the culture. A suspen-
sion equivalent to a McFarland No. 1 standard was prepared
in the same manner as a BACTEC positive vial, when
growth from a solid medium was used. Each vial was tested
immediately on a BACTEC instrument to provide CO2 in
the headspace. The vials were incubated at 37 ꢁC and tested
daily with a BACTEC instrument. When the GI in the
control read at least 30, the increase in GI (DGI) from
the previous day in the control was compared with that in
the drug vial. The following formula was used to interpret
results:
Data presentation and statistics. Statistical analyses were
carried out by the one-way analyses of variance (ANOVA)
test and criterion of the differences between means (ꢂSEM)
was P < 0.05.
Acknowledgements
Authors are thankful to the Tuberculosis Antimicrobial Ac-
quisition and Coordinating Facility (TAACF) in USA for the
in vitro evaluation of antimycobacterial activity.
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