Synthesis, characterization and biological evaluation of some thiourea derivatives bearing benzothiazole moiety as potential antimicrobial and anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2009.12.016

Five series of thiourea derivatives bearing benzothiazole moiety (20 compounds) were efficiently synthesized and evaluated for antimicrobial and anticancer activities. The results indicated that the compounds possessed a broad spectrum of activity against the tested microorganisms and showed higher activity against fungi than bacteria. Compounds 1b, 2b, 3b, 4b and 5b exhibited the greatest antimicrobial activity. Preliminary study of the structure-activity relationship revealed that electronic factors in benzothiazole rings had a great effect on the antimicrobial activity of these compounds. In preliminary MTT cytotoxicity studies, the thiourea derivatives (2d, 5c and 5d) were found most potent. In MCF-7 and HeLa cells, the IC₅₀ values were observed in the range of 18-26?μM and 38-46?μM, respectively.

Full text of DOI:10.1016/j.ejmech.2009.12.016

European Journal of Medicinal Chemistry 45 (2010) 1323–1331
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, characterization and biological evaluation of some thiourea derivatives
bearing benzothiazole moiety as potential antimicrobial and anticancer agents
,
a
b
c
d
Sohail Saeed a , Naghmana Rashid , Peter G. Jones , Muhammad Ali , Rizwan Hussain
*
a Department of Chemistry, Research Complex, Allama Iqbal Open University, Islamabad, Pakistan
b
¨
Institut fu¨r Anorganische und Analytische Chemie, Technische Universitat, Braunschweig, Postfach 3329, 38023 Braunschweig, Germany
c Department of Pharmacy, University of the Punjab, Lahore, Pakistan
d National Engineering & Scientific Commission, PO Box. 2801, Islamabad, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
Five series of thiourea derivatives bearing benzothiazole moiety (20 compounds) were efficiently
synthesized and evaluated for antimicrobial and anticancer activities. The results indicated that the
compounds possessed a broad spectrum of activity against the tested microorganisms and showed
higher activity against fungi than bacteria. Compounds 1b, 2b, 3b, 4b and 5b exhibited the greatest
antimicrobial activity. Preliminary study of the structure–activity relationship revealed that electronic
factors in benzothiazole rings had a great effect on the antimicrobial activity of these compounds. In
preliminary MTT cytotoxicity studies, the thiourea derivatives (2d, 5c and 5d) were found most potent. In
Received 19 August 2009
Received in revised form
3 December 2009
Accepted 7 December 2009
Available online 16 December 2009
Keywords:
MCF-7 and HeLa cells, the IC50 values were observed in the range of 18–26
respectively.
mM and 38–46 mM,
Thiourea derivatives
Antibacterial activity
Anti-fungal activity
Anticancer agents
Benzothiazole moiety
MCF-7
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
benzothiazoles have produced DNA topoisomerase [9,10] or HIV
reverse transcriptase inhibitors [11,12]. Thiourea derivatives
display a wide range of biological activity including antibacterial,
anti-fungal, antitubercular, antithyroid, antihelmintic, rodentici-
dal, insecticidal, herbicidal, and plant growth regulator proper-
ties [13–18].
The importance of such work lies in the possibility that the
next generation thiourea derivatives might be more efficacious as
antimicrobial and anticancer agents. However, a thorough inves-
tigation relating the structure and the activity of the thiourea
derivatives as well as their stability under biological conditions is
required. These detailed investigations could be helpful in
designing more potent antimicrobial and anticancer agents for the
therapeutic use.
Since varying substituents is a common method for drug
design in medicinal chemistry and a useful medical value of
substituted thiourea derivatives containing benzothiazole moiety,
we aimed to synthesize new thiourea derivatives and to investi-
gate their antimicrobial and anti-tumor activities. Based on these
reports, we herein report the synthesis, characterization, anti-
bacterial, anti-fungal and in vitro evaluation of anti-tumor activity
of five different series of novel thiourea derivatives bearing ben-
zothiazole moiety.
The development of new antimicrobial and anticancer thera-
peutic agents is one of the fundamental goals in medicinal chem-
istry. Cytotoxicity and genotoxicity of anticancer drugs to the
normal cells are major problems in cancer therapy and engender
the risk of inducing secondary malignancy [1]. A dose of anticancer
drug sufficient to kill tumor cells is often toxic to the normal tissue
and leads to many side effects, which in turn, limits its treatment
efficacy. In recent years, there has been a concerned search for the
discovery and development of novel selective anti-tumor agents,
devoid of many of the unpleasant side effects of conventional anti-
tumor agents.
In the efforts to develop drugs with such capabilities, scien-
tists have focused upon many different aspects of cancer biology
during their research. Among the anti-tumor drugs discovered in
the recent years, various benzothiazoles [2–4] as well as urea
and thiourea derivatives [5–8] possess potent anticancer prop-
erties. The combinations of urea and thiourea derivatives with
* Corresponding author. Tel.: þ92 51 9057225; fax: þ92 51 9250081.
E-mail address: sohail262001@yahoo.com (S. Saeed).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.016

1324
S. Saeed et al. / European Journal of Medicinal Chemistry 45 (2010) 1323–1331
2. Chemistry
3. Results and discussion
In the present study thiourea derivatives bearing benzothiazole
moiety were synthesized as presented in Scheme 1. These
compounds were prepared according to our published procedure
[19,20] with minor modifications. The use of phase transfer catalyst
(PTC) as a method of agitating a heterogeneous reaction system is
gaining recognition [21,22]. In search of improving methods to
prepare the target thiourea by reacting isothiocyanates with
nucleophiles, we have found the use of tetrabutyl ammonium
bromide (TBAB) as PTC can afford isothiocyanates in good yield. In
this paper, we have conducted our reaction using TBAB as PTC to
synthesize the thiophenoyl, butanoyl, benzoyl, para-nitrobenzoyl
and morpholinoyl thiourea derivatives bearing benzothiazole
moiety. All the structures of newly synthesized compounds were
assigned on the basis of their elemental analysis and spectroscopic
data, IR and 1H NMR. All the compounds were soluble in DMF,
DMSO, ethanol and ethyl acetate. IR (KBr) spectrum of all the
synthesized compounds had strong N–H absorptions at about
3.1. Antimicrobial activities
In the light, interesting antimicrobial activities of thiourea deriv-
atives were screened for antibacterial and anti-fungal activity against
Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus fae-
calis, Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae,
Proteus vulgaris, Enterobacter aerogenes, Candida albicans, Candida
glabrata and Candida tropicalis by the broth micro-dilutionprocedure.
The Gram-positive antibacterial agent, amikacin, the Gram-negative
antibacterial agent, gentamycin, and the anti-fungal agent, nystatin,
were used as controls. The in vitro antimicrobial properties against
a number of Gram-negative and Gram-positive bacteria, and yeasts
are presented in Tables 1–3, respectively.
All the compounds inhibited the growth of bacteria with MIC
values ranging between 10 and 100
activity with MICs between 5 and 50
showed MIC of >100 g/ml or above have not been included for the
m
g/ml and showed anti-yeast
mg/ml. Compounds which
m
3300 cmꢀ1, and displayed absorptions at about 1670–1710 cmꢀ1
,
discussion. According to the antimicrobial studies, all the
compounds showed such activity, albeit lower than their anti-yeast
efficacy. This difference may be due to the differences between the
cell structure of bacteria and yeast. While the cell wall of fungi
contain chitin, the cell wall of bacteria contains murein [17]. In
addition, fungi contain ergosterol in their cell membranes instead of
the cholesterol found in the cell membranes of animals [23]. When
all the anti-yeast MIC values are compared, four of twenty
compounds show good activity against C. glabrata and ten of twenty
compounds showed low activity against C. tropicalis. According to
the antibacterial studies, the efficacy against Gram-negative is
higher than Gram-positive bacteria. Seven of twenty compounds
showed good activity against S. epidermidis. In addition, 2b and 5b
showed high activityagainst Gram-positive, Gram-negativebacteria
and fungi. The value of the investigated compounds in this research
were higher than that reported for other thiourea derivatives
[24–26]. The main difference in the thiourea derivatives reported in
this paper is the presence of the benzothiazole moiety.
1440 cmꢀ1, which were assigned to C]O, and C]S functions
respectively. The medium strong yC]O band in the IR spectra of all
the compounds appeared at 1670–1710 cmꢀ1, which is lower than
that of the ordinary carbonyl absorption (1730 cmꢀ1). The formation
of H-bond leads an increase of their polarity, so the strength of their
double bond decreased, and absorption moved to lower wave
number. The 1H NMR spectrum exhibited broad signals at 10.50–
13.75 ppm, which were assigned to the N–H protons. 13C NMR
showed peaks at
d 168.29–169.09, 178.05–180.01 ppm for C]O
(amide) and C]S (thioamide), respectively for all the target
compounds.
R
NH
NH
R
NH
+
2
2
HCl
NH SCN
4
S
( I )
From the results of the antimicrobial activity of the synthesized
substituted thiourea derivatives, the following structure–activity
relationships (SAR) can be derived:
R
NH
NH
2
R
N
Br , CHCl
2
3
NH
2
S
S
a) In general, it was observed that most of the compounds with
substituted benzothiazole rings showed better antimicrobial
activity than the ones with a non-substituted benzothiazole ring.
b) The high antimicrobial activity of compounds 2b and 5b in
comparison to compounds 1a–5a may be attributed to the
( II )
C
O
O
S
TBAB
+
NH SCN
4
R
N
1
R
Cl
1
R
( III )
Table 1
MIC values (
negative bacteria.
mg/ml) of the synthesized thiourea derivatives against the tested Gram-
O
S
N
+
( III )
( II )
Compound
code
E. cloacae
P. aeruginosa
E. coli
P. vulgaris
S
R
N
N
1
(ATCC 13047) (ATCC 27853) (ATCC 25922) (ATCC 13315)
1b
1c
2a
2b
2c
2d
3b
3c
4b
4d
5a
5b
5c
5d
25
25
50
15
50
50
25
50
20
50
25
15
25
50
2
50
50
50
20
50
25
20
50
20
50
50
25
50
50
1
50
25
25
15
20
25
20
50
25
25
25
15
20
25
25
25
50
20
25
50
15
50
20
50
25
20
20
50
2
H
H
1a-1d: R1= 4-nitrophenyl
2a-2d: R1= 2- thiophene
1a- 5a: R= H
1b- 5b: R= NO2
1c- 5c: R= NH2
1d- 5d: R= Br
3a-3d
: R1 = phenyl
4a-4d: R1 = n-butyl
: R = 4-morpholine
5a-5d
1
Gentamycin
0.5
Scheme 1. Preparation of thiourea derivatives bearing benzothiazole moiety.

S. Saeed et al. / European Journal of Medicinal Chemistry 45 (2010) 1323–1331
1325
Table 2
Table 4
MIC values (
positive bacteria.
m
g/ml) of the synthesized thiourea derivatives against the tested Gram-
Preliminary MTTcytotoxicity screening of synthesized thiourea derivatives at 24 h of
drug exposure.
Compound S. epidermidis
S. aureus
E. faecalis
S. aureus
Compounds code
IC50 (mM)
code
(ATCC 12228) (ATCC 29213) (ATCC 29212) (ATCC 25923)
MCF-7
HeLa
1b
1c
1d
2a
2b
3b
3c
3d
4b
4c
4d
5a
5b
5c
5d
Amikacin
25
50
50
50
10
20
50
50
50
50
50
25
10
20
25
50
50
50
50
25
25
50
50
50
50
50
50
25
25
25
2
50
50
50
50
15
50
50
50
50
50
50
50
10
50
50
4
50
50
50
50
25
15
50
50
50
50
50
50
20
50
50
2
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
33.81
35.23
33.20
32.40
32.63
35.85
28.41
24.15
37.27
41.64
38.61
32.11
36.55
>100
44.73
45.72
29.31
31.00
26.43
18.10
2.62
62.56
68.47
63.11
56.53
68.25
72.56
56.10
46.46
57.47
75.13
70.09
64.56
56.70
NT
mM
NT
0.5
60.18
51.80
54.80
45.29
38.85
3.24
5a
5b
5c
5d
presence of nitro functional groups on benzothiazole ring. These
electron withdrawing groups may decrease electron density on
benzene ring through resonance effect. The synthesized
compounds containing nitro functional groups on benzothia-
zole ring are highly active not only against Gram-positive, but
also against Gram-negative bacteria and fungi (2b and 5b).
c) In contrast to point (b) mentioned above, the presence of
electron donating amino group also showed significant anti-
fungal activity against C. glabrata, C. albicans, and C. tropicalis in
case of 5c.Here the electronic factors is not justified. The other
Doxorubicin
The known numbers of cells (1.0 ꢂ 104) were incubated for 24 h in a 5% CO2
incubator at 37 ꢃC in the presence of different concentrations of test compounds.
After 24 h of drug incubation the MTT solution was added and supernatant was
discarded and 100 ml DMSO was added in each well and absorbance was recorded at
540 nm by ELISA reader.
NT: Not Tested.
lipophilicity. Compounds with 4-nitrophenyl (1b) and phenyl
group (3b) have relatively higher log Pow values of 3.37 ꢁ 0.91 and
3.33 ꢁ 0.92, respectively and hence show more lipophilic character
[27]. The calculated value of partition coefficient (log Pow) for
n-butyl derivative (4b) is 2.68 ꢁ 0.91. Its antibacterial activity is
comparatively lower than the compounds (5b) and (2b) due to its
higher partition coefficient (log Pow) value. The compounds with
a morpholine ring (5b) and thiophene ring (2b), which have the log
Pow value of 0.62 ꢁ 0.97 and 2.97 ꢁ 0.93, respectively show higher
antibacterial activity than other investigated compounds due
probably to their lower lipophilic character.
reason may be due to low lipophilic character (log Pow
0.39 ꢁ 0.97) of the compound.
w
However, the thiophene and morpholine based thiourea deriv-
atives (2b and 5b) containing nitro group as electron withdrawing
group on benzothiazole nucleus showed good antimicrobial
activity. These electron withdrawing groups are also present in
benzothiazole rings for 3b and 4b thiourea derivatives but their
antimicrobial activity is comparatively lower than 2b and 5b. Lip-
ophilicity is another factor, which correlates well with the bioac-
tivity of chemicals, is a very important molecular descriptor and
different lipophilic behavior of compounds plays an important role
in their biological activity mechanisms. The n-octanol/water
partition coefficient (log Pow) is widely used as a general measure of
3.2. Anticancer studies
3.2.1. Cytotoxicity studies on cancerous MCF-7 and HeLa cell lines
In vitro cytotoxicity of synthesized compounds was assessed by
standard 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) bioassay in different cancer cells at 24 h of drug
exposure. To assess the efficacy and to select the promising
compounds human cancer cells (HeLa cells and MCF-7) were used. In
MCF-7 cells, the IC50 values for thiourea derivatives bearing benzo-
Table 3
MIC values (mg/ml) of the synthesized thiourea derivatives against the tested fungi.
Compound
code
C. glabrata
(ATCC32554)
C. albicans
(ATCC 90028)
C. tropicalis
(ATCC 20336)
C. krusei
(ATCC 6268)
1b
1c
1d
2a
2b
2c
3a
3b
3c
3d
4a
4b
4c
4d
5a
25
50
50
25
10
20
25
15
25
25
50
25
50
50
25
5
25
50
50
50
15
25
50
25
25
25
20
20
20
25
25
15
20
50
1
25
25
50
50
15
25
50
50
50
50
50
50
50
50
25
5
25
50
50
25
20
25
50
50
25
50
25
25
50
50
25
20
25
50
0.5
thiazole moiety were in the range of 18.10–45.72
Similarly, in HeLa cells, the IC50 values for thiourea derivatives were
found in the range of 38.85–75.13 M. The compounds 4b and 4c do
mM (Table 4).
m
not show 50% inhibition even at a concentration of 100 mM; hence
Table 5
Cytotoxic activity of promising thiourea derivatives in MCF-7 cells at different time
points of drug exposure by MTT assay.
Compounds code
IC50 (mM)
24 h
48 h
72 h
5d
2d
5c
18.10
24.15
26.43
20.33
15.65
19.18
6.95
12.10
17.05
5b
5c
5d
Nystatin
15
25
2
20
25
4
The IC50 values of promising compounds at 48 h and 72 h were significantly reduced
in comparison with 24 h values.

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S. Saeed et al. / European Journal of Medicinal Chemistry 45 (2010) 1323–1331
Table 6
The effect of 2d and 5d on DNA damaging in MCF-7 cells at 24 h of drug exposure by
comet assay.a
Compounds Tail length
Tail DNA
Tail moment
OTM
2d
10
20
40
50
5d
10
20
40
50
m
m
m
m
M
M
M
M
103.25 ꢁ 7.016 15.36 ꢁ 1.42
107.56 ꢁ 7.87 17.65 ꢁ 1.85
134.10 ꢁ 5.24 22.31 ꢁ 1.63
16.87 ꢁ 2.21
18.09 ꢁ 2.89
86.63 ꢁ 4.26
21.87 ꢁ 1.09
23.35 ꢁ 1.85
102 ꢁ 15.57
305.30 ꢁ 32.12c 31.10 ꢁ 2.01c 105.52 ꢁ 14.75c 139.80 ꢁ 24.09c
m
m
m
m
M
M
M
M
97.10 ꢁ 4.78 14.63 ꢁ 1.105 15.01 ꢁ 1.96
110.27 ꢁ 6.46 16.10 ꢁ 1.41c 18.11 ꢁ 3.0
155.03 ꢁ 7.81 18.78 ꢁ 1.58c 31.87 ꢁ 4.21b
195.27 ꢁ 7.11c 23.62 ꢁ 2.21c 55.15 ꢁ 4.96c
18.35 ꢁ 9.95
21.01 ꢁ 5.98
24.20 ꢁ 7.25
32.33 ꢁ 2.24c
8.83 ꢁ 0.83
Controld
65.56 ꢁ 3.23
9.75 ꢁ 1.16
7.18 ꢁ 1.05
a
All values are means ꢁ SEM.
P < 0.05 compared to control.
P < 0.01 compared to control.
Doxorubicin as control.
b
c
d
they were not evaluated in HeLa cells. Present study reveals that
among the human cancer cell lines tested, MCF-7 cells are more
sensitive to all the tested compounds than HeLa cells. Many anti-
cancer drugs are effective against MCF-7 and HeLa cells by causing
apoptosis through the expression of caspase-3, generating reactive
oxygen species (ROS) and damaging DNA [28]. Cisplatin causes
cytotoxicity in MCF-7 and HeLa cells by a similar mechanism [29].
Chemotherapeutic agents such as doxorubicin, mitoxantrone and
bleomycin cause cytotoxicity by generating ROS [30]. Hence like
other cytotoxic drugs (doxorubicin, mitoxantrone and cisplatin), the
synthesized compounds may act as effective anticancer drugs by
similar mechanism. Previous studies have shown that strong elec-
tronegative atom substitution such as chloro/bromo at the para
position of the aromatic ring increases the lipophilicity of molecules
and is responsible for enhanced cytotoxicity in MTT model [31].
Similar substitutions are present in the compounds 1d, 2d, 3d, 4d
and 5d. We have also observed enhanced cytotoxicity in these
molecules. Hence, these molecules were taken up to assess the
cytotoxic potency at different intervals in MCF-7 cells.
In the MTT time course study, the selected compounds (2d, 5c,
5d) showed dose-dependent and time-dependent activities. The
previous study reported that the most potent fluorinated benzo-
thiazole, 5F 203, produced apoptosis and DNA damage in MCF-7
cells, which is characteristic of cytotoxic activity [32,33]. In our
present study, also MTT assays revealed substantial cytotoxicity in
MCF-7 cells with increasing exposure to drug concentration, the
IC50 values of promising compounds at 48 and 72 h were signifi-
cantly reduced as compared with 24 h values (Table 5)
Fig. 2. Packing diagram of 1-(1,3-benzothiazol-2-yl)-3-(thiophene-5-carbonyl)thiourea
(2a) viewed perpendicular to (101). Hydrogen bonds are indicated by thick dashed lines.
DNA damage-detecting methods, such as sister chromatid
exchange, alkali elution, and micronucleus assay, because of its high
sensitivity [34]. A number of cytotoxic compounds (doxorubicin,
cisplatin etc.) act as anticancer drugs by causing DNA damage and
subsequently inducing apoptosis in cancerous cells. 24 h treatment
with 2d and 5d produced a dose dependent increase in tail
moment. The damage produced by 2d was quite prominent as at
50 mM it produced apoptotic bodies and tail moment was 105.52.
Elongated tail length and reduced DNA content in head are suffi-
cient indicators of DNA damage, and higher degree of damage
would result in greater number of smaller fragments, ending up
Table 7
Crystallographic data, data collection and refinement of 2a structure.
CCDC
738482
Empirical formula
Formula weight
Temperature
Wavelength
C13H9N3OS3
319.41
100(2) K
0.71073 Å
Crystal system
Space group
Unit cell dimensions
Monoclinic
C2/c
a ¼ 24.6305(6) Å
b ¼ 5.88932(11) Å
c ¼ 18.7899(4) Å
2660.93(10) Å3
8
a
b
g
¼ 90ꢃ
3.2.2. DNA damaging studies
¼ 102.506(3)ꢃ
¼ 90ꢃ
To determine the DNA damaging activity of synthesized
compounds, alkaline comet assay was performed in MCF-7 cells.
Comet assays is a rapid and inexpensive method for measuring DNA
single-strand breaks (SSBs). It also has an advantage over the other
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Index ranges
Reflections collected
Independent reflections
Completeness to theta ¼ 30.03ꢃ
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2sigma (I)]
R indices (all data)
Largest diff. peak and hole
1.595 g/cm3
0.554 mmꢀ1
1312
0.20 ꢂ 0.15 ꢂ 0.05 mm3
2.22–30.03ꢃ
ꢀ34 ꢄ h ꢄ 34, ꢀ8 ꢄ k ꢄ 8, ꢀ26 ꢄ l ꢄ 26
33722
3851 [R (int) ¼ 0.0291]
98.9%
Semi-empirical from equivalents
1.00000 and 0.98475
Full-matrix least-squares on F2
3851/0/189
0.994
R1 ¼ 0.0258, wR2 ¼ 0.0670
R1 ¼ 0.0344, wR2 ¼ 0.0685
0.409 and ꢀ0.333 e Åꢀ3
Fig. 1. An ORTEP drawing of 1-(1, 3-benzothiazol-2-yl)-3-(thiophene-5-carbonyl)
thiourea (2a) with displacement ellipsoids plotted at 50% probability level. The dotted
line shows the intramolecular H-bonding interaction.

S. Saeed et al. / European Journal of Medicinal Chemistry 45 (2010) 1323–1331
1327
Table 8
6. Experimental protocols
Selected bond distances (Å), angles (ꢃ) and torsion angles (ꢃ).
6.1. Chemistry
S (1)-C (2)
C (2)-N (3)
C (2)-N (1)
N (3)-C (3A)
C (3A)-C (4)
S (3)-C (13)
S (3)-C (10)
C (9)-O
1.7528(11)
1.2973(14)
1.3913(14)
1.3898(15)
1.4024(16)
1.7018(13)
1.7171(12)
1.2249(14)
1.3804(16)
87.69(5)
Synthetic starting material, reagents and solvents were of
analytical reagent grade or of the highest quality commercially
available and were purchased from Aldrich Chemical Co., Merck
Chemical Co. and were dried when necessary. Melting points were
recorded on Electrothermal IA9000 series digital melting point
apparatus. The proton NMR and 13C spectra were recorded in DMSO-
d6 solvent on Jeol ECS-400 and 300 MHz spectrophotometer using
tetramethylsilane as an internal reference, respectively. The apparent
resonance multiplicity is described as s (singlet), br s (broad singlet),
d (doublet), dd (doublet of doublets), t (triplet), q (quartet) and m
(multiplet). Infrared measurements were recorded in the range
400–4000 cmꢀ1 on spectrum 2000 by Perkin Elmer. Elemental
analysis was carried out using Perkin Elmer CHNS/O 2400. Obtained
results were within 0.4% of the theoretical values. Mass spectra were
recorded onaMAT-112-Sspectrometerat70eV. X-raydiffractiondata
were collected on Oxford Diffraction Xcalibur Nova diffractometer.
Thin layer chromatography (TLC) analysis was carried out on
5 ꢂ 20 cm plate coated with silica gel GF254 type 60 (25–250 mesh)
using an ethyl acetate–petroleum ether mixture (1:2) as solvent.
C (10)-C (11)
C (7A)-S (1)-C (2)
N (3)-C (2)-N (1)
C (10)-C (11)-C (12)
C (13)-S (3)-C (10)
C (7A)-S (1)-C (2)-N (3)
S (1)-C (2)-N (1)-C (8)
N (1)-C (2)-N (3)-C (3A)
C (2)-N (3)-C (3A)-C (4)
N (3)-C (2)-N (1)-C (8)
C (7A)-S (1)-C (2)-N (1)
S (1)-C (2)-N (3)-C (3A)
N (1)-C (8)-N (2)-C (9)
117.47(10)
112.63(11)
91.71(6)
ꢀ0.65(10)
6.54(17)
ꢀ178.72(10)
ꢀ179.26(11)
ꢀ174.31(11)
178.49(10)
0.49(12)
ꢀ0.31(18)
with longer tails. The compound 5d significantly increased the tail
moment and tail length in MCF-7 cells. Previous studies have
shown that cytotoxicity of benzothiazoles is mediated via activa-
tion of the AhR signaling pathway in sensitive MCF-7 cells [35]. The
activation of AhR pathway leads to generation of reactive electro-
philic species by inducing CYP1A1 expression. The generated highly
reactive intermediates cause DNA damage, ultimately resulting in
cell death by activation of apoptotic machinery [36]. Hence our
compounds may be acting by similar mechanisms, which, however,
need to be confirmed by gene expression studies. (Table 6)
6.2. X-ray crystallography
A crystal of 2a in the form of a yellow lath was mounted in inert
oil on a glass fibre. The intensity data were collected at 100 K on an
Oxford Diffraction Xcalibur E diffractometer using
with graphite monochromatized MoKa radiation. The structure was
solved by direct methods and refined by full-matrix least-squares
techniques on F2 using the program SHELXL-97 [41]. Hydrogens of
NH groups were refined freely, other H atoms using a riding model.
u-scan mode
4. Crystal structure of 2a
6.3. General procedure for synthesis
The molecular structure and packing diagram of the compound
2a are shown in Figs. 1 and 2, respectively. Crystallographic data
and refinement are presented in Table 7. Selected bond distances
and angles are listed in Table 8. Compound 2a, C13H9N3OS3,
crystallizes in the thioamide forms. The hydrogens at N1 and N2
are anti and syn respectively with respect to the C ¼ S bond. Bond
lengths and angles may be regarded as normal. The entire mole-
cule is planar to within a mean deviation of 0.04 Å (for non-H
atoms) The C9–O and C8–S2 bonds show a typical double bond
character with bond lengths of 1.2249(14) and 1.6648(12) Å,
respectively. All the C–N bonds, C8–N1 ¼ 1.3426(15), C8–
N2 ¼ 1.3837(15), C2–N3 ¼ 1.2973(14) Å display at least partial
double bond character. Thiophene and benzimidazole units are
planner. There is a strong intramolecular hydrogen bond N1–
H01/O, with H01/O ¼ 1.962(16) Å, forming a 6-membered ring.
The molecular packing is three-dimensional, but can be inter-
preted reasonably well by viewing perpendicular to the (101)
plane (Fig. 2); the weak H bonds H02/LS2 2.92 Å and H11/LS2
2.78 Å can be recognised connecting columns molecules in the
centre of the Figure and H4/LN3 2.55 Å left and right of the
central column.
All the synthesized compounds were prepared according to the
following two-step procedures:
Step 01: To substituted aniline (25 mL), concentrated hydrochloric
acid (25 mL) was added and the solution was warmed for
30 min. A saturated solution of ammonium thiocyanate in
water (30 g in 60 mL) was added slowly in above solution.
The mixture was boiled until the solution got turbid. The
turbid solution was poured in cold water. The resulting
precipitate was filtered and re-crystallized from aqueous
ethanol (80%) to provide pure phenylthiourea. The
substituted phenylthiourea (26 mmol) in chloroform
(75 mL) was brominated by using bromine solution in
chloroform (5%) till the orange-yellow color appeared. The
slurry was kept overnight. The precipitate obtained was
filtered and washed with chloroform until the color dis-
appeared. The precipitate, as hydrobromide, was dissolved
in rectified spirit (150 mL) and basified with ammonia
solution. The precipitated was filtered, washed with water,
dried and re-crystallized using ethanol:dichloromethane
mixture (1:2). Substituted or unsubstituted 2-amino-
benzothiazole prepared by this method was named as (II)
Step 02: A solution of substituted carbonyl chloride (26 mmol) in
anhydrous acetone (80 ml) and 3% TBAB in acetone was
added drop wise to a suspension of ammonium thiocya-
nate in acetone (50 ml) and the reaction mixture was
refluxed for 30 min. After cooling at room temperature,
a solution of substituted or unsubstituted 2-amino-
benzothiazole (26 mmol) in acetone (25 ml) was added
5. Conclusions
An in vitro screen led to the identification of compounds 2d, 5c
and 5d as potential anticancer candidates worthy of further struc-
tural modification and pharmacological evaluation. Moreover, the
antimicrobial activity of this series suggests the benzothiazole-
thiourea core offers a novel template for the development of a new
class of antimicrobial agents.

1328
S. Saeed et al. / European Journal of Medicinal Chemistry 45 (2010) 1323–1331
and the resulting mixture refluxed for 1.5 h. The reaction
mixture was poured into five times its volume of cold
water when the thiourea precipitated as a solid. The solid
product was washed with water and purified by re-crys-
tallization from an ethanol–dichloromethane mixture
(1:2).
(300 MHz, DMSO-d6) in d (ppm): 179.02 (C]S),173.3 (C]N),168.29
(C]O), 150.9, 129.1, 128.4, 125.0, 124.1, 123.1, 116.5; EI MS (70 eV) m/
z (%): 437.30
6.3.5. 1-(1, 3-Benzothiazol-2-yl)-3-(thiophene-5-carbonyl)
thiourea (2a)
Elemental analysis for C13H9N3OS3 (MW ¼ 319.41) in wt% calc.
C ¼ 45.08, H ¼ 2.82, N ¼ 13.16, S ¼ 30.09 and found to be C ¼ 45.10,
H ¼ 2.85, N ¼ 13.15, S ¼ 30.20. m.pt. 185 ꢃC, yield 90%. IR (KBr pellet)
in cmꢀ1: 3309 (free NH), 3206 (assoc NH), 1672 (C]O), 1523
(benzene ring), 1405 (C–N stretching), 1140 (C]S), 1450, 1250, 1020
(benzothiazole stretching). 1H NMR (400 MHz, DMSO-d6) in
6.3.1. 1-(Benzo[d]thiazol-2-yl)-3-(4-nitrobenzoyl) thiourea (1a)
Elemental analysis for C15H10N4O3S2 (MW ¼ 358.39) in wt% calc.
C ¼ 50.27, H ¼ 2.79, N ¼ 15.64, S ¼ 17.87 and found to be C ¼ 50.35,
H ¼ 2.95, N ¼ 15.63, S ¼ 17.84. m.pt. 278 ꢃC, yield 89%. IR (KBr pellet)
in cmꢀ1: 3310 (free NH), 3206 (assoc NH), 1670 (C]O), 1525
(benzene ring), 1404 (C–N stretching), 1512 (NO2), 1140 (C]S). 1H
d
(ppm) and J (Hz): 12.40 (1H, s, broad, NH),10.25 (1H, s, broad, NH),
8.10(H, d, J ¼ 8.41 Hz), 7.34 (H, d, J ¼ 8.7 Hz), 7.31(H, d, J ¼ 8.0 Hz);
13C NMR (300 MHz, DMSO-d6) in
(ppm): 179.02(C]S),
NMR (400 MHz, DMSO-d6) in
d (ppm) and J (Hz): 13.53 (1H, s,
d
broad, NH), 10.25 (1H, s, broad, NH), 7.75 (2H, d, J ¼ 8.2 Hz), 7.65
172.2(C]N), 168.29(C]O), 149.4, 136.5, 128.5, 125.7, 122.3; EI MS
(70 eV) m/z (%): 319.40
(2H, d, J ¼ 6.9 Hz), 7.34 (H, d, J ¼ 8.7 Hz); 13C NMR (300 MHz, DMSO-
d6) in
d (ppm):179.02 (C]S), 173.3 (C]N), 168.29 (C]O), 151.8,
149.2, 144.5, 130.6, 128.2, 122.7, 120.9; EI MS (70 eV) m/z (%): 358.41
6.3.6. 1-(5-nitro[d]benzothiazol-2-yl)-3-(thiophene-5-carbonyl)
thiourea (2b)
6.3.2. 1-(5-Nitrobenzo[d]thiazol-2-yl)-3-(4-nitrobenzoyl)
thiourea (1b)
Elemental analysis for C13H8N4O3S3 (MW ¼ 364.37) in wt% calc.
C ¼ 42.85, H ¼ 2.19, N ¼ 15.38, S ¼ 26.37 and found to be
C ¼ 42.81, H ¼ 2.20, N ¼ 15.36, S ¼ 26.35. m.pt. 225 ꢃC, yield 85%.
IR (KBr pellet) in cmꢀ1: 3315 (free NH), 3204 (assoc NH), 1670
(C]O), 1525 (benzene ring), 1512 (NO2), 1404 (C–N stretching),
Elemental analysis for C15H9N5O5S2 (MW ¼ 403.39) in wt% calc.
C ¼ 44.66, H ¼ 2.23, N ¼ 17.36, S ¼ 15.88 and found to be C ¼ 44.63,
H ¼ 2.25, N ¼ 17.35, S ¼ 15.89. m.pt. 298 ꢃC, yield 73%. IR (KBr pellet)
in cmꢀ1: 3313 (free NH), 3203 (assoc NH), 1678 (C]O), 1520
(benzene ring), 1405 (C–N stretching), 1512 (NO2), 1141 (C]S). 1H
1142 (C]S). 1H NMR (400 MHz, DMSO-d6) in
d (ppm) and J (Hz):
12.40(1H, s, broad, NH), 10.25 (1H, s, broad, NH), 8.12 (d, H,
J ¼ 8.63 Hz), 7.34 (2H, d, J ¼ 8.7 Hz), 7.31(2H, d, J ¼ 8.0 Hz); 13C
NMR (400 MHz, DMSO-d6) d: 13.85 (1H, s, broad, NH), 10.40 (1H, s,
broad, NH), 8.12 (H, d, J ¼ 8.63 Hz), 7.74 (2H, d, J ¼ 8.1 Hz), 7.66 (2H,
NMR (300 MHz, DMSO-d6) in
d (ppm): 180.01(C]S), 174.2(C]N),
d, J ¼ 6.7 Hz); 13C NMR (300 MHz, DMSO-d6) in
d (ppm): 179.01
168.29(C]O), 149.7, 145.5, 137.0, 135.7, 130.3, 128.4, 123.5, 120.7; EI
MS (70 eV) m/z (%): 364.39
(C]S), 173.3 (C]N), 168.51 (C]O), 150.6, 148.3, 145.2, 128.8, 122.9,
114.5; EI MS (70 eV) m/z (%): 403.38
6.3.7. 1-(5-Aminobenzo[d]thiazol-2-yl)-3-(thiophene-5-carbonyl)
thiourea (2c)
6.3.3. 1-(5-Aminobenzo[d]thiazol-2-yl)-3-(4-aminobenzoyl)
thiourea (1c)
This compound was prepared from 2b by the following reduc-
tion procedure. 2b (3.65g, 10 mmol), 5 ml hydrazine monohydrate,
70 ml ethanol and 0.03 g of 10% Pd–C was transferred into 250 ml
two necked round bottom flask and refluxed for 18 h. The reaction
was monitored by TLC. After completion the reaction, it was
allowed to stand for one day and then filtered the reaction mixture.
Removed the solvent by rotary evaporator. The crude product was
re-crystallized in ethanol. Elemental analysis for C13H10N4OS3
(MW ¼ 334.12) in wt% calc. C ¼ 46.70, H ¼ 2.99, N ¼ 16.76, S ¼ 28.74
and found to be C ¼ 46.85, H ¼ 3.01, N ¼ 16.75, S ¼ 28.75. m.pt.
191 ꢃC, yield 80%. IR (KBr pellet) in cmꢀ1: 3315 (free NH), 3204
(assoc NH), 1671 (C]O), 1525 (benzene ring), 1407 (C–N stretch-
This compound was prepared from 1b by the following
reduction procedure. 1b (4.13 g, 10 mmol), 5 ml hydrazine mon-
ohydrate, 70 ml ethanol and 0.03 g of 10% Pd–C was transferred
into 250 ml two necked round bottom flask and refluxed for 18 h.
The reaction was monitored by TLC. After completion the reaction,
it was allowed to stand for one day and then filtered the reaction
mixture. Removed the solvent by rotary evaporator. The crude
product was re-crystallized in ethanol. Elemental analysis for
C15H11N5O3S2 (MW ¼ 373.42) in wt% calc. C ¼ 48.25, H ¼ 2.94,
N ¼ 18.76, S ¼ 17.15 and found to be C ¼ 47.98, H ¼ 2.96,
N ¼ 18.85, S ¼ 17.12. m.pt. 232 ꢃC, yield 70%. IR (KBr pellet) in
cmꢀ1
:
3310 (free NH), 3206 (assoc NH), 1670 (C]O), 1525
(benzene ring), 1404 (C–N stretching), 1140 (C]S). 1H NMR
(400 MHz, DMSO-d6) in (ppm) and J (Hz): 13.11 (1H, s, broad,
ing), 1142 (C]S). 1H NMR (400 MHz, DMSO-d6) in
d (ppm) and J
(Hz):12.40 (1H, s, broad, NH), 10.25(1H, s, broad, NH), 7.35 (2H, d,
J ¼ 8.5 Hz), 7.30 (2H, d, J ¼ 8.1 Hz), 6.60(H, d, J ¼ 8.42 Hz), 5.18(2H, s,
NH2), 4.28 (aromatic C–NH); 13C NMR (300 MHz, DMSO-d6) in
d
NH), 10.25 (1H, s, broad, NH), 7.73 (2H, d, J ¼ 8.0 Hz), 7.63 (2H, d,
J ¼ 6.8 Hz), 6.62(H, d, J ¼ 8.44 Hz), 5.18(2H, s, NH2); 13C NMR
d
(ppm): 180.01(C]S), 174.2(C]N), 168.29(C]O), 149.7, 145.5,
(300 MHz, DMSO-d6) in
d (ppm): 178.05 (C]S), 173.0 (C]N),
137.0, 135.7, 130.3, 128.4, 122.3, 120.7; EI MS (70 eV) m/z (%): 334.15
168.29 (C]O), 151.3, 148.5, 145.8, 130.7, 128.4, 122.1, 120.8, 116.9; EI
MS (70 eV) m/z (%): 373.45
6.3.8. 1-(5-Bromobenzo[d]thiazol-2-yl)-3-(thiophene-5-carbonyl)
thiourea (2d)
6.3.4. 1-(5-Bromobenzo[d]thiazol-2-yl)-3-(4-nitrobenzoyl)
thiourea (1d)
Elemental analysis for C13H8 BrN3OS3 (MW ¼ 398.68) in wt%
calc. C ¼ 39.19, H ¼ 2.01, N ¼ 10.55, S ¼ 24.12 and found to be
C ¼ 39.23, H ¼ 2.36, N ¼ 10.56, S ¼ 24.27. m.pt. 182 ꢃC, yield 69%. IR
(KBr pellet) in cmꢀ1: 3315 (free NH), 3204 (assoc NH), 1670 (C]O),
1525 (benzene ring), 1404 (C–N stretching), 1142 (C]S). 1H NMR
Elemental analysis for C15H9 BrN4O3S2 (MW ¼ 437.29) in wt%
calc. C ¼ 41.18, H ¼ 2.05, N ¼ 12.81, S ¼ 14.64 and found to be
C ¼ 41.17, H ¼ 2.08, N ¼ 12.83, S ¼ 14.63. m.pt. 247–248 ꢃC, yield
79%. IR (KBr pellet) in cmꢀ1: 3309 (free NH), 3206 (assoc NH), 1669
(C]O), 1525 (benzene ring), 1404 (C–N stretching), 1511 (NO2),
(400 MHz, DMSO-d6) in d (ppm) and J (Hz): 12.04 (1H, s, broad, NH),
10.32(1H, s, broad, NH), 7.34 (2H, d, J ¼ 8.7 Hz), 7.31(2H, d,
1140 (C]S). 1H NMR (400 MHz, DMSO-d6) in
d (ppm) and J (Hz):
J ¼ 8.0 Hz), 6.61(H, d, J ¼ 8.40 Hz); 13C NMR (300 MHz, DMSO-d6) in
13.75 (1H, s, broad, NH), 12.40 (1H, s, broad, NH), 7.75 (2H, d, J ¼ 8.2
d
(ppm): 180.01 (C]S), 174.2(C]N), 168.29(C]O), 149.5, 145.5,
Hz), 7.65 (2H, d, J ¼ 6.9 Hz), 6.61(H, d, J ¼ 8.40 Hz); 13C NMR
137.0, 135.7, 130.3, 128.4, 122.8, 120.1; EI MS (70 eV) m/z (%): 398.69

S. Saeed et al. / European Journal of Medicinal Chemistry 45 (2010) 1323–1331
1329
6.3.9. 1-(1, 3-Benzothiazol-2-yl)-3-benzoylthiourea (3a)
(400 MHz, DMSO-d6) in
d
(ppm) and J (Hz): 12.40 (1H, s, broad, NH),
Elemental analysis for C15H11N3OS2 (MW ¼ 313.39) in wt% calc.
C ¼ 57.50, H ¼ 3.51, N ¼ 13.41, S ¼ 20.44 and found to be C ¼ 57.43,
H ¼ 3.53, N ¼ 13.40, S ¼ 20.45. m.pt. 203–205 ꢃC, yield 78%. IR (KBr
pellet) in cmꢀ1: 3310 (free NH), 3206 (assoc NH), 1670 (C]O), 1525
(benzene ring), 1404 (C–N stretching), 1140 (C]S). 1H NMR
10.25 (1H, s, broad, NH), 8.13 (H, d, J ¼ 8.2 Hz), 2.17 (t, –CH2, J ¼ 7.3
Hz), 1.56 (m, –CH2), 0.95 (t, –CH3, J ¼ 7.1 Hz); 13C NMR (300 MHz,
DMSO-d6) in
d (ppm): 179.02 (C]S), 173.9 (C]N), 168.29 (C]O),
148.2, 126.6, 124.8, 121.2, 60.0, 45.3, 23.1; EI MS (70 eV) m/z (%):
279.36
(400 MHz, DMSO-d6) in d (ppm) and J (Hz): 12.40 (1H, s, broad, NH),
10.25 (1H, s, broad, NH), 7.75 (2H, d, J ¼ 8.2 Hz), 7.65 (2H, d,
6.3.14. 1-Butyryl-3-(5-nitrobenzo[d]thiazol-2-yl) thiourea (4b)
Elemental analysis for C12H12 N4O3S2 (MW ¼ 324.38) in wt%
calc. C ¼ 44.44, H ¼ 3.70, N ¼ 17.28, S ¼ 19.75 and found to be
C ¼ 44.21, H ¼ 3.87, N ¼ 17.26, S ¼ 19.76. m.pt. 197–198 ꢃC, yield
71%. IR (KBr pellet) in cmꢀ1: 3318 (free NH), 3205 (assoc NH), 1669
(C]O), 1525 (benzene ring), 1403 (C–N stretching), 1512 (NO2),
J ¼ 6.9 Hz), 7.34 (H, d, J ¼ 8.7 Hz), 7.31(H, d, J ¼ 8.0 Hz); 13C NMR
(300 MHz, DMSO-d6) in
d (ppm): 179.02 (C]S), 168.29 (C]O),
151.7, 148.9, 144.5, 142.1, 136.2, 128.4, 127.3; EI MS (70 eV) m/z (%):
313.37
6.3.10. 1-Benzoyl-3-(5-nitrobenzo[d]thiazol-2-yl) thiourea (3b)
Elemental analysis for C15H10N4O3S2 (MW ¼ 358.39) in wt% calc.
C ¼ 50.27, H ¼ 2.79, N ¼ 15.64, S ¼ 17.87 and found to be C ¼ 50.31,
H ¼ 2.84, N ¼ 15.64, S ¼ 17.85. m.pt. 203–205 ꢃC, yield 72%. IR (KBr
pellet) in cmꢀ1: 3310 (free NH), 3206 (assoc NH), 1671 (C]O), 1527
(benzene ring), 1406 (C–N stretching), 1512 (NO2), 1140 (C]S). 1H
1140 (C]S). 1H NMR (400 MHz, DMSO-d6) in
d (ppm) and J (Hz):
13.55 (1H, s, broad, NH), 10.28 (1H, s, broad, NH), 8.12 (d, H, J ¼ 8.63
Hz), 2.17 (t, –CH2, J ¼ 7.3 Hz), 1.56 (m, –CH2), 0.95 (t, –CH3, J ¼ 7.1
Hz); 13C NMR (300 MHz, DMSO-d6) in
d (ppm): 179.02 (C]S), 174.3
(C]N), 168.29 (C]O), 149.8, 146.2, 130.6, 122.9, 120.4, 117.5, 60.6,
45.3, 23.3; EI MS (70 eV) m/z (%): 324.37
NMR (400 MHz, DMSO-d6) in
d (ppm) and J (Hz): 12.40 (1H, s,
broad, NH), 10.20 (1H, s, broad, NH), 8.12 (d, H, J ¼ 8.63 Hz), 7.75
6.3.15. 1-Butyryl-3-(5-aminobenzo[d]thiazol-2-yl) thiourea (4c)
This compound was prepared from 4b by the following reduc-
tion procedure. 4b (3.30g, 10 mmol), 5 ml hydrazine monohydrate,
70 ml ethanol and 0.03 g of 10% Pd–C was transferred into 250 ml
two necked round bottom flask and refluxed for 18 h. The reaction
was monitored by TLC. After completion the reaction, it was
allowed to stand for one day and then filtered the reaction mixture.
The crude product was re-crystallized in ethanol. Elemental anal-
ysis for C12H14 N4OS2 (MW ¼ 294.39) in wt% calc. C ¼ 48.97,
H ¼ 4.76, N ¼ 19.04, S ¼ 21.76 and found to be C ¼ 48.84, H ¼ 4.89,
N ¼ 19.02, S ¼ 21.85. m.pt. 165–166 ꢃC, yield 69%. IR (KBr pellet) in
cmꢀ1: 3318 (free NH), 3210 (assoc NH), 1672 (C]O), 1525 (benzene
ring),1403 (C–N stretching),1140 (C]S). 1H NMR (400 MHz, DMSO-
(2H, d, J ¼ 8.2 Hz), 7.65 (2H, d, J ¼ 6.9 Hz), 7.34 (2H, d, J ¼ 8.7 Hz); 13C
NMR (300 MHz, DMSO-d6) in d (ppm): 179.02 (C]S), 168.29 (C]O),
151.8, 149.2, 144.0, 142.8, 134.6, 128.2, 127.9; EI MS (70 eV) m/z (%):
358.41
6.3.11. 1-Benzoyl-3-(5-aminobenzo[d]thiazol-2-yl) thiourea (3c)
This compound was prepared from 3b by the following reduc-
tion procedure. 3b (3.60 g, 10 mmol), 5 ml hydrazine monohydrate,
70 ml ethanol and 0.03 g of 10% Pd–C was transferred into 250 ml
two necked round bottom flask and refluxed for 18 h. The reaction
was monitored by TLC. After completion the reaction, it was
allowed to stand for one day and then filtered the reaction mixture.
Removed the solvent by rotary evaporator. The crude product was
re-crystallized in ethanol. Elemental analysis for C15H12N4OS2
(MW ¼ 328.41) in wt% calc. C ¼ 54.87, H ¼ 3.65, N ¼ 17.07, S ¼ 19.51
and found to be C ¼ 54.90, H ¼ 3.70, N ¼ 17.10, S ¼ 19.48. m.pt. 203–
205 ꢃC, yield 82%. IR (KBr pellet) in cmꢀ1: 3313 (free NH), 3206
(assoc NH), 1672 (C]O), 1523 (benzene ring), 1404 (C–N stretch-
d6) in
d (ppm) and J (Hz): 12.40 (1H, s, broad, NH), 10.25 (1H, s,
broad, NH), 8.13 (H, d, J ¼ 8.41 Hz), 5.13 (2H, s, NH2), 2.17 (t, –CH2,
J ¼ 7.3 Hz), 1.56 (m, –CH2), 0.95 (t, –CH3, J ¼ 7.1 Hz); 13C NMR
(300 MHz, DMSO-d6) in d (ppm): 179.02 (C]S), 174.0 (C]N),168.29
(C]O),149.0,145.5142.5, 122.6,115.8, 60.6, 45.0, 23.4; EI MS (70 eV)
m/z (%): 294.39
ing), 1141 (C]S). 1H NMR (400 MHz, DMSO-d6) in
d (ppm) and J
(Hz): 12.45 (1H, s, broad, NH); 10.20 (1H, s, broad, NH), 7.73 (2H, d,
6.3.16. 1-Butyryl-3-(5-bromobenzo[ d]thiazol-2-yl) thiourea (4d)
Elemental analysis for C12H12 BrN3OS2 (MW ¼ 358.27) in wt%
calc. C ¼ 40.22, H ¼ 3.35, N ¼ 11.73, S ¼ 17.87 and found to be
C ¼ 40.56, H ¼ 3.51, N ¼ 12.00, S ¼ 17.81. m.pt. 158 ꢃC, yield 79%. IR
(KBr pellet) in cmꢀ1: 3310 (free NH), 3206 (assoc NH), 1670 (C]O),
1525 (benzene ring), 1404 (C–N stretching), 1140 (C]S); 1H NMR
J ¼ 8.1 Hz), 7.62 (2H, d, J ¼ 6.8 Hz), 6.63(H, d, J ¼ 8.40 Hz), 5.18(2H, s,
NH2); 13C NMR (300 MHz, DMSO-d6) in
d (ppm): 179.02 (C]S),
168.29 (C]O), 151.8, 149.3, 144.5, 142.7, 125.4, 128.4, 127.0; EI MS
(70 eV) m/z (%): 328.45
6.3.12. 1-Benzoyl-3-(5-bromobenzo[d]thiazol-2-yl) thiourea (3d)
Elemental analysis for C15H10 BrN3OS2 (MW ¼ 392.29) in wt%
calc. C ¼ 45.91, H ¼ 2.55, N ¼ 10.71, S ¼ 16.32 and found to be
C ¼ 46.01, H ¼ 2.85, N ¼ 10.72, S ¼ 16.35. m.pt. 203–205 ꢃC, yield
83%. IR (KBr pellet) in cmꢀ1: 3310 (free NH), 3204 (assoc NH),
1670 (C]O), 1526 (benzene ring), 1402 (C–N stretching), 1140
(400 MHz, DMSO-d6) in d (ppm) and J (Hz): 12.40 (1H, s, broad, NH),
10.25 (1H, s, broad, NH), 8.12 (H, d, J ¼ 8.40 Hz), 2.17 (t, –CH2, J ¼ 7.3
Hz), 1.56 (m, –CH2), 0.95 (t, –CH3, J ¼ 7.1 Hz); 13C NMR (300 MHz,
DMSO-d6) in
d (ppm): 179.02 (C]S), 174.8 (C]N), 168.29 (C]O),
151.8, 129.1, 125.7, 124.1, 123.6, 116.8, 60.1, 45.1, 23.5; EI MS (70 eV)
m/z (%): 358.28
(C]S). 1H NMR (400 MHz, DMSO-d6) in
d (ppm) and J (Hz): 12.15
(1H, s, broad, NH), 10.70 (1H, s, broad, NH), 7.70 (2H, d, J ¼ 8.2
6.3.17. 1-(1, 3-Benzothiazol-2-yl)-3-(morpholine-4-carbonyl)
thiourea (5a)
Hz), 7.65 (2H, d, J ¼ 6.9 Hz), 6.60(H, d, J ¼ 8.41 Hz); 13C NMR
(300 MHz, DMSO-d6) in
d
(ppm): 179.02 (C]S), 168.29 (C]O),
Elemental analysis for C13H14 N4O2S2 (MW ¼ 322.40) in wt%
calc. C ¼ 48.48, H ¼ 4.34, N ¼ 17.39, S ¼ 19.87 and found to be
C ¼ 48.46, H ¼ 4.37, N ¼ 17.36, S ¼ 19.85. m.pt. 172–173 ꢃC, yield
88%. IR (KBr pellet) in cmꢀ1: 3310 (free NH), 3206 (assoc NH), 1670
(C]O), 1525 (benzene ring), 1404 (C–N stretching), 1140 (C]S). 1H
151.3, 149.2, 144.2, 142.2, 134.2, 128.6, 127.2; EI MS (70 eV) m/z
(%): 392.28
6.3.13. 1-(1, 3-Benzothiazol-2-yl)-3-butyryl thiourea (4a)
Elemental analysis for C12H13 N3OS2 (MW ¼ 279.38) in wt% calc.
C ¼ 51.61, H ¼ 4.65, N ¼ 15.05, S ¼ 22.93 and found to be C ¼ 51.56,
H ¼ 4.85, N ¼ 15.10, S ¼ 22.96. m.pt.156 ꢃC, yield 75%. IR (KBr pellet)
in cmꢀ1: 3311 (free NH), 3203 (assoc NH), 1670 (C]O), 1523
(benzene ring), 1401 (C–N stretching), 1140 (C]S). 1H NMR
NMR (400 MHz, DMSO-d6) in d (ppm) and J (Hz): 13.10 (1H, s, broad,
NH), 11.05 (1H, s, broad, NH), 7.75 (2H, d, J ¼ 8.2 Hz), 7.65 (2H, d,
J ¼ 6.9 Hz), 6.61(H, d, J ¼ 8.40 Hz); 13C NMR (300 MHz, DMSO-d6) in
d
(ppm): 179.02 (C]S), 168.29 (C]O), 151.8, 149.2, 144.5, 142.2,
134.8, 128.1, 127.3; EI MS (70 eV) m/z (%): 322.42

1330
S. Saeed et al. / European Journal of Medicinal Chemistry 45 (2010) 1323–1331
6.3.18. 1-(Morpholine-4-carbonyl)-3-(5-nitrobenzo[d]thiazol-2-yl)
thiourea (5b)
matched the Mc Farland Standard no: 0.5 Petri dishes containing
Mueller–Hinton agar for bacteria and Sabouraud Dextrose agar for
fungi were impregnated with these microbial suspensions. The
stock solutions of the synthesized compounds were prepared in
dimethyl sulfoxide (DMSO), which had no effect on the organisms
in the concentrations studied. The initial concentration was
200 mg/ml. All of the dilutions were done with distillated water.
The concentrations of tested compounds were 100, 50, 25, 12.5,
Elemental analysis for C13H13 N5O4S2 (MW ¼ 367.73) in wt% calc.
C ¼ 42.50, H ¼ 3.54, N ¼ 19.07, S ¼ 17.43 and found to be C ¼ 42.62,
H ¼ 3.85, N ¼ 18.97, S ¼ 17.47. m.pt. 217–218 ꢃC, yield 82%. IR (KBr
pellet) in cmꢀ1: 3313 (free NH), 3201 (assoc NH), 1670 (C]O), 1523
(benzene ring), 1512 (NO2), 1406 (C–N stretching), 1140 (C]S). 1H
NMR (400 MHz, DMSO-d6) in d (ppm) and J (Hz): 13.15 (1H, s, broad,
NH), 12.40 (1H, s, broad, NH), 8.12 (d, H, J ¼ 8.63 Hz), 7.75 (2H, d,
6.25, 3.125
mg/ml. DMSO was used as negative control. Gentamycin,
J ¼ 8.2 Hz), 7.65 (2H, d, J ¼ 6.9 Hz); 13C NMR (300 MHz, DMSO-d6) in
amikacin and nystatin were used as reference drugs for Gram-
negative antibacterial activity, Gram-positive antibacterial activity
and anti-fungal activity, respectively. All the inoculated plates were
incubated at 37 ꢃC and results were evaluated after 24 h for bacteria
and 48 h for fungi. The lowest concentration of the compounds that
prevented visible growth was considered minimal inhibitor
concentrations (MICs)
d
(ppm): 180.01 (C]S), 169.25 (C]O), 150.2, 149.1, 144.5, 142.1,
134.8, 128.2, 127.2; EI MS (70 eV) m/z (%): 367.74
6.3.19. 1-(Morpholine-4-carbonyl)-3-(5-aminobenzo[d]thiazol-2-
yl) thiourea (5c)
This compound was prepared from 5b by the following reduc-
tion procedure. 5b (3.70 g, 10 mmol), 5 ml hydrazine monohydrate,
70 ml ethanol and 0.03 g of 10% Pd–C was transferred into 250 ml
two necked round bottom flask and refluxed for 18 h. The reaction
was monitored by TLC. After completion the reaction, it was
allowed to stand for two days and then filtered the reaction
mixture. Removed the solvent by rotary evaporator. The crude
product was re-crystallized in ethanol. Elemental analysis for
C13H15 N5O2S2 (MW ¼ 337.83) in wt% calc. C ¼ 46.29, H ¼ 4.45,
N ¼ 20.77, S ¼ 18.99 and found to be C ¼ 46.31, H ¼ 4.49, N ¼ 20.65,
S ¼ 18.95. m.pt. 185–186 ꢃC, yield 81%. IR (KBr pellet) in cmꢀ1: 3310
(free NH), 3204 (assoc NH), 1670 (C]O), 1523 (benzene ring), 1402
(C–N stretching), 1140 (C]S). 1H NMR (400 MHz, DMSO-d6) in
6.5. Evaluation of anti-tumor activity
6.5.1. Preliminary in vitro cytotoxic activities (MTT assay)
In vitro cytotoxicity was determined using a standard MTT assay
[39] with protocol appropriate for the individual test system. Test
compounds were prepared prior to the experiment by dissolving in
0.1% DMSO and diluted with medium. The cells were then exposed
to different concentrations of the drugs (1–100
of 100 M/well. Cells in the control wells received the same volume
of medium containing 0.1% DMSO. After 24 h, the medium was
removed and cell cultures were incubated with 100 l MTT reagent
mM) in the volume
m
m
(1 mg/ml) for 5 h at 37 ꢃC. The suspension was placed on micro-
vibrator for 10 min and absorbance was recorded by the ELISA
reader. The experiment was performed in triplicate. Human cancer
cell lines, MCF-7 and HeLa cells were cultured in MEM medium
d
(ppm) and J (Hz): 13.02 (1H, s, broad, NH), 11.52 (1H, s, broad, NH),
7.75 (2H, d, J ¼ 8.2 Hz), 7.65 (2H, d, J ¼ 6.9 Hz), 6.61(H, d,
J ¼ 8.40 Hz), 5.15(2H, s, NH2); 13C NMR (300 MHz, DMSO-d6) in
d
(ppm): 179.08 (C]S),169.09 (C]O),151.5,148.9,144.5142.1,134.5,
128.6, 127.0; EI MS (70 eV) m/z (%): 337.85
supplemented with 10% FBS, 1% glutamine and 50 mM/ml genta-
micin sulphate in a CO2 incubator in a humidified atmosphere of 5%
CO2 and 95% air.
6.3.20. 1-(Morpholine-4-carbonyl)-3-(5-bromobenzo[d]thiazol-2-
yl) thiourea (5d)
Elemental analysis for C13H9 BrN4O2S2 (MW ¼ 397.51) in wt%
calc. C ¼ 39.29, H ¼ 2.26, N ¼ 14.10, S ¼ 16.12 and found to be
C ¼ 39.23, H ¼ 2.27, N ¼ 14.15, S ¼ 16.11. m.pt. 175 ꢃC, yield 78%. IR
(KBr pellet) in cmꢀ1: 3309 (free NH), 3205 (assoc NH), 1671 (C]O),
1521 (benzene ring), 1404 (C–N stretching), 1145 (C]S). 1H NMR
6.5.2. Alkaline comet assay
The effect of synthesized compounds on DNA was assessed by
comet assay as described by protocol [40] with slight modifications.
After the drug treatment, cells were harvested from each culture
flask and counted by trypan blue exclusion method. About 18,000
(400 MHz, DMSO-d6) in d (ppm) and J (Hz): 13.05 (1H, s, broad, NH),
cells in 50 ml medium were suspended in 150 ml of low melting
11.10 (1H, s, broad, NH), 7.76 (2H, d, J ¼ 8.1 Hz), 7.67 (2H, d,
agarose (0.75%) and layered onto slides pre-coated with an earlier
layer of agarose (1.5%). Slides were kept in lying solution (2.5 M
NaCl, 100 mM EDTA, 10 mM Tris; pH 10–10.5; 1% Triton X-100 and
10% DMSO) overnight at 4 ꢃC in dark, these were subjected to
unwinding under alkaline conditions (pH 13) for 1 h to allow DNA
supercoils to relax and express DNA single-strand breaks and
alkali-labile sites. Electrophoresis was then carried out under
highly alkaline (pH 13) conditions for 30 min at 16 V and 300 mA.
Three slides were prepared for each concentration. About 50 cells
were captured per slide using fluorescent microscope and the
images were analyzed using Komet 5.5 software (kinetic imaging
systems, UK). A variety of objective measurements like Head DNA,
Tail DNA, and Olive Tail moment were made. Tail moment was
calculated according to reported procedure [8].
J ¼ 6.9 Hz), 6.65(H, d, J ¼ 8.43 Hz); 13C NMR (300 MHz, DMSO-d6) in
d
(ppm): 179.42 (C]S), 168.58 (C]O), 151.4, 149.0, 144.5, 142.2,
134.2, 128.8, 127.6; EI MS (70 eV) m/z (%): 397.53
6.4. Evaluation of antimicrobial activity
For the bacterial organisms, both Gram-positive and Gram-
negative bacteria were used. Gram-positive and Gram-negative
bacteria can be differentiated in the physical appearance of their
cell envelopes. The compounds were screened for their in vitro
antibacterial and anti-yeast activities. Antimicrobial activities were
determined by the broth micro-dilution procedures and principles
of the Clinical and Laboratory Standards Institute (CLSI) [37,38].
Minimal inhibitory concentrations for each compound were
investigated against standard bacterial strains; S. aureus (ATCC
25923), S. aureus (ATCC 29213), E. faecalis (ATCC 29212), E. coli
(ATCC 25922), S. epidermidis (ATCC 12228), E. cloacae (ATCC 13047),
P. vulgaris (ATCC 13315), P. aeruginosa (ATCC 27853) and yeast-like
fungi, C. albicans (ATCC90028), C. glabrata (ATCC 32554),
C. tropicalis (ATCC 20336), C. krusei (ATCC 6268). Bacterial and
fungal colonies of the test organisms were suspended directly into
a small volume of 0.9% saline and further diluted until turbidity
Tail length ꢂ %Tail DNA
Tail moment ¼
100
Supplementary crystallographic data
Crystallographic data for the structure reported in this article
has been deposited with Cambridge Crystallographic Data Center,
CCDC 738482. Copies of this information may be obtained free of

S. Saeed et al. / European Journal of Medicinal Chemistry 45 (2010) 1323–1331
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