Synthesis and anticancer activity of (E)-2-benzothiazole hydrazones

Article abstract of DOI:10.1016/j.ejmech.2014.08.039

Benzothiazole hydrazones have been synthesized and evaluated for their in vitro antiproliferative activity against three human cancer cell lines: HL-60 (leukemia), MDAMB-435 (breast) and HCT-8 (colon). The good cytotoxicity for the three cancer cell lines

Full text of DOI:10.1016/j.ejmech.2014.08.039

European Journal of Medicinal Chemistry 86 (2014) 12e16
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and anticancer activity of (E)-2-benzothiazole hydrazones
Eric B. Lindgren a 1, Monique A. de Brito b, Thatyana R.A. Vasconcelos a,
,
c
d
e
a, *
ꢀ
Manuel O. de Moraes , Raquel C. Montenegro , Julliane D. Yoneda , Katia Z. Leal
a
ꢀ
ꢀ
~
~
~
ꢀ
Programa de Pos-Graduaçao em Química, Instituto de Química, Universidade Federal Fluminense, Outeiro de Sao Joao Batista s/n , 24020-141 Niteroi, RJ,
Brazil
b
c
ꢀ
ꢀ
ꢀ
Faculdade de Farmacia, Universidade Federal Fluminense, Rua Mario Viana 523, 24241-000 Niteroi, RJ, Brazil
ꢀ
ꢀ
Laboratorio de Oncologia Experimental, Universidade Federal do Ceara, Rua Coronel Nunes de Melo 1127, 60430-270 Fortaleza, CE, Brazil
Laboratorio de Citogenetica Humana, Instituto de Ciencias Biologicas, Universidade Federal do Para, Rua Augusto Correa 01, 66075-110 Belem, PA, Brazil
Departamento de Química, Instituto de Ciencias Exatas, Universidade Federal Fluminense, Rua Des. Ellis Hermydio Figueira 783, 27213-415, Volta
d
e
ꢀ
ꢀ
^
ꢀ
ꢀ
^
ꢀ
^
Redonda, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Benzothiazole hydrazones have been synthesized and evaluated for their in vitro antiproliferative activity
against three human cancer cell lines: HL-60 (leukemia), MDAMB-435 (breast) and HCT-8 (colon). The
good cytotoxicity for the three cancer cell lines and theoretical profile of compounds 3o and 3p pointed
them as promising lead molecules for anticancer drug design.
Received 5 June 2014
Received in revised form
11 August 2014
Accepted 11 August 2014
Available online 11 August 2014
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
(E)-2-benzothiazoles
Hydrazones
Anticancer
Lipinski's rule of five
1. Introduction
review article highlighting aspects of the chemistry and biological
properties of this heterocyclic core during the past few years [9].
Cancer still remains a threat to humanity's health, figuring
among the leading causes of death worldwide. In 2012, cancer was
responsible for 8.2 million deaths and it is expected that annual
cases will rise from 14 million in 2012 to 22 within the next two
decades [1,2]. In the last years, many efforts have been made to
develop new strategies for finding safe and effective ways of
treating this disease, which includes a better understanding of
biological processes involved in cancer cell survival, and also the
search for more selective and potent chemotherapeutic agents [3].
In this context, benzothiazoles represent an important class of
heterocyclic compounds that have attracted special attention due
to their diverse biological activities, including antitumor [4], anti-
microbial [5], antidiabetic [6], anticonvulsant [7] and anti-
inflammatory [8]. Recently, our research group has published a
For some time, we have been interested in structures of
aryleNHeN]CHearyl, especially those having some pharmaco-
logically important heterocycles [10e15]. In a previous work, we
have reported the synthesis and in vitro anticancer activities of
eleven (E)-2-benzothiazole hydrazones (3aek) [13]. In addition, we
have performed structural determinations and conformational
analysis of some of the synthesized compounds [14e15]. In the
course of our investigation on the anticancer properties of this class
of compounds, we report herein the synthesis of six hydrazones
(3leq), and the anticancer activities against three cancer cell lines:
HL-60 (leukemia), MDAMB-435 (breast) and HCT-8 (colon), for all
benzothiazole hydrazones obtained from these studies (3aeq).
2. Results and discussion
2.1. Chemistry
* Corresponding author.
E-mail addresses: jullianeyoneda@id.uff.br (J.D. Yoneda), kzleal@gmail.com
The target compounds 3aeq were synthesized from reactions
between 2-hydrazinyl-1,3-benzothiazole 1 with an appropriate
arenealdehyde 2aeq in ethanol at room temperature (1e2 h) in
good yields (Scheme 1). Spectral data of all compounds (1H NMR,
(K.Z. Leal).
1
Present address: School of Chemistry, University of Nottingham, University
Park, Nottingham NG7 2RD, United Kingdom.
http://dx.doi.org/10.1016/j.ejmech.2014.08.039
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

E.B. Lindgren et al. / European Journal of Medicinal Chemistry 86 (2014) 12e16
13
Scheme 1. Synthetic route for preparing the (E)-2-benzothiazole hydrazones.
13C NMR, IR and ESI-MS) are in full agreement with the proposed
structures. As an example, the IR spectra for compound 3o shows
the C]N peak at 1624 cmꢁ1 and the NeH stretching vibrations at
3358 cmꢁ1. The 1H NMR spectrum of compound 3o exhibits four
singlets at 12.13, 9.66, 8.95 and 8.36 ppm for NH, OH, OH and N]
CeH, respectively. For the benzothiazole core, the hydrogens H5
and H6 are shown as doubleedouble doublets at 7.29 and 7.09 ppm
and H4 and H7 as doublets at 7.75 and 7.38 ppm. On the aromatic
region we assigned phenyl hydrogens at 7.04, 6.74 and 6.68 for H60,
H30 and H40, respectively. The 13C NMR spectra exhibits the N]CeS
signal at 166.4 ppm and C]N signal at 126.1 ppm.
involved in cytotoxicity against cancer cell is not be related to
membrane damage (Table 1). In addition, none of the most active
compounds 3a, 3c, 3leq exhibited cytotoxicity against the human
lung fibroblast normal cells (MRC5).
2.3. Molecular modeling and Lipinski's rule of five
Calculations on the isomeric forms of the active compound 3o
were performed by using the Gaussian 09W program [19]. The two
isomers were fully optimized at the B3LYP/6-311þþG(d,p) level of
theory. Since no imaginary frequency was found, all the optimized
geometries were characterized as minima energy structures. As
shown in Table 2, the E-isomer is more stable than the Z-isomer,
which concurs with the isomeric form obtained synthetically.
In order to assess the potential oral bioavailability of the studied
compounds, they were submitted to the Lipinski's rule of five
analysis (Table 1), which states that an orally active molecule
should respect a molecular weight (MW) ꢂ 500 g/mol, a clogP ꢂ 5,
a number of hydrogen bond acceptors (HBA) ꢂ 10 and a number of
hydrogen bond donors (HBD) ꢂ 5 [20]. The related criteria Polar
Surface Area (PSA) ꢂ 140 A2, lately added by Veber et al. [21] was
also included in the analysis. Compounds 3aec, 3eef, 3h, 3jel,
3neq (encompassing all most active compounds) fulfilled all the
parameters which suggest that they may have a good oral bio-
disponibility (Table 1). Compounds 3d, 3g, 3i, 3k and 3m presented
clogP higher than 5, which indicates high hydrophobicity. Conse-
quently, they may have problems for penetrating the membrane of
the cancer cell. It is worth noting that the control compound Dox
does not fulfill three parameters: MW, PSA and HBA, which can
explains its poor bioavailability after oral administration and also
why Dox chemotherapy is limited to intravenous administration or
Dox-liposomes [22,23].
Additionally, the druglikeness and the drugscore factor were
obtained for all compounds in the Osiris Property Explorer (Fig. 1)
[24]. The results show that only compounds 3a, 3f and 3i presented
negative druglikeness score. Even though compound 3a has shown
antineoplastic activity, the negative value is probably due to the
nitro substituent, which is not an ordinary group found in currently
traded drugs. Regarding compounds 3f and 3i, the carboxyl group is
responsible for the negative value in compound 3f, while in com-
pound 3i it is probably due to the methyl groups as substituents.
The control compound, which is also the marketed and most active
molecule, presented the highest druglikeness value.
2.2. Biological activity
The in vitro anticancer activities of the title compounds were
assessed against three human cancer cell lines in comparison to
doxorubicin (Dox), the positive control, by using MTT assay [16].
The concentrations that induce 50% inhibition of cell growth (IC50
)
in M are reported in Table 1. Results showed that compounds 3a,
m
3c, 3leq exhibited good cytotoxicity against HL-60 leukemia cell
line, especially the substance 3n that showed a IC50 value of
0.35
mM. The novel compound 3o exhibited activity against all the
three cell lines, with IC50 ranging from 0.59 to 11.18
mM. Based on
data collected from three independent experiments, compounds
3a, 3l, 3m and 3q were found to be selective to leukemia cell line. It
should also be noted the significant cytotoxic activity of compounds
3n and 3p against HCT-8 cell line with IC50 values of 1.29 and
3.43
0.48
m
M, respectively. Dox exhibited IC50 ranging from 0.01 to
mg/mL (Table 1). These results are in agreement with the Na-
tional Cancer Institute (NCI) protocols, where compounds exhibit-
ing IC50 values <10 M or 15 M are considered active [17].
m
m
It is noteworthy that the most active compounds, for at least two
cancer cell lines, 3n and 3p, bear two hydroxyl groups in its
structure. It was also found a significant activity improved against
the three cancer cell lines from compound 3a, which bears a hy-
droxyl group at position 20 and a nitro group at position 50, to
compound 3o, with two hydroxyl groups in the same positions.
This fact suggests the importance of the hydroxyl for the biological
activity for this series.
The mechanical stability of red blood cells is a good parameter
for in vitro screening of unspecific cytotoxicity, since the membrane
of erythrocyte can suffer significant changes in its structural
properties [18]. The absence of hemolytic activity (EC50 > 200
mg/
mL) observed for all compounds suggests that the mechanism

14
E.B. Lindgren et al. / European Journal of Medicinal Chemistry 86 (2014) 12e16
Table 1
Cytotoxic activity expressed by IC50 in
m
M of compounds for cancer cell lines,a hemolytic activity and theoretical oral biodisponibility (Lipinski's rule of 5) for compounds 3aeq.
PSA (A2)
Lipinski RO5
Mw (Da)
b
Compound
IC50am
M
EC50
(
m
g/mL)
HL-60
MDAMB-435
HCT-8
HBA
HBD
cLogP
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
Dox
1.65
>16.70
13.60
>17.37
>16.87
>16.81
>15.52
>17.28
>17.77
>15.95
>15.05
5.51
11.47
0.35
0.59
1.89
4.60
14.00
>16.70
>17.52
>17.37
>16.87
>16.81
>15.52
>17.28
>17.77
>15.95
>15.05
>16.81
>15.13
>17.52
6.27
13.52
>16.70
>17.52
>17.37
>16.87
>16.81
>15.52
>17.28
>17.77
>15.95
>15.05
>16.81
>15.13
1.29
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
81.13
52.29
67.00
27.88
28.91
58.92
27.71
27.83
27.77
40.59
27.89
44.25
34.19
64.43
66.34
66.28
47.59
156.88
314.33
299.35
285.33
287.77
296.40
297.34
322.22
289.31
281.38
313.38
332.23
297.34
330.42
285.33
285.33
285.33
269.33
543.53
7
5
5
3
4
4
3
3
3
5
3
5
4
5
5
5
4
1
1
2
0
0
1
0
0
0
0
0
0
0
2
2
2
1
5
4.31
4.15
3.89
5.23
4.95
4.23
5.78
4.98
5.64
4.42
5.50
4.45
5.43
3.89
3.89
3.89
4.28
ꢁ0.68
11.18
3.43
>18.56
0.02
14.44
>18.56
0.88
0.04
12
a
Data are presented as IC50 values and 95% of confidence interval for leukemia (HL-60), breast (MDA-MB-435) and colon (HCT-8). Doxorubicin (Dox) was used as positive
control. Experiments were performed in triplicate.
b
EC50 ¼ effective concentration.
The drugscore, which combines druglikeness score, clogP, logS,
molecular weight and toxicity risks in one value, may be used to
predict the overall potential as a drug candidate of a particular
compound [24]. Results showed that Dox exhibited the highest
drugscore value, followed by three active compounds (3c, 3o and
3p) which have in common hydroxyl groups as substituents. The
low drugscore value observed for compound 3a is also probably
due to the presence of the nitro group (Fig. 1).
washed successively with cold alcohol and ethyl ether to give the
pure product 3. The spectral data of 3aek, 3m and 3q had been
already reported in our previous papers [13e15].
4.1.1.1. (E)-2-(2-(benzo[d][1,3]dioxol-5-ylmethylene)hydrazinyl)
benzo[d]thiazole (3l). Yield: 73%, m.p. 219e220 ꢀC, IR (ѵ cmꢁ1, KBr):
3433 (NH), 1610 (C]N); 1H NMR (300.00 MHz, DMSO-d6,
d ppm):
12.15 (s, 1H, NH), 8.04 (s, 1H, N]CeH), 7.74 (d, 1H, J ¼ 7.8 Hz, H4 or
H7), 7.41 (d, 1H, J ¼ 7.3 Hz, H7 or H4), 7.29 (dd, 1H, J ¼ 7.3 and 1.2 Hz,
0
3. Conclusion
H5 or H6), 7.26 (d, 1H, J ¼ 1.6 Hz, H4 ), 7.15 (dd, 1H, J ¼ 8.2 and 1.6 Hz,
0
H2 ), 7.09 (td, 1H, J ¼ 7.8 and 1.2 Hz, H6 or H5), 6.98 (d, 1H, J ¼ 8.0 Hz,
H3 ), 6.09 (s, 2H, CH2); 13C NMR (75.0 MHz DMSO-d6,
d
ppm): 167.0,
0
The synthesis of (E)-2-benzothiazole hydrazones was described
and their in vitro anticancer activities were evaluated against leu-
kemia (HL-60), breast (MDA-MB-435) and colon (HCT-8). The
cytotoxicity against all the three cancer cells lines of the dihydroxyl
compounds 3o and 3p suggests that they might be possible anti-
neoplastic lead molecules for further investigation.
148.8, 148.0, 128.9, 126.0, 122.6, 121.5, 108.6, 104.9, 101.6; ESI-MS:
m/z [MꢁH]ꢁ: 296.3; Anal. Calcd. For C15H11N3O2S: C, 60.59; H,
3.73; N, 14.13%, Found: C, 60.35; H, 3.83; N, 13.96%.
4.1.1.2. (E)-3-((2-(benzo[d]thiazol-2-yl)hydrazono)methyl)benzene-
1,2-diol (3n). Yield: 78%, m.p. 260e261 ꢀC, IR (ѵ cmꢁ1, KBr): 3248
(NH), 3051 (OH), 1614 (C]N); 1H NMR (500.13 MHz, DMSO-d6,
4. Experimental
d
ppm): 12.12 (s, 1H, NH), 9.99 (s, 1H, OH), 9.30 (s, 1H, OH), 8.44 (s,
4.1. Chemistry
1H, N]CeH), 7.73 (d, 1H, J ¼ 7.7 Hz, H4 or H7), 7.35 (d, 1H, J ¼ 7.1 Hz,
H7 or H4), 7.31e7.27 (m, 1H, H5 or H6), 7.11e7.05 (m, 2H, H6 or H5
0
0
Melting points were determined on a Fisatom 430 instrument
and are uncorrected. IR spectra were recorded on a PerkineElmer
1420 spectrometer using KBr pellets. Negative mode ESI-MS was
done on a ZQ-4000 single quadrupole mass spectrometer. NMR
spectra were recorded on a Varian Unity Plus 300 spectrometer
operating at 300.00 MHz (1H) and 75.0 MHz (13C) and on a Bruker
Avance 500 operating at 500.13 MHz (1H) and 125.75 MHz (13C) in
and H6 ), 6.84 (dd, 1H, J ¼ 7.8 and 1.5 Hz, H4 ), 6.73 (dd, 1H, J ¼ 7.8
and 7.8 Hz, H5 ); 13C NMR (125.75 MHz DMSO-d6,
145.5, 145.3, 126.2, 121.8, 121.5, 119.8, 119.2, 116.7; ESI-MS: m/z
[MꢁH]ꢁ: 284.6; Anal. Calcd. For C14H11N3O2S: C, 58.93; H, 3.89; N,
14.73%, Found: C, 58.59; H, 4.06; N, 14.36%.
d ppm): 166.2,
0
4.1.1.3. (E)-2-((2-(benzo[d]thiazol-2-yl)hydrazono)methyl)benzene-
1,4-diol (3o). Yield: 73%, m.p.: 265e266 ꢀC, IR (ѵ cmꢁ1, KBr): 3358
(NH), 3034 (OH), 1624 (C]N); 1H NMR (300.00 MHz, DMSO-d6,
DMSO-d6. Chemical shifts (d) are reported in ppm relative to tet-
ramethylsilane. Elemental analysis was performed at CA IQ-USP,
~
Sao Paulo, Brazil on a Perkin Elmer e CHN 2400 analyzer.
d
ppm): 12.13 (s, 1H, NH), 9.66 (s, 1H, OH), 8.95 (s, 1H, OH), 8.36 (s,
1H, N]CeH), 7.75 (d, 1H, J ¼ 7.6 Hz, H4 or H7), 7.38 (d, 1H, J ¼ 7.4 Hz,
4.1.1. General procedure for the synthesis of 2-benzo[d]thiazole
hydrazones (3aeq)
H7 or H4), 7.29 (ddd, 1H, J ¼ 7.2; 6.4 and 1.2 Hz, H5 or H6), 7.09 (ddd,
0
1H, J ¼ 7.2; 6.1 and 1.2 Hz, H6 or H5), 7.04 (d, 1H, J ¼ 2.2 Hz, H6 ), 6.74
0
0
1,3-Benzothiazole hydrazones 3aeq were prepared from re-
actions between 2-hydrazinyl-1,3-benzothiazole 1 (1 mmol) and
appropriate arenealdehydes 2aeq (1 mmol) in ethanol (10 ml) at
room temperature. After completion of reaction, 1e2 h (checked by
TLC), the resulting precipitate was collected by filtration and
(d, 1H, J ¼ 8.7 Hz, H3 ), 6.68 (dd, 1H, J ¼ 8.7 and 2.7 Hz, H4 ); 13C NMR
(75.0 MHz DMSO-d6,
d ppm): 166.4, 149.9, 149.5, 126.1, 121.6, 121.4,
119.9, 118.4, 116.9; ESI-MS: m/z [MꢁH]ꢁ: 284.3. Anal. Calcd. For
C
14H11N3O2S: C, 58.93; H, 3.89; N, 14.73%, Found: C, 58.64; H, 3.96;
N, 14.39%.

E.B. Lindgren et al. / European Journal of Medicinal Chemistry 86 (2014) 12e16
15
Table 2
Gibbs free energy for E- and Z-isomers of compound 3o calculated at the B3LYP/6-311þþG(d,p) level of theory [19].
E-isomer
Z-isomer
DG (kcal/mol)
ꢁ9.7
Fig. 1. Druglikeness and drugscore values for compounds 3aeq compared to Dox.
4.1.1.4. (E)-4-((2-(benzo[d]thiazol-2-yl)hydrazono)methyl)benzene-
1,3-diol (3p). Yield: 77%, m.p.: 219e220 ꢀC, IR (ѵ cmꢁ1, KBr): 3336
(NH), 3067 (OH), 1622 (C]N); 1H NMR (300.00 MHz, DMSO-d6,
4.4. Calculations
The Gibbs free energy for the two isomeric forms of compound
3o were obtained by using the Gaussian 09W program [19] at the
B3LYP/6-311þþG(d,p) level of theory. The druglikeness and drug-
score values were obtained by using the Osiris Property Explorer
[24]. The Lipinski's rule of five parameters e molecular weight
(MW), clogP, number of hydrogen bond acceptors (HBA), number of
hydrogen bond donors (HBD), and the related criteria polar su-
perficial area (PSA) e were determined in the SPARTAN'10 program
[25].
d
ppm): 9.99 (s, 1H, OH), 8.33 (s, 1H, N]CeH), 7.70 (d, 1H, J ¼ 7.5 Hz,
H4 or H7), 7.38 (d, 1H, J ¼ 8.2 Hz, H7 or H4), 7.29e7.24 (m, 2H, H5 or
0
0
H6 and H3 ), 7.09e7.02 (m, 1H, H6 or H5), 6.37e6.32 (m, 2H, H5 and
H6 ); 13C NMR (75.0 MHz DMSO-d6,
d ppm): 160.3, 158.6, 126.1,
0
121.8, 121.2, 111.2, 107.8, 102.4; ESI-MS: m/z [MꢁH]ꢁ: 284.5. Anal.
Calcd. For C14H11N3O2S: C, 58.93; H, 3.89; N, 14.73%, Found: C,
58.56; H, 3.94; N, 14.38%.
4.2. Cytotoxicity against cancer cell lines
Acknowledgments
Compounds (0.312e20 mM) were tested for cytotoxic activity
~
against three cancer cell lines: HL-60 (leukemia), MDAMB-435
(breast) and HCT-8 (colon). All cell lines were maintained in
DMEM medium supplemented with 10% fetal bovine serum, 2 mM
J.D.Yand T.R.A.V. thank Fundaçao Carlos Chagas Filho de Amparo
ꢁ
a Pesquisa do Estado do Rio de Janeiro for financial support. Fel-
lowships granted to E.B.L. by Coordenaçao de Aperfeiçoamento de
Pessoal de Nível Superior and to R.C.M. and T.R.A.V. by Conselho
Nacional de Desenvolvimento Científico e Tecnologico.
~
glutamine, 100 U/mL penicillin, and 100
with 5% CO2. Each compound was dissolved with DMSO and diluted
with water to obtain a concentration of 20 M. They were incu-
m
M streptomycin at 37 ꢀC
ꢀ
m
bated with the cells for 72 h. The negative control received the same
amount of DMSO (0.005% in the highest concentration). Doxoru-
bicin was used as a positive control. The cell viability was deter-
mined by reduction of the yellow dye 3-(4,5-dimethyl-2-thiazol)-
2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue formazan
product after 48 h as described by Mosmann [16].
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.08.039.
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The test was performed in 96-well plates using a 2% mouse
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