Synthesis of 2-anilinopyridyl linked benzothiazole hydrazones as apoptosis inducing cytotoxic agents

Article abstract of DOI:10.1039/c8nj06517a

A series of 2-anilinopyridyl linked benzothiazole-hydrazone conjugates (5a-aa) were designed, synthesized and evaluated for their in vitro cytotoxic potency against a panel of cancer cell lines like mouse skin melanoma (B16F10), lung adenocarcinoma (A549), breast adenocarcinoma (MCF-7), triple negative breast cancer (MDA-MB-231) and normal lung epithelial (L132) cell lines. Preliminary screening results revealed that some of these conjugates like 5i and 5l exhibited a significant antiproliferative effect against human breast cancer (MCF-7) with IC₅₀ values of 1.03 and 1.69 μM respectively. Further, detailed biological studies of this promising conjugate (5i) were carried out on MCF-7 cells. The flow cytometric analysis revealed that this conjugate induces cell-cycle arrest in the G2/M phase in a dose dependent manner. Furthermore, in order to determine the effect of the conjugate on cell viability, various cell based assays such as clonogenic assay, ethidium bromide staining, Hoechst staining, detection of ROS generation and annexin V-FITC/PI assays were performed. In these studies, apoptotic features were clearly observed indicating that this conjugate inhibited cell proliferation by apoptosis.

Full text of DOI:10.1039/c8nj06517a

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Saifi, S. Riyaz, G. S. Mani, S. P. Shaik, E. G. osas, C. Godugu, S. Shahjahan and A. Kamal, New J. Chem.,
2019, DOI: 10.1039/C8NJ06517A.
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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
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DOI: 10.1039/C8NJ06517A
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Synthesis of 2-anilinopyridyl linked benzothiazole hydrazones as
apoptosis inducing cytotoxic agents
Received 00th January 20xx,
Accepted 00th January 20xx
Faria Sultana,a,d Mohd Aslam Saifi,c Riyaz Syed,a Geeta Sai Mani,b Siddiq Pasha Shaik,a Egharevba
God’shelp Osas,a Chandraiah Godugu,c Syeda Shahjahand and Ahmed Kamal a, b, e*
DOI: 10.1039/x0xx00000x
Abstract: A series of 2-anilinopyridyl linked benzothiazole-hydrazone conjugates (5a-aa) were designed synthesized and
evaluated for their in vitro cytotoxic potency against a panel of cancer cell lines like mouse skin melanoma (B16F10), lung
adenocarcinoma (A549), breast adenocarcinoma (MCF-7), triple negative breast cancer (MDA-MB-231) and normal lung
epithelial (L132). Preliminary screening results revealed that some of these conjugates like 5i and 5l exhibited significant
antiproliferative effect against human breast cancer (MCF-7) with IC50 values of 1.03 and 1.69 µM respectively. Further, the
detailed biological studies of this promising conjugate (5i) were carried out on the MCF-7 cells. The flow cytometric
analysis revealed that this conjugate induce cell-cycle arrest in the G2/M phase in a dose dependent manner.
Furthermore, in order to determine the effect of the conjugate on cell viability various cell based assays such as clonogenic
assay, ethidium bromide staining, Hoechst staining, detection of ROS generation and annexin V–FITC/PI assays were
performed. In these studies, apoptotic features were clearly observed indicating that this conjugate inhibited cell
proliferation by apoptosis.
www.rsc.org/
fragmentation5,6 which finally leads to cell death. Accordingly,
proteins that regulate programmed cell death could represent
potential cancer drug targets and apoptosis inducers represent
potential anticancer agents, therefore their investigation
provides an attractive approach for the discovery and
development of anticancer agents.
Introduction
Considering the increasing incidence of cancers associated
with high mortality and tumour heterogeneity, its therapy is
always a real challenge. Moreover poor prognosis, non-
selectivity, acute toxicity and cellular drug resistance justifies
the growing interest for designing, developing and the
identification of newer chemotherapeutic agents for cancer
treatment.1 It is currently considered that deregulation of
apoptosis can disrupt the delicate balance between cell
proliferation and cell death and can lead to the development
of cancer.2,3 Literature reports are also suggestive of a link
between apoptosis-inducing ability and antitumor efficacy of
chemotherapeutic agents,4 therefore further establishing the
clinical significance of apoptosis towards overall tumour
sensitivity. Apoptosis generally occurs through two pathways,
the mitochondrial-intrinsic pathway and the death receptor
pathway both resulting in characteristic morphological cellular
changes such as cell shrinkage, blebbing of the plasma
membrane prior to cell lysis, chromatin condensation and DNA
Benzothiazole is a privileged bicyclic potent pharmacophore,
exhibiting wide spectrum of biological activities like
anticancer,7 antibacterial,8 antifungal,9 anti-inflammatory,10
anticonvulsant,11
anti-viral,12
antitubercular13
and
antidiabetic.14 Thus, this scaffold is highly important in the
medicinal chemistry and can be used as a template with new
chemical group insertions and modifications, thus providing
bioactive structural frameworks that could facilitate hit
exploration in the early stage of drug discovery. E7010 (2) is a
synthetic antimitotic sulphonamide based drug having
acceptable toxicity profile, oral bioavailability, and is
insensitive to multidrug resistance (MDR). Thus, proving to be
a promising scaffold that could be further explored for the
development of potential E7010 conjugates. Moreover, the
azomethine (-NHN=CH-) group is also bioactive and is observed
in many marketed anticancer drugs, e.g.: triapine (1) and some
representative benzothiazole, 2-anilinopyridyl and hydrazone
derivatives are illustrated in Figure. 1
aMedicinal Chemistry and Biotechnology Division, CSIR-Indian Institute of
Chemical Technology (IICT), Hyderabad-500007, India
bDepartment of Medicinal Chemistry, cDepartment of Regulatory Toxicology,
National Institute of Pharmaceutical Education and Research, Hyderabad-500037,
India
In continuation to our research for the development of
potential chemotherapeutic leads and considering the
promising antiproliferative profile of benzothiazoles,
hydrazones as well as E7010, and moreover based on our
previous reported potential leads.15,16 We herein, have
designed some 2-anilino pyridyl linked benzothiazole
dDepartment of chemistry, University College for Women,Hyderabad-500095, India
eSchool of Pharmaceutical Education and Research (SPER), Jamia Hamdard
University, New Delhi, 110062, India
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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hydrazone conjugates by replacing the sulphonamide group of report the synthesis, cytotoxicity and struVciteuwrAert-iaclcetOivnliitnye
the E7010 (2) with benzothiazole group of 2-anilinonicotinyl relationship (SAR) studies of 3-(D(2O-I(:b1e0.n10zo39[d/C]t8hNiJa0z6o5l1-72A-
linked 2-amino benzothiazoles (4), the aminopyridylhydrazine yl)hydrazono)methyl)-N-phenylpyridin-2-amine (5a-aa). In
linkage as observed in marketed drug triapine (1) is also addition, detailed biological investigations (for conjugate 5i)
incorporated in the conjugates (design strategy in Figure 2). such as clonogenic assay, Hoescht staining, acridine
Moreover this is in anticipation of hybridizing these bioactive orange/ethidium bromide staining, measurement of reactive
pharmacophores with complementary functions to result in a oxygen species (ROS), apoptosis detection assays and cell cycle
hybrid with enhanced therapeutic effects17 in comparison to analysis studies have been carried out and discussed herein.
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the parent structural components.
O
NH
S
O
Results and Discussion
Chemistry
O
NH2
N
H
N
NH
N
NH2
N
)
S
2
(
)
1
(
The E7010 linked benzothiazole hydrazone conjugates (5a-aa)
were synthesized as depicted in Scheme 1. The key
intermediates, substituted 2-(phenylamino)nicotinaldehydes
(10a–e) and 2-hydrazinylbenzo[d]thiazole (12a-e) were
synthesised in sequential steps as discussed below.
OH
X
X
H
N
O
S
O
S
H
N
S
O
N
N
N
N
Y
N
H
O
H
N
NH
R4
N
NH
N
NH
R1
R2
R2
O
R1
O
O
R3
OH
NH
R4
R2
5a aa
)
a
c
R3
b
OEt
OH
OEt
R1
N
(
-
3
(
)
4
(
)
NH
R2
N
Cl
N
Cl
8a e
-
Figure 1. Chemical structures of some anticancer molecules: Triapine (1), E7010 (2), 2-
anilino substituted nicotinyl arylsulfonylhydrazides (3),15 2-anilinonicotinyl linked 2-
aminobenzothiazoles (4),16 and synthesized 2-anilino pyridyl linked benzothiazole
hydrazone conjugates (5a-aa) .
6
7
R1
R3
R2
R1
R3
9a e
-
d
10a;R1=H;R2=Cl;R3=H
10b;R1=H;R2=CF3;R3=H
10c;R1=H;R2=F;R3=H
10d;R1=H;R2=H;R3=H
O
X
H
O
S
N
NH
O
S
N
N
NH
NH
O
10e;R1=OMe;R2=OMe;R3=OMe
H
NH2
N
O
H
N
NH
N
NH2
N
R1
R3
R1
R2
N
)
e
N
R2
10a e
N
S
12a;R4=OMe
NH2 12b;R4=OEt
12c;R4=Me
H
S
NH2
-
N
R4
1
(
S
11a e
-
R4
R4
2
(
)
R3
(
12a e
-
OH
4
)
12d;R4=H
12e;R4=F
O
H
N
S
N
N
H
H
N
f
N
Conjugation
H
R4
+
NH2
N
NH
N
NH
S
12a e
R4
-
S
N
R1
R3
R1
R3
N
R2
5a-5v
R2
10a e
-
N
N
NH
R4
e
S
S
N
R1
R3
NH2
NH
NH2
R2
5a 5aa
)
N
(
-
12F
11F
Synthesised conjugates
O
H
N
N
N
H
S
S
NH
NH2
+
g
N
NH
N
NH
N
12F
R1
R3
Apoptosis
R1
R3
R2
R2
10a e
-
5w 5aa
-
Figure 2. Design strategy of 2-anilino pyridyl linked benzothiazole hydrazone conjugates
(5a-aa): Triapine (1), E7010 (2), 2-anilinonicotinyl linked 2-amino benzothiazoles (4) .
Scheme 1. Synthesis of 2-anilinopyridyl linked benzothiazole hydrazone
conjugates; reagents and conditions: (a) Cat. H2SO4, EtOH, reflux, 2 h, 79%; (b)
substituted anilines, ethylene glycol, 120 °C, 6 h, 73–81%; (c) LAH, THF, 0 °C, 3 h,
71–78%; (d) IBX, DMSO, rt, 30 min, 73–82%; (e) Hydrazine monohydrate,
ethylene glycol, conc. HCl, reflux at 140 ° C, 3-4 h, 70-80%;(f) AcOH, EtOH, 3-4 h,
reflux, 70–85% (g) AcOH, EtOH, 3-4 h, rt, 70–85% .
A library of compounds corresponding to this template with
several substituents on both the pharmacophores has been
synthesized to establish the SAR and to explore the possibility
of developing new potent antitumor agents. Thus, we herein
2-(Phenylamino)nicotinaldehyde
chloronicotinic acid (6), which was converted to ethyl-2-
synthesis
involves
2-
2 | J. Name., 2012, 00, 1-3
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chloronicotinate (7), by refluxing in ethanol for 2 hours in
presence of catalytic amount of conc. H2SO4. The ester
obtained was heated with substituted anilines in ethylene
glycol at 140 °C for 6 h to provide the coupled product, ethyl 2-
(phenylamino)nicotinate (8a–e). These esters were reduced by
LAH in dry THF to give the corresponding alcohols (9a-e),
ARTICLE
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Biological activity
Antiproliferative activity
View Article Online
DOI: 10.1039/C8NJ06517A
All these conjugates 5a-aa were evaluated for their cytotoxic
activity against different cell lines including human breast
adenocarcinoma (MCF-7), triple negative breast cancer (MDA-
MB-231), lung adenocarcinoma (A549), mouse skin melanoma
(B16F10) and normal lung epithelial (L132) cell lines.
which
were
selectively
oxidized
to
2-
9
(phenylamino)nicotinaldehydes (10a–e) using 2-iodoxy benzoic
acid in DMSO. On the other hand, 2-hydrazinyl benzothiazoles
(12a-e) were synthesized by the reaction of hydrazine hydrate
with substituted 2-amino benzothiazoles (11a-e) in ethylene
glycol with catalytic amount of con. HCl.18
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Table 2. IC50 valuesa ± SD (in µM) for compounds in selected cancer cell lines.
Conjug
ates
5a
B16F10b
1.25±0.2
2.19±0.1
1.05±0.1
A549c
>20
MCF-7d
1.32±0.4
2.50±0.8
3.01±0.5
MDA-
MB-231e
15.68±1.
6
12.80±0.
2
19.18±1.
8
>20
L132f
Table 1. Generic structure of conjugates 5a-aa.
12.29±4.
5
13.82±4.
3
18.59±2.
7
H
N
H
N
S
N
N
5b
5c
6.56±0.21
2.38±0.31
N
N
S
R4
N
NH
N
NH
R1
R3
R1
R3
5d
5e
5f
1.19±0.3
4.00±0.9
2.04±0.6
2.19±0.46
>20
1.20±0.4
>20
>20
R2
R2
>20
>20
>20
5a-v
5w aa
-
>20
>20
17.58±5.
Compound
5a
R1
R2
R3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
R4
OMe
OMe
OMe
OMe
OEt
OEt
OEt
OEt
Me
Me
Me
Me
H
H
H
H
H
F
F
F
F
4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
OMe
H
H
Cl
CF3
F
H
Cl
CF3
F
H
Cl
CF3
F
H
Cl
CF3
F
H
OMe
Cl
CF3
F
H
OMe
Cl
5g
5h
5i
1.10±0.1
1.65±0.2
0.74±0.1
12.02±1.5
4.72±0.81
1.23±0.37
1.68±0.7
2.29±0.1
1.03±0.8
19.38±2.
7
>20
13.73±2.
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
9
15.44±5.
8
12.69±3.
5
6.22±0.1
5j
5k
5l
1.07±0.3
0.96±0.1
2.65±0.39
1.10±0.16
1.18±0.25
2.28±0.1
>20
18.61±15 12.37±14
15.03±01
4.54±0.2
13.0±4.3
0.93±0.0
3
1.69±0.4
19.64±0.
7
11.76±2.
7
>20
5m
5n
5o
5p
5q
0.96±0.1
4.97±0.84
16.72±1.89
>20
1.64±0.1
6.61±1.3
7
1
8.43±1.5
8
9.98±0.4
2
0.93±0.2
0.92±0.4
>20
0.96±0.0
4
2.38±0.4
14.56±3.
6
>20
5
1.73±0.1
8
2.49±0.3
4
OMe
>20
>20
H
H
H
H
OMe
H
>20
>20
>20
>20
5r
5s
1.13±0.5
0.92±0.3
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
F
-
-
-
1.17±0.2
>20
CF3
F
H
H
5t
>20
>20
5y
H
5u
>20
>20
>20
>20
5z
H
H
H
-
5v
3.01±0.8
>20
>20
>20
>20
5aa
OMe
OMe
OMe
-
5w
5x
>20
>20
>20
Finally, the desired 2-anilinopyridyl linked benzothiazole
hydrazone conjugates (5a-aa) were obtained by condensation
1.01±0.5
>20
>20
>20
>20
of
2-hydrazinyl
benzothiazoles
with
2-
5y
>20
>20
>20
(phenylamino)nicotinaldehydes in the presence of catalytic
amount of glacial acetic acid in ethanol and the product
obtained was purified by recrystallisation in ethanol and all the
conjugates were obtained in high yields.
Notably, they were synthesized with diverse substituents of
both electron donating as well as withdrawing nature on each
of the pharmacophores (Table 1). They were characterized by
spectral analysis such as 1H NMR, 13C NMR, mass and HRMS.
5z
>20
>20
>20
>20
5aa
E 7010
>20
>20
>20
>20
1.5±0.2
1.2±0.5
4.01±0.2
a50% Inhibitory concentration after 48 h of drug treatment and the values are
average of three individual experiments, bmouse skin melanoma, clung
adenocarcinoma, dbreast adenocarcinoma, etriple negative breast cancer, fnormal
lung epithelial cells).
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Inhibitory concentration (IC50), a concentration required to (ROS) generation, ethidium bromide staining, HoeVcihewstArstitcaleinOinnlinge,
inhibit proliferation of 50% cells after exposure to test change in mitochondrial membrane potential and apoptosis
compounds was determined using MTT assay. The IC50 values detection assay.
of different conjugates on the above cell lines are shown in Clonogenic assay
DOI: 10.1039/C8NJ06517A
Table 2 along with IC50 values of reference standard, E7010 (2).
The preliminary results indicated that some of these
conjugates were active and they (5d, 5i, 5n and 5s) showed
superior cytotoxic activity than E7010 (2), with IC50 values
Clonogenic assay was performed to assess the inhibitory effect
of conjugate on colony forming capability of MCF-7 cells. These
cells were allowed to grow in 6-well plate and treated with
conjugate 5i. The results clearly showed that there was a dose
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ranging from 0.9 to 1.3 μM. Wherein the conjugate 5i showed
dependent growth inhibition effect of conjugate 5i on MCF-7
maximum cell growth inhibition activity on MCF-7 cell line (IC50
cells. Moreover, the conjugate completely inhibited colony
1.03 ± 0.08 μM). In addition, conjugate 5l also showed
forming capability of single cancer cells at 5 and 10 μM as
significant activity on MCF-7 cells with an IC50 value of 1.69 ±
there were no visible colonies in the wells as shown in Figure
4.
0.24 μM. Based on the promising results obtained with
preliminary screening, conjugate 5i was selected for detailed
biological studies.
i)
a)
b)
c)
Several crucial structural requirements which contributed
towards the potency were revealed by the SAR study. By
exemplifying different substitution pattern on A and D ring,
conjugates possessing both electron withdrawing as well as
electron donating substituents were synthesized and their
cytotoxicity was evaluated. Substituent like OEt (5e), F (5t, 5u)
at the 6th position of ring D proved to be slightly less beneficial
towards the cytotoxicity, whereas presence of substituents like
H (5m, 5n), CH3 (5i, 5j, 5l) and OMe groups (5a, 5d) seems to
enhance the cytotoxicity. The observed order of improved
potency with the substituents on ring D may be represented as
CH3>H>OMe>OEt>F. It is noteworthy to observe that
substituents like Cl (5i, 5m) and H (5d, 5h, and 5n) at 4th
position of the ring A proved beneficial towards activity and
substituents like CF3 (5b, 5j and 5n) and F (5c, 5g, 5o) were
found to be well tolerated. However, the presence of
trimethoxy group (5q, and 5v) at ring A did not prove
beneficial and absence of ring D (5w-aa) leads to complete loss
of activity, thereby demonstrating the importance of benzene
ring or ring D in terms of cytotoxicity. Intriguingly, conjugates
5i and 5l, the most active compounds in the series exhibited
superior potency than the standard E7010 (2). The observed
order of improved potency with the substituents on ring A may
be represented as Cl>H>CF3>F>3,4,5-triOMe. Therefore, it can
be concluded that the presence of ring D and electron
donating substituent on ring D and electron withdrawing
substituent on ring A are beneficial for the cytotoxic activity
and are pictorially represented in Figure 3.
e)
d)
60
40
20
0
ii)
***
***
***

***

l
M
M
M
M
o
r


t
n
5
5
5
0
.
o
2
1
.
2
C
1
5j
5i
Figure 4. Effect of conjugate 5i on colony forming capabilities of MCF-7 cells. i) a)
Control cells (MCF-7), b) 5i (1.25 µM), c) 5i (2.5 µM), d) 5i (5 µM), e) 5i (10 µM) ii)
Number of colonies after treatment of the cells with increasing concentration of the
compound 5i.
Ring D is essential for cytotoxicity
H
N
S
C
N
B
N
N
R4
D
NH
Cytotoxicity order
CH3>H>OMe>OEt>F
A
Acridine orange/ethidium bromide (AO/EB) staining
R1
R3
Cytotoxicity order
Cl>H>CF3>F>3,4,5- triOMe
R2
To further confirm the cytotoxic activity of these conjugates,
acridine orange/ethidium bromide (AO/EB) dual staining was
performed to visualize nuclear changes and apoptotic body
formation, to differentiate between live and dead cells. The
exposure of cells with conjugate 5i resulted in alteration of
cellular morphology. Florescent microscopic images revealed
altered morphological characteristics in form of apoptotic
body formation, cell shrinkage, cell fragments and membrane
Figure 3. SAR analysis of 2-anilinopyridyl linked benzothiazole hydrazone conjugates.
Based on the promising results obtained with the preliminary
screening, two cytotoxic conjugates 5i and 5l show significant
activity on MCF-7 cells with an IC50 value of 1.03 and 1.69 μM.
Conjugate 5i was further subjected to investigations such as
clonogenic assay, cell cycle analysis, reactive oxygen species
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blebbing suggesting induction of apoptosis by treatment with Elevation in intracellular ROS is known to beVieiwnvAortilcvleeOdnlinine
DOI: 10.1039/C8NJ06517A
conjugate 5i in MCF-7 cells as shown in Figure 5.
induction of apoptosis in cancer cells. Intracellular levels of
ROS were estimated by using DCFH-DA staining method. MCF-
7 cells were treated with conjugate 5i at 0.625, 1.25, 2.5, 5,
and 10 μM for 6 hours.
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Figure 7. Determination of ROS generation in MCF-7. Fluorescence images are obtained
from the cells treated with the conjugate 5i for 24 h and then observed the production
of ROS by DCFH-DA. a) Control cells (MCF-7), b) 5i (0.625 µM), c) 5i (1.25 µM), d) 5i (2.5
µM), e) 5i (5 µM) and f) 5i (10 µM). There was a concentration dependent increase in
the bright green florescence as compared to untreated cells which indicated that the
Figure 5. Acridine orange ethidium bromide (AO/EB) dual staining in MCF-7 cell line
showing apoptosis. a) Untreated cells (MCF-7), b) 5i (1.25 µM), c) 5i (2.5 µM), d) 5i (5
µM). The treated cells showed induction in apoptosis as revealed by crescent shaped
cells stained with yellow green acridine orange (Early apoptotic) and orange colored
cells stained with ethidium bromide (Late apoptotic cells) as represented by arrows.
conjugate 5i increased intracellular ROS in MCF-7 in
a concentration dependent
manner. The increased ROS in the MCF-7 cells could be one of the potential
mechanisms behind cytotoxic potential of the conjugates.
Hoechst staining
The results showed increased intensity in green florescence in
a dose dependent manner when compared to control
indicating induction of increase in intracellular ROS production
by conjugate 5i (Figure 7) which could ultimately lead to
apoptosis.
Nuclear damage is a critical prerequisite for cellular death. To
examine nuclear damaging potential of these conjugates,
nuclear morphology was studied using Hoechst 33342 staining
method. Treated and untreated cells were washed with PBS
twice followed by staining with Hoechst dye to stain the nuclei.
The stained nuclei were observed under fluorescent
microscope using DAPI filter at 400X magnification. The
untreated control group showed no alteration in nuclear
morphology while exposure of cells with conjugate 5i
produced clearly visible nuclear fragments as shown in Figure
6 (marked by arrows). The results clearly demonstrated that
cells treated with conjugate 5i showed condensed, fragmented
and sickle shaped nuclei which are characteristic features of
cells undergoing apoptosis.
Measurement of mitochondrial membrane potential (ΔѰm)
Furthermore, we evaluated ROS-induced mitochondrial
membrane potential (MMP) alteration in MCF-7 cells, as
alteration in MMP (ΔѰm) is implicated as one of the major
pathways for cellular death. To further confirm changes in
MMP (ΔѰm) due to increased ROS we used JC-1 staining
method. JC-1 is a fluorescent dye which gives a red colour as
monomer while in the presence of altered MMP it gets
aggregated and imparts green coloration. Treatment of cells
for 24 hours showed increased green signal as shown in the
Figure 8 clearly indicating the alteration of MMP (ΔѰm) by
different concentrations of conjugate 5i. Moreover at 10 μM
concentration almost all cells stained with green colour
confirming alteration in MMP.
Figure 6. Hoechst staining in MCF-7 cell line. a) Control cells (MCF-7), b) 5i (0.625 µM),
c) 5i (1.25 µM), d) 5i (2.5 µM), e) 5i (5 µM) and f) 5i (10 µM). The cells treated with
compound 5i showed nuclear condensation and sickle shaped nuclei as represented by
arrows suggesting the nuclear damaging potential of the compound.
Detection of intracellular reactive oxygen species (ROS) by DCFH-
DA
Figure 8. MCF-7 cells treated with test compound 5i triggers mitochondrial injury. Drop
in membrane potential (ΔΨm) was assessed by JC-1 staining and the images were
captured by a fluorescent microscope. a) Control cells (MCF-7), b) 5i (0.625 µM), c) 5i
(1.25 µM), d) 5i (2.5 µM), e) 5i (5 µM) and f) 5i (10 µM). The treated cells showed a
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drop in mitochondrial membrane potential (ΔΨm) which was evident by increase in
green florescence after treatment of the cells with compound 5i. Further, the drop in
ΔΨm was found to be in a concentration dependent fashion.
fraction of cells present in G2/M phase from 15.94% to 22.78%
View Article Online
at 3 µM concentration respectively. TheDreOsI:u1l0ts.10o3b9t/Cai8nNeJd06w51i7thA
cell cycle analysis revealed that the growth inhibition induced
by these compounds was mainly due to G2/M phase arrest in
MCF-7 cells (Figure 10, Table 2).
Detection of apoptosis by annexin V-FITC/propidium iodide
staining
The arrest of cell growth in a specific phase of cell cycle may
lead to apoptosis of cells.24 To evaluate the extent of apoptosis
carried out by conjugate 5i, we used florescent conjugate of
annexin V with FITC along with PI. The results obtained with
annexin V assay showed 91.37% cells were alive in untreated
control group. On the other hand, population of early
apoptotic cells increased from 2.80% to 8.39 and 10.3% by
treatment with conjugate 5i with 1 and 3 μM, respectively.
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Figure 10. Cell cycle analysis of MCF-7 cells treated with conjugate 5i at indicated
concentrations for 24 h. a) Control cells (MCF-7), b) 5i (1µM), c) 5i (3 µM). The
untreated cells showed normal cell cycle behaviour while on the other hand, the
treated cells showed a G2/M specific cell growth arrest. The increase in the population
in G2/M phased indicated that the conjugate 5i halted the cell growth and arrested the
cell cycle in G2/M phase.
Table 2. Flow cytometric analysis in MCF-7 breast cancer cell line after treatment with
conjugate 5i.
Conjugate
Sub G1
G0/G1
S
G2/M
Control
2.76
0.66
3.42
77.04
76.61
64.56
4.43
5.00
7.02
14.03
15.94
22.78
5i( 1µm )
5i( 3µm )
Figure 9. Newly synthesized conjugates induce apoptosis in MCF-7 cells. Cells were
treated with 5i and E7010 (as positive control) for 24 h. Cells were labelled with
annexin V FITC, PI and analysed by flow cytometry. a) Control cells, b) 5i (1 µM), c) 5i (3
µM) and d) E7010 (3 µM). The compound 5i increased almost 3-fold and 3.5-fold
increase in the early apoptotic cells at a concentration of 1 and 3 μM, respectively. In
addition, 5i also caused almost 5-fold increase in the late apoptotic/necrotic cells at 3
μM as compared to untreated cells.
Conclusion
In summary, a library of 2-anilinopyridyl linked benzothiazole
hydrazone conjugates (5a-aa) were synthesised and evaluated
for their anticancer potential. The majority of these
compounds demonstrated significant antiproliferative
activities against tested cancer cell lines. SAR study revealed
the crucial structural requirements of the conjugates like
presence of ring D and electron donating substituents on ring
D and electron withdrawing substituents on ring A, which
contributed towards the potency. Amongst the synthesised
conjugates, 5i and 5l that contain CH3 group on ring D and Cl
and H group on ring A were found to be the most active with
an IC50 value of 1.03 and 1.69 µM against MCF-7 cells.
Moreover, cell cycle analysis indicates that conjugate 5i arrest
MCF-7 cells in S and G2/M phase. Subsequent cell viability
studies such as clonogenic assay, acridine orange/ethidium
bromide staining, Hoescht staining, generation of ROS,
changes in mitochondrial membrane potential, annexin V–
FITC/PI assay confirms that this conjugate induce apoptosis.
Based on these results, conjugate 5i could be considered as
potential lead in the development of chemical libraries that
would serve as a source of potential drugs for treating breast
cancer.
In addition, late apoptotic/necrotic cells increased from 5.74%
to 15.80 and 28.64% by exposure of cells to conjugate 5i at 1
and 3 μM, respectively. Moreover E7010 caused increase in
early apoptotic cells from 2.80 to 11.0% at concentrations of 3
μM. Therefore the percentage increase in number of early and
late apoptotic cells confirmed that conjugates induced
apoptosis in MCF-7 cells (Figure 9).
Cell cycle analysis
To evaluate the role of cell cycle arrest in imparting cytotoxic
potential to compounds, MCF-7 cells were treated with
different concentration of conjugate 5i followed by
observation of cell cycle distribution by flow cytometer. The
results clearly showed G2/M phase arrest when compared to
control as shown in Figure 10. Following treatment for 24
hours with conjugate 5i at 3 μM concentration reduced the
fraction of cells in G1 phase from 76.61% to 64.56%. However,
at the same time, an increased fraction of cells present in S
and G2/M phase was observed. Conjugate 5i increased the
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Compound 5b was prepared according to the above explained
Experimental section
Chemistry
View Article Online
DOI: 10.1039/C8NJ06517A
general
method
by
employing
2-((4-
(trifluoromethyl)phenyl)amino)nicotinaldehyde (10b, 100 mg,
0.37 mmol) and 2-hydrazinyl-6-methoxybenzo[d]thiazole (12a,
73 mg, 0.37 mmol) to afford 5b yellow solid, yield: 85%, Mp:
Materials and methods
All chemicals and reagents were obtained from Aldrich (Sigma-
Aldrich), St. Louis, MO, USA, Lancaster (Alfa Aesar, Johnson
Matthey Company, Ward Hill, MA, USA), or Spectrochem Pvt.
Ltd (Mumbai, India) and were used without further
purification. Reactions were monitored by TLC performed on a
silica gel glass plate containing 60 GF-254, and visualization
was done by iodine indicators or UV light. Column
chromatography was performed with Merck 60–120 mesh
o
324-327 C,1H NMR (300 MHz, TFA-d + CDCl3) δ 8.85 (m, 1H),
8.43 (m, 1H), 8.06 (m, 1H), 7.93 (m, 1H), 7.82 (m, 3H), 7.69 (m,
1H), 7.32 (m, 3H) 3.94 (s, 3H); 13C NMR (75 MHz, TFA-d + CDCl3)
δ 158.87, 157.91, 149.90, 149.45, 149.16, 138.44, 132.61,
130.06, 127.83, 124.11, 123.72, 117.95, 117.08, 115.30,
107.19, 56.48. MS (ESI): m/z 444 [M+H]+; HRMS calcd for
C21H16N5OF3S [M+H]+ 444.1100, found 444.1080.
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1
silica gel. H and 13C NMR spectra were recorded in TFA-D +
N-(4-Fluorophenyl)-3-((2-(6-methoxybenzo[d]thiazol-2-yl)
hydrazono)methyl)pyridin-2-amine (5c):
Compound 5c was prepared according to the above explained
CDCl3 by using Avance and Varian instruments. Chemical shifts
are expressed in parts per million (δ in ppm) downfield from
internal TMS and coupling constants are expressed in Hz. H
1
general
method
by
employing
2-((4-
NMR spectroscopic data are reported in the following order:
multiplicity (s, singlet; brs, broad singlet; d, doublet; dd,
doublet of doublets; t, triplet; m, multiplet), coupling
constants in Hz, number of protons. ESI mass spectra were
recorded on Micro mass Quattro LC using ESI+ software with
capillary voltage 3.98 kV and an ESI mode positive ion trap
detector. Melting points were determined with Electrothermal
melting point apparatus, and are uncorrected.
2-(Arylamino)nicotinaldehydes (10a–e) intermediates were
prepared by using the reported protocol.17 Likewise, 2-
hydrazinylbenzo[d]thiazoles (12a-e) were prepared by using
the reported protocol.18
fluorophenyl)amino)nicotinaldehyde (10c, 100 mg, 0.46 mmol)
and 2-hydrazinyl-6-methoxybenzo[d]thiazole (12a, 90 mg, 0.46
o
mmol) to afford 5c yellow solid, yield: 85%, Mp: 316-319 C,
1H NMR (300 MHz, TFA-d + CDCl3) δ 8.77 (s, 1H), 8.33 (d, J = 7.3
Hz, 1H), 7.98 (d, J = 5.4 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.51–
7.39 (m, 2H), 7.35–7.25 (m, 3H), 7.20 (d, J = 10.7 Hz, 2H), 3.89
(s, 3H); 13C NMR (75 MHz, TFA-d + CDCl3) δ 167.11, 158.67,
149.54, 148.52, 138.36, 131.47, 128.97, 128.85, 123.95,
118.80, 118.49, 117.60, 117.02, 116.45, 115.98, 114.55,
112.67, 106.79, 56.34. MS (ESI): m/z 394 [M+H]+; HRMS calcd
for C20H16N5OFS [M+H]+ 394.11324, found 394.11167
3-((2-(6-Methoxybenzo[d]thiazol-2-yl)hydrazono)methyl)-N-
phenylpyridin-2-amine (5d):
General procedure for the synthesis of 3-((2-(benzo[d]thiazol-
2-yl)hydrazono)methyl)-N-phenylpyridin-2-amines (5a-aa):
2-(arylamino)nicotinaldehyde
hydrazinylbenzo[d]thiazole (12a-e) were dissolved in EtOH (10
mL) and glacial acetic acid (2–3 drops) was added. The reaction
mixture was held at reflux for 3–4 h (reaction monitored by
TLC) and the precipitate formed on cooling was collected by
filtration and recrystallised in EtOH to obtain the pure product
(5a-aa).
Compound 5d was prepared according to the above explained
general method by employing 2-(phenylamino)nicotinaldehyde
(10d, 100 mg, 0.50 mmol) and 2-hydrazinyl-6-
methoxybenzo[d]thiazole (12a, 98 mg, 0.50 mmol) to afford 5d
(10a-e)
and
2-
o
1
yellow solid, yield: 92%, Mp: 336-338 C, H NMR (300 MHz,
TFA-d + CDCl3) δ 8.77 (s, 1H), 8.32 (d, J = 7.4 Hz, 1H), 7.97 (d, J
= 5.9 Hz, 1H), 7.67–7.57 (m, 4H), 7.45 (d, J = 7.7 Hz, 2H), 7.20
(td, J = 5.4, 2.5 Hz, 3H), 3.88 (s, 3H); 13C NMR (75 MHz, TFA-d +
CDCl3) δ 167.19, 158.65, 149.62, 149.32, 148.55, 138.31,
131.58, 131.48, 130.84, 126.23, 123.99, 117.59, 117.00,
115.98, 114.39, 108.90, 56.34. MS (ESI): m/z 376 [M+H]+;
HRMS calcd for C20H17N5OS [M+H]+ 376.12266, found
376.12159.
N-(4-Chlorophenyl)-3-((2-(6-methoxybenzo[d]thiazol-2-
yl)hydrazono)methyl)pyridin-2-amine (5a):
Compound 5a was prepared according to the above explained
general
method
by
employing
2-((4-
chlorophenyl)amino)nicotinaldehyde (10a, 100 mg, 0.43
mmol) and 2-hydrazinyl-6-methoxybenzo[d]thiazole (12a,
84mg, 0.43mmol) to afford 5a as yellow solid, yield: 82%, Mp:
N-(4-Chlorophenyl)-3-((2-(6-ethoxybenzo[d]thiazol-2-yl)
hydrazono)methyl)pyridin-2-amine (5e):
Compound 5e was prepared according to the above explained
o
1
336-339 C, H NMR (300 MHz, TFA-d + CDCl3) δ 8.76 (s, 1H),
8.34 (d, J = 7.0 Hz, 1H), 7.99 (d, J = 5.4 Hz, 1H), 7.62 (m, 3H),
7.42 (d, J = 7.7 Hz, 2H), 7.26 (m, 3H), 3.91 (s, 3H); 13C NMR (75
MHz, TFA-d + CDCl3) δ 167.10, 158.76, 149.72, 149.37, 148.85,
138.32, 137.44, 131.86, 131.40, 130.06, 127.79, 123.96,
117.80, 117.02, 116.15, 114.83, 106.96, 56.42. MS (ESI): m/z
410 [M+H]+; HRMS calculated for C20H17N5OClS [M+H]+
410.08369, found 410.08239.
general
method
by
employing
2-((4-
chlorophenyl)amino)nicotinaldehyde (10a, 100 mg, 0.43
mmol) and 6-ethoxy-2-hydrazinylbenzo[d]thiazole (12b, 90mg,
0.43mmol) to afford 5e yellow solid, yield: 85%, Mp: 312-
1
3150C , H NMR (300 MHz, TFA-d + CDCl3) δ 8.75 (s, 1H), 8.33
(d, J = 7.5 Hz, 1H), 7.99 (d, J = 6.3 Hz, 1H), 7.66–7.58 (m, 3H),
7.42 (d, J = 8.5 Hz, 2H), 7.25 (dt, J = 18.9, 5.6 Hz, 3H), 4.12 (q, J
= 6.8 Hz, 2H), 1.46 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, TFA-d +
CDCl3) δ 167.04, 158.11, 149.66, 148.84, 138.28, 137.43,
3-((2-(6-Methoxybenzo[d]thiazol-2-yl)hydrazono)methyl)-N-
(4-(trifluoromethyl)phenyl)pyridin-2-amine (5b):
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131.86, 131.27, 130.07, 127.79, 123.90, 118.41, 116.98, Compound 5i was prepared according to the above explained
View Article Online
DOI: 10.1039/C8NJ06517A
114.83, 107.57, 65.38, 14.65. MS (ESI): m/z 424 [M+H]+; HRMS general
method
by
employing
2-((4-
calcd for C21H18N5OClS [M+H]+ 424.09934, found 424.09808.
chlorophenyl)amino)nicotinaldehyde (10a, 100 mg, 0.43
mmol) and 2-hydrazinyl-6-methylbenzo[d]thiazole (12c, 77mg,
3-((2-(6-Ethoxybenzo[d]thiazol-2-yl)hydrazono)methyl)-N-(4- 0.43mmol) to afford 5i pale yellow solid, yield: 87%, Mp: 282-
o
(trifluoromethyl)phenyl)pyridin-2-amine (5f):
284 C, 1H NMR (300 MHz, TFA-d + CDCl3) δ 8.77 (s, 1H), 8.34
Compound 5f was prepared according to the above explained (d, J = 7.1 Hz, 1H), 7.99 (d, J = 5.6 Hz, 1H), 7.59 (d, J = 8.4 Hz,
general method by employing 2-((4- 4H), 7.43 (t, J = 10.3 Hz, 3H), 7.22 (t, J = 6.8 Hz, 1H), 2.49 (s,
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(trifluoromethyl)phenyl)amino)nicotinaldehyde (10b, 100 mg, 3H); 13C NMR (75 MHz, TFA-d + CDCl3) δ 167.82, 149.78,
0.37 mmol) and 6-ethoxy-2-hydrazinylbenzo[d]thiazole (12b, 149.29, 148.81, 138.33, 137.92, 137.23, 135.43, 131.76,
78mg, 0.37mmol) to afford 5f yellow solid, yield: 85%, Mp: 131.06, 130.12, 127.68, 122.70, 122.49, 116.10, 115.78,
242-244 C, H NMR (300 MHz, TFA-d + CDCl3) δ 8.78 (s, 1H), 114.76, 21.42. MS (ESI): m/z 394 [M+H]+; HRMS calcd for
8.37 (d, J = 6.8 Hz, 1H), 8.00 (d, J = 5.4 Hz, 1H), 7.70-7.85 (m, C20H16N5ClS [M+H]+ 394.08877, found 394.08711.
4H), 7.62 (d, J = 9.3 Hz, 1H), 7.26 – 7.17 (m, 3H), 4.09 (q, J = 7.0
Hz, 2H), 1.46 (t, J = 7.0 Hz, 3H); 13C NMR (75 MHz, TFA-d + 3-((2-(6-Methylbenzo[d]thiazol-2-yl)hydrazono)methyl)-N-(4-
CDCl3) δ 167.03, 158.08, 149.38, 149.17, 148.67, 138.49, (trifluoromethyl)phenyl)pyridin-2-amine (5j):
134.31, 132.72, 132.33, 131.28, 129.89, 123.89, 123.50, Compound 5j was prepared according to the above explained
o
1
118.21, 117.03, 115.15, 107.39, 65.13, 14.64. MS (ESI): m/z general
method
by
employing
2-((4-
458 [M+H]+; HRMS calcd for C22H18N5OF3S [M+H]+ 458.12569, (trifluoromethyl)phenyl)amino)nicotinaldehyde (10b, 100 mg,
found 458.12347.
0.37 mmol) and 2-hydrazinyl-6-methylbenzo[d]thiazole (12c,
67mg, 0.37mmol) to afford 5j pale yellow solid, yield: 82%,
o
3-((2-(6-Ethoxybenzo[d]thiazol-2-yl)hydrazono)methyl)-N-(4-
fluorophenyl)pyridin-2-amine (5g):
Mp: 296-298 C, 1H NMR (300 MHz, TFA-d + CDCl3) δ 8.79 (s,
1H), 8.38 (d, J = 7.2 Hz, 1H), 8.00 (d, J = 5.8 Hz, 1H), 7.83 (t, J =
Compound 5g was prepared according to the above explained 8.2 Hz, 1H), 7.7-7.8 (m, 3H), 7.63-7.54 (m, 2H), 7.45 (d, J = 8.5
general method by employing
2-((4- Hz, 1H), 7.24-7.3 (m, 1H), 2.49 (s, 3H); 13C NMR (75 MHz, TFA-d
fluorophenyl)amino)nicotinaldehyde (10c, 100 mg, 0.46 mmol) + CDCl3) δ 167.83, 149.71, 149.19, 148.87, 138.48, 137.95,
and 6-ethoxy-2-hydrazinylbenzo[d]thiazole (12b, 96mg, 135.41, 132.66, 132.36, 131.09, 129.83, 127.50, 123.46,
0.46mmol) to afford 5g yellow solid, yield: 87%, Mp: 272-275 122.71, 122.49, 116.22, 115.78, 115.12, 21.42. MS (ESI): m/z
1
oC , H NMR (300 MHz, TFA-d + CDCl3) δ 8.76 (s, 1H), 8.33 (d, J 428 [M+H]+; HRMS calcd for C21H16N5F3S [M+H]+ 428.11513,
= 7.4 Hz, 1H), 7.97 (d, J = 6.1 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), found 428.11233.
7.51 – 7.42 (m, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.27 (d, J = 2.1 Hz,
1H), 7.16-7.24 (m, 2H), 4.11 (q, J = 13.9, 6.9 Hz, 2H), 1.46 (t, J = N-(4-Fluorophenyl)-3-((2-(6-methylbenzo[d]thiazol-2-
6.9 Hz, 3H); 13C NMR (75 MHz, TFA-d + CDCl3) δ 167.02, 159.31, yl)hydrazono)methyl)pyridin-2-amine (5k):
158.74, 158.09, 149.64, 148.77, 138.16, 131.29, 129.01, Compound 5k was prepared according to the above explained
128.79, 127.40, 123.92, 120.33, 118.94, 118.67, 118.32, general
method
by
employing
2-((4-
116.93, 116.56, 116.22, 114.67, 112.79, 107.55, 65.32, 14.52. fluorophenyl)amino)nicotinaldehyde (10c, 100 mg, 0.46 mmol)
MS (ESI): m/z 408 [M+H]+; HRMS calcd for C21H18N5OFS [M+H]+ and 2-hydrazinyl-6-methylbenzo[d]thiazole (12c, 83mg,
408.12889, found 408.12663.
0.46mmol) to afford 5k yellow solid, yield: 85%, Mp: 302-304
oC, 1H NMR (300 MHz, TFA-d + CDCl3) δ 8.77 (s, 1H), 8.33 (d, J =
6.9 Hz, 1H), 7.97 (d, J = 5.5 Hz, 1H), 7.63-7.54 (m, 2H), 7.46 (dd,
J = 7.8, 5.6 Hz, 3H), 7.31 (t, J = 8.3 Hz, 2H), 7.23-7.16 (m, 1H),
3-((2-(6-Ethoxybenzo[d]thiazol-2-yl)hydrazono)methyl)-N-
phenylpyridin-2-amine (5h):
Compound 5h was prepared according to the above explained 2.49 (s, 3H); 13C NMR (75 MHz, TFA-d + CDCl3) δ 167.80 (d, J =
general method by employing 2-(phenylamino)nicotinaldehyde 253 Hz), 163.71, 149.84, 149.66, 148.73, 138.34, 137.88,
(10d,
100
mg,
0.50
mmol)
and
6-ethoxy-2- 135.43, 131.05, 128.91(d, J = 8.8 Hz), 122.69, 122.49, 120.24,
hydrazinylbenzo[d]thiazole (12b, 104 mg, 0.50 mmol) to afford 118.85, 116.00, 115.78, 114.59, 21.43. MS (ESI): m/z 378
5h yellow solid, yield: 92%, Mp: 274-276 oC, 1H NMR (300 MHz, [M+H]+; HRMS calcd for C20H16N5FS [M+H]+ 378.11832, found
TFA-d + CDCl3) δ 8.76 (s, 1H), 8.32 (d, J = 7.0 Hz, 1H), 7.97 (d, J 378.11728.
= 6.1 Hz, 1H), 7.70–7.56 (m, 4H), 7.48–7.41 (m, 2H), 7.18 (d, J =
10.5 Hz, 3H), 4.08 (q, J = 6.9 Hz, 2H), 1.45 (t, J = 7.0 Hz, 3H); 13
C
3-((2-(6-Methylbenzo[d]thiazol-2-yl)hydrazono)methyl)-N-
NMR (75 MHz, TFA-d + CDCl3) δ 167.15, 158.74, 158.18, phenylpyridin-2-amine (5l):
157.97, 149.42, 148.41, 138.30, 131.76, 131.53, 130.75, Compound 5l was prepared according to the above explained
126.22, 123.99, 118.02, 116.93, 116.53, 116.01, 114.35, general method by employing 2-(phenylamino)nicotinaldehyde
107.29, 64.98, 14.66. MS (ESI): m/z 390 [M+H]+; HRMS calcd (10d, 100 mg, 0.50 mmol) and 2-hydrazinyl-6-
for C21H20N5OS [M+H]+ 390.13831, found 390.13686.
methylbenzo[d]thiazole (12c, 91mg, 0.50mmol) to afford 5l
o
yellow solid, yield: 92%, Mp: 284-286 C , 1H NMR (300 MHz,
N-(4-Chlorophenyl)-3-((2-(6-methylbenzo[d]thiazol-2-
yl)hydrazono)methyl)pyridin-2-amine (5i):
TFA-d + CDCl3) δ 8.77 (s, 1H), 8.33 (d, J = 7.2 Hz, 1H), 7.96 (d, J
= 5.9 Hz, 1H), 7.55-7.67 (m, 5H), 7.45 (d, J = 7.7 Hz, 3H), 7.19 (t,
8 | J. Name., 2012, 00, 1-3
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J = 6.9 Hz, 1H), 2.49 (s, 3H); 13C NMR (75 MHz, TFA-d + CDCl3) δ Compound 5p was prepared according to the above explained
View Article Online
167.87, 149.90, 149.34, 148.74, 138.28, 137.83, 135.45, general method by employing 2-(phenylaDmOinI:o10)n.1i0c3o9t/iCn8aNldJe06h5y1d7Ae
131.62, 131.02, 130.88, 126.15, 122.71, 122.54, 116.00, (10d, 100 mg, 0.50 mmol) and 2-hydrazinylbenzo[d]thiazole
115.76, 114.43, 21.42. MS (ESI): m/z 360 [M+H]+; HRMS calcd (12d, 83 mg, 0.50 mmol) to afford 5p yellow solid, yield: 90%,
o
for C20H17N5S [M+H]+ 360.12774, found 360.12656.
Mp: 278-280 C, 1H NMR (300 MHz, TFA-d + CDCl3) δ 8.80 (s,
1H), 8.34 (d, J = 6.8 Hz, 1H), 7.96 (d, J = 5.4 Hz, 1H), 7.79-7.71
(m, 2H), 7.58-7.68 (m, 4H), 7.51 (t, J = 7.9 Hz, 1H), 7.45 (d, J =
6.5 Hz, 2H), 7.22-7.16 (t, J = 7.8 Hz, 1H); 13C NMR (75 MHz,
3-((2-(Benzo[d]thiazol-2-yl)hydrazono)methyl)-N-(4-
chlorophenyl)pyridin-2-amine (5m):
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compound 5m was prepared according to the above explained TFA-d + CDCl3) δ 168.35, 150.31, 149.38, 148.87, 138.38,
general
method
by
employing
2-((4- 137.66, 131.63, 130.90, 129.84, 126.92, 126.19, 122.88,
chlorophenyl)amino)nicotinaldehyde (10a, 100 mg, 0.43 122.49, 116.17, 115.94, 114.43. MS (ESI): m/z 346 [M+H]+;
mmol) and 2-hydrazinylbenzo[d]thiazole (12d, 71 mg, 0.43 HRMS calcd for C19H16N5S [M+H]+ 346.11227, found 346.11209.
o
mmol) to afford 5m yellow solid, yield: 85%, Mp: 296-298 C,
1H NMR (300 MHz, TFA-d + CDCl3) δ 8.79 (s, 1H), 8.36 (d, J = 7.3 3-((2-(Benzo[d]thiazol-2-yl)hydrazono)methyl)-N-(3,4,5-
Hz, 1H), 7.99 (d, J = 5.8 Hz, 1H), 7.81-7.48 (m, 6H), 7.42 (d, J = trimethoxyphenyl)pyridin-2-amine (5q):
8.2 Hz, 2H), 7.2-7.3 (m, 1H); 13C NMR (75 MHz, TFA-d + CDCl3) Compound 5q was prepared according to the above explained
δ 168.31, 150.23, 149.36, 148.98, 138.43, 137.62, 137.31, general
method
by
employing
2-((3,4,5-
131.80, 130.07, 129.92, 127.74, 127.03, 122.88, 122.44, trimethoxyphenyl)amino)nicotinaldehyde (10e, 100 mg, 0.34
116.19, 116.07, 114.80. MS (ESI): m/z 380[M+H]+; HRMS calcd mmol) and 2-hydrazinylbenzo[d]thiazole (12d, 57 mg, 0.34
o
for C19H15N5ClS [M+H]+ 380.07312, found 380.07409.
mmol) to afford 5q yellow solid, yield: 92%, Mp: 249-252 C ,
1H NMR (300 MHz, TFA-d + CDCl3) δ 8.78 (s, 1H), 8.35 (d, J = 7.0
Hz, 1H), 8.00 (d, J = 5.4 Hz, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.62-
7.72 (m, 2H), 7.43-7.53 (m, 1H), 7.15-7.25 (m, 1H), 6.71 (s, 2H),
3-((2-(Benzo[d]thiazol-2-yl)hydrazono)methyl)-N-(4-
(trifluoromethyl)phenyl)pyridin-2-amine (5n):
Compound 5n was prepared according to the above explained 3.97 (s, 3H), 3.87 (s, 6H); 13C NMR (75 MHz, TFA-d + CDCl3) δ
general
method
by
employing
2-((4- 168.15, 154.90, 153.80, 150.28, 149.56, 148.91, 138.49,
(trifluoromethyl)phenyl)amino)nicotinaldehyde (10b, 100 mg, 138.16, 137.58, 129.89, 127.87, 127.00, 122.92, 122.43,
0.37 mmol) and 2-hydrazinylbenzo[d]thiazole (12d, 62mg, 116.13, 115.87, 114.60, 104.06, 61.49, 56.58. MS (ESI): m/z
0.37mmol) to afford 5n yellow solid, yield: 85%, Mp: 264-266 436 [M+H]+; HRMS calcd for C22H22N5O3S [M+H]+ 436.14379,
oC, 1H NMR (300 MHz, TFA-d + CDCl3) δ 8.82 (s, 1H), 8.39 (d, J = found 436.14305.
6.8 Hz, 1H), 8.00 (d, J = 5.4 Hz, 1H), 7.83 (t, J = 8.0 Hz, 1H),
7.65-7.8(m, 6H), 7.52 (t, J = 7.6 Hz, 1H), 7.26 (t, J = 7.0 Hz, 1H); N-(4-Chlorophenyl)-3-((2-(6-fluorobenzo[d]thiazol-2-
13C NMR (75 MHz, TFA-d + CDCl3) δ 168.34, 150.10, 149.23, yl)hydrazono)methyl)pyridin-2-amine (5r):
148.84, 138.61, 137.65, 134.36, 133.91, 132.66, 132.36, Compound 5r was prepared according to the above explained
129.90, 127.47, 127.00, 123.49, 122.88, 122.45, 116.21, general
method
by
employing
2-((4-
115.13. MS (ESI): m/z 414 [M+H]+; HRMS calcd for C20H14N5F3S chlorophenyl)amino)nicotinaldehyde (10a, 100 mg, 0.43
[M+H]+ 414.09948, found 414.09889.
mmol) and 6-fluoro-2-hydrazinylbenzo[d]thiazole (12e, 79 mg,
0.43 mmol) to afford 5r yellow solid, yield: 87%, Mp: 318-320
oC, 1H NMR (300 MHz, TFA-d + CDCl3) δ 8.78 (s, 1H), 8.38-8.33
(m, 1H), 8.02-7.97 (m, 1H), 7.72 (dd, J = 9.0, 4.1 Hz, 1H), 7.59
3-((2-(Benzo[d]thiazol-2-yl)hydrazono)methyl)-N-(4-
fluorophenyl)pyridin-2-amine (5o):
Compound 5o was prepared according to the above explained (d, J = 8.7 Hz, 2H), 7.50 (dd, J = 7.2, 2.3 Hz, 1H), 7.40 (d, J = 8.7
general method by employing
2-((4- Hz, 3H), 7.26-7.18 (m, 1H); 13C NMR (75 MHz, TFA-d + CDCl3) δ
fluorophenyl)amino)nicotinaldehyde (10c, 100 mg, 0.46mmol) 168.25, 159.25, 150.44, 149.37, 149.04, 138.42, 137.39,
and 2-hydrazinylbenzo[d]thiazole (12d, 76 mg, 0.46 mmol) to 134.11, 131.82, 130.01, 127.74, 124.06, 123.78, 118.35,
o
1
afford 5o yellow solid, yield: 87%, Mp: 254-257 C , H NMR 118.03, 117.61, 117.49, 116.02, 114.83, 110.35, 109.98. MS
(300 MHz, TFA-d + CDCl3) δ 8.79 (s, 1H), 8.35 (d, J = 7.4 Hz, 1H), (ESI): m/z 398[M+H]+; HRMS calcd for C19H14N5FClS [M+H]+
7.97 (d, J = 5.7 Hz, 1H), 7.74 (dd, J = 11.5, 8.4 Hz, 2H), 7.65 (t, J 398.06374, found 398.06370.
= 7.7 Hz, 1H), 7.56-7.44 (m, 3H), 7.29 (dd, J = 14.5, 6.0 Hz, 2H),
7.21 (t, J = 7.0 Hz, 1H) 13C NMR (75 MHz, TFA-d + CDCl3) δ 3-((2-(6-Fluorobenzo[d]thiazol-2-yl)hydrazono)methyl)-N-(4-
168.30, 150.26, 149.71, 148.89, 138.40, 137.62, 129.89, 128.96 (trifluoromethyl)phenyl)pyridin-2-amine (5s):
(d, J = 9.0 Hz), 127.40 (d, J = 2.5 Hz), 126.99, 122.87, 122.45, Compound 5s was prepared according to the above explained
120.27, 118.56, 116.18, 115.96, 114.62. MS (ESI): m/z 364 general
method
by
employing
2-((4-
[M+H]+; HRMS calcd for C19H14N5FS [M+H]+ 364.10267, found (trifluoromethyl)phenyl)amino)nicotinaldehyde (10b, 100 mg,
364.10228.
0.37 mmol) and 6-fluoro-2-hydrazinylbenzo[d]thiazole (12e, 69
mg, 0.37 mmol) to afford 5s yellow solid, yield: 85%, Mp: 307-
o
3-((2-(Benzo[d]thiazol-2-yl)hydrazono)methyl)-N-
phenylpyridin-2-amine (5p):
309 C, 1H NMR (300 MHz, TFA-d + CDCl3) δ 8.79 (s, 1H), 8.38
(d, J = 7.2 Hz, 1H), 8.02 (d, J = 5.8 Hz, 1H), 7.81 (t, J = 7.8 Hz,
1H), 7.65-7.75 (m, 4H), 7.48 (dd, J = 7.2, 2.3 Hz, 1H), 7.37 (td, J
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= 8.7, 2.4 Hz, 1H), 7.23-7.28 (m, 1H). 13C NMR (75 MHz, TFA-d + m/z 454 [M+H]+; HRMS calcd for C22H21N5O3FS [M+H]+
CDCl3) δ 168.41, 160.64, 149.97, 149.20, 148.70, 138.82, 454.13436, found 454.13448.
134.45, 134.18, 133.73, 132.76, 132.27, 129.87, 127.39,
127.34, 124.01, 123.87, 123.45, 121.27, 120.43, 118.14, N-(4-Chlorophenyl)-3-((2-(thiazol-2-
117.81, 117.62, 116.10, 110.34, 109.91, 109.08. MS (ESI): m/z yl)hydrazono)methyl)pyridin-2-amine (5w):
432 [M+H]+; HRMS calcd for C20H14N5F4S [M+H]+ 432.09006, Compound 5w was prepared according to the above explained
View Article Online
DOI: 10.1039/C8NJ06517A
found 432.09001.
general
method
by
employing
2-((4-
chlorophenyl)amino)nicotinaldehyde (10a, 100 mg, 0.43
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3-((2-(6-Fluorobenzo[d]thiazol-2-yl)hydrazono)methyl)-N-(4-
mmol) and 2-hydrazinylthiazole (12f, 50 mg, 0.43 mmol) to
fluorophenyl)pyridin-2-amine 5t: Compound 5t was prepared afford 5w yellow solid, yield: 85%, Mp: 290-294 C ,1H NMR
according to the above explained general method by (300 MHz, TFA-d + CDCl3) δ 8.70 (s, 1H), 8.33 (d, J = 6.6 Hz, 1H),
employing 2-((4-fluorophenyl)amino)nicotinaldehyde (10c, 100 7.97 (d, J = 6.3 Hz, 1H), 7.52-7.62 (m, 3H), 7.37 (d, J = 8.6 Hz,
mg, 0.46 mmol) and 6-fluoro-2-hydrazinylbenzo[d]thiazole 2H), 7.21 (t, J = 6.7 Hz, 1H), 6.94 (d, J = 4.3 Hz, 1H); 13C NMR
(12e, 85mg, 0.46mmol) to afford 5t, yellow solid yield: 87%, (75 MHz, TFA-d + CDCl3) δ 168.95, 149.23, 148.94, 148.64,
o
o
Mp: 316-319 C, 1H NMR (300 MHz, TFA-d + CDCl3) δ 8.78 (s, 138.08, 137.28, 131.77, 130.03, 129.09, 127.52, 116.22,
1H), 8.34 (d, J = 6.9 Hz, 1H), 7.99 (d, J = 5.7 Hz, 1H), 7.72 (dd, J 114.78, 109.56. MS (ESI): m/z 330[M+H]+; HRMS calcd for
= 9.0, 4.1 Hz, 1H), 7.50-7.42 (m, 3H), 7.37 (td, J = 8.8, 2.4 Hz, C15H13N5ClS [M+H]+ 330.05747, found 330.05741.
1H), 7.33-7.26 (m, 2H), 7.24-7.16 (m, 1H); 13C NMR (75 MHz,
TFA-d + CDCl3) δ 168.35, 165.35, 162.36, 150.24, 149.66, 3-((2-(Thiazol-2-yl)hydrazono)methyl)-N-(4-(trifluoromethyl)
148.72, 138.58, 134.34, 128.93, 128.81, 127.46, 123.82, phenyl)pyridin-2-amine (5x):
118.48, 118.17, 117.84, 117.60, 117.48, 114.55, 112.82, Compound 5x was prepared according to the above explained
110.28, 109.91, 109.09. MS (ESI): m/z 410 [M+H]+; HRMS calcd general
method
by
employing
2-((4-
for C19H14N5F2S [M+H]+ 382.09325, found 382.09338.
(trifluoromethyl)phenyl)amino)nicotinaldehyde (10b, 100 mg,
0.37 mmol) and 2-hydrazinylthiazole (12f, 43mg, 0.37 mmol)
o
3-((2-(6-Fluorobenzo[d]thiazol-2-yl)hydrazono)methyl)-N-
phenyl pyridin-2-amine 5u:
to afford 5x yellow solid, yield: 82%, Mp: 280-282 C , 1H NMR
(300 MHz, TFA-d + CDCl3) δ 8.72 (s, 1H), 8.37 (d, J = 6.6 Hz, 1H),
Compound 5u was prepared according to the above explained 7.98 (d, J = 5.1 Hz, 1H), 7.80 (t, J = 8.9 Hz, 1H), 7.74-7.63 (m,
general method by employing 2-(phenylamino)nicotinaldehyde 3H), 7.59 (d, J = 4.4 Hz, 1H), 7.23 (d, J = 6.6 Hz, 1H), 6.94 (d, J =
(10d,
100
mg,
0.50
mmol)
and
6-fluoro-2- 4.3 Hz, 1H); 13C NMR (75 MHz, TFA-d + CDCl3) δ 168.95, 148.85,
hydrazinylbenzo[d]thiazole (12e, 92mg, 0.50mmol) to afford 148.70, 138.22, 133.93, 132.55, 132.37, 129.77, 129.07,
5u pale yellow solid, yield: 90%, Mp: 286-288 oC , 1H NMR (300 127.43, 123.37, 115.14, 109.62. MS (ESI): m/z 364 [M+H]+;
MHz, TFA-d + CDCl3) δ 8.79 (s, 1H), 8.34 (d, J = 6.5 Hz, 1H), 7.97 HRMS calcd for C16H13N5F3S [M+H]+ 364.08420, found
(d, J = 6.5, 1H), 7.71 (dd, J = 9.0, 4.0 Hz, 1H), 7.62 (d, J = 7.2 Hz, 364.08383.
2H), 7.49 (dd, J = 7.2, 2.3 Hz, 1H), 7.47-7.42 (m, 2H), 7.38 (td, J
= 8.8, 2.4 Hz, 1H), 7.20 (t, J = 8.8Hz, 1H); 13C NMR (75 MHz, N-(4-Fluorophenyl)-3-((2-(thiazol-2-
TFA-d + CDCl3) δ 168.33, 159.22, 150.62, 149.44, 149.06, yl)hydrazono)methyl)pyridin-2-amine 5y :
138.39, 134.07, 131.71, 131.48, 131.03, 126.21, 123.92, Compound 5y was prepared according to the above explained
123.78, 118.33, 118.01, 117.57, 117.45, 115.93, 114.52, general
method
by
employing
2-((4-
110.37, 110.00. MS (ESI): m/z 364 [M+H]+; HRMS calcd for fluorophenyl)amino)nicotinaldehyde (10c, 100 mg, 0.46 mmol)
C19H15N5FS [M+H]+ 364.10267, found 364.10271.
and 2-hydrazinylthiazole (12f, 53 mg, 0.46 mmol) to afford 5y
o
1
yellow solid, yield: 87%, Mp: 284-286 C, H NMR (300 MHz,
TFA-d + CDCl3) δ 8.70 (s, 1H), 8.32 (d, J = 7.4 Hz, 1H), 7.95 (d, J
= 6.2 Hz, 1H), 7.59 (d, J = 4.1 Hz, 1H), 7.46-7.37 (m, 2H), 7.27 (t,
3-((2-(6-Fluorobenzo[d]thiazol-2-yl)hydrazono)methyl)-N-
(3,4,5-trimethoxyphenyl)pyridin-2-amine (5v):
Compound 5v was prepared according to the above explained J = 8.1 Hz, 2H), 7.19 (t, J = 6.8 Hz, 1H), 6.93 (d, J = 3.8 Hz, 1H);
general method by employing
2-((3,4,5- 13C NMR (75 MHz, TFA-d + CDCl3) δ 168.95, 149.57, 148.94,
trimethoxyphenyl)amino)nicotinaldehyde (10e, 100 mg, 0.34 148.50, 138.07, 129.08, 128.75, 128.63, 118.81, 118.50,
mmol) and 6-fluoro-2-hydrazinylbenzo[d]thiazole (12e, 64mg, 116.10, 114.58, 109.49. MS (ESI): m/z 313 [M]+; HRMS calcd for
0.34mmol) to afford 5v yellow solid, yield: 92%, Mp: 264-266 C15H12N5FS [M+H]+ 313.07920, found 313.07722.
oC, 1H NMR (300 MHz, TFA-d + CDCl3) δ 8.77 (s, 1H), 8.35 (d, J =
7.4 Hz, 1H), 7.99 (d, J = 6.0 Hz, 1H), 7.70 (dd, J = 9.0, 4.0 Hz, N-Phenyl-3-((2-(thiazol-2-yl)hydrazono)methyl)pyridin-2-
1H), 7.52 (dd, J = 7.2, 2.2 Hz, 1H), 7.38 (td, J = 8.8, 2.3 Hz, 1H), amine (5z):
7.21 (t, J = 6.9 Hz, 1H), 6.71 (s, 2H), 3.97 (s, 3H), 3.87 (s, 6H). 13C Compound 5z was prepared according to the above explained
NMR (75 MHz, TFA-d + CDCl3) δ 169.13, 168.21, 160.59, general method by employing 2-(phenylamino)nicotinaldehyde
159.28, 154.95, 150.56, 149.62, 149.12, 138.44, 138.12, (10d, 100 mg, 0.50 mmol) and 2-hydrazinylthiazole (12f, 58
134.00, 127.93, 123.98, 123.84, 118.37, 118.10, 117.54, mg, 0.50 mmol) to afford 5z yellow solid, yield: 90%, Mp: 286-
117.40, 115.93, 114.72, 110.43, 110.05, 61.55, 56.49. MS (ESI): 290 C , 1H NMR (300 MHz, TFA-d + CDCl3) δ 8.70 (s, 1H), 8.31
o
(d, J = 7.6 Hz, 1H), 7.93 (d, J = 6.3 Hz, 1H), 7.55-7.65 (m, 4H),
10 | J. Name., 2012, 00, 1-3
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7.40 (d, J = 6.2 Hz, 2H), 7.17 (t, J = 6.5 Hz, 1H), 6.93 (d, J = 4.4 and incubated for next 4 hours at 37 °C. Subsequently,
View Article Online
Hz, 1H); 13C NMR (75 MHz, TFA-d + CDCl3) δ 168.96, 149.28, supernatant was discarded and formazan crystals were
DOI: 10.1039/C8NJ06517A
149.02, 148.58, 137.90, 131.63, 131.49, 130.88, 128.99, dissolved in 200 μL of DMSO. The absorbance was taken at 570
125.97, 116.15, 114.45, 109.60. MS (ESI): m/z 296[M+H]+; nm using spectrophotometer (Molecular devices, USA).
HRMS calcd for C15H14N5S [M+H]+ 296.09635, found 296.09644. Clonogenic assay
8
9
Clonogenic assay (colony formation assay) is an in vitro cell
survival assay technique which is based on the ability of a
single cell to grow and form colonies. The assay represents a
valuable technique to assess the retained capacity of cells after
treatment with cytotoxic agents, to eventually produce large
number of progeny. Clonogenic assay was performed to assess
the effect of compounds on colony forming capability of MCF-
7 cells as described by Franken et al with slight modifications.20
Briefly, the cells were plated at a density of 200 cells/well in a
6-well plate followed by their exposure to different
concentrations of conjugate 5i for 24 hours. Subsequently,
cells were allowed to grow for 14 days after giving fresh
media. The growth medium was changed with fresh medium
every 3rd day to supplement the cells with required nutrients
and growth factors. After 14 days, grown colonies were fixed
and stained with crystal violet (1% in methanol) and number of
stained colonies was counted.
3-((2-(Thiazol-2-yl)hydrazono)methyl)-N-(3,4,5-
trimethoxyphenyl) pyridin-2-amine (5aa):
Compound 5aa was prepared according to the above
explained general method by employing 2-((3,4,5-
trimethoxyphenyl)amino)nicotinaldehyde (10e, 100 mg, 0.34
mmol) and 2-hydrazinylthiazole (12f, 40 mg, 0.34 mmol) to
afford 5aa yellow solid, yield: 95%, Mp: 244-246 C , H NMR
(300 MHz, TFA-d + CDCl3) δ 8.68 (s, 1H), 8.32 (d, J = 7.5 Hz, 1H),
7.96 (d, J = 6.2 Hz, 1H), 7.57 (d, J = 4.4 Hz, 1H), 7.20 (dd, J =
13.2, 5.9 Hz, 1H), 6.95 (d, J = 4.3 Hz, 1H), 6.67 (s, 2H), 3.94 (s,
3H), 3.85 (s, 6H); 13C NMR (75 MHz, TFA-d + CDCl3) δ 168.85,
154.86, 149.43, 148.90, 148.55, 138.05, 128.95, 127.97,
116.01, 114.59, 109.65, 103.95, 61.50, 56.47. MS (ESI): m/z
386 [M+H]+; HRMS calcd for C18H20N5O3S [M+H]+ 386.12814,
found 386.12827.
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Detection of intracellular reactive oxygen species (ROS) by DCFDA
Experimental section
Biology
Increased intracellular levels of reactive oxygen species (ROS)
has been implicated as a major protagonist for induction of
apoptosis in cancer cells.21 To measure the levels of
intracellular ROS induced by test compound, an oxidant-
sensitive florescent probe, 2’,7’-dichlorofluorescindiacetate
(DCFH-DA) was used which gets converted to highly florescent
derivative when oxidized by intracellular ROS.22 MCF-7 cells
were plated in 12-well plate and allowed to grow for 24 hours.
Cells were treated with increasing concentration of conjugate
5i (0.625, 1.25, 2.5, 5, 10 μM) for 4 hours. After 4 hours of
incubation, treatment medium was discarded and cells were
washed with PBS followed by incubation of cells with DCFH-DA
(10 μM) at 37 °C for 20 min. The oxidation of DCFH-DA to
florescent DCF mediated by intracellular ROS was observed
under florescent microscope (Nikon, Japan) with excitation
(498 nm) and emission (530 nm) wavelength.
Cytotoxic activity
Cell culture
The human breast adenocarcinoma (MCF-7), triple negative
breast cancer (MDA-MB-231), lung adenocarcinoma (A549),
and mouse melanoma (B16F10) cells were obtained from
National Centre for Cell Science (NCCS, Pune). Dulbecco’s
Modified Eagle Medium (DMEM), Rosewell Park Memorial
Institute Medium 1640 (RPMI 1640), and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
were purchased from Sigma Aldrich (St. Louis, USA). Fetal
Bovine Serum (FBS), Penicillin-Streptomycin mixture and
Trypsin-EDTA were obtained from Gibco, USA. Cells were
grown in respective media supplemented with 10% FBS and
1% penicillin-streptomycin mixture and allowed to grow in an
incubator maintained at 37 ᵒC with 90% humidity and 5% CO2.
The cells were treated with compounds dissolved in DMSO
while untreated controls were exposed to corresponding
volumes of DMSO.
Measurement of mitochondrial membrane potential (ΔѰm)
Loss of mitochondrial membrane potential (ΔѰm) is implicated
as one of the major pathways for cellular death as
maintenance of mitochondrial membrane potential (ΔѰm) is
crucial to sustain mitochondrial integrity and its function [23].
To investigate the loss of mitochondrial membrane potential
(ΔѰm) MCF-7 cells (1 X 105 cells/well) were plated in 6-well
plate and allowed to grow for 24 hours. The cells were then
incubated with different concentrations (0.625, 1.25, 2.5, 5, 10
μM) of conjugate 5i for 24 hours. Subsequently, cells were
harvested, washed with PBS and resuspended in PBS
containing JC-1 dye (5 μg/ml) and incubated in dark for 30
minutes. Cells were washed with PBS twice and observed
under florescent microscope (Nikon, Japan).
MTT assay
Cytotoxicity of synthesized compounds was evaluated using
MTT assay which involves conversion of yellow colour MTT to
purple formazan crystals by live cells.19 Briefly, 3000-4000 cells
were seeded in 100 μl of DMEM supplemented with 10% FBS
and 1% antibiotic-antimycotic mixture in each well of 96
microtiter plate and incubated at 37 °C and 5% CO2
environment in an incubator. The seeded cells were exposed
to gradient concentrations (0.625-20 μM) of compounds along
with proper vehicle control for 48 hours. After 48 hours of
incubation, media was discarded and cells were washed twice
with PBS. 100 μL of MTT (0.5 mg/ml) was added to each well
Acridine orange/ethidium bromide (AO/EB) staining
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AO/EB staining method was used to visualize changes in incubated for 15 minutes in dark. After addition of 400 μl of 1X
View Article Online
cellular morphology such as chromatin condensation and binding buffer, cells were kept on iDceOI: a10n.d1039im/Cm8NeJd0i6a5t1e7lAy
apoptotic body formation which are characteristic features of analysed by flow cytometer (BD FACSVerse™, USA) for
cells undergoing apoptosis.24 MCF-7 cells were grown in 24- detection of population of cells undergoing apoptosis. The cell
well plate and treated with gradient concentration of population was divided into live (annexin V-negative, PI-
conjugate 5i (0.625, 1.25, 2.5 μM) for 24 hours at 37°C in an negative), early apoptotic (annexin V-positive, PI-negative),
atmosphere of 5% CO2. After 24 hours of treatment, cells were late apoptotic (annexin V-positive, PI-positive) and necrotic
washed with PBS after discarding treatment medium and were (annexin V-negative, PI-positive) cells after counting minimum
stained with acridine orange/ethidium bromide (10 μg/ml of 10,000 events.
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each) followed by observation under fluorescent microscope
(Nikon, Japan) with excitation (488 nm) and emission (550 nm)
at 200X magnification.
There are no conflicts to declare.
Hoechst staining
Conflicts of interest
Nuclear fragmentation and nuclear shrinkage are characteristic
features of cells undergoing apoptosis.25 To evaluate the
nuclear damaging potential of compounds, Hoechst 33342
staining method was used. Briefly, human breast
Acknowledgements
F.S. thanks UGC for the award of fellowship.
adenocarcinoma (MCF-7) cells were seeded at a density of 5 X
104 cells/well and allowed to adhere for 24 hours. Cells were
treated with conjugate 5i (0.625, 1.25, 2.5, 5, 10 μM) for 24
hours. Next, cells were washed and stained with Hoechst
33342 (5 μg/ml) for 30 minutes at room temperature. Stained
cells were examined under florescent microscope (Nikon,
Japan) using excitation (350 nm) and emission (460 nm) at
400X magnification.
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μM) of conjugate 5i. After 24 hours of incubation cells were
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resuspended in 100 μL of 1X binding buffer. Next, 5 μL of FITC
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DOI: 10.1039/C8NJ06517A
Graphical abstract
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Synthesis of 2-anilinopyridyl linked benzothiazole hydrazones as apoptosis
inducing cytotoxic agents
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Faria Sultana, Mohd Aslam Saifi, Riyaz Syed, Geeta Sai Mani, Siddiq pasha shaik,
Egharevba God’shelp Osas, Chandraiah Godugu, Syeda Shahjahan and Ahmed Kamal*
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2-Anilnopyridyl linked benzothiazole hydrazones
H
S
N
N
N
N
NH
CH3
Induces
ROS
Induces
apoptosis
Cl
0.36
32.04
12.83
Induces
G2/M arrest