Synthesis and biological evaluation of pyrazole linked benzothiazole-β-naphthol derivatives as topoisomerase I inhibitors with DNA binding ability

Article abstract of DOI:10.1016/j.bmc.2019.01.011

A series of new pyrazole linked benzothiazole-β-naphthol derivatives were designed and synthesized using a simple, efficient and ecofriendly route under catalyst-free conditions in good to excellent yields. These derivatives were evaluated for their cytotoxicity on selected human cancer cell lines. Among those, the derivatives 4j, 4k and 4l exhibited considerable cytotoxicity with IC₅₀ values ranging between 4.63 and 5.54 μM against human cervical cancer cells (HeLa). Structure activity relationship was elucidated by varying different substituents on benzothiazoles and pyrazoles. Further, flow cytometric analysis revealed that these derivatives induced cell cycle arrest in G2/M phase and spectroscopic studies such as UV–visible, fluorescence and circular dichroism studies showed that these derivatives exhibited good DNA binding affinity. Additionally, these derivatives can effectively inhibit the topoisomerase I activity. Viscosity studies and molecular docking studies demonstrated that the derivatives bind with the minor groove of the DNA.

Full text of DOI:10.1016/j.bmc.2019.01.011

Accepted Manuscript
Synthesis and Biological Evaluation of Pyrazole Linked Benzothiazole-β-Naph-
thol Derivatives as Topoisomerase I Inhibitors with DNA Binding Ability
Burri Nagaraju, Jeshma Kovvuri, C. Ganesh Kumar, Sunitha Rani Routhu, Md.
Adil Shareef, Manasa Kadagathur, Praveen Reddy Adiyala, Sateesh Alavala,
Narayana Nagesh, Ahmed Kamal
PII:
S0968-0896(18)31524-4
https://doi.org/10.1016/j.bmc.2019.01.011
BMC 14703
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
29 August 2018
10 January 2019
13 January 2019
Please cite this article as: Nagaraju, B., Kovvuri, J., Kumar, C.G., Routhu, S.R., Shareef, d.A., Kadagathur, M.,
Adiyala, P.R., Alavala, S., Nagesh, N., Kamal, A., Synthesis and Biological Evaluation of Pyrazole Linked
Benzothiazole-β-Naphthol Derivatives as Topoisomerase I Inhibitors with DNA Binding Ability, Bioorganic &
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.01.011
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Synthesis and Biological Evaluation of Pyrazole Linked Benzothiazole-β-
Naphthol Derivatives as Topoisomerase I Inhibitors with DNA Binding
Ability
a,b,Ϯ
a,b,Ϯ
*a,b
a
Burri Nagaraju,
Jeshma Kovvuri,
C. Ganesh Kumar,
Sunitha Rani Routhu, Md.
a,b
a
a
a
Adil Shareef, Manasa Kadagathur, Praveen Reddy Adiyala, Sateesh Alavala, Narayana
Nagesh and Ahmed Kamal
*c
*a,b,d
a
Medicinal Chemistry and Biotechnology Division, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500007, Telangana, India.
b
Academy of Scientific and Innovative Research (AcSIR), New Delhi 110025, India
c
CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500007, Telangana, India.
d
School of Pharmaceutical Education and Research, (SPER) Jamia Hamdard, New Delhi
1
10062, India
Ϯ
Both the authors contributed equally

a
Corresponding authors. Tel.: +91-40-27193157; Fax: +91-40-27193189;
E-mail: ahmedkamal@iict.res.in (Ahmed Kamal), nagesh@ccmb.res.in (Narayana Nagesh),
cgkumar@iict.res.in (C. Ganesh Kumar)
Abstract
A series of new pyrazole linked benzothiazole-β-naphthol derivatives were designed
and synthesized using a simple, efficient and ecofriendly route under catalyst-free conditions
in good to excellent yields. These derivatives were evaluated for their cytotoxicity on selected
human cancer cell lines. Among those, the derivatives 4j, 4k and 4l exhibited considerable
cytotoxicity with IC values ranging between 4.63 to 5.54 µM against human cervical cancer
5
0
cells (HeLa). Structure activity relationship was elucidated by varying different substituents
on benzothiazoles and pyrazoles. Further, flow cytometric analysis revealed that these
derivatives induced cell cycle arrest in G2/M phase and spectroscopic studies such as UV-
visible, fluorescence and circular dichroism studies showed that these derivatives exhibited
good DNA binding affinity. Additionally, these derivatives can effectively inhibit the
topoisomerase I activity. Viscosity studies and molecular docking studies demonstrated that
the derivatives bind with the minor groove of the DNA.
Keywords: Pyrazole; betanaphthol; benzothiazole; anticancer; DNA binding; topoisomerase.

1
. Introduction
Chemotherapeutic drugs are mainly aimed at rapid cell division since common feature
1
of cancer is sustained proliferation. DNA is known as important constituent of the cell and
essential for highly proliferative cells, therefore inhibition of DNA replication will induce
2
cell death. Hence DNA has been recognized as a primary intracellular target for designing
the new chemotherapeutic agents. DNA interactive drugs include intercalators, groove
3
binders and alkylating agents, DNA replication process involves many enzymes and one
such enzyme is topoisomerase. In this regard, the topoisomerase inhibitors are gaining
importance as most promising anticancer agents. Topoisomerase I cuts and rejoins the single
strand of double stranded DNA and is thus essential for transcription and replication. Minor
groove binder like netropsin, Hoechst 33258 (I, Fig. 1) and camptothecin are known to
4
inhibit topoisomerase I. Considering these facts, it is necessary to identify new
topoisomerase I inhibitors.
Pyrazole is well known nitrogen containing heterocyclic component finding prominent
5
-8
applications in the field of medicinal and pesticide chemistry. Many of the pyrazole
derivatives have obtained considerable attention as main building blocks for drug discovery.
Fig. 1. Structures of some biologically active compounds with DNA binding ability.
Additionally, the synthesis of pyrazole analogs has gained renewed interest due to their
9
10
11
various biological properties such as anti-depressant, anti-inflammatory, antimicrobial,
1
2
13
14
15,16
anti-obesity, antihypertensive, anti-inflammatory agents, analgesics and anticancer.
Their derivatives (II) are also reported as strong DNA binders. Therefore, a substantial
attention has been focused on the development of biologically important new pyrazole-fused
heterocycles.

On the other hand, benzothiazoles are privileged structures with greatest significance
which are fundamental and important scaffolds in several pharmaceutically active
1
7
18
molecules and exhibit broad range of biological applications such as anti-inflammatory,
analgesic, antimicrobial and anticancer activity. The benzothiazole derivatives exhibited good
DNA binding ability mainly through groove binding mode. 2,5-dibenzothiazolyl furans (III
and IV, Fig. 1) are active against different human cancer cell lines including MCF-7 and
1
9
HeLa exhibiting good DNA minor groove interactions. Considering the mechanism of
action, benzothiazoles can act as inhibitors of tyrosine kinase, selective HIV type-1
2
0
transcriptase and topoisomerases I and II. Whereas beta-naphthol moieties also possess
tremendous applications in synthetic and medicinal chemistry. They exhibit excellent
cytotoxicity towards breast cancer cells and find use as selective estrogen receptor
2
1
modulators in breast cancer therapy. Naphthalene derivatives such as amonafide (V)
exhibited good anticancer activity against a broad spectrum of cell lines, known as DNA
intercalator and topoisomerase inhibitor. Some of these derivatives have reached clinical
trials but not the market due to their dose-limited toxicity. Greater efforts are in progress to
improve the activity and decrease side effects by combining hetero-aromatic rings such as
imidazole, furan and pyrazine to naphthalene nucleus. Herein, pyrazole linked benzothiazole-
β-naphthol derivatives were synthesized and evaluated for their invitro anticancer activity.
2
2
. Results and discussion
.1 Chemistry
Pyrazole linked benzothiazole-β-naphthol derivatives were synthesized as shown in
scheme 1. Condensation of the acetophenones (1a-l) with phenylhydrazine (2) in ethanol
produced the corresponding acetophenone phenylhydrazones. This was followed by
cyclization of the acetophenone phenylhydrazones via the Vilsmeier-Haack reaction resulted
in the formation of 1,3-biphenyl pyrazole carboxaldehydes (3a-l). Finally, via a simple one
pot condensations of subsequent aldehydes 3a-l with β-naphthol and various 2-
aminobenzothiazole in the presence of methanol under reflex conditions afford the required
pyrazole linked benzothiazole-β-naphthol derivatives (4a-ad) in good to excellent yields.

Scheme 1. Reagents and conditions: a) Methanol, 2-napthol, 2-aminobenzothiazole reflux, 5 h.
2
.2. Biology
2
.2.1. Cytotoxicity
All the synthesized compounds (4a-4ad) were evaluated for their cytotoxicity against
selected human cancer cell lines such as A549 (lung cancer), HeLa (cervical cancer) and
MCF-7 (breast cancer) using MTT assay. The cytotoxicity results in comparison to the
positive control doxorubicin and Hoechst 33258 are expressed in IC50 values and are
tabulated in Table 1. Most of the derivatives exhibited considerable cytotoxicity values
against the tested cancer cell lines. Among the series of synthesized derivatives, the most
active compounds are 4j, 4k and 4l with IC values 5.20, 5.54 and 4.63 µM respectively, and
5
0
showed pronounced cytotoxicity on cervical cancer (HeLa) cell line as compared to other cell
lines. Specific killing of cancer cells without affecting normal cell growth is a key safety
feature of cancer chemotherapy. In this connection, compounds 4j, 4k and 4l were evaluated
for their possible cytotoxicity in normal cell line HEK-293 by employing MTT assay. The
assay results suggested that these compounds did not significantly affect the growth of
normal cells (As these compounds showed IC values >45), suggesting that these molecules
5
0
selectively inhibits the growth of cancer cells (Table 1).

a
Table 1. In vitro antiproliferative activity ( IC in µM) of synthesized compounds in human
5
0
cancer cell lines.
Test
IC values (µM)
5
0
b
c
d
e
Compound
A549
MCF 7
HeLa
HEK-293
4
a
32.02 ± 1.09
36.24 ± 1.76
24.81 ± 1.05
18.50 ± 1.02
26.20 ± 1.32
41.25 ± 1.64
20.40 ± 1.77
57.41 ± 1.89
23.10 ± 1.96
34.52 ± 1.10
22.50 ± 2.17
33.60 ± 2.51
28.12 ± 1.16
52.15 ± 1.19
39.30 ± 1.65
88.10 ± 2.34
44.64 ± 1.25
32.15 ± 1.54
42.50 ± 1.18
49.80 ± 1.17
54.14 ± 1.32
33.26 ± 1.76
51.10 ± 2.65
29.02 ± 1.02
57.20 ± 2.42
29.02 ± 2.06
57.41 ± 1.89
45.35 ± 1.64
31.20 ± 1.01
43.85 ± 1.94
48.87 ± 5.26
16.93 ± 0.99
17.34 ± 0.81
17.52 ± 0.85
14.72 ± 0.31
20.65 ± 0.91
13.07 ± 0.81
18.94 ± 0.51
23.66 ± 0.41
13.26 ± 0.91
16.92 ± 1.32
41.83 ± 1.24
26.80 ± 1.02
20.01 ± 1.32
62.58 ± 2.31
40.79 ± 2.31
21.64 ± 0.96
19.73 ± 0.43
36.00 ± 1.06
27.52 ± 1.42
17.47 ± 1.77
36.31 ± 0.84
65.00 ± 2.66
38.60 ± 1.07
20.70 ± 1.23
38.38 ± 1.96
49.73 ± 1.23
22.42 ± 1.46
8.22 ± 0.11
17.28 ± 0.16
14.94 ± 0.15
7.02 ± 0.51
9.34 ± 0.26
6.81 ± 2.08
6.83 ± 3.21
8.05 ± 5.62
6.59 ±1.02
--
--
--
--
--
--
--
--
4
b
4
c
4
d
4
e
4
f
4
4
g
h
4
4
i
j
5.20 ± 1.62
5.54 ± 0.99
4.63 ± 1.29
9.24 ± 0.66
6.14 ± 0.10
6.24 ± 0.48
10.53 ± 0.06
8.29 ± 0.51
30.12 ± 1.03
15.46 ± 0.23
30.02 ± 2.06
36.84 ± 1.06
18.27 ± 0.02
12.54 ± 0.86
12.06 ± 0.99
13.39 ± 1.78
7.04 ± 0.72
7.36 ± 1.29
9.43 ± 0.13
17.81 ± 0.02
55.99 ± 0.95
4
k
46.46 ± 0.85
4
l
58.06 ± 1.06
4
m
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
4
n
o
p
q
4
4
4
4
r
4
s
4
t
4
4
u
v
4
w
4
4
x
y
4
z
4aa
4ab
4ac

4
ad
41.85 ± 1.18
0.8 ± 0.09
ND
41.14 ± 2.42
0.7 ± 0.12
14.67 ± 0.01
0.6 ± 0.24
--
--
--
Doxorubicin
Hoechst 3325
48.1 ± 1.01
23.52 ± 0.32
[
a]
5
0% Inhibitory concentration; values are the mean ± SD of three individual experiments
[
b]
[c]
determined after 48 h of treatment.
Human lung cancer cells.
Human breast
[
d]
[e]
adenocarcinoma. Human epithelial cervical cancer cells. Human embryonic kidney cells.
Fig. 2. SAR analysis of pyrazole linked benzothiazole-β-naphthol derivatives
The cytotoxicity of the compounds is dependent on the nature of substituents on the 1,3-
disubstituted pyrazoles and benzothiazoles. We have varied the substitution pattern of the 2-
aminobenzothoazoles and 1,3-disubstituted pyrazole aldehydes consecutively to get insight
into the SAR (Fig. 2). The cytotoxic activities of derivatives 4a-4ad revealed that electron
rich substitution like methoxy and methyl on benzothiazole ring showed enhanced activity on
HeLa cell line compared to electron deficient substituent such as chloro on this ring. The
replacement of methoxy group (4j and 4l) by chloro 4r and 4u decreased the activity by 7
folds and 6.5 folds respectively. In addition, the influence of 1,3-biphenylpyrazoles on
activity was also examined, electron deficient substitutions like chloro, fluoro, nitro, iodo (4j,
4
k, 4l, 4n and 4o) on phenyl ring increased the activity in comparison to electron rich
substitution like methoxy (4m and 4p). The change of nitro substituent from position R (4f,
2
4
k, 4y and 4z) to R (4a, 4n, 4s and 4aa) did not show much difference in activity on HeLa
3
cell line. Based on the MTT assay results further detailed biological studies such as cell cycle
analysis, DNA binding studies, topoisomerase I inhibition and molecular docking studies
were investigated using the most active derivatives.
2
.2.2. Cell cycle analysis
Many anticancer compounds exert their growth inhibitory effect either by arresting the
cell cycle at a particular checkpoint of cell cycle or by induction of apoptosis or a

combination of both. The screening results revealed that compounds 4j and 4l showed
considerable cytotoxicity against human cervical cancer cell line (HeLa). A cell cycle
analysis study was performed, wherein HeLa cells were treated with these compounds (4j and
4
l) for 48 h at 2.5 µM and 5 µM concentrations, respectively.
A
B
C
D
E
Fig. 3. Flow cytometric analysis in HeLa cells after treatment with compounds 4j and 4l at
.5 and 5 µM concentrations for 48 h. A: Control cells, B: 4j (2.5 µM), C: 4j (5 µM), D: 4l
2.5 µM) and E: 4l (5 µM).
2
(
Table 2. Effect of compounds 4j and 4 l on cell cycle phase distribution in HeLa cells.
Entry
Untreated
4j (2.5 μM)
4j (5 μM)
4l (2.5 μM)
4l (5 μM)
SUB G1
G1
1.69
73.5
5.58
13.9
2.33
66.2
8.89
19.9
2.03
66.5
9.03
20
2.79
57.5
11.5
25.3
3.09
60
S
5.08
28.2
G2/M

The data obtained clearly indicated that these compounds showed cell cycle arrest at G2/M
phase as compared to the untreated control cells. G2/M phase cell cycle arrest is a common
2
2
cellular response to a variety of DNA-damaging agents. These compounds (4j and 4l)
showed 19.9% and 25.3% of cell accumulation in G2/M phase at 2.5 µM concentration,
whereas they exhibited 20% and 28.2% of cell accumulation at 5 µM concentration,
respectively (Fig. 3 and 4; Table 2).
Fig. 4. Graphical representation of FACS analysis
2
4
2
.2.3. DNA binding Studies
To further substantiate the biological activities of these active derivatives (4j, 4k and
l) and to know the nature of interaction with DNA, spectroscopic studies were performed.
.2.3.1. UV-visible studies
In order to investigate the nature of binding of these derivatives to DNA, the UV
absorption spectra of active derivatives 4j, 4k and 4l in the presence and absence of
increasing concentrations of CT-DNA were examined (Fig. 5). On addition of DNA to
derivative solution (4j, 4k and 4l), the absorption band at 336 nm intensity gradually reduced
without exhibiting any shift. Since the absorption band at around 336 nm shows
hypochromicity upon addition of DNA which indicates that the 4j, 4k and 4l derivatives
interact well with DNA. UV-visible titration studies alone were not sufficient to understand
the interaction between 4j, 4k and 4l derivatives and CT-DNA.

Fig. 5. UV-visible spectra indicates the interaction of compounds 4j, 4k and 4l (1 ml of 25.0
o
μM) with the addition of 5 μl of DNA (25.0 μM) each time at 25 C.
2
.2.3.2. Fluorescence spectroscopy
Fluorescence titration is another valuable technique to understand the binding mode of
2
3
small molecules to DNA at comparatively lower concentration. As these derivative
molecules showed fluorescence properties, their interaction with CT-DNA could provide
certain observations regarding the nature of their interactions with DNA. In this study, when
CT-DNA was added to the derivative, the fluorescence emission peak of 4j, 4k and 4l
exhibited hypochromicity. Hypochromicity of the fluorescence emission peak was observed
at 507 nm, 337 nm and 507 nm for the 4j, 4k and 4l derivatives, respectively, indicating that
the fluorescence of each derivative molecule gets quenched due to its interaction with CT-
DNA. In the emission spectra of the derivative, with increasing CT-DNA concentration the
emission intensity is decreased due to self - stacking of some free bases in the derivative
2
4
along the DNA surface. The quenching may be due to energy transfer from excited
derivative molecules to the surrounding water molecules. Fluorescence titration results
indicate that these derivative molecules may bind to the surface of DNA. The fluorescence
spectra recorded on the interaction of compounds 4j, 4k and 4l with CT-DNA is depicted in

Fig. 6. Among 4j, 4k and 4l, the extent of quenching of emission band intensities is more
with 4j, may be due to its higher level of interaction with CT-DNA.
Fig. 6. Fluorescence spectra depicts the interaction of compounds 4j (10 µM), 4k (10 µM)
o
and 4l (10 µM) with increasing concentrations of CT-DNA (multiples of 0.5 µM) at 25 C.
2
.2.3.3. Circular dichroism spectroscopy
The circular dichroism (CD) is a potential technique to study the nucleic acid
conformational changes due to interaction of DNA with molecules or changes in
environmental conditions. The CD spectrum of the calf thymus DNA (CT-DNA) exhibits a
positive band at 275 nm and a negative band at 245 nm due to π–π base stacking and right-
hand helicity. On the addition of 4j, 4k and 4l at a concentration of 10 µM to the same
concentration of CT-DNA solution (DNA: 4j/ 4k/ 4l, 1:1), positive band at 275 nm exhibited
25
slight hypochromicity, which is an indication of melting of the DNA–derivative complex.
On further increasing the concentration of 4j/ 4k/ 4l (DNA: 4j/4k/ 4l derivative, 1:2) the
positive band at 275 nm further decreased in its intensity, indicating further unwinding of
DNA on derivative interaction.

Fig. 7. Circular dichroism spectra represents the CT-DNA (10 μM) at 10 μM and 20 μM of
compounds 4j, 4k and 4l.
The negative band intensity at 245 nm altered with the addition of 4j/ 4k/ 4l
derivatives, indicating the ability of the derivative to bring change in the DNA helix. The
representative figures are shown in Fig. 7. Based on the DNA binding data, it was evident
that these derivatives interacted with DNA and melted the DNA upon interaction.
2
.2.3.4. Viscosity studies
In order to clearly understand the nature of interaction of these potential molecules with
CT DNA, viscosity studies were carried out. Spectroscopic studies indicated that 4j, 4k and
l complexes interacted well with CT-DNA and this may be due to binding to the DNA
4
groove. Relative viscosity of DNA increases when a molecule intercalates with DNA. The
enhancement of viscosity is due to increase in the axial length of DNA helix on molecule’s
intercalation. Hence, intercalation of a compound resulted in an increase of the viscosity of
2
6
DNA solutions however, reduction in the relative viscosity was typically observed with
2
7
covalent DNA binding. Viscosity changes will be less or will not change when molecules
2
8
bind to the surface of DNA. When complex intercalates with DNA, the axial length of DNA
helix will increase to accommodate the binding derivative molecules, which leads to an
increase in viscosity of DNA solution. With the addition of 4j, 4k and 4l derivatives, very

little increase in the viscosity of DNA solution was noticed. As shown in Fig. 8, on
interaction of 4j, 4k and 4l complexes with CT-DNA exhibited a slight increase in the
viscosity which is not as pronounced as observed for classical intercalators. As a control, 5
µM of ethidium bromide (Et Br) and Hoechst 33258 was added to CT-DNA and the viscosity
of DNA was measured under similar conditions. Viscosity has increased continuously with
EtBr and exhibited minimal or no change in viscosity with the addition of Hoechst 33258.
The graphs obtained with 4j, 4k and 4l were observed close to Hoechst 33258 (a well-known
groove binder). The results obtained from viscosity experiments indicated that these
1
/3
molecules may show groove binding to DNA. The graph plotted between (ŋ/ŋo) and
complex/CT-DNA was shown in Fig. 8.
Fig. 8. Graphical representation of viscosity studies of 4j, 4k and 4l (15 µM) compounds
o
with CT-DNA solution (150 µM) at 25 C, EtBr-CT-DNA and Hoechst 33258-CT-DNA
complexes were used as controls.
2
.2.4. Topoisomerase I inhibition study
Topo I has been identified as a potential target of several anticancer drugs used
clinically today. Since Topo I is involved in replication and proliferation process, over-
production of Topo I was observed in cancer cells as compared to normal cells. In general,
Topo I controls the changes in the DNA topology by cutting single stranded DNA and re-
joining the phosphate backbone during the normal cell cycle. Mechanism of Topo I inhibition
was known to occur via two ways. The inhibitors may bind topoisomerase directly or they
may bind to DNA and alter its structure, so that it cannot be recognised by topoisomerases. In
the present study, the potential of the synthesized 4j, 4k and 4l complexes towards moderate
inhibition of Topo I. Though the actual mechanism of Topo I inhibition by these derivatives
is not known, it is speculated that these derivatives has some effect on enzyme inhibition

which may be through DNA interaction, since these derivatives (4j, 4k and 4l) interacted well
with DNA. Cao and co-workers recently reported that camptothecin inhibited Topo I at 100
2
9
µM concentration, whereas harmine derivatives inhibited Topo I at 150 µM. Interestingly,
the present hybrids 4j, 4k and 4l have also shown Topo I inhibition at 100 µM concentration
like Camptothecin (Fig. 9). Since these complexes were found to be efficient in Topo-I
inhibition, they may act as potential anti-cancer molecules.
Fig. 9. Agarose gel picture showing Topoisomerase I inhibition: lane 1: pBR322 DNA alone;
lane 2: pBR322 DNA (0.5 µg) with topoisomerase I (1 unit); lane 3: pBR322 DNA with topo
I and 4j (20 µM); lane 4: pBR322 DNA with topo I and 4k (20 µM); lane 5: pBR322 DNA
with topo I and 4l (20 µM), lane 6: pBR322 DNA with topo I and camptothecin.
2
.3. Molecular docking studies
The protein structure of human DNA topoisomerase I (70 kDa) in complex with the
indenoisoquinoline, which is taken as the reference for defining active site, was selected for
the study. Molecular docking studies were performed by using Glide, keeping the grid box of
size 12 Å from the centroid to cover the entire vicinity of active site. For the validation of
docking protocol, the bound ligand-indenoisoquinoline was re-docked with the active site of
human DNA topoisomerase I which gave GLIDE score of -9.741 and showed hydrogen bond
with Arg364 and Asn722 along with π-stacking interaction with TGP11 and hydrophobic
interactions with Pro431 and Leu721 (Fig. 10A). After validating the docking protocol,
compounds 4j, 4k, 4l, 4n and 4o were docked into the active sites. All the five compounds
showed interaction with DNA as well as hydrogen bonding and hydrophobic interactions
with topoisomerase I. 4n gave the highest GLIDE score of -10.765, which was better than
that of the co-crystallized ligand. The O-atom of naphthol ring formed hydrogen bond with
TGP11, π-stacking interactions with TGP11, dA113 and hydrophobic interactions with
Ala351, Ile424, Tyr426, Ile427, Met428, Pro431 and Leu721 (Fig. 10B). 4j, 4k, 4l and 4o
also showed appreciable interactions with topoisomerase I as well as DNA, as shown in the
Fig. 10.

Fig. 10. Binding pose, hydrogen bonds and hydrophobic interactions of co-crystallized ligand
indenoisoquinoline (A), compounds 4j (B), 4k (C), 4l (D), 4n (E) and 4o (F) with DNA-
–
Topoisomerase I (PDB code: 1SC7). Co-crystallized ligand is indicated in green coloured ball
and stick, whereas compounds 4j, 4k, 4l, 4n and 4o are represented as orange, maroon, red,
blue purple and blue coloured ball and stick models, respectively. The pink colour lines
represent hydrogen bonding.
For better understanding of the DNA-binding nature of our compounds, the same
procedure was carried out for three-dimensional crystal structure of the A-tract DNA
dodecamer D(CGCAAATTTGCG) (PDB code: 264D). Initially the co-crystal, Hoechst
3
3258, was re-docked which showed minor-groove-binding. The N-atom of secondary amine
in 2-aminobenzimidazole ring showed hydrogen bonding with DT8, DA18 and DT7. Its
binding pose is shown in the Fig. 11A. 4l shows the highest docking score and the O-atom of
the naphthol ring shows hydrogen bond with DT19. Compounds 4j, 4k, 4l, 4n and 4o showed
minor-groove-binding (Fig. 11B, C, D, E and F), similar to that of Hoechst 33258. Due to a
difference in the spatial arrangement of the benzthiazol-2-amine and naphthol ring attached to
the tertiary carbon, deviated binding poses of few molecules are observed. However, this
change in spatial arrangement of the groups does not modify the interactions of the molecules
with the DNA, as all the molecules interact with the surface of the DNA by binding to its
minor groove.

Fig. 11. Binding poses of co-crystallized ligand–Hoechist 33258 (A), compounds 4j (B),
k (C), 4l (D), 4n (E) and 4o (F) with DNA (PDB code: 264D). Co-crystallized ligand is
4
indicated in green coloured ball and stick, whereas compounds 4j, 4k, 4l, 4n and 4o are
represented as orange, maroon, red, blue purple and blue coloured ball and stick models,
respectively.
3
. Conclusion
In conclusion, a series of thirty compounds were synthesized using efficient,
environmentally benign method under catalyst-free reaction conditions. The synthesized
derivatives were screened for their antiproliferative activity, among those derivatives 4j, 4k
and 4l showed considerable cytotoxicity against the human cervical cancer cell line (HeLa).
The flow cytometric analysis revealed that these derivatives cause cell cycle arrest at G2/M
phase. Furthermore, they efficiently bind to the DNA and can effectively inhibit the
topoisomerase I. The viscosity and molecular docking studies demonstrated that these
derivatives bind to the minor groove of the DNA. The SAR gives a useful insight for the
better cytotoxicity that these derivatives could be regarded as potential scaffolds which are
useful in the development of newer leads for the treatment of cervical cancer.
4
4
. Experimental data
.1. Chemistry
All chemicals and reagents were purchased from commercial sources and were used
without further purification. Reactions were monitored by TLC on silica gel glass plate
1
13
containing 60 GF-254, and visualization was done by UV light and iodine vapour. H and C
NMR spectra were recorded on Bruker UXNMR/XWIN-NMR (300 MHz) or Innova Varian-
VXR-unity (400, 500 MHz) instruments. Chemical shifts were expressed in parts per million
(in ppm) downfield from TMS expressed as internal standard and coupling constants are

1
expressed in Hz. H NMR spectral data were 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, and number of protons. ESI mass spectra were recorded on a
Micromass Quattro LC using ESI+ software with capillary voltage 3.98 kV and an ESI mode
positive ion trap detector. High resolution mass spectra were recorded on a QSTAR XL
Hybrid MS-MS mass spectrometer. Melting points were determined with an electro thermal
digital melting point apparatus IA9100 and are uncorrected. The purity of the compounds was
determined by HPLC performed on a Shimadzu LC-20AD apparatus equipped with a SPD-
M20A diode array detector and a Shimadzu SIL- 20AC auto injector using C18 column
(waters 5 μM C18, 4.6 mm 250 mm column). Elution conditions: mobile phase A (85%)-
H
acetonitrile; mobile phase B(15%)-water, P 4.3 adjusted with 0.1% formic acid + 10 mmol
NH OAc. The flow rate was 1.0 mL/min and the injection volume was 5 μL at 25 °C and
4
detection at 254 nm.
4
.1.1. General procedure for the synthesis of pyrazole linked benzothiazole-β-naphthol
derivatives (4a-ad)
To the solution of beta-naphthol (1 mmol) in methanol were added respective
3
1
substituted 2-aminobenzothiazole (1 mmol) and the synthesized pyrazole aldehyde (1
mmol). The reaction mixture was refluxed for 5 h. The reaction was monitored by TLC and
the advantage of this method is that a solid product (white ppt) was formed in the round
bottom flask after completion of the reaction. The solid compound was collected by filtration
and purified by column chromatography to obtain pure pyrazole linked benzothiazole-β-
naphthol derivatives in good to excellent yields.
4
.1.2.
yl)methyl)naphthalen-2-ol (4a)
Pale yellow solid; yield 87 %; mp: 230-234 °C; H NMR (500 MHz, DMSO-d ) δ 9.34 (s,
1-((benzo[d]thiazol-2-ylamino)(3-(3-nitrophenyl)-1-phenyl-1H-pyrazol-4-
1
6
1
H), 8.07 (s, 1H), 7.28 (s, 2H), 6.90 (d, J = 7.9 Hz, 1H), 6.85 (dd, J = 8.4, 3.8 Hz, 3H), 6.79
d, J = 8.0 Hz, 2H), 6.75 (d, J = 7.8 Hz, 1H), 6.59 (t, J = 7.9 Hz, 2H), 6.48 (dd, J = 16.9, 7.9
Hz, 2H), 6.42 (d, J = 7.4 Hz, 1H), 6.40 – 6.37 (m, 1H), 6.34 (s, 1H), 6.34 – 6.30 (m, 2H),
(
1
3
6
1
1
1
.28 (d, J = 7.9 Hz, 2H), 6.13 - 6.09 (m, 1H); C NMR (125 MHz, DMSO-d ) δ 162.07,
6
52.51, 151.34, 148.66, 144.31, 137.51, 131.08, 130.26, 129.60, 127.65, 127.40, 126.61,
26.18, 126.01, 125.08, 124.31, 124.14, 121.17, 120.44, 116.89, 116.53, 116.33, 111.04,
03.53, 77.44, 77.00, 76.56, 53.66, 45.39, 38.45, 38.17, 37.90, 37.62, 37.34, 37.09, 36.71;

+
HRMS (ESI): calcd for C H N O S [M+H] 570.1594, found 570.1596; HPLC: t 3.88 min,
33
24
5
3
R
purity 92.8%.
4
.1.2.1.
1-((benzo[d]thiazol-2-ylamino)(1,3-diphenyl-1H-pyrazol-4-
yl)methyl)naphthalen-2-ol (4b)
1
White solid; yield 94 %; mp: 209-213 °C; H NMR (500 MHz, DMSO-d ) δ 9.31 (s, 1H),
6
8
8
2
.08 (s, 1H), 7.28 (s, 2H), 6.91 (d, J = 7.9 Hz, 1H), 6.88 (dd, J = 8.4, 3.8 Hz, 3H), 6.72 (d, J =
.0 Hz, 2H), 6.70 (d, J = 7.8 Hz, 1H), 6.52 (t, J = 7.9 Hz, 2H), 6.43 (dd, J = 16.9, 7.9 Hz,
H), 6.42 (d, J = 7.4 Hz, 1H), 6.39 – 6.34 (m, 2H), 6.30 (s, 1H), 6.13 – 6.08 (m, 2H), 6.05 (d,
1
3
J = 7.9 Hz, 2H), 6.01 – 5.97 (m, 1H); C NMR (125 MHz, DMSO-d ) δ 164.49, 152.94,
6
1
1
4
49.49, 147.80, 146.09, 139.17, 138.71, 131.67, 130.26, 129.77, 129.26, 128.29, 128.10,
26.31, 126.12, 125.74, 123.27, 122.62, 121.95, 120.41, 118.07, 117.43, 117.07, 48.22,
+
6.60, 30.30, 20.32; HRMS (ESI): calcd for C H N OS [M+H] 525.1744, found 525.1746;
3
3
25
4
HPLC: t 4.62 min, purity 88.7%.
R
4
.1.2.2.
yl)methyl)naphthalen-2-ol (4c)
Off-white solid; yield 90 %; mp: 206-210 °C; H NMR (300 MHz, CDCl ) δ 10.01 (s, 1H),
1-((benzo[d]thiazol-2-ylamino)(3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
1
3
9
.08 (s, 1H), 8.22 (s, 1H), 8.02 (s, 1H), 7.94 (d, J = 8.1 Hz, 1H), 7.87 (s, 1H), 7.68 (d, J = 7.7
Hz, 4H), 7.63 (d, J = 8.8 Hz, 2H), 7.53 (t, J = 7.7 Hz, 4H), 7.48 – 7.37 (m, 4H), 7.02 (d, J =
1
3
8
.4 Hz, 1H), 6.94 (t, J = 7.9 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 3.86 (s, 3H); C NMR (75
MHz, DMSO-d ) δ 162.22, 152.67, 151.48, 148.84, 144.49, 137.67, 131.22, 130.39, 129.71,
6
1
1
5
27.81, 127.56, 126.77, 126.37, 126.17, 125.16, 124.52, 124.30, 121.33, 120.60, 117.05,
+
16.73, 116.49, 111.20, 103.63, 53.82, 45.55; HRMS (ESI): calcd for C H N O S [M+H]
3
4
27
4
2
55.1849, found 555.1851; HPLC: t 3.75 min, purity 96.3%.
R
4
.1.2.3.
1-((benzo[d]thiazol-2-ylamino)(1-phenyl-3-(m-tolyl)-1H-pyrazol-4-
yl)methyl)naphthalen-2-ol (4d)
White solid; yield 82 %; mp: 245-238 °C; H NMR (300 MHz, CDCl +DMSO-d ) δ 8.79 (s,
1
3
6
1
7
2
6
1
1
H), 8.29 (d, J = 8.5 Hz, 2H), 8.09 (s, 1H), 8.04 – 7.93 (m, 2H), 7.88 (d, J = 7.2 Hz, 1H),
.76 (d, J = 7.7 Hz, 1H), 7.65 (d, J = 7.8 Hz, 5H), 7.55 (d, J = 8.9 Hz, 4H), 7.49 – 7.34 (m,
H), 7.28 (dd, J = 11.5, 5.6 Hz, 1H), 7.18 (t, J = 7.3 Hz, 1H), 7.10 (d, J = 8.8 Hz, 1H), 7.03 –
1
3
.95 (m, 1H), 2.33 (s, 3H); C NMR (75 MHz, DMSO-d ) δ 164.36, 153.23, 149.75, 148.12,
6
46.34, 141.04, 139.44, 138.95, 131.66, 131.38, 129.39, 128.73, 128.25, 126.41, 125.83,
23.38, 122.72, 122.14, 121.62, 118.52, 118.21, 117.38, 113.15, 108.27, 55.74, 48.44, 46.93,

+
3
5
0.46; HRMS (ESI): calcd for C H N OS [M+H] 539.1900, found 539.1902; HPLC: t
34 27 4 R
.51 min, purity 91.8%.
4
.1.2.4. 1-((benzo[d]thiazol-2-ylamino)(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)naphthalen-2-ol (4e)
White solid; yield 86 %; mp: 221-224 °C; H NMR (300 MHz, CDCl ) δ 10.01 (s, 1H), 9.08
1
3
(s, 1H), 8.21 (s, 1H), 8.03 (s, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.87 (s, 1H), 7.62 (d, J = 7.7 Hz,
4
H), 7.63 (d, J = 8.8 Hz, 2H), 7.53 (t, J = 7.7 Hz, 4H), 7.48 – 7.37 (m, 4H), 7.02 (d, J = 8.4
1
3
Hz, 1H), 6.94 (t, J = 7.9 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H), 3.86 (s, 3H); C NMR (75 MHz,
DMSO-d ) δ 162.22, 152.65, 151.48, 148.84, 144.43, 137.67, 131.22, 130.33, 129.76, 127.81,
6
1
1
5
27.56, 126.77, 126.37, 126.17, 125.16, 124.53, 124.31, 121.33, 120.60, 117.05, 116.73,
+
16.49, 111.21, 103.69, 53.82, 45.55; HRMS (ESI): calcd for C H N O S [M+H]
3
4
27
4
2
55.1846, found 555.1848; HPLC: t 4.35 min, purity 95.2%.
R
4
.1.2.5.
1-((benzo[d]thiazol-2-ylamino)(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)naphthalen-2-ol (4f)
Yellow solid; yield 84 %; mp: 209-212 °C; H NMR (500 MHz, DMSO-d ) δ 9.21 (s, 1H),
1
6
8
8
2
.01 (s, 1H), 7.27 (s, 2H), 6.91 (d, J = 7.9 Hz, 1H), 6.82 (dd, J = 8.4, 3.8 Hz, 3H), 6.79 (d, J =
.0 Hz, 2H), 6.71 (d, J = 7.8 Hz, 1H), 6.59 (t, J = 7.9 Hz, 2H), 6.49 (dd, J = 16.9, 7.9 Hz,
H), 6.40 (d, J = 7.4 Hz, 1H), 6.39 – 6.24 (m, 1H), 6.20 (s, 1H), 6.18 – 6.14 (m, 2H), 6.09 (d,
1
3
J = 7.9 Hz, 2H), 6.05 – 6.01 (m, 1H); C NMR (125 MHz, DMSO-d ) δ 162.06, 152.51,
6
1
1
7
51.32, 148.63, 144.37, 137.51, 131.06, 130.23, 129.62, 127.65, 127.40, 126.61, 126.20,
26.04, 125.09, 124.31, 124.14, 121.17, 120.44, 116.89, 116.57, 116.34, 111.04, 103.53,
7.44, 77.06, 76.56, 53.66, 45.39, 38.45, 38.17, 37.90, 37.62, 37.34, 37.06, 36.78; HRMS
+
(
ESI): calcd for C H N O S [M+H] 570.1594, found 570.1596; HPLC: t 4.57 min, purity
3
3
24
5
3
R
9
2.8%.
4
.1.2.6.
1-((benzo[d]thiazol-2-ylamino)(3-(3-chlorophenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)naphthalen-2-ol (4g)
White solid; yield 80 %; mp: 205-209 °C; H NMR (300 MHz, CDCl ) δ 10.01 (s, 1H), 9.08
1
3
(s, 1H), 8.22 (s, 1H), 8.03 (s, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.86 (s, 1H), 7.69 (d, J = 7.7 Hz,
4
H), 7.63 (d, J = 8.8 Hz, 2H), 7.53 (t, J = 7.7 Hz, 4H), 7.48 – 7.37 (m, 3H), 7.02 (d, J = 8.4
13
Hz, 1H), 6.94 (t, J = 7.9 Hz, 1H), 6.86 (d, J = 8.3 Hz, 2H); C NMR (75 MHz, DMSO-d ) δ
6
1
1
62.22, 152.67, 151.48, 148.83, 144.47, 137.67, 131.22, 130.39, 129.76, 127.81, 127.56,
26.77, 126.37, 126.17, 125.16, 124.53, 124.33, 121.39, 120.61, 117.05, 116.73, 116.49,

+
1
11.24, 103.69; HRMS (ESI): calcd for C H ClN OS [M+H] 559.1354, found 559.1356;
33 24 4
HPLC: t 4.83 min, purity 92.8%.
R
4
.1.2.7.
yl)methyl)naphthalen-2-ol (4h)
Off white solid; yield 78 %; mp: 207-211 °C; H NMR (300 MHz, CDCl +DMSO-d ) δ 8.75
1-((benzo[d]thiazol-2-ylamino)(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-
1
3
6
(s, 1H), 8.29 (d, J = 19.5 Hz, 2H), 8.09 (s, 1H), 8.04 – 7.94 (m, 2H), 7.88 (d, J = 7.2 Hz, 1H),
7
1
6
1
1
.76 (d, J = 7.7 Hz, 1H), 7.63 (d, J = 7.8 Hz, 5H), 7.55 (d, J = 8.9 Hz, 4H), 7.49 – 7.34 (m,
H), 7.28 (dd, J = 11.5, 5.6 Hz, 1H), 7.18 (t, J = 7.3 Hz, 1H), 7.10 (d, J = 8.8 Hz, 1H), 7.03 –
1
3
.94 (m, 1H), 2.33 (s, 3H); C NMR (75 MHz, DMSO-d ) δ 162.27, 152.67, 151.48, 148.81,
6
44.47, 137.67, 131.22, 130.39, 129.76, 127.83, 127.54, 126.79, 126.37, 126.17, 125.16,
24.53, 124.30, 121.33, 120.60, 117.05, 116.73, 116.49, 111.23, 103.69, 53.82, 23.55; HRMS
+
(
ESI): calcd for C H N OS [M+H] 539.1896, found 539.1898; HPLC: t 5.30 min, purity
3
4
27
4
R
9
0.4%.
4
.1.2.8.
1-((benzo[d]thiazol-2-ylamino)(3-(4-iodophenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)naphthalen-2-ol (4i)
White solid; yield 80 %; mp: 214-219 °C; H NMR (300 MHz, DMSO-d ) δ 10.49 (s, 1H),
1
6
8
.92 (s, 1H), 8.25 (d, J = 15.7 Hz, 1H), 8.05 (d, J = 8.3 Hz, 1H), 7.86 – 7.66 (m, 6H), 7.61 (d,
1
3
J = 6.9 Hz, 2H), 7.47 (t, J = 7.6 Hz, 3H), 7.32 (m, 7H), 7.01 (t, J = 7.4 Hz, 1H); C NMR (75
MHz, DMSO-d ) δ 162.22, 152.67, 151.48, 148.84, 144.47, 137.65, 131.22, 130.39, 129.76,
6
1
1
27.81, 127.56, 126.77, 126.37, 126.17, 125.16, 124.63, 124.15, 121.33, 120.61, 117.05,
+
16.73, 116.49, 111.20, 103.69; HRMS (ESI): calcd for C H IN OS [M+H] 651.0710,
3
3
24
4
found 651.0710; HPLC: t 5.41 min, purity 93.2%.
R
4
.1.2.9. 1-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)((6-methoxybenzo[d]thiazol-2-
yl)amino)methyl)naphthalen-2-ol (4j)
White solid yield 80 %; mp: 207-211 °C; H NMR (300 MHz, CDCl +DMSO) δ 10.79 (s,
1
3
1
H), 8.20 (s, 1H), 7.93 (d, J = 7.6 Hz, 1H), 7.72 (dd, J = 16.9, 8.2 Hz, 4H), 7.53 (d, J = 7.9
Hz, 1H), 7.50 – 7.38 (m, 5H), 7.30 – 6.98 (m, 8H), 6.82 (dd, J = 8.8, 2.5 Hz, 1H), 3.76 (s,
1
3
3
1
1
H); C NMR (75 MHz, DMSO-d ) δ 162.59, 151.51, 148.06, 146.41, 144.63, 139.32,
6
37.73, 137.23, 129.67, 127.67, 126.53, 124.74, 124.11, 121.67, 121.01, 120.43, 119.91,
16.81, 116.51, 115.67, 111.43, 106.56, 54.03, 46.73, 45.26, 28.74; HRMS (ESI): calcd for
+
C H ClN O S [M+H] 589.0934, found 589.0936; HPLC: t 5.53 min, purity 98.3%
34
26
4
2
R
.

4
.1.2.10.
1-(((6-methoxybenzo[d]thiazol-2-yl)amino)(3-(4-nitrophenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-ol (4k)
1
Yellow solid; yield 82 %; mp: 230-234 °C; H NMR (300 MHz, CDCl +DMSO) δ 10.83 (s,
3
1
H), 8.23 (s, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.76 (dd, J = 16.9, 8.2 Hz, 4H), 7.57 (d, J = 7.9
Hz, 1H), 7.53 – 7.42 (m, 5H), 7.34 – 7.01 (m, 8H), 6.86 (dd, J = 8.8, 2.5 Hz, 1H), 3.79 (s,
1
3
3
1
1
4
H); C NMR (75 MHz, DMSO-d ) δ 161.97, 150.90, 147.45, 145.79, 144.01, 138.71,
6
37.12, 136.62, 129.33, 129.06, 127.06, 126.40, 125.92, 124.13, 123.50, 121.06, 120.40,
19.81, 119.29, 116.19, 115.90, 115.06, 110.82, 105.95, 76.67, 76.41, 76.14, 53.41, 46.12,
4.65, 37.52, 37.35, 37.19, 37.02, 36.85, 36.69, 36.52, 28.13; HRMS (ESI): calcd for
+
C H N O S [M] 599.0856, found 599.0858; HPLC: t 4.87 min, purity 96.8%.
34
25
5
4
R
4
.1.2.11. 1-((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)((6-methoxybenzo[d]thiazol-2-
yl)amino)methyl)naphthalen-2-ol (4l)
White solid; yield 76 %; mp: 217-219 °C; H NMR (300 MHz, CDCl +DMSO) δ 10.49 (s,
1
3
1
7
2
1
1
1
H), 8.10 (s, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.65 (dd, J = 16.7, 11.2 Hz, 5H), 7.57 (s, 1H),
.41 (dd, J = 7.0, 4.8 Hz, 4H), 7.30 – 7.19 (m, 5H), 7.16 (d, J = 8.8 Hz, 1H), 7.06 – 6.96 (m,
1
3
H), 6.84 (dd, J = 8.8, 2.6 Hz, 1H), 3.78 (s, 3H); C NMR (75 MHz, DMSO-d ) δ 162.73,
6
51.65, 148.20, 146.55, 144.77, 139.46, 137.87, 137.37, 130.08, 129.81, 127.81, 127.16,
26.67, 124.88, 124.25, 121.81, 121.15, 120.57, 120.05, 116.95, 116.65, 115.81, 111.57,
06.70, 77.42, 77.16, 76.90, 54.17, 46.87, 45.40, 28.88; HRMS (ESI): calcd for
+
C H FN O S [M+H] 573.1767, found 573.1769; HPLC: t 5.63 min, purity 97.6%
34
26
4
2
R
.
4
.1.2.12. 1-(((6-methoxybenzo[d]thiazol-2-yl)amino)(3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-ol (4m)
Light white solid; yield 84 %; mp: 214-218 °C; H NMR (500 MHz, DMSO-d ) δ 8.85 (s,
1
6
1
2
H), 8.24 (s, 1H), 8.00 (s, 1H), 7.80 – 7.67 (m, 7H), 7.65 (d, J = 8.8 Hz, 2H), 7.27 – 7.19 (m,
13
H), 7.26 – 7.19 (m, 4H), 6.93 (t, J = 7.9 Hz, 3H), 6.79 (d, J = 7.8 Hz, 1H), 3.85 (s, 6H); C
NMR (125 MHz, DMSO-d ) δ 162.22, 152.69, 151.42, 148.81, 144.47, 137.67, 131.22,
6
1
1
30.39, 129.76, 127.81, 127.56, 126.77, 126.37, 126.19, 125.14, 124.53, 124.31, 121.39,
20.60, 117.05, 116.73, 116.49, 111.22, 103.69, 54.82, 52.45; HRMS (ESI): calcd for
+
C H N O S [M+H] 585.1955, found 585.1957; HPLC: t 5.36 min, purity 96.3%
35
29
4
3
R
.

4
.1.2.13.
1-(((6-methoxybenzo[d]thiazol-2-yl)amino)(3-(3-nitrophenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-ol (4n)
1
Yellow solid; yield 81 %; mp: 220-224 °C; H NMR (300 MHz, CDCl +DMSO-d ) δ 10.51
3
6
(s, 1H), 8.10 (s, 1H), 7.77 (s, 1H), 7.65 (d, J = 5.5 Hz, 4H), 7.47 – 7.40 (m, 5H), 7.39 (d, J =
6
1
1
1
.6 Hz, 3H), 7.27 (s, 2H), 7.17 (s, 1H), 7.04 (d, J = 2.5 Hz, 2H), 7.00 (s, 1H), 6.86 – 6.82 (m,
1
3
H), 3.78 (s, 3H); C NMR (75 MHz, DMSO-d ) δ 162.76, 151.65, 148.20, 146.55, 144.77,
6
39.46, 137.87, 137.31, 130.08, 129.81, 127.81, 127.16, 126.67, 124.88, 124.25, 121.81,
21.15, 120.57, 120.05, 116.95, 116.65, 115.81, 111.57, 106.70, 52.17, 46.67, 54.44, 48.88;
+
HRMS (ESI): calcd for C H N O S [M+H] 600.1700, found 600.1702; HPLC: t 4.93 min,
34
26
5
4
R
purity 98.0%.
4
.1.2.14. 1-((3-(3-iodophenyl)-1-phenyl-1H-pyrazol-4-yl)((6-methoxybenzo[d]thiazol-2-
yl)amino)methyl)naphthalen-2-ol (4o)
Yellow solid; yield 78 %; mp: 213-217 °C; H NMR (300 MHz, CDCl +DMSO-d ) δ 8.27 (s,
1
3
6
1
H), 7.93 (d, J = 8.3 Hz, 1H), 7.67 (t, J = 9.4 Hz, 5H), 7.60 – 7.53 (m, 3H), 7.43 (d, J = 7.6
Hz, 1H), 7.40 (d, J = 2.9 Hz, 1H), 7.37 (s, 1H), 7.25 (dd, J = 7.8, 5.7 Hz, 2H), 7.18 (d, J = 6.9
Hz, 1H), 7.14 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H), 7.03 (s, 1H), 6.91 (t, J = 8.4 Hz,
1
3
1
1
1
1
H), 6.83 (dd, J = 8.8, 2.6 Hz, 1H), 3.78 (s, 3H); C NMR (75 MHz, DMSO-d ) δ 162.59,
6
51.51, 148.06, 146.41, 144.63, 139.32, 137.75, 137.43, 129.94, 129.69, 127.61, 127.02,
26.53, 124.74, 124.11, 121.67, 121.01, 120.43, 119.91, 116.81, 116.51, 115.67, 111.43,
+
06.56, 54.03, 46.73, 45.26, 28.74; HRMS (ESI): calcd for C H IN O S [M+H] 681.0814,
3
4
26
4
2
found 681.0816; HPLC: t 5.15 min, purity 90.6%.
R
4
.1.2.15. 1-(((6-methoxybenzo[d]thiazol-2-yl)amino)(3-(3-methoxyphenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-ol (4p)
Pale yellow solid; yield 80 %; mp: 219-222 °C; H NMR (500 MHz, DMSO-d ) δ 10.41 (s,
1
6
1
7
H), 8.85 (s, 1H), 8.23 (s, 1H), 8.01 (s, 1H), 7.80–7.67 (m, 7H), 7.27 – 7.19 (m, 2H), 7.26 –
1
3
.19 (m, 4H), 6.94 (t, J = 7.9 Hz, 3H), 6.79 (d, J = 7.8 Hz, 2H), 3.85 (s, 6H); C NMR (125
MHz, DMSO-d ) δ 162.22, 152.61, 151.48, 148.84, 144.47, 137.67, 131.22, 130.33, 129.76,
6
1
1
5
27.81, 127.56, 126.77, 126.37, 126.17, 125.16, 124.53, 124.30, 121.33, 120.61, 117.05,
+
16.73, 116.49, 111.21, 103.69, 54.82, 52.45; HRMS (ESI): calcd for C H N O S [M+H]
3
5
29
4
3
85.1942, found 585.1945; HPLC: t 5.61 min, purity 95.2%.
R

4
.1.2.16.
yl)amino)methyl)naphthalen-2-ol (4q)
Yellow solid; yield 86 %; mp: 219-223 °C; H NMR (300 MHz, DMSO-d ) δ 10.53 (s, 1H),
1-((1,3-diphenyl-1H-pyrazol-4-yl)((6-methoxybenzo[d]thiazol-2-
1
6
8
.92 (s, 1H), 8.24 (d, J = 15.7 Hz, 2H), 8.05 (d, J = 18.3 Hz, 1H), 7.86 – 7.65 (m, 6H), 7.61
(d, J = 6.9 Hz, 2H), 7.46 (t, J = 7.6 Hz, 3H), 7.32 (dd, J = 21.3, 12.6 Hz, 9H), 7.01 (t, J = 7.4
1
3
Hz, 1H); C NMR (75 MHz, DMSO-d ) δ 162.21, 152.67, 151.48, 148.84, 144.47, 137.67,
6
1
1
31.22, 130.39, 129.76, 127.81, 127.56, 126.77, 126.37, 126.17, 125.16, 124.53, 124.34,
21.29, 120.60, 117.05, 116.73, 116.49, 111.21, 103.69, 53.12, 45.15; HRMS (ESI): calcd for
+
C H N O S [M+H] 555.1849, found 555.1852; HPLC: t 3.86 min, purity 90.4%.
34
27
4
2
R
4
.1.2.17.
1-(((6-chlorobenzo[d]thiazol-2-yl)amino)(3-(4-fluorophenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-o (4r)
1
White solid; yield 80 %; mp: 210-213 °C; H NMR (300 MHz, DMSO-d ) δ 9.08 (s, 1H),
6
8
.12 (s, 1H), 6.93 (d, J = 7.9 Hz, 1H), 6.62 – 6.53 (m, 6H), 6.44 (d, J = 7.8 Hz, 1H), 6.30 (t, J
13
=
7.9 Hz, 2H), 6.06 (dd, J = 15.2, 8.1 Hz, 9H), 5.80 (t, J = 7.9 Hz, 1H); C NMR (75 MHz,
DMSO-d ) δ 162.27, 150.66, 147.24, 145.35, 143.47, 139.46, 137.87, 137.37, 130.08, 129.81,
6
1
1
5
27.81, 127.16, 126.67, 124.82, 124.25, 121.81, 121.15, 120.57, 120.05, 116.95, 116.65,
+
15.81, 111.57, 106.71, 52.17, 46.87; HRMS (ESI): calcd for C H ClFN OS [M+H]
3
3
23
4
77.1260, found 577.1260; HPLC: t 4.04 min, purity 87.2%.
R
4
.1.2.18.
1-(((6-chlorobenzo[d]thiazol-2-yl)amino)(3-(3-nitrophenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-ol (4s)
1
Yellow solid; yield 86 %; mp: 219-223 °C; H NMR (300 MHz, CDCl +DMSO-d ) δ 10.42
3
6
(s, 1H), 8.10 (s, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.64 (dd, J = 16.7, 11.2 Hz, 4H), 7.53 (s, 3H),
7
1
.41 (dd, J = 7.0, 4.8 Hz, 2H), 7.30 – 7.19 (m, 4H), 7.16 (d, J = 8.8 Hz, 1H), 7.06 – 6.96 (m,
1
3
H), 6.84 (dd, J = 8.8, 2.6 Hz, 1H), 3.76 (d, J = 10.0 Hz, 3H); C NMR (75 MHz, DMSO-d )
6
δ 161.13, 151.05, 147.10, 145.25, 144.07, 139.16, 137.27, 137.17, 130.18, 129.11, 127.21,
1
1
6
27.06, 126.17, 124.38, 124.25, 121.81, 121.15, 120.57, 120.25, 116.55, 116.65, 115.81,
+
11.27, 106.34, 52.07, 46.17, 54.31, 48.58; HRMS (ESI): calcd for C H ClN O S [M+H]
3
3
23
5
3
04.1205, found 604.1207; HPLC: t 4.88 min, purity 94.3%.
R
4
.1.2.19.
1-(((6-chlorobenzo[d]thiazol-2-yl)amino)(3-(3-methoxyphenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-ol (4t)
1
Cream solid; yield 83 %; mp: 217-221 °C; H NMR (400 MHz, CDCl ) δ 10.01 (s, 1H), 9.08
3
(s, 1H), 8.22 (s, 1H), 8.02 (s, 1H), 7.94 (d, J = 8.1 Hz, 1H), 7.87 (s, 1H), 7.69 (d, J = 7.7 Hz,

4
H), 7.63 (d, J = 8.8 Hz, 1H), 7.53 (t, J = 7.7 Hz, 4H), 7.48 – 7.37 (m, 4H), 7.02 (d, J = 8.4
1
3
Hz, 1H), 6.94 (t, J = 7.9 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H), 3.86 (s, 3H); C NMR (100 MHz,
DMSO-d ) δ 162.71, 151.64, 148.20, 146.59, 144.77, 139.46, 137.87, 137.37, 130.01, 129.81,
6
1
1
27.81, 127.16, 126.67, 124.83, 124.29, 121.81, 121.15, 120.55, 120.02, 116.95, 116.65,
15.81, 111.57, 106.70, 52.17, 46.67, 54.41, 48.88; HRMS (ESI): calcd for C H ClN O S
3
4
26
4
2
+
[
M+H] 589.1460, found 589.1462; HPLC: t 3.81 min, purity 91.5%.
R
4
.1.2.20.
1-(((6-chlorobenzo[d]thiazol-2-yl)amino)(3-(4-chlorophenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-ol (4u)
1
White solid; yield 74 %; mp: 219-222 °C; H NMR (300 MHz, CDCl +DMSO-d ) δ 8.17 (s,
3
6
1
H), 7.81 (d, J = 8.3 Hz, 1H), 7.67 (t, J = 9.4 Hz, 5H), 7.60 – 7.52 (m, 2H), 7.43 (d, J = 7.6
Hz, 1H), 7.40 (d, J = 2.9 Hz, 1H), 7.37 (s, 1H), 7.25 (dd, J = 7.8, 5.7 Hz, 3H), 7.19 (d, J = 6.9
Hz, 1H), 7.14 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H), 7.03 (s, 1H), 6.91 (t, J = 8.4 Hz,
1
3
1
1
1
4
5
H), 6.84 (dd, J = 8.8, 2.6 Hz, 2H); C NMR (75 MHz, DMSO-d ) δ 162.20, 152.65, 151.46,
6
48.82, 144.45, 137.65, 131.21, 130.37, 129.74, 127.79, 127.54, 126.75, 126.35, 126.15,
25.14, 124.51, 124.28, 121.31, 120.58, 117.09, 116.71, 116.26, 111.18, 103.67, 53.80,
+
5.53; HRMS (ESI): calcd for C H Cl N OS [M+H] 593.0964, found 593.0964; HPLC: t
3
3
23
2
4
R
.19 min, purity 90.4%.
4
.1.2.21. 1-(((6-chlorobenzo[d]thiazol-2-yl)amino)(3-(3-chlorophenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-ol (4v)
1
White solid; yield 70 %; mp: 220-223 °C; H NMR (300 MHz, CDCl ) δ 8.19 (s, 1H), 7.81
3
(d, J = 8.3 Hz, 1H), 7.64 (t, J = 9.4 Hz, 4H), 7.60 – 7.53 (m, 3H), 7.41 (d, J = 7.6 Hz, 1H),
7
7
.40 (d, J = 2.9 Hz, 1H), 7.32 (s, 1H), 7.25 (dd, J = 7.8, 5.7 Hz, 3H), 7.19 (d, J = 6.9 Hz, 1H),
.14 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H), 7.03 (s, 1H), 6.91 (t, J = 8.4 Hz, 1H), 6.84
1
3
(
dd, J = 8.8, 2.6 Hz, 2H); C NMR (75 MHz, CDCl ) δ 165.62, 153.01, 150.33, 148.54,
3
1
1
38.96, 132.59, 131.59, 131.47, 129.26, 128.09, 127.87, 127.68, 125.99, 125.32, 125.31,
22.01, 121.28, 121.09, 119.13, 118.22, 117.28, 48.16, 46.56, 30.19; HRMS (ESI): calcd for
+
C H Cl N OS [M+H] 593.0956, found 593.0958; HPLC: t 4.34 min, purity 85.7%.
33
23
2
4
R
4
.1.2.22.
1-(((6-chlorobenzo[d]thiazol-2-yl)amino)(1,3-diphenyl-1H-pyrazol-4-
yl)methyl)naphthalen-2-ol (4w)
White solid; yield 81 %; mp: 220-223 °C; H NMR (300 MHz, DMSO-d ) δ 10.51 (s, 1H),
1
6
8
.92 (s, 1H), 8.24 (d, J = 15.7 Hz, 1H), 8.05 (d, J = 18.3 Hz, 1H), 7.86 – 7.65 (m, 6H), 7.61
1
3
(
d, J = 6.9 Hz, 2H), 7.47 (t, J = 7.6 Hz, 3H), 7.32 (m, 7H), 7.01 (t, J = 7.4 Hz, 1H); C NMR

(
75 MHz, DMSO-d ) δ 162.06, 152.51, 151.32, 148.68, 144.31, 137.51, 131.06, 130.23,
6
1
1
3
29.60, 127.65, 127.41, 126.61, 126.21, 126.01, 125.07, 124.37, 124.14, 121.17, 120.44,
16.89, 116.57, 116.33, 111.04, 103.53, 77.34, 77.21, 76.56, 53.66, 45.39, 38.45, 38.19,
+
7.92, 37.62, 37.34, 37.06, 36.78; HRMS (ESI): calcd for C H ClN OS [M+H] 559.1354,
3
3
24
4
found 559.1356; HPLC: t 4.29 min, purity 88.4%.
R
4
.1.2.23.
1-(((6-chlorobenzo[d]thiazol-2-yl)amino)(3-(4-Iodophenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-ol (4x)
1
Cream solid; yield 79 %; mp: 211-214 °C; H NMR (300 MHz, DMSO-d ) δ 9.08 (s, 1H),
6
8
.12 (s, 1H), 6.93 (d, J = 7.9 Hz, 1H), 6.62 – 6.53 (m, 6H), 6.44 (d, J = 7.8 Hz, 1H), 6.30 (t, J
1
3
=
7.9 Hz, 2H), 6.06 (dd, J = 15.2, 8.1 Hz, 9H), 5.81 (t, J = 7.9 Hz, 1H); C NMR (75 MHz,
CDCl ) δ 165.62, 153.01, 150.33, 148.54, 138.96, 132.53, 131.59, 131.47, 129.26, 128.09,
3
1
4
6
27.87, 127.68, 125.99, 125.72, 125.31, 122.01, 121.18, 121.09, 119.53, 118.21, 117.88,
+
8.26, 46.76, 30.34; HRMS (ESI): calcd for C H ClIN OS [M+H] 685.0320, found
33
23
4
85.0322; HPLC: t 3.92 min, purity 85.7%.
R
4
.1.2.24. 1-(((6-chlorobenzo[d]thiazol-2-yl)amino)(3-(4-nitrophenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-ol (4y)
1
Yellow solid; yield 82 %; mp: 200-204 °C; H NMR (300 MHz, CDCl +DMSO-d ) δ 9.01 (s,
3
6
1
2
H), 8.34 (s, 1H), 8.06 (d, J = 8.2 Hz, 2H), 7.92 (d, J = 23.3 Hz, 4H), 7.75 (d, J = 8.0 Hz,
1
3
H), 7.61 (d, J = 8.1 Hz, 2H), 7.46 (s, 3H), 7.31 – 7.16 (m, 6H), 6.98 (t, J = 7.9 Hz, 1H); C
NMR (75 MHz, DMSO-d ) δ 162.12, 152.25, 151.26, 148.02, 144.45, 137.15, 131.21,
6
1
1
30.27, 129.14, 127.19, 127.04, 126.75, 126.35, 126.22, 125.14, 124.51, 124.28, 121.31,
20.56, 117.03, 116.71, 116.42, 111.14, 103.61, 53.74, 45.02; HRMS (ESI): calcd for
+
C H ClN O S [M+H] 604.1205, found 604.1205; HPLC: t 4.13 min, purity 92.8%.
33
23
5
3
R
4
.1.2.25.
1-(((6-methylbenzo[d]thiazol-2-yl)amino)(3-(4-nitrophenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-ol (4z)
1
Yellow solid; yield 82 %; mp: 212-217 °C; H NMR (300 MHz, CDCl +DMSO-d ) δ 8.17 (s,
3
6
1
H), 7.81 (d, J = 8.3 Hz, 1H), 7.67 (t, J = 9.4 Hz, 5H), 7.60 – 7.53 (m, 3H), 7.43 (d, J = 7.6
Hz, 1H), 7.40 (d, J = 2.9 Hz, 1H), 7.37 (s, 1H), 7.25 (dd, J = 7.8, 5.7 Hz, 3H), 7.19 (d, J = 6.9
Hz, 1H), 7.14 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H), 7.03 (s, 1H), 6.91 (t, J = 8.4 Hz,
1
3
1
1
1
H), 6.84 (dd, J = 8.8, 2.6 Hz, 1H), 2.43 (s, 3H); C NMR (75 MHz, DMSO-d ) δ 164.30,
6
53.23, 149.71, 148.19, 146.34, 141.04, 139.44, 138.95, 131.66, 131.38, 129.39, 128.73,
28.25, 126.46, 125.83, 123.38, 122.72, 122.14, 121.62, 118.52, 118.23, 117.31, 113.15,

+
1
08.27, 55.74, 48.44, 46.97, 30.46; HRMS (ESI): calcd for C H N O S [M+H] 584.1751,
34 26 5 3
found 584.1753; HPLC: t 4.39 min, purity 96.6%.
R
4
.1.2.26.
1-(((6-methylbenzo[d]thiazol-2-yl)amino)(3-(3-nitrophenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-ol (4aa)
1
Yellow solid; yield 82 %; mp: 212-217 °C; H NMR (300 MHz, CDCl +DMSO-d ) δ 8.23 (s,
3
6
1
H), 7.88 (d, J = 8.3 Hz, 1H), 7.61 (t, J = 9.4 Hz, 5H), 7.60 – 7.53 (m, 3H), 7.41 (d, J = 7.6
Hz, 1H), 7.39 (d, J = 2.9 Hz, 1H), 7.32 (s, 1H), 7.25 (dd, J = 7.8, 5.7 Hz, 3H), 7.19 (d, J = 6.9
Hz, 1H), 7.11 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H), 7.03 (s, 1H), 6.91 (t, J = 8.4 Hz,
1
3
1
1
1
1
H), 6.83 (dd, J = 8.8, 2.6 Hz, 1H), 2.43 (s, 3H); C NMR (75 MHz, DMSO-d ) δ 164.31,
6
53.23, 149.78, 148.12, 146.34, 141.04, 139.44, 138.95, 131.66, 131.38, 129.39, 128.73,
28.25, 126.46, 125.83, 123.34, 122.72, 122.14, 121.61, 118.52, 118.23, 117.35, 113.15,
+
08.27, 55.74, 48.44, 46.97, 30.26; HRMS (ESI): calcd for C H N O S [M+H] 584.1747,
3
4
26
5
3
found 584.1749 ; HPLC:t 4.83 min, purity 92.4%.
R
4
.1.2.27. 1-((3-(3-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)((6-methylbenzo[d]thiazol-2-
yl)amino)methyl)naphthalen-2-ol (4ab)
Yellow solid; yield 83 %; mp: 229-234 °C; H NMR (300 MHz, CDCl +DMSO-d ) δ 8.23 (s,
1
3
6
1
H), 7.92 (d, J = 8.3 Hz, 1H), 7.69 (t, J = 9.4 Hz, 5H), 7.60 – 7.53 (m, 3H), 7.41 (d, J = 7.6
Hz, 1H), 7.40 (d, J = 2.9 Hz, 1H), 7.35 (s, 1H), 7.25 (dd, J = 7.8, 5.7 Hz, 3H), 7.22 (d, J = 6.9
Hz, 1H), 7.18 (d, J = 8.8 Hz, 1H), 7.09 (d, J = 2.5 Hz, 1H), 7.03 (s, 1H), 6.90 (t, J = 8.4 Hz,
1
3
1
1
1
1
H), 6.82 (dd, J = 8.8, 2.6 Hz, 1H), 2.43 (s, 3H); C NMR (75 MHz, DMSO-d ) δ 162.71,
6
51.49, 148.24, 146.29, 144.31, 139.30, 137.91, 137.24, 129.92, 129.65, 127.65, 127.00,
26.51, 124.72, 124.09, 121.61, 120.19, 120.07, 119.82, 116.71, 116.29, 115.65, 111.48,
+
06.14, 54.01, 46.71, 45.24, 28.71; HRMS (ESI): calcd for C H N O S [M+H] 584.1744,
3
4
26
5
3
found 584.1746; HPLC: t 4.91 min, purity 90.7%.
R
4
4
.1.2.28. 1-(((5,6-dimethylbenzo[d]thiazol-2-yl)amino)(1-phenyl-3-(p-tolyl)-1H-pyrazol-
-yl)methyl)naphthalen-2-ol (4ac)
1
White solid; yield 71 %; mp: 210-213 °C; H NMR (400 MHz, DMSO-d ) δ 8.68 (s, 3H),
6
8
7
6
1
1
.04 (s, 3H), 7.66 (d, J = 8.0 Hz, 4H), 7.62 (dd, J = 8.2, 5.1 Hz, 1H), 7.52 (d, J = 7.9 Hz, 7H),
.36 (t, J = 7.9 Hz, 3H), 7.27 – 7.22 (m, 2H), 7.20 – 7.04 (m, 1H), 7.03 (d, J = 5.7 Hz, 1H),
1
3
.93 (s, 2H), 2.20 (s, 1H), 2.10 (d, J = 3.1 Hz, 2H); C NMR (100 MHz, DMSO-d ) δ
6
61.43, 151.25, 147.20, 145.55, 144.77, 139.46, 137.87, 137.37, 130.08, 129.81, 127.81,
27.16, 126.67, 124.88, 124.25, 121.81, 121.15, 120.57, 120.05, 116.95, 116.65, 115.81,

1
11.57, 106.70, 52.17, 46.67, 54.41, 23.41, 18.12, 18.14; HRMS (ESI): calcd for
+
C H N OS [M+H] 567.2213, found 567.2215; HPLC: t 5.61 min, purity 92.8%.
36
31
4
5
R
4
.1.2.29. 1-(((5,6-dimethylbenzo[d]thiazol-2-yl)amino)(3-(4-nitrophenyl)-1-phenyl-1H-
pyrazol-4-yl)methyl)naphthalen-2-ol (4ad)
1
Yellow solid; yield 76 %; mp: 233-237 °C; H NMR (400 MHz, DMSO-d ) δ 9.45 (s, 1H),
6
8
7
6
1
1
1
.04 (s, 2H), 7.66 (d, J = 8.0 Hz, 2H), 7.61 (dd, J = 8.2, 5.1 Hz, 1H), 7.55 (d, J = 7.9 Hz, 3H),
.36 (t, J = 7.9 Hz, 2H), 7.27 – 7.22 (m, 3H), 7.20 – 7.07 (m, 2H), 7.03 (d, J = 5.7 Hz, 2H),
1
3
.96 (s, 2H), 2.20 (s, 1H), 2.10 (d, J = 3.1 Hz, 6H); C NMR (100 MHz, DMSO-d ) δ
6
61.43, 151.25, 147.20, 145.55, 144.77, 139.46, 137.87, 137.37, 130.08, 129.81, 127.81,
27.16, 126.67, 124.88, 124.25, 121.81, 121.15, 120.57, 120.05, 116.91, 116.65, 115.81,
11.57, 106.73, 52.17, 46.61, 54.47, 23.41, 18.12.18.14; HRMS (ESI): calcd for
+
C H N O S [M+H] 598.1907, found 598.1909; HPLC: t 5.18 min, purity 90.5%.
35
28
5
3
R
4
.2. Biology
4
.2.1. Cytotoxicity evaluation (MTT assay)
The cytotoxicity of these pyrazole linked naphthalene-2-ol derivatives were determined
3
2
using MTT assay. Cell lines (A549, HeLa, MCF-7 and HEK293) were used in this assay. 1
4
×
10 cells/well were seeded in 200 µl Dulbecco’s modified Eagle’s medium (DMEM),
supplemented with 10% FBS in each well of 96-well micro culture plates and incubated for
o
2
4 h at 37 C in a CO incubator. All the derivatives diluted to the desired concentrations
2
(
500 nM, 1 µM, 5 µM, 10 µM, 25 µM, 50 µM, 75 µM, 100 µM and 150 µM) in culture
medium, were added to the wells with respective vehicle control. Doxorubicin and Hoechst
3258 treated cells, in the same concentration range were used as standards. After 48 h of
3
incubation, 10 µl MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) (5
mg/ml) was added to each well and the plates were further incubated for 4 h. Then the
supernatant from each well was carefully removed, formosan crystals were dissolved in 100
µl of DMSO and absorbance at 570 nm wavelength was recorded at a wavelength of 540 nm
using an ELx800 micro plate reader (BioTek, USA). Assay was repeated thrice and mean
values were considered.
4
.2.2. Cell cycle analysis
Based on the MTT assay, it was clear that compounds 4j and 4l exhibited cytotoxicity
at 4.2 and 5.6 μM concentration, respectively, in A549 cells. Flow cytometry analysis

(
FACS) was performed to evaluate the distribution of the cells under different cell cycle
phases. A549 cancer cells were incubated with a 2.5 and 5 μM concentration of 4j and 4l for
8 h. Untreated cells were used as control. Untreated and treated cells were harvested,
washed with PBS, fixed in ice-cold 70% ethanol and stained with Propidium Iodide (PI)
4
(Sigma Aldrich). Cell cycle was performed by flow cytometry (Becton Dickinson FACS
3
3
Caliber).
4
.2.3. UV-visible studies
UV-visible spectroscopy titrations were performed using ABI Lambda 40 UV-Vis
o
spectrophotometer (Foster City, USA) at 25 C using 1 cm path length quartz cuvette. Stock
solutions of 1 mM of CT DNA (calf thymus DNA, which can form perfect double stranded
DNA structure) were prepared in 100 mM Tris-HCl (pH 7.0). 1 mM synthesized 4j, 4k and 4l
derivatives, stock solution was prepared by dissolving them in 1:1 DMSO: Milli Q water.
UV-visible absorption titrations were done by adding 10 µM CT DNA solution in 100 mM
Tris-HCl (pH 7.0) each time to the quartz cuvette containing about 10 µM derivative
solution. Titrations were carried out until the complex absorption band remains at a fixed
wavelength upon five successive additions of CT DNA. Absorption spectra were recorded
from 200 nm to 400 nm.
4
.2.4. Fluorescence studies
Fluorescence emission spectra were measured at 25 C using a Hitachi F7000
o
spectrofluorimeter (Maryland, USA) using a 1 cm path length quartz cuvette. Throughout the
fluorescence experiment, the concentration of the 4j, 4k and 4l derivatives was kept constant
(10 µM) and titrated with increasing concentrations of CT DNA (each addition with an
increment of 10 µM CT DNA). Fluorescence spectra were recorded after each addition of CT
DNA to the fluorescent cuvette. After each experiment, the quartz cuvettes was thoroughly
washed with distilled water and dilute nitric acid (approximately 0.1 N, nitric acid) to remove
traces of derivative binding to the walls of quartz cuvette. 4j, 4k and 4l derivatives were
excited at 300 nm, and emission spectra for each titration were collected in the range from
3
10 nm to 600 nm. Each spectrum was recorded three times and the average of three scans
was taken.
4
.2.5. CD studies
Circular dichroism (CD) experiments were carried out using JASCO 815 CD
spectropolarimeter (Jasco, Tokyo, Japan). CD spectrum was recorded from 200 to 350 nm to

find the conformation of DNA after CT DNA-derivative interaction. For CD experiments, 10
-6
×
10 M of CT DNA was used. For characterizing derivative-CT DNA interaction, CD
spectra is recorded in 1: 0, 1: 1 and 1:2 molar ratio of CT DNA: derivative, respectively. CD
o
titrations were performed in 100 mM Tris-HCl (pH 7.0) at 25 C. Each CD spectrum was
recorded thrice and the average of three scans was considered.
4
.2.6. Viscosity studies
Viscosity experiments were conducted using Ostwald viscometer, immersed in a water
o
bath maintained at 25 C. Viscosity experiments were performed for each complex (15 µM),
after mixing them with CT-DNA solution (150 µM). Before mixing DNA and complexes,
viscosity measurements were performed with CT-DNA alone. EtBr-CT-DNA and Hoechst
3
3258-CT-DNA complexes were considered as control. DNA solution was prepared in 100
1
/3
mM Tris-HCl (pH 7.0). Graph was drawn by plotting (ŋ/ŋo) versus complex/CT-DNA,
where ŋ is the viscosity of CT-DNA in the presence of complexes and ŋ is the viscosity of
o
CT-DNA alone. Viscosity values were calculated according to the protocol mentioned by Tan
3
4
et al.
4
.2.7. Topoisomerase I inhibition
Topoisomerase I inhibitory activity was measured by a DNA cleavage assay as
3
5
described previously. The pBR 322 plasmid DNA was purchased from Sigma Aldrich, USA
and 0.5 µg of DNA was incubated with 1 unit of Topo I enzyme (Invitrogen) in 1X NEB
buffer (50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, 1 mM
DTT). All the synthetic derivatives (4j, 4k and 4l) at 100 µM was added to the Topo I-DNA
o
complex and incubated at 37 C for 30 min, allowing the formation of ternary enzyme-DNA-
ligand complex. Camptothecin was used as a control. Then the enzyme was inactivated by
o
increasing the temperature to 65 C. After the incubation, the samples were resolved using
1
% agarose gel electrophoresis which enabled the visualization of cleavage products. The
pBR322 DNA sample having no ligand was considered as control.
4
.3. Molecular docking studies
The protein structure of human DNA topoisomerase I (70 kDa) in complex with the
36
indenoisoquinoline (PDB code: 1SC7, resolution 3.0 Å) and three-dimensional crystal
structure of the A-tract DNA dodecamer D(CGCAAATTTGCG) complexed with the minor-
3
7
groove-binding drug Hoechst 33258 (PDB code: 264D, resolution 2.44 Å) were obtained
from the RCSB PDB and prepared using the Protein Preparation Wizard of the Maeströ 9.9.

Hydrogen atoms were added to the proteins including the protons necessary to define the
correct ionization and tautomeric states of amino acid residues. The missing side chains of
residues were corrected by using the Prime module incorporated in Maeströ 9.9. OPLS-2005
force field was used to attenuate steric clashes that may exist in the structures under study.
The minimization was stopped when the energy converged or the Root Mean Square
Deviation (RMSD) reached a maximum cut off of 0.30 Å. Water molecules beyond 5 Å from
hetero groups were deleted.
Acknowledgements
Authors J.K and B.N are thankful to Department of Science & Technology, New Delhi
for providing fellowships under DST-INSPIRE Fellowship programme. We also extend our
appreciation to the International Scientific Partnership Program ISPP at King Saud University
for funding this research work through ISPP#0054.
References and notes
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