Anticancer, antimicrobial activities of quinoline based hydrazone analogues: Synthesis, characterization and molecular docking

Article abstract of DOI:10.1016/j.bioorg.2019.103406

Based on the biologically active heterocycle quinoline, a series (18a-p) of quinoline hydrazone analogues were prepared, starting from 6-bromo/6-chloro-2-methyl-quinolin-4-yl-hydrazines. For all the newly synthesized compounds cytotoxic activities were carried out at the National Cancer Institute (NCI), USA, against full NCI 60 human cancer cell lines. Amongst all the tested compounds, nine compounds (18b, 18d, 18e, 18f, 18g, 18h, 18i, 18j, 18l) exhibited important anti-proliferative activity at 10 μM concentration and were further screened at 10-fold dilutions of five different concentrations (0.01, 0.1, 1, 10 and 100 μM) with GI₅₀ values ranging from 0.33 to 4.87 μM and LC₅₀ values ranging from 4.67 μM to >100j μM. Further, the mean values of GI₅₀, TGI and LC₅₀ of the most potent compound 18j were compared with the clinically used anticancer agents bendamustine and chlorambucil, revealed that the quinolyl hydrazones holds promise as a potential anticancer agents. Further all the newly prepared compounds were screened for their antimicrobial activity. All the quinolyl hydrazones displayed good to excellent antimicrobial activity with MIC values ranging from 6.25 to 100 μg/mL against the tested pathogenic strains. Molecular docking of the synthesized compounds into the active binding site of human DNA topoisomerase I (htopoI) was carried out to predict the binding mode to the DNA topoisomerase I inhibitors. Hopefully in future, compounds based on quinoline core could be used as a lead compounds for designing new anticancer agents.

Full text of DOI:10.1016/j.bioorg.2019.103406

Journal Pre-proofs
Anticancer, Antimicrobial Activities of Quinoline Based Hydrazone Ana-
logues: Synthesis, Characterization and Molecular Docking
Kanubhai D. Katariya, Shailesh R. Shah, Dushyanth Reddy
PII:
S0045-2068(19)31524-X
DOI:
https://doi.org/10.1016/j.bioorg.2019.103406
YBIOO 103406
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Bioorganic Chemistry
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23 October 2019
27 October 2019
Please cite this article as: K.D. Katariya, S.R. Shah, D. Reddy, Anticancer, Antimicrobial Activities of Quinoline
Based Hydrazone Analogues: Synthesis, Characterization and Molecular Docking, Bioorganic Chemistry (2019),
doi: https://doi.org/10.1016/j.bioorg.2019.103406
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Anticancer, Antimicrobial Activities of Quinoline Based
Hydrazone Analogues: Synthesis, Characterization and Molecular
Docking
Kanubhai D. Katariyaa*, Shailesh R. Shaha, Dushyanth Reddyb
a Department of Chemistry, Faculty of Science, The M S University of Baroda, Vadodara
390002 India.
b Department of Pharmaceutical Chemistry, KLE University College of Pharmacy,
Belagavi-590010 India.
Abstract
Based on the biologically active heterocycle quinoline, a series (18a-p) of quinoline hydrazone
analogues were prepared, starting from 6-bromo/6-chloro-2-methyl-quinolin-4-yl-hydrazines.
For all the newly synthesized compounds cytotoxic activities were carried out at the National
Cancer Institute (NCI), USA, against full NCI 60 human cancer cell lines. Amongst all the
tested compounds, nine compounds (18b, 18d, 18e, 18f, 18g, 18h, 18i, 18j, 18l) exhibited
important anti-proliferative activity at 10 µM concentration and were further screened at 10-
fold dilutions of five different concentrations (0.01, 0.1, 1, 10 and 100 µM) with GI50 values
ranging from 0.33-4.87 µM and LC50 values ranging from 4.67 µM to >100j µM. Further, the
mean values of GI50, TGI and LC50 of the most potent compound 18j were compared with the
clinically used anticancer agents bendamustine and chlorambucil, revealed that the quinolyl
hydrazones holds promise as a potential anticancer agents. Further all the newly prepared
compounds were screened for their antimicrobial activity. All the quinolyl hydrazones
displayed good to excellent antimicrobial activity with MIC values ranging from 6.25 - 100
µg/mL against the tested pathogenic strains. Molecular docking of the synthesized compounds
into the active binding site of human DNA topoisomerase I (htopoI) was carried out to predict
the binding mode to the DNA topoisomerase I inhibitors. Hopefully in future, compounds
based on quinoline core could be used as a lead compounds for designing new anticancer
agents.
Keywords: Quinoline; Hydrazones; Alkoxy aldehydes/acetophenones; Anticancer activity;
Antimicrobial activity; Docking studies.
1

1. Introduction
Cancer is a collection of various fatal diseases. Uncontrolled cell growth results in invasion of
surrounding tissue and spreading further to other parts of the human body are the characteristics
of the deadly disease cancer. According to the world cancer report released by WHO, in the
upcoming years the death rates due to cancer will upsurge twice its present-day percentage[1].
Consequently, discovery and development of novel effective drugs for the treatment of cancer
have become an urgent need and the key attention of lots of researchers and pharmaceutical
companies. Although previously reported chemotherapeutic agents efficiently kill cancer cells,
several times this closes to develop multi-drug resistance [2] and hence search and development
of novel anticancer drugs possessing heterocyclic core continues globally at a number of
research laboratories [3].
Compounds possessing heterocyclic core play significant role in designing and developing new
class of structural entities for pharmaceutical applications. Quinoline and its derivatives, as a
significant class of pharmaceutically active heterocyclic compounds, demonstrated different
pharmaceutical activities[4–10]. Quinoline framework played a crucial role in designing of
anticancer agents as their analogues have revealed tremendous results through various
mechanism involving disruption of cell migration, growth inhibitors by cell cycle arrest,
inhibition of angiogenesis, apoptosis, and modulation of nuclear receptor responsiveness[11].
Moreover, many novel derivatives of quinoline have been reported to display significant
anticancer activity through DNA intercalation, causing interference in the replication
process[12]. Because of the pharmacological significance of quinoline derivatives, their
structures and pharmaceutical properties have been well documented[11,13]. Even though a
number of quinoline derivatives and its applications have been reported so far, however only
few synthetic quinoline derivatives are available based on quinoline pharmacophore[14].
Halogen-substituted quinoline compounds have been of particular interest, because the halogen
atom plays a key role in the exhibition of biological activities[15,16]. Many halo-quinolines
have been studied for their potential anticancer activity (Figure 1(A)). Moreover, it was
reported that 2,4-disubstituted and 2,4,6-trisubstituted quinoline ring play a key role in the
designing of novel anti-cancer agents as these derivatives have shown outstanding results
through different mechanism of action[17].
2

O
S
N
N
HN
N
O
Cl
HN
N
HN
N
NH2
Cl
O
Cl
3
2
1
O
O
O
OCH3
N
N
S
NH
Br
Br
S
N
N
N
N
N
Br
4
5
Figure 1(A). Halo-quinolines as a potential anticancer agets.
Since the approval of pelitinib for cancer therapy, a number of the 4-aminoquinoline
derivatives have been developed as the skeleton, which is considered as promising framework
for antitumor drug development (Figure 1(B))[18]. Chloroquine (CQ), an immunostimulatory
agent with 4-aminoquinoline skeleton, has aroused increasing attention due to its
antiproliferative potency on different cancer cells[18]. Likewise, Bispo et al. prepared a series
of 4-quinolinylhydrazones derivatives (Figure 1(B)) as anticancer agents[19], which brought
us new faith in the design and study of quinoline based anticancer agents. Under the inspiration
of CQ and CQ-derivatives, we assumed that newly synthesized compounds with 2,4-
substituted-6-halogenated quinolines should possess promising anticancer activity as well.
F
HN
Cl
H
N
N
O
N
O
Pelitinib
R4
R3
R5
R3
R2
R1
R2
N
HN
N
N
N
HN
N
HN
N
R1
Cl/Br
Cl
Cl
Newly synthesized
Compounds, 18a-p
Chloroquine (CQ)
CQ- Derivatives
3

Figure 1(B). Structures of pelitinib, CQ, halo-quinoline derivatives and target compounds
18a-p.
These observations motivated us to synthesize new 2,4-substituted-6-halogenated quinoline
derivatives. In our efforts to synthesize compounds with improved electron affinity and better
biological interactions, keeping halo-quinoline ring intact, hydrazinyl (-NH-N=) group
introduced at C-4 positions. The introduction of halogen at the C-6 position might adjust
lipophilicity. All target molecules were screened for their in vitro anticancer activity against a
panel of sixty cancer cell lines in the Developmental Therapeutics Program (DTP) at the
National Cancer Institute (NCI). Further, all the newly synthesized compounds were also
screened for in vitro antifungal, antibacterial activities. Active compounds were also studied
for their molecular docking.
2. Results and Discussion
2.1 Chemistry
The synthesis of the target quinolyl hydrazones 18a-p is as represented in scheme 1-2. The
synthesis of the final quinolyl hydrazones was started with the preparation of the intermediate
6-bromo/6-chloro-2-methyl-quinolin-4-yl-hydrazines 9a/b from corresponding 4-
bromo/chloro anilines 6 by the reaction with ethyl acetoacetate in the presence of
polyphosphoric acid as per the reported procedure [20] (Scheme 1). The structures of the
quinolyl hydrazines 9a/b were confirmed using different spectroscopic techniques and are in
accordance with the reported once [21].
4

NH2
OH
N
Cl
N
HN
X
X
X
iii
i
ii
X
NH2
N
6
7 (78%)
8 (80%)
9 (72-75%)
9a; X = Cl, 9b; X = Br
O
O
iv
H
H
Br
+
n
OR
OH
10
11
12 (80-90%)
12a; R= -C2H5, 12b; R= -C3H7,
12c; R= -C4H9, 12d; R = -C5H11
n = 1, 2, 3, 4, 5, 7
,
12e; R = -C6H13, 12f; R = -C8H17
O
O
H
H
Br
v
+
7
OR
OH
OR
OH
13
14
15 (82%), R = -C8H17
O
O
OR
OH
OH
H
H
Br
v
+
7
OR
OH
OR
16
14
17 (89%), R = -C8H17
o
o
Reagents and conditions
: i) Ethyl acetoacetate, Polyphosphoric acid (PPA), 150 C, 2 h ii) POCl3, 80 C, 4 h,
o
o
o
v
iii) NH2NH2. H2O, Ethanol, 80 C, 4 h, iv) Acetone, K2CO3, 60-70 C, 6-7 h, ) DMF, K2CO3, 100 C,12h.
Scheme 1. Synthesis of intermediate compounds 9a/9b, 12a-e, 15 and 17.
5

R3
O
R1
R2
NH2
R3
R1
HN
N
H
N
X
I
HN
N
+
X
R2
9
12/15/17
18
(76-82%)
X = Br
18a R
X = Cl
18b R R R
; 1 = -OC2H5, 2 = -H, 3 = -H
R
R
;
1 = -OC2H5, 2 = -H, 3 = -H
18c R
R
R
18d R
R
R
;
1 = -OC3H7, 2 = -H, 3 = -H
;
1 = -OC3H7, 2 = -H, 3 = -H
18e R
R
R
18f R
R
R
;
1 = -OC4H9, 2 = -H, 3 = -H
;
1 = -OC4H9, 2 = -H, 3 = -H
18g R
R
R
18h R
R
R
;
1 = -OC5H11, 2 = -H, 3 = -H
;
1 = -OC5H11, 2 = -H, 3 = -H
18i R
R
R
18j R
R
R
;
1 = -OC6H13, 2 = -H, 3 = -H
;
1 = -OC6H13, 2 = -H, 3 = -H
18k R
R
R
18l R
R
R
;
1 = -OC8H17, 2 = -H, 3 = -H
;
1 = -OC8H17, 2 = -H, 3 = -H
18m R
R
R
18n R
R
R
;
1 = -OC8H17, 2 = -H, 3 = -OC8H17
;
1 = -OC8H17, 2 = -H, 3 = -OC8H17
18o R
R
R
18p R
R
R
;
1 = -OC8H17, 2 = -OC8H17, 3 = -OC8H17
;
1 = -OC8H17, 2 = -OC8H17, 3 = -OC8H17
Reagents and conditions
: i) Ethanol, Sulphuric acid, 80 °C, 4 h
Scheme 1. Synthetic protocol for quinolyl hydrazones, 18a-p.
Further, synthesis of several alkoxy substituted aromatic aldehydes 12/15/17 was carried out
by the reaction of 4-hydroxybenzaldehydes with varying alkyl chain lengths to have different
lipophilicity of the final compounds. The 4-alkyloxybenzaldehydes 12a-f were prepared by
alkylation of 4-hydroxy benzaldehyde 10 with different alkyl halides 11a-f with chain length
varying from 2 to 8 carbons in the presence of K2CO3 as a base in acetone following the
reported procedure [22] (Scheme 1). Similarly, 3,4-bis-octyloxy benzaldehyde 15 and 3,4,5-
tris-octyloxy benzaldehyde 17 were prepared by reacting 3,4-dihydroxy benzaldehyde 13 and
3,4,5-trihydroxybenzaldehyde 16 with 1-bromooctane 14 in dry DMF using K2CO3 as a base
to afford 3,4-bis-octyloxy benzaldehyde 15 and 3,4,5-tris-octyloxy benzaldehyde 17
respectively[23,24] (Scheme 1). The final target quinolyl hydrazones 18a-p were synthesized
by the condensation of various alkoxy benzaldehydes 12a-f/15/17 with 6-bromo/6-chloro-2-
methyl-quinolin-4-yl-hydrazine 9a/b in ethanol using a catalytic amount of sulphuric acid
following reported procedure[20] (Scheme 2).
All the quinolyl hydrazones were characterized by various spectroanalytical techniques. In the
IR spectrum of quinolyl hydrazones (18a-p), the N‒H stretching was observed between 3270-
3200 cm-1. The presence of long alkyl chains is marked by the C‒H stretching observed at
~2977 cm-1. The strong bands observed at ~1605 cm-1 and ~1571 cm-1 are corresponding to the
6

aromatic C=C stretching. Strong absorption bands at frequency range ~850 cm-1 and ~770 cm-1
are due to the presence of C-Cl and C-Br stretching. In proton NMR of the quinolyl hydrazones
(18a-p), the terminal methyl protons were observed most upfield near δ 0.9-1.1 ppm as a triplet
with coupling constant J = 7.2 Hz. The –CH3 group protons on quinoline ring were observed
at δ 2.73 ppm as a singlet. For compounds (18a/18b, R3 = -C2H5), –OCH2 protons were
observed at δ 4.1 ppm as a quartet with coupling constant J = 7.2 Hz while for other compounds
–OCH2 protons were appeared as triplet. The methylene proton of imine linkage (-N═CH-)
was observed as a singlet at δ 7.5 ppm. The –NH proton was observed most downfield at δ
13.0-14.0 ppm as a singlet. The other aromatic protons were observed in between δ 7.0-8.8
ppm value as per the substitution pattern at various positions of the aromatic ring. In 13C NMR
of the new quinolyl hydrazones (18a-p) the most downfield carbon was hydrazone carbon
observed near δ 160 ppm while the other aromatic carbons were observed between δ 152 to
100 ppm and the oxygen attached carbon was observed near δ 63 ppm and other aliphatic
carbons are observed up field with the chemical shift value up to δ 15 ppm. In the mass spectral
analysis of the newly synthesized quinolyl hydrazones (18a-p), the molecular ion peaks for
these compounds were observed as (M+H)+ for all the compounds. The physical data of all
the quinolyl hydrazones 18a-p are as summarized in Table 1.
Table 1. Physical data of the newly synthesized quinolyl hydrazones.
Substitution pattern
Molecular
formula
ID
NSC number
Yield
mp
X
R1
R2
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
R3
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
18a -Br -OC2H5
18b -Cl -OC2H5
18c -Br -OC3H7
18d -Cl -OC3H7
18e -Br -OC4H9
18f -Cl -OC4H9
18g -Br -OC5H11
18h -Cl -OC5H11
18i -Br -OC6H13
18j -Cl -OC6H13
18k -Br -OC8H17
18l -Cl -OC8H17
D-804234/1
D-804233/1
D-804236/1
D-804235/1
D-804238/1
D-804237/1
D-804240/1
D-804239/1
D-804242/1
D-804241/1
D-804271/1
D-804270/1
C19H18BrN3O
C19H18ClN3O
C20H20BrN3O
C20H20ClN3O
C21H22ClN3O
C21H22BrN3O
C22H24BrN3O
C22H24ClN3O
C23H26BrN3O
C22H24ClN3O
C25H30ClN3O
C25H30ClN3O
82 % 202-203 °C
74 % 210-211 °C
78 % 204-215 °C
74 % 200-201 °C
76 % 198-199 °C
80 % 186-187 °C
68 % 194-195 °C
72 % 190-191 °C
74 % 194-195 °C
76 % 188-189 °C
82 % 186-187 °C
79 % 182-183 °C
7

18m -Br -OC8H17
18n -Cl -OC8H17
-H
-H
-OC8H17
-OC8H17
D-804273/1
D-804272/1
D-804275/1
D-804274/1
C33H46BrN3O2 78 % 184-185 °C
C33H46ClN3O2 71 % 182-183 °C
C41H62ClN3O3 74 % 180-181 °C
C41H62BrN3O3 72 % 178-189 °C
18o -Br -OC8H17 -OC8H17 -OC8H17
18p -Cl -OC8H17 -OC8H17 -OC8H17
2.2 Anticancer activity
2.2.1 In vitro preliminary anticancer activity at a single dose (10 M) against full NCI 60
cell panel
The preliminary anticancer screening of the newly prepared quinolyl hydrazones 18a-p was
carried out at NCI, USA under the screening project at the National Cancer Institute (NCI),
USA. All the newly synthesized quinolyl hydrazones were selected and evaluated for
preliminary in vitro one dose anticancer screening at National Cancer Institute (NCI) against
full NCI 60 cell line panels representing nine different kinds of cancer comprising leukemia,
non-small cell lung cancer, melanoma, CNS cancer, ovarian cancer, renal cancer, prostate
cancer, breast cancer in agreement with the protocol of the NCI, USA (http://dtp.nci.nih.gov).
Results of each tested compound 18a-p were reported as a mean graph of the percent growth
of the treated cells when compared to the untreated control cells which gives both inhibition
values (between 0 and 100) and cytotoxicity values (less than 0). The single dose evaluation
results of all the quinolyl hydrazones 18a-p against sixty cancer cell lines were analysed by
COMPARE program. The screening results of all quinolyl hydrazones at 10 µM exhibited
excellent anticancer activity with broad spectrum of cytotoxic activity against several cancer
cell lines tested. Nine of the quinolyl hydrazones (18b, 18d, 18e, 18f, 18g, 18h, 18i, 18j, 18l)
were selected for further detailed study at five different dilutions due to outstanding results in
the single dose study and found to be lethal to a greater extent (see supporting information).
2.2.2 In vitro anticancer screening at 5 dose full NCI 60 cell panel
In the second stage, the selected nine compounds including 18b (D-804233/1), 18d (D-
804235/1), 18e (D-804238/1), 18f (D-804237/1), 18g (D-804240/1), 18h (D-804239/1), 18i
(D-804242/1), 18j (D-804241/1), 18l (D-804270/1) satisfied predetermined threshold
inhibition criteria, were screened and evaluated against all the 60 cell lines at 10-fold dilutions
of five different concentrations (0.01, 0.1, 1, 10 and 100 M)[25,26]. The percentage of growth
was examined spectrophotometrically against not treated with test agents and using SRB
8

(sulforhodamine-B) protein assay cell viability was determine according to the experimental
protocols described [25,26]. The results of the five dose assay are represented in terms of
response parameters GI50 (Required molar concentration to inhibit 50% of the growth of cancer
cell lines) and LC50 (Required molar concentration necessary to kill 50% of the cells at the end
of the incubation period of 48 h, cytotoxic activity) for each cell line tested [25,26]. The
calculated GI50 and LC50 values for each sixty cancer cell lines of these nine compounds over
the nine cancer types are summarized in Table 2. The five dose anticancer screening data
revealed that, all nine tested compounds (18b, 18d, 18e, 18f, 18g, 18h, 18i, 18j, 18l) exhibited
outstanding activity with great value of GI50 against several cancer cell lines, a number of them
lower than 2.0 M.
Compound 18b displayed outstanding activity with GI50 values from 1.12 to 2.37 μM (48 of
them < 2.00 μM) and LC50 values from 5.47 to 33.0 μM. The highest cytostatic activity of 18b
was observed against SR leukemia cell line with GI50 = 1.12 μM and the best cytotoxic activity
was shown against the UO-31 renal cancer cell line with LC50 = 5.47 μM. Compound 18d
displayed good activity with GI50 values from 1.17 to 2.31 μM (50 of them < 2.00 μM) and
LC50 values from 5.39 to 38.6 μM. The best cytostatic activity was observed against SR
leukemia cell line with GI50 values from 1.17 μM and the best cytotoxic activity was displayed
against the SK-MEL-5 melanoma cancer cell line with LC50 = 5.39 μM. Compound 18e
displayed potent activity with GI50 values ranging from 1.47 to 2.64 μM (52 of them < 2.00
μM) and LC50 values from 5.39 to >100 μM. Compound 18e displayed best cytostatic activity
against the HOP-62 non–small cell lung cancer cell line and SK-MEL-2 melanoma cancer cell
line with GI50 = 1.47 μM and the best cytotoxic potency was observed against the UO-31 renal
cancer cell line with LC50 = 5.39 μM. Compound 18f displayed noteworthy activity with GI50
values ranging from 0.50 to 2.13 μM (55 of them < 2.00 μM) and LC50 values from 5.34 to
>100 μM. Compound 18f displayed highest cytostatic activity against the SR leukemia cancer
cell line with GI50 = 0.50 μM and the best cytotoxic activity was observed against the RXF 393
renal cancer cell line with LC50 = 5.34 μM. Compound 18g showed good anticancer activity
with GI50 values ranging from from 0.44 to 2.13 μM (56 of them < 2.00 μM) and LC50 values
from 5.47 to >100 μM. Compound 18g displayed highest cytostatic activity against the SR
leukemia cancer cell line with GI50 = 0.44 μM and the best cytotoxic activity was observed
against the CAKI-1 and UO-31 renal cancer cell lines with LC50 = 5.47 μM. Compound 18h
exhibited excellent activity with GI50 values ranging from from 0.36 to 2.23 μM (53 of them <
2.00 μM) and LC50 values from 5.02 to 42.0 μM. Compound 18h displayed best cytostatic
9

activity against the SR and K-562 leukemia cancer cell lines with GI50 = 0.36 μM 0.69 μM
respectively and the best cytotoxic activity was observed against the HCT-116 colon cancer
cell lines with LC50 = 5.02 μM. Compound 18i exhibited GI50 values from 0.39 to 4.87 μM (50
of them < 2.00 μM) and LC50 values from 4.67 to >100 μM. Compound 18h displayed best
cytostatic activity against the SR leukemia and HCT-116 colon cancer cell lines with GI50 =
0.39 μM and 0.85 μM respectively and the best cytotoxic activity was observed against the
HCT-116 colon cancer cell lines with LC50 = 4.67 μM.
Among all new compounds, the highest activity was observed for compound 18j with GI50
values ranging from 0.33 to 2.05 μM (52 of them < 2.00 μM) and LC50 values from 5.15 to
24.4 μM (Table 2 and Figure 3). The plots of percentage growth in 60 cancer cell lines vs
sample concentration at five different dose levels (log dilution from 10-4 mol/L to 10-8 mol/L)
after treatment with compound 18j are shown in Figure 2. The dose response curve of
compound 18j plotted for six subpanels of leukaemia (Figure 3), illustrates the endpoint
calculations for GI50, TGI and LC50 at five dose concentrations. It displayed best cytostatic
activity against the some of the cancer cell lines including (SR leukemia cancer cell line with
GI50 = 0.33 μM), (K-562 leukemia cancer cell line with GI50 = 0.46 μM), (HCT-116 cancer cell
line with GI50 = 0.34 μM), (SW-620 cancer cell line with GI50 = 0.97 μM) and the best cytotoxic
activity was observed against the HCT-15 colon cancer cell lines with LC50 = 5.15 μM (Figure
2 and Figure 3). Compound 18l demonstrated good activity with GI50 values ranging from 1.48
to 2.21 μM (44 of them < 2.00 μM) and LC50 values from 5.53 to >100 μM. Compound 18l
displayed highest cytostatic activity against the HOP-62 non-small cell cancer cell line with
GI50 = 1.48 μM and the best cytotoxic activity was observed against the UO-31 renal cancer
cell line with LC50 = 5.53 μM.
Further, The in vitro anticancer activity of the most potent compound 18j, among the entire
series of newly synthesized compounds was also compared with the activity data obtained from
the NCI for the clinically used anticancer agents (Bendamustine and Chlorambucil)[27] in
terms of potency (µmol/L) by three response parameters and the results are illustrated in Table
3. The results revealed that the compound 18j has lower mean values of log molar concentration
for response parameters (GI50, TGI and LC50) as compared to clinically used anticancer drugs,
bendamustine and chlorambucil. Also, the mean graph midpoint (MG-MID) GI50 value of the
most active compound 18j was observed to be only 1.58 µM, which is considerably lesser than
the standard marketed anticancer drugs (60 and 52 µM, respectively) suggesting that the
quinolyl hydrazones holds promise as a potential anticancer agents.
10

Compound 18j was also studied for their cytotoxicity study in NIH/3T3 normal cell line. The
cytotoxicity of compound 18j was evaluated using MTT assay and IC50 value was measured.
MTT assay is based on the activity of mitochondrial dehydrogenase enzyme activity and
represent the metabolic rate or percentage viability of cell[28,29]. In MTT assay, the
percentage viability of cell line was decreased with increased concentration of compound 18j.
Compound 18j showed IC50 132 ± 0.05 μM against NIH/3T3 cell line, which was considerably
higher as compare to GI50 values of 18j against NCI 60 cancer cell lines, showed that compound
18j is very selective towards cancer cells at lower concentration as compared to healthy cell
line.
It has been reported in the literature that quinoline derivatives act by inhibiting DNA
topoisomerase complex[30,31]. As a result, there is a high probability that antiproliferative
potency of compound 18j, a quinolyl hydrazone could be due to DNA intercalation.
Nevertheless, the studies focusing on the mechanism of action of these derivatives are currently
under progress. In conclusion, it is conceivable that derivatization of such compounds will be
of attention with the hope to get newer effective and selective anticancer agents.
Table 2. Five dose in vitro anticancer activity resultsa (cytotoxic activities of compounds
expressed as GI50b (M) and LC50c (M) for compounds 18b-i against all sixty cancer cell
lines.
Compounds
Panel Cell
18b
18d
18e
18f
18g
lines
GI50 LC50 GI50 LC50 GI50 LC50 GI50 LC50 GI50 LC50
Leukemia
CCRF-CEM 1.14 9.60 1.78 9.32 1.88 29.0 1.86 >100 1.54 7.99
HL-60(TB)
K-562
1.86 7.46 1.83 7.78 2.02 8.19 1.76 7.60 1.91 7.48
1.36 6.52 1.48 6.63 1.71 9.20 1.09 1.24 6.45
-
MOLT-4
RPMI-8226
SR
1.89 7.84 1.81 7.88 1.91 8.82 1.70 7.66 1.73 7.42
1.94 9.62 1.81 8.79 2.03 17.2 1.92 >100 1.91 9.11
1.12 15.5 1.17 38.6 2.64 >100 0.50 >100 0.44 21.3
Non–small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
2.00 6.94 1.72 6.49 1.88 6.61 1.86 6.35 1.90 7.15
2.18 16.3 1.64 5.68 1.63 5.63 1.64 5.55 1.63 5.93
2.31 27.3 1.58 5.63 1.47 5.88 1.62 6.63 1.73 6.57
2.10 33.0 1.51 6.41 1.54 6.27 1.43 6.25 1.36 6.10
1.85 7.08 1.69 6.64 1.83 8.01 1.63 11.6 1.85 >100
1.76 6.23 1.81 6.82 1.86 7.61 1.77 6.63 1.85 7.89
NCI-H322M 1.83 5.95 1.69 5.56 1.76 5.65 1.74 5.63 1.74 5.72
NCI-H460
NCI-H522
2.06 8.85 2.04 7.97 2.08 7.94 1.99
1.73 6.24 1.75 6.68 1.72 6.00 1.72 6.27 2.06 15.1
-
1.94 7.08
11

Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
1.91 9.31 1.82 6.36 1.85 6.50 2.01 8.09 1.96 7.06
2.19 6.26 2.03 6.15 2.12 6.56 1.91 5.99 2.07 6.98
1.13
-
1.70 6.48 1.77 6.17 2.13 >100 1.78 6.35
1.55 5.59 1.52 5.67 1.61 5.80 1.35 5.54 1.57 6.25
1.90 6.33 180 6.77 1.55 5.69 1.24 5.51 1.94 9.25
1.89 7.04 1.80 6.22 1.95 7.62 1.93 7.73 1.74 5.87
KM12
SW-620
1.97 8.22 1.96 7.31 2.03
CNS Cancer
-
1.97
-
1.92 6.85
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
2.05 7.41 2.24 7.81 2.20
-
2.26
-
1.97 7.07
1.76 5.97 1.72 5.94 1.82 6.21 1.66 5.79 1.81 6.7
1.97 5.87 1.79 5.71 1.84 5.88 1.84 5.87 1.76 5.92
2.37 14.5 1.95 6.41 1.86 6.28 1.81 6.01 1.96 7.03
1.86 7.12 1.94 6.46 1.70 6.17 1.75 6.35 1.77 6.50
1.91 5.96 2.05 6.15 1.80 5.96 1.72 5.90 1.82 6.31
Melanoma
LOX IMVI
1.73 5.68 1.70 5.66 1.77 5.90 1.69 5.67 1.86 7.12
MALME-3M 2.02 6.40 1.79 5.93 1.91 6.06 1.85 6.18 1.88 6.37
M14 1.53 5.55 1.69 6.17 1.74 5.88 1.89 8.07 1.88 6.64
MDA-MB-435 1.75 6.05 1.80 5.83 1.80 5.94 1.73 5.80 1.76 5.74
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
1.90 6.17 1.73 5.99 1.47 5.56 1.85 6.34 1.98 7.69
1.78 5.73 1.73 5.74 1.72 5.71 1.66 5.72 1.65 5.84
1.72 5.56 1.54 5.39 1.68 5.53 1.61 5.47 1.74 5.71
1.81 6.10 1.79 6.16 1.66 6.09 1.61 6.01 1.88 6.89
1.79 6.17 1.76 5.94 1.74 9.09 1.61 5.69 1.66 5.90
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-
RES
1.93 6.87 1.92 6.85 1.91 6.78 1.93 6.84 1.72 6.60
1.93 6.36 2.13 6.44 2.06 7.18 2.03 7.04 1.92 5.92
1.79 6.90 1.71 6.01 1.67 5.62 1.56 5.76 1.76 6.06
1.97 6.22 1.86 5.76 1.86 5.71 1.82 5.76 1.83 5.88
2.34 14.1 2.31 8.88 1.77 7.07 1.78 6.74 2.13 14.0
1.90 7.83 1.90 7.65 1.96 7.56 1.73 6.50 1.83 9.59
SK-OV-3
2.86 25.0 1.78 5.71 1.66 5.61 1.80 5.94 1.75 5.77
Renal Cancer
786-0
A498
1.57 5.86 1.86 6.27 1.83 6.20 2.00 7.06 1.80 6.55
1.70 7.01 1.72 5.79 1.48 5.47 1.52 5.67 1.83 6.02
1.79 5.63 1.74 5.59 1.73 5.57 1.73 5.57 1.77 5.72
1.78 5.77 1.78 5.73 1.71 5.65 1.68 5.62 1.63 5.47
1.66 5.65 1.55 5.58 1.60 5.82 1.41 5.34 1.67 6.10
1.82 6.35 1.82 6.02 1.74 6.07 1.63 5.82 1.72 6.21
1.91 5.94 2.04 5.92 1.91 5.76 1.87 5.76 1.87 6.25
1.63 5.47 1.56 5.43 1.48 5.39 1.56 5.50 1.57 5.47
Prostate Cancer
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
1.51 6.54 1.53 6.39 1.75 6.49 1.71 6.55 1.55 5.88
1.82 6.30 1.90 6.15 1.94 7.73 1.82 7.57 1.81 5.85
Breast Cancer
MCF7
1.68 6.60 1.67 6.90 1.83 7.37 1.65 6.87 1.71 7.93
12

MDA-MB-
231/ATCC
HS 578T
BT-549
1.83 6.23 1.85 6.56 1.88 6.54 1.74 6.09 1.77 6.56
2.15 11.5 2.15 9.96 1.99 >100 1.86 2.33 >100
1.61 6.15 2.17 1.96 1.77 6.60 1.75 6.71 1.77 6.64
1.93 7.46 1.71 7.67 1.69 7.65 1.73 8.18 1.81 8.36
-
T-47D
MDA-MB-468 1.73 6.17 1.69 6.04 1.67 6.22 1.53 5.78 1.83 6.96
Table 2 Continue…..
Compounds
Panel Cell lines
18h
18i
18j
18l
GI50 LC50 GI50 LC50 GI50 LC50 GI50 LC50
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
1.74
-
1.71 9.20 1.53
-
2.04 47.0
1.64 6.90 1.29 8.25 1.39 8.21 2.00 9.95
0.69 6.20 1.02 5.21 0.46 24.4 1.66 18.3
1.65 6.85 1.20 8.42 1.85 8.88 2.05 52.4
1.84
-
1.86 8.87 1.81
-
2.01 19.4
0.36 4.20 0.39 >100 0.33 8.11 1.61 >100
Non–small cell lung cancer
A549/ATCC
EKVX
1.90 6.52 1.78 6.61 1.93 7.22 1.70 7.26
1.67 5.67 1.72 5.73 1.75
-
1.80 6.15
HOP-62
1.62 6.66 1.47 5.89 1.55 6.11 1.48 6.42
HOP-92
1.47 5.99 1.45 6.08 11.42 6.67 1.51 6.05
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
1.64 8.25 1.60 6.23 1.86
1.78 6.76 1.78 6.62 1.88 6.65 1.90 7.76
1.74 5.66 1.71 5.55 1.70 5.76 1.79 5.75
1.95 8.03 1.94 7.74 1.62
1.63 5.80 1.70 6.49 1.79 6.87 1.89 7.89
Colon Cancer
-
1.70 7.74
-
2.01 7.42
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
1.89 6.89 1.85 6.65 1.58 5.85 1.82 6.96
1.95 6.42 2.03 6.24 1.93 6.19 2.03 6.36
1.04 5.02 0.85 4.67 0.34
-
1.83 7.95
1.43 5.64 1.52 5.46 1.27 5.15 1.85 6.17
1.44 5.46 1.63 5.98 1.59 5.99 1.81 6.17
1.79 6.50 1.80 6.81 1.67 6.29 1.85 6.39
KM12
SW-620
1.73 7.18 1.56 8.16 0.97
CNS Cancer
-
1.89 6.96
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
2.23 8.94 2.07 8.70 2.10
-
2.21 7.90
1.77 6.28 1.72 5.95 1.73 6.07 1.83 6.50
1.86 5.87 1.87 5.75 1.84 5.78 1.86 5.81
1.87 6.22 1.83 6.43 1.88 6.12 2.00 7.34
1.63 6.36 1.84 6.50 1.81 6.50 1.86 6.58
1.70 5.87 1.87 6.02 1.90 6.20 1.82 6.30
Melanoma
LOX IMVI
MALME-3M
M14
1.69 5.90 1.53 5.50 1.26 5.25 1.66 6.06
1.85 6.21 4.87 6.10 1.91 6.22 1.85 6.08
1.76 5.91 1.67 5.67 1.71 6.26 1.75 7.06
1.75 5.91 1.73 5.77 1.71 5.80 1.73 5.79
MDA-MB-435
13

SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
1.70 5.90 1.78 6.17 1.72 6.10 1.97 6.53
1.69 5.66 1.81 5.81 1.76 5.74 1.78 5.75
1.63 5.54 1.67 5.51 1.59 5.54 1.73 5.79
1.72 6.22 1.95 6.83 1.61 6.59 1.75 9.30
1.68 5.89 1.74 6.01 1.78 6.01 1.87 6.53
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
1.91 6.79 1.75 6.80 1.79 6.96 1.84 7.34
1.97 6.51 1.94 6.85 2.05 6.83 2.17 6.70
1.72 6.03 1.81 6.59 1.75 5.78 1.82 6.92
1.82 5.71 1.94 5.84 1.92 5.82 1.79 5.70
1.98 9.08 2.07 8.23 1.91 9.28 2.14 15.0
1.89 8.14 1.90 7.73 1.90 7.20 1.97 8.39
1.65 5.78 1.65 5.70 1.70 5.70 1.65 5.83
Renal Cancer
786-0
A498
1.78 6.18 1.71 5.91 1.89
-
1.86 7.19
1.19 5.13 1.58 6.03 1.77 6.55 1.98 6.00
1.75 5.60 1.74 5.58 1.71 5.55 1.74 5.60
1.64 5.62 1.65 5.59 1.65 5.64 1.69 5.70
1.63 5.84 1.55 5.64 1.56 5.65 1.55 5.57
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
1.71 6.06 1.69 5.93 1.69
5.9 1.78 6.30
1.77 5.65 1.93 5.84 1.89 5.83 1.91 5.81
1.56 5.51 1.53 5.48 1.50 5.56 1.55 5.53
Prostate Cancer
PC-3
DU-145
1.61 5.98 1.64 6.29 1.57 6.56 1.50 6.16
1.88 7.28 1.92 7.13 1.86 6.87 1.76 6.24
Breast Cancer
MCF7
1.64 7.04 1.64 6.84 1.47 6.97 1.94 7.89
MDA-MB-231/ATCC 1.71 7.28 1.62 5.85 1.67 6.87 1.81 6.64
HS 578T
BT-549
T-47D
1.92
-
2.16 9.34 2.19
-
2.23 9.10
1.74 6.20 1.91 8.00 1.16 >100 1.42 6.91
1.76 7.83 1.82 7.60 1.98 8.00 1.92 8.71
1.70 6.67 1.70 6.11 1.65 5.95 1.79 6.54
MDA-MB-468
a Data obtained from the NCI’s in vitro human cancer cell lines screen.
b Required molar concentration to inhibit 50% of the growth of cancer cell lines.
c LC50: Required Molar concentration required to kill 50% of the cells.
The most active compounds (lowest GI50 and LC50 value) are highlighted in red.
14

Figure 2. Five dose assay graph of compound 18j (NSC: D-804241/1) against nine panel
cancer cell line at NCI.
Figure 3. Dose response curve of Leukemia subpanel of compound 18j (NSC: D-804241/1)
showing end point calculations for SR cell line at five dose concentrations (log dilution from
10-4 mol/L to 10-8 mol/L).
Table 3. In vitro anticancer activity comparison of compounds 18j and clinically used
anticancer agents (Bendamustine and Chlorambucil).
MIDe
GI50
1.58
60
Potency in log10(M) unit in µmol/L
Log (High
Conc.)
-5.0
No. of
Compd. NSC No.
N.C.L.d
GI50 EDPc
-5.80
TGI EDPc
-5.49
LC50 EDP
-5.14
expts
18j
804241/1
2
3
60
60
BENDAa 138783
-4.0
-4.153
-4.018
-4.004
15

CHLBb
3088
-5.0
-4.785
-4.282
-4.062
2
59
52
a Bendamustine hydrochloride (Treanda).
b Chlorambucil (Leukeran).
c Endpoint.
d Number of cell lines.
e Mean graph midpoint (MG-MID). Data were obtained from the Developmental Therapeutics
Program, NCI main web site for comparison purpose.
2.3 In vitro antimicrobial activity study
In continuation of biological activity, all newly prepared quinolyl hydrazones 18a-p, were
screened for their antimicrobial activity against (S. aureus (MTCC 96), B. subtilis (MTCC 619)
as Gram positive bacteria, E. coli (MTCC 739), P. aeruginosa (MTCC 741) as Gram negative
bacteria. For antifungal activity A. niger (MTCC 282) and C. albicans (MTCC 183) were used.
Zone of inhibition against all the six pathogenic strains were measured using paper disc
diffusion technique [32]. MIC of the test compound was determined by agar streak dilution
method [33]. The standard antibiotics, Ciprofloxacin and gresiofulvin were taken as the
standard references during for antibacterial and antifungal activity respectively.
The results revealed that, all the quinolyl hydrazones demonstrated outstanding antibacterial as
well as antifungal activity with MIC value ranging from 6.25 µg/mL to 100 µg/mL as
summarized in Table 4. Among all quinolyl hydrazones, compound 18p showed excellent
inhibitory activity (MIC = 6.25 µg/mL) against the Gram +ve bacteria S. aureus while
compounds 18g, 18i, 18j, 18o also displayed noteworthy inhibition with MIC = 12.5 µg/mL
against the same bacterial strain. Against B. subtilis compounds 18a, 18e, 18i, 18l and 18n
showed MIC = 12.5 µg/mL with remarkable inhibition (Table 4). In case of Gram -ve bacteria
E. coli, the best inhibitory activity was observed for compounds 18i with MIC = 6.25 µg/mL
while compounds 18k, 18n and 18p have shown potency with MIC = 12.5 µg/mL. In case of
P. aeruginosa, the best inhibitory activity was observed for compounds 18p with MIC = 6.25
µg/mL and compounds 18i, 18l and 18o demonstrated good potency against the same bacterial
strain with MIC = 12.5 µg/mL (Table 4). Against the fungal strain C. albicans, compounds
18c, 18e, 18j, 18 and 18r exhibited potent inhibitory activity (MIC = 12.5 µg/mL) while in the
case of A. niger, compounds 18a, 18g, 18i, 18l and 18p demonstrated excellent inhibitory
activity with MIC = 12.5 µg/mL (Table 4). In conclusion, all the quinolyl hydrazones showed
16

good to excellent antimicrobial activity and these kinds of compounds possessing quinoline as
a core unit along with hydrazone linkages could be develop as an effective antimicrobial agents.
Table 4. In vitro antimicrobial screening of compounds, 18a-p.
Zone of inhibition in mm and (MIC in µg/mL)
Gram(+ve) bacteria
S. aureus B. subtilis
Gram(-ve) bacteria
E. coli P. aeruginosa
Fungi
C. albicans
A. niger
ID
MIC
(µg/mL
)
MIC
(µg/mL
)
MIC
MIC
(µg/mL
)
MIC
(µg/mL
)
MIC
Zone
(mm)
Zone
(mm)
Zone
(mm)
Zone
(mm)
Zone
(mm)
Zone
(mm)
(µg/mL
)
(µg/mL
)
21±0.
1
25±0.
3
22±0.
1
19±0.
1
24±0.
4
25±0.
2
100
25
12.5
25
50
125
25
100
25
25
125
12.5
100
12.5
100
25
12.5
100
25
18a
18b
18c
18d
18e
18f
18g
18h
18i
24±0.
2
24±0.
2
19±0.
3
24±0.
3
19±0.
2
20±0.
3
25±0.
1
21±0.
1
24±0.
2
18±0.
2
25±0.
1
24±0.
1
12.5
100
50
100
25
125
50
21±0.
3
23±0.
2
20±0.
4
22±0.
3
21±0.
3
20±0.
4
100
100
125
100
125
6.25
25
100
100
25
22±0.
2
25±0.
3
20±0.
2
24±0.
1
25±0.
2
21±0.
3
12.5
100
50
25
20±0.
1
21±0.
1
18±0.
2
19±0.
2
20±0.
4
24±0.
2
100
12.5
25
125
100
50
25±0.
3
22±0.
3
21±0.
3
19±0.
3
24±0.
3
25±0.
1
12.5
100
12.5
25
24±0.
2
17±0.
2
20±0.
1
22±0.
2
18±0.
2
17±0.
2
125
12.5
25
125
50
26±0.
1
25±0.
1
27±0.
3
25±0.
3
22±0.
3
25±0.
3
12.5
12.5
25
12.5
25
25±0.
1
24±0.
1
24±0.
2
23±0.
1
25±0.
1
23±0.
2
12.5
100
25
18j
18k
18l
23±0.
3
22±0.
3
26±0.
1
24±0.
2
21±0.
2
22±0.
2
50
12.5
25
25
50
24±0.
2
25±0.
2
24±0.
3
25±0.
1
24±0.
3
25±0.
2
25
12.5
25
12.5
50
12.5
50
22±0.
4
23±0.
1
21±0.
2
22±0.
3
24±0.
1
22±0.
1
50
100
12.5
25
25
18m
18n
18o
18p
A
24±0.
2
26±0.
3
25±0.
3
23±0.
1
25±0.
3
23±0.
3
25
12.5
25
25
12.5
25
25
25±0.
1
24±0.
2
24±0.
3
26±0.
3
23±0.
2
24±0.
3
12.5
6.25
<3.12
12.5
6.25
<3.12
25
28±0.
3
24±0.
1
26±0.
2
27±0.
2
24±0.
1
25±0.
1
25
12.5
<3.12
25
12.5
33±0.
1
30±0.
2
32±0.
1
31±0.
2
<3.12
-----
----
31±0.
30±0.
----
----
----
----
<3.12
<3.12
B
1
2
DMS
O
----
----
----
----
----
----
A - Ciprofloxacin, standard reference drug for antibacterial screening.
B – Griseofulvin, standard reference drug for antifungal screening.
17

2.4 Molecular docking study
DNA topoisomerase I is a nuclear enzyme crucial for solving topological complications that
happen throughout DNA transcription, replication, and chromosome segregation [34]. DNA
topoisomerase 1 forms a single-stranded nick onto the DNA by a nucleophilic attack on the
DNA phosphodiester bond to make a “cleavage complex” in which the enzyme is covalently
linked to the 3′ end of the broken DNA strand followed by a “controlled rotation” of the broken
scissile strand nearby the unbroken strand, bring about in the DNA superhelical tension
relaxation. Ultimately, the 5′ end of the scissile strand facilitates a nucleophilic attack on the
phosphotyrosyl-DNA phosphodiester to transfer the DNA and release the enzyme, which ends
the catalytic cycle of the enzyme [35,36]. Trapped Top1ccs generate unfavourable lesions,
which lead to the formation of DNA double-strand breaks (DSBs) upon collision with
continuing replication forks and/or transcription machinery and are responsible for the killing
of proliferating malignant cancerous cells [37,38].
Quinoline containing compounds represent a large number of chemotherapeutic agents
demonstrating cytotoxic activity through DNA intercalation and hence affecting the replication
of DNA [39]. Topo I play a crucial role in cell proliferation and considered as chief target for
the prevention of prompt proliferation of cancerous cells [36]. A number of research studies
have been carried out on quinoline derivatives act by inhibiting human DNA topoisomerase
complex [11].
Subsequently, the molecular docking of all the active compounds (18b, 18d, 18e, 18f, 18g,
18h, 18i, 18j, 18l) with the target protein, human DNA topoisomerase-I enzyme (PDB: 1SC7)
has been carried out using QIAGEN CLC Drug Discovery Work Bench 3.0. Agreement of
Validation of Molecular Docking performed was emphasised by redocking of co-crystallized
ligand Indenoisoquinoline (M38 C990) on to the receptor pocket. Validated Docking results
are in good concurrence with existing co-crystal at the active site with an RMSD of 0.024
Ao[40]. Results of the docking study of the active compounds (18b, 18d, 18e, 18f, 18g, 18h,
18i, 18j, 18l) into the active site of enzyme revealed different molecular interactions exhibiting
hydrogen bonding,  interactions and hydrophobic interactions between the synthesized
ligands and topoisomerase enzyme (Table 5 and Figure 4).
All the docked molecules exhibited pronounced interactions both with Nucleotides (DNA,
TGP) and topoisomerase subunits Arg-364 (active site interaction) by hydrogen bonding
through the nitrogen atoms of hydrazone linkage. The CLC dock score for all the nine active
18

compounds was observed in between -35.54 to -57. 65. The highest dock score (-57.65) was
observed for compound 18l (Table 5).The most potent and active compound of the series 18j
interacted with the amino acid residue Arg-364 and TGP base pair of the DNA through
hydrogen bonding with dock score of -40.45 (Table 5). Compound 18j also showed
hydrophobic interaction with Asn-722, Leu-721, Thr-718, Lys-425 amino acid residues.
Molecules exhibited interactions both with DNA and topoisomerase subunits by hydrogen
bonding except compound 18l, which was intercalated only with DNA by hydrogen bonding
and compound 18i, interacted only with Arg-364 amino acid residue of topoisomerase enzyme
by forming a hydrogen bond between NH2 of Arg-364 and nitrogen atom of hydrazone linkage
(Figure 4). Compounds 18e and 18g interacted with NH2 of Lys-425 amino acid residue by
hydrogen bonding through oxygen of alkoxy side chain. Along with the hydrogen bonding all
active compounds showed number of hydrophobic interaction with different amino acid
residues including Asn-722, Leu-721, Pro-431, Asp-533 Thr-718 and Asp-533.
From the results of docking, all the active compounds approach to DNA towards the DNA
cleavage site in the DNA–topoisomerase-I and developing a stable ternary complex through
noncovalent interactions and thereby stabilizing it which leads to inhibitory effect on DNA–
topoisomerase-I. This prevents DNA re-ligation and therefore causes DNA damage which
results in apoptosis[41]. Binding interactions of some active compounds with the DNA–
topoisomerase-I receptor are shown in Figure 4.
Table 5. Molecular docking results of human DNA topoisomerase-I enzyme (PDB: 1SC7).
Hydrogen bonds
within 6A0
CLC
Dock
Score
Hydrophobic/Stearic interactions
within 6A0
Ligand
18b
Arg-364, (TGP)
Asn-722, Thr-718
-51.83
18d
18f
18e
Arg-364, (TGP)
Arg-364, (TGP)
Lys-425, (TGP)
Asn-722, Asp-533, Thr-718
Arg-316
Arg-364, Asn-352, Glu-356, Lys-425
Asn-722, Leu-721, Pro-31, Thr-718, Gln-748,
Lys-751, Thr-747
-36.11
-45.44
18h
Arg-364, (TGP)
-53.38
Arg-364, Asn-722, Asp-533, Glu-356, Lys-
374, Lys-425, Thr-718
Asn-722, Leu-721, Thr-718, Lys-425
Arg-364, Asn-722, Asp-533, Glu-356, Lys-
374, Lys-425, Thr-718
18g
18j
18i
Lys-425, (TGP)
Arg-364, (TGP)
Arg-364
-43.43
-40.45
-35.54
Asn-352, Glu-356, Gly-359, Ile-355, Lys-354,
Lys-374, Pro-357
18l
(TGP)
-57.65
Pro-358
19

Figure 4. Binding pose of the active compounds (A) 18b, (B) 18f, (C) 18h and (D) 18j in the
active site of DNA–topo-I receptor (PDB: 1SC7). Blue dotted lines represent the hydrogen
bond with amino acid residue of the enzyme and DNA base pairs and (E) represents the
validation of topo-I co-crystallised ligand interaction with Arg 364 and Leu 429.
3.0 Experimental section
3.1 Chemistry
The chemicals were used as received from local companies without further purification.
Organic solvents were purified by distillation prior to use. Column chromatography was carried
out using silica gel (60-120 mesh). Thin layer chromatography was performed on the pre-
coated silica gel 60 F254 aluminium sheets. Melting points are determined in open capillary and
are uncorrected. FT-IR spectra were recorded on Bruker Alpha FTIR spectrometer between
4000-400 cm-1 in solid state as KBr discs. The NMR spectra were recorded on 400 MHz Bruker
Avance-III instrument and chemical shifts are given in parts per million. In the NMR data for
20

1
19F decoupled H NMR experiments, the data for the affected signals only are included. 19F
chemical shift values are of 1H decoupled 19F signals. Thermo Fischer elemental analyser was
used for elemental analysis. ESI mass spectra were recorded on Waters’ Xevo G2-XS QToF
mass spectrometer at Zydus Research Centre, Ahmedabad. Compounds 9a/9b [20,42], 12a-f
[22], 15 and 17 [23,24] were synthesized according to the procedures reported in the literature
(see supporting information).
3.2 General Procedure for the Synthesis of 4-{ N’-arylidene-hydrazinyl}-6-bromo-2-
methylquinoline.
The final target compounds were synthesized by following the general method [20,42].
Alkyloxy benzaldehydes 12/15/17 (0.46 mmol) and 6-bromo-2-methyl-quinolin-4-yl-
hydrazine 9a (0.46 mmol) were dissolved in ethanol (5 mL). A catalytic amount of
concentrated sulphuric acid was added. The reaction mixture was heated at 80 °C for 4 h, and
then kept at room temperature overnight. The resultant solid separated was filtered, washed
with chilled ethanol and crystallized from ethanol to afford the pure final hydrazones 18. Yield:
76-82%.
3.2.1 [6-Bromo-4-{N’-(4-ethyloxybenzylidene)hydrazinyl}-2-methylquinoline], 18a.
Yield = 0.072g, 82%; Yellow Solid; MP = 202 °C; IR (KBr) cm-1: 3288, 2977, 1569, 1242,
846, 771; 1H NMR (400 MHz, DMSO-d6, δ ppm): 1.1 (t, 3H, terminal -CH3), 2.73 (s, 3H, -
CH3), 4.12 (q, 2H, alkoxy-OCH2), 7.06 (d, 2H, J = 8.8 Hz, Ar-H), 7.55 (s, 1H, -N=CH-), 7.87
(d, 2H, J = 8.8 Hz, Ar-H), 7.90 (d, 1H, J = 9.2 Hz, Ar-H), 8.01 (d, 1H, J = 9.2 Hz, Ar-H), 8.55
(s, 1H, Ar-H), 8.70 (d, 1H, J = 2 Hz, Ar-H), 12.22 (s, 1H, -NH); 13C NMR (100 MHz, DMSO-
d6, δ ppm): 15.5 (-CH3), 20.6 (-CH3), 63.9 (-OCH2), 101.0, 115.7, 115.9, 119.4, 122.6, 125.7,
126.1, 130.05, 136.6, 137.71, 150.6, 151.16 155.1, 161.4; Anal. Calcd. for C19H18BrN3O: C,
59.39; H, 4.72; N, 10.94; found C, 59.36; H, 4.78; N, 10.88; Mass (TOF MS ES+): m/z 384.07
(M+H)+, 386.06 (MH+2)+.
3.2.2 [6-Bromo-2-methyl-4-{N’-(4-propyloxybenzylidene)hydrazinyl}quinoline], 18c.
Yield = 0.071g, 78%; Yellow Solid; MP = 204 °C; IR (KBr) cm-1: 3211, 2964, 1605, 1249,
1168, 913, 845; 1H NMR (400 MHz, DMSO-d6, δ ppm): 0.99 (t, 3H, terminal -CH3), 1.75 (m,
2H, -CH2-), 2.71 (s, 3H, -CH3), 4.01 (t, 2H, alkoxy-OCH2), 7.06 (d, 2H, J = 8.8 Hz, Ar-H),
7.53 (s, 1H, -N=CH-), 7.81 (m, 3H, Ar-H), 8.11 (d, 1H, J = 9.2 Hz, Ar-H), 8.53 (s, 1H, Ar-H),
21

8.82 (s, 1H, Ar-H), 12.22 (s, 1H, -NH); 13C NMR (100 MHz, DMSO-d6, δ ppm): 10.8 (-CH3),
15.5 (-CH3), 22.4 (-CH2), 69.7 (-OCH2), 101.0, 115.4, 115.8, 119.5, 122.2, 125.7, 126.1, 130.0,
136.7, 137.7, 150.7, 150.9, 155.0, 161.6 ; Anal. Calcd. for C20H20BrN3O: C, 60.31; H, 5.06; N,
10.55; found C, 60.36; H, 5.01; N, 10.59; Mass (TOF MS ES+): m/z 398. 09 (M+H)+, 400.08
(MH+2)+.
3.2.3 [6-Bromo-4-{N’-(4-butyloxybenzylidene)hydrazinyl}-2-methylquinoline], 18e.
Yield = 0.075g, 80%; Yellow Solid; MP = 186 °C; IR (KBr) cm-1: 3211, 2935, 1604, 1445,
1251, 772; 1H NMR (400 MHz, DMSO-d6, δ ppm): 0.95 (t, 3H, terminal -CH3), 1.46 (m, 2H, -
CH2), 1.72 (m, 2H, -CH2), 2.72 (s, 3H, -CH3), 4.05 (t, 2H, alkoxy-OCH2), 7.06 (d, 2H, J = 8.4
Hz, Ar-H), 7.54 (s, 1H, -N=CH-), 7.82 (m, 3H, Ar-H), 8.11 (dd, 1H, J = 8.8 Hz, Ar-H), 8.54
(s, 1H, Ar-H), 8.83 (s, 1H, Ar-H), 12.23 (s, 1H, -NH); 13C NMR (100 MHz, DMSO-d6, δ ppm):
14.1 (-CH3), 15.5 (-CH3), 19.1 (-CH2), 31.0 (-CH2), 67.9 (-OCH2), 101.0, 115.3, 115.8, 119.5,
122.2, 125.6, 126.0, 130.0, 136.7, 137.7, 150.6, 150.9, 155.1, 161.6 ; Anal. Calcd. for
C21H22BrN3O: C, 61.17; H, 5.38; N, 10.19; found C, 61.15; H, 5.36; N, 10.16; Mass (TOF MS
ES+): m/z 412.10 (M+H)+, 414.10 (MH+2)+.
3.2.4 [6-Bromo-2-methyl-4-{N’-4-(pentyloxybenzylidene)hydrazinyl}quinoline], 18g.
Yield = 0.066g, 68%; Yellow Solid; MP = 194 °C; IR (KBr) cm-1: 3211, 2935, 1643, 1253,
899; 1H NMR (400 MHz, DMSO-d6, δ ppm): 0.90 (t, 3H, terminal -CH3), 1.52 (m, 4H, -CH2),
1.74 (m, 2H, -CH2), 2.70 (s, 3H, -CH3), 4.05 (t, 2H, alkoxy-OCH2), 7.06 (d, 2H, J = 8.4 Hz,
Ar-H), 7.52 (s, 1H, -N=CH-), 7.81 (m, 3H, Ar-H), 8.08 (d, 1H, J = 8.4 Hz, Ar-H), 8.53 (s, 1H,
Ar-H), 8.82 (s, 1H, Ar-H), 12.18 (s, 1H, -NH); 13C NMR (100 MHz, DMSO-d6, δ ppm): 14.3
(-CH3), 20.5 (-CH3), 22.3 (-CH2), 28.0 (-CH2), 28.6 (-CH2), 68.2 (-OCH2), 100.9, 115.3, 115.7,
119.6, 122.1, 125.6, 126.0, 130.0, 136.7, 137.5, 150.7, 150.8, 154.8, 161.5; Anal. Calcd. for
C22H24BrN3O: C, 61.98; H, 5.67; N, 9.86; found C, 61.96; H, 5.69; N, 9.89; Mass (TOF MS
ES+): m/z 426.08 (M+H)+, 428.08 (MH+2)+.
3.2.5 [6-Bromo-4-{N’-(4-hexyloxybenzylidene)hydrazinyl}-2-methylquinoline], 18i.
Yield = 0.072g, 72%; Yellow Solid; MP = 190 °C; IR (KBr) cm-1: 3210, 2925, 1604, 1211,
772;1H NMR (400 MHz, DMSO-d6, δ ppm): 0.86 (t, 3H, terminal-CH3), 1.29 (d(b), 3H, -CH2),
1.40 (t(b), 2H, -CH2), 1.72 (m, 2H, -CH2), 2.68 (s, 3H, -CH3), 4.02 (t, 2H, alkoxy-OCH2), 7.03
(d, 2H, J = 8.8 Hz, Ar-H), 7.47 (s, 1H, -N=CH-), 7.80 (t(b), 3H, Ar-H), 7.06 (d, 1H, J = 8.8
Hz, Ar-H), 8.50 (s, 1H, Ar-H), 8.78 (s, 1H, Ar-H), 12.17 (s, 1H, -NH); 13C NMR (100 MHz,
22

DMSO-d6, δ ppm): 14.3 (-CH3), 20.7 (-CH3), 22.4 (-CH2), 25.5 (-CH2), 28.9 (-CH2), 31.4 (-
CH2), 68.2 (-OCH2), 101.0, 115.4, 115.9, 119.4, 122.5, 122.7, 125.6, 126.1, 130.0, 136.6,
137.7, 150.5, 150.7, 154.8, 161.5; Anal. Calcd. for C23H26BrN3O: C, 62.73; H, 5.95; N, 9.54;
found C, 62.75; H, 5.92; N, 9.59; Mass (TOF MS ES+): m/z 440.02 (M+H)+.
3.2.6 [6-Bromo-2-methyl-4-{N’-(4-octyloxybenzylidene)hydrazinyl}quinoline], 18k.
Yield = 0.087g, 82%; Yellow Solid; MP = 186 °C; IR (KBr) cm-1: 3242, 2925, 1602, 897; 1H
NMR (400 MHz, DMSO-d6, δ ppm): 0.84 (t, 3H, terminal -CH3), 1.25 (d(b), 8H, -CH2), 1.39
(d(b), 2H, -CH2), 1.68-1.74 (m, 2H, -CH2), 2.67 (s, 3H, -CH3), 4.02 (t, 2H, alkoxy-OCH2), 7.03
(d, 2H, J = 8.8 Hz, Ar-H), 7.45 (s, 1H, -N=CH-), 7.79 (t(b), 3H, Ar-H), 8.05 (d, 1H, J = 8.8
Hz, Ar-H), 8.49 (s, 1H, Ar-H), 8.76 (s, 1H, Ar-H), 12.17 (s, 1H, -NH); 13C NMR (100 MHz,
DMSO-d6, δ ppm): 14.3 (-CH3), 20.4 (-CH3), 22.5 (-CH2), 25.8 (-CH2), 28.9 (-CH2), 29.0 (-
CH2), 29.1 (-CH2), 31.6 (-CH2), 68.1 (-OCH2), 100.9, 115.4, 115.9, 119.7, 122.3, 126.0, 130.0,
136.7, 137.6, 138.0, 150.9, 155.1, 156.5, 161.4 ; Anal. Calcd. for C25H30ClN3O: C, 64.10; H,
6.46; N, 8.97; found C, 64.14; H, 6.49; N, 8.95; Mass (TOF MS ES+): m/z 468.16 (M+H)+,
470.16 (MH+2)+.
3.2.7 4-[N’-{3,4-Bis-octyloxybenzylidene}hydrazinyl]-6-bromo-2-methylquinoline, 18m.
Yield = 0.106g, 78%; Yellow Solid; MP = 184 °C; IR (KBr) cm-1: 3212, 2923, 1607, 1270,
858; 1H NMR (400 MHz, DMSO-d6, δ ppm): 0.85 (t, 6H, terminal -CH3), 1.26 (d(b), 16H, -
CH2), 1.46 (d(b), 4H, -CH2), 1.74 (m, 4H, -CH2), 2.66 (s, 3H, -CH3), 4.01 (t, 2H, alkoxy-
OCH2), 4.06 (t, 2H, alkoxy-OCH2), 7.05 (d, 1H, J = 8.4 Hz, Ar-H), 7.34 (d, 1H, J = 8.0 Hz,
Ar-H), 7.42 (s, 1H, Ar-H), 7.46 (s, 1H, -N=CH-), 7.77 (d, 1H, J = 9.2 Hz, Ar-H), 8.01 (d, 1H,
J = 8.8 Hz, Ar-H), 8.45 (s, 1H, Qui-H), 8.76 (s, 1H, Qui-H), 11.97 (s, 1H, -NH); 13C NMR (100
MHz, DMSO-d6, δ ppm): 14.3 (-CH3), 20.6 (-CH3), 22.5 (-CH2), 26.0 (-CH2), 26.1 (-CH2),
29.2 (-CH2), 29.3 (-CH2), 31.7 (-CH2), 69.1 (-OCH2), 101.0, 111.8, 113.4, 115.4, 122.2, 123.4,
126.3, 131.3, 134.1, 137.3, 149.2, 151.1, 152.0, 155.0, 161.5; Anal. Calcd. for C33H46BrN3O2:
C, 66.43; H, 7.77; N, 7.04; found C, 66.43; H, 7.77; N, 7.04; Mass (TOF MS ES+): m/z 596.29
(M+H)+, 598.28 (MH+2) +.
3.2.8 6-Bromo-2-methyl-4-[N’-{3,4,5-tris-octyloxybenzylidene}hydrazinyl]quinoline, 18o.
Yield = 0.122g, 74%; Yellow Solid; MP = 180 °C; IR (KBr) cm-1: 3238, 2924, 1607, 1117,
722; 1H NMR (400 MHz, DMSO-d6, δ ppm): 0.85 (t, 9H, terminal -CH3), 1.27 (s(b), 26H, -
CH2), 1.55 (d(b), 6H, -CH2), 1.73 (m, 4H, -CH2), 2.73 (s, 3H, -CH3), 3.91 (t, 6H, alkoxy-
23

OCH2), 7.12 (s(b), 2H, Ar-H), 7.51 (s, 1H, -N=CH), 7.83 (d, 1H, J = 9.2 Hz, Ar-H), 7.11 (d,
1H, J = 9.2 Hz, Ar-H), 8.49 (s, 1H, Ar-H), 8.83 (s, 1H, Ar-H), 12.25 (s, 1H, -NH); 13C NMR
(100 MHz, DMSO-d6, δ ppm): 14.3 (-CH3), 20.6 (-CH3), 22.5 (-CH2), 26.0 (-CH2), 26.1 (-
CH2), 29.2 (-CH2), 30.3 (-CH2), 31.7 (-CH2), 68.4 (-OCH2), 101.2, 107.0, 119.7, 122.3, 122.3,
127.1, 129.2, 134.2, 137.6, 140.6, 150.9, 151.4, 153.4, 155.6; Anal. Calcd. for C41H62ClN3O3:
C, 67.94; H, 8.62; N, 5.80; found C, 67.91; H, 8.65; N, 5.84; Mass (TOF MS ES+): m/z 724.34
(M+H)+, 726.34 (MH+2)+.
3.3 General Procedure for the Synthesis of 4-{ N’-arylidene-hydrazinyl}-6-chloro-2-
methylquinoline.
The final target compounds were synthesized by following the general method [20,42].
Alkyloxy benzaldehydes 12/15/17 (0.46 mmol) and 6-chloro-2-methyl-quinolin-4-yl-
hydrazine 9b (0.46 mmol) were dissolved in ethanol (5 mL). A catalytic amount of
concentrated sulphuric acid was added. The reaction mixture was heated at 80 °C for 4 h, and
then kept at room temperature overnight. The resultant solid separated was filtered, washed
with chilled ethanol and crystallized from ethanol to afford the pure final hydrazones 18. Yield:
76-82%.
3.3.1 6-Chloro-4-{N’-(4-ethyloxybenzylidene)hydrazinyl}-2-methylquinoline], 18b.
Yield = 0.072g, 74%; Yellow Solid; MP = 210 °C; IR (KBr) cm-1: 3288, 2977, 1606, 1211,
847; 1H NMR (400 MHz, DMSO-d6, δ ppm): 1.10 (t, 3H, terminal -CH3), 2.73 (s, 3H, -CH3),
4.12 (q, 2H, alkoxy-OCH2), 7.05 (d, 2H, J = 8.8 Hz, Ar-H), 7.53 (s, 1H, -N=CH-), 7.87 (d, 2H,
J = 8.4 Hz, Ar-H), 7.89 (d, 1H, J = 9.2 Hz, Ar-H), 8.00 (d, 1H, J = 8.8 Hz, Ar-H), 8.54 (s, 1H,
Ar-H), 8.69 (s, 1H, Ar-H), 12.19 (s, 1H, -NH); 13C NMR (100 MHz, DMSO-d6, δ ppm): 15.5
(-CH3), 20.6 (-CH3), 63.9 (-OCH2), 101.1, 115.3, 115.5, 122.3, 122.7, 125.6, 126.1, 130.1,
131.3, 134.1, 150.7, 151.1, 155.2, 161.4; Anal. Calcd. for C19H18ClN3O: C, 67.16; H, 5.34; N,
12.37; found C, 67.16; H, 5.34; N, 12.37; Mass (TOF MS ES+): m/z 340.12 (M+H)+, 342.12
(MH+2)+.
3.3.2 [6-Chloro-2-methyl-4-{N’-(4-propyloxybenzylidene)hydrazinyl}quinoline], 18d.
Yield = 0.075g, 74%; Yellow Solid; MP = 200 °C; IR (KBr) cm-1: 3207, 2944, 1600, 1209,
772; 1H NMR (400 MHz, DMSO-d6, δ ppm): 1.10 (t, 3H, terminal -CH3), 1.75 (m, 2H, -CH2-
24

), 2.72 (s, 3H, -CH3), 3.73 (t, 2H, alkoxy-OCH2), 7.06 (d, 2H, J = 8.4 Hz, Ar-H), 7.53 (s, 1H, -
N=CH-), 7.88 (m, 3H, Ar-H), 8.00 (dd, 1H, J = 2.0 Hz, J = 8.8 Hz, Ar-H), 8.54 (s, 1H, Ar-H),
8.68 (d, 1H, J = 2.0 Hz, Ar-H), 12.21 (s, 1H, -NH); 13C NMR (100 MHz, DMSO-d6, δ ppm):
10.8 (-CH3), 15.5 (-CH3), 22.4 (-CH2), 69.7 (-OCH2), 101.1, 115.4, 115.5, 122.3, 122.7, 125.6,
126.1, 130.1, 131.3, 134.2, 150.7, 151.1, 155.2, 161.6; Anal. Calcd. for C20H20ClN3O: C, 67.89;
H, 5.70; N, 11.88; found C, 67.83; H, 5.75; Cl, 11.90; Mass (TOF MS ES+): m/z 354.13
(M+H)+, 356.13 (MH+2)+.
3.3.3 [4-{N’-(4-Butyloxybenzylidene)hydrazinyl}-6-chloro-2-methylquinoline], 18f.
Yield = 0.078g, 76%; Yellow Solid; MP = 198 °C; IR (KBr) cm-1: 3202, 2935, 1643, 1200,
772; 1H NMR (400 MHz, DMSO-d6, δ ppm): 0.94 (t, 3H, terminal -CH3), 1.46 (m, 3H, -CH2),
1.72 (m, 2H, -CH2), 2.71 (s, 3H, -CH3), 4.04 (t, 2H, alkoxy-OCH2), 7.06 (d, 2H, J = 8.4 Hz,
Ar-H), 7.52 (s, 1H, -N=CH-), 7.85 (d, 1H, J = 8.8 Hz, Ar-H), 7.88 (d, 2H, J = 9.2 Hz, Ar-H),
7.98 (d, 1H, J = 9.2 Hz, Ar-H), 8.53 (s, 1H, Ar-H), 8.68 (s, 1H, Ar-H), 12.17 (s, 1H, -NH); 13C
NMR (100 MHz, DMSO-d6, δ ppm): 14.1 (-CH3), 15.5 (-CH3), 19.1 (-CH2), 31.1 (-CH2), 67.9
(-OCH2), 101.9, 115.4, 115.8, 119.5, 122.6, 125.6, 126.1, 130.0, 131.3, 133.9, 150.6, 150.9,
155.1, 161.6; Anal. Calcd. for C21H22ClN3O: C, 68.56; H, 6.03; N, 11.42; found C, 68.51; H,
6.01; N, 11.44; Mass (TOF MS ES+): m/z 368.15 (M+H)+, 370.15 (MH+2)+.
3.3.4 [6-Chloro-2-methyl-4-{N’-(4-pentyloxybenzylidene)hydrazinyl}quinoline], 18h.
Yield = 0.079g, 72%; Yellow Solid; MP = 190 °C; IR (KBr) cm-1: 3235, 2932, 1604, 1253,
772; 1H NMR (400 MHz, DMSO-d6, δ ppm): 0.90 (t, 3H, terminal -CH3), 1.50 (m, 4H, -CH2),
1.74 (m, 2H, -CH2), 2.72 (s, 3H, -CH3), 4.05 (t, 2H, alkoxy-OCH2), 7.06 (d, 2H, J = 8.8 Hz,
Ar-H), 7.53 (s, 1H, -N=CH-), 7.88 (d, 2H, J = 8.8 Hz, Ar-H), 7.88 (d, 1H, J = 8.8 Hz, Ar-H),
8.00 (d, 1H, J = 8.8 Hz, Ar-H), 8.54 (s, 1H, Ar-H), 8.69 (s, 1H, Ar-H), 12.20 (s, 1H, -NH); 13C
NMR (100 MHz, DMSO-d6, δ ppm): 14.3 (-CH3), 20.7 (-CH3), 22.3 (-CH2), 28.1 (-CH2), 28.7
(-CH2), 68.2 (-OCH2), 101.0, 115.3, 115.5, 122.5, 122.6, 126.1, 130.0, 131.2, 134.0, 137.7,
150.5, 151.0, 155.2, 161.5; Anal. Calcd. for C22H24ClN3O: C, 69.19; H, 6.33; N, 11.00; found
C, 69.16; H, 6.35; N, 11.04; Mass (TOF MS ES+): m/z 382.13 (M+H)+, 384.13 (MH+2)+.
3.3.5 [6-Chloro-4-{N’-(4-hexyloxybenzylidene)hydrazinyl}-2-methylquinoline], 18j.
Yield = 0.086g, 76%; Yellow Solid; MP = 188 °C; IR (KBr) cm-1: 3242, 2928, 1604, 1252,
846; 1H NMR (400 MHz, DMSO-d6, δ ppm): 0.88 (t, 3H, terminal -CH3), 1.38 (m, 6H, -CH2),
1.73 (t(br), 2H, -CH2), 2.72 (s, 3H, -CH3), 4.05 (t, 2H, alkoxy-OCH2), 7.06 (d, 2H, J = 8.4 Hz,
25

Ar-H), 7.53 (s, 1H, -N=CH- proton), 7.86 (d, 2H, J = 8.8 Hz, Ar-H), 7.89 (d, 1H, J = 9.2 Hz,
Ar-H), 7.99 (d, 1H, J = 8.8 Hz, Ar-H), 8.54 (s, 1H, Ar-H), 8.69 (s, 1H, Ar-H), 12.20 (s, 1H, -
NH); 13C NMR (100 MHz, DMSO-d6, δ ppm): 14.3 (-CH3), 20.5 (-CH3), 22.4 (-CH2), 25.5 (-
CH2), 28.9 (-CH2), 31.4 (-CH2), 68.2 (-OCH2), 100.9, 115.3, 122.2, 122.6, 125.6, 126.0, 130.0,
131.4, 137.3, 150.7, 150.9, 155.0, 161.5; Anal. Calcd. for C22H24ClN3O: C, 72.71; H, 6.90; N,
11.06; found C, 72.74; H, 6.94; N, 11.09; Mass (TOF MS ES+): m/z 396.15 (M+H)+, 398.14
(MH+2)+.
3.3.6 [6-Chloro-2-methyl-4-{N’-(4-octyloxybenzylidene)hydrazinyl}quinoline], 18l.
Yield = 0.098g, 79%; Yellow Solid; MP = 182 °C; IR (KBr) cm-1: 3244, 2926, 1604, 1250,
772; 1H NMR (400 MHz, DMSO-d6, δ ppm): 0.84 (t, 3H, terminal -CH3), 1.25 (d(b), 8H, -
CH2), 1.39 (d(b), 2H, -CH2), 1.70 (m, 2H, -CH2), 2.69 (s, 3H, -CH3), 4.02 (t, 2H, alkoxy-
OCH2), 7.02 (d, 2H, J = 8.8 Hz, Ar-H), 7.46 (s, 1H, -N=CH-), 7.80 (d, 2H, J = 8.8 Hz, Ar-H),
7.86 (d, 1H, J = 9.2 Hz, Ar-H), 7.97 (d, 1H, J = 8.8 Hz, Ar-H), 8.50 (s, 1H, Ar-H), 8.63 (s, 1H,
Ar-H), 12.17 (s, 1H, Ar-H); 13C NMR (100 MHz, DMSO-d6, δ ppm): 14.3 (-CH3), 20.4 (-
CH3), 22.5 (-CH2), 25.9 (-CH2), 28.9 (-CH2), 29.0 (-CH2), 29.1 (-CH2), 31.6 (-CH2), 68.2 (-
OCH2), 100.9, 115.3, 115.8, 122.0, 122.5, 126.0, 130.0, 131.4, 134.1, 137.2, 150.7, 150.9,
154.9, 161.5; Anal. Calcd. for C25H30ClN3O: C, 70.82; H, 7.13; N, 9.91; found C, 70.80; H,
7.16; N, 9.94; Mass (TOF MS ES+): m/z 424.21 (M+H)+, 426.47 (MH+2)+.
3.3.7 4-[N’-{3,4-Bis-octyloxybenzylidene}hydrazinyl]-6-chloro-2-methylquinoline, 18n.
Yield = 0.112g, 71%; Yellow Solid; MP = 182 °C; IR (KBr) cm-1: 3242, 2919, 1604, 1239,
772;1H NMR (400 MHz, DMSO-d6, δ ppm): 0.85 (t, 6H, terminal -CH3), 1.30 (m, 16H, -CH2),
1.45 (t(b), 4H, -CH2), 1.74 (m, 4H, -CH2), 2.72 (s, 3H, -CH3), 4.03 (t, 2H, alkoxy-OCH2), 4.06
(t, 2H, J = 6.4 Hz, -OCH2), 7.06 (d, 1H, J = 8.4 Hz, Ar-H), 7.37 (d, 1H, J = 8.4 Hz, Ar-H), 7.50
(d(b), 2H, Ar-H), 7.89 (d, 1H, J = 8.8 Hz, Ar-H), 7.99 (d, 1H, J = 9.2 Hz, Ar-H), 8.50 (s, 1H,
Ar-H), 8.69 (s, 1H, Ar-H), 12.20 (s, 1H, -NH); 13C NMR (100 MHz, DMSO-d6, δ ppm): 14.3
(-CH3), 20.6 (-CH3), 22.5 (-CH2), 26.0 (-CH2), 26.1 (-CH2), 29.2 (-CH2), 29.3 (-CH2), 31.7 (-
CH2), 69.1 (-OCH2), 101.0, 111.8, 113.4, 115.4, 122.2, 122.6, 126.3, 131.3, 134.1, 137.3,
149.2, 151.1, 152.0, 155.0 ; Anal. Calcd. for C33H46ClN3O2: C, 73.92; H, 8.65; N, 7.84; found
C, 73.90; H, 8.61; N, 7.80; Mass (TOF MS ES+): m/z 552.33 (M+H)+, 554.34 (MH+2)+.
3.3.8
18p.
6-Chloro-2-methyl-4-[N’-{3,4,5-tris-octyloxybenzylidene}hydrazinyl]quinoline,
26

Yield = 0.141g, 72%; Yellow Solid; MP = 178 °C; IR (KBr) cm-1: 3247, 2923, 1606, 1206,
880; 1H NMR (400 MHz, DMSO-d6, δ ppm): 0.86 (t, 9H, terminal -CH3), 1.27 (s(b), 26H, -
CH2), 1.46 (d(b), 6H, -CH2), 1.70 (m, 4H, -CH2), 2.73 (s, 3H, -CH3), 3.91 (t, 2H, alkoxy-
OCH2), 4.05 (t, 4H, alkoxy-OCH2), 7.01 (s(b), 2H, Ar-H), 7.49 (s, 1H, -N=CH-), 7.90 (d, 1H,
J = 8.8 Hz, Ar-H), 8.00 (d, 1H, J = 9.2 Hz, Ar-H), 8.48 (s, 1H, Ar-H), 8.68 (s, 1H, Ar-H), 12.24
(s, 1H, -NH); 13C NMR (100 MHz, DMSO-d6, δ ppm): 14.3 (-CH3), 20.6 (-CH3), 22.5 (-CH2),
26.0 (-CH2), 26.1 (-CH2), 29.2 (-CH2), 30.3 (-CH2), 31.7 (-CH2), 69.0 (-OCH2), 101.0, 106.6,
115.5, 119.7, 122.3, 122.7, 128.8, 131.3, 134.2, 137.6, 140.4, 151.2, 153.4, 155.2; Anal. Calcd.
for C41H62BrN3O3: C, 72.38; H, 9.18; N, 6.18; found C, 72.34; H, 9.14; N, 6.21; Mass (TOF
MS ES+): m/z 680.45 (M+H)+, 682.45 (MH+2)+.
4. Conclusion
In summary, a series of new quinoline possessing hydrazones 18a-p have been prepared and
structure of the new compounds was elucidated by using various spectroanalytical techniques.
Sixty human cancer cell lines (NCI60) were used to screen and evaluate their anti-proliferative
activity. Among all the submitted and selected compounds (total 16) at single dose (10 M)
level, nine of the compounds (18b, 18d, 18e, 18f, 18g, 18h, 18i, 18j, 18l) showed prominent
% inhibition of various cancer cell lines and further screened for five dilution assay. Five
dilutions screening data revealed that, all nine compounds showed remarkable anti-
proliferative activity with GI50 values ranging from 0.33-4.87 µM and LC50 values ranging
from 4.67 µM to >100 µM. Among all compound, 18j emerged as potent anticancer agent
showed highest activity and selectivity for cancer cell lines. From the comparison of the mean
values of GI50, TGI and LC50 of compound 18j with the clinically used anticancer agents,
bendamustine and chlorambucil, clearly quinolyl hydrazones holds promise as potential
anticancer agents. Added to this, all quinolyl hydrazones 18a-p demonstrated outstanding
antimicrobial activity (MIC 6.25-100 µg/mL) against the tested pathogenic strains.
Furthermore, molecular docking studies were performed provided an understanding about the
binding and interactions of the compounds into the active sites of the DNA-topoisomerase-I
receptor. Finally, it is conceivable that, further derivatization and investigations of this class of
compounds may be carried in a quest of new potential and selective anticancer agents.
Conflict of interest
Authors have no conflicts of interest.
27

Acknowledgements
The authors are thankful to The Developmental Therapeutics Program (DTP) of the NCI, USA
for in vitro anticancer screening. Financial assistance from the University Grants Commission
(UGC) New Delhi in form of the Major Research Project MRP 39-897/2010 SR to KDK is
acknowledged. NMR facility from Department of Science and Technology under FIST
programme is gratefully acknowledged. Analytical support was extended by Zydus Research
Centre, Ahmedabad.
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