Arylidenes of Quinolin-2-one scaffold as Erlotinib analogues with activities against leukemia through inhibition of EGFR TK/ STAT-3 pathways

Article abstract of DOI:10.1016/j.bioorg.2020.103628

A new series of 6-substiuted-4-(2-(4-substituted-benzylidene)hydrazinyl)quinolin-2(1H)-one derivatives have been designed and synthesized. The structure of the synthesized compounds was proved by ¹H NMR, ¹³C NMR, 2D NMR, mass and elemental analyses. The target compounds were evaluated for their in vitro cytotoxic activity against 60 cancer cell lines according to NCI protocol. Consequently, the most active compounds were further examined against the most sensitive leukemia RPMI-8226 and on healthy cell lines. 6-Chloro-derivative was the most active one; with IC₅₀ = 15.72 ± 1.21 and 46.05 ± 2.36 μM against RPMI-8226 and normal cell lines, respectively. Also, it showed a remarkable inhibitory activity compared to gefitinib on the EGFR TK mutant, wild and on H-RAS in addition to STAT-3 with IC₅₀ = 695.49 ± 21.8, 263.15 ± 15.13, 10.61 ± 0.27 and 1.753 ± 0.81 nM, respectively. Cell cycle analysis of RPMI-8226 cells treated with the 6-chloro-derivative showed cell cycle arrest at G2/M phase (supported by Caspases-3,8, BAX and Bcl-2 studies) with a significant pro-apoptotic activity as indicated by annexin V-FITC staining. Moreover, the docking studies ROCS analysis and Tanimoto scores supported the results. The study illustrated the effect of several factors on compounds activity.

Full text of DOI:10.1016/j.bioorg.2020.103628

Journal Pre-proofs
Arylidenes of Quinolin-2-one scaffold as Erlotinib analogues with activities
against leukemia through inhibition of EGFR TK/ STAT-3 pathways
Mohammed A.I. Elbastawesy, Mohamed Ramadan, Yaseen A.M.M. El-
Shaier, Ashraf A. Aly, Gamal El-Din A. Abuo-Rahma
PII:
S0045-2068(19)31912-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.103628
YBIOO 103628
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
9 November 2019
21 December 2019
25 January 2020
Please cite this article as: M.A.I. Elbastawesy, M. Ramadan, Y.A.M. El-Shaier, A.A. Aly, G.E-D. Abuo-Rahma,
Arylidenes of Quinolin-2-one scaffold as Erlotinib analogues with activities against leukemia through inhibition
of EGFR TK/ STAT-3 pathways, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2020.103628
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Arylidenes of Quinolin-2-one scaffold as Erlotinib analogues with
activities against leukemia through inhibition of EGFR TK/ STAT-3
pathways
Mohammed A. I. Elbastawesy,a Mohamed Ramadan,a Yaseen A. M. M. El-Shaier,b
Ashraf A. Aly,c* Gamal El-Din A Abuo-Rahmad*
aDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, 71524
Assiut, Egypt bDepartment of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat
City, Sadat-City, Menufia, Egypt, c* Department of Chemistry, Faculty of Science, Minia University, 61519
Minia, Egypt,.d* Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519 Minia,
Egypt
:
*To whom correspondence should be addressed:
*A. A. Aly: ashrafaly63@yahoo.com, +201006268742
* G. E-D. A. Abuo-Rahma: gamal.aborahama@mu.edu.eg, +201003069431
Abstract
A new series of 6-substiuted-4-(2-(4-substituted-benzylidene)hydrazinyl)quinolin-2(1H)-one
derivatives have been designed and synthesized. The structure of the synthesized compounds
1
was proved by H NMR, 13C NMR, 2D NMR, mass and elemental analyses. The target
compounds were evaluated for their in vitro cytotoxic activity against 60 cancer cell lines
according to NCI protocol. Consequently, the most active compounds were further examined
against the most sensitive leukemia RPMI-8226 and on healthy cell lines. 6-Chloro-derivative
was the most active one; with IC50 = 15.72±1.21 and 46.05±2.36 M against RPMI-8226 and
normal cell lines, respectively. Also, it showed a remarkable inhibitory activity compared to
gefitinib on the EGFR TK mutant, wild and on H-RAS in addition to STAT-3 with IC50
=
695.49±21.8, 263.15±15.13, 10.61±0.27 and 1.753±0.81 nM, respectively. Cell cycle analysis of
RPMI-8226 cells treated with the 6-chloro-derivative showed cell cycle arrest at G2/M phase
(supported by Caspases-3,8, BAX and Bcl-2 studies) with a significant pro-apoptotic activity as
indicated by annexin V-FITC staining. Moreover, the docking studies (modeling, ROCS and
Tanimoto scores) supported the results; the study illustrated the effect of several factors on
compounds activity.
Keywords: Quinolin-2-one, Antiproliferative; Molecular docking, EGFR- TK; STAT, RAS,
apoptotic, Docking; Openeye.
1

1. Introduction
Quinolones represents an interesting class as one of the nitrogen containing heterocycles. They
also constitute indispensable structural units in medicinal chemistry. Among various
heterocyclic compounds, researchers all over the world have been interested in the biological
applications of quinolone derivatives [1,2]. In recent years, the quinolone bioisoster,
quinazoline derivatives like gefitinib and erlotinib (Figure 1) have been approved by the FDA
as EGFR kinase inhibitors for the treatment of non-small cell lung and breast cancer [3-5].
Modification of scaffold with different substituents is a general method for the drug design and
development [6,7] with the possibility of obtaining more successful next generation derivatives
[8]. Recently many groups [9-11] reported that bioisosteric replacement of the quinazoline core
with quinoline-2-one ring, afforded many new compounds (e.g. PQ-1 and PQ-2, Figure 1) with
potent EGFR kinase inhibitory activity and remarkable apoptotic activities [11,12].
Schiff base moiety which is characterized by the presence of azomethine group (-C=N-) is an
exceptionally adaptable drug-like format that is being utilized broadly in the structure of cancer
treatments and cellular apoptosis [13-16]. These compounds exhibit anticancer activity by the
restraint of various kinds of enzymes [17], proteins [18] and receptors [19] which assume basic
roles in cell division [20].
Research and development of new anticancer therapeutic agents are of paramount importance
because of the innate ability of tumor cells to develop resistance to existing therapies [21]. The
progression of multiple drug resistance to anticancer agents in human tumor cells has been
recognized as major impediment to unbeaten cancer chemotherapy [22]. Thus, studies for the
identification of novel drugs and targets for the management of such diseases are at the cutting
edge. The hybridization of biologically active molecules is a powerful tool for the drug
discovery used to target a variety of diseases [23, 24]. In continuation to our previous works on
2

the quinolone scaffold [11,25-27], the target compounds were elaborated utilizing molecular
modeling approaches [11]. The design strategy of the present study (Figure 1) is constructed
based on the well-known biologically active entities quinoline-2-one with Schiff base hopping
synergistic improvement of the designed molecules as antitumor agents. To investigate the
possible anticancer mechanism of the synthesized compounds; EGFR, STAT and RAS
enzymatic assay was performed. Molecular docking study of the most active compounds inside
the active site of EGFR enzyme was done. Cell cycle analysis and apoptosis through caspase-3,
and 8 expression levels were screened, in addition to expression level of Bax and Bcl-2.
Cl
left arm
F
right
arm
right
arm
left arm
O
HN
HN
N
N
O
O
left arm
N
O
O
O
O
N
N
Ring replacment
(core modification)
core
Erlotinib
Gefitinib
OH
S
HN
N
N
N
N
N
R1
N
O
H
N
PQ-1
PQ-2
N
HN
R2
rigt arm
R2= H, EDG, EWG
R1
left arm
R1= H, EDG, EWG
N
H
O
core
New designed series
Figure 1. Rational for the design of the target compounds.
2. Results and discussion
2.1. Chemistry
The target compounds 6a-o were prepared according to Scheme 1. Compounds 1-5 were
synthesized according to the reported method [28-30] and their structures were confirmed by
matching their spectral data with the reported ones [30] . The key intermediates 4-hydrazino-
3

quinolin-2-ones 5a-c were prepared by heating at reflux compounds 4a-c with hydrazine hydrate
for nearly 10-15 h [31]. Refluxing 5a-c with different aldehyde derivatives in presence of acetic
acid as catalyst furnished the target 6-substiuted-4-(2-(4-substituted-benzylidene)hydrazinyl)-
quinolin-2(1H)-ones 6a-o (Scheme 1).
OH
Cl
N
NH2
Cl
R1
R1
R1
a
c
b
N
H
O
Cl
N
H
O
R1
2a-c
3a-c
1
4a-c
d
N
NHNH2
O
HN
R2
R1
1
R1
5a:
R = H
e
1
5b
: R = CH3
1
N
H
O
5c
: R = Cl
N
H
O
R2
5a-c
6a-o
R2 = H, OCH3, N(CH3)2, F, Cl
1
2
6a:
R =H ; R = H
1
2
1
2
6i
: R = Cl ; R = N(CH3)2
6b
: R = CH3; R = H
1
2
1
2
6j
: R = H ; R = F
6c
6d
: R = Cl ; R = H
1
2
1
2
6k
: R = CH3 ; R = F
: R = H ; R = OCH3
1
2
1
2
6l
: R = Cl ; R = F
6e
: R = CH3 ; R = OCH3
1
2
1
2
6m
: R = H ; R = Cl
6f
: R = Cl ; R = OCH3
1
2
1
2
6n
: R = CH3 ; R = Cl
6g
: R = H ; R = N(CH3)2
1
2
1
2
6o
: R = Cl ; R = Cl
6h
: R = CH3 ; R = N(CH3)2
Scheme 1. Synthesis of target compounds 6a-o
Reagent and reaction conditions: a) Diethyl malonate, PPA, reflux 3 h; b) POCl3, reflux 2 h; c)
AcOH, reflux 10 h; d) NH2NH2·H2O (85%), EtOH, reflux 10-15 h; e) AcOH, EtOH, reflux 5-8 h.
The spectral and elemental data results revealed that all 6a-o derivatives underwent the reaction
smoothly to give the respective Schiff base-quinoline-2-one structure in 70-95% yield.1H NMR
spectra experienced the disappearance of NH2 signal and appearance of doublet of doublet
characteristic pattern in aromatic  6.70-8.00 ppm region which augments formation of Schiff
4

base. As a representative example, compound 6h, NMR spectra showed a singlet signals at
10.89 and 10.42 ppm for two NH groups. Also as mentioned; a characteristic pattern for the
additional four phenyl protons at  7.56 and 6.77 ppm represented di para substituted phenyl.
Moreover, 6H singlet signal at  2.98 which distinctive as dimethylamino protons. The
distinguished azomethine CH proton appeared at  8.26 ppm their attached carbons which
resonate at C 39.78 ppm gives HMBC correlation with the nitrogen at N 52.8, also with a non-
protonated carbon at C 151.14, assigned as para phenyl carbon. Moreover, the imine carbon
appears at C 144.60. represents (C=N) moiety giving HMBC correlation with the sp2 nitrogen
at N 308.2, assigned as N-4c, and with one of the two co-resonant nitrogens at N 140.4,
assigned as N-4b (Table 1).
Table 1: NMR spectroscopic assignments of compound 6h
1H NMR
10.89 (s; 1H)
COSY
6.14
Assignment
H-1
10.42 (s; 1H)
8.26 (s; 1H)
7.85 (s; 1H)
7.56 (d, J = 8.8; 2H)
7.31 (d, J = 8.4; 1H)
7.18 (d, J = 8.3; 1H)
6.77 (d, J = 8.8; 2H)
6.14 (s; 1H)
H-4b
H-4d
H-5
H-o
H-7
H-8
H-m
H-3
7.31, 2.38
6.77
7.85, 7.18, 2.38
7.31
7.56
10.89
m
p
4a
N(CH3)2
p' p"
o
4b
4c
N
4d
HN
4
i
6a
5
3
6
2.98 (s; 6H)
H-p”
H-6a
Assignment
N-4c
7
N
O
2
1
8a
8
H
2.38 (s; 3H)
7.85, 7.31
HMBC
10.42, 8.26
7.18, 6.14
8.26, 6.14
6.77, 2.98
15N NMR
308.2
HSQC
140.4
140.4
52.8
10.89
10.42
N-1
N-4b
N-p’
13C NMR
162.80
151.14
148.18
144.60
137.34
131.31
129.45
127.85
122.20
121.38
HSQC HMBC
Assignment
C-2
10.42
7.56, 2.98
10.42, 7.85, 7.18, 6.14
10.42, 7.56
7.85, 7.31, 7.18, 2.38
7.85, 2.38
7.85, 7.18, 2.38
7.56
8.26, 6.77
C-p
C-4
C-4d
C-8a
C-7
C-6
C-o
C-i
C-5
8.26
7.31
7.56
7.85
7.31, 7.18, 2.38
5

115.53
111.94
111.79
93.17
39.78
20.64
7.18
6.77
C-8
7.56, 6.77, 2.98
10.89, 10.42, 7.18, 6.14
10.89, 10.42
2.98
C-m
C-4a
C-3
C-p”
C-6a
6.14
2.98
2.38
7.85, 7.31
The mass spectra and elemental analyses indicated that the molecular weight is consistent with
1
the molecular formula of the target compounds. In another example, H NMR spectrum of 6j
showed a broad singlet signals at δ 11.17 and 10.83 ppm (disappeared upon deterioration)
represent both NH protons. A doublet of doublet pattern at δ 7.80, 7.30 ppm ascribed for four
phenyl protons.13C NMR spectrum of 6j showed peak at δ 162.25 ppm assigned for the C-F
carbon. In addition, other signals at δ142.08 ppm for (C=N) group and peak at δ 160.70 ppm
assigned to the (C=O) of the quinoline-2-one moiety.
2.2. Biology
2.2.1 Evaluation of in vitro antiproliferative activity for compounds 6a-o.
The target compounds 6a-o were selected by National Cancer Institute (NCI, Bethesda, ML,
USA (http://www.dtp.nci.nih.gov.) for evaluation at single concentration of 10 µM towards
panel of sixty cancer cell lines of nine diverse tissues according to NCI protocol, Appendix A.
The screening results were reported as the percent growth inhibition of treated cells compared to
untreated control cells. Results in Table 2 revealed that most compounds showed weak to
moderate activities on most tested cell lines specially leukemia and small cell lung cancer cell
lines. Compounds 6c showed promising activity on EKVX cell line achieving 53.96% of
growth inhibition. On other hand, compound 6f showed the widest activity on such cell line
achieving 59.77, 79.48, 76.11 and -42.75 % on A549/ATCC, NCI-H23, NCI-H460 and NCI-
H522 respectively. Compound 6l was the most active on non-small lung cancer cell line
achieving -48.31% against HOP-92 cell line. Moreover, the results on leukemia cell line was
6

remarkable; specially with compounds 6f and 6l which were the most active, achieving high
activity on all the tested cell lines.
It is obvious that the electronic effect on both position of substituent on the tested scaffolds had
a pronounced effect on their cytotoxic activity. Compound 6f with electron withdrawing group
on quinoline-2-one scaffold has showed higher antiproliferative activity on all tested cell lines
than compound 6d and 6e with electron donating groups (Table 2). Also, compound 6l with
chlorine substituted on quinoline-2-one 6 position showed approximately a complete inhibition
on all leukemia cell lines exceeding 6j and 6k with H or methyl substitution on the same
quinoline-2-one ring position. Substitutions on phenyl ring of Schiff bases also altering the
activity of such derivatives. Compounds 6j-l with florin group showed the highest activity
against leukemia cell line among the entire tested group. Also compounds 6j-o with the electron
withdrawing phenyl groups showed higher activities than 6d-6i with electron donating groups
and then 6a-c with no substitution at all. From the results, it is clear that substitution on the
quinoline-2-one is important for the antiproliferative activity especially with electron
withdrawing groups. Moreover, substitution on the phenyl terminal part increases the activity
also with electron withdrawing group like fluorine. The maximum biological effect has gained
in compound 6l with chlorine and fluorine atom in both positions, achieving the highest results
among all tested compounds with complete inhibition of the tested leukemia cancer cell lines
(Table 2).
7

Table 2. Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10 µM
concentration for compounds 6a-o.
Subpanel
cancer cell
Lines
% Growth Inhibition (GI %)a
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
Leukemia
-
-
-
-
-
30.88
-
CCRF-CEM 12.91 12.60
-
-
-
-
-
-
12.00 61.76
54.73
-
-
-
-
-
12.42
-
-
-
-
16.74 32.94 113.24
-
HL-60(TB) 22.34 21.04 13.81
-
35.07 24.43 109.23 10.73
33.23
22.98
21.30
K-562
23.02 29.20 25.20
20.87 22.81 10.41
20.54 79.05
20.98 69.71
18.39 63.41
17.12
47.64 34.73 97.62 28.87
MOLT-4
24.02 12.90 27.88 42.18 115.79 12.34
RPMI-8226 21.69 19.99 22.44
-small cell lung cancer
15.43
-
-
-
27.04 31.25 143.18
-
-
14.43
29.11
-
A549/ATCC 14.73 14.82 10.83
-
-
-
59.77
-
-
11.62 10.67
-
-
-
-
-
-
-
-
11.06
EKVX
10.86 24.67 53.96
18.03 36.19
-
14.45 14.29 13.01 42.63 25.83
-
HOP-62
-
10.78
24.22 30.13
16.25
-
-
-
11.17 13.74 28.10
11.97 12.05 29.23
-
11.34
23.23 17.81 22.99
17.09 25.03
15.66 17.21 21.29
-
-
14.33
-
-
18.37
-
HOP-92
21.02
148.31
-
18.72
22.26
-
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
-
-
18.55 19.62
24.01
-
22.65 23.87
-
13.93 24.27 13.87
16.07 29.96 79.48
-
-
-
-
14.75
-
-
-
-
-
-
-
-
13.24 33.71
76.11
-
-
-
-
-
11.02
-
-
-
18.18
43.89
13.22
-
14.56 15.59
14.88
NCI-H522 18.00 16.13 19.65
on cancer
COLO 205
24.50 24.76 142.75 12.89
12.89 13.95 21.16 33.78 27.19 20.71
-
-
-
-
-
-
-
-
-
16.72
11.16
-
-
-
-
-
-
-
14.49
-
-
-
-
-
-
18.42
-
HCC-2998
HCT-116
HCT-15
HT29
-
13.81
16.84
-
14.93
10.54
-
-
10.75 15.03
21.13 16.86 91.08
13.11
24.28 11.60 18.54 25.08 15.93
-
-
-
-
28.84
11.87
10.24
17.47
-
-
-
-
-
-
-
-
-
-
-
-
-
12.11 60.48
-
-
-
-
11.28
-
-
-
13.34 20.96 16.60
11.13
-
-
-
-
96.71
53.44
62.11
-
-
-
12.18 13.43 11.64 11.26
KM12
13.51
-
-
-
-
-
SW-620
10.10
14.02 11.47 14.05
S cancer
-
-
-
-
-
-
-
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
41.08
27.44
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
15.90
-
-
10.02
-
-
-
-
-
-
-
-
-
13.36
-
14.22
-
-
23.44
-
-
22.29 21.55
14.04
-
71.12
-
-
anoma
LOX IMVI
-
-
-
-
-
-
-
-
-
-
-
62.83
54.02
-
-
-
-
-
-
-
-
-
-
-
-
-
-
MALME-3M -
-
8

-
1 10.43
33.23
10.58
-
M14
15.44
13.65 11.85
11.93 13.75 45.19
10.98
10.40
-
-
-
-
-
-
19.78 20.55 18.85
35.94 27.68 21.37
16.67 22.23 22.78
-
-
-
-
-
MDA-MB-435 -
-
-
-
-
-
-
-
-
-
56.77
19.33 51.20
12.81
31.02 61.77
18.26
10.57 22.69
-
-
-
SK-MEL-2
-
-
14.68 22.20
-
20.67
-
SK-MEL-28
-
-
-
-
-
12.37
-
-
-
10.04
51.12
-
75.81
19.10
20.76
SK-MEL-5 12.07
UACC-257 10.43
UACC-62 12.12
22.56 31.14
-
-
12.45 45.26 88.45 10.86
12.08 15.81 14.22 15.39
-
-
-
-
10.65
13.25
13.76
10.71 17.87
-
18.18 19.56
-
rian cancer
IGROV1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
24.26
89.18
56.94
-
20.45
18.92 24.73
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
15.22
-
-
-
-
-
11.13 14.07
-
-
-
11.56
10.68
10.71
-
-
-
-
-
16.63
-
-
-
30.88
21.95
-
-
-
-
-
-
-
NCI/ADR-RE -
SK-OV-3 13.30
18.16
12.44
15.02
15.91 17.10 10.86 24.82 15.08 22.20
al cancer
786-0
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
37.86
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
10.42 10.50
-
-
-
-
-
-
-
-
-
A498
ACHN
11.07
18.87 12.24
12.33 19.29
-
-
25.84
-
-
-
-
-
-
14.96
-
18.25
-
-
-
-
-
-
-
-
27.31
13.84
14.37
-
-
-
15.04
CAKI-1
RXF 393
SN12C
15.27
10.21
-
12.76
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
11.68 37.67
-
-
10.48
-
TK-10
-
87.45
UO-31
17.40 25.42 12.81
24.25
-
34.45
32.27 32.11
14.84 20.99
state cancer
PC-3
12.07 22.91
21.04 25.22 21.40
-
-
20.93 40.92
35.34
13.63
-
19.60
-
13.65 22.84 24.14 143.04 12.31
-
-
DU-145
ast cancer
MCF7
-
-
-
-
-
-
-
-
-
-
-
31.81
25.43
23.74 29.09 13.26
-
-
21.93 64.82
-
23.10
14.48
19.04 14.67
-
-
15.4
-
-
-
MDA-MB-
231/ATCC
-
-
-
-
28.67
-
-
-
-
-
HS 578T
BT-549
T-47D
-
13.86 17.45
-
-
11.68
17.75
10.57
21.29
-
15.60
13.72
20.27
-
17.23
14.53
-
-
13.92
-
-
-
-
-
-
-
-
-
13.02
23.75
-
-
11.68 67.92
-
15.68
31.17
22.12 24.13 11.29 29.26 57.56
20.24 22.66 22.04
(–): Weak activity GI < 10%.
18.33 19.07 30.83 31.48
27.05
18.08
MDA-MB-468 -
-
-
-
-
-
2.2.2. In vitro five dose full NCI 60 cell panel assay
Compounds 6l was selected for five dose testing against the full panel of 60 human tumor cell lines
according to NCI protocol (http://www.dtp.nci.nih.gov.). All the 60 cell lines representing nine
9

tumor subpanels were incubated at five different concentrations (0.01, 0.1, 1, 10, and 100 µM). The
outcomes were used to form log concentration vs % growth inhibition curves and three response
parameters (GI50, TGI, and LC50) were calculated for each cell line. The GI50 value (growth
inhibitory activity) corresponds to the concentration of the compound causing 50% decrease in net
cell growth, the TGI value (cytostatic activity) is the concentration of the compound resulting in
total growth inhibition (TGI) and LC50 value (cytotoxic activity) is the concentration of the
compound causing net 50% loss of initial cells at the end of the incubation period of 48 h. The
principle for selectivity of compound depends upon the ratio obtained by dividing the full panel
MID (the average sensitivity of all cell lines toward the test agent) (µM) by their individual subpanel
MID (µM). Ratios between 3 and 6 refer to moderate selectivity; ratios > 6 indicate high selectivity
toward the corresponding cell line, whilst compounds not meeting either of these criteria rated
nonselective, Appendix A.
Compound 6l exhibited noteworthy antiproliferative activity against all cancer cell lines with low
selectivity ratios. Leukemia, melanoma, breast and small cell lung cancers were the most affected
tissues. Compound 6l achieved its highest results against MOLT-4 leukemia cell line with GI50 of 4.07
µM (Table 3) with selectivity ratio of 1.32 and GI50 against SK-MEL-5 melanoma cancer equal to
3.55 and selectivity ratio 1.30.
퐌퐈퐃퐚
Selective index
.
=
퐌퐈퐃퐛
10

Table 3. Results of in vitro five doses testing of nine human cancer types and selectivity for
compounds 6l.
6l
GI50
Conc.
Panel
Cell line
TGI
LC50
Selectivity
ratio
per cell MIDb
line
CCRF-CEM
HL-60(TB)
K-562
12.0
18.9
> 100
50.9
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
13.2
14.94
4.07
93.2
Leukemia
1.32
MOLT-4
RPMI-8226
SR
> 100
> 100
85.6
17.0
24.5
A549/ATCC
EKVX
23.5
16.8
20.8
14.0
> 100
> 100
> 100
45.9
HOP-62
HOP-92
Non-small cell lung cancer
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
14.6
19.5
22.2
20.3
17.4
18.2
32.4
17.4
19.9
23.1
22.0
26.8
33.6
21.9
23.6
23.5
12.8
21.1
25.3
14.2
10.2
18.79
1.05
> 100
76.1
> 100
> 100
74.3
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
43.5
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
78.2
Colon
Cancer
22.83
0.86
KM12
SW-620
SF-268
SF-295
SF-539
CNS
Cancer
22.75
15.23
0.87
1.30
SNB-19
SNB-75
U251
> 100
83.1
98.4
LOX IMVI
MALME-3M
M14
> 100
54.0
Melanoma
> 100
11

MDA-MB-
435
13.5
> 100
> 100
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
18.3
19.8
3.55
15.5
16.7
18.5
24.8
21.6
19.8
69.3
> 100
14.1
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
56.5
> 100
> 100
> 100
58.9
OVCAR-3
OVCAR-5
OVCAR-8
Ovarian Cancer
21.38
0.92
> 100
NCI/ADR-
RES
23.5
> 100
> 100
SK-OV-3
20.1
26.3
14.8
17.2
23.5
23.7
23.8
47.1
21.9
8.41
33.9
15.3
83.4
> 100
92.7
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
72.1
> 100
> 100
> 100
> 100
> 100
96.3
Renal Cancer
24.78
0.80
PC-3
Prostate Cancer
Breast Cancer
21.15
17.38
0.93
1.14
DU-145
MCF7
> 100
> 100
MDA-MB-
231/ATCC
16.0
51.7
> 100
HS 578T
BT-549
T-47D
25.3
15.8
18.6
> 100
44.5
> 100
> 100
> 100
> 100
MDA-MB-
468
13.3
49.1
> 100
MIDa for 6l = 19.75
12

Figure 2. Dose-antiproliferative response of compound 6l against nine different cancer cell lines
2.2.3. Cytotoxic activity evaluation of IC50
Independently, compound 6l was selected for further investigation, it was evaluated for its
anticancer activity against RPMI-8226 leukemia cancer and on normal healthy unaffected cell lines
by MTT assay (Appendix A) [32] (Table 4). Compound 6l achieved IC50=15.72±1.21 µM on the
selected RPMI-8226 leukemia cancer cell lines which is considered a comparable result with the
reference gefitinib IC50= 9.92±0.62 µM.
Same approach happened in normal healthy cells; 6l showed IC50= 46.05±2.36 µM and gefitinib
achived IC50= 33.49±1.73 uM, which indicate the relative safety of the tested compound on normal
cells.
13

Table 4. Antiproliferative IC50± SEM (µM) activity of compound 6l and gefitinib
Sample code
M.W
g/mol
Cytotoxicity
IC50± SEM (µM)
RPMI-8226
15.72±1.21
9.92±0.62
WI38
6l
315.06
446.90
46.05±2.36
33.49±1.73
Gefitinib
2.2.4. EGFR TK Inhibitory Activity
Epidermal growth factor receptor (EGFR) and its mediated signaling pathway regulate
many physiological processes such as cell growth, proliferation and differentiation In
recent years, EGFR has become a hot therapeutic target for cancer. The mechanism at
molecular level of the selected most active antiproliferative compound 6l was investigated
on mutant and wild EGFR TK kinase (Table 5) [33]. The findings of the cancer cell-based
assays have been powerfully complemented by the results from EGFR assay. Compound
6l showed moderated EGFR inhibition with IC50= 695.49±21.8 and 263.15±15.13 nM on
the mutant and wild EGFR respectively, while that of gefitinib was 358.78±17.2 and
157.54±4.85 nM.
Table 5. EGFR-TK Inhibitory activity of compounds 6l and gefitinib.
Compound
EGFR
IC50 nM
EGFR-T490M
IC50 nM
6l
695.49±21.8
358.78±17.2
263.15±15.13
157.54±4.85
Gefitinib
2.2.5. Human RAS Inhibitory Activity
Ras proteins are among the most frequently mutated drivers in human cancer and the quest for
compounds that bind to mutant RAS remains a major goal as elusive anti-cancer pharmaceutical
14

targeting A [34]. Mutations in the RAS family of proto-oncogenes (comprising H-RAS, N-RAS
and K-RAS) are very common, being found in 20% to 30% of all human tumors [35,36]. It is
reasonable to speculate that a pharmacological approach that curtails RAS activity may represent
a possible method to inhibit certain cancer types [37].
Compound 6l has been investigated on RPMI-8226 leukemia cell line as a possible H-RAS
inhibitor, using gefitinib as reference. The results showed that 6l achieved good activity with
10.61±0.27 ng/ml against gefitinib with 7.688±0.23 ng/ml on the same cell line (Table 6 and
Figure 3).
Table 6. RAS and STAT3 inhibitory activities of compounds 6l and gefitinib
Compound
H-RAS
conc.
STAT3
conc.
ng/ml
ng/ml
6l/RPMI-8226
Gefitinib/RPMI-8226
Cont.RPMI-8226
10.61±0.27
7.688±0.23
21.75±1.71
1.753±0.81
1.28±0.04
3.73±0.07
Figure 3. RAS and STAT3 inhibitory activities of compounds 6l and gefitinib
2.2.6. STAT3 Inhibitory Activity
STAT proteins also known as Signal Transducer and Activator of Transcription are transcription
factors involved in a huge repertoire of biological signal transduction cascades leading to
embryonic development, programmed cell death, organogenesis, innate immunity, adaptive
15

immunity and cell growth regulation in organisms ranging from insects to man [38]. There are
seven mammalian STAT family members that have been identified: STAT1, 2, 3, 4, 5 and 6.
STAT proteins are thought to be ideal targets for anti-cancer therapy since cancer cells are more
dependent on the STAT activity than their normal counterparts [39,40]. Compound 6l has been
investigated on RPMI-8226 leukemia cell line as a possible STAT3 inhibitor, using gefitinib as
reference. The results showed that 6l achieved excellent activity with result 1.753±0.81 ng/ml
comparable to gefitinib with 1.28±0.04 ng/ml on leukemia cell line (Table 6 and Figure 3).
2.2.7. Activation of caspases cascade
Activation of caspases plays a key role in the initiation and effecting of the apoptotic process
[41]. Among the most common caspases, caspase-3 is an essential player that cleaves multiple
proteins in the cells, leading to apoptotic cell death [42]. The effect of compound 6l on caspase 3
was evaluated and compared to gefitinib as a reference drug on RPMI-8226 cell line. Compound
6l showed an increase in the level of active caspase 3 by 5.31-fold compared to gefitinib (4.58-
fold) as shown in Table 7. Moreover, effect of compound 6l on caspase 8 was also evaluated;
compound 6l increases the levels of caspase-8 by 1.54-fold, while gefitinib was 1.85-fold. The
data indicate that 6l is potential activators of both intrinsic and extrinsic caspase.
Table 7. Effect of compound 6l and gefitinib on the active caspases-3 and 8 in RPMI-8226 cell line.
Comp. Number
Caspase-3
Caspase-8
Conc
Fold change
Conc
Fold change
(ng/ml)
(ng/ml)
6l
322.7±14.7
278.6±15.1
60.76±4.02
5.31
4.58
1
0.5475±0.03
0.6581±0.09
0.3541±0.02
1.546173
1.858515
1
Gefitinib
Control
2.2.8. Effects on BAX and Bcl-2 proteins
16

Bcl-2 (B-cell lymphoma 2) family members are significant regulators of apoptosis that can be
classified into three groups. The first group protects against apoptosis (Bcl-2 itself), the second
group of proteins is represented by Bax (Bcl-2 associated protein X) and Bak (Bcl-2
antagonist/killer). Bax and Bak are the key activators of the apoptosis machinery in response to
cellular stress stimuli. The third group is comprised of different categories of proteins such as
Bid (BH3 interacting death domain), Bim (Bcl-2 interacting mediator) and others [43]
.Overexpression of the antiapoptotic Bcl-2 members leading to antagonizing apoptosis and
development of resistance of tumor cells to common chemotherapeutic agents. Thus, the
development of new drugs that can inhibit the action of antiapoptotic Bcl-2 members is very
attractive strategy in design of anticancer agents [44]. The effect of compound 6l on the
expression levels of Bcl2 and BAX was determined after treatment of RPMI-8226 cell line with
the IC50 of compound 6l and the results were illustrated in Table 8. Compound 6l caused up-
regulation in the level of the proapoptotic protein BAX by approximately 6.5 folds while it
markedly down-regulated the levels of the antiapoptotic proteins Bcl2 up to 0.4 folds compared
to the control untreated cells. These findings proved the proapoptotic effect of compound 6l.
Table 8. Bax and Bcl-2 levels for compounds 6l and gefitinib on RPMI-8226 cell line.
Compound
Bax
Bcl-2
Fold change
Conc
(pg/ml)
Fold change
6.543221
Conc
(ng/ml)
6l
239.7±9.61
3.29±0.14
0.486471
Gefitinib
309.6±11.8
36.63±1.96
8.451319
1
3.747±0.22
6.763±0.46
0.554044
1
Control
2.2.9. Cell cycle analysis and apoptosis assay
2.2.9.1. Cell cycle analysis
17

The cell cycle includes four phases: The G1 phase where cell enlargement and preparation of
DNA duplication occurs; the S phase (synthesis) is the stage of DNA replication and chromatid
duplication; in G2 Phase, repairing of new DNA and more growth occurs while in the M stage
nuclear division takes place. Studies on the effect of compound 6l on cell cycle development and
induction of apoptosis in the RPMI-8226 were done. RPMI-8226 was incubated with IC50
concentration of compound 6l for 24 h. Consequently, the cell line was stained with PI/Annexin
V and analyzed by flow cytometry using BD FASCC alibur [45]. Investigation of the results in
Figure 4, exposed that percentage of pre G1 apoptosis induced by 6l on RPMI-8226 was
22.81%. A high percent of cell accumulation was observed in G2-M phase in RPMI-8226 treated
with compound 6l after 24 h incubation indicating arrest of cell cycle at G2-M phase.
Figure 4. Cell cycle analysis results for compound 6l
2.2.9.2. Apoptosis assay
Cell cycle analysis of RPMI-8226 after treatment with compound 6l showed presence of pre-G1
peak which is an indication of apoptosis. To confirm the ability of 6l to induce apoptosis, cells
were stained with Annexin V/PI, incubated for 24 h and analyzed. Analysis of early and late
apoptosis showed that compound 6l was able to induce significant levels of apoptosis percent
18

3.61 and 17.45, respectively (Figures 5 and 6) and that % of necrosis was 1.75, while the
reference gefitinib achieved 7.58 and 19.5 in apoptosis with 2.44 necrotic percentage.
Figure 5. Apoptosis induction analysis using Annexin V/PI for compound 6l and gefitinib
Figure 6. Apoptosis induction analysis using Annexin V/PI for compound 6l.
19

2.3. Docking study
The target compounds were docked with EGFR receptor which are cocrystallized standard ligand
gefitinib (ID: 2ITO) [46] extracted from protein data bank (PDB) using OpenEye scientific
software [47-49]. The standard gefitinib was docked in a similar mode as its reported
cocrystalized pose keeping the most important key interactions. Compound 6l interacts with the
receptor through formation of hydrogen bond (HB) with SER: 719: A (Figure 7A). Meanwhile
this compound overlay with gefitinib and erlotinib standards inside the receptor (Figures 7B,
7C) respectively with keeping. Meanwhile, the analogues compound 6c docked with EGFR
receptor without illustrating any HB interactions (Figure 7D). It’s clear that the kind of
substitution on aryl group or quinolone moiety effects on compounds binding mode and pose
which could give insight about the activity. In case of compounds 6a, 6g, 6d, and 6m docked
with the receptor with weak interactions without formation of HB. Compounds 6o and 6i
interacts with the receptor with formation of HBs with PHE 856: A, and THR 854: A
respectively (Appendix A).
A
B
20

C
D
Figure 7: A) compound 6l docked with EGFR receptor; B) compound 6l with gefitinib in same receptor; C)
compound 6l with erlotinib in the receptor, D) compound 6c docked without formation of HB interaction
In order to study the compound similarity to either gefitinib or erlotinib standards, the aim was
directed to apply Rapid overlay chemical similarity analysis (ROCS) analysis. ROCS is a method
that used to predict similarity between compounds based on their three dimensional [48]. Its
alignment needs a) query molecules and here are erlotinib and gefitinib. b) The database
molecules that our designed compounds especially compound 6l. The outputs of ROC analysis
are two forms: 1) the overlay between the query and database molecules as visualized by vROCS
and VIDA applications [50]. The query will be visualized in term of shape counter, shape atoms,
and color atoms labels.
The color shape of gefitinib showed the following atoms labels 4 rings, 2 donors, 5 acceptors and
1 cation site (Figure 8A). The color shape of erlotinib represented 3 rings, 1 donor, 6 acceptors
and one hydrophobic site (Figure 8B). Compounds 6l showed overlay with gefitinib, and
21

erlotinib as represented in Figures 8C and 8D, respectively. According to these mapping,
compound 6l illustrated high similarity to erlotinib drug. From these visualizations we can be
envisioned for elaboration a new derivative rom 6l through installing side chain to benzene ring
of quinoline-2-one moiety.
(A)
(B)
(C)
(D)
Figure 8. A) ROCS analysis overlay of gefitinib as query; B) erlotinib as query; C) 6l with
gefitinib; D) 6l with erlotinib
The second output of ROCs analysis is the Tanimoto Combo (TC) score, table 9. TC score
represents various aspects of that alignment. Tanimoto Combo is the sum of Shape Tanimoto
22

(Tanimoto coefficient) and Color Tanimoto scores. The scores are computed and the process is
repeated to each conformation of the query molecules (gefitinib and erlotinib) and each
conformation for the database molecule 6l (Table 9). The target compound showed higher score in
Tanimoto combo to lead erlotinib (0.84) rather than the gefitinib 0.76.
Table 9: Tanimoto combo score for compound 6l, and lead compounds Erlotinib and Gefitinib as
query
Erlotinb (Tanimato combo)
Gefitinb
(Tanimato combo)
Erlotinib
Gefitinib
6l
2
0.96
0.84
0.94
2
0.76
2.4. Structure activity relationship:
The final compounds have two variation sites either on position 6 of quinolin-one ring (R1) or on
para position of aryl hydrazine moiety (R2) as represented in Figure 1.
Installing R1 as electron withdrawal group (EWG) improved the activity of compounds rather
than the corresponding unsubstituted or those with electron donating group (EDG) as showed in
compound 6l and 6f versus compounds 6d, and 6e. In the same way, tethering the phenyl group
of hydrazone moiety with R2 as EWG increases the compounds activity. It was clear compounds
with both R1 and R2 as EWGs have higher activity (compounds 6l and 6f). The maximum
biological effect has gained in compound 6l with chlorine and fluorine atom in both positions,
achieving the highest results among all tested compounds with complete inhibition of the tested
leukemia cancer cell lines, Table 2.
3. Conclusions
A series of new 2-quinolone-4-arylidene derivatives 6a-o was prepared and characterized using
different spectroscopic techniques. Some of the target compounds have promising anticancer
23

activities. The 6-chloro derivative 6l was found to be the most active derivative among this
series. It showed outstanding inhibition on all leukemia cell lines tested with mean GI50 of 14.94
according to NCI protocol. The IC50 was determined as 15.72±1.21 compared to gefatinib
(9.92±0.62). Also, 6l showed inhibitory activity with comparable potency to gefatinib against
EGFR-TK mutant and wild cells. Similarly, 6l have inhibitory activity against H.RAS and
STAT-3 on leukemia cell lines. Moreover, 6l increased the level of Caspase-3 by 1.54 folds
compared to gefatinib (1.85) fold increase. Also, the effect of 6l on apoptotic regulators, BAX
and BCl-2 proved its proapoptotic effect. Results of cell cycle analysis showed that 6l can
accomplish cell cycle arrest at G2/M phas; 6l can induce apoptosis when stained with Annexin
V/PT. Results from molecular docking study showed that 6l can overlay with gefatinib and
erlotinib in binding to the protein (ID: 2ITO). The rapid overlay chemical similarity analysis
(ROCS) of 6l experienced similarity to erlotinib more than gefatinib in binding. Hence, 6l is
considered promising lead candidate erlotinib analogue effective in leukemia that require further
optimization.
4. Experimental
4.1. Chemistry
All materials were obtained from commercial suppliers and used without further purification.
Reactions were monitored by TLC (Kieselgel 60 F254precoated plates, E. Merck, Germany), the
spots were detected by exposure to UV lamp at 254 nm. Melting points were determined on an
electro thermal melting point apparatus (Stuart Scientific Co.) and were uncorrected. NMR
spectra were measured on a Bruker AV-400 spectrometer (Bruker Bio Spin Corp., Billerica, MA,
USA) (400 MHz for 1H, 101 MHz for 13C) at Florida Institute of Technology, USA. The 1H and
13C chemical shifts are given relative to internal standard TMS = 0., and external liquid ammonia
24

1
= 0 for 15N. Coupling constants are stated in Hz. Correlations were established using H-1H
1
1
COSY, and H-13C and H-15N HSQC and HMBC experiments. Vario EL III German CHN
Elemental analyzer model was used for Elemental analysis. Preparation and analytical data of
compounds 2-5 were as reported in lit [28-30,31].
4.1.1. General synthesis of compounds 6a-o
To a suspension of hydrazine- quinoline-2-one 5 (1.0 mmol) in ethanol (20 mL); an appropriate
aryl aldehyde derivative (1.0 mmol) with 2 drops of glacial acetic acid were added. The reaction
mixture was heated under reflux for 5-8 h. After cooling, the formed precipitate was filtered,
washed with diethyl ether and crystallized from ethanol.
(E)-4-(2-Benzylidenehydrazinyl)quinolin-2(1H)-one (6a)
1
Yield: 0.22 g (85%); mp: 180–182 °C, H NMR (400 MHz, DMSO-d6) δ ppm 11.08 (s, 1H,
O=C-NH), 10.79 (s, 1H, N-NH), 8.39 (s, 1H, N=CH), 8.05 (d, J = 8.1, 1H, quin-Ar-H), 7.75 (d,
J = 7.3 Hz, 2H, Ar-H), 7.50 (t, J = 7.6 Hz, 1H, quin-Ar-H), 7.46 (t, J = 7.6 Hz, 2H , Ar-H), 7.41
(t, J = 7.1 Hz, 1H, Ar-H), 7.30 (d, J = 8.2 Hz, 1H, quin-Ar-H), 7.19 (t, J = 7.6 Hz, 1H, quin-Ar-
H), 6.24 (s, 1H, C=CH).13C NMR (101 MHz, DMSO-d6) δ ppm 162.29, 147.88, 143.21, 138.91,
134.22, 129.93, 128.97, 128.38, 126.13, 121.43, 120.21, 115.23, 111.33, 93.75. LCMS: m/z
calcd: 263.11, found [M-H]-: 262.10. Anal. Calcd for C16H13N3O (263.11): C, 72.99; H, 4.88; N,
15.96. Found: C, 73.19; H, 4.66; N, 16.08.
(E)-4-(2-Benzylidenehydrazinyl)-6-methylquinolin-2(1H)-one (6b)
Yield: 0.23 g (83%); mp: 185–187 °C,1H NMR (400 MHz, DMSO-d6) δ ppm 11.09 (s, 1H, O=C-
NH), 10.77 (s, 1H, N-NH), 8.39 (s, 1H, N=CH), 7.88 (s, 1H, quin-Ar-H), 7.74 (d, J = 7.4 Hz, 2H,
Ar-H), 7.45 (t, J = 7.2 Hz, 2H , Ar-H), 7.39 (t, J = 7.1 Hz, 1H, Ar-H), 7.33 (d, J = 8.3 Hz, 1H,
quin-Ar-H), 7.22 (d, J = 8.3 Hz, 1H, quin-Ar-H), 6.26 (s, 1H, C=CH), 2.38 (s, 3H, CH3).13C
NMR (101 MHz, DMSO-d6) δ ppm 162.34, 147.76, 143.06, 136.89, 134.25, 131.07, 129.93,
25

128.92, 128.36, 126.09, 120.96, 115.16, 111.24, 93.74, 20.20. LCMS: m/z calcd: 277.12, found
[M-H]-: 276.10. Anal. Calcd for C17H15N3O (277.12): C, 73.63; H, 5.45; N, 15.15. Found: C,
73.96; H, 5.23; N, 15.40.
(E)-4-(2-Benzylidenehydrazinyl)-6-chloroquinolin-2(1H)-one (6c)
1
Yield: 0.23 g (79%); mp: 191–193 °C, H NMR (400 MHz, DMSO-d6) δ ppm 10.78 (s, 1H,
O=C-NH), 10.38 (s, 1H, N-NH), 7.91 (s, 1H, N=CH), 7.73 (s, 1H, quin-Ar-H), 7.29 (d, J = 7.2
Hz, 2H, Ar-H), 7.07 (d, J = 7.6 Hz, 1H, quin-Ar-H), 7.01-6.94 (m, 3H , Ar-H), 6.84 (d, J = 7.9
Hz, 1H, quin-Ar-H), 5.80 (s, 1H, C=CH). 13C NMR (101 MHz, DMSO-d6) δ ppm 162.07,
147.00, 143.58, 137.69, 134.07, 129.86, 129.08, 128.38, 126.20, 124.51, 120.92, 117.03, 112.54,
94.39. LCMS: m/z calcd: 297.07, found [M-H]-: 296.00. Anal. Calcd for C16H12ClN3O (277.12):
C, 64.54; H, 4.06; N, 14.11. Found: C, 64.63; H, 3.97; N, 14.04.
(E)-4-(2-(4-Methoxybenzylidene)hydrazinyl)quinolin-2(1H)-one (6d)
Yield: 0.21 g (73%); mp: 165-167 °C,1H NMR (400 MHz, DMSO-d6) δ ppm 10.60 (s, 1H, O=C-
NH), 10.21 (s, 1H, N-NH), 7.90 (s, 1H, N=CH), 7.58 (d, J = 7.8, 1H, quin-Ar-H), 7.29 (d, J =
7.2 Hz, 2H, Ar-H), 7.04 (t, J = 7.4, 1H, quin-Ar-H), 6.83 (d, J = 8.2 Hz, 1H, quin-Ar-H), 6.73 (t,
J = 7.6 Hz, 1H, quin-Ar-H), 6.57 (d, J = 7.6 Hz, 2H, Ar-H), 5.74 (s, 1H, C=CH), 3.34 (s, 3H,
OCH3).13C NMR (101 MHz, DMSO-d6) δ ppm 168.14, 165.62, 153.69, 148.96, 144.60, 135.57,
133.38, 132.53, 127.13, 125.88, 120.93, 119.57, 117.10, 98.91, 60.48. LCMS: m/z calcd: 293.12,
found [M-H]-: 292.10. Anal. Calcd for C17H15N3O2 (293.12): C, 69.61; H, 5.15; N, 14.33. Found:
C, 69.40; H, 5.13; N, 14.52.
(E)-4-(2-(4-Mthoxybenzylidene)hydrazinyl)-6-methylquinolin-2(1H)-one (6e)
1
Yield: 0.21 g (70%); mp: 205–207 °C, H NMR (400 MHz, DMSO-d6) δ ppm 11.05 (s, 1H,
O=C-NH), 10.63 (s, 1H, N-NH), 8.33 (s, 1H, N=CH), 7.86 (s, 1H, quin-Ar-H), 7.68 (d, J = 7.8
26

Hz, 2H, Ar-H), 7.32 (d, J = 8.1 Hz, 1H, quin-Ar-H), 7.21 (d, J = 8.3 Hz, 1H, quin-Ar-H), 7.45 (d,
J = 7.6 Hz, 2H , Ar-H), 6.22 (s, 1H, C=CH), 3.36 (s, 3H, OCH3), 2.36 (s, 3H, CH3).13C NMR
(101 MHz, DMSO-d6) δ ppm 162.42, 15.88, 147.83, 143.09, 136.87, 131.09, 129.16, 127.64,
126.87, 120.95, 115.18, 113.85, 111.28, 93.22, 54.73, 20.19. LCMS: m/z calcd: 307.13, found
[M-H]-: 306.10. Anal. Calcd for C18H17N3O2 (307.13): C, 70.34; H, 5.58; N, 13.67. Found: C,
70.01; H, 5.78; N, 13.48.
(E)-6-Chloro-4-(2-(4-methoxybenzylidene)hydrazinyl)quinolin-2(1H)-one (6f).
Yield: 0.30 g (93%); mp: 212–214 °C,1H NMR (400 MHz, DMSO-d6) δ ppm 10.74 (s, 1H, O=C-
NH), 10.22 (s, 1H, N-NH), 7.85 (s, 1H, N=CH), 7.72 (s, 1H, quin-Ar-H), 7.24 (d, J = 8.0 Hz, 2H,
Ar-H), 7.09 (d, J = 7.8 Hz, 1H, quin-Ar-H), 6.83 (d, J = 8.1 Hz, 1H, quin-Ar-H), 6.55 (d, J = 8.1
Hz, 2H , Ar-H), 5.77 (s, 1H, C=CH), 3.34 (s, 3H, OCH3).13C NMR (101 MHz, DMSO-d6) δ ppm
162.14, 159.99, 147.05, 143.59, 137.67, 129.77, 127.76, 126.68, 124.46, 120.90, 117.00, 113.86,
112.59, 93.85, 54.78. LCMS: m/z calcd: 327.08, found [M-H]-: 326.00. Anal. Calcd for
C17H14ClN3O2 (327.08): C, 62.30; H, 4.31; N, 12.82. Found: C, 62.18; H, 4.35; N, 12.64.
(E)-4-(2-(4-(Dimethylamino)benzylidene)hydrazinyl)quinolin-2(1H)-one (6g)
Yield: 0.27 g (89%); mp: 228-230 °C,1H NMR (400 MHz, DMSO-d6) δ ppm 10.95 (s, 1H, O=C-
NH), 10.47 (s, 1H, N-NH), 8.28 (s, 1H, N=CH), 8.04 (d, J = 7.8 Hz, 1H, quin-Ar-H), 7.57 (d, J =
7.2 Hz, 2H, Ar-H), 7.48 (t, J = 7.4 Hz, 1H, quin-Ar-H), 7.28 (d, J = 8.2 Hz, 1H, quin-Ar-H), 7.18
(t, J = 7.6 Hz, 1H, quin-Ar-H), 6.78 (d, J = 7.6 Hz, 2H , Ar-H), 6.15 (s, 1H, C=CH), 2.98 (s, 6H,
N(CH3)2).13C NMR (101 MHz, DMSO-d6) δ ppm 162.85, 151.16, 148.37, 144.78, 139.36,
130.18, 127.89, 122.15, 121.86, 120.50, 115.62, 111.94, 93.12, 39.51. LCMS: m/z calcd: 306.15,
27

found [M-H]-: 305.10. Anal. Calcd for C18H18N4O (306.15): C, 69.61; H, 5.15; N, 14.03. Found:
C, 69.30; H, 5.13; N, 13.92.
(E)-4-(2-(4-(Dimethylamino)benzylidene)hydrazinyl)-6-methylquinolin-2(1H)-one (6h)
Yield: 0.27 g (87%); mp: 176–178 °C,1H NMR (400 MHz, DMSO-d6) δ ppm 10.89 (s, 1H, O=C-
NH), 10.42 (s, 1H, N-NH), 8.26 (s, 1H, N=CH), 7.85 (s, 1H, quin-Ar-H), 7.56 (d, J = 8.8 Hz, 2H,
Ar-H), 7.31 (d, J = 8.4 Hz, 1H, quin-Ar-H), 7.18 (d, J = 8.3 Hz, 1H, quin-Ar-H), 6.77 (d, J = 7.6
Hz, 2H , Ar-H), 6.14 (s, 1H, C=CH), 2.98 (s, 6H, N(CH3)2), 2.38 (s, 3H, CH3).13C NMR (101
MHz, DMSO-d6) δ ppm 162.80, 151.14, 148.18, 144.60, 137.34, 131.31, 129.45, 127.85, 122.20,
121.38, 115.53, 111.94, 111.79, 93.17, 39.78, 20.64. LCMS: m/z calcd: 320.16, found [M-H]-:
319.10. Anal. Calcd for C19H20N4O (320.16): C, 71.23; H, 6.29; N, 17.49. Found: C, 71.47; H,
6.13; N, 17.27.
(E)-6-Chloro-4-(2-(4-(Dimethylamino)benzylidene)hydrazinyl)quinolin-2(1H)-one (6i).
Yield: 0.28 g (83%); mp: 210–212 °C,1H NMR (400 MHz, DMSO-d6) δ ppm 11.10 (s, 1H, O=C-
NH), 10.52 (s, 1H, N-NH), 8.25 (s, 1H, N=CH), 8.18 (d, J = 2.0 Hz, 1H, quin-Ar-H), 7.57 (d, J =
8.8 Hz, 2H, Ar-H), 7.54 (dd, J = 8.9,2.1 Hz, 1H, quin-Ar-H), 7.29 (d, J = 8.8 Hz, 1H, quin-Ar-
H), 6.77 (d, J = 8.8 Hz, 2H, Ar-H), 6.17 (s, 1H, C=CH), 2.98 (s, 6H, N(CH3)2).13C NMR (101
MHz, DMSO-d6) δ ppm 162.63, 151.24, 147.48, 145.15, 138.15, 130.11, 127.97, 124.83, 121.99,
121.35, 117.42, 113.15, 111.91, 93.74, 39.57. LCMS: m/z calcd: 340.11, found [M-H]-: 339.20.
Anal. Calcd for C18H17ClN4O (340.11): C, 63.44; H, 5.03; N, 16.44. Found: C, 63.62; H, 4.99; N,
16.27.
(E)-4-(2-(4-Fluorobenzylidene)hydrazinyl)quinolin-2(1H)-one (6j).
Yield: 0.26 g (95%); mp: 211-213 °C,1H NMR (400 MHz, DMSO-d6) δ ppm 11.17 (s, 1H, O=C-
NH), 10.83 (s, 1H, N-NH), 8.38 (s, 1H, N=CH), 8.06 (d, J = 8.1 Hz, 1H, quin-Ar-H), 7.80 (dd, J
= 7.7, 6.0 Hz, 2H, Ar-H), 7.50 (t, J = 7.6 Hz, 1H, quin-Ar-H), 7.30 (dd, J = 11.9, 8.5 Hz, 2H ,
28

Ar-H),7.26 (d, J = 8.6 Hz, 1H, quin-Ar-H), 7.18 (t, J = 7.5 Hz, 1H, quin-Ar-H), 6.27 (s, 1H,
C=CH). 13C NMR (101 MHz, DMSO-d6) δ ppm 162.42, 162.25, 147.95, 142.08, 138.89, 130.82,
129.93, 128.20, 121.43, 120.24, 115.37, 115.26, 111.35, 93.71. LCMS: m/z calcd: 281.10, found
[M-H]-: 280.10. Anal. Calcd for C16H12FN3O (281.10): C, 68.32; H, 4.30; N, 14.94. Found: C,
68.50; H, 4.16; N, 15.11.
(E)-4-(2-(4-Fluorobenzylidene)hydrazinyl)-6-methylquinolin-2(1H)-one (6k)
1
Yield: 0.24 g (83%); mp: 169-171 °C, H NMR (400 MHz, DMSO-d6) δ ppm 10.62 (s, 1H,
O=C-NH), 10.31 (s, 1H, N-NH), 7.92 (s, 1H, N=CH), 7.40 (s, 1H, quin-Ar-H), 7.33 (d, J = 8.0
Hz, 1H, quin-Ar-H), 6.84 (d, J = 7.8 Hz, 2H, Ar-H), 6.80 (d, J = 8.0 Hz, 1H, quin-Ar-H), 6.76 (d,
J = 7.8 Hz, 2H , Ar-H), 5.79 (s, 1H, C=CH), 1.91 (s, 3H, CH3).13C NMR (101 MHz, DMSO-d6)
δ ppm 163.45, 162.36, 160.99, 147.76, 141.89, 136.87, 131.05, 130.88, 129.21, 128.11, 120.94,
115.46, 115.24, 115.15, 111.23, 93.73, 20.18. LCMS: m/z calcd: 295.11, found [M+H]+: 296.00.
Anal. Calcd for C17H14FN3O (295.11): C, 69.14; H, 4.78; N, 14.23. Found: C, 68.95; H, 4.51; N,
14.18.
(E)-6-Chloro-4-(2-(4-fluorobenzylidene)hydrazinyl)quinolin-2(1H)-one (6l).
Yield: 0.25 g (80%); mp: 178-180 °C 1H NMR (400 MHz, DMSO-d6) δ ppm 11.28 (s, 1H, O=C-
NH), 10.82 (s, 1H, N-NH), 8.32 (s, 1H, N=CH), 8.16 (s, 1H, quin-Ar-H), 7.77 (d, J = 8.2 Hz, 1H,
quin-Ar-H), 7.52 (d, J = 8.0 Hz, 2H, Ar-H), 7.31 (d, J = 8.1 Hz, 1H, quin-Ar-H), 7.25 (d, J = 7.8
Hz, 2H , Ar-H), 6.28 (s, 1H, C=CH).13C NMR (101 MHz, DMSO-d6) δ ppm 163.54, 162.18,
161.07, 147.04, 142.39, 137.62, 130.71, 129.83, 128.30, 124.52, 120.92, 117.03, 115.49, 115.27,
112.55, 94.41. LCMS: m/z calcd: 315.06, found [M-H]-: 314.00. Anal. Calcd for C16H11ClFN3O
(315.06): C, 60.87; H, 3.51; N, 13.31. Found: C, 60.72; H, 3.69; N, 13.17.
(E)-4-(2-(4-Chlorobenzylidene)hydrazinyl)quinolin-2(1H)-one (6m)
29