Novel pyrrol-2(3H)-ones and pyridazin-3(2H)-ones carrying quinoline scaffold as anti-proliferative tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bioorg.2018.06.003

A novel quinolinyl pyrrolone and quinolinyl pyridazinone derivatives has been synthesized and characterized using different spectroscopic and elemental analysis techniques. Most of the target compounds displayed promising antiproliferative activity; In general, the pyrrolone derivatives 4a-f exhibited higher antiproliferative activity than their corresponding pyridazinone. The pyrrolone 4f showed outstanding antiproliferative activity with moderate selectivity against CNS and renal cancer with selectivity ratio of 3.49 and 3.56, respectively. Compound 4e and 5d experienced tubulin polymerization inhibitory activity comparable to that of vincristine while 4c, 4e and 4d showed good BRAF kinase inhibition compared to Erlotinib. Docking of compound 4e into colchicine binding site and biological assay results revealed that these compounds act mainly through tubulin polymerization inhibitory mechanism and can exhibit pre G1 apoptosis and cell cycle arrest at G2/M phase.

Full text of DOI:10.1016/j.bioorg.2018.06.003

Accepted Manuscript
Novel Pyrrol-2(3H)-ones and Pyridazin-3(2H)-ones Carrying Quinoline Scaf-
fold as Anti-proliferative Tubulin Polymerization Inhibitors
Mahmoud S Abdelbaset, Gamal El-Din A Abuo-Rahma, Mostafa H
Abdelrahman, Mohamed Ramadan, Bahaa G.M. Youssif, Syed Nasir Abbas
Bukhari, Mamdouh F.A. Mohamed, Mohamed Abdel-Aziz
PII:
S0045-2068(18)30365-1
DOI:
https://doi.org/10.1016/j.bioorg.2018.06.003
YBIOO 2383
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
14 April 2018
31 May 2018
1 June 2018
Please cite this article as: M.S. Abdelbaset, G. El-Din A Abuo-Rahma, M.H. Abdelrahman, M. Ramadan, B.G.M.
Youssif, S. Nasir Abbas Bukhari, M.F.A. Mohamed, M. Abdel-Aziz, Novel Pyrrol-2(3H)-ones and
Pyridazin-3(2H)-ones Carrying Quinoline Scaffold as Anti-proliferative Tubulin Polymerization Inhibitors,
Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.06.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

[
Type text]
Novel Pyrrol-2(3H)-ones and Pyridazin-3(2H)-ones Carrying Quinoline
Scaffold as Anti-proliferative Tubulin Polymerization Inhibitors
a
b
a
Mahmoud S Abdelbaset , Gamal El-Din A Abuo-Rahma *, Mostafa H Abdelrahman ,
a
c,d
d
Mohamed Ramadan , Bahaa G. M. Youssif , Syed Nasir Abbas Bukhari , Mamdouh
e
b
F.A. Mohamed and Mohamed Abdel-Aziz
a
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, 71524
Assiut, Egypt
Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt
b
c
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526,
Egypt
d
Department of Pharmaceutical Chemistry, College of Pharmacy, Aljouf University, Aljouf, Sakaka 2014,
Saudi Arabia.
e
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sohag University, 82524 Sohag, Egypt
Abstract
A novel quinolinyl pyrrolone and quinolinyl pyridazinone derivatives has been
synthesized and characterized using different spectroscopic and elemental analysis
techniques. Most of the target compounds displayed promising antiproliferative activity;
In general, the pyrrolone derivatives 4a-f exhibited higher antiproliferative activity than
their corresponding pyridazinone. The pyrrolone 4f showed outstanding antiproliferative
activity with moderate selectivity against CNS and renal cancer with selectivity ratio of
3
.49 and 3.56, respectively. Compound 4e and 5d experienced tubulin polymerization
inhibitory activity comparable to that of vincristine while 4c, 4e and 4d showed good
BRAF kinase inhibition compared to Erlotinib. Docking of compound 4e into colchicine
binding site and biological assay results revealed that these compounds act mainly
through tubulin polymerization inhibitory mechanism and can exhibit pre G1 apoptosis
and cell cycle arrest at G2/M phase.
Key words: Pyrrol-2(3H)-ones; Pyridazin-3(2H)-ones; Quinoline, Antiproliferative;
Tubulin polymerization; BRAF Kinase.
Introduction:
In recent years, the targeted anticancer therapy represent an important class of
anticancer agents[1]. Also, medicinal chemistry researches are focussing on selectivity of
-
1 -

[
Type text]
those therapies toward special cancer type [2]. The targeted anticancer drugs interfere
with specific enzyme, protein or blocking certain receptors preventing cancer cell division
and growth. The most famous examples for targeted cancer therapies are kinase inhibitors
[
3-6], topoisomerase inhibitors [7-9] and tubulin polymerization inhibitors [10,11].
Tubulin is a globular protein that plays an important role in mitosis. So, its inhibition is
considered one of the important targets in treatment of cancer [12]. Several naturally
occurring compounds inhibit tubulin polymerization such as
podophyllotoxins,
vincristine, colchicines [12] and combretastatin A-4 (CA-4) I through binding to
colchicine binding site leading to obvious shutdown of blood flow to solid tumour,
resulting in considerable necrosis for cancer cells [12-15].
The structural requirements of CA-4 includes a cis double bond, trimethoxy
phenyl ring A and ring B with small substitution having different steric, electronic and
lipophilicities. SAR studies revealed that replacement of ring A with halogenated phenyl
moiety retains the antitubulin activity [16] such as compound II. Similarly, replacement
of ring B with bicyclic moiety like naphthalene maintain the antitubulin activity [17] such
as compound III. Several literature reports provided an evidence about isomerization of
the cis double bond into trans lead to reduction of antitubulin and cytotoxicity [18, 19].
Consequently, Rigidification of the cis double bond by heterocyclic ring gave compounds
with potent cytotoxic activity[17] such as compounds II-IV, Fig.1.
Pyrrolone nucleus is considered as an important scaffold with variable biological
activities including antibacterial [20], anti-inflammatory [20], analgesic,
antimycobacterial [21] and antitumor activities [22]. Also, pyridazinones have wide
spread pharmacological actions such as antihypertensive [23], platelet aggregation
inhibitors [24], cardiotonic [25], antimicrobial [26], analgesic[27] and anti-inflammatory
[
27] activities. In addition, pyridazinones also have been reported as anticonvulsant [28],
anticancer [29] and PDEIII, IV inhibitors [25]. Some literature reports [30] referred to the
anticancer mechanism of pyridazinones and pyrrolones through tubulin polymerization
inhibition. A recent study revealed a pronounced inhibitory effect of pyridazinones and
pyrrolones against cellular localization of tubulin [31].
-
2 -

[
Type text]
Figure.1: Structure of CA-4 I and different synthetic analogues II, III and IV
Based on the above mentioned promising aspects, this work includes design of novel
pyrrolone and pyridazinone derivatives as CA-4 analogues which do not contain cis-
unstable double bond of CA-4. (Fig.2). In addition, replacement of trimethoxyphenyl
rings A in CA-4 with 6-substituted-quinoline ring to afford quinolinyl-pyrrolones
orpyridazinones. Quinoline moiety was introduced to increase water solubility through
hydrogen bonding, which is a major drawback of combretastatins in clinical trials. Also,
quinoline is bicyclic rings which may produce more fitting to colchicines-binding site like
naphthalene. Furthermore, quinoline ring was reported to be one of the kinase inhibitors
which may synergize the antiproliferative activity of the target compounds. The
substituent in position two of quinolone ring was designed to be with different electronic
and steric properties to investigate their effect on cytotoxic activities. The design of this
work was supported by docking study, which showed good fitting to tubulin target site.
The target compounds were tested for antiproliferative activity. Moreover, the
V6000
mechanistic study was evaluated on BRAF
, EGFR Tk kinases and tubulin
polymeization inhibitory activity. Furthermore, cell cycle analysis and apoptosis assay
were established for the most active compounds.
R1
N
R1
N
OCH3
A
A
OCH3
R
R
A
O
O
OCH3
C
C
C
N
OCH3
NH
NH
B
B
B
OCH3
Quinolinyl-Pyrrol-2(3H)-one
derivatives
Pyridazin-3(2H)-one
derivatives
Combretastatin A-4 (CA-4)
Figure.2: General structure for target compounds
-
3 -

[
Type text]
Results and Discussion
Chemistry
Scheme 1: Synthesis of the target quinolinyl-pyrrolones 4a-f and pyridazinones 5a-f
Reagents and reaction conditions: (a) Anh.AlCl
reflux; (c) NH (33%), EtOH, reflux; (d); NH NH
3
, Reflux; (b) TEA, acetic anhydride,
3
2
2
.H O (33%), EtOH, reflux.
2
The target compounds 4a-f and 5a-f were synthesized according to the synthetic route
outlined in Scheme 1. Heating at reflux of benzene and succinic anhydride in presence of
anhydrous aluminum chloride (1.1 eq.) under the condition of Friedl-Crafts acylation
afforded 3-benzoylpropionic acid 1 in good yield [32]. The intermediates quinoline
aldehydes 2a-f were synthesized through the reported methods [33-35]. Perkin
-
4 -

[
Type text]
condensation of 3-benzoylpropionic acid 1 with different quinoline aldehydes 2a-f in
presence of TEA and drops of acetic anhydride afforded the corresponding furanones [36]
1
3
a-f. H NMR spectra of furanones 3a,b showed a common singlet signal appeared at δ
1
1
2.11 and δ 12.06 ppm, respectively related to -OH group. Also, H NMR spectra of
furanones 3c,d showed a common signal appeared as singlet signal at δ 4.11 and 4.06
ppm, respectively related to -OCH group. Moreover, a characteristic singlet signal
appeared at δ 2.80 and δ 2.68 ppm related to -SCH group in the spectra of furanones 3e,
f, respectively. The C NMR spectra of furanone derivatives 3a-f showed a common
characteristic signal at δ 101.6 - 101.0 ppm related to C of furanone ring and another
3
3
1
3
4
signal at δ 168.6-168.0 ppm related to (C=O) of furanone. The olefenic and aromatic
carbons appeared at their expected chemical shifts.
The quinolin-3-yl-pyrrol-2(3H)-one derivatives 4a-f were synthesized by heating at reflux
1
3
a-f with ammonia (33%) in absolute ethanol. H NMR spectra of compounds 4a-f
showed a common singlet signal appeared at δ 10.75 - 9.12 ppm related to -NH group of
pyrrolone. Also, a common characteristic signal appeared as a singlet at δ 7.06 - 6.92 ppm
related to -H
.79 - 8.50 ppm related to -H
derivatives 4a-f showed a common characteristic signal at δ 101.6 - 97.0 ppm related to
of pyrrolone ring and another signal at δ 171.3 – 170.3 ppm related to (C=O) of
4
of pyrolone ring. Moreover, a characteristic singlet signal appeared at δ
1
3
8
4
of quinoline ring. The C NMR spectra of pyrrolone
C
4
pyrrolone. The olefinic and aromatic carbons appeared at their expected chemical shifts.
The quinolin-3-yl- pyridazinon-3(2H)-one derivatives 5a-f were synthesized by heating at
1
reflux 3a-f with hydrazine hydrate in absolute ethanol[37]. H NMR spectra of
compounds 5a-f showed a common singlet signal appeared at δ 13.29 - 12.96 ppm related
to -NH of pyridazinone. Also, a common characteristic signal appeared as a singlet at δ
4
.0 - 3.53 ppm related to the benzylic -CH
2
at the position 4 of pyridazinone ring.
of
Moreover, a characteristic singlet signal appeared at δ 8.00 - 7.62 ppm related to H
4
1
3
quinoline ring. The C NMR spectra of pyridazinone derivatives 5a-f showed the
characteristic benzylic carbon at δ 31.4 - 29.7 ppm and another characteristic signal of
(
C=O) at δ 161.6 – 160.5 ppm. The olefinic and aromatic carbons appeared at their
expected chemical shifts. Furthermore, DEPT135 spectra of compounds 5a-f exhibited a
characteristic signal at δ -29.74 to δ -30.45 ppm related to -CH benzylic carbon. The
2
purity of the newly prepared compounds was confirmed by elemental analysis; the results
are consistent with the molecular formula of the products. Mass spectra of the target
-
5 -

[
Type text]
compounds are consistent with the calculated ones. It is a worthy note that mass spectra
of compounds 3e,f, 4e,f and 5e,f exhibited {M+2} peak with relative intensity 4.38
related to sulphur isotope.
Biological evaluation
Evaluation of in vitro antiproliferative activity by NCI.
The present study investigates the antiproliferative activity of quinolinyl-furanones 3a-f,
quinolinyl-pyrrolones 4a-f, quinolinyl-pyridazinones 5a-f according to the protocol of the
drug evaluation branch of the national cancer institute (NCI), Bethesda, USA, for in vitro
anticancer activity. (http://www.dtp.nci.nih.gov). Results for each tested compound were
reported as the percentage of growth of the treated cells when compared to the untreated
control cells (see supplementary data).
The results indicated that quinolinyl furanones 3a-f that showed a weak anticancer
activity against different cancer cell lines. On the other hand, quinolinyl-pyrrolones 4a-f
exhibited noteworthy antiproliferative activity and growth inhibition percentage for these
compounds were outlined in Table 1. Compound 4a showed a moderate activity against
leukemia cancer cell line RPMI-8226, SR and non-small cell lung cancer cell line HOP-
9
2. Compound 4c revealed complete cell death against leukemia cancer cell line HL-60
and melanoma cancer cell line MDA-MB-435. Also, it exhibited remarkable growth
inhibition activity against leukemia cancer cell line K-562, MOLT-4, SR, non-small cell
lung cancer cell line NCI-H460, colon cancer cell line COLO 205, HCT-116, HT-29,
SW-620, CNS cancer cell line SF-295, SF-539, SNB-75, melanoma cancer cell line M-
1
4, SK-MEL-2, ovarian cancer cell line OVCAR-3, renal cancer cell line 786-0, breast
cancer cell line MCF-7, HS 578T and MDA-MB-468. Compound 4d showed complete
cell death against leukemia cancer cell line HL-60 and melanoma cancer cell line MDA-
MB-435. Also, it exhibited remarkable growth inhibition activity against leukemia cancer
cell line CCRF-CEM, K-562, MOLT-4, SR, non-small cell lung cancer cell line NCI-
H460, NCI-H522, colon cancer cell line HCT-116, HCT-15, HT-29, KM-12, CNS cancer
cell line SF-539, melanoma cancer cell line M-14, SK-MEL-2, UACC-62, ovarian cancer
cell line OVCAR-3, renal cancer cell line A498, breast cancer cell line MCF-7, BT-549
and MDA-MB-468. Compound 4e exhibited remarkable growth inhibition activity
-
6 -

[
Type text]
against leukemia cancer cell line SR and melanoma cancer cell line MDA-MB-435.
Additionally, it showed moderate growth inhibition activity against leukemia cancer cell
line K-562, MOLT-4, colon cancer cell line HCT-15, HT 29 and breast cancer cell line
MCF7. Compound 4f exhibited complete cell death against ovarian cancer cell line
OVCAR-4, renal cancer cell line ACHN, TK-10 and breast cancer cell line T-47D,
respectively. Also, it exhibited remarkable growth inhibition activity against leukemia
cancer cell line SR, non-small cell lung cancer cell line NCI-H460, colon cancer cell line
HCT-116, CNS cancer cell line SNB-75, U251, melanoma cancer cell line MDA-MB-
4
35, ovarian cancer cell line IGROV1, OVCAR-3, renal cancer cell line 786-0, CAKI-1,
UO-31 and breast cancer cell line HS 578T. Results of quinolinyl-pyridazinones 5a-f
indicated that compounds 5a,c,e,f has weak anticancer activity. On the other hand,
compound 5b and 5d revealed a noteworthy antiproliferative activity. Compound 5b
exhibited remarkable growth inhibition activity against non-small cell lung cancer cell
line HOP-92 and renal cancer cell line 786-0 Table 1. Moreover, Compound 5d showed
complete cell death against non-small cell lung cancer cell line HOP-92 and renal cancer
cell line 786-0. Also, it exhibited remarkable growth inhibition activity against CNS
cancer cell line SF-539, SNB-19, SNB-75, renal cancer cell line ACHN, CAKI-1, RXF
3
93 and breast cancer cell line HS 578T Table 1.
Table 1: Growth inhibition percentage of compounds 4a,c-f and 5b,d
Panel
Cell line
Growth inhibition percentage %
4
a
4c
-
108.46
86.75
78.47
-
4d
79.98
104.30
86.27
81.21
-
4e
4f
5b
5d
CCRF-CEM
HL-60
K-562
MOLT-4
RPMI-8226
SR
-
-
-
-
-
Leukemia
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
64.32
73.71
37.19
39.51
-
80.00
85.87
72.60
89.94
-
-
HOP-92
NCI-H460
NCI-H522
COLO-205
HCT-116
HCT-15
HT-29
KM-12
SW-620
SF-539
66.30
-
-
-
-
-
-
-
83.38
120.33
Non-small
lung cancer
-
-
-
-
-
-
-
-
-
-
-
-
-
84.27
74.36
73.03
90.79
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
70.76
87.29
Colon
cancer
82.34
23.35
92.33
81.73
-
-
90.74
58.72
62.07
-
-
-
-
-
89.96
-
-
-
-
-
-
72.34
80.39
71.43
-
75.41
112.53
91.06
-
86.88
80.15
CNS
cancer
SNB-75
U 251
M 14
73.60
82.74
-
-
-
-
-
82.20
101.51
Melanoma
MDA-MB-
96.73
69.05
435
SK-MEL-2
-
81.02
96.69
-
-
-
-
-
7 -

[
Type text]
UACC-62
IGROV1
OVCAR-3
OVCAR-4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
70.09
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
74.43
93.76
151.87
97.34
Ovarian
cancer
82.85
83.91
-
-
-
786-0
70.55
67.29
104.27
Renal
cancer
A498
-
-
-
75.31
-
-
-
-
-
-
-
-
-
-
ACHN
CAKI-1
TK-10
-
-
152.79
94.63
106.59
81.59
85.54
-
75.82
74.18
-
-
78.45
-
-
-
-
MCF7
50.19
Breast
cancer
HS 578T
BT-549
T-47D
-
-
-
-
71.10`
67.11
70.06
-
-
-
-
-
100.11
-
MDA-MB-
71.00
91.36
468
(
-): Weak activity.
Five dose testing for compound 4f
Compound 4f was selected for advanced five dose testing by NCI, where the cytotoxic
and/or growth inhibitory effects of the tested compound was tested in vitro against 60
human cancer cell lines derived from nine neoplastic diseases at 10 fold dilutions of five
-4
-8
concentrations ranging from 10 M to 10 M. Three dose response parameters were
calculated for each cell line, GI50 value (concentration causing 50% inhibition of net cell
growth), TGI value (concentration causing total growth inhibition) and LC50 value
(
concentration causing 50% loss of cells). The log10 GI50, log10 TGI, log10 LC50 were then
,
determined, defined as the means of the log10 s of the individual GI50, TGI, LC50 value.
Negative values indicated the most sensitive cell lines. Compound having log10 GI50
values -4 and < -4 is declared to be active. Selectivity of a compound can be evaluated
a
through the ratio obtained by dividing MID (the average sensitivity of all cell lines
b
toward the test agent µM) by their individual subpanel MID (µM). Compounds called
highly selective when this ratio > 6 and moderately selective if this ratio ranging from 3 -
6
, while ratios ˂ 3 refer to non-selective compounds. Results indicated that compound 4f
exhibited strong antiproliferative activity against all tested human cancer cell lines, where
the mid of log10 GI50 ranges from -5.35 to -4.06 (see supplementary data). Moreover,
results mentioned in Table 2 indicated that compound 4f exhibited moderate selectivity
toward the CNS cancer and renal cancer subpanels with selectivity ratio of 3.49 and 3.56,
respectively, at GI50level.
-
8 -

[
Type text]
Table 2. Selectivity of compound 4f on nine human cancer types
Panel
Cell line
GI50
TGI
LC50
b
Conc.per cell line MID Selectivity ratio
Leukemia CCRF-CEM
HL-60(TB)
26.40
100.00
8.83
15.50
˃100
˃100
˃100
˃100
˃100
˃100
˃100
˃100
K-562
MOLT-4
26.71
5.48
0.33
1.60
RPMI-8226
SR
Non-small A549/ATCC
8.90
0.60
3.54
13.1
2.42
2.85
7.30
10.00
5.94
0.65
3.53
5.90
10.00
0.73
14.00
3.44
3.13
2.62
3.16
1.44
2.35
4.6
˃100
˃100
30.7
˃100
˃100
˃100
˃100
94.50
˃100
˃100
˃100
˃100
79.80
˃100
˃100
˃100
40.30
˃100
˃100
˃100
˃100
˃100
67.30
36.30
˃100
˃100
25.1
cell lung
cancer
EKVX
˃100
11.70
13.40
˃100
˃100
˃100
10.90
52.50
˃100
˃100
4.07
˃100
˃100
82.90
92.10
48.00
15.00
9.66
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
Colon
cancer
5.69
2.49
1.52
3.49
KM12
SW-620
SF-268
SF-295
CNS
cancer
SF-539
SNB-19
SNB-75
U251
33.50
14.80
3.68
33.20
25.90
2.81
0.60
6.33
3.08
Melanoma LOX IMVI
˃100
˃100
MALME-
3M
M14
MDA-MB-
5.42
0.72
78.0
21.0
˃100
˃100
19.07
0.46
435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-
RES
6.31
23.10
21.30
94.60
10.80
2.71
1.78
0.30
19.30
3.60
3.99
30.4
˃100
˃100
˃100
˃100
˃100
˃100
82.8
˃100
˃100
˃100
48.2
24.10
15.90
1.03
˃100
23.20
87.50
Ovarian
Cancer
9.75
5.21
2.44
1.67
3.56
˃100
˃100
˃100
SK-OV-3
786-0
ACHN
CAKI-1
4.83
2.00
1.04
1.21
3.06
˃100
5.89
5.54
10.90
10.00
˃100
36.30
˃100
˃100
61.60
Renal
Cancer
RXF 393
-
9 -

[
Type text]
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
4.53
2.63
2.58
5.32
6.72
12.50
4.42
32.30
8.46
˃100
˃100
89.70
˃100
˃100
˃100
˃100
12.40
˃100
96.10
˃100
36.60
Prostate
Cancer
Breast
6.02
1.44
1.72
MDA-MB-
Cancer
231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-
1.92
3.94
1.66
5.77
7.46
˃100
96.80
˃100
˃100
5.03
22.90
24.50
61.30
468
a
MID
8.68
According to the above results, it is obvious that quinolinyl-furanones gave weak
anticancer activity which may be attributed to absence of amidic group present in both
active pyrrolones and pyridazinones. The docking study supported this idea and showed
that amidic group makes hydrogen bonding with amino acids ASN101 and ASN258 at the
colchicine site. Conversion of furanones into pyrrolones and pyridazinones increased the
anticancer activity, but the pyrrolones were found to be more potent than the pyridazinone
derivatives. Also, substitution of quinolinyl-pyrrolone at the position two of quinoline
ring with hydroxyl group decreased the anticancer activity, while substitution with
methoxy or methylthio groups at this position increased the anticancer activity.
Cell viability assay
Cell viability assay was carried out for quinolinyl-pyrrolones 4c,4e and 4f, quinolinyl-
pyridazinone 5d,e using human mammary gland epithelial cell line (MCF-10A).
Compounds were treated with MCF-10A cells for 4 days and 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium Bromide (MTT) assay was applied to evaluate the viability of
cells. Tested compounds were revealed to be non-toxic where exhibited more than 90 %
MCF-10A cell viability.
Cytotoxic activity evaluation of IC50
The selected pyrrolones and pyridazinones were evaluated according to standard
protocols for their in vitro antiproliferative activity against five human cancer cell lines
-
10 -

[
Type text]
including human pancreas cancer cell line (Panc-1), pancreatic carcinoma cells (PaCa-2),
colon cancer cells (HT-29), lung cancer cells (H-460) and melanoma cancer cell line
(
A375) using the propidium iodide (PI) fluorescence assay. The known anticancer drug
Erlotinib was used as a reference cytotoxic drug. GraphPad Prism software (GraphPad
Software, San Diego, CA, USA) was used to calculate the median inhibition
concentration (IC50) for all compounds. All of IC50 values in micromolar (µM) are listed
in Table 3.
The quinolinyl-pyrrolone 4c showed significant antiproliferative effects with IC50 of 2.5
and 2.8 µM against human pancreas cancer cell line panc-1 and pancreatic carcinoma cell
line paca-2, respectively. Also, it exhibited a moderate activity with IC50 values 5.9 and
4
.7 µM against lung cancer cell line H-460 and colon cancer cell line HT-29,
respectively. Moreover, the IC50 of this compound against melanoma cancer cell line
A375 is 8.8 µM, which a comparable to that of standard drug Erlotinib (IC50 = 5.12 µM).
Compounds 4e showed significant anticancer activity against all cancer cell lines with
IC50 of 2.0, 2.6, 1.9 and 1.9 µM against human pancreas cancer cell line panc-1,
pancreatic carcinoma cell line paca-2, lung cancer cell line H-460 and colon cancer cell
line HT-29, respectively. Moreover, results indicated that compound 4e is more potent
than Erolitinib with IC50 of 4.9 and 5.12 µM, respectively on melanoma cancer cell line
A375.
Quinolinyl-pyridazinone 5d showed significant activity with IC50 of 2.2 and 2.9 µM
against pancreatic carcinoma cell line paca-2 and human pancreas cancer cell line panc-1,
respectively. Also, it showed moderate activity against colon cancer cell line HT-29 and
lung cancer cell line H-460 with IC50 of 4.5 and 5.5 µM, respectively. Additionally, it
exhibited a good activity compared to Erlotinib against melanoma cancer cell line A375
with IC50 of 9.7 and 5.12 µM, respectively. On the other hand, compound 5e showed
moderate activity where IC50 range is 5.9 - 11.5 µM on different cancer cell lines.
Moreover, compounds 4c and 4e showed good activity compared to CA-4 against PaCa-
2
, A375, H-460 and Panc-1 cell lines. Also, compound 4e exhibited a noteworthy IC50
(
1.9 µM) against colon cancer cell line HT-29. Furthermore, compound 5d experienced
high antiproliferative activity against PaCa-2, H-460 and Panc-1 cell lines comparable to
CA-4.
-
11 -

[
Type text]
Table 3: Antiproliferative activities of synthetic pyrrolones 4c,e,f and pyridazinone 5d,e.
Antiproliferative activity IC50 ± SEM (µM)
Code
HT-29
PaCa-2
A375
H-460
Panc-1
NO.
4
4
c
e
4.7±1.6
1.9±0.9
2.8±0.9
2.6±1.2
8.8±2.5
4.9±1.5
>50
5.9±2.1
1.9±0.6
>50
2.5±1.2
2.0±0.5
1
2
3
4
5
.
.
.
.
.
4
f
19.5±2.4
4.5±0.7
9.2±1.7
9.9±2.4
5d
2.2±1.2
9.7±1.3
11.5±2.5
5.12±1.7
2.82±0.24
5.5±1.2
7.8±2.4
0.02±0.01
0.71±0.08
2.9±1.7
5
e
7.8±1.5
6.7±0.9
5.9±2.2
Erlotinib
CA-4
0.05±0.02
0.18±0.004
0.04±0.02
0.43±0.001
0.06±0.03
2.36±0.15
Tubulin polymerization inhibitory activity
The effect of selected newly synthetic, compounds 4c, 4e and 5d on tubulin
polymerization at concentration of 25 µM using vincristine and docetaxel (3 µM) as
reference compounds is outlined in fig.3 and table4. Compounds 4e and 5d are proved to
be the strongest inhibitors with tubulin polymerization inhibitory effect of (988) and
(
1002), respectively comparable to that of vincristine (805). From these results, it is
obvious that the molecular target of these compounds was most likely tubulin. On the
other hand, compound 4c did not inhibit the assembly of tubulin suggesting a different
mechanism for the observed cytotoxicity rather than tubulin polymerization inhibition.
-
12 -

[
Type text]
Figure 3: Effect of selected synthetic compounds on tubulin polymerization activity at
concentration of 25 µM. Results are the mean values of three experiments, n = 3, while
vincristine and docetaxel (3 µM) were used as reference compounds. The y-axis
demonstrates tubulin polymerization activity measured at 15 min (arbitrary units) in the
growth phase of the tubulin polymerization curve.
EGFR Inhibitory Activity:
EGFR is a protein which present on the surface of some cells that causes cells division
when occupied by epidermal growth factor. EGFR is found in high levels in cancer cells.
EGFR inhibitors like erlotinib bind to the tyrosine kinase domain in the epidermal growth
factor receptor and stop its activity. Table 4 shows the results of EGFR-TK assay for
tested compounds 4c, 4e and 5d. From the results, it is clear that quinolinyl-pyrrolones
and pyridazinones gave a weak EGFR inhibition with IC50 of 7.8 - 12.5 µM.
V600E
BRAF
inhibitory activity
BRAF is a kinase enzyme which controls cell growth and signaling. It may be found in a
mutated form in some cancer cells. Blocking mutated BRAF kinase proteins may prevent
the growth of cancer cells. Agents which blocks BRAF like Erlotinib are usually used in
treatment of cancer and called BRAF kinase inhibitors.
V600E
The activity of the newly synthesized compounds against BRAF
was tested using an
in vitro experiment and IC50 values were summarized in Table 4. Quinolinyl-pyrrolnes
and pyridazinones 4c, 4e and 5d exhibited good BRAF inhibitory activity with IC50
values of 3.4, 1.9 and 2.9 µM, respectively.
-
13 -

[
Type text]
Table 4: Effects of compounds 4c,e and 5d on Tubulin polymerization, EGFR and
V600E
BRAF
.
Compound
BRAF
EGFR
inhibition
(Tubulin)
Effect
inhibition
IC50 ± SEM
IC50 ± SEM
(arbitrary units)
(
µM)
(
µM)
3
1
2
.4±2.0
.9±0.7
.9±1.6
12.5±1.9
1635±248
988±160
1002±157
-
4
4
c
e
10.6±1.8
7.8±2.1
5d
0.05±0.03
0.06±0.03
Erlotinib
DPBS
-
-
-
-
-
-
2626±198
805±227
4625±290
Vincristine
Docetaxel
Cell cycle analysis and apoptosis
Cell cycle analysis was performed for the most active compounds 4e (2.6µM) and 5d
4.5µM) on Paca-2 and HT-29, respectively. In both cell lines, Paca-2 and HT-29 the
(
percentages of cells in G0/G1 phase of the cell cycle were 53.29% and 44.61% which
recorded a noteworthy decrease to 23.09% and 36.06% in G2/M phase for compounds 4e
and 5d, respectively, (Table:5, Fig.4 and fig.6). Furthermore, it is obvious that the
apoptotic cell percentage was increased from 2.17% for control untreated Paca-2 cells to
1
1
6
0.27% in treated cells with compound 4e and from 2.48% in control untreated HT-29 to
3.64% in cells treated with compound 5d, (Table.5). Moreover, from the results in table
it is clear that percentage of late apoptosis is more than that of early apoptosis which a
good evidence for irreversible apoptosis caused by both compounds 4e and 5d (Table:6 ,
Fig.5 and fig.6).
According to the above results, it can be concluded that the tested compounds exhibited
pre G1 apoptosis and cell cycle arrest at G2/M phase. Moreover, it is obvious that tested
compounds not cytotoxic but antiproliferative causing programed cell death and cell cycle
arrest.
-
14 -

[
Type text]
Table 5: Results of cell cycle analysis for compounds 4e, 5d and CA-4 on Paca-2 and
HT-29, respectively.
Sample data
Results
Ser
Sample
code
cells
%G0-G1
%S
%G2-M
%pre-G1
Comment
1
2
3
4e
5d
PaCa-2
HT-29
HT-29
53.29
44.61
39.33
22.81
19.33
14.82
23.09
36.06
45.85
10.27
13.64
18.62
PreG1apoptosis&Cell
growth arrest@G2/M
PreG1apoptosis&Cell
growth arrest@G2/M
PreG1apoptosis&Cell
growth arrest@G2/M
CA-4
4
5
Cont. PaCa-2
Cont. HT-29
59.25
56.65
29.24
35.99
11.51
7.36
2.17
2.48
Figure.4: Cell cycle analysis for compounds 4e (2.6µM) and 5d (4.5µM) on Paca-2 and
HT-29, respectively.
Table 6: Apoptosis % of compounds 4e, 5d and CA-4
Sample data
Apoptosis %
Necrosis
ser
Sample code
Total
10.27
13.64
18.62
2.17
early
3.07
5.29
5.83
0.82
1.36
Late
5.74
6.14
9.66
0.84
0.73
1
2
3
4
5
4e
5d
CA-4
1.46
2.21
3.13
0.51
0.39
Cont. PaCa-2
Cont. HT-29
2.48
-
15 -

[
Type text]
Figure.5: Apoptosis% of compounds 4e, 5d against Paca-2 and HT-29 cell lines at
concentration 2.6µM and 4.5µM, respectively.
-
16 -

[
Type text]
Figure.6: Cell cycle analysis and Apoptosis induction analysis using Annexin V/PI of
compounds 4e, 5d and CA-4 against Paca-2 and HT-29 at concentration 2.6µM and
4
.5µM, respectively.
Molecular docking
Tubulin is an attractive promising target in anti-tumor therapy due to the vital role of
microtubules in the cell cycle [38]. On the design and our preliminary data of the tubulin
polymerization assay, we hypothesized that the target compounds might bind to tubulin in
-
17 -

[
Type text]
the same way as colchicine. Therefore, compounds 4e and 4c were selected for molecular
docking studies into the active site of tubulin (PDB code: 1SA0) using Discovery Studio
software package with the aim of elucidating the binding mode of these compounds with
tubulin and investigate their similarity to the native ligand. As a first step and to confirm
the validity of the docking method, molecular docking studies of CA-4 with tubulin were
performed. As shown in Fig. 7A and 7B, CA-4 binds to the active pocket of tubulin by
interacting with amino acid residues YS 241 (a-tubulin, hydrogen bond, O----H–O: 2.06
Å) and many other hydrophobic interactions with D: VAL315, D: MET259, D: ASN258,
C: ALA180, D: LEU248, D: LYS352, D: ALA180, D: ALA354 and D: ALA317 as
previously reported indicating our docking method is reliable[10].
Figure.7: (A) 2D interaction diagram of the top docking pose of CA-4 into the active site
of tubulin (PDB: 1SA0) (B) Docking and binding pattern of CA-4 (green) into the active
site of tubulin (PDB: 1SA0) 3D structure. All hydrogens were removed for the purposes
of clarity.
Analysis of the docking results revealed that the docking studies were in consistence with
the tubulin polymerization assay. From the inspection of the docking results, it was found
that compound 4e showed comparable CDOCKER energy to the reference ligand and it
interacted with variable amino acids previously reported in molecular modeling studies of
CSIs[39]. As shown in Fig. 8A and 8B, a properly positioned NH group at pyrrole ring of
compound 4e was engaged in hydrogen bond interaction with C:ASN101 (O----H–N:
2
.27 Å) and the carbonyl group was engaged with another hydrogen bond with
D:ASN258 (C=O----H–O: 3.03 Å), this hydrogen bond was reported to increase the
-
18 -

[
Type text]
activity[40]. Of importance mentioning that although the hydrogen bonding to CYS241
was not reported in the docking poses of compounds 4e, a quinoline nucleus in this
compound was in the vicinity of this amino acid forming an important Pi interaction with
CYS241. Moreover, compound 4e shows other valuable interactions with hydrophobic
residues, such as D: ALA250, D: LEU255, D:LYS254, D:ALA316, D:ALA254,
D:VAL318, D:LEU248, C:ALA180.
Figure.8: (A) 2D interaction diagram of the top docking pose of the 4e into the active site
of tubulin (PDB: 1SA0) (B) Docking and binding pattern of compound 4e (magenta) into
the active site of tubulin (PDB: 1SA0) 3D structure. All hydrogens were removed for the
purposes of clarity.
On the other hand, compound 4c, the least active one, occupied the colchicine-binding
pocket without engaging in any hydrogen bond. However, compound 4c makes Pi-Sigma
interactions with D: Leu248 and Pi-Sulphur interaction with D: Cys241 and many other
hydrophobic interactions with C: THR178, C: SER178, D: LEU255, D: LYS254, D:
Ala250 and D: ALA316 Fig. 9A and 9B. Based on the docking results above, as a
promising novel tubulin-binding agent, compound 4e could occupy the colchicine binding
site of tubulin and had the potential to exhibit antitumor activity through inhibition of
tubulin polymerization, which merits further investigation.
-
19 -

[
Type text]
Figure.9: (A) 2D interaction diagram of the top docking pose of the 4c into the active site
of tubulin (PDB: 1SA0) (B) Docking and binding pattern of compound 4c (yellow) into
the active site of tubulin (PDB: 1SA0) 3D structure. All hydrogens were removed for the
purposes of clarity.
Conclusion
The target compounds, pyrrol-2(3H)-one 4a-f and pyridazin-3(2H)-one 5a-f carrying
quinoline moiety has been prepared through the intermediate furanones 3a-f. The
1
13
structural formula of the prepared compounds was proved by H NMR, C NMR, DEPT,
Mass and Elemental analysis. The target final compounds 4a-f and 5a-f were screened in
one dose testing for their cytotoxic activity against 60 cell lines according to NCI
protocol. The intermediate furanones 3a-f showed a weak anticancer activity against
different cancer cell lines. On the other hand, the target pyrrolones and pyridzinones
experienced promising anticancer activity. Compound 4f displayed outstanding activity in
five dose testing with moderate selectivity toward the CNS cancer and renal cancer
subpanels with selectivity ratio of 3.49 and 3.56, respectively From the results, it is
.
obvious that substitution of quinoline with 2-hydroxyl group reduce activity drastically
while substitution with 2-methoxy or 2-methylthio moiety can increase activity. Studying
the mechanism of action for compounds 4c, 4e and 5d revealed that the quinolinyl-
pyrrolones and pyridazinones has weak EGFR inhibition with IC50 of 7.8-12.5 µM and
good BRAF inhibitory activity with IC50 values of 3.4, 1.9 and 2.9 µM, respectively.
Moreover, compounds 4e and 5d can display tubulin polymerization inhibitory activities
-
20 -

[
Type text]
with arbitrary value (988) and (1002), respectively comparable to that of vincristine
805). The enzyme assay results and molecular docking study of compound 4e on
(
colchicine binding site suggest that tubulin is the main molecular target of these
compounds. In summary, the quinolinyl pyrrolone compounds represent promising
antiproliferative agents with tubulin polymerization inhibitory activity and can exhibit pre
G1 apoptosis and cell cycle arrest at G2/M phase.
Experimental
Chemistry:
Chemicals and solvents used in the preparation of the target compounds are of
commercial grade and purchased from Sigma Aldrich, Alfa Aesar, Merck, Fluka and El-
Nasr pharmaceutical chemicals companies. Chemicals and solvents were used without
purification. Thin layer chromatography (TLC); was used for monitoring chemical
reactions and was carried out using silica gel 60 F254 precoated sheet 20 * 20 cm,
layer thickening 0.2 mm (E, Merck , Germany), and were visualized using UV- lamp at
2
54 nm. Melting points (mp) were determined using Stuart electrochemical melting point
1
apparatus (Stuart scientific, England) and were uncorrected. HNMR spectra were carried
out on 400 MHz Brucker spectrometer, using TMS as an internal reference. Chemical
shift (δ) values are given in parts per million (ppm) relative to DMSO-d
6
(2.5) and
coupling constants (J) in Hertz (Hz). Splitting patterns are designated as follows: s,
1
3
singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet; m, multiplet. CNMR
spectra were carried out on Brucker spectrometer (100MHz). Shimadzu GC/MS was used
for mass spectroscopy. Vario EL III German CHN Elemental analyser model was used
,
for Elemental analysis. Compounds1[32] 2a,b[33] [41], 2c,d[35], 2e[34], 2f [42] are
prepared and their structures were confirmed by melting point as reported in literature.
General procedure for the synthesis of substituted furanones 3a-f:
A mixture of 3-benzoylpropionic acid 1 (0.53g, 0.003 mole) was fused with the
appropriate quinoline aldehyde 2a-f (0.003 mole) in acetic anhydride (8 drops) for 5 min.
TEA (3drops) and acetic anhydride (4drops) were added and the contents were heated at
-
21 -

[
Type text]
reflux for 15 minutes. The solution was left to cool and a solid mass was formed, filtered
off and crystallized from methylene chloride/ methanol (1:1), affording the target
furanones 3a-f.
3
-((2-Hydroxyquinolin-3-yl)methylene)-5-phenylfuran-2(3H)-one 3a
1
Yellowish brown crystals; yield: 69%; mp: 187 - 189°C, HNMR (400 MHz, DMSO-d6)
δ ppm 12.16 (s, 1H, OH), 8.66 (s, 1H, Ar-H), 7.93 - 7.90 (m, 3H, Ar-H), 7.78 (s, 1H, Ar-H), 7.62
(s, 1H, olefenic H), 7.59 - 7.55 (m, 4H, Ar-H), 7.34 (d, J = 8.2 Hz, 1H, Ar-H), 7.30 - 7.27(m, 1H,
1
3
Ar-H). CNMR (101 MHz, DMSO-d6) δ ppm 168.8, 160.8, 156.5, 140.0, 139.1, 132.0,
1
30.8, 129.3, 128.7, 127.9, 127.7, 126.3, 125.4, 125.3, 122.4, 119.4, 115.2, 101.6. GS
+
MS: m/z calcd: 315.09, found [M+H] : 316.00. Anal. Calcd for C20
H
13NO : (315.09): C,
3
7
6.18; H, 4.16; N, 4.44. Found: C, 76.34; H, 4.51; N, 4.68.
3
-((2-Hydroxy-6-methylquinolin-3-yl)methylene)-5-phenylfuran-2(3H)-one 3b
1
Yellowish brown crystals; yield: 65%; mp: 182 - 183°C, HNMR (400 MHz, DMSO-d6)
δ ppm 12.06 (s, 1H, OH), 8.56 (s, 1H, Ar-H), 7.90 (dd, J = 7.7 Hz, J = 1.7 Hz, 2H, Ar-H),
7
.75 (s, 1H, Ar-H), 7.69 (s, 1H, Ar-H), 7.60 - 7.53 (m, 4H, Ar-H, olefenic-H), 7.40 (dd, J
13
=
8.4 Hz, J = 1.4 Hz, 1H, Ar-H), 7.24 - 7.21 (m, 1H, Ar-H), 2.37 (s, 3H, CH ). CNMR
3
(
101 MHz, DMSO-d6) δ ppm 168.6, 160.7, 156.4, 139.8, 137.2, 133.2, 131.6, 130.1,
29.0, 128.6, 127.7, 126.2, 125.4, 125.1, 125.0, 119.3, 115.1,101.5, 20.2. GS MS: m/z
calcd: 329.11, found: 329.03. Anal. Calcd for C21 : (329.11): C, 76.58; H, 4.59; N,
1
H15NO
3
4
.25. Found: C, 76.80; H, 4.67; N, 4.34.
3
-((2-Methoxyquinolin-3-yl)methylene)-5-phenylfuran-2(3H)-one 3c
1
Yellowish brown crystals; yield: 71%; mp: 173 - 175°C, HNMR (400 MHz, DMSO-d6)
δ ppm 8.82 (s, 1H, Ar-H), 8.09 (d, J = 7.6 Hz, 1H, Ar-H), 7.92 (dd, J = 7.8 Hz, 1.6 Hz, 2H, Ar-
H), 7.80 - 7.71 (m, 3H, Ar-H), 7.58 - 7.50 (m, 5H, Ar-H, olefenic H), 4.09 (s, 3H,
1
3
OCH
3
). CNMR (101 MHz, DMSO-d6) δ ppm 168.4, 159.3, 156.9, 146.1, 138.6, 131.4,
1
30.9, 129.0, 128.9, 128.7, 127.6, 126.8, 126.5, 125.5, 124.9, 124.8, 119.1, 101.2, 53.9.
GS MS: m/z calcd: 329.11 found: 329.95. Anal. Calcd for C21
H, 4.59; N, 4.25. Found: C, 76.19; H, 4.62; N, 4.37.
H
15NO : (329.11): C, 76.58;
3
-
22 -

[
Type text]
3
-((2-Methoxy-6-methylquinolin-3-yl)methylene)-5-phenylfuran-2(3H)-one 3d
1
Yellowish brown crystals; yield: 66%; mp: 159 - 161°C, HNMR (400 MHz, DMSO-d6)
δ ppm 8.73 (s, 1H, Ar-H), 7.93 - 7.86 (m, 3H, Ar-H), 7.74 - 7.67 (m, 2H, Ar-H), 7.58 -
1
3
7
.54 (m, 5H, Ar-H, olefenic H), 4.06 (s, 3H, OCH
MHz, DMSO-d6) δ ppm 168.4, 158.9, 156.8, 144.5, 138.1, 134.0, 133.3, 130.9, 129.0,
27.9, 127.6, 127.0, 126.3, 126.2, 125.4, 124.9, 118.9, 101.2, 53.8, 20.8. GS MS: m/z
calcd: 343.12 found: 343.09. Anal. Calcd for C22 : (343.12): C, 76.95; H, 4.99; N,
3
), 2.49 (s, 3H, CH ). CNMR (101
3
1
H
17NO
3
4
.08. Found: C, 76.79; H, 5.12; N, 4.27.
3
-((2-(Methylthio)quinolin-3-yl)methylene)-5-phenylfuran-2(3H)-one 3e
1
Yellowish brown crystals; yield: 73%; mp: 147 - 149°C, HNMR (400 MHz, DMSO-d6)
δ ppm 8.65 (s, 1H, Ar-H), 8.10 (d, J = 8.0 Hz, 1H, Ar-H), 7.92 - 7.90 (m, 3H, Ar-H), 7.81
-
7.79 (m, 1H, Ar-H), 7.65 (s, 1H, olefenic H), 7.61 - 7.54 (m, 4H, Ar-H), 7.49 (s, 1H, Ar-
1
3
H), 2.62 (s, 3H, SCH ). CNMR (101 MHz, DMSO-d6) δ 168.5, 159.5, 157.9, 147.7,
3
1
1
36.4, 131.9, 131.5, 129.6, 129.5, 128.2, 128.0, 127.8, 127.7, 126.9, 126.5, 126.0, 125.8,
01.5, 13.3. GS MS: m/z calcd: 345.08 found 345.98. Anal. Calcd for C21 S:
H15NO
2
(
345.08): C, 73.02; H, 4.38; N, 4.06. Found: C, 72.89; H, 4.61; N, 4.19.
3
-((6-Methyl-2-(methylthio)quinolin-3-yl)methylene)-5-phenylfuran-2(3H)-one 3f
1
Yellowish brown crystals; yield: 76%; mp: 144 - 145°C, HNMR (400 MHz, DMSO-d6)
δ ppm 8.55 (s, 1H, Ar-H), 7.93 - 7.90 (m, 2H, Ar-H), 7.86 (s, 1H, olefinic H), 7.82 - 7.79
(
m, 1H, Ar-H), 7.64 - 7.61 (m, 2H, Ar-H), 7.55 - 7.53 (m, 3H, Ar-H), 7.48 (s, 1H, Ar-H),
1
3
2
1
1
.68 (s, 3H, SCH
3
) 2.49 (s, 3H, CH ). CNMR (101 MHz, DMSO-d6) δ ppm 168.0,
3
57.3, 145.8, 135.5, 133.1, 132.7, 130.5, 129.0, 128.9, 127.9, 127.5, 127.0, 126.4, 125.6,
+
25.5, 125.3, 124.9, 101.0, 20.9, 12.8. GS MS: m/z calcd: 359.10 found [M+H] : 360.03.
Anal. Calcd for C22
.89; N, 4.15.
H
17NO S: (359.10): C, 73.51; H, 4.77; N, 3.90. Found: C, 73.87; H,
2
4
General procedure for synthesis of substituted substituted-1H-pyrrol-2(3H)-ones 4a-
f:
A solution of substituted furanones 3a-f (0.003 mole) and ammonia 33% (10 mL) in
absolute ethanol (30 mL) was heated at reflux for 3hr. The reaction mixture was
-
23 -

[
Type text]
concentrated and the formed precipitate was filtered off affording the target substituted-
1
H- pyrrole-2(3H)-ones 4a-f.
3
-((2-Hydroxyquinolin-3-yl)methylene)-5-phenyl-1H-pyrrol-2(3H)-one 4a
1
Orang powder; yield: 85%; mp: 190 - 192°C, HNMR (400 MHz, DMSO-d6) δ ppm
1
-
2.02 (s, IH, OH), 10.62 (s, 1H, NH), 8.56 (s, 1H, Ar-H), 7.93 - 7.91 (m, 3H, Ar-H), 7.50
7.46 (m, 5H, Ar-H, olefinic H), 7.31 (d, J = 8.0 Hz, 1H, Ar-H), 7.25 - 7.22 (m, 1H, Ar-
1
3
H), 7.05 (s, 1H, Ar-H). CNMR (101 MHz, DMSO-d6) δ ppm 170.8, 161.2, 146.7, 138.6,
1
9
38.5, 131.7, 131.2, 129.9, 129.5, 129.0, 128.9, 127.3, 125.7, 122.7, 122.1, 119.6, 115.0,
+
7.6. GS MS: m/z calcd: 314.11, found [M+H] : 315.03. Anal. Calcd for C20
H
14
N
2
2
O :
(
314.11): C, 76.42; H, 4.49; N, 8.91. Found: C, 76.84; H, 4.64; N, 9.12.
3
-((2-Hydroxy-6-methylquinolin-3-yl)methylene)-5-phenyl-1H-pyrrol-2(3H)-one 4b
1
Orang powder; yield: 82%; mp: 195 - 197°C, HNMR (400 MHz, DMSO-d6) δ ppm
1
7
7
1.94 (s, 1H, OH), 10.61 (s, 1H, NH), 8.51 (s, 1H, Ar-H), 7.94 (d, J = 7.6 Hz, 2H, Ar-H),
.73 (s, 1H, Ar-H), 7.53 - 7.45 (m, 4H, Ar-H, olefinic H), 7.38 (d, J = 7.6 Hz, 1H, Ar-H),
13
.23 (d, J = 8.4 Hz, 1H, Ar-H), 7.06 (s, 1H, Ar-H), 2.38 (s, 3H, CH ). CNMR (101 MHz,
3
DMSO-d6) δ ppm 171.3, 161.6, 147.2, 138.8, 137.2, 133.1, 132.0, 131.6, 130.4, 130.0,
1
29.3, 128.9, 127.7, 126.2, 123.3, 120.1, 115.4, 98.1, 20.9. GS MS: m/z calcd: 328.12,
+
found [M+H] : 329.02. Anal. Calcd for C21
Found: C, 76.97; H, 4.98; N,8.69.
H
16
N
2
O : (328.12): C, 76.81; H, 4.91; N, 8.53.
2
3
-((2-Methoxyquinolin-3-yl)methylene)-5-phenyl-1H-pyrrol-2(3H)-one 4c
1
Orang powder; yield: 86%; mp: 182 - 180°C, HNMR (400 MHz, DMSO-d6) δ ppm
1
0.68 (s, 1H, NH), 8.79 (s, 1H, Ar-H), 8.11 (d, J = 8.0 Hz, 1H, Ar-H), 7.94 (d, J = 8.0 Hz,
H, Ar-H), 7.78 (d, J = 8.0 Hz, 1H, Ar-H), 7.72 - 7.69 (m, 1H, Ar-H), 7.53 - 7.45 (m, 5H,
2
1
3
Ar-H, olefinic H), 7.06 (s, 1H, Ar-H), 4.09 (s, 3H, OCH ). CNMR (101 MHz, DMSO-
3
d6) δ ppm 170.7, 159.6, 147.1, 145.6, 137.8, 132.6, 130.7, 130.0, 129.4, 128.8, 128.7,
1
3
7
26.4, 125.8, 125.1, 124.6, 121.7, 120.1, 97.3, 53.8. GS MS: m/z calcd: 328.12, found:
28.10. Anal. Calcd for C21
7.08; H, 5.08; N, 8.77.
H
16
N
2
2
O : (328.12): C, 76.81; H, 4.91; N, 8.53. Found: C,
3
-((2-Methoxy-6-methylquinolin-3-yl)methylene)-5-phenyl-1H-pyrrol-2(3H)-one 4d
-
24 -

[
Type text]
1
Orang powder; yield: 68%; mp: 178 - 179°C, HNMR (400 MHz, DMSO-d6) δ ppm
0.66 (s, 1H, NH), 8.69 (s, 1H, Ar-H), 7.95 (d, J = 7.1 Hz, 2H, Ar-H), 7.89 (s, 1H, Ar-
H), 7.69 (d, J = 8.5 Hz, 1H, Ar-H), 7.58 - 7.42 (m, 5H, Ar-H, olefinic H), 7.04 (s, 1H, Ar-
1
1
3
H), 4.07 (s, 3H, OCH
3
), 2.49 (s, 3H, CH
59.7, 147.5, 144.5, 137.8, 134.2, 133.1, 133.0, 130.5, 129.9, 129.3, 128.2, 126.7, 126.2,
25.6, 122.4, 120.5, 97.8, 54.2, 21.3. GS MS: m/z calcd: 342.14, found: 342.02. Anal.
: (342.14): C, 77.17; H, 5.30; N, 8.18. Found: C, 76.84; H, 5.47; N,
3
). CNMR (101 MHz, DMSO-d6) δ ppm 171.2,
1
1
Calcd for C22
H
18
N
2
O
2
8
.37.
3
-((2-(Methylthio)quinolin-3-yl)methylene)-5-phenyl-1H-pyrrol-2(3H)-one 4e
1
Orang powder; yield: 83%; mp: 172 - 174°C, HNMR (400 MHz, DMSO-d6) δ ppm
1
0.75 (s, 1H, NH), 8.61 (s, 1H, Ar-H), 8.11 (d, J = 8.0 Hz, 1H, Ar-H), 7.93 - 7.88 (m, 3H,
Ar-H), 7.77 - 7.73 (m, 1H, Ar-H), 7.57 - 7.54 (m, 1H, Ar-H), 7.52 - 7.44 (m, 3H, Ar-H),
1
3
7
.35 (s, 1H, olefinic H), 6.93 (s, 1H, Ar-H), 2.70 (s, 3H, SCH
3
). CNMR (101 MHz,
DMSO-d6) δ ppm 170.3, 159.2, 147.6, 146.8, 135.2, 133.8, 130.8, 130.1, 129.3, 128.9,
1
28.8, 127.5, 127.1, 125.8, 125.7, 125.5, 122.2, 97.0, 12.8. GS MS: m/z calcd: 344.10,
+
found [M+H] : 345.03. Anal. Calcd for C21
H
16
N OS: (344.10): C, 73.23; H, 4.68; N,
2
8
.13. Found: C, 73.56; H, 4.52; N, 8.37.
3
4
-((6-Methyl-2-(methylthio)quinolin-3-yl)methylene)-5-phenyl-1H-pyrrol-2(3H)-one
f
1
Orang powder; yield: 83%; mp: 170 - 172°C, HNMR (400 MHz, DMSO-d6) δ ppm
1
0.72 (s, 1H, NH), 8.51 (s, 1H, Ar-H), 7.93 - 7.88 (m, 3H, Ar-H), 7.81 - 7.79 (m, 1H, Ar-
H), 7.60 - 7.57 (m, 1H, Ar-H), 7.49 - 7.47 (m, 3H, Ar-H), 7.33 (s, 1H, olefinic H), 6.92 (s,
1
3
1
1
1
3
7
H, Ar-H), 2.67 (s, 3H, SCH
3
), 2.49 (s, 3H, CH ). CNMR (101 MHz, DMSO-d6) δ ppm
3
70.3, 158.1, 147.5, 145.4, 135.1, 134.8, 133.7, 132.8, 130.1, 129.3, 128.8, 127.7, 127.4,
+
26.9, 125.8, 125.5, 122.4, 97.0 , 21.0, 12.8. GS MS: m/z calcd: 358.11, found [M+H] :
59.01. Anal. Calcd for C22
3.52; H, 5.19; N, 8.04.
H
18
N OS: (358.11): C, 73.71; H, 5.06; N, 7.82. Found: C,
2
General procedure for the synthesis of substituted pyridazin-3(2H)-ones 5a-f.
A solution of substituted furanones 3a-f (0.003 mole) and hydrazine monohydrate 98 %
(
2 mL) in absolute ethanol (10 mL) was heated at reflux for 24 h. After cooling the
-
25 -

[
Type text]
reaction mixture poured onto crushed ice and the formed precipitate was filtered off,
washed, dried and crystalized from methanol to give the target pyridazinones [37] 5a-f.
4
-((2-Hydroxyquinolin-3-yl)methyl)-6-phenylpyridazin-3(2H)-one 5a
1
White powder; yield: 81%; mp: 211 - 213°C, HNMR (400 MHz, DMSO-d6) δ ppm 12.96
(
s, 1H, NH), 11.62 (s, 1H, OH), 7.62 (s, 1H, Ar-H), 7.58 - 7.57 (m, 2H, Ar-H), 7.52 (s,
1
1
H, Ar-H), 7.40 (d, J = 8.0 Hz, 1H, Ar-H), 7.24 - 7.21 (m, 4H, Ar-H), 7.08 (d, J = 8.0 Hz,
13
H, Ar-H), 6.92 (t, J = 8 Hz, 1H, Ar-H), 3.53 (s, 2H, CH ). CNMR (101 MHz, DMSO-
2
d6) δ ppm 161.6, 160.6, 143.9, 140.8, 138.0, 137.4, 134.9, 129.7, 129.6, 129.0, 128.9,
28.5, 127.5, 125.6, 121.7, 119.2, 114.8, 30.0. GS MS: m/z calcd: 329.12, found: 329.07.
Anal. Calcd for C20 : (329.12): C, 72.94; H, 4.59; N, 12.76. Found: C, 73.07; H,
1
H
15
N
3
O
2
4
.62; N, 12.88.
4
-((2-Hydroxy-6-methylquinolin-3-yl)methyl)-6-phenylpyridazin-3(2H)-one 5b
1
White powder; yield: 76%; mp: 214 - 215°C, HNMR (400 MHz, DMSO-d6) δ ppm 13.19
(
s, 1H, NH), 11.78 (s, 1H, OH), 7.82 (s, 1H, Ar-H), 7.81 - 7.79 (m, 2H, Ar-H), 7.69 (s,
1
H, Ar-H), 7.49 - 7.42 (m, 4H, Ar-H), 7.29 (dd, J = 8.4, J = 1.6 Hz, 1H, Ar-H), 7.21 (d, J
1
3
=
8.0 Hz, 1H, Ar-H), 3.75 (s, 2H, CH
2
), 2.31 (s, 3H, CH ). CNMR (101 MHz, DMSO-
3
d6) δ ppm 161.5, 160.6, 143.9, 140.9, 137.2, 136.0, 134.9, 130.8, 130.6, 129.5, 129.0,
1
3
7
28.9, 128.4, 127.0, 125.6, 119.2, 114.7, 30.0, 20.4. GS MS: m/z calcd: 343.13, found:
43.91. Anal. Calcd for C21
3.09; H, 5.13; N, 12.45.
H
17
N
3
2
O : (343.13): C, 73.45; H, 4.99; N, 12.24. Found: C,
4
-((2-Methoxyquinolin-3-yl)methyl)-6-phenylpyridazin-3(2H)-one 5c
1
White powder; yield: 74%; mp: 201 - 203°C, HNMR (400 MHz, DMSO-d6) δ ppm 13.23
(
s, 1H, NH), 8.00 (s, 1H, Ar-H), 7.84 - 7.76 (m, 5H, Ar-H), 7.63 - 7.60 (m, 1H, Ar-H),
13
7
.46 - 7.38 (m, 4H, Ar-H), 4.02 (s, 3H, OCH
3
), 3.94 (s, 2H, CH ). CNMR (101 MHz,
2
DMSO-d6) δ ppm 160.6, 160.2, 144.9, 143.9, 140.6, 137.6, 134.9, 129.1, 129.0, 128.9,
1
28.6, 127.4, 126.3, 125.6, 125.1, 124.1, 121.9, 53.5, 29.8. GS MS: m/z calcd: 343.13,
+
found [M+H] : 344.04. Anal. Calcd for C21
H
17
N
3
2
O : (343.13): C, 73.45; H, 4.99; N,
1
2.24. Found: C, 73.21; H, 5.13; N, 12.51.
-
26 -

[
Type text]
4
-((2-Methoxy-6-methylquinolin-3-yl)methyl)-6-phenylpyridazin-3(2H)-one 5d
1
White powder; yield: 70%; mp: 200 - 202°C, HNMR (400 MHz, DMSO-d6) δ ppm 13.23
(
s, 1H, NH), 7.91 (s, 1H, Ar-H), 7.80 - 7.77 (m, 3H, Ar-H), 7.67 (d, J = 8.0 Hz, 1H, Ar-
H), 7.60 (s, 1H, Ar-H), 7.49 - 7.42 (m, 4H, Ar-H), 4.00 (s, 3H, OCH ), 3.93 (s, 2H, CH ),
). CNMR (101 MHz, DMSO-d6) δ ppm 160.6, 159.7, 143.9, 143.2,
3
2
1
3
2
1
5
.42 (s, 3H, CH
3
40.7, 137.2, 134.9, 133.2, 131.0, 129.1, 128.9, 128.5, 126.4, 126.1, 125.6, 125.0, 121.7,
+
3.4, 29.7, 20.8. GS MS: m/z calcd: 357.15, found[M+H] : 358.07. Anal. Calcd for
C
22
H
19
N
3
2
O : (357.15): C, 73.93; H, 5.36; N, 11.76. Found: C, 74.29; H, 5.49; N, 11.50.
4
-((2-(Methylthio)quinolin-3-yl)methyl)-6-phenylpyridazin-3(2H)-one 5e
1
White powder; yield: 80%; mp: 187 - 189°C, HNMR (400 MHz, DMSO-d6) δ ppm 13.29
(
s, 1H, NH), 7.97 (s, 1H, Ar-H), 7.89 (t, J = 8.0 Hz, 2H, Ar-H), 7.80 - 7.77 (m, 3H, Ar-
H), 7.70 - 7.66 (m, 1H, Ar-H), 7.50 - 7.39 (m, 4H, Ar-H), 4.01 (s, 2H, CH ), 2.66 (s, 3H,
). CNMR (101 MHz, DMSO-d6) δ ppm 160.5, 159.0, 146.3, 143.8, 139.9, 134.9,
2
13
SCH
3
1
34.8, 129.4, 129.1, 129.0, 128.9, 127.7, 127.1, 125.6, 125.5, 125.4, 31.4, 12.6. GS MS:
+
m/z calcd: 359.11, found [M+H] : 360.01. Anal. Calcd for C21
H
17
3
N OS: (359.11): C,
7
0.17; H, 4.77; N, 11.69. Found: C, 70.41; H, 4.89; N, 11.95.
4
-((6-Methyl-2-(methylthio)quinolin-3-yl)methyl)-6-phenylpyridazin-3(2H)-one 5f
1
White powder; yield: 73%; mp: 192 - 194°C, HNMR (400 MHz, DMSO-d6) δ ppm 13.00
(
s, 1H, NH), 7.93 (s, 1H, Ar-H), 7.82 - 7.79 (m, 3H, Ar-H), 7.62 - 7.58 (m, 2H, Ar-H),
7
.44 - 7.38 (m, 4H, Ar-H), 3.99 (s, 2H, CH
2
), 2.63 (s, 3H, SCH
3
), 2.43 (s, 3H,
1
3
CH
3
). CNMR (101 MHz, DMSO-d6) δ ppm 160.5, 157.8, 144.9, 143.8, 140.0, 134.8,
1
1
34.7, 134.4, 131.4, 129.1, 129.0, 128.7, 128.5, 126.9, 126.5, 125.6, 125.5, 31.4, 21.0,
+
2.6. GS MS: m/z calcd: 373.12, found [M+H] : 374.05. Anal. Calcd for C22
H
19
3
N OS:
(
373.12): C, 70.75; H, 5.13; N, 11.25. Found: C, 71.03; H, 5.36; N, 11.49.
-
27 -

[
Type text]
Biology
The NCI-60 anticancer drug screen
The methodology of the NCI for primary anticancer assay was performed at sixty human
tumor cell lines panel derived from nine neoplastic diseases, in accordance with the
(....)
protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda . See
Appendix A
MTT assay
MTT assay was performed to investigate the effect of the synthesized compounds on
(….)
mammary epithelial cells (MCF-10A)
. See Appendix A
Antiproliferative Assay
Propidium iodide fluorescence assay was carried out on Panc-1(pancreas cancer), PaCa-2
pancreatic carcinoma), MCF-7 (breast cancer): A-549 (epithelial): HT-29 (colon cancer),
H-460 (lung cancer) and PC-3 (prostate cancer) cell lines to investigate the
(
(
….)
antiproliferative activity of compounds . See Appendix A
Tubulin polymerization assay
The activity of compounds 4c, 4e and 5d on tubulin polymerization was investigated by
(
----)
Tubulin Polymerization Assay Kit (Cytoskeleton Inc., Denver, CO, USA)
. See
Appendix A
EGFR inhibitory assay
The activity of compounds 4c, 4e and 5d against EGFR was investigated according to
(…..)
literature
. See appendix A
BRAF kinase assay
Compounds 4c, 4e and 5d in this study was subjected to V600E mutant BRAF kinase
(
…)
assay in triplicate .See appendix A
-
28 -

[
Type text]
Cell cycle analysis and apoptosis assay
Cell cycle analysis and apoptosis detection
Cell cycle analysis was performed for compounds 4e and 5d on Paca-2 and HT-29,
(…..)
respectively.
. See appendix A
Molecular docking:
The 3.5 Å 3D structure of tubulin in complex with DAMA-colchicine and with the
stathmin-like domain (SLD) of RB3 (PDB: 1SA0) was downloaded from protein data
bank[43]. All molecular modeling calculations and docking studies were carried out using
Discovery Studio software 2016 client v16.1.0.15350 (San Diego, CA) with CDOCKER
program. Removing of chains A, B and E of the protein together with co-crystallized
water molecules was performed. Automatic protein preparation module was used
applying CHARMm force field. The binding site sphere has been defined automatically
by the software. The docked compounds were built using Chem. 3D ultra 12.0 software
[
Chemical Structure Drawing Standard; Cambridge Soft corporation, USA (2010)], and
copied to Discovery Studio 2016 client v16.1.0.15350. Ligands were prepared using
Prepare Ligands” protocol in Discovery Studio where hydrogen atoms were added at
“
their standard geometry, optical isomers and 3D conformations were automatically
generated. Docking was performed using CDOCKER protocol in Discovery Studio
keeping the parameters at default. Each compound would retain 10 poses and the best
scoring pose of the docked compounds was recognized. Receptor–ligand interactions of
the complexes were examined in 2D and 3D styles.
-
29 -