Design, synthesis, and biological evaluation of new series of pyrrol-2(3H)-one and pyridazin-3(2H)-one derivatives as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104522

A potential microtubule destabilizing series of new thirty-five Pyrrol-2-one, Pyridazin-3(2H)-one and Pyridazin-3(2H)-one/oxime derivatives has been synthesized and tested for their antiproliferative activity against a panel of 60 human cancer cell lines. Compounds IVc, IVg and IVf showed a broad spectrum of growth inhibitory activity against cancer cell lines representing renal, cancer of lung, colon, central nervous system, ovary, and kidney. Among them, compound IVg was found to have broad spectrum anti-tumor activity against the tested nine tumor subpanels with selectivity ratios ranging between 0.21 and 3.77 at the GI₅₀ level. In vitro assaying revealed tubulin polymerization inhibition by all active compounds IVc, IVg and IVf. The results of the docking study revealed nice fitting of compounds IVc, IVf, and IVg into CA-4 binding site in tubulin. The three compounds exhibited high binding affinities (ΔG_b = ?12.49 to ?12.99 kcal/mol) toward tubulin compared to CA-4 (?8.87 kcal/mol). Investigation of the binding modes of the three compounds IVc, IVf, and IVg revealed that they interacted mainly hydrophobically with tubulin and similar binding orientations to that of CA-4. These observations suggest that tubulin is a possible target for these compounds.

Full text of DOI:10.1016/j.bioorg.2020.104522

Journal Pre-proofs
Design, Synthesis, and biological evaluation of new series of pyrrol-2(3H)-
one and pyridazin-3(2H)-one derivatives as tubulin polymerization inhibitors
Mahmoud S. Abdelbaset, Mostafa H. Abdelrahman, Syed Nasir Abbas
Bukhari, Ahmed M. Gouda, Bahaa G. M. Youssif, Mohamed Abdel-Aziz,
Gamal El-Din A. Abuo-Rahma
PII:
S0045-2068(20)31820-4
DOI:
https://doi.org/10.1016/j.bioorg.2020.104522
YBIOO 104522
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
2 September 2020
21 October 2020
27 November 2020
Please cite this article as: M.S. Abdelbaset, M.H. Abdelrahman, S. Nasir Abbas Bukhari, A.M. Gouda, B. G. M.
Youssif, M. Abdel-Aziz, G. El-Din A. Abuo-Rahma, Design, Synthesis, and biological evaluation of new series
of pyrrol-2(3H)-one and pyridazin-3(2H)-one derivatives as tubulin polymerization inhibitors, Bioorganic
Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104522
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Design, Synthesis, and biological evaluation of new series of pyrrol-2(3H)-one
and pyridazin-3(2H)-one derivatives as tubulin polymerization inhibitors
a
a
b
Mahmoud S. Abdelbaset , Mostafa H. Abdelrahman , Syed Nasir Abbas Bukhari , Ahmed
c
d
e
e,f
M. Gouda , Bahaa G. M. Youssif , Mohamed Abdel-Aziz , Gamal El-Din A. Abuo-Rahma
aDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar University,
b
Assiut 71524, Egypt; Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf
c
University, Aljouf, Sakaka 2014, Saudi Arabia; Department of Medicinal Chemistry, Faculty of
d
Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt; Department of Pharmaceutical
e
Organic Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt; Department
of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519-Minia, Egypt;
f
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Deraya University, New
Minia, Minia, Egypt .
Short running title: Synthesis and biological evaluation of new pyrrole and pyridazine
derivatives
*
To whom correspondence should be addressed:
Gamal El-Din A. Abuo-Rahma, Ph.D. Department of Medicinal Chemistry, Faculty of
Pharmacy, Minia University, 61519-Minia, Egypt.
Tel.:(002)-01003069431
E-mail address: gamal.aborahma@mu.du.eg
Bahaa G. M. Youssif, Ph.D. Pharmaceutical Organic Chemistry Department, Faculty of
Pharmacy, Assiut University, Assiut 71526, Egypt.
Tel.: (002)-01098294419
E-mail address: bahaa.youssif@pharm.aun.edu.eg, bgyoussif@ju.edu.sa
1

Abstract
A potential microtubule destabilizing series of new thirty-five Pyrrol-2-one, Pyridazin-3(2H)-one
and Pyridazin-3(2H)-one/oxime derivatives has been synthesized and tested for their
antiproliferative activity against a panel of 60 human cancer cell lines. Compounds IVc, IVg and
IVf showed a broad spectrum of growth inhibitory activity against cancer cell lines representing
renal, cancer of lung, colon, central nervous system, ovary, and kidney. Among them, compound
IVg was found to have broad spectrum anti-tumor activity against the tested nine tumor
subpanels with selectivity ratios ranging between 0.21 and 3.77 at the GI50 level. In vitro
assaying revealed tubulin polymerization inhibition by all active compounds IVc, IVg and IVf.
The results of the docking study revealed nice fitting of compounds IVc, IVf, and IVg into CA-4
binding site in tubulin. The three compounds exhibited high binding affinities (G = -12.49 to -
b
1
2.99 kcal/mol) toward tubulin compared to CA-4 (-8.87 kcal/mol). Investigation of the binding
modes of the three compounds IVc, IVf, and IVg revealed that they interacted mainly
hydrophobically with tubulin and similar binding orientations to that of CA-4. These
observations suggest that tubulin is a possible target for these compounds.
Key words: Pyrrol-2-one, Pyridazin-3(2H)-one, Anti-proliferative, Tubulin and CA-4.
2

Graphical Abstract
3

Highlights





A novel series of Pyrrol-2-one, Pyridazin-3(2H)-one derivatives has been synthesized
and tested by NCI for their antiproliferative activity.
The IVc and IVg compounds showed excellent activity and were further selected for a
five-dose assay.
In vitro antiproliferative activity of compounds IVc, IVf, and IVg was evaluated using
MTT assay.
Compounds IVc, IVf, and IVg have been evaluated against EGFR, BRAFV600E and
Tubulin anticancer targets.
A docking study of compounds IVc, IVf and IVg showed a strong fitting to the CA-4
binding sites in tubulin.
4

1
. INTRODUCTION
The main cytoskeletal filaments are microtubules composed by both α and Ꞵ -tubulin proteins
1]. Microtubules play a significant role in many aspects of cellular processes, including cell
[
division, shape maintenance, cell motility and vesicular transport [2-5]. The formation of
microtubules has been shown to be a complex mechanism for the polymerization and the
depolymerization of α/Ꞵ heterodimers [6]. The disruption of this complex equilibrium impedes
mitosizing cell division, leading to metaphase cell cycle arrest and cell mortality [7]. Tubulin
therefore became a promising target for the tumor therapy [8]. To date, several natural and
synthetic tubulin-targeted compounds have been developed to treat malignancies. Most of these
compounds have been classified into two major categories, one being the microtubule
destabilizing agent that bind to vinblastine and the colchicine site, the other one is microtubule-
stabilizing agents which bind to paclitaxel binding site and disturb tubulin disassembly [9-12].
Owing to the high toxicity to non-cancer cells, however, the FDA has not approved candidates
that are bind to a colchicine site [13].
Pyridazinone and pyrrolone constitute interesting heterocyclic compounds with variable
biological activities such as antibacterial [14,15], anti-inflammatory [16,17], vasodilators [18],
antihypertensive [19] and anticancer activities [20-22]. Furthermore, some literature reports
referred to the anticancer mechanism of pyridazinones and pyrrolones and provided an evidence
that anticancer mechanism of pyridazinones through tubulin polymerization inhibition [23]. Both
pyridazinones and pyrrolones could be synthesized by nucleophilic substitution from the
corresponding furanones [24]. Pyridazinones and pyrrolones are perfectly clinically appropriate
moieties. The most important examples are, Althiomycin 1 [25] which is a naturally occurring
5

alkaloid separated from Streptomyces Althioticus and used as antibiotic through inhibition of
protein synthesis. In addition, Pimobendan (2) [26] is a phosphodiesterase III (PDEIII) inhibitor
and used in management of congestive heart failure. Moreover, Emorfozan (3) [27] was
marketed in Japan as an analgesic and anti-inflammatory drug, Fig. 1.
N
O
N
OH
HO
S
HN
H
N
N
N
CH3
S
O
OCH3
N
N CH3
O
N
N
O
O
O
N
N
O
H
H C
3
H CO
3
Althiomycin (1)
Pimobendan (2)
Emorfozan (3)
Fig. 1. Structure of pyridazinone and pyrrolone -based clinically used drugs
Accordingly, based on the above data on the biological relevance of both pyridazinone and
pyrrolone [21, 22], we report herein the design and synthesis of novel two series of pyridazinone
and pyrrolone (Scaffolds A and B, Fig. 2) as promising anticancer candidates. The target
compounds were tested for antiproliferative action. In addition, a mechanistic study of
BRAFV6000, EGFR Tk kinases and tubulin polymerization inhibitor activity were evaluated. In
addition, cell cycle analysis and apoptosis assay have been established for the most active
compound.
6

1
.1.
Previously, we have reported compound 4e (Fig. 2) as CA-4 analog with potent cytotoxic activity
IC = 1.9-4.9 μM) among a series of pyrrol-2(3H)-one derivatives [22]. Mechanistic study of
Rational design of the new compounds
(
50
compound 4e revealed potent tubulin polymerization inhibitory activity. In attempt to optimize the
anticancer potential of 4e in the current study, we have performed a series of structural
modifications aiming to improve the binding affinity to tubulin protein. The structural
modifications in compound 4e included combining both alkylation of the pyrrole NH group with
aralkyl(benzyl/phenethyl) and replacement of the 2-(methylthio) quinoline by aryl ring. The result
of these improvements is the ability to build extra-binding hydrophobic pockets that can contribute
to improving binding affinity for tubulin protein, as assessed in a preliminary docking study. The
results of this study revealed improvement in binding affinities of the N-phenethyl derivatives (Gb
=
12.60-12.99 kcal/mol) compared to 11.82 kcal/mol for compound 4e.
Substitution with (R)
O
OH
R
a) OCH /(CH ) N (+)
3
3 2
N
A
b) 4-F/Br (-)
A
S
A
O
O
i) simplification
ii) N-alkylation
O
B
B
N
H
B
N
O
O
CA-4
2 2 4
X = CH /C H
X
4e
Scaffold A
Substitution with (R)
R
Additional binding interactions
increased binding affinity)
a) OCH /(CH ) N (+)
3
3 2
(
A
b) 4-F/Br (-)
i) Ring expansion
ii) N-alkylation
B
O
N N
Y
Y = CH2, CH2CO, CH2CNOH
Scaffold B
Additional binding interactions
increase binding affinity)
(
Fig. 2. Rational design and structural modifications of scaffolds A and B
7

The study of SAR of scaffold A also included substitution on ring A in scaffold A was done
using the electron-donating mono/di/tri-methoxy groups to mimic ring A in CA-4. In addition,
electron-withdrawing groups (4-F/Br) were also used to evaluate the impact of electronic effect
of different substituents on activity of the new compounds. Furthermore, scaffold B was
designed by expansion of the pyrrole ring into pyridazin-3(2H)-one ring with N-alkylation.
Scaffold B was also functionalized with hydrogen bond donner/acceptor groups to investigate
the impact of these groups on activity and binding affinities to tubulin protein.
2. RESULTS AND DISCUSSION
2
.1. Chemistry
The synthesis of N-benzyl and N-phenethyl-1H-pyrrol-2(3H)-ones IIIb-g and IVa-g was shown
in Scheme 1. The reflux of benzene and succinic anhydride in hood under the Friedl Craft
acylation produced 3-Benzoyl-propionic acid I with good yield [28]. Perkin condensation of I in
the presence of triethylamine (TEA) and drops of acetic anhydride with various aromatic
aldehydes gave corresponding furanones IIa-g [29].
8

Ar
O
O
O
O
O
a
b
c
O
+
OH
O
I
IIa-g
IIa, IVa, Ar = 4-OCH -C H
6 4
c
3
IIb, IIIb, IVb, Ar = 3-OCH -C H
6 4
3
IIc, IIIc, IVc, Ar = 3,4-di-OCH -C H
Ar
N
3
6
3
Ar
IId, IIId, IVd, Ar = 3,4,5-tri-OCH -C H
3
6
2
IIe, IIIe, IVe, Ar = 4-F-C H4
6
O
O
IIf, IIIf, IVf, Ar = 4-(CH ) N-C H
3
2
6
4
N
IIg, IIIg, IVg, Ar = 4-Br-C H4
6
IVb-g
IIIb-g
Scheme 1. Synthesis of N-benzyl and N-phenethyl-1H-pyrrol-2(3H)-ones IIIb-g and IVa-g
Reagent and reaction conditions: a) Anhydrous AlCl , reflux, 30 mins; b) Ar-CHO,
3
(
CH CO) O, TEA, fusion, 15 mins; c) Benzylamine or phenethylamine, EtOH, reflux, 3h.
3 2
The corresponding pyrrol-2-(3H)-ones IIIb-g and IVa-g were given respectively by heating
furanones IIa-g with benzylamine or phenethylamine in ethanol. The structure of IIIb-g and
1
IVa-g was confirmed by IR, NMR and HRESI-MS spectroscopy. H NMR spectra of
compounds IIIb-g showed a common peak appeared as doublet of doublet at δ 4.08-4.14 to 4.33-
4
.36 ppm with coupling constant (J = 15.3 Hz) associated with the geminal coupling of the
diastereotopic benzylic protons, which may be due to the effect of configuration around the
1
3
exocyclic double bond in C-3. The C NMR spectra of the pyrrolone derivatives IIIb-g showed
the characteristic benzylic carbons at δ 43.4 and 44.4 ppm and the (C=O) at 168.0 ppm. The
1
olefinic and aromatic carbons appeared at their expected chemical shifts. Similarly, the H NMR
9

spectra of compounds IVa-g showed four common signals appeared as doublet of doublet of
doublet at δ 3.49-2.55 ppm related to (CH CH ) of phenethyl group with coupling constant 13.4,
2
2
1
3
1
1.5, 5.2 Hz. The C NMR spectra of IVa-g showed the characteristic two signals at δ 44.1 ppm
related to (N-CH ) and 34.6 ppm related to the benzylic protons. Another significant signal
2
appeared at 168.0 ppm related to the (C=O) group. The olefinic and aromatic carbons appeared
at their expected chemical shifts. The purity of the prepared compounds was confirmed by
HRESI-MS spectroscopy and the results are in consistent with the molecular formula of the
products.
The synthesis of target derivatives VIa-f, VIIa-f and VIIIa-f was illustrated in Scheme 2. The
4
,6-disubstituted pyridazin-3(2H)-ones Va-f were prepared by heating furanone derivatives IIa-f
with hydrazine hydrate in absolute ethanol [30]. The IR spectra of pyridazinones Va-f showed
-1
medium stretching band at 3500-3269 cm related to (CONH) and strong stretching band at
-1
1
676-1652 cm related to (C=O), which are consistent with the proposed structure.
1
0

Ar
O
Ar
a
Ar
O
b
N
N
N
O
N
H
O
IIa-f
Va-f
VIa-f
c
Ar
Ar
Va -VIIIa, Ar = 4-OCH -C H
6 4
3
N
N
N
Vb -VIIIb, Ar = 3-OCH -C H
3
6
4
O
N
O
HO
Vc -VIIIc, Ar = 3,4-di-OCH -C H
3
6
3
O
N
d
Vd -VIIId, Ar = 3,4,5-tri-OCH -C H
6 2
3
Ve -VIIIe, Ar = 4-F-C H4
6
Vf -VIIIf, Ar = 4-(CH ) N-C H
6 4
3
2
VIIIa-f
VIIa-f
Scheme 2. Synthesis of pyridazinones and oximes
Reagent and reaction conditions: a) NH NH .H O, EtOH, reflux,12h; b) Benzyl bromide,
2
2
2
NaOEt, r.t, 12h.; c) Phenacyl bromide, NaOEt, r.t, 12 h.; d) NH OH, anhydrous sodium sulphate,
2
EtOH, reflux, 6 h.
1
In the H NMR of compounds Va-f, two common singlet signals one at 13.10-13.18 ppm related
to pyridazinone ring (NHCO) and the other at 3.73-3.86 ppm associated with the benzylic
1
3
protons. The C NMR spectra of Va-f showed the characteristic benzylic carbon at 35.1 ppm
and the (C=O) at 168 ppm. The olefenic and aromatic carbons appeared at their expected
chemical shifts. Stirring of pyridazin-3(2H)-ones Va-f with equimolar amount of benzyl or
phenethyl bromides at room temperature in sodium ethoxide, afforded the corresponding N-
benzylated pyridazin-3-ones VIa-f and N-phenethyl pyridazine-3-ones VIIa-f, respectively. It is
1
1

worth to note that carrying out the alkylation reaction in acetone/K CO for 24 h at room
2
3
temperature gave no product; the same results were obtained upon heating in DMF using K CO
2
3
as a base. Also, no product was formed upon using TEA as a base and acetonitrile as a solvent on
cold or reflux conditions. On the other hand, the use of NaOH and DMF gave a very low yield,
difficult to be purified. So, the presence of strong base like sodium ethoxide is important for this
type of alkylation reaction [28]. The IR spectra of compounds VIa-f showed strong stretching
-1
-1
bands at 1655-1648 cm related to (C=O) and 1632-1601 cm related to (C=C), which are in
1
consistent with the proposed structures. In the H NMR spectra of compounds VIa-f two
common singlet signals one at 3.77-3.89 ppm related to (CH benzylic) attached to the 4 position
2
on the pyridazinone ring and the second at 5.33-5.36 ppm related to (N-benzylic CH ). The IR
2
-1
spectra of compounds VIIa-f showed strong stretching bands at 1706-1696 cm related to
-1
(
CH C=O) and 1659-1632 cm related to (N C=O), which are consistent with the proposed
2
1
structures. In the H NMR spectra of compounds VIIa-f two singlet signals were common; one
at 5.74-5.78 ppm related to (NCH CO) on the 2 position of pyridazinone ring and the second at
2
3
.75-3.91 ppm related to (4-benzylic CH ) protons. Finally, heating N-alkylated pyridazinones
2
VIIa-f with hydroxylamine hydrochloride in absolute ethanol in presence of anhydrous sodium
sulfate as a dehydrating agent afforded the corresponding oximes VIIIa-f. The IR spectra of
-1
oximes VIIIa-f showed significant stretching bands at 3327-3219 cm related to (OH), 1647-
-1
-1
1
636 cm related to (NC=O) and absence of stretching band at 1706-1689 cm related to
(
CH C=O) characteristic of respective ketones VIIa-f which are in consistent with the proposed
2
1
structures. In the H NMR spectra of VIIIa-f a common singlet signals were observed at 11.64-
1
5
1.67 ppm related to (=N-OH), at 3.73-3.85 ppm associated with benzylic protons and at 5.46-
1
.50 of (NCH CO). Moreover, the H NMR results showed that oxime exist as two
2
1
2

diastereomers major in 82-88 % and minor in 12-18 %. These results are in agreement with a
literature results that refer to the presence of oxime in the two diastereomeric forms syn and anti
as illustrated [29]. The purity of the prepared compounds was confirmed by HRESI-MS
spectroscopy and the results are in consistent with the molecular formula of the products.
2
.2. Evaluation of biological Activities
2
.2.1. Screening of cytotoxic activity
For screening of the target compounds to detect their potential antiproliferative activity against a
panel of 60 cancer cell lines; compounds IIIb-d, IVa-g, VIb-f, VIIa-e and VIIIa-f were selected
for screening according to NCI-guidelines at concentration of 10 µM. The results for each
compound (Table 1) were reported as the percent of growth inhibition of treated cells compared
to untreated control cells. Generally, for Scaffold A, compounds IVa-g possessing N-phenethyl-
1
H-pyrrol-2(3H)-one were more active than IIIb-d possessing N-benzyl-1H-pyrrol-2(3H)-one
which directly reflects the influences of N-phenethyl moiety on the antiproliferative activity of
the title scaffold. Compounds IVc and IVg possessing 3,4-di-OCH and 4-Br substituent showed
3
a complete cell death against non-small cell lung cancer cell line HOP-62, CNS cancer cell line
SNB-75 and ovarian cancer cell line OVACAR-4 with growth inhibition percentage of -29.96, -
7
.78 and -5.49 for compound IVc, (Table 1) and -56.85, -34.27 and -17.07 for compound IVg,
respectively. Also, compound IVc exhibited a complete cell death against leukemia cancer cell
line SR, non-small cell lung cancer cell line HOP-92, ovarian cancer cell line SK-OV-3 and renal
cancer cell line A498, ACHN, RXF 393, TK-10 and UO-31 with growth inhibition percentage of
-
1.98, -4.05, -30.33, -75.37, -32.49, -3.29, -3.91 and -47.49, respectively (Table 1). Furthermore,
compound IVg, showed a complete cell death against non-small cell lung cancer cell line HOP-
2 and NCI-H226, CNS cancer cell line SF-295 and renal cancer cell line 786-0 with growth
9
1
3

percentage of -13.88, -1.06, -25.59 and -8.73, respectively (Table 1). In addition, the two
compounds recorded significant activity against non-small cell lung cancer cell line NCI-H460,
ovarian cancer cell line OVCAR-8 and breast cancer cell line HS 578T with growth inhibition
percentage 20.50, 25.86 and 13.10 for compound IVc and 25.57, 17.68 and 22.81 for compound
IVg, respectively. Moreover, compound IVf [(Ar = 4-(CH ) N-C H ] showed a complete cell
3
2
6
4
death against non-small cell lung cancer cell line HOP-62, CNS cancer cell line SNB-75, and
ovarian cancer cell line OVCAR-4 with growth inhibition percentage -13.97, -29.13 and -11.62.
On the other hand, compounds VIb-f, VIIa-e, and VIIIa-f (scaffold B) showed moderate
activity against the cancer cell lines studied. Compounds VIc and VId possessing N-benzylated
pyridazin-3-one moiety showed good activities against most of cancer cell lines with growth
inhibition percentage between 6 to 26, Table 1. Compound VIc exhibited a complete cell death
against leukemia cancer cell line SK-MEL-5 and renal cancer cell line A498 with growth
inhibition rate of -3.54 and -16.92, see supplementary data.
1
4

Table 1. One dose assay of nine different cancer cell types of compounds IVc, IVg, VIc, VId,
VIIIe and VIIIf.
Subpanel cancer
cell Lines
Growth% inhibition
IVc
IVg
VIc
Vid
VIIIe
VIIIf
Leukemia
CCRF-CEM
HL-60(TB)
K-562
57.58
19.64
47.56
-1.98
ND
100.75
83.78
75.54
58.56
ND
43.01
25.79
22.19
40.16
9.10
34.84
19.26
20.61
36.03
5.01
34.61
24.81
29.16
30.27
16.10
46.58
54.26
29.51
46.26
35.11
SR
MOLT4
RPMI-8226
70.57
101.47
15.20
24.84 25.63
28.24
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
32.86
56.08
8.19
20.00
14.44
19.88
14.65
24.22
29.13
30.24
49.54
40.11
HOP-62
-29.96
-4.05
-56.85
-13.88
-1.06
90.91
28.04
51.28
66.12
51.08
46.20
62.69
42.59
57.08
75.05
55.86
60.47
HOP-92
NCI-H226
Colon Cancer
COLO 205
HCT-116
HT-29
29.71
Nd
30.35
22.73
32.5
113.74 102.03
47.91
58.45
45.96
32.93
32.78
38.65
96.80
10.55
65.92 35.09
81.50
2
0.25
CNS Cancer
SF-295
26.78
-7.78
15.88
-25.59
-34.27
6.97
28.78
58.12
65.14
31.45
94.45
87.54
52.64
72.92
44.31
65.78
57.77
34.54
SNB-75
U251
Melanoma
LOX IMVI
MALME-3M
2.28
42.19
50.52
48.44
23.43
43.65
35.16
53.09
35.81
53.03
66.17
39.30
SK-MEL-5
38.47
54.48
70.06
-3.54
40.17
37.69
36.64
33.59
55.83
39.26
UACC-62
46.59 30.62
Ovarian Cancer
1
5

OVCAR-3
27.98
-5.49
42.10
-17.07
17.68
27.79
15.36
47.52
32.5
46.94
35.29
31.95
30.50
82.27
94.88
85.47
88.75
82.78
104.97
56.16
55.05
57.21
44.12
94.61
OVCAR-4
OVCAR-8
25.86
31.63
-30.33
32.32
30.16
74.06
NCI/ADR-RES
SK-OV-3
Renal Cancer
7
86-0
8.93
-75.37
-32.49
-3.29
-8.73
50.83
13.00
31.53
12.62
45.00
35.84
-16.92
27.12
42.38
49.81
14.71
49.93
51.14
27.90
31.68
45.21
21.57
69.22
9.60
75.37
-3.20
52.75
61.40
84.52
36.13
A498
ACHN
40.26
28.51
63.49
30.93
RXF 393
TK-10
-3.91
UO-31
-47.49
Breast Cancer
MCF7
2
9.63
24.79
ND
19.94
43.65
24.52
50.06
26.22
29.08
MDA-MB-231/ATC
ND
43.08 53.95
HS 578T
T-47D
13.10
8.00
22.81
50.52
66.62
7.51
74.90
6.19
73.84 83.09
11.83
11.78
1
6

2
.2.2 In vitro five dose full NCI 60 cell panel assay
Compound IVc and IVg were selected for advanced five dose testing, where the cytotoxic and/or
growth inhibitory effects of the tested compound was tested invitro against the full panel of 60
human tumor 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 parameter were calculated
for each cell line, GI50 (Growth inhibitory activity) corresponds to concentration of the
compound causing 50% inhibition of net cell growth, TGI value ( cytostatic activity) corresponds
to the concentration of the compound resulting in total growth inhibition and LC50 value
(cytotoxic activity) is the concentration of the compound causing net 50% loss of initial cells at
the end of incubation period of 48 h. Additionally, (MG_MID) is calculated for each mentioned
parameter, which displays an averaged activity parameter over all cell lines, as well as the delta
parameter and the range were also calculated. The log GI , log TGI, log LC were then
1
0
50
10
10
50
determined, defined as the means of the log10, s of the individual GI , TGI, LC value. Negative
50
50
values indicated the most sensitive cell lines. Compound having log GI values -4 and < -4 is
1
0
50
declared to be active. The results of the NCI-60 5-dose assay are shown in Tables 2 and 3, Fig. 3
and 4. IVc and IVg displayed strong antiproliferative properties. The best candidate for growth
inhibition properties was compound IVg, with GI50 values < 1 µM against 13 cell lines,
particularly for non-small cell lung cancer HOP 62 (GI = 0.35 µM) and HOP 92 (GI = 0.32
5
0
50
µM) cells, CNS SF-295 cells (GI = 0.33 µM), renal cancer 786-0, A498, and ACHN cells (GI
50
5
0
=
0.65 µM) and breast cancer HS578T cells (GI = 0.83 µM). Compound IVg showed good
50
antiproliferative activity against all melanoma cancer, ovarian cancer, and prostate cell lines with
GI ranging from 0.82 to 8.38 µM.
5
0
1
7

Compound IVc showed considerable antiproliferative activity against all CNS cancer, ovarian
cancer, and ovarian cell lines with GI ranging from 1.89 to 9.03 µM. but also on some cell lines
5
0
from non-small cell lung cancer, colon cancer, and breast cancer, respectively.
Evaluation of selectivity of a compound depends upon the ratio obtained by dividing the full
a
panel MID (the average sensitivity of all cell lines toward the test agent µM) by their individual
b
subpanel MID (µM). Ratios between 3 and 6 refer to moderate selectivity; ratios > 6 indicate
high selectivity toward the corresponding cell line, while compounds not meeting either of these
criteria rated non-selective. Compound IVc was found to have broad-spectrum antitumor activity
against the nine tumor subpanels tested with selectivity ratios ranging from 0.43 to 2.26 at the
GI level, Table 2, and Fig. 3. While compound IVg was found to have broad-spectrum
5
0
antitumor activity against the nine tumor subpanels examined, with selectivity ratios ranging
from 0.21 to 3.77 at GI level. IVg exhibited moderate selectivity toward the CNS cancer and
5
0
renal cancer subpanels with selectivity ratio of 3.77 and 3.13 respectively, at GI level. See
5
0
Table 3, Fig. 4, and supplementary data.
1
8

Table 2. Selectivity of compound IVc on sixty human cancer types
GI (µM)
5
0
Panel
Cell line
TGI
LC50
b
Conc.per cell line Subpanel MID Selectivity ratio
CCRF-CEM
HL-60(TB)
35.90
11.70
23.90
˃100
68.6
˃100
˃100
˃100
˃100
2
0.2
0.43
K-562
Leukemia
RPMI-8226
SR
18.4
11.10
5.06
10.40
2.72
2.54
4.60
17.00
8.77
7.34
84.9
57.7
17.7
54.0
10.0
8.17
30.2
˃100
33.2
30.7
36.0
˃100
64.1
11.6
˃100
48.9
˃100
˃100
26.0
24.0
23.5
21.0
6.28
9.31
19.0
61.9
˃100
˃100
˃100
68.1
94.5
86.8
40.9
52.1
17.8
87.0
73.1
22.5
68.5
˃100
˃100
54.7
˃100
˃100
57.5
˃100
˃100
˃100
˃100
˃100
˃100
˃100
34.0
˃100
˃100
˃100
˃100
76.5
70.4
63.4
71.4
45.7
32.3
45.1
˃100
˃100
˃100
˃100
˃100
˃100
˃100
˃100
˃100
48.5
A549/ATCC
EKVX
HOP-62
HOP-92
7.58
1.14
Non-small cell
lung cancer
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
9.80
17.30
12.90
3.09
11.10
8.92
Colon cancer
12.73
5.16
0.68
1.67
0.86
HT29
KM12
22.20
13.6
SW-620
SF-268
8
5
6
5
1
3
5
.18
.95
.68
.08
.89
.20
.94
SF-295
SF-539
CNS cancer
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
1
0.20
8
4
.84
.78
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
Melanoma
18.5
10.06
6
9
.76
.29
2
0.20
6
4
4
.01
.93
.82
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
Ovarian
Cancer
3.15
˃100
˃100
72.9
5
.49
1.57
6
6
9
.37
.09
.03
˃100
1
9

SK-OV-3
86-0
4
2
2
3
7
3
4
2
4
.04
.14
.25
.53
.53
.64
.88
.28
.35
18.8
5.85
5.32
14.0
˃100
15.6
21.8
4.91
20.6
˃100
˃100
57.2
17.9
94.6
20.8
42.0
48.3
˃100
42.0
85.4
16.0
63.1
˃100
˃100
˃100
65.6
7
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
3.83
2.26
Renal Cancer
Prostate
Cancer
21.1
3.2
4.18
17.15
0.50
1.47
DU-145
1
MCF7
MDA-MB-
4
.16
2
31/ATCC
HS 578T
3
.66
23.4
29.9
48.1
42.4
˃100
˃100
˃100
˃100
Breast Cancer
5
.86
BT-549
1
2.40
T-47D
3
7
.20
.57
MDA-MB-468
MIDa
8.64
2
0

Fig. 3. Concentration-antiproliferative response curves of compound IVc against sixty different
cancer cell lines
2
1

Table 3. Selectivity of compound IVg on sixty human cancer types.
GI (µM)
5
0
Panel
Cell line
TGI
LC50
b
Conc.per cell line Subpanel MID Selectivity ratio
CCRF-CEM
7.76
75.10
˃100
˃100
20.5
˃100
˃100
˃100
HL-60(TB)
K-562
2
.63
20.82
0.21
Leukemia
MOLT-4
RPMI-8226
SR
18.40
˃100
˃100
9.44
11.60
1.37
1.95
0.35
0.32
1.11
1.69
1.20
0.91
6.54
3.26
7.52
0.39
11.70
4.73
43.60
5.34
86.4
˃100
11.9
19.0
1.22
2.63
4.57
14.6
18.8
10.2
87.1
27.6
˃100
11.7
˃100
˃100
˃100
˃100
16.3
1.59
5.57
5.03
3.83
10.1
10.8
6.64
71.8
˃100
27.2
17.0
20.8
18.7
12.0
7.34
3.13
40.2
10.0
8.02
˃100
˃100
57.4
95.6
4.51
18.3
56.7
67.5
˃100
46.1
˃100
˃100
˃100
57.6
˃100
˃100
˃100
˃100
˃100
6.23
24.0
47.5
31.3
43.3
36.9
42.5
˃100
˃100
˃100
˃100
87.3
˃100
61.9
35.0
19.6
˃100
48.0
53.9
A549/ATCC
EKVX
HOP-62
HOP-92
1.72
2.54
Non-small cell
lung cancer
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
Colon cancer
5.12
1.16
3.52
1.71
0.82
3.77
1.24
2.56
HT29
KM12
SW-620
SF-268
2
0
1
1
0
0
0
1
3
8
4
2
3
2
1
1
.27
.33
.77
.21
.66
.76
.82
.50
.53
.38
.47
.75
.89
.82
.71
.78
SF-295
SF-539
CNS cancer
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
IGROV1
Melanoma
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
Ovarian
Cancer
0.55
4
1
1
.28
.04
.08
2
2

SK-OV-3
86-0
1
0
0
0
1
1
2
1
2
.55
.65
.65
.65
.23
.26
.15
.82
.82
5.00
2.80
5.44
11.0
7.60
3.78
20.4
6.06
19.8
5.06
15.8
32.7
24.7
26.0
10.5
69.6
65.8
40.7
13.7
˃100
35.6
˃100
27.4
82.3
˃100
˃100
7
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
1.14
1.87
3.13
Renal Cancer
Prostate
Cancer
1.20
.54
3.42
2.34
1.14
DU-145
2
MCF7
MDA-MB-
1
.67
2
31/ATCC
HS 578T
0
2
.83
.59
5.34
9.06
˃100
7.80
˃100
47.2
˃100
˃100
Breast Cancer
3
.85
BT-549
T-47D
1
1
3.30
.27
MDA-MB-468
MIDa
4.38
2
3

Fig. 4. Concentration-antiproliferative response curves of compound IVg against nine different
cancer cell lines
2
.2.3. Cell viability assay
Cell viability assay was carried out for compounds IVc, IVf, and IVg using human mammary
gland epithelial cell line (MCF-10A). MCF-10A cells were treated with the tested compounds for
4
days and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) assay was used
to assess the cell viability. For all compounds there are no cytotoxic effects and cell viability was
greater than 87% at 50 μM.
2
4

2
.2.4. Effect of compounds IVc, IVf, and IVg on cancer cell growth
Independently, propidium iodide (PI) fluorescence assay was performed in this analysis to assess
the effects of the most active compounds IVc, IVf, and IVg on Panc-1 (pancreas cancer cell
line), PaCa-2 (pancreatic carcinoma cell line), HT-29 (colon cancer cell line) and H-460 (lung
cancer cell line), while Erlotinib hydrochloride was used as a positive control. The results of the
anticancer activity are summarized in Table 4 and presented as IC50 values. These all
compounds were bearing N-phenethyl-1H-pyrrol-2(3H)-one moiety (Scaffold A). This class of
compounds was found to be more effective compared to other series of compounds having N-
benzyl-1H-pyrrol-2(3H)-one (Scaffold A) and pyridazin-3(2H)-one groups (Scaffold B).
Compound IVf bearing 4-(CH ) N-C H moiety was found to be least effective against cancer
3
2
6
4
cell lines. Attachment of 3,4-di-OCH -C H increased the considerable effect of compound IVc.
3
6
3
Compound IVg bearing 4-Br-C H on pyrrole ring showed the most potency and best
6
4
antiproliferative effects. Compounds IVc, IVf and IVg were therefore subject to further studies
to investigate the possible mechanism of action.
2
5

Table 4. Antiproliferative activities of compounds IVc, IVf, and IVg.
Antiproliferative activity IC ± SEM (µM)
5
0
Compd.
Ar
Cell
HT-29
PaCa-2
H-460
Panc-1
viability
IVc
IVf
3,4-di-OCH -C H
3
89
91
87
5.2±1.2
9.5±2.5
4.3±0.9
3.8±1.2
3.7±1.7
1.5±0.8
6.6±1.1
ND
3.2±1.3
5.8±1.6
3
6
4-(CH ) N-C H
4
3
2
6
IVg
4-Br-C H
2.1±0.7
0.02±0.01
1.8±0.4
6
4
Erlotinib
---
0.05±0.02 0.04±0.02
0.06±0.03
2
.2.5. Tubulin polymerization inhibitory activity
To confirm whether the observed anticancer activity of the above mentioned compounds is
conferred by a microtubule-targeting mechanism, the effect of compounds IVc, IVf, and IVg on
tubulin polymerization at concentration of 25 μM using two positive controls, vincristine (a
tubulin polymerization inhibitor) and docetaxel (a tubulin polymerization stabilizer) have been
evaluated at concentration of 3 μM (Table 5). Compounds IVc and IVg have been shown to be
the strongest inhibitors with tubulin polymerization inhibitory effect of (1012) and (929),
respectively comparable to that of vincristine (805). It is clear from these results that the
molecular target of these compounds was most likely tubulin.
2
.2.6. EGFR inhibitory activity
EGFR (Epidermal Growth Factor) is a protein located on the surface of certain cells that allows
cells to divide and is characterized by high levels in cancer cells. EGFR inhibitors such as
erlotinib bind to and interrupt the tyrosine kinase domain in the epidermal growth factor
receptors. Table 5. shows the results of EGFR-TK assay for tested compounds IVc, IVf, and
2
6

IVg. From the results, the tested compounds gave a weak EGFR inhibition with IC of 7.4–9.4
5
0
μM.
.2.7. BRAFV600E inhibitory activity
2
The activity of the newly synthesized compounds IVc, IVf, and IVg against BRAFV600E was
tested using an in vitro experiment and IC values were summarized in Table 5. N-phenethyl-
5
0
1
H-pyrrol-2(3H)-ones IVc, IVf, and IVg exhibited weak BRAFV600E inhibitory activity with IC50
values of 12.6±1.8, 14.8±2.9 and 16.2±1.7 μM, respectively.
Table 5. Effects of compounds IVc, IVf, and IVg on EGFR, BRAFV600E, and Tubulin
Polymerization.
BRAF
inhibition
(Tubulin)
Effect
EGFR inhibition
IC ± SEM (µM)
Comp.
5
0
IC ± SEM (µM)
(arbitrary units)
5
0
IVc
12.6±1.8
14.8±2.9
16.2±1.7
9.4±3.1
7.4±1.2
9.2±3.5
1012±244
1107±214
929±304
ND
IVf
IVg
Erlotinib
DPBS
0.05±0.03
ND
0.06±0.03
ND
2626±198
805±227
4625±290
Vincristine
ND
ND
Docetaxel
ND
ND
ND: Not determined
2
7

2
2
.2.8. Cell cycle analysis and apoptosis detection
.2.8.1. Cell cycle analysis
Studies have been conducted on the impact of IVg compound on the progression of the cell cycle
and induction of PaCa-2 apoptosis. PaCa-2 cell was incubated with an IC concentration of
5
0
compound IVg for 24 h. The cell line was stained with PI/Annexin V and analyzed by
flowcytometry using BD FASCC alibur. Results investigation (Fig. 5) showed that the
percentage of pre-G1 apoptosis induced by compound IVg after 24 h of incubation was 24.14%.
A high percent of cell accumulation was observed in G2/M phase (32.15%) in PaCa-2 treated
with compound IVg after 24 h incubation indicating arrest of cell cycle at G2/M phase. PaCa-2
cells treated with compound IVg also showed cell aggregation at phase G1 and phase G2 / M
after 24 h of incubation but more at phase G2 / M. This suggested that the cell cycle was also
arrested during the G2 / M process.
Fig. 5. Cell cycle analysis in MCF-7 cell line treated with compound IVg
2
8

2
.2.8.2. Apoptosis assay
After IVg treatment, analysis of the PaCa-2 cell cycle showed pre-G1 apoptosis signalling. Cells
were stained with Annexin V/PI and incubated for 24 h to test the ability of IVg to induce
apoptosis. Early and late apoptosis studies have shown that IVg is likely to cause significant
apoptosis with 1.98 percent necrosis, (Fig. 6).
Fig. 6. Percentage of apoptosis and necrosis for compound IVg in PaCa-2 cell line
2
.3. Docking study into tubulin
In the current work, compounds IVc, IVf, and IVg displayed inhibitory activity against tubulin
polymerization, Table 5. Accordingly, we have performed a molecular docking study to
investigate the potential mechanism which mediate their inhibitory activity against tubulin
polymerization. In this study, the binding modes, orientations, and interactions of the
compounds IVc, IVf, and IVg were evaluated into colchicine/combretastatin A-4 (CA-4) and
vinblastine binding sites. The results of the new compounds were evaluated against the
biological results and compared with those of the positive control/native ligands.
2
9