Synthesis, cytotoxic activity, and tubulin polymerization inhibitory activity of new pyrrol-2(3H)-ones and pyridazin-3(2H)-ones

Article abstract of DOI:10.1016/j.bioorg.2016.03.007

A series of new pyrrol-2(3H)-ones 4a-f and pyridazin-3(2H)-ones 7a-f were synthesized and characterized using different spectroscopic tools. Some of the tested compounds revealed moderate activity against 60 cell lines. The E form of the pyrrolones 4 show

Full text of DOI:10.1016/j.bioorg.2016.03.007

Accepted Manuscript
Synthesis, cytotoxic activity, and tubulin polymerization inhibitory activity of
new pyrrol-2(3H)-ones and pyridazin-3(2H)-ones
Samar Hafez Abbas, Gamal El-Din A.A. Abuo-Rahma, Mohamed Abdel-Aziz,
Omar M. Aly, Eman A. Beshr, Amira M. Gamal-Eldeen
PII:
S0045-2068(16)30026-8
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.03.007
YBIOO 1894
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 October 2015
13 March 2016
14 March 2016
Please cite this article as: S.H. Abbas, G.E-D. Abuo-Rahma, M. Abdel-Aziz, O.M. Aly, E.A. Beshr, A.M. Gamal-
Eldeen, Synthesis, cytotoxic activity, and tubulin polymerization inhibitory activity of new pyrrol-2(3H)-ones and
pyridazin-3(2H)-ones, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.03.007
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Synthesis, cytotoxic activity, and tubulin polymerization inhibitory
activity of new pyrrol-2(3H)-ones and pyridazin-3(2H)-ones
Samar Hafez Abbasa, Gamal El-Din A. A. Abuo-Rahmaa*, Mohamed Abdel-Aziza, Omar M. Alya, Eman A.
Beshra, b, Amira M. Gamal-Eldeenc
a Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia, 61519, Egypt.
a,b Department of Pharmaceutical Chemistry, College of Pharmacy, Umm Al-Qura University, Makkah
21955, Saudi Arabia.
cCancer Biology Laboratory, Center of Excellence for Advanced Sciences, National Research Center,
Dokki, Giza, Egypt.
*Corresponding author. Tel.: +201003069431. Fax.: +2086-2369075
E-mail address: gamal.aborahma@mu.edu.eg
Postal address: Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia, 61519,
Egypt.
_____________________________________________________________
Abstract
A series of new pyrrol-2(3H)-ones 4a-f and pyridazin-3(2H)-ones 7a-f were synthesized
and characterized using different spectroscopic tools. Some of the tested compounds
revealed moderate activity against 60 cell lines. The E form of the pyrrolones 4 showed
good cytotoxic activity than both the Z form and the corresponding open amide form.
Furthermore, the in vitro cytotoxic activity against HepG2 and MCF-7 cell lines revealed
that compounds (E)4b, 6f and 7f showed good cytotoxic activity against HepG2 with IC50
values of 11.47, 7.11 and 14.80 µM, respectively. Compounds (E)4b, 6f, 7d and 7f
showed a pronounced inhibitory effect against cellular localization of tubulin. Flow
cytometric analysis indicated that HepG2 cells treated with (E)4b showed a predominated
growth arrest at the S-phase compared to that of G2/M-phase. Molecular modeling study
using MOE® program indicated that most of the target compounds showed good binding
of β-subunit of tubulin with the binding free energy (dG) values about -10 Kcal/mole.
Keywords: Pyrrolone; pyridazinone; Cytotoxicity; Tubulin; immunofluorescence, docking
study.
_____________________________________________________________________
1. Introduction
Tubulin is a globular protein that represents an important target for anticancer
drug discovery. A variety of natural compounds including podophyllotoxin, colchicine,
steganacine and combretastatins (CAs) inhibit tubulin polymerization by binding to
1

colchicine binding site. In fact, CAs causes a pronounced shutdown in blood flow to solid
tumors, resulting in extensive tumor-cell necrosis. Among CAs products, combretastatin
A4 (I) which represents one of the most potent antitumor agents; it shows strong
cytotoxicity against a variety of cell lines and is considered one of the powerful vascular
disrupting agents.1
The structural feature of the CAs include: a cis double bond, a trimethoxy- phenyl
ring "A" and a small substituent on the ring B. Conversion of cis double bond into trans
one produces dramatic reduction in both antitubulin and cytotoxicity. Substitution of the
olefinic bond by a heterocyclic ring gave compounds with potent antimitotic properties
such as compounds II2, III3, IV4 and V5 (Fig. 1). This suggestion supports the fact that;
modification of the double bond could produce effective antimitotic agents. The structural
modifications to ring A had received very little attention. Most CAs derivatives retained
the trimethoxyphenyl moiety. Keira’s research group showed no necessity of the three
methoxy groups in ring A for optimum biological activity such as compound VI (Fig. 1).6
The potent cytotoxicity and structural simplicity of combretastatin, has been
envisaged as a very attractive lead. In fact, the cis configured C–C double bond of CA-4
is prone to isomerize to the trans-form during storage and administration thus producing
dramatic reduction in both antitubulin activity and cytotoxicity. Therefore, considerable
efforts have gone into modifying combretastatin and discovering its bioavailable cis-
restricted analogues based on the bioisosteric replacement of olefinic double bond of the
natural product with vicinal diaryl-substituted five membered heteroaromatic rings
including oxazole, isoxazole, thiazole, pyrazole, tetrazole and imidazole.4
Furanone ring system has interesting attraction because of the flexibility of its
conversion into several heterocyclic systems of biological importance such as pyrrolone,
pyridazine, oxadiazole and triazole derivatives.7 Moreover, furanone is widely recognized
as a component of natural products that possess a wide range of interesting biological
activities such as anti-inflammatory, anticancer. Also, different classes of synthetic
furanones and pyrrolones possess a wide spectrum of important pharmacological
10
11, 12
13, 14
activities such as antibacterial,8, 9 antifungal, anticancer,
anti-inflammatory,
vasodilation15 and anticonvulsant. 13 Also, pyridazinones represent an important class of
heterocyclic compounds, which have been extensive research due to their broad
biological activities, such as anti-inflammatory16, analgesic,17 antihypertensive,18
anticancer,19 anti-diabetic20, anticonvulsant,21 antimicrobial22 and antifungal activities23.
2

Fig. 1. Chemical structure of CA-4 (I) and it's analogues II-VI.
Promoted with the above mentioned promising aspects, and in order to further investigate
structural determinants of cytotoxic activity of this class of compounds, the present study
involves a flexible, concise, and highly convergent protocol for the synthesis of pyrrolone
and pyridazinone derivatives aiming at preparation of new combretastatin analogues with
restricted rotation of both ring A and ring B. The prepared compounds were evaluated for
both in vitro cytotoxic activity against liver carcinoma Hep-G2 and breast carcinoma
MCF-7 cell lines. Moreover, tubulin polymerization inhibitory activity expressed in the
localization of tubulin was explored in Hep-G2 cell line after treated with selected
promising compounds as indicated by ELISA through immunofluorescence labeling and
analysis under Apo time fluorescence microscope. Also, to explore the alteration in the cell
cycle phases after treatment with the active cytotoxic compounds (E) 4b, the cells were
subjected to flow cytometry analysis. In addition, Docking of the target compound was
done using MOE Program and X-ray crystallographic structure of tubulin complexes with
colchicines.
2. Results and Discussion
2.1. Chemistry
3-Arylidene-5-(4-methoxyphenyl)-2-(3H)-furanones 2a-f were synthesized by fusion of
the acid 1 and different appropriate aromatic aldehyde in the presence of TEA in acetic
anhydride (Scheme 1) according to modified Perkin reaction.14 The chemical structure of
1
furanones 2a-f was elucidated on the basis of their IR, H-NMR, mass spectra as well as
the elemental analyses. Calculation of the δ values of furanones 2a-f analogues using
3

incremental parameters for the hydrogen (semicyclic double bond) suggested the (E)
configuration as reported in the literature. 8-10
Heating at reflux of furanones 2a-f with benzylamine in equimolar ratio in dry benzene
afforded the corresponding amide derivatives15 3a-f. The chemical structures of the
1
amides 3a-f were elucidated on the basis of their IR, H-NMR, mass spectrum as well as
1
the elemental analysis. In the H-NMR of amides 3, it is noteworthy noting that the two
benzylic –CH2- protons are diastereotopic and appeared as two doublets. This may be
attributed to the expected tautomerism in these compounds (Scheme 1). Heating at reflux
of amides 3a-f with 6 M hydrochloric acid for 1 h afforded the corresponding pyrrolones
4a-f as reported in the literature.14 The chemical structure of the pyrrolones 4a-f was
1
confirmed by IR, H-NMR, 13C-NMR, mass spectra as well as elemental analyses. X-ray
crystallography of compound 4d (Figure 2) revealed that the proper configuration of this
compound is the kinetically stable (E) configuration.
MeO
MeO
Ar
CHO
COOH
Ar
Acetic anhydride, TEA
O
O
1
O
2a-f
MeO
MeO
CH2NH2
Ar
6 M HCl
Ar
reflux, 1hr
benzene,
reflux, 5hr
N
O
HN
O
O
(E) 4a-f
Rotomer A
MeO
Ar
MeO
Ar
OH
O
N
OH
HN
Hx
Hy
O
Rotomer B
(restricted rotation around C-N=C-OH)
Rotomer C
(free rotation around C-N-C=O)
3a-f
2a, 3a, 4a; Ar C6H5, 2b, 3b, 4b; Ar 4-OCH3-C6H4, 2c, 3c, 4c; Ar = 3,4,5-tri-OCH3-
=
=
C6H2, 2d, 3d, 4d; Ar = 2-OH-C6H4, 2e, 3e, 4e; Ar 4-OH-3-OCH3-C6H3 and 2f, 3f, 4f;
=
Ar = 4-Cl-C6H4.
Scheme 1. Synthesis of furanone derivatives 2a-f, amide derivatives 3a-f and (E)
pyrrolone derivatives 4a-f.
4

Fig. 2. The crystal structure of pyrrolone (E) 4d.
It is noteworthy noting that, the E configuration of 3a-f favors on Z configuration
because of ring closure of the amides 3a-f in (Z) configuration, don't occurred due to long
distance between oxygen and amide nitrogen.
Reaction of furanone derivatives 2a-f with hydrazine monohydrate afforded the
corresponding hydrazide 5a-f. Heating at reflux of p-methoxybenzaldehyde and
hydrazides 5a or 5f for 13-24 h in ethanol afforded the corresponding pyrrolones 6a & 6f.
Also, the pyrrolones 6g-j was prepared by stirring of hydrazide 5f with aromatic aldehyde
in ethanol at room temperature for 2 days. (Scheme 2). The chemical structure of the
1
pyrrolones 6a, 6f and 6g-j was confirmed by IR, H-NMR, mass spectra as well as
elemental analysis.
MeO
Ar
N
O
N
H
7a-f
6 M HCl
reflux,
1hr
Ar1
O
N
MeO
H
O
Ar
N
NH2NH2.H2O
Ar1
2a-f
Ar
Ethanol, RT, 1day
ethanol
13-24 h
O
HN
O
MeO
NH2
6a, 6f, 6g-j
5a-f
5a, 7a; Ar = C6H5, 5b, 7b; Ar = 4-OCH3-C6H4, 5c, 7c; Ar = 3,4,5-tri-OCH3-C6H2,
5d, 7d; Ar = 2-OH-C6H4, 5e, 7e; Ar = 4-OH-3-OCH3-C6H3, 5f, 7f; Ar = 4-Cl-C6H4.
In 6; 6a: Ar = Ar1 = 4-OCH3-C6H4, 6f: Ar = 4-Cl-C6H4, Ar1 = 4-OCH3-C6H4,
6g: Ar = 4-Cl-C6H4, Ar1 = C6H5, 6h: Ar = 4-Cl-C6H4, Ar1 = 3,4-di-OCH3-C6H3,
6i: Ar = 4-Cl-C6H4, Ar1 = 3,4,5-tri-OCH3-C6H2, 6j: Ar = Ar1 = 4-Cl-C6H4.
Scheme 2. Synthesis of hydrazide derivatives 5a-f, pyrrolone derivatives 6a, 6f, 6g-j
and pyridazinone derivatives 7a-f.
5

The pyridazinones 7a-f were prepared by heating at reflux of the appropriate
hydrazide derivatives 5a-f with 6 M hydrochloric acid for 1 h. Also, pyridazinones 7a-f
were obtained directly by heating at reflux of furanones 2a-f with hydrazine monohydrate
in absolute ethanol for 4-24 h.24 The chemical structure of compounds 7a-f was
confirmed by IR, 1H-NMR and 13C-NMR, mass spectra as well as elemental analyses.
2.2. Biology
2.2.1. Anticancer activity
The effect of the tested compounds on the viability of hepatocellular carcinoma
HepG2 cells and breast cancer MCF-7 cells were studied using MTT assay after 48 h of
incubation. Treatment of hepatocellular carcinoma HepG2 cells and breast cancer MCF-7
cells with gradual concentrations of tested compounds revealed that compounds (E)4b,
6f, 7d and 7f possessed promising cytotoxic effect on HepG2, with IC50 of 11.47, 7.11,
26.44 and 14.8 µM, respectively, while the other tested compounds showed weak
cytotoxic effect on HepG2. Compounds (E)4b and 6f possessed promising cytotoxic
effect on MCF-7 with IC50 of 18.58 and 19.49 µM, respectively, while the other tested
compounds showed weak cytotoxic effect on MCF-7, as shown in Table 1.
Table 1. Collective calculated IC50 (µM) from linear equation of dose response
curve for each tested compounds against breast adenocarcinoma MCF-7
and liver carcinoma Hep-G2 cells.
IC50(µM)
IC50(µM)
Compound
Compound
MCF-7HHep-G2
MCF-7Hep-G2
49.4
32.64
18.58
60.60
>100
39.37
66.64
40.0
70.34
43
43.74 37.82
19.49 7.11
34.91 103.17
2c
6a
3c
6f
11.47
>100
45.06
>100
>100
>100
E(4b)
Z(4b)
E(4c)
Z(4c)
E(4d)
E(4e)
E(4f)
7b
47.9
32.4
7c
37.45 26.44
50.53 55.6
37.12 14.8
7d
7e
7f
0.81
0.73
Paclitaxel
29.47 37.65
6

For more screening of antiproliferative activity, compounds 3c, 3e, 3f, (E)4d, (E)4f,
(Z)4f, 5e, 5f, 6f, 7c, 7e and 7f were selected by the National Cancer Institute (NCI)
according to the protocol of the Drug Evaluation Branch of the National Cancer Institute
25
Bethesda, USA for in vitro anticancer screening. Primary in vitro one dose anticancer
assay was performed in full NCI 60 cell lines derived from nine tumor subpanels,
including leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast
cancer cell lines. The selected compounds were added at a single concentration (10ˉ5 M)
and the culture was incubated for 48 h. End point determinations were made with a
protein binding dye sulforhodamine B (SRB). Results for each compound were reported
as a mean graph of the percent growth of the treated cells when compared to the untreated
control cells. The results of NCI screening for the tested compounds is illustrated in table
2. Compound 3c exhibited moderate cell growth inhibition activity against renal cancer
UO-31 and breast cancer T-47D, also amide 3e showed moderate cell growth inhibition
activity against non-small cell lung cancer NCI-H522 cell line and colon cancer HCT-15.
While the chloro amide 3f showed moderate cell growth inhibition activity against colon
cancer HCT-15, prostate cancer PC-3, non-small cell lung cancer NCI-H522 cell line,
leukemia K-562, SR, melanoma MDA-MB-435, UACC-62 and breast cancer T-47D,
MDA-MB-468 cell line.
On the other hand, the pyrrolone (E)4d exhibited moderate cell growth inhibition
activity against seventeen cell lines; among them leukemia HL-60(TB), SR, non-small
cell lung cancer NCI-H522, melanoma M14, SK-MEL-2, prostate cancer PC-3 and breast
cancer MCF7, T-47D, MDA-MB-468. A complete cell death was recorded for the
melanoma MDA-MB-435 cell line where the growth percent -4.21. Also, pyrrolone (E) 4f
exhibited moderate cell growth inhibition activity against fifteen cell lines; among them
leukemia SR, K-562, MOLT-4, non-small cell lung cancer NCI-H522, colon cancer HCT-
15, CNS cancer U251, melanoma MDA-MB-435, UACC-62, prostate cancer PC-3 and
breast cancer MDA-MB-468. In contrast, the cytotoxicity activity falls rapidly when the
configuration of 4f changed from E to Z against most of the cell lines although the Z form
exhibited remarkable cell growth inhibition activity against only five tested cell lines.
In addition, the hydrazide 5e showed significant cell growth inhibition activity
against melanoma MDA-MB-435, leukemia SR and breast cancer MDA-MB-468 where
the growth percent were 9.68, 26.93 and 27.58, respectively. Compound 5e revealed
moderate cell growth inhibition against fourteen tested cell line; among them leukemia K-
7

562, non-small cell lung cancer NCI-H460, NCI-H522, breast cancer MCF7, renal cancer
UO-31 and prostate cancer PC-3.
Compound 5f exhibited remarkable cell growth inhibition activity against renal
cancer UO-31, breast cancer T-47D and non-small cell lung cancer NCI-H522 only.
Also compound 6f exhibited remarkable cell growth inhibition activity against leukemia
HL-60(TB), non-small cell lung cancer HOP-92, NCI-H522, Melanoma UACC-257,
renal cancer UO-31 and Breast cancer T-47D.
The pyridazinone 7c exhibited remarkable cell growth inhibition activity against
non-small cell lung cancer NCI-H522, NCI-H226, HOP-62, A549/ATCC, CNS cancer
U251, Melanoma UACC-257, UACC-62, renal cancer UO-31 and breast cancer
MDA-MB-231/ATCC. A moderate cell growth inhibition was achieved against breast
cancer T-47D, MDA-MB-468 and leukemia RPMI-8226 cell lines. Compound 7e
exhibited remarkable cell growth inhibition activity against non-small cell lung cancer
NCI-H522, Melanoma UACC-257, renal cancer UO-31, CAKI-1 and breast cancer
T-47D while moderate cell growth inhibition was achieved against non-small cell lung
cancer HOP-92 cell line only.
Moreover, compound 7f exhibited moderate cell growth inhibition against breast
cancer T-47D and non-small cell lung cancer NCI-H522 cell lines only. A remarkable cell
growth inhibition was achieved against leukemia RPMI-8226, MOLT-4, non-small cell
lung cancer A549/ATCC, NCI-H226, Melanoma UACC-257, renal cancer UO-31, CAKI-
1 and breast cancer MDA-MB-468 cell lines.
The obtained results from NCI screening indicate that the presence of electron
withdrawing group on the benzylidene ring of the amide gives better cytotoxicity activity.
The E form of pyrrolones 4 have good cytotoxicity activity than the open amide form
while the Z form has less cytotoxicity activity, also the hydrazide have cytotoxicity
activity better than the cyclized pyridazinone form if the benzylidene ring have electron
donating group, while the reverse is true if the benzylidene ring have electron
withdrawing group.
8

Table 2. % of cell growth inhibition for compounds 3c, 3e, 3f, (E)4d, (E)4f, (Z)4f, 5e, 5f,6f,7c,7e
and 7f against different cell lines.
compound
Panel/Cell Line
3c
3e
3f
(E)4d
(E)4f
(Z)4f
5e
5f
6f
7c
7e
7f
% of Growth Inhibition
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
1.6
7.4
0.3
29.8
19.5
13.6
13.9
15.6
5.4
21.3
19.7
20.7
26.5
26.2
37.6
27.1
24.3
39.7
26.6
59.1
48.9
33.2
37
27.4
23.7
37.8
34.2
29
16.7
14.6
0
1.6
9.6
2.4
26.2
33.6
52.8
12.5
19.7
73.1
8.5
9.5
0.1
15.3
18.8
0
10
7.2
12.1
4
6.4
0
3.9
2.3
4.3
12.2
2
11.5 12.1 20.3
15.6 14.9 30.7 17.7 24.3
13.1 5.4 17.7 12.5 10.4
54.8
41.4
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
HOP-62
18.8
8.5
14.6
nda
21.5
9
13.9
0
19.7
18.7
6.9
9.5
22.6
13.7
11.1
18.8
0
15.3
4.4
17.4
13.8
26.6
9.4
15.5
25.6
39.3
26
15.7
4.1
26.1
24.7
33.8
7.3
19.1
34.7
38.2
18.3
1.8
9.5
20.6
11.7
0
0
0
22.2
20
6
32.6
30.5
8.3
31.1
18
56.4
47.9
13.4 18.5 24.3 15.5 19.9
6.7 14.4 5.3 12.9
12.4 16.7 24.5 18.9 14.3
nd 18.8 nd 32.8 nd
11.8 12.6 26.9 11.6 23.1
17.9
10.3
35.4
5.1
14.1
22.2
10
0
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
HCT-116
HCT-15
7.2
16.1 10.6 11.7
5.6
19.5 21.8 28.2
4
12
4.8
9.9
0
12.3
3
0
0
0
33.1
48.7
26
30.3
12.4
14
9.3
0
18.2 17.5
33.2 53
1.4
0.8
27.5
32.4
0
33.7
5.2
27.7
35.8
0
3.3
0
0
7.1
0.3
3.3
0
13.2 5.6
12.5 0.3
6
52.5
13.3
1.3
0
13.3
0
0
HT29
KM12
17.9
0.3
2.4
0
0
0
4.7
0
29.6
26.8
0
CNS cancer
U251
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer
CAKI-1
19
4.8
29.4
16.1
35.5
9.4
15.1
8.7
0
7.1
0
0
11.7
0
0
15
16.8
8.4
20.4 12.1 13.7
0
0
19.7
3
5.3
0
11.7
0
15.8
5.9
4
22.1
20.2
5.2
31.2
20.4
8.8
6.6
29.3
32.7
19
25.5
13.9
8.3
42.9
17
10.9
9.5
0
7.5
2.3
0
0
0
0.1
23.1
10
17.8
6.6
8.5
90.3
9.2
24.3
25
0
0
0
0
4.6
0
7.6
7.7
3.1
6.6
1.9
0
5.3
0
0
0
0
3.4
0
0
3
2.4
0
36.5
49.5
104.2
48.6
20.1
33.8
28.3
36.1
0.2
5.2
14
0
0
11.3
24.4
23.1
6.5
11.5
5.9
10.2
34.2
38.9
18.5
24.2
19.6 27.7 18.9 23.5
9.1
20
11.1 16.9
20.5
14.6
5.8
16.1
7.2
14.1
3.1
13.7
17.7
10.9
20.7
22.5
29.1
20.1
26.2
19.4
16.6
8.1
0.4
8.2
0
17.4
19.3
25.2
20.1
15.4
11.2
0
5.6
8.6
0
9.4
13.6
8.6
10.2
9.3
5.9
7.2
9.1
5.2
5.5
12.4
26.8
11.9
15.7 14.9 13.8 12.9
25.2
20.4
38.3
20.5
17.7
28.1
12.6
27.3
24
6.8
14.7
31.6
10
16.5
25.8
7
13.3
4
22.1
0
32.1
3.9
0
23.3 21.3
6.2
3.4
18
0
26
20.5 22.3
8.1
24.2 27.3
TK-10
UO-31
0
Prostate Cancer
PC-3
15
19.2
43.6
47.8
48.3
20.6
32.7
0
13.9 16.8
7.9
8.4
Breast Cancer
MCF7
MDA-
16.5
13
23.8
9.5
13.9
18.7
37.2
6.3
14.7
22.4
0
0.1
52.1
34.6
9.8
12.7
13.2
0.1
1.5
0
8
22.2 10.6 19.2
MB231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
nda: not detected
5.5
0
46.7
16.5
17.5
17.6
29.3
18
13.6
0
34.7
30.3
25.2
29.6
31.1
46.3
12.9
0
36.1
37.5
0
0
16.2
13.3
0
36.3
33
2.2
0
22
0.7
0
0
8.6
15.4
2.4
7.2
0
0
29.4 39.5 22.6 35.4
13.6 31.3 7.9 27.3
72.4
9

2.2.2. Immunofluorescence localization of tubulin in liver cancer cells
The localization of tubulin was explored in Hep-G2 cell line after treatment with
selected promising compounds that revealed an inhibitory effect in tubulin as indicated by
ELISA. Cells were treated with 25 µg/mL of each compound for 48 h, and then submitted
to immunofluorescence labeling and analysis under Apo time fluorescence microscope.
Compounds (E)4b, 6f, 7d and 7f showed a variable promising inhibitory effect of
cellular localization of tubulin (Fig. 3), as concluded from the inhibition of the
fluorescence intensities.
Fig. 3. Fluorescence intensity (IFU) of tubulin localization in HepG-2
cells after treatment for 48 h by tested compounds. Grey bar represent
control cells.
Treatment with compounds (E)4b, 6f, 7d and 7f resulted in a high degree of
cellular cytotoxicity and malformed cellular structure with shrinked nuclei. Abnormal
cytoplasm/ nucleus proportion with reduced cytoplasm and abnormal diminished tubulin
expression pattern. The cell-cell microtubules mesh disappeared, although the cells
possessed relatively high tubulin, but not distributed in a normal cellular distribution
pattern (Fig. 4).
10

Tubulin analysis in untreated HepG2
Tubulin analysis in combretastatin-
cells.
A4 treated HepG2 cells.
Tubulin analysis in 6f treated HepG2
Tubulin analysis in (E)4b treated
cells.
HepG2 cells.
Tubulin analysis in 7d treated HepG2
Tubulin analysis in 7f treated HepG2
cells.
cells.
Fig. 4. Fluorescence intensity (IFU) of tubulin localization in HepG-2 cells after
treatment with combretastatin-A4, (E)4b, 6f, 7d and 7f for 48 h compared to
control cells (photo X 200 ) as captured by Apotome fluorescence
microscope.
2.2.3. Cell cycle analysis
To explore the alteration in the cell cycle phases after treatment with the active
cytotoxic compounds (E)4b, the cells were subjected to flow cytometry analysis. The
analysis indicated that in HepG2 cells treated with (E)4b induced G2/M arrest of HepG2
cells and S-phase arrest, where a large proportion of cells were accumulated in S- and
G2/M-phase (P <0.05). HepG2 cells treated with (E)4b showed a predominated growth
11

arrest at the S- phase higher than that of G2/M-phase (P <0.01) as compared with control
cells, where the S-phase progression of HepG2 cells was considerably delayed (Fig. 5).
Control
E)4b
Fig.5. Flow cytometry analysis of HepG2 cells treated with (E)4b showed a
predominated growth arrest at the S- phase higher than that of G2/M-phase
(P<0.01) as compared with control cells, where the S-phase progression of
HepG2 cells were considerably delayed.
2.3. Docking of the target molecules on 1SA0, Tubulin-Colchicine: Stathmin-like domain
complex.
Molecular modeling study was carried out for the synthesized compounds including
3b, 3f, (E)4b, (E)4c, (E) 4d, (E)4e, (E)4f, (Z)4b, (Z)4c, (Z)4f, 5d, 5f, 6a, 6f-j and 7a-f,
DAMA-Colchicine and combretastatin A-4 in the active colchicine binding site of tubulin
polymerase enzyme using Molecular Operating Environment (MOE®) version 2014.09.
DAMA-Colchicine was bound in the active site through two H-bond interactions between
the ligand with Lys352 and Ala317, also there are strong hydrophobic interactions
between the ligand and Lys352, Leu255, Asn258, Leu248, Lys254 and Ala316 as
reported in the literature.26 On the other hand, Combretastatin A-4 showed only one
hydrogen bond between the hydroxyl group of the ligand and Lys-352. In addition, there
are strong hydrophobic interactions between the ligand and Lys352, Ala316, Leu255,
Asn258 and Leu248, while in the literature binding of CA-4 on 1AS0 revealed formation
12

of two hydrogen bonds with Cys241 and strong hydrophobic interaction with Leu242,
leu255, Ala250, Ala 318, Ala316, Lys352, Ile378 and Val 315.27
All the tested compounds showed inhibition of β-subunit of tubulin as the
binding free energy (dG) values of them above or around (-10) Kcal/mole (Table 3).
The data in Table 2 showed a rough correlation between the dG values of the target
compounds and their activity (expressed as IC50 values against liver cancer cells). All
compounds have dG value as Combretastatin A-4 dG value.
Table 3. Interaction energies of tested compounds, DAMA-Colchicine and combretastatin
A-4 in the active site β-subunit of tubulin.
Compound
dG
Compound
dG
Kcal/mole
Kcal/mole
-12.04
-10.01
-10.74
-10.26
-10.74
-11.07
-11.16
-12.81
DAMA-Colchicine
5f
6a
6f
Combretastatin A-4
3b
3f
6g
-10.45
-12.96
-10.69
-10.46
-11.29
-11.16
-10.94
-11.86
-11.51
-11.01
-9.94
-10.13
-10.75
-10.18
E(4b)
E(4c)
E(4d)
E(4e)
E(4f)
Z(4b)
Z(4c)
6h
6i
6j
7a
7b
7c
7d
-10.22
-10.79
-10.74
-10.74
-9.99
Z(4f)
5d
7e
7f
5f
Binding modes of tested compounds with tubulin binding site.
Molecular docking simulation of the amide 3b (Fig. 6) into tubulin active site,
revealed presence of one hydrogen bond between the hydroxyl group of the ligand and
Lys-352 at distance 3Å like combretastatinA-4, in addition to several hydrophobic
interactions between the ligand and critical amino acid residues including Leu248,
Lys254, Leu255, Lys352 and Asn258 like DAMA-colchicine.
On the other hand, molecular docking simulation of the amide 3f revealed one
hydrogen bond between the ligand but with Asn-258 and pi-cation interaction between
halophenyl ring of the ligand and Lys352. Also there are several hydrophobic interactions
between the ligand and Leu248, Leu255, Lys352, Ala316, Ile347, Asn349 and Asn258.
13

Fig. 6. 2D representation of docking of 3b in β-subunit of tubulin.
Molecular docking simulation of the cyclized form of the amide 3b the pyrrolone
E(4b) (Fig.7, 8) which showed inhibitory activity against MC7 and HepG2 cells revealed
presence of pi-cation interaction between the ligand and Lys-352 instead of hydrogen
bond in case of its amide 3b, in addition to several hydrophobic interactions between the
ligand and critical amino acid residues including Leu248, Lys254, Leu255, Ala316,
Lys352 and Asn258 like compound 3b.
It is reasonable to note the presence of hydrogen bond or pi-cation interaction
with Lys352 at distance like that of compound E(4b) around 3.81Å along with strong
hydrophobic interaction in the active site is essential for activity, therefore the lack of
activity in compound E(4c) due to the absence of interaction with Lys352. Compounds
E(4d) and E(4f) showed inhibitory activity because of presence of interaction with
Lys352. On the other hand compound E(4e) lacks activity due to it has less hydrophobic
interaction in the binding site although it have pi-cation interaction with Lys352 at 3.79Å.
14

Fig. 7. 2D representation of docking of E 4b in β-subunit of tubulin.
Fig. 8. Docking of E(4b) into the tubulin active site obtained from the crystal structure of
E(4b) complex.
Regarding the stereochemistry effect in binding on 1AS0, Molecular docking
simulation of compound E4b and Z4b revealed that the E form have strong hydrophobic
interaction with the critical amino acids in the binding site than that formed in case of Z
form, also the Z4b (Fig.9) lacks the pi-cation interaction with Lys352 as confirmed with
inhibitory activity. Also molecular docking simulation of compounds Z4c and Z4f
15

revealed absence of pi-cation interaction with Lys 352 or its presence but at distance 4.6Å
respectively.
Fig. 9. 2D representation of docking of Z (4b) in β-subunit of tubulin.
Also, molecular docking simulation of hydrazide 5d (Fig.10) revealed presence of
hydrogen bond and pi-cation interaction between the ligand with Asn258 and Lys352
respectively, in addition to hydrophobic interaction between the ligand and Leu248,
Leu255, Cys241, Lys352, Ala316 and Asn258, while hydrazide 5f was showed two
hydrogen bond interactions between the ligand and Cys241 and Lys254, in addition to
less hydrophobic interactions in the binding site than compound 5d.
Fig. 10. 2D representation of docking of 5d in β-subunit of tubulin.
16

Molecular docking simulation of compound 6a was showed no interaction with
the active site of tubulin these may be due to the steric effect as confirmed with the
biological activity., while binding of compounds 6f (Fig.11) which is the most active
compound against Hep-G2 carcinoma, revealed presence of one pi-cation interaction
between the ligand and Asn258 for 6f and hydrophobic interaction between the ligand and
Leu248, Leu255, Lys254 Lys352 and Asn258. This means presence of pi-cation or
hydrogen bond interaction with Asn258 along with strong hydrophobic interaction with
critical amino acid residues in the binding side produce active compounds. Compound 6i
expected to have similar activity as pyrrolone 6f because it has similar interaction like 6f
except formation of hydrogen bond with Asn258 instead of pi-cation interaction. On the
other hand compounds 6g, 6h expected to have good inhibitory activity through its pi-
cation interaction with Lys 352 with distance 3.73Å and 4.1Å as pyrrolone E4b, in
addition to the presence of hydrophobic interactions. Compounds 6j is expected to have
less inhibitory activity than pyrrolone 6f because of their formation of pi-cation
interaction with Lys352 with distance 4.98Å.
Fig. 11. 2D representation of docking of 6f in β-subunit of tubulin.
Regarding the pyridazinones 7a and 7e, their mode in binding may reflect that
they have no or little inhibitory activity because they have weak hydrophobic interaction,
also its binding revealed formation of pi-cation interaction with Lys352 at distance 4.51Å
and 3.11Å, respectively. Also compound 7c have weak inhibitory activity because it has
17

weak hydrophobic interaction in spite of its formation of pi-cation interaction with
Lys352 at distance 3.70Å.
On the other hand, pyridazinones 7d (Fig.12) and 7f which were shown moderate
inhibitory activity against Hep-G2 carcinoma, revealed presence of hydrophobic
interaction, in addition to hydrogen bond interaction with Cys241 at distance 3.88Å in
case of 7d while formation of pi-cation interaction with Lys352 at distance 3.89Å in
case of 7f. This means the contribution of inhibitory activity of binding with Lys 352 is
more than that of Cys241 because of the inhibitory effect of 7f more than that of 7d.
Again here the distance in binding is critical issue as well as the binding with critical
amino acid, therefore in case of compound 7b although it form four interaction with the
binding site, three of them H bond of the ligand with Cys 241 at distance 3.17Å, Ala 250
& Asp 251 and the fourth one is pi-cation between the ligand and Lys 254 but it has very
weak inhibitory activity.
Fig. 12. 2D representation of docking of 7d in β-subunit of tubulin.
3. EXPERIMENTAL
3.1: Chemistry
Melting points was determined using Stuart electrothermal melting point apparatus and
were uncorrected. The IR Spectra were carried out using Shimadzu IR-408
spectrophotometer, KBr pellets; Bruker Tensor 27 or Nicolet iS5 (ATR) FT-IR
spectrometer. For 1HNMR Spectra: Varian Germany NMR spectrophotometer (200 MHz)
18

or Varian Mercury VX-300 NMR spectrometer (300 MHz) or Bruker apparatus (DRX
400 MHz); or JEOL-JNM-LA-400 FT-spectrometer (400 MHz); chemical shift (δ) in
ppm relative to TMS (δ = 0 ppm) as internal standard and CDCl3 or DMSO-d6 as a
solvent. Coupling constant (J) in Hz, geminal coupling (2J), vicinal coupling (3J) and the
signals are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
For 13CNMR Spectra: Bruker 300 MHz NMR spectrometer (75 MHz), or Bruker
apparatus (100 MHz); For EI-MS: Shimadzu GC/ MS- QP5050A, or Shimadzu Qp-2010
Plus. for Elemental analyses: Vario EL III German CHN Elemental analyzer model, or
GmbH Vario EL V2.3 CHN Elemental analyzer model, X-ray crystallography: using
maXus, X-ray lab, National Research Centre, Dokki, Giza, Egypt. Chemicals and solvents
used in the preparation of the target compounds are of commercial grade, and purchased
from Alfa Aesar, Cambrian chemicals, Aldrich, Acros Organics, Fluka, Merck, and El-
Nasr pharmaceutical chemicals companies. Chemicals and solvents were used without
purification. The reactions progresses were monitored using TLC (Kiesslgel 60 F254
precoated plates, E. Merck, Dermastadt, Germany), the spots were detected by exposure
to UV lamp. Compounds 128, 2a29, 2b29, 2c30, 2d31, 2e32, 2f30, 3a33 and 4a33 are prepared
as in the literature.
3.1.1: General procedure for Synthesis of N-benzyl-2-benzylidene-4-oxo-4-(4-
methoxyphenyl)butanamides 3a-f.
Heating at reflux the appropriate furanone derivatives 2a-f (0.003 mol) and
benzylamine (0.004 mol, 0.43 g) in dry benzene for 5 h. The mixture leave to cool and the
obtained crystals were filtered off, washed with dry benzene.
3.1.1.1: (E)-N-Benzyl-2-benzylidene-4-(4-methoxyphenyl)-4-oxobutanamide 3a.33
White crystal ; (1.04 g, 90.4% yield); mp 129-131°C; IR (KBr) max = broad band 3264
1
cm-1 (NH), 1670 cm-1 (C=O benzoyl), 1636 cm-1 (C=O amide); H-NMR (400 MHz,
CDCl3)  (ppm) = 3.82 (s, 3H, OCH3), 4.84 (s, 2H, N-CH2), 6.21 (s, 1H, olefinic Ha),
6.86 (d, 2H, J = 8.0 Hz, ArH), 7.09 (d, 2H, J = 8.0 Hz, ArH), 7.18-7.22 (m, 2H, ArH),
7.24-7.28 (m, 3H; 2H, ArH and 1H, olefinic Hb, enol tautomer C), 7.34-7.39 (m, 2H,
ArH), 7.42 (d, 2H, J = 6.8Hz, ArH), 7.48 (s, 1H, NH), 7.64 (d, 2H, J = 6.8 Hz, ArH).
19

3.1.1.2: (E)-N-Benzyl-2-(4-methoxybenzylidene)-4-(4-methoxyphenyl)-4-
oxobutanamide 3b.
White crystals; (0.93 g, 74.3% yield); mp = 127-129°C; IR (KBr) max = broad band 3302
1
cm-1 (NH and OH), 1673 cm-1 (C=O benzoyl), 1645 cm-1 (C=O amide); H-NMR (300
MHz, CHCl3)  (ppm) = 3.37 (s, 2H, COCH2), 3.80 (s, 3H, OCH3), 3.81 (s, 3H, OCH3),
2
2
3.97 (s, 1H, OH) 4.08 (d, 1H, J =15.0 Hz, N-CH2), 4.70 (d, 1H, J =15.0 Hz, N-CH2),
6.81 (s, 1H, olefinic Ha, enol tautomer B ), 6.84 (d, 2H, J = 9.0 Hz, ArH), 7.17-7.28 (m,
2H, ArH), 7.30 (d, 2H, J = 9.0 Hz, ArH), 7.34-7.36 (m, 4H, ArH), 7.37-7.38 (m, 2H,
13
ArH), 7.48 (s, 1H, ArH); C NMR (100 MHz, CDCl3)  (ppm) = 169.7, 160.0, 159.4,
138.2, 134.2, 131.4, 131.4, 128.8, 128.2, 128.0, 127.2, 127.1, 125.7, 114.2, 113.6, 90.5,
55.3, 55.3, 45.4, 43.8; EI-MS (70 eV) m/z (%) 415 (M+, 14), 397 (100), 306 (35), 291
(15), 277 (2), 247 (4), 171 (4), 159 (3), 135 (78), 91 (76), 83 (27), 71 (44). Anal. Calcd.
For C26H25NO4 (415.18): C, 75.16; H, 6.06; N, 3.37. Found: C, 75.35; H, 6.09; N, 3.51.
3.1.1.3: (E)-N-Benzyl-4-(4-methoxyphenyl)-4-oxo-2-(3,4,5-trimethoxybenzylidene)-
butanamide 3c.
White crystals; (1.04 g, 77.5% yield); mp = 176-178°C; IR (KBr) max = broad band 3284
1
cm-1 (NH and OH), 1670 cm-1 (C=O benzoyl), 1643 cm-1 (C=O amide); H-NMR (300
MHz, CDCl3)  (ppm) = 3.83 (s, 3H, OCH3), 3.90 (s, 6H, 2OCH3), 3.91 (s, 3H, OCH3),
4.84 (s, 2H, N-CH2), 6.13 (s, 1H, olefinic Ha), 6.87-6.9 (m, 4H, ArH), 7.07-7.10 (m, 3H,
ArH), 7.23-7.26 (m, 5H; 4H, ArH and 1H, olefinic Hb, enol tautomer C), 7.41 (s, 1H,
NH); EI-MS (70 eV) m/z (%) 475 (M+, 17), 460 (9), 457 (100), 337 (3), 321 (6), 277 (15),
247 (2), 159 (2), 135 (67), 107 (11), 106 (15), 91 (76), 83 (5). Anal. Calcd. For
C28H29NO6 (475.20): C, 70.72; H, 6.15; N, 2.95. Found: 70.83; H, 6.22; N, 3.02.
3.1.1.4:
(E)-N-Benzyl-2-(2-hydroxybenzylidene)-4-(4-methoxyphenyl)-4-
oxobutanamide 3d.
White crystal; (0.5 g, 41.5% yield); mp = 178-180°C; IR (KBr) max = broad band 3356
1
cm-1 (NH and OH), 1681 cm-1 (C=O benzoyl), 1649 cm-1 (C=O amide); H-NMR (400
MHz, CDCl3)  (ppm) = 3.35 (s, 2H, COCH2, enol tautomer C), 3.84 (s, 3H, OCH3), 4.20
(d, 1H, 2J = 9.0 Hz, N-CH2, enol tautomer B), 4.87 (s, 1H, OH), 4.89 (d, 1H, 2J = 9.0Hz,
N-CH2), 6.23 (s, 1H olefinic Ha), 6.87 (d, 2H, J = 8.0 Hz, ArH), 6.96 (d, 2H, J = 8.0 Hz,
ArH), 7.15-7.24 (m, 2H; 1H, ArH, 1H and 1H, olefinic Hb), 7.26-7.35 (m, 2H, ArH),
20

7.37-7.47 (m, 2H; 1H, ArH, 1H, OH), 7.67-7.69 (m, 2H, ArH), 8.06-8.34 (m, 2H, ArH),
8.36 (d, 1H, J = 8.0 Hz, ArH), 9.30 (s, 1H, NH); EI-MS (70 eV) m/z (%) 401 (M+, 1),
383 (0.38), 294 (1), 159 (3), 135 (100), 107 (6), 92 (8), 83 (1), 77 (21). Anal. Calcd. For
C25H23NO4 (401.16): C, 74.79; H, 5.77; N, 3.49. Found: 74.88; H, 5.82; N, 3.54.
3.1.1.5:
(E)-N-Benzyl-2-(4-hydroxy-3-methoxybenzylidene)-4-(4-methoxyphenyl)-4-
oxobutanamide 3e.
Pale yellow crystals; (0.87 g, 67.3% yield); mp = 162-164°C; IR (KBr) max = broad
1
band 3401 cm-1 (NH and OH), 1666 cm-1 (C=O benzoyl), 1644 cm-1 (C=O amide); H-
NMR (300 MHz, CDCl3)  (ppm) = 3.84 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 4.85 (s,
2H, N-CH2), 6.30 (s, 1H, olefinic Ha), 6.87 (d, 2H, J = 9.0 Hz, ArH), 6.95 (d, 1H, J = 8.1
Hz, ArH), 7.08-7.11 (m, 2H, ArH), 7.14-7.15 (m, 1H, ArH), 7.20- 7.24
(m, 3H; 2H,
ArH and 1H, olefinic Hb, enol tautomer C), 7.25- 7.29 (m, 4H; 3H, ArH and OH), 7.37
(s, 1H, ArH), 7.43 (s, 1H, NH). Anal. Calcd. For C26H25NO5 (431.17): C, 72.37; H, 5.84;
N, 3.25. Found: C, 72.54; H, 5.90; N, 3.21.
3.1.1.6: (E)-N-Benzyl-2-(4-chlorobenzylidene)-4-(4-methoxyphenyl)-4-oxobutanamide
3f.
White crystals; (0.93 g, 74.0% yield); mp = 167-168oC; IR (KBr) max = broad band
1
3309 cm-1 (NH and OH), 1690 cm-1 (C=O benzoyl), 1645 cm-1 (C=O amide); H-NMR
(400 MHz, CDCl3)  (ppm) = 3.82 (s, 3H, OCH3), 4.84 (s, 2H, N-CH2), 6.14 (s, 1H,
olefinic Ha, enol tautomer C), 6.86 (d, 2H, J = 8.40 Hz, ArH), 7.08 (d, 2H, J = 8.00 Hz,
ArH), 7.19-7.24 (m, 3H, ArH), 7.25-7.27 (m, 3H, 2H, ArH and 1H, olefinic Hb), 7.36 (d,
1
2H, J = 8.0 Hz, ArH), 7.40 (s, 1H, NH), 7.56 (d, 2H, J = 8.4 Hz, ArH), H-NMR (400
MHz, CDCl3)  (ppm) = 3.36 (s, 2H, COCH2), 3.81 (s, 3H, OCH3), 3.94 (s, 1H, OH)
4.20 (d, 1H, 2J = 15.0 Hz, N-CH2), 4.71 (d, 1H, 2J = 15.0 Hz, N-CH2), 6.79-6.82 (m, 2H;
1H, ArH and 1H, olefinic Ha, enol tautomer B), 7.10-7.12 (m, 3H, ArH), 7.15-7.17 (m,
3H, ArH), 7.26-7.41 (m, 5H, ArH), 7.42 (s, 1H, ArH); EI-MS (70 eV) m/z (%) 419 (M+,
12), 401 (100), 310 (13), 282 (10), 281 (5), 275 (38), 247 (4), 232 (22), 172 (2), 158 (3),
135 (87), 91 (69), 83 (3), 77 (9). Anal. Calcd. For C25H22ClNO3 (419.13): C, 71.51; H,
5.28; N, 3.34. Found: C, 71.72; H, 5.25; N, 3.40.
21

3.1.2: General procedure for the synthesis of (E)-1-benzyl-3-benzylidene-5-(4-
methoxyphenyl)-1H-pyrrol-2(3H)-ones 4a-f.
Method A:-
Heating at reflux of the appropriate amide 3 (0.003 mol) in 6 M hydrochloric acid (20mL)
for 1 h, then The contents were left to cool and the obtained solid mass was filtered off,
washed with water and crystallized from ethanol to give compounds (E) 4a-f.
Method B:-
Heating at reflux a mixture of the appropriate amide 3 (0.003 mol) and hydroxylamine
hydrochloride (0.003 mol) in ethanol for 7 h, the reaction mixture was allowed to cool.
The obtained crystals were filtered off and crystallized from ethanol to give compounds
(E) 4a-f.
3.1.2.1: (E)-1-Benzyl-3-benzylidene-5-(4-methoxyphenyl)-1H-pyrrol-2(3H)-one 4a33
Red crystals; (method A; 0.74 g, 67.0% yield, method B; 0.84 g, 76.0% yield); mp = 142-
1
143°C; IR (KBr) max = 1694 cm-1 (C=O of pyrrolone); H-NMR (300 MHz, CDCl3) 
(ppm) = 3.84 (s, 3H, OCH3), 4.85 (s, 2H, N-CH2), 6.21 (s, 1H, olefinic H), 6.87 (d, 2H, J
= 8.7 Hz, ArH), 7.01 (d, 2H, J = 8.1Hz, ArH), 7.23-7.28 (m, 5H, ArH), 7.38-7.43 (m, 3H,
ArH), 7.50 (s, 1H, H-4 pyrrolone ring), 7.65 (d, 2H, J = 8.1 Hz, ArH); 13C NMR (75
MHz, CDCl3)  (ppm) = 160.7, 160.4, 149.7, 138.1, 136.3, 136.1, 132.4, 130.5, 130.4,
129.6, 129.1, 128.8, 127.4, 127.3, 114.8, 114.3, 100.2, 56.6, 44.8.
3.1.2.2: (E)-1-Benzyl-3-(4-methoxybenzylidene)-5-(4-methoxyphenyl)-1H-pyrrol-2-
(3H)-one 4b.
Red crystals; (method A; 0.65 g, 54.9% yield, method B; 0.94 g, 78.8% yield,); mp =
103-105°C; IR (KBr) max = 1690 cm-1 (C=O of pyrrolone); 1H-NMR (400 MHz, CDCl3)
 (ppm) = 3.77 (s, 3H, OCH3) , 3.78 (s, 3H, OCH3), 4.82 (s, 2H , N-CH2), 6.18 (s, 1H,
olefinic H), 6.68 (d, 2H, J = 8.8 Hz, ArH), 6.94 (d, 2H, J = 9.0 Hz, ArH), 7.07 (d, 2H, J =
9.0 Hz, ArH), 7.16-7.21(m, 2H, ArH) , 7.23-7.27 (m, 3H, ArH) , 7.46 (s, 1H, pyrrolone),
7.62 (d, 2H, J = 8.8 Hz, ArH); 13C NMR (75 MHz, CDCl3)  (ppm) = 160.5, 160.1,
159.9, 148.2, 137.7, 131.9, 131.8, 129.3, 129.2, 128.4, 128.3, 126.9, 125.9, 114.6, 113.4,
112.5, 99.9, 55.6, 55.5, 48.6; EI-MS (70 eV) m/z (%) 397 (M+, 100), 306 (36), 291 (18),
290 (3), 263 (23), 260 (4), 189 (7), 173 (1), 170 (1), 135 (29), 108 (1), 107 (4), 94 (1), 91
22

(68), 81 (1), 82 (1), 77 (9), 65 (14). For C26H23NO3 (397.17): C, 78.57; H, 5.83; N, 3.52.
Found: C, 78.74; H, 5.88; N, 3.59.
3.1.2.3: (E)-1-Benzyl-5-(4-methoxyphenyl)-3-(3,4,5-trimethoxybenzylidene)-1H-pyrrol-
2-(3H)-one 4c.
Red crystals; (method A; 0.75 g, 55.0% yield, method B; 0.69 g, 50.0% yield); mp = 101-
1
103°C; IR (KBr) max = 1687 cm-1 (C=O of pyrrolone); H-NMR (400 MHz, CDCl3) 
(ppm) = 3.81 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.91 (s, 6H, 2OCH3), 4.84 (s, 2H, N-
CH2), 6.15 (s, 1H, olefinic H), 6.24 (s, 2H, ArH), 6.89 -7.02 (m, 5H, ArH), 7.08 (d, 2H,
J = 6.4 Hz, ArH), 7.20 -7.40 (m, 2H, ArH), 7.41 (s, 1H, H-4 pyrrolone ring). Anal. Calcd.
For C28H27NO5 (457.19): C, 73.51; H, 5.95; N, 3.06. Found: C, 73.62; H, 5.96; N, 3.14.
3.1.2.4: (E)-1-Benzyl-3-(2-hydroxybenzylidene)- 5-(4-methoxyphenyl) -1H-pyrrol-2-
(3H)-one 4d.
Orange red crystals; (method A; 0.75 g, 65.5% yield, method B; 0.64 g, 55.5% yield); mp
1
= 222-224°C; IR (KBr) max = 1680 cm-1 (C=O of pyrrolone); H-NMR (400 MHz,
CDCl3)  (ppm) = 3.80 (s, 3H, OCH3), 4.88 (s, 2H, N-CH2), 6.21 (s, 1H, olefinic H), 6.84
(d, 2H, J = 7.6 Hz, ArH), 6.87-6.90 (m, 2H, Ar), 6.93 (s, 1H, OH), 7.01 (d, 2H, J = 7.6
Hz, ArH), 7.18-7.21 (m, 3H, ArH), 7.22-7.27 (m, 3H, ArH), 7.65 (d, 1H, ArH, J = 7.6
Hz), 8.33 (s, 1H, pyrrolone ring); 13C NMR (75 MHz, DMSO-d6)  (ppm) = 160.1, 160.1,
157.3, 148.1, 138.0, 131.3, 129.6, 129.1, 128.5, 127.8, 126.9, 126.3, 126.1, 123.1, 122.4,
119.7, 115.9, 114.2, 99.8, 55.3, 43.8. Anal. Calcd. For C25H21NO3 (383.15): C, 78.31; H,
5.52; N, 3.65. Found: C, 78.49; H, 5.61; N, 3.73.
Crystal and molecular structure of 4d (C25H21NO3)
The study of the 4d was undertaken to establish its three dimensional structure. All
diagrams and calculations were performed using maXus34 (Bruker Nonius, Delft and
MacScience, Japan). Atomic scattering factors from Waasmaier and Kirfel.35 Data
collection: KappaCCD. Cell refinement: HKL Scalepack (Otwinowski and Minor).36 Data
reduction: Denzo and Scalepak (Otwinowski & Minor).36 Programs used to solve
structure: SIR92 (Altomare et al).37 Programs used to refine structure: maXus (Mackay et
al.).31 Molecular graphics: ORTEP (Johnson).38 Software used to prepare material for
publication: maXus (Mackay et al.).34 The X-ray study of the pyrrolone 4d revealed that,
the pyrrolone 4d present in (E) configuration.
23

3.1.2.5: (E)-1-Benzyl-3-(4-hydroxy-3-methoxybenzylidene)-5-(4-methoxyphenyl)-1H-
pyrrol-2(3H)-one 4e.
Red crystals; (method A; 0.67 g, 56.2% yield, method B; 0.79 g, 64.0% yield); mp = 176-
1
177°C; IR (KBr) max = 1686 cm-1 (C=O of pyrrolone); H-NMR (400 MHz, CDCl3) 
(ppm) = 3.81 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 4.83 (s, 2H, N-CH2), 6.17 (s, 1H,
olefinic H), 6.87 (d, 2H, J = 6.8 Hz, ArH), 6.96 (d, 1H, J = 8.4 Hz, ArH), 7.09 (d, 2H, J =
6.8 Hz, ArH), 7.13-7.14 (m, 1H, ArH), 7.19-7.24 (m, 3H, ArH), 7.26-7.29 (m, 4H; 3H,
13
ArH and OH) 7.43 (s, 1H, H-4 pyrrolone ring); C NMR (75 MHz, CDCl3)  (ppm) =
160.1, 147.5, 147.4, 146.7, 138.0, 132.6, 129.6, 129.4, 128.5, 128.4, 127.5, 127.0, 124.7,
124.6, 123.9, 115.0, 114.1, 112.5, 99.9, 56.0, 55.3, 44.6. Anal. Calcd. C26H23NO4
(413.16): C, 75.53; H, 5.61; N, 3.39. Found: C, 75.65; H, 5.68; N, 3.44.
3.1.2.6: (E)-1-Benzyl-3-(4-chlorobenzylidene)-5-(4-methoxyphenyl)-1H-pyrrol-
2(3H)-one 4f.
Red crystals; (method A; 0.75 g, 62.6% yield, method B; 0.91 g, 75.3% yield); mp = 113-
1
115°C; IR (KBr) max = 1691 cm-1 (C=O of pyrrolone); H-NMR (400 MHz, CDCl3) 
(ppm) = 3.82 (s, 3H, OCH3), 4.84 (s, 2H , N-CH2), 6.14 (s, 1H, olefinic H), 6.87 (d, 2H, J
= 8.8 Hz, ArH), 7.08 (d, 2H, J = 8.0 Hz, ArH), 7.19-7.26 (m, 5H, ArH), 7.36 (d, 2H, J =
8.0 Hz, ArH), 7.40 (s, 1H, H-4 pyrrolone ring), 7.56 (d, 2H, J = 8.8 Hz, ArH); EI-MS (70
eV) m/z (%) 401 (M+, 72), 369 (4), 310 (5), 308 (24), 294 (4), 260 (5), 275 (45), 232
(20), 203 (17), 184 (7), 135 (10), 134 (10), 108 (1), 107 (7), 95 (10), 91 (100), 83 (4), 77
(13), 65 (18). Anal. Calcd. C25H20ClNO2 (401.12): C, 74.71; H, 5.02; N, 3.49. Found: C,
74.84; H, 5.08; N, 3.61.
3.1.3: General procedure for preparation of substituted-2-benzylidene-4-(4-
methoxyphenyl)-4-oxobutanehydrazide 5a-f.
To a solution of the appropriate furanone (0.003 mol) in absolute ethanol, hydrazine
monohydrate (0.006 mol) was added. The reaction mixture was left for 1 day at room
temperature. The solid mass that was formed was filtered off and recrystallized with
absolute ethanol afforded compounds 5a-f.
3.1.3.1: 2-Benzylidene-4-(4-methoxy phenyl)-4-oxobutanehydrazide 5a.
White powder; (0.42 g, 45.2% yield); mp = 144-145°C [reported39 176°C]; IR (KBr)

max = broad band 3200-3267 cm-1 (NHNH2), 1678 cm-1 (C=O benzoyl), 1656 cm-1 (C=O
24

1
2
hydrazide); H-NMR (400 MHz, DMSO-d6)
 (ppm) = 3.12 (d, 1H, J = 17.6 Hz,
2
COCH2), 3.21 (d, 1H, J = 17.6 Hz, COCH2), 3.75 (s, 3H, OCH3), 4.39 (s, 2H, NH2),
6.58 (s, H, olefinic H), 6.90 (d, 2H, J = 8.4 Hz, ArH), 7.28 (d, 2H, J = 8.4 Hz, ArH),
7.30-7.36 (m, 1H, ArH), 7.38(s, H, NH), 7.42 (d, 2H, J = 7.2 Hz, ArH), 7.50 (d, 2H, J =
7.2 Hz, ArH).
3.1.3.2: 2-(4-Methoxybenzylidene)-4-(4-methoxyphenyl)-4-oxobutanehydrazide 5b.
White powder; (0.57 gm, 55.9% yield); mp = 138-140°C; IR (KBr) max = broad band
1
3200-3319 cm-1 (NHNH2), 1682 cm-1 (C=O benzoyl), 1650 cm-1 (C=O hydrazide); H-
NMR (400 MHz, CDCl3)  (ppm) = 3.35 (d, 1H, 2J = 17.6 Hz, COCH2), 3.61 (d, 1H, 2J =
17.6 Hz, COCH2), 3.79 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.98 (s, 2H, NH2), 6.80 (d,
2H, J = 8.8 Hz, ArH), 6.88 (d, 2H, J = 8.8 Hz, ArH), 7.27 (m, 3H; 1H, ArH and 1H,
olefinic H), 7.34-7.37 (m, 3H; 2H, ArH and NH). Anal. Calcd. For C19H20N2O4 (340.14):
C, 67.05; H, 5.92; N, 8.23. Found: C, 67.39; H, 5.95; N, 8.40.
3.1.3.3: 4-(4-Methoxyphenyl)-4-oxo-2-(3,4,5- trimethoxybenzylidene)butanehydrzide
5c.
White powder; (0.48 g, 40.0% yield); mp = 93-95°C; IR (KBr) max = broad band 3200-
1
3318 cm-1 (NHNH2), 1679 cm-1 (C=O benzoyl), 1654 cm-1 (C=O hydrazide); H-NMR
2
2
(400 MHz, DMSO-d6)  (ppm) = 3.12 (d, 1H, J = 17.2 Hz, COCH2), 3.21 (d, 1H, J =
17.2 Hz, COCH2), 3.67 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.79 (s, 6H, 2OCH3), 4.36
(s, 2H, NH2), 6.57 (s, H, olefinic H), 6.80 (s, 2H, ArH), 6.91 (d, 2H, J = 8.7 Hz, ArH),
7.25 (s, 1H, NH), 7.28 (d, 2H, J = 8.7 Hz, ArH); EI-MS (70 eV) m/z (%) 400 (M+, 57),
308 (24), 275 (45), 232 (20), 203 (17), 135 (10), 91 (100), 77 (13). Anal. Calcd. For
C21H24N2O6 (400.16): C, 62.99; H, 6.04; N, 7.00. Found: C, 63.08; H, 6.09; N, 7.13.
3.5.4. 2-(2-Hydroxybenzylidene)-4-(4-methoxyphenyl)-4-oxobutanehydrazide 5d.
Buff powder; (0.39 g, 40.0% yield); mp = 129-130°C; IR (KBr) max = Broad band 3200-
3317 cm-1 (NHNH2 and OH), 1681 cm-1 (C=O benzoyl), 1650 cm-1 (C=O hydrazide); 1H-
2
NMR (400 MHz, DMSO-d6)  (ppm) = 3.07 (d, 1H, J = 16.0 Hz, COCH2), 3.18 (d, 1H,
2J = 16.0 Hz, COCH2), 3.77 (s, 3H, OCH3), 4.34 (s, 2H, NH2), 6.54 (s, H, olefinic H),
6.90-6.92 (m, 2H, ArH), 6.95-6.97 (m, 3H; 2H, ArH and OH), 7.23 (s, 1H, NH), 7.27 (d,
2H, J = 8.0 Hz, ArH), 7.45 (d, 2H, J = 8.0 Hz, ArH). Anal. Calcd. For C18H18N2O4
(326.13): C, 66.25; H, 5.56; N, 8.58. Found: C, 66.39; H, 5.63; N, 8.74.
25

3.5.5. 2-(4-Hydroxy-3-methoxybenzylidene)-4-(4-methoxyphenyl)-4-oxobutane-
hydrazide 5e.
Pale yellow powder; (0.45 g, 42.0% yield); mp = 120-122°C; IR (KBr) max = broad
band 3200-3276 cm-1 (NHNH2 and OH), 1671 cm-1 (C=O benzoyl), 1647 cm-1 (C=O
1
2
hydrazide); H-NMR (400 MHz, CDCl3)  (ppm) = 3.17 (d, 1H, J = 17.6 Hz, COCH2),
2
3.32 (d, 1H, J = 17.6 Hz, COCH2), 3.79 (s, 3H, OCH3), 3.83 (s, 5H, OCH3 and NH2),
6.83 (s, H, olefinic H), 6.85-6.95 (m, 4H, ArH), 7.27 (s, 2H, OH and NH), 7.34-7.36 (m,
3H, ArH); EI-MS (70 eV) m/z (%) 457 (M+1, 69), 292 (83), 201 (75), 135 (10), 110 (100),
78 (78). Anal. Calcd. For C19H20N2O5 (356.14): C, 64.04; H, 5.66; N, 7.86. Found: C,
64.17; H, 5.72; N, 7.94.
3.5.6. 2-(4-Chlorobenzylidene)-4-(4-methoxyphenyl)-4-oxobutanehydrazide 5f.
White powder; (0.44 g, 42.80% yield); mp = 99-101°C; IR (KBr) max = broad band
1
3200-3311 cm-1 (NHNH2), 1681 cm-1 (C=O benzoyl), 1654 cm-1 (C=O hydrazide); H-
NMR (400 MHz, CDCl3)  (ppm) = 3.17 (d, 1H, 2J = 18.0 Hz, COCH2), 3.32 (d, 1H, 2J =
18.0 Hz, COCH2), 3.84 (s, 3H, OCH3), 3.95 (s, 2H, NH2), 6.68 (m, 2H; 1H, ArH and
olefinic H), 7.05 (d, 1H, J = 8.4 Hz, ArH), 7.19-7.35 (m, 5H; 4H, ArH and 1H, NH),
7.37-7.51 (m, 1H, ArH), 7.70 (d, 1H, J = 8.4 Hz, ArH); EI-MS (70 eV) m/z (%) 344 (M+,
1), 327 (100), 311 (34), 297 (10), 233 (20), 203 (27), 135 (77), 92 (15), 76 (37). Anal.
Calcd. For C18H17ClN2O3 (344.09): C, 62.70; H, 4.97; N, 8.12. Found: C, 62.83; H, 5.01;
N, 8.25.
3.6. General procedure for synthesis of substituted 3-benzylidene-1-
((benzylidene)amino)-5-(4-methoxyphenyl)-1H-pyrrol-2(3H)-one (6a, 6f & 6g-j).
Preparation of 6a, 6f:- Heating at reflux a mixture of the appropriate hydrazide 5a or 5f
(0.001 mol) and p-methoxybenzaldehyde (0.001 mol) in ethanol for 13-24 h. The solvent
was evaporated under reduced pressure and the obtained residue was crystallized from
ethanol affording compound 6a or 6f.
Preparation of 6g-j: - A mixture of the hydrazide 5f (0.001 mol) and different aromatic
aldehyde (0.001 mol) in ethanol was left in room temperature for 2 days. The obtained
precipitate was filtered off and washed to give compounds 6g-j.
26

3.6.1. 3-(4-Methoxybenzylidene)-1-(-(4-methoxybenzylidene)amino)-5-(4-
methoxyphenyl)-1H-pyrrol-2(3H)-one 6a.
Orange powder; (0.23 g, 13 h, 51.7% yield); mp =191-193°C; IR (KBr) max = 1691 cm-1
(C=O of pyrrolone); 1H-NMR (400 MHz, CDCl3)  (ppm) = 3.82 (s, 3H, OCH3), 3.83 (s,
3H, OCH3), 3.85 (s, 3H, OCH3), 6.33 (s, 1H, olefinic H), 6.84 (d, 2H, J = 8.8 Hz, ArH),
6.93-6.96 (m, 4H, ArH), 7.37 (s, 1H, H-4 pyrrolone ring), 7.62 (d, 2H, J =5.2 Hz, ArH),
7.64 (d, 2H, J = 5.2 Hz, ArH), 7.66 (d, 2H, J = 8.8 Hz, ArH), 9.57 (s, 1H, CH=N); 13C
NMR (75 MHz, CDCl3)  (ppm) = 161.8, 161.0, 159.6, 159.5, 152.8, 147.2, 132.3, 129.8,
128.2, 127.8, 126.2, 122.6, 114.1, 113.9, 113.0, 103.3, 103.0, 98.3, 55.2, 55.1; EI-MS (70
eV) m/z (%): 440 (M+, 60), 379 (12), 322 (13), 308 (14), 307 (13), 286 (13), 248 (18),
186 (13), 171 (19), 141 (17), 144 (16), 135 (22), 133 (16), 121 (21), 120 (14), 108 (21),
94 (13), 80 (100), 79 (25), 77 (15), 64 (56), 55 (64). Anal. Calcd. For C27H24N2O4
(440.17): C, 73.62; H, 5.49; N, 6.36. Found: C, 73.70; H, 5.51; N, 6.45.
3.6.2. 3-(4-Chlorobenzylidene)-1-((4-methoxybenzylidene)amino)-5-(4-methoxyphenyl)-
1H-pyrrol-2(3H)-one 6f.
Orange crystal; (0.23 g, 24 h, 51.7% yield); mp = 184-186ºC; IR (KBr) max = 1694 cm-1
(C=O of pyrrolone); 1H-NMR (300 MHz, CDCl3)  (ppm) = 3.86 (s, 3H, OCH3), 3.89 (s,
3H, OCH3), 6.30 (s, 1H, olefinic H), 6.91 (d, 2H, J = 8.7 Hz, ArH), 6.96 (d, 2H, J = 9.0
Hz, ArH), 7.36 (s, 1H, H-4 pyrrolone ring), 7.40 (d, 2H, J = 8.4 Hz, ArH), 7.58 (d, 2H, J
= 8.4 Hz, ArH), 7.66 (d, 2H, J = 8.7 Hz, ArH), 7.69 (d, 2H, J = 9.0 Hz, ArH), 9.56 (s,
1H, CH=N); EI-MS (70 eV) m/z (%): 446 (M+2, 100), 311 (93), 296 (16), 268 (17), 247
(4), 205 (6), 189 (16), 148 (10), 134 (35), 107 (5), 79 (2), 91 (20), 77 (18), 64 (13). Anal.
Calcd. For C26H21ClN2O3 (444.12): C, 70.19; H, 4.76; N, 6.30. Found: C, 70.34; H, 4.82;
N, 6.43.
3.6.3. 1-(Benzylideneamino)-3-(4-chlorobenzylidene)-5-(4-methoxyphenyl)-1H-pyrrol-
2(3H)-one 6g.
Orange crystal; (0.16 g, 39.0% yield); mp = 146-148ºC; IR (KBr) max = 1697 cm-1 (C=O
of pyrrolone); 1H-NMR (400 MHz, CDCl3)  (ppm) = 3.90 (s, 3H, OCH3), 6.33 (s, 1H,
olefinic H), 6.99 (d, 2H, J = 8.0 Hz, ArH), 7.28 (s, 1H, H-4 pyrrolone ring), 7.39-7.44
(m, 5H, ArH), 7.61 (d, 2H, J = 8.0 Hz, ArH), 7.72 (d, 4H, J = 7.2 Hz, ArH), 9.70 (s, 1H,
27

CH=N). Anal. Calcd. For C25H19ClN2O2 (414.11): C, 72.37; H, 4.62; N, 6.75. Found: C,
72.45; H, 4.65; N, 6.83.
3.6.4. 3-(4-Chlorobenzylidene)-1-((3,4-dimethoxybenzylidene)amino)-5-(4-
methoxyphenyl)-1H-pyrrol-2(3H)-one 6h.
Red crystal; (0.25 g, 51.7% yield); mp = 176-178ºC; IR (KBr) max = 1702 cm-1 (C=O of
1
pyrrolone); H-NMR (400 MHz, CDCl3)  (ppm) = 3.89 (s, 3H, OCH3), 3.92 (s, 3H,
OCH3), 3.95 (s, 3H, OCH3), 6.33 (s, 1H, olefinic H), 6.90 (d, 1H, J = 8.4 Hz, ArH), 6.97
(d, 2H, J = 8.4 Hz, ArH), 7.29 (s, 1H, H-4 pyrrolone ring), 7.36 (d, 2H, J = 8.8 Hz, ArH),
7.43 (d, 2H, J = 8.4 Hz, ArH), 7.61 (d, 2H, J = 8.4 Hz, ArH), 7.73 (d, 2H, J = 8.8 Hz,
ArH), 9.58 (s, 1H, CH=N). Anal. Calcd. For C27H23ClN2O4 (474.13): C, 68.28; H, 4.88;
N, 5.90. Found: C, 68.39; H, 4.86; N, 6.04.
3.6.5. 3-(4-Chlorobenzylidene)-5-(4-methoxyphenyl)-1-((3,4,5-trimethoxy-
benzylidene)amino)-1H-pyrrol-2(3H)-one 6i.
Red needle crystals; (0.26 g, 52.0% yield); mp = 196-198ºC; IR (KBr) max = 1707 cm-1
(C=O of pyrrolone); 1H-NMR (400 MHz, CDCl3)  (ppm) = 3.89 (s, 6H, 2OCH3), 3.91 (s,
3H, OCH3), 3.98 (s, 3H, OCH3), 6.35 (s, 1H, olefinic H), 6.97-6.99 (m, 2H, ArH), 7.28
(s, 2H, ArH), 7.39 (s, 1H, H-4 pyrrolone ring), 7.43 (d, 2H, J = 8.0 Hz, ArH), 7.62 (d,
2H, J = 8.0 Hz, ArH), 7.73 (d, 2H, J =8.4 Hz, ArH), 9.61 (s, 1H, CH=N). Anal. Calcd.
For C28H25ClN2O5 (504.15): C, 66.60; H, 4.99; N, 5.55. Found: C, 66.75; H, 5.03; N,
5.67.
3.6.6. 3-(4-Chlorobenzylidene)-1-((4-chlorobenzylidene)amino)-5-(4-methoxy- phenyl)-
1H-pyrrol-2(3H)-one 6j.
Red crystals; (0.25 gm, 55.0 % yield); mp = 211-212ºC; IR (KBr) max = 1697 cm-1 (C=O
1
of pyrrolone); H-NMR (400 MHz, CDCl3)  (ppm) = 3.91 (s, 3H, OCH3), 6.34 (s, 1H,
olefinic H), 6.99 (d, 2H, J = 7.2 Hz, ArH), 7.39 (s, 1H, pyrrolone ring), 7.43-7.49 (m, 4H,
ArH), 7.61-7.63 (m, 2H, ArH), 7.64 (d, 2H, J = 8.0 Hz, ArH), 7.68 (d, 2H, J = 8.0 Hz,
ArH), 9.70 (s, 1H, CH=N). Anal. Calcd. For C25H18Cl2N2O2 (448.07): C, 66.83; H, 4.04;
N, 6.23. Found: C, 66.98; H, 4.11; N, 6.37.
28

3.7. General procedure for preparation of substituted 4-(benzyl)-6-(4-
methoxyphenyl)pyridazin-3(2H)-one 7a-f.
Method A: - Heating at reflux of the appropriate hydrazide 5 (0.001mol) in 6 M
hydrochloric acid (20 mL) for 1 h, the reaction mixture was cooled, filtered off, and
recrystallized afforded the corresponding pyridazinone 7a-f.
Method B: - Heating at reflux a mixture of the appropriate furanone 2 (0.001 mol) and
hydrazinemonohydrate (100%, 0.002 mol) in ethanol for 4-24 h, the solvent was
evaporated under reduced pressure. The obtained residue was recrystallized from ethanol
afforded compounds 7a-f.
3.7.1. 4-(Benzyl)-6-(4-methoxyphenyl)pyridazin-3(2H)-one 7a.
Buff powder; (method A; 0.19 g, 65.0% yield, method B; 10 h, 0.21 g, 70.6% yield); mp
1
= 191-193°C; IR (KBr) max = 1648 cm-1 (C=O of pyridazinone); H-NMR (400 MHz,
CDCl3)  (ppm) = 3.84 (s, 3H, OCH3), 4.12 (s, 2H, CH2), 6.85 (d, 2H, J = 8.8 Hz, ArH),
7.23-7.24 (m, 2H, ArH), 7.27-7.29 (m, 2H; 1H, ArH and 1H, H-5 pyridazinone ring),
7.33-7.37 (m, 2H, ArH), 7.58 (d, 2H, J = 8.8 Hz, ArH), 11.97 (s, 1H, NH). Anal. Calcd.
For C18H16N2O2 (292.12): C, 73.95; H, 5.52; N, 9.58. Found: C, 74.03; H, 5.59; N, 9.67.
3.7.2. 4-(4-Methoxybenzyl)-6-(4-methoxyphenyl)pyridazin-3(2H)-one 7b.
White crystals, (method A; 0.19 g, 60.0% yield, method B; 6 h, 0.24 g, 75.1% yield); mp
1
= 201-203°C; IR (KBr) max= 1650 cm-1 (C=O of pyridazinone); H-NMR (400 MHz,
CDCl3)  (ppm) = 3.82 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.94 (s, 2H, CH2), 6.90-6.94
(m, 4H, ArH), 7.21 (d, 2H, J = 8.4 Hz, ArH), 7.26 (s, 1H, H-5 pyridazinone ring), 7.61
13
(d, 2H, J = 8.4 Hz, ArH), 12.09 (s, 1H, NH); C NMR (75 MHz, DMSO-d6)  (ppm) =
160.7, 160.2, 160.2, 158.0, 143.9, 143.2, 130.2, 127.7, 127.4, 127.1, 114.4, 114.0, 55.4,
55.1, 34.4; EI-MS (70 eV) m/z (%): 323 (M+1, 100), 307 (43), 290 (1), 249 (4), 189 (9),
186 (1), 171 (2), 150 (6), 146 (35), 134 (11), 132 (22), 110 (4), 108 (7), 106 (2), 95 (1),
94 (2), 92 (10), 80 (2), 77 (22). Anal. Calcd. For C19H18N2O3 (322.13): C, 70.79; H, 5.63;
N, 8.69. Found: C, 70.92; H, 5.67; N, 8.84.
3.7.3. 6-(4-Methoxyphenyl)-4-(3,4,5-trimethoxybenzyl)pyridazin-3(2H)-one 7c.
White needle crystals; (method A; 0.27 g, 70.0% yield, method B; 5 h, 0.30 g, 78.7%
1
yield); mp = 199-201°C; IR (KBr) max = 1655 cm-1 (C=O of pyridazinone); H-NMR
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