Design, synthesis and molecular modeling of new 4-phenylcoumarin derivatives as tubulin polymerization inhibitors targeting MCF-7 breast cancer cells

Article abstract of DOI:10.1016/j.bmc.2018.05.022

A new set of 4-phenylcoumarin derivatives was designed and synthesized aiming to introduce new tubulin polymerization inhibitors as anti-breast cancer candidates. All the target compounds were evaluated for their cytotoxic effects against MCF-7 cell line, where compounds 2f, 3a, 3b, 3f, 7a and 7b, showed higher cytotoxic effect (IC₅₀ = 4.3–21.2 μg/mL) than the reference drug doxorubicin (IC₅₀ = 26.1 μg/mL), additionally, compounds 1 and 6b exhibited the same potency as doxorubicin (IC₅₀ = 25.2 and 28.0 μg/mL, respectively). The thiazolidinone derivatives 3a, 3b and 3f with potent and selective anticancer effects towards MCF-7 cells (IC₅₀ = 11.1, 16.7 and 21.2 μg/mL) were further assessed for tubulin polymerization inhibition effects which showed that the three compounds were potent tubulin polymerization suppressors with IC₅₀ values of 9.37, 2.89 and 6.13 μM, respectively, compared to the reference drug colchicine (IC₅₀ = 6.93 μM). The mechanistic effects on cell cycle progression and induction of apoptosis in MCF-7 cells were determined for compound 3a due to its potent and selective cytotoxic effects in addition to its promising tubulin polymerization inhibition potency. The results revealed that compound 3a induced cell cycle cessation at G2/M phase and accumulation of cells in pre-G1 phase and prevented its mitotic cycle, in addition to its activation of caspase-7 mediating apoptosis of MCF-7 cells. Molecular modeling studies for compounds 3a, 3b and 3f were carried out on tubulin crystallography, the results indicated that the compounds showed binding mode similar to the co-crystalized ligand; colchicine. Moreover, pharmacophore constructed models and docking studies revealed that thiazolidinone, acetamide and coumarin moieties are crucial for the activity. Molecular dynamics (MD) studies were carried out for the three compounds over 100 ps. MD results of compound 3a showed that it reached the stable state after 30 ps which was in agreement with the calculated potential and kinetic energy of compound 3a.

Full text of DOI:10.1016/j.bmc.2018.05.022

Accepted Manuscript
Design, synthesis and molecular modeling of new 4-phenylcoumarin derivatives
as tubulin polymerization inhibitors targeting MCF-7 breast cancer cells
Rasha Z. Batran, Asmaa F. Kassem, Eman M. Abbas, Samia A. El Seginy,
Marwa M. Mounier
PII:
S0968-0896(18)30745-4
https://doi.org/10.1016/j.bmc.2018.05.022
BMC 14364
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
17 April 2018
13 May 2018
15 May 2018
Please cite this article as: Batran, R.Z., Kassem, A.F., Abbas, E.M., El Seginy, S.A., Mounier, M.M., Design,
synthesis and molecular modeling of new 4-phenylcoumarin derivatives as tubulin polymerization inhibitors
targeting MCF-7 breast cancer cells, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2018.05.022
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Design, synthesis and molecular modeling of new 4-phenylcoumarin derivatives as tubulin
polymerization inhibitors targeting MCF-7 breast cancer cells
Rasha Z. Batran,a Asmaa F. Kassem,b* Eman M. Abbas,b Samia A. El Seginy,c
Marwa M. Mounierd
a Chemistry of Natural Compounds Department, Pharmaceutical and Drug Industries Research
Division, National Research Centre, 33 El Bohouth St., Dokki, Giza, P.O. Box 12622, Egypt
b
Chemistry of Natural and Microbial Products Department, Pharmaceutical and Drug
Industries Research Division, National Research Centre, 33 El Bohouth St., Dokki, Giza, P.O.
Box 12622, Egypt
c
Green Chemistry Department, Chemical Industries Research Division, National Research
Centre, 33 El Bohouth St., Dokki, Giza, p. o. box 12622, Egypt
d
Pharmacognosy Department, Drug Bioassay-Cell Culture Laboratory, Pharmaceutical and
Drug Industries Research Division, National Research Centre, 33 El Bohouth St., Dokki, Giza,
P.O. Box 12622, Egypt
Corresponding author: asmaa.kassem@yahoo.com
Abstract
A new set of 4-phenylcoumarin derivatives was designed and synthesized aiming to
introduce new tubulin polymerization inhibitors as anti-breast cancer candidates. All the target
compounds were evaluated for their cytotoxic effects against MCF-7 cell line, where compounds
2f, 3a, 3b, 3 f, 7a and 7b, showed higher cytotoxic effect (IC50=4.3-21.2 µg/mL) than the
reference drug doxorubicin (IC50= 26.1 µg/mL), additionally, compounds 1 and 6b exhibited the
same potency as doxorubicin (IC50= 25.2 and 28.0 µg/mL, respectively). The thiazolidinone
derivatives 3a, 3b and 3 f with potent and selective anticancer effects towards MCF-7 cells (IC50
= 11.1, 16.7 and 21.2 µg/mL) were further assessed for tubulin polymerization inhibition effects
which showed that the three compounds were potent tubulin polymerization suppressors with
IC50 values of 9.37, 2.89 and 6.13 µM, respectively, compared to the reference drug colchicine
(IC50= 6.93 µM). The mechanistic effects on cell cycle progression and induction of apoptosis in
MCF-7 cells were determined for compound 3a due to its potent and selective cytotoxic effects
1

in addition to its promising tubulin polymerization inhibition potency. The results revealed that
compound 3a induced cell cycle cessation at G2/M phase and accumulation of cells in pre-G1
phase and prevented its mitotic cycle, in addition to its activation of caspase-7 mediating
apoptosis of MCF-7 cells. Molecular modeling studies for compounds 3a, 3b and 3 f were
carried out on tubulin crystallography, the results indicated that the compounds showed binding
mode similar to the co-crystalized ligand; colchicine. Moreover, pharmacophore constructed
models and docking studies revealed that thiazolidinone, acetamide and coumarin moieties are
crucial for the activity. Molecular dynamics (MD) studies were carried out for the three
compounds over 100Ps. MD results of compound 3a showed that it reached the stable state after
30Ps which was in agreement with the calculated potential and kinetic energy of compound 3a.
Keywords: 4-phenylcoumarins; thiazole; MCF-7; tubulin polymerization; apoptosis; molecular
modeling.
1.
Introduction
The microtubule network is a fundamental element of the cytoskeleton of eukaryotic cells
which is involved in various biological processes. Microtubules are assembled from
heterodimers of α and β tubulin.1 The cycle of microtubule assembly and disassembly is
basically regulated by microtubule-associated proteins as MAP1A, MAP1A B, stathmin or tau.2-5
Colchicine, vinca-alkaloids and taxane are examples of therapeutic anticancer agents targeting
the microtubule cytoskeleton.6-8
The discovery of colchicine binding to tubulin was a crucial step in the development of
antimitotic drugs.9 Among the different antimitotic agents that inhibit tubulin polymerization via
interaction with the colchicine binding site, combretastatin derivatives represent the most widely
investigated class of antimitotic agents since the discovery of combretastatin A-4 (CA-4), a
natural antimitotic and anti-angiogenic agent isolated from the bark of the African willow tree
Combretum caffrum (Fig.1A). 10-13
The development of new antimitotic agents has led to new approaches of cancer
chemotherapy and advanced knowledge of microtubule biochemistry and pharmacology.
Recently, several studies have focused on the advanced clinical trial development of new
candidates as combretastatin AVE 8062A, combretastatin A-4 phosphate (CA-4P) and
combretastatin A1 diphosphate (OXi 4503) (Fig.1B).14-17
2

Structural modifications of CA-4 lead to the identification of new combretastatin-A4
thiazole/ thiazolidinone hybrids as tubulin targeting antimitotic agents acting through the
colchicine binding site of tubulin and causing disruption in microtubule assembly and dynamics.
(Fig.1C).18-21
On the other hand, coumarins are a class of naturally occurring compounds with diverse
pharmacological effects, especially in the field of anticancer therapy.22-28 4-Arylcoumarins,
known as neoflavones have been reported for their antimitotic effects.29-31 Due to structural
similarity, 4-arylcoumarins share the colchicine binding domain with the natural antimitotic
agent combretastatin A-4.30,32 Moreover, the rigid conformation of 4-phenylcoumarin backbone
avoids the biological inactivation resulting from the inherent conformational instability of
combretastatin scaffold due to the cis-trans isomerization about the ethylene linker (Fig. 1D).33,34
Hybridization of two or more scaffolds in one candidate molecule represents efficient
hybrid pharmacophore approach for the development of new antitumor agents. Previous structure
activity relationship studies of 4-arylcoumarin as tubulin inhibitors revealed that direct
substitution on ring A, particularly with methoxy groups, has an essential effect on tubulin
polymerization and antitumor effect.29-31 However, no reports have considered the conjugation of
the 4-phenylcoumarin scaffold and other bioactive hetrocyclic moieties.
With this aspect we focused on the synthesis of a new series of 4-phenylcoumarin
analogues hybridized with the heterocyclic thiazole and thiazolidinone moieties through
acetamide or acetohydrazide linker. All target compounds were assessed for their cytotoxic
effects against human breast cancer cell line MCF-7 and the potent antiproliferative candidates
were further examined as tubulin polymerization suppressors. Furthermore, the mechanistic
effects of the most active derivative were evaluated regarding cell cycle analysis, apoptosis and
caspase-7 detection. Molecular modeling study was carried out for the promising compounds to
explore the plausible binding modes in the tubulin active site and molecular dynamics simulation
was also performed to evaluate their binding stability.
3

O
O
A
O
O
O
A
O
B
O
O
OH
NH
CH3
O
O
Combretastatin A-4
O
Colchicine
B
O
O
O
P
O
O
OH
A
A
A
O
O
O
P
O
OH
B
B
B
OH
O
O
O
O
O
NH2
P
OH
OH
O
O
O
OH
CA-4P
OXi 4503
AVE 8062A
C
O
O
O
O
O
O
S
S
O
O
O
S
N
N
NH2
N
S
O
O
O
D
O
O
B
O
B
O
B
NH
OH
OH
OH
F
B
B
O
A
O
O
O
A
A
A
A
O
O
O
O
O
O
Cl
O
O
Cl
O
O
Fig.1. Tubulin polymerization inhibitors. A) Structures of known inhibitors, B) Structures of inhibitors
in clinical trials, C) Combretastatin-A4 thiazole/ thiazolidinone hybrids,
D) Structures of 4-arylcoumarin derivatives.
2.
2.1. Chemistry
The preparation of 4-phenyl-7-oxycoumarin derivatives 1, 2a-f, 3a-f, 4a,b, 5a,b, 6a,b,
Results and Discussion
7a,b, 8a,b, 9a,b and 10a,b was outlined in Schemes 1-3. The Schiff’s bases 2a-f were
synthesized through the reaction of hydrazide 1 with different aromatic aldehydes, namely; 4-
fluorobenzaldehyde, 4-chlorobenzaldehyde, 4-bromobenzaldehyde, 4-anisaldehyde, 3,4-
4

dimethoxybenzaldehyde and /or 3,4,5-trimethoxybenzaldehyde. Cyclocondensation of the
synthesized Schiff’s bases 2a-f with thioglycolic acid in refluxing benzene afforded the
corresponding thiazolidinone derivatives 3a-f (Scheme 1).
H
N
H
N
i)
N
O
O
O
H2N
O
O
O
(1)
O
O
(2a-f)
R
ii)
R
H
N
N
O
O
O
S
O
(3a-f)
O
R=4-F, 4-Cl,4-Br, 4-OCH3, 3,4-(OCH3)2, 3,4,5-(OCH3)3
Scheme 1. i) ArCHO, EtOH, CH3COOH, reflux, ii) HSCH2COOH, benzene, reflux
Condensation reaction of the hydrazide 1 with methyl isothiocyanate and/or ethyl
isothiocyanate yielded the thiosemicarbazide derivatives 4a,b. Heterocyclization of the prepared
thiosemicarbazide derivatives 4a,b with the appropriate α-halocarbonyl compounds, namely;
chloroacetone, 3-chloroacetylacetone and/ or phenacyl bromide, in refluxing ethanol and
catalytic amount of anhydrous sodium acetate afforded the corresponding thiazoline derivatives
5a,b -7a,b. Similarly, the thiazolidinones 8a,b were synthesized via refluxing 4a,b with ethyl
bromoacetate in the presence of anhydrous sodium acetate. These reactions were assumed to
proceed through S-alkylation reaction followed by dehydration or loss of an alcohol molecule
(Scheme 2).
5

S
H
N
H
N
R
i)
N
H
N
H
O
O
O
H2N
O
O
O
(1)
O
O
(4a,b)
R=CH3, C2H5
ii)
v)
S
S
H
N
H
N
iii)
O
iv)
N
N
N
O
O
O
N
O
O
O
R
R
O
O
(5a,b)
(8a,b)
H3COC
S
S
H
N
H
N
N
N
N
O
O
O
N
O
O
O
R
R
O
O
(6a,b)
7a,b)
Scheme 2. i) RNCS, EtOH, reflux, ii) CH3COCH2Cl, CH3COONa, EtOH, reflux, iii) CH3COCH(Cl)COCH3,
CH3COONa, EtOH, reflux, iv) PhCOCH2Br, CH3COONa, EtOH, reflux, v) CH3CH2OCOCH2Br, CH3COONa,
EtOH, reflux.
On the other hand, the reaction of the key intermediates 4a,b with ethyl-2-
chloroacetoacetate and ethyl-2-bromopropionate in absolute ethanol in the presence of anhydrous
sodium acetate proceeded via S-alkyaltion reaction without further dehydration or loss of alcohol
molecule to afford the unexpected open chain derivatives 9a,b and 10a,b, respectively (Scheme
1
3). The structures of the newly synthesized target compounds were elucidated by IR, H NMR,
13C NMR and EI-MS (Experimental section).
6

S
H
N
R
N
H
N
H
O
O
O
O
(4a,b)
R=CH3, C2H5
C2H5
O
N
i)
ii)
O
S
S
H
N
H
N
O
N
N
O
O
O
N
O
O
O
R
O
R
O
R
R
S
NH
O
O
O
NH
O
O
O
H
N
H
N
O
S
N
N
O
O
O
O
O
O
(9a,b)
(10a,b)
Scheme 3. i) CH3COCH(Cl)COOC2H5, CH3COONa, EtOH, reflux, ii) BrCH(CH3)COOC2H5,
CH3COONa, EtOH, reflux.
2.2.
Biology
2.2.1. In vitro anticancer activity against breast cancer cell line (MCF-7)
All the synthesized compounds were evaluated for their antiproliferative effect on human
breast carcinoma cell line MCF-7 at 100 µg/mL utilizing MTT assay (Fig.2). The compounds
which showed percentage of inhibition higher than 70% were further assessed for determination
of median growth inhibitory concentration (IC50) and doxorubicin was used as the standard drug
as shown in Table 1. Moreover, the active compounds were also screened for their cytotoxic
effects on human normal skin fibroblasts cell line BJ-1 at 100 µg/mL (Fig.3).
7

Cytotoxicity on MCF-7
120
100
80
60
40
20
0
1
2a 2b 2c 2d 2e 2f 3a 3b 3c 3d 3e 3f 4a 4b 5a 5b 6a 6b 7a 7b 8a 8b 9a 9b 10a10b
Fig.2. In vitro screening of the antiproliferative activities of compounds against human breast carcinoma cell line
(MCF-7). Preliminary concentration for screening was 100μg/mL. Each result is a mean of 3 replicate samples and
values are represented as % inhibition ( standard deviation)
.
Cytotoxicity on BJ-1
120
100
80
60
40
20
0
1
2b
2f
3a
3b
3c
3f
5a
5b
6a
6b
7a
7b
9a
9b 10a
Fig.3. In vitro cytotoxic screening of the active compounds against human normal skin cell line (BJ-1). Preliminary
concentration for screening was 100µg/mL. Each result is the mean of 3 replicate samples and values are represented
as % inhibition ( standard deviation)
The results revealed that the tested compounds showed IC50 values ranging from 4.3-78
µg/mL compared to doxorubicin (IC50 = 26.1 µg/mL) towards MCF-7 cells. The 3,4,5-
trimethoxyphenyl Schiff׳s base 2f, phenyl thiazoles 7a and 7b and the thiazolidinones 3a, 3b
and 3f showed remarkably potent anticancer effects (IC50 values of 4.3, 9.3, 10.6, 11.1, 16.7 and
8

21.2 µg/mL, respectively), higher than the reference doxorubicin (IC50 = 26.1 µg/mL). On the
other hand, compounds 1, 6b and 9a showed comparable potency to that of the reference
doxorubicin with IC50 values of 25.2, 28.0 and 30.6 µg/mL, respectively.
Table 1. IC50 of the active cytotoxic compounds exhibiting more than 70% cytotoxicity on MCF-7.
Compounds
1
IC50 (µg/mL)*
25.2 2.1
38.1 3.2
4.3 4.3
2b
2f
3a
11.1 1.9
16.7 2.3
42.0 4.8
21.2 3.2
36.9 1.4
78.0 5.1
42.0 4.4
28.0 1.8
9.3 4.2
3b
3c
3f
5a
5b
6a
6b
7a
10.6 3.2
30.6 1.3
41.9 4.9
37.0 3.5
26.1 1.3
7b
9a
9b
10a
doxorubicin
*Values of IC50 ( standard deviation) are calculated using SPSS statistical program.
Furthermore, it was obvious that the thiazolidinones 3a, 3b and 3f with promising
cytotoxic effect against MCF-7 revealed low cytotoxicity on normal BJ-1 cells with %
inhibition= 13.2, 26.9 and 16.8%, respectively, which makes them interesting candidates for
further biological evaluation (Figure 3).
2.2.2. Structure activity relationship
The 3,4,5-trimethoxyphenyl Schiff׳s base 2f was the most active derivative among all the
synthesized compounds with IC50 of 4.3 µg/mL. It showed higher anticancer effect than the
parent hydrazide compound (IC50 = 25.2 µg/mL). In addition, the 3,4,5-trimethoxyphenyl Schiff׳s
base 2f displayed higher percentage of inhibition of MCF-7 than the 3,4-dimethoxyphenyl and
the 4-methoxyphenyl derivatives 2e and 2d at 100 µg/mL (% inhibition= 90.7, 48.9 and 46.3%,
respectively). While the 4-chlorophenyl Schiff׳s base 2b showed moderate anticancer potency
(IC50 =38.1 µg/mL), it displayed higher percentage of MCF-7 inhibition at 100 µg/mL than the 4-
9

bromophenyl and 4-fluorophenyl congeners 2c and 2a (% inhibition= 76.5, 67.8 and 65.5%,
respectively).
The 3,4,5-trimethoxyphenyl thiazolidinone compound 3f showed lower anticancer effect
than the parent Schiff׳s base 2f (IC50=21.2 µg/mL). On the other hand the trimethoxyphenyl
thiazolidinone 3f displayed higher percentage of MCF-7 inhibition at 100 µg/mL than the 3,4-
dimethoxyphenyl and 4-methoxyphenyl derivatives 3e and 3d (% inhibition = 86.4, 63.3 and
64.4%, respectively). It could be observed that increasing the number of methoxy groups is
parallel to improving the anticancer affect as explored by Schiff׳s bases 2d, 2e and 2f and their
cyclized thiazolidinone compounds 3d, 3e and 3f. Cyclization of 4-halophenyl Schiff׳s bases into
the corresponding thiazolidinones improved the anticancer effect. The 4-fluorophenyl
thiazolidinone 3a was the most active thiazolidinone compound with IC50 of 11.1µg/mL. It was
more active than the 4-chlorophenyl derivative 3b (IC50 = 17.7 µg/mL) while a dramatic drop in
the anticancer effect was displayed for the 4-bromophenyl thiazolidinone 3c (IC50 = 42.0 µg/mL).
Both N-methyl and N-ethyl derivatives of coumarinyl-4-phenylthiazoloacetohydrazides
7a and 7b, respectively, showed significant equipotent anticancer effect (IC50 = 9.3 and 10.6
µg/mL, respectively) than the 4-methylthiazolyl congeners 5a and 5b (IC50 = 36.9 and 78 µg/mL,
respectively). On the other hand, the 5-acetyl-3-ethyl-4-methylthiazoline derivative 6b showed
higher anticancer potential against MCF-7 than the 3,4-dimethylthiazoline compound 6a (IC50 =
28.0 and 42.0 µg/mL, respectively). Generally the thiazolyl acetohydrazide derivatives 5a,b,
6a,b and 7a,b were more potent than the oxothiazolidinyl acetohydrazides 8a and 8b and their
parent thiosemicarbazide derivatives 4a and 4b.
Regarding the open chain derivatives, the N-methyl derivative of oxobutanoate and
propanoate compounds 9a and 10a (IC50 = 30.6 and 37.0 µg/mL, respectively) were more potent
than its N-ethyl congeners 9b (IC50 = 41.9 µg/mL) and 10b (% inhibition=67.1%).
2.2.3. Tubulin polymerization inhibiting effect
Among the common chemotherapeutic agents currently used in treating metastatic breast
35,36
cancer are the antimitotics drugs.
These agents bind primarily to β-tubulin, a major protein
in the mitotic spindles causing a reduction in the dynamics of microtubules in the mitotic
spindles, thus preventing spindle assembly and disturbing the normal movement of sister
chromatids towards the spindle poles.37-40
10

Accordingly, in order to evaluate the role of tubulin beta polymerization (TUBb) on
MCF-7 cells proliferation and to figure out the possible mode of action of the most cytotoxic and
selective compounds towards MCF-7 cells, the tubulin beta polymerization inhibitory effect of
the target compounds was assessed utilizing colchicine as a reference drug. This assay was
performed for compounds 3a, 3b and 3f which displayed the highest potential against MCF-7
cell line (IC50 = 11.1, 16.7 and 21.2 µg/mL) with the least cytotoxicity on normal BJ-1 cells.
Tubulin beta polymerization inhibiting effect of the target compounds 3a, 3b and 3f was
determined and IC50 values were calculated as summarized in table 2.
Table 2. Tubulin polymerization inhibiting effect of the target compounds.
Compound
IC50 (µM)
9.37 0.61
2.89 0.17
6.13 0.32
6.93 0.26
3a
3b
3f
colchicine
The three tested compounds showed significant TUBb polymerization inhibition activity
in accordance with the in vitro cytotoxic activity against MCF-7 cell line. Compound 3b showed
outstanding TUBb polymerization inhibiting effect with IC50 value of 2.89 µM. It was the most
active TUBb polymerization inhibitor and was about 2.4 fold more active than the standard
colchicine drug (IC50 = 6.93µM). Compound 3f was as potent inhibitor as colchicine with IC50
value of 6.13 µM and also compound 3a showed remarkable TUBb polymerization inhibiting
effect (IC50 = 9.37µM). Although compound 3a showed more cytotoxic effect against MCF-7
than 3b and 3f, it was not the most potent TUBb polymerization inhibitor which indicates that
inhibiting tubulin polymerization is not the only way for its antitumor activity.
2.2.4. Cellular mechanism of action
2.2.4.1. Cell apoptosis
Based on the well-balanced cytotoxic activity and tubulin polymerization inhibition
effect, compound 3a which demonstrated the most potent and selective cytotoxic effect against
MCF-7 cell line and exhibited significant inhibitory potency towards TUBb polymerization, was
chosen for further investigation of its cellular mechanism of action regarding its effects on cell
cycle progression and induction of apoptosis in MCF-7 cancer cells.
For evaluating the apoptosis process, flow cytometry was carried out using propidium
iodide (PI) and annexinV-FITC in MCF-7 cells.41 After treatment with IC50 concentration of
11

compound 3a (11.1µg/mL) for 24 hours, the cells were labeled with the two dyes. The
corresponding red (PI) and green (FITC) fluorescence were detected with the flow cytometry. In
comparison to DMSO as a negative control (Fig. 4A), it was observed that compound 3a induced
an increase in the late/secondary cellular apoptosis from 0.09% (DMSO control) to 10.63% (Fig.
4B). Also, an increase in the early/primary apoptosis was observed for compound 3a of 5.49%
(0.18% DMSO control). These data confirmed the apoptotic effect of compound 3a.
2.2.4.2. Cell cycle arrest
To further elucidate the molecular mechanism by which compound 3a exhibited its anti-
proliferative activity against MCF-7 cells, its effect on cell cycle distribution was analyzed by
flow cytometry.41 After exposure of MCF-7 cells to compound 3a at 11.1µg/mL for 24 hours, it
induced a significant increase in the percentage of cells at pre-G1 phase by 28.6 folds compared
to control (Fig.4. C and D). In addition, accumulation of cells was detected at G2/M phase by 5
folds compared to the control, from 8.59% in the vehicle group (Fig.4. C) to 43.59 % in the
group treated with the compound 3a (Fig.4. D). These results revealed that compound 3a
inhibited MCF-7 cells proliferation through the induction of G2/M phase arrest which led to cell
cycle cessation at G2/M phase and prevented its mitotic cycle. These results are in agreement
with the previously reported findings which investigated that the microtubule depolymerization
pathway is well established and plays a crucial role in the G2/M phase arrest and induction of
apoptosis.42-45
12

B
A
C
D
Fig.4. Cellular mechanism of action of compound 3a. (A, B) Induction of apoptosis by compound 3a.
Cells were exposed to 3a for 24h and analyzed by annexin V/ PI staining. The percentage of cells
undergoing apoptosis is defined as the sum of early apoptotic (annexin V+/ PI-) cell percentage and late
apoptotic (annexin V+/ PI+) cell percentage. (C, D) Cell cycle analysis of MCF-7 after incubation with
compound 3a for 24 h. DMSO diluent was used as a control.
2.2.4.3. Effect of compound 3a on the level of active caspase-7
Induction of apoptosis via either intrinsic (mitochondrial) or extrinsic (death receptor)
pathways is mediated by caspase cascade events. Caspases are cysteine proteases which paly an
essential role in the initiation of apoptosis due to proapoptotic signals, so it could be considered
that activating caspases is a significant step in apoptotic cell death.46,47 It has been demonstrated
that inhibition of executing caspase 3 and/or 7 is crucial for the programmed cell death of
multiple cell types.48 Because MCF-7 cells lack endogenous caspase-3, the level of active
13

caspase-7 was assessed time-dependently in MCF-7 cells treated with compound 3a at a
concentration of 11.1µg/mL for 24 hours. As shown in table 3, treatment with compound 3a
resulted in significant increase in caspase-7 level compared to doxorucicin.
Table 3. Caspase-7 concentration in MCF-7 cells after treatment with compound 3a for 24 hours.
Compound
Results
Cpd.
3a
Conc.
µg/mL
Casp7 conc.
ng/mL
FLD
11.1
1.162
1.813
0.359
3.236769
5.050139
1
26.1
Doxorubicin
Control
2.3. Molecular modeling studies
2.3.1. Molecular docking
In order to determine the possible binding modes of promising compounds 3a, 3b and 3f,
docking studies were carried out using the reported high-resolution crystal structure of the
tubulin/DAMA-colchicine complex (PDB ID: 1SA0). Compounds 3a, 3b and 3f were docked in
the colchicine binding pocket using MOE software. The colchicine binding pocket is a deep
pocket positioned at the α/β interface of tubulin heterodimer and consists of three zones, zone 1
located at α subunit interface, zone 2 which is the main zone situated at β subunit and most
structures of colchicine binding site inhibitors occupied this zone, and zone 3 which is buried
much deeper in β subunit.49 Interaction mode of colchicine showed that cyclohepatenone ring
interacts with Serα178, Valα181 and Valβ315 (zone 1) by ven der Waals while, the carbonyl
group forms H-bond with Valα181 at a distance of 3.56Å. The trimethoxy phenyl group is
embedded deeply in the hydrophobic portion of the pocket (zone 2) and surrounded by Leuβ242,
Alaβ250, Lysβ254, Leuβ255, Alaβ316, Asnβ350, Lysβ352 and Ileβ378. The methoxy group
behaves as H-bond acceptor and forms H-bond with thiol group of Cys241 at a distance of 3.31Å
(Fig. 5). Binding mode of compound 3a was nearly the same as that of colchicine and it mostly
occupied zone 2 in a similar manner to the binding mode of colchicine inhibitors. Closure look to
the docked pose of compound 3a within colchicine pocket illustrates the following features:
i)van der Waals interaction of oxothiazolidine acetamide with Serα178, Thrα179 Alaα180 and
Valα181 (zone1) in a similar manner to colchicine. ii) Carbonyl group of acetamide accepted two
14

hydrogen atoms, one from OH-Serα178 at a distance of 2.61Å and another one from OH -
Threα179 at a distance of 2.82Å. Interestingly, the acetamide carbonyl group exhibited the same
orientation of the carbonyl of cycloheptanone ring of colchicine (Fig. 6 and 7). iii) Coumarin
moiety located at zone 2, likewise the trimethoxyphenyl group of colchicine, showed
hydrophobic interactions with amino acids of zone 2 (Leuβ242, Alaβ250, Lysβ254, Leuβ255,
Alaβ316 and Lysβ352). iv) Carbonyl group of coumarin moiety formed H-bond with NH of
Valβ318 at a distance of 3.33Å. In addition, the 4-phenyl group linked to coumarin was
embedded deeply in the hydrophobic pocket in zone 2 facing Cysβ241.
Fig.5. The binding mode and molecular interactions of co-crystallized ligand; colchicine. Color code:
ligand (orange, ball) and black-line as hydrogen-bond interactions between residues and ligand
15

Fig.6. molecular interactions of compound 3a (orange, ball), within the colchicine binding site, hydrogen-
bonds are represented as black-line, C=O of acetamide formed two H-bonds with Serα178 and Thrα179.
C=O of coumarin formed H-bond with Valβ318.
.
Fig.7. The binding mode of compound 3a (orange, stick) within the active site of tubulin protein
(PDB:1SA0), showed that coumarin moiety located at zone 2, in similar manner to trimethoxyphenyl
group of colchicine (blue, stick), both C=O group of acetamide of compound 3a and C=O group of
colchicine showed the same orientation. The binding surface code colour, white; neutral, pink;
hydrophilic, green; hydrophobic.
16

Docking results of compound 3b showed that i) 4-Chlorophenyl thiazolidinone moiety
showed hydrophobic interactions with zone 1 amino acids (Serα178, Thrα179 and Valα181). ii)
Carbonyl group of oxothiazolidine received H atom from NH-Asnα101 and formed H-bond at a
distance of 2.56Å. in addition, carbonyl group of acetamide formed another H-bond with NH of
Lysβ254 at a distance of 2.70Å. Third H-bond was formed between C=O of coumarin and NH-
Alaβ354 at a distance of 3.68Å. iii) Coumarin moiety buried in the hydrophobic portion of the
pocket was restricted with residues of zone 2 (Leuβ242, Alaβ250, Lysβ254, Leuβ255 and
Lysβ352), additionally, phenyl group linked to coumarin moiety of compound 3b was embedded
deeply within the hydrophobic region of zone 2 in contact with Cysβ241(Fig.8 and 9).
Fig.8. Molecular interactions of compound 3b (orange, ball),at the colchicine binding site. C=O moiety
formed H-bond withAsnα101, C=O acetamide formed H-bond with Lysβ254 and C=O coumarin formed
H-bond with Alaβ354.Hydrogen-bond interactions between residues and ligand are represented as black
lines.
17

Fig 9.The binding mode of compound 3b (orange, stick). Coumarin moiety was buried in the
hydrophobic portion of the pocket restricted with residues of zone 2, in similar manner to trimethoxy
phenyl of colchicine (blue, stick).
Binding mode of compound 3f is highly similar to compounds 3a and 3b, as the same
interaction features were found i) Trimethoxy phenyl thiazolidinone moiety showed the same
hydrophobic interaction with zone 1 amino acids (Serα178 and Thrα179). ii) Carbonyl group of
acetamide formed two H- bonds with OH-Serα178 at a distance of 2.61Å and with OH -
Threα179 at a distance of 2.86Å. The acetamide carbonyl group of compound 3f exhibited the
same orientation of the carbonyl group of colchicine cycloheptenone ring. iii) Carbonyl moiety
of coumarin received H atom from NH-Val β318 and formed H-bond at a distance of 3.27Å.
Also, coumarin moiety showed the same hydrophobic interactions with zone 2 amino acids
((Leuβ242, Alaβ250, Lysβ254, Leuβ255, Alaβ316, Lysβ352 and Ile378). Finally, the 4-phenyl
group linked to coumarin moiety showed the same position within the hydrophobic pocket of
colchicine pocket site as in compounds 3a and 3b (Fig.10 and 11).
18

Fig.10. Molecular interactions of compound 3f (orange, ball) within the colchicine binding site, C=O
acetamide formed two H-bonds with Serα178 and Thrα179. C=O coumarin formed H- bond with
Valβ318. Colour code: Hydrogen-bond interactions between residues and ligand are represented as black
lines.
.
Fig.11. The binding mode of compound 3f (orange, stick) showed that coumarin moiety located at zone 2,
in similar manner to trimethoxy phenyl group of colchicine (blue, stick), both C=O group of acetamide of
compound 3f and C=O group of colchicine showed the same orientation.
19

In conclusion, the three compounds 3a, 3b and 3f showed the same binding mode within
the colchicine binding site, which is similar to colchicine. The thiazolidinone moiety showed two
types of interactions, hydrophobic interactions and H-bond formation between carbonyl group of
oxothiazolidine and amino acids of zone 1, these interactions are similar to that of
cycloheptanone moiety of colchicine. In addition, coumarin moiety of the three compounds
illustrated hydrophobic interactions with amino acids of zone 2 (important zone for colchicine
inhibitors). Besides, the H-bonds were formed between the C=O groups of the three compounds
and zone 2 residues. It is noticeable that these molecular interactions are the common
interactions of colchicine inhibitors, which explain the inhibition activity of compounds 3a, 3b
and 3f.
2.3.2. Pharmacophores
49
Nguyen et al.,
identified the common pharmacophore model of colchicine binding
inhibitors which consists of seven pharmacophore points: i) Three H-bond acceptors, A1 in
contact with Valβ181, A2 in contact with Cysβ241, and A3 establishing one contact mainly with
Alaβ250, Aspβ251, and Leuβ252. ii) One hydrogen bond donor, D1, which interacts with
Thrb179. iii) Two hydrophobic centers, H1 and H2. iv) A planar group R1. It should be noted
that, none of the colchicine binding inhibitors possessed all the 7-points of pharmacophore.50
With the aim of rationalizing the key common binding interactions of compounds 3a, 3b and 3f,
the pharmacophore hypotheses for these compounds were built and compared with Nuguen’s
model. Pharmacophore hypothesis of compound 3a (Fig.12A) showed four features, two
hydrogen bond acceptors (HBA) F1 in contact with Serα178 and Thrα179 and F2 in contact with
Valβ318 and two hydrophic centers F3 in contact with Leuβ248, Alaβ250, Leuβ255 and
Alaβ345 and F4 which showed hydrophobic interactions with Cysβ241 and Leuβ255. Compound
3b pharmacophore hypothesis showed five features, three H-bonds acceptors represented by
carbonyl groups of oxothiazoildine, acetamide and coumarin moieties, in addition to two
hydrophobic centers made up by coumarin moiety and phenyl group (Fig.12B). Compound 3f
pharmacophore model illustrated the same four features as compound 3a (Fig.12C). It is clear
from the results of the three pharmacophore models that there is an agreement with Nguyen’s
pharmacophore model, as the three pharmacophores showed four points common with the points
20

identified by Nguyen. Furthermore, there is a good correspondence between the pharmacophore
points and the docking results.
A)
B)
C)
Fig. 12. Pharmacophore models include hydrogen bond acceptors centres (dark blue), hydrophobic
centres (dark red). A) Represented compound 3a with 4 features. B) Represented compound 3b with
5 features. C) represented compound 3f with 4 features
2.3.3. Molecular dynamics studies (MD)
In order to study the stability of the docked poses produced by the molecular docking
studies and investigate their binding modes within colchicine binding site, compounds 3a, 3b
and 3f were subjected to MD studies. The ligand–receptor complexes of 3a, 3b and 3f that came
out from the docking simulation were used in the MD simulation study for 100 picoseconds (Ps).
It was noticeable from the study of the root mean square deviation (RMSD) of compound 3a
that, the compound fluctuated in the first 30Ps. Starting from the 35Ps, the compound become
21

stable and the complex (protein-ligand) reached the stable state and the average RMSD of
compound 3a during the simulation was 3Å which is a small distance indicating the effectiveness
and the strength of H-bonds between the ligand and protein (Fig.13 A). Compound 3b–protein
complex fluctuated in the first 25 Ps then became stable starting from the 25Ps. In the Ps 45-55,
compound 3a dropped down then retained the steady state for the remaining of the simulation
(Fig.13 B). Molecular dynamics simulation of compound 3f reached the balance after the 25Ps,
and RMSD of compound 3f was kept around a small distance of 1 Å (Fig.13 C).
Fig.13. A) Molecular dynamic simulation of compound 3a showed RMSD (Å) over 100Ps. B) Molecular
dynamic simulation of compound 3b showed the RMSD (Å) over 100Ps. C) Molecular Dynamic
simulation of compound 3f showed RMSD (Å) over 100Ps.
2.3.4. Energy study
Potential and kinetic energies in Kcal/mol were calculated for compound 3a during the
MD simulation,, the analysis of potential energy graph showed that the potential energy of the
system was so high in the first 10Ps, and the pattern reached the steady state at 25 Ps with
22

potential energy equal to 150 Kcal/mol (Fig. 14A). Kinetic energy figure of compound 3a
fluctuated in the first 25 Ps and it was so high in the first 10 Ps. The equilibrium was reached at
25Ps in a similar manner to the potential energy (Fig. 14B). The RMSD result of compound 3a
showed that compound 3a got stable after 30Ps. i.e. after the system reached to potential and
kinetic energy equilibrium as proved by potential and kinetic energy results which indicated the
reliability of MD results.
Fig14. A) Molecular dynamic simulation of compound 3a showed the potential energy (Kcal/mol) over
100Ps. B) Molecular dynamic simulation of compound 3a showed the Kinetic energy(Kcal/mol) over
100Ps.
23

2.3.5. Molecular property-based drug-likeness rules
Lipinski proposed the “Rule of Five”,51 the most popular drug-likeness filter, which
provides four rules to determine whether a molecule is well orally absorbed or not: octanol/water
partition coefficient (logP) ≤ 5, molecular weight (MW) ≤ 500, number of hydrogen bond
acceptors (HBA) ≤ 10 and number of hydrogen bond donors (HBD) ≤ 5. In this study the four
rules were calculated for compound 3a. In addition, polar surface area (PSA) was also
calculated, as it is thought that compounds with promising oral bioavailability have PSA ≤140
Å.52 Calculated molecular property of compound 3a based on drug likeness rules showed that
compound 3a fulfilled Lipinski rule, as logP = 5, Mwt = 490, number of hydrogen bond acceptor
(HBA) = 4, number of hydrogen bond donors (HBD) =1 and PSA = 84.9 (Table 4), this
indicates that compound 3a is well orally absorbed
In a similar manner to the purpose of Lipinski rules, the REOS (Rapid Elimination of
Swill) program was developed at Vertex,53 the drug-likeness criteria that are developed by REOS
include six rules: MW (200 ~ 500), number of hydrogen bond donors (0 ~ 5), number of
hydrogen bond acceptors (0 ~ 10), logP (−5 ~ 5), number of rotatable bonds (0 ~ 8) and number
of formal charge (−2 ~ 2). Calculated number of rotatable bonds and formal charge for
compound 3a were; 7 and 0 respectively, this reveals that compound 3a obeys REOS rules.
These results confirm the oral bioavailability of our promising compound 3a (Table 4).
Table 4.The calculated Lipinski and REOS criteria for compound 3a.
Parameters
Compound Mwt
HBA
4
HBD
1
logP Rotatable bonds Formal charge
PSA
84.9
3a
490
5
7
0
3.
Conclusion
In this study, new 4-phenylcoumarin derivatives were developed for cytotoxic assessment
against human breast cancer cell line MCF-7. Cytotoxic effect of the synthesized compounds
against MCF-7 revealed that most of the compounds showed promising antiproliferative effects
against MCF-7. Compounds 3a, 3b and 3f displayed remarkable cytotoxic effect on MCF-7 with
no significant cytotoxic effect on human normal skin cell line (BJ-1). Furthermore, tubulin
24

polymerization assay was performed for the most promising and selective cytotoxic compounds
3a, 3b and 3f. The results disclosed that the three derivatives demonstrated equal to higher
inhibitory potency of TUBb polymerization when compared to the reference drug colchicine of
IC50values (IC50 = 9.37, 2.89 and 6.13 µM, respectively, vs 6.93 µM for colchicine). Compound
3a was further subjected to cellular mechanistic studies on MCF-7 cells and revealed induction
of apoptosis and cell cycle arrest at G2/M phase, along with its significant activation of caspase-
7 that might mediate apoptosis of MCF-7 cancer cells. The combined analyses of molecular
docking, pharmacophore hypotheses and MD studies revealed significant information on the
structural features of the promising molecules, which are required for interactions and afforded a
guide for the design of novel lead compounds. Molecular modeling studies showed that
compounds 3a, 3b and 3f had the same binding mode of colchicine. In addition, molecular
modeling and pharmacophore models displayed that the most important features of interactions
are the carbonyl groups of thiazolidinone, acetamide and coumarin moieties, in addition to two
hydrophobic centers made up by coumarin moiety and phenyl group. MD illustrated that
compound 3a-complex became stable at 30Ps with very low RMSD value, also compounds 3b
and 3f reached the stable state at 25Ps with low RMSD value. MD results supported the
reliability of docking studies. Lipinski’s rule calculations showed that all the physical and
pharmacokinetic properties of compound 3a are within the accepted range indicating that
compound 3a is promising as drug like molecule.
4.
Experimental
4.1.
Chemistry
All melting points were uncorrected and measured using Electrothermal IA 9000
apparatus. Infrared spectra were measured by Nexus 670 FT-IR FT-Raman spectrometer using
KBr discs at National Research Centre, Egypt. The nuclear magnetic resonance NMR spectra
were determined utilizing Varian mercury 300 MHz spectrometer and using TMS as the internal
standard. The mass spectra were recorded on GCMS-QP 1000EX Shimadzu Gas
1
Chromatography MS Spectrometer. HNMR, 13CNMR, EI-MS and the elemental analyses were
performed at Micro-Analytical Laboratory, Central Services Laboratory, Faculty of Science,
Cairo University, Egypt. The reactions were followed by TLC (silica gel, aluminum sheets 60
F254, Merck) using chloroform-methanol (9.5:0.5 v/v) as eluent and sprayed with iodine-
potassium iodide reagent. The purity of the newly synthesized compounds was assessed by TLC
25

and elemental analysis and was found to be higher than 95%. Compounds 1,2b, 2d, 3b and 3d
were previously prepared.54
4.1.1. General procedure for the preparation of Schiff's bases 2a-f
To a mixture of the hydrazide compound 1 (3.1g, 0.01 mol) in absolute ethanol (20 ml)
containing glacial acetic acid (5mL), different aromatic aldehydes (0.01 mol), namely; 4-
fluorobenzaldehyde (1.07mL), 4-chlorobenzaldehyde (1.41g), 4-bromobenzaldehyde (1.85 g), 4-
methoxybenzaldehyde (1.22 mL), 3,4-dimethoxybenzaldehyde (1.66g) and / or 3,4,5,-
trimethoxybenzaldehyde (1.96 g) were added. The reaction mixture was refluxed for 6-8 hours.
The formed precipitate was filtered off and re-crystallized form acetic acid to afford the title
compounds.
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(4-fluorobenzylidene)acetohydrazide 2a
Yield 75%, mp 260-2°C. Anal. Calcd. for C24H17FN2O4 (416.4): C, 69.23; H, 4.12; N, 6.73.
Found: C, 69.36; H, 4.23; N, 6.89. IR (cm-1, KBr): 3210 (NH), 1719 (C=O), 1688 (C=O).
1HNMR (DMSO-d6, δ, ppm): 5.80 (2H, s, OCH2), 6.26 (1H, s, H-3 coumarin), 6.88-7.58 (12H,
m, Ar−H), 8.26 (1H, s, N=CH), 11.66 (1H, s, NH, D2O exchangeable). MS (EI, 70eV) m/z (%)::
416 (M+) (16.8 %), 293 (100.00%).
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(4-bromobenzylidene)acetohydrazide 2c
Yield 85%, mp 271-2°C. Anal. Calcd. for C24H17BrN2O4 (477.31): C, 60.39; H, 3.59; N, 5.87.
Found: C, 60.26; H, 3.42; N, 5.69. IR (cm-1, KBr): 3211 (NH), 1715(C=O), 1689 (C=O).
1HNMR (DMSO-d6, δ, ppm): 5.72 (2H, s, OCH2), 6.26 (1H, s, H-3 coumarin), 6.90-7.57 (12H,
m, Ar−H), 8.23 (1H, s, N=CH), 11.69 (1H, s, NH, D2O exchangeable). MS (EI, 70eV) m/z (%):
477,479 (M+, M++2) (14.83, 14.11 %), 295 (100.00%).
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(3,4-dimethoxybenzylidene)acetohydrazide 2e
Yield 80%, mp 238-40°C. Anal. Calcd. for C26H22N2O6 (458.46): C, 68.11; H, 4.84; N, 6.11.
Found: C, 68.27; H, 4.98; N, 6.26. IR (cm-1, KBr): 3210 (NH), 1720 (C=O), 1686 (C=O).
1HNMR (DMSO-d6, δ, ppm): 3.70 (3H, s, OCH3), 3.72 (3H, s, OCH3), 5.76 (2H, s, OCH2), 6.26
(1H, s, H-3 coumarin), 6.92-7.58 (11H, m, Ar−H), 8.24 (1H, s, N=CH), 11.41 (1H, s, NH, D2O
exchangeable). MS (EI, 70eV) m/z (%): 458 (M+) (13.9 %), 193 (100.00%).
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(3,4,5-trimethoxybenzylidene)acetohydrazide 2f
Yield 85%, mp 204-6°C. Anal. Calcd. for C27H24N2O7 (488.49): C, 66.39; H, 4.95; N, 5.73.
Found: C, 66.26; H, 4.82; N, 5.59. IR (cm-1, KBr): 3212 (NH), 1718 (C=O), 1684 (C=O).
26

1HNMR (DMSO-d6, δ, ppm): 3.62 (3H, s, OCH3), 3.75 (6H, s, 2OCH3), 5.77 (2H, s, OCH2),
6.25 (1H, s, H-3 coumarin), 6.72-7.55 (10H, m, Ar−H), 8.23 (1H, s, N=CH), 11.49 (1H, s, NH,
D2O exchangeable). MS (EI, 70eV) m/z (%): 488 (M+) (100.00%).
4.1.2. General procedure for the preparation of thiazolidinone compounds 3a-f
Thioglycolic acid (0.001 mol, 0.07 mL) was added to a well stirred solution of Schiff’s
bases 2a-f (0.001 mol) in dry benzene (20 mL) and the reaction mixture was refluxed for 7-9
hours. After completion of the reaction, excess solvent was evaporated under reduced pressure
and the residue was neutralized with cold dilute sodium bicarbonate solution, the formed product
was filtered off, washed with water and then re-crystallized from acetic acid to give the
thiazolidinone compounds 3a-f.
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)
acetamide 3a
Yield 65%, mp 198-200°C. Anal. Calcd. for C26H19FN2O5S (490.5): C, 63.66; H, 3.90; N, 5.71;
S, 6.54. Found: C, 63.56; H, 3.78; N, 5.59; S, 6.43. IR (cm-1, KBr): 3177 (NH), 1719 (C=O),
1683 (C=O), 1624 (C=O). 1HNMR (DMSO-d6, δ, ppm): 3.74-3.95 (2H, dd, CH2 thiazolidinone),
4.71 (2H, s, OCH2), 5.82 (1H, s, CH thiazolidinone), 6.27 (1H, s, H-3 coumarin), 6.90-7.59
(12H, m, Ar−H), 10.54 (1H, s, NH, D2O exchangeable). MS (EI, 70eV) m/z (%): 490 (M+) (1.74
%), 165 (100.00%).
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N-(2-(4-bromophenyl)-4-oxothiazolidin-3-yl)
acetamide 3c
Yield 67%, mp 230-2°C. Anal. Calcd. for C26H19BrN2O5S (551.41): C,56.63; H, 3.47; N, 5.08; S,
5.82. Found: C, 56.49; H, 3.38; N, 4.91; S, 5.73. IR (cm-1, KBr): 3182 (NH), 1717 (C=O), 1685
(C=O), 1623 (C=O). 1HNMR (DMSO-d6, δ, ppm): 3.75-3.94 (2H, dd, CH2 thiazolidinone), 4.72
(2H, s, OCH2), 5.79 (1H, s, CH thiazolidinone), 6.27 (1H, s, H-3 coumarin), 6.93-7.58 (12H, m,
Ar−H), 10.54 (1H, s, NH, D2O exchangeable). 13CNMR (DMSO-d6, δ ppm): 29.01, 60.88, 65.84,
101.98, 111.73, 112.55, 120.54, 122.02, 127.61, 128.15, 128.26, 128.74, 129.53, 129.80, 131.31,
134.82, 137.50, 154.84, 155.02, 159.70, 160.43, 166.06, 168.52. MS (EI, 70eV) m/z (%):
551,553 (M+, M++2) (0.50, 0.50%), 295 (100.00%).
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N-(2-(3,4-dimethoxyphenyl)-4-oxothiazolidin-3-yl)
acetamide 3e
27

Yield 70%, mp 135-7°C. Anal. Calcd. for C28H24N2O7S (532.56): C, 63.15; H, 4.54; N, 5.26; S,
6.02. Found: C, 63.27; H, 4.68; N, 5.37; S, 6.11. IR (cm-1, KBr): 3179 (NH), 1719 (C=O), 1689
1
(C=O), 1628 (C=O). HNMR (DMSO-d6, δ, ppm): 3.70 (3H, s, OCH3), 3.72 (3H, s, OCH3),
3.75-3.89 (2H, dd, CH2 thiazolidinone), 4.72 (2H, s, OCH2), 5.77 (1H, s, CH thiazolidinone),
6.26 (1H, s, H-3 coumarin), 6.82-7.58 (11H, m, Ar−H), 10.48 (1H, s, NH, D2O exchangeable).
MS (EI, 70eV) m/z (%): 532 (M+) (1.47 %), 182 (100.00%).
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N-(2-(3,4,5-trimethoxyphenyl)-4-oxothiazolidin-3-yl)
acetamide 3f
Yield 71%, mp 150-2°C. Anal. Calcd. for C29H26N2O8S (562.59): C, 61.91; H, 4.66; N, 4.98; S,
5.70. Found: C, 61.79; H, 4.52; N, 4.87; S, 5.61. IR (cm-1, KBr): 3183 (NH), 1716 (C=O), 1688
1
(C=O), 1625 (C=O). HNMR (DMSO-d6, δ, ppm): 3.62 (3H, s, OCH3), 3.75 (6H, s, 2OCH3),
3.77-3.91 (2H, dd, CH2 thiazolidinone), 4.75 (2H, s, OCH2), 5.78 (1H, s, CH thiazolidinone),
6.25 (1H, s, H-3 coumarin), 6.73-7.59 (10H, m, Ar−H), 10.51 (1H, s, NH, D2O exchangeable).
MS (EI, 70eV) m/z (%): 562 (M+) (4.41 %), 59 (100.00%).
4.1.3. General Procedure for the preparation of 1-(2-(2-oxo-4-phenyl-2H-chromen-7-
yloxy)acetyl)-4-methyl and / or ethylthiosemicarbazide 4a,b.
To a solution of the hydrazide 1 (3.1g, 0.01 mol) in hot absolute ethanol, methyl
isothiocyanate (0.68 mL, 0.01mol) and/or ethyl isothiocyanate (0.87g, 0.01mol) was added. The
mixture was refluxed for 5-7 hours and the separated solid was filtered, washed with ethanol and
crystallized from acetic acid.
1-(2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)acetyl)-4-methylthiosemicarbazide 4a.
Yield 85%, mp 217−9°C. Anal. Calcd. for C19H17N3O4S (383.42): C, 59.52; H, 4.47; N, 10.96; S,
8.36. Found: C, 59.64; H, 4.63; N, 11.19; S, 8.48. IR (cm-1, KBr): 3399, 3376, 3310 (3NH), 1702
(C=O), 1670 (C=O). 1HNMR (DMSO-d6, δ, ppm): 2.88-2.89 (3H, d, CH3), 4.74 (2H, s, OCH2),
6.26 (1H, s, H-3 coumarin), 6.81−7.57 (8H, m, Ar−H), 7.83-7.85 (1H, q, NHCH3, D2O
exchangeable), 10.18 (1H, s, NH, D2O exchangeable), 13.91(1H, s, NH, D2O exchangeable). MS
(EI, 70eV) m/z (%): 383 (M+) (13%), 163 (100%).
1-(2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)acetyl)-4-ethylthiosemicarbazide 4b.
Yield 80%, mp 219−21°C. Anal. Calcd. for C20H19N3O4S (397.45): C, 60.44; H, 4.82; N, 10.57;
S, 8.07. Found: C, 60.34; H, 4.71; N, 10.49; S, 7.91. IR (cm-1, KBr): 3385, 3376, 3325, (3NH),
1708 (C=O), 1675 (C=O).1HNMR (DMSO-d6, δ, ppm): 1.05-1.09 (3H, t, CH3), 3.47-3.49 (2H,
28

m, CH2), 4.73 (2H, s, OCH2), 6.26 (1H, s, H-3 coumarin), 6.99−7.57 (8H, m, Ar−H), 7.99-8.02
(1H, t, NHCH2, D2O exchangeable), 9.19 (1H, s, NH, D2O exchangeable), 10.08 (1H, s, NH,
D2O exchangeable). MS (EI, 70eV) m/z (%): 397 (M+) (3%), 161 (100%).
4.1.4. General Procedure for the preparation of 2-(2-oxo-4-phenyl-2H-chromen-7-yloxy)-N'-
(3,4-dialkylthiazol-2(3H)-ylidene)acetohydrazide (5a,b), 2-(2-oxo-4-phenyl-2H-chromen-7-
yloxy)-N'-(5-acetyl-3,4-dialkylthiazol-2(3H)-ylidene) acetohydrazide (6a,b) and 2-(2-Oxo-4-
phenyl-2H-chromen-7-yloxy)-N'-(3-alkyl-4-phenylthiazol-2(3H)-ylidene)acetohydrazide
(7a,b).
A mixture of compounds 4a,b (0.01 mol) and the appropriate α-halocarbonyl compounds
(0.01mol), namely, chloroacetone (0.82 mL), 3-chloroacetylacetone (1.13 mL) and / or phenacyl
bromide (1.99 g), in absolute ethanol (30 mL) containing sodium acetate anhydrous (1.64 g, 0.02
mol) was refluxed for 10-12 hours. After cooling the precipitate was filtered, washed with water,
dried, and re-crystallized from absolute ethanol to give compounds 5a,b, 6a,b and 7a,b,
respectively.
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(3,4-dimethylthiazol-2(3H)-ylidene)
acetohydrazide 5a
Yield 70%, mp 106-8°C. Anal. Calcd. for C22H19N3O4S (421.47): C, 62.69; H, 4.54; N,
9.97; S, 7.61. Found: C, 62.50; H, 4.39; N, 9.82; S, 7.51. IR (cm-1, KBr): 3465, (NH), 1713
(C=O), 1646 (C=O). 1HNMR (DMSO-d6, δ, ppm): 2.24 (3H, s, CH3), 3.61 (3H, s, N-CH3), 4.26
(2H, s, OCH2), 6.28 (1H, s, H-3 coumarin), 7.06−7.56 (10H, m, Ar−H, S-CH and NH). MS (EI,
70eV) m/z (%): 421 (M+) (12.06 %), 184 (100.00%).
2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(3-ethyl-4-methylthiazol-2(3H)-ylidene)
acetohydrazide 5b
Yield 75%, mp 80-2°C. Anal. Calcd. for C23H21N3O4S (435.5): C, 63.43; H, 4.86; N,
9.65; S, 7.36. Found: C, 63.59; H, 4.99; N, 9.83; S, 7.50. IR (cm-1, KBr): 3452, (NH), 1711
1
(C=O), 1641 (C=O). HNMR (DMSO-d6, δ, ppm): 1.05-1.08 (3H, t, CH3-CH2), 2.25 (3H, s,
CH3), 4.02-4.06 (2H, q, CH2-CH3), 4.29 (2H, s, OCH2), 6.27 (1H, s, H-3 coumarin), 6.98−7.56
(10H, m, Ar−H, S-CH and NH). MS (EI, 70eV) m/z (%): 435 (M+) (5.85 %), 57 (100.00%).
2-(2-oxo-4-phenyl-2H-chromen-7-yloxy)-N'-(5-acetyl-3,4-dimethylthiazol-2(3H)-ylidene)
acetohydrazide 6a
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