VEGFR-2 inhibiting effect and molecular modeling of newly synthesized coumarin derivatives as anti-breast cancer agents

Article abstract of DOI:10.1016/j.bmc.2020.115328

Twenty five newly synthesized coumarin scaffold based derivatives were assayed for their in vitro anticancer activity against MCF-7 breast and PC-3 prostate cancer cell lines and were further assessed for their in vitro VEGFR-2 kinase inhibitory activity. The in vitro cytotoxic studies revealed that most of the synthesized compounds possessed very promising cytotoxicity against MCF-7, particularly; compounds 4a (IC₅₀ = 1.24 μM) and 3d (IC₅₀ = 1.65 μM) exhibited exceptional activities superior to the positive control staurosporine (IC₅₀ = 8.81 μM). Similarly, the majority of the compounds exhibited higher antiproliferative activities compared to the reference standard with IC₅₀ values ranging from 2.07 to 8.68 μM. The two cytotoxic derivatives 4a and 3d were selected to evaluate their inhibitory potencies against VEGFR-2 kinase. Remarkably, compound 4a, exhibited significant IC₅₀ of 0.36 μM comparable to staurosporine (IC₅₀; 0.33 μM). Moreover, it was capable of inducing preG1 apoptosis, cell growth arrest at G2/M phase and activating caspase-9. On the other hand, insignificant cytotoxic activity was observed for all compounds towards PC-3 cell line. Molecular docking study was carried out for the most active anti-VEGFR-2 derivative 4a, which demonstrated the ability of the tested compound to interact with the key amino acids in the target VEGFR-2 kinase binding site. Additionally, the ADME parameters and physicochemical properties of compound 4a were examined in silico.

Full text of DOI:10.1016/j.bmc.2020.115328

Journal Pre-proofs
VEGFR-2 Inhibiting effect and Molecular Modeling of Newly Synthesized
Coumarin Derivatives as Anti-breast Cancer Agents
Eman Y. Ahmed, Nehad A. Abdel Latif, Mohamed F. El-Mansy, Weam S.
Elserwy, Omaima M. Abdelhafez
PII:
S0968-0896(19)31354-9
https://doi.org/10.1016/j.bmc.2020.115328
BMC 115328
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
8 August 2019
19 November 2019
13 January 2020
Please cite this article as: E.Y. Ahmed, N.A. Abdel Latif, M.F. El-Mansy, W.S. Elserwy, O.M. Abdelhafez,
VEGFR-2 Inhibiting effect and Molecular Modeling of Newly Synthesized Coumarin Derivatives as Anti-breast
Cancer Agents, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115328
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VEGFR-2 Inhibiting effect and Molecular Modeling of Newly Synthesized Coumarin
Derivatives as Anti-breast Cancer Agents.
Eman Y. Ahmed1, Nehad A. Abdel Latif1, Mohamed F. El-Mansy2, Weam S. Elserwy3
Omaima M. Abdelhafez1,*
1Chemistry of Natural Compounds Department, Pharmaceutical and Drug Industries Research
Division, National Research Centre. Dokki, Cairo, Egypt.
2Organometallic and Organometalloid Chemistry Department, Chemical Industries Division,
National Research Centre. Dokki, Cairo, Egypt.
3Chemistry of Natural and Microbial Products Department, Pharmaceutical and Drug Industries
Research Division, National Research Centre. Dokki, Cairo, Egypt.
Graphical abstract
4a
Abstract
Twenty five newly synthesized coumarin scaffold based derivatives were assayed for their in
vitro anticancer activity against MCF-7 breast and PC-3 prostate cancer cell lines and were
further assessed for their in vitro VEGFR-2 kinase inhibitory activity. The in vitro cytotoxic
studies revealed that most of the synthesized compounds possessed very promising cytotoxicity
against MCF-7, particularly; compounds 4a (IC50=1.24 µM) and 3d (IC50=1.65 µM) exhibited
exceptional activities superior to the positive control staurosporine (IC50=8.81 µM). Similarly,
the majority of the compounds exhibited higher antiproliferative activities compared to the
reference standard with IC50 values ranging from 2.07 to 8.68 µM. The two cytotoxic derivatives
1

4a and 3d were selected to evaluate their inhibitory potencies against VEGFR-2 kinase.
Remarkably, compound 4a, exhibited significant IC50 of 0.36 µM comparable to staurosporine
(IC50; 0.33 µM).Moreover, it was capable of inducing preG1 apoptosis, cell growth arrest at
G2/M phase and activating caspase-9. On the other hand, insignificant cytotoxic activity was
observed for all compounds towards PC-3 cell line. Molecular docking study was carried out for
the most active anti-VEGFR-2 derivative 4a, which demonstrated the ability of the tested
compound to interact with the key amino acids in the target VEGFR-2 kinase binding site.
Additionally, the ADME parameters and physicochemical properties of compound 4a were
examined in silico.
Key words: Coumarin; MCF-7; VEGFR-2 kinase; Molecular modeling.
1. Introduction
Despite the latest diagnostic and therapeutic advances, breast cancer remains one of the main
causes of female death globally, and the second most common cancer in female population.[1] In
recent years, the better understanding of breast cancer biology guided the evolution of drugs
specifically aimed against tumorigenesis-associated molecular pathways.[2,3] Angiogenesis, the
development of new blood vessels, is recognized as one of the malignancy hallmarks and breast
vasculature has been reported to play a crucial role in controlling breast tumors.[4,5]
Consequently, many anti-angiogenic drugs have been evaluated in breast cancer patients,
including the oral vascular endothelial growth factor-receptor (VEGFR-2) tyrosine kinase
inhibitor, sunitinib, which was approved by the Food and Drug Administration for treating
different types of cancers.[6,7] Different classes of natural products reported to have significant
effect in the treatment and prevention of breast cancer such as coumarins,[8] isoflavones,[9] and
curcuminoids.[10] Coumarins are characterized by the presence of benzopyrone platform in their
chemical structure and possess wide variety of biological properties.[11,12] The bio-
pharmacological activities of coumarin scaffold are based mainly on the chemical structure and
physicochemical properties of its heterocyclic ring, which permits easy binding to many target
proteins. The 2H-chromen-2-one ring is aromatic, planar and lipophilic, and hence is capable of
interacting with different biological counterparts. Furthermore, the lactone group of the coumarin
grants the molecule the ability to make strong polar binding, like hydrogen bonds and to acylate
protein targets.[13]
2

Recently, various studies reported the effect of coumarin derivatives in the treatment of breast
cancer, particularly by targeting VEGFR-2 kinase.[14–16] The goal of this study is to design and
develop novel derivatives of coumarin by incorporating specific functional groups of known
VEGFR-2 inhibition.
2. Results and discussion
2.1. Chemistry
Provoked by the gained insight into the correlation between breast cancer and coumarin
derivatives as VEGFR-2 inhibitors.[17–19] Literature survey revealed that certain types of
functional groups exhibit anti-VEGFR-2 activity such as hydrazides,[20,21] Schiff’s bases,[15]
sulfonamides,[22] and amide derivatives.[15] Moreover, general structure-activity relationship
(SAR) of coumarins divulged that alkylation at position 6 and ether linked substituent at position
7 augment the anticancer activity of the coumarin derivative, [23,24] as illustrated in Figure 1.
5
4
Alkylation at position 6 is crucial
for anticancer activity
3
6
7
2
O
1
O
7th position oxygen linked substituent is benificial
for anticancer potential
8
Bioactive coumarin scaffold
Figure 1. General structure-activity relationship of coumarins.
Considering this in mind, we designed novel coumarin derivatives bearing the aforementioned
functional moieties at the positions indicated in the SAR studies.[23,24] Previously our research
group disclosed the evaluation of coumarin-based hydrazides as VEGFR-2 inhibitors.[20,21]
Expanding our knowledge in this area, we utilized the chemical reactivity of the coumarin-based
hydrazides[25] toward diverse electrophiles to engender novel coumarin derivatives encompass
imine, amide and sulfonamide moieties to be evaluated as VEGFR-2 inhibitors. The coumarin
hydrazide derivative 1 was easily accessed from its ester derivative according to the reported
literature procedure.[26] The newly prepared hydrazone derivatives 2a-d were obtained in good
yield through the reaction of the hydrazide 1 with 4-nitrobenzaldehyde, 4-
dimethylaminobenzaldehyde, 4-fluorobenzaldehyde and 5- methyl furfural, respectively. The
3

structure assignment of 2b was based on 1H NMR data where the characteristic CH=N signal
appeared at 8.1 ppm, singlet signal for six protons was observed at 2.97 ppm (N(CH3)2) and 13C
NMR showed a signal at 145 ppm. Reaction of the hydrazide derivative 1 with phthalic
anhydride, maleic anhydride, isonicotinc anhydride and isatoic anhydride provided the
corresponding N- substituted acetamide 3a-d respectively as shown in Scheme 1.
Nucleophilic attack on the benzyl chloride proceeded smoothly to give the benzyl derivative 4a.
The 1H NMR spectra of 4a showed a characteristic signal at 4.78 ppm as a singlet for the CH2-N.
Reaction of hydrazide 1 with benzoyl chloride and chloroacetylchloride afforded the
corresponding hydrazides 4b and 5 respectively. The newly formed amid bonds were detected in
the 13C NMR at 169.4 ppm for 4b and 166.2 ppm for 5. The chloroacetohydrazide 5 was allowed
to react with different amines; piperidine, N-methyl piperazine and morphline to form the
corresponding tertiary amine derivatives 6a-c respectively, Scheme 1. The 1H NMR spectral data
for 6a revealed multiplet signal at δ 2.18-3.01 suggesting the presence of 5 CH2-piperidine, while
in 6b the appearance of a new singlet signal at 2.38 ppm indicates the presence of N-CH3 in
addition to the multiplet signal at 2.88-2.91 (10H, 4CH2-piperazine, N-CH2) and for the 6c
compound, a multiplet signal was detected at 2.48-3.66 ppm (10H, 4CH2-morpholine, N-CH2).
4

O
3a, Ar2 =
N
N
O
O
CH3
O
O
O
3b, Ar2 =
3d, Ar2 =
3c, Ar2 =
H
N
N
N
Ar2
O
O
O
O
(3)
O
N
N
O
N
H
(ii)
CH3
O
CH3
CH3
O
H
(iii)
O
Ar3
H
N
H
(i)
N
N
Ar1
N
O
O
N
O
O
H2N
O
O
H
O
O
O
(4)
(1)
(2)
(iv)
CH3
O
2a, Ar1 =
NO2
4a, Ar3 =
4b, Ar3 =
O
H
N
2b, Ar1 =
2c, Ar1 =
Cl
X
N(CH3)2
F
N
H
O
O
O
O
(5)
CH3
O
(v)
O
Me
O
6a
6b
O 6c
, X= H
, X= N-CH3
, X= O
2d, Ar1 =
H
N
N
N
H
O
O
(6)
Scheme 1: Reagents and reaction conditions; i) appropriate ArCHO, methanol, reflux, 1-5 h. ii)
appropriate acid anhydride, glacial AcOH, reflux 5-7 h iii) Acid chlorides, DMF, stirring, rt, 2 h
iv) Chloroacetyl chloride, DMF, stirring, rt, 2 h. v) secondary amines, DMF, anhyd. K2CO3,
stirring, rt, 1h.
Similarly, we envisioned new coumarin-based Schiff’s bases and amid derivatives that could be
prepared via treatment of 6-aminocoumarin derivative 7[27] with different electrophiles. Imine
derivatives 8a,b were successfully obtained in moderate to good yield through the reaction of
amine derivative 7 with benzaldehyde and/or 2, 4- dihydroxy benzaldehyde respectively. Also,
the reaction of amine 7 with acid anhydrides; phthalic anhydride and maleic anhydride was
5

investigated as shown in scheme 2 to produce 9a and 9b. The acetamide derivative 10a was
prepared according to Dawoud et al.,[27] by the reaction of amine 7 with acetyl chloride. In a
similar fashion, the benzamide derivative 10b was prepared in a decent yield.
CH3
CH3
CH3
O
Ar2
H2N
Ar1
N
(i)
(ii)
O
O
O
O
O
O
(9)
(7)
(8)
1
8a
8b
, Ar = Ph
2
N
1
9a
, Ar
=
, Ar = 2,4-di OH Ph
(iii)
O
O
N
CH3
O
O
Ar3
NH
2
9b
, Ar =
O
O
(10)
3
10a
, Ar = CH3
3
10b
, Ar = Ph
Scheme 2 : Reagents and reaction conditions; i) appropriate ArCHO, glacial AcOH , reflux 7-9
h. ii) appropriate acid anhydride, glacial AcOH , reflux 5-7 h. iii) dry DMF, appropriate acid
chloride, stirring 5-7 h.
To generate a series of sulfonamide-based coumarin derivatives, the commercially available
coumarin 11 was transformed into 3-bromocoumarin 12. The reported bromination of
coumarin,[28] was modified by using acetic acid as a solvent to cleanly convert the coumarin into
the brominated product which underwent elimination smoothly in presence of triethyl amine to
give 3-bromocoumarin 12 in an excellent yield and single purification step. The
chlorosulfonation of 12 was carried out according to the reported method,[29] to produce the
known chlorosulfonyl derivative 13. The possibility of synthesizing different sulfonamides was
feasible by reacting the prepared chlorosulfonyl compound 13 with various amines; namely:
morpholine, piperidine, N-methyl piperidine, 2-aminobenzoimidazole, piperonylamine and 2, 4,
6-trimethoxy benzyl amine, Scheme 3. The structure of the formed sulfonamides was confirmed
using 1H NMR data analysis particularly; in compound 14c N-Me signal appeared at 2.21 ppm as
a singlet while in 14e two signals at 3.95 ppm as doublet for N-CH2-Ar and singlet at 5.91 ppm
6

for CH2(O)2 were present. The presence of two singlet signals at 3.52 ppm (p-OMe) and the two
(o-OMe) at 3.67 ppm confirmed the structure of compound 14f.
RO2S
Br
O
Br
O
ClO2S
Br
O
(i)
(ii)
(iii)
O
O
O
O
O
O
(13)
(14)
(12)
(11)
N
14a, R =
14c, R =
N
14b, R =
H
N
H
N
NMe
N
14d, R =
14f, R =
N
MeO
NH
O
NH
14e, R =
OMe
O
MeO
Scheme 3: reagents and reaction conditions; i) bromine, AcOH, ii) chlorosulfonic acid, boiling;
iii) Toluene, Et3N, amine, stirring.
2.2. Biology
The new compounds were evaluated for their in vitro antitumor activities against MCF-7 and
PC-3 cancer cell lines. The derivatives of the best cytotoxic activity against MCF-7 breast cancer
cell line were subjected to VEGFR-2 kinase suppressing activity assay. Apoptosis, cell cycle
arrest and detection of caspase-9 intensity against MCF-7 were performed for the most active
compound as well.
2.2.1. In vitro cell proliferation assay:
We first investigated the growth inhibitory activities of compounds 2–14 against the two cancer cell
lines, MCF-7.and PC-3 using MTT assay.[30,31] The two cell lines were selected since they express the
target VEGFR-2 in this study.[15,32] The IC50 (μM) values of the cytotoxic compounds were calculated
as the average of at least three independent experiments, Table 1.
The results revealed that most of the newly prepared derivatives showed higher cytotoxicity than
the reference drug staurosporine (IC50=8.81 µM) against MCF-7 breast cancer cell line with IC50
values ranging from 1.24-8.68 M. In particular, the hydrazide 4a (IC50=1.24 µM) and the
7

acetamide 3d (IC50=1.65 µM) derivatives revealed the best cytotoxic activity against MCF-7 cell
line, Table 1. However, the tested compounds showed insignificant cytotoxic activity against
PC-3 cell line.
The anti-breast cancer activity of the synthesized compounds on MCF-7 cell line could be
explained with regard to their structures. Starting with the hydrazone derivatives 2a-d and the N-
substituted acetamides 3a-d, compounds 2d and 3d were the best in their series showing IC50 of
3.19 and 1.65 µM respectively, the rest of the compounds in the two series, except for
compounds 2c and 3a, showed as well remarkable cytotoxicity ranging from 2.11 to 8.68 µM
higher than the reference drug staurosporine (IC50=8.81 µM). As for the hydrazide series and its
amine derivatives 4-6, compound 4a (IC50=1.24 µM) revealed exceptional cytotoxicity when
compared to all the newly synthesized compounds. The aforementioned results, complies with
the structure-activity relationship of coumarins that reported the importance of ether linked
substitution, at the 7th position, for the anticancer activity.[23,24]
On the other hand, the acetamide derivatives 9a and 10b showed remarkable cytotoxicity
(IC50=4.17 and 4.60 µM) respectively and compounds 14b and 14d-f in the sulfonamide-based
coumarin derivatives series showed significant cytotoxicity with IC50 ranging from 2.07 to 7.79
µM. As observed, most of the acetamides and sulfonamides showed remarkable cytotoxic
activity conforming to the reported general SAR of coumarins which recognizes the significance
of substitution at the 6th position for the anticancer activity.[23,24]
Overall, the results elects most of the newly synthesized compounds as promising candidates that
could be utilized as lead compounds for optimizing new anticancer agents targeting breast
cancer.
2.2.2. In vitro VEGFR-2 kinase activity assay
The two compounds 4a and 3d which showed the most cytotoxic activity on MCF-7 were
evaluated for their effect on VEGFR-2 inhibition. The IC50 of the tested derivatives on VEGFR-2
as a marker for angiogensis is outlined in Table 1.
Compound 4a (IC50=0.36 µM) showed better anti-VEGFR-2 activity than compound 3d
(IC50=0.66 µM), which is consistet with the cytotoxic results.
8

Table 1. The growth inhibitory activities (IC50; M) of the tested compounds, VEGFR-2
inhibition (M) in MCF-7 breast cancer cell line.
Sample Code
cytotoxic activity IC50 (µM)
5.59±0.22
VEGFR-2 inhibition activity IC50 (µM)
2a
ND*
ND
2b
5.54±0.27
2c
9.78±0.38
ND
2d
3.19±0.14
ND
3a
14.68±0.57
2.11±0.07
ND
3b
ND
3c
8.68±0.34
ND
3d
1.65±0.07
0.66±0.019
0.36±0.011
ND
4a
1.24±0.06
4b
7.89±0.33
6b
6.68±0.26
ND
6c
37.33±1.6
ND
9a
4.17±0.19
ND
10b
4.60±0.18
ND
14a
13.20±0.55
2.07±0.11
ND
14b
ND
14c
31.38±1.31
3.65±0.17
ND
14d
14e
ND
7.79±0.36
ND
14f
5.76±0.23
ND
Staurosporine
8.81±0.39
0.33±0.011
ND*: Not Determined
9

2.2.3. Cellular mechanism of action
2.2.3.1. Cell apoptosis
Flow cytometry method was used to examine the effect of the most potent cytotoxic and anti-
VEGFR-2 derivative, 4a, on induction of apoptosis in MCF-7 cancer cells.[33] On subjecting the
cells to derivative 4a, there was an increase in the early and late cellular apoptosis, Figure 2.
(A)
(B)
(C)
(F)
(D)
(E)
Figure 2. Cellular mechanism of action of compound 4a. (A, B, C) Induction of apoptosis by
staurosporine and compound 4a. (D, E, F) Cell cycle analysis of MCF-7 after incubation with
staurosporine and compound 4a for 24 h.
2.2.3.2. Cell cycle inhibition
To examine the effect of derivative 4a on cell cycle, flow cytometry technique was utilized.[33]
Accumulation of cells at G2/M phase was observed, as shown in Figure 2. The results showed
that 4a induced PreG1apoptosis and G2/M phase arrest preventing cells mitosis.
2.2.3.3. Upregulation of caspase-9
An increase in caspase-9 concentration, involved in apoptosis,[34] was observed on treating MCF-
7 cells with 4a, as shown in Table 2.
10

Table 2: Caspase-9 concentration in MCF-7 cells after treatment with compound 4a in
comparison with staurosporine.
Sample Code
4a
Casp-9 (ng/ml)
8.015
Staurosporine
Cont.MCF-7
8.41
1.223
2.3. Docking
Molecular modeling studies were carried out for the most active anti-VEGFR-2 compound to
explore the significant interactions of the inhibitor and the amino acids residues in the VEGFR-2
kinase active site. The ability of compound 4a to interact with the key amino acids in the binding
site rationalizes its good activity as indicated by its docking score of −12.49 kcal/mol compared
to that of sorafenib of −15.20 kcal/mol.
The tested compound interacts through hydrogen bonding with the key amino acids Asp1046 of
the conserved DFG motif, the side chain carboxylate of Glu885 of the αC helix and Cys919 in
the hinge region of the protein. In addition, through hydrophobic interaction with hydrophobic
side chains lining the hydrophobic back pocket Ile888, Leu889, Ile892, Val898, Val899,
Leu1019 and Ile1044 by its benzyl moiety, Figure 3.
(A)
(B)
Figure 3. (A) 2D diagram, (B) 3D representation of compound 4a in the VEGFR-2 binding site.
11

2.4. In silico physicochemical properties and pharmacokinetic parameters assessment
The pharmacokinetic parameters and the physicochemical properties of the active derivative
were determined in silico to explore its drug like characteristics. Compound 4a manifested the
properties of a promising drug-like lead compound, Table 3. The compound showed zero
violations of the Lipinski’s rule of five, high GI absorption, 0.55 oral bioavailability score and
zero PAINS alerts (pain-assay interference structural alerts) confirming its reliability for future
optimization.
Table 3. Physicochemical and ADME characterization of compound 4a
#Heavy
atoms
#Rotatable
bonds
#H-bond
acceptors
Compd
#H-bond donors MR
TPSA
80.57
WLOGP
2.15
25
7
5
2
93.85
GI
BBB
permeant
Lipinski
#violations
Bioavailability PAINS Synthetic
SOL Class
4a
absorption
Score
#alerts
Accessibility
Soluble
High
No
0
0.55
0
3.32
3. Conclusion
In this study, new coumarin derivatives incorporating different functional groups were
synthesized and evaluated for their antiproliferative activity against MCF-7 breast cancer and
PC-3 prostate cell lines. Compounds 2a, 2b, 2d, 3b-d, 4a, 4b, 6b, 9a, 10b, 14b, 14d-f revealed
remarkable cytotoxic activity on MCF-7 with IC50 values, ranging from 1.24 to 8.68 µM, higher
than the reference standard staurosporine (IC50=8.81 µM). Particularly, compounds 4a
(IC50=1.24 µM) and 3d (IC50=1.65 µM) exhibited the best cytotoxic activities among the newly
synthesized derivatives. Moreover, VEGFR-2 kinase assay was performed on compounds 3d and
4a and the results showed that compound 4a, the most cytotoxic compound, had better anti-
VEGFR-2 kinase activity than 3d with IC50 value of 0.36 µM. Referring to both the cytotoxic
results and VEGFR-2 kinase assay results, one may relate the cytotoxicity of compound 4a to
suppression of the target enzyme. The cellular mechanism of action studies on 4a, revealed that
it was able to induce preG1 apoptosis and cell growth arrest at G2/M phase and to activate
caspase-9. However, none of the synthesized derivatives showed significant cytotoxic activity on
PC-3 cell line. On the other hand, the ability of the most active anti-VEGFR-2 derivative 4a to
interact with the key amino acids in VEGFR-2 binding site was demonstrated by the performed
12

molecular docking study. Finally, compound 4a was found to exhibit very good ADME
properties and drug-like characteristics without violating Lipinski’s rule of five.
4. Experimental
4.1. Chemistry
Melting points were determined on Electrothermal IA 9000 apparatus and were uncorrected.
Elemental analyses were carried out at the Micro-analytical Laboratory, Central Services
Laboratory, Faculty of Science, Cairo University, Egypt. 1HNMR spectra were determined on
Bruker 400 MHz at Faculty of Pharmacy, Cairo University, Egypt and 13CNMR spectra were
determined on Varian Mercury 100 MHz spectrometer at Faculty of Pharmacy, Cairo University,
Cairo, Egypt, using tetramethylsilane (TMS) as the internal standard. The reactions were
followed by TLC (silica gel, aluminum sheets 60 F254, Merck) using chloroform: methanol (9.5:
0.5 v/v) and toluene: ethyl acetate (8: 2 v/v) as eluents and sprayed with iodine-potassium iodide
reagent. All the reactions were monitored by TLC and the purity of the newly synthesized
compounds was assessed by TLC and elemental analysis.
4.1.1. General procedure for synthesis of (E)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N'-
(substituted) acetohydrazide (2a-d)
A solution of aromatic and heterocyclic aldehydes namely, 4-nitrobenzaldehyde, 4-
dimethylaminobenzaldehyde, 4-fluorobenzaldehyde and 5- methyl furfural, (0.01 mol) in
methanol (5 mL) was added dropwise to a well-stirred solution of compound 1 (0.01 mol) in
boiling methanol/ water (50.6 v/v). The reaction mixture was refluxed for 1-5 hours while
stirring, then concentrated and cooled. The solid product was filtered, washed with dilute
methanol and crystallized from ethanol.
4.1.1.1. (E)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N'-(4-nitrobenzylidene)acetohydrazide
(2a)
Yield 70 %; m.p. 265-267 ºC. 1H NMR (DMSO-d6, δ, ppm): 1.82 (3H, s, CH3), 4.52 (2H, s,
OCH2), 6.33 (1H, s, H-3 coumarin), 7.34-7.93 (7H, m, Ar-H), 8.1 (1H, s, CH=N), 10.54 (1H, s,
NH, D2O exchangeable). 13C NMR (DMSO-d6, δ, ppm): 19.31, 61.44, 103.01, 110.43,
111.19.112.13, 124.24, 124.96, 125.11, 138.98, 143.71, 149.52, 151.27, 153.33, 160.88, 161.96,
171.25. Anal. calcd. For C19H15N3O6 (381.33): C, 59.84; H, 3.96; N, 11.02 Found: C, 59.81; H,
3.93; N, 11.05
13

4.1.1.2. (E)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N'-(4-(dimethylamino)benzylidene)
acetohydrazide (2b)
Yield 70 %; m.p. 235-238 ºC; 1H NMR (DMSO-d6, δ, ppm): 2.40 (3H, s, CH3), 2.97 (6H, s,
N(CH3)2) , 4.76 (2H, s, OCH2), 6.21 (1H, s, H-3 coumarin), 6.9-7.7 (7H, m, Ar-H), 8.1 (1H, s,
CH=N), 11.38 (1H, s, NH, D2O exchangeable). 13C NMR (DMSO-d6, δ, ppm): 18.6, 41.69,
65.69, 102.14, 111.95, 113.76, 121.68, 126.84, 128.98, 145.31, 149.44, 151.89, 153.86, 155.03,
160.52, 161.90, 168.26. EIMS m/z (%):379 (M+, 15), 190 (2), 162 (5), 146(21), 84 (61), 82(100),
77 (12), 57 (33). Anal. calcd. For C21H21N3O4 (379.40): C, 66.48; H, 5.58; N, 11.08 Found: C,
66.44; H, 5.55; N, 11.06.
4.1.1.3. (E)-N'-(4-fluorobenzylidene)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)
acetohydrazide (2c)
Yield 75 %; m.p. 241-247 ºC; 1H NMR (DMSO-d6, δ, ppm): 1.97 (3H, s, CH3), 4.34 (2H, s,
OCH2), 6.27 (1H, s, H-3 coumarin), 7.34-7.93 (7H, m, Ar-H), 8.32 (1H, s, CH=N), 9.96 (1H, s,
NH, D2O exchangeable). 13C NMR (DMSO-d6, δ, ppm): 18.14, 68.43, 103.93, 111.78, 112.00,
112.96, 114.15, 125.83, 130.31, 131.18, 144.15, 152.76, 154.66, 159.88, 160.23, 165.27, 170.69.
Anal. calcd. For C19H15FN2O4 (354.33): C, 64.40; H, 4.27; N, 7.9. Found: C, 64.37; H, 4.24; N,
7.88
4.1.1.4. (E)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N'-((5-methylfuran-2-yl) methylene)
acetohydrazide (2d)
Yield 75 %; m.p. 268-274 ºC; 1H NMR (DMSO-d6, δ, ppm): 1.87 (3H,s , CH3), 2.01 (3H, s,
CH3), 4.45 (2H, s, OCH2), 6.38 (1H, s, H-3 coumarin), 7.34-7.93 (7H, m, Ar-H), 8.32 (1H, s,
CH=N), 9.96 (1H, s, NH, D2O exchangeable). 13C NMR (DMSO-d6, δ, ppm): 14.34, 19.64,
69.19, 104.67, 105.78, 110.39, 111.92, 112.14, 125.18, 133.64, 146.73, 152.01, 152.98, 154.23,
155.99, 161.82, 162.86, 170.94. Anal. calcd. For C18H16N2O5 (340.33): C, 63.52; H, 4.74; N,
8.23. Found: C, 63.49; H, 4.71; N, 8.20.
4.1.2. General procedure for the synthesis of N-(substituted)-2-(4-methyl-2-oxo-2H-
chromen-7-yloxy) acetamide (3a-d)
A mixture of compound 1 (0.01 mol) and the selected acid anhydride namely: phthalic
anhydride, maleic anhydride, isonicotinc anhydride and isatoic anhydride (0.01 mol) in glacial
acetic acid (50 mL) was heated under reflux for 5-7 hours. The obtained solid was filtered,
washed with acetic acid and crystallized from glacial acetic acid.
14

4.1.2.1. N-(1,3-dioxoisoindolin-2-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetamide (3a)
Yield 70 %; m.p. 208-210 ºC; 1H NMR (DMSO-d6, δ, ppm): 1.79 (3H, s, CH3), 4.50 (2H, s,
OCH2), 6.33 (1H, s, H-3 coumarin), 7.01-7.83 (7H, m, Ar-H), 9.96 (1H, s, NH, D2O
exchangeable). 13C NMR (DMSO-d6, δ, ppm): 18.94, 65.63, 103.05, 110.91, 111.82, 112.50,
122.37, 124.89, 131.78, 132.24, 151.27, 153.34, 161.80, 162.30, 164.73, 166.36. Anal. calcd. For
C20H14N2O6 (378.33): C, 63.49; H, 3.73; N, 7.40 Found: C, 63.45; H, 3.70; N, 7.37
4.1.2.2. N-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)
acetamide (3b)
Yield 75 %; m.p. 167-170 ºC; 1H NMR (DMSO-d6, δ, ppm): 1.88 (3H, s, CH3), 4.32 (2H, s,
OCH2), 6.25 (1H, s, H-3 coumarin), 7.23-7.99 (5H, m, Ar-H), 10.03 (1H, s, NH, D2O
exchangeable). 13C NMR (DMSO-d6, δ, ppm): 20.14, 65.63, 104.63, 110.23, 111.42, 113.35,
124.58, 132.99, 152.57, 154.54, 160.36, 162.63, 164.12, 166.44. Anal. calcd. For C16H12N2O6
(328.27): C, 58.54; H, 3.68; N, 8.53. Found: C, 58.51; H, 3.65; N, 8.50.
4.1.2.3. N-isonicotinoyl-N'-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetyl)
isonicotinohydrazide (3c)
Yield 75 %; m.p. 171-174 ºC; 1H NMR (DMSO-d6, δ, ppm): 2.40 (3H, s, CH3), 5.19 (2H, s,
OCH2), 6.2 (1H, s, H-3 coumarin), 7.10-8.17 (11H, m, Ar-H), 11.1 (1H, s, NH, D2O
exchangeable). 13C NMR (DMSO-d6, δ, ppm): 18.61, 66.8, 102.22, 111.78, 113.15, 113.94,
120.86, 126.43, 134.72, 146.78, 153.21, 153.85, 155.06, 160.57, 161.04, 161.81. Anal. calcd. For
C24H18N4O6 (458.42): C, 62.88; H, 3.96; N, 12.22. Found: C, 62.85; H, 3.93; N, 12.20.
4.1.2.4. N-(2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-(4-methyl-2-oxo-2H-chromen-7-
yloxy) acetamide (3d)
Yield 70 %; m.p. 139-141 ºC; 1H NMR (DMSO-d6, δ, ppm): 1.88 (3H, s, CH3), 4.74 (2H, s,
OCH2), 6.23 (1H, s, H-3 coumarin), 7.03-8.7 (7H, m, Ar-H), 9.8, 10.01 (2H, s, 2NH, D2O
exchangeable). 13C NMR (DMSO-d6, δ, ppm): 19.72, 66.67, 101.67, 111.94, 113.02, 113.80,
114.13, 114.21, 121.78, 126.94, 126.99, 139.77, 139.99, 150.93, 160.53, 161.06, 161.10, 164.43,
164.51, 166.66. Anal. calcd. For C20H15N3O6 (393.34): C, 61.07; H, 3.84; N, 10.68 Found: C, C,
61.04; H, 3.80; N, 10.63.
4.1.3. General procedure for the synthesis of 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N'-
substituted hydrazide (4a,b and 5)
15

To a well stirred solution of acetohydrazide 1 (0.0024 mol) in dry dimethylformamide (3 mL),
the appropriate acid chlorides namely: benzyl chloride, benzoyl chloride and
chloroacetylchloride (0.0024 mol) were dropwisely added with continuous stirring on cold at
20 ºC for 2 hours. The solution was poured onto ice/water; the formed precipitate was collected,
washed with water and crystallized from ethanol.
4.1.3.1. 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N'-benzylacetohydrazide (4a),
Yield: 85%; mp: 211–213 ˚C. 1H NMR (DMSO-d6, δ, ppm): 1.95 (3H, s, CH3), 4.78 (2H, s, CH2-
N), 5.12 (2H, s, OCH2), 6.20 (1H, s, H-3 coumarin), 6.85-7.71 (8H, m, Ar-H), 10.28, 10.48 (2H,
s, 2NH, D2O exchangeable). 13C NMR (DMSO-d6, δ, ppm): 18.58 (CH3), 65.81, 66.68, 101.89,
111.64, 111.85, 112.67, 112.84, 113.71, 113.99, 126.81, 126.98, 152.14, 153.82, 154.99, 158.03,
160.57, 161.84 (C=O coumarin), 168.86 (C=O). EIMS m/z (%):338 (M+, 0.14), 288 (23), 248
(2), 189(18), 148 (13), 113(100), 103 (35), 56 (19). Anal. calcd. for C19H18N2O4 (338.36): C,
67.44; H, 5.36; N, 8.28. Found: C, 67.51; H, 5.45; N, 8.29.
4.1.3.2. N'-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetyl)benzohydrazide (4b)
Yield (80 %); m.p. 94-96 ºC; 1H NMR (DMSO-d6, δ, ppm): 2.05 (3H, s, CH3), 5.56 (2H, s,
OCH2), 6.10 (1H, s, H-3 coumarin), 6.59-8.00 (8H, m, Ar-H), 9.77, 10.14 (2H, s, 2NH, D2O
exchangeable). 13C NMR (DMSO-d6, δ, ppm): 20.18 (CH3), 69.14, 109.17, 112.22, 114.64,
116.11, 126.42, 126.99, 127.68, 135.40, 137.11, 155.14, 157.82, 161.17, 162.61 (C=O
coumarin), 167.45 (C=O benzoyl), 169.32 (C=O). Anal. calcd. For C19H16N2O5 (352.34): C,
64.77; H, 4.58; N, 7.95, Found: C, 64.70; H, 4.48; N, 7.88.
4.1.3.3. N'-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetyl)-2-chloroacetohydrazide (5)
Yield (85 %); m.p. 233-235º C; 1H NMR (DMSO-d6, δ, ppm): 1.89 (3H, s, CH3), 4.56 (2H, s,
CH2Cl), 4.91 (2H, s, OCH2), 6.22 (1H, s, H-3 coumarin), 6.23 (1H, s, H-8 coumarin), 6.55 (1H,
d, H-6 coumarin), 7.00 (1H, d, H-5 coumarin),10.77, 10.98 (2H, s, 2NH, D2O exchangeable). 13C
NMR (DMSO-d6, δ, ppm): 19.42 (CH3), 45.66, 68.19, 108.19, 111.13, 113.55, 115.92, 128.11,
151.33, 152.98, 161.17, 161.93 (C=O coumarin), 166.14, 166.87. Anal. calcd. For C14H13ClN2O5
(324.72): C, 51.78; H, 4.04; N, 8.63, Found: C, 51.71; H, 4.09; N, 8.59.
4.1.4. General procedure for the synthesis of N'-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)
acetyl)-2-(substituted) acetohydrazide (6a-c)
16

To a well stirred solution of chloroacetohydrazide 5 (0.018 mol) in dry dimethylformamide (3
mL), different secondary amines namely: piperidine, N-methyl piperazine and morphline (0.002
mol) were added in the presence of (0.0022 mol) anhydrous potassium carbonate with
continuous stirring on cold at 20 ºC for 1 hour. The solution was poured onto ice/water to give a
precipitate which was collected, washed with water and crystallized from ethanol.
4.1.4.1. N'-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetyl)-2-(piperidin-1-
yl)acetohydrazide (6a)
Yield (81%); m.p. 175-177º C; 1H NMR (DMSO-d6, δ, ppm): 2.03 (3H, s, CH3), 2.18 - 3.01
(12H, m, 5CH2-piperidine, N-CH2), 4.80 (2H, s, OCH2), 6.25 (1H, s, H-3 coumarin), 6.63 (1H,
s, H-8 coumarin), 6.75 (1H, d, H-6 coumarin), 7.12 (1H, d, H-5 coumarin), 9.14,9.97 (2H, s,
2NH, D2O exchangeable).13C NMR(DMSO-d6, δ, ppm): 19.60 (CH3), 27.28, 28.00, 54.34, 60.10,
69.77, 109.63, 111.82, 112.65, 113.15, 128.46, 152.11, 153.28, 160.12, 160.98 (C=O pyrone),
165.66, 170.57. Anal. calcd. For C19H23N3O5 (373.4): C, 61.11; H, 6.21; N, 11.25, Found: C,
61.05; H, 6.24; N, 11.20.
4.1.4.2. N'-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetyl)-2-(4-methylpiperazin -1-yl)
acetohydrazide (6b)
Yield: 80%; mp: 220–222 ˚C; 1HNMR (DMSO-d6, δ, ppm): 2.15 (3H, s, CH3), 2.38 (3H, s, N-
CH3), 2.88 - 2.91 (10H, m, 4CH2-piperazine, N-CH2), 4.20 (2H, s, OCH2), 6.15 (1H, s, H-3
coumarin), 6.72 (1H, s, H-8 coumarin), 6.85 (1H, d, H-6 coumarin), 7.62 (1H, d, H-5 coumarin),
8.55,8.95 (2H, s, 2NH, D2O exchangeable).13C NMR(DMSO-d6, δ, ppm): 18.59 (CH3), 46.19
(N-CH3), 53.21, 53.42, 55.11, 68.90, 101.73, 110.91, 112.77, 113.06, 126.37, 154.02, 155.06,
160.80, 163.03, 166.18 (C=O pyrone), 169.48 (C=O). Anal. calcd. for C19H24N4O5 (388.42): C,
58.75; H, 6.23; N, 14.42. Found: C, 58.81; H, 6.32; N, 14.49.
4.1.4.3. N'-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetyl)-2-morpholinoacetohydrazide
(6c)
Yield (82%); m.p. 163-165º C; 1H NMR (DMSO-d6, δ, ppm): 1.99 (3H, s, CH3), 2.48-3.66 (10H,
m, 4CH2-morpholine, N-CH2), 4.86 (2H, s, OCH2), 6.11 (1H, s, H-3 coumarin), 6.54 (1H, s, H-8
coumarin), 6.85 (1H, d, H-6 coumarin), 7.23 (1H, d, H-5 coumarin), 10.01, 10.76 (2H, s, 2NH,
D2O exchangeable).13C NMR(DMSO-d6, δ, ppm): 20.63 (CH3), 52.34, 59.22, 65,71, 66.97,
119.72, 111.93, 112.97, 113.89, 127.55, 151.14, 152.26, 161.01, 161.98 (C=O pyrone), 166.56,
17

171.36. Anal. calcd. For C18H21N3O6 (375.38): C, 57.59; H, 5.64; N, 11.19, Found: C, 57.53; H,
5.66; N, 11.22.
4.1.5. General procedure for the synthesis of (E)-6-(substituted benzylideneamino)-4-
methyl-2H-chromen-2-one (8a,b)
To a well stirred solution of 6-amino coumarin 7 (0.001 mol) in chloroform, the appropriate
aldehydes namely: benzaldehyde and 2, 4- dihydroxy benzaldehyde, were dropwisely added,
then 2 mL glacial acetic acid was added. The reaction mixture was refluxed for 7- 9 hours. The
solid obtained was crystallized from ethanol.
4.1.5.1. (E)-6-(benzylideneamino)-4-methyl-2H-chromen-2-one (8a)
Yield 67 %; m.p. 215-217 º C; 1H NMR (DMSO-d6, δ, ppm): 2.12 (3H, s, CH3), 6.20 (1H, s, H-3
coumarin), 7.40-7.73 (8H, m, Ar-H), 8.14 (1H, s, HC=N). 13C NMR (DMSO-d6, δ, ppm): 18.40
(CH3), 110.51, 116.82, 120.78, 121.34, 129.18, 130.44, 132.31, 135.42, 149.81, 152.01, 153.17,
159.79 (C=N), 161.18 (C=O). Anal. calcd. For C17H13NO2 (263.29): C, 77.55; H, 4.98; N, 5.32,
Found: C, 77.51; H, 4.93; N, 5.35.
4.1.5.2. (E)-6-(2,4-dihydroxybenzylideneamino)-4-methyl-2H-chromen-2-one (8b)
Yield 74 %; m.p. 234-236º C; 1H NMR (DMSO-d6, δ, ppm): 2.12 (3H, s, CH3), 6.10 (1H, s, H-3
coumarin), 6.99-7.53 (6H, m, Ar-H), 8.87 (1H, s, HC=N), 9.72, 10.11 (2H, s, 2 OH, D2O
exchangeable). 13C NMR (DMSO-d6, δ, ppm): 19.11 (CH3), 102.27, 107.76, 111.52, 112.11,
117.88, 120.12, 121.29, 132.89, 148.14, 151.32, 152.07, 161.06 (C=N), 161.86 (C=O), 163.24,
164.50. Anal. calcd. For C17H13NO4 (295.28): C, 69.15; H, 4.44; N, 4.74, Found: C, 69.11; H,
4.38; N, 4.70.
4.1.6. General procedure for the synthesis (4-methyl-2-oxo-2H-chromen-6-yl) dione
derivatives (9a, b)
A solution of 6-amino coumarin 7 (0.1 mol) and the appropriate acid anhydrides namely phthalic
and glutaric anhydrides (0.1 mol) in glacial acetic acid (20 mL) were refluxed for 5-7 hours. The
reaction mixture was concentrated, and then water was added. The precipitate was filtered,
washed with water and crystallized from ethanol.
4.1.6.1. 2-(4-methyl-2-oxo-2H-chromen-6-yl)isoindoline-1, 3-dione (9a)
Yield 68 %; m.p. 233-235 º C; 1H NMR (DMSO-d6, δ, ppm): 1.91 (3H, s, CH3), 6.31 (1H, s, H-3
coumarin), 7.20- 8.44 (7H, m, Ar-H). 13C NMR (DMSO-d6, δ, ppm): 17.44 (CH3), 110.50,
18

114.41, 114.99, 121.32, 122.17, 125.74, 127.44, 130.22, 130.91, 148.82, 151.27, 160.08 (C=O),
168.47. Anal. calcd. For C18H11NO4 (305.28): 70.82; H, 3.63; N, 4.59, Found: , 70.80; H, 3.65;
N, 4.55.
4.1.6.1. 1-(4-methyl-2-oxo-2H-chromen-6-yl) piperidine-2, 6-dione (9b)
Yield 77 %; m.p. 240-142 º C; 1H NMR (DMSO-d6, δ, ppm): 1.99- 2.12 (6H, m, 3CH2 –
piperidine dione), 2.31 (3H, s, CH3), 6.12 (1H, s, H-3 coumarin), 7.15-7.90 (3H, m, Ar-H). 13C
NMR (DMSO-d6, δ, ppm): 16.1, 18.94(CH3), 30.19, 111.25, 115.64, 115.99, 120.32, 127.74,
131.09, 148.75, 152.19, 161.08 (C=O), 170.32. Anal. calcd. For C15H13NO4 (271.26): C, 66.41;
H, 4.83; N, 5.16, Found: C, 66.43; H, 4.80; N, 5.12.
4.1.7. General procedure for the synthesis of N-(4-methyl-2-oxo-2H-chromen-6-yl) amide
derivatives (10a, b)
To a well stirred solution of 6-amino coumarin 7 (0.1 mol) in dry dimethylformamide (5 mL),
the appropriate acid chloride namely: acetyl chloride and/or benzoyl chloride (0.1 mol) was
dropwisely added with continuous stirring on cold for 5-7 hours. The solution was poured onto
ice/water; the formed precipitate was collected, washed with water and crystallized from ethanol.
4.1.7.1. N-(4-methyl-2-oxo-2H-chromen-6-yl)benzamide (10b)
Yield 71 %; m.p. 250-252 º C; 1H NMR (DMSO-d6, δ, ppm): 2.32 (3H, s, CH3), 6.13 (1H, s, H-3
coumarin), 7.47- 7.95 (8H, m, Ar-H), 8.83 (1H, s, NH, D2O exchangeable). 13C NMR (DMSO-
d6, δ, ppm): 19.48 (CH3), 111.37, 111.60, 113.16, 114.93, 129.01, 129.70, 131.23, 133.30,
154.82, 160.61, 163.08, 164.61, 165.38, 167.80. Anal. calcd. For C17H13NO3 (279.29): C, 73.11;
H, 4.69; N, 5.02, Found: C, 73.06; H, 4.62; N, 5.05.
4.1.8. Procedure for the synthesis of 3-bromocoumarin (12)
To a stirred solution of the known coumarin 11 (0.0342 mmol) in acetic acid (20 mL) at room
temperature, a solution of bromine (0.0685 mmol) was added dropwisely over a time period of 1
hour. After the addition completed, the reaction was poured onto ice/water (300 mL) and
neutralized with NaOH solution (10%). The formed precipitate was filtered and dissolved in
DCM (150 mL) and was used in the next step without further purification. Triethyl amine was
added dropwise to the ice cold solution of the dibromocoumarin in DCM. After 1 hour the
reaction was quenched with HCl (10%, 100 mL), the bromocoumarin was extracted with DCM
(20 mL, 3x), dried (anh, CaCl2), concentracted in vacuo and the crude product was recrystallized
19

from ethanol (75%) to give 3-bromocoumarin 12 as colorless solid. (7.2 g, 94%); the spectral
data are consistent with that reported in literature.[35]
4.1.9. General procedure for the synthesis of Coumarin-6-Sulfonamides (14a-f)
To a stirred solution of 13 (0.001 mol) in toluene (10 mL) at room temperature, one drop of
triethyl amine was added followed by the addition of the selected amines, namely: morpholine,
piperidine, N-methyl piperidine, 2-aminobenzoimidazole, piperonylamine and 2,4,6-trimethoxy
benzyl amine (0.0011 mol). In a time period of 5 to 7 hrs, TLC showed the reaction is complete,
the formed solid was filtered and washed with toluene.
4.1.9.1. 3-bromo-6-(morpholinosulfonyl)-2H-chromen-2-one (14a)
Yield 86 %; m.p. 183-185 º C; 1H NMR (DMSO-d6, δ, ppm): 2.76 (4 H, s, 2 N-CH2 morpholine),
3. 7 (4 H, s, 2 O-CH2 morpholine), 7.60 (1 H, d, J = 8.7 Hz, H-8 coumarin), 8.15 (1H, dd, J = 1.4,
8.7 Hz, H-7 coumarin), 8.23 (1 H, d, J= 1.4 Hz, H-5 coumarin), 8.32 (1 H, s, H-4 coumarin). 13C
NMR (DMSO-d6, δ, ppm): 22.6, 32.5, 50.2, 56.6, 65.9, 120.9, 127.1, 127.2, 127.7, 128.25,
128.33, 128.5, 128.8, 138.8, 140.2, 146.7. Anal. calcd. For C13H12BrNO5S (374.21): C, 41.73; H,
3.23; N, 3.74, Found: C, 41.70; H, 3.17; N, 3.70
4.1.9.2. 3-bromo-6-(piperidin-1-ylsulfonyl)-2H-chromen-2-one (14b)
Yield 46 %; m.p. 221-224 ºC; 1H NMR (DMSO-d6, δ, ppm): 1.40- 1.60 (6 H, m, H-3, H-4
piperidine ), 3.17 (4 H, m, H-2 piperidine), 7.33 (1 H, d, J = 8.7 Hz, H-8 coumarin), 8.25 (1H,
dd, J = 1.4, 8.7 Hz, H-7 coumarin), 8.54 (1 H , d, J= 1.4 Hz, H-5 coumarin), 8.61(1 H , s, H-4
coumarin). 13C NMR (DMSO-d6, δ, ppm): 22.6, 32.5, 50.2, 56.6, 65.9, 120.9, 127.1, 127.2,
127.7, 128.25, 128.33, 128.5, 128.8, 138.8, 140.2, 146.7. Anal. calcd. For C14H14BrNO4S
(374.21): C, 45.17; H, 3.79; N, 3.76, Found: C, 45.13; H, 3.71; N, 3.70.
4.1.9.3. 3-bromo-6-((4-methylpiperazin-1-yl)sulfonyl)-2H-chromen-2-one (14c)
Yield 45%; m.p. 191-192 º C; 1H NMR (DMSO-d6, δ, ppm): 2.21 (3 H, s, CH3 ), 2.48 (4 H, s, H-
3 piperazine ), 2.97 (4 H, s, H-2 piperazine), 7.67 (1 H, d, J = 8.7 Hz, H-8 coumarin), 7.93 (1H,
dd, J = 1.4, 8.7 Hz, H-7 coumarin), 8.19 (1 H , d, J= 1.4 Hz, H-5 coumarin), 8.76 (1 H , s, H-4
coumarin ). 13C NMR (DMSO-d6, δ, ppm): 8.9 (CH3), 45.8, 53.7, 113.2, 118.1, 120.4, 128.3,
131.1, 131.8, 144.7, 155.7, 156.4. Anal. calcd. For C14H15BrN2O4S (387.25): C, 43.42; H, 3.90;
N, 7.23, Found: C, 43.44; H, 3.91; N, 7.18.
20

4.1.9.4. N-(1H-benzo[d]imidazol-2-yl)-3-bromo-2-oxo-2H-chromene-6-sulfonamide (14d)
Yield 54% %; m.p. 273-275 º C; 1H NMR (DMSO-d6, δ, ppm): 7.10- 7.32 (4 H, m, CH-
benzoimidazol), 7.70 (1 H, d, J = 8.7 Hz, H-8 coumarin), 8.12 (1H, dd, J = 1.4, 8.7 Hz, H-7
coumarin), 8.21 (1 H, d, J= 1.4 Hz, H-5 coumarin), 8.31(1 H, s, H-4 coumarin) , 8.55, 8.95 (2H,
s, 2NH, D2O exchangeable). 13C NMR (DMSO-d6, δ, ppm): 111.6, 111.8, 116.4, 119.1, 123.5,
125.4, 130.1, 145.2, 145.7, 151.0, 153.1, 157.0. Anal. calcd. For C16H10BrN3O4S (420.24): C,
45.73; H, 2.40; N, 10.00, Found: C, 45.70; H, 2.42; N, 10.05
4.1.9.5. N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-bromo-2-oxo-2H-chromene-6-sulfonamide
(14e)
Yield 60%; m.p. 173-176 º C; 1H NMR (DMSO-d6, δ, ppm): 3.95 (2 H , d, J= 6.1 Hz, CH2NH),
5.91 ( 2 H, s, CH2 dioxol ), 6.65 (1 H , d, J = 7.9 Hz, H-6 benzodioxol), 6.71 (1 H, s, H-4
benzodioxol), 6.74 (1H, d, J = 7.9 Hz, H-7 benzodioxol), 7.57 (1 H ,d, J= 8.7 Hz, H-8 coumarin),
7.93 (1 H , dd, J= 1.4, 8.7 Hz, H-7 coumarin), 8.09 (1 H , d, J= 1.4 Hz, H-5 coumarin), 8.30 (1 H
, t, J = 6.1 Hz, NH), 8.71 (1H , s, H-4 coumarin) ; 13C NMR (DMSO-d6, δ, ppm): 46.51, 101.34,
108.31, 108.62, 112.80, 117.86, 119.72, 121.62, 127.23, 130.15, 131.41, 137.82, 144.83, 146.72,
147.51, 154.99. Anal. calcd. For C17H12BrNO6S (438.25): C, 46.59; H, 2.76; N, 3.20, Found: C,
46.54; H, 2.72; N, 3.16
4.1.9.6. 3-bromo-2-oxo-N-(2, 4, 6-trimethoxybenzyl)-2H-chromene-6-sulfonamide (14f)
Yield 37%; m.p. 169-172 ºC; 1H NMR (DMSO-d6, δ, ppm): 3.52 (3 H , s, p-OCH3 ), 3.67 (6 H ,
s, 2 o-OCH3), 4.01 (2 H , d, J = 6.0 Hz, CH2N), 6.45 (2 H , s, H-3 phenyl), 7.53 (1H d, J = 8.7
Hz, H-8 coumarin), 7.91 (1 H , dd, J= 1.6, 8.7 Hz, H-7 coumarin), 8.10 (1 H , d, J= 1.6 Hz, H-5
coumarin ), 8.32 (1 H , t, J= 6.0 Hz, NH, D2O exchangeable ), 8.70 (1H , s, H-4 coumarin) ; 13C
NMR (DMSO-d6, δ, ppm): 47.01, 56.12, 60.23, 105.40, 112.74, 117.71, 119.58, 127.40, 130.29,
132.89, 136.71, 137.89, 144.80, 152.99, 154.87, 156.43. EIMS m/z (%): 485/483 (M+1, 15), 360
(86), 329 (66), 302(93), 288 (25), 224(18), 195 (93), 116(39), 88(100), 61 (65). Anal. calcd. For
C19H18BrNO7S (484.32): C, 47.12; H, 3.75; N, 2.89, Found: C, C, 47.09; H, 3.71; N, 2.83.
4.2. Biology
4.2.1. In vitro cytotoxicity assay on MCF-7
21

Cell viability was assessed by the mitochondrial dependent reduction of yellow MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to purple formazan.[30,31]
The cells used in cytotoxicity assay were cultured in RPMI 1640 medium supplemented with
10% fetal calf serum. Cells suspended in the medium (2 x 104 cells/mL) were plated in 96-well
culture plates and incubated at 37 °C in a 5% CO2 incubator. After 12 h, the test sample (2 μL)
was added to the cells (2 x 104) in 96-well plates and cultured at 37 °C for 3 days. The cultured
cells were mixed with 20 μL of MTT solution and incubated for 4 h at 37 °C. The supernatant
was carefully removed from each well and 100 μL of DMSO were added to each well to dissolve
the formazan crystals which were formed by the cellular reduction of MTT. After mixing with a
mechanical plate mixer, the absorbance of each well was measured by a microplate reader using
a test wavelength of 570 nm.
4.2.2. In vitro VEGFR-2 kinase activity assay by ELISA
ELISA (Enzyme Linked Immunosorbent Assay) kit according to manufacturer's instructions
(Cell Signaling Technology, Inc., USA) was used. The microtiter plate provided in this kit had
been pre‐coated with horseradish peroxidase (HRP) labeled secondary antibody (anti‐rabbit IgG)
specific to VEGFR‐2. Samples were added to the appropriate microtiter plate wells. Tetramethyl
benzidine (TMB) substrate was added and incubated at room temperature and the stop solution
was then added. The color change was measured colorimetrically at a wavelength of 450ꢀnm
using a microtiter plate reader. The concentration of test compounds causing 50% inhibition
(IC50) was calculated from the concentration inhibition response curve and compared to the
reference standard staurosporine.[36]
4.2.3. Cell cycle analysis and apoptosis assay
Cell cycle analysis and apoptosis detection were carried out by flow cytometry as reported.[33]
MCF-7 cells were seeded at 8x104 and incubated overnight at 37 °C and supplied with 5% CO2.
After 48 h of treatment, cell pellets were collected and centrifuged at 300 g for 5 min. For cell
cycle analysis, cell pellets were fixed in 70% ethanol on ice for 15 min. The collected pellets
were incubated with propidium iodide (PI) staining solution (50 μg/mL PI, 0.1 mg/mL RNaseA
and 0.05% Triton X-100) at room temperature for 1 h. Apoptosis detection was performed by
FITC Annexin-V/PI kit (Becton Dickenson, Franklin Lakes, NJ, USA) following the
manufacture’s protocol. The samples were analyzed by fluorescence-activated cell sorting
22

(FACS) with a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) within 1 h from the
staining. Data were analyzed using Kaluza v1.2 (Beckman Coulter).
4.2.4. Caspase-9 enzyme assay
The activity of caspase-9 was measured using Human CASP9 (Caspase- 9) ELISA Kit (Catalog#
MBS2505226, Invitrogen Corporation, USA) according to the manufacturer instructions.
4.3. Docking
All the molecular modeling studies were carried out using Molecular Operating Environment
(MOE, 10.2008) software. All minimizations were performed with MOE until an RMSD
gradient of 0.1 kcal∙mol−1Å−1 with MMFF94x force field and the partial charges were
automatically calculated. The X-ray crystallographic structure of vascular endothelial growth
factor receptor 2 (VEGFR-2) co-crystallized with the ureide derivative sorafenib [IC50 = 90nM]
(PDB ID: 4ASD) was downloaded from the protein data bank.[37] Water molecules and ligands
that are not involved in binding were removed. Then, the protein was prepared for docking study
using Protonate 3D protocol in MOE with default options. The co-crystalized ligand was used to
define the binding site for docking. Triangle Matcher placement method and London dG scoring
function were used for docking. Docking setup was first validated by re-docking of the co-
crystallized ligand (sorafenib) in the vicinity of the binding site of the enzyme with energy score
(S) = −15.20 kcal/mol and RMSD of 0.467 Å and by the ability of the docking pose to reproduce
all the key interactions accomplished by the co-crystallized ligand with the hot spots in the active
site (Glu885, Cys919 and Asp1046). The validated setup was then used in predicting the ligand-
receptor interactions at the binding site for the compound of interest.
4.4. In silico physicochemical properties and pharmacokinetic parameters assessment
The drug-likeness and ADME properties of the most active compound in this study, were tested
in silico with the online Swiss ADME web tool.[38] Swiss ADME employs a set of robust models
for prediction of physicochemical properties, pharmacokinetics, drug-likeness and medicinal
chemistry friendliness.
Acknowledgment
The authors extend their appreciation to the National Research Centre. Dokki, Cairo, Egypt, for
granting this work (fund number: AR110216). They also convey their thanks to Dr. Essam
Rashwan, head of confirmatory diagnostic unit, Vacsera-Egypt, for performing the biological
23

screening and to the Molecular Modeling Unit-Faculty of pharmacy – Cairo University as well
for performing the docking and pharmacokinetic studies.
Author Information
Corresponding Author
*Omaima M. Abdelhafez, +20 202 37608284. Fax: +20 202 33370931; Email:
dromaima45@gmail.com
Conflicts of Interest:
Authors declared that there are no actual or potential conflicts of interest, and have approved the
article.
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