Design, synthesis and biological evaluation of novel coumarin-based benzamides as potent histone deacetylase inhibitors and anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2017.03.024

Histone deacetylases (HDACs) are attractive therapeutic targets for the treatment of cancer and other diseases. It has four classes (I-IV), among them especially class I isozyme are involved in promoting tumor cells proliferation, angiogenesis, differentiation, invasion and metastasis and also viable targets for cancer therapeutics. A novel series of coumarin-based benzamides was designed and synthesized as HDAC inhibitors. The cytotoxic activity of the synthesized compounds (8a-u) was evaluated against six human cancer cell lines including HCT116, A2780, MCF7, PC3, HL60 and A549 and a single normal cell line (Huvec). We evaluated their inhibitory activities against pan HDAC and HDAC1 isoform. Four compounds (8f, 8q, 8r and 8u) showed significant cytotoxicity with IC₅₀ in the range of 0.53–57.59?μM on cancer cells and potent pan-HDAC inhibitory activity (consists of HDAC isoenzymes) (IC₅₀?=?0.80–14.81?μM) and HDAC1 inhibitory activity (IC₅₀?=?0.47–0.87?μM and also, had no effect on Huvec (human normal cell line) viability (IC₅₀?>?100?μM). Among them, 8u displayed a higher potency for HDAC1 inhibition with IC₅₀ value of 0.47?±?0.02?μM near equal to the reference drug Entinostat (IC₅₀?=?0.41?±?0.06?μM). Molecular docking studies and Molecular dynamics simulation of compound 8a displayed possible mode of interaction between this compound and HDAC1enzyme.

Full text of DOI:10.1016/j.ejmech.2017.03.024

Accepted Manuscript
Design, synthesis and biological evaluation of novel coumarin-based benzamides as
potent histone deacetylase inhibitors and anticancer agents
Tooba Abdizadeh, Mohammad Reza Kalani, Khalil Abnous, Zahra Tayarani, Bibi
Zahra Khashayarmanesh, Rahman Abdizadeh, Razieh Ghodsi, Farzin Hadizadeh
PII:
S0223-5234(17)30180-0
10.1016/j.ejmech.2017.03.024
EJMECH 9284
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 January 2017
Revised Date: 6 March 2017
Accepted Date: 14 March 2017
Please cite this article as: T. Abdizadeh, M.R. Kalani, K. Abnous, Z. Tayarani, B.Z. Khashayarmanesh,
R. Abdizadeh, R. Ghodsi, F. Hadizadeh, Design, synthesis and biological evaluation of novel coumarin-
based benzamides as potent histone deacetylase inhibitors and anticancer agents, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.024.
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ACCEPTED MANUSCRIPT
Graphical Abstract
A novel series of coumarin-based benzamides was designed and synthesized as HDAC
inhibitors. Compound 8u showed potent anti-proliferative activity and HDAC1 inhibitory
activity near equal to the reference drug Entinostat.
O
N
H
NH2
H
N
N
O
O
O
R3
R2
N
NH2
Chidamide
O
F
H
H
N
O
R1
O
O
N
H
NH2
H
N
Cap CU
linker
ZBG
N
Designed Structures
O
Entinostat
IC50 (µM)
compound
R
2
HDAC1
HCT116
Huvec (normal cell)
8
u
3,4-di-Cl-benzyloxy
-
0.47±0.02
0.41±0.06
1.76±0.30
2.03±0.26
>100
>100
Entinostat

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Design, Synthesis and Biological Evaluation of Novel Coumarin-Based Benzamides as
Potent Histone Deacetylase Inhibitors and Anticancer Agents
a,b
c,d
e
f
Tooba Abdizadeh,
Mohammad Reza Kalani, Khalil Abnous, Zahra Tayarani, Bibi Zahra
b
g
a,b
a,b
Khashayarmanesh, Rahman Abdizadeh, Razieh Ghodsi, * Farzin Hadizadeh *
a
Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
b
Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical
Sciences, Mashhad, Iran
c
School of Cell and Molecular Biology, University of Illinois at Urbana-Champaign, Urbana,
United States
d
Department of Molecular Medicine, Golestan University of Medical Sciences, Golestan, Iran
e
Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
f
Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of
Medical Sciences, Mashhad, Iran
g
Department of Medical Parasitology and Mycology, Faculty of Medicine, Shahrekord
University of Medical Sciences, Shahrekord, Iran
*
Corresponding authors:
Farzin Hadizadeh, Biotechnology Research Center, Mashhad University of Medical Sciences,
Mashhad, Iran; hadizadehf@mums.ac.ir
Razieh Ghodsi, Department of Medicinal Chemistry, School of Pharmacy Mashhad University
of Medical Sciences, Mashhad, Iran; ghodsir@mums.ac.ir

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Abstract
Histone deacetylases (HDACs) are attractive therapeutic targets for the treatment of cancer and
other diseases. It has four classes (I-IV), among them especially class I isozyme are involved in
promoting tumor cells proliferation, angiogenesis, differentiation, invasion and metastasis and
also viable targets for cancer therapeutics. A novel series of coumarin-based benzamides was
designed and synthesized as HDAC inhibitors. The cytotoxic activity of the synthesized
compounds (8a-u) was evaluated against six human cancer cell lines including HCT116, A2780,
MCF7, PC3, HL60 and A549 and a single normal cell line (Huvec). We evaluated their
inhibitory activities against pan HDAC and HDAC1 isoform. Four compounds (8f, 8q, 8r and
8
u) showed significant cytotoxicity with IC in the range of 0.53-57.59 µM on cancer cells and
50
potent pan-HDAC inhibitory activity (consists of HDAC isoenzymes) (IC = 0.80-14.81 µM)
5
0
and HDAC1 inhibitory activity (IC = 0.47-0.87 µM and also, had no effect on Huvec (human
5
0
normal cell line) viability (IC50 >100 µM). Among them, 8u displayed a higher potency for
HDAC1 inhibition with IC50 value of 0.47±0.02 µM near equal to the reference drug Entinostat
(
IC50 = 0.41±0.06 µM). Molecular docking studies and Molecular dynamics simulation of
compound 8a displayed possible mode of interaction between this compound and
HDAC1enzyme.
Key words:
Histone deacetylase inhibitors, coumarin, 2-Aminobenzamide, Docking,
Molecular dynamics
Abbreviations HDAC, Histone Deacetylases; HATs, Histone Acetyl Transferases; HSP 90,
Heat Shock Protein 90; SAHA, suberoylanilide hydroxamic acid; TSA, Trichostatin A; ZBG,
Zinc Binding Group; CU,
Carbonyldiimidazole.
Connect Unit; MD, Molecular Dynamics; CDI, N, N´-

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1
.
Introduction
Cancer, the uncontrolled, rapid and pathological proliferation of abnormal cells, is one of the
most life-threating diseases and cause of death worldwide. Cancer causes about 550,000 deaths a
year and is second leading cause of death in the world next to heart diseases. [1, 2] The
numerous drugs have been used for the cancer treatment but have severe side effects.
Consequently; increasing interest has been devoted to the design and discovery of more effective
anticancer agents with promising activity and high therapeutic index in current medicinal
chemistry. Over the recent years, a great many of important targets such as the histone
deacetylase (HDAC) has been considered for anticancer therapies. HDACs and histone acetyl
transferases (HATs) are crucial post-translational modification and play a pivotal role in the
epigenetic regulation of gene expression through chromatin modification. [3-6] Histone acetyl
transferase (HATs) catalyzes acetylation of positive charged lysine residues and leads to the
relaxation of chromatin and activates transcription. [7] Inversely, histone deacetylase (HDAC)
catalyze the removal of acetyl groups of the ε-amino of lysine residues on core histone and other
cellular proteins (e.g. HSP 90, tubulin), resulting in chromatin condensation and transcriptional
repression. [8, 9]
HDACs have an important effect on gene transcription, the cell cycle, differentiation, apoptosis
and tumourigenesis. Therefore, HDACs have been become as prominent therapeutic target for a
broad range of human disorder such as cancer. [10] There are 18 isoforms of human HDAC,
divided into four main classes based on their homology to yeast models, subcellular localization
and enzymatic activities. [11]
HDACI (HDAC 1, 2, 3 and 8), class IIa (HDAC 4, 5, 7 and 9), class IIb (HDAC 6 and 10) and
class IV (HDAC11) are zinc-dependent enzymes, whereas class III HDACs (sirtuins 1-7) are

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+
NAD dependent enzymes. [12-14] Class I HDACs are homologous to yeast Rpd3 and are
predominantly located in the nucleus (350-500 amino acids in length) and class II HDACs have
sequence homology to yeast Hda2 and shuttle between the cytoplasm and nucleus (about 1000
amino acids in length). [15-17] Class III are homologues of the yeast Sir2 and class IV shows the
characteristics of both class I and II HDACs. [18]
2
+
Zn -dependent HDACs, especially class I isozymes are involved in promoting tumor cells
proliferation, angiogenesis, differentiation, invasion and metastasis and also viable targets for
cancer therapeutics. [19] Among the class I HDAC isoforms, HDAC1 has a key role in cancer
[20] and overexpressed in prostate, ovarian, breast, colon, leukemia and pancreas cancers. [21]
HDAC inhibitors (HDACIs) are categorized into four classes according to their chemical
structure in clinical studies: hydroxamic acids (Trichostatine A (TSA)), [22] (Vorinostat
(SAHA)), [23] (Panobinostat (LBH-589)), [24] (Belinostat (PXD-101)), [25] benzamides
((Entinostat (MS-275)), [26] (Mocetinostat (MGCD-0103)), [27, 28] (Chidamide (CS-055)), [29]
(Tacedinaline (CI-994)), [30] short-chain fatty acids (valproic acid) [31, 32] and depsipeptides
(Romidepsin (FK-228)). [33] Up to now, five HDACIs Vorinostat, Romidepsin, Belinostat,
Chidamide and Panobinostat approved by FDA for the treatment of lymphoma or myeloma. [34]
The structures of several approved and clinical HDACI are shown in Fig.1.
Insert Fig. 1.
Development of selective isotope HDACIs is a significant way for prevention of the side effects
of HDACIs. Many hydroxamate HDACIs do not exert excellent selectivity toward a specific
isoform, while 2-aminobenzamides exert some isoform selectivity such as Entinostat and
Mocetinostat are relatively unique in inhibiting HDAC 1, 2 and 3. [35]

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Despite the huge structural diversity, the HDACIs generally have a general pharmacophore
model [36]: zinc binding group (ZBG) such as hydroxamic acid and 2-amino benzamide which
2
+
interacting to Zn at the bottom of active site and a requirement for the possession of HDAC
inhibitory activity, a hydrophobic linker occupying the narrow tunnel of HDACs and connect the
ZBG and the cap group, a polar connect unit (CU) that connecting cap group and linker, a
surface recognition group (cap), a hydrophobic and aromatic or heteroaromatic group, is
essential for recognizing and interacting with residues on the rim of active site of HDACs. [37]
A cap group determines the potency, stability, bioavailability and efficacy of the compounds.
[11, 38] Coumarins (2H-1-benzopyran-2-one or 2H-chromen-2-one), an important bicyclic
heterocycle as a part of flavonoid group of plant metabolite, are a wide class of natural and
synthetic compounds that show anticancer, [39] anti-HIV, [40] anti-Alzheimer,
[
41]antimicrobial, [42]antioxidant [43] and antiviral [44] activities. Coumarin and its derivatives
have rare nephrotoxicity, hepatotoxicity, cardio toxicity, dermal toxicity and other side effect.
45]
[
In the present study some new coumarin-based benzamides have been designed and synthesized
as HDAC inhibitors, as an attempt to check if the replacement of the benzyl carbamate moiety of
Entinostat or (E)-3-(pyridin-3-yl)acrylamide moiety of Chidamide with the coumarin
carboxamide are bioisosteric. The rationale for the design of these compounds was depicted in
Fig. 2. We selected Entinostat and chidamide as the lead compounds and our design strategy for
novel HDAC inhibitors was based on the common pharmacophore model of HDACs.
Insert Fig. 2.

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The synthesized compounds were evaluated for their cytotoxic activity against six different
cancer cell lines including HCT116 (human colon cancer cells), A2780 (human ovarian cancer
cells), MCF7 (human breast cancer cells), PC3 (human prostate cancer cells), HL60 (Human
promyelocytic leukemia cells) and A549 (adenocarcinoma human alveolar basal epithelial cells)
and normal Huvec cell line (Human Umbilical Vein Endothelial Cells). We evaluated their
inhibitory activities of pan HDAC and HDAC1 isoform. The structure activity relationships
(SAR) study performed on the cap moiety by investigating the electronic effects of the alkoxy or
benzyloxy ring with various substituents. Docking and molecular dynamics (MD) simulation
were performed for further investigating interaction of compounds with HDAC1.
2
2
. Results and discussion
.1. Chemistry
The synthetic routes to target compounds containing the coumarin ring were illustrated in
Schemes 1-3. Appropriately, ethyl coumarin-3-carboxylates 3a-e were synthesized through the
knoevenagel condensation of salicylaldehydes 1a-e with diethyl malonate in the presence of
catalytic amount of piperidine in ethanol. Then, O-alkylation or O-benzylation of the 7-hydroxy
derivative 3e with alkyl halides or benzyl halides was done in DMF in the presence of potassium
carbonate. The ethyl esters 3a-v were hydrolyzed with aqueous solution of sodium hydroxide to
the corresponding coumarin-3-carboxylic acids 4a-u (Scheme 1). [46]
Insert Scheme 1
The condensation of the carboxylic acids 4a-u with 4-(aminomethyl) benzoic acid using N, N´-
carbonyldiimidazole (CDI) and trifluoroacetic acid (TFA) in dry THF at room temperature led to
the formation of compounds 6a-u (Scheme 2). [30]

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Insert Scheme 2
Finally, compounds 6a-u were converted into imidazole intermediate with CDI at 55-60 °C in dry
THF, then reacted in situ with o- phenylene diamine in the presence of TFA at room temperature
to obtain the target benzamide analogs 8a-u as depicted in Scheme 3. The chemical structures of
1
13
final compounds were characterized by H NMR, C NMR, elemental analysis, IR and MS
spectroscopy.
Insert Scheme 3
2
2
.2. Biological Evaluation
.2.1. In Vitro Anticancer Activity
As depicted in Table 1-3, the antiproliferative activity of the target compounds (8a-u) was
evaluated against seven human cell lines including HCT116, A2780, MCF7, A549, PC3, HL60
and Huvec by 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
using Entinostat as the positive control. Most of the target compounds showed significant anti
proliferative activity with the IC values in micromolar range (0.27 to 80 µM) over all cell lines.
5
0
Compounds 8k was the most potent compound with IC values of 8.48, 10.14 and 16.6 µM in
5
0
MCF7, A2780 and PC3, respectively. This compound also showed superior activity against
HCT116, A549 and HL60 cells (IC = 0.27, 1.69 and 3.14 µM) compared with the reference
5
0
drug Entinostat (IC50 = 2.03, 3.11 and 4.53 µM). Overall, the majority of the synthesized
compounds were more potent against cell proliferation in HCT116 and A2780 cell, indicating
that our compounds exerted their cytotoxic activity with different mechanisms in different tumor
cells. Another interesting aspect of the results was that A549 and HL60 cell lines were sensitive

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to all the coumarin-based derivatives (IC50 < 80 µM). Some of the compounds showed promising
antiproliferative against PC3 (8i-k; 8n-u) and MCF7 cells (8e-u) with IC < 80 µM.
5
0
Insert Table 1
2
.2.2 Cellular HDAC Inhibition Assay of the Target Compounds
Since our compounds showed more cytotoxicity effects in human colon cancer HCT116 and
ovarian cancer A2780 cell lines in comparison to other cell lines, in vitro pan-HDAC inhibitory
activity of the synthesized target compounds 8a-u were evaluated on these two cell lines in
comparison with Entinostat as the reference drug, the results are presented in Table 2.
Insert Table 2
Since the linker and metal binding moieties of compounds 8a-u were identical with Entinostat;
differences in cellular effects were most likely due to the coumarin portion of the molecules as
cap group. In general benzyloxy coumarin derivatives showed stronger HDAC inhibitory activity
in comparison to alkoxy coumarin derivatives; this might be because of their more lipophilicity.
SAR data showed that the position and the length of alkoxy group have key roles in their HDAC
inhibitory and cytotoxicity activities. Compounds possessing methoxy or ethoxy groups on
position 7 of coumarin (8d and 8e) showed more cytotoxicity activity than their 8-alkoxy
isomers (8b and 8c). Among alkoxy coumarin derivatives, 7-propoxy derivative showed the most
HDAC inhibitory and antiproliferative activities in most of cancer cells. Compound 8k
possessing p-tolyloxy moiety was the most potent HDAC inhibitor with IC value of 0.25 and
5
0
2
.06 µM, in HCT116 and A2780 cell lines respectively. This compound displayed a higher
HDAC inhibitory activity (IC = 1.96 in HCT116) than Entinostat and IC =3.15 µM in A2780
5
0
50

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cell line). Compounds 8q and 8u also exhibited potent HDAC inhibitory activities with IC50
values of 0.42, 0.80 µM in HCT116 and 5.41, 4.90 µM in A2780 cancer cells, respectively.
We observed a significant correlation between HDAC inhibition and MTT data (r> 0.8). [47]
This correlation supported our hypothesis that HDAC inhibition mediates cytotoxicity (Fig. 3.)
Small differences between this assay and cytotoxicity values may be attributed to various
incubation times used in these tests - 48h for MTT against 18h for HDAC inhibition assay.
Insert Fig. 3.
From the point of view structure activity relationship, compound 8a as the simplest compound
with no substitution on the cap group displayed significant activity against HDAC in both cell
lines (11.41 µM in HCT116 and 54.92 µM in A2780). The substitutions at the different positions
on the benzene ring of coumarin affected the HDAC inhibition activity. Introduction of 6-bromo
substituent on coumarin ring increased inhibitory activity in compound 8g. Also, the presence of
8
-methoxy and 8-ethoxy substitutions showed higher inhibitory than parent compound 8a. 7-O-
alkyl or O-benzyl derivatives could increase the activity. In the 7-alkoxy derivatives 8d-f, 7-
propoxy coumarin derivative 8f displayed the significant activity with IC 1.09 and 14.81 µM
5
0
on cell lines. These results suggested that bulky R groups were beneficial for binding of these
molecules to HDAC, which may be related to the hydrophobic effect of the cap groups to
penetrate the cell membrane and also enlargement of cap group size facilitated occupation of the
active site and thus prevented the substrate from entering to the catalytic center.
The O-benzyl derivative 8h with IC values of 2.49 and 12.82 µM in HCT116 and A2780 cell
5
0
lines respectively was about four times more potent than compound 8a.

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The variety and position of substitutions on benzyl ring of coumarin played important role in
inhibitory activity. Potency in the O-benzyl derivatives could be further improved by the
introduction of electron-donating or electron-driving substitutions on the benzyl ring of the
coumarin. About these substitution, a trend of the inhibition enzymatic values is observed for
methyl > methoxy > bromo > Chloro > Fluoro > H. Compounds 8i-k with o, m or p- methyl
group on O-benzyl demonstrated significant HDAC inhibition with IC of 1.40 (o-), 1.31 (m-),
5
0
0
2
.25 µM (p-) in HCT116 in comparison with the values of IC50 in A2780 (20.84, 10.10±0.59 and
.06 µM respectively). Compound 8q with a 7-bromo substituent was the most potent inhibitor
between halo-substituted compounds (8l-q). This might be due to less electronegativity of bromo
in comparison to fluoro, so the π-electron density on the benzyl ring with bromo substituent was
a little dense. Thus, the π-π interaction between benzyl and the HDAC active site might be
slightly stronger. On the other, an o-substituent, in comparison with para or meta substituent, on
the benzyl decreased the HDAC inhibition activity (8i vs 8j and 8k; 8l vs. 8m and 8n; 8o vs 8p).
These findings confirmed that position of the substitutions on the O-benzyl group had significant
effect on enzyme activity. By comparing IC50 values of chloro derivatives, it could be concluded
that the introduction of second chloro atom onto the benzyl ring dramatically changed the anti-
HDAC activity and 3,4-dichloro derivative 8u with 0.80 and 4.90 µM in HCT116 and A2780
was more potent than 2,3-dichloro 8s and 2,4-dichloro 8t derivatives (IC =7.66, 38.72 (8s) and
5
0
IC =6.32, 27.57 (8t)). Also, SAR data suggested that the introduction of the O-benzyl group
5
0
into C-7 position of coumarin derivatives improved the HDAC inhibition potency than their O-
alkyl group. In general, modification of the lipophilic cap group led to increase the inhibitory
activity of the target compounds against HDAC.
Direct HDAC1 inhibition assay which will be discussed below, confirmed the latter (Table 3).

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ClogP is an important factor in membrane permeability and hence antiproliferative activity.
Theoretical logP (Clog P) values (http://www.organic-chemistry.org/prog/peo/) of compounds
also were measured. These compounds had ClogP values in range of 2.43-5. (Table 2). In
general there is a correlation between ClogP values and HDAC inhibition and MTT data.
Compounds with more ClogP values, showed more activity in comparison with those with less
ClogP.
2
.2.3. HDAC1 Inhibition Assay of the target Compounds
In effort for discovery of anticancer drugs, HDAC1 protein was targeted since it is widely
implicated in both transcriptional repression and chromatin remodeling. Thus, the most potent of
compounds were selected for HDAC1 inhibition assay which exhibited higher cytotoxic effect
and HDAC inhibitory potency (Table 2) and were not toxic to normal cells (IC >100µM).
5
0
Insert Table 3
As shown in Table 3, these compounds inhibited the deacetylase activity of HDAC1
significantly. Compound 8u inhibited HDAC1 with an IC value of 0.47 µM and showed near
5
0
equal effect to Entinostat (IC =0.41 µM). Compound 8u showed more selectivity of
5
0
HDAC1/HDAC than three other tested coumarins. Compounds 8f, 8q and 8r also, exhibited
significant inhibitory activity in the HDAC1 inhibition assay. These results demonstrated that
coumarin ring is an effective surface recognition cap for HDAC1 inhibition.
2
.2.4. In Vitro Cytotoxicity of Target Compounds in Normal Cells
To assess toxicity of target compounds in normal cells, Huvec cells were selected and treated
with all of the compounds under the same conditions. As shown in Table 4, lots of compounds
were nontoxic (IC > 100 µM). In vitro therapeutic index (IVTI) was calculated by comparing
5
0

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cytotoxicity activity (IC50) of compounds against HCT116 cells, which showed best sensitivity to
4
8
test compounds, with that of compounds against Huvec cell line . IVTI was found to be above
6
. This data suggested selective toxicity for our compounds.
Insert Table 4
2
.3. Molecular Docking Studies
In order to investigate the binding mode of the target compounds in the catalytic site of the
HDAC1 (PDB ID: 4BKX), docking studies was carried out using Auto Dock 4.2. The best pose
of each ligand in terms of free energy of binding was extracted from the 100 generated top poses
the binding mode of compounds 8a-g and the most potent compounds 8f, 8q, 8r, 8u and
Entinostat are shown in Fig. 4. Coumarins derivatives docked well to HDAC1 and bound similar
to Entinostat.
Insert Fig. 4.
The docking pose of 8a, the simplest coumarin in the series, in complex with HDAC1 was
shown in Fig. 5. and 6. HDAC1 is a zinc-dependent enzyme and its active site consisted of a
long, narrow tunnel leading to a cavity that contains the catalytic Zn ion.
Insert Fig. 5.
Zn ion was penta-coordinated with Asp176, His178 and Asp264 as well as the carbonyl and
aniline of benzamide of compound 8a. The protons of aniline formed hydrogen bonds with
His140 and His 141 while NH of amide could also form a hydrogen binding interaction with Gly

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1
49. The carbonyl oxygen of the benzamide group made hydrogen binding interaction with the
phenolic hydroxyl group of Tyr303. Nevertheless, the aryl linker region of compound 8a was
located in the lipophilic tube including Leu271, Phe 205, Phe150, Met39 and Glu98. There was
also π-π stacking interaction with benzene group of Phe150 which improved the binding affinity
of compound 8a to HDAC1.
Insert Fig. 6.
i
Linear regression analysis of calculated binding affinities, K versus experimental cytotoxicity
values, IC50 (Fig. 7.) showed that there was maximum correlation in case of HCT116 cells
2
2
(
r=0.88; r =0.78) and minimum correlation in PC3 cells (r=0.58; r =0.35).
Insert Fig. 7.
2
.4. Molecular Dynamic Simulation
Molecular dynamic simulation has been done to elucidate behavior of HDAC1 enzyme upon
binding to inhibitor and stability and interaction of structure throughout simulation. The dynamic
stability of secondary structure elements and conformational changes in ligand-bond protein
were compared with apo-form of HDAC1 by root-mean-square derivations (RMSD) and root
mean square fluctuation (RMSF) plots for 70 ns simulations.
RMSD is a measure of the stability of the structures. The Fig. 8. showed that the RMSD of
backbone (C , C, and N) of apo-form and ligand- bond protein 8a reached stability after about
α
◦
2
0ns of simulation. The average RMSD value of apo-form was 1.88±0.133 A , whereas the
ligand-bond protein was also converged very close to apo-form with average RMSD value of
◦
◦
2
.27±0.15 A . The average RMSD value of ligand 8a was 0.04±0.024 A (Fig. S1.) and ligand

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was stable during simulation. In RMSD of ligand-bond protein, the change was slightly larger
than apo-form. It seemed that binding of ligand with protein increased the conformation
flexibility of HDAC1 enzyme.
Insert Fig. 8.
A root mean square fluctuation (RMSF) is an indicator of the macromolecular flexibility and
indicates local changes in the protein structure. [49] The RMSF plots of apo-form and ligand-
bond protein was illustrated in Fig. 9. and Fig. S2 in the Supplementary data. The protein
structures of the two systems had similar RMSF distribution and same trends of dynamic
features. The average RMSF of ligand-bond protein and apo-form were 0.73±0.456 and
◦
0
.85±0.533 A , respectively. The relatively lower RMSF values of ligand-bond protein were
found for residues located in the binding site (Asp176, His 178, Gly149, Asp264, His 140, and
His 141and Tyr303). This could be explained by the relatively stronger binding of ligand with
these residues that led to their less flexibility. The high fluctuating residues indicated that they
were far from the active site or located in the loops (regions marked in bracket lines in Fig. 9.) or
terminals of protein.
Insert Fig. 9.
To analyze in a more quantitative way, contribution of different residues at the active site
(His140,His141, Gly149, Asp176, His178 , Asp264 and Tyr303) in electrostatic, VdW and total
interaction energy between ligand and HDAC1 was calculated (Fig. S3 in Supplementary data).
Radius of gyration (Rg) is a parameter that describes the equilibrium conformation of a total
system and is an indicator of the protein structure compactness. As shown in Fig. 10, Rg values
◦
of the apo-form and ligand-bond protein 8a were 20.53±0.095 and 20.48±0.085 A (mean±SD),

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respectively. The graphs showed that radius of gyration of apo-form was increased for the first
2
0 ns of simulation and then remained constant. It was observed that Rg of the ligand-bond
protein was constant and larger than apo-form.
Insert Fig. 10.
Hydrogen bonds play an essential role in stability ligand-protein complex. This stability of the
hydrogen-bond network formed by the ligand in the protein active site analyzed in Fig. 11. The
results showed that ligand formed three to four hydrogen bonds with protein. The benzamide
group of ligand made three hydrogen bonds with His 140, His 141 and Gly 149 and also, the
benzamide carbonyl of ligand could form strong bond with hydroxyl of Tyr 303. These hydrogen
bonds increased binding affinity of ligand with HDAC. The mean of intramolecular hydrogen
bonds of the protein were 80±7.19. These hydrogen bonds stabilized the secondary structure of
HDAC1 (Fig. S4 in Supplementary data).
Insert Fig. 11.
The percentage of protein secondary structures was investigated and it was found to be
unchanged during simulation (Fig. S5. and S6 in Supplementary data).
Another simple way to measure the stability of system is potential energy. The ligand bond
protein potential energy was found to be -33259.5±13.35 Kcal/mol indicating the stability of the
system (Fig. S7 in Supplementary data).
2
+
The average distance between coordinated Zn ion with active site residues (Asp176, His178
◦
and Asp264) was in the range 2.02±0.05 to 3.90±0.19 A (Fig. S8 in Supplementary data). In
2
+
addition, the distance between carbonyl and amino groups of ligand with Zn ion was in the

ACCEPTED MANUSCRIPT
◦
range of 2.09±0.07 to 2.21±0.08 A .These results improved the expected penta-coordination
complex between HDAC1 residues and ligand with zinc ion at the enzyme active site. To
discover the system condition during simulation, ligand bond protein structure was extracted
from trajectories for each 5 ns (Fig. S9 in Supplementary data). These snapshots proved fixed
orientation of ligand at the active site of the HDAC1 throughout simulation.
The 2D representation of the interaction between compound 8a after 70ns simulation by LigPlot
[50] has been depicted in Fig. 12.
Insert Fig. 12.
2
.5. ADME Analysis
The ADME parameters of coumarin compounds and Entinostat as drug reference were measured
using QikProp software (Table 5). All of the coumarins showed good partition coefficient
(QplogPo/w) in permeable range of 2.90-5.62. The Caco-2 cell permeability (QPPCaco) was in
the permissible range of 126.98 to 128.52. Also, the percentage human oral absorption for the
compounds ranged from 70 to 86%. All of the parameters were within the acceptable range
defined for human use and these compounds may exhibit significant pharmacokinetic and drug
likeliness properties.
3
. Conclusion
A new class of coumarin-based benzamides as HDAC inhibitors was designed, synthesized and
evaluated for their in vitro antiproliferative activities by MTT assay against human cancer
(HCT116, A2780, MCF7, PC3, HL60 and A549) and normal (Huvec) cell line as well as for pan
HDAC and HDAC1 isoform inhibitory activities. Most of compounds exhibited significant

ACCEPTED MANUSCRIPT
cytotoxicity and potent HDAC inhibition activity. Four compounds (8f, 8q, 8r and 8u) showed
superior cytotoxicity activities (IC = 0.53-57.59 µM) on cancer cells and higher enzymatic pan
5
0
HDAC (IC = 0.80-14.81 µM) and HDAC1 (IC = 0.47-0.87 µM) inhibitory activities and also,
5
0
50
had no effect on Huvec viability (IC >100 µM). Among them, 8u displayed a higher potency
5
0
for HDAC1 inhibition with IC50 value of 0.47±0.02 µM comparable with Entinostat (IC50=
.41±0.06 µM). Molecular docking studies of coumarin derivatives showed that they could bind
0
tightly to the active site of HDAC1. Molecular dynamics simulation showed interaction mode of
lead compound 8a with HDAC1 including zinc ion coordination, strong hydrophobic interactions
and formation of hydrogen bond with lead compound 8a.These findings suggested that coumarin
derivatives could be promising lead compounds for further development of anticancer agents
through HDACs inhibition.
4
4
. Experiment Section
.1. Chemistry
All reagents and solvents were purchased from Merck & Co., Inc. (Darmatadt, Germany) and
Acros Organics (Thermofisher, Belgium) and used without further purification. Entinostat and
@
vorinostat were purchased from EuroAsia Chemicals (www.euroasiarnd.com). The progress of
all reactions was monitored by thin-layer chromatography with 0.25mm Silica gel plates (60 GF-
2
54-Merck & Co (Darmatadt, Germany)) and visualized using UV light and iodine vapor.
Melting points were measured with an electrothermal melting point apparatus (Stafford, UK) and
were uncorrected. Infrared spectra were recorded on a Perkin Elmer Model 1420 spectrometer
1
13
(
KBr disks, Massachusetts, USA). H NMR and CNMR spectra were determined at 300 MHz
and 75 MHz, respectively (DMSO-d , TMS) on a Bruker FT-300 MHz instrument (Karlsruhe,
6

ACCEPTED MANUSCRIPT
Germany). The chemical shifts (δ) and coupling constants (J) are expressed in parts per million
and Hertz, respectively. Spin multiples are given as s (singlet), d (doublet), t (triplet), q (quartet),
s (sextet), m (multiplet). Mass spectra were obtained from a 6410 Agilent LC-MS triple
quadrupole mass spectrometer (LC-MS, Santa Clara, USA) with an electrospray ionization (ESI)
interface. Elemental analyses were performed on a Cos-Tec model EAS 4010 instrument
(Cernusco, Italy) and the results are within ±0.4% of the theoretical values. Cell lines, including
HCT116, A2780, MCF7, A549, HL60, PC3 and Huvec purchased from Pasteur Institute Cell
Bank of IRAN (Tehran, IRAN).
4
.2. General procedure for the synthesis of ethyl 2-oxo-2H-chromene-3-carboxylates (3a-u)
Diethyl malonate (2.11 mmol) and piperidine (2mL) were added to a solution of the appropriate
salicylaldehyde derivatives (1a-e) (10mmol) in ethanol (20 mL) and the resulting mixture was
refluxed for 14-15 h until the disappearance of the starting materials (monitored by TLC). After
cooling, the crude product was filtrated, washed with cold ethanol and recrystallized from
ethanol.
4
.3. General procedure for the synthesis of ethyl 7-alkyl or aryl-2-oxo-2H-chromene-3-
carboxylates (3f-v)
Potassium Carbonate (6 mmol) was added to ethyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate
(3e, 5mmol) in DMF (20 mL) and the mixture was stirred at rt for several minutes, then
appropriate alkyl halide or benzyl halide derivatives (14 mmol) were added dropwise to the
mixture and stirred for 10-12 h. Upon completion, water is added to the mixture and the
produced precipitate was filtrated, washed with water and used in the next step without further
purification.
4
.4. General procedure for the synthesis of 2-oxo-2H-chromene-3-carboxylic acids (4a-u)

ACCEPTED MANUSCRIPT
Esters (3a-v) were dissolved in aqueous sodium hydroxide (2N, 20 mL) and were stirred at rt for
5
-7 h. After cooling, solution was acidified with hydrochloric acid (2N) until a white precipitate
formed. The white solid was filtrated, washed with water and dried to yield acids (4a-u).
.5. General procedure for the synthesis of 4-((2-oxo-2H-chromene-3-carboxamido) methyl)
4
benzoic acid derivatives (6a-u)
the corresponding 2-oxo-2H-chromene-3-carboxylic acids (3a-v, 5 mmol) was added to a
suspension of 1, 1´-carbonyldimidazole (CDI, 25 mmol) in dry tetrahydrofuran (THF, 10 mL)
and the mixture was stirred for 2h at rt. Then 4-(aminomethyl) benzoic acid (5, 25 mmol) and
trifluoroacetic acid (TFA, 1.2 mL) were added and stirred for additional 10h at rt. The mixture
was evaporated to remove THF and extracted with EtOAc, dried over anhydrous Na SO and
2
4
concentrated under reduced pressure. Further purification was done with preparative thin layer
chromatography for some compounds (6p and 6q). Due to very low solubility of compounds 6g,
6
4
i and 6m, we could not report 13C-NMR data for these compounds.
.5.1. 4-((2-oxo-2H-chromene-3-carboxamido) methyl) benzoic acid (6a)
White solid; yield:78%; mp: 261-263°C; R = 0.56 (petroleum ether: ethyl acetate= 3:1) ; IR
f
-1
1
(
KBr): νmax (cm ) 3312 (NH), 1709 1602 (CO); H NMR (300 MHz, DMSO-d ) δ 4.63 (d, 2H,
6
2
J= 6.0 Hz,CH ), 7.44-7.54 (m, 4H, Ar-H), 7.77 (t, 1H, J= 6.0 Hz , Ar-H), 7.92 (d, 2H, J= 9.0 Hz
1
3
,
Ar-H), 7.99 (d, 1H, J= 9.0 Hz , Ar-H), 8.90 (s, 1H, CH), 9.23 (t, 1H, J= 6.0 Hz , NH); C NMR
(
75 MHz, DMSO-d ) δ 43.04, 116.62 , 118.94 , 119.57, 125.62, 127.78, 129.88, 130.06, 130.75,
6
1
34.61, 144.56, 148.08, 154.40, 160.73, 161.96, 167.60; Anal. calcd. for C H NO : C, 66.87;
18 13 5
H, 4.05; N, 4.33; found: C 66.80, H 4.09, N 4.24.
4
.5.2. 4-((8-methoxy-2-oxo-2H-chromene-3-carboxamido) methyl) benzoic acid (6b)

ACCEPTED MANUSCRIPT
f
White solid; yield: 73%; mp: 238-240°C; R = 0.49 (petroleum ether: ethyl acetate= 3:1); IR
-1
1
(
KBr): νmax (cm ) 3347 (NH), 1712 &1606 (CO); H NMR (300 MHz, DMSO-d ) δ 3.95 (s, 1H,
6
OCH ), 4.62 (d, 2H, J= 6.0 Hz, CH ), 7.35-7.54 (m, 5H, Ar-H) ,7.91 (d, 1H, J= 8.1 Hz, Ar-H),
3
2
1
3
8
6
.85 (s, 1H, CH), 9.24 (t, 1H, J= 5.7 Hz, NH); C NMR (75 MHz, DMSO-d ) δ 43.05, 56.65,
1
16.53, 119.46, 119.57, 121.62, 125.54, 127.81, 129.36, 137.81, 143.69, 144.56, 146.73, 148.30,
60.44, 161.83, 167.60; Anal. calcd. for C H NO : C, 64.59; H, 4.28; N, 3.96; found: C 64.66,
1
1
9
15
6
H 4.20, N 4.05.
4
.5.3. 4-((8-ethoxy-2-oxo-2H-chromene-3-carboxamido) methyl) benzoic acid (6c)
Pale yellow solid; yield: 79%; mp: 246-248 ˚C; R = 0.43 (petroleum ether: ethyl acetate= 3:1);
f
-1
1
IR (KBr): νmax (cm ) 3335 (NH) , 1707 and 1653 (CO); H NMR (300 MHz, DMSO-d ) δ 1.42
6
(
t, 3H, J=6.9Hz, CH ), 4.20 (q, 2H, J=6.9Hz,OCH ), 4.62 (d, 2H, J=6.0Hz, CH ), 7.34 (d, 1H,
3 2 2
J=7.5Hz, Ar-H), 7.42-7.48 (m, 3H, Ar-H), 7.52 (dd, 1H, J =7.5Hz, J =1.2Hz, Ar-H), 7.92 (d,
1
2
1
3
2
H, J=8.1Hz, Ar-H), 8.86 (s, 1H, CH), 9.23 (t, 1H, J=6.0Hz, NH); C NMR (75 MHz, DMSO-
d6) δ 15.04, 43.03, 65.02, 117.43, 119.56, 119.59, 121.62, 125.56, 127.82, 129.89, 137.82,
1
43.79, 144.63, 145.98, 148.37, 160.53, 161.96, 167.66; Anal. calcd. for C H NO : C, 65.39;
20 17 6
H, 4.66; N, 3.81; found: C 65.30, H 4.73, N 3.75.
4
.5.4. 4-((7-methoxy-2-oxo-2H-chromene-3-carboxamido) methyl) benzoic acid (6d)
Light brown; yield: 89%; mp: 242-244 °C; R
f
= 0.46 (petroleum ether: ethyl acetate= 3:1); IR
-1
1
(
KBr): νmax (cm ) 3346 (NH) , 1701, 1659 &1614 (CO); H NMR (300 MHz, DMSO-d ) δ 3.89
6
(
s, 3H, OCH ), 4.62 (d, 2H, J=6.0Hz, CH ), 7.02 (dd, 1H, J =8.7Hz, J =2.4Hz, Ar-H), 7.09 (d,
3
2
1
2
1
H, J=2.1Hz, Ar_H), 7.45 (d, 2H, J=8.1Hz, Ar-H), 7.90 (m, 3H, Ar-H), 8.83 (s, 1H, CH ), 9.17
1
3
(
t, 1H, J=6.0Hz, NH), 12.91 (brs, 1H, OH); C NMR (75 MHz, DMSO-d
6
) δ 42.98 , 56.67 ,
1
00.69, 112.54, 114.05, 115.16, 127.77, 129.84, 129.90, 131.99, 144.77, 148.47, 156.64, 161.24,

ACCEPTED MANUSCRIPT
1
62.20, 164.90, 167.65; Anal. calcd. for C19
H 4.34, N 3.89.
.5.5. 4-((7-ethoxy-2-oxo-2H-chromene-3-carboxamido) methyl) benzoic acid (6e)
6
H15NO : C, 64.59; H, 4.28; N, 3.96; found: C 64.48,
4
Pale yellow solid; yield: 85%; mp: 245-247 °C; R = 0.59 (petroleum ether: ethyl acetate= 3:1)
f
-1
1
IR (KBr): νmax (cm ) 3339 (NH), 1718, 1654 and 1613 (CO); H NMR (300 MHz, DMSO-d
6
) δ
1
2
.38 (t, 3H, J=6.6Hz, CH ), 4.18 (q, 2H, J=6.9Hz, OCH ), 4.67 (d, 2H, J=6.6Hz, CH ), 7.05 (m,
3
2
2
H, Ar-H), 7.50 (d, 2H, J=7.8Hz, Ar-H), 7.92 (d, 2H, J=6.9Hz, Ar-H), 8.10 (d, 1H, J=7.8Hz,
13
Ar-H), 8.86 (s, 1H, CH), 9.20 (t, 1H, J=7.8Hz, NH ), 12.90 (brs, 1H, OH); C NMR (75 MHz,
DMSO-d ) δ 14.82 , 42.84 , 64.89, 101.49, 112.49, 114.38, 115.14, 127.79, 129.70, 129.90,
31.03, 144.86, 148.54, 156.71, 161.28, 162.17, 164.22, 167.54; Anal. calcd. for C H NO : C,
6
1
6
2
0
17
6
5.39; H, 4.66; N, 3.81; found: C 65.29, H 4.75, N 3.74.
.5.6. 4-((2-oxo-7-propoxy-2H-chromene-3-carboxamido) methyl) benzoic acid (6f)
Yellow solid; Yield: 82%; mp: 259-261 °C; R = 0.55 (petroleum ether: ethyl acetate= 3:1)
4
f
-1
1
IR (KBr): νmax (cm ) 3333 (NH), 1701, 1659 and 1618 (CO); H NMR (300 MHz, DMSO-d
6
) δ
1
1
.00 (t, 3H, J=7.2Hz, CH ), 1.77 (se, 2H, J=6.9Hz, CH ), 4.10 (t, 2H, J=6.6Hz, OCH ), 4.62 (d,
3
2
2
H, J=6.0Hz, CH ), 7.04 (dd, 1H, J =8.7Hz, J =2.4Hz, Ar-H), 7.11 (d, 1H, J=2.1Hz, Ar-H), 7.45
2
1
2
(d, 2H, J=8.4Hz, Ar-H), 7.89-7.93 (m, 3H, Ar-H), 8.86 (s, 1H, CH), 9.19 (t, 1H, J=6.0Hz, NH);
1
3
6
C NMR (75 MHz, DMSO-d ) δ 10.75 , 22.23, 42.95, 70.55, 110.13, 112.49, 114.40, 115.12 ,
1
27.78, 129.72, 129.90, 132.08, 144.84, 148.54, 156.71, 161.26, 162.18,164.38, 167.56; Anal.
calcd. for C H NO : C, 66.13; H, 5.02; N, 3.67; found: C 65.20, H 4.93, N 3.73.
2
1
19
6
4.5.7. 4-((6-bromo-2-oxo-2H-chromene-3-carboxamido) methyl) benzoic acid (6g)
White solid; yield: 86%; mp: 295-297°C; R = 0.46 (petroleum ether: ethyl acetate= 3:1); IR
f
-1
1
(
6
KBr): νmax (cm ) 3349 (NH), 1713, 1653 and 1611 (CO); H NMR (300 MHz, DMSO-d ) δ

ACCEPTED MANUSCRIPT
4
.62 (d, 2H, CH
2
), 7.44-7.51 (m, 3H, Ar-H), 7.88-7. 93 (m, 3H, Ar-H), 8.26 (d, 1H, J=2.1Hz, Ar-
H), 8.84 (s, 1H, CH), 9.21 (t, 1H, J=6.0Hz, NH); Anal. calcd. for C H BrNO : C, 53.75; H,
1
8
12
5
3
.01; N, 3.48; found: C 53.68, H 3.10, N 3.37.
.5.8. 4-((2-oxo-7-phenoxy-2H-chromene-3-carboxamido) methyl) benzoic acid (6h)
4
Yellow solid; yield:85%; mp: 245-247°C; R = 0.34 (petroleum ether: ethyl acetate= 3:1); IR
f
-1
1
(
KBr): νmax (cm ) 3340 (NH), 1705, 1658 and 1613 (CO); H NMR (300 MHz, DMSO-d
6
) δ
4
.61 (d, 2H, J=6.0Hz, CH ), 5.28 (s, 2H, CH ), 7.12 (dd, 1H, J =8.7Hz, J =2.1 Hz, Ar-H), 7.21
2
2
1
2
(
d, 1H, J=2.1Hz, Ar-H), 7.35 (m, 7H, Ar-H), 7.91 (m, 3H, Ar-H), 8.85(s. 1H, CH ), 9.18(t, 1H,
1
1
3
NH), 12.89 (s, 1H, OH); C NMR (75 MHz, DMSO-d ) δ 42.96, 70.68, 101.66, 112.77, 114.65,
6
1
1
6
4
15.46, 127.07, 127.79, 128.51, 128.72, 129.05, 129.90, 132.12, 136.44, 144.83, 148.45, 156.58,
61.22, 162.25, 163.91, 167.59; Anal. calcd. for C H NO : C, 69.92; H, 4.46; N, 3.26; found: C
25
19
6
9.90, H 4.39, N 3.32.
.5.9. 4-((2-oxo-7-(o-tolyloxy)-2H-chromene-3-carboxamido) methyl) benzoic (6i)
Pink solid; yield: 83%; mp: 229-231°C; R = 0.45 (petroleum ether: ethyl acetate= 3:1) ; IR
f
-1
1
(
KBr): νmax (cm ) 3342 (NH), 1705, 1657 &1610 (CO); H NMR (300 MHz, DMSO-d
6
) δ 2.33
(
s, 3H, CH ), 4.61(d, 2H, J=5.7Hz, CH ), 5.24 (s, 2H, OCH ), 7.10 (dd, 1H, J =8.7Hz, J =2.1Hz,
3
2
2
1
2
CH ), 7.19-7.25 (m, 4H, Ar-H), 7.43-7.47 (m, 3H, Ar-H) 7.89-7.94 (m, 3H, Ar-H), 8.85(s, 1H,
2
CH), 9.18 (t, 1H, J=6.0Hz, NH), 12.89 (s, 1H, OH); Anal. calcd. for C H NO : C, 70.42; H,
2
6
21
6
4
.77; N, 3.16; found: C 70.50, H 4.73, N 3.25.
.5.10. 4-((2-oxo-7-(m-tolyloxy)-2H-chromene-3-carboxamido)methyl)benzoic acid (6j)
4
Pale yellow solid; Yield:85%; mp: 228-230°C; R
f
= 0.30 (petroleum ether: ethyl acetate= 3:1) ;
-1
1
IR (KBr): νmax (cm ) 3331 (NH), 1703, 1655 & 1618 (CO); H NMR (300 MHz, DMSO-d ) δ
6
2
.24 (s, 3H, CH ), 4.62 (d, 2H, J=6.0Hz,CH ), 5.23 (s, 2H, OCH ), 7.07-7.34 (m,6H, Ar-H), 7.44
3 2 2

ACCEPTED MANUSCRIPT
(d, 2H, J=8.1Hz, Ar-H), 7.90-7.94 (m, 3H, Ar-H), 8.86 (s, 1H, CH), 9.18 (t, 1H, J=6.0Hz, NH),
1
3
1
1
1
2.93 (s, 1H, OH); C NMR (400 MHz, DMSO-d ) δ 21.45, 42.97, 70.73, 101.61, 112.75,
6
14.63, 115.42 , 125.60, 127.78, 128.94, 129.05, 129.33, 129.82,132.10, 136.34,138.24, 144.81,
6
48.46, 156.58, 161.23, 162.23, 163.94, 167.63; Anal. calcd. for C26H21NO : C, 70.42; H, 4.77;
N, 3.16; found: C 70.52, H 4.70, N 3.23.
.5.11. 4-((2-oxo-7-(p-tolyloxy)-2H-chromene-3-carboxamido)methyl)benzoic acid (6k)
Pale yellow solid; Yield:89%; mp: 264-266°C; R = 0.35 (petroleum ether: ethyl acetate= 3:1)
4
f
-1
1
IR (KBr): νmax (cm ) 3321 (NH), 1700, 1651 & 1611(CO); H NMR (300 MHz, DMSO-d ) δ
6
2
.36 (s, 3H, CH
=2.4Hz, Ar-H), 7.24-7.28 (m, 3H, Ar-H), 7.41 (d, 2H, J=8.1Hz, Ar-H), 7.48 (d, 2H, J=8.1Hz,
Ar-H), 7.94-7.97 (m, 3H, Ar-H), 8.90 (s, 1H, CH), 9.22 (t, 1H, J =6.0Hz, NH ), 12.97 (s, 1H,
3 2 2 1
), 4.65 (d, 2H, J=6.0Hz, CH ) ,5.27 (s, 1H, OCH ) 7.14 (dd, 1H, J =8.7Hz,
J
2
1
3
OH); C NMR (75 MHz, DMSO-d ) δ 21.26 , 42.97, 70.60, 101.64 , 112.71, 114.66, 115.39,
6
1
1
27.78, 128.61, 129.36, 129.57, 129.89, 132.08, 133.39,138.02, 144.80, 148.46, 156.58, 161.23,
62.23, 163.94, 167.64; Anal. calcd. for C26 : C, 70.42; H, 4.77; N, 3.16; found: C 70.35,
H21NO
6
H 4.83, N 3.18.
.5.12. 4-((7-(2-fluorophenoxy)-2-oxo-2H-chromene-3-carboxamido) methyl) benzoic acid
6l)
Yellow solid; yield: 92%; mp: 238-240°C; R = 0.38 (petroleum ether: ethyl acetate= 3:1) ; IR
4
(
f
-1
1
(
KBr): νmax (cm ) 3340 (NH), 1713, 1695 and 1616 (CO); H NMR (300 MHz, DMSO-d
6
) δ
4
7
.61 (d, 2H, J =6.0 Hz,CH ), 5.31 (s, 2H, OCH ), 7.13 (dd, 1H, J = 8.7Hz, J = 2.4Hz, Ar-H),
2
2
1
2
.25-7.33 (m, 3H, Ar-H), 7.45 (m, 3H, Ar-H), 7.62 (t, 1H, J = 7.2Hz, Ar-H), 7.90-7.96 (m, 3H,
1
3
Ar-H), 8.86 (s, 1H, CH), 9.19 (t, 1H, J = 6.0Hz, NH), 12.93 (1H, OH); C NMR (75 MHz,
DMSO-d ) δ 42.98, 65.07, 101.55, 112.90, 114.44, 115.55, 115.86, 123.33, 125.14, 127.77,
6

ACCEPTED MANUSCRIPT
1
1
4
29.90, 131.45, 131.56, 131.61, 132.14, 144.77, 148.41, 156.55, 159.39, 161.21, 162.21, 163.67,
67.66; Anal. calcd. for C H FNO : C, 67.11; H, 4.06; N, 3.13; found: C 67.20, H 4.12, N 3.04.
25
18
6
.5.13. 4-((7-(3-fluorophenoxy)-2-oxo-2H-chromene-3-carboxamido) methyl) benzoic acid
(6m)
Pale yellow solid; yield: 80%; mp: 259-261°C; R = 0.39 (petroleum ether: ethyl acetate= 3:1); IR
f
-1
1
(
KBr): νmax (cm ) 3338 (NH), 1711, 1657 and 1614 (CO); H NMR (300 MHz, DMSO-d6) δ
4
7
.61 (d, 2H, J =6.0 Hz,CH ), 5.30(s, 2H, OCH ), 7.13-7.23 (m, 3H, Ar-H), 7.33 (d, 2H, J =
2 2
.2Hz, Ar-H), 7.44-7.52 (m, 3H, Ar-H), 7.90-7.96 (m, 3H, Ar-H), 8.86(s, 1H, CH), 9.18 (t, 1H, J
=
6.0Hz, NH), 12.94 (brs, 1H, OH); Anal. calcd. for C H FNO : C, 67.11; H, 4.06; N, 3.13;
25 18 6
found: C 67.22, H 4.00, N 3.20.
4
.5.14. 4-((7-(4-fluorophenoxy)-2-oxo-2H-chromene-3-carboxamido)methyl)benzoic acid
(6n)
Yellow solid; yield: 92%; mp: 266-268°C; R = 0.43 (petroleum ether: ethyl acetate= 3:1) ; IR
f
-1
1
(
KBr): νmax (cm ) 3338 (NH), 1704, 1653 and 1617 (CO); H NMR (300 MHz, DMSO-d
6
) δ
4
7
3
4
1
.61 (d, 2H, J =6.0 Hz,CH ), 5.25 (s, 2H, OCH ), 7.12 (dd, 1H, J1= 8.5Hz, J = 2.1Hz, Ar-H),
2
2
2
.21-7.29 (m, 3H, Ar-H), 7.44 (d, 2H, J = 8.1Hz, Ar-H), 7.53-7.60 (m, 2H, Ar-H), 7.90-7.94 (m,
1
3
H, Ar-H), 8.86(s, 1H, CH), 9.18 (t, 1H, J = 6.0Hz, NH); C NMR (75 MHz, DMSO-d6) δ
2.97, 69.94, 101.64, 112.79, 114.61, 115.48, 115.74, 116.02, 127.77, 129.89, 130.81, 131.79,
32.11, 132.66, 144.73, 148.43, 156.56, 161.21, 162.15, 163.78, 167.66; Anal. calcd. for
C H FNO : C, 67.11; H, 4.06; N, 3.13; found: C 67.04, H 3.97, N 3.23.
25
18
6
4.5.15. 4-((7-(2-chlorophenoxy)-2-oxo-2H-chromene-3-carboxamido) methyl) benzoic acid
(6o)

ACCEPTED MANUSCRIPT
f
Yellow solid; yield: 75%; m: 262-264°C; R = 0.33 (petroleum ether: ethyl acetate= 3:1) ; IR
-1
1
(
KBr): νmax (cm ) 3342 (NH), 1705, 1659 and 1616 (CO); H NMR (300 MHz, DMSO-d ) δ
6
4
7
7
.62 (d, 2H, J =6.0Hz, CH ), 5.37 (s, 2H, OCH ), 7.20 (dd, 1H, J =8.7Hz, J =2.4Hz, Ar-H),
2 2 1 2
.33 (d, 1H, J =2.4Hz, Ar-H), 7.44-7.53 (m, 4H, Ar-H), 7.61 (m, 1H, Ar-H), 7.71 (m, 1H, Ar-H),
1
3
.95-7-8.02 (m, 3H, Ar-H), 8.92 (s, 1H, CH), 9.24 (t, 1H, J =6.0Hz, NH), 12.95 (s, 1H, OH); C
NMR (75 MHz, DMSO-d ) δ 42.98, 68.42 , 101.63 , 112.97, 114.44, 115.63, 127.80, 127.98,
6
1
1
6
4
29.74, 129.90, 130.03, 130.90, 131.30, 132.19, 133.59, 133.73, 144.84, 148.42, 156.57, 161.20,
62.14, 163.70, 167.55; Anal. calcd. for C H ClNO : C, 64.73; H, 3.91; N, 3.02; found: C
2
5
18
6
4.67, H 4.01, N 3.09.
.5.16. 4-((7-(4-chlorophenoxy)-2-oxo-2H-chromene-3-carboxamido) methyl) benzoic acid
6p)
Yellow solid; yield: 80%; mp: 270-272°C; R = 0.36 (petroleum ether: ethyl acetate= 3:1)
(
f
-1
1
IR (KBr): νmax (cm ) 3340 (NH), 1703, 1656 and 1610 (CO); H NMR (300 MHz, DMSO-d
6
) δ
4
7
9
1
1
.61 (d, 2H, J =6.0Hz, CH ), 5.28 (s, 2H, OCH ), 7.12 (dd, 1H, J =8.7Hz, J =2.4Hz, Ar-H),
2
2
1
2
.22 (d, 1H, J =2.4Hz, Ar-H), 7.44-7.55 (m, 6H, Ar), 7.90-7.95 (m, 3H, Ar-H), 8.86 (s, 1H, CH),
13
.18 (t, 1H, J =6.0Hz, NH), 12.95 (s, 1H, OH); C NMR (75 MHz, DMSO-d ) δ 42.97, 69.78 ,
6
01.70 , 112.86, 114.53, 115.57, 127.78, 129.05, 129.83, 129.89, 130.33, 132.13, 133.30, 135.51,
6
44.79, 148.41, 156.55, 161.19, 162.22, 163.68, 167.63; Anal. calcd. for C25H18ClNO : C, 64.73;
H, 3.91; N, 3.02; found: C 64.80, H 3.83, N 3.10.
.5.17. 4-((7-(4-bromophenoxy)-2-oxo-2H-chromene-3-carboxamido) methyl) benzoic acid
6q)
Light brown solid; yield: 87%; mp: 258-260°C; R = 0.35 (petroleum ether: ethyl acetate= 3:1)
4
(
f

ACCEPTED MANUSCRIPT
-1
1
IR (KBr): νmax (cm ) 3346 (NH), 1705, 1652 and 1611 (CO); H NMR (300 MHz, DMSO-d ) δ
6
4
.61 (t, 2H, J =6.0Hz, CH ), 5.26 (s, 2H, OCH ), 7.07-7.20 (m, 2H, Ar-H), 7.20 (d, 1H, J
2 2
=
2.1Hz, Ar-H), 7.44 (d, 2H, J =6.9Hz, Ar-H), 7.61 (m, 3H, Ar-H), 7.91 (m, 3H, Ar-H), 8.85(s,
1
3
1
4
1
6
H, CH), 9.18 (t, 1H, J =6.0Hz, NH), 13.00 (s, 1H, OH); C NMR (75 MHz, DMSO-d ) δ
2.98, 69.78, 101.65, 112.86, 114.52, 115.55, 121.85, 127.78, 129.83, 129.89, 130.58, 131.97,
32.10, 135.93, 144.79, 148.41, 156.53, 161.19, 162.21, 163.66, 167.63; Anal. calcd. for
C H BrNO : C, 59.07; H, 3.57; N, 2.76; found: C 59.00, H 3.65, N 2.82.
25
18
6
4
.5.18. 4-((7-(4-methoxyphenoxy)-2-oxo-2H-chromene-3-carboxamido)methyl)benzoic acid
6r)
Yellow solid; Yield: 82%; mp: 220-222 °C; R
(
f
= 0.49 (petroleum ether: ethyl acetate= 3:1) ; IR
-1
1
(
KBr): νmax (cm ) 3342 (NH), 1701, 1659 and 1601 (CO); H NMR (300 MHz, DMSO-d ) δ
6
3
7
.77 (s, 3H, CH ), 4.62 (d, 2H, J =6.0Hz, CH ), 5.19 (s, 2H, CH ), 6.97 (d, 2H, J =8.4Hz, Ar-H),
3 2 2
1 2
.10 (dd, 1H, J =8.7Hz, J =2.4Hz, Ar-H), 7.21 (d, 1H, J=2.1Hz, Ar-H), 7.42-7.47 (m, 4H, Ar-
1
3
H), 7.90 (m, 3H, Ar-H), 8.86 (s, 1H, CH), 9.17 (t, 3H, J =6.0Hz, NH), 12.92 (S, 1H, OH); C
NMR (75MHz, DMSO-d ) δ 42.97, 55.60, 70.51, 101.61, 112.67, 114.40, 114.68, 115.34,
6
1
27.78, 128.27, 129.82, 129.90, 130.41, 132.06, 144.82, 148.47, 156.60, 159.75, 161.25, 162.24,
64.00, 167.63; Anal. calcd. for C H NO : C, 67.97; H, 4.61; N, 3.05; found: C 67.90, H 4.70,
1
2
6
21
7
N 3.13.
4
.5.19. 4-((7-(2, 3-dichlorophenoxy)-2-oxo-2H-chromene-3-carboxamido) methyl) benzoic
acid (6s)
Pale yellow solid; yield: 85%; mp: 252-254°C; R = 0.37 (petroleum ether: ethyl acetate= 3:1) ;
f
-1
1
IR (KBr): νmax (cm ) 3358 (NH), 1704, 1654 and 1611 (CO); H NMR (300 MHz, DMSO-d ) δ
6
4
2 2
.62 (t, 2H, J =6.0 Hz,CH ), 5.37 (s, 2H, OCH ), 7.10-7.21 (m, 3H, Ar-H), 7.29 (d, 1H, J =

ACCEPTED MANUSCRIPT
2
1
4
1
.1Hz, Ar-H), 7.45 (d, 2H, J = 7.8Hz, Ar-H ), 7.70 (m, 2H, Ar-H), 7.91 (m, 2H, Ar-H), 8.87 (s,
13
H, CH), 9.19 (t, 1H, J = 6.0Hz, NH), 12.83 (brs, 1H, OH); C NMR (75 MHz, DMSO-d ) δ
6
2.98, 68.65, 101.71, 113.09, 114.41, 115.76, 127.79, 128.96, 129.45, 129.84, 129.90, 131.09,
31.42, 132.58, 136.45, 144.79, 148.38, 156.54, 161.17, 162.20, 163.48, 167.63; Anal. calcd. for
C
25
H
17
C
l2NO
6
: C, 60.26; H, 3.44; N, 2.81; found: C 60.34, H 3.37, N 2.88.
4
.5.20. 4-((7-(2, 4-dichlorophenoxy)-2-oxo-2H-chromene-3-carboxamido) methyl) benzoic
acid (6t)
White solid; yield: 85%; mp: 245-247°C; R = 0.38 (petroleum ether: ethyl acetate= 3:1) ; IR
f
-1
1
(
KBr): νmax (cm ) 3341 (NH), 1706, 1652 and 1600 (CO); H NMR (300 MHz, DMSO-d6) δ
4
.62 (t, 2H, J =6.0 Hz,CH ), 5.32 (s, 2H, OCH ), 7.5 (dd, 1H, J = 8.7Hz, J = 2.1Hz, Ar-H), 7.28
2
2
(d, 1H, J = 1.8Hz, Ar-H), 7.45 (d, 2H, J = 8.1Hz, Ar-H ), 7.52 (dd, 1H, J = 8.1Hz, J = 1.8Hz, Ar-
H), 7.68 (d, 1H, J = 8.4Hz, Ar-H), 7.74 (d, 1H, J = 2.1Hz, Ar-H), 7.86-7.97 (m, 3H, Ar-H), 8.87
1
3
(
s, 1H, CH), 9.19 (t, 1H, J = 6.0Hz, NH), 12.88 (brs, 1H, OH); C NMR (75 MHz, DMSO-d
6
) δ
2.98, 67.78, 101.70, 113.06, 114.43, 115.74, 127.80, 128.15, 129.58, 129.74, 129.90, 131.09,
32.21, 132.45, 132.99, 134.56, 144.84, 148.39, 156.54, 161.17, 162.22, 163.51, 167.55; Anal.
4
1
calcd. for C H C NO : C, 60.26; H, 3.44; N, 2.81; found: C 60.19, H 3.52, N 2.75.
2
5
17 l2
6
4
.5.21. 4-((7-(3, 4-dichlorophenoxy)-2-oxo-2H-chromene-3-carboxamido) methyl) benzoic
acid (6u)
White solid; yield: 85%; mp: 249-251°C; R
f
= 0.53 (n-hexane: ethyl acetate= 3:1) ; IR (KBr):
-1
1
νmax (cm ) 3353 (NH), 1705, 1656 and 1617 (CO); H NMR (300 MHz, DMSO-d ) δ(ppm) δ
6
4
7
.61 (d, 2H, J = 6.0Hz, CH ), 5.30 (s, 2H, OCH ), 7.14 (dd, 1H, J = 8.7Hz, J = 2.4Hz, Ar-H),
2 2 1 2
.23 (d, 1H, J = 2.1Hz, Ar-H), 7.44-7.52 (m, 3H, Ar-H), 7.69 (d, 1H, J =8.1Hz, Ar-H), 7.79 (d,

ACCEPTED MANUSCRIPT
1
H, J = 1.8Hz, Ar-H), 7.93 (m, 3H, Ar-H), 8.86 (s, 1H, CH), 9.18 (t, 1H, J =6.0Hz, NH), 12.92
1
3
(
s, 1H, OH); C NMR (75 MHz, DMSO-d ) δ 42.97 , 69.01, 101.73, 112.99, 114.56, 115.70,
6
1
1
27.78 , 128.64, 129.89, 130.31, 131.27, 131.30, 131.68,132.17, 137.70, 144.79, 148.38, 156.52,
61.17, 162.20, 163.45, 167.63; Anal. calcd. for C25 : C, 60.26; H, 3.44; N, 2.81;
17 6
H Cl2NO
found: C 60.20, H 3.45, N 2.91.
.6. General procedure for the synthesis of N-(4-((2-aminophenyl) carbamoyl) benzyl)-2-
4
oxo-2H-chromene-3-carboxamides (8a-u)
To a suspension of 4-((2-oxo-2H-chromene-3-carboxamido) methyl) benzoic acids (6a-u) (5
mmol) in dry THF (10 mL) was added CDI (19mmol), and the mixture was stirred for 3 h at 60
°
C. The clear solution was cooled to rt. To this solution were added 1, 2-phenylenediamine (7,
5 mmol) and TFA (1.2 mL) and then stirred for 16 h. The reaction mixture was evaporated to
2
remove THF and the crude product was stirred in a mixture of hexane and water (2:5, v/v) for 1 h
and filtered and dried. The residue was triturated in dichloromethane twice to afford pure
compounds.
4
.6.1. N-(4-((2-aminophenyl) carbamoyl) benzyl)-2-oxo-2H-chromene-3-carboxamide (8a)
White solid; yield: 90%; mp: 207-209 °C; R = 0.76 (petroleum ether: ethyl acetate= 3:2) ; IR
f
-1
1
(
KBr): νmax (cm ) 3404 and 3322 (NH and NH ), 1720, 1653 and 1609 (CO); H NMR (300
2
MHz, DMSO-d
H, J= 6.6 Hz, Ar-H) , 6.79 (d, 1H, J= 7.5 Hz, Ar-H), 6.93 (t, 1H, J= 7.5 Hz, Ar-H), 7.18 (d, 1H,
J= 6.0 Hz, Ar-H), 7.48-7. 55 (m, 4H, Ar-H), 7.75 (t, 1H, J= 6.0 Hz, Ar-H), 7.98 (m, 2H, Ar-H),
.90 (s, 1H, CH), 9.22 (t, 1H, J= 6.0 Hz, NH D O exchangable), 9.64 (s, 1H, NH D O
6 2 2
) δ 4.64 (d, 2H, J= 6.0 Hz, CH ), 4.90 (s, 2H, NH2, D O exchangable), 6.62 (t,
1
8
,
2
,
2
1
3
exchangable); C NMR (75 MHz, DMSO-d
6
) δ 43.03, 116.63, 116.79, 118.95, 119.58, 123.70,
1
25.63, 126.93, 127.05, 127.63, 128.36, 129.93, 130.75, 133.75, 134.62, 143.00, 148.05, 154.39,