Synthesis, biological activity and multiscale molecular modeling studies of bis-coumarins as selective carbonic anhydrase IX and XII inhibitors with effective cytotoxicity against hepatocellular carcinoma

Article abstract of DOI:10.1016/j.bioorg.2019.03.003

A series of novel bis-coumarin derivatives containing triazole moiety as a linker between the alkyl chains was synthesized and their inhibitory activity against the human carbonic anhydrase (hCA) isoforms I, II, IX and XII were evaluated. In addition, cytotoxic effects of the synthesized compounds on renal adenocarcinoma (769P), hepatocellular carcinoma (HepG2) and breast adeno carcinoma (MDA-MB-231) cell lines were examined. While the hCA I and II isoforms were inhibited in the micromolar range, the tumor-associated isoform hCA IX and XII were inhibited in the high nanomolar range. 4-methyl-7-((1-(12-((2-oxo-2H-chromen-7-yl)oxy)dodecyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (5p) showed the strongest inhibitory activity against hCA IX with the K_i of 144.6 nM and 4-methyl-7-((1-(10-((2-oxo-2H-chromen-7-yl)oxy)decyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (5n) exhibited the highest hCA XII inhibition with the K_i of 71.5 nM. In order to better understand the inhibitory profiles of studied molecules, multiscale molecular modelling approaches were applied. Low energy docking poses of studied molecules at the binding sites of targets have been predicted. In addition, electrostatic potential surfaces (ESP) for binding sites were also generated to understand interactions between proteins and active ligands.

Full text of DOI:10.1016/j.bioorg.2019.03.003

Accepted Manuscript
Synthesis, Biological Activity and Multiscale Molecular Modeling Studies of
Bis-coumarins as Selective Carbonic Anhydrase IX and XII Inhibitors with Ef-
fective Cytotoxicity against Hepatocellular Carcinoma
Belma Zengin Kurt, Aydan Dag, Berna Doğan, Serdar Durdagi, Andrea Angeli,
Alessio Nocentini, Claudiu T. Supuran, Fatih Sonmez
PII:
S0045-2068(19)30171-3
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.003
YBIOO 2829
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
2 February 2019
14 February 2019
2 March 2019
Please cite this article as: B. Zengin Kurt, A. Dag, B. Doğan, S. Durdagi, A. Angeli, A. Nocentini, C.T. Supuran,
F. Sonmez, Synthesis, Biological Activity and Multiscale Molecular Modeling Studies of Bis-coumarins as Selective
Carbonic Anhydrase IX and XII Inhibitors with Effective Cytotoxicity against Hepatocellular Carcinoma,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.03.003
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Synthesis, Biological Activity and Multiscale Molecular Modeling Studies of
Bis-coumarins as Selective Carbonic Anhydrase IX and XII Inhibitors with
Effective Cytotoxicity against Hepatocellular Carcinoma
1
,
1
2
2,
3
Belma Zengin Kurt *, Aydan Dag , Berna Doğan , Serdar Durdagi *, Andrea Angeli , Alessio
3
3
4
Nocentini , Claudiu T. Supuran , Fatih Sonmez
1
Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34093,
Istanbul, Turkey
Computational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of
2
Medicine, Bahcesehir University, Istanbul, Turkey
3
Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di ScienzeFarmaceutiche e
Nutraceutiche, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
4
Sakarya University of Applied Sciences, Pamukova Vocational Highschool, Pamukova, Turkey
Abstract
A series of novel bis-coumarin derivatives containing triazole moiety as a linker between the alkyl
chains was synthesized and their inhibitory activity against the human carbonic anhydrase (hCA)
isoforms I, II, IX and XII were evaluated. In addition, cytotoxic effects of the synthesized
compounds on renal adenocarcinoma (769P), hepatocellular carcinoma (HepG2) and breast adeno
carcinoma (MDA-MB-231) cell lines were examined. While the hCA I and II isoforms were
inhibited in the micromolar range, the tumor-associated isoform hCA IX and XII were inhibited in
the high nanomolar range. 4-methyl-7-((1-(12-((2-oxo-2H-chromen-7-yl)oxy)dodecyl)-1H-1,2,3-
triazol-4-yl)methoxy)-2H-chromen-2-one (5p) showed the strongest inhibitory activity against
hCA IX with the K
H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (5n) exhibited the highest hCA XII inhibition
with the K of 71.5 nM. In order to better understand the inhibitory profiles of studied molecules,
i
of 144.6 nM and 4-methyl-7-((1-(10-((2-oxo-2H-chromen-7-yl)oxy)decyl)-
1
i
multiscale molecular modelling approaches were applied. Low energy docking poses of studied
molecules at the binding sites of targets have been predicted. In addition, electrostatic potential
surfaces (ESP) for binding sites were also generated to understand interactions between proteins
and active ligands.
Keywords: Coumarin; Carbonic Anhydrase; Cytotoxicity; Molecular Docking, Molecular
Dynamics (MD) Simulations
*
Corresponding authors: serdar.durdagi@med.bau.edu.tr (SD); bzengin@bezmialem.edu.tr
(
BZK).
1

1
. Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes, also known as carbonate
dehydratases, which carry a zinc (II) ion from metal ions which are fundamental to catalysis [1].
CA, explored in the beef erythrocytes for the first time, reversibly catalyzes the reactions of
-
hydration of CO
2
and dehydration of HCO
3
[2-3]. The hydration of CO occurs rather slowly
2
without catalyst [4]. Carbonic anhydrases have been developed as six different enzyme families,
ɑ-, β-, γ-, δ-, ζ- and η-CAs. In mammals, sixteen ɑ-CA isoenzymes or CA-related proteins
(
CARP) have been identified [5]. Their catalytic activities, intracellular localizations and tissue
distributions are different. CA I, CA II, CA III, CA VII and CA XIII cytosolic; CA IV, CA IX,
CA XII, CA XIV and CA XV attached to the membrane; CA VA and CA VB mitochondrial; CA
VI shows intracellular localization as isozymes when saliva, milk and urine are secreted [6-7]. The
three other isoenzymes are termed CA related proteins without catalytic activity; CARP VIII,
CARP X and CARP XI are found in the cytosol. Tissue distributions differ according to
physiologic/pathological conditions. While CA IX and XII isoenzymes are predominantly existing
in hypoxic tumors, VA and XIV mainly are found in the liver. Carbonic anhydrases play a role in
many pathological and physiological processes; transport of respiratory, CO
2
, and bicarbonate
between the metabolizing tissues and the lungs; pH and CO homeostasis; secretion of electrolytes
2
in various tissues and organs; biosynthesis reactions such as gluconeogenesis, lipogenesis; bone
resorption; calcification; and tumor formation. Inhibition of CA isoenzyme; is important in the
treatment of many diseases due to its role in diseases such as edema, glaucoma, obesity, cancer,
epilepsy and osteoporosis [2, 8].
Coumarins have a variety of pharmacological effects and they are mostly used in anticancer drug
design and discovery [9]. The coumarins are a relatively new class of inhibitors of the
metalloenzyme carbonic anhydrase [10-12]. Coumarin and its bioisosteres (thiocoumarins, 2-
thioxocoumarins) act as “prodrugs”, whereas their hydrolysis products (formed through CA-
mediated esterase activity) are the actually CA inhibitors [13-14]. Many studies have shown that
7
-hydroxycoumarin, 6-nitro-7-hydroxycoumarin, scopoletin and esculetin can be used in the
treatment of cancer [15]. Coumarins demonstrate different mechanisms of antitumor activity at
different stages of cancer formation. These mechanisms are; blockage of the cell cycle, apoptosis
of the cell, blockage of the estrogen receptor, or inhibition of DNA-related enzymes such as
topoisomerase [11].
2

The key feature of most tumors is hypoxic. Hypoxia is inadequate oxygenation, resulting in
pathologic consequences of tumor-damaged microcirculation and slowed oxygen diffusion [16-
1
7]. Hypoxic tumor is more prone to spread, malignant development, chemotherapy and
radiotherapy resistant. CA IX causes an increase in extracellular acidity catalyzed by the catalysis
of bicarbonate and protons hydration of CO , leads to the development of the acidic environment
2
around the tumor and thus to the development of the tumor in the metastatic phenotype and the
development of resistance to anticancer drugs [18-19]. Hypoxia inducible factor-1 (HIF-1) is
induced effectively under hypoxic conditions, leading to the degradation of the expression and pH
balance of CA IX and the development of classical chemotherapy and radiotherapy resistance
[
20]. In the studies performed, it was observed that continuous expression of IX in the Madin-
Darby canine kidney (MDCK) epithelial cell line reduced the extracellular pH (pHe). Low pH is
associated with tumor-specific changes, chromosomal rearrangements, extracellular matrix
breakdown, tumor migration and invasion, expression of cell growth factors and protease
activation [21]. Hypoxia regulates the expression of most genes. CA IX expression is increased by
hypoxia through HIF-1, while wild-type von Hippel-Lindau is reduced by tumor suppressor
protein (pVHL) [22]. This increased expression of CA IX may be detected in tumors developing
in glial and ependymal tissue, in mesothelioma, in papillary and follicular carcinomas, in bladder
carcinomas, in uterine and cervical carcinomas, in nasopharynx carcinomas, in head and neck
carcinomas, in breast, oesophagus, lung, brain, vulva, and kidney tumors [23]. In some cancer
cells, VHL gene mutation and HIF-1 activation were observed to be produced 150-fold more than
the resulting CA IX [24]. Thus, the inhibition of CA IX isoenzyme inhibits the tumor growth
activity of CA IX in hypoxic tumors, controlling pH irregularity in tumors and enabling new
applications in cancer diagnosis and treatment. Therefore, it is thought that inhibition of tumor
associated CA IX and CA XII isoenzymes may play an important role in the generation of new
generation anticancer drugs and may be effective against hypoxic tumors that cannot respond to
conventional chemotherapy and radiotherapy [25].
Many different studies have been performed previously for the identification of selective
inhibition of CA IX and XII enzymes. For example, Mollica et al [26] were synthesized novel
probenecid-based amide derivatives including different natural amino acids and assayed to test
their effect on the hCA IX and XII over the ubiquitous isoforms hCA I and II and most of the
synthesized compounds were presented a complete loss of hCA II inhibition (Kis >10,000 nM)
and strong inhibitory activity against hCA IX and XII in the nanomolar range with respect to the
3

parent compound. In another study D’Ascenzio et al [27] were stnthesized a large number of
novel secondary sulfonamides based on the open saccharin scaffold and evaluated as selective
inhibitors of four different hCA I, II, IX and XII.
Also, some natural compounds and peptides have been previously recognized as activators of
human and bacterial CAs. [28,29]
As a continuation of our studies on coumarin CAIs, in this work aimed to achieve a more effective
CA inhibition by performing double-ended synthesis of the coumarin molecule which is known to
be effective on carbonic anhydrases. Therefore, we focus on two types of the coumarins
possessing propargyl and azide moieties with different alkyl chains and they were combined via
Copper (I) catalysed-Azide-Alkyne-Cycloaddition (CuAAC) reactions, known as Click reactions.
Consequently, the novel sixteen bis-coumarin derivatives including different internal alkyl chains
between two peripheral coumarin rings were synthesized by simple and efficient methods and
their cytotoxic activity and effects on hCA isoforms were evaluated. Moreover, molecular
modeling studies were also applied for better understanding the structural, dynamical and binding
profiles of studied molecules at the active sites of targets. Specifically, molecular docking studies
were carried out to clarify the inhibition mode for the studied compounds at the active sites of the
target structures. Coumarin derivatives have been shown to hydrolyze with the increase of
incubation times [30]. Hence, in addition to parent coumarin derivatives, the possible hydrolysis
products shown in Fig. 1 were also considered. Predicted binding energies of these compounds
and structural and dynamical profiles of molecules at the target sites were estimated using GOLD
docking algorithm.
2
2
. Result and discussion
.1.Chemistry
The synthesis of the target compounds 5a-p is depicted in Scheme 1. 7-hydroxycoumarin (1a) or
-methyl-7-hydroxycoumarin (1b) was reacted with propargylbromide to obtain coumarin
4
derivatives including alkyne moiety (2a and 2b). On the other hand, 7-hydroxycoumarin (1a) was
also reacted with alkyl bromides and then it was treated with sodium azide to get other
components required for the synthesis of bis-coumarin. The coumarin derivatives containing
alkyne moiety (2a,b) and azide moiety (4a-h) were combined via Copper(I)-catalyzed Alkyne–
4

Azide Cycloaddition (CuAAC) reaction, known as Click reaction, to obtain bis-coumarin (5a-p)
derivatives containing triazole rings.
1
13
All of the compounds were synthesized here are new and characterized by H NMR, C NMR,
IR, MS and elemental analysis. According to the IR spectra of the synthesized bis-coumarin
-1
compounds, it was possible to observe the absorption peaks at about 2950 cm relating to CH
-1
stretch of aliphatic groups and absorption peaks at 1710-1740 cm from coumarin carbonyl
moiety stretch. Additionaly, because a triazole ring was generated along with the reaction of two
-1
coumarin molecules, the absorption peak of triazole ring at 3130 cm represented the click
1
reaction efficiency. From the H NMR spectra; the signals for aromatic hydrogens were observed
between 6.27 and 8.30 ppm, the signal of CH proton at triazole ring was detected at about 6.13-
6
.72 ppm and signals observed about 5.25 ppm belonged to -OCH proton. In addition, the signals
2
1
3
of aliphatic hydrogen atoms were determined between 1.00-4.50 ppm. From the C NMR spectra,
the signals can observed at about 161.5 and 162.5 ppm for carbonyl of lactone groups. The signals
of the aliphatic and aromatic carbons were observed at 18-69 ppm and 101-161 ppm, respectively.
1
13
H NMR, C NMR, and MS spectra of the synthesized compounds are given in supplementary
materials.
5

Scheme 1. Synthesis of new triazole ring substituted bis-coumarin derivatives. Reaction conditions: (i)
Propargylbromide, K CO , Acetone, 8h, reflux; (ii) Dibromide derivatives, K CO , CH CN, 2h, reflux; (iii)
NaN , DMF, 8h, rt; (iv) CuBr, PMDETA, 12h, rt.
2
3
2
3
3
3
2
.2.CA inhibition
i
The inhibition constants (K ) of the synthesized compounds 5a-p against hCA I, hCA II, hCA IX
and hCA XII isoforms are given in Table 1. The tumour-associated isoform hCA IX and hCA XII
were selectively inhibited by all the synthesized bis-coumarins with inhibition constants ranging
from 71.5 nM to 2737.1 nM, while the hCA I and II isoforms were inhibited in the μM range.
Among the synthesized bis-coumarins, 5p had the highest inhibitory activity against hCA IX with
a K
i
of 144.6 nM and 5n exhibited the strongest inhibition against hCA XII with a K
i
of 71.5 nM.
Table 1. In vitro inhibition K
i
values (nM) of 5a-p for the hCA I, II, IX and XII.
K
i
(nM)*
hCA IX
Compound
n
2
2
3
3
4
4
6
6
7
7
9
9
10
10
12
12
-
R
H
CH
H
CH
H
CH
H
CH
H
CH
H
CH
H
hCA I
hCA II
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
12.1
hCA XII
587.5
635.7
509.7
409.6
554.9
770.5
852.3
891.5
704.7
546.0
608.1
649.7
648.3
71.5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
250
645.0
3
3
3
3
3
3
3
3
1253.0
1312.3
1587.1
1247.6
1833.3
1054.5
1619.0
2737.1
2115.9
1969.7
1154.1
2675.4
160.9
CH
H
CH
-
211.1
144.6
415.3
785.2
5.7
AAZ
25.8
*
Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10 % of the reported
values).
6

Some notions about structure-activity relationship (SAR) emerged from these studies: (i) the
presence of the methyl group on the coumarin ring decreased the inhibitory activity against hCA
IX as the number of C in the linker is 2, 3, 4, or 6 (comparing 5a (n=2, R=H, K
b (n=2, R=CH , K = 1253.0 nM); comparing 5c (n=3, R=H, K = 1312.3 nM) with 5d (n=3,
R=CH , K = 1587.1 nM); comparing 5e (n=4, R=H, K = 1247.6 nM) with 5f (n=4, R=CH , K
833.3 nM); comparing 5g (n=6, R=H, K = 1054.5 nM) with 5h (n=6, R=CH , K = 1619.0 nM)),
whereas it increased the hCA IX inhibition as the C number is 7, 9, 10 or 12 (comparing 5i (n=7,
R=H, K = 2737.1 nM) with 5j (n=7, R=CH , K = 2115.9 nM); comparing 5k (n=9, R=H, K
969.7 nM) with 5l (n=9, R=CH , K = 1154.1 nM); comparing 5m (n=10, R=H, K = 2675.4 nM)
with 5n (n=10, R=CH , K = 160.9 nM); comparing 5o (n=12, R=H, K = 211.1 nM) with 5p
n=12, R=CH , K = 144.6 nM)). (ii) while R is H or CH3, and the linker is the dodecanyl (n=12),
the best hCA IX inhibitions were observed (5o (n=12, R=H, K = 211.1 nM) and 5p (n=12,
R=CH , K = 144.6 nM)). On the otherhand 5n (n=10, R=CH ) had the highest hCA XII inhibitory
activity (K = 71.5 nM). (iii) The increasing number of C in the linker moiety from two to three
and from six to seven led to a decline the inhibitory activity against hCA IX (comparing 5a (n=2,
R=H, K = 645.0 nM) with 5c (n=3, R=H, K = 1312.3 nM); comparing 5b (n=2, R=CH , K
253.0 nM) with 5d (n=3, R=CH , K = 1587.1 nM); comparing 5g (n=6, R=H, K = 1054.5 nM)
with 5i (n=7, R=H, K = 2737.1 nM); comparing 5h (n=6, R=CH , K = 1619.0 nM) with 5j (n=7,
R=CH , K = 2115.9 nM)), whereas it increased the inhibitory activity against hCA XII
comparing 5a (n=2, R=H, K = 587.5 nM) with 5c (n=3, R=H, K = 509.7 nM); comparing 5b
n=2, R=CH , K = 635.7 nM) with 5d (n=3, R=CH , K = 409.6 nM); comparing 5g (n=6, R=H,
= 852.3 nM) with 5i (n=7, R=H, K = 704.7 nM); comparing 5h (n=6, R=CH , K = 891.5 nM)
with 5j (n=7, R=CH , K = 546.0 nM)). Additionally, while R is CH , the increasing number of C
in the linker moiety from nine to ten dramatically increased the inhibitory activity against both
hCA IX and hCA XII (comparing 5l (n=9, R=CH , K = 1154.1 nM and 649.7 nm against hCA IX
and hCA XII, respectively) with 5n (n=10, R=CH , K = 160.9 nM and 71.5 nm against hCA IX
i
= 645.0 nM) with
5
3
i
i
3
i
i
3
i
=
1
i
3
i
i
3
i
i
=
1
3
i
i
3
i
i
(
3
i
i
3
i
3
i
i
i
3
i
=
1
3
i
i
i
3
i
3
i
(
(
i
i
3
i
3
i
K
i
i
3
i
3
i
3
3
i
3
i
and hCA XII, respectively)). Although there is no a linear relation between increasing C number
of the alkyl group and inhibitor activity, the best inhibitors against the tumor-associated hCA IX
and hCA XII have the longer alkyl chain (C number of 10 and 12). It can be hypothesized that the
long alkyl chain as a linker provides the molecule different conformations and intra-molecular
movement ability as shown in Figs. 2 and 4, therefore, both terminal coumarin moieties of the
synthesized compounds can effectively interact with the active sites of the enzyme.
7

It is known that coumarins are mechanism-based inhibitors, which undergo hydrolysis under the
influence of the zinc-hydroxide, nucleophilically active species of the enzyme, with generation of
substituted-2-hydroxycinnamic acids (Fig. 1) [30-34]. It was reported that coumarin/thiocoumarin
inhibitors and enzyme solutions were pre-incubated together before assay to form the E-I complex
or for the ultimate active site mediated hydrolysis of the inhibitor [35]. Based on these researches,
it can be considered that the synthesized bis-coumarin should hydrolase to cinnamic acid
derivative during pre-incubation on enzyme and inhibitor via three different possibilities as
opening the only coumarin A-ring, opening the only coumarin B-ring and opening both coumarin
rings (Fig. 1). If the pre-incubation time and the concentration of the zinc-hydroxide are sufficient
for hydrolyzing of the bis-coumarins, opening both coumarin rings can be considered as the
highest possibility.
Fig. 1. Formation of 2-hydroxy-cinnamic acids A1, B1 and HP-5a-p by the CA-mediated hydrolysis of
coumarin A, B and 5a-p.
2
.3.Cell Cytotoxicity
8

On basis of the screening results above, all of the synthesized compounds were examined to
specify their potential cytotoxicity effect towards many cancerous cell lines. Accordingly, the
renal cell adenocarcinoma (769P), liver hepatocellular carcinoma (HepG2) and breast
adenocarcinoma (MDA-MB-231) cell lines were treated with different doses of the synthesized
compounds (5a-p) for 24 h in complete medium. The cell viability was evaluated by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. The cytotoxicity effect (IC50) of the
synthesized compounds (5a-p) against 769P, HepG2 and MDA-MB-231 cell lines are given in
Table 2. In general, the IC50 values of the synthesized compounds were effective in the range
between 0.383 μM and 13.552 μM on cancer cell lines. Additionally, compound 5g showed the
strongest cytotoxic effect against 769P cell line with an IC50 value of 2.026 μM and 5k exhibited
the strongest cytotoxic effect against MDA-MB-231 with an IC50 value of 1.239 μM. Furthermore,
compound 5a showed the strongest cytotoxic effect against HepG2 with an IC50 value of 0.383
μM, which is almost 2-fold more than that of Doxorubicin used as a standard.
Table 2. Summary of IC50 values of 5a-p towards769P, HepG2 and MDA-MB-231 cell lines.
IC50 (μM)
HepG2
Compound
7
69P
MDA-MB-231
9.411±0.006
6.477±0.009
6.264±0.006
10.648±0.006
10.973±0.006
4.892±0.005
10.219±0.004
8.777±0.003
10.219±0.004
8.777±0.003
1.239±0.003
1.664±0.002
8.505±0.006
9.063±0.005
11.249±0.007
13.552±0.007
0.890±0.660
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
3.330±0.008
7.692±0.013
7.013±0.010
10.610±0.009
11.341±0.012
6.772±0.011
2.026±0.007
6.743±0.007
7.181±0.010
6.814±0.011
4.763±0.020
6.073±0.022
2.489±0.012
3.194±0.011
9.096±0.011
7.580±0.011
0.450±0.040
0.383±0.007
1.023±0.011
1.073±0.008
1.252±0.006
1.726±0.009
3.025±0.009
1.219±0.009
1.563±0.015
3.274±0.007
6.200±0.010
3.034±0.006
3.806±0.005
2.585±0.014
4.739±0.015
1.387±0.010
1.260±0.013
0.820±0.090
Doxorubicin
2
.4.Molecular Modeling
The docking scores of the studied compounds at the binding pockets of CA-IX and XII targets
along with their hydrolyzed products are shown in Table 3. Short MD simulations (10 ns) were
9

performed for the targets before the docking and the trajectories obtained by simulations were
clustered. The representative structure from the cluster with the largest size was taken as initial
complex structure for docking studies. In all cases except 5k for hCA IX, at least one of the
hydrolyzed derivatives, i.e. cinnamic acid derivatives are predicted to have higher fitness score
which means that more promising docking pose. In this sense, the docking results for all
compounds except 5k for hCA IX are in good agreement that hydrolyzed coumarin derivatives
have lower inhibition constants than that of coumarins as presented by Maresca et al. [30,31]. For
isoform hCA IX the highest fitness score is predicted for H3P-5p which is shown to be the most
promising inhibitor with lowest K experimentally (Table 1). In fact, the three compounds with the
i
lowest inhibition constant namely 5n, 5o, 5p and/or their hydrolyzed products were also the
highest scoring compounds based on the docking results. The electrostatic potential surfaces (EPS)
of these highest scoring docking poses for H3p-5p along with 2D ligand interaction diagram can
be found in Fig 2. Based on top-docking pose, Trp141 and Thr333 form hydrogen bonds with the
ligand. While the red colored regions show regions with negative partial atomic charges, blue
colored regions show regions with positive partial atomic charges. EPS of compound 5i, one of
the molecules that shows low inhibition against isoform IX is also shown in Fig. 3. It can be
argued that while compound 5p derivative H3P-5p fits into the target site without clashes and/or
repulsion between protein and ligand, there is some repulsion for H3p-5i ligand-protein complex
(
i.e., close red-colored regions can be interpreted as repulsive forces between ligand and receptor
sites). For isoform hCA XII, in vitro results showed that all compounds except 5n show similar
inhibition (Table 1). In our docking simulations we also found that mainly all compounds have
similar docking scores. However, surprisingly compound 5l’s H2P-5l form showed slightly higher
score compared to others. The 2D ligand interaction diagram of predicted docking pose for the 5n
product H1P-5n is shown in Fig. 4 along with the EPS of the complex. Based on interaction
diagram, H1P-5n forms following H-bonds with the target protein: Tyr6, Asn64, Lys69, Thr88,
and Gln89. Lys69 also forms salt bridge interactions with the ligand.
Table 3. Docking scores for compounds of 5a-p and their hydrolysis products HP-5a-p for the hCA IX and
XII.
Docking Score (CHEMPLP)
Compound
hCA IX
hCA XII
5
a
74.34
77.45
80.03
81.87
76.26
82.81
85.07
85.82
85.82
75.88
H1P-5a
H2P-5a
H3P-5a
5b
1
0

H1P-5b
H2P-5b
H3P-5b
78.72
78.74
82.86
75.20
79.29
77.59
81.62
78.01
76.86
83.62
76.56
81.52
78.32
78.48
86.73
79.16
81.85
83.11
83.24
83.78
87.65
83.47
85.18
82.63
86.97
83.65
80.57
84.56
84.25
85.47
84.03
79.15
84.29
83.15
88.19
86.44
84.92
78.84
84.17
85.38
84.96
86.06
90.52
75.48
85.23
79.12
96.12
79.64
87.06
85.54
90.28
78.25
87.06
82.73
85.06
80.39
90.56
89.41
92.77
81.83
92.08
85.19
97.76
86.32
91.36
92.23
100.24
88.39
95.04
84.03
93.38
82.13
5c
H1P-5c
H2P-5c
H3P-5c
5d
H1P-5d
H2P-5d
H3P-5d
5e
H1P-5e
H2P-5e
H3P-5e
5f
H1P-5f
H2P-5f
H3P-5f
5g
H1P-5g
H2P-5g
H3P-5g
5h
H1P-5h
H2P-5h
H3P-5h
5i
H1P-5i
H2P-5i
H3P-5i
5j
H1P-5j
H2P-5j
H3P-5j
5k
H1P-5k
H2P-5k
H3P-5k
82.09
84.57
85.25
86.18
86.84
84.57
85.25
82.88
83.64
87.18
84.49
87.34
85.25
82.14
90.40
90.04
90.49
90.22
88.09
87.99
91.02
93.58
94.15
84.69
98.68
104.87
91.25
84.79
88.25
92.23
88.01
90.53
96.01
87.74
92.62
82.31
90.90
94.57
95.12
86.00
5l
H1P-5l
H2P-5l
H3P-5l
5m
H1P-5m
H2P-5m
H3P-5m
5n
H1P-5n
H2P-5n
H3P-5n
5o
H1P-5o
H2P-5o
H3P-5o
5p
1
1

H1P-5p
H2P-5p
H3P-5p
90.56
89.04
92.13
91.26
92.35
91.75
Fig. 2. (left) Electrostatic potential map for H3P-5p and hCA IX complex highest scoring docking pose.
right) 2D ligand interaction diagram for docked ligand. The ligand is shown as CP2K representation,
binding site residues are shown as lines.
(
1
2

Fig. 3. Electrostatic potential map for H3P-5i and hCA IX complex. The ligand is shown as CP2K
representation, binding site residues are shown as lines. Protein is represented as ribbon. Compound 5i
shows lowest activity at the hCA IX, and its low activity can be due to repulsive forces between ligand and
target (labeled regions at the Figure).
Fig.
4. Electrostatic potential map for H1P-5n and hCA
XII complex. On the right, 2D ligand interaction diagram along for docked ligand. The ligand is shown as
CP2K representation, binding site residues shown as lines. Protein is represented as ribbon.
1
3

It is already well-established that designing inhibitors with selectivity against specific isoforms of
hCA is very challenging due to the sequence and structure similarity between isoforms [36,37]. In
Fig. 5, the sequence alignment of hCA isoforms I, II, IX and XII is shown. Here only the catalytic
domains of enzymes are considered for all isoforms, hence transmembrane domain of hCA IX and
hCA XII are ignored. Residue numbers are taken from PDB structures with codes 5E2M for hCA-
I, 3F8E for hCA-II, 5DVX for hCA-IX and 5MSA for hCA-XII. Secondary structures assignments
(
SSA) along with disulfide bonds (SSBOND) are also displayed in Fig. 5 for each protein
structures. The residues are colored based on their hydrophobicity. As it can be seen from this
figure, secondary structures of all considered isoforms are almost same and this situation is clearer
especially at the catalytic site (i.e., residue numbers 92, 94, 96, 106, 120-122, and 200-205 in Fig
5
.) where sulfonamide inhibitors bind, no selectivity could be achieved as the residues of the
catalytic sites are almost same in all considered isoforms. Hence, Maresca et al. suggested a new
binding site that is on the entrance to the enzyme binding site which is also the region with most
variation in amino acid sequences and it would be possible to design inhibitors with selectivity
against specific isoforms of hCA [38-41]. These coumarin binding sites are tentatively shown in
Fig. 6. The 3D structures of isoforms (from above mentioned PDB codes) are aligned and
specifically the coumarin binding site is assessed. It can be seen that there are important variations
in amino acid site chains on these coumarin sites of considered isoforms, specifically residues with
sequence numbers 67, 88, 133 in Fig. 5.
1
4

Fig. 5. Alignment of amino acid sequences for hCA I, II, IX and XII colored according to hydrophobicity
of amino acids.
1
5

Fig 6. (left) Alignment of isoforms I, II, IX and XII of hCA. (right) The detailed binding site is shown.
Binding site residues are represented by different colors: cyan for hCA I, orange for hCAII, green for hCA
IX and yellow for hCA XII. The blue colored dashed line shows the pi-pi stacking interaction between
Phe131 and phenyl ring of coumarin and yellow dashed lines show the hydrogen bonds.
3
. Conclusion
In conclusion, 16 novel bis-coumarin derivatives containing triazole ring and alkyl chain (5a-p)
are synthesized and their effects on the hCA I, II, IX and XII isoforms are evaluated. All the
synthesized compounds exhibited selective inhibitory activity in the high nM range against the
tumor-associated isoform hCA IX and hCA XII. Among them, 5p showed the strongest inhibitory
activity against hCA IX with the K
i
of 144.6 nM and 5n exhibited the highest hCA XII inhibition
with the K of 71.5 nM. Furthermore, cytotoxic effects of the synthesized compounds on renal
i
adenocarcinoma (769P), hepatocellular carcinoma (HepG2) and breast adeno carcinoma (MDA-
MB-231) cell lines are examined. The results revealed that they exhibited very good anticancer
properties especially in the hepatocellular carcinoma (HepG2) cell line between cytotoxicity
experiments performed on 3 different cell lines. 5a showed the strongest cytotoxic effect against
HepG2 with an IC50 value of 0.383 μM, which is almost 2-fold more than that of Doxorubicin
used as a standard.
Multiscale molecular modeling approaches are used to investigate binding poses and predicted
binding energies of studied compounds (both hydrolyzed and non-hydrolyzed forms) at the active
sites of the CA IX, and XII isoforms. The docking studies were in general agreement that
hydrolyzed products of synthesized compounds would interact with the target site better compared
to coumarin derivatives as for each compound, the highest docking scores are for compounds with
at least one coumarin ring open. Docking results showed the highest docking scores for 5n, 5o and
5
4
4
p compared to other compounds at the hCA IX.
. Experimental
.1. Material and Method
Melting points were taken on a STUART SMP40. IR spectra were measured on Alfa Bruker
1
13
spectrometer. H and C NMR spectra were measured on a Varian Infinity Plus spectrometer at
1
6

1
13
3
00 and at 75 Hz, respectively. H and C chemical shifts are referenced to the internal deuterated
solvent. Mass spectra were obtained using MICROMASS Quattro LC-MS-MS spectrometer. The
elemental analyses were carried out with a Leco CHNS-932 instrument. Spectrophotometric
analyses were performed by a BioTek Power Wave XS (BioTek, USA). The cell line was
purchased from American Type Culture Collection (ATCC). Dulbecco's Modified Eagle's
Medium-F12, RPMI Medium, fetal calf serum, and PBS were purchased from GIBCO BRL,
InVitrogen (Carlsbad, CA). The chemicals and solvents were purchased from Fluka Chemie,
Merck, Alfa Aesar and Sigma-Aldrich.
4
4
.2. General procedures and spectral data
.2.1. 4-methyl-/7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (2a-b)
To a solution of 7- hydroxycoumarin or 4-methyl-7- hydroxycoumarin (1.00 g, 6.17 mmol) in
acetone (40 mL), propargyl bromide (0.88 g, 7.40 mmol) and anhydrous K CO (1.00 g) were
2
3
added. The mixture was refluxed for 8h, and then it was hot filtered and concentrated under
reduced pressure. The crude was crystallized with petroleum ether, affording (95-97%) of 2a-b
[
42].
4
.2.2. General procedure for the synthesis of 3a-h
K
2
CO
CH CN (20 mL). The reaction mixture was refluxed for 2 h. Upon completion, K
removed by filtration and the solvent was concentrated under vacuum, the residue was dissolved
in CH Cl , washed with water, brine, dried over anhydrous Na SO and concentrated under
3
(12 mmol) and dibromide derivatives (6 mmol) were added to a solution of 1a (2 mmol) in
3
2
CO was
3
2
2
2
4
vacuum to give compounds 3a-h and purification coloumn chromatograph (Eluent; Hexane-
EtOAc; 4:1) [43].
7
1
5
6
9
1
-(2-bromoethoxy)-2H-chromen-2-one (3a). White powder, 42%yield; IR: 3081, 1727, 1711,
605, 1556, 1508, 1393, 1353, 1268, 1226, 1202, 1122, 1096, 1010, 990, 891, 831, 806, 760, 615,
-1 1
72, 546, 483, 461 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm: 3.84 (2H, s, br), 4.40 (2H, s, br),
.31 (1H, dd, J=2.6 Hz, 9.3 Hz), 6.97-7.04 (2H, m), 7.64 (1H, d, J=8.4 Hz), 8.0 (1H, dd, J=2.0 Hz,
1
3
.6 Hz); C NMR (DMSO-d , 75 MHz) δ/ppm: 31.7, 69.0, 102.1, 112.9, 113.4, 130.3, 144.9,
6
56.0, 160.9, 161.6, 162.6.
1
7

7
-(3-bromopropoxy)-2H-chromen-2-one (3b). White powder, 71% yield; IR: 3068, 2951, 2863,
-1
1
701, 1603, 1552, 1400, 1290, 1238, 1147, 1128, 1038, 833, 749, 717, 614, 572, 549, 460 cm ;
1
H NMR (DMSO-d , 300 MHz) δ/ppm: 2.23-2.28 (2H, m), 3.66 (2H, t, J=6.4 Hz), 4.17 (2H, t,
6
J=5.8 Hz), 6.28 (1H, d, J=9.4 Hz), 6.95 (1H, d, J=8.4 Hz), 7.0 (1H, s), 7.61 (1H, d, J=8.4 Hz),
1
3
7
1
.97 (1H, d, J=9.3 Hz); C NMR (DMSO-d , 75 MHz) δ/ppm: 57.7, 66.0, 66.7, 101.7, 101.9,
6
30.2, 145.0, 156.0, 160.9, 161.0, 162.5.
7
-(4-bromobutoxy)-2H-chromen-2-one (3c). White powder, 54% yield; IR: 3080, 2952, 2925,
-1
2
879, 1703, 1610, 1553, 1509, 1399, 1289, 1235, 1128, 1100, 1011, 826, 718, 614, 559, 457 cm ;
1
H NMR (DMSO-d
2H, d, J=9.3 Hz), 6.93-7.0 (2H, m), 7.63 (1H, d, J=8.7 Hz), 8.0 (1H, d, J=9.3 Hz); C NMR
DMSO-d , 75 MHz) δ/ppm: 25.7, 61.2, 68.5, 101.8, 112.9, 113.1, 113.4, 130.1, 145.0, 156.0,
61.0, 162.4.
6
, 300 MHz) δ/ppm: 1.91.(4H, s, br), 3.51 (2H, s, br), 4.16 (2H, s, br), 6.28
1
3
(
(
6
1
7
2
6
1
6
-((6-bromohexyl)oxy)-2H-chromen-2-one (3d). White powder, 60% yield; IR: 3080, 3049, 2942,
861, 1719, 1610, 1555, 1507, 1467, 1393, 1287, 1234, 1127, 1098, 1033, 1001, 890, 825, 716,
-1 1
40, 631, 615, 567, 515, 456 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm: 1.39- 1.44 (4H, m),
.69-1.82 (4H, m), 3.51 (2H, t, J=6.7 Hz), 4.04 (2H, t, J=6.4 Hz), 6.25 ( 1H, d, J=9.6 Hz), 6.89-
1
3
.95 (2H, m), 7.59 (1H, d, J=8.4 Hz), 7.96 (1H, d, J=9.3 Hz); C NMR (DMSO-d , 75 MHz)
6
δ/ppm: 32.8, 33.1, 35.8, 61.2, 68.8, 101.7, 112.9, 113.0, 130.1, 145.0, 156.0, 161.0, 162.5.
7
1
-((7-bromoheptyl)oxy)-2H-chromen-2-one (3e). White powder, 65% yield; IR: 3065, 2937, 1745,
-1 1
611, 1554, 1399, 1289, 1131, 1098, 1028, 865, 845, 724 cm ; H NMR (DMSO-d , 300 MHz)
6
δ/ppm: 1.26-1.37 (7H, m), 1.66-1.77 (3H,m), 3.34 (1H, t, J=6.4 Hz), 3.48 (1H, t, J=6.4 Hz), 4.01
(
2H, t, J=6.4 Hz), 6.23 (1H, d, J=9.6 Hz), 6.87-6.91 (2H,m), 7.57 (1H, d, J=8.4 Hz), 7.94 (1H, d,
1
3
J=9.3 Hz); C NMR (DMSO-d
6
, 75 MHz) δ/ppm: 26.1, 28.4, 29.3, 33.1, 35.8, 61.3, 68.8, 101.7,
1
12.8, 113.0, 113.3, 130.1, 145.0, 156.0, 161.0, 162.5.
7
1
1
-((9-bromononyl)oxy)-2H-chromen-2-one (3f). White powder, 74% yield; IR: 3080, 2918, 1729,
-1 1
615, 1552, 1470, 1293, 1237, 1131, 1013, 845, 722 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm:
.21-1.72 (11H, m), 2.47-2.48 (3H,m), 3.32 (1H, t, J=6.1 Hz), 3.46 (1H, t, J=6.7 Hz), 4.00 (2H, t,
J=6.1 Hz), 6.23 (1H, d, J=9.6 Hz), 6.86-6.91 (2H,m), 7.57 (1H, d, J=8.4 Hz), 7.95 (1H, d, J=9.3
1
3
Hz); C NMR (DMSO-d
6
, 75 MHz) δ/ppm: 26.1, 28.1, 29.1, 29.4, 29.7, 33.2, 35.8, 61.3, 68.8,
1
01.6, 112.8, 112.9, 113.3, 130.1, 145.0, 156.0, 161.0, 162.5.
1
8

7
2
7
1
-((10-bromodecyl)oxy)-2H-chromen-2-one (3g). White powder, 65% yield; IR: 3081, 2933,
916, 2852, 1707, 1617, 1556, 1508, 1472, 1394, 1289, 1234, 1129, 1098, 1034, 1014, 891, 840,
-1 1
58, 715, 685, 639, 568, 456 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm: 1.28-1.36 (14H, m),
.73-1.80 (4H,m), 3.52 (2H, t, J=6.7 Hz), 4.04-4.08 (2H, m), 6.28 (1H, d, J=9.3 Hz), 6.92-6.98
1
3
(
2H,m), 7.62 (1H, d, J=8.7 Hz), 7.98 (1H, d, J=9.6 Hz); C NMR (DMSO-d
6
, 75 MHz) δ/ppm:
2
1
6.1, 29.1, 29.4, 29.6, 29.7, 33.2, 61.3, 68.9, 101.7, 112.9, 113.0, 113.4, 130.1, 145.0, 156.0,
61.0, 162.5.
7
2
7
1
6
-((12-bromododecyl)oxy)-2H-chromen-2-one (3h). White powder, 74% yield; IR: 2934, 2916,
851, 1720, 1709, 1620, 1556, 1509, 1472, 1395, 1288, 1235, 1130, 1100, 1030, 1007, 891, 825,
-1 1
45, 716, 640, 616, 569, 457 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm: 1.24-1.37 (16H, m),
.69- 1.79 (4H, m), 3.51 (2H, t, J=6.4 Hz), 4.05 (2H, t, J=6.1 Hz), 6.27 (1H, d, J=9.3 Hz), 6.91-
1
3
.96 (2H, m), 7.60 (1H, d, J=8.4 Hz), 7.98 (1H, d, J=9.6 Hz); C NMR (DMSO-d , 75 MHz)
6
δ/ppm: 26.0, 26.1, 29.0, 29.3, 29.6, 29.7, 33.1, 61.4, 68.9, 101.7, 112.8, 113.0, 113.4, 130.1, 145.1,
1
56.0, 161.1, 162.5.
4
.2.3. General procedure for the of compounds 4a-h
NaN (2 mmol) were added to a solution of 3a-h (1 mmol) in DMF (15 mL). The reaction was
3
mixtured for 8 h at room temperature. The suspension was poured on crushed ice and the
precipitated product was filtered and washed with water. It was recrystallized from methanol [44].
7
2
7
-(2-azidoethoxy)-2H-chromen-2-one (4a). White powder, 97% yield; IR: 3075, 2956, 2160,
125, 2098, 1726, 1608, 1507, 1406, 1389, 1352, 1279, 1231, 1123, 1053, 994, 914, 892, 835,
-1 1
50, 616, 550, 522, 460 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 3.71 (2H, t, J=4.6 Hz), 4.29
6
(
2H, t, J=4.3 Hz), 6.32 (1H, d, J=9.3 Hz), 6.97 (1H, dd, J=2.6 Hz, 8.4 Hz), 7.04 (1H, d, J=2.3 Hz),
1
3
7
1
.65 (1H, d, J=8.4 Hz), 8.02 (1H, d, J=9.3 Hz); C NMR (DMSO-d , 75 MHz) δ/ppm: 50.0, 68.1,
6
01.9, 113.3, 113.4, 130.3, 145.0, 156.0, 160.9, 161.7.
7
2
5
-(3-azidopropoxy)-2H-chromen-2-one (4b). White powder, 98% yield; IR: 3069, 2951, 2882,
091, 1703, 1612, 1552, 1510, 1400, 1289, 1237, 1129, 1103, 1018, 992, 859, 833, 749, 717, 614,
-1 1
68, 549, 459 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 1.94-2.03 (2H, m), 3.50 (2H, t, J=6.7
6
Hz), 4.12 (2H, t, J=6.1 Hz), 6.26 (1H, dd, J=2.0 Hz, 9.3 Hz), 6.91-6.99 (2H, m), 7.0 (1H, d, J=8.4
13
Hz), 7.96 (1H, d, J=9.6 Hz); C NMR (DMSO-d
6
, 75 MHz) δ/ppm: 28.5, 48.2, 66.1, 101.8, 113.1,
1
13.2, 113.3, 118.6, 130.1, 145.0, 156.0, 160.9, 162.2, 162.3.
1
9

7
1
3
7
6
-(4-azidobutoxy)-2H-chromen-2-one (4c). White powder, 46% yield; IR: 3083, 2090, 1719,
-1 1
608, 1555, 1507, 1396, 1348, 1281, 1230, 1197, 1121, 992, 830, 751 cm ; H NMR (DMSO-d
6
,
00 MHz) δ/ppm: 1.91-2.10.(4H, m), 4.16 (2H, s, br), 6.28 (2H, d, J=9.3 Hz), 6.93-7.0 (2H, m),
1
3
.63 (1H, d, J=8.7 Hz), 8.0 (1H, d, J=9.3 Hz); C NMR (DMSO-d , 75 MHz) δ/ppm: 25.7, 51.2,
6
8.5, 101.8, 112.9, 113.1, 113.4, 130.1, 145.0, 156.0, 161.0, 162.4.
7
2
1
1
-((6-azidohexyl)oxy)-2H-chromen-2-one (4d). White powder, 62% yield; IR: 3092, 2946, 2930,
856, 2138, 2088, 1720, 1705, 1608, 1555, 1509, 1473, 1462, 1393, 1295, 1229, 1193, 1128,
-1 1
092, 1022, 1001, 846, 751, 723, 615, 569, 478, 461 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm:
.38-1.58 (5H, m), 1.74-1.77 (3H, m), 3.33 (2H, t, J=4.3 Hz), 4.04-4.10 (2H, m), 6.27(1H, d,
1
3
J=9.3 Hz), 6.91-6.96 (2H, m), 7.61 (1H, d, J=8.4 Hz), 7.97 (1H, d, J=9.3 Hz); C NMR (DMSO-
d
6
, 75 MHz) δ/ppm: 25.6, 26.5, 28.8, 29.0, 51.2, 68.8, 101.7, 112.9, 113.0, 113.3, 130.1, 145.0,
1
56.0, 161.0, 162.5.
7
1
-((7-azidoheptyl)oxy)-2H-chromen-2-one (4e). White powder, 80% yield; IR: 3060, 2942, 2097,
-1 1
729, 1614, 1555, 1472, 1290, 1239, 1129, 1028, 850, 720 cm ; H NMR (DMSO-d , 300 MHz)
6
δ/ppm: 1.29-1.35 (6H, m), 1.46-1.50 (2H,m), 1.65-1.70 (2H, m), 3.26 (2H, t, J=6.7 Hz), 4.00 (2H,
t, J=6.4 Hz), 6.24 (1H, d, J=9.6 Hz), 6.86-6.91 (2H,m), 7.56 (1H, d, J=8.4 Hz), 7.93 (1H, d, J=9.3
1
3
Hz); C NMR (DMSO-d
6
, 75 MHz) δ/ppm: 25.9, 26.7, 28.8, 28.9, 51.2, 68.8, 101.6, 112.8, 112.9,
1
13.3, 130.1, 145.0, 156.0, 161.0, 162.5.
7
1
1
-((9-azidononyl)oxy)-2H-chromen-2-one (4f). White powder, 86% yield; IR: 3061, 2920, 2096,
-1 1
739, 1611, 1556, 1471, 1238, 1097, 1016, 851, 721 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm:
.24-1.36 (10H, m), 1.45-1.49 (2H, m), 1.66-1.68 (2H, m), 3.26 (2H, t, J=7.0 Hz), 4.01 (2H, t,
J=6.1 Hz), 6.22 (1H, d, J=9.3 Hz), 6.87-6.92 (2H,m), 7.58 (1H, d, J=8.4 Hz), 7.95 (1H, d, J=9.3
1
3
Hz); C NMR (DMSO-d
6
, 75 MHz) δ/ppm: 26.0, 26.8, 28.9, 29.0, 29.1, 29.3, 29.5, 51.2, 68.9,
1
01.6, 112.8, 113.0, 113.3, 130.1, 145.0, 156.0, 161.0, 162.5.
7
2
8
-((10-azidodecyl)oxy)-2H-chromen-2-one (4g). White powder, 78% yield; IR: 3081, 2919, 2852,
096, 1722, 1709, 1619, 1556, 1508, 1472, 1393, 1378, 1350, 1289, 1236, 1128, 1101, 1018, 890,
-1 1
29, 758, 746, 631, 616, 569, 456 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 1.25-1.70 (14H,
6
m), 2.44-2.48 (2H,m), 3.26-3.30 (2H, m), 4.03 (2H, t, J=5.5 Hz), 6.25 (1H, d, J=9.3 Hz), 6.90-
1
3
6
.95 (2H, m), 7.59 (1H, d, J=8.4 Hz), 7.96 (1H, d, J=9.3 Hz); C NMR (DMSO-d
δ/ppm: 26.0, 26.8, 28.9, 29.1, 29.2, 29.3, 29.5, 51.2, 68.9, 101.7, 112.8, 113.0, 113.3, 130.1, 145.0,
56.1, 161.0, 162.5.
6
, 75 MHz)
1
2
0

7
2
6
-((12-azidododecyl)oxy)-2H-chromen-2-one (4h). White powder, 70% yield; IR: 2917, 2851,
096, 1722, 1710, 1621, 1557, 1508, 1472, 1393, 1290, 1236, 1130, 1100, 1030, 891, 828, 716,
-1 1
16, 569, 457 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 1.25-1.53 (18H, m), 1.67-1.74 (2H,
6
m), 3.30 (2H, t, J=6.7 Hz), 4.06 (2H, t, J=6.4 Hz), 6.27 (1H, d, J=9.9 Hz), 6.91-6.97 (2H, m), 7.62
1
3
(
1H, d, J=8.4 Hz), 7.98 (1H, d, J=9.3 Hz); C NMR (DMSO-d
6
, 75 MHz) δ/ppm: 26.0, 26.8,
2
1
8.8, 29.0, 29.1, 29.3, 29.5, 51.2, 68.9, 101.7, 112.8, 113.0, 113.4, 130.1, 145.0, 156.0, 161.0,
62.5.
4
.2.4. General procedure for the of compounds 5a-p
2
mmol 4a-h and 2 mmol 2a-b were dissolved in a 5 mL of nitrogen-purged DMF in a Schlenk
tube. CuBr (1mmol) and PMDETA (2 mmol) were added and the reaction mixture was degassed
by three cycles and stirred at room temperature for 12 h. After this specified time, the reaction
solution was passed through alumina column to remove copper salt and precipitated in water.
Next, the crude product was filtered. Finally, the crude product was dried in a vacuum oven at
o
3
5 C [45].
7
-((1-(2-((2-oxo-2H-chromen-7-yl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one
(
5a). White powder, 88% yield; mp. 187-189 °C; IR: 3110, 3083, 2954, 1740, 1704, 1607, 1555,
-
1 1
1
4
398, 1228, 1129, 829, 748 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm: 4.54 (2H, t, J=4.9 Hz),
.85 (2H, t, J=4.6 Hz), 5.29 (2H, s), 6.30 (2H, d, J=9.3 Hz), 6.90 (1H, d, J=9.8 Hz), 6.99-7.03
(
2H, m), 7.15 (1H, d, J=2.0 Hz), 7.61 (2H, t, J=8.4 Hz), 7.98 (2H, dd, J=9.6, 2.3 Hz), 8.39 (1H, s);
1
3
C NMR (DMSO-d , 75 MHz) δ/ppm: 49.5, 67.4, 79.6, 102.0, 102.1, 102.4, 113.2, 113.3, 113.4,
6
1
4
13.5, 113.6, 126.2, 130.1, 142.6, 144.9X2, 155.9X2, 160.8, 160.9, 161.5, 161.7;LC-MS (m/z):
+
54 [M +Na]. Anal.Calcd.for C23
H
17
N
3
6
O : C, 64.04; H, 3.97; N, 9.74; found: C, 64.06; H, 3.95;
N, 9.72.
4
-methyl-7-((1-(2-((2-oxo-2H-chromen-7-yl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5b). White powder, 92% yield; mp. 192-194 °C; IR: 3200, 3091, 2958, 1706,
-1 1
1
607, 1555, 1398, 1228, 1136, 832, 749 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 2.39 (3H,
6
s), 4.45 (2H, t, J=4.1 Hz), 4.85 (2H, t, J=4.3 Hz), 5.28 (2H, s), 6.13 (1H, s), 6.27 (1H, d, J=9.3
Hz), 6.76-6.81 (2H, m), 6.89-6.96 (2H, m), 7.38 (1H, d, J=8.4 Hz), 7.50 (1H, d, J=8.4 Hz), 7.63
1
3
(
1H, d, J=8.2 Hz), 7.88 (1H, s); C NMR (DMSO-d
6
, 75 MHz) δ/ppm: 18.9, 49.8, 62.3, 66.9,
1
01.9, 102.2, 112.4, 112.5, 112.7, 113.5, 114.0, 114.2, 124.4, 125.9, 129.3, 143.4, 152.7, 155.2,
2
1

+
1
6
55.8, 160.8, 161.0, 161.2, 161.3; LC-MS (m/z): 468 [M +Na]. Anal.Calcd.for C24
H
19
N
3
6
O : C,
4.72; H, 4.30; N, 9.43; found: C, 64.70; H, 4.33; N, 9.44.
7
-(3-(4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy)-2H-chromen-2-
one (5c). White powder, 94% yield; mp. 150-151 °C; IR: 3120, 3077, 2968, 1723, 1612, 1509,
-1 1
1
298, 1105, 890, 730 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm: 2.35-2.50 (2H, m), 4.04 (2H, t,
J=5.5 Hz), 4.64 (2H, t, J=6.7 Hz), 5.26 (2H, s), 6.27 (2H, dd, J=9.6, 4.1 Hz), 6.75-6.94 (4H, m),
1
3
7
7
1
.27 (1H, s), 7.38 (2H, d, J=8.4 Hz), 7.64 (1H, d, J=9.0 Hz), 7.69 (1H, s); C NMR (DMSO-d ,
6
5 MHz) δ/ppm:29.8, 47.3, 62.4, 64.8, 101.5, 101.7, 102.2, 112.7, 112.9, 113.1, 113.4, 113.7,
+
23.6, 129.0, 129.2, 143.3, 143.6, 155.9X2, 161.4, 161.6, 162.1; LC-MS (m/z): 468 [M +Na].
Anal.Calcd.for C24
H
19
N
3
6
O : C, 64.72; H, 4.30; N, 9.43; found: C, 64.71; H, 4.32; N, 9.45.
4
-methyl-7-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5d). White powder, 96% yield; mp. 163-164 °C; IR: 3117, 3089, 2983, 1715,
-
1 1
1
2
613, 1458, 1390, 1279, 1200, 1128, 1015, 853, 772 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm:
.29-2.33 (2H, m), 2.48 (3H, m), 4.08 (2H, t, J=5.8 Hz), 4.56 (2H, t, J=6.7 Hz), 5.25 (2H, s), 6.20
(
1H, s), 6.28 (2H, d, J=9.3 Hz), 6.85-7.03 (3H, m), 7.12 (1H, d, J=2.3 Hz), 7.60 (1H, t, J=4.6 Hz),
1
3
7
.67 (1H, d, J=8.7 Hz), 7.97 (1H, d, J=9.6 Hz), 8.33 (1H, s); C NMR (DMSO-d , 75 MHz)
6
δ/ppm:18.8, 29.9, 47.3, 62.3, 66.0, 101.8, 102.1, 111.9, 113.1, 113.3, 114.0, 125.7, 127.1, 130.1,
+
1
42.6, 144.9, 154.0, 155.3, 156.0, 160.8, 160.9, 161.6, 162.1, 162.2; LC-MS (m/z): 482 [M +Na].
Anal.Calcd.for C25
H
21
N
3
6
O : C, 65.35; H, 4.61; N, 9.15; found: C, 65.33; H, 4.62; N, 9.17.
7
-((1-(4-((2-oxo-2H-chromen-7-yl)oxy)butyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one
(
5e). White powder, 86% yield; mp. 111-113 °C; IR: 3114, 3082, 2950, 1721, 1708, 1613, 1554,
-
1 1
1
2
7
394, 1277, 1124, 986, 827 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm: 1.69-1.71 (2H, m), 1.95-
.00 (2H, m), 4.07 (2H, t, J=6.1 Hz), 4.45 (2H, t, J=6.4 Hz), 5.24 (2H, s), 6.25-6.29 (2H, m), 6.90-
1
3
.01 (3H, m), 7.13 (1H, s), 7.61 (2H, t, J=6.7 Hz), 7.96 (2H, d, J=9.3 Hz), 8.31 (1H, s); C NMR
, 75 MHz) δ/ppm:26.1, 27.1, 49.7, 62.3, 68.2, 101.8, 102.2, 113.0, 113.1, 113.2, 113.4,
(
DMSO-d
6
1
13.6, 125.5, 130.2, 142.6, 145.0, 155.9, 156.0, 160.9, 161.0, 161.7, 162.3; LC-MS (m/z): 482
+
[
M +Na]. Anal.Calcd.for C25
H
21
N
3
6
O : C, 65.35; H, 4.61; N, 9.15; found: C, 65.36; H, 4.60; N,
9
.16.
4
-methyl-7-((1-(4-((2-oxo-2H-chromen-7-yl)oxy)butyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5f). White powder, 80% yield; mp. 150-151 °C; IR: 3128, 3081, 2959, 1710,
-1 1
1
610, 1511, 1391, 1266, 1126, 989, 826 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 1.69-1.71
6
2
2

(
2H, m), 1.95-1.97 (2H, m), 2.37 (3H, s), 4.07 (2H, t, J=5.5 Hz), 4.45 (2H, t, J=6.7 Hz), 5.25 (2H,
s), 6.20 (1H, s), 6.26 (1H, d, J=9.3 Hz), 6.89-7.02 (3H, m), 7.12 (1H, s), 7.60 (1H, d, J=8.2 Hz),
1
3
7
.67 (1H, d, J=8.7 Hz), 7.96 (1H, d, J=9.3 Hz), 8.31 (1H, s); C NMR (DMSO-d
6
, 75 MHz)
δ/ppm:18.8, 26.1, 27.1, 49.7, 62.3, 68.2, 101.8, 102.2, 111.9, 113.0, 113.1, 113.3, 113.4, 114.0,
1
4
25.5, 127.1, 130.1, 142.6, 145.0, 154.1, 155.3, 156.0, 160.8, 161.0, 161.7, 162.3; LC-MS (m/z):
+
96 [M +Na]. Anal.Calcd.for C26
H
23
N
3
6
O : C, 65.95; H, 4.90; N, 8.87; found: C, 65.97; H, 4.92;
N, 8.85.
7
-((1-(6-((2-oxo-2H-chromen-7-yl)oxy)hexyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one
(
5g). White powder, 74% yield; mp. 111-112°C; IR: 3129, 3083, 2942, 1731, 1615, 1506, 1398,
-1 1
1
274, 1126, 985, 850 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 1.29-1.31 (2H, m), 1.41-1.46
6
(
2H, m), 1.68-1.73 (2H, m), 1.83-1.87 (2H, m), 4.04 (2H, t, J=6.4 Hz), 4.39 (2H, t, J=7.0 Hz),
.26 (2H, s), 6.29 (2H, t, J=7.6 Hz), 6.90-6.96 (2H, m), 7.00 (1H, d, J=8.4 Hz), 7.14 (1H, d, J=2.3
5
1
3
Hz), 7.62 (2H, t, J=7.0 Hz), 7.98 (1H, dd, J=9.6, 3.8 Hz), 8.30 (1H, s); C NMR (DMSO-d
6
, 75
MHz) δ/ppm:25.4, 26.1, 28.8, 30.2, 50.0, 62.3, 68.2, 101.7, 102.2, 112.9, 113.0, 113.2, 113.3,
1
13.6, 125.4, 130.1X2, 142.5, 144.9, 145.0, 155.9, 156.0, 160.9, 161.0, 161.7, 162.5; LC-MS
+
(
m/z): 510 [M +Na]. Anal.Calcd.for C27
H
25
N
3
6
O : C, 66.52; H, 5.17; N, 8.62; found: C, 66.50; H,
5
.18; N, 8.63.
4
-methyl-7-((1-(6-((2-oxo-2H-chromen-7-yl)oxy)hexyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5h). White powder, 72% yield; mp. 108-110°C; IR: 3120, 3069, 2936, 1710,
-
1 1
1
609, 1556, 1390, 1267, 1123, 990, 874 cm ; H NMR (DMSO-d
2H, m), 1.39-1.48 (2H, m), 1.68-1.75 (2H, m), 1.81-1.88 (2H, m), 2.39 (3H, s), 4.03 (2H, t, J=6.1
Hz), 4.39 (2H, t, J=7.0 Hz), 5.26 (2H, s), 6.21 (1H, s), 6.27 (1H, d, J=9.3 Hz), 6.90-6.95 (2H, m),
.01 (1H, dd, J=8.7, 2.6 Hz), 7.12 (1H, d, J=2.3 Hz), 7.60 (1H, d, J=8.4 Hz), 7.67 (1H, d, J=8.7
6
, 300 MHz) δ/ppm: 1.29-1.36
(
7
1
3
Hz), 7.97 (1H, d, J=9.3 Hz), 8.30 (1H, s); C NMR (DMSO-d
6
, 75 MHz) δ/ppm:18.7, 25.4, 26.1,
2
1
8.8, 30.2, 50.0, 62.3, 68.8, 101.7, 102.2, 111.9, 112.9, 113.0, 113.2, 113.3, 114.0, 125.4, 127.1,
+
30.1, 142.5, 144.9, 154.0, 155.3, 156.0, 160.7, 160.9, 161.6, 162.5; LC-MS (m/z): 524 [M +Na].
: C, 67.05; H, 5.43; N, 8.38; found: C, 67.07; H, 5.41; N, 8.36.
Anal.Calcd.for C28
H
27
N
3
O
6
7
-((1-(7-((2-oxo-2H-chromen-7-yl)oxy)heptyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-
one (5i). White powder, 74% yield; mp. 106-107°C; IR: 3127, 3083, 2943, 1726, 1617, 1506,
-
1 1
1
1
275, 1160, 1126, 850, 822cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm: 1.23-1.36 (6H, m), 1.68-
.70 (2H, m), 1.80-1.85 (2H, m), 4.04 (2H, t, J=6.4 Hz), 4.38 (2H, t, J=7.0 Hz), 5.26 (2H, s), 6.26-
2
3

6
.32 (2H, m), 6.91-7.03 (3H, m), 7.15 (1H, s), 7.62 (2H, t, J=7.6 Hz), 7.99 (2H, dd, J=9.6, 3.2
1
3
Hz), 8.29 (1H, s); C NMR (DMSO-d
6
, 75 MHz) δ/ppm: 25.9, 26.4, 28.6, 28.9, 30.2, 50.0, 62.3,
6
1
8.8, 101.7, 102.1, 112.2, 113.0, 113.2, 113.4, 113.6, 125.4, 130.1, 142.4, 145.0, 155.9, 156.0,
+
61.0, 161.7, 162.5; LC-MS (m/z): 524 [M +Na]. Anal.Calcd.for C28
H
27
N
3
6
O : C, 67.05; H, 5.43;
N, 8.38; found: C, 67.04; H, 5.42; N, 8.39.
4
-methyl-7-((1-(7-((2-oxo-2H-chromen-7-yl)oxy)heptyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5j). White powder, 68% yield; mp. 102-104°C; IR: 3127, 3083, 2943, 1726,
-
1 1
1
1
614, 1506, 1345, 1275, 1160, 1126, 985, 822 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm: 1.21-
.42 (6H, m), 1.77-1.81 (2H, m), 1.94-1.98 (2H, m), 2.37 (3H, s), 3.98 (2H, t, J=6.4 Hz), 4.42
(
2H, t, J=7.0 Hz), 5.24 (2H, s), 6.10 (1H, s), 6.22 (1H, d, J=9.3 Hz), 6.72 (1H, s), 6.80-6.95 (2H,
1
3
m), 7.36-7.38 (2H, m), 7.48 (1H, d, J=8.7 Hz), 7.65 (1H, d, J=9.3 Hz), 7.81 (1H, s); C NMR
DMSO-d , 75 MHz) δ/ppm:18.8, 25.9, 26.5, 28.8, 28.9, 30.3, 50.5, 62.3, 68.5, 101.3, 102.0,
12.1, 112.5, 112.6, 112.8, 112.9, 114.0, 123.4, 125.9, 129.0, 142.9, 143.8, 152.8, 155.1, 155.9,
(
6
1
1
5
+
61.2, 161.3, 161.4, 162.4; LC-MS (m/z): 538 [M +Na]. Anal.Calcd.for C29
H
29
N
3
6
O : C, 67.56; H,
.67; N, 8.15; found: C, 67.55; H, 5.68; N, 8.14.
7
-((9-(4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)nonyl)oxy)-2H-chromen-2-
one (5k). White powder, 78% yield; mp. 108-109°C; IR: 3127, 3083, 2919, 1729, 1614, 1506,
-
1 1
1
1
399, 1274, 1126, 995, 854 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm: 1.15-1.32 (10H, m),
.68-1.80 (4H, m), 4.03 (2H, t, J=3.22 Hz), 4.33 (2H, t, J=7.0 Hz), 5.23 (2H, s), 6.26 (2H, q, J=7.6
Hz), 6.84-7.00 (3H, m), 7.11 (1H, t, J=2.3 Hz), 7.59 (2H, t, J=7.6 Hz), 7.95 (2H, dd, J=9.3, 3.5
1
3
Hz), 8.26 (1H, s); C NMR (DMSO-d
6
, 75 MHz) δ/ppm: 26.0, 26.4, 28.9, 29.4, 30.3, 31.0, 50.0,
6
1
6
2.3, 68.9, 101.7, 102.2, 112.8, 113.0, 113.2, 113.3, 113.6, 125.4, 130.1, 142.5, 145.0, 155.9,
+
56.0, 160.9, 161.0, 161.7, 162.5; LC-MS (m/z): 552 [M +Na]. Anal.Calcd.for C30
H
31
N
3
6
O : C,
8.04; H, 5.90; N, 7.93; found: C, 68.06; H, 5.91; N, 7.90.
4
-methyl-7-((1-(9-((2-oxo-2H-chromen-7-yl)oxy)nonyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5l). White powder, 76% yield; mp. 118-119°C; IR: 3127, 3078, 2931, 1713,
-1 1
1
611, 1555, 1388, 1282, 1125, 825 cm ; H NMR (DMSO-d
m), 1.68-1.83 (4H, m), 2.39 (3H, s), 4.04 (2H, t, J=6.4 Hz), 4.37 (2H, t, J=6.7 Hz), 5.26 (2H, s),
.22 (1H, s), 6.27 (1H, d, J=9.3 Hz), 6.91-7.04 (3H, m), 7.13 (1H, d, J=2.3 Hz), 7.60 (1H, d, J=8.4
6
, 300 MHz) δ/ppm: 1.25-1.38 (10H,
6
1
3
Hz), 7.68 (1H, d, J=8.7 Hz), 7.98 (1H, d, J=9.3 Hz), 8.29 (1H, s); C NMR (DMSO-d
6
, 75 MHz)
δ/ppm:18.8, 26.0, 26.4, 28.9, 29.0, 29.2, 29.4, 30.3, 50.0, 62.3, 68.9, 101.7, 102.2, 111.9, 112.8,
2
4

1
1
7
13.0, 113.2, 113.3, 113.9, 125.4, 127.1, 130.1, 142.5, 145.0, 154.0, 155.3, 156.0, 160.8, 161.0,
+
61.6, 162.5; LC-MS (m/z): 566 [M +Na]. Anal.Calcd.for C31
H
33
N
3
6
O : C, 68.49; H, 6.12; N,
.73; found: C, 68.47; H, 6.10; N, 7.75.
7
-((1-(10-((2-oxo-2H-chromen-7-yl)oxy)decyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-
one (5m). White powder, 82% yield; mp. 115-116°C; IR: 3129, 3083, 2920, 1731, 1615, 1506,
-
1 1
1
1
6
399, 1276, 1098, 985, 850 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 1.20-1.35 (12H, m),
6
.66-1.79 (4H, m), 4.01 (2H, t, J=6.1 Hz), 4.33 (2H, t, J=7.0 Hz), 5.23 (2H, s), 6.23-6.29 (2H, m),
.87-7.00 (3H, m), 7.11 (1H, d, J=2.3 Hz), 7.56-7.61 (2H, m), 7.95 (2H, dd, J=9.6, 3.2 Hz), 8.26
1
3
(
1H, s); C NMR (DMSO-d
6
, 75 MHz) δ/ppm: 26.0, 26.4, 29.0, 29.1, 29.3, 29.4, 29.5, 30.3, 50.0,
6
1
2.3, 68.9, 101.7, 102.1, 112.8, 113.0, 113.2, 113.3, 113.6, 125.4, 130.1, 142.5, 144.9, 145.0,
+
55.9, 156.0, 160.9, 161.0, 161.7, 162.5; LC-MS (m/z): 566 [M +Na]. Anal.Calcd.for
: C, 68.49; H, 6.12; N, 7.73; found: C, 68.48; H, 6.11; N, 7.76.
C
31
H
33
N
3
O
6
4
-methyl-7-((1-(10-((2-oxo-2H-chromen-7-yl)oxy)decyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5n). White powder, 84% yield; mp. 106-107°C; IR: 3150, 3083, 2917, 1727,
-1
1
1
618, 1554, 1387, 1298, 1134, 998, 836 cm ; H NMR (CDCl
12H, m), 1.60-1.61 (2H, m), 1.78-1.93 (2H, m), 2.40 (3H, s), 4.01 (2H, t, J=6.4 Hz), 4.38 (2H, t,
J=7.3 Hz), 5.27 (2H, s), 6.16 (1H, s), 6.25 (1H, d, J=9.6 Hz), 6.79-6.85 (2H, m), 6.93-6.98 (2H,
3
, 300 MHz) δ/ppm: 1.26-1.46
(
1
3
m), 7.36 (1H, d, J=8.7 Hz), 7.51 (1H, d, J=8.7 Hz), 7.63-7.66 (2H, m); C NMR (CDCl
3
, 75
MHz) δ/ppm: 18.9, 26.1, 26.6, 29.1, 29.4, 29.5, 30.5, 50.7, 62.5, 68.8, 101.4, 102.3, 112.5, 112.6,
1
13.1, 113.2, 114.2, 122.9, 125.9, 128.9, 143.1, 143.7, 152.7, 155.3, 156.1, 161.3, 162.6; LC-MS
+
(
m/z): 580 [M +Na]. Anal.Calcd.for C32
H
35
N
3
6
O : C, 68.92; H, 6.33; N, 7.54; found: C, 68.93; H,
6
.35; N, 7.51.
7
-((1-(12-((2-oxo-2H-chromen-7-yl)oxy)dodecyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-
one (5o). White powder, 85% yield; mp. 116-117°C; IR: 3129, 3087, 2918, 1730, 1615, 1507,
-
1 1
1
1
397, 1232, 1126, 987, 822 cm ; H NMR (DMSO-d
6
, 300 MHz) δ/ppm: 1.16-1.35 (16H, m),
.65-1.78 (4H, m), 4.01 (2H, t, J=6.1 Hz), 4.33 (2H, t, J=7.0 Hz), 5.23 (2H, s), 6.25 (2H, t, J=9.3
Hz), 6.86-6.99 (3H, m), 7.11(1H, d, J=2.0 Hz), 7.58 (2H, t, J=8.4 Hz), 7.95 (2H, dd, J=9.3, 3.8
1
3
Hz), 8.26 (1H, s); C NMR (DMSO-d
6
, 75 MHz) δ/ppm: 26.1, 26.4, 29.0, 29.1, 29.4, 29.5, 29.6,
3
1
0.3, 50.0, 62.3, 68.9, 101.7, 102.1, 112.8, 113.0, 113.2, 113.3, 113.6, 125.4, 130.1, 142.5, 145.0,
+
55.9, 156.0, 160.9, 161.0, 161.7, 162.5; LC-MS (m/z): 594 [M +Na]. Anal.Calcd.for
: C, 69.33; H, 6.52; N, 7.35; found: C, 69.35; H, 6.51; N, 7.34.
C
33
H
37
N
3
O
6
2
5

4
-methyl-7-((1-(12-((2-oxo-2H-chromen-7-yl)oxy)dodecyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5p). White powder, 84% yield; mp. 103-105°C; IR: 3134, 3083, 2917, 1726,
-1 1
1
614, 1508, 1389, 1276, 1129, 836 cm ; H NMR (DMSO-d
m), 1.67-1.79 (4H, m), 2.37 (3H, s), 4.02 (2H, t, J=6.4 Hz), 4.33 (2H, t, J=7.0 Hz), 5.24 (2H, s),
.19 (1H, s), 6.25 (1H, d, J=9.3 Hz), 6.88-7.02 (2H, m), 7.11 (1H, d, J=2.3 Hz), 7.57 (1H, d, J=8.4
6
, 300 MHz) δ/ppm: 1.18-1.37 (16H,
6
1
3
Hz), 7.65 (1H, d, J=8.7 Hz), 7.95 (2H, d, J=9.6 Hz), 8.25 (1H, s); C NMR (DMSO-d
6
, 75 MHz)
δ/ppm: 18.8, 26.0, 26.4, 29.0, 29.1, 29.3, 29.5, 29.6, 30.3, 50.0, 62.3, 68.9, 101.7, 102.1, 111.9,
1
1
7
12.8, 113.0, 113.3, 113.4, 114.0, 125.4, 127.1, 130.1, 142.5, 145.0, 154.0, 155.3, 156.0, 160.8,
+
61.0, 161.6, 162.5; LC-MS (m/z): 586[M ]. Anal.Calcd.for C34
H
39
N
3
6
O : C, 69.72; H, 6.71; N,
.17; found: C, 69.74; H, 6.72; N, 7.15.
4
.3. CA inhibition assays
An SX.18V-R Applied Photophysics (Oxford, UK) stopped flow instrument has been used to
assay the catalytic/inhibition of various CA isozymes [47]. Phenol Red (at a concentration of 0.2
mM) has been used as an indicator, working at an absorbance maximum of 557 nm, with 10 mM
Hepes (pH 7.4) as a buffer, 0.1M Na
2
SO
4
or NaClO (for maintaining constant the ionic strength;
4
these anions are not inhibitory in the used concentration), following the CA-catalyzed CO
2
ᵒ
hydration reaction for a period of 5-10s. Saturated CO
2
solutions in water at 25 C were used as
substrate. Stock solutions of inhibitors were prepared at a concentration of 10mM (in DMSO-
water 1:1, v/v) and dilutions up to 0.01 nM done with the assay buffer mentioned above. At least 7
different inhibitor concentrations have been used for measuring the inhibition constant. Inhibitor
and enzyme solutions were pre-incubated together for 6 h at 4 °C prior to assay, in order to allow
for the formation of the E-I complex. Triplicate experiments were done for each inhibitor
concentration, and the values reported in this paper are the mean of such results. The inhibition
constants were obtained by non-linear least squares methods using the Cheng-Prusoff equation, as
reported earlier, and represent the mean from at least three different determinations [48]. All CA
isozymes used here were recombinant proteins obtained as reported earlier by our group [48-50].
4
.4. Molecular Modeling
Although there are some crystal structures of hCA IX and XII already deposited on protein data
bank (PDB) server, these structures are co-crystallized with sulfonamide CA inhibitors or apo
2
6

form (for isoform IX). However, coumarin derivatives were shown to plug the entrance to the
+
+
enzyme active site and make no interactions with Zn ion [30,31, 51]. Hence target binding site
of studied compounds were expected to be different than the ones of the crystal structures with
sulfonamide CA inhibitors. Therefore, the crystal structures with PDB codes 5DVX [52] and
5
MSA [53] for hCA IX and XII were retrieved, respectively. The crystal structure of coumarin
derivative bound hCA II that was resolved by Maresca et. al with PDB code 3F8E was also
downloaded [30]. Firstly, the mutated sequences in crystal structure (if there was any) were
corrected to match the wild type sequences in UniProt. The crystal structured were prepared using
Protein Preparation Wizard [54] implemented in the Schrodinger package with the default settings.
The ions, small molecules such as some derivatives of glycol that were used to help crystallization
+
+
and all but Zn ion coordinated water molecules were removed. Protonation states of amino acids
were predicted using PROPKA [55] whereas Epik [56] was used to predict ionization and
tautomeric states of the co-crystallized ligand at the physiological pH of 7.4. Restraint geometry
optimization and energy minimization with OPLS3 forcefield was the last step of protein
preparation [57]. To correctly assess the binding pocket of coumarins and hence the studied
compounds for isoforms IX and XII, the prepared hCA IX and hCA XII isoform structures were
aligned to prepared hCA II – coumarin derivative structures. Then, the co-crystallized ligands
inside isoforms hCA IX and XII were removed. The coumarin derivative of hCA II isoform as
+
+
well as Zn -coordinated water molecule was inserted into prepared hCA IX and XII isoform
structures. Maestro module of Schrodinger molecular modeling program was used to visualize and
alignment of structures [58]. As there may be some clashes in obtained complex structures,
molecular dynamics (MD) simulations were performed to adjust specifically side chains of
coumarin binding site. The complexes were placed in orthorhombic boxes of TIP3P water
molecules with box sizes of 10 Å along all three dimensions. The systems were all neutralized and
ionic concentration of 0.15 M NaCl solution was used to adjust concentration of the solvent
systems. The Desmond all atom MD simulations program was used [59]. Default settings of
Desmond package with initial temperature of 310 K were used along with default relaxation
protocol. MD simulations were performed for 10 ns. Then, the trajectories obtained by simulations
were clustered based on the average RMSD differences of ligand molecule. The representative
structure from the cluster with the largest size was taken as initial complex structure for docking
studies.
2
7

As mentioned before, coumarin derivatives could hydrolyze to form cinnamic acid derivatives
31]. Since there are two coumarin rings in the studied molecules as shown in Fig. 1, three
[
different hydrolysis products could be formed. The structures of studied compounds along with
their three possible hydrolysis products were drawn using Maestro module and 3D structures were
first subjected to restrained minimization. Then, LigPrep module [60] of Schrodinger with OPLS3
forcefield were used to prepare the compounds. The Schrodinger Epik module was used to
generate ionization for molecules in aqueous solution at pH 7.4.
Molecular docking studies were performed by GOLD docking program [61-63] that is based on a
genetic algorithm for docking flexible ligands into binding sites. Here, representative structures of
largest cluster of MD trajectories were used as protein-ligand complexed. The binding site was
shaped residues within 6 Å vicinity of ligands in complexes. While proteins binding pocket were
held rigid during docking, side chains of some amino acid such as Serine, Threonine and Tyrosine
+
hydroxyl groups and Lysine NH
3
groups were optimized (i.e., rotamers of these residues were
considered) throughout the docking in order to maximize the flexibility of binding pocket
residues. For ligand flexibility the default settings were considered, however protonated
carboxylic acid groups and ring-NHR or ring-NR1R2 groups were rotated instead of flipped. 50
docking poses were requested for each ligand and to increase the accuracy of binding pose
prediction, diverse solutions options with cluster size of 5 and RMSD differences of 3 Å were
chosen. Default settings were used for population (population size, 100) and genetic operations
(
number of operations, 100000) steps. Search efficiency were set as 200 % as recommended for
large highly flexible ligands. CHEMPLP fitness function of GOLD package was used for pose
prediction as it was found to give the highest success rates for pose prediction and virtual
screening experiments against diverse test sets and the default function of GOLD.
Supplementary Materials
1
13
H and C NMR and MS spectra of the synthesized compounds are given in Supplementary
Materials.
Acknowledgments
2
8

This work was supported by the Bezmialem Research Fund of the Bezmialem Vakıf University.
Project Number: 6.2016/11.
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