Inhibitory effects and structural insights for a novel series of coumarin-based compounds that selectively target human CA IX and CA XII carbonic anhydrases

Article abstract of DOI:10.1016/j.ejmech.2017.11.061

Coumarin derivatives are a peculiar class of inhibitors of the family of metalloenzymes carbonic anhydrases (CA, EC 4.2.1.1). Several coumarins display higher affinity and selectivity toward most relevant and druggable CA isoforms. By decorating the natur

Full text of DOI:10.1016/j.ejmech.2017.11.061

Accepted Manuscript
Inhibitory effects and structural insights for a novel series of coumarin-based
compounds that selectively target human CA IX and CA XII carbonic anhydrases
Laura De Luca, Francesca Mancuso, Stefania Ferro, Maria Rosa Buemi, Andrea
Angeli, Sonia del Prete, Clemente Capasso, Claudiu T. Supuran, Rosaria Gitto
PII:
S0223-5234(17)30964-9
10.1016/j.ejmech.2017.11.061
EJMECH 9936
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 July 2017
Revised Date: 13 October 2017
Accepted Date: 22 November 2017
Please cite this article as: L. De Luca, F. Mancuso, S. Ferro, M.R. Buemi, A. Angeli, S. del Prete, C.
Capasso, C.T. Supuran, R. Gitto, Inhibitory effects and structural insights for a novel series of coumarin-
based compounds that selectively target human CA IX and CA XII carbonic anhydrases, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.061.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Inhibitory Effects and Structural Insights for a Novel series of
Coumarin-based Compounds that Selectively Target human CA IX and
CA XII Carbonic Anhydrases
Laura De Luca1,* Francesca Mancuso,1,2 Stefania Ferro,1 Maria Rosa Buemi,1 Andrea Angeli,3
Sonia del Prete,4 Clemente Capasso,4 Claudiu T. Supuran,3 Rosaria Gitto1,*
1 Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali (CHIBIOFARAM),
Università degli Studi di Messina, Viale Annunziata, I-98168, Messina, Italy
2 Fondazione Prof. Antonio Imbesi, Piazza Pugliatti 1, 98100, Messina, Italy
3 Dipartimento NEUROFARBA, Università di Firenze, Via Ugo Schiff, I-50019, Sesto Fiorentino,
Italy
4Istituto di Bioscienze e Biorisorse - CNR ,Via Pietro Castellino 111- 80131, Napoli, Italy,
Corresponding authors:
Laura De Luca, PhD, Associate professor on Medicinal Chemistry, Dipartimento di Scienze
Chimiche, Biologiche, Farmaceutiche ed Ambientali (CHIBIOFARAM), Università degli Studi di
Messina, Viale Annunziata, I-98168, Messina, Italy
Tel 00390906766410, Fax 00390906766404, e-mail: ldeluca@unime.it
Rosaria Gitto, PhD, Associate professor on Medicinal Chemistry, Dipartimento di Scienze
Chimiche, Biologiche, Farmaceutiche ed Ambientali (CHIBIOFARAM), Università degli Studi di
Messina, Viale Annunziata, I-98168, Messina, Italy
Tel 00390906766412, Fax 00390906766404, e-mail: rgitto@unime.it
Keywords: coumarins; hCAs; hCA IX, hCA XII; antitumor agents; molecular docking
Abbreviations: hCA: human Carbonic Anhydrases; AAZ: acetazolamide; MBG: metal
binding groups; CARP: CA related-proteins; SG: sticky group; FC: Flash Chromatography;
PDB: RCSB Protein Data Bank;
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Abstract
Coumarin derivatives are a peculiar class of inhibitors of the family of metalloenzymes
carbonic anhydrases (CA, EC 4.2.1.1). Several coumarins display higher affinity and
selectivity toward most relevant and druggable CA isoforms. By decorating the natural
compound umbelliferone (1) we have identified a new series of coumarin-based
compounds demonstrating high CA inhibitory effects with nanomolar affinity for hCA IX
and hCA XII isoforms that were considered a target amenable to develop antitumor
agents. The most active tested compounds proved to be potent inhibitors with Ki values
equal to that of the well-known inhibitor acetazolamide (AAZ), that lacks selectivity over
ubiquitous hCA I and hCA II. As suggested by docking studies the coumarins, that are
lacking of the canonical metal binding groups, do not interact with Zinc ion within the
catalytic site as found for classical sulfonamide type inhibitors of CAs. Thus, the studied
inhibitors might possess a non-classical inhibitory mode of action preventing the carbon
dioxide to entry into catalytic cavity and its conversion into bicarbonate ion. Specifically,
the most active inhibitor of hCA XII compound 18i (Ki value of 5.5 nM) and its supposed
hydrolytic products could establish a web of H-bond interactions within the enzymatic
cavity.
1. Introduction
Coumarins are a very large series of polyphenolic derivatives widely diffuse throughout the
plant kingdom. Their structural diversity led to the classification into different categories
such as the simple coumarin prototype and many other groups of polycyclic analogues
(furanocoumarins and pyranocoumarins). The most widespread derivatives natural
compounds are umbelliferone (1), esculetin (2) and scopoletin (3), that belong to the
simple coumarin scaffold (see Chart 1). The wide range of biological functions attributed to
coumarin derivatives made them an attractive heterocyclic system as mould for further
chemical derivatization to identify novel therapeutic agents. To date, several studies have
proven multiple potential roles which include antimicrobial,[1] antiviral,[2-4] anti-
inflammatory,[5, 6] anti-oxidant,[5] anti-coagulant,[7] lipid lowering,[8] anti-proliferative
activity in some cancer cells lines,[9] and so on.
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HO
HO
HO
O
O
O
O
esculetin, 2
umbelliferone, 1
O
HO
O
O
scopoletin, 3
Chart 1. Chemical structures of coumarin
derivatives (1-3)
In the last decades, several coumarin derivatives have shown to constitute a new class of
inhibitors of the carbonic anhydrase (CA, EC 4.2.1.1) family [10-13]. The family of human
carbonic anhydrases (hCAs) comprises 16 α–CA isozymes that differ for kinetic properties,
tissue and cellular distribution. Among these isoforms, five are cytosolic (CA I-III, CA VII
and CA XIII), two are mitochondrial (CA VA and CA VB), four are membrane-bound (CA
IV, CA IX, CA XII and CA XIV) and one is secreted into saliva and milk (CA VI). Three
acatalytic isoforms are also known, the CA related-proteins (CARP), VIII, X and XI which
are located in the cytosol. CAs catalyze the simple, but crucial, reversible reaction of
hydration of carbon dioxide to bicarbonate ion and protons. Therefore, CAs are involved in
numerous physiological and pathological processes, including respiration and transport of
CO2/bicarbonate between metabolizing tissues and lungs, pH and CO2 homeostasis,
electrolyte secretion in a variety of tissues/organs, biosynthetic reactions (such as
gluconeogenesis, lipogenesis, and ureagenesis), bone resorption, calcification, and
tumorigenicity. On this basis, hCA inhibitors (hCAIs) could exert therapeutic potential for
the treatment of a plethora of pathologies. In addition to the validated role of the well-
known hCAIs as diuretic and anti-glaucoma agents, they also emerged as potential
anticonvulsant, antiobesity, anti-infective and anticancer drugs. However, the main
drawback of the clinically used CAIs is the lack of isoform selectivity, which may lead to
side effects due to the inhibition of the ubiquitous hCA I and II isoforms.
To obtain new theranostic tools useful in cancer, an attractive strategy is recently focused
on the development of new highly selective molecules targeting human CA IX/XII [14-17].
Specifically, the dimeric membrane bound hCA IX isoform is overexpressed in hypoxic
tumors, whilst it is not generally abundant in normal tissue. The catalytic activity of hCA IX
isoform is maximum at low pH values, that characterize hypoxic tumors not responsive to
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chemotherapy and radiotherapy. Therefore, it seems that hCA IX contributes to
acidification of the extracellular environment maintaining a moderately alkaline intracellular
pH. In a similar way, the transmembrane tumor-associated isozyme hCA XII decreases the
extracellular pH cooperating with hCA IX [14].
Typically, the CA family is inhibited by compounds anchoring the Zinc ion, through metal
binding groups (MBG) such as sulfonilamides and their bioisosteres (sulfamates,
sulfamides, N-substituted sulfonilamides, and so on) [18-22]. Due to the lack of the MBG,
the coumarins could represent a class of CAIs characterized by unusual mechanism of
enzyme inhibition [23]. The analysis of the X-ray crystal structure of the adduct hCA II with
the natural product 6-(1-S-hydroxy-3-methylbutyl)-7-methoxy-2H-chromen-2-one (4),
isolated from Leionema Ellipticum (Rutaceae), demonstrated the presence in to the
enzyme active site of its hydrolytic product as the substituted (Z)-2-hydroxycinnamic acid
derivative [12]. A similar behaviour it has been observed for the unsubstituted coumarin (5)
[10] for which the (E)-2-hydroxycinnamic acid is bound to the hCA II catalytic site.
Moreover, it has been demonstrated that several (thioxo)coumarins (e.g. 6) might occlude
the entrance of the enzyme active site [24].
OH
O
O
O
O
O
4
5
R 1
R 2
HO
R 3
O
O
O
S
R 4
R1= H, Me, Ph;
6
R2=R3=H, OH, OAc, OBz
R4= H, Me
Chart 2. Chemical structures of coumarin-based
carbonic anhydrase inhibitors 4-6 and new
designed compounds
Therefore, several coumarins bind the enzyme cavity through a sticky group (SG) attached
to the heterocyclic scaffold which can also incorporate a tail which makes additional
interaction away from the catalytic hole. It is well known that both the rim part of active site
cavity and the middle region are the most variable region of among the 16 isoforms
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controlling the selectivity profile of coumarin derivatives over abundant hCA I and hCA II
isoforms. Specifically, the 7-hydroxycoumarin (umbelliferone, 1) and some of its
derivatives [11] are inhibitors of the tumor-associated isoforms CA IX and XII.
In the last decade, we have been engaged in the development of potent and selective
inhibitors from synthetic source and we have identified a large class of isoquinoline-
sulfonamides being CAIs at nanomolar concentration [25-34]. With the aim to identify new
CAIs structurally related to natural products, we now report a novel series of coumarin
derivatives that were designed to study the influence of R1, R2, R3, R4-substituents on
coumarin scaffold (see Chart 2). To evaluate the inhibitory activity and selectivity toward
hCA IX/XII over ubiquitous hCA I and hCA II, all obtained coumarins were in vitro assayed
against these selected isoforms. Finally, to in-depth study the binding poses of the most
active inhibitors the docking simulations have been carried out.
2. Results and discussion
Firstly, we have focused our interest on 7-hydroxycoumarin (1), 6,7-dihydroxycoumarin (2)
and 6-hydroxycoumarin (7) that were also converted in to the corresponding esters 8-13
following previously reported procedures (Scheme 1) [35].
Scheme 1.
i
8, R1=OAc; R2=H;
9, R1=R2=OAc;
10, R1=H; R2=OAc;
1, R1=OH; R2=H;
2, R1=R2=OH;
ii
7, R1=H; R2=OH;
11, R1=OBz; R2=H;
12, R1=R2=OBz;
13, R1=H; R2=OBz;
Reagents and conditions: i) Ac2O, H2SO4, TEA,
rt; ii) BzCl, Et3N, DCM, rt.
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The inhibitory effects of this subseries of hydroxyl coumarins 1-2, 7 and corresponding
esters 8-13 were evaluated against the tumor-associated hCA IX and hCA XII and the
ubiquitous isoforms hCA I and hCA II. The Kis data are summarized in Table 1 in
comparison with well-known inhibitor acetazolamide (AAZ).
From the analysis of Ki values we observed that all the compounds show inhibitory activity
against hCA IX and hCA XII with affinity in the range of 23.1-116.2 nM and 9.3-814.4 nM,
respectively. Most of tested compounds were potent inhibitors which proved to be equi-
active when compared with AAZ. In contrast, the tested coumarins were ineffective
inhibitors of the hCA I and hCA II (Ki values > 10,000 nM), thus showing high selectivity
over these isoforms that are considered responsible of side-effects of CAIs. In terms of
structure-affinity relationships it could be observed that the natural compounds 7-hydroxy
and 6,7-dihydroxy-coumarins 1-2 were generally more active inhibitors when compared
with corresponding ester derivatives 8-9 and 11-12. Whereas, by masking the 6-hydroxyl
group of coumarin 7 we obtained esters 10 and 13 that showed a slight reduction of
inhibitory effects.
Table 1. Ki values of coumarin derivatives (1-2, 7-13) against
hCA I, hCA II, hCA IX, and hCA XII. For comparison purpose
data of AAZ were also reported
R 2
.
O
O
R 1
Ki(nM)a
R1
OH
OH
H
OAc
OAc
H
OBz
OBz
H
R2
H
OH
OH
H
OAc
OAc
H
OBz
OBz
-
hCA I
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
250
hCA II
hCA IX
24.9
36.4
30.5
40.2
116.2
23.1
51.4
38.4
29.9
25.0
hCA XII
45.1
9.3
1
2
7
8
9
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
12.1
33.9
573.2
48.2
47.0
814.4
292.4
87.4
5.7
10
11
12
13
AAZb
-
aErrors are in the range of ± 5-10% of the reported value, from 3
different assays. bData are taken from Reference 25.
From the first subseries of coumarin-based compounds, the umbelliferone (1) has been
selected for further structural optimization. Therefore, the second and third subseries of 4-
aryl-7-hydroxy-2H-chromen-2-ones (17a-i) and 4-aryl-7-hydroxy-8-methyl-2H-chromen-2-
ones (18a-i) have been designed by introducing aryl group as well as methyl group at C-4
and C-8 position of coumarin nucleus. To synthesize compounds 17a-h and 18 a-h, the
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classical Pechmann condensation has been employed (see Scheme 2). Thus, selected
commercially available ethyl benzoylacetates 14 a-h and the resorcinol 15 or 2-
methylresorcinol 16 reacted with in strong acid conditions to provide the desired coumarins
17a-h and 18 a-h, respectively. To study the influence of the introduction of a polar group
on phenyl ring, it was also performed the nitro-reduction of compounds 17h and 18h in to
the corresponding aniline derivatives 17i and 18i. All compounds were carefully
structurally characterized by spectroscopic techniques (see experimental section).
Scheme 2
O
O
O
+
HO
OH
R1
R2
15, R2 = H
14 a-h
16, R2 = Me
i
R1
R1
a
b
c
d
e
f
H
2-Cl
3-Cl
4-Cl
2-F
3-Me
4-Me
4-NO2
g
h
HO
O
O
ii
i
4-NH2
R2
R2= H, 17 a-h
R2= Me, 18 a-h
Reagents and conditions: i) H2SO4, rt; ii)
NH2NH2-H2O, EtOH, Pd/C, 70° C.
Table 2 collects the Ki inhibition data determined for coumarin-derived compounds
belonging to the 17a-i and 18 a-i subseries that differs for the presence of the C-8 methyl
substituent. As you can see in Table 2, the two series of 4-aryl-7-hydroxy-2H-chromen-2-
ones proved to be active and selective inhibitors over ubiquitous hCA I and hCA II
isoforms. Among the first subseries of compounds 17a-i we found that that the introduction
of an unsubstituted phenyl ring at C-4 position (i.e. 17a) resulted in the decrease of
inhibitory effects toward hCA IX and hCA XII isoforms when compared with Ki values of
prototype 1 (see Table 1). Concerning the inhibitory effects hCA IX isoform, with exception
of compound 18e (Ki value of 95.9 nM) a flat SAR generally characterizes the inhibitory
profile of the two subseries of compounds 17 and 18 for which the Ki values fall in a
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narrow range from 17.0 to 47.2 nM. Therefore, we can hypothesize that the 4-
arylsubstituent occupies a very large area lacking in specific hydrophilic/hydrophobic
contacts with crucial aminoacid residues.
Table 2. Ki values of coumarin derivatives 17a-i and 18
a-i against hCA I, hCA II, hCA IX, and hCA XII.
R 1
R2= H, 17 a-i
R2= Me, 18 a-i
HO
O
O
R 2
Ki(nM)a
hCA II
R1
H
2-Cl
3-Cl
4-Cl
2-F
3-Me
4-Me
4-NO2
4-NH2
H
2-Cl
3-Cl
4-Cl
2-F
R2
H
H
H
H
H
H
H
H
hCA I
hCA IX
47.2
30.8
31.0
31.5
31.8
17.0
35.8
20.3
21.6
39.5
27.5
36.6
35.3
95.9
37.4
38.2
30.9
24.2
hCA XII
90.8
7.8
17a
17b
17c
17d
17e
17f
17g
17h
17i
18a
18b
18c
18d
18e
18f
18g
18h
18i
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
>10000 >10000
7.6
8.9
8.0
7.8
64.7
6.5
7.7
402.2
31.0
369.2
293.7
9.0
89.1
44.9
9.3
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
3-Me
4-Me
4-NO2
4-NH2
5.5
aErrors are in the range of ± 5-10% of the reported value, from 3
different assays.
Notably significant inhibitory effects toward hCA XII are shown by the subseries of the 4-
aryl-7-hydroxy-2H-chromen-2-ones 17a-f and 17h-i (Ki values ranging from 6.5-8.9 nM). In
contrast, the 7-hydroxy-4-(4-methylphenyl)-2H-chromen-2-one (17g, R = 4-Me) displayed
moderate inhibitory effects toward this isoform. Introduction of a methyl group at C-8
position generally induces a decrease of hCA XII inhibitory effects for compounds
belonging to the subseries of 4-aryl-7-hydroxy-8-methyl-2H-chromen-2-ones 18 with
exception of derivatives 18e, 18h and 18i that were potent inhibitors sharing a similar
activity with analogs 17e, 17h and 17i. Surprisingly, for compounds 18a, 18c and 18d we
found up to 50-fold decrease of affinity. Among the two subseries of coumarins, the best
active hCA XII inhibitors were compounds 17f, 17h, 17i and 18i which displayed a very
similar potency when compared with well-known inhibitor AAZ and demonstrated high
selectivity over hCA I/II isoforms.
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To gain more information about the putative binding mode in to the hCA XII cavity for 4-
aryl-7-hydroxy-2H-chromen-2-one derivatives (17a-i and 18 a-i) we have performed
docking studies by means of AutoDock program and using the crystal structure of the hCA
XII (PDB code 1JCZ). Considering that CAs could exert an esterase activity which causes
the hydrolytic process of coumarin system, we chose to analyze the binding pose of the
tested coumarins as well as their corresponding 2-hydroxycinnamic acid derivatives as Z-
and E-isomers. Specifically, panels B-D in Figure 1 display the docking results for the most
active compound 18i and corresponding plausible hydrolytic products (Z)-18i-A and (E)-
18i-B, which were generated by a well-known pathway summarized in panel A of Figure 1.
NH2
NH2
CAs
-
COO
OH
HO
O
O
HO
Me
Me
18i
(Z)-18i-A
NH2
cis-trans
isomerization
-
COO
OH
(E)-18i-B
HO
B
Me
A
D
C
Figure 1. In panel A is shown the supposed CA-mediated hydrolysis of coumarin
derivative 18i leading to Z- or E-2-hydroxycinnamic acid derivatives (Z)-18i-A and (E)-18i-
B. Docked poses of compound 18i (B), isomer (Z)-18i-A (C) and isomer (E)-18i-B (D)
within hCA XII cavity retrieved from RCSB Protein Data Bank (PDB: 1JCZ). Key residues
of the hCAXII are presented. The hydrogen bonds are displayed in dotted lines. The Zinc
ion is shown as gray sphere.
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As expected for coumarin 18i as well as related compounds (Z)-18i-A and (E)-18i-B, there
was the lack of coordination of Zinc ion within catalytic site. From the comparison of the
docked poses for the three ligands it emerged that 18i, (Z)-18i-A and (E)-18i-B occupy the
hCA XII cavity and establish profitable contacts with hydrophilic/hydrophobic crucial
residues. A very similar binding mode was observed for coumarin derivative 18i (panel B)
and isomer (Z)-18i-A (panel C), for which the phenyl ring at C-4 position of coumarin
nucleus is located in proximity of the hydrophobic residue L198; whereas the 4’-amino-
functional group engages H-bond donor interaction with S135 in the top of the cavity.
Furthermore, for the 18i (panel B) and isomer (Z)-18i-A (panel C) there is a dense network
of hydrogen bonds with N62, H94, H119, and T199 residues.
As depicted in Figure 1 (panel D), the hypothetic (E)-18i-B isomer binds the hCA XII cavity
assuming a different orientation for the 4-aminophenyl substituent that is located in the
bottom of the cavity. The 2,4-dihydroxylphenyl ring assumes a peculiar disposition for
which two relevant hydrogen bond interactions with S135 and T200 mantain the aromatic
ring leaned against the hydrophobic wall (L198, P201 and P202) of the cavity. Finally, the
carboxylate moiety creates a strong bidentate interaction with H119 and T199 residues
and shows an orientation similar to that of precursor 18i.
3. Conclusions
Based on the proof-of-principle that coumarins derivatives could be selective inhibitors of
tumor-associated hCA IX and hCA XII isoforms, we have planned the synthesis of new
umbelliferone-based compounds that resulted potent inhibitors at low nanomolar
concentration. Moreover, all tested compounds showed relevant selectivity over ubiquitous
hCA I and hCA II isoforms. This experimental evidence confirms that coumarin derivatives
represent useful heterocyclic system for further chemical optimization to obtain CAIs
characterized by a peculiar mechanism of CA inhibition which does not involve the
coordination of Zinc ion within catalytic site.
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4. Experimental section
4.1 Chemistry
Starting materials and reagents were purchased from commercial sources (Sigma-Aldrich
Milan Italy and Alfa Aesar Karlsruhe Germany) and were used without further purification.
Melting points were determined on a BUCHI Melting Point B-545 (BUCHI Labortechnik AG
Flawil, Switzerland) apparatus and are uncorrected. Elemental analysis (C, H, N) were
carried out on a Carlo Erba Model 1106 Elemental Analyzer and the results were within ±
1
0.4% of the theoretical values. H-NMR spectra were recorded in CDCl3 and DMSO with
TMS as internal standard or DMSO-d6 on a Varian Gemini-300 spectrometer. Chemical
shifts were expressed in δ (ppm) and coupling constants (J) in hertz (Hz). Rf values were
determined on TLC plates using a mixture of Cy/EtOAc 8:2 as eluent.
4.1.1 General procedures for the synthesis of acetates 8-10
The compounds 1, 2 or 7 (respectively 1.2, 1.1 mmol and 1.1) were stirred with acetic
anhydride (3 mL) in ice bath and a catalytic amount of 96% sulfuric acid was added
dropwise. After that, 2.5 molar equivalents of Et3N were added to the mixture and agitated
until the disappereance of starting compounds (analyzed by TLC). After reaction was
completed it was cooled into ice and the solid was filtered off and dried to afford the
corresponding compounds 8-10. Chemical and spectral data for 8-9 are in good
agreement with literature [35].
4.1.1.1 (2-Oxochromen-6-yl)acetate (10) Mp: 143-145 °C (Et2O), yield 64%; reaction time:
1
1.5 hours; Rf: 0.16; H NMR (CDCl3): 2.32 (s, 3H, CH3), 6.45 (d, J=10.1, 1H, CH), 7.23-
7.25 (m, 2H, ArH), 7.36 (d, 1H, ArH), 7.65 (d, J=10.1, 1H, CH). Anal. Calcd for C11H8O4:
C:64.7; H:3.9. Found: C:64.3; H:3.6.
4.1.2 General procedures for the synthesis of benzoates 11-13
To a well-stirred solution of compounds 1, 2 or 7 (1mmol) in DCM (2 mL), Et3N (2.5 molar
equivalents) and benzoyl chloride (5 molar equivalents) were added. The mixture was
stirred until the end of the reaction (monitored by TLC) at room temperature and then
cooled into ice. The solid in suspension was filtered off and recrystallized from the suitable
solvent to give esters 11-13. Chemical and spectral data for 11-12 are in good agreement
with literature [35].
4.1.2.1 (2-Oxochromen-6-yl)benzoate (13)
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Mp: 137-139 °C (EtOH), yield 60%; reaction time: 20 h; Rf: 0.23; 1H NMR (DMSO-d6):
6.53 (s, J=2.1, 1H, CH), 6.56 (s, J=2.1, 1H, CH), 7.50-7.60 (m, 3H, ArH), 7.72-7.74 (m, 2H,
ArH), 8.02-8.06 (m, 1H, ArH), 8.11-8.14 (m, 2H, ArH). Anal. Calcd for C16H10O4: C:72.1;
H:3.7. Found: C:72.3; H:3.9.
4.1.3 General procedures for the synthesis of 4-arylcoumarins 17a-h and 18a-h
The title compounds were prepared as previously described [4] with slight modifications.
To a stirred solution of the appropriate ethylbenzoylacetate (14a-h) (1 mmol), resorcinol
(15) or 2-methylresorcinol (16) (1 mmol) and 96% sulfuric acid (1.5 ml) was added
dropwise at 0° C. The resulting mixture was stirred for different reaction times at room
temperature and monitored by TLC until completion. The reaction was quenched with ice
and extracted with EtOAc (3 x 10 mL). The organic phase was dried with Na2SO4 and
concentrated until dryness under reduced pressure. The residue was purified by
crystallization with suitable solvent to afford the desired compounds 17a-h and 18a-h.
Chemical and spectral data for 17a, 17e, 18a-b and 18d are in good agreement with
literature [4, 36].
4.1.3.1 4-(2-Chlorophenyl)-7-hydroxy-2H-chromen-2-one (17b)
Mp: 218-220°C (Et2O), yield 22%; reaction time: 2.5 hours; Rf: 0.16; H NMR (DMSO-d6):
1
6.16 (s, 1H, CH ), 6.72-6.77 (m, 3H, ArH), 7.45-7.50 (m, 3H, ArH), 7.52-7.62 (m, 1H, ArH),
10.69 (bs, 1H, OH). Anal. Calcd for C15H9ClO3: C:66.1; H:3.3. Found: 65.8; H: 3.0.
4.1.3.2 4-(3-Chlorophenyl)-7-hydroxy-2H-chromen-2-one (17c)
1
Mp: 237-239°C (EtOH), yield 31%; reaction time: 2 hours; Rf: 0.16; H NMR (DMSO-d6):
6.18 (s, 1H, CH), 6.74-6.78 (m, 2H, ArH), 7.18 (d, 1H, ArH), 7.40 (m, 1H, ArH), 7.55-7.58
(m, 3H, ArH). Anal. Calcd for C15H9ClO3: C:66.1; H:3.3. Found: C:65.9; H:3.4.
4.1.3.3 4-(4-Chlorophenyl)-7-hydroxy-2H-chromen-2-one (17d)
Mp: 280-282°C (EtOH), yield 33%; reaction time: 2.5 hours; Rf: 0.16; 1H NMR (DMSO-d6):
6.16 (s, 1H, CH ), 6.76- (d, J=8.8, 2H, ArH), 7.22 (d, 1H, ArH), 7,53 (d, J=8.8, 2H, ArH),
7.59 (m, 2H, ArH), 10.70 (bs, 1H, OH). Anal. Calcd for C15H9ClO3: C:66.1; H:3.3. Found:
C:65.8; H:3.1.
4.1.3.4 7-Hydroxy-4-(3-methylphenyl)-2H-chromen-2-one (17f)
1
Mp: 210-212 °C (Et2O), yield 32%;reaction time: 3 hours; Rf: 0.18; H NMR (DMSO-d6):
2.35 (s, 3H, CH3), 5.98 (s, 1H, CH), 6.65 (m, 2H, ArH), 7.19 (d, 1H, ArH), 7.24-7.28 (m,
12

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3H, ArH), 7.39 (m, 1H, ArH). Anal. Calcd for C16H12O3: C:76.2; H:4.8. Found: C:75.9;
H:4.5.
4.1.3.5 7-Hydroxy-4-(4-methylphenyl)-2H-chromen-2-one (17g)
Mp: 236-237 °C (Et2O/EtOH), yield 13%; reaction time: 4 hours; Rf: 0.16; 1H NMR (DMSO-
d6): 2.37 (s, 3H, CH3), 6.09 (s, 1H, CH), 6.74-6.76 (m, 2H, ArH), 7.35-7.37 (m, 5H, ArH).
Anal. Calcd for C16H12O3: C:76.2; H:4.8. Found: C:76.0; H:4.6.
4.1.3.6 7-Hydroxy-4-(4-nitrophenyl)-2H-chromen-2-one (17h)
Mp: 178-180 °C (EtOAc), yield 89%; reaction time: 1.5 hours; Rf: 0.33; 1H NMR (DMSO-
d6): 6.24 (s, 1H, CH), 6.77 (dd, J1=8.5, J2=2.1, 1H, CH), 6.84 (d,1H, CH), 7.15 (d, J=9.1
1H, CH), 7.79 (d, J=8.5, 2H, ArH), 8.35 (d, J=8.5, 2H, ArH). Anal. Calcd for C15H9NO5:
C:63.6; H:3.2 N:4.9. Found: C:63.4; H:3.3; N:4.7.
4.1.3.7 4-(3-Chlorophenyl)-7-hydroxy-8-methyl-2H-chromen-2-one (18c)
Mp: 315-317°C (EtOH), yield 79%; reaction time: 2.5 hours; Rf: 0.22; 1H NMR (DMSO-d6):
2.18 (s, 3H, CH3 ), 6.18 (s, 1H, CH), 6.83 (d, J= 8.5, 1H, ArH), 7.04 (d, J= 9.0, 1H, ArH),
7.44 (d, J= 8.5, 1H, ArH), 7.53-7.59 (m, 3H, ArH), 10.59 (bs, 1H, OH). Anal. Calcd for
C16H11ClO3: C:67.0; H:3.9. Found: C:66.7; H:3.6.
4.1.3.8 4-(2-Fluorophenyl)-7-hydroxy-8-methyl-2H-chromen-2-one (18e)
Mp: 256-258°C (EtOH), yield 60%; reaction time: 12 hours; Rf: 0.19; 1H NMR (DMSO-d6):
2.15 (s, 3H, CH3), 6.17 (s, 1H, CH), 6.78-6.87 (m, 2H, ArH), 7.36-7.43 (m, 3H, ArH), 7.57
(m, 1H, ArH), 10.58 (bs, 1H, OH). Anal. Calcd for C16H11FO3: C:65.0; H:4.0. Found:
C:64.7; H:3.75.
4.1.3.9 7-Hydroxy-8-methyl-4-(3-methylphenyl)-2H-chromen-2-one (18f)
Mp: 185-187° C (EtOAc), yield 99%; reaction time: 3 hours; Rf: 0.24; 1H NMR (DMSO-d6):
2.19 (s, 3H, CH3), 2.38 (s, 3H, CH3 ), 6.10 (s, 1H, CH ), 6.82 (d, J=8.8, 1H, ArH), 7.12 (d,
J=8.8, 1H, ArH), 7.27-7.43 (m, 4H, ArH), 10.56 (bs, 1H, OH). Anal. Calcd for C17H14O3: C:
76.7; H: 5.3. Found: C:76.6; H:5.1.
4.1.3.10 7-Hydroxy-8-methyl-4-(4-methylphenyl)-2H-chromen-2-one (18g)
Mp: 259-261°C (EtOH), yield 63%; reaction time: 2.5 hours; Rf: 0.22; 1H NMR (DMSO-d6):
2.19 (s, 3H, CH3), 2.38 (s, 3H, CH3), 6.09 (s, 1H, CH), 6.82 (d, J= 8.5, 1H, ArH), 7.13 (d,
J= 8.5, 1H, ArH), 7.35-7.37 (m, 4H, ArH), 10.53 (bs, 1H, OH). Anal. Calcd for C17H14O3: C:
76.7; H: 5.3. Found: C:76.8; H:4.9.
4.1.3.11 7-hydroxy-8-methyl-4-(4-nitrophenyl)chromen-2-one (18h)
1
Mp: 360 °C dec. (EtOAc), yield 98%; reaction time: 1.5 hours; Rf: 0.15; H NMR (DMSO-
d6): 2.17 (s, 3H, CH3), 6.22 (s, 1H, CH), 6.80 (d, J= 8.8, 1H, ArH), 6.90 (d, J= 8.8, 1H,
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ArH,) 7.75 (d, J= 7.0, 2H, ArH), 8.34 (d, J= 7, 2H, ArH). Anal. Calcd for C16H11NO5: C:64.6;
H:3.7; N:4.7. Found: C:64.2; H:3.3; N:4.4
4.1.4 Synthesis of 4-(4-aminophenyl)-7-hydroxychromen-2-one (17i) and 4-(4-
aminophenyl)-7-hydroxy-8-methyl-chromen-2-one (18i)
To a suspension of the arylnitro derivatives 17h or 18h (1 mmol) and a catalytic amount of
Pd/C in EtOH (15 mL), hydrazine hydrate (10 mmol) was slowly added. The reaction was
stirred and refluxed (70 °C) under nitrogen atmosphere. Then, the mixture was filtered
through Celite which was later washed with EtOH. The solution was evaporated in vacuo
to give the crude product. The residue was purified by crystallization from the suitable
solvent.
4.1.4.1 4-(4-Aminophenyl)-7-hydroxychromen-2-one (17i)
1
Mp: 307-309 °C (EtOH), yield 83%; reaction time: 1 hour; Rf: 0.03; H NMR (DMSO-d6):
5.60 (bs, 2H, NH2), 5.96 (s, 1H, CH), 6.65 (d, J=5.9, 2H, ArH), 6.73 (m, 2H, ArH), 7.17 (d,
J=5.9, 2H, ArH), 7.43 (m, 1H, ArH), 10.67 (bs, 1H, OH). Anal. Calcd for C15H11NO3:
C:71.1; H:4.4; N:5.5. Found C:70.8; H:4.1; N:5.2.
4.1.4.2 4-(4-Aminophenyl)-7-hydroxy-8-methyl-chromen-2-one (18i)
1
Mp: 250-252°C (EtOAc), yield 90%; reaction time: 1.5 hours; Rf: 0.03; H NMR (DMSO-
d6): 2.15 (s, 3H, CH3), 5.99 (bs, 2H, NH2), 5.96 (s, 1H, CH), 6.64 (d, J= 8.5, 2H, ArH), 6.81
(d, J= 9.0, 1H, ArH), 7.17 (d, J= 8.5, 2H, ArH), 7.29 (d, J= 9, 1H, ArH). Anal. Calcd for
C16H13NO3: C:71.9; H:4.9; N:5.2. Found: C:71.5; H:4.4; N:4.9.
4.2 CA Inhibition Assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA
catalysed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) has been used
as indicator, working at the absorbance maximum of 557 nm, with 10 – 20 mM Hepes (pH
7.5) or Tris (pH 8.3) as buffers, and 20 mM Na2SO4 or 20 mM NaClO4 (for maintaining
constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration
reaction for a period of 10-100 s. The CO2 concentrations ranged from 1.7 to 17 mM for
the determination of the kinetic parameters and inhibition constants. For each inhibitor at
least six traces of the initial 5-10% of the reaction have been used for determining the
initial velocity. The uncatalyzed rates were determined in the same manner and subtracted
from the total observed rates. Stock solutions of inhibitor (10 mM) were prepared in
distilled-deionized water and dilutions up to 0.01 nM were done thereafter with distilled-
deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min at
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room temperature prior to assay and to allow for the formation of the E-I complex. The
inhibition constants were obtained by non-linear least-squares methods using PRISM 3, as
reported earlier, and represent the mean from at least three different determinations. CA
isoforms were recombinant ones obtained as reported earlier by this group [11,12].
4.3 Molecular docking
Automated docking was carried out by means of the program AUTODOCK 4.2. [37] The
crystal structure of was retrieved from the RCSB Protein Data Bank (PDB: 1JCZ) [38]. The
ligand and water molecules were discarded, and hydrogen atoms were added to protein
with Discovery Studio 2.5.5. Structures of the ligands were constructed using Discovery
Studio 2.5.5 and energy minimized using the Powel protocol (1000 steps). The regions of
interest used by AUTODOCK were defined by considering the suitable ligand docked into
the CA XII receptor as the central group; in particular, a grid of 60, 60, and 60 points in the
x, y, and z directions was constructed centered on the center of the mass of Zn metal ion.
A grid spacing of 0.375 Å and a distance-dependent function of the dielectric constant
were used for the energetic map calculations. Using the Lamarckian Genetic Algorithm, all
docked compounds were subjected to 100 runs of the AUTODOCK search, in which the
default values of the other parameters were used. Cluster analysis was performed on the
docked results using an RMS tolerance of 2.0 Å. The Lamarckian genetic algorithm (LGA)
was applied to model the interaction between ligands and hCA XII active site. For the
Lamarckian genetic algorithm: 27,000 maximum generations; a gene mutation rate of 0.02
and; a crossover rate of 0.8 were used. Cluster analysis was performed on the docked
results using an RMSD (Root Mean Square Deviation) tolerance of 2 Å. All the compounds
were docked according to the afore mentioned parameters. The hCAXII/ligand complex
obtained by docking studies was minimized using 1000 iterations of SD and 1000
interaction of Polak-Ribiere Conjugate Gradient. Interactions were identified using the
LigPlot software [39] and the figures were prepared using PyMOL [40].
Acknowledgments
Financial support for this research by by Fondo di Ateneo per la Ricerca (PRA grant
number ORME09SPNC - Università degli Studi di Messina).
Author Contributions: The manuscript was written through contributions of all authors.
Notes The authors declare no competing financial interest.
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RESEARCH HIGHLIGHTS
• New coumarin-based derivatives as CA inhibitors were designed and synthesized.
• Several cumarins were effective inhibitors of tumor-associated hCA IX and hCA XII
• Developed cumarins were selective inhibitors over ubiquitous hCA I and hCA II.
• Docking studies suggested the binding mode of active compound (18i) and hCA XII