Novel 8-substituted coumarins that selectively inhibit human carbonic anhydrase IX and XII

Article abstract of DOI:10.3390/ijms20051208

A novel series of 8-substituted coumarin-based compounds, characterized by the presence of alkylpiperazine and arylpiperazine chains, were synthesized and tested for their inhibitory activity against four human carbonic anhydrase (hCA) isoforms. All compounds displayed nanomolar potency against the cancer-related hCA IX and hCA XII; moreover, they were shown to be devoid of any inhibitory activity toward the cytosolic hCA I and hCA II up to 10 μM concentration in the assay system. Therefore, the synthesized coumarin ligands demonstrated to be potent and selective hCA IX/XII inhibitors, and were shown to be as potent as the reference inhibitor acetazolamide against hCA XII, with single-digit nanomolar K_i values. Molecular modeling studies provided a rationale for explaining the selectivity profile of these non-classic hCA inhibitors and their interactions with the enzymes, according to their specific mechanism of action, thus paving the way for future structure-based lead optimization studies.

Full text of DOI:10.3390/ijms20051208

International Journal of
Molecular Sciences
Article
Novel 8-Substituted Coumarins That Selectively
Inhibit Human Carbonic Anhydrase IX and XII
Kerem Buran 1,†, Silvia Bua 2,† , Giulio Poli 3, F. Esra Önen Bayram 1,*, Tiziano Tuccinardi 3,
and Claudiu T. Supuran 2
*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Yeditepe University, Kayisdagi Cad.,
34755 Istanbul, Turkey; keremburan@gmail.com
NEUROFARBA Department, Pharmaceutical and Nutraceutical Section, University of Florence,
2
50019 Sesto Fiorentino, Italy; silvia.bua@unifi.it (S.B.); claudiu.supuran@unifi.it (C.T.S.)
Department of Pharmacy, University of Pisa, 56126 Pisa, Italy; giulio.poli@unipi.it
3
*
†
Correspondence: filizesraonen@gmail.com (F.E.Ö.B.); tiziano.tuccinardi@unipi.it (T.T.)
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 15 February 2019; Accepted: 6 March 2019; Published: 10 March 2019
Abstract: A novel series of 8-substituted coumarin-based compounds, characterized by the presence
of alkylpiperazine and arylpiperazine chains, were synthesized and tested for their inhibitory activity
against four human carbonic anhydrase (hCA) isoforms. All compounds displayed nanomolar
potency against the cancer-related hCA IX and hCA XII; moreover, they were shown to be devoid
of any inhibitory activity toward the cytosolic hCA I and hCA II up to 10 µM concentration in the
assay system. Therefore, the synthesized coumarin ligands demonstrated to be potent and selective
hCA IX/XII inhibitors, and were shown to be as potent as the reference inhibitor acetazolamide
against hCA XII, with single-digit nanomolar Ki values. Molecular modeling studies provided a
rationale for explaining the selectivity profile of these non-classic hCA inhibitors and their interactions
with the enzymes, according to their specific mechanism of action, thus paving the way for future
structure-based lead optimization studies.
Keywords: carbonic anhydrase inhibitor; metalloenzymes; coumarins; docking; molecular modeling
1. Introduction
Carbonic anhydrase (CA, EC 4.2.1.1) is a family of metalloenzymes that catalyze the reversible
interconversion of carbon dioxide and water to bicarbonate and a proton [1]. In humans, sixteen
CA isoforms are present and they all differ for kinetic properties, sub-cellular localization and tissue
distribution. The different hCAs are involved in many physiological processes such as pH regulation,
CO2 homeostasis, respiration, bone resorption and tumorigenesis [
pharmacological/clinical use for many decades as diuretics and antiglaucoma agents [
recently, they have also been evaluated for the imaging, for the treatment of hypoxic tumors [
and as anti-obesity agents [ ]. CAIs are generally compounds endowed with a suitable zinc-binding
2
]. hCA inhibitors (CAIs) are in
3,4]. More
5–7]
8
group (ZBG) able to chelate the prosthetic zinc ion placed within the binding site of hCAs, which
is critical for the catalytic activity of these enzymes. The most common ZBGs shared by classic
CAIs are represented by sulfonamide moieties or similar structural motifs (such as sulfamides and
sulfamates). These groups are particularly effective for endowing small-molecule ligands with high
affinity for hCAs, since they not only allow a proper coordination of the catalytic zinc ion but also
the formation of H-bond interactions with key protein residues placed in the surroundings of the
zinc-binding cavity. In fact, these compounds represent the most important classes of CAIs and
numerous ligands showing high inhibitory potency have been reported to date [9]. However, due to
Int. J. Mol. Sci. 2019, 20, 1208; doi:10.3390/ijms20051208
www.mdpi.com/journal/ijms

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the high amino acid conservation observed in the different hCA isoforms at the level of their catalytic
site and adjacent regions, these ligands often present the drawback of being insufficiently selective
toward specific therapeutically interesting hCAs. In particular, hCA IX and hCA XII have drawn special
attention in the medicinal chemistry field, since these isoforms have been designated as biomarkers
and pharmacological targets for various cancer types. As a consequence, a wide area of research is
currently focused on the inhibition of these two enzymes [10]. Nevertheless, the development hCA
IX/XII inhibitors with selectivity over hCA I/II, which are ubiquitously distributed and involved in
key physiological processes, is still a challenging task, although some example of selective ligands
have been reported [11,12]. In this context, non-classical CAIs constitute a particularly valuable
resource to seek for isoform specificity and prevent potential off-targets activities, side effects and/or
adverse reactions (such as sulphur allergies) connected to the use of sulfurated ligands [13]. Among
non-classical CAIs, coumarin derivatives represent a particularly atypical category of hCA ligands
due to their peculiar mechanism of inhibition, which does not involve the chelation of the prosthetic
zinc ion. In fact, upon binding to the catalytic site of hCAs, these ligands are hydrolyzed by the
esterase activity of the enzymes to the corresponding 2-hydroxy-cinnamic acid derivatives, which thus
represent the actual inhibitors. As demonstrated by crystallographic studies, these derivatives bind at
the entrance of hCAs catalytic pocket hampering the access to the site and blocking the catalytic activity
of the enzymes [14,15]. Coumarin-based inhibitors, particularly 7-hydroxycoumarin derivatives and
substituted analogues, were shown to be endowed with a good selectivity profile, demonstrating
nanomolar inhibition of hCA IX and XII, and a much lower potency against hCA I and II, with some
representative compounds demonstrating also cytotoxic activity in cancer cells [16–20]. With the
purpose of expanding the chemical space of available coumarin-based ligands and the corresponding
structure activity relationships, in the search for novel potent and selective CAIs, a novel series of
7-hydroxycoumarin derivatives has been synthesized and evaluated for their hCA inhibitory activity.
All compounds were found to selectively inhibit hCA IX and especially hCA XII, showing single-digit
nanomolar activity for this latter isoform, thus confirming the importance of the coumarin scaffold for
the development of selective CAIs.
2. Results and Discussion
2.1. Synthesis of 7-Hydroxycoumarin Derivatives
Our synthetic efforts aimed at the identification of new coumarin-based CAIs focused on the
substitution of position 8 of the central chromen-2-one scaffold. In particular, 4-methylumbelliferone
(7-hydroxy-4-methyl-chromen-2-one) (
acetoacetate according to the Pechmann reaction (Scheme 1). Subsequently, twelve derivatives of
compound functionalized at the position 8 of the bicyclic core were obtained through reaction with a
1) was initially synthesized starting from resorcinol and ethyl
1
series of five aliphatic and seven aromatic piperazine fragments (Scheme 2).
Scheme 1. Synthesis of 4-methylumbelliferone (compound 1).

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Scheme 2. Synthesis of piperazine derivatives 2–13.
Compound
1
was characterized with 1H-NMR, 13C-NMR and LC-MS. The 1H-NMR spectrum
3. Its neighboring
of this compound presented a doublet at 7.57 ppm (J = 8.7 Hz) for the aromatic
H
proton H4 gave a doublet of doublet (J1 = 8.7 Hz, J2 = 2.4 Hz) due to the o-coupling with H3 and
m-coupling with H6 that also gave a doublet at 6.68 ppm (J = 2.4 Hz). The doublet at 6.10 ppm
(J = 1.2 Hz) was obtained for the H1 proton, which presents a typical a cis-allylic coupling with the
methyl moiety bond to the cyclic double bond. Finally, the presence of the methyl was confirmed by
the quartet obtained at 2.34 ppm (J = 1.2 Hz) and the hydroxyl gave a signal for one proton at 10.5 ppm.
All these findings confirmed clearly the successful cyclization of resorcinol to generate the coumarin
ring. The synthesized compound 1 was derivatized with a series of five aliphatic and seven aromatic
piperazines. Targeted molecules were obtained by binding the heterocyclic moieties at the position 8
of compound
1 using formaldehyde. Yields obtained for the synthesized molecules (2–13) are given in
Table S1. The substitution of the coumarin heterocycle was easy to monitor via 1H-NMR as a successful
substitution gave a new singlet at around 4.0 ppm that integrated for two protons, corresponding to
the methylene spacer between the coumarin and piperazine heterocycles. However, interpreting the
signals of the piperazine cycle was not obvious as they can give multiplets or more commonly broad
singlets due to eight non-equivalent protons that, in addition to short-range correlation, can present
long-range correlations as mentioned in the literature [21]. The spectrum of each compound is detailed
in the experimental part and can also be found in the supplementary materials.

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2.2. Inhibitory Activity against hCA Isoforms
The thirteen total compounds were subjected to a stopped-flow CO2 hydrase assay, together with
the reference inhibitor acetazolamide (AAZ), in order to evaluate their inhibitory activity against hCA
I, II, IX and XII. As reported in Table 1, all compounds were found to inhibit the cancer-related hCA IX
and hCA XII with Ki values in the nanomolar range and showed to be devoid of any hCA I and hCA II
inhibitory activity up to 10 µM concentration in the assay system. The tested compounds showed a
higher affinity for hCA XII with respect to hCA IX (from 5-fold up to 40-fold). In fact, the activity of the
coumarin ligands against hCA XII was comparable to that observed for the AAZ, with single-digit
nanomolar Ki values determined for all compounds except for the o-fluorophenylpiperazine derivative
12 (Ki for hCA XII of 26.4 nM). It is interesting to note that the derivatization of 4-methylumbelliferone
(1
) with both aliphatic and aromatic piperazine fragments apparently seemed to have no influence
on hCA XII inhibitory activity; this suggests that the corresponding side chains of compounds 13
2
–
might not establish key interactions with hCA XII catalytic site able to affect their affinity for the
protein. As far as hCA IX inhibition is concerned, the activity of the ligands spanned a wider range,
with the alkylpiperazine derivatives showing better potencies, on average, than the arylpiperazine
compounds. In fact, the Ki values determined for the different derivatives ranged from around 300 nM
(for compounds
to be as potent as AAZ and represented the most active compound of the series. However, derivative
still showed an activity profile comparable to that of the parent compound , hence confirming
5 and 11) to the lowest value of 27.1 nM observed for compound 6, which was found
6
1
the relatively slight impact of the piperazine substituents on the hCA IX/XII inhibitory activity of
these ligands.
Table 1. Inhibition data of hCA I, hCA II, hCA IX, hCA XII with compounds here reported and the
standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped flow CO2 hydrase assay.
Ki (nM)
Compound
hCA I
hCA II
hCA IX
hCA XII
1
2
3
4
5
6
7
8
9
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
250
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
12
32.1
67.5
88.7
32.0
294.9
27.1
114.7
190.3
92.8
162.3
307.7
120.7
76.8
5.8
8.5
9.6
6.4
7.1
5.6
7.7
8.9
8.1
9.4
9.6
26.4
8.5
5.7
10
11
12
13
AAZ
25
* Mean from three different assays, by a stopped flow technique (errors were in the range of
reported values).
±
5–10% of the
2.3. Molecular Modeling Studies
With the aim of figuring out the ligand–protein interaction features at the basis of the selectivity
for hCA IX and XII over hCA I and II demonstrated by the synthesized coumarin derivatives, as well
as to get clues about the impact of their piperazine side chains on their activity profile, molecular
modeling studies including docking and molecular dynamics (MD) simulations were carried out.
Compound 13, which showed good potency on both hCA IX and XII, was taken into account for this
study as a reference ligand of the series. Coumarin derivatives are known to undergo hydrolysis
upon binding to the catalytic site of hCAs, which leads to the formation of 2-hydroxy-cinnamic acids.
These compounds keep binding to the enzymes and thus represent the real CAIs, as confirmed by

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X-ray crystallography [14
,
15]. For this reason, the 2,4-dihydroxy-cinnamic acid that can be obtained
from hydrolysis of compound 13 was docked into the X-ray structures of hCA I, II, IX and XII (PDB
codes 1AZM, 2AW1, 3IAI and 1JD0, respectively) by employing Autodock4 software (Autodock 4.2,
The Scripps Research Institute, La Jolla, CA, USA). Subsequently, the four ligand–protein complexes
generated by docking were further analyzed with MD simulation studies (see the Materials and
Methods section for details). In the binding modes generated through the whole computational
protocol, the ligand was predicted to interact with the four hCA isoforms showing a similar disposition
of the 2,4-dihydroxy-cinnamic moiety across the protein catalytic site. The carboxylic group of the
ligand was oriented toward the hydrophilic half of the enzyme, forming hydrogen bonds with the
polar residues located in this part of the binding site (Figure 1), in agreement with the experimentally
determined binding mode of 2-hydroxy-cinnamic acid bound to hCA II [15]. In particular, the ligand
was predicted to interact with all four hCA isoforms establishing two different H-bonds, one of which
always involved a histidine residue located in the hydrophilic side of the binding cavity, next to the
catalytic residues, i.e., H94, H200, H64 and H65 for hCA XII, IX, II and I, respectively (see Figure S1 in
the Supplementary Materials for the sequence alignment of the four hCAs). In hCA XII, the second
H-bond formed by the ligand was established with N92, while in hCA IX the compound was found to
interact with the zinc-bound water molecule. Finally, in hCA II and hCA I, the ligand was predicted
to form H-bonds with T199 and its homologue residue H201, respectively. On the contrary, the long
piperazine-containing chain connected to the cinnamic moiety showed to be highly solvent exposed
and to assume rather different orientations into the binding sites of the four different hCAs, without
forming significant interactions with the four proteins. In fact, the ligand terminal chain was predicted
to interact with the hydrophobic portion of hCA I and the polar side of hCA II catalytic pockets, while
when bound to hCA IX and hCA XII, the ligand oriented its chain in the middle between the two
sides. In all complexes, the piperazine-containing substituent did not show interactions that could
substantially contribute to the ligand’s affinity for the hCA isoforms. This is consistent with the results
of our experimental assays, highlighting the modest impact of the different piperazine fragments
on the activity profile of this series of compounds. Although the ligand did not protrude into the
inner part of the hCAs binding pocket where the zinc ion and the catalytic residues are located, MD
simulation results showed that the core scaffold of compound 13 was firmly anchored to the protein
thanks to the stable H-bonds formed by the carboxyl group (maintained for almost the whole MD
simulations) and could thus easily block the entrance of the catalytic pocket of the four hCAs.
Hence, the analysis of the binding modes calculated for the hydrolysis product of compound
13 into the four hCA isoforms suggested that the ligand could strongly bind to all the four hCAs.
Therefore, it was not possible to identify a rationale explaining the selective activity of the ligand and
its analogues against hCA IX and XII. These results led us to hypothesize that the ligand recognition
process, i.e., the binding interactions established by the coumarin derivatives prior to their hydrolysis,
could be implied in the selectivity of this class of compounds. It was already hypothesized by Supuran
and co-workers that these derivatives, before their hydrolysis, may bind within the CA active site
similarly to phenols i.e., by anchoring to the zinc-bound water molecule/hydroxide ion [15]. This
specific binding mode has indeed been confirmed for a 2-thioxocoumarine recently co-crystallized with
CA II [19]. For this reason, the whole computational protocol based on docking and MD simulation
studies was performed considering compound 13 in its native form. Figure 2 shows the binding mode
predicted for the ligand into the catalytic site of hCA XII. The carbonyl oxygen of the coumarin core
forms a hydrogen bond with the water molecule coordinating the prosthetic zinc ion, in agreement
with the mechanism of hydrolysis proposed for this series of derivatives [15]. The bicyclic scaffold
of the ligand is placed above the side chains of H119, Q117 and K97, forming interactions with these
residues. In particular, an NH–π interaction is observed with the amide group of Q117, while the
phenolic group of the ligand forms an H-bond with the charged nitrogen of K97. Finally, the 4-methyl
group of the coumarin core is placed in a hydrophobic pocket delimited by V147, L167 and L225, thus
taking lipophilic contacts with these residues, while the N-substituted piperazine chain primarily

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forms van der Waals interactions with residues constituting the hydrophilic site of the protein binding
site, such as W32, N92 and H94. A very similar binding pose was predicted for the ligand into hCA IX:
the compound essentially formed the same pattern of interactions observed with hCA XII, including
the key interaction with the zinc-coordinating structural water, but with the exception of the H-bond
with K97 (Figure 2B). In fact, K97 is a non-conserved residue of hCA XII, substituted by Q203 in hCA
IX; nevertheless, the ligand is still able to form an H-bond with the side chain of this latter residue
through the phenolic OH group and to form stacking interactions with Q224.
Figure 1. Minimized average structures of compound 13 in hydrolyzed form, in complex with hCA
XII (A); hCA IX (B); hCA II (C); and hCA I (D). Only residues coordinating the zinc ion or forming
H-bond with the ligands are shown in cyan. The protein surface is shown in gray. The zinc ion is
shown as a gray sphere.
Based on the binding mode predicted for compound 13 into hCA XII and IX, the non-conserved
residues K97 and Q203 seemed to play a substantial role in ligand–protein recognition, which could
also help explain the selectivity for hCA XII and IX of this class of compounds. In fact, the analysis of
the binding mode predicted for the ligand into hCA II revealed that, although the coumarin core of the
inhibitor assumes a disposition similar to that showed into the other two CA isoforms, the phenolic
OH group is not able to establish any H-bond interaction, due to the presence of the asparagine
residue N67 in place of the homolog residues K97 and Q203 present in hCA XII and IX, respectively.
Precisely because of the shorter side chain of N67 compared to Q203, the residue can only form an
NH–π interaction with the aromatic ring of the ligand, which thus loses a key anchoring point to

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the protein. This also determines a slight shift of the compound core away from the hydrophobic
side of the protein binding cavity, thus reducing the lipophilic interactions with V121, L140 and L197.
On the contrary, the presence of residue H68 in hCA I, in place of the homolog residues K97 and Q203
present in hCA XII and IX, respectively, would not only prevent the formation of H-bonds with the
ligand but also create a steric hindrance that determines its shift toward the hydrophobic side of the
catalytic site. In fact, compound 13 was predicted to bind hCA I with the coumarin core closer to the
lipophilic residues of the binding site such as V144, L199 and V208. However, this determines the
lack of both H-bonds and NH–π interactions with the residues belonging to the hydrophilic side of
the protein. The missing H-bonds with the polar residues of hCA II and hCA I binding sites could
be at the basis of the very weak activity demonstrated by compound 13 and its analogues against
the two hCA isoforms. Moreover, the higher potency against hCA XII compared to hCA IX observed
for this class of compounds could be explained considering the stronger H-bond interaction that the
coumarin derivatives can form with the charged residue K97 compared to Q203. Interestingly, these
considerations are in agreement with the observation that the structural element that seems to confer
hCA IX–XII selectivity to this class of compound is the 7-hydroxyl group of the coumarin core, which
should be implied in the H-bond with K97 of hCA XII and Q203 of hCA IX. In fact, compound
1
showed the same selectivity profile of the whole class of derivatives, despite the absence of the long
and charged substituent in position 8 of the bicyclic ring.
Figure 2. Minimized average structures of compound 13 (green) in complex with hCA XII (
IX ( ); hCA II ( ); and hCA I ( ). Ligand–protein H-bonds are shown as black dashed lines, and the
protein surface in the proximity of the ligands is shown in grey. The zinc ion is shown as a gray sphere.
A); hCA
B
C
D

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3. Materials and Methods
3.1. Chemistry
General procedure for 7-Hydroxy-4-methyl-chromen-2-one (
1
). Resorcinol (1.22 eq) was dissolved
◦
in concentrated H2SO4 (20 mL) at 0 C, then ethyl acetoacetate (1.0 eq) was slowly added and the
mixture was stirred at 0–5 ◦C for 2 h. At the end of the two hours, the reaction mixture was poured
onto ice-water. Then, the solid was washed with water and recrystallized from EtOH. Finally, pure
white solid 7-Hydroxy-4-methyl-chromen-2-one was obtained [22].
General procudure for synthesis of 7-hydroxy-4-methyl-8 (piperazines)chromen-2-ones (
2–13).
Compound (7-Hydroxy-4-methyl-chromen-2-one; 1.0 eq) was dissolved in 95% EtOH (5 mL), and
1
then piperazine derivative(s) (R) (1.0 eq) and formaldehyde (0.2 mL) were added to the reaction
medium. The reaction mixture was refluxed for 4–6 h. At the end of the 4–6 h, the reaction medium
was cooled and then the solvent was evaporated in vacuo. Pale-yellow oils were obtained and further
treated with a little amount of cool acetone. Then, the white solids were crystallized from acetone [23].
7-Hydroxy-4-methylchromen-2-one (
(aliphatic C–H); 1679 (C=O, lactone); 1599 (C=C, aromatic). H-NMR: (400 MHz, DMSO)
1
): FT-IR: (KBr), cm−1: 3150–3050 (OH); 3011 (aromatic C–H); 2958
1
δ 10.50 (s, 1H,
OH); 7.55 (d, J = 8.7, 1H, H5); 6.78 (dd, J1 =8.7 Hz, J2 = 2.4 Hz, 1H, H6); 6.67 (d, J = 2.4 Hz, 1H, H8); 6.08
(q, J = 1.2 Hz, 1H, H3); 3.33 (d, J = 1.2 Hz 3H, H9). 13C-NMR: (400 MHz, DMSO)
δ
161.5 (C2); 160.7,
155.2, 153.9, 127.0, 113.2, 112.4 (Ccoumarin-aromatic); 110.6, 102.6 (C3-4); 18.5 (C9). LC-MS: rt = 5.71 min,
m/z, 178.13 [M + H]+, Purity: 91%, UV (ACN/Water, λmax): 320 nm.
1,4-bis[(7-Hydroxy-4-methylcoumarin-8-yl)methyl] piperazine (2
): FT-IR: (KBr), cm−1: 3200–3050 (OH);
3048 (aromatic C–H); 2958 (aliphatic C–H); 1722 (C=O, lactone); 1627 (C=C, alkene); 1602 (C=C,
1
aromatic). H-NMR (400 MHz, CDCl3)
δ
7.41 (t, J = 9.70 Hz, 2H, H5); 6.77 (dd, J1 = 8.70 Hz, J2 = 15.3 Hz,
2H, H6); 6.08 (d, J = 6.50 Hz, 2H, H3); 4.08 (d, J = 15.80 Hz, 4H, H10); 2.49 (s, 16H, H11,12); 2.38 (s, 6H,
H9). 13C-NMR (400 MHz, CDCl3)
δ 162.5 (C2); 161.2, 153.3, 152.4, 126.8, 113.3, 112.1 (Ccoumarin-aromatic);
110.6, 107.6 (C3,4); 53.8, 52.6 (C11,12); 51.4 (C10); 18.8 (C10). LC-MS: rt = 5.10 min, m/z, 463.42 [M + H]+,
Purity: 93%, UV (ACN/Water, λmax): 320 nm.
7-Hydroxy-4-methyl-8-(4-methylpiperazin-1-ylmethyl)chromen-2-one (
(OH); 3071 (aromatic C–H), 1726 (C=O, lactone), 1609 (C=C, nonaromatic), 1580 (Ar C=C). 1H-NMR
(400 MHz, CDCl3) 7.40 (d, J = 8.7 Hz, 1H, H5); 6.75 (d, J = 8.7 Hz, 1H, H6); 6.07 (q, J = 1.1 Hz, 1H,
H3); 4.06 (s, 2H, H10); 2.90–2.40 (m, 8H, H11,12); 2.37 (d, J = 1.1 Hz, 3H, H9); 2.31 (s, 3H, H13). 13C-NMR
3
): FT-IR: (KBr), cm−1: 3300–3100
δ
(101 MHz, CDCl3)
δ 162.4 (C2); 161.2, 153.2, 152.5, 124.6, 113.3, 112.2 (Ccoumarin-aromatic); 110.6, 107.5
(C3,4); 54.7, 53.7 (C11,12); 52.5 (C10); 45.8 (C13); 18.7 (C9). LC-MS: rt = 0.9 min, m/z 289.33 [M + H]+.
Purity: 92%, UV (ACN/Water, λmax): 322 nm.
8-(4-Ethylpiperazin-1-ylmethyl)-7-hydroxy-4-methylchromen-2-one (
3075 (aromatic C–H), 1720 (C=O, lactone), 1600 (C=C, nonaromatic), 1567 (C=C, aromatic). H-NMR
4
): FT-IR: (KBr), cm−1: 3250–3050 (OH);
1
(400 MHz, CDCl3)
H3); 4.07 (s, 2H, H10); 3.77–2.50 (m, 8H, H11,12); 2.44 (q, J = 7.2 Hz, 2H, C13); 2.37 (d, J = 1.1 Hz, 3H, H9);
2.16 (s, 1H, OH); 1.08 (t, J = 7.2 Hz, 3H, H14). 13C-NMR (101 MHz, CDCl3)
162.5 (C2), 161.2, 153.7,
δ 7.41 (d, J = 8.7 Hz, 1H, H5); 6.76 (d, J = 8.7 Hz, 1H, H6); 6.07 (q, J = 1.1 Hz, 1H,
δ
153.3, 152.5, 124.6, 113.4 (Ccoumarin-aromatic); 110.6, 107.5 (C3,4); 58.9, 52.6 (C11,12); 52.0 (C10); 52.0 (C13);
18.9 (C9); 11.9 (C14). LC-MS: rt = 0.9 min, m/z 303.38 [M + H]+, Purity: 95%, UV (ACN/Water,
λmax):
322 nm.
7-Hydroxy-8-[4-(2-hydroxyethyl)piperazin-1-ylmethyl]-4-methylchromen-2-one (5 :
): FT-IR: (KBr), cm−1
3100–3400 (OH); 3222 (aromatic C–H), 1719 (C=O, lactone), 1597 (C=C, nonaromatic), 1567 (C=C,
1
aromatic). H-NMR: (400 MHz, CDCl3)
δ 7.41 (d, J = 8.7 Hz, 1H, H5); 6.76 (d, J = 8.7 Hz, 1H, H6); 6.07
(q, J = 1.1 Hz, 1H, H3); 4.07 (s, 2H, H10); 3.63 (t, J = 5.3 Hz, 2H, H14); 3.58–2.55 (m, 8H, H11,12); 2.58
(t, J = 5.3 Hz, 2H, H13); 2.38 (d, J = 1.1 Hz, 3H, H9), 2.17 (s, 1H, OH). 13C-NMR: (101 MHz, CDCl3)
162.2 (C2); 161.1, 153.2, 152.5, 124.6, 113.3, 112.3 (Ccoumarin-aromatic); 110.7, 107.5 (C3,4); 59.0, 57.8 (C11,12);
δ

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53.7 (C14); 52.6 (C10); 52.5 (C13); 18.8 (C9). LC-MS: rt = 0.91 min, m/z 319.37 [M + H]+, Purity: 94%, UV
(ACN/Water, λ max): 322 nm Purity: 97%.
8-(4-Allylpiperazin-1-ylmethyl)-7-hydroxy-4-methylchromen-2-one (6
): FT-IR: (KBr), cm−1: 3100–3300 (OH);
3067 (aromatic C–H), 3008 (aliphatic C-H); 1728 (C=O, lactone), 1599 (C=C, nonaromatic), 1495 (C=C,
1
aromatic). H-NMR: (400 MHz, CDCl3)
δ 7.40 (d, J = 8.7 Hz, 1H, H5); 6.75 (d, J = 8.7 Hz, 1H, H6);
6.07 (d, J = 1.2 Hz, 1H, H3); 5.84 (tt, J1 = 5.8 Hz, J2 = 17.0 Hz, H14); 5.18 (ddt, J1 = 1.5 Hz, J2 = 1.5 Hz,
J3 = 17 Hz, 2H, H15); 4.06 (s, 2H, H10); 3.02 (dt, J1 = 1.5 Hz, J2 = 5.8 Hz, 2H, H13); 2.98–2.40 (m, 8H,
H11,12); 2.37 (d, J = 1.2 Hz, 3H, H9). 13C-NMR: (101 MHz, CDCl3)
δ 162.4 (C2); 161.2, 153.2, 152.4, 124.6,
113.3, 112.2 (Ccoumarin-aromatic); 134.4, 118.4 (C14,15); 110.6, 107.5 (C3,4); 61.4 (C13); 53.8, 52.5 (C11,12); 52.5
(C10); 18.8 (C9). LC-MS: rt = 0.91 min, m/z 319.37 [M + H]+, Purity: 97%, UV (ACN/Water,
λmax):
322 nm Purity: 95%.
7-Hydroxy-4-methyl-8-(4-phenylpiperazin-1-ylmethyl)chromen-2-one (7
): FT-IR: (KBr), cm−1: 3200–3000
(OH); 3047 (aromatic C–H); 2956 (aliphatic C–H); 1715 (C=O, lactone); 1626 (C=C, nonaromatic); 1599
1
(C=C, aromatic). H-NMR (400 MHz, CDCl3)
δ 7.43 (d, J = 8.7 Hz, 1H, H5); 7.96–7.27 (m, 2H, H15);
6.96–6.87 (m, 3H, H14,16); 6.79 (d, J = 8.7 Hz, 1H, H6); 6.09 (q, J = 1.10 Hz, 1H, H3); 4.13 (s, 2H, H10);
3.04 (broad s, 8H, H11,12); 2.39 (d, J = 1.11 Hz, 3H, H9). 13C-NMR (400 MHz, CDCl3)
δ
162.2 (C2); 161.1,
153.2, 152.5, 129.1, 124.7, 113.3 (CCoumarin-aromatic); 150.7 (C13); 120.4 (C15); 116.4 (C16); 112.3, 110.7 (C3,4);
107.5 (C14); 53.8 (C10); 52.6, 49.1 (C11,12); 18.7 (C9). LC-MS: rt = 5.63 min, m/z 351.37 [M + H]+. Purity:
97%, UV (ACN/Water, λmax): 322 nm.
8-[4-(4-Chlorophenyl)piperazin-1-ylmethyl]-7-hydroxy-4-methylchromen-2-one (8 :
): FT-IR: (KBr), cm−1
3300–3050 (OH); 3043 (aromatic C–H); 2966 (aliphatic C–H); 1714 (C=O, lactone); 1625 (C=C,
1
nonaromatic); 1599 (C=C, aromatic). H-NMR: (400 MHz, CDCl3)
δ 7.43 (d, J = 8.75 Hz, 1H, H5); 7.22
(d, J = 9.0 Hz, 2H, H15); 6.84 (d, J = 9.0 Hz, 2H, H14); 6.79 (d, J = 8.75 Hz, 1H, H6); 6.10 (q, J = 1.2 Hz,
1H, H3); 4.13 (s, 2H, H10); 3.36–2.63 (br, 8H, H11,12); 2.39 (d, J = 1.1 Hz, 3H, H9). 13C-NMR: (101 MHz,
CDCl3) δ 162.2(C2); 161.1, 153.3, 152.5, 125.2, 117.6, 110.8 (Ccoumarin-aromatic); 149.3, 129.1, 124.8, 113.4,
(Cphenyl-arom); 112.4, 107.4 (C3,4); 53.7, 52.4 (C11,12); 49.1 (C10); 18.8 (C9). LC-MS: rt = 6.56 min, m/z
385.38 [M + H]+, Purity: 96%, UV (ACN/Water, λmax): 322 nm.
1
8-[(4-(4-Fluorophenyl)piperazin-1-yl)methyl]-7-hydroxy-4-methylchromen-2-one (
CDCl3)
1H, H6); 6.09 (q, J = 1.21 Hz, 1H, H3); 4.13 (s, 2H, H10); 3.29–2.65 (br, 8H, H11,12); 2.39 (d, J = 1.18 Hz,
3H, H9). 13C-NMR: (400 MHz, CDCl3)
162.2 (C2); 161.2, 153.3, 14.7, 118.3, 118.2, 115.7, 115.5, 113.4,
9): H-NMR: (400 MHz,
δ
7.43 (d, J = 8.7 Hz, 1H, H5); 7.01–6.92 (m, 2H, H15); 6.91–6.86 (m, 2H, H14); 6.78 (d, J = 8.7 Hz,
δ
112.4 (Carom); 110.8, 107.5 (C3,4); 53.7, 52.6 (C11,12); 50.1(C10); 18.8 (C9). LC-MS: rt = 6.02 min, m/z 369.32
[M + H]+, Purity: 98%, UV (ACN/Water, λmax): 239 nm, 321 nm.
7-Hydroxy-8-[4-(bromophenyl)-piperazin-1-ylmethyl]-4-methylchromen-2-one (10): FT-IR: (KBr), cm−1
:
3350–3100 (OH); 3038 (aromatic C–H); 2965 (aliphatic C–H); 1702 (C=O, lactone); 1625 (C=C,
1
nonaromatic); 1598 (C=C, aromatic). H-NMR (400 MHz, CDCl3)
δ
11.58 (s, 1H, OH); 7.41 (d, J = 8.8 Hz,
1H, H5); 7.32 (d, J = 8.8 Hz, 2H, H15); 6.77 (d, J = 8.8 Hz, 3H, H6,14); 6.07 (s, 1H, H3); 4.10 (s, 2H, H10);
3.21 (broad s, 4H, H12); 2.72 (broad s, 4H, H11); 2.37 (s, 3H, H9). 13C-NMR: (400 MHz, CDCl3)
δ
162.1
(C2); 161.0, 153.2, 152.5, 124.8, 113.3, 112.4 (CCoumarin-aromatic); 149.7 (C13); 131.9 (C15); 117.9 (C14); 112.5
(C16); 110.7, 107.5 (C3,4); 53.7, 52.4 (C11,12); 48.8 (C10); 18.8 (C9). LC-MS: rt= 6.60 min, m/z 432.24, 433.21
[M + H]+, Purity: 96%, UV (ACN/Water, λmax): 252 nm.
7-Hydroxy-4-methyl-8-(4-p-tolyl-piperazin-1-ylmethyl)chromen-2-one (11): FT-IR: (KBr), cm−1: 3200–3050
(OH); 3008 (aromatic C–H); 2962 (aliphatic C–H); 1703 (C=O, lactone); 1624 (C=C, nonaromatic); 1599
1
(C=C, aromatic). H-NMR: (400 MHz, DMSO)
δ 7.54 (d, J = 8.8 Hz, 1H, H5); 7.00 (d, J = 8.7 Hz, 2H,
H15); 6.82 (d, J = 8.7 Hz, 2H, H14); 6.78 (d, J = 8.7 Hz, 1H, H6); 6.12 (q, J = 1.2 Hz, 1H, H3); 3.92 (s, 2H,
H10); 3.08 (t, J = 4.5 Hz, 4H, H12); 2.67 (t, J = 4.5 Hz, 4H, H11); 2.36 (d, J = 1.2 Hz, 3H, H9); 2.17 (s, 3H,
H17). 13C-NMR: (400 MHz, DMSO-d6)
δ 162.2 (C2); 160.5, 154.5, 153.2, 149.1, 129.8, 128.3, 116.2, 113.3

Int. J. Mol. Sci. 2019, 20, 1208
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(Caromatics); 110.1, 108.8 (C3,4); 52.5, 49.1 (C11,12); 40.5 (C10); 20.3 (C17); 18.8 (C9). LC-MS: rt = 6.37 min,
m/z 365.22 [M + H]+, Purity: 95%, UV (ACN/Water, λmax): 322 nm.
8-[4-(2-Fluorophenyl)piperazin-1-ylmethyl]-7-hydroxy-4-methylchromen-2-one (12): FT-IR: (KBr), cm−1
3300–3150 (OH); 3063 (aromatic C–H); 2986 (aliphatic C-H); 1706 (C=O, lactone); 1625 (C=C,
nonaromatic); 1594 (C=C, aromatic). 1H-NMR (400 MHz, CDCl3)
7.41 (d, J = 8.78 Hz, 1H, H5);
:
δ
7.06–6.90 (m, 4H, H14,15,16,17); 6.77 (d, J = 8.79 Hz, 1H, H6); 6.07 (q, J = 1.22 Hz, 1H, H3); 4.11 (s, 2H, H10);
3.16 (broad s, 4H, H11); 2.82 (broad s, 4H, H12); 2.37 (d, J = 1.24 Hz, 3H, H9). 13C-NMR (400 MHz, CDCl3)
δ
162.2 (C2); 161.0, 156.9, 154.4, 124.7, 113.3, 112.3 (Ccoumarin-aromatic); 153.2, 152.5, 139.4, 122.9, 119.0,
116.3 (C13,14,15,16,17,18); 110.7, 107.5 (C3,4); 53.8, 52.6 (C11,12); 50.2 (C10); 18.7 (C9). LC-MS: rt = 6.17 min,
m/z 369.19 [M + H]+, Purity: 95%, UV (ACN/Water, λmax): 322 nm.
8-(4-Benzo[1,3]dioxol-5-ylmethyl-piperazin-1-ylmethyl)-7-hydroxy-4-methyl-chromen-2-one (13): FT-IR: (KBr),
cm−1: 3250–3110 (OH); 3047 (aromatic C–H); 2950 (aliphatic C-H); 1718 (C=O, lactone); 1595 (C=C,
1
aromatic). H-NMR (400 MHz, CDCl3)
δ
10.52 (s, 1H, OH); 7.37 (d, J = 8.7 Hz, 1H), 6.80–6.70 (m, 3H,
H6+15,17); 6.01 (broad s, 1H, H18); 5.90 (q, 1H, J = 1.1 Hz, H3); 4.02 (s, 2H, H10); 3.41 (s, 2H, H13); 2.52 (m,
8H, H11,12); 2.34 (s, 3H, H9). 13C-NMR (400 MHz, CDCl3)
δ
162.4 (C2); 161.2, 153.3, 152.4, 124.5, 122.1,
112.1, 110.5 (CCoumarin-aromatic); 147.6, 146.6 (Cphenyl); 131.4 (C14); 122.1 (C18); 113.3 (C15); 109.3 (C17);
107.8, 107.5 (C3,4); 100.8 (C16); 62.3 (C13); 53.8, 52.5 (C11,12); 52.4 (C10); 18.7 (C9). LC-MS: rt = 9.81 min,
m/z 409.38; [M + H]+, Purity: 96%, UV (ACN/Water, λmax): 220 nm, 321 nm.
3.2. Carbonic Anhydrase Inhibition Assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed
CO2 hydration activity [24]. Phenol red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM Tris (pH 8.3) as buffer, and 20 mM
Na2SO4 (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 (0.1 mM) were prepared in distilled-deionized water and dilutions
up to 0.005 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were
preincubated together for 6 hours at room temperature prior to assay, in order to allow for the
formation of the E-I complex. The inhibition constants were obtained by nonlinear least-squares
methods using PRISM 3 (GraphPad Software, San Diego, CA, USA) and the Cheng–Prusoff equation,
as reported by Maresca et al. [14], and represent the mean from at least three different determinations.
All CA isoforms were recombinant ones obtained in-house.
3.3. Molecular Modeling
Molecular docking. The crystal structures of hCA I (PDB code 1AZM), hCA II (PDB code 2AW1),
hCA IX (PDB code 3IAI) and hCA XII (PDB code 1JD0) were taken from the Protein Data Bank [25].
Molecular docking calculations were performed with Autodock 4.2 [26] using the improved force field
for the treatment of the zinc ion within hCAs binding site [27,28]. Autodock Tools were employed for
identifying the torsion angles in the ligand, add the solvent model and assign the Kollman atomic
charges to the protein. Ligand charges were calculated with the Gasteiger method. A grid spacing
of 0.375 Å and a distance-dependent function of the dielectric constant were used for the energetic
map calculations. The ligands were subjected to a docking procedure already used in pose prediction
studies [29]. The docked compounds were subjected to 100 runs of the Autodock search using the
Lamarckian Genetic Algorithm, performing 5,000,000 steps of energy evaluation. All other settings
were left as their defaults and the best docked conformation was taken into account for each ligand.

Int. J. Mol. Sci. 2019, 20, 1208
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Molecular dynamics simulations. Although Autodock scoring function was already successfully
used for analyzing the selectivity profile of small-molecule ligands [30], in this case, molecular
dynamics (MD) simulations were employed to study the reliability of the predicted ligand binding
poses. In fact, the presence of the zinc ion within the protein binding site and the multistep interaction
scheme typical of coumarin ligands (binding to the enzyme in both native and hydrolyzed form) would
hamper the reliability of ligand binding energy evaluations. Therefore, the ligand–protein complexes
generated by docking compound 13 in both native and hydrolyzed form into the catalytic sites of the
four hCA isoforms were studied through MD simulations with AMBER 16 (University of California,
San Francisco, CA, USA) [31]. Each complex was subjected to an MD protocol already applied in
pose prediction studies [32
5000 steps of steepest descent followed by conjugate gradient until a convergence of 0.05 kcal/(mol
,
33]. Prior to MD simulations, the system was energy minimized through
·
Å2)
was reached. The minimized complexes were then used as the starting point for three subsequent
MD steps, for a total of 30 ns of MD simulation. A 0.5 ns constant-volume simulation in which the
temperature of the system was raised from 0 to 300 K was initially performed. In the second step, the
system was equilibrated through a 29.5 ns constant-pressure simulation, maintaining the temperature
at the constant value of 300 K with the use of Langevin thermostat. All simulations were performed
using particle mesh Ewald electrostatics with a cutoff of 10 Å for non-bonded interactions and periodic
boundary conditions. A simulation step of 2.0 fs was employed, as all bonds involving hydrogen atoms
were kept rigid using the SHAKE algorithm, and General Amber force field (GAFF) parameters were
used for the ligand, whose partial charges were calculated with the AM1-BCC method as implemented
in the Antechamber suite of AMBER 16.
4. Conclusions
A novel series of coumarin derivatives, functionalized with aliphatic and aromatic piperazine
fragments in position 8 of the chromen-2-one scaffold, were synthesized and evaluated for their
inhibitory activity against hCA I, hCA II, hCA IX and hCA XII. All compounds demonstrated the
inhibition of the cancer-related hCA isoforms IX and XII with Ki values in the nanomolar range and
were shown to be devoid of any inhibitory activity toward the cytosolic hCA I and hCA II up to
10 µM concentration in the assay system. In particular, single-digit nanomolar Ki values for hCA XII
were determined for almost all tested compounds, which were found to be as potent as the reference
inhibitor acetazolamide. These results demonstrate that the synthesized compounds represent novel
potent and selective non-classic hCA IX/XII inhibitors and confirm the importance of the coumarin
scaffold for the development of selective CAIs.
Despite the presence of a coumarin core possibly being associated with side pharmacological
effects, our ligands should be endowed with a considerably safe profile. In particular, our compounds
should be less prone to form epoxide metabolites that are responsible for the hepatotoxic effect of
coumarin, which is, however, low (tolerable daily intake of coumarin = 0.1 mg/kg body weight).
In fact, the detoxification pathway of coumarin begins with its cytochrome-mediated 7-hydroxylation,
followed by urinary excretion either directly or after glucuronidation, a metabolic pathway that
determines low susceptibility to the hepatotoxic effect of coumarin in the majority of the human
population [34]. Notably, our compounds should be less likely to undergo epoxidation metabolism,
as they all present the 7-hydroxyl group that should promote their glucuronidation and excretion.
Moreover, our derivatives lack the bulky moiety in position 3 of the bicyclic scaffold that is typical
of coumarin-based pharmacologic agents with anticoagulant action, which antagonize the functions
of vitamin K. We indeed demonstrated that anticoagulant coumarin derivatives are devoid of hCA
inhibitory activity [14]. Finally, the peculiar mechanism of action of these ligands, requiring an in situ
CA-catalyzed hydrolysis for generating the cinnamic acids that constitute the real CAIs should prevent
any off-target effect that might be caused by their interaction with other zinc-dependent enzymes,
such as matrix metalloproteinases.

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Our molecular modeling studies addressed the ligand recognition process of the intact coumarin
compounds at the level of hCAs catalytic site, as well as the binding interactions of the corresponding
cinnamic acids produced by CA-mediated hydrolysis, according to mechanism of inhibition typical
of coumarin-based ligands. The results suggested that the selectivity of this series of compounds
should be ascribed to the ligand recognition process and the interactions of the coumarin scaffold.
Moreover, consistently with the experimental evidence, the piperazine-based substituents of the
ligands were shown to provide a modest contribution to the ligand–protein interactions predicted
for the compounds in both their coumarin and cinnamic form. Our analysis could thus be used to
guide lead optimization studies aimed at introducing modified structural moieties for either further
improving the ligand recognition process of the coumarin ligands or maximizing the ligand–protein
interactions that can be stablished by the hydrolyzed compounds.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/5/
1208/s1.
Author Contributions: K.B. and F.E.Ö.B. performed the syntethic work, G.P. and T.T. carried out the molecular
modeling studies; S.B. carried out biochemical experiments, and C.T.S. supervised the whole study.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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