Synthesis, computational studies and assessment of in vitro inhibitory activity of umbelliferon-based compounds against tumour-associated carbonic anhydrase isoforms IX and XII

Article abstract of DOI:10.1080/14756366.2020.1786821

Coumarins are widely diffused secondary metabolites possessing a plethora of biological activities. It has been established that coumarins represent a peculiar class of human carbonic anhydrase (hCA) inhibitors having a distinct mechanism of action involv

Full text of DOI:10.1080/14756366.2020.1786821

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis, computational studies and assessment
of in vitro inhibitory activity of umbelliferon-based
compounds against tumour-associated carbonic
anhydrase isoforms IX and XII
Francesca Mancuso, Laura De Luca, Andrea Angeli, Sonia Del Prete,
Clemente Capasso, Claudiu T. Supuran & Rosaria Gitto
To cite this article: Francesca Mancuso, Laura De Luca, Andrea Angeli, Sonia Del Prete,
Clemente Capasso, Claudiu T. Supuran & Rosaria Gitto (2020) Synthesis, computational studies
and assessment of in vitro inhibitory activity of umbelliferon-based compounds against tumour-
associated carbonic anhydrase isoforms IX and XII, Journal of Enzyme Inhibition and Medicinal
Chemistry, 35:1, 1442-1449, DOI: 10.1080/14756366.2020.1786821
To link to this article: https://doi.org/10.1080/14756366.2020.1786821
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1442–1449
https://doi.org/10.1080/14756366.2020.1786821
RESEARCH PAPER
Synthesis, computational studies and assessment of in vitro inhibitory activity of
umbelliferon-based compounds against tumour-associated carbonic anhydrase
isoforms IX and XII
Francesca Mancusoa , Laura De Lucaa , Andrea Angelib , Sonia Del Pretec , Clemente Capassoc
,
Claudiu T. Supuranb
and Rosaria Gittoa
a
ꢀ
Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali (CHIBIOFARAM), Universita degli Studi di Messina, Messina, Italy;
b
c
ꢀ
Dipartimento NEUROFARBA, Universita di Firenze, Sesto Fiorentino, Italy; Istituto di Bioscienze e Biorisorse – CNR, Napoli, Italy
ABSTRACT
ARTICLE HISTORY
Received 18 April 2020
Revised 29 May 2020
Accepted 18 June 2020
Coumarins are widely diffused secondary metabolites possessing a plethora of biological activities. It has
been established that coumarins represent a peculiar class of human carbonic anhydrase (hCA) inhibitors
having a distinct mechanism of action involving a non-classical binding with amino acid residues paving
the entrance of hCA catalytic site. Herein, we report the synthesis of a small series of new coumarin deriv-
atives 7-11, 15, 17 prepared via classical Pechmann condensation starting from resorcinol derivatives and
suitable b-ketoesters. The evaluation of inhibitory activity revealed that these compounds possessed nano-
molar affinity and high selectivity towards tumour-associated hCA IX and XII over cytosolic hCA I and hCA
II isoforms. To investigate the binding mode of these new coumarin-inspired inhibitors, the most active
compounds 10 and 17 were docked within hCA XII catalytic cleft.
KEYWORDS
Carbonic anhydrase
inhibitors (CAIs); tumour-
associated CA isoforms;
coumarin; Pechmann
condensation; Fries
rearrangement
Introduction
Human Carbonic anhydrases (hCAs) are a family of metalloen-
zymes belonging to a-class and comprising 12 catalytic isoforms
(cytosolic CA I, CA II, CA III, CA VII and CA XIII; membrane-associ-
ated CA IV, CA IX, CA XII, and CA XIV; mitochondria-associated CA
VA and CA VB; and secreted CA VI) that differ for kinetic proper-
ties, tissue distribution and subcellular localisation15,16. They cata-
lyse the simple reaction of CO2 hydration leading to the formation
of bicarbonate and proton. This reaction is a crucial physiological
process for different organisms in controlling pH as well as rele-
vant metabolic pathway. It has emerged that the CA-mediated pH
regulation can offer the opportunity to develop therapeutics able
Coumarin (2H-chromen-2-one 1, Figure 1) is classified as a mem-
ber of a large class of phenolic chemotypes occurring in higher
plants, bacteria and fungi. Apart from natural coumarins, a wide
collection of (semi)synthetic coumarins are endowed by various
pharmacological properties such as anticancer, anticoagulant, anti-
inflammatory, and antimicrobial activities1. From a structural point
of view, the coumarins are classified in different chemical classes
(simple coumarins, fused polycyclic coumarins, phenylcoumarins,
biscoumarins, and so on)1,2. The existence of an extensive library
of coumarins from (semi)synthetic source has been triggered by
the high versatility of benzo-a-pyrone system to be an excellent
scaffold to perform structural modifications3–6. Moreover, the
chemical diversity of the coumarin system implies that they might
play an important role in medicinal chemistry for drug discovery
processes, so that many coumarins are used currently in drug
development as vitamin K antagonists, choleretic and antibacterial
and antiviral agents7–11. It has been suggested that the benzopyr-
one structure enables its derivatives to readily interact with bio-
logical molecular targets through a network of non-covalent
interactions such as hydrophobic, p–p stacking, and cation–p
interactions as well as hydrogen and coordination bond interac-
tions. Specifically, coumarin-based compounds interact with
human carbonic anhydrases (CAs, EC 4.2.1.1), which possess sig-
nificant esterase activity and induce the opening of lactone ring;
due to the formation of the corresponding carboxylic derivative,
non-classical CA inhibitory effects have been evidenced for cou-
to inhibit the CA activity related to pathological processes16–18
.
Among them, there is growing evidence that the two membrane-
bound hCA IX and hCA XII isoforms might cause the extracellular
pH of tumour cell microenvironment (TME) to be acid, thus play-
ing a key role in the cancer progression and metastatic proc-
esses19. The dimeric hCA IX isoform is over-expressed in hypoxic
tumour cells, modulated by the hypoxia-inducible factor (HIF-1),
and results typically associated with aggressive tumour not
responsive to chemotherapy and radiotherapy20. In tandem with
the hCA IX isoform, the cancer-related hCA XII is involved in TME
regulation; for this reason, oncogenic hCA IX/XII isoforms repre-
sent promising antitumor targets for the development of selective
CA inhibitors able to reduce the growth of hypoxic cancers. The
proof-of-concept that hCA IX inhibition is a profitable target has
been confirmed by the therapeutically effective inhibitor SLC-
0111, which has recently entered clinical trials for breast and
marin-derived compounds (vide infra)12–14
.
brain cancer21–28
.
ꢀ
Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali (CHIBIOFARAM), Universita degli
CONTACT Rosaria Gitto
rosaria.gitto@unime.it
Studi di Messina, Viale Annunziata, I-98168 Messina, Italy
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1443
Figure 1. (A) Chemical structures of coumarin 1, 6-(1-S-hydroxy-3methylbutyl)-7-methoxy-2H-chromen-2-one (2) and corresponding hydrolytic products 2a and 2b; (B)
cocrystal structure of hydrolytic product of 6-(1-S-hydroxy-3methylbutyl)-7-methoxy-2H-chromen-2-one (2) in complex with hCA II (PDB code: 3F8E)12
.
Several coumarin-based compounds demonstrated to be
R1
potent hCA IX/XII inhibitors having a non-classical mechanism of
HO
O
O
HO
O
O
CA-inhibition by occlusion of the enzymatic site11,29–34 that dis-
plays a cone-shape cavity containing a zinc ion located in the bot-
tom and coordinated by three crucial histidine residues (His94,
His96, and His119). Their unusual binding mode has been demon-
strated for the coumarins such as the 6-(1-S-hydroxy-3methylbu-
tyl)-7-methoxy-2H-chromen-2-one
(2)
through
X-ray
crystallography for the corresponding hydrolytic product (see
Figure 1(B), PDB code: 3F8E)12; in detail, these studies evidenced a
hydrolytic pathway that led to the hydrolytic product 2-hydroxy-
cinnamic acid derivative bound at the entrance of the enzyme
pocket hindering the access of substrate to the catalytic active
site and blocking the catalytic activity.
R
7-hydroxy-4-phenyl-2H-chromen-2-one (
Umbelliferon
3, R=R =H
)
1
4-(4-aminophenyl)-7-hydroxy-8-methyl-2H-chromen-2-one
(4, R= NH2, R1= Me)
Figure 2. Chemical structures of umbelliferon, 3 and 4 as coumarin-based hCAIs.
As a result, the coumarin-based compounds might bind the
most variable region located in the top area of the protein cavity
for twelve catalytic isoforms, explaining the high selectivity
towards specific druggable isoforms such as hCA IX/XII in combin-
ation with low affinity for the ubiquitous hCA I and II. Based on
this evidence, we have recently synthesised a series of 4-phenyl-
coumarin derivatives (e.g. 3 and 4, Figure 2) and structurally
related to umbelliferon (7-hydroxy-2H-chromen-2-one, Figure 2)
which represents a selective and potent CA inhibitor29; notably,
some of the new synthesised derivatives exhibited high affinity
towards hCA IX and hCA XII with Ki values in the low nanomolar
range; additionally, similar to other well-known coumarin-based
compounds, they were inactive towards the ubiquitous hCA I and
hCA II up to 10 mM concentration29. By using the best active
and selective inhibitors 7-hydroxy-4-phenyl-2H-chromen-2-one (3,
R ¼ R1¼H) and 4-(4-aminophenyl)-7-hydroxy-8-methyl-2H-chro-
men-2-one (4, R ¼ NH2, R1¼Me) as templates, we now report the
synthesis of a further series of coumarins, which were tested as
inhibitors of selected hCA I, II, IX and XII isoforms. Moreover, dock-
ing simulations analysed the possible interactions within the cata-
lytic cavity of hCA XII29.
Material and methods
Chemistry
All reagents were used without further purification and bought
from common commercial suppliers. Melting points were deter-
mined on a Buchi B-545 apparatus (BUCHI Labortechnik AG Flawil,
Switzerland) and are uncorrected. By combustion analysis (C, H, N)
carried out on a Carlo Erba Model 1106-Elemental Analyser, we
determined the purity of synthesised compounds; the results con-
firmed a ꢀ 95% purity. Merck Silica Gel 60 F254 plates were used
for analytical TLC (Merck KGaA, Darmstadt, Germany). For detec-
1
tion, iodine vapour and UV light (254 nm) were used. H NMR and
13C NMR spectra were measured in dimethylsulfoxide-d6 (DMSO-
d6) with a Varian Gemini 300 or 500 spectrometer (Varian Inc. Palo
Alto, California USA); chemical shifts are expressed in d (ppm) and
coupling constants (J) in hertz. All exchangeable protons were
confirmed by the addition of D2O. Rf values were determined on
TLC plates using a mixture of CycloEx/EtOAc (60:40 v/v) as eluent.
For coumarins 7–11 and 15, the CAS registry numbers have been

1444
F. MANCUSO ET AL.
already assigned; however detailed information about chemical Anal. for (C22H14O4): C 77.18%, H 4.12%; Found: C 77.28%,
characterisation is not available in the literature; for selected com- H 4.22%.
pounds, representative 1H-NMR and 13C-NMR spectra are dis-
played in Supporting Material. Pharmacokinetics and drug-likeness
General synthesis for 8-acetyl-7-hydroxy-4-phenyl-2H-chromen-
2-one (10) and 7-hydroxy-4-phenyl-8-propanoyl-2H-chromen-2-
one (11)
prediction for the synthesised compounds (7-11, 15 and 17) were
performed by using the online tool SwissADME of Swiss Institute
Bioinformatics (http://www.sib.swiss) and the collected data are
shown in Supplemental Material.
A mixture of compounds 7 or 8 (1 mmol) and anhydrous AlCl3 (5
molar equivalents) was heated to 320 ꢂC and stirred for 2 h at the
same temperature. When the reaction mixture was cooled to
room temperature, a solution of 5% HCl (30 ml) was added. The
resulted suspension was stirred for 1 h at room temperature and
then heated under a steam bath for others 30 min. The precipitate
formed was filtered off and purified by crystallisation with EtOH to
yield compounds 10 and 11, for which detailed structural assign-
ments are not available in the literature.
Synthesis of 2-oxo-4-phenyl-2H-chromen-7-yl acetate (7)
To an ice-cold solution of resorcinol (6, 1 mmol) in the appropriate
ethyl benzoylacetate derivative (5, 1 mmol), 96% w/v sulphuric
acid (2 ml) was added dropwise. The mixture was brought to
room temperature and stirred at 350 rpm by a stirring magnet bar
for 24 h, then TLC showed the disappearance of both starting
materials. The reaction mixture was quenched with crushed ice
flakes, subsequently diluted with H2O (10 ml) and extracted with
EtOAc (3 ꢁ 10 ml). The organic layer was dried with Na2SO4 and
concentrated until dryness under reduced pressure. The targeted
compounds 3 was isolated from the crude by crystallisation with
EtOH. The spectroscopic data of compound 3 were consistent
with those previously reported in the literature29. Subsequently,
compound 3 (1 mmol) was stirred with acetic anhydride (3 ml) in
an ice bath and a catalytic amount of 96% sulphuric acid was
added dropwise. Then, Et3N (2.5 molar equivalents) was added to
the mixture and stirred until the disappearance of starting com-
pounds (TLC). After the reaction was completed, it was quenched
with ice and the solid was filtered off and dried to afford the cor-
responding desired compound 7 (CAS Number: 16299-27-7) for
which the structural assignments were in good agreement with
the literature35.
8-Acetyl-7-hydroxy-4-phenyl-2H-chromen-2-one (10) (CAS
Number: 54431-18-4)
Yield: 25%; m.p.: 164–166 ꢂC; Rf 0.76; 1H-NMR (DMSO-d6): (d)
1.10 (s, 3H, CH3), 6.14 (s, 1H, CH), 6.89 (d, J ¼ 8.4, 2H, ArH), 7.33 (d,
J ¼ 8.4, 2H, ArH), 7.51–7.56 (m, 5H, ArH). 13C NMR (126 MHz) (d):
200.37, 159.16, 159.00, 155.37, 151.98, 134.85, 129.62, 129.48,
128.80, 128.33, 115.99, 113.26, 110.65, 39.52, 32.35. Anal. for
(C17H12O4): C 72.85%, H 4.32%; Found: C 72.83%, H 4.20%.
7-Hydroxy-4-phenyl-8-propanoyl-2H-chromen-2-one (11)
Yield: 20%; m.p.: 172–174 ꢂC; Rf 0.80. 1H NMR (DMSO-d6): 1.10
(t, J ¼ 7.3, 3H, CH3), 2.90 (q, J ¼ 7.3, 2H, CH2), 6.14 (s, 1H, CH), 6.86
(d, J ¼ 8.4, 2H, ArH), 7.31 (d, J ¼ 8.4, 2H, ArH), 7.50–7.55 (m, 5H,
ArH). Anal. for (C18H14O4): C 73.46%, H 4.79%. Found: C 73.56%,
H 4.99%.
Yield: 79%; m.p.: 129–131 ꢂC; Rf 0.64; 1H-NMR (CDCl3) (d): 2.32
(s, 3H, CH3) 6.44 (s, 1H, CH), 7.17 (m, 1H, ArH), 7.40 (s, 1H, ArH),
7.48 (m, 1H, ArH), 7.55-7.60 (m, 5H, ArH). Anal. for (C17H12O4): C
72.85%, H 4.32%; Found: C 72.88%, H 4.45%.
General synthesis for 8-chloro-7-hydroxy-4-phenyl-2H-chromen-
2-one (15) and 8-chloro-7-hydroxy-4-(4-nitrophenyl)-2H-
chromen-2-one (16)
To an ice-cold mixture of 2-chlororesorcinol (14, 1 mmol) and
appropriate ethyl aroylacetate derivatives (13, 1 mmol), 96% w/v
sulphuric acid (2 ml) was added dropwise. The mixture was
brought to room temperature and stirred at 350 rpm by a stirring
magnet bar for 24 h, then TLC showed the disappearance of both
starting materials. The reaction mixture was quenched with
crushed ice flakes, subsequently diluted with H2O (10 ml) and
extracted with EtOAc (3 ꢁ 10 ml). The organic layer was dried with
Na2SO4 and concentrated until dryness under reduced pressure.
The targeted compounds 15 or 16 were isolated from the crude
by crystallisation with EtOH. The structural assignments of com-
pound 15 were in good agreement with the literature36.
General synthesis for 2-oxo-4-phenyl-2H-chromen-7-yl
propionate (8) and 2-oxo-4-phenyl-2H-chromen-7-yl
benzoate (9)
To a stirred solution of compound 3 (1 mmol) in DCM (2 ml) and
Et3N (2.5 molar equivalents), propionyl, or benzoyl chloride (5
molar equivalents) were slowly added in an ice bath. After 10 min,
the mixture was allowed to room temperature and stirred until
the complete conversion of starting material (TLC). Then, the solid
products were filtered off and recrystallized from Et2O. For com-
pounds 8 and 9, detailed structural assignments are not available
in the literature.
8-Chloro-7-hydroxy-4-phenyl-2H-chromen-2-one (15) (CAS
Number: 53391-82-5)
2-Oxo-4-phenyl-2H-chromen-7-yl propionate (8) (CAS
Number: 327048-31-7)
Yield: 50%; m.p.: 214-215 ꢂC; Rf 0.63; 1H-NMR (DMSO-d6) (d):
5.89 (s, 1H, CH), 6.66 (d, J ¼ 8.8, 1H, ArH), 6.95 (d, J ¼ 8.8, 1H, ArH),
7.15–7.25 (m, 5H, ArH). Anal. for (C15H9ClO3): C 66.04%, H 3.33%;
Found: C 66.14%, H 3.42%.
Yield: 20%; m.p.: 172–174 ꢂC; Rf 0.80; 1H-NMR (DMSO-d6) (d):
1.16 (t, J ¼ 7.3, 3H, CH3), 2.67 (q, J ¼ 7.3, 2H, CH2), 6.43 (s, 1H, CH),
7.17 (d, J ¼ 8.4, 2H, ArH), 7.40 (s, 1H, ArH), 7.48 (d, J ¼ 8.4, 2H,
ArH), 7.55–7.63 (m, 5H, ArH). 13C NMR (DMSO-d6) (d): 8.77, 26.99,
110.71, 114.22. 116.37, 118.71, 127.82, 128.58, 128.99, 129.85.
134.71, 153.17, 154.26, 154.64, 159.56, 172.20. Anal. For (C18H14O4):
C 73.46%, H 4.79%. Found: C 73.54%, H 4.77%.
8-Chloro-7-hydroxy-4-(4-nitrophenyl)-2H-chromen-2-one (16)
Yield: 55%; m.p.: 303–305 ꢂC; Rf 0.29; 1H-NMR (DMSO-d6) (d):
6.38 (s, 1H, CH), 6.98 (d, J ¼ 8.7, 1H, ArH), 7.15 (d, J ¼ 8.7, 1H, ArH),
7.82 (d, J ¼ 8.2, 2H, ArH), 8.39 (d, J ¼ 8.2, 2H, ArH). 13C NMR
(DMSO-d6) (d): 111.55, 112.08. 113.36, 124.32, 125.99, 130.60,
141.74, 146.71. 148.54, 151.42, 153.76, 157.83, 159.46. Anal.
2-Oxo-4-phenyl-2H-chromen-7-yl benzoate (9) (CAS Number:
94739-93)
Yield: 98%; m.p. 228–230 ꢂC; Rf 0.76; 1H NMR (DMSO-d6) (d): (C15H8ClNO5): C 56.71%, H 2.54%, N 4.41%; Found: C 56.75%, H
6.47 (s, 1H, CH), 7.32–7.79 (m, 11H, ArH), 8.16–8.18 (m, 2H, ArH). 2.27%, N 4.44%.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1445
Scheme 1. (i) H2SO4, 0 ꢂC to rt, 18–22h; (ii) A: (MeCO)2O, H2SO4, Et3N, 0 ꢂC to rt, 2 h; B: PheCOCl or EtCOCl, Et3N, DCM, rt, 24 h; (iii) AlCl3, 320 ꢂC, 6 h; (iv) NH2NH2-H2O,
Pd/C, EtOH, 70 ꢂC, 1 h.
Synthesis of 4-(4-aminophenyl)-8-chloro-7-hydroxy-2H-chromen-
2-one (17)
water and dilutions up to 0.01 nM were done thereafter with dis-
tilled-deionized water. Inhibitor and enzyme solutions were prein-
cubated together for 15 min at room temperature prior to assay
to allow for the formation of the E–I complex. The inhibition con-
stants 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
To a suspension of the nitro derivative 16 (1 mmol) and a catalytic
amount of Pd/C in EtOH (15 ml), hydrazine hydrate (NH2–NH2ꢃH2O,
10 molar equivalents) was slowly added. The reaction was stirred
and refluxed (70 ꢂC) under a nitrogen atmosphere for 1 h. Then,
the mixture was filtered through celite cake, which was later
washed with EtOAc. The obtained solution was evaporated in
vacuo to give the crude product, then diluted with EtOAc and
washed with H2O (3 ꢁ 10 ml). The organic layer was dried with
Na2SO4 and concentrated until dryness. The residue was purified
by crystallization with EtOH to give the corresponding amino
derivative 17.
ones obtained as reported earlier by this group37–40
.
Docking studies
Automated docking was carried out by means of the programme
AUTODOCK 4.241. The crystal structure of was retrieved from the
RCSB Protein Data Bank (PDB: 1JCZ)42. The ligand and water mole-
cules were discarded, and hydrogen atoms were added to protein
with Discovery Studio 2.5.5. Structures of the ligands were con-
structed using Discovery Studio 2.5.5 and energy was minimised
using the Powel protocol (1000 steps). The regions of interest
used by AUTODOCK were defined by considering the suitable lig-
and docked into the hCA XII receptor as the central group; the
docking box included the canonical binding site found for several
hydrolysed coumarins; in particular, a grid of 60, 60, and 60 points
in the x, y, and z directions was constructed centred on the centre
of the mass of metal as Zn2þ 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 (LGA), 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 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.
Yield: 40%; m.p.: 313–315 ꢂC; Rf 0.14; 1H-NMR (DMSO-d6) (d):
5.65 (bs, 2H, NH2), 6.06 (s, 1H, CH), 6.67 (d, J ¼ 8.2, 2H, ArH), 6.94
(m, 1H, ArH), 7.21 (d, J ¼ 8.2, 2H, ArH), 7.47 (m, 1H, ArH). Anal. for
(C15H10ClNO3):C 62.62%; H 3.50%; N 4.87%; Found: C 62.60%, H
3.68%, N 4.65%.
CA inhibitory 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 an 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), fol-
lowing the initial rates of the CA-catalysed 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 Cluster analysis was performed on the docked results using an

1446
F. MANCUSO ET AL.
Table 1. Inhibition data for hCA I, II, IX and XII with compounds 7–11, 15 and
17 as well as umbelliferon and 3–4 as reference compoundsa.
10,000 nM concentration in a good agreement with previously
reported coumarins 3–4 and umbelliferon.
R1
R2
R3
hCA I
hCA II
hCA IX hCA XII
A preliminary analysis of structure-affinity-relationship (SAR) for
the esters 7–9 suggested that the presence of acetyl, propionyl or
benzoyl group (e.g. R3CO), which masks the hydroxyl moiety of
the lead compound 3, gave an opposite kinetic behaviour against
hCA IX and XII. Specifically, the lack of the 7-hydroxyl group did
not significantly affect the inhibition potency of benzoate 9
against hCA IX so that its Ki value 21.8 nM was lower than that of
parent compound 3 (Ki ¼ 47.2 nM) and comparable to umbelli-
feron (Ki ¼ 24.9 nM). For acetate 7 and propionate 8, we found
less inhibitory effects towards hCA IX when compared with corre-
sponding hydroxyl derivative 3. Furthermore, all tested esters 7–9
were up to 8-fold less active towards hCA XII when compared
with parent compound 3. Thus, we can hypothesise that the 7-
hydroxyl polar group might play a crucial role in the binding rec-
ognition process within hCA XII. Moreover, the Ki values measured
for compounds 10, 11 and 15 highlighted that the introduction
of MeCO- or EtCO-moiety as well as a chlorine atom at the C8
position of coumarin scaffold was detrimental for the binding to
hCA IX isoform. On the contrary, compounds 10 and 15 displayed
an improved inhibitory effect towards hCA XII when compared to
unsubstituted parent compound 3, whereas as outlier the 7-
hydroxy-4-phenyl-8-propanoyl-2H-chromen-2-one (11) was 10-fold
less efficient than prototype 3. Finally, in the case of compound 4-
aminophenyl substituted compound 17, we found that the pres-
ence of a chlorine atom in place of methyl-substituent (i.e. com-
pound 4) significantly reduced the Ki affinity, especially towards
hCA IX isoform. Overall, coumarin 9 demonstrated the best inhibi-
tory effects and selectivity towards hCA IX; whereas, compounds
Umbelliferonb
3b
4
7
8
>10,000 >10,000
>10,000 >10,000
>10,000 >10,000
24.9
47.2
24.2
56.2 734.1
97.8 523.5
21.8 384.6
45.1
90.8
5.5
H
Me
H
H
H
COMe
COEt
Cl
H
OH
b
NH2 OH
H
H
H
H
H
H
OCOMe >10,000 >10,000
OCOEt >10,000 >10,000
OCOPh >10,000 >10,000
9
10
11
15
17
OH
OH
OH
>10,000 >10,000
>10,000 >10,000 3042
>10,000 >10,000
>10,000 >10,000
176.5
24.9
868.5
32.0
261.3
180.3
Cl
NH2 OH
29.3
aErrors in the range of 10% of the reported value, from 3 different assays.
bData from reference 29.
RMSD (Root Mean Square Deviation) tolerance of 2 Å. All the com-
pounds were docked according to the afore mentioned parame-
ters. The hCA XII/ligand complex obtained by docking studies was
minimised using 1000 iterations of SD and 1000 interaction of
Polak-Ribiere Conjugate Gradient. Interactions were identified
using the LigPlot software43 and the figures were prepared using
opensource software PyMOL (https://pymol.org/2/).
Results and discussion
To improve our knowledge on coumarin-based inhibitors from
synthetic sources and to study in depth their structure–affinity
relationships, we focussed our interest in the substitution pattern
at the C7 and C8 position of chromen-2-one nucleus (see
Figure 2) of prototypes 4-phenyl-coumarin derivatives 3 and 4 dis-
played in Figure 2. Firstly, we investigated the role of hydroxyl 10, 15 and 17 were efficacious hCA XII inhibitors having signifi-
cant potency and selectivity when compared with umbelliferon.
moiety located in the C-7 position of the coumarin system and
synthesised the corresponding acetate 7, propanoate 8 and
benzoate 9 by the reaction of acetic anhydride (pathway A) or
suitable chlorides (pathway B) with parent compound 3, which
was prepared in good yields through Pechmann reaction (Scheme
1)44 from resorcinol (6) and ethyl benzoylacetate (5). The obtained
esters 7 and 8 were further subjected to Fries rearrangement reac-
tion conditions by employing as Lewis acid AlCl3, thus giving the
desired 10 and 11 derivatives bearing 8-acetyl or 8-propionyl sub-
stituent, respectively. Unfortunately, we failed in all attempts to
obtain 8-benzoyl derivative 12 from corresponding benzoate 9.
Moreover, the Pechmann reaction allowed us to prepare further 4-
arylcoumarin analogs 15-16, thus introducing in 8-position a
chlorine atom in place of the hydrogen atom. As shown in
Scheme 1, the reaction of 2-chlorobenzene-1,3-diol (14) with the
suitable ethyl benzoylacetate (5) or ethyl 3-(4-nitrophenyl)-3-oxo-
propanoate (13) gave corresponding coumarins 15-16. In turn, by
nitroreduction of compound 16, we prepared the desired 4-ami-
nophenylderivative 17 as the analog of prototype 4.
Docking studies
In silico studies were carried out in order to elucidate the hypo-
thetical binding pose into the hCA XII catalytic cleft (PDB code
1JCZ)42 for the selected coumarins 10 (Ki value of 24.9 nM) and 17
(Ki value of 29.3 nM) displaying higher affinity than that of parent
compound umbelliferon (Ki value of 45.1 nM). A previously vali-
dated protocol29 was applied to perform docking simulations for
the newer coumarins studied in this work considering the couma-
rin system and its hydrolysed products. Firstly, we considered the
binding mode of closed forms of inhibitors 10 and 17; Figure 3
displays their ability to deeply occupy the middle area of the cata-
lytic cavity of hCA XII through two hydrogen bond contacts estab-
lished by 7-hydroxyl substituent and carbonyl group with
hydrophilic residues Asn62 and Thr199, respectively. As you can
see in Figure 3(A) the 4-phenyl-substituent is projected towards
hydrophobic half of the hCA XII cavity lined by Val121, Val141,
Leu142, Val143, and Leu198. Additionally, compound 17 is pre-
dicted to bind residue Ser135 by means of H-bond contact medi-
ated by aminophenyl moiety; by comparing the Ki values of
coumarins 10 and 17 we hypothesised that the contact with
Ser135 is not crucial to improve the hCA XII affinity. This hypoth-
esis was corroborated by comparing the Ki value measured for
compound 15 for which the amino group is not present.
Interestingly, the two studied coumarin 10 and 17 oriented the
lactone moiety towards the Zn2þ ion, creating a favourable condi-
tion for the hydrolysis. Keeping in mind these hypothetical bind-
ing modes, we can speculate that these selective inhibitors might
be not capable of accessing at the active site of hCA II, that is
Carbonic anhydrase inhibition
This small series of coumarin derivatives 7–11, 15–17 were sub-
jected to the stopped-flow CO2 hydrase assays to evaluate the
binding affinity against the tumour-associated hCA IX and XII over
ubiquitous isoforms hCA I and II. The data were collected in
Table 1 in comparison with previously reported coumarins 3 and
4 as well as umbelliferon. All tested compounds 7–11 and 15–16
displayed inhibitory effects towards hCA IX and XII with Ki values
in the wide range of 21.8–3042 nM; whereas, these compounds
did not inhibit off-target isoforms hCA I and II up to the characterised by the presence of Phe131 residue as the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1447
Figure 3. (A) Predicted binding mode of compound 10 (coloured in light blue) overlaid with compound 17 (coloured in wheat) into hCA XII cleft (PDB code 1JCZ).42
(B) Chemical structures of hydrolytic forms 10a and 10b. Predicted binding mode of (E)-10a (C, coloured in blue) and (Z)-10b (D, coloured in yellow) as hydrolysed
forms of coumarin 10 into hCA XII cleft (PDB Code: 1JCZ)42. Compounds and crucial residues are shown as sticks; dashed lines represent hydrogen bond interactions.
The protein structure is shown as pale-cyan surface and light grey cartoons. Zinc ion is depicted as a yellow sphere. Figures made by Pymol (https://pymol.org).
gatekeeper of enzymatic cleft; in the case of hCA IX or hCA XII iso- (coloured in yellow in Figure 3(D)) anchors the hCA XII cavity
forms, the residues Val131 or Ala131 replace the bulkier Phe131,
respectively, thus explaining the hCA IX/hCA XII selectivity of
tested coumarins 7-11, 15-16 over hCA II as found for other cou-
marin derivatives reported in the literature30,45. This issue could
justify the advantageous selectivity profile showed by this class of
coumarins that are lacking affinity against off-target isoforms hCA
II (Ki values> 10,000 nM).
through H-bond interactions with polar residues Gln92, Ser135,
and Thr199 and it assumes a different orientation even if it is
equally capable of occluding the entrance of the active site.
In conclusion, we identified a new series of coumarins structur-
ally related to umbelliferon possessing a high affinity towards
tumour-expressed hCA IX/XII and selectivity over hCA I/II isoforms.
The prediction of binding poses of some inhibitors suggested that
this series of compounds might anchor the residues outside of
the active site, so that these coumarins belong to non-classical
CAIs. Overall, this work might contribute to elucidate the mode of
interaction of coumarins against tumour-expressed hCA IX/
XII isoforms.
Considering that the Zn2þ–water-activated species can hydro-
lyse the lactone ring of the benzopyrone system of tested cou-
marins, we decided to compare the binding pose of selected
coumarin 10 with corresponding hydrolysed cis-/trans-hydroxycin-
namic acids (10a and 10 b, Figure 3(B)) that might be derived by
hCA esterase activity. Figures 3(C) and 3(D) show the predicted
binding mode for the two isomers (E)-10a and (Z)-10b into hCA
XII cleft. Docking simulations suggested that the isomer (E)-10a
(coloured in blue in Figure 3(C)) assumes a similar orientation of
parent coumarin 10. Indeed, the phenyl ring is orientated in the
proximity of the hydrophobic residue Leu198 located in the mid-
dle area of hCA XII cleft; whereas the 2,4-dihydroxyl phenyl ring is
leaned towards the entrance of the active site close to the hydro-
phobic wall, it engages a dense network of hydrogen bond inter-
actions by C4-hydroxyl and acetyl moiety with Asn62 and C2-
Author contributions
The manuscript was written through the contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Disclosure statement
hydroxyl group with Thr200. In contrast, the isomer (Z)-10b No potential conflict of interest was reported by the author(s).

1448
F. MANCUSO ET AL.
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Funding
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ORCID
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Francesca Mancuso
Laura De Luca
Andrea Angeli
Sonia Del Prete
Clemente Capasso
Claudiu T. Supuran
http://orcid.org/0000-0002-2209-957X
http://orcid.org/0000-0003-0614-5713
http://orcid.org/0000-0002-1470-7192
http://orcid.org/0000-0001-5291-8823
http://orcid.org/0000-0003-3314-2411
http://orcid.org/0000-0003-4262-0323
Rosaria Gitto
http://orcid.org/0000-0003-0002-2253
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