Selective inhibition of carbonic anhydrase IX and XII by coumarin and psoralen derivatives

Article abstract of DOI:10.1080/14756366.2021.1887171

A small library of coumarin and their psoralen analogues EMAC10157a-b-d-g and EMAC10160a-b-d-g has been designed and synthesised to investigate the effect of structural modifications on their inhibition ability and selectivity profile towards carbonic anhydrase isoforms I, II, IX, and XII. None of the new compounds exhibited activity towards hCA I and II isozymes. Conversely, both coumarin and psoralen derivatives were active against tumour associated isoforms IX and XII in the low micromolar or nanomolar range of concentration. These data further corroborate our previous findings on analogous derivatives, confirming that both coumarins and psoralens are interesting scaffolds for the design of isozyme selective hCA inhibitors.

Full text of DOI:10.1080/14756366.2021.1887171

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Selective inhibition of carbonic anhydrase IX and
XII by coumarin and psoralen derivatives
Rita Meleddu, Serenella Deplano, Elias Maccioni, Francesco Ortuso, Filippo
Cottiglia, Daniela Secci, Alessia Onali, Erica Sanna, Andrea Angeli, Rossella
Angius, Stefano Alcaro, Claudiu T. Supuran & Simona Distinto
To cite this article: Rita Meleddu, Serenella Deplano, Elias Maccioni, Francesco Ortuso, Filippo
Cottiglia, Daniela Secci, Alessia Onali, Erica Sanna, Andrea Angeli, Rossella Angius, Stefano
Alcaro, Claudiu T. Supuran & Simona Distinto (2021) Selective inhibition of carbonic anhydrase
IX and XII by coumarin and psoralen derivatives, Journal of Enzyme Inhibition and Medicinal
Chemistry, 36:1, 685-692, DOI: 10.1080/14756366.2021.1887171
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 685–692
https://doi.org/10.1080/14756366.2021.1887171
BRIEF REPORT
Selective inhibition of carbonic anhydrase IX and XII by coumarin and psoralen
derivatives
Rita Meleddua , Serenella Deplanoa, Elias Maccionia , Francesco Ortusob , Filippo Cottigliaa, Daniela Seccia,
Alessia Onalia, Erica Sannaa, Andrea Angelic , Rossella Angiusd, Stefano Alcarob , Claudiu T. Supuranc
Simona Distintoa
and
a
b
ꢀ
Department of Life and Environmental Sciences, University of Cagliari, Monserrato, Italy; Dipartimento di Scienze della Salute, Universita
c
ꢀ
Magna Graecia di Catanzaro, Catanzaro, Italy; Dipartimento NEUROFARBA, Sezione di Scienze Farmaceutiche, Universita degli Studi di Firenze,
Sesto Fiorentino, Italy; Laboratorio NMR e Tecnologie Bioanalitiche, Sardegna Ricerche, Pula, Italy
d
ABSTRACT
ARTICLE HISTORY
Received 8 January 2021
Revised 27 January 2021
Accepted 1 February 2021
A small library of coumarin and their psoralen analogues EMAC10157a-b-d-g and EMAC10160a-b-d-g
has been designed and synthesised to investigate the effect of structural modifications on their inhibition
ability and selectivity profile towards carbonic anhydrase isoforms I, II, IX, and XII. None of the new com-
pounds exhibited activity towards hCA I and II isozymes. Conversely, both coumarin and psoralen deriva-
tives were active against tumour associated isoforms IX and XII in the low micromolar or nanomolar range
of concentration. These data further corroborate our previous findings on analogous derivatives, confirm-
ing that both coumarins and psoralens are interesting scaffolds for the design of isozyme selective
hCA inhibitors.
KEYWORDS
hCAi; tumour; coumarin;
psoralen; docking
Introduction
druggable targets. In particular, coumarin derivatives have shown
a variety of biological activities such as CK2 inhibitors39, EGFR40,
PI3K-AKT-mTOR signalling inhibitors41–43. Furthermore, their anti-
cancer potential, tumour targets, diverse mechanisms of action as
well as their advantages and disadvantages have been recently
reviewed44. In continuation with our previous work and prompted
by these considerations, to further explore the influences of struc-
tural modifications on the coumarin and psoralene core on the
activity and selectivity towards membrane-bound hCA isozyme,
we have designed and synthesised a small library of methyl-2-[4-
methyl-2-oxo-7–(2-oxo-2-arylethoxy)-8-propylchromen-3-yl]acetate
The development of cancer is a complex multifactorial process,
involving many cellular adaptations and signal transduction path-
ways1–4. In solid tumours, cancer cells must survive in a low oxy-
gen concentration environment, due to the rapid cellular
proliferation and to the impossibility to promptly supply an
adequate vascularisation5,6. Indeed, many pathways are involved
in the hypoxia survival mechanism7–11 and they all concur in help-
ing cancer cells to escape from apoptosis. These pathways have
been investigated in depth and might be inhibited by relatively
new classes of anticancer drugs, to contrast the angiogenesis pro-
cess, such as VEGFR (sunitinib, sorafenib), VEGF directed monoclo-
nal antibodies (bevacizumab), and mTOR (everolimus,
temsirolimus) inhibitors12–17. In this contest, the key role of human
carbonic anhydrases, a class of metalloproteins that catalyse the
reversible conversion of carbon dioxide to bicarbonate and pro-
tons18–20, has been outlined. In particular, two membrane iso-
forms, namely hCA IX and XII, are mainly involved in cancer
proliferation and invasion21–31. Not surprisingly, several inhibitors
of membrane bounded hCA isozymes, with diverse structures and
mechanisms of action, have been designed and investigated so
far. In this respect, both synthetic and natural coumarin deriva-
tives have already demonstrated to possess high selectivity and
activity towards specific hCA isozymes32–36. Moreover, their inter-
action and binding mode on this class of metalloenzymes have
been investigated in depth37,38. Besides, it should be considered
and
2–(5-methyl-7-oxo-3-aryl-9-propyl-7H-furo[3,2-g]chromen-6-
yl)acetic acid derivatives were a propyl group in the position 8 or
9 has been introduced compared to the previously synthesised
derivatives.
Methods
Materials and apparatus
Starting materials and reagents were obtained from commercial
suppliers and were used without purification. All melting points
were determined on a Stuart SMP11 melting points apparatus and
are uncorrected. Melting points, the yield of reactions, and analyt-
ical data of derivatives EMAC10157a-b-d-g and EMAC10160a-b-
d-g are reported in Table 1.
1H-NMR and 13C-NMR spectra (Table 2) were registered on a
that coumarins have been reported to interact with several cancer Bruker AMX 400 MHz (chemical shifts in d values) operating at
ꢀ
CONTACT Claudiu T. Supuran
Schiff 6, Sesto Fiorentino 50019, Italy; Elias Maccioni
claudiu.supuran@unifi.it
Dipartimento NEUROFARBA, Sezione di Scienze Farmaceutiche, Universita degli Studi di Firenze, Via U.
maccione@unica.it Department of Life and Environmental Sciences, University of Cagliari, A Building-
Cittadella Universitaria, s.p 8 km 0.7, Monserrato 09042, Italy
Supplemental data for this article can be accessed here.
ß 2021 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.

686
R. MELEDDU ET AL.
Table 1. Chemical, analytical, and physical data of derivatives EMAC10157 a-b-d-g and EMAC10160 a-b-d-g.
C, %; H, %
Compound
R
Calc.
Found
M.P., ꢀC
Yield, %a
Aspect
EMAC10157a
EMAC10157b
EMAC10157d
EMAC10157g
EMAC10160a
EMAC10160b
EMAC10160d
EMAC10160g
4-CH3
4-OCH3
4-F
4-C6H5
4-CH3
4-OCH3
4-F
C, 71.07; H, 6.20
C, 68.48; H, 5.98
C, 67.60; H, 5.44
C, 74.36; H, 5.82
C, 74.24; H, 5.98
C, 71.41; H, 5.75
C, 70.58; H, 5.18
C, 77.24; H, 5.62
C, 71.10; H, 6.19
C, 68.74; H, 5.97
C, 67.79; H, 5.45
C, 74.55; H, 5.45
C, 74.19; H, 5.99
C, 71.68, H, 5.76
C, 70.30; H, 5.16
C, 77.52; H, 5.60
132–133
127–130
164–166
138–140
226–229
202–205
135–139
248–250
80
89
90
71
98
97
92
97
White crystals
Pale brown crystals
White crystals
White crystals
Grey crystals
Green crystals
Grey crystal
4-C6H5
Pale brown crystal
aYields are referred to the last step of the synthetic pathway.
Table 2. 1H NMR and 13C NMR data of derivatives EMAC10157a-b-d-g and EMAC10160a-b-d-g.
Compound
1H NMR and 13C NMR d (ppm)
EMAC10157a
1H NMR (400 MHz, DMSO) d 7.93 (d, 2H, J ¼ 8), 7.61 (d, 1H, J ¼ 9.2), 7.39 (d, 2H, J ¼ 8), 7.00 (d, 1H, J ¼ 8.8), 5.74 (s, 2H), 3.68 (s, 2H),
3.62 (s, 3H), 2.83–2.79 (m, 2H), 2.41 (s, 3H), 2.37 (s, 3H), 1.64–1.55 (m, 2H), 0.95–0.91 (t, 3H).
13C NMR (100 MHz, DMSO) d 193.70, 170.64, 160.88, 158.24, 150.61, 149.69, 144.37, 131.78, 129.34 (2C), 127.96 (2C), 123.92, 116.97,
115.87, 113.65, 108.77, 70.55, 51.82, 32.37, 24.34, 21.70, 21.22, 15.10, 13.96.
EMAC10157b
EMAC10157d
EMAC10157g
1H NMR (400 MHz, DMSO) d 8.01 (d, 2H, J ¼ 8.8), 7.61 (d, 1H, J ¼ 8), 7.09 (d, 2H, J ¼ 8), 6.98 (d, 1H, J ¼ 8), 5.70 (s, 2H), 3.86 (s, 3H), 3.68
(s, 2H), 3.62, (s, 3H), 2.82–2.79 (m, 2H), 2.36 (s, 3H), 1.64–1.55 (m, 2H), 0.95–0.91 (t, 3H)
13C NMR (100 MHz, DMSO) d 192.48, 170.64, 163.61, 160.89, 158.29, 150.60, 149.69, 130.23 (2C), 127.15 123.90, 116.96, 115.84,
114.05 (2C), 113.61, 108.76, 70.35, 55.60, 51.81, 32.36, 24.34, 21.70, 15.09, 13.96.
1H NMR (400 MHz, DMSO) d 8.13–8.10 (m, 2H), 7.62 (d, 1H, J ¼ 9.2), 7.45–7.40 (m, 2H), 7.03 (d, 1H, J ¼ 9.2), 5.77 (s, 2H), 3.68 (s, 2H),
3.62 (s, 3H), 2.83–2.79 (m, 2H), 2.37 (s, 3H), 1.64–1.55 (m, 2H), 0.95–0.91 (t, 3H).
13C NMR (100 MHz, DMSO) d 192.90, 170.65, 166.59, 164.08, 160.89, 158.15, 150.60, 149.70, 131.01 (2C), 123.94, 116.98, 116.01,
115.91 (2C), 113.70, 108.79, 70.55, 51.82, 32.36, 24.33, 21.70, 15.10, 13.95.
1H NMR (400 MHz, DMSO) d 8.12 (d, 2H, J ¼ 8.4), 7.88 (d, 2H, J ¼ 8.4), 7.78 (d, 2H, J ¼ 7.2), 7.65–7.61 (m, 1H), 7.53 (t, 2H, J ¼ 7.2), 7.45
(t, 1H, J ¼ 7.2), 7.06–7.02 (m, 1H), 5.82 (s, 2H), 3.68 (s, 2H), 3.62 (s, 3H), 2.85–2.81 (m, 2H),2.37 (s, 3H), 1.64–1.58 (m, 2H), 0.96–0.92
(t, 3H).
13C NMR (100 MHz, DMSO) d 193.78, 170.63, 160.88, 158.22, 150.62, 149.68, 145.17, 138.77, 133.07, 129.10 (2C), 128.61 (2C), 127.02
(2C), 126.96 (2C), 123.94, 116.99, 115.90, 113.68, 108.81, 73.94, 70.69, 51.81, 32.37, 24.37, 21.73, 15.11, 13.98.
1H NMR (400 MHz, DMSO) d 12.47 (bs, 1H), 8.41 (s, 1H), 8.03 (s, 1H), 7.69 (d, 2H, J ¼ 8), 7.34 (d, 2H, J ¼ 7.6),3.65 (s, 2H), 3.05–3.01 (m,
2H), 2.51 (s, 3H), 2.38 (s, 3H), 1.78–1.69 (m, 2H), 0.97–0.94 (t, 3H).
13C NMR (100 MHz, DMSO) d 171.49, 160.72, 154.93, 149.65, 147.36, 143.62, 137.11, 129.72 (2C), 127.82, 127.11 (2C), 122.21, 121.45,
117.83, 116.71, 114.20, 112.93, 32.90, 24.85, 21.93, 20.79, 15.64, 13.83.
EMAC10160a
EMAC10160b
EMAC10160d
EMAC10160g
1H NMR (400 MHz, DMSO) d 12.47 (bs, 1H), 8.37 (s, 1H), 8.01 (s,1H), 7.73 (d, 2H, J ¼ 8), 7.10 (d, 2H, J ¼ 8.8), 3.82 (s, 3H), 3.65 (s, 2H),
3.04–3.01 (m, 2H), 2.51 (s, 3H), 1.71–1.68 (m, 2H), 0.97–0.94 (t, 3H).
13C NMR (100 MHz, DMSO) d 171.50, 160.73, 158.92, 154.89, 149.66, 147.34, 143.17, 128.47 (2C), 123.00, 122.31, 121.18, 117.79,
116.66, 114.62 (2C), 114.16, 112.89, 55.17, 32.90, 24.84, 21.92, 15.65, 13.82.
1H NMR (400 MHz, DMSO) d 12.47 (bs, 1H), 8.46 (s, 1H), 8.03 (s, 1H), 7.87–7.85 (m, 2H), 7.39–7.35 (m, 2H), 3.65 (s, 2H), 3.05–3.01 (m,
2H), 2.52 (s, 3H), 1.78–1.69 (m, 2H), 0.97–0.94 (t, 3H).
13C NMR (100 MHz, DMSO) d 171.48, 162.91, 160.68, 160.51, 154.88, 149.67, 147.42, 129.23, 129.23, 127.21, 121.97, 120.59, 117.90,
116.81, 116.16, 115.94, 114.16, 112.98, 32.90, 27.45, 24.84, 21.92, 15.67, 13.82.
1H NMR (400 MHz, DMSO) d 12.49 (bs, 1H), 8.54 (s, 1H), 8.13 (s, 1H), 7.92 (d, 2H, J ¼ 8), 7.84 (d, 2H, J ¼ 8.4), 7.75 (d, 2H, J ¼ 7.2), 7.51
(t, 2H, J ¼ 7.2), 7.40 (t, 1H, J ¼ 7.2), 3.67 (s, 2H), 3.07–3.04 (m, 2H), 2.55 (s, 3H), 1.80–1.71 (m, 2H), 0.99–0.95 (t, 3H).
13C NMR (100 MHz, DMSO) d 171.49, 160.71, 155.01, 149.69, 147.44, 144.21, 139.56, 139.42, 129.93, 129.01 (2C), 127.73 (2C), 127.59,
127.37 (2C), 126.54 (2C), 122.04, 121.13, 117.92, 116.84, 114.35, 113,03, 32.92, 24.88, 21.94, 15.71, 13.85.
400 MHz and 100 MHz, respectively. All samples were measured in homogeneous sticky solid was obtained which was dissolved in
DMSO. Chemical shifts are reported referenced to the solvent in methanol and poured into ice water. The mixture was stirred until
which they were measured. Coupling constants J are expressed in ice melting and then filtered off to obtain a light yellow solid. The
hertz (Hz). Elemental analyses were obtained on a Perkin–Elmer crude product was washed with ethyl ether giving a white pow-
240 B microanalyser. Analytical data of the synthesised com- der that was crystallised from methanol.
pounds are in agreement within 0.4% of the theoretical values.
TLC chromatography was performed using silica gel plates (Merck
F 254), spots were visualised by UV light.
Synthesis of methyl-2-[4-methyl-2-oxo-7–(2-oxo-2-arylethoxyi)-8-
propylchromen-3-yl]acetate (EMAC 10157 a-b-d-g)
A hot solution of methyl 2–(7-hydroxy-4-methyl-2-oxo-8-propyl-
2H-chromen-3-yl)acetate (1 eq.) in dry acetone was treated with
K2CO3 (2.5 eq.), stirred vigorously, and treated with the appropri-
General procedure for the synthesis of compound EMAC10157
a-b-d-g and EMAC10160 a-b-d-g
Synthesis of methyl 2–(7-hydroxy-4-methyl-2-oxo-8-propyl-2H-
ate a-haloketone (1 eq.). The reaction mixture was heated to reflux
and stirred for 1–5 h (course of the reaction monitored by TLC
chromen-3-yl)acetate
A mixture of propylresorcinol (1 eq.), dimethylacetylsuccinate (1 using ethyl acetate/n-hexane 5:1). When the reaction was com-
eq.) and sulphuric acid 98% (2.8 eq.) was vigorously stirred at pleted, it was cooled at 0 ꢀC and the solution acidified with HCl
room temperature. The progression of the reaction was monitored conc. The resulting precipitate was filtered off and crystallised
by TLC, using ethyl acetate/n-hexane 2:1. After 30 min
a
if necessary.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
687
Synthesis of 2–(5-methyl-7-oxo-3-aryl-9-propyl-7H-furo[3,2-g]chro- Post docking protocol
To better consider, the induced fit phenomena, the most energy
favoured generated complexes were fully optimised with the
OPLS2005 force field in GB/SA implicit water. The optimisation
process was performed setting 10,000 steps interactions up to the
derivative convergence criterion equal to 0.05 kJ/(molÅ). The
resulting complexes were considered for the binding modes
graphical analysis with Pymol57 and Maestro.
men-6-yl)acetic acid (EMAC10160 a-b-d-g)
A solution or suspension of coumarin (1 eq., EMAC10157 a-b-d-g)
in propan-2-ol was treated with NaOH solution (4 eq., 1 N). The
reaction mixture was heated for 3–4 h, obtaining a dark solution.
The solution was cooled to room temperature and poured into ice
water. Concentrated HCl was added to the solution, obtaining a
suspension that was filtered and crystallised.
Analytical and spectral data of compounds EMAC10157 a-b-d-
g and EMAC10160 a-b-d-g are reported in Tables 1 and 2. 1H-
NMR and 13C-NMR spectra are reported in Supplementary material
(Figures S1–S16).
Results and discussion
As a continuation of our ongoing research in the field of carbonic
anhydrase and anticancer agents58–62 we have synthesised a new
series of methyl-2-[4-methyl-2-oxo-7–(2-oxo-2-arylethoxyi)-8-propyl-
chromen-3-yl]acetate and 2–(5-methyl-7-oxo-3-aryl-9-propyl-7H-
furo[3,2-g]chromen-6-yl)acetic acid to evaluate their activity and
selectivity towards hCA isozymes and to gain information on their
structure-activity relationships. The compounds are reported as
EMAC10157 a-b-d-g and EMAC10160 a-b-d-g and their struc-
tures are illustrated in Figure 1.
All the synthesised compounds show a propyl substituent in
the position 8 or 9 of the coumarin and psoralen nucleus, respect-
ively. As in previously reported derivatives, a methylene carboxylic
group was placed in position 3 of the chromene which may lead
to the formation of a bidentate chelator of the Zn2þ ion in the
catalytic pocket, due to the hCA esterase activity32,34,63 on the
dihydropyranone ring, as illustrated in Figure 2.
The synthesis of the new derivatives is reported in Scheme 1. It
consists of the H2SO4 mediated Pechman condensation of dime-
thylacetylsuccinate and 2-propylresorcinol at room temperature in
solvent-free conditions, followed by a Williamson reaction of the
phenolic group in the position 7 of the chromene ring with the
appropriate a-haloketone in dry acetone and K2CO3, to generate
the asymmetric ether which will lead to the furocoumarin forma-
tion by intramolecular electrophilic substitution, mediated by
refluxing in NaOH 1 N water solution.
Biological activity
Carbonic anhydrase inhibition assay
The CA catalysed CO2 hydration/inhibition was measured by using
a stopped-flow instrument (Applied Photophysics, Oxford, U.K.) as
the method described earlier45. Initial rates of the CA-catalysed
CO2 hydration reaction were followed for 10–100 s. The CO2 con-
centrations ranged from 1.7 to 17 mM for the determination of
the inhibition constants. For each inhibitor, at least six traces of
the initial 5–10% of the reaction were used for assessing the initial
velocity. The uncatalyzed rates were subtracted from the total
observed rates. Stock solutions of inhibitors (10 mM) and dilutions
up to 0.01 nM were prepared in distilled-deionized water. Inhibitor
and enzyme solutions were preincubated together for 15 min at
room temperature prior to assay, in order to allow for the forma-
tion 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 deter-
minations46–48. hCA I, hCA II, hCA IX (catalytic domain), and hCA
XII (catalytic domain) were recombinant proteins produced in-
house using our standardised protocol and their concentration in
the assay system was in the range of 3–10 nM (and even lower for
highly effective, sub-nanomolar inhibitors)46–48
.
EMAC10157 a-b-d-g-and EMAC10160 a-b-d-g were character-
ised employing analytical and spectroscopic methods and the
results are summarised in Tables 1 and 2. Compounds were then
submitted to enzymatic evaluation towards hCA I, II, IX, and XII.
The results are reported in Table 3. Interestingly, none of the new
EMAC derivatives exhibited any inhibition activity towards hCA I
and II isozymes. On the contrary, all of them are submicromolar
inhibitors of the hCA membrane isoforms IX and XII. This
behaviour is in agreement with our previous findings and
with the generally observed selectivity profile of coumarin
Molecular modelling
Ligand preparation
The ligands were built using the Maestro GUI49. The most stable
conformation has been determined by molecular mechanics con-
formational analysis performed by Macromodel software version
9.250. using the Merck Molecular Force Fields (MMFFs)51 and GB/
SA water implicit solvation model52, Polak–Ribier Conjugate
Gradient (PRCG) method, 5000 iterations, and a convergence cri-
terion of 0.05 kcal/(mol Å). All the other parameters were left
as default.
derivatives32,33,35,37,38,63
.
Within the tested compounds, EMAC10157 series was the
most active towards the hCA IX and XII isoforms. Therefore, its
mechanism of action was investigated in more detail by docking
Protein preparation
The coordinates for hCA isoforms enzymes were taken from the
RCSB Protein Data Bank53 (PDB codes 3k3454, for isoform II; 5fl455,
for isoform IX and 5msa, for isoform XII). The proteins were pre-
pared by using the Maestro Protein Preparation Wizard49. Original
water molecules were removed.
R
CH3
O
CH3
O
R
OH
O
O
O
CH3
O
O
O
O
O
Docking protocol
EMAC10157 a-b-d-g
EMAC10160 a-b-d-g
Molecular docking studies were performed using QMPL workflow
protocol56. Grids were defined around the refined structure by
centring on crystallised ligands. The other settings were left
as default.
R = a) 4-CH3; b) 4-OCH3; d) 4-F; g) 4-C6H5
Figure 1. Newly synthesised coumarin and psoralen derivatives.

688
R. MELEDDU ET AL.
Figure 2. (a) Esterase activity of carbonic anhydrase on compound EMAC10157g34. (b, c) Oral bioavailability radar profile.
Scheme 1. Synthetic pathway to compounds EMAC10157 a-b-d-g and EMAC10160 a-b-d-g. Reagents and conditions: (i) dimethylacetylsuccinate, H2SO4 98% R.T.; (ii)
a-halogeno arylketone, dry acetone, K2CO3, reflux; (iii) NaOH 1 N, reflux.
experiments followed by energy minimisation of the the chromene moiety could reach the bottom of the catalytic cav-
obtained complexes.
ity of hCA II, IX and XII and, therefore, be hydrolysed by the Zn2þ
In particular, considering that an interesting hCA esterase activated water molecule, which acts as a very potent nucleophile
mediated inhibition mechanism was recently reported for couma- (Figure 2(a)).
rin derivatives32,33,63, we firstly investigated this aspect. Hence, the
Confirming the selectivity profile already observed in previously
most promising compound, EMAC10157g, was submitted to investigated analogues docking experiment in hCA II enzyme
docking experiments to evaluate if the dihydropyranone ring of showed that the EMAC10157g tail is too bulky to access hCA II

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
689
Table 3. Inhibition data towards hCA I, II, IX, and XII of compounds EMAC10157a-b-d-g and EMAC10160 a-b-d-g.
R
CH3
CH3
R
O
O
O
OH
CH3
O
O
O
O
O
O
O
CH3
CH3
EMAC10157 a-b-d-g
EMAC10160 a-b-d-g
ꢁ
Ki (nM)
Compound
R
hCA I
hCA II
hCA IX
hCA XII
EMAC10157a
EMAC10157b
EMAC10157d
EMAC10157g
EMAC10160a
EMAC10160b
EMAC10160d
EMAC10160g
AAZ
4-CH3
4-OCH3
4-F
4-C6H5
4-CH3
4-OCH3
4-F
>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
12
247.7
352.7
239.5
135.2
467.3
489.3
379.7
397.7
25
350.3
324.2
257.1
283.1
758.1
859.4
460.0
550.0
5.7
4-C6H5
ꢁ
Standard error (from three different assays) were in the range of plus/minus 10% of the reported values.
Figure 3. 3D representation of the putative binding mode obtained by docking experiments. (a) hCA-IX – EMAC10157g, (b) hCA-IX – EMAC10157g-openE, and (c)
hCA-IX – EMAC10157g-openZ and the relative 2D representation of the complexes stabilising interactions with the binding site residues represented with different col-
our depending on their chemical-physical properties: green, hydrophobic; cyan, polar; violet, positive; red, negative charged residues; grey, metal atoms. Instead,
magenta arrows indicate the formation of a hydrogen bond between protein and ligand, while grey lines indicate the interaction with the complexed ion.
cavity, while, in isoforms IX and XII, the dihydropyranone portion the putative binding mode of the hydrolysed forms in hCA IX and
of EMAC10157g was able to bind close enough to the Zn2þ ion hCA XII (Figures 3(b,c) and 4(b,c).
(Figures 2(a) and 3(a)) cofactor to undergo the esterase reaction.
The predicted affinity of the open compounds was estimated
Hence, the two open cinnamic acid derivative (Figure 2(a)) con- to be better than that of the closed ones. What seems important
figurations (E/Z) were subjected to docking experiments to predict is the Zn2þ chelation and the interactions between the newly

690
R. MELEDDU ET AL.
Figure 4. 3D representation of the putative binding mode obtained by docking experiments. (a) hCA-XII – EMAC10157g, (b) hCA-XII – EMAC10157g-openE, and
(c) CA-XII- EMAC10157g-openZ and the relative 2D representation of the complexes stabilising interactions with the binding site residues.
formed carboxylate moiety and the methyl ester with the sur- prompted us to further investigate this scaffold to optimise both
rounding residues in the catalytic site.
the activity and the isozyme selectivity.
The Y shape of the compound, with propyl and -biphenyl-
2oxoethoxy moiety being the arms, helps to maintain the selectiv-
ity but, ultimately, decreases the activity, compared to previously
investigated psoralens32.
Acknowledgements
The authors wish to acknowledge the “Ufficio Valorizzazione dei
Risultati della Ricerca” of Sardegna Ricerche Technological Park,
Pula (CA) – Italy. The authors also acknowledge the PRIN 2017
research project “Novel anticancer agents endowed with multi-tar-
geting mechanism of action” (201744BN5T).
Biphenyl moiety accommodation is well tolerated due to the
wide cavity exposed to the solvent, as shown by other hCA inhibi-
tors with bulky tails. In fact, the interactions between this group
with adjacent hydrophobic residues stabilise the complexes.
In summary, the computational methods helped to rationalise
the good activity of the investigated compound towards the hCA
IX and hCA XII isoform and to suggest a reasonable mechanism of
action that could be further investigated to be confirmed. If veri-
fied, this compound can be considered as a new prodrug candi-
date with acceptable oral bioavailability properties (Figure 2(b)),
and with good drug-like properties. Nevertheless, considering the
encouraging predicted ADME properties64 and the activity data,
we are further optimising this scaffold.
Disclosure statement
No potential conflict of interest was reported by the author(s).
ORCID
Rita Meleddu
http://orcid.org/0000-0003-1629-7454
http://orcid.org/0000-0003-2175-2802
http://orcid.org/0000-0001-6235-8161
http://orcid.org/0000-0002-1470-7192
http://orcid.org/0000-0002-0437-358X
Elias Maccioni
Francesco Ortuso
Andrea Angeli
Stefano Alcaro
Conclusions
We have designed and synthesised a series of methyl-2-[4-methyl-
2-oxo-7–(2-oxo-2-arylethoxyi)-8-propylchromen-3-yl]acetate and 2–(5-
methyl-7-oxo-3-aryl-9-propyl-7H-furo[3,2-g]chromen-6-yl)acetic acid,
and evaluate their activity on hCA I, II, IX, and XII isozymes. As a
confirmation of the literature reported chromene derivatives
selectivity profile, none of the investigated compounds was able
to inhibit the off-target I and II isoforms of hCA. On the contrary,
they all inhibit the membrane isozymes hCA IX and XII and further
corroborate the reported data on chromene derivatives.
Considering the acceptable ADME prediction profile and the high
potential of coumarin derivatives, not only as hCA selective inhibi-
tors but also as potential multitarget anticancer agents, these data
Claudiu T. Supuran
Simona Distinto
http://orcid.org/0000-0003-4262-0323
http://orcid.org/0000-0003-1620-6225
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