Novel coumarins and benzocoumarins acting as isoform-selective inhibitors against the tumor-associated carbonic anhydrase IX

Article abstract of DOI:10.3109/14756366.2013.777334

A series of coumarins and benzocoumarins incorporating methyl and hydroxyl moieties in the heterocyclic ring were investigated for the inhibition of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). These coumarins were very weak or ineffective as inhibitors of the house-keeping, offtarget isoforms CA I and II, but showed effective, submicromolar inhibition of the transmembrane, tumor-associated isoforms CA IX and to a slightly less extent, CA XII. The nature and position of the groups substituting the coumarin ring influenced CA inhibitory properties. 4-Methyl-5,7-dihydroydroxycoumarin showed KIs >200 μM against CA I and II, of 0.19 μM against CA IX and of 6.4 μM against CA XII, being thus a selective, efficient inhibitor for the tumor-associated over cytosolic CA isoforms. These compounds are interesting leads for designing isoform-selective enzyme inhibitors.

Full text of DOI:10.3109/14756366.2013.777334

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J Enzyme Inhib Med Chem, Early Online: 1–5
2013 Informa UK Ltd. DOI: 10.3109/14756366.2013.777334
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SHORT COMMUNICATION
Novel coumarins and benzocoumarins acting as isoform-selective
inhibitors against the tumor-associated carbonic anhydrase IX
Aditi Sharma1, Meena Tiwari1, and Claudiu T. Supuran2
1
2
Department of Pharmacy, S.G.S.I.T.S, Indore, Madhya Pradesh, India and NEUROFARBA Department, Universita degli Studi di Firenze, Sezione di
`
Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
Abstract
Keywords
A series of coumarins and benzocoumarins incorporating methyl and hydroxyl moieties in the
heterocyclic ring were investigated for the inhibition of the zinc enzyme carbonic anhydrase
(CA, EC 4.2.1.1). These coumarins were very weak or ineffective as inhibitors of the house-
keeping, offtarget isoforms CA I and II, but showed effective, submicromolar inhibition of
the transmembrane, tumor-associated isoforms CA IX and to a slightly less extent, CA XII.
The nature and position of the groups substituting the coumarin ring influenced CA inhibitory
properties. 4-Methyl-5,7-dihydroydroxycoumarin showed KIs 4200 mM against CA I and II,
of 0.19 mM against CA IX and of 6.4 mM against CA XII, being thus a selective, efficient inhibitor
for the tumor-associated over cytosolic CA isoforms. These compounds are interesting leads
for designing isoform-selective enzyme inhibitors.
Benzocoumarin, carbonic anhydrase,
coumarin, hydroxycoumarin, selective
enzyme inhibitor, tumor-associated
isoforms IX and XII
History
Received 17 January 2013
Accepted 31 January 2013
Published online 14 March 2013
Introduction
substitution patterns, incorporating moieties of diverse
chemical nature, in different numbers of such moieties and
in diverse positions of the coumarin ring.
Coumarins are a class of widely spread natural compounds
which was only recently revealed to inhibit the zinc enzyme
carbonic anhydrase CA (EC 4.2.1.11)1–3. CAs are ubiquitous
enzymes in organisms throughout the tree of life, with several
genetically distinct families encoding them in prokaryotes and
eukaryotes4–11. CAs are inhibited by metal complexing anions,
sulfonamides and their isosteres (sulfamates, sulfamides, etc.),
phenols and polyamines, which bind either to the metal ion
within the enzyme active site or are anchored to the water
molecule coordinated to it11. However, the coumarin inhibition
mechanism of the a-CAs is totally different and was only recently
deciphered1,2. Coumarin derivatives were recently shown to
constitute a totally new class and type of CA inhibitors (CAIs),
being ‘‘prodrugs’’ which are hydrolyzed within the CA active
site with formation of 2-hydroxycinnamic acids.
A natural product coumarin, 6-(1S-hydroxy-3-methylbutyl)-
7-methoxy-2H-chromen-2-one, isolated from the Australian plant
Leionema ellipticum, was the first compound shown to possess
significant CA inhibitory activity1. By means of X-ray crystal-
lography of it’s adduct with the human (h) isoform hCA II, it was
evidenced the presence of a substituted 2-hydroxy-cinnamic acid
in the enzyme active site, which is the hydrolysis product
(mediated by the CA) of the original coumarin1. The same
situation has been thereafter observed for the simple, unsubsti-
tuted coumarin2. The 2-hydroxy-cinnamic acids formed from the
original coumarins occluded the entrance to the enzyme active
site, a mechanism never evidenced before for CA inhibition2,11
.
This region of the CA active site is on the other hand the most
variable one among the 16 CA isoforms present in mammals, and
this explains the observations that many coumarin derivatives
show a high selectivity ratio for inhibiting various CA isoforms,
The coumarins represent an interesting class of CAIs due to
several reasons:
(i) The inhibition mechanism is different from those of other
CAIs, and due to their binding at the entrance of the enzyme
active site, where there is a rather large variability in the
3D structure and amino acid sequence between the various
isoforms, may lead to compounds with isoform-selective
behavior1–3
(ii) Coumarins are widespread natural products and are also
easily prepared synthetically with large range of
many of which have pharmacologic applications1–3
.
In fact recently, some glycosyl-substituted coumarins with
potent inhibitory activity and selectivity for the tumor-associated
isozyme CA IX and XII, were shown to strongly inhibit the
growth of primary tumors and metastases overexpressing these
enzymes, in an animal model of breast cancer3. For this reason,
exploring novel coumarin derivatives as CAIs, or compounds
known in the literature but not yet investigated for this activity, is
of interest in the design of isoform-selective pharmacological
agents of this type. Indeed, CAIs have a range of applications as
.
a
Address for correspondence: Claudiu T. Supuran, NEUROFARBA
`
antiglaucoma, antiobesity, antiepileptic or diuretic agents12–26
.
Department, Universita degli Studi di Firenze, Sezione di Scienze
Furthermore, inhibition of CAs from various bacteria or
fungi may also lead to the development of antiinfectives
based on CAIs27,28. Here, we report the synthesis of several
Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence,
Italy. Tel: +39-055-4573005. Fax: +39-055-4573385. E-mail: claudiu.
supuran@unifi.it

2
A. Sharma et al.
J Enzyme Inhib Med Chem, Early Online: 1–5
coumarin/benzocoumarin derivatives and their inhibition against Yield 72%; Rf: 0.283 (ethyl acetate/hexane 3:7); m.p.: 260–262 ꢀC;
medicinally important CA isoforms, such as CA I, II, IX and XII. 1HNMR (400 MHz, DMSO): ꢀ ppm 2.339 (s, 3H, Me), 6.108 (s,
C¼CH), 6.783–7.090 (m, 2H, ArH), 9.644 (s, 1H, ArOH); IR
(KBr) 3412, 3233, 2865, 1663, 1609, 1597, 1236 cmꢁ1; EIMS:
Materials and methods
m/z: 192 (Mþ).
Chemistry
The Pechmann condensation is the most widely used method for
coumarin synthesis, as it uses simple starting materials and
gives good yields for variously substituted coumarins29,30. The
Pechmann condensation allows the synthesis of coumarins by
reaction of phenols with b-keto esters. The reaction was carried
out by addition of TiCl4 to the mixture of the reagents, with
stirring as exemplified below for the preparation of coumarins/
benzocoumarins 1–5.
Synthesis of 4-methyl-2H-benzo[h]chromen-2-one 4
a-Naphthol (10 mmol) and ethyl acetoacetate (20 mmol) were
mixed thoroughly in round bottom flask and titanium (IV)
chloride (10 mmol) was added to the mixture with constant
stirring at room temperature. Small amount of ethanol was added,
to facilitate the stirring of mixture. The reaction was completed
in 3–4 h. The thin layer chromatography was performed to check
the completion of reactions. On the completion of reactions as
indicated by TLC, solvent was evaporated and reaction mixture
was poured onto crushed ice and the resulting crude product was
Synthesis of 7-hydroxy-4-methyl-2H-2-one 1
Resorcinol (10 mmol, 1.10 g) and ethyl acetoacetate (20 mmol, filtered off and recystallized from ethanol–water mixture (1:1)
2.5 ml) were mixed thoroughly in round bottom flask and titanium and purified by column chromatography. Yield 60%; Rf: 0.352
(IV) chloride (10 mmol) was added to the mixture with constant (ethyl acetate/hexane 3:7); m.p.: 154–156 ꢀC; 1HNMR (400 MHz,
stirring at room temperature. Small amount of ethanol was added, DMSO): ꢀ ppm 1.827 (s, 3H, Me), 6.464 (s, 1H, C¼CH), 7.694–
to facilitate the stirring of mixture. The reaction was completed in 8.326 (m, 6H, ArH)); IR (KBr) 3059, 2920, 1707, 1609,
5–6 h. The thin layer chromatography was performed to check the 1273 cmꢁ1.; EIMS: m/z: 210 (Mþ).
completion of reactions. On the completion of reactions as
indicated by TLC, solvent was evaporated and reaction mixture
was poured onto crushed ice and the resulting crude product was
filtered off and recrystallized from ethanol–water mixture (1:1)
and then, purified by column chromatography. Yield 78%; Rf:
0.428 (ethyl acetate/hexane 3:7); m.p.: 188–190 ꢀC; 1HNMR
chloride (10 mmol) was added to the mixture with constant
Synthesis of 4-methyl-2H-benzo[g]chromen-2-one 5
b-Naphthol (10 mmol) and ethyl acetoacetate (20 mmol) were
mixed thoroughly in round bottom flask and titanium (IV)
stirring at room temperature. Small amount of ethanol was added,
to facilitate the stirring of mixture. The reaction was completed in
(400 MHz, DMSO): ꢀ ppm 2.257 (s, 3H, Me), 6.001(s, 1H,
C¼CH), 6.626–7.439 (m, 3H, ArH), 10.463 (s, 1H, ArOH); IR
(KBr) 3491, 3123, 2949, 1672, 1672, 1601, 1272 cmꢁ1. EIMS:
3–4 h. The thin layer chromatography was performed to check the
completion of reactions. On the completion of reactions as
indicated by TLC, solvent was evaporated and reaction mixture
m/z: 176 (Mþ).
was poured onto crushed ice and the resulting crude product
Synthesis of 5,7-dihydroxy-4-methyl-2H-chromen-2-one 2
was filtered off and recystallized from ethanol–water mixture
Phloroglucinol (10 mmol, 1.26 g) and ethyl acetoacetate (1:1) and purified by column chromatography. Yield 81%; Rf:
(20 mmol, 2.5 ml) were mixed thoroughly in round bottom flask 0.403 (ethyl acetate/hexane 3:7); m.p.: 150–150 ꢀC; 1HNMR
and titanium (IV) chloride (10 mmol) was added to the mixture (400 MHz, DMSO): ꢀ ppm 1.689 (s, 3H, Me), 7.052–7.749
with constant stirring at room temperature. Small amount (m, 6H, ArH); IR (KBr) 3273, 2866, 1674, 1630, 1277 cmꢁ1.;
of ethanol was added, to facilitate the stirring of mixture. The EIMS: m/z: 210 (Mþ).
reaction was completed in 18–20 h. The thin layer chromatog-
raphy was performed to check the completion of reactions.
On the completion of reactions as indicated by TLC, solvent
was evaporated and reaction mixture was poured onto crushed ice
CA inhibition
An applied photophysics stopped-flow instrument has been used
for assaying the CA catalyzed CO2 hydration activity. Phenol red
and the resulting crude product was filtered off and recystallized
(at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM
from ethanol–water mixture (1:1) and purified by column
chromatography. Yield 70%; Rf: 0.269 (ethyl acetate/hexane
3:7); m.p.: 260–262 ꢀC; 1HNMR (400 MHz, DMSO): ꢀ ppm
Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining
constant the ionic strength), following the initial rates of the
2.503 (s, 3H, Me), 5.848(s, 1H, C¼CH), 6.162–6.255 (d, 2H
CA-catalyzed CO2 hydration reaction for a period of 10–100 s.
ArH), 10.297 (s, 1H, ArOH), 10.523 (S, 1H, ArOH); IR
(KBr) 3421, 3124, 2922, 1663, 1618, 1296 cmꢁ1; EIMS: m/z:
The CO2 concentrations ranged from 1.7 to 17 mM for the
determination of the kinetic parameters and inhibition constants31.
For each inhibitor at least six traces of the initial 5–10% of the
192 (Mþ).
reaction have been used for determining the initial velocity.
Synthesis of 7,8-dihydroxy-4-methyl-2H-chromen-2-one 3
The uncatalyzed rates were determined in the same manner
Pyrogallol (benzene-1,2,3-triol, 10 mmol) and ethyl acetoacetate and subtracted from the total observed rates. Stock solutions of
(20 mmol) were mixed thoroughly in round bottom flask and inhibitor (0.1 mM) were prepared in distilled-deionized water and
titanium (IV) chloride (10 mmol) was added to the mixture dilutions up to 0.01 nM were done thereafter with distilled-
with constant stirring at room temperature. Small amount of deionized water. Inhibitor and enzyme solutions were preincu-
ethanol was added, to facilitate the stirring of mixture. The reaction bated together for 15 min to 72 h at room temperature (15 min) or
was completed in 8–9 h. The thin layer chromatography was 4 ꢀC (all other incubation times) prior to assay, in order to
performed to check the completion of reactions. On the completion allow for the formation of the E-I complex or for the eventual
of reactions as indicated by TLC, solvent was evaporated and active site mediated hydrolysis of the inhibitor. Data reported
reaction mixture was poured onto crushed ice and the resulting in Table 1 show the inhibition after 6 h incubation, which led
crude product was filtered off and recystallized from ethanol– to the completion of the in situ hydrolysis of the coumarin
water mixture (1:1) and purified by column chromatography. and formation of the 2-hydroxy-cinnamic acids. The inhibition

DOI: 10.3109/14756366.2013.777334
Novel coumarins and benzocoumarins acting as inhibitors
3
Table 1. hCA I, II, IX and XII inhibition data with coumarins 1–5 by a stopped flow, CO2 hydrase assay31
.
OH
HO
O
O
HO
O
O
HO
O
O
OH
2
3
1
O
O
O
O
5
4
Ki (mM)*
hCA II
Compounds
hCA I
hCA IX
hCA XII
1
2
3
4
5
4.8
8.4
8.7
9.0
8.2
4200
4200
4200
4200
4200
0.45
0.19
0.27
0.23
0.20
4.3
6.4
6.6
5.5
3.3
*Mean from three different assays, errors in the range of ꢂ10% of the reported values.
Scheme 1. Preparation of the coumarins/
benzocoumarins 1–5 from phenols A, C and
D and ethyl acetoacetate B.
OH
O
O
TiCl4
R
+
R
OEt
EtOH, r.t
O
O
A
B
1-3
OH
OH
TiCl4
O
O
+
+
EtOH, r.t
O
O
OEt
OEt
4
B
C
D
TiCl4
O
O
EtOH, r.t
O
O
5
B
constants were obtained by non-linear least-squares methods physico–chemical procedures which confirmed their structures
using PRISM 3, as reported earlier1–3, and represent the (‘‘Materials and methods’’ section for details).
mean from at least three different determinations. CA isoforms
were recombinant ones obtained in house as reported earlier1–3
Coumarins 1–5 were investigated as inhibitors31 of four
catalytically active hCA isoforms, hCA I, hCA II (cytosolic
isoforms) as well as hCA IX and hCA XII (transmembrane,
tumor-associated enzymes)32 (Table 1). The following structure–
activity relationship (SAR) can be evidenced from the data of
.
Discussion and conclusions
In previous contributions from our laboratory, we showed that Table 1:
even simple coumarins derivatives may lead to interesting
inhibition profiles against various CA isoforms, with the discov-
ery of a wide range of isoform-selective CAIs1,2,29,30. Thus, we
investigated here the synthesis of new coumarins and inhibition of
several pharmacologically important CA isoforms (e.g. CA I, II,
IX and XII) with a small series of such compounds, i.e. coumarins
(1–3) and benzocoumarins (4 and 5) (Scheme 1).
Compounds 1–5 were prepared by literature procedures, by the
Pechmann condensation of phenols A or naphthols C and D, with
ethyl acetoacetate B, in the presence of Ti(IV) chloride as
catalyst29,30. Compounds 1–5 have been characterized by standard
(i) The slow cytosolic isoform hCA I was moderately inhibited
by coumarins 1–5, with inhibition constants in the range of
4.8–9.0 mM. The bets inhibitor was 4-methyl-7-hydroxy-
coumarin 1 (KI of 4.8 mM), whereas the remaining 4
derivatives showed a flat, less potent inhibition (KIs
around 9 mM, Table 1).
(ii) As for other simple coumarins investigated earlier29,30, also
coumarins 1–3 and benzocoumarins 4 and 5 were devoid of
inhibitory activity against the dominant isoform hCA II,
which is a very interesting feature of this class of CAIs.
Indeed, the house-keeping, and quite abundant isoform hCA

4
A. Sharma et al.
J Enzyme Inhib Med Chem, Early Online: 1–5
11. Supuran CT. Structure-based drug discovery of carbonic anhydrase
inhibitors. J Enzyme Inhib Med Chem 2012;27:759–72.
II showed KIs4200 mM with all these compounds, which
constitutes a highly attractive feature for inhibitors that
should target isoforms involved in tumorigenesis (CA IX
and XII)32 or in other processes, such as for example obesity
´
12. Liu F, Martin-Mingot A, Lecornue F, et al. Carbonic Anhydrases
inhibitory effects of new benzenesulfonamides synthesized by
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(CA VA and VB)33,34
.
(iii) The tumor-associated hCA IX was well-inhibited by com-
pounds 1–5, with KIs in the submicromolar range, i.e. of
0.19–0.45 mM (Table 1). The best inhibitors were the linear
benzocoumarin 5 (KI of 0.20 mM) and the dihydroxy-
4-methyl-coumarin 2 (KI of 0.19 mM), but the remaining
derivatives were only slightly less effective CAIs than 2
and 5.
13. Alp C, Ozsoy S, Alp NA, et al. Sulfapyridine-like benzenesulfona-
mide derivatives as inhibitors of carbonic anhydrase isoenzymes I, II
and VI. J Enzyme Inhib Med Chem 2012;27:818–24.
14. Kolayli S, Karahalil F, Sahin H, et al. Characterization and
inhibition studies of an a-carbonic anhydrase from the endangered
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˘
15. Kazancıoglu EA, Gu¨ney M, Sentu¨rk M, Supuran CT. Simple
(iv) The second tumor-associated isoform, hCA XII, was also
inhibited by these compounds but less efficiently compared
to hCA IX. Indeed, the coumarins/benzocoumarins 1–5
showed inhibition constants in the range of 3.3–6.6 mM.
Again thus, a quite compact behavior, with little SAR, which
may be explained by the rather similar structures of the
investigated compounds.
In conclusion, a small series of coumarins/benzocoumarins
incorporating methyl and hydroxyl moieties in the heterocyclic
ring were investigated as CAIs. These coumarins were very
weak or ineffective as inhibitors of the house-keeping, offtarget
isoforms CA I and II, but showed effective, submicromolar
inhibition of the transmembrane, tumor-associated isoforms CA
IX and XII. The nature and position of the groups substituting
the coumarin ring influenced CA inhibitory properties. 5,7-
Dihydroydroxycoumarin showed KIs4200 mM against CA II, of
0.19 mM against CA IX and of 6.4 mM against CA XII, being thus
an isoform-selective, efficient inhibitor for the tumor-associated
over cytosolic CA isoforms. These compounds are interesting
leads for designing isoform-selective enzyme inhibitors.
methanesulfonates are hydrolyzed by the sulfatase carbonic
anhydrase activity. J Enzyme Inhib Med Chem 2012;27:880–5.
16. Cavdar H, Ekinci D, Talaz O, et al. a-Carbonic anhydrases are
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Chem 2012;27:148–54.
17. Ekinci D, Kurbanoglu NI, Salamcı E, et al. Carbonic
anhydrase inhibitors: inhibition of human and bovine isoenzymes
by benzenesulphonamides, cyclitols and phenolic compounds.
J Enzyme Inhib Med Chem 2012;27:845–8.
18. Ekinci D, Al-Rashida M, Abbas G, et al. Chromone containing
sulfonamides as potent carbonic anhydrase inhibitors. J Enzyme
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19. Singh S, Supuran CT. QSARs on human carbonic anhydrase VA and
VB inhibitors of some new not yet synthesized, substituted aromatic/
heterocyclic sulphonamides as anti-obesity agent. J Enzyme Inhib
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20. Fabrizi F, Mincione F, Somma T, et al.
A new approach
to antiglaucoma drugs: carbonic anhydrase inhibitors with
or without NO donating moieties. Mechanism of action and
preliminary pharmacology. J Enzyme Inhib Med Chem 2012;27:
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21. Sahin H, Can Z, Yildiz O, et al. Inhibition of carbonic anhydrase
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Acknowledgements
¨
22. Sentu¨rk M, Ekinci D, Goksu S, Supuran CT. Effects of dopamin-
We are grateful to Dr Alfonso Maresca and Dr Daniela Vullo for technical
support.
ergic compounds on carbonic anhydrase isozymes I, II, and VI.
J Enzyme Inhib Med Chem 2012;27:365–9.
¨
¨
23. Bootorabi F, Janis J, Hytonen VP, et al. Acetaldehyde-derived
modifications on cytosolic human carbonic anhydrases. J Enzyme
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Declaration of interest
24. Chohan ZH, Shad HA, Supuran CT. Synthesis, characterization
and biological studies of sulfonamide Schiff’s bases and some
of their metal derivatives. J Enzyme Inhib Med Chem 2012;27:
58–68.
25. Ozensoy O, Arslan M, Supuran CT. Carbonic anhydrase inhibitors:
purification and inhibition studies of pigeon (Columba livia var.
domestica) red blood cell carbonic anhydrase with sulfonamides.
J Enzyme Inhib Med Chem 2011;26:749–53.
The authors report no conflict of interest. This work was supported by an
EU FP7 research grant (Metoxia project).
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