Deciphering the mechanism of carbonic anhydrase inhibition with coumarins and thiocoumarins

Article abstract of DOI:10.1021/jm901287j

Coumarin derivatives were recently shown to constitute a totally new class of inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), being hydrolyzed within the CA active site to 2-hydroxycinnamic acids. We explore here a new series of variously substituted coumarins and a thiocoumarin for their interaction with 13 mammalian CA isoforms, detecting low nanomolar and isoform selective inhibitors. The mechanism of action of this class of inhibitors is delineated in detail by resolving the X-ray crystal structure ofCAII in complex with trans-2-hydroxy-cinnamic acid, the in situ hydrolysis product of simple coumarin. Thiocoumarins also act as efficient CAIs, similarly to coumarins. The versatility of the (thio)coumarin chemistry, the cis-trans isomerization evidenced here, and easy derivatization of the (thio)coumarin rings, coupled with the nanomolar inhibition range of several isozymes, afford isoform-selective CAIs with various biomedical applications, which render these classes of compounds superior to the clinically used sulfonamides.

Full text of DOI:10.1021/jm901287j

J. Med. Chem. 2010, 53, 335–344 335
DOI: 10.1021/jm901287j
Deciphering the Mechanism of Carbonic Anhydrase Inhibition with Coumarins and Thiocoumarins
Alfonso Maresca,‡ Claudia Temperini,‡ Lionel Pochet,§ Bernard Masereel,§ Andrea Scozzafava,‡ and Claudiu T. Supuran*,‡
‡
ꢀ
Universita degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze),
Italy and §University of Namur, Drug Design and Discovery Center, FUNDP, B-5000 Namur, Belgium
Received August 27, 2009
Coumarin derivatives were recently shown to constitute a totally new class of inhibitors of the zinc
metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), being hydrolyzed within the CA active site to 2-
hydroxycinnamic acids. We explore here a new series of variously substituted coumarins and a
thiocoumarin for their interaction with 13 mammalian CA isoforms, detecting low nanomolar and
isoform selective inhibitors. The mechanism of action of this class of inhibitors is delineated in detail by
resolving the X-ray crystal structure of CA II in complex with trans-2-hydroxy-cinnamic acid, the in situ
hydrolysis product of simple coumarin. Thiocoumarins also act as efficient CAIs, similarly to
coumarins. The versatility of the (thio)coumarin chemistry, the cis-trans isomerization evidenced
here, and easy derivatization of the (thio)coumarin rings, coupled with the nanomolar inhibition range
of several isozymes, afford isoform-selective CAIs with various biomedical applications, which render
these classes of compounds superior to the clinically used sulfonamides.
Introduction
Chart 1. Hydrolysis of Coumarins 1 and 2 to the Correspond-
ing 2-Hydroxy-cinnamic Acid Derivatives 3 and 4a/4b
In a recent paper,1 we reported a novel class of inhibitors of
the metalloenzyme carbonic anhydrase (CA,a EC 4.2.1.1)2
belonging to a completely new chemotype, the coumarins.3
The CA family of enzymes is widespread all over the phyloge-
netic tree (with 16 different R-CA isozymes presently known in
mammals) and is inhibited by compounds which bind to the
catalytically critical Zn(II) ion from the enzyme active site (or
the water/hydroxide ion coordinated to it): the sulfonamides,
their bioisosteres (sulfamates, sulfamides, N-substituted sulfo-
namides, etc), some metal complexing anions, and (thio)-
phenols among others,2,4 whereas the coumarins, such as the
natural product 1 for which this effect was initially reported,1,3
do not have any obvious functionality to confer them potent
CA inhibitory activity.
CAs are involved in numerous physiological and pathologi-
cal processes, including respiration and transport of CO2/
bicarbonate between metabolizing tissues and lungs, pH and
CO2 homeostasis, electrolyte secretion in a variety of tissues/
organs, biosynthetic reactions (e.g., gluconeogenesis, lipogen-
esis, and ureagenesis), bone resorption, calcification, tumori-
genicity, and many other physiological and pathological
processes in humans as well as the growth and virulence of
various fungal/bacterial pathogens.2,4-11 In addition to the
established role of CA inhibitors (CAIs) as diuretics and
antiglaucoma drugs, it has recently emerged that they have
potential as anticonvulsant, antiobesity, anticancer, and anti-
infective drugs.2,4-11 Many of the mammalian CA isozymes
involved in these processes are important therapeutic targets
with the potential to be inhibited or activated to treat a wide
range of disorders.2,4 However a critical barrier to the design of
CAIs as therapeutic agents is related to the high number of
isoforms in humans, their rather diffuse localization in many
tissues/organs, and the lack of isozyme selectivity of the pre-
sently available inhibitors of the sulfonamide/sulfamate type.2,4
However, in the preceding paper,1 we showed that the natural
product 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-chro-
men-2-one 1 as well as the simple, unsubstituted coumarin 2
are hydrolyzed within the CA active site with formation of the
2-hydroxy-cinnamic acids 3 and 4, respectively, which represent
the de facto enzyme inhibitors. At least two other interesting
facts emerged during this study:1 (i) this new class of CAIs, the
coumarins, binds (in hydrolyzed form) at the entrance of the
CA active site and does not interact with the metal ion,
constituting thus an entirely new category of mechanism-based
inhibitors, and (ii) for the specific case of compound 1, the
formed substituted-cinnamic acid 3 was observed bound within
the CA active site as the cis isomer, although these derivatives
are stable in solution as trans isomers.12 The tentative explana-
tion for this unusual geometry of the bound inhibitor was1 that
as 3 would be too bulky as trans isomer, in the restricted space
within the CA active site, in the enzyme-inhibitor adduct
characterized in detail by means of X-ray crystallography,1
the unstable in solution cis isomer is stabilized when bound
*To whom correspondence should be addressed. Phone: þ39-055-457
3005. Fax: þ39-055-4573385. E-mail: claudiu.supuran@unifi.it.
a Abbreviations: CA, carbonic anhydrase; CAI, carbonic anhydrase
inhibitor; hCA, human CA; SAR, structure-activity relationship.
r
2009 American Chemical Society
Published on Web 11/13/2009
pubs.acs.org/jmc

336 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Maresca et al.
within the enzyme cavity. In the same work,1 we prepared
(by chemical hydrolysis of coumarin 2 in the presence of
NaOH) the sodium salt of 2-hydroxy-cinnamic acid 4, stable
in solution as the trans isomer 4b, and showed it to possess the
same CA inhibitory properties as the parent coumarin 2. It
should be also mentioned that coumarins 1 and 2 were potent
inhibitors against all investigated human CA isoforms, i.e., CA
I-CA XV, which makes this entire class of derivatives of
paramount interest for designing novel applications for the
CAIs (Chart 1).
To understand in greater detail the CA inhibition mechan-
ism with the coumarins, which might be useful for the design
of newpharmacologicalapplications, we investigate here both
the simple lead compound 2 for its interaction with the CA
active site by means of high resolution X-ray crystallography
as well as a series of derivatives of 2 possessing various
moieties substituting the coumarin ring in the 3-, 6-, 7-, 3,6-,
4,7-, and 3,8-positions. A thiocoumarin derivative is also
investigated here for the first time for its interaction with the
CAs in order to understand whether this heterocyclic ring may
undergo the same transformation as the coumarin one and
thus lead to a novel class of CAIs. This is the first structure-
-activity relationship (SAR) study for this new type of CAIs,
bringing novel insights regarding the inhibition mechanism as
well as the structural requirements necessary to be present in
the molecules of such heterocyclic compounds for achieving
high affinity for various CA isoforms.
nature, represent important factors influencing the activity
of these CAIs. In the previous work,1 we also showed that the
simple, unsubstituted coumarin 2 already possesses interest-
ing enzyme inhibitory properties in addition to the more
complicated scaffold of the natural product coumarin 1.
Thus, we considered these core structures to which various
substituents were incorporated in order to delineate SAR for
the whole coumarin class as CAIs. The 7-monosubstituted
coumarins 5-10 investigated here possess various 7-alkoxy
moieties, such as the aliphatic C1-C4 groups, together with
the benzyl- and phenethyl- moieties. They were chosen to be
investigated as the 7-methoxy group was present in the lead
1, and X-ray crystal data for the hCA II-1 adduct, showed
the MeO group to participate in hydrogen bonds with
ordered water molecules present within the CA active
site, stabilizing thus the enzyme-inhibitor complex.1 Thus,
understanding how the length and nature of this moiety
influence enzyme inhibitory activity may constitute an im-
portant aspect of the SAR. Compounds 6-10 were prepared
from the commercially available 7-hydroxy-coumarin by
alkylation with alkyl/aralkyl halides, as described in the
literature12 (see Supporting Information for additional
details).
Disubstituted coumarins 11-16,13 possessing a carboxylic
acid as well as methyl-, methoxy-, and hydroxymethyl
moieties in various positions of the ring, were also included
in the study. Thiocoumarin 1714 was the only compound
incorporating this ring system in our study and was chosen
for understanding whether substitution of oxygen by sulfur
in the coumarin ring leads to CA inhibitory properties for
this class of compounds never before investigated, but also
because the corresponding carboxy-substituted coumarin 12
was present among the investigated derivatives. Disubstitut-
ed coumarins 18-2013 incorporate the ester moieties instead
of the corresponding carboxylic acid one present in the
parent compound 15, whereas 20 is an ester analogue of
the simple lead 5, investigated earlier.1 Because the lead 1
possessed a bulky moiety in position 6 of the coumarin ring,
derivatives 21-2313 were included in the study due to the
presence of both smaller, simpler such moieties (hydroxy-
methyl and aminomethyl), which can be easily derivatized,
and also of the much bulkier hexamethylene-tetramine one,
present in 23. Compounds 6-23 were reported earlier in the
search of serine protease inhibitors by Delarge’s and Maser-
eel’s groups.13,14
Results and Discussion
Chemistry and CA Inhibition. In addition to the previously
investigated simple coumarins 1, 2, and 5,1 a series of
diversely substituted coumarins and a thiocoumarin, of type
6-23, were included in this study.
Inhibition data for compounds 1-23 against all 13 cata-
lytically active mammalian (h = human, m = murine) CA
isoforms, CA I-IV, VA, VB, VI, VII, IX, and XII-XV, are
presented in Table 1. The data have been obtained by a
stopped flow technique monitoring the physiological reac-
tion catalyzed by these enzymes, i.e., CO2 hydration to
bicarbonate and protons.15 As for the previously studied
compounds 1, 2, and 5,1 coumarins 6-23 investigated here
were incubated with the enzymes for 6 h in order to allow for
the complete hydrolysis (within the enzyme active site) of the
(thio)coumarin ring and formation of the substituted 2-
hydroxy-/mercapto-cinnamic acids. Thus, data of Table 1
report the inhibition constants for these (thio)coumarins
1-23 following 6 h incubation with all the CA isoforms.
The following should be noted regarding inhibition data of
Table 1:
Derivatives 5-23 incorporate various moieties by substi-
tuting the (thio)coumarin ring in the 3-, 6-, 7-, 3,6-, 4,7-, and
3,8- positions, in order to delineate the main SAR features
for this class of CAI, considering the fact that the side chains
present in the natural product coumarin 1, the first CAI of
this family of compounds investigated in detail, were shown
to interact extensively with the enzyme active site when
bound (in hydrolyzed form) within it.1 Thus, the presence
of a different numbers of side chains, as well as their chemical
(i) Isoform hCA I was inhibited by all coumarins/thio-
coumarin 1-23, with inhibition constants in the
range of 78 nM-37.0 μM. The best inhibitors were

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 337
Table 1. Inhibition of Mammalian Isozymes CA I-XV (h = Human, m = Murine Isoform) with Coumarins/Thiocoumarins 1, 2, 5-23, by a Stopped-
Flow, CO2 Hydration Assay Method (6 h Incubation Time between Enzyme and (Thio)coumarin)15
KI (μM)c
isozymea
1d
0.078
0.059
>500
2d
3.1
9.2
>500
62.2
>500
5d
5.9
0.066
161
7.8
>500
6
7
8
9
10
11
3.72
0.099
>500
72.3
12
13
hCA I
hCA II
31.4
12.4
29.0
22.7
7.3
23.1
37.0
26.9
31.4
9.3
44.7
9.1
3.8
9.0
6.8
31.3
5.1
4.7
9.0
7.3
7.4
5.6
6.8
42.5
13.4
5.6
145
213
224
243
hCA III
hCA IV
hCA VA
hCA VB
hCA VI
hCA VII
hCA IXb
hCA XIIb
mCA XIII
hCA XIV
mCA XV
38.6
24.6
8.1
50.3
24.9
9.0
40.2
19.2
7.6
49.5
18.5
6.6
3.8
96.0
17.7
35.7
27.9
54.5
48.6
>500
>500
>500
8.6
8.3
>500
>500
>500
>500
>500
>500
>500
>500
48.6
61.2
9.1
60.9
5.8
71.7
37.4
7.7
76.4
26.8
15.0
4.5
65.1
9.7
69.4
8.5
31.7
29.6
7.7
7.2
1.6
11.8
1.7
15.9
3.9
9.4
>500
3.3
8.6
>500
>500
167.4
6.0
9.7
4.7
8.5
8.2
8.3
9.6
5.2
9.8
8.6
7.4
7.86
7.80
93.1
27.4
4.7
34.3
5.0
31.7
82.3
>500
4.3
65.2
2.0
43.1
5.5
44.5
13.0
6.7
>500
76.3
54.7
KI (μM)c
isozymea
14
15
16
17
18
19
20
21
22
7.1
23
hCA I
hCA II
6.6
15.3
14.5
4.9
9.3
44.5
9.7
1.3
30.1
17.1
5.8
0.100
6.2
9.0
0.098
0.032
8.9
3.3
50.5
9.6
2.4
31.4
9.1
7.8
32.4
9.8
5.1
8.9
6.8
9.9
6.9
4.1
3.7
9.6
5.4
9.7
6.2
9.2
8.1
6.7
8.6
3.2
8.1
6.2
8.2
5.6
8.9
3.1
0.048
3.2
3.8
7.7
8.2
hCA III
hCA IV
hCA VA
hCA VB
hCA VI
hCA VII
hCA IXb
hCA XIIb
mCA XIII
hCA XIV
mCA XV
4.3
6.2
4.1
8.4
6.5
8.4
0.048
3.0
5.7
9.5
8.7
7.6
9.4
6.5
8.6
7.5
8.8
7.1
9.5
2.9
1.5
8.0
10.5
4.9
15.6
6.6
25.6
6.1
42.0
20.3
6.5
7.3
0.047
8.7
7.3
0.045
5.6
0.045
0.047
8.4
6.6
9.0
5.6
8.9
7.4
8.6
0.093
8.2
8.8
6.2
3.8
0.048
8.4
8.6
7.8
1.0
0.042
7.4
4.8
0.041
7.3
7.4
6.1
4.6
0.046
9.5
7.4
0.040
8.8
7.1
39.6
7.2
9.6
6.6
6.4
0.046
a h = human; m = murine isozyme. b Catalytic domain. c Errors in the range of (5% of the reported data from three different assays. d From ref 1.
the natural coumarin 1, the thiocoumarin 17, as well
as the hydroxy-methyl-substituted ester 18 (KIs of
78-100 nM). Substitution patterns leading to reduced
hCA I inhibitory activity were those present in 6-10
(longer aliphatic/aralkyl chains in position 6 are thus
detrimental to the inhibitory activity of these com-
pounds compared to the unsubstituted 2 or methoxy-
substituted compound 5) and 11 (possessing a
4-carboxy moiety) because these derivatives showed
KIs > 9.3 μM. The remaining coumarins investigated
here were medium potency hCA I inhibitors, with
inhibition constants in the range of 1.3-9.3 μM
(Table 1).
between the carboxylic acid 15 and the corresponding
esters 18 and 19, with the methyl ester 18 being a very
potent, nanomolar inhibitor (KI of 32 nM), whereas
the free acid 15 and the ethyl ester 19 were 1390-1578
times less effective hCA II inhibitors. Such a sharp
SAR for minimal structural changes (i.e., a CH2
group) was never before evidenced for other classes
of CAIs, such as the sulfonamides or the sulfamates,16
and constitutes a valuable feature for this type of
inhibitor.
(iii) The muscle isoform hCA III, which is not easily
inhibited by sulfonamides,17 was weakly or not at
all inhibited by coumarins 1-3 and 11 (KIs of 161 f
500 μM), whereas coumarins 6-10 and 13, 14 showed
more efficient inhibitory activity, with KIs in the range
of 13.4-50.3 μM. Even better activity was observed for
the thiocoumarin 17 and coumarins 12, 15, 18-23,
with KIs in the range of 8.1-9.8 μM (Table 1).
(iv) The coumarin ester 19 was a nanomolar inhibitor of
the extracellular isoform hCA IV (KI of 48 nM),
whereas most other such derivatives (e.g., 1, 5,
12-18 and 20-23) showed effective inhibition, in
the low micromolar range, with KIs of 3.8-7.8 μM.
The lead 1 was the least effective hCA IV inhibitor
(KI of 62.2 μM), whereas compounds with bulky
groups in position 6 of the coumarin ring such
as 6-10 showed also weak inhibition (KIs of 18.5-
24.9 μM) compared to the methoxy-substituted cou-
marin 5.
(ii) The physiologically dominant cytosolic isozyme hCA
II was also inhibited by all these derivatives, with
inhibition constants in the range of 32 nM-243 μM.
The best inhibitor was again the methyl ester 18 (KI of
32 nM, the best coumarin inhibitor detected so far)
together with the natural product 1, the methoxyder-
ivative 5, and the carboxylic acid 11 (KIs of 59-99
nM). Again, the monosubstituted derivatives 6-10
incorporating bulky chains in position 6 of the cou-
marin ring showed reduced inhibitory activity com-
pared to the lead 5 (KIs of 12.4-243 μM), proving that
substituents other than methoxy are not tolerated in
that position for effective binding to hCA II. Not very
effective hCA II inhibitors were also derivatives
12-16, possessing the free COOH moiety in position
3. The thiocoumarin 17 was on the other hand a more
effective inhibitor (KI of 6.2 μM). It is, however, worth
noting that there is a very large difference of activity
(v) The mitochondrial isoform hCA VA was not inhibited
by 2, 5, and 11 (KI > 500 μM) and was weakly

338 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Maresca et al.
inhibited by the natural product 1 (KI of 96 μM). All
other coumarins investigated here and the thiocou-
marin 17 were on the other hand effective, low micro-
molar hCA VA inhibitors (KIs < 10 μM), with the
best inhibitor being the ester 19 (KI of 3.0 μM). Thus,
in the case of this isozyme, even the bulky derivatives
6-10 act as efficient CAIs.
CA IX inhibition, a large variations of structural
motifs are allowed in the 3- and 6-positions of the
(thio)coumarin ring. It is interesting to note that the
isostructural (to 21) amine 22, was 72 times less
inhibitory than the alcohol 21. It is not improbable
that the enhanced basicity of the amine 22 compared
to the alcohol 21 is detrimental to binding within the
enzyme active site due to a different pattern hydrogen
bonds between the two moieties and amino acid
residues at the entrance of the cavity, where pre-
sumably these moieties are found. However this
hypothesis should be checked by X-ray crystallogra-
phy, which will allow us to understand how the
various substituents of the coumarin ring interact
with amino acid residues within the enzyme active
site. The remaining coumarins, e.g., 6-10, 12-16, 20,
and 21, showed efficient, low micromolar inhibition
of CA IX, with inhibition constants in the range of
1.6-7.7 μM. It may be seen that many substitution
patterns on the (thio)coumarin ring lead to effective
nanomolar-low micromolar CA IX inhibitors, af-
fording for drug design campaigns for this important
drug target.2
(vi) The second mitochondrial isoform, hCA VB, showed
a very different inhibition profile with compounds
1-23 compared to hCA VA. Thus, 2 and 11 showed
no inhibitory activity (KI > 500 μM), the natural
product 1 as well as the monosubstituted derivatives
5-10 were weak inhibitors (KIs in the range of
17.7-76.4 μM), whereas the remaining coumarins
12-16 and 18-23, as well as the thiocoumarin 17,
were effective hCA VB inhibitors, with inhibition
constants in the range of 2.9-8.6 μM. It is obvious
that minimal structural changes in the scaffold of the
coumarins influence the inhibitory activity very
much.
(vii) The secreted (saliva, milk) isoform hCA VI was not
inhibited by 2 and 11 (KI > 500 μM), and was weakly
inhibited by the following coumarins: 1, 5, 7, 8, 12,
13, 15, 16, and 20 (KIs in the range of 15.6-61.2 μM).
Thus, SAR is less defined here compared to other CA
isoforms discussed above (e.g., for the bulky-substi-
tuted compounds 5-10, there is no linear correlation
between the length of the substituent present in the 6-
position and the hCA VI inhibitory activity, see
Table 1). Effective hCA VI inhibitory activity (KIs
in the range of 1.5-10.5 μM) was observed for
derivatives 6, 9, 10, 14, 17 (thiocoumarin), 18, 19,
and 21-23. The best inhibitor was the ester 19 (KI of
1.5 μM).
(x) hCA XII, another transmembrane isoform present in
tumors,18 was poorly inhibited by 2, 5, and 11,
whereas the natural product coumarin 1 was slightly
more inhibitory (KI of 48.6 μM). However, most of the
investigated (thio)coumarins were effective, low mi-
cromolar hCA XII inhibitors, with KIs in the range of
3.2-9.0 μM. Thus, a lot of substitution patterns
present in compounds 6-23 investigated here lead to
effective hCA XII inhibitors, although compounds
with nanomolar affinity for this isozyme were not
evidenced so far (Table 1).
(viii) Except 2, which was not inhibitory, the cytosolic
isoform hCA VII was inhibited by all compounds 1,
5-23 investigated here, with KIs in the range of
45 nM-29.6 μM. The only nanomolar inhibitor
was the ester 19 (KI of 45 nM), whereas most of
these derivatives were low micromolar inhibi-
tors. The least effective inhibitors were 1, 13, and
20 (KIs in the range 20.3-29.6 μM). Again small
structural variations in the coumarin scaffold lead to
important differences of activity (compare 12 and 13
which differ by a CH2 groups but by a factor of
almost 6 in their CA VII inhibitory activity).
(ix) The tumor-associated isoform hCA IX was weakly
inhibited by compound 1 and not inhibited by 2, 5,
and 11 (KI > 500 μM). However, a large number of
derivatives showed effective or very effective inhibi-
tory activity against this isoform, which represents a
new drug target for developing antitumor therapies
or diagnostic agents.7,9 Thus, thiocoumarin 17 and
several coumarins (18, 19, 21, and 23) showed low
nanomolar affinity for this enzyme, with inhibition
constants in the range of 45-98 nM. These coumarins
incorporate the 6-hydroxymethyl- and 3-ester moi-
eties (18 and 19), with no important differences of
activity between the methyl and ethyl esters in this
case. The monosubstituted derivatives 21 and 23 on
the other hand contained either a compact (CH2OH)
or a rather bulky (hexamethylenetetramine) group in
position 6 of the coumarin ring, which render our
findings quite important, as it is clear that for effective
(xi) Several low nanomolar mCA XIII inhibitors were
observed among the investigated compounds, such as
the thiocoumarin 17 and the coumarins 15, 18, 21,
and 22, possessing inhibition constants of 40-48 nM.
Together with CA IX, CA XIII is thus the isoform
leading to the highest number of nanomolar inhibi-
tors in this class of derivatives. Unlike other isoforms,
CA XIII was equally inhibited by the free carboxylic
acid 15 and its methyl ester 18 (KIs of 41-48 nM),
whereas the longer ethyl ester 19 was 127-148 times
less inhibitory than 15/18. Moderate inhibitory activ-
ity against CA XIII was observed with the coumarins
6, 7, and 11, whereas compounds 5, 8-10, 12-14, 16,
19, 20, and 23 were medium potency, low micromolar
inhibitors (KIs of 3.8-9.6 μM).
(xii) The transmembrane isoforms hCA XIV showed
an inhibition profile with the investigated deriva-
tives rather similar to that of hCA XII, with which
it shares some relevant sequence homology.19
Thus, 2, 11, and 14 were ineffective inhibitors
(KIs of 39.6 f 500 M), whereas all other coumarins
and the thiocoumarin investigated here showed
effective, low micromolar inhibitory activity (KIs
of 1.0-13.0 μM). The best hCA XIV inhibitor was
the 3,8-disubstituted coumarin 16 (KI of 1.0 μM),
which urges us to investigate also this substitu-
tion pattern represented here only by this unique
compound.
(xiii) mCA XV, the latest mammalian CA isoform de-
scribed so far,20 was not inhibited by 2, 5, and 11 and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 339
was weakly inhibited by coumarins 1, and 6-10 (KIs
of 43.1-93.1 μM). The ester 19 was on the other
hand a low nanomolar mCA XV inhibitor (KI of
46 nM), whereas the remaining coumarins and the
thiocoumarin 17 showed effective, low micromolar
inhibitory activity (KIs of 4.8-8.6 μM).
All these data show the potential of the coumarins/thio-
coumarins to selectively and potently inhibit some CA
isoforms among the 13 catalytically active ones described
in mammals, which are in fact undiscriminately inhibited by
sulfonamides and sulfamates. The complex SAR features
evidenced above afford the possibility to achieve this goal
of obtaining selective inhibitors for some of these en-
zymes. For example, 23 is a low nanomolar inhibitor of
only CA IX (KI of 48 nM), whereas it inhibits in the
micromolar range all other 12 CAs, a feature never evi-
denced before for a sulfonamide CAI.2,16 The same is true for
15 and 22, which act as nanomolar inhibitors against mCA
XIII (KIs of 40-48 nM), whereas the remaining 12 isoforms
inhibited in the micromolar range by these compounds
(Table 1).
Inhibition Mechanism and X-Ray Crystallography. To
better understand the inhibitory mechanism with this new
class of CAI, and hopefully the SAR discussed above, we
˚
resolved the X-ray crystal structure (at a resolution of 2.0 A)
of another coumarin,1 the simple, unsubstituted derivative 2
in adduct with the physiologically dominant CA isoform,
hCA II.2,21
Inspection of the electron density maps (Figure 1) at various
stages of the refinement showed features compatible with the
presence of one molecule of inhibitor bound within the active
site (Figure 2) as for the previously reported structure of
coumarin 1 in adduct with hCA II.1 However, again,1 in this
Figure 1. Electron density map at 1σ of the hydrolyzed coumarin 2
(i.e., trans-2-hydroxy-cinnamic acid 4b) bound within the hCA II
active site. The Zn(II) ion and its three protein ligands, (His94,
His96, His119, CPK colors) and the coordinated water molecule
(w, in red) are also shown.
Figure 2. Binding of the coumarin 2 hydrolysis product (trans-2-hydroxy-cinnamic acid 4b in yellow) and coumarin 1 hydrolysis product (cis-
2-hydroxycinnamic acid 3, magenta) to the hCA II active site. The protein backbone is shown as green ribbon, the catalytic Zn(II) ion as violet
sphere, with its three protein ligands (His94, 96, and 119, CPK colors) also evidenced. The proton shuttle residue (His64) is shown in red.

340 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Maresca et al.
electron density, we could not fit the structure of the coumarin 2.
Instead, its hydrolysis product, the trans-2-hydroxy-cinnamic
acid 4b (Chart 1) perfectly fitted within the electron density
shown in Figure 1. All atoms of the hydrolyzed inhibitor had an
average B factor of 47.0, proving that the occupancy of the
inhibitor was of 70%, which represents a good occupancy level
for the external part of the enzyme active site where the degree of
disorder is rather high.5 Thus, as for the similar case reported
earlier,1 the zinc hydroxide species of the enzyme, responsible
for the various catalytic activities of CAs2-4 including the
esterase one,2 appears likely to have hydrolyzed the lactone ring
of 2, leading to formation of the 2-hydroxycinnamic acid 4.
However, unlike the bulky derivative 3 investigated earlier,1
in this case the thermodynamically stable trans-2-hydroxy-
cinnamic acid 4b has been evidenced as bound within the CA
active site. Interactions between the protein and Zn2þ ion were
entirely preserved in the hCA II-4b adduct (data not shown).21
The inhibitor 4b was found bound at the entrance of the active
site cavity (Figure 2), in the same region where the cis-hydro-
xycinammic acid 3binds.1 However, as shown in Figure 2 where
the two substituted-2-hydroxycinnamic acids 3 and 4 are super-
posed (when bound to the enzyme active site), different orienta-
tions and conformations were adopted by the two inhibitors.
Thus, the bulky 2-hydroxycinnamic acid 3 has the cis geometry,
as mentioned above (probably due to steric impairment because
of the second bulky, hydroxypentyl chain present in its
molecule), whereas the less bulky compound 4 has the trans
geometry 4b (Figure 2). The second important difference
between the two coumarin hydrolysis products is related to
the orientation of the carboxyethylene moiety of the two
inhibitors 3 and 4. In fact, they are orientated toward opposite
parts of the active site, the one of 3 pointing toward the exit of
the cavity (and interacting with Glu238 from a symmetry-
related enzyme molecule),1 whereas the one of 4b points toward
the hydrophilic part of the CA II active site and interacting with
two amino acid residues (Asn62 and His64) by means of two
hydrogen bonds involving the COOH moiety of the inhibitor
(Figure 3). Thus, the carboxylate moiety of inhibitor 4b is
anchored by means of two hydrogen bonds (of 3.40 and 3.13
˚
A, respectively) to the NH of the imidazole of His64, and the
NH2 of Asn62, amino acid residues lining the hydrophilic half of
the hCA II active site (Figure 2 and 3). The phenolic moiety of
inhibitor 4b also participates in two hydrogen bonds, with
Figure 3. Hydrogen bonds in which 4b participates when bound to
the hCA II active site with residues Asn62, His64, Gln92, and a
˚
water molecule (w257). Figures represent distances (in A).
Figure 4. Superposition of the hCA II-3 adduct (gold, PDB file 3F8E), hCA II-4b adduct (violet, PDB file 4F8E) with the hCA II-phenol 24
adduct22 (file not available in PDB, sky blue) and hCA II-sulfonamide 25 adduct24 (magenta, PDB file 3EFT). The protein backbone is shown
as green ribbon, the catalytic Zn(II) ion as pink-violet sphere, with its three protein ligands (His94, 96, and 119) evidenced. Some of the amino
acid residues involved in the binding of hydrolyzed coumarins (His64, Asn67, and Phe131) are also shown (CPK colors).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 341
˚
the amide of Gln92 (of 2.84 A) and with a water molecule, w257
Chart 2. Proposed Inhibition Mechanism of CAs by Coumarins/
Thiocoumarins, Leading to cis- or trans-2-Hydroxy/mercapto-cin-
namic Acids
˚
(of 3.13 A), as shown schematically in Figure 3. Thus, this
hydrolyzed coumarin interacts with different amino acid resi-
dues (i.e., Asn62, His64, and Gln92) compared to the hydrolysis
product of 1, which primarily interacted with Asn67, Phe131,
and Glu238sym.1
To stress the novelty of this binding mode of coumarins
(hydrolyzed to cis-/trans-hydroxy-cinnamic acids) to the CA
active site, in Figure 4 we present a superposition of the hCA
II-3/4b adducts with those of phenol 2422,23 and a benzene
sulfonamide inhibitor recently reported by us, compound
25.24 Indeed, sulfonamides and phenols represent the other
classes of effective CAIs, with the sulfonamides and their
bioisosteres (sulfamates, sulfamides) having clinical
applications.2,16,22-24 It may be observed that sulfonamide
25 is coordinated to the Zn(II) ion within the hCA II active
site, whereas its organic scaffold fills the entire enzyme
cavity, making an extensive series of van der Waals and
polar interactions with amino acid residues both at the
bottom, middle, and entrance of the active site cavity. A
water molecule/hydroxide ion, which is the fourth zinc
ligand present in the noninhibited enzyme, is thus displaced
by the sulfonamidate nitrogen of the inhibitor when sulfo-
namides bind to CAs. In contrast, phenol 24 was shown to be
anchored to the zinc bound water molecule/hydroxide ion of
the enzyme, and to make two hydrogen bonds with Thr199,
an amino acid residue conserved in all R-CAs, by Christian-
son’s group.22 However, due to the limited number of
interactions, phenol is a quite weak CAI23 (24 has a KI of
5.5 μM against hCA II).23 Sulfonamide 25, which makes a
host of various interactions both with the metal ion and
amino acid residues within the enzyme active site, behaves as
a very potent CAI (KI of 12 nM against hCA II).24 As seen
from this superposition presented in Figure 4, the hydrolysis
product of coumarins 1 and 2, the cis/trans-2-hydroxy-
cinnamic acids derivative 3 and 4b bind very differently to
the hCA II active site compared both to sulfonamide 25 or
the phenol 24. It may be seen that the scaffolds of these
substituted-cinnamic acids are present in an active site region
where only the terminal tail moiety of sulfonamide 25 lies,
i.e., toward the entrance of the active site cavity, which is also
the region with most variation in amino acid side chains
among the various CA isoforms.2,16,19 This may explain the
very interesting inhibition profile of coumarins against the 13
CA isoforms investigated here.
15 min) similarly to phenols. We demostrated in an earlier
work23 that a bicyclic phenol, which has some structural
features in common with the above-mentioned zwitterionic
form of the (thio)coumarins, inhibits hCA II in the same
micromolar range as many (thio)coumarin derivatives
studied in the present paper. The benzo(thio)pyrylium
phenoxide shown in A may then undergo hydrolysis by
the zinc-activated water molecule/hydroxide ion from the
enzyme cavity, which acts as a very potent nucleophile.2
It is thus probable that a cis-2-hydroxy-cinnamic acid
intermediate is formed (Chart 2 step B), which cannot bind
effectively in the restricted space near the Zn(II) ion (where
simple phenols and sulfonamides bind), due to its bulky
pendant arms, being thus reoriented toward the exit of the
active site cavity (where it has been evidenced,1 as stable cis
derivative for the bulky coumarin 1). A rearrangement of
the enzyme-inhibitor adduct may then occur, leading to
the trans-hydroxy/mercapto-cinnamic acids shown in
Chart 2, step C, provided that the R and R0 moieties from
the initial (thio)coumarin are not too bulky to interfere
with the binding to the enzyme active site. Indeed, for
the case investigated here (R = R0 = H), the trans-2-
hydroxycinnamic acid 4b was evidenced bound to the CA
active site, and not the cis isomer observed for the bulky
coumarin 1.1 The proposed mechanism is a general one (for
coumarins and thiocoumarins) and may thus account for
both the kinetic and X-ray crystallographic data presented
here and in the earlier work.1
Considering these facts exposed above, and also the ob-
servation that thiocoumarins in addition to coumarins do act
as CAIs, we propose the following general inhibition me-
chanism with these two classes of CAIs (Chart 2).
Coumarins/thiocoumarins may possess various tauto-
meric forms, such as the zwitterionic benzo(thio)pyrylium
phenoxides, which may bind within the CA active site
similarly to phenols (Chart 2, step A),7 i.e., by anchoring
to the zinc-bound water molecule/hydroxide ion.22 Alterna-
tively, the chromenone tautomer may bind to the enzyme,
similarly to phenols, as illustrated in Chart 2A. In fact, as we
shown in the earlier work,1 coumarins initially bind CAs with
micromolar affinity (when incubated with the enzyme for

342 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Maresca et al.
Table 2. Crystallographic Parameters and Refinement Statistics for the
hCA II-4b Adduct
6-10 were prepared by alkylation of 7-hydroxycoumarin with
alkyl/aralkyl halides12 (see Supporting Information for details).
Compounds 12-23 were reported earlier by one of our
groups.13 CA isozymes were recombinant ones prepared in
our laboratory as reported earlier.1,6-9 1H spectra were recorded
using a Bruker Advance III 300 MHz spectrometer. The chemi-
cal shifts are reported in parts per million (ppm) and the
coupling constants (J) are expressed in hertz (Hz). Melting
points (mp) were measured in open capillary tubes, unless
parameter
value
Crystal Parameter
space group
cell parameters
P21
a = 42.2 A
˚
˚
b = 41.5 A
˚
c = 72.4 A
€
otherwise stated, using a Buchi melting point B-540 melting
β = 104.5°
point apparatus and are uncorrected. Thin layer chromatogra-
phy (TLC) was carried out on Merck Silica Gel 60 F254
aluminum backed plates. Elution of the plates was carried out
using MeOH/DCM or MeOH/CHCl3 systems. Visualization
was achieved with UV light at 254 nm by dipping into a 0.5%
aqueous potassium permanganate solution or by exposure to
iodine. Flash column chromatography was carried out using
silica gel (obtained from Aldrich Chemical Co.) as the adsor-
bent. The crude product was introduced into the column as a
solution in the same elution solvent system, alternatively as a
powder obtained by mixing the crude product with the same
weight of silica gel in acetone and then removing the solvent in
vacuo at room temperature or dissolved into a minimum
amount of DCM or carbon tetrachloride. All moisture or air
sensitive reactions were carried out in oven-dried glassware
under a positive pressure of nitrogen or argon using standard
syringe/septa techniques. All the inert gases used (nitrogen and
argon) were passed through jacket columns fitted with activated
silica gel containing cobalt(II) chloride adsorbed as humidity
indicator The purity of all compounds has been determined by
means of analytical HPLC, performed on a reversed-phase C18
Bondapack column, with a Beckman EM-1760 instrument.
Both conbustion and HPLC confirmed a purity of >99.5%
for the compounds reported/investigated here.
˚
Data Collection Statistics (20.0-2.0 A)
no. of total reflections
no. of unique reflections
completeness (%)a
F2/σ(F2)
27978
16696
99.4 (99.8)
17.2 (2.9)
14.3 (37.0)
R-sym (%)
˚
Refinement Statistics (20.0-2.0 A)
R factor (%)
R-free (%)b
22.0
29.0
0.012
1.53
˚
rmsd of bonds from ideality (A)
rmsd of angles from ideality (deg)
a Values in parentheses relate to the highest resolution shell (2.1-
2.0 A). b Calculated using 5% of data.
˚
Conclusions
We report here a detailed investigation of substituted
coumarins and a thiocoumarin derivative, as inhibitors of
the zinc enzyme CA. All 13 catalytically active mammalian
CA isoforms, i.e., CA I-VA, VB, VI, VII, IX, XII, and
XIII-XV, were inhibited by these classes of compounds with
activity in the low nanomolar-micromolar range. The sub-
stitution pattern at the (thio)coumarin ring is the main player
in controlling the inhibitory power. Several beneficial sub-
stitution patterns were detected, among some 3-, 6-, 7-mono-,
and 3,6-, 4,7-, and 3,8-disubstituted coumarins incorporating
alkyl- alkoxy-, carboxy-, ester, hydroxy/aminoethyl, or hexa-
methylenetetramine moieties. Importantly, various CA iso-
forms showed very different inhibition profiles with these
compounds, leading thus to many (thio)coumarins acting as
isozyme-selective inhibitor. An X-ray crystal structure for the
adduct of the simple, unsubstituted coumarin with CA II
showed a binding mode different of the one reported earlier
for a natural product possessing a 5,6-disubstitution pattern
incorporating a bulky 6-moiety. We show here that these two
classes of CAIs, the coumarins and the thiocoumarins, are
formed by the hydrolytic attack of the zinc hydroxide species
of the enzyme to the (thio)coumarin bound in its neighbor-
hood within the enzyme active site. The initially formed cis-2-
hydroxy/mercapto-cinnamic acid may be stabilized and binds
toward the exit of the active site cavity for (thio)coumarins
substituted with bulky moieties in the 6 position. Alterna-
tively, this intermediate isomerizes to the trans derivative (for
less bulky coumarins/thiocoumarins), being bound in the
same active site region, but with different orientations com-
pared to the cis-2-hydroxy-cinnamic acids. The versatility of
the (thio)coumarin chemistry, the cis-trans isomerization
evidenced here and easy derivatization of these heterocyclic
rings, coupled with the nanomolar inhibition range of several
isozymes with biomedical applications, may afford isoform-
selective CAIs which render these classes of compounds
superior to the clinically used sulfonamides/sulfamates.
7-Ethoxy-2H-chromen-2-one 6. δH (300 MHz, DMSO-d6)
1.37 (3H, t J = 13.8, 20-H3), 4.15 (2H, q J = 13.8, 10-H2), 6.30
(1H, d J = 9.5, 3-H), 6.95 (1H, dd J = 8.4 2.4, 6-H), 6.99 (1H, d
J = 2, 8-H2), 7.63 (1H, d, J = 8.4, 5-H), 8.00 (1H, d J = 9.5, 4-
H). m/z (ESIþ) 191.18 ([M þ H]þ 12%), 213.15 ([M þ Na]þ
25%), 403.17 ([2M þ Na]þ 100%); mp: 91.8 °C.
7-Propoxy-2H-chromen-2-one 7. δH (400 MHz, DMSO-d6)
0.99 (3H, t J = 14.6, 30-H3), 1.73-1.78 (2H, m J = 14.6, 20-H2),
4.04 (2H, t J = 12.8, 10-H2), 6.28 (1H, d J = 9.4, 3-H), 6.94 (1H,
dd J = 8.4 2.4, 6-H),; 6.97 (1H, d J = 2, 8-H), 7.62 (1H, d J =
8.4, 5-H), 7.99 (1H, d, J = 9.4, 4-H). m/z (ESIþ) 205.23 ([M þ
H]þ 5%), 227.20 ([M þ Na]þ 100%), 241.19 (4); mp: 67.6 °C.
CA Inhibition. An Applied Photophysics stopped-flow ins-
trument has been used for assaying the CA catalyzed CO2
hydration activity.15 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 Hepes (pH 7.5) 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 0.01
nM were done thereafter with distilled-deionized water. Inhi-
bitor and enzyme solutions were preincubated together for 15
min to 72 h at room temperature (15 min) or 4 °C (all other
incubation times) prior to assay in order to allow for the
formation of the E-I complex or for the eventual active site
mediated hydrolysis of the inhibitor. Data reported in Table 1
show the inhibition after 6 h incubation, which led to the
completion of the in situ hydrolysis of the (thio)coumarin
and formation of the hydroxy/mercapto-cinnamic acid.1
Experimental Protocols
Chemistry. Coumarins 2, 5, 11, and 6-hydroxycoumarin were
commercially available (Sigma-Aldrich, Milan, Italy). Coumarins

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 343
The inhibition constants were obtained by nonlinear least-
squares methods using PRISM 3, as reported earlier,1,6,7 and
represent the mean from at least three different determinations.
Crystallization, X-Ray Data Collection, and Refinement.
Crystals of the hCA II-4b complex were obtained by using
the hanging-drop method for cocrystallizing the protein with the
ligand, as previously described.1 A monochromatic experiment
at the CuR wavelength was performed on a crystal of hCA II
grown in the presence of 2 (10 μM) by the rotation method on a
inhibitors: X-ray and molecular modeling study for the interaction of
a fluorescent antitumor sulfonamide with isozyme II and IX. J. Am.
Chem. Soc. 2006, 128, 8329–8335. (c) Nishimori, I.; Onishi, S.;
Takeuchi, H.; Supuran, C. T. The R- and β-classes carbonic anhydrases
from Helicobacter pylori as novel drug targets. Curr. Pharm. Des.
2008, 14, 622–630.
(7) Ebbesen, P.; Pettersen, E. O.; Gorr, T. A.; Jobst, G.; Williams, K.;
Kienninger, J.; Wenger, R. H.; Pastorekova, S.; Dubois, L.; Lambin,
P.; Wouters, B. G.; Supuran, C. T.; Poellinger, L.; Ratcliffe, P.;
€
Kanopka, A.; Gorlach, A.; Gasmann, M.; Harris, A. L.; Maxwell,
P.; Scozzafava, A. Taking advantage of tumor cell adaptations to
hypoxia for developingnew tumor markers and treatment strategies.
J. Enzyme Inhib. Med. Chem. 2009, 24 (S1), 1–39.
PX-Ultra sealed-tube diffractometer (Oxford Diffraction) at
100 K. The crystal diffracted up to 2.0
˚
A resolution
˚
(resolution: 20.0-2.0 A), with 16696 unique reflections out of
27978 reflections, and belonged to the space group P21 (a =
(8) Schlicker, C.; Hall, R. A.; Vullo, D.; Middelhaufe, S.; Gertz, M.;
Supuran, C. T.; Muhlschlegel, F. A.; Steegborn, C. Structure and
inhibition of the CO2-sensing carbonic anhydrase Can2 from the
pathogenic fungus Cryptococcus neoformans. J. Mol. Biol. 2009,
385, 1207–1220.
˚
˚
˚
42.2 A, b = 41.5 A, c = 72.4 A and R = γ = 90°, β = 104.5°).
Data were processed with CrysAlis RED (Oxford Diffraction
2006).25 The structure was analyzed by difference Fourier
technique, using the PDB file 1CA2 as starting model. The
refinement was carried out with the program REFMAC5,26 and
model building and map inspections were performing using the
COOT program.27 The final model of the complex hCA II-4b
adduct had an R factor of 22.0% and R-free 29.0% in the
ꢁ
(9) (a) Thiry, A.; Dogne, J. M.; Masereel, B.; Supuran, C. T. Targeting
tumor-associated carbonic anhydrase IX in cancer therapy. Trends
Pharmacol. Sci. 2006, 27, 566–573. (b) Svastova, E.; Hulíkova, A.;
Rafajova, M.; Zatovicova, M.; Gibadulinova, A.; Casini, A.; Cecchi,
A.; Scozzafava, A.; Supuran, C. T.; Pastorek, J.; Pastorekova, S.
Hypoxia activates the capacity of tumor-associated carbonic anhydrase
IX to acidify extracellular pH. FEBS Lett. 2004, 577, 439–445.
(10) (a) Supuran, C. T. Diuretics: From classical carbonic anhydrase
inhibitors to novel applications of the sulfonamides. Curr. Pharm.
Des. 2008, 14, 641–648. (b) Supuran, C. T.; Di Fiore, A.; De Simone, G.
Carbonic anhydrase inhibitors as emerging drugs for the treatment of
obesity. Expert Opin. Emerging Drugs. 2008, 13, 383–392. (d) De
Simone, G.; Di Fiore, A.; Supuran, C. T. Are carbonic anhydrase
inhibitors suitable for obtaining antiobesity drugs? Curr. Pharm.
Des. 2008, 14, 655–660.
(11) (a) Minakuchi, T.; Nishimori, I.; Vullo, D.; Scozzafava, A.;
Supuran, C. T. Molecular cloning, characterization and inhibition
studies of the Rv1284 β-carbonic anhydrase from Mycobacterium
tuberculosis with sulfonamides and a sulfamate. J. Med. Chem.
2009, 52, 2226–2232. (b) Nishimori, I.; Minakuchi, T.; Vullo, D.;
Scozzafava, A.; Innocenti, A.; Supuran, C. T. Carbonic anhydrase
inhibitors. Cloning, characterization and inhibition studies of a new
β-carbonic anhydrase from Mycobacterium tuberculosis. J. Med.
Chem. 2009, 52, 3116–3120.
˚
resolution range 20.0-2.0 A, with a rms deviation from stan-
˚
dard geometry of 0.012 A in bond lengths and 1.53° in angles.
The correctness of stereochemistry was finally checked using
PROCHECK.28 Crystallographic parameters and refinement
statistics are shown in Table 2.
Acknowledgment. This research was financed in part by a
grantofthe 6thFrameworkProgramme (FP) of the European
Union (DeZnIT project) and by a grant of the 7th FP of EU
(Metoxia project).
Supporting Information Available: The synthesis and char-
acterization of compounds 6-10 are described in detail. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(12) Sethna, S. M.; Shah, N. M. The chemistry of coumarins. Chem.
Rev. 1945, 36, 1–62.
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Pirotte, B.; Jiang, K. Y.; Masereel, B.; de Tullio, P.; Delarge, J.;
Reboud-Ravaux, M. Esters and amides of 6-(chloromethyl)-2-oxo-
2H-1-benzopyran-3-carboxylic acid as inhibitors of alpha-chymo-
trypsin: significance of the “aromatic” nature of the novel ester-
type coumarin for strong inhibitory activity. J. Med. Chem. 1996,
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Delarge, J.; Reboud-Ravaux, M. 6-Substituted 2-oxo-2H-1-benzopyr-
an-3-carboxylic acid as a core structure for specific inhibitors of human
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