Novel coumarins and 2-thioxo-coumarins as inhibitors of the tumor-associated carbonic anhydrases IX and XII

Article abstract of DOI:10.1016/j.bmc.2012.02.014

A series of coumarins incorporating tert-butyl-dimethylsilyloxy- or allyoxy- moieties in positions 4-, 6 or 7 of the heterocyclic ring have been synthesized and then converted to the corresponding 2-thioxo-coumarins. Other derivatives incorporating hydrox

Full text of DOI:10.1016/j.bmc.2012.02.014

Bioorganic & Medicinal Chemistry 20 (2012) 2266–2273
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Bioorganic & Medicinal Chemistry
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Novel coumarins and 2-thioxo-coumarins as inhibitors
of the tumor-associated carbonic anhydrases IX and XII
⇑
Fabrizio Carta, Alfonso Maresca, Andrea Scozzafava, Claudiu T. Supuran
Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of coumarins incorporating tert-butyl-dimethylsilyloxy- or allyoxy- moieties in positions 4-, 6 or
7 of the heterocyclic ring have been synthesized and then converted to the corresponding 2-thioxo-
coumarins. Other derivatives incorporating hydroxyethyloxy-, tosylethoxy- and 2-fluroethyloxy- moie-
ties in position 7 of the coumarin ring were synthesized together with derivatives of 4-methyl-7-amino
coumarin incorporating acetamido, 3,5-dimethylphenylureido- and tert-butyloxycarbonylamido func-
tionalities. All these compounds were assayed as inhibitors of the metalloenzyme carbonic anhydrase
(CA, EC 4.2.1.1). The human (h) cytosolic isoforms hCA I and II were weakly inhibited (hCA I) or not inhib-
ited at all (hCA II) by these (thioxo)coumarins whereas the tumor-associated transmembrane isoforms
hCA IX and XII were inhibited with efficiencies from the submicromolar to the low micromolar range
by many of these derivatives. The structure–activity relationship for these classes of less investigated
CA inhibitors are delineated, with the potential of using them as leads to obtain isoform-selective inhib-
itors with excellent affinity for CA IX and XII (validated antitumor targets) which do not significantly inhi-
bit the cytosolic offtarget isoforms hCA I and II.
Received 14 December 2011
Revised 2 February 2012
Accepted 4 February 2012
Available online 13 February 2012
Keywords:
Carbonic anhydrase
Coumarin
Transmembrane isoform
Tumor-associated enzyme
Selective inhibition
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
to occlude the entrance to the enzyme active site, a mechanism
never evidenced before for CA inhibition.1,2 Thiocoumarins (pos-
Coumarins are a class of widely spread natural compounds pos-
sessing a variety of biological activities, which was only recently
discovered to inhibit the zinc enzyme carbonic anhydrase (CA, EC
4.2.1.1).1–5 CAs are ubiquitous enzymes in organisms throughout
the tree of life, with several genetically distinct families encoding
them in prokaryotes and eukaryotes.4 They are inhibited by metal
complexing anions, sulfonamides and their isosteres (sulfamates,
sulfamides, etc.), phenols and polyamines,4 which bind either to
the metal ion within the enzyme active site or are anchored to
sessing the endocyclic sulfur atom in position 1 of the heterocyclic
ring) were also shown to behave in a similar manner to the couma-
rins, but this class was scarcely investigated until now, with only
one such compound reported to possess CA inhibitory properties,2
whereas derivatives incorporating the sulfur atom in position 2
(exocyclic sulfur) were not investigated at all up to now as CA
inhibitors (CAIs). Several observations emerged during these stud-
ies of coumarins/thiocoumarins as CAIs: (i) they bind in hydrolyzed
form and do not interact with the catalytically crucial metal ion
from the active site cavity, constituting a new category of mecha-
nism-based inhibitors (they act as ‘prodrug inhibitors’);1–3 (ii) the
substituted-2-hydroxy-cinnamic acids formed from the original
coumarin by hydrolysis were observed bound within the active site
either as the cis isomer,1b as well as trans isomers2 (by means of
X-ray crystallography of enzyme-inhibitor adducts), depending
on the substitution pattern at the original coumarin prodrug; (iii)
these inhibitors were observed bound at the entrance of the en-
zyme active site, plugging its entrance, and blocking thus the cat-
alytic activity of the enzyme.1,2 An important observations was
also that this region of the CA active site is the most variable one
among the 16 CA isoforms present in mammals,3,8,9 and this may
explain why many coumarin derivatives show a high selectivity ra-
tio for inhibiting various CA isoforms (e.g., CA VA, VII, IX, XII, XIII
and XIV).1–3 Many of these isoforms have pharmacologic applica-
tions for obtaining antiglaucoma, antiobesity, anticonvulsant or
antitumor drugs/diagnostic agents.10
the water molecule coordinated to it.1–7 The coumarin
a-CA
inhibition mechanism is different from that of the other classes
of inhibitors, and was only recently deciphered.1–3 A natural
product coumarin, 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-
chromen-2-one A, isolated from the Australian plant Leionema
ellipticum,1a was shown to possess significant CA inhibitory activ-
ity.1 By means of X-ray crystallography of its 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 origi-
nal coumarin A.1b The same situation has been thereafter observed
for the simple, unsubstituted coumarin B.2 The 2-hydroxy-
cinnamic acids formed from the original coumarins were found
⇑
Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.014

F. Carta et al. / Bioorg. Med. Chem. 20 (2012) 2266–2273
2267
OH
tBuMe2SiCl, Et3N
HO
Si O
THF, r.t.
O
O
O
O
1a: 6-OH
2a: 6-OH; 64% yield
2b: 7-OH; 58% yield
1b: 7-OH
O
O
MeO
O
O
A
B
Lawesson's reagent
Tol., reflux
O
1.0M TBAF in THF
THF, r.t.
Si O
HO
R
R
O
S
O
S
O
O
O
O
O
O
3a: 6-OH;60% yield
4a:6-OH; 96% yield
4b: 7-OH; 55% yield
3b: 7-OH; 61% yield
O
D
C
Scheme 1. Preparation of silylated coumarins/2-thioxo-coumarins incorporating 6-
R = Me, Et, Pr, Bu, Bn, adamantyl
and 7-hydroxy moieties.
Ki > 100 uM (CA I and II)
Ki = 40-90 nM (CA IX)
Ki = 0.98 - 62 nM (CA XII)
Ki > 100 uM (CA I, II, XII
Ki = 60 - 8000 nM (CA IX)
fluorescein- or near infrared (NIR) tails are highly effective in visu-
alizing hypoxic tumor regions in experimental animals possessing
different xenografted tumors.12,13 Thus, 7-hydroxycoumarin 1b
was treated with chloroethanol in the presence of potassium car-
bonate and potassium iodide, leading to 7-hydroxyethyloxycoum-
arin 7. This compound was tosylated by reaction with tosyl
chloride in pyridine leading to the tosylate 8, which has been con-
verted to 7-(2-fluoroethyloxy)-coumarin 9 by a nucleophilic sub-
stitution reaction with TBAF in THF (Scheme 2). Another group of
derivatives, compounds 10–12, were obtained from 4-methyl-7-
amino-coumarin E (commercially available compound) which
was reacted with acetyl chloride, an arylisocyanate (i.e., 3,5-
dimethylphenylisocyanate) or Boc anhydride, in order to explore
the reactivity of the 7-amino group from coumarin E and its pro-
pensity to be derivatized for generating chemical diversity for this
type of CAIs. Indeed, both the acetamide 10, the urea derivative 11
and the carbamate 12 were obtained in a one step reaction with an
excellent yield (see Section 4). All compounds investigated here
were characterized by physico-chemical procedures (1H, 13C and
19F NMR, MS) and elemental analysis which confirmed their struc-
tures (see Section 4).
More recently we demonstrated3 that coumarins incorporating
hydroxy and acetylated moieties may lead to interesting CAIs,
some of which show high selectivity for inhibiting the tumor-asso-
ciated isoforms hCA IX and XII.3,4 For example, for the isomeric 7,8-
disubstituted- and 6-7-disubstituted coumarins C and D, only the
first substitution pattern led to highly effective CA IX/XII inhibitors
(in the subnanomolar–nanomolar range)3b whereas the corre-
sponding isomers D were ineffective as CA I, II and XII inhibitors
and much less effective (compared to C) as hCA IX inhibitors.3b
Considering that the substitution pattern and the nature (and
number) of moieties present in the various positions of the couma-
rin ring are the main factors influencing CA inhibitory properties as
well as selectivity profile in this class of inhibitors, we report here
the synthesis and enzyme inhibitory activity of a series of variously
substituted coumarins and 2-thioxo-coumarins incorporating sily-
lated, allyl, ether, arylureido and carbamate moieties. The rationale
of this study is based on the scarcity of data regarding this type of
substitution patterns in various positions of the coumarin ring sys-
tem. Furthermore, we obtained a rather large number of 2-thioxo-
coumarins (not investigated up until now as CAIs) which complete
our insights into the structure–activity relationship (SAR) for this
class of inhibitors.
2.2. Carbonic anhydrase inhibition studies
Inhibition data with compounds 2–12 reported here and the
simple coumarin B as standard, against four CA isozymes, that is,
hCA I, II, IX and XII, are shown in Table 1.14 The following struc-
ture–activity relationship (SAR) observations can be drawn from
data of Table 1 for these coumarin/2-thioxo-coumarin derivatives:
2. Results and discussion
2.1. Chemistry
In order to generate chemical diversity for obtaining coumarins/
2-thioxo-coumarins with CA inhibitory activity, the 6-/7-hydroxy-
substituted coumarins 1a and 1b were used as starting materials.
These compounds were silylated with tert-butyt-dimethylsilyl
chloride leading to the key intermediates 2a,b which were thereaf-
ter thionylated at the C2 position by treatment with Lawesson’s re-
agent (Scheme 1).11 The 2-thioxo-coumarins 3a,b thus obtained
afforded 6-/7-hydroxy-2-thioxo-coumarins 4a,b by treatment with
tetrabutyl ammonium fluoride (TBFA), as shown in Scheme 1.
Alternatively, 6-, 7- or 4-hydroxycoumarins 1a–1c were trans-
formed to the corresponding allyl ethers 5a–5c by treatment with
allyl bromide in the presence of cesium salts. Coumarins 5a–5c
were then converted to the corresponding 2-thioxo-coumarins
incorporating allylether functionalities, 6a–6c, by reaction with
Lawesson’s reagent (Scheme 2). Compounds 7–9 were then ob-
tained in order to devise a facile and rapid reaction for incorporat-
ing fluorine in the molecule of a coumarin CAI, with the scope of
eventually extending this reaction to 18F-containing compounds,
which may be useful as PET imaging agents. It has been in fact re-
ported by our group that fluorescent compounds incorporating
(i) The cytosolic isoform hCA I (h = human enzyme) was weakly
inhibited by the simple, hydroxyl-substituted coumarins
1a–1c investigated earlier,3a as well as by the variously
substituted (thioxo)coumarin derivatives 2–12, with inhibi-
tion constants in the range of 7.17–91.7 lM. Aminocouma-
rins E and the parent unsubstituted coumarin B were
slightly more effective as hCA I inhibitors (KIs of 3.10–
5.56 lM). The most effective hCA I inhibitors (but they are
all rather weak inhibitors) were the simple coumarins/2-thi-
oxo-coumarins 2a–4a incorporating the tert-butyl-dimeth-
ylsilyl moiety (or without such a substitution) and it may
be seen that there is no important difference of activity
between the coumarins/2-thioxo-coumarins, and also
between the compounds with or without the silyl moiety
(compare 3a/3b with 4a/4b, for example). Another group of
compounds showing a modest hCA I activity was 6a–6c
(KIs of 7.60–9.24 lM), which are 2-thioxo-coumarins incor-
porating allyl ether substitutions in various positions of
the ring. These compounds were much more effective

2268
F. Carta et al. / Bioorg. Med. Chem. 20 (2012) 2266–2273
Cs2CO3
Lawesson's reagent
Tol. , reflux
Br
DMF, 60°C, O.N.
O
O
HO
O
O
O
S
O
O
5a: 6-substituted; 62 %
5b: 7-substituted; 85 %
5c: 4-substituted; 70 %
6a: 6-substituted; 62 %
6b: 7-substituted; 87 %
6c: 4-substituted; 61 %
1a: 6-OH
1b: 7-OH
1c: 4-OH
Cl
HO
K2CO3; KI
DMF, 60°C, 5h
TsCl
Pyr., r.t.,
1.0M TBAF in THF
TsO
THF, r.t.
F
HO
O
O
O
O
O
O
O
O
O
9; 40 % yield
8; 35 % yield
7; 72 % yield
O
N
H
O
O
AcCl, Et3N
DCM, reflux, 7h
10; 73 % yield
O
ArNCO, Et3N
Acetone, reflux, 24 h
H2N
O
O
N
H
N
H
O
O
E
11; 23 %yield
(Boc)2O, Et3N
THF, reflux
O
O
N
H
O
O
12; 28 % yield
Scheme 2. Preparation of coumarin/2-thioxo-coumarin derivatives 5–12.
compared to the corresponding coumarins 5a–5c (KIs of
30.3–72.9 M). The 7-substituted derivatives were generally
very similar inhibitory behavior towards this isoform. The
allyl-substituted coumarins 5a–5c were also effective hCA
l
better inhibitors compared to the 6- or 4-substituted com-
pounds bearing the same substituent (Table 1). The remain-
ing substitution patterns, present in derivatives 7–12 led to
IX inhibitors with KIs in the range of 0.21–0.93 lM. For this
substitution pattern, the position of the allylether function-
ality strongly influenced the inhibitory power, with the
4-substituted compound 5c being 4.4-times a better inhibi-
tor compared to the 6-substituted derivative 5a. The
7-substituted compound 5b showed an intermediate behav-
ior between the two isomers mentioned above (Table 1).
Conversion of the allyl-substituted coumarins 5a–5c to the
corresponding 2-thioxo-coumarins 6a–6c led to an impor-
tant loss of hCA IX inhibitory effects, the thiocoumarins
being an order of magnitude weaker inhibitors compared
to the parent coumarins 5a–5c (KIs in the range of 3.04–
much less effective hCA I inhibitors (KIs of 24.4–91.7 lM). In
fact, a modest inhibition of this isoform is a desired feature
of such CAIs which should normally target selectively the
tumor-associated isoforms hCA IX and XII, and should not
possess potent hCA I and II inhibitory effects.
(ii) The ubiquitous, offtarget isoforms hCA II was not inhibited
by all of the coumarins investigated here (1–12 and E),
which showed KIs in the range of >200
ed coumarin B was a weak hCA II inhibitor, with a KI of
9.2
M, as reported earlier.1
(iii) The tumor associated isoform hCA IX was weakly or not at
all inhibited by coumarin B (KIs of >200 M), whereas the
compounds investigated here were effective or medium
potency inhibitors, with KIs in the range of 0.19–3.23
lM. The unsubstitut-
l
3.26
olar hCA IX inhibitory effects. The best inhibitor was the
tosylate 8 (KI of 0.53 M), followed by the fluoroderivative
9 (KI of 0.68 M), whereas the hydroxyethyl derivative 7
was a slightly weaker inhibitor (KI of 0.92 M). All three
lM). Compounds 7–9 showed interesting, submicrom-
l
l
l
lM
l
(Table 1). Thus, the silylated derivatives 2 and 3 as well as
the hydroxy-substituted 2-thioxo-coumarins 4a,b were sub-
micromolar inhibitors with KIs in the range of 0.78–0.96 lM,
amino-coumarin derivatives 10–12, incorporating acetam-
ido, ureido or carbamate functionalities were also effective,
submicromolar hCA IX inhibitors (KI in the range of 0.39-
but were slightly less effective compared to the parent
hydroxylated coumarins from which they were prepared
(Table 1) investigated earlier.3a The sylilation however did
not lead to a strong loss of CA IX inhibitory activity. The
transformation of the coumarin to the corresponding 2-thi-
oxo-coumarin, as well as the introduction of the tert-butyl-
dimethylsilyl moiety did not markedly influence the CA
inhibitory effects of these compounds which all showed a
0.97
to the parent, unsubstituted aminocoumarin
7.64 M).
l
M), being much better hCA IX inhibitors compared
E
(KI of
l
(iv) hCA XII is also present in many tumor types, being like hCA
IX a transmembrane isoform, with an extracellular active
site, and involved in many physiologic and pathologic pro-
cesses.4 It may be observed that again coumarin B was inef-
fective as hCA XII inhibitor (KI >200 lM), whereas the

F. Carta et al. / Bioorg. Med. Chem. 20 (2012) 2266–2273
2269
Table 1
heterocyclic ring, which have been obtained by a simple reaction
step and then converted to the corresponding 2-thioxo-coumarins,
a class of not investigated CAIs up until now. Other coumarin
derivatives incorporating hydroxyethyloxy-, tosylethoxy- and 2-
fluroethyloxy- moieties in position 7 of the coumarin ring were
synthesized in order to obtain compounds which may be labeled
with radioactive fluorine, for PET applications. Furthermore, deriv-
atives of 4-methyl-7-amino coumarin incorporating acetamido,
3,5-dimethylphenylureido- and tert-butyloxycarbonylamido func-
tionalities have also bee prepared from 7-aminocoumarin. All
these compounds have been assayed as inhibitors of several CA iso-
forms with medicinal chemistry applications. The cytosolic iso-
forms hCA I and II were weakly inhibited (hCA I) or not inhibited
at all (hCA II) by these (thio)coumarins whereas the tumor-
associated transmembrane isoforms hCA IX and XII were inhibited
with efficiencies from the submicromolar to the low micromolar
range by many of these derivatives. The structure–activity rela-
tionship of these classes of less investigated CA inhibitors are
delineated, with the potential of using them as leads to obtain iso-
form selective inhibitors with excellent affinity for CA IX and XII
(validated antitumor targets) which do not significantly inhibit
the cytosolic offtarget isoforms hCA I and II.
Inhibition data against hCA I, II, IX and XII with (thio)coumarins 2–12 by a stopped-
flow CO2 hydrase assay (incubation time of enzyme with inhibitor for 6 h)14
RO
RNH
O
X
O
X
1-9
E, 10-12
No.
X
R
hCA I
KIa
(
lM)
hCA XII
hCA II
hCA IX
1ªb
1bb
1cb
2a
3a
4a
2b
3b
4b
5a
5b
5c
6a
6b
6c
7
8
O
O
O
O
S
S
O
S
H
H
H
>100
58.4
95.0
8.78
7.57
7.17
8.32
8.18
8.02
30.3
72.9
43.2
8.51
7.60
9.24
24.4
66.3
42.9
47.7
38.8
91.7
5.56
3.1
>100
>100
>100
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
9.2
0.19
0.48
0.41
0.80
0.86
0.80
0.85
0.96
0.78
0.93
0.73
0.21
3.26
3.23
3.04
0.92
0.53
0.68
0.39
0.61
0.97
7.64
>200
0.68
0.75
6.30
0.28
0.31
0.34
0.83
0.35
0.32
0.80
0.64
0.88
1.25
2.83
1.27
0.63
0.90
0.59
0.91
0.64
0.58
9.13
>200
t-Bu(Me2)Si
t-Bu(Me2)Si
H
t-Bu(Me2)Si
t-Bu(Me2)Si
H
S
O
O
O
S
S
S
O
O
O
O
O
O
O
CH2@CH–CH2
CH2@CH–CH2
CH2@CH–CH2
CH2@CH–CH2
CH2@CH–CH2
CH2@CH–CH2
HOCH2CH2
Ts–OCH2CH2
FCH2CH2
4. Experimental
4.1. Chemistry
9
10
11
12
E
Ac
1H, 13C, DEPT, COSY, HMQC and HMBC spectra were recorded
using a Bruker Advance III 400 MHz spectrometer. The chemical
shifts are reported in parts per million (ppm) and the coupling con-
stants (J) are expressed in Hertz (Hz). For all new compounds DEPT,
COSY, HMQC and HMBC were routinely used to definitely assign
the signals of 1H and 13C. Infrared spectra were recorded on a Per-
kin Elmer Spectrum R XI spectrometer as solids on KBr plates.
Melting points (mp) were measured in open capillary tubes, unless
otherwise stated, using a Büchi Melting Point B-540 melting point
apparatus and are uncorrected. Thin layer chromatography (TLC)
was carried out on Merck Silica Gel 60 F254 aluminium backed
plates. Elution of the plates was carried out using ethyl acetate/
n-hexane or MeOH/DCM systems. Visualization was achieved with
UV light at 254 nm, by dipping into a 0.5% aqueous potassium per-
manganate solution, by Hanessian’s Stain solution and heating
with a hot air gun or by exposure to iodine.
3,5-Me2C6H3NHCO
t-Bu–OCO
H
Coumarin
a
Mean from 3 assays. Errors were in the range of 5–10% of the reported value
(data not sown).
b
From Ref. 3a.
derivatives 1–12 investigated here were low micromolar or
submicromolar inhibitors (KIs in the range of 0.28–
2.83 M). Again the silylated derivatives 2 and 3 as well as
l
the hydroxy-substituted 2-thioxo-coumarins 4a,b were sub-
micromolar inhibitors with similar KIs, in the range of 0.28–
0.83 lM, but in this case the sylilation improved the inhibi-
tory power compared to the parent hydroxyl-substituted
coumarins investigated earlier3a (Table 1). As for the case
of hCA IX inhibition discussed above, the transformation of
the coumarin to the corresponding 2-thioxo-coumarin, as
well as the introduction of the tert-butyl-dimethylsilyl moi-
ety did not influence significantly the CA XII inhibitory
effects of these compounds. For the allyl-substituted couma-
rins/2-thioxo-coumarins 5 and 6, the first group of deriva-
tives led to effective submicromolar inhibitors (KIs in the
Coumarins 1a–1c and B, E, solvents and other chemicals were
used as supplied from Aldrich Chemical Co., Acros, Fisher, Alfa Ae-
sar or Lancaster Synthesis.
4.1.1. Synthesis of 6-(tert-butyldimethylsilyloxy)-2H-chromen-
2-one (2a) and 7-(tert-butyldimethylsilyloxy)-2H-chromen-2-
one (2b)
range of 0.64–0.88
lM) whereas the 2-thioxo-coumarins
were less effective (KIs in the range of 1.25–2.83
l
M) (Table
O
HO
1). The remaining compounds 7–12, incorporating function-
alities derived from the hydroxyethyloxy derivative 7 or the
7-aminocoumarin E, were again quite effective as hCA XII
Si
O
O
O
O
inhibitors, with KIs in the range of 0.58–0.91 lM. As for
hCA IX, aminocoumarin E was a much weaker hCA XII inhib-
itor compared to its derivatives 10–12.
A solution of 6-hydroxy-2H-chromen-2-one or 7-hydroxy-2H-
chromen-2-one (0.5 g, 1.0 equiv) was treated at rt with tert-butyl-
dimethylsilyl chloride (1.1 equiv) and Et3N (1.0 equiv) in THF. The
reaction was stirred at rt until starting material was consumed
(TLC monitoring) then quenched with H2O (40 ml) and extracted
with ethyl acetate (3 ꢀ 15 ml). The combined organic layers were
washed with H2O (2 ꢀ 20 ml), dried over Na2SO4, filtered-off and
concentrated under vacuo to give a residue that was purified by sil-
ica gel column chromatography eluting with 20% ethyl acetate/n-
hexane v/v.
3. Conclusion
We extend our earlier investigations on coumarins as CAIs.1–3
We report here a series of coumarins incorporating tert-butyl-dim-
ethylsilyloxy- or allyoxy- moieties in positions 4-, 6 or 7 of the

2270
F. Carta et al. / Bioorg. Med. Chem. 20 (2012) 2266–2273
7.17 (1H, d, J 9.6, 3-H), 7.76 (1H, d, J 9.2, 5-H), 7.90 (1H, d, J 9.6, 4-
H); dC (100 MHz, DMSO-d6) 198.2 (C@S), 159.0, 158.2, 137.2, 130.9,
127.5, 126.1, 119.8, 115.9, 26.3, 18.9, ꢁ3.8.
5
8
4
6
O
3
Si
7
O
O
2
4.1.3. Synthesis of 6-hydroxy-2H-chromene-2-thione (4a) and
synthesis of 7-hydroxy-2H-chromene-2-thione (4b)
4.1.1.1. 6-(tert-Butyldimethylsilyloxy)-2H-chromen-2-one (2a).
Yield 64% yield; dH (400 MHz, DMSO-d6) 0.25 (6H, s, –Si-(CH3)2),
1.00 (9H, s, –Si-C(CH3)3), 6.51 (1H, d J 9.6, 3-H), 7.13 (1H, dd, J
9.4, 2.4, 7-H), 7.25 (1H, d, J 2.4, 5-H), 7.33 (1H, d, J 9.4, 8-H), 8.00
(1H, d, J 9.6, 4-H); dC (100 MHz, DMSO-d6) 161.0 (C@O), 152.2,
149.2, 144.8, 124.9, 120.3, 118.6, 118.3, 117.4, 26.4, 18.8, ꢁ3.8.
TBAF 1.0M
THF
O
HO
Si
O
S
O
S
6-(tert-Butyldimethylsilyloxy)-2H-chromene-2-thione (3a) or
7-(tert-butyldimethylsilyloxy)-2H-chromene-2-thione (3b) (0.3 g,
1.0 equiv) was dissolved in THF (2.0 ml) and treated at rt with TBAF
1.0 M in THF (1.1 equiv). The reaction was stirred at rt until start-
ing material was consumed (TLC monitoring) and then was
quenched with 3.0 M aqueous hydrochloric acid, extracted with
ethyl acetate (3 ꢀ 15 ml). The combined organic layers were
washed with H2O (3 ꢀ 20 ml), brine (3 ꢀ 20 ml) dried over Na2SO4,
filtered, concentrated under vacuo to give a residue that was puri-
fied by silica gel column chromatography eluting with 50% ethyl
acetate/n-hexane v/v.
5
4
6
3
O 7
O
O
Si
2
8
4.1.1.2. 7-(tert-Butyldimethylsilyloxy)-2H-chromen-2-one (2b).
Yield 58% yield; dH (400 MHz, DMSO-d6) 0.29 (6H, s, –Si-(CH3)2),
1.00 (9H, s, –Si-C(CH3)3), 6.34 (1H, d J 9.6, 3-H), 6.88 (1H, dd, J
9.4, 2.4, 6-H), 6.92 (1H, d, J 2.4, 8-H), 7.65 (1H, d, J 9.4, 5-H), 8.04
(1H, d, J 9.6, 4-H); dC (100 MHz, DMSO-d6) 162.2 (C@O), 156.4,
145.4, 130.6, 118.1, 114.2, 112.3, 107.9, 103.1, 26.7, 18.7, ꢁ2.3.
5
4
6
HO
3
4.1.2. Synthesis of 6-(tert-butyldimethylsilyloxy)-2H-chromene-
2-thione (3a) and 7-(tert-butyldimethylsilyloxy)-2H-chromene-
2-thione (3b)
7
O
S
2
8
4.1.3.1. 6-Hydroxy-2H-chromene-2-thione (4a). Yield 96% yield;
silica gel TLC Rf 0.35 (ethyl acetate/n-hexane 50% v/v); dH
(400 MHz, DMSO-d6) 7.12 (1H, d J 2.8, 5-H), 7.18 (1H, dd, J 9.2,
2.8, 7-H), 7.25 (1H, d, J 9.6, 3-H), 7.50 (1H, d, J 9.2, 8-H), 7.87
(1H, d, J 9.6, 4-H), 10.05 (1H, br s, exchange with D2O, OH); dC
(100 MHz, DMSO-d6) 197.8 (C@S), 155.8, 151.1, 137.0, 129.9,
122.1, 121.9, 118.2, 112.9.
Lawesson's Reagent
O
O
Si
Si
O
S
O
O
Tol. reflux
6-(tert-Butyldimethylsilyloxy)-2H-chromen-2-one (2a) or 7-
(tert-butyldimethylsilyloxy)-2H-chromen-2-one (2b) (0.5 g,
1.0 equiv) was dissolved in dry toluene (20 ml) and treated with
Lawesson’s reagents (1.5 equiv) at reflux for 3 h. The mixture was
cooled down to rt, solvent was removed under vacuo and the res-
idue was partitioned between H2O and ethyl acetate. The organic
layer was washed with H20 (3 ꢀ 15 ml), dried over Na2SO4, filtered
and concentrated in vacuo o give a residue that was purified by sil-
ica gel column chromatography eluting with 20% ethyl acetate/n-
hexane v/v.
5
4
6
7
3
HO
O
S
2
8
4.1.3.2. 6-Hydroxy-2H-chromene-2-thione (4b). Yield 55% yield;
silica gel TLC Rf 0.40 (ethyl acetate/n-hexane 50% v/v); dH
(400 MHz, DMSO-d6) 6.93 (2H, m, 6-H, 8-H), 7.09 (1H, d, J 9.6, 3-
H), 7.68 (1H, d, J 9.2, 5-H), 7.85 (1H, d, J 9.6, 4-H), 10.96 (1H, br
s, exchange with D2O, OH); dC (100 MHz, DMSO-d6) 198.1 (C@S),
163.3, 159.0, 137.8, 130.9, 126.0, 115.9, 114.1, 102.8.
5
4
6
O
3
Si
7
O
S
2
8
4.1.4. Synthesis of allyloxycoumarines 5a–c
Hydroxycoumarin (1.0 g, 1.0 equiv), Cs2CO3 (3.0 equiv) and ally-
lbromide (3.0 equiv) were dissolved in dry DMF (30 ml) and the
mixture was stirred at 60 °C O.N. The reaction was quenched with
slush and extracted with DCM (3 ꢀ 20 ml). The combined organic
layers were washed with brine (3 ꢀ 20 ml), H2O (5 ꢀ 20 ml), dried
over Na2SO4, filtered and concentrated under vacuo to give a resi-
due that was crystallized from MeOH/H2O.
4.1.2.1. 6-(tert-Butyldimethylsilyloxy)-2H-chromene-2-thione
(3a). Yield 60% yield; silica gel TLC Rf 0.40 (ethyl acetate/n-hexane
20% v/v); dH (400 MHz, DMSO-d6) 0.27 (6H, s, –Si-(CH3)2), 1.01 (9H,
s, –Si-C(CH3)3), 7.25 (1H, dd J 9.2, 2.8, 7-H), 7.29 (1H, d, J 9.6, 3-H),
7.32 (1H, d, J 2.8, 5-H), 7.55 (1H, d, J 9.2, 8-H), 7.90 (1H, d, J 9.6, 4-
H); dC (100 MHz, DMSO-d6) 198.0 (C@S), 153.3, 152.2, 136.8, 130.1,
126.0, 122.2, 118.4, 118.3, 26.4, 18.8, ꢁ3.8.
2'
5
4
5
4
6
6
3
O
3
3'
1'
Si
O
O
S
7
7
O
O
2
2
8
8
4.1.4.1. 6-(Allyloxy)-2H-chromen-2-one yield (5a). 62% yield; dH
(400 MHz, DMSO-d6) 4.64 (2H, d J 8.0, 10-H2), 5.32 (1H, dd, J 13.2,
4.8, 30-HH), 5.48 (1H, dd, J 15.6, 4.8, 30-HH), 6.10 (1H, m, 20-H),
6.16 (1H, d, J 9.6, 3-H), 7.25 (1H, dd, J 9.2, 2.4, 7-H), 7.41 (1H, d, J
4.1.2.2. 7-(tert-Butyldimethylsilyloxy)-2H-chromene-2-thione
(3b). Yield 61% yield; silica gel TLC Rf 0.38 (ethyl acetate/n-hexane
20% v/v); dH (400 MHz, DMSO-d6) 0.31 (6H, s, –Si-(CH3)2), 1.00 (9H,
s, –Si-C(CH3)3), 7.01 (1H, dd J 9.2, 2.8, 6-H), 7.03 (1H, d, J 2.8, 8-H),

F. Carta et al. / Bioorg. Med. Chem. 20 (2012) 2266–2273
2271
2.4, 5-H), 7.36 (1H, d, J 9.2, 8-H), 8.03 (1H, d, J 9.6, 4-H); dC
(100 MHz, DMSO-d6) 161.0 (C@O), 155.4, 148.8, 144.9, 134.3,
120.8, 120.1, 118.7, 118.3, 117.5, 112.7, 69.7.
158.9, 137.5, 133.7, 130.6, 127.1, 119.2, 115.8, 115.2, 102.0, 70.0;
Anal. Calcd C, 66.03; H, 4.62; S, 14.69. Found: C, 66.56; H, 4.32;
S, 14.68.
5
3'
4
3'
2'
6
3
2'
1'
O
1'
7
O
O
O
2
5
8
4
8
6
3
4.1.4.2. 7-(Allyloxy)-2H-chromen-2-one yield (5b). 85% yield; dH
(400 MHz, DMSO-d6) 4.73 (2H, d J 8.0, 10-H2), 5.32 (1H, dd, J 13.2,
4.8, 30-HH), 5.45 (1H, dd, J 15.6, 4.8, 30-HH), 6.09 (1H, m, 20-H),
6.33 (1H, d, J 9.6, 3-H), 7.00 (1H, dd, J 9.2, 2.4, 6-H), 7.05 (1H, d, J
2.4, 8-H), 7.67 (1H, d, J 9.2, 5-H), 8.03 (1H, d, J 9.6, 4-H); dC
(100 MHz, DMSO-d6) 162.0 (C@O), 155.0, 148.3, 144.2, 135.1,
119.2, 118.7, 118.5, 118.0, 117.2, 112.6, 69.5.
7
O
S
2
4.1.5.3. 4-(Allyloxy)-2H-chromene-2-thione (6c). 61% yield; dH
(400 MHz, DMSO-d6) 4.95 (2H, d J 6.0, 10-H2), 5.42 (1H, dd, J 13.2,
4.8, 30-HH), 5.59 (1H, dd, J 15.6, 4.8, 30-HH), 6.15 (1H, m, 20-H),
6.97 (1H, s, 3-H), 7.51 (1H, t, J 8.8, 7-H), 7.63 (1H, d, J 8.8, 8-H),
7.80 (1H, t, J 8.8, 6-H), 7.96 (1H, d, J 8.8, 5-H); dC (100 MHz,
DMSO-d6) 198.5 (C@S), 160.9, 157.2, 134.4, 132.5, 126.6, 123.8,
119.9, 117.5, 117.2, 107.6, 71.2.
2'
3'
1'
O
5
8
4
4.1.6. Synthesis of 7-(20-hydroxyethoxy)-2H-chromen-2-one (7)
A mixture of 7-hydroxy-2H-chromen-2-one (0.5 g, 1.0 equiv),
K2CO3 (5.0 equiv), KI (1.0 equiv) and chloroethanol (1.0 equiv) in
DMF dry (10 ml) was stirred at 60 °C for 5 h. The reaction mixture
was cooled down to 0 °C, quenched with 6 M aqueous hydrochloric
acid (50 ml) and extracted with ethyl acetate (3 ꢀ 20 ml). The com-
bined organic layers were washed several timed with H2O, dried
over Na2SO4, filtered-off and concentrated under vacuo to afford
a residue that was purified by silica gel column chromatography
eluting with 50% ethyl acetate/ n-hexane v/v.
6
3
7
O
O
2
4.1.4.3. 4-(Allyloxy)-2H-chromen-2-one yield (5c). 70% yield; dH
(400 MHz, DMSO-d6) 4.87 (2H, d J 8.0, 10-H2), 5.40 (1H, dd, J 13.2,
4.8, 30-HH), 5.59 (1H, dd, J 15.6, 4.8, 30-HH), 5.96 (1H, s, 3-H),
6.15 (1H, m, 20-H), 7.41 (1H, m, 7-H, 8-H), 7.22 (1H, dd, J 8.8, 8.4,
6-H), 7.89 (1H, d, J 8.8, 5-H); dC (100 MHz, DMSO-d6) 165.4
(C@O), 162.5, 153.7, 133.7, 132.6, 125.2, 123.8, 119.7, 117.4,
116.1, 92.0, 70.7.
4.1.6.1.
7-(20-Hydroxyethoxy)-2H-chromen-2-one
(7). 72%
yield; silica gel TLC Rf 0.10 (ethyl acetate/n-hexane 50% v/v); dH
(400 MHz, DMSO-d6) 3.78 (2H, m, 20-H2), 4.13 (2H, m, 10-H2),
4.97 (1H, t, J 5.6, exchange with D2O, O-H), 6.30 (1H, d, J 9.6, 3-
H), 6.97 (1H, dd, J 9.2, 2.4, 6-H), 7.02 (1H, d, J 2.4, 8-H), 7.65 (1H,
d, J 9.2, 5-H), 8.01 (1H, d, J 9.6, 4-H); dC (100 MHz, DMSO-d6)
162.8, 161.3, 156.3, 145.3, 130.4, 113.7, 113.4, 102.1, 71.3, 65.9,
60.3.
4.1.5. Synthesis of allyloxy-2H-chromene-2-thiones 6a–c
The proper allyloxycoumarine 5a-c (1.0 equiv) was dissolved in
dry toluene and treated with Lawesson’s reagent (2.0 equiv). The
reaction mixture was refluxed until consumption of the starting
material (TLC monitoring). Then solvent was removed in vacuo
and the residue obtained was purified by silica gel column chroma-
tography eluting with ethyl acetate in n-hexane to afford the cor-
responding thione.
4.1.7. Synthesis of 20-(2-oxo-2H-chromen-7-yloxy)ethyl
400-methylbenzenesulfonate (8)
2'
5
4
6
7-(20-Hydroxyethoxy)-2H-chromen-2-one (7) (0.2 g, 1.0 equiv)
was dissolved in dry pyridine (5 ml) and treated at 0 °C with TsCl
(1.1 equiv). The yellow solution was stirred at rt until starting
material was consumed (TLC monitoring) and then quenched with
a 1.0 M aqueous hydrochloric acid at 0 °C. The mixture was ex-
tracted with ethyl acetate (3 ꢀ 15 ml) and the combined organic
layers were washed with brine (3 ꢀ 20 ml), H2O (3 ꢀ 20 ml) dried
over Na2SO4, filtered-off and concentrated under vacuo to afford
a residue that was purified by silica gel column chromatography
eluting with 50% ethyl acetate/ n-hexane v/v.
O
3
3'
1'
7
O
S
2
8
4.1.5.1. 6-(Allyloxy)-2H-chromene-2-thione (6a). 62% yield; dH
(400 MHz, DMSO-d6) 4.68 (2H, d J 8.0, 10-H2), 5.32 (1H, dd, J 13.2,
4.8, 30-HH), 5.49 (1H, dd, J 15.6, 4.8, 30-HH), 6.09 (1H, m, 20-H),
7.30 (1H, d, J 9.6, 3-H), 7.36 (1H, dd, J 9.2, 2.4, 7-H), 7.38 (1H, d, J
2.4, 5-H), 7.59 (1H, d, J 9.2, 8-H), 7.89 (1H, d, J 9.6, 4-H); dC
(100 MHz, DMSO-d6) 197.9 (C@S), 156.3, 151.9, 145.7, 136.8,
134.0, 130.2, 121.9, 118.8, 118.4, 112.1, 69.8.
4.1.7.1. 20-(2-Oxo-2H-chromen-7-yloxy)ethyl 400-methylben-
zenesulfonate (8). 35% yield; silica gel TLC Rf 0.36 (ethyl ace-
tate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6) 2.44 (3H, s, CH3).
4.32 (2H, m, 10-H2), 4.41 (2H, m, 20-H2), 6.34 (1H, d, J 9.6, 3-H),
6.87 (1H, dd, J 9.2, 2.4, 6-H), 6.92 (1H, d, J 2.4, 8-H), 7.49 (2H, d J
8.4, 2 ꢀ 200-H/300-H), 7.64 (1H, d, J 9.2, 5-H), 7.83 (2H, d J 8.4,
2 ꢀ 200-H/300-H), 8.02 (1H, d, J 9.6, 4-H); dC (100 MHz, DMSO-d6)
161.7, 161.1, 156.1, 145.9, 145.1, 133.1, 131.0, 130.4, 128.6,
113.7, 113.6, 113.5, 102.3, 69.7, 66.9, 21.9.
5
4
3'
6
2'
3
1'
7
O
O
S
2
8
4.1.5.2. 7-(Allyloxy)-2H-chromene-2-thione 6b. 87% yield; silica
gel TLC Rf 0.32 (ethyl Acetate/n-hexane 20% v/v); mmax (KBr) cmꢁ1
1760, 1519, 1215; dH (400 MHz, DMSO-d6) 4.77 (2H, dt, J 5.6, 1.6,
10-H2), 5.35 (1H, dq, J 12.0, 1.6, 30-HH), 5.47 (1H, dq, J 17.2, 1.6,
30-HH), 6.11 (1H, m, 20-H), 7.11 (1H, dd, J 8.8, 2.4, 6-H), 7.12 (1H,
d, J 9.2, 3-H), 7.27 (1H, d, J 2.4, 8-H), 7.77 (1H, d, J 8.8, 5-H), 7.88
(1H, d, J 9.2, 4-H); dc (100 MHz, DMSO-d6), 198.2 (C@S), 162.9,
4.1.8. Synthesis of 7-(20-fluoroethoxy)-2H-chromen-2-one (9)
20-(2-Oxo-2H-chromen-7-yloxy)ethyl 400-methylbenzenesulfo-
nate (8) (0.1 g, 1.0 equiv) was dissolved in THF(1.0 ml) and treated

2272
F. Carta et al. / Bioorg. Med. Chem. 20 (2012) 2266–2273
with TBAF 1.0 M in THF (1.05 equiv). The yellow solution was stir-
red at rt for 15 min. Then solvents were removed in vacuo and the
residue was purified by silica gel column chromatography eluting
with 50% ethyl acetate/ n-hexane v/v.
4.1.11. Synthesis of tert-butyl 4-methyl-2-oxo-2H-chromen-7-
ylcarbamate (12)
5
8
4
6
3
(Boc)2O
THF
O
4.1.8.1. 7-(20-Fluoroethoxy)-2H-chromen-2-one (9). 40% yield;
silica gel TLC Rf 0.40 (ethyl acetate/n-hexane 50% v/v); dH
(400 MHz, DMSO-d6) 4.36 (1H, m, 10-HH), 4.44 (1H, m, 10-HH),
4.74 (1H, m, 10-HH), 4.87 (1H, m, 10-HH), 6.34 (1H, d, J 9.6, 3-H),
7.02 (1H, dd, J 9.2, 2.4, 6-H), 7.09 (1H, d, J 2.4, 8-H), 7.68 (1H, d, J
9.2, 5-H), 8.03 (1H, d, J 9.6, 4-H); dC (100 MHz, DMSO-d6) 162.2,
2'
O
N
H
O
O
2
7
1'
H2N
O
O
E
12
A suspension of 7-amino-4-methyl-2H-chromen-2-one (0.1 g,
1.0 equiv) in THF dry (2.0 ml) was treated at reflux with di-tert-bu-
tyl dicarbonate (1.0 equiv) and Et3N (1.1 equiv) for 24 h. Then the
solvents were removed in vacuo and the residue was purified by
silica gel column chromatography eluting with 50% ethyl acetate/
n-hexane v/v.
1
161.1, 145.2, 130.5, 113.63, 113.60, 113.5, 102.3, 82.0 (d, JC-F
2
166, C-20), 68.6 (d, JC-F 18, C-10), dF (376 MHz, DMSO-d6) –222.23
(1F, s).
4.1.9. Synthesis of N-(4-methyl-2-oxo-2H-chromen-7-yl)acet-
amide (10)
4.1.11.1. tert-Butyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate
(12). 28% yield; silica gel TLC Rf 0.42 (ethyl acetate/n-hexane 50%
v/v); dH (400 MHz, DMSO-d6) 1.54 (9H, s, 3 ꢀ 20-CH3), 2.42 (3H, s, 4-
CH3), 6.26 (1H, s, 3-H), 7.44 (1H, dd, J 9.2, 2.4, 6-H), 7.57 (1H, d, J
2.4, 8-H), 7.70 (1H, d, J 9.2, 5-H), 9.92 (1H, s, exchange with D2O,
N-H); dC (100 MHz, DMSO-d6) 161.0, 154.8, 154.2, 153.4, 144.1,
126.8, 115.1. 113.0, 105.2. 80.9, 28.9, 27.8, 18.9.
5
8
4
6
3
O
AcCl, Et3N
DCM
N
H
O
O
2
7
H2N
O
O
1'
10
A suspension of 7-amino-4-methyl-2H-chromen-2-one (0.1 g,
1.0 equiv) in DCM dry (5.0 ml) was treated with acetyl chloride
(1.0 equiv) and Et3N (1.0 equiv) under reflux for 7 h. Solvents were
removed under vacuo and the residue was purified by silica gel col-
umn chromatography eluting with 50% ethyl acetate/ n-hexane v/v.
4.2. CA inhibition
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalyzed CO2 hydration activity.14 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 con-
stant 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 determina-
tion 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 assay buffer. Inhibitor and enzyme solu-
tions were preincubated together for 15 min–72 h at room temper-
ature (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 even-
tual 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 coumarin and formation
of the 2-hydroxy-cinnamic acids. The inhibition constants were
obtained by non-linear least-squares methods using PRISM 3, as
reported earlier,1,2 and represent the mean from at least three
different determinations. CA isoforms were recombinant ones
obtained in house as reported earlier.2,3
4.1.9.1. N-(4-Methyl-2-oxo-2H-chromen-7-yl)acetamide (10).
73% yield; silica gel TLC Rf 0.11 (ethyl acetate/n-hexane 50% v/v);
dH (400 MHz, DMSO-d6) 2.14 (3H, s, 10-CH3), 2.43 (3H, s, 4-CH3),
6.29 (1H, s, 3-H), 7.50 (1H, dd, J 9.2, 2.4, 6-H), 7.74 (1H, d, J 9.2,
5-H), 7.79 (1H, d, J 2.4, 8-H), 10.40 (1H, br s, exchange with D2O,
N-H); dC (100 MHz, DMSO-d6) 170.0, 161.0, 154.6, 154.0, 143.5,
126.8, 115.9, 115.7, 113.0, 106.3, 25.1, 18.9.
4.1.10. Synthesis of 1-(30,50-dimethylphenyl)-3-(4-methyl-2-
oxo-2H-chromen-7-yl)urea (11)
O
C
5
8
4
6
3
N
4'
3'
O
Et3N
Acetone
O
O
N
H
N
H
O
2
H2N
7
1'
O
2'
E
11
7-amino-4-methyl-2H-chromen-2-one (0.1 g, 1.0 equiv) in ace-
tone (10 ml) was treated at reflux with 3,5-dimethyyl isocyanate
(1.0 equiv) and Et3N (1.1 equiv) for 24 h. Then the solvents were
removed in vacuo and the residue was purified by silica gel column
chromatography eluting with 50% ethyl acetate/ n-hexane v/v.
Acknowledgment
4.1.10.1. 1-(30,50-Dimethylphenyl)-3-(4-methyl-2-oxo-2H-chro-
men-7-yl)urea (11). 23% yield; silica gel TLC Rf 0.22 (ethyl ace-
tate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6) 2.28 (6H, s,
2 ꢀ 30-CH3), 2.43 (3H, s, 4-CH3), 6.25 (1H, s, 3-H), 6.69 (1H, s, 40-
H), 7.13 (2H, s, 2 ꢀ 20-H), 7.39 (1H, dd, J 9.2, 2.4, 6-H), 7.65 (1H,
d, J 2.4, 8-H), 7.71 (1H, d, J 9.2, 5-H), 8.72 (1H, s, exchange with
D2O, N-H), 9.20 (1H, s, exchange with D2O, N-H); dC (100 MHz,
DMSO-d6) 161.1, 254.2, 153.4, 144.5, 140.6, 140.0, 138.9, 138.6,
126.9, 124.9, 124.3, 117.3, 116.8, 115.3, 22.1, 19.0.
This research was financed by a 7 FP EU project (Metoxia).
References and notes
1. (a) Vu, H.; Pham, N. B.; Quinn, R. J. J. Biomol. Screen. 2008, 13, 265; (b) Maresca,
A.; Temperini, C.; Vu, H.; Pham, N. B.; Poulsen, S. A.; Scozzafava, A.; Quinn, R. J.;
Supuran, C. T. J. Am. Chem. Soc. 2009, 131, 3057.
2. Maresca, A.; Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava, A.; Supuran, C.
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