7,8-Disubstituted- but not 6,7-disubstituted coumarins selectively inhibit the transmembrane, tumor-associated carbonic anhydrase isoforms IX and XII over the cytosolic ones i and II in the low nanomolar/subnanomolar range

Article abstract of DOI:10.1016/j.bmcl.2010.10.094

Two series of disubstituted coumarins incorporating ether and acetyl/propionyl moieties in positions 6,7- and 7,8- of the heterocyclic ring were synthesized investigated for the inhibition of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). All these

Full text of DOI:10.1016/j.bmcl.2010.10.094

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7255–7258
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
7,8-Disubstituted- but not 6,7-disubstituted coumarins selectively inhibit the
transmembrane, tumor-associated carbonic anhydrase isoforms IX and XII
over the cytosolic ones I and II in the low nanomolar/subnanomolar range
⇑
Alfonso Maresca, Andrea Scozzafava, Claudiu T. Supuran
Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of disubstituted coumarins incorporating ether and acetyl/propionyl moieties in positions
6,7- and 7,8- of the heterocyclic ring were synthesized investigated for the inhibition of the zinc enzyme
carbonic anhydrase (CA, EC 4.2.1.1). All these coumarins were very weak or ineffective as inhibitors of the
housekeeping, offtarget isoforms CA I and II. The 6,7-disubstituted series showed ineffective inhibition
also for the transmembrane tumor-associated isoforms CA IX and XII, whereas the corresponding iso-
meric 7,8-disubstituted coumarins showed nanomolar/subnanomolar inhibition of CA IX/XII. The nature
and position of the groups substituting the coumarin ring in the 7,8-positions greatly influenced CA
inhibitory properties, with C1–C4 alkyl ethers being the most effective inhibitors.
Received 12 October 2010
Revised 18 October 2010
Accepted 19 October 2010
Available online 25 October 2010
Keywords:
Carbonic anhydrase
Tumor associated isoforms
Coumarin
Ó 2010 Elsevier Ltd. All rights reserved.
Isoform-selective inhibitor
Fries rearrangement
Coumarins were only recently discovered as a novel class of
inhibitors of the metalloenzyme carbonic anhydrase (CA, EC
4.2.1.1).1,2 Their mechanism of action has also been elucidated, this
class of CA inhibitors (CAIs) being prodrugs and differing of all
other known inhibitors of this enzyme. Coumarins in fact do not di-
rectly interact with the metal ion from the enzyme active site,
which is critical both for catalysis and inhibition of CAs with other
classes of compounds, such as sulfonamides, metal-complexing an-
ions, phenols or polyamines.1–7 Indeed, as shown by kinetic and
X-ray crystallographic studies, coumarins are mechanism based
inhibitors, which undergo hydrolysis under the influence of the
zinc-hydroxide, nucleophilically active species of the enzyme, with
generation of substituted-2-hydroxycinnamic acids.1,2 For exam-
ple, the natural product coumarin A or the simple non-substituted
derivative B (but also many of its congeners possessing various
substitution patterns at the coumarin ring)2 not only act as effec-
tive CAIs against many of the mammalian isoforms CA I–CA XV,
but the real enzyme inhibitor was detected to be the hydrolyzed
coumarins, A1 and B1 (Scheme 1), formed from the original
derivatives A and B.1 These adducts have been thoroughly charac-
terized by X-ray crystallography of enzyme-inhibitor adducts.1,2
The 2-hydroxy-cinnamic acids A1 and B1 were shown to bind in
an unprecedented way to the enzyme, at the entrance of the active
site cavity, plugging the entire entrance to it, and blocking thus the
catalytic activity of the enzyme.1,2 Only very recently, some fuller-
ene derivatives were shown to bind in a similar way to the CAs.8
Occlusion of the CA active site entrance, by hydrolyzed coumarins
(i.e., cis- or trans-2-hydroxycinnamic acids)1,2 or fullerenes8 thus
constitutes a totally novel mechanism of CA inhibition, which
started to be exploited to design compounds with various
applications.2
OH
MeO
O
O
O
O
HO
O
O
A
B
C
Indeed, more recently we shown that umbelliferone (7-
hydroxycoumarin) C and some of its derivatives are selective,
although not very active (submicromolar) inhibitors of the
tumor-associated isoforms CA IX and XII, whereas they basically
do not inhibit CA I and II, offtarget, highly abundant CA isoforms.9
In fact the CAIs of the sulfonamide type are clinically used for dec-
ades, for various classes of diuretics and systemically acting anti-
glaucoma agents, but their main drawback is just their rather
potent inhibition of CA I and II.3,4,10 However critical barriers to
the use of CAIs as therapeutic agents are related to the high num-
ber of isoforms in humans (i.e., 16 CAs, of which 13 have catalytic
activity), their rather diffuse localization in many tissues/organs,
and the lack of isozyme selectivity of the presently available
⇑
Corresponding author. Tel.: +39 055 457 3005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.094

7256
A. Maresca et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7255–7258
OH
I (KI of 58.4
lM), it does not inhibit significantly hCA II (KI
OH
>100 M), but it is a submicromolar inhibitor of the tumor-associ-
l
CA
ated isoforms hCA IX and XII, against which it has inhibition con-
stants in the range of 482–754 nM.9 We used thus this
compound to prepare novel coumarin derivatives, also because
its phenol moiety can be easily derivatized, leading to novel chem-
otypes which have not been investigated earlier as CAIs.1,2 Indeed,
disubstituted coumarins in the 6,7- and 7,8-positions have not
been included in the series of such compounds tested for their
interaction with various CAs in the previous work reported so
far.1,2,9
Umbelliferone C has been converted to the corresponding
acetyl and propionyl esters 3 and 4, by reaction with carboxylic
acids 1 and 2 in the presence of carbodimides (DCC, dicyclohexyl
carbodiimide and DMAP, dimethylaminopyridine), as shown in
Scheme 2. Acetyl-/propionyl esters 3 and 4 were subjected to
the Fries rearrangement,14 in the presence of AlCl3 at 180°, to give
the mixture key intermediates, isomeric acyl-umbelliferones 5–8.
These mixtures have been separated by flash-chromatography
leading to the corresponding pure pairs of derivatives 5/7 and
6/8, respectively. The ratio of 6,7-disubstituted versus 7,8-disub-
stituted coumarins obtained after the rearrangement was of 1:9.
Compounds 9–23 were the obtained in Mitsunobu conditions,15
using the appropriate aliphatic alcohol and the purified key inter-
mediates 5–8 mentioned above, in presence of DIAD and
triphenylphosphine.16
-
COO
r.t, pH 7.4
4-6 h
MeO
O
O
MeO
OH
A
A1
-
CA
COO
r.t, pH 7.4
4-6 h
O
O
OH
B1
B
Scheme 1. Formation of 2-hydroxy-cinnamic acids A1 and B1 by the CA-mediated
hydrolysis of coumarins A and B.
inhibitors of the sulfonamide/sulfamate type.3,4,10–13 Thus, there is
a stringent need of CAIs with a more selective inhibition profile
compared to the sulfonamides and their isosteres, and the couma-
rins represent an interesting class due to the fact that already a cer-
tain number of isoform-selective CAIs targeting CA IX, XII, XIII
among others, have been detected, even if the number of investi-
gated coumarins up to date is rather limited (less than 50 com-
pounds have been so far investigated for their interactions with
the mammalian CAs).1,2,9
Considering C as lead molecule, and continuing our interest in
investigating coumarins as CAIs, we report here the synthesis
and inhibition studies of two series of coumarin derivatives pre-
pared from umbelliferone C as lead, and their interaction with four
physiologically relevant isoforms, CA I, II, IX and XII.12,15
Inhibition data with compounds 5–23 reported here and A–C as
standards against four CA isozymes, that is, hCA I, II, IX and XII,17
are shown in Table 1. The following structure–activity relationship
(SAR) observations can be drawn from data of Table 1 for these
coumarins with 6,7- and 7,8-disubstitution pattern:
(i) The coumarins possessing 6,7-disubstituted moieties, of
types 5, 6, and 9–14, showed very weak or total lack of CA inhibi-
tory activity against all investigated isoforms. Thus, only 5 and 6
were micromolar hCA IX inhibitors; 9 was a micromolar inhibitor
Umbelliferone C was shown earlier to constitute an interesting
lead for designing novel CAIs based on the coumarin scaffold.9 In-
deed, this compound is an extremely weak inhibitor of isoform hCA
O
O
i
R'
OH
HO
O
O
R'
O
O
O
Umbelliferone
1: R' = Me
2: R'' = Et
3: R' = Me
4: R'' = Et
Table 1
hCA I, II, IX and XII inhibition data with coumarins 5–23 and A–C (as standard
inhibitors), by a stopped-flow, CO2 hydration assay method (6 h incubation time
between enzyme and coumarin)17
O
R'
ii
Compound
KI*
hCA IXb (nM)
HO
O
O
HO
O
O
hCA Ia
(
lM)
hCA IIa
(lM)
hCA XIIb (nM)
O
R'
A
B
C
0.078
3.1
58.4
0.059
9.2
54.5
>100
482
48.6
>100
5: R' = Me
6: R'' = Et
7: R' = Me
8: R'' = Et
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
754
5
6
7
8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
8030
8015
73.0
>100,000
>100,000
61.9
61.7
6540
>100,000
>100,000
>100,000
>100,000
77,700
60.9
1.0
0.98
8.8
22.4
31.5
26.3
33.2
38.4
iii
iii
58.2
O
9
7800
7400
7580
>100,000
>100,000
>100,000
78.3
70.8
56.7
61.2
72.3
R'
10
11
12
13
14
15
16
17
18
19
20
21
22
23
R''O
O
O
R''O
O
O
O
R'
R' = Me
9: R'' = Me
10: R'' = Et
R' = Me
15: R'' = Me
16: R'' = Et
17: R'' = n-Pr
18: R'' = n-Bu
19: R'' = Bn
20: R'' =
11: R'' = n-Pr
12: R'' = n-Bu
13: R'' = Bn
14: R'' =
63.9
37.8
46.7
50.2
R' = Et
21: R'' = Me
22: R'' = Et
23: R'' = n-Pr
a
Full length, cytosolic isoform.
Catalytic domain, recombinant enzyme.
i: dry DMF, DMAP, DCC, r.t, 16h; ii: AlCl3, 180°; iii: dry THF, PPh3, DIAD, r.t, 16h
b
*
Errors in the range of 5–10% of the reported value, from three different
determinations.
Scheme 2. Preparation of the two series of coumarins 3–23, investigated in this
study, starting from umbelliferone (7-hydroxycoumarin) C.

A. Maresca et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7255–7258
7257
of hCA IX and XII, whereas all other compounds generally showed
inhibition constants >100 M against all investigated isoforms.
Definitely, this substitution pattern leads to a total loss of CA inhib-
still among the best hCA XII coumarin CAIs reported so far, with
KI’s of 8.8–341.5 nM. For the propionyl series, the activity remained
good enough but the compounds 22 and 23 were less active com-
pared to the corresponding acetyl derivatives 16 and 17.
In conclusion, we report here that the 6,7-disubstituted couma-
rins prepared by Fries rearrangement from umbelliferone are de-
void of significant CA inhibitory properties against isoforms hCA
I, II, IX and XII. However, their isomers, incorporating 7,8-disubsti-
tuted moieties act as nanomolar or subnanomolar hCA IX and XII
inhibitors, whereas not inhibiting significantly the offtarget
isoforms hCA I and II. SAR for the inhibition of hCA IX and XII
was complex but the main features associated with low nanomolar
inhibitors have been delineated. This is the first report in which
subnanomolar inhibition of a CA isoform with a coumarin deriva-
tive is ever observed.
l
itory properties to the compounds incorporating it.
(ii) Compounds 7, 8 as well as 15–23, isomeric to the previously
discussed ones, but possessing the substituents in the 7,8-positions
of the coumarin ring, showed a totally different inhibition profile.
Thus, all these compounds were ineffective as hCA I and II inhibi-
tors, with KI’s >100 lM, similar to the lead molecule C. This is a
desirable feature for CAI, in order to target isoforms involved in
pathological processes, and not hCA I and especially hCA II (which
is the physiologically dominant isoform), whose inhibition may be
deleterious and lead to side effects of such a drug.3,4
(iii) The tumor-associated hCA IX was inhibited by umbellifer-
one C9 in the submicromolar range (KI of 482 nM), as discussed
above, but most of its derivatives 7, 8 and 15–23 were much better
inhibitors, with KI’s in the range of 37.8–78.3 nM (Table 1). Thus,
for the compounds obtained after the Fries rearrangement, the
activity increased from the acetyl 7 to the propionyl 8 derivatives.
In the case of the ethers 15–20, activity increased from the C1 to
the C3 derivative (the n-propyl derivative 17 was the best hCA IX
inhibitor for the acetyl subseries), to decrease then again for the
benzyl and adamantylethyl derivatives 19 and 20. However the
ethyl and adamantylethyl derivatives had quite similar derivatives,
proving that SAR (which generally for this class of CAIs is very
much sensitive to small modifications in the scaffold of the inhib-
itor) is less sharp for these two derivatives differing quite a lot by
the presence of such a bulky group in 20 (compared to 16). How-
ever, for the other derivatives investigated here the reverse is true,
with small modifications leading to a sharp increase or decrease of
activity (compare 7 and 8, 16 and 17, 17 and 18, respectively). For
the propionyl derivatives 21–23, activity was even more increased
compared to the corresponding acetyl derivatives 15–17, with
inhibition constants in the range of 37.8–50.2 nM. The best hCA
IX inhibitor was 21, which with a KI of 37.8 nM is the best couma-
rin hCA IX inhibitor reported so far (the previous best inhibitor had
a KI of 48 nM).2
(iv) The same behavior as that observed for hCA IX was also
detected for the inhibition of the second tumor-associated isoform,
hCA XII with coumarins 7, 8 and 15–23 investigated here. Thus, the
lead C was a moderate hCA XII inhibitor, with a KI of 754 nM,
whereas the new compounds reported here possessing the
7,8-disubstitution pattern, behaved as much more effective inhib-
itors, with KI’s in the range of 0.98–61.9 nM. It is the first time that
we detect subnanomolar inhibition with a coumarin derivative and
we think this is a very significant finding (many sulfonamides with
subnanomolar inhibition of various CA isozymes are known).3,4,10
The following SAR was evidenced for these new hCA XII inhibitors.
The two key intermediates 7 and 8 had the same potency as hCA
XII inhibitors (KI of 61.7–61.9 nM), irrespective whether an acetyl
or propionyl moiety was present in the 8-position of the coumarin
ring. This behavior is different from that observed with these two
compounds against hCA IX, as discussed above. The ethers 15–23
showed on the other hand enhanced inhibitory properties com-
pared to the parent phenols from which they were prepared. Thus,
the methoxy derivatives 15 and 21 were better inhibitors than the
parent phenols 7 and 8, but the increase in activity was not signif-
icant (KI’s of 60.9 nM for 15 and of 26.3 nM for 21 have been
measured, Table 1). However for the acetyl series, the increase of
the aliphatic chain in the ether moiety from one (in compound
15) to two and three carbon atoms led to an impressive increase
in the hCA XII inhibitory activity, the compounds 16 and 17 having
inhibition constants of 1 nM and of 0.98 nM, respectively (Table 1).
Further increasing the length of the aliphatic chain, as in 18, or
introduction of the benzyl or adamantylethyl moieties, as in 19
and 20, led to a decrease in activity, but these compounds were
Acknowledgment
This research was financed in part by a Grant of the 7th FP of EU
(Metoxia project).
References and notes
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Obtained in 60% yield; mp 164–166 °C; silica gel TLC Rf 0.16 (AcOEt/n-Hex 14%,
v/v); mmax (KBr) cmꢀ1, 2935 (C–H), 1732 (C@O), 1622 (C@C), 1533 (aromatic),
1240 (C–O), 1107 (O–C@O); dH (400 MHz, DMSO-d6) 1.47 (3H, t J = 6.8 Hz, 200-
H3), 2.60 (3H, s, 20H3), 4.29 (2H, q J = 6.8, 10-H2), 6.39 (1H, d J = 9.6 Hz, 3-H),
7.20 (1H, s, 8-H), 8.04 (1H, s, 5-H), 8.11 (1H, d J = 9.6 Hz, 4-H); dc (100 MHz,
DMSO-d6) 198.0 (C-10), 161.6 (C-7), 160.6 (C-2), 158.4 (C-9), 145.3 (C-4), 131.4
(C-5), 125.9 (C-6), 114.4 (C-3), 112.8 (C-10), 101.7 (C-8), 66.1 (C-100), 32.6 (C-
20), 15.4 (C-200).
14. Sabui, S. K.; Venkateswaran, R. V. Tetrahedron 2003, 42, 8375.
15. Kumara Swamy, K. C.; Bhuvan Kumar, N. N.; Balaraman, E.; Pavan Kumar, K. V.
P. Chem. Rev. 2009, 109, 2551.
16. 6-Acetyl-7-hydroxy-2H-chromen-2-one (5):
8-Propionyl-7-propoxy-2H-chromen-2-one (23):
O
4
5
6
10
9
5
4
3
6
1'
10
9
3
2'
2
7
3''
1''
2
7
HO
O
O
8
O
O
O
8
2''
1'
2'
3'
O
Obtained in 35% yield; mp 173–175 °C; silica gel TLC Rf 0.13 (AcOEt/n-Hex 20%,
v/v); mmax (KBr) cmꢀ1, 3071 (O–H), 2927 (C–H), 1736 (C@O), 1638 (C@C),1521
(aromatic), 1192 (O–CO); dH (400 MHz, DMSO-d6) 2.71 (3H, s, 20-H3), 6.38 (1H,
d J = 9.4 Hz, 3-H), 6.93 (1H, s, 8-H), 8.07 (1H, d J = 9.4 Hz, 4-H), 8.37 (1H, s, 5-
H); dc (100 MHz, DMSO-d6) 203.7 (C-10), 164.3 (C-7), 160.5 (C-2), 159.2 (C-9),
145.2 (C-4), 138.9 (C-5), 119.7 (C-6), 114.2 (C-3), 112.6 (C-10), 104.7 (C-8),
29.1 (C-20).
Obtained in 80% yield; mp 82–84 °C; silica gel TLC Rf 0.12 (AcOEt/n-Hex 20%, v/
v);
max (KBr) cmꢀ1, 2932 (C–H), 1747 (C@O), 1635 (C@C), 1534 (aromatic), 1287
m
(C–O), 1170 (O–C@O); dH (400 MHz, DMSO-d6) 0.98 (3H, t J = 7.2 Hz, 300-H3), 1.13
(3H, t J = 7.4 Hz, 30-H3), 1.74 (2H, six J = 7.2 Hz, 200-H2), 2.83 (2H, q J = 7.4 Hz,
20-H2), 4.13 (2H, t J = 7.2 Hz, 100-H2), 6.36 (1H, d J = 9.6 Hz, 3-H), 7.19 (1H, d
J = 8.8 Hz, 6-H), 7.77 (1H, d J = 8.8 Hz, 5-H), 8.06 (1H, d J = 9.6 Hz, 4-H); dc
(100 MHz, DMSO-d6) 202.9 (C-10), 160.3 (C-2), 158.5 (C-7), 151.3 (C-9), 145.2
(C-4), 131.2 (C-5), 119.4 (C-8), 113. (C-10), 113.5 (C-3), 110.2 (C-6), 71.24 (C-
100), 38.3 (C-20), 22.75 (C-200), 11.13(C300), 8.42 (C-30).
8-Acetyl-7-hydroxy-2H-chromen-2-one (7):
5
4
10
9
6
3
2
7
17. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-
flow instrument has been used for assaying the CA catalysed CO2 hydration
activity. Phenol red (at a concentration of 0.2 mM) has been used as 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. Inhibitor
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 shows 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.1,2
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 isofoms were recombinant ones obtained in
HO
O
O
8
1'
O
2'
Obtained in 65% yield; mp 169–171 °C; silica gel TLC Rf 0.27 (AcOEt/n-Hex 20%,
v/v); mmax (KBr) cmꢀ1, 3061 (O–H), 2927 (C–H), 1735 (C@O), 1615 (C@C),1526
(aromatic), 1178 (O–C@O); dH (400 MHz, DMSO-d6) 2.65 (3H, s, 20-H3), 6.33
(1H, d J = 9.4 Hz,
3-H), 6.95 (1H,
d J = 8.6 Hz, 6-H), 7.70 (1H, d J = 8.6 Hz, 5-H), 8.03 (1H, d
J = 9.4 Hz, 4-H); dc (100 MHz, DMSO-d6) 201.6 (C-10), 160.8 (C-2), 160.4 (C-7),
153.4 (C-9), 145.6 (C-4), 132.8 (C-5), 115.7 (C-8), 114.4 (C-6), 112.7 (C-10),
112.1 (C-3), 33.4 (C-20).
6-Acetyl-7-ethoxy-2H-chromen-2-one (10):
O
5
8
4
6
10
9
3
2'
1'
2
7
house as reported earlier.1,2
.
1''
O
O
O
2''