New chemotypes acting as isozyme-selective carbonic anhydrase inhibitors with low affinity for the offtarget cytosolic isoform II

Article abstract of DOI:10.1016/j.bmcl.2012.01.129

Considering phenols and coumarins as lead molecules for obtaining non-sulfonamide inhibitors of carbonic anhydrases (CAs, EC 4.2.1.1), we screened a large number of compounds possessing diverse chemotypes, but structural features which resemble the two chemical classes. Here we report an investigation of such derivatives which do not significantly inhibit CA II, but show interesting inhibition profiles against other isozymes. Pyridine-N-oxide-2-thiophenol, thiobenzoic acid, thimerosal, two oximes derived from a six-membered-ring lactone and from coumarin; 2-hydroxyquinoline and coumaphos, were investigated as inhibitors of CA I-XIV. All these compounds did not inhibit CA II, whereas the two oximes and 2-hydroxyquinoline were low nanomolar inhibitors of CA I, IX, XII, XIII and XIV, showing a very different inhibition profile compared to sulfonamides and sulfamates. Some other compounds showed low micromolar inhibition of other isoforms of interest, such as CA VA/VB, CA VI and VII. This study demonstrates that a rather wide range of structures show low nanomolar - micromolar inhibitory activity against many CA isozymes, without inhibiting significantly the offtarget isoform CA II.

Full text of DOI:10.1016/j.bmcl.2012.01.129

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2182–2185
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New chemotypes acting as isozyme-selective carbonic anhydrase inhibitors
with low affinity for the offtarget cytosolic isoform II
⇑
Fabrizio Carta, Daniela Vullo, 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:
Considering phenols and coumarins as lead molecules for obtaining non-sulfonamide inhibitors of
carbonic anhydrases (CAs, EC 4.2.1.1), we screened a large number of compounds possessing diverse
chemotypes, but structural features which resemble the two chemical classes. Here we report an
investigation of such derivatives which do not significantly inhibit CA II, but show interesting
inhibition profiles against other isozymes. Pyridine-N-oxide-2-thiophenol, thiobenzoic acid, thimerosal,
two oximes derived from a six-membered-ring lactone and from coumarin; 2-hydroxyquinoline and
coumaphos, were investigated as inhibitors of CA I-XIV. All these compounds did not inhibit CA II,
whereas the two oximes and 2-hydroxyquinoline were low nanomolar inhibitors of CA I, IX, XII, XIII
and XIV, showing a very different inhibition profile compared to sulfonamides and sulfamates. Some
other compounds showed low micromolar inhibition of other isoforms of interest, such as CA VA/VB,
CA VI and VII. This study demonstrates that a rather wide range of structures show low nanomolar—
micromolar inhibitory activity against many CA isozymes, without inhibiting significantly the offtarget
isoform CA II.
Received 1 December 2011
Revised 27 January 2012
Accepted 30 January 2012
Available online 6 February 2012
Keywords:
Carbonic anhydrase
Offtarget isoform
2-Hydroxyquinoline
Coumarin oxime
Coumaphos
Ó 2012 Elsevier Ltd. All rights reserved.
Sulfonamides and their bioisosteres (sulfamates, sulfamides,
etc) constitute the most investigated class of inhibitors of the
metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1).1–3 As they
generally efficiently inhibit most of the mammalian isoforms
known to date (16 in non-primate mammals and 15 in prima-
tes),1–3 sulfonamide-based CA inhibitors (CAIs) show many side ef-
fects when used as diuretic, antiglaucoma, antiobesity or
anticancer agents/diagnostic tools.4–8 Most of these side effects
are due to the inhibition of the cytosolic isoform CA II, which is a
catalytically highly active isozyme, abundant in many tissues/or-
gans, and involved in several ‘house-keeping’ physiological func-
tions, such as pH homeostay, secretion of electrolytes, transport
of anions, etc.1–4 Although CA II itself is a drug target for obtaining
antiglaucoma drugs,1,4 as it is involved among others in the secre-
tion of ocular fluid by the ciliary processes,4 this enzyme is most of
the time an offtarget, when isoforms such as CA VA/VB (for
antiobesity drugs),5 CA VII/XIV (anticonvulsants)6 or CA IX/XII
(antitumor agents)7,8 should be inhibited selectively. For this rea-
son, there is a constant search of isoform-selective CAIs which
should not act as potent CA II inhibitors. In the last period several
classes of such agents were detected. For example, some ureido-
substituted benzenesulfonamides9 are CA IX/XII low nanomolar
inhibitors being only micromolar CA II inhibitors, whereas many
different chemotypes, such as the hydroxamates,10 polyamines,11
coumarins12,13 and phenols14 show even better selectivity ratios
for inhibiting different CA isoforms with respect to CA II. Some of
these isoforms-selective inhibitors (targeting CA IX and XII) were
highly effective in vivo, showing anticancer and antimetastatic ef-
fects in an animal model of breast cancer.15
The inhibition mechanism with such non-sulfonamide chemo-
types are also well understood, since the X-ray crystal structures
of several adducts (with CA II) were reported.
For example, similar to phenol A, which was found anchored to
the water molecule/hydroxide ion coordinated to the zinc ion from
the CA II active site,16 spermine11 or the natural product phenol B,17
bind in the same manner. In the case of spermine a primary amino
group interacts with the water molecule/hydroxide ion acting as
zinc ligand,11 whereas for B, unexpectedly, the ester moiety was
the anchor to the zinc hydroxide from the enzyme active site.17
Even stranger was the behavior of the coumarin natural product
C which was shown to be a prodrug CAI,12a being hydrolyzed by
the enzyme to the actual inhibitor, the cis-hydroxycinnamic acid
D, which binds far away from the zinc, at the entrance of the active
site cavity, occluding it and assuring a highly efficient inhibition by
a completely new mechanism compared to the sulfonamides or
other inhibitors which are zinc binders.12 As the entrance to the
active site is the region which contains a high variability of amino
acids among the various CA isoforms, it is not surprising that
⇑
Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.129

F. Carta et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2182–2185
OH
2183
O
HOOC
HO
Cl
OMe
N
H
O
A
B
OH
OH
-
COO
MeO
O
O
MeO
OH
D
C
compounds binding in that region, such as the coumarins, led to
class of CAIs12,13 (compare the activity of 7 and 8 against this
highly isoform-selective CAIs (against
a
high number of CA
and other CA isoforms, Table 1).
isozymes).12,13,15b
(iii) isoform hCA III was not inhibited significantly by the simple
coumarin 8 (KI >200 M) but was inhibited in the low micro-
Considering phenols and coumarins as lead molecules for
obtaining non-sulfonamide CAIs, we screened a large number of
compounds possessing diverse chemotypes, but structural features
which resemble these two chemical classes. Here we report an
investigation of such derivatives which do not significantly inhibit
CA II, but show interesting inhibition profiles against other iso-
zymes. These compounds are rather heterogeneous from the
chemical point of view: a thiophenol derivative of pyridine-N-
oxide 1, thiobenzoic acid 2, thimerosal 3 (ethylmercutythiosalycilic
acid, an agent used as vaccine preservative)18; two oximes derived
l
molar range by compounds 1–7, which is a very interesting
result, considering that this sulfonamide-resistant isoform is
not inhibited strongly by sulfonamides and their isosteres.22
The compounds investigated here showed KIs in the range of
0.48–6.31 lM. The best hCA III inhibitors were the oximes 4
and 5 as well as the phenol 6 (Table 1).
(iv) The membrane-associated isoform hCA IV was inhibited by
compounds 1–8 with inhibition constants in the range of
from a six-membered-ring lactone (4) and from coumarin (5)19
;
the structurally similar 2-hydroxyquinoline 6 as well as a couma-
rin derivative incorporating a phosphorothioate moiety, couma-
phos 7, which is highly used in agriculture as an acaricide/
ectoparasiticide against the honey bee mite Varroa destructor.20
The simple coumarin 8 (which is hydrolyzed within the CA active
site to trans-2-hydroxycinnamic acid 9 which is the real CAI)12b
has been used as standard inhibitor in these studies.
Table 1
Inhibition of CA isoforms I-XIV with compounds 1–7 and the coumarin
standard24
8
as
S
COOH
OH
Hg
S
All catalytically active, human CA isoforms (hCA I-XIV) have
been screened for their inhibition profiles with compounds 1–8
(Table 1). The following structure–activity relationship (SAR) has
been observed:
N
SH
Et
-
O
1
2
3
OH
(i) the offtarget isoform hCA II was not inhibited at all (or
OH
O
N
O
N
OH
O
4
N
weakly inhibited with one compound, 2, KI of 51
these derivatives whih showed KIs >200 M. Coumarin 8 is
a medium potency hCA II inhibitor,12 with a KI of 9.2
(but most sulfonamide CAIs are low nanomolar CAIs against
this isoform)1–3
lM) by
6
l
5
lM
COOH
Cl
OEt
;
S
O
(ii) the oximes 4 and 5 were low nanomolar inhibitors of hCA I,
another highly abundant cytosolic isoform1 (KIs of 9 nM for
both compounds) whereas the isosteric compound (to 5) 6
was 692 times a weaker hCA I inhibitor, with a KI of
P
OH
O
EtO
O
O
8
9
7
Isoformb
KIa
(lM)
6.23 lM. If oximes 5 and 6 possess the same inhibition
Compound
1
2
3
4
5
6
7
8
mechanism as the coumarin 6 (which is hydrolyzed within
the enzyme active site to the carboxylic acid 9), after hydro-
lysis they should lead to the formation of hydroxamic acids,
which are known to bind the Zn(II) ion,10 at least in the hCA
II active site (but as mentioned above, these compounds, 4
and 5, do not act as CA II inhibitors, but we stress again, they
are low nanomolar hCA I inhibitors). It is thus not improba-
hCA I
hcA II
>200 10.8
>200 51.0
>200 0.009
>200 >200
0.009
>200
0.48
6.23
>200
0.57
0.36
61.7
>200 9.2
4.62
5.48
5.09
3.41
3.1
hCA III
hCA IV
hCA VA
hCA VB
hCA VI
hCA VII
hCA IX
hCA XII
hCA XIII
hCA XIV
1.62
6.13
5.12
3.55
2.41
7.76
1.72
5.40
4.15
2.91
5.91 6.31
8.10 5.61
2.25 7.25
2.35 5.87
0.71
0.26
>200
62.3
0.75
0.036 0.049 0.41
0.047 0.058 0.75
0.041 0.033 2.23
0.84 0.42 4.68
5.64 >200 0.016 0.058 0.082 6.41
>200
>200
>200
>200
>200
>200
>200
>200
5.10
5.12
0.008
0.27
5.74 10.9
ble that as coumarin 8 (a weak hCA I inhibitor, KI of 3.1 lM),
compounds 4 and 5 bind in hydrolyzed state within the cou-
marin-binding site, at the entrance of the CA active site.12,21
Thiobenzoic acid 2 was also a weak hCA I inhibitor (KI of
2.77 6.85
1.31 6.97
4.55 6.58
0.064 0.054 0.093 9.52
0.059 0.094 0.065 4.23
0.048 0.016 0.036 4.14
a
Mean from at least 3 different determinations, by a CO2 hydrase assay.24 Errors
10.8
(KI of 61.7
coumarin ring is critical for the biological activity of this
l
M) whereas the coumarin 7 was a very weak inhibitor
lM), proving that the substitution pattern at the
were in the range of 10% of the reported value (n = 3).
b
All enzymes were recombinant ones obtained in house.

2184
F. Carta et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2182–2185
0.26–62.3
pounds 4–6 (submicromolar inhibition) whereas the weak-
est one was 8 (KI of 62.3 M). As for isoforms III, the
l
M. The best hCA IV inhibitors were again com-
diverse structures do show low nanomolar—micromolar inhibitory
activity against many isoforms, without inhibiting significantly the
offtarget isoform CA II, which is a sulfonamide-avid isozyme.
l
oximes incorporating the six-membered ring found in 4, or
the coumarin oxime 5 (and its isosteric derivative, the phe-
nol 6) thus constitute highly interesting leads for obtaining
CAIs targeting this isoform.
Acknowledgment
This research was financed by a Grant of the 7th FP of EU
(Metoxia project).
(v) The two mitochondrial isoforms hCA VA and hCA VB were
inhibited in the nanomolar range by the oximes 4 and 5
(KIs in the range of 36–58 nM), and in the submicromolar
References and notes
one (KIs of 0.41–0.75
pounds were weak, micromolar inhibitors (KIs in the range
of 2.25–7.25 M for compounds 1–3 and 7, and >200
for the coumarin 8, Table 1).
lM) by the phenol 6. The other com-
1. (a) Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23, 146; (b)
Supuran, C. T. Nat. Rev. Drug Discov. 2008, 7, 168; (c) Supuran, C. T. Bioorg. Med.
Chem. Lett. 2010, 20, 3467.
2. (a) Alterio, V.; Vitale, R. M.; Monti, S. M.; Pedone, C.; Scozzafava, A.; Cecchi, A.;
De Simone, G.; Supuran, C. T. J. Am. Chem. Soc. 2006, 128, 8329; (b) Weber, A.;
Casini, A.; Heine, A.; Kuhn, D.; Supuran, C. T.; Scozzafava, A.; Klebe, G. J. Med.
Chem. 2004, 47, 550.
3. (a) Di Fiore, A.; Monti, S. M.; Hilvo, M.; Parkkila, S.; Romano, V.; Scaloni, A.;
Pedone, C.; Scozzafava, A.; Supuran, C. T.; De Simone, G. Proteins 2009, 74, 164;
(b) Winum, J. Y.; Temperini, C.; El Cheikh, K.; Innocenti, A.; Vullo, D.; Ciattini, S.;
Montero, J. L.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2006, 49, 7024; (c)
Innocenti, A.; Casini, A.; Alcaro, M. C.; Papini, A. M.; Scozzafava, A.; Supuran, C.
T. J. Med. Chem. 2004, 47, 5224.
4. Carta, F.; Supuran, C. T.; Scozzafava, A. Exper. Opin. Ther. Pat. 2012, 22, 79.
5. (a) Abbate, F.; Winum, J. Y.; Potter, B. V. L.; Casini, A.; Montero, J. L.; Scozzafava,
A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2004, 14, 231; (b) Nishimori, I.; Vullo,
D.; Innocenti, A.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. J. Med. Chem.
2005, 48, 7860.
l
lM
(vi) The secreted (in saliva and milk)1–3 isoform hCA VI was
inhibited in the low nanomolar range by compounds 4–6,
which showed KIs in the range of 8–41 nM, and in the micro-
molar one (KIs of 2.23–5.12
except 8 which was not inhibitory (KI >200
(vii) the cytosolic isoform hCA VII was inhibited in the submi-
cromolar range by 4–6 (KIs of 0.27–0.84 M) and in the
micromolar range by the remaining derivatives 1–3 and 7
(KIs of 4.68–10.9 M). Coumarin 8 was not inhibitory (KI
>200 M) also against this isoform.
lM by the remaining ones,
lM).
l
l
l
(viii) The tumor-associated hCA IX, a validated antitumor target,7
was inhibited in the low nanomolar range by 4 (KI of 16 nM)
and also significantly inhibited by compounds 5 and 6 (KIs of
58–82 nM) whereas 1, 2, and 7 were micromolar inhibitors
6. Bialer, M.; Johannessen, S. I.; Kupferberg, H. J.; Levy, R. H.; Loiseau, P.; Perucca,
E. Epilepsy Res. 2001, 43, 11.
7. Neri, D.; Supuran, C. T. Nat. Rev. Drug Discov. 2011, 10, 767.
8. 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.; Görlach, A.; Gasmann, M.; Harris,
(KIs of 1.72–6.41
lM). Thiomerosal 3 and coumarin 8 were
not inhibitory at all (KIs >200
l
M) against this isoform. A
A. L.; Maxwell, P.; Scozzafava, A. J. Enzyme Inhib. Med. Chem. 2009, 24, 1; (b)
´
rather similar behavior was observed also for the inhibition
of hCA XII, another tumor-associated CA,7 for which com-
pounds 4–6 showed inhibition constants of 54–93 nM, cou-
marin 8 was not inhibitory whereas derivatives 1–3 and 7
ˇ
Švastová, E.; Hulıková, A.; Rafajová, M.; Zat’ovicová, M.; Gibadulinová, A.;
Casini, A.; Cecchi, A.; Scozzafava, A.; Supuran, C.; Pastorek, J. FEBS Lett. 2004,
577, 439; (c) Dubois, L.; Lieuwes, N. G.; Maresca, A.; Thiry, A.; Supuran, C. T.;
Scozzafava, A.; Wouters, B. G.; Lambin, P. Radiother. Oncol. 2009, 92, 423; (d)
Swietach, P.; Wigfield, S.; Cobden, P.; Supuran, C. T.; Harris, A. L.; Vaughan-
Jones, R. D. J. Biol. Chem. 2008, 283, 20473.
were weak, micromolar inhibitors (KIs of 2.77–9.52 lM).
9. Pacchiano, F.; Carta, F.; McDonald, P. C.; Lou, Y.; Vullo, D.; Scozzafava, A.;
Dedhar, S.; Supuran, C. T. J. Med. Chem. 1896, 2011, 54.
10. Scolnick, L. R.; Clements, A. M.; Liao, J.; Crenshaw, L.; Hellberg, M.; May, J.;
Dean, T. R.; Christianson, D. W. J. Am. Chem. Soc. 1997, 119, 850.
11. Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C. T. J.
Med. Chem. 2010, 53, 5511.
12. (a) 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; (b) Maresca, A.;
Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava, A.; Supuran, C. T. J. Med.
Chem. 2010, 53, 335.
13. (a) Maresca, A.; Supuran, C. T. Bioorg Med. Chem. Lett. 2010, 20, 4511–4514; (b)
Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20,
7255.
(ix) although one is cytosolic (hCA XIII) and the other one trans-
membrane (hCA XIV), these two CAs showed a strikingly sim-
ilar inhibition profile with derivatives 1–8 (Table 1). Thus,
oximes 4, 5 and the isosteric to 5 phenol 6 were the best inhib-
itors (KIs in the range of 16–94 nM), whereas 8 was not at all
inhibitory (KI >200
thimerosal 3 and coumaphos 7 were weak, micromolar inhib-
itors, with inhibition constants in the range of 1.31–6.97 M.
lM). The mercaptan 1, thiobenzoic acid 2,
l
In addition of being CA II-sparing CAIs, the compounds investi-
gated here share another common feature: they can inhibit CAs by
various mechanisms. For example, most of these compounds pos-
sess groups which may coordinate to the Zn(II) ion from the CA ac-
tive site, such as the SH one in 1, the CSOH one in 2, COOH in 3, OH
(from the oxime in 4 and 5) or the phenolic moiety in 6, the C@S (or
even the ester one) in coumaphos 7. However, all these compounds
possess also moieties which render them similar to coumarins,12,13
or lactones23 which bind (in hydrolyzed state) at the entrance of
the active site, in the so-called coumarin-binding site.12,13,21
Although this study does not enable us to understand the inhibi-
tion mechanism of these compounds, as no good crystal was ob-
tained so far, the main conclusion is that a rather wide range of
14. (a) Davis, R. A.; Innocenti, A.; Poulsen, S. A.; Supuran, C. T. Bioorg. Med. Chem.
2010, 18, 14; (b) Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg.
Med. Chem. Lett. 2008, 18, 1583; (c) Innocenti, A.; Vullo, D.; Scozzafava, A.;
Supuran, C. T. Bioorg. Med. Chem. 2008, 16, 7424.
15. (a) Lou, Y.; McDonald, P. C.; Oloumi, A.; Chia, S. K.; Ostlund, C.; Ahmadi, A.;
Kyle, A.; Auf dem Keller, U.; Leung, S.; Huntsman, D. G.; Clarke, B.; Sutherland,
B. W.; Waterhouse, D.; Bally, M. B.; Roskelley, C. D.; Overall, C. M.; Minchinton,
A.; Pacchiano, F.; Carta, F.; Scozzafava, A.; Touisni, N.; Winum, J. Y.; Supuran, C.
T.; Dedhar, S. Cancer Res. 2011, 71, 3364; (b) Touisni, N.; Maresca, A.;
McDonald, P. C.; Lou, Y.; Scozzafava, A.; Dedhar, S.; Winum, J.-Y.; Supuran, C.
T. J. Med. Chem. 2011, 54, 8271.
16. Nair, S. K.; Ludwig, P. A.; Christianson, D. W. J. Am. Chem. Soc. 1994, 116, 3659.
17. Davis, R. A.; Hofmann, A.; Osman, A.; Hall, R. A.; Mühlschlegel, F. A.; Vullo, D.;
Innocenti, A.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2011, 54, 1682.
18. (a) Dorea, J. G. Neurochem. Res. 2011, 36, 927; (b) Geier, D. A.; Sykes, L. K.; Geier,
M. R. J. Toxicol. Environ. Health B. Crit. Rev. 2007, 10, 575.
Si O
NH2
MeOH
4
4
3
3
5
i)
ii) TBAF 1.0M, THF
S
5
Lawesson's Reagent
2
2
6
6
OH
O
N
O
Tol.
O
O
4; 30 % yield
10

F. Carta et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2182–2185
2185
19. Synthesis of 4,6-dimethyl-2H-pyran-2-one oxime 4
20. (a) Mao, W.; Schuler, M. A.; Berenbaum, M. R. Proc. Natl. Acad. Sci. U.S.A. 2011,
108, 12657; (b) Del Carlo, M.; Pepe, A.; Sergi, M.; Mascini, M.; Tarentini, A.;
Compagnone, D. Talanta 2010, 81, 76; (c) Maggi, M. D.; Ruffinengo, S. R.;
Damiani, N.; Sardella, N. H.; Eguaras, M. J. Exp. Appl. Acarol. 2009, 47, 317.
21. Temperini, C.; Innocenti, A.; Scozzafava, A.; Parkkila, S.; Supuran, C. T. J. Med.
Chem. 2010, 53, 850.
22. (a) Nishimori, I.; Minakuchi, T.; Onishi, S.; Vullo, D.; Cecchi, A.; Scozzafava, A.;
Supuran, C. T. Bioorg. Med. Chem. 2007, 15, 7229; (b) Nishimori, I.; Minakuchi,
T.; Onishi, S.; Vullo, D.; Cecchi, A.; Scozzafava, A.; Supuran, C. T. J. Enz. Inhib.
Med. Chem. 2009, 24, 70.
Preparation of 4,6-dimethyl-2H-pyran-2-thione 10: 4,6-dimethyl-2H-pyran-2-
one (0.1 g, 1.0 equiv) was treated with Lawesson’s reagent (1.5 equiv) dry
toluene (10 ml) and the yellow solution was refluxed until starting material
was consumed (TLC monitoring). Then the solvent was removed under vacuo
and the orange residue was partitioned between H2O and ethyl acetate. The
organic phase was washed with H2O (2 Â 20 ml), brine (3 Â 20 ml), dried over
Na2SO4, filtered-off and concentrated under vacuo to give a red sticky oil that
was purified by silica gel column chromatography eluting with 20% ethyl
acetate in n-hexane to afford 10 as a yellow solid.
4,6-Dimethyl-2H-pyran-2-thione 10: 75 % yield; mp 72–74 °C; silica gel TLC Rf
0.59 (Ethyl acetate/n-hexane 50% v/v);
23. Carta, F.; Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2012, 22, 267.
m
cmÀ1 (KBr) 1645, 1540; dH (400 MHz,
DMSO-d6) 2.13 (3H, s), 2.37 (3H, s), 6.60 (1H, s, 5-H), 7.06 (1H, s, 3-H); dC
(100 MHz, DMSO-d6) 197.5 (C@S), 168.2, 154.2, 127.4, 112.1, 21.3, 20.4. Data
are in agreement with reported data.23
24. 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–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 coumarin and formation of the 2-hydroxy-cinnamic acid type
of compounds for the coumarins (and probably oximes 4 and 5 too).1,2 For
Synthesis of 4,6-dimethyl-2H-pyran-2-one oxime 4
4,6-Dimethyl-2H-pyran-2-thione 10 (0.1 g, 1.0 equiv) was dissolved in MeOH
(10 ml) and O-(tert-butyldimethylsilyl)hydroxylamine (2.0 equiv) was added.
The solution was refluxed ON, then the solvents were removed under vacuo to
give a pale yellow oil that was dissolved in THF (3.0 ml) and treated with 1.0 M
TBAF in THF. The yellow solution was stirred at rt for 10 min, quenched with
H2O (20 ml) and extracted with ethyl acetate (3 Â 15 ml). The combined
organic layers were washed with brine (3 Â 15 ml), H2O (3 Â 15 ml), dried over
Na2SO4, filtered-off and concentrated under vacuo to give a residue that was
purified by silica gel column chromatography eluting with 50% ethyl acetate in
n-hexane to afford the titled compound as a pale yellow solid.
4,6-Dimethyl-2H-pyran-2-one oxime 4: 30% yield; silica gel TLC Rf 0.30 (ethyl
acetate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6) 2.13 (3H, s, CH3), 2.31 (3H,
s, CH3), 6.02 (1H, s, 3-H, 5-H), 6.22 (1H, s, 3-H, 5-H), 10.50 (1H, s, exchange with
D2O, O-H); dc (100 MHz, DMSO-d6), 159.6, 155.1 (C@N), 153.0, 108.2, 118.6,
24.2, 22.0.
Synthesis of (Z)-2H-chromen-2-one oxime 5
4
5
3
6
2
OH
7
O
N
E
8
Si O
NH2
4
5
MeOH
3
6
i)
ii) TBAF 1.0M, THF
S
12; traces
Lawesson's Reagent
2
4
5
7
3
2
6
O
Tol.
O
O
8
7
O
N
11
Z
8
OH
5; 60 % yield
(Z)-2H-Chromen-2-one oxime 5 was synthesized according to the previous
procedure. The geometric configuration of the oximes 12 and 5 was assigned
through NOESY experiments.
2H-Chromene-2-thione 11: 60% yield; silica gel TLC Rf 0.27 (Ethyl acetate/n-
hexane 20% v/v); mmax (KBr) cmÀ1 1765, 1518, 1220; dH (400 MHz, DMSO-d6)
7.31 (1H, d, J 10.0, 3-H), 7.61 (1H, dt, J 7.6, 1.2, 6-H), 7.64 (1H, d, J 8.4, 5-H), 7.74
(1H, dt, J 7.6, 1.2, 7-H), 7.85 (1H, d, J 8.4, 8-H), 7.96 (1H, d, J 10.0, 4-H); dc
(100 MHz, DMSO-d6), 198.5 (C@S), 157.0, 137.0, 133.6, 130.0, 129.6, 126.8,
121.2, 117.1; Anal. Calcd C, 66.64; H, 3.73; S, 19.77; Anal. Found. C, 66.15; H,
3.43; S, 12.38.
(E)-2H-Chromen-2-one oxime 12: traces amount; silica gel TLC Rf 0.34 (Ethyl
acetate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6) 6.84 (1H, d, J 9.8, 3-H), 7.14
(3H, m), 7.20 (1H, d, J 9.8, 4-H), 7.41 (1H, m), 10.04 (1H, s, exchange with D2O,
O-H).
(Z)-2H-Chromen-2-one oxime 5: 60% yield; silica gel TLC Rf 0.30 (Ethyl acetate/
n-hexane 50% v/v); dH (400 MHz, DMSO-d6) 6.34 (1H, d, J 9.6, 3-H), 7.03 (1H, d, J
9.6, 4-H), 7.17 (2H, m, 5-H, 8-H), 7.38 (2H, m, 6-H, 7-H), 10.34 (1H, s, exchange
with D2O, O-H); dc (100 MHz, DMSO-d6), 157.8, 153.1 (C@N), 132.7, 132.1,
128.6, 124.1, 120.7, 116.2, 111.4.
compounds 1–3 and 6, the inhibition constants were identical after 15 min or
6 h incubation time with the enzyme, proving that no hydrolytic processes
occur (data not shown). The inhibition constants were obtained by non-linear
least-squares methods using PRISM 3, as reported earlier,12–14 and represent
the mean from at least three different determinations. CA isofoms were
recombinant ones obtained in house as reported earlier.25 Compounds 1–3,
and 6–8 were commercially available from Sigma–Aldrich (Milan, Italy)
whereas oximes 4 and 5 were prepared as described in Ref. 19
25. (a) Supuran, C. T.; Scozzafava, A. Bioorg. Med. Chem. 2007, 15, 4336; (b)
Temperini, C.; Cecchi, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2008, 18, 2567; (c) Supuran, C. T. Curr. Pharm. Des. 2008, 14, 603; (d) Temperini,
C.; Cecchi, A.; Scozzafava, A.; Supuran, C. T. Org. Biomol. Chem. 2008, 6, 2499; (e)
Temperini, C.; Cecchi, A.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2009, 52,
322; (f) Temperini, C.; Cecchi, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med.
Chem. 2009, 17, 1214; (g) Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan,
P.; Parkkila, S.; Scaloni, A.; Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava,
A.; Monti, S. M.; De Simone, G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
16233.