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Özlen Güzel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2931–2934
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Chem. 2000, 43, 292; (d) Supuran, C. T.; Scozzafava, A.; Ilies, M. A.; Briganti, F. J.
Enzyme Inhib. 2000, 15, 381.
clinically used sulfonamides although showing effective CA IX
inhibitory activity (KIs in the range of 25–50 nM), are much more
potent hCA II (KIs in the range of 8–38 nM) than hCA IX inhibitors.
(iv) Against the second tumor-associated isoform, hCA XII (less
widespread as compared to hCA IX)2,3 the new compounds
reported here showed a mixed behavior, with three of them acting
as very effective inhibitors (5, 6 and 9, KIs in the range of 9.5–
12.6 nM), whereas 3, 4, 7 and 8 being moderate inhibitors (KIs in
the range of 38–97 nM). The remaining derivatives acted as very
weak hCA XII inhibitors (KIs in the range of 3980–8360 nM). It is
rather difficult to explain this SAR data, but it is obvious that small
structural variations in the nature, number and position of substit-
uents of the phenyl moiety in these pyridinium salts cause a large
variation of the hCA XII inhibitory activity.
9. (a) Supuran, C. T.; Manole, G.; Dinculescu, A.; Schiketanz, A.; Gheorghiu, M. D.;
Puscas, I.; Balaban, A. T. J. Pharm. Sci. 1992, 81, 716; (b) Supuran, C. T.;
Scozzafava, A.; Ilies, M. A.; Iorga, B.; Cristea, T.; Chiraleu, F.; Banciu, M. D. Eur. J.
Med. Chem. 1998, 33, 577.
10. Svastova, E.; Hulikova, A.; Rafajova, M.; Zat’ovicova, M.; Gibadulinova, A.;
Casini, A.; Cecchi, A.; Scozzafava, A.; Supuran, C. T.; Pastorek, J.; Pastorekova, S.
FEBS Lett. 2004, 577, 439.
11. Dubois, L.; Douma, K.; Supuran, C. T.; Chiu, R. K.; van Zandvoort, M. A. M. J.;
Pastorekova, S.; Scozzafava, A.; Wouters, B. G.; Lambin, P. Radiother. Oncol.
2007, 83, 367.
12. (a) Bayer, A. Ber. Dtsch. Chem. Ges. 1910, 43, 2337; (b) Bayer, A.; Piccard, T.
Liebigs Ann. Chem. 1911, 384, 208.
13. Balaban, A. T.; Dinculescu, A.; Dorofeenko, G. N.; Fischer, G. W.; Koblik, A. V.;
Mezheritskii, V. V.; Schroth, W.. Pyrylium Salts: Syntheses, Reactions and Physical
Properties; Academic Press: New York, 1982. pp 8–360.
14. (a) Balaban, A. T.; Toma, C. Tetrahedron 1966, 1–7; (b) Katritzky, A. R.; Manzo, R.
H. J. Chem. Soc., Perkin Trans. 2 1981, 571–575; (c) Katritzky, A. R.; Lloyd, J. M.;
Patel, R. C. J. Chem. Soc., Perkin Trans. 1982, 117–123.
In conclusion, we reported here a series of 2-(hydrazinocarbon-
yl)-3-aryl-1H-indole-5-sulfonamides possessing various 2-, 3- or
4- substituted phenyl groups with methyl-, halogeno- and meth-
oxy-, or the perfluorophenyl moieties in their molecule. They were
derivatized by reaction with 2,4,6-trimethylpyrylium perchlorate,
leading to the corresponding pyridinium derivatives. These com-
pounds were evaluated as inhibitors of four mammalian CA
isoforms, that is, CA I, II (cytosolic), CA IX and XII (transmembrane,
tumor-associated forms). Good inhibitory activity was observed
against hCA I and less effective inhibitors of hCA II, two cytosolic,
house-keeping enzymes. Most of these compounds were excellent
hCA IX inhibitors, whereas only some of them showed this feature
against hCA XII. Being membrane impermeant due to the presence
of the cationic moieties in their molecules, and thus restricted to
the extracellular space where the CA IX/XII active sites are situated,
these new positively-charged sulfonamides are interesting candi-
dates for targeting the tumor-associated enzymes as possible
diagnostic tools or therapeutic agents. In fact, very recently
Pouyssegur’s group22b demonstrated by using siRNA silencing, that
the dual inhibition of CA IX and XII leads to an impressive 85%
reduction of hypoxic tumor cell growth. Indeed, as concluded by
these authors, the hypoxia-induced CA IX and CA XII are major
tumor prosurvival pH-regulating enzymes, and their combined tar-
geting by inhibitors as those reported here shows that they hold
potential as interesting anticancer targets.
15. Güzel, Ö.; Innocenti, A.; Scozzafava, A.; Salman, A.; Parkkila, S.; Hilvo, M.;
Supuran, C. T. Bioorg. Med. Chem. 2008, 16, 9113.
16. 2,4,6-Trimethyl pyrylium perchlorate
methanol. After addition of 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-
indole-5-sulfonamide derivatives (3 mM), the solution was refluxed
2 (1.5 mM) was dissolved in 20 mL
1
overnight. The cold mixture was treated with 200 mL of 6% perchloric acid to
precipitate the pyridinium salts. The obtained products were recrystallized
from water with 6% perchloric acid. 1-({[5-(aminosulfonyl)-3-phenyl-1H-indol-
2-yl]carbonyl}amino)-2,4,6 trimethylpyridinium perchlorate 3. Yield 79%;
mp > 300 °C (dec.); 1H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.61 (9H, s,
pyridinium 2,4,6-(CH3)3), 7.24 (2H, s, SO2NH2), 7.42–7.68 (6H, m, Ar-H),
7.70–7.84 (2H, m, Ar-H), 7.90 (2H, s, Ar-H), 8.05 (1H, s, CONH), 12.50 (1H, s,
indole NH); LC/MS: m/z 436 (M+H)+.
17. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics (Oxford,
UK) stopped-flow instrument has been used for assaying the CA catalyzed 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 10 mM Hepes
(pH 7.5) as buffer, 0.1 M Na2SO4 (for maintaining constant the ionic strength),
following the CA-catalyzed CO2 hydration reaction. 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 (10 mM) were prepared in
distilled-deionized water with 10–20% (v/v) DMSO (which is not inhibitory at
Acknowledgements
This research was financed in part by a Grant of the 6th Frame-
work Programme (FP) of the European Union (DeZnIT project), and
by a Grant of the 7th FP of EU (Metoxia project). Ö.G. is grateful to
TUBITAK (Ankara, Turkey) for providing financing under the Con-
tract No. 2219/2008.
these concentrations) and dilutions up to 0.01 lM were done thereafter with
distilled-deionized water. Inhibitor and enzyme solutions were preincubated
together for 15 min at room temperature prior to assay, in order to allow for
the formation of the E-I complex. The inhibition constants were obtained by
non-linear least-squares methods using PRISM 3, and represent the mean from at
least three different determinations. CA isozymes were recombinant ones
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