Discovery of low nanomolar and subnanomolar inhibitors of the mycobacterial β-carbonic anhydrases Rv1284 and Rv3273

Article abstract of DOI:10.1021/jm9004016

A series of 2-(hydrazinocarbonyl)-3-aryl-1H-indole-5-sulfonamides has been derivatized by reaction with 2,4,6-trimethylpyrylium perchlorate, leading to pyridinium derivatives. The new sulfonamides were evaluated as inhibitors of two β-carbonic anhydrases

Full text of DOI:10.1021/jm9004016

J. Med. Chem. 2009, 52, 4063–4067
4063
Discovery of Low Nanomolar and Subnanomolar Inhibitors of the Mycobacterial ꢀ-Carbonic
Anhydrases Rv1284 and Rv3273
Ozlen Gu¨zel, Alfonso Maresca,‡ Andrea Scozzafava,‡ Aydın Salman,† Alexandru T. Balaban,§ and Claudiu T. Supuran*,‡
†,‡
¨
Istanbul UniVersity, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34116 Beyazıt, Istanbul, Turkey, UniVersita` degli Studi di
Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy,
Texas A&M UniVersity at GalVeston, 5007 AVenue U, GalVeston, Texas 77551
ReceiVed March 30, 2009
A series of 2-(hydrazinocarbonyl)-3-aryl-1H-indole-5-sulfonamides has been derivatized by reaction with
2,4,6-trimethylpyrylium perchlorate, leading to pyridinium derivatives. The new sulfonamides were evaluated
as inhibitors of two ꢀ-carbonic anhydrases (CAs, EC 4.2.1.1) from Mycobaterium tuberculosis, Rv1284
and Rv3273. The whole series showed excellent nanomolar inhibitory activity, with several subnanomolar
inhibitors being detected. Rv1284 and Rv3273 have potential for developing antimycobacterial agents with
an alternate mechanism of action.
Introduction
indisulam B were the most effective Rv1284 inhibitors,1 whereas
acetazolamide C and 2-amino-pyrimidin-4-yl-sulfanilamide D
were the best Rv3273 inhibitors),2 which are not easily amenable
to derivatization. Thus, we decided to explore different scaffolds
incorporating the sulfamoyl zinc-binding groups (ZBGs) for the
design of ꢀ-CA inhibitors targeting these mycobacterial enzymes.
Recently, we have shown that two of the three carbonic
anhydrases (CAs,a EC 4.2.1.1) present in Mycobacterium
tuberculosis1,2 may be targeted by inhibitors belonging to the
sulfonamide class. Indeed, the ꢀ-CAs encoded by the Rv12841,3
and Rv32732 genes of this widespread human pathogen4 were
shown to be catalytically active enzymes for the physiologic
reaction catalyzed by these proteins, i.e., CO2 hydration to
bicarbonate and protons,5 and to be susceptible to inhibition
with a wide range of aromatic and heteroaromatic sulfonamides
as well as the bioisosteric sulfamates.1,2 Indeed, M. tuberculosis
infection affects a large number of the world population, with
more than 9 million new cases diagnosed each year, of which
many are lethal, with multidrug resistant and extensively
multidrug resistant tuberculosis (TB) being more and more
widespread.6,7 The therapy used to treat nondrug resistant TB
is based on agents developed 30-40 years ago, with no new
drugs launched ultimately.8 Our preliminary work on myco-
bacterial CA inhibition mentioned above,1,2 as well as molecular
biology data showing these enzymes to be essential for the
growth/virulence of M. tuberculosis (based on mutagenesis
studies in strain H37Rv9 and up-regulation of the encoding genes
under the starvation conditions used to model persistent bac-
teria),10 suggest that inhibition of mycobacterial ꢀ-CAs may
be used for drug design campaigns aiming to find antimyco-
bacterial agents possessing a new mechanism of action.
Recently, we have reported the excellent inhibitory activity
against various mammalian CA isozymes (belonging to the
R-CA genetic family)5 of a series of of 2-(hydrazinocarbonyl)-
3-aryl-1H-indole-5-sulfonamides.11 The X-ray crystal structure
for the adduct of one of these compounds (the lead molecule
L) with the physiologically relevant isoform human (h) hCA II
has also been reported,11a evidencing a large number of
favorable interactions between the active site of the enzyme
and this inhibitor (Figure 1).
Considering this X-ray crystal structure of the hCA II - L
adduct as starting point11a and the good inhibitory activity of
this class of sulfonamides against many R-CA isoforms,11,12
we decided to investigate whether this scaffold may also lead
to effective ꢀ-CA inhibitors targeting the mycobacterial en-
zymes. We report here the synthesis and inhibitory properties
against the mycobacterial CAs of a series of sulfonamides
incorporating this new scaffold.
Although we showed that Rv12841 and Rv32732 may be
inhibited by many types of sulfonamides or sulfamates, the best
inhibitors detected so far showed only medium-low potency,
with the best Rv1284 inhibitors possessing KIs in the range of
100-200 nM and the best Rv3273 inhibitors having KIs in the
range of 90-500 nM. Furthemore, the best such compounds
possessed simple scaffolds (e.g., 3-bromosulfanilamide A and
* To whom correspondence should be addressed. Phone: +39-055-
4573005. Fax: +39-055-4573385. E-mail: claudiu.supuran@unifi.it.
†
Istanbul University, Faculty of Pharmacy, Department of Pharmaceuti-
cal Chemistry.
Results and Discussion
‡
Universita` degli Studi di Firenze, Polo Scientifico, Laboratorio di
Chimica Bioinorganica.
Chemistry. The reaction between pyrylium salts and amines,
leading to pyridinium derivatives, constitutes a versatile method
for the preparation of a large number of positively charged
derivatives, not readily accessible by other synthetic proce-
§
Texas A&M University at Galveston.
a Abbreviations: CA, carbonic anhydrase; CAI, CA inhibitor; hCA,
human CA; GST, glutathione-S-transferase; mtCA, Mycobacterium tuber-
culosis CA; TB, tuberculosis.
10.1021/jm9004016 CCC: $40.75  2009 American Chemical Society
Published on Web 05/13/2009

4064 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Brief Articles
so far,1,2 i.e., compounds A-D are also shown in Table 1 for
comparison reasons.
The following should be noted regarding the inhibition data
of Table 1:
(i) Rv1284 was effectively inhibited by the lead molecule L
possessing a new indolesulfonamide scaffold, with a KI of 48
nM. L is thus already two times a better Rv1284 CAI as
compared to the best such compound detected so far, indisulam
B (KI of 97 nM).1 Probably this new scaffold fits better within
the Rv1284 active site compared to the scaffolds of derivatives
investigated earlier for the inhibition of this enzyme. However,
the pyridinium derivatives obtained by reaction of L with 2,4,6-
trimethylpyrylium salts, of types 3-17, were much more
effective CAIs as compared to L or any other sulfonamide/
sulfamate investigated earlier.1 Thus, the new class of sulfona-
mides reported here showed inhibition constants in the range
of 0.92-35.3 nM against Rv1284. The least effective derivatives
were 7-10, which with KIs in the range of 20.9-35.3 nM were
anyhow much more effective Rv1284 CAIs compared to
compounds A and B investigated previously (KIs of 97-186
nM). These least effective CAIs incorporate 2-, 3-, 4-chloro-
and 2-bromophenyl moieties in position 3 of the indolesulfona-
mide scaffold. Another group of the new derivatives, among
which 3-6, 11, 12, and 14, showed an enhanced inhibitory
activity toward Rv1284, with KIs in the range of 1.5-9.7 nM.
These compounds incorporate the following substitution patterns
at the 3-phenyl moiety of the indolesulfonamide scaffold:
unsubstituted phenyl; 2-, 3-, and 4-fluorophenyl, 3-bromo- and
4-bromophenyl and 3-tolyl. The remaining derivatives 13 and
15-17 were subnanomolar inhibitors of Rv1284, with KIs of
0.92-0.98 nM. They incorporate 2- and 4-tolyl, 3-methoxyphe-
nyl, and perfluorophenyl moieties in the 3 position of the
indolesulfonamide scaffold. Thus, not only the 2-(hydrazinocar-
bonyl)-3-substituted-phenyl-1H-indole-5-sulfonamide deriva-
tives lead to highly effective Rv1284 CAIs but the pyridinium
derivatives obtained from this lead molecule show a very
interesting SAR, with the nature of the group substituting the
3-phenyl ring strongly influencing the enzyme inhibitory activity.
Many such favorable substitution patterns are reported here, such
as among others the perfluorophenyl, tolyl, or 3-methoxyphenyl
ones.
Figure 1. Schematic interactions to which L participates when bound
within the hCA II active site, as determined by X-ray crystallography
(PDB code 3B4F).11a Hydrogen bonds in which several moieties of
the inhibitor participate with amino acid residues Asn62, Asn67, and
Thr199 from the enzyme active site and a water molecule (Wat101)
are shown as dotted lines. The trimethypyridinium group of the newly
dsigned compounds 3-17 reported here presumably lies in the
hydrophilic half of the CA active site, between Asn62 and Asn67.
dures.13 Many biologically active compounds have been ob-
tained in this way,13-15 among which also different classes of
sulfonamide CAIs.14,15 The apparently simple reaction between
a pyrylium salt and an amine, leading to pyridinium salts, is in
reality a complicated process, as established by detailed
spectroscopic and kinetic data from Balaban’s and Katritzky’s
groups.16-18 Thus, the nucleophilic attack of a primary amine
RNH2 on pyrylium cations generally occurs in the R position,
with the formation of intermediates that are deprotonated in the
presence of bases leading to 2-amino-tetradehydropyran deriva-
tives and in the end to pyridinium salts.16,17 A supplementary
complication appears when the moiety substituting the 2- and/
or 6-position(s) of the pyrylium ring is a methyl, a case in which
a concurrent cyclization with formation of anilines in addition
to the pyridinium salts may also take place.16-18 These
concurrent reactions mentioned above are generally important
when the amine to be converted into pyridinium salt possesses
weak nucleophilicity or basicity, as it happens to be the case of
our sulfonamides 1,11 used as key intermediates for preparing
the new compounds reported here (Scheme 1).16-18
(ii) Rv3273 was also highly inhibited by L and its pyridinium
derivatives 3-17 investigated here, with KIs in the range of
0.88-31 nM (Table 1). L was the least effective such CAI (KI
of 31 nM) although being at least a 3 times better inhibitor
compared to compounds C and D investigated earlier2 and found
to be the most effective inhibitors of this mycobacterial CA.
All substitution patterns present in the pyridinium derivatives
3-17 were highly effective in inducing excellent Rv3273
inhibitory properties, as the entire class of compounds showed
a compact behavior of very potent CAIs, with inhibition
constants <8 nM. Thus, the unsubstituted compound 3, its 2-,
3-, 4-fluoro, 2- and 3-chloro, 2-, 3-, and 4-methyl, as well as
3-methoxy derivatives, showed KIs of 1.8-8.0 nM, whereas the
remaining compounds were even better CAIs, with inhibition
constants in the range of 0.88-1.01 nM (Table 1). These last
derivatives incorporate 4-chlorophenyl, 2-, 3-, and 4-bromophe-
nyl, and perfluorophenyl moieties in position 3 of the indole-
sulfonamide scaffold. It should be noted that the simple
sulfonamides A-D investigated earlier,2 as Rv3273 inhibitors
are several orders of magnitude weaker CAIs as compared to
the compounds investigated here.
The Bayer-Piccard synthesis13-18 was successfully applied
to sulfonamides 1,15 possessing the nucleophilic amino moiety
incorporated in the carbohydrazide functionality. These com-
pounds were reacted with 2,4,6-trimethylpyrylium perchlorate
2, leading to a series of new pyridinium salts of type 3-17
(Scheme 1). Yields were in the range of 41-79%, and no
complications for the synthesis and purification of these new
derivatives were encountered. The new compounds 3-17 were
characterized by standard physicochemical procedures, which
confirmed their structures (see Experimental Section and Sup-
porting Information for details) and possess a purity of >99.5%.
Mycobacterial CA Inhibition Studies. Inhibition data of the
two ꢀ-CAs of M. tuberculosis, encoded by the genes Rv1284
and Rv3273, are shown in Table 1. Data of Table 1 also present
the inhibitory activity against two physiologically relevant host
CA isozymes (hCA I and II, which are cytosolic, ubiquitous
isoforms)5 of the new compounds 3-17 reported here, as the
search of pathogen-selective CAIs is an important aspect in the
design of new applications for this class of pharmacological
agents. Inhibition data with the best mycobacterial CAIs detected
(iii) Compounds 3-17 were also investigated as inhibitors
of two human CAs, the cytosolic isoforms hCA I and II. Against

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4065
Scheme 1. Preparation of 1-(([5-(Aminosulfonyl)-3-substituted-phenyl-1H-indol-2-yl]carbonyl)amino)-2,4,6-trimethylpyridinium
Perchlorates 3-17a
a Reagents and conditions: MeOH (reflux), 6% HClO4.
Table 1. Inhibition of CA Human Isoforms hCA I, II, and
Mycobacterial Enzymes Rv1284 and Rv3273 with Sulfonamides 3-17,
L, as well as Simple Sulfonamides or Clinically Used CA Inhibitors
A-D as Standardsa
ineffective hCA II inhibitors have the substituent of the phenyl
moiety in the meta-position, probably provoking a clash with
some amino acid residues present in the hCA II active site, as
already documented by us earlier.19 However, in the absence
of detailed structural data for this class of inhibitors this remains
a hypothesis to be checked. The pentafluorophenyl derivative
17 on the other hand, is 7.7 times more effective as a hCA II
inhibitor as compared to the lead L, and it would be also of
great interest to resolve its high resolution X-ray crystal structure
in adduct with hCA II for understanding the elements leading
to this excellent inhibitory activity.
KIb (nM)
inhibitor
R
hCA Ic
hCA IIc
Rv1284d
Rv3273d
L
3
7.5
9.0
7.2
71
48
6.5
31
8.0
H
4
5
6
2-F
3-F
4-F
8.5
11.3
7.6
91
3380
65
9.3
7.6
9.7
6.9
6.0
6.5
7
8
9
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-Me
3-Me
4-Me
3-OMe
F5
25.1
113
3.2
43.4
30.8
12.3
10.5
110
5.1
8.6
9.7
6500
31
250
109
100
1800
77
38
74
35.3
31.8
20.9
25.1
3.2
5.2
0.98
1.5
0.97
0.92
0.93
186
97
6.7
6.6
(v) Many of the new sulfonamides reported here showed a
much better inhibition of the mycobacterial ꢀ-CAs Rv1284 and
Rv3273 than for the host enzymes hCA I and II belonging to
the R-CA genetic family. Furthermore, many of these com-
pounds showed appreciable inhibition of only hCA I, an enzyme
whose physiologic function is not well understood but seems
to be marginal,5 whereas the physiologically dominant hCA II
showed a weak inhibition with these compounds (except one
of them, 17, which behaved as a very strong hCA II inhibitor).
Thus, our finding is important not only for detecting low
nanomolar and subnanomolar inhibitors of the two mycobac-
terial CAs but also because these compounds showed a much
higher affinity for these ꢀ-CAs than for hCA II. Thus, the
selectivity ratios for the inhibition of the pathogen over the host
enzymes are indeed very favorable for the potential use of these
compounds for in vivo antimycobacterial studies. Furthermore,
two of the three mycobacterial CAs known so far are both highly
inhibited by this class of sulfonamides investigated here.
0.96
1.01
0.97
0.96
6.8
7.8
3.6
1.8
10
11
12
13
14
15
16
17
A
B
85
106
104
68
2840
0.93
40
15
12
33
0.88
7320
7840
104
91
C
D
481
750
a Subnanomolar inhibitors are highlighted in bold characters. b Errors
in the range of 5-10% of the shown data, from three different assays, by
a CO2 hydration stopped-flow assay.16 c Human, recombinant isozymes,
pH 7.5, 20 mM TRIS ·HCl buffer. d Bacterial recombinant enzyme, at 20
°C, pH 8.3 in 20 mM TRIS ·HCl buffer and 20 mM NaCl.
the cytosolic isoform hCA I, the new pyridinium sulfonamides
3-17 generally showed good inhibitory activity (Table 1), with
KIs in the range of 3.2-113 nM, being thus more active than
the clinically used sulfonamide acetazolamide C and having a
similar activity to indisulam C, a compound in clinical develop-
ment as an anticancer agent.5 Most of the new derivatives 3-17,
similarly to the lead L, were in fact low nanomolar hCA I
inhibitors (KIs in the range 3.2-30.8 nM), except for 8, 10,
and 14, which were less active (KIs in the range 43.4-113 nM).
SAR was thus rather flat except for the three less active
compounds mentioned earlier, proving that most of the substitu-
tion patterns present in the phenyl ring in position 3 are
beneficial for the hCA I inhibitory properties of these compounds.
(iv) Although the lead L showed excellent hCA II inhibitory
activity (KI of 7.2 nM), the derivatives 3-17 reported here were
generally much less effective inhibitors of this ubiquitous
isoform, with KIs in the range of 38-3380 nM, except for the
pentafluorophenyl derivative 17, which was a subnanomolar
hCA II inhibitor (KI of 0.93 nM). These data are indeed very
interesting, as they prove that the substitution pattern of the
phenyl moiety in the pyridinium salts 3-17 is crucial for their
hCA II inhibitory activity. Thus, a very active compound has
been detected (17), together with moderate inhibitors (such as
3, 4, 6, 7, 9-15, KIs in the range 38-106 nM), as well as three
very ineffective inhibitors (5, 8, and 16, possessing KIs in the
range 1800-3380 nM). It should be noted that all these
Conclusions
The ꢀ-CAs encoded by the genes Rv1284 and Rv3273 of
Mycobacterium tuberculosis, which show appreciable catalytic
activity for the physiological reaction, CO2 hydration to
bicarbonate, and a proton, play an important role in the pathogen
life cycle and are inhibited by sulfonamides. We report here a
series of 2-(hydrazinocarbonyl)-3-aryl-1H-indole-5-sulfonamides
possessing various 2-, 3-, or 4-substituted phenyl groups with
methyl-, halogeno-, and methoxy- functionalities as well as a
perfluorophenyl moieties in their molecule. They were deriva-
tized by reaction with 2,4,6-trimethylpyrylium perchlorate,
leading to pyridinium derivatives. These compounds were highly
effective, low nanomolar or subnanomolar inhibitors of the two
mycobacterial enzymes, having a good affinity for the host
enzyme hCA I but much lower inhibitory properties against the
major, physiologically dominant isoform hCA II. These new
compounds are several orders of magnitude better mycobacterial
CAIs compared to sulfonamides/sulfamates investigated earlier.
Rv1284 and Rv3273 have thus potential for developing anti-
mycobacterial agents with an alternate mechanism of action,
considering that many M. tuberculosis strains exhibit multidrug

4066 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Brief Articles
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Experimental Section
Materials and Methods. Sulfonamides 1 used in the synthesis
were reported earlier.11 2,4,6-Trimethylpyrylium perchlorate 2 and
standard sulfonamides were commercially available compounds
(from Sigma-Aldrich, Milan, Italy). The CA isozymes used in the
experiments were recombinant ones obtained and purified as
reported earlier by this group.1,2 1H, DEPT, NOESY, COSY,
HMQC, and HMBC spectra were recorded using a Bruker Advance
III 300 MHz spectrometer. The chemical shifts are reported in parts
per million (ppm). Melting points (mp) were measured in open
capillary tubes using a Bu¨chi melting point B-540 melting point
apparatus and are uncorrected. Thin layer chromatography (TLC)
was carried out on Merck silica gel 60 F254 aluminum backed plates.
Elution of the plates was carried out using MeOH/DCM or MeOH/
CHCl3 systems. Visualization was achieved with UV light at 254
nm. Elemental analysis was done in house by combustion. Electron
ionization mass spectra (30 eV) were recorded in positive or
negative mode on a Water MicroMass ZQ. The purity has been
determined by means of analytical HPLC, performed on a reversed-
phase C18 Bondapack column, with a Beckman EM-1760 instru-
ment. Both conbustion and HPLC confirmed a purity of >99.5%
for the new compounds reported here.
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culosis persistence by gene and protein expression profiling. Mol.
Microbiol. 2002, 43, 717–31.
General Procedure for the Preparation of Compounds
3-17. 2,4,6-Trimethylpyrylium perchlorate 2 (1.5 mM) was dis-
solved in 20 mL of methanol. After addition of 2-(hydrazinocar-
bonyl)-3-substituted-phenyl-1H-indole-5-sulfonamide derivatives 1
(3 mM), the solution was refluxed 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.
¨
(11) (a) Gu¨zel, O.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Salman,
A.; Supuran, C. T. Carbonic anhydrase inhibitors. Interaction of
2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide with twelve
mammalian isoforms: kinetic and X-Ray crystallographic studies.
¨
Bioorg. Med. Chem. Lett. 2008, 18, 152–158. (b) Gu¨zel, O.; Innocenti,
A.; Scozzafava, A.; Salman, A.; Parkkila, S.; Hilvo, M.; Supuran, C. T.
Carbonic anhydrase inhibitors. Synthesis and inhibition studies against
mammalian isoforms I - XV with a series of 2-(hydrazinocarbonyl)-
3-substituted-phenyl-1H-indole-5-sulfonamides. Bioorg. Med. Chem.
2008, 16, 9113–9120.
1-(([5-(Aminosulfonyl)-3-phenyl-1H-indol-2-yl]carbonyl)amino)-
2,4,6-trimethylpyridinium Perchlorate 3. Yield 79%; mp > 300
(12) Supuran, C. T.; Scozzafava, A.; Casini, A. Carbonic anhydrase
inhibitors. Med. Res. ReV. 2003, 23, 146–189.
1
°C (dec). H NMR (DMSO-d6, 300 MHz) δ (ppm): 2.61 (9H, s,
(13) (a) 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
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2). In Methoden der Organischen Chemie (Houben-Weyl), Vol. E7b;
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(c) Balaban, T. S.; Balaban, A. T. Pyrylium Salts. In Science of
Synthesis: Houben-Weyl Methods of Molecular Transformations,
Georg Thieme Verlag: Stuttgart, 2003; Vol. 14, pp 11-200.
(14) (a) Dinculescu, A.; Balaban, A. T. Reaction of pyrylium salts with
nucleophiles. XIV. New pyridinium salts with potential biological
activity. ReV. Roum. Chim. 1980, 25, 1505–1528. (b) Toma, C.;
Balaban, A. T. Reaction of pyrylium salts with nucleophiles. VI.
Reaction with amino acids. Tetrahedron 1966, (Suppl. 7), 27–34. (c)
Supuran, C. T.; Manole, G.; Dinculescu, A.; Schiketanz, A.; Gheo-
rghiu, M. D.; Puscas, I.; Balaban, A. T. Carbonic anhydrase inhibitors.
Part 5. Pyrylium salts in the synthesis of isozyme-specific inhibitors.
J. Pharm. Sci. 1992, 81, 716–719. (d) Supuran, C. T.; Scozzafava,
A.; Ilies, M. A.; Iorga, B.; Cristea, T.; Briganti, F.; Chiraleu, F.; Banciu,
M. D. Carbonic anhydrase inhibitors. Part 53. Synthesis of substituted-
pyridinium derivatives of aromatic sulfonamides: The first nonpoly-
meric membrane-impermeable inhibitors with selectivity for isozyme
IV. Eur. J. Med. Chem. 1998, 33, 577–595.
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)+. Elem anal. (C, H, N, S).
All other compounds (4-17) have been characterized as 3 (see
Supporting Information for details).
CA Catalytic Activity and Inhibition. An Applied Photophysics
stopped-flow instrument has been used for assaying the CA
catalyzed CO2 hydration activity20 as reported earlier.1,2
Acknowledgment. This research was financed in part by a
grant of the 6th Framework Programme of the European Union
¨
(DeZnIT project). O.G. is grateful to TUBITAK (Ankara,
Turkey) for the providing financing under contract no. 2219/
2008.
Supporting Information Available: The complete characteriza-
tion of compunds 3-17 is described in detail. This material is
available free of charge via the Internet at http://pubs.acs.org.
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