Carbonic anhydrase inhibitors. Diazenylbenzenesulfonamides are potent and selective inhibitors of the tumor-associated isozymes IX and XII over the cytosolic isoforms I and II

Article abstract of DOI:10.1016/j.bmc.2009.09.003

A series of diazenylbenzenesulfonamides, azo-dye derivatives of sulfanilamide or metanilamide incorporating phenol and amine moieties, were tested for inhibition of the tumor-associated isozymes of carbonic anhydrase (CA, EC 4.2.1.1), CA IX and XII. These compounds showed moderate-low inhibitory activities against the cytosolic isoforms CA I and II (offtargets) and excellent, low nanomolar inhibitory activity against the transmembrane CA IX and XII (K_Is in the range of 3.5-63 nM against CA IX and 5.0-69.4 nM against CA XII, respectively). The selectivity ratio for inhibiting the tumor-associated CA IX over the offtarget CA II was in the range of 15-104 for these diazenylbenzenesulfonamides, making them among the most isoform-selective inhibitors targeting tumor-associated CAs (over the ubiquitous CA II). Since CA IX/XII were recently shown to be both therapeutic and diagnostic targets for hypoxic solid tumors overexpressing these proteins, such compounds held promise for the management of hypoxic tumors, which are largely non-responsible to classical chemo- and radio-therapy.

Full text of DOI:10.1016/j.bmc.2009.09.003

Bioorganic & Medicinal Chemistry 17 (2009) 7093–7099
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Carbonic anhydrase inhibitors. Diazenylbenzenesulfonamides are potent
and selective inhibitors of the tumor-associated isozymes IX and XII over
the cytosolic isoforms I and II
*
Fabrizio Carta, Alfonso Maresca, Andrea Scozzafava, Daniela Vullo, 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:
Received 9 July 2009
Revised 29 August 2009
Accepted 3 September 2009
Available online 6 September 2009
A series of diazenylbenzenesulfonamides, azo-dye derivatives of sulfanilamide or metanilamide incorpo-
rating phenol and amine moieties, were tested for inhibition of the tumor-associated isozymes of car-
bonic anhydrase (CA, EC 4.2.1.1), CA IX and XII. These compounds showed moderate-low inhibitory
activities against the cytosolic isoforms CA I and II (offtargets) and excellent, low nanomolar inhibitory
activity against the transmembrane CA IX and XII (KIs in the range of 3.5–63 nM against CA IX and
5.0–69.4 nM against CA XII, respectively). The selectivity ratio for inhibiting the tumor-associated CA
IX over the offtarget CA II was in the range of 15–104 for these diazenylbenzenesulfonamides, making
them among the most isoform-selective inhibitors targeting tumor-associated CAs (over the ubiquitous
CA II). Since CA IX/XII were recently shown to be both therapeutic and diagnostic targets for hypoxic solid
tumors overexpressing these proteins, such compounds held promise for the management of hypoxic
tumors, which are largely non-responsible to classical chemo- and radio-therapy.
Keywords:
Carbonic anhydrase
Tumor-associated isoforms
CA IX
CA XII
Isoform-selective inhibitor
Diazenylbenzenesulfonamides
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
various pharmacological agents, such as diuretics, antiglaucoma,
antiobesity, antiepileptic or antitumor drugs/diagnostic agents.1
Carbonic anhydrases (CAs, EC 4.2.1.1) constitute an ubiquitous
family of metallo-enzymes found in prokaryotes and eukaryotes,
which catalyze the reversible hydration of carbon dioxide to the
Among all these applications, the potential use of CA inhibitors
(CAIs) for the management of solid hypoxic tumors which overex-
press two isoforms, CA IX and XII, became a hot topic, with many
investigations aiming to develop potent and isoform-selective CAIs
targeting these two isozymes.1,6–11 Indeed, hypoxia, through the
hypoxia inducible factor (HIF) cascade, leads to a strong overex-
pression of CA IX/XII in many tumors.8 The overall consequence
of these phenomena is a pH imbalance, with most hypoxic tumors
having acidic pH values around 6–6.5, in contrast to normal tissue
which have characteristic pH values around 7.4.8–10 Constitutive
expression of human CA IX (hCA IX) was recently shown to de-
crease extracellular pH (pHe) also in Madin-Darby canine kidney
(MDCK) epithelial cells (which normally do not express CA IX) by
this group.8 CA IX potent sulfonamide inhibitors were then shown
to reduce the medium acidity by inhibiting the catalytic activity of
the enzyme, and thus the generation of H+ ions, binding specifically
only to hypoxic cells expressing CA IX.8 Deletion of the CA active
site was demonstrated to reduce the medium acidity, but sulfon-
amide inhibitors did not bind to the active site of the mutant pro-
tein. Therefore, tumor cells decrease their pHe both by production
of lactic acid (due to the high glycolysis rates), and by CO2 hydra-
tion catalyzed by the tumor-associated CA IX/XII, possessing extra-
cellular catalytic domains.8–10 A low pHe has been associated with
tumorigenic transformation, chromosomal rearrangements,
+
1–5
ꢀ
bicarbonate ion and a proton (CO2 + H2O¡HCO3 + H ).
proteins are encoded by five evolutionarily unrelated gene fami-
lies: the -CAs (in vertebrates, bacteria, algae and cytoplasm of
green plants), the b-CAs (predominantly in bacteria algae and chlo-
These
a
roplasts), the c
-CAs (in archaea and some bacteria)1–3 and the d-
and n-CAs (in marine diatoms).2,3 There are no significant sequence
homologies between representatives of the different CA families,
but all members contain one Zn(II) or Cd(II) ion in their active site,
which are critical for the catalytic activity of the enzymes.1–3
a-CA isozymes are widely distributed in many tissues and or-
gans of vertebrates (including humans), as 16 different isoforms
(only 15 are found in primates, which lack the CA XV isoform).1
They play crucial roles in various physiological processes of such
organisms, such as CO2/HCO3ꢀ transport between metabolizing tis-
sues and lungs, pH and CO2 homeostasis, electrolyte secretion, bio-
synthetic reactions (gluconeogenesis, lipogenesis and ureagenesis),
bone resorption and tumorigenicity.1,6–10 As a consequence, many
of these CA isoforms are drug targets of interest for the design of
* Corresponding author. Tel.: +39 055 457 3005; fax: +39 055 457 3385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.003

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F. Carta et al. / Bioorg. Med. Chem. 17 (2009) 7093–7099
extracellular matrix breakdown, migration and invasion, induction
of the expression of cell growth factors and protease activation.10
CA IX also plays a role in providing bicarbonate to be used as a sub-
strate for cell growth, whilst it is established that bicarbonate is re-
quired in the synthesis of pyrimidine nucleotides.8–10 Thus, the
proof-of-concept study mentioned above8 showing the role of CA
IX in tumor acidification, also established that sulfonamide inhibi-
tors bind only to hypoxic cells overexpressing CA IX (and not to
their normal counterparts) and that CA IX inhibition with such
compounds reverts the tumor acidification processes, restoring a
more physiologically normal pHe. Dubois et al.8c also proved re-
cently the possibility to use sulfonamide CA IX—specific inhibitors
for imaging purposes in a xenograft tumor model in which CA IX is
overexpressed.
of sulfonamides, with weaker inhibitory properties against the
ubiquitous (offtarget) isozymes CA I and II, which may show inter-
esting inhibition of isoforms known to be medicinal chemistry tar-
gets (e.g., CA IX and XII).
SO2NH2
SO2NH2
SO2NH2
OH
N
N
AcNH
HO3S
N
NH2
N
R
SO3H
SA
Recently, Pouysségur and co-workers9 showed that in hypoxic
LS174Tr tumor cells expressing either CA IX or both CA IX and
XII, in response to a CO2 load, both enzymes contribute to extracel-
lular acidification and to maintaining a more alkaline resting intra-
cellular pH (pHi), an action that preserves ATP levels and cell
survival in a range of acidic outside pH (6.0–6.8) values, and low
bicarbonate medium. In vivo experiments in cell cultures showed
that silencing of CA IX alone leads to a 40% reduction in xenograft
tumor volume in mice, with up-regulation of the second gene, the
one encoding for CA XII. Silencing of both CA IX and CA XII gave an
impressive 85% reduction of tumor growth.9c Thus, hypoxia-in-
duced CA IX and CA XII are major tumor prosurvival pH-regulating
enzymes, and their combined targeting (i.e., inhibition) has great
potential for the design of anticancer drugs with a novel mecha-
nism of action.9 However, the in vivo proof-of-concept that sulfon-
amide CA IX inhibitors may indeed show antitumor effects, has
been only very recently published by Neri and co-workers,11 by
using membrane-impermeant derivatives based on the acetazola-
mide AAZ scaffold to which either fluoresceincarboxylic acid or
albumin-binding moieties were attached. This group demonstrated
the strong tumor growth retardation in mice with xenografts of a
renal clear cell carcinoma line (SK-RC-52) when the animals were
treated for one month with some of these CAIs. Such preliminary
data of our,8,10 Pouyssegur’s9 and Neri and co-workers11 groups
show indeed the great promise of tumor CA IX/XII inhibition with
sulfonamides or related agents for the development of alternative
anticancer drugs. Furthermore such compounds may be also used
for the imaging of hypoxic tumors.8c Here we investigate the inhi-
bition of the tumor-associated isoforms CA IX and XII with a series
of diazenylbenzenesulfonamides recently reported by our group,12
which showed modest CA I and II inhibitory activity, in the search
of tumor-isoforms selective inhibitors.
PR
A: R = OH
B: R = Me2N
We report here the preparation of a series of diazenylbenzene-
sulfonamides 1 and 2 structurally related to A, B and PR, obtained
from sulfanilamide or metanilamide (Scheme 1). The chemistry for
the preparation of these compounds is non-exceptional and in-
volves diazotization of the aminosulfonamide followed by coupling
with phenols or amines13–16 (see Section 4). We have chosen vari-
ous R moieties to be present in the molecules of the derivatives 1
and 2 (such as hydroxy, amino, methylamino, and dimethylamino,
as well as the sulfonate ones from 1e, 1f, 2e, 2f which may induce
enhanced water solubility to these compounds, as sodium salts).
The isomers 23 and 24 also differ by the para- or meta-bulky sub-
stituent (with respect to the sulfamoyl moiety). Finally, as sulfo-
nates have not been investigated earlier as CA inhibitors, we also
included in the study the intermediates 3 and 3 which have been
obtained in order to prepare the azo dyes 1e, 1f, 2e and 2f.
We decided to investigate whether the physiologically relevant
CA isoforms associated with tumors, that is, CA IX and XII, are
inhibited by these derivatives, a class of compounds not investi-
gated earlier for its interaction with the extracellular, tumor-asso-
ciated isoforms.
HN
N
N
CH3CONH
SO2NH2
S
SO2NH2
S
H3C
S
AAZ
O
O
DZA
2. Results and discussion
2.1. Chemistry
2.2. Carbonic anhydrase inhibition
The azo-sulfonamides 1a–1f and 2a–2f, as well as the sulfo-
nates 3 and 4 were assayed as inhibitors of the tumor-associated
isoforms hCA IX and XII (Table 1) by means of a stopped-flow as-
say.17 Standard sulfonamide drugs, such as acetazolamide AAZ
and dorzolamide DZA were also included in the assay for compar-
ison reasons. The following structure–activity relationship (SAR)
has been evidenced from data of Table 1:
In a recent work13 we have investigated the inhibition of the
cytosolic isoforms CA II (ubiquitous)1 and VII (present only in the
brain)1 with a series of simple benzenesulfonamides and two azo
dyes obtained by the coupling of diazotized sulfanilamide SA with
phenol or N,N-dimethylaniline, of types A and B. In fact, although
very important from the drug design point of view, as the antibac-
terial sulfa drugs have been discovered considering prontosil PR as
lead molecule,14 the sulfonamide azo dyes (of which PR is a repre-
sentative) were scarcely investigated for the inhibition of CAs.15
One of the interesting findings of our recent study13 was that the
azo dyes A and B showed moderate-weak CA II inhibitory activity
(in the range of 638–665 nM), but they were better inhibitors of
the cytosolic isoform with restricted expression in the brain, CA
VII. Thus, it appeared of interest to explore in more detail this class
(i) Unlike the cytosolic isoforms hCA I and II, which were
weakly inhibited by the sulfonamides 1 and 2,12 the transmem-
brane, tumor-associated hCA IX was strongly inhibited by all these
derivatives, with inhibition constants in the range of 3.5–63 nM.
For the para-substituted azo dyes 1, all substitution patterns at
the second phenyl ring were highly beneficial for the inhibitory
activity, with the phenol 1a, amines 1b–1d and sulfonates 1e, 1f

F. Carta et al. / Bioorg. Med. Chem. 17 (2009) 7093–7099
7095
SO2NH2
SO2NH2
R
SO2NH2
NH2
NaNO2
phenol/amine
coupling
+
HCl, 0ºC
N
N
N
N
-
Cl
4-NH2 : sulfanilamide
3-NH2 : metanilamide
R
1, 2
Scheme 1. Preparation of diazenylbenzenesulfonamides 1 and 2 by diazotization of sulfanilamide/metanilamide followed by coupling with phenols/amines.
Table 1
Inhibition of hCA isoforms I, II (cytosolic), and IX and XII (transmembrane, tumor-associated) with sulfonamides 1 and 2, the sulfonates 3 and 4, and standard sulfonamide
inhibitors (AAZ and DZA), by a stopped-flow CO2 hydrase assay17
SO2NH2
SO2NH2
SO3Na
SO3Na
N
N
N
HN
N
R
N
R
1
2
3
4
No.
R
KI (nM)a
Selectivity ratio
KI (CA II)/KI (CA IX)
hCA Ib
hCA IIb
hCA IXc
hCA XIIc
1a
1b
1c
1d
1e
1f
2a
2b
2d
2e
2f
OH
NH2
NHMe
NMe2
NHCH2SO3Na
N(Me)CH2SO3Na
OH
NH2
NMe2
NHCH2SO3Na
N(Me)CH2SO3Na
—
—
—
—
393
95
170
621
106
96
95
96
665
106
93
638
105
104
106
88
105
107
109
58,300
63,600
12
6.4
6.0
4.9
6.6
5.4
3.5
6.1
5.9
6.4
57
5.0
7.3
69.4
7.9
6.1
6.3
7.3
7.3
7.9
7.2
7.4
6250
5925
5.7
103.9
17.6
18.9
96.6
19.4
29.7
17.3
14.9
16.4
1.8
113
34,488
2435
25,240
56,030
250
63
1.7
3
4
5100
5850
25
11.4
10.8
0.48
0.17
AAZ**
DZA**
50,000
9
52
3.5
Data for hCA I and II inhibition from Ref. 12.
a
Mean value from at least 3 different measurements.17 Errors were in the range of 5% of the obtained value (data not shown).
Human cloned full length enzyme.
Catalytic domain of human recombinant enzyme.
b
c
acting as low nanomolar inhibitors (KIs of 3.5–6.6 nM). The sulfo-
63 nM. Similar inhibition was in fact observed with the clinically
used drugs AAZ and DZA (KIs of 25–52 nM). The simple sulfonates
3 and 4 were even less effective CA IX inhibitors, with inhibition
nates 1e and 1f showed good water solubility (as sodium salts),
which constitutes a favorable feature for a compound that should
target an extracellular CA such as CA IX (and XII). In addition,
due to the permanent negative charge present in their molecules,
these compounds are also membrane-impermeant, being thus
restricted to the extracellular space with no interaction with cyto-
solic or mitochondrial CAs in vivo.18 This may lead to a favorable
inhibition profile for these compounds in vivo, and thus potentially
less side effects due to undesired inhibition of other isozymes than
the extracellular ones.19 For the meta-substituted azo dyes 2, the
inhibitory profile was very good for the phenol 2a and amines
2b, 2c (KIs of 6.1–6.4 nM) whereas the sulfonates 2e and 2f showed
only moderate inhibitory activity, with inhibition constants of 57–
constants of 5.10–5.85 lM. However this is the first time that com-
pounds possessing the sulfonate moiety as zinc-binding group are
investigated for their interaction with CA IX. It may be observed
that CA IX has around 10-times higher affinity for these sulfonates
compared to CA II (Table 1).
(ii) The second tumor-associated isoform, CA XII, was also
strongly inhibited by sulfonamides 1 and 2, but with a slightly dif-
ferent inhibition profile compared to CA IX (Table 1). Thus, for the
para-subseries, the phenol 1a, the primary amine 1b and the ter-
tiary amine 1d showed similar, potent inhibitory properties, with

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F. Carta et al. / Bioorg. Med. Chem. 17 (2009) 7093–7099
KIs of 5.0–7.3 nM, whereas the secondary amine 1c was a much
weaker inhibitor (KI of 69.4 nM). This constitutes a nice example
of how a very small structural difference in the molecule of the
CAI leads to a strong (roughly a 10-times fold) decrease of inhibi-
tory power (if one compares the secondary amine 1c with the pri-
mary or tertiary ones 1b and 1d, respectively). It is rather difficult
to rationalize this behavior without X-ray crystal structures of
these adducts with the enzyme. The sulfonamides incorporating
sulfonate tails 1e and 1f on the other hand, showed again excellent
CA XII inhibitory properties, with KIs of 6.1–6.3 nM (Table 1). For
the meta-substituted azo dyes of type 2, all compounds showed a
compact behavior of highly effective inhibitors, with KIs of 7.2–
7.9 nM (but we were unable to prepare the secondary amine corre-
sponding to 1c). Thus, all substitution patterns are in this case
highly beneficial for obtaining metanilamide-derived azo dyes
with strong CA XII inhibitory activity. In fact the sulfonamides 1
and 2 reported here showed, similarly to AAZ and DZA, a very
potent CA XII inhibition. As for CA IX discussed earlier, the simple
sulfonates 3 and 4 were less effective CA XII inhibitors, with KIs of
isoform-selective inhibitors targeting tumor-associated CAs (over
the ubiquitous CA II). Since CA IX/XII were recently shown to be
both therapeutic and diagnostic targets for hypoxic solid tumors
overexpressing these proteins, such compounds held promise for
the management of hypoxic tumors, which are largely non-respon-
sible to classical chemo- and radio-therapy.
4. Experimental
4.1. Chemistry
4.1.1. General experimental details
1H, 13C, DEPT, NOESY, COSY, HMQC and HMBC spectra were re-
corded using a Bruker Advance III 400 MHz spectrometer. The chem-
ical shifts are reported in parts per million (ppm) and the coupling
constants (J) are expressed in hertz (Hz). For all new compounds
DEPT, COSY, HMQC and HMBC were routinely used to definitely as-
sign the signals of 1H and 13C. Infrared spectra were recorded on a
Perkin–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 aluminum 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 permanganate
solution, by Hanessian’s stain solution and heating with a hot air
gun or by exposure to iodine. Flash column chromatography was
carried out using silica gel (obtained from Aldrich Chemical Co.) as
the adsorbent. The crude product was introduced into the column
as a solution in the same elution solvent system, alternatively as a
powder obtained by mixing the crude product with the same weight
of silica gel in acetone and then removing the solvent in vacuo at
room temperature, or dissolved into a minimum amount of DCM
or carbon tetrachloride. All moisture or air sensitive reactions were
carried out in oven-dried glassware under a positive pressure of
nitrogen or argon using standard syringe/septa techniques. All the
inert gases used (nitrogen and argon) were passed through jacket
columns fitted with activated silica gel containing cobalt(II) chloride
adsorbed as humidity indicator. All other solvents and chemicals
were used as supplied from Aldrich Chemical Co., Acros, Fisher, Alfa
Aesar or Lancaster Synthesis. Sulfonamides used for the diazotiza-
tion, amines, phenols and AAZ–DZA are commercially available
from Sigma–Aldrich (Milan, Italy), and were used without further
purification. All CA isozymes were recombinant ones produced
and purified in our laboratory as described earlier.7,8
5.92–6.25 lM. Again, as for CA IX, this is the first report regarding
the interaction of CA XII with this type of compounds.
(iii) A very important aspect when designing CAIs is related to
their selectivity for the target isoform,20 due to the fact that at least
13 of the 16 mammalian CA isozymes show catalytic activity for
the CO2 hydration reaction, and affinity for sulfonamide inhibi-
tors.1,20 Thus, obtaining compounds with a good inhibition profile
for the target isoform, in this case the tumor-associated ones CA IX
and CA XII, but mainly with CA II-sparing (or CA II less avid) prop-
erties, is highly desirable. In fact CA II inhibition leads to a range of
unpleasant side effects due to the wide distribution of this isoform
all over the mammalian body and due to its high affinity for most
sulfonamides, such as the clinically used ones, which generally
show low nanomolar activity against CA II1,20 (see Table 1 for
AAZ and DZA inhibition data). It may be observed from data of
Table 1 that the selectivity ratio for inhibiting CA IX over CA II
for AAZ and DZA is in the range of 0.17–0.48, meaning that these
compounds act as much better CA II than CA IX inhibitors. How-
ever, the new sulfonamides investigated here of types 1 and 2
showed an excellent selectivity ratio for inhibiting the tumor-asso-
ciated isoforms CA IX/XII over the cytosolic ones CA I/II, as pre-
sented in detail for the CA IX versus CA II selectivity ratios from
Table 1. Indeed, except for 2e and 2f which were only weakly CA
IX-selective (selectivity ratios of 1.7–1.8) inhibitors, all other sul-
fonamides 1 and 2 have this parameter in the range of 14.9–
103.9, making them among the most isoform-selective CAIs
reported to date for targeting the tumor-associated isozymes CA
IX and XII. Highly CA IX-elective compounds were the phenol 1a
and the dimethylamino-aniline derivative 1d, with selectivity
ratios of 96.6–103.9. Overall, the para-substituted azo dyes 1 were
more isoform-selective inhibitors of the tumor-associated iso-
zymes, compared to the corresponding metanilamide azo dyes 2.
4.1.2. General procedure for preparation of diazonium chloride
solutions of 4-aminobenzenesulfonamide and 3-aminobenzene-
sulfonamide13
Aminobenenzenesulfonamide (sulfanilamide or metanilamide)
(0.20 g, 1.0 equiv) was dissolved in a freshly prepared 40% solution
of concentrated hydrochloric acid in deionised water (3.0 ml) and
then cooled down to ꢀ5 °C. Then a 2.3 M aqueous solution of NaNO2
(1.2 equiv) was added dropwise and the mixture was kept stirring at
the same temperature until a persistent pale yellow solution, for sul-
fanilamide, or pale orange solution, for metanilamide, was formed
(5–10 min). The solution was used freshly prepared prior to use.
3. Conclusions
A series of diazenylbenzenesulfonamides, azo dye derivatives of
sulfanilamide or metanilamide incorporating phenol and amine
moieties, were tested for inhibition of the tumor-associated iso-
zymes of carbonic anhydrase, CA IX and XII. These compounds
showed moderate-low inhibitory activities against the cytosolic
isoforms CA I and II (offtargets) and excellent, low nanomolar inhib-
itory activity against the transmembrane CA IX and XII (KIs in the
range of 3.5–63 nM against CA IX and 5.0–69.4 nM against CA XII,
respectively). The selectivity ratio for inhibiting the tumor-associ-
ated CA IX over the offtarget CA II was in the range of 15–104 for
these diazenylbenzenesulfonamides, making them among the most
4.1.3. Diazo coupling of the diazonium chloride of 4-aminoben-
zenesulfonamide 3 with N,N-dimethylaminobenzene13
A solution of diazotized sulfanilamide/metanilamide (prepared as
above) was added dropwise to a solution of N,N-dimethylaminoben-
zene (0.14 g, 0.15 ml, 1.0 equiv) in a saturated aqueous solution of
AcONa (3.0 ml) at ꢀ5 °C. The solution turned bright orange immedi-
ately and a precipitate was formed. The mixture was stirred at the

F. Carta et al. / Bioorg. Med. Chem. 17 (2009) 7093–7099
7097
same temperature for 15 min, then warmed to rt and the pH adjusted
to 7. The solid was collected by filtration, washed with a minimum
amount of H2O, dried under vacuo and purified by silica gel column
chromatography eluting with 5% MeOH in DCM to give 1d as an or-
ange solid in 79% yield. 4-(40-Dimethylaminophenyl)diazenylben-
zenesulfonamide 1d: mp 260–261 °C silica gel TLC Rf 0.38 (MeOH/
DCM 5%); mmax (KBr) cmꢀ1, 1604 (aromatic), 1520 (N@N), 1370
(SO2–N); dH (400 MHz, DMSO-d6) 3.13 (6H, s, 2 ꢁ CH3), 6.90 (2H, d, J
9.2, 2 ꢁ 30-H), 7.48 (2H, s, SO2NH2, exchange with D2O), 7.87 (2H, d,
J 9.2, 2 ꢁ 20-H), 7.93 (2H, d, J 7.8, 2 ꢁ 2-H), 7.99 (2H, d, J 7.8, 2 ꢁ 3-
H); dC (100 MHz, DMSO-d6) 155.9 (ipso), 154.8 (ipso), 145.0 (ipso),
144.0 (ipso), 128.5 (C-2), 127.0 (C-3), 123.6, 113.2, 30.8 (2 ꢁ CH3).
3-(40-Dimethylaminophenyl)diazenylbenzenesulfonamide 2d:
mp 198–200 °C; silica gel TLC Rf 0.35 (MeOH/DCM 5%); mmax
(KBr) cmꢀ1 1600 (aromatic), 1519 (N@N), 1367 (SO2–N); dH (400
MHz, DMSO-d6) 3.13 (6H, s, 2 ꢁ CH3), 6.90 (2H, d, J 9.4, 2 ꢁ 30-H),
7.50 (2H, s, SO2NH2, exchange with D2O), 7.76 (1H, t, J 7.7, 4-H),
7.88 (2H, d, J 9.4, 2 ꢁ 20-H), 7.90 (1H, ddd, J 7.7 2.0 1.2, 5-H), 8.02
(1H, ddd, J 7.7 2.0 1.2, 6-H), 8.21 (1H, dd, J 3.6 2.0, 2-H); dC
(100 MHz, DMSO-d6) 155.0 (C-3), 154.3 (C-40), 146.3 (C-10), 144.0
(C-1), 132.3 (C-4), 129.0, 128.0, 127.2 (C-20), 119.0 (C-2), 113.5
(C-30), 41.8 (2 ꢁ CH3).
formed and the mixture was stirred at 50 °C for 5 h. Then the reac-
tion was cooled down to rt and the solid was collected by filtration,
triturated with diethyl ether and dried under vacuo to give 3 as a
white solid in 80%yield. Sodium phenylaminomethanesulfonate
3: mp > 370 °C; mmax (KBr) cmꢀ1, 3468 (N–H), 1600 (aromatic),
1421 (SO2–O); dH (400 MHz, DMSO-d6) 3.86 (2H, d, J 6.6, 10-H2),
5.88 (1H, t, J 6.6, NH, exchange with D2O), 6.50 (1H, tt, J 7.2 1.2,
4-H), 6.72 (2H, dd, J 8.6 1.2, 2-H), 7.05 (2H, dd, J 8.6 7.2, 3-H); dC
(100 MHz, DMSO-d6) 149.0 (C-1), 129.4 (C-3), 116.6 (C-4), 113.4
(C-2), 61.5 (C-10).
4.1.6. Diazo coupling of the diazonium chloride of 4-aminoben-
zenesulfonamide with 313
A solution of diazonium salt of sulfanilamide (prepared as above)
was added dropwise to a suspension of 3 (0.24 g, 1.0 equiv) in a sat-
urated aqueous solution of AcONa (1.5 ml) at ꢀ5 °C. The reaction
turned orange immediately and was stirred at the same temperature
for 15 min, then warmed to rt and the pH adjusted to 7. The solid was
collected by filtration, dried under vacuo and purified by silica gel
column chromatography eluting with 20% MeOH in DCM to give
1e as an orange solid in 75% yield. Sodium 4-(40-N-methylensulf-
ophenyl)diazenyl)benzenesulfonamide 1e: mp 340 °C decomposi-
tion; silica gel TLC Rf 0.13 (MeOH/DCM 20%); m
max (KBr) cmꢀ1 3427
(N–H), 1604 (aromatic), 1515 (N@N), 1318 (SO2–N); dH (400 MHz,
DMSO-d6) 4.03 (2H, d, J 6.8, 50-H2), 6.90 (2H, d, J 9.2, 2 ꢁ 30-H), 7.42
(1H, t, J 6.8, NH, exchange with D2O), 7.45 (2H, s, SO2NH2, exchange
with D2O), 7.75 (2H, d, J 9.2, 2 ꢁ 20-H), 7.91 (2H, d, J 8.8, 2 ꢁ 2-H), 7.97
(2H, d, J 8.8, 2 ꢁ 3-H); dC (100 MHz, DMSO-d6) 155.2, 153.4, 144.7,
144.0, 127.8, 126.2, 122.8, 113.5, 60.6 (C-50).
4.1.4. Diazo coupling of the diazonium chloride of 3/4-amino-
benzenesulfonamide with phenol13
A solution of diazotized sulfanilamide/metanilamide (prepared
as above) was added dropwise to a solution of phenol (0.11 g,
0.10 ml, 1.0 equiv) in a 10 M aqueous solution of NaOH (3.0 ml) at
ꢀ5 °C. The solution turned orange immediately and a precipitate
was readily formed. The mixture was stirred at the same tempera-
ture for 15 min, then warmed to rt and the pH adjusted to 7. The so-
lid was collected by filtration, washed with a minimum amount of
H2O, dried under vacuo and purified crystallization from H2O/EtOH
to give the title compound as an orange solid in 52% yield. 4-(40-
Hydroxyphenyl)diazenylbenzenesulfonamide 1a: mp 259–261 °C;
silica gel TLC Rf 0.60 (MeOH/DCM 10%); mmax (KBr) cmꢀ1, 3349
(O–H), 1601 (aromatic), 1503 (N@N), 1399 (SO2–N); dH (400 MHz,
DMSO-d6) 7.00 (2H, d, J 8.8, 2 ꢁ 30-H), 7.53 (2H, s, SO2NH2, exchange
with D2O), 7.89 (2H, d, J 8.8, 2 ꢁ 20-H), 7.99 (2H, d, J 8.8, 2 ꢁ 2-H),
8.00 (2H, d, J 8.8, 2 ꢁ 3-H), 10.50 (1H, s, OH, exchange with D2O);
dC (100 MHz, DMSO-d6) 154.6 (ipso), 153.9 (ipso), 146.2 (ipso),
145.0 (ipso), 127.9 (C-2), 126.3 (C-3), 123.4, 117.
Sodium 3-(40-(N-methylensulfophenyl)diazenylbenzenesulfona-
mide 2e was prepared in a similar manner; mp 236–237 °C; silica
gel TLC Rf 0.12 (MeOH/DCM 20%); mmax (KBr) cmꢀ1 3427 (N–H),
1604 (aromatic), 1527 (N@N), 1326 (SO2–N); dH (400 MHz,
DMSO-d6) 4.04 (2H, d, J 6.8, 50-H2), 6.90 (2H, d, J 9.2, 2 ꢁ 30-H),
7.39 (1H, t, J 6.8, NH, exchange with D2O), 7.49 (2H, s,SO2NH2, ex-
change with D2O), 7.74 (1H, appt, J 7.6, 5-H), 7.75 (2H, d, J 9.2,
2 ꢁ 20-H), 7.86 (1H, ddd, J 7.6 2.0 1.2, 4-H), 8.00 (1H, ddd, J 7.6
2.0 1.2, 6-H), 8.20 (1H, appt, J 1.6, 2-H); dC (100 MHz, DMSO-d6)
153.4 (C-3), 153.3 (C-40), 146.1 (C-4), 143.7 (C-2), 130.9 (7C-10),
127.0, 126.6, 126.1, 118.3 (C-2), 113.5 (C-30), 60.6 (C-50).
4.1.7. Treatment of N-methylaniline with formaldehyde sodium
bisulfite adduct (preparation of 4)
3-(40-Hydroxyphenyl)diazenylbenzenesulfonamide 1b: mp 231–
232 °C with decomposition; silica gel TLC Rf 0.56 (MeOH/DCM
10%); mmax (KBr) cmꢀ1, 3372 (O–H), 1609 (aromatic), 1505 (N@N),
1320 (SO2–N); dH (400 MHz, DMSO-d6) 7.02 (2H, d, J 8.8, 2 ꢁ 30-H),
7.53 (2H, s, SO2NH2, exchange with D2O), 7.81 (1H, appt, J 8.0,
4-H), 7.89 (1H, d, J 8.8, 20-H), 7.98 (1H, ddd, J 8.0 2.0 1.2, 5-H),
8.08 (1H, ddd, J 8.0 2.0 1.2, 6-H), 8.25 (1H, appt, J 1.6, 2-H), 10.47,
(1H, s, OH, exchange with D2O); dC (100 MHz, DMSO-d6) 162.8
(C-40), 153.5 (C-3), 146.7 (C-10), 146.4 (C-1), 132.1, 128.6, 128.3,
126.9 (C-30), 119.2 (C-2), 117.6 (C-20).
Formaldehyde sodium bisulfite adduct (1.34 g, 1.0 equiv) was
dissolved in H2O (1.8 ml) at 40 °C and then N-methylaniline
(1.07 g, 1.09 ml, 1.0 equiv) was added dropwise. The solution was
stirred at 50 °C for 4 h. Then the reaction was cooled down to rt
and the solid formed was collected by filtration, triturated with
diethyl ether and dried under vacuo to give 4 as a white solid in
60% yield. Sodium N-methylphenylaminomethanesulfonate 4:
mp > 370 °C; mmax (KBr) cmꢀ1, 1603 (aromatic), 1369 (SO2–O); dH
(400 MHz, DMSO-d6) 3.02 (3H, s, CH3), 4.08 (2H, s, 10-H2), 6.62
(1H, t, J 7.2, 4-H), 6.84 (2H, d, J 8.8, 2 ꢁ 2-H), 7.14 (2H, dd, J 8.8
7.2, 2 ꢁ 3-H); dC (100 MHz, DMSO-d6) 149.7 (C-1), 129.4 (C-3),
116.8 (C-4), 113.2 (C-2), 69.6 (C-10), 39.3 (CH3).
4.1.5. Treatment of aniline with formaldehyde sodium bisulfite
adduct (preparation of 3)
1'
4.1.8. Diazo coupling of the diazonium chloride of 4-aminoben-
zenesulfonamide with 4
NH2
HN
SO3Na
1
H2O, 50 ºC
HO
A solution of 3 (prepared as above) was added dropwise to a
suspension of 4 (0.26 g, 1.0 equiv) in a saturated aqueous solution
of AcONa (1.0 ml) at ꢀ5 °C. The reaction turned orange immedi-
ately and was stirred at the same temperature for 15 min, then
warmed to rt and the pH adjusted to 7. The solid was collected
by filtration, dried under vacuo and purified by silica gel column
chromatography eluting with 20% MeOH in DCM to give 1f as an
orange solid in 76% yield.
2
3
SO3Na
3
Formaldehyde sodium bisulfite adduct (1.34 g, 1.0 equiv) was
dissolved in H2O (1.8 ml) at 40 °C and then aniline (1.0 g, 0.98 ml,
1.0 equiv) was added dropwise. A white precipitate was readily

7098
F. Carta et al. / Bioorg. Med. Chem. 17 (2009) 7093–7099
Sodium 4-(40-N-methylphenylaminomethanesulfonate)diaze-
(2H, d, J 8.8, 2 ꢁ 30-H), 6.95 (1H, br q, J 5.4, NH, exchange with
D2O), 7.47 (2H, s, SO2NH2, exchange with D2O), 7.81 (2H, d, J 8.8,
20-H), 7.90 (2H, d, J 8.8, 2 ꢁ 2-H), 7.97 (2H, d, J 8.8, 2 ꢁ 3-H); dC
(100 MHz, DMSO-d6) 154.8 (C-4), 154.2 (C-40), 144.3 (Ipso), 143.8
(Ipso), 127.5 (C-2), 124.9, 124.0, 114.2 (C-30), 39.3 (CH3).
nylbenzenesulfonamide 1f: mp 257–258 °C; silica gel TLC Rf 0.29
(MeOH/DCM 20%); mmax (KBr) cmꢀ1, 1604 (aromatic), 1514 (N@N),
1376 (SO2–N); dH (400 MHz, DMSO-d6) 3.19 (3H, s, CH3), 4.30 (2H,
s, 50-H2), 7.03 (2H, d, J 9.6, 2 ꢁ 30-H), 7.43 (2H, s, SO2NH2, exchange
with D2O), 7.82 (2H, d, J 9.6, 2 ꢁ 20-H), 7.94 (2H, d, J 8.8, 2 ꢁ 2-H),
7.98 (2H, d, J 8.8, 2 ꢁ 3-H); dC (100 MHz, DMSO-d6) 155.1 (C-4),
153.1 (C-40), 145.0 (ipso), 143.9 (ipso), 127.8 (C-20), 125.8, 123.0,
113.4 (C-30), 68.7 (C-50), 40.0 (CH3).
4.2. CA inhibition assay
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow
instrument has been used to assay the catalytic/inhibition of vari-
ous CA isozymes as reported by Khalifah.17 Phenol red (at a con-
centration of 0.2 mM) has been used as indicator, working at the
absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.4) as
buffer, 0.1 M Na2SO4 or NaClO4 (for maintaining constant the ionic
strength; these anions are not inhibitory in the used concentra-
tion),8 following the CA-catalyzed CO2 hydration reaction for a per-
iod of 5–10 s. Saturated CO2 solutions in water at 25 °C were used
as substrate. Stock solutions of inhibitors were prepared at a con-
centration of 10 mM (in DMSO–water 1:1, v/v) and dilutions up to
0.01 nM done with the assay buffer mentioned above. At least 7
different inhibitor concentrations have been used for measuring
the inhibition constant. Inhibitor and enzyme solutions were pre-
incubated together for 10 min at room temperature prior to assay,
in order to allow for the formation of the E–I complex. Triplicate
experiments were done for each inhibitor concentration, and the
values reported throughout the paper are the mean of such results.
The inhibition constants were obtained by non-linear least-squares
methods using PRISM 3, as reported earlier,8 and represent the
mean from at least three different determinations. All CA isozymes
used here were recombinant proteins obtained as reported earlier
by our group.7,8
Sodium 3-(40-N-methylphenylaminomethanesulfonate)diazenyl-
benzenesulfonamide 2f was prepared in a similar manner: mp
330 °C with decomposition; silica gel TLC Rf 0.22 (MeOH/DCM
20%); mmax (KBr) cmꢀ1, 1601 (aromatic), 1515 (N@N), 1377 (SO2–
N); dH (400 MHz, DMSO-d6) 3.19 (3H, s, CH3), 4.28 (2H, s, 50-H2),
7.03 (2H, d, J 9.2, 2 ꢁ 30-H), 7.49 (2H, s, SO2NH2, exchange with
D2O), 7.53 (1H, appt, J 7.6, 5-H), 7.82 (2H, d, J 9.2, 2 ꢁ 20-H), 7.88
(1H, ddd, J 7.6 2.0 1.2, 4-H), 8.025 (1H, ddd, J 7.6 2.0 1.2, 6-H),
8.22 (1H, appt, J 1.6, 2-H); dC (100 MHz, DMSO-d6) 153.4 (C-3),
153.0 (C-40), 146.2 (C-10), 143.7 (C-1), 131.0 (C-10), 127.1, 126.9,
125.6 (C-20), 118.4 (C-2), 113.4 (C-30), 68.7 (C-50), 40.0 (CH3).
4.1.9. Treatment of sodium 4-(40-N-methylensulfophenyl)diaze-
nylbenzenesulfonamide 1e with NaOH13
Sodium 4-(40-N-methylensulfophenyl)diazenylbenzenesulfona-
mide 1e (0.03 g, 1.0 equiv) was dissolved in a 10% aqueous solution
of NaOH (2.0 ml) and the reaction mixture was stirred overnight at
rt, the pH adjusted to
7 and extracted with ethyl acetate
(3 ꢁ 10 ml). The combined organic layers were washed with brine
(2 ꢁ 10 ml), dried over Na2SO4, filtered and concentrated in vacuo
to give a yellow residue that was purified by silica gel column chro-
matography eluting with 10% MeOH in DCM to give 1b as an or-
ange solid in 62% yield.
4-(40-Aminophenyl)diazenylbenzenesulfonamide 1b: mp 233–
235 °C (lit5 234–236 °C); silica gel TLC Rf 0.41 (MeOH/DCM 10%);
mmax (KBr) cmꢀ13342 (N–H), 1602 (aromatic), 1505 (N@N), 1301
(SO2–N); dH (400 MHz, DMSO-d6) 6.35 (2H, s, NH2 exchange with
D2O), 6.72 (2H, d, J 8.8, 2 ꢁ 30-H), 7.45 (2H, s, SO2NH2, exchange
with D2O), 7.74 (2H, d, J 8.8, 2 ꢁ 20-H), 7.90 (2H, d, J 8.8, 2 ꢁ 2-
H), 7.97 (2H, d, J 8.8, 2 ꢁ 3-H); dC (100 MHz, DMSO-d6) 155.1 (C-
4), 154.7 (C-40), 144.8 (Ipso), 143.7 (Ipso), 127.8 (C-2), 126.8,
122.8, 114.4 (C-30).
Acknowledgments
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).
References and notes
1. Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168.
3-(40-Aminophenyl)diazenylbenzenesulfonamide 2b was pre-
pared in a similar manner from 2e: mp 197–199 °C; silica gel TLC
2. (a) Supuran, C. T.; Scozzafava, A.; Conway, J. Carbonic Anhydrase—Its Inhibitors
and Activators; CRC: Boca Raton, New York, London, 2004; (b) Supuran, C. T.;
Scozzafava, A.; Casini, A. Development of Sulfonamide Carbonic Anhydrase
Inhibitors. In Carbonic Anhydrase—Its Inhibitors and Activators; Supuran, C. T.,
Scozzafava, A., Conway, J., Eds.; CRC: Boca Raton, 2004; pp 67–147.
3. Supuran, C. T. Carbonic Anhydrases as Drug Targets—General Presentation. In
Supuran, C. T., Winum, J. Y., Eds.; Drug Design of Zinc–Enzyme Inhibitors:
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15–38.
4. (a) Supuran, C. T. Curr. Pharm. Des. 2008, 14, 641; (b) Temperini, C.; Cecchi, A.;
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Rf 0.37 (MeOH/DCM 10%); m
max (KBr) cmꢀ1 3350 (N–H), 1601 (aro-
matic), 1504 (N@N), 1325 (SO2–N); dH (400 MHz, DMSO-d6) 6.31
(2H, s, NH2, exchange with D2O), 6.73 (2H, d, J 9.2, 2 ꢁ 30-H), 7.50
(2H, s, SO2NH2, exchange with D2O), 7.74 (1H, appt, J 7.6, 5-H),
7.75 (2H, d, J 9.2, 2 ꢁ 20-H), 7.86 (1H, ddd, J 7.6 2.0 1.2, 4-H), 7.99
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(100 MHz, DMSO-d6) 154.7 (C-3), 153.5 (C-40), 146.3 (C-10), 143.7
(C-1), 131.1, 127.1, 126.9, 126.8, 118.4, 114.5 (C-30).
4.1.10. Treatment of sodium 4-(40-N-methyl-N-methylensulfo-
phenyl)diazenylbenzenesulfonamide 1f with NaOH
Sodium 4-(40-N-methylensulfophenyl)diazenylbenzenesulfona-
mide 1f (0.03 g, 1.0 equiv) was dissolved in a 10% aqueous solution
of NaOH (2.0 ml) and the reaction mixture was stirred O.N. at rt,
the pH adjusted to 7 and extracted with ethyl acetate (3 ꢁ
10 ml). The combined organic layers were washed with brine
(2 ꢁ 10 ml), dried over Na2SO4, filtered and concentrated in vacuo
to give a yellow residue that was purified by silica gel column chro-
matography eluting with 10% MeOH in DCM to give 1c as a yellow
solid in 52% yield. 4-(40-N-Methylphenyl)diazenylbenzenesulfona-
mide 1c: mp 213–214 °C; silica gel TLC Rf 0.38 (MeOH/DCM 10%);
mmax (KBr) cmꢀ1 (3410 N–H), 1606 (aromatic), 1529 (N@N), 1390
(SO2–N); dH (400 MHz, DMSO-d6) 2.84 (3H, d, J 5.4, CH3), 6.72
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