N-Nitrosulfonamides: A new chemotype for carbonic anhydrase inhibition

Article abstract of DOI:10.1016/j.bmc.2016.05.072

A series of N¹-substituted aromatic sulfonamides was obtained by applying a selective sulfonamide nitration synthetic strategy leading to Ar-SO₂NHNO₂derivatives which were investigated as carbonic anhydrase (CA, EC 4.2.1.1

Full text of DOI:10.1016/j.bmc.2016.05.072

Accepted Manuscript
N-Nitrosulfonamides: a new chemotype for carbonic anhydrase inhibition
Alessio Nocentini, Daniela Vullo, Gianluca Bartolucci, Claudiu T. Supuran
PII:
S0968-0896(16)30416-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.05.072
BMC 13056
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
5 May 2016
29 May 2016
30 May 2016
Please cite this article as: Nocentini, A., Vullo, D., Bartolucci, G., Supuran, C.T., N-Nitrosulfonamides: a new
chemotype for carbonic anhydrase inhibition, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.bmc.2016.05.072
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N-Nitrosulfonamides: a new chemotype for carbonic anhydrase inhibition
Alessio Nocentini,1 Daniela Vullo, 2 Gianluca Bartolucci1 and Claudiu T. Supuran*1
1Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche,
Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
2Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica,
Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy.
Abstract. A series of N1-substituted aromatic sulfonamides was obtained by applying a
selective sulfonamide nitration synthetic strategy leading to Ar-SO2NHNO2 derivatives which
were investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Two human (h) hCA
isoforms, the cytosolic hCA II and the transmembrane hCA IX, in addition to the fungal
enzyme from Malassezia globosa, MgCA, were included in the study. Most of the new
compounds reported selectively inhibited hCA IX over hCA II and at the same time showed
effective MgCA inhibitory properties, with KIs ranging between 0.22 - 8.09 µM. The N-nitro
sulfonamides are a new chemotype with CA inhibitory effects. As hCA IX was recently
validated as antitumor/antimetastatic drug target, its selective inhibition could be exploited for
interesting biomedical applications. Moreover, due to the effective MgCAs inhibitory
properties of the N-nitro sulfonamides, of considerable interest in the cosmetics field as
potential anti-dandruff agents, the N-nitro sulfonamides may be considered as interesting
leads for the design of more efficient compounds targeting fungal enzymes.
Keywords: carbonic anhydrase; inhibitor; sulfonamide; N-nitro-sulfonamide; Malassezia
globosa
______
* Corresponding author: Phone: +39-055-4573729; fax: +39-055-4573385; E-mail:
claudiu.supuran@unifi.it.

1. Introduction
Sulfonamides have always been the most common zinc binding group (ZBG) used to
design inhibitors for the zinc-enzymes carbonic anhydrases (CA, EC 4.2.1.1).1-3 Their
structural features make the sulfonamide group the ideal moiety to fit within the deepest and
central part of the CA cavity active sites, represented by the Zn2+ ion and the residues nearby.1
Indeed the negatively charged nitrogen replaces the physiological zinc-bound water/hydroxide
ion, establishing a strong coordination bond with the positively charged metal ion, reinforced
by two strong H-bonds between one proton on the coordinated nitrogen and Thr199OG1 atom
and between one oxygen atom of the sulfonamide and Thr199 backbone amide group,
respectively (Figure 1).1
Figure 1. Schematic illustration of the key interactions between a generic sulfonamide CA inhibitor
(CAI) and the hCA II active site.
All CAs found in humans (h) belong to the α-class (α-CA) and are characterized as
fifteen isoforms, which differ by molecular features, oligomeric organization, cellular
localization, distribution in organs and tissues, expression levels, and kinetic
properties/affinity for inhibitors.1,2 In the last decade the research over hCAs has mainly
focused on the tumor-associated isoforms IX and XII.4,5 CA IX and XII are membrane
proteins that play a crucial role in the growth of solid tumors, tuning the pH to support an
extracellular hypoxic microenvironment suited for hypoxic tumor cell survival and
proliferation. The expression of CA IX and XII is upregulated in many hypoxic tumors. It
should be stressed that CA XII is constitutively expressed in a few normal tissues, while the
expression of CA IX is more strictly connected to hypoxia phenomena, being usually absent
in the corresponding normal tissue. 4-6

Many CAIs are clinically used for decades to treat several diseases (e.g. glaucoma, edema,
epilepsy) and actually they strongly inhibit the tumor associated isoforms, but at the same
time are strong inhibitors for the ubiquitous isoforms CA I and CA II, which are involved in
many physiological processes. This lack in selectivity prevents their therapeutic anti-tumor
applications, leading to many undesired side-effects of classical sulfonamides such as
acetazolamide, methazolamide, etc. 4-6
Many molecular scaffolds have been identified and thoroughly investigated in order to
avoid this problem and to obtain compounds showing better selectivity profiles.7-8 Indeed, the
sulfonamide moiety represents the most studied ZBG, as mentioned above. Two main
approaches have been applied to design sulfonamide derivatives able to inhibit selectively
different CA isoforms: the ring and the tail approaches.1-3 The first consists in modulating the
ring directly linked to the ZBG, whereas the second consists in appending different tails to the
aromatic/heterocyclic ring present in the scaffold of the CAI. A rather large number of
substitution patterns at the aromatic/ heterocyclic ring and a variety of diverse substituents
have been explored with the aim to obtain novel CAIs with the desired physico-chemical and
pharmacologic properties.1
Nowadays the generally previous accepted theory9 that only unsubstituted
sulfonamides may act as inhibitors has been extensively shown to be uncorrect, but in
comparison to the high number of tails appended at the aromatic ring incorporating the
sulfonamide ZBG 1,10, only few modifications have been carried out on this group itself.11
Indeed this could seem counterproductive, considering that the negative charge on nitrogen
and the presence of a proton on it themselves make this group the ideal ligand for CA, but on
the other hand a such functionalization could lead to novel inhibition mechanisms.
It should be considered that secondary sulfonamides maintain the possibility to
coordinate the Zn ion in the deprotonated form, as confirmed by X-ray crystallography. Di
Fiore et. al demonstrated that N-hydroxy and N-methoxy sulfonamides are able to bind the Zn
ion in the deprotonated form. 11d An analogous inhibition mechanism has been identified for
the cyclic secondary sulfonamide saccharin and its derivatives by several groups.12
On the other hand, tertiary sulfonamides lose their acid peculiarity, paving the way to
novel inhibition mechanism. Carradori et al.13 recently synthesized different series of tertiary
sulfonamides, among which probenecid and saccharin derivatives, obtaining interesting and
selective inhibition profiles, although the inhibition mechanisms of these compounds were not
been investigated in detail yet, as no X-ray crystallographic structures were obtained so far for
adducts of these CAIs with various CA isoforms.

Besides the hCAs, many other CAs have been investigated as potential therapeutic
targets.2,14 Indeed CAs are present in many pathogenic organisms such as fungi, bacteria and
protozoa, where they play crucial roles in the microbial virulence. Thus, compounds able to
inhibit such these enzymes represent potential novel chemotherapeutic agents, to be taken into
account in an environment where the serious drug resistance problems with the presently used
clinical agents are at the order of the day.14 Among the six CA classes (α, β, γ, δ, ζ and η)
discovered to date, only α - and β-CAs were reported so far in fungi.15 In particular, the β-
carbonic anhydrase from the dandruff producing fungus Malassezia globosa, MgCA, has been
recently validated as an anti-dandruff drug target.16 In a earlier work our group showed that
inhibition of MgCA with various sulfonamide inhibitors leads to an impairment of the fungal
growth, leading to non viable fragmented fungal hyphae.16 Up to now the inhibitory activity
of only few classes of compounds apart the sulfonamides was screened on MgCA.
Although few examples of N-nitro sulfonamide are present in literature,17 their CAs
inhibitory properties have never been investigated and studied. Herein we report a series of N-
nitro aromatic sulfonamide, which showed strong as well as selective inhibitory activity on
the tumor-associated isoform hCA IX over the cytosolic hCA II. In addition these derivatives
were shown to inhibit the pathogenic fungal MgCA in the low micromolar range.
H
O
S
O
S
O
N
O
NH
R
R = 2-NH ; 1
2
2
NH NO
NO
4
3
2
3-NH ; 2
2
H SO 95%
2
4
4-NH ; 3
2
R
4-N(CH ) ; 4
3 2
0°C
4-CH NH ; 5
2
2
4-NHCH C CH; 6
2
A-F
1-6
O
H
O
S
O
NH
2
O
N
NH NO
S
4
3
NO
2
H SO 95%
2
4
0°C
NH
2
O N
NH
2
2
OH
OH
7
G
NH NO
4
3
HCl
H N
H N
2
2
S
H SO 95%
O
S
2
4
SO NH
S
O
2
2
N
N
N
N
HN NO
0°C
2
8
H
Scheme 1. General synthetic procedure for compounds 1-8

2.Results and Discussion
2.1 Chemistry
The rationale of this work relies on the salient feaures of the nitro moiety, a strongly electro-
withdrawing group able to enhance the acidity of the sulfonamide, and thus the interaction
with the positive zinc ion. Furthermore, it allows for two additional H-bond acceptor atoms
for possible interactions with active site residues.
The general synthetic strategy purposed by Minksztym17a for the chemoselective
mononitration of aminosulfonamides has been applied and extended here (Scheme 1). The
strongly acidic reaction conditions deactivate the amino moiety against nitration ensuring the
exclusive functionalization of the sulfonamide nitrogen. The presence of the amino group
ensures the stability of the zwitterion adduct. In fact, in absence of a basic counterpart, the
strong acid N-nitrosulfonamide is not stable and rapidly decomposes to the corresponding
sulfonic acid releasing N2O.17a-b The presence of a basic function in such a molecule, not too
distant from the substituted sulfonamide, stabilizes it through the formation of a zwitterion.
The evidence for the formation of the zwitterion was demonstrated in the solid state by means
of IR spectroscopy by Minksztym.17a
The synthesis was carried out treating the amino sulfonamides A-H with NH4NO3 in
concentrated H2SO4 (Scheme 1) and the obtained nitro derivatives were purified by means of
crystallization in a polar solvent. The strong acidic character of the nitrosulfonamide reflects
1
in the H NMR spectra, where the signals were located with the signals of residual water
1
contained in DMSO-d6. The H NMR spectrum the 4-(2-aminomethyl)benzenesulfonamide
N1-nitro derivative 5 further reveals the presence of the zwitterion through a three protons
broad signal at 8.19 ppm representing the ammonium positive moiety. In the case of 3-amino-
4-hydroxybenzenesulfonamide G, the more activated aromatic ring undergoes an additional
nitration at the 5 position, affording derivative 7, which has two NO2 moieties in its molecule
(Scheme 1).
2.2 Carbonic anhydrase inhibition
N1-Nitro sulfonamides 1-8 were screened for the inhibition of the Malassezia globosa CA16
and two human (h) CA isoforms: the cytosolic hCA II, involved in important

physiologic/pathologic processes and the transmembrane, tumor-associated hCA IX (an
anticancer drug target).4-6 Table 1 shows inhibition data of compounds 1-8 in addition to
acetazolamide (AAZ) used as standard inhibitor and a clinically used drug.18
Table 1: Inhibition data of human CA isoforms hCA II, IX, Malassezia globosa CA (MgCA)
with sulfonamides AN reported here and the standard sulfonamide inhibitor acetazolamide
(AAZ) by a stopped flow CO2 hydrase assay.18
KI (µM)
Compound
hCA II
hCA IX
MgCA
1
>50
5.11
>50
>50
>50
0.42
>50
0.66
2.75
0.71
0.22
8.09
3.49
0.67
4.50
74.00
2
3
8.19
2.79
5.99
2.00
8.72
0.84
0.025
4
5
6
1.35
>50
4.52
0.012
7
8
AAZ
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of 5-10 % of
the reported values).
The following structure-activity relationship (SAR) can be extrapolated from the inhibition
data shown in Table 1:
(i) Most of the compounds of the series were efficient and selective inhibitors of the tumor-
associated isoform hCA IX over the cytosolic one hCAII. Indeed, only derivatives 2, 6 and 8
exhibited low micromolar inhibitory activity against hCA II, whereas the remaining ones did

not significantly inhibit the enzyme up to 50 µM inhibitor concentration. These data may
suggest that the N-nitrosulfonamide group does not fit well within the hCA II active site, but
the presence of some key elements on the molecular structure may improve the good contacts
with the enzyme active site, thus leading to an increased inhibitory activity. Repositioning of
the amino group in position 3 of the aromatic scaffold, a three carbon atoms alkyl substituent
on the para amino moiety or the substitution of the benzene ring with the heterocyclic 1,3,4-
thiadiazole, respectively contributed to make derivatives 2, 6 and 8 low micromolar inhibitors
for hCA II (KI values spanning between 1.35 and 5.11 µM, Table 1).
(ii) As mentioned above, the tumor-associated isoform hCA IX was inhibited in the low
micromolar range by all the new N1-nitro sulfonamides reported here with KIs ranging
between 0.42 and 8.72 µM, with the exception of compound 2, which did not significantly
inhibit the enzyme up to 50 µM inhibitor concentration. Thus, the peculiarity that enable
derivative 2 as a micromolar inhibitor against hCA II, that is the amino group in meta position
towards the N-nitro moiety, did not fit well within the hCA IX active site. Instead, the
analogous derivative incorporating an additional hydroxy and nitro group at the 4 and 5
positions of the benzene ring (derivative 7) acted as a micromolar inhibitor of hCA IX, with a
KI of 8.72 µM. The definition of a proper SAR for hCA IX is not feasible at the moment,
considering that the remaining derivatives showed similar affinities (Table 1). However we
can speculate that the presence of the aromatic amino moiety in position 2 with respect to the
substituted sulfonamide or appended to a hetearomatic core is better tolerated within the
active site cavity, as highlighted by the sub-micromolar KIs values of compounds 1 and 8.
(iii) The fungal enzyme MgCA was effectively inhibited by all N-nitro sulfonamides 1-8
investigated here, with KIs ranging between 0.22 and 8.09 µM (Table 1). These values were
from 10 to 300 times lower than the KI of the clinically used sulfonamide AAZ (KIs 74.0
µM).16a The presence of small hydrophilic substituents at position 4 of the benzene ring
(compounds 3, 4 and 7) positively influenced the MgCA inhibitory properties of this class of
derivatives. Indeed, compounds 3 and 4, which incorporate at that position an unsubstituted or
dimethyl substituted amino moiety, showed KIs of 0.71 - 0.22 µM, whereas derivative 7,
which incorporates a hydroxy moiety with an additional amino and nitro group in its ortho
positions, showed a KI of 0.67 µM. Thus, all the N-nitro sulfonamides reported here showed
interesting inhibitory properties against this fungal enzyme, making them interesting
candidates as lead molecules for fungal CAIs.
3. Conclusions

A series of N1-nitro aromatic sulfonamides was obtained by applying a selective
sulfonamide nitration synthetic strategy.17a Derivatives 1-8 were investigated for the
inhibition of two hCA isoforms, the cytosolic hCA II and the transmembrane hCA IX, in
addition to the fungal MgCA. Most of the new compounds reported here were shown to
inhibit selectively hCA IX over hCA II and at the same time to be able to inhibit the CA from
the dandruff producing fungus Malassezia globosa16, with KIs ranging between 0.22 and 8.09
µM (the clinically used sulfonamide AAZ showed a KI of 74.00 µM). Therefore, the N-nitro
sulfonamide moiety can be considered as a new chemotype for CA inhibition. Due to the ease
of preparation, additional series of derivatives should be prepared, in order to better
understand the SAR for this underinvestigated class of sulfonamides. As hCA IX was recently
validated as antitumor/antimetastatic drug target, its selective inhibition could be exploited for
interesting biomedical applications. Moreover, due to the effective MgCAs inhibitory
properties of the N-nitro sulfonamides, of considerable interest in the cosmetics field as
potential anti-dandruff drugs16, this group of N-substituted sulfonamides may be considered
as interesting leads for the design of more efficient CAIs targeting fungal enzymes.
4. Experimental Protocols
4.1. Chemistry. Anhydrous solvents and all reagents were purchased from Sigma-Aldrich,
Alfa Aesar and TCI. All reactions involving air- or moisture-sensitive compounds were
performed under a nitrogen atmosphere using dried glassware and syringes techniques to
13
transfer solutions. Nuclear magnetic resonance (1H-NMR, C-NMR) spectra were recorded
using a Bruker Advance III 400 MHz spectrometer in DMSO-d6. Chemical shifts are reported
in parts per million (ppm) and the coupling constants (J) are expressed in Hertz (Hz). Splitting
patterns are designated as follows: s, singlet; d, doublet; sept, septet; t, triplet; q, quadruplet;
m, multiplet; brs, broad singlet; dd, double of doubles, appt, aparent triplet, appq, aparent
quartet. The assignment of exchangeable protons (OH and NH) was confirmed by the addition
of D2O. Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel F-
254 plates. Flash chromatography purifications were performed on Merck Silica gel 60 (230-
400 mesh ASTM) as the stationary phase and ethyl acetate/n-hexane were used as eluents.
The solvents used in MS measures were acetone, acetonitrile (Chromasolv grade), purchased
from Sigma-Aldrich (Milan - Italy), and mQ water 18 MΩ, obtained from Millipore's
Simplicity system (Milan-Italy). The mass spectra were obtained using a Varian 1200L triple

quadrupole system (Palo Alto, CA, USA) equipped by Electrospray Source (ESI) operating in
both positive and negative ions. Stock solutions of analytes were prepared in acetone at 1.0
mg mL-1 and stored at 4°C. Working solutions of each analyte were freshly prepared by
diluting stock solutions in a mixture of mQ H2O/ACN 1/1 (v/v) up to a concentration of 1.0
µg mL-1 The mass spectra of each analyte were acquired by introducing, via syringe pump at
10 µL min-1, of the its working solution. Raw-data were collected an processed by Varian
Workstation Vers. 6.8 software.
General synthetic procedure of compounds 1-8.17a
NH4NO3 (1.1 eq) was added portion wise to a solution of aromatic sulfonamides A-H (1.2
mmol, 1.0 eq) in 95% H2SO4 (1 mL) at 0°C. The suspension was stirred at the same
temperature for 15 min, then quenched with slush (8 ml) and the formed precipitate was
filtered-off and washed with cold water.
Synthesis of N1-Nitro-2-amino-benzenesulfonamide 1.17a
O
S
O
S
O
NH2
O
N
NH4NO3
NO2
H2SO4 95%
H2N
H3N
0°C
A
1
N1-Nitro-2-amino-benzenesulfonamide 1 was obtained according the general procedure
earlier reported using 2-aminobenzenesulfonamide A (0.2g, 1.0 eq) and NH4NO3 (1.1 eq) in
conc H2SO4 (1 ml). The obtained residue was recrystallized from H2O to afford the titled
compound 1 as a white solid.
N1-Nitrosulfanilamide 1: 71 %yield; δH (400 MHz, DMSO-d6): 6.92 (t, J = 7.8, 1H), 6.97 (d,
J = 7.8, 1H), 7.37 (td, J = 1.0, 7.8, 1H), 7.66 (dd, J = 1.0, 7.8, 1H); δC (100 MHz, DMSO-d6):
121.6, 122.9, 129.6, 131.5, 134.0, 139.3; m/z (ESI negative) 216 [M-H]-
Experimental in agreement with reported data.17a
Synthesis of N1-nitro-3-amino-benzenesulfonamide 2.

O
S
O
S
O
NH
2
O
N
NH NO
NO
NH
4
3
2
3
H SO 95%
2
4
0°C
NH
2
B
2
N1-Nitrosulfanilamide 2 was obtained according the general procedure earlier reported using
3-aminobenzenesulfonamide B (0.2g, 1.0 eq) and NH4NO3 (1.1 eq) in conc H2SO4 (1 ml). The
obtained residue was recrystalized from H2O to afford the titled compound 2 as an orange
solid.
N1-Nitro-2-amino-benzenesulfonamide 2: 68% yield; δH (400 MHz, DMSO-d6): 7.33 (d, J =
7.4, 1H), 7.53 (t, J = 7.4, 1H), 7.77 (m, 2H); δC (100 MHz, DMSO-d6): 122.6, 126.1, 127.1,
130.8, 134.2, 145.0; m/z (ESI negative) 216 [M-H]-
Synthesis of N1-Nitrosulfanilamide 3.17a
O
S
O
S
O
N
O
NH
2
NH NO
NO
4
3
2
H SO 95%
2
4
0°C
NH
NH
3
2
C
3
N1-Nitrosulfanilamide 3 was obtained according the general procedure earlier reported using
sulfanilamide C (0.2g, 1.0 eq) and NH4NO3 (1.1 eq) in conc. H2SO4 (1 ml). The obtained
residue was recrystallized from H2O to afford the titled compound 3 as a white solid.
N1-Nitrosulfanilamide 3: 68% yield; δH (400 MHz, DMSO-d6): 7.01 (d, J = 8.4, 2H), 7.68
(d, J = 8.4, 2H); δC (100 MHz, DMSO-d6): 118.8, 130.7, 135.9, 143.7; m/z (ESI negative) 216
[M-H]-
Experimental in agreement with reported data.17a
Synthesis of N,N-dimethyl-N1-Nitrosulfanilamide 4. 17a

O
S
O
S
O
N
O
NH
2
NH NO
NO
4
3
2
H SO 95%
2
4
0°C
NH
N
D
4
N,N-Dimethyl-N1-nitrosulfanilamide 4was obtained according the general procedure reported
earlier using N,N-dimethylsulfanilamide D (0.24g, 1.0 eq) and NH4NO3 (1.1 eq) in conc.
H2SO4 (1 ml). The obtained residue was recrystallized from H2O to afford the titled
compound 4 as a white solid.
N,N-Dimethyl-N1-nitrosulfanilamide 4: 64 % yield; δH (400 MHz, DMSO-d6): 3.02 (s, 6H,
2 x CH3), 6.80 (d, J = 9.0, 2H), 7.62 (d, J = 9.0, 2H); δC (100 MHz, DMSO-d6): 41.2, 112.2,
129.0, 130.7, 152.6; m/z (ESI negative) 244 [M-H]-
Experimental in agreement with reported data.17a
Synthesis of N1-Nitro-4-aminomethyl-benzensulfonamide 5. 17a
O
S
O
S
O
N
O
NH2
NH4NO3
NO2
H2SO4 95%
0°C
NH3
NH2 HCl
E
5
N1-Nitrosulfanilamide 5 was obtained according the general procedure earlier reported using
4-aminomethyl-benzensulfonamide hydrochloride E (0.26g, 1.0 eq) and NH4NO3 (1.1 eq) in
conc. H2SO4 (1 ml). The obtained residue was recrystallized from H2O to afford the titled
compound 5 as a white solid.
N1-Nitro-4-aminomethyl-benzensulfonamide 5: 72 % yield; δH (400 MHz, DMSO-d6): 4.14
(s, 2H, CH2), 6.80 (d, J = 8.0, 2H), 7.85 (d, J = 8.0, 2H), 8.19 (bs, 3H, exchange with D2O,
+
NH3 ) ; δC (100 MHz, DMSO-d6):42.9, 129.0, 129.5, 138.1, 143.6; m/z (ESI negative) 230
[M-H]-
Experimental in agreement with reported data.17a
Synthesis of N1-Nitro-N-propargylsulfanilamide 6.

O
S
O
S
O
N
NH4NO3
NO2
O
NH2
H2SO4 95%
0°C
H2N
HN
F
6
N1-Nitro-N-propargylsulfanilamide 6 was obtained according the general procedure earlier
reported using N-propargylsulfanilamide F (0.25g, 1.0 eq) and NH4NO3 (1.1 eq) in conc.
H2SO4 (1 ml).
N1-Nitro-N-propargylsulfanilamide 6: 33% yield; δH (400 MHz, DMSO-d6): 3.12 (t, J =
2.2, 1H), 3.95 (d, J = 2.2, 2H, CH2), 6.66 (d, J = 8.8, 2H), 7.54 (d, J = 8.8, 2H); δC (100 MHz,
DMSO-d6): 32.6, 74.2, 82.3, 112.0, 129.1, 130.6, 151.3; m/z (ESI negative) 254 [M-H]-
Synthesis of N1-Nitro-3-amino-4-hydroxy-5-nitro-benzenesulfonamide 7.
O
S
O
S
O
NH
2
O
N
NH NO
NO
NH
4
3
2
3
H SO 95%
2
4
0°C
NH
O N
2
2
OH
OH
G
7
N1-Nitro-3-amino-4-hydroxy-5-nitro-benzenesulfonamide 7 was obtained according the
general procedure reported earlier, using 3-amino-4-hydroxy-benzenesulfonamide G (0.22g,
1.0 eq) and NH4NO3 (1.1 eq) in conc. H2SO4 (1 ml). The obtained residue was recrystallized
from acetonitrile to afford the titled compound 7 as a grey solid.
N1-Nitro-3-amino-4-hydroxy-5-nitro-benzenesulfonamide 7: 42 % yield; δH (400 MHz,
DMSO-d6): 7.49 (d, J = 2.4, 1H), 7.67 (d, J = 2.4, 1H); δC (100 MHz, DMSO-d6): 116.2,
121.3, 134.4, 135.8, 135.9, 145.3; m/z (ESI negative) 317 [M-H]-
Synthesis of N1-nitro-5-amino-1,3,4-thiadiazole-2-sulfonamide 8.
NH4NO3
H2SO4 95%
HCl
H2N
H3N
S
O
N
S
SO2NH2
S
O
NO2
N
N
N
N
0°C
8
H

N1-Nitro-5-Amino-1,3,4-thiadiazole-2-sulfonamide 8 was obtained according the general
procedure earlier reported using 5-amino-1,3,4-thiadiazole-2-sulfonamide hydrochloride H
(0.26g, 1.0 eq) and NH4NO3 (1.1 eq) in conc. H2SO4 (1 ml). The obtained residue was
recrystallized from EtOH to afford the titled compound 8 as a white solid.
N1-nitro-5-amino-1,3,4-thiadiazole-2-sulfonamide 8: 58% yield; δC (100 MHz, DMSO-d6):
158.8, 172.4; m/z (ESI negative) 224 [M-H]-
Synthesis of N,N-dimethyl sulfanilamide D.19
O
S
O
S
O
NH2
O
NH2
CH3I
K2CO3
DMF
N
NH2
C
D
Iodomethane (2.0 eq) was added dropwise to a suspension of sulfanilamide C (0.5g, 1.0 eq)
and potassium carbonate (2.0 eq) in dry DMF (2 ml). The mixture was stirred at 50°C for 1h,
then cooled and quenched with H2O (20 ml) and the formed precipitate was filtered-off,
washed with H2O and recrystallized from acetone to afford the titled compound D as a white
solid.
N,N-dimethyl-sulfanilamide D: 43% yield; silica gel TLC Rf 0.47 (EtOAc/n-hexane 50 %
v/v); δH (400 MHz, DMSO-d6): 3.34 (s, 6H, 2 x CH3), 6.79 (d, J = 9.0, 2H), 6.98 (s, 2H,
exchange with D2O, SO2NH2), 7.63 (d, J = 9.0, 2H); δC (100 MHz, DMSO-d6):38.6, 113.6,
125.2, 129.6, 153.4.
Experimental in agreement with reported data.19
Synthesis of N-propargylsulfanilamide F.
Br
O
O
O
S
NH2
O
S NH2
Py
DMF
HN
NH2
C
F

Propargyl bromide (1.2 eq) was added to a suspension of sulfanilamide C (0.5 g, 1.0 eq) and
pyridine (1.2 eq) in dry DMF (2 ml) under a nitrogen atmosphere and that was stirred at r.t.
overnight. The reaction mixture was quenched with H2O (10 ml) and the formed precipitate
was filtered-off and purified by silica gel column chromatography eluting with 60% ethyl
acetate in n-hexane to afford F as a yellow solid.
N-Propargylsulfanilamide F: 22% yield; silica gel TLC Rf 0.47 (MeOH/DCM 10 % v/v); δH
(400 MHz, DMSO-d6): 3.14 (t, J = 2.0, 1H), 3.97(dd, J = 2.0, 6.2, 2H), 6.73 (d, J = 8.8, 2H,
Ar), 6.76 (t, J = 6.2, 1H, exchange with D2O, NH), 6.99 (s, 2H, exchange with D2O,
SO2NH2), 7.59 (d, J = 8.8, 2H, Ar); δC (100 MHz, DMSO-d6): 37.1, 77.5, 77.7, 112.9, 122.6,
130.2, 154.5.
4.2. CA inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA
catalyzed CO2 hydration activity.18 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 the assay buffer. Inhibitor and enzyme solutions were pre-incubated 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 the Cheng-Prusoff equation, as reported earlier, and represent the mean from at
least three different determinations. All CA isoforms were recombinant ones obtained in-house
as reported earlier.20-24
Acknowledgments. This work was financed in part by two EU projects of the 7th Framework
Programme (Metoxia and Dynano).

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N-Nitrosulfonamides: a new chemotype for carbonic anhydrase inhibition
Alessio Nocentini, Daniela Vullo, Gianluca Bartolucci and Claudiu T. Supuran*
H
O
S
H
O
S
O
N
O
N
NO2
NH2
NO2
H2N
O
S
S
O
N
R
N
HN NO2
O2N
OH
R = o-NH2, m-NH2, p-NH2,
p-N(CH3)2, p-CH2NH2,
p-NHCH2C CH;
KI (hCA II) = 1.35 - >50 µM; KI (hCA IX) = 0.42 - >50 µM; KI (MgCA) = 0.22 – 8.09 µM.