A class of sulfonamide carbonic anhydrase inhibitors with neuropathic pain modulating effects

Article abstract of DOI:10.1016/j.bmc.2015.02.027

A series of benzene sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors which incorporate lipophilic 4-alkoxy- and 4-aryloxy moieties, together with several derivatives of ethoxzolamide and sulfanilamide are reported. These derivatives were investi

Full text of DOI:10.1016/j.bmc.2015.02.027

Accepted Manuscript
A class of sulfonamide carbonic anhydrase inhibitors with neuropathic pain
modulating effects
Fabrizio Carta, Lorenzo Di Cesare Mannelli, Melissa Pinard, Carla Ghelardini,
Andrea Scozzafava, Robert McKenna, Claudiu T. Supuran
PII:
S0968-0896(15)00121-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.02.027
BMC 12090
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 January 2015
10 February 2015
16 February 2015
Please cite this article as: Carta, F., Mannelli, L.D.C., Pinard, M., Ghelardini, C., Scozzafava, A., McKenna, R.,
Supuran, C.T., A class of sulfonamide carbonic anhydrase inhibitors with neuropathic pain modulating effects,
Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.02.027
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A class of sulfonamide carbonic anhydrase inhibitors with neuropathic pain modulating
effects
Fabrizio Carta,a Lorenzo Di Cesare Mannelli,b Melissa Pinard,c Carla Ghelardini,b Andrea
Scozzafava,a Robert McKennac and Claudiu T. Supurana,d*
a
Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della
Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy
b
Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Farmacologia, Viale G.
Pieraccini 6, 50139 Florence, Italy.
c Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida,
Box 100245, Gainesville, FL 32610, USA
d Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via
Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
Abstract. A series of benzene sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors which
incorporate lipophilic 4-alkoxy- and 4-aryloxy moieties, together with several derivatives of
ethoxzolamide and sulfanilamide are reported. These derivatives were investigated as inhibitors of
the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) of which multiple isoforms are known,
and some appear to be involved in pain. These sulfonamides showed modest inhibition against the
cytosolic isoform CA I, but were generally effective, low nanomolar CA II, VII, IX and XII
inhibitors. X-ray crystallographic data for the adduct of several such sulfonamides with CA II
allowed us to rationalize the good inhibition data. In a mice model of neuropathic pain induced by
oxaliplatin, one of the strong CA II/VII inhibitors reported here induced a long lasting pain
relieving effect, a fact never observed earlier. This is the first report of rationally designed
sulfonamide CA inhibitors with pain effective modulating effects.
Key words: sulfonamide, metalloenzyme, carbonic anhydrase, pain, analgesic, X-ray
crystallography.
__
Corresponding Author. Phone: +39-055-4573729; fax: +39-055-4573385; E-mail:
claudiu.supuran@unifi.it (CTS).

1. Introduction
The metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) catalyzes the interconversion between
carbon dioxide and bicarbonate, when H+ ions are also generated in the hydration reaction, being
thus one of the main players of pH regulation in many tissues, organs and organisms.1,2 Indeed, six
genetically different CA families are known to date, which are widespread in organisms all over the
tree of life.3,4 In humans, 15 different α-CA isoforms were described so far, which are involved in
many physiologic processes connected to pH regulation, secretion of electrolytes, biosynthetic
processes, tumorigenesis, etc.1,2 Interference with the activity of these enzymes, some of which are
extremely catalytically active for the physiologic reaction,1,2 leads to pharmacologic effects which
were exploited for obtaining diuretics,5 antiglaucoma agents,6 antiepileptics,7 antiobesity drugs,8
agents effective for treating high-altitude sickness,9 and ultimately anticancer/antimetastatic drugs
targeting hypoxic tumors.10 So many types of pharmacological effects are due to the fact that
different isoforms among the 15 human (h) hCAs are involved in diverse physiologic processes, and
are targeted by such drugs,1,2 which may be sometimes a rather challenging task considering the fact
that many of these isoforms are rather similar from the structural view point and even subcellular
localization.1b,c
There seem to be some connections between CA activity and pain, although this field has
been less investigated for the moment. Kaila and Price’s groups11a showed that CA inhibition with
the clinically used sulfonamide acetazolamide (AAZ) augments GABAA receptor-mediated
analgesia via a spinal mechanism of action. These authors explain their finding as being due to a
-
reduced HCO3 -dependent depolarization via GABAA receptors when the function of the neuron-
specific potassium-chloride (KCl) cotransporter KCC2 is compromised. CA inhibition with
acetazolamide thus mitigates the negative effects of loss of KCC2 function after nerve injury
resulting in an enhancement of analgesic effects for several GABAA allosteric modulators, being
proposed that this effect might be useful for designing agents effective in the management of
neuropathic pain. The same group showed earlier that acetazolamide and midazolam act
synergistically in inhibiting neuropathic pain,11b and the CA isoform thought to be involved in this
process was proposed to be the cytosolic, brain-specific CA VII.11c Although not exclusively found
in this organ, CA VII is in fact widely expressed in various brain tissues, and its physiologic
function is still elusive, being for example recently showed that it is involved not only in pH
regulation processes but also in protecting tissue from oxidative stress.12 Other recent studies
reported an enhanced expression of some CA isoforms (among which CA II) in states connected to

chronic pain, such as thrombus-induced ischemic pain,13a or chronic musculoskeletal pain in
14a
humans.13b Furthermore, acetazolamide AAZ
and celecoxib CLX,14b,c two sulfonamides with
known potent CA inhibitory properties,15 were shown to lead to analgesic effects in various animal
models of pain, such as chemical-stimulated pain, peripherally induced inflammatory pain, etc. The
mechanisms by which these drugs exert their analgesic effect seem to be correlated with CA
inhibition since the COX-2 inhibitor lumiracoxib LMC, which unlike CLX is not a CA inhibitor
(CAI), does not possess analgesic effects in the investigated animal models.14b
SO2NH2
Cl
N
N
NH
F
Me
AcNH
SO2NH2
S
COOH
N
N
AAZ
Me
F3C
CLX
LMC
Considering all these interesting and recent reports connecting CA and its inhibition to pain,
we report here the drug design and in vitro/in vivo investigations of a class of sulfonamides
possessing interesting pain modulating properties in an animal model of neuropathic pain. As far as
we know, this is the first structure-based drug design study of sulfonamide CAIs with pain-
modulating effects.
2. Results and Discussion
2.1. Chemistry and Drug Design. Apart heterocyclic derivatives such as acetazolamide AAZ, the
benzene sulfonamides (of which CLX is a clinically used representative)15 constitute a much
investigated class of CAIs,16 with many such compounds reported so far being derivatives of 3- or
4-amino-substituted benzenesulfonamides. A much less investigated class of CAIs is that derived
from 4-hydroxybenzene sulfonamide 1.17 In fact apart an earlier study of Vernier et al.,17a who
reported ethers of 1, recently we investigated the possibility to obtain 4-sulfamoylphenyl-ω-
aminoalkyl ethers incorporating 2-6 carbon atoms aliphatic chains, which proved to possess a good
water solubility and excellent in vivo antiglaucoma activity in an animal model of the disease.17b As
the chemistry of ethers of sulfonamide 1, which incorporate simple aliphatic and aromatic R
moieties in the RO-C6H4-SO2NH2 scaffold was poorly investigated to date, we report here an
extensive such study. In this paper, our main goal was to obtain compounds with an increased
lipophilicity in order to target brain-associated CA isoforms such as CA II and VII, unlike the

previous studies in which we were interested in obtaining water soluble derivatives for topical
antiglaucoma activity. Thus, we decided to incorporate small aliphatic, saturated and unsaturated R
moieties, such as Et, n-Pr, n-Bu, allyl or propargyl in the molecules of ethers derivated from
sulfonamide 1, which have been obtained as outlined in Scheme 1. Three different approaches were
used for obtaining the series of ethers 2a-2o reported here: (i) Williamson ether synthesis directly
from sulfonamide 1 and alkyl/alkenyl/alkynyl/benzyl halides (route A in Scheme 1); (ii) protection
of the sulfamoyl moiety of 1 (as N,N-dimethylformimidamide) followed by reaction of the phenolic
OH functionality with the corresponding substituted-benzyl halide and deprotection at the
sulfamoyl moiety (route B in Scheme 1); and (iii) Mitsunobu reaction of 1 and the corresponding
benzyl alcohol (route C in Scheme 1). Two ethers derived from ethoxzolamide 3, which was de-
alkylated to the corresponding ethoxzolamide phenol 4, were also obtained after protecting the
sulfamoyl moiety as described above, followed by O-alkylation and removal of the sulfonamide
protecting group (compounds 5a and 5b, route E, Scheme 1). We also prepared the 4-(bis-N,N-
propargyl) derivative of sulfanilamide 6, compound 7a, by reacting sulfanilamide at the aromatic
amino group with propargyl bromide (route D, Scheme 1). In order to increase the lipophilicity of
some of our derivatives, the compounds incorporating alkyne moieties, such as 2e and 7a, were
converted to the corresponding hexacarbonyl dicobalt(0) complexes 2f and 7b, respectively, as it
has been reported18 that such derivatives containing diverse alkynes as (bio)ligands showed
interesting potential for application as antitumor agents, hormonally active drugs or diagnostic
agents.18 Finally, sulfonamide 8 incorporating a poorly investigated scaffold up until now, the
indoline-5-sulfonamide one, was also prepared and derivatized with the trifluoroacetyl moiety in
order to increase its lipophilicity (route F, Scheme 1).
Scheme 1 here
All compounds 2a-2o, 5a, 5b, 7a, 7b and 8 reported here were completely characterized by
spectroscopic and chromatographic methods which confirmed their structures (see Experimental
protocols for details).
Table 1 here
2.2. Carbonic anhydrase inhibition. Inhibition data against five physiologically relevant CA
isoforms, i.e., h (human) CA I, II, VII, IX and XII; are shown in Table 1. We included these
isoforms in the study due to the fact that CA I and II are highly abundant cytosolic isoforms which
may be considered either drug targets (for antiglaucoma effects, diuretics, etc) or as offtargets
(when isoforms such as CA VA/B or CA IX/XII are targeted).1,2 hCA VII, as mentioned in the
Introduction, seems to be one of the isoforms involved in pain modulation and was included for this
purpose, whereas CA IX/XII are antitumor targets, these isoforms being present in high

concentrations in hypoxic, solid tumors.2 The following structure-activity relationship (SAR) can be
drawn from the data of Table 1:
(i) Except for the indoline-5-sulfonamide derivative 8a and the benzyloxy-derivative of
ethoxzolamide 5a, which were effective inhibitors of hCA I (KIs in the range of 34.3 – 48.1 nM),
the remaining sulfonamides investigated here were ineffective as inhibitors of the slow hCA I
isoform, with inhibition constants ranging between 143 and 6400 nM (Table 1).
(ii) The physiologically dominant hCA II was generally highly inhibited by the sulfonamides
reported here, except for the sulfanilamide propargyl derivative 7a and its dicobalt hexacarbonyl
complex 7b, which were rather ineffective CAIs (KIs of 162-1785 nM, Table 1). The best hCA II
inhibitors were the ethers derived from sulfanilamide 2c, 2k and 2l, incorporating n-Bu, 4-
nitrobenzyl and phenethyl moieties, and the ethoxzolamide derivative 5b incorporating benzyl
instead of ethyl moiety (KIs in the range of 1.4 – 7.8 nM). All these compounds are more effective
than acetazolamide AAZ (KI of 12 nM). The remaining derivatives showed effective hCA II
inhibitory properties, with a flat SAR and KIs in the range of 12.0 – 52.1 nM, rather similar with
that of celecoxib CLX (KI of 21 nM, Table 1).
(iii) A very interesting inhibition profile was registered against hCA VII. Except for three benzene
sulfonamide ethers (2m-2o) and one ethoxzolamide derivative 5a, all of which incorporate rather
bulky R moieties (1-phenyl-propargyl, diphenylethyl- and N-morpholyl-ethyl) and which were
medium potency hCA VII inhibitors (KIs of 70.7 – 97.2 nM), all other sulfonamides reported here
were highly effective hCA VII inhibitors. The SAR is in fact quite flat, with a variation of the KIs of
only between 0.9 and 10.1 nM. Thus, all compact aliphatic, aromatic and even organometallic
moieties were well tolerated in the molecules of these compounds, leading to low nanomolar hCA
VII inhibitors (Table 1). Interestingly, the sulfanilamide derivatives 7a and 7b showed a very
similar activity with the corresponding ethers 2e and 2f.
(iv) The transmembrane, tumor-associated isoform hCA IX was effectively inhibited by all
sulfonamides investigated here, with inhibition constants in the range of 0.86 – 34.1 nM (Table 1).
The most effective hCA IX inhibitors were 2i (incorporating the benzene sulfonamide ether and
pentafluorobenzyl moieties) and 5a (incorporating the ethoxzolamide and benzyl scaffolds), with
subnanomolar potency. Many derivatives with KIs < 10 nM were detected, such as 2a, 2e-2g, 2j-2l,
7a, 7b and 8a. Thus all scaffolds investigated here may lead to interesting inhibitors targeting CA
IX, with the nature of the R moiety from the ether being the crucial factor influencing biological
activity.
(v) The second transmembrane isoform investigated here, hCA XII; was also effectively inhibited
by most compounds reported in the paper. Thus, only 2m and 2n (incorporating the bulky moieties

1-phenyl- propargyl, diphenylethyl) were less effective hCA XII inhibitors (KIs of 33.8 – 50.4 nM)
with the remaining sulfonamides showing inhibition constants in the range of 0.72 – 13.6 nM
(Table 1). The most effective substitution patterns for hCA XII inhibition were propargyl and the
corresponding dicobalt hexacarbonyl complex in ethers 2e and 2f, pentafluorobenzyl (2i), 4-
nitrobenzyl (2k), phenethyl (2l), etc. The remaining scaffolds (ethoxzolamide in 5a, 5b; bis-
propargyl-sulfanilamide in 7a, 7b, or indoline-5-sulfonamide in 8a) also led to quite effective hCA
XII inhibitors.
Figs. 1-3 and Table 2 here
2.3. X-ray crystallography. In order to understand the high efficacy of this class of
benzenesulfonamides incorporating ether moieties for inhibition of various CA isoforms, we report
the high resolution X-ray crystallographic structure for the adduct of hCA II with sulfonamides 2a
(Figure 1a), 2b (Figure 1b), and 2d (Figure 1c),, all acting as highly efficient hCA II, VII, IX and
XII inhibitors (Table 2 and Fig. 1a).
Each inhibitor-soaked crystals crystallized in the monoclinic space group, P21 (Table 2). For
the CA II-2a complex, data was collected to a resolution of ~1.6 Å with unit cell dimensions: a =
42.3, b = 41.2, c = 71.9 Å and β = 104.3°. The final refined model had a Rcrys and Rfree value of
15.5% and 19.2%, respectively. The CA II-2b complex data was collected to a resolution of ~1.1 Å,
with the following unit cell dimensions: a = 42.3, b = 41.1, c = 71.7 Å and β = 104.3°. The final
refined model had a Rcrys and Rfree value of 20.1% and 21.7%, respectively. The CA II-2d complex
data was collected to a resolution of ~1.1 Å, with the following unit cell dimensions: a = 42.3, b =
41.2, c = 71.8 Å and β = 104.3°. The final refined model had a Rcrys and Rfree value of 15.2% and
16.2%,
The initial Fo–Fc electron density maps observed in Coot for each complex showed the
inhibitors bound in the active site of the enzyme (Fig. 2). The tetrahedral conformation about the
zinc(II) ion in the inhibitor bound state usually seen with sulfonamides was observed in each
enzyme-inhibitor complex. Like in most sulfonamides inhibitors the nitrogen atom of the
sulfonamide co-ordinated with the zinc in the active site at a distance of approximately 2.0 Å.
In the absence of an inhibitor the Zn(II) ion is tetrahedrally coordinated by His94, His96 and
His119 (CA II numbering) and a water molecule or hydroxyl ion (PDB ID: 3KS3). This Zn(II) ion
lies at the base of a deep conical cavity where the zinc bound solvent also forms hydrogen bond
interactions with the hydroxyl group of Thr199 which in turn interacts with the carboxylate group
of Glu106. These interactions form part of the conserved hydrogen bond network in the active site

of CA II, which allows the enzyme to increase the nucleophilcity of the zinc-bound solvent as well
as to proper orient the CO2 substrate in the hydrophobic pocket bordering the active site entrance.
In each protein-inhibitor complex, the conserved zinc-bound water molecule is displaced by
the inhibitor’s sulfonamide group. The NH of the same moiety also participated in a strong
hydrogen bond with the OH group of Thr199, again as in all sulfonamide complexes investigated so
far. 20,21 The aryl ring of 2a and the alkoxy moiety in para to the sulfonamide were stabilized by van
der Waals interactions with hydrophobic amino acid residues from the enzyme active site, such as
Val121, Val135, Val143, Phe131, Leu198 and Pro202. There were no polar interactions between
the inhibitor and the enzyme, apart the Zn(II) coordination and the anchoring to Thr199 mentioned
above, which probably explains the rather similar inhibitory properties of 2a against 4 out of 5 CA
isoforms (Table 1). However, in this work we were interested in a pain-modulating CA inhibitor as
the brain has high amounts of CA II, CA VII and CA XII and we do not know which of these
isoforms are targeted with respect to this phenomena involving the neuropathic pain.
Sulfonamides 2b and 2d were also co-crystallized with CA II, as they also contain a short
hydrophobic tail, similar to 2a. Derivatives 2b and 2d differ from 2a only by the presence of a –
CH2 group and a double bond in the tail of the inhibitor, respectively. Based on the crystallographic
structures both drugs bind in the CA II active site in a similar manner to 2a (Fig 1b and 2c). All
three inhibitors formed stable interactions with the same residues mentioned above for 2a. All three
inhibitors interacted with hydrophobic amino acid residues that are generally conserved among the
catalytically active CA isoforms (Fig. 3). Even the residues that are not conserved were also
hydrophobic in nature and this explains the modest change in binding affinity for the three
inhibitors among various CAs. In addition the high degree of amino acid conservation observed in
the active site of CA II, VII, IX and XII1 may provide some insight into the similar inhibition
profile among these isoforms.
Fig. 4 here
2.4. Pain modulating effects of CA inhibitors. We investigated the potent sulfonamide CAI 2a in
comparison to acetazolamide (AAZ), as possible pain relievers in a mice model of neuropathic pain
induced by oxaliplatin (Fig. 4). This third-generation platinum derivative has become a first-line
chemotherapy in metastatic colorectal cancer and a valid option as adjuvant therapy in several types
23
of cancer.22 The limiting side effect is a painful neuropathy that persists between cycles and is
correlated with characteristic alterations of the nervous system.24 Neuropathy development results
in therapy dose reduction or discontinuation and negatively influences quality of life on cancer
survivors.25

In mice repeatedly treated with oxaliplatin, a single administration of 2a reverted the
lowering of pain threshold to cold stimuli (cold plate test, Fig. 2). On day 14 of oxaliplatin
treatment, when neuropathy was well established,24,26 2a relieved pain dose-dependently (10 – 50
mg kg-1 p.o.), the higher dose was able to restore pain threshold up to the value of control group.
Significant pain relief was induced between 15 and 45 min after treatment (Figure 2). In the same
model AAZ was also effective but with less potency and efficacy compared to 2a. The minimum
effective dose of AAZ was 3 fold higher (30 mg kg-1 p.o.) in comparison to 2a. AAZ dosed at 100
mg kg-1 did not completely revert pain, and effectiveness was limited to 15 min (Figure 2).
The 2014 clinical practical guideline from the American Society of Clinical Oncology states
that there are no agents recommended for the prevention of chemotherapy-induced neuropathic
pain.25 The efficacy of CAIs against oxaliplatin-induced pain improves the knowledge about the
physiopathology of neuropathy and offers a new pharmacological target for this purpose.
3. Conclusions
A series of benzene sulfonamide ethers were obtained from 4-hydroxybenzenesulfonamide or the
phenol derivative of ethoxzolamide. They incorporate lipophilic moieties, such as alkyloxy,
alkenyloxy, alkynyloxy, substituted-benzyloxy, etc. Two dicobalt(0) hexacarbonyl complexes of the
obtained alkynes were also prepared. The new derivatives showed modest inhibition against the
cytosolic isoform CA I, but were generally effective, low nanomolar CA II, VII, IX and XII
inhibitors. X-ray crystallographic data for the adduct of one such sulfonamide with CA II allowed
us to rationalize the good inhibition data. In a mice model of neuropathic pain induced by
oxaliplatin, one of the strong CA II/VII inhibitors reported here induced a long lasting pain
relieving effect. This is the first report of rationally designed sulfonamide CA inhibitors with pain
modulating effects.
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 transfer solutions. Nuclear
magnetic resonance (1H-NMR, 13C-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; t, triplet; m, multiplet; brs, broad singlet; dd, double of doublets. 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 ethylacetate/n-hexane were used as eluents. Melting points (mp) were carried out in open
capillary tubes and are uncorrected.
4.1.1. General procedure for the synthesis of O-(N)-alkyl/benzyl-benzenesulfonamides 2a-o,
7a, and O-alkyl benzo[d]thiazole-2-sulfonamides 5a,b.
a) General procedure for (O)-N nucleophilic alkylations.
4-Hydroxybenzenesulfonamide
1
(1.0
eq)
(scheme
A),
N,N-dimethyl-N'-
(phenylsulfonyl)formimidamide (1.0 eq) (scheme B) or sulfanilamide (1.0 eq) (scheme D), were
treated with the appropriate base in dry DMF (10ml) and the suspension was stirred at r.t. for 20
min under a nitrogen atmosphere. Then the corresponding alkyl/benzyl halide was added and the
reaction was stirred at r.t. until starting material was consumed (TLC monitoring). The reaction
mixture was quenched with slush and the precipitate formed was collected by filtration, dried under
vacuo and purified by silica gel column chromatography eluting with ethyl acetate/n-hexane to
afford the desired product.
b) General procedure for (O)-alkylations via Mitsunobu coupling.
A solution of 4-hydroxybenzenesulfonamide (1.0g, 1.0 eq), alcohol (1.0 eq) and triphenylphospine
(1.0 eq) in dry THF (20 ml) was sonicated at 0°C for 5 min (scheme C). Then DIAD (1.1 eq) was
added drop-wise and the orange solution sonicated at the same temperature for 20 min, quenched
with H2O (20 ml) and extracted with ethyl acetate (3 x 15 ml). The combined organic layers were
washed with H2O (3 x 20 ml), dried over Na2SO4, filtered and concentrated in vacuo to give a sticky
residue that was purified by silica gel column chromatography eluting with 50 % ethyl acetate/n-
hexane 50 % v/v to and the white solid obtained was recrystallized from IPA.
Synthesis of 4-ethoxybenzenesulfonamide (2a).

SO NH
2
2
1
4
2
3
SO NH
2
2
Br
K CO
2
3
O
1'
DMF
OH
2'
2a
1
The synthesis was carried out according to the general procedure in scheme 1, route A to afford the
desired product as a white solid.
4-Ethoxybenzenesulfonamide 2a: 60 % yield; silica gel TLC Rf 0.60 (Ethyl acetate/n-hexane 50 %
v/v); m.p. 145 °C (lit.27 149° C); δH (400 MHz, DMSO-d6) 1.38 (3H, t, J 7.2, 2’-H3), 4.13 (2H, q, J
7.2, 1’-H2), 7.11 (2H, d, J 8.8, 2 x 3-H), 7.23 (2H, s, exchange with D2O, SO2NH2), 7.76 (2H, d, J
8.8, 2 x 2-H); δC (100 MHz, DMSO-d6) 161.8, 136.9, 128.6, 115.3, 64.5, 15.4; m/z (ESI positive)
202.05 [M+H]+.
Synthesis of 4-n-propoxybenzenesulfonamide (2b).
SO NH
2
2
1
2
3
SO NH
2
2
Br
K CO
2
3
4
O
1'
DMF
OH
2'
3'
2b
1
The synthesis was carried out according to the general procedure in scheme 1, route A to afford the
desired product as a white solid.
4-n-Propoxybenzenesulfonamide 2b: 57 % yield; silica gel TLC Rf 0.50 (Ethyl acetate/n-hexane 50
% v/v); m.p. 120 °C; δH (400 MHz, DMSO-d6) 1.02 (3H, t, J 7.6, 3’-H3), 1.78 (2H, m, 2’-H2), 4.04
(2H, t, J 7.6, 1’-H2), 7.10 (2H, d, J 8.8, 2 x 3-H), 7.24 (2H, s, exchange with D2O, SO2NH2), 7.76
(2H, d, J 8.8, 2 x 2-H); δC (100 MHz, DMSO-d6) 162.0, 136.9, 128.6, 115.3, 70.3, 22.8, 11.2; m/z
(ESI positive) 216.06 [M+H]+.
Synthesis of 4-n-butoxybenzenesulfonamide (2c).
SO NH
2
2
1
4
2
3
SO NH
2
2
Br
K CO
2
3
O
1'
DMF
OH
2'
3'
4'
2c
1

The synthesis was carried out according to the general procedure in scheme 1, route A to afford the
desired product as a white solid.
4-n-Butoxybenzenesulfonamide 2c: 67 % yield; silica gel TLC Rf 0.42 (Ethyl acetate/n-hexane 30
% v/v); m.p. 112 °C (lit.28 108 °C); δH (400 MHz, DMSO-d6) 0.97 (3H, t, J 7.2, 4’-H3), 1.48 (2H,
m), 1.76 (2H, m), 4.08 (2H, t, J 7.2, 1’-H2), 7.12 (2H, d, J 8.8, 2 x 3-H), 7.24 (2H, s, exchange with
D2O, SO2NH2), 7.77 (2H, d, J 8.8, 2 x 2-H); δC (100 MHz, DMSO-d6) 162.0, 136.9, 128.6, 115.3,
68.9, 31.5, 19.6, 14.6; m/z (ESI positive) 230.09 [M+H]+.
Synthesis of 4-(allyloxy)benzenesulfonamide (2d).
SO NH
2
2
1
2
3
SO NH
2
2
Br
K CO
2
3
4
O
1'
DMF
OH
2'
3'
1
2d
The synthesis was carried out according to the general procedure in scheme 1, route A to afford the
desired product as a white solid.
4-(Allyloxy)benzenesulfonamide 2d: 70 % yield; silica gel TLC Rf 0.47 (Ethyl acetate/n-hexane 50
% v/v); m.p. 128 °C (lit.29 132-133 °C); δH (400 MHz, DMSO-d6) 4.68 (2H, d, J 5.2, 1’-H2), 5.42
(2H, m, 3’-H2), 7.12 (2H, d, J 8.8, 2 x 3-H), 7.24 (2H, s, exchange with D2O, SO2NH2), 7.78 (2H, d,
J 8.8, 2 x 2-H); δC (100 MHz, DMSO-d6) 161.5, 137.2, 134.1, 128.6, 118.9, 115.6, 69.4; m/z (ESI
positive) 214.05 [M+H]+.
Synthesis of 4-(prop-2’-ynyloxy)benzenesulfonamide (2e). 30
SO2NH2
1
2
SO2NH2
3
OH
Ph3P, DIAD
4
O
1'
THF
2'
OH
3'
1
2e
The synthesis was carried out according to the general procedure in scheme C to afford the desired
product as a white solid.
4-(Prop-2’-ynyloxy)benzenesulfonamide 2e: 53 % yield; silica gel TLC Rf 0.31 (Ethyl acetate/n-
hexane 50 % v/v); m.p. 96 °C; δH (400 MHz, DMSO-d6) 3.66 (1H, t, J 1.9, 3’-H), 4.93 (2H, d, J 1.9,

1’-H2), 7.17 (2H, d, J 8.8, 2 x 3-H), 7.27 (2H, s, exchange with D2O, SO2NH2), 7.79 (2H, d, J 8.8,
Ar-H), 7.43 (8H, m, 2 x 2-H), 7.76 (2H, d, J 8.8, 2 x 2-H); δC (100 MHz, DMSO-d6) 160.5, 137.9,
128.7, 116.0, 79.8, 79.7, 56.8; m/z (ESI positive) 212.03 [M+H]+.
Synthesis of 4-(prop-2’-ynyloxy)benzenesulfonamidehexacarbonyldicobalt (2f).31
SO2NH2
SO2NH2
1
4
2
3
Co2(CO)8
THF
O
O
1'
2'
Co2(CO)6
3'
2e
2f
4-(Prop-2’-ynyloxy)benzenesulfonamide 2e (0.1g, 1.0 eq) was dissolved in THF (10ml) and then
cobalt carbonyl (1.05 eq) was added. The black solution was stirred at r.t. for 40 min. Then SiO2
(0.3g) was added and solvent removed under vacuo to give a black solid that was purified by silica
gel column chromatography eluting with 50 % ethyl acetate in n-hexane to give the title compound
as a red solid.
4-(Prop-2’-ynyloxy)benzenesulfonamidehexacarbonyldicobalt 2f: yield 82 % yield; silica gel TLC
Rf 0.56 (Ethyl acetate/n-hexane 50 % v/v); m.p. 174 °C; δH (400 MHz, DMSO-d6) 5.44 (2H, s, 1’-
H2), 6.86 (1H, s, 3’-H), 7.17 (2H, d, J 8.8, 2 x 3-H), 7.28 (2H, s, exchange with D2O, SO2NH2),
7.81 (2H, d, J 8.8, 2 x 2-H); δC (100 MHz, DMSO-d6) 200.9, 161.3, 137.7, 128.8, 115.7, 90.1, 74.0,
69.3; m/z (ESI positive) 512.90 [M+H]+.
Synthesis of 4-(benzyloxy)benzenesulfonamide (2g).
SO2NH2
1
2
SO2NH2
Br
3
K2CO3
4
O
5
DMF
6
OH
7
8
9
1
2g
The synthesis was carried out according to the general procedure in scheme A to afford the desired
product as a white solid.
4-(Benzyloxy)benzenesulfonamide 2g: 83 % yield; silica gel TLC Rf 0.38 (Ethyl acetate/n-hexane
50 % v/v); δH (400 MHz, DMSO-d6) 5.23 (2H, s, 5-H2), 7.19 (2H, d, J 8.8, 2 x 3-H), 7.24 (2H, s,
exchange with D2O, SO2NH2), 7.38-7.50 (5H, m, 2 x 7-H, 2 x 8-H, 9H), 7.79 (2H, d, J 8.8, 2 x 2-

H); δC (100 MHz, DMSO-d6) 161.6, 137.4, 137.3, 129.4, 128.9, 128.6, 128.6, 70.4; m/z (ESI
positive) 264.04 [M+H]+.
Experimental data are in agreement with reported data. 32
Synthesis of 4-(4’-fluorobenzyloxy)benzenesulfonamide (2h).
1''
N
SO NH
2
O
2
N
N
1
4
S
2
3
Cl
O
1
4
O
N
2
S
Cs CO
O
1.5M HCl/MeOH
2
3
3
O
DMF
1'
O
2'
3'
F
1'
4'
OH
2'
3'
4'
F
F
A
2h
B
The synthesis was carried out according to the general procedure in scheme B to afford the desired
product B as a white solid that was dissolved in a 1.5M HCl/MeOH solution (5 ml) and the reaction
was stirred at 60 °C in a sealed tube for 4 h, concentrated under vacuo to give a residue that was
purified by silica gel column chromatography eluting with ethyl acetate/n-hexane 50 % v/v
affording 2h as a white solid.
N'-(4-(4’-Fluorobenzyloxy)phenylsulfonyl)-N,N-dimethylformimidamide B: yield 55 % yield; silica
gel TLC Rf 0.24 (Ethyl acetate/n-hexane 70 % v/v); m.p. 150 °C; δH (400 MHz, DMSO-d6) 2.92
(3H, s, N-CH3), 3.17 (3H, s, N-CH3), 4.81 (2H, s, CH2), 7.04-7.29 (4H, m, Ar-H), 7.50-7.80 (4H, m,
Ar-H), 8.22 (1H, s, 1”-H); m/z (ESI positive) 337.10 [M+H]+.
4-(4’-Fluorobenzyloxy)benzenesulfonamide 2h: 42 % yield; silica gel TLC Rf 0.31 (Ethyl
acetate/n-hexane 50 % v/v); m.p. 182 °C; δH (400 MHz, DMSO-d6) 5.21 (2H, s, CH2), 7.25 (6H, m,
Ar-H, SO2NH2, exchange with D2O), 7.55 (2H, m, Ar-H), 7.78 (2H, d, J 8.8, Ar-H); δF (376 MHz,
DMSO-d6) –114.16; m/z (ESI positive) 282.05 [M+H]+.
Synthesis of 4-(perfluorobenzyloxy)benzenesulfonamide (2i).
1''
N
SO NH
O
2
2
N
N
1
4
S
2
3
Br
O
1
O
N
2
S
F
F
Cs CO
F
F
O
1.5M HCl/MeOH
2
3
3
O
4
DMF
1'
O
F
F
F
F
F
1'
2'
F
F
F
OH
2'
3'
4'
F
3'
F
4'
F
A
2i
C

The synthesis was carried out according to the general procedure in scheme B to afford the desired
product C as a white solid that was dissolved in a 1.5M HCl/MeOH solution (5 ml) and the reaction
was stirred at 60 °C in a sealed tube for 4 h, concentrated under vacuo to give a residue that was
purified by silica gel column chromatography eluting with ethyl acetate/n-hexane 50 % v/v
affording 2i as a pale yellow solid.
N,N-Dimethyl-N'-(4-(perfluorobenzyloxy)phenylsulfonyl)formimidamide C: yield 69 % yield;
silica gel TLC Rf 0.22 (Ethyl acetate/n-hexane 70 % v/v); m.p. 191 °C; δH (400 MHz, DMSO-d6)
2.93 (3H, s, N-CH3), 3.17 (3H, s, N-CH3), 5.33 (2H, s, CH2), 7.20 (2H, d, J 8.8, Ar-H), 7.56 (2H, d,
J 8.8, Ar-H), 8.24 (1H, s, 1”-H); m/z (ESI positive) 409.06 [M+H]+.
4-(Perfluorobenzyloxy)benzenesulfonamide 2i: yield 35 % yield; silica gel TLC Rf 0.75 (Ethyl
acetate/n-hexane 70 % v/v); m.p. 163 °C; δH (400 MHz, DMSO-d6) 5.34 (2H, s, CH2), 7.25 (2H, d,
J 8.8, Ar-H), 7.31 (2H, s, SO2NH2, exchange with D2O), 7.82 (2H, d, J 8.8, Ar-H); δF (376 MHz,
3
3
DMSO-d6) –142.8 (2F, d, J F-F 22.0, 2 x F-2’), -152.9 (1F, t, J F-F 21.0, F-4’), -164.21 (2F, m, 2 x
F-3’); m/z (ESI positive) 354.01 [M+H]+.
Synthesis of N'-(4-hydroxyphenylsulfonyl)-N,N-dimethylformimidamide (A).
Procedure 1
Procedure 2
N
1'
N
SO2NH2
SO2NH2
O
O
O
N
S
SOCl2, DMF
O
1
4
2
Solvents; Tol. = 10% yield
THF= 57% yield
DMF
3
OH
OH
65% yield
OH
A
Procedure 1:30 Thionyl chloride (3.43 g, 5.0 eq) was added to a suspension of 4-
hydroxybenzenesulfonamide (1.0g, 1.0eq) in toluene or THF (15 ml), followed by addition of DMF
(2.95g, 7.0 eq). The yellow suspension was stirred at 70°C O.N., quenched with slush, extracted
with ethyl acetate (3 x 20 ml) and the combined organic layers were washed with brine (3 x 20 ml),
H2O (3 x 20 ml), dried over Na2SO4, filtered and concentrated in vacuo to give a residue that was
purified by silica gel column chromatography eluting with ethyl acetate/n-hexane 70 % v/v to afford
the desired product as a white solid.
Procedure 2:33 Dimethoxy-N,N-dimethylmethanamine (4.54 g, 6.6 eq) was added to a solution of
4-hydroxybenzenesulfonamide (1.0 g, 1.0 eq) in DMF (0.5ml). The yellow solution was stirred at
40 °C 5h, quenched with slush, extracted with ethyl acetate (3 x 20 ml) and the combined organic

layers were washed with H2O (7 x 15 ml), dried over Na2SO4, filtered and concentrated in vacuo to
give a residue that was purified by silica gel column chromatography eluting with ethyl acetate/n-
hexane 70 % v/v to afford the desired product as a white solid.
N'-(4-Hydroxyphenylsulfonyl)-N,N-dimethylformimidamide A: silica gel TLC Rf 0.10 (Ethyl
acetate/n-hexane 70 % v/v); m.p. 178 °C; δH (400 MHz, DMSO-d6) 2.92 (3H, s, N-CH3), 3.16 (3H,
s, N-CH3), 7.12 (2H, d, J 6.8, Ar-H), 7.61 (2H, d, J 6.8, Ar-H), 8.18 (1H, s, 1’-H), 10.29 (1H, brs,
exchange with D2O, OH); δC (100 MHz, DMSO-d6) 161.3, 160.3, 134.2, 129.0, 116.2, 41.7, 35.9;
m/z (ESI positive) 229.10 [M+H]+.
Synthesis of 4-(5’-bromobenzyloxy)benzenesulfonamide (2j).
SO2NH2
1
2
Br
SO2NH2
3
K2CO3
DMF
4
O
1'
4'
OH
2'
Br
3'
Br
1
2j
The synthesis was carried out according to the general procedure in scheme A to afford the desired
product as a yellow solid.
4-(5’-bromobenzyloxy)benzenesulfonamide 2j: 63 % yield; silica gel TLC Rf 0.40 (Ethyl acetate/n-
hexane 50 % v/v); m.p. 178 °C; δH (400 MHz, DMSO-d6) 5.22 (2H, s, CH2), 7.19 (2H, d, J 8.0, Ar-
H), 7.25 (2H, s, exchange with D2O, SO2NH2), 7.46 (2H, d, J 8.0, Ar-H), 7.64 (2H, d, J 8.0, Ar-H),
7.78 (2H, d, J 8.0, Ar-H); δC (100 MHz, DMSO-d6) 161.4, 137.4, 136.9, 132.4, 130.8, 128.6, 122.1,
115.8, 69.6; m/z (ESI positive) 341.97 [M+H]+.
Synthesis of 4-(4’-nitrobenzyloxy)benzenesulfonamide (2k).
SO NH
2
2
1
2
Cl
SO NH
2
2
3
K CO
2
3
4
O
DMF
1'
OH
2'
3'
NO
2
4'
NO
2
1
2k
The synthesis was carried out according to the general procedure in scheme A to afford the desired
product as a yellow solid.

4-(4’-Nitrobenzyloxy)benzenesulfonamide 2k: 78 % yield; silica gel TLC Rf 0.35 (Ethyl acetate/n-
hexane 50 % v/v); m.p. 189 °C; δH (400 MHz, DMSO-d6) 4.54 (2H, s, CH2), 7.22 (2H, d, J 8.8, Ar-
H), 7.27 (2H, s, exchange with D2O, SO2NH2), 7.77 (2H, d, J 8.8, Ar-H), 7.78 (2H, d, J 8.8, Ar-H),
8.32 (2H, d, J 8.8, Ar-H); δC (100 MHz, DMSO-d6) 161.2, 148.0, 145.3, 137.7, 129.2, 128.7, 124.6,
115.9, 69.2; m/z (ESI positive) 309.05 [M+H]+.
Synthesis of 4-phenethoxybenzenesulfonamide (2l).
SO2NH2
1
2
Br
SO2NH2
3
K2CO3
4
O
5
DMF
6
OH
7
8
10
9
1
2l
The synthesis was carried out according to the general procedure in scheme A to afford the desired
product as a yellow solid.
4-Phenethoxybenzenesulfonamide 2l: 62 % yield; silica gel TLC Rf 0.41 (Ethyl acetate/n-hexane 50
% v/v); m.p. 112 °C; δH (400 MHz, DMSO-d6) 3.09 (2H, t, J 6.8, 6-H2), 4.31 (2H, t, J 6.8, 5-H2),
7.12 (2H, d, J 8.8, 2 x 3-H), 7.23 (2H, s, exchange with D2O, SO2NH2), 7.25-7.37 (5H, m, 2 x 8-H,
2 x 9-H, 10-H), 7.56 (2H, d, J 8.8, 2 x 2-H); δC (100 MHz, DMSO-d6) 161.7, 139.0, 137.1. 129.9,
129.3, 128.6, 127.3, 115.4. 69.5, 35.6; m/z (ESI positive) 278.08 [M+H]+.
Synthesis of 4-(3’-phenylprop-2’-ynyloxy)benzenesulfonamide (2m).
SO2NH2
1
2
Cl
3
SO2NH2
4
K2CO3
DMF
O
1'
2'
3'
OH
4'
5'
6'
7'
2m
1
The synthesis was carried out according to the general procedure in scheme A to afford the desired
product as a yellow solid.
4-(3’-Phenylprop-2’-ynyloxy)benzenesulfonamide 2m: 55 % yield; silica gel TLC Rf 0.36 (Ethyl
acetate/n-hexane 50 % v/v); m.p. 138 °C; δH (400 MHz, DMSO-d6) 5.19 (2H, s, 1’-H2), 7.23 (2H, d,
J 8.8, 2 x 3-H), 7.25 (2H, s, exchange with D2O, SO2NH2), 7.45 (5H, m, 2 x 5’-H, 2 x 6’-H, 7-H),

7.83 (2H, d, J 8.8, 2 x 2-H); δC (100 MHz, DMSO-d6) 160.5, 137.8, 132.4, 130.1, 129.7, 128.6,
122.3, 115.9, 87.7, 85.2, 57.3; m/z (ESI positive) 287.07 [M+H]+.
Synthesis of 4-(2’,2’-diphenylethoxy)benzenesulfonamide (2n).
SO2NH2
1
2
SO2NH2
HO
3
Ph3P, DIAD
4
O
1'
THF
2'
OH
3'
6'
4'
5'
2n
1
The synthesis was carried out according to the general procedure in scheme C to afford the desired
product as a yellow solid.
4-(2’,2’-Diphenylethoxy)benzenesulfonamide 2n: 53 % yield; silica gel TLC Rf 0.44 (Ethyl
acetate/n-hexane 50 % v/v); m.p. 176 °C; δH (400 MHz, DMSO-d6) 4.56 (1H, t, J 7.2, 2’-H), 4.68
(2H, d, J 7.2, 1’-H2), 7.12 (2H, d, J 8.8, Ar-H), 7.23 (2H, s, exchange with D2O, SO2NH2), 7.26
(2H, d, J 8.8, Ar-H), 7.43 (8H, m, Ar-H), 7.76 (2H, d, J 8.8, 2 x 2-H); δC (100 MHz, DMSO-d6)
161.7, 142.7, 137.2, 129.4, 129.0, 128.5, 127.5, 115.6, 71.2, 50.6; m/z (ESI positive) 354.11
[M+H]+.
Synthesis of 4-(2’-morpholinoethoxy)benzenesulfonamide (2o).
SO NH
2
2
1
2
3
Cl
SO NH
2
2
K CO
2
3
HCl
4
N
O
O
1'
DMF
OH
2'
N
O
3'
4'
2o
1
The synthesis was carried out according to the general procedure in scheme A using 2.2 eq. of
K2CO3 to afford the desired product as a white solid.
4-(2’-Morpholinoethoxy)benzenesulfonamide 2o: 62 % yield; silica gel TLC Rf 0.12 (Ethyl
acetate); m.p. 158 °C; δH (400 MHz, DMSO-d6) 2.49 (4H, m, 2 x 3’-H2), 2.74 (2H, t, J 5.6, 2’-H2),
3.61 (4H, m, 2 x 4’-H2), 4.20 (2H, t, J 5.6, 1’-H2), 7.12 (2H, d, J 6.8, 2 x 3-H), 7.23 (2H, s,
exchange with D2O, SO2NH2), 7.76 (2H, d, J 6.8, 2 x 2-H); δC (100 MHz, DMSO-d6) 161.8, 137.1,
128.6, 115.4, 67.1, 66.7, 57.7, 54.5; m/z (ESI positive) 286.10 [M+H]+.
Synthesis of 6-hydroxybenzo[d]thiazole-2-sulfonamide (4).34

4
N
S
5
N
S
2
AlCl
DCM
3
SO NH
2
SO NH
2
2
2
HO
O
6
7
4
3
Ethoxzolamide (2.0g, 1.0 eq) was suspended in DCM (40 ml) and the AlCl3 (3.61g, 3.5 eq) was
added portion-wise at 0°C. The reaction mixture was stirred vigorously at r.t. O.N. cooled down to
0° C and quenched with 3.0 M aqueus hydrochloric acid (100 ml). The solid formed was collected
by filtration, washed with H2O (150 ml), dried under vacuo and used as it is
6-Hydroxybenzo[d]thiazole-2-sulfonamide 4: 87 % yield; δH (400 MHz, DMSO-d6) 7.14 (1H, dd, J
8.8 2.4, 5-H), 7.53 (1H, d, J 2.4, 7-H), 7.99 (1H, d, J 8.8, 4-H), 8.23 (2H, s, exchange with D2O,
SO2NH2), 10.28 (1H, s, exchange with D2O, OH); δC (100 MHz, DMSO-d6) 166.1 (C-2), 158.1 (C-
6), 146.1 (C-4a), 138.5 C-7a), 125.9 (C-4), 118.5 (C-5), 107.9 (C-7); m/z (ESI positive) 230.98
[M+H]+.
Spectroscopic data are in agreement with reported data.34
Synthesis of N'-(6-hydroxybenzo[d]thiazol-2-ylsulfonyl)-N,N-dimethylformimidamide (4a).34
N
O
N
N
4
O
O
S
N
S
1'
5
N
S
2
SO NH
2
2
DMF
HO
O
HO
6
7
4a
4
The synthesis was carried out according to the general procedure 2 for the intermediate A to afford
the desired product as a white solid.
N'-(6-Hydroxybenzo[d]thiazol-2-ylsulfonyl)-N,N-dimethylformimidamide 4a: 55 % yield; silica gel
TLC Rf 0.36 (Ethyl acetate); δH (400 MHz, DMSO-d6) 3.12 (3H, s, N-CH3), 3.18 (3H, s, N-CH3),
7.12 (1H, dd, J 8.8 2.4, 5-H), 7.50 (1H, d, J 2.4, 7-H), 7.97 (1H, d, J 8.8, 4-H), 8.40 (1H, s, 1’-H),
10.35 (1H, brs, exchange with D2O, OH); δC (100 MHz, DMSO-d6) 165.5, 161.8, 158.1, 146.4,
138.5, 126.0, 118.4, 107.7, 42.2, 36.4; m/z (ESI positive) 286.02 [M+H]+.
Spectroscopic data are in agreement with reported data.34
Synthesis of 6-(benzyloxy)benzo[d]thiazole-2-sulfonamide (5a).
N
N
4
4
O
S
O
S
O
S
1'
N
S
N
S
5
N
S
5
BnBr
2
2
1.5M HCl/MeOH
N
N
NH
2
Cs CO
2
3
O
O
HO
BnO
6
O
BnO
6
7
7
DMF
4a
C
5a

The synthesis was carried out according to the general procedure in scheme E to afford the desired
product as a white solid.
N'-(6-(Benzyloxy)benzo[d]thiazol-2-ylsulfonyl)-N,N-dimethylformimidamide C: 64 % yield; silica
gel TLC Rf 0.20 (Ethyl acetate); δH (400 MHz, DMSO-d6) 3.03 (3H, s, N-CH3), 3.28 (3H, s, N-
CH3), 5.25 (2H, s, CH2), 7.30-7.50 (6H, m, Ar-H, 5-H), 7.91 (1H, d, J 2.4, 7-H), 8.08 (1H, d, J 8.8,
4-H), 8.41 (1H, s, 1’-H); m/z (ESI positive) 375.07 [M+H]+.
6-(Benzyloxy)benzo[d]thiazole-2-sulfonamide 5a: 40 % yield; silica gel TLC Rf 0.35 (Ethyl
acetate/n-hexane 50 % v/v); δH (400 MHz, DMSO-d6) 5.11 (2H, s, CH2), .6.97 (1H, dd, J 8.8 2.4, 5-
H), 7.05 (1H, d, J 8.8, 4-H), 7.32-7.50 (8H, m, Ar-H, 7-H, SO2NH2 exchange with D2O); δC (100
MHz, DMSO-d6) 171.8, 155.8, 138.3, 131.4, 130.0, 129.5, 129.3, 125.8, 115.8, 113.8, 110.4, 71.4;
m/z (ESI positive) 320.03 [M+H]+.
Synthesis of 6-(3’-phenylprop-2’-ynyloxy)benzo[d]thiazole-2-sulfonamide (5b).
Cl
N
4
4
O
S
O
S
N
S
N
S
5
5
1"
2
2
Cs2CO3
DMF
1.5M HCl/MeOH
N
NH2
N
N
O
O
O
O
6
6
O
S
O
7
7
N
S
1'
1'
2'
2'
HO
3'
3'
5b
D
4a
The synthesis was carried out according to the general procedure in scheme E to afford the desired
product as a white solid.
N,N-Dimethyl-N'-(6-(3’-phenylprop-2’-ynyloxy)benzo[d]thiazol-2-ylsulfonyl)formimidamide D: 24
% yield; silica gel TLC Rf 0.32 (Ethyl acetate); δH (400 MHz, DMSO-d6) 3.03 (3H, s, N-CH3), 3.27
(3H, s, N-CH3), 5.21 (2H, s, CH2), 7.37-7.50 (6H, m, Ar-H, 5-H), 7.94 (1H, d, J 2.4, 7-H), 8.11 (1H,
d, J 8.8, 4-H), 8.41 (1H, s, 1”-H); δC (100 MHz, DMSO-d6) 167.3, 161.9, 157.6, 147.8, 138.3,
132.5, 130.1, 129,7, 126.0, 122.3, 118.7, 107.4, 87.7, 85.3, 57.8, 42.2, 36.5; m/z (ESI positive)
400.07 [M+H]+.
6-(3’-Phenylprop-2’-ynyloxy)benzo[d]thiazole-2-sulfonamide 5b: 12 % yield; silica gel TLC Rf
0.28 (Ethyl acetate/n-hexane 10 % v/v); δH (400 MHz, DMSO-d6) 5.17 (2H, s, 1’-H2), 7.28 (1H, dd,
J 8.8 2.4, 5-H), 7.40-7.51 (7H, m, Ar-H, SO2NH2 exchange with D2O), 7.86 (1H, d, J 2.4, 7-H),

7.94 (1H, d, J 8.8, 4-H); δC (100 MHz, DMSO-d6) 156.6, 151.0, 146.1, 137.9, 132.5, 130.1, 129.7,
124.0, 122.4, 117.4, 107.7, 87.7, 85.5, 57.7; m/z (ESI positive) 345.03 [M+H]+.
Synthesis of 4-(diprop-2’-ynylamino)benzenesulfonamide (7a).
SO NH
2
2
1
4
SO NH
2
2
2
3
Br
K CO
2
3
DMF
N
1'
NH
2
2'
3'
6
7a
The synthesis was carried out according to the general procedure in scheme D using
propargylbromide (2.0 eq) and potassium carbonate (2.2 eq) to afford the desired product as a
yellow solid.
4-(Diprop-2’-ynylamino)benzenesulfonamide 7a: 78 % yield; silica gel TLC Rf 0.56 (Ethyl
acetate/n-hexane 30 % v/v); m.p. 124 °C; δH (400 MHz, DMSO-d6) 3.26 (2H, t, J 2.4, 2 x 3’-H),
4.03 (4H, d, J 1.9, 1’-H2), 6.12 (2H, s, exchange with D2O, SO2NH2), 6.63 (2H, d, J 8.8, 2 x 3-H),
7.46 (2H, d, J 8.8, 2 x 2-H); δC (100 MHz, DMSO-d6) 154.3, 130.5, 122.8, 113.5, 77.8, 77.4, 36.9;
m/z (ESI positive) 249.06 [M+H]+.
Synthesis of 4-(diprop-2’-ynylamino)benzenesulfonamidehexacarbonyldicobalt (7b).
SO2NH2
SO2NH2
1
4
2
3
Co2(CO)8
THF
N
N
1'
(OC)6Co2
2'
Co2(CO)6
3'
7a
7b
The synthesis was carried out according to the previous procedure for 2f using cobalt carbonyl (2.10
eq) to afford the desired product as a reddish solid.
4-(Diprop-2’-ynylamino)benzenesulfonamidedihexacarbonyldicobalt 7b: 79 % yield; silica gel TLC
Rf 0.16 (Ethyl acetate/n-hexane 20 % v/v); m.p. >300 °C; δH (400 MHz, DMSO-d6) 4.75 (4H, s, 2 x
1’-H2), 6.16 (2H, s, 2 x 3’-H), 6.54 (2H, s, exchange with D2O, SO2NH2), 6.69 (2H, d, J 8.8, 2 x 3-
H), 7.52 (2H, d, J 8.8, 2 x 2-H); δC (100 MHz, DMSO-d6) 200.3, 154.3, 130.1, 126.0, 113.6, 89.3,
75.5, 49.1; m/z (ESI positive) 850.80 [M+H]+.
Synthesis of 2,2,2-trifluoro-1-(indolin-1-yl)ethanone (8).34, 35

4
3
5
2
TFAA, Et3N
Et2O
N
N
6
H
7
CF3
O
8
TFAA (7.0g, 2.0eq) was added drop-wise to a solution of indoline (2.0g, 1.0 eq), Et3N (5.09g,
3.0eq) in Et2O (60 ml) at 0°C. The solution was stirred at r.t. until starting material was consumed
(TLC monitoring), quenched with H2O (50 ml) and the organic layer was washed with brine ( 3x 20
ml), dried over Na2SO4, filtered and concentrated in vacuo to give a residue that was purified by
silica gel column chromatography eluting with ethyl acetate/n-hexane 10 % v/v to afford the title
compound as a yellow solid.
2,2,2-Trifluoro-1-(indolin-1-yl)ethanone 8: 94 % yield; silica gel TLC Rf 0.29 (Ethyl acetate/n-
hexane 10 % v/v); δH (400 MHz, DMSO-d6) 3.29 (2H, t, J 8.0, 3-H2), 4.31 (2H, t, J 8.0, 2-H2), 7.21
(1H, t, J 7.2, Ar-H), 7.25 (1H, t, J 7.2, Ar-H), 7.41 (1H, d, J 7.2, 4-H), 8.1 (1H, d, J 7.2, 7-H); δF
(376 MHz, DMSO-d6) –71.49; m/z (ESI positive) 216.06 [M+H]+.
Spectroscopic data are in agreement with reported data.34
Synthesis of 1-(2,2,2-trifluoroacetyl)indoline-5-sulfonamide (8a).35
4
3
H2NO2S
5
6
2
i)ClSO3Cl, CCl4
ii) NH4OH
N
N
7
CF3
CF3
O
O
8
8a
A 0.1M solution of 2,2,2-trifluoro-1-(indolin-1-yl)ethanone 8 (0.5g, 1.0eq) in CCl4 was added drop-
wise to chlorosulfonic acid (0.81g, 3.0 eq) at 0°C. The brown solution was stirred at r.t. until
starting material was consumed (TLC monitoring), then added to slush and the mixture was
extracted with ethyl acetate (3 x 20 ml). The combined organic layers were washed with H2O (3 x
15ml), brine (2 x 20 ml), dried over Na2SO4, filtered and concentrated in vacuo to give a residue
that was treated with a concentrated ammonium hydroxide aqueous solution (2.0 eq) at 0°C. The
reaction mixture was allowed to warm to room temperature and was stirred overnight. The reaction was
quenched with H2O (30 ml), extracted with ethyl acetate (3 x 20 ml) and the combined organic layers
were washed with H2O (3 x 15ml), dried over Na2SO4, filtered and concentrated in vacuo to give a
residue that was purified by silica gel column chromatography eluting with ethyl acetate/n-hexane
50 % v/v to afford the title compound as a pale yellow solid.

1-(2,2,2-Trifluoroacetyl)indoline-5-sulfonamide 8a: 0.6 % yield; silica gel TLC Rf 0.25 (Ethyl
acetate/n-hexane 50 % v/v); δH (400 MHz, DMSO-d6) 3.32 (2H, t, J 8.0, 3-H2), 4.39 (2H, t, J 8.0, 2-
H2), 7.39 (2H, s, SO2NH2 exchange with D2O), 7.80 (2H, m, 4-H, 6-H), 8.19 (1H, d, J 7.2, 7-H); δF
(376 MHz, DMSO-d6) –71.69; m/z (ESI positive) 295.03 [M+H]+.
Spectroscopic data are in agreement with reported data.35
4.2. CA inhibition assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed
CO2 hydration activity19. 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 7.4) 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
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 the Cheng-Prusoff equation, as reported earlier,21 and represent the
mean from at least three different determinations. All CA isoforms were recombinant proteins
obtained in-house as reported earlier.8,9,12
4.3. X-ray Crystallography
Co-crystallization
In this study, 4-ethoxybenzenesulfonamide (2a), 4-propoxybenzenesulfonamide (2b) and 4-
(allyloxy)benzenesulfonamide (2d) were co-crystallized with CA II. A 600 mM concentration of
compound 2a was prepared and 2 µl of this solution was added to a 500 µl 1.6 M Sodium citrate, 50
mM Tris-HCL pH 7.8 reservoir solution. 1 µl of this reservoir solution was added to 5 µl of CA II at
a final concentration of 10 mg/ml so that the final drug concentration was at 0.24 mM. Hanging-
drops were set up and crystals were seen within 5 days. This was repeated for compound 2b and 2d.

Diffraction Data and Collection
Drug soaked CA II crystals were immersed in 20% (w/v) glycerol (~0.6 M) cryoprotectant for
approximately 5 seconds and immediately flash cooled at 100 K for data collection. Diffraction data
for CA II-2a, CA II-2b and CA II-2d complexes were collected at the F1 station at Cornell High
Energy Synchrotron Source (CHESS F1; λ = 0.9177 A˚) on an ADSC Q-270 detector. Images were
collected every 1° with an exposure time of 5 minutes at a detector distance of 100 mm. The crystal
data were integrated, merged and scaled using HKL2000.36
Structure Determination
Phasing of each complex was carried out in the PHENIX37 suite of programs using the Auto
Molecular Replacement procedure to obtain the initial phases using a previously solved CA II
structure with water molecules removed (PDB ID: 3KS338). The graphics program COOT39 was
used to view the electron density map, and each structure was adjusted based on the calculated
electron density. Topology files of the inhibitors were generated using the PRODRG40 server, and
these files were used to model the drug into the density generated; refinement was then continued
using PHENIX.REFINE until the Rcrys and Rfree were minimized. The geometric restraints of the
final model were analyzed using PROCHECK.41 The data diffraction and final model refinement
statistics are summarized in Table 2.
4.4. Biological data
Animals. CD-1 albino mice (23-25 g) were used in the experiments. The animals were fed with a
standard laboratory diet and tap water ad libitum, and kept at 23 1 °C with a 12 h light/dark cycle,
light on at 7 a.m. Animal handling was carried out according to the European Community
guidelines for animal care (DL 116/92, application of the European Communities Council Directive
86/609/EEC). The ethical policy of the University of Florence conforms with the Guide for the Care
and Use of Laboratory Animals of the U.S. National Institutes of Health (NIH Publication No. 85-
23, revised 1996; University of Florence assurance number: A5278-01). Formal approval to conduct
the experiments described herein was obtained from the Italian Ministry of Health (N°54/2014-B)
and from the Animal Subjects Review Board of the University of Florence. Experiments involving
animals have been reported according to ARRIVE guidelines.42
Oxaliplatin model and pharmacological treatments
Oxaliplatin neuropathy was induced in mice administering 2.4 mg kg-1 oxaliplatin, intraperitoneally
(i.p.) for 5 consecutive days every week for 2 weeks.26 Oxaliplatin was dissolved in 5% glucose

solution. Control animals received an equivalent volume of 5% glucose i.p. (vehicle). Behavioral
tests were performed on day 14. 2a (1 - 50 mg kg-1) and AAZ (10 - 100 mg kg-1) were suspended in
the vehicle (1% carboxymethylcellulose) and per os (p.o.) acutely administered on day 14.
Cold plate test
The animals were placed in a stainless box (12 cm x 20 cm x 10 cm) with a cold plate as floor. The
temperature of the cold plate was kept constant at 4°C 1°C. Pain-related behaviors (i.e. lifting and
licking of the hind paw) were observed and the time (s) of the first sign was recorded. The cut-off
time of the latency of paw lifting or licking was set at 60 s.43
Statistical analysis
Behavioral measurements were performed on 12 mice for each treatment carried out in 2 different
experimental sets. Results were expressed as means s.e.m. and the analysis of variance was
performed by one way ANOVA. A Bonferroni’s significant difference procedure was used as post-
hoc comparison. P values of less than 0.05 or 0.01 were considered significant. Data were analyzed
using the “Origin 9” software (OriginLab, Northampton, USA).
Acknowledgements. This research was financed by two EU grants of the 7th framework program
(Metoxia and Dynano projects to AS and CTS) and the National Institutes of Health grant
CA165284 (to RM).

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