Synthesis of different thio-scaffolds bearing sulfonamide with subnanomolar carbonic anhydrase II and IX inhibitory properties and X-ray investigations for their inhibitory mechanism

Article abstract of DOI:10.1016/j.bioorg.2018.09.028

Several new molecules with different thio-scaffolds were designed, synthesised, and evaluated biologically as inhibitors of Carbonic Anhydrases (CAIs). The structure-activity relationship analysis identified thioether derivatives, here reported, as a pote

Full text of DOI:10.1016/j.bioorg.2018.09.028

Accepted Manuscript
Synthesis of different thio-scaffolds bearing sulfonamide with subnanomolar
carbonic anhydrase II and IX inhibitory properties and X-ray investigations for
their inhibitory mechanism
Andrea Angeli, Damiano Tanini, Antonella Capperucci, Gianni Malevolti,
Francesca Turco, Marta Ferraroni, Claudiu T. Supuran
PII:
S0045-2068(18)30811-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.09.028
YBIOO 2525
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 August 2018
7 September 2018
18 September 2018
Please cite this article as: A. Angeli, D. Tanini, A. Capperucci, G. Malevolti, F. Turco, M. Ferraroni, C.T. Supuran,
Synthesis of different thio-scaffolds bearing sulfonamide with subnanomolar carbonic anhydrase II and IX inhibitory
properties and X-ray investigations for their inhibitory mechanism, Bioorganic Chemistry (2018), doi: https://
doi.org/10.1016/j.bioorg.2018.09.028
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1
Synthesis of different thio-scaffolds bearing sulfonamide with subnanomolar
carbonic anhydrase II and IX inhibitory properties and X-ray investigations for
their inhibitory mechanism
Andrea Angeli,a‡ Damiano Tanini,b‡ Antonella Capperucci,b Gianni Malevolti,b Francesca Turco,b
Marta Ferraroni b and Claudiu T. Supuran*a
a Department of University of Florence, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto
Fiorentino (Florence), Italy
b Department of University of Florence, Department of Chemistry "Ugo Schiff", Via della Lastruccia 13, I-50019 Sesto Fiorentino, Italy.
‡ These authors contributed equally to this work.
Abstract. Several new molecules with different thio-scaffolds were designed, synthesised, and
evaluated biologically as inhibitors of Carbonic Anhydrases (CAIs). The structure–activity relationship
analysis identified thioether derivatives, here reported, as a potent and selective CAIs against hCA II and
hCA IX. High resolution X-ray structure of inhibitor bound hCA II revealed extensive interactions with
the hydrophobic pocket of active site and provided molecular insight into the binding properties of these
new inhibitors
Keywords: carbonic anhydrase; inhibitor, metalloenzymes, sulfur
______
Corresponding author. Tel./fax:+39-055-4573729; e-mail: claudiu.supuran@unifi.it (CTS)

2
1. Introduction
The carbonic anhydrases (CAs, EC 4.2.1.1) belong to a superfamily of metalloenzymes present in all life
kingdoms and encoded by seven evolutionarily unrelated gene families: the α-, β-, γ-, δ-, ζ- ɳ- and the
last recently discover θ- CAs.1 These enzymes are involved in crucial physiological processes connected
with pH and CO2 homoeostasis/ sensing catalysing the hydration of carbon dioxide to produce
bicarbonate and protons.2 So far, in humans, 15 different α-CAs were described and implicated in
several physiological/ pathological processes.3 Carbonic anhydrase inhibitors (CAIs) like sulphonamide
derivatives are in clinical use for the treatment of various diseases such as diuretics4, glaucoma5,
epilepsy6 and more recently as antitumor and antimetastatic agents.7 Owing to the development of new
carbonic anhydrase inhibitors, we hypothesized that the poor explored disulphide, thioether and thioester
scaffolds, could allow for the development of novel inhibitors that are able to interact with the active site
region of carbonic anhydrase. In this study, the synthesis, kinetic analysis, biological evaluation and X-
ray structure of one ligand-protein complex were reported.
2. Results and discussion
2.1. Chemistry
Our group is involved for a long time in the study of synthetic procedures to access novel class of
functionalized organochalcogenides, mainly exploiting the reactivity of a wide range of sulfur-,
selenium- and tellurium-containing nucleophiles with different kind of electrophiles.8-12. Recently, we
became interested in the design and the synthesis of biologically active compounds and we started to
apply such procedures to access new chalcogen-containing small molecules as antioxidants13-15 and CAs
inhibitors.16,17 Very recently, we reported on the synthesis and the evaluation of inhibitory activity for a
series of β-functionalized selenides as CA I, II, IV, VII, and IX Inhibitors.18
In order to synthesize chemically novel sulfur-containing CAs inhibitors and to investigate the effect of
isosteric replacement of selenium with sulfur on such an activity, we explored the reactivity of the
disulfide 2, bearing the benzenesulfonamide moiety, with a plethora of electrophiles.
The disulfide 2 was conveniently obtained by treating disulfonylchloride 1 with an acqueous ammonium
hydroxide solution. Compound 1 was prepared from diphenyl disulfide and chlorosulfonic acid,
following the reported literature procedure (Scheme 1).19

3
Scheme 1: Synthesis of disulfide 2, bearing the benzenesulfonamide moiety
Thus, in order to synthesize unsymmetrical sulfides incorporating the benzenesulfonamide moiety, 2
was reduced with NaBH4, and the thiolate 3 was treated with the suitable electrophile 4 to achieve the
corresponding sulfides 5 in rather good yield, as reported in the Table 1.
Interestingly, product 5e (Table 1, entry 5) was selectively formed from methyl 3-bromopropionate 4e,
no traces of compounds arising from substitution or reduction at the carbonyl carbon being observed.
Table 1: Synthesis of sulfides bearing benzenesulfonamide moiety

4

5
Having explored the reactivity of 3 with alkyl halides, we turned our attention on the ring opening
reaction of strained heterocycles. Thus, monosubstituted epoxides 6a-d were treated with thiolate 3
under the reported conditions affording to β-hydroxysulfides 7a-d (Table 1, entries 6-9), bearing
saturated or unsaturated carbon chains, and protected or free hydroxyl groups. Furthermore, the
limonene derived sulfide 7e was synthesised from the disubstituted epoxide 6e. Finally, with the aim of
enlarging the scope of such a procedure to another class of three-membered heterocycles, the enantio-
enriched N-tosyl aziridine 8, obtained from L-phenylalanine, afforded the β-amino substituted sulfide 9
(Table 1, entry 11). All the ring opening reactions here described were demonstrated to be highly stereo-
and regio-selective, as only the isomer arising from the nucleophilic attack at the less hindered carbon of
the strained heterocycle was observed.8,11
Having obtained a number of sulfides through the reaction of 2 with different electrophiles, we extended
this procedure to the synthesis of thiol esters bearing the benzenesulfonamide moiety. Thus, the thiolate
3 was in situ treated with acyl chlorides (12a-e) to obtain thiol esters 10a-e. The reaction proved to be
efficient, allowing the synthesis of saturated and unsaturated fatty acid derived thiol esters 10a-c,
obtained from caprylic, stearic, and oleic acid, respectively. Furthermore, α,β-unsaturated thiol ester 10d
and aromatic thiol esters 10e were achieved from cinnamoyl chloride and 2,4-
bis(trifluoromethyl)benzoyl chloride as reported in Table 2.
Table 2: Synthesis of thiol esters bearing the benzenesulfonamide moiety

6
Having synthesised a representative series of functionalized sulfides and thiol esters encorporating the
benzenesulfonamide moiety, we also prepared disulfides 11a-c, bearing two primary and two secondary
sulfonamide groups. Compounds 11a-c could be easily achieved by reacting the disulphonylchloride 1
with the suitable amine (13a-c) in the presence of trimethylamine as outlined in Table 3.
Table 3: Synthesis of disulfides 11a-c

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2.2. Carbonic anhydrase inhibition
All compounds 2, 5a-e, 7a-e, 9, 10a-e and 11a-c were tested in vitro for their inhibitory activity against
the physiologically relevant hCA isoforms I, II, IV and IX by means of the stopped-flow carbon dioxide
hydration assay20 and their activities were compared to the standard CAI acetazolamide (AAZ) (Table
4).
Table 4. Inhibition data of human CA isoforms I, II, IV and IX with compounds 2, 5a-e, 7a-e, 9, 10a-e,
11a-c and AAZ by a stopped flow CO2 hydrase assay.20

8
KI (nM)*
hCAII
Cmp
2
hCA I
412.7
773.6
227.5
30.2
hCA IV
368.3
436.5
80.9
hCA IX
0.5
0.9
0.4
0.6
5a
0.4
0.9
5b
29.5
37.2
0.6
838.9
878.0
808.4
683.6
505.6
552.6
709.6
87.7
7.6
5c
135.6
41.7
36.7
6.6
5d
5e
138.1
139.9
74.8
0.8
2.0
7a
4.0
6.8
7b
31.0
3.7
41.7
6.0
7c
50.4
7d
9.8
1.4
0.6
7e
685.2
76.7
197.2
47.0
663.5
879.0
4.9
2045.7
5357.8
9500.0
9647.4
6888.2
4933.2
430.9
752.8
1985.8
74.0
969.0
166.3
4687.0
37.3
268.9
5.2
9
10a
10b
10c
10d
10e
11a
11b
11c
AAZ
513.3
519.3
89.3
32.3
4.9
88.1
53.1
69.0
8.3
5.6
542.5
244.4
250
55.4
139.3
25.8
12.1

9
* 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 drawn from the data obtained and reported in
Table 4:
i)
The cytosolic isoform, hCA I, was inhibited by disulfide 2 with inhibition constant in the
high nanomolar range (Ki 412.7 nM). Thioether scaffolds 5a-e play a crucial role for the
potency of inhibition. Compounds 5c and 5e showed an efficacy over 4 fold than the similar
derivatives. On the other hand, β-hydroxysulfide from natural product limonene 7e results the
most potent inhibitor against this isoform. Like as compounds mentioned above, thioester
tails lead to significant changes in the inhibition potency. Compounds 10b-c with high
hydrophobic moieties showed a significant decrease of the inhibitory potency in high
nanomolar range (Ki 513.3 and 519.3 nM respectively), instead, the efficacy increased when
the moiety was changed with short aliphatic chain (10a and 10e) or cinnamic scaffold (10d).
The potency of inhibition for disulfides 11a-c can be modulate changing the methylene chain
length between primary and secondary sulphonamide. Indeed, compound 11a showed a
potency over 2 times better than compound with an ethylene chain (11c) and over 6 times for
compound with a methylene chain (11b).
ii)
The dominant cytosolic isoform, hCA II, is strong inhibited by disulfide 2 and most thioether
compounds 5a-e in sub-nanomolar range (Ki 0.4 to 0.9 nM). An interesting case is
constituted by thioesther compounds 10a-e, for which the long hydrophobic tails led to a
decrease of potency over 100 fold compared to the other similar products (Ki 663.5 and 879.0
nM for 10b, c than 4.9 nM 10d, e). Unlike the other cytosolic isoform, the introduction of a
methylene linker in compounds 11a-c had diverse effects on the inhibition profile. A
decrease of the potency was observed for a methylene linker with one or without a carbon

10
atom (11b, a Ki of 69.0 nM and 11a a Ki of 53.1 nM), whereas, a further chain elongation, as
in 11c, led to an increase of the potency, with a KI of 8.2 nM.
iii)
The membrane-bound hCA IV was inhibited by most of the compounds investigated here in
the high nanomolar range or in the micromolar range. Thioethers 5a-e and β-hydroxysulfides
7a-e compounds showed an efficacy against this isoform, near ten times better than thioesters
derivatives 10a-e (Ki 0.4 to 37.2 nM for 5a-e and 7a-e than 4.9 to 879.0 nM for 10a-e).
However, the affinity of these inhibitors for CA IV is lower than to CA II. The side chain of
amino acids in the active site pocket of CA IV might be responsible for reduced affinity of
compounds here reported.
iv)
hCA IX, the tumor-associated isoform, was effectively inhibited by many of the compounds
reported here, especially thioether derivatives 5a-b, 7e and disulphide 2 in sub-nanomolar
range (Ki 0.5 to 09 nM). On the other hand, compounds 10a-e showed an over 100 times
decrease in potency against this isoform. Unlike the other isoforms, the introduction of
methylene chain in compounds 11a-c had an almost linear effect on efficacy of inhibition.
Indeed, increasing the linker led to loss of potency near ten fold with one atom of carbon
(11b Ki 55.4 nM) and over 20 fold with two atoms of carbon (11c Ki 139.3 nM).
2.3 Structure of hCA II/ Thioether Ligand Complex.
In order to identify the key interactions involved into protein-inhibitor interactions at the atomic level,
we solved the X-ray complex of hCA II/5b inhibitor at 1.5 Å resolution. Data collection and refinement
of adduct was performed as described in the Experimental Section (see Supporting Information for
statistics). Active site of hCA II showed the classic binding mode of sulfonamide inhibitors, where, the
deprotonated nitrogen atom displaces the hydroxyl ion/water molecule present in the native enzyme and
coordinates the zinc ion with a tetrahedral geometry by His94, His96 and His119. In addition, is present

11
another hydrogen bond interaction with residue Thr199 further contribute to stabilize the binding
(Figure 1).
Figure 1: Ligand 5b in the active site of hCA II (PDB: 6GOT) is shown in green. The zinc ion is the
gray sphere with its protein ligands (His94, 96 and 119 in blue) shown as stick model, in CPK colors.
Residues involved in the binding of inhibitors are also shown. Hydrophobic interactions and H-bonding
are shown as blue dashed lines, π-Stacking as magenta and water bridges as red.
The lipophilic tail of inhibitor 5b is located in the hydrophobic region of the hCA II active site in a small
pocket delimited by residues Phe131, Val135, Leu198, and Pro202 (Figure 1). and forms strong
hydrophobic interactions with these residues. Finally, we also evidenced a π-stacking interaction
between Phe131 and the aromatic ring scaffold of inhibitor.
3. Conclusions

12
Based on the well-known tail approach, we designed a new chemotype with CA inhibitory properties.
Many compounds here reported, showed a strong inhibition against hCA II (a well- established cytosolic
glaucoma target) and hCA IX (a transmembrane isoform believed to be involved in many hypoxic
tumors). However, the selectivity established for some compounds, such as 2, 5a, b, e and 7a, e for
inhibiting selectively hCA II over hCA I and hCA IV, constitutes a very significant finding in light of
isoform-selective CA targeting. Indeed, the X-ray structure of the hCA II complex with compound 5b
showed the effectiveness of building contacts with the lipophilic side of the hCA II active site, which
strongly correlates with the inhibition potency against this isoform.
4. Experimental Part
4.1. General
All reactions were carried out in an oven-dried glassware under inert atmosphere (N2). Ethanol was
dried using a solvent purification system (Pure-Solv™). All commercial materials were used as received
without further purification. Flash column chromatography purifications were performed with Silica gel
60 (230-400 mesh). Thin layer chromatography was performed with TLC plates Silica gel 60 F254. NMR
spectra were recorded in CDCl3 or DMSO-d6 with Mercury 400, and Bruker 400 Ultrashield
1
13
19
spectrometers operating at 400 MHz (for H), 100 MHz (for C) and 376 MHz (for F). NMR signals
1
were referenced to nondeuterated residual solvent signals (7.26 and 2.50 ppm for H, 77.0 and 40.5 ppm
13
1
for C). H NMR data are reported as follows: chemical shift, integration, multiplicity (s = singlet, d =
doublet, t = triplet, ap d = apparent doublet, m = multiplet, dd = doublet of doublet, bs = broad singlet,
bd = broad doublet, ecc.), coupling constant (J), and assignment.
Synthesis of 4,4'-Disulfanediyldibenzenesulfonyl chloride (1):
Disulfide 1 was synthesised following a reported procedure. A solution of 0.1 mol of diphenyldisulfide
in chloroform (100 mL) was added to a solution of 0.3 mol chlorosulfonic acid in chloroform at 10 °C.

13
Than, the mixture was stirred for one hour at 40 °C. After quenching on ice, the phases were separated,
the organic phase washed twice with sodium bicarbonate solution and dried with Na2SO4. The solvent
1
was eliminated and the product obtained as yellow powder with a yield of 74%. H NMR (400 MHz,
CDCl3) δ (ppm): 7.72 (4H, ap d, J = 8.7 Hz), 8.01 (4H, ap d, J = 8.7 Hz). 13C NMR (100 MHz, CDCl3) δ
(ppm): 127.1, 128.5, 143.6, 145.5.
Synthesis of 4,4'-Disulfanediyldibenzenesulfonamide (2):
4,4'-Disulfanediyldibenzenesulfonyl chloride 1 (831 mg, 2.0 mmol) was solubilized in THF (50 mL) and
treated with an excess of NH4OH (37% aq. Solution, 10 eq.). The reaction was stirred at ambient
temperature for 30 min; the solid was then filtered, washed with Et2O (20 mL) and dried under vacuum
1
to afford 4,4'-Disulfanediyldibenzenesulfonamide 2 (707 mg, 94%). H NMR (400 MHz, DMSO-d6) δ
13
(ppm): 7.45 (4H, bs), 7.78 (4H, ap d, J = 8.0 Hz), 7.88 (4H, ap d, J = 8.0 Hz). C NMR (100 MHz,
DMSO-d6) δ (ppm): 127.6, 127.7, 140.5, 144.0.
General Procedure for the preparation of sulfides 5a-e, 7a-e and 9 from disulfide 2:
NaBH4 (23 mg, 0.60 mmol, 3.0 eq.) was portionwise added to a solution of 4,4'-
disulfanediyldibenzenesulfonamide 2 (75 mg, 0.20 mmol, 1.0 eq.) in EtOH (2 mL) at ambient
temperature under inert atmosphere (N2). After 2 h, the suitable electrophile (0.42 mmol, 2.1 eq.) was
slowly added and the reaction mixture was stirred at room temperature for 3 h, until complete
consumption of the starting material was observed by TLC. The reaction was quenched by addition of
saturated aq. NH4Cl (2 mL) and diluted with EtOAc (5 mL), The layers were separated and the aqueous
layer was extracted with EtOAc (2 x 5 mL), dried over Na2SO4, filtered and concentrated under vacuum.
The crude material was purified by flash chromatography to yield sulfides 5, 7 and 9 bearing
benzenesulfonamide moiety.
4-(Pentylthio)benzenesulfonamide (5a):

14
Following the general procedure, disulfide 2 (75 mg, 0.20 mmol) and bromopentane 4a (63 mg, 0.42
1
mmol) gave 5a (76 mg, 74%) as a white solid. H NMR (400 MHz, DMSO-d6) δ (ppm): 0.88 (3H, t, J =
7.2 Hz), 1.26-1.35 (2H, m), 1.37-1.44 (2H, m), 1.59-1.67 (2H, m), 3.06 (2H, ap t, J = 7.3 Hz, CH2S),
7.31 (2H, bs, NH2), 7.46 (2H, ap d, J = 8.7 Hz), 7.73 (2H, ap d, J = 8.7 Hz). 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 14.9, 22.7, 29.0, 31.4, 32.0, 127.3, 127.6, 141.6, 143.2.
4-(Phenethylthio)benzenesulfonamide (5b):
Following the general procedure, disulfide 2 (56 mg, 0.15 mmol) and (2-bromoethyl)benzene 4b (58 mg,
1
0.32 mmol) gave 5b (54 mg, 61%) as a white solid. H NMR (400 MHz, DMSO-d6) δ (ppm): 2.92 (2H,
t, J= 7.6 Hz), 3.34 (2H, t, J = 7.6 Hz), 7.21-7.27 (1H, m), 7.28-7.35 (6H, m), 7.50 (2H, ap d, J = 8.5 Hz),
13
7.74 (2H, ap d, J = 8.5 Hz). C NMR (100 MHz, DMSO-d6) δ (ppm): 33.5, 35.4, 127.3, 127.5, 127.8,
129.4, 129.7, 140.8, 141.8, 142.8.
4-(Prop-2-yn-1-ylthio)benzenesulfonamide (5c):
Following the general procedure, disulfide 2 (75 mg, 0.20 mmol) and propargyl chloride 4c (31 mg, 0.42
1
mmol) gave 5c (75 mg, 83%) as a white solid. H NMR (400 MHz, DMSO-d6) δ (ppm): 3.21 (1H, t, J =
2.6 Hz, C≡CH), . 4.02 (2H, d, J = 2.6 Hz, CH2C≡C), 7.38 (2H, bs, NH2), 7.56 (2H, ap d, J = 8.6 Hz),
13
7.80 (2H, ap d, J = 8.6 Hz). C NMR (100 MHz, DMSO-d6) δ (ppm): 20.6, 75.1, 80.8, 127.2, 128.2,
141.3, 142.3.
4-((2-hydroxyethyl)thio)benzenesulfonamide (5d):
Following the general procedure, disulfide 2 (75 mg, 0.20 mmol) and 2-bromoethanol 4d (53 mg, 0.42
mmol) gave 5d (66 mg, 71%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.11 (2H, t, J =
6.7 Hz, CH2S), 3.55-3.62 (2H, m, CH2O), 4.98 (1H, bs, OH), 7.29 (2H, bs, NH2), 7.45 (2H, ap d, J =
8.6 Hz), 7.69 (2H, ap d, J = 8.6 Hz). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 34.4, 60.0, 126.6, 127.0,
141.0, 142.5. MS (ESI negative) m/z (%): 232 [M-H]-, required [M-H]- 232.02.
Methyl 3-((4-sulfamoylphenyl)thio)propanoate (5e):

15
Following the general procedure, disulfide 2 (56 mg, 0.15 mmol) and 3-bromopropionate 4e (54 mg,
1
0.32 mmol) gave 5e (71 mg, 86%) as a white solid. H NMR (400 MHz, CDCl3) δ (ppm): 2.69 (2H, t, J
= 7.3 Hz), 3.26 (2H, t, J = 7.3 Hz), 3.71 (3H, s), 5.01 (2H, bs, NH2), 7.37 (2H, ap d, J = 8.7 Hz), 7.81
(2H, ap d, J = 8.7 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm): 27.3, 33.6, 52.0, 127.0, 127.6, 138.9,
143.0, 171.8. MS (ESI negative) m/z (%): 298 [M+Na]+; required [M+Na]+ 298.02.
4-((2-Hydroxybutyl)thio)benzenesulfonamide (7a):
Following the general procedure, disulfide 2 (56 mg, 0.15 mmol) and 1,2-epoxybutane 6a (23 mg, 0.32
1
mmol) gave 7a (69 mg, 88%) as a white solid. H NMR (400 MHz, DMSO-d6) δ (ppm): 0.89 (3H, t, J =
7.4Hz), 1.36-1.47 (1H, m), 1.52-1.66 (1H, m), 3.04 (1H, dd, J = 6.7, 13.1 Hz, CHaHbS), 3.10 (1H, dd, J
= 5.4, 13.1 Hz, CHaHbS), 3.53-3.60 (1H, m, CHOH), 4.98 (1H, d, J = 5.3 Hz, CHOH), 7.32 (2H, bs,
NH2), 7.47 (2H, ap d, J = 8.5 Hz), 7.71 (2H, ap d, J = 8.5 Hz). 13C NMR (100 MHz, DMSO-d6) δ
(ppm): 11.0, 30.0, 39.4, 71.1, 127.2, 127.5, 141.4, 143.8. MS (ESI negative) m/z (%): 260 [M-H]-,
required [M-H]- 260.05.
4-((2-Hydroxyhex-5-en-1-yl)thio)benzenesulfonamide (7b):
Following the general procedure, disulfide 2 (75 mg, 0.20 mmol) and 1,2-Epoxy-5-hexene 6b (41 mg,
1
0.42 mmol) gave 7b (108 mg, 94%) as a white solid. H NMR (400 MHz, CDCl3) δ (ppm): 1.65-1.74
(2H, m), 1.92 (1H, bs, OH), 2.14-2.36 (2H, m), 3.02 (1H, dd, J = 8.2, 13.7 Hz, CHaHbS), 3.24 (1H, dd, J
= 3.7, 13.7 Hz, CHaHbS), 3.81-3.87 (1H, m, CHOH), 4.92 (2H, bs, NH2), 5.01-5.11 (2H, m), 5.78-5.90
(1H, m), 7.44 (2H, ap d, J = 8.4 Hz), 7.83 (2H, ap d, J = 8.4 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm):
30.5, 36.0, 40.9, 69.9, 116.0, 127.6, 128.4, 138.4, 139.5, 144.0. MS (ESI negative) m/z (%): 286 [M-H]-,
required [MH]- 286.06.
4-((2,3-Dihydroxypropyl)thio)benzenesulfonamide (7c):
Following the general procedure, disulfide 2 (75 mg, 0.20 mmol) and glycidol 6c (31 mg, 0.42 mmol)
1
gave 7c (80 mg, 76%) as a white solid. H NMR (400 MHz, DMSO-d6) δ (ppm): 2.98 (1H, dd, J = 7.2,

16
13.2 Hz, CHaHbS), 3.23 (1H, dd, J = 4.6, 13.2 Hz, CHaHbS), 3.38 (1H, dd, J = 6.0, 12.9 Hz, CHaHbO),
3.43 (1H, dd, J = 5.3, 10.9 Hz, CHaHbO), 3.62-3.69 (1H, m, CHOH), 4.74 (1H, bt, J = 5.7 Hz, CH2OH),
5.07 (1H, bd, J = 5.2 Hz, CHOH), 7.30 (2H, bs, NH2), 7.47 (2H, ap d, J = 8.6 Hz), 7.71 (2H, ap d, J =
8.6 Hz). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 36.6, 65.6, 71.3, 127.2, 127.5, 141.4, 143.9. MS (ESI
negative) m/z (%): 262 [M-H]-, required [M-H]- 262.03.
(E)-4-((2-Hydroxy-3-(prop-1-en-1-yloxy)propyl)thio) benzenesulfonamide (7d):
Following the general procedure, disulfide 2 (37.5 mg, 0.1 mmol) and allyl glycidyl ether 6d (24 mg,
1
0.21 mmol) gave 7d (54 mg, 89%) as a white solid. H NMR (400 MHz, CDCl3) δ (ppm): 2.79 (1H, bs,
OH), 3.09 (1H, dd, J = 6.7, 13.6 Hz, CHaHbS), 3.17 (1H, dd, J = 5.5, 13.6 Hz, CHaHbS), 3.49 (1H, dd, J
= 5.9, 9.4
Hz, CHaHbO), 3.54 (1H, dd, J = 3.7, 9.4 Hz, CHaHbO), 3.93-3.98 (1H, m, CHOH), 3.98-4.03 (2H, m),
5.17- 5.28 (2H, m, CH=CH2), 4.92 (2H, bs, NH2), 5.82-5.92 (1H, m, CH=CH2), 7.34-7.37 (2H, m), 7.73-
7.75 (2H, m). 13C NMR (100 MHz, CDCl3) δ (ppm): 35.7 (CH2S), 69.0, 72.2, 72.3, 117.7, 126.8, 127.3,
134.1, 138.7, 143.3. MS (ESI negative) m/z (%): 302 [M-H]-, required [M-H]- 302.06.
4-(((5R)-2-Hydroxy-2-methyl-5-(prop-1-en-2-yl)cyclohexyl) thio)benzenesulfonamide (7e):
Following the general procedure, disulfide 2 (37.5 mg, 0.1 mmol) and limonene oxide 6e (32 mg, 0.21
1
mmol) gave 7e (53 mg, 78%) as a white solid. H NMR (400 MHz, CDCl3) δ (ppm): 1.37 (3H, s), 1.58-
1.68 (3H, m), 1.69 (3H, s), 1.73-1.81 (2H, m), 1.84 (1H, bs), 2.16-2.23 (1H, m), 2.28-2.37 (1H, m),
3.48-3.52 (1H, m, CHS), 4.72 (2H, ap d, J = 11.5 Hz), 5.12 (2H, bs, NH2), 7.45 (2H, ap d, J = 8.4 Hz),
13
7.79 (2H, ap d, J = 8.4 Hz). C NMR (100 MHz, CDCl3) δ (ppm): 21.8, 26.7, 29.3, 33.2, 35.7, 39.3,
55.1, 72.9, 110.0, 127.5, 129.7, 139.6, 144.3, 149.2. MS (ESI positive) m/z (%): 364 [M+Na]+, required
[M+Na]+ 364.10.
(S)-4-Methyl-N-(1-phenyl-3-((4-sulfamoylphenyl)thio)propan-2-yl)benzenesulfonamide (9):

17
Following the general procedure, disulfide 2 (37.5 mg, 0.1 mmol) and (S)-2-benzyl-1-tosylaziridine 8
1
(60 mg, 0.21 mmol) gave 9 (60 mg, 62%) as a white solid. H NMR (400 MHz, DMSO-d6) δ (ppm):
2.30 (3H, s), 2.66 (1H, dd, J = 6.7, 13.5 Hz), 2.81 (1H, dd, J = 6.5, 13.5 Hz), 2.96 (1H, dd, J = 5.8, 13.3
Hz), 3.01 (1H,
dd, J = 5.1, 13.3 Hz), 3.28-3.39 (1H, m, CHNH, overlapped with H2O), 7.03-7.04 (2H, m), 7.10 (2H, ap
d, J = 8.5 Hz), 7.15-7.19 (5H, m), 7.32 (2H, bs, NH2), 7.43 (2H, ap d, J = 8.2 Hz), 7.60 (2H, ap d, J =
8.5 Hz), 7.97 (1H, d, J = 7.5 Hz, NH). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 21.0, 35.7, 40.1
(overlapped with DMSO-d6 signal), 54.2 (CHNH), 126.0, 126.3, 126.4, 126.5, 128.3, 129.2, 129.3,
137.4, 137.9, 140.8, 141.0, 142.4. MS (ESI negative) m/z (%): 475 [M-H]-, required [M-H]- 475.09.
General Procedure for the preparation of thiol esters 10a-e from disulfide 2:
NaBH4 (23 mg, 0.60 mmol, 3.0 eq.) was portionwise added to a solution of 4,4'-
disulfanediyldibenzenesulfonamide 2 (75 mg, 0.20 mmol, 1.0 eq.) in EtOH (2 mL) at ambient
temperature under inert atmosphere (N2). After 2 h, the suitable acyl chloride (0.42 mmol, 2.1 eq.) was
slowly added and the reaction mixture was stirred at room temperature for 1 h, until complete
consumption of the starting material was observed by TLC. The reaction was quenched by addition of
saturated aq. NH4Cl (2 mL) and diluted with EtOAc (5 mL). The layers were separated and the aqueous
layer was extracted with EtOAc (2 x 5 mL), dried over Na2SO4, filtered and concentrated under vacuum.
The crude material was purified by flash chromatography to yield thiol esters 10a-e.
S-(4-Sulfamoylphenyl) octanethioate (10a):
According to general procedure, using disulfide 2 (37.5 mg, 0.1 mmol) and capryloyl chloride (34 mg,
1
0.21 mmol) 10a (27 mg, 42%) was achieved as a white solid. H NMR (400 MHz, CDCl3) δ (ppm): 0.89
(3H, t, J = 6.8 Hz), 1.22-1.39 (8H, m), 1.66-1.76 (2H, m), 2.69 (2H, t, J = 7.5 Hz, CH2(CO)), 4.94 (2H,
bs, NH2), 7.56 (2H, ap d, J = 8.5 Hz), 7.94 (2H, ap d, J = 8.5 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm):

18
14.0, 22.6, 25.5, 28.9, 31.6, 44.1, 127.0, 134.0, 134.7, 142.4, 196.0. MS (ESI positive) m/z (%): 338
[M+Na]+, required [M+Na]+ 338.09.
S-(4-Sulfamoylphenyl) octadecanethioate (10b):
According to general procedure, using disulfide 2 (37.5 mg, 0.1 mmol) and stearoyl chloride (64 mg,
0.21 mmol) 10b (49 mg, 54%) was achieved as a white solid. 1H NMR (400 MHz, CDCl3) δ (ppm): 0.88
(3H, t, J = 6.7 Hz), 1.22-1.27 (28H, m), 1.67-1.78 (2H, m), 2.69 (2H, t, J = 7.5 Hz, CH2(CO)), 4.93 (2H,
bs, NH2), 7.56 (2H, ap d, J = 8.4 Hz), 7.94 (2H, ap d, J = 8.4 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm):
14.1, 22.7, 28.9, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 31.9, 44.1, 127.0, 128.7, 134.0, 134.2, 134.7, 142.4,
196.0. MS (ESI positive) m/z (%): 478 [M+Na]+, required [M+Na]+ 478.24.
S-(4-Sulfamoylphenyl) (Z)-octadec-9-enethioate (10c):
According to general procedure, using disulfide 2 (37.5 mg, 0.1 mmol) and oleoyl chloride (63 mg, 0.21
1
mmol) 10c (55 mg, 61%) was achieved as a waxy white solid. H NMR (400 MHz, CDCl3) δ (ppm):
0.88 (3H, t, J = 6.7 Hz), 1.21-1.41 (20H, m), 1.66-1.75 (2H, m), 1.95-2.07 (4H, m), 2.68 (2H, t, J = 7.5
Hz, CH2(CO)), 5.20 (2H, bs, NH2), 5.30-5.40 (2H, m), 7.53 (2H, ap d, J = 8.3 Hz), 7.92 (2H, ap d, J =
8.3 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm): 14.1, 27.1, 27.2, 28.9, 29.0, 29.1, 29.3, 29.5, 29.6, 29.7,
31.9, 44.0, 126.9, 129.6, 130.0, 133.7, 134.7, 142.5, 196.2. MS (ESI positive) m/z (%): 476 [M+Na]+,
required [M+Na]+ 476.23.
S-(4-Sulfamoylphenyl) (E)-3-phenylprop-2-enethioate (10d):
According to general procedure, using disulfide 2 (75 mg, 0.2 mmol) and cinnamoyl chloride (70 mg,
1
0.4 mmol) 10d (74 mg, 58%) was achieved as a white solid. H NMR (400 MHz, DMSO-d6) δ (ppm):
7.18 (1H, d, J = 15.8), 7.44-7.49 (3H, m), 7.51 (2H, bs), 7.70 (2H, ap d, J = 8.3 Hz), 7.72 (1H, d, J =
13
15.8), 7.81-7.83 (2H, m), 7.91 (2H, ap d, J = 8.3 Hz). C NMR (100 MHz, DMSO-d6) δ (ppm): 124.1,
126.3, 129.0, 131.2, 131.6, 133.6, 134.8, 142.2, 144.9, 186.2. MS (ESI negative) m/z (%): 318 [M-H]-,
required [M-H]- 318.03.

19
S-(4-Sulfamoylphenyl) 2,4-bis(trifluoromethyl)benzothioate (10e):
According to general procedure, using disulfide (37.5 mg, 0.1 mmol) and 2,4-
2
Bis(trifluoromethyl)benzoyl chloride (58 mg, 0.21 mmol), 10e (40 mg, 46%) was achieved as white
1
solid. H NMR (400 MHz, DMSO-d6) δ (ppm): 7.56 (2H, bs NH2), 7.80 (2H, ap d, J = 8.4 Hz), 7.99
13
(2H, ap d, J = 8.4 Hz), 8.26 (1H, ap d, J = 8.1 Hz), 8.34 (1H, ap d, J = 8.1 Hz), 8.31 (1H, s). C NMR
1
(100 MHz, DMSO-d6) δ (ppm): 123.0 (d, 1JC-F = 273 Hz, CF3), 125.2 (d, JC-F = 271 Hz, CF3), 125.6
2
2
(bs), 127.6 (q, JC-F = 22 Hz, CCF3), 127.8, 131.1, 131.2, 131.6 (bs), 133.4 (q, JC-F = 33 Hz, CCF3),
19
136.1, 140.7, 146.8, 190.3. F NMR (376 MHz, DMSO-d6) δ (ppm): -61.7, -57.5. MS (ESI negative)
m/z (%): 428 [M-H]-, required [M-H]- 427.99.
Synthesis of 4,4'-disulfanediylbis(N-(4-sulfamoylphenyl) benzenesulfonamide) (11a)
4,4'-Disulfanediyldibenzenesulfonyl chloride 1 (83 mg, 0.2 mmol) was solubilized in THF (5 mL) and
treated with sulfanilamide (69 mg, 0.4 mmol). The reaction was stirred at ambient temperature for 30
min; the solid was then filtered, washed with Et2O (5 mL) and dried under vacuum to afford 11a (49 mg,
36%). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.40 (4H, bs, NH2), 7.53-7.57 (10H, m), 7.85- 7.95 (8H,
m). 13C NMR (100 MHz, DMSO-d6) δ (ppm):123.7, 127.1, 128.1, 128.5, 130.4, 136.7, 140.3, 143.3.
Synthesis of 4,4'-Disulfanediylbis(N-(4-sulfamoylbenzyl) benzenesulfonamide) (11b):
4,4'-Disulfanediyldibenzenesulfonyl chloride 1 (83 mg, 0.2 mmol) was solubilized in THF (5 mL) and
treated with 4-aminomethylbenzenesulfonamide hydrochloride (89 mg, 0.4 mmol). The reaction was
stirred at ambient temperature for 30 min; the solid was then filtered, washed with Et2O (5 mL) and
1
dried under vacuum to afford 11b (75 mg, 53%). H NMR (400 MHz, DMSO-d6) δ (ppm): 4.07 (4H, d,
J = 6.2 Hz, CH2N), 7.31 (4H, bs, NH2), 7.43 (4H, d, J = 8.3 Hz), 7.75 (4H, d, J = 8.3 Hz), 7.75 (4H, d, J
13
= 8.6 Hz), 7.83 (4H, d, J = 8.6 Hz), 8.31 (2H, t, J = 6.2 Hz, NH). C NMR (100 MHz, DMSO-d6) δ
(ppm): 46.6, 126.7, 127.8, 128.7, 128.9, 140.6, 141.6, 142.9, 144.1.
Synthesis of 4,4'-Disulfanediylbis(N-(4-sulfamoylphenethyl) benzenesulfonamide) (11c):

20
4,4'-Disulfanediyldibenzenesulfonyl chloride 1 (42 mg, 0.1 mmol) was solubilized in THF (3 mL) and
treated with 4-(2-aminoethyl)benzenesulfonamide (40 mg, 0.2 mmol). The reaction was stirred at
ambient temperature for 30 min; the solid was then filtered, washed with Et2O (5 mL) and dried under
1
vacuum to afford 11c (24 mg, 32%). H NMR (400 MHz, DMSO-d6) δ (ppm): 2.76 (4H, t, J = 7.1 Hz),
2.97-3.02 (4H, m), 7.31 (4H, bs), 7.35 (4H, ap d, J = 8.3 Hz), 7.69-7.69 (12H, m), 7.80 (2H, bt, J = 5.6
Hz). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 36.0, 44.6, 126.7, 127.8, 128.7, 130.3, 140.3, 141.5,
143.3, 143.9.
4.2 Carbonic anhydrase inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2
hydration activity.20 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 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,21-26 and represent the mean from at least three different determinations. All
CA isofoms were recombinant ones obtained in-house as reported earlier.21-26

21
4.3 Crystallization and X-ray data collection
Crystals of hCAII were obtained using the hanging drop vapor diffusion method using 24 well Linbro
plate. 2 µl of 10 mg/ml solution of hCA II in Tris-HCl 20 mM pH 8.0 were mixed with 2 µl of a solution
of 1.5 M sodium citrate, 0.1 M Tris pH 8.0 and were equilibrated against the same solution at 296 K.
Crystals of the protein grew in one week. Afterwards hCAII crystals were soaked in 5mM inhibitor
solution for 3 days.
The crystals were flash-frozen at 100K using a solution obtained by adding 15% (v/v) glycerol to the
mother liquor solution as cryoprotectant. Data on crystals of the complexes were collected using
synchrotron radiation at the ID29 beamline at ESRF (Grenoble, France) with a wavelength of 0.827 Å
and a PILATUS 6M Dectris CCD detector. Data were integrated and scaled using the program XDS.27
Data processing statistics are shown in supporting information.
4.4 Structure determination
The crystal structure of hCA II (PDB accession code: 4FIK) without solvent molecules and other
heteroatoms was used to obtain initial phases of the structures using Refmac5.28 5% of the unique
reflections were selected randomly and excluded from the refinement data set for the purpose of Rfree
calculations. The initial |Fo - Fc| difference electron density maps unambiguously showed the inhibitor
molecules. The inhibitor was introduced in the model with 0.5 occupancy. In the |Fo - Fc| difference
electron density map a spheric density was present close to the benezensulphonamide moiety and was
interpreted as a solvent molecule at 0.5 occupancy.
Atomic models for inhibitors were calculated and energy minimized using the program JLigand 1.0.40.29
Refinements proceeded using normal protocols of positional, isotropic atomic displacement parameters
alternating with manual building of the models using COOT.30 Solvent molecules were introduced
automatically using the program ARP.31 The quality of the final models are assessed with COOT and

22
RAMPAGE.32 Crystal parameters and refinement data are summarized in Electronic Supplementary
Information (†ESI). Atomic coordinates were deposited in the Protein Data Bank (PDB accession code:
6GOT). Graphical representations were generated with Chimera.33
Acknowledgements
We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation
facilities and we would like to thank Guillaume Gotthard for assistance in using beamline ID29.
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25
Highlights
1. Synthesis of novel different thio-scaffolds bearing sulfonamide as inhibitors of carbonic
anhydrase are reported
2. In vitro studies reported potent and selective inhibition activity against hCA II and IX
isoforms.
3. Determined X-ray structure of hCA II in complex with one compound in order to obtain
ligand−protein interaction.

S
S
R/Ar
i) NaBH4
2
O
O
H2NO
2S
H2NO
2S
ii)
2
Cl
R/Ar
10a-e
S
S
i) NaBH4
E
2
Electrophile (E)
ii)
4,6,8
H2NO
2S
5a-e,7a-e,9
H2NO
2S
2