Discovery of new 2, 5-disubstituted 1,3-selenazoles as selective human carbonic anhydrase IX inhibitors with potent anti-tumor activity

Article abstract of DOI:10.1016/j.ejmech.2018.08.096

A series of disubstituted selenazole derivatives was synthetized and evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against the human (h) isoforms hCA I, II, IV, VA, VB and IX, involved in a variety of diseases including glaucoma, retinitis pigmentosa, epilepsy, arthritis and tumors. The investigated compounds showed potent inhibition against the tumor-associated transmembrane hCA IX, with K_Is in the subnanomolar – low nanomolar range, and were evaluated for their effects on cell viability against the human prostate (PC3) and breast (MDA-MB-231) cancer cell lines, showing effective anti-tumor activity. These selenazoles are interesting leads for the development of new, isoform-selective CA IX inhibitors.

Full text of DOI:10.1016/j.ejmech.2018.08.096

Accepted Manuscript
Discovery of new 2, 5-disubstituted 1,3-selenazoles as selective human carbonic
anhydrase IX inhibitors with potent anti-tumor activity
Andrea Angeli, Elena Trallori, Marta Ferraroni, Lorenzo Di Cesare Mannelli, Carla
Ghelardini, Claudiu T. Supuran
PII:
S0223-5234(18)30768-2
10.1016/j.ejmech.2018.08.096
EJMECH 10707
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 August 2018
Revised Date: 28 August 2018
Accepted Date: 31 August 2018
Please cite this article as: A. Angeli, E. Trallori, M. Ferraroni, L. Di Cesare Mannelli, C. Ghelardini, C.T.
Supuran, Discovery of new 2, 5-disubstituted 1,3-selenazoles as selective human carbonic anhydrase IX
inhibitors with potent anti-tumor activity, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.08.096.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of new 2, 5-disubstituted 1,3-selenazoles as selective human carbonic
anhydrase IX inhibitors with potent anti-tumor activity
Andrea Angeli,a Elena Trallori,b Marta Ferraroni,c Lorenzo Di Cesare Mannelli,b Carla
Ghelardini,b Claudiu T. Supurana*
a
Department of University of Florence, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto
Fiorentino (Florence), Italy
b
NEUROFARBA Department, Section of Pharmacology and Toxicology, Università degli Studi di Firenze, Viale Pieraccini 6, 50139
Florence, Italy
c Department of University of Florence, Department of Chemistry "Ugo Schiff", Via della Lastruccia 13, I-50019 Sesto Fiorentino, Italy.
Abstract. A series of disubstituted selenazole derivatives was synthetized and evaluated as carbonic
anhydrase (CA, EC 4.2.1.1) inhibitors against the human (h) isoforms hCA I, II, IV, VA, VB and IX,
involved in a variety of diseases including glaucoma, retinitis pigmentosa, epilepsy, arthritis and tumors.
The investigated compounds showed potent inhibition against the tumor-associated transmembrane hCA
IX, with KIs in the subnanomolar – low nanomolar range, and were evaluated for their effects on cell
viability against the human prostate (PC3) and breast (MDA-MB-231) cancer cell lines, showing
effective anti-tumor activity. These selenazoles are interesting leads for the development of new,
isoform-selective CA IX inhibitors.
Keywords: carbonic anhydrase; inhibitor, metalloenzymes, selenazole, selenium, tumors.
______
Corresponding author. Tel./fax:+39-055-4573729; e-mail: claudiu.supuran@unifi.it (CTS).

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1.Introduction
Selenium has a long history of association with human health and disease.[1, 2] Its toxicity is an old and
debated issue that for a long time hampered the idea that Se-containing compounds may have a potential
as therapeutic agents.[3] Today, the biochemistry and pharmacology of organoselenium compounds are
topics of intense research in medicinal chemistry, because it has been demonstrated that such compounds
may be used as antioxidants, antitumor, antiviral, antimicrobial, or antihypertensive agents as well as
enzyme modulators targeting nitric oxide synthase (NOS),[4] carbonic anhydrase (CA, EC 4.2.2.1)[5]
and lipoxygenase (LOX) among others [6]. Some of these enzymes are involved in serious diseases, thus
leading to possible applications as therapeutic agents of such derivatives.[7-9] Selenium compounds
have proved to be very potent anticarcinogenic agents in different models, such as spontaneous,
chemically induced, or transplanted tumours, as well as ex vivo cultures.[10-12] The unique redox
properties of selenium alter the proliferative potential of epithelial cancers by inducing apoptosis [13],
but selenium may also be considered as a double-edged sword in terms of possessing antioxidant as well
as pro-oxidant properties that may be beneficial or harmful, depending upon the form and dose being
used in a normal or cancerous tissue. Thus, the concept of the U-shape was introduced to reflect the
“good” and the “bad” effects of selenium as a function of dose.[14] Despite very promising research, to
date, no synthetic organoselenium compounds are in clinical use as anti-cancer agents. The applicability
of selenoheterocyclic compounds in tumor control has been demonstrated in compounds incorporating a
five-membered ring system.[15,16]
Many tumour types are characterized by an upregulated glucose metabolism, low levels of oxygen
(hypoxia) and a dysregulated acid base balance, with the extracellular pH more acidic than the normal
values.[17-19] In this particular contest, the metalloenzyme human (h) carbonic anhydrase (CA, EC
4.2.1.1) hCA IX is overexpressed in a wide range of hypoxic tumours as downstream target of the
transcription factor hypoxia-inducible factor-1α (HIF-1α) activation.[20,21] hCA IX participates in the
survival, proliferation, and metastasis processes of such hypoxic tumors. Indeed, hCA IX targeting has
received a lot of interest, and several efforts are being done towards developing novel inhibitors.[22]
Among the huge number of sulfonamide, sulfamate, sulfamide and coumarin hCA IX inhibitors reported
to date,[23-25] few compounds were investigated in detail in animal tumour models, and only SLC-
0111, an ureido-substituted benzenesulfonamide derivative, successfully ended Phase I clinical trials, in
2016, for the treatment of patients with advanced hypoxic tumors overexpressing the hCA IX and the
compound is currently scheduled to enter Phase II trials this year.[26]

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2. Results and discussion
2.1. Chemistry
Given our interest in the study of chalcogen-containing compounds, we have designed and synthetized a
series of selenazole derivatives with the aim to identify novel hCA IX selective inhibitors.[1,3]
Selenazoles are an important class of heterocycles with significant biological effects and considerable
pharmacological relevance.[15,16] Moreover, these five membered selenium heterocycles are easily
synthetized from primary selenoamide as starting materials. Thus, our attention focused on the synthesis
of primary selenoamide containing sulfonamide moiety, as shown in Scheme 1. First step was the
synthesis of 4-cyanobenzenesulfonamide (2) by reaction of the corresponding sulfonyl chloride (1) with
an aqueous solution of ammonium hydroxide. Successively, 4-sulfamoylbenzoselenoamide (3) was
prepared by the reaction of nitrile compound 2 with Na2Se as selenating reagent in reflux ethanol, for 6h.
SO2Cl
SO2NH2
SO2NH2
NH4OH
Na2Se
Et3N
THF
EtOH
reflux
CN
CN
H2N
Se
1
2
3
O
R
O
R
Cl
Br
EtOH
reflux
EtOH
reflux
4a-f
5a-c
SO2NH2
SO2NH2
R
Se
Se
R
N
N
6a-f
7a-c
6a
6d
7a
: R= H
: R= 4-Cl
: R= CH3
7b: R= COOH
7c
6b: R= 4-F 6e: R= 4-NO2
6c 6f
: R= 4-Br : R= 3-OCH3
: R= CH2Cl

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Scheme 1: Synthesis of primary selenoamide incorporating a sulfonamide moiety 3 and the
corresponding functionalized selenazoles 4a-f and 5a-c. Na2Se was generated in situ from elemental Se
(0.5 eq.) and NaBH4 (1.0 eq.).
Finally, the reaction of primary selenobenzamide (3) with different α-haloketones incorporating
aromatic 4a-f or aliphatic 5a-c moieties, in refluxing ethanol, gave various 2, 5-disubstituted 1, 3-
selenazoles 6a-f and 7a-c. In addition, herein, we report the synthesis of a variety of double-
functionalized and ionic 1, 3-selenazoles, by nucleophilic displacement reactions of 4-halomethyl- 1,3-
selenazoles 7c as shown in Scheme 2.
Scheme 2: Synthesis of substituted 2,5-selenazoles 10-13.

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Treatment of 7c with thiophenol and Et3N in acetonitrile afforded in excellent yields the corresponding
functionalized 1,3-selenazoles 10. A number of 1,3-selenazoles containing aromatic chalcogenide side
chains were also prepared (11a-c and 12a-c). The reaction started with the reduction of the appropriate
dichalcogenide and NaBH4 to afford the corresponding chalcogenate (8a-c and 9a-c), which was treated
in situ with 4-halomethyl- 1,3-selenazoles 7c leading to the corresponding functionalized 1,3-
selenazoles 11a-c and 12a-c in good yield (Scheme 2). Finally, 1, 3-selenazole containing the
pharmacologically relevant isothiouronium moiety (13) was prepared from 7c and thiourea.
2.2. Carbonic anhydrase inhibition
All compounds 3, 6a-f, 7a-c, 10, 11a-c, 12a-b and 13 were tested in vitro for their inhibitory activity
against the physiologically relevant hCA isoforms I, II, IV, VA, VB and IX by means of the stopped-
flow carbon dioxide hydration assay[27] and their activities were compared to the standard CAI
acetazolamide (AAZ) (Table 1).
Table 1. Inhibition data of human CA isoforms I, II, IV, VA, VB and IX with compounds 3, 6a-f, 7a-c,
10, 11a-c, 12a-c, 13 and AAZ by a stopped flow CO2 hydrase assay.[27]
KI (nM)
Cmp
hCA I
hCA II
hCA IV
hCA VA
hCA VB
hCA IX
3
44.2
33.5
637.9
84.8
29.6
85.8
941.1
652.3
6353.0
63.4
9.7
552.7
703.1
724.2
7.6
6.5
7.5
6a
6b
75.3

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6c
206.0
414.1
539.7
42.2
15.8
41.6
7.3
88.6
67.9
245.1
70.9
47.3
77.0
73.2
74.6
79.7
186.7
9.5
3098.0
873.1
9386.0
359.5
384.7
392.4
326.2
6860.0
5155.0
2695.0
853.9
751.0
203.9
120.8
216.5
74
86.6
82.2
85.7
82.5
82.3
42.5
58.4
55.8
56.3
56.8
40.0
68.3
75.6
47.1
57.3
63.0
620.4
730.0
757.3
808.7
600.0
460.0
90.9
0.87
8.4
6d
6e
8.2
6f
56.3
6.9
7a
7b
9.0
7c
2.8
10
935.0
135.2
8.2
550.0
81.0
5.4
11a
11b
11c
12a
12b
12c
13
7.2
809.5
85.0
2.6
47.7
289.1
30.3
7.1
0.89
9.1
9.8
517.0
54.1
9.6
7.1
7.7
319.1
700.0
54.0
0.55
7.0
14.6
250.0
80.4
12.1
AAZ
25.8
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5-10 % of the
reported values).
We have investigated a range of various kinds of 2, 5-disubstituted 1, 3-selenazole derivatives for their
interaction with the six hCAs here considered, after a period of 15 min of incubation of the enzyme and
inhibitor solutions.[28–31] The following structure activity relationship (SAR) may be noted regarding
the inhibition data of Table 1:

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i)
The cytosolic hCA I was inhibited by primary selenoamide 3 in the medium nanomolar range
(Ki 44.2 nM). On the other hand, the inhibition profile of selenazole derivatives varied
according to the substituent in position 5. The benzene moiety (6a) led to a slight increase in
the activity (Ki 33.5 nM) and substitutions in positions 3 or 4 led to a decrease of the
potency. An interesting inhibition profile was observed for compounds 6b-d incorporating
halogens. The dimension of this substituent proved to be crucial for the inhibition potency.
Indeed, replacement of fluorine (6b) by bromine (6c) caused an increase of activity for hCA I
(Ki 637.9 nM vs. Ki 206.0 nM). Moreover, substitution in position 3 (6f) led to a better
activity than the corresponding compounds with para groups (6b-e). Aliphatic moieties in
position 5 of selenazoles (7a-c) increased the efficacy compared to aromatic compounds (6a-
f). 7c showed an inhibition constant in the low nanomolar range (Ki 7.3 nM). Another
interesting point was the further substitution of the side chain in compound 7c with
chalcogen aromatic moiety. Going down along the chalcogen group of the periodic table
(from sulphur 10 to tellurium 11a) the activity increased (Ki 935.0 nM to Ki 135.2 nM) as the
chalcogen group element increased from Sulphur (10) to tellurium (11a). The second
dominant cytosolic isoform, hCA II, was inhibited by all these compounds in the medium
nanomolar range except for compounds 11c and 12a-c which showed low nanomolar
inhibition (Ki 7.7 to 9.8 nM). The different moieties in position 5 of the selenazole scaffold
did not influence significantly the inhibition constant. On the other hand, the further
functionalization of 4-halomethyl-1,3-selenazoles 7c with different selenites 12a-c and
tellurate 11c moieties led to an increase of potency near ten folds compared to the other
compounds here considered.
ii)
Almost all compounds investigated here, possessed low inhibitory activity for the membrane-
bound isoform hCA IV with ranges spanning between the high nanomolar to the micromolar

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(Ki 120.8 to 9386 nM). Aliphatic substituents in position 5 proved to be better than aromatic
ones, with an inhibition constants spanning from Ki 326.2 to 392.4 nM. Indeed, as for the
previously discussed isoform hCA II mentioned above, the functionalization of 4-
halomethyl-1,3-selenazoles 7c with selenolate (12a-c) and tellurate 11c moieties increased
the efficacy.
iii)
An interesting inhibition pattern was observed for the mitochondrial hCA VA and hCA VB
isoforms. All compounds here considered, except 12a, exhibited a preference of inhibition
for the isoform hCA VA over hCA VB and, selenazole 6a showed this preference with an
activity 70 folds more potent for hCA VA (Ki 9.7 nM) compared to hCA VB (Ki 703.1 nM).
On the other hand, the different moieties in position 5 of the selenazole scaffold did not
influence significantly the inhibition potency.
iv)
The membrane-bound, tumor-associated, hCA IX, is effectively inhibited by selenazoles in
low nanomolar to sub-nanomolar range (Ki 0.55 nM to 9 nM) except for compound 6f (Ki
56.3 nM). An interesting case was constituted by the isosteric substitution of the halogen
atom in the aromatic moiety of 6c-d and 6f. The transition from fluorine atom (6f) to
bromine (6b) led to a significant increase of the inhibitory potency (Ki 56.3 to 0.87 nM). The
activity was influenced also for the other chalcogen moieties (11a-c and 12a-c), especially
for methoxy substituent, which led to a subnanomolar inhibition (Ki 0.89 and 0.55 nM).
2.3 X-ray Crystallographic Results
X-ray crystallography (statistics summarized in ESI) was used to analyse the mode of binding of
compound 7c in complex with hCA II. Structural analysis of the initial Fo−Fc electron density map of the
active site showed well defined electron density, fully compatible with the presence of inhibitor 7c but,
electron density for chlorine atom was not present (Figure 1A). The sulfonamide moiety coordinates the

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catalytic zinc ion of hCA II with a tetrahedral geometry, by means of one nitrogen atom of the
sulfonamide group and displacing the zinc bound water molecule/hydroxide ion similarly to other
sulfonamide and sulfamide derivatives investigated earlier.[32,33]
Figure 1. Active site region of hCA II/7c adduct (PDB 6H3Q). Inhibitor showing the σA-weighted |Fo−
Fc| map (at 2.5 σ) (A). Hydrogen bonds, van der Waals interactions and Water Bridges are shown and
labelled in green, blue and red respectively (B).
The deprotonated nitrogen atom of the sulfonamide moiety forms a hydrogen bond with the NH moiety
of Thr199. Furthermore, a hydrogen bond is involved with a water molecule showing a well defined
solvent network that contributes to stabilize the inhibitor within the active site (Figure 1B). The phenyl
ring of inhibitor 7c is located in the active site channel, where it is involved in a number of hydrophobic
interaction with the side chains of residues Val121 and Leu198. The medium potency of inhibition
against hCA II (Ki 73 nM) could be explained with the rigid structure conferred by two consecutive
aromatic rings; this scaffold prevented the inhibitor from locating its tail in a small hydrophobic pocket
delimited by residues Phe131 and which strongly correlates with the inhibition potency against this

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isoform. On the other hand, nitrogen of selenazole moiety is involved in a water bridge with residue
Gln92 (Figure 1B).
2.4 Biological assays
We focused our attention on the ex vivo activity of compounds 6e, 11c and 12c, which were evaluated
for their effects on cell viability against the human prostate (PC3) and breast (MDA-MB-231) cancer
cell lines. All compounds were low/sub nanomolar hCA IX inhibitors, and were used at different
concentrations, being incubated for 48 h in both normoxic and hypoxic conditions, when overexpression
of high amounts of CA IX occurs.[17] In PC3 cells, selenazole derivative 6e, with a 4-NO2 phenyl
moiety, reduced the cell viability to 60% at 1µM. Its efficacy increased significantly at 10 µM reducing
the viability to 10% (Figure 2). In the hypoxic conditions, compound 6e showed significantly more
effects on cytotoxicity which reached 32% at 1µM. At higher concentration the compound was
comparable to its effects in normoxic condition. Chalcogenide atom in the side chain of compounds 11c
and 12c proved to play a crucial role for cytotoxicity. Compound 11c, whit tellurium atom in the side
chain, showed a strong activity against this cancer cell line in normoxic condition with a viability of
23% at 1 µM. On the other hand, in hypoxic condition compound 11c showed an efficacy two times
lower than in the normoxia. Finally, compound 12c, with selenium in the side chain, proved to be
inactive at lower doses against PC3 cell line, but the efficacy increased at 30 and 100 µM showing a cell
viability in normoxic condition of 59% and 11% respectively. In hypoxic condition compound 12c
exhibited more cytotoxic effects at low concentration (77% at 10 µM) whereas at higher doses exhibited
an efficacy comparable to that in normoxic condition.

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Figure 2. Effects of newly synthetized compounds 6e, 12c and 11c on viability of the human prostatic
cancer cell line PC3 following 48h treatment in normoxic and hypoxic (1% O2) conditions. ***p<0.001
versus control.
In the MDA-MB231 cell line, derivative 6e at the lower concentration showed a weak activity with
viability of 73%. On the other hand, at 10 µM the potency against this cancer cell line increased
drastically killing over 90% of the cells. Also for MDA-MB231 cell line the different chalcogenide in
the side chain of compound 11c and 12c played an important role on the viability (Figure 3). Indeed,
tellurium atom in derivative 11c exhibited a strong cytotoxicity in normoxic conditions, already at lower
concentration (16% at 1 µM). Also this time, the potency decreased over two time in hypoxic condition.
Compound 12c did not show any activity in this ex vivo normoxia assay at 1 and 10 µM concentration.
A reduced cell viability was observed for this compound only at high concentration (30 µM and 100
µM). In the hypoxic conditions the selenium derivative 12c did not show any significant activity, only at
higher concentration of 100 µM the cell viability arrived at 46%.

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Figure 3. Effects of the newly synthetized compounds 6e, 12c and 11c on viability of the human
adenocarcinoma breast cell line MDA-MB231 following 48h treatment in normoxic and hypoxic (1%
O2) conditions. *p<0.05, ** p<0.01, ***p<0.001 versus control.
3. Conclusions
We report here a novel series of selenazole derivatives as inhibitors on six α-carbonic anhydrases (CAs,
EC 4.2.1.1) of pharmacologic relevance i.e., hCA I, II, IV, VA, VB and IX. Most of the new derivatives
were medium potency inhibitors of the ubiquitous cytosolic hCA I and hCA II isoforms, but they
showed significant inhibition potency against the tumor-associated transmembrane hCA IX isoform,
with inhibition constants spanning from the sub-nanomolar to the low nanomolar range. These
compounds exhibited potent effects on cell viability against the human prostate (PC3) and breast (MDA-
MB-231) cancer cells lines, possessing thus effective anti-tumor activity. Moreover, we studied the
complex of one such selenazole with hCA II by X-ray crystallography, for better understanding the
mechanism of binding and the inhibition potency. Thus, this study clearly opens new perspectives in the
field of CA-dependent diseases and, the interesting leads detected here may be useful for the
development of more potent and hCA IX isoform-selective inhibitors.

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4. Experimental Part
4.1. General
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-
13
NMR, C-NMR, 19F-NMR, 77Se-NMR, 125Te-NMR) spectra were recorded using a Bruker Advance III
77
400 MHz spectrometer in DMSO-d6. (PhSe)2 and (PhTe)2were used as an external references for Se
NMR (δ = 461 ppm) and 125Te NMR (δ = 420 ppm). 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; q, quadruplet; 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 ethyl acetate/n-hexane were used as eluents. Melting points (mp) were measured in
open capillary tubes with a Gallenkamp MPD350.BM3.5 apparatus and are uncorrected. 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 and processed by Varian
Workstation Vers. 6.8 software.

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4.1.1 Procedure for the synthesis of 4-cyanobenzenesulfonamide (2).
4-cyanobenzenesulfonyl chloride 1 (10 mmol) was added to a solution of anhydrous THF (40 mL). An
aqueous solution of ammonium hydroxide (30%) (3mL) was added to the mixture at 0°C and stirred at
room temperature for 1 h. The mixture was extracted with ethyl acetate, dried with anhydrous Na2SO4,
and triturated with diethyl ether (1.6 g, 88%). 1H NMR (400 MHz, DMSO- d6) δ(ppm): 8.11 (2H, d,
13
J=8.58 Hz), 8.02 (2H, d, J=8.62 Hz), 7.69 (2H, bs, NH2, exchange with D2O). C NMR (100 MHz,
DMSO- d6) δ(ppm): 148.9, 134.2, 127.4, 118.8, 115.3.
4.1.2 Procedure for the synthesis of 4-sulfamoylbenzoselenoamide (3).
NaBH4 (626 mg, 16.47 mmol, 3.0 eq.) was portion wise added to a solution of elemental selenium
(650mg, 8.24 mmol, 1.5 eq.) in EtOH (20 mL) at 0°C under inert atmosphere (N2). After 30 min, 4-
cyanobenzenesulfonamide 2 (1 g, 5.49 mmol, 1.0 eq.) was added and the reaction mixture was stirred at
reflux for 6h. When the starting 4-cyanobenzenesulfonamide 2 had completely reacted (monitored by
TLC), cooling to room temperature and hydrochloric acid (6N, 2 ml) was added and the solution was
stirred for about an hour. The organic phase was extracted with EtOAc, washed with brine (1 x 5 mL),
dried over Na2SO4, filtered and concentrated under vacuum. The crude material was purified by flash
chromatography (1:1 hexane/ethyl acetate) to yield 4-sulfamoylbenzoselenoamide (3) as orange solid
(1.1 g, 76%). 1H NMR (400 MHz, DMSO- d6) δ(ppm): 11.05 (1H, bs, NH, exchange with D2O), 10.42 (1H,
bs, NH, exchange with D2O), 7.99 (2H, d, J=8.65 Hz), 7.85 (2H, d, J=8.65 Hz), 7.51 (2H, bs, NH2,
13
77
exchange with D2O). C NMR (100 MHz, DMSO- d6) δ(ppm): 203.7, 146.6, 146.3, 128.5, 126.1. Se
NMR (76 MHz, DMSO-d6) δ (ppm): 627.5. MS (ESI negative) m/z (%): 263.0 [M-H]-.
4.1.3 General procedure for the synthesis of 2, 5 substitutes 1, 3-selenazoles (6a-f and 7a-c).

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An EtOH solution (20 mL) of 4-sulfamoylbenzoselenoamide (3) (100 mg, 0.38 mmol) and appropriate
ω-halo derivatives 4a-f or 5a-c (0.38 mmol, 1 Eq.) was stirred at reflux for 20 min. After cooling, the
mixture was poured into H2O (20 mL), which resulted in the formation of a precipitate. This was filtered
off and recrystallized (EtOH) to give the corresponded 2,5 subtitutes 1, 3-selenazoles (6a-f and 7a-c).
4.1.4 4-(5-phenyl-1,3-selenazol-2-yl)benzenesulfonamide (6a)
Following the general procedure, 4-sulfamoylbenzoselenoamide (3) (100 mg, 0.38 mmol) and 2-
1
Bromoacetophenone 4a (76 mg, 0.38 mmol) gave 6a as white solid (115 mg, 83%). H NMR (400
MHz, DMSO- d6) δ (ppm): 8.83 (1H, s), 8.25 (2H, d, J = 8.62 Hz), 8.11 (2H, dd, J = 8.31, 1.21 Hz),
7.99 (2H, d, J = 8.63 Hz), 7.54-7.50 (4H, m), 7.42 (1H, t, J = 7.33 Hz). 13C NMR (100 MHz, DMSO-d6)
77
δ (ppm): 172.6, 156.9, 146.2, 139.2, 135.6, 129.7, 129.0, 128.1, 127.6, 127.3, 123.3; Se NMR (76
MHz, DMSO-d6) δ (ppm): 738.9. MS (ESI negative) m/z: 363.1 [M-H]-.
4.1.5 4-(5-(4-fluorophenyl)-1,3-selenazol-2-yl)benzenesulfonamide (6b)
Following the general procedure, 4-sulfamoylbenzoselenoamide (3) (100 mg, 0.38 mmol) and 2-Bromo-
4'-fluoroacetophenone 4b (83 mg, 0.38 mmol) gave 6b as pink solid (93 mg, 64%). 1H NMR (400 MHz,
DMSO- d6) δ (ppm): 8.81 (1H, s), 8.25 (2H, d, J = 8.42 Hz), 8.15 (2H, dd, J = 8.79, 5.54 Hz), 7.99 (2H,
13
d, J = 8.41 Hz), 7.54 (2H, bs, NH2, exchange with D2O), 7.35 (2H, t, J = 8.86 Hz). C NMR (100 MHz,
DMSO-d6) δ (ppm): 172.7, 162.8 (d, J = 245.19 Hz), 155.8, 146.3, 139.2, 132.2, 129.4 (d, J = 8.25 Hz),
77
128.1, 127.6, 123.0, 116.6 (d, J = 21.55 Hz); 19F-NMR (376 MHz, DMSO-d6) δ (ppm): -113.89; Se
NMR (76 MHz, DMSO-d6) δ (ppm): 741.0. MS (ESI negative) m/z: 381.1 [M-H]-.
4.1.6 4-(5-(4-bromophenyl)-1,3-selenazol-2-yl)benzenesulfonamide (6c)

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Following the general procedure, 4-sulfamoylbenzoselenoamide (3) (100 mg, 0.38 mmol) and 2-Bromo-
1
4'-bromoacetophenone 4c (106 mg, 0.38 mmol) gave 6c as white solid(101 mg, 60%). H NMR (400
MHz, DMSO- d6) δ (ppm): 8.90 (1H, s), 8.25 (2H, d, J = 8.21 Hz), 8.07 (2H, d, J = 8.38 Hz), 7.98 (2H,
d, J = 8.23 Hz), 7.71 (2H, d, J = 8.35 Hz), 7.55 (2H, bs, NH2, exchange with D2O). 13C NMR (100 MHz,
77
DMSO-d6) δ (ppm): 172.9, 155.6, 146.3, 139.1, 134.8, 132.7, 129.3, 128.1, 127.6, 124.2, 122.1; Se
NMR (76 MHz, DMSO-d6) δ (ppm): 744.8. MS (ESI negative) m/z: 441.1 [M-H]-.
4.1.7 4-(5-(4-chlorophenyl)-1,3-selenazol-2-yl)benzenesulfonamide (6d)
Following the general procedure, 4-sulfamoylbenzoselenoamide (3) (100 mg, 0.38 mmol) and 2-Bromo-
1
4'-chloroacetophenone 4d (89 mg, 0.38 mmol) gave 6d as white solid(92 mg, 61%). H NMR (400
MHz, DMSO- d6) δ (ppm): 8.89 (1H, s), 8.25 (2H, d, J = 8.40 Hz), 8.14 (2H, d, J = 8.55 Hz), 7.99 (2H,
13
d, J = 8.40 Hz), 7.59-7.54 (4H, m). C NMR (100 MHz, DMSO-d6) δ (ppm): 172.9, 155.6, 146.3,
139.1, 134.4, 133.5, 129.8, 129.1, 128.1, 127.6, 124.1; 77Se NMR (76 MHz, DMSO-d6) δ (ppm): 744.2.
MS (ESI negative) m/z: 397.1 [M-H]-.
4.1.8 4-(5-(4-nitrophenyl)-1,3-selenazol-2-yl)benzenesulfonamide (6e)
Following the general procedure, 4-sulfamoylbenzoselenoamide (3) (100 mg, 0.38 mmol) and 2-Bromo-
4'-nitroacetophenone 4e (93 mg, 0.38 mmol) gave 6e as yellow solid(85 mg, 55%). 1H NMR (400 MHz,
DMSO- d6) δ (ppm): 9.19 (1H, s), 8.38 (4H, aps), 8.28 (2H, d, J = 8.48 Hz), 8.00 (2H, d, J = 8.53 Hz),
13
7.56 (2H, bs, NH2, exchange with D2O). C NMR (100 MHz, DMSO-d6) δ (ppm): 173.6, 154.6, 147.6,
146.5, 141.5, 138.9, 128.3, 128.2, 127.9, 127.6, 125.2; 77Se NMR (76 MHz, DMSO-d6) δ (ppm): 757.4.
MS (ESI negative) m/z: 408.1 [M-H]-.
4.1.9 4-(5-(3-methoxyphenyl)-1,3-selenazol-2-yl)benzenesulfonamide (6f)

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Following the general procedure, 4-sulfamoylbenzoselenoamide (3) (100 mg, 0.38 mmol) and 2-Bromo-
1
3'-methoxyacetophenone 4f (87 mg, 0.38 mmol) gave 6f (120 mg, 80%). H NMR (400 MHz, DMSO-
d6) δ (ppm): 8.86 (1H, s), 8.25 (2H, d, J = 8.53 Hz), 7.99 (2H, d, J = 8.53 Hz), 7.68-7.66 (2H, m), 7.55
(
2H, bs, NH2, exchange with D2O), 7.43 (1H, t, J = 7.94 Hz), 7.00 (1H, ddd, J = 8.53 Hz), 3.88 (3H, s). 13C
NMR (100 MHz, DMSO-d6) δ (ppm): 172.4, 160.6, 156.7, 146.2, 139.2, 136.9, 130.8, 128.1, 127.6,
123.6, 119.8, 114.5,112.9, 56.1 77Se NMR (76 MHz, DMSO-d6) δ (ppm): 757.4. MS (ESI positive) m/z:
395.0 [M+H]+.
4.1.10 4-(5-methyl-1,3-selenazol-2-yl)benzenesulfonamide (7a)
Following the general procedure, 4-sulfamoylbenzoselenoamide (3) (100 mg, 0.38 mmol) and
1
Chloroacetone 5a (31 µl, 0.38 mmol) gave 7a as yellow solid (70 mg, 61%). H NMR (400 MHz,
DMSO- d6) δ (ppm): 8.12 (2H, d, J = 8.61 Hz), 7.94 (3H, m), 7.50 (2H, bs, NH2, exchange with D2O), 2.49
(3H, s). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.1, 154.8, 145.9, 139.3, 127.8, 127.5, 122.7, 18.9;
77Se NMR (76 MHz, DMSO-d6) δ (ppm): 711.9. MS (ESI negative) m/z: 301.0 [M-H]-.
4.1.11 2-(4-sulfamoylphenyl)-1,3-selenazole-5-carboxylic acid (7b)
Following the general procedure, 4-sulfamoylbenzoselenoamide (3) (100 mg, 0.38 mmol) and
1
Chloroacetic acid 5b (64 mg, 0.38 mmol) gave 7b as white solid (69 mg, 55%). H NMR (400 MHz,
DMSO- d6) δ (ppm): 13.07 (1H, bs, COOH), 9.22 (1H, apd), 8.20 (2H, d, J = 8.48 Hz), 7.98 (2H, d, J =
8.44 Hz), 7.54 (2H, bs, NH2, exchange with D2O). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 173.6, 173.2,
77
163.1, 149.1, 146.5, 138.8, 128.3, 127.6; Se NMR (76 MHz, DMSO-d6) δ (ppm): 763.7. MS (ESI
negative) m/z: 331.0 [M-H]-.
4.1.12 4-(5-(chloromethyl)-1,3-selenazol-2-yl)benzenesulfonamide (7c)

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Following the general procedure, 4-sulfamoylbenzoselenoamide (3) (100 mg, 0.38 mmol) and 1,3
1
Chloroacetone 5c (48 mg, 0.38 mmol) gave 7c as white solid (89 mg, 70%). H NMR (400 MHz,
DMSO- d6) δ (ppm): 8.47 (1H, s), 8.16 (2H, d, J = 8.62 Hz), 7.96 (2H, d, J = 8.62 Hz), 7.53 (2H, bs,
13
NH2, exchange with D2O), 4.93 (2H, s). C NMR (100 MHz, DMSO-d6) δ (ppm): 173.8, 154.5, 146.3,
77
138.9, 128.3, 128.0, 127.6, 42.7; Se NMR (76 MHz, DMSO-d6) δ (ppm): 728.4. MS (ESI negative)
m/z: 335.0 [M-H]-.
4.1.13 Procedure for the synthesis
benzenesulfonamide (10).
of
4-(5-((phenylthio)methyl)-1,3-selenazol-2-yl)
An acetonitrile solution (10 mL) of 2,5 substituted 1,3 selenazole (7c) (100 mg, 0.298 mmol), thiophenol
(36 µL, 1.2 Eq) and Et3N (79 µL, 2Eq) were stirred overnight at room temperature. The mixture was
extracted with ethyl acetate, dried with anhydrous Na2SO4 and the crude material was purified by flash
chromatography (1:1 hexane/ethyl acetate) to yield compound 10 as white solid (98 mg, 80%). 1H NMR
(400 MHz, DMSO- d6) δ (ppm): 8.16 (1H, s), 8.10 (2H, d, J = 8.36 Hz), 7.94 (2H, d, J = 8.34 Hz), 7.52
(2H, bs, NH2, exchange with D2O), 7.45 (2H, d, J = 7.62 Hz), 7.36 (2H, t, J = 7.66 Hz), 7.24 (1H, t, J =
13
7.33 Hz), 4.43 (2H, s). C NMR (100 MHz, DMSO-d6) δ (ppm): 172.9, 154.8, 146.1, 139.1, 136.8,
77
129.9, 129.4, 127.9, 127.5, 126.8, 125.5, 34.4; Se NMR (76 MHz, DMSO-d6) δ (ppm): 720.9. MS
(ESI positive) m/z: 411.0 [M+H]+.
4.1.14 General procedure for the synthesis of 2, 5 substitutes 1, 3-selenazoles 11a-c and 12a-c.
NaBH4 (43 mg, 1.14 mmol, 3.0 eq.) was portionwise added to a solution of appropriate chalcogenide 8a-
c or 9a-c (0.5 eq.) in EtOH (10 mL) at room temperature under inert atmosphere (N2). After 30 min, 2,5
substituted 1,3 selenazole 7c (127 mg, 0.38 mmol, 1.0 eq.) was slowly added and the reaction mixture
was stirred at room temperature for 3 h, until complete consumption of the starting material was

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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 (hexane/EtOAc 1:1) to yield the corresponded chalcogenide 11a-c or 12a-c.
4.1.15 4-(5-((phenyltellanyl)methyl)-1,3-selenazol-2-yl)benzenesulfonamide (11a).
Following the general procedure, 4-(5-(chloromethyl)-1,3-selenazol-2-yl)benzenesulfonamide 7c (127
mg, 0.38 mmol) and diphenyl ditelluride 8a (78 mg, 0.19 mmol) gave 11a as yellow solid (96 mg, 50%).
1H NMR (400 MHz, DMSO- d6) δ (ppm): 8.05 (2H, d, J = 8.52 Hz), 7.93 (2H, d, J = 8.54 Hz), 7.89
(1H, s), 7.75 (2H, dd, J = 1.32, 8.06 Hz), 7.52 (2H, bs, NH2, exchange with D2O), 7.33-7.25 (3H, m),
4.45 (2H, s). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.2, 157.5, 146.0, 139.2, 138.6, 130.1, 128.4,
127.8, 127.5, 122.5, 114.4, 8.0; 77Se NMR (76 MHz, DMSO-d6) δ (ppm): 717.6. 125Te NMR (126 MHz,
DMSO-d6) δ(ppm): 608.3. MS (ESI negative) m/z: 506.9 [M-H]-.
4.1.16 4-(5-((p-tolyltellanyl)methyl)-1,3-selenazol-2-yl)benzenesulfonamide (11b).
Following the general procedure, 4-(5-(chloromethyl)-1,3-selenazol-2-yl)benzenesulfonamide 7c (127
mg, 0.38 mmol) and p-tolyl ditelluride 8b (83 mg, 0.19 mmol) gave 11b as yellow solid (154 mg, 78%).
1H NMR (400 MHz, DMSO- d6) δ (ppm): 8.05 (2H, d, J = 8.59 Hz), 7.93 (2H, d, J = 8.60 Hz), 7.85
(1H, s), 7.63 (2H, d, J = 7.96 Hz), 7.51 (2H, bs, NH2, exchange with D2O), 7.09 (2H, d, J = 7.61 Hz),
4.40 (2H, s), 2.32 (3H, s). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.1, 157.6, 146.0, 139.2, 139.1,
77
138.1, 130.9, 127.8, 127.5, 122.5, 110.1, 21.6, 8.0; Se NMR (76 MHz, DMSO-d6) δ (ppm): 717.0.
125Te NMR (126 MHz, DMSO-d6) δ (ppm): 600.6. MS (ESI negative) m/z: 520.9 [M-H]-.
4.1.17 4-(5-(((4-methoxyphenyl)tellanyl)methyl)-1,3-selenazol-2-yl)benzenesulfonamide (11c)

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Following the general procedure, 4-(5-(chloromethyl)-1,3-selenazol-2-yl)benzenesulfonamide 7c (127
mg, 0.38 mmol) and 4-methoxyphenyl ditelluride 8c (89 mg, 0.19 mmol) gave 11c as yellow solid (153
1
mg, 75%). H NMR (400 MHz, DMSO- d6) δ (ppm): 8.05 (2H, d, J = 8.59 Hz), 7.93 (2H, d, J = 8.58
Hz), 7.85 (1H, s), 7.78 (1H, s), 7.64 (2H, d, J = 8.75 Hz), 7.51 (2H, bs, NH2, exchange with D2O), 6.84
(2H, d, J = 8.77 Hz), 4.35 (2H, s), 3.76 (3H, s). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.1, 160.3,
157.2, 146.0, 141.6, 139.2, 127.8, 127.5, 122.4, 116.1, 103.0, 55.9, 8.3; 77Se NMR (76 MHz, DMSO-d6)
δ (ppm): 716.1. 125Te NMR (126 MHz, DMSO-d6) δ (ppm): 600.9. MS (ESI negative) m/z: 536.9 [M-
H]-.
4.1.18 4-(5-((phenylselanyl)methyl)-1,3-selenazol-2-yl)benzenesulfonamide (12a)
Following the general procedure, 4-(5-(chloromethyl)-1,3-selenazol-2-yl)benzenesulfonamide 7c (127
mg, 0.38 mmol) and phenyl diselenide 9a (59 mg, 0.19 mmol) gave 12a as white solid (128 mg, 74%).
1H NMR (400 MHz, DMSO- d6) δ (ppm): 8.09-8.06 (3H, m), 7.94 (2H, d, J = 8.41 Hz), 7.57 (2H, dd, J
13
= 1.36, 8.01 Hz), 7.56 (2H, bs, NH2, exchange with D2O), 7.36-7.30 (3H, m), 4.41 (2H, s). C NMR
(100 MHz, DMSO-d6) δ (ppm): 172.7, 155.8, 146.1, 139.1, 132.9, 131.2, 130.0, 127.9, 127.8, 127.5,
124.8, 27.5; 77Se NMR (76 MHz, DMSO-d6) δ (ppm): 719.4, 347.3. MS (ESI positive) m/z: 459.0
[M+H]+.
4.1.19 4-(5-((p-tolylselanyl)methyl)-1,3-selenazol-2-yl)benzenesulfonamide (12b).
Following the general procedure, 4-(5-(chloromethyl)-1,3-selenazol-2-yl)benzenesulfonamide 7c (127
mg, 0.38 mmol) and p-tolyl diselenide 9b (65 mg, 0.19 mmol) gave 12b as white solid (143 mg, 80%).
1H NMR (400 MHz, DMSO- d6) δ (ppm): 8.08 (2H, d, J = 8.63 Hz), 8.01 (1H, s), 7.94 (2H, d, J = 8.63
Hz), 7.52 (2H, bs, NH2, exchange with D2O), 7.44 (2H, d, J = 8.05 Hz), 7.16 (2H, d, J = 7.81 Hz), 4.35
13
(2H, s), 2.31 (3H, s). C NMR (100 MHz, DMSO-d6) δ (ppm): 172.6, 156.0, 146.1, 139.2, 137.4,

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77
133.5, 130.7, 127.8, 127.5, 127.2, 124.7, 27.9, 21.5; Se NMR (76 MHz, DMSO-d6) δ (ppm): 722.2
MS (ESI positive) m/z: 473.0 [M+H]+.
4.1.20 4-(5-(((4-methoxyphenyl)selanyl)methyl)-1,3-selenazol-2-yl)benzenesulfonamide (12c).
Following the general procedure, 4-(5-(chloromethyl)-1,3-selenazol-2-yl)benzenesulfonamide 7c (127
mg, 0.38 mmol) and 4-methoxyphenyl diselenide 9c (71 mg, 0.19 mmol) gave 12c as white solid (129
mg, 70%). 1H NMR (400 MHz, DMSO- d6) δ (ppm): 8.08 (2H, d, J = 8.54 Hz), 7.95-7.92 (3H, m), 7.51
(2H, bs, NH2, exchange with D2O), 7.46 (2H, d, J = 8.80 Hz), 6.91 (2H, d, J = 8.80 Hz), 4.28 (2H, s),
3.77 (3H, s). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.6, 159.9, 156.0, 146.0, 139.2, 136.3, 127.8,
127.5, 124.5, 120.5, 115.7, 56.0, 28.7; 77Se NMR (76 MHz, DMSO-d6) δ (ppm): 717.8, 341.4. MS (ESI
negative) m/z: 485.1 [M-H]-.
4.1.21 Procedure for the synthesis of 2-((2-(4-sulfamoylphenyl)-1,3-selenazol-5-yl)methyl)
isothiouronium chloride (13)
4-(5-(chloromethyl)-1,3-selenazol-2-yl)benzenesulfonamide 7c (127 mg, 0.38 mmol) and Thiourea
(29mg, 0.38 mmol) were dissolved in dry acetone. The mixture was stirred at reflux for 1h and after
cooling, the precipitate was filtered off and washed with cold acetone to yield compound 13 as grey
1
solid (78 mg, 50%). H NMR (400 MHz, DMSO- d6) δ (ppm): 9.37 (2H, bs, NH2, exchange with D2O),
8.37 (1H, s), 8.14 (2H, d, J = 8.41 Hz), 7.96 (2H, d, J = 8.40 Hz), 7.56 (2H, bs, NH2, exchange with
D2O), 7.13 (2H, bs, NH2, exchange with D2O), 4.71 (2H, s). 13C NMR (100 MHz, DMSO-d6) δ (ppm):
77
174.2, 170.4, 152.3, 146.4, 138.7, 128.0, 127.6, 127.1, 32.0; Se NMR (76 MHz, DMSO-d6) δ (ppm):
731.5. MS (ESI positive) m/z: 377.0 [M+H]+.
4.2. Carbonic anhydrase inhibition

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An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2
hydration activity.[27] Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at
the absorbance maximum of 557 nm, with 20 mMHepes (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,[28-31] and represent the mean from at least three different determinations.
All CA isofoms were recombinant ones obtained in-house as reported earlier.[28-31]
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 ID23-2 beamline at ESRF (Grenoble, France) with a

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wavelength of 0.8731 Å and a PILATUS3 2M Dectris detector. Data were integrated and scaled using
the program XDS.[34] Data processing statistics were showed 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.[35] 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 1.0 occupancy. Atomic models for inhibitors
were calculated and energy minimized using the program JLigand 1.0.40.[36] Refinements proceeded
using normal protocols of positional, isotropic atomic displacement parameters alternating with manual
building of the models using COOT.[37] Solvent molecules were introduced automatically using the
program ARP.[38] The quality of the final models were assessed with COOT and RAMPAGE.[39]
Crystal parameters and refinement data are summarized in Electronic Supplementary Information (ESI).
Atomic coordinates were deposited in the Protein Data Bank (PDB accession code: 6H3Q). Graphical
representations were generated with Chimera.[40]
4.4. Biological Assays.
Human prostate cancer cell line PC3 and human breast cancer cell line MDA-MB-231 were obtained
from American Type Culture Collection (Rockville, MD). PC3 and MDA-MB-231 were cultured in
DMEM high glucose with 20% FBS in 5% CO2 atmosphere at 37° C. Media contained 2 mM L-
glutamine, 1% essential aminoacid mix, 100 IU ml-1 penicillin and 100 µg ml-1 streptomycin (Sigma,
Milan, Italy). Cells were plated in 960 wells cell culture (1.104/well) and, 24 h after, treated with the
tested compounds for 48 h. Low oxygen conditions were acquired in a hypoxic workstation

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(Concept 400 anaerobic incubator, Ruskinn Technology Ltd., Bridgend, UK). The atmosphere in the
chamber consisted of 1% O2 (hypoxia), 5% CO2, and residual N2. In parallel, normoxic (21% O2)
dishes were incubated in air with 5% CO2. Cell vitality was assessed via MTT assay. Viability is
expressed as % in comparison to the control cells (arbitrarily set 100 % of viable cells). Data are
presented as mean ± SEM. One-way ANOVA with a Bonferroni post-hoc test was used to compare the
treated samples to the control. A p-value less than 0.05 was considered to indicate a significant
difference.
Acknowledgments.
We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation
facilities and we would like to thank Max Nanao for assistance in using beamline ID23-2.
References
1. H.S. Olcott, W.D. Brown, J. Van Derveen, Seleno methionine as an Antioxidant. Nature 191
(1961) 1201.
2. R. Walter, I.L. Schwartz, J. Roy, Can selenoamino acids act as reversible biological
antioxidants? Ann N Y Acad Sci. 192 (1972) 175-180.
3. D.V. Frost, The Two Faces of Selenium Can Selenophobia Be Cured? Crit. Rev. Toxicol. 4
(1972) 467−513.
4. G. J. Southan, A. L. Salzman, C. Szabó, Potent inhibition of the inducible isoform of nitric oxide
synthase by aminoethylisoselenourea and related compounds. Life Sci. 58 (1996) 1139.
5. A. Angeli, D. Tanini, C. Viglianisi, L. Panzella, A. Capperucci, S. Menichetti, C.T. Supuran,
Evaluation of selenide, diselenide and selenoheterocycle derivatives as carbonic anhydrase I, II,
IV, VII and IX inhibitors. Bioorg Med Chem. 17 (2017) 30326-7.

ACCEPTED MANUSCRIPT
6. C. Schewe, T. Schewe, A. Wendel, Strong inhibition of mammalian lipoxygenases by the
antiinflammatory seleno-organic compound ebselen in the absence of glutathione. Biochem.
Pharmacol. 48 (1994) 65.
7. S.F. Wang, P. Komarov, H. Sies, H. de Groot, Inhibition of superoxide and nitric oxide release
and protection from reoxygenation injury by Ebselen in rat Kupffer cells. Hepatology 15 (1992)
1112.
8. P.C. Srivastava, R.K. Robins, Synthesis and antitumor activity of 2-beta-D-
ribofuranosylselenazole-4- carboxamide and related derivatives. J. Med. Chem. 26 (1983) 445.
9. A. Angeli, L. Di Cesare Mannelli, E. Lucarini, T.S. Peat, C. Ghelardini, C.T. Supuran, Design,
synthesis and X-ray crystallography of selenides bearing benzenesulfonamide moiety with
neuropathic pain modulating effects. Eur J Med Chem. 154 (2018) 210-219.
10. J.T. Salonen, Selenium and human cancer. Ann. Clin. Res. 18 (1986) 18.
11. H.J. Jung, Y.R. Seo, Current issues of selenium in cancer chemoprevention. Biofactors. 2 (2010)
153-158.
12. J. Squires, M.J. Berry, Selenium, selenoproteins, and cancer. Hawaii Med J. 8 (2006) 239-240.
13. R. Sinha, K. El-Bayoumy. Apoptosis is a critical cellular event in cancer chemoprevention and
chemotherapy by selenium compounds. Curr Cancer Drug Targets. 1 (2004) 13-28.
14. J. Brozmanová, D. Mániková, V. Vlčková, M. Chovanec, Selenium: a double-edged sword for
defense and offence in cancer. Arch Toxicol. 12 (2010) 919-38.
15. A.I. Ignat Grozav, L. Gaina, V. Kuete, L. Silaghi-Dumitrescu, T. Efferth, V. Zaharia,
Microwave-assisted synthesis of new selenazole derivatives with antiproliferative activity.
Molecules. 4 (2013) 4679-4688.

ACCEPTED MANUSCRIPT
16. H.C. Zhao, Y.P. Shi, Y.M. Liu, C.W. Li, L.N. Xuan, P. Wang, K. Zhang, B.Q. Chen, Synthesis
and antitumor-evaluation of 1,3-selenazole-containing 1,3,4-thiadiazole derivatives. Bioorg Med
Chem Lett. 24 (2013) 6577-6579.
17. P.C. McDonald, J.-Y. Winum, C.T. Supuran, S. Dedhar, Recent developments in targeting
carbonic anhydrase IX for cancer therapeutics. Oncotarget 3 (2012) 84.
18. D.P. Logsdon, M. Grimard, M. Luo, S. Shahda, Y. Jiang, Y. Tong, Z. Yu, N. Zyromski, E.
Schipani, F. Carta, C.T. Supuran, M. Korc, M. Ivan, M.R. Kelley, M.L. Fishel, Regulation of
HIF1α under Hypoxia by APE1/Ref-1 Impacts CA9 Expression: Dual Targeting in Patient-
Derived 3D Pancreatic Cancer Models. Mol Cancer Ther. 15 (2016) 2722.
19. D. Neri, C. T. Supuran, Interfering with pH regulation in tumours as a therapeutic strategy. Nat
Rev Drug Discov. 10 (2011) 767.
20. R.G. Gieling, M. Babur, L. Mamnani, N. Burrows, B.A. Telfer, S.R. Williams, K.E. Davies, F.
Carta, J.Y. Winum, A. Scozzafava, C.T. Supuran, K.J. Williams, Antimetastatic effect of
sulfamate carbonic anhydrase IX inhibitors in breast carcinoma xenografts. J Med Chem. 55
(2012) 5591.
21. C. Ward, J. Meehan, P. Mullen, C.T. Supuran, F. Carta, J.M. Dixon, J.S. Thomas, J.Y. Winum,
P. Lambin, L. Dubois, N. K. Pavathaneni, E.J. Jarman, L. Renshaw, I.H. Um, C. Kay, D.J.
Harrison, I.H. Kunkler, S.P. Langdon, Evaluation of carbonic anhydrase IX as a therapeutic
target for inhibition of breast cancer invasion and metastasis using a series of in vitro breast
cancer models. Oncotarget 6 (2015) 24856
22. A. Maresca, C. Temperini, H. Vu, N.B. Pham, S.A. Poulsen, A. Scozzafava, R.J. Quinn, C.T.
Supuran, Non-zinc mediated inhibition of carbonic anhydrases: coumarins are a new class of
suicide inhibitors. J Am Chem Soc. 8 (2009) 3057-3062.

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23. C.T. Supuran, How many carbonic anhydrase inhibition mechanisms exist? J. Enzyme Inhib.
Med. Chem. 31 (2016) 345–360.
24. C.T. Supuran, Advances in structure-based drug discovery of carbonic anhydrase inhibitors.
Expert Opin. Drug Discov. 12 (2017) 61–88.
25. C.T. Supuran, Structure-based drug discovery of carbonic anhydrase inhibitors. J. Enzyme Inhib.
Med. Chem. 27 (2012) 759–772.
26. See at: https://clinicaltrials.gov/ct2/show/NCT02215850
27. R.G. Khalifah, The carbon dioxide hydration activity of carbonic anhydrase. I. Stop flow kinetic
studies on the native human isoenzymes B and C. J. Biol. Chem. 246 (1971) 2561.
28. A. Angeli, D. Tanini, T. S. Peat, L. Di Cesare Mannelli, G. Bartolucci, A. Capperucci, C.
Ghelardini, C.T. Supuran, F. Carta, Discovery of New Selenoureido Analogues of 4-(4-
Fluorophenylureido)benzenesulfonamide as Carbonic Anhydrase Inhibitors. ACS Med Chem
Lett. 8 (2017) 963-968.
29. A. Angeli, D. Tanini, A. Capperucci, C.T. Supuran, Synthesis of novel selenides bearing
benzenesulfonamide moieties as carbonic anhydrase I, II, IV, VII and IX inhibitors. ACS Med.
Chem. Lett., 8 (2017) 1213-1217.
30. A. Angeli, D. Tanini, A. Capperucci, C.T. Supuran, First evaluation of organotellurium
derivatives as carbonic anhydrase I, II, IV, VII and IX inhibitors. Bioorg Chem. 76 (2018) 268-
272.
31. A. Angeli, T. S. Peat, G. Bartolucci, A. Nocentini, C.T. Supuran, F. Carta, Intramolecular
oxidative deselenization of acylselenoureas: a facile synthesis of benzoxazole amides and
carbonic anhydrase inhibitors. Org Biomol Chem. 14 (2016) 11353-11356.

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32. A. Angeli, L. di Cesare Mannelli, E. Trallori, T.S. Peat, C. Ghelardini, F. Carta, C.T. Supuran,
Design, Synthesis, and X-ray of Selenides as New Class of Agents for Prevention of Diabetic
Cerebrovascular Pathology. ACS Med. Chem. Lett., 9 (2018) 462–467
33. A. Di Fiore, G. De Simone, V. Alterio, V. Riccio, J.Y. Winum, F. Carta, C.T. Supuran, The
anticonvulsant sulfamide JNJ-26990990 and its S,S-dioxide analog strongly inhibit carbonic
anhydrases: solution and X-ray crystallographic studies. Org Biomol Chem. 14 (2016) 4853.
34. A.G.W. Leslie, H.R. Powell, Procesing diffraction data with mosflm. In: Read RJ, Sussman JL
(eds) Evolving methods for macromolecular crystallography, vol 245, NATO Science series,
Springer, Dordrecht, 2007, pp. 41-51.
35. G.N. Murshudov, A.A.Vagin, E.J. Dodson, Refinement of macromolecular structures by the
maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 53 (1997) 240-255.
36. A.A. Lebedev, P. Young, M.N. Isupov, O.V. Moroz, A.A. Vagin, G.N. Murshudov, JLigand: a
graphical tool for the CCP4 template-restraint library. Acta Crystallogr D Biol Crystallogr. 68
(2012) 431-440.
37. P. Emsley, B. Lohkamp, W. Scott, K. Cowtan, Features and development of Coot. Acta
Crystallogr D Biol Crystallogr. 66 (2010) 486-501.
38. V.S. Lamzin, A. Perrakis, K.S. Wilson, The ARP/wARP suite for automated construction and
refinement of protein models, M.G. Rossmann, E. Arnold (Eds.), Int. Tables for Crystallography.
Vol. F: Crystallography of Biological Macromolecules, Kluwer Academic Publishers, Dordrecht,
The Netherlands (2001), pp. 720-722
39. S.C. Lovell, I.W. Davis, W.B. Arendall III, P.I.W. de Bakker, J.M. Word, M.G. Prisant, J.S.
Richardson, D.C. Richardson, Structure validation by Cα geometry: ϕ,ψ and Cβ deviation,
Proteins, 50 (2003), pp. 437-450.