A novel library of saccharin and acesulfame derivatives as potent and selective inhibitors of carbonic anhydrase IX and XII isoforms

Article abstract of DOI:10.1016/j.bmc.2016.01.038

Small libraries of N-substituted saccharin and N-/O-substituted acesulfame derivatives were synthesized and tested as atypical and selective inhibitors of four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). Most of the

Full text of DOI:10.1016/j.bmc.2016.01.038

Accepted Manuscript
A novel library of saccharin and acesulfame derivatives as potent and selective
inhibitors of carbonic anhydrase IX and XII isoforms
Simone Carradori, Daniela Secci, Celeste De Monte, Adriano Mollica,
Mariangela Ceruso, Atilla Akdemir, Anatoly P. Sobolev, Rossella Codispoti,
Federica De Cosmi, Paolo Guglielmi, Claudiu T. Supuran
PII:
S0968-0896(16)30038-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.01.038
BMC 12781
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 November 2015
18 January 2016
19 January 2016
Please cite this article as: Carradori, S., Secci, D., De Monte, C., Mollica, A., Ceruso, M., Akdemir, A., Sobolev,
A.P., Codispoti, R., De Cosmi, F., Guglielmi, P., Supuran, C.T., A novel library of saccharin and acesulfame
derivatives as potent and selective inhibitors of carbonic anhydrase IX and XII isoforms, Bioorganic & Medicinal
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.01.038
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A novel library of saccharin and acesulfame derivatives as potent and
selective inhibitors of carbonic anhydrase IX and XII isoforms
Simone Carradoria, Daniela Seccib∗, Celeste De Monteb, Adriano Mollicaa, Mariangela Cerusoc,
Atilla Akdemird, Anatoly P. Soboleve, Rossella Codispotib, Federica De Cosmib, Paolo Guglielmib,
Claudiu T. Supuranc,f∗
aDepartment of Pharmacy, “G. D’Annunzio” University of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy
bDipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
cLaboratorio di Chimica Bioinorganica, Università degli Studi di Firenze, Via della Lastruccia 3, 50019 Sesto
Fiorentino (Florence), Italy
dBezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, Vatan Caddesi, 34093 Fatih,
Istanbul, Turkey.
eInstitute of Chemical Methodologies, Magnetic Resonance Laboratory “Annalaura Segre”, National Research Council,
Via Salaria km 29.300, 00015 Monterotondo (Rome), Italy
fNeurofarba Dept., Section of Pharmaceutical and Nutriceutical Sciences, Università degli Studi di Firenze, Via U.
Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
∗
Corresponding authors: Prof. Daniela Secci: Tel/fax: +39 06 49693242; e-mail:
daniela.secci@uniroma1.it; Prof. Claudiu T. Supuran: Tel: +39 055 4573005; Fax: +39 055
4573385; e-mail: claudiu.supuran@unifi.it.

Abstract
Small libraries of N-substituted saccharin and N-/O-substituted acesulfame derivatives were
synthesized and tested as atypical and selective inhibitors of four different isoforms of human
carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). Most of them inhibited hCA XII in the low
nanomolar range, hCA IX with KIs ranging between 19 and 2482 nM, whereas they were poorly
active against hCA II (KIs > 10 µM) and hCA I (KIs ranging between 318 nM to 50 µM). Since
hCA I and II are ubiquitous off-target isoforms, whereas the cancer-related isoforms hCA IX and
XII were recently validated as drug targets, these results represent an encouraging achievement in
the development of new anticancer candidates. Moreover, the lack of a classical zinc binding group
in the structure of these inhibitors opens innovative, yet unexplored scenarios for different
mechanisms of inhibition that could explain the high inhibitory selectivity. A computational
approach has been carried out to further rationalize the biological data and to characterize the
binding mode of some of these inhibitors.
Keywords: saccharin, acesulfame, carbonic anhydrase inhibitor, cancer-related isoforms, N/O-
substitution.

1.
Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are important metalloenzymes involved in many cellular
and physiological processes which require bicarbonate as substrate such as electrolyte secretion, pH
homoeostasis, respiration, and biosynthetic pathways including lipogenesis, glucogenesis, and
ureagenesis, as well as tumorigenicity.1,2 Human CAs (hCAs) exist in fifteen isoforms and differ in
their cellular localization (cytosol, mitochondria or cell membrane), sensibility to inhibitors and
catalytic activity. CAs are well established therapeutic targets to treat a wide range of disorders
through the use of inhibitors or activators,3-5 as well as recognized tools for drug delivery
purposes.6,7
Classical CA inhibitors (CAIs) usually act by complexing the metal ion (Zn+2) in the enzyme active
site such as primary sulfonamides and their bioisosters, inorganic anions, thiophenols,
hydroxamates and carboxylic acids.8 In this context, the emerging “non-zinc binding inhibitors”
have deeply increased over the last few years, especially in the quest of potent and selective anti-
cancer agents (targeting the tumor-associated isoforms hCA IX and XII).9-13 Our recent studies,
focused on the synthesis of saccharin- and acesulfame-based compounds,14,15 allowed identifying a
series of cyclic tertiary sulfonamides and their bioisosteric sulfamates acting as selective hCA
IX/XII inhibitors. The peculiar isoform selectivity displayed by these compounds was attributed to a
newly hypothesized mode of inhibition similar to other non-zinc binding inhibitors (e.g., probenecid
derivatives).16–18 As a consequence, this new class of CAIs might be considered as an innovative
tool in the design of selective CAIs for further applications in medicinal chemistry.19-21
Pursuing our efforts for the development of innovative treatment of cancer,22-25 in our previous
reports we investigated several N/O-substituted compounds exploring the chemical space around
these positions. In particular, among the reported derivatives we co-crystallized one of them with
hCA II demonstrating the interactions within the active site.26 In order to complete structure-activity
relationships (SARs) within these scaffolds, we designed and synthesized new derivatives
characterized by different substituents (CH3, CF3, CN, F, Cl, Br, NO2, OCH3, Ph) at the ortho, meta

and para positions of the benzylic or benzoylic portion keeping constant the saccharin/acesulfame
skeleton.27 In addition, we studied the change in the biological activity of bioisosteric replacement
of the methylene bridge (in the N-benzylic saccharin derivatives) with a sulfur atom and the
substitution of the SO2 group of the saccharin with C=O (phthalimide) and CH2 (isoindolinone),
respectively. This approach allowed us to obtain promising candidates not only potent and selective
inhibitors of the cancer-related hCA IX and hCA XII, but also showing appropriate physico-
chemical properties (water solubility, balanced hydro/liposolubility) for the development of
potential drugs.
2.
Chemistry
N-substituted saccharin derivatives (1a-13a) were synthesized according to the general procedure
reported in Scheme 1.28 Saccharin (1.0 eq.) was deprotonated using freshly grinded anhydrous
potassium carbonate and the corresponding salt was then reacted with a proper electrophile (2 eq.)
by stirring the reaction mixture in N,N-dimethylformamide at 80 °C overnight. Under these
conditions (polar aprotic solvent) and if the reaction reached thermal equilibrium, we observed the
formation of the more stable isomer (N-substituted saccharin) limiting the Chapman-Mumm
thermal rearrangement to the less stable O-substituted derivative.29 Conversely, the reaction
between potassium acesulfame and the electrophile reagents in N,N-dimethylformamide gave the
corresponding N-substituted derivatives (a series) or O-substituted derivatives (b series)
(compounds 14a-22b, Scheme 1). The formation of a mixture of amide and imidate compounds or
the generation of only one of them usually was influenced by the thermodynamic stability of the
products and by the steric hindrance of the electrophile reactant. In fact, the formation of the O-
substituted structure could compete with the more thermodynamically preferred N-substituted one
whether the C=N bond was contained in an aromatic or unsaturated nucleus and, furthermore, the
attack at the oxygen atom could also occur for steric effects.30 These considerations could explain
the formation of the derivatives belonging to a series and/or b series starting from the ambident
nucleophile (potassium acesulfame or saccharin). Moreover, compounds 23-25 (Scheme 1) were

purchased and used in the biological assays without further purification in order to explore the
structure-activity relationship within the saccharin scaffold.
O
S
O
S
O
O
R
K2CO3, 80 °C
DMF
N
+
NH
X
R
X = Cl, Br
O
O
a series
N
-substituted derivatives
1a 13a
-
K+
O
O
O
O
O
O
R
-
DMF
N
N
R
N
+
X
R
or
SO2
SO2
SO2
80 °C
X = Cl, Br
a series
b series
N
O
-substituted
-substituted derivatives
14a 17a
18b
22b
derivatives
-
and
and
19a 21a
-
O
O
N
O
O
O
S
NH
N
S
NO2
COOH
O
25
24
23
Scheme 1. Synthesis and structure of compounds 1-25.
3.
Biological evaluation
All the synthesized and purchased compounds of a and b series were tested to evaluate their
inhibitory activity towards the ubiquitous off-target isoforms, hCA I and II, and the cancer-related
ones, hCA IX and XII by a stopped-flow, CO2 hydrase assay method and their CA inhibition data
are summarized in Tables 1-3.

Table 1. Inhibitory activity of saccharin-based derivatives 1a-13a, against selected hCA isoforms
by a stopped-flow CO2 hydrase assay.
Ki (nM)
Compound
1a
R
hCA I
hCA II
hCA IX
hCA XII
1368
> 10000
221
16
2a
3579
> 10000
616
15
3a
4a
1503
> 10000
> 10000
184
19
26
17
> 10000
5a
6a
> 10000
1347
> 10000
> 10000
> 10000
> 10000
> 10000
> 10000
234
51
5.1
4.7
41
7a
2479
539
19
8a
2361
4.4
4.3
29
9a
> 10000
324
22
10a
1169
11a
12a
13a
1342
1106
1480
> 10000
> 10000
> 10000
1570
23
6.0
41
191
3.9
AAZ
(acetazolamide)
250
12
25
6.0

Table 2. Inhibitory activity of acesulfame-based derivatives 14a-22b, against selected hCA
isoforms by a stopped-flow CO2 hydrase assay.
Ki (nM)
Compound
14a
R
hCA I
hCA II
hCA IX
hCA XII
> 10000
> 10000
261
4.9
15a
> 10000
> 10000
239
4.5
16a
17a
> 10000
> 10000
> 10000
> 10000
210
161
4.3
5.6
18b
19a
20a
21a
9717
2047
> 10000
1749
196
2482
27
3.6
3.9
4.1
4.0
318
140
> 10000
> 10000
26
22b
> 10000
> 20000
> 10000
> 20000
265
5.5
potassium
acesulfame
2410
> 20000

Table 3. Inhibitory activity of saccharin analogues 23-27, against selected hCA isoforms by a
stopped-flow CO2 hydrase assay.
Ki (nM)
Compound
23
R
hCA I
hCA II
hCA IX
hCA XII
> 10000
> 10000
18
4.0
O
COOH
N
24
25
> 10000
> 10000
> 10000
> 10000
223
174
4.3
4.1
O
2615
> 50000
41
91
28
2726
110000
18540
78000
5950
310
103
42
Saccharin
633
4.
Results and discussion
4.1. New N-substituted saccharin derivatives (1a-13a).
All the tested compounds had no affinity for the common off-target hCA II isoform (KI > 10000
nM), and many of them were more active against the tumor-related ones with respect to the starting
compound saccharin. Compounds 4a, 5a and 9a displayed their effect exclusively against the
tumor-associated isoenzymes hCA IX and hCA XII (both transmembrane), without affecting either
hCA I or hCA II (both cytosolic). While compound 4a, containing a phenyl ring substituted in the
meta position, possessed a similar effect against hCA IX and XII (KI = 19 nM and KI = 17 nM,
respectively), compounds 5a and 9a, which had a substituent at C4 of the phenyl ring, were more
effective against hCA XII, with a KI of 5.1 and 4.3 nM, respectively. Compound 8a, bearing a
phenyl moiety with a chlorine at C4, was weakly active against hCA I (KI = 2361 nM), but it was
more selective towards hCA XII at very low nanomolar concentration (KI = 4.4 nM). Similarly, the

acetophenone derivative 13a, containing two chlorine atoms at C2 and C4 of the aromatic ring, was
weakly active towards hCA I (KI = 1480 nM) and more selective for hCA XII (KI = 3.9 nM).
4.2. New N- and O-substituted acesulfame derivatives (14a-22b).
This scaffold consists of compounds divided into two groups (a series and b series), according to
the N- or O- functionalization. Most of the compounds had more affinity for hCA IX and XII than
that registered with potassium acesulfame. Compounds 14a, 15a and 16a, bearing a phenyl ring
substituted at C4 with a halogen or a nitro group, had no effect towards the two off-target isoforms
and displayed the best biological activity selectively against the tumor-associated hCA XII (KI =
4.9, 4.5 and 4.3 nM, respectively). With regard to the acetophenone derivatives, they all inhibited
the cancer-related hCA XII at very low nanomolar concentrations. Compounds 19a and 20a were
not selective, while the N-substituted derivatives 17a, 21a and the O-substituted derivatives 18b and
22b were not active against the two off-target enzymes and more effective towards hCA XII with
respect to hCA IX.
Compound 23 allowed us to explore the biological change after the substitution of the methylene
bridge with a sulphur atom (see for comparison our previously synthesized compound 26). This
new derivative improved the inhibitory activity and selectivity against hCA IX and XII and lost its
interaction with hCA II. Among the other purchased compounds, both compound 25 (molecular
simplification of the saccharin skeleton by deletion of the SO2 moiety) and the N-alkylated
phthalimide 24 (bioisosteric replacement of the SO2 group with a C=O, see for comparison our
previously synthesized compound 27), which were analogues of saccharin but without the cyclic
sulfonamide moiety, were also active only towards the tumor-associated isoforms, without affecting
the other isoenzymes. These N-substituted saccharin derivatives and N- and O-substituted
acesulfame derivatives have proven to be promising lead compounds for further studies in the
discovery of new inhibitors of the tumor-associated hCA isoforms.
5.
Docking studies into the active site of hCA XII

All saccharin and acesulfame analogues (compounds 1a–25) inhibited hCA XII in the nanomolar
range and show approximately 11-fold difference between the lowest and highest values (KI values:
3.6-41 nM, Tables 1-3). Docking studies indicated that the sulfonamide oxygen atoms could form
interactions with the Zn2+-ion and the backbone of Thr199 (and occasionally also Thr200). The
para substituted bromophenyl group of saccharin-based compound 9a pointed either towards Asn62,
Ser65 and Lys67 or towards the solvent, while the carbonyl group was oriented towards the solvent
in both poses (Figure 1A). The carbonyl group of acesulfame-based compound 18b formed a
hydrogen bond with either Asn62 or Gln92, while the para substituted bromophenyl group pointed
towards the solvent (Figure 1B).
A
B
H64
N62
H64
W5
W5
K67
N62
P201
P201
T200
T200
H96
K67
P202
P202
T199
H96
H94
Q92
T199
H94
H119
V121
H119
L198
L198
Q92
V121
C
W5
P201
P202
T199
T200
H96
H64
L198
N62
V121
K67
Q92

Figure 1. Docked poses of compounds 9a (A), 18b (B) and 19a (C) in the active site of hCA XII.
Hydrogen bonds and interactions to the Zn2+-ion are depicted in red dashed lines. The Zn2+-ion is
depicted as a turquoise sphere.
The two docked poses observed for acesulfame-based compound 19a were different from the other
saccharin analogues and showed interactions between the nitro group and the Zn2+-ion (Figure 1C).
In the first pose, one of the sulfonamide oxygen atoms interacted with Asn62 and Lys67, while the
other sulfonamide oxygen and the carbonyl groups were water accessible. In the other docked pose,
the carbonyl group, embedded in the six-ring, interacted with Lys67 and Gln92. Docking studies
were not able to rationalize the difference between the Ki values measured for saccharin and
compound 25 (Table 3), as both compounds formed a direct interaction with the Zn2+-ion and
saccharin had additional hydrogen bonds. Probably, long ranged electrostatic interactions and
entropy were responsible for this unexpected effect.
6.
Docking studies into the active sites of hCA I, II and IX
Docking studies into hCA IX indicated that none of the compounds can directly interact with the
Zn2+-ion. Lys67 and Thr91 in hCA XII were replaced by Gln67 and Leu9, respectively, in hCA IX,
while the conserved Gln92 had a different conformation. Most likely Gln67 in hCA IX prevented
the ligands from interacting with the Zn2+-ion in hCA IX due to steric clashes with the ligand. In
addition, the cationic and flexible Lys67 could adopt a different conformation and approach the
ligands to form cation-π interactions with the aromatic groups (Figure 2).

W5
N62
H64
P201
P202
K67/Q67
T200
H96
T199
H94
Q92
H119
V121
L198
T91/L91
Figure 2. An overlay of the hCA IX active site (carbon atoms represented as light brown sticks)
upon the docked poses of acesulfame-based compound 18b (magenta and turquoise sticks) in the
active site of hCA XII (carbon atoms represented as grey sticks). Hydrogen bonds and interactions
to the Zn2+-ion are depicted in red dashed lines. The Zn2+-ion is depicted as a turquoise sphere.
Docking studies of hCA I and II showed that the ligands can form direct interactions with the Zn2+-
ion of both enzymes. However, enzyme inhibition assays showed in general high KI values for these
derivatives. We assume that this might be caused by long-ranged electrostatic interactions, enzyme
flexibility upon ligand binding (induced-fit) and entropic effects (such as the inability of the vicinal
Asn67 and Gln92 of hCA II to form hydrogen bonds with water or the ligand).
7.
Conclusion
A large library of N-substituted saccharin derivatives and N- and O-substituted acesulfame
derivatives were synthesized and tested against four human carbonic anhydrases: hCA I and II, the
most common off-targets in the discovery of antitumor drugs, and hCA IX and XII, the cancer-
related isoforms. Most of the compounds were not active towards hCA I and II, while they were
very effective against hCA IX and XII and, in particular, they all inhibited hCA XII in the low
nanomolar range. These large series of cyclic tertiary sulfonamide and sulfamate compounds could

represent an important starting point for the development of new antitumor agents based on the
inhibition of the cancer-related isoforms of human carbonic anhydrase overexpressed in hypoxic
tumors.31
8.
Experimental protocols
8.1. General
Solvents were used as supplied without further purification. Where mixtures of solvents are
specified, the stated ratios are volume:volume. Unless otherwise indicated, all aqueous solutions
used were saturated. Saccharin, potassium acesulfame and acetazolamide were purchased by
Sigma-Aldrich (Italy) and used in the syntheses and in the biological assays without further
purification. All synthesized compounds have been fully characterized by analytical and spectral
data. Column chromatography was carried out using Sigma-Aldrich® silica gel (high purity grade,
pore size 60 Å, 200-425 mesh particle size). Analytical thin-layer chromatography was carried out
on Sigma-Aldrich® silica gel on TLA aluminum foils with fluorescent indicator. Visualization was
carried out under UV irradiation (254 nm). 1H-NMR spectra were recorded on a Bruker AV400 (1H:
400 MHz, 13C: 101 MHz). 19F NMR spectra were recorded on a Bruker AVANCE 600 spectrometer
(19F: 564.7 MHz). Chemical shifts are quoted in ppm, based on appearance rather than
interpretation, and are referenced to the residual non deuterated solvent peak. In the case of 19F,
chemical shifts are referenced to external standard (CF3COOH, δ -76.55 ppm). Infra-red spectra
were recorded on a Bruker Tensor 27 FTIR spectrometer equipped with an attenuated total
reflectance attachment with internal calibration. Absorption maxima (νmax) are reported in
wavenumbers (cm-1). All melting points were measured on a Stuart® melting point apparatus SMP1
and are uncorrected. Temperatures are reported in °C. Elemental analyses were performed on a
Perkin Elmer series II, 2400 CHN analyzer. Where given, systematic compound names are those
generated by ChemBioDraw Ultra® 12.0 following IUPAC conventions.
8.2. Procedures for the synthesis of saccharin derivatives
8.2.1. 2-(2-Methylbenzyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (1a)

1.51 g of anhydrous potassium carbonate (1 eq.) were added to a stirring solution of saccharin (2.0
g, 1.0 eq.) in 20 mL of N,N-dimethylformamide at 80 °C. 2-Methylbenzyl bromide (2.01 g, 1 eq.)
was added and the reaction stirred at 80 °C for 48 h. The reaction was poured on ice and the
resulting suspension was filtered. Purification via column chromatography on silica gel (ethyl
acetate:n-hexane, 1:2) gave the title compound as a white solid (72% yield); mp 153-155 °C; IR
νmax 3073 (ν Csp2-H), 1726 (ν C=O), 1335 (νas S=O), 1244 (ν C-N), 1181 (νs S=O), 754 (δ Csp2-H),
676 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, CDCl3) δ 2.50 (3H, s, CH3), 4.98 (2H, s, CH2), 7.21-7.25
(m, 3H, CHAr), 7.44 (d, J = 7.2 Hz, 1H, CHAr), 7.83-7.91 (m, 2H, CHAr), 7.95 (d, J = 6.8 Hz, 1H,
CHAr), 8.10 (d, J = 8.0 Hz, 1H, CHAr); 13C-NMR (101 MHz, CDCl3) δ 19.3 (CH3), 40.6 (CH2),
121.0 (Ar), 125.3 (Ar), 126.3 (Ar), 127.3 (Ar), 128.3 (Ar), 128.7 (Ar), 130.5 (Ar), 132.1 (Ar), 134.4
(Ar), 134.9 (Ar), 136.3 (Ar), 137.9 (Ar), 159.0 (C=O). Anal. Calcd for C15H13NO3S: C, 62.70; H,
4.56; N, 4.87. Found: C, 62.52 ; H, 4.81; N, 5.06.
8.2.2. 2-(2-Trifluoromethylbenzyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (2a)
1.51 g of anhydrous potassium carbonate (1 eq.) were added to a stirring solution of saccharin (2.0 g,
1.0 eq.) in 20 mL of N,N-dimethylformamide at 80 °C. 2-Trifluoromethylbenzyl bromide (2.63 g, 1
eq.) was added and the reaction stirred at 80 °C for 48 h. The reaction was poured on ice and the
resulting suspension was filtered. Purification via column chromatography on silica gel (ethyl
acetate:n-hexane, 1:2) gave the title compound as a white solid (60% yield); mp 142-143 °C; IR
νmax 3095 (ν Csp2-H), 1733 (ν C=O), 1336 (νas S=O), 1241 (ν C-N), 1171 (νs S=O), 749 (δ Csp2-H),
1
676 (δ Csp2-H) cm-1; H-NMR (400 MHz, CDCl3) δ 5.20 (2H, s, CH2), 7.41-7.45 (t, 1H, CHAr),
7.51-7.57 (m, 2H, CHAr), 7.72 (d, J = 8.0 Hz, 1H, CHAr), 7.87-7.95 (m, 3H, CHAr), 8.13 (d, J = 7.6
Hz, 1H, CHAr); 13C-NMR (101 MHz, CDCl3) δ 39.0 (CH2), 121.2 (Ar), 122.9 (Ar), 125.5 (Ar),
126.2 (Ar), 127.0 (Ar), 128.0 (Ar), 128.3 (Ar), 132.3 (Ar), 133.0 (Ar), 134.6 (Ar), 135.1 (Ar), 137.9
(Ar), 159.1 (C=O). 19F-NMR (564.7 MHz, CDCl3) δ -57.34 (s, CF3). Anal. Calcd for
C15H10F3NO3S: C, 52.79; H, 2.95; N, 4.10. Found: C, 52.55 ; H, 3.17; N, 3.91.
8.2.3. 2-(3-Methylbenzyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (3a)

1.51 g of anhydrous potassium carbonate (1 eq.) were added to a stirring solution of saccharin (2.0 g,
1.0 eq.) in 20 mL of N,N-dimethylformamide at 80 °C. 3-Methylbenzyl bromide (2.01 g, 1 eq.) was
added and the reaction stirred at 80 °C for 48 h. The reaction was poured on ice and the resulting
suspension was filtered. Purification via column chromatography on silica gel (ethyl acetate:n-
hexane, 1:2) gave the title compound as a white solid (72% yield); mp 94-96 °C; IR νmax 3065 (ν
Csp2-H), 1729 (ν C=O), 1329 (νas S=O), 1260 (ν C-N), 1178 (νs S=O), 748 (δ Csp2-H), 676 (δ Csp2-
1
H) cm-1; H-NMR (400 MHz, CDCl3) δ 2.37 (3H, s, CH3), 4.90 (2H, s, CH2), 7.14 (d, J = 7.6 Hz,
1H, CHAr), 7.24-7.33 (m, 3H, CHAr), 7.82-7.88 (m, 2H, CHAr), 7.95 (d, J = 7.2 Hz, 1H, CHAr), 8.08
(d, J = 8.0 Hz, 1H, CHAr); 13C-NMR (101 MHz, CDCl3) δ 21.4 (CH3), 42.7 (CH2), 121.0 (Ar),
125.2 (Ar), 125.8 (Ar), 127.3 (Ar), 128.6 (Ar), 129.1 (Ar), 129.4 (Ar), 134.3 (Ar), 134.4 (Ar), 134.8
(Ar), 137.8 (Ar), 138.4 (Ar), 158.9 (C=O). Anal. Calcd for C15H13NO3S: C, 62.70; H, 4.56; N, 4.87.
Found: C, 62.99; H, 4.28; N, 4.69.
8.2.4. 2-(3-Trifluoromethylbenzyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (4a)
1.51 g of anhydrous potassium carbonate (1 eq.) were added to a stirring solution of saccharin (2.0 g,
1.0 eq.) in 20 mL of N,N-dimethylformamide at 80 °C. 3-Trifluoromethylbenzyl bromide (2.63 g, 1
eq.) was added and the reaction stirred at 80 °C for 48 h. The reaction was poured on ice and the
resulting suspension was filtered. Purification via column chromatography on silica gel (ethyl
acetate:n-hexane, 1:2) gave the title compound as a white solid (79% yield); mp 128-130 °C; IR
νmax 3092 (ν Csp2-H), 1720 (ν C=O), 1332 (νas S=O), 1263 (ν C-N), 1175 (νs S=O), 752 (δ Csp2-H),
1
680 (δ Csp2-H) cm-1; H-NMR (400 MHz, CDCl3) δ 4.97 (2H, s, CH2), 7.49-7.52 (m, 1H, CHAr),
7.60 (d, J = 8.0 Hz, 1H, CHAr), 7.72 (d, J = 7.6 Hz, 1H, CHAr), 7.79 (s, 1H, CHAr), 7.84-7.97 (m, 3H,
CHAr), 8.09 (d, J = 7.6 Hz, 1H, CHAr); 13C-NMR (101 MHz, CDCl3) δ 42.1 (CH2), 121.1 (Ar),
125.3 (Ar), 125.4 (Ar), 125.6 (Ar), 127.1 (Ar), 129.3 (Ar), 131.0 (Ar), 132.1 (Ar), 134.5 (Ar), 135.0
19
(Ar), 135.5 (Ar), 137.7 (Ar), 158.9 (C=O). F-NMR (564.7 MHz, CDCl3) δ -60.08 (s, CF3). Anal.
Calcd for C15H10F3NO3S: C, 52.79; H, 2.95; N, 4.10. Found: C, 52.98; H, 2.76; N, 4.32.
8.2.5. 2-(4-Methylbenzyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (5a)

1.51 g of anhydrous potassium carbonate (1 eq.) were added to a stirring solution of saccharin (2.0 g,
1.0 eq.) in 20 mL of N,N-dimethylformamide at 80 °C. 4-Methylbenzyl bromide (2.01 g, 1 eq.) was
added and the reaction stirred at 80 °C for 48 h. The reaction was poured on ice and the resulting
suspension was filtered. Purification via column chromatography on silica gel (ethyl acetate:n-
hexane, 1:2) gave the title compound as a white solid (67% yield); mp 111-113 °C; IR νmax 2920 (ν
Csp3-H), 1732 (ν C=O), 1329 (νas S=O), 1257 (ν C-N), 1174 (νs S=O), 747 (δ Csp2-H), 674 (δ Csp2-
1
H) cm-1; H-NMR (400 MHz, CDCl3) δ 2.35 (3H, s, CH3), 4.89 (2H, s, CH2), 7.18 (d, J = 8.0 Hz,
2H, CHAr), 7.42 (d, J = 8.0 Hz, 2H, CHAr), 7.81-7.88 (m, 3H, CHAr), 8.06 (d, J = 8.0 Hz, 1H, CHAr);
13C-NMR (101 MHz, CDCl3) δ 21.2 (CH3), 42.5 (CH2), 121.0 (Ar), 125.2 (Ar), 127.4 (Ar), 128.8
(Ar), 129.4 (Ar), 131.5 (Ar), 134.3 (Ar), 134.7 (Ar), 137.8 (Ar), 138.1 (Ar), 158.9 (C=O). Anal.
Calcd for C15H13NO3S: C, 62.70; H, 4.56; N, 4.87. Found: C, 62.57; H, 4.34; N, 4.58.
8.2.6. 2-(4-Trifluoromethylbenzyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (6a)
1.51 g of anhydrous potassium carbonate (1 eq.) were added to a stirring solution of saccharin (2.0 g,
1.0 eq.) in 20 mL of N,N-dimethylformamide at 80 °C. 4-Trifluoromethylbenzyl bromide (2.63 g, 1
eq.) was added and the reaction stirred at 80 °C for 48 h. The reaction was poured on ice and the
resulting suspension was filtered. Purification via column chromatography on silica gel (ethyl
acetate:n-hexane, 1:2) gave the title compound as a white solid (57% yield); mp 119-121 °C; IR
νmax 2963 (ν Csp3-H), 1741 (ν C=O), 1319 (νas S=O), 1264 (ν C-N), 1167 (νs S=O), 750 (δ Csp2-H),
1
679 (δ Csp2-H) cm-1; H-NMR (400 MHz, CDCl3) δ 4.97 (2H, s, CH2), 7.64 (bs, 4H, CHAr), 7.84-
7.98 (m, 3H, CHAr), 8.08 (d, J = 8.0 Hz, 1H, CHAr); 13C-NMR (101 MHz, CDCl3) δ 42.0 (CH2),
121.2 (Ar), 125.4 (Ar), 125.6 (Ar), 125.7 (Ar), 127.1 (Ar), 129.0 (Ar), 134.5 (Ar), 135.0 (Ar), 137.7
(Ar), 138.4 (Ar), 158.9 (C=O). 19F-NMR (564.7 MHz, CDCl3) δ -60.12 (s, CF3). Anal. Calcd for
C15H10F3NO3S: C, 52.79; H, 2.95; N, 4.10. Found: C, 53.04; H, 2.78; N, 3.95.
8.2.7. 2-(4-Fluorobenzyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (7a)
Anhydrous potassium carbonate (1.1 eq.) was added to a stirring solution of saccharin (1.0 eq.) in
10 mL of N,N-dimethylformamide. Then 4-fluorobenzyl bromide (1.5 eq.) was added and the

reaction mixture was stirred at 80 °C for 72 h. The mixture was poured on ice and the resulting
suspension was filtered and washed with water and petroleum ether. Purification by column
chromatography on silica gel (ethyl acetate:n-hexane, 1:3) gave compound 7a as a white powder
(89% yield); mp 125-126 °C; IR νmax 1726 (ν C=O), 1335 (νas S=O), 1295 (ν C-N), 1180 (νs S=O),
1158 (ν Csp2-F), 838 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 4.92 (s, 2H, CH2), 7.18 (t, J
= 8.8 Hz, 2H, CHAr), 7.47-7.50 (m, 2H, CHAr), 7.97-8.11 (m, 3H, CHAr), 8.31 (d, J = 7.6 Hz, 1H,
CHAr); 13C-NMR (101 MHz, DMSO-d6) δ 41.4 (CH2), 115.6 (Ar), 115.9 (Ar), 122.1 (Ar), 125.6
(Ar), 126.7 (Ar), 130.6 (Ar), 130.7 (Ar), 131.8 (Ar), 135.7 (Ar), 136.3 (Ar), 137.3 (Ar), 159.1
(C=O), 162.2 (d, JC-F = 244.9 Hz, Ar). 19F-NMR (564.7 MHz, CDCl3) δ -111.04 (tt, JF-H = 8.6 Hz
(ortho), 5.2 Hz (meta), CF). Anal. Calcd for C14H10FNO3S: C, 57.72; H, 3.46; N, 4.81. Found: C,
57.99; H, 3.67; N, 4.55.
8.2.8. 2-(4-Chlorobenzyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (8a)
Anhydrous potassium carbonate (1.1 eq.) was added to a stirring solution of saccharin (1.0 eq.) in
10 mL of N,N-dimethylformamide. Then 4-chlorobenzyl bromide (1.1 eq.) was added and the
reaction mixture was stirred at 80 °C for 48 h. The mixture was poured on ice and the resulting
suspension was filtered and washed with water, petroleum ether and diethyl ether. Purification by
column chromatography on silica gel (ethyl acetate:petroleum ether, 1:3) gave compound 8a as a
white powder (89% yield); mp 153-155 °C; IR νmax 3092 (ν Csp2-H), 1726 (ν C=O), 1331 (νas S=O),
1
1307 (ν C-N), 1197 (νs S=O), 749 (δ Csp2-H) cm-1; H-NMR (400 MHz, DMSO-d6) δ 4.93 (s, 2H,
CH2), 7.41-7.45 (m, 4H, CHAr), 7.98-8.08 (m, 3H, CHAr), 8.30-8.32 (m, 1H, CHAr); 13C-NMR (101
MHz, DMSO-d6) δ 41.4 (CH2), 122.1 (Ar), 125.6 (Ar), 126.7 (Ar), 128.9 (Ar), 130.3 (Ar), 133.0
(Ar), 134.6 (Ar), 135.6 (Ar), 136.3 (Ar), 137.2 (Ar), 159.1 (C=O). Anal. Calcd for C14H10ClNO3S:
C, 54.64; H, 3.28; N, 4.55. Found: C, 54.90; H, 3.47; N, 4.27.
8.2.9. 2-(4-Bromobenzyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (9a)
Anhydrous potassium carbonate (1.1 eq.) was added to a stirring solution of saccharin (1.0 eq.) in
10 mL of N,N-dimethylformamide. Then 4-bromobenzyl bromide (1.5 eq.) was added and the

reaction mixture was stirred at 80 °C for 72 h. The mixture was poured on ice and the resulting
suspension was filtered and washed with water and petroleum ether. Purification by column
chromatography on silica gel (ethyl acetate:petroleum ether, 1:3) gave compound 9a as a white
powder (89% yield); mp 158-160 °C; IR νmax 1725 (ν C=O), 1332 (νas S=O), 1303 (ν C-N), 1180 (νs
S=O or ν C-Br), 861 (δ Csp2-H) cm-1; 1H-NMR (400 MHz, CDCl3) δ 4.87 (s, 2H, CH2), 7.40 (d, J =
8.4 Hz, 2H, CHAr), 7.49 (d, J = 8.4 Hz, 2H, CHAr), 7.81-7.95 (m, 3H, CHAr), 8.06 (d, J = 7.2 Hz, 1H,
CHAr); 13C-NMR (101 MHz, CDCl3) δ 42.0 (CH2), 121.1 (Ar), 122.4 (Ar), 125.3 (Ar), 127.1 (Ar),
130.5 (Ar), 131.9 (Ar), 133.6 (Ar), 134.5 (Ar), 134.9 (Ar), 137.6 (Ar), 158.9 (C=O). Anal. Calcd for
C14H10BrNO3S: C, 47.74; H, 2.86; N, 3.98. Found: C, 47.92; H, 3.01; N, 4.15.
8.2.10. 2-(4-Nitrobenzyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (10a)
Anhydrous potassium carbonate (1.1 eq.) was added to a stirring solution of saccharin (1.0 eq.) in
10 mL of N,N-dimethylformamide. Then 4-nitrobenzyl bromide (1.1 eq.) was added and the
reaction mixture was stirred at 80 °C for 24 h. The mixture was poured on ice and the resulting
suspension was filtered and washed with water and n-hexane. Purification by column
chromatography on silica gel (ethyl acetate:petroleum ether, 1:3) gave compound 10a as a yellow
powder (91% yield); mp 178-180 °C; IR νmax 3092 (ν Csp2-H), 1727 (ν C=O), 1516 (νas N-O), 1338
1
(νas S=O), 1299 (νs N-O), 1248 (ν C-N), 1197 (νs S=O), 755 (δ Csp2-H) cm-1; H-NMR (400 MHz,
DMSO-d6) δ 5.10 (s, 2H, CH2), 7.69-7.71 (m, 2H, CHAr), 8.03-8.36 (m, 6H, CHAr); 13C-NMR (101
MHz, DMSO-d6) δ 41.3 (CH2), 122.2 (Ar), 124.1 (Ar), 125.7 (Ar), 126.7 (Ar), 129.3 (Ar), 135.8
(Ar), 136.4 (Ar), 137.2 (Ar), 143.3 (Ar), 147.5 (Ar), 159.1 (C=O). Anal. Calcd for C14H10N2O5S: C,
52.83; H, 3.17; N, 8.80. Found: C, 52.56; H, 3.41; N, 9.04.
8.2.11. 2-[2-(3-Methoxyphenyl)-2-oxoethyl]-1,2-benzothiazol-3(2H)-one 1,1-dioxide (11a)
Anhydrous potassium carbonate (1.1 eq.) was added to a stirring solution of saccharin (1.0 eq.) in
10 mL of N,N-dimethylformamide. Then, newly synthesized 2-bromo-3’-methoxyacetophenone32
(1.1 eq.) was added and the reaction mixture was stirred at 80 °C for 72 h. The mixture was poured
on ice and the resulting suspension was filtered and washed with water and petroleum ether.

Purification by column chromatography on silica gel (ethyl acetate:petroleum ether, 1:3) gave
compound 11a as a yellow powder (58% yield); mp 167-169 °C; IR νmax 1736 (ν C=O), 1694 (ν
C=O), 1328 (νas S=O), 1258 (ν C-N), 1182 (νs S=O), 864 (δ Csp2-H) 752 (δo Csp2-H) cm-1; 1H-NMR
(400 MHz, CDCl3) δ 3.86 (s, 3H, OCH3), 5.14 (s, 2H, CH2), 7.18-7.20 (m, 1H, CHAr), 7.41-7.58 (m,
3H, CHAr), 7.87-7.96 (m, 3H, CHAr), 8.11 (s, 1H, CHAr); 13C-NMR (101 MHz, CDCl3) δ 44.6 (CH2),
55.5 (OCH3), 112.4 (Ar), 120.7 (Ar), 120.9 (Ar), 121.2 (Ar), 125.5 (Ar), 127.3 (Ar), 129.9 (Ar),
134.5 (Ar), 134.9 (Ar), 135.3 (Ar), 137.9 (Ar), 159.1 (Ar), 160.0 (C=O), 188.7 (C=O). Anal. Calcd
for C16H13NO5S: C, 58.00; H, 3.95; N, 4.23. Found: C, 58.22; H, 4.11; N, 4.02.
8.2.12. 2-[2-(4-Fluorophenyl)-2-oxoethyl]-1,2-benzothiazol-3(2H)-one 1,1-dioxide (12a)
Anhydrous potassium carbonate (1.1 eq.) was added to a stirring solution of saccharin (1.0 eq.) in
10 mL of N,N-dimethylformamide. Then 2-chloro-4’-fluoroacetophenone (1.1 eq.) was added and
the reaction mixture was stirred at 80 °C for 72 h. The mixture was poured on ice and extracted with
chloroform (3 x 20 mL). The organics were reunited, dried over sodium sulfate and concentrated in
vacuo. Purification by column chromatography on silica gel (ethyl acetate:n-hexane, 1:3) gave
compound 12a as a light yellow powder (75% yield); mp 166-167 °C; IR νmax 1736 (ν C=O), 1692
1
(ν C=O), 1329 (νas S=O), 1305 (ν C-N), 1181 (νs S=O), 1156 (ν Csp2-F), 746 (δo Csp2-H) cm-1; H-
NMR (400 MHz, CDCl3) δ 5.14 (s, 2H, CH2), 7.19-7.23 (m, 2H, CHAr), 7.86-7.94 (m, 2H, CHAr),
7.97-7.99 (m, 1H, CHAr), 8.04-8.12 (m, 3H, CHAr); 13C-NMR (101 MHz, CDCl3) δ 44.3 (CH2),
116.2 (Ar), 116.4 (Ar), 121.2 (Ar), 125.5 (Ar), 127.2 (Ar), 130.6 (Ar), 130.9 (Ar), 131.0 (Ar), 134.5
(Ar), 135.0 (Ar), 137.9 (Ar), 159.1 (C=O), 166.3 (d, JC-F = 257.85 Hz, Ar), 187.4 (C=O). 19F-NMR
(564.7 MHz, DMSO-d6) δ -96.66 (tt, JF-H = 8.8 Hz (ortho), 5.5 Hz (meta), CF). Anal. Calcd for
C15H10FNO4S: C, 56.42; H, 3.16; N, 4.39. Found: C, 56.21; H, 2.91; N, 4.12.
8.2.13. 2-[2-(2,4-Dichlorophenyl)-2-oxoethyl]-1,2-benzothiazol-3(2H)-one 1,1-dioxide (13a)
Anhydrous potassium carbonate (1.1 eq.) was added to a stirring solution of saccharin (1.0 eq.) in
10 mL of N,N-dimethylformamide. Then 2,2’,4’-trichloroacetophenone (1.1 eq.) was added and the
reaction mixture was stirred at 80 °C for 72 h. The mixture was poured on ice and the resulting

suspension was filtered and washed with water, n-hexane and petroleum ether. Purification by
column chromatography on silica gel (ethyl acetate:petroleum ether, 1:3) gave compound 13a as a
yellow powder (92% yield); mp 117-119 °C; IR νmax 3082 (ν Csp2-H), 1736 (ν C=O), 1719 (ν C=O),
1
1332 (νas S=O), 1316 (ν C-N), 1184 (νs S=O), 883 (δ Csp2-H), 817 (δ Csp2-H) cm-1; H-NMR (400
MHz, DMSO-d6) δ 5.36 (s, 2H, CH2), 7.62-7.64 (m, 1H, CHAr), 7.76-7.77 (m, 1H, CHAr), 7.95-8.15
(m, 4H, CHAr), 8.35 (s, 1H, CHAr); 13C-NMR (101 MHz, DMSO-d6) δ 46.9 (CH2), 122.2 (Ar),
125.7 (Ar), 126.5 (Ar), 128.1 (Ar), 131.0 (Ar), 132.2 (Ar), 132.8 (Ar), 133.5 (Ar), 135.8 (Ar), 136.5
(Ar), 137.7 (Ar), 138.1 (Ar), 159.1 (C=O), 191.4 (C=O). Anal. Calcd for C15H9Cl2NO4S: C, 48.66;
H, 2.45; N, 3.78. Found: C, 48.24; H, 2.21; N, 4.01.
8.3. Procedures for the synthesis of acesulfame derivatives
8.3.1. 3-(4-Fluorobenzyl)-6-methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide (14a)
4-Fluorobenzyl bromide (1.5 eq.) was added to a stirring solution of potassium acesulfame (1.0 eq.)
in 10 mL of N,N-dimethylformamide. The reaction mixture was stirred at 80 °C for 72 h, poured on
ice and extracted with dichloromethane (3 x 20 mL). The organics were reunited, dried over sodium
sulfate and concentrated in vacuo. Purification by column chromatography on silica gel (ethyl
acetate:petroleum ether, 1:3) gave compound 14a as a yellow oil (45% yield); IR νmax 3089 (ν Csp2-
H), 1697 (ν C=O), 1397 (νas S=O), 1306 (ν C-N), 1198 (νs S=O), 1158 (ν Csp2-F), 924 (ν S-O), 839
1
(δp Csp2-H) cm-1; H-NMR (400 MHz, CDCl3) δ 2.18 (s, 3H, CH3), 4.96 (s, 2H, CH2), 5.83 (s, 1H,
CH=), 7.00-7.05 (m, 2H, CHAr), 7.41-7.44 (m, 2H, CHAr); 13C-NMR (101 MHz, CDCl3) δ 19.5
(CH3), 45.4 (CH2), 104.4 (CH=), 115.5 (Ar), 115.7 (Ar), 130.8 (Ar), 130.9 (Ar), 161.1 (d, JC-F
=
196.3, Ar), 161.4 (CH3C=), 163.9 (C=O). 19F-NMR (564.7 MHz, CDCl3) δ -110.63 (tt, JF-H = 8.6
Hz (ortho), 5.2 Hz (meta), CF). Anal. Calcd for C11H10FNO4S: C, 48.70; H, 3.72; N, 5.16. Found: C,
48.94; H, 3.97; N, 4.90.
8.3.2. 3-(4-Chlorobenzyl)-6-methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide (15a)
4-Chlorobenzyl bromide (1.1 eq.) was added to a stirring solution of potassium acesulfame (1.0 eq.)
in 10 mL of N,N-dimethylformamide. The reaction mixture was stirred at 80 °C for 72 h, poured on

ice and extracted with dichloromethane (3 x 20 mL). The organics were reunited, dried over sodium
sulfate and concentrated in vacuo. Purification by column chromatography on silica gel (ethyl
acetate:petroleum ether, 1:3) gave compound 15a as a white powder (45% yield); mp 54-56 °C; IR
νmax 3107 (ν Csp2-H), 1700 (ν C=O), 1398 (νas S=O), 1320 (ν C-N), 1199 (νs S=O), 922 (ν S-O), 795
(δp Csp2-H) cm-1; 1H-NMR (400 MHz, Methanol-d4) δ 2.14 (s, 3H, CH3), 4.94 (s, 2H, CH2), 5.95 (s,
1H, CH=), 7.29-7.33 (m, 4H, CHAr); 13C-NMR (101 MHz, Methanol-d4) δ 18.3 (CH3), 45.0 (CH2),
103.8 (CH=), 128.5 (Ar), 129.9 (Ar), 133.8 (Ar), 134.0 (Ar), 160.3 (CH3C=), 162.9 (C=O). Anal.
Calcd for C11H10ClNO4S: C, 45.92; H, 3.50; N, 4.87. Found: C, 46.11; H, 3.77; N, 4.68.
8.3.3. 6-Methyl-3-(4-nitrobenzyl)-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide (16a)
4-Nitrobenzyl bromide (1.1 eq.) was added to a stirring solution of potassium acesulfame (1.0 eq.)
in 10 mL of N,N-dimethylformamide. The reaction mixture was stirred at 80 °C for 72 h and poured
on ice. The resulting suspension was filtered and washed with water, n-hexane and petroleum ether
to give compound 16a as a light yellow powder (85% yield); mp 81-83 °C; IR νmax 3087 (ν Csp2-H),
1698 (ν C=O), 1549 (νas N-O), 1390 (νas S=O), 1349 (νs N-O), 1306 (ν C-N), 1200 (νs S=O), 926 (ν
1
S-O), 855 (δp Csp2-H) cm-1; H-NMR (400 MHz, CDCl3) δ 2.24 (s, 3H, CH3), 5.06 (s, 2H, CH2),
5.88 (s, 1H, CH=), 7.56 (d, J = 7.6 Hz, 2H, CHAr), 8.16 (d, J = 8.0 Hz, 2H, CHAr); 13C-NMR (101
MHz, CDCl3) δ 19.7 (CH3), 45.2 (CH2), 104.3 (CH=), 123.9 (Ar), 129.4 (Ar), 141.9 (Ar), 147.8
(Ar), 160.0 (CH3C=), 162.5 (C=O). Anal. Calcd for C11H10N2O6S: C, 44.29; H, 3.38; N, 9.39.
Found: C, 44.02; H, 3.67; N, 9.58.
8.3.4. 3-[2-(3-Methoxyphenyl)-2-oxoethyl]-6-methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide
(17a)
Newly synthesized 2-bromo-3’-methoxyacetophenone32 (1.1 eq.) was added to a stirring solution of
potassium acesulfame (1.0 eq.) in 10 mL of N,N-dimethylformamide. The reaction mixture was
stirred at 80 °C for 48 h, poured on ice and extracted with dichloromethane (3 x 20 mL). The
organics were reunited, dried over sodium sulfate and concentrated in vacuo. Purification by
column chromatography on silica gel (ethyl acetate:petroleum ether, 1:3) gave compound 17a as a

yellow powder (53% yield); mp 95-99 °C; IR νmax 2961 (ν Csp3-H), 1714 (ν C=O), 1696 (ν C=O),
1320 (νas S=O), 1200 (νs S=O), 930 (ν S-O), 861 (δm Csp2-H), 782 (δm Csp2-H), 679 (δm Csp2-H) cm-
1; 1H-NMR (400 MHz, CDCl3) δ 2.23 (s, 3H, CH3), 3.81 (s, 3H, OCH3), 5.24 (s, 2H, CH2), 5.89 (s,
1H, CH=), 7.15 (s, 1H, CHAr), 7.38-7.50 (m, 3H, CHAr); 13C-NMR (101 MHz, CDCl3) δ 19.7 (CH3),
49.0 (OCH3), 55.4 (CH2), 104.3 (CH=), 112.3 (Ar), 120.6 (Ar), 120.8 (Ar), 130.0 (Ar), 135.2 (Ar),
159.9 (Ar + CH3C=), 162.6 (C=O), 189.7 (C=O). Anal. Calcd for C13H13NO6S: C, 50.16; H, 4.21;
N, 4.50. Found: C, 49.91; H, 4.03; N, 4.72.
8.3.5. 1-(4-Bromophenyl)-2-[(6-methyl-2,2-dioxido-1,2,3-oxathiazin-4-yl)oxy]ethanone (18b)
Newly synthesized 2-bromo-4’-bromoacetophenone32 (1.1 eq.) was added to a stirring solution of
potassium acesulfame (1.0 eq.) in 10 mL of N,N-dimethylformamide. The reaction mixture was
stirred at 80 °C for 24 h, poured on ice and extracted with chloroform (3 x 20 mL). The organics
were reunited, dried over sodium sulfate and concentrated in vacuo. Purification by column
chromatography on silica gel (ethyl acetate:petroleum ether, 1:3) gave compound 18b as a yellow
powder (84% yield); mp 125-127 °C; IR νmax 3080 (ν Csp2-H), 1688 (ν C=O), 1651 (ν C=N), 1327
1
(νas S=O), 1199 (νs S=O), 1175 (ν C-O-C), 931 (ν S-O), 812 (δp Csp2-H) cm-1; H-NMR (400 MHz,
CDCl3) δ 2.29 (s, 3H, CH3), 5.22 (s, 2H, CH2), 5.92 (s, 1H, CH=), 7.65-7.67 (m, 2H, CHAr), 7.82-
7.84 (m, 2H, CHAr); 13C-NMR (101 MHz, CDCl3) δ 19.8 (CH3), 48.7 (CH2), 104.3 (CH=), 129.6
(Ar), 132.3 (Ar), 132.6 (Ar), 159.9 (Ar), 162.6 (CH3C= + C=N), 189.0 (C=O). Anal. Calcd for
C12H10BrNO5S: C, 40.02; H, 2.80; N, 3.89. Found: C, 39.84; H, 3.01; N, 4.05.
8.3.6. 6-Methyl-3-[2-(4-nitrophenyl)-2-oxoethyl]-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide (19a)
Newly synthesized 2-bromo-4’-nitroacetophenone32 (1.1 eq.) was added to a stirring solution of
potassium acesulfame (1.0 eq.) in 10 mL of N,N-dimethylformamide. The reaction mixture was
stirred at 80 °C for 24 h and poured on ice. The resulting suspension was filtered and washed with
water and petroleum ether. Purification by column chromatography on silica gel (ethyl
acetate:petroleum ether, 1:3) gave compound 19a as a brown powder (44% yield); mp 133-135 °C;
IR νmax 3071 (ν Csp2-H), 1707 (ν C=O), 1684 (ν C=O), 1524 (νas N-O), 1348 (νs N-O), 1333 (νas

S=O), 1201 (νs S=O), 929 (ν S-O), 857 (δp Csp2-H) cm-1; 1H-NMR (400 MHz, DMSO-d6) δ 2.33 (s,
3H, CH3), 5.60 (s, 2H, CH2), 6.37 (s, 1H, CH=), 8.31-8.39 (m, 4H, CHAr); 13C-NMR (101 MHz,
DMSO-d6) δ 19.5 (CH3), 49.7 (CH2), 104.1 (CH=), 124.4 (Ar), 130.3 (Ar), 138.7 (Ar), 150.9 (Ar),
160.1 (CH3C=), 163.8 (C=O), 191.11 (C=O). Anal. Calcd for C12H10N2O7S: C, 44.17; H, 3.09; N,
8.59. Found: C, 43.94; H, 3.31; N, 8.22.
8.3.7. 4-[(6-Methyl-2,2-dioxido-4-oxo-1,2,3-oxathiazin-3(4H)-yl)acetyl]benzonitrile (20a)
Newly synthesized 2-bromo-4’-cyanoacetophenone33 (1.1 eq.) was added to a stirring solution of
potassium acesulfame (1.0 eq.) in 10 mL of N,N-dimethylformamide. The reaction mixture was
stirred at 80 °C for 24 h, poured on ice and filtered. Purification by column chromatography on
silica gel (ethyl acetate:n-hexane, 1:3) gave compound 20a as a yellow powder (70% yield); mp
165-166 °C; IR νmax 3077 (ν Csp2-H), 2248 (ν CN), 1732 (ν C=O), 1687 (ν C=O), 1321 (νas S=O),
1199 (νs S=O), 918 (ν S-O), 825 (δp Csp2-H), 775 (δo Csp2-H) cm-1; 1H-NMR (400 MHz, DMSO-d6)
δ 2.31 (s, 3H, CH3), 5.58 (s, 2H, CH2), 6.36 (s, 1H, CH=), 8.20-8.28 (m, 4H, CHAr); 13C-NMR (101
MHz, DMSO-d6) δ 19.4 (CH3), 48.9 (CH2), 104.2 (CH=), 123.3 (CN), 131.4 (Ar), 138.9 (Ar), 141.2
(Ar), 149.1 (Ar), 160.3 (CH3C=), 163.7 (C=O), 191.5 (C=O). Anal. Calcd for C13H10N2O5S: C,
50.98; H, 3.29; N, 9.15. Found: C, 51.19; H, 3.42; N, 8.92.
8.3.8. 3-[2-(Biphenyl-4-yl)-2-oxoethyl]-6-methyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide (21a)
Newly synthesized 2-bromo-4’-phenylacetophenone33 (1.1 eq.) was added to a stirring solution of
potassium acesulfame (1.0 eq.) in 10 mL of N,N-dimethylformamide. The reaction mixture was
stirred at 80 °C for 48 h, poured on ice and filtered. Purification by column chromatography on
silica gel (ethyl acetate:petroleum ether, 1:3) gave compound 21a as a grey powder (77% yield); mp
170-172 °C; IR νmax 3085 (ν Csp2-H), 1731 (ν C=O), 1684 (ν C=O), 1328 (νas S=O), 1197 (νs S=O),
1
929 (ν S-O), 829 (δp Csp2-H), 778 (δo Csp2-H) cm-1; H-NMR (400 MHz, DMSO-d6) δ 2.32 (s, 3H,
CH3), 5.54 (s, 2H, CH2), 6.29 (s, 1H, CH=), 8.13-8.35 (m, 9H, CHAr); 13C-NMR (101 MHz,
DMSO-d6) δ 20.0 (CH3), 48.2 (CH2), 104.9 (CH=), 122.8 (Ar), 123.6 (Ar), 123.9 (Ar), 125.1 (Ar),

125.6 (Ar), 132.8 (Ar), 133.9 (Ar), 148.9 (Ar), 161.8 (CH3C=), 160.2 (C=O), 190.9 (C=O). Anal.
Calcd for C18H15NO5S: C, 60.49; H, 4.23; N, 3.92. Found: C, 60.21; H, 4.45; N, 4.11.
8.3.9. 1-(2,4-Dichlorophenyl)-2-[(6-methyl-2,2-dioxido-1,2,3-oxathiazin-4-yl)oxy]ethanone
(22b)
2,2’,4’-Trichloroacetophenone (1.1 eq.) was added to a stirring solution of potassium acesulfame
(1.0 eq.) in 10 mL of N,N-dimethylformamide. The reaction mixture was stirred at 80 °C for 24 h,
poured on ice and extracted with dichloromethane (3 x 20 mL). The organics were reunited, dried
over sodium sulfate and concentrated in vacuo. Purification by column chromatography on silica
gel (ethyl acetate:petroleum ether, 1:3) gave compound 22b as an orange powder (83% yield); mp
90-94 °C; IR νmax 3080 (ν Csp2-H), 1737 (ν C=O), 1647 (ν C=N), 1369 (νas S=O), 1259 (ν C-O-C),
1
1198 (νs S=O), 921 (ν S-O), 827 (δp Csp2-H), 777 (δo Csp2-H) cm-1; H-NMR (400 MHz, CDCl3) δ
2.22 (s, 3H, CH3), 5.45 (s, 2H, CH2), 5.94 (s, 1H, CH=), 7.34-7.35 (m, 1H, CHAr), 7.43 (s, 1H,
CHAr), 7.61-7.63 (m, 1H, CHAr); 13C-NMR (101 MHz, CDCl3) δ 20.6 (CH3), 71.0 (CH2), 95.2
(CH=), 127.9 (Ar), 130.6 (Ar), 131.6 (Ar), 132.8 (Ar), 133.2 (Ar), 139.3 (Ar), 168.8 (CH3C=),
169.9 (C=N), 191.5 (C=O). Anal. Calcd for C12H9Cl2NO5S: C, 41.16; H, 2.59; N, 4.00. Found: C,
40.91; H, 2.78; N, 4.25.
8.4. Enzyme inhibition assays
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed
CO2 hydration activity. Phenol red (0.2 mM) has been used as indicator, working at the absorbance
maximum of 557 nm, with 20 mM Hepes (pH 7.5, for α-CAs) as buffer, and 20 mM NaClO4 (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 (1 µM) were prepared in distilled-deionized water and dilutions up to

0.1 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 or for the eventual active site mediated hydrolysis of the inhibitor.
The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the
Cheng-Prusoff equation,34 and represent the average from at least three different determinations. All
recombinant CA isoforms were obtained in-house as previously reported.35
8.5. Molecular modelling studies
8.5.1. Preparation of saccharin- and acesulfame-based structures
The saccharin- and acesulfame-based analogues 1-25 were prepared in 3D with the MOE software
package (v2013.08.02, Chemical Computing Group Inc., Montreal, Canada) as previously
reported.36 All possible structural isomers of compounds were constructed. Strong acids were
deprotonated and strong bases were protonated. Finally, the ligands were energy minimized using a
steepest-descent protocol (MMFF94x force field).
8.5.2. Preparation of hCA crystal structures for docking studies
The structures of hCA I (PDB: 3LXE, 1.90 Å), hCA II (PDB: 4E3D, 1.60 Å), hCA IX (PDB: 3IAI,
2.20 Å) and hCA XII (PDB: 1JD0, 1.50 Å) were obtained from the protein databank (PDB). The
protein atoms, the active site zinc ions and the zinc-bound water molecule of hCA II were retained
and all other atoms were omitted. The remaining structure was protonated using the MOE software
package and subsequently the obtained structure was energy-minimized (AMBER99 force field).
Finally, the obtained protein models were superposed on the hCA I structure using the backbone
Cα-atoms. The zinc-bound water molecule of hCA II coordinated well to the zinc ions of the other
hCAs.
8.5.3. Docking studies
The GOLD Suite software package (v5.2, CCDC, Cambridge, UK) and the GoldScore scoring
function were used to dock the derivatives into the hCA structures with and without the zinc-bound
water molecule (25 dockings per ligand). The binding pocket was defined as all residues within 13

Å of a centroid (x: -17.071, y: 35.081, 43.681; corresponding approximately to the position of the
thiadiazole ring of acetazolamide in the 1JD0 structure).
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This work was financed by two FP7 EU projects (Metoxia and Dynano) to CTS.
References
1.
2.
3.
Neri, D.; Supuran, C. T. Nat. Rev. Drug Discov. 2011, 10, 767–777.
Supuran, C. T. Bioorg. Med. Chem. 2013, 21, 1377−1378.
Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem. Rev. 2012,
112, 4421–4468.
4.
5.
6.
Supuran, C. T.; Scozzafava, A. Bioorg. Med. Chem. 2007, 15, 4336−4350.
Guler, O. O.; De Simone, G.; Supuran, C. T. Curr. Med. Chem. 2010, 17, 1516−1526.
a) Annesini, M. C.; Di Marzio, L.; Finazzi-Agrò, A.; Serafino, A. L.; Mossa, G. Biochem.
Mol. Biol. Int. 1994, 32, 87–94; b) Annesini, M. C.; Di Giorgio, L.; Di Marzio, L.; Finazzi-Agrò,
A.; Serafino, A. L.; Mossa, G. J. Liposome Res. 1993, 3, 639-648.
7.
8.
9.
Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013, 28, 229−230.
Supuran, C. T. Nat. Rev. Drug Discov. 2008, 7, 168–181.
Carradori, S.; De Monte, C.; D’Ascenzio, M.; Secci, D.; Celik, G.; Ceruso, M.; Vullo, D.;
Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2013, 23, 6759–6763.
10. D’Ambrosio, K.; Carradori, S.; Monti, S. M.; Buonanno, M.; Secci, D.; Vullo, D.; Supuran,
C. T.; De Simone, G. Chem. Commun. 2015, 51, 302–305.
11. De Monte, C.; Carradori, S.; Gentili, A.; Mollica, A.; Trisciuoglio, D.; Supuran, C. T. Curr.
Med. Chem. 2015, 22, 2812–2818.
12. Gidaro, M. C.; Alcaro, F.; Carradori, S.; Costa, G.; Vullo, D.; Supuran, C. T.; Alcaro, S.
Planta Med. 2015, 81, 533–540.
13. Carradori, S.; Mollica, A.; De Monte, C.; Granese, A.; Supuran, C. T. Molecules 2015, 20,
5667–5679.
14.
De Monte, C.; Carradori, S.; Secci, D.; D’Ascenzio, M.; Vullo, D.; Ceruso, M.; Supuran, C.
T. Eur. J. Med. Chem. 2014, 84, 240–246.
15.
D’Ascenzio, M.; Carradori, S.; De Monte, C.; Secci, D.; Ceruso, M.; Supuran, C. T. Bioorg.
Med. Chem. 2014, 22, 1821–31.

16.
Ceruso, M.; Supuran, C. T. Bioorg. Med. Chem. 2015, 23, 5311–5318.
17. D’Ascenzio, M.; Carradori, S.; Secci, D.; Vullo, D.; Ceruso, M.; Akdemir, A.; Supuran, C.
T. Bioorg. Med. Chem. 2014, 22, 3982–3988.
18. Carradori, S.; Mollica, A.; Ceruso, M.; D’Ascenzio, M.; De Monte, C.; Chimenti, P.; Sabia,
R.; Akdemir, A.; Supuran, C. T. Bioorg. Med. Chem. 2015, 23, 2975–2981.
Mollica, A.; Costante, R.; Akdemir, A.; Carradori, S.; Stefanucci, A.; Macedonio, G.;
19.
20.
21.
Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759−772.
De Simone, G.; Alterio, V.; Supuran, C. T. Expert Opin. Drug Discov. 2013, 8, 793−810.
Alterio, V.; Pan, P.; Parkkila, S.; Buonanno, M.; Supuran, C. T.; Monti, S. M.; De Simone,
G. Biopolymers 2014, 101, 769−778.
22. De Monte, C.; Carradori, S.; Secci, D.; D’Ascenzio, M.; Guglielmi, P.; Mollica, A.;
Morrone, S.; Scarpa, S.; Aglianò, A. M.; Giantulli, S.; Silvestri, I. Eur. J. Med. Chem. 2015, 105,
245-262.
23.
Agents Med. Chem. 2012, 12, 589-603.
24. Secci, D.; Carradori, S.; Bizzarri, B.; Bolasco, A.; Ballario, P.; Patramani, Z.; Fragapane, P.;
Vernarecci, S.; Canzonetta, C.; Filetici, P. Bioorg. Med. Chem. 2014, 22, 1680-1689.
25. Trisciuoglio, D.; Ragazzoni, Y.; Pelosi, A.; Desideri, M.; Carradori, S.; Gabellini, C.;
Carradori, S.; Cristini, C.; Secci, D.; Gulia, C.; Gentile, V.; Di Pierro, G. B. Anti-Cancer
Maresca, G.; Nescatelli, R.; Secci, D.; Bolasco, A.; Bizzarri, B.; Cavaliere, C.; D'Agnano, I.;
Filetici, P.; Ricci-Vitiani, L.; Rizzo, M. G.; Del Bufalo, D. Clinical Cancer Res. 2012, 18, 475-486.
26.
Simone, G. Chem. Eur. J. 2016, 22, 97-100.
27. De Simone, G.; Pizika, G.; Monti, S. M.; Di Fiore, A.; Ivanova, J.; Vozny, I.; Trapencieris,
P.; Zalubovskis, R.; Supuran, C. T.; Alterio, V. BioMed Res. Int. 2014, 2014, 1−11.
28. Soler, L.; Cerrada, V.; Matía, M. P.; Novella, J. L.; Alvarez-Builla, J. Arkivoc 2007, 2007,
312–319.
Langella, E.; D’Ambrosio, K.; D’Ascenzio, M.; Carradori, S.; Monti, S.; Supuran, C. T.; De
29.
30.
31.
32.
Hettler, H. Tetrahedron Lett. 1968, 9, 1793–1796.
Mayr, H.; Breugst, M.; Ofial, A. R. Angew. Chemie - Int. Ed. 2011, 50, 6470–6505.
Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2015, DOI:10.3109/14756366.2015.1122001.
Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Granese, A.; Carradori, S.; Yáñez, M.;
Orallo, F.; Sanna, M. L.; Gallinella, B.; Cirilli, R. J. Med. Chem. 2010, 53, 6516-6520.
33. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Granese, A.; Carradori, S.; D’Ascenzio,
M.; Yáñez, M.; Orallo, F. Med. Chem. Commun. 2010, 1, 61-72.
34. Cheng, H. C. J. Pharmacol. Toxicol. Methods 2001, 46, 61–71.

35.
Y.; Scozzafava, A.; Supuran, C. T.; Williams, K. J. J. Med. Chem. 2012, 55, 5591–5600.
36. Akdemir, A.; De Monte, C.; Carradori, S.; Supuran, C. T. J. Enzyme Inhib. Med. Chem.
2015, 30, 114–118.
Gieling, R. G.; Babur, M.; Mamnani, L.; Burrows, N.; Telfer, B. A.; Carta, F.; Winum, J.

Graphical Abstract
A novel library of saccharin and acesulfame
derivatives as potent and selective inhibitors
of carbonic anhydrase IX and XII isoforms
Simone Carradori, Daniela Secci, Celeste De Monte, Adriano Mollica, Mariangela Ceruso, Atilla Akdemir,
Anatoly P. Sobolev, Rossella Codispoti, Federica De Cosmi, Paolo Guglielmi, Claudiu T. Supuran