N-Substituted and ring opened saccharin derivatives selectively inhibit transmembrane, tumor-associated carbonic anhydrases IX and XII

Article abstract of DOI:10.1016/j.bmc.2017.04.007

A series of N-substituted saccharins incorporating aryl, alkyl and alkynyl moieties, as well as some ring opened derivatives were prepared and investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The widespread cytosolic is

Full text of DOI:10.1016/j.bmc.2017.04.007

Accepted Manuscript
N-Substituted and ring opened saccharin derivatives selectively inhibit trans-
membrane, tumor-associated carbonic anhydrases IX and XII
Jekaterīna Ivanova, Fabrizio Carta, Daniela Vullo, Janis Leitans, Andris Kazaks,
Kaspars Tars, Raivis Žalubovskis, Claudiu T. Supuran
PII:
S0968-0896(17)30525-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.007
BMC 13677
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
15 March 2017
5 April 2017
Please cite this article as: Ivanova, J., Carta, F., Vullo, D., Leitans, J., Kazaks, A., Tars, K., Žalubovskis, R., Supuran,
C.T., N-Substituted and ring opened saccharin derivatives selectively inhibit transmembrane, tumor-associated
carbonic anhydrases IX and XII, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.
2017.04.007
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N-Substituted and ring opened saccharin derivatives selectively inhibit
transmembrane, tumor-associated carbonic anhydrases IX and XII
Jekaterīna Ivanova,1,2 Fabrizio Carta,3 Daniela Vullo,4 Janis Leitans,5 Andris
Kazaks,5 Kaspars Tars,5,6 Raivis Žalubovskis,1,2 Claudiu T. Supuran3*
1 Latvian Institute of Organic Synthesis, Aizkraukles Str. 21, Riga LV-1006, Latvia.
2 Institute of Technology of Organic Chemistry, Faculty of Materials Science and Applied
Chemistry, Riga Technical University, Paula Valdena Str. 3/7, Riga LV-1048, Latvia.
3Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di
ScienzeFarmaceutiche e Nutraceutiche, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence,
Italy
4Università degli Studi di Firenze, Dipartimento di Chimica, Laboratorio di Chimica
Bioinorganica, Polo Scientifico, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence,
Italy
5Latvian Biomedical Research and Study Center, Rātsupītes 1, LV-1067, Riga, Latvia.
6Faculty of Biology, Department of Molecular Biology, University of Latvia, Jelgavas 1, LV-
1004, Riga, Latvia
Abstract. A series of N-substituted saccharins incorporating aryl, alkyl and alkynyl
moieties, as well as some ring opened derivatives were prepared and investigated as
inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The
widespread cytosolic isoforms CA I and II were not inhibited by these sulfonamides
whereas transmembrane, tumor-associated ones were effectively inhibited, with KIs in
the range of 22.1 – 481 nM for CA IX and of 3.9 – 245 nM for hCA XII. Although
the inhibition mechanism of these tertiary/secondary sulfonamides is unknown for the
moment, the good efficacy and especially selectivity for the inhibition of the tumor-
associated over the cytosolic, widespread isoforms, make these derivatives of
considerable interest as enzyme inhibitors with various pharmacologic applications.
Keywords: carbonic anhydrase; inhibitor; saccharin; secondary/tertiary sulfonamide;
isoform selectivity
_____
* Corresponding authors: E-mail: raivis@osi.lv (RZ); claudiu.supuran@unifi.it (CTS)

1. Introduction
The carbonic anhydrases (CAs, EC 4.2.1.1) catalyze CO2 hydration to
bicarbonate and protons and the corresponding reverse reaction between bicarbonate
and protons, being among the most effective catalysts known in Nature.1,2 Since these
small molecules/ions are involved in many physiologic processes connected to pH
regulation, general homeostasis of cell/organisms, electrolyte secretion, ion transport,
metabolic pathways involving among others gluconeogenesis, urea and lipid
biosynthesis, as well as tumorigenesis, their production through the CA-catalyzed
reaction leads to pharmacologic responses which have been exploited for decades.1-3
Indeed, the primary sulfonamides, the most investigated class of CA inhibitors (CAIs)
has applications as diuretics, antiglaucoma, anticonvulsant and antiobesity drugs,
whereas in the last period anticancer/antimetastatic properties of these pharmacologic
agents were also investigated in detail.1-3
Unlike the primary sulfonamides, the secondary/tertiary ones, although much
investigated from the synthetic viewpoints,4-7 have found less pharmacologic
applications, although we and Klebe’s group reported the interesting CA inhibitory
effects of saccharin 1,8 an acylated secondary sulfonamide widely used as a
sweetening agent.
Considering the fact that CAs are widespread enzymes not only in humans,
but in most organisms on earth, with seven distinct genetic families known to date,9-11
a large number of novel applications of their inhibitors and activators were
investigated in the last decade, being shown that CAIs have a great potential in
pathologies for which their role was not recognized till recently, such as neuropathic
pain,12 arthritis,13 and cerebral ischemia.14 In fact, several CA isoforms of the 12
catalytically active known so far in humans,1,9,10 have recently been validated for
these new pharmacologic applications. Thus, the development of isoform-selective
CAIs is a challenging task, which has been successfully addressed through a variety
of methods in the last period, starting with structure-based drug design of sulfonamide
inhibitors and ending with the discovery of totally new chemotypes with such
properties, among which the coumarins,15 dithiocarbamates,16 polyamines,17 etc.
Many of them show diverse inhibition mechanisms apart Zn(II) ion coordination, as
shown schematically in Fig. 1. Among such new chemotypes with efficient CA
inhibitory properties and isoform-selective inhibition profiles, the secondary/tertiary

sulfonamides were reported only several years ago.18 As a particular example of such
derivatives are also the saccharins,2a,3 of which many examples have been
investigated in detail by this and Carradori’s group.3a,b Here we extend these earlier
studies,3a and report a novel series of N-substituted saccharins and the corresponding
opened ring compounds, which show interesting, isoform-selective CA inhibitory
properties.
Hydrophilic
part of active site
Hydrophilic
part of active site
TAIL
TAIL
O
Glu 106
O
Glu 106
SCAFFOLD
SCAFFOLD
N
O
N
O
AG
H
O
H
O
O
O
ZBG
H
H
OH2
Hydrophobic part
of active site
Hydrophobic part
of active site
N
N
Thr 199
Thr 199
Zn2+
Zn2+
His 119
His 119
His 94
His 94
His 96
His 96
A
B
INHIBITOR
Hydrophilic
part of active site
Hydrophilic
part of active site
TAIL
O
Glu 106
O
Glu 106
SG
SCAFFOLD
N
O
N
O
H
O
O
H
O
O
H
OH2
Hydrophobic part
of active site
H
OH2
N
Hydrophobic part
of active site
N
Thr 199
Thr 199
Zn2+
Zn2+
His 119
His 119
His 94
His 94
His 96
His 96
C
D
Fig. 1. CA inhibition mechanisms: A. Zinc-binders; B. Compounds which anchor to
the metal ion coordinated water/hydroxide ion; C. Occlusion of the active site
entrance. D. Out of the active site binding.1,9
2. Results and Discussions
2.1. Chemistry
Continuing our drug design of saccharin-type CAIs,3 the synthesis of N-substituted
saccharin derivatives (2–11) was performed according to the general procedure
presented in Scheme 1. Considering the fact that several such derivatives were
reported earlier (e.g., 3 and 11) and that they showed an interesting biological activity,

we decided to increase the number of compounds bearing a similar substitution
pattern as 3 and 11, including both aromatic R moieties with a diverse substitution
pattern, as well as aliphatic ones, which were less investigated to date for N-
substituted saccharin CAIs.2,3a
O
O
O
O
R
O
O
N
S
N
H
S
S
(i)
(ii)
NH
R
OH
O
O
O
1
2 (74%)
3 (65%)
R=
12 (56%)
R=
Ph
Ph
13 (79%)
Br
Br
Me
4 (76%)
5 (76%)
14 (70%)
15 (60%)
NO2
Me
Me
Me
16 (70%)
17 (68%)
Me
6 (65%)
7 (32%)
Me
Me
Me
8 (60%)
9 (78%)
Me
Ph
Cl
10 (32%)
O
11 (69%)
Cl
Scheme 1. Reagents and conditions: (i) 1) K2CO3, H2O, 2) R-Br, DMF, 90 ºC, 16h;
(ii) NaOH, THF/MeOH/water (2:1:2), RT, 16h.
The procedure involves preparation of saccharin potassium salt by reaction saccharin
(1) (1 equiv) with potassium carbonate (0.5 equiv) in water with following
replacement of solvent and reaction with appropriate benzyl or alkyl bromide. N-
benzyl (2-4) and alkyl (5-10) derivatives were mostly obtained in good yields. N-
benzoyl derivative 11 was obtained by reaction of saccharin potassium salt prepared
us above with corresponding benzoyl chloride in 69% yield. For the synthesis of
derivatives of open-saccharin (12-17) we applied previous developed strategy, where
corresponding saccharin derivative was hydrolyzed by treatment with sodium
hydroxide with following acidic work-up. The desired carboxylic acids (12-17) were
obtained in moderate to good yields.
2.2. Carbonic anhydrase inhibition

We assessed the CA inhibitory activity of compounds 1 - 17, using the clinical drug
acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide, AAZ) as positive
control, for the inhibition of four CA isoforms of medical importance, hCA I and II
(cytosolic, widely distributed enzymes) as well as hCA IX and XII (transmembrane,
tumor-associated enzymes) – Table 1.
Table 1. CA inhibition data against isoforms hCA I, II, IX and XII with compounds
1-17 and acetazolamide (AAZ) as standard, by a stopped-flow CO2 hydrase assay.19
_____________________________________________________________________
Compound
KI (nM)*
hCA IX
_____________________________________________________________________
hCA I
hCA II
hCA XII
1 (SAC)
2
3 3a
43a
5
18,540
>10,000
>10,000
324
5950
103
89.2
22.1
1169
236
209
164
102
563
76.1
191
342
277
481
436
392
250
25
633
18.7
4.3
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
12
29.0
45.1
37.9
36.5
34.0
43.7
10.4
3.9
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
1480
6
7
8
9
10
113a
12
13
14
15
16
17
AAZ
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
250
125
86.4
245
227
151
74.6
5.6
_____________________________________________________________________
*Errors in the range of 5 % of the reported values, from three different assays.
Compounds 3, 4 and 11 were already reported in ref. 3a

Data of Table 1 show the following structure-activity relationship (SAR) for the
inhibition of CAs with sulfonamides 2-17 investigated here:
(i) The cytosolic isoforms hCA I and II are not inhibited by the new sulfonamides up
to 10 µM concentration of inhibitor in the assay system, a feature which has already
been observed for other saccharin derivatives investigated earlier.3 In fact, only two
compounds show a weak hCA I inhibitory action, 4 and 11, with KIs in the range of
324-1480 nM (Table 1). This behavior of poor inhibitory action against these
widespread cytosolic enzymes is a favorable feature for compounds for which
isoform-selectivity is searched for, such as for example derivatives which should
target the tumor-associated isoforms hCA IX and XII.1,9-11
(ii) Indeed, hCA IX was rather efficiently inhibited by most secondary/tertiary
sulfonamides 2-17, with KIs in the range of 22.1 – 1169 nM (Table 1). It should be
observed that saccharin itself is a medium potency CA IX inhibitor, with an inhibition
constant of 103 nM,8 whereas acetazolamide AAZ is roughly 4-time a better inhibitor
compared to 1, with a KI of 25 nM. Compounds 2, 3 and 10 reported here were the
best hCA IX inhibitors, with KIs in the range of 22.1 – 89.2 nM. They incorporate
benzyl, 4-bromobenzyl and phenethyl R moieties. It is interesting to note that a
bromine in para (as in 3) leads to a 4-times increase of the CA IX inhibitory action
compared to the unsubstituted benzyl derivative 2, whereas the replacement of the
bromine by a nitro moiety greatly reduces inhibitory action (as in 4, which is 53 times
a weaker CA IX inhibitor compared to 3). The remaining compounds, irrespective of
the nature of R (aromatic, aliphatic or unsaturated) showed a behavior of medium-
weak CA IX inhibitor, with inhibition constants in the range of 103 – 481 nM (Table
1). The “opened” saccharins (compounds 12-17) showed a rather modest activity as
hCA IX inhibitors (Table 1).
(iii) hCA XII was on the other hand better inhibited by the investigated sulfonamides
compared to hCA IX, with KIs in the range of 3.9 – 245 nM. All these derivatives
were much better inhibitors compared to saccharin 1 (KI of 633 nM against this
isoform). Derivatives 3 and 11 were the best inhibitors (KIs in the range of 3.9 – 4.3
nM) and they incorporate 4-bromobenzyl and 2,4-dichlorobenzoyl moieties as R
group. Other efficient hCA XII inhibitors were 2, and 4-10, which had KIs in the
range of 10.4 – 45.1 nM. It may be observed that they incorporate a variety of R
groups, among which benzyl, 4-nitrobenzyl, as well as aliphatic and unsaturated
moieties with 2-5 carbon atoms in the chain. The least effective hCA XII inhibitors

were 14 and 15, which are “opened” saccharins incorporating a secondary
sulfonamide and a COOH moiety.
3. Conclusions
Continuing our research of isoform-selective CAIs belonging to the secondary/tertiary
sulfonamide chemotype, we investigated here a series of N-substituted saccharins
incorporating aryl, alkyl and alkynyl moieties, as well as some ring opened
derivatives, all of which were prepared from saccharin. The widespread cytosolic
isoforms CA I and II were not inhibited by these sulfonamides whereas
transmembrane, tumor-associated ones were effectively inhibited, with KIs in the
range of 22.1 – 481 nM for CA IX and of 3.9 – 245 nM for hCA XII. Although the
inhibition mechanism of these tertiary/secondary sulfonamides is unknown for the
moment, the good efficacy and especially selectivity for the inhibition of the tumor-
associated over the cytosolic, widespread isoforms, make these derivatives of
considerable interest as enzyme inhibitors with various pharmacologic applications.
4. Experimental part
4.1. Chemistry
Reagents, starting materials and solvents were obtained from commercial sources and
used as received. Thin-layer chromatography was performed on silica gel, spots were
visualized with UV light (254 and 365 nm). Melting points were determined on an
OptiMelt automated melting point system. NMR spectra were recorded on Varian
Mercury (400 MHz) spectrometer with chemical shifts values (δ) in ppm relative to
TMS using the residual DMSO-d6 signal (1H 2.50; 13C 39.52) as an internal standard.
In the IR spectra of the new compounds the strong SO2 vibrations at 1170 and 1340
cm-1 were present, together with the CON band at 1745 cm-1 (data not shown)
General procedure for the synthesis of 2-alkyl-1,2-benzothiazol-3(2H)-one 1,1-
dioxides (2-10)
Saccharin (1) (1 equiv) was suspended in H2O (20 mL), potassium carbonate
(0.5 equiv) was slowly added, and solvent was driven off in vacuum. The dry residue
was suspended in DMF (10 mL) and corresponding bromide (1 equiv) was added. The

reaction mixture was stirred at 90 ºC for 16 h. After cooling to room temperature
CH2Cl2 (20 mL), H2O (20 mL), and 2 M HCl (20 mL) were added, and it was
extracted with CH2Cl2 (3×20 mL). Organic layer was washed with brine (30 mL) and
dried over Na2SO4. CH2Cl2 was driven off in vacuum. Addition of H2O to the
remaining DMF solution resulted in precipitate formation, which were collected and
dried.
O
O
S
N
O
2-Benzyl-1,2-benzothiazol-3(2H)-one 1,1-dioxide (2)4
Compound 2 was prepared according to the general procedure from saccharin
(1) (0.50 g, 2.73 mmol), potassium carbonate (0.19 g, 1.37 mmol) and benzyl bromide
1
(0.32 mL, 2.73 mmol) as white solid (0.55 g, 74%). Mp 123-124 ºC. H NMR (400
MHz, DMSO-d6) δ= 8.36-8.32 (m, 1H), 8.14-8.11 (m, 1H), 8.10-7.99 (m, 2H), 7.45-
7.40 (m, 2H), 7.39-7.28 (m, 3H), 4.92 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ=
158.6, 136.9, 135.9, 135.3, 135.1, 128.5, 127.9, 127.8, 126.2, 125.2, 121.6, 41.7.
O
O
S
N
O
Br
2-(4-Bromobenzyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (3) 3a
Compound 3 was prepared according to the general procedure from saccharin
(1) (0.20 g, 1.09 mmol), potassium carbonate (0.08 g, 0.55 mmol) and 4-bromo
benzyl bromide (0.27 g, 1.09 mmol) as white solid (0.25 g, 65%). Mp 168-169 ºC. 1H
NMR (400 MHz, DMSO-d6) δ= 8.36-8.33 (m, 1H), 8.14-8.10 (m, 1H), 8.07 (td, 1H, J
= 7.6, 1.2 Hz), 8.01 (td, 1H, J = 7.6, 1.2 Hz), 7.59-7.54 (m, 2H), 7.41-7.37 (m, 2H),
4.91 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ= 158.6, 136.8, 136.0, 135.4, 134.7,
131.4, 130.1, 126.2, 125.2, 121.7, 121.0, 41.0.
O
O
S
N
O
NO
2

2-(4-Nitrobenzyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (4) 3a
Compound 4 was prepared according to the general procedure from saccharin
(1) (0.50 g, 2.73 mmol), potassium carbonate (0.19 g, 1.37 mmol) and 4-nitrobenzyl
1
bromide (0.59 g, 2.73 mmol) as white solid (0.66 g, 76%). Mp 190-191 ºC. H NMR
(400 MHz, DMSO-d6) δ= 8.39-8.35 (m, 1H), 8.26-8.21 (m, 2H), 8.16-8.13 (m, 1H),
8.09 (td, 1H, J = 7.6, 1.2 Hz), 8.03 (dt, 1H, J = 7.6, 1.2 Hz), 7.72-7.67 (m, 2H), 5.10
(s, 2H); 13C NMR (100 MHz, DMSO-d6) δ= 158.7, 147.1, 142.9, 136.8, 136.0, 135.4,
128.9, 126.2, 125.3, 123.7, 121.8, 40.9.
O
O
S
N
Me
O
2-Ethyl-1,2-benzothiazol-3(2H)-one 1,1-dioxide (5) 5
Compound 5 was prepared according to the general procedure from saccharin
(1) (0.50 g, 2.73 mmol), potassium carbonate (0.19 g, 1.37 mmol) and ethyl bromide
1
(0.20 mL, 2.73 mmol) as white solid (0.36 g, 63%). Mp 104-105 ºC. H NMR (400
MHz, DMSO-d6) δ= 8.32-8.27 (m, 1H), 8.13-8.08 (m, 1H), 8.08-8.02 (m, 1H), 8.01-
7.97 (m, 1H), 3.77 (q, 2H, J = 7.2 Hz), 1.31 (t, 3H, J = 7.2 Hz); 13C NMR (100 MHz,
DMSO-d6) δ= 158.2, 136.9, 135.7, 135.1, 126.4, 124.9, 121.4, 33.8, 13.7.
O
O
S
N
Me
O
2-Propyl-1,2-benzothiazol-3(2H)-one 1,1-dioxide (6) 4
Compound 6 was prepared according to the general procedure from saccharin
(1) (0.50 g, 2.73 mmol), potassium carbonate (0.19 g, 1.37 mmol) and n-propyl
1
bromide (0.25 mL, 2.73 mmol) as white solid (0.40 g, 65%). Mp 87-88 ºC. H NMR
(400 MHz, DMSO-d6) δ= 8.32-8.29 (m, 1H), 8.13-8.09 (m, 1H), 8.05 (td, 1H, J = 7.6,
1.3 Hz), 8.00 (td, 1H, J = 7.6, 1.3 Hz), 3.68 (t, 2H, J = 7.3 Hz), 1.75 (sextet, 2H, J =
7.3 Hz), 0.94 (t, 3H, J = 7.3 Hz); 13C NMR (100 MHz, DMSO-d6) δ= 158.6, 136.8,
135.7, 135.2, 126.3, 125.0, 121.5, 40.3, 21.3, 11.0.
O
O
S
N
Me
O

2-Butyl-1,2-benzothiazol-3(2H)-one 1,1-dioxide (7) 6
Compound 7 was prepared according to the general procedure from saccharin
(1) (0.50 g, 2.73 mmol), potassium carbonate (0.19 g, 1.37 mmol) and butyl bromide
(0.29 mL, 2.73 mmol). Additional purification by column chromatography (silica gel,
1
hexane/EtOAc 4:1) afforded 7 as colorless oil (0.21 g, 32%). H NMR (400 MHz,
DMSO-d6) δ= 8.28-8.25 (m, 1H), 8.09-8.05 (m, 1H), 8.03 (td, 1H, J = 7.5, 1.2 Hz),
7.97 (td, 1H, J = 7.5, 1.2 Hz), 3.69 (t, 2H, J = 7.3 Hz), 1.74-1.65 (m, 2H), 1.40-1.29
(m, 2H), 0.89 (t, 3H, J = 7.3 Hz); 13C NMR (100 MHz, DMSO-d6) δ= 158.6, 136.8,
135.7, 135.1, 126.3, 125.0, 121.4, 38.4, 29.9, 19.3, 13.3.
O
O
S
N
Me
O
6
2-Pentyl-1,2-benzothiazol-3(2H)-one 1,1-dioxide (8)
Compound 8 was prepared according to the general procedure from saccharin
(1) (0.30 g, 1.64 mmol), potassium carbonate (0.11 g, 0.82 mmol) and pentyl bromide
1
(0.20 mL, 1.64 mmol) as white solid (0.25 g, 60%). Mp 62-63 ºC. H NMR (400
MHz, DMSO-d6) δ= 8.32-8.28 (m, 1H), 8.12-8.09 (m, 1H), 8.05 (td, 1H, J = 7.5, 1.2
Hz), 8.00 (td, 1H, J = 7.5, 1.2 Hz), 3.70 (t, 2H, J = 7.3 Hz), 1.78-1.68 (m, 2H), 1.36-
1.28 (m, 4H), 0.90-0.83 (m, 3H); 13C NMR (100 MHz, DMSO-d6) δ= 158.6, 136.8,
135.7, 135.2, 126.3, 125.0, 121.5, 38.6, 28.2, 27.5, 21.5, 13.7.
O
O
S
N
O
Me
2-(But-2-yn-1-yl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (9)
Compound 9 was prepared according to the general procedure from saccharin
(1) (0.30 g, 1.64 mmol), potassium carbonate (0.11 g, 0.82 mmol) and 1-bromo-2-
1
butyne (0.14 mL, 1.64 mmol) as light brown solid (0.30 g, 78%). Mp 127-128 ºC. H
NMR (400 MHz, DMSO-d6) δ= 8.34-8.31 (m, 1H), 8.15-8.12 (m, 1H), 8.07 (td, 1H, J
= 7.6, 1.2 Hz), 8.01 (td, 1H, J = 7.6, 1.2 Hz), 4.52 (q, 2H, J = 2.5 Hz), 1.80 (t, 3H, J =
2.5 Hz); 13C NMR (100 MHz, DMSO-d6) δ= 157.9, 137.0, 136.0, 135.3, 126.0, 125.2,
121.6, 80.3, 72.4, 27.9, 3.0.

O
O
N
S
O
2-(2-Phenylethyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (10)
Compound 10 was prepared according to the general procedure from saccharin
(1) (0.30 g, 1.64 mmol), potassium carbonate (0.11 g, 0.82 mmol) and phenylethyl
1
bromide (0.22 mL, 1.64 mmol) as white solid (0.15 g, 32%). Mp 139-140 ºC. H
NMR (400 MHz, DMSO-d6) δ= 8.33-8.30 (m, 1H), 8.09-8.03 (m, 2H), 7.99 (td, 1H, J
= 7.5, 1.1 Hz), 7.33-7.27 (m, 4H), 7.25-7.19 (m, 1H), 3.98-3.92 (m, 2H), 3.07-3.02
(m, 2H); 13C NMR (100 MHz, DMSO-d6) δ= 158.3, 137.6, 136.8, 135.8, 135.2,
128.7, 128.4, 126.6, 126.2, 125.0, 121.5, 39.7, 33.9.
O
O
S
O
N
Cl
Cl
O
2-(2,4-Dichlorobenzoyl)-1,2-benzothiazol-3(2H)-one 1,1-dioxide (11) 3a
Saccharin (1) (0.30 g, 1.64 mmol) was suspended in acetone (5 mL), reaction
mixture was cooled to 0 ºC, triethylamine (0.25 mL, 1.80 mmol ) was added followed
by 2,4-dichlorobenzoyl chloride (0.25 mL, 1.80 mmol). It was allowed to reach room
temperature and stirred at room temperature for 16 h. Reaction mixture was poured
onto ice, precipitate were collected and washed with water. Recrystallization from
DCM/MeOH afforded 11 (0.40 g, 69%) as white solid. Mp 221-222 ºC. 1H NMR (400
MHz, DMSO-d6) δ= 8.46 (dt, 1H, J = 7.8, 0.8 Hz), 8.22-8.14 (m, 2H), 8.11-8.06 (m,
1H), 7.84 (d, 1H, J = 2.0 Hz), 7.78 (d, 1H, J = 8.3 Hz), 7.63 (dd, 1H, J = 8.3, 2.0 Hz);
13C NMR (100 MHz, DMSO-d6) δ= 163.2, 156.1, 137.7, 137.1, 136.7, 136.0, 132.1,
130.8, 130.6, 129.2, 127.9, 126.5, 124.3, 121.9.
General procedure for the synthesis of 2-(alkylsulfamoyl)benzoic acids (12-17)
The appropriate 1,2-benzothiazol-3(2H)-one 1,1-dioxide (1 equiv) was dissolved
in a mixture of THF/MeOH/water (2:1:2). NaOH (5 equiv) was added to the reaction
mixture. The reaction mixture was stirred at room temperature for 16 h, acidified with
HCl (conc.), the solvent was evaporated. The crude product was re-crystallized from
water and dried in vacuum.

O
O
S
N
H
OH
O
2-(Benzylsulfamoyl)benzoic acid (12) 7
Compound 12 was prepared according to the general procedure from compound
2 (0.20 g, 0.73 mmol) and NaOH (0.15 g, 3.66 mmol) as white solid (0.12 g, 56%).
Mp 143-144 ºC. 1H NMR (400 MHz, DMSO-d6) δ= 7.85-7.82 (m, 1H), 7.72 (t, 1H, J
13
= 6.4 Hz), 7.68-7.59 (m, 3H), 7.28-7.17 (m, 5H), 4.10 (d, 2H, J = 6.4 Hz); C NMR
(100 MHz, DMSO-d6) δ= 169.0, 138.0, 137.7, 133.0, 132.4, 130.6, 128.9, 128.3,
128.2, 127.6, 127.1, 46.2.
O
O
S
N
H
Br
OH
O
2-[(4-Bromobenzyl)sulfamoyl]benzoic acid (13)
Compound 13 was prepared according to the general procedure from compound
3 (0.12 g, 0.34 mmol) and NaOH (0.07 g, 1.70 mmol) as white solid (0.10 g, 79%).
Mp 181-182 ºC. 1H NMR (400 MHz, DMSO-d6) δ= 7.86-7.79 (m, 2H), 7.70-7.58 (m,
3H), 7.45-7.41 (m, 2H), 7.22-7.17 (m, 2H), 4.08 (d, 2H, J = 5.8 Hz); 13C NMR (100
MHz, DMSO-d6) δ= 169.0, 137.9, 137.4, 133.1, 132.4, 131.0, 130.6, 129.8, 128.9,
128.3, 120.2, 45.4.
O
Me
O
S
N
H
OH
O
2-(Propylsulfamoyl)benzoic acid (14)
Compound 14 was prepared according to the general procedure from compound
6 (0.25 g, 1.11 mmol) and NaOH (0.22 g, 5.55 mmol) as white solid (0.19 g, 70%).
Mp 106-107 ºC. 1H NMR (400 MHz, DMSO-d6) δ= 7.92-7.86 (m, 1H), 7.72-7.65 (m,
3H), 7.11 (t, 1H, J = 6.0 Hz), 2.84-2.77 (m, 2H), 1.38 (sextet, 2H, J = 7.4 Hz), 0.77 (t,
3H, J = 7.4 Hz); 13C NMR (100 MHz, DMSO-d6) δ= 169.0, 137.8, 133.2, 132.4,
130.7, 129.0, 128.3, 44.5, 22.5, 11.0.

O
Me
O
S
N
H
OH
O
2-(Butylsulfamoyl)benzoic acid (15)
Compound 15 was prepared according to the general procedure from compound
7 (0.20 g, 0.84 mmol) and NaOH (0.17 g, 4.20 mmol) as white solid (0.13 g, 60%).
Mp 112-113 ºC. 1H NMR (400 MHz, DMSO-d6) δ= 7.93-7.86 (m, 1H), 7.72-7.65 (m,
3H), 7.09 (t, 1H, J = 6.0 Hz), 2.87-2.80 (m, 2H), 1.39-1.30 (m, 2H), 1.26-1.15 (m,
2H), 0.77 (t, 3H, J = 7.4 Hz); 13C NMR (100 MHz, DMSO-d6) δ= 158.6, 136.8,
135.7, 135.2, 126.3, 125.0, 121.5, 38.4, 29.9, 19.3, 13.3.
Me
O
O
S
N
H
OH
O
2-(Pentylsulfamoyl)benzoic acid (16)
Compound 10 was prepared according to the general procedure from compound
8 (0.16 g, 0.63 mmol) and NaOH (0.13 g, 3.16 mmol) as white solid (0.12 g, 70%).
Mp 133-134 ºC. 1H NMR (400 MHz, DMSO-d6) δ= 7.91-7.86 (m, 1H), 7.73-7.64 (m,
3H), 7.09 (t, 1H, J = 5.8 Hz), 2.87-2.80 (m, 2H), 1.40-1.32 (m, 2H), 1.21-1.13 (m,
4H), 0.81-0.75 (m, 3H); 13C NMR (100 MHz, DMSO-d6) δ= 169.0, 137.8, 133.2,
132.4, 130.6, 129.0, 128.2, 42.6, 28.6, 28.1, 21.5, 13.7.
O
O
S
N
H
Me
OH
O
2-(But-2-yn-1-yl-sulfamoyl)benzoic acid (17)
Compound 10 was prepared according to the general procedure from compound
9 (0.15 g, 0.64 mmol) and NaOH (0.13 g, 3.20 mmol) as brown solid (0.11 g, 68%).
Mp 145-146 ºC. 1H NMR (400 MHz, DMSO-d6) δ= 7.96-7.91 (m, 1H), 7.74-7.68 (m,
3H), 7.38 (t, 1H, J = 6.1 Hz), 3.77-3.72 (m, 2H), 1.49 (t, 3H, J = 2.4 Hz); 13C NMR
(100 MHz, DMSO-d6) δ= 168.9, 138.3, 132.8, 132.5, 130.6, 129.2, 128.8, 80.1, 74.5,
32.6, 2.8.
CA inhibition assay

An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument has
been used to assay the catalytic/inhibition of various CA isozymes.19 Phenol Red (at a
concentration of 0.2 mM) has been used as indicator, working at the absorbance
maximum of 557 nm, with 10 mM Hepes (pH 7.4) as buffer, 0.1 M Na2SO4 or
NaClO4 (for maintaining constant the ionic strength; these anions are not inhibitory in
the used concentration),20 following the CA-catalyzed CO2 hydration reaction for a
period of 5-10 s. Saturated CO2 solutions in water at 25 °C were used as substrate.
Stock solutions of inhibitors were prepared at a concentration of 10 mM (in DMSO-
water 1:1, v/v) and dilutions up to 1 nM done with the assay buffer mentioned above.
At least 7 different inhibitor concentrations have been used for measuring the
inhibition constant. Inhibitor and enzyme solutions were preincubated together for 10
min at room temperature prior to assay, in order to allow for the formation of the E-I
complex. Triplicate experiments were done for each inhibitor concentration, and the
values reported throughout the paper are the mean of such results. The inhibition
constants were obtained by non-linear least-squares methods using the Cheng-Prusoff
equation, as reported earlier,20 and represent the mean from at least three different
determinations. All CA isozymes used here were recombinant proteins obtained as
reported earlier by our group.21-25
Acknowledgments. This work was financed in part by two EU FP7 projects,
InnovaBalt and Dynano.

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N-Substituted and ring opened saccharin derivatives selectively inhibit
transmembrane, tumor-associated carbonic anhydrases IX and XII
Jekaterīna Ivanova,1,2 Fabrizio Carta,3 Daniela Vullo,4 Janis Leitans,5 Andris
Kazaks,5 Kaspars Tars,5,6 Raivis Žalubovskis,1,2 Claudiu T. Supuran3*
O
O
O
O
R
O
O
N
S
N
H
S
S
(i)
(ii)
NH
R
OH
O
O
O
1
2 (74%)
3 (65%)
R=
12 (56%)
R=
Ph
Ph
13 (79%)
Br
Br
Me
4 (76%)
5 (76%)
14 (70%)
15 (60%)
NO2
Me
Me
Me
16 (70%)
17 (68%)
Me
6 (65%)
7 (32%)
Me
Me
Me
8 (60%)
9 (78%)
Me
Ph
Cl
10 (32%)
O
11 (69%)
Cl