Synthesis and carbonic anhydrase inhibition of a series of SLC-0111 analogs

Article abstract of DOI:10.1016/j.bmc.2017.03.027

SLC-0111 is a sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor (CAI) in Phase I/II clinical trials for the treatment of advanced hypoxic tumors complicated with metastases. Its antitumor effects are due to inhibition of the enzymatic activity of CA IX, an isoform predominantly found in tumors/metastases, but it also reduces the cancer stem cells population. Here we report the synthesis of analogs of SLC-0111, both of the sulfanilamide and metanilamide series, which possess diverse substitution patterns at the terminal ureido-phenyl moiety, thus including one or more halogens, trifluoromethyl, perchloro-/perfluorophenyl groups instead of the 4-fluorophenyl present in SLC-0111. Most of the sulfanilamide ureido derivatives were highly effective inhibitors of the tumor associated isoform and some showed selective CA IX/XII inhibitory profiles. Most of the sulfanilamide ureido derivatives were highly effective and in some cases selective CA IX/XII inhibitors, whereas the metanilamide ureido derivatives were less effective as transmembrane CA isoforms inhibitors. Structure activity relationship for this class of sulfonamides is discussed in detail.

Full text of DOI:10.1016/j.bmc.2017.03.027

Accepted Manuscript
Synthesis and carbonic anhydrase inhibition of a series of SLC-0111 analogs
Fabrizio Carta, Daniela Vullo, Sameh M. Osman, Zeid AlOthman, Claudiu T.
Supuran
PII:
S0968-0896(17)30352-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.027
BMC 13625
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 February 2017
9 March 2017
15 March 2017
Please cite this article as: Carta, F., Vullo, D., Osman, S.M., AlOthman, Z., Supuran, C.T., Synthesis and carbonic
anhydrase inhibition of a series of SLC-0111 analogs, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.03.027
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.

Synthesis and carbonic anhydrase inhibition of a series of SLC-0111 analogs
Fabrizio Carta,1 Daniela Vullo,2 Sameh M. Osman,3 Zeid AlOthman,3 and
Claudiu T. Supuran*1,3
1Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e
Nutraceutiche, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy.
2Università degli Studi di Firenze, Dipartimento di Chimica Ugo Schiff, Laboratorio di Chimica
Bioinorganica, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy.
3Department of Chemistry, College of Science, King Saud University, P.O. Box 2455 Riyadh
11451, Saudi Arabia.
Abstract. SLC-0111 is a sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor (CAI) in
Phase I/II clinical trials for the treatment of advanced hypoxic tumors complicated with metastases.
Its antitumor effects are due to inhibition of the enzymatic activity of CA IX, an isoform
predominantly found in tumors/metastases, but it also reduces the cancer stem cells population.
Here we report the synthesis of analogs of SLC-0111, both of the sulfanilamide and metanilamide
series, which possess diverse substitution patterns at the terminal ureido-phenyl moiety, thus
including one or more halogens, trifluoromethyl, perchloro-/perfluorophenyl groups instead of the
4-fluorophenyl present in SLC-0111. Most of the sulfanilamide ureido derivatives were highly
effective inhibitors of the tumor associated isoform and some showed selective CA IX/XII
inhibitory profiles. Most of the sulfanilamide ureido derivatives were highly effective and in some
cases selective CA IX/XII inhibitors, whereas the metanilamide ureido derivatives were less
effective as transmembrane CA isoforms inhibitors. Structure activity relationship for this class of
sulfonamides is discussed in detail.
Keywords: carbonic anhydrase; sulfonamide; inhibitor; tumor; urea derivative; SLC-0111
______
*
Corresponding author: Phone: +39-055-4573005; Fax: +39-055-4573385; e-mail:
claudiu.supuran@unifi.it (Claudiu T. Supuran).

1. Introduction
Many tumor 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 (pHe of around 6.0 – 6.5) and the intracellular pH more alkaline than the normal
(pHi of around 7.5).1-3 This is known for decades as the Warburg effect,4 but only in the last period
it has been demonstrated that a transcription factor, the hypoxia-inducible factor (HIF) 1α, is the
main player in these processes, through the overexpression of proteins involved in pH regulation,
glucose metabolism, and erythropoiesis.1-5 Several studies showed that the metalloenzyme carbonic
anhydrase (CA, EC 4.2.1.1),6-10 also involved in several processes related to tumorigenesis, tumor
progression and metastases formation, is also highly overexpressed in many tumors, as a
downstream target of HIF-1α activation.11,12
CAs are highly effective catalysts for the reversible hydration of carbon dioxide with
formation of bicarbonate and protons, being widespread in all life kingdoms, with seven genetically
distinct families known to date.6-10,13,14 Thus, CAs are involved in many physiological processes
connected with pH regulation,1-3 electrolyte secretion,15 metabolic processes,16 tumorigenesis,11,12
etc., and their inhibition leads to pharmacological effects.17,18 Indeed, sulfonamide CA inhibitors
(CAIs) are clinically used as diuretics, antiglaucoma, anticonvulsant, antiobesity and antitumor
agents for decades.17-20 Many drug design strategies are presently available for obtaining effective
and isoform-selective such agents belonging to various chemotypes, but the primary sulfonamides
still remain among the most investigated CAIs due to their high affinity for many CA isoforms of
pharmacologic interest, rather convenient pharmacology and ease of preparation.21,22
A sulfonamide CAI reported by one of our groups, SLC-0111,23a (Fig. 1) was shown to be a
highly effective inhibitor of CA IX/XII, two isoforms predominantly found in tumors,1-4 and to
possess a significant antitumor/antimetastatic effect in animal models of cancer.23-25 Furthermore,
the cancer stem cell population was also diminished after treatment with this drug.26
SO2NH2
O
NH
NH
F
Fig. 1. Structure of SLC-0111, a sulfonamide CAI in Phase I/II clinical trials as antitumor drug.24

SLC-0111 entered in Phase I clinical trials in 2014 for the treatment of advanced, metastatic
solid tumors overexpressing CA IX/XII.24 The Phase I clinical trial finished successfully at the end
of 2016, and the compound is currently scheduled to enter Phase II trials this year. 24
Being the first-in-class CAI with anticancer/antimetastatic action (and also acting on the
cancer stem cell population in a favorable manner, diverse from all other anticancer agents used
clinically, which favor the increase of this stem cell population),26 SLC-0111 has been used as a
lead compound for designing other agents with a similar pharmacologic action.27 Here we present a
new study, reporting a series of derivatives structurally related to SLC-0111, which were
investigated as inhibitors of four physiologically important CA isoforms, the human (h) hCA I and
II (cytosolic, off-target isoforms) and hCA IX/XII (anticancer drug targets).1-3,11,12
2. Results and Discussion
2.1. Chemistry
The rationale of this work is straightforward and simple. We used SLC-0111 as lead compound, and
we modified its 4-fluorophenylureido fragment. (i) A first series of sulfanilamide ureido derivatives
were obtained by coupling commercially available sulfanilamide A with aryl-isocyanates, leading to
ureas 1-15, which are structurally very similar to SLC-0111 except for the various substituents
present on the aromatic tail section. Indeed, the fluorine atom present in the lead was replaced by
one or more halogens (identical or different ones), trifluoromethyl and nitro moieties (Scheme 1).
1; Ar= 4-Chloro-2-fluorophenyl
2; Ar= 4-Bromo-2-fluorophenyl
3; Ar= 2-Fluoro-5-nitrophenyl
4; Ar= 2,4,5-Trifluorophenyl
5; Ar= 2-Fluoro-5-trifluorophenyl
O
C
6; Ar= 2-Fluoro-3-trifluorophenyl
7; Ar= 2,3,4-Trifluorophenyl
8; Ar= 2-Fluorophenyl
9; Ar= 2,4-Difluorophenyl
10; Ar= 3-Chlorophenyl
11; Ar= 2,5-Dichlorophenyl
12; Ar= 2-Chloro-5-nitrophenyl
13; Ar= 2-Chloro-4-trifluorophenyl
14; Ar= 2,6-Difluorophenyl
15; Ar=Perchlorophenyl
H NO S
2
H NO S
2
2
2
Ar
N
O
Ar
NH
ACN
N
H
N
H
2
1-15
A
O
SO2NH2
16; Ar= 4-Iodophenyl
17; Ar= 4-Fluorophenyl
18; Ar= 3-Nitrophenyl
19; Ar= 4-Acetylphenyl
20; Ar= 2-Isopropylphenyl
21; Ar= Perflurophenyl
C
H2NO2S
NH2
Ar
N
O
Ar
N
H
N
H
ACN
B
16-21
Scheme 1. Synthesis of ureido-benzenesulfonamides 1-21 from sulfanilamide A, metanilamide B
and aryl-isocyanates.

Fig. 2: X-ray crystal structure of the hCA II – SLC-0111 adduct.23b The zinc ion is shown as a gray
sphere, its three His ligands and amino acid residues involved in the binding of the inhibitor (one
letter amino acid code) are represented in yellow. The electron density of the bound inhibitor is
shown.
The reasons for these changes are due to the fact that the X-ray crystal structure of SLC-0111 (and
some of its analogs) bound to hCA II, 23b showed variable orientations of the terminal aromatic ring,
which, in turn, were correlated with the efficacy/selectivity as CAIs of these compounds against the
various isoforms.23
(ii) We explored the synthesis of SLC-0111 regioisomers, of the type 16-21, which were obtained
from metanilamide B and arylisocyanates (Scheme 1). The main difference between these
derivatives and SLC-0111/1-15 is the meta position of the primary sulfonamide (-SO2NH2) moiety.
As for the previously mentioned compounds (1-15), one or more halogens (identical or different
ones), trifluoromethyl, nitro, acetyl and iso-propyl moieties were present in the aryl-isocyanates
used as starting materials, in order to increase the explored chemical space. We stress again, X-ray
crystallographic data of enzyme – SLC-0111 and analogs complexes demonstrated that the nature
of the second aryl moiety is the most important factor governing CA inhibitory activity as well as
selectivity towards various isoforms of pharmacological interest for this class of sulfonamide
CAIs.23
2.2. CA inhibition
We assessed the CA inhibitory activity of compounds 1 - 21, employing the clinically used
drug acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide, AAZ) and SLC-0111 as positive

controls, 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 with ureidosubstituted benzenesulfonamides 1-21, and acetazolamide
(AAZ)/SLC-0111 as standard drugs, by a stopped-flow CO2 hydrase assay.28
Compound
KI (nM)*
hCA IX
hCA I
hCA II
hCA XII
1
436
373
519
569
363
395
484
550
275
158
747
307
192
158
538
1680
3260
2590
4800
1560
318
5080
250
162
418
276
215
170
196
335
33.0
60.6
10.0
58.2
0.94
0.97
0.93
362
24.9
17.2
97.4
258
410
263
960
12.0
5.1
6.3
7.3
7.6
7.4
8.7
9.6
7.7
3.7
8.4
8.9
8.2
8.4
6.1
6.3
116
219
154
178
349
162
45
7.9
2
7.4
3
7.6
4
6.2
5
10.1
8.5
6
7
6.4
8
7.3
9
7.1
10
8.5
11
6.8
12
6.1
13
8.2
14
8.3
15
7.6
16
96.4
113
78.5
66.9
83.6
90.2
4.5
17
18
19
20
21
SLC-0111
AAZ
25.2
5.6

* Errors in the range of 5 % of the reported values, from 3 different determinations (data not
shown).
The following structure-activity relationship (SAR) can be drawn from data of Table 1:
(i) hCA I, a widely abundant cytosolic isoform in the gastro-intestinal tract and red blood cells,6-8
was moderately inhibited by sulfonamides 1-21, with KIs in the range of 158-4800 nM (Table 1). It
should be observed that AAZ is also a moderate hCA I inhibitor (KI of 250 nM) whereas SLC-0111
is a much weaker one (KI of 5080 nM), which is a positive feature of the candidate drug, as hCA I
is in this case an off-target isoform. The sulfanilamide urea derivatives 1-15 were better hCA I
inhibitors compared to the metanilamide ureas 16 – 21. Furthermore, the presence of additional
groups on the second aromatic ring from SLC-0111, as in derivatives 1-15, had as a consequence
the increase of the affinity for hCA I from the micromolar range (SLC-0111) to the high nM range
for the new derivatives reported here. For example 7, which has two more fluorine atoms compared
to SLC-0111, was 10.5 times a better hCA I inhibitor compared to the lead, and this is a general
trend for the other derivatives 1-15 reported here. Only the metanilamide 16-21 maintained the
micromolar affinity for hCA I, similar to SLC-0111 (Table 1).
(ii) The physiologically dominant cytosolic isoform, hCA II, showed a very interesting inhibition
profile with the new sulfonamides reported here. Thus, unlike SLC-0111 which is a poor inhibitor
of this isoform (KI of 960 nM), many of the new sulfonamides were low nanomolar or even
subnanomolar hCA II inhibitors, others had a medium potency inhibitory profile whereas some
others were medium-weak inhibitors. The KIs against hCA II ranged between 0.93 and 418 nM
(Table 1). AAZ is also an efficient hCA II inhibitor, with a KI of 12.0 nM. The most efficient hCA
II inhibitors in the series were 10, 12-14, 16 and 17. The last two derivatives are metanilamide ureas
whereas the first four belong to the sulfanilamide series. It is very interesting to note the substitution
patterns present in the second aromatic ring of these compounds and to compare them to the one
found in SLC-0111, which is a rather ineffective hCA II inhibitor, as mentioned above. Thus, 10, 12
and 13 incorporate at least one chlorine as substituent on the second aromatic ring, and eventually
other groups, such as nitro and trifluoromethyl. On the other hand 14 has two fluorine atoms but in
a different position compared to SLC-0111 (2,6-difluoro). This is in fact the best inhibitor in this
series, being 1032 times a better hCA II inhibitor compared to the clinical candidate drug. This is a
huge difference of activity, explained only by the different orientation/contacts that the 2,6-
difluorophenyl moiety can do when bound to the active site, compared to the 4-fluorophenyl
functionality of SLC-0111. Another interesting case is 17, which is the meta-regiomer of SLC-
0111. This compound is also an effective hCA II inhibitor, being 55.8 times more effective than

SLC-0111 in inhibiting hCA II. All these data show that very small structural changes in the
scaffold of these inhibitors leads to huge differences of activity. Four other derivatives, 8, 9, 11 and
18, had KIs < 100 nM, whereas the remaining ones were medium potency hCA II inhibitors (KIs
ranging between 162 and 418 nM. The main conclusion of the SAR, mentioned above, is reinforced
by these data: small structural changes in the scaffold, mainly at the second aromatic ring, influence
significantly the CA inhibitory effects of these sulfonamides.
(iii) hCA IX, the main drug target for anticancer effects of the CAIs, was effectively inhibited by
the sulfanilamide ureas 1-15, with KIs in the range of 3.7 – 9.6 nM, whereas the metanilamide 16-
21 derivatives were much weaker inhibitors (KIs in the range of 116 – 349 nM, Table 1). The SAR
is almost absent in this case, as all sulfanilamide ureas showed a very compact behavior as potent
CA IX inhibitors (being more effective than the lead, SLC-0111), whereas all metanilamide ureas
were less effective. It is thus clear that the additional substituents present in the second aromatic
ring of SLC-0111 led to an increase of affinity for hCA IX, but with a major decrease of selectivity
for inhibiting hCA IX over hCA II and hCA I.
(iv) In analogy to hCA IX, the second transmembrane isoform connected to tumors, hCA XII, was
also effectively inhibited by the sulfanilamide derivatives 1-15 (KIs in the range of 6.2 – 10.1 nM),
and less effectively inhibited by the metanilamides 16-21 (KIs in the range of 66.9 – 113 nM, Table
1). SAR is thus similar to what discussed above for hCA IX.
(v) Some of the sulfonamides reported here, such as 1-7 and 15 are selective inhibitors of hCA
IX/XII over hCA I/II; but the selectivity indexes are lower compared to those of the lead compound
SLC-0111.
3. Conclusions
SLC-0111 is a sulfonamide CAI in Phase I/II clinical trials for the treatment of advanced hypoxic
tumors complicated with metastases. Its antitumor effects are due to inhibition of the enzymatic
activity of CA IX, an isoform predominantly found in tumors/metastases, but it also reduces the
cancer stem cells population. Here we report the synthesis of analogs of SLC-0111, which possess
diverse substitution patterns at the ureido-phenyl moiety of the candidate drug. Both sulfanilamide
and metanilamide ureido derivatives were obtained, possessing one or more halogen,
trifluoromethyl, perchloro-/perfluorophenyl moieties instead of the 4-fluorophenyl group found in
SLC-0111. Most of the sulfanilamide ureido derivatives were highly effective and sometimes
selective CA IX/XII inhibitors, whereas the amide ureido derivatives were less effective as

transmembrane CA isoforms inhibitors. Structure activity relationship for this class of sulfonamides
is discussed in detail.
4. Experimental
4.1. Chemistry
Anhydrous solvents and all reagents were purchased from Sigma-Aldrich, Alfa Aesar and
TCI. All reactions involving air- or moisture-sensitive compounds were performed under a nitrogen
atmosphere using dried glassware and syringes techniques to transfer solutions. Infrared (IR)
spectra were recorded as KBr plates and are expressed in ν (cm-1). Nuclear magnetic resonance (1H-
NMR, 13C-NMR) spectra were recorded using a Bruker Advance III 400 MHz spectrometer in
DMSO-d6. The 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;
sept, septet; 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.
General procedure for the synthesis of compounds 1-21. 23a
Sulfanilamide A or metanilamide B (1.0 eq) was dissolved in acetonitrile (ACN) and then treated
with a stoichiometric amount of the proper, commercially available isocyanate ArNCO. The
reaction mixture was stirred at room temperature until completion (TLC monitoring). The heavy
precipitate formed was filtered-off, washed with diethyl ether, and dried under vacuo to afford the
title compounds 1-21 which did not require further purification.
4-(3-(4’-chloro-2-fluorophenyl)ureido)benzenesulfonamide 1

5'
2
1
6'
H2NO2S
Cl
3
4
O
4'
N
H
N
1'
H
3'
2'
F
4-(3-(4’-Chloro-2-fluorophenyl)ureido)benzenesulfonamide 1: νmax (KBr) cm-1 3270, 1650, 1560;
δH (400 MHz, DMSO-d6) 7.26-7.30 (3H, brs, SO2NH2, exchange with D2O, 5’/6’-H), 7.52 (1H, dd,
J, 8.2, 2.1, 5’/6’-H), 7.64 (2H, d, J 8.2, 2 x 2/3-H), 7.78 (2H, d, J 8.2, 2 x 2/3-H), 8.19 (1H, dd, J
9.0, 8.2 3’-H), 8.79 (1H, s, NH, exchange with D2O), 9.47 (1H, s, NH, exchange with D2O); δC (100
MHz, DMSO-d6) 152.8 (d, J1 C-F 245, C-2’), 152.7 (C=O), 143.2, 138.2, 127.8, 127.4, (d, J C-F 10),
126.7 (d, J C-F 10), 125.6, 122.5, 118.4, 116.6 (d, J C-F 23); δF (376 MHz, DMSO-d6) –126.38; Anal.
Elem. Cal.: C, 45.42; H, 3.23; N, 12.22), Found: C, 45.39; H, 3.18; N, 12.08.
4-(3-(4’-bromo-2’-fluorophenyl)ureido)benzenesulfonamide 2
5'
2
1
6'
H2NO2S
Br
3
4
O
4'
N
H
N
1'
H
3'
2'
F
4-(3-(4’-Bromo-2’-fluorophenyl)ureido)benzenesulfonamide 2: νmax (KBr) cm-1, 3272, 3265, 1643,
1590; δH (400 MHz, DMSO-d6) 7.27 (2H, s, SO2NH2, exchange with D2O), 7.40 (1H, d, J, 9.2
5’/6’-H), 7.64 (3H, m, 2 x 2/3-H, 5’/6’-H), 7.78 (2H, d, J 9.2, 2 x 2/3-H), 8.16 (1H, t, J 8.8 3’-H),
8.79 (1H, s, NH, exchange with D2O), 9.48 (1H, s, NH, exchange with D2O); δC (100 MHz, DMSO-
d6) 152.8 (d, J1
245, C-2’), 152.7 (C=O), 143.2, 138.2, 128.5, 127.9, 127.7 (d, J
6), 122.8,
C-F
C-F
119.2 (d, J C-F 22), 118.4, 114.0 (d, J C-F 9); δF (376 MHz, DMSO-d6) –126.38; Anal. Elem. Cal.: C,
40.22; H, 2.86; N, 10.82), Found: C, 39.97; H, 2.64; N, 10.99.
4-(3-(2’-fluoro-5’-nitrophenyl)ureido)benzenesulfonamide 3
NO2
5'
2
1
6'
H2NO2S
3
4
4'
O
N
H
N
H
3'
1'
2'
F
4-(3-(2’-Fluoro-5’-nitrophenyl)ureido)benzenesulfonamide 3: νmax (KBr) cm-1, 3300, 3269, 1648,
1600; δH (400 MHz, DMSO-d6) 7.28 (2H, s, SO2NH2, exchange with D2O), 7.60 (1H, dd, J, 9.2 6.8,

6’-H), 7.70 (2H, d, J 9.2, 2 x 2/3-H), 7.80 (2H, d, J 9.2, 2 x 2/3-H), 7.96 (1H, m, 4’-H), 9.13 (1H, s,
NH, exchange with D2O), 9.18 (1H, m, 6’-H), 9.57 (1H, s, NH, exchange with D2O); δC (100 MHz,
DMSO-d6) 156.0 (d, J1 C-F 251, C-2’), 154.8 (C=O), 144.9, 142.9, 138.6, 129.3 (d, J C-F 12), 127.9,
119.0 (d, J C-F 9), 118.7, 117.0 (d, J C-F 22), 115.7; δF (376 MHz, DMSO-d6) –119.43; Anal. Elem.
Cal.: C, 44.07; H, 3.13; N, 15.81), Found: C, 43.82; H, 2.95; N, 15.95.
4-(3-(2’,4’,5’-trifluorophenyl)ureido)benzenesulfonamide 4
F
5'
2'
2
1
6'
H2NO2S
F
3
4
O
4'
3'
N
H
N
1'
H
F
4-(3-(2’,4’,5’-Trifluorophenyl)ureido)benzenesulfonamide 4: νmax (KBr) cm-1, 3270, 3163, 1638,
1580; δH (400 MHz, DMSO-d6) 7.27 (2H, s, SO2NH2, exchange with D2O), 7.68 (3H, m, 2 x 2/3-H,
3’-H), 7.78 (2H, d, J 9.2, 2 x 2/3-H), 8.22 (1H, m, 6’-H), 8.58 (1H, s, NH, exchange with D2O),
9.47 (1H, s, NH, exchange with D2O); δC (100 MHz, DMSO-d6) 152.7 (C=O), 148.2 (dd, J1 C-F 240,
12.2), 146.4 (d, J1 C-F 237, 15.8), 144.4 (dd, J1 C-F 250, 12.0), 143.3, 138.3, 127.8, 125.0 (m), 118.5,
109.5 (dd, J C-F 24.5, 2.9), 106.4 (dd, J C-F 25.6, 22.0); δF (376 MHz, DMSO-d6) –130.64 (d, J
F-F
13.9), –141.75 (m), –143.06 (d, J F-F 24.4); Anal. Elem. Cal.: C, 45.22; H, 2.92; N, 12.17), Found:
C, 45.41; H, 2.84; N, 12.25.
4-(3-(2’-fluoro-5’-(trifluoromethyl)phenyl)ureido)benzenesulfonamide 5
CF3
5'
2
1
6'
H2NO2S
3
4
O
4'
3'
N
H
N
1'
H
2'
F
4-(3-(2’-Fluoro-5’-(trifluoromethyl)phenyl)ureido)benzenesulfonamide 5: νmax (KBr) cm-1 3270,
1660, 1560; δH (400 MHz, DMSO-d6) 7.28 (2H, s, SO2NH2, exchange with D2O), 7.47 (1H, m, 4’-
H), 7.55 (1H, dd, J 10.8 8.8, 3’-H), 7.67 (2H, d, J 8.8, 2 x 2/3-H), 7.79 (2H, d, J 8.8, 2 x 2/3-H),
8.63 (1H, m, 6’-H), 9.03 (1H, s, NH, exchange with D2O), 9.56 (1H, s, NH, exchange with D2O); δC
(100 MHz, DMSO-d6) 154.0 (d, J1 C-F 247, C-2’), 152.8 (C=O), 143.0, 138.5, 129.30 (d, J C-F 11.3),
127.9, 126.2 (dd, J C-F 31.8, 3.3), 123.5, 120.7 (m), 118.6, 117.7, 117.0 (d, J C-F 20.5); δF (376 MHz,

DMSO-d6) –60.7 (3F, C-F3), -123.8 (1F, 2’-F); Anal. Elem. Cal.: C, 44.56; H, 2.94; N, 11.14),
Found: C, 44.63; H, 2.86; N, 11.00.
4-(3-(2’-fluoro-3’-(trifluoromethyl)phenyl)ureido)benzenesulfonamide 6
5'
2
1
6'
H2NO2S
3
4
4'
3'
O
N
H
N
H
CF3
1'
2'
F
4-(3-(2’-fluoro-3’-(trifluoromethyl)phenyl)ureido)benzenesulfonamide 6: νmax (KBr) cm-1, 3320,
1645, 1570; δH (400 MHz, DMSO-d6) 7.27 (2H, s, SO2NH2, exchange with D2O), 7.43 (2H, m, 5’-
H, 6’-H), 7.65 (2H, d, J 8.8, 2 x 2/3-H), 7.79 (2H, d, J 8.8, 2 x 2/3-H), 8.46 (1H, m, 4’-H), 8.96 (1H,
s, NH, exchange with D2O), 9.53 (1H, s, NH, exchange with D2O); δC (100 MHz, DMSO-d6) 152.7
(C=O), 150.0 (d, J1
250, C-2’), 143.1, 138.3, 129.4 (d, J
9), 127.8, 125.9 (d, J
43.9),
C-F
C-F
C-F
124.9, 122.2, 120.4, 118.7, 117.5 (dd, J
32.1, 3.2); δF (376 MHz, DMSO-d6) –59.8 (3F,d, JF-F
C-F
13.2, C-F3), -132.2 (1F, q, J 13.2, 2’-F); Anal. Elem. Cal.: C, 44.56; H, 2.94; N, 11.14), Found: C,
44.18; H, 2.78; N, 11.21.
4-(3-(2’,3’,4’-trifluorophenyl)ureido)benzenesulfonamide 7
5'
2
4'
1
6'
H2NO2S
F
3
4
O
3'
N
H
N
H
F
1'
2'
F
4-(3-(2’,3’,4’-Trifluorophenyl)ureido)benzenesulfonamide 7: νmax (KBr) cm-1 3270, 1640, 1592; δH
(400 MHz, DMSO-d6) 7.27 (2H, s, SO2NH2, exchange with D2O), 7.34 (2H, m, 5’/6’-H), 7.65 (2H,
d, J 8.8, 2 x 2/3-H), 7.77 (2H, d, J 8.8, 2 x 2/3-H), 7.88 (1H, m, 5’/6’-H), 8.82 (1H, s, NH, exchange
with D2O), 9.45 (1H, s, NH, exchange with D2O); δC (100 MHz, DMSO-d6) 152.9 (C=O), 146.6 (d,
J1 C-F 237), 143.2, 141.8 (d, J1 C-F 240), 139.6 (d, J1 C-F 242), 138.3, 127.8, 126.0 (m), 118.5, 116.7
(m), 112.6 (dd, J C-F 18, 4); δF (376 MHz, DMSO-d6) –143.1 (1F,d, JF-F 21.7, 2’/4’-F), -148.4 (1F,d,
JF-F 21.7, 2’/4’-F), -161.1 (1F,t, JF-F 21.7, 3’-F); Anal. Elem. Cal.: C, 45.22; H, 2.92; N, 12.17),
Found: C, 45.59; H, 2.65; N, 12.53.
4-(3-(2’-fluorophenyl)ureido)benzenesulfonamide 8

5'
2
1
6'
H2NO2S
4'
3
4
O
3'
N
H
N
1'
H
2'
F
4-(3-(2’-Fluorophenyl)ureido)benzenesulfonamide 8: νmax (KBr) cm-1 3272, 1643, 1600; δH (400
MHz, DMSO-d6) 7.07 (1H, m, 3’-H), 7.20 (1H, m, 4’-H), 7.26 (3H, brs, SO2NH2, exchange with
D2O, 5’-H), 7.64 (2H, d, J 8.8, 2 x 2/3-H), 7.80 (2H, d, J 8.8, 2 x 2/3-H), 8.20 (1H, m, 6’-H)8.70
(1H, s, NH, exchange with D2O), 9.46 (1H, s, NH, exchange with D2O); δC (100 MHz, DMSO-d6)
153.1 (d, J1 C-F 240), 152.9 (C=O), 143.4, 138.1, 128.1, 127.8, 125.4, 123.8, 121.7, 118.3, 116.6 (d,
J C-F 19); δF (376 MHz, DMSO-d6) –129.59; Anal. Elem. Cal.: C, 50.48; H, 3.91; N, 13.58), Found:
C, 50.32; H, 4.13; N, 13.68.
4-(3-(2’,4’-difluorophenyl)ureido)benzenesulfonamide 9
5'
2
1
4'
6'
H2NO2S
F
3
4
O
3'
N
H
N
1'
H
2'
F
4-(3-(2’,4’-Difluorophenyl)ureido)benzenesulfonamide 9: νmax (KBr) cm-1 3270, 1642, 1598; δH
(400 MHz, DMSO-d6) 7.08 (1H, m, 3’/5’-H), 7.25 (2H, s, SO2NH2, exchange with D2O), 7.36 (1H,
m, 3’/5’-H), 7.64 (2H, d, J 8.8, 2 x 2/3-H), 7.78 (2H, d, J 8.8, 2 x 2/3-H), 8.11 (1H, m, 6’-H), 8.64
(1H, s, NH, exchange with D2O), 9.41 (1H, s, NH, exchange with D2O); δC (100 MHz, DMSO-d6)
158.1 (dd, J C-F 243, 13), 153.4 (dd, J C-F 244, 12), 153.0 (C=O), 143.4, 138.1, 128.1, 127.8, 124.5
(d, J C-F 9), 123.2 (d, J C-F 8), 118.4, 112.0 (d, J C-F 21), 104.8 (t, J C-F 25); δF (376 MHz, DMSO-d6)
–117.52, -124.3; Anal. Elem. Cal.: C, 47.70; H, 3.39; N, 12.84), Found: C, 47.60; H, 3.09; N, 13.14.
4-(3-(3’-chlorophenyl)ureido)benzenesulfonamide 10
5'
2
1
6'
H2NO2S
3
4
4'
3'
O
N
H
N
H
Cl
1'
2'
4-(3-(3’-chlorophenyl)ureido)benzenesulfonamide 10: νmax (KBr) cm-1, 3274, 1641, 1591; δH (400
MHz, DMSO-d6) 7.07 (1H, d, J 8.8, 4’-H), 7.25 (2H, s, SO2NH2, exchange with D2O), 7.33 (2H, m,

5’-H, 6’-H), 7.64 (2H, d, J 8.8, 2 x 2/3-H), 7.77 (3H, m, J 8.8, 2 x 2/3-H, 2’-H), 9.04 (1H, s, NH,
exchange with D2O), 9.17 (1H, s, NH, exchange with D2O); δC (100 MHz, DMSO-d6) 153.1 (C=O),
143.5, 141.8, 138.0, 134.1, 131.3, 127.7, 122.7, 118.7, 118.6, 117.8; Anal. Elem. Cal.: C, 47.93; H,
3.71; N, 12.90), Found: C, 47.50; H, 3.44; N, 13.13.
4-(3-(2’,5’-dichlorophenyl)ureido)benzenesulfonamide 11
Cl
2
5'
6'
1'
1
H2NO2S
3
4
4'
3'
O
N
H
N
H
2'
Cl
4-(3-(2’,5’-Dichlorophenyl)ureido)benzenesulfonamide 11: νmax (KBr) cm-1 3275, 1650, 1578; δH
(400 MHz, DMSO-d6) 7.15 (1H, dd, J 8.8 2.8, 4’-H), 7.28 (2H, s, SO2NH2, exchange with D2O),
7.55 (1H, dd, J 8.8 2.8, 3’-H), 7.64 (2H, d, J 8.8, 2 x 2/3-H), 7.79 (2H, d, J 8.8, 2 x 2/3-H), 8.35
(1H, d, J 2.8, 6’-H), 8.60 (1H, s, NH, exchange with D2O), 9.90 (1H, s, NH, exchange with D2O);
δC (100 MHz, DMSO-d6) 152.7 (C=O), 143.1, 138.4, 137.8, 132.9, 131.5, 127.9, 124.0, 121.3,
121.1, 118.7; Anal. Elem. Cal.: C, 43.35; H, 3.08; N, 11.67), Found: C, 43.64; H, 3.08; N, 11.66.
4-(3-(2’-Chloro-5’-nitrophenyl)ureido)benzenesulfonamide 12
NO2
5'
2
6'
1'
1
H2NO2S
3
4
4'
3'
O
N
H
N
H
2'
Cl
4-(3-(2’-Chloro-5’-nitrophenyl)ureido)benzenesulfonamide 12: νmax (KBr) cm-1, 3164, 3271, 1641,
1592; δH (400 MHz, DMSO-d6) 7.29 (2H, s, SO2NH2, exchange with D2O), 7.70 (2H, d, J 8.8, 2 x
2/3-H), 7.82 (3H, m, 2 x 2/3-H, 3’-H), 7.93 (1H, dd, J 8.9 2.2, 4’-H), 8.84 (1H, s, NH, exchange
with D2O), 9.20 (1H, d, J 2.2, 6’-H), 9.99 (1H, s, NH, exchange with D2O); δC (100 MHz, DMSO-
d6) 152.7 (C=O), 147.5. 142.9, 138.6, 137.7, 131.3, 128.9, 127.9, 118.8, 118.5, 115.6; Anal. Elem.
Cal.: C, 42.11; H, 2.99; N, 15.11), Found: C, 42.38; H, 3.24; N, 15.43.
4-(3-(2’-Chloro-4’-(trifluoromethyl)phenyl)ureido)benzenesulfonamide 13

2
5'
2'
6'
1'
1
H2NO2S
CF3
3
4
O
4'
3'
N
H
N
H
Cl
4-(3-(2’-Chloro-4’-(trifluoromethyl)phenyl)ureido)benzenesulfonamide 13: νmax (KBr) cm-1, 3169,
1639, 1560; δH (400 MHz, DMSO-d6) 7.29 (2H, s, SO2NH2, exchange with D2O), 7.69 (2H, d, J
8.8, 2 x 2/3-H), 7.74 (1H, dd, J 8.8 4.0, 5’-H), 7.80 (2H, d, J 8.8, 2 x 2/3-H), 7.92 (1H, dd, J 4.0, 3’-
H), 8.50 (1H, d, J 8.8, 6’-H), 8.76 (1H, s, NH, exchange with D2O), 10.0 (1H, s, NH, exchange with
D2O); δC (100 MHz, DMSO-d6) 152.5, 143.0, 140.4, 138.5, 128.7, 127.9, 127.2 (m), 125.8 (m),
124.0 (d, J
33.0), 13.2, 122.7, 121.4; δF (376 MHz, DMSO-d6) –60.42; Anal. Elem. Cal.: C,
C-F
42.70; H, 2.82; N, 10.67), Found: C, 42.92; H, 2.76; N, 11.70.
4-(3-(2’,6’-difluorophenyl)ureido)benzenesulfonamide 14
2
1
6'
1'
H2NO2S
F
3
4
4'
3'
O
N
H
N
H
2'
F
4-(3-(2’,6’-Difluorophenyl)ureido)benzenesulfonamide 14: νmax (KBr) cm-1, 3164, 3270, 1641,
1590; δH (400 MHz, DMSO-d6) 7.33 (4H, m, SO2NH2, exchange with D2O, 2 x 5’-H), 7.37 (1H, m,
4’-H), 7.64 (2H, d, J 8.8, 2 x 2/3-H), 7.76 (2H, d, J 8.8, 2 x 2/3-H), 8.30 (1H, s, NH, exchange with
D2O), 9.38 (1H, s, NH, exchange with D2O); δC (100 MHz, DMSO-d6) 158 (d, J1 C-F 247, C-2’, C-
6’), 153.27 (C=O), 143.7, 138.0, 128.3 (m), 127.7, 118.5, 115.9 (t, J C-F 16, C-4’), 112.6 (d, J C-F 23,
2 x C-3’); δF (376 MHz, DMSO-d6) –118.73; Anal. Elem. Cal.: C, 47.70; H, 3.39; N, 12.84), Found:
C, 47.45; H, 2.91; N, 13.17.
4-(3-(perchlorophenyl)ureido)benzenesulfonamide 15
Cl
2
1
H2NO2S
Cl
Cl
Cl
3
4
O
N
H
N
H
Cl

4-(3-(perchlorophenyl)ureido)benzenesulfonamide 15: νmax (KBr) cm-1, 3168, 3275, 1641, 1590; δH
(400 MHz, DMSO-d6) 7.24 (2H, m, SO2NH2, exchange with D2O), 7.64 (2H, d, J 8.8, 2 x 2/3-H),
7.75 (2H, d, J 8.8, 2 x 2/3-H), 8.29 (1H, s, NH, exchange with D2O), 9.38 (1H, s, NH, exchange
with D2O); δC (100 MHz, DMSO-d6) 153.2 (C=O), 142.0, 139.2, 130.1 (overlapping signals), 127.4,
125.5; Anal. Elem. Calc.: C, 33.68; H, 1.74; N, 9.06), Found: C, 34.01; H, 1.81; N, 9.10.
3-(3-(4’-Iodophenyl)ureido)benzenesulfonamide 16
SO2NH2
1
I
2
6
O
4'
3'
3
5
N
H
N
1'
H
4
2'
3-(3-(4’-Iodophenyl)ureido)benzenesulfonamide 16: m.p. 256-258 °C; νmax (KBr) cm-1, 3165, 3265,
1643, 1589; δH (400 MHz, DMSO-d6) 7.34-7.66 (9H, m, Ar-H, SO2NH2, exchange with D2O), 8.10
(1H, d, J, 2.1, 2-H), 8.79 (1H, s, NH, exchange with D2O), 9.08 (1H, s, NH, exchange with D2O);
δC (100 MHz, DMSO-d6) 153.1 (C=O), 145.6, 140.9, 140.3, 138.3, 130.3, 122.1, 121.6, 119.9,
116.1, 85.9; Elem. Anal. Calc. C, 37.42; H, 2.90; N, 10.07; Found; C, 37.36; H, 2.79; N, 9.82; m/z
(ESI+) 418 (M+H)+.
3-(3-(4’-Fluorophenyl)ureido)benzenesulfonamide 17
SO2NH2
1
F
2
6
O
4'
3
5
3'
N
H
N
1'
H
4
2'
3-(3-(4’-Fluorophenyl)ureido)benzenesulfonamide 17: m.p. 233-235 °C; νmax (KBr) cm-1, 3377,
3352, 1685, 1557; δH (400 MHz, DMSO-d6) 7.17 (1H, dd, J 8.8, Ar-H), 7.45 (2H, s, SO2NH2,
exchange with D2O), 7.47-7.60 (5H, m, Ar-H), 8.10 (1H, d, J, 2.1, 2-H), 8.78 (1H, s, NH, exchange
with D2O), 9.04 (1H, s, NH, exchange with D2O); δC (100 MHz, DMSO-d6) 158.4 (d, JC-F 237, C-
4’), 153.4 (C=O), 145.6, 141.1, 136.6, 130.3, 122.0, 121.2, 119.8, 116.3, 116.1; δF (376 MHz,
DMSO-d6) –121.14; Elem. Anal. Calc. C, 50.48; H, 3.91; N, 13.58; Found: C, 49.98; H, 3.79; N,
13.49]; m/z (ESI+) 311 (M+H)+.
3-(3-(3’-Nitrophenyl)ureido)benzenesulfonamide 18

SO2NH2
5'
1
6'
2
3
6
5
4'
O
N
H
N
H
NO2
3'
1'
4
2'
3-(3-(3’-Nitrophenyl)ureido)benzenesulfonamide 18: m.p. 252-255 °C; νmax (KBr) cm-1, 3380,
3350, 1689, 1550; δH (400 MHz, DMSO-d6) 7.41 (2H, s, SO2NH2, exchange with D2O), 7.48-7.64
(4H, m, Ar-H), 7.77 (1H, dd, J 7.2, 2.1, Ar-H), 7.87 (1H, dd, J 7.2, 2.1, Ar-H), 8.14 (1H, d, J, 2.1,
2-H), 8.63 (1H, d, J, 2.1, 2’-H), 9.23 (1H, s, NH, exchange with D2O), 9.31 (1H, s, NH, exchange
with D2O); δC (100 MHz, DMSO-d6) 153.3 (C=O), 149.1, 145.7, 141.7, 140.6, 131.0, 130.3, 125.4,
122.4, 120.2, 117.4, 116.4, 113.2; Elem. Anal. Calc. C, 46.78; H, 3.60; N, 16.66; Found C, 46.91;
H, 3.55; N, 16.94; m/z (ESI+) 337 (M+H)+.
3-(3-(4’-Acetylphenyl)ureido)benzenesulfonamide 19
SO2NH2
O
1
2
3
6
5
O
4'
3'
N
H
N
1'
H
4
2'
3-(3-(4’-Acetylphenyl)ureido)benzenesulfonamide 19: m.p. 267-269 °C; νmax (KBr) cm-1, 3402,
3351, 2014, 1933, 1912, 1593; δH (400 MHz, DMSO-d6) 2.56 (3H, s, CH3), 7.41 (2H, s, SO2NH2,
exchange with D2O), 7.48-7.55 (3H, m, 4-H, 5-H, 6-H), 7.60 (2H, d, J 7.2, 2 x 2’-H), 7.97 (2H, d, J
7.2, 2 x 3’-H), 8.13 (1H, t, J 2.0, 2-H), 9.18 (1H, s, NH, exchange with D2O), 9.19 (1H, s, NH,
exchange with D2O); δC (100 MHz, DMSO-d6) 197.2 (CH3C=O), 153.0 (C=O), 145.7, 145.0, 140.7,
131.6, 130.5, 130.4, 122.2, 120.2, 118.2, 116.2, 27.2; Elem. Anal. Calc. C, 54.04; H, 4.54; N, 12.60;
Found C, 54.78; H, 4.47; N, 12.96; m/z (ESI+) 334 (M+H)+.
3-(3-(2’-Isopropylphenyl)ureido)benzenesulfonamide 20
SO2NH2
5'
1
6'
2
6
5
4'
O
3
N
H
N
1'
3'
2'
H
4
7'
8'
3-(3-(2’-Isopropylphenyl)ureido)benzenesulfonamide 20: m.p. 175-176 °C; νmax (KBr) cm-1, 3328,
3300, 1690, 1556; δH (400 MHz, DMSO-d6) 1.23 (6H, d, J 6.2, 2 x 8’-H3), 3.19 (1H, sept, J 6.2, 7’-
H), 7.14 (2H, m, Ar-H), 7.35 (1H, d, J 7.2, Ar-H), 7.37 (2H, s, SO2NH2, exchange with D2O), 7.59-

7.70 (4H, m, Ar-H), 8.00 (1H, s, NH, exchange with D2O), 8.10 (1H, t, J 2.0, 2-H), 9.30 (1H, s, NH,
exchange with D2O); δC (100 MHz, DMSO-d6) 153.9 (C=O), 145.6, 141.3, 140.7, 136.1, 130.3,
126.7, 126.2, 125.2, 124.7, 121.7, 119.5, 115.8, 27.8, 24.0; Elem. Anal. Calc. C, 57.64; H, 5.74; N,
12.60; Found C, 58.14; H, 5.73; N, 12.70; m/z (ESI+) 334 (M+H)+.
3-(3-(Perfluorophenyl)ureido)benzenesulfonamide 21
SO2NH2
F
F
1
2
3
F
F
6
5
O
4'
3'
N
H
N
H
F
1'
2'
4
3-(3-(Perfluorophenyl)ureido)benzenesulfonamide 21: m.p. 224-227 °C; νmax (KBr) cm-1, 3390,
3287, 1785, 1560; δH (400 MHz, DMSO-d6) 7.38 (2H, s, SO2NH2, exchange with D2O), 7.50 (2H,
m, Ar-H), 7.63 (1H, d, J 7.2, Ar-H), 8.09 (1H, s, 2-H), 8.63 (1H, s, ArNHCONH, exchange with
D2O), 9.47 ((1H, s, ArNHCONH, exchange with D2O); δC (100 MHz, DMSO-d6) 152.9 (C=O),
145.6, 144.1 (d, J1 C-F 245), 140.7, 139.6 (d, J1 C-F 253), 138.2 (d, J1 C-F 245), 130.4, 122.3, 120.4,
116.4, 114.7 (t, J C-F 12); δF (376 MHz, DMSO-d6) –146.3 (2F, dd, J 19.2 4.8, 2 x 2’-F), -159.2 (1F,
t, J 22.9, 4’-F), -164.0 (2F, dt, J 22.6 4.8, 2 x 3’-F); Elem. Anal. Calc. C, 40.95; H, 2.11; N, 11.02;
Found C, 40.85; H, 1.89; N, 11.00; m/z (ESI+) 382 (M+H)+.
4.2. Carbonic anhydrase inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2
hydration activity.28 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working
at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4
(for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO2
hydration reaction for a period of 10-100 s. The CO2 concentrations ranged from 1.7 to 17 mM for
the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six
traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The
uncatalyzed rates were determined in the same manner and subtracted from the total observed rates.
Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to
0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were
preincubated together for 15 min at room temperature prior to assay, in order to allow for the
formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares

methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier,29 and represent the
mean from at least three different determinations. All CA isofoms were recombinant ones obtained
in-house as reported earlier. 30
Acknowledgments. This work was financed in part by a Distinguished Scientist Fellowship
Program (DSFP) of King Saud University, Riyadh, Saudi Arabia.
References
1. a) P.C. McDonald, J.-Y. Winum, C.T. Supuran, S. Dedhar, Oncotarget 3 (2012) 84; b) S. Fais, G.
Venturi, B. Gatenby, Cancer Met Rev. 33 (2014) 1095; c) C. Ward, S.P. Langdon, P. Mullen, A.L.
Harris, D.J. Harrison, C.T. Supuran, I. Kunkler, Cancer Treatm Rev. 39 (2013) 171.
2. a) N. Raghunand, B.P. Mahoney, R.J. Gillies, Biochem Pharmacol. 66 (2003) 1219; b) 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, Mol Cancer Ther. 15 (2016)
2722.
3. D. Neri, C. T. Supuran, Nat Rev Drug Discov. 10 (2011) 767.
4. L. Schwartz, C.T. Supuran, K.O. Alfarouk, Anticancer Agents Med Chem. 17 (2017) 164.
5. a) P. Ebbesen, E.O. Pettersen, T.A. Gorr, G. Jobst, K. Williams, J. Kienninger, R.H. Wenger, S.
Pastorekova, L. Dubois, P. Lambin, B.G. Wouters, C.T. Supuran, L. Poellinger, P. Ratcliffe, A.
Kanopka, A. Görlach, M. Gasmann, A.L. Harris, P. Maxwell, A. Scozzafava, J Enzyme Inhib Med
Chem. 24 Sup1 (2009) 1; b) E.O. Pettersen, P. Ebbesen, R.G Gieling, K.J Williams, L. Dubois, P.
Lambin, C. Ward, J. Meehan, I.H Kunkler, S.P Langdon, A.H. Ree, K. Flatmark, H. Lyng, M.J.
Calzada, L. del Peso, M.O. Landazuri, A. Görlach, H. Flamm, J. Kieninger, G. Urban, A. Weltin, D.
Singleton, F.M. Buffa, A. Harris, A. Scozzafava, C.T. Supuran, I. Moser, G. Jobst, J. Pouyssegur, J.
Chiche, N. Mazure, I. Marchiq, S. Parks, A. Ahmed, M. Ashcroft, S. Pastorekova, Y. Cao, K.M.
Rouschop, B. Wouters, M. Koritzinsky, H. Mujcic, D. Cojocari, J Enzyme Inhib Med Chem. 30
(2015) 689.
6. C.T. Supuran, Biochem J. 473 (2016) 2023; b) C.T. Supuran, J Enzyme Inhib Med Chem. 31
(2016) 345; c) C. T. Supuran, Bioorg Med Chem. 21 (2013) 1377; d) C.T. Supuran, Expert Opin
Drug Discov. 12 (2017) 61; e) V. Alterio, A. Di Fiore, K. D’Ambrosio, C.T. Supuran, G. De
Simone, Chem Rev. 112 (2012) 4421; f) C.T. Supuran, Pathogens 5 (2016) E44.
7. C.T. Supuran, Nat Rev Drug Discov. 7 (2008) 168; b) S. Durdagi, D. Vullo, P. Pan, N.
Kähkönen, J.A. Määttä, V.P. Hytönen, A. Scozzafava, S. Parkkila, C.T. Supuran, J Med Chem. 55

(2012) 5529; c) L. Di Cesare Mannelli, L. Micheli, F. Carta, A. Cozzi, C. Ghelardini, C.T. Supuran,
J Enzyme Inhib Med Chem. 31 (2016) 894.
8. a) G. De Simone, V. Alterio, C.T. Supuran, Expert Opin Drug Discov. 8 (2013) 793; b) E.
Masini, F. Carta, A. Scozzafava, C.T. Supuran, Expert Opin Ther Pat. 23 (2013) 705; c) C.T.
Supuran, Bioorg Med Chem Lett. 20 (2010) 3467; d) C.T. Supuran, J Enzyme Inhib Med Chem. 28
(2013) 229; e) K. D’Ambrosio, S. Carradori, S.M. Monti, M. Buonanno, D. Secci, D. Vullo, C.T.
Supuran, G. De Simone, Chem Commun. 51 (2015) 302.
9. a) C.T. Supuran, J Enzyme Inhib Med Chem. 27 (2012) 759; b) C.T. Supuran, Expert Opin Ther
Pat. 23 (2013) 677; c) J.Y. Winum, C.T. Supuran, J Enzyme Inhib Med Chem. 30 (2015) 321; d) V.
De Luca, S. Del Prete, C.T. Supuran, C. Capasso, J Enzyme Inhib Med Chem. 30 (2015) 277; e)
C.L. Lomelino, C.T. Supuran, R. McKenna, Int J Mol Sci. 17 (2016) E1150.
10. a) M.A. Pinard, C.D. Boone, B.D. Rife, C.T. Supuran, R. McKenna, Bioorg Med Chem. 21
(2013) 7210; b) K. Chegaev, L. Lazzarato, Y. Tamboli, D. Boschi, M. Blangetti, A. Scozzafava, F.
Carta, E. Masini, R. Fruttero, C.T. Supuran, A. Gasco, Bioorg Med Chem. 22 (2014) 3913; c) D.
Vullo, M. Durante, F.S. Di Leva, S. Cosconati, E. Masini, A. Scozzafava, E. Novellino, C.T.
Supuran, F. Carta, J Med Chem. 59 (2016) 5857; d) F. Fabrizi, F. Mincione, T. Somma, G.
Scozzafava, F. Galassi, E. Masini, F. Impagnatiello, C.T. Supuran, J Enzyme Inhib Med Chem. 27
(2012) 138.
11. 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, J Med Chem. 55 (2012) 5591.
12. a) L. Dubois, N.G. Lieuwes, A. Maresca, A. Thiry, C.T. Supuran, A. Scozzafava, B.G. Wouters,
P. Lambin, Radiother Oncol. 92 (2009) 423; b) M. Rami, L. Dubois, N.K. Parvathaneni, V. Alterio,
S. van Kuijk, S.M. Monti, P. Lambin, G. De Simone, C.T. Supuran, J.Y. Winum, J Med Chem. 56
(2013) 8512; c) 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, Oncotarget 6 (2015) 24856.
13. a) P. Pan, A.B. Vermelho, G. Capaci Rodrigues, A. Scozzafava, M.E. Tolvanen, S. Parkkila, C.
Capasso,C.T. Supuran, J Med Chem. 56 (2013) 1761; b) L. Syrjänen, A.B. Vermelho, I. de Almeida
Rodrigues, S. Corte-Real, T. Salonen, P. Pan, D. Vullo, S. Parkkila, C. Capasso, C.T. Supuran, J
Med Chem. 56 (2013) 7372; c) S. Del Prete, D. Vullo, G.M. Fisher, K.T. Andrews, S.A. Poulsen,
C. Capasso, C.T. Supuran, Bioorg Med Chem Lett. 24 (2014) 4389; d) C.T. Supuran, C. Capasso,
Expert Opin Ther Targets 19 (2015) 551; e) C.T. Supuran, Future Med Chem. 8 (2016) 311.
14. a) C. Capasso, C.T. Supuran, Expert Opin Ther Pat. 23 (2013) 693; b) S. Del Prete, D. Vullo, V.
De Luca, V. Carginale, A. Scozzafava, C.T. Supuran, C. Capasso, Bioorg Med Chem Lett. 23

(2013) 4067; c) S. Del Prete, V. De Luca, D. Vullo, A. Scozzafava, V. Carginale, C.T. Supuran, C.
Capasso, J Enzyme Inhib Med Chem. 29 (2014) 532; d) C. Capasso, C.T. Supuran, J Enzyme Inhib
Med Chem. 30 (2015) 325; e) J.K. Modak, Y.C. Liu, M.A. Machuca, C.T. Supuran, A.
Roujeinikova, PLoS One 10 (2015) e0127149; e) F. Carta, S.M. Osman, D. Vullo, Z. AlOthman, S.
Del Prete, C. Capasso, C.T. Supuran, Bioorg Med Chem. 23 (2015) 6794.
15. a) S.L. Alderman, T.S. Harter, J.M. Wilson, C.T. Supuran, A.P. Farrell, C.J. Brauner, J Exp
Biol. 219 (2016) 719; b) T.J. Warren, M.J. Van Hook, C.T. Supuran, W.B. Thoreson, J Physiol. 594
(2016) 6661.
16. a) C.T. Supuran, Expert Opin Drug Metab Toxicol. 12 (2016) 423; b) R.L. Arechederra, A.
Waheed, W.S. Sly, C.T. Supuran, S.D. Minteer, Bioorg Med Chem. 21 (2013) 1544.
17. a) C.T. Supuran, M.A. Ilies, A. Scozzafava, Eur J Med Chem. 33 (1998) 739; b) C.T. Supuran,
Curr Pharm Des. 2008, 14, 641; c) F. Carta, C.T. Supuran, Expert Opin Ther Pat. 23 (2013) 681; d)
C. Temperini, A. Cecchi, A, Scozzafava, C.T. Supuran, Org Biomol Chem. 6 (2008) 2499.
18. H. S. Lee, T. Bae, J.-H. Lee, D. G. Kim, Y. S. Oh, Y. Jang, J.-T. Kim, J.-J. Lee, A. Innocenti,
C.T. Supuran, L. Chem, K. Rho, S. Kim, BMC Syst Biol. 6 (2012) 80.
19. a) N. Lounnas, C. Rosilio, M. Nebout, D. Mary, E. Griessinger, Z. Neffati, H. Spits, T.
Hagenbeek, V. Asnafi, S.A. Poulsen, C.T. Supuran, J.F. Peyron, V. Imbert, Cancer Lett. 333 (2013)
76; b) F. Margheri, M. Ceruso, F. Carta, A. Laurenzana, L. Maggi, S. Lazzeri, G. Simonini, F.
Annunziato, M. Del Rosso, C.T. Supuran, R. Cimaz, J Enzyme Inhib Med Chem 31(sup4) (2016)
60; c) S. Bua, L. Di Cesare Mannelli, D. Vullo, C. Ghelardini, G. Bartolucci, A. Scozzafava, C.T.
Supuran, F. Carta, J Med Chem 60 (2017) 1159.
20. a) S. Carradori, A. Mollica, C. De Monte, A. Granese, C.T. Supuran, Molecules 20 (2015)
5667; b) C.T. Supuran, Expert Rev Neurother. 16 (2016) 961.
21. a) C.T. Supuran, F. Carta, A. Scozzafava, Expert Opin Ther Pat. 23 (2013) 777; b) C. Capasso,
C.T. Supuran, J Enzyme Inhib Med Chem. 29 (2014) 379; c) M. Ceruso, S. Del Prete, Z.
AlOthman, S.M. Osman, C. Capasso, C.T.Supuran, Bioorg Med Chem Lett. 22 (2014) 4006; d) C.
Capasso, C.T. Supuran, Expert Opin Ther Targets 19 (2015) 1689; e) C.T. Supuran, A. Scozzafava,
A. Mastrolorenzo, Expert Opin Ther Pat. 11 (2001) 221.
22. a) F. Carta, A. Scozzafava, C.T. Supuran, Expert Opin Ther Pat. 22 (2012) 747; b) Y. Akbaba,
A. Akincioglu, H. Göçer, S. Göksu, I. Gülçin, C.T. Supuran, J Enzyme Inhib Med Chem. 29 (2014)
35; c) S.M. Monti, C.T. Supuran, G. De Simone, Expert Opin Ther Pat. 23 (2013) 737; d) S.
Carradori, C. De Monte, M. D’Ascenzio, D. Secci, G. Celik, M. Ceruso, D. Vullo, A. Scozzafava,
C.T. Supuran, Bioorg Med Chem Lett. 23 (2013) 6759; e) M. D’Ascenzio, S. Carradori, D. Secci,
D. Vullo, M. Ceruso, A. Akdemir, C.T. Supuran, Bioorg Med Chem. 22 (2014) 3982; f) L. Puccetti,

G. Fasolis, D. Vullo, Z.H. Chohan, A. Scozzafava, C.T. Supuran, Bioorg Med Chem Lett. 15
(2005) 3096; g) J.Y. Winum, A. Scozzafava, J.L. Montero, C.T. Supuran, Med Res Rev. 26 (2006)
767.
23. a) F. Pacchiano, F. Carta, P. C. McDonald, Y. Lou, D. Vullo, A. Scozzafava, S. Dedhar, C.T.
Supuran, J Med Chem. 54 (2011) 1896; b) F. Pacchiano, M. Aggarwal, B. S. Avvaru, A.H.
Robbins, A. Scozzafava, R. McKenna, C.T. Supuran, Chem. Commun. 46 (2010) 8371.
24. See at: https://clinicaltrials.gov/ct2/show/NCT02215850
25. Y. Lou, P.C. McDonald, A.Oloumi, S. Chia, C. Oslund, A. Ahmadi, A. Kyle, U. auf dem
Keller, S. Leung, D. Huntsman, B. Clarke, B.W. Sutherland, AD. Waterhouse, M. Bally, C.
Roskelley, C.M. Overall, A. Minchinton, F. Pacchiano, F. Carta, A. Scozzafava, N. Touisni, J.Y.
Winum, C.T. Supuran,S. Dedhar, Cancer Res. 71 (2011) 3364.
26. F.E. Lock, P.C. McDonald, Y. Lou, I. Serrano, S.C. Chafe, C. Ostlund, S. Aparicio, J.-Y.
Winum, C.T. Supuran, S. Dedhar, Oncogene 32 (2013) 5210.
27. a) C.L. Lomelino, B.P. Mahon, R. McKenna, F. Carta, C.T. Supuran, Bioorg Med Chem. 2016,
24, 976; b) C. Congiu, V Onnis, A. Deplano, G. Balboni, M. Ceruso, C.T. Supuran, Bioorg Med
Chem. 23 (2015) 5619; c) C. Congiu, V Onnis, A. Deplano, G. Balboni, N. Dedeoglu, C.T.
Supuran, Bioorg Med Chem Lett. 25 (2015) 3850.
28. R. G. Khalifah, J. Biol. Chem. 246 (1971) 2561.
29. a) D. Vullo, J. Voipio, A. Innocenti, C. Rivera, H. Ranki, A. Scozzafava, K. Kaila, C.T.
Supuran, Bioorg. Med. Chem. Lett. 15 (2005) 971; b) I. Nishimori, D. Vullo, A. Innocenti, A.
Scozzafava, A. Mastrolorenzo, C.T. Supuran, J. Med. Chem. 48 (2005) 7860; c) A. Scozzafava, L.
Menabuoni, F. Mincione, C.T. Supuran, J. Med. Chem. 45 (2002) 1466; d) C.T. Supuran, M.A.
Ilies, A. Scozzafava, Eur. J. Med. Chem. 33 (1998) 739; e) Z.H. Chohan, A. Scozzafava, C.T.
Supuran, J. Enzyme Inhib. Med. Chem. 17 (2002) 261.
30. a) C.T. Supuran, M. Barboiu, C. Luca, E. Pop, M.E. Brewster, A. Dinculescu, Eur. J. Med.
Chem. 31 (1996) 597; b) F. Carta, M. Aggarwal, A. Maresca, A. Scozzafava, R. McKenna, E.
Masini, C.T. Supuran, J. Med. Chem. 55 (2012) 1721; c) L. Puccetti, Fasolis G., D. Vullo, Z.H.
Chohan, A. Scozzafava, C.T. Supuran, Bioorg. Med. Chem. Lett. 15 (2005) 3096; d) C.T. Supuran,
A. Nicolae, A. Popescu, Eur. J. Med. Chem. 31 (1996) 431; e) S. Bilginer, E. Unluer, H.I. Gul, E.
Mete, S. Isik, D. Vullo, S. Beyaztas, O. Ozensoy-Guler, C. Capasso, C.T. Supuran, J Enzyme Inhib
Med Chem. 29 (2014) 495.

Synthesis and carbonic anhydrase inhibition of a series of SLC-0111 analogs
Fabrizio Carta,1 Daniela Vullo,2 Sameh M. Osman,3 Zeid AlOthman,3 and
Claudiu T. Supuran*1,2,3
1; Ar= 4-Chloro-2-fluorophenyl
2; Ar= 4-Bromo-2-fluorophenyl
3; Ar= 2-Fluoro-5-nitrophenyl
4; Ar= 2,4,5-Trifluorophenyl
5; Ar= 2-Fluoro-5-trifluorophenyl
O
C
6; Ar= 2-Fluoro-3-trifluorophenyl
7; Ar= 2,3,4-Trifluorophenyl
8; Ar= 2-Fluorophenyl
9; Ar= 2,4-Difluorophenyl
10; Ar= 3-Chlorophenyl
11; Ar= 2,5-Dichlorophenyl
12; Ar= 2-Chloro-5-nitrophenyl
13; Ar= 2-Chloro-4-trifluorophenyl
14; Ar= 2,6-Difluorophenyl
15; Ar=Perchlorophenyl
H NO S
2
H NO S
2
2
2
Ar
N
O
Ar
NH
ACN
N
H
N
H
2
1-15
A