Isoform-selective inhibitory profile of 2-imidazoline-substituted benzene sulfonamides against a panel of human carbonic anhydrases

Article abstract of DOI:10.1080/14756366.2016.1178248

A series of novel benzene sulfonamides (previously evaluated as selective cyclooxygenase-2 inhibitors) has been profiled against human carbonic anhydrases I, II, IV and VII in an attempt to observe the manifestation of the well established “tail” approach

Full text of DOI:10.1080/14756366.2016.1178248

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Isoform-selective inhibitory profile of 2-
imidazoline-substituted benzene sulfonamides
against a panel of human carbonic anhydrases
Claudiu T. Supuran, Stanislav Kalinin, Muhammet Tanç, Pakornwit Sarnpitak,
Prashant Mujumdar, Sally-Ann Poulsen & Mikhail Krasavin
To cite this article: Claudiu T. Supuran, Stanislav Kalinin, Muhammet Tanç, Pakornwit
Sarnpitak, Prashant Mujumdar, Sally-Ann Poulsen & Mikhail Krasavin (2016): Isoform-selective
inhibitory profile of 2-imidazoline-substituted benzene sulfonamides against a panel of human
carbonic anhydrases, Journal of Enzyme Inhibition and Medicinal Chemistry
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1178248
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–6
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1178248
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SHORT COMMUNICATION
Isoform-selective inhibitory profile of 2-imidazoline-substituted
benzene sulfonamides against a panel of human carbonic anhydrases
Claudiu T. Supuran1, Stanislav Kalinin2, Muhammet Tanc¸1, Pakornwit Sarnpitak3, Prashant Mujumdar3,
Sally-Ann Poulsen3, and Mikhail Krasavin4
1Neurofarba Department, Universita Degli Studi Di Firenze, Florence, Italy, 2The Ushinsky Yaroslavl State Pedagogical University, Yaroslavl, Russian
4
Federation, 3Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia, and Institutes of Chemistry and Translational
Biomedicine, Saint Petersburg State University, Saint Petersburg, Russian Federation
Abstract
Keywords
A series of novel benzene sulfonamides (previously evaluated as selective cyclooxygenase-2
inhibitors) has been profiled against human carbonic anhydrases I, II, IV and VII in an attempt to
observe the manifestation of the well established ‘‘tail’’ approach for designing potent, isoform-
selective inhibitors of carbonic anhydrases (CAs, EC 4.2.1.1). The compounds displayed an
excellent (pKi 7–8) inhibitory profile against CA II (a cytosolic anti-glaucoma and anti-edema
biological target) and CA VII (also a cytosolic target believed to be involved in epilepsy and
neuropathic pain) and a marked (1–2 orders of magnitude) selectivity against cytosolic isoform
CA I and membrane-bound isoform CA IV. The separation of the CA II and CA IV (both of which
are catalytically active isoforms, highly sensitive to sulfonamide-type inhibitors) is particularly
remarkable and is adding significantly to the global body of data on the chemical biology of
carbonic anhydrases.
Carbonic anhydrases, cyclooxygenase-2
inhibitors, dual active site architecture, 2-
imidazolines, isoform selectivity, lipophilic
appendages, tail approach
History
Received 24 March 2016
Revised 9 April 2016
Accepted 11 April 2016
Published online 3 May 2016
Introduction
the protein environment where two distinct parts have been
delineated8. By grafting appendages of various nature (hydro-
philic and lipophilic alike) onto the benzenesulfonamide scaffold, it
has become possible to impart significant potency and selectivity to
the resulting CAIs. The effectiveness of building contacts with the
lipophilic side of the CA active site has been shown to correlate
with the potency of the CAIs (exemplified by compounds 1–3)9. A
well-designed and informative comparison of hydrophilic versus
lipophilic (as well as mixed) appendage design by the so-called
‘‘tail approach’’10 (illustrated by compounds 4–6) has been recently
published by Poulsen and colleagues11.
Inspired by these findings, we became interested in assessing
CAI profile of a series of benzene sulfonamides decorated with 2-
aryl-2-imidazoline appendages (7). These compounds have been
recently described in the context of selective cyclooxygenase-2
inhibition12. We were interested to see if these compounds
(somewhat akin to ureido compounds 1–3)9 would be effective as
CAIs in principle (Figure 1). Furthermore, assessing their isoform
selectivity profile against CAs with COX-2 inhibition data12
would offer an opportunity to consider developing dual-action
therapeutic agents (e.g. endowed with antiglaucoma and anti-
inflammatory activity manifested via inhibition of CAs and COX-
2, respectively)13. This article describes the details of our findings
in this area.
The goal of developing isoform-selective inhibitors of human
carbonic anhydrases (hCA) has stimulated extensive medicinal
chemistry activity worldwide1. Selective inhibition of a particular
isoform (in contrast to nonselective carbonic anhydrase inhibitors
(CAIs), such as acetazolamide2) is likely to improve therapeutic
benefit over side-effects and toxicity for a well-established hCA-
disease relationship (e.g. hCA II for glaucoma)3. Enhancing the
selectivity of CAIs may even validate CAs in fundamentally new
therapeutic areas. For instance, selective targeting of membrane-
bound isoforms hCA IX and XII has been proposed as a novel
approach to halt the growth of solid tumors by suppressing tumor
survival mechanism in hypoxic environment4. A significant body
of pre-clinical proof-of-concept data5 as well as one front-runner
compound (SLC-0111) in clinical trials for cancer6 offers much
hope that selective hCA isoform targeting will receive clinical
validation in the future.
A well-established, rational approach to designing isoform-
selective CAIs exists in perhaps the most investigated class of
inhibitors, namely, benzenesulfonamides. Anchoring of the sul-
fonamide group in this class of compounds to the prosthetic zinc
ion defines the position of the organic portion of the molecule
within the CA binding cavity7 and offers significant opportunity
to build, somewhat serendipitously, additional interactions with
Materials and methods
Address for correspondence: Claudiu T. Supuran, Neurofarba
Department, Universita Degli Studi Di Firenze, Florence, Italy. E-mail:
claudiu.supuran@unifi.it; Mikhail Krasavin, Institutes of Chemistry and
Translational Biomedicine, Saint Petersburg State University, Saint
Petersburg 199034, Russia. E-mail: m.krasavin@spbu.ru
Chemical syntheses – general
All chemicals and solvents were used as received from the
suppliers. The crude reaction mixtures were concentrated under

2
C. T. Supuran et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
R
Ki (CA II), nM
96
Hydrophobic
appendage
1:
2:
3:
4-FC6H4
O
R
HN
HN
O
S NH2
O
C6F5
50
15
Zinc-binding group (ZBG)
3-O2NC6H4
1 - 3
OH
O
HO
HO
N
N
N
N
N
N
N
N
N
OH
OH
N
N
N
N
OH
O
N
N
N
N
N
N
N
N
O
O
O
S O
NH2
S
N
S
N
O
S
N
HO
HO
HO
HO
S
O
S
O
N
N
N
OH
NH2
NH2
OH
4 ("HYDRO/LIPO")
5 ("LIPO/LIPO")
6 ("HYDRO/HYDRO")
R
Hydrophobic
appendage
O
N
N
S NH2
O
Zinc-binding group (ZBG)
5
Figure 1. Structures of benzenesulfonamide CAIs containing hydrophobic ureido tails (1–3), the ‘‘dual-tail’’ compounds (4–6) and the design rationale
for 2-imidazoline-substituted compounds 7.
reduced pressure by removing organic solvents on rotary 4-bromo-N,N-bis(2,4-dimethoxybenzyl)benzenesulfonamide15
evaporator. Column chromatography was performed using silica (3.0 mmol), Cs2CO3 (3.0 mmol), and toluene (15 mL).
gel 60 (particle size 0.040–0.063 mm, 230–400 mesh ASTM). Pd(OAc)2 (0.12 mmol) and BINAP (0.24 mmol) were weighed
Analytical thin-layer chromatography (TLC) was performed with as solids into a 10 mL screw-capped vial and toluene (8 mL) was
silica gel 60 F254 aluminum sheets. Proton and carbon-13 nuclear added. The resulting suspension was heated at 110 ꢀC until a clear
magnetic resonance (1H NMR and 13C NMR) spectra were purple solution was evident. The solution was rapidly transferred,
recorded on a Varian Unity INOVA 500 MHz spectrometer while still hot, to the screw-capped pressure tube using a Pasteur
(NMR Laboratory, Urbana, IL). Chemical shifts for nuclear pipette. The reaction vessel was filled with argon, sealed, and the
magnetic resonance (NMR) spectra were reported in parts per reaction mixture vigorously stirred at 110 ꢀC for 20 h. The
million (ppm, d) using the residual solvent peak(s) as internal reaction mixture was cooled to ambient temperature and filtered
standard. Splitting patterns are described as singlet (s), doublet through a Celite pad (Beaver Chemicals, Burlington Ltd., Ontario,
(d), triplet (t), quartet (q), multiplet (m), broad (br) and doublets Canada), washed with copious amounts of EtOAc, and the
of doublet (dd). High-resolution mass spectra (HRMS) were combined filtrate and washings evaporated to dryness. The crude
acquired using a Bruker Daltonics Fourier Transform Ion material was fractionated on silica (CH2Cl2:MeOH, 95:5) and the
Cyclotron Resonance Spectrometer (Bruker Daltonics Inc., fractions containing the N-aryl 2-imidazolines were collected and
Billerica, MA) fitted with an electrospray source operating in evaporated to dryness. The residue was taken forward to the next
positive ion mode.
step without further purification.
TFA (3 mL) was added drop wise to a solution of DMB-
protected sulfonamides (obtained as described above) in CH2Cl2
(10 mL) at 0 ꢀC and stirring continued at the same temperature for
30–60 min. The excess TFA was neutralized with sat. aq.
NaHCO3, the organic layer separated and dried over anhydrous
MgSO4. The solution was filtered and concentrated in vacuo. The
resulting crude product was purified by column chromatography
on silica using an appropriate gradient of MeOH in CH2Cl2 as
eluent to provide sulfonamide products 5a–o.
General procedure 1 for preparation of 2-imidazolines 6a–o14
Ethylenediamine (10.5 mmol, 1.05 equiv.) was added to a stirred
solution of aldehyde (10.0 mmol, 1.0 equiv.) in CH2Cl2 (50 mL) at
0 ꢀC, and the resulting solution stirred for 20 min. N-bromosucci-
nimide (10.5 mmol, 1.05 equiv.) was then added in portions over
10 min. The reaction mixture was warmed to room temperature
and stirred for an additional 12 h, during which time a precipitate
formed. A solution of KOH (1.0 M, 50 mL, 50 mmol, 5.0 equiv.)
was added causing the precipitate to disappear and a clear
biphasic solution was formed. The organic layer was separated,
dried over anhydrous MgSO4, filtered, and concentrated at
reduced pressure to afford the crude product. The analytically
pure imidazolines 6 were obtained by crystallization from EtOAc/
hexane. All imidazolines 6a–o used in subsequent Pd-catalyzed
N-arylation are known compounds12.
4-(2-(4-Fluorophenyl)-4,5-dihydroimidazol-1-yl)benzenesulfona-
mide (5a). Prepared according to General Procedure 2 from 2–
(4-fluorophenyl)-4,5-dihydro-1H-imidazole. Yield 700 mg (73%),
1
light yellow solid, m. p. 94–96 ꢀC; H NMR (500 MHz, CDCl3)
d 7.69 (d, J ¼ 8.8 Hz, 2 H), 7.50–7.45 (m, 2 H), 7.07–7.02
(m, 2 H), 6.75 (d, J ¼ 8.8 Hz, 2 H), 4.91 (br s, 2 H), 4.15–4.03
(m, 4 H); 13C NMR (125 MHz, CDCl3) d 163.9 (d, JC-F
¼ 251.4 Hz), 160.1, 146.2, 134.9, 130.6 (d, JC-F ¼ 8.6 Hz),
127.4, 126.8 (d, JC-F ¼ 3.4 Hz), 120.5, 115.8 (d, JC-F ¼ 21.9 Hz),
General procedure 2 for preparation of compounds 5a–o12
A
thick-glass screw-capped pressure tube (50 mL) was 53.13, 53.11; HRMS m/z calcd for C15H15FN3O2S
charged with a suspension of starting imidazoline (3.0 mmol), (M + H)+ 320.0864 found 320.0860.

DOI: 10.1080/14756366.2016.1178248
Isoform-selective inhibitory profile of 2-imidazoline-substituted benzene sulfonamides
3
4-(2-(4-Fluorophenyl)-4-methyl-4,5-dihydroimidazol-1-yl)benze-
nesulfonamide (5b). Prepared according to General Procedure 2 147.1, 140.2, 134.9 (d, JC-F ¼ 7.8 Hz), 132.6 (d, JC-F ¼ 8.1 Hz),
from
NMR (125 MHz, CD3OD) d 164.7 (d, JC-F ¼ 244.6 Hz), 163.6,
2-(4-fluorophenyl)-4-methyl-4,5-dihydro-1H-imidazole. 128.9, 126.5 (d, JC-F ¼ 3.2 Hz), 123.5, 117.4 (d, JC-F ¼ 23.1 Hz),
1
Yield 360 mg (36%), yellow solid, m. p. 157–159 ꢀC; H NMR 119.4 (d, JC-F ¼ 21.1 Hz), 54.9, 54.2; HRMS m/z calcd for
(500 MHz, CD3OD) d 7.86–7.82 (m, 2 H), 7.57–7.53 (m, 2 H), C15H15FN3O2S (M + H)+ 320.0864 found 320.0854.
7.26–7.19 (m, 4 H), 4.63–4.53 (m 2 H), 4.14–4.06 (m 1 H), 1.56–
1.51 (m, 3 H), NH2 protons in exchange. 13C NMR (125 MHz,
4-(2-(3-Chlorophenyl)-4,5-dihydroimidazol-1-yl)benzenesulfona-
CD3OD) d 166.7 (d, JC-F ¼ 254.0 Hz), 164.2, 143.3, 142.8, 133.1
mide (5h). Prepared according to General Procedure 2 from 2-
(d, JC-F ¼ 9.4 Hz), 128.6, 125.5, 117.5 (d, JC-F ¼ 22.6 Hz), 61.3,
(3-chlorophenyl)-4,5-dihydro-1H-imidazole. Yield 794 mg (79%),
white solid, m. p. 188–190 ꢀC; 1H NMR (500 MHz, CD3OD)
d 7.78–7.76 (m, 2 H), 7.54–7.56 (m, 2 H), 7.44–7.38 (m, 2 H),
55.8, 20.9. HRMS m/z calcd for C16H17FN3O2S
(M + H)+ 334.1020 found 334.1016.
6.98–6.96 (m, 2 H), 4.25 (t, J ¼ 9.4 Hz, 2 H), 4.10 (t, J ¼ 9.7 Hz,
4-(2-(4-Chlorophenyl)-4,5-dihydroimidazol-1-yl)benzenesulfona-
2 H), NH2 protons in exchange; 13C NMR (125 MHz, CD3OD)
mide (5c). Prepared according to General Procedure 2 from 2- d 163.5, 147.1, 140.2, 136.5, 134.7, 132.6, 132.2, 130.5, 129.0,
(4-chlorophenyl)-4,5-dihydro-1H-imidazole. Yield 663 mg (66%), 128.9, 123.5, 55.0, 54.2; HRMS m/z calcd for C15H15ClN3O2S
yellow solid, m. p. 141–143 ꢀC; 1H NMR (500 MHz, CD3OD) (M + H)+ 336.0568 found 336.0560.
d 7.81 (d, J ¼ 8.8 Hz, 2 H), 7.54–7.46 (m, 4 H), 7.08 (d,
4-(2-m-Tolyl-4,5-dihydroimidazol-1-yl)benzenesulfonamide
(5i). Prepared according to General Procedure 2 from 2-(m-
J ¼ 8.8 Hz, 2 H), 4.36 (t, J ¼ 9.9 Hz, 2 H), 4.14 (t, J ¼ 9.9 Hz,
2 H), NH2 protons in exchange; 13C NMR (125 MHz, CD3OD)
tolyl)-4,5-dihydro-1H-imidazole. Yield 350 mg (37%), cream
d 163.9, 145.0, 140.7, 1387, 131.5, 130.1, 128.4, 128.3, 123.7,
54.3, 51.1; HRMS m/z calcd for C15H15ClN3O2S
solid, m. p. 244–246 ꢀC; 1H NMR (500 MHz, CD3OD) d 7.73
(M + H)+ 336.0568 found 336.0564.
(d, J ¼ 8.8 Hz, 2 H), 7.37–7.26 (m, 3 H), 7.22–7.18 (m, 1 H), 6.99
(d, J ¼ 8.8 Hz, 2 H), 4.28 (t, J ¼ 10.3 Hz, 2 H), 4.07 (t, J ¼ 10.3 Hz,
2 H), 2.33 (s, 3H), NH2 protons in exchange; 13C NMR (125 MHz,
4-(2-(4-Chlorophenyl)-4-methyl-4,5-dihydroimidazol-1-yl)benze-
nesulfonamide (5d). Prepared according to General Procedure 2
CD3OD) d 164.7, 145.5, 140.1, 140.0, 133.1, 130.3, 130.2, 129.8,
128.1, 126.9, 123.1, 54.1, 51.4, 21.3; HRMS m/z calcd for
C16H18N3O2S (M + H)+ 316.1114 found 316.1107.
from
2-(4-chlorophenyl)-4-methyl-4,5-dihydro-1H-imidazole.
Yield 534 mg (51%), light brown solid, m. p. 160–162 ꢀC; 1H
NMR (500 MHz, CD3OD) d 7.85 (d, J ¼ 8.7 Hz, 2 H), 7.53–7.47
(m, 4 H), 7.26 (d, J ¼ 8.7 Hz, 2 H), 4.67–4.58 (m, 2 H), 4.17–4.09
(m, 1 H), 1.58–1.50 (m, 3 H), NH2 protons in exchange; 13C NMR
(125 MHz, CD3OD) d 164.3, 143.5, 142.5, 140.5, 132.0, 130.6,
128.6, 125.7, 124.5, 61.3, 55.6, 20.8; HRMS m/z calcd for
C16H17ClN3O2S (M + H)+ 350.0724 found 350.0719.
4-(2-(3-Fluoro-4-methoxyphenyl)-4,5-dihydroimidazol-1-yl)ben-
zenesulfonamide (5j). Prepared according to General Procedure
2 from 2-(3-fluoro-4-methoxyphenyl)-4,5-dihydro-1H-imidazole.
1
Yield 712 mg (68%), yellow solid, m. p. 195–197 ꢀC; H NMR
(500 MHz, CD3OD) d 7.78–7.76 (m, 2 H), 7.25 (t, J ¼ 7.3 Hz,
2 H), 7.14 (t, J ¼ 7.6 Hz, 1 H), 7.00–6.98 (m, 2 H), 4.22 (t,
J ¼ 9.6 Hz, 2 H), 4.06 (t, J ¼ 9.6 Hz, 2 H), 3.95 (s, 3H), NH2
protons in exchange; 13C NMR (125 MHz, CD3OD) d 163.6,
153.9 (d, JC-F ¼ 245.3 Hz), 152.0 (d, JC-F ¼ 10.6 Hz), 147.5,
140.1, 128.9, 127.4 (d, JC-F ¼ 3.5 Hz), 124.8 (d, JC-F ¼ 7.0 Hz),
123.7, 118.1 (d, JC-F ¼ 20.0 Hz), 115.3, 57.6, 55.1, 53.8; HRMS
m/z calcd for C16H17FN3O3S (M + H)+ 350.0969 found 350.0960.
4-(2-(3-Bromophenyl)-4,5-dihydroimidazol-1-yl)benzenesulfona-
mide (5e). Prepared according to General Procedure 2 from 2-
(3-bromophenyl)-4,5-dihydro-1H-imidazole. Yield 409 mg (36%),
white solid, m. p. 194–194 ꢀC; 1H NMR (500 MHz, CD3OD)
d 7.73 (d, J ¼ 8.8 Hz, 2 H), 7.68–7.64 (m, 2 H), 7.40–7.36 (m,
1 H), 7.31 (t, J ¼ 7.8 Hz, 1 H), 6.98 (d, J ¼ 8.8 Hz, 2 H), 4.20 (td,
J ¼ 9.3, 1.2 Hz, 2 H), 4.05 (td, J ¼ 9.3, 1.2 Hz, 2 H), NH2 protons
in exchange; 13C NMR (125 MHz, CD3OD) d 162.6, 146.2, 139.4,
134.8, 134.0, 132.3, 131.5, 128.5, 128.1, 123.5, 122.6, 54.1, 53.3;
HRMS m/z calcd for C15H15BrN3O2S (M + H)+ 380.0063 found
380.0057.
4-(2-(4-Fluoro-3-methoxyphenyl)-4,5-dihydroimidazol-1-yl)ben-
zenesulfonamide (5k). Prepared according to General Procedure
2 from 2-(4-fluoro-3-methoxyphenyl)-4,5-dihydro-1H-imidazole.
Yield 691 mg (66%), white solid, m. p. 248–249 ꢀC; 1H NMR
(500 MHz, CD3OD) d 7.79–7.76 (m, 2 H), 7.24 (dd, J ¼ 8.1,
1.8 Hz, 1 H), 7.14 (dd, J ¼ 13.7, 8.3 Hz, 1 H), 7.01–7.04 (m, 1 H),
7.00 (d, J ¼ 8.8 Hz, 2 H), 4.25 (t, J ¼ 9.4 Hz, 2 H), 4.09 (t,
J ¼ 10.3 Hz, 2 H), 3.83 (s, 3 H), NH2 protons in exchange; 13C
NMR (125 MHz, CD3OD) d 164.1, 155.8 (d, JC-F ¼ 249.1 Hz),
150.0 (d, JC-F ¼ 11.3 Hz), 147.3, 140.1, 130.7, 128.8, 123.7,
123.6, 118.0 (d, JC-F ¼ 19.0 Hz), 116.2 (d, JC-F ¼ 1.8 Hz), 57.6,
54.9, 53.8; HRMS m/z calcd for C16H17FN3O3S
(M + H)+ 350.09693 found 350.0960.
4-(2-(3-Bromophenyl)-4-methyl-4,5-dihydroimidazol-1-yl)benze-
nesulfonamide (5f). Prepared according to General Procedure 2
from
2-(3-bromophenyl)-4-methyl-4,5-dihydro-1H-imidazole.
Yield 755 mg (64%), white solid, m. p. 180 ꢀC (decomp.); 1H
NMR (500 MHz, CD3OD) d 7.91 (d, J ¼ 8.7 Hz, 2 H), 7.86–7.83
(m, 1 H), 7.81–7.78 (m, 1 H), 7.48–7.37 (m, 4 H), 4.75–4.68 (m,
2 H), 4.32–4.24 (m, 1 H), 1.63–1.60 (m, 3 H), NH2 protons in
exchange; 13C NMR (125 MHz, CD3OD) d 164.7, 145.1, 141.0,
138.0, 133.2, 132.2, 129.3, 128.8, 126.7, 125.9, 124.0, 51.5, 54.0,
20.4; HRMS m/z calcd for C16H17BrN3O2S (M + H)+ 394.0219
found 394.0214.
4-(2-(3-Chloro-4-methoxyphenyl)-4,5-dihydroimidazol-1-yl)ben-
zenesulfonamide (5l). Prepared according to General Procedure
2 from 2-(3-chloro-4-methoxyphenyl)-4,5-dihydro-1H-imidazole.
Yield 712 mg (63%), white solid, m. p. 255–257 ꢀC; 1H NMR
(500 MHz, CD3OD) d 7.78 (d, J ¼ 8.6 Hz, 2 H), 7.55 (s, 1 H), 7.38
4-(2-(3-Fluorophenyl)-4,5-dihydroimidazol-1-yl)benzenesulfona-
mide (5g). Prepared according to General Procedure 2 from 2– (d, J ¼ 8.6 Hz, 1 H), 7.11 (d, J ¼ 8.4 Hz, 1 H), 6.99 (d, J ¼ 8.6 Hz,
(3-fluorophenyl)-4,5-dihydro-1H-imidazole. Yield 584 mg (61%), 2 H), 4.23 (t, J ¼ 9.6 Hz, 2 H), 4.06 (t, J ¼ 9.2 Hz, 2 H), 3.97 (s,
white solid, m. p. 225–227 ꢀC; 1H NMR (500 MHz, CD3OD) 3 H), NH2 protons in exchange; 13C NMR (125 MHz, CD3OD)
d 7.77–7.75 (m, 2 H), 7.48–7.44 (m, 1 H), 7.29 (t, J ¼ 7.4 Hz, 2 H), d 163.5, 159.3, 147.5, 140.1, 132.3, 130.8, 129.0, 125.4, 124.6,
7.25 (d, J ¼ 8.0 Hz, 1 H), 6.97–6.96 (m, 2 H), 4.24 (t, J ¼ 9.6 Hz, 123.6, 114.0, 57.7, 55.1, 53.8; HRMS m/z calcd for
2 H), 4.09 (t, J ¼ 9.6 Hz, 2 H), NH2 protons in exchange; 13C C16H17ClN3O3S (M + H)+ 366.0674 found 366.0664.

4
C. T. Supuran et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
4-(2-(3,4-Difluorophenyl)-4,5-dihydroimidazol-1-yl)benzenesul-
the kinetic parameters and inhibition constants. For each inhibitor
fonamide (5m). Prepared according to General Procedure 2 at least six traces of the initial 5–10% of the reaction have been
(alternatively, the recently reported microwave-assisted protocol used for determining the initial velocity. The uncatalyzed rates
was used from 2-(3,4-difluorophenyl)-4,5-dihydro-1H-imidazole. were determined in the same manner and subtracted from the total
Yield 465 mg (46%), white solid, m. p. 177–179 ꢀC; 1H NMR observed rates. Stock solutions of inhibitor (0.1 mM) were
(500 MHz, CD3OD) d 7.78 (d, J ¼ 7.9 Hz, 2 H), 7.46–7.44 (dd, prepared in distilled-deionized water and dilutions up to
J ¼ 10.6, 8.9 Hz, 1 H), 7.38–7.30 (m, 2 H), 6.98 (d, J ¼ 8.0 Hz, 0.005 nM were done thereafter with the assay buffer. Inhibitor
2 H), 4.24 (t, J ¼ 9.8 Hz, 2 H), 4.09 (t, J ¼ 9.7 Hz, 2 H), NH2 and enzyme solutions were pre incubated together for 15 min at
protons in exchange; 13C NMR (125 MHz, CD3OD) d 162.9, room temperature prior to assay, in order to allow for the
153.8 (dd, JC-F ¼ 251.8, 12.5 Hz), 152.2 (dd, JC-F ¼ 248.6, formation of the E-I complex. The inhibition constants were
12.8 Hz), 147.1, 140.3, 129.8 (dd, JC-F ¼ 6.3, 4.2 Hz), 129.0, obtained by non-linear least-squares methods using PRISM 3 and
127.7 (dd, JC-F ¼ 6.7, 3.7 Hz), 123.7, 119.9 (d, JC-F ¼ 19.0 Hz), the Cheng–Prusoff equation, as reported earlier, and represent the
119.7 (d, JC-F ¼ 18.0 Hz), 55.1, 54.2; HRMS m/z calcd for mean from at least three different determinations. All CA
C15H14F2N3O2S
(M + H)+ 338.0769
found
338.0760. isoforms were recombinant ones obtained in-house17–20
.
Alternatively, the recently reported microwave-assisted protocol15
was used to obtain the same compound in 68% yield.
Results and discussion
Compounds 5a–o were synthesized as outlined in Scheme 112.
Imidazolines 6 were prepared by condensation of a series of
benzaldehydes with ethylenediamine or 1,2-diaminopropane
using a well-established protocol13. Pd-Catalyzed N-arylation of
6a–o with 8 proved facile and, without further purification, the
DMB (2,4-dimethoxybenzyl) protecting group from 7a–o was
removed with TFA and the target sulfonamides were obtained in
fair to good yields (see Supplementary material).
4-(2-(3,4-Dichlorophenyl)-4,5-dihydroimidazol-1-yl)benzenesul-
fonamide (5n). Prepared according to General Procedure 2 from
2-(3,4-dichlorophenyl)-4,5-dihydro-1H-imidazole. Yield 886 mg
(80%), white solid, m. p. 232–234 ꢀC; 1H NMR (500 MHz,
CD3OD) d 7.79–7.78 (m, 2 H), 7.70 (s, 1 H), 7.59–7.58 (m, 1 H),
7.36–7.38 (m, 1 H), 6.99–6.98 (m, 2 H), 4.29–4.19 (m, 2 H), 4.15–
4.03 (m, 2 H), NH2 protons in exchange; 13C NMR (125 MHz,
CD3OD) d 155.8, 147.0, 140.5, 136.7, 134.7, 133.0, 132.8, 132.6,
130.3, 129.1, 123.7, 55.2, 54.3; HRMS m/z calcd for
C15H14Cl2N3O2S (M + H)+ 370.0178 found 370.0169.
Following our earlier reported synthesis of compounds 5a–o
and their evaluation as selective COX-2 inhibitors12, we have
since developed an operationally convenient protocol21 for
microwave-assisted, Pd-catalyzed N-arylation of 2-imidazolines
which significantly shortened reaction times and, occasionally,
improved isolated product yields. In this work, we demonstrated
the same microwave-promoted procedure to be applicable to the
synthesis of a representative compound (5m) and to lead to a
noticeable improvement of the isolated yield (Scheme 2).
When tested for CA I, II, IV and VII inhibition, compounds
5a–o, reassuringly, displayed an interesting inhibition profile
(Table 1), which is likely a result of the sulfonamide moiety acting
as a zinc-binding group (ZBG)22. A double-digit nanomolar
inhibition was reliably achieved for CA II, which is a well-
established biological target for anti-glaucoma and anti-edema
drugs2. The same level of activity was also achieved against CA
VII, while the lower activity against CA I is perhaps not very
unusual considering the generally lower inhibitory potency of
sulfonamides toward this isoform1. What constitutes an especially
valuable finding in this work was, in our opinion, the marked
selectivity (SI) between CA II and CA IV, which are both highly
catalytically active CA isoforms and known to be very sensitive to
sulfonamides. While the structural understanding of the observed
separation of the CA II versus IV activity remains to be gained via
crystallographic studies, the body of biological data found here
contributes quite substantially to the global knowledge about
isoform-selective CAIs.
4-(2-(4-Chloro-3-(trifluoromethyl)phenyl)-4,5-dihydroimidazol-1-
yl)benzenesulfonamide (5o). Prepared according to General
Procedure
dihydro-1H-imidazole. Yield 786 mg (65%), white solid, m. p.
2 from 2-[4-chloro-3-(trifluoromethyl)phenyl]-4,5-
1
215–217 ꢀC; H NMR (500 MHz, CD3OD) d 7.98 (s, 1 H), 7.79
(d, J ¼ 8.6 Hz, 2 H), 7.66–7.58 (m, 2 H), 7.00 (d, J ¼ 8.6 Hz, 2 H),
4.27 (t, J ¼ 9.7 Hz, 2 H), 4.12 (t, J ¼ 9.6 Hz, 2 H), NH2 protons
in exchange; 13C NMR (125 MHz, CD3OD) d 162.7, 147.0, 140.7,
136.2 (unresolved q, JC-F ¼ 1.9 Hz), 135.5, 133.9, 132.1,
130.5 (q, JC-F ¼ 31.8 Hz), 130.0 (q, JC-F ¼ 5.1 Hz), 129.1, 124.7
(q, JC-F ¼ 271.1 Hz), 123.9, 55.2, 54.4; HRMS m/z calcd for
C16H14ClF3N3O2S (M + H)+ 404.0442 found 404.0431.
Carbonic anhydrase inhibition assay
An Applied Photophysics Stopped-Flow instrument (Applied
Photophysics Ltd., Leatherhead, Surrey, UK) has been used for
assaying the CA catalyzed CO2 hydration activity16. 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 Tris
(pH 8.3) 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
O O
S
O
NH2
S
O
S
N(DMB)2
O
O
Br
(DMB)2N
NH2
8
H2N
Pd(OAc)2 (5 mol%)
H
N
TFA, DCM
0 oC --> r.t.
BINAP (10 mol%)
R
N
Ar CHO
N
Ar
Ar
Ar
Cs2CO3 (1.0 equiv.)
toluene, 100 oC, 20 h
DCM
N
R'
0 oC --> r.t.
R'
N
N
R'
5a-o
7a-o
6a-o
Scheme 1. Synthesis of 2-imidazoline-substituted benzene sulfonamides 5a–o.

DOI: 10.1080/14756366.2016.1178248
Isoform-selective inhibitory profile of 2-imidazoline-substituted benzene sulfonamides
5
O O
S
O
S
O
S
NH2
N(DMB)2
O
O
Br
(DMB)2N
Pd(OAc)2 (2 mol%)
BINAP (4 mol%)
F
TFA, DCM
0 oC --> r.t.
F
F
H
N
N
N
N
F
F
F
Cs2CO3 (1 equiv.)
toluene, MW
N
N
6m
130 oC, 45 min
5m
68% (2 steps)
7m
Scheme 2. Microwave-assisted synthesis of 5m.
Table 1. Inhibitory activity data for compound 5a–o obtained against hCA I, II, IV and VII.
Ar
O
N
N
H2N S
O
R
Ki, nM
SI (hCAIV/hCAII)
Compound
Ar
4-FC6H4
4-FC6H4
4-ClC6H4
4-ClC6H4
3-BrC6H4
3-BrC6H4
3-FC6H4
3-ClC6H4
3-MeC6H4
3-F,4-MeOC6H3
4-F,3-MeOC6H3
3-Cl,4-MeOC6H3
3,4-diFC6H3
3,4-diClC6H3
4-Cl,3-CF3C6H3
R
hCA I
hCA II
hCA IV
hCA VII
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
H
Me
H
Me
H
Me
H
H
H
H
H
H
2 343.0
816.5
960.5
76.1
52.4
46.7
54.1
53.0
55.3
75.9
185.1
111.9
80.7
61.6
227.1
34.6
30.2
84.3
12.0
9 726.0
8 705.0
8 142.0
6 785.0
5 010.0
5 272.0
410 000
5 701.0
9 778.0
8 266.0
4 228.0
410 000
5 381.0
932.0
303.5
86.6
269.0
69.6
204.7
45.8
311.6
204.2
906.7
709.4
258.6
103.9
71.3
35.5
32.6
2.5
127.8
166.1
174.3
125.4
94.5
897.9
1 844.0
1 281.0
3 230.0
1 668.0
3 943.0
1 231.0
2 166.0
3 987.0
746.8
95.3
131.8
30.8
87.4
102.4
68.6
44.0
155.5
30.9
45.6
H
H
H
787.7
3 685.0
250.0
3 845.0
74.0
Acetazolamide (AAZ)
6.2
Currently, the absence of the crystallographic information on
the potent 2-imidazoline CAIs described herein does not allow
drawing substantiated conclusions on the role of the 2-aryl-2-
imidazoline tails grafted onto benzenesulfonamide pharmaco-
phore. However, in our view, it appears unlikely that the polar 2-
imidazoline moiety participates in active interactions with the
protein target. It is rather the lipophilic aromatic tail responsible
for interacting with the hydrophobic half of the CA binding side
which is likely to result in the observed potency and selectivity.
Should the polar 2-imidazoline core be contributing significantly
(e.g. by building hydrogen-bonding interactions with the hydro-
philic half of the binding site), the introduction of a methyl
substituent on the 2-imidazoline scaffold (as in 5b, 5d and 5f)
would be expected to disrupt this vital interaction. However, such
a modification only seems to improve the CA VII affinity and
virtually does not affect the CA I, II and IV inhibitory profile.
This strongly argues for 2-imidazoline being a generally indiffer-
ent element of the periphery, responsible for a correct projection
of the lipophilic periphery toward the target rather than building
any specific interactions with the latter, in a direct analogy to 4-
ureido benzenesulfonamides 1–3 (vide supra). The analogy is
illustrated (Figure 2) quite well by the effective spatial superpos-
ition (in energy-minimized conformations) of compound 3, a
Figure 2. Spatial alignment of compound 5a (green) with its ureido
analog 3 (red).
Interestingly, compound 5n which we had earlier identified as
the lead selective COX-2 inhibitor12, turned out to be a rather
potent CA II and CA VII inhibitor which confirms the potential of
using this series as a dual CA/COX-2 inhibitors.
Conclusion
potent CA II inhibitor (Ki ¼ 96 nM) with compound 5a which The polypharmacology of primary aromatic sulfonamides
inhibits CA II with roughly the same potency (Ki ¼ 76 nM).
prompted us to evaluate the profile of compounds 5a–o (earlier

6
C. T. Supuran et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
8. Krasavin M, Korsakov M, Dorogov M, et al. Probing the ‘bipolar’
nature of the carbonic anhydrase active site: aromatic sulfonamides
containing 1,3-oxazol-5-yl moiety as picomolar inhibitors of cyto-
solic CA I and CA II isoforms. Eur J Med Chem 2015;101:334–47.
9. Pacchiano F, Aggarwal M, Avvaru BS, et al. Selective hydrophobic
pocket binding observed within the carbonic anhydrase II active
site accommodate different 4-substituted-ureidobenzenesulfona-
mides and correlate to inhibitor potency. Chem Commun 2010;46:
8371–3.
10. Supuran CT. Structure-based drug discovery of carbonic anhydrase
inhibitors. J Enzyme Inhib Med Chem 2012;27:759–72.
11. Tanpure RP, Ren B, Peat TS, et al. Carbonic anhydrase inhibitors
with dual-tail moieties to match the hydrophobic and hydrophilic
halves of the carbonic anhydrase active site. J Med Chem 2015;58:
1494–501.
described in the context of selective cyclooxygenase-2 inhibition)
against a panel of hCA (CA I, II, IV and VII). The compounds
showed a strong inhibition of CA II (a well-established cytosolic
glaucoma target) and CA VII (also a cytosolic isoform believed to
be involved in the onset of epilepsy). The absence of activity
against CA I is perhaps unsurprising (considering the documented
lower affinity of CA I toward sulfonamides). However, the
selectivity established for some of compounds 5a–o with respect
to CA II versus CA IV (both of which are highly active isoforms
of the enzyme, strongly inhibited by sulfonamides in general)
constitutes a very significant finding in light of isoform-selective
CA targeting. For compound 5n, strong inhibition of COX-2 and
some CA isoforms could be a basis for designing dual-action
therapeutic agents.
12. Sarnpitak P, Mujumdar P, Morisseau C, et al. Potent, orally
available, selective COX-2 inhibitors based on 2-imidazoline core.
Eur J Med Chem 2014;84:160–72.
13. De Monte C, Carradori S, Gentili A, et al. Dual cyclooxygenase and
carbonic anhydrase inhibition by nonsteroidal anti-inflammatory
drugs for the treatment of cancer. Curr Med Chem 2015;22:
2812–18.
Acknowledgements
This research was supported by the Russian Scientific Fund (project grant
14-50-00069).
14. Krasavin M. Novel diversely substituted 1-heteroaryl-2-imidazolines
for fragment-based drug discovery. Tetrahedron Lett 2012;53:
2876–80.
Declaration of interest
15. Krasavin M. Pd-catalyzed N-arylation of 2-imidazolines provides
convenient access to selective cyclooxygenase-2 inhibitors. Lett Org
Chem 2013;10:235–9.
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
16. Khalifah RG. The carbon dioxide hydration activity of carbonic
anhydrase. I. Stop-flow kinetic studies on the native human
isoenzymes B and C. J Biol Chem 1971;246:2561–73.
17. Maresca A, Carta F, Vullo D, Supuran CT. Dithiocarbamates
strongly inhibit the b-class carbonic anhydrases from Mycobacterium
tuberculosis. J Enzyme Inhib Med Chem 2013;28:407–11.
18. Ekinci D, Kurbanoglu NI, Salamci E, et al. Carbonic anhydrase
inhibitors: inhibition of human and bovine isoenzymes by
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Supplementary material available online