Synthesis of novel benzenesulfamide derivatives with inhibitory activity against human cytosolic carbonic anhydrase I and II and Vibrio cholerae α- and β-class enzymes

Article abstract of DOI:10.1080/14756366.2018.1467901

The synthesis of a new series of sulfamides incorporating ortho-, meta, and para-benzenesulfamide moieties is reported, which were investigated for the inhibition of two human (h) isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA I and II, and two Vibrio cholerae enzymes, belonging to the α- and β-CA classes (VchCAα, VchCAβ). The compounds were prepared by using the “tail approach”, aiming to overcome the scarcity of selective inhibition profiles associated to CA inhibitors belonging to the zinc binders. The built structure–activity relationship showed that the incorporation of benzhydryl piperazine tails on a phenyl sulfamide scaffold determines rather good efficacies against hCA I and VchCAα, with several compounds showing K _Is < 100 nM. The activity was lower against hCA II and VchCAβ, probably due to the fact that the incorporated tails are quite bulky. The obtained evidences allow us to continue the investigations of different tails/zinc binding groups, with the purpose to increase the effectiveness/selectivity of such inhibitors against bacterial CAs from pathogens, affording thus potential new anti-infectives.

Full text of DOI:10.1080/14756366.2018.1467901

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis of novel benzenesulfamide derivatives
with inhibitory activity against human cytosolic
carbonic anhydrase I and II and Vibrio cholerae α-
and β-class enzymes
Silvia Bua, Emanuela Berrino, Sonia Del Prete, Vallabhaneni S. Murthy,
Vijayaparthasarathi Vijayakumar, Yasinalli Tamboli, Clemente Capasso,
Elisabetta Cerbai, Alessandro Mugelli, Fabrizio Carta & Claudiu T. Supuran
To cite this article: Silvia Bua, Emanuela Berrino, Sonia Del Prete, Vallabhaneni S.
Murthy, Vijayaparthasarathi Vijayakumar, Yasinalli Tamboli, Clemente Capasso, Elisabetta
Cerbai, Alessandro Mugelli, Fabrizio Carta & Claudiu T. Supuran (2018) Synthesis of novel
benzenesulfamide derivatives with inhibitory activity against human cytosolic carbonic anhydrase
I and II and Vibrioꢀcholerae α- and β-class enzymes, Journal of Enzyme Inhibition and Medicinal
Chemistry, 33:1, 1125-1136, DOI: 10.1080/14756366.2018.1467901
To link to this article: https://doi.org/10.1080/14756366.2018.1467901
© 2018 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 10 Jul 2018.
Article views: 60
Submit your article to this journal
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 1125–1136
https://doi.org/10.1080/14756366.2018.1467901
RESEARCH PAPER
Synthesis of novel benzenesulfamide derivatives with inhibitory activity against
human cytosolic carbonic anhydrase I and II and Vibrio cholerae a- and
b-class enzymes
a
a
a
b
b
ꢀ
ꢀ
Silvia Bua , Emanuela Berrino , Sonia Del Prete , Vallabhaneni S. Murthy , Vijayaparthasarathi Vijayakumar ,
Yasinalli Tambolib, Clemente Capassoc, Elisabetta Cerbaid,e, Alessandro Mugellid,e, Fabrizio Cartaa and
Claudiu T. Supurana
a
ꢀ
NEUROFARBA Department, Sezione di Scienze Farmaceutiche e Nutraceutiche, Universita degli Studi di Firenze, Sesto Fiorentino (Florence),
b
c
Italy; School of Advanced Sciences, Center for Organic and Medicinal Chemistry, VIT University, Vellore, India; Istituto di Bioscienze e
Biorisorse, CNR, Napoli, Italy; Department of Neurosciences, Psychology, Drug's Research and Child's Health (NEUROFARBA), University of
Florence, Firenze, Italy; Section of Pharmacology and Toxicology, Firenze, Italy
d
e
ABSTRACT
ARTICLE HISTORY
Received 15 March 2018
Revised 17 April 2018
Accepted 18 April 2018
The synthesis of a new series of sulfamides incorporating ortho-, meta, and para-benzenesulfamide moi-
eties is reported, which were investigated for the inhibition of two human (h) isoforms of the zinc enzyme
carbonic anhydrase (CA, EC 4.2.1.1), hCA I and II, and two Vibrio cholerae enzymes, belonging to the a-
and b-CA classes (VchCAa, VchCAb). The compounds were prepared by using the “tail approach”, aiming
to overcome the scarcity of selective inhibition profiles associated to CA inhibitors belonging to the zinc
binders. The built structure–activity relationship showed that the incorporation of benzhydryl piperazine
tails on a phenyl sulfamide scaffold determines rather good efficacies against hCA I and VchCAa, with sev-
eral compounds showing KIs < 100nM. The activity was lower against hCA II and VchCAb, probably due
to the fact that the incorporated tails are quite bulky. The obtained evidences allow us to continue the
investigations of different tails/zinc binding groups, with the purpose to increase the effectiveness/select-
ivity of such inhibitors against bacterial CAs from pathogens, affording thus potential new anti-infectives.
KEYWORDS
Carbonic anhydrase
inhibitors (CAIs); sulfamides;
structure–activity relation-
ship (SAR); Vibrio cholerae
1. Introduction
suggested as potential targets for anti-infectives development
with a novel mechanism of action9,10
.
Cholera is an infectious human disease of the small intestine and
is caused by the gram-negative bacterium Vibrio cholera1,2. It is
characterised by a massive loss of water and electrolytes which
leads to severe dehydration and hypovolemic shock followed by
death if the disease is not well treated2–4. In developing countries,
cholera spreads among victims mainly through contaminated
water sources, and countries without proper sanitation techniques
have greater incidence of this disease3,5. In this regard, WHO
reported 132,121 cases in 38 countries during 2016, which also
included 2420 deaths6. It has been demonstrated that a potential
inducer of virulence gene expression is sodium bicarbonate, which
is present at a high concentration in the upper small intestine2.
Consequently, bicarbonate is considered the first positive effector
for ToxT, the major direct transcription activator of the viru-
lence genes2.
Up to now, inhibition studies performed against VchCAs mainly
considered sulfonamide-bearing derivatives, with sulfamates (R-
OSO2NH2) and sulfamides (R-NHSO2NH2) being less investigated.
These latter derivatives are the closest bioisosters and congeners
to the primary sulfonamides (R-SO2NH2), which constitute the
most important, clinically used class of CA inhibitors (CAIs)11–14
.
We recently reported a set of benzhydrylpiperazine benzene-
sulfamide showing effective and selective inhibition profiles
against the cytosolic isoform hCA I15. In the present study, we
have oriented our efforts in developing sulfamide CAIs as poten-
tial antibacterial agents, continuing the development of the previ-
ously reported series of sulfamides15, and report the synthesis of
new series of such derivatives. In addition to the bacterial enzyme
VchCAa and b, these novel compounds were investigated for their
property to inhibit the physiologically most important human
Many studies conducted by some of us have shown that spe- cytosolic isoforms CA I and II16,17
cific carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, may control
.
the bicarbonate-mediated virulence induction, suggesting the con-
2. Materials and methods
version of CO2 into bicarbonate by CA plays a crucial role as a
2.1. General
virulence factor for Vibrio cholerae7,8. Three different CAs have
been found in Vibrio cholerae, belonging to the three enzyme
Anhydrous solvents and all reagents were purchased from Sigma-
classes found in bacteria, VchCAa, b, and c. VchCAs have been Aldrich (Milan, Italy), Alfa Aesar (Milan, Italy), and TCI (Milan, Italy).
CONTACT Fabrizio Carta
fabrizio.carta@unifi.it; Claudiu T. Supuran
claudiu.supuran@unifi.it
NEUROFARBA Department, Sezione di Scienze Farmaceutiche e
ꢀ
Nutraceutiche, Universita degli Studi di Firenze, Via Ugo Schiff 6, Sesto Fiorentino (Florence) 50019, Italy
ꢀ
Both authors equally contributed to this work.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1126
S. BUA ET AL.
All reactions involving air- or moisture-sensitive compounds were 2.2.3. Tert-butyl (2-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-2-
performed under a nitrogen atmosphere using dried glassware
and syringes techniques to transfer solutions. Nuclear magnetic
oxoethyl)carbamate (2c)
Using 1b and N-Boc-glycine as starting materials, compound 2c
resonance spectra (1H NMR: 400 MHz; 13C NMR: 100 MHz; 19F was obtained with yield: 99%, off white solid. 1H NMR (DMSO-d6,
400 MHz): d 1.36 (9H, s, 3 ꢃ CH3), 2.27 (4H, m, 2 ꢃ piperazine-CH2),
NMR: 376 MHz) were recorded in DMSO-d6 using an Avance III
400 MHz spectrometer (Bruker, Milan, Italy). Chemical shifts are
reported in parts per million (ppm) and the coupling constants (J)
are expressed in Hertz (Hz). Splitting patterns are designated as
3.42 (4H, m, 2 ꢃ piperazine-CH2), 3.72 (2H, d, J ¼ 5.7, COCH2), 4.32
(1H, s, CH), 6.71 (1H, m, NH), 7.14 (4H, m, J ¼ 7.2, 4 ꢃ Ar-H), 7.43
(4H, m, J ¼ 7.4, 4 ꢃ Ar-H).
follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet;
brs, broad singlet; dd, double of doublets. The assignment of
2.2.4. Tert-butyl 4-(4-(bis(4-fluorophenyl)methyl)piperazine-1-car-
exchangeable protons (OH and NH) was confirmed by the add-
bonyl)piperidine-1-carboxylate (2d)
ition of D2O. Analytical thin-layer chromatography (TLC) was car-
ried out on silica gel F-254 plates (Merck, Milan, Italy). Melting
points (m.p.) were carried out in open capillary tubes and are
uncorrected. The solvents used in MS measures were acetone,
acetonitrile (Chromasolv grade), purchased from Sigma-Aldrich
(Milan, Italy) and mQ water 18 MX, obtained from Millipore’s
Simplicity system (Milan, Italy). The mass spectra were obtained
using a 1200 L triple quadrupole system (Varian, Palo Alto, CA)
equipped by electrospray source (ESI) operating in both positive
Using 1b and N-Boc-isonipecotic acid as starting materials, com-
pound 2d was obtained with yield: 71%, off white solid. 1H NMR
(DMSO-d6, 400 MHz): d 1.38 (11H, s, 3 ꢃ CH3, piperidine-CH2), 1.55
(2H, m, piperidine-CH2), 2.25 (4H, br s, 2 ꢃ piperazine-CH2), 2.70
(3H, m, piperidine-CH2, COCH), 3.48 (4H, br d, 2 ꢃ piperazine-CH2),
3.87 (2H, m, piperidine-CH2), 4.40 (1H, s, CH), 7.14 (4H, t, J ¼ 8.7,
4 ꢃ Ar-H), 7.44 (4H, t, J ¼ 8.1, 4 ꢃ Ar-H).
and negative ions. Stock solutions of analytes were prepared in
acetone at 1.0 mg mlꢁ1 and stored at 4 ꢂC. Working solutions of
each analyte were freshly prepared by diluting stock solutions in a
mixture of mQ H2O/ACN 1:1 (v/v) up to a concentration of
1.0 mg mlꢁ1. The mass spectra of each analyte were acquired by
introducing, via syringe pump at 10 ml minꢁ1, its working solution.
Raw-data were collected and processed by Varian Workstation
Vers. 6.8 software (Palo Alto, CA).
2.2.5. General procedure for the synthesis of compounds 4a–p
A stirred solution of compounds 2a–d (1.0 eq) in DCM (10.0 ml)
was treated with TFA (3.0 eq) and stirred at r.t. for 2 h. The reac-
tion mixture was concentrated to dry and co-distilled twice with
DCM to afford the corresponding amines 3a–d as TFA salts (not
isolated), which were readily dissolved in DCM (10.0 ml) and
treated with DIPEA (5.0 eq) and the appropriate sulfonyl chloride
(1.2 eq). The reaction solutions were stirred at r.t. for 1 h, then
concentrated to dry and the residue obtained was purified by sil-
ica gel column chromatography using ethyl acetate in n-hexane
(20–40% v/v) as eluent to afford the titled compounds 4a–p as
off-white solids.
2.2. General procedure for the synthesis of compounds 2a–d
Compounds 1a,b (1.0 eq) and the appropriate N-Boc-protected
carboxylic acid (1.1 eq) in DMF (10.0 ml) were treated with DIPEA
(2.0 eq), and HATU (1.5 eq) at r.t. for 30 min. When the reaction
was complete (TLC: monitoring), it was quenched with ice cold
water and extracted with ethyl acetate (3 ꢃ 15 ml). The combined
organic layers were washed with H2O (3 ꢃ 15 ml), dried over
Na2SO4, filtered-off and concentrated under reduced pressure to
afford the title compounds 2a–d as off-white solids.
2.2.6. 1-Benzhydryl-4-((3-nitrophenyl)sulfonyl)piperazine (4a)
Using 1a and 3-nitrobenzenesulfonyl chloride as starting materials,
compound 4a was obtained with yield 75%, off white solid; m.p.
170–172 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 2.36 (4H, br s,
2 ꢃ piperazine-CH2), 3.00 (4H, br s, 2 ꢃ piperazine-CH2), 4.31 (1H, s,
CH), 7.14 (2H, t, J ¼ 7.2, 2 ꢃ Ar-H), 7.23 (4H, t, J ¼ 7.4, 4 ꢃ Ar-H),
7.33 (4H, d, J ¼ 7.3, 4 ꢃ Ar-H), 7.96 (1H, t, J ¼ 8.0, Ar-H), 8.15 (1H, d,
J ¼ 7.8, Ar-H), 8.35 (1H, s, Ar-H), 8.56 (1H, m, Ar-H); 13C NMR
(DMSO-d6, 100 MHz): d 46.0, 50.3, 74.1, 122.2, 126.9, 127.4, 127.8,
128.5, 131.5, 133.4, 136.5, 142.2, 148.2; MS (ESI positive)
m/z ¼ 437.9 [M þ H]þ.
2.2.1. Tert-butyl (2-4-benzhydrylpiperazin-1-yl)-2-oxoethyl)carba-
mate (2a)
Using 1a and N-Boc-glycine as starting materials, compound 2a
was obtained with yield: 99%, off white solid. 1H NMR (DMSO-d6,
400 MHz): d 1.36 (9H, s, 3 ꢃ CH3), 2.27 (4H, m, 2 ꢃ piperazine-CH2),
3.42 (4H, m, 2 ꢃ piperazine-CH2), 3.72 (2H, d, J ¼ 5.7, COCH2), 4.32
(1H, s, CH), 6.71 (1H, m, NH), 7.19 (2H, t, J ¼ 7.2, 2 ꢃ Ar-H), 7.30
(4H, t, J ¼ 7.5, 4 ꢃ Ar-H), 7.43 (4H, d, J ¼ 7.4, Ar-H).
2.2.7. 1-Benzhydryl-4-((4-nitrophenyl)sulfonyl)piperazine (4b)
Using 1a and 4-nitrobenzenesulfonyl chloride as starting materials,
compound 4b was obtained with yield 46%, off white solid; m.p.
216–218 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 2.36 (4H, br s,
2 ꢃ piperazine-CH2), 2.99 (4H, br s, 2 ꢃ piperazine-CH2), 4.31 (1H, s,
CH), 7.14 (2H, t, J ¼ 7.2, 2 ꢃ Ar-H), 7.23 (4H, t, J ¼ 7.4, 4 ꢃ Ar-H),
7.33 (4H, d, J ¼ 7.3, 4 ꢃ Ar-H), 7.98 (2H, d, J ¼ 8.8, 2 ꢃ Ar-H), 8.45
(2H, d, J ¼ 8.8, 2 ꢃ Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 46.0,
50.2, 74.0, 124.7, 126.9, 127.4, 128.5, 129.1, 140.5, 142.1, 150.1; MS
(ESI positive) m/z ¼ 437.9 [M þ H]þ.
2.2.2. Tert-butyl 4-4-benzhydrylpiperazine-1-carbonyl)piperidine-1-
carboxylate (2b)
Using 1a and N-Boc-isonipecotic acid as starting materials, com-
pound 2b was obtained with yield: 76%, off white solid. 1H NMR
(DMSO-d6, 400 MHz): d 1.38 (11H, s, 3 ꢃ CH3, piperidine-CH2), 1.55
(2H, m, piperidine-CH2), 2.25 (4H, br s, 2 ꢃ piperazine-CH2), 2.70
(3H, m, piperidine-CH2, COCH), 3.48 (4H, br d, 2 ꢃ piperazine-CH2),
3.87 (2H, m, piperidine-CH2), 4.40 (1H, s, CH), 7.19 (2H, t, J ¼ 7.2,
2 ꢃ Ar-H), 7.30 (4H, t, J ¼ 7.5, 4 ꢃ Ar-H), 7.43 (4H, d, J ¼ 7.4,
4 ꢃ Ar-H).
2.2.8. N-(2-(4-Benzhydrylpiperazin-1-yl)-2-oxoethyl)-2-nitrobenzene-
sulfonamide (4c)
Using 3a and 2-nitrobenzenesulfonyl chloride as starting materials,
compound 4c was obtained with yield 61%, off white solid; m.p.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1127
188–192 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 2.20 (4H, br d, piperidine-CH2), 1.64 (2H, m, piperidine-CH2), 2.20 (4H, br s,
2 ꢃ piperazine-CH2), 3.37 (4H, br s, 2 ꢃ piperazine-CH2), 3.89 (2H, d,
J ¼ 4.8, CH2), 4.28 (1H, s, CH), 7.17 (2H, t, J ¼ 7.3, 2 ꢃ Ar-H), 7.28
(4H, t, J ¼ 7.4, 4 ꢃ Ar-H), 7.40 (4H, d, J ¼ 7.3, 4 ꢃ Ar-H), 7.81 (2H, m,
2 Ar-H), 7.93 (2H, m, Ar-H, NH), 8.01 (1H, m, Ar-H); 13C NMR
(DMSO-d6, 100 MHz): d 41.5, 43.9, 44.0, 50.9, 51.3, 74.6, 124.4,
126.9, 127.5, 128.5, 129.8, 132.6, 133.1, 133.8, 142.3, 147.4, 165.4;
MS (ESI positive) m/z ¼ 494.9 [M þ H]þ.
2 ꢃ piperazine-CH2), 2.37 (2H, m, piperidine-CH2), 2.57 (1H, m,
COCH), 3.41 (4H, br s, 2 ꢃ piperazine-CH2), 3.62 (2H, m, piperidine-
CH2), 4.27 (1H, s, CH), 7.16 (2H, t, J ¼ 7.3, 2 ꢃ Ar-H), 7.27 (4H, t,
J ¼ 7.4, 4 ꢃ Ar-H), 7.39 (4H, d, J ¼ 7.5, 4 ꢃ Ar-H), 7.99 (2H, d, J ¼ 6.9,
2 ꢃ Ar-H), 8.42 (2H, d, J ¼ 6.9, 2 ꢃ Ar-H); 13C NMR (DMSO-d6,
100 MHz): d 26.5, 35.6, 41.1, 44.7, 45.1, 51.2, 51.9, 74.6, 124.6,
126.9, 127.5, 128.5, 128.9, 141.2, 142.3, 149.9, 171.5; MS (ESI posi-
tive) m/z ¼ 548.9 [M þ H]þ.
2.2.9. N-(2-(4-Benzhydrylpiperazin-1-yl)-2-oxoethyl)-3-nitrobenzene-
sulfonamide (4d)
2.2.13. 1-(Bis(4-Fluorophenyl)methyl)-4-((2-nitrophenyl)sulfonyl)pip-
erazine (4h)
Using 3a and 3-nitrobenzenesulfonyl chloride as starting materials,
compound 4d was obtained with yield 56%, off white solid; m.p.
208–212 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 2.13 (2H, s, pipera-
zine-CH2), 2.20 (2H, s, piperazine-CH2), 3.30 (4H, br s,
2 ꢃ piperazine-CH2), 3.81 (2H, d, J ¼ 5.5, CH2), 4.24 (1H, s, CH), 7.17
(2H, t, J ¼ 7.3, 2 ꢃ Ar-H), 7.28 (4H, t, J ¼ 7.4, 4 ꢃ Ar-H), 7.39 (4H, d,
J ¼ 7.2, 4 ꢃ Ar-H), 7.84 (1H, t, J ¼ 8.0, Ar-H), 8.17 (2H, m, Ar-H, NH),
8.43 (1H, m, Ar-H), 8.54 (1H, m, Ar-H); 13C NMR (DMSO-d6,
100 MHz): d 41.4, 43.6, 44.1, 50.9, 51.5, 74.7, 121.5, 126.8, 126.9,
127.5, 128.5, 130.9, 132.8, 142.3, 147.6, 165.3; MS (ESI positive) m/z ¼
494.9 [M þ H]þ.
Using 1b and 2-nitrobenzenesulfonyl chloride as starting materials,
compound 4h was obtained with yield: 77%, off white solid; m.p.
120–124 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 2.34 (4H, br s,
2 ꢃ piperazine-CH2), 3.18 (4H, br s, 2 ꢃ piperazine-CH2), 4.43 (1H, s,
CH), 7.09 (4H, t, J ¼ 8.7, 4 ꢃ Ar-H), 7.39 (4H, t, J ¼ 8.2, 4 Ar-H), 7.91
(4H, m, 4 ꢃ Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 45.9, 50.3, 72.0,
115.2, 115.4, 124.1, 128.4, 129.3, 129.4, 130.4, 132.2, 134.9, 138.1,
147.9, 159.9, 162.3; MS (ESI positive) m/z ¼ 474.04 [M þ H]þ.
2.2.14. 1-(Bis(4-Fluorophenyl)methyl)-4-((3-nitrophenyl)sulfonyl)pip-
erazine (4i)
2.2.10. (4-Benzhydrylpiperazin-1-yl)(1-((2-nitrophenyl)sulfonyl)pipe-
ridin-4-yl)methanone (4e)
Using 3b and 2-nitrobenzenesulfonyl chloride as starting materials,
compound 4e was obtained with yield 49%, off white solid; m.p.
158–160 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 1.48 (2H, m, piperi-
dine-CH2), 1.66 (2H, m, piperidine-CH2), 2.22 (4H, br s,
Using 1b and 3-nitrobenzenesulfonyl chloride as starting materials,
compound 4i was obtained with yield: 87%, off white solid; m.p.
168–172 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 2.34 (4H, br s,
2 ꢃ piperazine-CH2), 2.99 (4H, br s, 2 ꢃ piperazine-CH2), 4.40 (1H, s,
CH), 7.07 (4H, t, J ¼ 8.8, 4 ꢃ Ar-H), 7.35 (4H, q, J ¼ 5.7, 4 ꢃ Ar-H),
2 ꢃ piperazine-CH2), 2.72 (3H, m, piperidine-CH2, COCH), 3.44 (4H, 7.96 (1H, t, J ¼ 8.0, Ar-H), 8.15 (1H, d, J ¼ 7.8, Ar-H), 8.35 (1H, s, Ar-
H), 8.56 (1H, d, J ¼ 8.1, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 45.9,
50.0, 71.9, 115.2, 115.4, 124.7, 129.1, 129.2, 129.4, 138.1, 143.5,
155.1, 159.8, 162.2; MS (ESI positive) m/z ¼ 473.9 [M þ H]þ.
br d, 2 ꢃ piperazine-CH2), 3.66 (2H, d, piperidine-CH2), 4.29 (1H, s,
CH), 7.17 (2H, t, J ¼ 7.3, 2 ꢃ Ar-H), 7.28 (4H, t, J ¼ 7.4, 4 ꢃ Ar-H),
7.40 (4H, d, J ¼ 7.5, 4 ꢃ Ar-H), 7.85 (2H, m, 2 ꢃ Ar-H), 7.97 (2H, m,
2 ꢃ Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 27.7, 35.7, 41.3, 44.7,
45.0, 51.2, 52.0, 074.6, 124.0, 126.9, 127.5, 128.5, 129.2, 130.3,
132.1, 134.7, 142.4, 147.8, 171.6; MS (ESI positive) m/z ¼
548.9 [M þ H]þ.
2.2.15. 1-(Bis(4-Fluorophenyl)methyl)-4-((4-nitrophenyl)sulfonyl)pip-
erazine (4j)
Using 1b and 4-nitrobenzenesulfonyl chloride as starting materials,
compound 4j was obtained with yield: 73%, yellow solid; m.p.
222–226 ꢂC; 1H NMR (DMSO-d6, 400 MHZ): d 2.34 (4H, br s,
2 ꢃ piperazine-CH2), 2.99 (4H, br s, 2 ꢃ piperazine-CH2), 4.40 (1H, s,
CH), 7.07 (4H, t, J ¼ 8.8, 4 Ar-H), 7.34 (4H, m, J ¼ 5.7, 4 ꢃ Ar-H), 7.98
(2H, d, J ¼ 8.7, 2 ꢃ Ar-H), 8.45 (2H, d, J ¼ 8.7, 2 ꢃ Ar-H); 13C NMR
(DMSO-d6, 100 MHz): d 46.0, 50.0, 71.9, 115.2, 115.4, 124.7, 129.1,
129.2, 129.3, 138.1, 140.5, 150.1, 159.8, 162.2; MS (ESI positive) m/z ¼
473.9 [M þ H]þ.
2.2.11. (4-Benzhydrylpiperazin-1-yl)(1-((3-nitrophenyl)sulfonyl)pipe-
ridin-4-yl)methanone (4f)
Using 3b and 3-nitrobenzenesulfonyl chloride as starting materials,
compound 4f was obtained with yield 48%, off white solid; m.p.
222–226 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 1.52 (2H, m, piperi-
dine-CH2), 1.64 (2H, m, piperidine-CH2), 2.20 (4H, br s,
2 ꢃ piperazine-CH2), 2.37 (2H, m, piperidine-CH2), 2.53 (1H, m,
COCH), 3.41 (4H, 4 s, 2 ꢃ piperazine-CH2), 3.63 (2H, d, piperidine-
CH2), 4.27 (1H, s, CH), 7.17 (2H, t, J ¼ 7.3, 2 ꢃ Ar-H), 7.27 (4H, t,
J ¼ 7.4, 4 ꢃ Ar-H), 7.39 (4H, d, J ¼ 7.5, 4 ꢃ Ar-H), 7.93 (1H, t, J ¼ 8.0,
Ar-H), 8.16 (1H, d, J ¼ 7.8, Ar-H), 8.34 (1H, br s, Ar-H), 8.52 (1H, d,
J ¼ 8.1, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 27.5, 35.6, 41.1,
44.6, 45.2, 51.2, 52.0, 74.6, 121.8, 126.8, 126.9, 127.6, 128.5, 131.5,
133.4, 137.1, 142.3, 147.9, 171.5; MS (ESI positive) m/z ¼
548.9 [M þ H]þ.
2.2.16. N-(2-(4-(bis(4-Fluorophenyl)methyl)piperazin-1-yl)-2-oxoethyl)-
2-nitrobenzenesulfonamide (4k)
Using 3c and 2-nitrobenzenesulfonyl chloride as starting materials,
compound 4k was obtained with yield: 69%, off white solid; m.p.
136–140 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 2.20 (4H, br d,
2 ꢃ pipeazine-CH2), 3.37 (4H, br s, 2 ꢃ piperazine-CH2), 3.91 (2H, d,
J ¼ 5.38, CH2), 4.39 (1H, s, CH), 7.13 (4H, t, J ¼ 8.7, 4 ꢃ Ar-H), 7.42
2.2.12. (4-Benzhydrylpiperazin-1-yl)(1-((4-nitrophenyl)sulfonyl)pipe- (4H, q, J ¼ 5.8, 4 ꢃ Ar-H), 7.83 (2H, m, 2 ꢃ Ar-H), 8.02 (3H, m, Ar-H,
NH), 8.01 (1H, m, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 41.5, 43.9,
ridin-4-yl)methanone (4g)
43.9, 50.9, 51.3, 72.5, 115.2, 115.5, 124.4, 129.4, 129.5, 129.9, 132.6,
Using 3b and 4-nitrobenzenesulfonyl chloride as starting materials,
compound 4g was obtained with yield 59%, off white solid; m.p.
133.1, 133.9, 138.3, 159.5, 162.7, 165.4; MS (ESI positive) m/z ¼
182–186 ꢂC; 1H NMR (DMSO-d6, 400 MHz):
d
1.49 (2H, m, 531.12 [M þ H]þ.

1128
S. BUA ET AL.
2.2.17. N-(2-(4-(bis(4-Fluorophenyl)methyl)piperazin-1-yl)-2-oxoethyl)- 2.2.21. (4-(bis(4-Fluorophenyl)methyl)piperazin-1-yl)(1-((4-nitrophe-
3-nitrobenzenesulfonamide (4l) nyl)sulfonyl)piperidin-4-yl)methanone (4p)
Using 3c and 3-nitrobenzenesulfonyl chloride as starting materials, Using 3d and 4-nitrobenzenesulfonyl chloride as starting materials,
compound 4l was obtained with yield: 58%, off white solid; m.p. compound 4p was obtained with yield: 46%, off white solid; m.p.
202–206 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 2.15 (4H, br s, 232–236 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 1.54 (2H, m, piperi-
2 ꢃ piperazine-CH2), 3.30 (4H, br s, 2 ꢃ piperazine-CH2), 3.82 (2H, d, dine-CH2), 1.66 (2H, m, CH2), 2.21 (4H, br s, 2 ꢃ piperazine-CH2),
J ¼ 5.16, CH2), 4.34 (1H, s, CH), 7.11 (4H, t, J ¼ 8.7, 4 ꢃ Ar-H), 7.40 2.37 (2H, m, piperidine-CH2), 2.80 (1H, m, COCH), 3.41 (4H, br s,
(4H, t, J ¼ 6.1, 4 ꢃ Ar-H), 7.84 (1H, t, J ¼ 8.01, Ar-H), 8.15 (1H, t, 2 ꢃ piperazine-CH2), 3.65 (2H, m, piperdine-CH2), 4.30 (1H, s, CH),
J ¼ 5.09, NH), 8.19 (1H, d, J ¼ 7.78, Ar-H), 8.44 (1H, d, J ¼ 8.16, Ar- 7.12 (4H, t, J ¼ 8.8, 4 ꢃ Ar-H), 7.44 (4H, t, J ¼ 5.7, 4 ꢃ Ar-H), 7.98
H), 8.53 (1H, s, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 23.6, 26.6, (2H, d, J ¼ 9.2, 2 ꢃ Ar-H) 8.42 (2H, d, J ¼ 9.2, 2 ꢃ Ar-H); 13C NMR
37.3, 41.1, 44.8, 45.9, 48.1, 51.0, 51.8, 72.6, 115.3, 115.6, 124.5, (DMSO-d6, 100 MHz): d 26.6, 35.6, 41.2, 44.7, 45.2, 51.3, 51.9, 74.7,
129.4, 129.5, 129.9, 132.6, 133.2, 133.8, 138.3, 159.5, 162.8, 165.5; 115.2, 115.4, 124.7, 128.7, 129.3, 129.4, 138.3, 141.4, 149.9, 159.8,
MS (ESI positive) m/z ¼ 531.04 [M þ H]þ.
162.3, 170.3; MS (ESI positive) m/z ¼ 585.10 [M þ H]þ.
2.2.22. General procedure for the synthesis of compounds 5a–p
The appropriate nitrobenzenesulfonamides 4a–p (1.0 eq) in a solu-
tion of H2O (0.4 ml) and EtOH (0.3 ml) was treated with glacial
AcOH (0.05 ml) and Fe (0) (12.0 eq). The reaction mixture was
stirred at 75 ꢂC for 1 h (TLC monitoring), then cooled to r.t. and
diluted with EtOAc (10.0 ml). The mixture was filtered through
2.2.18. N-(2-(4-(bis(4-Fluorophenyl)methyl)piperazin-1-yl)-2-oxoethyl)-
4-nitrobenzenesulfonamide (4m)
Using 3c and 4-nitrobenzenesulfonyl chloride as starting materials,
compound 4m was obtained with yield: 47%, off white solid; m.p.
176–180 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 2.17 (4H, br d,
2 ꢃ piperazine-CH2), 3.33 (4H, br s, 2 ꢃ piperazine-CH2), 3.81 (2H, d,
J ¼ 5.5, CH2), 4.37 (1H, s, CH), 7.11 (4H, t, J ¼ 8.6, 4 ꢃ Ar-H), 7.41
(4H, t, J ¼ 6.1, 4 ꢃ Ar-H), 8.03 (2H, d, J ¼ 8.6, 2 ꢃ Ar-H), 8.15 (1H, t,
J ¼ 5.4, NH), 8.36 (2H, d, J ¼ 8.6, 2 ꢃ Ar-H); 13C NMR (DMSO-d6,
100 MHz): d 41.4, 43.7, 44.1, 50.7, 51.2, 72.5, 115.5, 124.2, 129.3,
132.4, 138.2, 146.3, 149.4, 159.8, 162.3, 165.3; MS (ESI positive)
m/z ¼ 531.04 [M þ H]þ.
R
Celite 521V, washed with a saturated NaHCO3 aqueous solution
(3 ꢃ 15 ml), brine (3 ꢃ 10 ml) and dried over Na2SO4. The organic
solvent was evaporated in vacuo to give an oil residue, which was
triturated from Et2O, to afford the titled compounds 5a–p as
white solids.
2.2.23. 3-((4-Benzhydrylpiperazin-1-yl)sulfonyl)aniline (5a)
Compound 5a was obtained in 70% yield; m.p. 120–122 ꢂC; TLC:
Rf ¼ 0.17 (ethyl acetate/n-hexane 20% v/v); 1H NMR (DMSO-d6,
400 MHZ) (DMSO-d6, 400 MHZ) (DMSO-d6, 400 MHz): d 2.40 (4H, m,
2 ꢃ piperazine-CH2), 2.92 (4H, m, 2 ꢃ piperazine-CH2), 4.34 (1H, s,
CH), 5.70 (2H, s, exchange with D2O, NH2), 6.81 (1H, m, Ar-H), 6.91
(2H, m, Ar-H), 7.20 (2H, t, J ¼ 7.2, Ar-H), 7.29 (5H, m, Ar-H), 7.40
(4H, m, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 47.0, 51.4, 75.3,
114.5, 117.3, 118.4, 127.9, 128.4, 129.4, 130.9, 138.0, 142.7, 148.2;
MS (ESI positive) m/z ¼ 408.2 [M þ H]þ.
2.2.19. (4-(bis(4-Fluorophenyl)methyl)piperazin-1-yl)(1-((2-nitrophe-
nyl)sulfonyl)piperidin-4-yl)methanone (4n)
Using 3d and 2-nitrobenzenesulfonyl chloride as starting materials,
compound 4n was obtained with yield: 59%, off white solid; m.p.
170–174 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 1.48 (2H, m, piperi-
dine-CH2), 1.66 (2H, m, piperidine-CH2), 2.20 (4H, br s,
2 ꢃ piperazine-CH2), 2.71 (3H, m, piperidine-CH2, COCH), 3.44 (4H,
br d, 2 ꢃ piperazine-CH2), 3.67 (2H, m, piperidine-CH2), 4.38 (1H, s,
CH), 7.11 (4H, t, J ¼ 8.7, 4 ꢃ Ar-H), 7.41 (4H, m, 4 ꢃ Ar-H), 7.91 (4H,
m, 4 ꢃ Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 27.7, 35.7, 40.1, 44.8,
44.9, 51.0, 51.8, 72.5, 115.2, 115.4, 124.0, 129.2, 129.3, 129.4, 130.3,
132.1, 134.7, 138.3, 147.8, 159.8, 162.2, 171.6; MS (ESI positive) m/z ¼
585.11 [M þ H]þ.
2.2.24. 4-((4-Benzhydrylpiperazin-1-yl)sulfonyl)aniline (5b)
Compound 5b was obtained in 80% yield; m.p. 196–198 ꢂC; TLC:
Rf ¼ 0.25 (ethyl acetate/n-hexane 20% v/v); 1H NMR (DMSO-d6,
400 MHz):
d
2.38 (4H, m, 2 ꢃ piperazine-CH2), 2.85 (4H, m,
2 ꢃ piperazine-CH2), 4.33 (1H, s, CH), 6.16 (2H, s, exchange with
D2O, NH2), 6.70 (2H, m, Ar-H), 7.20 (2H, m, Ar-H), 7.29 (4H, t,
J ¼ 7.4, Ar-H), 7.40 (6H, m, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d
47.0, 51.4, 75.3, 113.6, 126.8, 127.8, 128.4, 129.3, 130.6, 143.4,
154.2; MS (ESI positive) m/z ¼ 408.2 [M þ H]þ.
2.2.20. (4-(bis(4-Fluorophenyl)methyl)piperazin-1-yl)(1-((3-nitrophe-
nyl)sulfonyl)piperidin-4-yl)methanone (4o)
Using 3d and 3-nitrobenzenesulfonyl chloride as starting materials,
compound 4o was obtained with yield: 48%, off white solid; m.p.
180–184 ꢂC; 1H NMR (DMSO-d6, 400 MHz): d 1.52 (2H, m, piper-
2.2.25. 2-Amino-N-(2-(4-benzhydrylpiperazin-1-yl)-2-oxoethyl)ben-
zenesulfonamide (5c)
dine-CH2), 1.65 (2H, d, piperidine-CH2), 2.20 (4H, s, 2 ꢃ piperazine- Compound 5c was obtained in 85% yield; m.p. 140–142 ꢂC; TLC:
CH2), 2.37 (2H, m, piperidne-CH2), 2.54 (1H, m, COCH), 3.41 (4H, br Rf ¼ 0.39 (ethyl acetate/n-hexane 50% v/v); 1H NMR (DMSO-d6,
s, 2 ꢃ piperazine-CH2), 3.64 (2H, m, piperidine-CH2), 4.29 (1H, s, 400 MHz):
d
2.25 (4H, m, 2 ꢃ piperazine-CH2), 3.42 (4H, m,
CH), 7.12 (4H, t, J ¼ 8.7, 4 ꢃ Ar-H), 7.43 (4H, t, J ¼ 5.7, 4 ꢃ Ar-H), 2 ꢃ piperazine-CH2), 3.66 (2H, d, J ¼ 5.4, COCH2NH), 4.33 (1H, s,
7.94 (1H, t, J ¼ 8.7, Ar-H), 8.16 (1H, d, J ¼ 8.8, Ar-H), 8.34 (1H, s, Ar- CH), 5.99 (2H, s, exchange with D2O, NH2), 6.59 (1H, t, J ¼ 7.4, Ar-
H), 8.53 (1H, d, J ¼ 8.7, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 27.5, H), 6.80 (1H, d, J ¼ 8.0, Ar-H), 7.24 (3H, m, Ar-H), 7.33 (4H, m, Ar-H),
35.7, 41.1, 44.7, 45.2, 51.2, 51.9, 74.6, 115.3, 115.5, 121.9, 127.8, 7.47 (6H, m, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 42.4, 45.1,
129.4, 129.3, 129.4, 129.5, 131.5, 133.3, 137.5, 138.4, 138.4, 148.1, 52.2, 75.5, 115.7, 117.8, 120.3, 127.8, 128.4, 129.4, 130.0, 134.4,
159.5, 162.7, 170.6; MS (ESI positive) m/z ¼ 585.09 [M þ H]þ.
143.3, 147.3, 166.43; MS (ESI positive) m/z ¼ 465.2 [M þ H]þ.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1129
2.2.26. 3-Amino-N-(2-(4-benzhydrylpiperazin-1-yl)-2-oxoethyl)ben- (DMSO-d6, 400 MHz): d 2.41 (4H, m, 2 ꢃ piperazine-CH2), 3.03 (4H,
m, 2 ꢃ piperazine-CH2), 4.44 (1H, s, CH), 6.07 (2H, s, exchange with
zenesulfonamide (5d)
Compound 5d was obtained in 78% yield; m.p. 135–137 ꢂC; TLC:
D2O, NH2), 6.69 (1H, t, J ¼ 8.0, Ar-H), 6.91 (1H, d, J ¼ 8.0, Ar-H), 7.12
(4H, m, Ar-H), 7.39 (6H, m, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d
Rf ¼ 0.33 (ethyl acetate/n-hexane 50% v/v); 1H NMR (DMSO-d6,
2
47.0, 51.1, 73.2, 115.4, 116.2 (d, JC–F 21), 116.4, 118.2, 130.2
400 MHz): d 2.26 (4H, m, 2 ꢃ piperazine-CH2), 3.33 (4H, overlap
3
1
(d, JC–F 8), 130.8, 135.2, 139.3, 148.4, 161.9 (d, JC–F 242); 19F NMR
(DMSO-d6, 376 MHz): d –115.6 (2F, s); MS (ESI positive) m/z ¼
444.2 [M þ H]þ.
with water peak, 2 ꢃ piperazine-CH2), 3.67 (2H, s, COCH2NH), 4.34
(1H, s, CH), 5.58 (2H, s, exchange with D2O, NH2), 6.76 (1H, d,
J ¼ 6.8, Ar-H), 6.90 (1H, d, J ¼ 7.0, Ar-H), 6.99 (1H, m, Ar-H), 7.21
(3H, m, Ar-H), 7.33 (4H, m, Ar-H), 7.45 (5H, m, 4 ꢃ Ar-H, exchange
with D2O, CH2NHSO2); 13C NMR (DMSO-d6, 100 MHz): d 42.2, 44.9,
51.9, 75.7, 116.4, 120.7, 122.5, 126.2, 127.6, 128.2, 129.4, 141.1,
141.6, 148.3, 164.3; MS (ESI positive) m/z ¼ 465.2 [M þ H]þ.
2.2.31. 3-((4-(Bis(4-Fluorophenyl)methyl)piperazin-1-yl)sulfonyl)ani-
line (5i)
Compound 5i was obtained in 53% yield; m.p. 183–186 ꢂC (dec);
TLC: Rf ¼ 0.21 (ethyl acetate/n-hexane 30% v/v); 1H NMR (DMSO-
d6, 400 MHz): d 2.39 (4H, m, 2 ꢃ piperazine-CH2), 2.91 (4H, m,
2 ꢃ piperazine-CH2), 4.42 (1H, s, CH), 5.69 (2H, s, exchange with
D2O, NH2), 6.81 (1H, d, J ¼ 7.8, Ar-H), 6.90 (2H, m, Ar-H), 7.12 (4H,
m, Ar-H), 7.30 (1H, t, J ¼ 7.8, Ar-H), 7.42 (4H, m, Ar-H); 13C NMR
2.2.27. (1-((2-Aminophenyl)sulfonyl)piperidin-4-yl)(4-benzhydrylpi-
perazin-1-yl)methanone (5e)
Compound 5e was obtained in 47% yield; m.p. 202–234 ꢂC; TLC:
Rf ¼ 0.54 (ethyl acetate/n-hexane 60% v/v); 1H NMR (DMSO-d6,
400 MHz): d 1.52 (2H, m, piperidine-CH2), 1.67 (2H, m, piperidine-
CH2), 2.26 (4H, m, 2 ꢃ piperazine-CH2), 2.54 (3H, overlap with
DMSO peak, COCH, piperidine-CH2), 3.47 (4H, m, 2 ꢃ piperazine-
CH2), 3.63 (2H, m, piperidine-CH2), 4.32 (1H, s, CH), 6.07 (2H, s,
exchange with D2O, NH2), 6.65 (1H, m, Ar-H), 6.86 (1H, d, J ¼ 7.6,
Ar-H), 7.22 (2H, m, Ar-H), 7.32 (5H, m, Ar-H), 7.38 (1H, t, J ¼ 7.6, Ar-
H) 7.42 (4H, m, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 28.6, 36.8,
42.0, 45.9, 53.0, 75.6, 116.6, 127.8, 128.5, 129.4, 130.9, 132.5, 134.6,
134.7, 135.5, 143.3, 167.8; MS (ESI positive) m/z ¼ 519.7 [M þ H]þ.
2
(DMSO-d6, 100 MHz): d 47.0, 51.2, 73.2, 112.7, 115.1, 116.2 (d, JC–F
3
21), 118.8, 130.2 (d, JC–F 8), 130.5, 135.7, 139.2, 148.4, 161.9 (d,
1JC–F 242); 19F NMR (DMSO-d6, 376 MHz): d –115.6 (2F, s); MS (ESI
positive) m/z ¼ 444.2 [M þ H]þ.
2.2.32. 4-((4-(Bis(4-Fluorophenyl)methyl)piperazin-1-yl)sulfonyl)ani-
line (5j)
Compound 5j was obtained in 64% yield; m.p. 155–157 ꢂC (dec);
TLC: Rf ¼ 0.18 (ethyl acetate/n-hexane 30% v/v); 1H NMR (DMSO-
d6, 400 MHz): d 2.37 (4H, m, 2 ꢃ piperazine-CH2), 2.85 (4H, m,
2 ꢃ piperazine-CH2), 4.46 (1H, s, CH), 6.16 (2H, s, exchange with
D2O, NH2), 6.71 (2H, d, J ¼ 8.8, Ar-H), 7.12 (4H, m, Ar-H), 7.38 (2H,
d, J ¼ 8.8, Ar-H), 7.42 (4H, m, Ar-H); 13C NMR (DMSO-d6, 100 MHz):
2.2.28. (1-((3-Aminophenyl)sulfonyl)piperidin-4-yl)(4-benzhydrylpi-
perazin-1-yl)methanone (5f)
Compound 5f was obtained in 30% yield; m.p. 124–126 ꢂC; TLC:
Rf ¼ 0.13 (ethyl acetate/n-hexane 60% v/v); 1H NMR (DMSO-d6,
400 MHz): d 1.54 (2H, m, piperidine-CH2), 1.68 (2H, m, piperidine-
CH2), 2.29 (6H, m, 2 ꢃ piperazine-CH2, piperidine-CH2), 2.54 (1H,
overlap with DMSO peak, COCH) 3.33 (4H, m, overlap with water
peak, 2 ꢃ piperazine-CH2), 3.56 (2H, m, piperidine-CH2), 4.32 (1H, s,
CH), 5.64 (2H, s, exchange with D2O, NH2) 6.81 (2H, m, Ar-H), 6.91
(1H, m, Ar-H), 7.23 (3H, m, Ar-H), 7.30 (4H, t, J ¼ 7.6, Ar-H), 7.44
(4H, d, J ¼ 7.2, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 28.6, 36.8
(overlap with DMSO peak), 40.4 (overlap with DMSO peak), 46.2,
65.8, 75.6, 112.6, 114.9, 118.7, 127.8, 128.5, 129.5, 130.5, 136.5,
143.4, 150.4, 172.7; MS (ESI positive) m/z ¼ 519.2 [M þ H]þ.
2
3
d 47.0, 51.1, 73.2, 113.6, 116.2 (d, JC–F 21), 125.6, 130.2 (d, JC–F 8),
1
130.5, 139.2, 154.1, 161.9 (d, JC–F 242); 19F NMR (DMSO-d6,
376 MHz): d –115.6 (2F, s); MS (ESI positive) m/z ¼ 444.2 [M þ H]þ.
2.2.33. 2-Amino-N-(2-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-
2-oxoethyl)benzenesulfonamide (5k)
Compound 5k was obtained in 79% yield; m.p. 137–139 ꢂC; TLC:
Rf ¼ 0.16 (ethyl acetate/n-hexane 60% v/v); 1H NMR (DMSO-d6,
400 MHz): d 2.24 (4H, m, 2 ꢃ piperazine-CH2), 3.33 (4H, m, overlap
with water peak, 2 ꢃ piperazine-CH2), 3.66 (2H, s, COCH2NH) 4.42
(1H, s, CH), 5.99 (2H, s, exchange with D2O, NH2), 6.59 (1H, t,
2.2.29. (1-((4-Aminophenyl)sulfonyl)piperidin-4-yl)(4-benzhydrylpi- J ¼ 7.2, Ar-H), 6.79 (2H, d, J ¼ 8.0, Ar-H), 7.17 (4H, m, Ar-H), 7.28
(1H, m, Ar-H), 7.36 (1H, t, J ¼ 7.2, exchange with D2O, SO2NHCH2),
perazin-1-yl)methanone (5g)
7.46 (4H, m, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d 42.4, 45.1,
Compound 5g was obtained in 70% yield; m.p. 174–176 ꢂC; TLC:
Rf ¼ 0.40 (ethyl acetate/n-hexane 70% v/v); 1H NMR (DMSO-d6,
400 MHz): d 1.54 (2H, m, piperidine-CH2), 1.68 (2H, m, piperidine-
CH2), 2.29 (6H, m, 2 ꢃ piperazine-CH2, piperidine-CH2), 2.54 (1H,
overlap with DMSO peak, COCH), 3.51 (6H, m, 2 ꢃ piperazine-CH2,
piperidine-CH2), 4.32 (1H, s, CH), 6.06 (2H, s, exchange with D2O,
NH2) 6.65 (2H, m, Ar-H), 6.91 (1H, m, Ar-H), 7.23 (3H, m, Ar-H), 7.30
(4H, t, J ¼ 7.6, Ar-H), 7.44 (4H, d, J ¼ 7.2, Ar-H); 13C NMR (DMSO-d6,
100 MHz): d 28.6, 36.8, 40.4 (overlap with DMSO peak), 46.2, 62.2,
75.6, 113.6, 127.8, 128.5, 129.5, 130.5, 132.5, 143.4, 153.9, 172.7;
MS (ESI positive) m/z ¼ 519.2 [M þ H]þ.
2
3
52.2, 75.5, 116.1, 116.3 (d, JC–F 21), 116.6, 118.2, 130.2, (d, JC–F 9),
130.8, 135.6, 139.2, 148.1, 162.1 (d, JC–F 242), 171.3; 19F NMR
1
(DMSO-d6, 376 MHz): d –115.6 (2F, s); MS (ESI positive) m/z ¼
501.2 [M þ H]þ.
2.2.34. 3-Amino-N-(2-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-
2-oxoethyl)benzenesulfonamide (5l)
Compound 5l was obtained in 32% yield; m.p. 162–164 ꢂC; TLC:
Rf ¼ 0.40 (ethyl acetate/n-hexane 70% v/v); 1H NMR (DMSO-d6,
400 MHz): d 2.24 (4H, m, 2 ꢃ piperazine-CH2), 3.41 (4H, overlap
with water peak, 2 ꢃ piperazine-CH2), 3.66 (2H, s, COCH2NH) 4.42
(1H, s, CH), 6.0 (2H, s, exchange with D2O, NH2), 6.77 (1H, d,
J ¼ 7.4, Ar-H), 6.90 (1H, d, J ¼ 7.4, Ar-H), 6.99 (1H, s, Ar-H), 7.16 (5H,
2.2.30. 2-((4-(bis(4-Fluorophenyl)methyl)piperazin-1-yl)sulfonyl)ani-
line (5h)
Compound 5h was obtained in 94% yield; m.p. 176–179 ꢂC m, Ar-H), 7.46 (5H, m, 4 ꢃ Ar-H, exchange with D2O, SO2NHCH2);
(dec); TLC: Rf ¼ 0.46 (ethyl acetate/n-hexane 30% v/v); 1H NMR 13C NMR (DMSO-d6, 100 MHz): d 42.4, 44.9, 51.9, 73.5, 112.0, 114.2,

1130
S. BUA ET AL.
2
3
116.9 (d, JC–F 21), 118.1, 130.3 (d, JC–F 8), 130.4, 139.2, 141.4, (DMSO-d6, 100 MHz): d 28.6, 36.8, 41.9, 46.2, 52.4, 73.5, 113.5,
1
150.1, 161.9 (d, JC–F 242), 166.3; 19F NMR (DMSO-d6, 376 MHz): d
2
3
116.2 (d, JC–F 21), 120.6, 130.2, 130.3 (d, JC–F 8), 139.2, 153.9,
–115.6 (2F, s); MS (ESI positive) m/z ¼ 501.2 [M þ H]þ.
1
162.4 (d, JC–F 242), 172.7; 19F NMR (DMSO-d6, 376 MHz): d –115.6
(2F, s); MS (ESI positive) m/z ¼ 555.2 [M þ H]þ.
2.2.35. 4-Amino-N-(2-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-
2-oxoethyl)benzenesulfonamide (5m)
2.3. General procedure for the synthesis of compounds 6a–p
Compound 5m was obtained in 44% yield; m.p. 182–184 ꢂC; TLC:
Rf ¼ 0.28 (methanol/dichloromethane 5% v/v); 1H NMR (DMSO-d6,
400 MHz): d 2.21 (4H, m, 2 ꢃ piperazine-CH2), 3.37 (4H, m, overlap
with water peak, 2 ꢃ piperazine-CH2), 3.59 (2H, s, COCH2NH) 4.40
(1H, s, CH), 6.0 (2H, s, exchange with D2O, NH2), 6.61 (2H, d,
J ¼ 8.8, Ar-H), 7.16 (4H, m, Ar-H), 7.28 (1H, m, exchange with D2O,
SO2NHCH2), 7.45 (6H, m, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d
42.4, 44.9, 51.7, 73.5, 113.4, 116.3 (2JC–F 21), 125.6, 129.6, 130.3
The appropriate aminobenzensulfonamides 5a–p (1.0 eq) dis-
solved in dry DMA (5.0 ml) at 0 ꢂC were treated with Et3N (1.3 eq)
and freshly prepared sulfamoyl chloride until consumption of
starting material was confirmed (TLC monitoring). Then the solu-
tion was quenched with slush and extracted with EtOAc
(3 ꢃ 20 ml). The combined organic layers were washed with
NaHCO3 aqueous solution (3 ꢃ 10 ml), HCl aqueous solution 1.0 M
(1 ꢃ 10 ml), brine (3 ꢃ 10 ml), dried over Na2SO4, filtered-off and
concentrated under vacuo. The obtained residue was purified by
trituration from Et2O to afford the titled sulfamides 6a–p as
white solids.
3
1
(d, JC–F 8), 139.2, 153.5, 162.0 (d, JC–F 242), 166.6; 19F NMR
(DMSO-d6, 376 MHz): d –115.6 (2F, s); MS (ESI positive) m/z ¼
501.2 [M þ H]þ.
2.2.36. (1-((2-Aminophenyl)sulfonyl)piperidin-4-yl)(4-(bis(4-fluoro-
phenyl)methyl)piperazin-1-yl)methanone (5n)
Compound 5n was obtained in 70% yield; m.p. 140–142 ꢂC; TLC:
Rf ¼ 0.35 (methanol/dichloromethane 5% v/v); 1H NMR (DMSO-d6,
400 MHz): d 1.54 (2H, m, piperidine-CH2), 1.66 (2H, m, piperidine-
2.3.1. 1-Benzhydryl-4-((3-sulfamoylaminophenyl)sulfonyl)piperazine
(6a)
Compound 6a was obtained in 14% yield; m.p. 125–127 ꢂC; TLC:
CH2), 2.47 (4H, m, 2 ꢃ piperazine-CH2), 2.54 (3H, m, overlap with Rf ¼ 0.55 (ethyl acetate/n-hexane 60% v/v); 1H NMR (DMSO-d6,
DMSO peak, piperidine-CH2, COCH), 3.46 (4H, m, 2 ꢃ piperazine-
CH2), 3.64 (2H, m, piperidine-CH2), 3.42 (1H, s, CH), 6.06 (2H s,
exchange with D2O, NH2), 6.66 (1H, t, J ¼ 8.0, Ar-H), 6.87 (1H, d,
J ¼ 8.4, Ar-H), 7.16 (4H, t, J ¼ 8.8, Ar-H), 7.38 (1H, m, Ar-H), 7.55
(4H, m, Ar-H), 7.59 (1H, m, Ar-H); 13C NMR (DMSO-d6, 100 MHz): d
400 MHz):
d
2.40 (4H, m, 2 ꢃ piperazine-CH2), 2.94 (4H, m,
2 ꢃ piperazine-CH2), 4.34 (1H, s, CH), 7.20 (2H, t, J ¼ 7.2, Ar-H), 7.29
(5H, m, Ar-H), 7.40 (5H, m, Ar-H), 7.53 (3H, m, Ar-H, exchange with
D2O, NHSO2NH2), 7.61 (1H, t, J ¼ 7.6, Ar-H), 10.04 (1H, s, exchange
with D2O, NHSO2NH2); 13C NMR (DMSO-d6, 100 MHz): d 47.0, 51.4,
75.3, 117.3, 121.6, 122.7, 127.9, 128.4, 129.5, 129.6, 136.2, 141.3,
143.3; MS (ESI positive) m/z ¼ 487.0 [M þ H]þ.
2
28.6, 36.8, 42.0, 45.9, 53.0, 75.6, 116.1, 116.3 (d, JC–F 21), 116.6,
3
1
118.2, 130.2, (d, JC–F 9), 130.8, 135.6, 139.2, 148.1, 162.1 (d, JC–F
242), 171.3; 19F NMR (DMSO-d6, 376 MHz): d –115.6 (2F, s); MS (ESI
positive) m/z ¼ 555.2 [M þ H]þ.
2.3.2. 1-Benzhydryl-4-((4-sulfamoylaminophenyl)sulfonyl)piperazine
(6b)
2.2.37. (1-((3-Aminophenyl)sulfonyl)piperidin-4-yl)(4-(bis(4-fluoro-
phenyl)methyl)piperazin-1-yl)methanone (5o)
Compound 5o was obtained in 52% yield; m.p. 142–144 ꢂC; TLC:
Rf ¼ 0.31 (ethyl acetate/n-hexane 70% v/v); 1H NMR (DMSO-d6,
400 MHz): d 1.54 (2H, m, piperidine-CH2), 1.68 (2H, m, piperidine-
CH2), 2.23 (4H, m, 2 ꢃ piperazine-CH2), 2.33 (2H, m, piperidine-
Compound 6b was obtained in 42% yield; m.p. 207–210 ꢂC; TLC:
Rf ¼ 0.30 (ethyl acetate/n-hexane 50% v/v); 1H NMR (DMSO-d6,
400 MHz):
d
2.40 (4H, m, 2 ꢃ piperazine-CH2), 2.91 (4H, m,
2 ꢃ piperazine-CH2), 4.33 (1H, s, CH), 7.19 (2H, t, J ¼ 7.2, Ar-H), 7.28
(4H, t, J ¼ 7.2, Ar-H), 7.38 (8H, m, 6 ꢃ Ar-H, exchange with D2O,
CH2), 2.60 (1H, m, COCH), 3.52 (4H, m, 2 ꢃ piperazine-CH2), 3.60 NHSO2NH2), 7.65 (2H, d, J ¼ 8.4, Ar-H), 10.35 (1H, s, exchange with
D2O, NHSO2NH2); 13C NMR (DMSO-d6, 100 MHz): d 47.0, 51.3, 75.3,
(2H, m, piperidine-CH2), 4.42 (1H, s, CH), 5.64 (2H, s, exchange
with D2O, NH2), 6.82 (2H, m, Ar-H), 6.92 (1H, s, Ar-H), 7.16 (4H, m,
Ar-H), 7.25 (1H, m, Ar-H); 7.46 (4H, m, Ar-H); 13C NMR (DMSO-d6,
100 MHz): d 28.6, 36.8, 41.9, 46.2, 52.4, 73.5, 112.6, 114.9, 116.2 (d,
117.5, 127.3, 127.9, 128.4, 129.5, 130.0, 143.3, 144.9; MS (ESI posi-
tive) m/z ¼ 487.0 [M þ H]þ.
3
2JC–F 21), 118.7, 130.3 (d, JC–F 8), 130.4, 136.5,139.2, 150.4, 162.0
1
(d, JC–F 242), 172.6; 19F NMR (DMSO-d6, 376 MHz): d –115.6 (2F, s);
2.3.3. N-(2-(4-Benzhydrylpiperazin-1-yl)-2-oxoethyl)-2-(sulfamoyla-
mino)benzenesulfonamide (6c)
MS (ESI positive) m/z ¼ 555.2 [M þ H]þ.
Compound 6c was obtained in 10% yield; m.p. 150–152 ꢂC; TLC:
Rf ¼ 0.64 (ethyl acetate/n-hexane 60% v/v); 1H NMR (DMSO-d6,
2.2.38. (1-((4-Aminophenyl)sulfonyl)piperidin-4-yl)(4-(bis(4-fluoro-
phenyl)methyl)piperazin-1-yl)methanone (5p)
400 MHz):
d
2.26 (4H, m, 2 ꢃ piperazine-CH2), 3.40 (4H, m,
Compound 5p was obtained in 74% yield; m.p. 162–164 ꢂC (dec);
TLC: Rf ¼ 0.43 (ethyl acetate/n-hexane 70% v/v); 1H NMR (DMSO-
d6, 400 MHz): d 1.55 (2H, m, piperidine-CH2), 1.66 (2H, m, piperi-
dine-CH2), 2.22 (6H, m, 2 ꢃ piperazine-CH2, piperidine-CH2), 2.54
(1H, m, overlap with DMSO peak, COCH), 3.51 (6H, m,
2 ꢃ piperazine-CH2, piperidine-CH2), 4.41 (1H, s, CH), 5.64 (2H, s,
exchange with D2O, NH2), 6.66 (2H, d, J ¼ 8.8, Ar-H), 7.16 (4H, m,
Ar-H), 7.35 (2H, d, J ¼ 8.8, Ar-H), 7.46 (4H, m, Ar-H); 13C NMR
2 ꢃ piperazine-CH2), 3.80 (2H, s, COCH2NH), 4.35 (1H, s, CH), 7.21
(3H, m, Ar-H), 7.33 (4H, m, Ar-H), 7.45 (4H, m, Ar-H), 7.50 (2H, s,
exchange with D2O, NHSO2NH2), 7.64 (2H, m, Ar-H), 7.81 (1H, d,
J ¼ 8.0, Ar-H), 8.22 (1H, s, exchange with D2O, SO2NHCH2), 8.84
(1H, s, exchange with D2O, NHSO2NH2); 13C NMR (DMSO-d6,
100 MHz): d 42.2, 45.3, 52.0, 75.6, 117.8, 123.1, 127.2, 127.9, 128.5,
129.5, 130.2, 134.6, 137.4, 143.3, 166.2; MS (ESI positive) m/z ¼
487.0 [M þ H]þ.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1131
2.3.4. N-(2-(4-Benzhydrylpiperazin-1-yl)-2-oxoethyl)-3-(sulfamoyl- 2.3.8. 1-(Bis(4-Fluorophenyl)methyl)-4-((2-sulfamoylaminophenyl)-
sulfonyl)piperazine (6h)
amino)benzenesulfonamide (6d)
Compound 6h was obtained in 34% yield; m.p. 170–172 ꢂC (dec);
TLC: Rf ¼ 0.56 (methanol/dichloromethane 10% v/v); 1H NMR
(DMSO-d6, 400 MHz): d 2.41 (4H, m, 2 ꢃ piperazine-CH2), 3.03 (4H,
m, 2 ꢃ piperazine-CH2), 4.44 (1H, s, CH), 7.13 (4H, m, Ar-H), 7.29
(1H, t, J ¼ 7.4, Ar-H), 7.43 (5H, m, Ar-H) , 7.75 (4H, m, 2 ꢃ Ar-H,
exchange with D2O, NHSO2NH2), 8.95 (1H, s, exchange with D2O,
NHSO2NH2); 13C NMR (DMSO-d6, 100 MHz): d 46.8, 51.1, 73.1,
Compound 6d was obtained in 29% yield; m.p. 154–156 ꢂC; TLC:
1
Rf ¼ 0.18 (methanol/dichloromethane 10% v/v); H NMR (DMSO-d6,
400 MHz):
d
2.26 (4H, m, 2 ꢃ piperazine-CH2), 3.13 (4H, m,
2 ꢃ piperazine-CH2), 3.72 (2H, s, COCH2NH), 4.34 (1H, s, CH), 7.24
(3H, m, Ar-H, exchange with D2O, SO2NHCH2), 7.33 (6H, m, Ar-H),
7.45 (7H, m, 5 ꢃ Ar-H, exchange with D2O, NHSO2NH2), 7.62 (1H,
m, Ar-H), 8.99 (1H, s, exchange with D2O, NHSO2NH2); 13C NMR
(DMSO-d6, 100 MHz): d 42.2, 44.9, 51.9, 75.7, 116.4, 120.7, 122.5,
127.9, 128.6, 129.5, 130.5, 141.1, 141.6, 143.3, 171.3; MS (ESI posi-
tive) m/z ¼ 544.0 [M þ H]þ.
2
3
115.9, 116.2 (d, JC–F 21), 118.3, 123.0, 130.2 (d, JC–F 7), 130.8,
135.1, 138.6, 139.1, 161.9 (d, JC–F 241); 19F NMR (DMSO-d6,
1
376 MHz): d –115.4 (2F, s); MS (ESI positive) m/z ¼ 523.0 [M þ H]þ.
2.3.9. 1-(Bis(4-Fluorophenyl)methyl)-4-((3-sulfamoylaminophenyl)-
sulfonyl)piperazine (6i)
2.3.5. (4-Benzhydrylpiperazin-1-yl)(1-((2-sulfamoylaminophenyl)sul-
fonyl)piperidin-4-yl)methanone (6e)
Compound 6i was obtained in 43% yield; m.p. 132–135 ꢂC; TLC:
Rf ¼ 0.30 (ethyl acetate/n-hexane 50% v/v); 1H NMR (DMSO-d6,
Compound 6e was obtained in 65% yield; m.p. 162–164 ꢂC; TLC:
1
Rf ¼ 0.41 (methanol/dichloromethane 10% v/v); H NMR (DMSO-d6,
400 MHz):
d
2.39 (4H, m, 2 ꢃ piperazine-CH2), 2.96 (4H, m,
400 MHz): d 1.58 (2H, m, piperidine-CH2), 1.70 (2H, m, piperidine-
CH2), 2.26 (4H, m, 2 ꢃ piperazine-CH2), 2.54 (2H, overlap with
DMSO peak, piperidine-CH2), 2.60 (1H, overlap with DMSO peak,
COCH), 3.47 (4H, m, 2 ꢃ piperazine-CH2), 3.65 (2H, m, piperidine-
2 ꢃ piperazine-CH2), 4.43 (1H, s, CH), 7.12 (4H, t, J ¼ 8.8, Ar-H), 7.37
(7H, m, 5 ꢃ Ar-H, exchange with D2O, NHSO2NH2), 7.52 (2H, m, Ar-
H), 7.59 (1H, m, Ar-H), 10.01 (1H, s, exchange with D2O,
NHSO2NH2); 13C NMR (DMSO-d6, 100 MHz): d 47.0, 51.2, 73.2, 116.2
2
3
CH2), 4.33 (1H, s, CH), 7.23 (3H, m, Ar-H), 7.32 (5H, m, Ar-H), 7.43 (d, JC–F 21), 117.2, 121.5, 122.7, 130.2 (d, JC–F 8), 130.8, 136.1,
139.2, 141.3, 161.9 (d, JC–F 242); 19F NMR (DMSO-d6, 376 MHz):
1
(5H, m, Ar-H), 7.70 (3H, m, Ar-H, exchange with D2O, NHSO2NH2),
d –115.6 (2F, s); MS (ESI positive) m/z ¼ 523.0 [M þ H]þ.
8.95 (1H, s, exchange with D2O, NHSO2NH2); 13C NMR (DMSO-d6,
100 MHz): d 28.5, 36.6, 41.9, 46.0, 53.0, 75.6, 118.8, 121.7, 127.9,
128.6, 129.5, 130.9, 135.5, 138.3, 141.3, 143.3, 172.5; MS (ESI posi-
tive) m/z ¼ 598.0 [M þ H]þ.
2.3.10. 1-(Bis(4-Fluorophenyl)methyl)-4-((4-sulfamoylaminophenyl)-
sulfonyl)piperazine (6j)
Compound 6j was obtained in 42% yield; m.p. 176–179 ꢂC (dec);
TLC: Rf ¼ 0.28 (methanol/dichloromethane 10% v/v); 1H NMR
2.3.6. (4-Benzhydrylpiperazin-1-yl)(1-((3-sulfamoylaminophenyl)sul-
(DMSO-d6, 400 MHz): d 2.38 (4H, m, 2 ꢃ piperazine-CH2), 2.90 (4H,
fonyl)piperidin-4-yl)methanone (6f)
m, 2 ꢃ piperazine-CH2), 4.42 (1H, s, CH), 7.12 (4H, m, Ar-H), 7.41
Compound 6f was obtained in 65% yield; m.p. 230–232 ꢂC; TLC:
(8H, m, 6 ꢃ Ar-H, exchange with D2O, NHSO2NH2), 7.65 (2H, d,
Rf ¼ 0.13 (methanol/dichloromethane 5% v/v); 1H NMR (DMSO-d6,
400 MHz): d 1.58 (2H, m, piperidine-CH2), 1.68 (2H, m, piperidine-
CH2), 2.25 (4H, m, 2 ꢃ piperazine-CH2), 2.33 (2H, m, piperidine-
CH2), 2.54 (1H, overlap with DMSO peak, COCH), 3.4 (4H, m,
2 ꢃ piperazine-CH2), 3.59 (2H, m, piperidine-CH2), 4.32 (1H, s, CH),
7.21 (2H, m, Ar-H), 7.32 (7H, m, 5 ꢃ Ar-H, exchange with D2O,
NHSO2NH2), 7.45 (5H, m, Ar-H), 7.54 (2H, m, Ar-H), 9.97 (1H, s,
exchange with D2O, NHSO2NH2); 13C NMR (DMSO-d6, 100 MHz): d
28.6, 36.8, 40.4 (overlap with DMSO peak), 46.2, 52.4, 75.6, 117.1,
121.3, 122.5, 126.2, 127.9, 128.5, 129.5, 130.8, 136.9, 141.3, 172.7;
MS (ESI positive) m/z ¼ 598.0 [M þ H]þ.
J ¼ 8.4, Ar-H), 10.33 (1H, s, exchange with D2O, NHSO2NH2); 13C
2
NMR (DMSO-d6, 100 MHz): d 47.0, 51.1, 73.2, 116.2 (d, JC–F 21),
3
1
117.5, 127.2, 129.9, 130.2 (d, JC–F 8), 139.2, 144.8, 161.9 (d, JC–F
242); 19F NMR (DMSO-d6, 376 MHz): d –115.6 (2F, s); MS (ESI posi-
tive) m/z ¼ 523.0 [M þ H]þ.
2.3.11. N-(2-(4-(bis(4-Fluorophenyl)methyl)piperazin-1-yl)-2-oxoethyl)-
2-(sulfamoylamino)benzenesulfonamide (6k)
Compound 6k was obtained in 80% yield; m.p. 183–186 ꢂC (dec);
TLC: Rf ¼ 0.23 (methanol/dichloromethane 5% v/v); 1H NMR
(DMSO-d6, 400 MHz): d 2.20 (4H, m, 2 ꢃ piperazine-CH2), 3.45 (4H,
m, 2 ꢃ piperazine-CH2), 3.75 (2H, s, COCH2NH) 4.43 (1H, s, CH),
7.17 (5H, m, Ar-H), 7.46 (6H, m, 4 ꢃ Ar-H, exchange with D2O,
NHSO2NH2), 7.63 (2H, m, Ar-H), 7.80 (1H, d, J ¼ 8.0, Ar-H), 8.23 (1H,
m, exchange with D2O, SO2NHCH2), 8.84 (1H, s, exchange with
D2O, NHSO2NH2); 13C NMR (DMSO-d6, 100 MHz): d 42.4, 44.9, 51.9,
2.3.7. (4-Benzhydrylpiperazin-1-yl)(1-((4-sulfamoylaminophenyl)sul-
fonyl)piperidin-4-yl)methanone (6g)
Compound 6 g was obtained in 44% yield; m.p. 160–162 ꢂC; TLC:
Rf ¼ 0.26 (ethyl acetate/n-hexane 70% v/v); 1H NMR (DMSO-d6,
400 MHz): d 1.55 (2H, m, piperidine-CH2), 1.67 (2H, m, piperidine-
CH2), 2.25 (6H, m, 2 ꢃ piperazine-CH2, piperidine-CH2), 2.54 (1H, m,
overlap with DMSO peak, COCH), 3.47 (4H, m, 2 ꢃ piperazine-CH2),
3.58 (2H, m, piperidine-CH2), 4.31 (1H, s, CH), 7.21 (2H, t, J ¼ 8.6,
Ar-H), 7.32 (7H, m, Ar-H), 7.44 (4H, m, 2 ꢃ Ar-H, exchange with
2
3
73.5, 116.3 (d, JC–F 21), 116.7, 118.2, 123.1, 130.3 (d, JC–F 10),
130.5, 134.9, 138.2, 139.3, 162.0 (d, JC–F 242), 171.4; 19F NMR
1
(DMSO-d6, 376 MHz): d –115.6 (2F, s); MS (ESI positive) m/z ¼
580.0 [M þ H]þ.
D2O, NHSO2NH2), 7.64 (2H, d, J ¼ 8.2, Ar-H), 10.28 (1H, s, exchange 2.3.12. N-(2-(4-(bis(4-Fluorophenyl)methyl)piperazin-1-yl)-2-oxoethyl)-
with D2O, NHSO2NH2); 13C NMR (DMSO-d6, 100 MHz): d 28.6, 36.9,
3-(sulfamoylamino)benzenesulfonamide (6l)
Compound 6l was obtained in 29% yield; m.p. 155–157 ꢂC (dec);
40.4 (overlap with DMSO peak), 46.2, 62.2, 75.6, 113.6, 127.8,
TLC: Rf ¼ 0.28 (methanol/dichloromethane 5% v/v); 1H NMR
(DMSO-d6, 400 MHz): d 2.27 (4H, m, 2 ꢃ piperazine-CH2), 3.41 (4H,
128.4, 129.5, 130.2, 132.6, 143.1, 153.9, 172.70; MS (ESI positive) m/z ¼
598.0 [M þ H]þ.

1132
S. BUA ET AL.
overlap with water peak, 2 ꢃ piperazine-CH2), 3.76 (2H, s, 2.3.16. (4-(bis(4-Fluorophenyl)methyl)piperazin-1-yl)(1-((4-sulfamo-
COCH2NH) 4.43 (1H, s, CH), 7.16 (5H, m, Ar-H), 7.26 (2H, s,
exchange with D2O, NHSO2NH2), 7.36 (1H, m, exchange with D2O,
ylaminophenyl)sulfonyl)piperidin-4-yl)methanone (6p)
Compound 6p was obtained in 46% yield; m.p. 150–152 ꢂC (dec);
SO2NHCH2), 7.42 (6H, m, Ar-H), 7.61 (1H, s, Ar-H), 9.93 (1H, s, TLC: Rf ¼ 0.16 (methanol/dichloromethane 5% v/v); 1H NMR
exchange with D2O, NHSO2NH2); 13C NMR (DMSO-d6, 100 MHz): d
(DMSO-d6, 400 MHz): d 1.55 (2H, m, piperidine-CH2), 1.67 (2H, m,
2
piperidine-CH2), 2.26 (6H, m, 2 ꢃ piperazine-CH2, piperidine-CH2),
42.4, 44.9, 51.9, 73.5, 116.9 (d, JC–F 21), 118.4, 122.3, 123.2, 130.4
3
1
2.54 (1H, m, overlap with DMSO peak, COCH), 3.46 (4H, m,
2 ꢃ piperazine-CH2), 3.59 (2H, m, piperidine-CH2), 4.41 (1H, s, CH),
7.15 (4H, t, J ¼ 8.6, Ar-H), 7.32 (2H, d, J ¼ 8.2, Ar-H), 7.46 (6H, m,
4 ꢃ Ar-H, exchange with D2O, NHSO2NH2), 7.64 (2H, d, J ¼ 8.2, Ar-
H) 10.28 (1H, s, exchange with D2O, NHSO2NH2); 13C NMR (DMSO-
(d, JC–F 8), 130.9, 137.2, 139.1, 141.2, 161.9 (d, JC–F 242), 167.2;
19F NMR (DMSO-d6, 376 MHz): d –115.6 (2F, s); MS (ESI positive)
m/z ¼ 580.0 [M þ H]þ.
2
d6, 100 MHz): d 28.6, 36.8, 41.9, 46.2, 52.4, 73.5, 116.2 (d, JC–F 21),
3
1
2.3.13. N-(2-(4-(bis(4-Fluorophenyl)methyl)piperazin-1-yl)-2-oxoethyl)-
4-(sulfamoylamino)benzene sulfonamide (6m)
116.6, 129.7, 130.2, 130.3 (d, JC–F 8), 139.2, 143.1, 162.4 (d, JC–F
242), 172.70; 19F NMR (DMSO-d6, 376 MHz): d –115.6 (2F, s); MS
(ESI positive) m/z ¼ 634.0 [M þ H]þ.
Compound 6m was obtained in 35% yield; m.p. 157–159 ꢂC; TLC:
Rf ¼ 0.12 (methanol/dichloromethane 5% v/v); 1H NMR (DMSO-d6,
400 MHz): d 2.22 (4H, m, 2 ꢃ piperazine-CH2), 3.37 (4H, m, overlap
with water peak, 2 ꢃ piperazine-CH2), 3.68 (2H, s, COCH2NH), 4.42
(1H, s, CH), 7.16 (4H, m, Ar-H), 7.22 (2H, d, J ¼ 8.8, Ar-H), 7.40 (2H,
s, exchange with D2O, NHSO2NH2), 7.47 (5H, m, 4 ꢃ Ar-H, exchange
2.4. Carbonic anhydrase inhibition assay
An Applied Photophysics (Leatherhead, UK) stopped-flow instru-
ment has been used for assaying the CA-catalysed CO2 hydration
with D2O, SO2NHCH2), 7.76 (2H, d, J ¼ 8.8, Ar-H), 10.18 (1H, s, activity18. Phenol red (at a concentration of 0.2 mM) has been used
exchange with D2O, NHSO2NH2); 13C NMR (DMSO-d6, 100 MHz): d
as indicator, working at the absorbance maximum of 557 nm, with
20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintain-
ing constant the ionic strength), following the initial rates of the
42.4, 44.9, 51.7, 73.5, 116.3 (2JC–F 21), 116.6, 129.6, 130.1, 130.3 (d,
1
3JC–F 8), 139.3, 142.9, 162.0 (d, JC–F 242), 166.6; 19F NMR (DMSO-
CA-catalysed CO2 hydration reaction for a period of 10–100 s. The
d6, 376 MHz):
d
–115.6 (2F, s); MS (ESI positive) m/z ¼
580.0 [M þ H]þ.
CO2 concentrations ranged from 1.7 to 17 mM for the determin-
ation 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 uncata-
lysed 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-deionised 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, and represent the mean from at least
three different determinations19,20. All CA isoforms were recombin-
ant ones obtained in-house as reported earlier21.
2.3.14. (4-(bis(4-Fluorophenyl)methyl)piperazin-1-yl)(1-((2-sulfamo-
ylaminophenyl)sulfonyl)piperidin-4-yl)methanone (6n)
Compound 6n was obtained in 44% yield; m.p. 140–142 ꢂC; TLC:
Rf ¼ 0.50 (methanol/dichloromethane 5% v/v); 1H NMR (DMSO-d6,
400 MHz): d 1.56 (2H, m, piperidine-CH2), 1.70 (2H, m, piperidine-
CH2), 2.24 (4H, m, 2 ꢃ piperazine-CH2), 2.58 (2H, m, piperidine-
CH2), 2.68 (1H, m, COCH), 3.48 (4H, m, 2 ꢃ piperazine-CH2), 3.66
(2H, m, piperidine-CH2), 4.42 (1H, s, CH), 7.22 (5H, m, Ar-H), 7.43
(4H, m, Ar-H), 7.60 (2H, s, exchange with D2O, NHSO2NH2), 7.65
(2H, m, Ar-H), 7.76 (1H, m, Ar-H), 8.95 (1H, s, exchange with D2O,
NHSO2NH2); 13C NMR (DMSO-d6, 100 MHz): d 28.6, 36.8, 41.9, 46.2,
2
3
52.4, 73.5, 115.1, 115.4 (d, JC–F 21), 118.0, 123.0, 129.4 (d, JC–F 8),
130.8, 135.4, 138.4, 139.6, 161.1 (d, JC–F 242), 168.4; 19F NMR
1
3. Results and discussion
(DMSO-d6, 376 MHz): d –115.6 (2F, s); MS (ESI positive) m/z ¼
634.0 [M þ H]þ.
3.1. Chemistry
The sulfamide moiety is able to establish a more extended net-
work of hydrogen bonds within the enzymatic pocket compared
to classical sulfonamides, due to the presence of the additional
nitrogen atom, but unfortunately this feature does not confer
selectivity against different CA isoforms16,17. Therefore, the pur-
pose of this study is not only to explore the activity of the sulfa-
mides against the two CA isoforms of Vibrio cholerae, but also to
propose specific structural changes to obviate to the problem of
selectivity.
The novel sulfamides reported here feature the sulfamides
groups (in ortho-, meta-, or para positions on the benzene ring),
connected into a highly flexible alkyl-aryl scaffolds. In particular,
we designed two series of compounds which differ by the spacer
connecting the 1-benzhydrylpiperazin tail with the benzene-sulfa-
mide zinc binding functionality (Scheme 1)16.
2.3.15. (4-(bis(4-Fluorophenyl)methyl)piperazin-1-yl)(1-((3-sulfamo-
ylaminophenyl)sulfonyl)piperidin-4-yl)methanone (6o)
Compound 6o was obtained in 35% yield; m.p. 160–162 ꢂC (dec);
TLC: Rf ¼ 0.12 (methanol/dichloromethane 5% v/v); 1H NMR
(DMSO-d6, 400 MHz): d 1.54 (2H, m, piperidine-CH2), 1.68 (2H, m,
piperidine-CH2), 2.23 (4H, m, 2 ꢃ piperazine-CH2), 2.33 (2H, m,
piperidine-CH2), 2.54 (1H, m, overlap with DMSO peak, COCH),
3.47 (4H, m, 2 ꢃ piperazine-CH2), 3.60 (2H, m, piperidine-CH2), 4.42
(1H, s, CH), 7.16 (5H, m, Ar-H), 7.32 (2H, s, exchange with D2O,
NHSO2NH2), 7.54 (7H, m, Ar-H), 9.81 (1H, s, exchange with D2O,
NHSO2NH2); 13C NMR (DMSO-d6, 100 MHz): d 28.6, 36.8, 41.9, 46.2,
2
3
52.4, 73.5, 116.2 (d, JC–F 21), 118.7, 122.0, 122.9, 130.3 (d, JC–F 8),
130.9, 135.3, 137.4, 139.6, 162.0 (d, JC–F 242), 172.6; 19F NMR
1
The planned synthesis of the compounds 6a–p provides a first
step during which the coupling of the benzhydryl piperazines
1a,b with the appropriate acids takes place to give the
(DMSO-d6, 376 MHz):
d
–115.6 (2F, s); MS (ESI positive)
m/z ¼ 634.0 [M þ H]þ.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1133
Scheme 1. General synthetic scheme of compounds 6a–p.
intermediates 2a–d, followed by treatment with trifluoroacetic
acid (TFA) to afford the alkylamines 3a–d. In the second step, the
free amines were coupled with commercially available nitrobenze-
nesulfonyl chlorides and the obtained nitro derivatives 4a–p were
reduced with iron, Fe(0), in acidic media, to afford the amines
5a–p. Finally, the desired compounds 6a–p (Figure 1) were
obtained by treatment of amines 5a–p with freshly prepared sul-
famoyl chloride16. All compounds were purified by trituration from
Et2O to afford products with high purity in yields between 10 and
80%; the analytical and spectroscopic data of the purified com-
pounds are in agreement with the purposed structures (see sec-
tion 2 for details).
aromatic rings of compound 6i (KI ¼ 75.0 nM), did not have a
significant impact on its activity, both compound having a
similar activity to that of 6a (KI ¼ 66.2 nM). The inhibition
potencies were dependent not only on the presence of halo-
gens but also on the sulfamide regioisomer considered. In
fact, the potency ranking for compounds 6j, 6i, and 6h
(KIs ¼ 64.6, 75.0, 842.6 nM), 6m, 6l, and 6k (KIs ¼ 47.8, 71.1,
337.8 nM) and for compounds 6p, 6o, and 6n (KIs ¼ 49.1,
72.0, 682.6 nM) was para>meta>ortho.
ii. The ubiquitous hCA II was inhibited by sulfamides 6a–p,
with KIs spanning between range 181.7 and 1430.7 nM.
Likewise hCA I, the derivatives with sulfamide group in para
position of the aromatic ring showed better activity than the
corresponding meta and ortho analogues. In fact, compound
6g shows a lower KI ¼ 226.7 nM than its meta-substituted
analogue 6f (KI ¼ 576.4 nM) or ortho-substituted derivative 6e
(KI ¼ 1360.1 nM). The introduction of fluorine atoms on the
phenyl rings of compounds 6g, 6f, and 6e to give derivatives
6o, 6p, and 6n, caused a slight change in the inhibition
trend, with a better KI of the ortho-substituted compared to
the meta-substituted derivative. It is interesting to underline
the case of the compound 6p (KI ¼ 181.7 nM), which is the
best inhibitor against hCA II. The substitution of the isonipe-
cotic acid spacer of 6p with the glycine spacer of 6m did
not show any impact on the inhibitory activity. The removal
of the fluorine atoms in 6g did not substantially altered the
activity compared to the perhydro derivative. Equally to hCA
I, for hCA II the most considerable differences of inhibitory
activity concerned the position of the ZBG in the phenyl
ring, as demonstrated for example by compounds 6d (meta-
substituted) and 6c (ortho-substituted) with KIs of 396.4 and
850.2 nM, respectively.
3.2. Carbonic anhydrase inhibition
All synthesised compounds 6a–p, were tested in vitro for their
inhibitory activity against the cytosolic isoenzymes hCA I and II
and bacterial enzymes VchCAa and VchCAb by means of a
stopped-flow carbon dioxide hydration assay18, and their activities
were compared to the standard CAI acetazolamide (AAZ). The fol-
lowing structure–activity–relationship (SARs) can be drawn from
the data of Table 1:
i. hCA I was effectively inhibited by most compounds investi-
gated here, with inhibition constants spanning between 43.1
and 839.9 nM. It is interesting to note the case of compounds
6m and 6p that demonstrate the best activity (KIs ¼ 47.8 and
49.1 nM), both having the sulfamide group in para position
of the aromatic ring but differing for the glycine and isonipe-
cotic acid spacer, respectively. The removal of the fluorine
atoms in the aromatic rings of compound 6g (corresponding
to the non-halogenated analogue of 6d), decreased the
activity (KI ¼ 601.3 nM), suggesting that substitution with hal-
ogens as –F, in this case, is advantageous for the inhibitory
activity. The same trend concerned compounds 6j and its
corresponding non-halogenated 6b, which showed the fol-
lowing inhibition data KIs of 64.6 and 555.1 nM, respectively.
On the other hand, the presence of fluorine atoms in
iii. The general tendencies described above were also applicable
for the VchCAa, with this latter being moderately inhibited
by benzenesulfamide derivatives prepared in this study (KIs
values in the range of 91.4 and 3748.6 nM). As reported for
the human isoforms discussed above, also for VchCAa, the
worst inhibitors are reconfirmed to be the compounds that

1134
S. BUA ET AL.
Figure 1. Structures of the compounds 6a–p.
present the ZBG in the ortho position of the phenyl ring (6c, iv. VchCAb was the least inhibited among the enzymes herein
6e, 6h, 6k, and 6n with KIs ¼ 930.8, 879.3, 2797.0, 1747.0,
and 3748.6 nM, respectively). The most active compounds
were the halogen derivative 6l and the non-halogen ana-
logue 6d, which exhibited KI values of 91.4 and 95.7 nM,
respectively. It is interesting to note that the introduction of
the rigid linker of isonipecotic acid determined a lower effi-
cacy against this isoform, as shown by the compounds 6f
(KI ¼ 654.2 nM) and 6o (KI ¼ 497.2 nM), compared with the
above-mentioned derivatives 6d and 6l. This improvement of
VchCAa inhibition potency might be due to the flexible gly-
cine spacer, which probably increases the interactions of the
inhibitor with active site amino acids residues.
considered and showed KIs spanning between 1578.2 and
4676.1 nM. Interestingly, the meta derivatives 6o and 6i
(KIs ¼ 1578.2 and 1742.2 nM) were the most potent inhibitors
against the VchCAb, compared to the corresponding para
analogues 6p and 6j (KIs ¼ 4533.7 and 4349.2 nM). On the
contrary, the meta derivatives 6a and 6f (KIs ¼ 4676.1 and
3792.0 nM) were less effective with respect to para derivatives
6b and 6g (KIs ¼ 1880.7 and 2538.1 nM). It is rather difficult to
explain this result but we could speculate that the introduc-
tion of fluorine atoms leads to a decrease in the inhibitory
potency of sulfamides against VchCAb. In fact the fluorination
of compounds 6g, 6b, and 6d (KIs ¼ 2538.1, 1880.7, and

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1135
Table 1. Inhibition data of hCA I, hCA II, VchCA a, VchCA b
with compounds reported here and the standard sulfonamide
References
inhibitor acetazolamide (AAZ) by
a stopped flow CO2
1. Silva AJ, Benitez JA. Vibrio cholerae biofilms and cholera
pathogenesis. PLoS Negl Trop Dis 2016;10:e0004330.
2. Abuaita BH, Withey JH. Bicarbonate induces Vibrio cholerae
virulence gene expression by enhancing ToxT activity. Infect
Immun 2009;77:4111–20.
3. Alafeefy AM, Ceruso M, Al-Tamimi AM, et al.
Quinazoline–sulfonamides with potent inhibitory activity
against the a-carbonic anhydrase from Vibrio cholerae.
Bioorg Med Chem 2014;22:5133–40.
4. Unterweger D, Miyata ST, Bachmann V, et al. The Vibrio chol-
erae type VI secretion system employs diverse effector mod-
ules for intraspecific competition. Nat Commun 2014;5:3549.
5. Zuckerman JN, Rombo L, Fisch A. The true burden and risk
of cholera: implications for prevention and control. Lancet
Infect Dis 2007;7:521–30.
6. Cholera Annual Report 2017. Weekly Epidemiological Record
8 September 2017; 92, 36:521–36. Available from: http://
www.who.int/wer/2017/wer9236/en/.
hydrase assay18
.
ꢀ
KI (nM)
Cmp
hCA I
hCA II
VchCAa
VchCAb
6a
6b
6c
6d
6e
6f
6g
6h
6i
66.2
555.1
839.9
58.1
578.1
43.1
601.3
842.6
75.0
64.6
337.8
71.1
47.8
682.6
417.4
359.2
850.2
396.4
1360
576.4
226.7
1171
389.2
352.9
1431
217.5
207.8
589.8
663.1
181.7
12
333.7
236.4
930.8
95.7
4676
1881
>10,000
3783
879.3
654.2
140.3
2797.0
588.0
273.8
1747.0
91.4
105.6
3748.6
497.2
398.2
6.8
>10,000
3792
2538
>10,000
1742
6j
6k
6l
4349
>10,000
>10,000
4666
6m
6n
6o
6p
AAZ
ꢀ
>10,000
72.0
49.1
250
1578
4534
451
7. Thomson JJ, Withey JH. Bicarbonate increases binding affin-
ity of Vibrio cholerae ToxT to virulence gene promoters.
J Bacteriol 2014;196:3872–80.
Mean from three different assays, by a stopped flow tech-
nique (errors were in the range of
reported values).
5–10% of the
8. Del Prete S, Isik S, Vullo D, et al. DNA cloning, characteriza-
tion, and inhibition studies of an a-carbonic anhydrase from
the pathogenic bacterium Vibrio cholerae. J Med Chem
2012;55:10742–8.
9. Del Prete S, De Luca V, Scozzafava A, et al. Biochemical
properties of a new a-carbonic anhydrase from the human
pathogenic bacterium, Vibrio cholerae. J Enzyme Inhib Med
Chem 2014;29:23–7.
10. Del Prete S, Vullo D, De Luca V, et al. Sulfonamide inhibition
studies of the b-carbonic anhydrase from the pathogenic
bacterium Vibrio cholerae. Bioorg Med Chem 2016;24:
1115–20.
3782.8 nM), to afford 6p, 6j, and 6l, respectively, decreased
the inhibition efficacy (KIs ¼ 4533.7, 4349.2, and >10,000 nM,
respectively), with the potency of compound 6l being very
low. Finally, it is important to emphasise the selectivity of
some compounds against VchCAa with respect to the b-bac-
terial isoform. For instance, compounds 6l and 6d showed a
nanomolar efficacy against VchCAa (KIs of 91.4 and 95.7 nM)
but 6l was totally ineffective against the b-isoform, whereas
compound 6d showed a KI of 3782.8 nM against VchCAb.
11. Nocentini A, Moi D, Balboni G, et al. Discovery of thiazolin-4-
one-based aromatic sulfamates as a new class of carbonic
anhydrase isoforms I, II, IV, and IX inhibitors. Bioorg Chem
2018;77:293–9.
4. Conclusions
In this work, we reported the investigation of benzenesulfamide
derivatives (–NH-SO2NH2) as an alternative zinc binding class of
inhibitors to the primary aromatic sulfonamides in the design of
new potential bacterial CAIs17. The “tail approach”, the method
that consists in varying the terminal portions of the molecule to
selectively enhance the inhibition of the target isoform(s), was
applied to the phenyl sulfamide scaffold. All compounds were
synthesised by a convenient and efficient method and were
screened for in vitro inhibition against cytosolic isoenzymes hCA I,
II, VchCAa and VchCAb by using a stopped-flow CO2 hydrase
assay method. The obtained results suggest that the benzhydryl
piperazine moiety appended to benzene-sulfamide functionalities
are not favourable for the interaction with Vibrio cholerae CA iso-
forms. Conversely, the same moiety is slightly more favourable for
the inhibition of hCA I, as demonstrated by compounds 6f, 6m,
6p, and 6d that showed interesting activities against this isoform,
with a nanomolar inhibition constant (KIs ¼ 43.1, 47.8, 49.1, and
58.1 nM, respectively), being thus more potent than AAZ (as
standard inhibitor).
12. (a) Carta F, Supuran CT, Scozzafava A. Sulfonamides and
their isosters as carbonic anhydrase inhibitors. Future Med
Chem 2014;6:1149–65. (b) Carta F, Supuran CT. Diuretics
with carbonic anhydrase inhibitory action: a patent and lit-
erature review (2005–2013). Expert Opin Ther Pat
2013;23:681–91. (c) Masini E, Carta F, Scozzafava A, Supuran
CT. Antiglaucoma carbonic anhydrase inhibitors: a patent
review. Expert Opin Ther Pat 2013;23:705–16. (d) Scozzafava
A, Supuran CT, Carta F. Antiobesity carbonic anhydrase
inhibitors: a literature and patent review. Expert Opin Ther
Pat 2013;23:725–35. (d) Supuran CT. Carbonic anhydrases
and metabolism. Metabolites 2018;8:E25.
13. (a) Supuran CT. Advances in structure-based drug discovery
of carbonic anhydrase inhibitors. Expert Opin Drug Discov
2017;12:61–88. (b) Supuran CT. Structure and function of
carbonic anhydrases. Biochem J 2016;473:2023–32. (c) Neri
D, Supuran CT. Interfering with pH regulation in tumours as
a therapeutic strategy. Nat Rev Drug Discov 2011;10:767–77.
14. (a) D'Ambrosio K, Smaine FZ, Carta F, et al. Development of
potent carbonic anhydrase inhibitors incorporating both sul-
fonamide and sulfamide groups.
J
Med Chem
2012;55:6776–83. (b) Supuran CT. How many carbonic anhy-
drase inhibition mechanisms exist? J Enzyme Inhib Med
Chem 2016;31:345–60. (c) Alterio V, Di Fiore A, D'Ambrosio
Disclosure statement
The authors do not report conflict of interest.

1136
S. BUA ET AL.
K, et al. Multiple binding modes of inhibitors to carbonic
anhydrases: how to design specific drugs targeting 15 differ-
ent isoforms? Chem Rev 2012;112:4421–68. (d) Abbate F,
Winum JY, Potter BV, et al. Carbonic anhydrase inhibitors: X-
ray crystallographic structure of the adduct of human iso-
zyme II with EMATE, a dual inhibitor of carbonic anhydrases
and steroid sulfatase. Bioorg Med Chem Lett 2004;14:231–4.
et al. Carbonic anhydrase inhibitors: novel sulfonamides
incorporating 1,3,5-triazine moieties as inhibitors of the
cytosolic and tumour-associated carbonic anhydrase iso-
zymes I, II and IX. Bioorg Med Chem Lett 2005;15:3102–8.
_
€
€
(d) S¸enturk M, Gulc¸in I, Beydemir S¸, et al. In vitro inhibition
of human carbonic anhydrase I and II isozymes with natural
phenolic compounds. Chem Biol Drug Des 2011;77:494–9.
(e) Fabrizi F, Mincione F, Somma T, et al. A new approach to
antiglaucoma drugs: carbonic anhydrase inhibitors with or
without NO donating moieties. Mechanism of action and
15. Berrino E, Bua S, Mori M, et al. Novel sulfamide-containing
compounds as selective carbonic anhydrase I inhibitors.
Molecules 2017;22:pii:1049.
16. (a) Supuran CT. Structure-based drug discovery of carbonic
preliminary pharmacology.
J Enzyme Inhib Med Chem
anhydrase inhibitors.
J
Enzyme Inhib Med Chem
2012;27:138–47. (f) Dogne JM, Hanson J, Supuran C, Pratico
D. Coxibs and cardiovascular side-effects: from light to
shadow. Curr Pharm Des 2006;12:971–5.
2012;27:759–72. (b) Supuran CT. Carbonic anhydrase inhib-
ition and the management of hypoxic tumors. Metabolites
2017;7:E48. (c) Ward C, Langdon SP, Mullen P, et al. New
strategies for targeting the hypoxic tumour microenviron-
ment in breast cancer. Cancer Treat Rev 2013;39:171–9. (d)
Garaj V, Puccetti L, Fasolis G, et al. Carbonic anhydrase
inhibitors: novel sulfonamides incorporating 1,3,5-triazine
moieties as inhibitors of the cytosolic and tumour-associated
carbonic anhydrase isozymes I, II and IX. Bioorg Med Chem
Lett 2005;15:3102–8. (e) Casey JR, Morgan PE, Vullo D, et al.
Carbonic anhydrase inhibitors. Design of selective, mem-
brane-impermeant inhibitors targeting the human tumor-
associated isozyme IX. J Med Chem 2004;47:2337–47.
20. (a) Supuran CT, Nicolae A, Popescu A. Carbonic anhydrase
inhibitors. Part 35. Synthesis of Schiff bases derived from
sulfanilamide and aromatic aldehydes: the first inhibitors
with equally high affinity towards cytosolic and membrane-
bound isozymes. Eur
J Med Chem 1996;31:431–8. (b)
Pacchiano F, Aggarwal M, Avvaru BS, et al. Selective hydro-
phobic pocket binding observed within the carbonic anhy-
drase II active site accommodate different 4-substituted-
ureido-benzenesulfonamides and correlate to inhibitor
potency. Chem Commun (Camb) 2010;46:8371–3. (c)
Ozensoy Guler O, Capasso C, Supuran CT. A magnificent
enzyme superfamily: carbonic anhydrases, their purification
17. (a) Supuran CT. Carbonic anhydrases: novel therapeutic
applications for inhibitors and activators. Nat Rev Drug
Discov 2008;7:168–81. (b) Capasso C, Supuran CT. An over-
view of the alpha-, beta-and gamma-carbonic anhydrases
from bacteria: can bacterial carbonic anhydrases shed new
light on evolution of bacteria? J Enzyme Inhib Med Chem
2015;30:325–32. (c) Capasso C, Supuran CT. Bacterial, fungal
and protozoan carbonic anhydrases as drug targets. Expert
Opin Ther Targets 2015;19:1689–704. (d) Vermelho AB, da
Silva CV, Ricci JE, et al. Nanoemulsions of sulfonamide car-
bonic anhydrase inhibitors strongly inhibit the growth of
and characterization.
J
Enzyme Inhib Med Chem
2016;31:689–94. (d) De Simone G, Langella E, Esposito D,
et al. Insights into the binding mode of sulphamates and
sulphamides to hCA II: crystallographic studies and binding
free energy calculations.
J Enzyme Inhib Med Chem
2017;32:1002–11. (e) Di Fiore A, De Simone G, Alterio V,
et al. The anticonvulsant sulfamide JNJ-26990990 and its
S,S-dioxide analog strongly inhibit carbonic anhydrases:
solution and X-ray crystallographic studies. Org Biomol
Chem 2016;14:4853–8.
Trypanosoma cruzi.
J
Enzyme Inhib Med Chem
21. (a) Krall N, Pretto F, Decurtins W, et al. A small-molecule
drug conjugate for the treatment of carbonic anhydrase IX
expressing tumors. Angew Chem Int Ed Engl
2014;53:4231–5. (b) Rehman SU, Chohan ZH, Gulnaz F,
Supuran CT. In-vitro antibacterial, antifungal and cytotoxic
activities of some coumarins and their metal complexes.
J Enzyme Inhib Med Chem 2005;20:333–40. (c) Clare BW,
Supuran CT. Carbonic anhydrase activators. 3: structure–ac-
tivity correlations for a series of isozyme II activators.
J Pharm Sci 1994;83:768–73. (d) Dubois L, Peeters S, Lieuwes
NG, et al. Specific inhibition of carbonic anhydrase IX activity
enhances the in vivo therapeutic effect of tumor irradiation.
Radiother Oncol 2011;99:424–31. (e) Chohan ZH, Munawar
A, Supuran CT. Transition metal ion complexes of Schiff-
bases. Synthesis, characterization and antibacterial proper-
ties. Met Based Drugs 2001;8:137–43. (f) Zimmerman SA,
Ferry JG, Supuran CT. Inhibition of the archaeal b-class (Cab)
and c-class (Cam) carbonic anhydrases. Curr Top Med Chem
2007;7:901–8. (g) De Simone G, Supuran CT. (In)organic
anions as carbonic anhydrase inhibitors. J Inorg Biochem
2012;111:117–29.
2018;33:139–46. (e) de Menezes DR, Calvet CM, Rodrigues
GC, et al. Hydroxamic acid derivatives: a promising scaffold
for rational compound optimization in Chagas disease. J
Enzyme Inhib Med Chem 2016;31:964–73. (f) Nocentini A,
Cadoni R, Dumy P, et al. Carbonic anhydrases from
Trypanosoma cruzi and Leishmania donovani chagasi are
inhibited by benzoxaboroles. J Enzyme Inhib Med Chem
2018;33:286–9.
18. Khalifah RG. The carbon dioxide hydration activity of car-
bonic anhydrase. I. Stop-flow kinetic studies on the native
human isoenzymes B and C. J Biol Chem 1971;246:2561–73.
19. (a) Menchise V, De Simone G, Alterio V, et al. Carbonic anhy-
drase inhibitors: stacking with Phe131 determines active site
binding region of inhibitors as exemplified by the X-ray crys-
tal structure of a membrane-impermeant antitumor sulfona-
mide complexed with isozyme II.
J
Med Chem
2005;48:5721–7. (b) Supuran CT, Mincione F, Scozzafava A,
et al. Carbonic anhydrase inhibitors—part 52. Metal com-
plexes of heterocyclic sulfonamides: a new class of strong
topical intraocular pressure-lowering agents in rabbits. Eur J
Med Chem 1998;33:247–54. (c) Garaj V, Puccetti L, Fasolis G,