7-Aryl-triazolyl-substituted sulfocoumarins are potent, selective inhibitors of the tumor-associated carbonic anhydrase IX and XII

Article abstract of DOI:10.3109/14756366.2015.1115401

Sulfocoumarins behave as interesting inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Here, we report a new series of 7-substituted derivatives which were obtained by the click chemistry approach from 7-propargyloxy-sulfocoumarin and aryl azides incorporating halogens, hydroxy, methoxy and carboxyl moieties in their molecules. The new compounds were screened for the inhibition on four physiologically relevant human CA (hCA) isoforms, the cytosolic hCA I and II and the transmembrane tumor-associated hCA IX and XII. The new compounds did not inhibit the cytosolic isoforms but were low nanomolar inhibitors of the tumor-associated ones hCA IX and XII.

Full text of DOI:10.3109/14756366.2015.1115401

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
7-Aryl-triazolyl-substituted sulfocoumarins
are potent, selective inhibitors of the tumor-
associated carbonic anhydrase IX and XII
Alessio Nocentini, Mariangela Ceruso, Fabrizio Carta & Claudiu T. Supuran
To cite this article: Alessio Nocentini, Mariangela Ceruso, Fabrizio Carta & Claudiu T. Supuran
(2015): 7-Aryl-triazolyl-substituted sulfocoumarins are potent, selective inhibitors of the
tumor-associated carbonic anhydrase IX and XII, Journal of Enzyme Inhibition and Medicinal
Chemistry, DOI: 10.3109/14756366.2015.1115401
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1115401
Published online: 18 Dec 2015.
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
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2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1115401
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RESEARCH ARTICLE
7-Aryl-triazolyl-substituted sulfocoumarins are potent, selective
inhibitors of the tumor-associated carbonic anhydrase IX and XII
Alessio Nocentini1, Mariangela Ceruso2, Fabrizio Carta1,2, and Claudiu T. Supuran1,2
2
1University of Florence, Neurofarba Department, Sesto Fiorentino, Firenze, Italy and University of Florence, Dipartimento di Chimica ‘‘U. Schiff’’,
Laboratorio di Chimica Bioinorganica, Sesto Fiorentino, Firenze, Italy
Abstract
Keywords
Sulfocoumarins behave as interesting inhibitors of the metalloenzyme carbonic anhydrase (CA,
EC 4.2.1.1). Here, we report a new series of 7-substituted derivatives which were obtained by
the click chemistry approach from 7-propargyloxy-sulfocoumarin and aryl azides incorporating
halogens, hydroxy, methoxy and carboxyl moieties in their molecules. The new compounds
were screened for the inhibition on four physiologically relevant human CA (hCA) isoforms, the
cytosolic hCA I and II and the transmembrane tumor-associated hCA IX and XII. The new
compounds did not inhibit the cytosolic isoforms but were low nanomolar inhibitors of the
tumor-associated ones hCA IX and XII.
Carbonic anhydrase, click chemistry, human
carbonic anhydrase IX, human carbonic
anhydrase XII, sulfocoumarin,
tumor-associated isoforms
History
Received 16 October 2015
Accepted 28 October 2015
Published online 16 December 2015
Introduction
The highly selective CA inhibition profiles shown by the
coumarins and sulfocoumarins rely on this particular inhibition
mechanism, which in turn depends from different binding modes of
the hydrolysis products within the CAs catalytic cavities10. Such a
feature is highly desired in the current CAIs research as the main
problem associated to the sulfonamide-type clinically used CAIs is
represented by their indiscriminate inhibition of the hCAs leading
thus to a plethora of side effects10,12–17. Since many CA isoforms
are involved in diverse physio/pathological conditions such as
glaucoma (hCA I, II, IV and XII), edema (hCA II, IV, XIV as the
most important), central nervous system (CNS)-related pathologies
(hCAVII and XIV are particularly involved in the epilepsy) and
tumors (hCA IX and hCA XII are strictly associated with hypoxic
tumors), it is not surprising that CAIs are used in the clinic for some
applications for almost 70 years18–24. Many synthetic efforts have
been made for the development of specific CAIs: in the last 15
years, in addition to the classical ‘‘tail approach’’10–17,25–28 which
is mainly applied to classical sulfonamide inhibitors and their
isosters, novel CAIs scaffolds have been also identified such as the
polyamines29, phenols30–32, dithiocarbamates33–36, xanthates37,
coumarins, thiocoumarins, 2-thioxocoumarins, coumarine
oximes4,38,39. Sulfocoumarins are the latest CAI class identified
and similarly to the coumarins showed the most selective inhibition
1,2-Benzoxathiines-2,2-dioxides, also referred as sulfocoumarins,
were recently validated as a novel class of inhibitors of the zinc
enzyme carbonic anhydrase (CA, EC 4.2.1.1)1. The design of
sulfocoumarins as CA inhibitors (CAIs) was inspired by their
corresponding bioisosters coumarins2–9, whose inhibition mech-
anism relies on the CA-mediated esterase activity. Indeed the
sulfocoumarins, as well as coumarins, undergo a CA-mediated
hydrolysis within the active site cavity with the generation of the
inhibitory species. The inhibition mechanism of such hydrolysis
products is different at the molecular level when compared to the
classical sulfonamide-based CAIs10,11, that coordinate in the
deprotonated form to the zinc metal ion, thus substituting the
fourth ligand represented by a water molecule/hydroxide ion. As
demonstrated by means of X-ray crystallographic and kinetic
studies, the hydrolyzed coumarine derivative (i.e. cinnamic acid)
is placed at the rim of the enzymatic cavity thus occluding it. The
resulting ligand-enzyme complex is stabilized by interactions with
the amino acids present therein that constitute the most variable
region within CA isoforms known to date (15 human CA (hCA)
isoforms are reported)2–8,10. Conversely, 2-hydroxyphenyl-!-ethe-
nyl sulfonic acids which are formed from the original sulfocou-
marins, tightly bind to the zinc-coordinated water molecule by
means of hydrogen bonding, whereas the scaffold of the inhibitor
establishes additional favorable interactions within the cavity
(Figure 1)1.
profiles against the pathologically valuable CA isoforms1,9,40–44
.
In analogy to coumarins, the substitution pattern at the
sulfocoumarin scaffolds strongly influences the potency and
selectivity profile against different hCA isoforms1,40–44. Indeed, in
our previous reports40–43, we investigated large series of
sulfocoumarins bearing the tetrazolyl, triazolyl or aryl/alkyl
moieties at 6 position, which showed low nanomolar hCAIX/
XII inhibitory potencies, without particular effects on the
cytosolic hCAI/II. On the contrary, derivatives bearing small
Address for correspondence: Fabrizio Carta, University of Florence,
Neurofarba Department, Via Ugo Schiff 6, Polo Scientifico, 50019 –
Sesto Fiorentino, Firenze, Italy. Tel: +39-055-4573005. Fax: +39-055-
4573385. E-mail: fabrizio.carta@unifi.it

2
A. Nocentini et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
General synthetic procedure of compounds 7–1445
N
3
R= H; A
= p-F; E
= p-CF ; F
= p-OH; G
= m-Cl; B
3
O
S
O
-
R
m
Br; C
O
O
= p-CO H; H
m-OCH ; D
R
2
3
Cu(0) nanosized, TMACl
H2O/tBuOH 1/1
N
N
O
S
O
N
O
O
R= H; 7
= m-Cl; 8
= p-F; 11
6
= p-CF ; 12
3
-
= p-OH; 13
m
Br; 9
= p-CO H; 14
m-OCH ; 10
2
3
7-Prop-2-ynyloxy-benzo-[e][1,2]-oxathiine 2,2-dioxide
6
(1.0 eq) was added to a suspension of aryl azide A–H (1.1 eq)
in H2O/tBuOH 1/1 (3.5 ml) at room temperature (r.t.), fol-
lowed by addition of copper (0) nanosized (0.1 eq) and
tetramethylammoniun chloride (TMACl) (1.0 eq). The suspension
was stirred at 60 ꢀC until starting materials were consumed
(TLC monitoring), then quenched with H2O (20 ml) and the
formed precipitate was filtered off and washed with H2O.
The solid was dissolved in a minimal amount of acetone; the
obtained solution was filtered through Celite 521Õ and then
concentrated under vacuo to give a residue that was triturated with
Et2O or dichloromethane (DCM) to afford the titled compounds
7–14.
Figure 1. CA inhibition mechanism of sulfocoumarins. (A) The
sulfocoumarin undergoes an enzyme-mediated hydrolysis with the forma-
tion of the trans-2-hydroxy-phenyl-!-ethenylsulfonic acid 3. (B) The
sulfonic acid 3 binds to the hCA II active site, by anchoring to the zinc-
coordinated water molecule. The Zn(II) ion (central larger sphere), its three
His ligands (His94, 96 and 119), the water molecule coordinated to the zinc
(small sphere) as well as active site residues Thr200 and Pro201 involved
Synthesis of 4-(2,2-dioxo-2H-2l6-benzo-[e][1,2]-oxathiin-7-ylox-
in the binding of the hydrolyzed sulfocoumarin are shown, as determined ymethyl)-1-phenyl-1H-[1,2,3]-triazole 7
by X-ray crystallography (PDB file 4BCW)1.
O
S
O
substituents as well as benzyl esters at the 7 position act as low
nanomolar hCAII inhibitors40.
O
O
N
N
N
To date, sulfocoumarins incorporating bulky and flexible
moieties at 7 position were not reported, thus herein we report the
synthesis, characterization and in vitro inhibition profiles against
four relevant hCA isoforms (hCAI, II, IX and XII) of a small
series of 7-substituted sulfocoumarins bearing the aryl-triazolyl
moieties linked through an oxymethylene group, which were
synthesized by means of the click chemistry approach.
7
4-(2,2-Dioxo-2H-2ꢀ6-benzo-[e][1,2]-oxathiin-7-yloxymethyl)-
1-phenyl-1H-[1,2,3]-triazole 7 was obtained according to the
general procedure earlier reported using phenylazide A (1.1 eq),
7-prop-2-ynyloxy-benzo-[e][1,2]-oxathiine 2,2-dioxide 6 (0.05 g,
1.0 eq) in BuOH/H2O (1/1, 3.5 ml), TMACl (1.0 eq) and copper
nanosize (0.1 eq). The reaction mixture was stirred for 7 h to give
the titled compound 7 as a white solid.
t
Materials and methods
4-(2,2-dioxo-2H-2l6-benzo-[e][1,2]-oxathiin-7-yloxymethyl)-1-
phenyl-1H-[1,2,3]triazole 7. Seventy-two percent yield; m.p.
141–143 ꢀC; silica gel TLC Rf 0.39 (EtOAc/n-hexane 50% v/v);
dH (400 MHz, DMSO-d6): 5.41 (s, 2H, CH2), 7.16 (dd, J ¼ 2.4,
8.8, 1H), 7.30 (d, J ¼ 2.4, 1H), 7.37 (d, J ¼ 10.4, 1H), 7.55 (t,
J ¼ 7.6, 1H), 7.67 (m, 4H), 7.95 (d, J ¼ 7.6, 2H), 9.05 (s, 1H); dC
(100 MHz, DMSO-d6): 62.7, 105.6, 113.2, 114.4, 120.3, 121.2,
124.2, 129.8, 130.7, 132.2, 137.4, 137.4, 143.9, 153.2, 161.9; m/z
(ESI positive) 356.0 [M + H]+.
Chemistry
Anhydrous solvents and all reagents were purchased from Sigma-
Aldrich (St. Louis, MO), Alfa Aesar (Haverhill, MA) and TCI
(Eschborn, Germany). All reactions involving air- or moisture-
sensitive compounds were performed under a nitrogen atmos-
phere using dried glassware and syringe techniques to transfer
solutions. Nuclear magnetic resonance (1H-NMR, 13C-NMR)
spectra were recorded using a Bruker Avance III 400 MHz
spectrometer in deuterated dimethyl sulfoxide (DMSO-d6).
Chemical shifts are reported in parts per million and the coupling
constants (J) are expressed in Hertz (Hz). Splitting patterns are
designated as follows: s, singlet; d, doublet; sept, septet; t, triplet;
q, quartet; m, multiplet; brs, broad singlet; dd, doublet of
doublets; appt, apparent triplet; appq, apparent quartet. The
assignment of exchangeable protons (OH and NH) was confirmed
by the addition of D2O. Analytical thin-layer chromatography
(TLC) was carried out on Merck silica gel F-254 plates. Flash
chromatography purifications were performed on Merck Silica gel
60 (230–400 mesh American Society for Testing and Materials
(ASTM)) as the stationary phase and ethyl acetate/n-hexane were
used as eluents. Melting points (m.p.) were measured in open
capillary tubes with a Gallenkamp MPD350.BM3.5 apparatus and
are uncorrected. All compounds were495% pure by high
performance liquid chromatography (HPLC).
Synthesis of 1-(3-chloro-phenyl)-4-(2,2-dioxo-2H-2l6-
benzo-[e][1,2]-oxathiin-7-yloxymethyl)-1H-[1,2,3]-triazole 8
Cl
O
S
O
O
N
N
O
N
8
1-azido-3-chlorobenzene
ynyloxy-benzo-[e][1,2]-oxathiine 2,2-dioxide 6 (0.05 g, 1.0 eq)
B
(1.1 eq) and 7-prop-2-
t
were dissolved in BuOH/H2O 1/1 (3.5 ml) and then TMACl
(1.0 eq) and copper (0) nanosize (10% mol) were added. The
mixture was stirred at 60 ꢀC for 3.5 h, and then treated as
described in general procedure earlier reported to afford 8 as a
white solid.

DOI: 10.3109/14756366.2015.1115401
Sulfocoumarins
3
1-(3-Chloro-phenyl)-4-(2,2-dioxo-2H-2l6-benzo-[e][1,2]-
oxathiin-7-yloxymethyl)-1H-[1,2,3]-triazole 8. Seventy-one per- ymethyl)-1-(4-fluoro-phenyl)-1H-[1,2,3]-triazole 11
Synthesis of 4-(2,2-dioxo-2H-2l6-benzo-[e][1,2]-oxathiin-7-ylox-
cent yield; m.p. 174–176 ꢀC; silica gel TLC Rf 0.26 (EtOAc/n-
hexane 50% v/v); dH (400 MHz, DMSO-d6): 5.41 (s, 2H, CH2),
7.16 (dd, J ¼ 2.4, 8.8, 1H), 7.28 (d, J ¼ 2.4, 1H), 7.37 (d,
J ¼ 10.4, 1H), 7.66 (m, 4H), 7.97 (d, J ¼ 8.8, 1H), 8.10 (s, 1H),
9.11 (s, 1H); dC (100 MHz, DMSO-d6): 62.6, 105.6, 113.2,
114.4, 119.7, 120.3, 121.0, 124.4, 129.6, 132.2, 132.6, 135.2,
137.4, 138.5, 144.1, 153.2, 161.8; m/z (ESI positive) 390.0
[M + H]+.
O
S
O
F
O
O
N
N
N
11
1-Azido-4-fluorobenzene E (1.1 eq) and 7-prop-2-ynyloxy-
benzo-[e][1,2]-oxathiine 2,2-dioxide 6 (0.05 g, 1.0 eq) were
t
dissolved in BuOH/H2O 1/1 (3.5 ml) and then TMACl (1.0 eq)
Synthesis of 1-(3-bromo-phenyl)-4-(2,2-dioxo-2H-2l6-benzo-
[e][1,2]-oxathiin-7-yloxymethyl)-1H-[1,2,3]-triazole 9
and copper (0) nanosize (10% mol) were added. The mixture was
stirred at 60 ꢀC for 2.5 h and then treated as described in general
procedure earlier reported to afford 11 as a white solid.
Br
O
S
4-(2,2-Dioxo-2H-2l6-benzo-[e][1,2]-oxathiin-7-yloxymethyl)-1-
(4-fluoro-phenyl)-1H-[1,2,3]-triazole 11. Sixty-three percent
yield; m.p. 168–170 ꢀC; silica gel TLC Rf 0.33 (EtOAc/n-
hexane 50% v/v); dH (400 MHz, DMSO-d6): 5.41 (s, 2H, CH2),
7.16 (dd, J ¼ 2.4, 8.8, 1H), 7.29 (d, J ¼ 2.4, 1H), 7.37 (d, J ¼ 10.4,
1H), 7.51 (t, J ¼ 8.8, 2H), 7.69 (m, 2H), 8.00 (m, 2H), 9.01 (s,
1H); dF(376 MHz, DMSO-d6): ꢁ112.92 (s, 1F); dC (100 MHz,
DMSO-d6): 62.7, 105.6, 113.2, 114.4, 117.7 (d, J2CF ¼ 23.0),
120.3, 123.6 (d, J1CF ¼ 8.9), 124.5, 132.2, 134.0, 137.4, 144.0,
153.2, 161.9, 162.7 (d, J1CF ¼ 245.2); m/z (ESI positive) 374.0
[M + H]+.
O
O
N
N
O
N
9
1-Azido-3-bromobenzene C (1.1 eq) and 7-prop-2-ynyloxy-
benzo-[e][1,2]-oxathiine 2,2-dioxide 6 (0.05 g, 1.0 eq) were
t
dissolved in BuOH/H2O 1/1 (3.5 ml) and then TMACl (1.0 eq)
and copper (0) nanosize (10% mol) were added. The mixture was
stirred at 60 ꢀC for 3.5 h and then treated as described in general
procedure earlier reported to afford 9 as a white solid.
1-(3-Bromo-phenyl)-4-(2,2-dioxo-2H-2l6-benzo-[e][1,2]-
Synthesis of 4-(2,2-dioxo-2H-2l6-benzo-[e][1,2]-oxathiin-7-
yloxymethyl)-1-(4-trifluoromethyl-phenyl)-1H-[1,2,3]-triazole 12
oxathiin-7-yloxymethyl)-1H-[1,2,3]-triazole
9. Seventy-nine
percent yield; m.p. 171–173 ꢀC; silica gel TLC Rf 0.49
(EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 5.41 (s,
2H, CH2), 7.16 (dd, J ¼ 2.4, 8.8, 1H), 7.30 (d, J ¼ 2.4, 1H), 7.37
(d, J ¼ 10.4, 1H), 7.61 (t, J ¼ 8.4, 1H), 7.69 (m, 2H), 7.76 (d,
J ¼ 8.4, 1H), 8.01 (d, J ¼ 8.4, 1H), 8.23 (t, J ¼ 2.0, 1H), 9.11 (s,
1H); dC (100 MHz, DMSO-d6): 62.7, 105.6, 113.3, 114.4, 120.2,
120.4, 123.4, 123.8, 124.4, 132.2, 132.6, 132.8, 137.4, 138.6,
144.1, 153.2, 161.9; m/z (ESI positive) 434.0 [M + H]+.
O
S
O
F3C
O
O
N
N
N
12
1-Azido-4-trifluoromethylbenzene F (1.1 eq) and 7-prop-2-
ynyloxy-benzo-[e][1,2]-oxathiine 2,2-dioxide 6 (0.05 g, 1.0 eq)
Synthesis of 4-(2,2-Dioxo-2H-2l6-benzo-[e][1,2]-oxathiin-7-
yloxymethyl)-1-(3-methoxy-phenyl)-1H-[1,2,3]-triazole 10
t
were dissolved in BuOH/H2O 1/1 (3.5 ml) and then TMACl (1.0
eq) and copper (0) nanosize (10% mol) were added. The mixture
was stirred at 60 ꢀC for 5 h and then treated as described in general
procedure earlier reported to afford 12 as a white solid.
MeO
O
S
O
O
N
N
O
N
4-(2,2-Dioxo-2H-2l6-benzo-[e][1,2]-oxathiin-7-yloxymethyl)-1-
(4-trifluoromethyl-phenyl)-1H-[1,2,3]-triazole 12. Seventy-four
percent yield; m.p. 210–212 ꢀC; silica gel TLC Rf0.40 (EtOAc/n-
hexane 50% v/v); dH (400 MHz, DMSO-d6): 5.43 (s, 2H, CH2),
7.16 (dd, J ¼ 2.4, 8.8, 1H), 7.29 (d, J ¼ 2.4, 1H), 7.37 (d, J ¼ 10.4,
1H), 7.69 (m, 2H), 8.04 (d, J ¼ 8.4, 2H), 8.22 (d, J ¼ 8.4, 2H),
9.19 (s, 1H); dC (100 MHz, DMSO-d6): 62.7, 105.6, 113.3, 114.4,
120.4, 121.7, 124.5, 124.8 (d, J1CF ¼ 270.4), 128.2, 129.8 (d,
J2CF ¼ 32.0), 132.2, 137.4, 140.3, 144.4, 153.3, 161.9; m/z (ESI
positive) 324.0 [M + H]+.
10
4-(2,2-dioxo-2H-2ꢀ6-benzo-[e][1,2]-oxathiin-7-yloxymethyl)-
1-(3-methoxy-phenyl)-1H-[1,2,3]-triazole 10 was obtained
according the general procedure earlier reported using 1-azido-
3-methoxybenzene D (1.1eq), 7-prop-2-ynyloxy-benzo-[e][1,2]-
t
oxathiine 2,2-dioxide 6 (0.05 g, 1.0 eq) in BuOH/H2O (1/1,
3.5 ml), TMACl (1.0 eq) and copper nanosize (0.1 eq). The
reaction mixture was stirred for 6.5 h to give the titled compound
10 as a white solid.
Synthesis of 4-(2,2-dioxo-2H-2l6-benzo-[e][1,2]-oxathiin-7-ylox-
ymethyl)-1-(4-hydroxy-phenyl)-1H-[1,2,3]-triazole 13
4-(2,2-Dioxo-2H-2l6-benzo-[e][1,2]-oxathiin-7-yloxymethyl)-1-
(3-methoxy-phenyl)-1H-[1,2,3]-triazole 10. Sixty-eight percent
yield; m.p. 129–131 ꢀC; silica gel TLC Rf 0.40 (EtOAc/n-hexane
50% v/v); dH (400 MHz, DMSO-d6): 3.90 (s, 3H, CH3), 5.41 (s,
2H, CH2), 7.11 (d, J ¼ 7.2, 1H), 7.16 (dd, J ¼ 2.4, 8.8, 1H), 7.30
(d, J ¼ 2.4, 1H), 7.37 (d, J ¼ 10.4, 1H), 7.54 (m, 3H), 7.69 (m,
2H), 9.06 (s, 1H); dC (100 MHz, DMSO-d6): 56.6, 62.7, 105.6,
106.8, 113.1, 113.2, 114.3, 115.5, 120.3, 124.3, 131.8, 132.1,
137.4, 138.5, 143.9, 153.2, 161.1, 161.9; m/z (ESI positive) 386.0
[M + H]+.
O
S
HO
O
O
N
N
O
N
13
1-Azido-4-hydroxybenzene G (1.1 eq) and 7-prop-2-yny-
loxy-benzo-[e][1,2]-oxathiine 2,2-dioxide 6 (0.05 g, 1.0 eq)

4
A. Nocentini et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
t
were dissolved in BuOH/H2O 1/1 (3.5 ml) and then TMACl Synthesis of phenylazides A–H46
(1.0 eq) and copper (0) nanosize (10% mol) were added. The
mixture was stirred at 60 ꢀC for 4 h and then treated as
described in general procedure earlier reported to afford 13 as
a white solid.
NH
2
N
3
R= H; A
= p-F; E
NaNO , NaN
= p-CF ; F
= m-Cl; B
2
3
3
-
= p-OH; G
m Br; C
R
R
HCl 4M (aq)
= p-CO H; H
m-OCH ; D
2
3
4-(2,2-Dioxo-2H-2l6-benzo-[e][1,2]-oxathiin-7-yloxymethyl)-1-
(4-hydroxy-phenyl)-1H-[1,2,3]-triazole 13. Eighty-two percent
yield; m.p. 244–246 ꢀC; silica gel TLC Rf 0.35 (EtOAc/n-hexane
50% v/v); dH (400 MHz, DMSO-d6): 5.38 (s, 2H, CH2), 6.98 (d,
J ¼ 8.8, 2H), 7.15 (dd, J ¼ 2.4, 8.8, 1H), 7.28 (d, J ¼ 2.4, 1H),
7.37 (d, J ¼ 10.4, 1H), 7.69 (m, 4H), 8.84 (s, 1H), 10.00 (s, 1H,
exchange with D2O, OH); dC (100 MHz, DMSO-d6): 62.7, 105.6,
113.2, 114.3, 117.0, 120.3, 123.0, 124.1, 129.6, 132.1, 137.42,
143.5, 153.2, 158.8, 161.9; m/z (ESI positive) 372.0 [M + H]+.
The proper aniline (0.5 g, 1.0eq) was dissolved in a 4 M HCl
aqueous solution (5 ml) at 0 ꢀC. NaNO2 (1.2 eq) was slowly added and
the resulting solution was stirred at the same temperature for 0.5 h.
Then NaN3 (1.5 eq) was added portion-wise and the mixture was
stirred at r.t. for 0.5 h. The reaction mixture was filtered off or extracted
with Et2O (2ꢂ 15 ml) and the combined organic layers were dried over
Na2SO4, filtered off and the solvent evaporated in vacuo to afford the
corresponding phenylazide which was used without further purification.
Synthesis of phenylazide A
Synthesis of 1-(4-carboxy-phenyl)-4-(2,2-dioxo-2H-2l6-benzo-
[e][1,2]-oxathiin-7-yloxymethyl)-1H-[1,2,3]-triazole 14
N
3
O
O
S
O
O
N
N
O
HO
A
N
14
Phenylazide A was obtained according to the general proced-
ure reported earlier.
1-azido-carboxybenzene H (1.1 eq) and 7-prop-2-ynyloxy-
benzo-[e][1,2]-oxathiine 2,2-dioxide 6 (0.05 g, 1.0 eq) were
Phenylazide A. Sixty percent yield; silica gel TLC Rf 0.76
(EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-d6):7.12 (d,
J ¼ 7.6, 2H), 7.20 (t, J ¼ 7.6, 1H), 7.42 (t, J ¼ 7.6, 2H); dC
(100 MHz, DMSO-d6): 120.0, 126.1, 131.0, 140.3.
t
dissolved in BuOH/H2O 1/1 (3.5 ml) and then TMACl (1.0 eq)
and copper (0) nanosize (10% mol) were added. The mixture was
stirred at 60 ꢀC for 12.5 h and then treated as described in general
procedure earlier reported to afford 14 as a white solid.
Experimental in agreement with reported data46.
1-(4-Carboxy-phenyl)-4-(2,2-dioxo-2H-2l6-benzo-[e][1,2]-
Synthesis of 3-chlorophenylazide B
oxathiin-7-yloxymethyl)-1H-[1,2,3]-triazole
14. Twenty-two
percent yield; m.p.4300 ꢀC; silica gel TLC Rf 0.42 (MeOH/
DCM 10% v/v); dH (400 MHz, DMSO-d6): 5.43 (s, 2H, CH2), 7.17
(dd, J ¼ 2.4, 8.8, 1H), 7.30 (d, J ¼ 2.4, 1H), 7.38 (d, J ¼ 10.4, 1H),
7.69 (m, 2H), 8.11 (d, J ¼ 8.4, 2H), 8.19 (d, J ¼ 8.4, 2H), 9.16 (s,
1H), 13.31 (bs, 1H, exchange with D2O, COOH); m/z (ESI
positive) 400.0 [M + H]+.
N
3
Cl
B
3-Chlorophenylazide B was obtained according to the general
procedure reported earlier.
Synthesis 7-prop-2-ynyloxy-benzo-[e][1,2]-oxathiine 2,
2-dioxide 6
3-Chlorophenylazide B. Eighty-six percent yield; silica gel TLC
Rf 0.84 (EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-
d6):7.14 (dd, J ¼ 2.2, 8.0, 1H), 7.24 (t, J ¼ 2.2, 1H), 7.30 (d,
J ¼ 2.2, 1H), 7.46 (t, J ¼ 8.0, 1H); dC (100 MHz, DMSO-d6):
118.9, 120.1, 126.0, 132.4, 135.1, 142.2.
O
S
O
O
O
6
Experimental in agreement with reported data47.
Propargyl bromide (1.2 eq) was added to a suspension of
compound 5 (0.5 g, 1.0 eq) and K2CO3 (2.0 eq) in dry DMF
(4 ml) under a nitrogen atmosphere and the mixture was stirred
at r.t. for 2 h. The reaction mixture was quenched with H2O
(20 ml) and extracted with EtOAc (3 ꢂ 15 ml). The organic layer
was washed with brine (4 ꢂ 15 ml), dried over Na2SO4, filtered
off and concentrated under vacuo to give a residue that was
triturated with Et2O to afford the titled compound 6 as a white
powder.
Synthesis of 3-bromophenylazide C
N
3
Br
C
3-Bromophenylazide C was obtained according to the general
procedure reported earlier.
7-Prop-2-ynyloxy-benzo-[e][1,2]-oxathiine 2,2-dioxide 6. Fifty-
four percent yield; m.p. 150–151 ꢀC; silica gel TLC Rf 0.83
(EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-d6): 3.70 (t,
J ¼ 2.4, 1H), 4.98 (d, J ¼ 2.4, 2H), 7.07 (dd, J ¼ 2.4, 8.4, 1H),
7.15 (d, J ¼ 2.4, 1H), 7.37 (d, J ¼ 10.4, 1H), 7.68 (m, 2H); dC
(100 MHz, DMSO-d6): 57.21, 79.28, 80.03, 105.70, 113.43,
114.42, 120.51, 132.11, 137.40, 153.07, 161.03.
3-Bromophenylazide C. Eighty-eight percent yield; silica gel
TLC Rf 0.83 (EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-
d6):7.18 (dt, J ¼ 1.6, 7,2, 1H), 7.38 (m, 3H); dC (100 MHz,
DMSO-d6): 119.3, 122.8, 123.5, 128.9, 132.7, 142.3.
Experimental in agreement with reported data47.

DOI: 10.3109/14756366.2015.1115401
Sulfocoumarins
5
Synthesis of 3-methoxyphenylazide D
4-hydroxyphenylazide G was obtained according to the
general procedure reported earlier.
N
3
4-Hydroxyphenylazide G. Forty-six percent yield; silica gel
TLC Rf 0.84 (EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-
d6): 6.84 (d, J ¼ 8.8, 2H), 6.97 (d, J ¼ 8.8, 2H), 9.56 (bs, 1H,
exchange with D2O, OH); dC (100 MHz, DMSO-d6): 117.5, 121.1,
130.6, 156.0.
O
D
Experimental in agreement with reported data49.
3-Methoxyphenylazide D was obtained according to the
general procedure reported earlier.
Synthesis of 4-azidobenzoic acid H
3-Methoxyphenylazide D. Seventy-four percent yield; silica gel
TLC Rf 0.78 (EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-
d6): 3.80 (s, 3H, CH3), 6.66 (t, J ¼ 2.4, 1H), 6.74 (ddd, J ¼ 0.8,
2.4, 8.2, 1H), 6.81 (ddd, J ¼ 0.8, 2.4, 8.2, 1H), 7.36 (t, J¼ 8.2,
1H); dC (100 MHz, DMSO-d6): 56.3, 105.7, 112.0, 112.1, 131.7,
141.5, 161.5.
N
3
OH
O
Experimental in agreement with reported data46.
H
4-azidobenzoic acid H was obtained according to the general
procedure reported earlier. 4-aminobenzoic acid was treated with
NaNO2 and NaN3 in a 4 M HCl aqueous solution and the formed
precipitate was filtered-off to afford the title compound H as a
yellow solid.
Synthesis of 4-fluorophenylazide E
N
3
4-Azidobenzoic acid H. Seventy-three percent yield; m.p. 188–
190 ꢀC dec; silica gel TLC Rf 0.71 (EtOAc/n-hexane 50% v/v); dH
(400 MHz, DMSO-d6): 7.25 (d, J ¼ 8.4, 2H), 7.99 (d, J ¼ 8.4, 2H),
13.02 (bs, 1H, exchange with D2O, COOH); dC (100 MHz,
DMSO-d6): 120.2, 128.3, 132.2, 144.9, 167.6. Experimental in
agreement with reported data50.
F
E
4-Fluorophenylazide E was obtained according to the general
procedure reported earlier.
4-Fluorophenylazide E. Eighty-nine percent yield; silica gel
TLC Rf 0.79 (EtOAc/n-hexane 50% v/v); dH (400 MHz, DMSO-
d6): 67.19 (m, 2H), 7.29 (t, J ¼ 8.8, 2H); dF (376 MHz, DMSO-
d6): ꢁ117.77 (s, 1F); dC (100 MHz, DMSO-d6): 117.7 (d,
J2 ¼ 23), 121.8 (d, J3 ¼ 9.0), 136.4, 160.3 (d, J1 ¼ 241.0).
CA inhibition
An applied photophysics stopped-flow instrument has been used
for assaying the CA catalyzed CO2 hydration activity51. Phenol
red (at a concentration of 0.2 mm) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mm
Hepes (pH 7.5) as buffer, and 20 mm Na2SO4 (for maintaining
constant the ionic strength), following the initial rates of the CA-
catalyzed CO2 hydration reaction for a period of 10–100 s. The
CO2 concentrations ranged from 1.7 to 17 mm for the 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
uncatalyzed rates were determined in the same manner and
subtracted from the total observed rates. Stock solutions of
inhibitor (0.1 mm) were prepared in distilled-deionized water and
CF
CF
CF
Experimental in agreement with reported data47.
Synthesis of 4-trifluoromethyl-phenylazide F
N
3
CF
3
F
4-Trifluoromethylphenylazide F was obtained according to the
general procedure reported earlier.
Table 1. Inhibition data of human CA isoforms hCA I, II, IX and XII with
sulfocoumarins 7–14 reported here and the standard sulfonamide inhibitor
acetazolamide (AAZ) by a stopped-flow CO2 hydrase assay51
.
4-Trifluoromethylphenylazide F. Sixty-seven percent yield; silica
gel TLC Rf 0.84 (EtOAc/n-hexane 50% v/v); dH (400MHz, DMSO-
d6): 7.35 (d, J ¼ 8.8, 2H), 7.78 (d, J ¼ 8.8, 2H); dF(376MHz,
DMSO-d6):ꢁ56.18 (s, 3F); dC (100 MHz, DMSO-d6): 120.8, 125.0
(d, J1 ¼ 269.6), 126.2 (q, J2 ¼ 32.0), 130.0, 144.7.
KI (nM)*
Compound
R
–
hCAI
hCA II
hCA IX
hCA XII
CF
CF
Experimental in agreement with reported data48.
7
8
9
410 000 410 000
2301 410 000
3-Br 410 000 410 000
25.3
25.6
36.5
24.1
26.8
19.0
19.6
19.2
25
4.5
4.5
5.5
4.3
5.5
5.2
19.1
4.6
5.7
3-Cl
Synthesis of 4-hydroxy-phenylazide G
10
11
12
13
14
AAZ
3-OCH3 410 000 410 000
4-F
984 410 000
4-CF3 410 000 410 000
4-OH 410 000 410 000
4-COOH 410 000 410 000
N
3
–
250
12
OH
*Mean from three different assays, by a stopped-flow technique (errors
were in the range of 5–10% of the reported values).
G

6
A. Nocentini et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
N3
= p-F; E
= p-CF3; F
= p-OH; G
R = H; A
O
S
O
= m-Cl; B
= m- Br; C
= m-OCH3; D
O
O
R
R
O
Br
K2CO3
DMF
= p-CO H; H
N
N
2
N
ref. 40
H
O
S
O
Cu(0) nanosized, TMACl
H2O/tBuOH 1/1
O
S
O
HO
O
O
O
HO
OH
= p-F; 11
R = H; 7
= p-CF3; 12
= p-OH; 13
= m-Cl; 8
= m- Br; 9
6
4
5
= p-CO2H; 14
= m-OCH3; 10
Scheme 1. Synthesis of 7-substituted sulfocoumarins 7–14.
dilutions up to 0.01 nm were done thereafter with the assay buffer.
Inhibitor and enzyme solutions were preincubated together for
15 min to 6 h at room temperature (15 min) or 4 ꢀC (6 h) prior to
assay, in order to allow for the formation of the E-I complex. Data
from Table 1 were obtained after 6 h incubation of enzyme and
inhibitor, as for the sulfocoumarins and coumarins reported
earlier1–4,7. The inhibition constants were obtained by non-linear
least-squares methods using PRISM 3 and the Cheng–Prusoff
considering that all these compounds showed similar
enzyme affinities. However the data we reported clearly
showed that the nature of the substitution pattern -R on the
phenyl moiety has a weak influence on the inhibitory
properties, whereas the substitution itself within the
sulfocoumarin scaffold at 7 position plays a pivotal role
addressing the selectivity profiles.
equation, as reported earlier2,5, and represent the mean from at Chemistry
least three different determinations. All CA isoforms were
recombinant ones obtained in-house as reported earlier52–56
The general strategy of Zabulovski’s group1,9 for the preparation
of 6-substituted sulfocoumarins was recently validated by us for
the synthesis of 7-substituted such derivatives. As reported40, the
regioselective protection of the commercially available 2,4-
dihydroxybenzaldehyde 4, followed by intramolecular cyclization
and aromatization, afforded the desired 7-hydroxysulfocoumarin
5, which finally was converted to the derivative 6 through reaction
with propargyl bromide (Scheme 1). Then, 6 was reacted with
various freshly prepared aryl azides A–H in the presence of
copper (0) nanosized as catalyst45 to afford the desired 1,2,3-
triazolyl derivatives 7–14 (Scheme 1).
.
Results and discussion
CA inhibition
Sulfocoumarins 7–14 were screened in vitro for the inhibition
against four physiologically relevant hCA isoforms, the cytosolic
hCAI and II and the trans-membrane tumor-associated hCA IX
and XII10,12,24,57–60. Table 1 shows the inhibition data obtained
and compared to the standard clinically used sulfonamide
acetazolamide (AAZ) after a period of incubation of 6 h of the
enzyme and inhibitors. Noteworthy the assay inhibition performed
within the usual 15 min incubation period (as for the sulfona-
mides)51 led to the very weak inhibition constants (data not
shown). For this reason, herein is reported a 6 h incubation time
instead.
Conclusion
Herein, we report a series of 7-substituted sulfocoumarins bearing
the 1,2,3-triazolyl moieties via a CH2O linker obtained by the
click chemistry approach. Unlike 7-substituted sulfocoumarins
investigated earlier,40 which were potent and selective hCA II
inhibitors and ineffective as hCA I, IX and XII inhibitors,
compounds from this new series exhibit effective and selective
inhibitory properties for the tumor-associated isoforms over hCA
IX and XII.
As hCA IX and hCA XII were recently validated as antitumor/
antimetastatic drug targets, with one inhibitor in Phase I clinical
trials61, this prodrug, isoform-selective CAIs, reported here might
be considered of interest for such biomedical applications.
The following structure-activity relationship (SAR) should be
noted:
(i) According to our previous reports40–44, isoform hCA I was
not or was poorly inhibited by sulfocoumarins 7–14.
Compounds 8 and 11 are high micromolar inhibitors,
whereas the remaining ones did not significantly inhibit
the enzyme below 10 mm inhibitor concentration.
(ii) The inhibition profile shown by sulfocoumarins 7–14 on the
remaining three CA isoforms was quite surprising and
unexpected. Indeed the 7-substituted sulfocoumarins ear-
lier40 reported were effective and selective inhibitors against
the hCA II over the tumor-associated isoforms IX and XII.
Surprisingly derivatives 7–14 herein reported did not exhibit
any activity against the cytosolic isoform hCA II and were
low nanomolar inhibitors against the transmembrane iso-
forms IX and XII (Table 1). Such a behavior might be
ascribed that the previously synthesized derivatives incor-
porated small moieties or benzyl esters at the 7 position,
whereas sulfocoumarins 7–14 bear flexible and bulky aryl-
triazolyl moieties via a CH2O linker.
(iii) As mentioned, the transmembrane isoforms were strongly
inhibited by the new sulfocoumarins 7–14 here reported,
with KI values spanning between 19.0 and 36.5 nM for the
hCA IX and 4.3–19.1 nM for the hCA XII, respectively.
These values were comparable with the clinically used
sulfonamide AAZ (KI: 2.5–5.7 nM). The definition of a
proper SAR for hCA IX and hCA XII is not feasible,
Declaration of interest
This work was financed in part by a Marie Curie ITN FP7 project
(Dynano). The authors report no conflicts of interest. The authors
alone are responsible for the content and writing of this article.
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