Fluorescent sulfonamide carbonic anhydrase inhibitors incorporating 1,2,3-triazole moieties: Kinetic and X-ray crystallographic studies

Article abstract of DOI:10.1016/j.bmc.2015.11.031

Fluorescent sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) were essential for demonstrating the role played by the tumor-associated isoform CA IX in acidification of tumors, cancer progression towards metastasis and for the development

Full text of DOI:10.1016/j.bmc.2015.11.031

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluorescent sulfonamide carbonic anhydrase inhibitors
incorporating 1,2,3-triazole moieties: Kinetic and X-ray
crystallographic studies
Fabrizio Carta a,b, Marta Ferraroni a, Andrea Scozzafava a, Claudiu T. Supuran a,b,
⇑
a Università degli Studi di Firenze, Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy
b Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche, Polo Scientifico, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorescent sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) were essential for
demonstrating the role played by the tumor-associated isoform CA IX in acidification of tumors, cancer
progression towards metastasis and for the development of imaging and therapeutic strategies for the
management of hypoxic tumors which overexpress CA IX. However, the presently available such com-
pounds are poorly water soluble which limits their use. Here we report new fluorescent sulfonamides
7, 8 and 10 with increased water solubility. The new derivatives showed poor hCA I inhibitory properties,
but were effective inhibitors against the hCA II (KIs of 366–127 nM), CA IX (KIs of 8.1–36.9 nM), CA XII (KIs
of 4.1–20.5 nM) and CA XIV (KIs of 12.8–53.6 nM). A high resolution X-ray crystal structure of one of these
compounds bound to hCA II revealed the factors associated with the good inhibitory properties.
Furthermore, this compound showed a three-fold increase of water solubility compared to a similar
derivative devoid of the triazole moiety, making it an interesting candidate for ex vivo/in vivo studies.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 31 October 2015
Revised 21 November 2015
Accepted 24 November 2015
Available online xxxx
Keywords:
Carbonic anhydrase
Sulfonamide
Fluorescence
Click chemistry
1,2,3-Triazole
1. Introduction
in principle can be achieved in several ways: either by changing
the linker between the benzenesulfonamide head and the
Fluorescent sulfonamide carbonic anhydrase (CA, EC 4.2.1.1)
inhibitors (CAIs)1–3 such as compounds A and B reported earlier
by our group,4 were essential for demonstrating the role played
by the tumor-associated isoform CA IX in acidification of tumors,5
cancer progression towards metastasis,6 and for the development
of imaging7 and therapeutic strategies8 for the management of
hypoxic tumors which overexpress high amounts of CA IX.9 Indeed,
starting from such proof-of-concept experiments,5,7,8 many inter-
esting, CA IX-selective inhibitors were developed,10,11 with one
such compound, SLC-0111 currently in advanced Phase I clinical tri-
als for the treatment of hypoxic, metastatic solid tumors (Fig. 1).12
However one of the main drawbacks associated with the previ-
ously fluorescent sulfonamide CAIs, that is, compounds A and B, is
related to their relatively low water solubility at pH values in the
range of 6.5–7.4.4 This limits their extensive use in many biological
situations in which working with higher concentrations of inhibi-
tor is required. As a consequence, there is urgent need to develop
fluorescent sulfonamides with increased water solubility, which
fluorescent tail present in A and B, or by changing the nature of
the fluorescent moiety to incorporate hydrophilic moieties, not
present in the fluorescein fragment. Herein we report new com-
pounds 7, 8 and 10 obtained using both synthetic strategies men-
tioned above, their inhibition potencies and selectivity profiles
against the physiologically relevant human (h) CA isoforms such
as hCA I, II, IX, XII and XIV, as well an X-ray crystallographic
structure at 1.3 Å resolution of one selected compound in adduct
with the physiologically dominant isoform hCA II.
2. Results and discussion
2.1. Drug design and chemistry
The first synthetic approach used here consisted of replacing
the alkyl spacers between the benzenesulfonamide head and the
thioureido-fluorescent moiety (methylene or ethylene for A and
B, respectively) by a 1,2,3-triazolyl spacer, which due to the pres-
ence of the heterocyclic ring system rich in heteroatoms should
increase water solubility. Since the click chemistry methodology13
has been successfully applied for obtaining large series of
sulfonamide, sulfocoumarin or coumarin CAIs,14–16 also in this case
was the preferred choice.
⇑
Corresponding author at: Università degli Studi di Firenze, Dipartimento di
Chimica, Laboratorio di Chimica Bioinorganica, Via della Lastruccia 3, 50019 Sesto
Fiorentino, Florence, Italy. Tel.: +39 055 4573005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
http://dx.doi.org/10.1016/j.bmc.2015.11.031
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Carta, F.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.11.031

2
F. Carta et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
SO2NH2
SO2NH2
HN
HN
HN
S
SO2NH2
HN
HN
HN
S
O
CO2H
CO2H
F
O
HO
O
HO
O
O
SLC-0111
B
A
Figure 1. Structures of fluorescent sulfonamides A, B and the advanced Phase I SLC-0111.
O
N
H
O
NH HCl
2
N
N
N
3
N
N
O
Cu (0) nanosized, TMACl
HCl aq.
N
N
O
H O/tBuOH 1/1
2
NH
SO NH
2
2
SO NH
2
2
SO NH
2 2
4a= 4-amino
4b= 3-amino
3a,b
5a= 4-amino
5b= 3-amino
1
S
C
N
S
C
N
N
N
N
N
HN
HN
N
N
HN
S
HN
S
SO2NH2
Et3N
DMF
5a
Et3N
DMF
5b
CO2H
CO2H
CO2H
CO2H
SO2NH2
HO
O
O
HO
O
O
HO
O
O
HO
O
O
8
7
6
6
S
C
N
N
HN
HN
N
N
S
SO2NH2
Et3N
DMF
5b
CO2H
CO2H
Cl
FC9-395A
Cl
N
O
N
N
O
N
10
9
Scheme 1. Synthesis of fluorescent-tagged compounds 7, 8 and 10.
The 1,2,3-triazolyl-benzenesulfonamide key intermediates 5a
6-hydroxy-xanthen-3-one moiety from fluorescein by the
(6-dimethylamino-xanthen-3-ylidene)-dimethyl-ammonium pre-
sent in 10, is supposed to increase the water solubility as well as
the membrane impermeability of compound 10 compared to the
structurally-related derivative 8 (Scheme 1). The synthesis of 10
was achieved again by using the amino sulfonamide 5b and the
fluorescent isothiocyanate 9 (see Section 4 for details).
We have compared the phosphate buffer (pH 7.4) solubility of
A, one of the most investigated fluorescent CAI for a variety of
in vitro and in vivo studies,4–9 with compound 7 designed here.
A reported a solubility of 1.2 mg/mL whereas 7 had a more than
3-fold increased solubility of 3.8 mg/mL in the same conditions
(25 °C, pH 7.4 phosphate buffer).
and 5b were prepared by routine procedures as outlined in
Scheme 1. The propargylamine Boc-protected 1 was reacted in the
presence of copper(0) nanosized catalysts with freshly prepared
azidobenzenesulfonamides 3a and 3b to afford the desired inter-
mediates 5a and 5b upon deprotection of the amino moiety with
aqueous 12 M hydrochloric acid. These amines were then coupled
with fluorescein isothiocyanate (FITC) leading to the thioureas 7
and 8, which are analogs of the leads A and B, but incorporate the
water-solubilizing triazole moieties in their molecule (Scheme 1).
The alternative procedure was to replace the fluorescein
moiety present in A and B by a fluorescent tag which promotes a
better water solubility itself. Indeed, the replacement of the
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F. Carta et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
2.2. Carbonic anhydrase inhibition
(iii) hCA IX, the target isoform for anticancer CAIs,4–8 was well
inhibited by the compounds reported here, which showed a
similar activity with the leads A, B. The inhibition constants
ranged between 8.1 and 36.9 nM, with the best inhibitor
being the sulfanilamide derivative 7 (Table 1). A similar
behavior was observed for the inhibition of the second
tumor-associated, transmembrane isoform, hCA XII, but the
inhibitory power was even stronger (KIs in the range of
4.1–20.5 nM), with the SAR similar to what mentioned above
for the inhibition of hCA IX. Noteworthy the selectivity pro-
file (hCA IX/hCA II) of compound 10 was slightly improved
when compared to 8 (3.44 and 3.62, respectively) but
resulted decreased for the second tumor associated CA iso-
form (hCA XII/hCA II 12.0 and 8.22 for 8 and 10, respectively).
(iv) The other transmembrane isoform (not connected with
tumors), hCA XIV,18 was also effectively inhibited by sulfon-
amides investigated here, with KIs in the range of 12.8–
53.6 nM. Again the best inhibitor was the fluorescein-tria-
zolyl derivative of sulfanilamide 7, whereas the worst one
was the corresponding metanilamide derivative 8.
Inhibition data against five physiologically relevant isoforms,
that is, hCA I, II (cytosolic), IX, XII and XIV (trans-membrane) with
the fluorescent sulfonamides A and B (lead molecules) and the
newly prepared 7, 8 and 10, are shown in Table 1.
The structure–activity relationship (SAR) for the inhibition of
these isoforms is rather well delineated:
(i) The cytosolic isoform, hCA I, was poorly inhibited by these
sulfonamides with KIs ranging between 1300 and 7630 nM.
Introduction of the triazole moiety in compounds 7–10 led
to a decrease of the inhibitory power compared to the lead
molecules A and B, which is favorable feature of the new
compounds as hCA I is an off-target, highly abundant in
the red blood cells and gastrointestinal tract enzyme.1
(ii) In the case of hCA II, one of the physiologically dominant iso-
forms, the nature of the benzenesulfonamide head strongly
influenced the inhibitory activity. Thus, the sulfanilamide
derivative 7, similar to the related benzenesulfonamide A
and B (possessing a p-sulfamoyl functional group) were mod-
erate-effective inhibitors (KIs of 36–45 nM) whereas the two
metanilamide derivatives 8 and 10 showed a decreased inhi-
bitory power (KIs of 88–127 nM). The hydrophilic fluorescent
moiety present in 10, led to a more efficient CAI (1.44 fold)
when compared to the fluorescein-containing, structurally
similar derivative 8.
2.3. X-ray crystallography
Considering the interesting CA inhibitory and solubility features
of 7, we co-crystallized this compound with hCA II and solved its
crystal structure by means of X-ray crystallography. It should be
mentioned that the hCA II–B adduct has also been characterized
by X-ray crystallography earlier, by Alterio et al.4b
The electron density of 7 bound within the hCA II active site
(Fig. 2) shows density well defined for all atoms of the inhibitor,
which was found coordinated to the Zn(II) ion from the active site
cavity by means of the deprotonated sulfonamide moiety, as
reported for all CA–sulfonamide adducts characterized by this
technique to date (see Table 2 in Section 4 for details).4a,19–22 The
NH coordinated to zinc also makes a strong H-bond with the OH
of Thr199, an interactions also observed in all CA–sulfonamide
adducts investigated so far.4a,19–22 The scaffold of the inhibitor
occupies all the enzyme active site, with the benzenesulfonamide
moiety in van der Waals contacts with Gln92, Asn67, Thr199 and
Thr200 residues, whereas the 1,2,3-triazole one in van der Waals
contacts with Phe131, Thr200 and Leu204 residues. No polar
interactions between the inhibitor heteroatoms and the active
site residues were observed, as also reported for the hCA II–B
adduct mentioned above.4a
Table 1
Inhibition data of isoforms hCA I, II, IX, XII and XIV with fluorescent sulfonamides, by
a stopped-flow, CO2 hydrase assay17
Entry
KI* (nM)
hCA I
hCA II
hCA IX
hCA XII
hCA XIV
Aa
Bb
7
8
10
AAZ
1450
1300
2560
7630
6425
0.25
44
45
36
127
88
26
24
8.1
36.9
24.3
0.06
7.0
5.0
4.1
20.5
10.7
25
41
33
12.8
53.6
31.4
5.7
0.012
*
Mean from three different assays. Errors were in the range of 10% of the
reported values, data not shown.
a
From Ref. 4a, except CA XII and XIV data which are reported here for the first
time.
b
From Refs. 1,4a.
Figure 2. Crystallographic adduct of 7 with hCA II (PDB accession code: 4RH2). An Fo À Fc omit electron density map is also shown contoured at 2
r.
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F. Carta et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 2
3. Conclusions
Summary of data collection and atomic model refinement statistics*
hCAII + 7
New fluorescent CAIs are reported which possess enhanced
water solubility and high affinity for the transmembrane isoforms
hCA IX, XII and XIV compared to previously investigated such
derivatives. The synthesis has been achieved by click chemistry,
which allowed the introduction of 1,2,3-triazole moieties which
enhanced hydrosolubility. X-ray crystallography of hCA II com-
plexed with one of these sulfonamides evidenced interesting inter-
actions between the inhibitor scaffold and residues involved in the
binding of inhibitors. Furthermore, the tail of the new fluorescent
inhibitor had a very different orientation compared to a struc-
turally similar compound devoid of the triazole moiety.
PDB ID
Wavelength (Å)
Space group
4RH2
0.980
P21
Unit cell (a, b, c, ) (Å, °)
42.362, 41.282, 72.138, 104.30
29.1–1.3 (1.38–1.30)
50,556 (4078)
0.022 (0.063)
2.07 (1.58)
Limiting resolution (Å)
Unique reflections
Rsym (%)
Redundancy
Completeness overall (%)
<I/(I)>
84.7 (44.8)
23.8 (8.7)
Refinement statistics
Resolution range (Å)
Unique reflections, working/free
Rfactor (%)
29.1–1.3
50,556/2526
0.156
0.171
2177
385
45
0.006
1.331
4. Experimental protocols
4.1. Chemistry
Rfree (%)
No. of protein atoms
No. of water molecules
No. of heterogen atoms
r.m.s.d. bonds (Å)
r.m.s.d. angles (°)
Anhydrous solvents and all reagents were purchased from
Sigma–Aldrich, Alfa Aesar and TCI. All reactions involving air- or
moisture-sensitive compounds were performed under a nitrogen
atmosphere using dried glassware and syringes techniques to
transfer solutions. Nuclear magnetic resonance (1H NMR, 13C
NMR) spectra were recorded using a Bruker Advance III 400 MHz
spectrometer in DMSO-d6. Chemical shifts are reported in parts
per million (ppm) and the coupling constants (J) are expressed in
Hertz (Hz). Splitting patterns are designated as follows: s, singlet;
d, doublet; sept, septet; t, triplet; q, quadruplet; m, multiplet; br s,
broad singlet; dd, double of doubles, 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 ASTM) as the stationary
phase and ethyl acetate/n-hexane were used as eluents. Melting
points (mp) were measured in open capillary tubes with a Gal-
lenkamp MPD350.BM3.5 apparatus and are uncorrected. All com-
pounds reported here were >95% HPLC pure.
Ramachandran statistics (%)2
Most favored
Additionally allowed
Generously outlier regions
Average B factor (Å2)
All atoms
Compound
96.1
3.9
0
13.12
25.74
26.59
Solvent
*
Values in parentheses are for the highest resolution shell.
A superimposition of the two structures is in fact shown in Fig-
ure 3. From this figure it may be observed that only the benzene-
sulfonamide moieties of the two inhibitors and are
superimposable. The ethylene spacer of B and the 1,2,3-triazole
moiety of 7 have comparable lengths in the two inhibitors, but
they do not completely superimpose, and this also leads to differ-
ent orientations of the two thioureido fragments and the corre-
B
7
sponding fluorescein tails present in
B (Fig. 3). In B the
fluorescein tail is closer to Phe131, an amino acid residue which
delineates two sub-pockets within the hydrophobic half of the
CA II active site.19 On the contrary, in 7 the tail is in a totally differ-
ent orientation, being far from Phe131 and closer to Leu204,
Val135 hydrophobic pocket. This is probably also the reason why
7 is a three times better hCA II inhibitor compared to B (Table 1).
4.1.1. Synthesis of prop-2-ynyl-carbamic acid tert-butyl ester 123
(Boc)2O, 1.0 M, Et3N
O
DCM
O
NH2
NH
1
Figure 3. Superimposition of adducts 7 and B with hCA II (PDB accession code: 2F14).
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F. Carta et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Propargylamine (1.0 g, 1.0 equiv) and triethylamine (1.1 equiv)
4.2.2. Synthesis of 3-azido-benzenesulfonamide 3b
were dissolved in DCM (80 ml). The solution was cooled to 0 °C
and tert-butyloxycarbonylcarbonate (1.1 equiv) dissolved in
20 ml of DCM was added drop-wise. The solution was stirred at
rt for 5 h then was quenched with aqueous HCl 1.0 M (100 ml)
and the organic layer was washed with H2O (3 Â 50 ml), brine
(3 Â 20 ml) and dried over Na2SO4, filtered off and concentrated
under vacuo to give a brown oil that was purified by silica gel col-
umn chromatography eluting with 10% v/v Ethyl acetate/n-hexane
to give 1 as a colorless oil.
N
3
SO NH
2
2
3b
3-Azido-benzenesulfonamide 3b was obtained according the
general procedure earlier reported as a pale yellow solid.
3-Azido-benzenesulfonamide 3b: 60% yield; mp 110 °C dec.; sil-
ica gel TLC Rf 0.47 (MeOH/DCM 10% v/v); dH (400 MHz, DMSO-d6):
7.37 (d, J = 8.8, 2H), 7.48 (s, 2H, exchange with D2O, SO2NH2), 7.52
(s, 1H), 7.63 (m, 1H); dC (100 MHz, DMSO-d6): 117.2, 123.1, 123.5,
131.8, 141.4, 146.9.
Prop-2-ynyl-carbamic acid tert-butyl ester 1: silica gel TLC Rf
0.20 (Ethyl Acetate/n-hexane 10% v/v); mmax (KBr) cmÀ1 3350
(C„CAH), 2170 (C„CH), 1760 (C@O); dH (400 MHz, DMSO-d6)
1.42 (9H, s, 3 Â CH3), 3.08 (1H, t, J 4.0, 4-H), 3.73 (2H, m, 2-H2),
7.29 (1H, br s, 1-H); dC (100 MHz, DMSO-d6) 156.6 (C@O), 82.6,
79.1, 73.6, 30.3 (C-2) and 29.9 (3 Â CH3).
4.3. General synthetic procedure of compounds 5a,b25
Experimental in agreement with reported data.23
O
N
H
O
NH TFA
2
N
N
N
3
N
N
O
Cu (0) nanosized, TMACl
H O/tBuOH 1/1
HCl
N
N
O
2
NH
SO NH
2
2
SO NH
2
2
SO NH
2 2
4a= 4-amino
4b= 3-amino
3a,b
5a= 4-amino
5b= 3-amino
1
4.2. General synthetic procedure of compounds 3a,b24
Freshly prepared azidobenzenesulfonamide 3a,b (0.12 g,
1.1 equiv) was added to a suspension of alkyne 3a (1.0 equiv) in
H2O/tBuOH 1:1 (3 ml) at rt. Then copper(0) nanosized (0.1 equiv)
and TMACl (1.0 equiv) were added to the suspension and the reac-
tion mixture was stirred at rt until starting materials were con-
sumed (TLC monitoring). The reaction was quenched with H2O
(20 ml) and the mixture was extracted with ethyl acetate
(3 Â 15 ml). The combined organic layers were washed with H2O
(3 Â 15 ml), dried over Na2SO4, filtered-off and concentrated under
vacuo to give a pale yellow residue that was purified by silica gel
column chromatography eluting 50% EtOAc/n-hexane to afford 4a
and b which were treated with concentrated aqueous hydrochloric
acid for 4 h at rt. The reaction was treated with Na2CO3 to pH 5 and
then extracted with ethyl acetate (3 Â 15 ml). The combined
organic layers were washed with H2O (3 Â 15 ml) dried over Na2-
SO4, filtered and concentrated under vacuo to give a residue that
was purified by silica gel column chromatography eluting with
MeOH/DCM 10% to afford the titled compounds.
NH
2
N
3
NaNO NaN
2
3
HCl 4M (aq)
SO NH
2
2
SO NH
2 2
2a,b
3a,b
3a= 4-amino
3b= 3-amino
2a= 4-amino
2b= 3-amino
The appropriate aminobenzenesulfonamide 2a,b (0.5 g,
1.0 equiv) was dissolved in a 4 M HCl aqueous solution (5 ml) at
0 °C. Then NaNO2 5 M aqueous solution (1.2 equiv) was added
drop-wise to the solution followed by a NaN3 5 M aqueous solution
(1.5 equiv). The reaction mixture was stirred for 0.5 h at rt and the
solid formed was filtered-off, dried under vacuo and used as it is.
4.2.1. Synthesis of 4-azido-benzenesulfonamide 3a
4.3.1. Synthesis of [1-(4-sulfamoyl-phenyl)-1H-[1,2,3]-triazol-4-
ylmethyl]-carbamic acid tert-butyl ester 4a25c
N3
O
N
H
O
SO2NH2
N
N
3a
N
4-Azido-benzenesulfonamide 3a was obtained according the
general procedure earlier reported as a yellow solid.
4-Azido-benzenesulfonamide 3a: 65% yield; mp 120–121 °C;
silica gel TLC Rf 0.50 (MeOH/DCM 10% v/v); dH (400 MHz, DMSO-
d6): 7.33 (d, J = 8.8, 2H), 7.41 (s, 2H, exchange with D2O, SO2NH2),
7.87 (d, J = 8.8, 2H); dC (100 MHz, DMSO-d6): 120.5, 128.6, 141.5,
143.9.
SO NH
2
2
4a
1-(4-Sulfamoyl-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-carbamic
acid tert-butyl ester 4 was obtained according the general procedure
earlier reported as a pale yellow solid.
Experimental in agreement with reported data.24
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6
F. Carta et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
[1-(4-Sulfamoyl-phenyl)-1H-[1,2,3]-triazol-4-ylmethyl]-carbamic
(1H, s, Ar-H triazole); dC (100 MHz, DMSO-d6): 34.8, 121.4, 123.8,
acid tert-butyl ester 4a: 79% yield; silica gel TLC Rf 0.24 (Ethyl
acetate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6) 1.44 (9H, s),
4.31 (2H, d, J = 6.0, CH2), 7.42 (1H, t, J 6.0, exchange with D2O,
–NH), 7.55 (2H, s, exchange with D2O, SO2NH2), 8.04 (2H, d,
J = 8.8, Ar-H), 8.16 (2H, d, J = 8.8, Ar-H), 8.73 (1H, s, Ar-H triazole);
dC (100 MHz, DMSO-d6): 29.1, 36.8, 79.5, 121.1, 121.9, 128.4, 139.6,
144.6, 148.0, 156.8.
128.6, 139.4, 142.9, 145.1.
4.3.4. Synthesis of 3-(4-aminomethyl-[1,2,3]-triazol-1-yl)-ben-
zenesulfonamide hydrochloride 5b
NH2 HCl
N
N
N
4.3.2. Synthesis of [1-(3-sulfamoyl-phenyl)-1H-[1,2,3]-triazol-4-
ylmethyl]-carbamic acid tert-butyl ester 4b
SO2NH2
O
5b
N
H
O
N
3-(4-Aminomethyl-[1,2,3]-triazol-1-yl)-benzenesulfonamide 5b
was obtained according the general procedure earlier reported as a
white solid.
N
N
3-(4-Aminomethyl-[1,2,3]-triazol-1-yl)-benzenesulfonamide
hydrochloride 5b: 63% yield; silica gel TLC Rf 0.10 (MeOH/DCM 10%
v/v); dH (400 MHz, DMSO-d6) 4.29 (2H, s, CH2), 7.66 (2H, s,
exchange with D2O, SO2NH2), 7.87 (1H, m, Ar-H), 7.91 (1H, m,
Ar-H), 8.15 (1H, m, Ar-H), 8.36 (1H, d, J = 2.8, Ar-H), 8.46 (3H, br
s, exchange with D2O, –NH3), 8.96 (1H, s, Ar-H triazole); dC
(100 MHz, DMSO-d6): 35.3, 118.2, 123.8, 124.1, 126.7, 132.0,
137.3, 142.5, 146.8.
SO NH
2
2
4b
[1-(3-Sulfamoyl-phenyl)-1H-[1,2,3]-triazol-4-ylmethyl]-carbamic
acid tert-butyl ester 4b was obtained according the general proce-
dure earlier reported as a pale yellow solid.
[1-(3-Sulfamoyl-phenyl)-1H-[1,2,3]-triazol-4-ylmethyl]-carbamic
acid tert-butyl ester 4b: 79% yield; silica gel TLC Rf 0.24 (Ethyl acet-
ate/n-hexane 50% v/v); dH (400 MHz, DMSO-d6) 1.42 (9H, s), 4.28
(2H, d, J = 6.2, CH2), 7.40 (1H, t, J 6.2; exchange with D2O, –NH),
7.54 (2H, s, exchange with D2O, SO2NH2), 7.80 (1H, appt, J = 8.2,
Ar-H), 7.90 (1H, d, J = 8.2, Ar-H), 8.13 (1H, dd, J = 8.2 3.7, Ar-H),
8.33 (1H, t, J 3.7), 8.69 (1H, s, Ar-H triazole); dC (100 MHz,
DMSO-d6): 29.3, 36.6, 79.8, 118.2, 123.8, 124.1, 126.7, 130.4,
132.0, 137.3, 142.5, 146.8.
4.4. General procedure for synthesis of fluorescent sulfona-
mides 7, 8 and 1026
4-(4-Aminomethyl-[1,2,3]triazol-1-yl)-benzenesulfonamide
hydrochloride salt 5a (1.0 equiv) or 3-(4-aminomethyl-[1,2,3]-tria-
zol-1-yl)-benzenesulfonamide hydrochloride salt 5b (1.0 equiv)
and the appropriate 5-isothiocyanato-benzoic acid fluorescent
tag 6 or 9 (1.0 equiv) were poured in a two neck flask and dry
DMA (1.0 ml) was added, followed by addition of TEA (1.5 equiv).
The reaction was stirred at rt until starting materials were con-
sumed (TLC monitoring), and then it was quenched with slush,
treated with a 6.0 M aqueous hydrochloric acid solution and
extracted with ethyl acetate (3 Â 15 ml). The combined organic
layers were washed with H2O (3 Â 20 ml), dried over Na2SO4, fil-
tered and concentrated in vacuo to give a solid residue that was
purified by silica gel column chromatography eluting with ethyl
acetate afford the titled compounds.
4.3.3. Synthesis of 4-(4-aminomethyl-[1,2,3]-triazol-1-yl)-ben-
zenesulfonamide hydrochloride 5a25c
NH2 HCl
N
N
N
SO2NH2
5a
4.4.1. Synthesis of 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-{3-
[1-(4-sulfamoyl-phenyl)-1H-[1,2,3]-triazol-4-ylmethyl]-thiour-
eido}-benzoic acid 7
4-(4-Aminomethyl-[1,2,3]-triazol-1-yl)-benzenesulfonamide
hydrochloride 5a was obtained according the general procedure
earlier reported as a pale yellow solid.
S
C
N
N
HN
N
N
NH HCl
2
HN
S
N
N
Et N
3
DMF
N
CO H
2
CO H
2
SO NH
2
2
SO NH
2
HO
O
O
HO
O
O
2
5a
6
7
4-(4-Aminomethyl-[1,2,3]-triazol-1-yl)-benzenesulfonamide
hydrochloride 5a: 72% yield; silica gel TLC Rf 0.02 (MeOH/DCM 10%
v/v); dH (400 MHz, DMSO-d6) 4.29 (2H, s, CH2), 7.60 (2H, s,
exchange with D2O, SO2NH2), 8.10 (2H, d, J = 8.8, Ar-H), 8.15 (2H,
d, J = 8.8, Ar-H), 8.42 (3H, br s, exchange with D2O, –NH3), 9.0
3-(4-Aminomethyl-[1,2,3]triazol-1-yl)-benzenesulfonamide
hydrochloride salt 5a (1.0 equiv) and 2-(6-hydroxy-3-oxo-3H-
xanthen-9-yl)-5-isothiocyanato-benzoic acid 6 (1.0 equiv) were
treated according to the general procedure previously reported
afford the titled compound 7 as an orange powder.
Please cite this article in press as: Carta, F.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.11.031

F. Carta et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5-{3-[1-(4-sulfamoyl-
phenyl)-1H-[1,2,3]-triazol-4-ylmethyl]-thioureido}-benzoic acid
7: 38% yield; silica gel TLC Rf 0.24 (Ethyl acetate); mp 194–
198 °C; dH (400 MHz, DMSO-d6) 4.96 (2H, d, J 5.2, CH2), 6.61 (5H,
m), 6.71 (2H, s), 7.24 (1H, d, J 8.8, Ar-H), 7.56 (2H, s, exchange with
D2O, SO2NH2), 7.80 (1H, dd, J 8.8 2.3, Ar-H), 8.06 (2H, d, J 8.8, Ar-H),
8.19 (2H, d, J 8.8, Ar-H), 8.31 (1H, d, J 2.0, Ar-H), 8.62 (1H, br s,
exchange with D2O, NH), 8.91 (1H, s, Ar-H triazole), 10.14 (4H, br
s, exchange with D2O, 2 Â OH, COOH, NH); dC (100 MHz, DMSO-
d6) 45.4, 103.2, 110.0, 113.7, 115.4, 117.1, 126.0, 128.1, 128.7,
129.8, 130.0, 137.1, 141.9, 148.9, 153.1, 160.8, 161.9, 168.6, 168.4
(C@O), 182.6 (C@S); m/z (ESI positive) 642.2 [M+H]+.
then-9-yl)-5-isothiocyanato-benzoic acid 9 (1.0 equiv) were trea-
ted according to the general procedure previously reported afford
the titled compound 10 as light pink powder.
[9-(2-Carboxy-4-{3-[1-(3-sulfamoyl-phenyl)-1H-[1,2,3]-triazol-
4-ylmethyl]-thioureido}-phenyl)-6-dimethylamino-xanthen-3-
ylidene]-dimethyl-ammonium chloride 10: 18% yield; silica gel
TLC Rf 0.05 (Ethyl acetate); mp >200 °C; dH (400 MHz, DMSO-d6)
3.30 (12H, s), 4. 32 (2H, d, J 5.4, CH2), 7.00 (4H, m), 7.14 (3H, m),
7.24 (2H, s, exchange with D2O, SO2NH2), 7.41 (1H, m), 7.72 (1H,
d, J 8.8, Ar-H), 7.82 (1H, m, Ar-H), 7.89 (1H, m, Ar-H), 8.13 (1H,
m, Ar-H), 8.32 (1H, d, J 2.0, Ar-H), 8.42 (1H, br s, exchange with
D2O, NH), 8.90 (1H, s, Ar-H triazole), 10.38 (2H, br s, exchange with
D2O, COOH, NH); dC (100 MHz, DMSO-d6) 35.1, 45.4, 103.4, 111.0,
113.8, 115.2, 117.0, 125.9, 126.0, 126.2, 128.0, 128.6, 129.9,
130.1, 131.4, 132.0, 137.0, 142.0, 148.8, 153.0, 160.9, 162.0,
168.2, 170.0 (C@O), 182.1 (C@S); m/z (ESI positive) 732.27 [M+H]+.
4.4.2. Synthesis of 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-{3-
[1-(3-sulfamoyl-phenyl)-1H-[1,2,3]-triazol-4-ylmethyl]-thiour-
eido}-benzoic acid 8
S
C
N
HN
N
N
NH2 HCl
N
HN
S
N
N
SO2NH2
Et3N
DMF
N
CO2H
CO2H
SO2NH2
HO
O
O
HO
O
O
6
5b
8
3-(4-Aminomethyl-[1,2,3]triazol-1-yl)-benzenesulfonamide
hydrochloride salt 5b (1.0 equiv) and 2-(6-hydroxy-3-oxo-3H-
xanthen-9-yl)-5-isothiocyanato-benzoic acid 6 (1.0 equiv) were
treated according to the general procedure previously reported
afford the titled compound 8b as an orange powder.
4.5. Kinetic assay inhibition
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalyzed CO2 hydration activity. 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 determina-
tion of the kinetic parameters and inhibition constants. For each
inhibitor at least six traces of the initial 5–10% of the reaction have
been used for determining the initial velocity. The uncatalyzed
rates were determined in the same manner and subtracted from
the total observed rates. Stock solutions of inhibitor (0.1 mM) were
prepared in distilled–deionized water and dilutions up to 0.01 nM
were done thereafter with the assay buffer. Inhibitor and enzyme
solutions were preincubated together for 15 min at room temper-
ature (15 min) or 4 °C (6 h) prior to assay, in order to allow for the
formation of the E–I complex. The inhibition constants were
obtained by non-linear leastsquares methods using PRISM 3, as
reported earlier,20f and represent the mean from at least three
2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5-{3-[1-(3-sulfamoyl-
phenyl)-1H-[1,2,3]-triazol-4-ylmethyl]-thioureido}-benzoic acid
8: 26% yield; silica gel TLC Rf 0.33 (Ethyl acetate); mp 180 °C; dH
(400 MHz, DMSO-d6) 4.94 (2H, d, J 5.4, CH2), 6.62 (5H, m), 6.68
(2H, s), 7.24 (1H, d, J 8.8, Ar-H), 7.54 (2H, s, exchange with D2O,
SO2NH2), 7.80 (1H, dd, J 8.8 2.3, Ar-H), 7.84 (1H, m, Ar-H), 7.92
(1H, m, Ar-H), 8.13 (1H, m, Ar-H), 8.32 (1H, d, J 2.0, Ar-H), 8.65
(1H, br s, exchange with D2O, NH), 8.89 (1H, s, Ar-H triazole),
10.12 (4H, br s, exchange with D2O, 2 Â OH, COOH, NH); dC
(100 MHz, DMSO-d6) 45.4, 103.2, 110.0, 113.7, 115.4, 117.1,
125.8, 126.0, 126.1, 128.1, 128.7, 129.8, 130.0, 131.4, 132.0,
137.1, 141.9, 148.9, 153.1, 160.8, 161.9, 168.6, 169.7 (C@O),
182.2 (C@S); m/z (ESI positive) 643.1 [M+H]+.
4.4.3. Synthesis of [9-(2-carboxy-4-{3-[1-(3-sulfamoyl-phenyl)-
1H-[1,2,3]-triazol-4-ylmethyl]-thioureido}-phenyl)-6-dimethy-
lamino-xanthen-3-ylidene]-dimethyl-ammonium chloride 10
S
C
N
N
HN
N
N
NH HCl
2
HN
S
N
N
SO NH
Et N
3
DMF
2
2
N
CO H
2
CO H
2
SO NH
2
2
Cl
Cl
N
O
N
N
O
N
9
5b
10
3-(4-Aminomethyl-[1,2,3]triazol-1-yl)-benzenesulfonamide
hydrochloride salt 5b (1.0 equiv) and 2-(6-hydroxy-3-oxo-3H-xan-
different determinations. All CA isoforms were recombinant ones
obtained in-house as reported earlier.15,20
Please cite this article in press as: Carta, F.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.11.031

8
F. Carta et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9. (a) Krall, N.; Pretto, F.; Decurtins, W.; Bernardes, G. J. L.; Supuran, C. T.; Neri, D.
Angew. Chem., Int. Ed. 2014, 53, 4231; (b) Buller, F.; Steiner, M.; Frey, K.; Mircsof,
D.; Scheuermann, J.; Kalisch, M.; Bühlmann, P.; Supuran, C. T.; Neri, D. ACS
Chem. Biol. 2011, 6, 336.
10. (a) Vu, H.; Pham, N. B.; Quinn, R. J. J. Biomol. Screen. 2008, 13, 265; (b) Maresca,
A.; Temperini, C.; Vu, H.; Pham, P. B.; Poulsen, S. A.; Scozzafava, A.; Quinn, R. J.;
Supuran, C. T. J. Am. Chem. Soc. 2009, 131, 3057; (c) Carta, F.; Temperini, C.;
Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C. T. J. Med. Chem. 2010, 53,
5511.
4.6. Co-crystallization and X-ray data collection
Crystals of hCA II complexed with compound 7 were obtained
using the sitting drop vapor diffusion method. An equal volume
of 0.8 mM solution of hCA II in Tris pH = 8.0 and 1.6 mM of 7 in
Hepes 20 mM pH = 7.4 was mixed and incubated for 15 minutes.
2 ll of the complex solution were mixed with 2 ll of a solution
11. (a) Maresca, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 4511; (b)
Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20,
7255; (c) Bozdag, M.; Ferraroni, M.; Carta, F.; Vullo, D.; Lucarini, L.; Orlandini,
E.; Rossello, A.; Nuti, E.; Scozzafava, A.; Masini, E.; Supuran, C. T. J. Med. Chem.
2014, 57, 9152; (d) Touisni, N.; Maresca, A.; McDonald, P. C.; Lou, Y.;
Scozzafava, A.; Dedhar, S.; Winum, J. Y.; Supuran, C. T. J. Med. Chem. 2011,
54, 8271; (e) Bonneau, A.; Maresca, A.; Winum, J. Y.; Supuran, C. T. J. Enzyme
Inhib. Med. Chem. 2013, 28, 397; (f) Sharma, A.; Tiwari, M.; Supuran, C. T. J.
Enzyme Inhib. Med. Chem. 2014, 29, 292.
12. (a) Pacchiano, F.; Carta, F.; McDonald, P. C.; Lou, Y.; Vullo, D.; Scozzafava, A.;
Dedhar, S.; Supuran, C. T. J. Med. Chem. 2011, 54, 1896; (b) Pacchiano, F.;
Aggarwal, M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; McKenna, R.;
Supuran, C. T. Chem. Commun. 2010, 8371.
of 1.6 M sodium citrate, 50 mM Tris pH 8.0 and were equilibrated
against the same solution at 296 K. Crystals of the complex grew in
a few days. The crystals were flash-frozen at 100 K using a solution
obtained by adding 25% (v/v) glycerol to the mother liquor solution
as cryoprotectant. A data set on a crystal of the complex hCAII–
compound 7 was collected using synchrotron radiation at the Xaloc
beamline at ALBA (Barcelona, Spain) with a wavelength of 0.980 Å
and a DECTRIS Pilatus 6 M detector. Data were integrated and
scaled using the program XDS.27 Data processing statistics are
showed in Table 2.
13. a) Wilkinson, B. L.; Bornaghi, L. F.; Houston, T. A.; Innocenti, A.; Supuran, C. T.;
Poulsen, S. A. J. Med. Chem. 2006, 49, 6539; (b) Wilkinson, B. L.; Bornaghi, L. F.;
Houston, T. A.; Innocenti, A.; Vullo, D.; Supuran, C. T.; Poulsen, S. A. J. Med.
Chem. 2007, 50, 1651; (c) Wilkinson, B. L.; Innocenti, A.; Vullo, D.; Supuran, C.
T.; Poulsen, S. A. J. Med. Chem. 2008, 51, 1945.
4.7. Structure determination
The crystal structure of hCA II (PDB accession code: 4FIK) with-
out solvent molecules and other heteroatoms was used to obtain
initial phases of the structures using Refmac5.28 5% of the unique
reflections were selected randomly and excluded from the refine-
ment data set for the purpose of Rfree calculations. The initial
|Fo À Fc| difference electron density maps unambiguously showed
the inhibitors. Atomic models for inhibitors were calculated and
energy minimized using the program JLigand 1.0.39. Refinements
proceeded using normal protocols of positional, isotropic atomic
displacement parameters alternating with manual building of the
models using COOT.29 Solvent molecules were introduced auto-
matically using the program ARP.30 Final rounds of refinement
for all the models included hydrogen at calculated positions and
refined using a riding model. The quality of the final models was
assessed with PROCHECK.31 Crystal parameters and refinement
data are summarized in Table 2. Atomic coordinates were depos-
ited in the Protein Data Bank (PDB accession code: 4RH2). Graph-
ical representations were generated with Chimera.32
14. (a) Lopez, M.; Paul, B.; Hofmann, A.; Morizzi, J.; Wu, Q.; Charman, S. A.;
Innocenti, A.; Vullo, D.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2009, 52,
6421; (b) Lopez, M.; Salmon, A. J.; Supuran, C. T.; Poulsen, S. A. Curr. Pharm. Des.
2010, 16, 3277; (c) Lopez, M.; Trajkovic, J.; Bornaghi, L. F.; Innocenti, A.; Vullo,
D.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2011, 54, 1481.
15. (a) Tanc, M.; Carta, F.; Bozdag, M.; Scozzafava, A.; Supuran, C. T. Bioorg. Med.
Chem. 2013, 15, 4502; (b) Grandane, A.; Tanc, M.; Zalubovskis, R.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2014, 5, 1256; (c) Grandane, A.; Tanc, M.; Zalubovskis,
R.; Supuran, C. T. Bioorg. Med. Chem. 2014, 5, 1522; (d) Tars, K.; Vullo, D.;
Kazaks, A.; Leitans, J.; Lends, A.; Grandane, A.; Zalubovskis, R.; Scozzafava, A.;
Supuran, C. T. J. Med. Chem. 2013, 56, 293.
16. (a) Tanc, M.; Carta, F.; Scozzafava, A.; Supuran, C. T. Org. Biomol. Chem. 2015, 13,
77; (b) Grandane, A.; Tanc, M.; Di Cesare Mannelli, L.; Carta, F.; Ghelardini, C.;
ˇ
Zalubovskis, R.; Supuran, C. T. J. Med. Chem. 2015, 58, 3975; (c) Sßentürk, M.;
Gülçin, I.; Dasßtan, A.; Küfreviog˘lu, Ö. I.; Supuran, C. T. Bioorg. Med. Chem. 2009,
17, 3207.
17. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561.
18. Alterio, V.; Pan, P.; Parkkila, S.; Buonanno, M.; Supuran, C. T.; Monti, S. M.; De
Simone, G. Biopolymers 2014, 101, 769.
19. (a) Avvaru, B. S.; Wagner, J. M.; Maresca, A.; Scozzafava, A.; Robbins, A. H.;
Supuran, C. T.; McKenna, R. Bioorg. Med. Chem. Lett. 2010, 20, 4376; (b) Wagner,
J.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; Supuran, C. T.; McKenna, R.
Bioorg. Med. Chem. 2010, 18, 4873; (c) Carta, F.; Garaj, V.; Maresca, A.; Wagner,
J.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; McKenna, R.; Supuran, C. T.
Bioorg. Med. Chem. 2011, 19, 3105; (d) Hen, N.; Bialer, M.; Yagen, B.; Maresca,
A.; Aggarwal, M.; Robbins, A. H.; McKenna, R.; Scozzafava, A.; Supuran, C. T. J.
Med. Chem. 2011, 54, 3977.
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