Carbonic anhydrase inhibitors. Inhibition of human cytosolic isoforms i and II with (reduced) Schiff's bases incorporating sulfonamide, carboxylate and carboxymethyl moieties

Article abstract of DOI:10.1016/j.bmc.2014.03.041

A library of Schiff bases was synthesized by condensation of aromatic amines incorporating sulfonamide, carboxylic acid or carboxymethyl functionalities as Zn²⁺-binding groups, with aromatic aldehydes incorporating tert-butyl, hydroxy and/or methoxy groups. The corresponding amines were thereafter obtained by reduction of the imines. These compounds were assayed for the inhibition of two cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isoenzymes, hCA I and II. The K_i values of the Schiff bases were in the range of 7.0-21,400 nM against hCA II and of 52-8600 nM against hCA I, respectively. The corresponding amines showed K_i values in the range of 8.6 nM-5.3 μM against hCA II, and of 18.7-251 nM against hCA I, respectively. Unlike the imines, the reduced Schiff bases are stable to hydrolysis and several low-nanomolar inhibitors were detected, most of them incorporating sulfonamide groups. Some carboxylates also showed interesting CA inhibitory properties. Such hydrosoluble derivatives may show pharmacologic applications.

Full text of DOI:10.1016/j.bmc.2014.03.041

Bioorganic & Medicinal Chemistry 22 (2014) 2867–2874
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Carbonic anhydrase inhibitors. Inhibition of human cytosolic
isoforms I and II with (reduced) Schiff’s bases incorporating
sulfonamide, carboxylate and carboxymethyl moieties
b
c
Gihane Nasr a, Alina Cristian a, Mihail Barboiu a, , Daniella Vullo , Jean-Yves Winum ,
⇑
Claudiu T. Supuran b,d,
⇑
a Adaptive Supramolecular Nanosystems Group, Institut Européen des Membranes, ENSCM/UMII/UMR-CNRS 5635, Place Eugène Bataillon, CC 047, 34095 Montpellier, Cedex 5, France
b Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
c Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CNRS-UM1-UM2 Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier,
8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France
d Università degli Studi di Firenze, Polo Scientifico, Dipartimento NEUROFABA, Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino
(Firenze), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of Schiff bases was synthesized by condensation of aromatic amines incorporating sulfonamide,
carboxylic acid or carboxymethyl functionalities as Zn2+-binding groups, with aromatic aldehydes
incorporating tert-butyl, hydroxy and/or methoxy groups. The corresponding amines were thereafter
obtained by reduction of the imines. These compounds were assayed for the inhibition of two cytosolic
human carbonic anhydrase (hCA, EC 4.2.1.1) isoenzymes, hCA I and II. The Ki values of the Schiff bases
were in the range of 7.0–21,400 nM against hCA II and of 52–8600 nM against hCA I, respectively.
Received 14 February 2014
Revised 21 March 2014
Accepted 27 March 2014
Available online 4 April 2014
Keywords:
Carbonic anhydrase
Inhibitor
Sulfonamide
Schiff’s base
Amine
The corresponding amines showed Ki values in the range of 8.6 nM–5.3 lM against hCA II, and of
18.7–251 nM against hCA I, respectively. Unlike the imines, the reduced Schiff bases are stable to
hydrolysis and several low-nanomolar inhibitors were detected, most of them incorporating sulfonamide
groups. Some carboxylates also showed interesting CA inhibitory properties. Such hydrosoluble
derivatives may show pharmacologic applications.
Imine
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Many of the CAs are therapeutic targets with the potential to be
inhibited or activated. CA II and CA I inhibitors are used for the
Carbonic anhydrase CA, the catalyst for the interconversion be-
tween carbon dioxide and bicarbonate, is an essential enzyme in
bacteria, archaea and eukaryotes. In humans, the CA-catalysed
reaction involves four simple chemical entities, water, CO2, HCO3À
and H+ essential in a host of physiological and pathological pro-
cesses, such as respiration, pH and CO2 homeostasis, electrolyte
secretion lipogenesis.1–3 Sixteen isozymes have been described so
far in mammals. Some of these isoenzymes are cytosolic (CA I,
CA II, CA III, CA VII and CA XIII), other are membrane bound (CA
IV, CA IX, CA XII and CA XIV), two are mitochondrial (CA VA, CA
VB) and one is secreted in saliva and milk (CA VI). CA I and CA II
are the two major CA isozymes present at high concentrations in
the cytosol in erythrocytes, and CA II is the most active of all
CAs.1–3
treatment of glaucoma, epilepsy, or as diuretics. For such reasons,
the design of new, potent inhibitors, may lead to clinical applica-
tions for treating a multitude of diseases.1–3
Schiff bases incorporating aromatic/heterocyclic sulfonamide
moieties in their molecules have been extensively investigated as
CA inhibitors (CAIs).4,5 Some of these derivatives were the first
ones reported to have some selectivity for the inhibition of some
human (h) CA isoforms, such as hCA I, II or IV.4,5 Furthermore,
the reversible reaction between an amine and an aldehyde/ketone
has been employed to generate Dynamic Combinatorial Libraries-
DCLs of sulfonamide CAIs, in which the enzyme acts as a template
for the imine reaction formation, ‘selected’ for the most effective
inhibitors.6
Here we reported two new libraries of sulfonamide CAIs, that is,
imines (Schiff bases obtained by reacting aromatic amines also
incorporating zinc-binding functions of the sulfonamide, carboxyl-
ate and carboxymethyl type) and the corresponding amines
(obtained by the reduction of the Schiff bases). Many of the new
derivatives showed a good inhibition against the physiologically
⇑
Corresponding authors. Tel.: +33 467149195; fax: +33 467149119 (M.B.);
tel.: +39 0554573005; fax: +39 0554573385 (C.T.S.).
E-mail addresses:
mihail-dumitru.barboiu@univ-montp2.fr (M. Barboiu),
claudiu.supuran@unifi.it (C.T. Supuran).
http://dx.doi.org/10.1016/j.bmc.2014.03.041
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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G. Nasr et al. / Bioorg. Med. Chem. 22 (2014) 2867–2874
dominant and relevant isoforms hCA I and hCA II. We also report
here some CAIs presenting higher affinity for hCA I as compared
to hCA II, which is an unusual trend, since most sulfonamides are
better hCA II than hCA I inhibitors.
versus cytosolic CA isoforms.4–6 Thus, we have investigated the li-
braries of prepared compounds for the inhibition of the physiolog-
ically relevant cytosolic isoforms hCA I and II (Table 1).7
3.1. Compounds from aldehydes A and B
3.1.1. Imines
2. Chemistry
The rationale of this study was to use ‘amines’ also incorporating
potential zinc-binding functions of the sulfonamide, carboxylate
and carboxymethyl type, which have been condensed with alde-
hydes possessing both lipophilic (tert-butyl-phenyl) or hydrophilic
(hydroxyphenyl) or mixed-type (hydro- and lipophilic, e.g., 2-hy-
droxy-5-methoxy-phenyl) moieties in their molecule in order to
generate a library of Schiff bases (which will be called imines in
the following). These imines were then reduced to afford the corre-
sponding secondary amines (which will be called amines in the
discussion).
The following amines were included in the study:
4-aminobenzoic acid (A), methyl 4-aminobenzoate (B), sulfanil-
amide (C), 4-aminomethylbenzene-sulfonamide hydrochloride
(D), 4-(2-aminoethyl) benzenesulfonamide (E), and the follow-
ing aldehydes: benzaldehyde (1), 4-tert-butylbenzaldehyde (2),
4-hydroxybenandehyde (3) and 2-hydroxy-5-methoxybenzalde-
hyde (4). The 20 Schiff bases obtained were then reduced in the
presence of an excess of sodium borohydride affording the corre-
sponding 20 amines (Scheme 1).
(i) The substituted carboxy and carboxymethyl Schiff bases
(1–4)Aim and (1–4)Bim show Ki values in the range of
1040–8600 nM and 780–21,400 nM against hCA I and hCA
II, respectively (Scheme 3). These data show less effective
inhibition comparing to sulfonamide Schiff bases (see later).
Some reports described the inhibition of CAs with carboxyl-
ate, acetate or salicylic acid.2,3 It has been shown that
the cytosolic hCA I and II were weakly inhibited by all the
investigated anions, with inhibition constants in the range
of millimolar. The hCA II was crystallized in an acetate-
bound complex and the crystallographic data show that
the acetate group has replaced the zinc-bound water,
forming a tetrahedral coordination, and thus disrupts the
hydrogen-bonded solvent network. From the other side,
acetate interacts with Thr199 with the methyl group
directed away from the hydrophobic patch.8
(ii) All these imines expressed very different inhibition straight
against hCA I and hCA II. In general the benzyl moiety or
organic scaffold lay in the hydrophobic part of the hCAs
and involved in many interactions (if the geometry is
suitable) and the COOH or CO2CH3 bind weakly in the
precatalytic complex. As it had been known, the two
metalloenzymes hCA I and hCAII present many structure
differences based on the disposition, the number and
the nature of amino acids (hydrophilic/hydrophobic) inside
3. CA inhibition data
As mentioned in the introductory part, many of the Schiff base
sulfonamide CAIs showed not only excellent inhibitory power
against many physiologically relevant isoforms, but also interest-
ing selectivity profiles for the inhibition of membrane-associated
Scheme 1. Synthesis of imines and secondary amines as CA inhibitors by reaction of amines A–E with aldehydes 1–4, followed by reduction of the resulting Schiff bases.
Coding of the Schiff’s bases is done by considering the aldehyde reagent and the amine one, such as for example the aldehyde, reacting with amine A leads to imine 1Aim
When the reduction was also done, the compound (amine) is labeled as 1Aam
.
.

G. Nasr et al. / Bioorg. Med. Chem. 22 (2014) 2867–2874
2869
Table 1
Inhibition constant of imines (1–4)(A–E)im and the corresponding amines (1–4)
(A–E)am reported in the paper, by a stopped-flow, CO2 hydrase assay (Kis are mean
from 3 different assays)7
Inhibitors
Ki (nM) hCA II
Ki (nM) hCA I
1Aim
1Aam
2Aim
2Aam
3Aim
3Aam
4Aim
4Aam
1Bim
1Bam
2Bim
2Bam
21,400
123.2
10,400
184.5
N.D.
200.5
13,300
142.5
1160
22,171
780
158.4
1300
141.5
11,250
5329
105
1230
56.7
8270
266
N.D.
116.4
1290
189.5
8600
200.5
7460
251.5
1040
209.5
7450
228.3
311
116.6
85
18.75
80
29
52
23.5
93
30.3
72
25.5
76
59.5
92
11250
21400
13300
10400
1300
7450
1040
780
7460
8600
1160
8270
3B
im
1290
Ki hCAI
3Bam
4Bim
4Bam
1Cim
1Cam
2Cim
2Cam
3Cim
3Cam
4Cim
4Cam
1Dim
1Dam
2Dim
2Dam
3Dim
3Dam
4Dim
4Dam
1Eim
1Eam
2Eim
2Eam
3Eim
3Eam
4Eim
4Eam
AZA
1230
Ki hCAII
21.4
78
32.4
11.1
701.8
7.0
Figure 1. Inhibition constant values against hCA II and hCA I for aromatic Schiff
bases with acid and ester substituents (see also Table 1 for the precise Ki values).
86.6
7.1
38.5
11.0
24.0
10.3
79.7
9.4
130.5
11.0
160.0
11.2
8.6
43
236
12.6
51.5
250
than –CO2CH3. The monosubstitued tert-butyl compounds
2Aam–2Bam and 3Aam–3Bam express small differences of Ki
values againt hCA I and hCA II (Fig. 1). The selectivity ratio
for the inhibition of hCA I over hCA II is very good for
1Bam and 4Bam, with hCA II/hCA I = 111 and 23, respectively.
The X-ray structures of such adducts can afford better
understanding of interaction between certain moieties of
the inhibitors and amino acid residues within the enzyme
active site and can explain why a minor structural difference
between two compounds may lead to very different inhibi-
tory effects against various CAs.2
51.9
678
62.4
665
50.3
105
92.1
70
(ii) The 8 amines 1Aam–2Aam–3Aam–4Aam–1Bam–2Bam–3Bam
–
56.05
12
14
4Bam express high inhibition power compared to their corre-
sponding imines, especially for hCA I. It seems that the
stereochemically rigid imino compounds would result in
poorer binding, decreasing inhibitory properties related to
the saturated mobile amino compounds. The difference in
inhibition is very important reaching 93 times better for
4Aam:4Aim.This binding is less valid in the case of very
selective hCA II. This selectivity is an advantage in order to
avoid side effects of drugs based on them.
MZA
DCP
50
1200
38
the enzyme pocket so that they induce large differences in Ki
values for the same compound (e.g. Ki of 2Bim is 7460 nM
against hCA I and only 780 nM againt hCA II). In this case
the selectivity ratio is very good, in the range of 10. Further-
more, it may observed that even very small alterations in the
structure of these compounds lead to a drastic change in
CAII inhibitory capacity, for example the incorporation of
an additional substitutent OCH3 in 4Bim structure comparing
to 3Bim structure drastically decreases the inhibition power
from 1300 to 11,250 nM.
3.2. Compounds obtained from aldehydes C, D, E
3.2.1. Imines
(i) The 3 aldehydes C, D and E possess 0, 1 or 2 CH2 groups
respectively, in para to the sulfonamide group. It has been
demonstrated that an additional CH2 moiety may lead to
important change in inhibition constant value:9–11 for exam-
ple the Ki value increase from 72 nM (2Dim) to 665 nM
(2Eim) by adding one CH2 group. All these derivatives
showed higher hCA II affinity (7–105 nM) than hCA I (52–
678 nM) (Fig. 2 and Chart 1). This result is expected because
sulfonamides are known to be better inhibitors for hCA I
than hCA II.
3.1.2. Amines
(i) The reduced imines 1Aam and its homologue 1Bam are the
best inhibitors against hCA I from all acid and ester com-
pounds, with Ki = 56.6 and 200.5 nM, respectively. The
unsubstituted benzyl scaffold 1 fits well inside the bulky
enzyme pocket of hCA I (bulky space due of the presence
of His200 and His67 among others).1,2 The acid 1Aam (56.6
for hCA I and 123.2 for hCA II nM) is more potent than the
ester 1Bam (200.5 for hCA I and 22171 for hCA II nM) prob-
ably due to the presence of better binding function –COOH
(ii) Very good inhibitor properties have been detected: the best
compounds are 4Cim and 1Dim (7 and 7.1 nM) againt hCA II
and seven other potent hCA II inhibitors (such as 3Cim
,

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G. Nasr et al. / Bioorg. Med. Chem. 22 (2014) 2867–2874
141
158
22171
200
251
209
142
266
184
189
5329
123
116
228
Ki hCAII
56
3Bam
Ki hCAII
4Bam
2Bam
200
4Aam
Ki hCAI
3Aam
Ki hCAI
2Aam
1Bam
1Aam
H2
C
H2
C
H2
OH
H2
C
HO
HO
C
N
COOH
N
COOH
N
H
COOH
H
H
N
COOH
H
H3CO
4Aam
2Aam
1Aam
3Aam
H2
C
H2
H2
OH
C
C
H2
N
COOCH3
N
COOCH3
N
COOCH3
C
H1Bam
2HBam
3HBam
N
H
COOCH3
H3CO
4Bam
Figure 2. Inhibition constant values against hCA II and hCA I for aromatic amines with carboxylic acid and ester substituents.
1Eim, 2Eim, 4Eim, 2Dim, 3Dim, 4Dim with Ki ranging between
9.4 and 12.6 nM. Imine 4Cim (52 nM) was the best inhibitor
against hCA I. These data clearly indicate that these com-
pounds act as efficient CA inhibitors with potencies compa-
rable to those of reference sulfonamide drugs, or clinically
used CA inhibitors as diuretics, antiglaucoma agents, anti-
epileptics, eg: AZA, MZA, DCP (Table 1).12
substituted benzyl tert-butyl 2 seem to be more active than
the non-substituted phenyl 1 and the substituted phenyl 4
express better activity by the presence of OH and OCH3
attached to the benzyl group (Fig. 3 and Scheme 2).
3.2.2. Amine
(iii) All sulfonamides with a benzylamino group D (n = 1) express
better inhibition properties than compound with phenyleth-
ylamino group E (n = 2) or aniline group C (n = 0), following
(i) The imines and the corresponding amines are more effective
hCA I and hCA II inhibitors as compared to the parent sulfon-
amide amines C, D and E (Table 2).15 The data of reduced sul-
fonamide imines are very different from their corresponding
imines. Certainly the presence of –CH2–NH– groups (in the
case of amines) instead of –CH@N– groups (in the case of
imines) induces flexibility and different interactions with
amino acid residues of the isozymes. The best inhibitor
againt hCA I is 2Cam with Ki = 18.75 nM followed by 4Cam
with Ki = 23.5 nM, and 2Eam against hCA II with Ki = 8.6 nM.
(ii) Compounds with tert-butylbenzyl group, 2 possess good
activity against hCA I and hCA II with Ki in the range of
8.6–50.3 nM and are slightly more potent than compound
from non-substituted benzyl 1: 2C 18.75 > 1C 116.65 nM
(except against hCA II), 2E 665–11.2 > 1E 678–11, 2D
25.4–24 > 1D 30.3–38.5 nM (hCA I and hCA II, respectively).
Certainly the bulky tert-butyl fills the pocket space and
stabilizes this hCAs.
this order 1Dim > 1Cim > 1Eim
,
2Dim ꢀ 2Eim > 2Cim, 3Dim
ꢀ 3Cim > 3Eim. The phenethylamine group E seems to be ste-
rically long and did not bind tightly to the active site of the
enzyme. The extended linker would result in poorer binding
because of a higher entropic loss upon binding, especially in
hCA I. With the compounds originating from aldehyde 4, the
Ki values of 4Cim, 4Eim and 4Dim are close and are the lowest
because the hydroxy and methoxy groups participate in H-
bonds, strongly stabilizing the enzyme–inhibitor adduct.
There are in fact several amino acid residues situated in
the middle of the active site cavity of hCA II (such as
Asn62, His64, Gln92, Th200) responsible of this kind of inter-
action.13,14 Finally due to the good inhibition properties of
compounds related to 4, there is no notable difference for
this kind of potent inhibitors 4Cim ꢀ 4Dim ꢀ 4Eim
.
(iv) Comparing Ki for all compounds derived from C or E, the
order of Ki values for such compounds are similar except
for 3Eim (against hCA II): 1Cim 311–105 > 2Cim 85–
78 > 3Cim 80–11.1 > 4Cim 52–7 nM (against hCA II and hCA
I respectively) and 1Eim 678 > 2Eim 665 > 3Eim 105 > 4Eim
70 nM (only against hCA I) and 1Eim 11 > 2Eim 11.2 > 4Eim
12.6 > 3Eim 43 nM (against hCA II). Based on this result, the
(iii) Compounds with 2-hydroxy-5-methoxybenzyl group 4 are
more potent than compounds from 4-hydroxybenzyl 3
except for 4D against hCA II. It is obvious that the disubsti-
tuted aromatic moiety 4 assures more H-bond interaction
and tighter bonding with amino acid residues of the hydrop-
obic pocket.

G. Nasr et al. / Bioorg. Med. Chem. 22 (2014) 2867–2874
2871
H
OH
C
HO
H
C
H
N
SO2NH2
H2
C
HO
H2
C
C
OH
N
SO2NH2
N
SO2NH2
2
H3CO
SO2NH2
2
H
C
H
C
N
N
SO2NH2
H3CO
120
100
80
60
40
20
0
105
92
93
85
80
76
78
72
70
hCAII
52
hCAI
43
7
11
10,3
11,1
12
9,4
7,1
OH
H
H2
C
H
C
C
H2
C
N
SO2NH2
H2
N
SO2NH2
H3CO
H
C
H
C
H2
C
HO
N
SO2NH2
N
SO2NH2
C
H
C
H
C
H
C
H2
C
H2
C
N
SO2NH2
N
SO2NH2
N
SO2NH2
2
2
678
665
hCAII
hCAI
700
600
500
400
300
200
100
0
311
105
11,2
11
Figure 3. Ki (nM) for sulfonamide inhibitors againt hCA I and hCA II.
fouraromatic aldehydes substitued by tert-butyl, hydroxy or hy-
droxy and methoxy groups, followed by reduction of the imino
to the amino group.
t-Bu
OH, MeO
Hydrophilic
interaction
Hydrophobic
interaction
OH
MeO
(i) Moderate inhibition constants in the range of micromolar
were assayed for compounds with carboxy, A or methoxy,
B substituents. The flexible amines express higher inhibition
compared to their more rigid imine (for hCA I). High selectiv-
ity of 102 is expressed for hCA II over hCA.
OH
>
>
Scheme 2. Interaction sites of incorporated substituents.
(ii) Low nanomolar Ki values of sulfonamide imines and amines
(with C, D, E) reaching 7 and 8.6 nM respectively.
(iii) Better inhibition power was determined against hCA II ver-
sus hCA I for sulfonamide imines.
(iv) We discovered new amine and imine derivatives with inhib-
itory properties against hCA I and hCA II and good water sol-
ubility. These compounds are potential candidates in
ophtalmologic studies l in which eye drops containing CAIs
are useful for the treatment of glaucoma and ocular
hypertension.
Table 2
Ki values of sulfonamide amines C–E
Sulfonamide mines
Ki (nM)
hCA I
hCA II
C
D
E
28,000
25,000
21,000
300
170
160
5. Experimental
5.1. Chemistry
4. Conclusions
Two libraries of CA inhibitors of (20 imines and 20 amines) have
been synthesised by condensation between five amines incorpo-
rating carboxylic acid, carboxymethyl or sulfonamide groups, with
All reagents were obtained from Aldrich and used without fur-
ther purification. All organic solutions were routinely dried by

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G. Nasr et al. / Bioorg. Med. Chem. 22 (2014) 2867–2874
using sodium sulfate. 1H NMR spectra were recorded on an ARX
300 MHz Bruker spectrometer in CDCl3 with the use of the residual
solvent CHCl3 peak as reference. Elemental analyses were per-
formed at Service d’Analyse de l’Université Montpellier II.
5.1.2.8. 4Bim: Methyl 4-(2-hydroxy-5-methoxybenzylideneimi-
no)benzoate.
1H NMR (300 MHz, 9:1 CDCl3:CD3OD) d: 3.725
(s, 3H, OCH3), 3.846 (s, 3H, COOCH3), 6.816–6.925 (m, 3H, Ar-H);
7.240–7.268, 7.984–8.012 (d, 2H, Ar-H from methyl benzoate),
8.953 (s, 1H, CH@N). Elem. anal calcd for C16H15NO4: C, 67.36; H,
5.30; N, 4.91; O, 22.43; found C, 67.12; H, 5.29; N, 4.7.
5.1.1. General synthesis of imine compounds
1 mmol of amine A–E and 1.1 mmol of aldehyde 1–4 were
introduced in 50 ml of CHCl3:MeOH (5:1, v/v). The solution was
stirred at reflux for 18 h. The solvent was then evaporated and
the pure imine compounds were obtained after precipitated by
ether.
5.1.2.9. 1Cim: 4-(Benzylidene-imino)-benzenesulfonamide. 1H
NMR (300 MHz, 9:1 CDCl3:CD3OD) d: 7.128–7.155 (d, 2H, Ar-H
from benzenesulfonamide) 7.38 (m, 3H, Ar-H), 7.79 (m, 4H, 2
Ar-H and 2 Ar-H from benzenesulfonamide), 8.319 (s, 1H, CH=N).
Elem. anal calcd for C13H12N2O2S: C, 59.98; H, 4.65; N, 10.76; O,
12.29; S, 12.32; found C, 59.95; H, 4.91; N, 10.38; S, 12.14.
5.1.2. Imines
5.1.2.1. 1Aim: 4-(Benzylideneimino) benzoic acid.
1H NMR
(300 MHz, CDCl3) d: 7.251–7.279 (d, 2H, Ar-H from benzoic acid
moiety), 7.491–7.585 (m, 3H, Ar-H), 7.92 (dd, 2H, Ar-H), 8.168–
8.191 (d, 2H, Ar-H from benzoic acid), 8.472 (s, 1H, CH@N). Elem.
anal. calcd for C14H11NO2: C, 74.65; H, 4.92; N, 6.22; O, 14.21;
found C, 74.36; H, 5.11; N, 6.09.
5.1.2.10. 2Cim
sulfonamide.
:
4-[(4-tert-Butyl-benzylidene)-imino]-benzene-
1H NMR (300 MHz, 9:1 CDCl3: CD3OD) d:
1.189 (s, 9H, tBu). 7.079–7.108 (d, 2H Ar-H from benzenesulfon-
amide), 7.346–7.374, 7.650–7.678 (d, 2H, Ar-H), 7.741–7.770 (d,
2H, Ar-H from benzenesulfonamide), 8.255 (s, 1H, CH@N). Elem.
anal. calcd for C17H20N2O2S: C, 64.53; H, 6.37; N, 8.85; O, 10.11;
S, 10.13; found C, 64.63; H, 6.47; N, 8.55; S, 9.70.
5.1.2.2.
acid.
2Aim
:
4-(4-tert-Butylbenzylideneimino)
benzoic
1H NMR (300 MHz, CDCl3) d: 1.191 (s, 9H, tBu), 7.042–
7.07 (d, 2H, Ar-H from benzoic acid), 7.349–7.377 (d, 2H, Ar-H),
7.656–7.684 (d, 2H, Ar-H), 7.894–7.922 (d, 2H, Ar-H from benzoic
acid), 8.282 (s, 1H, CH@N). Elem. anal. calcd for C18H19NO2: C,
76.84; H, 6.81; N, 4.98; O, 11.37; found C, 75.90; H, 6.60; N, 4.75.
5.1.2.11. 3Cim: 4-[(4-Hydroxy-benzylidene)-imino]-benzenesul-
fonamide.
6.567 (d, 2H, Ar-H), 6.895–6.924 (d, 2H, Ar-H from benzene sulfon-
amide), 7.402–7.431 (d, 2H, Ar-H), 7.543–7.571 (d, 2H, Ar-H from
benzene sulfonamide), 8.017 (s, 1H, CH@N). Elem. anal. calcd for
1H NMR (300 MHz, 9:1 CDCl3:CD3OD) d: 6.538–
5.1.2.3.
acid.
3Aim
:
4-(4-Hydroxybenzylideneimino)
benzoic
1H NMR (300 MHz, CDCl3) d: 6.880–6.852 (d, 2H, Ar-
C13H12N2O3S: C, 56.51; H, 4.38; N, 10.14; O, 17.37; S, 11.60; found
C, 56.31; H, 4.46; N, 10.05; S, 11.75.
H), 7.147–7.120 (d, 2H, Ar-H from benzoic acid), 7.727–7.700 (d,
2H, Ar-H), 7.981–7.953 (d, 2H, Ar-H from benzoic acid), 8.316 (s,
1H, CH@N). Elem. anal calcd for C14H11NO3: C, 69.70; H, 4.60; N,
5.81; O, 19.90; found C, 69.76; H, 4.96; N, 5.80.
5.1.2.12. 4Cim: 4-[(2-Hydroxy-5-methoxy-benzylidene)-imino]-
benzenesulfonamide.
1H NMR (300 MHz, 9:1 CDCl3:CD3OD)
d: 3.684 (s, 3H, OCH3), 6.918–7.049 (m, 3H, H-Ar), 7.352–7.381,
7.936–7.965 (d, 2H, Ar-H from benzenesulfonamide), 8.642 (s,
1H, CH@N). Elem. anal calcd for C14H14N2O4S: C, 54.89; H, 4.61;
N, 9.14; O, 20.89; S, 10.47; found C, 55.00; H, 4.87; N, 8.71; S, 10.22.
5.1.2.4. 4Aim: 4-(2-Hydroxy-5-methoxybenzylideneimino)ben-
zoic acid.
1H NMR (300 MHz, CDCl3) d: 3.772 (s, 3H, OCH3),
6.891–6.900 (d, 1H, Ar-H), 6.910–6.940 (d, 1H, Ar-H), 6.973–
6.982, 7.004–7.012 (dd, 1H, Ar-H), 7.252–7.279 (d, 2H, Ar-H from
benzoic acid), 8.055–8.082 (d, 2H, Ar-H from benzoic acid), 8.569
(s, 1H, CH@N). Elem. anal. calcd for C15H13NO4: C, 66.41; H, 4.83;
N, 5.16; O, 23.59; found C, 66.30; H 5.2; N 4.93.
5.1.2.13. 1Dim: 4-[(Benzylidene-imino)-methyl]-benzenesulfon-
amide.
1H NMR (300 MHz, 9 CDCl3:1 CD3OD) d: 5 (s, 2H, CH2),
7.066–7.095 (d, 2H, Ar-H from benzenesulfonamide), 7.275–7.375
(m, 3H, Ar-H), 7.692–7.760 (m, 4H, 2Ar-H from benzenesulfon-
amide and 2Ar-H), 8.25 (s, 1H, CH@N). Elem. anal calcd for C14H14-
N2O2S: C, 61.29; H, 5.14; N, 10.21; O, 11.66; S, 11.69; found C,
61.08; H, 5.17; N, 9.87; S, 11.38.
5.1.2.5. 1Bim
:
Methyl 4-(benzylideneimino)benzoate.
1H
NMR (300 MHz, 9:1 CDCl3:CD3OD) d: 3.930 (s, 3H, COOCH3),
7.198–7.227 (d, 2H, Ar-H from methyl benzoate), 7.5 (m, 3H, Ar-
H), 7.901–7.928 (d, 2H, Ar-H), 8.066–8.095 (d, 2H, Ar-H from
methyl benzoate), 8.441 (s, 1H, CH@N). Elem. Anal calcd for
5.1.2.14. 2Dim: 4-{[(4-tert-Butyl-benzylidene)-imino]-methyl}-
benzenesulfonamide.
1H NMR (300 MHz, 9 CDCl3:1 CH3OD)
C15H13NO2: C, 75.30; H, 5.48; N, 5.85; O, 13.37; found C, 75.25;
d: 1.177 (s, 9H, tBu), 3.162 (s, 2H, CH2), 7.073–7.102 (d, 2H, Ar-H
from benzenesulfonamide), 7.337–7.365, 7.639–7.667 (d, 2H, Ar-
H), 7.732–7.760 (d, 2H, Ar-H from benzenesulfonamide), 8.248 (s,
1H, CH@N). Elem. anal calcd for C18H22N2O2S: C, 66.26; H, 8.34;
N, 7.73; O, 8.83; S, 8.85; found C, 66.53; H, 8.84; N, 7.96.
H, 5.69; N, 5.61.
5.1.2.6. 2Bim: Methyl 4-(4-tert-butylbenzylideneimino)benzo-
ate.
1H NMR (300 MHz, 9:1 CDCl3/CD3OD) d: 1.363 (s, 9H,
tBu), 3.889 (s, 3H, COOCH3), 7.244–7.271 (d, 2H, Ar-H from methyl
benzoate), 7.564–7.592, 7.859–7.886 (d, 2H, Ar-H), 8.025–8.053 (d,
2H, Ar-H from methyl benzoate), 8.497 (s, 1H, CH@N). Elem. anal
calcd for C19H21NO2: C, 77.26; H, 7.17; N, 4.74; O, 10.83; found C,
77.07; H, 7.38; N, 4.43.
5.1.2.15. 3Dim
benzenesulfonamide.
:
4-{[(4-Hydroxy-benzylidene)-imino]-methyl}-
1H NMR (300 MHz, 9 CDCl3: 1 CD3OD)
d: 4.77 (s, 2H, CH2), 6.584–6.613 (d, 2H, Ar-H), 6.714–6.742 (d, 2H,
Ar-H from benzenesulfonamide), 7.157–7.185 (d, 2H, Ar-H), 7.524–
7.554 (d, 2H, Ar-H from benzenesulfonamide), 8.266 (s, 1H, CH@N).
Elem. anal calcd for C14H14N2O3S: C, 57.92; H, 4.86; N, 9.65; O,
16.53; S, 11.04; found C, 56.26; H, 4.98; N, 8.81; S, 11.40.
5.1.2.7. 3Bim
: Methyl 4-(4-hydroxybenzylideneimino)benzo-
ate.
1H NMR (300 MHz, 9:1 CDCl3:CD3OD) d: 3.946 (s, 3H,
COOCH3), 6.940–6.968 (d, 2H, Ar-H), 7.196–7.224 (d, 2H, Ar-H from
methyl benzoate), 7.828–7.857 (d, 2H, Ar-H), 8.070–8.098 (d, 2H,
Ar-H from methyl benzoate), 8.377 (s, 1H, CH@N). Elem. anal calcd
for C15H13NO3: C, 70.58; H, 5.13; N, 5.49; O, 18.80; found C, 69.81;
H, 5.01; N, 5.31.
5.1.2.16. 4Dim: 4-{[(2-Hydroxy-5-methoxy-benzylidene)-imino]-
methyl}-benzenesulfonamide.
1 CD3OD) d: 3.821 (s, 3H, OCH3), 6.914–6.923 (d, 1H, Ar-H),
6.977–7.007 (d, 1H, Ar-H), 7.043–7.053, 7.073–7.083 (dd, 1H,
1H NMR (300 MHz, 9 CDCl3:

G. Nasr et al. / Bioorg. Med. Chem. 22 (2014) 2867–2874
2873
Ar-H), 7.358–7.386, 7.980–8.008 (d, 2H, Ar-H from benzenesulfon-
amide), 8.586 (s, 1H, CH@N). Elem. anal calcd for C15H16N2O4S: C,
56.24; H, 5.03; N, 8.74; O, 19.98; S, 10.01; found C, 55.16; H,
5.14; N, 8.07; S, 9.59.
5.1.4.4. 4Aam: 4-(2-Hydroxy-5-methoxybenzylideneamino)ben-
zoic acid.
1H NMR (300 MHz, CD3OD) d: 3.633 (s, 3H, OCH3),
4.323–4.339 (d, 2H, CH2), 6.593–6.650 (m, 3H, Ar-H), 6.711–
6.794 (m, 2H, Ar-H from benzoic acid), 7.740–7.767 (d, 2H, Ar-H
from benzoic acid).
5.1.2.17. 1Eim: 4-[2-(Benzylidene-imino)-ethyl]-benzenesulfon-
amide.
1H NMR (300 MHz, CD3CN) d: 3.059–3.105 (t, 2H,
5.1.4.5. 1Bam: Methyl 4-(benzylideneamino)benzoate.
1H
CH2), 3.858–3.905 (td, 2H, CH2), 5.636 (s, 2H, SO2NH2), 7.448–
7.475 (m, 5H, 2 Ar-H from benzenesulfonamide and 3 Ar-H),
7.700–7.732 (m, 2H, Ar-H), 7.780–7.808 (d, 2H, Ar-H from ben-
zenesulfonamide), 8.249 (s, 1H, CH@N). Elem. anal calcd for C15H16-
N2O2S: C, 62.48; H, 5.59; N, 9.71; O, 11.10; S, 11.12; found C, 62.23;
H, 6.02; N, 9.41; S, 9.15.
NMR (300 MHz, CD3OD) d: 3.231 (s, 3H, OCH3), 3.771 (s, 2H,
CH2), 5.962–6.002 (t, 2H, Ar-H), 6.734 (m, 5H, Ar-H), 7.261–7.274
(d, 2H, Ar-H).
5.1.4.6. 2Bam: Methyl 4-(4-tert-butylbenzylideneamino)benzo-
ate.
1H NMR (300 MHz, CDCl3) d: 1.321 (s, 9H, tBu), 3.845 (s,
2H, OCH3), 4.334–4.352 (d, 2H, CH2), 6.578–6.607 (d, 2H, Ar-H from
methyl-benzoate), 7.259–7.294, 7.374–7.396 (d, 2H, Ar-H), 7.849–
7.878 (d, 2H, Ar-H from methyl-benzoate). 13C (75 Hz, CDCl3) d:
18.2, 31, 34.5, 47.2, 51.4, 58.3, 111.8, 118.5, 125.7, 127, 131.7,
135, 150.6, 151.8, 167.2.
5.1.2.18. 2Eim: 4-{2-[(4-tert-Butyl-benzylidene)-imino]-ethyl}-
benzenesulfonamide.
1H NMR (300 MHz, CD3CN) d: 1.338
(s, 9H, tBu), 3.049–3.095 (t, 2H, CH2), 3.840–3.889 (td, 2H, CH2),
5.625 (s, 2H, SO2NH2), 7.441–7.469 (d, 2H, Ar-H from benzenesul-
fonamide), 7.476–7.505, 7.623–7.651 (d, 2H, Ar-H), 7.775–7.802 (d,
2H, Ar-H from benzenesulfonamide), 8.206 (s, 1H, CH@N).1 Elem.
anal calcd for C19H24N2O2S: C, 66.25; H, 7.02; N, 8.13; O, 9.29; S,
9.31; found C, 64.44; H, 7.52; N, 7.55; S, 10.34.
5.1.4.7. 3Bam
ate.
: Methyl 4-(4-hydroxybenzylideneamino)benzo-
1H NMR (300 MHz, CDCl3) d: 3.845 (s, 2H, OCH3),
6.578–6.607 (d, 2H, Ar-H from methyl-benzoate), 7.259–7.274,
7.374–7.396 (d, 2H, Ar-H), 7.849–7.878 (d, 2H, Ar-H from
methyl-benzoate).
5.1.2.19. 3Eim
benzenesulfonamide.
:
4-{2-[(4-Hydroxy-benzylidene)-imino]-ethyl}-
1H NMR (300 MHz, 9 CDCl3: 1 CH3OH)
d: 3.251–3.298 (t, 2H, CH2), 4.022–4.069 (t, 2H, CH2), 5.06 (s, 1H,
OH), 7.029–7.057, 7.598–7.625 (d, 2H, Ar-H from benzenesulfon-
amide), 7.749–7.777, 7.994–8.022 (d, 2H, Ar-H), 8.318 (s, CH@N).
Elem.anal. calcd for C15H16N2O3S: C, 59.19; H, 5.30; N, 9.20; O,
15.77; S, 10.54; found C, 58.50; H, 5.46; N, 9.75; S, 9.93.
5.1.4.8. 4Bam: Methyl 4-(2-hydroxy-5-methoxybenzylideneami-
no)benzoate.
CH3–O–Ar), 3.730 (s, 3H, COOCH3), 4.220 (s, 2H, CH2N)6.589–
6.657 (m, 3H, 3H from methyl-benzoate and 1H from Ar-H),
6.719–6.760 (m, 2H, Ar-H), 7.651–7.678 (d, 2H, Ar-H from
methyl-benzoate).
1H NMR (300 MHz, DMSO-d6) d: 3.59 (s, 3H,
5.1.2.20.
4Eim
:
4-{2-[(2-Hydroxy-5-methoxy-benzylidene)-
1H NMR (300 MHz,
imino]-ethyl}-benzenesulfonamide.
5.1.4.9. 1Cam: 4-(Benzylidene-amino)-benzenesulfonamide. 1H
NMR (300 MHz, DMSO-d6) d: 4.410 (s, 2H, CH2), 6.698 (s, 2H,
Ar-H), 7.058 (s, 1H, Ar-H), 7.313–7.552 (m, 6H, Ar-H).
CDCl3) d: 3.051–3.096 (t, 2H, CH2), 3.739 (t, 3H, O-CH3), 3.849–
3.895 (t, 2H, CH2), 6.739–6.748, 6.795–6.825 (d, 1H, Ar-H),
6.867–6.877, 6.897–6.907 (dd, 1H, Ar-H), 7.352–7.381, 7.786–
7.813 (d, 1H, Ar-H from benzenesulfonamide), 8.238 (s, 1H, CH@N).
Elem. anal calcd for C16H18N2O4S: C, 57.47; H, 5.43; N, 8.38; O,
19.14; S, 959; found C, 57.26; H, 5.13; N, 8.65.
5.1.4.10. 2Cam: 4-[(4-tert-Butyl-benzylidene)-amino]-benzene-
sulfonamide.
1H NMR (300 MHz, DMSO-d6) d: 1.262 (s, 9H,
tB), 4.275–4.294 (d, 2H, CH2), 6.621–6.645 (d, 2H, Ar-H from ben-
zenesulfonamide), 6.889 (s, 2H, SO2NH2), 6.932–6.970 (t, 1H, NH),
7.251–7.274, 7.335–7.363 (dd, 2H, Ar-H), 7.465–7.494 (d, 2H, Ar-H
from benzenesulfonamide).
5.1.3. General synthesis of amine compounds
One mole of imine was dissolved in 30 ml of MeOH. 10 equiva-
lent of NaBH4 was added during one hour. The mixture is left under
stirring for 3 more hours. The mixture was evaporated than dis-
solved in acid solution and the amine is extracted in ether. Some
amines were purified in flash column to eliminate all salt residues.
We may note that all the amino compounds reported bellow are
chromatographically pure following a study reported previously:
see Ref.6b
5.1.4.11. 3Cam: 4-[(4-Hydroxy-benzylidene)-amino]-benzenesul-
fonamide.
1H NMR (300 MHz, DMSO-d6) d: 6.760–6.789 (d,
2H, Ar-H from benzenesulfonamide), 7.032–7.061 (d, 2H), 7.124–
7.153 (d, 2H), 7.718–7.748 (d, 2H, Ar-H from benzenesulfonamide).
5.1.4.12. 4Cam: 4-[(2-Hydroxy-5-methoxy-benzylidene)-amino]-
benzenesulfonamide.
1H NMR (300 MHz, DMSO-d6) d: 3.581
5.1.4. Amines
5.1.4.1. 1Aam: 4-(Benzylideneamino) benzoic acid.
(300 MHz, CDCl3) d: 4.434 (s, 2H, CH2-Ar), 6.652–6.681 (d, 2H,
Ar-H from benzoic acid), 7.372–7.385 (m, 5H, Ar-H), 7.927–7.955
(d, 2H, Ar-H).
(s, 3H, CH3), 4.208 (s, 2H, CH2), 6.519–6.579 (m, 3H, 2 Ar-H from
benzenesulfonamide and 1 Ar-H), 6.665–6.694 (m, 2H, Ar-H),
7.470–7.499 (d, 2H, Ar-H from benzenesulfonamide).
1H NMR
5.1.4.13. 1Dam
fonamide.
:
4-[(Benzylidene-amino)-methyl]-benzenesul-
1H NMR (300 MHz, CD3OD/CDCl3) d: 3.734–3.797
5.1.4.2. 2Aam: 4-(4-tert-Butylbenzylideneamino)benzoic acid. 1H
NMR (300 MHz, CDCl3) d: 1.234 (s, 9H, tBu), 4.239 (s, 2H, CH2),
6.472–6.501 (d, 2H, Ar-H from benzoic acid), 7.221–7.293 (m, 4H,
Ar-H), 7.652–7.681 (d, 2H, Ar-H from benzoic acid).
(d, 4H, CH2), 7.305 (m, 5H, Ar-H), 7.414–7.438, 7.8–7.85 (d, 2H,
Ar-H from benzenesulfonamide). 13C (75 Hz, CDCl3) d: 5, 56, 57,
130, 131, 132.1, 132.2, 132.3, 145.2, 146, 148.
5.1.4.14. 2Dam: 4-{[(4-tert-Butyl-benzylidene)-amino]-methyl}-
benzenesulfonamide.
9H, tBu), 3.641–3.744 (d, 4H, CH2), 7.175–7.202 (d, 2H, Ar-H),
7.294–7.316, 7.434–7.462 (d, 2H, Ar-H from benzenesulfonamide),
7.790–7.818 (d, 2H, Ar-H).
5.1.4.3. 3Aam: 4-(4-Hydroxybenzylideneamino)benzoic acid. 1H
NMR (300 MHz, CD3OD) d: 6.592–6.621 (d, 2H, Ar-H from benzoic
acid), 6.746–6.773, 7.170–7.197 (d, 2H, Ar-H), 7.751–7.774 (d, 2H,
Ar-H from benzoic acid).
1H NMR (300 MHz, CD3OD) d: 1.246 (s,

2874
G. Nasr et al. / Bioorg. Med. Chem. 22 (2014) 2867–2874
5.1.4.15. 3Dam
benzenesulfonamide.
:
4-{[(4-Hydroxy-benzylidene)-amino]-methyl}-
1H NMR (300 MHz, CD3OD) d: 3.574,
3
and represent the mean from at least three different
determinations.
3.721 (s, 2H, CH2), 6.637–6.665 (d, 2H, Ar-H), 7.051–7.078 (d, 2H,
Ar-H from benzenesulfonamide), 7.417–7.444 (d, 2H, Ar-H),
7.782–7.809 (d, 2H, Ar-H from benzenesulfonamide).
Acknowledgements
This work was supported by funds from ITN Marie Curie Net-
work-DYNANO, PITN-GA-2011-289033 (www.dynano.eu).
5.1.4.16. 4Dam: 4-{[(2-Hydroxy-5-methoxy-benzylidene)-amino]-
methyl}-benzenesulfonamide.
1H NMR (300 MHz, CD3OD) d:
3.580 (s, 3H, CH3),, 3.721 (s, 2H, CH2), 6.637–6.665 (d, 2H, Ar-H),
7.051–7.078 (d, 2H, Ar-H from benzenesulfonamide), 7.417–
7.444 (d, 2H, Ar-H), 7.782–7.809 (d, 2H, Ar-H from
benzenesulfonamide).
References and notes
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5.1.4.17. 1Eam: 4-[2-(Benzylidene-amino)-ethyl]-benzenesulfon-
amide.
1H NMR (300 MHz, D2O) d: 2.109, 3.701 (t, 2H, CH2),
4.219 (s, 2H, CH2N), 7.152 (m, 7H, Ar-H), 7.731–7.759 (2H, Ar-H
from benzenesulfonamide).
5.1.4.18. 2Eam: 4-{2-[(4-tert-Butyl-benzylidene)-amino]-ethyl}-
benzenesulfonamide.
1H NMR (300 MHz, CDCl3) d: 1.132 (s,
9H, tBu), 2.171 (s, 4H, CH2), 3.574 (s, 2H, CH2N), 7.000–7.028 (d,
2H, Ar-H from benzenesulfonamide), 7.136–7.191 (td, 4H, Ar-H),
7.636–7.664 (d, 2H, Ar-H from benzenesulfonamide).
4. (a) Supuran, C. T.; Nicolae, A.; Popescu, A. Eur. J. Med. Chem. 1996, 31, 431; (b)
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C.; Barboiu, M.; Supuran, C. T. Revue Roum. Chim. 1991, 36, 1169.
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Creanga, A.; Bobes-Tureac, G.; Banciu, M. D. Eur. J. Med. Chem. 1997, 32, 445; (b)
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7. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561.
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2008, F64, 163.
5.1.4.19. 3Eam
: 4-{2-[(4-Hydroxy-benzylidene)-amino]-ethyl}-
benzenesulfonamide. 1H NMR (300 MHz, CD3OD) d: 3.035, 4.044
(t, 2H, CH2), 6.739–6.768 (d, 2H, Ar-H from benzenesulfonamide),
7.230–7.259, 7.359–7.387 (d, 2H, Ar-H), 7.767–7.795 (t, 2H, Ar-H
from benzenesulfonamide).
5.1.4.20. 4Eam
:
4-{2-[(2-Hydroxy-5-methoxy-benzylidene)-
amino]-ethyl}-benzenesulfonamide. 1H NMR (300 MHz, D2O)
d: 3.082, 3.291 (t, 2H, CH2), 3.708 (s, 3H, OCH3), 4.163 (s, 2H,
CH2N), 6.795 (m, 3H, Ar-H), 7.391–7.419, 7.779–7.807 (d, 2H, Ar-
H from benzenesulfonamide).
9. (a) Casini, A.; Antel, J.; Abbate, F.; Scozzafava, A.; David, S.; Waldeck, H.;
Schäfer, S.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2003, 13, 841; (b) Borras, J.;
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Karch, S. B. J. R. Soc. Med. 2004, 97, 397.
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Houston, T. A.; Innocenti, A.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2006,
49, 6539; (c) Singh, S.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 666.
12. (a) Abbate, F.; Coetzee, A.; Casini, A.; Ciattini, S.; Scozzafava, A.; Supuran, C. T.
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247.
5.2. CA inhibition assay
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-
flow instrument has been used to assay the catalytic/inhibition
of various CA isozymes as reported by Khalifah.7 Phenol Red (at
a concentration of 0.2 mM) has been used as indicator, working
at the absorbance maximum of 557 nm, with 10 mM Hepes (pH
7.4) as buffer, 0.1 M Na2SO4 or NaClO4 (for maintaining constant
the ionic strength; these anions are not inhibitory in the used
concentration), following the CA-catalyzed CO2 hydration reaction
for a period of 5–10 s. Saturated CO2 solutions in water at 25 °C
were used as substrate. Stock solutions of inhibitors were pre-
pared at a concentration of 10 mM (in DMSO–water 1:1, v/v)
and dilutions up to 1 nM done with the assay buffer mentioned
above. At least 7 different inhibitor concentrations have been
used for measuring the inhibition constant. Inhibitor and enzyme
solutions were preincubated together for 10 min at room
temperature prior to assay, in order to allow for the formation
of the E–I complex. Triplicate experiments were done for each
inhibitor concentration, and the values reported throughout
the Letter are the mean of such results. The inhibition constants
were obtained by non-linear least-squares methods using PRISM
14. (a) Abbate, F.; Casini, A.; Owa, T.; Scozzafava, A.; Supuran, C. T Bioorg. Med.
Chem. Lett. 2004, 14, 217; (b) Fabrizi, F.; Mincione, F.; Somma, T.; Scozzafava,
G.; Galassi, F.; Masini, E.; Impagnatiello, F.; Supuran, C. T. J. Enzyme Inhib. Med.
Chem. 2012, 27, 138; (c) Bertol, E.; Mari, F.; Milia, M. G.; Politi, L.; Furlanetto, S.;
Karch, S. B. J. Pharm. Biomed. Anal. 2011, 55, 1186.
15. (a) Vullo, D.; Franchi, M.; Gallori, E.; Antel, J.; Scozzafava, A.; Supuran, C. T. J.
Med. Chem. 2004, 47, 1272; (b) Alp, C.; Özsoy, S.; Alp, N. A.; Erdem, D.; Gültekin,
_
M. S.; Küfreviog˘lu, Ö. I.; Sßentürk, M.; Supuran, C. T. J. Enzyme Inhib. Med. Chem.
2012, 27, 818.