Mono-/dihydroxybenzoic acid esters and phenol pyridinium derivatives as inhibitors of the mammalian carbonic anhydrase isoforms I, II, VII, IX, XII and XIV

Article abstract of DOI:10.1016/j.bmc.2012.05.019

Using hydroxy-/dihydroxybenzoic acids as leads, a series of methyl, ethyl and iso-propyl esters of 4-hydroxy-benzoic acid, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dihydroxybenzoic acids and of coumaric acid, were obtained and investigated for the inhibition of six mammalian carbonic anhydrase (CA, EC 4.2.1.1) isoforms, that is, the cytosolic CA I, II and VII, and the transmembrane CA IX, XII and XIV, many of which are established drug targets. Other compounds incorporating phenol/catechol moieties were obtained from dopamine by reaction with fluorescein isothiocyanate or with 2,4,6-trisubstituted pyrylium salts. Some aminophenols were also derivatized in a similar manner, by using pyrylium salts. Many of these compounds showed increased inhibitory action compared to the lead compounds from which they were obtained, with efficacy in the submicromolar range against most investigated CA isoforms. As phenols are a class of less investigated CA inhibitors (CAIs) compared to the sulfonamides, and their mechanism of inhibition is less well understood, compounds of the type designed here may be helpful in gaining more insights into these phenomena.

Full text of DOI:10.1016/j.bmc.2012.05.019

Bioorganic & Medicinal Chemistry 21 (2013) 1564–1569
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Mono-/dihydroxybenzoic acid esters and phenol pyridinium derivatives
as inhibitors of the mammalian carbonic anhydrase isoforms I, II, VII, IX, XII
and XIV
⇑
⇑
Fabrizio Carta , Daniela Vullo, Alfonso Maresca, Andrea Scozzafava, Claudiu T. Supuran
Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 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:
Available online 17 May 2012
Using hydroxy-/dihydroxybenzoic acids as leads, a series of methyl, ethyl and iso-propyl esters of
4-hydroxy-benzoic acid, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dihydroxybenzoic acids and of coumaric acid, were
obtained and investigated for the inhibition of six mammalian carbonic anhydrase (CA, EC 4.2.1.1) iso-
forms, that is, the cytosolic CA I, II and VII, and the transmembrane CA IX, XII and XIV, many of which
are established drug targets. Other compounds incorporating phenol/catechol moieties were obtained
from dopamine by reaction with fluorescein isothiocyanate or with 2,4,6-trisubstituted pyrylium salts.
Some aminophenols were also derivatized in a similar manner, by using pyrylium salts. Many of these
compounds showed increased inhibitory action compared to the lead compounds from which they were
obtained, with efficacy in the submicromolar range against most investigated CA isoforms. As phenols are
a class of less investigated CA inhibitors (CAIs) compared to the sulfonamides, and their mechanism of
inhibition is less well understood, compounds of the type designed here may be helpful in gaining more
insights into these phenomena.
Keywords:
Carbonic anhydrase
Phenol
Hydroxy-/dihydroxybenzoic acid
Dopamine
Aminophenol
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
metal complexing anions,16 thiols and dithiocarbamates.17 They
coordinate to the Zn(II) ion from the enzyme active site, in tetrahe-
Non-sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors
(CAIs) were investigated in great detail in the last period. Many
new such chemotypes were identified, among which phenols1–4
and thiols,5 coumarins, thiocoumarins, 2-thioxo-coumarins and
5/6-ring membered (thio)lactones,6–8 the antiepileptic drug lacosa-
mide,9 polyamines,10 some fullerene derivatives11 and even com-
pounds expected to bind as CA activators (pyridinium histamine
derivatives),12 which were observed deeply bound within the en-
zyme active site (by means of high resolution X-ray crystallogra-
phy) where usually inhibitors are located.13–15
dral or trigonal bipyramidal geometries of the Zn(II) ion;13–17 (ii)
Compounds anchoring to the non-protein zinc ligand, such as phe-
nols and polyamines. They bind to the zinc-coordinated water mol-
ecule/hydroxide ion from the active site, through a network of two
hydrogen bonds;8 and (iii) Coumarins/thiocoumarins and similar
compounds (lactones, coumarin oximes, 2-thioxocoumarins, etc.)
acting as ‘prodrug inhibitors’. They bind after in situ hydrolysis
to 2-hydroxycinnamic acids (or the corresponding compounds
formed from thiocoumarins, coumarin oximes or lactones/thiolac-
tones, respectively) in the same active site region as the CA activa-
tors,18 occluding the entrance to the active site cavity.10
Fullerenes13 and lacosamide10 bind in a similar manner to the
hydrolyzed coumarins,10 that is, by occluding the entrance to the
enzyme active site cavity.
CAIs investigated in detail so far, by means of high resolution X-
ray crystallography, belong to three main classes: (i) Zinc binders,
such as sulfonamides (and their bioisosteres, including sulfamates,
sulfamides, N-hydroxysulfonamides, and similar derivatives),13–15
OMe
COOH
OMe
COOH
OMe
OMe
MeO
MeO
MeO
OMe
OMe
OMe
OMe
A
B
C
D
E
⇑
Corresponding authors. Tel.: +39 055 457 3005; fax: +39 055 4573385.
E-mail addresses: fabrizio.carta@unifi.it (F. Carta), claudiu.supuran@unifi.it (C.T. Supuran).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.019

F. Carta et al. / Bioorg. Med. Chem. 21 (2013) 1564–1569
1565
Table 1
Recently, by means of kinetic measurements and docking
experiments, we identified a fourth possible CA inhibition mecha-
nism.1 We observed that some hydroxy-/methoxy-substituted
benzenes and benzoic acids, of types A–E, acting as low micromo-
lar inhibitors of several CA isozymes,1 bind differently of all other
CAIs known to date. These compounds were observed to be located
between the phenol-binding site and the coumarin-binding site
within the enzyme cavity, by using molecular docking. They are
thus situated more or less in the middle of the cavity,1 exploiting
different interactions with amino acid residues and water mole-
cules from the active site compared to other classes of inhibitors
investigated so far and mentioned above. In this way, they offer
an interesting possibility to design CAIs with a different inhibition
profile compared to the clinically used sulfonamides/sulfamates, or
the other classes of inhibitors investigated in some detail ulti-
mately, such as the coumarins, dithiocarbamates, etc. However,
only very few such derivatives have been investigated so far, and
only for their interaction with four CA isoforms (CA I, II, IX and
XII) in addition to A–E mentioned above. Thus, by using those com-
pounds as leads, we report here a series of esters of hydroxy-/dihy-
droxy benzoic acids (5 regiomers of dihydroxybenzoic acids were
investigated, i.e., 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dihydroxybenzoic
acids), as well as other compounds incorporating phenol or cate-
chol moieties and pyridinium/fluorescein functionalities in their
molecules, which were investigated for the inhibition of 6 physio-
logically relevant hCA (h = human enzyme) isoforms, i.e., hCA I, II,
VII (cytosolic isoforms) and hCA IX, XII and XIV (transmembrane
isoforms), many of which are established drug targets.
Inhibition data with compounds 1–7, against CA isozymes I, II, VII, IX, XII, and XIV by a
stopped-flow technique monitoring the CO2 hydration reaction38
COOR
COOR
OH
COOR
OH
COOR
OH
HO
HO
OH
1a-1d
OH
2a-2d
3a-3d
4a-4d
COOR
COOR
COOR
HO
OH
HO
OH
OH
5a-5d
7a-7d
6a-6d
No.
R
KIb
(lM)
hCA I
hCA II
hCA VII
hCA IX
hCA XII
hCA XIV
1aa
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
5ab
5b
5c
5d
6ab
6b
6c
6d
7ªc
7b
7c
7d
Ab
Bb
H
9.8
10.6
3.4
4.8
4.4
4.9
0.76
0.52
0.51
4.1
0.77
0.69
0.58
5.2
0.70
0.56
0.53
0.47
0.58
0.61
0.42
0.51
0.69
0.66
0.70
0.98
0.80
0.71
0.52
0.50
3.35
1.43
6.18
1.02
716
0.78
8.7
0.94
72
0.85
0.81
0.54
68
118
5.5
8.2
7.4
68
0.89
0.82
0.31
6.6
0.84
0.86
0.81
5.9
0.81
0.78
0.79
4.45
0.79
0.78
0.80
4.41
0.81
0.91
0.90
5.33
0.85
0.61
0.68
8.63
4.15
4.06
8.25
9.07
4.8
0.82
8.6
34.3
4.1
7.7
Me
Et
2.6
7.9
i-Pr
H
7.3
5.2
8.5
7.5
0.82
70
Me
Et
i-Pr
H
0.83
0.63
0.39
4.2
0.95
0.77
0.50
7.3
0.86
0.74
0.33
67
Me
Et
i-Pr
H
0.82
0.71
0.97
5.7
8.3
0.83
0.75
0.82
7.2
0.80
0.77
0.84
6.5
0.83
0.84
6.6
2. Chemistry
Me
Et
i-Pr
H
Me
Et
i-Pr
H
Me
Et
i-Pr
H
Me
Et
i-Pr
—
0.68
0.67
0.72
1.08
0.71
0.65
0.83
0.55
0.77
0.69
0.92
1.07
0.78
0.32
0.54
10.4
8.21
5.96
6.83
9.67
0.71
0.76
0.80
7.87
0.81
0.75
0.91
7.17
0.78
0.90
0.89
5.23
0.82
0.85
0.69
7.25
3.88
3.70
7.15
3.22
0.72
0.84
0.88
4.09
0.96
0.83
0.86
3.67
0.84
0.85
0.89
8.01
0.81
0.49
0.76
8.36
4.02
7.80
6.70
8.23
0.76
0.82
0.83
0.69
0.89
0.86
0.77
0.65
0.78
0.83
0.85
6.68
0.83
0.80
0.73
6.83
6.79
0.63
0.74
0.55
The lead molecules for the present drug design study were the
hydroxy-/methoxy-substituted benzenes and benzoic acids, of
types A–E investigated in the earlier work.1 The phenol moiety
from a non-sulfonamide CAI is important for the possible anchor-
ing to the zinc coordinated water molecule/hydroxide ion,1,2
whereas the COOH moiety (as carboxylate) when present in a com-
pound, may coordinate to this metal ion.16 We thus decided to use
hydroxy-/dihydroxy benzoic acids as well as p-coumaric acid as
starting materials for obtaining a first group of compounds. The
working hypothesis was the following: the OH moiety/moieties
of these compounds will be left free, whereas the COOH one will
be derivatized (as esters) in order to evaluate the structure–
activity relationship (SAR) in a series of compounds which are
not able any longer to coordinate the Zn(II) ion as carboxylates
(Table 1). Thus, carboxylic acids 1a–7a have been converted to
the corresponding methyl-, ethyl and isopropyl esters 1b–7d, by
a routine esterification procedure involving the reaction between
the carboxylic acid and the corresponding alcohol (see Section 5
for details). Some of these compounds were reported earlier in
the literature.19–28
—
—
—
—
Cb
Db
Eb
a
b
c
From Ref.3c
.
Data for hCA I, II, IX and XII from Ref. 1, data for hCA VII and XIV this work.
From Ref.4b
Another group of derivatives were prepared in such a way as to
include pyridinium moieties attached to scaffolds possessing one or
two phenolic OH moieties (Schemes 1 and 2).29,30 The presence of
the substituted pyridinium moieties was desired because of at least
two reasons: sulfonamides incorporating this substitution pattern
showed excellent CA inhibitory activity against several isoforms,31
and in the X-ray crystal structure of adducts with CA II, a stacking
interaction between the 2,4,6-trisubstituted pyridinium ring and
the phenyl moiety of Phe131 from the enzyme cavity has been
evidenced.32 Such a stacking interaction would be of interest to
investigate for non-sulfonamide CAIs, such as the phenols. For
introducing chemical diversity, both 2,4,6-trimethylpyrylium per-
chlorate and 4-phenyl-2,6-dimethyl-pyrylium perchlorate were
used to derivatize dopamine 8, leading to the pyridinium salts 10
R
OH
NH2
HO
H
N
HO
FITC
O+
S
HO
-
ClO4
HN
COOH
OH
N+
OH
-
ClO4
8
R
HO
O
O
10: R = Me
11: R = Ph
9
Scheme 1. Preparation of derivatized dopamine derivatives 9–11.

1566
F. Carta et al. / Bioorg. Med. Chem. 21 (2013) 1564–1569
VII, IX and XIV, with KIs of 68–72 lM; 3a against hCA VII
and XIV, etc—see Table 1). Typically, the esterification led
to a net increase of the inhibitory power compared to the
free carboxylic acid. For example, esters of 4-hydroxybenzo-
NH2
R
N
ClO4
R
MeOH
O
ic acid 1a, a very weak hCA VII inhibitor (KI of 716
showed inhibition constants against the same isoform in
the range of 0.78–8.7 M. The 2,6-dihydroxybenzoic acid
4a was a micromolar hCA I inhibitor (KI of 5.7 M) whereas
its esters showed KIs in the range of 0.68–0.72 M against
lM)
OH
BF4
OH
l
l
12: R = H
13: R = Cl
14: R = H
15: R = Cl
l
this isoform. This situation was the one encountered for
most of the investigated carboxylic acids/esters from this
work. It should be also noted that all esters were more effec-
tive CAIs compared to the lead compounds A–E investigated
earlier (Table 1).1
Scheme 2. Preparation of pyridinium phenol derivatives 14 and 15.
Table 2
Inhibition data with compounds 9–15, against CA isozymes I, II, VII, IX, XII, and XIV by
(ii) The nature of the R group present in the investigated esters
2b–7d was one of the most important factors influencing CA
inhibitory activity, but SAR was not always straightforward.
For example, in the case of 2,4-dihydroxybenzoic acid esters
2b–2d, against all isoforms, inhibitory activity increased
from the methyl to the ethyl and then the isopropyl deriva-
tive, in a quasi linear manner (Table 1). However, this simple
situation was seen only rarely, for example for the deriva-
tives 3b–3d against isoforms hCA II and VII; 4b–4d against
isoforms hCA II and IX or 7b–7d against isoforms hCA II
and XIV (Table 1). In most other cases all esters showed a
rather compact and similar inhibitory activity. For example,
a stopped-flow technique monitoring the CO2 hydration reaction37
No.
R
KI (lM)
hCA I
hCA II
hCA VII
hCA IX
hCA XII
hCA XIV
9
—
4.8
7.4
5.1
4.9
6.3
0.39
0.50
4.4
6.8
4.7
4.7
4.9
0.32
0.49
6.8
8.7
7.2
5.9
6.4
0.72
0.73
6.7
0.73
0.76
0.40
7.7
7.5
0.54
0.78
0.71
0.81
0.74
7.2
6.4
0.70
0.74
10
11
12
13
14
15
Me
Ph
H
Cl
H
0.83
0.71
4.9
4.7
0.68
0.70
Cl
and 11, or aminophenols 12 and 13, leading to the pyridinium salts
14 and 15 (Schemes 1 and 2). Dopamine 8 was also reacted with
fluorescein isothioyanate (FITC),33 leading to the fluorescent thio-
urea compound 9 (Scheme 1). This compound was prepared consid-
ering the sulfonamides tagged with fluorescent tails derived from
FITC, which were essential in demonstrating the role of CA IX in
the tumor acidification processes.34 Thus, obtaining a fluorescent
non-sulfonamide CAI may be useful in biological studies in which
the fluorescence is used as a marker of hypoxia or binding to a CA
isoform present in that tissue.35 The compounds 1b–7d, 9–11 and
14, 15 investigated here were characterized by standard physico-
chemical and spectral procedures which confirmed their structures
(see Section 5 for details).
the esters 5b–5d showed KIs in the range of 0.65–0.83
lM
against hCA I, of 0.75–0.91 M against hCA VII, etc. This
l
was the most typical situation for the compounds investi-
gated here, that is, slight and disordered variation of the
inhibitory activity when passing from the methyl to the
ethyl and isopropyl derivatives.
(iii) The number of hydroxyl moieties and their positions for the
regiomeric dihydroxybenzoic acids also influenced activity
of these esters, as observed from data of Table 1. Thus, the
monohydroxy phenols 1b–1d were the least active CAIs
against all isoforms investigated here whereas the longer
molecule esters of coumaric acid 7a, together with the
diphenols 2b–2d to 6b-6d were stronger, submicromolar
CAIs compared to the corresponding esters of 4-hydroxy-
benzoic acid. The position of the two hydroxyl moieties from
these last derivatives with respect to each other and the
ester group also influenced activity but to a less extent, since
the biological activity of the diphenols was in the same
range for the various isomers investigated here (Table 1).
(iv) The dopamine derivatives 9–11 were also effective, low
micromolar or submicromolar CAIs, although dopamine
itself is a CA activator.12–14 Indeed, the activating properties
of this amine is due to the proton shuttling capacity of the
amino group when the activator is bound within the cavity,
which offers an additional pathway for the release of the
proton from the water coordinated to zinc to the environ-
3. CA inhibition studies
Data of Tables 1 and 2 show the CA inhibition data with com-
pounds 1b–7d, 9–11 and 14, 15 reported here and standard inhib-
itors of types A–E. Six physiologically relevant mammalian CA
isoforms have been included in the study: a group of cytosolic iso-
forms, CA I, II and VII, involved both in important pathologies (e.g.,
glaucoma (CA II)14 or epilepsy (CA VII)14c), and also considered as
offtargets when for example the cancer-associated or mitochon-
drial enzymes must be inhibited.14 The second group of investi-
gated isoforms includes the three transmembrane ones, CA IX, XII
and XIV, two of which (CA IX and XII) are associated with the tu-
mor progression and invasion.14a,b,f,36 The following structure–
activity relationship (SAR) can be observed by inspection of data
from Tables 1 and 2:
ment, with formation of the zinc hydroxide species of the
12–14
enzyme.
Blocking the moiety responsible for this pro-
cess, for example by acylation of the amine group, was
shown earlier to lead to the loss of the CA activating effects.
12–14
(i) Esters 1b–1d to 7b–7d were generally much more inhibitory
(in several cases even orders of magnitude) compared to the
corresponding parent carboxylic acid 1a–7a, against most
CA isoforms investigated here. The only exceptions to this
rule were five esters, that is, 5b and 5c against hCA II and
6b–6d against hCA I and II (Table 1). The parent carboxylic
acid 1a–7a usually showed micromolar inhibitory activity
against the CA isoforms investigated here, although in sev-
eral cases their activity was much lower (e.g., 1a against
This was also the case for compounds 9-11 reported
here, which acquired CA inhibitory activity. It may be seen
from data of Table 2 that 9 showed KIs in the range of
0.71–6.8
in the range of 0.81–8.7
7.2 M, respectively. Thus, compounds 9-11 behave as a typ-
l
M against the investigated six CA isoforms; 10
lM, and 11 in the range of 0.40–
l
ical phenol CAI. The cytosolic isoforms hCA I, II and VII were
slightly less sensitive to these compounds compared to the
transmembrane isoforms hCA IX, XII and XIV which were
generally inhibited almost one order of magnitude more
hCA VII and IX, with KIs of 118–716 lM; 2a against hCA

F. Carta et al. / Bioorg. Med. Chem. 21 (2013) 1564–1569
1567
efficiently by most of the dopamine derivatives 9-11. Among
the pyridinium salts 10 and 11, the phenyl-dimethyl-pyrid-
inium derivative 11 was slightly more effective as a CAI
compared to the 2,4,6-trimethylpyridinium derivative 10.
(v) The aminophenols 12 and 13 were low micromolar CAIs
against all six CA isoforms investigated here but their deriv-
atization by means of 2,4,6-trimethylpyridinium moieties, as
in compounds 14 and 15, led to an intensification of these
properties, the new compounds being more than one order
of magnitude better CAIs compared to the parent aminophe-
atmosphere using dried glassware and syringe techniques to trans-
fer solutions. Infrared (IR) spectra were recorded as KBr plates and
are expressed in
m
(cmꢀ1). Nuclear magnetic resonance (1H NMR,
13C NMR, DEPT, HSQC, HMBC) spectra were recorded using a Bru-
ker Advance III 400 MHz spectrometer in MeOH-d4 or in DMSO-
d6. The 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, sep-
tet; t, triplet; q, quadruplet; m, multiplet; br s, broad singlet; dd,
double of doubles, appt, aparent triplet, appq, aparent quartet.
The assignment of exchangeable protons (OH and NH) was con-
firmed by the addition of D2O. Analytical thin-layer chromatogra-
phy (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 or MeOH/DCM were used as eluents. Melting
points (mp) were carried out in open capillary tubes and are
uncorrected.
nols. Indeed, 14 showed KIs in the range of 0.32–0.72
lM,
whereas 15 in the range of 0.49–0.78 M. The presence of
l
the chlorine moiety in 15 was thus slightly detrimental to
the CA inhibitory properties of the compound with respect
to the corresponding derivative without the halogen, 14.
It should be mentioned that recently Martin and Cohen37 re-
ported the X-ray crystal structure of the adducts of three carbox-
ylic acids investigated earlier by us,1 1a, 3a and 4a, bound to hCA
II (these compounds were used as leads in the present study). In
this interesting crystallographic work, our hypothesis was con-
firmed, since these carboxylates were observed anchored to the
zinc bound hydroxide ion/water molecule37 (without the necessity
to term this already known binding mode by a new, not very happy
term, i.e., ‘nucleophile recognition’).37 Thus, similar to spermine,
phenol and some carboxylic acid esters investigated earlier by
X-ray crystallography and solution studies,8–10 these compounds
are not zinc binders, but possess a diverse inhibition mechanism.
The interesting study by Martin and Cohen37 seems to be flawed
by the unusually high inhibition constants reported for these com-
pounds (in the millimolar range) which would in fact not allow the
formation of the adducts observed crystallographically. Although
an esterase method was used,37 which normally gives higher inhi-
bition constants compared to the CO2 hydrase one used by us, we
consider that the reported data in the Martin and Cohen’s paper37
are inconsistent with their own crystallographic study.
(2E)-3-(4-hydroxyphenyl)-2-propenoic acid, 3-aminophenol,
dopamine, FITC, pyrylium salts, 3-amino-4-chlorophenol and the
hydroxy/dihydroxybenzoic acids were purchased from Sigma–
Aldrich (Milan, Italy) and used as they are (highest purity
available).
5.2. General procedure for preparation of esters 1b–7d19,20
O
OH
O
O
R2
SOCl2
R2OH
R
R1
R
R1
R= H, OH
R1= H, OH
R2= Me, Et, iPr
4. Conclusions
Thionyl chloride (1.16 g, 1.5 equiv) was added drop-wise to a
solution of acid (1.0 g, 1.0 equiv) in the corresponding alcohol
(15 ml) at 0 °C. The solution was refluxed under a nitrogen atmo-
sphere until all starting material was consumed (TLC monitoring).
Then the solvent was removed under vacuo and the residue was
purified by silica gel column chromatography eluting with ethyl
acetate/n-hexane to afford the corresponding carboxylic esters.
By using hydroxy-/dihydroxybenzoic acids as leads, we report
here
a series of methyl, ethyl and isopropyl esters of 4-
hydroxybenzoic, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dihydroxybenzoic
acids and of coumaric acid. They were investigated for the inhibi-
tion of six mammalian CA isoforms, that is, the cytosolic CA I, II
and VII, and the transmembrane CA IX, XII and XIV, many of which
are established drug targets. Other compounds incorporating phe-
nol/diphenol moieties were obtained from dopamine by reaction
with fluorescein isothiocyanate or with 2,4,6-trisubstituted pyryli-
um salts. Some aminophenols were also derivatized in a similar
manner, by using pyrylium salts. Many of these compounds
showed increased inhibitory action compared to the lead com-
pounds from which they were obtained, with efficacy in the submi-
cromolar range against most investigated CA isoforms. As phenols
are a class of less investigated CA inhibitors (CAIs) compared to the
sulfonamides, and their mechanism of inhibition is less well under-
stood, compounds of the type designed here may be helpful in
gaining more insights into these phenomena.
5.3. Synthesis of 5-(3-(3,4-dihydroxyphenethyl)thioureido)-2-
(3-hydroxy-6-oxo-6H-xanthen-9-yl)benzoic acid 9
HO
O
O
CO2H
HO
O
O
NH3Cl
OH
Et3N
DMF
NH
CO2H
S
HN
S
OH
2'
1'
1
N
C
2
6
5
5. Experimental protocols
5.1. General
3
OH
4
OH
Anhydrous solvents and all reagents were purchased from Sig-
ma–Aldrich, Alfa Aesar and TCI. All reactions involving air- or mois-
Dopamine hydrochloride (0.02 g, 1.0 equiv) and fluoresceine
isothiocyanate (0.04 g, 1.0 equiv) were dissolved in dry DMF
ture-sensitive compounds were performed under
a nitrogen

1568
F. Carta et al. / Bioorg. Med. Chem. 21 (2013) 1564–1569
(3.0 ml) and triethylamine (0.01 g, 1.0 equiv) was added drop-wise
at 0 °C. The red solution was stirred at rt under a nitrogen atmo-
sphere for 4 h (TLC monitoring), quenched with slush and the or-
ange precipitate formed was collected by filtration, washed with
H2O (3 ꢁ 10 ml) and triturated with diethyl ether to afford the de-
sired product as an orange solid in 55% yield.
5.4.2. 10-(3,4-Dihydroxyphenethyl)-200,600-dimethyl-400-
phenylpyridinium tetrafluoroborate 11
Mp 200–203 °C; m
max (KBr) cmꢀ1, 3220 (O–H), 1560 (aromatic);
dH (400 MHz, DMSO-d6) 2.90 (6H, s, 2 ꢁ 200-CH3), 3.05 (2H, appt, J
5.6, 10-H2), 4.64 (2H, appt, J 5.6, 20-H2) 6.55 (1H, dd, J 8.0, 1.6, 5-
H), 6.67 (1H, d, J 1.6, 2-H), 6.71 (1H, d, J 8.0, 6-H), 7.69 (3H, m,
Ar-H), 8.09 (2H, m, Ar-H), 8.38 (2H, s, 2 ꢁ 300-H), 9.01 (2H, br s, ex-
change with D2O, 2 ꢁ OH); dc (100 MHz, DMSO-d6) 156.5, 154.2,
146.4, 145.4, 134.4, 132.9, 130.6, 128.8, 128.2, 125.1, 120.6,
117.3, 116.7, 54.3 (C-20), 33.7 (C-10), 21.6 (CH3).
5.3.1. 5-(3-(3,4-Dihydroxyphenethyl)thioureido)-2-(3-hydroxy-
6-oxo-6H-xanthen-9-yl)benzoic acid 9
Mp 170–175 °C; m
max (KBr) cmꢀ1, 3270 (O–H), 1535 (aromatic);
dH (400 MHz, DMSO-d6) 2.79 (2H, appt, J 6.0, 10-H2), 3.67 (2H, q, J
6.0, 10-H2), 6.60-6.80 (7H, m, Ar-H), 7.20 (1H, d, J 8.0, Ar-H), 7.80
(1H, br s), 8.05 (1H, br s, exchange with D2O, CH2NH–), 8.30 (1H,
s), 8.80 (1H, s), 8.90 (1H, s), 10.0 (1H, br s), 10.20 (2H, s, exchange
with D2O, CO2H and Ar-NH); dc (100 MHz, DMSO-d6) 181.2 (C@S),
172.0 (C@O), 170.0 (C@O acid), 158.6, 157.3, 155.0, 149.0, 148.1,
145.1, 136.0, 135.6, 135.7, 134.2, 132.0, 129.2, 128.8, 128.6,
126.4, 124.9, 122.0, 118.0, 116.6, 116.2, 110.0, 109.7, 105.6, 98.3,
48.0, 34.2.
5.5. Synthesis of 1-(3-hydroxyphenyl)-20,40,60-trimethylpyridi
nium 1431 and 1-(2-chloro-5-hydroxyphenyl)-20,40,60-trim
ethylpyridinium 15
4'
3'
NH2
2'
R
N
ClO4
R
MeOH
5.4. Synthesis of 10-(3,4-dihydroxyphenethyl)-200,400,600-trimethyl
pyridinium tetrafluoroborate 10 and 10-(3,4-dihydroxypheneth
yl)-200,600-dimethyl-400-phenylpyridinium tetrafluoroborate 1130
O
OH
BF4
OH
R= H 14
R= Cl 15
R
4"
3"
The appropriate aminophenol (0.5 g, 1.0 equiv) and 2,4,6-
trimethylpyrylium tetrafluoroborate (1.2 equiv) were dissolved in
dry methanol (10 ml) and the solution was refluxed for 5 h. Then
solvent was partially removed, the mixture was cooled to rt and
treated with a 1.0 M aqueous solution of HClO4 (3.0 equiv). The
precipitate formed was collected by filtration, and crystallized
from H2O to afford the corresponding pyridinium perchlorate salts
14 and 15 as white solids in 38% and 32% yield respectively.
2"
NH3Cl
OH
R
N
Et3N
EtOH
2'
1'
1
BF4
O
2
3
6
5
OH
BF4
OH
4
OH
R= Me 10
R= Ph 11
5.5.1. 1-(3-Hydroxyphenyl)-2,4,6-trimethylpyridinium 14
Mp 158-161 °C (Lit.31 156 °C); mmax (KBr) cmꢀ1, 3300 (O–H),
1580 (aromatic); dH (400 MHz, DMSO-d6) 2.37 (6H, s, 2 ꢁ 20-CH3),
2.63 (3H, s, 40-CH3), 6.99 (2H, m, 2-H and 4-H), 7.12 (1H, dd, J
8.2, 1.6, 6-H), 7.76 (1H, appt, J 8.2, 5-H), 7.94 (2H, s, 2 ꢁ 30-H),
10.34 (1H, br s, exchange with D2O, OH); dc (100 MHz, DMSO-d6),
160.0, 155.2, 137.0, 133.1, 128.6, 120.0, 118.2, 115.7, 112.2, 22.4
(40-CH3), 21.5 (20-CH3).
Dopamine hydrochloride (0.5 g, 1.0 equiv) and pyrylium tetra-
fluoroborate salt (1.2 equiv) were suspended in dry ethanol
(10 ml) and the mixture was refluxed for 2 h. Then acetic acid
was added (0.48 g, 3.0 equiv), followed after 1 h by addition of
30% aqueous NH4OH (2.0 ml). The mixture was cooled down to rt
and added to diethyl ether (30 ml). For 10 a solid was readily ob-
tained and it was collected by filtration, crystallized from H2O to
afford the desired product as a pale yellow solid in 40% yield. For
11 a sticky gum was formed which was triturated with diethyl
ether and petroleum ether until a semisolid residue was obtained
and then recrystallized from H2O to afford the desired product as
a white solid in 32% yield.
5.5.2. 1-(2-Chloro-5-hydroxyphenyl)-20,40,60-trimethylpyridi
nium 15
Mp 145 °C; mmax (KBr) cmꢀ1, 3360 (O-H), 1560 (aromatic); dH
(400 MHz, DMSO-d6) 2.38 (6H, s, 2 ꢁ 20-CH3), 2.67 (3H, s, 40-CH3),
7.19 (1H, dd, J 8.0, 1.6, 4-H), 7.27 (1H, d, J 1.6, 6-H), 7.23 (1H, d, J
8.0, 3-H), 8.04 (2H, s, 2 ꢁ 30-H), 10.74 (1H, s, exchange with D2O,
OH); dc (100 MHz, DMSO-d6), 161.4, 159.3, 155.4, 136.6, 132.9,
128.5, 121.1, 117.9, 115.5, 22.4 (40-CH3), 21.5 (20-CH3).
5.4.1. 10-(3,4-Dihydroxyphenethyl)-200,400,600-trimethylpyridinium
tetrafluoroborate 10
5.6. CA inhibition
Mp 215–220 °C; m
max (KBr) cmꢀ1, 3200 (O–H), 1520 (aromatic);
dH (400 MHz, DMSO-d6) 2.52 (3H, s, 400-CH3), 2.76 (6H, s, 2 ꢁ 200-
CH3), 3.00 (2H, appq, J 8.0, 10-H2), 4.59 (2H, appq, J 8.0, 20-H2) 6.48
(major rotamer, 1H, dd, J 8.0, 1.6, 5-H), 6.63 (major rotamer, 1H, d,
J 1.6, 2-H), 6.71 (major rotamer, 1H, d, J 8.0, 6-H), 7.77 (2H, s,
2 ꢁ 300-H), 8.89 (2H, br s, exchange with D2O, 2 ꢁ OH); dc (100
MHz, DMSO-d6) 159.2, 155.8, 152.0, 146.0, 129.8, 129.1, 127.3,
121.6, 117.5, 55.0 (C-20), 38.8 (C-10), 22.0 (200-CH3), 20.8 (400-CH3)
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 7.4) as buffer, and 20 mM Na2SO4 (for maintaining con-
stant the ionic strength), following the initial rates of the CA-cata-
lyzed CO2 hydration reaction for a period of 10–100 s.38 The CO2

F. Carta et al. / Bioorg. Med. Chem. 21 (2013) 1564–1569
1569
10. Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C. T. J.
Med. Chem. 2010, 53, 5511.
11. Innocenti, A.; Durdagi, S.; Doostdar, N.; Strom, T. A.; Barron, A. R.; Supuran, C. T.
Bioorg. Med. Chem. 2010, 28, 2822.
concentrations ranged from 1.7 to 17 mM for the determination 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 to-
tal observed rates. Stock solutions of inhibitor (10 mM) were pre-
pared in distilled–deionized water and dilutions up to 0.001 nM
were done thereafter with the assay buffer. Inhibitor and enzyme
solutions were preincubated together for 15 min at room temper-
ature prior to assay, in order to allow for the formation of the E–I
complex (the proteins were incubated also for longer periods, of
1–24 h, but no differences of activity have been detected). The inhi-
bition constants were obtained by non-linear least-squares meth-
ods using PRISM 3, as reported earlier,1,6 and represent the mean
from at least three different determinations. All CA isoforms were
recombinant ones obtained in house as reported earlier.6–9
12. (a) Dave, K.; Ilies, M. A.; Scozzafava, A.; Temperini, C.; Vullo, D.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2011, 21, 2764; (b) Dave, K.; Scozzafava, A.; Vullo, D.;
Supuran, C. T.; Ilies, M. A. Org. Biomol. Chem. 2011, 9, 2790.
13. (a) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. X-Ray
Crystallography of CA Inhibitors and its Importance in Drug Design. In Drug
Design of Zinc-Enzyme Inhibitors: Functional, Structural, and Disease Applications;
Supuran, C. T., Winum, J. Y., Eds.; Wiley: Hoboken, 2009; pp 73–138; (b)
Alterio, V.; Vitale, R. M.; Monti, S. M.; Pedone, C.; Scozzafava, A.; Cecchi, A.; De
Simone, G.; Supuran, C. T. J. Am. Chem. Soc. 2006, 128, 8329.
14. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Neri, D.; Supuran, C. T.
Nat. Rev. Drug Disc. 2011, 10, 767; (c) Supuran, C. T. Bioorg. Med. Chem. Lett.
2010, 20, 3467; (d) Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003,
23, 146; (e) Supuran, C. T.; Vullo, D.; Manole, G.; Casini, A.; Scozzafava, A. Curr.
Med. Chem. Cardiovasc. Hematol. Agents 2004, 2, 49; (f) Supuran, C. T. Curr.
Pharm. Des. 2010, 16, 3233.
15. (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. M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; Supuran, C. T.; McKenna, R.
Bioorg. Med. Chem. 2010, 18, 4873; (c) Pacchiano, F.; Aggarwal, M.; Avvaru, B. S.;
Robbins, A. H.; Scozzafava, A.; McKenna, R.; Supuran, C. T. Chem. Commun.
2010, 46, 8371.
Acknowledgments
16. (a) De Simone, G.; Supuran, C.T. J. Inorg. Biochem. in press.; (b) Parkkila, S.;
Vullo, D.; Maresca, A.; Carta, F.; Scozzafava, A.; Supuran, C. T. Chem. Commun.
2012, 48, 3551.
17. (a) Carta, F.; Aggarwal, M.; Maresca, A.; Scozzafava, A.; McKenna, R.; Supuran,
C. T. Chem. Commun. 1868, 2012, 48; (b) Maresca, A.; Carta, F.; Vullo, D.;
Supuran, C.T. J. Enzyme Inhib. Med. Chem. in press.; (c) Monti, S. M.; Maresca, A.;
Carta, F.; De Simone, G.; Mühlschlegel, F. A.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2012, 22, 859; (d) Carta, F.; Aggarwal, M.; Maresca, A.;
Scozzafava, A.; McKenna, R.; Masini, E.; Supuran, C. T. J. Med. Chem. 2012, 55,
1721.
This research was financed by a 7th FP EU project (Metoxia).
Thanks are addressed to Dr. Alessio Innocenti who was involved
in the first phases of this research.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.019.
18. Temperini, C.; Vullo, D.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2006, 49,
3019.
19. Gianolio, E.; Giovenzana, G. B.; Ciampa, A.; Lanzardo, S.; Imperio, D.; Aime, S.
ChemMedChem 2008, 3, 60.
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