Carbonic anhydrase inhibitors: Copper(II) complexes of polyamino-polycarboxylamido aromatic/heterocyclic sulfonamides are very potent inhibitors of the tumor-associated isoforms IX and XII

Article abstract of DOI:10.1016/j.bmcl.2007.11.025

Reaction of EDTA/DTPA dianhydride with aromatic/heterocyclic sulfonamides afforded a series of derivatives incorporating polyaminopolycarboxylate tails and benzenesulfonamide or 1,3,4-thiadiazole-2-sulfonamide heads. These compounds have been used as liga

Full text of DOI:10.1016/j.bmcl.2007.11.025

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 836–841
Carbonic anhydrase inhibitors: Copper(II) complexes
of polyamino-polycarboxylamido aromatic/heterocyclic
sulfonamides are very potent inhibitors
of the tumor-associated isoforms IX and XII
Marouan Rami,a Jean-Yves Winum,a,* Alessio Innocenti,b
Jean-Louis Montero,a Andrea Scozzafavab and Claudiu T. Supuranb,*
a
ˆ
Institut des Biomole´cules Max Mousseron (IBMM) UMR 5247 CNRS-UM1-UM2 Batiment de Recherche Max Mousseron,
Ecole Nationale Supe´rieure de Chimie de Montpellier, 8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France
b
`
Universita degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Room 188,
Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy
Received 26 September 2007; revised 6 November 2007; accepted 8 November 2007
Available online 13 November 2007
Abstract—Reaction of EDTA/DTPA dianhydride with aromatic/heterocyclic sulfonamides afforded a series of derivatives incorpo-
rating polyaminopolycarboxylate tails and benzenesulfonamide or 1,3,4-thiadiazole-2-sulfonamide heads. These compounds have
been used as ligands to prepare Cu(II) complexes. Both parent sulfonamides as well as their copper complexes behaved as potent
inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, and transmembrane CA IX and XII.
Some Cu(II) complexes showed subnanomolar affinities and some selectivity for the inhibition of the tumor-associated isoforms IX
and XII and might be used as PET hypoxia markers of tumors.
Ó 2007 Elsevier Ltd. All rights reserved.
Although sulfanilamide SA and acetazolamide AZA
metal complexes were used for the gravimetric determi-
nation of silver for a long time,1 only recently such sul-
fonamide coordination compounds were investigated in
more detail by our and Borras’ groups.2–5 Indeed,
many transition metal ion acetazolamide complexes
were synthesized by the two groups and their 3D struc-
ture determined by X-ray crystallography, allowing a
detailed understanding of the multiple possible
interactions between this sulfonamide and transition
di-/tri-valent cations.2–5 However, the most interesting
property of such derivatives was their unexpectedly
strong inhibitory activity against the zinc enzyme car-
bonic anhydrase (CA, EC 4.2.1.1).2 Thus, metal com-
plexes of the various clinically used sulfonamide CA
inhibitors (CAIs), such as acetazolamide AZA, metha-
zolamide MZA, ethoxzolamide EZA, and saccharin
SAC with different main group and transition metal
ions, were shown to act as very powerful inhibitors
of these enzymes (more precisely of isoforms CA I
and II), being generally 10–100 times more effective
as compared to the parent sulfonamides from which
they were synthesized.2–6 This was thereafter explained6
as being due to a dual inhibition of the enzyme by the
metal complexes, by means of both sulfonamide anions
(which bind to the catalytic Zn(II) ion within the CA
active site) as well as metal cations (which bind to crit-
ical histidine residues for the catalytic cycle, such as
His64, within the enzyme active site).6–11 Metal com-
plexes of such ligands as the sulfonamides mentioned
above were also shown to be not very stable in aque-
ous solution,6 being easily dissociated to metal ions
and sulfonamidate anions which both bind to different
binding sites within the enzyme cavity, explaining thus
the enhanced inhibitory power of the complexes as
compared to the parent sulfonamides.2–7
Keywords: Carbonic anhydrase; Sulfonamide; Metal complex; Cu(II);
Tumor-associated isoform; CA IX; Isozyme selective inhibitor.
*
Corresponding authors. Tel.: +39 055 4573005; fax: +39 055
4573385; e-mail addresses: winumj@univ-montp2.fr; claudiu.
supuran@unifi.it
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.025

M. Rami et al. / Bioorg. Med. Chem. Lett. 18 (2008) 836–841
837
H3C
N
N
N
N
H2N
SO2NH2
CH3CONH
SO2NH2
S
CH3CON
SO2NH2
S
SA
AZA
MZA
O
N
NH
S
EtO
S
SO2NH2
O
O
EZA
SAC
Sulfonamide metal complexes have been assayed up to
now only for the inhibition of three CA isoforms (i.e.,
CA I, II, and IV)2–9 of the 16 presently known in mam-
mals.12 Many of these isoforms were shown to be in-
volved in a host of physiological and pathological
processes, such as among others: gluconeogenesis, lipo-
genesis, ureagenesis, tumorigenicity, and the growth and
virulence of various pathogens.12 Thus, in the search of
derivatives showing some specificity for various CA iso-
forms, and more precisely the tumor-associated ones CA
IX and XII,13 we report here the preparation and inhibi-
tion assay of some Cu(II) complexes of aromatic/hetero-
cyclic sulfonamides incorporating EDTA and DTPA
tails. In addition, such copper(II) derivatives with potent
CA IX/XII inhibitory activity might also be important
for developing positron emission tomography (PET)
imaging agents for tumor hypoxia. Indeed, recently,
The strong in vitro enzyme inhibitory properties of such
sulfonamide metal complexes2–6 against CA I and II, as
well as their excellent in vivo activity as intraocular pres-
sure (IOP) lowering agents in animal models of glau-
coma,7–9 led to further investigations of such enzyme
inhibitors. Indeed, in addition to the metal complexes
of the rather simple aromatic/heterocyclic sulfonamides
SA–SAC mentioned above, new ligands were designed
incorporating benzenesulfonamide or 1,3,4-thiadiazole-
sulfonamide heads as well as polyamino-polycarboxyl-
ate moieties present in classical metal-complexing agents
such as EDTA, DTPA, IDA (iminodiacetic acid),
etc.7b,9,10 These metal-complexing moieties (EDTA,
DTPA, IDA, etc.) were incorporated in the molecules
of sulfonamide ligands used to prepare the metal com-
plexes in order to both enhance the stability of the metal
complex itself, as well as the CA inhibitory properties of
such larger molecules, which presumably may lead to
additional favorable interactions when bound within
the enzyme cavity, a hypothesis largely confirmed by
our studies.7b,9,10 Some Zn(II) and Cu(II) metal com-
plexes of such sulfonamides incorporating polyamino-
polycarboxylated tails have also been reported, which
indeed showed very good in vitro CA inhibitory activity
against isoforms CA I, II, and IV, and some of them
were also effective in vivo IOP lowering agents in hyper-
tensive rabbits as a glaucoma animal model.7b,9 Srivast-
ava’s group10 proposed then that such ‘two-prong’
inhibitors may also lead to the design of isozyme selec-
tive CAIs, due to the presence of various histidine resi-
dues on the surface of different CA isoforms (an
observation initially reported by our group)11 which
can interact with the metal ions present in the complex
inhibitor. However, this observation has not been con-
firmed thereafter, since the metal ion of all copper com-
plexes investigated up to now by X-ray crystallography
binds only to His64 in CA II and His200 in CA I, irre-
spective of the length of their spacers separating the sul-
fonamide head of the IDA–Cu(II) moiety.10b In
addition, the weak electron density reported as a third
binding site for such sulfonamide metal complexes was
found at the interface between two protein molecules,
toward the amino terminal part of the enzyme, and
might be due to the very high concentration of copper
complex (1.4–2 mM) present in the crystallographic
experiments,10b which are not at all comparable with
the experiments reported here in which micro-nanomo-
lar concentrations of inhibitor are used (see later in
the text).
Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone),
a
compound with no CA inhibitory activity, was shown
to be a valuable PET hypoxia marker in some but not
all tumor types.14 As hypoxia is present in many tumors,
where it triggers a strong overexpression of CA IX and
XII via the HIF-1a transcription factor activation,15,16
compounds containing Cu(II) and also possessing a high
affinity for these CA isozymes might be even more useful
than Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone)
as PET imaging agents.
It should be also stressed that the levels of CA IX and
XII in most organs are rather low, whereas hypoxia trig-
gers a very strong overexpression of these enzymes,15
which attain quite high concentrations in such tumors.
Being absent (or present in very low concentrations)
within the normal tissues, and abundant in tumors, as
shown by extensive immunohistological work from
Pastorekova’s and Harris’ groups among others,15,16
CA IX and XII become very attractive targets for both
diagnosis and treatment of hypoxic tumors.12,13
Sulfonamides A1–A5 and C1–C5 were prepared as pre-
viously described7b,9 by reacting EDTA dianhydride or
DTPA dianhydride with amino-sulfonamides such as
sulfanilamide, metanilamide, homosulfanilamide, 4-
aminoethyl-benzenesulfonamide, and 5-amino-1,3,4-
thiadiazole-2-sulfonamide (Scheme 1). The copper com-
plexes B1–B5 and D1–D5 were then obtained by react-
ing the corresponding bis-sulfonamides (as carboxylate
disodium salts) with Cu(II) chloride (Scheme 1) as re-
ported earlier by this group.7b,9,17 The ligands were re-

838
M. Rami et al. / Bioorg. Med. Chem. Lett. 18 (2008) 836–841
O
O
O
O
O
O
N
O
N
N
O
N
O
N
O
O
OH
O
O
R-NH2
DMF
R-NH2
DMF
O
O
O
R
R
R
O
N
OH
N
N
N
H
H
H
N
N
N
N
H
N
HO
O
HO
O
HO
HO
O
R
O
O
C
A
CuCl2, 2H2O
CH3OH
CuCl2, 2H2O
CH3OH
O
O
O
R
R
R
O
N
OH
Cu2+
N
H
N
N
H
H
N
N
N
N
H
N
HO
O
HO
O
HO
HO
O
R
Cu2+
O
O
D
B
SO2NH2
SO2NH2
R=
N
N
1
3
SO2NH2
S
SO2NH2
5
2
4
SO2NH2
Scheme 1. Preparation of sulfonamides A1–A5 and C1–C5 and their corresponding Cu(II) complexes B1–B5 and D1–D5.
ported earlier,9 whereas the Cu(II) complexes are new
compounds, except for B5 and D5 which were obtained
in the previous study.9 However, all these compounds
(ligands A1–A5 and C1–C5 and metal complexes B1–
B5 and D1–D5) were never assayed for the inhibition
of the tumor-associated isoforms CA IX and XII. Their
testing for the inhibition of CA I, II, and IV has been
reported earlier by using an esterase assay,9 whereas
here we used a stopped-flow, CO2 hydration assay for
reinvestigating the inhibition of the physiological reac-
tion with all these derivatives.18
sulfanilamide derivative A3 as a weaker inhibitor, but
probably this compound has a clash with some amino
acid(s) from the hCA I active site, a situation we recently
evidenced for inhibitors of both hCA II19 and hCA I.20
Work is in progress in our laboratories to better under-
stand the interactions between these inhibitors and var-
ious CAs by means of X-ray crystallography. The
DTPA bis-sulfonamides C1–C5 were on the other hand
medium potency hCA I inhibitors, with KI-s in the range
of 386–578 nM, except for C4 which is a potent inhibitor
(KI of 9.5 nM). The copper complexes D1–D5 were
stronger inhibitors as compared to the corresponding
parent bis-sulfonamides, with KI-s in the range of 6.5–
144 nM (Table 1). It is thus clear that the more compact
EDTA moiety generally leads to better hCA I inhibiting
bis-sulfonamides as compared to the bulkier DTPA
moiety, probably due to the restricted hCA I active site
as compared to that of isoforms hCA II, IX, and XII.
Indeed, some bulky amino acid residues (among others
His200 and His67) are found only in isoform
Data of Table 1 show that bis-sulfonamides A1–A5 and
C1–C5, as well as their Cu(II) complexes B1–B5 and
D1–D5, generally act as very potent inhibitors of all four
CA isozymes investigated here, the cytosolic, ubiquitous
human CA I and II,12 as well as the transmembrane, tu-
mor-associated hCA IX and hCA XII.13,15 The follow-
ing structure–activity relationship (SAR) data are
evident: (i) against hCA I, the EDTA bis-sulfonamides
A1–A5 show effective inhibitory activity, with KI-s in
the range of 8.6–9.4 nM except for A3 which is a med-
ium potency inhibitor (KI of 438 nM). The correspond-
ing metal complexes are on the other hand much better
inhibitors, with KI-s in the range of 1.7–7.4 nM. It is
rather difficult to rationalize the behavior of the homo-
I
10,12,19,20; (ii) against the ubiquitous hCA II, the
EDTA-derived bis-sulfonamides A1–A5 generally be-
haved as very potent inhibitors, with KI-s in the range
of 0.77–11.3 nM. The corresponding copper complexes
were also good inhibitors (generally better than the par-
ent sulfonamides, except for B3 and B4 which are

M. Rami et al. / Bioorg. Med. Chem. Lett. 18 (2008) 836–841
839
Table 1. Inhibition of CA isoforms hCA I, II, IX, and XII with
sulfonamides A1–A5, C1–C5 and their corresponding Cu(II) com-
plexes B1–B5, D1–D5, as well as clinically used inhibitors
CAIs, as already reported earlier for many sulfonamide
complexes investigated by us,22 such as AZA, MZA,
EZA, etc. However, here are the first ever reported
subnanomolar hCA XII inhibitors (e.g., B5 and D5).
It may be also observed that generally the EDTA deriv-
atives and their copper complexes are slightly better
CAIs as compared to the corresponding DTPA deriva-
tives and their metal complexes (Table 1). It should also
be noted that the compounds investigated here of type
A1–A5, C1–C5, B1–B5, and D1–D5 are generally much
better CAIs than the clinically used drugs AZA, MZA,
EZA, SAC, etc. The observed specificity for inhibiting
CA IX/XII over the cytosolic isoforms with the com-
pounds reported here is probably due to the fact that
the copper-polyamino-polycarboxylate tails interact in
a distinct manner with the active site cavities of these
isoforms, as shown already for some structurally related
derivatives by Christianson’s group10b; (v) one of the
important issues regarding the design of CAIs regards
the selectivity of such compounds for the inhibition of
the target isoform over that of the ubiquitous ones CA
I and II.23 As seen from data of Table 1, clinically used
sulfonamides such as AZA, MZA, EZA do indiscrimi-
nately inhibit both the cytosolic isoforms CA I and II
as well as the tumor-associated ones CA IX and XII
with rather similar potency. Furthermore, as observed
from Table 2, many times the selectivity ratios for inhib-
iting the tumor-associated isoform CA IX over the cyto-
solic ubiquitous one CA II are in the range of 0.23–0.51
for these compounds, meaning that all of them are bet-
ter CA II than CA IX inhibitors. However, data of Ta-
ble 2 also show that some of the compounds investigated
here show good selectivity ratios for the inhibition of the
transmembrane over the cytosolic isozymes. The most
tumor-associated CA selective such inhibitors are C1,
C3, and the copper complex of this derivative, D3,
which showed excellent selectivity ratios for the inhibi-
tion of CA IX over CA I, CA XII over CA I and II
(in the range of 10.4–143.7), and acceptable ones for
the inhibition of CA IX over CA II, in the range of
5.4–7.3.
Inhibitor
KIa (nM)
hCA IXc
hCA Ib
hCA IIb
hCA XIIc
A1
B1
9.0
1.7
10.7
7.8
11.3
1.4
0.89
1.2
0.77
2.6
11.0
5.6
46
7.8
3.7
7.9
3.9
7.3
6.2
8.2
4.1
8.5
6.4
8.4
8.1
9.6
9.2
8.7
4.5
7.5
5.5
8.3
4.8
3.2
1.4
4.8
2.0
3.0
1.3
2.8
1.2
2.1
0.7
4.4
2.1
6.8
3.5
4.0
1.7
3.9
1.0
2.8
0.6
37
A2
B2
9.0
7.4
A3
B3
438
4.3
9.4
A4
B4
4.6
8.6
A5
B5
2.0
C1
386
111
578
12.2
575
144
9.5
D1
C2
1.2
69
D2
C3
1.4
62
D3
C4
33
11.4
2.9
13.4
4.5
240
10
D4
C5
6.5
487
62
D5
SA
AZA
MZA
EZA
SAC
25000
250
50
294
25
27
5.7
3.4
22
14
25
18540
8
5950
34
103
633
a Errors in the range of 5–10% of the shown data, from three different
assays, by a CO2 hydration stopped-flow assay.20
b Human, recombinant isozymes.
c Catalytic domain of human cloned isoforms.13,21
slightly less effective than A3 and A4, respectively), with
KI-s in the range of 1.2–7.8 nM. The DTPA bis-sulfon-
amides C1–C5 were on the other hand less effective
hCA II inhibitors as compared to the corresponding
EDTA derivatives, with KI-s in the range of 11.4–
69 nM. However, all their copper complexes D1–D5
showed enhanced hCA II inhibitory properties, with
KI-s in the range of 1.2–33 nM (Table 1); (iii) both the
EDTA- as well as DTPA-based sulfonamides A1–A5
and C1–C5 showed excellent hCA IX inhibitory activity,
with inhibition constants in the range of 7.3–9.6 nM,
whereas all their corresponding copper complexes B1–
B5 and D1–D5 were even better inhibitors, with KI-s
in the range of 3.7–9.2 nM (Table 1). Thus, for hCA
IX which has a larger active site as compared to hCA
I and II,21 all these sulfonamide ligands and their Cu(II)
complexes showed a very compact behavior of extremely
potent CAIs, a rather positive feature for compounds
possibly useful to target hypoxic tumors in which CA
IX is usually highly overexpressed13,15,16; (iv) an even
better inhibition profile has been observed for hCA
XII22 with the derivatives reported here. Thus, the li-
gands A1–A5 and C1–C5 showed inhibition constants
in the range of 1.2–6.8 nM, whereas the corresponding
copper derivatives B1–B5 and D1–D5 in the range of
0.6–3.5 nM. Again the behavior of these derivatives
was very compact, all of them being extremely potent
In conclusion, we report here some polyamino-poly-
carboxylated sulfonamide derivatives incorporating
EDTA and DTPA tails as well as benzenesulfon-
amide-/1,3,4-thiadiazole-5-sulfonamide heads, together
Table 2. Selectivity ratios for the inhibition of the tumor-associated
(CA IX and XII) over the cytosolic (CA I and II) isozymes with
selected CAIs reported here
Compound
Selectivity ratio
hCA
hCA
hCA
I/hCA IX II/hCA IX I/hCA XII II/hCA XII
hCA
AZA
MZA
EZA
B4
10
1.85
0.48
0.51
0.23
0.63
5.4
43.8
14.7
2.10
4.11
0.36
2.1
0.73
1.1
1.13
3.8
87.7
143.7
84.7
6.5
C1
C3
45.9
10.4
66.0
32.0
1.1
7.1
7.3
15.5
19.4
2.9
D3
D4
0.52
0.93
D5
12.9
103.3
7.5

840
M. Rami et al. / Bioorg. Med. Chem. Lett. 18 (2008) 836–841
11. Briganti, F.; Mangani, S.; Orioli, P.; Scozzafava, A.;
Vernaglione, G.; Supuran, C. T. Biochemistry 1997, 36,
10384.
12. (a) Supuran, C. T. Curr. Top. Med. Chem. 2007, 7, 825; (b)
Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev.
2003, 23, 146; (c) Supuran, C. T.; Scozzafava, A. Bioorg.
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with their corresponding Cu(II) complexes. Both parent
sulfonamides as well as the copper complexes behaved
as potent inhibitors of four CA isoforms, the cytosolic
CA I and II, and transmembrane, tumor-associated
CA IX and XII. Some Cu(II) complexes showed subn-
anomolar affinities and some selectivity for the inhibi-
tion of the tumor-associated isoforms IX and XII over
that of the cytosolic ones, and might be used as PET hy-
poxia markers of tumors.
13. (a) Winum, J. Y.; Rami, M.; Scozzafava, A.; Montero, J.
L., Supuran, C. Med. Res. Rev. 2007, 27, in press; (b)
´
Thiry, A.; Dogne, J.-M.; Masereel, B.; Supuran, C. T.
Trends Pharmacol. Sci. 2006, 27, 566; (c) Pastorekova, S.;
Kopacek, J.; Pastorek, J. Curr. Top. Med. Chem. 2007, 7,
865.
Acknowledgments
14. Yuan, H.; Schroeder, T.; Bowsher, J. E.; Hedlund, L. W.;
Wong, T.; Dewhirst, M. W. J. Nucl. Med. 2006, 47, 989.
This research was financed in part by two grants of the
6th Framework Programme of the European Union
(EUROXY and DeZnIT projects).
ˇ
´
15. (a) Svastova, E.; Hulıkova, A.; Rafajova, M.; Zatovicova,
´
M.; Gibadulinova, A.; Casini, A.; Cecchi, A.; Scozzafava,
´
´
´
´
A.; Supuran, C. T.; Pastorek, J.; Pastorekova, S. FEBS
´
Lett. 2004, 577, 439; (b) Dubois, L.; Douma, K.; Supuran,
C. T.; Chiu, R. K.; van Zandvoort, M. A. M. J.;
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17. Synthesis of derivatives A and C: 1 equiv of EDTA
dianhydride or DTPA dianhydride was mixed with 2 equiv
of amino-sulfonamide in DMF. The mixture was stirred at
room temperature during the night. Methylene chloride
was then poured in the mixture and the precipitate was
filtered and washed several times with acetone and
acetonitrile. In the case of compound, A3, A5, C3, and
C5 the starting amine (chlorohydrate form) was previously
treated with 2 equiv of triethylamine. Synthesis of copper
complexes B and D: 1 equiv of compound A or C was
dissolved in methanol, and 1.1 equiv of CuCl2, 2H2O was
added. The mixture was stirred for 15 min and evaporated.
The residue is washed several times with acetone and
chloroform and filtered. A1: yield: 84%; mp 193–195 °C;
MS (ESI+): 600.94 [M+H]+, 623.16 [M+Na]+; 1H NMR
(DMSO-d6, 400 MHz): d 3.25 (m, 4H), 3.90 (s, 4H), 3.93
(s, 4H), 7.67 (s, 4H), 8.15 (d, 4H, J = 9.1 Hz), 8.21 (d, 4H,
J = 9.1 Hz), 10.86 (s, 2H); 13C NMR (DMSO-d6,
101 MHz): d 42.13, 55.37, 58.01, 118.66, 126.66, 138.30,
141.63, 170.47, 173.04. A2: Yield: 83%; mp 179–181 °C;
MS (ESI+): 601.07 [M+H]+, 623.04 [M+Na]+, 639.03
[M+K]+; 1H NMR (DMSO-d6, 400 MHz): d 3.25 (m, 4H),
3.90 (s, 4H), 3.91 (s, 4H), 7.77 (s, 4H), 7.91 (m, 6H), 8.67
(s, 2H), 10.80 (s, 2H). 13C NMR (DMSO-d6, 101MHz): d
52.17, 55.54, 58.07, 116.18, 122.19, 129.55, 139.12, 144.62,
170.23, 173.12. A3: Yield: 84%; mp 110–112 °C; MS
(ESI+): 629.15 [M+H]+, 650.99 [M+Na]+; 1H NMR
(DMSO-d6, 400 MHz): d 3.16 (s, 4H), 3.36 (m, 4H), 3.71
(s, 4H), 3.75 (s, 4H), 7.73 (s, 4H), 7.82 (d, 4H,
J = 8.33 Hz), 8.16 (d, 4H, J = 8.33 Hz), 9.21 (s, 2H); 13C
NMR (DMSO-d6, 101 MHz): d 45.37, 52.51, 56.12, 57.51,
125.83, 127.56, 142.55, 143.76, 170.87, 172.98. A4: Rdt:
81%; mp 175–177 °C; MS (ESI+): 657.23 [M+H]+, 679.20
[M+Na]+.; 1H NMR (DMSO-d6, 400 MHz): d 3.09 (m,
4H), 3.21 (m, 4H), 3.62 (m, 4H), 3.76 (m, 8H), 7.71(s, 4H),
7.80 (d, 4H, J = 8.2 Hz), 8.14 (d, 4H, J = 8.2 Hz), 8.56 (s,
2H).; 13C NMR (DMSO-d6, 101 MHz): d 52.29, 55.45,
57.45, 125.83, 129.24, 143.77, 170.40, 172.57. A5: Rdt:
69%; mp 182–184 °C; MS (ESI+): 639.85 [M+Na]+. 1H
3. (a) Alzuet, G.; Casella, L.; Perotti, A.; Borras, J. J. Chem.
Soc. Dalton Trans. 1994, 2347; (b) Ferrer, S.; Alzuet, G.;
Borras, J. J. Inorg. Biochem. 1989, 39, 297; (c) Ferrer, S.;
Borras, J.; Miratvilles, C.; Fuertes, A. Inorg. Chem. 1989,
28, 160; (d) Ferrer, S.; Jimenez, A.; Borras, J. Inorg. Chim.
Acta 1987, 129, 103.
4. (a) Ko¨hler, K.; Hillebrecht, A.; Schulze Wischeler, J.;
Innocenti, A.; Heine, A.; Supuran, C. T.; Klebe, G.
Angew. Chem., Int. Ed. Engl. 2007, in press; (b) Supuran,
C. T. Rev. Roum. Chim. 1992, 37, 849; (c) Supuran, C. T.;
Loloiu, G.; Manole, G. Rev. Roum. Chim. 1993, 38, 115.
5. (a) Alzuet, G.; Ferrer, S.; Borras, J.; Supuran, C. T. Roum.
Chem. Quart. Rev. 1994, 2, 283; (b) Borja, P.; Alzuet, G.;
`
Server-Carrio, J.; Borras, J.; Supuran, C. T. Main Group
Met. Chem. 1998, 21, 279; (c) Alzuet, G.; Casanova, J.;
´
Borras, J.; Garcia-Granda, S.; Gutierrez-Rodriguez, A.;
Supuran, C. T. Inorg. Chim. Acta 1998, 273, 334.
6. (a) Luca, C.; Barboiu, M.; Supuran, C. T. Rev. Roum.
Chim. 1991, 36, 1169; (b) Supuran, C. T.; Manole, G.;
Andruh, M. J. Inorg. Biochem. 1993, 49, 97; (c) Supuran,
C. T. Metal Based Drugs 1995, 2, 331.
7. (a) Supuran, C. T.; Mincione, F.; Scozzafava, A.; Briganti,
F.; Mincione, G.; Ilies, M. A. Eur. J. Med. Chem. 1998, 33,
247; (b) Scozzafava, A.; Menabuoni, L.; Mincione, F.;
Mincione, G.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2001, 11, 575.
8. (a) Mastrolorenzo, A.; Scozzafava, A.; Supuran, C. T.
Eur. J. Pharm. Sci. 2000, 11, 99; (b) Briganti, F.; Tilli, S.;
Mincione, G.; Mincione, F.; Menabuoni, L.; Scozzafava,
A.; Supuran, C. T. J. Enzyme Inhib. 2000, 15, 185.
9. Scozzafava, A.; Menabuoni, L.; Mincione, F.; Supuran,
C. T. J. Med. Chem. 2002, 45, 1466.
10. (a) Banerjee, A. L.; Eiler, D.; Roy, B. C.; Jia, X.; Haldar,
M. K.; Mallik, S.; Srivastava, D. K. Biochemistry 2005, 44,
3211; (b) Jude, K. M.; Banerjee, A. L.; Haldar, M. K.;
Manokaran, S.; Roy, B.; Mallik, S.; Srivastava, D. K.;
Christianson, D. W. J. Am. Chem. Soc. 2006, 128, 3011.

M. Rami et al. / Bioorg. Med. Chem. Lett. 18 (2008) 836–841
841
NMR (DMSO-d6, 400 MHz): d 2.09 (s, 2H), 3.47 (m, 4H),
3.53 (s, 4H), 3.72 (s, 4H), 8.32(s, 4H). 13C NMR (DMSO-
d6, 101 MHz): d 45.56, 45.59, 161.16, 164.24, 172.01,
172.67. B1: Yield: 86%; mp 170–172 °C; MS (ESI+):
661.96 [M+H]+, 698.04 [M+K]+; 1H NMR (DMSO-d6,
400 MHz): d 2.06 (s, 2H), 2.06 (m, 4H), 3.41 (s, 4H), 3.90 (s,
4H), 7.57 (s, 4H), 8.44 (m, 8H). B2: Yield: 82%; mp 165–
MS (ESI+): 791.12 [M+H]+. 1H NMR (DMSO-d6,
400 MHz): d 1.18 (s, 4H), 2.07 (s, 2H), 3.42 (m), 7.35 (s,
4H), 7.85 (m, 4H). D4: Yield: 71%; mp 160-162 °C; MS
(ESI+): 819.11 [M+H]+; 1H NMR (DMSO-d6, 400 MHz): d
2.07 (2H). 3.38 (m, 24H), 7.24 (s, 4H), 7.77 (m, 8H). D5:
Yield: 82%; mp 180–182 °C; MS (ESI+): 778.98 [M+H]+,
1
800.95 [M+Na]+; H NMR (DMSO-d6, 400 MHz): d 2.07
1
167 °C; MS (ESI+): 661.96 [M+H]+, 700.09 [M+K]+.; H
(s, 2H), 3.06 (s, 4H), 3.16 (m,4H), 3.57 (m, 8H), 3.82 (s, 2H),
8.35 (s, 4H). The Cu(II) complexes B and D afforded
elemental analysis (C, Cu, H, and N) within 0.4% of the
theoretical, calculated values for the proposed formulas
(data not shown).
NMR (DMSO-d6, 400 MHz): d 2.07 (s, 2H), 2,07 (m, 4H),
3.43 (s, 2H), 3.74 (m, 4H), 7.68 (m, 12H). B3: Yield: 83%;
mp 163–165 °C; MS (ESIÀ): 688.02 [M–H]À.; 1H NMR
(DMSO-d6, 400 MHz): d 1.18 (s, 2H, NH), 1.18 (m, 4H),
2.07 (s, 4H), 3.04 (s, 4H), 3.43 (s, 4H), 7.04 (s, 4H), 8.0 (m,
4H). B4: Yield: 78%; mp 187–189 °C; MS (ESI+): 718.16
[M+H]+, 740.06 [M+Na]+.; 1H NMR (DMSO-d6,
400 MHz): d 2.07 (s, 2H), 3.38 (m, 20H), 7.27 (s, 4H), 7.9
(m, 8H). B5: Yield: 77%; mp 176-178 °C; MS (ESI+): 677.92
[M+H]+, 699.96 [M+Na]+; 1H NMR (DMSO-d6,
400 MHz): d 1.17 (m, 4H), 2.06 (s, 2H), 3.04 (s, 4H), 3.9
(s, 4H), 8.35 (s, 4H). C1: Yield: 89%; mp 145–147 °C; MS
(ESI+): 702.18 [M+H]+, 724.22 [M+Na]+. 1H NMR
(DMSO-d6, 400 MHz): d 3.22 (m, 4H), 3.37 (m, 4H), 3.65
(s, 4H), 3.76 (s, 4H), 3.83 (s, 2H), 7.73 (s, 4H), 7.82 (d, 4H,
J = 7.9Hz), 8.16 (d, 4H, J = 7.9 Hz), 8.68 (s, 2H); 13C NMR
(DMSO-d6, 101 MHz): d 49.64, 49.67, 51.69, 54.64, 56.77,
128.81, 125.37, 141.56, 143.31, 168.27, 169.88, 172.37.C2:
Yield: 91%; mp 142–144 °C; MS (ESI+): 702.12 [M+H]+,
18. Khalifah, R. G.. J. Biol. Chem. 1971, 246, 2561; An
applied photophysics stopped-flow instrument has been
used for assaying the CA-catalyzed CO2 hydration activ-
ity. 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 7.5) as buffer, 0.1 M
Na2SO4 (for maintaining constant the ionic strength),
following 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 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 uncat-
alyzed 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 0.1 nM were done thereafter
with distilled-deionized water. Inhibitor and enzyme
solutions were preincubated together for 15 min at room
temperature prior to assay, in order to allow for the
formation of the E–I complex. The inhibition constants
were obtained by non-linear least-squares methods using
PRISM 3, as reported earlier,9 and represent the mean
from at least three different determinations. Enzyme
concentrations in the assay system were: 9.2 nM for
hCA I, 7.6 nM for hCA II, 12 nM for hCA IX, and
13 nM for hCA XII.7–9
19. Winum, J.-Y.; Temperini, C.; El Cheikh, K.; Innocenti,
A.; Vullo, D.; Ciattini, S.; Montero, J.-L.; Scozzafava, A.;
Supuran, C. T. J. Med. Chem. 2006, 49, 7024.
20. Temperini, C.; Innocenti, A.; Guerri, A.; Scozzafava, A.;
Rusconi, S.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2007, 17, 2210.
21. (a) Vullo, D.; Scozzafava, A.; Pastorekova, S.; Pastorek,
J.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2004, 14,
2351; (b) Pastorekova, S.; Casini, A.; Scozzafava, A.;
Vullo, D.; Pastorek, J.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2004, 14, 869.
22. Vullo, D.; Innocenti, A.; Nishimori, I.; Pastorek, J.;
Scozzafava, A.; Pastorekova, S.; Supuran, C. T. Bioorg.
Med. Chem. Lett. 2005, 15, 963.
1
724.15 [M+Na]+.; H NMR (DMSO-d6, 400 MHz): d 3.30
(m, 4H), 3.47 (m, 4H), 3.88 (s, 4H), 3.91 (s, 4H), 3,97 (s,
2H), 7.87 (s, 4H), 7.88 (m, 6H), 8.71 (s, 2H), 10.77 (s, 2H);
13C NMR (DMSO-d6, 101 MHz): d 50.37, 52.48, 55.31,
55.51, 58.11, 116.44, 120.51, 122.52, 129.41, 139.15, 144.54,
168.93, 169.88, 173.27. C3: Yield: 89%; mp 149–151 °C; MS
(ESIÀ): 728.11 [MÀH]À.; 1H NMR (DMSO-d6, 400 MHz):
d 2.89 (m, 4H), 3.01 (m, 4H), 3.32 (s, 4H), 3.34 (s, 4H), 3.42
(s, 2H), 4.33 (m, 4H), 7.32 (s, 4H), 7.42 (d, 4H, J = 8.1Hz),
7.74 (d, 4H, J = 8.1 Hz), 8.82 (s, 2H); 13C NMR(DMSO-d6,
101 MHz): d 41.57, 45.29, 50.37, 52.19, 55.21, 55.45, 125.61,
127.46, 142.41, 143.65, 168.96, 170.62, 172.82. C4: Yield:
86%; mp 127–129°C; MS (ESI+): 758.18 [M+H]+, 780.16
[M+Na]+, 796.28 [M+K]+. 1H NMR (DMSO-d6,
400 MHz): d 2.81 (m, 8H), 2.93 (m, 8H), 3.23 (s, 4H),
3.33 (s, 4H), 3,38 (s, 2H), 7.31 (s, 4H), 7.38 (d, 4H,
J = 8.1 Hz), 7.72 (d, 4H, J = 8.1 Hz), 8.20 (s, 2H); 13C
NMR (DMSO-d6, 101 MHz): d 35.85, 50.46, 52.41, 52.49,
55.22, 55.29, 58.14, 118.93, 126.57, 138.31, 141.67, 160.01,
170.09, 173.14. C5: Yield: 75%; mp 147–149 °C; MS (ESIÀ):
716.04 [M–H]À; 1H NMR (DMSO-d6, 400 MHz): d 2.09 (s,
2H), 3.04 (m, 4H), 3.53 (s, 4H), 3.71 (s, 4H), 3.81 (s, 2H),
8.32 (s, 4H); 13C NMR (DMSO-d6, 101 MHz): d 45.36,
45.4, 51.55, 55.31, 55.391, 161.48, 164.19, 171.51, 172.52,
172.97.
D1: Yield: 85%; mp 165–167 °C; MS (ESI+): 819.16
[M+H2O+K]+. 1H NMR (DMSO-d6, 400 MHz): d 2.08
(s, 2H), 3.42 (m, 18H), 7.28 (s, 4H), 7.81 (m, 4H). D2: Yield:
84%; mp 174–176 °C; MS (ESI+): 763.09 [M+H]+. 1H
NMR (DMSO-d6, 400 MHz): d 2.76 (s, 2H), 3.44 (m, 18H),
7.61 (s, 4H), 7.95 (m, 4H). D3: Yield: 81%; mp 171–173 °C;
23. Supuran, C. T.; Scozzafava, A.; Casini, A. Development
of sulfonamide carbonic anhydrase inhibitors. In Carbonic
Anhydrase–Its Inhibitors and Activators; Supuran, C. T.,
Scozzafava, A., Conway, J., Eds.; CRC Press: Boca
Raton, 2004; pp 67–147.