Carbonic anhydrase-encoded dynamic constitutional libraries: Toward the discovery of isozyme-specific inhibitors

Article abstract of DOI:10.1021/jm900449v

A constitutional dynamic library (CDL) was generated under thermodynamic control by using the amino-carbonyl/imine interconversion as reversible chemistry, combined with noncovalent bonding within the active site of the metalloenzyme carbonic anhydrase (C

Full text of DOI:10.1021/jm900449v

J. Med. Chem. 2009, 52, 4853–4859 4853
DOI: 10.1021/jm900449v
Carbonic Anhydrase-Encoded Dynamic Constitutional Libraries: Toward the Discovery of
Isozyme-Specific Inhibitors
Gihane Nasr,† Eddy Petit,† Daniela Vullo,‡ Jean-Yves Winum,§ Claudiu T. Supuran,*,‡ and Mihail Barboiu*,†
†
ꢀ
Adaptative Supramolecular Nanosystems Group, Institut Europeen des Membranes, ENSCM/UMII/UMR-CNRS 5635, Place Eugene
Bataillon, CC 047, 34095 Montpellier, Cedex 5, France, Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica
ꢁ
‡
ꢁ
§
ꢀ
Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy, and Institut des Biomolecules Max Mousseron (IBMM),
UMR 5247, CNRS-UM1-UM2, Batiment de Recherche Max Mousseron, Ecole Nationale Superieure de Chimie de Montpellier, 8 Rue de l’Ecole
^
ꢀ
Normale, 34296 Montpellier Cedex, France
Received April 7, 2009
A constitutional dynamic library (CDL) was generated under thermodynamic control by using the
amino-carbonyl/imine interconversion as reversible chemistry, combined with noncovalent bonding
within the active site of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Considering the
pharmacological importance to find isoform-selective CA inhibitors (CAIs), two of the 15 human (h)
isoform, i.e., hCAI and hCA II, have been subjected to a parallel screening of the same CDL. The use of
parallel constitutional screening of CDL chemistry for the discovery of enzyme inhibitors is straightfor-
ward and it might provide initial insights toward the generation of efficient classes of selective, high
affinity inhibitors. We demonstrate here that the high selectivity and specificity of inhibiting the hCA I
and hCA II isozymes with some of the detected hits may be used to describe a complex constitutional
behavior through component selection from the dynamic library, driven by the selective binding to the
specific isoform active site. These results also point to the possibility of modulating the drug discovery
methods by constitutional recomposition induced by a specific enzymatic target.
Introduction
most selective isoforms. Their inhibition has already offered
important biomedical options in the development of antiglau-
coma, antiepileptic, antiobesity, or anticancer drugs.
Carbonic anhydrases (CAa) represent an important class of
ubiquitously expressed zinc metalloenzymes catalyzing the
reversible hydration of carbon dioxide to bicarbonate and a
proton.1-3 The clinically available pharmacological agents
known to date present weak inhibition selectivity toward of
different CA isoforms inducing important side effects. Thus,
the development of isozyme-specific inhibitors is currently a
great challenge for obtaining novel types of drugs acting in
specific physiologic/pathologic processes.1 Muchprogress has
been achieved in the past decade for identifying selective CA
inhibitors (CAIs) by means of rational drug design.2,3 The
emergence of numerous families of selective CA inhibitors
against several pharmacologically relevant isozymes are based
on specific strategies including X-ray crystal structures for
some enzyme-inhibitor complexes.4 They mostly address
major basic structural elements such as the zinc coordinating
function5,6 or the nature of the hydrophobic residue7-10 lying
to the hydrophobic pocket standing above active metal ion
binding site. Among the 13 catalytically active R-CA isozymes
currently known and studied as the drug targets, human
carbonic anhydrases hCA I and hCA II are considered the
Dynamic combinatorial chemistry (DCC)11-13 has been
extensively implemented during the past decade as a powerful
approach in drug discovery14 that gives access to rapid and
attractive identification of ligands and inhibitors for receptors
and enzymes. The dynamic combinatorial approach is based
on a shift of chemical equilibrium of a library of reversibly
connected molecular components encompassing all possible
combinations, driven by a biomolecular (molecular) target
that favors the amplification of the fittest constituent forming
the most stable noncovalent supramolecular entities with the
target. DCC has been successfully implemented in a variety of
biological systems nonexhaustively including lectins,15,16 acet-
ylcholinesterase,17,18 neuraminidase,19,20 galactosyltransfer-
ase,21 glycosidase,22 DNA,23,24 etc.
Carbonic anhydrases (CA) have been one of the early
addressed biological targets for which the DCC25-29 may
offer a complementary route to high-throughput combinator-
ial methods.30 The first example in this field has been pio-
neeredby Lehnet al., who reported a library of12constituents
containing different Zn2þ complexing groups and various
aromatic moieties connected by the reversible imino-bond,
generating thus a hydrophobic sulphonamide inhibitor pos-
sessing high affinity toward the bovine carbonic anhydrase
(bCA II, EC 4.2.1.1).25 Then, the feasibility of this concept has
been extended by Nguyen et al.26 and Poulsen et al.,27-29
including a kinetic approach and a thermodynamic approach
based of cross-metathesis reversible reaction, all of which
addressing the same challenge: discovery of small molecule
*To whom correspondence should be addressed. For C.T.S.: phone,
þ390554573005; fax, þ390554573385; E-mail, claudiu.supuran@unifi.
it. For M.B.: phone, þ33467149195; fax, þ33467149119; E-mail, mihai.
barboiu@iemm.univ-montp2.fr.
a Abbreviations: CDL, constitutional dynamic library; DCC, dy-
namic combinatorial chemistry; DCL, dynamic combinatorial libraries;
CA, carbonic anhydrase; CAI, carbonic anhydrase inhibitors; hCA I,
human carbonic anydrase I; hCA II, human carbonic anydrase II;
bCAII, bovine carbonic anydrase II; ZBG, zinc-binding groups.
r
2009 American Chemical Society
Published on Web 07/06/2009
pubs.acs.org/jmc

4854 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Nasr et al.
inhibitors of bCA II, an easily accessible and inexpensive
enzyme but not very useful for discovering human CA
inhibitors (the two enzymes differ a lot from the point of view
of binding inhibitors, crystallographically and kinetically).1
One major advantage with reversible dynamic combinator-
ial libraries (DCLs) over their irreversible counterparts is their
potential adaptability to express the sorting constituent in
response to an external selection pressure.11,12 Change in the
output constituents of a DCL under the pressure of internal
and external factors (stimuli) based on constitutional dy-
namics31 express the system adaptation to a given situation.32
Herein, we report a such CDL that is susceptible to change its
composition (output expression) through component selection
driven by the selective binding to human hCAI and hCA II
isozymes. Considering the pharmacological importance to
find selective CAIs isozymes for a specific inhibitor, the hu-
man hCAI and hCA II isozymes, known to be more selective
(specific) than bovine carbonic anhydrase (bCA II), are
subjected to a parallel screening of the same CDL21 by using
the aminocarbonyl/imine interconversion as reversible chem-
istry. In particular, the useof CDL chemistry for the inhibitors
discovery might provide initial insights toward the strategy of
generation of efficient classes of selective active compounds,
against human carbonic anhydrases, for instance.
counterparts potentially interacting with the hydrophobic
pocket of the enzyme. Subsequent reduction reaction with
NaBH3CN stopped the dynamic exchange at equilibrium,
leading thus to the corresponding libraries of amines (Figure 1).
To generate dynamic combinatorial library, a set of three
aldehydes, 1-3, and two amines, A and B (Figure 1), were
selected. Aromatic aldehydes bearing electron withdrawing
moieties and primary amines are known to form high yield
Schiff’s bases in water. They are specifically chosen to arrange
the interactional components in a given geometry, favorably
interacting within the enzyme active site of the different CA
isozymes, leading to effective inhibitors.
The structural variability involving different substitution
patterns at the aromatic rings is essential for obtaining DCC
libraries with diverse binding affinities to the enzyme. Thus,
the structural core components were chosen to contain a
sulfonamide group (amine A) as one of the best ZBG allowing
to generate potent CAIs,37 the sulfonic acid (aldehyde 2),
which is a weak ZBG compared to the sulfamoyl one. For
example p-toluenesulfonate has a KI of 460 μM against
hCA II, whereas the corresponding sulfonamide of 11 μM38
and carboxylic acid (aldehyde 3) moieties also have weak zinc
binding functions.39 Two aromatic hydrophobic moieties
such as phenyl (amine B) and 2-fluorophenyl (aldehyde 1)
have been selected in order to probe the hydrophobic inter-
actions on the hydrophobic pocket above the active enzyme
site (Figure 1).
Results and Discussion
For achieving this objective, to design a constitutional
dynamic library of inhibitors against the ubiquitous and
pharmacologically highly relevant isoforms hCAI and hCA II,
imine generation and exchange has been used to produce a
prototypical DCL.32-35 In the present study, our choice went
to imine libraries of inhibitors resulting from the reversi-
ble assembly of a series of amines bearing specific zinc-
binding groups (ZBGs) with a set of hydrophobic aldehyde
Equilibration of amines A-B with aldehydes 1-3 was
expected to produce a set of six imines in equilibrium with
the six initial amines components. The distribution of this
mixture has been strongly altered by the addition of human
hCAI and hCA II isozymes. After reduction, most of initial
primary and secondary amines of the DCL together with
the corresponding alcohols resulted by reduction of initial
Figure 1. Constitutional dynamic chemistry applied to human carbonic anhydrase hCAI and hCA II isozymes and elaboration of
constitutional dynamic library (CDL).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4855
Figure 2. HPLC analysis of generation and screening of the CDL library: (a) in the presence and (b) in the absence of human hCA I isozyme
and (c) in the presence and (d) in the absence of human hCA II isozyme.
aldehydes have been identified by comparison of the retention
time in HPLC and UV profiles with those of each pure
compound. Surprisingly, compound 1B was not detected in
the mixture.
being respectively 3 and 2 times better hCA I inhibitors as
compared to imino compounds. This rule is no more valuable
in the case of very selective hCA II isozyme for which both
imino/amino compounds present almost the same the inhibi-
tory properties.
Simultaneously, the six imines 1Aim-3Aim, 1Bim-3Bim and
six secondary amines 1A-3A, 1B-3B compounds have been
synthesized separately and their inhibition constant KI against
catalytically active human hCA I and hCA II cytosolic
isozymes have been determined. Inhibition data correlated
with the results obtained from this library generation and
screening process, as well as the standard sulphonamide
acetazolamideAZA, are shownin Figures2 and3 andTable1.
As a general rule, there are also differences of hCA I
inhibitory activity between the stereochemically rigid imino
compounds and the corresponding saturated mobile amino
compounds. It is interesting to note that a rigid imino linker
would result in poorer binding, decreasing the inhibitory
properties. The difference in inhibition is very important, for
compounds 1Aim/1A and 3Aim/3A with the amino derivatives
The following structure-activity/CDL evolution relation-
ship can be drawn by considering data of Table 1:
(a) In the presence of hCA I only two amines: 3A and 2B
were amplified (Figures 2 and 3). The amine 3A prese-
nted an important amplification above 350% and very
good inhibition constants, confirming the strong in-
hibition power of sulfonamide group combined to
hydrophobic/H bonding effects of the carboxylic
group of the component 3 present in hydrophobic
pocket. There are several cases of compounds contain-
ing COOH moieties, which act as very effective hCA I/II
inhibitors, the best studied one being furosemide, for
which an X-ray crystal structure is available. This com-
pound is a good inhibitor of both hCA I (KI of 62 nM)

4856 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Nasr et al.
Figure 3. Inhibition constants KI and the amplification of the constitutional dynamic library (CDL) against catalytically active human hCA I
and hCA II cytosolic isosymes.
Table 1. Inhibition Constants KI against hCA I and hCA II of Pure Compounds 1A-3A and 1B-3B, the Relative Peak Area, and the Amplification of
the CDL in the Presence of hCA I and hCA II
KI (nM)a
relative peak area, RPAb
amplification factorc %
inhibitors
hCA I
620
hCA II
7.2
hCA I
hCA II
hCA I
hCA II
1Aim
1A
260
35
6.9
4.9
7.6
0.6
4.5
2.8
5.2
4.4
3.5
420
340
250
2Aim
2A
39
65
3Aim
3A
51
39
57
>100000
>100000
>100000
8025
350
180
1Bim
1B
>100000
>100000
8960
2Bim
2B
3490
0.7
3Bim
3B
AZAd
>100000
>100000
250
>100000
>100000
12
a From 3 different determinations. Errors: (5-10% of the shown value. b Relative value of the peak area is calculated using the ratio between the
experimental peak area in the presence and in the absence of CA: RPA=SCA/S0 c We speak about amplification when the peak area in the presence of CA
is higher than in the absence of CA. It is calculated as following: A % = [(SCA-S0)/S0] ꢀ 100. d Acetazolamide (AZA) was used as standard drug for
inhibition constant determination.
and of hCA II (KI of 65 nM).40 It is very interesting to
note the selective formation of amine 3A in the pre-
sence of 1A and 2A compounds. The presence of 2-
fluoride substituent in aldehyde component 1 leads to
a dramatic decrease of enzyme inhibitory activity,
resulting in poorer binding and thus reduced reactivity
into the enzyme hydrophobic site. Moreover, the
amine 2A,41 presenting the similar inhibitory activity
against hCA I with 3A, is practically not formed in this
CDL, probably as a result of the weak reactivity of the
furansulfonic aldehyde 2.
sterically better fitting into the enzyme active site than
1B-3B (Table 1). Surprisingly, the hydrophobic com-
ponent 1A with the same inhibition against hCA II
like 2A (amplification of 340%), presented the higher
amplification of 420%. It is in fact documented that
the fluorine atoms present in CAIs may form hydro-
gen bonds and other favorable interactions with ami-
no acid residues from the active site, leading thus to
enhanced stability of the enzyme-inhibitor com-
plex.42,43 In the same time, the presence of 3-carboxyl
substituent in aldehyde component 3 leads to a 10
times weaker hCA II inhibitory activity, resulting in
poorer enzyme binding and thus reduced reactivity
(amplification of 250%), probably due to a clash
between this moiety and some amino acid residue
form the hCA II active site, as evidenced earlier by
this group for a sugar sulfamide derivative.44 We
conclude that the specific hydrophobic or H bonding
binding sites present in the hydrophobic pocket of
the enzyme connected to the sulfonamide group of
(b) The strong inhibition power of sulfonamide group is
confirmed by the strong amplification of compounds
1A-3A in the presence of hCA II one of the most
active (selective) isoforms of CA family. The selective
amplification of all the possible combinations of
component A against the similar combinations of
component B, which are not present in the reactional
mixture in the presence of hCA II, is probably due
to the strong inhibitory power of amines 1A-3A,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4857
component A control the composition of the resulted
CDL by the synergistic inhibition/reactional phenom-
ena as a result of the optimized enthalpic/entropic
factors. The strong enthalpic effect of the sulphona-
mide binding seems to be not reliable to the com-
pounds 1B-3B presenting low inhibition activity.
used without further purification. 1H and 13C NMR spectra
were recorded on an ARX 300 MHz Bruker spectrometer in
CD3OD and D2O with the use of the residual solvent peak as
reference. The assignments were made on the base of the COSY
and NOESY spectra. Mass spectrometric studies were per-
formed in the positive ion mode using a quadrupole mass
spectrometer (Micromass, Platform II). Samples were continu-
ously introduced into the mass spectrometer through a Waters
616HPLC pump. The temperature (60 °C) and the extraction
cone voltage (Vc=5-10 V) were usually set to avoid fragmenta-
tions. The purity of synthesized imine and amine compounds
was determined by HPLC method (see below and Supporting
Information), confirming >95% purity.
Conclusion
In this study, it has been shown that a fine analysis can be
performed to identify new inhibitors and to evaluate their
relative affinities toward the physiologically relevant human
carbonic anhydrase hCA I and hCA II isozymes. A dynamic
combinatorial library of six components can be generated
under thermodynamic control by imine formation and ex-
change combined with noncovalent bonding within the en-
zymebinding site. The observed high selectivityand specificity
of hCA I and hCA II isozymes may be used to describe the
complex behavior displayed by the constitutional recomposi-
tion of a dynamic library under the distinct and specific
templating effect of the two enzymes.21,31,45 In this context,
the constitutional dynamic library (CDL) presented in this
paper is susceptible to change its composition (output ex-
pression) through component selection driven by the selective
binding to human hCAI and hCA II isozymes. Among all
possible imines formed, active compounds of appropriate
geometry can be easily identified in competitive reactional
conditions. This method enables the identification of a series
of three sulfonamide inhibitors 1A-3A presenting a good
inhibition and potent formation with high amplification
factors (250-420%) in the presence of hCA II isosyme.
Moreover, the dynamic screening process was beneficial to
finely identify compound 2B, as potent inhibitor of hCA I
isosyme. The compound 3A, which might represent the better
compromisebetweenentropic/enthalpic factorsasaresultof a
combined binding effects such as the sulphonamide zinc-
coordinating function of the component A and the hydro-
phobic/H-bonding of the component 4 present in hydropho-
bic pocket. Finally, once the fittest structural features have
been found, the results illustrate the response of such a
dynamic constitutional system to the presence of two different
enzymatic effects, demonstrating thus the adaptive behavior
of the system under the pressure of external factors. Such
features are of great interest for the development of drugs
targeting enzymes family with many isoforms such as the
family of the carbonic anhydrases. Indeed, a large number of
representatives (13 catalytically active isoforms in mammals)
playing fundamental physiological and pathological func-
tions are known for the mammalian CAs, and these findings
can be used as a paradigm in nonconventional drug design
studies aimed at obtaining compounds with selectivity for
some isoforms and thus drug candidates with reduced side
effects. Our findings may be thus relevant to the general drug
design research, especially when enzyme families with a multi-
tude of members and with similar active site features are
targeted.
General Procedure of Imine 1-3Aim and 1-3Bim Synthesis.
One mmol of the starting aldehyde (1-3) and 1 mmol of
corresponding amine (A, B) were dissolved in a CH3CN and
stirred at 70 °C for 18 h. The reaction mixture was than
evaporated and the crude product was dissolved in a minimum
of CH3CN and precipitated in hexane or ethylic ether. The
purity of synthesized imine compounds was determined by
HPLC method (see below and Supporting Information), con-
firming >95% purity.
General Procedure of Amine 1-3A and 1-3B Synthesis. To a
solution of imine (1 mmol) in methanol, we added drop by drop
a
methanolic solution of sodium borohydride (NaBH4)
(10 mmol). After 4 h, the product was purified by a semipre-
parative C18 silica column by using the methanol as eluent. The
purity of synthesized imine compounds was determined by
HPLC method (see below and Supporting Information), con-
firming >95% purity.
1H NMR and ESI MS characterization data of the most
amplified compounds 1-3A and 2B are the following:
N-(2-Fluorobenzyl)(4-sulphonamide phenyl)ethanamine (1A).
Yield 32%. 1H NMR (300 MHz, D2O) δ=7.78 (td, 1H, CH-50),
7.71 (d, 2H, CH-3,5), 7.38 (td, 1H, CH-20), 7.32 (d, 2H, CH-6,2),
6.95-7.01 (m, 2H, CH-30,40), 3.3 (t, 2H, CH2-N), 2.90 (t, 2H,
CH2-Ar). C17H15FN2O2S, mol wt: 308.36. ES-MS: m/z (%)
m/z 309.2 (90) MHþ, 349.1(90) [MCH3CNH]þ.
N-(5-Methane-2-furansulfonicacid)(4-sulfonamidephenyl)-
ethanamine (2A). Yield 46%. 1H NMR (300 MHz, D2O) δ=7.81
(d,2H,CH-3,5),7.44(d,2H, CH-2,4),6.78(d,1H,CHfurane),6.61
(d, 1H, CH furane), 3.29 (t, 2H, CH2-N), 3.07 (t, 2H,
CH2-Ar). C15H13N2O6S2, mol wt: 359.38. ES--MS: m/z 359
(100) M-.
N-(3-Carboxybenzyl)(4-sulphonamidephenyl)ethanamine (3A).
Yield 36%. 1H NMR (300 MHz, D2O) δ=7.90 (s, 1H, CH-60),
7.83-7.03 (m, 3H, CH-40,3,5), 7.45 (m, 4H, CH-20,30,2,6), 3.72 (t,
2H, CH2-N), 2.79 (t, 2H, CH2-Ar), 2.03 (s, 2H, NH2).
C18H16N2O4S, mol wt: 334.38. ES--MS: m/z 333.3 (100) MH-.
N-(5-Methane-2-furansulfonicacid)(phenyl)methanamine (2B).
Yield 46%. 1H NMR (300 MHz, D2O) δ=7.39 (m, 5H, CH Ar
2-6), 6.78 (d, 1H, CH furane), 6.60 (d, 1H, CH furane), 4.16 (s,
2H, CH2). C14H10NO4S, mol wt: 266.28. ESþ-MS: m/z 267.1
(100) MHþ. ES--MS: m/z 266.2 (100) M-.
For 1H NMR and ESI MS characterization data of the
nonamplified compounds, see Supporting Information.
Synthesis of DCL Libraries. The reactions were performed in
the presence of hCA I or hCA II in water at pH 6.5. A 10-fold
excess of the starting amines was used to limit side reactions
between the aldehydes and the terminal amino residues groups
of CA. The aldehydes and amines reacted reversibly to form
imines with percentages that vary according to the reactivity of
the starting amines and aldehydes and to their affinity for the
casting site on CA.46 Three reactions were performed with and
without enzyme (hCAI or hCAII) in a sodium phosphate
solution at pH 6.5 (20 mM phosphate buffer). Stock solution
in DMSO of three aldehydes (10 mM) and the two amines
(100 mM) were added to three3 aqueous solutions in order to
reach the final concentration of 0.08 mM for aldehydes and
0.8 mM for amines, respectively. The clear mixture was
Experimental Section
Materials and Methods. Carbonic anhydrase hCA I or hCA II
isosymes from human erythrocytes (Sigma), benzylamine
99.5% (Sigma), 4-(2-aminoethyl)benzenzsulfonamide, 99%
(Across Organics), 2-fluorobenzaldehyde 98%, 3-carboxy-ben-
zaldehyde 95% (Fluka), and 5-formyl-2-furansulfonic acid
sodium salt hydrates 97% (Alfa Aesar) were purchased and

4858 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Nasr et al.
incubated at 25 °C for 3 days before addition 0.8 mM of
NaBH3CN and incubated for one more week. Prior to analyzing
the library, the mixture was left for 2 h to be separated from CA
by decantation. The thermal denaturation of the enzyme (2 min
at 80 °C) was also tested to ensure the release from casting site of
some possible tightly bound ligands.
(2) Carbonic Anhydrase: Its Inhibitors and Activators; Supuran,
C. T., Scozzafava, A., Conway, J., Eds.; CRC Press: Boca Raton,
FL, 2004, pp 1-376, and references cited therein.
(3) Supuran C. T.; Winum J.-Y. Selectivity issues in the design of CA
inhibitors. In Drug Design of Zinc-Enzyme Inhibitors: Func-
tional, Structural, and Disease Applications; Supuran, C. T., Winum,
J.-Y., Eds.; Wiley: New York, 2009.
HPLC Screening Method. The chromatography was per-
formed on a diode array detector. An Atlantis C18 reverse-phase
column was used (4.6 mmꢀ100 mm, 3 μm), under a binary
gradient of CH3CN and buffer solution at pH 3.1. The purity of
synthesized imine and amine compounds have been determined
by HPLC. The buffer solution was prepared by dissolving
KH2PO4 in distillated water. The pH was adjusted at 3.1 by
addition H3PO4. The total concentration of the buffer was fixed
at 6.8 g/L. The optimized gradient begins by 100% of buffer
solution then reached 45%ofbuffer solutionand55% of CH3CN
in 60 min. After the reduction, the chromatographic analysis of
the resulted mixtures in the absence or in the presence of hCAI or
hCAII showed exclusively the presence of reduced secondary
imines and the excess of starting amines. We noticed the absence
of related alcohols produced by direct reduction of the aldehydes.
The overall conversion of the initial aldehydes is 100%, probably
determined by the 10-fold excess of starting amines. High con-
version for imine formation in water has been already observed at
this stoichiometry as previously reported.25,46
CA Inhibition Assay. An SX.18MV-R Applied Photophysics
(Oxford, UK) stopped-flow instrument was used to assay the
catalytic/inhibition of various CA isozymes as reported by Kha-
lifah.36 Phenol Red (at a concentration of 0.2 mM) was 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 (see Supporting Information for the synthesis of
pure compounds details) for were prepared 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 seven different
inhibitor concentrations have been used for measuring the in-
hibition constant. Inhibitor and enzyme solutions were preincu-
bated 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 paper are the mean of such
results. The inhibition constants were obtained by nonlinear
least-squares methods using PRISM 3 and represent the mean
from at least three different determinations.
(4) 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: New York, 2009.
(5) Winum, J.-Y.; Montero, J.-L.; Scozzafava, A.; Supuran, C.-T. New
zinc binding motifs in the design of selective carbonic anhydrase
inhibitors. Mini-Rev. Med. Chem. 2006, 6, 921–936.
(6) Winum, J.-Y.; Scozzafava, A.; Montero, J.-L.; Supuran, C.-T.
Design of zinc binding functions for carbonic anhydrase inhibitors.
Curr. Pharm. Des. 2008, 14, 615–21.
(7) Supuran, C. T.; Scozzafava, A.; Casini, A. Carbonic anhydrase
inhibitors. Med. Res. Rev. 2003, 23, 146–189.
(8) Barboiu, M.; Supuran, C. T.; Menabuoni, L.; Scozzafava, A.;
Mincione, F.; Briganti, F.; Mincione, G. Carbonic anhydrase
inhibitors, synthesis of topically effective intraocular pressure
lowering agents derived from 5-(aminoalkyl-carboxamido)-1,3,4-
thiadiazole-2-sulfonamide. J. Enzyme. Inhib. 1999, 15, 23–46.
(9) Supuran, C. T.; Barboiu, M.; Luca, C.; Pop, E.; Dinculescu, A.
Carbonic Anhydrase Activators. Part 14. Syntheses of positively
charged derivatives of 2-amino-5-(2-aminoethyl) and 2-amino-
5-(2-aminopropyl)-1,3,4 thiadiazole and their interaction with
isozyme II. Eur. J. Med. Chem. 1996, 31, 597–606.
(10) Winum, J.-Y.; Rami, M.; Scozzafava, A.; Montero, J.-L.; Supuran,
C. Carbonic anhydrase IX: a new druggable target for the design of
antitumor agents. Med. Res. Rev. 2008, 28, 445–463.
(11) Lehn, J.-M. Dynamic combinatorial chemistry and virtual combi-
natorial libraries. Chem.;Eur. J. 1999, 5, 2455–2463.
(12) Lehn, J.-M. Toward complex matter: Supramolecular chemistry
and self-organization. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4763–
4768.
(13) Corbett, P. T.; Leclaire, J.; Vial, L.; West, K. R.; Wietor, J.-L.;
Sanders, J. K. M.; Otto, S. Dynamic combinatorial chemistry.
Chem. Rev. 2006, 106, 3652–3711.
(14) Ramstrom, O.; Lehn, J.-M. Drug Discovery by Dynamic Combi-
natorial Libraries. Nat. Rev. Drug. Discovery 2002, 1, 26–36.
(15) Ramstrom, O.; Lehn, J.-M. In situ generation and screening of a
dynamic combinatorial carbohydrate library against Concanava-
lin A. ChemBioChem 2000, 1, 41–48.
(16) Ramstrom, O.; Lohman, S.; Bunyapaiboonsri, T.; Lehn, J.-M.
Dynamic combinatorial carbohydrate libraries: probing the bind-
ing site of the Concanavalin A lectin. Chem.;Eur. J. 2004, 10,
1711–1715.
(17) Bunyapaiboonsri, T.; Ramstrom, O.; Lohman, S.; Lehn, J.-M.
Dynamic deconvolution of a preequilibrated dynamic combinator-
ial library of acetylcholinesterase inhibitors. ChemBioChem 2001,
2, 438–444.
(18) Larsson, R.; Pei, Z.; Ramstrom, O. Catalytic Self-Screening of
Cholinesterase Substrates from a Dynamic Combinatorial Thioe-
ster Library. Angew. Chem., Int. Ed. 2004, 43, 3716–3718.
(19) Hochgurtel, M.; Kroth, H.; Piecha, D.; Hofmann, M. W.; Nicolau,
C.; Krause, S.; Schaaf, O.; Sonnenmoser, G.; Eliseev, A. V. Target-
induced formation of neuraminidase inhibitors from in vitro
virtual combinatorial libraries. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 3382–3387.
(20) Hochgurtel, M.; Biesinger, R.; Kroth, H.; Piecha, D.; Hofmann,
M. W.; Krause, S.; Schaaf, O.; Nicolau, C.; Eliseev, A. V. Ketones
as building blocks for dynamic combinatorial libraries: highly
active neuraminidase inhibitors generated via selection pressure
of the biological target. J. Med. Chem. 2003, 46, 356–358.
(21) Valade, A.; Urban, D.; Beau, J.-M. Two galactosyltransferases’
selection of different binders from the same uridine-based dynamic
combinatorial library. J. Comb. Chem. 2007, 9, 1–4.
(22) Gerber-Lemaire, S.; Popowycz, F.; Rodriguez-Garcia, E.; Asenjo,
A. T. C.; Robina, I.; Vogel , P. An efficient combinatorial method
for the discovery of glycosidase inhibitors. ChemBioChem 2002, 3,
466–470.
Acknowledgment. This work, conducted as part of the
award “Dynamic adaptative materials for separation and
sensing Microsystems” made under the European Heads of
Research Councils and European Science Foundation EUR-
YI (European Young Investigator) Awards scheme in 2004,
was supported by funds from the participating organizations
of EURYI and the EC Sixth Framework Programme. See
www.esf.org/euryi. This research was also financed in part by
a grant of the 6th Framework Programme of the European
Union (DeZnIT project) and one belonging to the 7th Frame-
work Programme (Metoxia project).
Supporting Information Available: HPLC screening method
details; UV, NMR, and ESI-MS spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
(23) Whitney, A. M.; Ladame, S.; Balasubramanian, S. Templated
ligand assembly by using G-quadruplex DNA and dynamic cova-
lent chemistry. Angew. Chem., Int. Ed 2004, 43, 1143–1146.
(24) Tsujita, S.; Tanada, M.; Kataoka, T.; Sasaki, S. Equilibrium shift
by target DNA substrates for determination of DNA binding
ligands. Bioorg. Med. Chem. Lett. 2007, 17, 68–72.
References
(25) Huc, I.; Lehn, J.-M. Virtual combinatorial libraries: dynamic
generation of molecular and supramolecular diversity by self-
assembly. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2106–2110.
(1) Supuran, C. T. Carbonic anhydrases: novel therapeutic applica-
tions for inhibitors and activators. Nat. Rev. Drug. Discovery 2008,
7, 168–181.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4859
(26) Nguyen, R.; Huc, I. Using and enzyme’s active site to template
inhibitors. Angew. Chem., Int. Ed 2001, 40, 1774–1776.
(27) Poulsen, S.-A.; Bornaghi, L. F. Fragment-based drug discovery of
carbonic anhydrase II inhibitors by dynamic combinatorial chem-
istry utilizing cross metathesis. Bioorg. Med. Chem. 2006, 14, 3275–
3284.
(28) Poulsen, S.-A. Direct screening of a dynamic combinatorial library
using mass spectrometry. J. Am. Soc. Mass Spectrom. 2006, 17,
1074–1080.
(38) Briganti, F.; Pierattelli, R.; Scozzafava, A.; Supuran, C. T. Carbo-
nic anhydrase inhibitors. Part 37. Novel classes of carbonic anhy-
drase inhibitors and their interaction with the native and cobalt-
substituted enzyme: kinetic and spectroscopic investigations. Eur.
J. Med. Chem. 1996, 31, 1001–1010.
(39) Innocenti, A.; Vullo, D.; Scozzafava, A.; Casey, J. R.; Supuran
C. T. Carbonic anhydrase inhibitors. Interaction of isozymes I, II,
IV, V, and IX with carboxylates. Bioorg. Med. Chem. Lett. 2005,
15, 573–578.
(29) Poulsen, S.-A.; Davis, R. A.; Keys, T. G. Screening natural
product-based combinatorial library using FTICR mass spectro-
metry. Bioorg. Med. Chem. 2006, 14, 510–515.
(30) Combinatorial Chemistry-Synthesis and Applications Wilson,
S. R., Czarnik, A. W., Eds.; Wiley: New York, 1997.
(31) Lehn, J.-M. From supramolecular chemistry towards constitu-
tional dynamic chemistry and adaptative chemistry. Chem. Soc.
Rev. 2007, 36, 151–160.
(32) Giusepponne, N.; Lehn, J.-M. Protonic and Temperature Mod-
ulation of Constituent Expression by Component Selection in a
Dynamic Combinatorial Library of Imines. Chem.;Eur. J. 2006,
12, 1715–1722.
(33) Dumitru, F.; Petit, E.; van der Lee, A.; Barboiu, M. Homoduplex
and Heteroduplex Complexes Resulted from Terpyridine-Type
Ligands and Zn2þ Metal Ions. Eur. J. Inorg. Chem. 2005, 21,
4255–4262.
(40) Temperini, C.; Cecchi, A.; Scozzafava, A.; Supuran, C. T. Carbo-
nic anhydrase inhibitors. Comparison of chlorthalidone, indapa-
mide, trichloromethiazide and furosemide X-ray crystal structures
in adducts with isozyme II, when several water molecules make the
difference. Bioorg. Med. Chem. 2009, 17, 1214–1221.
(41) The amine 2A which are not formed in the absence of carbonic
anhydrase present weak amplifications in the presence of the
enzyme but its enrichment can not be evaluated from the experi-
mental results.
(42) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.; Min-
cione, V.; Supuran, C. T. Carbonic anhydrase inhibitors: Perfluor-
oalkyl/aryl-substituted derivatives of aromatic/heterocyclic
sulfonamides as topical intraocular pressure lowering agents with
prolonged duration of action. J. Med. Chem. 2000, 43, 4542–4551.
(43) Abbate, F.; Casini, A.; Scozzafava, A.; Supuran, C. T. Carbonic
anhydrase inhibitors: X-ray crystallographic structure of the ad-
duct of human isozyme II with the perfluorobenzoyl analogue of
methazolamide. Implications for the drug design of fluorinated
inhibitors. J. Enzyme Inhib. Med. Chem. 2003, 18, 303–308.
(44) Winum, J. Y.; Temperini, C.; El Cheikh, K.; Innocenti, A.; Vullo,
D.; Ciattini, S.; Montero, J. L.; Scozzafava, A.; Supuran, C. T.
Carbonic anhydrase inhibitors: clash with Ala65 as a means for
designing inhibitors with low affinity for the ubiquitous isozyme II,
exemplified by the crystal structure of the topiramate sulfamide
analogue. J. Med. Chem. 2006, 49, 7024–7031.
(34) Legrand, Y. M.; van der Lee, A.; Barboiu, M. Self-Optimizing
Charge Transfer Energy Phenomena in Metallosupramolecular
Complexes by Dynamic Constitutional Self-Sorting. Inorg. Chem.
2007, 46, 9540–9547.
(35) Barboiu, M.; Petit, E.; van der Lee, A.; Vaughan, G. Constitutional
Self-Selection of [2 ꢀ 2] Homonuclear Grids from a Dynamic
Combinatorial Library. Inorg. Chem. 2006, 45, 484–486.
(36) Khalifah, R. G. The carbon dioxide hydration activity of carbonic
anhydrase. I. Stop-flow kinetic studies on the native human
isoenzymes B and C. J. Biol. Chem. 1971, 246, 2561–2573.
(37) Puccetti, L.; Fasolis, G.; Vullo, D.; Chohan, Z. H.; Scozzafava, A.;
Supuran, C. T. Carbonic anhydrase inhibitors. Inhibition of
cytosolic/tumor-associated carbonic anhydrase isozymes I, II,
IX, and XII with Schiff’s bases incorporating chromone and
aromatic sulfonamide moieties, and their zinc complexes. Bioorg.
Med. Chem. Lett. 2005, 15, 3096–3101.
(45) Barboiu, M.; Lehn, J.-M. Dynamic chemical devices. Modulation
of contraction/extension molecular motion by coupled ion bind-
ing/pH change induced structural switching. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 5201–5206.
(46) Godoy-Alcantar, C.; Tatsimirsky, A. K.; Lehn, J. M. Structure
stability correlations for imine formation in aqueous solution. J.
Phys. Org. Chem. 2005, 18, 979–985.