Probing Molecular Interactions between Human Carbonic Anhydrases (hCAs) and a Novel Class of Benzenesulfonamides

Article abstract of DOI:10.1021/acs.jmedchem.7b00264

On the basis of X-ray crystallographic studies of the complex of hCA II with 4-(3,4-dihydro-1H-isoquinoline-2-carbonyl)benzenesulfonamide (3) (PDB code 4Z1J), a novel series of 4-(1-aryl-3,4-dihydro-1H-isoquinolin-2-carbonyl)benzenesulfonamides (23-33) wa

Full text of DOI:10.1021/acs.jmedchem.7b00264

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Article
Probing Molecular Interactions between human Carbonic
Anhydrases (hCAs) and a Novel Class of Benzenesulfonamides
Elvira Bruno, Maria Rosa Buemi, Anna Di Fiore, Laura De Luca, Stefania
Ferro, Andrea Angeli, Roberto Cirilli, Daniele Sadutto, Vincenzo Alterio, Simona
Maria Monti, Claudiu T Supuran, Giuseppina De Simone, and Rosaria Gitto
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017
Downloaded from http://pubs.acs.org on April 29, 2017
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Probing Molecular Interactions between human
Carbonic Anhydrases (hCAs) and a Novel Class of
Benzenesulfonamides
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Elvira Bruno,a Maria Rosa Buemi,a Anna Di Fiore,b Laura De Luca,a Stefania Ferro,a Andrea
Angeli,c Roberto Cirilli,d Daniele Sadutto,d Vincenzo Alterio,b Simona Maria Monti,b Claudiu T.
Supuran,c Giuseppina De Simone,b Rosaria Gittoa*
aDipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali (CHIBIOFARAM),
Università degli Studi di Messina, Viale Annunziata, Iꢀ98168, Messina.
bIstituto di Biostrutture e BioimmaginiꢀCNR, Via Mezzocannone 16, 80134, Napoli
cDipartimento NEUROFARBA, Università di Firenze, Via Ugo Schiff 6, Iꢀ50019 ꢀ Sesto
Fiorentino.
dCentro nazionale per il controllo e la valutazione dei farmaci, Istituto Superiore di Sanità, V.le
Regina Elena, 299, 00161 Roma.
Keywords: human Carbonic Anhydrases; isoquinolineꢀsulfonamides; HPLC; Xꢀray
crystallography; docking studies
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Abstract: On the basis of Xꢀray crystallographic studies of the complex of hCA II with 4ꢀ(3,4ꢀ
dihydroꢀ1Hꢀisoquinolineꢀ2ꢀcarbonyl)benzenesulfonamide (3) (PDB code 4Z1J), a novel series of 4ꢀ
(1ꢀarylꢀ3,4ꢀdihydroꢀ1Hꢀisoquinolinꢀ2ꢀcarbonyl)benzenesulfonamides (23-33) was designed.
Specifically, our idea was to improve the selectivity toward druggable isoforms through the
introduction of additional hydrophobic/hydrophilic functionalities. Among the synthesized and
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tested
compounds,
the
(R,S)ꢀ4ꢀ(6,7ꢀdihydroxyꢀ1ꢀphenylꢀ3,4ꢀtetrahydroisoquinolineꢀ1Hꢀ2ꢀ
carbonyl)benzenesulfonamide (30) exhibited a remarkable inhibition for the brainꢀexpressed hCA
VII (Ki = 0.20 nM) and selectivity over wider distributed hCA I and hCA II isoforms. By
enantioselective HPLC we solved the racemic mixture and ascertained that the two enantiomers
(30a and 30b) are equiactive inhibitors for hCA VII. Crystallographic and docking studies revealed
the main interactions of these inhibitors into the carbonic anhydrase (CA) catalytic site, thus
highlighting the relevant role of nonꢀpolar contacts for this class of hCA inhibitors.
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Introduction
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Human carbonic anhydrases (hCAs, EC 4.2.1.1) are metalloenzymes that catalyse the reversible
hydration of carbon dioxide to bicarbonate and proton. The hCA family comprises 15 different αꢀ
CA isoforms, of which five are cytosolic (CA IꢀIII, CA VII and CA XIII), four are membraneꢀ
bound (CA IV, CA IX, CA XII, and CA XIV), two are mitochondrial (CA VA and CA VB), and
one is secreted into saliva (CA VI). hCAs are involved in various physiological processes
(gluconeogenesis, lipogenesis, and ureagenesis). However, their abnormal levels or activities have
been often associated with several diseases such as cancer, epilepsy, obesity, glaucoma, etc.1ꢀ6 For
this reason, CA isoforms have become relevant targets for the design of inhibitors with biomedical
applications.5, 7, 8
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hCA VII, hCA IX, hCA XII and hCA XIV are among the most interesting hCA isoforms. hCA
VII is mainly expressed in the cortex, hippocampus and thalamus regions within the mammalian
brain where it is involved in generating neuronal excitation and seizures4, 9 as well as in neuropathic
pain control.10 hCA IX is overexpressed in many solid tumours associated with the hypoxic
phenotype, where it is involved in critical processes connected with cancer progression.11,12ꢀ22
Recently, it was demonstrated that also hCA XII is a tumorꢀassociated isoform, thus emerging as
innovative target for the development of anticancer agents.22 Finally, hCA XIV is a transmembrane
isozyme with an extracellularly oriented active site; it is highly abundant in neurons and axons in
the murine and human brain, where it seems to play an important role in modulating excitatory
synaptic transmission.23, 24
Although, the first generation of CA inhibitors (CAIs), such as acetazolamide (AAZ, 1) and
topiramate (TPM, 2) (Chart 1), were able to strongly bind hCA VII, hCA IX, hCA XII and hCA
XIV, they were also strong inhibitors of the ubiquitous hCA I and hCA II isoforms, displaying
many undesired sideꢀeffects. Consequently, many research efforts have been recently dedicated to
the design of new CAIs able to target selectively hCA VII, hCA IX, hCAXII and hCA XIV.25
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NH2
O
S
O
O
O
N N
S
O
O
S
HN
NH2
O
O
O
O
O
9
AAZ, 1
TPM, 2
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NH2
O
O
S
NH2
O
O
S
N
O
*
N
O
R 1 R2
R 3
3
23-33
Chart 1. Chemical structures of selected hCAIs (1-3) and designed compounds (23-33)
Among the CAIs so far developed, sulfonamide based compounds are the most studied. These
compounds bind the catalytic zinc ion through the deprotonated nitrogen of the sulfonamide moiety,
blocking the CA enzymatic activity.26ꢀ40 The hCA isoform selectivity of sulfonamideꢀbased CAIs is
tuned by the remaining molecular fragment, which can interact with hydrophobic/hydrophilic
residues delimiting the CAꢀcatalytic site.38, 41ꢀ47 So the high selectivity towards several isoforms is
controlled by profitable interactions between the hydrophobic/hydrophilic residues on the CA cleft
and suitable functional groups of the most selective hCAIs.24, 48, 49
We have recently reported a new series of heteroarylꢀNꢀcarbonylbenzenesulfonamides as CAIs.50
Among them, the 4ꢀ(3,4ꢀdihydroꢀ1Hꢀisoquinolineꢀ2ꢀcarbonyl)benzenesulfonamide (3, Chart 1) had
significant inhibitory effects against hCA VII, hCA IX, and hCA XIV. Herein we report the
synthesis, structural characterization and evaluation of inhibitory effects of eleven (R,S)ꢀ4ꢀ(1ꢀarylꢀ
3,4ꢀdihydroꢀ1Hꢀisoquinolineꢀ2ꢀcarbonyl)benzenesulfonamides (23-33, Chart 1) inspired by the
prototype 3. In particular, we investigated the introduction of an alkoxy or hydroxyl substituent on
6/7ꢀpositions of isoquinoline system combined with the presence of an arylꢀsubstituent at the Cꢀ1
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position. Specifically, the 6 and/or 7 substituents (R1R2) could furnish new Hꢀbonding contacts;
whereas the Cꢀ1 fragment could add a new hydrophobic anchoring moiety, that might be useful to
strengthen the network of nonꢀpolar interactions with hydrophobic residues within catalytic site. To
enhance the hydrophobic interactions we also examined the effects of an additional haloꢀsubstituent
(R3) at para position of Cꢀ1 phenyl ring. The introduction of Cꢀ1 phenyl ring generates a chiral
center that could furnish a couple of enantiomers with different inhibitory effects. Thereby, we
decided to perform the resolution of racemic mixtures of selected inhibitors to explore the impact of
stereochemistry on CA inhibition. Moreover, experimental and computational analyses have been
performed to highlight the interactions of these molecules within the active site. Specifically, we
studied the crystallographic structures of the adducts of the most active compounds of the series
with hCA II and performed docking simulations to identify the binding poses of these molecules
within the hCA VII catalytic site. Finally, we have compared the solved crystal structures with
docking poses to search for structural information about the isoformꢀselectivity profile.
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Results and discussion
Synthesis.
The
designed
(R,S)ꢀ4ꢀ(1ꢀarylꢀ3,4ꢀdihydroꢀ1Hꢀisoquinolineꢀ2ꢀ
carbonyl)benzenesulfonamides (23-33) were synthesized by the procedure described in Scheme 1.
previously reported method,51 we prepared the 1ꢀphenylꢀ1,2,3,4ꢀ
Following
a
tetrahydroisoquinolines 17-22 in Microwave Assisted Organic Synthesis conditions by a Pictetꢀ
Spengler approach from phenylethylamine derivatives 4-6 and suitable benzaldehyde derivatives 7-
10. Specifically, in the first step we obtained the imine intermediates 11-16, which were treated
with trifluoroacetic acid (TFA) to provide the desired isoquinolines 17-22 as racemic mixtures.
Successively, amines 17-22 reacted with the 4ꢀ(aminosulfonyl)benzoic acid to give desired Nꢀ
substituted isoquinolines 23-28. The 6,7ꢀdimethoxy and 6ꢀmethoxy derivatives 23, 25-28 were
further transformed into the corresponding hydroxyl analogues 29-33 by treatment with BBr3 in the
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presence of anhydrous CH2Cl2 and under an atmosphere of N2. The chemical characterization of
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intermediates and final compounds was supported HꢀNMR (see Experimental section). The purity
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of tested compounds was confirmed by elemental analysis.
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R1
R2
R1
R2
R1
R2
O
H
i
ii
+
R 3
NH2
NH
N
4-6
4, R1 = OCH3 ; R2 = H
7-10
11-16
17-22
7, R3 = H
8, R3 = F
R 3
R 3
5, R1 = O(CH2)2CH3 ; R2 = H
6, R1 = R2 = OCH3
9, R3 = Cl
10, R3 = Br
iii
O
NH2
R1
R2
S
O
N
O
11, 17, 23, 29 R1 = OCH3 ; R2 = R3= H
12, 18, 24 --- R1 = O(CH2)2CH3; R2 = R3 = H
13, 19, 25, 30 R1 = R2 = OCH3 ; R3 = H
14, 20, 26, 31 R1 = R2 = OCH3 ; R3 = F,
15, 21, 27, 32 R1 = R2 = OCH3 ; R3 = Cl
16, 22, 28, 33 R1 = R2 = OCH3 ; R3 = Br
23-28
iv
R 3
23, 25-28
29-33
Scheme 1. Reagents and conditions: i) MW: 10 min, 90°C, 150 W; ii) TFA, MW: 10 min, 90 °C,
150 W; iii) 4ꢀ(aminosulfonyl)benzoic acid, HBTU, TEA, DCM, rt, overnight; iv) BBr3(1 M in
DCM), DCM, rt, overnight.
Carbonic Anhydrase Inhibition. The CA inhibitory effects of obtained 4ꢀ(1ꢀarylꢀ3,4ꢀdihydroꢀ1Hꢀ
isochinolinꢀ2ꢀcarbonyl)benzenesulfonamides (23-33) were measured for human CA I, CA II,
CAVII, CA IX, CA XII and CA XIV; results are summarized in Table 1 and compared with Ki
values of prototype 3, AAZ (1) and TPM (2) as reference compounds. For the preliminary
investigation, we have tested the racemic mixtures of all new synthesized compounds.
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O
NH2
S
R1
R2
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O
N
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23-33
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R 3
Table 1. Ki values of arylsulfonamide derivatives 3, and 23-33 against hCA I, hCA II, hCA VII,
hCA IX, hCA XII and hCA XIV. For comparison purpose, data of AAZ and TPM were also
reported.
Ki(nM)a
R1
R2
R3 hCA I hCA II hCA VII hCA IX hCA XII hCA XIV
3b
H
H
ꢀꢀꢀ 76.2
7.7
9.20
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84.4
7.30
0.53
0.77
3.80
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0.20
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0.40
0.33
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7.50
39.2
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4.90
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91.4
90.9
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36.9
8.20
9.00
81.5
58
n.t.
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6.7
60.3
4.3
933
847
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4.7
4.3
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9.3
3.8
5.7
9.6
OCH3
OC3H7
OCH3
OCH3
OCH3
OCH3
OH
H
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H
H
H
F
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850
16.4
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5.3
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H
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33.2
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25.6
4.3
25
OCH3
OCH3
OCH3
OCH3
H
26
0.84
5.5
374
325
4010
2.9
27
Cl 10.9
Br 38.9
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16.9
4.5
29
H
H
F
6.2
9.3
2.2
OH
OH
OH
OH
OH
30
3.5
3.3
OH
31
0.21
0.29
0.28
10
346
2915
3930
1460
41
OH
32
Cl 3.5
Br 3.4
250
OH
33
TPMb
AAZb
250
12.1
25.
aErrors are in the range of ± 10 % of the reported value, from 3 different assays. Data are taken from Reference 50.
b
n.t. = not tested
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Firstly, we can observe that the introduction of a methoxyꢀgroup at Cꢀ6 position and a phenyl ring
at Cꢀ1 was well tolerated except for hCA IX, so that compound 23 displayed similar inhibitory
effects than those of prototype 3. In turn, the replacement of the methoxy group with propoxy one
(see compound 24) generally resulted in reduction of inhibition against studied hCAs. The presence
of an additional methoxy group at Cꢀ7 position (i.e. compound 25 vs 23) resulted in about 3ꢀfold
drop in potency of inhibition against hCA XII isoform and a 5ꢀfold improvement against hCA IX
(see Table 1). Secondly, the introduction of a fluorine atom at para position of Cꢀ1 phenyl ring
(compound 26) resulted in a remarkable inhibition of hCA II and hCA VII isoforms for compound
26; whereas this modification led to a loss of activity toward hCA XIV. Then, the replacement of
the fluorine atom with chlorine or bromine atoms induced a remarkable reduction of potency for
hCA XII and hCA XIV isoforms for compounds 27 and 28 compared with fluorine analogue 26. It
was possible to observe that the increasing size of the halogen atom was detrimental for inhibition
of all studied isoforms. The degree of CA inhibitory potency was the following 4ꢀF>4ꢀCl>4ꢀBr.
Finally, we examined the impact on CA inhibition of the removal of methyl group of 6/7
methoxysubstituents on isoquinoline nucleus. The monohydroxyꢀderivative 29 and dihydroxyꢀ
derivative 30 displayed a very similar inhibitory potency against studied hCA isoforms.
Specifically, they showed remarkable potency as hCA VII inhibitors (Ki values of 0.20 and 0.35
nM, respectively for compound 30 and 29). Interestingly, the 4ꢀhalophenyl derivatives 31-33
proved to be very active inhibitors displaying Ki values ranging from 0.21 to 0.51 nM against hCA
II and hCA VII. By analysing the hCA VII Ki values shown by hydroxyl compounds 29-33, we
found a flat influence of the steric and electronic effects of the paraꢀphenyl substituent, thus
suggesting that this substituent does not establish crucial contacts within catalytic site. Moreover,
compounds 31ꢀ33 showed high inhibitory potency for hCA I, hCA IX and hCA XII. The
replacement of chlorine atom of compound 32 (Ki value of 9.0 nM) with bromine atom gave about
tenꢀfold reduction of potency (i.e. compound 33, Ki value of 81.5 nM) exclusively for hCA IX
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inhibition. On the contrary, the presence of halogen atoms gave up to 1200ꢀfold drop of potency
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against hCA XIV (i.e. compound 33 vs compound 30). These data might suggest that the increase in
atomic radius impair the binding recognition within catalytic site of hCA XIV.
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Results displayed in Table 1 can be summarized as follows. By combining the hydroxyl
substituents on 6/7 positions of isoquinoline nucleus with the introduction of aryl substituent at Cꢀ1
position, a series of very active CA inhibitors was obtained. These structural modifications
generally led to the enhancement of activity up to subnanomolar concentration especially toward
hCA VII isoform when compared with prototype 3 and wellꢀknown reference compound AAZ.
Interestingly, the presence of 4ꢀhalophenyl substituent at Cꢀ1 generally resulted in very low
inhibition of hCA XIV isoform.
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Enantiomeric resolution. Since all synthesized compounds contain a stereogenic centre and it is
recognized that the molecular chirality adds an additional level of specificity and complexity in
achieving of biological activity, the enantiomers of most promising derivatives (compounds 23, 25,
29 and 30) were separated by enantioselective HPLC. The potential inhibitory activity against CAs
was evaluated for eight compounds 23a, 23b, 25a, 25b, 29a, 29b, 30a, and 30b.
Specifically, the complete HPLC resolution was achieved on polysaccharideꢀbased chiral stationary
phases (CSPs) using normalꢀphase eluents. Typical chromatograms of the HPLC enantioseparation
on an analytical scale are shown in Figure 1. The optimized analytical enantioselective conditions
were successfully scaled up to a multiꢀmilligram level using semiꢀpreparative 250 mm x 10 mm
I.D. columns. Both enantiomers of each compound were isolated with high enantiomeric purity
(enantiomeric excess > 99 %).
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Figure 1. Analytical HPLC resolution of 23, 25, 29 and 30. Column: Chiralpak ASꢀH 250 mm x
4.6 mm i.d. (23, 25, 29) and Chiralpak IA 250 mm x 4.6 mm i.d. (30); mobile phase: nꢀ
hexane/ethanol/TFA 20/100/0.1 (23, 25, 29) an nꢀhexane/ethanol/TFA 40/60/0.1 (30); flow rate: 1
mL/min; temperature: 25 (30) and 40 °C (23, 25, 29); detector: UV (black line) and CD (red line) at
280 nm. The letters a and b denote the enantiomer elution order.
The enantiomeric nature of the samples obtained on mgꢀscale was demonstrated by circular
dichroism (CD) analysis (see Supporting Information). The assignment of absolute configuration of
the enantiomerically pure 30a and 30b has been determined by Xꢀray studies (vide infra). For each
single enantiomer CA inhibitory effects were screened. By analysing the Ki values summarized in
Table 2 we can observe that there is not significant influence of the stereochemistry on the CA
inhibitory effects. With the exception of 23b, which proved to be slightly less potent than 23a, both
enantiomers were found to be equiactive inhibitors at low nanomolar concentrations. This evidence
suggests that the two enantiomers could assume binding poses, which are effective for CA
inhibition.
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Table 2. Ki values against hCA I, hCA II, hCA VII, hCA IX, hCA XII and hCA XIV by each single
enantiomer of selected arylsulfonamides 23, 25, 29 and 30
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Ki nM
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R1
R2
H
hCA I hCA II
hCA VII hCA IX hCA XII hCA XIV
OCH3
OCH3
OCH3
OCH3
OH
15.6
77.8
4.8
6.0
4.9
8.9
5.0
3.2
3.3
3.6
0.77
4.1
25.3
58.7
5.0
13.0
30.6
19.7
45.5
8.6
2.4
6.1
4.3
4.3
6.3
1.7
3.2
3.4
23a
23b
25a
25b
29a
29b
30a
30b
H
OCH3 16.6
OCH3 16.0
9.2
5.4
4.8
H
7.5
5.6
9.3
9.3
0.80
0.49
0.21
0.19
73.2
39.8
32.7
39.2
OH
H
3.0
OH
OH
OH
5.8
OH
3.3
aErrors in the range of ± 10 % of the reported value, from 3 different assays.
X-ray Crystallography. To identify the key interactions involved into protein/inhibitor recognition
of this CAI class, the crystal structure of the adduct which hCA II forms with one of its best
inhibitors, namely compound 30, was solved. hCA II was selected as model isoform for
crystallization, since it easily forms crystals and many studies have been reported on its adducts
with different classes of inhibitors.40 In particular, the two 30 enantiomers, namely (R)ꢀ30a and (S)ꢀ
30b, were singularly used for soaking experiments. Data collection and refinement of both adducts
were performed as described in the experimental section (see Supporting Information for statistics).
In both complexes, inspection of the initial Fo − Fc electron density maps immediately revealed
the binding of the inhibitor molecule in the active site. The inhibitor binding does not alter hCA II
threeꢀdimensional structure. Indeed, the r.m.s.d. values calculated by superposition of all the Cα
atoms of the hCA II/30a and hCA II/30b adducts with those of the native protein were 0.26 Å and
0.25 Å, respectively.
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Active site analysis of the two structures showed that the sulfonamide moiety of the two
inhibitors maintains a binding mode to the protein similar to that observed for other CAIs
containing the same group.40 Indeed, the deprotonated nitrogen atom displaces the hydroxyl
ion/water molecule present in the native enzyme and coordinates the zinc ion with a tetrahedral
geometry, while additional hydrogen bond interactions with residue Thr199 further contribute to
stabilize the binding (Figure 2A and 2B).
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Figure 2. Active site region of the hCA II/(R)ꢀ30a (A), hCA II/(S)30b (B) adducts showing the σAꢀ
weighted |2FoꢀFc| OMIT map (contoured at 1.0 σ) relative to the inhibitor molecule. The zinc ion
coordination and polar interactions are also reported.
The orientation of the organic scaffold of the two inhibitors is instead rather peculiar. Indeed, in
both cases, the phenylꢀsubstituent at the Cꢀ1 position is located in the hydrophobic region of the
active site, in a small pocket delimited by residues Phe131, Val135, Leu198 and Pro202, whereas
the isoquinoline ring is oriented toward a poorly explored area of the active site, located at the
border between the hydrophobic and hydrophilic regions (Figure 3).40
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Figure 3. Solvent accessible surface of hCA II/inhibitor adducts. The hydrophobic region of the
active site is shown in red, whereas the hydrophilic one in blue. Residues delimiting the small
pocket interacting with the Cꢀ1 phenyl substituent of both inhibitors (Phe131, Val135, Leu198 and
Pro202) are shown in stick representation.
By the superposition of the hCA II/30a and hCA II/30b structures, it is evident that small
differences are present in the orientation of the organic scaffold of the two inhibitors, due to the
diverse chirality of the Cꢀ1 atom (Figure 3). As a consequence (R)ꢀ30a establishes with the enzyme
two hydrogen bonds, involving both the carbonyl oxygen and one of the two hydroxyl groups
(Figure 2A), which instead are not present in the hCA/(S)ꢀ30b structure (Figure 2B). Nevertheless,
the two inhibitors retain the same affinity for the enzyme (see Table 2), thus indicating that the
influence of these two polar interactions in the binding is negligible with respect to the contribution
of the sulfonamide binding and of the high number of van der Waals contacts established by the
bulky organic scaffold.
Interestingly, the superposition of both hCA II/30a and hCA II/30b structures (see Figure 4A and
Figure 4B) with that of the lead compound 3 in complex with the same enzyme50 reveals that the
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introduction of the Cꢀ1 phenyl substituent on the isoquinoline ring causes a dramatic rearrangement
of the latter within the active site cavity. Indeed, such ring in the hCA II/3 structure is located in the
small hydrophobic pocket occupied by the phenyl ring in hCA II/30a and hCA II/30b adducts.
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Figure 4. Structural superposition between (R)ꢀ30a and 3 (A) and (S)ꢀ30b and 3 (B) bound to the
hCA II active site. 30a, 30b and 3 are colored in green, cyan and magenta respectively
These data suggest that the introduction of suitable substituents on the isoquinoline ring of
compound 3 is a proper strategy to modulate the interactions of these inhibitors with different
regions of the active site.
Molecular Modelling. To gain structural insights about the binding recognition into the catalytic
pocket of hCA VII we performed molecular docking simulations selecting the structure of
C183S/C217S mutant in complex with acetazolamide (PDB code 3ML5).52 Specifically, we
modelled the binding poses of derivatives (R)ꢀ30a and (S)ꢀ30b for which relevant and very similar
inhibitory effects were found (Ki values of 0.21 and 0.19 nM, respectively).
Figure 5 displays the binding poses within hCA VII catalytic site for the inhibitors (R)ꢀ30a (pale
green) and (S)ꢀ30b (orange). As found for the crystallographic structures of hCA II/30a and hCA
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II/30b adducts, both (R)ꢀ30a and (S)ꢀ30b fit nicely the enzymatic pocket of hCA VII assuming two
specular binding poses (Figure 5). In detail, the two arylsulfonamide moieties display similar
binding orientation for which the deprotonated nitrogen atom coordinates the zinc ion within
catalytic site. The Cꢀ1 phenyl substituents were buried in the hydrophobic cleft (area colored in red)
defined by residues Phe131, Ala135, Leu 141, Leu198, Pro202, Leu204. Specifically, compound
30a established van der Waals interactions with Phe131 generating a crucial Tꢀshaped πꢀstacking;
whereas for compound 30b hydrophobic contact with Pro202 were found. The two isoquinoline
rings are surrounded by the hydrophilic region (coloured area in blue) defined by residues Lys91,
Asn62, His64, Thr199, Thr200 and the top portion of benzeneꢀfused rings are exposed to solvent
front (Figure 5). Moreover, enantiomer 30a established an additional hydrogen bond interaction
Lys91 through the 6ꢀhydroxyꢀsubstituent.
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Figure 5. Compounds 30a (orange) and 30b (pale green) in stick representation docked into hCA
VII active site; the hCA VII hydrophobic region is defined by residues Phe131 Ala135, Leu198,
Pro202 (red); the hCA VII hydrophilic region is defined by residues Lys91, Asn62, His64, Thr199,
Thr200 (blue). Active site residues involved in the interactions are also shown as sticks. Polar
interaction is represented by yellow dotted lines. Zinc ion is represented by gray sphere.
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To guide the design of further analogues and gain more information about isoform selectivity, we
attempted to explain the slight difference of CA affinity of (R)ꢀ30a and (S)ꢀ30b toward hCA VII
over hCA II (see Table 1). At this aim, the Xꢀray structures of hCA II/30a (5NOD) and hCA II/30b
(5NOE) adducts were overlaid with the docking structures of the corresponding complexes with
hCA VII (see Figure 6).
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Figure 6. Comparison of binding modes for inhibitors 30a and 30b into active site cavities of hCA
II (gray) and hCA VII (violet). (A) Docking pose of 30a (orange) into hCA VII superimposed with
its crystal structure in hCA II (green). (B) Docking pose of 30b (pale green) into hCA VII
superimposed with its crystal structure in hCA II (cyan). The Zinc ion is depicted as a gray sphere
and dotted line indicates enzyme–ligand interaction. The not conserved residues in hCA VII are
labeled in parentheses.
From the superposition it is evident that the (S)ꢀenantiomer 30b assumed a quite similar orientation
in the active site of hCA VII and hCA II, whereas small differences were observed in the binding
mode of (R)ꢀ30a to the two enzymes, particularly in the orientation of the dihydroxyisoquinoline
ring. This moiety in hCA VII was oriented toward the hydrophilic pocket formed by residues
Gln62, Gln67 and Lys91, establishing a hydrogen bond interaction with the polar residue Lys91,
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which corresponds to the nonpolar residue of Ile91 of hCA II. Considering that the 30a and 30b
share the same isoform selectivity profile, we can hypothesize that the hydrogen bond interaction
with the residue Lys91 does not exert a crucial role for the affinity to hCA VII over hCA II.
Overall, the lack of high isoform selectivity is probably due to the large hydrophobic/hydrophilic
pocket that does not allow a good discrimination of each enantiomer for both enzymes for this class
of isoquinolinesulfonamide inhibitors.
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Conclusions
In this work we rationally designed and synthesized a new class of (R,S)ꢀ4ꢀ(1ꢀarylꢀ3,4ꢀdihydroꢀ
1Hꢀisoquinolineꢀ2ꢀcarbonyl)benzenesulfonamide derivatives (23-33) as CAIs. These compounds
were designed by introducing additional hydrophobic/hydrophilic moieties on the known inhibitor 3
displaying low CA isoform selectivity. This approach proved to be efficacious to obtain new
arylsulfonamides that proved to be active up to subnanomolar concentration. Specifically, the most
active inhibitor 30 exerted excellent Ki value against hCA VII (0.20 nM). We carried out HPLC
enantiomeric resolution and analyzed the proper binding position of the two enantiomers (R)ꢀ30a
and (S)ꢀ30b into hCA II catalytic site. Moreover we docked (R)ꢀ30a and (S)ꢀ30b within hCA VII
active site, thus obtaining insights into their binding positions. By collecting these structural studies,
obtained by computational and experimental procedures, we confirmed that the presence of Cꢀ1
phenyl ring on isoquinoline ring of (R,S)ꢀ30 generates extensive nonꢀpolar contacts within catalytic
site. Overall, we have clearly demonstrated that highly potent CAIs should entirely occupy the two
halves (hydrophilic and hydrophobic regions) of the catalytic site of hCA II/hCA VII isoforms.
Experimental section
Chemistry. Microwaveꢀassisted reactions were carried out in a Focused Microwawe TM Synthesis
System, Model Discover (CEM Technology Ltd Buckingham, UK). Melting points were
determined on a Buchi Bꢀ545 apparatus (BUCHI Labortechnik AG Flawil, Switzerland) and are
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uncorrected. By combustion analysis (C, H, N) carried out on a Carlo Erba Model 1106ꢀElemental
Analyzer we determined the purity of synthesized compounds; the results confirmed a ≥95% purity.
Merck Silica Gel 60 F254 plates were used for analytical TLC (Merck KGaA, Darmstadt,
Germany,). Flash Chromatography (FC) was carried out on a Biotage SP1 EXP (Biotage AB
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1
Uppsala, Sweden). H NMR spectra were measured in dimethylsulfoxideꢀd6 (DMSOꢀd6) with a
Varian Gemini 300 spectrometer (Varian Inc. Palo Alto, California USA); chemical shifts are
expressed in δ (ppm) and coupling constants (J) in hertz. All exchangeable protons were confirmed
by addition of D2O. Rf values were determined on TLC plates using a mixture of CH2Cl2/CH3OH
(96/4) as eluent.
General procedure for the synthesis of (R,S)ꢀ1-aryl-1,2,3,4-tetrahydroisoquinolines (17-22)
Following a previously reported procedure, the appropriate benzaldehyde (7-10) (1.2 mmol) and
suitable phenylethylamine (4-6) (1.0 mmol,) were placed in a cylindrical quartz tube (Ø 2 cm), then
stirred and irradiated in a microwave oven at 150W for 10 min at 90 °C. After cooling to room
temperature, trifluoroacetic acid (3,3 ml) was added to crude intermediates 11-16 obtained in the
previous step and the mixture was irradiated at 150W for 10 min at 90 °C. The reaction was basified
(pH=̃9) with sodium hydroxide, 10 N. The mixture was quenched by addition of H2O (5 mL) and
extracted with EtOAc (3 x 5 mL). The organic layer was dried over Na2SO4 and concentrated until
dryness under reduced pressure. The crude product was collected by filtration and purified by
crystallization with Et2O and EtOH to afford compounds 17-22. Analytical data for compounds 17,
19-22 are in good agreement with literature.51, 53, 54
1
1-Phenyl-6-propoxy-1,2,3,4-tetrahydroisoquinoline (18). Yield: 58%; M.p.:138ꢀ140°C; HNMR
(DMSOꢀd6): δ 0.93 (t, 3H, CH3, J= 7.0, J= 7.6), 1.66 (m, 2H, CH2), 3.64ꢀ 3.06 (m, 4H, CH2), 3.84
(t, 2H, CH2, J= 6.4, J= 6.5), 4.88 (s, 1H, CH), 6.47 (d, J= 8.2, 1H, ArH), 6.55 (d, J= 8.2, 1H, ArH),
6.64 (s, 1H, ArH), 7.21ꢀ7.28 (m, 5H, ArH); Anal. Calcd for C18H21NO: C 80.86% H 7.92% N
5.24%; Found: C 80.96; H 7.83; N 5.16.
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General procedure for the synthesis of 6/7-alkoxy-substituted derivatives of (R,S)-4-(1-aryl-
4
5
6
7
8
3,4-dihydro-1
To a solution of 4ꢀ(aminosulfonyl)benzoic acid (2 mmol) dissolved in dimethylformamide (DMF)
(2mL) was added the N,N,N’,N’ꢀtetramethylꢀOꢀ(1Hꢀbenzotriazolꢀ1ꢀyl)ꢀ
H-isoquinoline-2-carbonyl)benzenesulfonamides (23-28)
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uraniumhexafluorophosphate (HBTU) (2 mmol). The mixture was stirred at room temperature for 1
h. Then TEA (2 mmol) and appropriate (R,S)ꢀ1ꢀarylꢀ1,2,3,4ꢀtetrahydroisoquinolines (17-22) (2
mmol) were added. The reaction mixture was left overnight at room temperature and then quenched
with H2O (10 mL) and extracted with EtOAc (3 x 5 mL). The organic phase was washed with an
aqueous saturated solution of NaHCO3 (2 x 5 mL), dried with Na2SO4 and concentrated until
dryness under reduced pressure. The residue was purified by flash chromatography (DCM/MeOH
96:4), crystallized by treatment with Et2O and EtOH to give the desired final compounds 23-28 as
white crystals.
4-(6-Methoxy-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carbonyl)benzenesulfonamide(23)
Yield: 31%; M.p.:239ꢀ241°C; Rf:0.36; 1HNMR (DMSOꢀd6): δ 2.71ꢀ3.38 (m, 4H, CH2), 3.74 (s, 3H,
OCH3), 6.76 (s, 1H, CH), 6.81 (m, 2H, ArH), 7.05 (m, 1H, ArH), 7.24ꢀ7.33 (m, 5H, ArH), 7.46 (s,
2H, NH2), 7.58 (d, J= 8.2, 2H, ArH); 7.87 (d, J= 8.2, 2H, ArH). Anal. Calcd for C23H22N2O4S: C
65.38%, H 5.25%, N 6.63%; Found: C 65.01, H 5.06, N 6.23.
4-(1-Phenyl-6-propoxy-3,4-dihydro-1
H
-isoquinoline-2-carbonyl)benzenesulfonamide (24)
1
Yield: 61%; M.p.:231ꢀ233°C; Rf:0.41; HNMR (DMSOꢀd6) δ 0.96 (t, 3H, J= 7.0, CH3), 1.70ꢀ1.75
(m, 2H, CH2), 2.69ꢀ2.94 (m, 4H, CH2), 3.91 (t, 2H, J= 6.5, OCH2), 6.76 (s, 1H, CH), 6.81 (m, 1H,
ArH), 7.04ꢀ7.07 (m, 2H, ArH), 7.25ꢀ7.34 (m, 5H, ArH), 7.47 (s, 1H, NH2), 7.59 (d, J= 8.0, 2H,
ArH), 7.86 7.88 (d, J= 8.0, 2H, CH2). Anal. Calcd for C25H26N2O4S: C 66.64%, H 5.82%, N 6.22%;
Found: C 66.60, H 5.80, N 6.20.
4-(6,7-Dimethoxy-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carbonyl)benzenesulfonamide (25)
1
Yield: 66%; Mp:248ꢀ250°C; Rf:0.41 HNMR (DMSOꢀd6): δ 2.63ꢀ3.25 (m, 4H, CH2), 3.63 (s, 3H,
OCH3), 3.74 (s, 3H, OCH3), 6.72 (s, 1H, CH), 6.78 (s, 1H, ArH), 6.80 (s, 1H, ArH), 7.22ꢀ7.34 (m,
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5H, ArH), 7.46 (s, 2H, NH2), 7.55 (d, J= 8.2, 2H, ArH), 7.85 (d, J= 8.2, 2H, ArH); Anal. Calcd for
4
5
C24H24N2O5S: C 63.70%, H 5.35%, N 6.19%; Found: C 63.52, H 5.21, N 6.02.
6
7
8
4-[1-(4-Fluorophenyl)-6,7-dimethoxy-3,4-dihydro-1H-isoquinoline-2-
9
carbonyl]benzenesulfonamide (26)
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1
Yield: 41%; M.p.:220ꢀ222°C; Rf:0.47; HNMR (DMSOꢀd6) δ 2.63ꢀ3.40 (m, 4H, CH2), 3.63 (s, 3H,
OCH3), 3.75 (s, 3H, OCH3), 6.72 (s, 1H, CH), 6.78 (s, 1H, ArH), 6.81 (s, 1H, ArH), 7.14ꢀ7.27 (m,
4H, ArH), 7.46 (bs, 2H, NH2), 7.57 (d, J= 8.6, 2H, ArH), 7.87 (d, J= 8.6, 2H, ArH); Anal. Calcd
for C24H23FN2O5S: C 61.27%, H 4.93%, N 5.95%; Found: C 61.37, H 4.80, N 5.80.
4-[1-(4-Chlorophenyl)-6,7-dimethoxy-3,4-dihydro-1H-isoquinoline-2-
carbonyl]benzenesulfonamide (27)
1
Yield: 50%; M.p.:204ꢀ205°C; Rf:0.40; HNMR (DMSOꢀd6) δ 2.64ꢀ3.23 (m, 4H, CH2), 3.64 (s, 3H,
OCH3), 3.75 (s, 3H, OCH3), 6.73 (s, 1H, CH), 6.77 (s, 1H, ArH), 6.81 (s, 1H, ArH), 7.28 (d, J= 8.2,
2H, ArH), 7.45 (bs, 2H, NH2) 7.57 (d, J= 8.2, 2H, ArH), 7.87 (d, J= 8.2, 2H, ArH); Anal. Calcd for
C24H23ClN2O5S: C 59.20%, H 4.76%, N 5.75%; Found: C 59.30, H 4.66, N 8.85.
4-[1-(4-Bromophenyl)-6,7-dimethoxy-3,4-dihydro-1H-isoquinoline-2-
carbonyl]benzenesulfonamide (28)
1
Yield: 51%; M.p 191ꢀ192°C; Rf:0.41; HNMR (DMSOꢀd6) δ 2.63ꢀ3.38 (m, 4H, CH2), 3.64 (s, 3H,
OCH3), 3.75 (s, 3H, OCH3), 6.74 (m, 2H, ArH), 6.81 (s, 1H, CH), 7.19 (d, J= 8.2, 2H, ArH), 7.46
(bs, 2H, NH2), 7.53ꢀ7.58 (m, 4H, ArH), 7.88 (d, J= 7.6, 2H, ArH). Anal. Calcd for C24H23BrN2O5S:
C 54.24%, H 4.36%, N 5.27%; Found: C 54.34, H 4.46, N 5.20
.
General procedure for the synthesis of 6/7-hydroxy-substituted derivatives of (R,S)-4-(1-aryl-
3,4-dihydro-1 -isoquinoline-2-carbonyl)benzenesulfonamides (29-33)
H
The methoxyꢀderivatives 23, 25-28 (1 mmol) were dissolved in methylene chloride (DCM) (5 mL),
treated with BBr3 (1M in DCM) (6 mmol) under nitrogen atmosphere and stirred overnight. After
completion of the reaction, MeOH (7 mL) was carefully added at 0°C and the solvents removed
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under reduced pressure. The residue was dissolved in EtOAc (10 mL) and washed with H2O (3 x 10
mL). The organic layer was dried (Na2SO4) and concentrated in vacuo. The crude products were
crystallized from Et2O to give the desired corresponding hydroxyꢀderivatives 29-33.
9
4-(6-Hydroxy-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carbonyl)benzenesulfonamide (29)
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Yield: 86%; Mp: 282ꢀ284°C; Rf:0.23; 1HNMR (DMSOꢀd6): δ 2.62ꢀ3.39 (m, 4H, CH2), 6.62 (s, 1H,
ArH), 6.72 (s, 1H, CH), 6.93 (m, 1H, ArH), 7.22 (d, 1H, ArH), 7.25ꢀ7.34 (m, 5H, ArH), 7.46 (s, 2H,
NH2), 7.58 (d, J= 8.2, 2H, ArH), 7.87 (d, J=8.2, 2H, ArH), 9.42 (bs, 1H, OH). Anal. Calcd for
C22H20N2O4S: C 64.69%, H 4.94%, N 6.86%; Found: C 64.49, H 4.65, N 6.75.
4-(6,7-Dihydroxy-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carbonyl)benzenesulfonamide (30)
Yield: 83%; Mp: 239ꢀ240°C; Rf: 0.10; 1HNMR (DMSOꢀd6): δ 2.71ꢀ3.28 (m, 4H, CH2), 6.41 (s, 1H,
CH), 6.57 (s, 1H, ArH), 6.60 (s, 1H, ArH), 7.24ꢀ7.33 (m, 5H, ArH), 7.46 (s, 2H, NH2), 7.54 (d, J=
8.2, 2H, ArH), 7.85 (d, J= 8.2, 2H, ArH), 8.86 (s, 1H, OH), 8.95 (s, 1H, OH). Anal. Calcd for
C22H20N2O5S: C 62.25%, H 4.75%, N 6.60%; Found: C 62.01, H 4.45, N 6.52.
4-[6,7-Dihydroxy-1-(4-fluorophenyl)-3,4-dihydro-1
carbonyl]benzenesulfonamide (31)
H-isoquinoline-2-
Yield: 31%; M.p.:240ꢀ243°C; Rf: 0.11; 1HNMR (DMSOꢀd6) δ 2.57ꢀ3.23 (m, 4H, CH2), 3.64 (s, 3H,
OCH3), 3.76 (s, 3H, OCH3), 6.82 (s, 1H, CH), 6.85 (s, 1H, ArH), 6.88 (s, 1H, ArH), 7.47 (bs, 2H,
NH2), 7.58ꢀ7.70 (m, 4H, ArH), 7.88 (d, J= 8.2, 2H, ArH), 8.07 (s, 1H, ArH); 8.16 (m, 1H,ArH).
Anal. Calcd for C24H23N3O7S: C 57.94%, H 4.66%, N 8.45%; Found: C 57.84, H 4.76, N 8.55.
4-[1-(4-Chlorophenyl)-6,7-dihydroxy-3,4-dihydro-1
carbonyl]benzenesulfonamide (32)
H-isoquinoline-2-
Yield: 67%; M.p.:185ꢀ186°C; Rf: 0.12; 1HNMR (DMSOꢀd6) δ 2.42ꢀ3.23 (m, 4H, CH2), 6.42 (s, 1H,
CH), 6.57ꢀ6.58 (m, 2H, ArH), 7.27 (d, J= 8.2, 2H, ArH), 7.40 (d, J= 8.2, 2H, ArH), 7.46 (bs, 2H,
NH2) 7.55 (d, J= 8.2, 2H, ArH), 7.85 (d, J= 8.2, 2H, ArH), 8.88 (bs, 1H, OH), 8.98 (bs, 1H, OH).
Anal. Calcd for C22H19ClN2O5S: C 57.58%, H 4.17%, N 6.10%; Found: C 57.68, H 4.10, N 6.20.
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4-[1-(4-Bromophenyl)-6,7-dihydroxy-3,4-dihydro-1
carbonyl]benzenesulfonamide (33)
H-isoquinoline-2-
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Yield: 42%; M.p.:189ꢀ191°C; Rf:0.13; 1HNMR (DMSOꢀd6) δ 2.07 (m, 4H, CH2), 6.41 (s, 1H, CH),
6.57 (s, 2H, ArH), 7.20 (d, J= 8.3, 2H, ArH), 7.46 (bs, 2H, NH2), 7.52ꢀ7.56 (m, 4H, ArH), 7.85 (d,
J= 8.3, 2H, ArH), 9.00 (bs, 2H, OH). Anal. Calcd for C22H19BrN2O5S: C 52.49%, H 3.80%, N
5.57%; Found: C 52.52, H 3.84, N 5.60.
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CA Inhibition Assay An Applied Photophysics stoppedꢀflow instrument has been used for
assaying the CA catalysed 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 10 – 20 mM Hepes
(pH 7.5) or Tris (pH 8.3) as buffers, and 20 mM Na2SO4 or 20 mM NaClO4 (for maintaining
constant the ionic strength), following the initial rates of the CAꢀcatalyzed CO2 hydration reaction
for a period of 10ꢀ100 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
5ꢀ10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates
were determined in the same manner and subtracted from the total observed rates. Stock solutions
of inhibitor (10 mM) were prepared in distilledꢀdeionized water and dilutions up to 0.01 nM were
done thereafter with distilledꢀdeionized 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 EꢀI
complex. The inhibition constants were obtained by nonꢀlinear leastꢀsquares methods using PRISM
3, as reported earlier, and represent the mean from at least three different determinations. CA
isoforms were recombinant ones obtained as reported earlier by this group.55ꢀ57
Enantiomeric resolution. Enantioselective HPLC analyses were performed by using stainlessꢀsteel
Chiralpak IA (250 mm x 4.6 mm I.D. and 250 mm x 10 mm I.D.) and Chiralpak ASꢀH (250 mm x
4.6 mm I.D. and 250 mm x 10 mm I.D.) (Daicel, Chemical Industries, Tokyo, Japan) columns.
HPLCꢀgrade solvents were used as supplied by Aldrich (Milan, Italy). The HPLC apparatus
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consisted of a Perkin Elmer (Norwalk, CT, USA) 200 lc pump equipped with a Rheodyne (Cotati,
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CA, USA) injector, a 1000ꢀꢀl sample loop, a HPLC Perkin Elmer oven and a Perkin Elmer 290
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detector. The signal was acquired and processed by Clarity software (DataApex, Prague, The Czech
Republic). HPLC resolutions were carried out by using polysaccharideꢀbased chiral stationary
phases (CSPs). The CD spectra of the enantiomers isolated at semipreparative scale, dissolved in
dichloromethane (concentration about 0.2 mg/ml) in a quartz cell (0.1 cmꢀpath length) at 25 °C,
were measured by using a Jasco (Jasco, Ishikawaꢀcho, Hachioji City, Tokyo, Japan) Model Jꢀ700
spectropolarimeter in the 400ꢀ200 nm spectral range. The spectra are average computed over three
instrumental scans and the intensities are presented in terms of terms of ellipticity values (mdeg).
X-ray crystallography. Crystals of hCA II adducts with inhibitors (R)ꢀ30a and (S)ꢀ30b were
prepared by the soaking technique. In particular, hCA II native crystals were grown at room
temperature using the vapor diffusion hanging drop method. Equal volumes of protein (10 mg/ml in
0.02 M TrisꢀHCl pH 8.0) and precipitant solutions (1.3 M sodium citrate and 0.1 M TrisꢀHCl, pH
8.5) were mixed and equilibrated against 1 mL reservoir containing the same precipitant solution. A
few native enzyme crystals were then transferred in a 3 ꢁl drop of freshly prepared precipitant
solution, containing 10% (v/v) glycerol and 20 mM inhibitor. These crystals were kept in the
soaking solution for about 12 hours. Crystals were frozen in a gaseous nitrogen stream prior to the
diffraction experiment. Complete Xꢀray data set were collected at 100 K by a copper rotating anode
generator developed by Rigaku and equipped with a Rigaku Saturn CCD detector. Diffraction data
up to 1.70 Å resolution for hCA II/30a and 1.75 Å resolution for hCA II/30b were processed and
scaled using program HKL2000 (HKL Research).58 Data collection statistics are reported in Table
1S. The initial phases of hCA II/inhibitor structures were calculated using the atomic coordinates of
the native enzyme with waters removed (PDB entry 1CA2).59 Clear electron density for the two
compounds was observed in the difference maps after a single round of refinement. A model for
both 30a and 30b was then built and introduced into the atomic coordinates set for further
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refinement, which proceeded to convergence with alternating cycles of water addiction, manual
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rebuilding with the O program,60 and energy minimization and Bꢀfactor refinement with the CNS
program.61 Crystallographic refinement was carried out against 95% of the measured data. The
remaining 5% of the observed data, which was randomly selected, was used for Rfree calculations
to monitor the progress of refinement. Topology files for both compounds were obtained using the
PRODRG server.62 The final Rꢀwork and Rꢀfree values were 0.173 and 0.209 for hCA II/30a and
0.166 and 0.198 for hCA II/30b. Refinement statistics are summarized in Table 1S (see Supporting
Information). Coordinates and structure factors have been deposited in the Protein Data Bank
(accession codes 5NOD and 5NOE).
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Molecular docking. The crystal structures of hCA VII in complex with the inhibitor acetazolamide
was retrieved from the RCSB Protein Data Bank (PDB: 3ML5).52 Ligand and water molecules were
discarded, and hydrogen atoms were added to protein with Discovery Studio 2.5.5.63 Structures of
the ligands were constructed using Discovery Studio 2.5.5 and energy minimized using the Smart
Minimizer protocol (1000 steps) which combines the Steepest Descent and the Conjugate Gradient
methods. The ligands minimized in this way were docked in their corresponding proteins by means
of Gold Suite 5.0.1.64 The region of interest used by Gold program was defined in order to contain
residues within 10 Å from the original position of the ligand in the Xꢀray structure. A scaffold
constraint (penalty = 10.0) was used to restrict the solutions in which the sulfonamide moiety was
able to coordinate the metal within the catalytic binding site. GoldScore was chosen as fitness
function, the standard default setting was used in all calculations and ligands were submitted to 100
genetic algorithm runs. The “allow early termination” command was deactivated. Results differing
by less than 0.75 Å in ligandꢀall atom RMSD, were clustered together. The best GOLDꢀcalculated
conformation was used both for analysis and representation.
Acknowledgments
Financial support for this research by by Fondo di Ateneo per la Ricerca (PRA grant number
ORME09SPNC ꢀ Università degli Studi di Messina)
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Associated content
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Supporting Information. The Supporting Information is available free of charge on the ACS
Publications website at DOI:
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Diffraction Data and Refinement Statistics, CD spectra of the enantiomers (PDF)
Molecular formula strings (XLSX)
Accession Codes. The final models of hCA II bound to inhibitors 30a and 30b are deposited to the
Protein Databank (5NOD and 5NOE). Authors will release the atomic coordinates and
experimental data upon article publication
Author information
Corresponding author: Rosaria Gitto, PhD, Associate professor on Medicinal Chemistry,
Dipartimento Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali (CHIBIOFARAM),
Università degli Studi di Messina, Viale Annunziata, Iꢀ98168, Messina. Tel 00390906766413, Fax
00390906766404, eꢀmail: rgitto@unime.it ORCID: Gitto Rosaria 0000ꢀ0003ꢀ0002ꢀ2253
Author Contributions: The manuscript was written through contributions of all authors.
Notes The authors declare no competing financial interest.
Abbreviation Used
AAZ acetazolamide
CA
carbonic anhydrase
CSPS chiral stationary phases
DCM dichloromethane
FC
Flash Chromatography
HBTU N,N,N′,N′-TetramethylꢀO-(1Hꢀbenzotriazolꢀ1ꢀyl)uronium hexafluorophosphate
MW Microwave
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TEA triethylamine
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TFA Trifluoroacetic acid
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TLC Thin Layer Chromatography
TPM topiramate
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