Monothiocarbamates Strongly Inhibit Carbonic Anhydrases in Vitro and Possess Intraocular Pressure Lowering Activity in an Animal Model of Glaucoma

Article abstract of DOI:10.1021/acs.jmedchem.6b00462

A series of monothiocarbamates (MTCs) were prepared from primary/secondary amines and COS as potential carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, using the dithiocarbamates, the xanthates, and the trithiocarbonates as lead compounds. The MTCs effectively inhibited the pharmacologically relevant human (h) hCAs isoforms I, II, IX, and XII in vitro and showed K_Is spanning between the low and medium nanomolar range. By means of a computational study, the MTC moiety binding mode on the CAs was explained. Furthermore, a selection of MTCs were evaluated in a normotensive glaucoma rabbit model for their intraocular pressure (IOP) lowering effects and showed interesting activity.

Full text of DOI:10.1021/acs.jmedchem.6b00462

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Article
Monothiocarbamates strongly inhibit carbonic
anhydrases in vitro and possess intraocular pressure
lowering activity in an animal model of glaucoma
Daniela Vullo, Mariaconcetta Durante, Francesco Saverio Di Leva, Sandro Cosconati,
Emanuela Masini, Andrea Scozzafava, Ettore Novellino, Claudiu T Supuran, and Fabrizio Carta
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00462 • Publication Date (Web): 02 Jun 2016
Downloaded from http://pubs.acs.org on June 7, 2016
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Monothiocarbamates strongly inhibit carbonic anhydrases in vitro and possess intraocular
pressure lowering activity in an animal model of glaucoma
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Daniela Vullo, Mariaconcetta Durante, Francesco Saverio Di Leva, Sandro Cosconati,
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Emanuela Masini, Andrea Scozzafava, Ettore Novellino, Claudiu T. Supuran and
1
*
Fabrizio Carta,
1
Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm.
188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy.
2
Università degli Studi di Firenze, Dipartimento NEUROFARBA, Sezione di Farmacologia, Viale
Pieraccini 6, 50139 Florence, Italy.
3
Department of Pharmacy, University of Naples "Federico II", Via D. Montesano 49, 80131
Naples, Italy.
4
DiSTABiF, Seconda Università di Napoli, Via Vivaldi 43, 81100 Caserta, Italy.
5
Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via
Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
Keywords: Monothiocarbamates (MTCs), carbonic anhydrase inhibitors (CAIs), metalloenzymes,
glaucoma.
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Abstract: A series of monothiocarbamates (MTCs) were prepared from primary/secondary amines
and COS, as potential carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, using the dithiocarbamates,
the xanthates and the trithiocarbonates as lead compounds. The MTCs effectively inhibited the
I
pharmacologically relevant human (h) hCAs isoforms I, II, IX and XII in vitro, and showed K s
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spanning between the low and medium nanomolar range. By means of a computational study the
MTC moiety binding mode on the CAs was explained. Furthermore, a selection of MTCs were
evaluated in a normotensive glaucoma rabbit model for their intraocular pressure (IOP) lowering
effects, and showed interesting activity.
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Introduction.
The metalloenzyme Carbonic Anhydrases (CAs, EC 4.2.1.1) are widely expressed within all living
organisms and are genetically encoded by six unrelated families indicated with the Greek letters α−,
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1
ꢀ3
β−, γ−, δ−, ζ− and η−CAs. Among the various reactions catalyzed by the CAs, the reversible
hydration of carbon dioxide to afford bicarbonate and protons, represents the unique one with
1
c,d
physiological consequences.
Such a simple reaction is deeply connected to fundamental biological processes, which include
respiration, electrolyte secretions, pH homeostasis, bone resorption, biosynthesis of lipids, nucleic
1
ꢀ13
acids and glucose.
Thus the modulation of such an equilibrium through the CAs catalytic
4
ꢀ13
activities, definitely represents a powerful approach for the treatment of various diseases.
To
date, the use of small molecules CA activators (CAAs) as anticonvulsant/antiepileptic appear quite
promising. Even better, CAAs were demonstrated to be efficient for the treatment of central nervous
4
ꢀ7
system (CNS) affecting diseases such as Alzheimer's. Conversely the use of CA inhibitors (CAIs)
8
ꢀ
is more common, since their introduction in the 60’s as antihypertensive and antiglaucoma agents.
1
0
Nowadays CAIs are employed for the treatment of a vast array of diseases, including epilepsy,
metabolic related pathologies (e.g., obesity), as well as for the management or diagnosis of hypoxic
1
ꢀ16
tumors.
Pharmacological therapies based on the use of CA modulators (i.e.,
inhibitors/activators), mainly take advantage of the essential role played by the carbon dioxide
catalyzed reactions within the cells life cycles. In addition, the variety of CAs expressed within
organisms is strictly dependent from their physio/pathological conditions. Thus since all isozymes
differ for their kinetic properties as well as for cellular, subꢀcellular and tissue distributions it is
clear that a successful CA modulation approach therapy resides on its ability to properly address
1c,12
such a biochemical diversity .
Such a scenario clearly introduces to the main problem to be faced within the CAs field, which is
11a, 12
represented by the search of selective modulators targeting the preferred isoforms.
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The application of the tail approach to the classical zinc binding CAIs, such as the sulfonamides and
their bioisosters, represented the preferred medicinal chemistry approach to address the isoform
3d,12b,17
selectivity issues.
In addition, the many efforts conducted towards the identification of new
18a,b
chemical classes acting as CAIs, drove to the discovery of the coumarins
and their congeners,
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8cꢀh
19
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the phenols
and the polyamines.
All of them were demonstrated to possess different
inhibition mechanisms by means of kinetics as well as Xꢀray crystallographic experiments in adduct
17ꢀ20
with various hCA isoforms.
In particular in vitro screening of inorganic anions allowed to identify the trithiocarbonate (CS
a low micromolar (ꢁM) inhibitor of the highly abundant cytosolic hCAs (I, II) and of the tumor
ꢀ
3
) as
2
1
ꢀ
associated isoform IX.
3
The CS enzymatic zincꢀbinding ability was disclosed through its
22
ꢀ
crystallographic adduct with hCA II. The pioneering work on the CS ion as CAI, triggered the
3
investigation towards small molecules containing the zincꢀbinding dithioꢀmoiety, such as the
2
3
24
24
dithiocarbamates the xanthates and the thioxanthates or trithiocarbonates. All these classes
showed excellent inhibition potencies against the αꢀ as well as the pathogenous βꢀCAs with K
spanning in the lowꢀmedium nanomolar (nM) range.
I
s
Herein, as an extension of our studies we report for the first time the synthesis, characterization as
well as in vitro kinetic profiling against the hCAs I, II, IX and XII of the monothiocarbamates
(MTCs). A plausible enzymatic binding mode is also proposed by means of molecular modeling
studies. A selection of such compounds was evaluated in a normotensive glaucoma rabbit model for
the possible effect as IOP lowering agents.
Results and Discussion.
Compounds Design and Synthesis.
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The drug design rationale of this project is based on the replacement of the sulphur atom of the
DTCs moiety with an oxygen instead (Figure 1A). As reported below the synthetic methodology
consisted of a direct carbamoylation between primary/secondary amines with carbonyl monosulfide
(COS) in the presence of sodium hydroxide as a base (Figure 1B).
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A)
B)
S
O
O
COS, NaOH
R
R
H
R
N
S
Na
N
S
Na
S
Na
N
N
R
R1
R1
R1
R1
1ꢀ15
1aꢀ15a
DTC
MTC
Figure 1A) Rational design of the MTCs; B) General synthetic procedure for MTCs 1aꢀ15a
The selected amines contained alkyl/aralkyl moieties allowing us to obtain a series of 15 MTC
1
13
derivatives as sodium salts (Table 1). All compounds were characterized by means of H, C NMR
spectroscopy and HRMS and were >98 % pure, as determined by HPLC (see the Experimental
1
3
Section for details). The insertion of the mothiocarbamate moiety was confirmed by C NMR,
which gave the characteristic signal at 180ꢀ186 ppm using dmso-d
6
as solvent.
CA Inhibition.
All compounds synthesized, 1aꢀ15a, were tested in vitro for their inhibitory properties against the
physiological relevant hCA isoforms I, II, IX and XII, by means of the stoppedꢀflow carbon dioxide
25
hydration assay. Their activities were compared to the standard CAI acetazolamide (AAZ) as well
their structurally related moieties (i.e. the Dithiocarbamate 16, Xanthate 17) and to the inorganic
2
ꢀ
CS3 ion (Table 1).
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Table 1. hCA I, II, IX, and XII inhibition data with MTCs 1a−15a, Dithiocarbamate 16, Xanthate
25
1
7 and Trithiocarbonate by a StoppedꢀFlow CO Hydrase Assay.
2
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a
No.
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K (nM)
b
R
R
1
hCA I
hCA II
hCA IX
hCA XII
1
2
3
4
5
a
a
a
a
a
H
O
891
26.7
15.2
21.5
7.7
3.3
4.7
9.2
7.9
7.5
3.4
7.2
1.9
44.5
O
H
>2000
>2000
>2000
909
43.7
H
N
N
NH
35.0
nꢀPr
nꢀBu
iꢀBu
Et
nꢀPr
nꢀBu
iꢀBu
nꢀBu
46.7
12.5
6.8
>2000
43.0
6a
681
9.2
7
a
700
>2000
44.5
6.2
O
OEt
8a
Me
Me
827
22.1
11.7
9
a
Bn
>2000
>2000
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10a
O
NH
569
>2000
22.4
13.5
10.4
6.5
2.8
1
1
1
1
1
1
1a
2a
3a
4a
5a
HN
NH
876
6.8
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F
N
NH
895
46.8
3.8
F3C
N
NH
>2000
686
43.6
11.8
4.9
7.5
Cl
N
NH
>2000
45.9
23.0
7.3
H
N
N
949
14.0
19.2
58.0
9.7 (µM)
25
O
NH
c
6
H
Bn
4.1
0.70
11.5
63.3
120 (µM)
5.7
d
17
64.1
8.7 (µM)
250
5.4
2ꢀ
e
e
f
f
CS3
8.8 (µM)
12
AAZ
a
Means from three different assays. Errors were within ±5−10% of the reported values (data not
b
c
d
e
f
shown). Catalytic domain. From ref. 23c. From ref. 24. From ref. 21a. From ref. 21b.
The data reported above clearly showed the MTCs 1aꢀ15a act as highꢀmedium potency CAIs
against the abundantly expressed isoforms I and II, with K values spanning between 0.95ꢀ0.57 ꢁM
I
and 46.8ꢀ22.4 nM respectively. On the other hand, the inhibition constants against the tumor
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associated isoforms IX and XII were comprised in the low nM range and comparable with the
standard sulfonamide AAZ (K 5.7 nM). Overall the MTCs showed to be more potent CAIs when
I
2ꢀ
3
compared to the CS
I
ion (K s in the µM range), and definitely more selective in inhibiting the
tumor associated isoforms (hCA IX and XII) over the cytosolic ones (I, II) when compared to the
Dithiocarbamate 16 and Xanthate 17. Such compounds were considered as comparison since their
binding modes with hCAs were all similar (see later in the text).
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The following StructureꢀActivity Relationships (SAR) are reported:
i)
started with the insertion of the MTC moiety into primary amines such as the 2,4ꢀdimethoxy
phenylethylamine, which showed a high nM inhibition value (compound 1a K 891 nM). The
The hCA I is the less inhibited isoform from the MTCs herein reported. Our investigation
I
removal of the methoxy substituents or the use of the 4ꢀmethylꢀpiperazinꢀ1ꢀylamine as primary
amine instead resulted in a complete loss (K >2000) of the inhibition activity (compound 2a and 3a
I
respectively). Insertion of the MTC moiety into secondary amines bearing linear or branched alkyl
I
chains, such as in compounds 5aꢀ8a, resulted in K s values between 681ꢀ909 nM, with the only
I
exception of 4a and 9a which were ineffective (K s >2000 nM). Interestingly merging of the
smallest alkyl substituent into a heterocyclic moiety, such as in the morpholine10a, resulted in a
slight increase of the inhibition potency against the hCA I (K 569 nM), which was the highest
I
within the series. Surprisingly, the replacement of the morpholine oxygen in 10a with a nitrogen
and insertion of two MTC functionalities to afford compound 11a, caused a 1.5 fold decrease in the
I
inhibition potency (K 876 nM). Substitution of a piperazinic proton in 11a with the pꢀfluorophenyl
moiety, as in 12a, did not sensibly affect the inhibition potency (K 895 nM). However the
I
introduction of the pꢀtrifluoromethyl instead, as in compound 13a, suppressed the inhibition activity
(
K
I
>2000) which was restored with the mꢀchlorophenyl moiety (compound 14a K
Exploitation of the piperazine scaffold was also accomplished by introducing the MTC moiety into
the antiulcer drug Hexaprazole to give the derivative 15a (K of 949 nM).
I
686 nM).
I
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ii)
Interesting inhibition data were obtained for the highly abundanthCA II isoform which is of
particular interest for the pharmacological treatment of glaucoma. The MTCs 1aꢀ3a, showed good
affinity data with K values comprised between 26.7 and 43.7 nM. Opposite to the hCA I, the
removal of the methoxy substituents in 1a to afford 2a resulted only in a 1.63 fold enhancement of
the K (26.7 and 43.7 nM respectively). Among the substituted secondary amines bearing the MTC
I
10
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13
14
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16
17
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I
moiety only the symmetrical nꢀpropyl 4a, iꢀpropyl 6a and the asymmetric 8a derivatives resulted
good inhibitors of the hCA II (K s 46.7, 43.0 and 44.5 nM respectively), whereas the remaining
I
ones were ineffective (K
against the hCA II (K >2000), whilst the disubstituted piperazine 11a is a rather effective CAI with
a K of 22.4 nM, which makes it the most potent hCA II inhibitor within the series. Interestingly the
I
s >2000). Interestingly the MTC morpholine10a showed no inhibition
I
I
piperazine monosubstituted MTCs containing the fluoroꢀaryl moieties, such as 13a and 12a, or an
αꢀamidoalkyl chain, as for the Hexaprazole 15a, showed K s spanning between 43.6ꢀ45.9 nM and
I
comparable with those of 4a, 6a and 8a. Switch of the fluorine to chlorine and change of its position
from para to meta, as in compound 14a, resulted in loss of the inhibition potency (K
iii) All the synthesized MTCs showed excellent affinities against the tumor associated hCA IX
with K values comprised between 6.2 and 22.1 nM, and thus comparable to the standard AAZ (KI
I
>2000).
I
2
5 nM). Again, removal of the methoxy substituents such as in 1a to afford 2a resulted in a
lowering of their inhibition potencies (K s of 15.2 and 21.5 nM, respectively). Conversely, the
hydrazinoꢀmethyl piperazine MTC 3a resulted highly potent CAI (K 7.7 nM). Secondary amine
MTC derivatives 4aꢀ11a were all good hCA IX inhibitors with K s spanning between 6.2 and 22.1
I
I
I
nM. Interestingly the introduction into the piperazine scaffold of the paraꢀfluorophenyl moiety, as
in compound 12a, resulted in a strengthening of the inhibition activity (K 6.5 nM), which
I
conversely showed a 1.81 and 2.15 fold K
I
increase when the trifluoromethyl and the alfaꢀ
amidoalkyl moiety were inserted instead (compounds 13a and 15a respectively). Noteworthy the
I
metaꢀchorophenyl derivative 14a reported a sensible increase of the potency with a K of 4.9 nM,
which is the most active in inhibiting the hCA IX among all the MTCs tested.
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iv)
The second transmembrane hCA isoform (hCA XII) was also strongly inhibited by all
synthesized compounds. In particular MTCs 1aꢀ3a, obtained from primary amines, were low
nanomolar inhibitors (K s 3.3, 4.7, 9.2 nM respectively) and comparable to the standard CAI
sulfonamide AAZ (K 5.7 nM). The secondary symmetrical MTC containing amines, such as 4a
and 5a, showed similar inhibition potencies (K s 7.9 and 7.5 nM respectively). Interestingly the
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I
substitution of the of the linear n-butyl chains in 5a with the branched isomers, as in compound 6a,
resulted in a 2.2 fold increase of the inhibition potency (K 3.4 nM). Deꢀsymmetrization of the open
I
alkyl moieties, compounds 7a and 9a, resulted in a sensible increase of the K
respectively, with the only exception represented from the compound 8a which showed the highest
inhibition potency against the hCA XII (K 1.9 nM). The MTCs bearing the secondary cyclic
I
s to 7.2 and 44.3 nM
I
scaffolds, such as the morpholine10a and the piperazine11a, also resulted quite efficient inhibitors
of the hCA XII showing K s of 2.8 and 6.8 nM. Among the various substituents inserted into the
I
piperazine scaffold, the paraꢀfluorophenyl containing MTC 12a was the most potent with a K
.8 nM, while its trifluoromethyl derivative 13a resulted 2 folds less potent (K 7.5 nM) and
comparable to the Hexaprazole MTC 15a (K 7.3 nM). The metaꢀchlorophenyl14a was the less
I
of
3
I
I
effective hCA XII inhibitor of the piperazine series having a K of 23 nM.
I
Thus, in general, all the synthesized compounds showed interesting inhibition properties against the
physiological relevant hCAs. Whilst the hCA I was poorly inhibited, the catalytically more efficient
hCA II showed a better profile with the bis substituted piperazine derivative 11aas the most potent
I
within the series (K 22.4 nM). The tumor associated hCA isoforms hCA IX and XII were strongly
inhibited from the MTCs herein reported with K values in the low nanomolar range and by far
I
lower to the standard CAI AAZ. In particular the piperazine derivative 14a showed a K of 4.9 nM
I
against the hCA IX and the asymmetric MTC 8a a K
I
of 1.9 nM against the hCA XII.
Molecular Modeling.
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To elucidate the binding mode of our MTCs at the αꢀCA active site, molecular docking studies were
performed on compound 2a, which was demonstrated to have good inhibitory properties against
hCAs II, IX and XII (Table 1). Docking of 2a into the Xꢀray crystal structure of hCA II (PDB code
3
P5L) predicts that the ligand binds deep into the enzyme catalytic site in its anionic form, with its
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2
+
MTC moiety chelating the Zn ion in a monodentate manner. In this regards, calculations suggest
that the metal ion can be alternatively coordinated through the sulfur or the oxygen atom. However,
previous spectroscopic and crystallographic studies indicated that MTC derivatives coordinate
metal ions through sulfur, since the negative charge distribution resulted mainly shifted towards this
2
6
2+
atom. Thus the lowest energy docking pose among those showing the sulfurꢀZn coordination was
considered for our investigations (Figure 2a). In this pose, the MTC oxygen atom in compound 2a
interacts through a hydrogen bond with the T200 residue, and in analogy with many zincꢀbinding
7
CAIs. The phenylethyl moiety extends from the enzymatic cavity towards the hydrophobic region
of the CA binding pocket, establishing a Tꢀshaped stacking interaction with F131, and favorable
van der Waals contacts with F131, V135, L141, L198 and P202. Noteworthy this binding pose
resulted highly superimposable with a DTC inhibitor in complex with hCA II of comparable
23a
potency and early reported by some of us (see Figure S1 in Supporting Information).
Additional docking calculations were also attained for compounds 4a, 11a and 12a (see Figure S2
in Supporting Information). These ligands were chosen because of their different steric demands in
the interaction with the enzyme counterpart thereby allowing to rationalize the different hCA II
inhibitory potencies of our MTCs. Dockings of the latter compounds would in fact suggest that: i)
in the case of Nꢀalkyl diꢀsubstituted MTCs (4aꢀ7a), long linear chains may not be hosted in the
enzyme cavity for steric reasons, explaining why compounds featuring a nꢀbutyl chain (5a and 7a)
are inactive towards the hCA II enzyme; ii) more hydrophilic compounds (8a, 10a, 11a and 15a)
may compensate the loss of lipophilic contacts by establishing an additional hydrogen bond with
the Q92 side chain (see Figure S2b). This interaction is not established by the morpholine ring of
1
0a (data not shown), which is indeed inactive towards hCAII; iii) for aromatic substituted
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derivatives (1a, 2a, 9a, and 12aꢀ14a), a short chain linking the ring and the MTC moiety or meta
substitutions on the terminal aryl ring should hamper the interactions with the residues lining the
hydrophobic pocket (i.e. F131), thereby explaining the inactivity of compounds 9a and 14a.
Docking calculations also helped to elucidate the molecular bases for the slight selectivity profile of
our MTC derivatives towards the tumor associated hCA IX and XII. Multiple sequence alignment
of hCAs I, II, IX and XII showed that single point mutations occur at the binding cavities of these
enzymes (see Figure S3 in Supporting Information). Specifically, the bulky F131 residue in hCA II
resulted replaced by the smaller valine (F131V) and alanine (F131A) in hCA IX and XII
respectively. Interestingly, docking of compound 2a into hCA IX and hCA XII (Figures 2b and 2c)
reveals that these substitutions, although causing the loss of the highly stabilizing Tꢀshaped π−π
interaction, allow the ligand to penetrate deeper into the hydrophobic region of the active site, thus
enhancing the contribution of desolvation to the overall ligand binding free energy. Finally, in hCA
I the N67 and T200 amino acids are both replaced by a histidine residue, which might affect ligand
binding for steric reasons. Moreover, the T200H substitution results in the loss of the key hydrogen
bond established by the ligand MTC moiety in hCA II, IX and XII (Figure 3). This would explain
the inactivity of compound 2a, and more generally the lower activity of our MTCs, against the hCA
I compared to the other three isoforms considered in this study.
0
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23a
Figure 2. Binding mode of compound 2a at the active site of a) hCA II (PDB code 3P5L),
b)
1
e
1f
hCA IX (PDB code 3IAI) and c) hCA XII (PDB code 1JD0). The ligand is shown as pink sticks.
hCA II, IX and XII are represented as surface and cartoons, respectively. Amino acids important for
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2+
ligand binding are highlighted as sticks. The Zn ion is depicted as a purple sphere. Hydrogen
bonds are displayed as dashed yellow lines.
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Figure 3. Superposition between the predicted hCA IIꢀ2a complex and the crystal structure of hCA
27
I (PDB code 2NMX). The ligand is shown as pink sticks. hCA II and I (only key substitutions) are
represented as cyan and yellow cartoon and surfaces, respectively. In both enzymes, residues
2
+
important for selectivity are highlighted as sticks. The Zn ion is depicted as a purple sphere.
Hydrogen bonds are displayed as dashed yellow lines.
IOP lowering activity.
The main clinical approach for the treatment of glaucoma or glaucoma related diseases, consists in
tackling the intraꢀocularꢀpressure (IOP) increase, which is the typical symptom. Nowadays such a
result can be achieved through laser therapy or surgical operation. However, the preferred way still
remains the topical pharmacological administration of drugs comprising para/sympaticomimetics,
8
ꢀ10
βꢀblockers and CAIs alone or in combination.
The use of CAIs, such as Dorzolamide or
Brinzolamide, is by far the most common since their lesser side effects allow for chronic
8ꢀ10
treatments.
Among the MTC series herein reported, we investigated some of the most active
compounds against the hCA II (2a, 3a, 11a and 15a) for their IOP lowering ability in vivo. Such
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compounds were considered also for their excellent water solubility properties. All MTCs were
formulated at 1% eye drop solution at neutral pH (due to their salt character), whereas the standard
Dorzolamide was formulated at pH 5.5, as a hydrochloride salt. The water solubility of eye drugs is
usually a problem of particular relevance. Actually the majority of drugs used for the topical
treatment of glaucoma usually possess acceptable solubility for the obtainment of the
pharmacological effect only as salts with strong acids, thus causing eye irritation and Dorzolamide
0
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ꢀ10
is a wellꢀknown case.
In our experiments we induced a transient ocular hypertention by the injection of 50 µl of sterile
hypertonic saline in the vitreous of New Zealand white rabbits, then the MTCs were administered
and compared both to the clinically used DRZ and vehicle (Figure 4).
Figure 4. Intraocular pressure (ꢂIOP, in mmHg) lowering versus time (min) of hypertonic salineꢀ
induced ocular hypertension in rabbits, treated with 50 ꢁL of 1% solution of compounds 2a, 3a, 11a
and 15a using DRZ as standard drug and vehicle. Errors were within 10ꢀ15% of the reported IOP
values (from three different measurements for each of the four animals in the study group) and were
statistically significant (p = 0.045 by the Student’s t test)
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As reported above all the compounds tested clearly showed IOP reduction potencies up to 120
minutes. In particular the MTC 11a showed the strongest biological effect already at 60 minutes
after injection (3.8 mmHg ꢂIOP) and reached the maximum up to 120 minutes (4.9 mmHg ꢂIOP).
The Hexaprazole MTC 15a provided an IOP reducing outcome comparable to the standard
Dorzolamide up to 120 minutes and then its effect resulted increased over the time of the
experiment (data not shown). In analogy the MTC 2a showed an IOP reducing pattern similar to
Dorzolamide even if lower in magnitude. Interestingly 2a and 15a retained their IOP reduction
activities up to 240 minutes. Compound 3a didn’t show significant IOP lowering ability and
reached its maximum at 120 minutes with ꢂIOP value comparable to the standard Dorzolamide.
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Conclusions
We report for the first time MTCs as a new class of CAIs. All compounds 1aꢀ15a were prepared
according to known reported synthetic methods and characterized by means of spectroscopic, mass
and elemental analyses. The MTCs were evaluated for their ability to inhibit the most relevant
physio/pathological hCA isoforms (I, II, IX and XII) in vitro. They exhibited K values comprised
I
between the low and medium nanomolar range. Noteworthy the MTCs reported here were highly
potent against the transmembrane isoforms hCA IX and XII. Molecular modeling studies elucidated
the binding mode of our inhibitors to the distinct hCA isoforms, thus revealing at the molecular
bases their different activity and selectivity profiles.
The best performing compounds against the highly abundant hCA II isoform and showing excellent
aqueous solubility properties, were tested in an acute glaucoma animal model for their ability to
lower the IOP values in comparison to the standard drug Dorzolamide at the same concentrations.
Interestingly the Hexaprazole MTC 11a was the most potent in lowering the IOP but within a short
period (120 min), whereas 2a and 15a were respectively able to retain their biological response up
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to 240 minutes. Thus we can consider the MTCs as a new class of CAIs which may constitute
interesting candidates for the future development of novel antiglaucoma drugs, a field in which no
new drug has emerged in the last 15 years. Moreover our study reports on an unusual zinc binding
motif the medicinal chemistry field, the MTCs, which can be successfully considered for its activity
on different metalloenzymes of pharmaceutical interest.
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Experimental protocols
General. Anhydrous solvents and all reagents were purchased from SigmaꢀAldrich, Alfa Aesar and
TCI. All reactions involving airꢀ or moistureꢀsensitive compounds were performed under a nitrogen
atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear magnetic
1
13
resonance ( HꢀNMR, CꢀNMR) spectra were recorded using a Bruker Advance III 400 MHz
spectrometer in DMSOꢀd . Chemical shifts are reported in parts per million (ppm) and the coupling
6
constants (J) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d,
doublet; t, triplet; m, multiplet; brs, broad singlet; dd, double of doubles. The assignment of
exchangeable protons (OH and NH) was confirmed by the addition of D O. Analytical thinꢀlayer
2
chromatography (TLC) was carried out on Merck silica gel Fꢀ254 plates. Flash chromatography
purifications were performed on Merck Silica gel 60 (230ꢀ400 mesh ASTM) as the stationary phase
and MeOH/DCM were used as eluents. Melting points (m.p.) were carried out in open capillary
tubes and are uncorrected. ESIꢀMS spectra were recorded by direct introduction at 5 mL/min flow
rate in an LTQ linear ion trap (Thermo, San Jose, CA, USA), equipped with a conventional ESI
source. The spectra were acquired in both positive and negative ion mode; the specific conditions
used for these experiments were as follows: the spray voltage was 5 kV in both polarity; the
capillary voltage were 49 V in positive ion mode and ꢀ15 V in negative ion mode; the capillary
temperature was kept at 280C. The sheath gas was set at 10 (arbitrary units), the sweep gas and
auxiliary gas were kept at 5 (arbitrary units). Scan Time was 2 microscans and the maximun
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injection time was 50ms. The ESI spectra were acquired using Xcalibur 2.0 (Thermo) the spectrum
range was 150ꢀ500 m/z. A Gallenkamp MPD350.BM3.5 apparatus was used to measure the melting
points.Amines 1ꢀ15 were commercially available (from SigmaꢀAldrich, Milan, Italy). All
compounds reported 1aꢀ15a were >98% pure by HPLC
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General procedure for the synthesis of monothiocarbamates 1aꢀ15a.
O
COS, NaOH 3.0 M aq.
H
N
R
N
S
Na
R
R1
Diethyl ether
R1
1ꢀ15
1aꢀ15a
The appropriate amines 1ꢀ15 (1.0 eq.) were dissolved in diethyl ether and treated with a freshly
prepared 3.0 M aqueous solution of NaOH (1.2 eq). The mixture was stirred at r.t. for 10 minutes,
then freshly produced COS was bubbled until the formation of a heavy precipitate. The reaction
mixture was stirred for 10 min and then all solvents were removed under vacuo to give a solid
residue, which was dissolved in MeOH and filtered through a Celite pad. The filtrate was
concentrated under vacuo to afford the titled compounds 1aꢀ15a.
28
Synthesis of Carbonyl Sulfide (COS).
N
S
K
S C O
H SO conc.
2
4
28
COS was generated by modification of the procedures reported in the literature. A saturated
aqueous solution of potassium thiocyanate was carefully added through a pressure equalized
dropping funnel to a concentrate sulfuric acid solution at –15 °C contained into an Erlenmeyer
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flask. The gas evolved was purified by bubbling into a 33% aqueous sodium hydroxide and
concentrated sulfuric acid solutions prior to be injected into the reaction vessel.
Synthesis of 3,4ꢀdimethoxyphenethylcarbamothioate sodium salt 1a.
0
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O
O
O
N
S Na
H
1a
3
,4ꢀDimethoxyphenethylcarbamothioate sodium salt 1a was obtained according to the general
procedure previously reported using 2ꢀ(3’,4’ꢀdimethoxyphenyl)ethanamine 1.
,4ꢀDimethoxyphenethylcarbamothioate sodium salt 1a: 72% yield; δ (400 MHz, DMSOꢀd
2H, d, J 6.7, CH ), 3.12 (2H, d, J 6.7, CH NH), 3.74 (3H, s, OCH ), 3.76 (3H, s, OCH ), 6.72 (1H,
appt, J 7.8, ArH), 6.81 (1H, m, ArH), 6.88 (1H, d, J 7.8, ArH); δ (100 MHz, DMSOꢀd ) 36.7, 44.7,
3
H
6
) 2.73
(
2
2
3
3
C
6
5
5
6.5, 56.6, 113.0, 113.6, 121.4, 134.0, 148.1, 149.6, 184.4; Elemental analysis: calc: C, 50.18; H,
.36; N, 5.32; S, 12.18; found: C, 50.20; H, 5.40; N, 5.35; S, 12.14; m/z (ESI negative) 240.07 [Mꢀ
ꢀ
Na] .
Synthesis of phenethylcarbamothioate sodium salt 2a.
O
N
S Na
H
2
a
Phenethylcarbamothioate sodium salt 2a was obtained according to the general procedure
previously reported using 2ꢀphenylethanamine 2.
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Phenethylcarbamothioate sodium salt 2a: 65% yield; δ (400 MHz, DMSOꢀd ) 2.74 (2H, d, J 6.8,
H
6
CH ), 3.14 (2H, d, J 6.8, CH NH), 7.27 (2H, d, J 7.8, ArH), 7.31 (2H, d, J 7.8, ArH), 7.38 (1H,
2
2
appt, J 7.8, ArH); δ
C
6
(100 MHz, DMSOꢀd ) 36.6, 45.8, 123.0, 134.0, 146.2, 148.8, 185.0; Elemental
analysis: calc: C, 53.19; H, 4.96; N, 6.89; S, 15.78; found: C, 53.17; H, 4.98; N, 6.87; S, 15.74; m/z
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ꢀ
(
ESI negative) 180.05 [MꢀNa] .
Synthesis of 4ꢀmethylpiperazinꢀ1ꢀylcarbamothioate sodium salt 3a.
N
N
HN
S
Na
O
3a
4
ꢀMethylpiperazinꢀ1ꢀylcarbamothioate sodium salt 3a was obtained according to the general
procedure previously reported using 4ꢀmethylpiperazinꢀ1ꢀamine 3.
ꢀMethylpiperazinꢀ1ꢀylcarbamothioate sodium salt 3a: 60% yield; δ
3H, s, CH ), 2.52 (4H, m, 2 x CH ), 2.63 (4H, m, 2 x CH ); δ (100 MHz, DMSOꢀd ) 44.2, 54.2,
4
H 6
(400 MHz, DMSOꢀd ) 2.32
(
3
2
2
C
6
5
6.9, 184.9; Elemental analysis: calc: C, 36.54; H, 6.13; N, 21.30; S, 16.26; found: C, 36.56; H,
ꢀ
6.12; N, 21.28; S, 16.22; m/z (ESI negative) 174.07 [MꢀNa] .
Synthesis of N,Nꢀdipropylcarbamothioate sodium salt 4a.
O
N
S Na
4a
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N,NꢀDipropylcarbamothioate sodium salt 4a was obtained according to the general procedure
previously reported using N,Nꢀdipropylamine 4.
N,NꢀDipropylcarbamothioate sodium salt 4a: 60% yield; δ
.8, CH ), 1.00 (3H, t, J 6.8, CH ), 1.48 (4H, m, 2 x CH ), 2.63 (4H, m, 2 x CH
DMSOꢀd
H
(400 MHz, DMSOꢀd
6
) 0.92 (3H, t, J
); δ (100 MHz,
6
3
3
2
2
C
0
1
2
3
4
5
6
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
) 15.2, 18.6, 52.4, 185.1; Elemental analysis: calc: C, 45.88; H, 7.70; N, 7.64; S, 17.50;
ꢀ
found: C, 46.01; H, 7.74; N, 7.58; S, 17.46; m/z (ESI negative) 160.08 [MꢀNa] .
Synthesis of N,Nꢀdibutylcarbamothioate sodium salt 5a.
O
N
S Na
5a
N,NꢀDibutylcarbamothioate sodium salt 5a was obtained according to the general procedure
previously reported using N,Nꢀdibutylamine 5.
N,NꢀDibutylcarbamothioate sodium salt 5a: 58% yield; δ
H
(400 MHz, DMSOꢀd
), 1.50 (4H, m, 2 x CH
) 15.2, 18.6, 52.4, 185.1; δ (100 MHz, DMSOꢀd
6
6
) 0.98 (3H, t, J 6.8,
), 2.69 (4H, m, 2 x
) 14.8, 15.0, 20.2,
CH
3
2
), 1.00 (3H, t, J 6.8, CH
); δ (100 MHz, DMSOꢀd
3
), 1.32 (4H, m, 2 x CH
2
2
CH
C
6
C
2
8
7.8, 47.9, 184.6; Elemental analysis: calc: C, 51.16; H, 8.59; N, 6.63; S, 15.18; found: C, 51.14; H,
ꢀ
.55; N, 6.60; S, 15.14; m/z (ESI negative) 188.11 [MꢀNa] .
Synthesis of N,Nꢀdiisobutylcarbamothioate sodium salt 6a.
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O
N
S Na
6a
10
11
12
13
14
15
16
17
18
19
20
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22
23
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31
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37
38
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40
41
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44
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N,NꢀDiisobutylcarbamothioate sodium salt 6a was obtained according to the general procedure
previously reported using N,Nꢀdiisobutylamine 6.
2
9
N,NꢀDiisobutylcarbamothioate sodium salt 6a: 32% yield; m.p. 214°C (Lit. : 210ꢀ212°C; δ
H
(400
);
MHz, DMSOꢀd ) 0.96 (12H, m, 4 x CH ), 2.56 (2H, m, 2 x CH), 2.73 (4H, d, J 6.8, 2 x CH
6
3
2
δ
C
(100 MHz, DMSOꢀd
6
) 21.3, 27.8, 51.9, 186.0; Elemental analysis: calc: C, 51.16; H, 8.59; N,
ꢀ
6
.63; S, 15.18; found: C, 51.18; H, 8.62; N, 6.58; S, 15.02; m/z (ESI negative) 211.10 [MꢀNa] .
Synthesis of N,Nꢀbutyl(ethyl)carbamothioate sodium salt 7a.
O
N
S Na
7
a
N,NꢀButyl(ethyl)carbamothioate sodium salt 7a was obtained according to the general procedure
previously reported using Nꢀethylbutanꢀ1ꢀamine 7.
H 6
N,NꢀButyl(ethyl)carbamothioate sodium salt 7a: 66% yield; δ (400 MHz, DMSOꢀd ) 0.90 (3H, t, J
6
.8, CH ), 1.01 (3H, t, J 6.8, CH ), 1.24 (2H, m, CH ), 1.48 (2H, m, CH ), 3.40 (4H, m, 2 x CH );
3
3
2
2
2
δ
C
6
(100 MHz, DMSOꢀd ) 14.7, 15.0, 20.8, 31.6, 48.8, 180.8; Elemental analysis: calc: C, 45.88; H,
7
.70; N, 7.64; S, 17.50; found: C, 45.90; H, 7.72; N, 7.68; S, 17.44; m/z (ESI negative) 160.08 [Mꢀ
ꢀ
Na] .
Synthesis of ethylꢀNꢀoxoethylmethylcarbamothioate sodium salt 8a.
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1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
O
O
N
S Na
O
8a
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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2
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4
5
6
7
8
9
0
EthylꢀNꢀoxoethylmethylcarbamothioate sodium salt 8a was obtained according to the general
procedure previously reported using ethyl 2ꢀ(methylamino)acetate 8.
EthylꢀNꢀoxoethylmethylcarbamothioate sodium salt 8a: 22% yield; δ
H 6
(400 MHz, DMSOꢀd ) 1.27
(3H, t, J 6.4, CH CH ), 2.82 (3H, s, NCH ), 3.95 (2H, s, CH ), 4.28 (2H, q, J 6.4, CH CH ); δ (100
2
3
3
2
2
3
C
MHz, DMSOꢀd
6
) 13.8, 36.7, 54.2, 63.0, 168.8, 185.4; Elemental analysis: calc: C, 36.18; H, 5.06;
ꢀ
N, 7.03; S, 16.10; found: C, 36.20; H, 5.09; N, 7.00; S, 16.07; m/z (ESI negative) 176.04 [MꢀNa] .
Synthesis of NꢀbenzylꢀNꢀmethylcarbamothioate sodium salt 9a.
O
N
S Na
9a
NꢀBenzylꢀNꢀmethylcarbamothioate sodium salt 9a was obtained according to the general procedure
previously reported using Nꢀmethyl(phenyl)methanamine 9.
NꢀBenzylꢀNꢀmethylcarbamothioate sodium salt 9a: 32% yield; δ
H 6
(400 MHz, DMSOꢀd ) 3.10 (3H,
s, CH ), 4.20 (2H, s, CH ), 7.23 (1H, appt, J 8.4, ArH), 7.31 (2H, d, J 8.4, ArH), 7.28 (2H, d, J 8.4,
3
2
ArH); δ (100 MHz, DMSOꢀd ) 33.2, 55.6, 127.2, 128.3, 129.0, 141.4, 186.3; Elemental analysis:
C
6
calc: C, 53.19; H, 4.96; N, 6.89; S, 15.78; found: C, 53.17; H, 4.94; N, 6.88; S, 15.75; m/z (ESI
ꢀ
negative) 180.05 [MꢀNa] .
Synthesis of morpholineꢀ4ꢀcarbothioate sodium salt 10a.
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O
N
O
S Na
10a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
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27
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29
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31
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33
34
35
36
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39
40
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43
44
45
46
47
48
49
50
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52
53
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56
57
58
59
60
Morpholineꢀ4ꢀcarbothioate sodium salt 10awas obtained according to the general procedure
previously reported using morpholine 10.
Morpholineꢀ4ꢀcarbothioate sodium salt 10a: 82% yield; δ
H 6
(400 MHz, DMSOꢀd ) 2.67 (4H, m,
CH ), 3.3.53 (4H, m, 2 x CH ); δ (100 MHz, DMSOꢀd ) 45.9, 68.3, 185.3; Elemental analysis: calc:
2
2
C
6
C, 35.50; H, 4.77; N, 8.28; S, 18.95; found: C, 35.52; H, 4.76; N, 8.30; S, 18.92; m/z (ESI negative)
ꢀ
1
46.03 [MꢀNa] .
Synthesis of piperazineꢀ1,4ꢀcarbothioate sodium salt 11a.
O
O
S
Na
N
N
S Na
1a
1
Piperazineꢀ1,4ꢀcarbothioate sodium salt 11a was obtained according to the general procedure
previously reported using piperazine 11.
Piperazineꢀ1,4ꢀcarbothioate sodium salt 11a: 38% yield; δ (400 MHz, DMSOꢀd ) 4.80 (8H, brs, 4
H
6
x CH
2
); δ
C
(100 MHz, DMSOꢀd
6
) 46.9, 185.2; Elemental analysis: calc: C, 38.28; H, 4.28; N, 14.88;
ꢀ
S, 34.06; found: C, 38.25; H, 4.30; N, 14.86; S, 34.01; m/z (ESI negative) 188.01 [MꢀNa] .
30
Experimental data in agreement with reported data.
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2
2
2
2
2
2
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4
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5
5
5
5
5
5
5
5
6
Synthesis of 4ꢀ(4ꢀfluorophenyl)piperazineꢀ1ꢀcarbothioate sodium salt 12a.
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
N
N
S
Na
12a
4ꢀ(4ꢀFluorophenyl)piperazineꢀ1ꢀcarbothioate sodium salt 12a was obtained according to the general
procedure previously reported using 1ꢀ(4ꢀfluorophenyl)piperazine 12.
4
H 6
ꢀ(4ꢀFluorophenyl)piperazineꢀ1ꢀcarbothioate sodium salt 12a: 56% yield; δ (400 MHz, DMSOꢀd )
2
.86 (4H, m, 2 x CH
2
), 3.81 (4H, brs, 2 x CH
2
C
), 6.94 (2H, m, ArH), 7.19 (2H, m, ArH); δ (100
2
1
MHz, DMSOꢀd
6
) 45.3, 50.7, 116.1 (d, J C-F 22.0), 118.5, 149.5, 156.0 (d, J C-F 234), 185.0;
δ (376.6 MHz, DMSOꢀd ) ꢀ125.75 (1F, s); Elemental analysis: calc: C, 50.37; H, 4.61; N, 10.68; S,
F
6
ꢀ
1
2.23; found: C, 50.40; H, 4.59; N, 10.66; S, 12.19; m/z (ESI negative) 239.07 [MꢀNa] .
Synthesis of 4ꢀ(4ꢀtrifluorophenyl)piperazineꢀ1ꢀcarbothioate sodium salt 13a.
O
F3C
N
N
S
Na
13a
4
ꢀ(4ꢀTrifluorophenyl)piperazineꢀ1ꢀcarbothioate sodium salt 13a was obtained according to the
general procedure previously reported using 1ꢀ(4ꢀtrifluorophenyl)piperazine 13.
4
H
ꢀ(4ꢀTrifluorophenyl)piperazineꢀ1ꢀcarbothioate sodium salt 13a: 48% yield; δ (400 MHz, DMSOꢀ
d
6
2
) 2.84 (4H, m, 2 x CH ), 3.79 (4H, brs, 2 x CH
2 C
), 6.96 (2H, m, ArH), 7.22 (2H, m, ArH); δ (100
2
1
MHz, DMSOꢀd ) 45.7, 48.3, 115.1, 118.3 (q, J
31.0), 126.0 (d, J
270), 127.3, 154.4, 185.0;
C-F
6
C-F
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δ (376.6 MHz, DMSOꢀd ) ꢀ59.9 (1F, s); Elemental analysis: calc: C, 46.15; H, 3.87; N, 8.97; S,
F
6
ꢀ
1
0.27; found: C, 46.18; H, 3.89; N, 8.95; S, 10.24; m/z (ESI negative) 289.06 [MꢀNa] .
Synthesis of 4ꢀ(3ꢀchlorophenyl)piperazineꢀ1ꢀcarbothioate sodium salt 14a.
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11
12
13
14
15
16
17
18
19
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Cl
O
N
N
S
Na
1
4a
4ꢀ(3ꢀChlorophenyl)piperazineꢀ1ꢀcarbothioate sodium salt 14a was obtained according to the general
procedure previously reported using 4ꢀ(3ꢀchlorophenyl)piperazine 14.
4
3
7
ꢀ(3ꢀChlorophenyl)piperazineꢀ1ꢀcarbothioate sodium salt 14a: 70% yield; δ
.06 (4H, t, J 6.8, 2 x CH ), 3.80 (4H, brs, 2 x CH ), 6.78 (1H, d J 8.4, ArH), 6.93 (2H, m, ArH),
.23 (1H, appt, J 8.4, ArH); δ (100 MHz, DMSOꢀd ) 46.4, 49.7, 114.4, 115.6, 118.8, 131.3, 134.7,
H 6
(400 MHz, DMSOꢀd )
2
2
C
6
153.5, 185.4; Elemental analysis: calc: C, 45.88; H, 7.70; N, 7.64; S, 17.50; found: C, 45.90; H,
ꢀ
7
.72; N, 7.68; S, 17.44; m/z (ESI negative) 160.08 [MꢀNa] .
Synthesis of 4ꢀ(2ꢀ(cyclohexylamino)ꢀ2ꢀoxoethyl)piperazineꢀ1ꢀcarbothioate sodium salt 15a.
O
HN
O
S
N
N
Na
1
5a
4ꢀ(2ꢀ(Cyclohexylamino)ꢀ2ꢀoxoethyl)piperazineꢀ1ꢀcarbothioate sodium salt 15a was obtained
according to the general procedure previously reported using 4ꢀ(2ꢀ(cyclohexylamino)ꢀ2ꢀ
oxoethyl)piperazine 15.
4
H
ꢀ(2ꢀ(Cyclohexylamino)ꢀ2ꢀoxoethyl)piperazineꢀ1ꢀcarbothioate sodium salt 15a: 54% yield; δ (400
MHz, DMSOꢀd ) 1.18 (4H, m), 1.38 (4H, m), 1.56 (2H, m), 2.65 (4H, m), 3.14 (4H, m), 3.36 (2H,
6
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1
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2
2
2
2
2
2
2
2
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3
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
s, CH2), 3.52 (1H, m); δ (100 MHz, DMSOꢀd ) 25.4, 26.1, 33.2, 46.4, 47.8, 54.2, 62.4, 169.0,
C
6
1
1
85.0; Elemental analysis: calc: C, 50.80; H, 7.21; N, 13.67; S, 10.43; found: C, 50.81; H, 7.23; N,
ꢀ
3.65; S, 10.40; m/z (ESI negative) 284.14 [MꢀNa] .
0
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2
3
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8
9
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0
CA Inhibition
An Applied Photophysics stoppedꢀflow instrument has been used for assaying the CAꢀcatalyzed
CO
working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as the buffer, and 20
mM Na SO (for maintaining constant the ionic strength), following the initial rates of the CAꢀ
catalyzed CO hydration reaction for a period of 10−100 s. The CO concentrations ranged from
2
hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as a pH indicator,
2
4
25
2
2
1
.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each
inhibitor, at least six traces of the initial 5−10 % of the reaction have been used for determining the
initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the
total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled−deionized
water, and dilutions up to 0.01 nM were done thereafter with distilled−deionized water. Inhibitor
and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, to
allow for the formation of the E−I complex. The inhibition constants were obtained by nonlinear
20
least squares methods using PRISM 3, as reported earlier, and represent the mean from at least
three different determinations. All CA isofoms were recombinant ones obtained in house as
20ꢀ24
reported earlier.
Molecular Modelling.
Ligand setup. The ligand’s tridimensional structures were generated with Maestro v. 10.4
(Maestro, version 10.4, Schrödinger, LLC, New York, NY, 2015). Protonation states, including the
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potential metalꢀbinding forms, were predicted using Epik 3.4 as implemented in Maestro (Epik,
version 3.4, Schrödinger, LLC, New York, NY, 2015). Ligands geometry were then optimized
ꢀ1
ꢀ1
through a Polak−Ribiere conjugate gradient minimization (convergence = 0.0005 kJ Å mol )
using the MacroModel v.11.0 module in Maestro (MacroModel, version 11.0, Schrödinger, LLC,
New York, NY, 2015). Due to the inaccuracies in the assignment of partial charges of the MTC
moiety by the empirical force fields implemented in Maestro, ligands charges were computed using
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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59
60
31
the Restrained Electrostatic Potential (RESP) fitting procedure. The ESP was first calculated with
Gaussian 09 Rev. D.01 (Gaussian 09, Revision E.01, Gaussian, Inc., Wallingford CT, 2009) using a
6
–31G* basis set at the Hartree–Fock level of theory, and then the RESP charges were obtained by
3
2
a twoꢀstages fitting procedure using Antechamber.
2
3a
Proteins setup. The crystal structures of hCA II (PDB code 3P5L), and of the catalytic domains
1e
1f
of hCA IX (PDB code 3IAI) and hCA XII (PDB code 1JD0) were prepared for docking
calculations using the “Protein Preparation Wizard” panel of Schrödinger 2015 molecular modeling
package (Maestro, version 10.4, Schrödinger, LLC, New York, NY, 2015). First, missing side
chains or loops were added using Prime 4.2 (Prime, version 4.2, Schrödinger, LLC, New York, NY,
2
015). Then, bond orders were assigned and all the hydrogen atoms were added. The prediction of
the side chains hetero groups ionization and tautomeric states was performed using Epik 3.4 (Epik,
version 3.4, Schrödinger, LLC, New York, NY, 2015). Finally, an optimization of the hydrogenꢀ
bonding network was performed, and the positions of the hydrogen atoms were optimized. All the
water molecules were deleted prior to docking calculations.
33
Molecular docking. Docking studies were carried out with the gridꢀbased program Glide 6.9. For
the grid generation, a box of 22Å x 22Å x 22Å, surrounding the ligand binding cavity site, was
considered for each enzyme. The standard precision (SP) mode of the GlideScore function was used
3
4
to score the predicted binding poses. The force field used for the docking was OPLSꢀ2005. All of
the pictures were rendered with PyMOL (www.pymol.org).
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Transient ocular hypertensive rabbit model.
The increase in IOP was induced by the injection of 50
µl of sterile hypertonic saline (5%) into the
vitreous bilaterally in preꢀanesthetized New Zealand white rabbits with tiletamine/zolazepam
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
10d
(Zoletil 50/50 mg/mL, Virbac) i.m. 0.05 ml/kg, plus xylazine (Xylor 2%,) i.m. 0.02 ml/kg. Drugs
were dissolved in pirogenꢀfree appropriate vehicle (buffered aqueous solution pH 7, polyoxyl 40,
dimethylsulphoxyde (DMSO) 2%, hydrogenated castor oil 5 mg/ml, tromethamine 10 mg/ml,
edetate disodium 0.5mg/ml and benzalkonium chloride 0.15 mg/mL) and administered as eye drops
at different concentrations immediately after hypertonic saline injection. IOP was measured using a
®
TonoꢀPen AVIA (Reichert Inc. Depew, NY) prior (basal) and post hypertonic saline injection 60,
1
20, 240 min after drug or vehicle treatment. One drop of 0.2% oxybuprocaine hydrochloride, 4
mg/ml, was instilled in each eye immediately before each set of pressure measurements. The ocular
hypotensive activity of studied compounds is expressed in mmHg as average difference in IOP
10d
between drugꢀ and vehicleꢀtreated eyes.
Acknowledgements. This research was financed by two EU grants of the 7th framework program
Metoxia and Dynano projects to AS and CTS).
(
Corresponding Authors. FC, Phone: +39ꢀ055ꢀ4573005; fax: +39ꢀ055ꢀ4573385; Eꢀmail:
fabrizio.carta@unifi.it; CTS, Phone: +39ꢀ055ꢀ4573005; fax: +39ꢀ055ꢀ4573385; Eꢀmail:
claudiu.supuran@unifi.it
Abbreviations Used. MTCs, monothiocarbamates; CAI(s), carbonic anhydrase inhibitor(s); AAZ,
I
acetazolamide; (h)CA, (human)carbonic anhydrase; K , inhibition constant;
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Supporting Informations. Supporting Informations are available free of charge on the ACS
Publications website. Superposition of a predicted binding pose of compound 2a with a known
DTC at the hCA II active site (Figure S1), binding mode of compounds 4a, 11a and 12a at the
active site of hCA II (Figure S2), multiple sequence alignment of hCA I, II, IX and XII (Figure S3)
and molecular formula strings.
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55
56
57
58
59
60
ACS Paragon Plus Environment