Alessandro Bonardi1,2, Alessio Nocentini1,2, Silvia Bua1, Jacob Combs3, Carrie Lomelino3, Jacob Andring3, Laura Lucarini4, Silvia Sgambellone4, Emanuela Masini4, Robert McKenna3, Paola Gratteri1,2, Claudiu T Supuran1. 1. Department NEUROFARBA - Pharmaceutical and nutraceutical section, University of Firenze, via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence Italy. 2. Department NEUROFARBA - Pharmaceutical and nutraceutical section; Laboratory of Molecular Modeling Cheminformatics & QSAR, University of Firenze, via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy. 3. Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Box 100245, Gainesville, Florida 32610, United States. 4. Department NEUROFARBA - Pharmaceutical and nutraceutical section, University of Firenze, viale Gaetano Pieraccini 6, 50139 Firenze, Florence, Italy.
Abstract
The "tail approach" has become a milestone in human carbonic anhydrase inhibitor (hCAI) design for various therapeutics, including antiglaucoma agents. Besides the classical hydrophobic/hydrophilic division of hCAs active site, several subpockets have been identified at the middle/outer active sites rim, which could be targeted to increase the CAI isoform selectivity. This postulate is explored here by three-tailed benzenesulfonamide CAIs (TTI) to fully exploit such amino acid differences among hCAs. In this proof-of-concept study, an extensive structure-activity relationship (SAR) study was carried out with 32 such benzenesulfonamides differing in tails combination that were assayed for hCAs I, II, IV, and XII inhibition. A structural study was undertaken by X-ray crystallography and in silico tools to assess the ligand/target interaction mode. The most active and selective inhibitors against isoforms implicated in glaucoma were assessed in a rabbit model of the disease achieving an intraocular pressure-lowering action comparable to the clinically used dorzolamide.
The "tail aplass="Chemical">proach" has become a mlass="Chemical">pan class="Chemical">ilestone in humancarbonic anhydrase inhibitor (hCAI) design for various therapeutics, including antiglaucoma agents. Besides the classical hydrophobic/hydrophilic division of hCAs active site, several subpockets have been identified at the middle/outer active sites rim, which could be targeted to increase the CAI isoform selectivity. This postulate is explored here by three-tailed benzenesulfonamideCAIs (TTI) to fully exploit such amino acid differences among hCAs. In thisproof-of-concept study, an extensive structure-activity relationship (SAR) study was carried out with 32 such benzenesulfonamides differing in tails combination that were assayed for hCAs I, II, IV, and XII inhibition. A structural study was undertaken by X-ray crystallography and in silico tools to assess the ligand/target interaction mode. The most active and selective inhibitors against isoforms implicated in glaucoma were assessed in a rabbit model of the disease achieving an intraocular pressure-lowering action comparable to the clinically used dorzolamide.
lass="Chemical">Carbonic
anhydrases (lass="Chemical">pan class="Gene">CAs, EC 4.2.1.1) are among the most efficient
catalysts, speeding up the simple yet physiologically essential reaction
in all kingdoms: the reversible hydration of carbon dioxide to bicarbonate
and protons.[1] Among the eight genetically
unrelated CA families α, β, γ, δ, η,
ζ, θ, and ι,[2−9] α-CAs are uniquely present in higher vertebrates.[2,10] In particular, humans express 15 α-CA isoforms (hCAs) which
differ in catalytic activity, subcellular/tissue localization, and
physiological role.[11] Therefore, hCAs are
involved in multiple physiological processes and their levels of activities
are linked to many human disorders such as glaucoma, retinal/cerebral
edema, retinitis pigmentosa, other retinopathies, stroke, epilepsy,
sterility, osteoporosis, altitude sickness, cariogenesis, neurodegeneration,
obesity, and cancer.[12−14] As a result, almost all catalytically active hCAs
have generated great interest for the design of inhibitors (carbonic
anhydrase inhibitors, CAIs) or activators (CAAs) with biomedical applications.[15] Although initially CAIs were used as diuretics,
antiglaucoma agents, antiepileptics, and for the management of altitude
sickness,[2] a new generation of CAIs are
being developed for the treatment of cancers, obesity, inflammation,
neuropathic pain, infections, and neurodegenerative disorders.[16−21] CAAs are also of interest in the field of cognition, aging, and
neurodegeneration.[22]
Nevertheless,
the use as antilass="Disease">glaucoma agents is still the main
theralass="Chemical">peutic alass="Chemical">plass="Chemical">plilass="Chemical">pan class="Gene">cation of CAIs. In fixed-drugs combinations (mainly
with prostaglandin analogues and β-blockers), CAIs continue
to be marketed worldwide and widely used.[23] Acetazolamide (AAZ), methazolamide (MTZ), and dichlorophenamide (DCP) are first-generation
CAIs used as systemic drugs for the management of this disease (Figure ). Dorzolamide (DRZ) and brinzolamide (BRZ) represent second-generation
inhibitors used topically, as eye drops, with less side effects compared
to first-generation drugs.[24] However, none
of these drugs possess a selective inhibition profile against the
hCA isoforms mainly implicated in the disease that are hCA II (main
isoform), IV, and XII. Considering that the current therapies are
overall often inadequate given that multiple classes of medications
have to be coadministered to control intraocular pressure (IOP) efficiently,[25] it might be of crucial importance to optimize
the single CAI agents, by increasing their efficacy (against the target
CAs) and decreasing adverse events (improving their selectivity of
action).
Figure 1
Clinically used antiglaucoma CAIs.
Clinipan class="Gene">cally used antilass="Chemical">pan class="Disease">glaucoma CAIs.
The 12 lass="Gene">catalytilass="Chemical">pan class="Gene">cally active hCAs (isoforms VIII, X, and XI are
catalytically inactive) are characterized by a Zn(II) ion, which is
tetrahedrally coordinated by three histidine residues and a solvent
molecule that are situated at the base of a 13 Å deep conical
cavity portioned into hydrophobic and hydrophilic sides.[11,15,26] As the hCAscatalytic domains
are structurally homologous and conserved in amino acid sequence identity,
it is rather challenging to achieve targeted inhibition of a specific
hCA isozyme over others. Despite this, many new approaches have been
developed for this purpose, especially over the last two decades.[15]
So far, four unique lass="Gene">CA inhibition mechanisms
have been lass="Chemical">pan class="Chemical">validated
by both kinetic and structural assessments:[15,27] (1) zinc binding, which consists of the direct coordination of a
catalytical Zn(II) ion with a tetrahedral or trigonal bipyramidal
coordination geometry (sulfonamides, sulfamides, sulfonates, anions,
mono-dithiocarbamates, xanthates, thioxanthates, carboxylates, hydroxamates,
benzoxaboroles, selenols); (2) anchorage to the zinc-bound water molecule/hydroxide
ion (phenols, thiophenols, polyphenols, carboxylates, polyamines,
2-thioxocoumarins, sulfocoumarins); (3) occlusion of the active site
entrance (coumarins and bioisosters); and (4) binding out of the active
site (a unique carboxylic acid derivative exhibited this inhibition
mode to date).
Undoubtedly, zinc binders, such as pan class="Chemical">sulfonamides
and their bioisosters
lass="Chemical">pan class="Chemical">sulfamates and sulfamides in a prominent position, are among the most
effective and investigated derivatives in the field of CA inhibition
as well as in the related clinical context.[11,15]
In fact, most efforts have been made on tlass="Chemical">his class of lass="Chemical">pan class="Gene">CAIs
to achieve
isozyme selectivity of action, to lower the side effects consequent
to promiscuous inhibition.[28] As simple
as effective, the so-called “tail approach” made its
appearance in the field of CA inhibition in 1999 and led to the development
of a large number of studies and compounds that expanded the database
of CA isoform-selective inhibitors by appending a wide spectrum of
chemical functionalities, named tails, to the main zinc-binding scaffold.[29−35] The original aim was to increase the water solubility[29] and subsequently membrane (im)permeability of
aromatic sulfonamide derivatives.[32] Afterward,
the design was shifted toward the modulation of the interactions between
the ligand and the middle and outer rims of the hCAs active sites,
which contain the most variable polypeptide regions among the various
isoforms, to increase isoform specificity. Simple tailed CAIs are
composed of the following elements: (i) a zinc-binding function, (ii)
a main scaffold that can include a linker, and (iii) the tail (Figure A).
Figure 2
Schematic representation
of the (A) “tail”, (B) “two-tails”,
and (C) “three-tails” approach for the design of zinc-binding
CAIs.
Schematic representation
of the (A) “tail”, (B) “two-tails”,
and (C) “pan class="Chemical">three-tails” alass="Chemical">plass="Chemical">pan class="Chemical">proach for the design of zinc-binding
CAIs.
An extension of tlass="Chemical">his alass="Chemical">plass="Chemical">pan class="Chemical">proach
was proposed in 2015 by Tanpure et
al.,[36] with the simultaneous inclusion
of two tails of diverse nature onto aromatic sulfonamide scaffolds,
at a nitrogen atom branching point, allowing distinct binding to the
hydrophobic and hydrophilic sections of the hCAs active site (Figure B). However, a limited
number of compounds were reported (three), and an in vitro assay was performed solely on hCA II, which makes this pioneering
study rather unfulfilled. More recently, Fares et al. have used a
similar approach proposing a diverse type of dual tails to benzenesulfonamideCAIs.[37]
The detalass="Chemical">iled knowledge of
the active site comlass="Chemical">position and architecture
of lass="Chemical">pan class="Gene">hCAs (mostly available by X-ray crystallographic studies, except
for CAs VA and VB) derived from many previous studies[38−40] led to the conclusion that the simple hydrophobic/hydrophilic division
of the isoforms binding pocket may no longer be sufficient. In fact,
some CA isozymes do not exhibit such a precise distinction as originally
noted in hCA I, II, and IX,[13] and a bulk
of accessory subpockets exist, which differentiate the various CA
isoforms. Here, the inclusion of a third tail is proposed as an approach
to improve the matching and fitting of the target–ligand interaction
within the different hCAs active sites (Figure C).
As a first lass="Chemical">proof of concelass="Chemical">pt of
tlass="Chemical">pan class="Chemical">his improved approach, a diverse
array of tail combinations were investigated with the aim of identifying
suitable isoform imprints. Described here is the screening of hCA
isozymes I, II, IV, and XII with 32 benzenesulfonamide derivatives
incorporating three tails. In the context of the antiglaucomaCAI
application, hCA I is the main off-target isoform as it is widespread
in red blood cells and many other tissues.[2] A comprehensive structural study was also undertaken by X-ray crystallography
with hCA II and in silico with isozymes hCA I, IV,
and XII, to assess the ligand–target interaction modes. A selection
of the three-tailed inhibitors most active against hCAs implicated
in glaucoma was assessed in vivo in a rabbit model
of the diseases and compared to classical clinically used CAIs.
Results
and Discussion
Drug Design and Chemistry
Currently,
the tail appan class="Chemical">proach
has been a focus of lass="Chemical">pan class="Gene">CAIs research area with most design studies adopting
the p-substituted benzenesulfonamide scaffold as
a main foothold to include a variety of chemical frameworks.[15] In fact, avoiding heteroaromatic sulfonamide
scaffolds markedly eases the synthesis procedures, moving the focus
on the inclusion of pendants on the inhibitor structure.[36] Likewise, to converge efforts and attention
on studying the three-tailing effects on CA inhibition, a p-substituted benzenesulfonamide was here adopted as a CAI
scaffold.
It should be stressed that it is not possible to easily
include lass="Chemical">three chemilass="Chemical">pan class="Gene">cally diverse tails on a single branching atom
(e.g., a nitrogen atom, as proposed by Tanpure et al. in the two-tails
approach),[36] unless obtaining an ammonium
salt or a chiral center. As a result, among several identified alternatives
to branch a spacer attached to the main scaffold into three tails,
the general structure TTI (Figure ) was selected to combine easy and versatile
chemistry with the possibility to extend it to many diverse chemical
groups, which is relevant for producing a range of tail combinations.
As a result, TTI was designed in the following manner:
(i) a benzenesulfonamide scaffold (blue), which assures the interaction
with the zinc ion and the bottom of the active site; (ii) an ethylenic
spacer (red), which has the function to allow sufficient space between
the main scaffold and the tails; (iii) a first ramification point
(N atom, in black) from which the first tail T1 (green)
branches off; (iv) an amide-based spacer (red); and (v) a second intersection
point (N atom, in black) by which T2 and T3 (green)
branch off. Having the benzenesulfonamide bound to the Zn(II) at the
bottom of the active site, the linkers in red (Figure ) were chosen in such a way as to explore
a vast chemical space at the middle and outer rims of the binding
clefts.
Figure 3
General structure of the designed three-tailed inhibitors (TTIs).
General structure of the designed pan class="Chemical">three-talass="Chemical">pan class="Chemical">iled inhibitors (TTIs).
The synthesis strategies adopted
to yield the TTI derivatives
are reported in Schemes –3. T1 was introduced on the lass="Chemical">4-(2-aminoethyl)benzenesulfonamide by reductive
amination with the lass="Chemical">pan class="Chemical">proper aromatic aldehyde and sodium borohydride
in MeOH or, alternatively, by nucleophilic substitution with the appropriate
halides in anhydrous N,N-dimethylformamide
(DMF) and in the presence of tetraethylammonium (TEA) to furnish secondary
amines 1–5 and 6 and 7, respectively. The latter were reacted with chloroacetyl chloride
or chloropropionyl chloride in acetone and in the presence of K2CO3 to provide amides 8–17.
T2 and T3 were finally included through a nucleophilic
substitution with commercially available or synthesized secondary
amines in anhydrous ACN and TEA as a base to produce TTIs 18–33. The nitrile derivatives 33–37 were further
converted to the corresponding amines 40–44 through
a Ni/Raney-catalyzed hydrogenation or hydrolyzed in NaOH(aq) into the corresponding carboxylic acids 45–49 (Scheme ). Additionally,
the markedly hydrophobic oleylamide derivative 50 was
yielded by coupling the carboxylic acid 46 with oleylamine
in the presence of EDC and 4-dimethylaminopyridine (DMAP) in anhydrous
DMF.
All derivatives were
purified by pan class="Chemical">silica gel chromatogralass="Chemical">phy eluting
with lass="Chemical">pan class="Chemical">MeOH/DCM gradients and fully characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS) (Supporting Information).
Carbonic Anhydrase Inhibition
In tlass="Chemical">his first screening,
mono-talass="Chemical">pan class="Chemical">iled (1–7) and three-tailed (18–50) compounds were analyzed by a stopped-flow kinetic assay with hCA
isoforms I, II, IV, and XII.[41] HCAs II,
IV, and XII are involved in glaucoma with the last isoform being reported
to be upregulated in the eyes of glaucomapatients. Thus, all of them
are involved in this disease, both in the elevation of intraocular
pressure (IOP) and the decrease of blood flow and oxygen supply within
the hypoxic neovascular retinic tissues.[42] HCA IV was reported to be involved in stroke, glaucoma, retinitis
pigmentosa, astrocytomas, and gliomas.[12] HCA XII is also validated as an anticancer target (being overexpressed
on the membrane of hypoxic tumor cells),[17] and recently, overexpression of this isoform has also been linked
to inflammation.[19] HCA I is a main off-target
isoform for the therapeutic application of CAIs in ocular diseases,
as this isoform is widespread in red blood cells and many other tissues.[2]
Generally, the inhibition data reported
in Table highlighted
that mono-tapan class="Chemical">iled comlass="Chemical">pounds 1–7 were medium to
high nanomolar inhibitors of lass="Chemical">pan class="Gene">hCA I (KI = 68.4–458.1 nM), II (KI = 62.8–153.7
nM), and XII (KI = 55.4–113.2 nM),
and weak inhibitors of hCA IV with inhibition constant (KI) values in the low micromolar range (1.1–6.2
μM).
Table 1
Inhibition Data of Human CA Isoforms
CA I, II, IV, and XII with Sulfonamides 1–7, 18–50 Reported Here and the Standard Sulfonamide Inhibitor
Acetazolamide (AAZ) by a Stopped-Flow CO2 Hydrase
Assay[41]
KIa (nM)
cmpd
n
R1
R2
R3
CA I
CA II
CA IV
CA XII
1
C6H5
95.3
98.4
2854.4
65.4
2
4-NO2-C6H4
224.3
120.9
1685.3
77.4
3
4-F-C6H4
112.8
78.5
1196.7
60.1
4
2-Naph
458.1
87.1
6248.1
78.6
5
Fu
68.4
62.8
1584.5
55.4
6
CH2CN
105.3
153.7
5547.2
113.2
7
CH2C6H5
278.4
89.1
3587.4
104.3
18
1
C6H5
CH2CH3
CH2CH3
786.6
8.3
4147.5
43.9
19
1
C6H5
CH2CH3
CH2C6H5
4210.4
391.6
>10000
82.6
20
1
C6H5
CH2C6H5
CH2C6H5
865.9
412.3
>10000
98.8
21
1
C6H5
(CH2)4CH3
(CH2)4CH3
506.1
124.5
>10000
69.4
22
1
C6H5
(CH2)5CH3
(CH2)5CH3
878.7
237
>10000
92.8
23
1
C6H5
(CH2)7CH3
(CH2)7CH3
946.7
843.8
>10000
99.4
24
2
C6H5
CH2CH3
CH2CH3
184.7
8.9
3928.8
61.1
25
2
C6H5
CH2CH3
CH2C6H5
544.3
79.6
>10000
90.4
26
2
C6H5
CH2C6H5
CH2C6H5
692.3
559.2
4640.8
302.5
27
2
C6H5
(CH2)4CH3
(CH2)4CH3
563.6
522.6
3244.8
100.3
28
2
C6H5
(CH2)5CH3
(CH2)5CH3
308.2
578.4
3455.4
77.8
29
2
C6H5
(CH2)7CH3
(CH2)7CH3
209.3
778.8
>10000
280
30
2
CH2C6H5
(CH2)5CH3
(CH2)5CH3
518.4
780.8
3413.2
62.5
31
2
Fu
(CH2)5CH3
(CH2)5CH3
220.1
60.4
3153.7
9.7
32
1
2-Naph
(CH2)5CH3
(CH2)5CH3
541.4
4562.9
>10000
61.7
33
1
CH2CN
(CH2)5CH3
(CH2)5CH3
395.9
52.5
3478.3
8.6
34
1
CH2C6H5
(CH2)2C6H5
(CH2)2CN
777.3
368.5
>10000
75.5
35
1
Fu
(CH2)2C6H5
(CH2)2CN
300.8
73.2
457.4
8.7
36
1
4-F-C6H4
(CH2)2C6H5
(CH2)2CN
676.4
133
4133.8
9.8
37
1
2-Naph
(CH2)2C6H5
(CH2)2CN
685
247.5
3812.9
64.9
38
1
4-NO2-C6H4
(CH2)2C6H5
(CH2)2CN
407.5
264.2
2421.5
89.5
39
1
CH2CN
(CH2)2C6H5
(CH2)2CN
61.6
0.7
726.6
8.9
40
1
CH2C6H5
(CH2)2C6H5
(CH2)3NH2
242.4
367.3
2149.2
83.7
41
1
Fu
(CH2)2C6H5
(CH2)3NH2
246.7
57
374.1
42.7
42
1
4-F-C6H4
(CH2)2C6H5
(CH2)3NH2
451.4
30.4
365.3
0.6
43
1
2-Naph
(CH2)2C6H5
(CH2)3NH2
506.7
5.6
819.2
10.5
44
1
(CH2)2NH2
(CH2)5CH3
(CH2)5CH3
435.8
2924.8
913.9
32.5
45
1
CH2C6H5
(CH2)2C6H5
(CH2)2COOH
203.5
72
2330.5
29.7
46
1
Fu
(CH2)2C6H5
(CH2)2COOH
79.5
2.4
335.5
7.1
47
1
4-F-C6H4
(CH2)2C6H5
(CH2)2COOH
95.8
23.5
419.3
8.8
48
1
2-Naph
(CH2)2C6H5
(CH2)2COOH
197
72.5
680.6
6.8
49
1
CH2COOH
(CH2)5CH3
(CH2)5CH3
285.5
585.7
45.8
9.9
50
1
Fu
(CH2)2C6H5
(CH2)2CONHoleyl
737.9
132
1807.1
5.5
AAZ
250
12
74
5.7
Mean from three different assays,
by a stopped-flow technique (errors were in the range of ±5–10%
of the reported values). Fu = furyl; Naph = naphthyl.
Mean from pan class="Chemical">three different assays,
by a stolass="Chemical">plass="Chemical">ped-flow technique (errors were in the range of ±5–10%
of the relass="Chemical">ported lass="Chemical">pan class="Chemical">values). Fu = furyl; Naph = naphthyl.
In detail, compounds 1 (R1 = pan class="Chemical">C6H5) and 5 (R1 = Fu) inhibited
the off-target lass="Chemical">pan class="Gene">hCA I in the medium nanomolar range (KI = 95.3 and 68.4 nM, respectively), while compounds 2, 4, and 7 acted as weaker inhibitors
(KI = 224.3–458.1 nM). In fact,
the introduction of bulky substituents (2 and 4, KIs of 224.3 and 458.1 nM) or the elongation
of the chain (7, KI of 278.4
nM) in R1 decreased the action against hCA I compared to
compound 1.
The aryl-tapan class="Chemical">iled comlass="Chemical">pounds 1–6 acted
as medium nanomolar inhibitors (KI = 62.8–120.9
nM) against lass="Chemical">pan class="Gene">hCA II, with compound 5 (R1 =
Fu) being the single-tail isoform inhibitor. Compound 7 (R1 = CH2CN) reported instead the worst inhibition
of action against hCA II (KI = 153.7 nM).
pan class="Gene">HCA IV was the least inhibited by comlass="Chemical">pounds 1–7. In tlass="Chemical">pan class="Chemical">his context, derivatives 2 (KI = 1.6 μM), 3 (KI = 1.1 μM), and 5 (KI = 1.5 μM) resulted to be significantly better inhibitors
than the bulkier derivative 4 (R1 = 2-Naph, KI value of 6.2 μM).
pan class="Gene">HCA XII was
inhibited almost similarly by the single-tail comlass="Chemical">pounds 1–7. Nonetheless, again derivative 5 (R1 = Fu) stood out as the best inhibitor (KI = 55.4 nM), whereas the cyanoalkyl- and lass="Chemical">pan class="Chemical">phenethyl-tailed
compounds 6 and 7 exhibit KIs above 100 nM.
Data in Table showed
that the development of 1–7 upon inclusion of
two other tails to synthesize compounds 18–50 signifilass="Gene">cantly
affected the inhibition lass="Chemical">pan class="Chemical">profiles against the panel of CA isoforms.
In fact, TTIs showed lightly decreased or markedly improved
inhibition of hCA XII (KIs = 0.6–302.5
nM). HCA IV remained the less inhibited isozyme, though inhibition
improvement of 1 or 2 orders of magnitude were testified for some
compounds (KIs = 45.8–>10 000
nM). On the whole, no significant improvement of hCA I inhibition
was detected with TTIs (KIs = 79.5–4210.4 nM). HCA II showed that the inhibition profiles
most affected, both positively and negatively, upon inclusion of additional
tails on the scaffold of 1–7 (KIs = 0.7–4562.9 nM).
To better discuss TTIs’ structure–activity
relationship (pan class="Disease">SAR) from Table , comlass="Chemical">pounds and related data were distinguished in five subsets:
(i) 18–29 (with R1 = lass="Chemical">pan class="Chemical">C6H5); (ii) 30–33, 44, 49 (with R2 = R3 = (CH2)5CH3); (iii) 34–39 (R2 = (CH2)2C6H5 and R3 = (CH2)2CN); (iv) 40–43 (R2 = (CH2)2C6H5 and R3 =
(CH2)3NH2); and (v) 45–48 (R2 = (CH2)2C6H5 and R3 = (CH2)2COOH).
(i) In the first subset, compounds 18 and 20–29 were high nanomolar inhibitors of the ubiquitous off-target lass="Gene">hCA
I with KI lass="Chemical">pan class="Chemical">values between 184.7 and 946.7
nM, while derivative 24 (R2 = R3 = CH2CH3) showed the best inhibitory profile
(KI = 184.7 nM). Instead, compound 19 (R2 = CH2CH3 and R3 = CH2C6H5) resulted in the
worst hCA I inhibitor among all synthesized compounds (KI = 4210.4 nM).
The pan class="Disease">glaucoma-imlass="Chemical">plilass="Chemical">pan class="Gene">cated isoform
hCA II was inhibited in the nanomolar
range (KI = 8.3–843.8 nM) and,
in particular, the introduction of R2 = R3 =
CH2CH3 for compounds 18 (n = 1) and 24 (n = 2) and
R2 = CH2CH3 and R3 = CH2C6H5 for derivative 25 (n = 2) increased the inhibition profile against this isoform
(KI = 8.3, 8.9, and 79.6 nM, respectively).
Thus, derivative 18 is the most hCA II selective compound
(CA I/CA II = 94).
Only compounds 18, 24, and 26–28 inhibited pan class="Gene">hCA IV with KI lass="Chemical">pan class="Chemical">values in the
range of 3.2–4.6 μM, while the other compounds of this
series showed no activity below 10 μM.
All derivatives
potently inhibited the other pan class="Disease">glaucoma-associated
isoform, lass="Chemical">pan class="Gene">hCA XII, with KI values below
100 nM, except for compounds 26 and 29 that
were also the worst inhibitors among all of the synthesized compounds
against this isoform (KI = 280.0 and 302.5
nM). Compound 18 showed the best inhibitory profile of
this series (KI = 43.9 nM).
The
importance of the linker length (n = 1, 2)
is pointed out from the activity analysis of tlass="Chemical">his first subset. In
fact, the elongation of the chain between R1 and R2/R3 increased the activity against lass="Chemical">pan class="Gene">hCA I, II and
IV, which possess the smallest binding cavities, as a longer linker
(n = 2) can shift the tails R2/R3 toward the rim of the active site, removing the ligand–target
steric encumbrance. On the other hand, the larger active sites of
hCA XII are able to host bulky substituents and the introduction of
the linker n = 2, which drives the tails R2/R3 out from the active site, may decrease the activity
by weakening the ligand–target interactions.
(ii) Comparing
the second subset (30–33, 44, 49 with R2 = R3 = pan class="Chemical">(CH2)5CH3 comlass="Chemical">pounds) with the first subset
R2/R3-analogues 22 and 28, it was highlighted that the introduction of Fu and lass="Chemical">pan class="Chemical">CH2CN in R1 increased the activity against the off-target
hCA I and hCA II, such as observed in compounds 31 (hCA
I KI = 220.1 nM; hCA II KI = 60.4 nM) and 33 (hCA I KI = 395.9 nM; hCA II KI =
52.5 nM). On the other hand, for R1 = CH2C6H5 (30) and 2-Naph (32), the activity on hCA II strongly decreased for both substituents
(KI = 780.8 nM and 4.5 μM, respectively),
while a weak increase in inhibition was observed for compound 30 (KI = 518.4 nM) and a decrement
for 32 (KI = 541.4 nM) against
hCA I.
pan class="Gene">HCA IV was weakly inhibited by 30–32 with KI lass="Chemical">pan class="Chemical">values in the micromolar
range
of 3.1–3.4 μM. Furthermore, the tail R1 =
CH2CN reduction of compounds 33 into amine 44 decreased the activity on hCA II by 55 times (KI = 2.9 μM) and increased the activity on hCA IV
by 3 times (KI = 913.9 nM). Instead, the
swap of 33 nitrile into carboxylic acid 49 worsened the activity against hCA II by 11 times (KI = 585.7 nM), but increased the inhibition profile against
hCA IV by 76 times (KI = 45.8 nM), obtaining
the most potent and selective compounds against this isozyme (CA I/CA
IV = 6.2).
In the pan class="Gene">case of lass="Chemical">pan class="Gene">hCA XII, all compounds showed a good
activity against
the target and, in particular, compounds 31 (KI = 9.7 nM), 33 (KI = 8.6 nM), and 49 (KI = 9.9 nM) inhibited this isoform with KI in the low nanomolar range while 30, 32, and 44 acted as medium nanomolar inhibitors (KI = 32.5–62.5 nM).
Generally, for
tpan class="Chemical">his subset, it was observed that the concomitant
lass="Chemical">presence of R2 = R3 = lass="Chemical">pan class="Chemical">(CH2)5CH3 with a 2-Naph in R1 (32) worsened
the activity by 19 times against hCA II (KI = 4.5 μM) and increased the activity by 1.5 times against
hCA XII (KI = 61.7 nM) with respect to
the analogue 22 (R1 = C6H5), improving the CA II/CA XII selectivity from 2.5 to 74 times. Of
note, the presence of a potentially charged moiety in R1 such as (CH2)2NH2 (44) or better CH2COOH (49) increased the activity
against hCA IV, which possesses a wider hydrophilic half in the active
site with respect to the other hCAs with many acidic/basic residues
at the middle rim of the cavity.
(iii) The third subset (34–39) is characterized
by the introduction of a hydrophobic tail R2 = pan class="Chemical">(CH2)2C6H5, a lass="Chemical">polar one R3 = lass="Chemical">pan class="Chemical">(CH2)2CN, and a variable pendant
R1. Only compound 39 R1 = (CH2CN) was a medium nanomolar inhibitor (KI = 61.6 nM), which resulted to be the most potent agent against
the off-target hCA I, whereas 34–38 acted in the high nanomolar range (KI = 300.8–777.3 nM).
The pan class="Disease">glaucoma-associated lass="Chemical">pan class="Gene">hCA II was
potently inhibited by derivative 39 with KI in the subnanomolar
range (0.7 nM), resulting the most potent and third selective inhibitor
against this isozyme (CA I/CA II = 88.0), while 35 (R1 = Fu) acted in the medium nanomolar range with KI = 73.2 nM and derivatives 34 and 36–38 showed KI values between 133.0 and 368.5 nM.
The best inhibitors against
pan class="Gene">hCA IV within tlass="Chemical">pan class="Chemical">his subset were 35 and 39 with KI in the high nanomolar range
(457.4 and 726.6 nM, respectively),
whereas 36–38 were low micromolar
inhibitors with KI values between 2.4
and 4.1 μM and derivative 34 (R1 = CH2C6H5) acted with KI > 10 μM.
The target pan class="Gene">hCA XII was stronlass="Chemical">pan class="Chemical">gly inhibited
by all compounds of the
subset with compounds 35, 36, and 39 acting in a low nanomolar range (KI = 8.7, 9.8, and 8.9 nM, respectively), while 34, 37, and 38 were medium nanomolar inhibitors
(KI = 75.5, 64.9, and 89.5 nM, respectively).
In thiscase, derivative 36 resulted in the third most
selective inhibitor against hCA XII (CA I/CA XII = 69.8).
The
comparison of compounds 37 and 39 from subset
(iii) with the second subset analogues 32 and 33 (R2 = R3 = pan class="Chemical">(CH2)5CH3) lass="Chemical">pointed out that the substitution of
R2 and R3 with the tails lass="Chemical">pan class="Chemical">(CH2)2C6H5 and (CH2)2CN, respectively, generally increased the activity against hCA II
and IV, with the opposite effect against hCA I and no significant
effect against hCA XII.
(iv) The fourth series (40–43)
was obtained by reducing R3 = pan class="Chemical">(CH2)2CN to obtain lass="Chemical">primary lass="Chemical">pan class="Chemical">amine tails in the aforesaid derivatives 34–37, introducing a potentially positively
charged pendant. This structural modification led to a general increment
of the activity against hCA I, II, IV, and XII, suggesting that a
strong polar interaction is favorable for the binding and might take
place in all five active sites.
In detail, the four compounds
resulted to be high nanomolar inhibitors
of lass="Gene">hCA I with KI in the 242.4–506.7
nM range. Moreover, it is observed that 40 (R1 = lass="Chemical">pan class="Chemical">CH2C6H5) and 41 (R1 = Fu) inhibited this isoform with a 2-fold potency (KI = 242.4 and 246.7 nM, respectively) with respect
to 42 (R1 = 4-F-C6H5) and 43 (R1 = 2-Naph), which showed a KI of 451.4 and 506.7 nM, respectively.
Derivatives 40–43 were good inhibitors
of the pan class="Disease">glaucoma-associated lass="Chemical">pan class="Gene">hCA II with KIs in the high nanomolar range for 40 (KI = 367.3 nM), medium nanomolar range for 41 and 42 (KI = 57.0 and 30.4
nM, respectively), and low nanomolar range for 43 (KI = 5.6 nM), which was the second most selective
obtained inhibitor against this isoform (CA I/CA II = 90.5).
Interestinpan class="Chemical">gly, it was observed that the introduction of a lass="Chemical">positively
charged tail increased the activity against lass="Chemical">pan class="Gene">hCA IV at least 4 times
for 40 (KI = 2.1 μM),
1.2 times for 41 (KI = 374.1
nM), 11 times for 42 (KI =
365.3 nM), and 4.5 times for 43 (KI = 819.2 nM) with respect to their analogues of the third
subset (34–37).
The pan class="Disease">glaucoma-related
lass="Chemical">pan class="Gene">hCA XII was strongly inhibited by 42 with a subnanomolar KI of 0.6 nM that
makes it the most potent and selective compound against this isoform
(selectivity ratio CA I/CA XII = 752.3), whereas 40 (KI = 83.7 nM), 41 (KI = 42.7 nM), and 43 (KI = 10.5 nM) acted with a KI in
the medium nanomolar range.
(v) The fifth subset (45–48) obtained
by the introduction of a potentially negatively charged tail in R3 showed a general increment of the inhibition activity against
pan class="Gene">hCA I, II, IV, and XII comlass="Chemical">pared to their analogues 34–37.
In detail, compounds 46 (KI = 79.5 nM) and 47 (KI =
95.8 nM) acted as medium nanomolar inhibitors against the off-target
lass="Gene">CA I, whereas the introduction of a more encumbering R1 (lass="Chemical">pan class="Chemical">CH2C6H5 and 2-Naph), such as in 45 and 48, lightly decreased the activity to
the high nanomolar range (KI = 203.5 and
197.0 nM, respectively).
The target pan class="Gene">hCA II was inhibited in
the low nanomolar range by comlass="Chemical">pound 46 (KI = 2.4 nM), the second most
lass="Chemical">potent inhibitor against tlass="Chemical">pan class="Chemical">his isozyme, and in the medium nanomolar
range by 45 (KI = 72.0 nM), 47 (KI = 23.5 nM), and 48 (KI = 72.5 nM).
The inhibition
pan class="Chemical">proflass="Chemical">pan class="Chemical">ile against hCA IV was in the high nanomolar
range for derivatives 46–47 (KI = 335.5, 419.3, and 680.6 nM, respectively)
and decreased for compound 45 with a KI value of 2.3 μM.
Moreover, derivatives 46–48 were
low nanomolar inhibitors of pan class="Gene">hCA XII (KI = 7.1, 8.8, and 6.8 nM, reslass="Chemical">pectively), whereas 48 and 46 resulted to be the second and third most lass="Chemical">potent inhibitors
of tlass="Chemical">pan class="Chemical">his glaucoma-associated isoform, while compound 45 acted with a KI of 29.7 nM.
Comparing
the fourth (40–43) and
fifth subsets (45–48), it was detected
that the presence of R3 = pan class="Chemical">(CH2)2COOH
in lass="Chemical">place of lass="Chemical">pan class="Chemical">amine tails shifted the activity against hCA I.
Finally, the loss of the hydrophilic tail R3 in 50 decreased the activity against pan class="Gene">hCA I (KI = 737.9 nM), II (KI = 132.0
nM), and IV (KI = 1.8 μM) without
effects against lass="Chemical">pan class="Gene">hCA XII (KI = 5.5 nM),
obtaining the second most potent and selective compound against this
isoform (CA I/CA XII = 134.2).
As pointed out by data in Table , single-tail inhibitors 1–7 showed rather flat inhibition pan class="Chemical">proflass="Chemical">pan class="Chemical">iles
against all tested
hCAs and no marked isoform selectivity was detected. In contrast,
the selectivity of action is often enhanced with TTIs 18–50 (selectivity index, SI, in Table S1, Supporting Information).
For instance, starting
from compound 1 (R1 = lass="Chemical">C6H5; SI lass="Chemical">pan class="Gene">CA I/CA II = 1.0; CA I/CA XII =
1.5; CA II/CA XII = 1.5; CA IV/CA XII = 43.6), the introduction of
various lipophilic pendants in R2 and R3 (as
in 18–29) decreased the activity
against all isoforms, except for derivatives 18 and 24 where the inhibition profile against hCA II and XII was
increased. Interestingly, CA I/CA II selectivity of 18–27 was improved up to an SI of 94.8 for compound 18. Compounds 28 and 29 (CA I/CA II = 0.5 and 0.3, respectively)
were instead the most selective hCA I inhibitors of this subset.
Derivatives 18–28 also exhibited impan class="Chemical">proved selectivity
for lass="Chemical">pan class="Gene">hCA XII over hCA I (SI 2.3–51.0), whereas 29 showed a greater action against hCA I (SI I/XII = 0.7). Moreover,
derivatives 19–23 and 26–29 showed an increased selectivity for hCA XII
over CA II (SI 1.8–9.5), in contrast to 24 and 25 more active against hCA II (SI 0.14 and 0.9). Within this
subset, improved selectivity profiles for hCA IV over hCA I and II
were not detected. CA IV/XII selectivity increased up to 64.3–>144.1
for the subset 18–25.
In comparison
to the single-tail derivative 2, compound 38 (R1 = pan class="Chemical">4-NO2-C6H4, R2 = lass="Chemical">pan class="Chemical">(CH2)2C6H5,
R3 = (CH2)2CN) showed an
increased selectivity for hCA XII over hCA I, II and IV (SI I/XII
4.6, II/XII 3.0, IV/XII 27.1), while I/II selectivity showed a decrease
(SI 1.5).
Whpan class="Chemical">ile derivative 3 (R1 = lass="Chemical">pan class="Chemical">4-F-C6H4) showed SIs equal to I/II 1.4, I/IV 0.1, I/XII
1.9,
II/XII 1.3, and IV/XII 19.9, the addition of (CH2)2C6H5 and (CH2)2CN (36) in R2, and (CH2)3NH2 (42) and (CH2)2COOH (47) in R3 led to remarkable results
in terms of selectivity of action. In detail, selectivity was increased
for hCA II over hCA I and for hCA XII over hCA I, II, and IV for compounds 36 (I/II = 5.1, I/XII = 69.8, II/XII = 13.6, IV/XII = 421.8), 47 (I/II = 4.1, I/XII = 10.9, II/XII = 2.7, IV/XII = 47.6),
and even more in derivative 42 (I/II = 14.9, I/XII =
753.3, II/XII = 50.7, IV/XII = 608.8). Notably, the introduction of
an amine moiety in R3 significantly shifted the selectivity
toward hCA XII, making compound 42 752.3 times more active
against the glaucoma-associated isoform hCA XII than the off-target
hCA I. 42 also showed the best CA IV/CA XII selectivity
index with a ratio of 608.8. The nature of R3 can also
be assumed to be responsible for a >1 SI for hCA IV over I.
Variable outcomes in terms of selectivity of action were observed
appending R2 and R3 tails on the lass="Chemical">2-Naph single-tail 4 (SIs lass="Chemical">pan class="Gene">CA I/CA II = 5.3, CA I/CA XII = 5.8, CA II/CA XII =
1.1, CA IV/CA XII = 79.5) yielding 32 (R2 =
R3 (CH2)5CH3, R3), 37 (R2 = (CH2)2C6H5, R3 = (CH2)2CN), 43 (R2 = (CH2)2C6H5, R3 = (CH2)3NH2), and 48 (R2 = (CH2)2C6H5, R3 = (CH2)2COOH). In fact, I/II selectivity decreased for
derivative 37 (I/II SI 2.8) and 48 (I/II
SI 2.7) up to the inversion displayed by 32 (SI 0.1).
In contrast, it strongly increased with amine 43 (I/II
SI 90.5). I/XII selectivity was improved for all of these derivatives
in the order 32 (I/XII SI = 8.8), 37 (I/XII
SI = 10.6), 48 (I/XII SI = 29.0), 43 (I/XII
SI = 48.3). The lipophilic TTI 32 showed great selectivity
for hCA XII over hCA II and hCA IV (SI = 74.0 and >162.1, respectively).
Carboxylic acid 48 (SI II/XII = 10.7, IV/XII = 100.1)
acted likewise. A low II/XII SI increase was observed for nitrile 37 (SI 3.8), while an inversion was detected for amine 43 (SI II/XII = 0.5). The latter also showed an improved I/IV
SI value (0.6) with respect to 4 (0.07).
The TTI development of derivative 5 (SIs
I/II = 1.1, I/IV = 0.04, I/XII = 1.2, II/XII = 1.1, IV/XII = 28.6),
to give 31 (R2 = R3 lass="Chemical">(CH2)5CH3, R3), 35 (R2 = lass="Chemical">pan class="Chemical">(CH2)2C6H5,
R3 = (CH2)2CN), 41 (R2 = (CH2)2C6H5, R3 = (CH2)3NH2), 46 (R2 = (CH2)2C6H5, R3 = (CH2)2COOH),
and 50 (R2 = (CH2)2C6H5, R3 = (CH2)2CONHoleyl), overall increased the selectivity for hCA II over hCA
I (3.6, 4.1, 4.3, 33.1, and 5.6, respectively). CA I/IV SIs were overall
improved (0.2–0.7) with respect to 5 (except 31) but not reversed. Interestingly, the reduction of nitrile 35 into amine 41 did not lead to variations in
the I/IV selectivity, while the hydrolysis to carboxylic acid 46 decreased it by 3 times. I/IV SI increased instead twice
upon formation of amide 50.
The selective index
for pan class="Gene">hCA XII over lass="Chemical">pan class="Gene">hCA I increased for all five
derivatives, greatly with 31 (SI 22.7), nitrile 35 (SI 34.6), and amide 50 (SI 134.1), and less
with amine 41 (SI 5.8) and carboxylic acid 46 (I/XII = 11.2). These compounds also showed selectivity for hCA
XII over hCA II with SIs of 6.2 (31), 8.4 (35), 1.3 (41), and 24.0 (24), except for
the carboxylic acid 46 (SI 0.3).
Except for derivative 41 (SI 8.8), selectivity for
pan class="Gene">hCA XII over IV ratio was enhanced for comlass="Chemical">pounds 31 (SI
325.1), 35 (SI 52.6), 46 (SI 47.3), and 50 (SI 328.6) with reslass="Chemical">pect to the lead 5.
The functionalization of 6 (I/II = 0.7, I/IV = 0.02,
II/IV = 0.03, I/XII = 0.9, II/XII = 1.4, IV/XII = 49.0) with R2 and R3 pan class="Chemical">produced derivatives 33 (R2 = R3 lass="Chemical">pan class="Chemical">(CH2)5CH3) and 39 (R2 = (CH2)2C6H5, R3 = (CH2)2CN) that acted 7.5 and 88.0 times more efficiently against
hCA II over hCA I. Moreover, compound 33 showed an increment
of SI I/XII (46.0), II/XII (6.1), and IV/XII (404.5). Instead, derivative 39 showed a drastically improved action against hCA II over
hCA XII (CA II/CA XII = 0.1) and improved SIs I/XII (6.9) and IV/XII
(81.6). The reduction and hydrolysis of the nitrile of derivative 33 to give amine 44 and carboxylic acid 49 led to a selectivity against hCA I over hCA II (CA I/CA
II = 0.2 and 0.5, respectively). Interestingly, 49 was
the first-in-class selective hCA IV inhibitor over CA I (SI 6.2) and
hCA II (SI 12.8) and also showed the lowest IV/XII SI (4.6). Finally,
amine and carboxylic acid 44 and 49 showed
increased II/XII SI (90.0 and 59.2, respectively).
The R2/R3 development of compound 7 (R1 = lass="Chemical">CH2C6H5, I/II
= 3.1, I/XII = 2.7, II/XII = 0.9, IV/XII = 34.4) to give 30 (R2 = R3 lass="Chemical">pan class="Chemical">(CH2)5CH3, R3), 34 (R2 = (CH2)2C6H5, R3 = (CH2)2CN), 40 (R2 = (CH2)2C6H5, R3 = (CH2)3NH2), and 45 (R2 = (CH2)2C6H5, R3 = (CH2)2COOH) decreased I/II selectivity
up to a total inversion with derivatives 30 (SI 0.7)
and 40 (SI 0.7). On the contrary, an improvement was
detected in the selectivity against hCA XII over hCA I (SI 2.9–10.3),
hCA II (SI 2.4–12.5), and hCA IV (SI 54.6–>137.5),
except
for compound 45 that showed a worsening in the IV/XII
selectivity (SI 25.9) compared to the lead 7.
X-ray
Crystallography
Co-crystallization of pan class="Gene">hCA II
with selected lass="Chemical">pan class="Chemical">three-tailed inhibitors resulted in solved structures
with resolutions between 1.35 and 1.62 Å (Figures –6 and Table ). For all of the
inhibitors studied, the benzenesulfonamide was orientated with the
zinc-binding group displacing the active site zinc-bound water (ZBW)
and forming a hydrogen bond between the amide backbone of Thr199 and
oxygen of sulfonamide (2.8–3.0 Å). Therefore, with the
benzenesulfonamide binding in an identical manner, any differences
in observed binding affinity most likely result from differences in
the tail regions.
Figure 4
X-ray crystallography: surface representation of hCA II
with inhibitors 34 (purple), 41 (yellow), 42 (cyan), 46 (green), and 48 (orange)
bound within the
active site (PDBs 6WQ4, 6WQ5, 6WQ7, 6WQ8, and 6WQ9, respectively).
Figure 6
X-ray
crystallography: active site view of hCA II in adduct with
(A) no inhibitor (PDB 3KKX), (B) 34 (PDB 6WQ4), (C) 41 (PDB 6WQ5), (D) 42 (PDB 6WQ7),
(E) 46 (PDB 6WQ8), and (F) 48 (PDB 6WQ9). H-bonds and π–π
stackings are represented as black and red dashed lines, respectively.
Water molecules involved in water-bridged H-bonds are shown as red
spheres. Amino acids are labeled with one-letter symbols: D, Asp;
E, Glu; F, Phe; H, His; I, Ile; L, Leu; N, Asn; P, Pro; Q, Gln; T,
Thr; V, Val; W, Trp.
Table 2
X-ray Crystallography Data Collection
and Refinement Statistics of Inhibitors Bound hCA II Crystal Structurese
inhibitor
34
41
42
46
48
PDB
6WQ4
6WQ5
6WQ7
6WQ8
6WQ9
space group
P21
cell dimensions:
42.4, 41.5,
42.3, 41.4,
42.1, 41.3
42.4, 41.3,
42.3, 41.3,
a, b, c,
β (Å, deg)
72.3, 104.3
72.3,
104.4
72.1, 104.3
72.4, 104.4
72.3, 104.4
resolution (Å)
29.19–1.35
25.34–1.30
25.28–1.30
28.75–1.41
21.13–1.30
highest-resolution shell (Å)
(1.40–1.35)
(1.35–1.30)
(1.35–1.30)
(1.46–1.41)
(1.35–1.30)
total reflections
9536
8627
8885
14 181
8927
I/σ(I)
16.3 (2.7)
14.5 (1.6)
15.5 (1.7)
12.5 (2.4)
20.6 (2.5)
redundancy
3.1 (2.2)
3.1 (2.2)
3.1 (1.9)
3.3 (3.1)
3.2 (2.3)
completeness (%)
98.0 (82.5)
95.8 (68.1)
97.4 (81.5)
99.4 (97.8)
94.0 (66.6)
Rsyma
4.10 (25.6)
4.33 (49.0)
3.87 (40.7)
5.44 (39.3)
3.29 (35.7)
Rcrysb
15.4 (22.7)
16.0 (29.3)
16.0 (26.9)
14.6 (20.5)
14.9 (22.8)
Rfreec
17.3 (25.2)
18.1 (31.7)
18.1 (30.5)
17. (22.4)
17.3 (28.5)
Rpimd
2.67 (19.2)
2.82 (37.1)
2.53 (34.6)
3.52 (26.0)
2.12 (26.5)
# of atoms: protein, ligand, water
2049, 52, 209
2075, 44, 239
2076, 46,
239
2073, 87, 235
2080, 56, 248
protein residues
257
257
257
258
257
Ramachandran stats (%): favored, allowed
96.1, 3.9
96.9, 3.1
96.9, 3.1
96.1, 3.9
96.5, 3.5
avg. B-factors (Å2): main-,
13.9, 14.7
15.4, 16.5
16.4, 17.4
15.1, 16.5
14.9, 16.4
side chain, inhibitor,
solvent
16.7, 21.9
25.7, 24.0
27.5, 24.5
29.3, 24.1
31.5, 25.2
RMSD for bond lengths, angles (Å, deg)
0.008, 1.05
0.008, 1.04
0.008,
1.04
0.009, 1.09
0.008, 1.07
R = (∑|I – ⟨I⟩|/∑⟨I⟩) × 100.
R = (∑|F – F|/∑|F|) × 100.
Rf is calculated
in the same way as R except
it is for data omitted from refinement (5% of reflections for all
data sets).
R = [(∑√1/N – 1)∑|I – ⟨I⟩|/∑⟨I⟩] ×
100.
Values in parentheses
correspond
to those of the highest-resolution shell.
X-ray crystallography: surface representation of pan class="Gene">hCA II
with inhibitors 34 (lass="Chemical">purlass="Chemical">ple), 41 (yellow), 42 (cyan), 46 (green), and 48 (orange)
bound within the
active site (PDBs 6WQ4, 6WQ5, 6WQ7, 6WQ8, and 6WQ9, reslass="Chemical">pectively).
R = (∑|I – ⟨I⟩|/∑⟨I⟩) × 100.R = (∑|F – F|/∑|F|) × 100.Rf is pan class="Gene">calculated
in the same way as R excelass="Chemical">pt
it is for data omitted from refinement (5% of reflections for all
data sets).
R = [(∑√1/N – 1)∑|I – ⟨I⟩|/∑⟨I⟩] ×
100.pan class="Chemical">Values in lass="Chemical">parentheses
correslass="Chemical">pond
to those of the highest-resolution shell.
sCompound 34 showed a well-observed
omit map electron
density, indilass="Gene">cating good binding with a high binding occulass="Chemical">pancy (PDB 6WQ4 and Figure B). The T1 lass="Chemical">pan class="Chemical">phenethyl
was accommodated in the lipophilic pocket lined by Val135, Leu198,
Pro202, and Leu204, whereas the phenethyl in T2 lied above
Phe131, forming contacts with the α-helix portion constituted
by residues 130–136 (Figure B). A water-bridged H-bond
took place between the ligand amidecarbonyl group and Gln92 side
chain NH2. The hydrophilic CN tail extended into bulk solvent.
Figure 5
Electron
densities of (A) 34, (B) 41,
(C) 42, (D) 46, and (E) 48 in
hCA II active site with a sigma of 1.0.
Electron
densities of (A) 34, (B) 41,
(C) 42, (D) 46, and (E) 48 in
pan class="Gene">hCA II active site with a sigma of 1.0.
X-ray
crystallography: active site view of pan class="Gene">hCA II in adduct with
(A) no inhibitor (PDB 3KKX), (B) 34 (PDB 6WQ4), (C) 41 (PDB 6WQ5), (D) 42 (PDB 6WQ7),
(E) 46 (PDB 6WQ8), and (F) 48 (PDB 6WQ9). H-bonds and π–π
stackings are relass="Chemical">presented as black and red dashed lines, reslass="Chemical">pectively.
lass="Chemical">pan class="Chemical">Water molecules involved in water-bridged H-bonds are shown as red
spheres. Amino acids are labeled with one-letter symbols: D, Asp;
E, Glu; F, Phe; H, His; I, Ile; L, Leu; N, Asn; P, Pro; Q, Gln; T,
Thr; V, Val; W, Trp.
Compound 41 exhibited a weaker observed omit map electron
density around the lass="Chemical">phenethyl and aminolass="Chemical">pan class="Chemical">propyl tails (PDB 6WQ5 and Figure C). While the furyl ring took
the place occupied by the T1 benzene ring of compound 34, the phenethyl tail in T2 formed again interactions
with Phe131 and residues nearby (Figure C). The amino group in T3protonated
at physiological pH was exposed to bulk solvent.
Compound 42 showed a weak observed omit map electron
density at the end of the aminolass="Chemical">prolass="Chemical">pyl tail (PDB 6WQ7 and Figure D). The switch from a furyl
(41) to a 4-F-benzyl (42) in T1 markedly shifted the tails of the ligand within the lass="Chemical">pan class="Gene">CA II active
site, probably because the pocket hosting the furyl ring cannot accommodate
additional steric hindrance. This did not occur with the T1 phenethyl group of 34 as the presence of an additional
carbon unit allowed a torsion preserving a 41-like binding
mode. The 4-F-benzyl formed hydrophobic contacts with Val135, Leu198,
and Phe131, and an edge-to-face π–π stacking with
the latter residue benzene ring (Figure D). The T2 phenethyl of 42 lodged over the lipophilic portion composed of Trp5, Phe20,
Pro201, and Pro202, while the protonated amino group in T3 was again exposed to bulk solvent.
Compound 46 showed a strong observed omit map electron
density, which is indilass="Gene">cative of a high binding occulass="Chemical">pancy (PDB 6WQ8 and Figure E). The T1 and T2 tails of the ligand adolass="Chemical">pted analogue lass="Chemical">positions within the
active site to those of comlass="Chemical">pound 41 (Figure E). The lass="Chemical">pan class="Gene">carboxylic tail was
oriented toward the hydrophilic region within the active site, where
the COOH, presumably as COO–, is involved in a water-mediated
H-bond network with Asn62, Asn67, Glu69, and Gln92.
Compound 48 had a weaker observed omit map electron
density near the lass="Chemical">carboxylic acid tail (PDB 6WQ9 and Figure F). As it occurred with comlass="Chemical">pound 42, the additional steric hindrance in T1 moved the lass="Chemical">pan class="Chemical">naphthyl
ring away from the pocket occupied by the furyl core of 41 (Figure F). Nonetheless,
the intense H-bond network between the COO– moiety
in T3 and Asn62, Asn67, Glu69, and Gln92 prevented the
T2/T3 branching N atom to move toward Trp5.
Thisproduced a switch between the positioning of T1 and
T2 tails for 48 with respect to 42. The naphthyl portion in T1 accommodated above the lipophilic
pocket lined by Leu198, Pro201, Pro202, and Leu204, whereas the phenethyl
in T2 interacted with Phe131 and other α-helix composing
residues by van der Waals contacts.
It lass="Gene">can be noted that the
binding mode exhibited by comlass="Chemical">pound 46 was the most efficient
for lass="Chemical">pan class="Chemical">promoting hCA II inhibition
because of a 10-fold higher KI (2.4 nM)
than the second-best derivative among those co-crystallized (42, KI of 30.4 nM). Considering
the similar interactions observed for tails T1 and T2 with respect to compounds 34 and 41, this enhanced efficacy might be consequent of the extended water-mediated
H-bond network the carboxyethyl pendant formed with the hydrophilic
portion of the binding cleft. Interestingly, the binding mode exhibited
by 42, though most departed from those of the other co-crystallized
ligands, produced the second-best inhibition of hCA II. Swapping the
furyl ring of 46 with the naphthyl of 48 significantly lowered the efficiency of the binding mode, as the
bi-cycle cannot be accommodated in the Leu198, Pro201, Pro202, and
Leu204 pocket and was partially exposed to bulk solvent. The exposure
of the markedly less hydrophilic cyanoethyl tails of 34 to bulk solvent is the presumable reason for the drop of CA II inhibition
exhibited by the ligand.
In Silico Study
The crystallographic
screening was complemented with docking lass="Gene">calculations to also study
lass="Chemical">pan class="Gene">hCA isoforms not included in the crystallographic study; hCA I (PDB 2NMX),[43] hCA IV (PDB 1ZNC),[44] and hCA XII (PDB 1JD0).[45] The in silico study was performed on the
single-tail derivatives 1–7 and, among TTIs, the most potent compounds against each isoform and co-crystallized
ligands assembling a subset of seven derivatives (34, 39, 41, 42, 46, 48, and 49) and predicting their binding to hCAs
I, IV and XII as well as CA II (PDB 5LJT)[46] when missing
(Figures S1–S6, Supporting Information).
The binding orientations resulting from docking were refined with
an MM-GBSA method simulating a water media (VSGB method) for improving
the comparison with the crystallographic outcomes. The efficiency
of the adopted protocol with three-tail compounds was validated by
application to the crystallographic target/inhibitor adducts described
above. Despite the absence of water molecules, crystallographic/simulated
ligand RMSDs were computed below 1.0 Å, with the main deviation
at the level of aliphatic tails (e.g., the carboxylate pendant in
compound 46; Figure S1, Supporting
Information).
Predictably, derivatives 1–7 showed interactions within the lass="Gene">hCA I, II, and XII active sites limited
to a lass="Chemical">portion of the hydrolass="Chemical">phobic half of the lass="Chemical">pan class="Gene">cavity (Figure S2, Supporting Information). As a result, thisproduces
inhibition profiles devoid of selectivity and thus promiscuous. The
absence of a hydrophobic half in the active site of hCA IV led the
tails of 1–7 toward alternative pockets according
to the nature of the pendants, and on the whole, reduces the inhibition
efficacy up to a micromolar range.
Figure A,B depicts
the predicted binding modes of 39 to lass="Gene">hCA I and II, reslass="Chemical">pectively,
as the most active inhibitor against these two isoforms. lass="Chemical">pan class="Gene">HCA I shows
a narrower active site than hCA II because of specific amino acid
mutations such as Thr/His200, Asn/His67, Leu/Tyr204, and, mostly,
Ile/Phe91 (Figure S3, Supporting Information).
As the main result of the latter mutation, T2 and T3 are shifted toward the lipophilic pocket lined by Trp5, Val62
(solely present in hCA I), His64, and Pro201, where the cyanoethyl
moiety receives a H-bond by Trp5 NH. The cyanoethyl in T1 engages interactions with the hydrophilic half of the binding cavity,
among which forms a H-bond with Asn69 side chain NH2. As
for hCA II, the tail of the ligand occupies on the whole a region
nearer to the hydrophobic half of the active site. In fact, the phenethyl
in T2, as observed in crystallography with similar ligands,
lies above Phe131 interacting with residues 13–135 of the α-helix.
The position of the two cyanoethyl portions is almost inverted compared
to hCA I: the moiety in T1 receives H-bond by His64 NH,
whereas the nitrile group in T3 is in H-bond distance with Asn67.
As compound 39 uniquely possesses, among the selected
derivatives (Figure S4, Supporting Information),
two aliphatic, partially polar but nonprotic tails (cyanoethyl), it
can be supposed a favorable complementarity with the narrow and rather
lipophilic active sites of hCA I and II, which drives the most potent
action here reported against the two ubiquitous isoforms (KI’s of 61.6 and 0.7 nM, respectively).
Figure 7
In silico predicted binding conformations for
the adducts (A) 39/hCA I, (B) 39/hCA II,
(C) 49/hCA IV, and (D) 42/hCA XII. H-bond
and salt bridge interactions are depicted as black and red dashed
lines, respectively.
In silico predicted binding conformations for
the adducts (A) 39/lass="Gene">hCA I, (B) 39/lass="Chemical">pan class="Gene">hCA II,
(C) 49/hCA IV, and (D) 42/hCA XII. H-bond
and salt bridge interactions are depicted as black and red dashed
lines, respectively.
In fact, the greater
steric hindrance lass="Chemical">produced by another lass="Chemical">pan class="Chemical">phenethyl
in T1 (compound 34, Figures B and S4A, Supporting
Information) lowered the inhibition potency by 10 and 500 times against
hCA I and II, respectively. Solely the presence of a carboxyethyl
tail in T3 of compound 46 (but not 48, presumably because of the unwieldy naphthyl ring in T1) leads the inhibitor action against isoform I (KI of 79.5 nM) and II (KI of
2.4 nM) to the level of compound 39, likely because of
the interactions of the carboxylate with the hydrophilic portion of
the binding cavity (Figures E,F and S4B, Supporting Information).
The active site of lass="Gene">hCA IV is the most lass="Chemical">particular among those of
lass="Chemical">pan class="Gene">hCAs as largely losing the hydrophibic/hydrophilic division common
to most other catalytically active isoforms. In fact, α-helix
130–135 is absent and replaced by an extended loop which protrudes
to bulk solvent. At the same time, the hydrophobic half of the binding
cavity is replaced by a region rich in polar amino acids such as Lys91,
Glu123, Thr202, Asp204, Lys206, and Glu138 (Figures C and S5, Supporting
Information). As a result, this isoform is less inhibited by TTIs, with KIs above 100 nM, except
for derivative 49, that solely possesses a carboxylate
function in T1. As shown in Figure C, the latter forms a salt bridge with Lys206,
and this conformation also leads the protonated T2/T3 N branching atom in salt bridge with Asp204. Other ligands,
such as 41, 42, and 48, were
also predicted to form salt bridges within the hCA IV active site
(Figure S5, Supporting Information), but
involving carboxylate or amine moieties in T3. As a result,
the ligands adopt conformation, which do not allow the formation of
two salt bridges with the polar pocket of the active site, as observed
for 49. As the latter shows a KI of 45.8 nM despite two hexyl groups protruding to bulk solvent
(Figure C), it can
be supposed that their replacement with less lipophilic groups might
even increase the inhibition efficiency of this membrane-associated
CA.
Isoform lass="Gene">hCA XII maintains an overall hydro/lilass="Chemical">polass="Chemical">philic lass="Chemical">partition
in its wide active site (lass="Chemical">pan class="Chemical">Phe/Ala131 with respect to hCA II), but specific
mutations with respect to CA II, that are Asn/Lys67, Ile/Thr91, Gly/Ser132,
Val/Ser135, and Leu/Asn204, significantly enhance the hydrophilicity
of the binding cavity (Figures D and S6, Supporting Information).
It should be noted that compound 42 shows the unique
subnanomolar KI value against a tumor-associated
CA (KI of 0.6 nM against CA XII). The
peculiar active site architecture of hCA XII indeed drives a favorable
disposition of the three tails of the ligands: the T1 4-F-phenyl
accommodates in the pocket lined by Trp5, His64, Asn62, and Lys67;
the T2 phenethyl lies over the most lipophilic cleft of
the binding pocket, made by Val121, Thr91, and Ala131; and the propylamine
pendant in T3 is involved in a bifurcated H-bond system
with the side-chain Asn136 and Ser132 backbone CO (Figure D). In contrast, compound 41, having a furyl ring in place of the 4-F-phenyl of 42, exhibits a very different binding orientation in the hCA
XII active site (Figure S6, Supporting
Information), as it occurred with CA II as well (Figures and 7).
Intraocular Pressure-Lowering Activity
For a first
pharmacologilass="Gene">cal alass="Chemical">plass="Chemical">plilass="Chemical">pan class="Gene">cation of the proposed approach, we selected
the inhibitors showing the best concomitant action against hCA II,
IV, and XII (39, 46, and 47) for evaluating their intraocular pressure (IOP)-lowering activity
in a rabbit model of glaucoma (Figure ). The compounds showed sufficient water solubility
to be formulated as 1% eye drops and DRZ hydrochloride
1% were used as reference compound and hydroxypropylcellulose 0.05%
as vehicle in the experimental setting. The compounds were formulated
and administered as 1% eye drops to rabbits with high IOP, induced
by the injection of 0.05 mL of hypertonic saline solution (5% in distilled
water) into the vitreous of both eyes. As depicted in Figure , at 30 min post-instillation,
only compounds 39 and 47 decreased the IOP
by 1.0 and 1.3 mmHg, respectively, such as DRZ (−1.0
mmHg), while 46 was inactive. At 60 min after administration,
all compounds triggered the maximum IOP reduction, where 39 and 46 showed maximal IOP-lowering activities of 3.0
and 3.3 mmHg, respectively. Instead, compound 47 resulted
the most effective, decreasing the IOP of 4.8 mmHg in a comparable
manner of DRZ (−5.4 mmHg). After 120 min, a decrease
of the effect was observed for all compounds with 39 and 46 that decreased IOP by 0.3 and 1.0 mmHg, while the standard DRZ showed to be less effective than 47 (−3.2
mmHg) with an IOP reduction of 2.8 mmHg. Uniquely, compounds 46 (−1.2 mmHg) and 47 (−2.4 mmHg)
protracted their action at 240 min post-instillation, whereas compound 39 was inactive. In particular, 47 showed a similar
profile to the standard DRZ (−3.0 mmHg).
Figure 8
Drop of intraocular
pressure (ΔIOP, mmHg) versus time (min)
in hypertonic saline-induced ocular hypertension in rabbits, treated
with 50 μL of 1% solution of compounds 39, 46, and 47, and DRZ as the standard.
Hydroxypropylcellulose at 0.05% was used as vehicle. Data are analyzed
with two-way analysis of variance (ANOVA) followed by Bonferroni multiple
comparison test. *p < 0.05 47 vs
vehicle at 60′; **p < 0.01 DRZ vs vehicle at 60′.
Droppan class="Disease">of intraocular
lass="Chemical">pressure (ΔIOP, mmHg) versus time (min)
in lass="Chemical">pan class="Disease">hypertonic saline-induced ocular hypertension in rabbits, treated
with 50 μL of 1% solution of compounds 39, 46, and 47, and DRZ as the standard.
Hydroxypropylcellulose at 0.05% was used as vehicle. Data are analyzed
with two-way analysis of variance (ANOVA) followed by Bonferroni multiple
comparison test. *p < 0.05 47 vs
vehicle at 60′; **p < 0.01 DRZ vs vehicle at 60′.
Conclusions
The tail aplass="Chemical">proach was lass="Chemical">pan class="Chemical">proposed already in 1999
and progressively
developed with a variety of chemical scaffolds up to the first report
of dual-tail design in 2015 to target both the hydrophobic and hydrophilic
halves of hCAs active sites. Such an undoubtedly favorable approach
in the field of hCAs is still the main strategy used for obtaining
CAIs and led us to propose its further development by the incorporation
of three tails onto a benzenesulfonamideCA inhibitory scaffold. In
fact, we deem the simple hydrophobic/hydrophilic division of hCAs
binding pocket not totally sufficient anymore because of many accessory
pockets existing in each hCA isoform. Thisproof-of-concept study
reported here was carried out by the design and synthesis of 32 benzenesulfonamide
derivatives of the TTI type (Figure ) screened against a first set of hCAs that
are I, II, IV, and XII, and comparing the results with the corresponding
single-tail derivatives 1–7 (Table ).
Our results showed
that the development of 1–7 upon inclusion of
two other tails to give compounds 18–50 signifilass="Gene">cantly
affected the inhibition lass="Chemical">pan class="Chemical">profiles in terms of potency
and selectivity of action. On the whole, it should be noted that the
inclusion of three lipophilic tails in the TTI structure,
such as in compounds 18–32, did not produce noteworthy
outcomes in terms of potency and selectivity against the tested hCAs,
with very flat SAR within the subset, except for a few exceptions.
In contrast, increasing the polarity of at least one tail (starting
from compound 33) resulted in a great variability of
potencies and selectivities according to the type of tails included
in T1, T2, and T3.
The structural
study made by X-ray crystallography with pan class="Gene">hCA II
and in silico tools with the other isozymes lass="Chemical">pointed
out the limited and almost sulass="Chemical">perimlass="Chemical">posable interactions that the tail
of 1–7 lass="Chemical">pan class="Gene">can establish within the CAs active site.
In contrast, we demonstrated that the TTI derivatives
show a greater occupancy of the binding cavities with a great variability
among isoforms that contribute to the development of improved selectivity
of action.
Structural studies and lass="Disease">SAR analysis showed how different
tail combinations
lass="Chemical">pan class="Gene">can distinctly promote the binding of benzenesulfonamide derivatives
to the various hCAs active site. As an outcome of this preliminary
investigation, we can infer that inhibition of hCA I and II, possessing
narrow and markedly lipophilic active sites, can be promoted by inclusion
of a lipophilic tail and two small half-polarity pendants in the TTI structure (e.g., compound 39) or alternatively
two not too bulky tails and a polar one (e.g., compound 46). The marked polarity of hCA IV active site makes a significantly
polar tail in T1 (nearby the main CA inhibitory scaffold)
necessary to attain a low nanomolar inhibition (e.g., compound 49). HCA XII and its wide hydrophobic binding pocket better
accommodate almost all ligands with respect to hCA II, and thus most
tail combinations produce efficient inhibition of the isozyme. The
combination of lipophilic and polar tails coexisting with a medium
polarity one even led to a subnanomolar hCA XII inhibitor (compound 42).
For a first pharmacologilass="Gene">cal alass="Chemical">plass="Chemical">plilass="Chemical">pan class="Gene">cation of the
proposed approach,
three TTIs were selected because of their potent and
concomitant inhibition of hCA II, IV, and XII (CAs implicated in glaucoma)
and were assessed in vivo in a rabbit model of the
disease. Compound 47 showed the capability of lowering
IOP as efficiently as the clinically used DRZ up to 120
min post-administration.
The outcomes of tpan class="Chemical">his lass="Chemical">pan class="Chemical">proof-of-concept
study represent a firm starting
point for optimizing the general TTI design as well as
to produce a wider set of tail combinations to even improve the ligand/isoforms
matching in search of new CAIcandidates in the treatment of spreading
diseases such as glaucoma, tumors, neuropathic pain, and inflammation.
It should be stressed that an analogous approach might be extended
to other multi-isoform metalloenzymes to improve the outcomes in terms
of selectivity of action.
Experimental Section
Chemistry
Anhydrous solvents and all reagents were
purchased from Sigma-Aldrich, Fluorochem, and lass="Gene">TCI Chemilass="Chemical">pan class="Gene">cals. All reactions
involving air- or moisture-sensitive compounds were performed under
a nitrogen atmosphere using dried glassware, and syringes were used
to transfer solutions. Nuclear magnetic resonance (1H NMR, 13C NMR) spectra were recorded using a Bruker Advance III 400
MHz spectrometer in DMSO-d6. Chemical
shifts are reported in parts per million (ppm), and the coupling constants
(J) are expressed in hertz (Hz). Splitting patterns
are designated as follows: s, singlet; d, doublet; t, triplet; q,
quadruplet; m, multiplet; bs, broad singlet; dd, double of doublets.
The assignment of exchangeable protons (OH and NH) was confirmed by the addition of D2O. Two
tautomeric forms of the amide bond were detected for compounds 8–50, which partially double the signals in the 1H and 13C NMR spectra. Analytical thin-layer chromatography
(TLC) was carried out on Sigma-Aldrich silica gel F-254 plates. Flash
chromatography purifications were performed on Sigma-Aldrich silica
gel 60 (230–400 mesh ASTM) as the stationary phase, and ethyl
acetate/n-hexane or MeOH/DCM was used as eluents. Melting points (mp)
were measured in open capillary tubes with a Gallenkamp MPD350.BM3.5
apparatus and are uncorrected.
Compounds 1–7 and 18–50 were ≥95% pure. The purity
of the final compounds was determined by HPLC analysis performed using
an Aglass="Chemical">ilent 1200 Series equilass="Chemical">plass="Chemical">ped with an autosamlass="Chemical">pler, a binary lass="Chemical">pumlass="Chemical">p
system, and a diode array detector (DAD). The column used was a Luna
PFP with 30 mm length, 2 mm internal diameter, and 3 μm lass="Chemical">particle
size (lass="Chemical">pan class="Chemical">Phenomenex, Bologna, Italy) at a constant flow of 0.25 mL min–1, employing a binary mobile phase elution gradient.
The eluents used were 10 mM formic acid and 5 mM ammonium formate
in an mQ water solution (solvent A) and 10 mM formic acid and 5 mM
ammonium formate in methanol (solvent B) according to the elution
gradient as follows: initial at 90% solvent A, which was then decreased
to 10% in 8 min, kept for 3 min, returned to initial conditions in
0.1 min, and maintained for 3 min for reconditioning, to a total run
time of 14 min. The stock solution of each analyte was prepared in
methanol at 1.0 mg mL–1 and stored at 4 °C.
The sample solution of the analyte was freshly prepared by diluting
its stock solution up to a concentration of 10 μg mL–1 in a mixture of mQ water:methanol 50:50 (v/v), and 5 μL was
injected into the HPLC system. The solvents used in HPLC measurements
were methanol (Chromasolv grade), purchased from Sigma-Aldrich (Milan,
Italy), and mQ water 18 MΩ cm, obtained from Millipore’s
Simplicity system (Milan, Italy).
The high-resolution mass spectrometry
(HRMS) analysis was performed
with a Thermo Finnigan LTQ Orbitrap mass spectrometer equipped with
an electrospray ionization (ESI) source. The analysis was lass="Gene">carried
out by introducing, via a syringe lass="Chemical">pumlass="Chemical">p at 10 μL min–1, the samlass="Chemical">ple solution (1.0 μg lass="Chemical">pan class="Gene">mL–1 in mQ
water/acetonitrile 50:50), and it acquired the signal of the positive
ions. These experimental conditions allow the monitoring of protonated
molecules of the studied compounds ([M + H]+ species) that
were measured with a proper dwell time to achieve 60 000 units
of resolution at full width at half-maximum (FWHM). Elemental compositions
of compounds were calculated on the basis of their measured accurate
masses, accepting only results with an attribution error less than
2.5 ppm and a noninteger RDB (double bond/ring equivalents) value,
to consider only the protonated species.[47] None of the screened derivatives reported PAINS alerts determined
by SwissADME server (www.swissadme.ch).
General Synthesis Procedures for Preparation of 4-(2-(arylalkyl)aminoethyl)benzenesulfonamides
(1–7)
pan class="Chemical">Procedure 1: To
a solution of lass="Chemical">pan class="Chemical">4-(2-aminoethyl)benzenesulfonamide (9.99 mmol, 1.0 equiv)
in dry MeOH (40 mL), the appropriate aldehyde (1.1 equiv) was added
and the mixture was heated at reflux temperature under stirring for
0.5–4 h. Sodium borohydride (1.6 equiv) was added portionwise
at 0 °C, and the reaction mixture was stirred at reflux temperature
for 0.5–3 h. The solvent was evaporated under vacuum, and water was added (25 mL). pH was taken to 7 with 1 M HCl. The
suspension was filtered, and the collected powder was purified by
flash silica chromatography (5% MeOH in DCM) to give compounds 1–5.
pan class="Chemical">Procedure 2: To a solution
of lass="Chemical">pan class="Chemical">4-(2-aminoethyl)benzenesulfonamide (9.99 mmol, 1.0 equiv) in dry
DMF (5 mL), triethylamine (1.2 equiv) and the appropriate halide (1.1
equiv) were added at room temperature, and the mixture was stirred
at room temperature for 0.5 h (6) or 60 °C for 8
h (7). The reaction mixture was quenched by addition
of water (20 mL) and extracted with DCM (30 mL × 3). The organic
layer was collected, washed with brine (40 mL × 3), dried over
Na2SO4, filtered, and evaporated under vacuum to give compounds 6–7 as powders.
Compound 1 was obtained
according to the generalpan class="Chemical">procedure 1 earlier relass="Chemical">ported using lass="Chemical">pan class="Chemical">4-(2-aminoethyl)benzenesulfonamide
(9.99 mmol, 1.0 equiv) and benzaldehyde (1.1 equiv) in dry MeOH (40
mL). The reaction mixture was initially stirred at reflux temperature
for 4 h, and after the addition of sodium borohydride (1.6 equiv),
it was stirred at reflux temperature for another 2 h. Yield 96%; mp
173–175 °C; silica gel TLC R 0.08 (TFA/MeOH/DCM 3/5/92% v/v). δH (400 MHz,
DMSO-d6): 7.76 (d, J =
8.1 Hz, 2H, Ar-H), 7.42 (m, 7H, Ar-H), 7.32 (s, 2H, exchange with D2O, SO2NH2, overlap with signal at 7.42), 4.04 (s, 2H,
CH2), 3.07 (m, 2H, CH2), 2.97 (m, 2H, CH2). δC
(100 MHz, DMSO-d6): 145.87, 142.74, 141.80,
129.99, 129.05, 128.86, 127.46, 126.55, 53.77, 50.91, 36.50. ESI-MS
(m/z) [M + H]+: calcd
for C15H19N2O2S 291.1;
found 291.2.
Compound 3 was obtained according
to the generalpan class="Chemical">procedure 1 earlier relass="Chemical">ported using lass="Chemical">pan class="Chemical">4-(2-aminoethyl)benzenesulfonamide
(9.99 mmol, 1.0 equiv) and 4-fluorobenzaldehyde (1.1 equiv) in dry
MeOH (40 mL). The reaction mixture was initially stirred at reflux
temperature for 2 h, and after the addition of sodium borohydride
(1.6 equiv), it was stirred at reflux temperature for another 2 h.
Yield 95%; mp 145–147 °C; silica gel TLC R 0.21 (TFA/MeOH/DCM 3/5/92% v/v). δH
(400 MHz, DMSO-d6): 7.73 (d, J = 8.2 Hz, 2H, Ar-H), 7.38 (m, 4H, Ar-H), 7.28 (s, 2H, exchange with D2O, SO2NH2, overlap with signal at 7.38), 7.12 (t, J = 8.8 Hz, 2H, Ar-H), 3.73 (s, 2H, CH2), 2.79 (m, 4H, 2 × CH2). δF (376 MHz, DMSO-d6): −116.18. δC (100 MHz, DMSO-d6): 145.61, 142.94, 131.06, 130.98, 130.10, 126.73, 115.96,
115.75, 52.74, 50.62, 36.18. ESI-MS (m/z) [M + H]+: calcd for C15H18FN2O2S 309.1; found 309.1.
Compound 6 was obtained according
to the
generalpan class="Chemical">procedure 2 earlier relass="Chemical">ported using lass="Chemical">pan class="Chemical">4-(2-aminoethyl)benzenesulfonamide
(9.99 mmol, 1.0 equiv) and 3-chloropropionitrile (1.1 equiv) in dry
DMF (5 mL) and at rt stirring for 0.5 h. Yield 85%; mp 85–87
°C; silica gel TLC R 0.15 (TFA/MeOH/DCM 3/5/92% v/v). δH (400 MHz, DMSO-d6): 7.72 (d, J = 8.0 Hz, 2H,
Ar-H), 7.41 (d, J = 8.0 Hz, 2H,
Ar-H), 7.27 (s, 2H, exchange with D2O,
SO2NH2), 2.76 (m, 6H, 3 ×
CH2), 2.57 (t, J = 6.6 Hz, 2H, CH2). δC (100 MHz, DMSO-d6): 145.72, 142.88, 130.14, 126.68, 121.19, 50.83, 45.66,
36.59, 18.88. ESI-MS (m/z) [M +
H]+: calcd for C11H16N3O2S 254.1; found 254.0.
4-(2-(Phenethylamino)ethyl)benzenesulfonamide
(7)
Compound 7 was obtained according
to the
generalpan class="Chemical">procedure 2 earlier relass="Chemical">ported using lass="Chemical">pan class="Chemical">4-(2-aminoethyl)benzenesulfonamide
(9.99 mmol, 1.0 equiv) and (2-bromoethyl)benzene (1.1 equiv) in dry
DMF (5 mL) and at 60 °C stirring for 8 h. Yield 73%; mp 213–215
°C; silica gel TLC R 0.02 (TFA/MeOH/DCM 3/5/92% v/v). δH (400 MHz, DMSO-d6): 7.78 (d, J = 8.2 Hz, 2H,
Ar-H), 7.44 (d, J = 8.2 Hz, 2H,
Ar-H), 7.34 (m, 4H, Ar-H), 7.26
(s, 2H, exchange with D2O, SO2NH2, overlap with signal at 7.25), 7.25 (m, 1H, Ar-H), 2.89 (m, 8H, 4 × CH2). δC (100 MHz, DMSO-d6): 145.71,
142.27, 141.02, 130.04, 129.47, 129.26, 126.88, 126.59, 51.35, 50.91,
36.29, 36.05. ESI-MS (m/z) [M +
H]+: calcd for C16H21N2O2S 305.1; found 305.1.
General Synthesis Procedure
of Chloro-amides (8–17)
To a suspension
of 4-(2-(arylalkyl)aminoethyl)lass="Chemical">benzenesulfonamide 1–7 (6.89 mmol, 1.0 equiv) and lass="Chemical">pan class="Chemical">K2CO3 (1.2 equiv)
in acetone (40 mL) cooled to 0 °C, the appropriate
chloroacylchloride (1.2 equiv) was added dropwise and the mixture
was stirred for 0.5 h. The solvent was evaporated under vacuum, then slush (50 mL) was added, and the basic suspension was neutralized
with 1 M HCl. The precipitate was collected by filtration and purified
with flash chromatography (1% MeOH in DCM) to give compounds 8–17.
To a
solution of pan class="Chemical">phenethylamine (16,5 mmol, 1.0 equiv) in lass="Chemical">pan class="Disease">dry DMF (5 mL),
triethylamine (1.2 equiv) and 3-chloropropionitrile (1.1 equiv) were
added, and the mixture was stirred at room temperature for 0.5 h.
The reaction was quenched by addition of water (20 mL) and extracted
with EtOAc (30 mL × 3). The organic layer was collected, washed
with brine (40 mL × 3), dried over Na2SO4, filtered off, and evaporated under vacuum to give
3-(phenethylamino)propanenitrile as an orange oil. Yield 92%; silica
gel TLC R 0.42 (TFA/MeOH/DCM
1.5/1.5/97% v/v). 7.23 (m, 5H, Ar-H), 2.85 (m, 6H,
3 × CH2), 2.57 (t, J = 6.6 Hz, 2H, CH2), 1.89 (bs, 1H, exchange
with D2O, NH). δC (100 MHz, DMSO-d6): 141.30, 129.64, 129.28, 126.90, 120.99,
51.41, 45.80, 37.04, 18.99. ESI-MS (m/z) [M + H]+: calcd for C11H15N2 175.1; found 175.0.
General Synthesis Procedure of Three-Tail
Compounds 18–39
To a solution of chloroalkylpan class="Chemical">amide 8–17 (0.69 mmol, 1.0 equiv) and lass="Chemical">pan class="Chemical">triethylamine (1.2
equiv) in MeCN dry
(5 mL), the proper secondary amine (1.1 equiv) was added, and the
mixture was heated at reflux temperature for 4–24 h under stirring.
The solvent was evaporated under vacuum, and the
crude was treated with NaHCO3 saturated solution (5 mL)
and extracted with EtOAc (10 mL × 3). The organic layer was dried
over Na2SO4, filtered, and evaporated under
vacuum. The obtained residue was purified by flash chromatography
(1% MeOH in DCM) to give compounds 18–39 as an
oil or powder.
Compound 39 was obtained according to the generallass="Chemical">procedure
earlier relass="Chemical">ported using lass="Chemical">pan class="Chemical">2-chloro-N-(2-cyanoethyl)-N-(4-sulfamoylphenethyl)acetamide 15 and 3-(phenethylamino)propanenitrile
(1.1 equiv) in MeCN dry (5 mL) and stirring for 14 h at reflux temperature.
The sticky residue was purified by flash chromatography (1% MeOH in
DCM) to give 39 as an oil. Yield 75%; silica gel TLC R 0.24 (TFA/MeOH/DCM 3/5/92%
v/v). δH (400 MHz, DMSO-d6): 7.76
(m, 2H, Ar-H), 7.35 (m, 7H, Ar-H), 7.30 (s, 2H, exchange with D2O, SO2NH2, overlap with signal at 7.35), 3,55 (m, 6H,
3 × CH2), 2.82 (m, 10H, 5 ×
CH2). δC (100 MHz, DMSO-d6): 167.38, 167.09, 144.29, 144.09, 143.83,
143.50, 143.47, 143.24, 138.62, 130.54, 130.50, 130.43, 130.28, 130.23,
129.77, 129.33, 129.10, 126.84, 126.82, 126.58, 120.21, 120.04, 56.21,
50.23, 49.92, 49.14, 47.96, 47.42, 44.10, 43.46, 43.21, 43.03, 42.82,
42.17, 34.99, 33.84, 21.84, 18.01, 17.88, 16.59, 16.43. ESI-HRMS (m/z) [M + H]+: calcd for C24H30N5O3S 468.2069; found
468.2073.
General Synthesis Procedure of Amine Derivatives 40–44
To a solution of pan class="Chemical">nitrile derivatives 33–39 (0.5 mmol, 1.0 equiv) and 5 M lass="Chemical">pan class="Chemical">NaOH(aq) (3.0 equiv) in
EtOH (10 mL), Ni/Raney (0.5 mL) was added, and the mixture was stirred
o.n. under H2 pressure (50 psi). The solution was filtered
off, and the solvent was evaporated under vacuum.
The residue was purified by flash chromatography (5–15% MeOH
in DCM) to give compounds 40–44.
Compound 44 was obtained according to the generalpan class="Chemical">procedure
earlier relass="Chemical">ported using lass="Chemical">pan class="Chemical">N-(2-cyanoethyl)-2-(dihexylamino)-N-(4-sulfamoylphenethyl)acetamide 33. The obtained
solid was purified by flash chromatography to give 44 as an oil. Yield 31%; silica gel TLC R 0.43 (TFA/MeOH/DCM 3/5/92% v/v). δH (400 MHz,
DMSO-d6): 7.78 (d, J =
8.0 Hz, 2H, Ar-H), 7.49 (d, J =
8.0 Hz, 2H, Ar-H), 7.35 (s, 2H, exchange with D2O, SO2NH2), 3.48 (m,
2H, CH2), 3.20 (m, 2H, CH2), 2.88 (m, 8H, 4 × CH2), 1.85 (m, 4H, 2 × CH2), 1.45 (m,
4H, 2 × CH2), 1.25 (m, 12H, 6 ×
CH2), 0.86 (m, 6H, 2 × CH3). δC (100 MHz, DMSO-d6): 169.55, 169.26, 143.81, 143.54, 135.94, 133.31, 131.36, 130.47,
130.20, 126.81, 55.06, 55.00, 48.79, 47.51, 37.69, 37.45, 37.41, 34.96,
34.74, 33.94, 33.80, 31.96, 31.94, 28.69, 27.89, 27.31, 27.17, 27.03,
26.88, 26.30, 23.05, 23.02, 14.93, 14.92. ESI-HRMS (m/z) [M + H]+: calcd for C25H47N4O3S 483.3369; found 483.3374.
General Synthesis Procedure of Carboxylic Acid Derivatives 45–49
To a solution of the aplass="Chemical">prolass="Chemical">priate lass="Chemical">pan class="Chemical">nitrile
derivatives 33–39 (0.5 mmol, 1.0 equiv) in EtOH
(5 mL), 5 M NaOH(aq) (3.0 equiv) was added, and the mixture
was heated at reflux temperature under stirring o.n.. The solution
was cooled to 0 °C and 12 M HCl (2.0 equiv) was added dropwise
until precipitation of a powder that was collected by filtration.
The solid was purified by flash chromatography (5–15% MeOH
in DCM) to give the compounds 45–49.
Compound 49 was obtained
according to the generallass="Chemical">procedure earlier relass="Chemical">ported using lass="Chemical">pan class="Chemical">N-(2-cyanoethyl)-2-(dihexylamino)-N-(4-sulfamoylphenethyl)acetamide 33. The obtained solid was purified by flash chromatography
to give 49 as a powder. Yield 33%; mp > 300 °C;
silica gel TLC R 0.36
(TFA/MeOH/DCM 3/5/92% v/v). δH (400 MHz, DMSO-d6): 12.14 (brs, 1H, exchange with D2O, COOH), 7.73 (d, J = 8.0 Hz, 2H, Ar-H), 7.40 (d, J = 8.0 Hz, 2H, Ar-H), 7.31 (s, 2H, exchange with D2O, SO2NH2), 3.66 (m, 2H, CH2), 3.48 (m, 2H, CH2), 3.25
(s, 1.1H, CH2), 2.99 (s, 0.9H, CH2), 2.91 (m, 1H, CH2), 2.79 (m, 1H, CH2), 2.42 (m, 2H, CH2), 2.32 (m, 2H, CH2), 2.10 (m, 2H, CH2), 1.30 (m, 16H, 8
× CH2), 0.84 (m, 6H, 2 × CH3). δC (100 MHz, DMSO-d6): 175.95, 175.28, 170.94, 170.61, 144.84, 144.49, 143.37,
143.20, 130.27, 130.11, 126.80, 126.76, 59.22, 57.99, 54.91, 54.80,
48.91, 47.61, 38.89, 35.54, 34.23, 32.27, 32.20, 27.67, 27.64, 27.52,
27.35, 25.62, 23.19, 23.14, 14.99, 14.95. ESI-HRMS (m/z) [M + H]+: calcd for C25H44N3O5S 498.3001; found 498.2997.
Synthesis of (Z)-3-((2-((furan-2-ylmethyl)(4-sulfamoylphenethyl)amino)-2-oxoethyl)(phenethyl)amino)-N-(octadec-9-en-1-yl)propanamide (50)
To a solution of 46 (0.5 mmol, 1.0 eq) in pan class="Chemical">DMF dry (1
lass="Chemical">pan class="Gene">mL), oleylamine (1.1 eq), EDC·HCl (1.2 eq), and DMAP (catalytic)
were added, and the reaction mixture was stirred at r.t. for 6 h.
The reaction was quenched with water and extracted with EtOAc (15
mL × 3). The organic layers were washed with brine (20 mL ×
4), dried over Na2SO4, filtered off, and evaporated
under vacuum. The obtained residue was purified by
flash chromatography (3% MeOH in DCM) to give compound 50 as an oil. Yield 73%; silica gel TLC R 0.29 (TFA/MeOH/DCM 3/5/92% v/v). δH (400 MHz,
DMSO-d6): 7.93 (s, 1H, exchange with D2O, CONH), 7.73 (t, J = 7.2 Hz, 2H, Ar-H), 7.63 (s, 0.5H, Ar-H), 7.57 (s, 0.5H,
Ar-H), 7.27 (m, 7H, Ar-H), 7.21
(s, 2H, exchange with D2O, SO2NH2, overlap with signal at 7.27), 6.39 (m, 2H, Ar-H), 5.31 (m, 2H, 2 × =CH), 4.55 (s,
2H, CH2) 3.48 (m, 3.1 H, 2 × CH2), 3.21 (s, 0.9H, CH2), 2.99 (m, 2H, CH2), 2.73 (m, 10H, 5
× CH2), 2.21 (m, 2H, CH2), 1.97 (m, 4H, 2 × CH2), 1.29 (m, 22H, 11 × CH2), 0.83
(m, 3H, CH3). δC (100 MHz, DMSO-d6): 171.94, 171.93, 171.89, 171.88, 170.88,
170.76, 170.75, 170.69, 152.29, 152.07, 144.44, 144.16, 143.40, 143.12,
141.47, 141.33, 131.14, 130.69, 130.34, 130.04, 129.68, 129.63, 129.23,
126.84, 126.81, 111.61, 111.57, 109.42, 109.39, 65.97, 57.81, 57.55,
56.03, 55.96, 55.41, 50.91, 50.82, 48.61, 47.91, 44.54, 39.49, 34.27,
34.20, 34.18, 33.81, 33.55, 33.52, 33.49, 32.94, 32.32, 30.17, 30.14,
30.13, 30.08, 30.05, 29.94, 29.88, 29.82, 29.73, 29.63, 29.51, 27.67,
27.61, 27.50, 23.13, 16.23, 15.00. ESI-HRMS (m/z) [M + H]+: calcd for C44H67N4O5S 763.4832; found 763.4826.
Carbonic
Anhydrase Inhibition
An Applied Photophysics
stopped-flow instrument has been used for assaying the lass="Gene">CA-lass="Chemical">pan class="Gene">catalyzed
CO2 hydration activity.[34] Phenol
red (at a concentration of 0.2 mM) has been used as an indicator,
working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH
7.5) as a buffer and 20 mM Na2SO4 (for maintaining
the ionic strength constant), following the initial rates of the CA-catalyzed
CO2 hydration reaction for a period of 10–100 s.
The CO2 concentrations ranged from 1.7 to 17 mM for the
determination of the kinetic parameters and inhibition constants.
For each inhibitor, at least six traces of the initial 5–10%
of the reaction have been used for determining the initial velocity.
The 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 the assay buffer. Inhibitor and
enzyme solutions were preincubated together for 15 min at room temperature
prior to assay to allow the formation of the E–I complex. The
inhibition constants were obtained by nonlinear least-squares methods
using PRISM 3 and the Cheng–Prusoff equation, as reported earlier,[36] and represent the mean from at least three different
determinations. All hCA isofoms were recombinant ones obtained in-house
as reported earlier.[49]
X-ray Crystallography
Protein
Expression and Purification
Competent lass="CellLine">BL21lass="Chemical">pan class="Species">Escherichia coli cells were transformed separately
with plasmid DNA containing the hCA II gene using standard protocols.[50,51] An overnight culture in LB was started with large-scale growth the
following day until OD600 reached ∼0.6. Isopropyl
β-d-1-thiogalactoside (IPTG, 0.5 mM) and zinc sulfate
(1 mM) were used to induce protein expression for 3 h. The cells were
pelleted and lysed via a microfluidizer set to 18 000 PSI.
Supernatant was filtered with a 0.4 μm filter before being run
through an affinity column with p-aminomethyl-benzenesulfonamideagarose. Enzyme was eluted with azide and buffer-exchanged into storage
buffer (50 mM Tris pH 7.8) to remove azide. The purity of the protein
was determined by a 12% sodium dodecyl sulfatepolyacrylamide gel
electrophoresis (SDS-PAGE) and UV/vis spectroscopy at a 280 nm measured
protein concentration.
Crystallization
Inhibitors were
successfully co-crystallized
with lass="Gene">hCA II via the hanging-drolass="Chemical">p valass="Chemical">por diffusion method. Mother liquor
(500 μL) consisting of 1.6 M lass="Chemical">pan class="Chemical">sodium citrate and 50 mM Tris at
pH 7.8 was used in setting up crystal trays for each well. Each drop
contained a 1:1 ratio of 10 mg/mLprotein to mother liquor. DMSO was
used to dissolve inhibitors to 1 mM, with the drops’ final
concentration ∼100 μM. Co-crystals of hCA II formed within
a week.
Data Collection and Processing
Diffraction data were
collected via the F1 bealass="Gene">mline at Cornell High Energy Synchrotron Source
(lass="Chemical">pan class="Species">CHESS) at 0.977 Å wavelength. A Pilatus 6M detector collected
data sets with a crystal-to-detector distance of 270 mm, 1° oscillation,
and 4 s image exposure, for a total of 180 images. Diffraction data
were indexed and integrated with XDS.[52] Data were scaled in space group P21 via
AIMLESS[53] from the CCP4program suite.[54] Phases were determined via molecular replacement
using PDB: 3KS3(55) as a search model. Modifications to
the model such as addition of inhibitor, ligand (glycerol), zinc,
and water to the active site were executed in Coot[56] along with ligand PDB file modifications. Refinements were
completed and ligand restraint files were created in Phenix.[57] Figures were generated with PyMol (Schrödinger).
Protein–ligand bond lengths and active site interactions were
observed with LigPlot Plus.[58]
Computational
Study
lass="Gene">HCA I (PDB: 2NMX),[43] lass="Chemical">pan class="Gene">hCA II (PDB: 5LJT),[46] hCA IV (PDB: 1ZNC),[44] and hCA XII (PDB: 1JD0)[45] crystal structures were
prepared according to the Protein Preparation module in Maestro-Schrödinger
suite, assigning bond orders, adding hydrogens, deleting water molecules,
and optimizing H-bonding networks.[59] Finally,
energy minimization with a root-mean-square deviation (RMSD) value
of 0.30 was applied using an Optimized Potentials for Liquid Simulation
(OPLS-3) force field. Input 3D ligand structures were prepared by
Maestro[59a] and evaluated for their ionization
states with Epik.[59b] Sulfonamides were
considered in their deprotonated form on the basis of evidence from
neutron crystallography. OPLS-3 force field in Macromodel[59c] was used for energy minimization for a maximum
number of 2500 conjugate gradient iteration and setting a convergence
criterion of 0.05 kcal mol–1 Å–1. The docking grid was generated using Glide[59d] with default settings, with the center located on the center
of mass of the co-crystallized ligand. Ligands were docked with the
standard precision (SP) mode of Glide and the five top-scoring poses
of each molecule retained as output. The best pose for each compound,
evaluated in terms of coordination, hydrogen-bond interactions, and
hydrophobic contacts, was refined by Prime MM-GBSA methods using a
VSGB solvation model.[60−63]
Hypertensive Rabbit IOP Lowering Studies
Male New Zealand
albino lass="Species">rabbits weighing 1500–2000 g were used in these studies.
Animals were anesthetized using lass="Chemical">pan class="Chemical">Zoletil (tiletamine chloride plus
zolazepam chloride, 3 mg/kg body weight, im), and elevated IOP was
induced by the injection of 0.05 mL of hypertonic saline solution
(5% in distilled water) into the vitreous of both eyes. IOP was determined
using a pneumo-tonometer Reichert, model 30 (Reichert, Inc., Depew,
NY) prior to hypertonic saline injection (basal), and at 1, 2, 3,
and 4 h after administration of the different drugs. Vehicle (hydroxypropylcellulose
at 0.05%) or drugs were instilled immediately after the injection
of hypertonic saline. Eyes were randomly assigned to different groups.
Vehicle or drug (0.05 mL) was directly instilled into the conjunctive
pocket at the desired doses (1–2%).[64] Four different animals were used for each tested compound. All animal
manipulations were carried out according to the European Community
guidelines for animal care [DL 116/92, application of the European
Communities Council Directive of 24 November 1986 (86/609/EEC)]. The
ethical policy of the University of Florence complies with the Guide
for the Care and Use of Laboratory Animals of the US National Institutes
of Health (NIH Publication no. 85–23, revised 1996; University
of Florence assurance number A5278-01). Formal approval to conduct
the experiments described was obtained from the Animal Subjects Review
Board of the University of Florence and upon authorization of the
National Ethics Committee of the Italian Ministry of Health (number
1179/2015-PR). Experiments involving animals have been reported according
to ARRIVE, Animal Research: Reporting of in Vivo Experiments, guidelines.[65] All efforts were made to minimize animal suffering
and to reduce the number of animals used.
Authors: Stanislav Kalinin; Alessio Nocentini; Alexander Kovalenko; Vladimir Sharoyko; Alessandro Bonardi; Andrea Angeli; Paola Gratteri; Tatiana B Tennikova; Claudiu T Supuran; Mikhail Krasavin Journal: Eur J Med Chem Date: 2019-08-26 Impact factor: 6.514
Authors: Melissa A Pinard; Christopher D Boone; Brittany D Rife; Claudiu T Supuran; Robert McKenna Journal: Bioorg Med Chem Date: 2013-08-28 Impact factor: 3.641
Authors: Mohamed A Said; Wagdy M Eldehna; Alessio Nocentini; Alessandro Bonardi; Samar H Fahim; Silvia Bua; Dalia H Soliman; Hatem A Abdel-Aziz; Paola Gratteri; Sahar M Abou-Seri; Claudiu T Supuran Journal: Eur J Med Chem Date: 2019-11-02 Impact factor: 6.514
Authors: Martyn D Winn; Charles C Ballard; Kevin D Cowtan; Eleanor J Dodson; Paul Emsley; Phil R Evans; Ronan M Keegan; Eugene B Krissinel; Andrew G W Leslie; Airlie McCoy; Stuart J McNicholas; Garib N Murshudov; Navraj S Pannu; Elizabeth A Potterton; Harold R Powell; Randy J Read; Alexei Vagin; Keith S Wilson Journal: Acta Crystallogr D Biol Crystallogr Date: 2011-03-18
Authors: Alessio Nocentini; Chad S Hewitt; Margaret D Mastrolorenzo; Daniel P Flaherty; Claudiu T Supuran Journal: J Enzyme Inhib Med Chem Date: 2021-12 Impact factor: 5.051
Authors: Sarah L Mueller; Panagiotis K Chrysanthopoulos; Maria A Halili; Caryn Hepburn; Tom Nebl; Claudiu T Supuran; Alessio Nocentini; Thomas S Peat; Sally-Ann Poulsen Journal: Molecules Date: 2021-05-18 Impact factor: 4.411
Authors: Galina F Makhaeva; Nadezhda V Kovaleva; Natalia P Boltneva; Sofya V Lushchekina; Tatiana Yu Astakhova; Elena V Rudakova; Alexey N Proshin; Igor V Serkov; Eugene V Radchenko; Vladimir A Palyulin; Sergey O Bachurin; Rudy J Richardson Journal: Molecules Date: 2020-08-27 Impact factor: 4.411
Figure 1
Figure 2
Figure 3
Scheme 1
Scheme 3
Scheme 2
Figure 4
Figure 6
Figure 5
Figure 7
Figure 8