Ninhydrins inhibit carbonic anhydrases directly binding to the metal ion

Article abstract of DOI:10.1016/j.ejmech.2020.112875

Ninhydrins show extensive application in organic chemistry and agriculture whereas they have been poorly investigated as bioactive molecules for medicinal chemistry purposes. A series of ninhydrin derivatives was here investigated for the inhibition of human carbonic anhydrases (CAs, EC 4.2.1.1), based on earlier evidence that gem diols are able to coordinate the metal ion from the CA active site. Ninhydrins demonstrated a micromolar inhibitory action against CA I and IX (K_Is in the range 0.57–68.2 μM) and up to a nanomolar efficacy against CA II and VII (K_Is in the range 0.025–78.2 μM), validated isoforms as targets in several CNS-related diseases. CA IV was instead weakly or poorly inhibited. A computational protocol based on docking, MM-GBSA and metadynamics calculations was used to elucidate the putative binding mode of this type of inhibitors to CA II and CA VII. The findings of this study testify that such pharmacologically underestimated ligands may represent interesting lead compounds for the development of CA inhibitors possessing an innovative mechanism of action, i.e., mono- or bis-coordination to the zinc ion through the diol moiety.

Full text of DOI:10.1016/j.ejmech.2020.112875

European Journal of Medicinal Chemistry 209 (2021) 112875
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Ninhydrins inhibit carbonic anhydrases directly binding to the metal
ion
,
b
,
, d,
Abdeslem Bouzina a 1, Malika Berredjem b, Alessio Nocentini c 1, Silvia Bua c,
Zouhair Bouaziz a, Joachim Jose e, Marc Le Borgne a f, Christelle Marminon a
,
,
, f, **
Paola Gratteri c *, Claudiu T. Supuran c
, d,
a
ꢀ
ꢀ
ꢀ
EA 4446 Bioactive Molecules and Medicinal Chemistry, Faculte de Pharmacie-ISPB, SFR Sante Lyon-Est CNRS UMS3453-INSERM US7, Universite de Lyon,
ꢀ
Universite Claude Bernard Lyon 1, 8 Avenue Rockefeller, F-69373, Lyon Cedex 8, France
b Laboratory of Applied Organic Chemistry, Synthesis of Biomolecules and Molecular Modelling Group, Badji-Mokhtar-Annaba University, Box 12, Annaba,
23000, Algeria
c Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Florence, Italy
d Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, Laboratory of Molecular Modeling Cheminformatics & QSAR,
University of Florence, Florence, Italy
e
€
€
Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westfalische WilhelmsUniverstitat Münster, 48149, Münster, Germany
f
ꢀ
ꢀ
ꢀ
ꢀ
Small Molecules for Biological Targets Team, Centre de recherche en cancerologie de Lyon, Centre Leon Berard, CNRS 5286, INSERM 1052, Universite Claude
Bernard Lyon 1, Univ Lyon, Lyon, 69373, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Ninhydrins show extensive application in organic chemistry and agriculture whereas they have been
poorly investigated as bioactive molecules for medicinal chemistry purposes. A series of ninhydrin de-
rivatives was here investigated for the inhibition of human carbonic anhydrases (CAs, EC 4.2.1.1), based
on earlier evidence that gem diols are able to coordinate the metal ion from the CA active site. Ninhydrins
Received 30 July 2020
Received in revised form
27 August 2020
Accepted 22 September 2020
Available online 5 October 2020
demonstrated a micromolar inhibitory action against CA I and IX (KIs in the range 0.57e68.2
mM) and up
to a nanomolar efficacy against CA II and VII (KIs in the range 0.025e78.2 M), validated isoforms as
m
targets in several CNS-related diseases. CA IV was instead weakly or poorly inhibited. A computational
protocol based on docking, MM-GBSA and metadynamics calculations was used to elucidate the putative
binding mode of this type of inhibitors to CA II and CA VII. The findings of this study testify that such
pharmacologically underestimated ligands may represent interesting lead compounds for the develop-
ment of CA inhibitors possessing an innovative mechanism of action, i.e., mono- or bis-coordination to
the zinc ion through the diol moiety.
Keywords:
Carbonic anhydrases
Ninhydrins
Microwave synthesis
Inhibitors
Binding mode
Zinc binder
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
have been identified and characterized in human (h) so far [1,2]. A
chorus of chemotypes have been evaluated for the design of hCAs
Carbonic anhydrases (CAs, EC 4.2.1.1), a group of ubiquitously
expressed metalloenzymes, are implicated in numerous physio-
logical and pathological processes. Fifteen different CA isoforms
inhibitors or activators with biomedical applications [3]. On the
basis of previous exploratory work on the inhibitory action of a gem
diol derivative (bearing a trifluoro-dihydroxy-propanone moiety)
which was observed to coordinated to the metal ion from the CA
active site [3] and in order to explore a new molecular scaffold
capable of interacting with CAs in the same manner, ninhydrin (2,2-
dihydroxyindane-1,3-dione, CAS number 485-47-2) and some of its
substituted derivatives were selected for detailed investigations. It
should be stressed here that ninhydrins were not investigated so far
as inhibitors of CAs.
* Corresponding author. Department of NEUROFARBA, Section of Pharmaceutical
and Nutraceutical Sciences, University of Florence, Florence, Italy.
** Corresponding author. EA 4446 Bioactive Molecules and Medicinal Chemistry,
ꢀ
ꢀ
Faculte de Pharmacie-ISPB, SFR Sante Lyon-Est CNRS UMS3453-INSERM US7, Uni-
ꢀ
ꢀ
versite de Lyon, Universite Claude Bernard Lyon 1, 8 Avenue Rockefeller, F-69373,
Lyon Cedex 8, France.
E-mail addresses: christelle.marminon-davoust@univ-lyon1.fr (C. Marminon),
paola.gratteri@unifi.it (P. Gratteri).
On 1910, S. Ruhemann described for the very first time the
synthesis of ninhydrin and its particular reactivity towards amines
1
These investigators equally contributed to the work.
https://doi.org/10.1016/j.ejmech.2020.112875
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

A. Bouzina, M. Berredjem, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112875
using potassium carbonate (K2CO3) and 2-iodopropane in acetone,
while indanone 1s was commercially available. The synthesis of
indanones 1o and 1p was achieved by electrophilic aromatic sub-
stitution in the para position of the 4-methoxyindan-1-one 1e [20].
Introduction of the isopropyl (iPr) group was more difficult to
introduce, attempts by using 2-halogenopropane and Lewis acid
remaining unsuccessful. Compound 1o was finally obtained with
41% yield, adding isopropanol (2 eq) to a solution of 1e (1 eq) in
sulfuric acid (2 eq) at 60 ꢀC [21]. For 1p, the amidomethylation
conditions [22] were adapted to both reduce the time reaction and
permit its completion. So, a mixture of 1e (1 eq), 2-chloro-N-
(hydroxymethyl)acetamide (1.1 eq) in acetic acid/sulfuric acid (ratio
9:1) was microwave irradiated at 70 ꢀC for 10 min to afford 1p with
84% yield.
Abbreviations
AAZ
BCS
BP
acetazolamide
Binding CompScore
Binding Persistence
Binding pose
BP
BPS
CAs
h
Binding PoseScore
Carbonic anhydrases
human
HMBC
Heteronuclear Multiple Bond Correlation
MM-GBSA Molecular Mechanics-Generalized Born Surface
Area
NOE
Nuclear Overhauser Effect
Regiochemistry was confirmed by 1H NMR data. Indeed, the
presence of the doublet in the aromatic area involved a ortho or
para substitution as expected, due to the orientation effect of the
methoxy group. Moreover the chemical shift lower than 7.00 ppm,
that is to say at 6.98 ppm for 1o and 6.95 ppm for 1p, related to the
methoxy effect, was in favour of the para substitution. This sup-
position was confirmed by NOESY and HMBC experiments for 1o
(Fig. 1). In fact, large correlations were observed on the one hand
between the methoxy group and H-5 (at 6.98 ppm), and on the
other hand between the methyl of the isopropyl group and H-6 (at
7.25 ppm). The same observations were noted in the HMBC spec-
trum: the carbonyl group only correlated with H-6 (4JH,C) while the
methylene group C-2 only correlated with H-5 (4JH,C).
A synthesis path was reported for ninhydrin 2t [23,24], even if
no NMR data was described. In this strategy, ninhydrin 2t was
obtained in two steps from the tetrabromoindane-1,3-dione 3 by
using first p-toluenesulfonyl azide in the presence of triethylamine
and then tert-butylhypochlorite in 10% aqueous acetonitrile. So, we
decided to adapt our previously reported method [18] to rapidly
and easily oxidize indane-1,3-dione 3 into ninhydrin 2t. Actually,
5 min of microwave irradiation of 3 at 180 ꢀC by using 1.5 eq se-
lenium dioxide afforded ninhydrin 2t with a good yield of 70%
(Scheme 1). Tetrabromoindane-1,3-dione 3 was prepared in two
steps from the commercially available tetrabromophtalic anhydride
and tert-butylacetoacetate [25,26].
NOESY
RMDS
RMSD
SAR
TBID
VSGB
Nuclear Overhauser Effect Spectroscopy
Reactive Molecular Dynamics Simulation
Root Mean Square Deviation
structure-activity relationship
tetrabromoindane-1,3-dione
variable dielectric surface generalized born
and ammonia, yielding colored products [4]. Ninhydrin is sensitive
to prolonged exposure to light and then fluorescent ternary de-
rivatives can be formed by reaction with aldehydes and primary
amines. Over the decades many works and reviews have presented
the chemistry developed around ninhydrin [5]. Until now the
studies around ninhydrin and its derivatives remained numerous
and major, in many areas (e.g. agriculture, biomedical research,
applied chemistry) [6,7]. Among all applications using ninhydrin as
reagent, quantification of amino acids [8] and fingerprint detection
are the most known [9]. Ninhydrin also remains a major reagent for
test development [10]. In parallel, ninhydrin is a very important
starting material in the design of new polyheterocycles [11].
Moreover, intensive developments in the field of green chemistry
(e.g. multicomponent reactions, solvent-free synthesis) use
ninhydrin as starting material [12,13]. As shown in Refs. [6c,12],
ninhydrin is at the heart of the development of indeno[1,2-b]in-
doles [14]. The use of ninhydrin and substituted ones allowed ac-
cess to a large library of functionalized indeno[1,2-b]indoles with
diverse biological activities [15].
2.2. Carbonic anhydrase inhibition
In this particular context, some of us has developed numerous
ninhydrins to prepare corresponding indeno[1,2-b]indoles. To date,
ninhydrins have been poorly considered as bioactive molecules as
such. Herein, it is for the first time highlighted that such molecules
can inhibit hCAs and can be used as lead compounds for the
development of inhibitors possessing an innovative mechanism of
action. Docking, MM-GBSA and metadynamics calculations [16,17]
were used to assess the putative binding mode of this class of
underestimated compounds to hCA II and hCA VII, the most phys-
iologically relevant and the most affected CA isoforms, respectively.
Ninhydrins 2a-t were investigated as inhibitors of the human
CAs I, II, IV, VII and IX by a stopped flow CO2 hydrase assay. The
inhibition data, compared to those of the standard sulfonamide
inhibitor acetazolamide (AAZ), are reported in Table 1.
The inhibition profiles measured for derivatives 2a-t indicate
that the substitution pattern at the aromatic portion has a great
impact on the CA inhibition efficacy of ninhydrins. In fact, com-
pounds 2a-t resulted to act as low nanomolar to medium micro-
molar inhibitors depending on the CA isozyme. Interesting
structure-activity relationship (SAR) can be worked out. CA II and
VII are mostly inhibited in a submicromolar range by ninhydrins
2a-t. CA II is a cytosolic ubiquitous isoform, whose inhibition is
responsible of most side effects of non-selective CAIs. Nonetheless,
CA II is abundantly expressed in the brain, as CA VII, and together
have been validated as targets for the treatment of CNS-related
diseases (e.g. neuropathic pain) [17b]. In detail, inhibition con-
stants (KIs) against CA VII reach two-digits nanomolar values,
making it the most affected CA among those evaluated (KIs in the
range 25e21,300 nM). Notably, only a small pattern of sub-
stitutions, that are 5-NO2 (2k), 5-OH (2m), 4-OCH3-7-iPr (2o), 5,6-
diOCH3 (2r), 4,5,6,7-tetraBr (2t), increase two to three-fold the in-
hibition efficacy of ninhydrin 2a (KIs in the range 25e65 nM).
Surprisingly, all other substitutions lead to a more or less marked
2. Results and discussion
2.1. Chemistry
While ninhydrin 2a was commercially available, all the other
tested ninhydrins were synthesised by microwave selenium diox-
ide oxidation of either the corresponding indan-1-ones 1b-s [18]
for ninhydrins 2b-s or the tetrabromoindane-1,3-dione 4 for
ninhydrin 2t (Scheme 1). The five new ninhydrins 2d, 2o, 2p, 2s and
2t, described herein, were also obtained in good to excellent yields
(one at 60% for 2p, and more than 70% for the other four).
Indan-1-one 1d [19] was prepared by O-alkylation of 1c [20] by
2

A. Bouzina, M. Berredjem, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112875
Scheme 1. General ninhydrins 2b-t syntheses from indan-1-ones 1b-s or indane-1,3-dione 3. New compounds described in this article are highlighted blue. Reagent and con-
ditions: a). SeO2, dioxane/water, MW, 5 min, 180 ꢀC, 60e87%; b). (CH3)2CHI, K2CO3, acetone, 60 ꢀC, 4 h; 60%; c). (CH3)2CHOH, H2SO4, 60 ꢀC, 2 h; 41%; d). ClCH2NHCOCH2Cl, AcOH,
H2SO4, MW, 70 ꢀC, 10 min, 84%; e). SeO2, dioxane, MW, 5 min, 180 ꢀC, 70%.
5,6-diOCH3 (2r), 4,5,6,7-tetraBr (2t). A few substitutions produced
instead a worsening of CA II inhibition, such as in compounds 2i, 2l,
2n, and 2s (KIs in the range 26.7-78.2 mM).
CA I and IX are slightly less inhibited by ninhydrins 2a-t than CA
II. In fact, most KIs against CA I and IX settle in a low micromolar
range (1.34e68.2
that showed a submicromolar CA I inhibition (KIs of 0.87 and
0.57 M). Surprisingly, ninhydrin 2a resulted the second-best CA IX
inhibitor, overcome only by the 4-OiPr derivative 2d (KIs 2.37 vs
1.34 M). Also the bromo-derivatives 2l and 2t showed significant
mM), with the exception of compounds 2e and 2l
m
m
single digit micromolar KIs against CA IX (2.85 and 2.81
respectively).
mM,
Fig. 1. Significant NOE interactions (in blue) and 1He13C correlations (in red) observed
in the NOESY and HMBC spectra of 2o. (For interpretation of the references to color in
this figure legend, the reader is referred to the Web version of this article.)
Interestingly, ninhydrins 2a-t weakly inhibit (2i, 2l, 2n, 2t
showed KIs in the range 39.7e84.6 M) or did not inhibit CA IV up
to a 100 M concentration.
m
m
drop of inhibition efficacy up to more than 2-orders of magnitude
(compounds 2d, 2e, 2h, 2i, 2n, 2p are micromolar CA VII inhibitors).
In contrast, ninhydrin 2a acts as a weak CA II inhibitor (KIs of
2.2.1. Molecular modelling
The unsubstituted derivative 2a was taken as reference com-
pound to investigate the ninhydrin binding mode of the studied
derivatives within CAs. Quantum mechanics (QM) derived atom
charges were used for docking 2a within CA II (pdb 5JLT) and CA VII
(pdb 3MDZ) active sites. Three top-scored binding poses (bp) for
22.7
matic ring increase ninhydrin CA II inhibitory action to a nanomolar
range (KIs in the range 0.20-0.84 M), that are 4-OH (2c), 4-OCH3
(2e), 4-NO2 (2g), 5-F (2j), 5-NO2 (2k), 5-OH (2 m), 4,7-diOCH3 (2q),
mM) and only certain investigated substitutions on the aro-
m
3

A. Bouzina, M. Berredjem, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112875
Table 1
pocket of CA II and VII, lined by L198, F131, L141, V121, and V143, up
to the edge with the hydrophilic active site area represented by
Q92.
Inhibition data of human CA I, CA II, CA IV, CA VII, CA IX with ninhydrins 2a-t re-
ported here and the standard sulfonamide inhibitor acetazolamide (AAZ) by a
stopped flow CO2 hydrase assay.
In order to improve the confidence of the binding mode pre-
diction MM-GBSA score refinement and metadynamics simulations
were performed.
First, a MM-GBSA procedure was applied to optimize all six
binding modes within the respective CAs active site using the VSGB
solvation model and setting the target flexible within 3 Å around
the ligand. In CA II, the stability of the binding conformation,
measured in term of the computed binding free energy value, re-
flects the order bp1>bp2>bp3 (Table 2) while bp3 represents the
energetically most favored binding orientation in CA VII
(ꢁ24.53 kcal/mol). The pose stability was assessed with Desmond
metadynamics simulations monitoring the fluctuations of the
ligand RMSD (Root Mean Square Deviation) over the course of the
MD, and the persistence of important contacts, such as hydrogen
Cmpd
R
KI (m
M)a
CA I CA II
CA IV CA VII CA IX
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
H
4-F
5.38 22.7
8.57 18.4
16.7 0.73
23.7 6.36
0.87 0.84
4.86 14.3
26.8 0.41
6.56 6.39
7.92 49.6
7.79 0.58
28.4 0.61
0.57 37.4
19.7 0.44
7.10 78.2
36.4 3.12
>100
>100
>100
>100
>100
>100
>100
>100
84.6
0.074
0.62
0.083
21.3
5.36
1.84
0.28
1.26
2.46
2.37
44.3
17.1
1.34
34.1
51.4
31.0
61.2
16.8
38.7
29.6
2.85
27.8
13.4
54.3
8.41
68.2
30.4
2.81
46.7
0.025
4-OH
4-OiPr
4-OCH3
4-CH3
4-NO2
4-Br
5-CH3
5-F
5-NO2
5-Br
bonds and
p-p interactions, between the ligand and the counter-
part including the receptor and any other cofactors or solvent
molecules. The RMSD of the simulated ligand with respect to the
starting pose, evaluated after the target alignment, was used as the
collective variable for the metadynamics simulation. To improve
the statistics, 10 simulations have been performed for each bp and
the results were averaged over the simulations. Table 2 and Fig. 4
show the outcome of the metadynamics calculations.
The outcomes of the metadynamics (Table 2 and Fig. 4) suggested
similar but different 2a conformations are the most energetically
favorable binding poses in CAII (bp1) and CA VII (bp3). This evidence
is also confirmed by both the binding persistence (BP) and binding
PoseScore (BPS) values, which represent, respectively, the average
persistence of contacts and the expectation of the RMSD of the pose
over the course of the metadynamics trajectories. The linear com-
bination of these parameters, i. e the Binding CompScore (BCS),
corroborates the complex stability trend bp1>bp2>bp3 for CA II and
bp3>bp1>bp2 for CA VII. These findings lead to the identification of
the putative binding conformations of 2a within the enzyme iso-
forms (Fig. 5). The narrower active site of CA VII as compared to that
one of CA II, well accommodates and stabilizes the bp3 preferred
conformation of compound 2a, embedding the ligand within its
lipophilic cavity better than for bp2 conformation in CA II.
>100
>100
71.3
0.27
0.030
0.39
5-OH
5-OCH3
4-OCH3-7-iPr
>100
74.3
0.065
2.06
>100
>100
>100
>100
>100
39.7
0.026
9.71
0.19
0.029
0.51
0.025
4-OCH3-7-(CH2NHCOCH2Cl) 8.10 8.90
4,7-diOCH3
5,6-diOCH3
4,6-diBr
4,5,6,7-tetraBr
e
8.23 0.35
41.4 0.20
36.7 26.7
4.79 0.26
2s
2t
AAZ
0.25 0.012 0.074 0.006
a
Mean from 3 different assays, by a stopped flow technique (errors were in the
range of 5e10% of the reported values).
each CA isoform were computed which exhibit both a tetrahedral
zinc-binder character (namely bp1 and bp2 in CA II and bp1 and
bp3 in CA VII) and a dual coordination (as trigonal bipyramid)
around the metal atom (bp3 in CA II and bp2 in CA VII) (Figs. 2 and
3). Notably, the tetra-coordinated bp establish a series of H-bonds
with the residues Thr199 and Thr200 which may involve either the
ligand hydroxyl or carbonyl (OH or the CO) groups or both. Addi-
tionally, bp1 in CA II features a H-bond between one] CO group of
the ligand and H64 side chain. In contrast, a less extended network
of H-bonds features the trigonal bipyramid bp where only one (bp3
in CA II, Fig. 2) or two (bp2 in CA VII, Fig. 3) H-bonds are formed
with residues Thr199 and Thr200. In all bp, the aromatic portion of
ninhydrin 2a favorably accommodates within the lipophilic binding
As shown in Table 1, the incorporation of substituents on the
ninhydrin scaffold can thin the inhibition differences existing for 2a
between CA II and VII. Moreover, significant amino acid mutations
existing at the edge of CA II and CA VII binding sites (i.e. N67Q,
E69D, I91K, L204S) could influence the entrance of the geminal diol
derivative having a role in CA VII improved inhibition of 2a with
respect to CA II.
Fig. 2. Top-ranked binding poses for ninhydrin 2a within CA II active site (pdb 5JLT).
4

A. Bouzina, M. Berredjem, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112875
Fig. 3. Top-ranked binding poses for ninhydrin 2a within CA VII active site (pdb 3MDZ).
Table 2
MM-GBSA derived binding
average) outcomes with ninhydrin 2a.
related diseases. CA IV was instead the least affected isoform. A
computational protocol composed by docking, MM-GBSA and
metadynamics simulations suggested the most putative binding
mode of ninhydrin into the active site of CA II and CA VII. The above
findings testify that such pharmacologically underestimated li-
gands may represent interesting lead compounds for the devel-
opment of CA inhibitors possessing an innovative mechanism of
action, which is not very common to other classes of CAIs, i.e., co-
ordination of one or two OH moieties from a diol to the zinc ion
from the enzyme active site. In particular, the inhibition profile of
ninhydrins, i.e. low nanomolar CA VII inhibition vs high nanomolar/
low micromolar CA II inhibition, make them of relevant interest in
the field of CNS-related pathologies that implicate CA (e.g. neuro-
pathic pain). In fact, CA II is widespread and ubiquitous in human
tissues, whereas CA VII might be considered a CNS-associated
enzyme, and its inhibition is expected to induce a minor number
of side effects than CA II.
DG values and binding pose metadynamics (10 trials
Binding pose (bp)
DG binding
BPa
BPSb
BCSc
CA II
bp1
bp2
bp3
CA VII
bp1
ꢁ28.56
ꢁ26.14
ꢁ15.58
0.377
0.295
0.109
1.793
2.245
2.112
ꢁ0.094
0.768
1.566
ꢁ19.26
ꢁ19.84
ꢁ24.53
0.094
0.150
0.164
2.429
3.045
1.938
1.959
2.295
1.120
bp2
bp3
a
BP: Binding Persistence, the average persistence of contacts over the course of
the metadynamics trajectories. Higher values equate to more stable complexes.
b
BPS: Binding PoseScore, the expectation of the RMSD of the pose over the course
of the metadynamics. Lower values equate to more stable complexes.
c
BCS: Binding CompScore, the composite score linearly combining the BPS and
BP scores. Lower values equate to more stable complexes.
3. Conclusion
4. Experimental section
Ninhydrin derivatives have an extensive application in organic
and applied chemistry and agriculture but have been scarcely
investigated in the medicinal chemistry field. Ninhydrin and a se-
ries of derivatives substituted on its aromatic ring are here pro-
posed as inhibitors of human carbonic anhydrases (CAs, EC 4.2.1.1),
based on earlier evidence that gem diol compounds can coordinate
the metal ion from the CA active site. Ninhydrins act as micromolar
4.1. Chemistry
4.1.1. Methods
Chemicals are named according to IUPAC nomenclature. All of
the reagents were purchased from Sigma-Aldrich and Thermo-
Fisher Scientific. Microwave reactions were done on a Biotage
Initiator Microwave synthesizer 2.0440 W. Melting points were
determined on an Electrothermal 9200 capillary apparatus. The IR
spectra were recorded on a PerkinElmer Spectrum Two IR spec-
trometer. The 1H and 13C NMR spectra were recorded at 400 MHz
inhibitors of CA I and IX (KIs in the range 0.57e68.2
low nanomolar inhibitors against CA II and VII (KIs in the range
0.025e78.2 M), recently validated as targets in several CNS-
mM) and up to
m
Fig. 4. RMSD average value (10 trials) for 2a from each of the three initial binding pose per isoform along the simulation course with CA II (left) and CA VII (right).
5

A. Bouzina, M. Berredjem, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112875
- cyclohexane or dichloromethane - acetone) to afford the corre-
sponding ninhydrin.
4.1.3. Spectral data of ninhydrins 2d, 2o, 2p and 2s
4.1.3.1. 2,2-Dihydroxy-4-isopropoxyindane-1,3-dione
(2d). Rf
(cyclohexane - ethyl acetate 1:1): 0.28; pink solid; yield: 79%; mp
119e121 ꢀC; IR
y
(cmꢁ1): 3270 (OH), 1749 (C¼O]), 1709 (C¼O]);
1H NMR (400 MHz, DMSO‑d6):
d
7.92 (dd, 1H, J ¼ 8.3 Hz, J ¼ 7.3 Hz,
H-6), 7.61 (d, 1H, J ¼ 8.3 Hz, H-7), 7.47 (d, 1H, J ¼ 7.3 Hz, H-5), 7.37 (s,
2H, OH), 4.87 (sept, 1H, J ¼ 6.0 Hz, CH(CH3)2), 1.35 (d, 6H, J ¼ 6.0 Hz,
CH3); 13C NMR (100.62 MHz, DMSO‑d6):
d
197.35 (C¼O]), 194.27
(C¼O]),156.38 (Cquat),140.14 (CH), 138.80 (Cquat),126.15 (Cquat),
121.52 (CH), 114.67 (CH), 87.33 (C(OH)2), 71.29 (OCH), 21.62 (2CH3);
HRMS calcd for C12H12NaO5 [MþNa]þ 259.0577, found: 259.0571.
4.1.3.2. 2,2-Dihydroxy-4-isopropyl-7-methoxyindane-1,3-dione (2o).
Rf (dichloromethane - acetone 9:1): 0.43; yellow solid; yield: 78%;
mp 127e129 ꢀC; IR
y
(cmꢁ1): 3390 (OH), 1740 (C¼O]), 1704
(C¼O]); 1H NMR (400 MHz, DMSO‑d6):
d
7.90 (d, 1H, J ¼ 8.8 Hz),
7.55 (d, 1H, J ¼ 8.8 Hz), 7.33 (bs, 2H, OH), 4.03 (m, 1H, CH(CH3)2),
3.94 (s, 3H, OCH3), 1.21 (d, 6H, J ¼ 7.1 Hz); 13C NMR (100.62 MHz,
DMSO‑d6):
d
198.41 (C¼O]), 194.31 (C¼O]), 155.97 (Cquat),
140.66 (Cquat), 136.04 (CH), 134.98 (Cquat), 125.73 (Cquat), 119.91
(CH), 87.01 (C(OH)2), 56.21 (OCH3), 26.88 (CH(CH3)2), 22.91 (2CH3);
HRMS calcd for C13H13O4 [M-H2O þ H]þ 233.0808, found 233.0812.
4.1.3.3. 2-Chloro-N-((2,2-dihydroxy-7-methoxy-1,3-dioxoindan-4-yl)
methyl)acetamide (2p). Rf (dichloromethane - acetone 7:3): 0.40;
Fig. 5. Superimposition of most stable 2a (light green)/CA II (white) and 2a (dark
green)/CA VII (blue) adducts predicted on the basis of MM-GBSA and metadynamics
simulations. L204S, V207A, E69D notations indicate CA II/VII mutated residues. (For
interpretation of the references to color in this figure legend, the reader is referred to
the Web version of this article.)
beige solid; yield: 60%; mp 118e120 ꢀC; IR
y
(cmꢁ1): 3359 (OH),
3218 (NH), 1763 (C¼O]), 1715 (C¼O]), 1669 (C¼O]); 1H NMR
(400 MHz, DMSO‑d6):
d
8.75 (t, 1H, J ¼ 5.9 Hz, NH), 7.79 (d, 1H,
J ¼ 8.6 Hz, HeAr), 7.58 (d, 1H, J ¼ 8.7 Hz, HeAr), 7.41 (bs, 2H, 2OH),
4.70 (d, 2H, J ¼ 5.9 Hz, CH2NH), 4.17 (s, 2H, CH2CO), 3.95 (s, 3H,
on a Bruker DRX 400 spectrometer or at 300 MHz on a Bruker AM
300 spectrometer. Chemical shifts are expressed in ppm (d)
OCH3); 13C NMR (100.62 MHz, DMSO‑d6):
d
198.34 (C¼O]), 194.31
(C¼O]), 166.47 (Cquat), 156.81 (Cquat), 137.44 (CH), 135.59
(Cquat), 130.08 (Cquat), 125.75 (Cquat), 119.69 (CH), 87.11 (C(OH)2),
56.40 (OCH3), 42.65 (CH2), 38.55 (CH2); HRMS calcd for
downfield from internal tetramethylsilane and coupling constants J
are reported in hertz (Hz). The following abbreviations are used: s,
singlet; bs, broad singlet; d, doublet; t, triplet; dd, doubled doublet;
q, quartet; qui, quintuplet; sept, septuplet; m, multiplet; Cquat,
quaternary carbons. The mass spectra were performed by direct
ionization (EI or CI) on a ThermoFinnigan MAT 95 XL apparatus.
Chromatographic separations were performed on silica gel col-
umns by column chromatography (Kieselgel 300e400 mesh). All
reactions were monitored by TLC on GF254 plates that were visu-
alized under a UV lamp (254 nm). Evaporation of solvent was
performed in vacuum with rotating evaporator. The purity of the
final compounds (greater than 95%) was determined by uHPLC/MS
on an Agilent 1290 system using a Agilent 1290 Infinity ZORBAX
C
13H11ClNO5 [M-H2O þ Hþ]þ 296.0320, found 296.0329.
4.1.3.4. 4,6-Dibromo-2,2-dihydroxyindane-1,3-dione (2s). Rf (cyclo-
hexane - ethyl acetate 7:3): 0.57; white solid; yield: 76%; mp
147e149 ꢀC; IR
y
(cmꢁ1): 3394 (2OH), 1761 (C¼O]), 1730 (C¼O]),
1182 (CeO); 1H NMR (400 MHz, DMSO‑d6):
d
8.47 (d, 1H, J ¼ 1.6 Hz,
H-5), 8.17 (d, 1H, J ¼ 1.6 Hz, H-7), 7.50 (bs, 2H, OH); 13C NMR
(100.62 MHz, DMSO‑d6):
d
194.11 (C¼O]), 193.66 (C¼O]), 142.97
(CH), 141.19 (Cquat), 130.83 (Cquat), 129.84 (Cquat), 125.76 (CH),
119.70 (Cquat), 87.13 (C(OH)2); HRMS calcd for C9H4Br2NaO4
[MþNa]þ 356.8369, found 356.8371.
Eclipse Plus C18 column (2.1 mm ꢂ 50 mm,1.8
m
m particle size) or a
Poroshell 120 Agilent infinity lab (2.1 mm ꢂ 50 mm, 2.7
mm particle
4.1.4. Synthetic procedure of 4,5,6,7-tetrabromo-2,2-
dihydroxyindane-1,3-dione (2t)
size) with a gradient mobile phase of H2O/CH3CN (90:10, v/v) with
0.1% of formic acid to H2O/CH3CN (10:90, v/v) with 0.1% of formic
acid at a flow rate of 0.5 mL/min, with UV monitoring at the
wavelength of 254 nm with a run time of 10 min.
A sealed-pressurised reaction vessel (20 mL) equipped with a
magnetic stirrer and charged with 4 (1.85 g, 4.01 mmol), selenium
dioxide (667 mg, 6.01 mmol) and dioxane (15 mL), was irradiated
5 min at 180 ꢀC with a maximum of 400 W. Then, the vessel was
rapidly forced-air cooled to room temperature. The mixture was
transferred into a round bottom flask, and the vessel washed with
acetone. Silica was added to prepare a solid deposit. The volatile
solvents were then evaporated in vacuo before purification by flash
chromatography (ethyl acetate 1/cyclohexane 1) to afford the tet-
rabromoninhydrin 2t (1.39 g, 2.81 mmol, 70%).
4.1.2. Synthesis of ninhydrins 2d, 2o, 2p and 2s
A sealed-pressurised reaction vessel (5 mL) equipped with a
magnetic stirrer was charged with indan-1-one (1 eq), selenium
dioxide (3.1 eq) and dioxane/water (3 mL/0.3 mL). It was then
irradiated with microwave heating to 180 ꢀC with a maximum of
400 W for 5 min. Then, the vessel was rapidly forced-air cooled to
room temperature. The mixture was transferred into a round bot-
tom flask, and the vessel washed with acetone. Silica was added to
prepare a solid deposit. The volatile solvents were then evaporated
in vacuo before purification by flash chromatography (ethyl acetate
Rf (cyclohexane - ethyl acetate 2:1): 0.26; red solid; yield: 70%;
mp > 160 ꢀC decomposition; IR
y
(cmꢁ1): 3347 (OH, 1763 (C¼O]),
1729 (C¼O]), 1163 (CeO); 1H NMR (300 MHz, DMSO‑d6):
d 7.75
(bs, 2H, OH); 13C NMR (75 MHz, DMSO‑d6): 192.55 (2]C¼O),
6

A. Bouzina, M. Berredjem, A. Nocentini et al.
European Journal of Medicinal Chemistry 209 (2021) 112875
139.80 (2Cquat), 137.08 (2Cquat), 121.97 (2Cquat), 86.59 (C(OH)2);
15 min (standard assay at room temperature) prior to assay, to allow
for the formation of the E-I complex. The inhibition constant (KI) was
subsequently obtained from the Michaelis-Menten equation, which
has been fitted by non-linear least squares using software PRISM 3.
Enzyme concentrations in the assay system were in the range of
5e12 nM. The CA isoforms employed were obtained in-house and
were recombinant proteins reported earlier by our group [28e30].
MS-ESI calcd for C9H2Br4O4 [MþH]þ 490.67, found: 490.70.
4.1.5. Synthetic procedure of 7-isopropyl-4-methoxyindan-1-one
(1o)
To a stirring solution under argon atmosphere of sulfuric acid
(5 mL) at 0 ꢀC, was added portion wise 1e (500 mg, 3.09 mmol). The
mixture was then heated to 60 ꢀC and isopropanol (0.48 mL,
6.18 mmol) rapidly added and left 2 h more stirring. The reaction
mixture was then poured into crushed ice and the pH adjusted to
approximatively 7 with a 20% aqueous solution of NaHCO3. After
extraction of the aqueous layer, with dichloromethane (3 ꢂ 30 mL),
the combined layers were washed with H2O, dried over Na2SO4,
filtered and evaporated under reduced pressure. The obtained
residue was purified by flash chromatography (cyclohexane - ethyl
acetate 9:1) to afford 1o (220 mg, 1.08 mmol, 41%).
4.3. In silico studies
The crystal structure of CA II (pdb 5JLT) [58] and CA VII (pdb
3MDZ) [59] were prepared using the Protein Preparation Wizard
€
tool implemented in Maestro - Schrodinger suite, assigning bond
orders, adding hydrogens, deleting water molecules, and opti-
mizing H-bonding networks [46]. Energy minimization protocol
with a root mean square deviation (RMSD) value of 0.30 was
applied using an Optimized Potentials for Liquid Simulation
(OPLS3e) force field. The structure of 2a was submitted to QM
optimization (B3LYP/6-31G*þ) and ESP charges calculation with the
Rf (cyclohexane - ethyl acetate 9:1): 0.71; yellow solid; yield:
41%; mp 59e61 ꢀC; IR
y
(cmꢁ1): 1692 (]C¼O); 1H NMR (400 MHz,
CDCl3):
d
7.25 (dd, 1H, J ¼ 8.3 Hz, J ¼ 0.5 Hz, H-6), 6.98 (d, 1H,
J ¼ 8.3 Hz, H-5), 4.10 (sept, 1H, J ¼ 6.9 Hz, CH(CH3)2), 3.88 (s, 3H,
€
Jaguar module of Schrodinger [6e]. Grids were centered on the
OCH3), 3.00e2.93 (m, 2H, CH2CO), 2.69e2.62 (m, 2H, CH2), 1.23 (d,
centroids of the co-crystallized ligands and 2a was docked using
QM-computed charges and the standard precision mode (SP). The
best poses (bp1-3) within CA II and CA VII, evaluated in terms of
score, anchorage, hydrogen bond interactions and hydrophobic
contacts, were submitted to optimization and their binding free
energies was evaluated by the Prime MM-GBSA protocol [6a], using
QM charges and a VSGB solvation model.
6H, J ¼ 6.9 Hz, CH(CH3)2); 13C NMR (100.62 MHz, CDCl3):
d 207.99
(]C¼O), 154.78 (Cquat), 144.80 (Cquat), 141.45 (Cquat), 134.38
(Cquat), 124.89 (CH), 114.73 (CH), 55.48 (OCH3), 36.94 (CH2), 26.55
(CH iPr), 23.40 (2CH3), 21.82 (CH2); LC-MS calcd for C13H17O2
[MþH]þ 205.12, found: 205.10.
4.1.6. Synthetic procedure of 2-chloro-N-((7-methoxy-3-oxo-
indan-4-yl)methyl)acetamide (1p)
Additionally, bp1-3 for both CA II and VII were submitted to
metadynamics simulations using Desmond [50] and the OPL3e
force field (10 simulations have been performed for each bp).
Specifically, the default parameters of the binding pose metady-
namics tool implemented in Desmond were used.
A solution of 1e (2.00 g, 12.3 mmol), acetic acid (9 mL), sulfuric
acid (1 mL) and 2-chloro-N-(hydroxymethyl)acetamide (1.28 g,
13.5 mmol) was microwave irradiated at 70 ꢀC for 10 min. After
cooling, the reaction mixture was transferred in a round bottom
flask, the vessel washed with ethyl acetate. Then, silica was added
to prepare a solid deposit. The volatile solvents were then evapo-
rated in vacuo before purification by flash chromatography (cyclo-
hexane - ethyl acetate 7:3) to afford 1p (2.76 g, 10.3 mmol, 84%).
Rf (cyclohexane - ethyl acetate 7:3): 0.40; beige solid; yield:
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
84%; mp 119 ꢀC; IR
y
(cmꢁ1): 3283 (NH), 1702 (]C¼O), 1645 (]
C¼O); 1H NMR (400 MHz, CDCl3):
d 7.99 (bs, 1H, NH), 7.32 (d,
Acknowledgements
J ¼ 8.1 Hz, 1H, H-6), 6.95 (d, J ¼ 8.1 Hz, 1H, H-5), 4.62 (d, J ¼ 6.6 Hz,
2H, CH2NH), 3.97 (s, 2H, CH2Cl), 3.89 (s, 3H, OCH3), 3.07e2.99 (m,
2H, CH2CO), 2.76e2.68 (m, 2H, CH2); 13C NMR (100.62 MHz, CDCl3):
This work was financially supported in part by the General
Directorate for Scientific Research and Technological Development
(DG-RSDT), the Algerian Ministry of Scientific Research, the Applied
Organic Chemistry Laboratory (FNR 2000) and by the Italian Min-
istry for University and Research (MIUR), grant PRIN: 2017XYBP2R.
d
209.30 (]C¼O), 165.98 (N]C¼O), 156.98 (Cquat), 146.25 (Cquat),
136.04 (Cquat), 130.31 (CH), 128.31 (Cquat), 114.86 (CH), 55.89
(OCH3), 43.04 (CH2), 40.65 (CH2), 36.86 (CH2), 22.88 (CH2); HRMS
calcd for C13H15ClNO3 [MþH]þ 268.0735, found 268.0733.
Appendix A. Supplementary data
4.2. Carbonic anhydrase inhibition
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112875.
An Applied Photophysics stopped-flow instrument was used for
assaying the CA catalyzed CO2 hydration activity [27]. Phenol red (at
a concentration of 0.2 mM) was used as indicator, working at the
absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5) as
buffer, 0.1 M Na2SO4 (for maintaining constant ionic strength),
following the CA-catalyzed CO2 hydration reaction for a period of
10 s at 25 ꢀC. The CO2 concentrations ranged from 1.7 to 17 mM for
the determination of the kinetic parameters and activation con-
stants. For each inhibitor at least six traces of the initial 5e10% of the
reaction have been used for determining the initial velocity. The
uncatalyzed rates were determined in the same manner and sub-
tracted from the total observed rates. Stock solutions of activators
(10 mM) were prepared in distilled-deionized water and dilutions up
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