Discovery of novel dual-active 3-(4-(dimethylamino)phenyl)-7-aminoalcoxy-coumarin as potent and selective acetylcholinesterase inhibitor and antioxidant

Article abstract of DOI:10.1080/14756366.2019.1571270

A series of 3-substituted-7-aminoalcoxy-coumarin was designed and evaluated as cholinesterase inhibitors and antioxidants. All compounds were effective in inhibiting AChE with potencies in the nanomolar range. The 3-(4-(dimethylamino)phenyl)-7-aminoethoxy-coumarin (6a) was considered a hit, showing good AChE inhibition potency (IC₅₀ = 20 nM) and selectivity (IC₅₀ BuChE/AChE = 354), quite similar to the reference drug donepezil (IC₅₀ = 6 nM; IC₅₀ BuChE/AChE = 365), also presenting antioxidant properties, low citotoxicity and good-predicted ADMET properties. The mode of action (mixed-type) and SAR analysis for this series of compounds were described by means of kinetic and molecular modeling evaluations.

Full text of DOI:10.1080/14756366.2019.1571270

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Discovery of novel dual-active 3-(4-
(dimethylamino)phenyl)-7-aminoalcoxy-coumarin
as potent and selective acetylcholinesterase
inhibitor and antioxidant
Gabriela Alves de Souza, Soraia John da Silva, Catarina de Nigris Del Cistia,
Paulo Pitasse-Santos, Lucas de Oliveira Pires, Yulli Moraes Passos, Yraima
Cordeiro, Cristiane Martins Cardoso, Rosane Nora Castro, Carlos Mauricio R.
Sant’Anna & Arthur Eugen Kümmerle
To cite this article: Gabriela Alves de Souza, Soraia John da Silva, Catarina de Nigris Del Cistia,
Paulo Pitasse-Santos, Lucas de Oliveira Pires, Yulli Moraes Passos, Yraima Cordeiro, Cristiane
Martins Cardoso, Rosane Nora Castro, Carlos Mauricio R. Sant’Anna & Arthur Eugen Kümmerle
(2019) Discovery of novel dual-active 3-(4-(dimethylamino)phenyl)-7-aminoalcoxy-coumarin as
potent and selective acetylcholinesterase inhibitor and antioxidant, Journal of Enzyme Inhibition
and Medicinal Chemistry, 34:1, 631-637, DOI: 10.1080/14756366.2019.1571270
To link to this article: https://doi.org/10.1080/14756366.2019.1571270
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2019 The Author(s). Published by Informa
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
019, VOL. 34, NO. 1, 631–637
2
https://doi.org/10.1080/14756366.2019.1571270
SHORT COMMUNICATION
Discovery of novel dual-active 3-(4-(dimethylamino)phenyl)-7-aminoalcoxy-
coumarin as potent and selective acetylcholinesterase inhibitor and antioxidant
a,b
a
a
a
Gabriela Alves de Souza , Soraia John da Silva , Catarina de Nigris Del Cistia , Paulo Pitasse-Santos ,
a
c
c
a
a
Lucas de Oliveira Pires , Yulli Moraes Passos , Yraima Cordeiro , Cristiane Martins Cardoso , Rosane Nora Castro ,
Carlos Mauricio R. Sant’Anna and Arthur Eugen K u€ mmerle
a
a
a
b
Programa de P ꢀo s-Gradu c¸ ~a o em Qu ꢀı mica (PPGQ), Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro, Brazil; Laborat ꢀo rio de
Diversidade Molecular e Qu ꢀı mica Medicinal (LaDMol-QM, Molecular Diversity and Medicinal Chemistry Laboratory), Departament of Chemistry,
c
Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro, Brazil; Faculdade de Farm ꢀa cia, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, Brazil
ABSTRACT
ARTICLE HISTORY
A series of 3-substituted-7-aminoalcoxy-coumarin was designed and evaluated as cholinesterase inhibitors
and antioxidants. All compounds were effective in inhibiting AChE with potencies in the nanomolar range.
The 3-(4-(dimethylamino)phenyl)-7-aminoethoxy-coumarin (6a) was considered a hit, showing good AChE
inhibition potency (IC50 ¼ 20 nM) and selectivity (IC50 BuChE/AChE ¼ 354), quite similar to the reference
Received 24 October 2018
Revised 10 December 2018
Accepted 11 December 2018
KEYWORDS
Coumarins; cholinesterase;
antioxidant; bioisosterism
drug donepezil (IC50 ¼ 6 nM; IC BuChE/AChE ¼ 365), also presenting antioxidant properties, low citotox-
50
icity and good-predicted ADMET properties. The mode of action (mixed-type) and SAR analysis for this ser-
ies of compounds were described by means of kinetic and molecular modeling evaluations.
Introduction
however, PTau becomes hyperphosphorylated, denaturing and
resulting in its dissociation of microtubules, followed by formation
of neurofibrillary filaments that aggregate, acting as physical bar-
Alzheimer’s disease (AD) is the most frequent case of age-related
neurodegenerative dementia, characterized by progressive loss of
4
1
,2
riers to microtubules . In addition, the occurrence of glial cell neu-
memory and other cognitive functions . AD is a heterogeneous
disease, driven by the interaction between multiple deleterious
factors. However, the exact mode of how these factors contribute
to impair neuronal functions and neuronal survival still remains
undetermined. One of the main markers of AD is the accumula-
roinflammation, synaptic loss, and specific neuronal death is
5
6
common in AD and can be aggravated by oxidative stress .
The knowledge of neurotransmitter disorders in AD has led to
7
the approval of drugs with symptomatic effects . The cholinergic
tion of b-amyloid plaques (Ab) in nerve cells. In healthy brain, hypothesis of AD states that the degeneration of cholinergic neu-
3
these aggregates of proteins are degraded and eliminated . rons in basal forebrain nuclei causes disorders in the presynaptic
However, in AD the aggregates accumulate to form insoluble pla- cholinergic terminals in the hippocampus and neocortex, which
3
ques . Another characteristic is the presence of insoluble neurofib- are regions of extreme importance for memory disorders and
4
8
rillary filaments that is associated with tau protein (PTau) . In AD, other cognitive symptoms . Because of neurodegeneration, the
CONTACT Arthur Eugen K u€ mmerle
Serop ꢀe dica, Rio de Janeiro, Brazil
akummerle@hotmail.com
Programa de P ꢀo s-Gradu c¸ ~a o em Qu ꢀı mica (PPGQ), Universidade Federal Rural do Rio de Janeiro,
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

6
32
G. A. DE SOUZA ET AL.
activity of cholinergic neurons and levels of neurotransmitter was precipitated in hexanes under ultrasound irradiation and fil-
ACh are reduced. One approach to improve cholinergic neuro- tered off.
transmission is to increase the availability of ACh by inhibition of
9
acetylcholinesterase .
General procedure for the synthesis of 3a–d
Acetyl (AChE) and butyrylcholinesterase (BuChE) inhibitors are
the main drugs for the clinical treatment of AD in the initial to
moderate stage . Galantamine and donepezil are selective inhibi-
To a stirred solution of 1.7 mmol of the respective O-alkyl couma-
rin derivative (2a–d), 5 mmol (3 eq.) of sodium acetate in 8 ml of
1
0
tors of AChE, whereas rivastigmine inhibits AChE and BuChE with glacial acetic acid and 2.1 mmol (1.3 eq.) of Br were slowly added
2
similar affinities. Selective AChE inhibitors have demonstrated bet- (Scheme 2). The reaction was stirred at room temperature for 2 h.
1
1
ter therapeutic effects when compared to nonselective inhibitors
After reagent consumption, the reaction mixture was poured to a
since BuChE is also associated with drug metabolism and detoxifi- beaker containing crushed ice. The formed precipitate was filtered
12
cation, lipoprotein metabolism and diseases . Thus, our objectives off under vacuum and purified by silica gel column chromatog-
herein were the design, synthesis and pharmacological evaluation raphy (hexanes: dichloromethane mixture, 50–90% gradi-
of novel 3-substituted-7-aminoalcoxy-coumarins as selective inhibi- ent elution).
tors of AChE and antioxidant, based on a previously described
1
3
indanone series .
General procedure for the synthesis of 4a–d
In a reactional vessel, 1.3 mmol of the respective 3-bromo-7-(bro-
moalkoxy)coumarin derivatives (3a–d) and 3.9 mmol (3 eq.) of
Materials and methods
General procedure for the synthesis of 2a–d
piperidine were dissolved in 8 ml CH CN (Scheme 2). The reaction
was kept under stirring at 60 C for 3–8 h. Acetonitrile was evapo-
rated in a rotary evaporator and the respective products purified
by silica gel column chromatography (dichloromethane: methanol,
3
ꢀ
In a reactional borosilicate tube, 10–15 mmol of dibromoalkanes
(
4–6 eq.) and 5 mmol (2 eq.) of K
acetone (Scheme 2). To this stirred suspension a solution of
.5 mmol of 7-hydroxycoumarin (1) in 8 ml of acetone was added
2 3
CO were solubilized in 2 ml of
0
–25% gradient elution).
2
dropwise. Thereafter, the reactional tube was sealed and the reac-
ꢀ
tion was kept at 60 C and stirred for 6–12 h. After reaction com-
General procedure for the synthesis of 5a–c and 6a–c
pletion, acetone was evaporated and the crude reaction
partitioned with distilled water and ethyl acetate. The final slurry
In a reaction borosilicate tube, 0.14 mmol of the corresponding
derivative (4a, 4b and 4d), 0.20 mmol (1.4 eq.) of appropriate phe-
nylboronic acid and 0.42 mmol (3 eq.) of K CO were solubilized in
2
3
4
2
ml of a solvent mixture (water: ethanol: toluene (2:1:1)) (Scheme
). The reaction was degassed with N then 0.01 mmol (7 mol%) of
2
Pd(PPh
3
)
4
catalyst added. The reaction tubes were sealed and the
ꢀ
mixtures were subjected to magnetic stirring and heating at 65 C
for 3–5 h. At the end of the reaction, the solvent mixture was
evaporated in a rotary evaporator and the respective products
purified by silica gel column chromatography (dichloromethane:
methanol, 0–25% mixture gradient elution).
Cholinesterase inhibition and kinetics assays
Activity of enzymes and inhibition kinetics were determined using
a Bio-Rad iMark microplate reader based on a modification of the
1
4,15
Ellman method.
Compounds were dissolved in DMSO. The
0
assay solution which contained 60 mL 5,5 -Dithiobis(2-nitrobenzoic
acid) (DTNB) at 1.1 mM, 30 mL AChE/BuChE at 0.20 U/mL (initial
concentration) and 150 mL tested compound solution with
Scheme 1. Design of alkylamino-coumarin cholinesterase inhibitors series.
ꢀ
Scheme 2. Reagents and conditions: (i) Br(CH
2
)
n
Br (n ¼ 2–5), K
2
CO
3
, acetone, 60 C, 2-8h, 68–78%; (ii) Br
2
, AcOH, NaOAc, r.t., 2h, 79–84%; (iii) piperidine, acetonitrile,
ꢀ
ꢀ
6
0 C, 2-5h, 95–99%; (iv) Ph-B(OH)
2
or 4-(Me)
2
N-Ph-B(OH) , Na CO
2
2
3
, Pd(PPh
3 4
) , H
2
O, EtOH, PhMe, 80 C, 3h, 70–75%.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
633
different concentrations. Absorbance was then recorded at coumarins (2a–d) using Br
2
in buffered medium of sodium acet-
ꢀ
k ¼ 415 nm. After 10-min incubation at 30 C, 24 mL acetylthiocho- ate/acetic acid at room temperature, furnishing the brominated
line iodide/S-butyrylthiocholine iodide (at 2.75 mM for activity derivatives (3a–d) in yields ranging between 79–84%. These inter-
inhibition assay and 2.75–0.44 mM for kinetic study assay) were mediates (3a–d) were then subjected to amination reactions with
added and the absorbance recorded after a 10-min incubation (for piperidine in acetonitrile, leading to the formation of the desired
activity inhibition assay) or after 0–20 min incubation (for kinetic 7-amino-alkoxy-3-bromo-coumarin derivatives (4a–d) as yellow
ꢀ
study assay) at 30 C.
solids in 95–99% yields after purification by flash chromatography.
From the 7-amino-alkoxy-3-bromo-coumarin derivatives with 2, 3
and 5 methylene spacers (4a, 4b, 4d), Suzuki cross coupling reac-
Molecular modeling
3 4
tions were then carried out using Pd(PPh ) catalyst and phenyl
and 4-dimethylamino-phenyl boronic acids to obtain the final ary-
lated 3-substituted coumarins (5a–c and 6a–c) in yields ranging
between 70–75% after purification by flash chromatography
For EeAChE (Electrophorus electricus), the PDB structure 1C2O was
used; for EqBuChE (Equus caballus), a 3 D homology model was
necessarily built from a sequence available in the UniProtKB/
Swiss-Prot (entry Q9N1N9) with the automated mode of the pro-
(Scheme 2).
1
6
tein structure homology-modeling server, Swiss-Model , using as
17
template the human BuChE (PDB 4TPK) . Spartan’14 program
Wavefunction, Inc.] was utilized to construct and optimize the
Cholinesterase inhibitory activity, biological profile, and
SAR analyses
[
18
inhibitors with the PM6 method . The program GOLD 5.6 (CCDC
Software Ltd., Cambridge, UK) was used to for the docking study The inhibitory activities of the coumarin compounds (4a–d, 5a–c
19
with the GoldScore scoring function .
and 6a–c) on AChE and BuChE were determined by the Ellman’s
1
4,15
method
using donepezil as the reference compound. As
depicted in Table 1, compounds presented potent inhibitory activ-
ities against AChE with IC50 values varying from 0.02 to 0.92 mM
for compounds 6a and 6c respectively. On the other hand, the
tested coumarins were not so efficient in inhibiting BuChE with
IC50 ranging from 0.90 to 15.87 mM, demonstrating a good select-
Evaluation of the antioxidant activity by the ferric reducing
ability of plasma (FRAP) method
A 0.5 ml solution of coumarin compounds in methanol (50 mM
final concentration) was mixed with 4.5 ml of the FRAP reagent.
ꢀ
After 10 min of incubation at 37 C, absorbance at 593 nm was ivity for AChE. The inhibition behavior of the simplest bromo-cou-
20,21
measured using methanol as blank.
The calibration curve was marins (4a–d) was quite similar of that related in the literature for
13
prepared with quercetin and the results expressed as: antioxidant AChE , the bigger the methylene chain the lower the activity.
index based on quercetine (Q) (mmol Q/mol). The analyses were However, we were surprised by compound 4d with a five-methy-
performed in triplicate.
lene spacer link that was equipotent to 4a (IC50=0.14 mM for 4d
and IC =0.18 mM for 4a). Conversely, the inhibitions of BuChE was
5
0
in general inverse to those of AChE, and compounds with longest
linker chains were more potent in inhibiting BuChE (IC50=8.37 mM
for 4a and IC50=5.00 mM for 4d). By this way, we decided to evalu-
ate the 3-aryl substituted coumarins with 2, 3 and 5 methylene
spacers in the 7-amino-alkoxy group. In general, the substitution
of bromine for phenyl (5a–c) or 4-dimethylamine-phenyl (6a–c)
led to compounds with better potencies on the inhibition of both
Murine neuroblastoma cell (N2a) culture and cell viability assay
N2a cells were cultured in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum and 0.1% gentamicin
in a 5% CO atmosphere. N2a cells were transferred to a 96-well
2
2
plate (ꢁ10,000 cells/cm ) and incubated for 24 h, before treatment
with the compounds at 10 or 50 mM. Cell viability was evaluated
by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium brom-
ide) assay.
Table 1. AChE and BuChE inhibitory activities of coumarin compounds.
Results and discussion
Compounds design and synthesis
a
IC50(lM)±SD
The design of the novel alkylamino-coumarin derivatives (Scheme
b
c
d
FRAP value e
1
) was based on the structural requirements for mixed-type select-
Compound
R
n AChE
BuChE
SI (mmol Q/mol)
ive AChE inhibition present in alkylamino-indanone inhibitor
recently described , as well as on the widespread use of coumar-
ins for this pharmacological activity
based on: 1- the maintenance of the cyclic alkylamino group, 4d
which is responsible for the interaction with the cationic catalytic 5a
site (CAS) of AChE, exploring different lengths of methylene link-
ers (2–6); 2-exchange of the indanone nucleus by the coumarin
through non-classical isosterism of ring expansion ; 3- use of
1
3
4a
4b
Br
Br
Br
1
2
3
4
1
2
4
0.18 ± 0.009 8.37 ± 0.167 47
0.37 ± 0.008 15.87 ± 0.007 42
0.55 ± 0.010 4.92 ± 0.095
0.15 ± 0.005 5.01 ± 0.253 33
0.14 ± 0.009 2.50 ± 0.177 18
0.24 ± 0.014 1.86 ± 0.024
0.45 ± 0.036 0.90 ± 0.001
NA
NA
NA
NA
NA
NA
NA
22,23
. The coumarin series was
4c
9
Br
Ph
Ph
Ph
5
5
6
6
b
c
a
b
8
2
4-(CH
4-(CH
3
)
)
2
N-Ph 1 0.02 ± 0.001 6.73 ± 0.040 354 7.49 ± 0.61
N-Ph 2 0.33 ± 0.011 7.27 ± 0.273 22 2.42 ± 0.19
24
3
2
hydrophobic groups at position 3 of coumarin, targeting interac- 6c
4-(CH ) N-Ph 4 0.96 ± 0.036 3.85 ± 0.190
4
2.77 ± 0.00
3
2
Donepezil
–
– 0.007 ± 0.0002 2.39 ± 0.105 365
–
tions with the peripheral anionic site (PAS) of AChE.
a
The synthesis of the desired compounds started with the 7-
OH-coumarin (1). In the first step, through an O-alkylation reaction
of 1 with diverse dibromo-alkanes, the 7-bromoalkoxy-coumarin
products (2a–d) were obtained in yields of 68–78%. The second
step consisted of a bromination reaction of the bromoalkoxy- acetate (1.2
Concentration required for 50% inhibition of ChEs, data were shown in
b
mean
± SD of triplicate independent experiments; AChE from electric eel;
d
c
BuChE from horse serum; Selectivity index (SI) is defined as BuChE IC50/AChE
e
IC50. Antioxidant index based on quercetine (Q); FRAP value (mmol Q/mol). 7,8-
dimethoxy-coumarin (NA) and ethyl 2–(7,8-dimethoxy-2-oxo-2H-chromen-3-yl)
25
2
5
±
0.1) .

6
34
G. A. DE SOUZA ET AL.
AChE and BuChE, and a reduction in the selectivity index (IC50 3-substituted coumarins, i.e. 4d and 6a, were selected for kinetic
BuChE/AChE). However, one compound behavior itself differently studies. The linear Lineweaver–Burk equation of the
and presented an interesting profile, the 4-dimethylamine-phenyl Michaelis–Menten was applied to evaluate the inhibition profile.
substituted coumarin (6a) with the best inhibition of AChE Increasing concentrations of both compounds were able to
(
IC =0.02 mM) and selectivity (IC BuChE/AChE = 354), quite simi- increase Km and decrease Vmax, presenting a mixed-type inhibition
5
0
50
lar to the reference drug donepezil (IC50 AChE = 0.007 mM and in AChE as well as in BuChE, as exemplified in Figure 1 for com-
selectivity = 365) (Table 1).
The antioxidant evaluation of coumarin compounds showed
that only 6a–c presented activity in Ferric Ion Reduction Method
pound 6a (complete analysis in Supplementary material). The
competitive inhibitory constant (Ki) and the noncompetitive con-
stant (Ki’) for 6a and 4d are described in Table
3 at
(FRAP) with values from 2.42 to 7.49 mmol Q/mol (Table 1). Series
Supplementary material. As example, the best Ki values against
AChE were obtained for compound 6a: Ki = 0.001 mM (competi-
tive) and Ki’=0.010 mM (noncompetitive).
4a–d and 5a–c did not demonstrate any considerable result, similar
2
5
to other 7-alkoxy coumarins described in the literature . Probably,
the antioxidant effect is coming from dimethylamino-phenyl moi-
ety and this feature could be explored in a forthcoming series.
Aiming at discovery the mode of action of coumarins described
herein, the most potent compounds from the bromo and aryl
With the complete inhibitory profile of the target compounds,
we proceeded with a molecular modeling evaluation to under-
stand the importance of changing the methylene size spacer and
nature of substituents in position 3 of coumarins. Thus, we
selected compounds 4a and 6a, the strongest 2-methylene spacer
bromo and aryl substituted coumarins inhibitors of AChE; and 6c,
the weakest inhibitor. Docking results in EeAChE and EqBuChE are
presented in Table 4 at Supplementary material. All inhibitors
were generally predicted as better ligands of EeAChE, being 6a
3
(Goldscore = 78.1) better than 6c (Goldscore = 71.4) and 4a
2
(Goldscore = 64.9), whereas 6c (Goldscore = 67.9) was better than
6a (Goldscore = 63.0) and 4a (Goldscore = 57.6) as a ligand of
EqBuChE, in qualitative accordance to our experimental results.
The molecular docking results of compound 4a, 6a and 6c
showed that all were able to occupy the peripheral (PAS) and the
catalytic (CAS) sites simultaneously in the EeAChE (Figure 2) (and
Figure 5 at Supplementary material), as previewed by kinetic eval-
uations. In the CAS, they interact similarly by means of their pro-
tonated piperidinyl group with Trp86 (a cation-p interaction). In
the PAS, both 6a and 6c molecules were involved in p-stacking
interactions with Trp286, which was more effective for 6c, involv-
ing its coumarin ring (Figure 2). On the other hand, 4a was only
capable of doing weak hydrophobic interactions with Trp286
[
[
[
I] = 0
I] = 0,01 µM
I] = 0,03 µM
1
0
−
10
0
10
20
30
1/[S]
Figure 1. Lineweaver-Burk plots of EeAChE inhibition kinetics of compound 6a. (Figure 5 at Supplementary material). The presence of a narrower
Inset: concentrations used for 6a are depicted with [I] graphic symbol.
spacer in 6a makes its coumarin ring to be best located in the
Figure 2. Superposition of the interaction poses of compounds 6a (A, carbon atoms in yellow) and 6c (B, carbon atoms cyan) with EeAChE obtained by molecular
docking (Goldscore function). H-bond distances (Å) are shown in yellow. Figure generated with PyMol 0.99 (DeLano Scientific LLC).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
635
Figure 3. Neuroblastoma cell viability after compound treatment. Samples containing compounds were added to the culture 48 h before MTT addition. The com-
pounds were tested at the final concentration of 50 mM. MTT reduction was evaluated as described in Experimental Procedures. Data are expressed as the percentage
ꢂꢂ
ꢂꢂꢂ
p < .001;
ꢂꢂꢂꢂ
p < .0001.
of MTT reduction relative to the value for control cells (cells without treatment). Error bars represent standard deviations. p < .01;
27
Figure 4. BOILED-Egg ADMET model for coumarin compounds 4a–d, 5a–c, and 6a–c. (HIA) gastrointestinal absorption; (BBB) brain penetration; (PGPþ) substrate for
P-glycoprotein; (PGP-) Not a substrate for P-glycoprotein.
gorge, where it is involved in H-bonds with Tyr337 and the pep- (neuroblastoma), after 48 h incubation at concentrations of 10 and
tide group of Phe295. These H-bonds, that had no counterparts in 50 mM (Figure 3 and Supplementary material). The most potent
the 6c/enzyme complex, were probably the reason for the most compounds in inhibiting AChE, i.e. 4a, 5a, and 6a, were not cyto-
effective interaction between compounds with short spacers and toxic at the maximum tested concentration (50 mM) (Figure 3). As
EeAChE, which could be related to their greater inhibitory action a rule, long methylene chains (three or five spacers) in phenyl-
over the enzyme.
substituted coumarins (5b, 5c, 6b and 6c) could not be useful for
further developments due to increase in toxicity.
Finally, in silico evaluations showed a good ADMET profile for
coumarin compounds. Parameters as topological polar surface
area (TPSA), consensus Log P, Log S, human intestinal absorption
Cell cytotoxicity and in silico ADMET physico-chemical
profile analysis
(HIA), blood–brain barrier permeation (BBB), and P-glycoprotein
In order to accede the drugability of tested coumarins, we first (P-gP) substrate and drug-likeness profile (Supplementary mater-
2
6
proceeded with the cytotoxicity evaluation against N2a cells ial) . TPSA values and consensus Log P ranged from 42.68 to 45.

6
36
G. A. DE SOUZA ET AL.
9
2 and 3.35 to 5.01, respectively. The moderate polarity (PSA < 79
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Å ) and relative lipophilicic characteristics put our compounds in
the yellow compartment of BOILED-Egg model (Figure 4), having
a high probability to access the CNS , which is fundamental for
the distribution of central-acting molecules. Additionally, the most
potent compounds 4a, 5a and 6a were not considered as P-gP
substrate and having a good drug-likeness profile with no one
27
7. Anand R, Gill KD, Mahdi AA, et al. Therapeutics of
Alzheimer’s
disease:
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and
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Conclusions
The designed and synthesized coumarin compounds were able to
potently inhibit cholinesterases in the nanomolar range. In gen-
eral, compounds with narrow methylene linkers were more potent
and selective for AChE (with IC50 and selectivity of up to 20 nM
and 354 times, respectively), and less toxic as well. The introduc-
tion of aromatic substituents in position 3 of coumarins led to
compounds with better potencies on the inhibition of both AChE
and BuChE. As highlighted, compound 6a could be elected as a
hit for in vivo studies, showing good AChE inhibition potency and
selectivity (IC =20 nM and 354 times), antioxidant properties, low
2
112–7.
1
0. Mushtaq G, Greig NH, Khan JA, et al. Status of acetylcholin-
esterase and butyrylcholinesterase in Alzheimer’s disease
and type 2 diabetes mellitus. CNS Neurol Disord Drug
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cytotoxicity and good predict ADMET profile.
Acknowledgments
13. Huang L, Miao H, Sun Y, et al. Discovery of indanone deriva-
tives as multi-target-directed ligands against Alzheimer’s dis-
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The authors would like to thank Marco E. F. de Lima for furnishing
donepezil standard and Marina Amaral from Laborat oꢀ rio de Apoio
ao Desenvolvimento Tecnol oꢀ gico (LADETEC, UFRRJ) for the mass
spectrometry analysis.
1
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Disclosure statement
derivatives:
Phytochemistry 2012;81:24–30.
a
kinetics-molecular modeling approach.
No potential conflict of interest was reported by the authors.
1
6. Biasini M, Bienert S, Waterhouse A, et al. SWISS-MODEL:
modelling protein tertiary and quaternary structure using
evolutionary information. Nucleic Acids Res 2014;42:W252–8.
Funding
Fellowship and financial support for this study was provided by 17. Brus B, Kosak U, Turk S, et al. Discovery, biological evalu-
the CNPq (BR), FAPERJ (BR) and Coordena c¸ ~a o de Aperfei c¸ oamento
de Pessoal de N ꢀı vel Superior - Brasil (CAPES) - Finance Code 001.
ation, and crystal structure of a novel nanomolar selective
butyrylcholinesterase inhibitor. Med Chem 2014;57:
167–79.
J
8
1
8. Stewart JJP. Optimization of parameters for semiempirical
methods V: modification of NDDO approximations and
application to 70 elements. J Mol Model 2007;13:1173–213.
9. Jones G, Willett P, Glen RC, et al. Development and valid-
ation of a genetic algorithm for flexible docking. J Mol Biol
ORCID
Arthur Eugen K u€ mmerle
http://orcid.org/0000-0002-8458-0975
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1
997;267:727–48.
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