Synthesis and biological assessment of KojoTacrines as new agents for Alzheimer’s disease therapy

Article abstract of DOI:10.1080/14756366.2018.1538136

In view of the multifactorial nature of Alzheimer’s disease (AD), multitarget small molecules (MTSM) represent the most potent and attractive therapeutic strategy to design new drugs for Alzheimer’s disease therapy. The new MTSM KojoTacrines (KTs) were designed and synthesized by juxtaposition of selected pharmacophoric motifs from kojic acid and tacrine. Among them, 11-amino-2-(hydroxymethyl)-12-(3-methoxyphenyl)-7,9,10,12-tetrahydropyrano [2',3':5,6] pyrano[2,3-b]quinolin-4(8H)-one (KT2d) was identified as less-hepatotoxic than tacrine, at higher concentration, a moderate, but selective human acetylcholinesterase inhibitor (IC₅₀ = 4.52 ± 0.24 μM), as well as an antioxidant agent (TE = 4.79) showing significant neuroprotection against Aβ_1–40 at 3 μM and 10 μM concentrations. Consequently, KT2d is a potential new hit-ligand for AD therapy for further biological exploration.

Full text of DOI:10.1080/14756366.2018.1538136

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and biological assessment of
KojoTacrines as new agents for Alzheimer’s
disease therapy
Youssef Dgachi, Hélène Martin, Rim Malek, Daniel Jun, Jana Janockova,
Vendula Sepsova, Ondrej Soukup, Isabel Iriepa, Ignacio Moraleda, Emna
Maalej, M. Carmo Carreiras, Bernard Refouvelet, Fakher Chabchoub, José
Marco-Contelles & Lhassane Ismaili
To cite this article: Youssef Dgachi, Hélène Martin, Rim Malek, Daniel Jun, Jana Janockova,
Vendula Sepsova, Ondrej Soukup, Isabel Iriepa, Ignacio Moraleda, Emna Maalej, M. Carmo
Carreiras, Bernard Refouvelet, Fakher Chabchoub, José Marco-Contelles & Lhassane Ismaili
(
2019) Synthesis and biological assessment of KojoTacrines as new agents for Alzheimer’s
disease therapy, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 163-170, DOI:
0.1080/14756366.2018.1538136
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
019, VOL. 34, NO. 1, 163–170
2
https://doi.org/10.1080/14756366.2018.1538136
SHORT COMMUNICATION
Synthesis and biological assessment of KojoTacrines as new agents for Alzheimer’s
disease therapy
a,b
c
b
d
e
d
Youssef Dgachi , H ꢀe l ꢁe ne Martin , Rim Malek , Daniel Jun
, Jana Janockova
, Vendula Sepsova , Ondrej
e
f
f
g
h
b
Soukup
, Isabel Iriepa
Fakher Chabchoub , Jos ꢀe Marco-Contelles
, Ignacio Moraleda
, Emna Maalej , M. Carmo Carreiras , Bernard Refouvelet ,
a
i
b
and Lhassane Ismaili
a
b
Laboratory of Applied Chemistry, Heterocycles, Lipids and Polymers, Faculty of Sciences of Sfax, University of Sfax, Sfax, Tunisia; Laboratoire
de Chimie Organique et Th ꢀe rapeutique, Neurosciences Int ꢀe gratives et Cliniques EA 481, Univ. Bourgogne Franche-Comt ꢀe , Besan c¸ on, France;
c
d
Laboratoire de Toxicologie Cellulaire, Univ. Bourgogne Franche-Comt ꢀe , Besan c¸ on, France; Department of Toxicology and Military Pharmacy,
e
Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic; Biomedical Research Center, University Hospital
Hradec Kralove, Hradec Kralove, Czech Republic; Department of Organic Chemistry and Inorganic Chemistry, School of Biology, Environmental
Sciences and Chemistry, University of Alcal ꢀa , Alcal ꢀa de Henares, Spain; Laboratoire Mat ꢀe riaux, Traitement et Analyse (LMTA), Institut National
de Recherche et d’Analyse Physico-chimique Technopole, Ariana-Tunis, Tunisia; Research Institute for Medicines (iMed.ULisboa), Faculty of
Pharmacy, Universidade de Lisboa, Lisboa, Portugal; Laboratory of Medicinal Chemistry, IQOG, CSIC, Madrid, Spain
f
g
h
i
ABSTRACT
ARTICLE HISTORY
In view of the multifactorial nature of Alzheimer’s disease (AD), multitarget small molecules (MTSM) repre-
sent the most potent and attractive therapeutic strategy to design new drugs for Alzheimer’s disease ther-
apy. The new MTSM KojoTacrines (KTs) were designed and synthesized by juxtaposition of selected
pharmacophoric motifs from kojic acid and tacrine. Among them, 11-amino-2-(hydroxymethyl)-12-(3-
methoxyphenyl)-7,9,10,12-tetrahydropyrano [2’,3’:5,6] pyrano[2,3-b]quinolin-4(8H)-one (KT2d) was identified
as less-hepatotoxic than tacrine, at higher concentration, a moderate, but selective human acetylcholin-
esterase inhibitor (IC50 ¼ 4.52 ± 0.24 mM), as well as an antioxidant agent (TE ¼ 4.79) showing significant
neuroprotection against Ab1–40 at 3 mM and 10 mM concentrations. Consequently, KT2d is a potential new
hit-ligand for AD therapy for further biological exploration.
Received 29 May 2018
Revised 10 October 2018
Accepted 15 October 2018
KEYWORDS
Alzheimer disease; kojic
acid; multitarget small
molecules; tacrine
GRAPHICAL ABSTRACT
Neuroprotective
against A1-40
O
Drug-like
properties
Less hepatoxic
than tacrine
NH2
N
O
O
HO
O
Antioxidant
.29 Trolox eq
hAChE inhibitor
IC50 = 4.52
KT2d
4
Introduction
3,4
impairments . Although the aetiology of the disease is still not fully
5
understood , low levels of acetylcholine (ACh), the aggregation of
The World Health Organization claims that 46 million people world-
amyloid-beta peptide (Ab), hyperphosphorylation of tau protein, oxi-
wide suffer from Alzheimer’s disease (AD). It is likely that this number
1,2
may significantly rise as the life expectancy increases . AD is a dative stress, and accumulation of biometals (Cu, Fe, Zn) are thought
multifactorial neurodegenerative disorder, inducing a progressive to play key roles in the pathophysiology, progress and development
6,7
memory loss, decline in language skills, and other cognitive of AD . AD patients are currently treated either with the N-methyl-
CONTACT Fakher Chabchoub
University of Sfax, B. P 802, 3000 Sfax, Tunisia; Jos ꢀe Marco-Contelles
8006 Madrid, Spain; and Lhassane Ismaili lhassane.ismaili@univ-fcomte.fr
fakher.chabchoub@yahoo.fr
Laboratory of Applied Chemistry, Heterocycles, Lipids and Polymers, Faculty of Sciences of Sfax,
iqoc21@iqog.csic.es Laboratory of Medicinal Chemistry, IQOG, CSIC, Juan de la Cierva 3,
Laboratoire de Chimie Organique et Th ꢀe rapeutique, Neurosciences Int ꢀe gratives et
2
Cliniques EA 481, Univ. Bourgogne Franche-Comt ꢀe , UFR Sant ꢀe , 19, rue Ambroise Par ꢀe , F-25000 Besan c¸ on, France.
Supplemental data for this article can be accessed at https://doi.org/10.1080/14756366.2018.1538136.
ß 2018 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.

1
64
Y. DGACHI ET AL.
Figure 1. Design of KojoTacrine hybrids.
8
D-aspartate receptor antagonist, memantine , or donepezil, rivastig- Amino-2-(hydroxymethyl)-12–(3-methoxyphenyl)-7,9,10,12-tetra
9
mine, and galantamine , three acetylcholinesterase inhibitors hydropyrano[2’,3’:5,6] pyrano[2,3-b]quinolin-4(8H)-one (2d)
24
(AChEIs), but with limited therapeutic success. Consequently, there is
A
solution of previously described compound 1d
(0.5 g,
1
2
2
.5 mmol) in 1,4-dioxane (30 ml) with cyclohexanone (0.23 g,
an urgent need for new and more efficient drugs for AD therapy.
Because of the complex nature of AD, multitarget small mole-
cules (MTSM), able to interact simultaneously with the different
enzymatic systems or receptors involved in the pathology, have
emerged as one of the most promising therapeutic strategies in
ꢀ
.25 mmol) and AlCl (0.31 g, 2.25 mmol) was heated at 110 C for
3
h and monitored by TLC (CH Cl /MeOH, 8/2, v/v). When the
2
2
reaction was complete, the reaction mixture was diluted with a
solution of CH Cl /water (1/1) and treated with an aqueous solu-
tion of sodium hydroxide (10%) until pH 11–12. The mixture was
stirred for 20 min and then extracted with CH Cl , dried over
anhydrous sodium sulfate, filtered, and the solvent was evapo-
rated to generated the desired product 2d (0.36 g, 59%): mp
2
2
10–15
order to design and identify new drugs for AD
. Following this
2
2
paradigm, we have prepared a number of new MTSM by multi-
16–19
component reactions (MCR)
as AChEIs, showing neuroprotec-
tive effect and strong antioxidant activity.
ꢀ
>
260 C; IR (KBr) ꢀ 3470 (OH), 3330 and 3250 (NH ), 1725 (C ¼ O)
2
In this article, we report the synthesis and biological evaluation
of novel MTSM, called KojoTacrines (KTs), resulting from the com-
bination of selected motifs present in kojic acid (KA) and tacrine
ꢁ
1 1
cm ; H NMR (DMSO-d , 400 MHz) d 1.72 (m, 4H), 2.18–2.22 (m,
6
1H), 2.33–2.37 (m, 1H), 2.55–2.66 (m, 2H), 3.72 (s, 3H, OCH
4.20–4.28 (m, 2H), 5.32 (s, 1H), 5.69 (br s, 2H, NH ), 6.32 (s, 1H),
6.77–6.40 (m, 2Harom), 6.97 (s, 1Harom), 7.21–7.25 (m, 1Harom);
3
),
2
(Figure 1). Tacrine was the first marketed and approved AChEI for
13
C
AD therapy although discontinued shortly after its approval due
NMR (DMSO-d
6
, 100 MHz) d 22.4 (CH
2 2 2
), 22.7 (CH ), 23.4 (CH ), 32.5
2
0
to its hepatotoxicity . Despite this, tacrine has been largely used
as a template to design new non-hepatotoxic analogues for
(
(
(
CH ), 40.6 (CH), 55.5 (OCH ), 59.8 (CH ), 96.7 (C), 111.7 (CH), 112.7
CH), 112.8 (C), 114.9 (CH), 120.2 (CH), 130.4 (CH), 137.8 (C), 142.6
C), 150.0 (C), 152.1 (C), 153.7 (C), 154.3 (C), 159.7 (C), 168.1 (C),
2
3
2
1
3,15
AD
. Our current choice for tacrine to build the new anti-
Alzheimer agents is based on the fact that (1) tacrine derivatives
are easily available by simple synthetic protocols; (2) tacrine is a
potent ChE inhibitor, able thus to increase the level of neurotrans-
mitter ACh in the brain, and consequently, it has been assumed
1
71.04 (C). Anal. Calcd for C H N O : C, 67.97; H, 5.46; N, 6.89.
23 22 2 5
Found: C, 68.21; H, 5.38; N, 7.01.
that (new) tacrine derivatives should behave similarly with particu- Biological evaluation
lar effects depending on their structure; (3) we have demonstrated
The methods for the in vitro cytotoxicity in HepG2 cells, the inhib-
ition of EeAChE, eqBuChE, and hChE, the neuroprotective assay, as
well as the ORAC assay have been briefly described
that it is possible to design and prepare non-hepatotoxic tacrine
21
derivatives showing ChEs inhibition ; and (4) last but not least,
the number of published papers dealing with tacrine derivatives
as anti-Alzheimer should be also considered as a strong point in
order to support our choice. So, the purpose of this preliminary com- In vitro cytotoxicity of KojoTacrines in HepG2 cells1
munication is to identify a new tacrine-based scaffold and its deriva- The culture of HepG2 cells were incubated at 37 C in a humidi-
7,18,25
ꢀ
tives. KA, a naturally occurring fungal metabolite, is a recognized fied atmosphere of 5% CO and 95% air. The culture medium was
2
22,23
antioxidant agent
. Thus, considering KA ability to scavenge react- refreshed after 24 h of incubation and 100 ml of six different con-
ive oxygen species, 12 new kojo tacrines (KTs) were designed, syn- centrations (1–1000 mM) of test compounds or DMSO (0.1%) were
thesized, and evaluated as potential cholinesterase inhibitors (ChEIs) then added. The cell viability was monitored by colourimetric
enhanced with antioxidant properties. As a result, 11-amino-2- assay with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium
2
6
(hydroxymethyl)-12–(3-methoxyphenyl)-7,9,10,12-tetrahydropyrano[2’,
bromide (MTT)
.
3’:5,6]pyrano[2,3-b]quinolin-4(8H)-one (KT2d) was identified as a hit-
agent, less hepatotoxic than tacrine, completely selective against
AChE, showing potent antioxidant properties and displaying a very
significant neuroprotection against amyloid beta (Ab).
Inhibition of EeAChE and eqBuChE of compounds KTs 2a–d, 2f–h,
1
7,27
and 2l
Ellman’s protocol was followed to perform the anticholinesterasic
2
8
activity . Briefly, the reaction was carried out in a final volume of
3 ml of a phosphate-buffered solution (0.1 M) at pH ¼8.0 contain-
ing (2.625 ml, 0.35 mM) of 5,5’-dithiobis-2-nitrobenzoic acid (DTNB),
Methods
Chemistry
4
0 ml 1% w/v Bovine Albumin Serum phosphate-buffered solution
The synthetic method of only compound KT2d is described here, (BSA), (29 ml, 0.035 U/ml) of EeAChE or (60 ml, 0.05 U/ml) of
for the rest of compounds readers are invited to refer to eqBuChE, and 3 ml of tested compound, which were incubated for
Supporting Information section.
10 min. Then, the acetylthiocholine iodide or butyrylthiocholine

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
165
Scheme 1. Synthesis of racemic KojoTacrines 2a-l
was added, and the reaction was incubated for additional 15 min. (10 mM)/rotenone (30 mM) (Sigma, France) or Ab1–40 (30 mM) with
The activity in absence of compound was used as control. The or without the tested compounds 2b–d and 2f at nontoxic con-
absorbances were measured in a spectrometric plate reader at centrations against cells for an additional period of 24 h. MTT
4
12 nm and the IC50 were determined using GraphPad Prism 5.
assay was then performed to determine the % of cell viability.
1
7,27
30,31
Inhibition of human cholinesterases
Oxygen radical absorbance capacity assay
Inhibitory activities of the tested compounds were expressed as The antioxidant activity of KTs was determined using the ORAC-FL
3
0
IC50, i.e., concentration of compound required for 50% reduction method described by D ꢀa valos et al. using fluorescein as a fluor-
28
in cholinesterase activity using Ellman’s method . The assay was escent probe with minor modification. Briefly, fluorescein and anti-
carried out in medium (100 ml) containing 10 ml of cholinesterase oxidant were incubated in a black 96-well microplate (Nunc) for
ꢀ
obtained from human erythrocyte hemolyzate, out of blood sam- 15 min at 37 C. The 2,2’-azobis(amidinopropane) dihydrochloride
(
AAPH) was then added quickly using the built-in injector of
ples, 40 ml of 0.1 M PBS (pH 7.4), 20 ml of 0.01 M DTNB, 10 ml of dif-
ferent concentrations of tested compound (10 –10 M). This
blend was preincubated for 5 min and 20 ml of 0.01 M substrate
ꢁ
3
ꢁ9
Varioskan Flash plate reader (Thermo Scientific). The fluorescence
was measured at 485 nm (excitation wavelength) and 535 nm
(emission wavelength) every min for 60 min.
(ATCh or BTCh) was added to initiate the reaction. The activity
was determined by measuring the increase in absorbance at
ꢀ
4
12 nm at 37 C in 2 min intervals using Multi-mode microplate
Molecular modelling
reader Synergy 2 (Vermont, USA). The IC50 values are expressed as
a mean ± SEM.
Molecular modelling of compound KT2d
3
2
For docking experiments, Autodock Vina software was employed.
Compounds (R)-KT2d and (S)-KT2d were prepared with Discovery
Studio, version 2.1, software package, using standard bond lengths
2
9
Neuroprotection assay
33
SH-SY5Y cell culture and treatment with compounds 2b–d and 2f.
The cultures of human SH-SY5Y neuroblastoma cells kept under a
CO /air (5%/95%) humidified atmosphere at 37 C for 24 h were
treated with 100 ml of the test compounds or DMSO (0.1%) in
and bond angles. With the CHARMm force field and partial atomic
charges, the molecular geometries of the compounds were energy-
minimized using the adopted-based Newton–Rapson algorithm until
ꢀ
2
ꢁ
1
the rms gradient was below 0.01 kcal (mol-Å) . Three-dimensional
DMEM/F12 (1:1) medium supplemented with 1% fetal bovine crystal structure of hAChE complexed with fasciculin-II (PDB: 1B41)
serum, 1X non-essential amino acids, 100 units/ml penicillin and was retrieved from the Protein Data Bank (PDB). Before docking the
1
0 mg/ml streptomycin. Treatment medium was renewed every ligands into the protein, this was prepared by removing all water mol-
day for up to 48 h of treatment. MTT assay was then performed.
ecules, heteroatoms, any co-crystallized solvent, and the ligand.
Protein model tool in Discovery Studio, version 2.1, software package
was used to assign proper bonds, bond orders, hybridization, and
charges. CHARMm force field was applied using the receptor-ligand
interactions tool in Discovery Studio, version 2.1, software package.
Effect of 2b–d and 2f on O/R, Ab1–40 peptide-induced cell death in
SH-SY5Y cells
SH-SY5Y cells were seeded in 96-well culture plates at a density of AutoDockTools (ADT; version 1.5.4) was used to add hydrogens and
4
8
ꢂ 10 cells per well in DMEM/F12 (1:1) medium supplemented partial charges for proteins and ligands using Gasteiger charges.
with 10% fetal bovine serum, 1X non-essential amino acids, 100 Selected side chains into the target macromolecule are allowed to
units/mL penicillin and 10 mg/mL streptomycin (Dutscher, France). change their conformations at the same time as the ligand that is
After 24 h of incubation, the cultures were treated with 100 ml of being docked. Using the AutoTors module, the macromolecule side
the test compounds or DMSO (0.1%) in the same medium. chains chosen to be flexible are Trp286, Tyr124, Tyr337, Tyr72, Asp74,
Following 24 h, the cells were co-incubated with oligomycin A Thr75, Trp86 and Tyr341. The docking box was displayed using ADT

1
66
Y. DGACHI ET AL.
a
Table 1. Effects of KojoTacrines 2a-l on human hepatocyte HepG2 cell viability.
1
mM
3 mM
10 mM
30 mM
100 mM
300 mM
1000 mM
ꢃꢃꢃ
ꢃꢃꢃ
Tacrine
112.2 ± 4.0
96.8 ± 1.0
105.1 ± 3.1
94.7 ± 2.4
100.8 ± 2.5
95.5 ± 5.5
108.0 ± 6.2
101.2 ± 7.1
97.4 ± 2.3
98.6 ± 1.4
97.6 ± 2.0
101.4 ± 4.8
99.9 ± 2.7
113.6 ± 4.8
94.9 ± 2.9
109.5 ± 3.2
103.4 ± 2.8
98.0 ± 4.1
96.1 ± 7.1
96.1 ± 2.5
96.5 ± 7.4
95.9 ± 3.8
100.1 ± 1.9
101.4 ± 5.4
104.0 ± 8.9
102.1 ± 2.3
107.8 ± 3.4
99.3 ± 1.7
105.4 ± 1.9
99.0 ± 1.1
101.5 ± 3.2
95.9 ± 5.6
99.2 ± 2.8
99.1 ± 3.8
100.5 ± 1.5
99.4 ± 1.2
105.1 ± 5.5
94.9 ± 2.6
102.2 ± 2.6
109.6 ± 4.2
105.2 ± 5.3
107.0 ± 2.9
98.2 ± 2.2
101.7 ± 1.8
98.3 ± 3.8
102.2 ± 7.1
108.8 ± 6.1
101.4 ± 0.6
104.7 ± 2.6
99.6 ± 2.4
95.8 ± 2.5
99.9 ± 3.2
101.9 ± 2.4
108.1 ± 5.0
112.9 ± 8.4
96.1 ± 1.5
99.0 ± 1.1
86.1 ± 3.8
100.9 ± 9.2
103.1 ± 6.4
102.2 ± 1.8
95.2 ± 4.7
102.1 ± 6.9
100.7 ± 3.1
88.9 ± 8.0
42.4 ± 2.0
95.0 ± 3.4
108.5 ± 5.7
88.5 ± 7.1
87.2 ± 6.1
70.9 ± 4.5
95.3 ± 4.1
90.5 ± 9.6
87.2 ± 6.7
64.4 ± 6.3
52.7 ± 8.3
10.9 ± 2.0
50.3 ± 5.7
87.9 ± 5.2
70.7 ± 7.7
65.5 ± 1.3
41.7 ± 3.0
64.0 ± 8.0
62.6 ± 9.7
55.3 ± 6.0
23.1 ± 6.0
14.5 ± 6.4
37.2 ± 5.9
50.5 ± 7.5
ꢃꢃ
2
2
2
2
2
2
2
2
2
2
2
2
a
a
b
c
d
e
f
g
h
i
ꢃ
ꢃ
ꢃ
ꢃꢃꢃ
ꢃ
ꢃ
ꢃꢃ
ꢃꢃꢃ
ꢃꢃꢃ
ꢃꢃꢃ
ꢃꢃ
ꢃ
ꢃꢃ
ꢃ
j
k
l
60.1 ± 10.4
ꢃ
68.3 ± 9.3
Data are expressed as percent of cell viability as compared to control cultures (DMSO-treated cells). Means ± SEM of triplicates from three different cultures.
ꢃ
ꢃꢃ ꢃꢃꢃ
p < 0.05, p < 0.01, p < 0.001, as compared to the control cultures (one-way ANOVA).
a
and it is big enough to include whole protein target (“blind docking”). Table 2. Inhibition of EeAChE, hACHE, eqBuChE, (IC50, lM) , and ORAC-FL values
ꢀ
for KTs 2a-d, 2f-h, 2l, tacrine, and ferulic acid (FA).
A grid box of 60ꢂ 60ꢂ 72 with grid point spacing of 1 Å was posi-
tioned at the middle of the protein (x ¼ 116.546; y ¼ 110.33; z ¼
ꢁ
134.181). Default parameters were used except num_modes, which
was set to 40. The AutoDock Vina docking procedure used was previ-
2
5
ously validated . The scoring function of AutoDock Vina was chosen,
and the docking poses for each ligand were analyzed by examining
their relative total energy score. The more energetically favorable
conformation was selected as the best pose. The structures of the
macromolecule and ligands, as well as the docking results, were
processed using Discovery Studio software.
EeAChE
eqBuChE
hAChE
KT
R
(IC50, lM)
(IC50, lM)
(IC50 mM)
ORAC
Results and discussion
2
2
2
2
2
2
a
b
c
d
f
H
4-CH
2-OCH
3-OCH
2-F
3-F
4-F
2,6-diCl
3.30 ± 0.09
1.40 ± 0.00
2.39 ± 0.02
0.64 ± 0.06
2.11 ± 0.01
2.14 ± 0.12
3.44 ± 0.04
2.12 ± 0.09
0.031 ± 0.006
–c
12.15 ± 0.09
10.09 ± 0.09
–b
13.7 ± 1.7
2.58 ± 0.22
2.96 ± 0.18
6.05 ± 0.41
4.79 ± 0.39
3.85 ± 0.42
4.34 ± 0.17
4.69 ± 0.18
6.14 ± 0.40
0.2 ± 0.1
3
Chemistry
3
–b
–b
–c
3
4.52 ± 0.24
The synthesis of racemic KTs 11-amino-2-(hydroxymethyl)-12-aryl-
6.69 ± 0.10
4.54 ± 0.20
–b
–c
–c
7
,9,10,12-tetrahydropyrano[2’,3’:5,6]pyrano[2,3-b]quinolin-4(8H)-
g
ones (2a–l) was carried out in two steps by using easily available 2h
precursors, as shown in Scheme 1. Thus, KA was reacted with
selected arylidenemalonitriles, in the presence of catalytic
amounts of triethylamine, in ethanol, at 80 C, for 5 min, to give
the known 2-amino-6-(hydroxymethyl)-8-oxo-4-phenyl-4,8-dihydro-
–b
–b
2
l
–c
0.13 ± 0.00
–c
Tacrine
–
–
–
0.005 ± 0.001
FA
KA
a
–c
–c
3.74 ± 0.22
2.51 ± 0.17
ꢀ
–c
–c
Inhibition curves were obtained by nonlinear regression. Each IC50 value is the
mean ± SEM of quadruplicate of at least three different experiments.
3
4
pyrano[3,2-b]pyran-3-carbonitriles 1a–l in very good yields . Next,
b
c
Friedl €a nder-type reaction35 of compounds 1a–l with cyclohexa-
%
Inhibition under 50% at 10 lM.
35
Not determined.
none, under the usual experimental conditions , afforded the
expected racemic KTs 2a–l (Scheme 1).
All new KT ligands showed analytical and spectroscopic data in
good agreement with their structures (see Experimental Part).
cell death but not as much as tacrine, remaining from 5- to 7-fold
less toxic than tacrine.
Based on the hepatotoxicity results, the next step was to inves-
tigate the capacity KTs 2a–d, 2f–h, and 2l, showing values >50%
in the HepG2 cell viability test, for their ChE inhibition capacity
and ORAC power.
Biological evaluation
In vitro cytotoxicity of KTs 2a–l in HepG2 cells
Hepatotoxicity is the most critical biological concern when dealing
with new tacrine derivatives. In this sense, all new KTs were sub- Evaluation of AChE and BuChE inhibition of KTs 2a–d, 2f–h,
mitted to the in vitro cell viability test (MTT assay) using human
hepatocellular carcinoma cell line (HepG2) , as the most accepted
probe to evaluate hepatotoxic effects. For this experiment, six dif-
and 2l
26
The inhibition of Electrophorus electricus AChE (EeAChE) and serum
horse BuChE (eqBuChE) was carried out following Ellman’s assay ,
2
8
ferent concentrations from 1 to 1000 mM were used. As shown in using the synthesized KTs and tacrine, as a positive control for
Table 1, it is important to note that no significant toxicity at con- comparative purposes.
centrations up to 1000 mM was observed for KT2b; therefore, this
As shown in Table 2, all KTs investigated are less potent ChEIs
compound can be considered as non-hepatotoxic. As shown, tac- than tacrine. However, they are moderate, but selective AChEIs, in
rine and KTs 2e, 2i–l start to exert a marked cytotoxicity at the micromolar range, showing IC50 (mM) values from 0.64 ± 0.06
3
00 mM, in contrast to KTs 2a–d and 2f–h, which showed low tox- (2d) to 3.44 ± 0.04 (2h). The most potent EeAChEIs was ligands 2d
icity at this concentration; however, at 1000 mM, these KTs induce with IC50 values equal to 0.64, being only approximately 20-fold less

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
167
active than tacrine (IC50 ¼ 0.031 mM). For the inhibition of eqBuChE, Evaluation of antioxidant power of KTs 2a–d, 2f–h, and 2l
the KTs showed micromolar inhibition ranging from 4.54 to The antioxidant ability of KTs 2a–d, 2f–h, and 2l was then eval-
3
0,36
1
2.15 mM. The most potent was KT 2 g (IC50 ¼ 4.54 mM). Regarding uated using the Oxygen Radical Capacity (ORAC-FL) method
29,37
the structure-activity relationships, no evident and clear trends with tacrine and ferulic acid (FA)
as negative and positive con-
could be observed for the inhibition of EeAChE and eqBuChE.
trols, respectively. Results are expressed in the Trolox equivalents
Next, the most balanced KTs 2b–d and 2f, based on the hep- (TE) unit in relation to radical scavenging properties of Trolox. As
atotoxicity data and inhibitory activities against EeAChE and shown in Table 2, all evaluated KTs exhibited good radical scav-
eqBuChE, were evaluated for their ability to inhibit human AChE
enging properties, the ORAC values ranging from 2.58 TE (2a) to
.14 TE (2l), comparing favourably with KA (2.51 TE) and ferulic
(hAChE) and human BuChE (hBuChE). Regarding hBuChE the four
6
products showed a percentage of inhibition under 50% at 10 mM
and, consequently, their IC50 values were not determined.
However, for hAChE, compound KT2d showed significant inhib-
ition (IC50 ¼ 4.52 mM), whereas the non-hepatotoxic KT2b showed
a lower inhibitory activity equal to 13.7 mM, as it is shown in
Table 2. At this point of our project, KT 2d had been identified as
a promising compound on diverse biological tests, in spite that
this ligand shows lower AChE inhibition than tacrine, because its
hepatotoxic effects are lower, still showing a quite good, in the
low micromolar range, and selective, AChE inhibition. A very finely
balanced situation has been found. Next, as planned at the begin-
acid (FA) (3.74 TE). The most potent antioxidant KTs were those
bearing a methoxy group (2c) in position C2, and two chloro
atoms in positions C2 and C6 (2l) showing 6.05 and 6.14 TE,
respectively. Interestingly, the most balanced KT2d, regarding its
hepatotoxicity, neuroprotection, cytotoxicity, and ChE inhibition,
showed a very high 4.79 TE, 1.3-fold more potent than FA. Based
on these interesting results, next, we decided to investigate the
neuroprotective properties of some selected non-hepatotoxic KTs.
ning of the project, the analysis of the antioxidant capacity of KT Neuroprotective capacity of KojoTacrines 2b–d and 2f
2a–d, 2f–h, and 2l was carried out.
The ability to prevent the human neuroblastoma cell line SH-SY5Y
cell death induced by toxic insults such as Ab1–40, implicated in
3
8
Table 3. Neuroprotective activity of KTs 2b-d and 2f on Oligomycin/Rotenone ROS production and apoptosis-related signalling pathways , and
3
9
(
O at 10 mM)/Rotenone (R at 30 mM), and Ab1–40 (30 mM) peptide-induced cell oligomycin/rotenone (O/R) , was then assayed to determine the
death in SH-SY5Y cells.
neuroprotective capacity of the four less hepatotoxic KTs 2b–d and
f at nontoxic concentration against SH-SY5Y 3 and 10 mM, using
KojoTacrine
Dose
O/R (%)
Ab1–40 (%)
27.85 ± 0.15ꢃ
2
2
2
2
2
b
c
d
f
3 mM
19.66 ± 0.07
3.65 ± 0.07
12.18 ± 0.08
16.56 ± 0.02
24.35 ± 0.05
12.53 ± 0.09
16.50 ± 0.07
np
melatonin and KA as reference molecules. For the evaluation of the
neuroprotective effect, the cytotoxicity of O/R (10 mM/30 mM) on SH-
SY5Y cell viability was determined as well as the toxicity of the
selected KTs 2b–d and 2f at. As shown in Table 3, only KT2d showed
a significant and similar neuroprotection against Ab1–40 insult at 3
and 10 mM and, therefore, slightly less active than KA. It must also be
recognized that the non-hepatotoxic KT2b showed very poor neuro-
protective activity on any of the toxic insults investigated.
Finally, based on the results described above, KT2d was
selected as our hit-compound. Thus, its docking analysis was car-
ried out, using the program Autodock Vina and the crystal struc-
ture (PDB: 1B41) of hAChE available as a complex with fasciculin
II, in order to justify its binding properties on hAChE.
1
0 mM
3 mM
0 mM
3 mM
0 mM
3 mM
0 mM
1 mM
np
np
np
1
1
1
ꢃ
ꢃꢃꢃ
42.92 ± 0.20
41.19 ± 0.18
25.10 ± 0.06
4.1 ± 0.04
35.5 ± 4.1
60.2 ± 3.57
51.56 ± 0.18
65.11 ± 0.20
ꢃꢃꢃ
ꢃ
17
ꢃꢃ
ꢃꢃ
ꢃꢃ
Melatonin
Kojic acid
50.1 ± 3.42
59.1 ± 4.50
np
ꢃꢃꢃ
ꢃꢃꢃ
ꢃꢃꢃ
3
mM
1 mM
mM
3
np
Neuroprotection was expressed in percentage viable cells of control condition.
Data are the means ± SEM of quadruplicates from three different cultures;
32
ꢃ
ꢃꢃ
ꢃꢃꢃ
p < 0.05, P ꢄ 0.01, p < 0.001, as compared to the control cultures (one-
way ANOVA); np: not protective.
Figure 2. Binding mode of inhibitor (R)-KT2d at the active site of hAChE. Compound (R)-KT2d is pictured as ball and sticks in pink and the side chains of the
mobile residues are represented in the pale pink. The sub-sites of the active site were coloured: oxyanion hole (OH) in fuchsia, anionic sub-site (AS) in orange, except
Trp86, acyl binding pocket (ABP) in yellow, catalytic triad (CT) in green and PAS in light violet. Hydrogen bond interactions are represented as dashed black lines.

1
68
Y. DGACHI ET AL.
Figure 3. Docking pose of inhibitor (S)-KT2d at the active site of hAChE. Compound (S)-KT2d is illustrated as ball and sticks in blue and the side chains of the
mobile residues are represented in pale blue. The subsites of the active site were coloured: Oxyanion hole (OH) in fuchsia, anionic sub-site (AS) in orange, except
Trp86, acyl binding pocket (ABP) in yellow, catalytic triad (CT) in green, and peripheral anionic subsite (PAS) in light violet. Hydrogen bond interactions are represented
as dashed black lines.
Molecular modelling of compound KT2d
Since compound KT2d has a chiral centre at the pyran ring, the between the (R)-enantiomer [Figure 2 and Figure S1 (Supporting
reported biological data refer to the racemic mixture, and the Information)], the (S)-enantiomer [Figure and Figure S2
On the basis of the set of interactions, which were identified
3
observed whole result is a combination of the effects of both (Supporting Information)] and the protein residues within the
enantiomers. Nevertheless, to obtain more information about the active site gorge of both enzymes, we conclude that the OH
binding properties, docking studies were performed on both (R)- group and the phenyl ring are likely to be important features for
and (S)-enantiomers of compound KT2d. The docking procedure these derivatives to exhibit hAChE inhibitory activities.
was applied to the whole protein target, without imposing the
binding site (“blind docking”). Flexible docking experiments have
been performed for (R)- and (S)-enantiomers of KT2d. To address
ADME of compounds KT2a–l
Theoretical calculations of the ADME (Absorption, Distribution,
receptor flexibility in hAChE, the rearrangement of the side chains
Metabolism, and Excretion) properties of both enantiomers of tac-
of eight residues, Trp286, Tyr124, Tyr337, Tyr72, Asp74, Thr75,
rine derivatives KT2a–l were accomplished using the QikProp
module of Schrodinger suite (QikProp, version 3.8, Schrodinger,
LLC, New York, NY, 2013) running in normal mode, for assessing
Trp86, and Tyr341 was allowed. The movement of these residues
may increase the size of the gorge and thus facilitate the access
25,40
of bulky ligands to the catalytic site
.
the druggability. Drug kinetics and exposure of tissues to drug
influences the pharmacological activity and the performance of a
drug, which is ultimately determined by its ADME properties.
Nearly 45 physically significant descriptors and pharmacologically
relevant properties were predicted and investigated (Table S1, see
Supporting Information).
In particular, for compound KT2d, the more significant observed
data were the following: total Solvent Accessible Surface Area, in
square angstroms, using a probe with a 1.4 Å radius (SASA 5
Both (R)- and (S)-enantiomers of compound KT2d were studied
in docking simulations. The binding energy of (S)-enantiomer
(
ꢁ10.4 Kcal/mol) was lower than that of the (R)-enantiomer
(ꢁ9.6 Kcal/mol). As depicted in Figures 2 and 3, both enantiomers
are positioned in the enzyme active site in such way to interact with
the aminoacids at the rim of the cavity and act as a barrier prevent-
ing the entry of the substrate into the active site. The fused-rings,
part of compound KT2d, which has an approximately planar struc-
ture, make p–p-stacking with the aromatic aminoacids at the rim of
the peripherial active site (PAS), including Trp286 and Tyr341. The
phenyl ring showed p-alkyl hydrophobic interaction with Leu76
653.798 and 670.621 for (R)- and (S)-enantiomer, respectively; lim-
its 300.0–1000.0); estimated number of hydrogen bonds that would
be accepted by the solute (donorHB 52.5 for both enantiomers;
limits: 2.0–20.0); predicted octanol/water partition coefficient
(Figures S1 and S2, see Supporting Information).
Furthermore, the binding is also supported by the formation of
(QPlogPo/w 5 2.716 and 2.662 for (R)- and (S)-enantiomer, respect-
hydrogen bonds. For compound (R)-KT2d, a hydrogen bond
between the Tyr124-OH and the nitrogen atom of the pyranota-
crine moiety is established (Figure 2). For compound (S)-KT2d, the
amino group established hydrogen bond interactions with Asp74
carboxylate group (key residue of PAS) and the oxygen atom of
ively; limits ꢁ2.0–6.5); predicted aqueous solubility. S, in mol/dm3, is
the concentration of the solute’s saturated solution that is in equilib-
rium with crystalline solid (QPlogS 5 24.852 and 25.125 for (R)-
and (S)-enantiomer, respectively; limits ꢁ6.5–0.5); Van der Waals sur-
face area of polar nitrogen and oxygen atoms (PSA 5107.282 and
the methoxy group establishes a hydrogen bond interaction with 107.713 for (R)- and (S)-enantiomer, respectively; limits 7.0–200.0);
the backbone-NH-group of Thr75 (Figure 3). Hydrogen bonding predicted brain/blood partition coefficient (QPlog BB 5 21.083
interactions with residue Tyr72 were observed for both enantiom- and 21.289 for (R)- and (S)-enantiomer, respectively; limits
ers. As can be seen in Figure 5, another p-anion interaction has ꢁ3.0–1.2); number of violations of Lipinski’s Rule Of Five (molecular
been established for (S)-KT2d enantiomer between the phenyl weight <500, QPlogPo/w < 5, number of hydrogen bond donor ꢄ5,
moiety and the Asp74.
number of hydrogen bond acceptors HB ꢄ10; ROF 50 for both

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
169
enantiomers); number of violations of Jorgensen’s rule of three
QPlogS > ꢁ5.7, QPCaco >22 nm/s, number of primary metabolites
7; ROT 51 for both enantiomers).
4. Finder VH. Alzheimer’s disease: a general introduction and
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(
<
8
:856–65.
Conclusions
6
.
Bush AI. Drug development based on the metals hypothesis
We are persuaded that the MTSM approach is the most appropriate
therapeutic strategy to design new drugs for AD therapy. In this con-
text, Lipinski’s rules must be kept in mind during drug discovery to
ensure molecular properties concerning pharmacokinetics will enable
candidate drugs to have a greater opportunity of achieving the market.
Based on this concept, new single dual-targeted chemical entities have
been designed and synthesized by juxtaposition of molecular frame-
works from KA and tacrine. The new KTs are small molecules showing
ChE inhibitory activity, antioxidant, and neuroprotective properties.
Particularly, KT2d [IC50 (hAChE) ¼ 4.52 ± 0.24 mM; TE ¼ 4.79], is six-fold
less-hepatotoxic than tacrine at 1000 mM, nontoxic for SH-SY5Y cells at
of Alzheimer’s disease. J Alzheimers Dis 2008;15:223–40.
7. Opazo C, Huang X, Cherny RA, et al. Metalloenzyme-like
activity of Alzheimer’s disease beta-amyloid. Cu-dependent
catalytic conversion of dopamine, cholesterol, and biological
reducing agents to neurotoxic H(2)O(2). J Biol Chem 2002;
277:40302–8.
8. Wilkinson D, Wirth Y, Goebel C. Memantine in patients with
moderate to severe Alzheimer’s disease: meta-analyses using
realistic definitions of response. Dement Geriatr Cogn Disord
2014;37:71–85.
9. Bond M, Rogers G, Peters J, et al. The effectiveness and
cost-effectiveness of donepezil, galantamine, rivastigmine
and memantine for the treatment of Alzheimer’s disease
10 mM, exhibiting significant neuroprotection against Ab1–40 at 3 mM
and 10 mM; thus, KT2d is a potential new hit agent that deserves fur-
ther investigation for AD therapy. Work is now in progress in our
laboratory to separate the enantiomers of the racemic mixture of com-
pound KT2d in order to analyze their corresponding in vitro and in
vivo biological properties. Results will be reported elsewhere.
(
review of Technology Appraisal No. 111): a systematic
review and economic model. Health Technol Assess 2012;16:
–470. [10.3310/hta16210]
1
1
0. Perez D, Martinez A, Gil C, Campillo N. From bitopic inhibi-
tors to multitarget drugs for the future treatment of
Alzheimer’s disease. Curr Med Chem 2015;22:3789–806.
1. Agis-Torres A, Sollhuber M, Fernandez M, Sanchez-Montero
JM. Multi-target-directed ligands and other therapeutic strat-
egies in the search of a real solution for Alzheimer’s disease.
Curr Neuropharmacol 2014;12:2–36.
2. Cavalli A, Bolognesi ML, Minarini A, et al. Multi-target-
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Disclosure statement
1
The authors declare no competing financial interest.
Funding
1
LI thanks the Regional Council of Franche-Comte (2016YC-04540
and 04560) for financial support. OS thanks Faculty of Military
Health Sciences, University of Defense, MH CZ – DRO (UHHK, 13. Le oꢀ n R, Garcia AG, Marco-Contelles J. Recent advances in
0
0179906) and by Long Term Development Plan – 1011 for sup-
the multitarget-directed ligands approach for the treatment
port. JMC thank MINECO (Spain) for grant SAF2012-33304 and EU
of Alzheimer’s disease. Med Res Rev 2013;33:139–89.
(COST Action CA15135: “Multi-target paradigm for innovative 14. Rosini M, Simoni E, Caporaso R, Minarini A. Multitarget strat-
ligand identification in the drug discovery process”) for support.
egies in Alzheimer’s disease: benefits and challenges on the
road to therapeutics. Future Med Chem 2016;8:697–711.
5. Ismaili L, Refouvelet B, Benchekroun M, et al. Multitarget
compounds bearing tacrine- and donepezil-like structural
and functional motifs for the potential treatment of
Alzheimer’s disease. Prog Neurobiol 2017;151:4–34.
1
ORCID
Daniel Jun
http://orcid.org/0000-0002-0882-6304
Jana Janockova
Ondrej Soukup
Isabel Iriepa
http://orcid.org/0000-0002-2034-1860
http://orcid.org/0000-0001-6376-8701
http://orcid.org/0000-0003-3475-9624
16. Benchekroun M, Ismaili L, Pudlo M, et al. Donepezil-ferulic
acid hybrids as anti-Alzheimer drugs. Future Med Chem
Ignacio Moraleda
Jos ꢀe Marco-Contelles
Lhassane Ismaili
http://orcid.org/0000-0001-8032-5689
http://orcid.org/0000-0003-0690-0328
http://orcid.org/0000-0002-6013-3150
2
015;7:15–21.
1
7. Benchekroun M, Romero A, Egea J, et al. The antioxidant
additive approach for Alzheimer’s disease therapy: new
ferulic (lipoic) acid plus melatonin modified tacrines as choli-
nesterases inhibitors, direct antioxidants, and nuclear factor
(
9
erythroid-derived 2)-like 2 activators. J Med Chem 2016;59:
967–73.
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