Design, synthesis and biological evaluation of novel indano- and thiaindano-pyrazoles with potential interest for Alzheimer's disease

Article abstract of DOI:10.1039/c3md00041a

The synthesis of eighteen novel (thia)indanopyrazole derivatives was achieved starting from amino(thia)indanones. Some of them displayed a dual binding site acetylcholinesterase inhibition which makes them potentially interesting for Alzheimer's disease treatment.

Full text of DOI:10.1039/c3md00041a

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Design, synthesis and biological evaluation of novel
indano- and thiaindano-pyrazoles with potential
interest for Alzheimer’s disease†
Cite this: DOI: 10.1039/c3md00041a
ab
ab
David Genest,ab Christophe Rochais,
Cedric Lecoutey, Jana Sopkova-de Oliveira
*
´
Santos,ab Celine Ballandonne, Sabrina Butt-Gueulle,ab Remi Legay,ab Marc Sinceab
and Patrick Dallemagneab
ab
´
Received 7th February 2013
Accepted 4th April 2013
The synthesis of eighteen novel (thia)indanopyrazole derivatives was achieved starting from amino(thia)
indanones. Some of them displayed a dual binding site acetylcholinesterase inhibition which makes
them potentially interesting for Alzheimer’s disease treatment.
DOI: 10.1039/c3md00041a
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performed a study which led to the design of a DBS AChE
inhibition pharmacophore.28 The latter appears particularly
1 Introduction
Since a cholinergic hypothesis has been postulated as a crucial
element in Alzheimer’s Disease (AD) symptoms etiology, cata-
lytic acetylcholinesterase (AChE) inhibitors have constituted
until today the main drugs used against AD.1 It seems however
necessary today to complete this symptomatic approach with a
more disease-modifying one, these two activities being able to
be associated in a single chemical compound. Such Multi-
Target-Directed Ligands (MTDL) have been proposed.2–10
Among them, Dual Binding Site (DBS) AChE inhibitors appear
as promising anti-AD agents11–13 and several novel inhibitors
have been developed.14–25 In fact, they have the potential to
restore the cholinergic decit by inhibiting the catalytic site
(CAS) of AChE and at the same time to reduce the Ab deposition
and aggregation through their interaction with the peripheral
anionic site (PAS)26 of the enzyme. Indeed the PAS is implicated
in a stable complex with Ab and promotes its aggregation.
Donepezil itself, a widely used AD drug, is an AChE DBS
inhibitor, inhibits brillogenesis (%inhibition ¼ 22% at
100 mM)12 and appears more efficient than the other catalytic
AChE inhibitors in the latest stages of the disease.27
useful to design some novel DBS inhibitors starting from CAS
AChE inhibitors.
Fig. 1 Structure of donepezil and general structure of (thia)indano eeAChE
inhibitors.
˚
The distance separating the two AChE sites is about 14 A and
consequently it is possible to design appropriate DBS inhibitors
with relatively small compounds conserving a correct phar-
maco-kinetic prole. For this purpose, we recently have
a
´
Normandie Universite, France
b
´
Universite de Caen Basse-Normandie, Centre d’Etudes et de Recherche sur le
´
Medicament de Normandie (EA 4258 - FR CNRS 3038 INC3M - SF 4206 ICORE)
UFR des Sciences Pharmaceutiques, Bd Becquerel, F-14032 Caen, France. E-mail:
christophe.rochais@unicaen.fr; Fax: +33-231-566-803; Tel: +33-231-566-803
† Electronic supplementary information (ESI) available: Full experimental
procedures and characterization data for reported compounds including X-ray
structures of compounds 2b and 7a. CCDC 899872 and 899873. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c3md00041a
Fig. 2 The AChE binding sites from the X-ray structure of complexes with (A)
donepezil (PDB ID: 4EY7) and (B) with propidium (PDB ID: 1N5R). The protein is
represented as a ribbon, the side chains of binding site residues are in stick
representation and red balls represent water molecules. This figure was made
with PYMOL (DeLano Scientific, 2002, San Carlo, USA).
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yields.33 The E geometry of the latter was established by NOE
experiments and conrmed by the X-ray structure of 2b.34 The
pyrazole derivatives 3a–c, 4a–c and 5a–c were quantitatively
obtained by treating 2a–c in boiling acetic acid with hydrazine
sulfate, methylhydrazine and phenylhydrazine respectively.35–38
Acidic hydrolysis yielded the hydrochloric salts of the (thia)
indano-pyrazolamines 6 to 8a–c. Concerning the N-substituted
derivatives 4a–c, 5a–c, 7a–c and 8a–c the methyl group and
phenyl ring were selectively borne by the N-1 atom as observed
through NMR experiments and conrmed by X-ray diffractom-
etry of 7a.34
The amino group of the synthesized tricyclic compounds was
then converted into a haloalkyl carboxamide one with chlor-
oacetyl chloride in DCM at 0 ꢀC in the presence of TEA. The
sequence led to the carboxamides 9 to 11a–c in good yields.
Finally the targeted structures 12 to 17a–c were obtained by
treating 9 to 11a–c with N-benzyl and 2-uorobenzyl piperazines
respectively in the presence of K2CO3 in reuxing acetone.
Fig. 3 Targeted structures.
A few years ago, we performed the synthesis of numerous
(thia)indano derivatives, among which some analogs of done-
pezil exhibited potent catalytic electric eel (ee) AChE inhibition
(Fig. 1).29
In order to obtain a DBS inhibition prole for this series, we
performed a molecular modeling study in which we compared
the AChE binding sites from the X-ray structure of complexes
with donepezil and propidium, a reference PAS inhibitor which
strongly inhibits brillogenesis (%inhibition ¼ 82% at 100 mM)
(Fig. 2).12 This study suggested that the introduction of a third
fused ring on the thiaindane moiety of MR 26086 could enhance
its interaction with the PAS of AChE. We wish to report herein
the results obtained with the synthesis of some indano and
thiaindanopyrazole derivatives (Fig. 3).
3 Biology
Compounds 12 to 17a–c were rst evaluated as potential
inhibitors of electric eel (ee) AChE according to Ellman’s test,39
donepezil being used as a control (Table 1). Some of them were
further evaluated against human (h) AChE and equine (eq)
butyrylcholinesterase (BuChE). Finally, displacement of propi-
dium iodide from the PAS of eeAChE was performed for selected
compounds using donepezil as a control.40
3.1 Results
2 Chemistry
Concerning the catalytic inhibition of eeAChE, among the
The access to targeted compounds was achieved starting from eighteen tested compounds, ve of them (13c, 17a, 16c, 14c and
triuoroacetylamino(thia)indanones (1a–c) that we previously 16a) exhibited submicromolar IC50, whereas donepezil inhibi-
described (Scheme 1).30–32 The latter were treated by DMF–DMA ted the CAS of the enzyme with an IC50 of 20 nM. Compounds
in reuxing toluene to give the enamines 2a–c in quantitative were further evaluated against hAChE. The inhibitory activities
Scheme 1 Chemical pathway to targeted structures 12 to 17a–c. Reagents and conditions: (i) DMF–DMA, toluene, reflux, 8 h; (ii) NH2NH2, H2SO4 (R ¼ H) or NH2NHR
(R ¼ Me, C6H5), AcOH, reflux, 4 h; (iii) 6 M HCl, reflux, 2 h; (iv) ClCH2COCl, TEA, DCM, 0 ꢀC to rt, 1 h; (v) benzylpiperazine, 2HCl (X ¼ H) or 2-fluorobenzylpiperazine (X ¼ F),
K2CO3, acetone, oil bath reflux or microwave irradiation.
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Table 1 Inhibition of eeAChE, hAChE, and eq BuChE for compounds 12 to 17a–c and propidium iodide competition assay for selected compounds
eeAChE
hAChE
Eq BuChE
Propidium iodide
displacement
IC50 (mM)
IC50 (mM)
IC50 (mM)
Compound
X
R
or (%) inh 10ꢁ5
M
or (%) inh 10ꢁ5
M
or (%) inh 10ꢁ5
M
(%) inh 10ꢁ5
M
12a
13a
14a
15a
16a
17a
12b
13b
14b
15b
16b
17b
12c
13c
14c
15c
16c
H
2.94
1.28
1.36
3.98
0.72
0.21
49%
1.91
31%
4.59
1.31
4.06
8.82
0.10
0.29
4.53
0.24
27%
0.020
49%
1.84
2.99
50%
2.44
2.43
9.18
0.93
36%
42%
1.32
42%
1.13
0.33
0.87
42%
0.88
15%
0.011
NTa
NT
NT
NT
1%
8%
NT
NT
NT
NT
NT
NT
NT
7.76
37%
NT
30%
NT
NT
NTa
NT
NT
NT
H
F
Me
C6H5
H
Me
C6H5
H
Me
C6H5
H
Me
C6H5
H
Me
C6H5
H
Me
C6H5
—
11% ꢂ 1, n ¼ 3
12% ꢂ 2, n ¼ 4
NT
H
F
NT
NT
NT
NT
NT
NT
H
F
15% ꢂ 1, n ¼ 3
14% ꢂ 3, n ¼ 3
NT
7% ꢂ 3, n ¼ 3
17c
Donepezil
NT
—
—
19% ꢂ 3, n ¼ 2
a
NT: not tested.
of the ve selected compounds were globally correlated between
the two enzymes with IC50 three times higher against hAChE,
except for 17a whose IC50 differs by a factor twelve between
hAChE and eeAChE. This difference could be explained by the
fact that the hAChE aromatic gorge is narrower than in other
species which could lead to conformational ip of the piperi-
dine ring in donepezil.41 As described by Cheung et al.41 these
differences in the gorge shape could affect generally the binding
of long inhibitors such as DBS inhibitors. Furthermore, all of
them acted as a selective inhibitor of AChE over BuChE.
The ve selected compounds were further tested to evaluate
their capacity to displace propidium iodide from the PAS of
eeAChE. Two of them (13c and 14c) displaced propidium in a
slightly weaker manner (%inhibition of the uorescence of
propidium bound to AChE ¼ 14–15% at 10 mM) than donepezil
(19%), whereas 16c appeared weaker (7%).
3.2 SAR discussion
These results show rstly that the benzylpiperazine–carbox-
amide moiety borne by a (thia)indanopyrazole system is
likely to conserve the catalytic AChE inhibition exerted by the
(thia)indane series and to display further interactions with
the PAS of the enzyme. However, in a similar manner to
that of the (thia)indane series,28 the nature and the substi-
tutions of the aromatic ring appear crucial for the catalytic
inhibition of the enzyme since the members of the cyclo-
penta[b]thiophene series (b) appear less active than those of
the dibromocyclopenta[c]thiophene (c) and dimethoxy-
indane (a) series. The presence of a uorine atom on the
benzyl ring seems likely to maintain or to slightly improve
the activity in regard to the corresponding unsubstituted
compounds, except for 17c, which has totally lost the activity
expressed by 14c.
Consequently, we studied the mechanism of AChE inhibi-
tion for compounds 13c and 16c by means of a kinetic study, the
results of which are reported in a typical Lineweaver–Burk plot
(Fig. 4). For 16c, the study accounted for a non-competitive
inhibition, its inhibition constant (Ki) being equal to 429 nM.
For 13c, for which Ki was equal to 235 nM, the kinetic prole
accounted for a reversible, mixed-type model of inhibition, in
accordance with a likely DBS mode of interaction with AChE.42
On the other hand, substitution of the N-1 atom of the
pyrazole ring by a methyl group appears important for the
catalytic inhibition of AChE since N-methyl compounds (13a–c
and 16b,c) are generally more potent than the unsubstituted or
phenyl substituted corresponding compounds. The most potent
catalytic inhibitor in this series is 13c (eeAChE-IC50 ¼ 100 nM,
hAChE-IC50 ¼ 330 nM).
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Fig. 4 Lineweaver–Burk plots of inhibition kinetics for 13c (A) and 16c (B): reciprocals of enzyme activity (eeAChE) vs. reciprocal of substrate (acetylthiocholine)
concentration in the presence of different concentrations of tested compounds.
5 Conclusion
In conclusion, during this work we have synthesized several
novel (thia)indanopyrazole derivatives and some of them
strongly inhibit the catalytic activity of electric eel and human
AChE in a submicromolar range of magnitude and further
displace propidium from the PAS of the enzyme in a similar
manner to that of donepezil. The latter result accounts for a
potential inhibitory effect against AChE-induced Ab aggregation
likely to complete the capacity of these MTDL to preserve
cholinergic neuro-transmission in the AD experimental model.
This work is under investigation.
Fig. 5 Compound 13c (orange) and donepezil (magenta) positioned in the
AChE binding site from docking studies. The protein is represented as a ribbon,
6 Experimental part
6.1 General
the side chains of binding site residues are in stick representation. This figure was
made with PYMOL (DeLano Scientific, 2002, San Carlo, USA).
All commercial solvents and reagents were used as-received.
The microwave reactions were performed using a Biotage
Concerning the DBS inhibition, the latter is observed espe-
Initiator Microwave oven using 2–5 mL sealed vials; tempera-
cially in dibromocyclopenta[c]thiophene series (compounds 13c
tures were measured with an IR-sensor and reaction times given
and 14c) which displace propidium from the PAS of AChE with
as hold times. Flash chromatography was realized on a spot 2
the same range of activity than donepezil. Compound 16c, the
apparatus. Melting points were determined on a Koer melting
uoro analog of 13c, however does almost not interact with the
point apparatus and were uncorrected. IR spectra were recorded
PAS of the enzyme. These results are supported by the kinetic
on KBr discs; only selected absorbances were quoted. TLC was
study of these compounds.
carried out on 5 ꢃ 10 pre-coated plates with silica gel GF254
type 60; LC/MS (ESI) analyses were realized with a separating
module using the following gradient: A (95%)/B (5%) to A (5%)/
4 Molecular modeling
B (95%) in 5 min; this ratio was held for 2 min before returning
The docking of 13c, the best synthesized ligand in our series, to initial conditions in 1 min. Initial conditions were then
showed that its position in the AChE binding site remains very maintained for 2 min (A: H2O, B: CH3CN; each containing
close to the donepezil one, in spite of the presence of the pyr- HCOOH: 0.1%; column: C18, ow: 0.4 mL minꢁ1). MS detection
azole ring (Fig. 5). Along the docking pose, 13c appears to be was performed by positive or negative ESI. High resolution mass
capable of interacting with the AChE CAS and PAS and it can be spectra were recorded at 40 eV by electronic impact (HREIMS) or
1
considered as a DBS inhibitor. However the introduction of the positive or negative electrospray (HRESIMS). H and 13C NMR
pyrazole ring led to a different position of the tricycle which did spectra were recorded, respectively, at 400 and 100 MHz using
not improve its interaction with the PAS. These observations are CDCl3, d6-DMSO, CD3OD or (CD3)2CO as solvents. 2D NMR
correlated with the experimental results showing the same level spectra and 1D NOESY experiments were recorded at 500 MHz.
in the propidium displacement for 13c and donepezil.
The apparent multiplicity is described as s (singlet), d (doublet),
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dd (doublet of doublets), t (triplet), q (quadruplet), m (multi-
plet); chemical shis d are reported in parts per million with the
solvent resonance as the internal standard; coupling constants
J are given in Hertz.
6.2.4 2-Chloro-N-(6,7-dimethoxy-1,4-dihydroindeno[1,2-c]
pyrazol-4-yl)acetamide (9a). To a suspension of 6a (500 mg,
1.64 mmol) in DCM (10 mL) was added TEA (0.71 mL,
5.26 mmol, 3.2 equiv.). Aer 5 min, the mixture was cooled in an
ice bath and a solution of chloroacetyl chloride (144 mL,
1.80 mmol, 1.1 equiv.) in DCM (10 mL) was added drop-wise.
When the addition was over, the ice bath was removed and the
mixture was stirred for 1 h. The solvent was then evaporated
under reduced pressure and the crude powder was triturated in
water, ltered and dried under air. This led to 9a as a white
powder (364 mg, 72%); mp 200–202 ꢀC; IR (KBr) l (cmꢁ1) 2933,
1655, 1592, 1518, 1485, 1465, 1434, 1384, 1281, 1215, 1186,
6.2 Chemistry
6.2.1 N-{(2E)-2-[(Dimethylamino)methylene]-5,6-dimethoxy-
3-oxo-2,3-dihydro-1H-inden-1-yl}-2,2,2-triuoroacetamide (2a).
To a suspension of 10 g of 1a (33 mmol) in toluene (150 mL) was
added DMF–DMA (8.8 mL, 2 equiv.). The suspension was then
heated at reux for 8 h, then cooled to room temperature and
concentrated under reduced pressure. The resulting orange
powder obtained was triturated in ice-cold ether and ltered,
which led to 2a as a yellow powder (11.1 g, 94%); mp > 260 ꢀC; IR
(KBr) l (cmꢁ1) 2999, 2972, 2937, 2839, 1702, 1663, 1589, 1533,
1501, 1472, 1436, 1360, 1310, 1283, 1207, 1184, 1145, 1128, 1097,
1033, 999; 1H NMR (400 MHz, CDCl3) d 8.26 (d, J ¼ 7.83 Hz, 1H,
NH), 7.05 (s, 1H, CHNMe2), 7.04 (s, 1H, H phenyl), 7.01 (s, 1H, H
phenyl), 6.25 (1H, d, J ¼ 8.79 Hz, CHNH), 3.92 (3H, s, OCH3), 3.90
(3H, s, OCH3), 3.06 (6H, s, NMe2); 13C NMR (100 MHz, CDCl3) d
191.0, 157.8 (q, J ¼ 37.4 Hz, COCF3), 153.9, 150.2, 147.4 (2C),
141.8, 131.3, 116.2 (q, J ¼ 287.8 Hz, COCF3), 106.4, 103.4, 103.0,
56.4, 56.0, 50.0 (2C); HREIMS [M+] m/z 359.1219 (calcd for
C16H17F3N2O4 359.1219).
6.2.2 N-(6,7-Dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-4-yl)
2,2,2-triuoroacetamide (3a). To a suspension of 5 g of 2a
(14 mmol) in AcOH (30 mL) was added hydrazine sulfate (2.18 g,
16.7 mmol, 1.2 equiv.). The suspension was then heated at reux
for 4 h, then cooled to room temperature and concentrated under
reduced pressure. The crude mixture was then triturated in water,
ltered and dried underꢀair. This led to 3a as a yellow powder
(4.34 g, 95%); mp > 260 C; IR (KBr), l (cmꢁ1) 3271, 1702, 1550,
1486, 1386, 1284, 1212, 1187, 1156, 1034; 1H NMR (400 MHz, d6-
DMSO), d 12.67 (s, 1H, NH pyrazole), 10.00 (d, J ¼ 5.8 Hz,
NHCOCF3), 7.66 (s, 1H, H pyrazole), 7.18 (s, 1H, H phenyl), 7.05 (s,
1H, H phenyl), 5.74 (d, J ¼ 5.8 Hz, 1H, CHNH), 3.83 (s, 3H, OCH3),
3.76 (s, 3H, OCH3); 13C NMR (100 MHz, d6-DMSO) d 157.6, 157.5
(q, J ¼ 35.7 Hz, COCF3), 149.8, 148.4, 140.7, 127.2, 124.8, 123.5,
116.1 (q, J ¼ 287.4 Hz, COCF3), 109.7, 103.4, 55.9 (2C), 48.0;
HRESIMS [M + H] m/z 328.0984 (calcd for C14H12F3N3O3
328.0909).
1
1173, 1136, 1034; H NMR (400 MHz, CDCl3) d 7.50 (s, 1H, H
pyrazole), 7.29 (s, 1H, H phenyl), 7.08 (s, 1H, H phenyl), 6.78 (d,
J ¼ 7.79 Hz, 1H, NH acetamide), 5.92 (d, J ¼ 7.79 Hz, 1H,
CHNH), 4.19 (d, J ¼ 15.6 Hz, 1H, Ha), 4.14 (d, J ¼ 15.6 Hz, 1H,
Hb), 3.96 (s, 3H, OCH3), 3.93 (s, 3H, OCH3); 13C NMR (100 MHz,
CDCl3) d 166.7, 159.2, 150.3, 149.5, 141.4, 126.9, 124.8, 124.5,
109.2, 103.8, 56.4, 56.3, 48.6, 42.6; HREIMS [M+] m/z 307.0709
(calcd for C14H14ClN3O3 307.07234).
6.2.5 2-(4-Benzylpiperazin-1-yl)-N-(6,7-dimethoxy-1,4-dihy-
dro-indeno[1,2-c]pyrazol-4-yl)acetamide (12a). Chloroacetamide
9a (100 mg, 0.32 mmol), benzylpiperazine dihydrochloride
(89 mg, 0.36 mmol, 1.1 equiv.) and K2CO3 (144 mg, 1.04 mmol,
3.2 equiv.) were reuxed in acetone (2 mL) for 12 h. Aer being
cooled to ambient temperature, the crude mixture was ltered
and the insoluble product was washed with DCM. The ltrates
were combined and the solvents were evaporated under reduced
pressure. The crude powder obtained was then puried by
column chromatography (Al2O3, gradient from MeOH/DCM
0/100 to 10/90). This led to 14a as a yellow powder (51 mg, 35%);
mp 152 ꢀC; IR (KBr) l (cmꢁ1) 2929, 2834, 1662, 1581, 1492, 1445,
1380, 1288, 1223, 1134, 1031; 1H NMR (400 MHz, CDCl3) d 7.45
(s, 1H, H pyrazole), 7.27 (m, 6H, H phenyl), 7.04 (s, 1H, H
phenyl), 5.96 (d, J ¼ 7.79 Hz, 1H, CHNH), 3.94 (s, 3H, OCH3),
3.88 (s, 3H, OCH3), 3.44 (s, 2H, CH2 phenyl), 3.15 (d, J ¼ 16.6 Hz,
1H, Ha), 3.10 (d, J ¼ 16.6 Hz, 1H, Hb), 2.54 (broad m, 4H, H
piperazine), 2.38 (broad m, 4H, H piperazine); 13C NMR
(100 MHz, CDCl3) d 171.1, 159.1, 149.9, 149.2, 142.4, 137.8,
129.1 (2C), 128.3 (2C), 127.2, 126.7, 125.5, 124.2, 108.8, 103.6,
62.8, 61.6, 56.3, 56.2, 53.5 (2C), 52.9 (2C), 47.7; HRESIMS [M + H]
m/z 448.2339 (calcd for C25H29N5O3 448.2349).
6.2.3 6,7-Dimethoxy-1,4-dihydroindeno[1,2-c]pyrazol-4-amine
dihydrochloride (6a). Triuoroacetamide 3a (2 g, 6.11 mmol) was
suspended in 6 M HCl solution (15 mL) and reuxed for 2 h. The
6.3 Biology
6.3.1 In vitro tests of AChE and BuChE biological activity.
solvent was evaporated under reduced pressure and the crude Inhibitory capacity of compounds on AChE biological activity
powder was triturated in ice-cold EtOH, ltered, triturated in ice- was evaluated through the use of the spectrometric method of
cold ether and ltered again. This led to 6a as a yellow powder Ellman. Acetyl- or butyrylthiocholine iodide and 5,5-dithiobis-
(1.52 g, 82%); mp 227–229 ꢀC; IR (KBr) l (cmꢁ1) 2915, 2599, 1624, (2-nitrobenzoic) acid (DTNB) were purchased from Sigma
1
1585, 1505, 1461, 1441, 1280, 1221, 1129, 1058, 1027; H NMR Aldrich. Lyophilized electric eel AChE (Type III, Sigma Aldrich)
+
(400 MHz, d6-DMSO) d 8.90 (d, J ¼ 5.2 Hz, 3H, NH3 Clꢁ), 7.72 (s, or BuChE from equine serum (Sigma Aldrich) was dissolved in
1H, H pyrazole), 7.70 (s, 1H, H phenyl), 7.22 (s, 1H, H phenyl), 5.07 0.2 M phosphate buffer pH 7.4 to obtain enzyme stock solu-
+
(q, J ¼ 5.2 Hz, 1H, CHNH3 Clꢁ), 3.84 (s, 3H, OCH3), 3.79 (s, 3H, tions with 2.5 units per mL enzyme activity. AChE from human
OCH3); 13C NMR (100 MHz, d6-DMSO) d 157.5, 150.0, 148.2, 138.0, erythrocytes (buffered aqueous solution, $500 units per mg
127.2, 126.3, 121.3, 110.9, 103.5, 55.9, 47.3, 34.2; HRESIMS [M + H] protein (BCA), Sigma Aldrich) was diluted in 20 mM HEPES
m/z 232.1085 (calcd for C12H13N3O2 232.1086).
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buffer pH 8, 0.1% Triton X-100 to obtain enzyme stock solution
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with 2.5 units per mL enzyme activity. In this procedure, 100
mL of 0.3 mM DTNB dissolved in phosphate buffer pH 7.4 were
added into the 96 well plate followed by 50 mL of test
compound solution and 50 mL of enzyme solution. Aer 5 min
of preincubation, the reaction was then initiated by the
injection of 50 mL of 10 mM acetyl- or butyrylthiocholine
iodide solution. The hydrolysis of acetyl- or butyrylthiocholine
was monitored by the formation of a yellow 5-thio-2-nitro-
benzoate anion as the result of the reaction of DTNB with
thiocholine, released by the enzymatic hydrolysis of acetyl- or
butyrylthiocholine, at a wavelength of 412 nm every minute for
10 min using a 96-well microplate plate reader (TECAN Innite
M200, Lyon, France). Test compounds were dissolved in
analytical grade DMSO. Donepezil was used as a reference
standard.
6.4 Molecular modeling
The docking of the compound 13c into human AChE (PDB ID:
4EY7)41 was carried out with the GOLD program (v5.0) using the
default parameters.43,44 This program applies a genetic algo-
rithm to explore conformational spaces and ligand binding
modes. To evaluate the proposed ligand poses the ChemScore
tness function was applied in the docking studies.
˚
The binding site in the AChE was dened as a 12 A sphere
centred at the donepezil ligand present in the 4EY7 crystal
structure. Furthermore, we paid special attention during the
docking procedure to water molecules in the binding site and
water HOH931 was included in the binding site during docking.
Acknowledgements
First screening of AChE and BuChE activity was carried out at
a 10ꢁ5 M concentration of compounds under study. For the
compounds with signicant inhibition ($50%) aer 4 min of
reaction, IC50 values were determined graphically from 6 point
inhibition curves using the Origin soware.
`
This work was supported through funding from the Ministere
´
Français de l’Enseignement Superieur et de la Recherche and
the Conseil Regional de Basse Normandie (Dispositif de Soutien
´
aux Projets de Recherche Emergents).
6.3.2 Propidium competition assay. Propidium exhibits an
increase in uorescence on binding to the AChE peripheral site,
making it a useful probe for competitive ligand binding to the
enzyme. Fluorescence was measured in a Tecan Innite M200
plate reader. Measurements were carried out in 200 mL solution
volume, in 96-well plates. The buffer used was 1 mM Tris–HCl,
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ꢀ
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