Synthesis and evaluation of tacrine-E2020 hybrids as acetylcholinesterase inhibitors for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2004.07.005

Tacrine-E2020 hybrids and some related compounds were prepared and their bioactivities on the Alzheimer's Disease were assayed. The optimum hybrid inhibitor 3 is much more potent and selective than tacrine in vitro. Tacrine-E2020 hybrids and some related compounds were prepared and their bioactivities on the Alzheimer's disease were assayed. The optimum hybrid inhibitor 3 is 37-fold more potent and 31-fold more selective than tacrine in vitro.

Full text of DOI:10.1016/j.bmcl.2004.07.005

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4639–4642
Synthesis and evaluation of tacrine–E2020 hybrids as
acetylcholinesterase inhibitors for the treatment of
Alzheimerꢀs disease
Dong Shao, Chunyan Zou, Cheng Luo, Xican Tang and Yuanchao Li*
Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences,
Graduate School of the Chinese Academy of Sciences, 555 Zhu Chong Zhi Road, Zhangjiang Hi-Tech Park,
Shanghai 201203, China
Received 26 May 2004; revised 29June 2004; accepted 1 July 2004
Available online 24 July 2004
Abstract—Tacrine–E2020 hybrids and some related compounds were prepared and their bioactivities on the Alzheimerꢀs disease
were assayed. The optimum hybrid inhibitor 3 is 37-fold more potent and 31-fold more selective than tacrine in vitro.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
pounds, the optimum inhibitor 3 is much more potent
and selective than tacrine (1) (Fig. 1).
Alzheimerꢀs disease (AD), the most common form of
dementia among the elderly, is a progressive, degenera-
tive disorder of the brain with a loss of memory, and
cognition.1 The social and economic consequences of
AD are alarming due to the notable increase in life
expectancy. Tacrine (1) is a reversible inhibitor of acetyl-
cholinesterase (AChE) that was launched in 1993 as the
first drug for the treatment of AD.2 The evaluation of
the clinical effects of tacrine has shown efficacy in delay-
ing the deterioration of the symptoms of AD, while con-
firming the adverse events consisting mainly in the
elevated liver transaminase levels.3 The study of tacrine
analogs is still of interest to medicinal chemists involved
in AD research. In fact, the possibility to design potent
and selective bis-tetrahydroaminoacridine4 and tacrine–
huperzine A Hybrids5 was demonstrated. The other
modification of tacrine has been reported.6–12 E2020
(2) is a member of a large family of N-benzylpiperidine
based AChE inhibitors, which were developed, synthe-
sized, and evaluated by the Eisai Company in Japan,
and has been approved for use in the US for the treat-
ment of AD.13 In this communication we report a new
class of tacrine–E2020 hybrids and other related com-
2. Chemistry
The synthesis of the compound 3 was accomplished as
shown in Scheme 1. 11 can be prepared from commer-
cially available 1-benzyl-4-piperidone 8 and malononit-
rile in 66.4% over three steps, the synthetic method
has not been reported before, it can be synthesized on
large scale. Reduction of 11 with LiAlH4 provided
amine 12 (83%). Intermediate 12 was then converted
to the corresponding 13 (72.3%) with the active p-ni-
tro-phenyl ester in THF at room temperature, deprotec-
tion of 13 with CF3COOH in CH2Cl2 to give 14 in
76.4% yield. 9-Chloro-1,2,3,4-tetrahydroacridine 15
can be prepared from anthranilic acid and cyclohexa-
none in 89%,4b treatment of 15 with 14 in refluxing
1-pentanol provided the hybrid 3 in 81.1%, then it was
treated with fumaric acid–EtOH, and the resulting solid
were crystallized from EtOH–Et2O to give the fumaric
salt of 3 (70.6%).14
The synthesis of the compound 4 was accomplished as
shown in Scheme 2. Ethyl piperidine-4-carboxylate 16
reacted with benzoyl chloride and hydrolyzed in 10%
NaOH to give 17 (84.6%), Esterification of 17 with 4-
nitrophenol in DCC and aminated to provide the amide
18 (71.5%). Reduction of 18 with LiAlH4 provided
Keywords: Tacrine–E2020; Hybrids; Acetylcholinesterase inhibitor;
Alzheimerꢀs disease.
*
Corresponding author. Tel./fax: +86-21-50807288; e-mail: ycli@mail.
shcnc.ac.cn
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.005

4640
D. Shao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4639–4642
N
O
N
MeO
MeO
N
N
H
N
( )n
NH
NH2
O
1
2
4
n=1
3
n=2
Tacrine
E-2020
Figure 1. Structure of tacrine, E-2020, and their hybrids.
CN
CN
CN
a
b
PhCH2
N
C
PhCH2
N
O
PhCH2
N
PhCH2
N
CH
COOCH3
9
10
8
d
c
PhCH2
N
CH2CH2NH2
N
CH2CN
11
12
14
f
e
PhCH2
N
CH2CH2NHCOCH2NHBoc
PhCH2
N
CH2CH2NHCOCH2NH2
13
N
g
H
N
HN
Cl
O
N
3
15
Scheme 1. Synthesis of the hybrid 3. Reagents and conditions: (a) CH2(CN)2, CH2Cl2, rt, 90.3%; (b) NaBH4, HCl/CH3OH, 85.5%; (c) 10% NaOH/
HCl, DMF, reflux, 86%; (d) LiAlH4, rt, 83%; (e) BocNHCH2 COO–Ph–NO2-p, THF, 72.3%; (f) CF3COOH/CH2Cl2, rt, 76.4%; (g) 14, 1-pentanol,
reflux, 81.1%.
O
O
O
h
i
HN
COOC2H5
16
Ph
N
COOH
Ph
N
NH2
17
18
k
j
Ph
N
PhCH2
N
CH2NHCOCH2NH2
N H2
19
20
N
N
l
N
H
N
NH
Cl
O
15
4
Scheme 2. Synthesis of the hybrid 4. Reagents and conditions: (h) C6H5COCl/Et3N, 10% NaOH, 84.6%; (i) p-NO2–Ph–OH/DCC, NH3ÆH2O, 71.5%;
(j) LiAlH4/THF, reflux, 73.6%; (k) BocNHCH2COO–Ph–NO2-p/THF, CF3COOH/CH2Cl2, 52.4%; (l) 20, 1-pentanol, reflux, 76.9%.
amine 19 (73.6%). Intermediate 19 reacted with the
active p-nitro-phenyl ester in THF, then deprotection
of Boc group with CF3COOH in CH2Cl2 to give 20
(52.4%). Treatment of 15 with 20 in refluxing 1-pentanol
provided the hybrid 4 in 76.9%, which was treated with
fumaric acid–EtOH to give the fumaric salt of 4 (73.2%).
As can be seen (Table 1), tacrine–E2020 hybrids 3 and 4
are more potent for AChE inhibition than tacrine (1).5c
The optimum 3 is easily synthesized, has nanomolar
affinity for AChE (IC50 =6.0nM), and is 37-fold more
potent and 31-fold more selective than tacrine (1). In
the related compounds, 6 is similar to tacrine for AChE
inhibition, 5 and 7 are weak inhibitors. However, their
selectivity is higher than tacrine (Scheme 3).
3. Biological results and discussion
With completion of the synthesis, in vitro AChE and Bu-
ChE inhibitory activities of the hybrids and some related
compounds as compared with tacrine (1) were measured
according to the method of Ellman et al.15 using rat cor-
tex homogenate (AChE) and rat serum (BuChE).
4. Molecular modeling
To understand the recognition of 3 and to enable
rational design of new derivatives, we examined the
binding modes of 3 in AChE, the hybrid 3 is a long

D. Shao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4639–4642
Table 1. Cholinesterase inhibition IC50 (nM)
4641
Compd
AChE
BuChE
Selectivity for AChE
3
6.0 0.7
10.2 1.1
662 38
230 18
869 41
223 11
76 8.2
12.7
1.2
4
12.4 1.3
19,200 134
6920 57
5
29.0
30.1
20.5
0.4
6
7
Tacrine (1)
17,800 113
92
2
O
O
H
N
m
n
MeO
MeO
MeO
MeO
N
H
Cl
O
N
5
21
O
N
N
H
N
O
N
H
6
22
O
O
o
N
HN
H
N
H
N
H
23
7
Scheme 3. Synthesis of some related compounds 5–7. Reagents and conditions: (m) 14, Et3N/CH2Cl2, 82.0%; (n) 14, Ti(O-iPr)4/NaBH4, 46.0%; (o)
14, Ti(O-iPr)4/NaBH4, 35.2%.
chain molecule, our hypothesis was to assume that its
binding to AChE shares some or all of the features that
modulate the binding of E-2020, certainly there maybe
the tacrine ring of the hybrid 3 at the catalytic site and
the phenyl group at the peripheral site. Binding free
energies were calculated through autodock soft. We
found the free energy of binding DG=À15.84kcal/mol
with the tacrine ring at the peripheral site and
DG=À14.42kcal/mol with the tacrine ring at the
catalytic site, which exhibited the former state with
AChE much more stable than the latter. Accordingly,
modeling of the interaction 3 with the enzyme was based
on the crystallographic structures of AChE complexes
with E-2020.16 The figure was plotted by insight II soft
(left) and ligplot soft (right), respectively.
As seen in Figure 2, the hybrid 3 makes principal inter-
actions along the active-site gorge of the enzyme
Figure 2. Plot of the main interactions between AChE and ligand 3.

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D. Shao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4639–4642
Lieu, Y. P.; Wong, H. S.; Pang, Y. P. Bioorg. Med. Chem.
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through its four major functional groups: the benzyl
moiety, the piperidine nitrogen, the nitrogen–hydrogen
of the amide, and the acridine moiety. Near the bottom
of the gorge, one face of the benzyl ring displays classic
parallel p–p stacking with the six-membered ring of the
˚
Trp84 indole, the ring-to-ring distance is 3.62A between
Trp84 and the benzyl ring. In the constricted region,
halfway up the gorge, the charged nitrogen of the piperi-
dine ring makes a cation–p interaction with the phenyl
˚
ring of Phe330, with the distances of 4.61A between
the nitrogen and the aromatic ring. The nitrogen–hydro-
gen of the amide makes an hydrogen bond with the oxy-
˚
gen of Tyr121, the distance is 3.4A. At the top of the
gorge the acridine ring stacks against the indole of
Trp279, in the peripheral binding site, by a classical p–
˚
p interaction with the distances of 3.75A.
˚
In the homolog the molecular chain length of 3 is 16.8A,
it approaches in the distance of the two tacrine-binding
sites,4a which is 16A, 3 derives its potency and selectivity
˚
from simultaneous binding to the peripheral and cata-
lytic sites of AChE.
11. Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.;
Carlier, P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 1053.
5. Conclusion
12. Marco, J. L.; Rios, C.; Garcia, A. G.; Villarroya, M.;
Carreiras, M. C.; Martins, C.; Eleuterio, A.; Morreale, A.;
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2199.
13. Kawakami, Y.; Inoue, A.; Kawai, T.; Wakita, M.;
Sugimoto, H.; Hopfinger, A. Bioorg. Med. Chem. 1996,
4, 1429.
In summary, the hybrids of tacrine–E2020 and some re-
lated compounds were synthesized and their bioactivi-
ties on AD were assayed. The optimum inhibitor 3 is
much more potent and selective than tacrine. The mode-
ling studies clearly indicate that ligand 3 is nicely accom-
modated by AChE, engaging in appropriate hydrogen
bond interactions. It is useful for the modification of
the hybrids and design of new AChE inhibitors. Further
studies on hybrids are in progress and will be reported in
due course.
14. 3 (fumaric salt) mp 186–188°C (d) 1H NMR (400MHz,
DMSO-d6, ppm): d 8.24 (t, 1H, J=5.6Hz), 8.18 (d, 1H,
J=8.4Hz), 7.88 (d, 1H, J=8.5Hz), 7.73 (t, 1H, J=7.2Hz),
7.45 (t, 1H, J=7.2Hz), 7.37–7.26 (m, 5H), 7.14 (br s, 1H),
6.58 (s, 2H), 4.35 (s, 2H), 3.68 (s, H), 3.14 (q, 2H,
J=6.5Hz), 2.97 (br s, 2H), 2.90 (br d, 2H, J=12.1Hz),
2.69(br s, 2H), 2.15 (t, 2H, J=10.0Hz), 1.83–1.81 (m, 4H),
1.64 (d, 2H, J=11.7Hz), 1.35–1.21 (m, 5H) 13C NMR
(100MHz DMSO-d6, ppm): 168.3, 166.8, 154, 152.8,
140.4, 134.8, 134.5, 131.3, 129.9, 128.3, 127.9, 124.5,
124.2, 121.8, 116.7, 112.3, 60.7, 52.1, 49.4, 36.2, 35.3, 31.6,
30.1, 29.4, 23.6, 21.7, 20.9; IR (KBr): 3291, 2942, 1714,
1677, 1276, 1188, 983, 630cmÀ1: EI-MS MS (m/z): 456
(M+). Anal. Calcd for C29H36N4OÆC4H4O4: C 69.21, H
7.04, N 9.78. Found: C 68.92, H 7.01, N 9.74.
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