Design, synthesis and evaluation of novel heterodimers of donepezil and huperzine fragments as acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.11.044

Four series of novel heterodimers comprised of donepezil and huperzine A (HupA) fragments were designed, synthesized, and evaluated in search of potent acetylcholinesterase (AChE) inhibitors as potential therapeutic treatment for Alzheimer's disease. Hete

Full text of DOI:10.1016/j.bmc.2012.11.044

Bioorganic & Medicinal Chemistry 21 (2013) 676–683
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of novel heterodimers of donepezil
and huperzine fragments as acetylcholinesterase inhibitors
Yueqing Hu a,c, Jun Zhang a,c, Oormila Chandrashankra a,c, Fanny C. F. Ip a,b,c, Nancy Y. Ip a,b,c,
⇑
a Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
b State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
c Biotechnology Research Institute, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Four series of novel heterodimers comprised of donepezil and huperzine A (HupA) fragments were
designed, synthesized, and evaluated in search of potent acetylcholinesterase (AChE) inhibitors as poten-
tial therapeutic treatment for Alzheimer’s disease. Heterodimers comprised of dimethoxyindanone (from
donepezil), hupyridone (from HupA), and connected with a multimethylene linker, were identified as
potent and selective inhibitors of AChE. Diastereomeric heterodimers (RS,S)-17b (with a tetramethylene
linker) exhibited the highest potency of inhibition towards AChE with an IC50 value of 9 nM and no
detectable inhibitory effect on butyrylcholinesterase at 1 mM.
Received 12 October 2012
Revised 26 November 2012
Accepted 27 November 2012
Available online 6 December 2012
Keywords:
Alzheimer’s disease
Acetylcholinesterase
Heterodimer
Ó 2012 Elsevier Ltd. All rights reserved.
Huperzine A
1. Introduction
two lead compounds of interest have proven to be successful strat-
egies in the discovery of novel AChE inhibitors. Dimeric AChE
Alzheimer’s disease (AD) is a common age-related neurodegen-
erative disorder characterized by progressive cognitive dysfunction
and memory loss, neuropsychiatric and behavioral disturbances,
and restrictions in daily activities.1 Recent report indicates that
approximately 36 million people worldwide suffer from AD, and
this number is projected to reach 115 million by the year 2050.2
Although tremendous progress has been made in understanding
the pathophysiological mechanisms underlying AD, at present
there is no cure for this disease. The current medications used
for the symptomatic treatment of AD mainly belong to a class of
acetylcholinesterase (AChE) inhibitors which enhance central cho-
linergic neurotransmission by inhibiting the degradation of acetyl-
choline.3 Figure 1 depicts the structures of current therapeutic
agents for AD. Out of the five medications on the market, four of
them (tacrine, donepezil, rivastigmine and galantamine) are AChE
inhibitors, while memantine is a NMDA receptor antagonist. How-
ever, all of these medicines show unwanted side effects in some of
the patients and their effectiveness diminishes after prolonged
treatment.4 Due to the rapidly increasing aging population glob-
ally, there is a huge demand for unmet medical need.
inhibitors exhibit improved pharmacological profiles as they can
interact with both the active and peripheral sites of AChE, or simul-
taneously interact with AChE and another biological target in-
volved in Alzheimer’s disease.5 Since the discovery of tacrine
homodimers in the mid-1990s,6 a variety of dimeric AChE inhibi-
tors have been identified over the years.7–15 For example, homodi-
mers of two inactive hupyridones from huperzine A (HupA),
heterodimers of hupyridone and tacrine, and heterodimers of
donepezil and tacrine, have been reported to be potent AChE inhib-
itors.7–11
We report here our efforts in search of novel potent AChE inhib-
itors using the dimerization strategy. A number of heterodimers,
with structural moieties derived from two known AChE inhibitors,
donepezil and HupA, were designed, synthesized, and evaluated.
2. Results and discussion
2.1. Design and chemistry
The heterodimers were designed based on the structures of two
potent AChE inhibitors, donepezil and HupA (Fig. 2). The binding
modes of donepezil and HupA with AChE have been revealed by
3D X-ray structures, molecular docking, and mutagenesis stud-
ies.16,17 Donepezil interacts with AChE at both the active-site gorge
and the peripheral anionic site. While the dimethoxyindanone
moiety of donepezil binds to the peripheral site, the piperidine
The search for novel AChE inhibitors with improved biological
profiles continues to be of great interest to medicinal chemists.
Hybridization and dimerization of two structural units of one or
⇑
Corresponding author. Tel.: +852 2358 7267; fax: +852 2358 1552.
E-mail address: boip@ust.hk (N.Y. Ip).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.044

Y. Hu et al. / Bioorg. Med. Chem. 21 (2013) 676–683
677
Figure 1. Current therapeutic agents for AD.
Figure 2. Design strategy for heterodimers of donepezil and huperzine A fragments.
and benzyl moieties bind to the mid and lower gorge, the active site
of AChE. Donepezil does not bind to the catalytic triad.18 HupA, how-
ever, binds to the AChE active-site gorge and the catalytic triad but
not the peripheral site, either directly or indirectly via the carbonyl
oxygen, the ring amine, the 5-amino and the ethylidene methyl
groups.19
In view of the superior potency of donepezil and HupA, a total of
four series of heterodimers were designed. These heterodimers con-
sisted of a dimethoxybenzene unit from donepezil and a hupyridone
fragment from HupA linked by a chain of methylenes. Based on the
crystallographic findings on the complexes comprised of Torpedo
californica AChE (TcAChE) with either donepezil, HupA or bis-
hupyridones,18–20 it was speculated that the dimethoxybenzene
unit would interact with the peripheral site while the hupyridone
fragment would bind to the active site of the AChE. The hupyridone
fragment could act as a bioisosteric structure of benzylpiperidine as
both fragments contain basic nitrogen and aromatic units. Since the
carbonyl oxygen of donepezil was involved in AChE binding, an oxy-
gen atom at a similar position was preserved in the heterodimer
structures. In series A, an amide bond was used to connect the dime-
thoxyphenyl to the multimethylene linker whereas in series B and C,
an ester and ether bond were put in place, respectively. In series D,
the dimethoxyindanone ring was preserved, and was directly con-
nected to the multimethylene linker. Since the number of carbons
between the carbonyl and basic nitrogen in donepezil is 5, heterodi-
mers with a linker consisting of 3–9 methylenes were synthesized
and evaluated. Heterodimers of series A, B, and C were enantiomeri-
cally pure, while compounds of series D were expected to be mix-
tures comprising of a pair of diastereomers due to the racemic
nature of the C2 stereogenic center of the indanone ring. The config-
uration of C2 indanone system in series D was expected to intercon-
vert via a keto-enol intermediate based on studies of donepezil.21

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Y. Hu et al. / Bioorg. Med. Chem. 21 (2013) 676–683
Scheme 1. Synthesis of hupyridone building blocks.
The benzyl protected hupyridone building block (S)-3 and its
a,x-multimethylene hydroxylamine to produce amides 5a/5c.
enantiomer (R)-3 were synthesized from a readily available start-
ing material—dihydroresorcinol 1 through intermediate 2 in 6
steps according to literature (Scheme 1).7,22 The enantiomeric ex-
cess of (S)-3 and (R)-3 were both >97%, as determined by optical
rotation analysis.
Synthesis of heterodimers with A, B, and C scaffolds was accom-
plished as shown in Scheme 2. The S-enantiomer of hupyridone
benzyl ether 3 was utilized to prepare these heterodimers. The
starting material for both series A and B was 3,4-dimethoxybenzoic
acid 4. Treatment of acid 4 with thionyl chloride generated
3,4-dimethoxybenzoyl chloride, which was then reacted with
The terminal hydroxyl groups of 5a/5c were then converted to
the corresponding chlorides with thionyl chloride to generate com-
pounds 6a/6c. Mixing of the chlorides 6a/6c with amine (S)-3 with-
out addition of any base, and subsequently heating the mixture to
130 °C yielded heterodimers (S)-7a/7c. Deprotection of the benzyl
ether by catalytic hydrogenation generated the target heterodi-
mers (S)-8a/8c of series A. Treatment of 4 with a,x-dibromoalkane
in the presence of potassium carbonate generated bromides 9a–g.
Reaction of bromides 9a–g with amine (S)-3, followed by deprotec-
tion of the benzyl group resulted in heterodimers (S)-10a–g of
series B. For series C, combination of the dimethoxyphenol with
Scheme 2. Synthesis of heterodimers A, B and C series.

Y. Hu et al. / Bioorg. Med. Chem. 21 (2013) 676–683
679
Scheme 3. Synthesis of heterodimers of D series.
a
,
x
-dibromoalkanes in the presence of sodium hydride produced
IC50 and selectivity of AChE over BuChE for donepezil, HupA, and
tacrine resulting from the assay were within the range reported
in literature.7,9,18,19 Across the different series—series A with the
amido-multimethylene linker, series B with the carboxy-multim-
ethylene linker, and series C with the oxy-multimethylene lin-
ker—all of the compounds exhibited poor AChE inhibition
potency with IC50 in the micromolar range. They were much less
potent than either of the two original compounds. Of the three ser-
ies, compound (S)-12c of series C, consisting of a pentamethylene
linker, exhibited the best activity towards AChE inhibition with
an IC50 of 590 nM and an affinity for AChE 70 times greater than
for BuChE. Importantly, a tether length-dependent AChE potency
was observed in series B. Compound (S)-10b, which contained 4
methylenes, most effectively inhibited AChE within the series,
indirectly indicating a dual binding mode for these compounds.
Interestingly, compounds that exhibited the most promising
inhibitory effect on AChE belonged to series D, which were hetero-
dimers of dimethoxyindanone and hupyridone. Diastereomeric
mixture (RS,S)-17b with a linker of 4 methylenes showed the
strongest inhibition towards AChE with an IC50 of 9 nM. It was
equally potent to donepezil (reported IC50 = 5.7 nM)24, and ꢀ7-fold
and ꢀ28-fold more potent towards AChE inhibition than HupA and
tacrine. Diastereomers (RS,S)-17a and (RS,S)-17c with only one
bromides 11c/11d. Target products (S)-12c/12d were then ob-
tained by reacting the bromides 11c/11d with amine (S)-3 followed
by benzyl deprotection.
The heterodimers of series D were prepared as shown in
Scheme 3. Synthesis was initiated by treating dimethoxyindanone
13 with sodium hydride and diethyl carbonate to incorporate an
ethoxycarbonyl group at the
ating 14. Mono-alkylation of 14 with
a
-position of the carbonyl thus gener-
-dibromoalkanes in the
a,x
presence of sodium hydride provided 15a–g with a terminal bro-
mide. Diastereomeric mixtures (RS,S)-16a–g were then prepared
by reacting racemic bromides 15a–g with chiral amine (S)-3 by
heating toluene to form heterodimers with the carboxyl ethyl ester
attached, followed by saponification with sodium hydroxide and
decarboxylation to remove the carboxyl ethyl ester. Catalytic
hydrogenation removed the benzyl protecting group in (RS,S)-
16a–g, thus generating the desired heterodimers (RS,S)-17a–g.
Alternatively, another pair of diastereomers (RS,R)-17b were syn-
thesized from racemic intermediate 15b (n = 4) and chiral amine
(R)-3 following the same methodology.
Although the heterodimers of series D are a pair of diastereo-
mers due to the racemic nature of the C2 position on the indanone
ring, there were no detectable differences in 1H NMR and 13C NMR
of the heterodimers with a linker comprised of more than 5 meth-
ylenes. For diastereomers (RS,S)-17a and (RS,S)-17b that consisted
of shorter linkers (3 or 4 methylenes), duplicated doublets were
detected in the 1H NMR at ꢀ7.45 ppm and in a ratio of 1:1. The
configuration of the stereogenic center at C2 position of the inda-
none ring could interconvert via a keto–enol intermediate as ob-
served for donepezil,21 but no effort was made to separate the
two diastereomers in this study.
methylene different from (RS,S)-17b were ꢀ4600- (41
lM) and
ꢀ20-fold (187 nM) less potent than (RS,S)-17b towards AChE inhi-
bition, indicating the importance of the length of the linker in AChE
inhibition potency. Furthermore, the heterodimers in series D,
especially (RS,S)-17a–d, exhibited high selectivity for AChE as they
did not show any inhibitory effect on BuChE at the maximum con-
centration tested (1 mM). In particular, (RS,S)-17b exhibited great-
er than 100,000-fold selectivity for AChE inhibition (assuming the
IC50 for BuChE was ꢀ1 mM), which was a substantial increase in
specificity when compared with donepezil, Hup A, and tacrine
(1344, 1138, and 0.6, Table 1).
2.2. Biological activity
The AChE and BuChE inhibitory activities of the heterodimers
were determined by a modified version of the Ellman’s method.23
AChE and BuChE were freshly prepared from the cortices and ser-
um of adult rats, respectively. At least three independent experi-
ments were performed to evaluate the efficacies of the
heterodimers on cholinesterase inhibition.
The data is summarized in Table 1. Serving as controls, donepe-
zil hydrochloride, HupA, and tacrine exhibited inhibitory effects
against AChE with IC50 of 32, 65, and 249 nM, respectively. The
The dimethoxyindanone unit of donepezil binds to Trp279 lo-
cated at the peripheral site of TcAChE. Due to the lack of a corre-
sponding aromatic residue in BuChE, the binding of
dimethoxyindanone unit to the indole ring of Trp279 accounts
for the higher selectivity on AChE of donepezil.19 The presence of
a dimethoxyindanone unit in our heterodimers might preserve this
advantage on the selectivity. In accordance with Cardozo’s re-
port,25 the carbonyl group of the indanone ring of donepezil is
important for its higher potency in AChE inhibition, since our series

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Y. Hu et al. / Bioorg. Med. Chem. 21 (2013) 676–683
Table 1
In vitro inhibitory activities of heterodimers against AChE and BuChE
Compounda
Series
n
AChE IC50 (nM)
BuChE IC50 (nM)
BuChE IC50/AChE IC50
(S)-8a
(S)-8c
(S)-10a
(S)-10b
(S)-10c
(S)-10e
(S)-10g
(S)-12c
A
A
B
B
B
B
B
C
C
D
D
D
D
D
D
D
3
5
3
4
5
7
9
5
6
3
4
5
6
7
9
4
88,000 10,000
12,000 1800
162,000 46,000
4100 2000
7200 700
8200 1000
9670 30
590 10
139,000 16,000
33,000 11,000
860,000 160,000
15,000 1000
460,000 57,000
194,000 67,000
308,000 48,000
42,000 2000
1.6
2.7
5.3
3.7
64
24
32
71
4.5
NDc
ND
ND
ND
222
261
ND
1344
1138
0.6
(S)-12d
3950 60
41,000 3000
17,700 9700
b
(RS,S)-17a
(RS,S)-17b
(RS,S)-17c
(RS,S)-17d
(RS,S)-17e
(RS,S)-17g
(RS,R)-17b
Donepezil
(ꢂ)-Huperzine A
Tacrine
—
9
1
—
—
—
187 33
37
8
1860 40
2600 40
530 120
32 16
65
413,000 49,000
678,000 52,000
—
43,000 7200
74,000
249 19
137 19
a
b
c
All of the compounds were assayed as their HCl salts.
No inhibition at 1 mM.
Not detectable.
A–C lacking an intact indanone ring showed poorer inhibitory
effect on AChE when compared to series D. Furthermore, when
comparing (RS,S)-17b with (RS,R)-17b, (RS,R)-17b inhibited AChE
ꢀ60-fold less potently with an IC50 of 530 nM. The observations
for (RS,S)-17b and (RS,R)-17b were also seen in the homodimer
of hupyridones. The (S,S)-enantiomer exhibited a ꢀ60-fold greater
potency than its (R,R)-form.7
Our data further supports the notion that the absolute configu-
ration of the stereogenic center near the basic nitrogen greatly af-
fects the potency of the AChE inhibitor.7 The optimal tether length
between indanone and hupyridone of heterodimer D series is 4
methylenes. In the structure of donepezil, the space between the
indanone ring and basic nitrogen is also four carbon units. These
results indicate that our design strategy of replacing N-benzylpi-
peridine with the bioisosteric hupyridone moiety successfully
resulted in generating potent and selective AChE inhibitors. How-
ever, since the heterodimers in series D are a pair of diastereomers,
data analysis for pharmacological potency of these compounds
should be taken with caution. It will be of further interest to exam-
ine their individual potency towards cholinesterase inhibition.
400 MHz spectrometer. Chemical shifts of 1H NMR spectra were
measured in parts per million referenced to residual solvent (chlo-
roform at 7.26, TMS at 0.00, methanol at 3.30 ppm). Coupling con-
stants were measured in hertz. Chemical shifts of 13C NMR were
referenced to residual solvent (chloroform at 77.0, methanol at
49.0 ppm). HRMS (ESI) was obtained using API QSTAR XL Mass
Spectrometer system. Optical rotations were measured using a
JASCO P-2000 digital polarimeter. Final compounds in HCl salt
form were used for measuring the optical rotation.
4.1.1. 5,6–Dimethoxy-1-oxo-indan-2-carboxylic acid ethyl ester
(14)
To a solution of diethyl carbonate (1.89 mL, 15.6 mmol), NaH
(0.46 g, 15.6 mmol, 80% dispersion in mineral oil) in toluene
(10 mL), a solution of 5,6-dimethoxy-indan-1-one 13 (1.50 g,
7.8 mmol) in toluene (20 mL) was added dropwise over a 1 h per-
iod, and the mixture was stirred at reflux for 5 h. After cooling to
room temperature, the reaction was quenched with water
(30 mL) and acetic acid (1.5 mL). The resulting mixture was ex-
tracted with dichloromethane (3 ꢁ 30 mL), and the combined or-
ganic layers were washed with brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. Purification by flash column
chromatography (silica gel, EtOAc/PE, 3/7) generated the title com-
pound (1.76 g, 85%) as a white solid. 1H NMR (300 MHz, CDCl3): d
7.18 (s, 1H), 6.92 (s, 1H), 4.24 (q, J = 7.0 Hz, 2H), 3.98 (s, 3H), 3.91 (s,
3H), 3.69–3.72 (m, 1H), 3.45 (dd, J = 17.0, 3.5 Hz, 1H), 3.28 (dd,
J = 17.0, 7.9 Hz, 1H), 1.31 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz,
CDCl3) d 198.1, 169.5, 156.0, 149.8, 149.3, 128.0, 107.3, 104.8,
61.7, 56.3, 56.1, 53.6, 30.1, 14.2.
3. Conclusion
In conclusion, based on the superior binding properties of
donepezil and HupA with AChE,18–20 we have designed and synthe-
sized four different series of heterodimers consisting of donepezil
and HupA fragments, and have evaluated their effectiveness as
anti-cholinesterase agents. Potent and selective AChE inhibitors
were identified from novel heterodimers comprising of dimeth-
oxyindanone from donepezil and hupyridone from HupA (series
D). Heterodimers (RS,S)-17b linked by 4 methylenes exhibited
the highest potency of inhibition towards AChE with an IC50 value
of 9 nM and no detectable inhibitory effect on BuChE at 1 mM.
4.1.2. 2-(3-Chloropropyl)-5,6-dimethoxy-1-oxo-indan-2-
carboxylic acid ethyl ester (15a, n = 3)
A
mixture of 5,6-dimethoxy-1-oxo-indan-2-carboxylic acid
ethyl ester 14 (235 mg, 0.88 mmol) and NaH (79.5 mg, 2.65 mmol,
80% dispersion in mineral oil) in DMF (8 mL) was stirred at room
temperature for 30 min, after which 1-chloro-3-iodo propane
(0.185 mL, 1.76 mmol) was added. The reaction mixture was stir-
red at room temperature for 20 h and quenched by adding water
(15 mL) and aqueous HCl (1 mL, 1.0 N). The mixture was extracted
with dichloromethane (3 ꢁ 20 mL), and the combined organic lay-
ers were washed with brine, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The resulting residue was
4. Experimental section
4.1. Chemistry
All reagents and starting materials were purchased from com-
mercial sources and used without further purification. 1H NMR
and 13C NMR were recorded using a Varian 300 MHz or Joel

Y. Hu et al. / Bioorg. Med. Chem. 21 (2013) 676–683
681
purified by column chromatography (silica gel, EtOAc/PE, 1/4) to
generate the title compound (200 mg, 66%) as pale yellow oil. 1H
NMR (300 MHz, CDCl3): d 7.09 (s, 1H), 6.85 (s, 1H), 4.07 (q,
J = 7.1 Hz, 2H), 3.91 (s, 3H), 3.83 (s, 3H), 3.55 (d, J = 17.0 Hz, 1H),
3.44 (t, J = 6.5 Hz, 2H), 2.91 (d, J = 17.0 Hz, 1H), 2.14 (m, 1H), 1.95
(m, 1H), 1.67 (m, 2H), 1.16 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz,
CDCl3): d 200.5, 171.0, 156.0, 149.7, 148.3, 127.6, 107.0, 104.7,
61.4, 60.1, 56.2, 55.9, 44.7, 36.5, 32.0, 27.8, 13.9.
0.5 mL) was refluxed for 21 h. The solvent was removed under re-
duced pressure and ice water (20 mL) was added. After extraction
with dichloromethane (3 ꢁ 20 mL), the combined organic layers
were washed with brine, dried over anhydrous Na2SO4, and con-
centrated under reduced pressure. The residue was purified by col-
umn chromatography (silica gel, MeOH/DCM, 3/97) to generate the
title compound (90 mg, 32% for two steps). 1H NMR (300 MHz,
CDCl3): d 7.54–7.58 (m, 1H), 7.42–7.44 (m, 2H), 7.28–7.36 (m,
3H), 7.15 (s, 1H), 6.84 (s, 1H), 6.57 (d, J = 8.5 Hz, 1H), 5.31 (s, 2H),
3.93 (s, 3H), 3.88 (s, 3H), 3.69 (br s, 1H), 3.20 (dd, J = 16.7, 7.0 Hz,
1H), 2.63–2.81 (m, 6H), 1.56–1.98 (m, 8H). 13C NMR (75 MHz,
CDCl3): d 207.4, 161.7, 155.4, 154.6, 149.3, 148.9, 139.7, 137.5,
129.3, 128.2, 128.2, 128.0, 128.0, 127.6, 127.0, 108.2, 107.3,
104.2, 67.3, 56.1, 56.0, 54.5, 47.3, 46.8, 32.4, 32.2, 29.3, 28.1,
28.0, 18.7.
4.1.3. 2-(4-Chlorobutyl)-5,6-dimethoxy-1-oxo-indan-2-
carboxylic acid ethyl ester (15b, n = 4)
Yield 65%. Pale yellow oil. 1H NMR (300 MHz, CDCl3): d 7.08 (s,
1H), 6.84 (s, 1H), 4.07 (q, J = 7.0 Hz, 2H), 3.90 (s, 3H), 3.82 (s, 3H),
3.54 (d, J = 17.0 Hz, 1H), 3.43 (t, J = 6.5 Hz, 2H), 2.91 (d,
J = 17.0 Hz, 1H), 1.95–2.05 (m, 1H), 1.65–1.82 (m, 3H), 1.16–1.35
(m, 2H), 1.13 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz, CDCl3): d
200.7, 171.0, 155.8, 149.5, 148.4, 127.6, 107.0, 104.6, 61.3, 60.6,
56.1, 55.8, 44.3, 36.1, 33.6, 32.4, 21.7, 13.9.
4.1.9. (2RS)-2-{4-[(5S)-2-Benzyloxy-5,6,7,8-tetrahydro-quinolin-
5-ylamino]-butyl}-5,6-dimethoxy-indan-1-one [(RS,S)-16b,
n = 4]
4.1.4. 2-(5-Bromopentyl)-5,6-dimethoxy-1-oxo-indan-2-
carboxylic acid ethyl ester (15c, n = 5)
Yield 45% for two steps. 1H NMR (300 MHz, CDCl3): d 7.56 (d,
J = 8.5 Hz, 1H), 7.44–7.46 (m, 2H), 7.27–7.37 (m, 3H), 7.17 (s, 1H),
6.86 (s, 1H), 6.59 (d, J = 8.5 Hz, 1H), 5.33 (s, 2H), 3.95 (s, 3H), 3.90
(s, 3H), 3.70 (br s, 1H), 3.22 (dd, J = 16.7, 7.7 Hz, 1H), 2.64–2.84
(m, 6H), 1.48–2.03 (m, 10H). 13C NMR (75 MHz, CDCl3): d 207.5,
161.7, 155.4, 154.6, 149.3, 148.9, 139.6, 137.5, 129.4, 128.2,
128.2, 128.0, 128.0, 127.6, 127.1, 108.2, 107.3, 104.2, 67.3, 56.1,
56.0, 54.5, 47.5, 46.7, 32.5, 32.2, 31.5, 30.4, 28.0, 25.1, 18.7.
Yield 50%. Pale yellow oil. 1H NMR (300 MHz, CDCl3): d 7.11 (s,
1H), 6.86 (s, 1H), 4.10 (q, J = 7.0 Hz, 2H), 3.93 (s, 3H), 3.85 (s, 3H),
3.56 (d, J = 17.0 Hz, 1H), 3.31 (t, J = 7.0 Hz, 2H), 2.92 (d,
J = 17.0 Hz, 1H), 1.99–2.09 (m, 1H), 1.72–1.88 (m, 3H), 1.34–1.44
(m, 2H), 1.14–1.27 (m, 5H). 13C NMR (75 MHz, CDCl3): d 200.9,
171.2, 155.9, 149.5, 148.4, 127.8, 107.0, 104.7, 61.4, 60.7, 56.2,
56.0, 36.3, 34.4, 33.5, 32.2, 28.2, 23.6, 13.9.
4.1.10. (2RS)-2-{5-[(5S)-2-Benzyloxy-5,6,7,8-tetrahydro-
quinolin-5-ylamino]-pentyl}-5,6-dimethoxy-indan-1-one
[(RS,S)-16c, n = 5]
4.1.5. 2-(6-Bromohexyl)-5,6-dimethoxy-1-oxo-indan-2-
carboxylic acid ethyl ester (15d, n = 6)
Yield 61%. Pale yellow oil. 1H NMR (300 MHz, CDCl3): d 7.11 (s,
1H), 6.85 (s, 1H), 4.11 (q, J = 7.0 Hz, 2H), 3.93 (s, 3H), 3.88 (s, 3H),
3.55 (d, J = 17.0 Hz, 1H), 3.31 (t, J = 6.7 Hz, 2H), 2.92 (d,
J = 17.0 Hz, 1H), 1.98–2.05 (m, 1H), 1.70–1.86 (m, 3H), 1.14–1.36
(m, 6H), 1.16 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz, CDCl3) d:
201.1, 171.2, 155.9, 149.5, 148.5, 127.8, 107.0, 104.7, 61.3, 60.8,
56.2, 56.0, 36.3, 34.5, 33.7, 32.5, 28.9, 27.7, 24.3, 13.9.
Yield 57% for two steps. 1H NMR (300 MHz, CDCl3): d 7.56 (d,
J = 8.5 Hz, 1H), 7.42–7.45 (m, 2H), 7.27–7.36 (m, 3H), 7.15 (s, 1H),
6.84 (s, 1H), 6.57 (d, J = 8.5 Hz, 1H), 5.32 (s, 2H), 3.93 (s, 3H), 3.88
(s, 3H), 3.70 (br s, 1H), 3.22 (dd, J = 16.8, 7.2 Hz, 1H), 2.62–2.86
(m, 6H), 1.78–2.01 (m, 6H), 1.41–1.54 (m, 6H). 13C NMR (75 MHz,
CDCl3): d 207.6, 161.7, 155.3, 154.6, 149.3, 148.9, 139.6, 137.5,
129.4, 128.3, 128.3, 128.0, 128.0, 127.6, 126.9, 108.3, 107.3,
104.2, 67.3, 56.1, 56.0, 54.4, 47.5, 46.7, 32.5, 32.2, 31.5, 30.1,
28.0, 27.3, 27.2, 18.7.
4.1.6. 2-(7-Bromoheptyl)-5,6-dimethoxy-1-oxo-indan-2-
carboxylic acid ethyl ester (15e, n = 7)
Yield 54%. Pale yellow oil. 1H NMR (300 MHz, CDCl3): d 7.13 (s,
1H), 6.87 (s, 1H), 4.13 (q, J = 7.0 Hz, 2H), 3.95 (s, 3H), 3.87 (s, 3H),
3.57 (d, J = 17.0 Hz, 1H), 3.34 (t, J = 6.7 Hz, 2H), 2.94 (d,
J = 17.0 Hz, 1H), 2.02–2.06 (m, 1H), 1.73–1.84 (m, 3H), 1.14–1.37
(m, 8H), 1.18 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz, CDCl3): d
201.2, 171.4, 155.9, 149.6, 148.6, 127.9, 107.1, 104.8, 61.4, 60.9,
56.3, 56.1, 36.4, 34.7, 33.9, 32.7, 29.6, 28.4, 27.9, 24.4, 14.0.
4.1.11. (2RS)-2-{6-[(5S)-2-Benzyloxy-5,6,7,8-tetrahydro-
quinolin-5-ylamino]-hexyl}-5,6-dimethoxy-indan-1-one
[(RS,S)-16d, n = 6)]
Yield 47% for two steps. 1H NMR (300 MHz, CDCl3): d 7.57 (d,
J = 8.5 Hz, 1H), 7.44–7.47 (m, 2H), 7.27–7.39 (m, 3H), 7.17 (s, 1H),
6.87 (s, 1H), 6.59 (d, J = 8.5 Hz, 1H), 5.33 (s, 2H), 3.96 (s, 3H), 3.91
(s, 3H), 3.73 (br s, 1H), 3.22 (dd, J = 16.8, 7.2 Hz, 1H), 2.62–2.88
(m, 6H), 1.74–2.02 (m, 6H), 1.36–1.51 (m, 8H). 13C NMR (75 MHz,
CDCl3): d 207.7, 161.7, 155.3, 154.6, 149.3, 148.9, 139.6, 137.5,
129.4, 128.3, 128.3, 128.0, 128.0, 127.6, 126.9, 108.3, 107.3,
104.2, 67.3, 56.1, 56.0, 54.4, 47.6, 46.8, 32.5, 32.2, 31.5, 30.2,
29.5, 28.0, 27.3, 27.2, 18.7.
4.1.7. 2-(9-Bromononyl)-5,6-dimethoxy-1-oxo-indan-2-
carboxylic acid ethyl ester (15g, n = 9)
Yield 46%. Pale yellow oil. 1H NMR (300 MHz, CDCl3): d 7.16 (s,
1H), 6.89 (s, 1H), 4.14 (q, J = 7.0 Hz, 2H), 3.97 (s, 3H), 3.89 (s, 3H),
3.60 (d, J = 17.0 Hz, 1H), 3.38 (t, J = 6.7 Hz, 2H), 2.96 (d,
J = 17.0 Hz, 1H), 2.03–2.07 (m, 1H), 1.77–1.86 (m, 3H), 1.17–1.40
(m, 15H). 13C NMR (75 MHz, CDCl3): d 201.3, 171.4, 155.9, 149.6,
148.6, 128.0, 107.1, 104.9, 61.5, 61.0, 56.3, 56.1, 36.4, 34.8, 34.0,
32.7, 29.8, 29.3, 29.2, 28.6, 28.1, 24.6, 14.1.
4.1.12. (2RS)-2-{7-[(5S)-2-Benzyloxy-5,6,7,8-tetrahydro-
quinolin-5-yl-amino]-heptyl}-5,6-dimethoxy-indan-1-one
[(RS,S)-16e, n = 7]
Yield 59% for two steps. 1H NMR (300 MHz, CDCl3): d 7.56 (d,
J = 8.5 Hz, 1H), 7.42–7.44 (m, 2H), 7.26–7.36 (m, 3H), 7.15 (s, 1H),
6.84 (s, 1H), 6.57 (d, J = 8.5 Hz, 1H), 5.31 (s, 2H), 3.93 (s, 3H), 3.88
(s, 3H), 3.70 (br s, 1H), 3.20 (dd, J = 16.8, 7.2 Hz, 1H), 2.57–2.82
(m, 6H), 1.75–1.98 (m, 6H), 1.31–1.55 (m, 10H). 13C NMR
(75 MHz, CDCl3): d 207.7, 161.7, 155.3, 154.6, 149.3, 148.9, 139.6,
137.5, 129.4, 128.2, 128.2, 128.0, 128.0, 127.6, 126.9, 108.3,
107.3, 104.2, 67.3, 56.1, 56.0, 54.4, 47.6, 46.8, 32.5, 32.2, 31.5,
30.2, 29.5, 29.3, 28.0, 27.3, 27.2, 18.7.
4.1.8. (2RS)-2-{3-[(5S)-2-Benzyloxy-5,6,7,8-tetrahydro-quinolin-
5-ylamino]-propyl}-5,6-dimethoxy-indan-1-one [(RS,S)-16a,
n = 3]
A mixture of 15a, (200 mg, 0.58 mmol) and (S)-3 (223.8 mg,
0.88 mmol) in toluene (0.5 mL) was refluxed for 20 h. The solvent
was removed in vacuo to generate a residue. A mixture of the res-
idue and KOH (240 mg, 4.28 mmol) in ethanol/water (3 mL /

682
Y. Hu et al. / Bioorg. Med. Chem. 21 (2013) 676–683
4.1.13. (2RS)-2-{9-[(5S)-2-Benzyloxy-5,6,7,8-tetrahydro-
quinolin-5-ylamino]-nonyl}-5,6-dimethoxy-indan-1-one
[(RS,S)-16g, n = 9]
4.1.18. (5S)-5-{7-[(2RS)-5,6-Dimethoxy-1-oxo-indan-2-yl]-
heptylamino}-5,6,7,8-tetrahydro-1H-quinolin-2-one [(RS,S)-
17e]
Yield 42% for two steps. 1H NMR (300 MHz, CDCl3): d 7.55 (d,
J = 8.5 Hz, 1H), 7.42–7.44 (m, 2H), 7.26–7.36 (m, 3H), 7.15 (s, 1H),
6.84 (s, 1H), 6.57 (d, J = 8.5 Hz, 1H), 5.31 (s, 2H), 3.93 (s, 3H), 3.88
(s, 3H), 3.70 (br s, 1H), 3.20 (dd, J = 16.8, 7.2 Hz, 1H), 2.58–2.80
(m, 6H), 1.76–2.02 (m, 6H), 1.19–1.52 (m, 14H). 13C NMR
(75 MHz, CDCl3): d 207.8, 161.7, 155.4, 154.7, 149.3, 148.9, 139.7,
137.5, 129.4, 128.3, 128.3, 128.0, 128.0, 127.6, 126.9, 108.3,
107.3, 104.2, 67.3, 56.1, 56.0, 54.4, 47.6, 46.8, 32.5, 32.2, 31.6,
30.2, 29.6, 29.5, 29.4, 29.4, 27.9, 27.3, 27.3, 18.7.
Yield 90%. 1H NMR (300 MHz, CDCl3): d 7.42 (d, J = 9.0 Hz, 1H),
7.09 (s, 1H), 6.80 (s, 1H), 6.32 (d, J = 9.0 Hz, 1H), 3.89 (s, 3H), 3.83
(s, 3H), 3.48 (br s, 1H), 3.15 (dd, J = 16.8, 7.2 Hz, 1H), 2.49–2.68
(m, 6H), 1.69–1.86 (m, 6H), 1.26–1.47 (m, 10H). 13C NMR
(75 MHz, CDCl3): d 207.6, 165.0, 155.2, 149.2, 148.9, 144.2, 143.2,
129.3, 117.3, 116.7, 107.2, 104.1, 56.0, 55.9, 52.8, 47.5, 46.9, 32.4,
31.5, 30.3, 29.4, 29.2, 27.3, 27.2, 27.2, 26.6, 17.2. MS (HRCI) calcd
for C27H37N2O4 [M+H]+ 453.2748; Found 453.2743. ½a 2D3
ꢂ29.5°
ꢄ
(c 1.00, MeOH).
4.1.14. (5S)-5-{3[(2RS)-5,6-Dimethoxy-1-oxo-indan-2-yl]-
propylamino}-5,6,7,8-tetrahydro-1H-quinolin-2-one [(RS,S)-
17a]
4.1.19. (5S)-5-{9-[(2RS)-5,6-Dimethoxy-1-oxo-indan-2-yl]-
nonylamino}-5,6,7,8-tetrahydro-1H-quinolin-2-one [(RS,S)-17g]
Yield 94%. 1H NMR (300 MHz, CDCl3): d 7.47 (d, J = 9.0 Hz, 1H),
7.15 (s, 1H), 6.85 (s, 1H), 6.37 (d, J = 9.0 Hz, 1H), 3.94 (s, 3H), 3.88
(s, 3H), 3.54 (br s, 1H), 3.20 (dd, J = 16.8, 7.2 Hz, 1H), 2.55–2.73
(m, 6H), 1.70–1.91 (m, 6H), 1.26–1.47 (m, 14H). 13C NMR
(75 MHz, CDCl3): d 207.8, 165.0, 155.4, 149.3, 149.0, 144.3, 143.3,
129.5, 117.5, 116.9, 107.4, 104.3, 56.2, 56.1, 53.0, 47.7, 47.0, 32.6,
31.7, 30.3, 29.6, 29.5, 29.5, 29.4, 27.3, 27.3, 27.3, 26.8, 17.4. MS
(HRCI) calcd for C29H41N2O4 [M+H]+ 481.3061; Found 481.3061.
To a solution of (RS,S)-16a (68 mg, 0.14 mmol) in MeOH (2 mL),
10% Pd/C (40 wt%, 27 mg) was added, and the mixture was stirred
under hydrogen atmosphere at room temperature for 3 h. The cat-
alyst was removed by filtration on Celite and the filtrate was con-
centrated under vacuum. The resulting residue was purified by
column chromatography (silica gel, MeOH/DCM/NH3ꢃH2O, 1/19/
0.2) to generate the title compound (50 mg, 90%). 1H NMR
(300 MHz, CDCl3): d 7.43–7.48 (m, 1H), 7.12 (s, 1H), 6.82 (s, 1H),
6.35 (d, J = 8.7 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 3.51 (br s, 1H),
3.20 (dd, J = 16.8, 7.2 Hz, 1H), 2.58–2.73 (m, 6H), 1.54–1.94 (m,
8H). 13C NMR (75 MHz, CDCl3): d 207.4, 165.0, 155.4, 149.4,
148.9, 144.3, 143.3, 129.3, 117.3, 116.9, 107.3, 104.2, 56.1, 56.0,
53.0, 47.4, 46.9, 32.4, 29.3, 28.1, 27.5, 26.7, 18.7. MS (HRCI) calcd
½
a 2D3
ꢄ
ꢂ24.3° (c 0.82, MeOH).
4.1.20. (5R)-5-{4-[(2RS)-5,6-Dimethoxy-1-oxo-indan-2-yl]-
butylamino}-5,6,7,8-tetrahydro-1H-quinolin-2-one [(RS,R)-17b]
Procedures were the same as (RS,S)-17b except that (R)-3 was
the starting material instead of (S)-3. HRMS (ESI) calcd for
for C23H29N2O4 [M+H]+ 397.2122; Found 397.2136. ½a 2D5
ꢄ
ꢂ27.7°
C
24H31N2O4 [M+H]+ 411.2284; Found 411.2291.
½
a 2D3
+36.6°
ꢄ
(c 0.57, MeOH)
(c 1.16, MeOH).
4.1.15. (5S)-5-{4-[(2RS)-5,6-Dimethoxy-1-oxo-indan-2-yl]-
butylamino}-5,6,7,8-tetrahydro-1H-quinolin-2-one [(RS,S)-17b]
Yield 91%. 1H NMR (300 MHz, CDCl3): d 7.44 (d, J = 9.0 Hz, 1H),
7.13 (s, 1H), 6.83 (s, 1H), 6.36 (d, J = 9.0 Hz, 1H), 3.93 (s, 3H), 3.87
(s, 3H), 3.51 (br s, 1H), 3.20 (dd, J = 16.8, 7.2 Hz, 1H), 2.56–2.72
(m, 6H), 1.68–1.90 (m, 6H), 1.45–1.54 (m, 4H). 13C NMR (75 MHz,
CDCl3): d 207.6, 165.0, 155.4, 149.4, 148.9, 144.3, 143.3, 129.4,
117.4, 116.9, 107.3, 104.2, 56.1, 56.0, 52.9, 47.5, 46.7, 32.5, 31.5,
30.4, 27.5, 26.7, 25.0, 17.3. MS (HRCI) calcd for C24H31N2O4
4.2. In vitro cholinesterase inhibition studies
Sprague–Dawley rats of 3–6 week-old were supplied by the
Animal Care Facility and the procedures used were approved by
the Ethics Committee of The Hong Kong University of Science
and Technology. All the reagents were purchased from Sigma.
Brain cortices and serum collected from rats immediately after
decapitation were used as sources of AChE and BuChE, respec-
tively. The traditional colorimetric method (Ellman’s method)23
was used for the detection of cholinesterase activity and modified
[M+H]+ 411.2278; Found 411.2284. ½a 2D3
ꢂ39.3° (c 2.62, MeOH).
ꢄ
for use in 96-well-plates (NUNC) with a final volume of 200 lL.
4.1.16. (5S)-5-{5-[(2RS)-5,6-Dimethoxy-1-oxo-indan-2-yl]-
pentylamino}-5,6,7,8-tetrahydro-1H-quinolin-2-one [(RS,S)-
17c]
Briefly, the cholinesterase inhibitory activity of the potential com-
pounds inhibits the breakdown of the substrate (acetyl thiocholine
iodide) to acetic acid and choline, a reaction catalyzed by the
enzymatic action of AChE. Acetic acid and the chromogen 5,
50-dithio-bis(2-nitrobenzoic) acid react to form yellow anions of
5-thio-2-nitro-benzoic acid that are then detected in a plate reader
at an absorbance of 405 nm (ref k 630 nm). Ethopropazine–HCl
Yield 90%. 1H NMR (300 MHz, CDCl3): d 7.43 (d, J = 9.3 Hz, 1H),
7.11 (s, 1H), 6.82 (s, 1H), 6.33 (d, J = 9.3 Hz, 1H), 3.90 (s, 3H), 3.84
(s, 3H), 3.49 (br s, 1H), 3.17 (dd, J = 16.8, 7.2 Hz, 1H), 2.51–2.69
(m, 6H), 1.70–1.87 (m, 6H), 1.42–1.49 (m, 6H). 13C NMR (75 MHz,
CDCl3): d 207.6, 165.0, 155.3, 149.2, 148.9, 144.2, 143.2, 129.3,
117.3, 116.8, 107.3, 104.2, 56.1, 56.0, 52.8, 47.5, 46.8, 32.5, 31.5,
30.2, 27.3, 27.2, 27.1, 26.6, 17.3. MS (HRCI) calcd for C25H33N2O4
(100 lM) and BW284c51 (10 lM) were added to the brain homog-
enate and serum, respectively. The inhibitory curve and the
IC50 were determined from series of drug concentrations using
GraphPad Prism 5. Assays were performed in duplicate and the
values were presented as the mean SEM from three independent
experiments. The specificity of the inhibition was calculated as a
ratio by comparing the inhibition potency of BuChE over AChE.
[M+H]+ 425.2435; Found 425.2446. ½a 2D3
ꢂ38.6° (c 0.66, MeOH).
ꢄ
4.1.17. (5S)-5-{6-[(2RS)-5,6-Dimethoxy-1-oxo-indan-2-yl]-
hexylamino}-5,6,7,8-tetrahydro-1H-quinolin-2-one [(RS,S)-17d]
Yield 94%. 1H NMR (300 MHz, CDCl3): d 7.49 (d, J = 9.3 Hz, 1H),
7.18 (s, 1H), 6.88 (s, 1H), 6.40 (d, J = 9.3 Hz, 1H), 3.97 (s, 3H), 3.91
(s, 3H), 3.54 (br s, 1H), 3.22 (dd, J = 16.8, 7.2 Hz, 1H), 2.58–2.76
(m, 6H), 1.77–1.92 (m, 6H), 1.41–1.49 (m, 8H). 13C NMR (75 MHz,
CDCl3): d 207.7, 165.1, 155.3, 149.2, 148.9, 144.2, 143.3, 129.4,
117.4, 116.8, 107.3, 104.2, 56.1, 56.0, 52.8, 47.5, 47.0, 32.5, 31.5,
30.3, 29.4, 27.4, 27.2, 27.2, 26.7, 17.3. MS (HRCI) calcd
Acknowledgements
We would like to thank Dr. Guangmiao Fu for his critical read-
ing of the manuscript and members of the Ip laboratory for their
helpful comments and suggestions. This study was supported by
the Area of Excellence Scheme of the University Grants Committee
of HKSAR (AoE/B-15/01), Hong Kong Jockey Club Charities Trust
for C26H35N2O4 [M+H]+ 439.2591; Found 439.2608. ½a 2D3
ꢄ
ꢂ14.0o
(c 0.29, MeOH).

Y. Hu et al. / Bioorg. Med. Chem. 21 (2013) 676–683
683
12. Bolea, I.; Juarez-Jimenez, J.; Rios, C.; Chioua, M.; Pouplana, R.; Luque, F. J.;
Unzeta, M.; Marco-Contelles, J.; Samadi, A. J. Med. Chem. 2011, 54, 825.
13. Tumiatti, V.; Minarini, A.; Bolognesi, M. L.; Milelli, A.; Rosini, M.; Melchiorre, C.
Curr. Med. Chem. 1825, 2010, 17.
and the Innovation and Technology Fund of Hong Kong (ITS/246/
09FP) with sponsorship by Axis Biotech Pty Limited.
14. Viayna, E.; Gomez, T.; Galdeano, C.; Ramirez, L.; Ratia, M.; Badia, A.; Clos, M. V.;
Verdaguer, E.; Junyent, F.; Camins, A.; Pallas, M.; Bartolini, M.; Mancini, F.;
Andrisano, V.; Arce, M. P.; Rodríguez-Franco, M. I.; Bidon-Chanal, A.; Luque, F.
J.; Camps, P.; Munoz-Torrero, D. ChemMedChem 1855, 2010, 5.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.044.
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