A novel series of tacrine-selegiline hybrids with cholinesterase and monoamine oxidase inhibition activities for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2013.01.039

A novel series of tacrine-selegiline hybrids was synthesised and evaluated for application as inhibitors of cholinesterase (AChE/BuChE) and monoamine oxidase (MAO-A/B). The results demonstrate that most of the synthesised compounds exhibit high inhibitory activity. Among these compounds, compound 8g provided a good balance of activity towards all targets (with IC₅₀ values of 22.6 nM, 9.37 nM, 0.3724 μM, and 0.1810 μM for AChE, BuChE, MAO-A and MAO-B, respectively). These results indicated that 8g has the potential to be a multi-functional candidate for Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2013.01.039

European Journal of Medicinal Chemistry 62 (2013) 745e753
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A novel series of tacrineeselegiline hybrids with cholinesterase
and monoamine oxidase inhibition activities for the treatment
of Alzheimer’s disease
,
,
,
a,
Chuanjun Lu a 1, Qi Zhou a 1, Jun Yan a, Zhiyun Du b, Ling Huang a , Xingshu Li
**
*
a Institute of Drug Synthesis and Pharmaceutical Processing, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
b Faculty of Light Industry and Chemical Engineering, Guang Dong University of Technology, Guang Dong 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of tacrineeselegiline hybrids was synthesised and evaluated for application as inhibitors of
cholinesterase (AChE/BuChE) and monoamine oxidase (MAO-A/B). The results demonstrate that most of
the synthesised compounds exhibit high inhibitory activity. Among these compounds, compound 8g
Received 5 December 2012
Received in revised form
24 January 2013
Accepted 30 January 2013
Available online 8 February 2013
provided a good balance of activity towards all targets (with IC50 values of 22.6 nM, 9.37 nM, 0.3724
and 0.1810 M for AChE, BuChE, MAO-A and MAO-B, respectively). These results indicated that 8g has the
potential to be a multi-functional candidate for Alzheimer’s disease.
mM,
m
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Tacrine
Selegiline
Cholinesterase inhibitor
Monoamine oxidase inhibitor
Alzheimer’s disease
1. Introduction
patients in clinical trials. Although this compound suffers from
therapy-limiting liver toxicity, tacrine has been widely used as
a scaffold for the development of new multi-functional agents with
additional biological properties beyond AChE inhibition. Recent
studies have demonstrated that homo- and hetero-dimers can
improve and enlarge the biological profile of tacrine with fewer
side effects [4]. The homo- and hetero-dimers that have been
investigated include bis-tacrine [5], tacrine-8-hydroxyquino-
line hybrids [6], mercapto-tacrine derivatives [7], tacrinee
fluorobenzoic acid hybrids [8], and tacrineemultialkoxybenzene
hybrids [9], among others [10].
Alzheimer’s disease (AD) is an age-related progressive neuro-
degenerative disorder. It has been estimated that there were 35.6
million people with dementia in 2010, and the number is expected to
nearly double every 20 years, resulting in more than 115 million in
2050 [1]. Although the aetiology of AD is not fully understood, several
conditions play significant roles in the pathogenesis of AD, including
lowlevelsofacetylcholine(ACh), theformationof
-protein, oxidative stress and dyshomeostasis of biometals [2].
In the past decade, treatment strategies for AD have mainly been
b-amyloiddeposits,
s
aimed at improving cholinergic neurotransmission in the brain,
which were mostly based on the ‘cholinergic hypothesis’. Many
approaches have been investigated based on this hypothesis [3].
These approaches include the use of cholinesterase inhibitors
(ChEIs) to improve the endogenous levels of ACh in the brain of AD
patients. ChEIs represent the only species that have demonstrated
some promise in the treatment of AD. Tacrine was the first chol-
inesterase inhibitor approved by the FDA for the treatment of AD
Monoamine oxidases (MAOs) are flavin adenine dinucleotide
(FAD)-containing enzymes localised in the outer mitochondrial
membrane in various cells found in nerve terminals, liver, intestinal
mucosa and other tissues [11]. MAOs are responsible for the oxi-
dative deamination of neurotransmitters. Based on their substrate
and inhibitor specificities, MAOs exist in two distinct enzymatic
isoforms, MAO-A and MAO-B [12]. Selective inhibitors of MAO-A
have been shown to be effective antidepressants, whereas MAO-B
inhibitors are useful in several neurodegenerative disorders such
as Parkinson’s disease (PD), AD, Huntington chorea and amyo-
trophic lateral sclerosis [13]. It is well known that high expression
levels of MAO-B in neuronal tissue could result in an increase in the
level of free radicals, which play a major role in the aetiology of AD.
Therefore, MAO inhibitors are considered to be potential candidates
* Corresponding author. Tel./fax: þ86 20 39943050.
** Corresponding author.
E-mail address: lixsh@mail.sysu.edu.cn (X. Li).
C. Lu and Q. Zhou contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.039

746
C. Lu et al. / European Journal of Medicinal Chemistry 62 (2013) 745e753
for anti-Alzheimer drugs due to their capacity to inhibit oxidative
damage [14]. Among the MAO inhibitors, selegiline, an irreversible
and selective MAO-B inhibitor, acts as a neuroprotective agent in
cellular and animal models of AD [15].
Numerous highly selective compounds which target a single risk
factor have been developed in the past decade. However, very few
of them exhibited efficient therapy in clinic trials, which suggests
limitations in the traditional “one molecule, one target” paradigm.
Given the complex nature of this disease, multi-functional mole-
cules with two or more complementary biological activities may
represent an important advance for the treatment of this disease.
Because both ChE and MAOs are important targets for the treat-
ment of AD, some studies have been devoted to finding multi-
functional agents that target both ChE and MAOs. Ladostigil (Fig. 1),
a bifunctional drug developed from the carbamate moiety of riva-
stigmine and the indolamine moiety of rasagiline, was approved for
phase IIb clinical trial recently. This fact suggests that the combi-
nation of AChEIs and MAO inhibitors is an effective route for
developing new multi-functional anti-AD drugs. Meanwhile, a se-
ries of multipotent inhibitors that can simultaneously inhibit MAO
and ChE, such as heterocyclic substituted alkyl and cycloalkyl
propargyl amines, has been reported in recent years (Fig. 1) [16].
Inspired by these drug design strategies, and our previous work
on tacrineebenzylamine hybrids as multi-functional anti-AD
agents [17], we combined the tacrine moiety (ChE inhibitory ac-
tivity) with the selegiline moiety (MAO inhibitory activity) using
carbon spacers of different lengths (Fig. 2). Herein, we describe the
synthesis and biological evaluation of a series of tacrineeselegiline
hybrids as multi-functional anti-AD agents with cholinesterase and
MAO inhibition activities.
Fig. 2. Design strategy for the new series of tacrineeselegiline hybrids.
using the spectroscopic method described by Ellman et al. with
tacrine as the standard [14]. AChE and BuChE were obtained from
electric eel and equine serum, respectively. The results provided
in Table 1 show that all target compounds are potent inhibitors of
AChE and BuChE with IC50 values in the sub-micromolar range. In
addition, the length of the alkylene linkage affects the inhibition
of both AChE and BuChE. Compounds 8aed containing two,
three, four and five carbon spacers between the tacrine and
selegiline moiety provide stronger inhibitory activities of AChE
than tacrine (8a, IC50 ¼ 83.1 nM; 8b, IC50 ¼ 36.1 nM; 8c,
IC50 ¼ 53.0 nM; 8d, IC50 ¼ 77.6 nM; Tacrine, IC50 ¼ 110.2 nM,
respectively). However, compounds 8e and 8f, with six and
eight-carbon spacers, only exhibited IC50 values of 138 and
147 nM, respectively. It is interesting that the inhibitory activities
significantly increased when the compounds possess nine and
ten methylene spacers (8g, IC50 ¼ 22.6 nM; 8h, IC50 ¼ 23.2 nM).
Generally, the BuChE inhibitory activity increases as the number
of methylene groups increases. Compound 8h, which has ten-
carbon spacers, exhibited the best IC50 value (2.03 nM) in this
series. All target compounds derived from 6-chlorotacrine and
selegiline moiety were also potent inhibitors of AChE and
BuChE with the same trend, wherein compound 9a, possessing
2. Results and discussion
2.1. Chemistry
The selegiline analogue 6, a key intermediate in the synthesis of
target compounds 8 and 9, was prepared according to the pathway
described in Scheme 1. Beginning from the commercially available
1-(4-methoxyphenyl)propan-2-one 1, chiral amine 3 was prepared
via a chiral induction followed by debenzylation [18]. Then, 3 was
reacted with propargylbromide in the presence of potassium car-
bonate to yield the chiral propargyl amine 4, which was converted
to 6 by reacting with formaldehyde/formic acid followed the
demethylation in high yield.
a
three-carbon linker, exhibited the best results for AChE
(IC50 ¼ 14.2 nM) and good activity for BuChE (IC50 ¼ 66.0 nM) in
this series. In contrast, compound 11, wherein the tacrine is
linked by a six-carbon spacer to the N position of selegiline
moiety, provides the weakest inhibition activity of AChE
(IC50 ¼ 456 nM) and good activity for BuChE (IC50 ¼ 28.7 nM).
These results indicate that steric effects may play an important
role in the inhibition of AChE. Some evidence suggests that the
inhibition of BuChE could raise ACh levels and improve cognition
for AD patients [19]. Dual AChE/BuChE inhibitors may have better
activity and show clinical efficacy without remarkable side ef-
fects [19,20]. Consequently, most of the hybrids, especially 8aec
and 8g, exhibited a good balance of AChE/BuChE inhibitory ac-
tivity and may represent good dual AChE/BuChE inhibitors to
treat Alzheimer’s disease.
The synthetic route for the preparation of tacrineeselegiline hy-
brids is provided in Scheme 2. Briefly, the reaction between com-
pound 6, an analogue of selegiline, and
a,u-dibromoalkanes in the
presence of K2CO3 affords compound 7 in good yield. Subsequently,
compound 7 was reacted with tacrine or 6-chlorotacrine in the
presenceofpotassium hydroxideand DMSO to obtaintargetproducts
8aeh and 9aed, respectively. For comparative purposes, compound
11, wherein tacrine is linked to the selegiline moiety via the propargyl
amine nitrogen, was also synthesised according to Scheme 3.
2.2. In vitro inhibition studies of AChE and BuChE
To evaluate the potential application of target hybrids in the
treatment of AD, the inhibition of AChE and BuChE was evaluated
Fig. 1. Chemical structures of Ladostigil and heterocyclic substituted alkyl and cycloalkyl propargyl amine I and II.

C. Lu et al. / European Journal of Medicinal Chemistry 62 (2013) 745e753
747
Scheme 1. Synthesis of 6. Reagents and conditions: (a) (i) (R)-1-phenylethanamine, Pt/C, H2; (ii) L-(þ)-Tartaric acid, ethanol; (iii) NaOH, H2O; (b) Pd/C, H2; (c) Propargylbromide,
K2CO3, MeCN; (d) HCHO, HCOOH, 90 ꢀC; (e) BBr3, CH2Cl2, ꢁ20 ꢀC.
2.3. Kinetic study of AChE
inhibitory activity against both isoforms. The results in Table 1
show that most of the target compounds were effective in inhib-
iting MAO-A and MAO-B in the sub-micromolar range. Among the
synthesised compounds, 9d, with an eight-carbon spacer between
tacrine and selegiline, gave the best results for both MAO-A
To study the inhibitory mechanism of this class of tacrinee
selegiline hybrids, compound 8g, which provided a good balance
of AChE/BuChE inhibitory activity, was chosen for further kinetic
studies. The LineweavereBurk reciprocal plots of 8g against AChE
(Fig. 3) revealed an increasing slope and an increasing intercept
with higher inhibitor concentrations, indicating a mixed-type
inhibitory behaviour for compound 8g in the presence of AChE.
(IC50 ¼ 0.1926
m
M) and MAO-B (IC50 ¼ 0.1290
mM). SAR analysis
showed that the MAO inhibitory potency was closely related to the
length of the alkylene chain. Compounds with 3e5 carbon alkylene
chain are poor MAO-A/B inhibitors (8aec, 9a and 9b with inhibitory
activities in the micromolar range). Surprisingly, when the alkylene
chain contains six carbons, the compounds are potent MAO in-
hibitors exhibiting sub-micromolar activities (8e, 9c). Generally,
a longer linker resulted in better inhibitory activity (except 8a). A
comparison of compounds 8 and 9, where the length of the alky-
lene chain is same, reveals no significant change in the inhibitory
activities. Therefore, introducing chlorine in the tacrine ring did not
improve the inhibition of MAO as it did for AChE. It should be noted
that hybrid 8e exhibited much better inhibitory activity for MAO-A
2.4. Molecular modelling studies
To evaluate the binding modes of this class of tacrineeselegiline
hybrids with respect to AChE, docking simulations of compound 8g
were performed using the CDOCKER program in the Discovery
studio 2.1 software, based on the structure of the Torpedo californica
enzyme complex (TcAChE; PDB entry 2CMF). The full image of the
interaction between 8g and TcAChE is provided in Fig. 4. The
docking results in Fig. 5 show that the tacrine ring binds to the CAS,
which is sandwiched between Trp84 (4.28 A) and Phe330 (4.15 A).
The carbon linker spans across the aromatic gorge. The selegiline
moiety stacks against the indole ring of Trp279 (5.07 A) through the
and MAO-B (IC50 ¼ 0.3978 and 0.2090
m
M, respectively) than its
ꢀ
ꢀ
isomer 11 (IC50 ¼ 33.79
m
M for MAO-A and 18.11 m
M for MAO-B).
These results indicate that steric factors may produce the main
inhibitory effect. Overall, among the synthesised compounds, 8g
exhibited best balance of inhibition for both ChE and MAO.
ꢀ
pep interaction bind the PAS. In addition, the O atom links the
benzene ring of the selegiline moiety is hydrogen-bonded to the
hydroxyl group of Tyr121.
2.6. Reversibility and irreversibility study of hMAO-B
2.5. In vitro inhibition studies of hMAO-A and hMAO-B
The reversibility and irreversibility of inhibitory activities of 8g
was assessed according to a method in the literature [22]. Pargyline,
with known irreversibility for the MAO-B inhibitor [23], was used
as a reference compound. The results provided in Table 2 demon-
strate that 8g is an irreversible MAO-B inhibitor because no re-
covery of the enzyme activity was observed even after repeated
washing [22].
To confirm the multipotent biological profile of tacrinee
selegiline hybrids, inhibitory activity against MAO was deter-
mined. Because AD patients exhibit depressive symptoms, dual
inhibition of MAO-A and MAO-B, rather than inhibition of MAO-B
alone, may be of value for AD therapy [21]. Thus, we tested the
Scheme 2. Synthesis of T series tacrineeselegiline hybrids. Reagents and conditions: (f) Br(CH2)nBr, K2CO3, MeCN, reflux; (g) Tacrine, KOH, DMSO; (h) 6-chlorotacrine, KOH, and
DMSO.

748
C. Lu et al. / European Journal of Medicinal Chemistry 62 (2013) 745e753
Scheme 3. Synthesis of N series tacrineeselegiline hybrid. Reagents and conditions: (i) Br(CH2)6Br, K2CO3, MeCN, reflux; (g) Tacrine, KOH, and DMSO.
3. Conclusion
IT-TOF mass spectrometer. The purity of the synthesised com-
pounds was evaluated using a high-performance liquid chroma-
tography (HPLC) equipped with an Eclipse Plus C8 column
In summary, we have designed, synthesised and evaluated
a series of tacrineeselegiline and 6-chlorotacrineeselegiline hy-
brids as multi-functional anti-AD agents with cholinesterase and
MAOs inhibitory activities. Most of these compounds were potent
inhibitors of ChE and MAO. The tacrineeselegiline hybrid 8
exhibited better or parallel activity to the reference compound
tacrine but slightly lower MAO inhibitory activity than selegiline.
Among all the synthesised compounds, 8g exhibited a good balance
of AChE, BuChE, hMAO-A and hMAO-B inhibition activities. Addi-
tional experiments on 8g indicate that it is a mixed-type inhibitor
of AChE and an irreversible inhibitor of hMAO-B. Molecular mod-
elling studies demonstrate that 8g could interact with both CAS and
PAS of AChE. Thus, it is expected that this hybrids could enhance
patient cognition by increasing levels of acetylcholine and protect
neurons by keeping the activities of selegiline. Therefore, this series
of tacrineeselegiline hybrids should be considered as novel “one-
compound-multi-targets” candidates for anti-Alzheimer therapy.
(4.6 mm ꢂ 150 mm, 5
mm). IR spectra (KBr disks) were recorded on
a Bruker Tensor 27 FT-IR. Compound 2, tacrine and 6-chlorotacrine
were prepared according to the method reported previously
[18,24].
4.1.1. Preparation of (R)-1-(4-methoxyphenyl)propan-2-amine (3)
To a solution of compound 2 (13.47 g, 50.0 mmol) in methanol
(100 mL), 10% Pd/C (1.35 g) was added, and the mixture was stirred
under hydrogen atmosphere (1.5 MPa pressure) at 40 ꢀC for 24 h.
The catalyst was filtered off and the solvent was evaporated to give
compound 3 as a white solid (8.24 g, 99.8%). IR (KBr): 3294, 2958,
2923, 2847, 1610, 1512, 1458, 1246, 1220, 1025 cmꢁ1
(400 MHz, CDCl3)
;
1H NMR
d
7.10 (d, J ¼ 7.3 Hz, 2H), 6.84 (d, J ¼ 7.1 Hz, 2H),
3.79 (s, 3H), 3.12 (dd, J ¼ 12.4, 6.0 Hz, 1H), 2.66 (dd, J ¼ 13.4, 5.2 Hz,
1H), 2.47 (dd, J ¼ 13.1, 7.8 Hz, 1H), 1.11 (d, J ¼ 7.6 Hz, 3H). 13C NMR
(101 MHz, CDCl3)
d 158.06, 131.74, 130.14, 113.43, 55.22, 48.56,
45.71, 23.46; LCeMS (ESI) m/z [M]þ 166.2.
4. Experimental section
4.1.2. Preparation of (R)-N-(1-(4-methoxyphenyl)propan-2-yl)
prop-2-yn-1-amine (4)
4.1. General
To a solution of compound 3 (8.24 g, 49.9 mmol), prop-
argylbromide (80% in toluene, 7.42 g, 49.9 mmol) in acetonitrile
(150 mL), potassium carbonate (8.48 g, 61.4 mmol) was added. After
the mixture was stirred under nitrogen atmosphere at room tem-
perature for 16 h, the solid was filtered, the solvent was evaporated
to provide the crude product which was purified on silica gel
NMR spectra were obtained on a Bruker Avance III spectrometer
with TMS as the internal standard. Low-resolution MS spectra were
obtained on an Agilent LCeMS 6120 instrument with an ESI mass
detector, and the data were obtained in the positive ion mode.
High-resolution ESI-MS data were obtained on a Shimadzu LCeMS-
Table 1
Cholinesterases and human recombinant MAO isoforms inhibitory activity of tested compounds and reference compounds.
Compd.
n
IC50(nM) ꢃ SDa
AChEb
SIe
IC50
(
m
M) ꢃ SDa
SIf
BuChEc
hMAO-A
hMAO-B
8a
8b
8c
8d
8e
8f
8g
8h
9a
9b
9c
9d
2
3
4
5
6
8
9
10
3
5
6
8
6
e
e
e
83.1 ꢃ 1.13
36.1 ꢃ 1.50
53.0 ꢃ 2.21
77.6 ꢃ 5.90
138 ꢃ 9.54
147 ꢃ 3.50
22.6 ꢃ 3.04
23.2 ꢃ 0.23
14.2 ꢃ 1.75
82.0 ꢃ 6.35
79.3 ꢃ 1.86
55.4 ꢃ 3.04
456 ꢃ 11.00
110.2 ꢃ 7.30
nt.
50.6 ꢃ 6.20
41.4 ꢃ 3.04
39.6 ꢃ 3.10
23.5 ꢃ 2.31
16.1 ꢃ 3.20
35.5 ꢃ 0.28
9.37 ꢃ 0.75
2.03 ꢃ 0.32
66.0 ꢃ 2.90
18.6 ꢃ 1.83
18.8 ꢃ 1.20
17.6 ꢃ 1.90
28.7 ꢃ 0.35
21.6 ꢃ 1.77
nt.
0.6
1.1
0.7
0.3
0.1
0.2
0.4
0.1
4.6
0.2
0.2
0.3
0.1
0.2
e
4.86 ꢃ 0.43
0.6930 ꢃ 0.0001
4.46 ꢃ 0.49
7.0
9.7
0.8
0.5
1.9
3.5
2.1
0.9
0.8
0.5
1.0
1.5
1.9
e
43.38 ꢃ 2.53
7.00 ꢃ 0.52
8.55 ꢃ 0.63
1.26 ꢃ 0.20
2.40 ꢃ 0.05
0.3978 ꢃ 0.0338
0.5910 ꢃ 0.0177
0.3724 ꢃ 0.0249
0.3130 ꢃ 0.1143
9.05 ꢃ 0.67
0.2090 ꢃ 0.0050
0.1670 ꢃ 0.0007
0.1810 ꢃ 0.0300
0.3460 ꢃ 0.0240
11.07 ꢃ 1.35
6.21 ꢃ 0.68
2.86 ꢃ 0.35
0.5214 ꢃ 0.0264
0.1926 ꢃ 0.0269
33.79 ꢃ 3.87
nt.d
0.5190 ꢃ 0.0109
0.1290 ꢃ 0.0050
18.11 ꢃ 2.53
nt.
11
Tacrine
Clorgyline
Pargyline
0.0041 ꢃ 0.0002
nt.
e
nt.
nt.
e
nt.
0.1880 ꢃ 0.0160
e
a
Results are the mean of three independent experiments (n ¼ 3) ꢃ SD.
AChE from electric eel was used.
BuChE from equine serum was used.
b
c
d
e
f
nt. ¼ not tested.
Selectivity ratio: IC50 (BuChE)/IC50 (AChE).
Selectivity ratio: IC50 (MAO-A)/IC50 (MAO-B).

C. Lu et al. / European Journal of Medicinal Chemistry 62 (2013) 745e753
749
Fig. 3. Steady state inhibition of AChE by 8g via the hydrolysis of ACh. Reciprocal plots
of initial velocity and substrate concentration: the plots show mixed-type inhibition of
8g on AChE.
chromatography to afford compound 4 as a colourless oil (6.15 g,
60.6%). IR (KBr): 3298, 2960, 2925, 2843, 1610, 1512, 1460, 1247,
Fig. 4. Binding model for inhibitor 8g (purple) and TcAChE (grey). Some key residues
are coloured yellow. The image was generated using PyMOL.(For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)
1220, 1036 cmꢁ1
;
1H NMR (400 MHz, CDCl3)
d
7.12 (d, J ¼ 8.4 Hz,
2H), 6.84 (d, J ¼ 8.5 Hz, 2H), 3.79 (s, 3H), 3.43 (q, J ¼ 17.2 Hz, 2H),
3.11 (dd, J ¼ 12.8, 6.4 Hz,1H), 2.62 (t, J ¼ 6.5 Hz, 2H), 2.17 (s, 1H),1.05
(d, J ¼ 6.2 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 158.12, 131.00,
130.20, 113.8, 82.05, 71.19, 55.22, 52.60, 42.54, 35.56, 19.54; LCeMS
(ESI) m/z [M]þ 204.1.
130.08,129.86,114.82, 80.74, 74.93, 58.89, 42.56, 37.88, 36.55,14.46;
LCeMS (ESI) m/z [M]þ 204.1.
4.1.3. Preparation of (R)-N-(1-(4-methoxyphenyl)propan-2-yl)-N-
methylprop-2-yn-1-amine (5)
4.1.5. General procedure for preparation of compound 7
A mixture of compound 4 (6.15 g, 30.3 mmol), formalin (7.4 mL),
formic acid (7.4 mL) was stirred for 12 h at 90 ꢀC. After the reaction
finished (monitored by TLC), the mixture was poured into water
(50 mL), basified with aqueous 10 M NaOH and extracted with
EtOAc. The organic layer was washed with water and brine, dried
with anhydrous Na2SO4, filtered, and evaporated. The residue was
purified on silica gel chromatography to afford compound 5 as
a yellow oil (5.23 g, 79.6%). IR (KBr): 3294, 2958, 2930, 2843, 2791,
1611, 1512, 1457, 1246, 1175, 1038 cmꢁ1; 1H NMR (400 MHz, CDCl3)
A mixture of compound 6 (0.20 g,1.0 mmol), a,g-dibromoethane
(2.5 mmol), potassium iodide (16.6 mg, 0.1 mmol) and potassium
carbonate (0.41 g, 3.0 mmol) in acetonitrile (5 mL) was refluxed for
16 h under nitrogen atmosphere. After the mixture was filtered,
solvent was evaporated and the residue was purified on silica gel
chromatography to afford the desired compound 7 respectively.
4.1.5.1. (R)-N-(1-(4-(2-bromoethoxy)phenyl)propan-2-yl)-N-methyl-
prop-2-yn-1-amine (7a). Colourless oil (96.8 mg, 31.2%). IR (KBr):
3294, 2964, 2928, 2858, 2792, 1609, 1510,1457, 1240, 1039 cmꢁ1; 1H
d
7.09 (d, J ¼ 8.4 Hz, 2H), 6.82 (d, J ¼ 8.5 Hz, 2H), 3.78 (s, 3H), 3.41 (s,
2H), 3.02e2.87 (m, 2H), 2.41 (s, 3H), 2.34 (dd, J ¼ 12.6, 9.2 Hz, 1H),
NMR (400 MHz, CDCl3)
d
7.09 (d, J ¼ 8.5 Hz, 2H), 6.89e6.80 (m, 2H),
2.23 (s, 1H), 0.95 (d, J ¼ 6.4 Hz, 3H). 13C NMR (101 MHz, CDCl3)
4.27 (t, J ¼ 6.3 Hz, 2H), 3.62 (t, J ¼ 6.3 Hz, 2H), 3.42 (s, 2H), 3.02e
d
157.86, 132.26, 130.14, 113.70, 80.43, 72.49, 59.51, 55.21, 43.14,
2.87 (m, 2H), 2.41 (s, 3H), 2.37e2.29 (m, 1H), 2.24 (s, 1H), 0.95 (d,
38.90, 37.48, 14.99; LCeMS (ESI) m/z [M]þ 218.1.
J ¼ 6.5 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 156.32, 133.10, 130.22,
114.59, 80.39, 72.56, 67.88, 59.38, 43.10, 38.87, 37.41, 29.28, 14.96;
4.1.4. Preparation of (R)-4-(2-(methyl(prop-2-ynyl)amino)propyl)
phenol (6)
LCeMS (ESI) m/z [M]þ 310.1.
To a solution of compound 5 (5.23 g, 24.1 mmol) in dry CH2Cl2
(100 mL) was dropwise added BBr3 (18.11 g, 72.3 mmol) under
nitrogen atmosphere at ꢁ20 ꢀC. The mixture was stirred overnight
at room temperature and then quenched with water, basified with
saturated sodium bicarbonate solution and extracted with CH2Cl2.
The organic layer was washed with water and brine, dried with
anhydrous Na2SO4, filtered, and evaporated. The residue was pu-
rified on silica gel chromatography to afford compound 6 as
a brown solid (4.26 g, 87.0%). IR (KBr): 3676, 3284, 965, 2928, 2857,
4.1.5.2. (R)-N-(1-(4-(3-bromopropoxy)phenyl)propan-2-yl)-N-meth-
ylprop-2-yn-1-amine (7b). Colourless oil (35.4%). IR (KBr): 3295,
2963, 2930, 2869, 2791, 1610, 1510, 1463, 1240, 1036 cmꢁ1; 1H NMR
(400 MHz, CDCl3)
d
7.09 (d, J ¼ 8.6 Hz, 2H), 6.82 (d, J ¼ 8.6 Hz, 2H),
4.08 (t, J ¼ 5.8 Hz, 2H), 3.60 (t, J ¼ 6.5 Hz, 2H), 3.42 (s, 2H), 3.00e
2.86 (m, 2H), 2.41 (s, 3H), 2.38e2.26 (m, 3H), 2.24 (t, J ¼ 2.4 Hz,
1H), 0.95 (d, J ¼ 6.7 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 132.57,
130.19, 114.36, 80.45, 72.53, 65.29, 59.49, 43.15, 38.91, 37.47, 32.45,
30.11, 15.01; LCeMS (ESI) m/z [M]þ 324.1.
2803, 1595, 1515, 1457, 1248, 1138, 1034 cmꢁ1
;
1H NMR (400 MHz,
CDCl3)
d
7.01 (d, J ¼ 8.3 Hz, 2H), 6.74 (d, J ¼ 8.4 Hz, 2H), 3.46 (s, 2H),
4.1.5.3. (R)-N-(1-(4-(4-bromobutoxy)phenyl)propan-2-yl)-N-meth-
3.02e2.91 (m, 2H), 2.44 (s, 3H), 2.33 (dd, J ¼ 12.8, 9.7 Hz, 1H), 2.26
ylprop-2-yn-1-amine (7c). Colourless oil (60.8%). IR (KBr): 3296,
(s, 1H), 0.98 (d, J ¼ 6.6 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
155.14,
2927, 2861, 2792, 1610, 1511, 1462, 1242, 1040 cmꢁ1 1H NMR
;

750
C. Lu et al. / European Journal of Medicinal Chemistry 62 (2013) 745e753
Fig. 5. Compound 8g (coloured by atom type) docked into the active site of TcAChE (PDB code: 2CMF). Hydrogen bonds are highlighted by green dashed lines.(For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)
(400 MHz, CDCl3)
d
7.08 (d, J ¼ 8.3 Hz, 2H), 6.80 (d, J ¼ 8.1 Hz, 2H),
4.1.5.6. (R)-N-(1-(4-(8-bromooctyloxy)phenyl)propan-2-yl)-N-meth-
3.97 (t, J ¼ 5.9 Hz, 2H), 3.49 (t, J ¼ 6.5 Hz, 2H), 3.42 (s, 2H), 3.02e
2.86 (m, 2H), 2.41 (s, 3H), 2.33 (dd, J ¼ 12.6, 9.3 Hz, 1H), 2.23 (s,
1H), 2.12e2.01 (m, 2H), 1.99e1.87 (m, 2H), 0.95 (d, J ¼ 6.2 Hz, 3H).
ylprop-2-yn-1-amine (7f). Colourless oil (61.8%). IR (KBr): 3300,
2929, 2858, 2792, 1610, 1511, 1463, 1244, 1038 cmꢁ1 1H NMR
;
(400 MHz, CDCl3)
d
7.07 (d, J ¼ 8.1 Hz, 2H), 6.81 (d, J ¼ 8.2 Hz, 2H),
13C NMR (101 MHz, CDCl3)
d
157.15, 132.37, 130.16, 114.27, 80.44,
3.93 (t, J ¼ 6.4 Hz, 2H), 3.41 (t, J ¼ 5.2 Hz, 4H), 2.93 (ddd, J ¼ 16.3,
14.0, 5.1 Hz, 2H), 2.41 (s, 3H), 2.33 (dd, J ¼ 12.5, 9.6 Hz, 1H), 2.24 (s,
1H), 1.86 (dt, J ¼ 13.8, 6.7 Hz, 2H), 1.76 (dt, J ¼ 13.5, 6.6 Hz, 2H),
1.49e1.31 (m, 8H), 0.95 (d, J ¼ 6.2 Hz, 3H). 13C NMR (101 MHz,
72.49, 66.78, 59.51, 43.15, 38.91, 37.49, 33.51, 29.51, 27.95, 15.01;
LCeMS (ESI) m/z [M]þ 338.2.
4.1.5.4. (R)-N-(1-(4-(5-bromopentyloxy)phenyl)propan-2-yl)-N-
methylprop-2-yn-1-amine (7d). Colourless oil (59.8%). IR (KBr):
3297, 2932, 2866, 2792, 1610, 1511, 1463, 1244, 1041 cmꢁ1; 1H NMR
CDCl3) d 157.39, 132.07, 130.11, 114.29, 80.47, 72.52, 67.84, 59.54,
43.15, 38.91, 37.49, 33.95, 32.79, 29.25, 28.70, 28.10, 25.99, 15.04;
LCeMS (ESI) m/z [M]þ 394.2.
(400 MHz, CDCl3)
d
7.07 (d, J ¼ 8.4 Hz, 2H), 6.81 (d, J ¼ 8.4 Hz, 2H),
3.94 (t, J ¼ 6.3 Hz, 2H), 3.46e3.40 (m, 4H), 2.93 (ddt, J ¼ 13.0, 10.5,
5.2 Hz, 2H), 2.41 (s, 3H), 2.33 (dd, J ¼ 12.6, 9.3 Hz, 1H), 2.23 (s, 1H),
1.97e1.90 (m, 2H), 1.84e1.75 (m, 2H), 1.62 (dt, J ¼ 15.2, 7.6 Hz, 2H),
4.1.5.7. (R)-N-(1-(4-(9-bromononyloxy)phenyl)propan-2-yl)-N-
methylprop-2-yn-1-amine (7g). Colourless oil (60.7%). IR (KBr):
3300, 2930, 2857, 2792, 1611, 1511, 1464, 1244, 1039 cmꢁ1; 1H NMR
0.95 (d, J ¼ 6.4 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
157.26, 132.24,
(400 MHz, CDCl3)
d
7.07 (d, J ¼ 8.5 Hz, 2H), 6.81 (d, J ¼ 8.5 Hz, 2H),
130.14, 114.29, 80.46, 72.50, 67.52, 59.52, 43.15, 38.91, 37.49, 33.62,
3.92 (t, J ¼ 6.5 Hz, 2H), 3.46e3.36 (m, 4H), 2.94 (ddt, J ¼ 12.9, 10.3,
5.0 Hz, 2H), 2.42 (s, 3H), 2.33 (dd, J ¼ 12.7, 9.4 Hz, 1H), 2.24 (t,
J ¼ 2.3 Hz, 1H), 1.89e1.81 (m, 2H), 1.80e1.72 (m, 2H), 1.48e1.31 (m,
32.51, 28.50, 24.87, 15.02; LCeMS (ESI) m/z [M]þ 352.1.
4.1.5.5. (R)-N-(1-(4-(6-bromohexyloxy)phenyl)propan-2-yl)-N-
methylprop-2-yn-1-amine (7e). Colourless oil (60.3%). IR (KBr):
3299, 2934, 2862, 2793, 1611, 1511, 1464, 1243, 1043 cmꢁ1; 1H NMR
10H), 0.96 (d, J ¼ 6.5 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 157.41,
132.07, 130.10, 114.30, 80.46, 72.46, 67.91, 59.54, 43.14, 38.91, 37.50,
33.99, 32.80, 29.34, 29.31, 29.27, 28.68, 28.14, 26.03, 15.02; LCeMS
(ESI) m/z [M]þ 408.2.
(400 MHz, CDCl3)
d
7.07 (d, J ¼ 8.6 Hz, 2H), 6.81 (d, J ¼ 8.6 Hz, 2H),
3.93 (t, J ¼ 6.4 Hz, 2H), 3.42 (dd, J ¼ 7.8, 5.7 Hz, 4H), 3.00e2.86 (m,
2H), 2.41 (s, 3H), 2.33 (dd, J ¼ 12.7, 9.4 Hz, 1H), 2.23 (t, J ¼ 2.4 Hz,
1H), 1.94e1.85 (m, 2H), 1.78 (dd, J ¼ 13.4, 6.6 Hz, 2H), 1.56e1.44 (m,
4.1.5.8. (R)-N-(1-(4-(10-bromodecyloxy)phenyl)propan-2-yl)-N-
methylprop-2-yn-1-amine (7h). Colourless oil (65.1%). IR (KBr):
3301, 2929, 2856, 2792, 1611, 1511, 1463, 1244, 1039 cmꢁ1; 1H NMR
4H), 0.95 (d, J ¼ 6.7 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 157.34,
132.13, 130.12, 114.30, 80.37, 72.52, 67.68, 59.54, 43.13, 38.87, 37.47,
33.80, 32.68, 29.13, 27.92, 25.31, 14.99; LCeMS (ESI) m/z [M]þ
366.2.
(400 MHz, CDCl3)
d
7.07 (d, J ¼ 8.5 Hz, 2H), 6.81 (d, J ¼ 8.6 Hz, 2H),
3.92 (t, J ¼ 6.5 Hz, 2H), 3.41 (dd, J ¼ 9.0, 4.6 Hz, 4H), 3.01e2.87 (m,
2H), 2.41 (s, 3H), 2.33 (dd, J ¼ 12.7, 9.4 Hz, 1H), 2.23 (t, J ¼ 2.4 Hz,
1H), 1.89e1.81 (m, 2H), 1.77 (dd, J ¼ 14.2, 7.3 Hz, 2H), 1.46e1.29 (m,
12H), 0.95 (d, J ¼ 6.5 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 157.42,
Table 2
132.05, 130.09, 114.30, 80.46, 72.47, 67.92, 59.54, 43.15, 38.91, 37.49,
33.99, 32.82, 29.39, 28.74, 28.16, 26.05,15.03; LCeMS (ESI) m/z [M]þ
422.2.
Reversibility and irreversibility of hMAO-B inhibition of 8g and reference compound
pargyline.a
Compound
% hMAO-B inhibition
Before washing
After repeated washing
4.1.6. Preparation of (R)-6-bromo-N-(1-(4-methoxyphenyl)propan-
2-yl)-N-(prop-2-ynyl)hexan-1-amine (10)
A mixture of compound 4 (0.10 g, 0.5 mmol),1,6-dibromohexane
(0.30 g, 1.25 mmol), and potassium carbonate (0.21 g, 1.5 mmol) in
8g (200 nM)
Pargyline (200 nM)
70.75 ꢃ 5.00
60.30 ꢃ 6.25
81.57 ꢃ 5.43
56.60 ꢃ 5.56
a
Each value is a mean ꢃ SD from three experiments.

C. Lu et al. / European Journal of Medicinal Chemistry 62 (2013) 745e753
751
acetonitrile (5 mL) was refluxed for 16 h. After the mixture was
filtered, solvent was evaporated and the residue was purified on
silica gel chromatography to afford compound 10 as a colourless oil
(70.1 mg, 38.3%). IR (KBr): 3296, 2931, 2858, 1608, 1212, 1459, 1245,
(KBr): 3297, 2932, 2865, 2792, 1612, 1582, 1507, 1241, 1123,
1043 cmꢁ1; 1H NMR (400 MHz, CDCl3)
7.96 (d, J ¼ 8.4 Hz, 1H), 7.90
d
(d, J ¼ 8.4 Hz, 1H), 7.54 (t, J ¼ 7.6 Hz, 1H), 7.30 (t, J ¼ 7.6 Hz, 1H), 7.10
(d, J ¼ 8.5 Hz, 2H), 6.84 (d, J ¼ 8.4 Hz, 2H), 4.11 (t, J ¼ 5.6 Hz, 2H),
3.70 (d, J ¼ 4.7 Hz, 2H), 3.42 (s, 2H), 3.05 (t, J ¼ 5.9 Hz, 2H), 2.95
(ddd, J ¼ 9.8, 9.2, 3.3 Hz, 2H), 2.74 (t, J ¼ 5.7 Hz, 2H), 2.41 (s, 3H),
2.35 (dd, J ¼ 12.6, 9.2 Hz, 1H), 2.24 (s, 1H), 2.15e2.08 (m, 2H), 1.94e
1.85 (m, 4H), 0.96 (d, J ¼ 6.4 Hz, 3H). 13C NMR (101 MHz, CDCl3)
1037 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d
7.08 (d, J ¼ 8.5 Hz, 2H), 6.82
(d, J ¼ 8.5 Hz, 2H), 3.79 (s, 3H), 3.44 (d, J ¼ 2.0 Hz, 2H), 3.40 (t,
J ¼ 6.8 Hz, 2H), 3.04e2.91 (m, 2H), 2.61 (t, J ¼ 7.2 Hz, 2H), 2.37 (dd,
J ¼ 13.1, 9.1 Hz, 1H), 2.18 (s, 1H), 1.90e1.80 (m, 2H), 1.45 (ddd,
J ¼ 14.8, 11.0, 7.4 Hz, 4H), 1.35e1.29 (m, 2H), 0.98 (d, J ¼ 6.5 Hz, 3H).
d
158.61, 156.91, 150.55, 147.58, 132.77, 130.26, 128.86, 128.20,
13C NMR (101 MHz, CDCl3)
d
157.79, 132.59, 130.10, 113.61, 81.47,
123.66, 122.74, 120.39, 116.40, 114.26, 80.44, 72.48, 66.44, 59.47,
47.24, 43.15, 38.94, 37.46, 34.11, 30.83, 25.00, 23.07, 22.82, 15.00.
HRMS calcd for C29H35N3O [M þ H]þ: 442.2853, found: 442.2863.
72.03, 58.99, 55.23, 49.20, 39.37, 39.02, 33.94, 32.80, 28.06, 26.48,
15.56; LCeMS (ESI) m/z [M]þ 366.2.
4.1.7. Preparation of (R)-N1-(1-(4-methoxyphenyl)propan-2-yl)-
N1-(prop-2-ynyl)-N6-(1,2,3,4-tetrahydroacridin-9-yl)hexane-1,6-
diamine (11)
4.1.8.3. (R)-N-(4-(4-(2-(methyl(prop-2-ynyl)amino)propyl)phenoxy)
butyl)-1,2,3,4-tetrahydroacridin-9-amine
(8c). Colourless
oil
(52.6%); Purity 96% (by HPLC); LCeMS (ESI) m/z [M]þ 456.2; IR
A mixture of tacrine (56.5 mg, 0.29 mmol), KOH (31.9 mg,
0.57 mmol) in DMSO (5 mL) was stirred for 20 min, and then
compound 10 (70.1 mg, 0.19 mmol) was added. The mixture was
stirred under nitrogen atmosphere at room temperature for 24 h
(monitored by TLC) and extracted with n-butanol. After the solvent
was removed in vacuo, the residue was purified on silica gel
chromatography to afford compound 11 as a colourless oil (54.3 mg,
59.1%); Purity 97% (by HPLC); LCeMS (ESI) m/z [M]þ 484.4. IR (KBr):
3303, 2929, 2857, 1728, 1609, 1583, 1508, 1246, 1141, 1035 cmꢁ1; 1H
(KBr): 3300, 2930, 2864, 2792, 1725, 1609, 1582, 1507, 1244, 1128,
1038 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d
7.96 (d, J ¼ 8.4 Hz, 1H), 7.90
(d, J ¼ 8.4 Hz, 1H), 7.54 (t, J ¼ 7.6 Hz, 1H), 7.33 (t, J ¼ 7.6 Hz, 1H), 7.08
(d, J ¼ 8.5 Hz, 2H), 6.80 (d, J ¼ 8.5 Hz, 2H), 3.97 (t, J ¼ 5.4 Hz, 2H),
3.56 (s, 2H), 3.41 (s, 2H), 3.05 (d, J ¼ 6.0 Hz, 2H), 3.00e2.89 (m, 2H),
2.72 (t, J ¼ 5.3 Hz, 2H), 2.41 (s, 3H), 2.35e2.30 (m, 1H), 2.24 (s, 1H),
1.95e1.88 (m, 4H), 1.88e1.83 (m, 4H), 0.95 (d, J ¼ 6.6 Hz, 3H). 13C
NMR (101 MHz, CDCl3)
d 158.56, 157.12, 150.57, 147.53, 132.45,
130.18, 128.82, 128.25, 123.67, 122.71, 120.31, 116.14, 114.30, 80.42,
72.47, 67.41, 59.48, 49.12, 43.14, 38.92, 37.46, 34.06, 28.57, 26.78,
24.87, 23.06, 22.79, 14.99. HRMS calcd for C30 H37N3O [M þ H]þ:
456.3009, found: 456.3003.
NMR (400 MHz, CDCl3)
d
7.96 (d, J ¼ 8.4 Hz, 1H), 7.90 (d, J ¼ 8.4 Hz,
1H), 7.57e7.51 (m, 1H), 7.34 (t, J ¼ 7.6 Hz, 1H), 7.07 (d, J ¼ 8.5 Hz,
2H), 6.81 (d, J ¼ 8.5 Hz, 2H), 3.77 (s, 3H), 3.52e3.40 (m, 4H), 3.06 (s,
2H), 3.00e2.88 (m, 2H), 2.71 (s, 2H), 2.60 (dd, J ¼ 13.1, 6.0 Hz, 2H),
2.35 (dd, J ¼ 13.0, 9.2 Hz, 1H), 2.17 (t, J ¼ 2.0 Hz, 1H), 1.96e1.87 (m,
4H), 1.65 (dd, J ¼ 14.6, 7.4 Hz, 2H), 1.49e1.43 (m, 2H), 1.42e1.36 (m,
2H), 1.33 (d, J ¼ 6.7 Hz, 2H), 0.97 (d, J ¼ 6.5 Hz, 3H). 13C NMR
4.1.8.4. (R)-N-(5-(4-(2-(methyl(prop-2-ynyl)amino)propyl)phenoxy)
pentyl)-1,2,3,4-tetrahydroacridin-9-amine
(8d). Colourless
oil
(47.8%); Purity 99% (by HPLC); LCeMS (ESI) m/z [M]þ 470.3. IR
(101 MHz, CDCl3)
d
158.50, 157.81, 150.74, 147.55, 132.57, 130.09,
(KBr): 3299, 2930, 2861, 2793, 1722, 1611, 1581, 1508, 1243, 1133,
128.80, 128.22, 123.56, 122.82, 120.29, 115.91, 113.63, 81.45, 72.02,
59.00, 55.22, 49.51, 49.23, 39.36, 39.04, 34.08, 31.78, 28.15, 27.10,
26.89, 24.82, 23.09, 22.82, 15.56. HRMS calcd for C32H41N3O
[M þ H]þ: 484.3322, found: 484.3337.
1038 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d
7.95 (d, J ¼ 8.5 Hz, 1H), 7.90
(d, J ¼ 8.4 Hz,1H), 7.55 (dd, J ¼ 8.2, 7.0 Hz,1H), 7.34 (t, J ¼ 7.6 Hz,1H),
7.07 (d, J ¼ 8.4 Hz, 2H), 6.79 (d, J ¼ 8.5 Hz, 2H), 3.93 (t, J ¼ 6.2 Hz,
2H), 3.51 (t, J ¼ 6.9 Hz, 2H), 3.41 (s, 2H), 3.06 (s, 2H), 2.98e2.88 (m,
2H), 2.72 (s, 2H), 2.41 (s, 3H), 2.33 (dd, J ¼ 12.6, 9.4 Hz, 1H), 2.23 (s,
1H), 1.95e1.86 (m, 4H), 1.80 (dd, J ¼ 14.2, 6.7 Hz, 2H), 1.76e1.70 (m,
2H), 1.59 (dd, J ¼ 15.0, 7.9 Hz, 2H), 0.95 (d, J ¼ 6.7 Hz, 3H). 13C NMR
4.1.8. General procedure for preparation of 8
A
mixture of 1,2,3,4-tetrahydroacridin-9-amine (89.2 mg,
0.45 mmol), KOH (50.5 mg, 0.90 mmol) in DMSO (5 mL) was stirred
for 20 min, then compound 7 (0.30 mmol) was added. The mixture
was stirred under nitrogen atmosphere at room temperature for
24 h and then extracted with n-butanol. After the solvent was
removed in vacuo, the residue was purified on silica gel
chromatography.
(101 MHz, CDCl3) d 158.55, 157.26, 150.63, 147.55, 132.29, 130.14,
128.83, 128.23, 123.64, 122.74, 120.35, 116.13, 114.28, 80.44, 72.44,
67.52, 59.50, 49.37, 43.14, 38.92, 37.46, 34.09, 31.47, 29.05, 24.82,
23.59, 23.07, 22.80, 15.00. HRMS calcd for C31H39N3O [M þ H]þ:
470.3166, found: 470.3176.
4.1.8.5. (R)-N-(6-(4-(2-(methyl(prop-2-ynyl)amino)propyl)phenoxy)
4.1.8.1. (R)-N-(2-(4-(2-(methyl(prop-2-ynyl)amino)propyl)phenoxy)
hexyl)-1,2,3,4-tetrahydroacridin-9-amine
(8e). Colourless
oil
ethyl)-1,2,3,4-tetrahydroacridin-9-amine
(8a). Colourless
oil
(42.7%); Purity 98% (by HPLC); LCeMS (ESI) m/z [M]þ 484.2. IR
(29.6 mg, 23.1%); Purity 96% (by HPLC); LCeMS (ESI) m/z [M]þ 428.2.
(KBr): 3308, 2929, 2857, 2795, 1612, 1582, 1511, 1219, 1134,
IR (KBr): 3296, 2930, 2863, 2792, 1724, 1611, 1583, 1506, 1242, 1126,
1037 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d
7.95 (d, J ¼ 8.5 Hz, 1H), 7.90
1043 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d
7.98 (d, J ¼ 8.3 Hz, 1H), 7.92
(d, J ¼ 8.5 Hz, 1H), 7.54 (t, J ¼ 7.6 Hz, 1H), 7.33 (t, J ¼ 7.6 Hz, 1H), 7.07
(d, J ¼ 8.3 Hz, 2H), 6.83e6.75 (m, 2H), 3.92 (t, J ¼ 6.3 Hz, 2H), 3.48 (t,
J ¼ 6.7 Hz, 2H), 3.41 (s, 2H), 3.06 (s, 2H), 2.93 (ddd, J ¼ 11.6, 10.7,
3.8 Hz, 2H), 2.71 (s, 2H), 2.40 (s, 3H), 2.33 (dd, J ¼ 12.5, 9.4 Hz, 1H),
2.23 (s, 1H), 1.95e1.86 (m, 4H), 1.76 (dd, J ¼ 13.0, 6.5 Hz, 2H), 1.72e
1.65 (m, 2H),1.48 (d, J ¼ 3.2 Hz, 4H), 0.95 (d, J ¼ 6.3 Hz, 3H). 13C NMR
(d, J ¼ 8.3 Hz, 1H), 7.59e7.51 (m, 1H), 7.36 (t, J ¼ 7.3 Hz, 1H), 7.10 (d,
J ¼ 7.6 Hz, 2H), 6.82 (d, J ¼ 8.0 Hz, 2H), 4.02 (s, 2H), 3.82 (d, J ¼ 4.0 Hz,
2H), 3.42 (s, 2H), 3.05 (d, J ¼ 5.7 Hz, 2H), 2.95 (dd, J ¼ 17.0, 9.9 Hz, 2H),
2.77 (d, J ¼ 5.0 Hz, 2H), 2.41 (s, 3H), 2.34 (d, J ¼ 9.7 Hz, 1H), 2.24 (s,
1H), 1.94e1.79 (m, 4H), 0.95 (d, J ¼ 5.0 Hz, 3H). 13C NMR (101 MHz,
CDCl3)
d
158.82, 156.69, 150.07, 147.42, 133.06, 130.29, 128.85,
(101 MHz, CDCl3) d 158.49, 157.32, 150.69, 147.52, 132.20, 130.12,
128.33, 124.05, 122.67, 120.90, 118.01, 114.44, 80.40, 72.49, 67.40,
59.43, 48.47, 43.15, 38.93, 37.44, 34.02, 24.56, 22.96, 22.78, 14.98.
HRMS calcd for C28H33N3O [M þ H]þ: 428.2696, found: 428.2695.
128.78, 128.23, 123.60, 122.79, 120.30, 115.99, 114.29, 80.45, 72.47,
67.67, 59.50, 49.41, 43.14, 38.91, 37.46, 34.07, 31.70, 29.21, 26.72,
25.91, 24.81, 23.07, 22.80, 15.00. HRMS calcd for C32H41N3O
[M þ H]þ: 484.3322, found: 484.3342.
4.1.8.2. (R)-N-(3-(4-(2-(methyl(prop-2-ynyl)amino)propyl)phenoxy)
propyl)-1,2,3,4-tetrahydroacridin-9-amine
(8b). Colourless
oil
4.1.8.6. (R)-N-(8-(4-(2-(methyl(prop-2-ynyl)amino)propyl)phenoxy)
octyl)-1,2,3,4-tetrahydroacridin-9-amine (8f). Colourless oil (44.6%);
(36.5%); Purity 99% (by HPLC); LCeMS (ESI) m/z [M]þ 442.3. IR

752
C. Lu et al. / European Journal of Medicinal Chemistry 62 (2013) 745e753
Purity 99% (by HPLC); LCeMS (ESI) m/z [M]þ 512.3. IR (KBr): 3302,
2.36 (dd, J ¼ 12.4, 9.0 Hz, 1H), 2.24 (d, J ¼ 1.6 Hz, 1H), 2.12 (dt,
J ¼ 11.8, 5.9 Hz, 2H), 1.94e1.82 (m, 4H), 0.96 (d, J ¼ 6.3 Hz, 3H). 13C
2926, 2855, 1727, 1611, 1580, 1508, 1244, 1135, 1034 cmꢁ1; 1H NMR
(400 MHz, CDCl3)
d
7.96 (d, J ¼ 8.5 Hz, 1H), 7.91 (d, J ¼ 8.5 Hz, 1H),
NMR (101 MHz, CDCl3) d 159.67, 156.81, 150.67, 148.20, 133.91,
7.58e7.51 (m, 1H), 7.34 (t, J ¼ 7.6 Hz, 1H), 7.07 (d, J ¼ 8.4 Hz, 2H),
6.80 (d, J ¼ 8.5 Hz, 2H), 3.91 (t, J ¼ 6.4 Hz, 2H), 3.49 (d, J ¼ 6.8 Hz,
2H), 3.42 (s, 2H), 3.06 (s, 2H), 2.99e2.91 (m, 2H), 2.71 (s, 2H), 2.41 (s,
3H), 2.35e2.30 (m, 1H), 2.23 (s, 1H), 1.96e1.87 (m, 4H), 1.78e1.72
(m, 2H), 1.67 (dd, J ¼ 13.6, 6.8 Hz, 2H), 1.44e1.34 (m, 8H), 0.95 (d,
132.88, 130.29, 127.64, 124.48, 124.26, 118.54, 116.23, 114.23, 80.42,
72.49, 66.48, 59.44, 47.48, 43.15, 38.94, 37.44, 34.05, 30.78, 24.79,
22.92, 22.66, 14.99. HRMS calcd for C29H34N3OCl [M þ H]þ:
476.2463, found: 476.2454.
J ¼ 6.4 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
158.40, 157.40, 150.80,
4.1.9.2. (R)-6-chloro-N-(5-(4-(2-(methyl(prop-2-ynyl)amino)
147.44, 132.12, 130.10, 128.69, 128.27, 123.57, 122.82, 120.22, 115.83,
114.3, 80.44, 72.44, 67.87, 59.51, 49.51, 43.13, 38.91, 37.46, 33.99,
31.76, 29.28, 29.25, 26.86, 25.98, 24.79, 23.06, 22.78, 15.00. HRMS
calcd for C34H45N3O [M þ H]þ: 512.3635, found: 512.3644.
propyl)phenoxy)pentyl)-1,2,3,4-tetrahydroacridin-9-amine
(9b).
Colourless oil (40.5%); Purity 97% (by HPLC); LCeMS (ESI) m/z [M]þ
504.2. IR (KBr): 3306, 2934, 2864, 2796, 1723, 1609, 1582, 1510,
1243, 1219, 1125, 1041 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d 7.89 (dd,
J ¼ 5.5, 3.2 Hz, 2H), 7.29e7.23 (m, 1H), 7.07 (d, J ¼ 8.5 Hz, 2H), 6.79
(d, J ¼ 8.6 Hz, 2H), 3.94 (t, J ¼ 6.2 Hz, 2H), 3.51 (t, J ¼ 7.1 Hz, 2H), 3.42
(s, 2H), 3.03 (s, 2H), 2.94 (ddd, J ¼ 9.9, 9.4, 3.2 Hz, 2H), 2.67 (s, 2H),
2.41 (s, 3H), 2.33 (dd, J ¼ 12.6, 9.3 Hz, 1H), 2.24 (s, 1H), 1.94e1.86 (m,
4H), 1.83e1.78 (m, 2H), 1.76e1.70 (m, 2H), 1.63e1.54 (m, 2H), 0.95
4.1.8.7. (R)-N-(9-(4-(2-(methyl(prop-2-ynyl)amino)propyl)phenoxy)
nonyl)-1,2,3,4-tetrahydroacridin-9-amine (8g). Colourless oil (41.8%);
Purity 97% (by HPLC); LCeMS (ESI) m/z [M]þ 526.3. IR (KBr): 3304,
2927, 2856, 2794, 1610, 1584, 1507, 1219, 1134, 1037 cmꢁ1; 1H NMR
(400 MHz, CDCl3)
d
7.95 (d, J ¼ 8.5 Hz, 1H), 7.90 (d, J ¼ 8.5 Hz, 1H),
(d, J ¼ 6.6 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 159.64, 157.23,
7.54 (t, J ¼ 7.6 Hz, 1H), 7.34 (t, J ¼ 7.6 Hz, 1H), 7.07 (d, J ¼ 8.4 Hz, 2H),
6.81 (d, J ¼ 8.5 Hz, 2H), 3.92 (t, J ¼ 6.5 Hz, 2H), 3.48 (t, J ¼ 7.2 Hz, 2H),
3.41 (s, 2H), 3.06 (s, 2H), 2.99e2.89 (m, 2H), 2.71 (s, 2H), 2.41 (s, 3H),
2.33 (dd, J ¼ 12.7, 9.3 Hz,1H), 2.23 (s,1H),1.95e1.87 (m, 4H),1.75 (dd,
J ¼ 14.5, 6.8 Hz, 2H), 1.65 (dd, J ¼ 14.5, 7.4 Hz, 2H), 1.45e1.29 (m,
150.70, 148.21, 133.94, 132.33, 130.15, 127.67, 124.48, 124.27, 118.52,
115.99, 114.27, 80.44, 72.46, 67.46, 59.49, 49.47, 43.14, 38.92, 37.45,
34.07, 31.46, 29.00, 24.60, 23.56, 22.93, 22.65, 14.99. HRMS calcd for
C31H38N3OCl [M þ H]þ: 504.2776, found: 504.2792.
10H), 0.95 (d, J ¼ 6.4 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
158.49,
4.1.9.3. (R)-6-chloro-N-(6-(4-(2-(methyl(prop-2-ynyl)amino)propyl)
phenoxy)hexyl)-1,2,3,4-tetrahydroacridin-9-amine (9c). Colourless oil
(35.1%); Purity 98% (by HPLC); LCeMS (ESI) m/z [M]þ 518.2. IR (KBr):
157.42, 150.74, 147.57, 132.10, 130.10, 128.82, 128.20, 123.54, 122.82,
120.29, 115.89, 114.31, 80.46, 72.44, 67.92, 59.52, 49.53, 43.14, 38.92,
37.47, 34.10, 31.78, 29.42, 29.31, 29.28, 29.27, 26.92, 26.03, 24.81,
23.09, 22.82, 15.01. HRMS calcd for C35H47N3O [M þ H]þ: 526.3792,
found: 526.3793.
1
3304, 2932, 2860, 2794, 1607, 1580, 1510, 1243, 1126, 1039 cmꢁ1; H
NMR (400 MHz, CDCl3)
d
7.89 (d, J ¼ 7.6 Hz, 2H), 7.26 (d, J ¼ 6.4 Hz,
1H), 7.07 (d, J ¼ 7.8 Hz, 2H), 6.79 (d, J ¼ 7.8 Hz, 2H), 3.93 (d, J ¼ 5.9 Hz,
2H), 3.49 (s, 2H), 3.41 (s, 2H), 3.02 (s, 2H), 2.94 (dd, J ¼ 17.9, 8.9 Hz,
2H), 2.66 (s, 2H), 2.41 (s, 3H), 2.37e2.29 (m,1H), 2.23 (s, 1H), 1.95e1.86
(m, 4H), 1.82e1.74 (m, 2H), 1.72e1.65 (m, 2H), 1.57e1.41 (m, 4H), 0.95
4.1.8.8. (R)-N-(10-(4-(2-(methyl(prop-2-ynyl)amino)propyl)phe-
noxy)decyl)-1,2,3,4-tetrahydroacridin-9-amine (8h). Colourless oil
(40.7%); Purity 96% (by HPLC); LCeMS (ESI) m/z [M]þ 540.3. IR
(KBr): 3303, 2927, 2855, 2793, 1727, 1611, 1581, 1507, 1243, 1125,
(d, J ¼ 5.9 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 159.61, 157.31, 150.75,
148.21, 133.95, 132.24, 130.13, 127.65, 124.51, 124.24, 118.49, 115.87,
114.28, 80.44, 72.45, 67.64, 59.50, 49.52, 43.13, 38.91, 37.46, 34.06,
31.71, 29.19, 26.68, 25.90, 24.59, 22.93, 22.66, 14.99. HRMS calcd for
C32H40N3OCl [M þ H]þ: 518.2933, found: 518.2959.
1039 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d
7.96 (d, J ¼ 8.4 Hz, 1H), 7.91
(d, J ¼ 8.3 Hz, 1H), 7.54 (t, J ¼ 7.5 Hz, 1H), 7.34 (t, J ¼ 7.5 Hz, 1H), 7.07
(d, J ¼ 7.6 Hz, 2H), 6.81 (d, J ¼ 7.6 Hz, 2H), 3.92 (t, J ¼ 6.2 Hz, 2H),
3.48 (t, J ¼ 6.7 Hz, 2H), 3.41 (s, 2H), 3.07 (s, 2H), 2.94 (dd, J ¼ 18.0,
9.9 Hz, 2H), 2.71 (s, 2H), 2.41 (s, 3H), 2.36e2.29 (m, 1H), 2.23 (s, 1H),
1.97e1.86 (m, 4H), 1.79e1.72 (m, 2H), 1.69e1.62 (m, 2H), 1.47e1.28
(m, 12H), 0.95 (d, J ¼ 6.0 Hz, 3H). 13C NMR (101 MHz, CDCl3)
4.1.9.4. (R)-6-chloro-N-(8-(4-(2-(methyl(prop-2-ynyl)amino)propyl)
phenoxy)octyl)-1,2,3,4-tetrahydroacridin-9-amine (9d). Colourless oil
(33.6%); Purity 97% (by HPLC); LCeMS (ESI) m/z [M]þ 546.2. IR (KBr):
3306, 2930, 2858, 2796, 1725, 1608, 1580, 1510, 1244, 1219, 1123,
d
158.33, 157.42, 150.86, 147.38, 132.08, 130.09, 128.62, 128.29,
123.56, 122.87, 120.18, 115.75, 114.31, 80.45, 72.45, 67.94, 59.52,
49.52, 43.13, 38.91, 37.46, 33.96, 31.77, 29.43, 29.33, 26.92, 26.05,
24.77, 23.06, 22.77, 15.00. HRMS calcd for C36H49N3O [M þ H]þ:
540.3948, found: 540.3953.
1040 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d 7.95e7.85 (m, 2H), 7.29e7.24
(m, 1H), 7.07 (d, J ¼ 8.4 Hz, 2H), 6.80 (d, J ¼ 8.5 Hz, 2H), 3.92 (t,
J ¼ 6.4 Hz, 2H), 3.48 (t, J ¼ 7.1 Hz, 2H), 3.41 (s, 2H), 3.03 (s, 2H), 2.93
(ddd, J ¼ 11.6, 10.5, 3.9 Hz, 2H), 2.66 (s, 2H), 2.41 (s, 3H), 2.33 (dd,
J ¼ 12.6, 9.3 Hz,1H), 2.23 (s,1H), 1.96e1.86 (m, 4H),1.79e1.72 (m, 2H),
1.65 (dd, J ¼ 14.1, 7.2 Hz, 2H), 1.47e1.33 (m, 8H), 0.95 (d, J ¼ 6.4 Hz,
4.1.9. General procedure for preparation of 9
A
mixture of 6-chloro-1,2,3,4-tetrahydroacridin-9-amine
3H). 13C NMR (101 MHz, CDCl3)
d 159.44, 157.40, 150.88, 148.04,
(104.7 mg, 0.45 mmol), KOH (50.5 mg, 0.90 mmol) in DMSO
(5 mL) was stirred for 20 min, then compound 7 (0.30 mmol) was
added. The mixture was stirred under nitrogen atmosphere at room
temperature for 24 h, and then was extracted with n-butanol. After
the solvent was removed in vacuo, the residue was purified on silica
gel chromatography.
134.02, 132.15, 130.10, 127.49, 124.58, 124.22, 118.38, 115.66, 114.31,
80.46, 72.43, 67.86, 59.51, 49.58, 43.14, 38.92, 37.45, 33.95, 31.75,
29.28, 29.24, 26.82, 25.97, 24.56, 22.92, 22.62, 15.01. HRMS calcd for
C34H44N3OCl [M þ H]þ: 546.3246, found: 546.3244.
4.2. Biological assay
4.1.9.1. (R)-6-chloro-N-(3-(4-(2-(methyl(prop-2-ynyl)amino)propyl)
4.2.1. In vitro inhibition of AChE and BuChE
phenoxy)propyl)-1,2,3,4-tetrahydroacridin-9-amine
(9a).
Acetylcholinesterase (AChE, from the electric eel), butylcholi-
nesterase (BuChE, from equine serum), 5,50-dithiobis-(2-
nitrobenzoic acid) (Ellman’s reagent, DTNB), acetylthiocholinech-
loride (ATC) and butylthiocholine chloride (BTC) were purchased
from SigmaeAldrich. All in vitro AChE assays were performed in
0.1 M KH2PO4/K2HPO4 buffer (pH 8.0), using a Shimadzu UV-2450
Spectrophotometer. The ChE solutions with a concentration of 2.0
Colourless oil (45.1 mg, 31.6%); Purity 95% (by HPLC); LCeMS (ESI)
m/z [M]þ 476.2. IR (KBr): 3303, 2932, 2866, 2793, 1607, 1581, 1509,
1240, 1222, 1124, 1043 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d 7.89 (dd,
J ¼ 7.6, 5.5 Hz, 2H), 7.22 (dd, J ¼ 9.0, 1.5 Hz, 1H), 7.11 (d, J ¼ 8.3 Hz,
2H), 6.83 (d, J ¼ 8.3 Hz, 2H), 4.12 (t, J ¼ 5.4 Hz, 2H), 3.71 (dd, J ¼ 11.6,
5.8 Hz, 2H), 3.42 (s, 2H), 3.05e2.89 (m, 4H), 2.70 (s, 2H), 2.41 (s, 3H),

C. Lu et al. / European Journal of Medicinal Chemistry 62 (2013) 745e753
753
units/mL were prepared in 2 mL aliquots. The assay medium (1 mL)
consisted of phosphate buffer (pH 8.0), 50 L of 0.01 M DTNB, 10
of enzyme and 50 L of 0.01 M substrate. Test compounds were
Discovery studio 2.1 package (Accelrys Inc., San Diego, CA). The
CDOCKER program in the Discovery studio 2.1 software was used to
perform docking simulations, which provided full flexibility for the
ligand.
m
mL
m
added to the assay solution and pre-incubated at 37 ꢀC with the
enzyme for 15 min, followed by the addition of substrate. Inhibitory
activity was determined at 37 ꢀC by measuring the increase in
absorbance at 412 nm at 1 min intervals. Calculations were per-
formed according to the method of Ellman et al. [25]. Each con-
centration was assayed in triplicate.
Acknowledgements
We thank the Natural Science Foundation of China (20972198)
and Guangdong Engineering Research Center of Chiral Drugs for
the financial support of this study.
4.2.2. Determination of MAO activity
Adequate amounts of recombinant hMAO-A or hMAO-B
References
(SigmaeAldrich) were acquired and adjusted to 12.5 and 75
mL for hMAO-A and hMAO-B, respectively. Test drugs (20 L) and
MAO (80
L) were incubated for 15 min at 37 ꢀC in a flat-black-
bottom 96-well microtest plate in the dark. The reaction was ini-
tiated by adding 200 M of Amplex Red reagent, 2 U/mL of
mg/
m
[1] M. Prince, J. Jackson, Alzheimer’s Dis. Int. World Alzheimer Rep. 2009 (2009)
1e93.
[2] E. Scarpini, P. Schelterns, H. Feldman, Lancet Neurol. 2 (2003) 539e547.
[3] B. Tasso, M. Catto, O. Nicolotti, F. Novelli, M. Tonelli, I. Giangreco, L. Pisani,
A. Sparatore, V. Boido, A. Carotti, F. Sparatore, Eur. J. Med. Chem. 46 (2011)
2170e2184.
[4] V. Tumiatti, A. Minarini, M.L. Bolognesi, A. Milelli, M. Rosini, C. Melchiorre,
Curr. Med. Chem. 17 (2010) 1825e1838.
[5] (a) Y.-P. Pang, P. Quiram, T. Jelacic, F. Hong, S. Brimijoin, J. Biol. Chem. 271
(1996) 23646e23649;
(b) N.C. Inestrosa, A. Alvarez, F. Calderon, Mol. Psychiatry 1 (1996) 359e360.
[6] M.I. Fernández-Bachiller, C. Pérez, G.C. González-Muñoz, S. Conde, M.G. López,
M. Villarroya, A.G. García, M.I. Rodríguez-Franco, J. Med. Chem. 53 (2010)
4927e4937.
[7] Y. Wang, X.-L. Guan, P.-F. Wu, C.-M. Wang, H. Cao, L. Li, X.-J. Guo, F. Wang,
N. Xie, F.-C. Jiang, J.-G. Chen, J. Med. Chem. 55 (2012) 3588e3592.
m
m
horseradish peroxidase, and 2 mM of p-tyramine for hMAO-A or
2 mM benzylamine for hMAO-B, and the reactions were incubated
at 37 ꢀC for 20 min. The results were quantified in a multidetection
microplate fluorescence reader based on the fluorescence gen-
erated (excitation, 545 nm; emission, 590 nm).
The specific fluorescence emission was calculated after sub-
tracting the background, which was determined by using a solution
containing all the components except hMAO.
ꢁ
[8] P. Szymanski, A. Karpinski, E. Mikiciuk-Olasik, Eur. J. Med. Chem. 46 (2011)
4.2.3. Kinetic characterisation of AChE inhibition
3250e3257.
The kinetic characterisation of AChE was performed using
a reported method [26]. Test compounds and AChE were pre-
incubated at 37 ꢀC for 15 min, followed by the addition of sub-
strate. Kinetic characterisation of the hydrolysis of ATC catalysed by
AChE was recorded by spectrometrically at 412 nm. A parallel
control was made for an assay solution with no inhibitor. The plots
were assessed by a weighted least square analysis that assumed the
variance of V to be a constant percentage of V for the entire data set.
The reciprocal plot slopes were plotted as a function of the con-
centration of the inhibitors in a weighted analysis.
[9] (a) W. Luo, Y.-P. Li, Y. He, S.-L. Huang, D. Li, L.-Q. Gu, Z.-S. Huang, Eur. J. Med.
Chem. 46 (2011) 2609e2616;
(b) W. Luo, Y.-P. Li, Y. He, S.-L. Huang, J.-H. Tan, T.-M. Ou, D. Li, L.-Q. Gu, Z.-
S. Huang, Bioorg. Med. Chem. 19 (2011) 763e770.
[10] (a) S. Rizzo, A. Bisi, M. Bartolini, F. Mancini, F. Belluti, S. Gobbi, V. Andrisano,
A. Rampa, Eur. J. Med. Chem. 46 (2011) 4336e4343;
(b) A. Wie˛ckowska, M. Bajda, N. Guzior, B. Malawska, Eur. J. Med. Chem. 45
(2010) 5602e5611.
[11] N.D. Chaurasiya, S. Ganesan, N.P.D. Nanayakkara, L.R.S. Dias, L.A. Walker,
B.L. Tekwani, Bioorg. Med. Chem. Lett. 22 (2012) 1701e1704.
[12] J.W. Greenawalt, C. Schnaitman, J. Cell Biol. 46 (1970) 173e179.
[13] J. Saura, J.M. Luque, A.M. Cesura, M.D. Prada, V. Chan-Palay, G. Huber, J. Löffler,
J.G. Richards, Neuroscience 62 (1994) 15e30.
[14] A.S. Kalgutkar, D.K. Dalvie, N. Castagnoli, T.J. Taylor, Chem. Res. Toxicol. 14
(2001) 1139e1162.
[15] O. Bar-Am, T. Amit, O. Weinreb, M.B.H. Youdim, S. Mandel, J. Alzheimer’s Dis.
21 (2010) 361e371.
[16] A. Samadi, C. de los Ríos, I. Bolea, M. Chioua, I. Iriepa, I. Moraleda, M. Bartolini,
V. Andrisano, E. Gálvez, C. Valderas, M. Unzeta, J. Marco-Contelles, Eur. J. Med.
Chem. 52 (2012) 251e262.
[17] F. Mao, L. Huang, Z. Luo, A. Liu, C. Lu, Z. Xie, X. Li, Bioorg. Med. Chem. 20 (2012)
5884e5892.
4.2.4. Reversibility and irreversibility study
The experiment was performed accordance to a previously
reported method [27]. Adequate amounts of recombinant mono-
amine oxidase with or without test drugs were incubated for
15 min at 37 ꢀC. An aliquot of this incubated solution was stored at
4
ꢀC for the subsequent measurement of MAO activity. Another
aliquot was transferred to an Amicon Ultra-0.5 Centrifugal Filter
Unit with Ultracel-30 membrane (Millipore) and centrifuged (4 ꢀC,
9000g, 20 min) 3 times. The enzyme was obtained and then used
for the subsequent measurement of activity using a similar method
to the one described above (in section Determination of MAO
Activity). The corresponding values of the percent (%) hMAO-B in-
hibition were separately calculated for samples with and without
repeated washing.
[18] L. Huang, J. Liu, W. Shan, B. Liu, A. Shi, X. Li, Chirality 22 (2010) 206e211.
[19] (a)S.Darvesh,C.MacKnight,K.Rockwood, Int.Psychogeriatr.13(2001)461e464;
(b) N.H. Greig, T. Utsuki, D.K. Ingram, Y. Wang, G. Pepeu, C. Scali, Q.-S. Yu,
J. Mamczarz, H.W. Holloway, T. Giordano, D. Chen, K. Furukawa, K. Sambamurti,
A. Brossi, D.K. Lahiri, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 17213e17218;
(c) F. Marcelo, F.V.M. Silva, M. Goulart, J. Justino, P. Sinaÿ, Y. Blériot, A.P. Rauter,
Bioorg. Med. Chem. 17 (2009) 5106e5116.
[20] R.M. Lane, S.G. Potkin, A. Enz, Int. J. Neuropsychopharmacol. 9 (2006) 101e124.
[21] I. Bolea, J. Juárez-Jiménez, C. de los Ríos, M. Chioua, R. Pouplana, F.J. Luque,
M. Unzeta, J. Marco-Contelles, A. Samadi, J. Med. Chem. 54 (2011) 8251e8270.
[22] F. Chimenti, R. Fioravanti, A. Bolasco, P. Chimenti, D. Secci, F. Rossi, M. Yáñez,
F. Orallo, F. Ortuso, S. Alcaro, J. Med. Chem. 52 (2009) 2818e2824.
[23] T. Izumi, N. Iwamoto, Y. Kitaichi, A. Kato, T. Inoue, T. Koyama, Eur. J. Phar-
macol. 565 (2007) 105e112.
[24] J. Tang, J. Li, L. Zhang, S. Ma, D. Shi, Q. Zhang, L. Yang, X. Wang, X. Liu, C. Liu,
J. Heterocycl. Chem. 49 (2012) 533e542.
[25] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Featherstone, Biochem. Phar-
macol. 7 (1961) 88e95.
[26] (a) L. Huang, Z. Luo, F. He, J. Lu, X. Li, Bioorg. Med. Chem. 18 (2010) 4475e4484;
(b) L. Huang, A. Shi, F. He, X. Li, Bioorg. Med. Chem. 18 (2010) 1244e1251.
[27] N. Desideri, A. Bolasco, R. Fioravanti, L. Proietti Monaco, F. Orallo, M. Yáñez,
F. Ortuso, S. Alcaro, J. Med. Chem. 54 (2011) 2155e2164.
4.2.5. Molecular modelling
The molecular modelling was performed according to the pre-
viously reported method [26b]. The simulation system was built on
the X-ray structure of the bis-tacrineeAChE complex, which was
obtained from the Protein Data Bank (PDB entry 2CMF). The orig-
inal ligand was removed, while the water molecules present in the
PDB file were maintained in their native positions. The 3D struc-
tures of the derivatives were generated and optimised by the