Inhibition of cholinesterase and monoamine oxidase-B activity by Tacrine-Homoisoflavonoid hybrids

Article abstract of DOI:10.1016/j.bmc.2013.09.050

A series of Tacrine-Homoisoflavonoid hybrids were designed, synthesised and evaluated as inhibitors of cholinesterases (ChEs) and human monoamine oxidases (MAOs). Most of the compounds were found to be potent against both ChEs and MAO-B. Among these hybrids, compound 8b, with a 6 carbon linker between tacrine and (E)-7-hydroxy-3-(4-methoxybenzylidene)chroman-4-one, proved to be the most potent against AChE and MAO-B with IC₅₀ values of 67.9 nM and 0.401 μM, respectively. This compound was observed to cross the blood-brain barrier (BBB) in a parallel artificial membrane permeation assay for the BBB (PAMPA-BBB). The results indicated that compound 8b is an excellent multifunctional promising compound for development of novel drugs for Alzheimer's disease (AD).

Full text of DOI:10.1016/j.bmc.2013.09.050

Bioorganic & Medicinal Chemistry 21 (2013) 7406–7417
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of cholinesterase and monoamine oxidase-B activity
by Tacrine–Homoisoflavonoid hybrids
a,
Yang Sun a, Jianwen Chen a, Xuemin Chen b, Ling Huang a, , Xingshu Li
⇑
⇑
a School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
b Shenzhen Salubris Pharmaceuticals Co., Ltd, Shenzhen 518102, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of Tacrine–Homoisoflavonoid hybrids were designed, synthesised and evaluated as inhibitors of
cholinesterases (ChEs) and human monoamine oxidases (MAOs). Most of the compounds were found to
be potent against both ChEs and MAO-B. Among these hybrids, compound 8b, with a 6 carbon linker
between tacrine and (E)-7-hydroxy-3-(4-methoxybenzylidene)chroman-4-one, proved to be the most
Received 9 August 2013
Revised 19 September 2013
Accepted 19 September 2013
Available online 1 October 2013
potent against AChE and MAO-B with IC50 values of 67.9 nM and 0.401 lM, respectively. This compound
was observed to cross the blood–brain barrier (BBB) in a parallel artificial membrane permeation assay
for the BBB (PAMPA-BBB). The results indicated that compound 8b is an excellent multifunctional prom-
ising compound for development of novel drugs for Alzheimer’s disease (AD).
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Alzheimer’s disease
Cholinesterase
Monoamine oxidase-B
1. Introduction
and metabolism of monoamine neurotransmitters in the brain and
peripheral tissues.6 Recent studies have revealed that MAOs are
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder leading to the most common form of dementia in elderly
people.1 The pathogenesis of the disease is complex; decades after
Alzheimer’s original description, there is still limited progress in
defining the pathogenesis for AD.2 Although several diverse hall-
associated with psychiatric and neurological disorders, including
depression, Parkinson’s disease (PD) and AD. Because inhibition
of MAOs could increase the level of neurotransmitters in the cen-
tral nervous system, development of MAO inhibitors has become
an important approach to the treatment of such illnesses.7 Two iso-
forms of MAO (MAO-A and MAO-B) have been identified in
humans.8 MAO-A inhibitors are used as antidepressants and anti-
anxiety agents in the clinic, while MAO-B inhibitors are used as
therapeutics for AD and PD.9 Moreover, MAO-B activity increases
in association with gliosis, which can result in higher levels of
H2O2 and oxidative free radicals.10 Thus, MAO-B inhibitors are po-
tential candidates as anti-AD drugs due to their regulation of neu-
rotransmitters and capacity to inhibit oxidative damage in the
central nervous system.
More recently, a novel dual AChE and MAO-B inhibitor, ladosti-
gil (Fig. 1), was developed by combining the carbamate moiety of
rivastigmine (an AChE inhibitor) and the propargyl group of rasag-
iline (an MAO inhibitor, Fig. 1).11 This multi-functional anti-AD
drug was recently approved for Phase II clinical trials by the
FDA; this outcome demonstrates the benefit of developing drugs
that simultaneously target AChE and MAO-B.12 In light of this drug
design strategy, we combined tacrine (an AChE inhibitor) and
homoisoflavonoids (which are known to be efficient MAO-B selec-
tive inhibitors13) by carbon spacers of different lengths (Fig. 2) to
yield a series of Tacrine–Homoisoflavonoid hybrids. In this study,
we describe the preparation and preliminary in vitro screening of
these hybrids as inhibitors of ChEs and MAOs. To estimate the effi-
cacy of these hybrids, the permeability of the blood–brain barrier
marks, such as b-amyloid (Ab) deposits,
s (tau)—protein aggrega-
tion, oxidative stress and low levels of acetylcholine (ACh) have
been thought to play significant roles in the pathophysiology of
AD, acetylcholinesterase inhibitors (AChEIs) are the main drugs
used clinically for the treatment of this disease.3 The drug design
strategy of AChEIs is based on the cholinergic hypothesis, which
proposes that insufficient cholinergic neurotransmission in AD is
responsible for the progressive loss of cognition and memoryca-
pacities.4 Currently, there are four AChEIs (i.e., tacrine, donepezil,
rivastigmine, and galantamine) that have been approved by the
U.S. Food and Drug Administration (FDA) for the treatment of AD,
with tacrine being the first. Although the therapeutic benefit of ta-
crine is limited by its liver toxicity and is not used in US and many
other countries, this drug has been widely used as a scaffold for the
development of new multifunctional agents with additional bio-
logical properties aside from AChE inhibition.5
Monoamine oxidases (MAOs; EC 1.4.3.4), which are found in the
outer mitochondrial membrane, are responsible for the regulation
⇑
Corresponding authors. Tel./fax: +86 20 3994 3051 (L.H.); tel./fax: +86 20 3994
3050 (X.S.).
E-mail addresses: Huangl72@mail.sysu.edu.cn (L. Huang), lixsh@mail.sysu.
edu.cn (X. Li).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.09.050

Y. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7406–7417
7407
spectroscopic method described by Ellman et al.14 using tacrine
as the standard. The results summarised in Table 1 showed that
most of these compounds have good inhibitory activity against
AChE and BuChE compared to tacrine, with IC50 values in the nano-
molar range. In particular, compound 8b, with a para-methoxy
group substituent at the homoisoflavonoid moiety, provided the
most potent inhibition activity for AChE (IC50 = 67.9 nM).
Figure 1. Chemical structures of Ladostigil and Rasagiline.
The IC50 values of hybrids revealed that the length of the alkyl
chain has an effect on the inhibition of AChE. When the length of
the alkyl chain was changed from 5 to 8, the activity against AChE
did not change significantly (Table 1, 8a, 8b and 8c, n = 5, 6 and 8,
IC50 = 78.3, 67.9 and 68.5 nM, respectively). However, the activity
decreased dramatically when the chain length increased to 9 or
10 (Table 1, 8d and 8e, n = 9 and 10, IC50 = 168.9 and 887.9 nM,
respectively). These results suggested that an alkyl chain length
of 5–8 between the two pharmacophore is suitable. Moreover,
changes in substituent at the homoisoflavonoid moiety did not af-
fect the inhibition activity obviously, except in the case of the
diethylamine group (8j, IC50 = 693.3 nM). It is surprising that the
6-chlorotacrine derivatives (8o and 8p) displayed weaker activity
than the tacrine derivatives, which was inconsistent with some
other 6-chlorotacrine derivatives in literatures. Meanwhile, homoi-
soflavonoid 7a, a parent compound of hybrids, did not show any
activity against ChEs, which could be concluded that the tacrine
moiety plays a crucial role in inhibiting ChEs. Notably, only com-
pound 8e exhibited higher selectivity for BuChE than AChE, which
indicated that most of these hybrids could be useful as dual AChE/
BChE inhibitors for treatment of AD.
(BBB) was assayed by the parallel artificial membrane permeation
assay for the BBB (PAMPA-BBB) method.
2. Results and discussion
2.1. Chemistry
The synthetic routes for 8a–8p and 10 are shown in Scheme 1.
First, tacrine (2a) and 6-chlorotacrine (2b) were synthesised using
cyclohexanone and 2-aminobenzonitrile or 2-amino-4-chloroben-
zonitrile as the starting materials. These two compounds were
then reacted with different dibromoalkanes to yield intermediate
3a–3g. Synthesis of the homoisoflavonoid intermediate began with
resorcinol (4), which was reacted with 3-chloropropionic acid via
Fries rearrangement to yield compound 5. Following cyclization
of compound 5 in aqueous NaOH, the key intermediate 7-hydrox-
ychroman-4-one (6) was obtained. The aldol reaction of 6 with dif-
ferently substituted benzaldehydes was catalyzed by piperidine to
afford compounds 7a–7j. The 1H NMR spectrums of 7a–7j showed
that the olefinic proton (=CH) in (E)-isomers appeared as a singlet
in the range of d 7.56 and 8.32, confirming that a single stereo iso-
mer (E) was obtained. Finally, the two intermediates 3a–3g and
7a–7j were refluxed in acetone to afford the desired compounds
8a–8p.
2.3. In vitro inhibition of hAChE
Considering human AChE (hAChE) is more appropriate, we also
evaluated the inhibition activity of compounds 8a, 8b, 8c and 8h
against hAChE. The IC50 values were listed in Table 2. The results
indicated that all the four tested compounds are potent to inhibit
the hAChE, with IC50 values at nanomolar range. Among the tested
hybrids, 8a showed the best inhibition activity against hAChE, with
an IC50 of 98.0 nM, which was 2.9-fold better than tacrine
(IC50 = 285.0 nM). Similar to eeAChE inhibition activity, the length
of the alkyl chain did not affect the activity obviously. In addition,
hybrid 8h (IC50 = 105.3 nM), with a fluoro group at the para-posi-
tion of homoisoflavonoid moiety, provided better activity than hy-
To investigate the necessity of the double bond in compound
7a, we synthesised the single bond compound 10. This consisted
of hydrogenation of 7a in the presence of 10% Pd/C to afford com-
pound 9, which was subsequently reacted with 3b in refluxing ace-
tone to obtain the desired compound 10.
2.2. In vitro inhibition of AChE and BuChE
The AChE (Electrophorus electricus, eeAChE) and BuChE (equine
serum) inhibitory effects of the hybrids were determined by the
brid 8b (IC50 = 156.8 nM), with
a methoxy group at the
Figure 2. Design strategy for Tacrine–Homoisoflavonoid hybrids.

7408
Y. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7406–7417
Scheme 1. Reagents and conditions: (a) (i) cyclohexanone, POCl3, 120 °C, (ii) cyclohexanone, ZnCl2, 140 °C; (b) different dibromoalkanes, KOH, CH3CN; (c) 3-chloropropionic
acid, trifluoromethanesulfonic acid; (d) NaOH, 0 °C; (e) substituted benzaldehyde, piperidine, 80 °C; (f) Compounds 3a–3g, K2CO3, acetone, reflux; (g) Pd/C, H2; (h) Compound
3b, K2CO3, acetone, reflux.
para-position of homoisoflavonoid moiety. However, considering
the results in inhibiting MAO-B, compound 8b seems to be a better
dual AChE and MAO-B inhibitor, so we choose 8b for kinetic study.
indicating a mixed-type inhibition. This result revealed that com-
pound 8b was able to bind to both the catalytic anionic site
(CAS) and the peripheral anionic site (PAS) of AChE.
Recent study shows that the PAS region of the AChE involved in
the interaction with Ab peptide, which may induce the deposition
of neurotoxic Ab fibrils.15 It is possible that the mixed-type inhib-
itor might be able to decrease amyloid plaque formation and to
alleviate cognitive deficits in AD patients. Donepezil, a mixed-type
inhibitor, exhibited some extent of effect on decreasing AChE in-
duced Ab aggregation (around 22%), which can be attributed to
its higher affinity for the PAS.16 Compound 8b, with a long linker
2.4. Kinetic study of AChE
The inhibition type for eeAChE was investigated by graphical
analysis of steady state inhibition data (Fig. 3) using compound
8b as a typical example. Reciprocal plots (Lineweaver–Burk plots)
showed both increasing slopes (decreased Vmax) and increasing
intercepts (higher Km) at increasing concentration of the inhibitor,

Y. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7406–7417
7409
Table 1
Inhibition of ChEs and MAOs by Tacrine–Homoisoflavonoid hybrids 8a–8p and 10, (E)-7-hydroxy-3-(4-methoxybenzylidene)chroman-4-one (7a), Tacrine (2a)
Compd
n
R
R1
R2
R3
R4
IC50 SD (nM)a
BuChEd
BuChE/AChE Selectivity
MAO-Ab inhibition (%)
IC50 MAO-Ba
1.16 0.01
0.401 0.011
0.518 0.010
0.437 0.039
3.43 0.22
1.03 0.02
(lM)
AChEc
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
5
6
8
9
10
6
6
6
6
6
6
6
6
6
6
8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
—
—
—
—
—
—
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
N(CH3)2
CH3
F
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
78.3 6.7
67.9 4.1
68.5 7.6
168.9 9.4
887.9 6.4
115.4 5.8
129.1 0.3
70.1 0.2
100.5 1.0
693.3 48.0
141.6 4.5
151.4 1.0
116.8 5.4
106.1 2.4
123.1 4.7
188.3 11.8
91.7 5.1
>100,000
111.0 2.5
—
23.6 0.3
33.0 2.7
93.1 4.1
30.9 1.3
6.2 0.5
65.7 4.3
54.6 3.1
44.2 1.9
44.5 4.4
235.2 14.6
47.1 0.6
29.9 3.0
34.7 2.6
108.8 6.1
25.9 0.5
612.5 44.8
27.4 2.6
>100,000
19.2 1.4
—
0.31
0.49
1.35
0.18
0.007
0.57
0.43
0.63
0.45
0.34
0.33
0.20
0.30
1.02
0.21
3.26
0.29
—
58.4 1.4
48.3 0.6
64.8 3.3
60.2 1.3
69.6 1.2
31.5 1.5
60.5 1.3
58.4 2.3
23.3 1.2
22.4 1.2
54.1 1.9
70.3 2.3
51.6 3.1
25.7 0.6
15.3 0.3
33.1 2.3
78.2 4.2
24.1 1.2
31.2 5.1
4.1 0.2 nMg
nt.h
1.24 0.16
1.09 0.09
H
H
Cl
15.4 0.35
e
N(C2H5)2
H
H
OCH3
OCH3
OCH3
OCH3
—
—
—
—
—
OCH3
H
OCH3
OCH3
H
H
—
—
—
2.20 0.38
1.79 0.04
19.73 0.08
e
1.36 0.07
1.56 0.01
1.11 0.01
H
10
7a
2a
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1.06 0.01
f
0.17
—
Clorgyline
Pargyline
Ladostigil
—
—
—
nt.
—
—
—
—
0.188 0.016
37.1 3.1
—
—
—
nt.
a
b
c
Data are the mean SD of three independent experiments.
Test concentration is 100
AChE from electric eel.
BuChE from horse serum.
lM.
d
e
f
Inactive at 20
lM.
Inactive at 100
l
M (highest concentration tested).
g
h
The IC50 of Clorgyline.
nt. = not tested.
between tacrine and homoisoflavonoid, can simultaneously inter-
act with the CAS and PAS of AChE, which has potential activity to
AChE-induced Ab aggregation.
2.5. In vitro inhibition of hMAO-A and hMAO-B
To further study the multipotent biological profile of the target
compounds, the inhibitory activity against hMAO-A and hMAO-B
(recombinant human enzyme) was determined17 and compared
with that of ladostigil, which was an hMAO-B inhibitor approved
to carry out phase II clinical trial by FDA. The results were depicted
in Table 1. Most of these hybrids exhibited selective inhibition to
hMAO-B in the micromolar range. A structure–activity relationship
analysis showed that hybrids with a methoxy group in the R3 posi-
tion were favorable for inhibitory activity against hMAO-B. For
example, compound 8b, featuring a methoxy group in R3 position,
displayed the most potent inhibitory activity with an IC50 of
Table 2
Inhibition of human AChE by 8a, 8b, 8c, 8h and tacrine (2a)
Compd
n
R
R1
R2
R3
R4
IC50 SDa (nM)
h-AChEb
8a
8b
8c
8h
2a
5
6
8
6
—
H
H
H
H
—
H
H
H
H
—
H
H
H
H
—
OCH3
OCH3
OCH3
F
H
H
H
H
—
98.0 5.9
156.8 12.9
121.6 11.6
105.3 9.4
285.0 29.4
—
a
Data are the mean SD of three independent experiments.
AChE (EC 3.1.1.7) from human erythrocytes.
0.401 lM, which was approximately 2.5-fold activity as the lead
b

7410
Y. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7406–7417
Figure 3. Steady state inhibition of AChE-catalyzed ACh hydrolysis by compound 8b.
Table 4
compound 7a (IC50 = 1.06
lM). All other analogues gave weaker
Permeability (Pe ꢁ 10ꢀ6 cm sꢀ1) in the PAMPA-BBB assay for 13 commercial drugs,
activities when the methoxygroup was substituted by other groups
or at other positions. Dramatically, compound 8n, which featured
three methoxygroups at the R2, R3 and R4 positions, provided al-
most no inhibitory activity; the result can be attributed to steric
hindrance. In order to investigate whether the double bond is nec-
essary in inhibiting hMAO-B, we prepared compound 10, the
reduction product of 8b. The IC50 value of compound 10 had in-
used in the experiment validation
Commercial drugs
Bibla
PBS:EtOH (70:30)b
Testosterone
Verapamil
Desipramine
Progesterone
Promazine
Chlorpromazine
Clonidine
Piroxicam
Hydrocortisone
Lomefloxacin
Atenolol
Ofloxacin
Theophylline
17
16
12
22.3 1.4
21.2 1.9
16.4 1.2
17.7 1.2
14.3 0.5
6.0 0.3
9.3
8.8
6.5
5.3
2.5
1.9
1.1
0.8
0.8
0.1
creased to 1.11
to the activity.
lM, which indicated that the double bond is crucial
5.1 0.3
0.24 0.01
0.65 0.01
0.37 0.02
0.78 0.02
0.37 0.02
0.26 0.01
Based on the results of ChEs and MAOs inhibitory activity, we
chose compound 8b (eeAChE: IC50 = 67.9 nM; hAChE: IC50 = 158.8 -
nM; BuChE: IC50 = 33.0 nM; MAO-B: IC50 = 0.401
ing multi-functional inhibitor for further study.
lM) as a promis-
2.6. Reversibility and irreversibility of hMAO-B inhibition
a
Taken from Ref. 20
Data are the mean SD of three independent experiments.
b
To determine the reversibility or irreversibility of the hybrids’
inhibitory activity, 8b and pargyline were used for study according
to a literature method.17 Pargyline, with known irreversibility for
the hMAO-B inhibitor, was used as a reference compound.18 Data
shown in Table 3 indicated that hMAO-B inhibition of 8b is irre-
versible as evidenced by the lack of enzyme activity restoration
after repeated washing. A similar result was obtained for pargyline,
a well-known irreversible hMAO-B inhibitor.
Di et al.20 Assay validation was made by comparing experimental
permeabilities of 13 commercial drugs with reported values
(Table 4).
A plot of experimental data versus bibliographic values gave a
good linear correlation, Pe (exp.) = 1.4574 Pe (bibl.) ꢀ1.0773
(R2 = 0.9427). From this equation and considering the limit estab-
lished by Di et al. for blood–brain barrier permeation, we estab-
lished that compounds with permeability values over
4.7 ꢁ 10ꢀ6 cm sꢀ1 should be able to cross the BBB. Six compounds
(8a, 8b, 8c, 8h, 8i and 10) that exhibited good activity against AChE
and MAO-B were chosen as the test compounds. The results sum-
2.7. In vitro blood–brain barrier permeation assay
Brain penetration is very important for successful anti-AD
drugs.19 To evaluate the potential for these hybrids to cross the
blood–brain barrier (BBB), we used a parallel artificial membrane
permeation assay for BBB (PAMPA-BBB), which was described by
Table 5
Permeability (Pe ꢁ 10ꢀ6 cm sꢀ1) in the PAMPA-BBB assay for Tacrine–Homoisoflavo-
noid hybrids and their predictive penetration in the CNS
Table 3
Reversibility and irreversibility of hMAO-B inhibition of hybrid 8b and Pargylinea
Compd
Permeabilitya (Peꢁ10ꢀ6 cm sꢀ1
)
Prediction
Compd
% hMAO-B inhibitionb
Before washing After repeated washing
8a
8b
8c
8h
8i
6.6 0.6
6.8 0.6
4.9 0.4
4.0 0.3
1.1 0.05
2.8 0.2
CNS +
CNS +
CNS +
CNS +/ꢀ
CNS ꢀ
CNS +/ꢀ
8b (400 nM)
8b (1
Pargyline (200 nM)
47.06 2.73
78.39 0.97
60.3 6.25
33.47 1.03
67.50 2.41
56.6 5.56
l
M)
10
a
Pargyline is an irreversible hMAO-B inhibitor.
Data are the mean SD of three independent experiments.
b
a
Data are the mean (n = 3) SD.

Y. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7406–7417
7411
marised in Table 5 indicated that three of the hybrids (8a, 8b, and
8c) should be able to cross the BBB to target the enzyme in the cen-
tral nervous system, two of them (8h and 10) were located in the
uncertain area, and one (8i) was classified as low BBB permeation.
After stirring for 2 h, the mixture was filtered, and the filter cake
was washed with water and extracted with methanol. The com-
bined extracts were concentrated to afford the desired product
(4.7 g) in 67% yield. 1H NMR (400 MHz, DMSO)
d 8.17 (d,
J = 9.0 Hz, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.28 (s, 1H), 6.46 (s, 2H),
2.81 (d, J = 5.5 Hz, 2H), 2.52 (s, 2H), 1.80 (d, J = 4.7 Hz, 4H). LC/MS
(ESI): m/z 233.2 [M+H]+.
3. Conclusion
We developed a new series of Tacrine–Homoisoflavonoid hy-
brids as dual inhibitors capable of targeting ChEs and MAOs. Most
of these compounds were potent ChEIs with IC50 values in the
nanomolar range. These hybrids were also potent and selective
hMAO-B inhibitors and interact irreversibly with hMAO-B. Com-
pared to lead compound 7a, several of these hybrids (8a, 8b, 8c)
exhibited greater potency to inhibit hMAO-B. Among the hybrids,
compound 8b, with a methoxygroup in para-position of the homoi-
soflavonoid moiety and a six carbon linker between the two phar-
macophores, provided the best results with an IC50 of 67.9 nM for
4.1.3. General procedure for the synthesis of 3a–3g
Compound 2a or 2b (2 mmol) and KOH (0.34 g, 6 mmol) were
dissolved in acetonitrile. The solution was stirred for 1 h under ar-
gon at room temperature. Then, different dibromoalkanes
(4 mmol) were added to the mixture. After stirring for 48 h, the
mixture was filtered and evaporated under reduced pressure. The
residue was chromatographed on silica gel, with petroleum
ether/ethyl acetate, 10:1, plus 10 mL triethylamine per 1000 mL
as the eluent to afford the compounds.
eeAChE, 158.8 nM for hAChE and 0.401 lM for hMAO-B. The PAM-
4.1.4. N-(5-Bromononyl)-1,2,3,4-tetrahydroacridin-9-amine
(3a)
PA-BBB assay indicated that compound 8b is able to cross the BBB.
Based on these results, compound 8b is a promising candidate to
target both ChEs and hMAO-B for AD treatment and warrants fur-
ther study.
Compound 2a (1,2,3,4-tetrahydroacridin-9-amine) was treated
with 1,5-dibromopentane according to the general procedure to
give the desired product 3a as a yellow oil, 13% yield. 1H NMR
(400 MHz, CDCl3) d 7.94 (d, J = 8.5 Hz, 1H), 7.91 (d, J = 8.5 Hz, 1H),
7.55 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 3.93 (s, 1H), 3.49
(dd, J = 13.0, 6.5 Hz, 2H), 3.40 (t, J = 6.6 Hz, 2H), 3.06 (s, 2H), 2.72
(s, 2H), 1.94–1.84 (m, 6H), 1.71–1.65 (m, 2H), 1.58–1.52 (m, 2H).
LC/MS (ESI): m/z 347.2 [M+H]+.
4. Experimental section
4.1. Chemistry
1H NMR and 13C NMR spectra were recorded on a Bruker Bio-
Spin GmbH spectrometer at 400.132 and 100.614 MHz, respec-
tively, using TMS as the internal standard. Coupling constants are
given in Hz. High-resolution mass spectra were obtained using a
Shimadzu LCMS-IT-TOF mass spectrometer. Flash column chroma-
tography was performed with silica gel (200–300 mesh) purchased
from Qingdao Haiyang Chemical Co. Ltd. All reactions were moni-
tored by thin layer chromatography using silica gel. The purity of
compounds 8a–8p, 10 (greater than 95%) were confirmed by HPLC
(Agilent Technologies 1200 series system, TC-C18 column
4.1.5. N-(6-Bromononyl)-1,2,3,4-tetrahydroacridin-9-amine
(3b)
Compound 2a (1,2,3,4-tetrahydroacridin-9-amine) was treated
with 1,6-dibromohexane according to the general procedure to
give the desired product 3b as a yellow oil, 22% yield. 1H NMR
(400 MHz, CDCl3) d 7.95 (t, J = 7.9 Hz, 2H), 7.60–7.54 (m, 1H),
7.36 (t, J = 7.6 Hz, 1H), 4.03 (s, 1H), 3.51 (d, J = 4.4 Hz, 2H), 3.40
(t, J = 6.7 Hz, 2H), 3.08 (s, 2H), 2.72 (s, 2H), 1.93 (dt, J = 6.5,
3.4 Hz, 4H), 1.89–1.84 (m, 2H), 1.73–1.67 (m, 2H), 1.51–1.43 (m,
4H). LC/MS (ESI): m/z 361.2 [M+H]+.
(4.6 ꢁ 250 mm, 5
lm), eluted with methanol/water (0.1%TFA),
70:30, at a flow rate of 0.8 mL/min).
4.1.6. N-(8-Bromononyl)-1,2,3,4-tetrahydroacridin-9-amine (3c)
Compound 2a (1,2,3,4-tetrahydroacridin-9-amine) was treated
with 1,8-dibromooctane according to the general procedure to give
the desired product 3c as a yellow oil, 26% yield. 1H NMR
(400 MHz, CDCl3) d 7.95 (dd, J = 8.5, 0.8 Hz, 1H), 7.90 (dd, J = 8.5,
0.7 Hz, 1H), 7.54 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.33 (ddd, J = 8.2,
6.8, 1.2 Hz, 1H), 3.93 (s, 1H), 3.47 (dd, J = 12.8, 6.9 Hz, 2H), 3.39
(t, J = 6.8 Hz, 2H), 3.09–3.01 (m, 2H), 2.70 (s, 2H), 1.94–1.88 (m,
4H), 1.86–1.80 (m, 2H), 1.68–1.61 (m, 2H), 1.43–1.36 (m, 4H),
1.34–1.29 (m, 4H). LC/MS (ESI): m/z 389.2 [M+H]+.
4.1.1. Synthesis of 2a (1,2,3,4-tetrahydroacridin-9-amine)
To a stirred solution of 2-aminobenzonitrile (6.8 g, 50 mmol)
and cyclohexanone (4.9 g, 50 mmol) at 0 °C, phosphorusoxychlo-
ride (41.5 mL) was added in dropwise. The solution was warmed
to 120 °C for 2 h. The mixture was cooled to room temperature
and the phosphorusoxychloride was evaporated under reduced
pressure. The residue was diluted with ethyl acetate, neutralised
with an aqueous solution of K2CO3 to pH 7, and extracted with
ethyl acetate (3 ꢁ 100 mL). The combined organic layers were
washed with brine, dried over K2CO3, and evaporated to dryness
under reduced pressure. The residue was recrystallised in acetone
to obtain a yellow solid (6.4 g) in 65% yield. 1H NMR (400 MHz,
DMSO) d 8.12 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.51–
7.43 (m, 1H), 7.26 (ddd, J = 8.1, 6.8, 1.2 Hz, 1H), 6.29 (s, 2H), 2.81
(t, J = 5.7 Hz, 2H), 2.54 (t, J = 5.9 Hz, 2H), 1.87–1.75 (m, 4H). LC/
MS (ESI): m/z 199.1 [M+H]+.
4.1.7. N-(9-Bromononyl)-1,2,3,4-tetrahydroacridin-9-amine
(3d)
Compound 2a (1,2,3,4-tetrahydroacridin-9-amine) was treated
with 1,9-dibromononane according to the general procedure to
give the desired product 3d as a yellow oil, 29% yield. 1H NMR
(400 MHz, CDCl3) d 7.96 (d, J = 8.5 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H),
7.55 (t, J = 7.1 Hz, 1H), 7.34 (t, J = 7.2 Hz, 1H), 4.04 (s, 1H), 3.50
(dd, J = 12.5, 6.4 Hz, 2H), 3.40 (t, J = 6.8 Hz, 2H), 3.07 (s, 2H), 2.70
(s, 2H), 1.91 (d, J = 3.1 Hz, 4H), 1.87–1.80 (m, 2H), 1.70–1.62 (m,
2H), 1.39 (s, 4H), 1.30 (s, 6H). LC/MS (ESI): m/z 403.2 [M+H]+.
4.1.2. Synthesis of 2b (6-chloro-1,2,3,4-tetrahydroacridin-9-
amine)
Cyclohexanone (36 mL), 2-amino-4-chlorobenzonitrile (4.6 g,
30 mmol) and zinc chloride (4.1 g, 30 mmol) were mixed in a flask
and the mixture was heated to 120 °C for 3 h. Following cooling to
room temperature and evaporation of the solvent, the residue was
diluted with ethyl acetate (30 mL) and the solid was collected by
filtration. A 10% aqueous solution of NaOH (50 mL) was added.
4.1.8. N-(10-Bromodecyl)-1,2,3,4-tetrahydroacridin-9-amine
(3e)
Compound 2a (1,2,3,4-tetrahydroacridin-9-amine) was treated
with 1,10-dibromodecane according to the general procedure to

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Y. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7406–7417
give the desired product 3e as a yellow oil, 44% yield. 1H NMR
(400 MHz, CDCl3) d 7.97 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H),
7.56 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 4.01 (s, 1H), 3.50 (s,
2H), 3.41 (t, J = 6.8 Hz, 2H), 3.08 (s, 2H), 2.72 (s, 2H), 1.97–1.91
(m, 4H), 1.88–1.82 (m, 2H), 1.71–1.63 (m, 2H), 1.41 (d, J = 5.8 Hz,
4H), 1.30 (s, 8H). LC/MS (ESI): m/z 417.2 [M+H]+.
4.1.14. (E)-7-Hydroxy-3-(4-methoxybenzylidene)chroman-4-
one (7a)
7-Hydroxychroman-4-one (6) was treated with 4-methoxy-
benzaldehyde according to the general procedure to give the de-
sired product 7a as a yellow solid, 44% yield. 1H NMR (400 MHz,
DMSO) d 10.64 (s, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.63 (s, 1H), 7.40
(d, J = 8.6 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H), 6.54 (dd, J = 8.6, 2.0 Hz,
1H), 6.31 (d, J = 1.9 Hz, 1H), 5.35 (s, 2H), 3.81 (s, 3H). LC/MS
(ESI): m/z 283.1 [M+H]+.
4.1.9. N-(6-Bromohexyl)-6-chloro-1,2,3,4-tetrahydroacridin-9-
amine (3f)
Compound 2b (6-chloro-1,2,3,4-tetrahydroacridin-9-amine)
was treated with 1,6-dibromohexane according to the general pro-
4.1.15. (E)-3-(4-(Dimethylamino)benzylidene)-7-
hydroxychroman-4-one (7b)
cedure to give the desired product 3f as a yellow oil, 23% yield. 1
H
NMR (400 MHz, CDCl3) d 7.94–7.86 (m, 2H), 7.32–7.27 (m, 1H),
3.93 (s, 1H), 3.49 (d, J = 6.1 Hz, 2H), 3.41 (td, J = 6.6, 1.3 Hz, 2H),
3.04 (s, 2H), 2.68 (s, 2H), 1.93 (d, J = 1.4 Hz, 4H), 1.89–1.85 (m,
2H), 1.71–1.66 (m, 2H), 1.51–1.42 (m, 4H). LC/MS (ESI): m/z
395.2 [M+H]+.
7-Hydroxychroman-4-one (6) was treated with 4-(dimethyl-
amino)benzaldehyde according to the general procedure to give
the desired product 7b as a red solid, 33% yield. 1H NMR
(400 MHz, DMSO) d 10.58 (s, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.58 (s,
1H), 7.29 (d, J = 8.6 Hz, 2H), 6.76 (d, J = 8.6 Hz, 2H), 6.56 – 6.49
(m, 1H), 6.31 (t, J = 4.2 Hz, 1H), 5.37 (s, 2H), 2.97 (d, J = 8.5 Hz,
6H). LC/MS (ESI): m/z 296.2 [M+H]+.
4.1.10. N-(8-Bromooctyl)-6-chloro-1,2,3,4-tetrahydroacridin-9-
amine (3g)
Compound 2b (6-chloro-1,2,3,4-tetrahydroacridin-9-amine)
was treated with 1,8-dibromooctane according to the general pro-
cedure to give the desired product 3g as a yellow oil, 12% yield. 1H
NMR (400 MHz, CDCl3) d 7.84–7.76 (m, 2H), 7.18 (d, J = 2.9 Hz, 1H),
3.85 (s, 1H), 3.44–3.37 (m, 2H), 3.32 (t, J = 6.7 Hz, 2H), 2.95 (s, 2H),
2.60 (s, 2H), 1.84 (d, J = 2.9 Hz, 4H), 1.76 (d, J = 7.2 Hz, 2H), 1.57 (d,
J = 7.1 Hz, 2H), 1.37–1.30 (m, 8H). LC/MS (ESI): m/z 423.2 [M+H]+.
4.1.16. (E)-7-Hydroxy-3-(4-methylbenzylidene)chroman-4-one
(7c)
7-Hydroxychroman-4-one (6) was treated with 4-methylbenz-
aldehyde according to the general procedure to give the desired
product 7c as a yellow solid, 31% yield. 1H NMR (400 MHz, DMSO)
d 10.73 (s, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.71 (s, 1H), 7.37 (q,
J = 8.4 Hz, 4H), 6.62 (dd, J = 8.7, 2.1 Hz, 1H), 6.38 (d, J = 2.2 Hz,
1H), 5.41 (d, J = 1.6 Hz, 2H), 2.42 (s, 3H). LC/MS (ESI): m/z 267.2
[M+H]+.
4.1.11. Synthesis of 3-chloro-1-(2,4-dihydroxyphenyl)propan-1-
one (5)
To a stirred mixture of resorcinol (2.2 g, 20 mmol) and 3-chloro-
propionic acid (2.2 g, 20 mmol), trifluoromethanesulphonic acid
(10 g, 66.4 mmol) was added in one portion. The solution was
warmed to 80 °C, stirred for 1 h, then cooled to room temperature,
and poured into water (100 mL). The aqueous layer was extracted
with CHCl3 (3 ꢁ 100 mL). The combined organic layers were
washed with brine and dried over Na2SO4. Concentration in vacuo
gave 1.9 g of an orange solid (47% yield) which was used in the
next step without purification. 1H NMR (400 MHz, CDCl3) d 12.48
(s, 1H), 7.63 (d, J = 8.6 Hz, 1H), 6.47–6.38 (m, 2H), 3.91 (t,
J = 6.7 Hz, 2H), 3.40 (t, J = 6.7 Hz, 2H). LC/MS (ESI): m/z 201.2
[M+H]+.
4.1.17. (E)-3-(4-Fluorobenzylidene)-7-hydroxychroman-4-one
(7d)
7-Hydroxychroman-4-one (6) was treated with 4-fluorobenzal-
dehyde according to the general procedure to give the desired
product 7d as a orange solid, 24% yield. 1H NMR (400 MHz, DMSO)
d 10.69 (s, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.66 (s, 1H), 7.49 (dd, J = 8.6,
5.6 Hz, 2H), 7.31 (t, J = 8.8 Hz, 2H), 6.55 (dd, J = 8.7, 2.2 Hz, 1H), 6.32
(d, J = 2.2 Hz, 1H), 5.33 (d, J = 1.7 Hz, 2H). LC/MS (ESI): m/z 271.2
[M+H]+.
4.1.18. (E)-3-(4-Chlorobenzylidene)-7-hydroxychroman-4-one
(7e)
7-Hydroxychroman-4-one (6) was treated with 4-chlorobenzal-
dehyde according to the general procedure to give the desired
product 7e as a yellow solid, 24% yield. 1H NMR (400 MHz, DMSO)
d 10.72 (s, 1H), 7.76 (dd, J = 8.7, 4.1 Hz, 1H), 7.66 (s, 1H), 7.58–7.52
(m, 2H), 7.50–7.42 (m, 2H), 6.59–6.54 (m, 1H), 6.36–6.30 (m, 1H),
5.34 (s, 2H). LC/MS (ESI): m/z 287.2 [M+H]+.
4.1.12. Synthesis of 7-hydroxychroman-4-one (6)
To a stirred solution of 2 M NaOH (20 mL) at 0 °C, 5 (1.9 g,
9.5 mmol) was added in one portion. The solution was warmed
to room temperature for 2 h and then cooled to 0 °C. The pH was
adjusted to 2 with 6 M H2SO4. The mixture was extracted with
ethyl acetate (3 ꢁ 50 mL), washed with brine twice, dried over Na2-
SO4 and concentrated in vacuo to give a brown solid. Recrystallisa-
tion from water gave the product (1.0 g, 67% yield). 1H NMR
(400 MHz, CDCl3) d 7.84 (d, J = 8.4 Hz, 1H), 6.55 (d, J = 8.6 Hz, 1H),
6.42 (s, 1H), 4.52 (s, 2H), 2.78 (s, 2H). LC/MS (ESI): m/z 165.1
[M+H]+.
4.1.19. (E)-3-(4-(Diethylamino)benzylidene)-7-
hydroxychroman-4-one (7f)
7-Hydroxychroman-4-one (6) was treated with 4-(diethyl-
amino)benzaldehyde according to the general procedure to give
the desired product 7f as a yellow solid, 24% yield. 1H NMR
(400 MHz, DMSO) d 10.53 (s, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.56 (s,
1H), 7.28 (d, J = 8.7 Hz, 2H), 6.73 (d, J = 8.7 Hz, 2H), 6.52 (dd,
J = 8.6, 2.1 Hz, 1H), 6.31 (d, J = 2.0 Hz, 1H), 5.38 (s, 2H), 3.43 (d,
J = 6.9 Hz, 2H), 3.39 (d, J = 7.0 Hz, 2H), 1.12 (t, J = 6.9 Hz, 6H). LC/
MS (ESI): m/z 324.2 [M+H]+.
4.1.13. General procedure for the synthesis of 7a–7j
A mixture of 6 (0.2 g, 1.22 mmol), substituted benzaldehyde
(1.76 mmol), and piperidine (0.25 mL) was heated at 80 °C for
2 h. Upon cooling to room temperature, the mixture was diluted
with water (50 mL), acidified with HCl, and extracted with ethyl
acetate (3 ꢁ 50 mL). The combined organic layers were washed
with water and brine and dried over Na2SO4. Evaporation of the
solvent afforded the residue, which was purified by column chro-
matography over silica gel using petroleum ether and ethyl acetate
(10:1) as eluent to yield the homoisoflavonoid products (23–61%
yield).
4.1.20. (E)-7-Hydroxy-3-(3-methoxybenzylidene)chroman-4-
one (7g)
7-Hydroxychroman-4-one (6) was treated with 3-methoxy-
benzaldehyde according to the general procedure to give the de-
sired product 7g as a yellow solid, 34% yield. 1H NMR (400 MHz,
DMSO) d 10.69 (s, 1H), 7.76 (d, J = 8.6 Hz, 1H), 7.66 (s, 1H), 7.40

Y. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7406–7417
7413
(t, J = 7.7 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 8.1 Hz, 2H),
6.57 (d, J = 8.5 Hz, 1H), 6.33 (s, 1H), 5.36 (s, 2H), 3.80 (s, 3H). LC/
MS (ESI): m/z 283.1 [M+H]+.
J = 8.5 Hz, 2H), 7.33 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 8.4 Hz, 2H), 6.63
(d, J = 8.9 Hz, 1H), 6.50 (s, 1H), 5.40 (s, 3H), 4.00 (t, J = 6.0 Hz,
2H), 3.83 (s, 3H), 3.41 (d, J = 6.7 Hz, 2H), 2.89 (s, 2H), 2.71 (s, 2H),
1.80 (d, J = 4.8 Hz, 4H), 1.73–1.66 (m, 2H), 1.65–1.57 (m, 2H),
1.43 (d, J = 6.9 Hz, 2H); 13C NMR (101 MHz, DMSO) d 179.53,
164.85, 162.35, 160.26, 157.88, 150.26, 146.89, 135.49, 132.15,
128.88, 128.44, 128.23, 127.75, 126.37, 123.11, 122.93, 120.20,
115.75, 115.01, 114.18, 110.43, 101.11, 67.93, 67.63, 55.19, 47.83,
33.45, 30.23, 28.09, 24.99, 22.77, 22.69, 22.37; HRMS (ESI) m/z
calcd for C35H36N2O4, 549.2748; found, 549.2726. Purity: 99.2%
(by HPLC).
4.1.21. (E)-7-Hydroxy-3-(2-methoxybenzylidene)chroman-4-
one (7h)
7-Hydroxychroman-4-one (6) was treated with 2-methoxy-
benzaldehyde according to the general procedure to give the de-
sired product 7h as a yellow solid, 40% yield. 1H NMR (400 MHz,
DMSO) d 10.67 (s, 1H), 7.80–7.72 (m, 2H), 7.45 (t, J = 7.8 Hz, 1H),
7.16 (d, J = 7.4 Hz, 1H), 7.13 (d, J = 8.3 Hz, 1H), 7.04 (t, J = 7.5 Hz,
1H), 6.56 (d, J = 8.7 Hz, 1H), 6.32 (s, 1H), 5.21 (s, 2H), 3.85 (s, 3H).
LC/MS (ESI): m/z 283.1 [M+H]+.
4.1.27. (E)-3-(4-Methoxybenzylidene)-7-(6-(1,2,3,4-
tetrahydroacridin-9-ylamino)hexyloxy)chroman-4-one (8b)
(E)-7-Hydroxy-3-(4-methoxybenzylidene)chroman-4-one (7a)
was treated with 3b (N-(6-bromononyl)-1,2,3,4-tetrahydroacri-
din-9-amine) according to the general procedure to give the de-
sired product 8b as a yellow oil, 80% yield. 1H NMR (400 MHz,
DMSO) d 8.10 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.69 (d,
J = 8.2 Hz, 1H), 7.66 (s, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.41 (d,
J = 8.8 Hz, 2H), 7.31 (t, J = 7.6 Hz, 1H), 7.04 (d, J = 8.7 Hz, 2H), 6.64
(dd, J = 8.8, 2.3 Hz, 1H), 6.49 (d, J = 2.3 Hz, 1H), 5.39 (d, J = 1.6 Hz,
2H), 5.32 (t, J = 6.3 Hz, 1H), 3.98 (t, J = 6.4 Hz, 2H), 3.82 (s, 3H),
3.39 (d, J = 7.1 Hz, 2H), 2.88 (t, J = 6.1 Hz, 2H), 2.70 (t, J = 5.9 Hz,
2H), 1.87–1.73 (m, 4H), 1.71–1.61 (m, 2H), 1.58–1.54 (m, 2H),
1.40–1.30 (m, 4H); 13C NMR (101 MHz, DMSO) d 179.63, 164.93,
162.42, 160.31, 157.78, 150.39, 146.71, 135.56, 132.23, 128.92,
128.50, 128.06, 127.87, 126.38, 123.16, 122.98, 120.11, 115.69,
115.00, 114.27, 110.58, 101.17, 67.98, 67.65, 55.28, 47.71, 33.35,
30.39, 28.22, 25.91, 25.05, 24.99, 22.66, 22.35; HRMS (ESI) m/z
calcd for C36H38N2O4, 563.2904; found, 563.2903. Purity: 97.4%
(by HPLC).
4.1.22. (E)-3-(3,4-Dimethoxybenzylidene)-7-hydroxychroman-
4-one (7i)
7-Hydroxychroman-4-one (6) was treated with 3,4-dimethoxy-
benzaldehyde according to the general procedure to give the de-
sired product 7i as a yellow solid, 44% yield. 1H NMR (400 MHz,
DMSO) d 7.77 (dd, J = 8.6, 3.1 Hz, 1H), 7.67 (s, 1H), 7.06 (d,
J = 7.3 Hz, 2H), 6.99 (d, J = 7.8 Hz, 1H), 6.58 (dd, J = 5.7, 2.2 Hz,
1H), 6.35 (d, J = 2.2 Hz, 1H), 5.41 (s, 2H), 3.83 (d, J = 2.7 Hz, 6H).
LC/MS (ESI): m/z 313.2 [M+H]+.
4.1.23. (E)-7-Hydroxy-3-(3,4,5-
trimethoxybenzylidene)chroman-4-one (7j)
7-Hydroxychroman-4-one (6) was treated with 3,4,5-trimeth-
oxybenzaldehyde according to the general procedure to give the
desired product 7j as
a
yellow solid, 61% yield. 1H NMR
(400 MHz, DMSO) d 10.68 (s, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.64 (s,
1H), 6.73 (s, 2H), 6.56 (dd, J = 8.7, 2.2 Hz, 1H), 6.33 (d, J = 2.1 Hz,
1H), 5.42 (s, 2H), 3.83 (s, 6H), 3.72 (s, 3H). LC/MS (ESI): m/z 343.3
[M+H]+.
4.1.28. (E)-3-(4-Methoxybenzylidene)-7-(8-(1,2,3,4-
4.1.24. Synthesis of 7-hydroxy-3-(4-methoxybenzyl)chroman-4-
one (9)
tetrahydroacridin-9-ylamino)hexyloxy)chroman-4-one (8c)
(E)-7-Hydroxy-3-(4-methoxybenzylidene)chroman-4-one (7a)
was treated with 3c (N-(8-bromononyl)-1,2,3,4-tetrahydroacri-
din-9-amine) according to the general procedure to give the de-
sired product 8c as a yellow oil, 80% yield. 1H NMR (400 MHz,
DMSO) d 8.10 (d, J = 8.4 Hz, 1H), 7.79 (dd, J = 8.8, 1.0 Hz, 1H), 7.69
(d, J = 8.4 Hz, 1H), 7.67 (s, 1H), 7.53–7.48 (m, 1H), 7.42 (d,
J = 8.2 Hz, 2H), 7.35–7.29 (m, 1H), 7.06 (d, J = 7.9 Hz, 2H), 6.67 (d,
J = 8.9 Hz, 1H), 6.53 (s, 1H), 5.41 (s, 2H), 5.38 (s, 1H), 4.01 (t,
J = 6.2 Hz, 2H), 3.83 (s, 3H), 3.40–3.36 (m, 2H), 2.89 (t, J = 5.9 Hz,
2H), 2.70 (t, J = 5.6 Hz, 2H), 1.85–1.75 (m, 4H), 1.71–1.64 (m, 2H),
1.59–1.51 (m, 2H), 1.36–1.24 (m, 8H); 13C NMR (101 MHz, DMSO)
d 179.59, 164.94, 162.41, 160.30, 157.77, 150.30, 146.77, 135.52,
132.21, 128.92, 128.50, 128.12, 127.81, 126.39, 123.11, 122.97,
120.12, 115.64, 115.00, 114.25, 110.56, 101.17, 68.09, 67.66,
55.27, 47.73, 33.39, 30.45, 28.58, 28.53, 28.30, 26.15, 25.20,
To a stirred solution of (E)-7-hydroxy-3-(4-methoxybenzyli-
dene)chroman-4-one (7a, 0.1 g) in THF at 0 °C was added 10% Pd/
C. The solution was charged with H2 at 180 psi for 4 h. The mixture
was filtered and evaporated under reduced pressure. The residue
was purified by column chromatography (petroleum ether/ethyl
acetate, 5:1) to give
a white solid 9
(90% yield). 1H NMR
(400 MHz, DMSO) d 10.56 (s, 1H), 7.64 (d, J = 8.6 Hz, 1H), 7.15 (d,
J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 6.50 (dd, J = 8.6, 1.7 Hz,
1H), 6.30 (d, J = 1.8 Hz, 1H), 4.29 (dd, J = 11.4, 4.5 Hz, 1H), 4.09
(dd, J = 11.1, 9.2 Hz, 1H), 3.73 (s, 3H), 3.04 (dd, J = 14.0, 4.9 Hz,
1H), 2.93–2.85 (m, 1H), 2.59 (dd, J = 13.9, 9.5 Hz, 1H). LC/MS
(ESI): m/z 285.0 [M+H]+.
4.1.25. General procedure for the synthesis of 8a–8p and 10
A mixture of 7a–7j or 9 (0.2 mmol) and K2CO3 (0.4 mmol) was
dissolved in acetone and stirred at room temperature for 30 min.
Compounds 3a–3f (0.2 mmol) were added to the mixture and then
refluxed for 12 h. The mixture was filtered and evaporated to re-
move the solvent. The residue was purified by column chromatog-
raphy (petroleum ether/ethyl acetate, 5:1, plus 10 mL
triethylamine per 1000 mL).
25.00, 22.68, 22.37; HRMS (ESI) m/z calcd for
C38H42N2O4,
591.3217; found, 591.3244. Purity: 99.4% (by HPLC).
4.1.29. (E)-3-(4-Methoxybenzylidene)-7-(9-(1,2,3,4-
tetrahydroacridin-9-ylamino)hexyloxy)chroman-4-one (8d)
(E)-7-Hydroxy-3-(4-methoxybenzylidene)chroman-4-one (7a)
was treated with 3d (N-(9-bromononyl)-1,2,3,4-tetrahydroacri-
din-9-amine) according to the general procedure to give the
desired product 8d as a yellow oil, 83% yield. 1H NMR (400 MHz,
DMSO) d 8.10 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.72–7.64
(m, 2H), 7.50 (t, J = 7.6 Hz, 1H), 7.41 (d, J = 8.8 Hz, 2H), 7.32 (t,
J = 7.6 Hz, 1H), 7.05 (d, J = 8.7 Hz, 2H), 6.66 (dd, J = 8.8, 2.3 Hz,
1H), 6.52 (d, J = 2.3 Hz, 1H), 5.40 (d, J = 1.6 Hz, 2H), 5.35 (t,
J = 6.4 Hz, 1H), 4.00 (t, J = 6.5 Hz, 2H), 3.82 (s, 3H), 3.41–3.36 (m,
2H), 2.89 (t, J = 6.0 Hz, 2H), 2.70 (t, J = 5.8 Hz, 2H), 1.86–1.76 (m,
4H), 1.71–1.62 (m, 2H), 1.57–1.50 (m, 2H), 1.34–1.20 (m, 10H);
4.1.26. (E)-3-(4-Methoxybenzylidene)-7-(5-(1,2,3,4-
tetrahydroacridin-9-ylamino)pentyloxy)chroman-4-one (8a)
(E)-7-Hydroxy-3-(4-methoxybenzylidene)chroman-4-one (7a)
was treated with 3a (N-(5-bromononyl)-1,2,3,4-tetrahydroacri-
din-9-amine) according to the general procedure to give the de-
sired product 8a as a yellow oil, 91% yield. 1H NMR (400 MHz,
DMSO) d 8.11 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.70 (d,
J = 8.3 Hz, 1H), 7.67 (s, 1H), 7.51 (t, J = 7.5 Hz, 1H), 7.42 (d,

7414
Y. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7406–7417
13C NMR (101 MHz, DMSO) d 179.65, 164.95, 162.39, 160.29,
157.60, 150.54, 146.46, 135.56, 132.19, 128.93, 128.44, 127.96,
127.78, 126.34, 123.16, 122.99, 119.94, 115.46, 114.96, 114.24,
110.55, 101.13, 68.08, 67.62, 55.25, 47.81, 33.17, 30.46, 28.72,
28.51, 28.44, 28.28, 26.12, 25.23, 24.92, 22.61, 22.26; HRMS (ESI)
m/z calcd for C39H44N2O4, 605.3374; found, 605.3379. Purity:
99.8% (by HPLC).
20.93; HRMS (ESI) m/z calcd for C36H38N2O3, 547.2955; found,
547.2949. Purity: 98.4% (by HPLC).
4.1.33. (E)-3-(4-Fluorobenzylidene)-7-(6-(1,2,3,4-
tetrahydroacridin-9-ylamino)hexyloxy)chroman-4-one (8h)
(E)-3-(4-Fluorobenzylidene)-7-hydroxychroman-4-one (7d) was
treated with 3b (N-(6-bromononyl)-1,2,3,4-tetrahydroacridin-9-
amine) according to the general procedure to give the desired
product 8h as a yellow oil, 36% yield. 1H NMR (400 MHz, DMSO)
4.1.30. (E)-3-(4-Methoxybenzylidene)-7-(10-(1,2,3,4-
tetrahydroacridin-9-ylamino)hexyloxy)chroman-4-one (8e)
(E)-7-Hydroxy-3-(4-methoxybenzylidene)chroman-4-one (7a)
was treated with 3e (N-(10-bromononyl)-1,2,3,4-tetrahydroacri-
din-9-amine) according to the general procedure to give the de-
sired product 8e as a yellow oil, 75% yield. 1H NMR (400 MHz,
DMSO) d 8.09 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.69 (d,
J = 8.4 Hz, 1H), 7.65 (s, 1H), 7.49 (t, J = 7.4 Hz, 1H), 7.40 (d,
J = 8.4 Hz, 2H), 7.31 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 8.4 Hz, 2H), 6.66
(d, J = 8.7 Hz, 1H), 6.51 (s, 1H), 5.39 (s, 2H), 5.35 (t, J = 5.5 Hz,
1H), 4.00 (t, J = 6.0 Hz, 2H), 3.81 (s, 3H), 3.37 (d, J = 4.4 Hz, 2H),
2.88 (s, 2H), 2.69 (s, 2H), 1.78 (s, 4H), 1.69–1.63 (m, 2H), 1.55–
1.48 (m, 2H), 1.33 (s, 2H), 1.23–1.14 (m, 10H); 13C NMR
d 8.10 (d, J = 8.6 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.69 (d,
J = 5.5 Hz, 2H), 7.55–7.47 (m, 3H), 7.33 (t, J = 8.6 Hz, 3H), 6.66 (d,
J = 8.8 Hz, 1H), 6.52 (s, 1H), 5.38 (s, 2H), 5.36 (s, 1H), 4.00 (t,
J = 6.4 Hz, 2H), 3.40 (d, J = 6.6 Hz, 2H), 2.89 (t, J = 5.6 Hz, 2H), 2.71
(t, J = 5.4 Hz, 2H), 1.80 (d, J = 5.5 Hz, 4H), 1.71–1.64 (m, 2H),
1.60–1.54 (m, 2H), 1.36 (s, 4H); 13C NMR (101 MHz, DMSO) d
179.63, 165.11, 163.71, 162.55, 161.23 (d, J = 250.48 Hz), 157.46,
150.66, 146.26, 134.57, 132.56, 132.48 (d, J = 8.08 Hz), 130.49,
130.38, 130.35 (d, J = 3.03 Hz), 129.00, 128.12, 127.57, 123.26,
123.05, 119.84, 115.84, 115.62 (d, J = 22.22 Hz), 115.43, 114.83,
110.75, 101.17, 68.01, 67.39, 47.76, 33.03, 30.36, 28.17, 25.85,
25.00, 24.89, 22.56, 22.20; HRMS (ESI) m/z calcd for C35H35N2O3F,
551.2704; found, 551.2702. Purity: 98.2% (by HPLC).
(101 MHz, DMSO)
d 179.51, 164.92, 162.37, 160.27, 157.76,
150.26, 146.83, 135.47, 132.16, 128.89, 128.44, 128.16, 127.74,
126.37, 123.04, 122.93, 120.15, 115.61, 115.00, 114.20, 110.47,
101.11, 68.07, 67.64, 55.22, 47.77, 33.42, 30.54, 30.50, 28.80,
28.77, 28.64, 28.35, 26.22, 25.31, 25.00, 22.69, 22.38; HRMS (ESI)
m/z calcd for C40H46N2O4, 619.3530; found, 619.3537. Purity:
99.7% (by HPLC).
4.1.34. (E)-3-(4-Chlorobenzylidene)-7-(6-(1,2,3,4-
tetrahydroacridin-9-ylamino)hexyloxy)chroman-4-one (8i)
(E)-3-(4-Chlorobenzylidene)-7-hydroxychroman-4-one
(7e)
was treated with 3b (N-(6-bromononyl)-1,2,3,4-tetrahydroacri-
din-9-amine) according to the general procedure to give the de-
sired product 8i as a yellow oil, 47% yield. 1H NMR (400 MHz,
DMSO) d 8.10 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.68 (s,
2H), 7.58–7.46 (m, 5H), 7.35–7.29 (m, 1H), 6.66 (d, J = 8.9 Hz,
1H), 6.52 (s, 1H), 5.37 (s, 3H), 4.00 (t, J = 6.5 Hz, 2H), 3.41 (s, 2H),
2.89 (t, J = 6.0 Hz, 2H), 2.71 (t, J = 5.5 Hz, 2H), 1.79 (s, 4H), 1.70–
1.64 (m, 2H), 1.60–1.54 (m, 2H), 1.36 (s, 4H); 13C NMR (101 MHz,
DMSO) d 179.48, 165.16, 162.60, 157.81, 150.29, 146.76, 134.28,
134.15, 132.74, 131.87, 131.29, 129.01, 128.73, 128.12, 127.84,
123.15, 122.97, 120.13, 115.72, 114.83, 110.81, 101.21, 68.05,
67.41, 47.72, 33.38, 30.40, 28.21, 25.91, 25.05, 25.00, 22.67,
22.36; HRMS (ESI) m/z calcd for C35H35N2O3Cl, 567.2409; found,
567.2426. Purity: 96.1% (by HPLC).
4.1.31. (E)-3-(4-(Dimethylamino)benzylidene)-7-(6-(1,2,3,4-
tetrahydroacridin-9-ylamino)hexyloxy)chroman-4-one. (8f)
(E)-3-(4-(Dimethylamino)benzylidene)-7-hydroxychroman-4-one
(7b) was treated with 3b (N-(6-bromononyl)-1,2,3,4-tetrahydroac-
ridin-9-amine) according to the general procedure to give the
desired product 8f as a yellow oil, 88% yield. 1H NMR (400 MHz,
DMSO) d 8.10 (d, J = 8.5 Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H), 7.69
(d, J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.50 (t, J = 7.2 Hz, 1H), 7.33 (s,
1H), 7.29 (d, J = 8.9 Hz, 2H), 6.76 (d, J = 8.9 Hz, 2H), 6.61
(dd, J = 8.8, 2.3 Hz, 1H), 6.47 (d, J = 2.2 Hz, 1H), 5.40 (s, 2H), 5.32
(t, J = 6.2 Hz, 1H), 3.97 (t, J = 6.4 Hz, 2H), 3.39 (d, J = 6.3 Hz, 2H),
2.98 (s, 6H), 2.88 (t, J = 6.1 Hz, 2H), 2.70 (t, J = 5.8 Hz, 2H), 1.79
(d, J = 5.7 Hz, 4H), 1.68–1.62 (m, 2H), 1.59–1.53 (m, 2H), 1.34
4.1.35. (E)-3-(4-(Diethylamino)benzylidene)-7-(6-(1,2,3,4-
tetrahydroacridin-9-ylamino)hexyloxy)chroman-4-one (8j)
(E)-3-(4-(Diethylamino)benzylidene)-7-hydroxychroman-4-one
(7f) was treated with 3b (N-(6-bromononyl)-1,2,3,4-tetrahydroac-
ridin-9-amine) according to the general procedure to give the
desired product 8j as a yellow oil, 43% yield. 1H NMR (400 MHz,
DMSO) d 8.10 (d, J = 8.3 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.69 (d,
J = 8.4 Hz, 1H), 7.58 (s, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.33 (d,
J = 7.5 Hz, 1H), 7.29 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 8.4 Hz, 2H),
6.63 (t, J = 8.3 Hz, 1H), 6.48 (s, 1H), 5.42 (s, 2H), 5.38 (s, 1H), 3.98
(t, J = 6.3 Hz, 2H), 3.40 (d, J = 6.8 Hz, 6H), 2.89 (t, J = 5.6 Hz, 2H),
2.70 (t, J = 5.5 Hz, 2H), 1.79 (s, 4H), 1.66 (s, 2H), 1.59–1.54 (m,
2H), 1.35 (s, 4H), 1.11 (t, J = 6.8 Hz, 6H); 13C NMR (101 MHz, DMSO)
d 179.39, 164.56, 162.14, 157.81, 150.31, 148.48, 146.75, 136.56,
132.86, 128.76, 128.11, 127.85, 124.74, 123.17, 122.98, 120.41,
120.13, 115.73, 115.32, 111.08, 110.25, 101.14, 68.05, 67.92,
47.72, 43.71, 33.38, 30.40, 28.25, 25.92, 25.07, 24.99, 22.67,
22.37, 12.36; HRMS (ESI) m/z calcd for C39H45N3O3, 604.3534;
found, 604.3546. Purity: 99.2% (by HPLC).
(s, 4H); 13C NMR (101 MHz, DMSO)
d
179.42, 164.61,
162.16, 157.83, 150.96, 150.28, 146.80, 136.52, 132.41, 128.78,
128.15, 127.82, 125.33, 123.15, 122.97, 121.18, 120.15, 115.73,
115.27, 111.65, 110.27, 101.13, 67.99, 67.92, 47.73, 33.41, 30.45,
30.41, 28.25, 25.93, 25.07, 25.00, 22.67, 22.37; HRMS (ESI) m/z
calcd for C37H41N3O3, 576.3221; found, 576.3242. Purity: 95.6%
(by HPLC).
4.1.32. (E)-3-(4-Methylbenzylidene)-7-(6-(1,2,3,4-
tetrahydroacridin-9-ylamino)hexyloxy)chroman-4-one (8g)
(E)-7-Hydroxy-3-(4-methylbenzylidene)chroman-4-one
(7c)
was treated with 3b (N-(6-bromononyl)-1,2,3,4-tetrahydroacri-
din-9-amine) according to the general procedure to give the de-
sired product 8g as a yellow oil, 42% yield. 1H NMR (400 MHz,
DMSO) d 8.09 (d, J = 8.3 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.70–7.66
(m, 2H), 7.52–7.47 (m, 1H), 7.35–7.29 (m, 5H), 6.65 (d, J = 8.8 Hz,
1H), 6.50 (s, 1H), 5.38 (s, 2H), 5.35 (d, J = 6.2 Hz, 1H), 3.99 (t,
J = 6.3 Hz, 2H), 3.43 (s, 2H), 2.88 (t, J = 5.8 Hz, 2H), 2.70 (t,
J = 5.7 Hz, 2H), 2.36 (s, 3H), 1.79 (d, J = 5.7 Hz, 4H), 1.68–1.63 (m,
2H), 1.58–1.53 (m, 2H), 1.34 (s, 4H); 13C NMR (101 MHz, DMSO)
d 179.71, 165.04, 162.54, 157.73, 150.37, 146.63, 139.48, 135.73,
131.08, 130.25, 129.89, 129.36, 128.98, 127.99, 127.92, 123.20,
123.01, 120.08, 115.67, 114.96, 110.70, 101.22, 68.03, 67.58,
47.70, 33.31, 30.37, 28.21, 25.89, 25.05, 24.98, 22.65, 22.33,
4.1.36. (E)-3-(3-Methoxybenzylidene)-7-(6-(1,2,3,4-
tetrahydroacridin-9-ylamino)hexyloxy)chroman-4-one (8k)
(E)-7-Hydroxy-3-(3-methoxybenzylidene)chroman-4-one (7g)
was treated with 3b (N-(6-bromononyl)-1,2,3,4-tetrahydroacri-
din-9-amine) according to the general procedure to give the de-

Y. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7406–7417
7415
sired product 8k as a yellow oil, 54% yield. 1H NMR (400 MHz,
DMSO) d 8.10 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.69 (d,
J = 8.2 Hz, 2H), 7.52–7.47 (m, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.34–
7.29 (m, 1H), 7.02 (d, J = 7.4 Hz, 1H), 6.98 (d, J = 8.1 Hz, 2H), 6.65
(dd, J = 8.6, 1.9 Hz, 1H), 6.51 (d, J = 2.0 Hz, 1H), 5.38 (s, 2H), 5.36
(d, J = 6.2 Hz, 1H), 3.99 (t, J = 6.3 Hz, 2H), 3.80 (s, 3H), 3.39 (d,
J = 6.9 Hz, 2H), 2.88 (t, J = 5.9 Hz, 2H), 2.70 (t, J = 5.7 Hz, 2H), 1.79
(d, J = 5.6 Hz, 4H), 1.69–1.64 (m, 2H), 1.59–1.53 (m, 2H), 1.34 (s,
(t, J = 6.1 Hz, 1H), 4.00 (t, J = 6.9 Hz, 2H), 3.83 (s, 6H), 3.72 (s, 3H),
3.40 (d, J = 6.9 Hz, 2H), 2.89 (t, J = 5.9 Hz, 2H), 2.71 (t, J = 5.6 Hz,
2H), 1.80 (d, J = 5.4 Hz, 4H), 1.70–1.64 (m, 2H), 1.60–1.54 (m,
2H), 1.36 (s, 4H); 13C NMR (101 MHz, DMSO) d 179.60, 165.03,
162.55, 157.84, 152.81, 150.32, 146.80, 138.70, 136.04, 130.00,
129.40, 128.99, 128.15, 127.80, 123.13, 122.95, 120.15, 115.73,
114.95, 110.58, 107.83, 101.20, 68.00, 67.62, 60.07, 55.99, 47.84,
33.40, 30.45, 28.23, 25.92, 25.06, 24.99, 22.67, 22.37; HRMS (ESI)
m/z calcd for C38H42N2O6, 623.3116; found, 623.3131. Purity:
97.7% (by HPLC).
4H); 13C NMR (101 MHz, DMSO)
d 179.61, 165.09, 162.60,
159.28, 157.86, 150.24, 146.85, 135.61, 135.22, 130.92, 129.75,
128.99, 128.21, 127.77, 123.12, 122.94, 122.20, 120.18, 115.75,
115.30, 115.27, 114.91, 110.70, 101.19, 68.02, 67.53, 55.15, 47.74,
33.44, 30.42, 28.23, 25.93, 25.06, 25.00, 22.69, 22.39; HRMS (ESI)
m/z calcd for C36H38N2O4, 563.2904; found, 563.2879. Purity:
95.7% (by HPLC).
4.1.40. (E)-7-(6-(6-Chloro-1,2,3,4-tetrahydroacridin-9-
ylamino)hexyloxy)-3-(4-methoxybenzylidene)chroman-4-one
(8o)
(E)-7-Hydroxy-3-(4-methoxybenzylidene)chroman-4-one (7a)
was treated with 3f (N-(6-bromohexyl)-6-chloro-1,2,3,4-tetrahy-
droacridin-9-amine) according to the general procedure to give
the desired product 8o as a yellow oil, 84% yield. 1H NMR
(400 MHz, DMSO) d 8.14 (d, J = 9.1 Hz, 1H), 7.79 (d, J = 8.8 Hz,
1H), 7.70 (d, J = 2.2 Hz, 1H), 7.67 (s, 1H), 7.42 (d, J = 8.8 Hz, 2H),
7.32 (dd, J = 9.1, 2.3 Hz, 1H), 7.06 (d, J = 8.8 Hz, 2H), 6.65 (dd,
J = 8.8, 2.3 Hz, 1H), 6.51 (d, J = 2.3 Hz, 1H), 5.55 (t, J = 6.2 Hz, 1H),
5.41 (s, 2H), 4.00 (t, J = 6.4 Hz, 2H), 3.83 (s, 3H), 3.44–3.39 (m,
2H), 2.88 (t, J = 6.0 Hz, 2H), 2.68 (t, J = 5.7 Hz, 2H), 1.79 (d,
J = 5.5 Hz, 4H), 1.70–1.64 (m, 2H), 1.61–1.53 (m, 2H), 1.40–1.29
(m, 4H); 13C NMR (101 MHz, DMSO) d 179.47, 164.83, 162.31,
160.23, 159.20, 150.42, 147.51, 135.44, 132.40, 132.11, 128.85,
128.39, 126.61, 126.34, 125.24, 123.22, 118.44, 115.69, 114.96,
114.15, 110.38, 101.04, 67.90, 67.60, 55.16, 47.81, 33.42, 30.42,
28.24, 25.91, 25.04, 24.89, 22.50, 22.18; HRMS (ESI) m/z calcd for
4.1.37. (E)-3-(2-Methoxybenzylidene)-7-(6-(1,2,3,4-
tetrahydroacridin-9-ylamino)hexyloxy)chroman-4-one (8l)
(E)-7-Hydroxy-3-(2-methoxybenzylidene)chroman-4-one (7h)
was treated with 3b (N-(6-bromononyl)-1,2,3,4-tetrahydroacri-
din-9-amine) according to the general procedure to give the de-
sired product 8l as a yellow oil, 38% yield. 1H NMR (400 MHz,
DMSO) d 8.08 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 7.1 Hz, 2H), 7.69 (d,
J = 8.3 Hz, 1H), 7.51–7.46 (m, 1H), 7.43 (t, J = 7.8 Hz, 1H), 7.34–
7.28 (m, 1H), 7.14 (d, J = 7.5 Hz, 1H), 7.09 (d, J = 8.3 Hz, 1H), 7.02
(d, J = 7.3 Hz, 1H), 6.62 (d, J = 8.8 Hz, 1H), 6.46 (s, 1H), 5.35 (d,
J = 5.6 Hz, 1H), 5.21 (s, 2H), 3.94 (t, J = 6.1 Hz, 2H), 3.82 (s, 3H),
3.37 (d, J = 6.4 Hz, 2H), 2.87 (s, 2H), 2.67 (s, 2H), 1.77 (s, 4H), 1.63
(s, 2H), 1.54 (s, 2H), 1.32 (s, 4H); 13C NMR (101 MHz, DMSO) d
179.88, 165.04, 162.68, 157.87, 157.76, 150.27, 146.82, 131.66,
131.42, 130.40, 130.33, 128.98, 128.17, 127.81, 123.15, 122.96,
122.41, 120.25, 120.15, 115.74, 115.04, 111.31, 110.70, 101.22,
68.01, 67.77, 55.51, 47.72, 33.41, 30.45, 28.21, 25.90, 25.04,
C36H37N2O4Cl, 597.2515; found, 597.2507. Purity: 98.4% (by HPLC).
4.1.41. (E)-7-(8-(6-Chloro-1,2,3,4-tetrahydroacridin-9-
ylamino)hexyloxy)-3-(4-methoxybenzylidene)chroman-4-one
(8p)
25.00, 22.67, 22.37; HRMS (ESI) m/z calcd for
C36H38N2O4,
563.2904; found, 563.2911. Purity: 98.2% (by HPLC).
(E)-7-Hydroxy-3-(4-methoxybenzylidene)chroman-4-one (7a)
was treated with 3g (N-(8-bromohexyl)-6-chloro-1,2,3,4-tetrahy-
droacridin-9-amine) according to the general procedure to give
the desired product 8p as a yellow oil, 75% yield. 1H NMR
(400 MHz, DMSO) d 8.12 (d, J = 9.1 Hz, 1H), 7.78 (d, J = 8.8 Hz,
1H), 7.71–7.64 (m, 2H), 7.41 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 9.1 Hz,
1H), 7.04 (d, J = 8.5 Hz, 2H), 6.65 (d, J = 8.7 Hz, 1H), 6.51 (s, 1H),
5.54 (s, 1H), 5.39 (s, 2H), 3.99 (t, J = 6.4 Hz, 2H), 3.81 (s, 3H),
3.42–3.39 (m, 2H), 2.86 (s, 2H), 2.66 (s, 2H), 1.77 (s, 4H), 1.70–
1.62 (m, 2H), 1.53 (s, 2H), 1.31 (s, 2H), 1.23 (s, 6H); 13C NMR
4.1.38. (E)-3-(3,4-Dimethoxybenzylidene)-7-(6-(1,2,3,4-
tetrahydroacridin-9-ylamino)hexyloxy)chroman-4-one (8m)
(E)-3-(3,4-Dimethoxybenzylidene)-7-hydroxychroman-4-one
(7i) was treated with 3b (N-(6-bromononyl)-1,2,3,4-tetrahydroac-
ridin-9-amine) according to the general procedure to give the de-
sired product 8m as a yellow oil, 38% yield. 1H NMR (400 MHz,
DMSO) d 8.11 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.70 (d,
J = 8.6 Hz, 1H), 7.67 (s, 1H), 7.52 (d, J = 7.2 Hz, 1H), 7.33 (d,
J = 8.0 Hz, 1H), 7.07 (d, J = 7.6 Hz, 2H), 7.00 (d, J = 8.4 Hz, 1H),
6.65 (d, J = 8.8 Hz, 1H), 6.51 (s, 1H), 5.44 (s, 2H), 5.37 (s, 1H), 4.00
(t, J = 6.3 Hz, 2H), 3.82 (s, 3H), 3.81 (s, 3H), 3.40 (d, J = 6.6 Hz,
2H), 2.89 (t, J = 5.6 Hz, 2H), 2.71 (t, J = 5.4 Hz, 2H), 1.85–1.76 (m,
4H), 1.70–1.64 (m, 2H), 1.61–1.52 (m, 2H), 1.36 (s, 4H); 13C NMR
(101 MHz, DMSO)
d 179.40, 164.89, 162.38, 160.28, 158.99,
150.59, 147.44, 135.38, 132.47, 131.98, 128.80, 128.46, 126.43,
126.39, 125.15, 123.12, 118.42, 115.54, 115.03, 114.06, 110.28,
100.97, 67.97, 67.60, 55.02, 47.86, 33.30, 30.96, 30.49, 28.61,
28.36, 26.16, 25.24, 24.90, 22.53, 22.20; HRMS (ESI) m/z calcd for
(101 MHz, DMSO)
d 179.56, 164.90, 162.39, 157.86, 150.31,
150.10, 148.59, 146.84, 135.96, 128.91, 128.62, 128.18, 127.78,
126.59, 123.66, 123.11, 122.94, 120.17, 115.72, 115.01, 113.81,
111.50, 110.46, 101.14, 67.95, 67.68, 55.49, 47.86, 33.42, 30.47,
28.25, 25.94, 25.07, 24.99, 22.68, 22.37; HRMS (ESI) m/z calcd for
C38H41N2O4Cl, 625.2828; found, 625.2832. Purity: 96.3% (by HPLC).
4.1.42. 3-(4-Methoxybenzyl)-7-(6-(1,2,3,4-tetrahydroacridin-9-
ylamino)hexyloxy)chroman-4-one (10)
C
37H40N2O5, 593.3010; found, 593.3009. Purity: 95.9% (by HPLC).
7-Hydroxy-3-(4-methoxybenzyl)chroman-4-one (9) was trea-
ted with 3b (N-(6-bromononyl)-1,2,3,4-tetrahydroacridin-9-
amine) according to the general procedure to give the desired
product 10 as a colorless oil, 66% yield. 1H NMR (400 MHz, DMSO)
d 8.09 (d, J = 8.4 Hz, 1H), 7.69 (d, J = 4.6 Hz, 1H), 7.67 (d, J = 5.1 Hz,
1H), 7.49 (t, J = 7.6 Hz, 1H), 7.34–7.28 (m, 1H), 7.14 (d, J = 8.4 Hz,
2H), 6.85 (d, J = 8.4 Hz, 2H), 6.59 (dd, J = 8.7, 2.0 Hz, 1H), 6.48 (d,
J = 2.1 Hz, 1H), 5.35 (s, 1H), 4.31 (dd, J = 11.4, 4.4 Hz, 1H), 4.12
(dd, J = 11.3, 9.1 Hz, 1H), 3.95 (t, J = 6.4 Hz, 2H), 3.71 (s, 3H), 3.41
(s, 2H), 3.03 (dd, J = 13.9, 5.0 Hz, 1H), 2.94–2.90 (m, 1H), 2.88 (t,
J = 5.5 Hz, 2H), 2.69 (t, J = 5.8 Hz, 2H), 2.59 (dd, J = 13.9, 9.4 Hz,
1H), 1.78 (d, J = 5.5 Hz, 4H), 1.68–1.61 (m, 2H), 1.58–1.52 (m,
4.1.39. (E)-7-(6-(1,2,3,4-Tetrahydroacridin-9-
ylamino)hexyloxy)-3-(3,4,5-trimethoxybenzylidene)chroman-
4-one (8n)
(E)-7-Hydroxy-3-(3,4,5-trimethoxybenzylidene)chroman-4-one
(7j) was treated with 3b (N-(6-bromononyl)-1,2,3,4-tetrahydroac-
ridin-9-amine) according to the general procedure to give the
desired product 8n as a yellow oil, 48% yield. 1H NMR (400 MHz,
DMSO) d 8.10 (d, J = 8.5 Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.71–7.63
(m, 2H), 7.53–7.47 (m, 1H), 7.32 (t, J = 7.6 Hz, 1H), 6.74
(s, 2H), 6.66 (d, J = 8.8 Hz, 1H), 6.51 (s, 1H), 5.46 (s, 2H), 5.36

7416
Y. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7406–7417
2H), 1.33 (s, 4H); 13C NMR (101 MHz, DMSO) d 191.56, 164.80,
162.99, 157.89, 157.75, 150.30, 146.89, 130.13, 129.86, 128.38,
128.23, 127.75, 123.10, 122.94, 120.21, 115.74, 113.81, 113.75,
110.06, 101.00, 69.53, 67.90, 54.88, 47.87, 46.31, 33.45, 30.87,
30.48, 28.23, 25.93, 25.06, 25.01, 22.68, 22.38; HRMS (ESI) m/z
calcd for C36H40N2O4, 565.3061; found, 565.3061. Purity: 99.4%
(by HPLC).
incubated at 37 °C for 15 min in a flat-black-bottom 96-well micro-
test plate in dark. The reaction was started by adding 200
l
M
Amplex Red reagent, 2 U/mL horseradish peroxidase, and 2 mM
p-tyramine for hMAO-A or 2 mM benzylamine for hMAO-B, at
37 °C for 20 min. The reaction was quantified in a multidetection
microplate fluorescence reader based on the fluorescence gener-
ated (excitation, 545 nm; emission, 590 nm).
The specific fluorescence emission was calculated after subtrac-
tion of the background activity, which was determined from vials
containing all components except the hMAO isoforms, which were
replaced by a sodium phosphate buffer solution.
4.2. Biological activity
4.2.1. In vitro inhibition studies on AChE and BuChE14
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel), hu-
man acetylcholinesterase (hAChE, EC 3.1.1.7, from human erythro-
cytes), butylcholinesterase (BuChE, E.C. 3.1.1.8, from equine
serum), 5,50-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent,
DTNB), acetylthiocholine chloride (ATC), and butylthiocholine
chloride (BTC) were purchased from Sigma–Aldrich. Compounds
were dissolved in DMSO (10 mM) and diluted in 0.1 M KH2PO4/
K2HPO4 buffer (pH 8.0) to the desired final concentration. All the
compounds are soluble at the tested concentration. DMSO was di-
luted to a concentration of less than 0.01%, and no inhibitory action
on either AChE or BuChE was detected in separate prior experi-
ments at this concentration.
4.2.4. Reversibility and irreversibility experiments17
To evaluate whether compound 8b is a reversible or irreversible
hMAO-B inhibitor, an effective centrifugation ultrafiltration meth-
od (so-called ‘repeated washing’) was used. The experiment was
performed according a reported method.13 Adequate amounts of
recombinant monoamine oxidase with or without test drugs were
incubated at 37 °C for 15 min. An aliquot from this incubation was
stored at 4 °C for later use in the measurement of MAO activity. An-
other 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 used
for the subsequent measurement of activity using a method similar
to that described above (the section ‘Inhibition of MAO activity’).
The corresponding values of percent hMAO-B inhibition were sep-
arately calculated for samples with and without repeated washing.
The in vitro AChE assay was performed as follows: all assays
were conducted in 0.1 M KH2PO4/K2HPO4 buffer (pH 8.0) using a
Shimadzu UV-2450 spectrophotometer. Enzyme solutions were
prepared to give 2.0 U/mL in 2 mL aliquots. The assay medium
(1 mL) consisted of phosphate buffer (pH 8.0), 50
lL of 0.01 M
DTNB, 10 L of enzyme, and 50 L of 0.01 M substrate (ACh chlo-
l
l
4.2.5. In vitro blood–brain barrier permeation assay.20
ride solution). Test compounds were added to the assay solution
and pre-incubated with the enzyme at 37 °C for 15 min, followed
by addition of substrate. The activity was determined by measur-
ing the increase in absorbance at 412 nm at 1 min intervals at
37 °C. Calculations were performed according to the method of
the equation in Ellman et al. Each concentration was assayed in
triplicate.
Brain penetration of compounds was evaluated using a parallel
artificial membrane permeation assay (PAMPA) in a similar man-
ner as described by Di et al. Commercial drugs were purchased
from Sigma and Alfa Aesar. The porcine brain lipid (PBL) was ob-
tained from Avanti Polar Lipids. The donor microplate (PVDF mem-
brane, pore size 0.45 mm) and the acceptor microplate were both
from Millipore. The 96-well UV plate (COSTARÒ) was from Corning
Incorporated. The acceptor 96-well microplate was filled with
The in vitro BuChE and hAChE assay was performed similarly to
the method described above.
300
nated with 4
dissolved in DMSO at 5 mg/mL and diluted 50-fold in PBS/EtOH
(7:3) to achieve a concentration of 100 mg/mL, 200 L of which
lL of PBS/EtOH (7:3), and the filter membrane was impreg-
lL of PBL in dodecane (20 mg/mL). Compounds were
4.2.2. Kinetic characterization of AChE inhibition
Kinetic characterization of AChE was performed using a re-
ported method. Test compound was added into the assay solution
and pre-incubated with the enzyme at 37 °C for 15 min, followed
by addition of substrate. Kinetic characterization of the hydrolysis
of ATC catalyzed by AChE was performed spectrometrically at
412 nm. A parallel control with no inhibitor in the mixture allowed
the change in activities to be measured at various times. 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 slopes of these reciprocal plots were subsequently
plotted against the concentration of the inhibitors in a weighted
analysis, and Ki was determined as the ratio of the replot intercept
to the replot slope.
l
was added to the donor wells. The acceptor filter plate was care-
fully placed on the donor plate to form a sandwich, which was left
undisturbed for 10 h at 25 °C. After incubation, the donor plate was
carefully removed and the concentration of compound in the
acceptor wells was determined using a UV plate reader (Flexsta-
tionÒ 3). Every sample was analyzed at five wavelengths, in four
wells, in at least three independent runs, and the results are given
as the mean standard deviation. In each experiment, 13 quality
control standards of known BBB permeability were included to val-
idate the analysis set.
Acknowledgements
4.2.3. Inhibition of MAO activity17
This work was supported by National Natural Science Founda-
tion of China (No. 21302235), Distinguished Young Talents in High-
er Education of Guangdong (2012LYM_003), Ph.D. Programs
Foundation of Ministry of Education of China (20120171120045),
and the Guangdong Engineering Research Center of Chiral Drugs.
The potential effects of the test compounds on hMAO activity
were investigated by measuring their effects on the production
of H2O2 from p-tyramine, using the Amplex Red MAO assay kit
(Molecular Probes, Inc.) and recombinant human MAO-A or
MAO-B (Sigma–Aldrich) according to published procedures. Com-
pounds were dissolved in DMSO (10 mM) and diluted in 0.1 M KH2-
PO4/K2HPO4 buffer (pH 8.0) to the desired final concentration. All
the compounds are soluble at the tested concentration. Adequate
amounts of recombinant hMAO-A or hMAO-B (Sigma–Aldrich)
References and notes
1. de Blennow, K.; Leon, M. J.; Zetterberg, H. Lancet 2006, 368, 387.
2. Selkoe, D. J. Physiol. Rev. 2001, 81, 741.
3. (a) Scarpini, E.; Schelterns, P.; Feldman, H. Lancet Neurol. 2003, 2, 539; (b)
Winkler, J.; Thal, L. J.; Gage, F. H.; Fisher, L. J. J. Mol. Med. 1998, 76, 555; (c)
Cummings, J. L. Am. J. Psychiatry 2000, 157, 4.
were acquired and adjusted to 12.5
75 g/mL for hMAO-B. Test drugs (20
l
g/mL for hMAO-A and
l
l
L) and MAO (80 L) were
l

Y. Sun et al. / Bioorg. Med. Chem. 21 (2013) 7406–7417
7417
4. Francis, P. T.; Palmer, A. M.; Snape, M.; Wilcock, G. K. J. Neurol. Neurosurg.
Psychiatry 1999, 66, 137.
12. Youdim, M.; Amit, T.; Bar-Am, O.; Weinreb, O.; Yogev-Falach, M. Neurotox. Res.
2006, 10, 181.
5. Villarroyaa, M.; García, A. G.; Marco, J. L. Curr. Pharm. Des. 2004, 10, 3177.
6. Walker, W. H.; Kearney, E. B.; Seng, R. L.; Singer, T. P. Eur. J. Biochem. 1971, 24,
328.
7. (a) Binda, C.; Newton-Vinson, P.; Hubalek, F.; Edmondson, D. E.; Mattevi, A. Nat.
Struct. Biol. 2002, 9, 22; (b) Edmondson, D. E.; DeColibus, L.; Binda, C.; Li, M.;
Mattevi, A. J. Neural Trans. (Vienna, Austria: 1996) 2007, 114, 703.
8. Bach, A. W.; Lan, N. C.; Johnson, D. L.; Abell, C. W.; Bembenek, M. E.;
Kwan, S. W.; Seeburg, P. H.; Shih, J. C. Proc. Natl. Acad. Sci. U.S.A. 1988,
85, 4934.
13. Desideri, N.; Bolasco, A.; Fioravanti, R.; Proietti Monaco, L.; Orallo, F.; Yáñez,
M.; Ortuso, F.; Alcaro, S. J. Med. Chem. 2011, 54, 2155.
14. Ellman, G. L.; Courtney, K. D.; Andres, B. J.; Featherstone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
15. Inestrosa, N. C.; Alvarez, A.; Perez, C. A.; Moreno, R. D.; Vicente, M.; Linker, C.;
Casanueva, O. I.; Soto, C.; Garrido, J. Neuron 1996, 16, 881.
16. Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. Biochem. Pharmacol. 2003,
65, 407.
17. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Bizzarri, B.; Granese, A.;
Carradori, S.; Yáñez, M.; Orallo, F.; Ortuso, F.; Alcaro, S. J. Med. Chem. 2009, 52,
1935.
9. Saura, J.; Luque, J. M.; Da Cesura, A. M.; Prada, M.; Chan-Palay, V.; Huber, G.;
Loffler, J.; Richards, J. G. Neuroscience 1994, 62, 15.
10. Nebbioso, M.; Pascarella, A.; Cavallotti, C.; Pescosolido, N. Int. J. Exp. Pathol
2012, 93, 401.
18. Izumi, N. I. T.; Kitaichi, Y.; Kato, A.; Inoue, T.; Koyama, T. Eur. J. Pharmacol. 2007,
565, 105.
11. Sterling, J.; Herzig, Y.; Goren, T.; Finkelstein, N.; Lerner, D.; Goldenberg, W.;
Miskolczi, I.; Molnar, S.; Rantal, F.; Tamas, T.; Toth, G.; Zagyva, A.; Zekany, A.;
Lavian, G.; Gross, A.; Friedman, R.; Razin, M.; Huang, W.; Krais, B.; Chorev, M.;
Youdim, M. B.; Weinstock, M. J. Med. Chem. 2002, 45, 5260.
19. (a) Clark, D. E.; Pickett, S. D. Drug Discovery Today 2000, 5, 49; (b) Kerns, E. H.;
Di, L. Drug Discovery Today 2003, 8, 316.
20. Di, L.; Kerns, E. H.; Fan, K.; McConnell, O. J.; Carter, G. T. E. J. Med. Chem. 2003,
38, 223.