O-Hydroxyl- or o-amino benzylamine-tacrine hybrids: Multifunctional biometals chelators, antioxidants, and inhibitors of cholinesterase activity and amyloid-β aggregation

Article abstract of DOI:10.1016/j.bmc.2012.07.045

In an effort to identify novel multifunctional drug candidates for the treatment of Alzheimer's disease (AD), a series of hybrid molecules were synthesised by reacting N-(aminoalkyl)tacrine with salicylic aldehyde or derivatives of 2-aminobenzaldehyde. Th

Full text of DOI:10.1016/j.bmc.2012.07.045

Bioorganic & Medicinal Chemistry 20 (2012) 5884–5892
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Bioorganic & Medicinal Chemistry
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O-Hydroxyl- or o-amino benzylamine-tacrine hybrids: Multifunctional
biometals chelators, antioxidants, and inhibitors of cholinesterase activity
and amyloid-b aggregation
⇑
⇑
Fei Mao, Ling Huang, Zonghua Luo, Anqiu Liu, Chuanjun Lu, Zhiyong Xie , Xingshu Li
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to identify novel multifunctional drug candidates for the treatment of Alzheimer’s disease
(AD), a series of hybrid molecules were synthesised by reacting N-(aminoalkyl)tacrine with salicylic alde-
hyde or derivatives of 2-aminobenzaldehyde. These compounds were then evaluated as multifunctional
anti-Alzheimer’s disease agents. All of the hybrids are potential biometal chelators, and in addition, most
of them were better antioxidants and inhibitors of cholinesterases and amyloid-b (Ab) aggregation than
the lead compound tacrine. Compound 7c has the potential to be a candidate for AD therapy: it is a much
better inhibitor of acetylcholinesterase (AChE) than tacrine (IC50: 0.55 nM vs 109 nM), has good biometal
chelation ability, is able to inhibit Ab aggregation and has moderate antioxidant activity (1.22 Trolox
equivalents).
Received 15 June 2012
Revised 24 July 2012
Accepted 27 July 2012
Available online 15 August 2012
Keywords:
Alzheimer’s disease
Multifunctional drug candidates
Biometals chelators
Inhibitors of cholinesterases
Antioxidants
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
(M30, Fig. 1) have been used for the treatment of AD.7 Recently,
the small molecules N-(pyridin-2-ylmethyl) aniline (1, Fig. 1) and
Acetylcholinesterase inhibitors (AChEIs), such as tacrine,
donepezil, rivastigmine and galantamine, which increase neuro-
transmission at cholinergic synapses in the brain and temporarily
reduce cognitive deficits, are the current therapeutic options for
the treatment of Alzheimer’s disease (AD).1,2 However, many facts
strongly suggest that AD is a multifaceted illness. In addition to the
hallmark low levels of acetylcholine, the following are important in
N1,N1-dimethyl-N4-(pyridin-2-ylmethyl)benzene-1,4-diamine (2,
Fig. 1) were designed to regulate metal-induced Ab aggregation
and neurotoxicity.8 Many studies have been devoted to the search
for multifunctional agents that simultaneously inhibit cholinester-
ase, decrease Ab levels and chelate redox-active metals because of
the potential uses of these agents for the treatment of AD.9
Tacrine, the first ChE inhibitor approved by the FDA for the
treatment of AD, suffers from therapy-limiting liver toxicity. De-
spite this limitation, tacrine has been widely used as scaffold for
the development of new multifunctional agents with additional
biological properties other than AChE inhibition.10–13 For example,
Pang et al. developed bis(7)tacrine, which is composed of two ta-
crine units linked by a seven-carbon linker. This compound exhib-
ited a 1000-fold greater AChE inhibition potency than tacrine.14,15
Rodríguez-Franco reported that tacrine-8-hydroxyquinoline hy-
brids are potential multifunctional drugs for the treatment of AD
because these compounds exhibited more potent AChE inhibitory
activities than tacrine and had good copper-complexing proper-
ties.16 Other compounds, such as compounds containing both mer-
capto groups and tacrine,17 hybrids of tacrine and 4-fluorobenzoic
acid,18 heterobivalent tacrine derivatives and tacrine-multialkoxy-
benzene hybrids,19,20 were also developed recently.
the aetiology of AD: b-amyloid (Ab) deposits, s-protein aggrega-
tion, oxidative stress and the dyshomeostasis of biometals. Recent
studies have indicated that biometals, such as Cu(II), Zn(II), and
Fe(II, III), play a very important role in many critical aspects of
AD. Raman spectra demonstrated that Zn(II) and Cu(II) are coordi-
nated to histidine residues in senile plaque (SP) cores. Treatment of
SPs with the chelator EDTA reverses Cu(II) binding to SP histidines
and leads to a broadening of the amide features, indicating a ‘loos-
ening’ of the b-structure.3 Furthermore, Cu(II) and Fe(II, III) have
been linked to the production of reactive oxygen species (ROS)
and oxidative stress (OS).4,5 Thus, the modulation of these biomet-
als in the brain has been proposed as a potential therapeutic strat-
egy for the treatment of AD.6 In view of this proposal, the biometal
chelators desferrioxamine (DFO, Fig. 1), EDTA, clioquinol (Fig. 1),
and 5-[N-methyl-N-propargylaminomethyl]-8-hydroxyquinoline
In our previous work, we designed and synthesised a series of
berberine derivatives as multifunctional anti-AD agents.21 Herein,
we describe the design, synthesis, and evaluation a series of o-hy-
droxyl and o-amino benzylamine-tacrine hybrids 6a-6g, 7a–7e and
⇑
Corresponding authors. Tel./fax: +86 20 39943050.
E-mail address: lixsh@mail.sysu.edu.cn (X. Li).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.045

F. Mao et al. / Bioorg. Med. Chem. 20 (2012) 5884–5892
5885
of 2-amino benzoic acid or 2-amino-4-chlorobenzoic acid with
cyclohexanone and phosphorus oxychloride at a temperature of
0 °C to reflux provided the intermediate 2.22 Compound 2 was then
reacted with diamine in the presence of a catalytic amount of
potassium iodide to give 3. o-Fluorobenzaldehyde was reacted
with dimethylamine or diethylamine in the presence of K2CO3 to
provide the substituted o-amino benzaldehyde 5. Finally, com-
pound 3 was reacted with salicylaldehyde or 5 to give the corre-
sponding Schiff base, which was then reduced by sodium
borohydride to afford the target product 6 or 7 in good chemical
yields. Compound 7e, the hybrid consisting of tacrine and pyri-
dine-2-yl methanamine, was obtained by reacting compound 3
with pyridine-2-carboxaldehyde under the same reaction
conditions.
2.2. Biological activities
Figure 1. Chemical structure of metal-chelating agents.
2.2.1. In vitro inhibition studies of AChE and BuChE
The AChE and BuChE inhibitory activities of all the o-hydroxyl
and o-amino benzylamine-tacrine hybrids were examined by the
method of Ellman et al.23 using AChE from the electric eel and
BuChE from equine serum. Tacrine was used as the reference stan-
dard. The IC50 values are summarised in Table 1. Relative to tacrine,
which had an IC50 value of 109 nM, fifteen of the nineteen hybrids
were more potent AChEIs, with IC50 values ranging from 0.55 to
43.3 nM. A structure-activity relationship analysis showed that
the AChE inhibitory potency was closely related to the length of
the alkylene chain. Hybrids 6a, 6b, and 6c, with two, three and four
carbon spacers between tacrine and an o-hydroxyl unit had weak
inhibitory activities than tacrine (6a, two methylene groups, with
an IC50 value of 146.19 nM; 6b, three methylene groups,
131.13 nM; 6c, four methylene groups, 252.5 nM). Generally, the
o-hydroxyl benzylamine-(7-chloro tacrine) hybrids 6h-6n (their
leading compound exhibited better inhibitive activity than ta-
crine22) as multifunctional anti-AD agents with metal-chelating
ability, antioxidative activity, and cholinesterase and Ab aggrega-
tion inhibition activities.
2. Results and discussion
2.1. Chemistry
The synthetic pathway of the o-hydroxyl and o-amino benzyl-
amine-tacrine hybrids is shown in Scheme 1. First, the reaction
3a:R=H,n=2 3b:R=H,n=3
Cl
NH(CH2)nNH2
3c:R=H,n=4 3d:R=H,n=6
COOH
b
3e:R=H,n=7 3f:R=H,n=8
3g:R=H,n=9 3h:R=Cl,n=2
3i:R=Cl,n=3 3j:R=Cl,n=4
a
R
NH2
R
N
2
R
N
3k:R=Cl,n=6 3l:R=Cl,n=7
3m:R=Cl,n=8 3n:R=Cl,n=9
3
N(R1)2
CHO
F
c
CHO
4
5a:R1=Me
5b:R1=Et
HO
6a:R=H,n=2 6b:R=H,n=3
6c:R=H,n=4 6d:R=H,n=6
6e:R=H,n=7 6f:R=H,n=8
6g:R=H,n=9 6h:R=Cl,n=2
6i:R=Cl,n=3 6j:R=Cl,n=4
6k:R=Cl,n=6 6l:R=Cl,n=7
6m:R=Cl,n=8 6n:R=Cl,n=9
NH(CH2)nNH
N
CHO
OH
R
NH(CH2)nNH2
N
d,e
R
(R1)2N
NH(CH2)nNH
5
3
7a:R1=Me, n=7
7b:R1=Me, n=8
7c:R1=Me, n=9
7d:R1=Me, n=8
d,e
R
N
CHO
N
NH(CH2)8NH2
N
NH(CH2)8NH
N
d,e
R
N
3f
7e
Scheme 1. Synthesis of o-hydroxyl- or o-amino benzylamine tacrine. Reagents and conditions: (a) cyclohexanone, POCl3, 0 °C?reflux; (b) diamine, KI, 1-pentanol, 160 °C; (c)
NHMe2 or NHEt2, K2CO3, DMF, 70 °C; (d) ethanol, rt; (e) NaBH4, ethanol, rt.

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F. Mao et al. / Bioorg. Med. Chem. 20 (2012) 5884–5892
Table 1
In vitro inhibition and selectivity of AChE and BuChE and Oxygen Radical Absorbance Capacity (ORAC, Trolox Equivalents) by tacrine and tacrine derivatives 6a-6n and 7a-7e
Compound
R
R1
n
IC50a(nM)
AChEb
Selectivity for AChEd
Trolox equive
BuChEc
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
7a
7b
7c
7d
7e
Tacrine
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2
146.19 4.53
131.13 28.6
252.5 3.54
25.15 3.49
9.55 1.15
6.17 1.19
7.52 0.41
22.49 2.64
43.32 3.05
125.0 2.83
43 2.55
10.48 0.23
6.77 0.66
7.78 0.14
3.50 0.19
1.55 0.067
0.55 0.034
1.36 0.046
2.95 0.02
109 10.3
10.00 0.76
46.13 1.11
20.25 1.64
9.65 0.46
5.84 0.16
6.25 0.29
8.20 0.45
9.97 0.6
76.51 8.11
28.09 0.34
11.1 0.35
17.79 1.72
18.48 0.68
22.98 2.2
2.92 0.36
2.11 0.11
2.65 0.17
2.82 0.30
2.99 0.1
0.07
0.35
0.08
0.38
0.61
1.01
1.09
0.44
1.77
0.22
0.26
1.7
2.73
2.95
0.83
1.36
4.78
2.07
1.01
0.14
11.86
13.81
11.76
11.14
7.91
5.45
4.41
8.76
11.32
8.04
7.87
5.83
4.79
4.54
2.16
1.97
1.22
0.88
n.t.f
3
4
6
7
8
9
2
3
4
6
7
8
9
7
8
9
8
—
—
Me
Me
Me
Et
—
f
—
15.8 1.32
n.t.
a
b
c
d
e
f
Mean SD of at least three independent measurements.
AChE from the electric eel.
BuChE from equine serum.
Selectivity for AChE = IC50 (BuChE)/ IC50 (AChE).
Data are expressed as (lmol trolox)/(lmol tested compound).
n.t. = not tested.
AChE inhibitory activity intensifies as the number of methylene
groups increases. Compound 6f, which has an eight-carbon linker,
had the best IC50 value (6.17 nM). The same trend was also ob-
served for the hybrids derived from salicylaldehyde and 7-chloro
tacrine, among which compound 6m, possessing an eight-carbon
linker, also gave the best result in this series. Substituted amino
groups seem to have a favourable effect on the AChE inhibitory
activity. For example, hybrids 7a-d, which were derived from ta-
crine and substituted o-amino benzaldehyde, gave better results
than the corresponding hybrids of salicylaldehyde and tacrine.
Among 7a-d, compound 7c, in which tacrine was linked with o-
(N,N-dimethylamino) benzylamine by a 9-carbon spacer, exhibited
the greatest inhibitory potency towards AChE (IC50 value: 0.55
nM).
the enzymes should be attributed to a slightly better fit of the com-
pound to the active sites of electric eel.
Most of the hybrids also exhibited very good BuChE inhibitory
activities. Compounds 6f and 7c, which had the best AChE inhibi-
tory activities in the two series, also had very good BuChE inhibi-
tory activities (IC50 values: 6.25 and 2.63 nM, respectively).
However, the trend for the structure-activity relationship found
for the AChE inhibitory activity was not found for the BuChE inhib-
itory activity.
2.3. Kinetics of AChE inhibition
To study the inhibitory mechanism for this class of tacrine-de-
rived hybrids, the most potent AChE inhibitor among these com-
pounds, compound 7c, was chosen for further kinetic studies.
The graphical presentation of the steady-state inhibition data of
compound 7c for AChE is shown in Figure 2. The results showed
that there was an increasing slope and an increasing intercept at
higher inhibitor concentrations, indicating a mixed-type inhibitory
behaviour for compound 7c as the result of binding to both the cat-
alytic active site (CAS) and peripheral anionic site(PAS) of AChE.
A selection of the compounds (7a-7c) was evaluated as inhibi-
tors of human AChE and the results were listed in table 2. All tested
derivatives gave IC50 values in the nano- and subnanomolar range
(5.18, 4.04, 3.52 nM), which showed the same tendency as that of
electric eel (Table 1, 3.50, 1.55, 0.55 nM). The different activities to
Table 2
2.4. Molecular modelling studies
Inhibition of human AChE by selected tacrine derivatives 7a-7c
Compound
R1
n
IC50a(nM) hAChEb
To explore the possible interaction mode of the tacrine hybrids
with TcAChE, molecular docking simulations for the derivative 7c
onto TcAChE were performed using the CDOCKER program in
Discovery Studio 2.1. These simulations were based on the X-ray
crystal structure of the TcAChE-bis-(7)-tacrine complex (PDB
entry 2CMF), and the result is shown in Figure 3a and b. These
7a
7b
7c
Me
Me
Me
7
8
9
5.18 0.24
4.04 0.028
3.52 0.057
a
Mean SD of at least three independent measurements.
AChE from human recombinant.
b

F. Mao et al. / Bioorg. Med. Chem. 20 (2012) 5884–5892
5887
inhibitory activity, the antioxidant activities of the hybrids de-
creased as the length of the carbon spacer increased. Compounds
6a-d, which have 2- to 6-carbon spacers linking tacrine to o-hydro-
xyl benzylamine, had activities of more than 10 Trolox equivalents,
whereas compound 6g, a hybrid with a similar structure apart
from its nine-carbon spacer, had an activity of only 4.41 Trolox
equivalents. Substituted amino groups seem to have an unfavour-
able effect on the antioxidant activity, and compounds 7a-d all had
lower activities than their relatives 6e-g.
2.6. Inhibition of Ab1–42 aggregation
On the basis of the results of the in vitro inhibition studies for
AChE and BuChE, the ability of tacrine-derived hybrids to reduce
Ab1–42 self-aggregation was studied through a thioflavin T-based
fluorimetric assay with curcumin (Cur) as the standard compound.
The results in Figure 4 indicate that these hybrids showed moder-
ate to good potencies (25.8–40.5% at 20
cumin (52.1% at 20 M). Among the hybrids, compound 7c, which
exhibited the most potent AChE inhibition activity, had good Ab
aggregation inhibition properties (39.4% at 20 M). In contrast,
lM) relative to that of cur-
l
Figure 2. Steady state inhibition by compound 7c of the AChE hydrolysis of Ach.
The plots show mixed-type AChE inhibition for compound 7c.
l
compound 6m, which was the most potent AChE inhibitor in the
series of o-hydroxyl benzylamine-tacrine hybrids, exhibited 25.8%
inhibition at 20 lM, indicating that substituted amino groups have
a favourable effect on Ab1–42 peptide aggregation inhibition.
simulations indicated that compound 7c occupies the entire enzy-
matic CAS, the mid-gorge sites and the PAS and could simulta-
neously bind to both the central pocket and peripheral sites. The
tacrine moiety of the hybrid was observed to bind to the CAS via
a parallel
p–p interaction between Trp84 (4.195 Å) and Phe330
2.7. Metal-chelating properties of compounds 6f, 7c and 7e
(4.048 Å) in a ‘sandwich’ form. The protonated nitrogen atom of
benzylamine interacted with Trp279 of the PAS with an atom-to-
ring distance of 3.935 Å. In addition, the methylene moiety of the
long chain, bound to the gorge, could fold in a conformation in
the gorge that allows the linker to interact with Tyr334, Gly335,
Phe330, Tyr121, Asp72 and Tyr70 through hydrophobic interac-
tions. The benzyl moiety, bound to the rim of the gorge, also could
interact with Leu282 and Ile287 through hydrophobic interactions.
The complexation abilities of compounds 6f, 7c and 7e for bio-
metals such as Cu2+, Fe2+ and Zn2+ in ethanol were studied by UV–
vis spectrometry,26,27 and the results are shown in Figures 5, 6
and 7. All the electronic spectra of 6f, 7c and 7e exhibited a red shift
(6f: the peak at 323 nm shifted to 337 nm; 7c: the peak at 325 nm
shifted to 336 nm; 7e: the peak at 323 nm shifted to 337 nm). After
the addition of Cu2+. These results indicated that the two hybrids
could interact with the Cu2+ ion effectively. Two other biometals,
Fe2+ and Zn2+, also expressed similar results; the peak in the elec-
tronic spectra of 6f at 323 nm shifted to 335 nm when Fe2+ was
added, and the peak changed from 323 nm to 337 nm when Zn2+
was added. The peaks of 7c changed from 325 nm to 336 nm and
from 325 nm to 336 nm, respectively, in response to the addition
of the two biometals. The peaks of 7e changed from 323 nm to
336 nm and from 323 nm to 336 nm, respectively.
2.5. Antioxidant activity
The antioxidant activities of all of the hybrids were determined
by measuring the oxygen radical absorbance capacity of fluores-
cein (ORAC-FL), and the results are presented in Table 1.24,25 A vita-
min E analogue, Trolox was used as a standard (the activities are
expressed as Trolox equivalents, lmol of Trolox equivalents/lmol
of tested compound). It could be observed from the results that
most of the hybrids had very potent peroxyl radical absorbance
capacities, ranging from 1.22- to 13.81-fold the value of Trolox.
The exception was compound 7d, o-(N,N-diethylamino) benzyl-
amine linked with tacrine by an eight-carbon spacer, which had
an activity of 0.88 Trolox equivalents. In contrast to the AChE
3. Conclusion
In conclusion, our study involved the synthesis of a new series
of tacrine-derived hybrids incorporating o-hydroxyl- or o-amino
benzylamine and tacrine as synthons. These compounds were
Figure 3. Docking models of the compound–enzyme complex. (a) The binding pattern of compound 7c (yellow stick) to the TcAChE. (b) Representation of compound 7c
interacting with residues in the binding site of TcAChE, highlighting the protein residues that participate in the main interactions with the inhibitor.

5888
F. Mao et al. / Bioorg. Med. Chem. 20 (2012) 5884–5892
Figure 7. (a) UV spectrum of compound 7e (40
7e (40 M) and CuSO4 (40 M). (c) Spectrum of a mixture of 7e (40
(40 M). (d) Spectrum of a mixture of 7e (40 M) and ZnCl2 (40 M).
l
M). (b) Spectrum of a mixture of
l
l
lM) and FeSO4
Figure 4. Inhibition of self-mediated Ab1-42 aggregation by compounds 6m, 7a, 7b,
7c, 7d and curcumin. The measurements were carried out in the presence of 20 lM
l
l
l
test compound. The thioflavin-T fluorescence method was used. The values
represent the mean SD from at least two independent measurements.
evaluated as potential multi-valent inhibitors of ChE and Ab aggre-
gation, as antioxidant agents, and as chelators of biometals. Among
the synthesised compounds, compound 7c, in which tacrine was
linked with o-(N,N-dimethylamino) benzylamine by a 9-carbon
spacer, exhibited the greatest inhibitory potency towards AChE
(IC50 value: 0.55 nM), good biometal-chelating ability, good inhibi-
tion of Ab aggregation (39.4% at 20 lM) and moderate antioxidant
activity (1.22 Trolox equivalents). Further investigations of AD can-
didates based on these results are in progress.
4. Material and methods
4.1. Chemistry
The 1H NMR and 13C NMR spectra were recorded using TMS as
the internal standard on a Bruker BioSpin GmbH spectrometer at
400.132 MHz and 100.614 MHz, respectively. Coupling constants
are given in Hz. MS spectra were recorded on an Agilent LC–MS
6120 instrument with an ESI mass selective detector. High-resolu-
tion mass spectra were obtained using a Shimadzu LCMS-IT-TOF
mass spectrometer. Flash column chromatography was performed
with silica gel (200–300 mesh) purchased from Qingdao Haiyang
Chemical Co. Ltd or alumina from Sinopharm Chemical Reagent
Co. Ltd All the reactions were monitored by thin layer chromatog-
raphy on silica gel. The purities of synthesized compounds were
confirmed to be higher than 95% by analytical HPLC performed
with a dual pump Shimadzu LC-20AB system equipped with a
Figure 5. (a) UV spectrum of compound 6f (40
l
M). (b) Spectrum of a mixture of 6f
M). (c) Spectrum of a mixture of 6f (40 M) and FeSO4
M). (d) Spectrum of a mixture of 6f (40 M) and ZnCl2 (40 M).
(40
(40
l
l
M) and CuSO4 (40
l
l
l
l
Eclipse Plus XDB-C8 column (4.6 ꢀ 150 mm, 5
lm) and eluted with
methanol/water (55:45–40:60) containing 0.1% TFA at a flow rate
of 0.5 mL/min. Compound 3 and compound 5 was prepared accord-
ing to the reported procedure.28–30
4.2. General procedures for the preparation of compound 6a–
6n, 7a–7e
Aromatic aldehydes 5 or salicylic aldehyde or pyridine-2-car-
boxaldehyde (0.5 mmol) and intermediate 3 (0.5 mmol) were stir-
red in EtOH for 5 h and then NaBH4 (1 mmol) was added at 0 °C.
The solution was stirred for 5 min at 0 °C. After 2 h at room temper-
ature, the solvent was evaporated and the residue was quenched by
water (10 mL) followed by extraction with EtOAc (3 ꢀ 10 mL). The
organic layer was washed with brine (3 ꢀ 10 mL), and then dried
over MgSO4 and then concentrated, the compounds were purified
by flash chromatography with CH2Cl2/MeOH/NH4OH (10:1:1%)
elution.
Figure 6. (a) UV spectrum of compound 7c (40
l
M). (b) Spectrum of a mixture of 7c
M). (c) Spectrum of a mixture of 7c (40 M) and FeSO4
M). (d) Spectrum of a mixture of 7c (40 M) and ZnCl2 (40 M).
(40
(40
l
l
M) and CuSO4 (40
l
l
l
l

F. Mao et al. / Bioorg. Med. Chem. 20 (2012) 5884–5892
5889
1
4.2.1. 2-(((2-((1,2,3,4-Tetrahydroacridin-9-yl)amino)ethyl)
amino)methyl)phenol (6a)
(400 MHz, CDCl3) d 7.96 (d, J = 8.5 Hz, 2H), 7.56 (t, J = 7.6 Hz, 1H),
7.35 (t, J = 7.6 Hz, 1H), 7.15 (t, J = 7.7 Hz, 1H), 6.97 (d, J = 7.2 Hz,
1H), 6.82 (d, J = 8.0 Hz, 1H), 6.76 (t, J = 7.4 Hz, 1H), 3.98 (s, 2H),
3.51 (t, J = 7.2 Hz, 2H), 3.09 (s, 2H), 2.67 (dd, J = 15.7, 8.7 Hz, 4H),
1.92 (s, 4H), 1.65 (dd, J = 14.2, 7.1 Hz, 2H), 1.51 (d, J = 6.6 Hz, 2H),
1.43–1.30 (m, 6H). 13C NMR (101 MHz, CDCl3) d 158.31, 157.76,
151.19, 146.65, 128.59(2H), 128.26, 127.91, 123.69, 122.96,
122.61, 119.85, 118.88, 116.26, 115.40, 52.64, 49.27, 48.57, 33.45,
31.56, 29.39, 29.05, 26.94, 26.75, 24.71, 22.95, 22.58. Purity:
97.5% by HPLC; LC/MS (ESI) m/z:[M+H]+ 418.2. HRMS m/z [M+H]+
Calcd for C27H35N3O 418.2853, found 418.2839.
Salicylic aldehyde was treated with 3a according to general pro-
cedure to give the desired product as light yellow solid (59% yield).
1
m.p. 119.5–121.1 °C; (400 MHz, CDCl3) d 7.93 (t, J = 8.5 Hz, 2H),
7.57 (t, J = 7.4 Hz, 1H), 7.42–7.34 (m, 1H), 7.20 (t, J = 7.7 Hz, 1H),
7.01 (d, J = 7.3 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 6.81 (t, J = 7.4 Hz,
1H), 4.06 (s, 2H), 3.62 (s, 2H), 3.08 (s, 2H), 2.97 (t, J = 5.9 Hz, 2H),
2.73 (s, 2H), 1.92 (s, 4H). 13C NMR (101 MHz, CDCl3) d 158.01,
157.71, 150.78, 146.55, 128.89, 128.79, 128.72, 127.91, 124.07,
122.81, 122.70, 120.02, 119.25, 116.47, 116.30, 52.18, 49.36,
48.28, 33.40, 24.70, 22.87, 22.53. Purity: 98.0% by HPLC; LC/MS
(ESI) m/z:[M+H]+ 348.2. HRMS m/z [M+H]+ Calcd for C22H25N3O
348.2070, found 348.2075.
4.2.6. 2-(((8-((1,2,3,4-Tetrahydroacridin-9-yl)amino)octyl)
amino)methyl)phenol (6f)
Salicylic aldehyde was treated with 3f according to general pro-
cedure to give the desired product as light yellow oil (70% yield).
(400 MHz, CDCl3) d 8.01–7.91 (m, 2H), 7.55 (t, J = 7.6 Hz, 1H), 7.34
4.2.2. 2-(((3-((1,2,3,4-Tetrahydroacridin-9-yl)amino)propyl)
amino)methyl)phenol (6b)
1
Salicylic aldehyde was treated with 3b according to general pro-
cedure to give the desired product as light yellow solid (68% yield).
m.p. 144.3-146.2 °C; 1 (400 MHz, CDCl3) d 7.90 (dd, J = 12.1, 8.5 Hz,
2H), 7.55 (t, J = 7.4 Hz, 1H), 7.38–7.30 (m, 1H), 7.18 (t, J = 7.5 Hz,
1H), 6.98 (d, J = 7.2 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.78 (t,
J = 7.4 Hz, 1H), 4.01 (s, 2H), 3.55 (t, J = 6.7 Hz, 2H), 3.06 (s, 2H),
2.78 (t, J = 6.9 Hz, 2H), 2.68 (s, 2H), 1.98–1.83 (m, 6H). 13C NMR
(t, J = 7.6 Hz, 1H), 7.15 (t, J = 7.7 Hz, 1H), 6.97 (d, J = 7.3 Hz, 1H),
6.82 (d, J = 8.0 Hz, 1H), 6.76 (t, J = 7.3 Hz, 1H), 3.97 (s, 2H), 3.51
(t, J = 7.9 Hz, 2H), 3.08 (s, 2H), 2.66 (dd, J = 16.0, 8.9 Hz, 4H), 1.91
(s, 4H), 1.72–1.60 (m, 2H), 1.50 (d, J = 6.4 Hz, 2H), 1.41–1.27 (m,
8H). 13C NMR (101 MHz, CDCl3) d 158.31, 157.71, 151.24, 146.65,
128.59, 128.54, 128.29, 127.85, 123.65, 123.07, 122.68, 119.79,
118.85, 116.22, 115.30, 52.60, 49.33, 48.62, 33.47, 31.65, 29.47,
29.25, 29.19, 26.97, 26.79, 24.69, 22.94, 22.59. Purity: 99.3% by
HPLC; LC/MS (ESI) m/z:[M+H]+ 432.2. HRMS m/z [M+H]+ Calcd for
(101 MHz, DMSO)
d 157.77, 157.16, 150.26, 146.79, 128.55,
128.11, 127.82, 127.77, 124.08, 123.14, 123.01, 120.10, 118.38,
115.67, 115.26, 50.26, 46.29, 45.99, 33.41, 30.17, 24.99, 22.68,
22.35. Purity: 99.8% by HPLC; LC/MS (ESI) m/z:[M+H]+ 362.2. HRMS
m/z [M+H]+ Calcd for C23H27N3O 362.2227, found 362.2216.
C28H37N3O 432.3009, found 432.2997.
4.2.7. 2-(((9-((1,2,3,4-Tetrahydroacridin-9-yl)amino)nonyl)
amino)methyl)phenol (6g)
4.2.3. 2-(((4-((1,2,3,4-Tetrahydroacridin-9-yl)amino)butyl)
amino)methyl)phenol (6c)
Salicylic aldehyde was treated with 3g according to general pro-
cedure to give the desired product as light yellow oil (65% yield).
(400 MHz, CDCl3) d 8.00–7.89 (m, 2H), 7.55 (t, J = 7.6 Hz, 1H), 7.34
1
Salicylic aldehyde was treated with 3c according to general pro-
cedure to give the desired product as light yellow oil (62% yield).
(t, J = 7.6 Hz, 1H), 7.15 (t, J = 7.7 Hz, 1H), 6.97 (d, J = 7.4 Hz, 1H),
6.81 (d, J = 8.1 Hz, 1H), 6.76 (t, J = 7.4 Hz, 1H), 3.97 (s, 2H), 3.54–
3.42 (m, 2H), 3.07 (s, 2H), 2.66 (dd, J = 16.2, 9.0 Hz, 4H), 1.91 (d,
J = 2.9 Hz, 4H), 1.64 (dd, J = 13.6, 6.6 Hz, 2H), 1.50 (d, J = 6.5 Hz,
2H), 1.28 (s, 10H). 13C NMR (101 MHz, CDCl3) d 158.35, 158.01,
151.05, 146.99, 128.54, 128.43, 128.22(2H), 123.60, 122.94,
122.68, 120.01, 118.85, 116.26, 115.52, 52.67, 49.40, 48.69, 33.68,
31.68, 29.52, 29.34, 29.26, 29.22, 27.05, 26.85, 24.73, 23.00,
22.67. Purity: 99.7% by HPLC; LC/MS (ESI) m/z:[M+H]+ 446.2. HRMS
m/z [M+H]+ Calcd for C29H39N3O 446.3166, found 446.3165.
1
(400 MHz, CDCl3) d 7.96 (dd, J = 16.1, 8.4 Hz, 2H), 7.57 (t, J = 7.7 Hz,
1H), 7.36 (t, J = 7.7 Hz, 1H), 7.17 (t, J = 7.8 Hz, 1H), 6.97 (d,
J = 7.2 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.77 (t, J = 7.4 Hz, 1H), 3.99
(s, 2H), 3.51 (d, J = 7.0 Hz, 2H), 3.10 (s, 2H), 2.80–2.56 (m, 4H),
1.92 (s, 4H), 1.73 (d, J = 7.0 Hz, 2H), 1.66 (d, J = 6.8 Hz, 2H). 13C
NMR (101 MHz, CDCl3) d 158.12, 157.66, 151.15, 146.35, 128.76,
128.69, 128.41, 127.67, 123.89, 122.93, 122.49, 119.80, 119.01,
116.26, 115.67, 52.51, 48.94, 48.19, 33.30, 29.21, 26.94, 24.75,
22.88, 22.50. Purity: 99.7% by HPLC; LC/MS (ESI) m/z:[M+H]+
376.2. HRMS m/z [M+H]+ Calcd for C24H29N3O 376.2383, found
376.2388.
4.2.8. 2-(((2-((6-chloro-1,2,3,4-Tetrahydroacridin-9-yl)
amino)ethyl)amino)methyl)phenol (6h)
4.2.4. 2-(((6-((1,2,3,4-Tetrahydroacridin-9-yl)amino)hexyl)
amino)methyl)phenol (6d)
Salicylic aldehyde was treated with 3h according to general
procedure to give the desired product as light yellow solid (62%
1
Salicylic aldehyde was treated with 3d according to general pro-
cedure to give the desired product as light yellow oil (76% yield).
yield). m.p. 151.3–152.6 °C;
(400 MHz, CDCl3) d 7.89 (dd,
J = 21.5, 5.5 Hz, 2H), 7.29 (dd, J = 9.1, 2.1 Hz, 1H), 7.24–7.16 (m,
1H), 7.02 (d, J = 7.2 Hz, 1H), 6.87 (d, J = 7.9 Hz, 1H), 6.81 (t,
J = 7.4 Hz, 1H), 4.06 (s, 2H), 3.63 (s, 2H), 3.04 (s, 2H), 2.98 (t,
J = 5.8 Hz, 2H), 2.68 (s, 2H), 1.96–1.85 (m, 4H). 13C NMR
1
(400 MHz, CDCl3) d 7.98 (t, J = 8.6 Hz, 2H), 7.56 (t, J = 7.6 Hz, 1H),
7.35 (dd, J = 8.2, 7.0 Hz, 1H), 7.15 (t, J = 7.7 Hz, 1H), 6.97 (d,
J = 7.4 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 6.76 (dd, J = 7.9, 6.8 Hz,
1H), 3.98 (s, 2H), 3.54 (t, J = 7.2 Hz, 2H), 3.10 (s, 2H), 2.72–2.61
(m, 4H), 1.91 (s, 4H), 1.74–1.61 (m, 2H), 1.59–1.47 (m, 2H), 1.39
(d, J = 2.1 Hz, 4H). 13C NMR (101 MHz, CDCl3) d 158.27, 157.01,
151.64, 145.80, 128.97, 128.57, 128.34, 127.05, 123.82, 123.21,
122.56, 119.39, 118.88, 116.21, 114.92, 52.55, 49.07, 48.41, 32.94,
31.48, 29.34, 26.79, 26.63, 24.65, 22.83, 22.38. Purity: 99.7% by
HPLC; LC/MS (ESI) m/z:[M+H]+ 404.2. HRMS m/z [M+H]+ Calcd for
(101 MHz, CDCl3)
d 159.08, 157.59, 150.73, 147.08, 134.50,
129.02, 128.71, 126.78, 124.75, 124.28, 122.40, 119.42, 118.22,
116.48, 116.36, 52.38, 49.36, 48.40, 33.42, 24.54, 22.75, 22.37. Pur-
ity: 98.9% by HPLC; LC/MS (ESI) m/z:[M+H]+ 382.3. HRMS m/z
[M+H]+ Calcd for C22H24ClN3O 382.1681, found 382.1680.
4.2.9. 2-(((3-((6-chloro-1,2,3,4-Tetrahydroacridin-9-yl)
amino)propyl)amino)methyl)phenol (6i)
C26H33N3O 404.2696, found 404.2686.
Salicylic aldehyde was treated with 3i according to general pro-
cedure to give the desired product as light yellow solid (63% yield).
m.p. 153.6–155.2 °C; 1 (400 MHz, CDCl3) d 7.86 (dd, J = 17.4, 5.5 Hz,
2H), 7.20 (dd, J = 9.1, 2.0 Hz, 1H), 7.18–7.13 (m, 1H), 6.98 (d,
J = 7.1 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.78 (t, J = 7.4 Hz, 1H), 3.99
4.2.5. 2-(((7-((1,2,3,4-Tetrahydroacridin-9-yl)amino)heptyl)
amino)methyl)phenol (6e)
Salicylic aldehyde was treated with 3e according to general pro-
cedure to give the desired product as light yellow oil (73% yield).

5890
F. Mao et al. / Bioorg. Med. Chem. 20 (2012) 5884–5892
(s, 2H), 3.61 (t, J = 6.8 Hz, 2H), 2.99 (d, J = 5.7 Hz, 2H), 2.78 (t,
J = 6.7 Hz, 2H), 2.58 (d, J = 5.5 Hz, 2H), 1.94–1.76 (m, 6H). 13C
NMR (101 MHz, DMSO) d 159.26, 157.02, 150.36, 147.56, 132.35,
128.64, 127.83, 126.61, 125.41, 123.95, 123.27, 118.45, 118.41,
115.76, 115.23, 50.03, 46.25, 45.92, 33.47, 29.99, 24.92, 22.52,
22.20. Purity: 99.8% by HPLC; LC/MS (ESI) m/z:[M+H]+ 396.3. HRMS
m/z [M+H]+ Calcd for C23H26ClN3O 396.1837, found 396.1831.
29.41, 29.22, 29.16, 26.95, 26.75, 24.47, 22.78, 22.38. Purity:
98.6% by HPLC; LC/MS (ESI) m/z:[M+H]+ 466.3. HRMS m/z [M+H]+
Calcd for C28H36ClN3O 466.2620, found 466.2615.
4.2.14. 2-(((9-((6-Chloro-1,2,3,4-tetrahydroacridin-9-
yl)amino)nonyl)amino)methyl)phenol (6n)
Salicylic aldehyde was treated with 3n according to general
procedure to give the desired product as light yellow oil (74%
1
4.2.10. 2-(((4-((6-Chloro-1,2,3,4-tetrahydroacridin-9-
yl)amino)butyl)amino)methyl)phenol (6j)
yield). (400 MHz, CDCl3) d 7.90 (dd, J = 5.5, 3.3 Hz, 2H), 7.29–
7.23 (m, 1H), 7.18–7.12 (m, 1H), 6.97 (d, J = 6.9 Hz, 1H), 6.82 (d,
J = 8.1 Hz, 1H), 6.76 (td, J = 7.4, 1.0 Hz, 1H), 3.98 (s, 2H), 3.49 (t,
J = 7.2 Hz, 2H), 3.03 (s, 2H), 2.66 (t, J = 7.0 Hz, 4H), 1.90 (t,
J = 3.1 Hz, 4H), 1.69–1.60 (m, 2H), 1.56–1.48 (m, 2H), 1.39–1.26
(m, 11H). 13C NMR (101 MHz, CDCl3) d 159.14, 158.28, 151.03,
147.73, 134.06, 128.62, 128.35, 127.05, 124.78, 124.12, 122.51,
118.88, 118.16, 116.26, 115.36, 52.52, 49.49, 48.61, 33.71, 31.71,
29.43, 29.35, 29.27, 29.21, 27.04, 26.82, 24.52, 22.87, 22.55. Purity:
99.8% by HPLC; LC/MS (ESI) m/z:[M+H]+ 480.3. HRMS m/z [M+H]+
Calcd for C29H38ClN3O 480.2776, found 480.2788.
Salicylic aldehyde was treated with 3j according to general pro-
cedure to give the desired product as light yellow oil (65% yield).
1
(400 MHz, CDCl3) d 7.87 (dd, J = 13.3, 5.5 Hz, 2H), 7.30–7.23 (m,
1H), 7.16 (dd, J = 11.1, 4.4 Hz, 1H), 6.97 (d, J = 7.0 Hz, 1H), 6.84–
6.73 (m, 2H), 3.98 (s, 2H), 3.47 (t, J = 7.0 Hz, 2H), 3.03 (s, 2H),
2.69 (dd, J = 15.7, 9.0 Hz, 4H), 1.97–1.84 (m, 4H), 1.76–1.67 (m,
2H), 1.63 (dd, J = 14.4, 7.7 Hz, 2H). 13C NMR (101 MHz, CDCl3) d
159.32, 158.08, 150.78, 147.67, 134.03, 128.67, 128.45, 127.07,
124.63, 124.27, 122.53, 119.02, 118.30, 116.20, 115.93, 52.48,
49.08, 48.17, 33.75, 29.26, 26.92, 24.59, 22.83, 22.51. Purity:
99.4% by HPLC; LC/MS (ESI) m/z:[M+H]+ 410.3. HRMS m/z [M+H]+
Calcd for C24H28ClN3O 410.1994, found 410.1992.
4.2.15. N1-(2-(Dimethylamino)benzyl)-N7-(1,2,3,4-
tetrahydroacridin-9-yl)heptane-1,7-diamine (7a)
Aromatic aldehyde 5a was treated with 3e according to general
procedure to give the desired product as light yellow oil (51%
yield). (400 MHz, CDCl3) d 8.02–7.89 (m, 2H), 7.54 (d, J = 6.8 Hz,
4.2.11. 2-(((6-((6-Chloro-1,2,3,4-tetrahydroacridin-9-
yl)amino)hexyl)amino)methyl)phenol (6k)
1
Salicylic aldehyde was treated with 3k according to general pro-
cedure to give the desired product as light yellow oil (58% yield).
(400 MHz, CDCl3) d 7.95–7.84 (m, 2H), 7.28 (d, J = 2.1 Hz, 1H), 7.16
1H), 7.43–7.28 (m, 2H), 7.25–7.18 (m, 1H), 7.12 (dd, J = 7.6,
4.0 Hz, 1H), 7.04 (dd, J = 11.1, 7.2 Hz, 1H), 3.88 (s, 2H), 3.49 (d,
J = 4.1 Hz, 2H), 3.07 (s, 2H), 2.69 (s, 6H), 2.61 (dt, J = 11.6, 5.9 Hz,
2H), 2.16 (s, 4H), 1.93 (d, J = 3.9 Hz, 4H), 1.66 (d, J = 4.7 Hz, 2H),
1.52–1.51 (m, 2H), 1.34–1.30 (m, 6H). 13C NMR (101 MHz, CDCl3)
d 158.00, 152.63, 150.98, 147.01, 133.61, 129.84, 128.37, 128.25,
127.90, 123.58, 123.51, 122.89, 120.02, 119.47, 115.56, 50.19,
49.34, 49.01, 44.93(2C), 33.69, 31.61, 29.52, 29.16, 27.13, 26.82,
24.73, 22.99, 22.66. Purity: 97.9% by HPLC; LC/MS (ESI) m/
z:[M+H]+ 445.4. HRMS m/z [M+H]+ Calcd for C29H40N4 445.3253,
found 445.3336.
1
(t, J = 7.1 Hz, 1H), 6.98 (d, J = 7.2 Hz, 1H), 6.86–6.73 (m, 2H), 3.98
(s, 2H), 3.49 (dd, J = 8.4, 6.0 Hz, 2H), 3.04 (s, 2H), 2.67 (t,
J = 6.9 Hz, 4H), 1.94–1.88 (m, 4H), 1.69–1.63 (m, 2H), 1.57–1.50
(m, 2H), 1.43–1.36 (m, 4H). 13C NMR (101 MHz, CDCl3) d 158.14,
157.79, 151.80, 146.03, 134.96, 128.81, 128.51, 125.56, 124.95,
124.54, 122.13, 119.00, 117.47, 116.34, 114.74, 52.32, 49.06,
48.27, 32.68, 31.42, 29.09, 26.70, 26.55, 24.50, 22.67, 22.15. Purity:
99.9% by HPLC; LC/MS (ESI) m/z:[M+H]+ 438.3. HRMS m/z [M+H]+
Calcd for C26H32ClN3O 438.2307, found 438.2318.
4.2.16. N1-(2-(Dimethylamino)benzyl)-N8-(1,2,3,4-
4.2.12. 2-(((7-((6-Chloro-1,2,3,4-tetrahydroacridin-9-
yl)amino)heptyl)amino)methyl)phenol (6l)
tetrahydroacridin-9-yl)octane-1,8-diamine (7b)
Aromatic aldehyde 5a was treated with 3f according to general
procedure to give the desired product as light yellow oil (57%
Salicylic aldehyde was treated with 3l according to general pro-
cedure to give the desired product as light yellow oil (50% yield).
1
yield). (400 MHz, CDCl3) d 8.01–7.89 (m, 2H), 7.54 (dd, J = 15.9,
1
(400 MHz, CDCl3) d 7.99 (s, 1H), 7.93 (d, J = 9.1 Hz, 1H), 7.29 (d,
8.0 Hz, 1H), 7.38–7.27 (m, 2H), 7.23 (d, J = 7.2 Hz, 1H), 7.13 (d,
J = 7.8 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H), 3.92 (s, 2H), 3.50 (t,
J = 7.1 Hz, 2H), 3.08 (s, 2H), 2.69 (s, 6H), 2.64–2.57 (m, 4H), 1.92
(s, 4H), 1.71–1.63 (m, 2H), 1.53–1.51 (m, 2H), 1.38–1.30 (m 8H).
13C NMR (101 MHz, CDCl3) d 157.92, 152.58, 151.00, 146.98,
133.65, 129.80, 128.37, 128.18, 127.85, 123.54, 123.46, 122.97,
119.95, 119.41, 115.46, 50.20, 49.35, 49.12, 44.93(2C), 33.69,
31.65, 29.62, 29.33, 29.24, 27.16, 26.82, 24.71, 22.97, 22.66. Purity:
98.5% by HPLC; LC/MS (ESI) m/z:[M+H]+ 459.4. HRMS m/z [M+H]+
Calcd for C30H42N4 459.3409, found 459.3493.
J = 1.8 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 6.98 (d, J = 7.3 Hz, 1H), 6.82
(d, J = 8.1 Hz, 1H), 6.77 (t, J = 7.4 Hz, 1H), 3.99 (s, 2H), 3.55 (t,
J = 7.1 Hz, 2H), 3.07 (s, 2H), 2.73–2.61 (m, 5H), 1.91 (s, 4H), 1.67
(dd, J = 14.2, 7.2 Hz, 2H), 1.57–1.48 (m, 2H), 1.37 (d, J = 17.6 Hz,
6H). 13C NMR (101 MHz, CDCl3) d 158.22, 158.17, 151.58, 146.57,
134.65, 128.71, 128.44, 126.00, 124.96, 124.37, 122.31, 118.94,
117.65, 116.26, 114.90, 52.45, 49.24, 48.44, 33.03, 31.49, 29.23,
28.99, 26.89, 26.70, 24.48, 22.73, 22.28. Purity: 98.1% by HPLC;
LC/MS (ESI) m/z:[M+H]+ 452.3. HRMS m/z [M+H]+ Calcd for
C
27H34ClN3O 452.2463, found 452.2478.
4.2.17. N1-(2-(Dimethylamino)benzyl)-N9-(1,2,3,4-
tetrahydroacridin-9-yl)nonane-1,9-diamine (7c)
Aromatic aldehyde 5a was treated with 3g according to general
4.2.13. 2-(((8-((6-Chloro-1,2,3,4-tetrahydroacridin-9-
yl)amino)octyl)amino)methyl)phenol (6m)
Salicylic aldehyde was treated with 3m according to general
procedure to give the desired product as light yellow oil (53%
yield). (400 MHz, CDCl3) d 7.90 (dd, J = 5.5, 3.3 Hz, 2H), 7.29–
7.23 (m, 1H), 7.18–7.12 (m, 1H), 6.97 (d, J = 6.9 Hz, 1H), 6.82 (d,
J = 8.1 Hz, 1H), 6.76 (td, J = 7.4, 1.0 Hz, 1H), 3.98 (s, 2H), 3.49 (t,
J = 7.2 Hz, 2H), 3.03 (s, 2H), 2.66 (t, J = 7.0 Hz, 4H), 1.90 (t,
J = 3.1 Hz, 4H), 1.69–1.60 (m, 2H), 1.56–1.48 (m, 2H), 1.40–1.27
(m, 10H). 13C NMR (101 MHz, CDCl3) d 158.57, 158.27, 151.40,
147.02, 134.50, 128.67, 128.33, 126.44, 124.85, 124.33, 122.44,
118.93, 117.84, 116.29, 115.07, 52.60, 49.41, 48.59, 33.29, 31.64,
procedure to give the desired product as light yellow oil (53%
1
yield).
(400 MHz, CDCl3) d 8.00–7.88 (m, 2H), 7.58–7.48 (m,
1
1H), 7.38–7.28 (m, 2H), 7.22 (d, J = 7.7 Hz, 1H), 7.12 (d, J = 7.9 Hz,
1H), 7.04 (t, J = 7.2 Hz, 1H), 3.90 (s, 2H), 3.49 (t, J = 7.1 Hz, 2H),
3.07 (s, 2H), 2.69 (s, 6H), 2.61 (t, J = 7.2 Hz, 4H), 1.92 (s, 4H),
1.69–1.60 (m, 2H), 1.531.51 (m, 2H), 1.38–1.28 (m, 10H). 13C
NMR (101 MHz, CDCl3) d 157.93, 152.63, 151.08, 146.91, 133.17,
129.95, 128.44, 128.18, 128.04, 123.63, 123.61, 122.92, 119.96,
119.55, 115.48, 50.12, 49.40, 48.93, 44.93(2C), 33.64, 31.68,
29.42, 29.37, 29.30, 29.23, 27.18, 26.85, 24.71, 22.99, 22.65. Purity:

F. Mao et al. / Bioorg. Med. Chem. 20 (2012) 5884–5892
5891
98.6% by HPLC; LC/MS (ESI) m/z:[M+H]+ 473.4. HRMS m/z [M+H]+
Calcd for C31H44N4 473.3566, found 473.3656.
was added to the assay solution and incubated with the enzyme at
37 °C for 15 min, then substrate was added. Kinetic characteriza-
tion of the hydrolysis of ATC catalyzed by AChE was performed
spectrophotometrically at 412 nm. A parallel control, with no
inhibitor in the mixture, allowed activities to be measured at var-
ious times. The plots were analyzed using a weighted least square
analysis that assumed the variance of V to be a constant percentage
of V for the entire data set. Slopes of these reciprocal plots were
then 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.
4.2.18. N1-(2-(Diethylamino)benzyl)-N8-(1,2,3,4-
tetrahydroacridin-9-yl)octane-1,8-diamine (7d)
Aromatic aldehyde 5b was treated with 3f according to general
procedure to give the desired product as light yellow oil (48%
1
yield). (400 MHz, CDCl3) d 7.96 (d, J = 8.1 Hz, 2H), 7.61–7.51 (m,
1H), 7.29 (s, 2H), 7.23 (d, J = 7.4 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H),
7.07 (t, J = 7.3 Hz, 1H), 3.91 (s, 2H), 3.55–3.44 (m, 2H), 3.09 (s,
2H), 2.98 (dd, J = 13.9, 6.8 Hz, 4H), 2.70 (s, 2H), 2.58 (t, J = 7.1 Hz,
2H), 1.92 (s, 4H), 1.71–1.62 (m, 2H), 1.54–1.51 (m, 2H), 1.35-1.31
(m, 8H), 1.00 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) d
157.87, 151.11, 149.73, 146.83, 135.73, 130.02, 128.47, 128.10,
127.64, 124.16, 123.62, 122.92, 122.75, 119.92, 115.44, 50.47,
49.37, 48.83, 48.08 (2C), 33.58, 31.65, 29.52, 29.30, 29.21, 27.18,
26.81, 24.70, 22.96, 22.62, 12.60 (2C). Purity: 97.6% by HPLC; LC/
MS (ESI) m/z:[M+H]+ 487.4. HRMS m/z [M+H]+ Calcd for
C32H46N4O 487.3795, found 487.3788.
4.3.3. The antioxidant activity assay
The antioxidant activity was determined by the oxygen radical
absorbance capacity-fluorescein (ORAC-FL) assay.24,25,32 All the as-
says were under 75 mM phosphate buffer (pH 7.4) and the final
reaction mixture was 200
(120 L, 300 nM final concentration) were placed in the wells of
a black 96-well plate and the mixture was incubated for 10 min
at 37 °C. Then, AAPH (Aldrich) solution (60 L; 12 mM final con-
lL. Antioxidant (20 lL) and fluorescein
l
l
4.2.19. N1-(Pyridin-2-ylmethyl)-N8-(1,2,3,4-tetrahydroacridin-
9-yl)octane-1,8-diamine (7e)
centration) was added rapidly. The plate was immediately placed
into a Spectrafluor Plus plate reader (Tecan, Crailsheim, Germany)
and the fluorescence was measured every 60 s for 4 h with exita-
tion at 485 nm and emission at 535 nm. Trolox was used as stan-
Pyridine-2-carboxaldehyde was treated with 3f according to
general procedure to give the desired product as light yellow oil
1
(42% yield). (400 MHz, CDCl3) d 8.55 (d, J = 4.7 Hz, 1H), 7.97 (d,
dard (1–10 lM, final concentration). A blank (FL + AAPH) using
J = 8.6 Hz, 2H), 7.63 (td, J = 7.7, 1.7 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H),
7.35 (t, J = 7.7 Hz, 1H), 7.29 (d, J = 7.8 Hz, 1H), 7.20–7.11 (m, 1H),
3.90 (s, 2H), 3.52 (t, J = 7.1 Hz, 2H), 3.09 (s, 2H), 2.70 (s, 2H), 2.64
(t, J = 7.2 Hz, 2H), 1.92 (d, J = 3.0 Hz, 4H), 1.66 (dd, J = 14.5, 7.4 Hz,
2H), 1.52 (d, J = 6.9 Hz, 2H), 1.41–1.30 (m, 8H). 13C NMR
phosphate buffer instead of antioxidant and Trolox calibration
were carried out in each assay. The samples were measured at dif-
ferent concentrations (0.5–10 lM). All reaction mixtures were pre-
pared fourfold and at least four independent runs were performed
for each sample. Fluorescence measurements were normalized to
the curve of the blank (without antioxidant). The ORAC-FL values
were calculated as described in the reference31 and the final results
(101 MHz, CDCl3)
d 159.72, 157.72, 151.19, 149.22, 146.66,
136.44, 128.57, 127.93, 123.64, 122.99, 122.31, 121.91, 119.78,
115.30, 55.24, 49.61, 49.39, 33.48, 31.67, 30.01, 29.37, 29.23,
27.18, 26.82, 24.65, 22.92, 22.57. Purity: 99.7% by HPLC; LC/MS
(ESI) m/z:[M+H]+ 431.3. HRMS m/z [M+H]+ Calcd for C27H36N4O
417.3013, found 417.3013.
were in lM of Trolox equivalent/lM of pure compound.
4.3.4. Inhibition of Ab1–42 peptide aggregation33
HFIP pretreated Ab1–42 samples (AnaSpec) were resolubilized
with a 50 mM phosphate buffer (pH = 7.4) in order to have a stable
4.3. Biological activity
stock solution ([Ab] = 200
50 mM phosphate buffer (pH = 7.4) at 37 °C for 48 h (final Ab con-
centration 50 M) with and without the tested compound at
20 M. After incubation, the samples were diluted to a final vol-
ume of 200 L with 50 mM glycine-NaOH buffer (pH 8.0) contain-
ing thioflavin T. Then, a 300-seconds-time scan of fluorescence
intensity was performed (kexc = 450 nm; kem = 485 nm), and values
at plateau were averaged after subtracting the background fluores-
cence of thioflavin T solution.
lM). The peptide was incubated in
4.3.1. In vitro inhibition of AChE and BuChE
l
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from the electric eel),
butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum),
5,50-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), acet-
ylthiocholine chloride (ATC), and butylthiocholine chloride (BTC)
were purchased from Sigma Aldrich. Tested compounds were dis-
solved in DMSO and then diluted in 0.1 M KH2PO4/K2HPO4 buffer
(pH 8.0) to the final concentration.
l
l
All the in vitro AChE assays were carried out in 0.1 M KH2PO4/
K2HPO4 buffer, pH 8.0, using a Shimadzu UV-2450 Spectrophotom-
eter. AChE and BuChE solutions were prepared to give 2.0 units/ml
in 2 ml aliquots. The assay medium (1 ml) consisted of phosphate
4.3.5. Metal-chelating properties of compound 6f and 7c
The complexing studies were made in ethanol at 298 K using a
UV–vis spectrophotometer (SHIMADZC UV-2450PC) with wave-
length ranging from 200 to 500 nm. CuSO4, FeSO4 or ZnCl2 solution
buffer (pH 8.0), 50
l
l of 0.01 M DTNB, 10
l
l of enzyme, and 50
l
l
were prepared in 200
tested compounds were dissolved in DMSO and then diluted with
ethanol to 400 M concentration. To a mixture of 100 L tested
compound solution and 700 L ethanol, 200 L CuSO4 solution
(FeSO4, or ZnCl2) was added. The solution was incubated at 298 K
for 30 min and then the absorption spectra were recorded at
298 K in a 1 cm quartz cell. The control was prepared by mixting
lM using ethanol using volumetric flask. The
of 0.01 M substrate (ACh chloride solution). Test compounds were
added to the assay solution and preincubated at 37 °C with the en-
zyme for 15 min, followed by the addition of substrate. Activity
was determined by measuring the increase in absorbance at
412 nm at 1 min intervals at 37 °C. Calculations were performed
according to the method of Ellman et al23 Each concentration
was assayed in triplicate.
l
l
l
l
100
lL tested compound solution and 900
lL ethanol. The blank
The in vitro BuChE assay (BuChE or ACh as the enzyme sub-
strate) was performed according to a method similar to that de-
scribed above.
was 4
l
L DMSO in 996 L ethanol. All absorption spectra sub-
l
tracted the blank absorption spectra.
Acknowledgment
4.3.2. Kinetic characterization of AChE inhibition
The kinetic characterization of AChE inhibitory activity was per-
We thank the Natural Science Foundation of China (20972198
and 81274028) for financial support of this study.
formed according to a published protocol.31 Briefly, test compound

5892
F. Mao et al. / Bioorg. Med. Chem. 20 (2012) 5884–5892
17. Wang, Y.; Guan, X.-L.; Wu, P.-F.; Wang, C.-M.; Cao, H.; Li, L.; Guo, X.-J.; Wang,
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