Tacrine-flurbiprofen hybrids as multifunctional drug candidates for the treatment of alzheimer's disease

Article abstract of DOI:10.1002/ardp.201300074

Five tacrine-flurbiprofen hybrid compounds (3a-e) were synthesized as multi-target-directed compounds for the treatment of Alzheimer's disease. Compared to tacrine, two compounds (3d and 3e) showed better acetylcholinesterase (AChE) inhibitory activity and others (3b-e) better or the same butyrylcholinesterase (BuChE) inhibitory activity. Notably, 3d showed a mixed-type inhibitory action for AChE, indicating a dual-binding site action of both toward the catalytic active site (CAS) and the peripheral anionic site (PAS), whereas for BuChE, a competitive inhibitory action was observed. Furthermore, a cell-based assay on amyloid-β inhibition demonstrated that the selected target compound 3d effectively inhibits the formation of amyloid-β in vitro. Tacrine-flurbiprofen hybrid compound 3d inhibits acetylcholinesterase by a dual-binding site action both toward the catalytic active site and the peripheral anionic site. Furthermore, it inhibits the formation of amyloid-β in vitro.

Full text of DOI:10.1002/ardp.201300074

Arch. Pharm. Chem. Life Sci. 2013, 346, 865–871
865
Full Paper
Tacrine–Flurbiprofen Hybrids as Multifunctional Drug
Candidates for the Treatment of Alzheimer’s Disease
Yao Chen1, 2, 3, Jianfei Sun4, Sixun Peng1, 2, Hong Liao4, Yihua Zhang1, 2, and Jochen Lehmann3
1
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P. R. China
Center of Drug Discovery, China Pharmaceutical University, Nanjing, P. R. China
Lehrstuhl für Pharmazeutische/Medizinische Chemie, Institut für Pharmazie, Friedrich-Schiller-Universität
2
3
Jena, Jena, Germany
4
Neurobiology Lab, New Drug Screening Center, China Pharmaceutical University, Nanjing, P. R. China
Five tacrine–flurbiprofen hybrid compounds (3a–e) were synthesized as multi-target-directed
compounds for the treatment of Alzheimer’s disease. Compared to tacrine, two compounds (3d
and 3e) showed better acetylcholinesterase (AChE) inhibitory activity and others (3b–e) better or the
same butyrylcholinesterase (BuChE) inhibitory activity. Notably, 3d showed a mixed-type inhibitory
action for AChE, indicating a “dual-binding site action” of both toward the catalytic active site (CAS) and
the peripheral anionic site (PAS), whereas for BuChE, a competitive inhibitory action was observed.
Furthermore, a cell-based assay on amyloid-b inhibition demonstrated that the selected target
compound 3d effectively inhibits the formation of amyloid-b in vitro.
Keywords: Alzheimer’s disease / Amyloid-b / Cholinesterase inhibitor / Flurbiprofen / Tacrine
Received: February 27, 2013; Revised: August 14, 2013; Accepted: August 15, 2013
DOI 10.1002/ardp.201300074
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
In addition to the cholinergic strategy, the Ab hypothesis
has been widely accepted [5]. This asserts that the accumula-
tion and aggregation of Ab, 40/42 amino acid peptides, which
are generated by the “amyloidogenic” proteolysis of the
b-amyloid precursor protein (APP), catalyzed by b- and
g-secretase, is the trigger for progression of AD [6]. As a
consequence, inhibiting the generation of Ab could be
beneficial for the treatment of AD. Flurbiprofen (Fig. 1) has
been used as a non-steroidal anti-inflammatory drug (NSAID)
and was then identified with the ability to lower Ab40/42
peptide production by an allosteric modulation of presenilin-1
(PS-1), the major component of the g-secretase complex [7].
A strategy named multi-target-directed ligands (MTDLs) has
been widely applied in the research of novel anti-AD drugs,
due to the limited and transient benefits of the traditional
approach of single-target-molecule [8]. In this strategy, hybrid
compounds composed of two distinct parts with different
biological activities have been proved to exhibit multifunc-
tional activities [9, 10].
Alzheimer’s disease (AD), the most common form of
dementia, is characterized by loss of memory and progressive
cognitive impairment [1]. Many factors are involved in the
complicated pathogenesis, such as cholinergic dysfunction,
deposits of amyloid-b (Ab) and t-protein, oxidative stress, and
poor blood supply in the brain [2]. To date, the cholinergic
strategy is the most common and effective therapeutic
approach for the treatment of AD. It asserts that cholinester-
ase (ChE) inhibitors, aiming to increase the acetylcholine
(ACh) level in the brain, can reduce the cognitive deficit in
AD [3]. Therefore, acetylcholinesterase (AChE) and butyryl-
cholinesterase (BuChE), two types of enzymes responsible for
hydrolyzing ACh, are therapeutic targets [4], and inhibitors of
both, such as tacrine (Fig. 1) and rivastigmine, have been
clinically applied.
Correspondence: Prof. Jochen Lehmann, Lehrstuhl für Pharmazeuti-
sche/Medizinische Chemie, Institut für Pharmazie, Friedrich-Schiller-
Universität Jena, Philosophenweg 14, D-07743 Jena, Germany.
E-mail: j.lehmann@uni-jena.de
Additional correspondence: Hong Liao, E-mail: hliao@cup.edu.en;
Prof. Yihua Zhang, E-mail: zyhtgd@hotmail.com
Fax: þ49 3641949802
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Arch. Pharm. Chem. Life Sci. 2013, 346, 865–871
Table 1. Inhibition of AChE and BuChE (IC50 values) and
selectivity expressed as the ratio of the resulting IC50 values.
IC50 (nM) ꢀ SEMa)
Selectivity
Compd.
AChE
BChE
ratiob)
Tacrine
3a
3b
3c
3d
61.7 ꢀ 14.9
714.9 ꢀ 251.7
344.8 ꢀ 45.5
193.1 ꢀ 8.5
19.3 ꢀ 3.4
9.0 ꢀ 1.8
58.7 ꢀ 7.3
13.9 ꢀ 3.4
11.0 ꢀ 1.6
3.7 ꢀ 0.4
2.1 ꢀ 0.3
6.9
12.2
24.8
17.6
5.2
Figure 1. Structures of tacrine and flurbiprofen.
3e
34.5 ꢀ 3.3
16.4
a)
In this study, a series of tacrine–flurbiprofen hybrids (3a–e),
in which the tacrine-like heterocycle was connected to
racemic flurbiprofen via alkylenediamine linkers, were
designed and synthesized, in order to achieve potential
multi-potent anti-AD drugs.
Data are the mean of at least three determinations.
Selectivity ratio ¼ (IC50 of AChE)/(IC50 of BuChE).
b)
BuChE from equine serum, following Ellman’s method [12]
(Table 1). Among them, 3d and 3e showed better inhibitory
activity for AChE than tacrine. For BuChE, all compounds
exhibited the same or better inhibitory activity compared to
tacrine with the exception of 3a. Interestingly, the results
revealed that the length of the diamine side chain might
influence the ability to inhibit ChE. Compounds 3d and 3e
showed high activity toward both AChE and BuChE (IC50 of
AChE ¼ 19.3 and 34.5 nM; IC50 of BuChE ¼ 3.7 and 2.1 nM),
which suggests the optimal spacer length to be six atoms.
Notably, compound 3b showed much higher activity toward
BuChE (IC50 ¼ 13.9 nM) than AChE (IC50 ¼ 344.8 nM), demon-
Results and discussion
The intermediate 9-chloro-1,2,3,4-tetrahydroacridine 1 was
synthesized according to a previously reported protocol [11]
using anthranilic acid and cyclohexanone as starting
materials. Then different alkylenediamines were introduced
to 1 to give the 9-aminoalkylamino-1,2,3,4-tetrahydroacri-
dines 2a–e, which were finally connected to racemic
flurbiprofen in the presence of DCC/DMAP, yielding the
target compounds 3a–e (Scheme 1).
Target compounds 3a–e were evaluated in vitro as inhibitors
of the AChE from Electrophorus electricus (eeAChE) and of the
strating 3b as
ratio ¼ 24.8).
a selective BuChE inhibitor (selectivity
Scheme 1. General method for the synthesis of 3a–e. Reagents: (a) POCl3 reflux, 3 h; (b) pentanol NH2(CH2)mNH2, reflux, 18 h; and
(c) DCC, DMAP, anhydrous CH2Cl2 room temp., 24 h.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 865–871
Tacrine–Flurbiprofen Hybrids
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Lineweaver-Burk plot
AChE inhibition
by compound 3d
Lineweaver-Burk plot
BuChE inhibition
by compound 3d
30
20
10
90
80
70
60
50
40
30
20
10
[3d] = 10 nM
[3d] = 10 nM
[3d] = 4 nM
[3d] = 2 nM
[3d] = 1 nM
[3d] = 0 nM
[3d] = 4 nM
[3d] = 2 nM
[3d] = 1 nM
[3d] = 0 nM
-0.03 -0.02
0.01
0.02
0.03
0.04
0.05
-10
-20
-30
-40
-0.03
-0.02
0.01
0.02
_
0.03
0.04
0.05
_
1
µ
M
1/[S] in
1
µ
1/[S] in
M
-10
Figure 2. Lineweaver–Burk plots resulting from sub-velocity curves
of AChE activity with different substrate concentrations (25–450 mM)
in the absence and presence of 1, 2, 4, and 10 nM 3d.
Figure 3. Lineweaver–Burk plots resulting from sub-velocity curves
of BuChE activity with different substrate concentrations (25–
450 mM) in the absence and presence of 1, 2, 4, and 10 nM 3d.
Based on the good performance on both AChE and BuChE,
3d was selected for kinetic measurements to study the
binding mode of the novel compound. The mechanism was
analyzed by Lineweaver–Burk reciprocal plots, which were
reciprocal rates versus reciprocal substrate concentrations for
different concentrations of 3d. For AChE, the plot showed
both increased slopes (decreased Vmax) and intercepts (higher
Km) at increasing concentration of 3d (Fig. 2), indicating a
mixed-type inhibition. This demonstrates that the compound
not only competes for the same catalytic active site (CAS) as
the substrate ACh does, but also interacts with a second
binding site. In contrast, a different plot for BuChE was
observed, with different Km and constant Vmax at different
concentrations of 3d (Fig. 3). This suggested a competitive
inhibition, indicating that this compound at this target
competes orthosterically for the same binding site (CAS) as the
substrate ACh does.
In order to further explore the binding mode of 3d with
AChE, a molecular modeling study using CDOCKER within
Discovery Studio (DS, Accelrys) was performed (Fig. 4). As can
be seen from the result, the binding conformation suggested
that 3d can cover the binding gorge in a satisfied orientation
and mode, thus leading to the high inhibitory potency. The
binding model also indicated that the tacrine fragment of 3d
was bound to near the bottom of the gorge (CAS) through
strong parallel p–p stacking against the indole ring of Trp86.
At the mouth of the gorge, the benzene ring of flurbiprofen
group in 3d showed hydrophobic interaction with the
residues Tyr286, a key residue in the peripheral anionic site
ꢁ
(PAS) [13], which is around 18 A away from CAS and has been
Figure 4. Representation of the binding mode of compound 3d (purple) with AChE (PDB id: 2X8B).
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Arch. Pharm. Chem. Life Sci. 2013, 346, 865–871
proved to have close relation to the neurotoxic cascade of AD
initiated by AChE-induced Ab aggregation [14]. According to
all the results, we postulated that compound 3d might be a
dual-binding site inhibitor of both PAS and CAS.
3d was chosen for the cell-based Ab inhibition assay
and showed significant potency in inhibiting the formation
of Ab40. In recent years, several authors have postulated that
neither the presence of monomeric nor polymeric but
rather the accumulation of oligomeric b-amyloid causes
the pathogenesis of Alzheimer disease. Nevertheless, in this
study we looked at the inhibition of b-amyloid production
because a lower level of the monomeric should also
consequently reduce the quantity of oligomeric and polymer-
ic aggregations of b-amyloid.
Before performing further pharmacology, toxicity was
determined by using
a
3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl-tetrazolium bromide (MTT) assay, and the result
demonstrated that there was no significant toxicity when the
concentration of 3d was <1 mM, which is beyond the
concentration needed for significant effects in inhibiting
CHEs and reducing the formation of Ab40
.
All the results characterized the novel tacrine–flurbiprofen
hybrids as multi-potent anti-AD drug candidates, and 3d
might be a promising lead compound for further research.
To elucidate the potency of the compounds in inhibiting
the formation of Ab in vitro, the cell-based Ab40 reducing
activity of compound 3d was assessed by an enzyme-linked
immune sandwich assay (ELISA) [15]. As can be seen from the
result (Fig. 5), 3d showed a significant 31% reduction of Ab40
formation at the concentration 0.25 mM, demonstrating that
the target hybrid compound can inhibit the generation of Ab,
probably by inhibition of g-secretase. However, reducing the
production of b-amyloid affords much more higher concen-
tration than inhibiting ChE activity. Flurbiprofen and tacrine
did not show any Ab reduction at this concentration.
In conclusion, we have designed and synthesized five novel
tacrine–flurbiprofen hybrids 3a–e. Two of the compounds (3d
and 3e) inhibit AChE more potently than tacrine and all
compounds (except 3a) exhibited better or the same BuChE
inhibitory activity compared to tacrine. In addition, 3c
was found to be a significantly selective BuChE inhibitor. The
inhibitory mechanism of 3d was analyzed by Lineweaver–
Burk reciprocal plots and docking and was recognized as a
dual site (CAS and PAS) binding mode for AChE, whereas a
competitive inhibition is performed for BuChE. Additionally,
Experimental
Chemistry
General methods
Melting points are uncorrected and were measured in open
capillary tubes, using a Gallenkamp melting point apparatus. 1H
and 13C NMR spectral data were obtained from a Bruker Advance
250 spectrometer (250 MHz). HRMS data were recorded using
Agilent technologies LC/MSD TOF. TLC was performed on silica gel
F254 plates (Merck). Column chromatography was carried out
with silica gel 60, 63–200 mm (Baker). Elemental analyses were
performed on a Heraeus Vario EL III apparatus (Firma Elementar
Analysensysteme GmbH, Germany).
9-Aminoalkylamino-1,2,3,4-tetrahydroacridines
(2a–e) – General procedure I
To a solution of 1 (9-chlorotetrahydroacridine) in 10 mL of
pentanol 3 molar equiv. of diaminolkane were added. After
refluxing for 18 h under nitrogen, the solution was cooled to 0°C
and then acidified to pH 2 with HCl/ether solution. The deposit
was separated and dissolved in 10 mL of water; then the solution
was basified to pH 10 with a saturated aqueous solution of
Na2CO3 and extracted with CH2Cl2 (3 ꢂ 10 mL). The combined
organic layers were washed with brine, dried over anhydrous
Na2SO4, and evaporated in vacuo. The residue was purified by
column chromatography (CH2Cl2/MeOH ¼ 7:3 v/v, plus 5 mL
triethylamine per 1000 mL).
9-(2-Aminoethylamino)-1,2,3,4-tetrahydroacridine (2a)
Following general procedure I, using 1 (2.17 g, 10 mmol) and 1,2-
diaminoethane (2.00 mL, 30 mmol). Brownish oil (1.45 g, 60%).
1H NMR (CDCl3): d 8.02–7.99 (d, 1H, arom), 7.93–7.89 (d, 1H, arom),
7.58–7.51 (t, 1H, arom), 7.37–7.31 (t, 1H, arom), 4.83 (br, 1H,
NHCH2), 3.50–3.48 (t, 2H, NHCH2), 3.06–3.03 (t, 2H, C4-H2), 2.98–
2.93 (m, 2H, CH2NH2), 2.80–2.76 (t, 2H, C1-H2), 1.94–1.89 (m, 4H,
C3-H2, C2-H2). HRMS (ESI) m/z calcd. for C15H19N3 [Mþ] 241.1579;
found 241.1579.
9-(3-Aminopropylamino)-1,2,3,4-tetrahydroacridine (2b)
From 1 (2.17 g, 10 mmol) and 1,3-diaminopropane (2.49 mL,
30 mmol). Brownish oil (1.37 g, 54%). 1H NMR (CDCl3) d 7.98–7.95
(d, 1H, arom), 7.89–7.86 (m, 1H, arom), 7.54–7.48 (t, 1H, arom),
7.33–7.27 (t, 1H, arom), 3.59–3.53 (t, 2H, NHCH2), 3.03 (br, 2H,
Figure 5. Ab40 reduction of 3d, tacrine, and flurbiprofen (0.25 mM)
in the cell-based assay. A known g-secretase inhibitor, {1S-benzyl-
4R-[1-(1S-carbamoyl-2-phenyl-ethyl-carbamoyl)-1S-3-methylbutyl-
carbamoyl]-2R-hydrox-5-phenylpentyl}carbamic acid tert-butyl es-
ter (L-685458), was used as a positive control.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 865–871
Tacrine–Flurbiprofen Hybrids
869
0
0
0
C4-H2), 2.88–2.83 (t, 2H, CH2NH2), 2.69 (br, 2H, C1-H2), 1.89–1.87
(m, 4H, C3-H2, C2-H2), 1.83–1.71 (m, 2H, CH2CH2NH2). HRMS (ESI)
m/z calcd. for C16H21N3 [Mþ] 255.1735; found 255.1737.
(arom, C1 ), 131.06, 130.99 (arom, C5), 128.87 (arom, C6 , C2 ),
0
0
128.81 (arom), 128.55 (arom), 128.47 (arom, C3 , C5 ), 128.00,
0
127.75 (arom, C4), 127.78 (arom, C4 ), 123.88 (arom), 123.58,
123.53 (arom, C6), 122.58 (arom), 119.82 (arom), 116.07 (arom),
115.45, 115.07 (arom, C2), 49.42 (NHCH2), 46.40, 46.39 (CHCH3),
40.72 (CH2NHCO), 33.59 (C4), 24.98 (C1), 22.91 (C3), 22.58 (C2),
18.58 (CHCH3). Anal. calcd. for (C30H30FN3O · 1/2H2O): found C,
75.5; H, 6.52; N, 8.8; requires C, 75.6; H, 6.56; N, 8.8.
9-(4-Aminobutylamino)-1,2,3,4-tetrahydroacridine (2c)
From
1 (2.17 g, 10 mmol) and 1,4-diaminobutane (3.01 mL,
30 mmol). Brownish oil (1.35 g, 50%). 1H NMR (CDCl3) d 7.96–
7.88 (m, 2H, arom), 7.57–7.51 (t, 1H, arom), 7.36–7.30 (t, 1H,
arom), 3.52–3.46 (t, 2H, NHCH2), 3.05 (br, 2H, C4-H2), 2.75–2.70 (m,
4H, CH2NH2, C1-H2), 1.92–1.89 (m, 4H, C3-H2, C2-H2), 1.76–1.64 (m,
2H, CH2CH2NH2), 1.59–1.50 (m, 2H, NHCH2CH2). HRMS (ESI) m/z
calcd. for C17H23N3 [Mþ] 269.1892; found 269.1892.
2-(2-Fluorobiphenyl-4-yl)-N-[3-(1,2,3,4-tetrahydroacridin-
9-ylamino)propyl]propanamide (3b)
Compound 2b (0.26 g, 1 mmol) reacted with flurbiprofen (0.24 g,
1 mmol) to furnish 3b as a yellow solid (0.21 g, 43%); m.p. 67–
69°C. 1H NMR (CDCl3): d 7.97–7.87 (m, 2H, arom), 7.55–7.29
(m, 8H, arom), 7.13–7.08 (m, 2H, arom), 6.22–6.18 (t, 1H, NHCO),
4.63 (br, 1H, NH), 3.64–3.55 (q, 1H, CHCH3), 3.42–3.40 (m, 4H,
NHCH2CH2CH2), 3.02–2.98 (m, 2H, C4-H2), 2.71 (br, 2H, C1-H2),
1.86 (br, 4H, C3-H2, C2-H2), 1.80–1.70 (m, 2H, NHCH2CH2), 1.57–
1.54 (d, 3H, CHCH3); 13C NMR (CDCl3): d 174.34 (NHCO), 161.73,
9-(5-Aminopentylamino)-1,2,3,4-tetrahydroacridine (2d)
From
1 (2.17 g, 10 mmol) and 1,6-diaminohexane (3.48 g,
30 mmol). Brownish oil (1.97 g, 66%). 1H NMR (CDCl3): d 7.97–
7.89 (m, 2H, arom), 7.58–7.51 (m, 1H, arom), 7.37–7.30 (m, 1H,
arom), 3.97 (br, 1H, NHCH2), 3.51–3.45 (m, 2H, NHCH2), 3.06 (br,
2H, C4-H2), 2.70–2.64 (m, 4H, CH2NH2, C1-H2), 1.94–1.90 (m, 4H,
C3-H2, C2-H2), 1.72–1.61 (m, 2H, NHCH2CH2), 1.47–1.37 (m, 6H,
CH2CH2CH2CH2NH2). HRMS (ESI) m/z calcd. for C19H27N3 [Mþ]
297.2205; found 297.2203.
157.77 (arom, C3), 158.44 (arom), 150.63 (arom), 147.08 (arom),
0
142.68, 142.56 (arom, C1), 135.020 (arom, C1 ), 131.03, 130.96
0
(arom,0 C5), 128.84 (arom, C6 , C2 ), 128.79 (arom), 128.45 (arom,
0
0
C3 , C5 ), 128.40 (arom), 127.97, 127.75 (arom, C4, C4 ), 123.93
(arom), 123.60, 123.54 (arom, C6), 122.56 (arom), 120.41 (arom),
116.82 (arom), 115.46, 115.09 (arom, C2), 46.59 (CHCH3), 45.69
(NHCH2), 37.07 (CH2NHCO), 33.82 (C4), 31.26 (NHCH2CH2), 24.99
(C1), 22.98 (C3), 22.68 (C2), 18.57 (CHCH3). Anal. calcd. for
(C31H32FN3O · 1/2H2O): found C, 75.6; H, 6.63; N, 8.7; requires C,
75.9; H, 6.78; N, 8.6.
9-(8-Aminooctylamino)-1,2,3,4-tetrahydroacridine (2e)
From
1 (2.17 g, 10 mmol) and 1,8-diaminooctane (4.32 g,
30 mmol). Brownish oil (1.52 g, 48%). 1H NMR (CDCl3): d 7.95–
7.86 (m, 2H, arom), 7.55–7.49 (m, 1H, arom), 7.34–7.28 (m, 1H,
arom), 3.92 (br, 1H, NHCH2), 3.47–3.42 (t, 2H, NHCH2), 3.04
(br, 2H, C4-H2), 2.68–2.61 (m, 4H, CH2NH2, C1-H2), 1.94–1.88 (m,
4H, C3-H2, C2-H2), 1.65–1.57 (m, 2H, NHCH2CH2), 1.45–1.28 (m,
10H, CH2CH2CH2CH2CH2CH2NH2). HRMS (ESI) m/z calcd. for
C21H31N3 [Mþ] 325.2518; found 325.2518.
2-(2-Fluorobiphenyl-4-yl)-N-[4-(1,2,3,4-tetrahydroacridin-
9-ylamino)butyl]propanamide (3c)
Compound 2c (0.27 g, 1 mmol) reacted with flurbiprofen (0.24 g,
1 mmol) to furnish 3c as a yellow solid (0.23 g, 46%); m.p. 63–65°C.
1H NMR (CDCl3): d 7.94–7.91 (d, 2H, arom), 7.55–7.31 (m, 8H,
arom), 7.14–7.09 (m, 2H, arom), 6.22–6.18 (t, 1H, NHCO), 4.31 (br,
1H, NH), 3.64–3.49 (m, 3H, CHCH3, NHCH2), 3.30–3.23 (m, 2H,
CH2NHCO), 3.02 (br, 2H, C4-H2), 2.63 (br, 2H, C1-H2), 1.85 (br, 4H,
C3-H2, C2-H2), 1.66–1.49 (m, 7H, NHCH2CH2CH2, CHCH3); 13C NMR
(CDCl3): d 173.76 (NHCO), 161.69, 157.42 (arom, C3), 157.74 (arom),
151.20 (arom), 146.13 (arom), 143.04, 142.92 (arom, C1), 135.31
2-(2-Fluorobiphenyl-4-yl)-N-[(1,2,3,4-tetrahydroacridin-
9-ylamino)alkyl]propanamides (3a–e) – General
procedure II
Flurbiprofen and an equiv. molar amount of N,N0-dicyclohex-
ylcarbodiimide (DCC) were dissolved in 20 mL of dry CH2Cl2
and the mixture was stirred at room temperature for 0.5 h. Then a
solution of an equiv. molar amount of 2 in 5 mL of dry CH2Cl2 was
added, followed by a catalytic amount of 4-dimethylaminopyr-
idine (DMAP). After being stirred for 24 h at room temperature,
the reaction was quenched by adding 10 mL of H2O. Then the
resulting mixture was filtrated and the filtrate was extracted with
CH2Cl2 (3 ꢂ 15 mL). The combined organic phases were washed
with brine, dried over anhydrous Na2SO4, and evaporated
in vacuo. The residue was purified by column chromatography
(CH2Cl2/MeOH ¼ 10:1 v/v).
0
0
0
(arom, C1 ), 130.93, 130.87 (arom, C5), 128.86 (arom, C6 , C2 ),
0
0
128.81 (arom), 128.45 (arom, C3 , C5 ), 127.81, 127.60 (arom, C4),
0
127.71 (arom), 127.39, (arom, C4 ), 123.92 (arom), 123.60, 123.54
(arom, C6), 122.94 (arom), 119.63 (arom), 115.49 (arom), 115.42,
115.04 (arom, C2), 48.66 (NHCH2), 46.43 (CHCH3), 39.16
(CH2NHCO), 33.17 (C4), 28.73 (CH2CH2NHCO), 26.99 (C1), 24.67
(NHCH2CH2), 22.81 (C3), 22.41 (C2), 18.56 (CHCH3). Anal. calcd. for
(C32H34FN3O · H2O): found C, 74.8; H, 6.87; N, 8.0; requires C, 74.8;
H, 7.06; N, 8.2.
2-(2-Fluorobiphenyl-4-yl)-N-[2-(1,2,3,4-tetrahydroacridin-
9-ylamino)ethyl]propanamide (3a)
2-(2-Fluorobiphenyl-4-yl)-N-[6-(1,2,3,4-tetrahydroacridin-
9-ylamino)hexyl]propanamide (3d)
Following general procedure I, compound 2a (0.24 g, 1 mmol)
reacted with flurbiprofen (0.24 g, 1 mmol) to furnish 3a as a
yellow solid (0.19 g, 41%); m.p. 76–78°C. 1H NMR (CDCl3): d 7.89–
7.84 (m, 2H, arom), 7.50–7.23 (m, 8H, arom), 7.10–7.06 (d, 2H,
arom), 6.68–6.64 (t, 1H, NHCO), 4.74 (br, 1H, NH), 3.63–3.52 (m,
5H, NHCH2CH2, CHCH3), 2.98 (br, 2H, C4-H2), 2.61 (br, 2H, C1-H2),
1.81 (br, 4H, C3-H2, C2-H2), 1.53–1.50 (d, 3H, CHCH3); 13C NMR
(CDCl3): d 175.00 (NHCO), 161.70, 157.74 (arom, C3), 158.15 (arom),
150.65 (arom), 146.80 (arom), 142.61, 142.48 (arom, C1), 135.24
Compound 2d (0.30 g, 1 mmol) reacted with flurbiprofen (0.24 g,
1 mmol) to furnish 3d as a yellow solid (0.23 g, 44%); m.p. 57–
59°C. 1H NMR (CDCl3): d 8.01–7.97 (m, 2H, arom), 7.57–7.52
(m, 3H, arom), 7.48–7.30 (m, 5H, arom), 7.17–7.10 (m, 2H, arom),
5.98–5.94 (t, 1H, NHCO), 4.44 (br, 1H, NH), 3.64–3.48 (m, 3H,
CHCH3, NHCH2), 3.26–3.18 (m, 2H, CH2NHCO), 3.07 (br, 2H,
C4-H2), 2.65 (br, 2H, C1-H2), 1.88 (br, 4H, C3-H2, C2-H2), 1.68–1.57
(m, 2H, NHCH2CH2), 1.53–1.25 (m, 9H, CH2CH2CH2CH2NHCO,
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870
Y. Chen et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 865–871
CHCH3); 13C NMR (CDCl3): d 173.57 (NHCO), 161.69, 156.92
of DTNB was added. The reaction was started by addition of 20 mL
of the substrate solution (ATC/BTC). The solution was mixed
immediately, and exactly 2 min after substrate addition, the
absorption was measured at 25°C at 412 nm. For the reference
value, 100 mL of water replaced the test compound solution. For
determining the blank value, additionally 100 mL of water
replaced the enzyme solution. Each concentration was measured
in triplicate at 25°C. The inhibition curve was obtained by
plotting percentage enzyme activity (100% for the reference)
versus logarithm of test compound concentration. Calculation of
the IC50 values was performed with Graph Pad PrismTM 4.
Kinetic measurements were performed in the same manner,
while the substrate (ATC/BTC) was used in concentrations of 25,
50, 90, 150, 226, and 452 mM for each test compound
concentration and the reaction was extended to 4 min before
measurement of the absorption. Vmax and Km values of the
Michaelis–Menten kinetics were calculated by nonlinear regres-
sion from substrate–velocity curves using Graph Pad PrismTM 4.
Linear regression was used for calculating the Lineweaver–Burk
plots.
(arom, C3), 157.74 (arom), 151.69 (arom), 145.65 (arom), 143.14,
0
143.02 (arom, C1), 135.34 (arom, C1 ), 0130.91, 130.85 (arom, C50),
0
0
128.86 (arom), 128.81 (arom, C6 , C2 ), 128.45 (arom, C3 , C5 ),
0
127.78, 127.56 (arom, C4), 127.69 (arom), 127.00 (arom, C4 ),
123.89 (arom), 123.63, 123.58 (arom, C6), 123.15 (arom), 119.29
(arom), 115.41 (arom), 115.04, 114.84 (arom, C2), 48.93 (NHCH2),
46.48 (CHCH3), 39.33 (CH2NHCO), 32.85 (NHCH2CH2), 31.40
(CH2CH2NHCO), 29.39 (C4), 26.32 (NHCH2CH2CH2), 26.29 (C1),
24.56 (CH2CH2CH2NHCO), 22.79 (C3), 22.32 (C2), 18.56 (CHCH3).
Anal. calcd. for (C34H38FN3O · H2O): found C, 75.6; H, 7.21; N, 7.8;
requires C, 75.4; H, 7.44; N, 7.8.
2-(2-Fluorobiphenyl-4-yl)-N-[8-(1,2,3,4-tetrahydroacridin-
9-ylamino)octyl]propanamide (3e)
Compound 2e (0.31 g, 1 mmol) reacted with flurbiprofen (0.24 g,
1 mmol) to furnish 3e as a yellow solid (0.22 g, 40%); m.p. 50–52°C.
1H NMR (CDCl3): d 7.96–7.89 (t, 2H, arom), 7.56–7.50 (m, 3H,
arom), 7.44–7.30 (m, 5H, arom), 7.15–7.09 (t, 2H, arom), 5.78 (br,
1H, NHCO), 3.99 (br, 1H, NH), 3.60–3.41 (m, 3H, CHCH3, NHCH2),
3.24–3.16 (m, 2H, CH2NHCO), 3.05 (br, 2H, C4-H2), 2.68 (br, 2H,
C1-H2), 1.90 (br, 4H, C3-H2, C2-H2), 1.64–1.51 (m, 5H, NHCH2CH2,
CHCH3), 1.44–1.24 (m, 10H, CH2CH2CH2CH2CH2CH2NHCO);
13C NMR (CDCl3): d 173.39 (NHCO), 161.72, 157.77 (arom, C3),
Cell-based Ab inhibition assay
Ab40 were measured in culture medium of primary cortical
neuron cells (pregnant Wistar rats). Cells were seeded onto 48-
well plates and allowed to grow for 3–5 days in 5% CO2/95% air in
a humidified atmosphere. Cells were incubated for 24 h with test
compound at concentration 0.25 mM. {1S-Benzyl-4R-[1-(1S-carba-
moyl-2-phenyl-ethyl-carbamoyl)-1S-3-methylbutylcarbamoyl]-2R-
hydrox-5-phenylpentyl}carbamic acid tert-butyl ester (L-685458)
was used as positive control. DMSO (0.3%) was used as negative
control. Following incubation with the test compounds the
supernatant was collected and the Ab40 levels were determined
using a sandwich human/rat b-amyloid (40) ELISA kit II,
performed in 96-well microtiter plates. Conditioned media was
incubated with capture antibody for 16 h at 4°C, followed by
washing. The detecting antibody incubation was performed for
2 h at 4°C, again followed by the wash steps. Then, 100 mL of TMB
ultrasubstrate was added and incubated for 30 min at room
temperature in the dark, and the reaction was stopped by adding
100 mL of 2 M H2SO4. Absorbance at 450 nm was recorded with a
Microplate Reader TECAN Safire2 (Tecan Group Ltd., Maennedorf,
Switzerland). Ab40 was used to generate standard curves. The
percent of control Ab40 as a function of the concentration of the
test compound was recorded. Triplicate measurements from each
drug concentration were averaged.
158.22 (arom), 150.93 (arom), 147.22 (arom), 143.11, 143.00
0
(arom, C1), 135.39 (arom, 0C1 ),0 130.95, 130.89 (arom, C50 ), 128.88
0
(arom), 128.84 (arom, C6 , C2 ), 128.45 (arom, C3 , C5 ), 128.37
0
(arom), 127.82, 127.60 (arom, C4), 127.69 (arom, C4 ), 123.60
(arom), 123.60, 123.55 (arom, C6), 122.92 (arom), 120.08 (arom),
115.66 (arom), 115.42, 115.04 (arom, C2), 49.38 (NHCH2),
46.61, 46.59 (CHCH3), 39.63 (CH2NHCO), 33.87 (NHCH2CH2),
31.68 (CH2CH2NHCO), 29.44 (NHCH2CH2CH2CH2), 29.15
(CH2CH2CH2CH2NHCO), 29.03 (C4), 26.75 (NHCH2CH2CH2),
26.63 (CH2CH2CH2NHCO), 24.73 (C1), 23.01 (C3), 22.72 (C2),
18.60 (CHCH3). Anal. calcd. for (C36H42FN3O · 3/4H2O): found C,
76.6; H, 7.67; N, 7.3; requires C, 76.5; H, 7.76; N, 7.4.
Pharmacology
Cholinesterase inhibition assay in vitro
The assay followed the method of Ellman et al., using a Shimadzu
160 Spectrophotometer. AChE (E.C.3.1.1.7, Type VI-S, from electric
eel) and BChE (E.C.3.1.1.8, from equine serum), 5,50-dithiobis(2-
nitrobenzoic acid) (Ellman’s reagent, DTNB), acetylthiocholine
(ATC), and butyrylthiocholine (BTC) iodides were purchased from
Sigma–Aldrich (Steinheim, Germany). AChE/BChE stock solution
was prepared by adjusting 500 units of the enzyme and 1 mL of
gelatine solution (1% in water) to 100 mL with water. This enzyme
solution was further diluted before use, to give 2.5 units/mL in
1.4 mL aliquots. 0.075 M ATC/BTC iodide solution was prepared in
water. 0.01 M DTNB solution was prepared in water containing
0.15% w/v sodium bicarbonate. For buffer preparation, potassium
dihydrogen phosphate (1.36 g, 10 mmol) was dissolved in 100 mL
of water and adjusted with KOH to pH 8.0 ꢀ 0.1. Stock solutions of
the test compounds were prepared in ethanol, 100 mL of which
gave a final concentration of 10ꢃ4 M when diluted to the final
volume of 3.32 mL. For each compound, a dilution series of at
least five different concentrations (normally 10ꢃ4–10ꢃ9 M) were
prepared.
The cytotoxicity potential of test compound was assessed in the
same cells of the Ab assay using the 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT) assay. After adding
increasing concentrations of test compound (ranging from 0.01
to 100 mM), cells were incubated for 24 h. Then cells with 100 mL of
culture medium containing 20mL (0.5 mg/mL) of MTT were
incubated for another 4 h at 37°C, 5% CO2, and saturated
humidity. After removal of the medium, an amount of 120 mL of
100% DMSO was added to each well. The amount of formed
formazan was determined by reading the samples at 570 nm.
Molecular docking
The docking study was performed by using CDOCKER module
inbuilt in Discovery Studion 3.0. In general, CDOCKER generated
ligand “seeds” to populate the binding pocket. Each seed was then
subjected to high-temperature molecular dynamics (MD) using a
For measurement, a cuvette containing 3.0 mL of phosphate
buffer, 100 mL of the respective enzyme, and 100 mL of the test
compound solution was allowed to stand for 5 min before 100 mL
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 865–871
Tacrine–Flurbiprofen Hybrids
871
modified version of CHARMm force field. Each of the structures
from the MD run were then located and fully minimized. The
solutions were then clustered according to position and
conformation and ranked by energy. CHARMm charges were
used for the protein structure. The crystal structure of human
AChE (HuAChE, PDB id: 2 ꢂ 8B) was selected for molecular
docking. As there was no small molecule binding to 2 ꢂ 8B, the co-
crystal structure of bis(7)-tacrine with TcAChE (PDB id: 2CKM) was
selected as the reference structure. The 2 ꢂ 8B was firstly aligned
and superimposed onto the 2CKM (see Supporting Information
Fig. S1). The RMSD between the two structures was 1.202.
Additionally, the CAS site and the PAS site of the two kinds of
AChE exhibit high similarity, indicating that the reference
[2] E. Scarpini, P. Scheltens, H. Feldman, Lancet Neurol. 2003, 2,
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29, 12787–12794.
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G. Dondio, L. Fontanella, P. Misiano, C. Bigogno, A. Rizzi,
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structure is reliable in this study. After the superimposition, the
ꢁ
residues around bis(7)-tacrine in HuAChE (in 10 A radius) was set
as binding site. For CDOCKER simulation, the heating step,
cooling steps, and cooling temperature were set to 5000, 10,000,
and 310, respectively. Other parameters were kept as default.
[8] M. B. Youdim, J. J. Buccafusso, Trends Pharmacol. Sci. 2005, 26,
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M. Decker, Bioorg. Med. Chem. Lett. 2008, 18, 2905–2909.
The support by “DAAD Sandwich Program” Scholarship (April 2010 to
March 2012) for Chen Yao is very gratefully acknowledged. The authors
also thank National Science Foundation of China (NSFC, No. 30973608),
National Natural Science Foundation of China (No. 81070967), and the
Natural Science Foundation of Jiangsu Province of China (No.
BK2009296) for further funding.
[10] M. I. Fernández-Bachiller, C. Pérez, G. C. González-Muñoz,
S. Conde, M. G. López, M. Villarroya, A. G. García,
M. S. Rodríguez-Franco, J. Med. Chem. 2010, 53, 4927–4937.
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351–357.
[12] G. L. Ellman, K. D. Courtney, V. Andres, Jr., R. M. Feather-
Stone, Biochem. Pharmacol. 1961, 7, 88–95.
The authors have declared no conflict of interest.
[13] Y. Bourne, P. Taylor, Z. Radić, P. Marchot, EMBO J. 2003, 22, 1–12.
[14] M. Bartolini, C. Bertucci, V. Cavrini, V. Andrisano, Biochem.
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