Design and synthesis of tacrine-phenothiazine hybrids as multitarget drugs for Alzheimer's disease

Article abstract of DOI:10.1007/s00044-014-0931-2

Tacrine is well-known drug for Alzheimer's disease as an acetylcholinesterase inhibitor. Rember is a bright and promising AD drugs targeting tau protein, and it is currently in Phase III clinical trials. Phenothiazine, the key pharmacophore of Rember, can prevent tau filament formation. In this work, several tacrine-phenothiazine hybrids (T1-T26) were designed for inhibiting acetylcholinesterase and tau protein involved in Alzheimer's disease. After initial screening with the help of computational chemistry software and Molegro Virtual Docker, three molecules (T5, T18, and T22) were selected for further synthesis and biological evaluation. Next, T5, T18, and T22 were synthesized and evaluated for their acetylcholinesterase and tau hyperphosphorylation inhibition. All the tested compounds had better acetylcholinesterase inhibitory activity compared with tacrine. Among them, compound T5 was found to be the most potent compound with IC₅₀ 89 nM. Meanwhile, T5 markedly prevented tau hyperphosphorylation induced by okadaic acid in N2α cell. Its P-tau level was decreased with 39.5 % inhibition when tested at 10^-5 M, lower than that Rember (55.7 %). Besides acetylcholinesterase and tau hyperphosphorylation inhibition, T5 can also interact with fibrill beta amyloid using surface plasmon resonance, the data of K_D were 5.51 × 10^-8 M. All the above results indicated that tacrine-phenothiazine hybrids are potential multitarget directed ligands targeting acetylcholinesterase, tau protein, and beta amyloid.

Full text of DOI:10.1007/s00044-014-0931-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-0931-2
ORIGINAL RESEARCH
Design and synthesis of tacrine-phenothiazine hybrids
as multitarget drugs for Alzheimer’s disease
Ai-ling Hui
•
Yan Chen
•
Shi-jing Zhu
•
Chang-sheng Gan
•
Jian Pan An Zhou
•
Received: 30 August 2013 / Accepted: 27 January 2014
Ó Springer Science+Business Media New York 2014
Abstract Tacrine is well-known drug for Alzheimer’s
disease as an acetylcholinesterase inhibitor. Rember is a
bright and promising AD drugs targeting tau protein, and it is
currently in Phase III clinical trials. Phenothiazine, the key
pharmacophore of Rember, can prevent tau filament for-
mation. In this work, several tacrine-phenothiazine hybrids
fibrill beta amyloid using surface plasmon resonance, the
-
8
data of KD were 5.51 9 10 M. All the above results
indicated that tacrine-phenothiazine hybrids are potential
multitarget directed ligands targeting acetylcholinesterase,
tau protein, and beta amyloid.
(
T1–T26) were designed for inhibiting acetylcholinesterase
Keywords Alzheimer’s disease ꢀ
Acetylcholinesterase inhibitor (AChEI) ꢀ
Tau phosphorylation ꢀ Beta amyloid ꢀ
Tacrine-phenothiazine hybrids
and tau protein involved in Alzheimer’s disease. After initial
screening with the help of computational chemistry software
and Molegro Virtual Docker, three molecules (T5, T18, and
T22) were selected for further synthesis and biological
evaluation. Next, T5, T18, and T22 were synthesized and
evaluated for their acetylcholinesterase and tau hyper-
phosphorylation inhibition. All the tested compounds had
better acetylcholinesterase inhibitory activitycomparedwith
tacrine. Among them, compound T5 was found to be the
most potent compound with IC50 89 nM. Meanwhile, T5
markedly prevented tau hyperphosphorylation induced by
okadaic acid in N2a cell. Its P-tau level was decreased with
Introduction
Alzheimer’s disease (AD), the most common dementia in
elderly people, is a complex neurodegenerative disorder of
the central nervous system (CNS). It is characterized by the
progressive deterioration of cognition and the emergence of
behavioral and psychological symptoms. In AD brains, the
cholinergic system is the most dramatically affected. Besides
cholinergic deficiency, the abnormal formation of extracel-
lular senile plaques (SP) consisting of aggregated beta
amyloid (Ab) peptide deposits and intracellular neurofibril-
lary tangles (NFTs) consisting of hyper-phosphorylated
forms of the microtubule-associated tau protein is the most
characteristic pathological hallmarks present in AD brain.
Current clinical therapy for AD is mainly palliative treat-
ment targeting acetylcholinesterase (AChE) or N-methyl-D-
aspartate (NMDA) receptor. AChE inhibitors (AChEIs) can
increase the level of neurotransmitter in synaptic cleft, thereby
improving cognition level of mild to moderate AD patients
-
5
3
9.5 % inhibition when tested at 10 M, lower than that
Rember (55.7 %). Besides acetylcholinesterase and tau hy-
perphosphorylation inhibition, T5 can also interact with
A. Hui (&) ꢀ S. Zhu ꢀ C. Gan ꢀ J. Pan
Institute of Natural Medicine, Hefei University of Technology,
No. 193, Tunxi Road, Hefei 230009, Anhui,
People’s Republic of China
e-mail: haling@mail.ustc.edu.cn; haling@ustc.edu
Y. Chen
School of Life Science, Anhui University, Hefei,
People’s Republic of China
(
Massoud and Guathier 2010). So far, four AChEIs (tacrine,
A. Zhou
donepezil, galamantine, and rivastigmine) and one NMDA
receptor antagonist (memantine) have been approved by FDA.
However, their role in the course of preventing AD is limited;
Anhui Province Key Laboratory of R&D of Chinese Medicine,
Anhui University of Traditional Chinese Medicine, Hefei,
People’s Republic of China
123

Med Chem Res
none of them have proven effective against the progression of
AD. Given that AChEIs and NMDA receptor antagonist have
still been questioned and multifactorial nature of AD, the
classic ‘‘one molecule, one target’’ solution may not be effec-
tive enough. Researchers are focusing on the development of
new therapy strategies for arresting and reverting its progres-
sion. One general concept is multitarget directed ligand
Nevertheless, many recent clinical studies showed that
the treatment specially targeting Ab had no effects on AD.
Many drugs failed in phase III clinical trials, for example,
Tarenflurbil developed by Myriad Genetics Company and
Tramiprosate developed by Neurochem Company. On the
other hand, the treatment targeting tau protein shows a
bright future, a representative example is Rember devel-
oped by Taurx Company. 321 patients had participated in
phase II clinical trials, it showed better efficacy. Phase III
clinical trials are ongoing.
(MTDL) approach (Cavalli et al., 2008; Morphy and Rankovic,
2009), inwhichdrugs aredesigned to address selectedactivities
toward selected targets involved in the AD.
In line with MTDL paradigm, several multifunctional
agents capable of hitting different biological targets have
been developed and their biological profiles seem to be
promising (Fern a´ ndez-Bachiller et al., 2010; Bajda et al.,
The aim of this study was to develop novel tacrine
hybrids for their mainly inhibiting AChE and tau protein
involved in the AD. It is reported that Rember (methylene
blue, MB) can reduce soluble tau and regenerate cognition
(O’Leary et al., 2010). Phenothiazine, the key pharmaco-
phore of Rember also can prevent tau filament formation
(Taniguchi et al., 2005). Herein, a series of tacrine-phe-
nothiazine hybrids via an alkylenediamine-type spacer will
be designed according to the principle of multitarget drugs
design. These hybrids are expected to inhibit AChE and tau
hyperphosphorylation and other targets involved in AD.
2
011; Minarini et al., 2012; Rizzo et al., 2012).
It is the case of tacrine dimeric derivatives paved the way
for the development of very interesting homodimer and
heterodimer to prevent AD. An heptylene-tethered dimer of
tacrine named bis (7)-tacrine (Fig. 1) is able to simulta-
neously contact the central anionic site (CAS) and the
peripheral anionic site (PAS) (Pang et al., 1996), prevent
glutamate-induced neuronal apoptosis by blocking NMDA
receptor (Li et al., 2005; Luo et al., 2007), and reduce Ab
generation by directly inhibiting beta-site APP cleavage
enzyme (BACE1) activity (Fu et al., 2008). As for the hybrid
structure, it would reduce individual toxicity by special
metabolic pathway compared with the combinational drugs.
The recent studies following this strategy have led to the
synthesis of several chemically diverse structures with dual
or multiple biological profiles of AD, including AChE and
monoamine oxidase B (MAO-B) dual inhibitors (Sterling
et al., 2002); AChE and Ab aggregation inhibitors (Kapkova9
et al., 2006; Tumiatti et al., 2008; Ouberai et al., 2011); Ab
aggregation and peroxidase activity inhibitors (Ghadami
et al., 2013); AChE and BACE1 dual inhibitors (Zhu et al.,
Results and discussion
Design and screen
On the basis of tacrine and phenothiazine, two series of
new hybrids (T1–T26) are designed, as shown in Fig. 2.
ADME properties of the designed 26 molecules were
predicted using the web-based PreADMET (v2.0) (http://
preadmet.bmdrc.org/) (Chitranshi et al., 2013; Lee et al.,
2003). This gave an estimate of physiochemical properties
and the bioavailability of the molecule. Fundamental phys-
iochemical features of CNS drugs are related to their ability
2
009); AChE, Ab aggregation inhibiting, and antioxidant
multiple functional compounds (Cavalli et al., 2007); and
AChE, Ab aggregation, and NMDA receptor inhibitor (Ro-
sini et al., 2008). The biological activities of hybrids men-
tioned above have been improved by the introduction of the
second or the third pharmacophores targeting Ab, BACE1,
NMDA, or some oxidases and others.
N
N
N
NH(CH ) NH
2
n
N
m
m
O
O
N
S
N
S
H
NH
N
N
N
n = 2-9, m = 1-3
m = 1-5
T22-T26)
(
Bis-(7)-tacrine
(T1-T21)
Fig. 1 Chemical structure of bis-(7)-tacrine
Fig. 2 General structure of the designed molecules
1
23

Med Chem Res
to penetrate the blood–brain barrier (BBB) and exhibit CNS
activity. The molecule would regard as CNS penetrant with
Cbrain/Cblood [0.1. The drug-likeness prediction of the 26
molecules is tabulated in Table 1. As could be seen from
Table 1, all the molecules (T1–T26) exhibited general drug
likeness. Their Cbrain/Cblood values are[0.1 besides T23 and
T24, which reveal their potential BBB penetrability.
glycogen synthase kinase-3b (GSK-3b) is the most impli-
cated (Hanger et al., 2009b; Takashima, 2006). Herein,
GSK-3b was selected as another target protein in the
present work.
Once the two mainly target protein was established,
MolegroVirtual Docker (MVD) 2009 trial was applied for
further molecular docking studies. Docking procedures
basically aim to identify the correct conformation of the
molecule ligands in the binding pocket of the protein and to
predict the affinity between the molecule ligand and the
protein (Su, 2011).
After initial ADME screening, the molecular docking
technique was undertaken for the best tacrine-phenothia-
zine hybrids inhibiting AChE and tau hyperphosphoryla-
tion among 26 molecules. In addition to AChE as a target
protein in the process of molecular docking, another target
protein which affects tau hyperphosphorylation must be
found. It has been recognized that an imbalanced regula-
tion between protein kinases and protein phosphatases is a
direct result of AD-like tau hyperphosphorylation (Hanger
et al., 2009a). Among various kinases and phosphatases,
The crystal structure of AChE complex with donepezil
(Aricept) was retrieved from the Protein Data Bank (PDB
ID: 1EVE). Aricept and water molecule were firstly
removed followed with enzyme optimization. Similarly,
the crystal structure of GSK-3b (PDB ID: 3GB2) was also
retrieved. Finally, the minimized poses were recorded
Table 1 Physiochemical descriptors calculated for the selected compounds using PreADMET
Compound
name
n, m
Human intestinal
absorption (HIA, %)
In vivo skin
permeability
In vitro plasma
protein
binding (%)
In vivo blood–brain
barrier penetration
(C. brain/C. blood)
(
Log Kp, cm/h)
T1
n = 2, m = 1
n = 3, m = 1
n = 4, m = 1
n = 5, m = 1
n = 6, m = 1
n = 7, m = 1
n = 8, m = 1
n = 9, m = 1
n = 2, m = 2
n = 3, m = 2
n = 4, m = 2
n = 5, m = 2
n = 6, m = 2
n = 7, m = 2
n = 8, m = 2
n = 2, m = 3
n = 3, m = 3
n = 4, m = 3
n = 5, m = 3
n = 6, m = 3
n = 7, m = 3
m = 1
97.072429
97.141426
97.205072
97.263624
97.317235
97.366064
97.410275
97.450030
97.141834
97.205610
97.264175
97.317798
97.366640
97.410864
97.450634
97.205497
97.264182
97.317808
97.366650
97.410875
97.450645
98.168642
98.206526
98.242005
98.275096
98.305831
96.517346
95.742616
97.951353
-2.64907
-2.57576
-2.3987
85.503982
86.078610
86.104074
85.985029
86.743558
86.241982
86.613634
87.126810
86.414089
84.459587
84.940830
84.864895
85.788631
85.189595
85.594753
84.687633
84.599574
85.659313
85.826236
86.997540
86.583695
85.131086
86.590993
84.645431
84.477508
87.828355
95.393497
98.064683
84.615816
0.403176
0.412231
1.00739
1.7899
T2
T3
T4
-2.26372
-2.14617
-2.04459
-1.95696
-1.88099
-2.5321
T5
2.8854
T6
4.12672
5.31387
6.41301
0.55582
0.609947
1.38431
2.2915
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
T17
T18
T19
T20
T21
T22
T23
T24
T25
T26
Tacrine
Bis(7)-tacrine
Donepezil
-2.46422
-2.30185
-2.17936
-2.07338
-1.98203
-1.9031
3.59994
4.79546
5.95378
0.902598
0.973773
1.98348
3.16381
4.52704
5.68517
0.482569
0.0688374
0.0847133
0.130199
0.237945
0.866481
15.6351
0.187923
-2.4124
-2.35089
-2.20486
-2.09553
-2.00129
-1.91998
-2.65986
-2.54326
-2.42453
-2.29217
-2.17302
-3.04445
-2.08538
-3.04165
m = 2
m = 3
m = 4
m = 5
123

Med Chem Res
using MVD scoring function. Only top scoring molecule
ligand was taken for further synthesis and biological
evaluation.
Table 2 MolDock scores of putative AChE and GSK-3b binders for
the selected compounds using MVD
Compound name
AChE (1EVE)
MolDock score
GSK-3b (3GB2)
MolDock score
(kJ/mol)
The molecular docking studies of the 26 ligands are
illustrated as representative case in Table 2. The present
molecular docking studies showed relatively low MolDock
Score (-82.259 kJ/mol) for tacrine, which indicates low
affinity toward AChE. In general, the affinity of the ligands
(
kJ/mol)
T1
-192.250
-180.754
-199.500
-179.776
-183.585
-195.865
-183.213
-192.805
-195.721
-180.123
-179.422
-168.312
-164.267
-188.631
-157.321
-169.631
-166.319
-202.166
-178.387
-184.302
-161.103
-170.388
-180.631
-179.188
-190.774
-189.001
-82.259
-123.573
-138.015
-143.087
-133.536
-148.821
-145.270
-147.834
-139.603
-133.434
-142.973
-135.664
-144.246
-124.311
-146.16
T2
T3
(
T1–T26) toward AChE or GSK-3b was better than tacrine
T4
or donepezil. Among the 26 molecules, T5 had a significant
affinity toward GSK-3b and a moderate affinity toward
AChE. The MolDock Score of T5 toward GSK-3b was
T5
T6
T7
-
148.821 kJ/mol; also its MolDock Score toward AChE
T8
(
(
-183.585 kJ/mol) was slightly lower than bis (7)-tacrine
-190.66 kJ/mol). It was reported that bis (7)-tacrine had
T9
T10
T11
T12
T13
T14
T15
T16
T17
T18
T19
T20
T21
T22
T23
T24
T25
T26
Tacrine
Bis(7)-tacrine
Donepezil
much better AChE inhibitory activity (IC50 = 1.5 nM) than
tacrine (IC50 = 223 nM) (Carlier et al., 1997). Meanwhile,
T18 was also found as a potential molecule, which had a
significant affinity toward AChE (-202.166 kJ/mol) and
good affinity toward GSK-3b. As for the affinity with GSK-
-138.955
-124.935
-114.792
-143.581
-145.071
-146.763
-146.817
-126.539
-122.621
-131.680
-133.290
-139.057
-71.497
3
b, the MolDock Score of T18 (-143.581 kJ/mol) was
slightly lower than the best T5 (-148.821 kJ/mol).
On the other hand, it is also found from Table 2 that the
affinity of the hybrid molecules via piperazine connection
(
T22–T26) was generally worse than those of molecules
via diamine connection (T1–T21). For example, T22 bin-
ded to AChE and GSK-3b with lower MolDock Score
(
-170.388 and -126.538 kJ/mol); even so, its MolDock
Score was greater than tacrine or donepezil. Therefore, T5,
T18, and T22 were selected as representative case for
further synthesis and biological evaluation.
Chemistry
-190.660
-158.715
-161.823
-111.367
The synthesis route of T5 or T18 is shown in Scheme 1.
Intermediates A, B, and C were prepared according to the
literature (Hu et al., 2002; Tang et al., 2011; Darvesh et al.,
2
005) and slightly modified, respectively. Once the inter-
the increase and the proportion of the two new products
was gradually reduced. T5 or T18 was not finally realized
as unique product or main product even if the ratio of B
and C was also changed. In addition, the type of solvents
and bases had certain impact on the composition of the
products. There are other by-products in the reaction
mixture when we use triethylamine and CH COCH
mediate was available, the resulting diamine (B1 or B2)
was reacted with C1 or C2 in the presence of base (K CO
2
3
or Et N) to yield the desired T5 and T18. However, another
3
new product (T5-B or T18-B) was also obtained, respec-
tively, when we use CH Cl or CH COCH as reaction
2
2
3
3
solvent under reflux condition. The two new products (T5/
T5-B, T18/T18-B) were characterized as one or two molar
amine displacement with slightly difference in molar ratio.
Subsequently, the reactant ratio of B and C, reaction tem-
perature, and reaction time were investigated for the opti-
mal conditions of T5 or T18. Reducing temperature or
shortening time was negative on the conversion of reactant
and product yields. With the reaction processing and
reactant consuming, the amount of T5-B or T18-B was on
3
3
instead of CH Cl and K CO .
2
2
2
3
The synthesis procedure of T22 is outlined in Scheme 2.
Chloroacetyl phenothiazine (C1) was reacted with exces-
sive piperazine in the presence of K CO /KI, 1-(10H-
2
3
phenothiazin-10-yl)-2-(piperazin-1-yl) ethanone (D) was
obtained as the unique product. Next, the intermediate D
was converted to the desired T22 using similar synthesis
method of B1 or B2.
1
23

Med Chem Res
Scheme 1 Synthesis of T5 and
T18
O
Cl
8
5
1
NH2
OH
9
14
13
1
1
2
POCl3
7
+
3
6
Reflux
12
N
4
10
O
A
n
NH2
1
Cl
N
HN
8
9
1
4
11
2
7
6
NaI
+
H N nNH2
2
Phenol
3
1
3
12
N
5
4
1
0
B
(
B1 n = 6; B2 n = 4)
5
6
4
1
S
12
7
8
13
14
3
2
S
O
1
0
N
Et N
3
+
Cl
11
Cl
CH Cl
9
m
2
2
N
H
O
m
Cl
C
(
C1 m = 1; C2 m =3)
7
′
8′
6′
1
3′
S5
9
′
′
1
4′
H
1
0′
12′
N
n
m
4′
3′
HN
N
11′
1′
K CO
8
2
3
9
1
O
B + C
14
11
12
2
3
7
CH Cl
2 ′
2
2
1
3
6
N
5
4
T5 n = 6, m = 1
T18 n = 4, m = 3
1
0
+
S
N
m
O
N
n
N
O
N
N
H
m
S
T5-B n = 6, m = 1
T18-B n = 4, m = 3
123

Med Chem Res
H
N
Cl
N
O
H
N
O
K CO , KI
2
3
N
S
9
'
1'
+
10'
1
4'
N
1
1'
2'
3'
8
'
DMF
N
H
7'
1
3'
12'
S
4
'
6
'
5'
C1
D
2'
3
'
1
1
'
O
4'
1'
H
N
10'
1
2'
N
4'
S
13'
5'
1
Cl
N
N
9
'
N
6
'
PhOH, NaI
O
+
8'
7
'
8
1
N
9
N
2
7
1
4
11
12
1
3
A
6
3
S
N
5
4
10
D
T22
Scheme 2 Synthesis of T22
Table 3 Inhibitory activity on rain brain homogenate AChE
Table 4 Inhibition of P-Tau for test compounds in OA-treated N2a
cells
Compound name
IC50 (nM)
Group
Concentration
P-Tau content (ng/L)
Inhibition (%)
T5
89 ± 19
Control
OA
–
22.8 ± 2.5
–
T5-B
T18
305 ± 29
109 ± 21
283 ± 41
217 ± 30
275 ± 32
33 ± 8
#
#
60 nM
68.6 ± 4.1
41.5 ± 0.9
44.8 ? 1.3
50.9 ? 1.5
45.3 ± 0.8
49.3 ± 1.3
54.1 ± 0.6
50.5 ± 4.3
56.6 ± 2.7
61.8 ± 2.0
30.4 ± 3.5
36.8 ± 4.2
44.3 ± 1.0
53.4 ± 1.6
61.3 ± 4.7
*
–
-
-
-
-
-
-
5
6
7
5
6
7
**
**
**
**
**
**
**
*
T5
10
M
M
M
M
M
M
M
M
M
M
M
M
M
39.5 ± 1.1
34.7 ± 2.0
25.8 ± 1.9
34.0 ± 0.7
26.7 ± 1.0
21.1 ± 1.3
26.4 ± 2.1
17.5 ± 1.3
9.9 ± 0.8
55.7 ± 2.3
46.4 ± 1.0
35.4 ± 1.7
22.2 ± 1.4
10.6 ± 1.1
T18-B
T22
1
1
0
0
Tacrine
Donepezil
T18
10
1
1
0
0
Biological evaluation
-5
T22
10
-
-
-
-
-
-
6
7
5
6
7
8
1
1
0
0
The preliminary anti-acetylcholinesterase activity for the
synthesized tacrine derivatives (T5, T5-B, T18, T18-B,
T22) was performed according to AChE enzyme-linked
immunosorbent assay (ELISA) Kit using tacrine and do-
nepezil as reference compound. AChE was obtained from
homogenates of rat brain. Results of anti-AChE activity of
the compounds are illustrated in Table 3. It is easily seen
that all the tacrine derivatives exhibited better inhibitory
activity against AChE than tacrine. From the biological
activity studies, we also found that T5-B and T18-B
demonstrated weaker AChE inhibition as compared to T5
or T18. Their bulk structure (T5-B, T18-B) was partially
responsible for the lower AChE inhibition, which cannot
**
**
**
**
Rember
10
1
1
1
0
0
0
Tacrine
20 lM
#
#
**
p \ 0.01 versus Control group; p \ 0.05, p \ 0.01 versus OA
group
efficiently enter into binding pocket of AChE. And as for
the three desired tacrine hybrids (T5, T18, and T22), T5
was found to be the most potent compounds with IC50 89
1
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Med Chem Res
RU
7
6
5
4
3
2
1
00
00
00
00
00
00
00
0
T5 50µM
CR 200µM
T5 25µM
T5 12.5µM
-
-
-
100
200
300
0
500
1000
1500
2000
2500
s
Time
0
0
μΜ
.625 μΜ
6
5
4
3
2
1
00
00
00
00
00
00
0
1
2
5
10 μΜ
2
.25 μΜ
.5 μΜ
μΜ
0 μΜ
40 μΜ
-
-
100
200
0
1
2
3
4
5
6
Time(min)
Fig. 3 Analysis of the affinities of T5 for fAb(1–40) by SPR
-
the concentration of T5 was reduced to 10 concentration
7
nM, lower than donepezil with IC50 33 nM. T22 showed
the lowest activity in the three compounds, its IC50 was 217
nM, slightly greater than tacrine (275 nM). The above-
mentioned AChE inhibition results are consistent with the
inhibition sequence (Table 2) predicted by MVD.
of T5 wasM, the P-Tau inhibition (25.8 %) was almost
-
equal to that of T22 at 10 M. At the same conditions,
5
Rember displayed a percentage of inhibition ranged from
-
8
22.2 to 55.7 % when tested at 10 M–10 M.
-5
The effect of the tacrine hybrids (T5, T18, T22) on OA-
induced P-Tau accumulation is shown in Table 4. The
results were given by measuring the sum of P-Tau in OA-
treated N2a cells with an ELISA assay. 12 h exposure of
N2a cells to OA resulted in a significant increase in
intracellular P-Tau generation, the P-Tau level reached to
On the basis of these promising results, T5 was also
tested for its ability to bind with Ab fibril (fAb). Interaction
of T5 with fAb(1–40) was investigated by surface plasmon
resonance (SPR) in grade concentrations at 0.625–40 lM
for generation of binding curves. As shown in Fig. 3, dis-
tinct association and dissociation reactions were observed
after the addition of freshly prepared T5 to immobilized
fAb(1–40), indicating that T5 could react with the immo-
bilized fAb. The responses of T5 reached equilibrium
(Req) quickly and returned to baseline rapidly after dis-
sociation. The binding response was calculated as the Req
value divided by the molecular weight. The dose–response
6
8.6 ng/L compared with control group (22.8 ng/L). As
shown in Table 4, a treatment with tacrine hybrids obvi-
ously prevented OA induced increase in the P-Tau level,
which displayed inhibition higher than 25 % when tested at
-
5
1
0
M. Among the three hybrids, T5 was the most potent,
-
which inhibited P-tau level by 39.5 % at 10 M. Even if
5
123

Med Chem Res
curve for T5 appeared to be monophasic, and it reached a
saturation level at higher concentrations. The dissociation
mixture of o-aminobenzoic acid (22 mmol, 3 g) and cyclo-
hexanone (20 mmol, 2 g) 15 ml POCl at ice bath was
3
constant (K ) was calculated from data at dose ranging
D
carefully added. The mixture was heated under reflux for 4 h,
then cooled to room temperature, and concentrated to give
the slurry. The residue was diluted with ethyl acetate, neu-
tralized with aqueous K CO , and washed with brine. The
from 0.625 to 40 lM by steady-state analysis using BIA
evaluation software (version 4.1). The K of T5 to
D
-
8
fAb(1–40) was 5.51 9 10 M, lower than Congo Red
2
3
-
CR, positive control) with K 6.78 9 10 M, while ta-
7
(
organic layer was dried over anhydrous K CO and evapo-
2 3
D
crine have no binding with fAb(1–40) under the same
condition.
rated. The crude product was purified by flash chromatog-
raphy to give the desired product. It was obtained as pale
yellow solid, 3.4 g, 78 % yield); mp 69.0–69.8 °C; IR (KBr)
-
1
1
tmax 2,936.1, 1,577.7, 1,552.6, 1,481.2, 754.0 cm ; H
Conclusion
NMR (CDCl , 300 MHz): d = 8.16 (1H, d, J = 8.4 Hz,
3
H-8), 7.97 (1H, d, J = 8.4 Hz, H-5), 7.66 (1H, dd, J = 8.4,
9.6 Hz, H-6), 7.53 (1H, dd, J = 8.3, 9.6 Hz, H-7), 3.13 (2H,
t, J = 6.0 Hz, H-4), 3.02(2H, t, J = 5.4 Hz, H-1), 1.92–1.97
The tacrine-phenothiazine hybrid has already been suc-
cessful in the design of MTDL for combating the multi-
factorial nature of AD. In addition to its classic anti-AChE
effects, tacrine-phenothiazine hybrid has been proven to be
effective in inhibiting P-Tau and binding with Ab fibrils
when presenting in a multimeric form. These results
expand the pharmacological profile of tacrine hybrids and
may also help to design new chemical entities targeting tau
protein.
1
3
(4H, m, H-2 and H-3); C (CDCl , 75 MHz): d = 159.4 (C,
3
C-12), 146.6 (C, C-13), 141.4 (C, C-9), 129.2 (C, C-11),
128.8 (CH, C-6), 128.5 (CH, C-5), 126.4 (CH, C-7), 125.3
(C, C-14), 123.6 (CH, C-8), 34.1 (CH , C-4), 27.4 (CH ,
2
2
?
C-1), 22.6 (CH , C-3, C-2). ESI–MS: m/z [M ? 1] 218.25.
2
N-(1, 2, 3, 4-tetrahyoacridin-9-yl) hexane-1, 6-diamine
(B1, Scheme 1): brown oily liquid (This compound was
prepared as follows: In a round-bottomed flask (100 ml)
equipped with a magnetic stirrer, 9-chloro-1, 2, 3, 4-tetrahy-
droacridine (9 mmol, 1.95 g), sodium iodine (1.8 mmol,
Experimental section
300 mg), and phenol (54 mmol, 5.1 g) were stirred at 90 °C
Chemistry
for 2 h and then hexane-1, 6-diamine (63 mmol, 7.32 g) was
added. The reaction temperature was raised to 120 °C and
maintained 4 h. The mixture was cooled to room temperature
The reagents used in the synthesis were purchased from
Sinopharm Chemical Reagent Co. Ltd., and were used
without further purification unless otherwise indicated. All
the reactions were monitored using TLC. Column chro-
matography was performed using silica gel (200–300
mesh). All yields of the products refer to isolated yield.
Melting points were determined on YRT-3 apparatus
and uncorrected. IR spectra were obtained on a Thermo
and diluted with CHCl and made basic with 10 % NaOH
3
solution. The organic layer was washed with water and brine
and dried over anhydrous MgSO . The filtrate was evaporated
4
and the residue was purified on silica gel column chroma-
tography (CH Cl /MeOH/NH ꢀH O = 9:1:0.1) to give the
2
2
3
2
brown oily product, 1.86 g, 70 % yield); IR (film) t
max
3,359.0, 2,929.6, 1,563.3, 1,131.8, 764.4 cm ; H NMR
-1 1
1
Scientific Nicolet 8700 system. H NMR spectra were
(CDCl , 300 MHz): d = 7.90–7.97 (2H, m, H-5, H-8),
3
obtained on a Bruker Avance 300 (300 MHz) spectra or
Agilent VNMRS 600 (600 MHz) with TMS as the internal
standard, and chemical shifts were recorded in ppm along
with coupling constants (J values) in Hz. Multiplicities
were designated as singlet (s), broad singlet (br), doublet
7.52–7.58 (1H, m, H-6), 7.32–7.37 (1H, m, H-7), 3.98 (2H, br,
NH ), 3.45–3.52 (2H, m, CH NH), 3.07 (2H, m, H-4),
2
2
2.66–2.72 (4H, m, H-1, CH NH ), 1.91–1.95 (4H, m, H-2,
2
2
H-3), 1.62–1.72 (2H, m, CH CH NH ), 1.48–1.69 (6H, m,
2
2
2
1
3
(CH ) CH CH NH ); C (CDCl , 75 MHz): d = 158.4 (C,
2
3
2
2
2
3
(
d), triplet (t), and multiplet (m). Mass spectra (MS) were
C-12), 150.7 (C, C-9), 147.1 (C, C-13), 128.6 (CH, C-6),
128.3 (CH, C-5), 123.6 (CH, C-7), 122.7 (CH, C-8), 120.1 (C,
C-14), 115.9 (C, C-11), 51.8 (CH , CH NH), 49.1 (CH ,
obtained by electrospray ionization (ESI) at 70 eV in a
Finnigan LCQAdvantage MAX spectrometer with direct
insertion probe. Elemental analysis (EA) was performed on
an Elementar Analysensysteme GmbH Vario EL.
2
2
2
CH NH ), 33.9 (CH , C-4), 32.1 (CH , CH CH NH ), 31.2
2
2
2
2
2
2
2
(CH , CH CH NH), 26.8 (CH , CH CH CH NH ), 25.6
2
2
2
2
2
2
2
2
(CH , CH CH CH NH), 24.5 (CH , C-1), 22.9 (CH , C-3,
2 2 2 2 2 2
Synthesis of staring materials (compound A, B, C
in Scheme 1 and compound D in Scheme 2)
C-2).
N-(1, 2, 3, 4-tetrahyoacridin-9-yl) butane-1, 4-diamine
(B2, Scheme 1): brown oily liquid (Prepared following the
9
-chloro-1, 2, 3, 4-tetrahydroacridine (A, Scheme 1): pale
general procedure of B1 and starting from butane-1,
4-diamine (63 mmol, 5.55 g). The brown oily product B2
yellow solid (This compound was prepared as follows: To a
1
23

Med Chem Res
was obtained by chromatography (CH Cl /MeOH/NH ꢀ
124.9 (C, C-12, C-13), 42.9 (CH , CH Cl), 31.3(CH ,
2 2 2
2
2
3
H O = 10:1:0.1), 1.62 g, 67 % yield); IR (film) t
COCH ), 27.3 (CH , CH CH Cl).
2 2 2 2
2
max
-1 1
,358.1, 2,931.2, 1,561.5, 1,129.8, 768.4 cm ; H NMR
3
1-(10H-phenothiazin-10-yl)-2-(piperazin-1-yl) ethanone
(D, Scheme 2): white solid (This compound was prepared
as followings: To a solution containing chloroacetyl phe-
nothiazine (7.2 mmol, 2.0 g), piperazine (36 mmol, 3.1 g),
and potassium iodine (7.2 mmol, 1.2 g) in N, N-dimethyl
formamide (100 ml) was carefully added anhydrous
K CO (7.2 mmol, 0.995 g) at room temperature. After
(
CDCl , 300 MHz): d = 7.89–7.96 (2H, m, H-5, H-8),
3
7
.53–7.57 (1H, m, H-6), 7.32–7.36 (1H, m, H-7), 4.01 (2H,
br, NH ), 3.46–3.50 (2H, m, CH NH), 3.11 (2H, m, H-4),
2
2
2
.73–2.79 (4H, m, H-1, CH NH ), 1.97–1.99 (4H, m, H-2,
2 2
1
H-3), 1.39–1.63 (4H, m, (CH ) CH NH ); C (CDCl3,
3
2
2
2
2
7
5 MHz): d = 158.0 (C, C-12), 150.1 (C, C-9), 147.1 (C,
C-13), 128.7 (CH, C-6), 128.1 (CH, C-5), 123.6 (CH, C-7),
22.3 (CH, C-8), 120.1 (C, C-14), 115.9 (C, C-11), 51.5
CH , CH NH), 48.9 (CH , CH NH ), 33.6 (CH , C-4),
2
3
about 10 h reaction period with stirring, TLC analysis
revealed that chloroacetyl phenothiazine was consumed. N,
N-dimethyl formamide and excess piperazine were
removed under vacuum condition. The residue was poured
into water and extracted with ethyl acetate; the organic
layer was combined and washed with water and brine,
dried over anhydrous Na SO , filtered, and evaporated. The
1
(
2
2
2
2
2
2
3
1.0 (CH , CH CH NH ), 29.8 (CH , CH CH NH), 24.8
2 2 2 2 2 2 2
(
CH , C-1), 22.8 (CH , C-3, C-2).
2 2
2
-chloro-1-(10H-phenothiazin-10-yl) ethanone (C1,
Scheme 1): off-white solid (This compound was prepared as
follows: A solution containing phenothiazine (26 mmol,
2
4
desired product D was obtained as white solid, 1.99 g,
5
.248 g), 2-chloroacetyl chloride (66 mmol, 7.45 g, 5.5 ml),
and triethylamine (26 mmol, 4 ml) in dichloromethane
100 ml) was refluxed with stirring. After about 2–3 h reflux
85 % yield); mp 140.8–141.5 °C; IR (KBr) tmax 3,316.6,
1,668.6, 1,460.0, 1,325.1, 1,261.2, 1,132.3, 773.7 cm ; H
-
1 1
0
(
NMR (CDCl3, 300 MHz): d = 7.51–7.56 (2H, m, H-1 ,
0
0
0
period, TLC analysis revealed that all phenothiazine were
consumed. The reaction mixture was cooled to room tem-
perature and then washed successively with 5 % aqueous
sodium bicarbonate, 5 % hydrochloric acid, and water. The
H-9 ), 7.41–7.46 (2H, m, H-4 , H-6 ), 7.19–7.34 (4H, m,
0
0
0
0
H-2 , H-3 , H-7 , H-8 ), 3.29(2H, s, CH CO), 2.71–2.74
2
(4H, m, CH NCH ), 2.39–2.32 (4H, m, CH NHCH ), 1.99
2
2
2
2
1
3
(1H, br, NH); C NMR (CDCl , 75 MHz): d = 168.6 (C,
3
0 0 0 0
solution was dried over MgSO , filtered, and evaporated.
4
CON), 138.7 (C, CH, C-11 , C-14 , C-4 , C-6 ), 127.8 (CH,
0 0 0 0 0 0
C-2 , C-8 , C-1 , C-9 ), 126.9 (CH, C-3 , C-7 ), 126.7 (C,
The crude product was purified by flash column chroma-
tography with dichloromethane as elute and recrystallized
from petroleum ether/dichloromethane (2:1). Finally, the
desired product C1 was obtained as off-white solid, 3.6 g,
0
0
C-12 , C-13 ), 60.9 (CH , COCH ), 54.0 (CH , NCH ),
2
2
2
2
?
45.9 (CH , NHCH ). ESI–MS: m/z [M ? 1] 326.1. Anal.
2
2
Calcd. for C H N OS: C, 66.43; H, 5.88; N, 12.91.
18 19 3
5
0 % yield.); mp 110.3-111.0 °C; IR (KBr) tmax 1,693.1,
Found: C, 66.40; H, 5.78; N, 12.87.
-
1 1
1
,671.7, 1,335.1, 1,247.9, 1,170.3, 1,125.6, 761.6 cm ; H
NMR (CDCl , 300 MHz): d = 7.60 (2H, d, J = 7.8 Hz,
Synthesis of the desired products (T5, T5-B, T18,
3
H-1, H-9), 7.48 (2H, d, J = 7.8 Hz, H-4, H-6), 7.33-7.38
T18-B in Scheme 1 and T22 in Scheme 2)
(
2H, m, H-2, H-8), 7.24–7.30 (2H, m, H-3, H-7), 4.18 (2H,
3
1
s, CH Cl); C (CDCl , 75 MHz): d = 165.4 (C, CON),
1-(10H-phenothiazin-10-yl)-2-(6-(5, 6, 7, 8-tetrahydroacridin-
9-ylamino) hexylamino) ethanone (T5, Scheme 1): yellow
foam (This compound was prepared as follows: To a solution
of B1 (3 mmol, 891 mg) in dry dichloromethane, potassium
carbonate(3 mmol, 416 mg)andpotassiumiodine(0.3 mmol,
50 mg) were added, the resulting mixture was stirred at room
temperature for 1 h. Then, C1 (3 mmol, 828 mg) was added
and the reaction mixture was refluxed for 10–12 h. Then, the
mixture was cooled to room temperature and water was
poured, the organic layerwas combined andwashedwith water
and brine, dried over Na SO , filtered, and evaporated. The
2
3
1
37.8 (C, CH, C-11, C-14, C-4, C-6), 128.1 (CH, C-2, C-8),
1
27.3 (CH, C-1, C-9, C-3, C-7), 126.5 (C, C-12, C-13), 41.8
(
CH , CH Cl).
2 2
4-chloro-1-(10H-phenothiazin-10-yl) butan-1-one (C2,
Scheme 1): off-white solid (Prepared following the general
procedure of C1 and starting from 4-chlorobutanoyl chlo-
ride (66 mmol, 9.3 g). The crude product was purified by
column chromatography with petroleum ether/ethyl acetate
(
4:1) as elute, C2 was obtained as off-white solid, 3.3 g,
4
1
2 % yield); mp 118.0–118.8 °C; IR (KBr) tmax 1,693.4,
,672.0, 1,336.1, 1,248.5, 1,170.3, 1,126.8, 762.0 cm ; H
2
4
-1 1
crude product was purified by column chromatography
NMR (CDCl , 300 MHz): d = 7.58 (2H, d, J = 7.6 Hz,
H-1, H-9), 7.47 (2H, d, J = 7.6 Hz, H-4, H-6), 7.36–7.38
(CH Cl /MeOH/NH ꢀH O = 15:1:0.1). The desired product
3
2
2
3
2
T5 was obtained as yellow foam, 338 mg, 21 % yield); IR
-1 1
(
2H, m, H-2, H-8), 7.26–7.30 (2H, m, H-3, H-7), 3.43 (2H,
(KBr) tmax 3,352.9, 3,061.3, 1,679.8, 1,457.8, 763.1 cm ; H
t, J = 7.5 Hz, CH Cl), 2.26 (2H, t, J = 4.8 Hz, COCH ),
2
NMR (CDCl , 300 MHz): d = 8.09–8.21 (1H, m, H-5),
2
3
1
3
2
.01 (2H, m, CH CH Cl);
2
C (CDCl , 75 MHz):
3
7.93–7.96 (1H, m, H-8), 7.54–7.63 (1H, m, H-6), 7.14–7.48
(9H, m, H-7, Ar–H in phenothiazine), 4.30 (1H, br, NH), 3.63
(2H, s, COCH ), 3.45–3.49 (2H, m, CH NHAr), 3.11 (2H, m,
2
d = 172.5 (C, CON), 135.9 (C, CH, C-11, C-14, C-4, C-6),
28.2 (CH, C-2, C-8), 126.1 (CH, C-1, C-9, C-3, C-7),
1
2
2
123

Med Chem Res
H-4), 2.53–2.64 (4H, m, H-1, CH NH), 1.89–1.91 (4H, m,
2
128.1 (C, Ar), 124.5 (C, Ar), 118.9 (C, Ar), 115.3 (C, Ar),
50.9 (CH , NHCH ), 49.3 (CH , CH NH), 45.0 (CH ,
H-2, H-3), 1.62–1.73 (2H, m, CH CH NH), 1.18–1.26 (6H,
2
2
2
2
2
2
2
1
m, (CH ) CH CH NH); C NMR (CDCl , 75 MHz):
3
CH NHAr), 36.9 (CH , C-4), 31.7 (CH , COCH ), 27.9
2 2 2 2
2
3
2
2
3
d = 164.7 (C, C-12), 163.5 (C, CON), 148.3 (C, C-9), 145.2
(COCH CH CH NHCH CH CH ), 24.9 (CH , C-1), 23.0
2 2 2 2 2 2 2
0
0
0
0
?
(CH , C-2, C-3); ESI–MS: m/z [M ? H] 537.3. Anal.
(
C, C-13), 133.1 (C, C-11 , C-14 ), 132.0 (CH, C-4 , C6 )
2
1
5
28.1 (C, Ar), 124.3 (C, Ar), 118.5 (C, Ar), 114.9 (C, Ar),
0.9 (CH , COCH ), 48.6 (CH , CH NHAr), 45.7 (CH ,
Calcd. for C H N OS: C, 73.85; H, 6.76; N, 10.44. Found
33 36 4
C, 73.83; H, 6.71; N, 10.39.
0
2
2
2
2
2
CH NH), 35.2 (CH , C-4), 31.2 (CH , CH CH NHAr), 30.3
2
4,4 -(4-(5,6,7,8-tetrahydroacridin-9-ylamino)butylazanediyl)
2
2
2
2
(
CH , CH CH NH), 26.9 (CH , CH CH CH CH NH), 24.0
2
bis(1-(10H-phenothiazin-10-yl)butan-1-one)
(T18-B,
2
2
2
2
2
2
2
?
(
CH , C-1), 22.5 (CH , C-2, C-3); ESI–MS: m/z [M ? 1]
2
Scheme 1): yellow foam (In the preparation process of T18,
another product T18-B was obtained as yellow foam by
column chromatography, 675 mg, 28 % yield); IR (KBr)
2
5
37.3; Anal. Calcd. for C H N OS: C, 73.85; H, 6.76; N,
33 36 4
1
0.44. Found C, 73.80; H, 6.65; N, 10.43.
0
-1
1
2,2 -(6-(5,6,7,8-tetrahydroacridin-9-ylamino)hexylazanediyl)
tmax 3,360.5, 3,058.2, 1,675.7, 1,463.5, 765.1 cm ; H
bis(1-(10H-phenothiazin-10-yl)ethanone)(T5-B, Scheme 1):
yellow foam (In the preparation process of T5, another
product T5-B was obtained as a yellow foam by column
NMR (CDCl , 600 MHz): d = 8.58 (1H, d, J = 8.5 Hz,
3
H-5), 8.09 (1H, d, J = 8.6 Hz, H-8), 7.68–7.72 (1H, m, H-6),
7.33-7.48 (9H, m, H-7, Ar–H in phenothiazine), 7.16–7.28
(8H, m, Ar–H in phenothiazine), 5.48 (1H, m, NHAr),
chromatography, 628 mg, 27 % yield); IR (KBr) t
max
1 1
-
3
,356.9, 3,059.7, 2,929.7, 1,678.3, 1,460.6, 760.8 cm ; H
3.45–3.60 (2H, m, CH NHAr), 3.11-3.19 (2H, m, H-4),
2
NMR (CDCl , 600 MHz): d = 8.63 (1H, d, J = 8.5 Hz,
2.48–2.60 (8H, m, H-1, (CH ) N), 2.25–2.34 (4H, m,
2 3
3
H-5), 8.11 (1H, d, J = 8.6 Hz, H-8), 7.68–7.72 (1H, m, H-6),
CH CO), 1.66–1.70 (8H, m, H-2, H-3, CH CH CO),
2
2
2
1
3
7
.37–7.47 (9H, m, H-7, Ar–H in phenothiazine), 7.16–7.29
8H, m, Ar–H in phenothiazine), 5.57 (1H, m, NHAr),
.61–3.75 (6H, m, COCH , COCH , CH NH), 3.26–3.38
1.35–1.57 (m, 4H, CH CH CH NH); C NMR (CDCl3,
2 2 2
(
150 MHz): d = 175.0 (C, CON), 164.9 (C, C-12), 146.3 (C,
C-9, C-13), 130.9 (C, Ar), 129.1 (C, Ar), 125.3 (C, Ar), 119.5
(C, Ar), 54.9 (CH , CH N), 53.1 (CH , CH N), 46.8 (CH ,
3
2
2
2
(
2H, m, H-4), 2.48–2.60 (4H, m, H-1, CH N), 1.86–1.89 (4H,
2
2
2
2
2
2
m, H-2, H-3), 1.66–1.70 (2H, m, CH CH NH), 1.29–1.35
2
CH NH), 34.2 (CH , C-4), 30.8 (CH , CH CO), 27.6 (CH ,
2 2 2 2 2
2
(
2H, m, CH CH N), 1.17–1.22 (4H, m, CH CH CH
2
CH CH NHAr), 25.9 (CH , CH CH N), 24.3 (CH , C-1),
2 2 2 2 2 2
2
2
2
2
1
3
?
21.9 (CH , C-2, C-3); ESI–MS: m/z [M ? 1] 804.3. Anal.
CH NH); C NMR (CDCl , 150 MHz): d = 169.8 (C, C-12,
2
3
2
CON), 138.3 (C, Ar), 127.9 (C, Ar), 126.9 (C, Ar), 126.8 (C,
Calcd. for C H N O S : C, 73.19; H, 6.14; N, 8.71. Found
9 49 5 2 2
4
Ar), 124.0 (C, Ar), 123.4 (C, Ar), 55.0 (CH , COCH ), 53.9
2
C, 73.15; H, 6.11; N, 8.70.
2
(
(
(
CH , NCH ), 48.9 (CH , NHCH ), 31.2 (CH , C-4), 29.6
2
1-(10H-phenothiazin-10-yl)-2-(4-(5, 6, 7, 8-tetrahydro-
acridin-9-yl) piperazin-1-yl) ethanone (T22, Scheme 2):
brown oily liquid (Prepared following the general proce-
dure for the synthesis of B1 or B2. The starting material of
the reaction included 9-chloro-1, 2, 3, 4-tetrahydroacridine
(2 mmol, 435 mg), sodium iodine (0.3 mmol, 45 mg),
phenol (8 mmol, 752 mg), and Compound D (2.5 mmol,
814 mg). The brown oily product T22 was obtained by
column chromatography (CH Cl /MeOH/NH ꢀH O =
2
2
2
2
CH , NHCH CH ), 28.9 (CH , NHCH CH CH ), 27.6
2
2
2
2
2
2
2
CH , NCH CH ), 26.6 (CH , NCH CH CH ), 24.3 (CH ,
2
2
2
2
2
2
2
2
C-1), 22.5 (CH , C-3), 21.9 (CH , C-2); ESI–MS: m/z
2
2
?
[
M ? 1] 776.4; Anal. Calcd. for C H N O S : C, 72.74;
47 45 5 2 2
H, 5.84; N, 9.02. Found C, 72.70; H, 5.73; N, 9.00.
-(10H-phenothiazin-10-yl)-4-(4-(5, 6, 7, 8-tetra-
1
hydroacridin-9-ylamino) butylamino)butan-1-one (T18,
Scheme 1): yellow foam (Prepared following the general
procedure of T5 and starting from B2 (3 mmol, 807 mg) and
C2 (3 mmol, 914 mg). The crude products were purified
by column chromatography (CH Cl /MeOH/NH ꢀH O =
2
2
3
2
15:1:0.1), 679 mg, 67 % yield); IR (KBr) t
2,925.6,
max
-1 1
1,682.6, 1,460.1, 1,262.6, 1,126.6, 761.3 cm ; H NMR
(CDCl , 600 MHz): d = 8.05–8.09 (1H, m, H-5), 7.93-
2
2
3
2
3
1
5:1:0.1), the desired product T18 was obtained as yellow
7.99 (1H, m, H-8), 7.48–7.60 (6H, m, Ar–H), 7.25–7.38
foam, 306 mg, 19 % yield); IR (KBr) t
1
3,357.7, 3,058.2,
max
-1 1
,680.7, 1,460.0, 760.1 cm ; H NMR (CDCl , 600 MHz):
(4H, m, Ar–H in phenothiazine), 3.47 (2H, s, CH CO),
2
3.19–3.27 (4H, m, CH NAr), 3.09–3.11 (2H, t, J = 6.6 Hz,
2
3
d = 8.07–8.15 (1H, m, H-5), 7.95–7.99 (1H, m, H-8),
.60–7.68 (2H, m, H-6, H-7), 7.11–7.51 (8H, m, Ar–H in
H-4), 2.86–2.89 (2H, t, J = 6.6 Hz, H-1), 2.58–2.69 (4H,
7
m, CH N), 1.91–1.93 (2H, m, H-2), 1.82–1.85 (2H, m,
2
1
3
phenothiazine), 4.36 (1H, br, ArNH), 3.35–3.43 (2H, m,
H-3); C NMR (CDCl , 150 MHz): d = 168.9 (C, C-12),
3
CH NHAr), 2.98–3.11 (2H, m, H-4), 2.54–2.63 (6H, m,
2
160.5 (C, CON), 153.0 (C, C-9), 147.9 (C, C-13), 138.9 (C,
0
0
CH NHCH , H-1), 2.21–2.35 (2H, m, COCH ), 1.75–1.91
2
C-11 , C-14 ), 128.8 (C, Ar), 128.5 (C, Ar), 128.1 (C, Ar),
127.9 (C, Ar), 127.2 (C, Ar), 127.0 (C, Ar), 126.0 (C, Ar),
125.0 (C, Ar), 124.1 (C, Ar), 60.8(CH , CH CO), 53.9
2
2
(
6H, m, H-2, H-3, CH CH CO), 1.42–1.61 (4H, m,
2
2
1
3
CH CH CH NHAr);
2
C
NMR (CDCl3, 150 MHz):
2
2
2
2
d = 174.5 (C, CON), 164.0 (C, C-12), 147.0 (C, C-9), 145.2
(CH , CH N), 50.5 (CH , CH N), 34.2 (CH , C-4), 32.1
2 2 2 2 2
0
0
0
0
?
(
C, C-13), 133.1 (C, C-11 , C-14 ), 132.1 (CH, C-4 , C-6 ),
(CH , C-1), 22.9 (CH , C-2, C-3); ESI–MS: m/z [M ? 1]
2 2
1
23

Med Chem Res
5
07.3. Anal. Calcd. for C H N OS: C, 73.49; H, 5.97; N,
31 30 4
Ab fibril (fAb) binding study by surface plasmon resonance
(SPR)
1
1.06. Found: C, 73.40; H, 5.86; N,11.05.
A BIAcore 3,000 (Biacore Inc., USA) equipped with four
flow cell channels on sensor chip was used for real-time
binding studies. Carboxymethylated dextran 5 (CM5) sensor
chip was used in this experiment for fAb(1–40). fAb(1–40)
stored at 4 °C was centrifuged at 4 °C for 2 h at 16,000 g.
fAb(1–40) precipitated completely, and the pellet was
resuspended in water, sonicated, diluted with 10 mM sodium
acetate (pH 4.0), and immobilized immediately on CM5
sensor chip by amine coupling. fAb(1–40) was immobilized
on the CM5 chip in the second channel, and the first channel
was used as a reference(without immobilization of
fAb(1–40)). The carboxymethyl dextran surface was acti-
Biological evaluation
In vitro estimation of acetylcholinesterase inhibitory
activity
The preliminary anti-acetylcholinesterase activity for the
synthesized compounds was performed according to AChE
enzyme-linked immunosorbent assay (ELISA) Kit (96T,
R&D system) using tacrine and donepezil as reference
compound. AChE was obtained from homogenates of rat
brain. Test compounds (T5, T5-B, T18, T18-B, T22) were
0
vated using 70lL of a mixture of 0.4 M N-ethyl-N -[3-
dissolved in DMSO and diluted with DMSO-H O and rat
2
(dimethylamino)propyl] carbodiimide hydrochloride(EDC)
and 0.1 M N-hydroxysuccinimide (NHS) at an injection rate
of 10 lL/min, followed by the injection of fAb(1–40) into
channel 2 (70 lL of fAb diluted with 600 lL of PH 4.0
sodium acetate buffer and injected at a rate of 5 lL/min).
After fAb immobilization, the remaining activated groups
were blocked with 70 lL of 1.0 M ethanolamine (pH 9.0).
The reference cell was prepared by the above described
amine coupling without the addition of fAb. The running
buffer solution contained 50 mM phosphate buffer (pH 7.5),
brain homogenates, the final concentration ranged from 0.01
to 100 lM. Blank was prepared using rat brain homogenates
instead of the test sample, and all other procedures were
similar to those used in the case of the sample mixture.
Absorbance (OD) was measured at 450 nm using microplate
reader (DG5033A, Nanjing Huadong Electronics Group Co.
Ltd.,). The AChE content in sample is positively correlated
with OD. AChE content in the negative control hole as
1
00 %, the reduction of AChE content in test samples hole is
the enzyme inhibition rate. Experiments were done in tri-
plate. Data are expressed as mean ± standarddeviation (SD)
and were analyzed using SPSS 19.0 statistical software.
1
00 mM NaCl, 0.3 mM EDTA, and 0.05 % Tween 20, and
the washing solvent contained 1 % DMSO.
After immobilization, test compounds with different
concentrations were injected into the cell, and binding was
observed. Each binding experiment was repeated three
times, and the reaction was performed 25 °C using running
buffer solution mentioned above. Test compound (T5) dis-
solved in DMSO was diluted with running buffer to a final
concentration of 0.625–40 lM and injected over the refer-
ence and fAb-immobilized flow cells at a flow rate of 30 lL/
min. The surface of sensor chip immobilized with fAb was
regenerated with a mixture of 0.025 % sodium laurel sul-
fated (SDS) and 25 mM NaOH. Data from the reference cell
without fAb immobilization were subtracted from raw data.
The running buffer injection using the same method was also
subtracted as a double reference (Yan et al., 2007).
In vitro estimation of P-Tau inhibitory activity
The N2a cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10 % fetal bovine serum
(
GIBCO). Cells were grown in a 100 % humidified incubator
at 37 °C with 5 % CO and passaged 2–3 days prior to use.
2
Logarithmic phase’s N2a cells were seeded in 96-well plates
at a density of 10 cells per well. After growing for 12 h, the
4
cells were incubated in DMEM containing 60 nM okadaic
acid (OA, Sigma Chemical Co.) for 12 h. Then, test com-
-
4
pound (or other compound) with final concentration of 10
-7
-
5
-6
M, 10 M, 10 M, 10 M, or 20 lM methylene blue
Rember) were added to the culture medium and incubated
(
with the cells for 24 h. Cells were washed twice with ice-cold
PBS, homogenized in RIPA Lysis buffer (Solarbio Science
Acknowledgments This research was financially supported by the
Anhui Province Science and Technology Project (No. 1101041025)
and Hefei University of Technology Major Pre-research Special
Project (No. 2010HGZY0011).
&
Technology Co.) for 5 min on ice, and the lysate was
centrifugated at 14,000g for 5 min at 4 °C prior to ELISA
detection. For quantitative analysis of P-Tau in cell lysates,
the assay was performed as described in the instructions of
the P-Tau ELISA Kit (Innogenetics NV Co.). Experiments
were done in triplate. Data are expressed as mean ± stan-
dard deviation (SD) and were analyzed using SPSS 19.0
statistical software.
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