Novel multipotent phenylthiazole-tacrine hybrids for the inhibition of cholinesterase activity, β-amyloid aggregation and Ca2+ overload

Article abstract of DOI:10.1016/j.bmc.2012.08.040

In this study, a series of multipotent phenylthiazole-tacrine hybrids (7a-7e, 8, and 9a-9m) were synthesized and biologically evaluated. Screening results showed that phenylthiazole-tacrine hybrids were potent cholinesterase inhibitors with pIC₅₀

Full text of DOI:10.1016/j.bmc.2012.08.040

Bioorganic & Medicinal Chemistry 20 (2012) 6513–6522
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Bioorganic & Medicinal Chemistry
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Novel multipotent phenylthiazole–tacrine hybrids for the inhibition
of cholinesterase activity, b-amyloid aggregation and Ca2+ overload
Yue Wang a, Fang Wang a,d, Jun-Ping Yu c, Feng-Chao Jiang b, Xin-Lei Guan a, Can-Ming Wang a, Lei Li b,
Hui Cao a, Ming-Xing Li a, Jian-Guo Chen a,d,
⇑
a Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China
b Department of Medicinal Chemistry, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China
c State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China
d The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a series of multipotent phenylthiazole–tacrine hybrids (7a–7e, 8, and 9a–9m) were synthe-
sized and biologically evaluated. Screening results showed that phenylthiazole–tacrine hybrids were
potent cholinesterase inhibitors with pIC50 (ꢀlogIC50) value ranging from 5.78 0.05 to 7.14 0.01 for
acetylcholinesterase (AChE), and from 5.75 0.03 to 10.35 0.15 for butyrylcholinesterase (BuChE).
The second series of phenylthiazole–tacrine hybrids (9a–9m) could efficiently prevent Ab1–42 self-aggre-
gation. The structure–activity relationship revealed that their inhibitory potency relied on the type of
middle linker and substitutions at 40-position of 4-phenyl-2-aminothiazole. In addition, 7a and 7c also
displayed the Ca2+ overload blockade effect in the primary cultured cortical neurons. Consequently, these
compounds emerged as promising molecules for the therapy of Alzheimer’s disease.
Received 14 June 2012
Revised 9 August 2012
Accepted 10 August 2012
Available online 30 August 2012
Keywords:
Alzheimer’s disease
Phenylthiazole–tacrine hybrids
Cholinesterase inhibition
b-Amyloid aggregation
Ca2+ overload
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
disease. Thus, efficient therapy is more likely to be achieved by
drugs that incorporate several pharmacological effects into a single
Alzheimer’s disease (AD), the most common form of dementia
in the elderly, is characterized by deposits of b-amyloid peptide
(Ab), neurofibrillary tangles, massive loss of neurons and severe
cognition impairment.1 Since cholinergic dysfunction contributes
to the symptoms of AD patients, most of the commercialized drugs
are mainly based on increasing cholinergic neurotransmission by
inhibiting cholinesterase (doepenzil, rivastigmine, tacrine and
galantanmine).2
molecular entity.7–9 Tacrine is the first approved cholinesterase
inhibitor by the FDA. Although, its side effects, its modification is
still of great interest.10,11 Some multipotent compounds have been
developed by conjugating tacrine with other active groups, such as
acetylcholinesterase/b-amyloid-directed compounds (Fig. 1A and
B),12,13 dual-target inhibitors of cholinesterase/muscarinic M1
receptors (Fig. 1C),14 and dual-target inhibitors of cholinesterase/
Ca2+ channels (Fig. 1D).15,16
An abnormal extracellular accumulation of b-amyloid peptides
(Ab) is another major neuropathological feature in AD. Ab peptides
are the main component of amyloid plaques and are accumulated
Recently, it has been reported that phenylthiazolyl–hydrazide
derivatives could block the pathological aggregation of tau pro-
tein and disturbed the formation of paired helical filaments
(PHFs).17,18 Given that the phenylthiazole scaffold could inhibit
tau protein aggregation, we thought that its derivatives might
also display pharmacological action on blockade of Ab self-aggre-
gation. In addition, phenylthiazole derivatives have attracted our
interest because of their extensive biological actions, such as
protecting neurons from ischemic damage and anti-inflammatory
activity.19–21 In this context, we designed and synthesized a ser-
ies of novel multifunctional compounds by conjugating tacrine
with 4-phenyl-2-aminothiazole group through different middle
linkers (Fig. 2). The pharmacological effects of these novel com-
pounds were investigated by measurement of acetylcholinester-
ase (AChE) and butyrylcholinesterase (BuChE) inhibition, Ab1–42
through hydrolyzing Ab precursor protein (APP) by b- and c-secre-
tase.3,4 Ab soluble oligomers and the assembly of Ab aggregates
into fibrils are toxic to neurons.5 Compounds that are able to slow
or block the Ab polymerization process will serve as potential ther-
apeutic drugs for AD treatment.6
AD is a multifaceted neurodegenerative disorder. It is unlikely
that a unitary mechanism of action will provide a comprehensive
therapeutic approach to such multifaceted neurodegenerative
⇑
Corresponding author. Tel.: +86 27 83692636; fax: +86 27 83692608.
E-mail address: chenj@mails.tjmu.edu.cn (J.-G. Chen).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.040

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Y. Wang et al. / Bioorg. Med. Chem. 20 (2012) 6513–6522
Figure 1. Multipotent tacrine derivatives.
Figure 2. Design strategy of phenylthiazole–tacrine hybrids.
self-aggregation inhibitory activity, and Ca2+ overload blockade
effect.
thiazole derivatives reacting with succinic anhydride or glutaric
anhydride in THF. Intermediate 1 and 2 were synthesized accord-
ing to the published method.22 Reaction of 3a–3c or 4 with 1 in
the presence of 1-ethyl-3-(3-diethylaminopropyl) carbodiimide
(EDCꢁHCl) and hydroxybenzotriazole (HOBt) in DMF/CH2Cl2
yielded compound 7a–7e and 8. Compound 9a–9m were synthe-
sized following the convergent synthetic approach in Scheme 2.
2-Aminothiazole derivatives reacted with chloroacetyl chloride or
3-bromopropionyl chloride to produce 5a–5f. Reaction of 5a–5f
2. Results and discussion
2.1. Chemistry
The synthetic strategies to 7a–7e and 8 were illustrated in
Scheme 1. Intermediate 3a–3c and 4 were prepared from 2-amino-
Scheme 1. Synthesis of the first series of phenylthiazole–tacrine hybrids (7a–7e, 8). Reagents and conditions: (a) succinic anhydride or glutaric anhydride, THF, 65 °C, 7 h; (b)
EDCꢁHCl/HOBt, DMF/CH2Cl2, 25 °C, 12 h.

Y. Wang et al. / Bioorg. Med. Chem. 20 (2012) 6513–6522
6515
Scheme 2. Synthesis of the second series of phenylthiazole–tacrine hybrids (9a–9m). Reagents and conditions: (a) chloroacetyl chloride or 3-bromopropionyl chloride, Et3N,
THF/CH2Cl2, rt; (b) K2CO3/KI, DMF, 70 °C, 2 h.
Table 1
Inhibition of AChE and BuChE (pIC50 values) by phenylthiazole–tacrine hybrids and tacrine
Compd
pIC50 SEMa
BuChEb
Selectivity ratioc
Compd
pIC50 SEMa
Selectivity ratioc
AChEb
AChEb
BuChEb
7a
7b
7c
7d
7e
8
9a
9b
9c
9d
6.31 0.01
6.42 0.06
7.14 0.01
6.55 0.03
5.87 0.14
5.90 0.03
6.98 0.03
5.86 0.05
6.80 0.08
5.78 0.05
9.22 0.13
5.75 0.03
9.45 0.08
9.26 0.05
7.78 0.04
6.44 0.02
10.35 0.15
6.56 0.03
7.83 0.05
7.69 0.07
747
0.2
200
514
90
3.5
2117
5
9e
9f
9g
9h
9i
9j
9k
9l
9m
Tacrine
5.79 0.09
6.49 0.04
7.01 0.02
5.92 0.03
6.96 0.06
6.54 0.04
6.92 0.04
7.09 0.09
6.80 0.09
7.19 0.03
7.11 0.07
8.22 0.05
9.32 0.28
8.38 0.09
7.74 0.03
8.18 0.20
7.34 0.06
6.33 0.06
6.23 0.01
8.42 0.11
21
53
150
277
6.3
42
2.6
0.2
0.3
17
11
81
a
b
c
Data are the mean values of at least three determinations standard error of mean. pIC50 = ꢀlogIC50
AChE from electric eel and BuChE from equine serum were used.
Selectivity ratio = (IC50 of AChE)/(IC50 of BuChE).
.
with 2 in the presence of K2CO3/KI in DMF produced compound
9a–9m.
2.2. Cholinesterase inhibition
Inhibition of AChE and BuChE were determined by the Ellman
method, selecting tacrine as reference compound.23 All phenyl-
thiazole–tacrine hybrids (Table 1) were potent inhibitors of AChE
with pIC50 (ꢀlogIC50) ranging from 5.78 0.05 to 7.14 0.01. In
the first series (7a–7e, and 8), the length of middle linker affected
the AChE inhibitory potency. Compound 7c displayed the optimal
AChE inhibitory potency (AChE pIC50 = 7.14 0.01), characteristic
of the length of 10 spacer atoms (no counting N atoms). Compared
with H at 40-position of 4-phenyl-2-aminothiazole (7a, AChE
pIC50 = 6.31 0.01), substituting with Cl at 40-position afforded a
slight reduction of AChE inhibition (7e, pIC50 = 5.87 0.14). When
changing 4-phenyl-2-aminothiazole (7a) with 4,5,6,7-tetra-
hydrobenzo[d]thiazol-2-amine (8), both of the AChE and BuChE
pIC50 were decreased. In the second series (9a–9m), the AChE
inhibitory potency was sensitive to substitutions at 40-position of
4-phenyl-2-aminothiazole. Compared with H at 40-position of 4-
phenyl-2-aminothiazole (9a, 9c, 9f, 9i, 9l), substituting with
OCH3 or Cl at 40-position (9b, 9d, 9e, 9h, 9j, 9k, 9m) were less
favorable, except 9g (pIC50 = 7.01 0.02) which was slightly potent
than 9f (pIC50 = 6.49 0.04). Many reports revealed previously that
introduction a diamine side chain to 9-position of tacrine unit or
presence of Cl atom at 6-position of tacrine unit could improve
the AChE inhibitory activity. However, in this study, phenylthiaz-
ole–tacrine hybrids showed a decrease in the inhibitory activity
Figure 3. Kinetic study on the mechanism of AChE inhibition by 7c. Lineweaver–
Burk reciprocal plots of AChE initial velocity at increasing substrate concentration
(0.067–0.5 mM) in the absence of inhibitor and in the presence of 7c (20–80 nm)
are shown.

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Y. Wang et al. / Bioorg. Med. Chem. 20 (2012) 6513–6522
Table 2
Inhibition of Ab1–42 self-aggregation by phenylthiazole–tacrine hybrids
Compd
Inhibition of Ab aggregation % SEMa
Compd
Inhibition of Ab aggregation % SEMa
7a
7b
7c
7d
7e
8
9a
9b
9c
9d
12.5 3.6
23.7 1.6
35.8 5.8
9.7 3.0
16.4 3.1
ꢀ7.8 4.4
49.4 3.8
63.8 4.1
62.4 1.2
66.1 2.6
9e
9f
9g
9h
9i
9j
9k
9l
63.3 2.6
51.8 4.1
59.2 3.6
57.1 1.3
54.2 2.8
58.2 0.8
39.6 1.2
69.6 1.1
72.0 3.4
57.1 5.7
9m
Propidium iodide
H3CO
N
N
11.2 3.6
11.6 2.2
NH2
NH2
S
S
a
Values are expressed as mean standard error of the mean from three independent measurements, each performed in duplicate. Inhibition of Ab1–42 self-aggregation
with the tested compounds at concentration of 20 M.
l
against AChE. The reduced potency of phenylthiazole–tacrine hy-
brids against AChE might result from an unsuitable linker type or
conformation, which wasn’t in favor for the nitrogen atom interac-
tion with the mid-gorge recognition site of AChE or the phenyl-
thiazole moiety binding tightly with the peripheral site of AChE.
Considering the therapeutic benefits of BuChE, it was notewor-
thy that a possible role of BuChE inhibition could contribute to dis-
ease modification.24–26 Phenylthiazole–tacrine hybrids inhibited
BuChE with pIC50 value ranging from 5.75 0.03 to 10.35 0.15.
In some cases, their inhibitory potency (7a, 7c, 7d, 9a, 9g, 9h) were
higher than or comparable to tacrine. Regarding the selectivity pro-
file, phenylthiazole–tacrine hybrids were more potent inhibitors of
BuChE than that of AChE, except 7b, 9l and 9m. Particularly, com-
pound 9a (BuChE pIC50 = 10.35 0.15) showed the highest potency
and selectivity for BuChE.
2.3. Kinetic characterization of AChE inhibition
Figure 4. The concentration–effect curves of 9l (4) and 9m (N) determined by the
ThT assay.
The mechanism of AChE inhibition was investigated in vitro by
using compound 7c, the most potent inhibitor of the two series. An
analysis of the LineweaverꢀBurk reciprocal plots of 7c against
AChE revealed that there were an increasing slope and an increas-
ing intercept with higher inhibitor concentration (Fig. 3). This re-
sult indicate that 7c is characterized by a linear mixed-type of
enzyme inhibition and could bind simultaneously with the cata-
lytic and the peripheral sites of AChE. This inhibitory pattern was
similar to that of tacrine and other reported tacrine
derivatives.10,12,13
than the positive control propidium iodide. Among them, 9m
(72.0 3.4% at 20 lM) displayed the highest potency. The second
series (9a–9m) were generally more potent than the first series
(7a–7e, and 8), indicating that the inhibition efficacy seemed to de-
pend on the type of middle linker. Compared with H at 40-position
of 4-phenyl-2-aminothiazole (9a, 9c, 9f, 9i, 9l), substituting with
OCH3 at 40-position (9b, 9d, 9g, 9j, 9m) enhanced the potency of
Ab1–42 self-aggregation inhibition. 4-Phenyl-2-aminothiazole and
4-(4-methoxyphenyl)thiazol-2-amine, the monomeric parent
structure of phenylthiazole–tacrine hybrids, also displayed some
2.4. Inhibition of self-mediated Ab1–42 aggregation
extent of Ab1–42 self-aggregation inhibitory potency at 20 lM
(11.2 3.6% and 11.6 2.2%). Besides, we further investigated 9l
and 9m on Ab1–42 aggregation at different concentrations. The con-
centration-effect curves of 9l and 9m in Figure 4 indicated that
they could inhibit Ab1–42 self-aggregation in a concentration-
dependent manner.
2.4.1. Thioflavin T fluorescence assay
The ability of phenylthiazole–tacrine hybrids to reduce Ab1–42
self-aggregation was investigated by Thioflavin T (ThT) fluores-
cence assay, selecting propidium iodide as reference compound.
ThT can selectively concentrate in amyloid deposits and display
intensive fluorescence emission at 485 nm.27 As shown in Table 2,
the first series (7a–7e and 8) were found to be less pronounced to
2.4.2. Atomic force microscopy assay
Due to the nanoscale dimensions and the varied morphology of
aggregates, atomic force microscopy (AFM) has emerged as an
ideal tool for distinguishing structural features of the aggregate
forms.28 We used AFM as an imaging technique to measure the
aggregation process in the absence or presence of 9m. When
inhibit Ab1–42 self-aggregation at 20
lM, except 7c (35.8 5.8% at
20 M). When replacing 4-phenyl-2-aminothiazole (7a) with
l
4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine (8), 8 turned out to be
completely inactive. The second series (9a–9m) can efficiently
block Ab1–42 self-aggregation at 20
lM. Remarkably, nine of syn-
50
lM Ab1–42 was incubated alone in 10% DMSO phosphate buffer
thesized derivatives (9b–9e, 9g, 9h, 9j, 9l, 9m) were more potent
(pH 7.4) at 30 °C for 4 days, Ab1–42 converted into robust fibrils and

Y. Wang et al. / Bioorg. Med. Chem. 20 (2012) 6513–6522
6517
Figure 5. Morphological study of Ab1–42 aggregation by AFM: (A) AFM scan image of 50
l
M Ab1–42 incubated alone for 4 days at 30 °C; (B) AFM scan image of 50
l
M Ab1–42
incubated with 100 lM 9m for 4 days at 30 °C. Scale bars are 1 lm.
Figure 6. Effect of 1 l
M 7a, 7c, and nifedipine on the intracellular Ca2+ concentration induced by high K+ (30 mM) in the primary cultured cortical neurons. (A) Representative
curves of the F340/F380 ratio in the absence or in the presence of 7a, 7c, and nifedipine; (B) statistical analysis of the F340/F380 ratio in the absence (control, n = 20) or in the
presence of 7a (n = 19), 7c (n = 21), or nifedipine (n = 20). Experiments were performed from at least three different cell cultures. Data were expressed as mean SD. ##p <0.01
versus baseline, ⁄⁄p <0.01 versus control group.
large ordered aggregates (Fig. 5A). In contrast, no fibrils but some
small aggregates were detected when 100 M 9m was added to
50 M Ab1–42 solution at the same condition (Fig. 5B). Obviously,
9m effectively prevented the Ab1–42 self-aggregation. The AFM re-
sult of 9m was consistent with the above results of ThT.
ratio of F340/F380 from 0.52 0.04 to 1.31 0.24. In this experi-
ment, we found that 7a (n = 19) and 7c (n = 21) displayed the
Ca2+ overload blockade effect (Fig. 6). Compound 7c could decrease
the F340/F380 ratio from 1.31 0.24 to 0.64 0.06. 7a changed the
ratio of F340/F380 from 1.31 0.24 to 0.83 0.14. Unfortunately,
9m (n > 20) failed to block the increase of F340/F380 ratio, but
caused the ratio of F340/F380 persistent rise. Nifedipine, a calcium
channel antagonist, was selected as a positive control. Pretreated
l
l
2.5. Inhibition of Ca2+ overload
Dysregulation of intracellular calcium signaling has been impli-
cated in the pathogenesis of AD. Ca2+ overload can cause mitochon-
drial dysfunction, accumulation of Ab and tau phosphorylation.
Hence, inhibition of Ca2+ overload could serve as potential thera-
peutic approaches for AD prevention and treatment.29,30 To inves-
tigate the potential of Ca2+ overload blockade effect, we studied
whether 7a, 7c, and 9m displayed any effect on Ca2+ overload in-
duced by high K+ (30 mM) in the primary cultured cortical neurons.
The Fluo-2/AM-loaded neurons were incubated in the absence or
with 1 lM Nifedipine (n = 20) brought down the F340/F380 ratio
from 1.31 0.24 to 0.88 0.13.
3. Conclusion
In this study, a series of phenylthiazole–tacrine hybrids were
synthesized and biologically evaluated. Phenylthiazole–tacrine hy-
brids were potent inhibitors of cholinesterase with pIC50 value
ranging from 5.78 0.05 to 7.14 0.01 for AChE, and from
5.75 0.03 to 10.35 0.15 for BuChE. They were more potent
inhibitors of BuChE than that of AChE, except 7b, 9l and 9m. Com-
pound 9a (BuChE pIC50 = 10.35 0.15) was proved to be the high-
est potency and selectivity for BuChE. The kinetic characterization
presence of compounds (1 lM) for 5 min and then stimulated with
30 mM KCl solution. The F340/F380 fluorescence ratio was re-
corded and used as an indicator of the intracellular Ca2+ concentra-
tion. 30 mM KCl (n = 20) alone induced a significant increase in the

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Y. Wang et al. / Bioorg. Med. Chem. 20 (2012) 6513–6522
of 7c was analyzed by using Lineweaver–Burk plots, which re-
vealed that 7c was a mixed-type inhibitor and could bind simulta-
neously at the catalytic and the peripheral sites of AChE. Most of
phenylthiazole–tacrine hybrids showed a good inhibitory potency
on Ab1–42 self-aggregation. Their inhibitory effect depended on the
type of middle linker and substitutions at 40-position of 4-phenyl-
2-aminothiazole. Among them, 9m exhibited the highest inhibi-
tory potency. Additionally, 7a and 7c displayed the blockade effect
on Ca2+ overload in the primary cultured cortical neurons.
178.6, 175.5, 163.3, 163.0, 138.3, 137.6, 133.6 (2C), 132.2 (2C),
112.8, 35.1, 33.6.
4.2.4. 4-Oxo-4-(4,5,6,7-tetrahydrobenzo[d]thiazol-2-ylamino)
butanoic acid (4)
Milk-white powder, yield: 45%. 1H NMR (DMSO-d6, 400 MHz, d
ppm): 11.94 (br s, 2H), 2.62–2.58 (m, 4H), 2.54–2.50 (m, 4H), 1.78–
1.68 (m, 4H). 13C NMR (DMSO-d6, 100 MHz, d ppm): 174.0, 170.3,
155.1, 144.3, 121.4, 30.3, 28.9, 26.5, 23.4, 23.1, 22.7.
4.3. Compound 5a–5f were obtained according to the following
general procedure
4. Experimental
4.1. Chemistry
To a solution of 1 molar equiv of 2-aminothiazole derivatives
and 1.5 molar equiv of triethylamine in THF, 1.2 molar equiv of
chloroacetyl chloride or 3-bromopropionyl chloride in THF/
CH2Cl2 was added dropwise at 0 °C. The reaction mixture was
stirred at room temperature, and monitored by TLC (petroleum
ether/ethyl acetate = 2/1). After reaction complete, the solvent
was removed and the residue was purified by flash chromatog-
raphy (petroleum ether/ethyl acetate = 10/1) to yield the
product.
1H and 13C NMR spectra were recorded using TMS as the
internal standard in CDCl3 or DMSO-d6 with a Bruker spectrom-
eter at 400 and 100 MHz. The MS spectra were recorded on a
Finnigan LCQ Deca XPTM instrument with an ESI mass selective
detector. Reactions were monitored by thin layer chromatogra-
phy and MS spectrometer. Flash column chromatography was
performed with silica gel (200–300 mesh) purchased from Qing-
dao Haiyang Chemical Co. Ltd. The purities of targeted com-
pounds were confirmed by analytical HPLC on a HITACHI L-
2000 instrument and
a Diamonsil ODS-C18 HPLC column
4.3.1. 2-Chloro-N-(4-phenylthiazol-2-yl)acetamide (5a)
Off-white powder, yield: 45%. 1H NMR (CDCl3, 400 MHz, d
ppm): 10.6 (br s, 1H), 7.83 (d, J = 7.2 Hz, 2H), 7.45 (t, J = 7.2 Hz,
2H), 7.37 (t, J = 7.2 Hz, 1H), 7.02 (s, 1H), 4.06 (s, 2H). 13C NMR
(CDCl3, 100 MHz, d ppm): 164.3, 157.4, 150.2, 134.1, 128.9 (2C),
128.4, 126.2 (2C), 108.5, 41.9.
(250 ꢂ 4.6 mm, 5 m) at 340 nm, eluted with a gradient of 90%
l
solvent B (methanol) in solvent A (water) containing 0.2% phos-
phate buffer (pH 7.23) at a speed of 1 mL/min. The purities of
target compounds were determined to be P95% by HPLC unless
otherwise indicated.
4.3.2. 3-Bromo-N-(4-phenylthiazol-2-yl)propanamide (5b)
Off-white powder, yield: 52%. 1H NMR (CDCl3, 400 MHz, d
ppm): 11.0 (br s, 1H), 7.74 (d, J = 7.2 Hz, 2H), 7.45 (t, J = 7.2 Hz,
2H), 7.38 (t, J = 7.2 Hz, 1H), 7.13 (s, 1H), 3.58 (t, J = 6.8 Hz, 2H),
2.82 (t, J = 6.8 Hz, 2H). 13C NMR (CDCl3, 100 MHz, d ppm): 168.3,
159.8, 149.4, 133.7, 129.0(2C), 128.8, 126.4 (2C), 108.2, 38.8, 25.5.
4.2. Compound 3a–3c and 4 were obtained according to the
following general procedure
One molar equivalent of 2-aminothiazole derivatives in 50 mL
THF were mixed with 1 molar equiv of succinic anhydride or glu-
taric anhydride. The reaction solution was refluxed at 65 °C for
7 h, and then evaporated under vacuum to yield crude product.
The crude product was dissolved in a cooled aqueous solution of
K2CO3/KOH, and then filtered to discard the insoluble solid. The
combined aqueous phase was acidified by 1 M HCl to pH 1.0 to ob-
tain milk-white deposits. The deposits were then recrystallized
from ethyl acetate/ethanol to obtain a pure milk-white powder.
4.3.3. 2-Chloro-N-(4-(4-methoxyphenyl)thiazol-2-yl)acetamide
(5c)
Off-white powder, yield: 35%. 1H NMR (CDCl3, 400 MHz, d
ppm): 10.3 (br s, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.07 (s, 1H), 6.96
(d, J = 8.8 Hz, 2H), 4.13 (s, 2H), 3.86 (s, 3H). 13C NMR (CDCl3,
100 MHz, d ppm): 164.1, 159.8, 157.1, 150.0, 127.5 (2C), 126.9,
114.2 (2C), 106.7, 55.4, 41.9.
4.2.1. 4-Oxo-4-(4-phenylthiazol-2-ylamino)butanoic acid (3a)
Milk-white powder, yield: 86%. 1H NMR (DMSO-d6, 400 MHz, d
ppm): 12.29 (br s, 1H), 12.21 (br s, 1H), 7.88 (d, J = 7.2 Hz, 2H), 7.59
(s, 1H), 7.42 (t, J = 7.2 Hz, 2H), 7.31 (t, J = 7.2 Hz, 1H), 2.69 (t,
J = 6.4 Hz, 2H), 2.58 (t, J = 6.4 Hz, 2H). 13C NMR (DMSO-d6,
100 MHz, d ppm): 174.0, 171.0, 158.3, 149.2, 134.8, 129.2 (2C),
128.2, 126.1 (2C), 108.3, 30.3, 28.8.
4.3.4. 3-Bromo-N-(4-(4-methoxyphenyl)thiazol-2-yl)propana-
mide (5d)
Off-white powder, yield: 26%. 1H NMR (CDCl3, 400 MHz, d
ppm): 11.90 (br s, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.07 (s, 1H), 6.96
(d, J = 8.8 Hz, 2H), 3.84 (s, 3H), 3.4 (t, J = 7.6 Hz, 2H), 2.46 (t,
J = 7.6 Hz, 2H).
4.2.2. 5-Oxo-5-(4-phenylthiazol-2-ylamino)pentanoic acid (3b)
Milk-white powder, yield: 36%. 1H NMR (DMSO-d6, 400 MHz, d
ppm): 12.24 (br s, 1H), 12.12 (br s, 1H), 7.88 (d, J = 7.2 Hz, 2H), 7.58
(s, 1H), 7.41 (t, J = 7.2 Hz, 2H), 7.30 (t, J = 7.2 Hz, 1H), 2.49 (t,
J = 7.2 Hz, 2H), 2.28 (t, J = 7.2 Hz, 2H), 1.83 (m, J = 7.2 Hz, 2H). 13C
NMR (DMSO-d6, 100 MHz, d ppm): 174.5, 171.6, 158.3, 149.2,
134.8, 129.2 (2C), 128.2, 126.1 (2C), 108.3, 34.4, 33.3, 20.4.
4.3.5. 2-Chloro-N-(4-(4-chlorophenyl)thiazol-2-yl)acetamide
(5e)
Off-white powder, yield: 52%. 1H NMR (CDCl3, 400 MHz, d
ppm): 9.73 (br s, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz,
2H), 7.20 (s, 1H), 4.31 (s, 2H).
4.3.6. 3-Bromo-N-(4-(4-chlorophenyl)thiazol-2-yl)propanamide
(5f)
4.2.3. 4-(4-(4-Chlorophenyl) thiazol-2-ylamino)-4-oxobutanoic
acid (3c)
Off-white powder, yield: 21%. 1H NMR (CDCl3, 400 MHz, d
ppm): 11.30 (br s, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.41(d, J = 8.4 Hz,
2H), 7.17 (s, 1H), 3.53 (t, J = 6.8 Hz, 2H), 2.70 (t, J = 6.8 Hz, 2H).
13C NMR (CDCl3, 100 MHz, d ppm): 168.1, 159.1, 148.5, 134.3,
132.4, 129.2 (2C), 127.5 (2C), 108.6, 38.9, 25.3.
Milk-white powder, yield: 30%. 1H NMR (DMSO-d6/CDCl3,
400 MHz, d ppm): 7.85 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 8.8 Hz, 2H),
7.36 (s, 1H), 3.33 (br s, 1H), 2.76 (t, J = 6.8 Hz, 2H), 2.63 (t,
J = 6.8 Hz, 2H). 13C NMR (DMSO-d6/CDCl3, 100 MHz, d ppm):

Y. Wang et al. / Bioorg. Med. Chem. 20 (2012) 6513–6522
6519
4.4. Compound 7a–7e, 8 were obtained according to the
following general procedure
7.41 (t, J = 7.6 Hz, 2H), 7.32 (td, J = 6.8 and 0.8 Hz, 1H), 7.30 (m,
1H), 5.42 (t, J = 5.6 Hz, 1H), 3.30 (q, J = 6.8 Hz, 2H), 3.01 (m, 2H),
2.88 (m, 2H), 2.69 (m, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.08 (t,
J = 7.6 Hz, 2H), 1.80 (m, 6H), 1.53 (m, J = 7.2 Hz, 2H), 1.41 (m,
J = 7.2 Hz, 2H). 13C NMR (DMSO-d6, 100 MHz, d ppm): 172.6,
171.1, 158.5, 158.3, 150.7, 149.7, 147.0, 134.4, 128.7 (2C), 128.5,
128.2, 128.1, 126.1 (2C), 123.8, 122.8, 120.2, 116.2, 107.6, 48.8,
39.1, 35.1, 34.8, 33.7, 28.9, 27.1, 24.8, 22.9, 22.6, 21.2. ESI-MS
[M+H]+ (m/z): 542.3. C31H35N5O2S [541.25].
To a stirred solution of 1.2 molar equiv of compound 3 or 4 in
DMF/CH2Cl2 at 25 °C was added 1.5 molar equiv of 1-ethyl-3-(3-
diethylaminopropyl)carbodiimide (EDCꢁHCl) and 1.5 molar equiv
of hydroxybenzotriazole (HOBt). The resulting mixture was stirred
at room temperature for 30 min before a solution of 1.0 molar
equiv of 1 in CH2Cl2 was added. The resulting mixture was stirred
at 25 °C for 12 h before it was quenched with brine and diluted
with CH2Cl2. The combined organic phase was dried over Na2SO4
and filtered. After removal of the solvent under vacuum, the resi-
due was purified by flash column chromatography with petroleum
ether/ethyl acetate containing 0.5% triethylamine to yield the
products.
4.4.5. N1-(4-(4-Chlorophenyl)thiazol-2-yl)-N4-(4-(1,2,3,4-
tetrahydroacridin-9-ylamino)butyl)succinamide (7e)
Off-white powder, yield: 18%. Purity: 95.7%, tR = 8.31 min. 1H
NMR (DMSO-d6, 400 MHz, d ppm): 12.24 (br s, 1H), 8.09 (d,
J = 8.4 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.86 (t, J = 5.6 Hz, 1H), 7.69
(d, J = 8.8 Hz, 1H), 7.63 (s, 1H), 7.50 (td, J = 7.6 and 0.8 Hz, 1H),
7.47 (d, J = 8.4 Hz, 2H), 7.32 (td, J = 7.4 and 1.2 Hz, 1H), 5.36 (t,
J = 6.0 Hz, 1H), 3.37 (q, J = 6.8 Hz, 2H), 3.1 (m, 2H), 2.88 (t,
J = 6.0 Hz, 2H), 2.70–2.63 (m, 4H), 2.40 (t, J = 6.8 Hz, 2H), 1.81–
1.74 (m, 4H), 1.57–1.50 (m, 2H), 1.44–1.35 (m, 2H). 13C NMR
(DMSO-d6, 100 MHz, d ppm): 171.4, 171.1, 158.6, 158.3, 150.7,
147.9, 147.3, 133.7, 132.6, 129.2(2C), 128.7, 128.3, 127.8(2C),
123.7, 123.5, 120.7, 116.3, 109.0, 48.1, 38.7, 34.0, 30.8, 30.1, 28.5,
27.0, 25.5, 23.2, 22.9. ESI-MS [M+H]+ (m/z): 562.2. C30H32ClN5O2S
[561.20].
4.4.1. N1-(4-Phenylthiazol-2-yl)-N4-(4-(1,2,3,4-
tetrahydroacridin-9-ylamino)butyl)succinamide (7a)
White powder, yield: 57%. Purity: 97.4%, tR = 6.19 min. 1H NMR
(DMSO-d6, 400 MHz, d ppm): 7.94 (d, J = 9.6 Hz, 1H), 7.91 (d,
J = 9.6 Hz, 1H), 7.80 (d, J = 7.2 Hz, 2H), 7.54 (m, 1H), 7.37 (t,
J = 7.2 Hz, 2H), 7.31 (m, 2H), 7.07 (s, 1H), 5.93 (br s, 1H), 4.04 (br
s, 1H), 3.47 (t, J = 6.8 Hz, 2H), 3.33 (m ,2H), 3.06 (m, 2H), 2.75 (m,
2H), 2.67 (m, 2H), 2.58 (m, 2H), 1.89 (m, 4H), 1.64 (m, 4H). 13C
NMR (DMSO-d6, 100 MHz, d ppm): 171.9, 170.3, 158.4, 157.6,
150.5, 149.9, 147.2, 134.4, 128.6 (2C), 128.5, 128.4, 128.0, 126.1
(2C), 123.8, 122.7, 120.3, 116.3, 107.5, 48.8, 39.4, 33.9, 31.5, 31.1,
28.8, 27.1, 24.8, 23.0, 22.7. ESI-MS [M+H]+ (m/z): 528.4.
4.4.6. N1-(4-(1,2,3,4-Tetrahydroacridin-9-ylamino)butyl)-N4-
(4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)succinamide (8)
Off-white powder, yield: 44%. Purity: 98.9%, tR = 6.17 min. 1H
NMR (DMSO-d6, 400 MHz, d ppm): 11.81 (br s, 1H), 8.08 (d,
J = 8.0 Hz, 1H), 7.83 (t, J = 5.6 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.50
(td, J = 7.8 and 1.2 Hz, 1H), 7.32 (td, J = 7.8 and 1.2 Hz, 1H), 5.33
(t, J = 6.4 Hz, 1H), 3.36 (q, J = 6.8 Hz, 2H), 3.00 (q, J = 6.4 Hz, 2H),
2.89 (t, J = 6.4 Hz, 2H), 2.69 (t, J = 6.0 Hz, 2H), 2.59–2.55 (m, 4H),
2.52 (m, 2H), 2.36 (t, J = 6.8 Hz, 2H), 1.82–1.77 (m, 4H), 1.76–1.73
(m, 4H), 1.57–1.49 (m, J = 7.2 Hz, 2H), 1.43–1.35(m, J = 7.2 Hz,
2H). 13C NMR (DMSO-d6, 100 MHz, d ppm): 171.2, 170.6, 158.4,
155.2, 150.7, 147.4, 144.3, 128.8, 128.3, 123.7, 123.5, 121.3,
120.7, 116.3, 48.1, 38.7, 34.0, 30.8, 30.2, 28.5, 27.0, 26.4, 25.5,
23.4, 23.2, 23.1, 22.9, 22.7. ESI-MS [M+H]+ (m/z): 506.3.
C28H35N5O2S [505.25].
C30H33N5O2S [527.24].
4.4.2. N1-(4-Phenylthiazol-2-yl)-N5-(3-(1,2,3,4-
tetrahydroacridin-9-ylamino)propyl)glutaramide (7b)
Colorless powder, yield: 39%. Purity: 99.1%, tR = 6.34 min. 1H
NMR (DMSO-d6, 400 MHz, d ppm): 12.21 (br s, 1H), 8.10 (d,
J = 8.0 Hz, 1H), 7.88 (m, 3H), 7.68 (d, J = 8.0 Hz, 1H), 7.59 (s, 1H),
7.49 (t, J = 7.2 Hz, 1H), 7.42 (t, J = 7.2 Hz, 2H), 7.32 (t, J = 7.2 Hz,
1H), 7.31 (t, J = 6.8 Hz, 1H), 5.52 (t, J = 6.8 Hz, 1H), 3.87 (m, 2H),
3.09 (m, 2H), 2.88 (t, J = 6.0 Hz, 2H), 2.70 (t, J = 6.0 Hz, 2H), 2.44
(t, J = 7.2 Hz, 2H), 2.11 (t, J = 7.2 Hz, 2H), 1.82 (m, 6H), 1.64 (m,
2H) 13C NMR (DMSO-d6, 100 MHz, d ppm): 172.2, 171.6, 158.4,
158.3, 150.6, 149.2, 147.4, 134.8, 129.2, 128.8(2C), 128.2,
126.1(2C), 123.8, 123.3, 120.8, 116.5, 108.3, 45.6, 36.4, 34.9, 34.7,
34.0, 31.2, 25.6, 23.2, 22.9, 21.3. ESI-MS [M+H]+ (m/z): 528.4.
4.5. Compound 9a–9m were obtained according to the
following general procedure
C30H33N5O2S [527.24]..
4.4.3. N1-(4-Phenylthiazol-2-yl)-N4-(6-(1,2,3,4-
tetrahydroacridin-9-ylamino)hexyl)succinamide (7c)
To a solution of 1 molar equiv of 2 in 10 mL DMF was added
1 molar equiv of compound 5 or 6 in presence of K2CO3/KI. The
reaction mixture was stirred at 70 °C for 2 h, and then diluted with
CH2Cl2, washed with brine to remove DMF. The combined organic
phase was dried over Na2SO4 and filtered. After removal of the sol-
vent under vacuum, the residue was purified by flash column chro-
matography with petroleum ether/ethyl acetate 0.5% triethylamine
to yield the products.
Off-white powder, yield: 17%. Purity: 94.2%, tR = 8.35 min. 1H
NMR (DMSO-d6, 400 MHz, d ppm): 7.96 (d, J = 8.4 Hz, 1H), 7.94
(d, J = 8.4 Hz, 1H), 7.80 (d, J = 7.2 Hz, 2H), 7.56 (td, J = 7.6 and
0.8 Hz, 1H), 7.38 (t, J = 7.2 Hz, 2H), 7.36 (m, 1H), 7.30 (m, 1H),
7.08 (s, 1H), 5.82 (m, 1H), 4.13 (br s, 1H), 3.47 (t, J = 7.2 Hz, 2H),
3.28 (q, J = 7.2 Hz, 2H), 3.08 (m, 2H), 2.79 (m, 2H), 2.67 (m, 2H),
2.61 (m, 2H), 1.91 (m, 4H), 1.62 (m, J = 7.2 Hz, 2H), 1.50 (m, 2H),
1.35 (m, 4H). 13C NMR (DMSO-d6, 100 MHz, d ppm): 171.8, 170.4,
158.4, 157.7, 150.7, 149.9, 147.4, 134.4, 128.6 (2C), 128.6, 128.3,
127.9, 126.1 (2C), 123.6, 122.8, 120.2, 115.9, 107.4, 49.2, 39.6,
33.9, 31.6, 31.1, 29.5, 26.5 (2C), 24.8, 23.0, 22.7. ESI-MS [M+H]+
(m/z): 556.4. C32H37N5O2S [555.27].
4.5.1. 2-(Ethyl(3-(1,2,3,4-tetrahydroacridin-9-ylamino)propyl)
amino)-N-(4-phenylthiazol-2-yl)acetamide (9a)
Pale yellow oil, yield: 82%. Purity: 99.1%, tR = 10.50 min. 1H NMR
(CDCl3, 400 MHz, d ppm): 10.54 (br s, 1H), 7.96 (d, J = 8.8 Hz, 1H),
7.94 (d, J = 8.8 Hz, 1H), 7.72 (d, J = 7.6 Hz, 2H), 7.54 (t, J = 7.6 Hz,
1H), 7.39 (m, 2H), 7.32 (m, 2H), 7.15 (s, 1H), 3.57 (t, J = 6.8 Hz,
2H), 3.34 (s, 2H), 3.05 (t, J = 6.8 Hz, 2H), 2.72 (m, 6H), 1.91 (t,
J = 7.2 Hz, 2H), 1.86 (m, 4H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3,
100 MHz, d ppm): 169.9, 158.4, 156.9, 150.6, 150.2, 147.1, 134.3,
128.7 (3C), 128.5, 128.0, 126.0 (2C), 124.1, 122.5, 120.3, 116.7,
107.7, 57.5, 52.8, 49.3, 47.4, 33.7, 29.3, 24.9, 22.9, 22.6, 11.9. ESI-
MS [M+H]+ (m/z): 500.3. C29H33N5OS [499.24].
4.4.4. N1-(4-Phenylthiazol-2-yl)-N5-(4-(1,2,3,4-
tetrahydroacridin-9-ylamino)butyl)glutaramide (7d)
Off-white powder, yield: 49%. Purity: 99.7%, tR = 6.55 min. 1H
NMR (DMSO-d6, 400 MHz, d ppm): 12.22 (br s, 1H), 8.10 (d,
J = 8.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.78 (t, J = 5.6 Hz, 1H), 7.68
(d, J = 8.0 Hz, 1H), 7.58 (s, 1H), 7.50 (td, J = 7.6 and 0.8 Hz, 1H),

6520
Y. Wang et al. / Bioorg. Med. Chem. 20 (2012) 6513–6522
4.5.2. 2-(Ethyl(3-(1,2,3,4-tetrahydroacridin-9-ylamino)propyl)
amino)-N-(4-(4-methoxyphenyl)thiazol-2-yl)acetamide (9b)
Pale yellow oil, yield: 61%. Purity: 96.6%, tR = 10.58 min. 1H NMR
(CDCl3, 400 MHz, d ppm): 10.53 (br s, 1H), 7.95 (d, J = 8.0 Hz, 1H),
7.92 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 8.8 Hz, 2H), 7.54 (t, J = 8.0 Hz,
1H), 7.34 (t, J = 8.0 Hz, 1H), 7.01 (s, 1H), 7.65 (d, J = 8.8 Hz, 2H),
4.00 (br s, 1H), 3.85 (s, 3H), 3.55 (t, J = 7.2 Hz, 2H), 3.33 (s, 2H),
3.06 (t, J = 6.0 Hz, 2H), 2.71 (m, 6H), 1.88 (m, 6H), 1.12 (t,
J = 7.2 Hz, 3H). 13C NMR (CDCl3, 400 MHz, d ppm): 169.8, 159.5,
158.7, 156.8, 150.4, 150.0, 147.4, 128.9, 128.4, 127.3 (2C), 127.2,
124.0, 122.5, 120.5, 116.9, 114.1 (2C), 106.0, 57.6, 55.3, 52.8,
49.4, 47.5, 34.0, 29.3, 24.9, 22.9, 22.7, 11.8. ESI-MS [M+H]+ (m/z):
530.3. C30H35N5O2S [529.25].
(2C), 123.7, 122.4, 120.3, 116.3, 107.4, 50.5, 48.9, 46.9, 46.2, 33.9,
31.8, 28.8, 24.9, 22.9, 22.7, 10.6. ESI-MS [M+H]+ (m/z): 514.3.
C30H35N5OS [513.26].
4.5.7. 3-(Ethyl(3-(1,2,3,4-tetrahydroacridin-9-ylamino)
propyl)amino)-N-(4-(4-methoxyphenyl)thiazol-2-yl)
propanamide (9g)
Pale yellow foam, yield: 49%. Purity: 98.7%, tR = 9.69 min. 1H
NMR (CDCl3, 400 MHz, d ppm) : 12.71 (br s, 1H), 7.92 (d, J = 8.4 Hz,
2H), 7.72 (d, J = 8.8 Hz, 2H), 7.52 (m, 1H), 7.27 (m, 1H), 7.25 (m,
1H), 6.99 (s, 1H), 6.82 (d, J = 8.8 Hz, 2H), 4.48 (br s, 1H), 3.80 (s,
3H), 3.75 (m, 2H), 3.01 (t, J = 6.0 Hz, 2H), 2.83 (t, J = 6.0 Hz, 2H),
2.73 (q, J = 7.2 Hz, 2H), 2.66 (t, J = 6.4 Hz, 2H), 2.58 (m, 4H), 1.96 (t,
J = 7.2 Hz, 2H), 1.79 (m, 4H), 1.16 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3,
100 MHz, d ppm): 170.2, 159.4, 158.5, 157.5, 150.2, 149.8, 147.3,
128.7, 128.3, 127.4, 127.2(2C), 123.7, 122.5, 120.3, 116.3, 114.0
(2C), 105.6, 55.3, 50.6, 48.9, 47.0, 46.2, 33.9, 31.9, 28.8, 24.9, 22.9,
22.7, 10.6. ESI-MS [M+H]+ (m/z): 544.3. C31H37N5O2S [543.27].
4.5.3. 2-(Ethyl(4-(1,2,3,4-tetrahydroacridin-9-ylamino)butyl)
amino)-N-(4-phenylthiazol-2-yl)acetamide (9c)
Pale yellow oil, yield: 63%. Purity: 96.1%, tR = 11.58 min. 1H NMR
(CDCl3, 400 MHz, d ppm): 10.50 (br s, 1H), 7.92 (d, J = 7.6 Hz, 1H),
7.91 (d, J = 7.6 Hz, 1H), 7.83 (d, J = 7.2 Hz, 2H), 7.54 (m, 1H), 7.40
(t, J = 7.6 Hz, 2H), 7.33 (m, 2H), 7.16 (s, 1H),3.50 (t, J = 7.2 Hz,2H),
3.29 (s, 2H), 2.69 (m, 4H), 2.61 (t, J = 7.2 Hz, 2H), 1.88 (m, 4H),
1.73 (m, 2H), 1.62 (m, 2H), 1.11 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3,
100 MHz, d ppm): 170.1, 158.5, 157.0, 150.5, 150.2, 147.4, 134.4,
128.7 (3C), 128.3, 128.0, 126.1 (2C), 123.8, 122.6, 120.4, 116.4,
107.8, 57.4, 54.9, 49.3, 49.1, 40.0, 29.6, 24.8, 24.7, 23.0, 22.7,
11.9. ESI-MS [M+H]+ (m/z): 514.3. C30H35N5OS [513.26].
4.5.8. N-(4-(4-Chlorophenyl)thiazol-2-yl)-3-(ethyl(3-(1,2,3,4-
tetrahydroacridin-9-yl amino)propyl)amino)propanamide (9h)
Pale yellow foam, yield: 37%. Purity: 99.8%, tR = 12.66 min. 1H
NMR (CDCl3, 400 MHz, d ppm): 12.77 (br s, 1H), 7.92 (m, 2H),
7.72 (d, J = 8.8 Hz, 2H), 7.51 (t, J = 7.2 Hz, 1H), 7.27 (m, 2H), 7.25
(m, 2H), 7.11 (s, 1H), 4.41 (br s, 1H), 3.73 (m, 2H), 3.02 (t,
J = 6.0 Hz, 2H), 2.84 (t, J = 6.0 Hz, 2H), 2.75 (q, J = 7.2 Hz, 2H), 2.68
(t, J = 6.8 Hz, 2H), 2.59 (m, 4H), 1.96 (m, 2H), 1.81 (m, 4H), 1.17
(t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 100 MHz, d ppm): 170.2,
158.6, 157.6, 150.1, 148.8, 147.4, 133.5, 132.9, 128.8, 128.7 (2C),
128.3, 127.2 (2C), 123.8, 122.3, 120.3, 116.4, 107.7, 50.5, 48.9,
47.0, 46.2, 34.0, 31.9, 28.8, 24.9, 22.9, 22.7, 10.6. ESI-MS [M+H]+
(m/z): 548.2. C30H34ClN5OS [547.22].
4.5.4. 2-(Ethyl(4-(1,2,3,4-tetrahydroacridin-9-ylamino)butyl)
amino)-N-(4-(4-methoxyphenyl)thiazol-2-yl)acetamide (9d)
Pale yellow oil, yield: 43%. Purity: 98.1%, tR = 11.50 min. 1H NMR
(CDCl3, 400 MHz, d ppm): 10.48 (br s, 1H), 7.95 (d, J = 7.6 Hz, 1H),
7.93 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.55 (m ,1H), 7.34
(m, 1H), 7.02 (s, 1H), 6.92 (d, 2H), 3.84 (s, 3H), 3.52 (t, J = 7.2 Hz,
2H), 3.29 (s, 2H), 3.07 (t, J = 6.0 Hz, 2H), 2.68 (m, 4H), 2.61 (t,
J = 7.2 Hz, 2H), 1.88 (m, 4H), 1.74 (m, 2H), 1.62 (m, 2H), 1.11 (t,
J = 7.2 Hz, 3H). 13C NMR (CDCl3, 100 MHz, d ppm): 170.0, 159.5,
158.2, 156.9, 150.7, 149.9, 128.6, 128.4, 128.3, 127.4 (2C), 127.7,
123.9, 122.7, 120.1, 116.1, 114.1 (2C), 106.1, 57.4, 55.3, 54.9,
49.3, 49.1, 33.7, 29.6, 24.8, 24.7, 22.9, 22.6, 11.9. ESI-MS [M+H]+
(m/z): 544.3. C31H37N5O2S [543.27].
4.5.9. 3-(Ethyl(4-(1,2,3,4-tetrahydroacridin-9-ylamino)butyl)
amino)-N-(4-phenylthiazol-2-yl)propanamide (9i)
Pale yellow oil, yield: 96%. Purity: 99.1%, tR = 9.68 min. 1H NMR
(CDCl3, 400 MHz, d ppm) : 12.95 (br s, 1H), 7.85 (d, J = 8.0 Hz, 1H),
7.84 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 2H), 7.47 (t, J = 8.0 Hz,
2H), 7.22 (m, 4H), 7.07 (s, 1H), 3.47 (t, J = 7.2 Hz, 2H), 2.96 (t,
J = 6.4 Hz, 2H), 2.75 (t, J = 6.0 Hz, 2H), 2.64 (q, J = 7.2 Hz, 2H), 2.53
(m, 4H), 2.42 (t, J = 6.4 Hz, 2H), 1.78 (m, 4H), 1.67 (m, 4H), 1.11
(t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 100 MHz, d ppm): 170.4,
158.2, 157.5, 150.7, 149.8, 147.1, 134.5, 128.5 (2C), 128.4, 128.3,
127.8, 125.8 (2C), 123.6, 122.8, 120.1, 116.0, 107.2, 52.3, 49.2,
48.9, 46.2, 33.8, 31.8, 29.6, 24.5, 24.3, 22.9, 22.6, 10.8. ESI-MS
[M+H]+ (m/z): 528.3. C31H37N5OS [527.27].
4.5.5. N-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(ethyl(4-(1,2,3,4-
tetrahydroacridin-9-ylamino)butyl)amino)acetamide (9e)
Light brown oil, yield: 94%. Purity: 98.3%, tR = 14.81 min. 1H
NMR (CDCl3, 400 MHz,
d ppm): 10.47 (br s, 1H), 7.90 (d,
J = 7.6 Hz, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.74 (d, J = 8.4 Hz, 2H),
7.52 (m, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.29 (m, 1H), 7.11 (s, 1H),
3.47 (t, J = 7.2 Hz, 2H), 3.27 (s, 2H), 3.03 (t, J = 6.0 Hz, 2H), 2.66
(m, 4H), 2.59 (m, 2H), 1.85 (m, 4H), 1.69 (m, 2H), 1.59 (m, 2H),
1.08 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 100 MHz, d ppm): 170.1,
158.3, 157.2, 150.5, 148.9, 147.2, 133.7, 132.9, 128.8 (2C), 128.6,
128.4, 127.3 (2C), 123.8, 122.6, 120.2, 116.3, 108.1, 57.4, 54.9,
49.3, 49.1, 33.8, 29.5, 24.8, 24.7, 22.9, 22.7, 11.9. ESI-MS [M+H]+
(m/z): 548.3. C30H34ClN5OS [547.22].
4.5.10. 3-(Ethyl(4-(1,2,3,4-tetrahydroacridin-9-ylamino)butyl)
amino)-N-(4-(4-methoxyphenyl)thiazol-2-yl)propanamide (9j)
Pale yellow foam, yield: 85%. Purity: 98.7%, tR = 9.54 min. 1H
NMR (CDCl3, 400 MHz,
d ppm): 12.96 (br s, 1H), 7.87 (d,
J = 8.4 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 7.51 (m, 1H), 7.27 (m,
1H), 6.98 (s, 1H), 6.79 (d, J = 8.4 Hz, 2H), 3.92 (br s, 1H), 3.74 (s,
3H), 3.51 (t, J = 6.8 Hz, 2H), 2.99 (t, J = 6.4 Hz, 2H), 2.80 (t,
J = 6.4 Hz, 2H), 2.71 (q, J = 7.2 Hz, 2H), 2.59 (m, 4H), 2.46 (t,
J = 6.4 Hz, 2H), 1.83 (m, 4H), 1.73 (m, 4H), 1.17 (t, J = 7.2 Hz,
3H). 13C NMR (CDCl3, 100 MHz, d ppm): 170.3, 159.3, 158.4,
157.4, 150.6, 149.7, 147.4, 128.7, 128.2, 127.5, 127.2 (2C), 123.6,
122.7, 120.3, 116.3, 113.9 (2C), 105.5, 55.2, 52.4, 49.3, 48.9,
46.1, 33.9, 31.8, 29.7, 24.5, 24.3, 22.9, 22.7, 10.7. ESI-MS [M+H]+
(m/z): 558.3. C32H39N5O2S [557.28].
4.5.6. 3-(Ethyl(3-(1,2,3,4-tetrahydroacridin-9-ylamino)propyl)
amino)-N-(4-phenylthiazol-2-yl)propanamide (9f)
Pale yellow oil, yield: 45%. Purity: 95.7%, tR = 8.82 min. 1H NMR
(CDCl3, 400 MHz, d ppm): 12.73 (br s, 1H), 7.91 (d, J = 8.4 Hz, 1H),
7.89 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 7.6 Hz, 2H), 7.51 (t, J = 7.6 Hz,
1H), 7.31 (t, J = 7.6 Hz, 2H), 7.25 (m, 2H), 7.13 (s, 1H), 4.33 (br s,
1H), 3.71 (t, J = 6.8 Hz, 2H), 2.99 (t, J = 6.4 Hz, 2H), 2.82 (t,
J = 6.0 Hz, 2H), 2.72 (q, J = 7.2 Hz, 2H), 2.65 (t, J = 6.8 Hz, 2H), 2.58
(m, 4H), 1.95 (t, J = 6.8 Hz, 2H), 1.79 (m, 4H), 1.15 (t, J = 7.2 Hz,
3H). 13C NMR (CDCl3, 100 MHz, d ppm): 170.2, 158.5, 157.5,
150.2, 150.0, 147.3, 134.4, 128.7, 128.6(2C), 128.2, 127.8, 126.0
4.5.11. N-(4-(4-Chlorophenyl)thiazol-2-yl)-3-(ethyl(4-(1,2,3,4-
tetrahydroacridin-9-ylamino)butyl)amino)propanamide (9k)
Pale yellow oil, yield: 60%. Purity: 97.6%, tR = 13.74 min. 1H NMR
(CDCl3, 400 MHz, d ppm): 13.04 (br s, 1H), 7.89 (d, J = 8.0 Hz, 1H),

Y. Wang et al. / Bioorg. Med. Chem. 20 (2012) 6513–6522
6521
7.84 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 1H),
7.27 (m, 2H), 7.24 (m, 1H), 7.10 (s, 1H), 3.91 (br s, 1H), 3.51 (t,
J = 6.8 Hz, 2H), 3.01 (t, J = 6.4 Hz, 2H), 2.81 (t, J = 6.4 Hz, 2H), 2.72
(q, J = 7.2 Hz, 2H), 2.60 (m, 4H), 2.48 (t, J = 6.4 Hz, 2H), 1.84 (m,
4H), 1.75 (m, 4H), 1.18 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3,
100 MHz, d ppm):170.4, 158.3, 157.7, 150.6, 148.8, 147.2, 133.5,
133.0, 128.7 (2C), 128.6, 128.3, 127.1 (2C), 123.7, 122.6, 120.2,
116.2, 107.6, 52.4, 49.3, 48.9, 46.2, 33.9, 31.7, 29.7, 24.6, 24.3, 22.9,
22.7, 10.8. ESI-MS [M+H]+ (m/z): 562.3. C31H36ClN5OS [561.23].
were constructed at relatively low concentration of substrate
(0.067–0.5 mM) by using Ellman’s method. Three concentrations
of compound 7c were selected for this study: 20, 40, and 80 nm.
The assay medium containing 20
and 20 l compound solution was allowed to incubate for 5 min,
and then 100 l of 1.5 mM DTNB were added for 5 min. The reac-
tion was started by addition of 10 l different concentration of
ll PBS (pH 7.4), 50 ll of enzyme,
l
l
l
ATC. Kinetic characterization of the hydrolysis of ATC catalyzed
by AChE was measured at 415 nm at 3 min intervals by a Biotek
ELx 800 microplate reader.
4.5.12. 2-(Ethyl(6-(5,6,7,8-tetrahydroacridin-9-ylamino)hexyl)
amino)-N-(4-phenylthiazol-2-yl)acetamide (9l)
4.8. Thioflavin T (ThT) fluorescence assay
Pale yellow oil, yield: 64%. Purity: 97.4%, tR = 18.96 min. 1H NMR
(CDCl3, 400 MHz, d ppm): 10.52 (br s, 1H), 7.96 (d, J = 8.0 Hz, 1H),
7.92 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 7.6 Hz, 2H), 7.56 (t, J = 7.6 Hz,
1H), 7.40 (t, J = 7.6 Hz, 2H), 7.32 (m, 2H), 7.14 (s, 1H), 4.06 (br s,
1H), 3.49 (t, J = 7.2 Hz, 2H), 3.29 (s, 2H), 3.08 (m, 2H), 2.68 (q,
J = 7.2 Hz, 2H), 2.63 (m, 2H), 2.57 (t, J = 7.2 Hz, 2H), 1.89 (m, 4H),
1.70 (m, 2H), 1.53 (m, 2H), 1.42 (m, 4H), 1.12 (t, J = 7.2 Hz, 3H).
13C NMR (CDCl3, 100 MHz, d ppm): 170.2, 157.6, 157.1, 151.1,
150.1, 143.3, 134.4, 128.7 (3C), 128.0 (2C), 126.0 (2C), 123.8,
122.9, 119.7, 116.4, 107.7, 57.5, 55.1, 49.3 (2C), 33.4, 31.7, 27.2
(2C), 26.8, 24.6, 22.9, 22.5, 12.0. ESI-MS [M+H]+ (m/z): 542.3.
Ab1–42 was dissolved in 100% 1,1,1,3,3,3-hexafluoro-2-propanal
(HFIP) to a concentration of 1 mg/mL, sonicated in a water bath for
10 min, aliquoted into microcentrifuge tubes, dried under vacuum,
and stored at ꢀ20 °C. Immediately prior to use, the HFIP-treated
Ab1–42 was dissolved in dimethylsulfoxide (DMSO) as the stored
solution. A screening assay for all compounds that inhibited Ab1–
aggregation was performed by measuring the ThT fluorescence
42
emission. Amounts of
200 M Ab1–42 were added into 40
(PBS at pH 7.4). After incubation for 24 h at room temperature,
100 l of 5 M ThT solution (in PBS at pH 7.4) was added to the
reaction solution. Fluorescence was measured at 485 nm with an
5
l
l
compound (200
lM) and 5 ll of
l
ll of phosphate-buffered saline
C32H39N5OS [541.29].
l
l
4.5.13. 2-(Ethyl(6-(5,6,7,8-tetrahydroacridin-9-ylamino)hexyl)
amino)-N-(4-(4-methoxyphenyl)thiazol-2-yl)acetamide (9m)
Pale yellow oil, yield: 61%. Purity: 99.1%, tR = 17.67 min. 1H NMR
(CDCl3, 400 MHz, d ppm): 10.50 (br s, 1H), 7.91 (d, J = 8.4 Hz, 2H),
7.76 (d, J = 8.8 Hz, 2H), 7.54 (t, J = 8.4 Hz, 1H), 7.32 (t, J = 8.4 Hz,
1H), 7.00 (s, 1H), 6.92 (d, J = 8.8 Hz, 2H), 3.92 (br s, 1H), 3.82 (s,
3H), 3.46 (t, J = 7.2 Hz, 2H), 3.28 (s, 2H), 3.06 (m, 2H), 2.67 (q,
J = 7.2 Hz, 2H), 2.66 (m, 2H), 2.56 (t, J = 7.2 Hz, 2H), 1.90 (m, 4H),
1.68 (m, 2H), 1.52 (m, 2H), 1.41 (m, 4H), 1.11 (t, J = 7.2 Hz, 3H).
13C NMR (CDCl3, 100 MHz, d ppm): 170.3, 159.5, 158.3, 156.9,
150.7, 149.9, 147.4, 128.6, 128.3, 127.4, 127.3(2C), 123.6, 122.8,
120.2, 115.8, 114.1 (2C), 106.0, 57.5, 55.3, 55.1, 49.3, 49.2, 33.9,
31.7, 27.2, 27.1, 26.9, 24.7, 23.0, 22.7, 12.0. ESI-MS [M+H]+ (m/z):
572.3. C33H41N5O2S [571.3].
excitation wavelength of 435 nm on
fluorospectrophotometer.
a perkinelmer LS-55
4.9. Atomic force microscopy (AFM) assay
The HFIP-treated Ab1–42 was dissolved in DMSO to 2 mM as
stock solution. Phosphate buffer (PBS) was dissolved by milli-
porewater, and filtered by 0.22
Ab1–42 (1 mM, 2.5 l), PBS (pH 7.4, 45
2.5 l) were incubated for 4 days at 30 °C. As a control, 2 mM
Ab1–42 (2.5 l), PBS (pH 7.4, 45 l), and DMSO (2.5 l) were incu-
bated for 4 days at 30 °C. AFM experiments were performed as
follows: Take an aliquot of 10 l from the incubating solution
lM microfiltration membrane.
l
l
l), and 9 m (2 mM,
l
l
l
l
l
and deposit it on the mica surface directly. The deposited droplet
is left on the mica surface for 10 min and wash the sample with
ultra-pure water to remove excess salts and unbound peptide.
Gently blow the sample dry under a stream of nitrogen. Finally,
scan samples with tapping mode on a PicoScan 2500 PicoSPM II
controller with a silicon probe of k = 40 N/m and 300 kHz reso-
nant frequency.
4.6. AChE and BuChE inhibition assay
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from 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 butylthiocholinlrine chloride
(BTC) were purchased from sigma. Synthesized derivatives were
dissolved in DMSO and then diluted in PBS (pH 7.4) to provide a fi-
nal concentration range. AChE enzyme solution was prepared to
give 0.22 units/ml in 10 mL aliquots. The assay medium contained
4.10. Cell culture of rat cortical neurons
Neonatal Sprague-Dawley (SD) rats (day 0–3) were obtained
from the Experimental Animal Center of Tongji Medical College,
Huazhong University of Science & Technology. All experiments
conformed to the local and international guidelines on ethical
use of animals. The University Animal Welfare Committee ap-
proved the used animal protocol. Briefly, the cortex of newborn
SD rats were dissected and rinsed in ice-cold Dulbecco’s phos-
phate-buffered saline. The dissected tissues were treated with
0.125% trypsin in Hanks’ balanced salt solution for 25 min at
37 °C and mechanically dissociated using a fire-polished Pasteur
pipettes. Cells were collected by centrifugation and resuspended
in DMEM/F12 (1:1) with 10% fetal bovine serum. For calcium
imaging, cells (20,000–40,000) were seeded on poly-D-lysine
coated coverslips and kept at 37 °C in 5% CO2 incubator. After
24 h, the culture medium was changed into DMEM medium sup-
plemented with 2% B27 and the cortical neurons were fed with
fresh medium twice weekly. Microscopically, glial cells were not
apparent by employing this protocol. The neurons were main-
20
100
ATC. The assay medium containing 20
zyme, and 20 l compound solution was allowed to incubate for
5 min, and then 100 l of 1.5 mM DTNB were added for 5 min.
The reaction was started by addition of 10 l of 30 mM ATC. The
l
l
l PBS (pH 7.4), 20
l of 1.5 mM DTNB, 50
l
l compound solution or blank solution,
l of enzyme, and 10 l of 30 mM
l PBS (pH 7.4), 50 l of en-
l
l
l
l
l
l
l
activity was determined by measuring the increase in absorbance
at 415 nm at 3 min intervals by a Biotek ELx 800 microplate reader.
The similar method described above was applied to determine the
BuChE activity (0.3 units/mL BuChE enzyme solution, 1.5 mM
DTNB, 30 mM BTC).
4.7. Kinetic characterization of acetylcholinesterase inhibition
To obtain kinetic study on the mechanism of AChE inhibition by
compound 7c, reciprocal plots of 1/velocity versus 1/[substrate]

6522
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Acknowledgments
This work was supported by Grants from the Key Project of Na-
tional Natural Science Foundation of China (No.30930104), Inter-
national Science & Technology Cooperation Program of China
(No. 2011DFA32670) and the Important National Science & Tech-
nology Specific Projects (No. 2012ZX09103-101-045) to Dr. J.G
Chen.
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Supplementary data
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Supplementary data associated with this article can be found, in
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These data include MOL files and InChiKeys of the most important
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