Design, synthesis,: In vitro and in vivo evaluation of tacrine-cinnamic acid hybrids as multi-target acetyl- and butyrylcholinesterase inhibitors against Alzheimer's disease

Article abstract of DOI:10.1039/c7ra04385f

Previously, we have reported tacrine-ferulic acid hybrids as multi-target cholinesterase inhibitors against Alzheimer's disease. However, the detailed structure-activity relationship (SAR), especially regarding the ferulic acid moiety, has yet to be eluci

Full text of DOI:10.1039/c7ra04385f

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Design, synthesis, in vitro and in vivo evaluation of
tacrine–cinnamic acid hybrids as multi-target
acetyl- and butyrylcholinesterase inhibitors against
Alzheimer's disease†
Cite this: RSC Adv., 2017, 7, 33851
Yao Chen,abc Hongzhi Lin,e Jie Zhu,e Kai Gu,e Qi Li,e Siyu He,e Xin Lu,e Renxiang Tan,b
a
b
d
e
*
*
Yuqiong Pei, Liang Wu, Yaoyao Bian and Haopeng Sun
Previously, we have reported tacrine–ferulic acid hybrids as multi-target cholinesterase inhibitors against
Alzheimer's disease. However, the detailed structure–activity relationship (SAR), especially regarding the
ferulic acid moiety, has yet to be elucidated. Herein we report the structural modification of the ferulic
acid moiety, which is replaced by cinnamic acid with different substitutions. The target compounds are
synthesized and evaluated for their in vitro cholinesterase inhibitory activities, inhibition of amyloid b-
protein self-aggregation, cyto-protective effects against hydrogen peroxide and antiproliferative activity
in PC-12 cells. The optimal compounds 35 and 36 are subsequently selected for in vivo assays. 36 shows
much better performance in ameliorating the scopolamine-induced cognition impairment and less
hepatotoxicity than tacrine. The compound serves as a good lead compound for further optimization.
Received 19th April 2017
Accepted 27th June 2017
DOI: 10.1039/c7ra04385f
rsc.li/rsc-advances
factor hypothesis.9 Additionally, oxidative stress,10 free radical
formation,11 metal dyshomeostasis,12 and mitochondrial
dysfunction,13 are also reported to be tightly correlated to the
development of AD by supplying an inammatory micro-
environmental condition. These theories increase under-
standing of the basic mechanism of AD, and, also depict a more
complex AD scenario.
The cholinergic hypothesis of the pathogenesis of AD asserts
that dysfunction of cholinergic system, mainly decline of
acetylcholine (ACh) level, results in the cognitive and memory
decits. Therefore, recovering cholinergic function is believed
to be benecial for the treatment of AD.14 Generally, ACh can be
hydrolyzed by two types of cholinesterases (ChEs), namely
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).
Although the elucidation of the pathophysiology of AD provides
multiple potential drug targets for designing effective drugs,
acetylcholinesterase inhibitors (AChEIs) still serve as the main
therapeutic agents applied clinically for AD.
Introduction
Alzheimer's disease (AD) is the most prevalent form of late-life
mental failure in humans and it affects about 6% of the pop-
ulation aged over 65.1 It is estimated that more than 18 million
people presently suffer from AD, and the number is predicted to
sharply increase to 70 million by 2050.2 The cardinal features of
AD include progressive memory impairment, disordered
cognitive function, altered behavior such as depression, hallu-
cination, delusion, and agitation, and a progressive decline in
language function.3 So far, it is well accepted that AD is
a multifactorial syndrome deriving from a complex array of
neurochemical factors. During the process of AD, cholinergic
neurons and synapses of the basal forebrain are selectively lost,
causing cognitive impairment.4 These ndings inspired several
theories about AD pathogenesis, including cholinergic
dysfunction,5 amyloid cascade,6 hyperphosphorylation of s-
protein,7 cell cycle hypothesis,8 and brain-derived neurotrophic
The enzymatic site of human AChE is a narrow gorge with
˚
a length of approximate 20 A, which contains two binding sites:
aSchool of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China
bState Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing
University of Chinese Medicine, Nanjing, 210023, China
the catalytic active site (CAS) at the bottom and the peripheral
anionic site (PAS) near the entrance of the gorge.15,16 CAS is in
charge of the hydrolysis of ACh and is consisted of key residues
including Ser203, Glu334, and His447, which are referred to as
the catalytic triad.17 PAS is composed of several aromatic resi-
dues such as Trp86, Trp286.18 It has been proved that PAS is
closely related to both hydrolysis of ACh and neurotoxic cascade
of AD through AChE-induced b-amyloid (Ab) aggregation.19
Under normal condition, AChE is more active and can hydrolyze
cJiangsu Key Laboratory for Functional Substance of Chinese Medicine, Nanjing
University of Chinese Medicine, Nanjing, 210023, China
dSchool of Nursing, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
E-mail: 1691@163.com; Tel: +86-15952007562
eDepartment of Medicinal Chemistry, China Pharmaceutical University, Nanjing,
210009, China. E-mail: sunhaopeng@163.com; Tel: +86-25-85863169
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra04385f
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about 80% of ACh in human brains.20 However, both the level Ab1–42 self-aggregation, cyto-protective effects against hydrogen
and the activity of AChE in AD patients are found to be peroxide and antiproliferative activity in PC-12 cells. Addition-
remarkably reduced, leading to the compensative upregulation ally, we also report the in vivo behavioral and hepatotoxic eval-
of BuChE, which further modulates the ACh levels.21 Therefore, uations for the optimal compound selected from in vitro assays.
inhibitors of both AChE and BuChE, such as tacrine and riva- Based on these results, we hope to supply more useful infor-
stigmine, are expected to exert potent therapeutic effect on AD. mation of structure–activity relationship (SAR) that can guide
Unfortunately, instead of curing or preventing the neuro- further discovery of new MTDLs against AD.
degeneration, these drugs can only enable a palliative treat-
ment.22 Considering the multifactorial nature of AD, the
traditional agents designed by one-molecule one-target
Results and discussions
Compound design and chemistry
approach is insufficient to provide enough benets. Thus,
designing compounds that can simultaneously regulate
multiple signicant targets in the development of AD, has
emerged as a new strategy. These compounds, which are
referred to as multi-target-directed ligands (MTDLs),23 are
considered to offer additional properties other than cholines-
terase inhibition. Substantial studies have been performed to
achieve different types of MTDLs, many of which have been
proved to show promising pharmacological effects on AD.24–39
These results encourage medicinal chemists to continue this
work. In recent years, designing MTDLs based on tacrine has
attracted the attentions of medicinal chemists throughout the
world and numerous related publications are disclosed to
describe the efforts on this eld. Compared to other AChEIs,
tacrine is a good scaffold for the design of MTDLs due to its
simple structure and high ligand efficiency (LE), which means
tacrine can potently inhibit AChE with small number of non-
hydrogen atoms. Moreover, tacrine has a good endurance
against substantial structural modication while retaining the
target-based activity, further provides a sound basis for the
Compound CY-1, which was previously reported by our group,
was used as the lead compound for structural modication.41
The ferulic acid moiety of CY-1 was replaced by cinnamic acid
with various substitutions at different positions of the phenyl
ring (Fig. 1).
The synthesis of the tacrine–cinnamic acid hybrids is
described in Fig. 1. Anthranilic acid (1) was condensed with
cyclohexanone (2) to yield chloro acridine 3.42 Treatment of 3
with ethane-1,2-diamine led to 4, which was condensed with
different cinnamic acid analogs (5-8–5-32) to result in the target
compounds 8–32. Hydroxyl substituted cinnamic acids (5-33–5-
36) were rst protected with benzyl to obtain intermediates 6-
33–6-36, whose benzyl ester moiety was subsequently hydro-
lyzed to result in 7-33–7-36. They were condensed with 4 to
acquire target compounds 33–36. Reduction of the nitro group
of 27–29 resulted in 37–39.
Cholinesterase inhibitory activity and SAR analysis
design of MTDLs. However, there is no newly approved small The inhibitory effects of the synthesized compounds against
molecular agent for the treatment of AD in recent years, and AChE from electrophorus electricus (eeAChE) and BuChE from
most of the MTDLs remain at the stage of preclinical study. equine serum (eqBuChE) were determined, following Ellman's
Therefore, it is still urgently needed for us to design new MTDLs method.43 The data were expressed as IC50 values (Table 1).
and to fully understand the structural requirement through Most of the compounds were proved to be potent inhibitors of
detailed structure–activity relationship (SAR) study.
ChEs, with IC50 values lower than 100 nM. The AChE IC50 value
Our group has been dedicated to the discovery of new MTDLs of 8, an analog without any substitution at the cinnamic acid
for nearly a decade. Previously, Fang L. et al.40 and Chen Y. moiety, was higher than most of the substituted ones. Methyl
et al.41 have disclosed a series of tacrine–ferulic acid hybrids as substitution (9–11) led to an increase of AChE inhibitory
multifunctional potent ChEs inhibitors, most of which effec- activity. The position of the methyl group was also considered,
tively inhibited ChEs in vitro in nanomolar range. These showing that the activity was para- > meta- > ortho-. Interest-
compounds were also proved to exert multiple functions, ingly, 11 showed much improved activity on AChE (IC50 ¼ 34.3
including antioxidant activities, vasorelaxation effects, and NO- ꢀ 1.8 nM), while its inhibitory effect on BuChE remarkably
donating behavior. In vivo studies by using the scopolamine- reduced (IC50 ¼ 86.9 ꢀ 6.6 nM). When the methyl was replaced
induced cognition impairment mouse model conrmed that by a 4-methyl carbonate substitution (32), the compound
these compounds can ameliorate the cognitive impairment and exhibited a higher selectivity on AChE (AChE IC50 ¼ 71.2 ꢀ
reduce the hepatotoxicity compared to the reference compound 2.4 nM, BuChE IC50 ¼ 342.0 ꢀ 61.5 nM). The results suggested
tacrine. These studies provided us promising lead compounds that bulky functional groups were well tolerated by AChE,
for further research. However, structural modication, espe- while they were restricted by BuChE. Therefore, bulky groups
cially on the ferulic acid moiety, is still limited and needs to be at para-position can be a good choice to enhance the target
further elucidated. To deepen the understanding of the struc- selectivity on AChE. Next, we designed a series of methoxy
tural requirement for tacrine–ferulic acid hybrids, here we group analogs to evaluate the impact of this group on activity.
report the structural modication of ferulic acid moiety, which For mono-substituted compounds (12–19), the activity on
is replaced by cinnamic acid with different substitutions. The AChE was para- > meta- > ortho-, the same to that of methyl
target compounds are synthesized and evaluated for their in analogs. Substitution of methoxy group at meta-position (13)
vitro and in vivo activities related to the treatment of AD, seemed have no impact on the target selectivity (AChE IC50
including in vitro assays for cholinesterase catalytic activity, 47.4 ꢀ 2.0 nM, BuChE IC50 ¼ 57.4 ꢀ 5.6 nM). It was noticeable
¼
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when methoxy group was at ortho-position (12), it showed ortho-substitution (15 and 16) was preferred by BuChE, while
a 7.99-fold selectivity on BuChE (AChE IC50 ¼ 123.8 ꢀ 7.6 nM, para-substitution was better for AChE (19). Interestingly, for
BuChE IC50 ¼ 15.5 ꢀ 1.5 nM). Similar results were also triOCH3 analogs, 17 was very potent on both AChE and BuChE
observed on 9 with 2-methyl substitution (AChE IC50 ¼ 80.6 ꢀ (IC50 ¼ 17.3 ꢀ 0.6 and 23.3 ꢀ 2.3 nM, respectively), while 18
9.8 nM, BuChE IC50 ¼ 37.3 ꢀ 3.3 nM). We inferred that the showed considerably high selectivity on AChE.
shape difference of the binding site between AChE and BuChE
Next, we evaluated the impact of halogen atoms on the ChEs
led to such phenomenon. The binding site of AChE is narrow activity. When substituted by Cl (20–22), the activity on AChE
and long, while BuChE is broad and short. As a result, was para- > meta- > ortho-, while it showed an opposite manner
substitution at the ortho-position can lead to steric hindrance on BuChE. The results were in accordance with those from
to the narrow binding site of AChE but was tolerated by methyl and methoxy group substitution. When substituted by
BuChE. Oppositely, para-substitution resulted in the elonga- different halogen atoms, the activity on AChE was –Cl (22) z
tion of the molecular shape of the inhibitors, and was more –Br (24) > –F (23). Meanwhile, the three compounds with para-
suitable for the binding site of AChE. For multi-substituted substitution were more selective on AChE than BuChE, a similar
compounds (15–19), we could also observe a trend that manner as mentioned above.
Fig. 1 Design strategy and synthetic route of the target compounds. Reagents and conditions: (a) POCl3, reflux, 3 h; (b) pentanol, NH2(CH2)2NH2,
NaI, reflux, 18 h; (c) PyBop, DIPEA, anhydrous CH2Cl2, room temp, 24 h; (d) benzyl bromide, K2CO3, DMF; (e) NaOH, MeOH, H2O, 5 h; (f)
SnCl2$2H2O, EtOH, reflux, 8 h.
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Then –NO2 (27–29) and –CF3 (25–26) and –OCF3 (31), three
electron-withdrawing groups, were introduced as the R group. It
was noteworthy that analogs with –NO2 substitution were the
most active compounds on AChE among all the derivatives, with
IC50 values in a single-digit nanomolar range. Meanwhile, they
Table 1 Inhibition of AChE and BuChE (IC50 values), and selectivity
expressed as the ratio of the resulting IC50 values
Fig. 2 Lineweaver–Burk plots resulting from subvelocity curves of
huAChE activity with different substrate concentrations (25–450 mM)
in the absence and presence of 20, 60, 100, 200 nM 36.
all exhibited a remarkable selectivity toward AChE than BuChE
(SI ¼ 0.04–0.15). The impact of –CF3 and –OCF3 was lower than
–NO2, however, they also showed the same target selective rule
to other groups mentioned above.
IC50 (nM) ꢀ SEMa
Cpd.
R
AChEb
BuChEc
SIf
We subsequently introduced amino (37–39) as hydrogen-
bond donating group. We found that such groups resulted in
a remarkably reduced activity on AChE (IC50 ¼ 54.7 ꢀ 8.8–173.3
ꢀ 41.2 nM) except 37, which was active toward both AChE and
BuChE (IC50 ¼ 28.7 ꢀ 2.7, 18.7 ꢀ 2.2 nM, respectively).
Considering the hydrophobic nature of the binding site, espe-
cially for AChE, introduction of polar substitutions may lead to
the improper intermolecular recognition, thus reducing the
activity. Inspired by this, we replaced the hydroxyl to benzyloxyl.
The ortho-substitution (33) exhibited high selectivity on BuChE,
while meta- (34) and para-substitution (35 and 36) were prefer-
able to AChE. These results further suggested the steric
hindrance of the groups on the target selectivity.
8
9
H
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
2,3-diOCH3
2,5-diOCH3
2,3,4-triOCH3
3,4,5-triOCH3
3,4-OCH2O–
2-Cl
3-Cl
4-Cl
4-F
4-Br
2-CF3
4-CF3
2-NO2
3-NO2
92.5 ꢀ 6.9
80.6 ꢀ 9.8
78.9 ꢀ 2.3
34.3 ꢀ 1.8
123.8 ꢀ 7.6
47.4 ꢀ 2.0
22.5 ꢀ 1.1
47.8 ꢀ 2.5
72.3 ꢀ 6.2
17.3 ꢀ 0.6
20.3 ꢀ 0.5
49.3 ꢀ 2.5
50.4 ꢀ 4.1
34.9 ꢀ 4.3
21.3 ꢀ 1.6
68.6 ꢀ 5.7
27.4 ꢀ 1.7
52.1 ꢀ 1.5
33.0 ꢀ 1.3
7.1 ꢀ 0.5
23.3 ꢀ 2.7
37.3 ꢀ 3.3
28.3 ꢀ 2.0
86.9 ꢀ 6.6
15.5 ꢀ 1.5
57.4 ꢀ 5.6
61.3 ꢀ 4.3
23.2 ꢀ 3.1
26.1 ꢀ 4.5
23.3 ꢀ 2.3
270.6 ꢀ 21.9
80.7 ꢀ 9.3
19.4 ꢀ 1.4
21.8 ꢀ 1.4
39.1 ꢀ 3.7
125.8 ꢀ 11.5
92.5 ꢀ 4.1
28.4 ꢀ 2.1
170.6 ꢀ 15.2
48.2 ꢀ 2.8
34.7 ꢀ 2.2
154.3 ꢀ 20.3
64.4 ꢀ 5.4
33.4 ꢀ 4.9
342.0 ꢀ 61.5
13.6 ꢀ 1.5
15.0 ꢀ 1.1
42.6 ꢀ 3.0
86.1 ꢀ 15.5e
52.6 ꢀ 5.6
55.9 ꢀ 3.3e
18.7 ꢀ 2.2
115.2 ꢀ 12.5
68.5 ꢀ 7.9
37.5 ꢀ 9.7
10.6 ꢀ 1.1
4.0
2.2
2.8
0.4
8.0
0.8
0.4
2.1
2.8
0.7
0.1
0.6
2.6
1.6
0.5
0.6
0.3
1.8
0.2
0.2
0.1
0.04
0.1
1.7
0.2
7.6
2.7
0.7
0.7
0.3
1.0
1.5
0.5
2.5
1.7
6.6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
To further validated the inhibitory activities of synthesized
compounds on human ChEs, the representative compounds, 35
and 36 were selected for determination (Table 1). 35 exhibited
huAChE IC50 ¼ 60.6 ꢀ 5.6 nM, huBuChE IC50 ¼ 86.1 ꢀ 15.5 nM;
36 exhibited huAChE IC50 ¼ 55.1 ꢀ 4.9 nM, huBuChE IC50
¼
3.8 ꢀ 0.1
55.9 ꢀ 3.3 nM. The results showed that the synthesized
compounds efficiently inhibited the activities of human ChEs,
further conrmed their activities as ChEs inhibitors.
4-NO2
6.8 ꢀ 0.7
4-Cl-3-NO2
3-OCF3
4-Methyl carbonate
2-OBn
3-OBn
4-OBn
5.5 ꢀ 0.2
56.5 ꢀ 1.7
71.2 ꢀ 2.4
103.2 ꢀ 9.9
40.1 ꢀ 1.2
29.5 ꢀ 1.0
60.6 ꢀ 5.7d
15.8 ꢀ 0.7
55.1 ꢀ 4.9d
28.7 ꢀ 2.7
54.7 ꢀ 8.8
173.3 ꢀ 41.2
62.0 ꢀ 10.5
69.8 ꢀ 11.1
Kinetic study of AChE inhibition
To further analyze the binding manner of the synthesized
compounds to huAChE, the potent inhibitor 36 was selected as
36
3-OMe-4-OBn
37
38
39
CY-1
Tacrine
2-NH2
3-NH2
4-NH2
—
Table 2 The Vmax and Km values for compound 36 in kinetic studies.
Data are shown in mean ꢀ SD of three experiments
Concentration
(nM)
Vmax (mM minꢁ1
)
Km (mM)
R square
—
a
Concentration of the compound required for 50% inactivation of
0
5
10
15
40
0.9 ꢀ 0.1
0.8 ꢀ 0.0
0.5 ꢀ 0.0
0.3 ꢀ 0.0
0.2 ꢀ 0.0
147.8 ꢀ 18.8
139.7 ꢀ 16.3
145.9 ꢀ 22.4
141.4 ꢀ 25.2
148.7 ꢀ 27.5
0.99
0.99
0.99
0.99
0.99
b
ChEs, data were shown in mean ꢀ SEM of three experiments. AChE
c
(EC 3.1.1.7) from electric eel. BuChE (EC 3.1.1.8) from horse serum.
d
e
AChE (EC 3.1.1.7) from human. BuChE (EC 3.1.1.8) from human.
Selectivity index (SI) ¼ AChE IC50/BuChE IC50
f
.
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Fig. 3 Binding mode prediction of 35 (A), and 36 (B) with huAChE (PDB id: 4EY7). Compounds were shown in stick mode colored in yellow. Key
residues were labeled as thin stick mode colored in white. Intermolecular interactions were shown as dot lines with different colors according to
the type of the interaction: light green, hydrophobic contact; pink, p–p stacking; purple, p–alkyl contact.
representative compound to perform the kinetic studies as known to inhibit the Ab1–42 self-aggregation, was used as the
described previously.34 Lineweaver–Burk reciprocal plots was reference compound. Most of the analogs only showed poor or
applied to elucidate the type of inhibition of the test moderate inhibition on Ab1–42 self-aggregation (ranging from
compounds. Briey, Lineweaver–Burk plot can be described by 4.2 ꢀ 0.1–37.8 ꢀ 3.9%, Table 3). Two compounds, 35, and 36,
reciprocal rates versus reciprocal substrate concentrations for exhibited inhibitory rate over 40% (40.7 ꢀ 1.9, 42.2 ꢀ 2.6%,
the different inhibitor concentrations resulting from the respectively). Interestingly, 36 was potent on both AChE and
substrate–velocity curves for ChEs. According to the results self-induced Ab1–42 aggregation, indicating its potential for
(Fig. 2), increasing concentration of the compounds (20, 60, acting as a multi-target compound. It seemed that a methyl
100, and 200 nM) resulted in both increased slopes (decreased group was preferred in inhibiting Ab1–42 self-aggregation,
Vmax) and intercepts (higher Km), indicating a mixed-type inhi- especially when substituted at the para-position. Larger
bition of 36. The detailed values of Vmax and Km at different groups such as t-Bu led to a greater than 2-fold decrease in
concentrations are shown in Table 2.
activity. Mono-substitution of methoxy group led to the
completely loss of the inhibitory effect, however, di- or tri-
substitution of methoxy groups (16, 18, 19) supplied moderate
Molecular modeling studies
To investigate the binding pattern of the synthesized
compounds with huAChE, molecular docking studies were
performed using Discovery Studio (DS). 35, and 36 were selected
as representative compounds. As shown in Fig. 3A and B, both
the compounds bound to AChE in a dual-site manner by
occupying both CAS and PAS. The 1,2,3,4-tetrahydroacridin
moiety of the two compounds inserted into the CAS. The tet-
rahydroacridin moiety formed multiple p–p stacking contacts
with the aromatic sidechains of Trp86 and Tyr124. These
hydrophobic contacts provided driving force for the binding of
the compounds to CAS of huAChE. The substituted phenyl ring
of cinnamic acid moiety was located at the PAS of huAChE
binding groove.44 It formed p–p stacking contacts with the
sidechain of Trp286, Tyr341. It was noticeable that the methoxy
group at meta-position of the phenyl ring (36) formed an addi-
tional p–alkyl interaction with the sidechain of Trp286. The
binding difference may explain the slightly better huAChE
inhibitory activity of 35 than 36.
Table
3 Inhibition of self-induced Ab1–42 aggregation and anti-
proliferative activities of the synthesized compounds
Cpd. Ab IRa %
IC50b (mM)
Cpd.
Ab IRa %
IC50b (mM)
8
9
7.0 ꢀ 0.6 55.7 ꢀ 5.8
33.2 ꢀ 0.8 71.7 ꢀ 6.8
32.9 ꢀ 2.1 39.9 ꢀ 4.0
40.9 ꢀ 1.6 27.1 ꢀ 3.2
4.2 ꢀ 0.1 63.0 ꢀ 9.2
8.4 ꢀ 0.6 56.3 ꢀ 5.1
7.9 ꢀ 0.9 51.6 ꢀ 9.4
24
25
26
27
28
29
30
21.6 ꢀ 3.6 34.2 ꢀ 4.7
7.0 ꢀ 0.1 19.8 ꢀ 2.4
22.4 ꢀ 1.7 10.1 ꢀ 1.0
6.0 ꢀ 0.8 34.5 ꢀ 2.5
5.6 ꢀ 0.2 23.7 ꢀ 1.2
13.6 ꢀ 1.2 55.0 ꢀ 6.5
19.4 ꢀ 2.8 13.5 ꢀ 1.7
30.0 ꢀ 5.1 10.0 ꢀ 1.0
17.6 ꢀ 1.3 59.6 ꢀ 8.0
40.7 ꢀ 1.9 14.5 ꢀ 1.9
35.9 ꢀ 2.1 43.8 ꢀ 3.9
37.8 ꢀ 3.9 92.2 ꢀ 8.8
42.2 ꢀ 2.6 84.6 ꢀ 7.3
19.9 ꢀ 0.3 34.8 ꢀ 2.6
14.4 ꢀ 1.0 41.8 ꢀ 3.9
25.1 ꢀ 3.1 37.3 ꢀ 4.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
5.1 ꢀ 0.2 79.6 ꢀ 11.5 31
34.5 ꢀ 2.3 55.2 ꢀ 4.4
21.4 ꢀ 3.8 26.3 ꢀ 2.9
28.8 ꢀ 4.0 18.7 ꢀ 1.4
31.3 ꢀ 4.0 34.9 ꢀ 4.0
30.1 ꢀ 2.7 29.4 ꢀ 1.9
28.9 ꢀ 2.1 30.9 ꢀ 4.2
35.3 ꢀ 3.8 20.1 ꢀ 3.0
22.7 ꢀ 2.9 28.7 ꢀ 2.1
32
33
34
35
36
37
38
39
In summary, the binding mode of the selected compounds
supported the mixed-type of binding manner revealed by the
kinetic study.
Curcumin 46.2 ꢀ 3.2
—
a
IR stands for inhibitory rate. Inhibition of self-induced Ab1–42
aggregation was determined by thioavin-T uorescence method, and
Inhibition of self-induced Ab1–42 aggregation
the data were shown in mean
experiments. The experiments were performed in the presence of 20
mM target compounds. The antiproliferative activities of the target
compounds on PC12 cells. The IC50 values were calculated based on
ꢀ
SEM of three independent
All compounds were evaluated for their inhibitory capacity on
self-induced Ab1–42 aggregation based on a thioavin T-based
uorometric assay. Curcumin, a natural product that is
b
three independent experiments and were shown in mean ꢀ SEM.
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Although strong electron-withdrawing groups were preferred
for AChE inhibition, it seemed that they were prone to cause
a strong cytotoxicity. Similar manner was observed in 3-OCF3
substituted analog 31 (IC50 ¼ 10.0 ꢀ 1.0 mM). Highly toxic
compounds also included 18, 22, and 33 (IC50 ¼ 18.7 ꢀ 1.4, 20.1
ꢀ 3.0, and 14.5 ꢀ 1.9 mM, respectively).
Then, we evaluated the cytoprotective effects of 35 and 36 on
H2O2-induced cell damage. Treatment with 500 mM H2O2 for
24 h caused over 60% death rate of PC12 cells compared with
the control group (Fig. 4). When pretreated with 35 and 36 for
24 h, the mortality rate of PC-12 cells caused by H2O2 was
signicantly attenuated. Such protective effect exhibited dose-
dependent manner for both the two compounds. 36 showed
a better cytoprotective effect than 35. It increased the cell
viability to 63.1 ꢀ 2.1% and 72.5 ꢀ 2.1% at the concentration of
20 and 40 mM, respectively. These results indicated that 36 had
a potential in antagonizing the oxidative stress.
Fig. 4 Protective effects of compound 35 or 36 against H2O2-induced
cell death in PC-12 cells. PC12 cells were plated in a 96-well plate for
12 h and then treated with various concentrations of 35 or 36 for
another 24 h. After replacing the medium containing 500 mM H2O2,
incubation of the cells was continued for 12 h, then the cell viability
was measured by MTT assay. All data represent the means ꢀ SD of
three independent experiments. Data were shown as mean ꢀ SD (n ¼
3). p### < 0.001 compared to control, p* < 0.05, p** < 0.01 compared
to H2O2-treated cells.
Behavioral studies
Improvement of cognitive ability is the most signicant prole
of anti-AD agents. Based on the multiple evaluations mentioned
above, compound 36 with the best multipotent activity prole
was selected for in vivo behavioral study by using a Morris water
maze test. The animal model was built on the basis of
scopolamine-induced cognition-impaired adult ICR mice and
was applied for the cognitive improvement effects of 36.
Compound 35 was also evaluated with the aim to understand
the importance of the methoxy group. Tacrine (20 mmol kgꢁ1
body weight) was used as positive control. 35, 36, and tacrine
were orally administered to the ICR mice 30 min before intra-
peritoneal (ip) administration of scopolamine (1 mg kgꢁ1) or
saline solution for 10 consecutive days to adapt the apparatus.
The test included 5 days of learning and memory training and
a probe trial on the sixth day. The mean escape latency values of
all the groups on the sixth day were shown in Table 4 and
Fig. 5A. Compared to the control group, scopolamine led to
a remarkable delay of the latency to target (8.9 ꢀ 4.0 seconds vs.
activity. Electron-withdrawing group, such as –NO2 and –CF3,
remarkably reduced the activity. Substituent groups such as
halogen, hydroxyl or amino group had no impact on the inhi-
bition of Ab1–42 self-aggregation, no matter what position they
were at. It was noticeable that benzyl substituted compounds
(33–36) exhibited the best activity among all the derivatives,
suggesting this benzyl group was not only important for AChE
inhibition, but also acted as a preferred moiety for designing
new MTDLs.
Cell toxicity and cyto-protection effects of the compounds in
PC-12 neuroblastoma cells
We next focused on the cell toxicities of the synthesized 45.2 ꢀ 11.6 seconds), indicating that the cognitive impairment
compounds. They were evaluated for the anti-proliferative mouse model was successfully built. Treatment of tacrine
effects against neuroblastoma PC-12 cell line. Most of the ameliorated the impairment and the latency to target reduced
synthesized compounds exhibited IC50 values above 30 mM to 36.3 ꢀ 11.6 seconds (*p < 0.05). 35 exhibited a comparable
(Table 3). 35 and 36 showed the best safety on PC-12 cells (IC50 activity to tacrine (36.4 ꢀ 14.3 seconds, *p < 0.05). Compared to
¼ 92.2 ꢀ 8.8 mM and 84.6 ꢀ 7.3, respectively). Considering their tacrine and 35, 36 signicantly reduced the latency to target
good inhibitory activity on ChEs and self-induced Ab1–42
aggregation, especially 36, they were selected for further in vivo
evaluations. It was noteworthy that the nitro-substituted Table 4 Effects of oral administration of 35 and 36 (15 mg kgꢁ1) on
scopolamine-induced memory impairment in ICR mice evaluated by
compounds 27, 28, and 30 showed much stronger anti-
proliferative activity against PC-12 cells than most of the
the Morris water maze test. Tacrine (15 mg kgꢁ1) was used as positive
control. Data are presented as the mean ꢀ SEM (n ¼ 6; *p < 0.05, ***p
derivatives (IC50 ¼ 34.5 ꢀ 2.5, 23.7 ꢀ 1.2, 13.5 ꢀ 1.7 mM,
< 0.001, ****p < 0.0001 vs. scopolamine model group)
respectively), indicating their potential cytotoxicity. Although
Group
Latency to target (s)
Distance to target (cm)
these nitro-substituted compounds showed the best inhibitory
effects on AChE, they only exerted poor activity on self-induced
Ab1–42 aggregation. Taken together, these compounds were not
investigated for their in vivo activity. Besides, CF3-substituted
compounds 25 and 26 also potently inhibited the proliferation
of PC-12 cells (IC50 ¼ 19.8 ꢀ 2.4 and 10.1 ꢀ 1.0 mM, respectively).
Control
Model
Tacrine
36
8.9 ꢀ 4.0
292.8 ꢀ 206.4****
1637.3 ꢀ 517.1
45.2 ꢀ 11.6
36.3 ꢀ 11.6*
13.2 ꢀ 7.6***
36.4 ꢀ 14.3*
1125.3 ꢀ 367.1
469.5 ꢀ 278.8****
1274.9 ꢀ 452.6
35
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Fig. 5 Effects of oral administration of tacrine (15 mg kgꢁ1), 35 (15 mg kgꢁ1), and 36 (15 mg kgꢁ1) on scopolamine-induced cognitive impairment
in ICR mice determined by the Morris water maze test. (A) The latency to target; (B) the distance to target. Data are presented as the mean ꢀ SEM
(n ¼ 6; *p < 0.05, ***p < 0.001, ****p < 0.0001 vs. scopolamine group).
Fig. 6 The trajectories of mice in control (A), model (B), tacrine (C), 35 (D), and 36 (E) group in the Morris water maze test.
(13.2 ꢀ 7.6 seconds, ***p < 0.001), indicating that 36 consid-
The distance to target (Table 4 and Fig. 6B) and the trajec-
erably ameliorated the cognitive impairment of the treated mice tories of the mice in each group were also analyzed. Compared
and was much better than tacrine. The results also suggested to the control group, administration of scopolamine remark-
the critical role of the methoxy group of ferulic acid moiety of ably led to the extended distance to target (1637.3 ꢀ 517.1 cm vs.
compound 36. Removal of this group led to the markedly 292.8 ꢀ 206.4 cm, ****p < 0.0001). Tacrine and 35 reduced the
decrease of the in vivo activity as compared to 35. We confer distance to target (1125.3 ꢀ 367.1 cm, 1274.9 ꢀ 452.6 cm,
there may be two reasons for the results: (1) the methoxy group respectively). When treated with 36, the distance to target was
may enhance the ability of 36 to penetrate the blood–brain signicantly shortened (469.5 ꢀ 278.8, ****p < 0.0001). These
barrier (BBB) and target the central nervous system (CNS); (2) results were supported by trajectory analysis. As shown in
the methoxy group may prevent the metabolism at meta-posi- Fig. 6B, the trajectory of the mice in scopolamine model group
tion of the phenyl ring, thus enhance the concentration of the was very long and disordered, while tacrine and 35 groups
compound to target CNS.
(Fig. 6C and E) showed shortened distances, but still much
Table 5 ALT and AST activity after the administration of 35 and 36. Tacrine (30 mg kgꢁ1) was used as the reference compound. Values were
expressed as the mean ꢀ SD (n ¼ 6)
Group
ALT (U Lꢁ1
Control
Tacrine
35
8 h
22 h
36 h
72 h
96 h
)
31.4 ꢀ 2.3
32.9 ꢀ 5.3
30.5 ꢀ 3.0
32.4 ꢀ 4.8
29.0 ꢀ 8.0
27.8 ꢀ 7.5
28.2 ꢀ 3.8
30.5 ꢀ 3.4
29.7 ꢀ 3.4
30.7 ꢀ 4.0
29.1 ꢀ 6.0
30.5 ꢀ 6.0
30.7 ꢀ 4.0
27.5 ꢀ 5.7
28.3 ꢀ 5.4
36.3 ꢀ 6.9
32.0 ꢀ 7.7
34.9 ꢀ 8.6
40.9 ꢀ 6.9
27.8 ꢀ 5.0
36
AST (U Lꢁ1
Control
Tacrine
35
)
91.1 ꢀ 16.0
89.3 ꢀ 22.1
102.6 ꢀ 14.3
91.2 ꢀ 19.8
91.5 ꢀ 21.4
105.4 ꢀ 13.6
93.7 ꢀ 12.0
96.7 ꢀ 15.6
95.6 ꢀ 9.3
87.8 ꢀ 22.5
73.0 ꢀ 10.0
78.0 ꢀ 14.6
83.9 ꢀ 16.9
91.6 ꢀ 18.5
78.1 ꢀ 10.3
88.9 ꢀ 24.1
86.4 ꢀ 18.2
87.7 ꢀ 14.4
82.1 ꢀ 16.7
77.9 ꢀ 16.2
36
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Fig. 7 ALT (A) and AST (B) activity after the administration of tacrine, 35, and 36. Values are expressed as mean ꢀ SEM (n ¼ 6; t test, compared to
the control of the same time after administration).
longer than the control group (Fig. 6A). Mice treated with 36 and 36 h and 96 h for AST, compared to those of tacrine group.
almost recovered to the normal cognition (Fig. 6D), with The results indicated the hepatic safety prole of 36. Interest-
a similar orientation and distance to that of the normal mice. ingly, 35 exhibited an induction of the ALT (36.3 ꢀ 6.9 vs. 31.4 ꢀ
Taken together, these results supported that 36 remarkably 2.3 at 8 h, 40.9 ꢀ 6.9 vs. 32.9 ꢀ 5.3 at 22 h) and AST level (102.6 ꢀ
ameliorated the cognition impairment caused by scopolamine. 14.3 vs. 91.1 ꢀ 16.0 at 8 h) compared to the control group,
indicating a potential toxic effect of this compound. When the
time extended to 72 h and 96 h, the levels of ALT and AST were
comparable to those of control group. Considering the struc-
Hepatotoxicity studies
Given that the serious hepatotoxicity of tacrine has been the
tural difference between 35 and 36, we can speculate that the
primary limitation for its clinical use, to ensure the safety of 35
methoxy group was benecial to the hepatic safety. The results
and 36 for further development, we next investigated the
were also in accordance with those from the behavioral studies.
possible drug-induced hepatotoxicity by comparing their toxic
Therefore, it is important to introduce proper groups at this
prole to tacrine. Heparinized serum was collected 8 h, 22 h,
36 h, 72 h, and 96 h aer the administration of tacrine, 35 and
36 for the evaluation of the levels of alanine aminotransferase
(ALT) and aspartate aminotransferase (AST), two known
position in order to avoid undesired metabolism.
To further analyze the hepatotoxicity of 35 and 36,
morphologic studies by immunohistochemical staining were
applied. Treatment of tacrine (Fig. 8B), 35 (Fig. 8C) or 36
biomarkers of live damage (Table 5 and Fig. 7). Compared to the
(Fig. 8D) did not result in remarkable morphologic changes in
control group, aer the treatment of tacrine, the levels of ALT
liver compared to the control group (Fig. 8A). Taken together, 36
and AST were slightly induced at 22 h, but in general, no
remarkable damage was observed. As expected, 36 did not
showed any hepatotoxicity at all the time points, the level of ALT
exhibited the highest safety among all the test compounds,
ensuring its further development.
and AST even slightly reduced at 22 h, 36 h, and 96 h for ALT
Conclusions
CY-1 was a tacrine–ferulic acid hybrid reported by our group
previously. Guided by this compound, in the present studies,
a series of tacrine–cinnamic acid hybrids were designed and
synthesized so as to identify the optimal substitution on the
phenyl ring of the cinnamic acid moiety. Although there are
several publications about tacrine–ferulic acid hybrid, as far as
we concerned, this is the rst medicinal chemistry study on the
ferulic acid moiety. In vitro assays proved that most of the
compounds effectively inhibited ChEs in the nanomolar range.
Additionally, some interesting information was summarized
from the SAR study and can guide the further optimization of
this series of compounds. 36 was one of the most potent
analogs, which was about 4-fold more active than the parent
compound CY-1 against AChE. Kinetic studies and molecular
docking indicated that 36 inhibited AChE in a mixed-type
manner by simultaneously binding to CAS and PAS of AChE.
This compound effectively inhibited the self-induced Ab1–42
aggregation, and exhibited cytoprotective effects against H2O2
induced cell damage. Meanwhile, it was proved to be non-toxic
to PC-12 cells when it exerted its biological functions, indicating
its good safety. The in vitro assays conrmed the
Fig. 8 Histomorphological appearance of livers of male mice after
treatment with the solvent only (control, A), or 22 h after administration
of tacrine (B), 35 (C), or 36 (D). HE staining, original magnification
ꢂ200.
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multifunctional potent manner of 36 as potential anti-AD agent. was added. Aer being stirred for overnight at room tempera-
Therefore, it was subjected to in vivo evaluation including ture. The solvent was removed under vacuo, 20 mL ethyl acetate
Morris water maze test and hepatotoxicity studies. 36 remark- was added, the solid was ltered. Then the solid was puried by
ably reduced the scopolamine-induced cognitive impairment in column chromatography (CH2Cl2/MeOH ¼ 50 : 1, v/v, with 0.1%
animal model and showed very low hepatotoxicity under the triethylamine) to give a white powder 0.12 g (39.1%). Mp 186–
therapeutic concentration. Altogether, 36 can be considered as 187 ꢃC. 1H NMR (300 MHz, DMSO-d6): d 8.53 (t, J ¼ 6.10 Hz, 1H,
a promising lead compound for further identication of new ArH), 8.47 (d, J ¼ 8.64 Hz, 1H, ArH), 7.96 (s, 1H, CONH), 7.86 (t, J
anti-AD agents.
¼ 7.58 Hz, ArH), 7.60–7.75 (m, 3H, ArH and NH), 7.40 (d, J ¼
6.84 Hz, 3H, ArH and COCH]CH), 6.57 (d, J ¼ 15.81 Hz, 1H,
COCH]CH), 4.02 (d, J ¼ 5.40 Hz, 2H, NHCH2), 3.57 (d, J ¼
5.40 Hz, 2H, CH2NHCO), 2.94 (br, 2H, C4–H2), 2.68 (br, 2H, C1–
H2), 1.82 (br, 4H, C2–H2 and C3–H2). 13C NMR (125 MHz,
Experimental sections
Chemistry
General experimental. Melting points were determined on CDCl3): d 167.61, 157.94, 151.06, 146.59, 141.52, 134.68, 129.85,
a Mel-TEMP II melting point apparatus and are uncorrected. 1H- 128.86, 128.66, 127.86, 127.67, 123.86, 122.85, 120.37, 119.73,
NMR spectra were recorded with a Bruker Avance 300 MHz 115.85, 63.96, 49.84, 40.68, 33.43, 25.01, 22.95, 22.53. HRMS
spectrometer at 300 K, using TMS as an internal standard. MS (ESI): calcd for C24H25N3O [M + H]+ 372.207, found 372.2074.
spectra were recorded on a Shimadzu GC-MS 2010 (EI) or HPLC (70% methanol in water with 0.5% H3PO4): tR ¼ 3.56 min,
a Mariner Mass Spectrum (ESI) or a LC/MSD TOF HR-MS 97.077%.
Spectrum. All compounds were routinely checked by TLC and
(E)-N-(2-((1,2,3,4-Tetrahydroacridin-9-yl)amino)ethyl)-3-(o-tolyl)-
1H NMR. TLCs and preparative thin-layer chromatography were acrylamide (9). According to the procedure used to synthesize 8, 9
performed on silica gel GF/UV 254 supported by glass plate, and was synthesized from (E)-3-(o-tolyl)acrylic acid (0.14 g, 0.83
the chromatograms were performed on silica gel (200–300 mmol) to give a white powder 0.14 g (43.89%). Mp 202–204 ꢃC.
mesh) visualized under UV light at 254 and 365 nm. Purity for 1H NMR (300 MHz, DMSO-d6): d 8.83 (s, 1H, ArH), 8.53 (d, J ¼
nal compounds was greater than 95% and was measured by 8.53 Hz, 1H, ArH), 8.00 (d, J ¼ 8.19 Hz, 2H, ArH and CONH), 7.84
HPLC with Agilent Technologies 1260 innity C18 4.60 mm ꢂ (t, J ¼ 7.59 Hz, 1H, ArH), 7.65 (d, J ¼ 15.72 Hz, 1H, COCH]CH),
150 mm column using a mixture of solvent methanol/water or 7.56 (t, J ¼ 7.68 Hz, 1H, ArH), 7.45 (d, J ¼ 6.93 Hz, 1H, ArH), 7.24
acetonitrile/water at the ow rate of 0.5 mL minꢁ1 and peak (br, 2H, ArH and NH), 6.53 (d, J ¼ 15.66 Hz, COCH]CH), 4.02 (d,
detection at 254 nm under UV. All solvents were reagent grade J ¼ 6.18 Hz, 2H, NHC H2 ), 3.56 (d, J ¼ 5.22 Hz, 2H, C H2 NHCO),
and, when necessary, were puried and dried by standards 3.00 (s, 3H, CH3) 2.71 (s, 2H, C4–H2 ), 2.35 (s, 2H, C1–H2 ), 1.82 (s,
methods. Concentration of solutions aer reactions and 4H, C2–H2 , C3–H2 ). 13C NMR (125 MHz, DMSO-d6): d 166.78,
extractions involved the use of a rotary evaporator operating at 156.12, 148.29, 137.22, 137.00, 134.12, 132.79, 131.15, 129.76,
a reduced pressure of ca. 20 Torr. Organic solutions were dried 126.87, 126.45, 125.50, 123.47, 119.56, 115.95, 111.91, 48.70,
over anhydrous sodium sulfate. Analytical results are within 28.44, 24.31, 21.98, 21.44, 20.81. HRMS (ESI): calcd for
(0.40% of the theoretical values).
N-(1,2,3,4-Tetrahydroacridin-9-yl)ethane-1,2-diamine (4). To methanol in water with 0.5% H3PO4): tR ¼ 4.53 min, 95.828%.
a solution of 3 (9-chlorotetrahydroacridine) (2 g, 9.2 mmol) in 10
(E)-N-(2-((1,2,3,4-Tetrahydroacridin-9-yl)amino)ethyl)-3-(m-tolyl)-
C25H27N3O [M + H]+ 386.2227, found 386.2226. HPLC (70%
mL of pentanol, diaminoethane (2.76 g, 45.94 mmol) were acrylamide (10). According to the procedure used to synthesize 8,
added. Aer reuxing for 24 h, the solution was cooled to room 10 was synthesized from (E)-3-(m-tolyl)acrylic acid (0.14 g, 0.83
temperature and then acidied with hydrochloric acid. The mmol) to give a white powder 0.14 g (43.89%). Mp 200–202 ꢃC. 1H
solution was extracted with acid water (4 ꢂ 10 mL). The NMR (300 MHz, DMSO-d6): d 8.51–8.46 (m, 2H, ArH), 7.97 (br,
combined aqueous phase was basied by NaOH and then 1H, CONH), 7.87 (t, J ¼ 7.56 Hz, 1H, ArH), 7.78 (d, J ¼ 7.65 Hz, 1H,
extracted with CH2Cl2 (4 ꢂ 15 mL). The water phase was ArH), 7.58 (t, J ¼ 7.49 Hz, 1H, ArH), 7.40 (d, J ¼ 15.84 Hz, 1H,
combined and washed with brine, dried over anhydrous Na2SO4 COCH]CH), 7.35–7.26 (m, 3H, ArH and NH), 7.19 (d, J ¼
overnight, and evaporated in vacuo. The residue was puried by 7.26 Hz, 1H, ArH) 6.56 (d, J ¼ 15.81 Hz, 1H, COCH]CH), 4.02 (d,
column chromatography (CH2Cl2/MeOH ¼ 20 : 1, v/v, with 0.1% J ¼ 5.46, 2H, NHC H2 ), 3.57 (d, J ¼ 5.61 Hz, 2H, C H2 NHCO),
triethylamine). 1.5 g yellow oil was given (67.6%). 1H NMR 2.95 (br, 2H, C4–H2 ), 2.68 (br, 2H, C1–H2 ), 2.31 (s, 3H, CH3), 1.83
(CD3OD): d 8.13–8.10 (m, 1H, ArH), 7.78–7.75 (m, 1H, ArH), (br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 MHz, DMSO-d6):
7.60–7.54 (m, 1H, ArH), 7.42–7.36 (m, 1H, ArH), 3.61 (t, J ¼ d 167.06, 156.66, 151.00, 139.99, 138.64, 138.33, 135.08, 133.31,
7.50 Hz, 2H, NH–CH2), 2.96–2.91 (m, 4H, C4–H2, CH2–NH2), 130.86, 129.35, 128.61, 125.79, 125.60, 125.26, 121.68, 119.54,
2.76 (br, 2H, C1–H2), 1.91–1.88 (m, 4H, C3–H2, C2–H2); MS (GC) 116.00, 111.92, 48.82, 28.42, 24.21, 21.92, 21.38, 20.79. HRMS
m/z (% rel. Int.) 241 (M+, 45), 197 (100) (found C, 74.99; H, 8.18; (ESI): calcd for C25H27N3O [M + H]+ 386.2227, found 386.2215.
N, 16.98; C15H19N3 requires C, 74.65; H, 7.94; N, 17.41).
N-(2-((1,2,3,4-Tetrahydroacridin-9-yl)amino)ethyl)cinnamamide 99.188%.
(8).45 Cinnamic acid (0.123 g, 0.83 mmol), pyBOP (0.44 g, 0.99
HPLC (70% methanol in water with 0.5% H3PO4): tR ¼ 4.97 min,
(E)-N-(2-((1,2,3,4-Tetrahydroacridin-9-yl)amino)ethyl)-3-(p-tolyl)-
mmol), and DIPEA (0.14 g, 1.08 mmol) were dissolved in 3 mL of acrylamide (11). According to the procedure used to synthesize 8,
CH2Cl2, and the mixture was stirred at room temperature for 11 was synthesized from (E)-3-(p-tolyl)acrylic acid (0.14 g, 0.83
40 min. Then a solution of 4 (0.2 g, 0.83 mmol) in 2 mL CH2Cl2 mmol) to give a white powder 0.17 g (53.29%). Mp 212–214 ꢃC.
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1H NMR (300 MHz, DMSO-d6): d 8.47 (d, J ¼ 7.41 Hz, 2H, ArH), 1H, CONH), 7.72 (t, J ¼ 7.01 Hz, 1H, ArH), 7.52 (d, J ¼ 7.97 Hz,
7.95 (br, 1H, CONH), 7.86 (t, J ¼ 7.32 Hz, 1H, ArH), 7.78 (d, J ¼ 2H, ArH), 7.46 (d, J ¼ 15.93 Hz, 1H, COCH]CH), 7.16–7.12 (m,
7.37 Hz, 1H, ArH), 7.57 (t, J ¼ 7.56 Hz, 1H, ArH), 7.45–7.42 (m, 3H 2H, ArH and NH), 6.99 (d, J ¼ 7.98 Hz, 2H, ArH), 6.50 (d, J ¼
ArH and NH and COCH]CH), 7.22 (d, J ¼ 7.08 Hz, 1H, ArH), 15.94 Hz, 1H, COCH]CH), 4.05 (d, J ¼ 5.75 Hz, 2H, NHC H2 ),
6.81 (d, J ¼ 15.69 Hz, 1H, COCH]CH), 4.02 (br, 2H, NHC H2 ), 3.83 (s, 3H, OCH3), 3.50 (d, J ¼ 5.74 Hz, 2H, C H2 NHCO), 2.89
3.57 (br, 2H, C H2 NHCO), 2.94 (br, 2H, C4–H2 ), 2.68 (br, 2H, (br, 2H, C4–H2 ), 2.72 (br, 2H, C1–H2 ), 1.86 (br, 4H, C2–H2 and
C1–H2 ), 2.31 (s, 3H, CH3), 1.83 (br, 4H, C2–H2 and C3–H2 ). 13
C
C3–H2 ). 13C NMR (125 MHz, DMSO-d6): d 167.40, 158.11,
NMR (125 MHz, DMSO-d6): d 167.19, 156.69, 150.99, 139.98, 156.46, 151.26, 138.67, 135.01, 133.11, 131.55, 128.50, 125.62,
139.88, 138.32, 133.32, 132.38, 130.06, 128.07, 125.80, 125.60, 123.51, 122.24, 121.22, 119.87, 116.17, 112.16, 56.06, 48.86,
120.78, 119.52, 116.00, 111.91, 48.88, 28.41, 24.21, 21.92, 21.43, 46.35, 28.62, 26.39, 24.24, 21.96, 20.86. HRMS (ESI): calcd for
20.79. HRMS (ESI): calcd for C25H27N3O [M + H]+ 386.2227,
C
25H27N3O2 [M + H]+ 402.2176, found 402.2182. HPLC (70%
found 386.2219. HPLC (70% methanol in water with 0.5% methanol in water with 0.5% H3PO4): tR ¼ 5.44 min, 96.122%.
H3PO4): tR ¼ 4.75 min, 98.085%. (E)-3-(2,3-Dimethoxyphenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)-
(E)-3-(2-Methoxyphenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)- amino)ethyl)acrylamide (15). According to the procedure used to
amino)ethyl)acrylamide (12). According to the procedure used to synthesize 8, 15 was synthesized from (E)-3-(2,3-dimethoxyphenyl)
synthesize 8, 12 was synthesized from (E)-3-(2-methoxyphenyl) acrylic acid (0.17 g, 0.83 mmol) to give a white powder 0.2 g
acrylic acid (0.15 g, 0.83 mmol) to give a white powder 0.15 g (55.87%). Mp 195–197 ꢃC. 1H NMR (300 MHz, DMSO-d6): d 8.53–
(45.05%). Mp 173–175 ꢃC. 1H NMR (300 MHz, DMSO-d6): d 8.48– 8.46 (m, 2H, ArH), 7.94 (br, 1H, CONH), 7.86 (t, J ¼ 7.02 Hz, 1H,
8.45 (m, 2H, ArH), 7.85 (t, J ¼ 7.02 Hz, 2H, ArH and CONH), 7.78 ArH), 7.78 (d, J ¼ 7.71 Hz, 1H, ArH), 7.65–7.55 (m, 2H, COCH]CH
(d, J ¼ 7.77 Hz, 1H, ArH), 7.66 (d, J ¼ 15.96 Hz, 1H, COCH]CH), and ArH), 7.11–7.07 (m, 3H, NH and ArH), 6.59 (d, J ¼ 15.96 Hz,
7.57 (t, J ¼ 6.93 Hz, 1H, ArH), 7.49 (d, J ¼ 7.23, 1H, ArH), 7.36 (t, J 1H, COCH]CH), 4.05–3.98 (m, 2H, NHC H2 ), 3.81 (s, 3H, OCH3),
¼ 7.62 Hz, 1H, ArH), 7.07 (d, J ¼ 8.25 Hz, 1H, ArH), 6.97 (t, J ¼ 3.73 (s, 3H, OCH3), 3.56 (d, J ¼ 5.37 Hz, 2H, C H2 NHCO), 2.87 (br,
7.35 Hz, ArH), 6.60 (d, J ¼ 15.96 Hz, 1H, COCH]CH), 4.00 (d, J 2H, C4–H2 ), 2.68 (br, 2H, C1–H2 ), 1.83 (br, 4H, C2–H2 and C3–
¼ 5.56 Hz, 2H, NHC H2 ), 3.85 (s, 3H, OCH3), 3.55 (d, J ¼ 5.71 Hz, H2 ). 13C NMR (125 MHz, DMSO-d6): d 167.10, 156.69, 153.32,
2H, C H2 NHCO), 2.94 (br, 2H, C4–H2 ), 2.68 (br, 2H, C1–H2 ), 147.97, 138.34, 134.39, 133.30, 128.70, 125.79, 125.61, 124.89,
1.83 (br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 MHz, DMSO- 123.09, 119.34, 116.03, 114.51, 111.96, 61.12, 60.23, 56.27, 48.73,
d6): d 167.40, 158.11, 156.48, 151.24, 138.65, 135.01, 133.12, 46.36, 28.43, 26.42, 24.23, 21.92, 21.01. HRMS (ESI): calcd for
131.55, 128.50, 125.63, 123.51, 122.25, 121.22, 119.85, 116.16, C26H29N3O3 [M + H]+ 432.2282, found 432.2278. HPLC (70%
111.15, 56.06, 48.86, 46.35, 28.61, 26.39, 24.24, 21.96, 20.85. methanol in water with 0.5% H3PO4): tR ¼ 3.21 min, 98.413%.
HRMS (ESI): calcd for C25H27N3O2 [M + H]+ 402.2176, found
402.2169. HPLC (70% methanol in water with 0.5% H3PO4): tR ¼ yl)-amino)ethyl)acrylamide (16). According to the procedure
3.63 min, 97.648%. used to synthesize 8, 16 was synthesized from (E)-3-(2,5-dime-
(E)-3-(2,5-Dimethoxyphenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-
(E)-3-(3-Methoxyphenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)- thoxyphenyl)acrylic acid (0.17 g, 0.83 mmol) to give a white
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amino)ethyl)acrylamide (13). According to the procedure used to powder 0.14 g (39.11%). Mp 190–191 C. H NMR (300 MHz,
synthesize 8, 13 was synthesized from (E)-3-(3-methoxyphenyl) DMSO-d6): d 8.48–8.46 (m, 2H, ArH), 7.95 (br, 1H, CONH), 7.86
acrylic acid (0.15 g, 0.83 mmol) to give a white powder 0.11 g (t, J ¼ 7.50 1H, ArH), 7.78 (d, J ¼ 8.13 Hz, 1H, ArH), 7.65–7.55
(33.03%). Mp 169–172 ꢃC. 1H NMR (300 MHz, DMSO-d6): d 8.49– (m, 2H, COCH]CH and ArH), 7.04–6.93 (m, 3H, NH and ArH),
8.46 (m, 2H, ArH), 7.96 (br, 1H, CONH), 7.87 (t, J ¼ 6.87 Hz, 1H, 6.61 (d, J ¼ 15.87 Hz, 1H, COCH]CH), 4.01 (d, J ¼ 4.92 Hz, 2H,
ArH), 7.78 (d, J ¼ 7.50 Hz, 1H, ArH), 7.57 (t, J ¼ 7.08 Hz, 1H, NHC H2 ), 3.79 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.56 (d, J ¼
ArH), 7.40 (d, J ¼ 15.81 Hz, 1H, COCH]CH), 7.32 (t, J ¼ 7.95 Hz, 5.22 Hz, 2H, C H2 NHCO), 2.94 (br, 2H, C4–H2 ), 2.68 (br, 2H,
1H, ArH), 7.13–7.10 (m, 2H, ArH and NH), 6.95 (dd, J1 ¼ 7.95 Hz, C1–H2 ), 1.83 (br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 MHz,
J2 ¼ 2.04 Hz, 1H, ArH), 6.57 (d, J ¼ 15.81 Hz, 1H, COCH]CH), DMSO-d6): d 167.09, 156.69, 153.32, 147.96, 138.34, 134.39,
4.02 (d, J ¼ 5.65 Hz, 2H, NHC H2 ), 3.77 (s, 3H, OCH3), 3.57 (d, J 133.31, 128.70, 125.79, 125.61, 124.89, 123.09, 119.54, 119.14,
¼ 5.55 Hz, 2H, C H2 NHCO), 2.95 (br, 2H, C4–H2 ), 2.68 (br, 2H, 116.03, 114.51, 111.96, 61.12, 60.23, 56.27, 48.73, 46.36, 28.43,
C1–H2 ), 1.83 (br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 MHz, 26.42, 24.23, 21.92, 21.22, 20.80. HRMS (ESI): calcd for
CDCl3): d 167.64, 159.86, 157.81, 151.14, 146.49, 141.27, 136.12,
129.83, 128.67, 127.50, 123.83, 122.92, 120.84, 120.39, 119.67, methanol in water with 0.5% H3PO4): tR ¼ 3.63 min, 97.736%.
C
26H29N3O3 [M + H]+ 432.2282, found 432.2277. HPLC (70%
115.72, 115.40, 113.16, 55.26, 49.87, 40.67, 33.37, 25.00, 22.93,
(E)-N-(2-((1,2,3,4-Tetrahydroacridin-9-yl)amino)ethyl)-3-(2,3,4-
22.50. HRMS (ESI): calcd for C25H27N3O2 [M + H]+ 402.2176, trimethoxyphenyl)acrylamide (17). According to the procedure
found 402.2174. HPLC (70% methanol in water with 0.5% used to synthesize 8, 17 was synthesized from (E)-3-(2,3,4-tri-
H3PO4): tR ¼ 3.68 min, 98.401%.
methoxyphenyl)acrylic acid (0.20 g, 0.83 mmol) to give a white
(E)-3-(4-Methoxyphenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)- powder 0.13 g (33.94%). Mp 206–208 ꢃC. 1H NMR (300 MHz,
amino)ethyl)acrylamide (14). According to the procedure used to DMSO-d6): d 8.50–8.47 (m, 2H, ArH), 8.00 (br, 1H, CONH), 7.88
synthesize 8, 14 was synthesized from (E)-3-(4-methoxyphenyl) (t, J ¼ 7.46 Hz, 1H, ArH), 7.79 (d, J ¼ 8.07 Hz, 1H, ArH), 7.62–7.51
acrylic acid (0.15 g, 0.83 mmol) to give a white powder 0.16 g (m, 2H, ArH and COCH]CH), 7.28 (d, J ¼ 8.82 Hz, 1H, ArH),
1
1
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(48.05%). Mp 180–182 C. H NMR (300 MHz, DMSO-d6): d H 6.88 (d, J ¼ 8.88 Hz, 1H, ArH), 6.53 (d, J ¼ 15.84 Hz, 1H, COCH]
NMR (300 MHz, DMSO-d6): d 8.47–8.44 (m, 2H, ArH), 7.93 (br, CH), 4.03 (d, J ¼ 5.25 Hz, 2H, NHC H2 ), 3.83 (s, 3H, OCH3), 3.80
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(s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.57 (d, J ¼ 5.82 Hz, 2H, 132.97, 131.60, 130.50, 128.32, 128.13, 125.77, 125.64, 125.01,
C H2 NHCO), 2.96 (br, 2H, C4–H2 ), 2.69 (br, 2H, C1–H2 ), 1.84 119.56, 116.05, 112.01, 48.51, 28.45, 24.24, 21.92, 20.81.
(br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 MHz, DMSO-d6): HRMS (ESI): calcd for C24H24ClN3O [M + H]+ 406.1681, found
d 167.77, 158.45, 155.06, 152.73, 150.63, 147.46, 142.40, 134.25, 406.1675. HPLC (70% methanol in water with 0.5% H3PO4):
128.81, 128.31, 123.73, 123.48, 123.04, 121.74, 121.29, 120.56, tR ¼ 4.69 min, 97.027%.
116.32, 108.89, 61.61, 60.87, 56.37, 48.79, 46.18, 34.05, 25.46,
(E)-3-(3-Chlorophenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)-
23.25, 22.91. HRMS (ESI): calcd for C27H31N3O4 [M + H]+ amino)ethyl)acrylamide (21). According to the procedure used
462.2387, found 462.2386. HPLC (80% methanol in water with to synthesize 8, 21 was synthesized from (E)-3-(3-chlor-
0.5% H3PO4): tR ¼ 3.39 min, 96.333%.
ophenyl)acrylic acid (0.15 g, 0.83 mmol) to give a yellow
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(E)-N-(2-((1,2,3,4-Tetrahydroacridin-9-yl)amino)ethyl)-3-(3,4,5- powder 0.17 g (50.53%). Mp 192–193 C. H NMR (300 MHz,
trimethoxyphenyl)acrylamide (18). According to the procedure DMSO-d6): d 8.50–8.45 (m, 2H, ArH) 7.94–7.78 (m, 2H, CONH
used to synthesize 8, 18 was synthesized from (E)-3-(3,4,5-tri- and ArH), 7.78 (d, J ¼ 8.07 Hz, 1H, ArH) 7.62–7.51 (m, 3H, ArH
methoxyphenyl)acrylic acid (0.20 g, 0.83 mmol) to give a white and COCH]CH), 7.44–7.39 (m, 3H, ArH and NH), 6.63 (d, J ¼
powder 0.17 g (44.39%). Mp 201–202 ꢃC. 1H NMR (300 MHz, 15.81 Hz, 1H, COCH]CH), 4.02 (d, J ¼ 4.80 Hz, 2H, NHC H2 ),
DMSO-d6): d 8.49–8.46 (m, 2H, ArH), 7.96–7.77 (m, 3H, CONH 3.57 (d, J ¼ 5.31 Hz, 2H, C H2 NHCO), 2.95 (br, 2H, C4–H2 ),
and ArH), 7.60–7.50 (m, 2H, COCH]CH and ArH), 7.27 (d, J ¼ 2.68 (br, 2H, C1–H2 ), 1.83 (br, 4H, C2–H2 and C3–H2 ). 13C
8.67 Hz, 1H, ArH), 6.86 (d, J ¼ 8.79 Hz, 1H, NH) 6.51 (d, J ¼ NMR (125 MHz, DMSO-d6): d 166.59, 156.65, 150.99, 138.32,
15.75 Hz, 1H, COCH]CH), 4.02 (br, 2H, NHC H2 ), 3.82–3.54 137.43, 134.17, 133.34, 131.29, 129.78, 127.77, 126.56, 125.80,
(m, 11H, 3*OCH3 and C H2 NHCO), 2.94 (br, 2H, C4–H2 ), 2.68 125.61, 123.51, 119.52, 115.96, 111.91, 48.61, 28.39, 24.20,
(br, 2H, C1–H2 ), 1.83 (br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 21.89, 20.78. HRMS (ESI): calcd for C24H24ClN3O [M + H]+
MHz, DMSO-d6): d 166.43, 158.46, 153.57, 150.65, 150.63, 406.1681, found 406.1671. HPLC (70% methanol in water
147.47, 139.61, 139.24, 130.97, 128.81, 128.32, 123.74, 123.48, with 0.5% H3PO4): tR ¼ 5.15 min, 95.756%.
121.75, 120.55, 116.31, 105.48, 60.55, 56.34, 48.76, 34.05, 25.48,
(E)-3-(4-Chlorophenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)-
23.25, 22.92. HRMS (ESI): calcd for C27H31N3O4 [M + H]+ amino)ethyl)acrylamide (22). According to the procedure used to
462.2387, found 462.2388. HPLC (70% methanol in water with synthesize 8, 22 was synthesized from (E)-3-(4-chlorophenyl)
0.5% H3PO4): tR ¼ 2.96 min, 98.112%.
acrylic acid (0.15 g, 0.83 mmol) to give a yellow powder 0.2 g
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-N-(2-((1,2,3,4-tetrahydroacridin- (59.45%). Mp 200–202 ꢃC. 1H NMR (300 MHz, DMSO-d6): d 8.52–
9-yl)amino)ethyl)acrylamide (19).45 According to the procedure 8.45 (m, 2H, ArH), 7.95 (br, 1H, CONH), 7.89–7.76 (m, 2H, ArH
used to synthesize 8, 19 was synthesized from (E)-3-(benzo[d] and COCH]CH), 7.57 (br, 2H, ArH and NH), 7.48–7.40 (m, 3H,
[1,3]dioxol-5-yl)acrylic acid (0.16 g, 0.83 mmol) to give a white ArH), 6.57 (d, J ¼ 15.81 Hz, 1H, COCH]CH), 4.02 (d, J ¼
powder 0.21 g (60.99%). Mp 197–199 ꢃC. 1H NMR (300 MHz, 5.07 Hz, 2H, NHC H2 ), 3.57 (d, J ¼ 5.37 Hz, 2H, C H2 NHCO),
DMSO-d6): d 8.48–8.42 (m, 2H, ArH), 7.95 (br, 1H, CONH), 7.80– 2.94 (br, 2H, C4–H2 ), 2.68 (br, 2H, C1–H2 ), 1.83 (br, 4H, C2–H2
7.77 (m, 2H, ArH), 7.56 (t, J ¼ 7.32 Hz, 1H, ArH) 7.35 (d, J ¼ and C3–H2 ). 13C NMR (125 MHz, DMSO-d6): d 166.72, 156.63,
15.69 Hz, 1H, COCH]CH), 7.14 (s, 1H, NH), 7.06 (d, J ¼ 7.98 Hz, 150.96, 138.53, 134.59, 134.08, 133.31, 129.80, 129.50, 125.80,
1H, ArH), 6.94 (d, J ¼ 7.98 Hz, 1H, ArH), 6.41 (d, J ¼ 15.72 Hz, 125.60, 122.61, 119.59, 116.00, 111.94, 48.65, 28.43, 24.21,
1H, COCH]CH), 6.05 (s, 2H, OCH2O), 4.00 (d, J ¼ 4.71 Hz, 2H, 21.90, 20.79. HRMS (ESI): calcd for C24H24ClN3O [M + H]+
NHC H2 ), 3.55 (d, J ¼ 5.34 Hz, 2H, C H2 NHCO), 2.94 (br, 2H, 406.1681, found 406.1673. HPLC (70% methanol in water with
C4–H2 ), 2.69 (br, 2H, C1–H2 ), 1.82 (br, 4H, C2–H2 and C3–H2 ). 0.5% H3PO4): tR ¼ 5.23 min, 95.101%.
13C NMR (125 MHz, DMSO-d6): d 167.25, 156.50, 151.05, 149.12,
(E)-3-(4-Fluorophenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)-
148.43, 139.75, 138.45, 133.19, 129.47, 125.76, 125.53, 123.96, amino)ethyl)acrylamide (23). According to the procedure used to
119.81, 119.64, 116.00, 111.89, 109.07, 106.70, 101.97, 48.87, synthesize 8, 23 was synthesized from (E)-3-(4-uorophenyl)
28.46, 24.21, 21.91, 20.80. HRMS (ESI): calcd for C25H25N3O3 [M acrylic acid (0.14 g, 0.83 mmol) to give a white powder 0.14 g
+ H]+ 416.1969, found 416.1964. HPLC (70% methanol in water (43.37%). Mp 198–199 ꢃC. 1H NMR (300 MHz, DMSO-d6): d 8.50–
with 0.5% H3PO4): tR ¼ 3.44 min, 98.605%.
8.45 (m, 2H, ArH), 7.96 (br, 1H, CONH), 7.89–7.76 (m, 2H, ArH),
(E)-3-(2-Chlorophenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)- 7.64–7.55 (m, 3H, ArH and NH), 7.43 (d, J ¼ 15.78 Hz, 1H,
amino)ethyl)acrylamide (20). According to the procedure used COCH]CH), 7.24 (t, J ¼ 8.64 Hz, 2H, ArH), 6.52 (d, J ¼ 15.81 Hz,
to synthesize 8, 20 was synthesized from (E)-3-(2-chlor- 1H, COCH]CH), 4.02 (d, J ¼ 4.80 Hz, 2H, NHC H2 ), 3.57 (d, J ¼
ophenyl)acrylic acid (0.15 g, 0.83 mmol) to give a yellow 5.07 Hz, 2H, C H2 NHCO), 2.94 (br, 2H, C4–H2 ), 2.68 (br, 2H,
1
powder 0.18 g (53.51%). Mp 201–203 C. H NMR (300 MHz, C1–H2 ), 1.83 (br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 MHz,
ꢃ
DMSO-d6): d 8.60 (t, J ¼ 5.70 Hz, 1H, ArH), 8.47 (d, J ¼ 8.64 Hz, DMSO-d6): d 166.90, 164.09, 162.45, 156.65, 150.96, 138.71,
1H, ArH) 7.89–7.84 (m, 2H, CONH and ArH), 7.80–7.66 (m, 138.30, 133.32, 131.74, 130.28, 125.69, 121.68, 119.51, 116.51,
3H, ArH and COCH]CH), 7.60–7.51 (m, 2H, ArH), 7.43–7.38 116.36, 115.95, 111.88, 48.74, 46.22, 28.38, 24.19, 21.89, 20.77.
(m, 2H, ArH and NH), 6.60 (d, J ¼ 15.69 Hz, 1H, COCH]CH), HRMS (ESI): calcd for C24H24FN3O [M + H]+ 390.1976, found
4.02 (d, J ¼ 5.31 Hz, 2H, NHC H2 ), 3.58 (d, J ¼ 5.67 Hz, 2H, 390.1968. HPLC (70% methanol in water with 0.5% H3PO4): tR ¼
C H2 NHCO), 2.94 (br, 2H, C4–H2 ), 2.67 (br, 2H, C1–H2 ), 1.83 5.30 min, 95.458%.
(br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 MHz, DMSO-d6):
d 166.36, 156.71, 151.06, 138.33, 135.12, 133.80, 133.32, amino)ethyl)acrylamide (24). According to the procedure used to
(E)-3-(4-Bromophenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)-
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synthesize 8, 24 was synthesized from (E)-3-(4-bromophenyl) and COCH]CH), 6.65 (d, J ¼ 15.84 Hz, 1H, COCH]CH), 4.00 (d,
acrylic acid (0.19 g, 0.83 mmol) to give a brown powder 0.19 g J ¼ 4.62 Hz, 2H, NHC H2 ), 3.57 (d, J ¼ 5.64 Hz, 2H, C H2 NHCO),
(50.91%). Mp 203–205 ꢃC. 1H NMR (300 MHz, DMSO-d6): d 8.53 2.94 (br, 2H, C4–H2 ), 2.68 (br, 2H, C1–H2 ), 1.83 (br, 4H, C2–H2
(br, 1H, ArH), 8.41 (d, J ¼ 8.61 Hz, 1H, ArH), 7.79 (br, 2H, CONH and C3–H2 ). 13C NMR (125 MHz, DMSO-d6): d 166.01, 156.51,
and ArH), 7.62 (d, J ¼ 8.31 Hz, 2H, ArH), 7.52 (d, J ¼ 8.25 Hz, 4H, 151.16, 148.77, 138.53, 135.02, 134.38, 133.23, 130.90, 130.30,
ArH and NH), 7.41 (d, J ¼ 15.59 Hz, 1H, COCH]CH), 6.60 (d, J ¼ 129.21, 126.44, 125.75, 125.59, 125.16, 119.76, 116.08, 112.07,
15.81 Hz, 1H, COCH]CH), 3.93 (d, J ¼ 5.70 Hz, 2H, NHC H2 ), 48.48, 28.53, 24.21, 21.91, 20.82. HRMS (ESI): calcd for
3.55 (d, J ¼ 5.34 Hz, 2H, C H2 NHCO), 2.94 (br, 2H, C4–H2 ), 2.69 C24H24N4O3 [M + H]+ 417.1921, found 417.192. HPLC (70%
(br, 2H, C1–H2 ), 1.82 (br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 methanol in water with 0.5% H3PO4): tR ¼ 2.97 min, 99.720%.
MHz, DMSO-d6): d 166.05, 158.46, 156.65, 150.59, 147.47,
(E)-3-(3-Nitrophenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
138.16, 134.62, 132.33, 129.93, 128.84, 128.31, 123.75, 123.47, ethyl)acrylamide (28). According to the procedure used to
123.27, 123.13, 120.60, 116.41, 116.36, 48.63, 46.20, 34.05, synthesize 8, 28 was synthesized from (E)-3-(3-nitrophenyl)acrylic
25.48, 23.26, 22.93. HRMS (ESI): calcd for C24H24BrN3O [M + H]+ acid (0.16 g, 0.83 mmol) to give a yellow powder 0.22 g (63.74%).
450.1176, found 450.1181. HPLC (70% methanol in water with Mp 210–213 ꢃC. 1H NMR (300 MHz, DMSO-d6): d 8.57 (t, J ¼
0.5% H3PO4): tR ¼ 5.79 min, 95.458%.
5.55 Hz, 1H, ArH), 8.47 (d, J ¼ 8.76 Hz, 1H, ArH), 8.37 (s, 1H,
(E)-N-(2-((1,2,3,4-Tetrahydroacridin-9-yl)amino)ethyl)-3-(2-(tri- CONH), 8.21 (d, J ¼ 6.87 Hz, 1H, ArH), 8.01 (d, J ¼ 7.86 Hz, 1H,
uoromethyl)phenyl)acrylamide (25). According to the procedure ArH) 7.94 (br, 1H, NH), 7.87 (t, J ¼ 7.59 Hz, 1H, ArH), 7.77 (d, J ¼
used to synthesize 8, 25 was synthesized from (E)-3-(2-(tri- 8.43 Hz, 1H, ArH), 7.70 (t, J ¼ 8.00 Hz, 1H, ArH), 7.60–7.53 (m, 2H,
uoromethyl)phenyl)acrylic acid (0.18 g, 0.83 mmol) to give ArH and COCH]CH), 6.75 (d, J ¼ 15.81 Hz, 1H, COCH]CH),
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a white powder 0.22 g (60.41%). Mp 183–184 C. H NMR (300 4.03 (d, J ¼ 5.31 Hz, 2H, NHC H2 ), 3.59 (d, J ¼ 5.58 Hz, 2H,
MHz, DMSO-d6): d 8.62 (t, J ¼ 5.43 Hz, 1H, ArH), 8.46 (d, J ¼ C H2 NHCO), 2.94 (br, 2H, C4–H2 ), 2.68 (br, 2H, C1–H2 ), 1.83 (br,
8.7 Hz, 1H, ArH) 7.89–7.84 (m, 2H, CONH and ArH), 7.80–7.73 4H, C2–H2 and C3–H2 ). 13C NMR (125 MHz, DMSO-d6): d 166.25,
(m, 4H, ArH and COCH]CH), 7.70–7.68 (m, 1H, ArH), 7.62–7.55 156.54, 151.16, 148.76, 137.46, 137.01, 134.38, 133.22, 131.02,
(m, 2H, ArH and NH), 6.62 (d, J ¼ 15.60 Hz, 1H, COCH]CH), 125.76, 125.59, 124.80, 124.40, 122.16, 119.59, 116.03, 111.94,
4.03 (d, J ¼ 5.40 Hz, 2H, NHC H2 ), 3.59 (d, J ¼ 5.76 Hz, 2H, 48.40, 28.41, 24.24, 21.92, 20.78. HRMS (ESI): calcd for
C H2 NHCO), 2.95 (br, 2H, C4–H2 ), 2.67 (br, 2H, C1–H2 ), 1.83 C24H24N4O3 [M + H]+ 417.1921, found 417.1921. HPLC (70%
(br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 MHz, DMSO-d6): methanol in water with 0.5% H3PO4): tR ¼ 3.19 min, 98.916%.
d 165.95, 156.56, 138.43, 134.64, 133.61, 133.26, 130.23, 128.30,
(E)-3-(4-Nitrophenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
127.40, 127.20, 126.64, 126.41, 125.74, 125.59, 123.73, 119.66, ethyl)acrylamide (29). According to the procedure used to
48.40, 46.33, 28.47, 26.41, 26.38, 24.23, 21.88, 20.78. HRMS synthesize 8, 29 was synthesized from (E)-3-(4-nitrophenyl)acrylic
(ESI): calcd for C25H24F3N3O [M + H]+ 440.1944, found 440.1936. acid (0.16 g, 0.83 mmol) to give a yellow powder 0.24 g (69.54%).
1
ꢃ
HPLC (70% methanol in water with 0.5% H3PO4): tR ¼ 4.72 min, Mp 215–217 C. H NMR (300 MHz, DMSO-d6): d 8.64 (br, 1H,
97.899%.
ArH), 8.46 (d, J ¼ 8.73 Hz, 1H, ArH), 8.04 (d, J ¼ 7.92 Hz, 1H, ArH),
(E)-N-(2-((1,2,3,4-Tetrahydroacridin-9-yl)amino)ethyl)-3-(4-(tri- 7.86 (t, J ¼ 7.25 Hz, 2H, CONH and ArH), 7.79–7.74 (m, 3H, ArH
uoromethyl)phenyl)acrylamide (26). According to the procedure and COCH]CH), 7.68–7.55 (m, 3H, ArH and NH), 6.55 (d, J ¼
used to synthesize 8, 26 was synthesized from (E)-3-(4-(tri- 15.54 Hz, 1H, COCH]CH), 4.02 (d, J ¼ 5.43 Hz, 2H, NHC H2 ),
uoromethyl)phenyl)acrylic acid (0.18 g, 0.83 mmol) to give 3.59 (d, J ¼ 5.58 Hz, 2H, C H2 NHCO), 2.96 (br, 2H, C4–H2 ), 2.67
a white powder 0.18 g (49.42%). Mp 182–184 C. H NMR (300 (br, 2H, C1–H2 ), 1.83 (br, 4H, C2–H2 and C3–H2 ). 13C NMR (125
1
ꢃ
MHz, DMSO-d6): d 8.62 (br, 1H, ArH), 8.47 (d, J ¼ 8.61 Hz, 1H, MHz, DMSO-d6): d 166.14, 156.43, 151.24, 148.06, 141.68, 138.55,
ArH), 7.92–7.84 (m, 2H, CONH and ArH), 7.77 (br, 4H, ArH and 137.43, 133.16, 129.13, 126.08, 125.68, 125.55, 124.59, 123.22,
NH), 7.58 (t, J ¼ 7.44 Hz, 1H, ArH), 7.50 (d, J ¼ 15.84 Hz, 1H, 119.80, 116.09, 112.05, 48.43, 28.56, 24.26, 21.92, 20.84. HRMS
COCH]CH), 6.62 (d, J ¼ 15.60 Hz, 1H, COCH]CH), 4.03 (d, J ¼ (ESI): calcd for C24H24N4O3 [M + H]+ 417.1921, found 417.1914.
5.40 Hz, 2H, NHC H2 ), 3.59 (d, J ¼ 5.76 Hz, 2H, C H2 NHCO), HPLC (70% methanol in water with 0.5% H3PO4): tR ¼ 3.27 min,
2.95 (br, 2H, C4–H2 ), 2.67 (br, 2H, C1–H2 ), 1.83 (br, 4H, C2–H2 97.468%.
and C3–H2 ). 13C NMR (125 MHz, DMSO-d6): d 166.40, 156.60,
(E)-3-(4-Chloro-3-nitrophenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-
151.16, 139.21, 133.28, 138.38, 138.16, 133.28, 128.72, 126.34, yl)amino)ethyl)acrylamide (30). According to the procedure used
126.31, 125.76, 125.60, 125.45, 124.65, 123.65, 119.61, 116.02, to synthesize 8, 30 was synthesized from (E)-3-(4-chloro-3-
111.96, 48.50, 28.44, 24.24, 21.90, 20.80. HRMS (ESI): calcd for nitrophenyl)acrylic acid (0.19 g, 0.83 mmol) to give a white
C
25H24F3N3O [M + H]+ 440.1944, found 440.1948. HPLC (70% powder 0.19 g (50.84%). Mp 208–210 ꢃC. 1H NMR (300 MHz,
methanol in water with 0.5% H3PO4): tR ¼ 5.36 min, 98.618%. DMSO-d6): d 8.56 (br, 1H, ArH), 8.47 (d, J ¼ 8.55 Hz, 1H, ArH),
(E)-3-(2-Nitrophenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)- 8.30–8.26 (m, 1H, ArH), 7.92–7.77 (m, 5H, CONH and ArH and
ethyl)acrylamide (27). According to the procedure used to NH), 7.58 (t, J ¼ 7.52 Hz, 1H, ArH), 7.48 (d, J ¼ 15.87 Hz, 1H,
synthesize 8, 27 was synthesized from (E)-3-(2-nitrophenyl)acrylic COCH]CH), 6.71 (d, J ¼ 15.84 Hz, 1H, COCH]CH), 4.02 (d, J ¼
acid (0.16 g, 0.83 mmol) to give a yellow powder 0.23 g (66.64%). 5.52 Hz, 2H, NHC H2 ), 3.58 (d, J ¼ 5.64 Hz, 2H, C H2 NHCO),
1
ꢃ
Mp 216–218 C. H NMR (300 MHz, DMSO-d6): d 8.63 (br, 1H, 2.95 (br, 2H, C4–H2 ), 2.68 (br, 2H, C1–H2 ), 1.83 (br, 4H, C2–H2
ArH), 8.45 (d, J ¼ 8.55 Hz, 1H, ArH), 8.25 (d, J ¼ 8.70 Hz, 2H, ArH), and C3–H2 ). 13C NMR (125 MHz, DMSO-d6): d 166.16, 156.61,
7.87–7.76 (m, 4H, CONH and ArH and NH), 7.59–7.51 (m, 2H, ArH 151.00, 148.44, 138.28, 136.52, 135.84, 133.31, 132.79, 132.64,
33862 | RSC Adv., 2017, 7, 33851–33867
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125.76, 125.60, 125.25, 124.67, 119.52, 115.94, 111.91, 48.47, synthesize 8, 34 was synthesized from 7–34 (0.21 g, 0.83 mmol) to
1
ꢃ
46.34, 28.39, 24.21, 21.88, 20.78. HRMS (ESI): calcd for give a white powder 0.18 g (45.48%). Mp 112–115 C. H NMR
C24H24ClN4O3 [M + H]+ 451.1532, found 451.1528. HPLC (70% (300 MHz, DMSO-d6): d 8.49 (br, 2H, ArH), 7.97 (br, 1H, CONH),
methanol in water with 0.5% H3PO4): tR ¼ 4.23 min, 98.389%.
7.89 (t, J ¼ 7.59 Hz, 1H, ArH), 7.79 (d, J ¼ 7.95 Hz, 1H, ArH), 7.58
(E)-N-(2-((1,2,3,4-Tetrahydroacridin-9-yl)amino)ethyl)-3-(4-(triuo- (t, J ¼ 7.05 Hz, 1H, ArH), 7.46–7.32 (m, 7H, ArH and COCH]CH),
romethoxy)phenyl)acrylamide (31). According to the procedure 7.20 (br, 1H, NH), 7.14 (d, J ¼ 7.53 Hz, 1H, ArH), 7.03 (d, J ¼
used to synthesize 8, 31 was synthesized from (E)-3-(4-(tri- 6.57 Hz, 1H, ArH), 6.57 (d, J ¼ 15.87 Hz, 1H, COCH]CH), 5.13 (s,
uoromethoxy)phenyl)acrylic acid (0.19 g, 0.83 mmol) to give 2H, OCH2Ar), 4.02 (d, J ¼ 6.81 Hz, 2H, NHC H2 ), 3.57 (d, J ¼
1
ꢃ
a white powder 0.13 g (34.44%). Mp 188–189 C. H NMR (300 5.25 Hz, 2H, C H2 NHCO), 2.95 (br, 2H, C4–H2 ), 2.68 (br, 2H, C1–
MHz, DMSO-d6): d 8.54–8.48 (m, 2H, ArH), 7.94–7.79 (m, 3H, H2 ), 1.83 (br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 MHz,
CONH and ArH), 7.56–7.38 (m, 5H, ArH and NH and COCH]CH), DMSO-d6): d 166.74, 159.13, 155.58, 152.06, 139.50, 137.37,
6.70–6.62 (m, 1H, COCH]CH), 4.02 (br, 2H, NHC H2 ), 3.58 (br, 136.66, 132.36, 130.51, 128.93, 128.36, 128.24, 125.42, 125.27,
2H, C H2 NHCO), 2.95 (br, 2H, C4–H2 ), 2.68 (br, 2H, C1–H2 ), 1.82 122.46, 120.87, 120.53, 116.61, 114.23, 112.43, 69.67, 48.43, 29.18,
(br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 MHz, DMSO-d6): 24.51, 22.13, 21.05. HRMS (ESI): calcd for C31H31N3O2 [M + H]+
d 166.51, 156.62, 151.02, 149.28, 138.33, 138.18, 137.65, 133.32, 478.2489, found 478.2483. HPLC (70% methanol in water with
131.47, 127.10, 125.78, 125.60, 123.85, 122.37, 121.38, 120.28, 0.5% H3PO4): tR ¼ 8.69 min, 96.547%.
119.68, 119.55, 115.97, 111.92, 48.59, 28.41, 24.20, 21.89, 20.78.
(E)-3-(4-(Benzyloxy)phenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)-
HRMS (ESI): calcd for C25H24FN3O2 [M + H]+ 456.1893, found amino)ethyl)acrylamide (35). According to the procedure used to
456.1886. HPLC (70% methanol in water with 0.5% H3PO4): tR ¼ synthesize 8, 35 was synthesized from 7–35 (0.21 g, 0.83 mmol) to
1
ꢃ
6.05 min, 98.933%.
give a white powder 0.21 g (53.06%). Mp 120–122 C. H NMR
(E)-Methyl-(4-(3-oxo-3-((2-((1,2,3,4-tetrahydroacridin-9-yl)amino)- (300 MHz, DMSO-d6): d 8.49 (br, 2H, ArH), 7.99 (br, 1H, CONH),
ethyl)amino)prop-1-en-1-yl)phenyl)-carbonate (32). According to the 7.86 (t, J ¼ 7.58 Hz, 1H, ArH), 7.78 (d, J ¼ 7.74 Hz, 1H, ArH), 7.59–
procedure used to synthesize 8, 32 was synthesized from (E)-3-(4- 7.32 (m, 9H, ArH and COCH]CH and NH), 7.04 (d, J ¼ 8.71 Hz,
((methoxycarbonyl)oxy)phenyl)acrylic acid (0.18 g, 0.83 mmol) to 2H, ArH), 6.43 (d, J ¼ 15.78 Hz, 1H, COCH]CH), 5.13 (s, 2H,
give a white powder 0.2 g (54.17%). Mp 201–203 ꢃC. 1H NMR (300 OCH2Ar), 4.01 (d, J ¼ 5.01 Hz, 2H, NHC H2 ), 3.55 (d, J ¼ 5.37 Hz,
MHz, DMSO-d6): d 8.56–8.46 (m, 2H, ArH), 7.95 (br, 1H, CONH), 2H, C H2 NHCO), 2.94 (br, 2H, C4–H2 ), 2.68 (br, 2H, C1–H2 ),
7.87 (t, J ¼ 7.47 Hz, 1H, ArH), 7.78 (d, J ¼ 8.16 Hz, 1H, ArH), 7.63– 1.82 (br, 4H, C2–H2 and C3–H2 ). 13C NMR (75 MHz, CDCl3):
7.58 (m, 3H, ArH and NH), 7.45 (d, J ¼ 15.78 Hz, 1H, COCH]CH), d 167.39, 159.64, 157.91, 150.29, 146.63, 140.48, 135.98, 128.91,
7.28 (d, J ¼ 8.37 Hz, 1H, ArH), 6.55 (d, J ¼ 15.78 Hz, 1H, COCH] 128.15, 127.92, 127.64, 127.11, 126.96, 123.24, 122.28, 119.47,
CH), 4.02 (d, J ¼ 4.74 Hz, 2H, NHC H2 ), 3.83 (s, 3H, OCH3), 3.58 117.61, 115.52, 114.64, 69.55, 49.43, 40.15, 33.29, 24.58, 22.51,
(br, 2H, C H2 NHCO), 2.95 (br, 2H, C4–H2 ), 2.68 (br, 2H, C1–H2 ), 22.15. HRMS (ESI): calcd for C31H31N3O2 [M + H]+ 478.2489,
1.83 (br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 MHz, DMSO-d6): found 478.2488. HPLC (70% methanol in water with 0.5%
d 166.82, 156.64, 153.85, 151.98, 138.79, 133.31, 133.14, 129.36, H3PO4): tR ¼ 8.40 min, 96.871%.
125.80, 125.59, 122.36, 122.13, 119.53, 115.97, 111.91, 56.05,
(E)-3-(4-(Benzyloxy)-3-methoxyphenyl)-N-(2-((1,2,3,4-tetrahydroa-
54.06, 48.69, 42.31, 36.93, 28.41, 24.20, 21.89, 20.79, 18.55, 17.19, cridin-9-yl)amino)ethyl)acrylamide (36). According to the proce-
12.98. HRMS (ESI): calcd for C26H27N3O4 [M + H]+ 446.2074, dure used to synthesize 8, 36 was synthesized from 7–36 (0.24 g,
found 446.2077. HPLC (70% methanol in water with 0.5% 0.83 mmol) to give a white powder 0.25 g (59.43%). Mp 109–
H3PO4): tR ¼ 3.06 min, 95.785%.
111 ꢃC. 1H NMR (300 MHz, CDCl3): d 7.99–7.90 (m, 2H, ArH), 7.59
(E)-3-(2-(Benzyloxy)phenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)- (d, J ¼ 15.57 Hz, 1H, COCH]CH), 7.50–7.29 (m, 7H, ArH), 7.04–
amino)ethyl)acrylamide (33). According to the procedure used to 7.01 (m, 2H, ArH and NH), 6.86 (d, J ¼ 8.04 Hz, 1H, ArH), 6.61 (br,
synthesize 8, 33 was synthesized from 7–33 (0.21 g, 0.83 mmol) to 1H, CONH), 6.35 (d, J ¼ 15.60 Hz, 1H, COCH]CH), 5.18 (s, 2H,
1
ꢃ
give a white powder 0.19 g (48.00%). Mp 116–118 C. H NMR OCH2Ar), 3.90 (s, 3H, OCH3), 3.76–3.71 (m, 4H, NHC H2 and
(300 MHz, DMSO-d6): d 8.48 (br, 2H, ArH), 7.95 (br, 1H, CONH), C H2 NHCO), 3.02 (br, 2H, C4–H2 ), 2.66 (br, 2H, C1–H2 ), 1.85
7.88–7.75 (m, 3H, ArH), 7.57–7.51 (m, 2H, ArH), 7.46–7.32 (m, 6H, (br, 4H, C2–H2 and C3–H2 ). 13C NMR (75 MHz, CDCl3): d 167.50,
ArH and NH and COCH]CH), 7.14 (d, J ¼ 8.25 Hz, 1H, ArH), 156.41, 151.86, 149.32, 149.18, 140.60, 136.09, 128.42, 128.11,
6.98 (t, J ¼ 7.41 Hz, 1H, ArH), 6.58 (d, J ¼ 15.87 Hz, 1H, COCH] 127.61, 127.52, 126.77, 126.16, 123.31, 122.55, 121.24, 118.61,
CH), 5.21 (s, 2H, OCH2), 4.02 (d, J ¼ 4.92 Hz, 2H, NHC H2 ), 3.56 118.04, 114.53, 113.05, 110.04, 70.37, 55.46, 49.52, 39.99, 31.02,
(d, J ¼ 5.22 Hz, 2H, C H2 NHCO), 2.94 (br, 2H, C4–H2 ), 2.66 (br, 24.36, 22.31, 21.77. HRMS (ESI): calcd for C32H34N3O3 [M + H]+
2H, C1–H2 ), 1.80 (br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 508.2595, found 508.2591. HPLC (70% methanol in water with
MHz, DMSO-d6): d 167.28, 156.92, 156.61, 150.94, 138.33, 137.35, 0.5% H3PO4): tR ¼ 5.97 min, 96.738%.
134.71, 133.29, 131.53, 129.00, 128.41, 128.00, 127.88, 125.80,
(E)-3-(2-Aminophenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
125.55, 123.86, 121.93, 121.48, 119.52, 115.94, 113.64, 111.88, ethyl)acrylamide (37). 27 (0.2 g, 0.48 mmol) and stannous chloride
70.01, 48.91, 28.39, 24.17, 21.87, 20.77. HRMS (ESI): calcd for dehydrate (0.54 g, 2.40 mmol) were dissolved in 10 mL EtOH, the
C31H31N3O2 [M + H]+ 478.2489, found 478.2476. HPLC (70% mixture was stirred at 80 C for 6 h. Then the reaction cooled to
ꢃ
methanol in water with 0.5% H3PO4): tR ¼ 7.96 min, 95.046%.
room temperature and acidized by AcOH. The reaction was ltered
(E)-3-(3-(Benzyloxy)phenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)- and the concentrated the ltrate. Then the ltrate was puried by
amino)ethyl)acrylamide (34). According to the procedure used to column chromatography (CH2Cl2/MeOH ¼ 40 : 1, v/v, with 0.1%
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triethylamine) to give an orange powder 0.07 g (37.63%). Mp 205– purchased from Sigma-Aldrich (St. Louis, MO, USA). AChE/BuChE
206 ꢃC. 1H NMR (300 MHz, DMSO-d6): d 8.81 (br, 1H, ArH), 8.55 (d, stock solution was diluted before use to give 2.5 units per mL (for
J ¼ 7.71 Hz, 1H, ArH), 8.05 (br, 2H, CONH and ArH), 7.83 (br, 1H, eeAChE, eqBuChE and huAChE) or 0.5 units per mL for huBuChE.
ArH), 7.69 (d, J ¼ 15.54 Hz, 1H, COCH]CH), 7.55 (br, 1H, ArH), ATC/BTC iodide solution (0.075 M) was prepared in deionized
7.26 (d, J ¼ 7.50 Hz, 1H, ArH), 7.02 (br, 1H, NH), 6.68 (d, J ¼ water. DTNB solution (0.01 M) was prepared in water containing
7.71 Hz, 1H, ArH), 6.53 (br, 1H, ArH), 6.40 (d, J ¼ 15.57 Hz, 1H, 0.15% (w/v) sodium bicarbonate. For buffer preparation, potas-
COCH]CH), 5.42 (br, 2H, NH2), 4.02 (br, 2H, NHC H2 ), 3.54 (br, sium dihydrogen phosphate (1.36 g, 10 mmol) was dissolved in
2H, C H2 NHCO), 2.73 (br, 2H, C1–H2 ), 1.82 (br, 4H, C2–H2 and 100 mL of water. The pH of the solution was adjusted to 8.0 ꢀ 0.1
C3–H2 ). 13C NMR (125 MHz, DMSO-d6): d 166.11, 156.23, 151.50, by KOH. Stock solutions of the test compounds were dissolved in
148.09, 141.72, 137.47, 132.98, 130.80, 129.15, 126.14, 125.61, ethanol to give a nal concentration of 10ꢁ4 M when diluted to the
125.49, 124.62, 123.39, 123.24, 116.30, 112.25, 48.41, 28.78, 24.32, nal volume of 3.32 mL. For each compound, a dilution series of
21.98, 20.92, 20.80. HRMS (ESI): calcd for C24H26N4O2 [M + H]+ at least ve different concentrations (normally 10ꢁ5 to 10ꢁ9 M)
387.2179, found 387.2186. HPLC (70% methanol in water with were prepared.
0.5% H3PO4): tR ¼ 4.42 min, 96.101%.
For measurement, a cuvette containing 3.0 mL of phosphate
(E)-3-(3-Aminophenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)- buffer, 100 mL of AChE or BuChE, 100 mL of DTNB and 100 mL of
amino)ethyl)acrylamide (38). According to the procedure used to the test compound solution were added. Aer the addition of 20
synthesize 37, 38 was synthesized from 28 (0.2 g, 0.48 mmol) to mL of ATC or BTC, the reaction was initiated and solution was
give an orange powder 0.06 g (32.26%). Mp 207–209 ꢃC. 1H NMR mixed immediately. Two minutes (eeAChE and eqBuChE) or
(300 MHz, DMSO-d6): d 8.78 (br, 1H, ArH), 8.58 (d, J ¼ 7.60 Hz, een minutes (huAChE and huBuChE) aer substrate addi-
1H, ArH), 8.10 (br, 2H, CONH and ArH), 7.78 (br, 1H, ArH), 7.70 tion, the absorption was determined at 25 ꢃC (eeAChE and
(d, J ¼ 15.90 Hz, 1H, COCH]CH), 7.59 (br, 1H, ArH), 7.22 (d, J ¼ eqBuChE) or 37 ꢃC (huAChE and huBuChE) at 412 nm. For the
7.66 Hz, 1H, ArH), 7.03 (br, 1H, NH), 6.66 (d, J ¼ 7.80 Hz, 1H, reference value, 100 mL of water replaced the test compound
ArH), 6.49 (br, 1H, ArH), 6.38 (d, J ¼ 15.88 Hz, 1H, COCH]CH), solution. For determining the blank value, additionally 100 mL
5.37 (br, 2H, NH2), 4.00 (br, 2H, NHC H2 ), 3.59 (br, 2H, of water replaced the enzyme solution. The measurement for
C H2 NHCO), 2.70 (br, 2H, C1–H2 ), 1.81 (br, 4H, C2–H2 and C3– each concentration was performed in triplicate. The inhibition
H2 ). 13C NMR (125 MHz, DMSO-d6): 167.68, 162.35, 148.16, curve was tted by plotting percentage enzyme activity (100%
140.69, 135.83, 132.91, 130.78, 128.33, 127.04, 125.76, 125.49, for the reference) versus logarithm of test compound concen-
124.39, 122.36, 122.19, 116.62, 111.75, 48.39, 28.39, 24.44, tration. The IC50 values were calculated by GraphPad Prism 5
21.98, 21.82, 20.74, 20.56. HRMS (ESI): calcd for C24H26N4O2 [M and the data were shown in mean ꢀ SEM.
+ H]+ 387.2179, found 387.2183. HPLC (70% methanol in water
with 0.5% H3PO4): tR ¼ 4.69 min, 98.131%.
Kinetic studies of AChE inhibition
(E)-3-(4-Aminophenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)-
Kinetic studies were performed in the same manner to the
ethyl)acrylamide (39). According to the procedure used to synthe-
determination of ChEs inhibition, while the substrate (ATC/BTC)
size 37, 39 was synthesized from 29 (0.2 g, 0.48 mmol) to give an
was used in concentrations of 25, 50, 90, 150, 226, and 452 mM.
1
orange powder 0.07 g (37.63%). Mp 208–209 ꢃC. H NMR (300
The concentrations of test compounds were set to 0, 20, 60, 100,
MHz, DMSO-d6): d 8.46 (d, J ¼ 8.61 Hz, 1H, ArH), 8.38 (br, 1H,
200 nM for 36. The enzymatic reaction was extended to 4 min
ArH), 7.87–7.79 (m, 4H, CONH and ArH and NH), 7.54 (t, J ¼
(eeAChE and eqBuChE) or 20 min (huAChE and huBuChE) before
6.68 Hz, 1H, ArH), 7.30–7.20 (m, 3H, COCH]CH and ArH), 6.54 (d,
the determination of the absorption. Vmax and Km values of the
J ¼ 8.28 Hz, 2H, ArH), 6.24 (d, J ¼ 15.66 Hz, 1H, COCH]CH), 5.61
Michaelis–Menten kinetics were calculated by nonlinear regres-
(br, 2H, NH2), 3.97 (d, J ¼ 4.32 Hz, 2H, NHC H2 ), 3.52 (d, J ¼
sion from substrate–velocity curves using GraphPad Prism 5.
5.16 Hz, 2H, C H2 NHCO), 2.95 (br, 2H, C4–H2 ), 2.68 (br, 2H, C1–
Linear regression was used for tting the Lineweaver–Burk plots.
H2 ), 1.81 (br, 4H, C2–H2 and C3–H2 ). 13C NMR (125 MHz, DMSO-
d6): d 166.12, 156.27, 148.09, 145.91, 141.72, 137.48, 133.03, 130.80,
129.15, 126.13, 125.64, 125.51, 124.62, 123.25, 120.10, 116.27,
Molecular modeling studies
112.21, 48.41, 28.74, 24.32, 21.97, 20.91, 20.79. HRMS (ESI): calcd The docking study was performed by CDOCKER module
for C24H26N4O2 [M + H]+ 387.2179, found 387.2175. HPLC (70% implemented in Discovery Studio (version 3.0, BIOVIA, USA).
methanol in water with 0.5% H3PO4): tR ¼ 4.33 min, 97.356%.
CDOCKER is a grid-based molecular docking method that
employs CHARMm.46 Random ligand conformations are
generated from the initial ligand structure through high
temperature molecular dynamics, followed by random rota-
In vitro inhibitory evaluations on AChE and BuChE
The investigation of the inhibitory effects of the test compounds tions. The random conformations are rened by grid-based
was performed followed the method of Ellman et al., using a Shi- simulated annealing and a nal grid-based or full forceeld
madzu 160 spectrophotometer. AChE (EC 3.1.1.7, Type VI-S, from minimization. The solutions are then clustered according to
Electric Eel, C3389; from human, C1682) and BuChE (EC 3.1.1.8, position and conformation and ranked by energy.
from equine serum, C0663; from human, B4186), 5,50-dithiobis(2-
The co-crystal structure of huAChE bound with donepezil
nitrobenzoic acid) (DTNB, D218200), acetylthiocholine iodide was selected for molecular docking. The structure was down-
(ATC, A5751), and butyrylthiocholine iodide (BTC, B3253) were load from Protein Data Bank (PDB, ID: 4EY7). It was prepared by
33864 | RSC Adv., 2017, 7, 33851–33867
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“Prepare Protein” module in DS for further docking. Missed mL of MTT solution was added into each well of the plate and
sidechains were added and the water molecules were removed, incubated for 4 h. Then the solution was removed and 150.0 mL
then it was protonated at pH 7.4. For the test compounds of DMSO was added into each well to dissolve the MTT-
docked into huAChE, they were rst sketched in DS, and then formazan crystals. DMSO was used as a negative control. The
prepared by using “Prepare Ligands” module to protonate at pH absorbance values (OD value) were read at 570 nm by Elx800
7.4. The resulted molecules were minimized by “Minimize Absorbance Microplate Reader (BioTek, Vermont, USA). The
Ligands” module. The “Smart Minimizer” algorithm was used inhibitory rate for each concentration of the test compound was
to perform the minimization, with max steps set to 2000, RMS calculated by the equation as follows:
Gradient set to 0.01. Other parameters were set as default.47
IR ¼ [1 ꢁ (ODtest ꢁ ODblank)/(ODcontrol ꢁ ODblank)] ꢂ 100%.
˚
A sphere (in 10 A radius) around donepezil was determined
as the binding site, including both CAS and PAS of huAChE. For
Here, ODtest, ODblank, and ODcontrol stand for the OD value from
the simulated annealing, heating steps and cooling steps was
test compound, background, and DMSO, respectively. The IC50
set to 2000 and 5000, respectively, while heating and cooling
values were calculated by GraphPad Prism 5 and the data were
temperature was set to 700, and 310, respectively. Other
shown in mean ꢀ SEM.
parameters were kept as default. Aer docking, ten top-ranked
conformations were retained for analysis. Binding patterns of
the docked molecules were described by DS visualizer.48
A statement identifying the institutional and/or licensing
committee experimental approval
Inhibition of self-induced Ab1–42 aggregation
All animal studies were performed in compliance with the
Chinese Academy of Medical Sciences and the Animal Ethics
Committees of the Institute of Materia Medica. The animal
experiments were performed in accordance with the National
Institutes of Health Guide for the Care and Use of Laboratory
Animals with the approval of the Center for New Drug Evalua-
tion and Research of China Pharmaceutical University (Nanjing,
China).
Inhibition of self-induced Ab1–42 aggregation was measured
using a Thioavin T (ThT)-(T3516, Sigma-Aldrich, St. Louis, MO,
USA) binding assay as previously described.49 Aliquots of 2.0 mL
of Ab1–42 (AS-64129-05 Anaspec Inc.), lyophilized from 2 mg
mLꢁ1 HFIP (1,1,1,3,3,3-hexauoro-2-propanol, 52517, Sigma-
Aldrich, St. Louis, MO, USA) and dissolved in DMSO, were
incubated for 24 h at room temperature in 0.215 M sodium
phosphate buffer (pH 8.0) at a nal concentration of 500 mM.
Test compounds were dissolved in DMSO and then diluted by
buffer to a nal concentration of 20 mM. Aer incubation, the
samples were diluted to a nal volume of 150 mL with 50 mM
glycine–NaOH buffer (pH 8.5) containing 5 mM thioavin T.
Fluorescence signal was determined (excitation wavelength
450 nm, emission wavelength 485 nm) on a SpectraMax Para-
digm Multimode Reader (Molecular Device, USA).
The inhibitory rate of Ab1–42 aggregation was calculated
according to the following equation: (1-IFi/IFc) ꢂ 100%. Here,
IFi and IFc were the uorescence intensities obtained for
absorbance in the presence and absence of inhibitors, respec-
tively, aer subtracting the background uorescence of the
5 mM thioavin T solution. Each measurement was measured
in triplicate. The inhibitory rate of the test compound was
shown in mean ꢀ SD.
Behavioral studies
Behavioral studies were performed by using adult male ICR
mice (8–10 weeks old, weight 20–25 g), which were purchased
from the Yangzhou University Medical Center (Yangzhou,
China). Scopolamine hydrobromide was supplied by Aladdin
Reagents (H1507073, Shanghai, China). Tacrine was synthe-
sized in our lab with >95% purity as determined by HPLC.
The mice were separated into ve groups as follows: (i)
vehicle as blank control, (ii) scopolamine as model group, (iii)
tacrine plus scopolamine as positive control, (iv) compound 36
plus scopolamine as test group, and (v) compound 35 plus
scopolamine as test group. Tacrine, 36 and 35 (20 mmol kgꢁ1
body weight) were orally administered to mice in groups (iii),
(iv), and (v), respectively, 30 min before the ip administration of
scopolamine (1 mg kgꢁ1) or saline for 10 consecutive days.
Cognitive function was evaluated by the Morris water maze
analysis-management system (Panlab SMART 3.0, America),
according to the method previously described.37 The maze was
Cyto-protection and cell toxicity against PC-12 neuroblastoma
cells
Cytotoxicity was determined by using 3-(4,5-dimethylthiazol-2- placed in a lit room with visual cues at 25 ꢃC. An escape platform
yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. The PC-12 (10 cm diameter) was located in the center of one quadrant of the
cell line was purchased from Cell Culture Center at the Insti- circular pool (120 cm diameter, 60 cm height) with a depth of
tute of Basic Medical Sciences, Chinese Academy of Medical 40 cm water. The behavioral study of each mouse included 5 days
Sciences. MTT was purchased from Sigma (M2128, St. Louis, of learning and memory training and a probe trial on day 6. The
MO). It was dissolved in phosphate buffered saline (PBS) to animal starting positions faced to the pool wall, and were pseu-
a stock concentration of 5 mg mLꢁ1 and stored at ꢁ20 ꢃC. PC-12 dorandomized for each trial. For the cognitive evaluation, each
cells were plated in 96-well plates, raised to a population of 1 ꢂ mouse was individually evaluated on both visible-platform (days
104 cells per well, and incubated overnight. Aer cells were 1–2) and hidden-platform (days 3–5) versions of the water maze.
treated with density gradient of test compounds or DMSO for All mice received nonspatial pretraining during the rst two
24 h at 37 ꢃC or treated with 500 mM H2O2 for another 12 h, 20.0 training days, which prepared them for the subsequent spatial
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learning test. During the two days, mice were trained to nd the materials of the manuscript. Z. J., G. K., L. Q., H. Y. and L. X.
platform that was labeled by a small ag (5 cm tall). The hidden- were responsible for the synthesis of the inhibitor. P. Q. was
platform version was used to determine the retention of memory responsible for the characterization of the compounds. B. Y.
to nd the platform. During the hidden-platform training trials, and W. L. were responsible for the biological determination. T.
the escape platform was placed 1 cm below the surface of the X. revised the manuscript. All authors reviewed the manuscript.
water. On each day, the animal was subjected to two trials, each
of which lasted for 90 s. The time for the mouse to nd the
platform (a successful escape) was recorded. If a mouse failed to
Conflict of interest
reach the platform within 90 s, the test was terminated and the
animal was gently navigated to the platform by hand. Whether
The authors declare no competing nancial interests.
a mouse was successful or failed to reach the platform within
90 s, it was kept on the platform for 30 s. On the last day (day 6), Acknowledgements
the platform was removed from its location and the animals were
We gratefully thank the support from the grants 81402851 and
given a probe trial in which they had 90 s to search for the plat-
81573281 of National Natural Science Foundation of China,
form. The time taken to reach the missing platform and the
BK20140957 of Natural Science Foundation of Jiangsu Province.
number of times the animals crossed the platform location were
We also thank the support from Fundamental Research Funds
recorded.
for the Central Universities (2015ZD009), Jiangsu Qing Lan
Data for the time of escape latency, the trajectory traveled,
Project, Top-notch Academic Programs Project of Jiangsu
and the number of platform location crossings were recorded by
Higher Education Institutions (TAPP-PPZY2015A070) and
Panlab SMART 3.0 and processed by Graphpad Prism 5.
Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD).
Hepatotoxicity studies
Hepatotoxicity was evaluated according to the method previ-
References
ously described32 by using adult male ICR mice (8–10 weeks old,
weighing 20–25 g) obtained from the Yangzhou University
Medicine Centre (Yangzhou, China). Tacrine and the test
compounds were dissolved in a sodium carboxymethyl cellulose
(CMC-Na) solution (0.5 g CMC-Na in 100 mL distilled water).
Concentration of 3 mg/100 g body wt of tacrine, corresponding
to 151.5 mmol kgꢁ1 body wt, was administered intragastrically
(ig). Equimolar dose of test compounds to that of tacrine was
administered ig. 8, 22, and 36 h aer the administration,
heparinized serum was collected from the retrobulbar plexus
and subjected to hepatotoxicity evaluation. The activity of
aspartate aminotransferase (AST) and alanine aminotransferase
(ALT), two indicators of liver damage, was determined using
corresponding assay kit (EF551 and EF550 for ALT, EH027 and
EF548 for AST, Wako, Japan). The data were processed by
Biochemical Analyzer (HITACHI 7020, Japan).
1 V. Tumiatti, A. Minarini, M. L. Bolognesi, A. Milelli,
M. Rosini and C. Melchiorre, Curr. Med. Chem., 2010, 17,
1825–1838.
2 H. Tang, H. T. Zhao, S. M. Zhong, Z. Y. Wang, Z. F. Chen and
H. Liang, Bioorg. Med. Chem. Lett., 2012, 22, 2257–2261.
3 D. J. Selkoe, Physiol. Rev., 2001, 81, 741–766.
4 J. Hardy, Neuron, 2006, 52, 3–13.
5 P. T. Francis, A. M. Palmer, M. Snape and G. K. Wilcock, J.
Neurol., Neurosurg. Psychiatry, 1999, 66, 137–147.
6 E. Karran, M. Mercken and B. De Strooper, Nat. Rev. Drug
Discovery, 2011, 10, 698–712.
7 C. Ballatore, V. M. Lee and J. Q. Trojanowski, Nat. Rev.
Neurosci., 2007, 8, 663–672.
8 K. M. Webber, A. K. Raina, M. W. Marlatt, X. Zhu, M. I. Prat,
L. Morelli, G. Casadesus, G. Perry and M. A. Smith, Mech.
Ageing Dev., 2005, 126, 1019–1025.
9 L. Tapia-Arancibia, E. Aliaga, M. Silhol and S. Arancibia,
Brain Res. Rev., 2008, 59, 201–220.
10 B. Su, X. Wang, A. Nunomura, P. I. Moreira, H. G. Lee,
G. Perry, M. A. Smith and X. Zhu, Curr. Alzheimer Res.,
2008, 5, 525–532.
11 K. J. Barnham, C. L. Masters and A. I. Bush, Nat. Rev. Drug
Discovery, 2004, 3, 205–214.
1 h aer the collection of retrobulbar blood, mice were
sacriced and livers were harvested for morphological studies
by using immunohistochemical method. Two 3 mm sections of
each liver extending from the hilus to the margin of the le
lateral lobe were isolated by Ultra-Thin Semiautomatic Micro-
tome (Leica RM2245, Germany) and immediately placed in 10%
buffered formaldehyde, xed for two days, and embedded
together in one paraffin block by using Paraffin Embedding
Station (Leica EG1150H, Germany). Subsequently,
5 mm
12 K. Jomova and M. Valko, Toxicology, 2011, 283, 65–87.
13 L. Devi and H. K. Anandatheerthavarada, Biochim. Biophys.
Acta, 2010, 1802, 11–19.
14 U. Holzgrabe, P. Kapkova, V. Alptuzun, J. Scheiber and
E. Kugelmann, Expert Opin. Ther. Targets, 2007, 11, 161–179.
15 J. L. Sussman, M. Harel, F. Frolow, C. Oefner, A. Goldman,
L. Toker and I. Silman, Science, 1991, 253, 872–879.
sections were prepared from these paraffin blocks. They were
deparaffinated and stained with hematoxylin and eosin or by
means of the periodic acid-Schiff procedure for glycogen.
Author contributions
C. Y. wrote the result and discussion of main manuscript text. S. 16 Y. Bourne, P. Taylor, Z. Radic and P. Marchot, EMBO J., 2003,
P. wrote the introduction part. L. Z. wrote the methods and
22, 1–12.
33866 | RSC Adv., 2017, 7, 33851–33867
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
17 J. Cheung, M. J. Rudolph, F. Burshteyn, M. S. Cassidy, 33 S. Liao, H. Deng, S. Huang, J. Yang, S. Wang, B. Yin,
E. N. Gary, J. Love, M. C. Franklin and J. J. Height, J. Med.
Chem., 2012, 55, 10282–10286.
T. Zheng, D. Zhang, J. Liu, G. Gao, J. Ma and Z. Deng,
Bioorg. Med. Chem. Lett., 2015, 25, 1541–1545.
18 M. Bartolini, C. Bertucci, V. Cavrini and V. Andrisano, 34 J. Misik, J. Korabecny, E. Nepovimova, A. Kracmarova and
Biochem. Pharmacol., 2003, 65, 407–416. J. Kassa, Neurosci. Lett., 2016, 612, 261–268.
19 N. C. Inestrosa, J. P. Sagal and M. Colombres, Subcell. 35 E. Nepovimova, J. Korabecny, R. Dolezal, K. Babkova,
Biochem., 2005, 38, 299–317.
20 S. Darvesh, D. A. Hopkins and C. Geula, Nat. Rev. Neurosci.,
2003, 4, 131–138.
A. Ondrejicek, D. Jun, V. Sepsova, A. Horova,
M. Hrabinova, O. Soukup, N. Bukum, P. Jost, L. Muckova,
J. Kassa, D. Malinak, M. Andrs and K. Kuca, J. Med. Chem.,
2015, 58, 8985–9003.
21 C. Chianella, D. Gragnaniello, P. Maisano Delser,
M. F. Visentini, E. Sette, M. R. Tola, G. Barbujani and 36 B. N. Saglik, S. Ilgin and Y. Ozkay, Eur. J. Med. Chem., 2016,
S. Fuselli, Eur. J. Clin. Pharmacol., 2011, 67, 1147–1157. 124, 1026–1040.
22 A. V. Terry Jr and J. J. Buccafusco, J. Pharmacol. Exp. Ther., 37 X. Zha, D. Lamba, L. Zhang, Y. Lou, C. Xu, D. Kang, L. Chen,
2003, 306, 821–827.
23 M. Bajda, N. Guzior, M. Ignasik and B. Malawska, Curr. Med.
Chem., 2011, 18, 4949–4975.
Y. Xu, L. Zhang, A. De Simone, S. Samez, A. Pesaresi,
J. Stojan, M. G. Lopez, J. Egea, V. Andrisano and
M. Bartolini, J. Med. Chem., 2016, 59, 114–131.
24 S. Leone, A. Ottani and A. Bertolini, Curr. Top. Med. Chem., 38 C. Zhang, Q. Y. Du, L. D. Chen, W. H. Wu, S. Y. Liao, L. H. Yu
2007, 7, 265–275. and X. T. Liang, Eur. J. Med. Chem., 2016, 116, 200–209.
25 A. Minarini, A. Milelli, E. Simoni, M. Rosini, M. L. Bolognesi, 39 B. Sameem, M. Saeedi, M. Mahdavi and A. Shaee, Eur. J.
C. Marchetti and V. Tumiatti, Curr. Top. Med. Chem., 2013,
13, 1771–1786.
26 R. A. de Aquino, L. V. Modolo, R. B. Alves and A. de Fatima,
Curr. Drug Targets, 2013, 14, 378–397.
Med. Chem., 2017, 128, 332–345.
40 L. Fang, B. Kraus, J. Lehmann, J. Heilmann, Y. Zhang and
M. Decker, Bioorg. Med. Chem. Lett., 2008, 18, 2905–2909.
41 Y. Chen, J. Sun, L. Fang, M. Liu, S. Peng, H. Liao, J. Lehmann
and Y. Zhang, J. Med. Chem., 2012, 55, 4309–4321.
27 M. Benchekroun, M. Bartolini, J. Egea, A. Romero,
E. Soriano, M. Pudlo, V. Luzet, V. Andrisano, M. L. Jimeno, 42 P. R. Carlier, Y. F. Han, E. S. Chow, C. P. Li, H. Wang,
M. G. Lopez, S. Wehle, T. Gharbi, B. Refouvelet, L. de
Andres, C. Herrera-Arozamena, B. Monti, M. L. Bolognesi,
T. X. Lieu, H. S. Wong and Y. P. Pang, Bioorg. Med. Chem.,
1999, 7, 351–357.
M. I. Rodriguez-Franco, M. Decker, J. Marco-Contelles and 43 G. L. Ellman, K. D. Courtney, V. Andres Jr and R. M. Feather-
L. Ismaili, ChemMedChem, 2015, 10, 523–539. Stone, Biochem. Pharmacol., 1961, 7, 88–95.
28 M. A. Ceschi, J. S. da Costa, J. P. Lopes, V. S. Camara, 44 S. L. Cockro, C. A. Hunter, K. R. Lawson, J. Perkins and
L. F. Campo, A. C. Borges, C. A. Goncalves, D. F. de Souza, C. J. Urch, J. Am. Chem. Soc., 2005, 127, 8594–8595.
E. L. Konrath, A. L. Karl, I. A. Guedes and L. E. Dardenne, 45 C. Quintanova, R. S. Keri, S. M. Marques, M. G. Fernandes,
Eur. J. Med. Chem., 2016, 121, 758–772.
S. M. Cardoso, M. L. Serralheiro and M. A. Santos,
29 M. Digiacomo, Z. Chen, S. Wang, A. Lapucci, M. Macchia,
MedChemComm, 2015, 6, 1969–1977.
X. Yang, J. Chu, Y. Han, R. Pi and S. Rapposelli, Bioorg. 46 G. Wu, D. H. Robertson, C. L. Brooks 3rd and M. Vieth, J.
Med. Chem. Lett., 2015, 25, 807–810. Comput. Chem., 2003, 24, 1549–1562.
30 R. S. Keri, C. Quintanova, S. M. Marques, A. R. Esteves, 47 Y. Chen, X. Xu, T. Fu, W. Li, Z. Liu and H. Sun, RSC Adv.,
S. M. Cardoso and M. A. Santos, Bioorg. Med. Chem., 2013,
21, 4559–4569.
31 J. S. Lan, S. S. Xie, S. Y. Li, L. F. Pan, X. B. Wang and
L. Y. Kong, Bioorg. Med. Chem., 2014, 22, 6089–6104.
2015, 5, 90288–90294.
48 M. Bartolini, C. Bertucci, M. L. Bolognesi, A. Cavalli,
C. Melchiorre and V. Andrisano, ChemBioChem, 2007, 8,
2152–2161.
32 S. Y. Li, N. Jiang, S. S. Xie, K. D. Wang, X. B. Wang and 49 L. L. Xu, X. Zhang, Z. Y. Jiang and Q. D. You, Bioorg. Med.
L. Y. Kong, Org. Biomol. Chem., 2014, 12, 801–814.
Chem., 2016, 24, 3540–3547.
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