Synthesis and evaluation of 1,2,3,4-tetrahydro-1-acridone analogues as potential dual inhibitors for amyloid-beta and tau aggregation

Article abstract of DOI:10.1016/j.bmc.2018.08.007

Amyloid-β (Aβ) and tau protein are two crucial hallmarks in Alzheimer's disease (AD). Their aggregation forms are thought to be toxic to the neurons in the brain. A series of new 1,2,3,4-tetrahydro-1-acridone analogues were designed, synthesized, and evaluated as potential dual inhibitors for Aβ and tau aggregation. In vitro studies showed that compounds 25–30 (20 μM) with N-methylation of the quinolone ring effectively inhibited Aβ_1-42 aggregation by 84.7%–99.5% and tau aggregation by 71.2%–101.8%. Their structure-activity relationships are discussed. In particular, 30 could permeate the blood-brain barrier, bind to Aβ_1-42 and tau, inhibit Aβ_1-42 β-sheets formation, and prevent tau aggregation in living cells.

Full text of DOI:10.1016/j.bmc.2018.08.007

Accepted Manuscript
Synthesis and evaluation of 1,2,3,4-tetrahydro-1-acridone analogues as poten-
tial dual inhibitors for amyloid-beta and tau aggregation
Peng Lv, Chun-Li Xia, Ning Wang, Zhen-Quan Liu, Zhi-Shu Huang, Shi-Liang
Huang
PII:
S0968-0896(18)31114-3
https://doi.org/10.1016/j.bmc.2018.08.007
BMC 14494
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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15 June 2018
27 July 2018
4 August 2018
Please cite this article as: Lv, P., Xia, C-L., Wang, N., Liu, Z-Q., Huang, Z-S., Huang, S-L., Synthesis and evaluation
of 1,2,3,4-tetrahydro-1-acridone analogues as potential dual inhibitors for amyloid-beta and tau aggregation,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.08.007
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Title page
Synthesis and evaluation of 1,2,3,4-tetrahydro-1-acridone analogues as potential dual
inhibitors for amyloid-beta and tau aggregation
Peng Lv1, Chun-Li Xia1, 2, Ning Wang1, Zhen-Quan Liu1, Zhi-Shu Huang1, Shi-Liang Huang1, *
1 School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People′s Republic of China
2
Department of Bioengineering, Zhuhai Campus of Zunyi Medical University, Zhuhai 519041, People′s
Republic of China
*
To whom correspondence should be addressed. Tel. 8620-39943052and Fax: 8620-39943056, E-mail:
lsshsl@mail.sysu.edu.cn (S.-L. Huang).
Abstract
Amyloid-β (Aβ) and tau protein are two crucial hallmarks in Alzheimer’s disease (AD). Their aggregation
forms are thought to be toxic to the neurons in the brain. A series of new 1,2,3,4-tetrahydro-1-acridone
analogues were designed, synthesized, and evaluated as potential dual inhibitors for Aβ and tau aggregation.
In vitro studies showed that compounds 25-30 (20 M) with N-methylation of the quinolone ring effectively
inhibited Aβ1-42 aggregation by 84.7% to 99.5% and tau aggregation by 71.2% to 101.8%. Their
structure-activity relationships are discussed. In particular, 30 could permeate the blood-brain barrier, bind to
Aβ1-42 and tau, inhibit Aβ1-42 β-sheets formation, and prevent tau aggregation in living cells.
Keywords
Alzheimer’s disease; amyloid β; tau protein; aggregation inhibitors; 1,2,3,4-tetrahydro-1-acridone analogues
Abbreviations
AD, Alzheimer’s disease
Aβ, amyloid-β
BBB, blood-brain barrier
CAs, cinnamic acid derivatives
CD, circular dichroism
DAPI, 4′,6-diamidino-2-phenylindole
DMED, Dulbecco’s modified Eagle’s medium
DMSO, dimethylsulfoxide
HRMS, high resolution mass spectra
LMTM, leucomethylene blue
NFT, intracellular neurofibrillary tangles
PBL, porcine brain lipid
SPR, surface plasmon resonance
TEM, transmission electron microscope
ThS, Thioflavin S
ThT, Thioflavin T
1

1. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is characterized by cognitive
decline and associated dementia for a period ranging from years to decades before death. However, memory
can be retrieved at the early stage of AD.1 Extracellular amyloid-β (Aβ) deposits and intracellular
neurofibrillary tangles (NFT) composed of highly phosphorylated tau proteins are two major
neuropathological lesions described in patients with AD. Both Aβ and tau aggregation could emerge far more
years before cognitive decline, thus making them potential targets for AD treatment.
Aβ and tau have been studied independently and therapies based on Aβ cascade hypothesis and tau
hyperphosphorylation have been examined in clinical trials; however, few breakthroughs were achieved.
Recent studies determined that there are links between Aβ and tau. Some evidence suggests that Aβ formation
can drive tau pathology, even though tau deposition in the brain might precede the formation of Aβ plaques.2
Removal of extracellular and intraneuronal Aβ via immunization has been shown to reduce neural
accumulation of tau.3 On the other hand, crossing of APP transgenic mice with knockout mice lacking tau
could prevent cognitive deficits associated with the presence of Aβ.4 However, whether Aβ is necessary for tau
neurotoxicity or whether the reverse is true remains a matter of debate.5
Due to the complex multifactorial nature of AD, molecules that modulate the activity of a single protein
target are incapable of significantly altering the progression of the disease. In contrast, multifunctional agents
with two or more correlative biological activities, which correspond with the multifactors of AD, have been
widely designed for the treatment of AD.6-8 Although some progress in this area has been made, the design of
multifunctional drugs is still very difficult. Selecting a promising lead compound becomes the key point to
explore potential multifunctional drugs.
2

Figure 1. (A) Chemical structures of tacrine, clioquinol, and 4a1. (B) Chemical structures of oleocanthal,
cinnamaldehyde, and asperbenzaldehyde. (C) Chemical structures of methylene blue and crystal violet.
Recently, some studies have suggested that the quinoline moiety is the pharmacophore of many anti-AD
drugs, such as tacrine 9 and clioqunol.10 Additionally, our previous research proved that 4a1, which contains a
quinoline moiety, is a potential multifunctional agent for AD treatment (Figure 1A).11
In addition, many studies have suggested that α,β-unsaturated carbonyl compounds could act as covalent
13
inhibitors in the aggregation pathway of tau, including oleocanthal,12 cinnamaldehyde
and
asperbenzaldehyde 14 (Figure 1B).
Compounds containing highly conjugated π-electron networks with high polarizability such as methylene
blue and crystal violet are both the most potent tau aggregation inhibitors reported (Figure 1C),15 and all share
a centrosymmetric structure with extensive delocalization of their fixed cationic charge.
In the present study, we fused the structure of tacrine and cinnamaldehyde in a single molecular entity to
3

obtain compound 5 to make the quinoline group and α,β-unsaturated ketone group work in a synergistic
manner (Figure 2). It has been reported that the polar substitutions on the aromatic rings of the compounds
are essential for their binding with Aβ.16 Therefore, we introduced a series of aromatic rings with different
substituents to the D ring to obtain compounds 6-17. The amino substituent at the 9-position of the B ring was
also modified to obtain compounds 18-24. In addition, a cationic charge was introduced at the N-position of
the quinolone ring to obtain compounds 25-30. To study the impact of the chain between the C ring and D ring,
compounds 31-44 were also designed. With the above considerations,
a
series of new
1,2,3,4-tetrahydro-1-acridone analogues were synthesized, and their inhibitory activities against Aβ and tau
aggregation were evaluated.
Figure 2. Design of the new compounds derived from tacrine and cinnamaldehyde.
2. Chemistry
The synthetic pathway for 1,2,3,4-tetrahydro-1-acridone analogues is shown in Scheme 1. The treatment
of 1,3-cyclohexanedione with different substrates (for example, anthranilonitrile, 2-nitrobenzaldehyde or
4

2-aminoacetophenone) yielded the intermediates 1, 2,17 3,18 or 4.19 The target compounds 5-24 and 31 were
obtained by the aldol reaction of intermediates 2, 3, and 4 with different aromatic aldehydes in the presence of
sodium hydroxide. Compound 32 was obtained by the catalytic hydrogenation of compound 5 in the presence
of Pd/C. Compounds 33-38 were obtained by the exposure of compounds 5, 7, 9, 11, 16 or 17 under UV light
for 24 h in dimethylsulfoxide (DMSO). Compounds 5, 7, 9, 11, 16 or 17 and 33-38, via interaction with
iodomethane under sealed conditions at 80 ºC for 48 h, yielded the target compounds 25-30 and 39-44. The
1
13
structures of the target compounds were validated by using H NMR, C NMR and HRMS. The E/Z isomers
can be identified by using NOESY spectra. As an example, the NOESY and HMBC spectra of the compounds
5(E-isomer) and 33(Z-isomer) were add to SI file. In addition, their purities were determined to be above 95%
by using HPLC assay.
5

Scheme 1. (A) Synthetic route of compounds 5-17 and 25-44. Reagents and reaction conditions: (i) toluene, 120 ºC, 3 h;
(ii) copper (I) chloride, potassium carbonate, THF, reflux, 4 h; (iii) aromatic aldehyde, NaOH, ethanol, r.t.~80 ºC, 3~24 h;
(iv) Pd/C, ethanol, H2, r.t., 96 h; (v) CH3I, acetone, 80 ºC, 48 h; (vi) λ = 365 nm, DMSO, r.t., 24 h. (B) Synthetic route of
compounds 18-23. Reagents and reaction conditions: (i) methanol, acetic acid, H2O, iron, 100 ºC, 1 h; (ii) aromatic
aldehyde, NaOH, ethanol, r.t.~50 ºC, 10~60 min. (C) Synthetic route of compound 24. Reagents and reaction conditions:
(i) H2O, 70 ºC, overnight; (ii) aromatic aldehyde, NaOH, ethanol, reflux, 48 h.
6

3. Results and Discussion
3.1. Screening of the potent inhibitors for Aβ1-42 and tau aggregation
To investigate the inhibition activity of the synthesized compounds against the self-induced Aβ1-42
aggregation, the Thioflavin T (ThT) fluorescence assay was performed with resveratrol as the reference
compound. To investigate the inhibition of tau aggregation, the Thioflavin S (ThS) fluorescence assay was
performed with methylene blue as the reference compound. To measure the capacity of the synthesized
compounds to penetrate the blood-brain barrier (BBB), a method described in our previous work was
employed.11 Compound 5 was first synthesized and revealed as much a weaker inhibitor of Aβ1-42 aggregation
(7.7%) compared with resveratrol (78.9%), and of tau aggregation (18.7%) compared with methylene blue
(80.1%) (Table 1).
A focused exploration of 5 was conducted that aimed to identify key structural elements for biological
potency. In the first instance, the amino group at the 9-position did not appear to facilitate the inhibitory
activities of Aβ1-42 and tau aggregation; however, introduction of the hydrophilic amino group (R group in
Table 1) led to a better permeability than introduction of hydrogen (18) or methyl (24) at the 9-position, as 5
exhibited a good permeability through the BBB (Pe = 26.4 × 10-6 cm/s).
Next, variation around the aromatic D ring was explored (Ar group in Table 1). The study revealed an
interesting and unexpected SAR: introducing a halogen atom (F or Cl) to the phenyl ring (compounds 6-10)
exhibited a better inhibition of Aβ1-42 aggregation compared to compound 5; however, the inhibitory activity
against tau aggregation decreased, except for with compound 6. A similar result was found when comparing
compound 18 with compounds 19-22. In addition, introducing a methoxyl group (13 and 14) exhibited better
inhibition of tau aggregation but not of Aβ1-42 aggregation. Introduction of a heterocyclic group, such as
7

furan-2-yl (15), improved inhibition of both Aβ1-42 and tau aggregation. However, none of the compounds
described above exhibited better inhibitory activities than the reference compounds resveratrol and methylene
blue.
Table 1. Inhibition of Self-induced Aβ1-42 and Tau Aggregation and Permeability for Compounds 5-24
Aβ1-42 aggregation
inhibition (%)a
NDd
Tau aggregation
inhibition (%)b
80.1 ± 1.6
NDd
Pe
Comp.
R
Ar
(× 10-6 cm/s)c
NDd
-
-
-
MB
Res
5
-
78.9 ± 1.7
7.7 ± 0.1
37.0 ± 0.3
20.3 ± 1.7
14.1 ± 0.2
34.2 ± 0.3
24.8 ± 1.2
8.2 ± 0.1
NDd
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
Ph
18.7 ± 0.5
25.3 ± 0.8
4.9 ± 0.6
26.4 ± 1.2
6.9 ± 0.5
5.3 ± 0.4
7.4 ± 0.4
1.1 ± 0.1
30.3 ± 4.9
3.3 ± 0.2
11.2 ± 0.6
7.5 ± 0.4
10.5 ± 0.5
16.2 ± 0.7
7.2 ± 0.5
0.5 ± 0.02
8.6 ± 0.6
8.3 ± 0.9
17.2 ± 0.1
7.1 ± 0.1
2.0 ± 0.1
2-F-Ph
6
3-F-Ph
7
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
3-NO2-Ph
4-NO2-Ph
3-OCH3-Ph
4-OCH3-Ph
furan-2-yl
thiophen-2-yl
naphthalen-1-yl
Ph
-3.6 ± 0.4
-3.1 ± 0.1
7.1 ± 0.3
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
-10.3 ± 0.3
-3.3 ± 0.1
44.3 ± 3.5
43.4 ± 0.4
30.7 ± 1.1
11.9 ± 0.2
-6.7 ± 0.3
20.5 ± 0.6
2.9 ± 0.2
2.4 ± 0.2
7.9 ± 0.1
-2.0 ± 0.1
15.2 ± 3.5
14.5 ± 0.6
7.8 ± 0.2
-3.2 ± 1.3
-0.5 ± 0.5
7.2 ± 0.4
H
2-F-Ph
H
3-F-Ph
14.7 ± 0.1
14.9 ± 0.2
4.2 ± 0.8
H
2-Cl-Ph
3-Cl-Ph
3.5 ± 1.2
H
46.2 ± 1.7
8

H
4-NO2-Ph
Ph
2.7 ± 0.2
6.3 ± 2.2
3.2 ± 0.3
23
24
CH3
16.6 ± 0.2
15.7 ± 2.3
3.1 ± 0.04
a The evaluation of inhibition of Aβ1-42 aggregation by ThT test. The concentration of Aβ1-42 is 40 μM, while the concentrations of the
b
compounds are 20 μM. The evaluation of inhibition of tau aggregation by ThS test. The concentration of tau is 10 μM while the
c
concentrations of the compounds are 20 μM. Compounds with Pe > 3.8 × 10-6 cm/s are able to permeate the BBB. d ND: Not
Determined.
Our results confirmed that the quaternary amine might be crucial for these types of molecules to act as
excellent dual inhibitors of Aβ1-42 and tau aggregation (Table 2) because compounds 25-30 showed
significantly higher inhibitory activities (Aβ1-42 aggregation inhibitory ratio: 84.7-99.5%; tau aggregation
inhibitory ratio: 71.2-101.8%) than 5, coming close to the inhibitory activity of the reference compounds
resveratrol and methylene blue or exhibiting even better inhibition. Compound 27 and 30 were especially
predicted to exhibit a good permeability through the BBB (27, Pe = 4.1 × 10-6 cm/s, 30, Pe = 4.6 × 10-6 cm/s).
Table 2. Inhibition of Self-induced Aβ1-42 and Tau Aggregation and Permeability for Compounds 25-30
Aβ1-42 aggregation
inhibition (%)a
84.7 ± 7.8
Tau aggregation
inhibition (%)b
71.2 ± 2.7
Pe
Y
Ar
Comp.
(× 10-6 cm/s)c
2.4 ± 0.1
3.6 ± 0.2
4.1 ± 0.3
2.1 ± 0.2
1.7 ± 0.1
4.6 ± 0.4
N+CH3
N+CH3
N+CH3
N+CH3
N+CH3
Ph
25
26
27
28
29
30
3-F-Ph
98.7 ± 1.6
86.3 ± 1.1
3-Cl-Ph
91.9 ± 2.9
98.3 ± 2.2
3-NO2-Ph
thiophen-2-yl
86.4 ± 1.2
98.2 ± 2.2
99.5 ± 3.8
95.3 ± 4.6
N+CH3 naphthalen-1-yl
86.7 ± 3.5
101.8 ± 3.0
a The evaluation of inhibition of Aβ1-42 aggregation by ThT test. The concentration of Aβ1-42 is 40 μM, while the concentrations of the
b
compounds are 20 μM. The evaluation of inhibition of tau aggregation by ThS test. The concentration of tau is 10 μM while the
concentrations of the compounds are 20 μM. c Compounds with Pe > 3.8 × 10-6 cm/s are able to permeate the BBB.
It was reported that cinnamic acid derivatives (CAs) could photoisomerize from the trans-forms to
20
cis-CAs under sunlight and ultraviolet light.21, 22 Compounds 5-17 contained such groups. To explore the
9

impacts of isomers on their biological activities, compounds 33-38 were synthesized. As shown in Table 3, it
was revealed that the Z-forms compounds showed opposite effects on Aβ1-42 and tau aggregation compared to
the corresponding E-forms compounds (5, 7, 9, 11, 16 and 17). In other words, compounds 33-38 seem to
accelerate the aggregation of Aβ1-42 and tau. In addition, variation of the length (31) and flexibility (32) were
explored, which revealed that both exhibited disadvantages (Table 3).
Table 3. Inhibition of Self-induced Aβ1-42 and Tau Aggregation and Permeability for Compounds 31-38
Aβ1-42 aggregation
inhibition rate (%)a
2.0 ± 0.5
Tau aggregation
inhibition rate (%)b
-3.8 ± 0.5
Pe
Ar
Comp.
(× 10-6 cm/s)c
1.3 ± 0.1
-
-
31
32
33
34
35
36
37
38
-4.0 ± 0.1
10.0 ± 0.1
13.9 ± 3.8
13.3 ± 1.3
20.3 ± 1.1
15.6 ± 1.2
9.1 ± 1.2
Ph
-18.9 ± 2.6
-28.5 ± 2.6
-40.8 ± 3.0
-23.5 ± 4.5
-25.0 ± 1.5
5.9 ± 6.9
3-F-Ph
-27.7 ± 2.0
3-Cl-Ph
3-NO2-Ph
thiophen-2-yl
naphthalen-1-yl
-26.5 ± 3.7
-42.6 ± 6.9
-11.1 ± 0.9
10.8 ± 1.9
49.5 ± 6.0
-6.8 ± 7.5
-30.5 ± 9.2
a The evaluation of inhibition of Aβ1-42 aggregation by ThT test. The concentration of Aβ1-42 is 40 μM, while the concentrations of the
b
compounds are 20 μM. The evaluation of inhibition of tau aggregation by ThS test. The concentration of tau is 10 μM while the
concentrations of the compounds are 20 μM. c Compounds with Pe > 3.8 × 10-6 cm/s are able to permeate the BBB.
Since Z-E isomers could exhibit totally opposite effects, compounds 39-44, which were the Z-forms of
compounds 33-38, were additionally explored (Table 4). Compounds 39-44 also showed strong inhibitory
activities against Aβ1-42 aggregation (89.1-104.6%). However, they exhibited a significant decrease in
inhibition of tau aggregation (7.2-31.6%), except 44 (79.2%).
10

Table 4. Inhibition of Self-induced Aβ1-42 and Tau Aggregation and Permeability for Compounds 39-44
Aβ1-42 aggregation
inhibition rate (%)a
97.6 ± 1.8
Tau aggregation
inhibition rate (%)b
7.2 ± 5.1
Pe
Ar
Comp.
(× 10-6 cm/s)c
1.8 ± 0.2
2.7 ± 0.1
4.6 ± 0.1
1.8 ± 0.01
1.7 ± 0.1
3.5 ± 0.1
Ph
39
40
41
42
43
44
3-F-Ph
89.1 ± 4.4
23.3 ± 3.6
3-Cl-Ph
104.6 ± 3.3
94.4 ± 2.0
15.4 ± 6.5
3-NO2-Ph
thiophen-2-yl
naphthalen-1-yl
32.0 ± 5.5
94.9 ± 4.0
31.6 ± 3.8
91.2 ± 1.5
79.2 ± 7.3
a The evaluation of inhibition of Aβ1-42 aggregation by ThT test. The concentration of Aβ1-42 is 40 μM, while the concentrations of the
b
compounds are 20 μM. The evaluation of inhibition of tau aggregation by ThS test. The concentration of tau is 10 μM while the
concentrations of the compounds are 20 μM. c Compounds with Pe > 3.8 × 10-6 cm/s are able to permeate the BBB.
Among them, compound 30 could inhibit both Aβ1-42 aggregation (86.7%) and tau aggregation (101.8%),
and it was predicted to permeate the BBB. Compound 30 was selected as a candidate for further evaluation.
3.2. Effect of compound 30 on abundance of Aβ1-42 and tau fibrils
To avoid false positives and further confirm the ability of compound 30 in inhibiting Aβ1-42 aggregation
and tau aggregation, the inhibitory activity of compound 30 was monitored by transmission electron microscope
(TEM) with resveratrol or methylene blue as the control, respectively. Substantial changes in the morphology
of the aggregates were observed when Aβ1-42 or tau was incubated with compound 30. At 0 h, there was no
aggregation for Aβ1-42 or tau (Figure 3A, 4A). After a 48 h incubation at 37 ºC, Aβ1-42 alone aggregated into
many long, well-defined network Aβ fibrils (Figure 3B), while a few thick fibrils were observed for the
sample of Aβ1-42 in the presence of resveratrol (Figure 3C). Meanwhile, when Aβ1-42 was incubated with
compound 30 under identical conditions, fewer short fibrils were found (Figure 3D). After 48 h incubation at
11

37 ºC, a large number of fibrils were observed in the sample of tau alone (Figure 4B). Compared to methylene
blue, a few short tau fibrils were observed in the presence of 30 (Figure 4C and 4D). The TEM experimental
results were in agreement with the results of the ThT and ThS studies, which further proved that compound 30
could inhibit and slow down the formation of Aβ1-42 and tau fibrils.
Figure 3. TEM image analysis of Aβ1-42 aggregation in the presence and absence of compound 30. (A) Aβ1-42 (50 μM), 0
h. (B) Aβ1-42 (50 μM) was incubated at 37 ºC for 48 h. (C) Aβ1-42 (50 μM) and resveratrol (Res, 20 μM) were incubated at
37 ºC for 48 h. (D) Aβ1-42 (50 μM) and 30 (20 μM) were incubated at 37 ºC for 48 h.
Figure 4. TEM image analysis of tau aggregation in the presence and absence of compound 30. (A) Tau (10 μM), 0 h. (B)
Tau (10 μM) was incubated at 37 ºC for 48 h. (C) Tau (10 μM) and methylene blue (MB, 20 μM) were incubated at 37 ºC
for 48 h. (D) Tau (10 μM) and 30 (20 μM) were incubated at 37 ºC for 48 h.
3.3. Effect of compound 30 on formation of Aβ1-42 β-sheet structure
Aβ adopted α-helix, β-sheet, and random coil conformations in the aqueous solution and underwent a
conformational transition to form an intramolecular β-sheet structure in the fibrillation.23 The effect of 30 on
the secondary structure of aggregated Aβ1-42 was examined by using circular dichroism (CD) spectroscopy. In
12

the absence of potassium, sodium, or any other cations, the CD spectrum of monomeric Aβ1-42 exhibited a
negative peak at 210 nm, as shown in Figure 5A. Incubation at 37 ºC for 24 h induced the formation of
β-sheets, exhibiting a positive peak at 195-200 nm and a negative peak at 218 nm (Figure 5B).24,25
Coincubation of Aβ1-42 with 30 resulted in fewer sharp peaks, which indicated that our compounds restrained
the generation of Aβ fibrils by preventing the formation of a β-sheet structure in the course of Aβ aggregation.
Combining with the previous data, 30 inhibited self-induced Aβ1-42 aggregation by inhibiting the formation of
β-sheets.
Figure 5. Inhibition of Aβ1-42 β-sheets formation by 30 in vitro. (A) CD spectra of Aβ1-42 (50 μM) before incubation with
or without 30 (20 μM); (B) CD spectra of Aβ1-42 (50 μM) after 24 h incubation at 37 ºC with or without 30(20 μM).
3.4. Evaluation of 30 as Aβ1-42 and tau ligands
The binding of compounds to Aβ1-42 and tau is also an important parameter for identifying an effective
protein ligand. We further evaluated the binding ability of 30 with Aβ1-42 and tau by using surface plasmon
resonance (SPR) assay. After fitting the original SPR data, the binding constants (KD) could be calculated. As
shown in Figure 6, 30 demonstrated certain binding abilities with Aβ1-42 and tau. The KD value of 30 with
Aβ1-42 was 1.60 × 10-5 mol/L (Figure 6A), while the KD values of resveratrol with Aβ1-42 was 3.85 × 10-5
mol/L (Figure 6B). The KD value of 30 with tau was 3.37 × 10-5 mol/L (Figure 6C), while the KD value of
13

methylene blue with tau was 2.82 × 10-5 mol/L (Figure 6D). The binding curve further indicated that 30
interacted with Aβ1-42 and tau through noncovalent interactions.
Figure 6. SPR studies for binding of 30 to Aβ1-42 and tau. (A) SPR sensorgrams for binding of 30 to Aβ1-42; the
equilibrium binding constant (KD) for the binding of 30 with Aβ1-42 was 1.60 × 10-5 mol/L. (B) SPR sensorgrams for the
binding of resveratrol (Res) to Aβ1-42; the equilibrium binding constants (KD) for the binding of Res with Aβ1-42 was 3.85
× 10-5 mol/L. (C) SPR sensorgrams for the binding of 30 to tau; the equilibrium binding constant (KD) for the binding of
30 with tau was 3.37 × 10-5 mol/L. (D) SPR sensorgrams for the binding of methylene blue (MB) to tau; the equilibrium
14

binding constant (KD) for the binding of MB with tau was 2.82 × 10-5 mol/L.
3.5. Inhibition of tau aggregation in living cells by 30
To further explore the inhibitory ability of compound 30 on tau aggregation, a cellular tau-aggregation
assay was performed. A DNA fragment containing coding sequence of EGFP-tau P301L was transfected and
expressed in HEK-293T cells; 6 h after transfection, 30 (1 μM) or leucomethylene blue (LMTM,
Medchemexpress, CN) (0.5 μM) was added to the cells. Tau aggregation was promoted by treatment with 10
μM Congo red, which is a known small-molecule agonist of tau aggregation, and was incubated for another 24
h. Analysis is performed by cytological analysis following stringent extraction to eliminate soluble forms of
tau.26,27 As shown in Figure 7, the tau aggregation foci declined significantly after the administration of 30
(62.6%) and LMTM (74.4%), while the 4′,6-diamidino-2-phenylindole (DAPI) foci did not change
considerably. Collectively, these results confirmed that 30 could effectively inhibit tau aggregation in living
cells without restraining cell proliferation at 1 μM.
Figure 7. Inhibition of tau aggregation in living cells by 30. (A) Imaging of HEK-293T cells incubated with or without
LMTM (0.5 μM) and 30 (1 μM). (B) Quantification of the GFP fluorescence intensity with or without LMTM (0.5 μM)
and 30 (1 μM) for A. For each sample, approximately 35,000 cells were measured. The data are expressed as the mean ±
SEM (standard error of mean): (***) P < 0.0001, significantly different from the control; (**) P < 0.001, significantly
different from the control.
15

4. Conclusion
In summary, forty new 1,2,3,4-tetrahydro-1-acridone analogues were synthesized and were characterized
as potential dual inhibitors for Aβ1-42 and tau aggregation. Compounds 25-30 displayed high effective
inhibitory potencies against Aβ1-42 and tau aggregation. Among them, compounds 27 and 30 were also
predicted to be able to permeate the BBB. SAR analysis for all these synthesized compounds was investigated,
and the analysis suggested that the introduction of cationic charge in the quinoline ring, such as the
introduction of a quaternary amine group, is crucial to the inhibitory potency of the compounds against both
Aβ1-42 and tau aggregation. Further investigation of compound 30 through TEM confirmed that 30 could
inhibit Aβ1-42 and tau fibril formation. In addition, an SPR experiment suggested that 30 could bind Aβ1-42 and
tau well and act as a noncovalent inhibitor. A CD experiment revealed that 30 could inhibit Aβ1-42 β-sheet
formation. It was also proven that 30 could inhibit tau aggregation in living cells. These results indicated that
these new 1,2,3,4-tetrahydro-1-acridone analogues could be further developed as dual functional agents for
AD treatment.
5. Experimental Section
5.1. Chemistry
All reagents (chemicals) were purchased from commercial companies without further purification. The
progress of the reaction was monitored by using analytical thin layer chromatography (TLC) on silica gel
GF254. 1H and 13C NMR spectra were recorded using TMS as the internal standard in CDCl3 or DMSO-d6 with
a Bruker BioSpin GmbH spectrometer at 400 MHz and 101 MHz, respectively. High resolution mass spectra
(HRMS) were recorded on Shimadzu LCMS-IT-TOF spectrometer. Flash colum chromatography was
16

performed with silica gel (200-300 mesh) purchased from Qingdao Haiyang Chemical Co.Ltd. Melting points
(m.p.) were determined using an SRS-OptiMelt automated melting point instrument without correction. The
purity of synthesized compounds was confirmed to be higher than 95% through analytical HPLC performed
with a dual pump Shimadzu LC-20AB system equipped with an Ultimate XB-C18 column (4.6 × 250 mm, 5
μm) and eluted with methanol/water (60:40-80:20) containing 0.1% TFA at a flow of 0.5 mL/min.
5.1.1. Synthesis of 2-((3-oxocyclohexyl)amino)benzonitrile (1)
A suspension of anthranilonitrile (5.0 g, 42.32 mmol) in 25 mL of toluene containing 1,3-cyclohexanedione
(5.2 g, 46.58 mmol) and p-toluenesulfonic acid monohydrate (257.0 mg, 1.35 mmol) was refluxed for 2.5 h.
Then the reaction mixture was chilled to ca. 0 ºC and the product was filtered off and washed with cold
toluene. The crude product obtained in this manner was slurried with 25 mL water and stirred for 1 h, after
1
which it was filtered off and dried under reduced pressure to afford 1 as white powder in 90% yield. H NMR
(400 MHz, CDCl3) δ 7.57 (d, J = 7.7 Hz, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.17 (t, J =
7.6 Hz, 1H), 6.44 (s, 1H), 5.53 (s, 1H), 2.51 (t, J = 6.1 Hz, 2H), 2.33 (t, J = 6.5 Hz, 2H), 2.07 – 1.96 (m, 2H).
5.1.2. Synthesis of 9-amino-3,4-dihydroacridin-1(2H)-one (2)
In 175 mL of THF was suspended 1 (8.0 g, 37.49 mmol), CuCl (130.5 mg, 1.32 mmol) and anhydrous K2CO3
(941.1 mg, 6.81 mmol). The reaction mixture was refluxed for 4 h and then the hot mixture was filtered into
175 mL of hexane. The precipitate was filtered off, washed with water, and dried to afford 2 as canary yellow
powder in 69% yield. 1H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 8.3 Hz, 1H), 7.74 (d, J = 8.3 Hz, 1H), 7.64 (t,
J = 7.5 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 3.06 (t, J = 6.2 Hz, 2H), 2.67 (t, J = 6.4 Hz, 2H), 2.09 (dt, J = 12.3,
6.3 Hz, 2H).
17

5.1.3. Synthesis of 3,4-dihydroacridin-1(2H)-one (3)
Iron powder (5.0 g, 90 mmol) was added to a stirred solution of 2-nitrobenzaldehyde (4.5 g, 30 mmol) and
1,3-cyclohexanedione (4.0 g, 36 mmol) in 300 mL of mixed solvent (methanol/acetic acid/water: 2/2/1). This
mixture was refluxed for 1 h. After completion of the reaction, the reaction mixture was cooled to room
temperature, poured into 300 mL of ice water, and filtered to remove the iron impurities. The mixture was
extracted by using EtOAc. The organic phase was washed by sodium bicarbonate solution and brine,
successively, and then dried by MgSO4. The solvent was removed under reduced pressure and the crud
product thus obtained was purified by column chromatography with PE/DCM: 1/1 to afford 3 as white powder
1
in 48% yield. H NMR (400 MHz, CDCl3) δ 8.87 (s, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.1 Hz, 1H),
7.82 (t, J = 7.7 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H), 3.34 (t, J = 6.2 Hz, 2H), 2.87 – 2.73 (m, 2H), 2.34 – 2.22 (m,
2H).
5.1.4. Synthesis of 9-methyl-3,4-dihydroacridin-1(2H)-one (4)
In 100 mL of water was suspended 2-aminoacetophenone (1.2 mL, 10 mmol) and 1,3-cyclohexanedione (1.2 g,
11 mmol). The reaction mixture was stirred under 70 ºC overnight. After completion of the reaction, the
reaction mixture was cooled to room temperature, extracted by using EtOAc. The organic phase was washed
by brine, and dried by MgSO4. The solvent was removed under pressure and the crud product thus obtained
was purified by column chromatography with DCM/methanol: 100/1 to afford 4 as brown powder in 44%
1
yield. H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 8.5 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.73 – 7.64 (m, 1H),
7.47 (t, J = 7.7 Hz, 1H), 3.22 – 3.16 (m, 2H), 2.95 (s, 3H), 2.72 (t, J = 6.6 Hz, 2H), 2.17 – 2.08 (m, 3H).
5.1.5. General procedure A: Preparation of compounds 5-17 and 31
To a mixture of compound 2 (212.3 mg, 1 mmol), aromatic aldehyde (1.1 mmol) and 10 mL of ethanol,
18

400 μL of 10% NaOH aqueous solution was added slowly at room temperature, and the resulting mixture was
stirred under room temperature to 80 ºC for 3 to 24 h. After completion of the reaction, the reaction mixture
was cooled to room temperature, and poured into ice water (50 mL). The precipitate was then separated from
the solvent through filtration, washed with ethanol, and purified by using flash column chromatography with
dichloromethane or dichloromethane/methanol: 200/1 elution to give the desired products.
5.1.5.1. (E)-9-amino-2-benzylidene-3,4-dihydroacridin-1(2H)-one (5)
Compound 2 was reacted with benzaldehyde according to general procedure A under room temperature for 24
1
h to afford 5 as yellow powder in 60% yield. Mp: 226.9-228.4 ºC. H NMR (400 MHz, DMSO) δ 10.24 (s,
1H), 8.55 (s, 1H), 8.38 (d, J = 8.3 Hz, 1H), 7.77 – 7.64 (m, 3H), 7.52 (d, J = 7.6 Hz, 2H), 7.50 – 7.42 (m, 3H),
7.39 (t, J = 7.0 Hz, 1H), 3.07 (t, J = 6.2 Hz, 2H), 3.02 – 2.91 (m, 2H). 13C NMR (101 MHz, DMSO) δ 189.4,
162.5, 155.8, 148.4, 136.9, 136.0, 134. 8, 132. 5, 130.3, 129.0, 129.0, 129.0, 124.9, 123.8, 118.6, 106.1, 33.4,
26.1. ESI-HRMS: calcd for C20H16N2O [M+H]+: 301.1335; found: 301.1337. HPLC purity: 97.1%.
5.1.5.2. (E)-9-amino-2-(2-fluorobenzylidene)-3,4-dihydroacridin-1(2H)-one (6)
Compound 2 was reacted with 2-fluorobenzaldehyde according to general procedure A under 50 ºC for 4 h to
1
afford 6 as yellow powder in 36% yield. Mp: 223.4-224.6 ºC. H NMR (400 MHz, DMSO) δ 10.23 (s, 1H),
8.61 (s, 1H), 8.39 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 3.7 Hz, 2H), 7.65 (s, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.50 –
7.42 (m, 2H), 7.35 – 7.26 (m, 2H), 3.07 – 2.87 (m, 4H). 13C NMR (101 MHz, DMSO) δ 188.84, 162.59,
160.56 (d, J = 247.5 Hz), 156.0, 148.4, 139.1, 132.6, 131.3 (d, J = 2.0 Hz), 131.2, 129.0, 126.9 (d, J = 3.1 Hz),
125.0, 124.9, 123.9, 123.7 (d, J = 13.8 Hz), 118.6, 116.2 (d, J = 21.7 Hz), 106.0, 33.4, 26.3. ESI-HRMS: calcd
for C20H15N2OF [M+H]+: 319.1241; found: 319.1232. HPLC purity: 96.5%.
19

5.1.5.3. (E)-9-amino-2-(3-fluorobenzylidene)-3,4-dihydroacridin-1(2H)-one (7)
Compound 2 was reacted with 3-fluorobenzaldehyde according to general procedure A under 80 ºC for 4 h to
1
afford 7 as yellow powder in 49% yield. Mp: 236.4-238.1 ºC. H NMR (400 MHz, CDCl3) δ 10.46 (s, 1H),
7.92 – 7.81 (m, 2H), 7.77 (s, 1H), 7.72 (t, J = 7.4 Hz, 1H), 7.45 (t, J = 7.4 Hz, 1H), 7.38 (t, 1H), 7.21 (d, J =
7.6 Hz, 1H), 7.14 (d, J = 9.7 Hz, 1H), 7.05 (t, J = 8.1 Hz, 1H), 6.16 (s, 1H), 3.13 (s, 4H). 13C NMR (101 MHz,
CDCl3) δ 189.0, 161.7 (d, J = 246.5 Hz), 161.4, 153.9, 147.3, 137.1 (d, J = 7.9 Hz), 136.2, 133.3 (d, J = 2.1
Hz), 131.2, 129.0 (d, J = 8.4 Hz), 128.3, 124.6 (d, J = 2.7 Hz), 124.0, 120.0, 116.8, 115.4 (d, J = 21.8 Hz),
114.4 (d, J = 21.0 Hz), 105.7, 32.6, 25.0. ESI-HRMS: calcd for C20H15N2OF [M+H]+: 19.1241; found:
319.1237. HPLC purity: 99.4%.
5.1.5.4. (E)-9-amino-2-(2-chlorobenzylidene)-3,4-dihydroacridin-1(2H)-one (8)
Compound 2 was reacted with 2-chlorobenzaldehyde according to general procedure A under 50 ºC for 4 h to
1
afford 8 as yellow powder in 76% yield. Mp: 240.6-241.4 ºC. H NMR (400 MHz, CDCl3) δ 10.51 (s, 1H),
7.94 – 7.83 (m, 3H), 7.72 (t, J = 7.6 Hz, 1H), 7.51 – 7.41 (m, 2H), 7.39 – 7.28 (m, 3H), 6.20 (s, 1H), 3.13 (t,
2H), 3.01 (t, 2H). 13C NMR (101 MHz, CDCl3) δ 190.1, 162.7, 155.0, 148.3, 137.8, 134.8, 134.5, 132.6, 132.2,
130.5, 129.8, 129.6, 129.3, 126.4, 125.0, 120.9, 117.8, 106.8, 33.8, 26.2. ESI-HRMS: calcd for C20H15N2OCl
[M+H]+: 335.0946; found: 335.0939. HPLC purity: 98.2%.
5.1.5.5. (E)-9-amino-2-(3-chlorobenzylidene)-3,4-dihydroacridin-1(2H)-one (9)
Compound 2 was reacted with 3-chlorobenzaldehyde according to general procedure A under 50 ºC for 4 h to
1
afford 9 as yellow powder in 53% yield. Mp: 224.3-227.3 ºC. H NMR (400 MHz, CDCl3) δ 10.44 (s, 1H),
7.89 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.78 – 7.68 (m, 2H), 7.45 (t, J = 7.6 Hz, 1H), 7.42 (s, 1H),
7.38 – 7.28 (m, 3H), 6.15 (s, 1H), 3.21 – 3.06 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 190.0, 162.4, 154.9,
20

148.3, 137.7, 137.4, 134.4, 134.2, 132.2, 129.8, 129.6, 129.3, 128.5, 127.9, 125.0, 120. 9, 117.8, 106.7, 33.6,
26.1. ESI-HRMS: calcd for C20H15N2OCl [M+H]+: 335.0946; found: 335.0938. HPLC purity: 98.6%.
5.1.5.6. (E)-9-amino-2-(4-chlorobenzylidene)-3,4-dihydroacridin-1(2H)-one (10)
Compound 2 was reacted with 4-chlorobenzaldehyde according to general procedure A under 50 ºC for 6 h to
afford 10 as yellow powder in 56% yield. Mp: 220.5-221.6 ºC. 1H NMR (400 MHz, DMSO) δ 10.22 (s, 1H),
8.57 (s, 1H), 8.37 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 3.7 Hz, 2H), 7.64 (s, 1H), 7.57 – 7.48 (m, 4H), 7.47 – 7.42
(m, 1H), 3.08 – 3.00 (m, 2H), 3.00 – 2.90 (m, 2H). 13C NMR (101 MHz, DMSO) δ 189.1, 162.5, 155.8, 148.3,
137.5, 134.9, 133.6, 133.4, 132.5, 132.1, 129.1, 128.9, 125.0, 123.9, 118.6, 106.1, 33.3, 26.0. ESI-HRMS:
calcd for C20H15N2OCl [M+H]+: 335.0946; found: 335.0941. HPLC purity: 98.0%.
5.1.5.7. (E)-9-amino-2-(3-nitrobenzylidene)-3,4-dihydroacridin-1(2H)-one (11)
Compound 2 was reacted with 2-nitrobenzaldehyde according to general procedure A under 50 ºC for 3 h to
1
afford 11 as yellow powder in 83% yield. Mp: 225.7-228.9 ºC. H NMR (400 MHz, CDCl3) δ 10.41 (s, 1H),
8.29 (s, 1H), 8.21 (d, J = 8.2 Hz, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.83 (s, 1H), 7.73 (t, J
= 7.0 Hz, 2H), 7.61 (t, J = 7.9 Hz, 1H), 7.47 (t, J = 7.3 Hz, 1H), 6.20 (s, 1H), 3.19 – 3.10 (m, 4H). 13C NMR
(101 MHz, CDCl3) δ 190. 0, 162.2, 155.1, 148.4, 138.7, 137.6, 135.5, 132.7, 132.4, 129.5, 129.4, 125.2, 124.3,
123.0, 120.9, 117.8, 106.7, 33.5, 26.0. ESI-HRMS: calcd for C20H15N3O3 [M+H]+: 346.1186; found: 346.1179.
HPLC purity: 99.2%.
5.1.5.8. (E)-9-amino-2-(4-nitrobenzylidene)-3,4-dihydroacridin-1(2H)-one (12)
Compound 2 was reacted with 4-nitrobenzaldehyde according to general procedure A under 50 ºC for 6 h to
1
afford 12 as yellow powder in 19% yield. Mp: 218.3-219.6 ºC. H NMR (400 MHz, CDCl3) δ 10.43 (s, 1H),
8.29 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.83 (s, 1H), 7.74 (t, J = 7.6 Hz,
21

1H), 7.58 (d, J = 8.5 Hz, 2H), 7.47 (t, J = 7.6 Hz, 1H), 6.20 (s, 1H), 3.25 – 3.04 (m, 4H). 13C NMR (101 MHz,
DMSO) δ 188.2, 161.9, 155.5, 147.9, 146.7, 142.4, 139.5, 132.1, 131.8, 130.8, 128. 5, 124.5, 123.5, 123.4,
118.1, 105.6, 32.7, 25.6. ESI-HRMS: calcd for C20H15N3O3 [M+H]+: 346.1186; found: 346.1178. HPLC purity:
96.4%.
5.1.5.9. (E)-9-amino-2-(3-methoxybenzylidene)-3,4-dihydroacridin-1(2H)-one (13)
Compound 2 was reacted with 3-methoxybenzaldehyde according to general procedure A under 80 ºC for 3 h
1
to afford 13 as yellow powder in 22% yield. Mp: 212.8-216.5 ºC. H NMR (400 MHz, CDCl3) δ 7.89 (d, J =
8.3 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.81 (s, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.35 (t, J
= 7.9 Hz, 1H), 7.05 (d, J = 7.4 Hz, 1H), 6.98 (s, 1H), 6.91 (d, J = 8.1 Hz, 1H), 3.85 (s, 3H), 3.22 – 3.08 (m,
4H). 13C NMR (101 MHz, DMSO) δ 189.4, 162.5, 159.7, 155.8, 148.3, 137.3, 137.1, 134.7, 132.5, 130.1,
128.9, 124.9, 123.8, 122.5, 118.6, 115.6, 114.1, 106.1, 55.6, 33.4, 26.1. ESI-HRMS: calcd for C21H18N2O2
[M+H]+: 331.1441; found: 331.1435. HPLC purity: 98.2%.
5.1.5.10. (E)-9-amino-2-(4-methoxybenzylidene)-3,4-dihydroacridin-1(2H)-one (14)
Compound 2 was reacted with 4-methoxybenzaldehyde according to general procedure A under 50 ºC for 4 h
to afford 14 as yellow powder in 40% yield. Mp: 202.5-203.4 ºC. 1H NMR (400 MHz, DMSO) δ 10.22 (s, 1H),
8.49 (s, 1H), 8.36 (d, J = 8.3 Hz, 1H), 7.70 (d, J = 3.8 Hz, 2H), 7.66 (s, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.47 –
7.40 (m, 1H), 7.02 (d, J = 8.7 Hz, 2H), 3.80 (s, 3H), 3.06 (t, J = 6.2 Hz, 2H), 3.00 – 2.85 (m, 2H). 13C NMR
(101 MHz, DMSO) δ 189.4, 162.5, 160.1, 155.6, 148.4, 134.9, 134.7, 132.3, 132.2, 128.9, 128.4, 124.8, 123.8,
118.7, 114.6, 106.2, 55.7, 33.4, 26.1. ESI-HRMS: calcd for C21H18N2O2 [M+H]+: 331.1441; found: 331.1437.
HPLC purity: 96.1%.
22

5.1.5.11. (E)-9-amino-2-(furan-2-ylmethylene)-3,4-dihydroacridin-1(2H)-one (15)
Compound 2 was reacted with furan-2-carbaldehyde according to general procedure A to afford 15 as yellow
powder in 80% yield. Mp: 212.0-213.8 ºC. 1H NMR (400 MHz, DMSO) δ 10.19 (s, 1H), 8.51 (s, 1H), 8.35 (d,
J = 8.3 Hz, 1H), 7.90 (d, J = 1.4 Hz, 1H), 7.70 (d, J = 3.8 Hz, 2H), 7.49 – 7.39 (m, 2H), 6.97 (d, J = 3.4 Hz,
1H), 6.71 – 6.63 (m, 1H), 3.22 (t, J = 6.4 Hz, 2H), 3.09 – 2.95 (m, 2H). 13C NMR (101 MHz, DMSO) δ 188.7,
162.6, 155.6, 152.2, 148.3, 145.8, 133.0, 132.4, 128.9, 124.9, 123.8, 121.4, 118.6, 117.0, 113.1, 106.2, 32.8,
25.6. ESI-HRMS: calcd for C18H14N2O2 [M+H]+: 291.1128; found: 291.1120. HPLC purity: 99.6%.
5.1.5.12. (E)-9-amino-2-(thiophen-2-ylmethylene)-3,4-dihydroacridin-1(2H)-one (16)
Compound 2 was reacted with thiophene-2-carbaldehyde according to general procedure A under 80 ºC for 3 h
to afford 16 as yellow powder in 74% yield. Mp: 236.7-238.8 ºC. 1H NMR (400 MHz, CDCl3) δ 10.41 (s, 1H),
8.02 (s, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.71 (t, J = 7.6 Hz, 1H), 7.52 (d, J = 4.9 Hz,
1H), 7.44 (t, J = 7.6 Hz, 1H), 7.39 (d, J = 3.1 Hz, 1H), 7.14 (t, J = 4.2 Hz, 1H), 6.13 (s, 1H), 3.29 – 3.16 (m,
4H). 13C NMR (101 MHz, CDCl3) δ 189.5, 162.4, 154.6, 148.3, 139.2, 132.9, 132.5, 132.0, 129.3, 128.7,
127.6, 124.9, 120.9, 117.9, 107.0, 33.0, 25.9. ESI-HRMS: calcd for C24H18N2O [M+H]+: 351.1492; found:
351.1498. HPLC purity: 96.1%.
5.1.5.13. (E)-9-amino-2-(naphthalen-1-ylmethylene)-3,4-dihydroacridin-1(2H)-one (17)
Compound 2 was reacted with 1-naphthaldehyde according to general procedure A under 50 ºC for 12 h to
1
afford 17 as yellow powder in 52% yield. Mp: 231.8-233.0 ºC. H NMR (400 MHz, CDCl3) δ 10.56 (s, 1H),
8.35 (s, 1H), 8.09 – 8.00 (m, 1H), 7.95 – 7.82 (m, 4H), 7.73 (t, J = 7.6 Hz, 1H), 7.58 – 7.49 (m, 3H), 7.46 (t, J
= 7.5 Hz, 2H), 6.21 (s, 1H), 3.15 – 3.06 (m, 2H), 3.06 – 2.98 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 190.4,
162.9, 155.0, 148.3, 138.1, 133.9, 133.6, 133.2, 132.2, 132.0, 129.3, 128.9, 128.6, 126.8, 126.5, 126.2, 125.2,
23

125.0, 124.8, 120.9, 120.9, 117.8, 106.8, 34.1, 26.5. ESI-HRMS: calcd for C24H18N2O [M+H]+: 351.1492;
found: 351.1498. HPLC purity: 98.5%.
5.1.5.14. (E)-9-amino-2-((E)-3-phenylallylidene)-3,4-dihydroacridin-1(2H)-one (31)
Compound 2 was reacted with cinnamaldehyde according to general procedure A to afford 31 as yellow
powder in 60% yield. Mp: 240.0-242.3 ºC. 1H NMR (400 MHz, DMSO) δ 10.25 (s, 1H), 8.49 (s, 1H), 8.36 (d,
J = 8.2 Hz, 1H), 7.71 (d, J = 3.5 Hz, 2H), 7.67 (d, J = 7.5 Hz, 2H), 7.48 – 7.36 (m, 5H), 7.35 – 7.27 (m, 1H),
7.13 (d, J = 13.3 Hz, 1H), 3.15 – 2.94 (m, 4H). 13C NMR (101 MHz, DMSO) δ 189.2, 162.8, 155.7, 148.2,
140.4, 137.1, 136.2, 134.6, 132.4, 129.3, 128.9, 127.8, 124.9, 124.5, 123.8, 118.6, 106.3, 33.5, 24.7.
ESI-HRMS: calcd for C22H18N2O [M+H]+: 327.1492; found: 327.1485. HPLC purity: 95.8%.
5.1.6. General procedure B: Preparation of compounds 18-23
To a mixture of compound 3 (197.2 mg, 1 mmol), aromatic aldehyde (1.1 mmol) and 10 mL of ethanol,
400 μL of 10% NaOH aqueous solution was added slowly at room temperature, and the resulting mixture was
stirred under room temperature to 50 ºC for 10 to 150 min. After completion of the reaction, the reaction
mixture was cooled to room temperature, and poured into ice water (50 mL). The precipitate was then
separated from the solvent through filtration, washed with ethanol, and purified by using flash column
chromatography or recrystallization from ethanol to give the desired products.
5.1.6.1. (E)-2-benzylidene-3,4-dihydroacridin-1(2H)-one (18)
Compound 3 was reacted with benzaldehyde according to general procedure B under 50 ºC for 60 min to
afford 18 as yellow powder in 83% yield. Mp: 134.9 ºC. 1H NMR (400 MHz, CDCl3) δ 8.95 (s, 1H), 8.07 (d, J
= 8.5 Hz, 1H), 7.98 (d, J = 8.6 Hz, 2H), 7.82 (t, J = 7.6 Hz, 1H), 7.57 (t, J = 7.5 Hz, 1H), 7.50 (d, J = 7.4 Hz,
2H), 7.45 (t, J = 7.4 Hz, 2H), 7.39 (t, 1H), 3.40 – 3.24 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 187.6, 160.8,
24

149.5, 138.2, 138.0, 135.5, 134.6, 132.3, 130.1, 129.7, 129.0, 128.6, 128.6, 127.2, 127.1, 126.7, 32.7, 26.1.
ESI-HRMS: calcd for C20H15NO [M+H]+: 286.1226; found: 286.1230. HPLC purity: 99.8%.
5.1.6.2. (E)-2-(2-fluorobenzylidene)-3,4-dihydroacridin-1(2H)-one (19)
Compound 3 was reacted with 2-fluorobenzaldehyde according to general procedure B under room
temperature for 60 min to afford 19 as yellow powder in 48% yield. Mp: 146.3-147.0 ºC. 1H NMR (400 MHz,
CDCl3) δ 8.97 (s, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 9.3 Hz, 2H), 7.82 (t, J = 7.7 Hz, 1H), 7.58 (t, J =
7.5 Hz, 1H), 7.45 – 7.33 (m, 2H), 7.21 (t, J = 7.5 Hz, 1H), 7.16 (t, J = 9.2 Hz, 1H), 3.32 (t, J = 6.5 Hz, 2H),
3.16 (t, J = 6.4 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 187.2, 160.9, 160.8 (d, J = 250.8 Hz), 149.6, 138.2,
136.8, 132.4, 130.9, 130.8, 129.7, 128.7, 127.2, 126.9, 126.8, 124.0 (d, J = 3.5 Hz), 123.5 (d, J = 14.1 Hz),
116.0 (d, J = 21.9 Hz), 32.7, 26.5 (d, J = 2.0 Hz). ESI-HRMS: calcd for C20H14NOF [M+H]+: 304.1132; found:
304.1127. HPLC purity: 96.6%.
5.1.6.3. (E)-2-(3-fluorobenzylidene)-3,4-dihydroacridin-1(2H)-one (20)
Compound 3 was reacted with 3-fluorobenzaldehyde according to general procedure B under room
temperature for 60 min to afford 20 as yellow powder in 41% yield. Mp: 130.9-134.6 ºC. 1H NMR (400 MHz,
CDCl3) δ 8.95 (s, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.91 (s, 1H), 7.83 (t, J = 7.7 Hz, 1H),
7.58 (t, J = 7.5 Hz, 1H), 7.46 – 7.37 (m, 1H), 7.26 (d, J = 6.0 Hz, 1H), 7.18 (d, J = 9.7 Hz, 1H), 7.09 (t, J = 9.1
Hz, 1H), 3.36 – 3.29 (m, 2H), 3.29 – 3.23 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 186.3, 161.7 (d, J = 246.7
Hz), 159.6, 148.5, 1371, 136.5 (d, J = 7.8 Hz), 135.5 (d, J = 2.2 Hz), 134.6, 131.4, 129.1 (d, J = 8.3 Hz), 128.7,
127.6, 126.1, 125.9, 125.8, 124.8 (d, J = 2.8 Hz), 115.5 (d, J = 21.8 Hz), 114.9 (d, J = 21.2 Hz), 31.5, 25.0.
ESI-HRMS: calcd for C20H14NOF [M+H]+: 304.1132; found: 304.1125. HPLC purity: 95.8%.
25

5.1.6.4. (E)-2-(2-chlorobenzylidene)-3,4-dihydroacridin-1(2H)-one (21)
Compound 3 was reacted with 2-chlorobenzaldehyde according to general procedure B under room
1
temperature for 10 min to afford 21 as white powder in 46% yield. Mp: 154.0-155.3 ºC. H NMR (400 MHz,
CDCl3) δ 8.98 (s, 1H), 8.06 (d, J = 8.5 Hz, 1H), 8.04 (s, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.83 (t, J = 7.7 Hz, 1H),
7.58 (t, J = 7.5 Hz, 1H), 7.50 – 7.46 (m, 1H), 7.40 – 7.30 (m, 3H), 3.35 – 3.28 (m, 2H), 3.17 – 3.10 (m, 2H).
13C NMR (101 MHz, CDCl3) δ 186.2, 159.9, 148.6, 137.1, 135.3, 134.0, 133.8, 133.1, 131.4, 129.4, 128.9,
128.9, 128.7, 127.6, 126.2, 125.9, 125.7, 125.5, 31.8, 25.2. ESI-HRMS: calcd for C20H14NOCl [M+H]+:
320.0837; found: 320.0832. HPLC purity: 97.4%.
5.1.6.5. (E)-2-(3-chlorobenzylidene)-3,4-dihydroacridin-1(2H)-one (22)
Compound 3 was reacted with 3-chlorobenzaldehyde according to general procedure B under room
1
temperature for 10 min to afford 22 as white powder in 35% yield. Mp: 134.2-136.1 ºC. H NMR (400 MHz,
CDCl3) δ 8.95 (s, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.89 (s, 1H), 7.85 – 7.80 (m, 1H),
13
7.63 – 7.54 (m, 1H), 7.46 (s, 1H), 7.40 – 7.33 (m, 3H), 3.36 – 3.29 (m, 2H), 3.29 – 3.22 (m, 2H). C NMR
(101 MHz, CDCl3) δ 186.3, 159.6, 148.5, 137.1, 136.2, 135.4, 134.7, 133.5, 131.4, 128.8, 128.7, 128.7, 127.9,
127.6, 127.1, 126.1, 125.8, 125.8, 31.5, 25.0. ESI-HRMS: calcd for C20H14NOCl [M+H]+: 320.0837; found:
320.0835. HPLC purity: 98.9%.
5.1.6.6. (E)-2-(3-methoxybenzylidene)-3,4-dihydroacridin-1(2H)-one (23)
Compound 3 was reacted with 4-nitrobenzaldehyde according to general procedure B under 50 ºC for 10 min
to afford 23 as yellow powder in 60% yield. Mp: 227.1-229.0 ºC. 1H NMR (400 MHz, CDCl3) δ 8.97 (s, 1H),
8.31 (d, J = 8.7 Hz, 2H), 8.08 (d, J = 8.5 Hz, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.96 (s, 1H), 7.85 (t, J = 7.3 Hz,
13
1H), 7.68 – 7.57 (m, 3H), 3.38 – 3.31 (m, 2H), 3.30 – 3.23 (m, 2H). C NMR (101 MHz, CDCl3) δ 186.0,
26

159.3, 148.6, 146.5, 140.9, 137.4, 136.6, 134.0, 131.7, 129.6, 128.8, 127.6, 126.1, 126.0, 125.6, 122.8, 31.4,
25.2. ESI-HRMS: calcd for C20H14N2O3 [M+H]+: 331.1077; found: 331.1069. HPLC purity: 96.2%.
5.1.7. Synthesis of (E)-2-benzylidene-9-methyl-3,4-dihydroacridin-1(2H)-one (24)
To a mixture of compound 4 (100.0 mg, 0.5 mmol), benzaldehyde (104 μL, 1 mmol) and 6 mL of ethanol, 200
μL of 10% NaOH aqueous solution was added slowly at 0 ºC, and the resulting mixture was reflux for 48 h.
The reaction mixture was cooled to room temperature, and poured into ice water (50 mL). The precipitate was
then separated from the solvent through filtration, washed with ethanol, and purified by using flash column
chromatography or recrystallization from ethanol to give 24 as green powder in 11% yield. Mp > 200 ºC. 1H
NMR (400 MHz, CDCl3) δ 8.22 (d, J = 8.5 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.94 (s, 1H), 7.78 (t, J = 7.7 Hz,
1H), 7.59 (t, J =7.2 Hz, 1H), 7.50 (d, J = 7.6 Hz, 2H), 7.44 (t, J = 7.6 Hz, 2H), 7.38 (t, J = 7.2 Hz, 1H), 3.26 –
3.21 (m, 2H), 3.21 – 3.16 (m, 2H), 3.06 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 189.6, 161.0, 149.7, 148.0,
137.2, 136.4, 135.8, 131.5, 130.2, 129.4, 129.0, 128.7, 128.3, 126.6, 126.5, 125.6, 33.8, 26.1, 16.1.
ESI-HRMS: calcd for C21H17NO [M+H]+: 300.1383; found: 300.1382. HPLC purity: 99.4%.
5.1.8. Synthesis of 9-amino-2-benzyl-3,4-dihydroacridin-1(2H)-one (32)
In 5 mL of ethanol was suspended 5 (300.0 mg, 1 mmol) and Pd/C (60 mg). The reaction mixture was stirred
under room temperature for 96 h. After completion of the reaction, the reaction mixture was filtered to remove
Pd/C. The solvent was removed under pressure and the crude product thus obtained was purified by column
chromatography with DCM/methanol: 100/1 to afford 32 as yellow powder in 41% yield. Mp: 178.3-179.1 ºC.
1H NMR (400 MHz, CDCl3) δ 10.08 (s, 1H), 8.43 (s, 1H), 8.35 (d, J = 8.3 Hz, 1H), 7.74 – 7.64 (m, 2H), 7.47
– 7.39 (m, 1H), 7.34 – 7.23 (m, 4H), 7.20 (t, J = 7.0 Hz, 1H), 3.38 – 3.30 (m, 1H), 3.06 – 2.81 (m, 3H), 2.72 –
2.62 (m, 1H), 1.98 – 1.86 (m, 1H), 1.75 – 1.59 (m, 1H). 13C NMR (101 MHz, DMSO) δ 201.9, 163.5, 155.4,
27

148.4, 140.5, 132.3, 129.6, 128.8, 128.8, 126.4, 124.7, 123.8, 118.4, 105.5, 49.2, 36.0, 33.1, 26.0. ESI-HRMS:
calcd for C20H18N2O [M+H]+: 303.1492; found: 303.1486. HPLC purity: 95.9%.
5.1.9. General procedure C: Preparation of compounds 25-30
To a 15 mL sealed tube was added compound 5, 7, 9, 11, 16 or 17 (0.3 mmol), 2 mL of acetone and 0.3
mL of iodomethane. The reaction mixture was stirred under 80 ºC for 48 h. After completion of the reaction,
the product was filtered off and washed with diethyl ether, and the crude product thus obtained was purified
by column chromatography with DCM/methanol: 20/1 to give the desired products.
5.1.9.1. (E)-9-amino-2-benzylidene-10-methyl-1-oxo-1,2,3,4-tetrahydroacridin-10-ium iodide (25)
Compound 5 was reacted according to general procedure C to afford 25 as yellow powder in 18% yield. Mp:
194.6-198.8 ºC. 1H NMR (400 MHz, DMSO) δ 11.18 (s, 1H), 10.09 (s, 1H), 8.73 (d, J = 8.2 Hz, 1H), 8.27 (d,
J = 8.7 Hz, 1H), 8.12 (t, J = 7.9 Hz, 1H), 7.84 (t, J = 7.6 Hz, 1H), 7.78 (s, 1H), 7.58 (d, J = 7.3 Hz, 2H), 7.52
(t, J = 7.3 Hz, 2H), 7.46 (t, J = 7.1 Hz, 1H), 4.07 (s, 3H), 3.42 (t, J = 6.5 Hz, 2H), 3.22 – 3.14 (m, 2H). 13C
NMR (101 MHz, DMSO) δ 188.3, 164.1, 157.7, 139.7, 136.8, 136.2, 135.2, 133.6, 130.5, 129.8, 129.3, 127.9,
125.5, 119.7, 118.4, 106.9, 38.2, 28.5, 24.4. ESI-HRMS: calcd for C21H18N2O [M+H]+: 315.1492; found:
315.1486. HPLC purity: 95.2%.
5.1.9.2. (E)-9-amino-2-(3-fluorobenzylidene)-10-methyl-1-oxo-1,2,3,4-tetrahydroacridin-10-ium iodide (26)
Compound 7 was reacted according to general procedure C to afford 26 as incarnadine powder in 73% yield.
1
Mp: 200.0-204.0 ºC. H NMR (400 MHz, DMSO) δ 11.17 (s, 1H), 10.13 (s, 1H), 8.73 (d, J = 6.9 Hz, 1H),
8.28 (d, J = 7.6 Hz, 1H), 8.13 (s, 1H), 7.85 (s, 1H), 7.74 (s, 1H), 7.55 (s, 1H), 7.44 (d, J = 10.2 Hz, 2H), 7.31
(s, 1H), 4.07 (s, 3H), 3.42 (s, 2H), 3.17 (s, 2H). 13C NMR (101 MHz, DMSO) δ 188.1, 164.2, 162.6 (d, J =
244.2 Hz), 157.7, 139.7, 137.6 (d, J = 8.4 Hz), 136.2, 135.3, 134.8, 131.2 (d, J = 8.8 Hz), 127.9, 126.6 (d, J =
28

2.2 Hz), 125.6, 119. 7, 118.4, 116.9 (d, J = 21.8 Hz), 116.5 (d, J = 21.1 Hz), 106.8, 38.2, 28.5, 24.3.
ESI-HRMS: calcd for C21H17N2OF [M+H]+: 333.1398; found: 333.1394. HPLC purity: 98.2%.
5.1.9.3. (E)-9-amino-2-(3-chlorobenzylidene)-10-methyl-1-oxo-1,2,3,4-tetrahydroacridin-10-ium iodide (27)
Compound 9 was reacted according to general procedure C to afford 27 as pink powder in 68% yield. Mp:
208.7-208.9 ºC. 1H NMR (400 MHz, DMSO) δ 11.16 (s, 1H), 10.12 (s, 1H), 8.73 (d, J = 8.3 Hz, 1H), 8.27 (d,
J = 8.8 Hz, 1H), 8.12 (t, J = 7.8 Hz, 1H), 7.84 (t, J = 7.6 Hz, 1H), 7.73 (s, 1H), 7.64 (s, 1H), 7.54 (s, 3H), 4.06
(s, 3H), 3.42 (t, J = 6.3 Hz, 2H), 3.16 (d, J = 5.8 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 188.1, 164.2, 157.8,
139.7, 137.4, 136.2, 135.1, 135.0, 133.9, 131.1, 129.9, 129.4, 129.0, 127.9, 125.6, 119.7, 118.4, 106.8, 38.2,
28.5, 24.4. ESI-HRMS: calcd for C21H17N2OCl [M+H]+: 349.1102; found: 349.1106. HPLC purity: 97.0%.
5.1.9.4. (E)-9-amino-10-methyl-2-(3-nitrobenzylidene)-1-oxo-1,2,3,4-tetrahydroacridin-10-ium iodide (28)
Compound 11 was reacted according to general procedure C to afford 28 as orange powder in 66% yield. Mp >
1
300 ºC. H NMR (400 MHz, DMSO) δ 11.17 (s, 1H), 10.15 (s, 1H), 8.74 (d, J = 8.2 Hz, 1H), 8.37 (s, 1H),
8.29 (d, J = 8.7 Hz, 2H), 8.13 (t, J = 7.8 Hz, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.83 (dt, J = 16.3, 8.2 Hz, 3H),
4.07 (s, 3H), 3.44 (t, J = 6.4 Hz, 2H), 3.19 (t, J = 5.8 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 187.9, 164.2,
157.8, 148.5, 139.7, 136.9, 136.7, 136.3, 136.0, 134.2, 130.8, 128.0, 125.6, 124.7, 124.1, 119.7, 118.4, 106.78,
38.3, 28.5, 24.3. ESI-HRMS: calcd for C21H17N3O3 [M+H]+: 360.1343; found: 360.1339. HPLC purity:
98.7%.
5.1.9.5. (E)-9-amino-10-methyl-1-oxo-2-(thiophen-2-ylmethylene)-1,2,3,4-tetrahydroacridin-10-ium
iodide
(29)
Compound 16 was reacted according to general procedure C to afford 29 as orange powder in 88% yield. Mp >
300 ºC.1H NMR (400 MHz, DMSO) δ 11.16 (s, 1H), 10.06 (s, 1H), 8.71 (d, J = 8.1 Hz, 1H), 8.28 (d, J = 8.8
29