Molecular design, synthesis and biological research of novel pyridyl acridones as potent DNA-binding and apoptosis-inducing agents

Article abstract of DOI:10.1016/j.ejmech.2015.02.003

A series of novel pyridyl acridone derivatives comprised of a pseudo-five-cyclic system to extend the π-conjugated acridone chromophore, were designed and synthesized as potent DNA binding antitumor compounds. Most synthesized compounds displayed good act

Full text of DOI:10.1016/j.ejmech.2015.02.003

Accepted Manuscript
Molecular design, synthesis and biological research of novel pyridyl acridones as
potent DNA-binding and apoptosis-inducing agents
Bin Zhang, Kang Chen, Ning Wang, Chunmei Gao, Qinsheng Sun, Lulu Li, Yuzong
Chen, Chunyan Tan, Hongxia Liu, Yuyang Jiang
PII:
S0223-5234(15)00088-4
10.1016/j.ejmech.2015.02.003
EJMECH 7684
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 August 2014
Revised Date: 19 December 2014
Accepted Date: 5 February 2015
Please cite this article as: B. Zhang, K. Chen, N. Wang, C. Gao Q. Sun, L. Li, Y. Chen, C. Tan, H. Liu,
Y. Jiang Molecular design, synthesis and biological research of novel pyridyl acridones as potent DNA-
binding and apoptosis-inducing agents, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.02.003.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Molecular design, synthesis and biological research of novel pyridyl acridones as
DNA-binding and apoptosis-inducing agents
A series of novel pyridyl acridone derivatives comprised of a pseudo-five-cyclic
system to extend the π-conjugated acridone chromophore were designed and
synthesized as potent DNA binding and apoptosis-inducing agents.
IC50 (K562) = 0.46 µM

ACCEPTED MANUSCRIPT
Molecular design, synthesis and biological research of novel pyridyl acridones as
potent DNA-binding and apoptosis-inducing agents
a,b
a,b
a,b
b,c,*
b
b
Bin Zhang , Kang Chen , Ning Wang , Chunmei Gao , Qinsheng Sun , Lulu Li ,
d
b,c
b
b,c,e,*
Yuzong Chen , Chunyan Tan , Hongxia Liu , Yuyang Jiang
a
Tsinghua University, Department of Chemistry, Beijing 100084, PR China
b
The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key
Laboratory of Chemical Biology, Shenzhen 518055, P. R. China
c
Shenzhen Anti-Tumor Drug Development Engineering Laboratory, the Graduate
School at Shenzhen, Tsinghua University, Shenzhen 518055, P. R. China
d
Bioinformatics and Drug Design Group, Department of Pharmacy, Centre for
Computational Science and Engineering, 117543, Singapore
e
School of Medicine, Tsinghua University, Beijing 100084, PR China
*
Corresponding author. Tel.: +86 755 2603 2094; fax: +86 755 2603 2094;
Email address: jiangyy@sz.tsinghua.edu.cn; chunmeigao@sz.tsinghua.edu.cn
1

ACCEPTED MANUSCRIPT
Abstract
A series of novel pyridyl acridone derivatives comprised of a pseudo-five-cyclic
system to extend the π-conjugated acridone chromophore, were designed and
synthesized as potent DNA binding antitumor compounds. Most synthesized
compounds displayed good activity against human leukemia K562 cells in MTT tests,
with compound 6d exhibiting the highest activity with IC50 value at 0.46 ꢀM.
Moreover, 6d showed potent activities against solid tumor cell lines (0.16-3.79 ꢀM).
Several experimental studies demonstrated that the antitumor mode of action of
compound 6d involves DNA intercalation, topoisomerase I inhibition, and apoptosis
induction through the mitochondrial pathway. In summary, compound 6d represents a
novel and promising lead structure for the development of new potent anticancer
DNA-binding agents.
Keywords: Acridone; pyridyl; molecular docking; DNA; topoisomerase I; antitumor.
2

ACCEPTED MANUSCRIPT
1. Introduction
Cancer is a challenging disease with the second highest mortality rate[1]. While
targeted anticancer drugs have been developed and clinically used, chemotherapy
remains an important first-line treatment option for various cancers[2-5]. Therefore
efforts have been continuously made for the development of more potent and less
toxic anticancer chemotherapy drugs[6-11]. As part of the efforts in the discovery of
potent antitumor agents, we have developed several series of new compounds such as
aminopyrrolidine[12], tricyclic thiophene analogues[13] and acridine/acridone
derivatives[14-19] with good antitumor activity. The polycyclic acridine/acridone
derivatives have π-conjugated planar structure, which makes them useful in many
applications[20-22], such as pigments and dyestuffs, antibacterial agents, antipsoriatic
agents[23, 24], anticancer drugs[20, 25, 26]. Acridine/acridones can intercalate into
the double stranded DNA base pairs and then inhibit the activity of DNA-related
enzymes such as topoisomerases[27, 28] and telomerases[29, 30]. Recently, a number
of acridine/acridone drugs have entered clinical trials or preclinical studies[1, 20],
such as Imidazoacridone (C-1311), Pyrazoloacridine (PZA), N-[2-(dimethylamino)-
ethyl]-acridine-4-carboxamide (DACA), 9-[4-(N,N-dimethylamino)phenylamino]-
3,6-bis(3-pyrrolodino-propionamido)acridine (BRACO-19) and Amsacrine (m-AMSA)
(Figure 1), which suggests the usefulness of the relevant molecular scaffolds for
developing effective anticancer chemotherapy drugs and indicates the possibility of
further exploring these molecular scaffolds for developing agents with more improved
properties.
3

ACCEPTED MANUSCRIPT
Acridone, which is shown in Figure 2, has three planar rings. Earlier studies
have suggested that, by adding rings, the resulting acridine/acridone derivatives such
as C-1311[31] and PZA[32] may have enhanced DNA-binding capability. Because of
the difficulties in adding more rings to acridine/acridone, it is desirable to rationally
introduce ring additions that likely increase the activities. By analyzing the structure
of acridone, the introduction of amino group at 1 or 8 position and carboxy group at 4
or 6 position may enable the formation of intramolecular hydrogen bonds (IMHBs)
with the C=O and N-H groups on the acridone ring respectively. These two IMHBs
could form a pseudo-five-cyclic system, which might extend the π-conjugated
acridone chromophore and improve the DNA binding ability.
Moreover, the carboxamide group with terminal amino substituents on the
acridine ring are beneficial for the DNA binding, as found in molecules such as
DACA[33, 34], BRACO-19[35]. We therefore introduced this group to acridones to
improve the antitumor activity. We further noted that m-AMSA achieves potent
anticancer activity by the interaction with DNA and topoisomerase and the
substitution pattern of its 9-anilino group participates in the interaction with
topoisomerase. Based on the structure of m-AMSA, we hypothesized that anilino
group at 1 or 8 position may be beneficial for the interaction with topoisomerase.
Molecular docking in our study (Figure 3) indicated that acridone derivatives with
pyridylalkyl amino group have better DNA-topoisomerase I (DNA-topo I) binding
affinity than those with anilino groups (Molecular docking studies of the
representative acridone compound with anilino group, compound A with topo I-DNA
4

ACCEPTED MANUSCRIPT
complex model, Figure 1S, supporting information).
Therefore, two series of acridone derivatives, I and II (Figure 2) may be
introduced. As compound I have two big substituents on the two sides of acridone
ring (Molecular docking studies of the representative compound B, Figure 2S,
supporting information), which are not good for DNA-topo I interaction, compound II
was selected to be synthesized and evaluated for their antitumor activity. Denny’s
group[33] revealed that the substitution pattern of C5 position on acridine ring played
an important role in the antitumor activity. Therefore, a series of groups including
electron-donating and electron-withdrawing groups were further introduced on C5
position on the acridone scaffold to study the structure-activity relationship (SAR).
Based on the above described rational analysis, we designed and synthesized a
series of novel pyridyl acridone derivatives 6a-w and 7a-c as our target compounds
based on the following attributes: (a) the presence of 4-carboxamide side chain, (b)
the presence of 5-substituted group, and (c) introducing pyridinylalkyl group on the C
ring on the acridone scaffold. The antiproliferative effects of the pyridyl acridone
derivatives on K562 cells were evaluated and the SAR was discussed. The most active
compound 6d was selected to investigate the mode of action. The results showed that
the synthesized pyridyl acridones exhibits broad antitumor activity, interacts with
DNA and inhibits topo I activity and induces apoptosis through mitochondrial
pathway.
2
. Results and discussion
.1. Chemistry
2
5

ACCEPTED MANUSCRIPT
The synthesis of compounds 6a-w and 7a-c is shown in Scheme 1. First,
compounds 3a-e were obtained from the Ullmann reaction of 2,4-dichlorobenzoic
acid 2 with anthranilic acid 1a or its derivatives 1b-e in DMF using Cu as the
catalyst[16, 17]. Subsequent Friedel-Crafts acylation was carried out in concentrated
sulfuric acid at 80 °C for 5 h to give acridone-4-carboxylic acid derivatives 4a-e in
high yields[36], which were then reacted with the corresponding primary aliphatic
amines using N,N′-carbonyldiimidazole (CDI) as the condensation agent[37] to afford
the intermediates acridone-4-carboxamides 5a-j. The desired compounds 6a-w and
7a-c were produced by the nucleophilic substitution between the corresponding
acridone-4-carboxamides and various picolylamines or 4-pyridineethanamine under
1
13
argon atmosphere. Structures were confirmed by H NMR, C NMR and high
resolution mass spectral data.
2.2. In vitro cytotoxicity
As most acridone and acridine derivatives showed better antitumor activity
against leukemia tumor cells than solid tumor cells[20], the vitro cytotoxicity of 26
desired pyridyl acridone derivatives against human leukemia K562 cells was first
assayed by MTT reduction method. Adriamycin and cisplatin were used as the
positive controls. The structures and bioactivity results were shown in Table 1. Most
of these compounds had better antitumor activity than cisplatin. Compound 6d
showed the highest activity with IC50 value at 0.46 ꢀM, which was comparable to
adriamycin.
The structure-activity analysis of the data in Table 1 indicated that the
6

ACCEPTED MANUSCRIPT
substitution of the acridone ring played an important role on the antitumor activity.
First, the length of alkyl chains between the N,N-dimethylamino group and
4-carboxamide group had great influence on the antitumor activity. Compounds
containing two methylene units displayed better antiproliferative activity than those
containing three methylene units, suggested by the IC50 values of 6b vs. 6e, 6c vs. 6f,
6
d vs. 6g, 6k vs. 6o. However, when four methylene units existed, the cytotoxicity
increased, for example, compound 6q displayed approximately 2-fold more active
than 6u. In addition, R group on the 4-carboxamide side chain had a great effect on
2
the antitumor activity. Compounds with N,N-dimethylamino group displayed better
antitumor activity than those with methoxy group, which can be seen from the IC50
values of 6b and 6h, 6c and 6i. Most compounds with methoxy group displayed no
cytotoxicity (IC50 > 50 µM).
From the biological data of these pyridyl acridone derivatives shown in Table 1,
most of them had a gratifying antiproliferative activity against K562 cells. These
suggested that the position of nitrogen atom on pyridine ring (2-pyridyl, 3-pyridyl,
and 4-pyridyl) may have little important influence on the cytotoxicity. In order to
evaluate whether the number of pyridyl group has an effect on antitumor activity, we
also introduced 2,2′-bispyridyl group to the acridone scaffold (Table 2). However, the
results led reduction of antitumor activity compared to mono-pyridyl acridone
derivatives with N,N-dimethylamino group. For instance, 6k had significant antitumor
activity with IC50 of 2.28 µM, while the IC50 of 7b was 13.90 µM. Moreover, the
length between the pyridyl group and 1-amino group on the acridone scaffold also had
7

ACCEPTED MANUSCRIPT
an important influence on the antiproliferative activity, which can be seen from the the
IC50 values of 6n (m = 2) and 6m (m = 1).
The substitution on C5 position of acridone or acridine ring reportedly plays an
important role on the antitumor activity[33]. We therefore synthesized several
compounds containing electron-donating or electron-withdrawing groups on C5
position. The testing results suggested that the introduction of methyl group on C5
position of acridone significantly increased the cytotoxicity. Compounds with methyl
group (6d) showed more than 14-fold better antitumor activity than 6a. When the
more electron-donating group methoxyl was introduced, 6m resulted in reduced
antiproliferative activity compared with 6d. Introduction of electron-withdrawing
group in the case of 6v (5-trifluoromethyl) and 6w (5,7-dichloro) also produced less
cytotoxicity compared with 6d. The results indicated that electron-negativity and
steric effect on C5 position of acridone may change the cytotoxic profile.
In order to study whether the highly potent compound 6d have broad antitumor
activities against solid tumor cells, we tested it on various cancer cell lines including
NCI-H520, U251, A375, A172, Hela, CNE-2, U118-MG, HepG2 and MCF-7 cells
with the results shown in Table 3. Our testing results showed that 6d also have potent
activities against these solid tumor cells. For example, the IC50 values against A375
cells and HepG2 cells were 0.16 ꢀM and 0.32 ꢀM, respectively. Therefore, 6d was
selected to study the antitumor mechanism.
2.3. Molecular modeling
Molecular docking studies of the representative compound 6d with topo I-DNA
8

ACCEPTED MANUSCRIPT
complex model (PDB ID: 1K4T) were conducted using the Discovery Studio
3.1.1/Libdock protocol. The LibDockScore is 194.834. As shown in Figure 3a and
Figure 3b, pi-pi interactions were formed between the acridone ring (A-ring, B-ring
and C-ring) of 6d and DNA base pairs (DT10, TGP11, DC112 and DA113). Moreover,
specific interactions of 6d with topo I included: (a) three hydrogen bonds, one formed
between the nitrogen atom (N) at the pyridyl ring and Arg488 (N···H-NH), with the
distance of 3.0 Å; another formed between C=O at B-ring of acridone and NH
Arg364 (C=O···H-NH), with the distance of 2.9 Å; the other formed between N(CH
on the 4-carboxamide side chain and NH of Lys751 (N···H-NH), with the distance of
2
of
3 2
)
2
3.1 Å; (b) three pi-pi interactions forming between the three rings at acridone
backbone (A-ring, B-ring and C-ring) and Asp533; (c) two cation-pi interaction, one
existing between the pyridyl ring and an ammonium cation of Arg364, the other
2
existing between C-ring of acridone and ε-NH of Lys751. Figure 3c showed
interactions between the receptor and the ligand on 2D diagram. These analyses
suggest that 6d may intercalate into DNA and bind with topo I to contribute or partly
contribute to its observed anti-tumor activity.
2.4. UV-visible spectral absorbance
UV-visible spectral absorbance has been universally employed to examine the
binding mode of DNA with small molecules [17, 38-40]. In this study, the interactions
of 6d with Calf thymus DNA (ct DNA) were firstly investigated using absorption
spectroscopy. The absorption spectra of 6d in the absence and presence of ct DNA at
constant concentration are given in Figure 4a. In the absence of ct DNA, 6d had
9

ACCEPTED MANUSCRIPT
strong absorbance peak at about 425 nm. Increasing ct DNA concentration, the
absorption bands of 6d displayed clear hypochromism with a concomitant minor red
shift. These phenomena indicated that 6d probably interact with DNA by intercalation
mode, involving a stacking interaction between acridone ring of 6d and DNA base
pairs[38].
In order to further elucidate the binding capability of 6d, the binding constant K
b
was determined by monitoring the changes in absorbance at 425 nm with increasing
concentrations of ct DNA. The equation (1) was used to calculate the binding constant
K [41] (Figure 4b). In this equation, [DNA] represents the ct DNA concentrations in
b
base pair, while ε
band at a given ct DNA concentration, the complex free in solution and the complex
when fully bound to DNA, respectively. The K is calculated by the gradient ratio to
the intercept. The value of K was derived to be 5.1 × 10 M .
a f b
, ε and ε are the extinction coefficient of the compound absorption
b
4
-1
b
[
DNA] / (ε
a
- ε
f
) = [DNA] / (ε
b
- ε
f
) + 1 / K
b
(ε
b
- ε
f
)
(1)
2.5. Fluorescence emission spectra
Fluorescence spectral technique is an effective method to study the interaction of
organic compounds with DNA[17, 38, 42]. The binding mode of DNA with
compound 6d was further investigated using fluorescence emission spectroscopy.
Fixed amount of 6d was titrated with increasing amounts of ct DNA. The results can
be seen in Figure 5a. Enhanced fluorescence intensity was obtained with increasing
10

ACCEPTED MANUSCRIPT
ct DNA concentration indicating a deep intercalation of 6d with DNA. This result
may suggest efficient protection of 6d from water by the hydrophobic environment
inside the DNA helix[41]. In addition, the fluorescence intensity reached saturation
gradually with increasing ct DNA concentration, and the terminal concentration of
DNA was ten-fold higher than 6d, the increasing fluorescence trend can be seen in
Figure 5b.
2.6. DNA-EB displacements
The intercalation mode of 6d binding to DNA was further investigated using the
competitive binding displacement experiment[41]. Ethidium Bromide (EB) is a
conjugate planar molecule, and its fluorescence intensity is very weak in the absence
of duplex DNA. However, the fluorescence intensity increases greatly when EB
intercalates into the base pairs of double-stranded DNA. If there has a compound that
can compete with EB for DNA-binding sites, the fluorescence quenching of DNA-EB
system is observed. In order to investigate whether 6d can intercalate into DNA, the
competitive binding displacement experiments were carried out, which were shown in
Figure 6a. The emission intensity of DNA-EB system at about 600 nm decreased
distinctly when the concentration of 6d increased and an isoactinic point appeared at
about 500 nm. The phenomenon suggested that 6d can intercalate into DNA. To
confirm the binding affinity of 6d with DNA quantitatively, the classical Stern-Volmer
equation (2) was used to calculate the binding constant[17] (Figure 6b).
F
0
/ F = 1 + K
q
[Q]
(2)
11

ACCEPTED MANUSCRIPT
0
In the equation above, F is the fluorescence emission intensity in the absence of
the quencher, while F is the fluorescence emission intensity in the presence of the
quencher and [Q] is the concentration of the quencher. The quenching constant K of
q
4
-1
6
d was 1.0×10 M , which is in accordance with the absorption spectra.
2.7. Viscosity experiments
Viscosity experiment is considered as one of the least ambiguous and the most
critical tests of the binding modes of compounds to DNA[39, 41]. The viscosity of a
DNA solution was sensitive to the addition of compounds bound by intercalation[38].
The relative changes in viscosity were measured using ct DNA with increasing
concentrations of 6d, which were shown in Figure 7. In the presence of increased 6d,
DNA viscosity increased, which further confirmed that 6d could bind to DNA through
intercalation binding mode.
2.8. DNA topo I inhibition assay
Most acridine/acridone derivatives can interact with DNA and inhibit the activity
of topoisomerases, it is of interest to evaluate whether the synthesized acridone
derivatives can also inhibit the activity of topoisomerases. Figure 8 showed the
relative affinity of all synthesized compounds on the relaxtion of plasmid pBR322
DNA mediated by topo I. The results indicated that most of the compounds displayed
good topo I inhibitory activity, however, neither pyridyl acridone compounds with
methoxy group on the 4-carboxamide side chain (e.g. 6h-j and 6r-t) nor bispyridyl
acridone derivatives (7a-c) showed any topo I inhibitory activities, which were in
12

ACCEPTED MANUSCRIPT
accordance with their antitumor activities. All these data suggested that these
compounds may have antiproliferative activity by interacting with DNA and then
inhibiting topo I.
2.9. Apoptosis induced by compound 6d
The above data indicated that 6d may bind with DNA and inhibit topo I activity,
which was expected to subsequently lead to apoptosis in cancer cells. In order to
evaluate this hypothesis, an Annexin-V/PI binding assay was conducted in K562 cells
as shown in Figure 9. The lower right-hand quadrants (R4) displayed the early stage
of apoptotic cells. The upper right-hand quadrants (R2) displayed the late stage of
apoptotic or necrotic cells. K562 cells were treated with 6d at the concentrations of 0,
0.05, 0.25, 0.5, 1 and 2.5 ꢀM for 48 h. As the concentration of 6d increased, the
percentage of early stage apoptotic cells increased from 8.98% to 54.93%. From the
results we conclude that 6d can effectively induce K562 cells apoptosis in a
dose-dependent manner.
Apoptosis can be triggered by either intrinsic mitochondrial or death receptor
pathways. In order to understand which pathway that 6d induced apoptosis, the
activity of cleaved caspase-9, caspase-3 and caspase-7 was first tested. As shown in
Figure 10, 6d at 1 ꢀM for 36 h induced significant activation of cleaved caspase-9,
caspase-3 and caspase-7, suggesting that the mitochondrial pathway was involved in
the 6d-induced apoptosis.
PARP (poly ADP-ribose polymerase) is the substrate of caspases. During
apoptosis, caspases mediated PARP cleavage, which inactivates the enzyme by
13

ACCEPTED MANUSCRIPT
destroying its ability to respond to DNA strand breaks. The results in Figure 10
revealed the generation of the cleaved PARP. Additionally, Bcl-XL, an important
member of Bcl-2 family proteins, plays an important role in extending cellular
survival. Compound 6d at 1 ꢀM effectively decreased the expression of Bcl-XL
protein as shown in Figure 10, which further confirmed that cell death through
apoptosis.
Moreover, the mitochondrial membrane potential (MMP)[43] was tested to
confirm compound 6d induced apoptosis through the mitochondrial pathway. The
K562
cells
were
stained
with
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1)
probe and imaged by fluorescent microscope. JC-1 can aggregate in normal
mitochondria and presents red fluorescence, while it produces increased green
fluorescence when the potential of mitochondrial membrane is low.
Carbonylcyanide-m-chlorophenylhydrazone (CCCP) was used as the positive control.
As shown in Figure 11, the red fluorescence decreased and the obvious green
fluorescence increased with 6d added. From the results above, 6d could induce
apoptosis through mitochondrial pathway.
3. Conclusion
Based on rational analysis and molecular modeling studies, a series of novel
acridone derivatives were synthesized by combination of pyridyl group and the
acridone-4-carboxamide scaffold, most of which showed good cytotoxicity against
K562 cells. In particular, compound 6d showed potent activities against K562 cells
14

ACCEPTED MANUSCRIPT
(0.46 ꢀM) and nine solid tumor cell lines (0.16-3.79 ꢀM). The DNA-binding
properties of 6d have been investigated by spectrophotometric methods, viscosity
measurements and topo I inhibition assay, which demonstrated that 6d interacts with
DNA, inhibits topo I activity and induces apoptosis through the mitochondrial
pathway. Our study showed the promising potential of compound 6d as a potent DNA
binding anticancer lead compound and further optimization works were planned for
developing it and other analogs into effective anticancer agents.
4
4
. Experimental section
.1. Synthesis and characterization
See supporting information for synthetic methods and the preparation of compounds
3
4
a-e, 4a-e and 5a-j.
.1.1. General procedure for compounds 6a-w and 7a-c.
9-oxo-9,10-dihydroacridine-4-carboxamide derivatives (5a-j, 0.28mmol) was
dissolved in various picolylamines (or 4-Pyridineethanamine, 2.80mmol). The
apparatus was flushed with argon and the mixture was stirred at 90 °C until the TLC
showed the disappearance of the starting material. The mixture was cooled to room
2 2
temperature and partitioned between CH Cl (50 mL) and water (50 mL). The organic
layer was worked up to give a residue which was purified by column-chromatography
eluted with EtOAC/EtOH (9:1 v/v) to give pure product.
4.1.1.1.
N-(2-(dimethylamino)ethyl)-9-oxo-1-((pyridin-4-ylmethyl)amino)-9,10-
dihydroacridine-4-carboxamide (6a) yellow solid powder, Yield 10.3%; mp
15

ACCEPTED MANUSCRIPT
1
1
8
7
8
3
83-185℃; H NMR (400 MHz, CDCl ) δ 13.46 (s, 1H), 11.52 (t, J = 5.7 Hz, 1H),
.56 (dd, J = 4.6, 1.4 Hz, 2H), 8.37 (d, J = 7.8 Hz, 1H), 7.64 (dd, J = 11.8, 5.0 Hz, 2H),
.38 (d, J = 8.2 Hz, 1H), 7.33 – 7.28 (m, 2H), 7.25 (s, 1H), 6.98 (s, 1H), 6.03 (d, J =
.9 Hz, 1H), 4.59 (d, J = 6.0 Hz, 2H), 3.52 (dd, J = 10.9, 5.2 Hz, 2H), 2.59 (t, J = 5.8
13
Hz, 2H), 2.31 (s, 6H); C NMR (100 MHz, CDCl
3
) δ 181.10, 169.12, 154.96, 150.19,
1
1
47.39, 144.88, 139.41, 133.60, 133.39, 126.06, 122.01, 121.90, 117.37, 107.14,
+
01.94, 99.74, 57.67, 45.72, 45.05, 36.65; HR-MS(ESI): Calcd for [M+H] 416.2087;
Found: 416.2097.
4.1.1.2. N-(2-(dimethylamino)ethyl)-5-methyl-9-oxo-1-((pyridine-2-ylmethyl)
amino)-9,10-dihydroacridine-4-carboxamide (6b) yellow solid powder, Yield
1
3
5
6.1%; mp 168-170℃; H NMR (400 MHz, DMSO-d ) δ 13.97 (s, 1H), 11.39 (t, J =
6
.3 Hz, 1H), 8.62 (d, J = 4.2 Hz, 1H), 8.41 (t, J = 5.3 Hz, 1H), 8.06 (dd, J = 17.4, 8.4
Hz, 2H), 7.80 (td, J = 7.7, 1.7 Hz, 1H), 7.60 (d, J = 7.0 Hz, 1H), 7.44 (d, J = 7.8 Hz,
1
4
2
1
1
H), 7.33 (dd, J = 6.7, 5.1 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 6.35 (d, J = 9.0 Hz, 1H),
.71 (d, J = 5.4 Hz, 2H), 3.41 (dd, J = 12.5, 6.5 Hz, 2H), 2.53 (s, 3H), 2.49-2.42 (m,
1
3
6
H), 2.22 (s, 6H); C NMR (100 MHz, DMSO-d ) δ 180.28, 169.33, 157.84, 154.40,
49.67, 144.58, 138.46, 137.44, 135.09, 134.16, 125.18, 123.78, 122.93, 122.10,
21.77, 121.54, 106.33, 101.52, 100.46, 58.61, 48.19, 45.69, 37.60, 17.02;
+
HR-MS(ESI): Calcd for [M+H] 430.2243; Found: 430.2242.
4.1.1.3. N-(2-(dimethylamino)ethyl)-5-methyl-9-oxo-1-((pyridin-3-ylmethyl)
16

ACCEPTED MANUSCRIPT
amino)-9,10-dihydroacridine-4-carboxamide (6c) yellow solid powder, Yield
1
3
5
7
6.1%; mp 149-151℃; H NMR (400 MHz, DMSO-d ) δ 13.93 (s, 1H), 11.22 (t, J =
6
.5 Hz, 1H), 8.67 (s, 1H), 8.57 – 8.42 (m, 2H), 8.05 (d, J = 8.6 Hz, 2H), 7.82 (d, J =
.8 Hz, 1H), 7.60 (d, J = 6.9 Hz, 1H), 7.40 (dd, J = 7.6, 4.9 Hz, 1H), 7.19 (t, J = 7.6
Hz, 1H), 6.37 (d, J = 9.0 Hz, 1H), 4.67 (d, J = 5.6 Hz, 2H), 3.47-3.43 (m, 2H),
1
3
2
1
1
4
6
.70-2.60 (m, 2H), 2.52 (s, 3H), 2.32 (s, 6H). C NMR (100 MHz, DMSO-d ) δ
80.43, 169.42, 154.51, 149.40, 148.95, 144.53, 138.45, 135.57, 135.21, 134.56,
34.23, 125.18, 124.19, 123.74, 121.83, 121.47, 106.30, 101.76, 100.35, 58.27, 45.22,
+
3.81, 37.15, 16.95; HR-MS(ESI): Calcd for [M+H] 430.2243; Found: 430.2244.
4.1.1.4. N-(2-(dimethylamino)ethyl)-5-methyl-9-oxo-1-((pyridin-4-ylmethyl)
amino)-9,10-dihydroacridine-4-carboxamide (6d) yellow solid powder, Yield
1
5
9.4%; mp 220-222℃; H NMR (400 MHz, CDCl
3
) δ 13.62 (s, 1H), 11.53 (t, J = 5.7
Hz, 1H), 8.56 (d, J = 5.3 Hz, 2H), 8.25 (d, J = 8.1 Hz, 1H), 7.64 (d, J = 8.9 Hz, 1H),
7
1
2
1
1
1
.50 (d, J = 7.0 Hz, 1H), 7.31 (d, J = 5.2 Hz, 2H), 7.19 (t, J = 7.6 Hz, 1H), 6.99 (s,
H), 6.03 (d, J = 8.9 Hz, 1H), 4.59 (d, J = 6.0 Hz, 2H), 3.52 (dd, J = 10.7, 5.2 Hz, 2H),
1
3
3
.63 (s, 3H), 2.55 (t, J = 5.7 Hz, 2H), 2.28 (s, 6H); C NMR (100 MHz, CDCl ) δ
81.38, 169.23, 154.87, 150.13, 147.50, 144.60, 138.54, 133.79, 133.53, 125.02,
23.81, 121.92, 121.75, 121.60, 106.92, 102.08, 99.71, 57.68, 45.70, 45.09, 36.79,
+
7.14; HR-MS(ESI): Calcd for [M+H] 430.2243; Found: 430.2246.
4.1.1.5. N-(3-(dimethylamino)propyl)-5-methyl-9-oxo-1-((pyridin-2-ylmethyl)
17

ACCEPTED MANUSCRIPT
amino)-9,10-dihydroacridine-4-carboxamide (6e) yellow solid powder, Yield
1
5
6.8%; mp 162-164℃; H NMR (400 MHz, CDCl ) δ 13.85 (s, 1H), 11.61 (t, J = 5.7
3
Hz, 1H), 8.78 (s, 1H), 8.64 (d, J = 4.2 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 7.65 (td, J =
7
.7, 1.7 Hz, 1H), 7.53 (d, J = 8.9 Hz, 1H), 7.49 (d, J = 7.0 Hz, 1H), 7.41 (d, J = 7.9 Hz,
H), 7.20 (dd, J = 8.0 Hz, 1H), 7.16 (t, J = 7.9 Hz, 1H), 6.17 (d, J = 8.9 Hz, 1H), 4.72
1
(d, J = 5.9 Hz, 2H), 3.58 (dd, J = 10.6, 5.7 Hz, 2H), 2.63 (s, 3H), 2.57-2.45 (m, 2H),
1
3
2
1
1
4
3
.33 (s, 6H), 1.79 (dt, J = 11.6, 5.9 Hz, 2H); C NMR (100 MHz, CDCl ) δ 181.35,
69.37, 158.16, 154.86, 149.47, 144.78, 138.64, 136.96, 133.59, 133.26, 125.02,
23.87, 122.24, 121.78, 121.34, 121.09, 106.98, 101.97, 99.94, 59.59, 48.66, 45.41,
+
0.69, 24.83, 17.23; HR-MS(ESI): Calcd for [M+H] 444.2400; Found: 444.2383.
4.1.1.6. N-(3-(dimethylamino)propyl)-5-methyl-9-oxo-1-((pyridin-3-ylmethyl)
amino)-9,10-dihydroacridine-4-carboxamide (6f) yellow solid powder, Yield
1
5
6.2%; mp 143-145℃; H NMR (400 MHz, CDCl
3
) δ 13.80 (s, 1H), 11.43 (s, 1H),
8.71 (s, 1H), 8.66 (s, 1H), 8.53 (d, J = 3.9 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.72 (dd,
J = 15.6, 8.4 Hz, 2H), 7.47 (d, J = 6.9 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 6.14 (d, J =
8
2
1
1
4
.9 Hz, 1H), 4.57 (d, J = 5.5 Hz, 2H), 3.59 (d, J = 5.1 Hz, 2H), 2.79 – 2.65 (m, 2H),
1
3
3
.60 (s, 3H), 2.46 (s, 6H), 1.90 (d, J = 5.2 Hz, 2H); C NMR (100 MHz, CDCl ) δ
81.34, 169.33, 154.71, 149.00, 148.84, 144.74, 138.61, 134.81, 133.71, 133.66,
33.33, 125.05, 123.75, 121.73, 121.43, 106.92, 102.22, 99.58, 59.29, 45.26, 44.31,
+
0.37, 24.80, 17.18; HR-MS(ESI): Calcd for [M+H] 444.2400; Found: 444.2405.
18

ACCEPTED MANUSCRIPT
4.1.1.7. N-(3-(dimethylamino)propyl)-5-methyl-9-oxo-1-((pyridin-4-ylmethyl)
amino)-9,10-dihydroacridine-4-carboxamide (6g) yellow solid powder, Yield
1
3
8
7
5
1
1
1
6.7%; mp 199-201℃; H NMR (400 MHz, CDCl
3
) δ 13.89 (s, 1H), 11.53 (s, 1H),
.85 (s, 1H), 8.57 (d, J = 4.2 Hz, 2H), 8.25 (d, J = 7.6 Hz, 1H), 7.52-7.50 (m, 2H),
.33 (d, J = 3.7 Hz, 2H), 7.18 (t, J = 7.3 Hz, 1H), 6.03 (d, J = 8.8 Hz, 1H), 4.61 (d, J =
.3 Hz, 2H), 3.60-3.50 (m, 2H), 2.64 (s, 3H), 2.55-2.45 (m, 2H), 2.33 (s, 6H),
13
3
.80-1.70 (m, 2H); C NMR (100 MHz, CDCl ) δ 181.44, 169.26, 154.74, 150.21,
47.49, 144.75, 138.66, 133.75, 133.21, 125.13, 123.81, 121.95, 121.74, 121.51,
06.99, 102.47, 99.60, 59.69, 45.70, 45.45, 40.81, 24.75, 17.24; HR-MS(ESI): Calcd
+
for [M+H] 444.2400; Found: 444.2397.
4
9
2
4
.1.1.8. N-(2-methoxyethyl)-5-methyl-9-oxo-1-((pyridin-2-ylmethyl)amino)-
,10-dihydroacridine-4-carboxamide (6h) yellow solid powder, Yield 57.9%; mp
1
06-207℃; H NMR (400 MHz, CDCl
3
) δ 13.49 (s, 1H), 11.65 (s, 1H), 8.65 (d, J =
.2 Hz, 1H), 8.27 (d, J = 8.1 Hz, 1H), 7.66 (td, J = 7.8, 1.5 Hz, 1H), 7.61 (d, J = 8.9
Hz, 1H), 7.50 (d, J = 6.9 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.20 (dt, J = 15.1, 7.5 Hz,
2
4
H), 6.56 (s, 1H), 6.17 (d, J = 8.9 Hz, 1H), 4.73 (d, J = 5.7 Hz, 2H), 3.67 (dd, J = 9.7,
13
.8 Hz, 2H), 3.62-3.53 (m, 2H), 3.41 (s, 3H), 2.62 (s, 3H); C NMR (100 MHz,
CDCl ) δ 181.28, 169.29, 157.95, 155.13, 149.46, 144.68, 138.50, 137.01, 133.68,
3
1
7
4
33.33, 124.92, 123.92, 122.30, 121.85, 121.51, 121.12, 106.92, 101.49, 100.00,
+
1.26, 58.90, 48.63, 39.41, 17.14; HR-MS(ESI): Calcd for [M+H] 417.1927; Found:
17.1927.
19

ACCEPTED MANUSCRIPT
4.1.1.9. N-(2-methoxyethyl)-5-methyl-9-oxo-1-((pyridin-3-ylmethyl)amino)-9,10-
dihydroacridine-4-carboxamide (6i) yellow solid powder, Yield 57.9%; mp
1
1
8
84-185℃; H NMR (400 MHz, CDCl
3
) δ 13.51 (s, 1H), 11.50 (t, J = 5.5 Hz, 1H),
.67 (d, J = 1.7 Hz, 1H), 8.55 (dd, J = 4.8, 1.4 Hz, 1H), 8.23 (d, J = 8.1 Hz, 1H), 7.74
(
d, J = 7.8 Hz, 1H), 7.61 (d, J = 8.9 Hz, 1H), 7.50 (d, J = 7.0 Hz, 1H), 7.31-7.28 (m,
H), 7.19 (t, J = 7.6 Hz, 1H), 6.54 (s, 1H), 6.14 (d, J = 8.9 Hz, 1H), 4.60 (d, J = 5.7
Hz, 2H), 3.67 (dd, J = 9.9, 4.8 Hz, 2H), 3.59 (t, J = 4.8 Hz, 2H), 3.41 (s, 3H), 2.62 (s,
1
1
3
3
1
1
H); C NMR (100 MHz, CDCl ) δ 181.31, 169.21, 154.96, 149.01, 148.90, 144.64,
3
38.49, 134.80, 133.78, 133.58, 133.33, 124.97, 123.81, 123.76, 121.79, 121.63,
06.88, 101.76, 99.65, 71.22, 58.90, 44.34, 39.41, 17.12; HR-MS(ESI): Calcd for
+
[
M+H] 417.1927; Found: 417.1920.
4
9
2
4
1
.1.1.10. N-(2-methoxyethyl)-5-methyl-9-oxo-1-((pyridin-4-ylmethyl)amino)-
,10-dihydroacridine-4-carboxamide (6j) yellow solid powder, Yield 82.7%; mp
1
28-230℃; H NMR (400 MHz, CDCl
3
) δ 13.53 (s, 1H), 11.59 (s, 1H), 8.59 (d, J =
.7 Hz, 2H), 8.27 (d, J = 8.1 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.53 (d, J = 7.0 Hz,
H), 7.33 (d, J = 4.5 Hz, 2H), 7.22 (t, J = 7.6 Hz, 1H), 6.51 (s, 1H), 6.05 (d, J = 8.9
Hz, 1H), 4.62 (d, J = 5.7 Hz, 2H), 3.68 (d, J = 4.7 Hz, 2H), 3.60 (d, J = 4.5 Hz, 2H),
1
3
3
1
1
3
.41 (s, 3H), 2.64 (s, 3H); C NMR (100 MHz, CDCl ) δ 181.39, 169.17, 155.02,
50.17, 147.42, 144.64, 138.52, 133.86, 133.29, 125.01, 123.85, 121.93, 121.80,
21.69, 106.94, 101.95, 99.72, 71.18, 58.90, 45.72, 39.41, 17.13; HR-MS(ESI): Calcd
20

ACCEPTED MANUSCRIPT
+
for [M+H] 417.1927; Found: 417.1932.
4
9
1
8
.1.1.11. 1-(bis(pyridin-2-ylmethyl)amino)-N-(2-methoxyethyl)-5-methyl-9-oxo-
,10-dihydroacridine-4-carboxamide (7a) yellow solid powder, Yield 33.9%; mp
1
80-182℃; H NMR (400 MHz, CDCl
3
) δ 13.19 (s, 1H), 8.47 (d, J = 4.1 Hz, 2H),
.25 (d, J = 8.0 Hz, 1H), 7.65 – 7.55 (m, 3H), 7.52 (d, J = 7.6 Hz, 2H), 7.47 (d, J = 6.9
Hz, 1H), 7.19 – 7.13 (m, 1H), 7.13 – 7.06 (m, 2H), 6.78 (s, 1H), 6.60 (d, J = 8.7 Hz,
1H), 4.72 (s, 4H), 3.72 – 3.60 (m, 2H), 3.60 – 3.47 (m, 2H), 3.37 (s, 3H), 2.59 (s, 3H);
1
3
C NMR (100 MHz, CDCl ) δ 178.11, 169.01, 158.25, 154.91, 148.91, 145.30,
3
138.14, 136.89, 133.46, 131.57, 124.60, 124.57, 122.89, 122.81, 122.10, 121.36,
112.39, 109.21, 107.03, 71.08, 59.71, 58.84, 45.86, 39.47; HR-MS(ESI): Calcd for
+
[
M+H] 508.2349; Found: 508.2363.
4.1.1.12. N-(2-(dimethylamino)ethyl)-5-methoxy-9-oxo-1-((pyridin-2-ylmethyl)
amino)-9,10-dihydroacridine-4-carboxamide (6k) yellow solid powder, Yield
1
4
8
1
1.9%; mp 201-203℃; H NMR (400 MHz, CDCl
3
) δ 13.60 (s, 1H), 11.64 (s, 1H),
.65-8.64 (m, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.68-7.64 (m, 2H), 7.41 (d, J = 7.6 Hz,
H), 7.19 (d, J = 7.8 Hz, 2H), 7.10 (d, J = 7.4 Hz, 1H), 6.97 (s, 1H), 6.18 (d, J = 8.8
Hz, 1H), 4.73 (d, J = 5.7 Hz, 2H), 4.10 (s, 3H), 3.56 (d, J = 4.9 Hz, 2H), 2.74-2.46 (m,
1
3
2
H), 2.31 (s, 6H); C NMR (100 MHz, CDCl
49.51, 147.94, 144.15, 136.93, 133.65, 131.02, 122.46, 122.23, 121.15, 121.05,
17.17, 111.31, 107.37, 102.06, 99.94, 57.76, 56.28, 48.70, 45.08, 36.73;
3
) δ 180.91, 169.09, 158.11, 155.09,
1
1
21

ACCEPTED MANUSCRIPT
+
HR-MS(ESI): Calcd for [M+H] 446.2192; Found: 446.2190.
4.1.1.13. N-(2-(dimethylamino)ethyl)-5-methoxy-9-oxo-1-((pyridin-3-ylmethyl)
amino)-9,10-dihydroacridine-4-carboxamide (6l) yellow solid powder, Yield 53.9%;
1
mp 222-224℃; H NMR (400 MHz, DMSO-d
6
) δ 13.83 (s, 1H), 11.21 (t, J = 5.7 Hz,
1
H), 8.66 (s, 1H), 8.50 (d, J = 4.6 Hz, 1H), 8.31 (s, 1H), 8.00 (d, J = 8.9 Hz, 1H), 7.82
d, J = 7.6 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.40 (dd, J = 7.6, 4.9 Hz, 1H), 7.31 (d, J
7.8 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 6.36 (d, J = 8.9 Hz, 1H), 4.66 (d, J = 5.7 Hz,
(
=
13
2
H), 4.04 (s, 3H), 3.41-3.38 (m, 2H), 2.43 (t, J = 6.8 Hz, 2H), 2.19 (s, 6H); C NMR
(
100 MHz, DMSO-d ) δ 180.05, 168.87, 154.45, 149.40, 148.93, 147.94, 143.94,
6
1
1
4
35.57, 135.12, 134.59, 130.71, 124.18, 122.05, 121.80, 116.95, 112.84, 106.73,
+
02.30, 100.25, 58.69, 56.88, 45.77, 43.83, 37.72; HR-MS(ESI): Calcd for [M+H]
46.2192; Found: 446.2207.
4.1.1.14. N-(2-(dimethylamino)ethyl)-5-methoxy-9-oxo-1-((pyridin-4-ylmethyl)
amino)-9,10-dihydroacridine-4-carboxamide (6m) yellow solid powder, Yield
1
8
8
3.8%; mp 190-192℃; H NMR (400 MHz, CDCl ) δ 13.61 (s, 1H), 11.54 (s, 1H),
3
.57 (d, J = 4.8 Hz, 2H), 7.95 (d, J = 8.2 Hz, 1H), 7.69 (d, J = 8.9 Hz, 1H), 7.32 (d, J
=
5.2 Hz, 2H), 7.21 (t, J = 8.0 Hz, 1H), 7.12 (t, J = 7.0 Hz, 1H), 7.08 (s, 1H), 6.03 (d,
J = 8.8 Hz, 1H), 4.59 (d, J = 5.9 Hz, 2H), 4.10 (s, 3H), 3.58 (dd, J = 10.5, 5.1 Hz, 2H),
13
2
.62 (t, J = 5.6 Hz, 2H), 2.34 (s, 6H); C NMR (100 MHz, CDCl
3
) δ 180.98, 169.02,
154.92, 150.15, 147.96, 147.50, 144.07, 133.70, 131.02, 122.38, 121.94, 121.33,
22

ACCEPTED MANUSCRIPT
117.07, 111.44, 102.50, 100.00, 99.69, 57.82, 56.30, 45.73, 45.00, 36.63;
+
HR-MS(ESI): Calcd for [M+H] 446.2192; Found: 446.2209.
4.1.1.15. 1-(bis(pyridin-2-ylmethyl)amino)-N-(2-(dimethylamino)ethyl)-5-
methoxy-9-oxo-9,10-dihydroacridine-4-carboxamide (7b) yellow solid powder,
1
Yield 38.7%; mp 97-98℃; H NMR (400 MHz, CDCl
3
) δ 13.25 (s, 1H), 8.46 (s, 2H),
7
7
.93 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 8.7 Hz, 1H), 7.65 (s, 1H), 7.61 – 7.43 (m, 4H),
.14 (t, J = 7.8 Hz, 1H), 7.07 (dd, J = 12.4, 6.7 Hz, 3H), 6.61 (d, J = 8.6 Hz, 1H), 4.70
1
3
(
s, 4H), 4.04 (s, 3H), 3.66 (br, 2H), 2.81 (br, 2H), 2.48 (s, 6H); C NMR (100 MHz,
CDCl ) δ 177.61, 168.97, 158.35, 155.02, 149.02, 147.76, 144.88, 136.75, 132.29,
30.55, 123.47, 122.73, 122.02, 120.95, 117.86, 112.62, 111.27, 109.22, 107.13, 59.73,
3
1
5
5
+
8.08, 56.27, 44.63, 36.11; HR-MS(ESI): Calcd for [M+H] 537.2614; Found:
37.2595.
4.1.1.16. N-(2-(dimethylamino)ethyl)-5-methoxy-9-oxo-1-((2-(pyridin-4-yl)ethyl)
amino)-9,10-dihydroacridine-4-carboxamide (6n) yellow solid powder, Yield
1
2
8
5.4%; mp 208-210℃; H NMR (400 MHz, CDCl ) δ 13.57 (s, 1H), 11.17 (s, 1H),
3
.54 (d, J = 5.7 Hz, 2H), 7.90 (d, J = 8.1 Hz, 1H), 7.78 (d, J = 8.9 Hz, 1H), 7.23 (d, J
=
5.6 Hz, 2H), 7.17 (t, J = 8.0 Hz, 1H), 7.13 (s, 1H), 7.07 (d, J = 7.7 Hz, 1H), 6.23 (d,
J = 8.9 Hz, 1H), 4.07 (s, 3H), 3.65-3.55 (m, 4H), 3.06 (t, J = 7.3 Hz, 2H), 2.72-2.61
1
3
(
m, 2H), 2.39 (s, 6H); C NMR (100 MHz, CDCl
3
) δ 180.74, 169.16, 155.02, 149.98,
147.99, 147.91, 144.26, 133.83, 130.97, 124.10, 122.44, 121.14, 117.12, 111.34,
23

ACCEPTED MANUSCRIPT
106.99, 101.62, 99.03, 58.11, 56.26, 44.96, 43.23, 36.51, 34.73; HR-MS(ESI): Calcd
+
for [M+H] 460.2349; Found: 460.2362.
4.1.1.17. N-(3-(dimethylamino)propyl)-5-methoxy-9-oxo-1-((pyridin-2-ylmethyl)
amino)-9,10-dihydroacridine-4-carboxamide (6o) yellow solid powder, Yield
1
6
7.4%; mp 204-206℃; H NMR (400 MHz, CDCl
3
) δ 13.87 (s, 1H), 11.53 (t, J = 5.8
Hz, 1H), 8.76 (s, 1H), 8.57 (dd, J = 4.5, 1.5 Hz, 2H), 7.94 (d, J = 7.9 Hz, 1H), 7.52 (d,
J = 8.9 Hz, 1H), 7.33 (d, J = 5.9 Hz, 2H), 7.19 (t, J = 8.0 Hz, 1H), 7.09 (dd, J = 7.8,
0.9 Hz, 1H), 6.02 (d, J = 8.9 Hz, 1H), 4.60 (d, J = 6.0 Hz, 2H), 4.09 (s, 3H), 3.60 (dd,
J = 10.5, 5.7 Hz, 2H), 2.58-2.51 (m, 2H), 2.32 (s, 6H), 1.79 (dt, J = 11.5, 5.9 Hz, 2H);
1
3
3
C NMR (100 MHz, CDCl ) δ 180.92, 169.02, 158.25, 154.86, 149.45, 148.01,
1
1
44.23, 136.90, 133.19, 131.13, 122.41, 122.18, 121.06, 120.99, 117.14, 111.24,
07.43, 102.49, 99.82, 59.79, 56.24, 48.71, 45.50, 40.81, 24.99; HR-MS(ESI): Calcd
+
for [M+H] 460.2349; Found: 460.2343.
4.1.1.18. N-(3-(dimethylamino)propyl)-5-methoxy-9-oxo-1-((pyridin-3-ylmethyl)
amino)-9,10-dihydroacridine-4-carboxamide (6p) yellow solid powder, Yield
1
5
8
6.2%; mp 220-222℃; H NMR (400 MHz, CDCl
3
) δ 13.83 (s, 1H), 11.46 (s, 1H),
.70 (s, 1H), 8.67 (s, 1H), 8.54 (d, J = 3.8 Hz, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.75 (d, J
7.6 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.30 (s, 1H), 7.17 (t, J = 7.9 Hz, 1H), 7.08 (d,
J = 7.5 Hz, 1H), 6.14 (d, J = 8.9 Hz, 1H), 4.59 (d, J = 5.3 Hz, 2H), 4.08 (s, 3H), 3.60
=
13
(
d, J = 4.5 Hz, 2H), 2.65-2.55 (m, 2H), 2.38 (s, 6H), 1.86-1.76 (m, 2H); C NMR
24

ACCEPTED MANUSCRIPT
(
100 MHz, CDCl
3
) δ 180.93, 169.06, 154.70, 149.01, 148.84, 147.99, 144.17, 134.84,
1
9
4
33.72, 133.45, 131.05, 123.78, 122.32, 121.16, 116.98, 111.30, 107.33, 102.59,
+
9.56, 59.17, 56.26, 45.19, 44.32, 40.17, 24.80; HR-MS(ESI): Calcd for [M+H]
60.2349; Found: 460.2343.
4.1.1.19. N-(3-(dimethylamino)propyl)-5-methoxy-9-oxo-1-((pyridin-4-ylmethyl)
amino)-9,10-dihydroacridine-4-carboxamide (6q) yellow solid powder, Yield
1
6
5.8%; mp 212-214℃; H NMR (400 MHz, CDCl
3
) δ 13.84 (s, 1H), 11.62 (t, J = 5.8
Hz, 1H), 8.73 (s, 1H), 8.66-8.59 (m, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.64 (td, J = 7.7,
.8 Hz, 1H), 7.52 (d, J = 8.9 Hz, 1H), 7.41 (d, J = 7.9 Hz, 1H), 7.18 (dd, J = 15.3, 7.5
Hz, 2H), 7.08 (dd, J = 7.8, 1.0 Hz, 1H), 6.16 (d, J = 8.9 Hz, 1H), 4.72 (d, J = 5.9 Hz,
1
2
1
1
H), 4.08 (s, 3H), 3.59 (dd, J = 10.6, 5.7 Hz, 2H), 2.58-2.48 (m, 2H), 2.34 (s, 6H),
13
.83-1.72 (m, 2H); C NMR (100 MHz, CDCl
49.46, 147.99, 144.23, 136.94, 133.28, 131.09, 122.38, 122.21, 121.06, 121.02,
17.10, 111.21, 107.40, 102.41, 99.87, 59.67, 56.25, 48.69, 45.44, 40.69, 24.88;
3
) δ 180.93, 169.05, 158.22, 154.85,
1
+
HR-MS(ESI): Calcd for [M+H] 460.2349; Found: 460.2336.
4.1.1.20. 1-(bis(pyridin-2-ylmethyl)amino)-N-(3-(dimethylamino)propyl)-5-
methoxy-9-oxo-9,10-dihydroacridine-4-carboxamide (7c) yellow solid powder,
1
Yield 39.9%; mp 90-92℃; H NMR (400 MHz, CDCl
3
) δ 13.55 (s, 1H), 8.85 (s, 1H),
8.50 (d, J = 4.4 Hz, 2H), 7.96 (d, J = 8.0 Hz, 1H), 7.63-7.54 (m, 4H), 7.52 (d, J = 8.8
Hz, 1H), 7.16 (t, J = 7.9 Hz, 1H), 7.13-6.99 (m, 3H), 6.60 (d, J = 8.7 Hz, 1H), 4.73 (s,
25

ACCEPTED MANUSCRIPT
4H), 4.07 (s, 3H), 3.58 (d, J = 4.8 Hz, 2H), 2.58-2.46 (m, 2H), 2.29 (s, 6H), 1.82-1.65
1
3
(
m, 2H); C NMR (100 MHz, CDCl
47.87, 144.89, 136.80, 131.35, 130.66, 123.38, 122.81, 122.03, 120.88, 117.76,
12.91, 111.17, 109.20, 108.00, 59.75, 59.47, 56.24, 45.41, 40.68, 24.86;
3
) δ 177.70, 168.66, 158.47, 154.59, 148.94,
1
1
+
HR-MS(ESI): Calcd for [M+H] 551.2771; Found: 551.2793.
4
9
2
4
1
6
=
.1.1.21. 5-methoxy-N-(2-methoxyethyl)-9-oxo-1-((pyridin-2-ylmethyl)amino)-
,10-dihydroacridine-4-carboxamide (6r) yellow solid powder, Yield 83.5%; mp
1
18-220℃; H NMR (400 MHz, CDCl
3
) δ 13.48 (s, 1H), 11.65 (s, 1H), 8.64 (d, J =
.6 Hz, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.66 (t, J = 7.1 Hz, 1H), 7.60 (d, J = 8.9 Hz,
H), 7.40 (d, J = 7.7 Hz, 1H), 7.20 (dd, J = 16.1, 7.9 Hz, 2H), 7.09 (d, J = 7.7 Hz, 1H),
.47 (s, 1H), 6.17 (d, J = 8.9 Hz, 1H), 4.73 (d, J = 5.5 Hz, 2H), 4.08 (s, 3H), 3.67 (d, J
13
3
4.9 Hz, 2H), 3.58 (d, J = 4.8 Hz, 2H), 3.39 (s, 3H); C NMR (100 MHz, CDCl ) δ
1
1
5
80.90, 168.95, 157.96, 155.13, 149.28, 147.90, 144.11, 137.15, 133.37, 130.97,
22.45, 122.32, 121.22, 121.16, 117.15, 111.35, 107.36, 101.97, 99.93, 71.29, 58.88,
+
6.29, 48.56, 39.35; HR-MS(ESI): Calcd for [M+H] 433.1876; Found: 433.1862.
4
9
2
8
8
.1.1.22. 5-methoxy-N-(2-methoxyethyl)-9-oxo-1-((pyridin-3-ylmethyl)amino)-
,10-dihydroacridine-4-carboxamide (6s) yellow solid powder, Yield 75.2%; mp
1
52-253℃; H NMR (400 MHz, CDCl
3
) δ 13.48 (s, 1H), 11.49 (s, 1H), 8.66 (s, 1H),
.54 (d, J = 3.9 Hz, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.60 (d, J =
.9 Hz, 1H), 7.29 – 7.27 (m, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.08 (d, J = 7.7 Hz, 1H),
26

ACCEPTED MANUSCRIPT
6
3
1
1
5
.50 (s, 1H), 6.11 (d, J = 8.9 Hz, 1H), 4.58 (d, J = 5.5 Hz, 2H), 4.08 (s, 3H), 3.75 –
13
3
.62 (m, 2H), 3.62 – 3.50 (m, 2H), 3.39 (s, 3H); C NMR (100 MHz, CDCl ) δ
80.88, 168.90, 154.95, 149.03, 148.89, 147.92, 144.06, 134.78, 133.59, 133.34,
30.97, 123.72, 122.41, 121.30, 117.06, 111.43, 107.31, 102.29, 99.54, 71.27, 58.85,
+
6.28, 44.35, 39.37; HR-MS(ESI): Calcd for [M+H] 433.1876; Found: 433.1881.
4
9
2
8
.1.1.23. 5-methoxy-N-(2-methoxyethyl)-9-oxo-1-((pyridin-4-ylmethyl)amino)-
,10-dihydroacridine-4-carboxamide (6t) yellow solid powder, Yield 79.3%; mp
1
35-236℃; H NMR (400 MHz, CDCl
3
) δ 13.48 (s, 1H), 11.56 (t, J = 5.7 Hz, 1H),
.57 (d, J = 5.1 Hz, 2H), 7.94 (d, J = 8.2 Hz, 1H), 7.58 (d, J = 8.9 Hz, 1H), 7.34 (d, J
5.2 Hz, 2H), 7.19 (d, J = 8.0 Hz, 1H), 7.10 (d, J = 7.8 Hz, 1H), 6.48 (s, 1H), 6.01 (d,
J = 8.9 Hz, 1H), 4.60 (d, J = 5.9 Hz, 2H), 4.09 (s, 3H), 3.67 (dd, J = 10.0, 5.1 Hz, 2H),
=
1
3
3
.57 (t, J = 4.9 Hz, 2H), 3.39 (s, 3H); C NMR (100 MHz, CDCl
54.99, 150.15, 147.94, 147.42, 144.04, 133.30, 130.99, 122.41, 121.92, 121.36,
17.09, 111.48, 107.36, 102.48, 99.59, 71.24, 58.85, 56.30, 45.74, 39.37;
3
) δ 180.95, 168.85,
1
1
+
HR-MS(ESI): Calcd for [M+H] 433.1876; Found: 433.1881.
4.1.1.24. N-(4-(dimethylamino)butyl)-5-methoxy-9-oxo-1-((pyridin-4-ylmethyl)
amino)-9,10-dihydroacridine-4-carboxamide (6u) yellow solid powder, Yield
1
7
5
4.2%; mp 213-215℃; H NMR (400 MHz, DMSO-d
6
) δ 13.86 (s, 1H), 11.26 (t, J =
.8 Hz, 1H), 8.54 (d, J = 4.0 Hz, 2H), 8.43 (s, 1H), 7.98 (d, J = 9.0 Hz, 1H), 7.77 (d, J
7.9 Hz, 1H), 7.39 (d, J = 5.2 Hz, 2H), 7.32 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 8.0 Hz,
=
27

ACCEPTED MANUSCRIPT
1
2
=
H), 6.22 (d, J = 9.0 Hz, 1H), 4.69 (d, J = 5.8 Hz, 2H), 4.04 (s, 3H), 3.30-3.20 (m,
H), 2.21 (t, J = 7.0 Hz, 2H), 2.11 (s, 6H), 1.54 (dd, J = 14.2, 7.1 Hz, 2H), 1.46 (dd, J
13
3
14.2, 7.4 Hz, 2H); C NMR (100 MHz, CDCl ) δ 180.98, 169.16, 154.67, 150.13,
1
1
47.99, 147.45, 143.94, 133.51, 131.06, 122.35, 121.96, 121.23, 117.04, 111.38,
07.40, 103.20, 99.25, 59.34, 56.26, 45.72, 45.32, 39.90, 27.40, 25.69; HR-MS(ESI):
+
Calcd for [M+H] 474.2505; Found: 474.2495.
4.1.1.25. N-(2-(dimethylamino)ethyl)-9-oxo-1-((pyridin-4-ylmethyl)amino)-5-
(trifluoromethyl)-9,10-dihydroacridine-4-carboxamide (6v) yellow solid powder,
1
Yield 18.2%; mp 178-180℃; H NMR (400 MHz, MeOD) δ 8.58 (d, J = 8.2 Hz, 1H),
8.50 (d, J = 5.9 Hz, 2H), 8.05 (d, J = 7.5 Hz, 1H), 7.93 (d, J = 9.0 Hz, 1H), 7.49 (d, J
=
5.4 Hz, 2H), 7.38 (t, J = 7.9 Hz, 1H), 6.28 (d, J = 9.1 Hz, 1H), 4.73-4.76 (m, 2H),
1
9
3
.56 (t, J = 6.7 Hz, 2H), 2.64 (t, J = 6.6 Hz, 2H), 2.37 (s, 6H). F NMR (376 MHz,
+
DMSO-d ) δ -61.63; HR-MS(ESI): Calcd for [M+H] 484.1960; Found: 484.1940.
6
4.1.1.26. 5,7-dichloro-N-(2-(dimethylamino)ethyl)-9-oxo-1-((pyridin-4-ylmethyl)
amino)-9,10-dihydroacridine-4-carboxamide (6w) yellow solid powder, Yield
1
2
8
8.1%; mp 114-116℃; H NMR (400 MHz, CDCl
3
) δ 14.01 (s, 1H), 11.23 (s, 1H),
.57 (d, J = 5.7 Hz, 2H), 8.23 (d, J = 2.2 Hz, 1H), 7.74 (d, J = 9.0 Hz, 1H), 7.69 (d, J
2.2 Hz, 1H), 7.46 (s, 1H), 7.29 (d, J = 5.5 Hz, 2H), 6.04 (d, J = 9.0 Hz, 1H), 4.56 (d,
=
13
J = 5.6 Hz, 2H), 3.64 (d, J = 5.2 Hz, 2H), 2.80-2.70 (m, 2H), 2.44 (s, 6H); C NMR
100 MHz, CDCl ) δ 179.24, 169.03, 154.69, 150.20, 146.97, 144.41, 135.25, 134.56,
(
3
28