Novel multi-substituted benzyl acridone derivatives as survivin inhibitors for hepatocellular carcinoma treatment

Article abstract of DOI:10.1016/j.ejmech.2017.02.027

Sorafenib was the only small-molecule drug approved by FDA for treatment of the advanced hepatocellular carcinoma (HCC). Recent study indicated that YM155 was a promising agent for HCC cells with high survivin expression, however, the antitumor activity needs to be further improved. Based on molecular docking and rational design method, a series of multi-substituted benzyl acridone derivatives were designed and synthesized. MTT assay indicated that some of the synthesized compounds displayed better antiproliferative activity against HepG2 cells than YM155. Later study indicated that the representive compound 8u may directly interact with survivin protein and induce HepG2 cells apoptosis, which is different from YM155. In addition, ADME property was predicted in silico, and it performed well. Moreover, in vivo preliminary experiments showed that 8u may be a good lead compound in the treatment of HCC.

Full text of DOI:10.1016/j.ejmech.2017.02.027

Accepted Manuscript
Novel multi-substituted benzyl acridone derivatives as survivin inhibitors for
hepatocellular carcinoma treatment
Bin Zhang, Ning Wang, Cunlong Zhang, Chunmei Gao, Wei Zhang, Kang Chen,
Weibin Wu, Yuzong Chen, Chunyan Tan, Feng Liu, Yuyang Jiang
PII:
S0223-5234(17)30092-2
10.1016/j.ejmech.2017.02.027
EJMECH 9223
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 January 2017
Revised Date: 7 February 2017
Accepted Date: 10 February 2017
Please cite this article as: B. Zhang, N. Wang, C. Zhang, C. Gao, W. Zhang, K. Chen, W. Wu, Y. Chen,
C. Tan, F. Liu, Y. Jiang, Novel multi-substituted benzyl acridone derivatives as survivin inhibitors for
hepatocellular carcinoma treatment, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.02.027.
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ACCEPTED MANUSCRIPT
Novel multi-substituted benzyl acridone derivatives as survivin inhibitors for
hepatocellular carcinoma treatment
Based on molecular docking and rational design method, a series of
multi-substituted benzyl acridone derivatives were designed and synthesized as
survivin inhibitors for the treatment of hepatocellular carcinoma.

ACCEPTED MANUSCRIPT
Novel multi-substituted benzyl acridone derivatives as survivin inhibitors for
hepatocellular carcinoma treatment
a,b,†
c,
b
c*
c
†
Bin Zhang , Ning Wang , Cunlong Zhang , Chunmei Gao , Wei Zhang , Kang
c
a,b
b,e
c
c
a,c,d*
Chen , Weibin Wu , Yuzong Chen , Chunyan Tan , Feng Liu , Yuyang Jiang
a
b
c
d
e
Department of Pharmaceutical Engineering, Shenyang Pharmaceutical University,
Shenyang, Liaoning 110016, P. R. China
Shenzhen Kivita Innovative Drug Discovery Institute, Shenzhen 518057, P. R.
China
National & Local United Engineering Lab for Personalized anti-tumor drugs, the
Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China
Department of Pharmacology and Pharmaceutical Sciences, School of Medicine,
Tsinghua University, Beijing, 100084, P. R. China
Bioinformatics and Drug Design Group, Department of Pharmacy, Centre for
Computational Science and Engineering, National University of Singapore, 117543,
Singapore
*
Corresponding author. Tel.: +86 755 2603 2094; fax: +86 755 2603 2094;
Email address: jiangyy@sz.tsinghua.edu.cn; chunmeigao@sz.tsinghua.edu.cn
†
Authors with equal contributions.

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Abstract
Sorafenib was the only small-molecule drug approved by FDA for treatment of
the advanced hepatocellular carcinoma (HCC). Recent study indicated that YM155
was a promising agent for HCC cells with high survivin expression, however, the
antitumor activity needs to be further improved. Based on molecular docking and
rational design method, a series of multi-substituted benzyl acridone derivatives were
designed and synthesized. MTT assay indicated that some of the synthesized
compounds displayed better antiproliferative activity against HepG2 cells than
YM155. Later study indicated that the representive compound 8u may directly
interact with survivin protein and induce HepG2 cells apoptosis, which is different
from YM155. In addition, ADME property was predicted in silico, and it performed
well. Moreover, in vivo preliminary experiments showed that 8u may be a good lead
compound in the treatment of HCC.
Key words: Acridones, Survivin, YM155, Molecular docking, Antitumor

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1
. Introduction
Currently, hepatocellular carcinoma (HCC) is one of the most common
potentially lethal human malignancies worldwide [1]. The only approved
small-molecule drug for advanced HCC is the multikinase inhibitor Sorafenib,
however, it has only little survival advantage in the presence of major side effects [2].
Therefore, the development of novel small molecule inhibitors against HCC is
urgently required. Survivin is the smallest member of Inhibitor of Apoptosis Proteins
(
IAPs) [3, 4], which is highly expressed in most human solid tumors including HCC,
but with little expression in most normal terminally differentiated adult tissues [5-8].
Therefore, survivin has become a putative drug target for cancer therapy [9-14]. Now,
it has been regarded as an attractive therapeutic target for the treatment of HCC
[
15-19].
YM155 (Sepantronium Bromide), a small-molecule survivin suppressant, has
entered clinical trials for the treatment of lymphoma, melanoma, prostate and
non-small cell lung carcinoma [20, 21]. Additionally, preclinical studies have also
shown that YM155 was a promising agent for HCC cells with high survivin
expression [17], and it yielded significantly better therapeutic effect than sorafenib in
an orthotopic mouse model [16]. Although the anti-cancer mechanisms of YM155
have not yet been fully elucidated [17, 22], a library of YM155 analogs have been
designed and synthesized as anti-cancer small molecule inhibitors in recent years
because of its excellent anti-cancer activities [23]. The United States National Cancer
Institute also developed a novel dioxonaphthoimidazolium derivative NSC80467, the

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chemical structure of which is similar to YM155, and it displayed similar antitumor
activity against NCI-60 cell lines compared to YM155 [24]. Although series of
YM155 derivatives have been developed, few compounds displayed better antitumor
activity against HCC have been found. Therefore, it is important to develop new
YM155 analogues to treat HCC.
Comparing the structures of YM155 and NSC80467, both of them contain a
tricyclic conjugated system (Figure 1a, D, red ellipses) and two main side chains, one
of which contains an aromatic ring group (Figure 1a, blue ellipses) and the other is an
alkyl or alkoxy side chain (Figure 1a, green ellipses). We found that the D part of
YM155 and NSC80467 is similar to acridone structure, which have been developed as
good HCC inhibition agents by our group and other researches [23, 25-34]. We think
that if two appropriate side chains were introduced to the acridone ring, acridone
derivatives can be developed as survivin inhibitors similar to YM155 for the treatment
of HCC. The 3D structural superposition of the representive compound 8u and
YM155 is shown in Figure 1b, which indicated that 8u and YM155 might exhibit
similar bioactivity. As the main targets of acridones are DNA and its related enzymes
(such as topoisomerases, telomerase, etc.), it is very interesting and reasonable to
found their new mechanisms of action.
In this paper, a library of novel multi-substituted benzyl acridone derivatives
were designed and synthesized based on the chemical structural features of YM155
and NSC80467. The antiproliferative activity of the compounds were tested and their

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SAR were established. The typical compound 8u could inhibit the expression of
survivin to display HCC inhibition activity in vitro and in vivo.
2
2
. Results and discussion
.1. Chemistry
The synthesis of targets compounds 5a-b, 7a-b and 8a-w was shown in Scheme
. Compounds 3a-d were obtained from the Ullmann reaction of anthranilic acid
1
derivatives 1a-d with 2,4-dichlorobenzoic acid 2 in DMF using Cu as the catalyst [35,
6]. Subsequent Friedel-Crafts acylation in concentrated sulfuric acid at 80 � for 3-5
3
h gave acridone-4-carboxylic acid derivatives 4a-d in high yields [37], which were
then reacted with the corresponding primary aliphatic amines using
N,N′-carbonyldiimidazole (CDI) as the condensation agent [38] at room temperature
to afford the intermediates acridone-4-carboxamides 6a-h. It is noteworthy that when
the reaction temperature raised to 70 � , it will generate a small amount of 5a-b. The
desired compounds 7a-b and 8a-v were produced by the nucleophilic substitution
between the corresponding acridone-4-carboxamides and hydrazine hydrate or various
multi-substituted benzyl amines. Additionally, compound 8w was obtained by the
reduction reaction of 8u under the iron and ammonium chloride conditions [39]. The
1
13
structures of all target compounds were established via H NMR, C NMR and high
resolution mass spectral data.
2
.2. In vitro cytotoxicity
The in vitro cytotoxicity of 30 desired acridone derivatives against HepG2 cells
were firstly assayed by MTT reduction method, and YM155 was used as the positive

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control. In order to verify the inhibitory activity of the synthesized compounds on
other solid tumor cells, human breast cancer MCF-7 cells were also tested. The
structures and bioactivity results were shown in Table 1 and Table 2. Most of these
compounds had better antitumor activity than YM155. Table 1 showed the MTT
results for the benzylamino-substituted acridone compounds. It can be seen that the R2
group on the 4-carboxamide side chain had a great effect on the antitumor activity.
Acridones with N,N-dimethylamino group displayed better antitumor activity than
those with methoxy group except 8n, which can be seen from the IC values of 8e, 8f,
5
0
8
g, 8h and 8i. These compounds with methoxy group displayed no cytotoxicity (IC >
50
5
0 µM). Interestingly, when the R substituent was 4-methoxy group, the activities
3
were also outstanding. For example, 8c had an IC value of 2.11 µM against HepG2
50
cells, and 8r was 1.05 µM against MCF-7 cells. In addition, as shown in Table 1, four
compounds (8b, 8c, 8t, and 8u) had IC values of less than 4 µM against HepG2 cells,
5
0
and these compounds also exhibited good inhibitory activity against MCF-7 cells
2.92 to 6.84 µM). These results suggested that benzylamino acridones had almost the
(
same inhibitory effects on both two solid tumor cells. It should be noted that the
difference in chemical structure between 8r and 8u was only the length of the alkyl
chains between the N,N-dimethylamino group and 4-carboxamide group (the number
of methylene units, n), and the IC values of 8r and 8u on HepG2 cells were almost
5
0
the same. Thus, the length of the amide side chain had little effect on the antitumor
activity. Additionally, the difference between 8w and 8u was only the substituent at
the C-5 position (8w:5-NH , 8u: 5-NO ), but the IC values against HepG2 cells
2
2
50

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were 5.68 µM and 3.56 µM, respectively. Moreover, the antiproliferative activity of
8
w against MCF-7 cells was about 3 times lower than that of 8u.
The electronic effects of the 5-position substituent of acridone scaffold were also
discussed, including different electron-donating and electron-withdrawing groups on
C5 position, such as 5-methyl, 5-methoxy, 5-amino, 5-nitro and 5,7-dichloro
substituents. From the biological data shown in Table 1, compounds with good
activities generally had methyl or nitro groups at the C5 position (8d vs. 8u). The
results indicated that electron-negativity and steric effect on C5 position of acridone
scaffold might have little change in the cytotoxic profile. However, when 5,7-dichloro
substituent was introduced (e.g. 8o), the antiproliferative activities of HepG2 cells and
MCF-7 cells were significantly decreased compared to the other substituents at the
5
-position.
Table 2 showed the structure-activity relationship of no benzyl substituted
acridone derivatives (the benzylamino substituents are replaced by -Cl, -NHNH and
2
-
N(CH ) substituents). From the data we can see, most of non-benzylamino
3 2
acridinone compounds showed poor antiproliferative activities, for instance, 5b had
poor antitumor activity against HepG2 cells with an IC value of 36.82 µM. These
5
0
results indicated that the introduction of the benzylamino group in the target
compounds was important for the anticancer activity.
For the most active compounds (8b, 8c, 8t and 8u) against HepG2 cells, we
further tested their in vitro toxicity against human liver cells QGY-7701 [40, 41].
From Table 3 we can see that only 8u was less toxic than 8b, 8c and 8t to normal

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hepatocytes, and its IC value is 11 times lower than for HepG2 cells. However, the
5
0
other three compounds are still toxic to QGY-7701. Therefore, 8u was selected for
subsequent biological research.
2
.3. In silico predictions of druggability, pharmacokinetic properties and toxicity
As compound 8u displayed good antiproliferative activity and low toxic side
effect, the in silico predictions of drugability and pharmacokinetics of 8u using
ACD/Percepta Platform (Table 4, 5), and toxicity prediction using Osiris soft (Table
6
) were performed. It is well-known that the Lipinski's rule of five [42] based on the
pharmacokinetic properties of small molecule drugs, is important for the prediction of
ligand druggability. It can be seen from Table 4 that the number of hydrogen bond
donors and acceptors of 8u were 10 and 3, respectively; the calculated log P values
was 4.77, the molecular weight was 503.55 (Slightly higher); the number of rotatable
2
bonds was 10, the number of rings were 4 and the surface area were 131.53 Å , all of
which except the slightly larger molecular weight were within the range specified by
the rule. Therefore, the Lipinski's violations of 8u was 1, which are accorded with the
limit of Lipinski's rule (≤1).
The evaluation of pharmacokinetic properties of 8u was carried out using
ACD/Percepta software 14.0.0 (Table 5). In silico prediction of the passive absorption
of drugs based on Caco-2 cell permeability, provides a useful drug discovery tool by
predicting human oral absorption of new compounds. Yazdanian et al. [43] reported
–
6
that compounds with values greater than 7×10 cm/s had excellent oral absorption.
–
6
The permeability coefficient of 8u was greater than 7×10 cm/s, which indicated

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good absorption characteristics. In the analysis of a new drug, it is very important to
know the basic pharmacokinetic parameters such as protein binding. For the
estimation of binding of compounds to human plasma proteins, e.g., albumin,
lipoproteins and alpha-1-acid glycoprotein, we used the %PPB parameter as the
cumulative percentage. An ability to cross the blood brain barrier (BBB) is an asset
for compounds to be used for therapies of central nervous system (CNS) afflictions,
which depends on the lipid-solubility and molecular weight of the drug itself. BBB
transport parameters contain the rate of brain penetration (LogPS), extent of brain
penetration (LogBB) and the brain/plasma equilibration rate (Log(PS*fu, brain)).
These values were used to classify the compounds as CNS permeable or
non-permeable. However, these results showed that 8u had low brain penetration,
which may indicated inactive to CNS.
P-glycoprotein (P-gp) is a well characterized transporter, which moves a variety
of substrates across extra- and intracellular membranes. It is an important drug efflux
pump, therefore, predicting P-gp transport is important aiming to develop anti-cancer
drugs. However, the prediction result is inconclusive. Similarly, the first-pass
metabolism also failed to give a prediction. In addition, the predicted solubility of 8u
was only 0.1mg/mL, apparently this result was not good. To this end, we synthesized
8
u hydrochloride, which its solubility in water had a large extent improved. Therefore,
we used its hydrochloric acid salt to study in vivo experiments.
The toxicity prediction for 8u was based on the online software Osiris Property
Explorer ver. 2. The software was used to predict the toxicity from mutagenic,

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tumorigenic, irritant and reproductive aspects. It should be noted that whether
compounds are mutagenic, based on their ability to induce DNA damage. The toxicity
prediction results were shown in Table 6，in mutagenic, tumorigenic, irritant and
reproductive aspects, 8u has shown low toxicity, which was in accordance to the MTT
values.
2
.4. DNA binding and Topo I/II inhibition experiments
Literature has proved that YM155 is a DNA damaging agent while suppression
of survivin is a secondary event [21, 24]. As the structure of 8u is very similar to
YM155, therefore, we firstly studied the DNA binding ability of 8u, which was shown
in Figure 1s. The results showed that 8u was not a DNA binding agent compared to
our published DNA binders [25-27, 35, 44].
As most of acridine/acridone derivatives had the inhibitory activity of
topoisomerases, it is of interest to evaluate whether our synthesized novel acridone
derivative 8u can also inhibit the activity of topoisomerases. Figure 2a showed the
relative affinity of 8u on the relaxation of plasmid pBR322 DNA mediated by Topo I,
which indicated that 8u displayed no Topo I inhibitory activity. Figure 2b showed the
human Topo II decatenation assay, and two concentrations (1 and 10 µM) of
Doxorubicin (DOX) were used as the positive controls. From the results we can see,
8
u (50 µM) seemed to be weakly bound to Topo II, however, the inhibition rate was
only about 10.9% while Doxorubicin (10 µM) was 74.4%. Therefore, Topo II is not
the main drug target of 8u. The above results indicated that DNA and topoisomerases
are not the main targets of 8u, which is different from YM155.

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.5. Identification of YM155 and 8u targeting to survivin protein
.5.1 Molecular modeling
2
2
As survivin small-molecule inhibitor, YM155 displays an obvious
antiproliferative activity in a large panel of tumor cellular model [45], however, its
molecular mechanisms of action needs to be further evaluated [17, 22]. Survivin
protein exists as a homodimer in most tumor cells, and the presence of a homodimer
is a necessary condition for its physiological function. Targeting of the small molecule
into a domain that restricts the dimerization hydrophobic interface may cause
destabilization and degradation of the protein, thereby further leading to cancer cells
apoptosis [14]. To this end, we used molecular docking methods to verify whether 8u
can be directly binding to survivin hydrophobic surface.
There is a high hydrophobicity surface in survivin dimerization domain, its key
residues comprised of Leu6, Pro7, Pro8, Ala9, Trp10, Phe93, Glu94, Glu95, Leu96,
Thr97, Leu98, Gly99, Phe101, Leu102, and it is considered to be a good target site for
drug discovery [14]. Therefore, molecular docking studies of the representative
compound 8u and YM155 with survivin protein model were conducted using
SYBYL-X v1.3 program. As seen from Figure 3, 8u can effectively target the
dimerization hydrophobic domain of survivin, and the hydrophobic effect between the
ligand and receptor might be due to π-conjugated planar acridone scaffold of 8u.
Moreover, three hydrogen bonds of 8u were also formed in the hydrophobic domain
(residues Phe93, Glu94 and Leu96). Therefore, it was confirmed that 8u can
effectively bind to the critical hydrophobic core, which suggested that survivin protein

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may be the main drug target of 8u. However, there was no interaction between
YM155 and survivin, which can be seen in Figure 2s.
2
.5.2 Fluorogenic titration assay
To verify that 8u indeed binds to survivin, we performed a fluorogenic titration
assay in the presence or absence of survivin. Figure 4 showed that the fluorescence of
u dramatically increased in the presence of survivin, indicating that 8u likely
8
interacted with survivin protein. Meanwhile, we also carried out the fluorescence
experiments of YM155 and survivin protein (Figure 3s) and the results showed that
with the YM155 concentration increased, the fluorescence trend was not changed,
indicating that YM155 may not target survivin protein directly.
2
.6. 8u causes survivin degradation and induces cells apoptosis
We next determined if 8u causes survivin degradation by western blot analysis.
For this purpose, HepG2 cells were treated with 8u with different concentration
gradients. Figure 5 showed that the content of survivin protein in HepG2 cells was
significantly decreased with the increase of the concentration of 8u from 1 µM to 10
µM.
In order to understand whether 8u induced cancer cells apoptosis, the activity of
cleaved caspase-7 was firstly tested. As shown in Figure 5, 8u at 10 µM for 24 h
induced significant activation of cleaved caspase-7, suggesting that 8u induced
HepG2 cells apoptosis. PARP (poly ADP-ribose polymerase) is the substrate of
caspases. During apoptosis, caspases mediated PARP cleavage, which inactivates the
enzyme by destroying its ability to respond to DNA strand breaks. The results

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revealed the generation of the cleaved PARP. Additionally, Bcl-2, an important
member of Bcl-2 family proteins, plays an important role in extending cellular
survival. 8u at 10 µM effectively decreased the expression of Bcl-2 protein as shown
in Figure 5, which further confirmed that 8u could induce HepG2 cells apoptosis.
2
.7. In vivo preliminary experiments
We finally evaluated the in vivo anti-tumor effect of compound 8u
(hydrochloride salt) on subcutaneously xenografts tumor models of human
hepatocarcinoma cell line HepG2 in nude mice, and measured the body weight and
tumor size of the nude mice once every two days after the first administration. Figure
6
a showed the effect of 8u on tumor size in the HepG2 implanted model with the days
of administration increased. From the data we can see that treatment with 8u (3 mg/kg)
significantly reduced HepG2 tumor volume by 54% after 30 days of treatment, which
was comparable to the positive control (64%, 25 mg/kg). In addition, little toxicity of
8
u was observed, as demonstrated by the absence of weight loss (Figure 6b) and by
the normal growth and behavior of all mice tested. Moreover, 8u had no significant
effect on the organ weight of nude mice (Table 7). 8u could obviously increase the
spleen weight in a dose-dependent manner, which suggested that 8u might be able to
enhance the body's immune capacity. All the results indicated that 8u had efficacious
antitumor effect and low adverse effects, which may constitute a novel lead in the
search for anticancer drug candidates with new mechanisms of action.
3
. Conclusion
Based on the structural characteristics of survivin inhibitors YM155 and
NSC80467, 30 multi-benzylamino acridone derivatives were designed and

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synthesized. The synthesized compounds were evaluated through MTT screening,
most of them showed good in vitro anti-cancer activities against HepG2 cells and
MCF-7 cells. Among them, compound 8u with high activity showed low toxicity to
hepatocyte QGY-7701 cells in vitro. Druggability and toxicity prediction showed that
8
u conformed to the rules of Lipinski and showed low toxicity in mutagenic,
tumorigenic, irritant and reproductive aspects. In addition, the ADME property
prediction indicated that 8u has good absorption characteristics, but it was ineffective
to CNS. Molecular docking showed that 8u restricts the survivin dimerization
hydrophobic domain, and the fluorescence experiments further confirmed that 8u can
binding with the survivin protein. Interestingly, DNA binding experiments and
topoisomerase inhibition experiments (Topo I/II) found that 8u did not cause direct or
indirect DNA damage, which is different from YM155 and NSC80467. In addition,
we found that 8u can significantly decrease the content of survivin in HepG2 cells
with the increase of drug concentration, which proved the possible mechanism
proposed in the molecular docking. The apoptosis mechanism of hepatocellular
carcinoma cells further showed that 8u could induce the apoptosis of HepG2 cells. In
vivo preliminary experiments exhibited that 8u effectively suppressed the growth of
HepG2 xenograft tumors, and the tumor inhibition rate of 8u reached 54% after 30
days of administration. Moreover, 8u had not affected the body weight, organ weight
and organ index of nude mice, which was consistent with in vitro anticancer activity
and toxicity assay. Therefore, 8u may be a promising lead compound in the treatment
of HCC, moreover, metabolomics and proteomics research of 8u on HepG2 cells is in
progress.
4
4
. Experimental section
.1. Synthesis and characterization

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See supporting information for synthetic methods and the preparation of
compounds 3a-d, 4a-d, 5a-b and 6a-h.
4
.1.1. General procedure for compounds 7a-b and 8a-v.
-oxo-9,10-dihydroacridine-4-carboxamide derivatives (6a-h, 0.28 mmol) and
9
hydrazine hydrate or various multi-substituted benzyl amines (0.84 mmol) were
dissolved in 2-ethoxyethanol, and the mixture was stirred at 90-100°C until the TLC
showed the disappearance of the starting material. The mixture was cooled to room
temperature and partitioned between CH Cl (50 mL) and water (50 mL). The organic
2
2
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 7a-b and 8a-v.
4
.1.1.1. N-(2-(dimethylamino)ethyl)-1-hydrazinyl-5-methyl-9-oxo-9,10-dihydro
1
acridine-4-carboxamide (7a) yellow solid powder, Yield 20.2%; mp 190-192℃; H
NMR (400 MHz, CDCl ) δ 13.57 (s, 1H), 11.55 (s, 1H), 8.21 (d, J = 8.1 Hz, 1H), 7.72
3
(d, J = 9.0 Hz, 1H), 7.48 (d, J = 7.0 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 6.95 (s, 1H),
6
2
1
3
4
.88 (d, J = 9.0 Hz, 1H), 3.78 (br, 2H), 3.55 (dd, J = 10.7, 5.2 Hz, 2H), 2.62 (s, 3H),
1
3
.60-2.52 (m, 2H), 2.32 (s, 6H); C NMR (100 MHz, CDCl ) δ 180.9, 169.4, 157.6,
3
44.5, 138.6, 133.6, 133.4, 124.9, 123.9, 121.7, 121.3, 105.8, 101.4, 99.1, 57.8, 45.2,
+
6.9, 17.2; HR-MS(ESI): Calcd for [M+H] 354.1930; Found: 354.1928.
.1.1.2.N-(3-(dimethylamino)propyl)-1-hydrazinyl-5-methoxy-9-oxo-9,10-dihydro
1
acridine-4-carboxamide (7b) yellow solid powder, Yield 40.4%; mp 236-238℃; H
NMR (400 MHz, CDCl ) δ 13.58 (s, 1H), 11.58 (s, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.74
3
(d, J = 9.0 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.08 (d, J = 7.7 Hz, 1H), 6.91 (d, J = 8.8

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Hz, 2H), 4.09 (s, 3H), 3.78 (br, 2H), 3.62-3.51 (m, 2H), 2.58 (t, J = 5.8 Hz, 2H), 2.32
1
3
(
s, 6H); C NMR (100 MHz, CDCl ) δ 180.5, 169.1, 157.7, 148.0, 144.0, 133.5,
3
1
31.1, 122.4, 121.0, 117.2, 111.3, 106.3, 101.9, 99.0, 57.8, 56.3, 45.2, 36.9;
+
HR-MS(ESI): Calcd for [M+H] 370.1879; Found: 370.1870.
4
.1.1.3. 1-(benzylamino)-N-(2-(dimethylamino)ethyl)-5-methyl-9-oxo-9,10-
dihydroacridine-4-carboxamide (8a) yellow solid powder, Yield 60.9%; mp
1
1
8
2
6
2
1
1
44-146℃; H NMR (400 MHz, CDCl ) δ 13.58 (s, 1H), 11.41 (s, 1H), 8.22 (d, J =
3
.1 Hz, 1H), 7.60 (d, J = 8.9 Hz, 1H), 7.46 (d, J = 6.9 Hz, 1H), 7.39 (d, J = 7.3 Hz,
H), 7.34 (t, J = 7.5 Hz, 2H), 7.28-7.24 (m, 1H), 7.15 (t, J = 7.6 Hz, 1H), 6.89 (s, 1H),
.14 (d, J = 8.7 Hz, 1H), 4.55 (d, J = 5.5 Hz, 2H), 3.50 (dd, J = 10.6, 5.2 Hz, 2H),
13
.60 (s, 3H), 2.53 (t, J = 5.8 Hz, 2H), 2.27 (s, 6H); C NMR (100 MHz, d -DMSO) δ
6
80.4, 169.3, 154.6, 144.6, 138.9, 138.5, 135.1, 134.1, 129.1, 127.8, 127.6, 125.1,
23.7, 121.7, 121.5, 106.2, 101.5, 100.3, 58.6, 46.4, 45.7, 37.6, 17.0; HR-MS(ESI):
+
Calcd for [M+H] 429.2291; Found: 429.2300.
4
9
.1.1.4. N-(2-(dimethylamino)ethyl)-1-((3-methoxybenzyl)amino)-5-methyl-
-oxo-9,10-dihydroacridine-4-carboxamide (8b) yellow solid powder, Yield 27.3%;
1
mp 164-166℃; H NMR (400 MHz, CDCl ) δ 13.58 (s, 1H), 11.43 (s, 1H), 8.24 (d, J
3
=
8.1 Hz, 1H), 7.63 (d, J = 8.9 Hz, 1H), 7.48 (d, J = 7.0 Hz, 1H), 7.28-7.24 (m, 1H),
.16 (t, J = 7.6 Hz, 1H), 6.99 (d, J = 7.6 Hz, 1H), 6.93 (s, 2H), 6.81 (dd, J = 8.1, 2.0
Hz, 1H), 6.17 (d, J = 8.9 Hz, 1H), 4.55 (d, J = 5.7 Hz, 2H), 3.80 (d, J = 13.0 Hz, 3H),
7
1
3
3
.53 (dd, J = 10.8, 5.2 Hz, 2H), 2.62 (s, 3H), 2.57 (t, J = 5.8 Hz, 2H), 2.30 (s, 6H); C
NMR (100MHz, CDCl ) δ 181.2, 169.4, 160.1, 155.3, 144.7, 139.8, 138.6, 133.6,
3

ACCEPTED MANUSCRIPT
1
5
4
4
9
33.4, 129.8, 124.9, 123.9, 121.9, 121.4, 119.4, 112.8, 112.8, 106.8, 101.3, 100.0,
+
7.7, 55.2, 46.8, 45.1, 36.7, 17.1; HR-MS(ESI): Calcd for [M+H] 459.2396; Found:
59.2408.
.1.1.5. N-(2-(dimethylamino)ethyl)-1-((4-methoxybenzyl)amino)-5-methyl-
-oxo-9,10-dihydroacridine-4-carboxamide (8c) yellow solid powder, Yield 54.6%;
1
mp 225-227℃; H NMR (400 MHz, CDCl ) δ 13.59 (s, 1H), 11.34 (s, 1H), 8.22 (d, J
3
=
8.0 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.47 (d, J = 7.0 Hz, 1H), 7.31 (d, J = 8.5 Hz,
H), 7.15 (t, J = 7.6 Hz, 1H), 6.98 (s, 1H), 6.88 (d, J = 8.5 Hz, 2H), 6.18 (d, J = 9.0
Hz, 1H), 4.48 (d, J = 5.5 Hz, 2H), 3.79 (s, 3H), 3.53 (dd, J = 10.7, 5.2 Hz, 2H), 2.61 (s,
2
1
3
3
1
1
H), 2.56 (t, J = 5.7 Hz, 2H), 2.29 (s, 6H); C NMR (100 MHz, CDCl ) δ 181.2,
3
69.4, 158.9, 155.1, 144.8, 138.5, 133.5, 133.5, 130.0, 128.4, 124.9, 123.8, 121.9,
21.4, 114.2, 106.7, 101.1, 99.9, 57.8, 55.3, 46.3, 45.1, 36.8, 17.1; HR-MS(ESI):
+
Calcd for [M+H] 459.2396; Found: 459.2401.
4
9
.1.1.6. N-(3-(dimethylamino)propyl)-1-((4-methoxybenzyl)amino)-5-methyl-
-oxo-9,10-dihydroacridine-4-carboxamide (8d) yellow solid powder, Yield 22.4%;
1
mp 185-187℃; H NMR (400 MHz, CDCl ) δ 13.82 (s, 1H), 11.32 (s, 1H), 8.68 (s,
3
1
H), 8.22 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.47 (d, J = 6.1 Hz, 1H), 7.33
d, J = 7.7 Hz, 2H), 7.16 (d, J = 7.2 Hz, 1H), 6.89 (d, J = 7.6 Hz, 2H), 6.20 (d, J = 8.8
Hz, 1H), 4.49 (s, 2H), 3.80 (s, 3H), 3.59 (br, 2H), 2.62 (br, 5H), 2.39 (s, 6H), 1.84 (br,
(
1
3
2
1
4
H); C NMR (100 MHz, CDCl ) δ 181.2, 169.5, 159.0, 155.0, 144.9, 138.59, 133.5,
3
33.4, 130.0, 128.5, 124.9, 123.8, 121.8, 121.3, 114.2, 106.7, 101.4, 99.8, 59.1, 55.3,
+
6.3, 45.1, 40.1, 24.9, 17.2; HR-MS(ESI): Calcd for [M+H] 473.2552; Found:

ACCEPTED MANUSCRIPT
4
73.2566.
.1.1.7. 1-(benzylamino)-N-(2-methoxyethyl)-5-methyl-9-oxo-9,10-dihydro
4
1
acridine-4-carboxamide (8e) yellow solid powder, Yield 62.1%; mp 191-193℃; H
NMR (400 MHz, CDCl ) δ 13.50 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.8 Hz,
3
1
H), 7.50 (d, J = 7.0 Hz, 1H), 7.41 (d, J = 7.3 Hz, 2H), 7.36 (t, J = 7.4 Hz, 2H), 7.30
d, J = 7.2 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 6.49 (s, 1H), 6.18 (d, J = 8.8 Hz, 1H),
.58 (s, 2H), 3.67 (br, 2H), 3.59 (t, J = 4.7 Hz, 2H), 3.42 (d, J = 8.1 Hz, 3H), 2.62 (s,
(
4
3
1
4
4
9
1
8
7
1
3
1
3
H); C NMR (100 MHz, CDCl ) δ 181.2, 169.3, 155.2, 144.7, 138.5, 137.9, 133.7,
3
33.3, 128.8, 127.4, 127.2, 124.9, 123.9, 121.9, 121.5, 106.8, 101.1, 100.0, 71.3, 58.9,
+
6.88, 39.4, 17.1; HR-MS(ESI): Calcd for [M+H] 416.1974; Found: 416.1980.
.1.1.8. 1-((3-methoxybenzyl)amino)-N-(2-methoxyethyl)-5-methyl-9-oxo-
,10-dihydroacridine-4-carboxamide (8f) yellow solid powder, Yield 61.8%; mp
1
38-140℃; H NMR (400 MHz, CDCl ) δ 13.50 (s, 1H), 11.46 (s, 1H), 8.24 (d, J =
3
.0 Hz, 1H), 7.59 (d, J = 8.9 Hz, 1H), 7.50 (d, J = 6.9 Hz, 1H), 7.31-7.23 (m, 1H),
.18 (t, J = 7.6 Hz, 1H), 7.00 (d, J = 7.6 Hz, 1H), 6.94 (s, 1H), 6.83 (d, J = 8.0 Hz,
H), 6.48 (s, 1H), 6.17 (d, J = 8.9 Hz, 1H), 4.56 (d, J = 5.6 Hz, 2H), 3.80 (s, 3H),
13
.72-3.64 (m, 2H), 3.59 (t, J = 4.7 Hz, 2H), 3.41 (s, 3H), 2.63 (s, 3H); C NMR (100
MHz, CDCl ) δ 181.3, 169.3, 160.0, 155.3, 144.7, 139.7, 138.5, 133.7, 133.3, 129.9,
3
1
4
4
9
24.9, 123.8, 121.8, 121.5, 119.4, 112.8, 112.7, 106.8, 101.1, 100.0, 71.3, 58.9, 55.3,
+
6.8, 39.4, 17.2; HR-MS(ESI): Calcd for [M+H] 446.2080; Found: 446.2082.
.1.1.9. 1-((4-methoxybenzyl)amino)-N-(2-methoxyethyl)-5-methyl-9-oxo-
,10-dihydroacridine-4-carboxamide (8g) yellow solid powder, Yield 38.6%; mp

ACCEPTED MANUSCRIPT
1
2
7
1
32-234℃; H NMR (400 MHz, CDCl ) δ 13.49 (s, 1H), 8.22 (d, J = 7.5 Hz, 1H),
3
.60 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 5.4 Hz, 1H), 7.32 (d, J = 7.6 Hz, 2H), 7.16 (s,
H), 6.89 (d, J = 7.5 Hz, 2H), 6.50 (s, 1H), 6.20 (d, J = 8.1 Hz, 1H), 4.49 (s, 2H), 3.80
1
3
(
s, 3H), 3.66 (br, 2H), 3.59 (br, 2H), 3.41 (s, 3H), 2.61 (s, 3H); C NMR (100 MHz,
CDCl ) δ 181.2, 169.3, 159.0, 155.1, 144.7, 138.5, 133.6, 133.2, 129.8, 128.5, 124.9,
3
1
23.8, 121.9, 121.5, 114.2, 106.7, 101.1, 100.0, 71.3, 58.9, 55.31, 46.4, 39.4, 17.1;
+
HR-MS(ESI): Calcd for [M+H] 446.2080; Found: 446.2078.
4
.1.1.10. N-(2-methoxyethyl)-5-methyl-9-oxo-1-((4-(trifluoromethyl)benzyl)
amino)-9,10-dihydroacridine-4-carboxamide (8h) yellow solid powder, Yield
1
3
5.6%; mp 260-262℃; H NMR (400 MHz, CDCl ) δ 13.51 (s, 1H), 8.24 (d, J = 8.1
3
Hz, 1H), 7.61-7.58 (m, 3H), 7.52-7.50 (m, 3H), 7.22-7.15 (m, 1H), 6.49 (s, 1H), 6.08
(d, J = 8.9 Hz, 1H), 4.64 (s, 2H), 3.66 (d, J = 4.6 Hz, 2H), 3.60-3.53 (m, 2H), 3.40 (s,
1
3
3
1
9
H), 2.62 (s, 3H); C NMR (100 MHz, CDCl ) δ 181.3, 169.2, 155.1, 144.7, 142.2,
3
38.5, 133.8, 133.3, 127.3, 125.8, 125.7, 125.0, 123.8, 121.8, 121.6, 106.9, 101.7,
1
9
9.7, 71.2, 58.9, 46.4, 39.4, 17.1; F NMR (376 MHz, CDCl ) δ -62.45; HR-MS(ESI):
3
+
Calcd for [M+H] 484.1848; Found: 484.1856.
4
9
2
7
2
3
.1.1.11. N-(2-methoxyethyl)-5-methyl-9-oxo-1-((3,4,5-trimethoxybenzyl)amino)-
,10-dihydroacridine-4-carboxamide (8i) yellow solid powder, Yield 68.0%; mp
1
30-232℃; H NMR (400 MHz, CDCl ) δ 13.53 (s, 1H), 8.25 (d, J = 7.8 Hz, 1H),
3
.64 (d, J = 8.6 Hz, 1H), 7.52 (d, J = 6.6 Hz, 1H), 7.20 (t, J = 7.4 Hz, 1H), 6.64 (s,
H), 6.54 (s, 1H), 6.21 (d, J = 8.5 Hz, 1H), 4.52 (s, 2H), 3.86 (s, 9H), 3.69 (br, 2H),
1
3
.61 (br, 2H), 3.43 (s, 3H), 2.64 (s, 3H); C NMR (100 MHz, CDCl ) δ 181.2, 169.3,
3

ACCEPTED MANUSCRIPT
1
1
55.1, 153.6, 144.7, 138.5, 137.2, 133.7, 133.7, 133.3, 125.0, 123.8, 121.8, 121.6,
06.8, 104.1, 101.5, 100.2, 71.3, 60.9, 58.9, 56.2, 47.4, 39.4, 17.1; HR-MS(ESI):
+
Calcd for [M+H] 506.2291; Found: 506.2293.
4
.1.1.12. N-(2-(dimethylamino)ethyl)-5-methyl-9-oxo-1-((3,4,5-trimethoxybenzyl)
amino)-9,10-dihydroacridine-4-carboxamide (8j) yellow solid powder, Yield 33.1%;
1
mp 195-197℃; H NMR (400 MHz, CDCl ) δ 13.61 (s, 1H), 11.38 (s, 1H), 8.23 (d, J
3
=
8.1 Hz, 1H), 7.71 (d, J = 8.9 Hz, 1H), 7.49 (d, J = 6.9 Hz, 1H), 7.23-7.13 (m, 1H),
7
9
.06 (s, 1H), 6.62 (s, 2H), 6.18 (d, J = 8.9 Hz, 1H), 4.50 (d, J = 5.5 Hz, 2H), 3.84 (s,
1
3
H), 3.56 (dd, J = 10.6, 5.1 Hz, 2H), 2.62 (br, 5H), 2.35 (s, 6H); C NMR (100 MHz,
CDCl ) δ 181.3, 169.4, 155.2, 153.6, 144.7, 138.5, 137.3, 133.8, 133.6, 133.6, 124.9,
3
1
23.8, 121.8, 121.4, 106.8, 104.1, 101.4, 100.1, 60.8, 57.8, 56.2, 47.2, 45.0, 36.6, 17.1;
+
HR-MS(ESI): Calcd for [M+H] 519.2607; Found: 519.2623.
4
.1.1.13. 1-(benzylamino)-N-(2-(dimethylamino)ethyl)-5-methoxy-9-oxo-9,10-
dihydroacridine-4-carboxamide (8k) yellow solid powder, Yield 57.1%; mp
1
2
8
2
30-231℃; H NMR (400 MHz, CDCl ) δ 13.61 (s, 1H), 11.44 (s, 1H), 7.94 (d, J =
3
.1 Hz, 1H), 7.67 (d, J = 8.9 Hz, 1H), 7.42 (d, J = 7.2 Hz, 2H), 7.36 (t, J = 7.3 Hz,
H), 7.30 (d, J = 7.2 Hz, 1H), 7.18 (t, J = 7.9 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H), 6.97
(s, 1H), 6.17 (d, J = 8.9 Hz, 1H), 4.57 (d, J = 5.4 Hz, 2H), 4.09 (s, 3H), 3.56 (d, J =
1
3
5
1
1
.2 Hz, 2H), 2.56 (t, J = 5.5 Hz, 2H), 2.29 (s, 6H); C NMR (100 MHz, CDCl ) δ
3
80.9, 169.1, 155.2, 148.0, 144.2, 138.1, 133.6, 131.0, 128.8, 127.3, 127.2, 122.5,
21.1, 117.1, 111.3, 107.2, 101.8, 99.8, 57.9, 56.3, 46.9, 45.1, 36.8; HR-MS(ESI):
+
Calcd for [M+H] 445.2240; Found: 445.2252.

ACCEPTED MANUSCRIPT
4
.1.1.14. N-(2-(dimethylamino)ethyl)-5-methoxy-1-((4-methoxybenzyl)
amino)-9-oxo-9,10-dihydroacridine-4-carboxamide (8l) yellow solid powder, Yield
1
7
7
8
0.8%; mp 245-246℃; H NMR (400 MHz, CDCl ) δ 13.57 (s, 1H), 11.36 (s, 1H),
3
.92 (d, J = 8.1 Hz, 1H), 7.67 (d, J = 8.9 Hz, 1H), 7.32 (d, J = 8.4 Hz, 2H), 7.17 (t, J =
.0 Hz, 1H), 7.07 (d, J = 7.5 Hz, 1H), 6.96 (s, 1H), 6.89 (d, J = 8.5 Hz, 2H), 6.20 (d, J
=
8.9 Hz, 1H), 4.49 (d, J = 5.4 Hz, 2H), 4.08 (s, 3H), 3.80 (s, 3H), 3.61-3.48 (m, 2H),
13
2
.60 (t, J = 5.6 Hz, 2H), 2.33 (s, 6H); C NMR (100 MHz, CDCl ) δ 180.8, 169.1,
3
1
58.9, 155.2, 147.9, 144.2, 133.6, 131.0, 130.0, 128.4, 122.5, 121.0, 117.1, 114.2,
111.3, 107.2, 101.6, 99.8, 57.9, 56.3, 55.3, 46.4, 45.0, 36.6; HR-MS(ESI): Calcd for
+
[
M+H] 475.2345; Found: 475.2336.
4
9
.1.1.15. N-(3-(dimethylamino)propyl)-5-methoxy-1-((4-methoxybenzyl)amino)-
-oxo-9,10-dihydroacridine-4-carboxamide (8m) Yellow solid powder, Yield 63.4%;
1
mp 260-261℃; H NMR (400 MHz, CDCl ) δ 13.84 (s, 1H), 11.32 (s, 1H), 8.66 (s,
3
1
H), 7.91 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 8.9 Hz, 1H), 7.33 (d, J = 8.5 Hz, 2H), 7.16
t, J = 8.0 Hz, 1H), 7.07 (d, J = 7.5 Hz, 1H), 6.89 (d, J = 8.5 Hz, 2H), 6.18 (d, J = 8.9
Hz, 1H), 4.49 (d, J = 5.4 Hz, 2H), 4.07 (s, 3H), 3.80 (s, 3H), 3.60 (dd, J = 10.4, 5.4 Hz,
(
1
3
2
H), 2.59-2.50 (m, 2H), 2.33 (s, 6H), 1.84-1.72 (m, 2H); C NMR (100 MHz, CDCl )
3
δ 180.8, 169.1, 158.9, 154.9, 148.0, 144.3, 133.3, 131.0, 130.0, 128.5, 122.4, 121.0,
17.0, 114.2, 111.1, 107.2, 101.9, 99.6, 59.7, 56.2, 55.3, 46.4, 45.5, 40.7, 25.0;
1
+
HR-MS(ESI): Calcd for [M+H] 489.2502; Found: 489.2493.
.1.1.16. N-(2-(dimethylamino)ethyl)-5-methoxy-9-oxo-1-((3,4,5-trimethoxy
benzyl)amino)-9,10-dihydroacridine-4-carboxamide (8n) yellow solid powder,
4

ACCEPTED MANUSCRIPT
1
Yield 57.3%; mp 257-259℃; H NMR (400 MHz, CDCl ) δ 13.60 (s, 1H), 11.39 (t, J
3
=
5.4 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.19 (t, J = 8.0 Hz,
H), 7.09 (d, J = 7.7 Hz, 1H), 6.98 (s, 1H), 6.63 (s, 2H), 6.19 (d, J = 8.9 Hz, 1H), 4.50
d, J = 5.6 Hz, 2H), 4.09 (s, 3H), 3.85 (s, 9H), 3.57 (dd, J = 10.8, 5.2 Hz, 2H), 2.60 (t,
1
(
13
J = 5.7 Hz, 2H), 2.33 (s, 6H); C NMR (100 MHz, CDCl ) δ 180.9, 169.1, 155.2,
3
1
1
53.6, 148.0, 144.1, 137.1, 133.8, 133.7, 131.0, 122.4, 121.2, 117.0, 111.3, 107.2,
04.0, 101.8, 100.0, 60.9, 57.8, 56.3, 56.2, 47.3, 45.1, 36.7; HR-MS(ESI): Calcd for
+
[
M+H] 535.2557; Found: 535.2556.
4
9
.1.1.17. 5,7-dichloro-N-(2-(dimethylamino)ethyl)-1-((4-methoxybenzyl)amino)-
-oxo-9,10-dihydroacridine-4-carboxamide (8o) yellow solid powder, Yield 69.6%;
1
mp 238-240℃; H NMR (400 MHz, CDCl ) δ 11.11 (d, J = 5.1 Hz, 1H), 8.23 (d, J =
3
2
.3 Hz, 1H), 7.70-7.67 (m, 2H), 7.32 (d, J = 8.6 Hz, 2H), 7.02 (s, 1H), 6.91 (dd, J =
.7, 4.8 Hz, 2H), 6.25 (d, J = 9.0 Hz, 1H), 4.50 (d, J = 5.5 Hz, 2H), 3.81 (s, 3H), 3.56
6
1
3
(
dd, J = 10.7, 5.3 Hz, 2H), 2.62-2.55 (m, 2H), 2.32 (s, 6H); C NMR (100 MHz,
CDCl ) δ 179.2, 168.9, 159.1, 154.9, 144.6, 135.4, 134.0, 132.5, 129.6, 128.4, 126.7,
3
1
24.4, 123.4, 122.5, 114.3, 106.7, 101.5, 100.9, 57.7, 55.3, 46.4, 45.1, 36.7;
+
HR-MS(ESI): Calcd for [M+H] 513.1460; Found: 513.1467.
4
.1.1.18. 1-(benzylamino)-N-(2-(dimethylamino)ethyl)-5-nitro-9-oxo-9,10-dihydro
1
acridine-4-carboxamide (8p) Red solid powder, Yield 33.8%; mp 243-245℃; H
NMR (400 MHz, d -DMSO) δ 15.00 (s, 1H), 10.92 (s, 1H), 8.65 (d, J = 7.2 Hz, 1H),
6
8
.57 (d, J = 7.2 Hz, 1H), 8.46 (s, 1H), 8.03 (d, J = 8.8 Hz, 1H), 4.40-7.37 (m, 5H),
.29 (d, J = 6.5 Hz, 1H), 6.45 (d, J = 8.6 Hz, 1H), 4.61 (s, 2H), 2.53 (br, 2H), 2.27 (s,
7

ACCEPTED MANUSCRIPT
1
3
6
1
3
4
9
1
7
6
1
2
1
1
4
4
9
2
6
2
H); C NMR (100 MHz, d -DMSO) δ 178.8, 168.2, 154.3, 138.7, 135.9, 135.2,
6
34.4, 131.7, 129.2, 127.8, 127.7, 124.2, 120.7, 106.5, 103.2, 102.5, 58.4, 46.4, 45.4,
+
7.3; HR-MS(ESI): Calcd for [M+H] 460.1985; Found: 460.2000.
.1.1.19. N-(2-(dimethylamino)ethyl)-1-((3-methoxybenzyl)amino)-5-nitro-9-oxo-
,10-dihydroacridine-4-carboxamide (8q) Red solid powder, Yield 19.8%; mp
1
55-157℃; H NMR (400 MHz, CDCl ) δ 15.02 (s, 1H), 11.08 (s, 1H), 8.71 (d, J =
3
.6 Hz, 1H), 8.66 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.30-7.26 (m, 2H),
.98 (d, J = 7.4 Hz, 2H), 6.92 (s, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.29 (d, J = 8.9 Hz,
H), 4.54 (d, J = 5.3 Hz, 2H), 3.80 (s, 3H), 3.59 (d, J = 4.6 Hz, 2H), 2.65-2.56 (m,
1
3
H), 2.31 (s, 6H); C NMR (100 MHz, CDCl ) δ 179.4, 168.2, 160.1, 154.7, 144.1,
3
39.3, 135.0, 134.9, 134.5, 134.4, 131.2, 130.0, 124.6, 119.8, 119.3, 112.9, 112.8,
+
07.1, 103.3, 102.2, 57.8, 55.3, 46.9, 45.1, 36.8; HR-MS(ESI): Calcd for [M+H]
90.2090; Found: 490.2091.
.1.1.20. N-(2-(dimethylamino)ethyl)-1-((4-methoxybenzyl)amino)-5-nitro-9-oxo-
,10-dihydroacridine-4-carboxamide (8r) Red solid powder, Yield 27.7%; mp
1
28-230℃; H NMR (400 MHz, CDCl ) δ 15.01 (s, 1H), 10.99 (s, 1H), 8.69 (d, J =
3
.8 Hz, 1H), 8.65 (d, J = 7.5 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.31 (d, J = 7.5 Hz,
H), 7.12 (s, 1H), 6.90 (d, J = 7.3 Hz, 2H), 6.31 (d, J = 8.2 Hz, 1H), 4.48 (s, 2H), 3.80
1
3
(
s, 3H), 3.62 (br, 2H), 2.65 (br, 2H), 2.37 (s, 6H); C NMR (100 MHz, CDCl ) δ
3
1
1
79.3, 168.2, 159.1, 154.6, 144.1, 135.0, 134.8, 134.5, 134.4, 131.1, 129.5, 128.5,
24.6, 119.7, 114.3, 106.9, 103.0, 102.1, 57.9, 55.3, 46.4, 45.0, 36.6; HR-MS(ESI):
+
Calcd for [M+H] 490.2090; Found: 490.2099.

ACCEPTED MANUSCRIPT
4
.1.1.21. N-(2-(dimethylamino)ethyl)-5-nitro-9-oxo-1-((3,4,5-trimethoxybenzyl)
amino)-9,10-dihydroacridine-4-carboxamide (8s) Red solid powder, Yield 49.4%;
1
H NMR (400 MHz, CDCl ) δ 15.04 (s, 1H), 11.01 (s, 1H), 8.68 (dd, J = 15.5, 7.3 Hz,
3
2
H), 7.71 (d, J = 8.7 Hz, 1H), 7.30-7.28(m, 1H), 6.95 (s, 1H), 6.61 (s, 2H), 6.30 (d, J
8.7 Hz, 1H), 4.48 (d, J = 4.0 Hz, 2H), 3.85 (s, 9H), 3.59 (br, 2H), 2.57 (br, 2H), 2.31
=
1
3
(
s, 6H); C NMR (100 MHz, CDCl ) δ 179.4, 168.1, 154.6, 153.7, 144.0, 137.4,
3
1
6
5
4
35.0, 134.8, 134.4, 134.4, 133.2, 131.2, 124.6, 119.8, 107.0, 104.2, 103.4, 102.1,
+
0.9, 57.7, 56.2, 47.4, 45.1, 36.8; mp 243-244℃; HR-MS(ESI): Calcd for [M+H]
50.2302; Found: 550.2308.
.1.1.22. 1-(benzylamino)-N-(3-(dimethylamino)propyl)-5-nitro-9-oxo-9,10-
dihydroacridine-4-carboxamide (8t) Red solid powder, Yield 73.7%; mp 222-224℃;
1
H NMR (400 MHz, CDCl ) δ 15.25 (s, 1H), 11.06 (s, 1H), 8.71 (d, J = 7.6 Hz, 2H),
3
8
2
2
1
1
.65 (d, J = 7.9 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.46-7.33 (m, 4H), 7.33-7.26 (m,
H), 6.31 (d, J = 8.9 Hz, 1H), 4.57 (d, J = 5.3 Hz, 2H), 3.64 (br, 2H), 2.58 (br, 2H),
1
3
.35 (s, 6H), 1.82 (br, 2H); C NMR (100 MHz, CDCl ) δ 179.4, 168.1, 154.5, 144.2,
3
37.6, 135.1, 134.9, 134.4, 134.1, 131.1, 128.9, 127.5, 127.2, 124.6, 119.6, 107.1,
+
03.6, 102.0, 59.4, 46.9, 45.3, 40.5, 24.9; HR-MS(ESI): Calcd for [M+H] 474.2141;
Found: 474.2126.
4
9
.1.1.23. N-(3-(dimethylamino)propyl)-1-((4-methoxybenzyl)amino)-5-nitro-
-oxo-9,10-dihydroacridine-4-carboxamide (8u) Red solid powder, Yield 63.9%;
1
mp 251-252℃; H NMR (400 MHz, CDCl ) δ 15.27 (s, 1H), 10.97 (s, 1H), 8.76 (s,
3
1
H), 8.70 (d, J = 7.6 Hz, 1H), 8.65 (d, J = 7.9 Hz, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.33

ACCEPTED MANUSCRIPT
(d, J = 8.2 Hz, 2H), 7.27-7,24 (m, 1H), 6.91 (d, J = 8.3 Hz, 2H), 6.32 (d, J = 8.9 Hz,
1
1
1
5
5
4
9
2
1
7
H), 4.49 (d, J = 5.1 Hz, 2H), 3.81 (s, 3H), 3.64 (br, 2H), 2.56 (br, 2H), 2.35 (s, 6H),
1
3
.80 (br, 2H); C NMR (100 MHz, CDCl ) δ 179.4, 168.1, 1591, 154.4, 144.2, 135.1,
3
34.85, 134.4, 134.1, 131.1, 129.5, 128.5, 124.6, 119.6, 114.3, 107.0, 103.4, 101.9,
+
9.6, 55.3, 46.5, 45.4, 40.7, 24.9; HR-MS(ESI): Calcd for [M+H] 504.2247; Found:
04.2245.
.1.1.24. N-(3-(dimethylamino)propyl)-1-((4-ethylbenzyl)amino)-5-nitro-9-oxo-
,10-dihydroacridine-4-carboxamide (8v) Red solid powder, Yield 24.0%; mp
1
66-267℃; H NMR (400 MHz, CDCl ) δ 15.27 (s, 1H), 11.03 (s, 1H), 8.79-8.71 (m,
3
H), 8.66 (dd, J = 8.1, 1.4 Hz, 1H), 7.69 (d, J = 9.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 2H),
.28 (d, J = 8.1 Hz, 1H), 7.21 (d, J = 8.0 Hz, 2H), 6.35 (d, J = 9.1 Hz, 1H), 4.53 (d, J
=
5.5 Hz, 2H), 3.66-3.65 (m, 2H), 2.68-2.66 (m, 2H), 2.65-2.63 (m, 2H), 2.41 (s, 6H),
1
3
1
1
1
.86-1.85 (m, 2H), 1.24 (t, J = 7.6 Hz, 3H); C NMR (100 MHz, CDCl ) δ 179.4,
3
68.3, 154.6, 144.3, 143.6, 135.1, 134.9, 134.7, 134.4, 134.31, 131.1, 128.4, 127.2,
24.6, 119.6, 107.0, 103.3, 102.1, 59.0, 46.8, 45.0, 40.0, 28.5, 24.8, 15.5;
+
HR-MS(ESI): Calcd for [M+H] 502.2454; Found: 502.2467.
4
.1.2. General procedure for compound 8w.
Compound 8u (0.18 mmol) and iron powder (0.89 mmol) was stirred in
anhydrous ethanol (20 mL) and heated at 50℃ for 20 minutes. Subsequently, an
aqueous solution of ammonium chloride was added to the reaction system (0.014 g
ammonium chloride in 3 mL water, 0.27 mmol), the temperature was raised to 80 ° C
and stirring for 3 hours. The suspension was cooled to room temperature and the

ACCEPTED MANUSCRIPT
mixture was filtered on cellite to remove the iron. The filtrate 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 8w.
5
-amino-N-(3-(dimethylamino)propyl)-1-((4-methoxybenzyl)amino)-9-oxo-9,10-d
ihydroacridine-4-carboxamide (8w) yellow solid powder, Yield 47.3%; mp
1
2
20-222℃; H NMR (400 MHz, CDCl ) δ 13.91 (s, 1H), 11.31 (s, 1H), 8.68 (br, 1H),
3
7
.87 (br, 1H), 7.58 (br, 1H), 7.33 (d, J = 8.0 Hz, 2H), 7.06 (br, 2H), 6.90 (br, 2H), 6.19
(d, J = 8.0 Hz, 1H), 4.49 (br, 2H), 3.80 (br, 5H), 3.58 (br, 2H), 2.59 (br, 2H), 2.38 (s,
1
3
6
1
9
H), 1.82 (br, 2H); C NMR (100 MHz, CDCl ) δ 181.1, 169.5, 159.0, 155.0, 144.7,
3
34.0, 133.2, 130.9, 130.0, 128.5, 122.5, 121.6, 118.9, 117.1, 114.2, 106.8, 101.3,
+
9.7, 59.2, 55.3, 46.3, 45.2, 40.2, 24.9; HR-MS(ESI): Calcd for [M+H] 474.2505;
Found: 474.2514.
4
.2. Molecular docking
The molecular modeling of small molecule compounds were performed with the
molecular modeling package SYBYL-X 1.3 (Tripos associate Inc., St. Louis, MO,
USA) according to the reported process [46]. Briefly, the three-dimensional
coordinates of survivin protein were acquired from PDB (PDB ID: 1F3H). Only one
of the two chains was kept and the protein chain was prepared for docking. The
general procedure is as followed: (a) removing the water molecules which
co-crystallized with the original protein structure; (b) preparing ligand and receptor
and then finding the candidate binding site; (c) docking the test compounds; (d)
analysis of results.

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4
.3. In silico physicochemical, pharmacokinetics and toxicity analysis
Physicochemical, pharmacokinetic and toxicity parameters for selected
compound were predicted in silico using Advanced Chemistry Development, Inc.
(
ACD/Labs, ACD/Percepta Platform, version 14.0.0), and Osiris Property Explorer
http://www.organic-chemistry.org/prog/peo).
(
4
4
.4. Bioassay
.4.1. Cell culture
HepG-2, MCF-7 and QGY-7701 (adherent cell lines) were cultured in DMEM,
with 10% fetal bovine serum (FBS) in humidified air at 37 ºC with 5% CO2.
4
.4.2. Cell growth inhibition assay
HepG2 cells, MCF-7 cells and QGY-7701 cells were seeded into 96-well plates at
3
6
×10 cells/well, treated with the synthesized compounds at different concentrations
after these adherent cells hatched for 12 hours at 37 ºC, 5% CO . (The final
2
concentrations of these compounds were 50, 25, 10, 1 and 0.1 µM). After 48 h
treatment, the cells were incubated with 10 µL MTT solution (5 mg/mL) for 4 h at 37
ºC, 5% CO .The formazan precipitates were dissolved in 100 µL DMSO. At 490 nm,
2
the absorbance was measured by Infinite M1000 PRO (TECAN).
4
4
.5. Biophysical evaluation
.5.1. DNA binding experiment
Concentrated stock solutions of compounds were prepared by dissolving them in
DMSO. Calf thymus DNA (ct DNA) was obtained from Sigma Chemical Co. All the
measurements involving the interactions of tested compound 8u with ct DNA were

ACCEPTED MANUSCRIPT
carried out in doubly distilled water buffer containing 5 mM Tris and 50 mM NaCl,
and adjusted to pH 7.2 with hydrochloric acid. Stock solutions of ct DNA were
prepared in buffer and concentration was determined by UV absorbance by employing
-1
-1
an extinction coefficient of 6600 M cm at 260 nm.
The emission spectra were carried out on Fluorolog spectrometer. 16 µL of tested
compound 8u (hydrochloride salt, 5 mM in DMSO) was incubated in Tris-HCl buffer
solution (the final concentration of the tested compound was 40 µM), and then ct
DNA solution (2 mM in Tris-HCl buffer) was also added with the final concentration
from 0 to 120 µM. The excitation wavelength was set at 280 nm. The incubating time
before testing was 5 min.
4
.5.2. DNA Topo I inhibition assay
The solutions of a mixture of 100 ng of plasmid DNA pBR322 (commercial
available from Takara), 1.0 units of recombinant human DNA Topo I (from Takara)
and with compounds of different amount were incubated at 37 ºC for 30 min in the
relaxation buffer (35 mM Tris-HCl (pH 8.0), 5 mM MgCl , 72 mM KCl, 0.01%
2
bovine serum albumin, 2 mM spermidine, 5 mM dithiothreitol). DNA samples were
then electrophoresed on a 1% agarose gel at 100 V for 25 min with a running Buffer
(Tris-Acetic acid-EDTA). Gels were visualized by EB staining under ultraviolet light.
4
.5.3. DNA Topo II inhibition assay
DNA Topo II inhibition assay were performed by HD Biosciences Corporation,
Shanghai. The main steps as follows: (a) Transfer 1 µl of compound or 50% DMSO
into 96-well plates; (b) Add 19 µl reaction mix; (c) Add 5 µl of diluted enzyme in the