Synthesis and antiproliferative activity of 9-benzylamino-6-chloro-2-methoxy-acridine derivatives as potent DNA-binding ligands and topoisomerase II inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.03.066

A series of 9-benzylamino acridine derivatives were synthesized as an extension of our discovery of acridine antitumor agents. Most of these acridine compounds displayed good antiproliferative activity with IC₅₀ values in low micromole range and structure-activity relationships were studied. Topo I- and II- mediated relaxation studies suggested that all of our compounds displayed strong Topo II inhibitory activity at 100 μM, while only four exhibited moderate Topo I inhibitory activity. The typical compound 8p could penetrate A549 cancer cells efficiently. Compound 8p could intercalate within the double-stranded DNA structure and induce DNA damage. Moreover, compound 8p could induce A549 cells apoptosis through caspase-dependent intrinsic pathway and arrest A549 cells at the G2/M phase.

Full text of DOI:10.1016/j.ejmech.2016.03.066

Accepted Manuscript
Synthesis and Antiproliferative Activity of 9-Benzylamino-6-chloro-2-methoxy-acridine
Derivatives as Potent DNA-binding Ligands and Topoisomerase II Inhibitors
Wei Zhang, Bin Zhang, Wei Zhang, Ti Yang, Ning Wang, Chunmei Gao, Chunyan
Tan, Hongxia Liu, Yuyang Jiang
PII:
S0223-5234(16)30248-3
10.1016/j.ejmech.2016.03.066
EJMECH 8491
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 December 2015
Revised Date: 11 March 2016
Accepted Date: 25 March 2016
Please cite this article as: W. Zhang, B. Zhang, W. Zhang, T. Yang, N. Wang, C. Gao, C. Tan, H.
Liu, Y. Jiang, Synthesis and Antiproliferative Activity of 9-Benzylamino-6-chloro-2-methoxy-acridine
Derivatives as Potent DNA-binding Ligands and Topoisomerase II Inhibitors, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.066.
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ACCEPTED MANUSCRIPT
Synthesis and Antiproliferative Activity of
9-Benzylamino-6-chloro-2-methoxy-acridine Derivatives as Potent DNA-binding
Ligands and Topoisomerase II Inhibitors
A series of 9-benzyl acridine derivatives were synthesized and evaluated as good
antiproliferative inhibitors. All of them displayed strong Topo II inhibitory activity at
100 µM. The typical compound 8p showed potent DNA-binding ability, caused DNA
double-strand breaks and induced A549 cells apoptosis through caspase-dependent
intrinsic pathway.

ACCEPTED MANUSCRIPT
Synthesis and Antiproliferative Activity of
9-Benzylamino-6-chloro-2-methoxy-acridine Derivatives as Potent DNA-binding
Ligands and Topoisomerase II Inhibitors
Wei Zhanga, b, c, Bin Zhangc, Wei Zhangb, c, Ti Yanga, Ning Wanga, b, Chunmei Gaoa,b*
Chunyan Tan b, d, Hongxia Liu b, Yuyang Jianga, b, d*
,
aTsinghua University, Department of Chemistry, Beijing 100084, PR China
bThe Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of
Chemical Biology, the Graduate School at Shenzhen, Tsinghua University, Shenzhen 518-55, P. R.
China
cShenzhenAnti-Tumor Drug Development Engineering Laboratory, the Graduate School at Shenzhen,
Tsinghua University, Shenzhen 518055, P. R. China
dDepartment of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University,
Beijing, 100084, P. R. China
Keywords
Acridine derivatives; Topoisomerase II inhibitor; Antiproliferative activity; DNA
damage
* Corresponding authors. Tel./Fax: +86 (755) 26032094
e-mail:chunmeigao@sz.tsinghua.edu.cn, jiangyy@sz.tsinghua.edu.cn
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Abstract
A series of 9-benzylamino acridine derivatives were synthesized as an extension of
our discovery of acridine antitumor agents. Most of these acridine compounds
displayed good antiproliferative activity with IC50 values in low micromole range and
structure-activity relationships were studied. Topo I- and II- mediated relaxation
studies suggested that all of our compounds displayed strong Topo II inhibitory
activity at 100 ꢀM, while only four exhibited moderate Topo I inhibitory activity. The
typical compound 8p could penetrate A549 cancer cells efficiently. Compound 8p
could intercalate within the double-stranded DNA structure and induce DNA damage.
Moreover, compound 8p could induce A549 cells apoptosis through
caspase-dependent intrinsic pathway and arrest A549 cells at the G2/M phase.
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1. Introduction
About a century ago, biologists have suggested that there might be a relationship
between the pathogenesis of cancer and “specific and abnormal chromosome
constitution” [1, 2]. The alterations in DNA structure as well as changes in
chromosome structure and number are collectively known as genomic instability,
which has been recognized as the most pervasive characteristic of most cancers
nowadays [3]. As DNA damages such as single-strand breaks (SSBs), double-strand
breaks (DSBs) might lead to cell death, cells possess complex mechanisms to respond
to those damages to maintain genomic integrity and cell alive [4]. For the sake of
killing cancer cells, both DNA and DNA replication related enzymes can serve as
targets for cancer therapy and practiced clinically for decades [3, 5-7]. Among those
enzymes, topoisomerases are studied for many years with more prominent
effectiveness. Both topoisomerase I (Topo I) and topoisomerase II (Topo II) with the
ability to catalyze DNA breakage and religation, facilitate the replication and
transcription of DNA, which is essential to mammalian cell division cycle [8, 9]. The
following steps were involved in the action of topo enzymes. Firstly, enzymes bind
DNA duplex to their DNA-binding and cleavage core, then produce DNA breakage
and form a covalent transient DNA-enzyme complex to change the topological
structure of DNA. Topo I and Topo II are different here in that Topo I mediates single
strand break of a DNA double helix to let another strand across, while Topo II
mediates DNA double strand break to pass another noncleaved duplex. At last
enzymes get reset after reseal the cleaved DNA backbone [10]. Numerous anticancer
drugs used in clinic are those which can interact with DNA and topoisomerases to
induce DNA damage and prevent DNA lesion repairment [5, 11]. For example,
camptothecin causes SSBs by stabilizing DNA-Topo I-drug complex, etoposide
causes DSBs by stabilizing DNA-Topo II-drug complex [12-14].
Acridines (Figure 1, acridine) are firstly used as antibacterial and antiparasite
agents with planar aromatic structures that are capable to intercalate into DNA base
pairs. Since its anticancer effect has been noticed, considerable researches have been
conducted to synthesize more effective and less toxic acridines as anticancer agents
[15-17]. Amsacrine (Figure 1, m-AMSA) was firstly been proved to interact with
Topo II-DNA complex exhibiting clinical efficiency as an antileukaemic agent [15].
For years, m-AMSA and its analogues were intensely studied [18, 19]. Most of the
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acridine derivatives are focused on the 9-anilino acridine scaffold, paying more
attention on the substituents effects in the acridine and anilino rings on the DNA
binding ability and cytotoxicity [20-24]. Recently we found that the linkers between
acridine and benzene groups affected the bioactivity greatly, among which –NHCH2
was the best. In addition, our work as well as references indicated that acridines with
chloro and methoxy groups substituted at C-2 and C-6 positions of acridine ring gave
a favorable antiproliferative activity in vitro (Figure 1, compound X) [25, 26]. As our
continuous efforts for developing new anticancer compounds [26-32], we try to
modify the benzyl ring of 3 to generate compounds with more biological activity,
since the substitution pattern on the 9-anilinogroup played an important role on the
antitumor activity [18, 19, 21, 33-35]. In this work, various 9-benzylamine acridine
derivatives (Table 1; 8 (a–w)) with different substituents in the benzyl group were
synthesized for the discovery of potent Topo II targeted anticancer agents and
evaluated the structure-activity relationships.
Figure 1. Chemical structures: 1, acridine, 2, m-AMSA, 3, compound X.
2. Results and discussion
2.1. Chemistry
Synthesis of the 9-benzylamine acridine derivatives 8 (a-w) were carried out as
shown in Scheme 1 as previously described [25]. Utilizing the Ullmann reaction the
anthralic acid 6 was obtained in high yields using Cu as the catalyst refluxed in DMF
under basic condition, which was cyclized to the 9-chloroacridine derivative 7.
Reactions of compounds 7 and various substituted benzyl amines were carried out in
the presence of a catalytic amount of KI under basic conditions in absolute ethanol to
give the desired corresponding 8 (a-w).
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Scheme 1. Reagents and conditions: (i) Cu, K2CO3, DMF, reflux; (ii) POCl3, reflux; (iii) various
benzyl amines, KI, K2CO3, C2H5OH, reflux.
2.2. In vitro cytotoxicity
With the chemical library of 8 (a-w) in hand, the cell proliferation inhibitory
activities of all compounds were evaluated using human lung cancer cell line A549,
among which, five compounds 8n, 8p, 8s, 8u and 8v displayed significant
antiproliferative activity with IC50 values less than 1 ꢀM (Table 1). As shown from
Table 1, although the cytotoxicities typically did not differ more than factor 10, the
observed antiproliferative activities were dependent on the nature of the substituents
and the substitution pattern on the benzyl ring.
As fluorinated compounds have a remarkable record in medicinal chemistry,
compounds 8a-8d were firstly synthesized to evaluate their antiproliferative activity.
The results indicated that the meta- or para-position is less preferable than
ortho-position (8a vs 8b and 8c). To our surprise, the cytotoxicity was lost when both
meta- and para-position were substituted with fluorines (8d). The replacement of
fluorine by chlorine or bromine resulted in little change in the antiproliferative
activity (8b vs 8e, 8c vs 8f and 8h, 8a vs 8g).
In addition, alkyl or alkoxyl groups were also played an important role in the
activity. The introduction of methyl or ethyl group resulted in a little increase in the
antiproliferative activity than the corresponding halogen substituted acridine
derivatives (8i vs 8b and 8e, 8j and 8k vs 8c and 8f). The replacement of ethyl by
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butyl on para-position led to a significant decrease of activity (8l vs 8k). The
excrescent methoxy(s) was (were) not help to improve the activity (8p vs 8q, 8r, 8t),
and the replacement of two hydrogens of methoxy group (8p) by two fluorines (8u)
did not change the IC50 value so much. Thus we surmised that substituents with
higher steric hindrance were not preferred. The replacement of polar methoxy group
(8p) by methyl (8j) / ethyl group (8k) or the replacement of trifluoromethyl group (8n)
by methyl group (8i) led to obvious decrease of activities, suggesting that polar
substitutions on meta- or para-position are preferred. What’s more, the introduction of
fluorine(s) to compound 8p led to a decrease of activity (8p vs 8v and 8w). We
speculate that the subtle electronic effects might play an important role in the
cytotoxicity.
As compounds 8n, 8p, 8s, 8u and 8v displayed the best antiproliferative activity
against A549 cells, they were selected to test broad-spectrum antitumor activity.
Table 2 indicated that the five compounds displayed good antiproliferative activity
against the human breast cancer cell line MCF-7, human cervical cancer cell line
Hela, human colon cancer cell line HCT-116 and human acute lymphoblastic cancer
cell line CCRF-CEM. Compound 8p displayed the best antiproliferative activity
against MCF-7 and Hela cells and good activity against CCRF-CEM cells. While
compound 8n and 8v exhibited better antiproliferative activity against HCT-116 cells.
Table 1 IC50 values of 8 (a-w) in A549 Cancer Cells.
Compd
Tail
C50 (µM)
Compd
Tail
IC50 (µM)
8a
2.82±0.02
8m
6.32±0.24
8b
8c
8d
8e
4.19±0.15
4.05±0.10
>25
8n
8o
8p
8q
0.16±0.07
6.04±0.01
0.61±0.06
2.56±0.15
4.64±0.18
8f
4.20±0.13
8r
9.20±0.05
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8g
8h
3.00±0.08
8s
0.65±0.04
2.18±0.05
3.26±0.02
8t
8i
8j
2.22±0.08
2.63±0.24
2.58±0.40
8u
8v
0.72±0.13
0.95±0.04
1.82±0.36
8k
8w
8l
7.03±0.36
3.82±0.05
Doxorubicin
Amsacrine
0.09±0.004
5.96±0.14
Etoposide
Table 2 IC50 values of typical compounds in several cancer cell lines.
IC50 (µM)
Compd
Tail
MCF-7
10.50±0.34
6.95±0.94
10.33±0.39
9.95±0.88
7.60±0.24
0.81±0.01
ND
Hela
HCT-116
0.76±0.01
6.32±0.49
1.62±0.02
1.16±0.10
0.86±0.03
0.07±0.03
6.03±0.70
0.93±0.21
CCRF-CEM
5.11±0.73
2.54±0.07
2.00±0.04
6.87±0.37
3.08±0.28
0.052±0.006
0.12±0.05
0.18±0.04
8n
8p
10.07±0.43
1.70±0.26
7.00±1.68
4.41±0.24
4.08±0.31
0.78±0.10
41.21±4.45
0.34±0.04
8s
8u
8v
Doxorubicin
Etoposide
Amsacrine
26.76±0.59
2.3. Topo I and IIα Inhibitory Activities
As the structure of our compounds was similar to that of m-AMSA, we anticipate
that our compounds might inhibit the activity of topoisomerases. All our newly
synthesized compounds were subjected to Topo I and Topo IIα inhibitory assay.
Compounds 8c, 8k, 8p and 8q exhibited apparent Topo I inhibitory activity at 100
ꢀM, further investigation showed that these four compounds also inhibit Topo I
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activity at both 75 and 50 ꢀM (Figure 2A). Camptothecin was used as a positive
control as Topo I inhibitor. All of our compounds displayed strong Topo IIα
inhibitory activity at 100 ꢀM. Amsacrine and etoposide, two well-known DNA
intercalating and DNA binding Topo II inhibitors, were used as positive controls.
Moreover, studies on five compounds 8n, 8p, 8s, 8u and 8v showed that these
compounds possessed better Topo IIα inhibitory activities than etoposide at 50 ꢀM
and 10 ꢀM (except 8n) (Figure 2B). This data suggested that most of these acridine
compounds served as Topo II specific inhibitors. In addition, compounds 8c, 8k, 8p
and 8q served as both Topo I and Topo II dual inhibitors. Compound 8p was selected
to further investigate the action mechanism due to its better antiproliferative activity
against tested tumor cell lines and Topo inhibitory activity.
Figure 2. Topo I and Topo IIα inhibitory activities. (A) Topo I: lane D, pBR322 DNA; lane T,
Topo I + pBR322 DNA; lane C, camptothecin + Topo I + pBR322 DNA; the others, tested
compounds + Topo I + pBR322 DNA. (B) Topo IIα: lane D, pBR322 DNA; lane T, Topo IIα +
pBR322 DNA; lane A, amsacrine + Topo IIα + pBR322 DNA; lane E, etoposide + Topo IIα +
pBR322 DNA; the others, tested compounds + Topo IIα + pBR322 DNA.
2.4.Uptake of 8p in A549cells
It is important for a designed anticancer drug to penetrate cell membranes to bind to
the genomic target with a high efficiency. Therefore, the representative compound 8p
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was subjected to the cellular uptake assay with lung cancer cell line A549 by
fluorescence-activated cell sorting. As shown in Figure 3, after A549 cells incubated
with compound 8p for 4 h, as the concentration increased from 1 ꢀM or 5 ꢀM, the
fluorescence associated with the cells strongly increased, indicating the ability of 8p
to penetrate cell membranes. In addition, extension incubated time led to no big
difference in fluorescence intensity, illustrating that the uptake is up to a maximum
after 4 h of incubation.
Figure 3. FACS analysis of A549 cancer cells treated with 8p for 4, 8, 16 h. (a) control, (b) 1 ꢀM,
(c) 5 ꢀM.
2.5. DNA interaction studies
As compound 8p could penetrate cell membranes, it is feasible to study whether the
interaction with DNA contributed to its cytotoxicity. UV-visible spectral absorbance
was firstly carried out to determine whether compound 8p could interact with DNA.
The results showed that compound 8p exhibited absorption bands in the region of
365-480 nm (Figure 4A). The maximal absorptions were around 420 nm. When
ctDNA was up to 64 ꢀM, the absorption spectra were hypochromicity (60%) and
slight bathochromic shifts (10 nm) (Figure 4A), indicating that compound 8p could
intercalate into DNA [36]. The formula (1) was used to calculate the DNA binding
constant Kb [37]. The Kb of compound 8p was 1.68×105 M-1, suggesting its good
DNA binding capability which was comparable to amsacrine (Kb = 1.36×105 M-1,
Figure 1s).
[DNA]/(εa-εf) = [DNA]/(εb-εf) + 1/Kb(εb-εf)
(1)
The spectrofluorimetric study was also performed to further evaluate the DNA
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binding ability. 8p (10 ꢀM) exhibited an emission band in the range of 420-750 nm
with an excitation wavelength at 400 nm. The fluorescence of 8p was gradually
quenched with increasing concentrations of ctDNA suggesting the interaction between
compound 8p and DNA. The quenching constant K was calculated according to the
classical Stern-Volmer Eq. (2) [38]. The quenching constant K of compound 8p was
1.02×105 M-1. Which was in accordance with the absorption spectra. The data
indicated that 8p might intercalate into DNA, since references reported that the
intercalation complexes exhibited K from 104 to 106 M-1, which are conspicuously
smaller than the K of groove binders (105 -109 M-1) [39]. These results indicated that
compound 8p could intercalate within the double-stranded DNA structure and thus
might cause DNA damage.
F0/F=1+ K[Q]
(2)
Figure 4. (A) UV-visible absorption spectra of 8p 10 ꢀM with various concentrations of ctDNA in
Tris-HCl buffer (pH 7.4). (B) The plot of absorption data, [DNA], DNA concentration, Kb =
1.68
ctDNA in Tris-HCl buffer (pH 7.4) after excitation at λex = 400 nm. (D) The plots of the
fluorescence titration, [Q], DNA concentration, K = 1.02
105 M-1.
×
105 M-1. (C) Fluorescence emission spectra of 8p 10 ꢀM with various concentrations of
×
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2.6. DNA damage induced by 8p
Considering that compounds 8p functioned as an intercalative catalytic inhibitor,
we further investigated the action mode of compound 8p with A549 cells by
observing comet tails in comet assay. As shown in Figure 5A and 5B, untreated
control A549 cells had almost no detectable comet tails, while cells displayed
significant DNA tails after treatment with compound 8p at 5 ꢀM, as well as etoposide,
the positive control. The results indicated that 8p could induce severe DNA damage in
A549 cells. To gain more insight into the induction of DNA damage of compound 8p,
we monitored the levels of γH2AX in A549 cells treated with 8p. γH2AX is known as
a biomarker for double strand breaks in DNA [40-44]. As shown in Figure 5C, the
levels of γH2AX in A549 cells treated 8p were clearly increased, indicating that
compound 8p caused DSBs.
Figure 5. Compound 8p induce DNA double strands break in A549 cells. (A) Comet assay.
Images of A549 cells after treatment with the negative control (nontreated), etoposide, and
compound 8p. (B) Histogram representation of the percentage of tail DNA. (C) 8p induced the
increased expression of γH2AX in A549 cells. Cells were incubated with 8p at indicated
concentrations for 48 h. β-Actin was used as an equal loading control.
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2.7. Apoptosis induced by compound 8p
Given compound 8p could cause severe DNA damage, we further investigated the
mechanism of compound 8p inducing A549 cells death using annexin-V (AV)/PI
binding assay. In early apoptotic cells, phosphatidyl serine (PS) on cell
membrane turned inside-out due to the phospholipid asymmetry lost. As AV possesses
a high affinity to PS, AV-FITC could stain early apoptotic cells. While in late
apoptotic or necrotic cells, both AV and PI are permeable and stained cells on PS and
nucleus, respectively. The results of fluorescence-activated cell sorting analysis
performed after treatment of A549 cells with 8p at the concentrations of 0, 5, 10 ꢀM
for 36 or 48 h were shown in Figure 6A. In comparison with the control, A549 cells
treated with compounds 8p exhibited an accumulation of early and late apoptotic cells
in a time and dose-dependent manner. For example, the percentage of late apoptotic
cells was 0.29% in untreated cells, while it was 10.47% and 88.13% in the cells
treated with 8p at 5, 10 ꢀM for 36 h, respectively. The corresponding percentage was
0.62%, 21.23% and 82.46% in cells treated with 8p for 48 h, respectively. Also, the
percentage of early apoptotic cells showed the same change trend (Figure 6A).
In order to further validate the mechanism of the induction of cellular apoptosis, we
monitored the expressions of regulatory proteins related to apoptotic pathway such as
cleaved caspase-3, cleaved caspase-7 and cleaved caspase-9 in A549 cells by
immunoblotting. Caspase-9 is involved in the mitochondrial pathway. Activated
caspase-9 further cleaves downstream caspases including caspase-3 and -7 to initiate
the caspase cascade. Caspase-3 and caspase-7 play irreplaceable roles in cell
apoptosis once activated by caspase-9 [45]. The activation of caspases is required for
the induction and execution of cellular apoptosis [46]. As shown in Figure 6B, the
treatment with compound 8p increased the expression of proapoptotic marker cleaved
caspase-9 and caspase-3 and caspase-7. Altogether, our results suggested that
compound 8p was able to induce caspase-dependent intrinsic pathway of apoptosis in
A549 cells.
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Figure 6. Compound 8p induced apoptosis in A549 cells. (A) AV/PI assay for the detection of
apoptotic A549 cells after treatment with 8p at the indicated concentrations for 36 or 48 h. (B)
Treatment with compound 8p increased the expression of cleavage-caspase3/7/9 in A549 cells at
indicated concentrations after 48 h. β-Actin was used as an equal loading control.
2.8. G2/M phase arrest induced by 8p
The cell cycle including four phases G1, S, G2, and M. Cell cycle checkpoints are
essential safe guard systems to trigger arrest of cell cycle progression, thus provide
time for the cell to correct the dysregulations of cell cycle events before proceeding to
the next phase [6, 47, 48]. It is typical for cancer cells that abnormal events such as
SSBs, DSBs occur in the cell cycle progression. The cell cycle might be arrested only
if the accidental damage corrected. Therefore, we investigated the effect of 8p on cell
cycle distribution of cancer cells using flowcytometry-based total DNA content
analysis. After treatment of 8p at 0.5, 1, and 5 ꢀM in A549 cells for 36 h, as shown in
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Figure 7, the cells were arrested in the G2/M phase of the cell cycle in a
dose-dependent manner. In response to agents that cause DNA damaging or target
cytoskeleton assembly, G2/M checkpoint arrest cells at G2/M phase
before entering mitosis [49]. In fact, after 36 h, the cell population in G2/M increases
from 7.62% in the untreated cells to 8.12%, 9.62% and 16.52% in the cells treated
with 8p at 0.5, 1, 5 ꢀM, respectively.
Figure 7. Flowcytometric analysis of compounds 8p induce G2/M phase arrest in A549 cells.
A549 cells were treated with compound 8p at the indicated concentrations for 36 h.
3. Conclusions
A new series of 9-benzylamine acridine derivatives 8 (a-w) were simply
synthesized and their biological activity were determined. Most of the compounds
displayed significant antiproliferative effects against A549 cells. All of our
compounds strongly inhibited topo II activity and some of them also exhibited Topo I
inhibitory activity. The typical compound 8p displayed broad antitumor activity,
strong DNA binding ability. Further study indicated that compound 8p significantly
induced DNA damage. Compound 8p arrested cancer cells at G2/M phase and
induced them apoptosis potentially by stirring up DNA damage response. The results
of this study demonstrated that chemical modification of the 9-benzylamine acridine
was a feasible way to develop potent antitumor agents. Further work to optimize the
bioactivity is ongoing.
4. Experimental
4.1. Chemistry
Generally, the procedure for the synthesis of 8 (a-w) was as follows. A mixture of
substituted acridine (1) and benzylamine (2 equiv) in ethanol (8 mL) was added
potassium carbonate (2 equiv) and potassium iodide (2% equiv). The reaction mixture
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was stirred for 30 min at room temperature and refluxed in oil bath for 24-48 h. Then
the mixture was extracted with absolute ethanol to give the crude products, which
were purified by column chromatography with petroleum ether and ethyl acetate (5:1
v/v).
4.1.1. 6-chloro-N-(2-fluorobenzyl)-2-methoxyacridin-9-amine (8a). Yield 43%; mp
168.6-179.8 °C; 1H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 7.99 (dd, J = 9.2, 7.6 Hz,
2H), 7.40 (dd, J = 9.2, 2.0 Hz, 1H), 7.33-7.26 (m, 3H), 7.18 (d, J = 2.4 Hz, 1H),
7.11-7.04 (m, 2H), 4.89 (s, 1H), 4.80 (s, 2H), 3.84 (s, 3H); 13C NMR (101 MHz,
CDCl3) δ 162.18, 159.74, 156.52, 148.97, 148.17, 134.81, 131.61, 129.88, 129.85,
129.77, 128.49, 126.23, 126.09, 125.44, 125.03, 124.57, 124.53, 123.63, 119.45,
117.38, 115.76, 115.54, 98.90, 55.37, 48.76, 48.73; HR-MS(ESI): calcd for
C21H16ClFN2O [M+H]+ 367.1013; found: 367.1021.
4.1.2. 6-chloro-N-(3-fluorobenzyl)-2-methoxyacridin-9-amine (8b). Yield 52%; mp
172.4-173.5 °C; 1H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 2.0 Hz, 1H), 8.01 (d, J =
9.2 Hz, 1H), 7.93 (d, J = 9.2 Hz, 1H), 7.41 (dd, J = 9.6, 2.8 Hz, 1H), 7.37-7.29 (m,
2H), 7.16 (d, J = 7.6 Hz, 2H), 7.11 (d, J = 2.4 Hz, 1H), 7.03 (dt, J = 7.2, 2.0 Hz, 1H),
4.94 (s, 1H), 4.81 (s, 2H), 3.77 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 164.46, 162.00,
156.41, 149.03, 142.03, 141.96, 134.87, 130.67, 130.59, 125.30, 124.92, 123.53,
122.90, 122.87, 118.58, 116.58, 114.93, 114.73, 114.41, 114.19, 99.00. 55.36, 54.04;
HR-MS(ESI): calcd for C21H16ClFN2O [M+H]+ 367.1013; found: 367.1012.
4.1.3. 6-chloro-N-(4-fluorobenzyl)-2-methoxyacridin-9-amine (8c). Yield 48%; mp
1
189.2-191.4 °C; H NMR (400 MHz, DMSO-d6) δ 8.29 (d, J = 9.2 Hz, 1H), 7.81 (s,
1H), 7.83 (s, 1H), 7.61 (s, 1H), 7.55 (s, 1H), 7.48 (dd, J = 8.0, 2.4 Hz, 2H), 7.39 (d, J
= 8.8, 1H), 7.31-7.26 (m, 1H), 7.20-7.11 (m, 3H), 4.95 (s, 2H), 3.75 (s, 3H); 13C NMR
(101 MHz, DMSO-d6) δ 168.92, 162.25, 159.83, 154.83, 149.97, 135.97, 133.36,
128.62, 128.54, 125.96, 124.12, 122.71, 115.10, 114.89, 104.88, 100.64, 55.16, 51.25,
22.33; HR-MS(ESI): calcd for C21H16ClFN2O [M+H]+ 367.1013; found: 367.1018.
4.1.4. 6-chloro-N-(3,4-difluorobenzyl)-2-methoxyacridin-9-amine (8d). Yield 63%;
1
mp 205.5-206.4 °C; H NMR (400 MHz, DMSO-d6) δ 8.26 (d, J = 9.2 Hz, 1H),
7.86-7.82 (m, 2H), 7.57-7.53 (m, 3H), 7.43-7.38(m, 2H), 7.30 (d, J = 6.8 Hz, 2H),
15

ACCEPTED MANUSCRIPT
4.94 (s, 2H), 3.78 (s, 3H); 13C NMR (400 MHz, DMSO-d6) δ 154.90, 150.47, 150.34,
149.86, 149.48, 149.36, 148.03, 147.90, 147.05, 146.93, 133.44, 123.36, 123.33,
117.31, 117.14, 115.83, 115.65, 55.16, 51.15; HR-MS(ESI): calcd for C21H15ClF2N2O
[M+H]+ 385.0919; found: 385.0927.
4.1.5. 6-chloro-N-(3-chlorobenzyl)-2-methoxyacridin-9-amine (8e). Yield 59%; mp
171.5-171.8 °C; 1H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 2.0 Hz, 1H), 8.00 (d, J =
9.6 Hz, 1H), 7.92 (d, J = 9.2 Hz, 1H), 7.46 (s, 1H), 7.40 (dd, J = 9.6, 2.8 Hz, 1H),
7.31-7.28 (m, 3H), 7.25-7.23 (m, 1H), 7.10 (d, J = 2.8 Hz, 1H), 4.95 (s, 1H), 4.77 (s,
2H), 3.77 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 156.44, 149.02, 148.22, 147.02,
141.45, 134.98, 134.89, 131.63, 130.29, 128.47, 128.07, 127.48, 125.44, 125.30,
124.96, 123.58, 118.61, 116.59, 98.98, 55.38, 53.98; HR-MS(ESI): calcd for
C21H16Cl2N2O [M+H]+ 383.0718; found: 383.0708.
4.1.6. 6-chloro-N-(4-chlorobenzyl)-2-methoxyacridin-9-amine (8f). Yield 62%; mp
1
190.3-192.5 °C; H NMR (400 MHz, DMSO-d6) δ 8.27 (d, J = 9.2 Hz, 1H), 7.88 (s,
1H), 7.60 (d, J = 7.6 Hz, 1H), 7.54 (s, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.42 – 7.38 (m,
3H), 7.30 (d, J = 8.0 Hz, 1H), 4.96 (s, 2H), 3.76 (s, 3H); 13C NMR (101 MHz,
DMSO-d6) δ 154.85, 149.80, 138.99, 133.29, 131.30, 128.53, 128.20, 125.84, 124.27,
122.69, 120.86, 119.07, 116.83, 116.01, 114.52, 104.87, 100.46, 55.16, 51.26;
HR-MS(ESI): calcd for C21H16Cl2N2O [M+H]+ 383.0718; found: 383.0727.
4.1.7. N-(2-bromobenzyl)-6-chloro-2-methoxyacridin-9-amine (8g). Yield 60%; mp
179.1-180.0 °C; 1H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 1.6 Hz, 1H), 7.98 (d, J =
9.2 Hz, 2H), 7.59 (dd, J = 9.6, 1.6 Hz, 1H), 7.37 (dd, J = 9.2, 2.0 Hz, 1H), 7.31 (dd, J
= 9.2, 2.0 Hz, 1H), 7.24 (dd, J = 7.2, 2.0 Hz, 1H), 7.19-7.11 (m, 3H), 5.13 (s, 1H),
4.83 (s, 2H), 3.78 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 156.45, 148.82, 148.16,
147.09, 138.13, 134.79, 133.08, 131.59, 129.89, 129.58, 128.50, 127.92, 125.41,
125.04, 123.74, 123.40, 119.39, 117.34, 98.91, 55.36, 54.66; HR-MS(ESI): calcd for
C21H16BrClN2O [M+H]+ 427.0213; found: 427.0224.
4.1.8. N-(4-bromobenzyl)-6-chloro-2-methoxyacridin-9-amine (8h). Yield 48%; mp
1
167.3-169.3 °C; H NMR (400 MHz, DMSO-d6) δ 8.26 (d, J = 9.6 Hz, 1H), 7.86 (s,
1H), 7.82 (d, J = 6.8 Hz, 1H), 7.56-7.38 (m, 6H), 7.29 (d, J = 8.4 Hz, 1H), 4.94 (s,
16

ACCEPTED MANUSCRIPT
2H), 3.76 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 171.73, 154.86, 149.85, 139.41,
133.34, 131.51, 131.27, 131.12, 130.88, 129.25, 128.91, 128.43, 125.96, 122.63,
119.78, 55.17, 51.38, 20.82; HR-MS(ESI): calcd for C21H16BrClN2O [M+H]+
427.0213; found: 427.0211.
4.1.9. 6-chloro-2-methoxy-N-(3-methylbenzyl)acridin-9-amine (8i). Yield 68%; mp
161.1-161.5 °C; 1H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 2.0 Hz, 1H), 8.01 (d, J =
9.6 Hz, 1H), 7.96 (d, J = 9.2 Hz, 1H), 7.41 (dd, J = 9.2, 2.4 Hz, 1H), 7.31 – 7.26 (m,
13
2H), 7.22 – 7.14 (m, 4H), 4.93 (s, 1H), 4.80 (s, 2H), 3.78 (s, 3H), 2.35 (s, 3H); C
NMR (101 MHz, CDCl3) δ 156.21, 149.52, 148.41, 147.16, 139.34, 138.84, 134.81,
131.70, 128.99, 128.71, 128.52, 128.21, 124.98, 124.80, 124.51, 123.82, 118.33,
116.34, 99.26, 55.38, 54.78, 21.42; HR-MS(ESI): calcd for C22H19ClN2O [M+H]+
363.1264; found: 363.1265.
4.1.10. 6-chloro-2-methoxy-N-(4-methylbenzyl)acridin-9-amine (8j). Yield 79%; mp
1
170.6-171.8 °C; H NMR (400 MHz, DMSO-d6) δ 8.30 (d, J = 9.6 Hz, 1H), 7.87 (s,
1H), 7.82 (d, J = 8.8 Hz, 1H), 7.59 (d, J = 1.6 Hz, 2H), 7.38 (d, J = 8.4 Hz, 1H), 7.33
(d, J = 8 Hz, 2H), 7.27 (d, J = 9.2 Hz, 1H), 7.15 (d, J = 8 Hz, 2H), 4.93 (s, 2H), 3.75
13
(s, 3H); C NMR (101 MHz, DMSO-d6) δ 154.90, 150.07, 136.85, 135.96, 133.30,
130.70, 128.93, 127.12, 126.64, 124.16, 122.68, 114.46, 100.69, 55.28, 51.62, 20.55;
HR-MS(ESI): calcd for C22H19ClN2O [M+H]+ 363.1264; found: 363.1265.
4.1.11. 6-chloro-N-(4-ethylbenzyl)-2-methoxyacridin-9-amine (8k). Yield 55%; mp
1
104.8-105.9 °C; H NMR (400 MHz, DMSO-d6) δ 8.32 (d, J = 7.6 Hz, 1H), 7.87 (s,
1H), 7.82 (d, J = 8.0 Hz, 1H), 7.57 (s, 1H), 7.39-7.35 (m, 3H), 7.28 (d, J = 8.8 Hz,
1H), 7.18 (d, J = 8.0 Hz, 2H), 4.93 (s, 2H), 2.57 (q, J = 7.6 Hz, 2H), 1.14 (t, J = 7.6
13
Hz, 3H); C NMR (400 MHz, DMSO-d6) δ 154.80, 150.08, 142.33, 137.11, 133.25,
130.56, 127.65, 126.63, 125.95, 124.05, 122.56, 116.77, 114.50, 100.79, 55.14, 55.10,
51.78, 27.60, 15.39; HR-MS(ESI): calcd for C23H21ClN2O [M+H]+ 377.1421; found:
377.1415.
4.1.12. N-(4-(tert-butyl)benzyl)-6-chloro-2-methoxyacridin-9-amine (8l). Yield 69%;
mp 143.5-145.1 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.35 (d, J = 9.6 Hz, 1H), 7.85
(d, J = 2.0 Hz, 1H), 7.83 (d, J = 9.2 Hz, 1H), 7.57-7.54 (m, 2H), 7.40-7.37 (m, 4H),
17

ACCEPTED MANUSCRIPT
7.31 (dd, J = 2.8 Hz, 2H), 4.92 (d, J = 7.2 Hz 2H), 3.68 (s, 3H). 13C NMR (400 MHz,
DMSO-d6) δ 154.74, 150.10, 149.26, 147.84, 146.18, 136.84, 133.18, 130.63, 127.07,
126.33, 125.80, 124.99, 124.12, 122.68, 116.68, 114.47, 100.71, 55.01, 51.59, 33.97,
30.93; HR-MS(ESI): calcd for C25H25ClN2O [M+H]+ 405.1734; found: 405.1741.
4.1.13. 6-chloro-2-methoxy-N-(2-(trifluoromethyl)benzyl)acridin-9-amine (8m). Yield
5%; mp 195.8-196.9 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 9.2 Hz, 1H),
8.09 (d, J = 8.0 Hz, 1H), 7.88-7.77 (m, 3H), 7.60-7.56 (m, 2H), 7.42-7.40 (m, 2H),
7.30 (d, J = 9.2 Hz, 1H), 5.09 (s, 2H), 3.60 (s, 3H). 13C NMR (400 MHz, DMSO-d6) δ
154.93, 149.85, 149.26, 146.16, 136.84, 132.82, 130.80, 129.32, 128.30, 127.66,
127.23,125.24, 124.31, 123.04,114.42,100.12,54.76, 48.77,28.78; HR-MS(ESI): calcd
for C22H16ClF3N2O [M+H]+ 417.0982; found: 417.0970.
4.1.14. 6-chloro-2-methoxy-N-(3-(trifluoromethyl)benzyl)acridin-9-amine (8n). Yield
53%; mp 116.7-118.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.30 (d, J = 9.2 Hz, 1H),
7.89 (s, 1H), 7.87 (s, 1H), 7.81 (d, J = 9.2 Hz 1H), 7.75 (d, J = 7.2Hz, 1H), 7.65-7.57
(m, 3H), 7.51 (d, J = 2Hz, 1H), 7.39 (dd, J = 9.2, 2.4 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H),
5.05 (s, 2H), 3.72 (s, 3H); 13C NMR (400 MHz, DMSO-d6) δ 154.89, 150.10, 141.61,
133.53, 130.83, 129.41, 129.28, 129.12, 128.80, 125.99, 125.40, 124.00, 123.52,
123.39, 123.35, 122.69, 55.03, 51.76; HR-MS(ESI): calcd for C22H16ClF3N2O
[M+H]+ 417.0982; found: 417.0987.
4.1.15. 6-chloro-2-methoxy-N-(4-(trifluoromethyl)benzyl)acridin-9-amine (8o). Yield
42%; mp 141.3-142.7 °C; 1H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 8.00 (d, J = 9.6
Hz, 1H), 7.90 (d, J = 9.2 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H),
7.40 (dd, J = 9.2, 2.4 Hz, 1H), 7.28 (dd, J = 9.6, 2.0 Hz, 1H), 7.26 (s, 1H)ꢀ 7.08 (d, J
= 2.4 Hz, 1H), 4.97 (s, 1H), 4.85 (s, 2H), 3.74 (s, 3H); 13C NMR (101 MHz, CDCl3) δ
156.47, 148.96, 148.19, 143.43, 134.94, 130.75, 130.43, 130.10, 129.78, 127.59,
126.01, 125.98, 125.94, 125.90, 125.37, 124.94, 123.47, 122.67, 118.62, 116.58,
98.98, 55.32, 53.99; HR-MS(ESI): calcd for C22H16ClF3N2O [M+H]+ 417.0981; found:
417.0986.
4.1.16. 6-chloro-2-methoxy-N-(4-methoxybenzyl)acridin-9-amine (8p). Yield 65%;
18

ACCEPTED MANUSCRIPT
1
mp 156.6-157.7 °C; H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 9.2 Hz, 1H), 7.86
(s, 1H), 7.81 (d, J = 9.2 Hz, 1H), 7.60 (s, 1H), 7.39 (d, J = 9.6 Hz, 1H), 7.35 (d, J = 8.0
Hz, 2H), 7.28 (d, J = 9.2 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 6.90 (d, J = 8.0 Hz, 2H),
13
4.90 (s, 2H), 3.77 (s, 3H), 3.71 (s, 3H); C NMR (101 MHz, DMSO-d6) δ 158.25,
154.91, 150.15, 147.71, 133.38, 131.73, 130.45, 127.94, 126.81, 126.21, 124.11,
122.61, 116.98, 114.61, 113.80, 100.93, 55.33, 54.95; 51.49; HR-MS(ESI): calcd for
C22H19ClN2O2 [M+H]+ 379.1213; found: 379.1205.
4.1.17. 6-chloro-N-(3,4-dimethoxybenzyl)-2-methoxyacridin-9-amine (8q). Yield
61%; mp 159.8-160.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.34 (d, J = 9.6 Hz, 1H),
7.86 (s, 1H), 7.82 (d, J = 9.2 Hz, 1H), 7.62 (d, J = 1.6 Hz, 1H), 7.39 (dd, J =9.2, 2.0
Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.04 (s, 1H), 6.934-6.875 (m, 2H), 4.89 (s, 2H),
13
3.78 (s, 3H), 3.70 (s, 3H), 3.67 (s, 3H); C NMR (101 MHz, DMSO-d6) δ 154.82,
150.18, 148.55, 147.63, 133.29, 132.14, 130.42, 129.42, 126.84, 126.10, 124.11,
122.63, 118.63, 114.58, 111.57, 110.59, 100.63, 55.29, 55.20; 55.16; 51.69;
HR-MS(ESI): calcd for C23H21ClN2O3 [M+H]+ 409.1319; found: 409.1314.
4.1.18. 6-chloro-N-(3,5-dimethoxybenzyl)-2-methoxyacridin-9-amine (8r). Yield 79%;
1
mp 147.9-149.5 °C; H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 9.2 Hz, 1H), 7.86
(s, 1H), 7.81 (d, J = 9.2 Hz, 1H), 7.60 (d, J = 2.4 Hz, 1H), 7.40 (dd, J = 9.2, 2.8
Hz,1H), 7.31 (dd, J = 9.2, 2.0 Hz, 1H), 6.63 (s, 2H), 6.39 (t, J = 2.0 Hz, 1H), 4.90 (s,
13
2H), 3.76 (s, 3H), 3.69 (s, 6H); C NMR (400 MHz, DMSO-d6) δ 160.45, 154.82,
150.29, 142.40, 133.48, 126.05, 124.05, 122.61, 116.81, 114.43, 104.57, 101.05,
98.56, 55.38, 55.15, 54.91, 51.94; HR-MS(ESI): calcd for C23H21ClN2O3 [M+H]+
409.1319; found: 409.1328.
4.1.19. N-(benzo[d][1,3]dioxol-5-ylmethyl)-6-chloro-2-methoxyacridin-9-amine (8s).
Yield 44%; mp 158.6-160.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.30 (d, J = 9.6 Hz,
1H), 7.86 (s, 1H), 7.81 (d, J = 9.6 Hz, 1H), 7.60 (d, J = 2.4 Hz,1H), 7.40 (dd, J = 9.2,
2.4 Hz, 1H), 7.30 (dd, J = 9.2, 2.0 Hz, 1H), 7.00 (s, 1H),6.92-6.86 (m, 2H), 4.88 (s,
13
2H),3.80 (s, 3H). C NMR (400 MHz, DMSO-d6) δ 154.85, 150.17, 147.22, 146.02,
133.57, 133.50, 129.50, 126.16, 124.06, 122.57, 119.75, 116.85, 114.42, 107.95,
107.14, 101.05, 100.69, 100.71, 55.25, 51.69; HR-MS(ESI): calcd for C22H17ClN2O3
[M+H]+ 393.1006; found: 393.1008.
19

ACCEPTED MANUSCRIPT
4.1.20. 6-chloro-2-methoxy-N-(3,4,5-trimethoxybenzyl)acridin-9-amine (8t). Yield
27%; mp 200.7-202.3 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.35 (d, J = 9.2 Hz, 1H),
7.88 (s, 1H), 7.82 (d, J = 9.2 Hz, 1H), 7.59 (d, J = 2.4 Hz ,1H), 7.40 (dd, J = 9.6, 2.4
Hz, 1H), 7.34 (dd, J = 9.2, 2.0 Hz, 1H), 6.77 (s, 2H), 4.89 (s, 2H), 3.75 (s, 3H) , 3.69
(s, 6H) , 3.63 (s, 3H). 13C NMR (400 MHz, DMSO-d6) δ 154.94, 152.86, 150.53,
136.30, 135.79, 133.52, 126.19, 124.21, 122.89, 117.22, 114.93, 104.03, 100.99,
59.92, 55.71, 55.21, 52.47; HR-MS(ESI): calcd for C24H23ClN2O4 [M+H]+ 439.1425;
found: 439.1423.
4.1.21. 6-chloro-N-(4-(difluoromethoxy)benzyl)-2-methoxyacridin-9-amine (8u).
Yield 10%; mp 121.8-123.1 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 9.2 Hz,
1H), 7.86 (s, 1H), 7.81 (d, J = 9.2 Hz, 1H), 7.57 (d, J = 2 Hz, 1H), 7.51 (d, J = 8.4,
2H), 7.43-7.02 (m,4H), 4.99 (s, 2H), 3.75 (s, 3H). 13C NMR (400 MHz, DMSO-d6) δ
154.88, 150.46, 149.70, 146.98, 136.63, 133.79, 128.26, 126.17, 125.80, 124.19,
122.66, 118.38, 116.84, 116.08, 114.27, 113.52, 100.12, 55.20, 51.27; HR-MS(ESI):
calcd for C22H17ClF2N2O2 [M+H]+ 415.1025; found: 415.1019.
4.1.22. 6-chloro-N-(3-fluoro-4-methoxybenzyl)-2-methoxyacridin-9-amine (8v).
Yield 71%; mp 145.4-147.2 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.29 (d, J = 9.2 Hz,
1H), 7.89 (s, 1H), 7.84 (d, J = 9.6 Hz, 1H), 7.57 (s, 1H), 7.51 (s, 1H), 7.40 (d, J = 8.4
Hz, 1H), 7.30 (dd, J = 12.6,1.6 Hz 1H), 7.18 (d, J = 8.8 Hz, 1H), 7.12 (t, J = 8.4 Hz,
1H), 4.89 (s, 2H), 3.79 (s, 3H), 3.78 (s, 3H). 13C NMR (400 MHz, DMSO-d6) δ
154.88, 152.45, 150.02, 149.95, 145.90, 145.80, 133.37, 132.81, 126.05, 123.99,
122.75, 122.72, 117.08, 114.67, 114.39, 114.21, 113.66, 100.82, 55.80, 55.20, 51.12;
HR-MS(ESI): calcd for C22H18ClFN2O2 [M+H]+ 397.1119; found: 397.1109.
4.1.23. 6-chloro-N-(3,5-difluoro-4-methoxybenzyl)-2-methoxyacridin-9-amine (8w).
Yield 81%; mp 195.2-196.9 °C; 1H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 8.03 (d, J
= 9.2 Hz, 1H), 7.92 (d, J = 9.2 Hz, 1H), 7.43 (dd, J = 9.2, 2.4 Hz, 1H), 7.34 (dd, J =
9.2, 2.0 Hz, 1H), 7.12 (s, 1H), 7.00 (d, J = 4.8 Hz, 1H), 4.73 (s, 2H), 4.01 (s, 3H), 3.84
(s, 3H); 13C NMR (400 MHz, CDCl3) δ 157.25, 157.19, 156.60, 154.77, 154.70,
148.75, 135.01, 134.62, 125.51, 124.96, 123.46, 118.73, 116.67, 111.18, 111.11,
111.01, 110.95, 99.87, 61.90, 55.44, 55.43, 29.72; HR-MS(ESI): calcd for
20

ACCEPTED MANUSCRIPT
C22H17ClF2N2O2 [M+H]+ 415.1025; found: 415.1024.
4.2. Cell Proliferation Assay
Cell proliferation assays based on MTT were performed with 6 cancer cell lines.
Briefly, cells were seeded in 96-well plates at a density of 4-8 × 103 cells per well
with incubation overnight in a 5% CO2 incubator at 37 °C, the growth medium in
each well was then exchanged with 0.1 mL of fresh medium containing graded
concentrations of compounds to be tested or equal DMSO and incubated continuously
for 48 h. Then 10 ꢀL MTT solution (5 mg/mL) was added to each well, and the cells
were incubated for additional 4 h. The MTT-formazan crystals were dissolved in 100
ꢀL of DMSO, the absorbance of each well was measured at 490 nm using
anautomatic ELISA reader system (TECAN, CHE). Etoposide and Doxorubicin were
used as positive controls.
4.3. Human Topo I and Topo IIα relaxation Assay
The inhibition of compounds to Topoisomerases I and Topo IIα were determined by
assessing the relaxation of supercoiled pBR322 plasmid DNA. The assay was
performed in a final volume of 20 ꢀL reaction volume containing 500 ng of
supercoiled DNA pBR322 (Takara Biotechnology, Japan) and 1 unit of human Topo I
(Takara Biotechnology, Japan) or Topo IIα (USB Corp., USA) with or without our
compounds in the reaction buffer. The reaction mixtures were incubated at 37 °C for
10-15 min and terminated by adding 4 ꢀL of DNA loading buffer. Electrophoresis was
performed on a 0.8% agarose gel at 80 V for 30 min in TAE buffer. Then stained gels
for 10 min with ethidium bromide (2.5 ꢀg/mL) and destain in water for 5 min. DNA
bands were visualized with UV light. Camptothecin (Sigma, USA) was used as a
positive control as topo I inhibitor and etoposide (Sigma, USA) were used as a Topo
II inhibitor, respectively.
4.4. Absorption spectra
Compound 8p was diluted into Tris-HCl buffer (pH 7.4) to a final concentration of
10 ꢀM and then ctDNA was added with concentration of 0-44 ꢀM. After 5 min
21

ACCEPTED MANUSCRIPT
standing the solution was subjected to UV-vis absorption spectra measurement on a
Beckman Coulter DU 800 spectrophotometer.
4.5. Fluorescence emission spectra
Compounds 8p was diluted to a final concentration of 10 ꢀM in Tris-HCl buffer (10
mM, pH 7.4), followed adding ctDNA into the system. The final concentration of
ctDNA was ranged from 0 to 64 ꢀM. Fluorescence mission spectra were determined
on Fluorolog spectrometer with Xenon arc lamp used as excitation light source. The
emission spectra were detected from 420-750 nm with an excitation wavelength of
400 nm.
4.6. FACS Analysis
For uptake studies, the cells have been treated for 4 h with 8p (1 and 5 ꢀM). After
incubation, the cells were trypsinized and pelleted. The pellets were resuspended in
200 ꢀL of PBS and immediately analyzed by FACS. A minimum of 10000 cells for
each sample were acquired and analyzed with an excited wavelength of 400 nm.
For cell cycle Analysis, approximately 2 × 105 cells per well were seeded in six-well
plate and incubated for 12 h in serum-free medium following treated with graded
concentrations of compounds for 24 h. Cells were harvested by trypsinization, then
fixed in ice-cold absolute methanol. Cells were stained with 4 ꢀg/mL PI and 0.1
mg/mL RNaseA in PBS. After incubated in the dark at room temperature for 30 min,
simples were subjected to flowcytometric analysis.
For Annexin V-propidium iodide assay, A549 cells were seeded in a six-well plates
in a density of 2 × 105 cells per well, incubated overnight and then treated with graded
concentrations of compounds for 24 h. Cellular apoptosis was determined by annexin
V-PI apoptosis detection kit (Beyotime, CN) following its instructions. Cells in early
or late apoptotic phases were represented as cumulative percentage compared with
control.
4.7. Comet Assay
Approximately 105 A549 cells per well were seeded in a 6-well plate with
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incubation for 12 h. Cells were then treated with etoposide or compounds for 24 h in
serum free media and DMSO group was set as control. The collected cells were
resuspended in ice-cold PBS at a density of 105/mL. 7 ꢀL of the prepared cells were
mixed with 70 ꢀL of low-melting agarose at 37 °C and spread on coagulated
normal-melting agarose, then covered with low-melting agarose after it was
coagulated. Then the slides were submerged in lysis buffer for 2 h followed in the
alkaline solution for 0.5 h at 4 °C. Electrophoresis was performed at 300 mA in
alkaline solution for 25 min in dark. Slides were neutralized thrice with Tris-HCl
buffer then stained with PI for 10 min in dark at 4 °C. Comet images were captured
with a fluorescent microscope microscope (Olympus, Japan). A total of 50 A549 cells
were randomly selected for statistical analysis. The results were represented in the
extent of DNA damage.
4.8. Western Blot Assay
A549 cells were grown on 60 mm plates at 1×106 cells until reaching 80%
confluency. The cells were then treated with graded concentration of compounds to be
tested and incubated for 48 h and harvested by trypsinization. Collected Cells were
lysed in lysis buffer solution containing 50 mM Tris-HCl, 300 mM NaCl, 1% Triton
X-100, 10% glycerol, 1.5 mM MgCl2, 1 mM CaCl2, 1 mM PMSF, and 1% protease
inhibitor cocktail. Then 40 ꢀg of protein per sample was resolved by 12 or 15%
SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). The membranes
were blocked with 5% skim milk in Tris buffered saline containing 0.1% Tween 20
(TBST) and probed with primary antibodies in a dilution ratio of 1:1000 for 6-8 h.
The blots were washed, exposed to HRP-conjugated anti rabbit IgG (Cell Signaling
Technology Inc. USA) in a dilution ratio of 1:2000 for 2 h, and detected with ECL
Western blotting detection reagent (Animal Genetics Inc., Korea). All primary
antibodies used were purchased from Cell Signaling Technology Inc. (USA). Western
blot images were taken by LAS-3000 (Fuji Photo Film Co., Ltd., Japan) and analyzed
using Multi-Gauge Software (Fuji Photo Film Co. Ltd., Japan).
Acknowledgments
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The authors would like to thank the financial supports from Shenzhen Municipal
Development and Reform Commission (Disciplinary Development Program for
Chemical Biology), the Chinese National Natural Science Foundation (21272134 and
21372141), and Shenzhen Sci& Tech Bureau (JCYJ20130402164027386,
JCYJ20150331151358131, CXZZ20150529165045064, and CXB201104210014A).
Conflict of Interest The authors declare no conflicts of interest.
Appendix A. Supplementary material
NMR spectrum and high resolution mass spectrometry can be found in supplementary
material online.
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