Cryptolepine and aromathecin based mimics as potent G-quadruplex-binding, DNA-cleavage and anticancer agents: Design, synthesis and DNA targeting-induced apoptosis

Article abstract of DOI:10.1016/j.ejmech.2019.02.072

Thirty Cryptolepine and Aromathecin based mimics were designed and synthesized. Their cytotoxicity was evaluated in four human cancer cell lines (HepG-2, T24, NCI-H460 and MGC-803) and one normal human cell line (HL-7702). Most compounds exhibited potent anticancer activity with IC₅₀ values from 0.31 to 11.97 μM. 8-Fluoro-10-(N-3-dimethylaminopropyl)amino-11H-indeno[1,2-b]quinoline (5b) was identified as the most promising candidate in view of its anticancer activity. Molecular mechanism studies suggested that 5b not only could strongly bind to G-quadruplex, but intercalate into supercoil DNA and resulted in significant DNA double-strand break as well. Furthermore, 5b caused cell cycle arrest at S/G2 phase and induced apoptosis. After treatment with 5b, pro-apoptotic proteins Bak, Bax and Bim were up-regulated, anti-apoptotic proteins Bcl-2 and Bcl-xL were down-regulated, and the effector caspase-3/9 was activated to initiate apoptosis. The anticancer activity of 5b was finally validated in a MGC-803 xenograft tumor model with tumor growth inhibition (TGI) up to 53.2%, while displaying no obvious toxicity. Taken together, these results suggest that 5b may be a potential candidate of cytotoxic antineoplastic drugs for cancer therapy.

Full text of DOI:10.1016/j.ejmech.2019.02.072

Accepted Manuscript
Cryptolepine and aromathecin based mimics as potent G-quadruplex-binding, DNA-
cleavage and anticancer agents: Design, synthesis and DNA targeting-induced
apoptosis
Jing-Mei Yuan, Kai Wei, Guo-Hai Zhang, Nan-Ying Chen, Xin-Wei Wei, Cheng-Xue
Pan, Dong-Liang Mo, Gui-Fa Su
PII:
S0223-5234(19)30196-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.072
EJMECH 11164
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 October 2018
Revised Date: 26 February 2019
Accepted Date: 26 February 2019
Please cite this article as: J.-M. Yuan, K. Wei, G.-H. Zhang, N.-Y. Chen, X.-W. Wei, C.-X. Pan, D.-L. Mo,
G.-F. Su, Cryptolepine and aromathecin based mimics as potent G-quadruplex-binding, DNA-cleavage
and anticancer agents: Design, synthesis and DNA targeting-induced apoptosis, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.02.072.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Cryptolepine and Aromathecin based Mimics as Potent
G-Quadruplex-binding, DNA-cleavage and Anticancer
Agents: Design, Synthesis and DNA Targeting-Induced
Apoptosis
†
†
Jing-Mei Yuan , Kai Wei , Guo-Hai Zhang, Nan-Ying Chen, Xin-Wei Wei,
Cheng-Xue Pan*, Dong-Liang Mo*, Gui-Fa Su*
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources,
School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004,
PR China
†
These authors contributed equally to this work.
*
Corresponding Authors: Professors Cheng-Xue Pan, Dong-Liang Mo, and Gui-Fa Su
Tel. Fax: +86 773 5826869, Email: chengxuepan@163.com (C.-X. Pan);
&
moeastlight@gxnu.edu.cn (D.-L. Mo); gfysglgx@163.com (G.-F. Su)
1

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ABSTRACT
Thirty Cryptolepine and Aromathecin based mimics were designed and
synthesized. Their cytotoxicity was evaluated in four human cancer cell lines
(HepG-2, T24, NCI-H460 and MGC-803) and one normal human cell line (HL-7702).
Most compounds exhibited potent anticancer activity with IC values from 0.31 to
5
0
11.97 µM. 8-Fluoro-10-(N-3-dimethylaminopropyl)amino-11H-indeno[1,2-b]quino-
-line (5b) was identified as the most promising candidate in view of its anticancer
activity. Molecular mechanism studies suggested that 5b not only could strongly bind
to G-quadruplex, but intercalate into supercoil DNA and resulted in significant DNA
double-strand break as well. Furthermore, 5b caused cell cycle arrest at S/G2 phase
and induced apoptosis. After treatment with 5b, pro-apoptotic proteins Bak, Bax and
Bim were up-regulated, anti-apoptotic proteins Bcl-2 and Bcl-xL were
down-regulated, and the effector caspase-3/9 was activated to initiate apoptosis. The
anticancer activity of 5b was finally validated in a MGC-803 xenograft tumor model
with tumor growth inhibition (TGI) up to 53.2%, while displaying no obvious toxicity.
Taken together, these results suggest that 5b may be a potential candidate of cytotoxic
antineoplastic drugs for cancer therapy.
Keywords:
Natural
products
mimics;
Aromathecins;
Cryptolepine;
G-Quadruplex-binding and DNA-cleavage agent; Topo I inhibitor
2

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1
. Introduction
Cancer has become the leading global health burden with high mortality rate.
Due to the urgent need for more effective anti-cancer drugs, the search for novel
antitumor scaffolds, in the hope of identifying new chemistry entities (NCEs) with
promising anticancer activities, had been one of the most active research fields in
medicinal chemistry [1–12].
Natural products had been a rich source and the leading contributor to the
development of new anticancer drugs [13–18]. More than 60% of the clinically used
anticancer drugs are either natural products or their derivatives [19]. The success of
Camptothecin (CPT), Paclitaxol, Podophyllotoxin, Vincristine and their derivatives
demonstrates the validity to make more research efforts in natural products–based
drug discovery.
(
)n
R
HN
N
X
N
(
)n
R
N
HN
N
CH3
B
A
Gu and Ou's work
n = 2, 3; X=NH, O
Cryptolepine
X
R
E
( )n
O
our work
n = 2, 3
O
N
R
N
N
N
D
C
Cushman's work
R = H, Aromathecin
R = CH OH, Hydroxyacuminatin
n = 1-3; X = CH , NH
2
2
Fig. 1. The design of Cryptolepine and Aromathecin based mimics based on previous studies
Cryptolepine (Fig. 1A), a pharmacologically active alkaloid isolated from the
roots of central and west Africa shrub Cryptolepis sanguinolenta, exhibits diverse
pharmacological and biological properties [20]. Its synthesis, structural modification
and structure–activity relationship have been explored by many groups [21-27]. Gu’s
and Ou’s groups reported that Cryptolepine derivatives with a side chain (Fig. 1B)
interact with the G-quadruplex form of DNA and show an inhibitory effect on
telomerase to exert their potent anticancer activities [25-27].
Aromathecin, a derivative of natural product 22-hydroxyacuminatin [28] (Fig.
1
C), presents a pentacyclic skeleton and exhibits similar antitumor activity as CPT by
3

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targeting the Topo I [29]. Since 2008, the structural modifications of Aromathecin had
been reported by Cushman’s group [29–33]. By adding a side chain on its polycyclic
skeleton (Fig. 1D), they found several target compounds exhibiting more than 50–60
fold higher potency in anti-proliferative activities than aromathecins. Inspired by the
results of previous work, herein we report the design and synthesis of Cryptolepine
and Aromathecin based mimics (Fig. 1E), and their interactions with DNA and
DNA/Topo I, anticancer activity in vitro and in vivo, as well as their mechanisms of
actions.
2
. Results and discussion
2
.1 Chemistry
Scheme 1. Reactants and conditions: i) neat, 180 ℃ , 1–1.5 h; ii) POCl , 110 ℃ , 4 h;
3
iii) RNH , HOCH CH OH, 150 ℃ , 15–24 h.
2
2
2
The preparation of the mimics was outlined in Scheme 1. The target compounds
were readily accomplished in 3 steps starting from 1-indanones 1 and anthranilic acid
derivatives 2. First, 1-indanones 1 reacted with anthranilic acid derivatives 2
according to the literature methods [34] under solvent–free condition to provide the
tetracyclic intermediates 3. The 3 was then refluxed in excess POCl to afford the
3
chloro-indenoquinoline 4 in high yield. Finally compound 4 reacted with amines in
4

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glycol to furnish the target compounds 5–7.
2
2
.2. Biological evaluation
.2.1. Anti-proliferative activity
The in vitro cytotoxicity of the target compounds 5–7 against four human tumor
cell lines (HepG-2, T24, MGC-803, NCI-H460) as well as one human normal liver
cell line HL-7702 were evaluated by MTT assay and their IC values are shown in
5
0
Table 1. The in vitro cytotoxic activity indicated that almost all the tested compounds
exhibited potent cytotoxic activity against the tested cell lines, with the IC values
5
0
ranging from 0.31 to 11.97 µM, much more potent than the chemotherapeutic agent
Camptothecin.
Table 1
Structures and in vitro cytotoxicity of compounds 5–7
a
IC50 (µM)
1
2
Cmpd.
R , R
n, R
HepG-2
T-24
MGC-803
NCI-H460
HL-7702
5
5
a
H, H
3, N(CH
3, N(CH
3, N(CH
3, N(CH
3, N(CH
3, N(CH
3, N(CH
3, N(CH
3, N(CH
3, N(CH
2, N(CH
3
3
3
3
3
3
3
3
3
3
3
)
)
)
)
)
)
)
)
)
)
)
2
2
2
2
2
2
2
2
2
2
2
2.60±0.19
1.77±0.11
2.14±0.25
3.12±0.09
1.19±0.09
4.60±0.51
5.23±0.61
4.18±0.18
6.38±1.07
0.31±0.09
0.84±0.26
0.91±0.23
1.36±0.07
0.84±0.07
2.47±0.30
2.86±0.18
2.09±0.29
4.97±0.33
0.95±0.20
0.97±0.21
1.11±0.19
1.80±0.04
0.86±0.06
5.17±0.55
2.11±0.14
1.58±0.08
3.66±0.84
5.08±0.42
0.93±0.13
1.78±0.17
3.49±0.21
2.98±0.21
0.92±0.04
1.36±0.16
1.66±0.21
1.96±0.29
2.31±0.14
2.02±0.05
1.16±0.10
3.92±0.39
3.52±0.22
1.95±0.11
5.73±0.42
5.52±0.44
1.23±0.01
2.89±0.20
4.96±0.25
2.80±0.20
1.03±0.09
3.10±0.18
0
.91±0.07
1.57±0.08
2.30±0.25
1.64±0.20
0.58±0.04
4.35±0.26
3.07±0.14
2.60±0.16
7.06±0.61
4.94±0.32
b
H, F
H, Cl
5
c
5
d
H, Br
H, CH3
5
e
5
f
5
,6-(OMe) , H
2
5
5
g
5
,6-(OMe) , F
2
h
5
,6-(OMe) , Cl
2
5
i
5
5
,6-(OMe) , Br
2
5
j
11.97±0.96 4.64±0.33
,6-(OMe) , Me
2
6
6
a
H, H
2.31±0.10
2.59±0.13
6.71±0.37
4.18±0.19
1.71±0.10
3.14±0.19
0.61±0.11
1.15±0.14
1.74±0.15
2.21±0.25
0.31±0.02
1.82±0.20
1
.31±0.08
b
H, F
H, Cl
2, N(CH3)2
2, N(CH3)2
2, N(CH3)2
2, N(CH3)2
2, N(CH3)2
3.22±0.43
3.60±0.09
6.43±0.45
1.15±0.07
2.06±0.17
6
c
6
d
H, Br
H, CH3
6
e
6
f
5
,6-(OMe) , H
2
5

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6
6
g
1.77±0.19
3.12±0.14
1.61±0.25
2.57±0.15
4.48±0.11
4.46±0.22
5.90±0.13
6.32±0.40
1.88±0.13
3.94±0.20
8.74±0.63
7.60±0.44
3.97±0.26
7.46±0.22
5.69±1.85
2.20±0.33
2.21±0.33
1.53±0.26
2.39±0.22
1.43±0.18
1.90±0.31
4.32±0.17
9.41±0.53
0.58±0.05
2.77±0.16
3.74±0.18
2.23±0.16
2.64±0.24
4.62±0.19
3.09±2.13
2.35±0.24
2.22±0.27
2.73±0.23
2.46±0.22
1.95±0.12
2.79±0.27
6.89±0.74
7.50±0.22
1.42±0.15
2.52±0.13
3.70±0.16
4.13±0.22
3.38±0.24
3.50±0.28
4.27±1.76
5.73±0.37
2.55±0.19
2.83±0.23
4.99±0.34
4.60±0.23
6.66±0.32
5.21±0.47
8.57±0.94
0.89±0.07
5.00±0.23
6.42±0.61
2.40±0.24
4.25±0.37
5.64±0.67
5.37±2.74
5
5
5
5
,6-(OMe) , F
2, N(CH3)2
2, N(CH3)2
2, N(CH3)2
4.38±0.34
2.56±0.25
6.95±0.45
3.01±0.10
2
h
,6-(OMe) , Cl
2
6
i
,6-(OMe) , Br
2
6
j
,6-(OMe) , Me
2, N(CH3)2
2
7
7
a
H, H
3, Morpholinyl
4
.73±0.19
b
H, F
H, Cl
3, Morpholinyl
3, Morpholinyl
3, Morpholinyl
3, Morpholinyl
3.05±0.11
7.12±0.14
5.71±0.46
1.67±0.11
4.97±0.28
3.71±0.13
3.80±0.27
5.02±0.19
7
c
7
d
H, Br
H, CH3
7
e
7
f
5
,6-(OMe) , H 3, Morpholinyl
2
7
7
g
5
,6-(OMe) , F
3, Morpholinyl
3, Morpholinyl
3, Morpholinyl
2
h
5
5
5
,6-(OMe) , Cl
2
7
i
,6-(OMe) , Br
2
7
j
b
,6-(OMe) , Me 3, Morpholinyl
7.72±0.29
2.15±0.31
2
CPT
a
b
IC50 values are presented as the means ± SD (standard deviation) from three independentexperiments. Camptothecin.
The IC values suggested that the substituents at the benzene ring of indene or
5
0
quinolone, as well as the side chain attached to the N-4 position of the quinoline did
not have too much impact on the anticancer activity. 5,6-Dimethoxyl substituents at
indene ring (f–j series) slightly decreased the cytotoxicity, especially the products
with 5,6-dimethoxyl substitution and a side chain with the ω-substituent of
morpholinyl group (compounds 7f–7j). Halo-substituents at quinoline ring (b, c, d
series) also slightly decreased the potency. Among them, fluoro substitution (b series)
seemed more favorable than bromo (d series) or chloro substitution (c series). In
contrast, products with a methyl substituent (e series) generally resulted in the highest
anticancer activity.
The side chain also had slight effect on cytotoxicity. N-ω-dimethylamino-propyl
amino group (compounds 5) was more favorable than the N-ω-dimethylamino-ethyl
amino group (compounds 6), especially against the T-24, MGC-803 and NCI-H460
cell lines. When the ω-dimethyl on the side chain was replaced by morpholinyl group
(5 vs 7), the cytotoxicity of the products decreased slightly. In general, compound 5b
exhibited promising anti-cancer effects in MGC-803 cells while showing relatively
less toxicity to the normal cell HL-7702. Therefore, compound 5b was chosen for the
further investigation of the mechanisms of biological action.
2
.2.2. Compound 5b inhibited the proliferation of MGC-803 cells in a dose– and
6

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time–dependent manner
The antitumor activity of 5b was systematically studied in MGC-803 cells. As
indicated in Fig. 2, 5b showed a dose– and time–dependent proliferation inhibition
activity on MGC-803 cells. The cell inhibition rate increased remarkably at
concentrations lower than 2.5 µM, but only changed slightly when the concentration
was higher than 5 µM. Moreover, the cell inhibition rates were from 40% to 95% after
MGC-803 cells were treated with 5b for different periods of time at the concentration
of 10 µM. These data indicated that 5b exhibited potential anticancer activity both in a
dose–dependent and time–dependent manner.
Fig. 2. Effects of 5b on MGC-803 cell proliferation. Cell inhibition ratios of MGC-803 cells
treated with 5b for 12 h, 24 h, 48 h and 72 h. Values were presented as means ± SD; results are
representative of three independent experiments.
2
.2.3. Compound 5b directly interacts with DNA to induce DNA double-strand break
and Topo I inhibition at low concentration
Because Aromathecin and Cryptolepine exert their biological activity by
interaction with DNA or DNA/Topo I, the Topo I inhibitory activity of 5b was firstly
evaluated by measuring its ability to convert the supercoiled pBR322 DNA to its
relaxed form [35,36]. CPT, a well–known Topo I inhibitor was used as the positive
control. As depicted in Fig. 3A, the supercoiled form of DNA prominently increased
after treatment with 5b at 5.0 µM. To gain further insight into the mechanisms of Topo
I inhibition by 5b, we then investigated whether the Topo I inhibition induced by 5b
was due to DNA intercalation via agarose-gel electrophoresis. Ethidium bromide (EB),
a well-known DNA intercalator, was used as the positive control and CPT used as the
negative control. As shown in Fig. 3B, 5b possessed high DNA intercalation activity,
7

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as indicating by its ability to induce noticeable retardation of DNA movement
comparing with the positive control EB. 5b could efficiently cleave the supercoiled
form of DNA (Form I) to an open circular form (Form II) at a concentration as low as
2
.5 µM. However, the negative control CPT displayed no obvious activity to cleave
DNA in a Topo I-independent manner.
To further confirm its activity in inducing DNA cleavage, the level of γ-H2AX, a
marker of DNA double-strand break, was measured by Western blotting assay in
5
b-treated MGC-803 cells. As depicted in Fig. 3C and D, 2.5 µM of 5b substantially
increased the level of γ-H2AX in MGC-803 cells, suggesting that 5b induced DNA
cleavage, which may be responsible in part for its potent anticancer activity.
Fig. 3. Topo I inhibitory activity and DNA cleavage of 5b. (A) Topo I inhibitory activity was
evaluated by agarose gel electrophoresis, Camptothecin (CPT) is a positive control. Supercoiled
pBR322 DNA was incubated with Topo I in the absence or presence of 5b at 37 ℃ for 30 min.
Lane 1 is pBR322 DNA only; Lane 2 is the mixture of pBR322 DNA and Topo I; Line 3 is the
mixture of pBR322 DNA, Topo I and CPT (5 µM); Lines 4–7 are the mixture of pBR322 DNA,
Topo I and 5b (1, 5, 10, 20 µM, respectively). (B) DNA cleavage assay of 5b, Camptothecin (CPT)
and ethidium bromide (EB) are negative and positive controls, respectively. Lane 1 is pBR322
DNA only; Lane 2 is the mixture of pBR322 DNA and CPT (5 µM); Lane 3 is the mixture of
pBR322 DNA and EB (5 µM); Lanes 4–8 are the mixture of pBR322 DNA and 5b (0.5, 1.0, 2.5,
5
.0, 10 µM, respectively). (C) Immunoblotting analysis of γ-H2AX related to 5b–induced DNA
damage. (D) γ-H2AX bands quantified by Image J. Results are representative of three independent
experiments and shown as the mean ± SD, error bars represented SD, *p < 0.05, ** p < 0.01, ***
p < 0.001 compared with control group.
8

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2
.2.4. Investigation of the DNA binding properties of 5b by spectroscopic analyses
To obtain more detailed information about the DNA binding properties of 5b.
UV–Vis, fluorescence and CD spectroscopic analyses were used to investigate the
interaction of 5b with ct-DNA. The UV–Vis spectra of 5b in the absence (dashed line)
and presence of ct-DNA were shown in Fig. 4A. The characteristic absorption peak of
5
b at ca. 270 nm is ascribed to the π–π* electron transition of the aromatic scaffold of
quinoline. With the [DNA]/[5b] ratio increased from 1: 1 to 10: 1, the corresponding
increase in the absorption intensity was observed. The peak absorbance (Amax) also
showed obvious hyperchromic effect and 10 nm of blue shift, suggesting the
intercalation of 5b into ct-DNA [37,38].
Fig. 4. (A) UV–Vis absorption spectra of 5b in the absence (dashed line) and presence (solid lines)
of increasing [ct-DNA]/[5b] ranging from 1: 1 to 10: 1. (B) Fluorescence quenching curves of EB
bound to DNA in the absence (dashed line) and presence (solid lines) of 5b with [5b]/[EB] ratios
ranging from 1: 1 to 10: 1. Inset: Plot of I /I versus [5b] the quenching constant (Kq) of 5b
0
towards ct-DNA–EB solution. (C) CD spectrum of ct-DNA with the addition of 5b, [DNA]/[5b] =
+
1
0: 1. (D-F). Fluorescence emission spectra of 5b (10 µM) in 100 mM K solution in the absence
(
dashed line) and presence of HTG21, Pu27, c-kit-1 G-quadruplex DNA (solid line), respectively.
G-I). CD spectra of HTG21, Pu27, c-kit-1 G-quadruplex DNA (dashed line) and the spectra of
(
them with 5b (2.0 µM, solid lines)
9

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To further confirm the intercalation mode of interaction of 5b with DNA, a
competitive binding assay was performed using the classic DNA intercalator ethidium
bromide (EB) as a probe [39]. As shown in Fig. 4B, the addition of 5b significantly
quenched the ct-DNA–EB fluorescence intensity. The quenching constant Kq,
4
according to the Stern–Volmer quenching equation, was calculated to be 5.92×10 .
The result indicated that 5b efficiently competed with EB at the same binding sites in
ct-DNA and the fluorescence quenching of DNA-EB complex by 5b belonged to a
dynamic quenching type.
Since many quinoline derivatives [40], especially some Cryptolepine derivatives
had been reported to be able to bind to G-quadruplex DNA [25-27, 41]. Fluorometric
titration assays were used to investigate the binding affinity of 5b to three different
sequences of G-quadruplex DNA, including human telomeric G4-DNA HTG21,
c-myc promoter Pu27 and c-kit-1. As shown in Fig. 4D-F, with the addition of
G4-DNA HTG21, Pu27 or c-kit-1 to the solutions of 5b, remarkably decrease in
fluorescence intensity were observed, indicating that 5b could strongly bind to all of
the three tested G-quadruplex DNA. By comparing the quenching constant (Kq) of
7
7
6
HTG21, Pu27 or c-kit-1 (1.56×10 , 1.50×10 and 6.26×10 , respectively, Fig. 4D-F)
4
that are nearly 300 folds to that of the ct-DNA (5.92×10 , Fig. 4B), suggesting that 5b
is more preferentially binding to G-quadruplex than to ct-DNA.
CD spectroscopy is also commonly used to detect the interaction between DNA
with small molecule [42,43]. CD spectral titration was therefore carried out to further
investigate the interaction of 5b with ct-DNA and G-quadruplex DNA. As shown in
Fig. 4C, the CD spectrum of ct-DNA (B-form right–handed helicity) usually shows a
positive band at 285 and a negative band at 248 nm; with the addition of 5b to the
ct-DNA solution, the decrease of positive and negative peaks’ intensity was observed,
indicated that ct-DNA maintained the B-form helices when interacting with 5b. As in
Fig. 4G-I, the spectra Pu27, c-kit-1 possessed a negative peak at 240 nm and a
positive one around 260 nm, the typical peak of parallel quadruplex DNA [44], while
the spectra of HTG21 also showed another positive peak at 290 nm, indicating the
coexistence of parallel and anti-parallel forms in the HTG21. With the addition of 5b,
a hyperchromic effect on the positive peak and red shift of the negative peak was
observed in the HTG21. While in the Pu27, 5b produce a hypochromic effect on the
1
0

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negative peak and red shift of the positive one. And in the c-kit-1, only hypochromic
effect on both the peaks was observed, indicating that 5b might adopt divers mode to
bind with different G-quadruplex. The results of spectroscopic analyses suggested the
5
b can preferentially bind to G-quadruplex, interfering the function of DNA which
might result in the cell cycle arrest.
2
.2.5. Compound 5b arrested cell cycle in S and G2/M phases
Fig. 5. Cell cycle analysis of 5b in MGC-803 cells. (A) Effects of 5b on cell cycle distribution in
MGC-803 cells. Cells were treated with different concentrations (0.5, 1.0, 2.5 µM) of 5b for 48 h
to determine cell cycle phase distribution. (B) The percentages of MGC-803 cells in the different
phases of cell cycle were presented. (C) Immunoblotting analysis of proteins related to 5b–
induced cell cycle arrest, β-actin was used as the loading reference. (D) The expression levels of
p21, p27 in MGC-803 cells treated with 5b for 24 h were detected by western blot, β-actin was
used as the loading reference.
To investigate whether 5b inhibits the proliferation of MGC-803 cells through
cell cycle arrest, the cell cycle distribution of 5b-treated MGC-803 cells was
1
1

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examined by flow cytometry. As shown in Fig. 5A and B, the percentage of MGC-803
cells at S phase was markedly increased from 32.97% (0 µM) to 44.73% (0.5 µM),
5
2.87% (1.0 µM) and 59.44% (2.5 µM) after incubation with 5b. Meanwhile, cells in
G2/M phase increased from 3.76% (0.5 µM) to 23.02% (1.0 µM) and 31.82% (2.5
µM), and the G1 phase decreased from 51.51% in control to 8.74% (2.5 µM),
indicating that 5b caused a remarked G2/M phase arrest in a dose–dependent manner.
Taken into consideration of the effects of 5b blocking cell cycle in S and G2/M
phase, cell cycle–related proteins were analyzed by Western blot assay to elucidate the
mechanisms involved in the cell cycle arrest in MGC-803 cells. As shown in Fig. 5C,
5
b-treatment slightly down-regulated the expression levels of cyclin B1 and CDK2,
but did not affect the levels of cyclin A2 and cyclin E2. However, the levels of cyclin–
dependent kinase (CDK) inhibitors p21 and p27, especially that of p21, were
obviously increasing, suggesting that the increases in the levels of p21 and p27 were
predominantly responsible for the S and G2/M cell cycle arrest induced by 5b.
2
.2.6. Compound 5b induced mitochondria–mediated apoptosis in MGC-803 cells
To determine whether the anti-proliferation effect of 5b is via apoptosis, the
apoptotic effect of 5b was evaluated by Annexin V–FITC/PI staining assay. MGC-803
cells were treated with 5b at the concentrations of 0, 0.5, 1.0, and 2.5 µM for 24 h. As
shown in Fig. 6A and Table 2, the percentages of apoptotic cells were increased
markedly from 1.66% (Control) to 17.95% (0.5 µM), 37.00% (1.0 µM) and 58.65%
(2.5 µM), indicating the extraordinary apoptosis-inducing effects of 5b. The
morphology of 5b–treated MGC-803 cells was further explored. 5b–treated MGC-803
cells (0.5, 1.0, 2.5 µM, respectively, for 24 h) were stained with Hoechst 33258 and
observed under a fluorescence microscope. As shown in Fig. 6B, cell shrinkage
occurred and the fluorescence intensity in MGC-803 cells was increased significantly,
suggesting a typical apoptotic morphology. Notably, when the concentration of 5b
increased to 2.5 µM, the contours of many cells became irregular, the nuclei
condensed and the apoptotic bodies appeared.
To explore which death effectors were the dynamic participants in the apoptosis
signaling, the levels of Bcl-2 family proteins were measured before and after 5b
1
2

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treatment. As shown in Fig. 6D, the expression levels of pro-apoptotic proteins Bim,
Bak, and Bax were up–regulated while the levels of anti-apoptotic proteins Bcl-2 and
Bcl-xL were down–regulated significantly. In addition, the levels of cytochrome-c and
Apaf-1 (Fig. 6E) were significantly increased. These results collectively indicated that
5
b–induced cell apoptosis was mainly through mitochondria–mediated intrinsic
apoptotic pathway.
Fig. 6. 5b induces cell apoptosis through the intrinsic apoptotic pathway. (A) The induction of
apoptosis in MGC-803 cells by 5b was examined by flow cytometer analysis with Annexin V–
FITC and PI staining after treatment with 0.5, 1.0, 2.5 µM for 24 h. (B) Morphological changes in
1
3

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the nuclei of cultured MGC-803 cells by 5b treatment. MGC-803 cells were treated with 5b at
indicate concentration for 24 h then stained with Hoechst 33258. (C) Activated ratio of
Caspase-3/9 in MGC-803 cells with 5b (2.5 µM) for 24 h. Caspase-3/9 was evaluated by
Caspase-3/9 staining kits. (D) Immunoblotting analysis of proteins related to the apoptotic
pathway evoked by 5b. (E) Western blotting was performed for the detection of cytochrome c,
Apaf-1 and AIF.
Table 2
Quantitative apoptosis assay of MGC-803 cell treated with 5b
Conc.
Viable cells
(Q4, %)
Early apoptotic
cells (Q3, %)
0.97±0.10
Late apoptotic
cells (Q2, %)
0.69±0.05
Necrotic
cells (Q1, %)
1.81±0.09
1.76±0.21
4.69±1.33
4.01±1.57
Apoptotic cells
(Q2 + Q3, %)
1.66±0.05
(µM)
0
96.53±2.11
80.29±2.03
58.31±1.52
37.30±1.44
**
**
**
0.5
1.0
2.5
13.80±1.13
22.00±2.02
51.20±3.41
4.15±0.44
17.95±1.62
37.00±3.51
58.65±3.84
15.00±3.18
7.45±1.09
Data were presented as the mean ± SD. *p < 0.05, and **p < 0.01, vs control.
Since Caspase-9 and Caspase-3 play crucial roles as an initiator and an executor
in cell apoptosis, respectively [45,46], whether the proapoptotic effect of 5b in
MGC-803 cells was involved with activated Caspase-3/9 was also investigated. As
shown in Fig. 6C, the proportion of Caspase-3/9 activated cells after 5b-treatment
increased to 45.1% and 46.9%, respectively, confirming that 5b was the efficient
activator of Caspase-3/9 and it induced cell apoptosis via a caspase-dependent
pathway. Substantial increase of the AIF expression was also observed in Fig. 6E,
suggesting that the AIF-mediated pathway to induce apoptosis probably existed.
Fig. 7. 5b triggers apoptosis through mitochondrial pathway. (A) Collapse of mitochondrial
membrane potential of MGC-803 cells treated with 5b for 24 h stained by JC-1. Images were
acquired using a BioTek Cytation microscope (magnification 20×). (B) Collapse of mitochondrial
1
4

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membrane potential of MGC-803 cells untreated and treated with 5b for 24 h by flow cytometry
2
+
analysis with JC-1 staining. Effects of 5b on the intracellular free Ca level in MGC-803 cells.
After treated with 5b at the dose of 0.5, 1.0, 2.5 µM for 24 h, cells stained with Fluo-3 AM for 30
min were analyzed by flow cytometry. (D) Effects of 5b on intracellular ROS generation.
MGC-803 cells were treated with 5b at the dose of 0.5, 1.0, 2.5 µM for 24 h. Results are expressed
as relative fluorescent intensity.
Mitochondria–mediated intrinsic pathway is commonly involved in
chemotherapeutic agent induced cell apoptosis [47]. During the process, fluctuations
2
+
of mitochondrial membrane potential (∆Ψm), Ca and reactive oxygen species (ROS)
occur. To further investigate the apoptosis–inducing effect of 5b, the changes of ∆Ψm,
2
+
Ca and ROS in MGC-803 cells were investigated via flow cytometry. The
fluorescent probe JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-
-carbocyanine), DCFH–DA (2′,7′-dichlorodihydrofluorescein diacetate) and fluo-3
2
+
AM were used to detect the changes of ∆Ψm, ROS and Ca , respectively.
As shown in Fig. 7A, MGC-803 cells in the control group displayed mainly the
red fluorescence, while the 5b-treated cells showed the green fluorescence, indicating
a remarkable decrease of ∆Ψm. Flow cytometry was used to further analyze the
changes in red/green fluorescence intensity quantitatively. As shown in Fig. 7B, flow
cytometric analysis clearly indicated the increase in depolarized cell population (p2)
from untreated control group (3.37%) to 0.5 µM (23.60%), 1.0 µM (89.10%) and 2.5
µM (97.5%) 5b-treated cells. Since the maintenance of ∆Ψm is important for
mitochondrial integrity and bioenergetic function [48], the intensive decrease in ∆Ψm
indicated mitochondrial dysfunction and occurrence of apoptosis.
In Fig. 7C and 7D, fluorescence intensities were all increased in a dose–
dependent manner when comparing with those in control group, indicating the
2
+
increase of Ca level and ROS in 5b-treated cells. ROS generation has been reported
to be involved in early stages of apoptosis in cellular systems [49,50]. The massive
2
+
generation of ROS, the intensive fluctuation of Ca level and the ∆Ψm are usually the
indicator of the collapse of mitochondrial function and the occurrence of cell
apoptosis.
2
.2.7. Compound 5b exhibited high anticancer potency in vivo
The anticancer potency of 5b in vivo was evaluated in a MGC-803 xenograft
1
5

ACCEPTED MANUSCRIPT
tumor model. The MGC-803 xenograft tumor-bearing mice with tumors at the volume
3
of 80 mm were randomly divided into the vehicle control group and treatment group
(n = 6/group) and treated via intraperitoneal injection of 5b or CPT (positive control),
respectively, every other day. As shown in Fig. 8, 5b intraperitoneally administered to
MGC-803 xenograft tumor-bearing mice at 6 or 12 mg/kg/2 days for two weeks
significantly inhibited the tumor growth in the MGC-803 xenograft model. The two
dosage groups showed tumor growth inhibition (TGI) of 31.2% and 53.2%,
respectively, which was slightly lower than that of CPT (62.3%). There were no
significant changes in body weight and other adverse effects during the experimental
period in both the vehicle control group and treatment group, confirming that 5b
possessed potent anticancer efficacy in vivo with low toxicity.
Fig. 8. In vivo antitumor efficacy of 5b in human gastric carcinoma xenograft models. (A)
Changes in tumor volume of mice from different groups over the observation period. (B) Body
weight of mice treated with 5b (6 or 12 mg/kg) or CPT (6 mg/kg) for two weeks. (C) Tumor
weight and tumor growth inhibition after treating with 5b at the end of treatment. (D) Photographs
of excised tumor from vehicle group and treatment groups. Results are expressed as the mean ±
SD, error bars represented SD, n = 6, (∗) p < 0.05 and (∗∗) p < 0.01 vs vehicle.
3
. Conclusion
In this study, a series of novel natural product mimics were designed based on
1
6

ACCEPTED MANUSCRIPT
the skeleton of Cryptolepine and Aromathecin, and 30 target compounds were
synthesized, characterized, and their antitumor activities, interaction with DNA and
the molecular mechanism of apoptosis induction were investigated. The results
demonstrated that the representative compound 5b exhibited strong G-quadruplex
binding, potent DNA cleavage and Topo I inhibitory activity, causing cell cycle arrest
at S and G2/M phase. It also induced cell apoptosis at low concentrations via a
caspase-dependent intrinsic mitochondria-mediated pathway (Fig. 9) in MGC-803
cells. The in vivo study demonstrated that 5b could effectively inhibit tumor growth in
the MGC-803 xenograft nude mouse model with low side effects. Compound 5b is a
promising candidate for further exploration as a cancer chemotherapeutic agent.
Fig. 9. Proposed molecular mechanism and in vivo anticancer activity of 5b
4
. Experimental section
4
.1. General information
The major reagents employed in this work are commercially available and were
used without further purification unless otherwise noted. All reactions were carried
out under an atmosphere of dry nitrogen. Melting points were recorded on WRS–IA
apparatus and uncorrected. Thin layer chromatography (TLC) was performed on silica
gel plates 60 F–254 and column chromatography on silica gel (200–300 mesh) from
Branch of Qingdao Marine Chemical Co., Ltd. NMR spectra were recorded in CDCl3,
CD OD, or DMSO-d on Bruker Advance (500 MHz, 600 MHz) with TMS as internal
3
6
standard. Chemical shifts were recorded in δ values. HRMS were measured in ESI
1
7

ACCEPTED MANUSCRIPT
mode and the mass analyzer of the HRMS was TOF. The NMR spectra and HRMS
spectra could be found in Supporting Information.
Topoisomerase I and pBR322 DNA were purchased from TaKaRa Biotechnology
(
Dalian) Co., Ltd. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MTT) and propidium iodide (PI) were purchased from Sigma (St. Louis, MO).
(
Anti-cyclinA2, anti-cyclinB1, anti-cyclinE2, anti-CDK2, anti-p21, anti-p27, anti-Bim,
anti-Bax, Anti-Bak, anti-Bcl-xl, anti-Bcl-2, anti-AIF, anti-Apaf-1, anti-cytochrome c,
anti-β-actin antibodies were purchased from Abcam (Cambridge, MA). Caspase-3/9
activity fluorescence detection kits were purchased from Biovision. Anti-rabbit and
-
mouse secondary antibodies were purchased from Santa Cruz Biotechnology, Inc.
Dallas, TX, USA).
(
4
4
4
3
.2. Compounds synthesis and characterization
.2.1. Preparation of compounds 4
.2.1.1 General procedure
The mixture of indenoquinolines 3 (5 mmol) in 15 mL of POCl was refluxed for
3
–4 h. The excess POCl was removed under reduced pressure. The residue was
3
neutralized with saturated NaHCO solution and extracted with CH Cl (3×100 mL).
3
2
2
The organic layer was combined, washed with brine and dried over anhydrous
Na SO . The solvent was removed under reduced pressure and the residue was
2
4
purified on silica gel (eluent: VEtOAc/VPE =1: 4) to provide the chloro-indeno-
quinoline derivatives 4.
-
4.2.1.2 10-Chloro-11H-indeno[1,2-b]quinoline (4a). Pale yellow solid, 86%
1
yield, m.p. 152–153 ℃ . H NMR (500 MHz, CDCl ) δ 8.27–8.21 (m, 2H), 8.20–8.17
3
1
3
(
m, 1H), 7.77–7.73 (m, 1H), 7.62–7.57 (m, 2H), 7.53–7.47 (m, 2H), 4.02 (s, 2H); C
NMR (125 MHz, CDCl ) δ 161.3, 149.2, 144.2, 140.2, 137.7, 133.0, 130.4, 129.5,
3
1
29.4, 127.7, 126.5, 125.4, 125.2, 123.6, 122.3, 34.1.
4.2.1.3. 10-Chloro-8-fluoro-11H-indeno[1,2-b]quinoline (4b). Pale yellow solid,
1
9
8
3
1
0% yield, m.p. 169–170 ℃ . H NMR (500 MHz, CDCl ) δ 8.17–8.14 (m, 1H), 8.13–
3
.11 (m, 1H), 7.78 (dd, J = 9.7, 2.9 Hz, 1H), 7.56–7.52 (m, 1H), 7.48–7.43 (m, 3H),
1
3
.94 (s, 2H); C NMR (125 MHz, CDCl ) δ 160.8, 160.7 (d, J = 246.3 Hz), 146.0,
3
43.9, 139.9, 136.7 (d, J = 6.3 Hz), 133.7, 131.8 (d, J = 8.8 Hz), 130.4, 127.7, 126.09
1
8

ACCEPTED MANUSCRIPT
(d, J = 10.0 Hz), 125.4, 122.2, 119.4 (d, J = 12.5 Hz), 107.6 (d, J = 12.5 Hz), 34.1.
4.2.1.4. 8,10-Dichloro-11H-indeno[1,2-b]quinoline (4c). Pale yellow solid, 91%
1
yield, m.p. 208–209 ℃ . H NMR (500 MHz, CDCl ) δ 8.22–8.14 (m, 2H), 8.08 (d, J =
3
8
2
1
.9 Hz, 1H), 7.65 (d, J = 8.9 Hz, 1H), 7.60–7.56 (m, 1H), 7.52–7.48 (m, 2H), 4.01 (s,
1
3
H); C NMR (125 MHz, CDCl ) δ 161.6, 147.5, 144.2, 139.8, 136.5, 133.9, 132.5,
3
31.0, 130.7, 130.4, 127.8, 126.0, 125.5, 122.7, 122.4, 34.2.
4.2.1.5. 8-Bromo-10-chloro-11H-indeno[1,2-b]quinoline (4d). Pale yellow solid,
1
9
0% yield, m.p. 201–202 ℃ . H NMR (500 MHz, CDCl ) δ 8.32 (s, 1H), 8.19–8.15
3
(m, 1H), 7.99 (d, J = 8.9 Hz, 1H), 7.76 (d, J = 8.9, 1H), 7.57–7.56 (m, 1H), 7.51–7.47
13
(
m, 2H), 3.98 (s, 2H); C NMR (125 MHz, CDCl ) δ 161.7, 147.7, 144.2, 139.8,
3
1
36.4, 133.8, 132.9, 131.0, 130.7, 127.8, 126.3, 126.0, 125.5, 122.4, 120.6, 34.2.
4.2.1.6. 10-Chloro-8-methyl-11H-indeno[1,2-b]quinoline (4e). Pale yellow solid,
1
8
5% yield, m.p. 164–166 ℃ . H NMR (500 MHz, CDCl ) δ 8.21–8.18 (m, 1H), 8.05
3
(d, J = 8.6 Hz, 1H), 7.94 (s, 1H), 7.56–7.52(m, 2H), 7.48–7.44 (m, 2H), 3.95 (s, 2H),
1
3
2
.56 (s, 3H); C NMR (125 MHz, CDCl ) δ 160.4, 147.6, 144.1, 140.3, 137.0, 136.6,
3
1
32.9, 131.7, 130.1, 129.1, 127.6, 125.4, 125.1, 122.6, 122.1, 34.1, 21.8.
4.2.1.7. 4f Pale yellow solid, yield 70%, m.p. 185–187 ℃ ; 4g Pale yellow solid,
yield 65%, m.p. 250–251 ℃ ; 4h Pale yellow solid, yield 75%, m.p. 234–235 ℃ , 4i
Pale yellow solid, 70% yield, m.p. 232–233 ℃ , 4j Pale yellow solid, 89% yield, m.p.
2
09–210 ℃ . These products hardly dissolved in the Commonly used solvents and
can’t be further structurally characterize by NMR.
4
4
.2.2. Preparation of compounds 5, 6 and 7
.4.2.1 General procedure
To a solution of chlor-indenoquinoline derivatives 4 (1 mmol) and amines (5
equiv.) in glycol (3 mL) was heated at 150 ℃ for 15–20 h (monitored by TLC) until
completed. The mixture was neutralized with saturated NaHCO solution and
3
extracted with CH Cl (3×100 mL). The organic layer was combined, washed with
2
2
brine and dried over anhydrous Na SO , filtered, the filtrate was concentrated under
2
4
reduced pressure. The residue was purified on silica gel (eluent: VMeOH/VEtOAc = 1: 3)
to furnish the corresponding product 5–7.
1
3
3
4
.2.2.2. N -(11H-Indeno[1,2-b]quinolin-10-yl)-N ,N -dimethylpropane-1,3-
1
diamine (5a). Pale yellow solid, 63% yield, m.p. 83–84 ℃ . H NMR (500 MHz,
1
9

ACCEPTED MANUSCRIPT
CDCl ) δ 8.31–8.28 (m, 1H), 8.08 (dd, J = 8.4, 1.0 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H),
3
7
.57–7.53 (m, 1H), 7.46–7.40 (m, 3H), 7.39–7.35 (m, 1H), 7.31–7.28 (m, 1H), 4.02 (s,
2
H), 3.78 (t, J = 5.5 Hz, 2H), 2.51(t, J = 5.5 Hz, 2H), 2.33 (s, 6H), 1.84–1.78 (m, 2H);
1
3
C NMR (125 MHz, CDCl ) δ 161.7, 148.2, 147.5, 144.6, 140.4, 129.1, 128.8, 128.5,
3
1
27.0, 124.6, 123.8, 121.9, 120.6, 118.9, 111.8, 59.1, 45.9, 45.5, 35.2, 26.0. HRMS
+
(
ESI) m/z calcd for C H N [M+H] 318.1970, found 318.1958.
2
1
24
3
1
3
3
4
.2.2.3. N -(8-Fluoro-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -dimethylpropane-
1
1
,3-diamine (5b). Pale yellow solid, 67% yield, m.p. 99–100 ℃ . H NMR (500 MHz,
CDCl ) δ 8.21 (d, J = 7.5 Hz, 1H), 8.05–7.99 (m, 1H), 7.59 (s, 1H), 7.45 (d, J = 7.1
3
Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 7.39 (d, J = 7.3 Hz, 1H), 7.35 (s, 1H), 7.33 (s, 1H),
4
.09 (s, 2H), 3.85 (t, J = 5.5 Hz, 2H), 2.55 (t, J = 5.5 Hz, 2H), 2.38 (s, 6H), 1.87–1.83
1
3
(
m, 2H); C NMR (125 MHz, CDCl ) δ 163.3 (d, J = 241.3 Hz), 161.9, 147.0, 145.7,
3
1
44.4, 140.6, 131.3 (d, J = 8.8 Hz), 129.0, 127.1, 124.7, 121.6, 119.6 (d, J = 15.8 Hz),
117.8 (d, J = 25.0 Hz), 112.3, 105.1 (d, J = 23.8 Hz), 59.5, 46.4, 45.5, 35.0, 25.5.
+
HRMS (ESI) m/z calcd for C H FN [M+H] 336.1876, found 336.1863.
2
1
23
3
1
3
3
4
.2.2.4. N -(8-Chloro-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -dimethylpropane-
1
1
,3-diamine (5c). Pale yellow solid, 63% yield, m.p. 163–164 ℃ . H NMR (500 MHz,
CDCl ) δ 8.21 (d, J = 7.8 Hz, 1H), 8.04 (s, 1H), 7.97 (dd, J = 8.9, 1.2 Hz, 1H), 7.68–
3
7
.65 (m, 1H), 7.50–7.47 (m, 1H), 7.45–7.40 (m, 2H), 7.40–7.35 (m, 1H), 4.07–4.03
(m, 2H), 3.85 (t, J = 4.9 Hz, 2H), 2.58 (t, J = 4.9 Hz, 2H), 2.41 (d, J = 1.5 Hz, 6H),
1
3
1
1
2
.88–1.82 (m, 2H); C NMR (125 MHz, CDCl ) δ 162.3, 146.9, 144.6, 140.4, 130.4,
3
29.2, 129.1, 128.9, 127.1, 124.7, 121.8, 120.7, 119.9, 112.3, 59.6, 46.6, 45.4, 35.1,
+
5.2. HRMS (ESI) m/z calcd for C H ClN [M+H] 352.1581, found 352.1576.
2
1
23
3
1
3
3
4
.2.2.5. N -(8-Bromo-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -dimethylpropane-
1
1
,3-diamine (5d). Yellow solid, 56% yield, m.p. 173–174 ℃ . H NMR (500 MHz,
CDCl ) δ 8.23 (d, J = 6.7 Hz, 1H), 8.15 (s, 1H), 7.92–7.89 (m, 1H), 7.85–7.81 (m,
3
1
H), 7.61–7.57 (m, 1H), 7.44–7.37 (m, 3H), 4.08–4.01 (m, 2H), 3.86 (t, J = 3.1 Hz,
1
3
2
H), 2.59 (t, J = 4.9 Hz, 2H), 2.42 (d, J = 2.4 Hz, 6H), 1.89–1.83 (m, 2H); C NMR
(
125 MHz, CDCl ) δ 162.0, 146.9, 144.6, 140.0, 131.5, 130.2, 129.3, 127.1, 124.6,
3
1
24.0, 121.9, 120.3, 117.1, 112.1, 59.6, 46.5, 45.4, 35.1, 25.1. HRMS (ESI) m/z calcd
+
for C H BrN [M+H] 396.1075, found 396.1062.
2
1
23
3
1
1
3
4
.2.2.6. N ,N -Dimethyl-N -(8-methyl-11H-indeno[1,2-b]quinolin-10-yl)propane-
1
1
,3-diamine (5e). Pale yellow solid, 76% yield, m.p. 139–140 ℃ . H NMR (500 MHz,
CDCl ) δ 8.26 (d, J = 7.5 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.51 (br, 1H), 7.48 (s, 1H),
3
2
0

ACCEPTED MANUSCRIPT
7
.47–7.42 (m, 2H), 7.42–7.37 (m, 2H), 4.09 (s, 2H), 3.85 (t, J = 5.6 Hz, 2H), 2.54 (t, J
1
3
=
5.6 Hz, 2H), 2.48 (s, 3H), 2.37 (s, 6H), 1.88–1.83 (m, 2H); C NMR (125 MHz,
CDCl ) δ 161.3, 147.1, 146.8, 144.5, 140.8, 133.2, 130.3, 128.8, 127.0, 124.6, 121.7,
3
1
20.1, 118.9, 112.0, 59.3, 46.2, 45.6, 35.2, 25.9, 21.9. HRMS (ESI) m/z calcd for
+
C H N [M+H] 332.2127, found 332.2113.
22
26
3
1
3
3
4
.2.2.7. N -(2,3-Dimethoxy-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -dimethyl-
1
propane-1,3-diamine (5f). Pale yellow solid, 41% yield, m.p. 167–168 ℃ . H NMR
(
500 MHz, DMSO-d ) δ 8.13 (d, J = 8.3 Hz, 1H), 7.84 (dd, J = 8.4, 1.1 Hz, 1H),
6
7
.59–7.55 (m, 1H), 7.51 (s, 1H), 7.38–7.36 (m, 1H), 7.25 (s, 1H), 7.11 (t, J = 5.5 Hz,
H), 4.16 (s, 2H), 3.89 (s, 3H), 3.86 (s, 3H), 3.77 (t, J = 6.6 Hz, 2H), 2.40 (t, J = 6.6
1
13
Hz, 2H), 2.19 (s, 6H), 1.82–1.79 (m, 2H); C NMR (125 MHz, DMSO-d ) δ 162.4,
6
1
1
51.2, 149.3, 149.0, 146.6, 138.1, 133.2, 129.0, 128.7, 123.5, 122.1, 119.1, 111.6,
08.7, 104.0, 57.7, 56.2, 56.1, 45.7, 43.6, 35.0, 28.2. HRMS (ESI) m/z calcd for
+
C H N O [M+H] 378.2182, found 378.2166.
23
28
3
2
1
3
3
4
.2.2.8. N -(8-Fluoro-2,3-dimethoxy-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -
1
dimethylpropane-1,3-diamine (5g). Pale yellow solid, 50% yield, m.p. 175–176 ℃ . H
NMR (600 MHz, DMSO-d ) δ 7.97 (dd, J = 11.7, 2.6 Hz, 1H), 7.90–7.87 (m, 1H),
6
7
3
6
.50 (s, 1H), 7.48–7.45 (m, 1H), 7.23 (s, 1H), 6.91 (t, J = 5.5 Hz, 1H), 4.15 (s, 2H),
.89 (s, 3H), 3.85 (s, 3H), 3.73 (t, J = 6.7 Hz, 2H), 2.39 (t, J = 6.7 Hz, 2H), 2.19 (s,
1
3
H), 1.82–1.77 (m, 2H); C NMR (150 MHz, DMSO-d ) δ 162.2, 158.8 (d, J = 237.0
6
Hz), 151.2, 149.3, 146.1 (d, J = 3.8 Hz), 146.0, 137.9, 132.9, 131.2 (d, J = 7.5 Hz),
19.4 (d, J = 6.3 Hz), 118.0 (d, J = 21.3 Hz), 112.0, 108.6, 106.5 (d, J = 18.8 Hz),
1
1
03.8, 57.5, 56.2, 56.1, 45.6, 43.3, 34.9, 28.3. HRMS (ESI) m/z calcd for
+
C H FN O [M+H] 396.2087, found 396.2072.
23
27
3
2
1
3
3
4
.2.2.9. N -(8-Chloro-2,3-dimethoxy-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -
1
dimethylpropane-1,3-diamine (5h). Yellow solid, 31% yield, m.p. 125–126 ℃ . H
NMR (500 MHz, DMSO-d ) δ 8.26 (d, J = 1.5 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.57
6
(dd, J = 8.8, 1.7 Hz, 1H), 7.49 (s, 1H), 7.23 (s, 1H), 7.16 (t, J = 4.9 Hz, 1H), 4.16 (s,
2
H), 3.88 (s, 3H), 3.85 (s, 3H), 3.75 (t, J = 4.9 Hz, 2H), 2.50 (t, J = 4.9 Hz, 2H), 2.27
1
3
(
s, 6H), 1.87–1.81 (m, 2H); C NMR (125 MHz, DMSO-d ) δ 163.2, 150.5, 149.6,
6
1
5
47.6, 145.8, 138.6, 131.7, 130.8, 128.9, 127.6, 121.5, 119.9, 112.3, 112.0, 104.4,
7.7, 56.2, 45.7, 43.6, 34.6, 31.4, 28.1. HRMS (ESI) m/z calcd for C H ClN O
2
3
27
3
2
+
[
M+H] 412.1792, found 412.1778.
1
3
3
4
.2.2.10. N -(8-Bromo-2,3-dimethoxy-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -
2
1

ACCEPTED MANUSCRIPT
1
dimethylpropane-1,3-diamine (5i). Pale yellow solid, 53% yield, m.p. 206–207 ℃ . H
NMR (500 MHz, DMSO-d ) δ 8.36 (s, 1H), 7.77 (d, J = 8.9 Hz, 1H), 7.67 (dd, J = 8.7,
6
1
.5 Hz, 1H), 7.49 (s, 1H), 7.26 (t, J = 5.6 Hz, 1H), 7.23 (s, 1H), 4.15 (s, 2H), 3.89 (s,
H), 3.86 (s, 3H), 3.75 (t, J = 6.5 Hz, 2H), 2.42 (t, J = 6.5 Hz, 2H), 2.22 (s, 6H), 1.84–
3
1
3
1
.79 (m, 2H); C NMR (125 MHz, DMSO-d ) δ 163.0, 151.4, 149.4, 147.8, 145.8,
6
1
38.3, 132.8, 131.6, 131.1, 124.7, 120.5, 116.1, 112.3, 108.6, 103.9, 57.7, 56.2, 56.1,
+
4
5.6, 43.6, 35.0, 28.0. HRMS (ESI) m/z calcd for C H BrN O [M+H] 456.1287,
2
3
27
3
2
found 456.1271.
1
3
3
4
.2.2.11. N -(2,3-Dimethoxy-8-methyl-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -
1
dimethylpropane-1,3-diamine (5j). Pale yellow solid, 30% yield, m.p. 143–144 ℃ . H
NMR (500 MHz, DMSO-d ) δ 7.89 (s, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.50 (s, 1H),
6
7
.41 (d, J = 8.2 Hz, 1H), 7.22 (s, 1H), 7.09 (s, 1H), 4.13 (s, 2H), 3.89 (s, 3H), 3.85 (s,
H), 3.76 (t, J = 6.3 Hz, 2H), 2.48 (s, 3H), 2.40 (t, J = 6.3 Hz, 2H), 2.21 (s, 6H), 1.83–
3
1
3
1
.76 (m, 2H); C NMR (125 MHz, DMSO-d ) δ 161.7, 151.0, 149.3, 147.3, 146.1,
6
1
37.9, 133.3, 132.6, 130.5, 128.8, 121.2, 118.9, 111.6, 108.6, 103.8, 57.9, 56.2, 56.1,
+
4
5.7, 43.8, 34.9, 28.0, 21.8. HRMS (ESI) m/z calcd for C H N O [M+H] 392.2338,
2
4
30
3
2
found 392.2323.
1
2
2
4
.2.2.12. N -(11H-Indeno[1,2-b]quinolin-10-yl)-N ,N -dimethylethane-1,2-
1
diamine (6a). Pale yellow solid, 75% yield, m.p. 172–173 ℃ . H NMR (500 MHz,
CDCl ) δ 8.26 (d, J = 7.4 Hz, 1H), 8.09 (dd, J = 8.4, 1.0 Hz, 1H), 7.79 (d, J = 8.4 Hz,
3
1
1
2
1
H), 7.63–7.59 (m, 1H), 7.50 (d, J = 7.3 Hz, 1H), 7.48–7.44 (m, 1H), 7.43–7.41 (m,
H), 7.40–7.38 (m, 1H), 5.90 (s, 1H), 4.14 (d, J = 1.7 Hz, 2H), 3.75 (t, J = 5.3 Hz,
1
3
H), 2.63 (t, J = 5.3 Hz, 2H), 2.32 (s, 6H); C NMR (125 MHz, CDCl ) δ 162.3,
3
48.9, 146.7, 144.5, 140.9, 129.5, 129.1, 128.5, 127.1, 124.7, 124.0, 121.8, 120.2,
+
119.1, 113.0, 58.2, 45.1, 42.1, 34.9. HRMS (ESI) m/z calcd for C H N [M+H]
20 22 3
3
04.1814, found 304.1801.
1
2
2
4
.2.2.13. N -(8-Fluoro-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -dimethylethane-
1
1
,2-diamine (6b). Pale yellow solid, 60% yield, m.p. 156–157 ℃ . H NMR (500 MHz,
CDCl ) δ 8.20 (d, J = 7.4 Hz, 1H), 8.05–8.02 (m, 1H), 7.47–7.46 (m, 1H), 7.44–7.43
3
(m, 1H), 7.42–7.39 (m, 1H), 7.39–7.36 (m, 1H), 7.36–7.32 (m, 1H), 5.64 (d, J = 3.4
Hz, 1H), 4.07 (d, J = 6.1 Hz, 2H), 3.71 (t, J = 4.8 Hz, 2H), 2.65 (t, J = 4.8 Hz, 2H),
1
3
2
.32 (s, 6H); C NMR (125 MHz, CDCl ) δ 161.9, 159.4 (d, J = 242.5 Hz), 146.2 (d,
3
J = 3.8 Hz), 145.9, 144.3, 140.7, 131.6 (d, J = 8.8 Hz), 129.2, 127.2, 124.7, 121.6,
19.5 (d, J = 8.8 Hz), 118.0 (d, J = 25.0 Hz), 113.6, 104.6 (d, J = 22.5 Hz), 58.2, 45.0,
1
2
2

ACCEPTED MANUSCRIPT
4
2.0, 34.7. HRMS (ESI) m/z calcd for C H N F 322.1720, found 322.1708.
2
0
21
3
1
2
2
4
.2.2.14. N -(8-Chloro-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -dimethylethane-
1
1
,2-diamine (6c). Pale yellow solid, 62% yield, m.p. 178–180 ℃ . H NMR (500 MHz,
CDCl ) δ 8.20 (d, J = 7.1 Hz, 1H), 7.98 (d, J = 8.9 Hz, 1H), 7.69 (d, J = 2.1 Hz, 1H),
3
7
.51 (dd, J = 8.9, 2.2 Hz, 1H), 7.47–7.43 (m, 2H), 7.41 (dd, J = 7.2, 1.4 Hz, 1H), 5.73
(s, 1H), 4.07 (s, 2H), 3.73 (t, J = 5.8 Hz, 2H), 2.63 (t, J = 5.8 Hz, 2H), 2.33 (s, 6H);
1
3
C NMR (125 MHz, CDCl ) δ 162.4, 147.2, 145.9, 144.4, 140.4, 130.8, 129.5, 129.4,
3
1
29.2, 127.2, 124.8, 121.8, 119.8, 113.7, 58.1, 45.1, 42.1, 34.9. HRMS (ESI) m/z
calcd for C H ClN 338.1424, found 338.1410.
2
0
21
3
1
2
2
4
.2.2.15. N -(8-Bromo-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -dimethylethane-
1
1
,2-diamine (6d). Pale yellow solid, 62% yield, m.p. 187–188 ℃ . H NMR (500 MHz,
CDCl ) δ 8.20 (d, J = 7.0 Hz, 1H), 7.91 (d, J = 8.9 Hz, 1H), 7.89–7.88 (m, 1H), 7.64
3
(dd, J = 8.9, 2.0 Hz, 1H), 7.48 (d, J = 7.2 Hz, 1H), 7.47–7.41 (m, 2H), 5.70 (s, 1H),
1
3
4
.09 (s, 2H), 3.75 (t, J = 5.8 Hz, 2H), 2.63 (t, J = 5.8 Hz, 2H), 2.33 (s, 6H); C NMR
(
125 MHz, CDCl ) δ 162.7, 147.7, 145.8, 144.4, 140.6, 131.7, 131.2, 129.4, 127.2,
3
1
24.8, 123.1, 121.8, 120.5, 117.4, 113.8, 58.2, 45.1, 42.2, 34.9. HRMS (ESI) m/z
calcd for C H BrN 382.0919, found 382.0904.
2
0
21
3
1
1
2
4
.2.2.16. N ,N -Dimethyl-N -(8-methyl-11H-indeno[1,2-b]quinolin-10-yl)ethane
1
-
1,2-diamine (6e). Pale yellow solid, 56% yield, m.p. 176–177 ℃ . H NMR (500 MHz,
CDCl ) δ 8.27 (d, J = 7.5 Hz, 1H), 7.99 (d, J = 8.5 Hz, 1H), 7.50 (s, 1H), 7.48–7.43
3
(m, 2H), 7.43–7.39 (m, 2H), 5.84 (s, 1H), 4.08 (s, 2H), 3.76 (t, J = 6.0 Hz, 2H), 2.64 (t,
1
3
J = 5.8 Hz, 2H), 2.50 (s, 3H), 2.33 (s, 6H); C NMR (125 MHz, CDCl ) δ 161.0,
3
1
46.6, 146.5, 144.4, 140.5, 133.7, 130.7, 129.1, 128.7, 127.1, 124.7, 121.9, 119.5,
18.8, 113.0, 58.4, 45.1, 42.2, 34.9, 21.9. HRMS (ESI) m/z calcd for C H N
3
1
3
2
1
24
18.1970, found 318.1958.
1
2
2
4
.2.2.17. N -(2,3-Dimethoxy-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -dimethyl-
1
ethane-1,2-diamine (6f). Pale yellow solid, 46% yield, m.p. 215–216 ℃ . H NMR
(
500 MHz, DMSO-d ) δ 8.12 (d, J = 8.3 Hz, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.60–7.58
6
(m, 1H), 7.52 (s, 1H), 7.40–7.36 (m, 1H), 7.25 (s, 1H), 6.62 (t, J = 5.3 Hz, 1H), 4.18
(s, 2H), 3.89 (s, 3H), 3.86 (s, 3H), 3.80 (t, J = 6.6 Hz, 2H), 2.58 (t, J = 6.6 Hz, 2H),
1
3
2
1
4
.26 (s, 6H); C NMR (125 MHz, DMSO-d ) δ 162.2, 151.3, 149.4, 148.7, 148.3,
6
46.3, 138.2, 133.0, 128.8, 123.6, 122.0, 118.9, 111.8, 108.7, 104.0, 59.5, 56.2, 56.1,
5.7, 42.2, 34.9. HRMS (ESI) m/z calcd for C H N O 364.2025, found 364.2012.
2
2
26
3
2
1
2
2
4
.2.2.18. N -(8-Fluoro-2,3-dimethoxy-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -
2
3

ACCEPTED MANUSCRIPT
1
-
dimethylethane-1,2-diamine (6g). Yellow solid, 63% yield, m.p. 232–234 ℃ . H
NMR (500 MHz, DMSO-d ) δ 8.02 (dd, J = 11.5, 2.6 Hz, 1H), 7.92–7.87 (m, 1H),
6
7
3
6
1
.51 (s, 1H), 7.50–7.47 (m, 1H), 7.25 (s, 1H), 6.59 (t, J = 5.8 Hz, 1H), 4.17 (s, 2H),
.89 (s, 3H), 3.86 (s, 3H), 3.81 (t, J = 6.6 Hz, 2H), 2.63 (t, J = 6.6 Hz, 2H), 2.30 (s,
1
3
H); C NMR (125 MHz, DMSO-d ) δ 162.1, 158.8 (d, J = 238.8 Hz), 151.3, 149.4,
6
45.9 (d, J = 2.5 Hz), 138.0, 132.8, 131.1 (d, J = 8.8 Hz), 119.4 (d, J = 7.5 Hz), 118.1
(d, J = 25.0 Hz), 112.2, 108.6, 106.6 (d, J = 23.8 Hz), 103.8, 59.5, 56.2, 56.1, 45.6,
4
2.1, 34.8. HRMS (ESI) m/z calcd for C H FN O 382.1931, found 382.1914.
2
2
25
3
2
1
2
2
4
.2.2.19. N -(8-Chloro-2,3-dimethoxy-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -
1
-
dimethylethane-1,2-diamine (6h). Yellow solid, 58% yield, m.p. 181–183 ℃ . H
NMR (500 MHz, DMSO-d ) δ 8.30 (d, J = 2.1 Hz, 1H), 7.85 (d, J = 8.9 Hz, 1H), 7.58
6
(dd, J = 8.9, 2.1 Hz, 1H), 7.50 (s, 1H), 7.28 (s, 1H), 6.73 (t, J = 5.8 Hz, 1H), 4.19 (s,
2
H), 3.89 (s, 3H), 3.87 (s, 3H), 3.79 (t, J = 6.7 Hz, 2H), 2.57 (t, J = 6.7 Hz, 2H), 2.26
1
3
(
s, 6H); C NMR (125 MHz, DMSO-d ) δ 162.9, 151.4, 149.4, 147.6, 145.6, 138.2,
6
1
4
32.7, 130.9, 129.1, 127.9, 121.5, 119.9, 112.3, 108.6, 103.9, 59.8, 56.2, 56.1, 45.9,
2.4, 34.9. HRMS (ESI) m/z calcd for C H ClN O 398.1635, found 398.1619.
2
2
25
3
2
1
2
2
4
.2.2.20. N -(8-Bromo-2,3-dimethoxy-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -
-dimethylethane-1,2-diamine (6i). Yellow solid, 40% yield, m.p. 236–237 ℃ . This
product hardly dissolved in the commonly used solvents and can’t be further
structurally characterize by NMR. HRMS (ESI) m/z calcd for C H BrN O
2
2
2
25
3
4
42.1130, found 442.1117.
1
2
2
4
.2.2.21. N -(2,3-Dimethoxy-8-methyl-11H-indeno[1,2-b]quinolin-10-yl)-N ,N -
1
-
dimethylethane-1,2-diamine (6j). Yellow solid, 64% yield, m.p. 213–214 ℃ . H NMR
(
500 MHz, DMSO-d ) δ 7.95 (s, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.56 (s, 1H), 7.43 (dd,
6
J = 8.5, 1.4 Hz, 1H), 7.25 (s, 1H), 6.62 (s, 1H), 4.16 (s, 2H), 3.89 (s, 3H), 3.86 (s, 3H),
1
3
3
.81 (t, J = 6.8 Hz, 2H), 2.61 (t, J = 6.8 Hz, 2H), 2.49 (s, 3H), 2.28 (s, 6H); C NMR
125 MHz, DMSO-d ) δ 161.2, 154.1, 151.2, 149.3, 146.0, 138.0, 132.9, 130.8, 128.3,
(
6
1
27.7, 121.2, 118.7, 111.7, 108.6, 104.0, 59.6, 56.2, 56.1, 45.7, 42.2, 34.9, 21.8.
HRMS (ESI) m/z calcd for C H N O 378.2182, found 378.2165.
2
3
28
3
2
4.2.2.22. N-(3-Morpholinopropyl)-11H-indeno[1,2-b]quinolin-10-amine (7a).
1
Yellow solid, 87% yield, m.p. 163˗164 ℃ . H NMR (500 MHz, CDCl ) δ 8.25 (d, J =
3
7
.4 Hz, 1H), 8.06 (dd, J = 8.3, 0.7 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.59 (t, J = 7.6
Hz, 1H), 7.47–7.43 (m, 2H), 7.41–7.37 (m, 1H), 7.35–7.30 (m, 1H), 6.64 (s, 1H), 4.07
1
3
(
s, 2H), 3.81–3.75 (m, 6H), 2.53–2.50 (m, 6H), 1.82 (s, 2H); C NMR (125 MHz,
2
4

ACCEPTED MANUSCRIPT
CDCl ) δ 162.4, 148.8, 147.0, 144.6, 140.8, 129.5, 129.1, 128.5, 127.1, 124.7, 123.5,
3
1
21.8, 120.8, 118.9, 112.2, 66.8, 58.7, 541, 45.8, 35.3, 25.0. HRMS (ESI) m/z calcd
for C H N O 360.2076, found 360.2064.
2
3
26
3
4
.2.2.23. 8-Fluoro-N-(3-morpholinopropyl)-11H-indeno[1,2-b]quinolin-10-amine
1
(
7b). Pale yellow solid, 67% yield, m.p. 186–187 ℃ . H NMR (500 MHz, CDCl ) δ
3
8.19 (d, J = 7.4 Hz, 1H), 8.05–8.01 (m, 1H), 7.56 (dd, J = 10.5, 2.6 Hz, 1H), 7.47–
7.43 (m, 2H), 7.40 (d, J = 7.2 Hz, 1H), 7.38–7.33 (m, 1H), 6.57 (s, 1H), 4.08 (s, 2H),
3.86 (t, J = 4.6 Hz, 4H), 3.83–3.78 (m, 2H), 2.58–2.51 (m, 6H), 1.88–1.82 (m, 2H);
1
3
C NMR (125 MHz, CDCl ) δ 162.1, 159.4 (d, J = 241.3 Hz), 146.5 (d, J = 3.8 Hz),
3
1
45.8, 144.3, 140.7, 131.6 (d, J = 8.8 Hz), 129.2, 127.1, 124.7, 121.6, 119.3 (d, J= 7.5
Hz), 117.9 (d, J = 23.8 Hz), 112.6, 105.1 (d, J = 22.5 Hz), 66.7, 59.1, 54.2, 46.1, 35.1,
2
4.6. HRMS (ESI) m/z calcd for C H FN O 378.1982, found 378.1965.
23 25 3
4
.2.2.24. 8-Chloro-N-(3-morpholinopropyl)-11H-indeno[1,2-b]quinolin-10-amine
1
(
7c). Pale yellow solid, 60% yield, m.p. 219–220 ℃ . H NMR (500 MHz, CDCl ) δ
3
8
.20 (d, J = 7.4 Hz, 1H), 7.98 (d, J = 8.9 Hz, 1H), 7.89 (d, J = 2.1 Hz, 1H), 7.52 (dd, J
8.9, 2.2 Hz, 1H), 7.48–7.44 (m, 2H), 7.43–7.39 (m, 1H), 6.87 (s, 1H), 4.12 (s, 2H),
=
3
.91 (t, J = 4.7 Hz, 4H), 3.88–3.84 (m, 2H), 2.63–2.56 (m, 6H), 1.90–1.85 (m, 2H);
1
3
C NMR (125 MHz, CDCl ) δ 162.7, 147.3, 146.3, 144.5, 140.5, 131.0, 129.3, 129.1,
3
1
27.2, 124.7, 121.7, 120.0, 119.6, 112.6, 66.6, 59.5, 54.4, 46.4, 35.4, 24.4. HRMS
(
ESI) m/z calcd for C H ClN O 394.1686, found 394.1672.
23 25 3
4
.2.2.25. 8-Bromo-N-(3-morpholinopropyl)-11H-indeno[1,2-b]quinolin-10-amine
1
(
7d). Pale yellow solid, 55% yield, m.p. 233–234 ℃ . H NMR (500 MHz, CDCl ) δ
3
8
.21 (d, J = 7.1 Hz, 1H), 8.05 (d, J = 2.0 Hz, 1H), 7.91 (d, J = 8.9 Hz, 1H), 7.65 (dd, J
8.9, 2.1 Hz, 1H), 7.49 (d, J = 7.2 Hz, 1H), 7.45 (d, J = 6.7 Hz, 1H), 7.43–7.42 m,
H), 6.86 (s, 1H), 4.16 (s, 2H), 3.93 (t, J = 4.7 Hz, 4H), 3.91–3.87 (m, 2H), 2.63–2.54
=
1
1
3
(
m, 6H), 1.92–1.87 (m, 2H); C NMR (125 MHz, CDCl ) δ 162.8, 147.5, 146.3,
3
1
44.5, 140.5, 131.8, 131.2, 129.4, 127.2, 124.7, 123.1, 121.8, 120.2, 117.30, 112.7,
6
6.7, 59.4, 54.4, 46.5, 35.5, 24.5. HRMS (ESI) m/z calcd for C H BrN O 438.1181,
2
3
25
3
found 438.1167.
4
.2.2.26. 8-Methyl-N-(3-morpholinopropyl)-11H-indeno[1,2-b]quinolin-10-amine
1
(
7e). Pale yellow solid, 45% yield, m.p. 219–220 ℃ . H NMR (500 MHz, CDCl ) δ
3
8
2
3
.24 (d, J = 7.4 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.59 (s, 1H), 7.46 (t, J = 6.9 Hz,
H), 7.43–7.39 (m, 2H), 6.37 (s, 1H), 4.09 (s, 2H), 3.83–3.78 (m, 6H), 2.57–2.53 (m,
13
H), 2.51 (s, 6H), 1.88–1.81 (m, 2H); C NMR (125 MHz, CDCl ) δ 161.5, 147.0,
3
2
5

ACCEPTED MANUSCRIPT
1
46.5, 144.4, 140.7, 133.4, 130.7, 129.2, 129.0, 127.1, 124.6, 121.7, 119.6, 118.,
112.21, 66.8, 58.7, 54.2, 45.8, 35.4, 25.4, 21.8. HRMS (ESI) m/z calcd for C H N O
24 28 3
3
74.2232, found 374.2216.
4.2.2.27. 2,3-Dimethoxy-N-(3-morpholinopropyl)-11H-indeno[1,2-b]quinolin-
1
1
0-amine (7f). Pale yellow solid, 46% yield, m.p. 147–148 ℃ . H NMR (500 MHz,
DMSO-d ) δ 8.23 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.61–7.58 (m, 2H),
6
7
.40–7.36 (m, 1H), 7.22 (s, 1H), 7.07 (s, 1H), 4.16 (s, 2H), 3.89–3.84 (m, 6H), 3.78–
.73 (m, 2H), 3.59–3.56 (m, 4H), 2.45–2.38 (m, 3H), 2.36 (s, 3H), 1.85–1.78 (m, 2H);
3
1
3
C NMR (125 MHz, DMSO-d ) δ 151.4, 149.4, 148.3, 147.0, 138.4, 129.1, 123.6,
6
1
4
22.4, 118.8, 112.0, 111.7, 111.5, 111.3, 108.5, 104.2, 66.6, 56.6, 56.2, 56.1, 53.9,
3.2, 35.2, 27.4. HRMS (ESI) m/z calcd for C H N O 420.2287, found 420.2271.
2
5
30
3
3
4.2.2.28. 8-Fluoro-2,3-dimethoxy-N-(3-morpholinopropyl)-11H-indeno[1,2-b]
1
quinoline-10-amine (7g). Yellow solid, 48% yield, m.p. 175–177 ℃ . H NMR (500
MHz, DMSO-d ) δ 8.06 (dd, J = 11.6, 2.6 Hz, 1H), 7.91–7.86 (m, 1H), 7.51–7.44 (m,
6
2
H), 7.23 (s, 1H), 6.76 (t, J = 5.8 Hz, 1H), 4.17 (s, 2H), 3.89 (s, 3H), 3.85 (s, 3H),
.77–3.71 (m, 2H), 3.58 (t, J = 4.4 Hz, 4H), 2.42 (t, J = 6.7 Hz, 3H), 2.36 (s, 3H),
3
13
1
.84–1.77 (m, 2H); C NMR (125 MHz, CDCl ) δ 162.2, 158.8 (d, J = 238.8 Hz),
3
1
51.2, 149.4, 146.1 (d, J = 3.8 Hz), 137.9, 133.0, 131.3 (d, J = 8.8 Hz), 119.4 (d, J =
.8 Hz), 117.9 (d, J = 8.8 Hz), 112.1, 108.6, 106.6 (d, J = 23.8 Hz), 103.9, 66.6, 56.2,
6.1, 54.0, 49.1, 43.0, 34.9, 27.5. HRMS (ESI) m/z calcd for C H FN O 438.2190,
8
5
2
5
29
3
3
found 438.2176.
4.2.2.29. 8-Chloro-2,3-dimethoxy-N-(3-morpholinopropyl)-11H-indeno[1,2-b]
1
quinolin-10-amine (7h). Yellow solid, 60% yield, m.p. 208–210 ℃ . H NMR (500
MHz, CDCl ) δ 7.93 (d, J = 8.9 Hz, 1H), 7.88 (d, J = 2.2 Hz, 1H), 7.68 (s, 1H), 7.50
3
(dd, J = 8.9, 2.2 Hz, 1H), 6.92 (s, 1H), 6.80 (s, 1H), 4.04–3.97 (m, 6H), 3.93–3.89 (m,
1
3
6
H), 3.84–3.79 (m, 2H), 2.61–2.57 (m, 3H), 2.55 (s, 3H), 1.89–1.83 (m, 2H). C
NMR (125 MHz, CDCl ) δ 163.0, 151.1, 149.3, 147.1, 145.7, 137.8, 132.8, 130.5,
3
1
2
29.0, 129.0, 120.1, 119.5, 112.7, 107.1, 103.9, 66.6, 59.4, 56.2, 56.0, 54.3, 46.4, 35.0,
4.4. HRMS (ESI) m/z calcd for C H ClN O 438.2190 454.1898, found 454.1882.
2
5
29
3
3
4.2.2.30. 8-Bromo-2,3-dimethoxy-N-(3-morpholinopropyl)-11H-indeno[1,2-b]
1
quinolin-10-amine (7i). Yellow solid, 63% yield, m.p. 232–233 ℃ . H NMR (500
MHz, CDCl ) δ 8.04 (d, J = 2.1 Hz, 1H), 7.87 (d, J = 8.9 Hz, 1H), 7.67 (s, 1H), 7.64
3
(dd, J = 8.9, 2.1 Hz, 1H), 6.95 (s, 1H), 6.79 (s, 1H), 4.03 (s, 2H), 4.03 (s, 3H), 3.95–
3
.91 (m, 7H), 3.86–3.81 (m, 2H), 2.64–2.58 (m, 3H), 2.56 (s, 3H), 1.90–1.85 (m, 2H).
2
6

ACCEPTED MANUSCRIPT
1
3
C NMR (125 MHz, CDCl ) δ 163.2, 151.1, 149.3, 147.6, 145.6, 137.8, 132.9, 131.7,
3
1
2
30.9, 123.1, 120.1, 116.8, 112.7, 107.1, 103.8, 66.7, 59.5, 56.2, 56.1, 54.4, 46.5, 35.1,
4.5. HRMS (ESI) m/z calcd for C H BrN O 498.1392, found 498.1372.
2
5
29
3
3
4.2.2.31. 2,3-Dimethoxy-8-methyl-N-(3-morpholinopropyl)-11H-indeno[1,2-b]
quinolin-10-amine (7j). Pale yellow solid, 75% yield, m.p. 200–202 ℃ . This product
hardly dissolved in the commonly used solvents and can’t be further structurally
characterize by NMR. HRMS (ESI) m/z calcd for C H N O 434.2444, found
2
6
32
3
3
4
34.2429.
4
.3. Cell viability assay
The anti-proliferative activities of target compounds were determined using MTT
4
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay 1×10
cells/well (MGC-803, HepG2, NCI-H460, T-24, HL-7702) were seeded in 180 µL
RPMI 1640 supplemented with 10% FBS in each well of 96–well microculture plates
and incubated for 24 h at 37 °C in 5% CO incubator. Target compounds were
2
prepared in DMSO and were diluted to the desired concentrations in culture medium
and treated with indicated concentrations of samples for 48 h. After the period of
incubation, MTT was added and the plates were further incubated for 4 h. Removal
the supernatant from each well and dissolved the purple formazan crystals by adding
DMSO in each well. The plates were swirled gently for 10 min to dissolve the
precipitate and quantified by measuring the optical density (OD) of the plates at a
wavelength of 570 nm on plate reader (TECAN, infinite, M1000). Each concentration
was repeated in five wells and the same experimental conditions were provided for all
compounds and MTT analysis was repeated three times for each cell line.
4
.4. Topoisomerase I inhibition activity and DNA cleavage assay
In Topoisomerase I inhibition experiments, the compounds were prepared as 2.0
mM DMSO stock solutions and diluted to gradient concentrations from 1.0, 5.0, 10,
0 µM by TAE buffer. The gradient concentrations of 5b were mixed with 0.25 µg
2
DNA, 1% BSA, Topoisomerase I buffer and 1U Topoisomerase I brought to 10 µL by
TAE buffer, and then all samples were incubated at 37 ℃ in dark for 30 min.
Following that the samples were separated by electrophoresis at 80 V of constant
2
7

ACCEPTED MANUSCRIPT
voltage on 1% agarose gel, and images of the DNA gel were captured using a BIO–
RAD imaging system after staining with EB staining reagent.
In plasmid DNA cleavage experiments, the pBR322 DNA were incubated with a
range of concentrations of 5b (0.5, 1.0, 2.5, 5.0,10 µM) in TAE buffer for 3 h. The
samples were electrophoresed in 1% agarose gel and stained with ethidium bromide
before detection. The gel was then visualized and captured on a BIO–RAD imaging
system under a UV–Vis transilluminator.
4
.5. Spectroscopic studies on DNA binding
The ct-DNA was dissolved in Tris–HCl buffer (pH 7.4) for DNA binding studies,
and its concentration was determined from the absorption intensity at 260 nm with a
−1
−1
molar extinction coefficient of 6600 M cm [51]. The test 5b was prepared as 10
mM DMSO stock solution for all the experiments, and then stored at 4 ℃ for no more
than 3 days before use.
UV–Vis spectroscopy. For UV–Vis absorption spectral analysis, the concentration
of the 5b was kept constant at 10 µM and changed the concentration of calf-thymus
DNA in Tris–HCl buffer (pH 7.4). The ct-DNA stock solutions were added gradually
to solutions containing 5b (10 µM), wherein the [DNA]/[5b] ration ranging from 1 to
1
0 at every 1 interval. For each addition, the mixed solutions were shaken and
incubated for 5 min at equilibrium before recording absorption spectrum, and then the
absorption spectrum were recorded in the range of 200–400 nm.
Fluorescence competitive binding spectroscopy. The competitive binding
research was performed with a mixed solution of the ethidium bromide (EB, 10 µM)
and ct-DNA (100 µM) in Tris–HCl buffer (pH 7.4), while gradually adding the
concentrations of 5b (10–100 µM). For every addition, the test sample was shaken
and equilibrated for 5 min, and then the fluorescence emission spectra were recorded
between 530 and 750 nm with an excitation wavelength set at 510 nm.
Fluorometric titration spectroscopy. To obtain G-quadruplexes, oligonucleotides
were annealed in 10 mM Tris-HCl buffers containing 100 mM KCl by heating at
9
5 °C for 5 min, followed by gradual cooling to room temperature. Sequences of
oligomers
used
′-GGGTTAGGGTTAGGGTTAGGG-3′;
′-TGGGGAGGGTGGGGAGGGTGGGGAAGG-3′;
in
this
work
as
list
below:
HTG21:
Pu27:
5
5
c-kit-1:
5′-CGGGCG-
2
8