Identification of 3-(benzazol-2-yl)quinoxaline derivatives as potent anticancer compounds: Privileged structure-based design, synthesis, and bioactive evaluation in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2019.01.004

Inspired by the common structural characteristics of numerous known antitumor compounds targeting DNA or topoisomerase I, 3-(benzazol-2-yl)-quinoxaline-based scaffold was designed via the combination of two important privileged structure units —quinoxaline and benzazole. Thirty novel 3-(benzazol-2-yl)-quinoxaline derivatives were synthesized and evaluated for their biological activities. The MTT assay indicated that most compounds possessed moderate to potent antiproliferation effects against MGC-803, HepG2, A549, HeLa, T-24 and WI-38 cell lines. 3-(Benzoxazol- -2-yl)-2-(N-3-dimethylaminopropyl)aminoquinoxaline (12a) exhibited the most potent cytotoxicity, with IC₅₀ values ranging from 1.49 to 10.99 μM against the five tested cancer and one normal cell line. Agarose-gel electrophoresis assays suggested that 12a did not interact with intact DNA, but rather it strongly inhibited topoisomerase I (Topo I) via Topo I-mediated DNA unwinding to exert its anticancer activity. The molecular modeling study indicated that 12a adopt a unique mode to interact with DNA and Topo I. Detailed biological study of 12a in MGC-803 cells revealed that 12a could arrest the cell cycle in G2 phase, inducing the generation of reactive oxygen species (ROS), the fluctuation of intracellular Ca²⁺, and the loss of mitochondrial membrane potential (ΔΨm). Western Blot analysis indicated that 12a-treatment could significantly up-regulate the levels of pro-apoptosis proteins Bak, Bax, and Bim, down-regulate anti-apoptosis proteins Bcl-2 and Bcl-xl, and increase levels of cyclin B1 and CDKs inhibitor p21, cytochrome c, caspase-3, caspase-9 and their activated form in MGC-803 cells in a dose-dependent manner to induce cell apoptosis via a caspase-dependent intrinsic mitochondria-mediated pathway. Studies in MGC-803 xenograft tumors models demonstrated that 12a could signi?cantly reduce tumor growth in vivo at doses as low as 6 mg/kg with low toxicity. Its convenient preparation and potent anticancer efficacy in vivo makes the 3-(benzazol-2-yl)quinoxaline scaffold a promising new chemistry entity for the development of novel chemotherapeutic agents.

Full text of DOI:10.1016/j.ejmech.2019.01.004

Accepted Manuscript
Identification of 3-(benzazol-2-yl)quinoxaline derivatives as potent anticancer
compounds: Privileged structure-based design, synthesis, and bioactive evaluation in
vitro and in vivo
Qing-Qing Liu, Ke Lu, Hai-Miao Zhu, Shi-Lin Kong, Jing-Mei Yuan, Guo-Hai Zhang,
Nan-Ying Chen, Chen-Xi Gu, Cheng-Xue Pan, Dong-Liang Mo, Gui-Fa Su
PII:
S0223-5234(19)30004-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.004
EJMECH 11011
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 October 2018
Revised Date: 27 December 2018
Accepted Date: 2 January 2019
Please cite this article as: Q.-Q. Liu, K. Lu, H.-M. Zhu, S.-L. Kong, J.-M. Yuan, G.-H. Zhang, N.-Y. Chen,
C.-X. Gu, C.-X. Pan, D.-L. Mo, G.-F. Su, Identification of 3-(benzazol-2-yl)quinoxaline derivatives as
potent anticancer compounds: Privileged structure-based design, synthesis, and bioactive evaluation
in vitro and in vivo, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.01.004.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Identification of 3-(benzazol-2-yl)quinoxaline derivatives as
potent anticancer compounds: privileged structure-based design,
synthesis, and bioactive evaluation in vitro and in vivo
†
†
Qing-Qing Liu , Ke Lu , Hai-Miao Zhu, Shi-Lin Kong, Jing-Mei Yuan, Guo-Hai Zhang,
Nan-Ying Chen, Chen-Xi Gu, Cheng-Xue Pan*, Dong-Liang Mo*, Gui-Fa Su*
State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Ministry
of Science and Technology of China; School of Chemistry and Pharmaceutical Sciences, Guangxi
Normal University, 15 Yu Cai Road, Guilin 541004, 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

ACCEPTED MANUSCRIPT
ABSTRACT
Inspired by the common structural characteristics of numerous known antitumor
compounds targeting DNA or topoisomerase I, 3-(benzazol-2-yl)-quinoxaline-based
scaffold was designed via the combination of two important privileged structure units
—quinoxaline and benzazole. Thirty novel 3-(benzazol-2-yl)-quinoxaline derivatives
were synthesized and evaluated for their biological activities. The MTT assay
indicated that most compounds possessed moderate to potent antiproliferation effects
against MGC-803, HepG2, A549, Hela, T-24 and WI-38 cell lines. 3-(Benzoxazol-
-2-yl)-2-(N-3-dimethylaminopropyl)aminoquinoxaline (12a) exhibited the most potent
cytotoxicity, with IC values ranging from 1.49 to 10.99 µM against the five tested
5
0
cancer and one normal cell line. Agarose-gel electrophoresis assays suggested that
2a did not interact with intact DNA, but rather it strongly inhibited topoisomerase I
Topo I) via Topo I-mediated DNA unwinding to exert its anticancer activity. The
1
(
molecular modeling study indicated that 12a adopt a unique mode to interact with
DNA and Topo I. Detailed biological study of 12a in MGC-803 cells revealed that
1
2a could arrest the cell cycle in G2 phase, inducing the generation of reactive
2
+
oxygen species (ROS), the fluctuation of intracellular Ca , and the loss of
mitochondrial membrane potential (∆Ψm). Western Blot analysis indicated that
1
2a-treatment could significantly up-regulate the levels of pro-apoptosis proteins Bak,
Bax, and Bim, down-regulate anti-apoptosis proteins Bcl-2 and Bcl-xl, and increase
levels of cyclin B1 and CDKs inhibitor p21, cytochrome c, caspase-3, caspase-9 and
their activated form in MGC-803 cells in a dose-dependent manner to induce cell
apoptosis via a caspase-dependent intrinsic mitochondria-mediated pathway. Studies
in MGC-803 xenograft tumors models demonstrated that 12a could significantly
reduce tumor growth in vivo at doses as low as 6 mg/kg with low toxicity. Its
convenient preparation and potent anticancer efficacy in vivo makes the
3
-(benzazol-2-yl)quinoxaline scaffold a promising new chemistry entity for the
development of novel chemotherapeutic agents.
Keywords: Topoisomerase I inhibitor, DNA unwinding, Quinoxaline derivatives,
Privileged structure, Anticancer
2

ACCEPTED MANUSCRIPT
1
. Introduction
Cancer has become the leading cause of death worldwide, especially in
developing countries [1]. In 2015, there were about 4.3 million newly diagnosed cases
of invasive cancer in China, leading to more than 2.8 million deaths in that year alone
[
2]. Thus, the investigation and identification of more effective agents to treat cancer
is urgently needed.
H
N
O
O
O
O
O
N
HN
O
OH
N
N
N
H
H
O
camptothecin (CPT)
Indenoisoquinolines
indolocarbazole
(A)
O
O
X
HN
N
N
N
HN
N
Isoindoloquinoxalines
quinolone-benzazole
dibenzonaphthyridinone
Privileged Structure
Combination
Y
N
Poly(hetero)aromatic
Ring Skeleton
N
HN ( )
(B)
N
PS-based design
n
(
)n R
R
structural characteristics
of known scaffold
novel scaffold
Fig. 1. Representative Topo I inhibitors and strategy to design novel scaffold
DNA and DNA Topo I are important pharmacologic targets in the development
of anti-cancer therapeutics [3–5]. Topo I inhibitors are important chemotherapeutic
agents. They can interfere with the Topo I through two different mechanisms, namely
pensioner or catalytic inhibitor[6-9]. To date, the most intensive studies scaffold of
Topo
I
inhibitors including the derivatives of camptothecin(CPT),
indolocarbazole[7–8], dibenzonaphthyridinone[7–8], Indenoisoquinoline[9]. Some
quinolone-benzazoles[10] and isoindoloquinoxalines[11] also were identified as
promising Topo I inhibitors(Fig. 1 A).
Even though Topo I inhibitors reported in literatures are very structurally diverse,
most of them possess s a polycyclic skeleton with a side chain that usually contain an
ω-dialkyl-amino or nitrogen-containing heterocycle (Fig. 1 B). Considering that
3

ACCEPTED MANUSCRIPT
drug-likeness such as ideal physicochemical and pharmacokinetic profiles, are crucial
factors for drug development. And it had been well known that introduction
privileged structure into the scaffold might help to improve the drug-likeness of the
proposed compounds [12–13]. We envisioned that a privileged structure-based design,
as depicted in Fig. 1, might serve as a useful strategy to help us to find novel
anticancer scaffolds with suitable physicochemical and pharmacokinetic profiles.
Herein, we report the design, synthesis, and bioactive evaluation of a novel
anticancer scaffolds in which quinoxaline and benzazole were selected as the
privileged structure motifs, since quinoxaline [14–17] and benzazole [18–32]
derivatives are very important in drug development and widely present in many
clinical drugs or bioactive compound. The novel scaffold also can be views as the
combination of two important units of Topo I inhibitors quinolone-benzazoles and
isoindoloquinoxalines reported in literature (Fig. 1 A).
2
. Results and discussion
2
.1. Chemistry
NH
2
N
2
CO Et
Y
a, b
c
N
d
6
7%
76%~94%
77%~91%
N
NH2
N
H
O
2 steps
N
H
O
4-6
1
2
Y
N
a-d; n=3, R=N(CH ) , ~N
O
Y
N
3 2
N
e
5
8%~77%
N
,
~N
OH
N
N
N
NH(CH ) R
Cl
-9
2 n
7
10-12
, 7, 10 (Y=NH); 5, 8, 11 (Y = S); 6, 9, 12 (Y = O)
e-f; n=2, R=N(CH ) , ~N
3
2
O
4
Scheme 1. Reagents and conditions: (a) CHBr(CO
2
Et)
2
, 1.1 eq, EtOH , r.t.; (b) O
2
, CeCl
3 2
-7H O,
(
CH ) CHOH, r.t.; (c) 3 (benzene-1,2-diamine, 2-amino-benzenethiol or 2-amino-phenol, 1.1 eq),
3 2
Ph O, reflux; (d) POCl , reflux; (e) R(CH ) NH , toluene , 90 oC
2
3
2 n
2
R2
R1
R2
R1
O
O
a
N
b
N
2
3
8%~75%
N
77%~91%
N
N
H
N
O
Cl
13
14
R2
O
O
c
N
R1
N
N
N
5
8%~77%
N
or
N
NH(CH ) R
NH(CH
) R
2 3
2
3
1
5a-19a; 15g-19g
20a, 20g
1
2
1
2
1
5-17: R =Cl, CH , t-Bu; R =H; 18-19: R =H; R =Cl, CH ; a: R = N(CH ) ; g: R = N(Et)
3
3
3 2
2
Scheme 2. Reagents and conditions: (a) 1.1 eq 2-amino-phenol derivatives or 1-amino-
naphthalen-2-ol, Ph O, reflux; (b) POCl , reflux; (c) R(CH ) NH , toluene, 90 oC
2
3
2 3
2
4

ACCEPTED MANUSCRIPT
The preparation of the 3-(benzazol-2-yl)quinoxaline derivatives (10–12, 15–20)
was outlined in Scheme 1 and Scheme 2. Benzene-1,2-diamine 1 and diethyl
2
-bromomalonate in ethanol were refluxed to give the intermediate 3-oxo-1,2,3,4-
tetrahydro-quinoxaline-2-carboxylate, which was further dehydrogenated under 1 atm
oxygen atmosphere promoted by cerium (� ) chloride heptahydrate to provide the
3
1
4
-oxo-3,4-dihydro-quinoxaline-2-carboxylate 2. Then 2 condensated with 3 (benzene-
,2-diamine, or 2-amino-benzenethiol, or 2-amino-phenol) in diphenyl ether to afford
–6 in high yield. 4–6 were refluxed in excess POCl to provide the 2-chloro-3-
3
(benzazol-2-yl)quinoxaline 7–9, followed by reacted with different amines [33] to
furnish the targeted compounds 10–12 (Scheme 1). Synthesis of 15–20 was followed
the same procedure as the synthesis of compounds 10–12 starting from 2 and
substituted 2-amino-phenol or 1-amino-naphthalen-2-ol (Scheme 2).
2
2
.2. Biological evaluation
.2.1. Antiproliferative activity
The in vitro anti-proliferative activity of the target compound 10–12 and 15–20
against five human tumor cell lines (MGC-803, HepG-2, A549, HeLa and T24) and
one normal cell (WI-38) was evaluated by the MTT assay. The IC values derived
5
0
from the dose-response curves are summarized in Table 1.
Table 1 Structures and in vitro cytotoxicity of compounds 10–12 and 15–20
N
Ar
R
N
N
H
IC (µM)
5
0
Cmpd
Ar
R
WI-38 MGC-803 HepG2 A549 HeLa T-24
Me
26.01
3.85
36.45 67.99 67.54 60.82
±4.08 ±11.1 ±9.99 ±9.04
HN
10a
10b
10c
N
>100
>100
>100
(
)3
N
Me
±
N
O
>100
>100
>100
>100
>100 >100 >100
>100 >100 >100
()
3
5

ACCEPTED MANUSCRIPT
10d
10e
10f
>100
>100
>100
>100 >100 >100
1
4.29
1.15
10.36
±1.02
14.17 15.04 69.92 10.82
±1.87 ±1.70 ±12.4 ±0.98
±
>100
>100
>100
>100 >100 >100
Me
7.41
8.26
11.79
5.34 12.55 11.81
11a
N
(
)3
Me
±0.87
±0.92
±1.07 ±0.73 ±1.09 ±1.27
1
1b
N
O
>100
>100
>100
>100
>100
>100 >100 >100
()
3
1
9.81
1.90
11.67 52.71 39.74 34.29
±1.06 ±8.05 ±7.13 ±7.17
11c
±
11d
>100
>100
>100 >100 >100
9
.74
5.86
±0.77
12.95
5.67 12.65 6.81
11e
±0.99
±1.11 ±0.75 ±1.10 ±0.79
11f
>100
>100
>100
>100 >100 >100
Me
10.99
1.49
5.27
6.91
6.38
4.49
12a
12b
12c
12d
12e
N
(
)3
Me
±1.06
±0.18
±0.72 ±0.84 ±0.81 ±0.65
N
O
>100
>100
>100
>100
>100
>100
>100 >100 >100
>100 >100 >100
()
3
>100
>100
>100
8.52
>100 >100 >100
5.39 15.34 4.21
6
.82
2.93
±0.47
±0.83
±0.93 ±0.73 ±1.05 ±0.62
6

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
2
1
2f
>100
>100
>100
>100 >100 >100
6
Me
11.09
±1.07
8.72
13.83 11.89 10.01 5.66
±1.50 ±1.52 ±0.94 ±0.23
O
Cl
5a
6a
7a
8a
9a
0a
5g
5
N
(
(
(
(
(
)3
)3
)3
)3
)3
N
Me
±1.13
6
Me
13.54
±1.68
12.20
±1.17
16.45 13.52 12.52 10.87
±1.24 ±1.79 ±1.55 ±1.09
O
O
5
N
N
N
Me
6
Me
14.80
13.87
±1.53
18.40
5
N
>100
>100 >100
Me
±1.04
±0.94
Cl
6
Me
15.33
1.04
14.01
±1.21
14.47 15.40 7.83
±1.24 ±1.99 ±0.51
O
5
N
>100
8.82
Me
±
N
6
Me
10.95
0.83
8.96
8.35 11.98 5.99
O
5
N
Me
±
±1.55
±0.65 ±0.41 ±0.91 ±0.22
N
Me
16.45
1.88
11.29
±1.15
10.76
4.66 10.54 5.75
O
N
N
()3
Me
±
±1.32 ±0.71 ±0.92 ±0.31
6
5
Et
11.20
±0.01
9.20
13.74
9.32 10.15 5.49
O
Cl
N
(
)3
N
Et
±1.68
±1.62 ±1.17 ±1.36 ±0.62
6
5
Et
12.57
±0.79
7.88
12.74
7.98
8.71
6.96
O
O
16g
17g
18g
19g
20g
N
(
)3
N
N
Et
±0.48
±0.12 ±1.15 ±1.08 ±0.81
6
5
Et
25.15
10.53
±0.83
14.39 19.50 7.58
>100
N
(
)3
Et
±3.71
±1.08 ±2.08 ±0.69
Cl
6
Et
13.57
1.66
11.81
±1.77
13.47 12.98 12.51 9.32
±1.01 ±1.18 ±1.18 ±0.99
O
5
N
(
)3
Et
±
N
6
Et
12.26
0.94
9.01
13.09
7.88 10.30 6.13
O
5
N
(
)3
Et
±
±1.14
±2.36 ±1.09 ±1.46 ±0.34
N
Et
14.71
11.51
±2.28
12.73 11.34 10.85 8.00
±2.36 ±2.61 ±1.02 ±0.87
O
N
(
)3
N
Et
±2.36
8
.37
2.96
4.79
4.43
6.51
6.80
b
CPT
±0.52
±0.23
±0.54 ±0.85 ±0.83 ±0.54
a
Results were expressed as means ± SD (standard deviation) from triplicate assay in a single experiment, P < 0.05.
CPT: Camptothecin
b
7

ACCEPTED MANUSCRIPT
As shown in Table 1, compounds 10e, 11a, 11e, 12a, 12e and 15–20 exhibited
moderate to potent cytotoxicity against all the tested cell lines. The data indicated that
the side chains at the 2-position of the quinoxaline ring played an important role in
bioactivity. Only side chains with ω-dialkylamino substitution resulted in significant
cytotoxic potency (series a, e and g), and a ω-dimethylamino substitution (series a
and e) was more favorable than ω-diethylamino ones (series g). The length of the side
chain also affected the anti-proliferative activity. The aminopropyl group (series a)
generally yielded greater potency than the aminoethyl group (series e).
The benzazol group on 3-position of quinoxaline also contributed to the
anti-proliferative activity of the target compound. In general, the benzoxazole gave
the most potent activity (series 12). When the benzoxazole was replaced by
benzoimidazole, benzothiazole, or naphthoxazole (series 10, 11 or 20, respectively),
the cytotoxicities of the compounds were slightly decreased.
The data in Table 1 suggested that the substituents on the benzoxazole ring and
their position did not significantly impact the anti-proliferative activity of the targeted
compounds. Hydrogen (H) atom (12a) appeared to be more favorable than any other
common substituent such as halogen atoms (15 and 18) or alkyls (16, 17and 19).
However, when the substituent was bulky, such as t-butyl (17a, 17g), the cytotoxicity
of the compounds would significant decrease.
Among the tested compound listed in table 1, 12a not only displayed exceptional
efficacy against MGC-803 cancer cell lines with IC values as low as 1.49 ±0.18
5
0
µM, but exhibited less toxicity (IC = 10.99 ± 1.06 µM) to normal cell WI-38 from
5
0
human lung tissue. To further test the safety of compound 12a, its cytotoxicity against
normal human liver cell 7702 was further evaluated. The results indicated that 12a
also exhibit more potent cytotoxicity against cancer cell MGC-803 (IC =1.63±0.25
5
0
µM) than to normal human liver cell 7702 (IC =5.2 ±0.67 µM).
5
0
2
.2.2. The cytotoxic compounds and 12a did not interact with intact DNA
Since the targeted compounds were designed based on common structural
characteristics of known scaffolds able to interact with DNA or DNA/topoisomerase I.
To gain insight into the drug-target interaction of these novel scaffolds, we first
explored whether DNA was the direct target of these compounds. DNA intercalation
8

ACCEPTED MANUSCRIPT
of fifteen selected compounds with potent cytotoxicity were evaluated via an
agarose-gel electrophoresis assay [33–34]. Ethidium bromide (EB), a well-known
DNA intercalator was used as the positive control. Compared with the EB which
demonstrated strong DNA intercalation activity, as indicated by its ability to induce
noticeable retardation of DNA migration, the tested compounds did not result in
obvious retardation of DNA migration (Fig. 2A & 2B), suggesting that the tested
compounds were not directly interacting with intact DNA.
Fig. 2. (A) DNA intercalation assay of the selected compounds in 50 µM. Lane 1(A): DMSO +
DNA, lane 2: EB (10 µM) + DNA, lanes 3–16: selected cytotoxic compounds (50 µM,
respectively) + DNA. (B) DNA intercalation assay of 12a in different concentration. Lane 1(A):
DMSO + DNA, lane 2: EB (10 µM) + DNA, lanes 3–8: 12a (75, 100, 125, 150, 200, 250 µM,
respectively) + DNA.
2
.2.3. The cytotoxic compounds and 12a induced potent topoisomerase I inhibition via
Topo I-mediated DNA unwinding
Having determined that the tested compounds had low binding affinity with
intact DNA, we then performed a topoisomerase I inhibition assay [35–37] to
determine whether they acted as topoisomerase I inhibitors. The inhibitory effects of
these selected compounds against Topo I was determined by measuring the migration
of supercoiled plasmid DNA via agarose-gel electrophoresis, which indicated the
degree of DNA relaxation. The well-known Topo I inhibitor camptothecin (CPT) was
used as a positive control.
As depicted in Fig. 3A, most of the tested compounds exhibited potent Topo I
inhibitory activity at 50
ring at the 3-position and demonstrated no apparent inhibitory activity, indicating that
0a and 20g might exert their cytotoxic effect via a different mechanism. The Topo I
µM, except for 20a and 20g which contain a naphthoxazole
2
inhibitory activity of 12a, which demonstrated the strongest cytotoxicity activity, was
9

ACCEPTED MANUSCRIPT
further tested at concentrations from 5 to 60 µΜ and found that 40 µM of 12a was
sufficient to completely inhibit the activity of Topo I (Fig. 3B).
Fig. 3. (A) Topo I inhibitory activity assay of the selected compounds in 50 µM. Lane 1(A): DNA,
lane 2(B): DNA + Topo I, lane 3(C): CPT (100 µM) + DNA + Topo I, lanes 4–17: selected
cytotoxic compounds (50 µM, respectively) + DNA + Topo I. (B) Topo I inhibitory activity assay
of the 12a in different concentration. Lane 1(A): DNA, lane 2(B): DNA + Topo I, lanes 3–4: CPT
(100, 200 µM, respectively) + DNA + Topo I, lanes 5–9: 12a (5, 10, 20, 40, 60 µM, respectively)
+
DNA + Topo I.
By comparison with the bands in Fig. 3, it could be determined that the Topo I
inhibition mode of the tested compounds was different to that of the positive control
CPT, a Topo I inhibitor acting by forming a drug-enzyme-DNA ternary complex [6]
and cleaving DNA, which was indicated by the production of nicked DNA in the CPT
bands.
According to the catalytic cycle of Topo I [5], DNA intercalating or unwinding
agents also could inhibit Topo I by unwinding closed circular DNA [38]. To gain
further insight into the Topo I inhibition mechanism of the 3-(benzazol-2-yl)quin-
-oxaline derivatives, we investigated whether they could produce DNA unwinding in
the presence of excess Topo I. EB, a DNA intercalating agent, was used as a positive
control, and CPT, which inhibits Topo I by forming the drug-enzyme-DNA covalent
ternary complex, was used as a negative control.
As shown in Fig. 4A, EB and most of the tested 3-(benzazol-2-yl)-quinoxaline
derivatives exhibited clear DNA unwinding at 50 µM, except for compounds 20a and
2
0g which demonstrated no Topo I inhibitory activity. The most cytotoxic compound
2a was further tested at concentrations from 5 to 60 µM. We found that 12a could
1
exert its significant Topo I-mediated unwinding effect at 20 µM (Fig. 4B), indicating
that 12a exerts its Topo I inhibitory activity via Topo I-mediated DNA unwinding.
1
0

ACCEPTED MANUSCRIPT
Fig. 4. (A) Topo I inhibitory activity assay of the selected compounds in 50 µM. Lane 1(A): DNA,
lane 2(B): DNA + Topo I, lane 3(C): CPT (100 µM) + DNA + Topo I, lane 4: EB (50 µM) + DNA
+
Topo I, lanes 5–18: selected compounds (50 µM, respectively) + DNA + Topo I. (B) Lane 1(A):
DNA, lane 2(B): DNA + Topo I, lanes 3–4: CPT (100, 200 µM, respectively) + DNA + Topo I,
lanes 5–6: EB (10, 20 µM, respectively) + DNA + Topo I, lanes 7–11: 12a (5, 10, 20, 40, 60 µM,
respectively) + DNA + Topo I.
2
.2.4. Molecular modeling
Since the mechanism studies indicated that 12a might blocks the combination
between DNA and Topo I via Topo I-mediated DNA unwinding, a molecular docking
study was performed using C-DOCKER in Discovery Studio 2017R2 to predict and to
better understand the binding mode of 12a with DNA and Topo I. The structure of the
ternary complex, containing topoisomerase I, DNA, and CPT, was downloaded from
the Protein Data Bank (PDB code 1T8I).
(A)
(B)
Fig. 5. The 2D(A) and 3D(B) diagram of hypothetical binding mode for 12a with DNA-Topo I.
As shown in Fig. 5A and B, The docking results suggest that 12a binds with the
DNA-Topo I complex in a unsimilar manner to that of the Topo I poisoner [39] or the
11

ACCEPTED MANUSCRIPT
Topo I catalytic inhibitor [40]. In the 12a -DNA-Topo I ternary complex. The
dimethyl amino group on the side chain was protonation and hydrogen bonding to
ASP533 in Topo I, while the quinoxaline and benzoxazole ring interact by pi-pi
interaction through the insertion of planar aromatic rings between DNA base pairs.
The electron-rich nature of the benzoxazole ring also furnish it to interact with
LYS425 via pi-cation interaction.
The results provide evidence for the mechanism with which 12a interfere with
the Topo I/DNA. The non-coplanar feature of the quinoxaline and benzoxazole ring
make this scaffold not easily direct intercalate into intact DNA. But if the supercoiled
DNA was relaxed by Topo I, the compounds will be able to insert into DNA and
interact with Topo I/DNA. The results also well explain how the length and the ω-
substituent of the side chain, as well as the bulk of substituents on the aryl ring affect
the activities of the target compounds.
2
.2.5. Compound 12a induced G2 phase cell cycle arrest
As 12a displayed the most promising activity not only in cytotoxicity but also in
Topo I inhibition, the mechanism of its cytotoxic effect was further studied. Whether
2a inhibited the proliferation of MGC-803 cells through cell cycle arrest was first
examined by flow cytometry. As shown in Fig. 5, 12a treatment (doses from 0, 1.0,
1
2
8
.0 to 8.0 µM, 48 h) clearly increased the proportion of cells in G2 phase from 3.77%,
.09%, 11.67%, to 26.09%, whereas the S phase population decreased from 41.88% in
the control to 25.65% (8.0 µM). These results indicated that 12a caused G2 arrest in a
dose-dependent manner and that its anti-proliferation in cancer cell lines was
potentially due to G2 phase arrest.
Fig. 5. Cell cycle analysis of compound 12a in MGC-803 cells. Cells were treated with 12a (0, 1.0,
2
.0 and 8.0 µM, respectively) for 48 h.
1
2

ACCEPTED MANUSCRIPT
The formation of cyclin B1-CDK1 complexes is required for the cell cycle
transition from G2 to M phase during cell division [41]. To further confirm the G2
cell cycle arrest caused by 12a, we investigated the levels of Cyclin B1 and
cyclin-dependent kinase (CDK) inhibitor p21 in MGC-803 cells. Immunoblotting
assay revealed that 12a-treament would increase the amount of Cyclin B1 and p21 in
MGC-803 cells (Fig. 6), suggesting that 12a might not only could up-regulate the
levels of CDK inhibitor p21, but also hinder cyclin B1 binding to CDK1 to form the
cyclin B1-CDK1 complex, resulting in G2 arrest induced by 12a.
5
Control
2
5
.5 µM
µM
4
3
2
1
0
1
0 µM
Cyclin B1
P21
Fig. 6. Effect of 12a on the level of Cyclin B1 and CDK inhibitor p21 in cell detected by western
blot. Protein was from MGC-803 cell line treated by 12a (0, 2.5, 5.0 and 10 µM, respectively) for
2
4 h. Whole-cell extracts were prepared and analyzed using antibodies against proteins indicated.
The data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001
vs the negative control.
2
.2.6. Compound 12a induced cell apoptosis via caspase-dependent intrinsic
mitochondrial pathway
To further examine whether the observed cytotoxicity was due to induction of
apoptosis, Annexin V FITC/PI dual staining assay was performed to evaluate the
apoptosis-inducing effect of 12a. MGC-803 cells were treated with 12a at a dose of 0,
2
.5, 5.0, 10.0 µM for 24 h. As shown in Fig. 7, the percentage of apoptotic cells was
increased from 1.82% (0 µM) to 10.77% (2.5 µM), 17.94% (5.0 µM) and 26.38%
10.0 µM), respectively, indicating that 12a could induce MGC-803 cell apoptosis in a
dose-dependent manner.
(
1
3

ACCEPTED MANUSCRIPT
Fig. 7. The induction of apoptosis effects of 12a in MGC-803 cells. Cell was treated with 12a (0,
2
.5, 5.0 and 10 µM, respectively) for 24 h and the results were examined by FACS analysis with
PI and FITC-Annexin V staining.
Fig. 8. ROS generation assay of compound 12a in MGC-803 cells. MGC-803 cells were treated
with 12a (0, 2.5, 5.0 and 10 µM, respectively) for 24 h.
2
+
Fig. 9. Effect of 12a on intracellular Ca level in MGC-803 cells. MGC-803 cells were treated
with 12a (0, 2.5, 5.0 and 10 µM, respectively) for 24 h.
1
4

ACCEPTED MANUSCRIPT
2
+
As reactive oxygen species (ROS) [42] and Ca [43–44] play key roles in the
regulation of apoptosis, to determine whether 12a treatment would induce the
2
+
production of ROS and fluctuation of Ca , MGC-803 cells were treated with 12a at
doses from 0, 2.5, 5.0 to 10.0 µM for 24 h in the presence of 2′,7′-dichloro-
2
+
-
fluorescein (DCF) or fura-3 AM, which are fluorescent indicators for ROS and Ca ,
respectively.
As depicted in Fig. 8 and Fig. 9, the fluorescence intensities were significantly
2
+
increased when the dose of 12a increased, indicating that intracellular ROS and Ca
levels increased in a dose-dependent manner after 12a-treament, which may be
responsible for apoptosis induction by 12a.
100
80
60
40
20
0
Control 2.5 µM
5 µM
10 µM
1
5

ACCEPTED MANUSCRIPT
Fig. 10. Effect of 12a on ∆Ψm of MGC-803 cells. MGC-803 cells were treated
with 12a (0, 2.5, 5.0 and 10 µM, respectively) for 24 h.
To further determine the pathway that mediated the induction of cell apoptosis by
1
2a, the mitochondrial membrane potential (∆Ψm) of 12a-treated MGC-803 cells was
measured by the fluorescent probe JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-
benzimidazolylcarbocyanine). As shown in Fig. 10, the fluorescence intensity in
MGC-803 cells was obviously decreased after treatment with 12a (doses 0, 2.5, 5.0,
1
0.0 µM) for 24 h, especially when the dose of 12a was increased from 0 to 2.5 µM,
suggesting that the 12a-treament could result in the loss of ∆ψm that usually indicate
the mitochondrial dysfunction associated with intrinsic mitochondrial apoptosis
pathway [45].
In order to get more detailed information about the apoptotic-induction of 12a,
the levels of apoptosis-related Bcl-2 family proteins which are regarded as key
regulators of cell apoptosis [46–47], and the indicators of activation of the
mitochondrial apoptosis pathway [48–49], such as cytochrome c, caspase-9, caspase-3
and their cleaved forms were measured by Western blot.
As shown in Fig. 11, the expression of anti-apoptosis proteins Bcl-2 and Bcl-xl,
especially the later, were obviously down-regulated, whereas the expression of
pro-apoptotic proteins Bak, Bax and Bim were up-regulated, confirming that
apoptosis was induced by 12a. Meanwhile, the level of cytochrome c, caspase-3 and
caspase-9, and the cleaved form of these proteins in the tumor cell were increased
significantly after 12a-treatment (Fig. 12), indicating that 12a induced cell apoptosis
via a caspase-dependent intrinsic mitochondria-mediated pathway.
Fig. 11. Effect of 12a on the level of Bcl-2, Bcl-xl, Bak, Bax and Bim in cell detected by Western
blot. Protein was from MGC-803 cell line treated by 12a (0, 2.5, 5.0 and 10 µM, respectively) for
1
6

ACCEPTED MANUSCRIPT
2
4 h. Whole-cell extracts were prepared and analyzed using antibodies against proteins indicated.
β-Actin was used as a loading control. The data are representative of three independent
experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs the negative control.
Fig. 12. (A) Effect of 12a on the level of cytochrome c, caspase-3/9 in cell detected by Western
blot. Protein was from MGC-803 cell line treated by 12a (0, 2.5, 5.0 and 10 µM, respectively) for
2
4 h. (B) 12a induced apoptosis by triggering Caspase-3, Caspase-9 activities in MGC-803 cells.
MGC-803 cells were incubated with 10 µM of 12a for 24 h. Arrow showed the Caspase-3 or
Caspase-9 activated cells.
2
.2.7. Compound 12a demonstrated potential anticancer potency in vivo with low
toxicity
The in vivo anticancer efficiency of 12a was further evaluated using MGC-803
xenograft tumors models. Twenty-four nude mice with tumors at a volume of 80−200
3
mm were randomized to vehicle, CPT-treatment (6 mg/kg/2 days), low dose
1
2a-treatment (6 mg/kg/2 days), and high dose 12a-treatment (12 mg/kg/2 days)
groups (6 mice/group). The treatment groups were ip injection every other day of CPT
or 12a. As shown in Fig. 13a, b, d, as compared with the vehicle control group, the in
vivo tumor growth inhibition (TGI) of 12a was 41.0% (high doses), only 10% lower
than that of CPT (51.5%). This confirmed that 12a possessed significant tumor
growth inhibition in vivo. The potential toxicity of 12a was also investigated by
monitoring the weight loss of all tested nude mice. As depicted in Fig. 13c, no
differences were found between the vehicle control and the 12a-treated group in body
weight loss, suggesting that 12a at the therapeutic dosage does not possess apparent
toxicity.
1
7

ACCEPTED MANUSCRIPT
Fig. 13. In vivo anticancer activity of 12a in MGC-830 xenograft mice. (a) The tumor volume of
mice from each group during the observation period. Tumor-bearing mice were administered the
vehicle (negative control), 12a (6 or 12 mg/kg per 2 days) or CPT (6 mg/kg per 2 days, positive
control). (b) Images of the excised tumors from each group. (c) Body weights of the mice recorded
at the end of the treatments. (d) Weight of the excised tumors from MGC-803 xenograft model.
Data are presented as the mean ± SD. Error bars represent SD, n = 6, *p < 0.05 and **p < 0.01, vs
control.
3
. Conclusion
In summary, based on the privileged structure combination, 30 novel
-(benzazol-2-yl)quinoxaline derivatives were designed, synthesized, and studied for
3
their cytotoxicities. The result indicated that many target compounds display potent
anticancer activity in vitro. Agarose-gel electrophoresis assay and molecular docking
indicated that most of the cytotoxic compounds could strongly inhibit Topo I activity
via Topo I-mediated DNA unwinding. The most potent molecule, 3-(benzo[d]oxazol-
-2-yl)-2-(N-3-dimethylaminopropyl)aminoquinoxalin 12a, was selected for further
evaluation of its bioactivity. The results indicated that 12a-treament would increase
the levels of CDK inhibitor p21 and trigger a G2 phase cell cycle arrest, induce the
2
+
generation of reactive oxygen species (ROS), cause fluctuation of intracellular Ca ,
loss of mitochondrial membrane potential (∆Ψm), up-regulation the pro-apoptosis
proteins Bak, Bax and Bim, down-regulation the anti-apoptosis proteins Bcl-2 and
1
8

ACCEPTED MANUSCRIPT
Bcl-xl, increases the level of cytochrome c, caspase-3 and caspase-9 and their
activated forms in MGC-803 cells in a dose-dependent manner, and finally triggering
a caspase-dependent intrinsic mitochondria-mediated cell apoptosis. Compound 12a
also demonstrated potent anticancer efficiency in vivo with low toxicity and could be
served as a promising lead compound for further drug development.
4
. Experimental section
4
.1. Chemistry
All reagents were purchased from commercial sources and were used without
further purification. Melting points were recorded on a X-5 apparatus without
correction. NMR spectra were recorded in CDCl or in DMSO-d on a Bruker
3
6
Advance (500 or 400 MHz) with TMS as the internal standard. HRMS were measured
in ESI mode and the mass analyzer was TOF.
4
.1.1. Synthesis of ethyl 3-oxo-3,4-dihydroquinoxaline-2-carboxylate (2).
The solution of benzene-1,2-diamine (1.09 g, 10 mmol) and diethyl 2-bromo-
-malonate (1.9 mL, 11 mmol) in ethanol (20 mL) was stirred at room temperature for
4
h until the reaction completed (monitored by TLC). Then the solvent was
evaporated under reduced pressure, washed with petroleum ether (10 mL× 3) to give a
pale-yellow solid. The solid was dissolved in 100 mL of isopropanol, cerium
trichloride heptahydrate (1.86 g, 5 mmol) and water (0.5 mL) were added and the
mixture was stirred under 1 atm oxygen at room temperature for 6 h until the reaction
completed (monitored by TLC). The solvent was evaporated under reduced pressure
and the solid was washed with water (10 mL × 3) to provide a yellow solid 2 (2.86 g),
yield 67%, mp 175–177 °C, which was used in next steps without further purification.
4
.1.2. General Procedure for Synthesis of 4–6 and 13.
Compound 2 (2.18 g, 10 mmol) and benzene-1,2-diamine (1.30 g, 12 mmol) in
Ph O (10 mL) was heated to reflux for 2 h until the reaction completed (monitored by
2
TLC). Then the mixture was cooled, filtered, and the solid was washed with
1
9

ACCEPTED MANUSCRIPT
petroleum ether (15 mL × 3) to give 2.28 g of grey solid 4, which was used in next
step without further purification. Compounds 5–6 and 13 were synthesized followed
the same procedure.
4
.1.3. General Procedure for Synthesis of 7–9 and 14.
The mixture of compound 4 (0.52 g, 2 mmol) and redistilled POCl (5 mL) was
3
heated in 90 °C for about 2 h under nitrogen atmosphere. After completion (monitored
by TLC), the excess POCl was removed under reduced pressure and ice-water was
3
poured in. The solid was filtered and washed with water. The dry crude product was
further purified by column chromatography on silica gel eluted with ethyl
acetate/petroleum ether (V: V = 1:8) to afford pale-yellow solid 7 (0.49 g), yield 88%,
mp 216–218 °C. Compounds 8–9 and 14 were synthesized followed the same
procedure.
4
.1.4. Preparation of compounds 10–12 and 15–20.
.1.4.1. General procedure
4
A solution of compounds 7 (or 8 or 9 or 14) (0.5 mmol) and amine derivatives
(2.5 mmol) in toluene (5 mL) was heated in 90 °C for 1.5 h until the reaction
completed (monitored by TLC). Then the solvent was removed under reduced
pressure and the residue was purified by column chromatography on silica gel eluted
with ethyl acetate/petroleum ether to provide 10–12 or 15–20.
4
.1.4.2.
3-(1H-Benzo[d]imidazol-2-yl)-2-(N-3-dimethylaminopropyl)aminoquin-
1
-
oxaline (10a), 0.14 g, yield 92%, pale yellow solid, m.p. 175–176 °C, H NMR (500
MHz, DMSO-d ) δ 13.39 (s, 1H), 9.87 (t, J = 5.4 Hz, 1H), 7.94 (dd, J = 8.5, 0.8 Hz,
6
1
H), 7.73–7.62 (m, 4H), 7.45–7.42 (m, 1H), 7.34 (s, 2H), 3.71–3.67 (m, 2H), 2.41 (t,
13
J = 6.9 Hz, 2H), 2.20 (s, 6H), 1.89–1.83 (m, 2H). C NMR (125 MHz, DMSO-d ) δ
6
1
50.8, 149.8, 142.7, 135.6, 132.7, 131.3, 128.9, 126.1, 124.7, 57.3, 45.7, 38.9, 26.9.
+
HRMS (ESI) m/z calcd for C H N [M+H] 347.1984, found 347.1986.
2
0
23
6
4
.1.4.3.
3-(1H-Benzo[d]imidazol-2-yl)-2-(N-3-morpholino-4-ylpropyl)aminoquin-
1
-
oxaline (10b), 0.176 g, yield 91%, yellow solid, m.p. 210–211 °C, H NMR (400
MHz, DMSO-d ) δ 13.40 (s, 1H), 9.84 (t, J = 3.6 Hz, 1H), 7.94 (d, J = 5.4 Hz, 1H),
6
7
.79 (d, J = 5.3 Hz, 1H), 7.66–7.63 (m, 3H), 7.45–7.41 (m, 1H), 7.38–7.35 (m, 1H),
2
0

ACCEPTED MANUSCRIPT
7
.30 (t, J = 5.1 Hz, 1H), 3.71–3.68 (m, 2H), 3.59 (t, J = 3.0 Hz, 4H), 2.46 (t, J = 4.6
1
3
Hz, 2H), 2.40 (s, 4H), 1.91–1.86 (m, 2H). C NMR (100 MHz, DMSO-d ) δ 150.8,
6
1
49.8, 143.1, 142.6, 135.7, 134.6, 132.7, 131.3, 128.9, 126.1, 124.9, 124.7, 123.1,
1
19.9, 112.8, 66.7, 56.4, 53.9, 38.9, 25.8. HRMS (ESI) m/z calcd for C H N O
22
25
6
+
[
M+H] 389.2090, found 389.2075.
4
.1.4.4. 3-(1H-benzo[d]imidazol-2-yl)-2-(N-3-(1H-Imidazol-1-yl)propyl)aminoquin-
1
-
oxaline (10c), 0.165 g, yield 89%, pale yellow solid, m.p. 205–206 °C, H NMR (500
MHz, DMSO-d ) δ 13.47 (s, 1H), 9.89 (t, J = 5.6 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H),
6
7
1
.73 (s, 1H), 7.65–7.63 (m, 4H), 7.47–7.43 (m, 1H), 7.28 (s, 1H), 7.02 (s, 2H), 6.94 (s,
1
3
H), 4.16 (t, J = 6.9 Hz, 2H), 3.64–3.61 (m, 2H), 2.23–2.20 (m, 2H). C NMR (125
MHz, DMSO-d ) δ 150.7, 149.8, 143.1, 142.4, 137.8, 135.8, 135.6, 134.6, 132.7,
6
1
31.3, 128.9, 126.2, 125.0, 124.9, 123.1, 120.0, 119.8, 112.8, 44.4, 38.0, 30.6. HRMS
+
(
ESI) m/z calcd for C H N [M+H] 370.1780, found 370.1767.
2
1
20
7
4
.1.4.5. 3-(3-(1H-Benzo[d]imidazol-2-yl)-2-(N-3-hydroxylpropyl)aminoquinoxaline
1
(
10d), 0.147 g, yield 92%, pale yellow solid, m.p. 208–209 °C, H NMR (400 MHz,
DMSO-d ) δ 13.39 (s, 1H), 9.86 (t, J = 3.6 Hz, 1H), 7.95–7.93 (m, 1H), 7.83 (d, J =
6
5
1
2
1
.4 Hz, 1H), 7.67–7.62 (m, 3H), 7.45–7.42 (m, 1H), 7.38–7.35 (m, 1H), 7.32–7.29 (m,
H), 4.66 (t, J = 3.4 Hz, 1H), 3.75–3.71 (m, 2H), 3.65–3.62 (m, 2H), 1.94–1.90 (m,
1
3
H). C NMR (100 MHz, DMSO-d ) δ 150.8, 149.8, 143.1, 142.6, 135.7, 134.5,
6
32.7, 131.3, 128.9, 126.1, 124.9, 124.7, 123.0, 120.0, 112.8, 59.1, 38.1, 32.5. HRMS
+
(
ESI) m/z calcd for C H N O [M+H] 320.1511, found 320.1497.
1
8
18
5
4
.1.4.6. 3-(1H-Benzo[d]imidazol-2-yl)-2-(N-2-dimethylaminoethyl)aminoquinoxaline
1
(
10e), 0.136 g, yield 82%, pale yellow solid, m.p. 164–165 °C, H NMR (400 MHz,
DMSO-d ) δ 13.36 (s, 1H), 9.96 (t, J = 3.4 Hz, 1H), 7.94 (d, J = 5.3 Hz, 1H), 7.78 (d,
6
J = 4.9 Hz, 1H), 7.66–7.62 (m, 3H), 7.45–7.42 (m, 1H), 7.36 (d, J = 4.3 Hz, 1H), 7.31
1
3
(
d, J = 4.8 Hz, 1H), 3.74–3.71 (m, 2H), 2.64 (t, J = 8.4 Hz, 2H), 2.30 (s, 6H). C
NMR (100 MHz, DMSO-d ) δ 150.7, 149.7, 143.1, 142.6, 135.7, 134.6, 132.7, 131.2,
6
1
28.9, 126.1, 124.9, 124.7, 123.0, 120.0, 112.8, 58.2, 45.8, 39.1. HRMS (ESI) m/z
+
calcd for C H N [M+H] 333.1827, found 333.1811.
1
9
21
6
4
.1.4.7.
3-(1H-Benzo[d]imidazol-2-yl)-2-(N-2-morpholino-4-ylethyl)aminoquin-
2
1

ACCEPTED MANUSCRIPT
1
-
oxaline (10f), 0.179 g, yield 96%, pale yellow solid, m.p. 257–258 °C, H NMR (400
MHz, DMSO-d ) δ 13.36 (s, 1H), 10.05 (t, J = 3.2 Hz, 1H), 7.94 (d, J = 5.4 Hz, 1H),
6
7
7
2
1
.80 (d, J = 5.3 Hz, 1H), 7.67–7.63 (m, 3H), 7.45–7.43 (m, 1H), 7.38–7.36 (m, 1H),
.34–7.31 (m, 1H), 3.78–3.75 (m, 2H), 3.71 (t, J = 3.0 Hz, 4H), 2.70 (t, J = 4.1 Hz,
13
H), 2.55 (s, 4H). C NMR (100 MHz, DMSO-d ) δ 150.7, 149.8, 143.2, 142.6,
6
34.6, 131.2, 128.9, 126.1, 124.9, 124.7, 123.0, 119.8, 112.8, 67.0, 56.8, 53.6, 37.9.
+
HRMS (ESI) m/z calcd for C H N O [M+H] 375.1933, found 375.1917.
2
1
23
6
4
.1.4.8.
3-(Benzo[d]thiazol-2-yl)-2-(N-3-dimethylaminopropyl)aminoquinoxaline
1
(
11a), 0.117 g, yield 81%, pale yellow solid, m.p. 134~135 °C, H NMR (400 MHz,
DMSO-d ) δ 9.28 (t, J = 5.4 Hz, 1H), 8.18 (t, J = 7.8 Hz, 2H), 7.87–7.86 (m, 1H),
6
7
.68–7.60 (m, 3H), 7.57–7.54 (m, 1H), 7.44–7.41 (m, 1H), 3.73–3.69 (m, 2H), 2.66 (t,
1
3
J = 6.9 Hz, 2H), 2.38 (s, 6H), 1.99–1.94 (m, 2H). C NMR (100 MHz, DMSO-d ) δ
6
1
1
69.2, 153.4, 149.9, 143.0, 135.7, 135.2, 134.5, 132.2, 129.2, 127.4, 127.3, 126.2,
25.2, 124.0, 122.9, 56.5, 44.5, 40.5, 38.8, 25.8. HRMS (ESI) calcd for C H N S
2
0
22
5
+
[
M+H] 364.1596, found 364.1584.
4
.1.4.9.
3-(Benzo[d]thiazol-2-yl)-2-(N-3-morpholino-4-ylpropyl)aminoquinoxaline
1
(
11b), 0.197 g, yield 97%, pale yellow solid, m.p. 137–138 °C, H NMR (400 MHz,
DMSO-d ) δ 9.23 (t, J = 5.3 Hz, 1H), 8.20–8.13 (m, 2H), 7.86 (d, J = 8.2 Hz, 1H),
6
7
2
.69–7.54 (m, 4H), 7.44–7.40 (m, 1H), 3.72–3.67 (m, 2H), 3.58 (t, J = 4.4 Hz, 4H),
1
3
.47–2.44 (m, 2H), 2.40 (s, 4H), 1.93–1.86 (m, 2H). C NMR (100 MHz, DMSO-d )
6
δ 169.3, 153.3, 149.9, 143.1, 135.7, 135.2, 134.5, 132.3, 129.2, 127.4, 127.3, 126.1,
1
25.2, 123.9, 122.9, 66.7, 56.4, 53.9, 39.1, 25.7. HRMS (ESI) m/z calcd for
+
C H N OS [M+H] 406.1701, found 406.1686.
22
24
5
4
.1.4.10. 3-(Benzo[d]thiazol-2-yl)-2-(N-3-(1H-Imidazol-1-ylpropyl)aminoquinoxaline
1
(
11c), 0.187 g, yield 97%, pale yellow solid, m.p. 145–146 °C, H NMR (400 MHz,
DMSO-d ) δ 9.22 (t, J = 3.6 Hz, 1H), 8.17 (t, J = 5.4 Hz, 2H), 7.85 (dd, J = 5.5, 0.7
6
Hz, 1H), 7.71 (s, 1H), 7.67–7.64 (m, 1H), 7.62–7.58 (m, 2H), 7.55–7.52 (m, 1H),
7
3
1
1
.42–7.40 (m, 1H), 7.27 (t, J = 0.7 Hz, 1H), 6.93 (s, 1H), 4.15 (t, J = 4.6 Hz, 2H),
1
3
.62–3.59 (m, 2H), 2.23–2.19 (m, 2H). C NMR (100 MHz, DMSO-d ) δ 169.2,
6
53.3, 149.8, 142.9, 137.8, 135.8, 135.2, 134.5, 132.2, 129.1, 128.9, 127.3, 127.2,
26.1, 125.2, 124.1, 122.8, 119.8, 44.4, 38.1, 30.4. HRMS (ESI) m/z calcd for
2
2

ACCEPTED MANUSCRIPT
+
C H N S [M+H] 387.1392, found 387.1373.
21
19
6
4
0
9
.1.4.11. 3-(3-(Benzo[d]thiazol-2-yl)-2-(N-3-hydroxylpropyl)aminoquinoxaline (11d),
1
.151 g, yield 90%, yellow solid, m.p. 178–179 °C, H NMR (400 MHz, DMSO-d ) δ
6
.37 (t, J = 3.4 Hz, 1H), 8.18 (t, J = 5.8 Hz, 2H), 7.87 (d, J = 5.4 Hz, 1H), 7.68–7.63
(m, 2H), 7.62–7.59 (m, 1H), 7.57–7.54 (m, 1H), 7.43–7.41 (m, 1H), 3.74–3.71 (m,
1
3
2
1
1
3
H), 3.66 (t, J = 4.0 Hz, 2H), 1.94–1.89 (m, 2H). C NMR (100 MHz, DMSO-d ) δ
6
69.2, 153.3, 149.9, 143.1, 135.7, 135.2, 134.5, 132.2, 129.2, 127.3, 127.2, 126.1,
+
25.1, 124.1, 122.9, 59.4, 38.6, 32.2. HRMS (ESI) m/z calcd for C H N OS [M+H]
1
8
17
4
37.1123, found 337.1106.
4
.1.4.12.
3-(Benzo[d]thiazol-2-yl)-2-(N-2-dimethylaminoethyl)aminoquinoxaline
1
(
11e), 0.154 g, yield 88%, yellow solid, m.p. 77–79 °C, H NMR (400 MHz,
DMSO-d ) δ 9.53 (t, J = 3.0 Hz, 1H), 8.19 (d, J = 5.2 Hz, 1H), 8.08 (d, J = 5.4 Hz,
6
1
H), 7.86 (d, J = 5.4 Hz, 1H), 7.67–7.60 (m, 3H), 7.56–7.54 (m, 1H), 7.43–7.40 (m,
1
3
1
H), 3.69– 3.66 (m, 2H), 2.63 (t, J = 4.0 Hz, 2H), 2.33 (s, 6H). C NMR (100 MHz,
DMSO-d ) δ 169.0, 153.3, 149.8, 143.2, 135.7, 135.3, 134.6, 132.2, 129.2, 127.4,
6
1
27.3, 126.1, 125.0, 123.9, 122.9, 57.8, 45.6, 38.9. HRMS (ESI) m/z calcd for
+
C H N S [M+H] 350.1439, found 350.1423.
19
20
5
4
.1.4.13.
3-(Benzo[d]thiazol-2-yl)-2-(N-2-morpholino-4-ylethyl)aminoquinoxaline
1
(
11f), 0.182 g, yield 93%, pale yellow solid, m.p. 151–152 °C, H NMR (400 MHz,
DMSO-d ) δ 9.49 (t, J = 2.8 Hz, 1H), 8.23 (d, J = 5.2 Hz, 1H), 8.16 (d, J = 5.4 Hz,
6
1
H), 7.90 (d, J = 5.5 Hz, 1H), 7.70–7.65 (m, 3H), 7.60–7.57 (m, 1H), 7.46–7.43 (m,
1
3
1
H), 3.78–3.75 (m, 2H), 3.71–3.70 (m, 4H), 2.72 (t, J = 4.0 Hz, 2H), 2.55 (s, 4H). C
NMR (100 MHz, DMSO-d ) δ 169.2, 153.5, 149.8, 143.2, 135.7, 135.3, 134.6, 132.3,
6
1
29.2, 127.5, 127.3, 126.2, 125.2, 123.7, 123.0, 67.0, 56.8, 53.6, 37.8. HRMS (ESI)
+
m/z calcd for C H N OS [M+H] 392.1545, found 392.1526.
2
1
22
5
4
.1.4.14.
3-(Benzo[d]oxazol-2-yl)-2-(N-3-dimethylaminopropyl)aminoquinoxaline
1
(
12a), 0.159 g, yield 91%, pale yellow solid, m.p. 61–63 °C, H NMR (400 MHz,
DMSO-d ) δ 8.99 (t, J = 5.4 Hz, 1H), 7.94–7.91 (m, 3H), 7.69–7.65 (m, 1H),
6
7
3
.64–7.62 (m, 1H), 7.58–7.54 (m, 1H), 7.52–7.49 (m, 1H), 7.44–7.42 (m, 1H),
13
.69–3.65 (m, 2H), 2.41 (t, J = 6.7 Hz, 2H), 2.21 (s, 6H), 1.87–1.82 (m, 2H). C
2
3

ACCEPTED MANUSCRIPT
NMR (100 MHz, DMSO-d ) δ 160.0, 150.5, 150.1, 143.0, 140.6, 135.9, 132.5, 129.7,
6
1
29.5, 127.7, 126.2, 126.0, 125.2, 120.8, 112.0, 57.4, 45.7, 39.4, 26.6. HRMS (ESI)
+
m/z calcd for C H N O [M+H] 348.1824, found 348.1809.
2
0
22
5
4
.1.4.15. 3-(Benzo[d]oxazol-2-yl)-2-(N-3-morpholino-4-ylpropyl)aminoquinoxaline
1
(
12b), 0.182 g, yield 93%, pale yellow solid, m.p. 118–119 °C, H NMR (400 MHz,
DMSO-d ) δ 8.91 (t, J = 5.5 Hz, 1H), 7.97–7.93 (m, 3H), 7.71–7.68 (m, 1H), 7.64 (d,
6
J = 8.3 Hz, 1H), 7.59–7.57 (m, 1H), 7.53–7.51 (m, 1H), 7.47–7.44 (m, 1H), 3.72–3.69
(m, 2H), 3.60 (t, J = 4.5 Hz, 4H), 2.45 (t, J = 6.9 Hz, 2H), 2.40 (s, 4H), 1.91–1.86 (m,
1
3
2
1
H). C NMR (100 MHz, DMSO-d ) δ 160.1, 150.6, 150.1, 143.0, 140.6, 132.6,
6
29.7, 127.7, 126.2, 126.1, 125.3, 120.8, 112.0, 66.7, 56.3, 53.9, 39.1, 25.6. HRMS
+
(
ESI) m/z calcd for C H N O [M+H] 390.1930, found 390.1912.
2
2
24
5
2
4
.1.4.16. 3-(Benzo[d]oxazol-2-yl)-2-(N-3-(1H-Imidazol-1-ylpropyl)aminoquinoxaline
1
(
12c), 0.169 g, yield 90%, pale yellow solid, m.p. 177–178 °C, H NMR (400 MHz,
DMSO-d ) δ 8.88 (t, J = 5.5 Hz, 1H), 7.98–7.95 (m, 3H), 7.71–7.69 (m, 2H),
6
7
1
.65–7.64 (m, 1H), 7.59–7.56 (m, 1H), 7.53–7.51 (m, 1H), 7.47–7.45 (m, 1H), 7.26 (s,
13
H), 6.91 (s, 1H), 4.15 (t, J = 6.9 Hz, 2H), 3.65–3.62 (m, 2H), 2.23–2.19 (m, 2H). C
NMR (100 MHz, DMSO-d ) δ 160.1, 150.5, 150.1, 142.8, 140.6, 137.8, 136.0, 132.6,
6
1
29.7, 129.6, 128.9, 127.7, 126.3, 126.0, 125.4, 121.0, 119.8, 112.0, 44.4, 38.2, 30.5.
+
HRMS (ESI) calcd for C H ON [M+H] 371.1620, found 371.1603.
2
1
19
6
4
0
8
1
.1.4.17. 3-(3-(Benzo[d]oxazol-2-yl)-2-(N-3-hydroxylpropyl)aminoquinoxaline (12d),
1
.151 g, yield 94%, yellow solid, m.p. 188–189 °C, H NMR (400 MHz, DMSO-d ) δ
6
.90 (t, J = 4.8 Hz, 1H), 7.93–7.91 (m, 3H), 7.67–7.62 (m, 2H), 7.55 (t, J = 7.7 Hz,
H), 7.49 (t, J = 7.5 Hz, 1H), 7.43–7.41 (m, 1H), 3.71–3.68 (m, 2H), 3.62 (t, J = 6.1
1
3
Hz, 2H), 1.92–1.87 (m, 2H). C NMR (100 MHz, DMSO-d ) δ 160.0, 150.5, 150.1,
6
1
5
3
42.9, 140.6, 135.8, 132.5, 129.7, 129.5, 127.6, 126.2, 125.9, 125.2, 120.9, 111.9,
+
9.2, 38.5, 32.3. HRMS (ESI) m/z calcd for C H N O [M+H] 321.1351, found
1
8
17
4
2
21.1335.
4
.1.4.18. 3-(Benzo[d]oxazol-2-yl)-2-(N-2-dimethylaminoethyl)aminoquinoxaline (12e),
1
0
.131 g, yield 78%, pale yellow solid, m.p. 86–87 °C, H NMR (400 MHz, DMSO-d )
6
δ 9.07 (t, J = 4.8 Hz, 1H), 7.95–7.93 (m, 2H), 7.90 (d, J = 7.8 Hz, 1H), 7.69–7.67 (m,
2
4

ACCEPTED MANUSCRIPT
1
H), 7.64 (dd, J = 5.5, 0.6 Hz, 1H), 7.58–7.55 (m, 1H), 7.52–7.49 (m, 1H), 7.45–7.42
1
3
(
m, 1H), 3.70–3.67 (m, 2H), 2.62 (t, J = 6.2 Hz, 2H), 2.30 (s, 6H). C NMR (100
MHz, DMSO-d ) δ 160.0, 150.5, 150.1, 143.0, 140.6, 135.9, 132.5, 129.7, 129.5,
6
1
27.7, 126.2, 126.0, 125.2, 120.9, 112.0, 57.9, 45.7, 39.1. HRMS (ESI) m/z calcd for
+
C H N O [M+H] 334.1668, found 334.1651.
19
20
5
4
.1.4.19.
3-(Benzo[d]oxazol-2-yl)-2-(N-2-morpholino-4-ylethyl)aminoquinoxaline
1
(
12f), 0.155 g, yield 82%, pale yellow solid, m.p. 152–153 °C, H NMR (500 MHz,
DMSO-d ) δ 9.22 (s, 1H), 7.94 (d, J = 8.1 Hz, 2H), 7.88 (d, J = 7.7 Hz, 1H), 7.70–
6
7
.63 (m, 2H), 7.59–7.52 (m, 2H), 7.44 (t, J = 7.4 Hz, 1H), 3.70 (d, J = 3.8 Hz, 6H),
1
3
2
.69 (t, J = 6.0 Hz, 2H), 2.53 (s, 4H). C NMR (125 MHz, DMSO-d ) δ 160.0, 150.5,
6
1
50.1, 143.0, 140.7, 123.5, 129.7, 129.6, 127.7, 126.2, 126.1, 125.2, 120.6, 112.0,
+
6
7.0, 56.5, 53.5, 37.9. HRMS (ESI) m/z calcd for C H N O [M+H] 376.1773,
2
1
22
5
2
found 376.1757.
4
.1.4.20. 3-(5-Chlorobenzo[d]oxazol-2-yl)-2-(N-3-dimethylaminopropyl)aminoquin-
1
-
oxaline (15a), 0.085 g, yield 51 %, pale yellow solid, m.p. 126–127 °C, H NMR
(
400 MHz, CDCl ) δ 8.90 (s, 1H), 8.04–8.02 (m, 1H), 7.82–7.81 (m, 1H), 7.72–7.61
3
(m, 3H), 7.45–7.38 (m, 2H), 3.83–3.87 (m, 2H), 2.51 (t, J = 7.1 Hz, 2H), 2.32 (s, 6H),
1
3
2
1
2
.02–1.95 (m, 2H). C NMR (100 MHz, CDCl ) δ 161.2, 150.7, 148.8, 143.6, 141.9,
3
36.1, 132.2, 130.8, 129.7, 128.7, 127.2, 126.3, 124.8, 120.3, 112.3, 57.6, 45.6, 39.4,
+
7.1. HRMS (ESI) m/z calcd for C H ClN O [M+H] 382.1429, found 382.1418.
2
0
21
5
4
.1.4.21. 3-(5-Methylbenzo[d]oxazol-2-yl)-2-(N-3-dimethylaminopropyl)aminoquin-
1
-
oxaline (16a), 0.043 g, yield 27 %, pale yellow solid, m.p. 122–124 °C, H NMR
(
400 MHz, CDCl ) δ 9.08 (s, 1H), 8.05 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H),
3
7
.64–7.60 (m, 3H), 7.42–7.38 (m, 1H), 7.28 (s, 1H), 3.84 (dd, J = 12.4, 6.7 Hz, 2H),
1
3
2
.58 (t, J = 7.3 Hz, 2H), 2.52 (s, 3H), 2.36 (s, 6H), 2.05–2.02 (m, 2H). C NMR (100
MHz, CDCl ) δ 160.0, 150.7, 148.6, 143.3, 141.0, 136.1, 135.2, 131.8, 129.7, 129.5,
3
1
28.2, 126.2, 124.7, 120.3, 111.0, 57.4, 45.3, 39.2, 26.9, 21.6. HRMS (ESI) m/z calcd
+
for C H N O [M+H] 362.1975, found 362.1968.
2
1
24
5
4
.1.4.22. 3-(5-(Tert-butyl)benzo[d]oxazol-2-yl)-2-(N-3-dimethylaminopropyl)amino-
1
-
quinoxaline (17a), 0.092 g, yield 52 %, pale yellow solid, m.p. 111–112 °C, H NMR
2
5

ACCEPTED MANUSCRIPT
(
400 MHz, CDCl ) δ 9.07 (t, J = 5.2 Hz, 1H), 8.03 (dd, J = 8.3, 1.0 Hz, 1H), 7.82 (d, J
3
=
=
1.5 Hz, 1H), 7.69–7.67 (m, 1H), 7.65–7.63 (m, 1H), 7.60–7.56 (m, 1H), 7.52 (dd, J
8.7, 1.9 Hz, 1H), 7.38–7.34 (m, 1H), 3.80 (dd, J = 12.4, 6.9 Hz, 2H), 2.30 (s, 6H),
1
3
2
1
5
4
.02–1.95 (m, 2H), 1.40 (s, 9H). C NMR (100 MHz, CDCl ) δ 159.0, 149.6, 147.8,
3
47.3, 142.2, 139.7, 135.0, 130.6, 128.6, 128.4, 125.1, 123.7, 123.5, 115.8, 109.7,
+
6.5, 44.5, 38.2, 34.0, 30.7, 26.1. HRMS (ESI) m/z calcd for C H N O [M+H]
2
4
30
5
04.2445, found 404.2440.
4
.1.4.23. 3-(6-Chlorobenzo[d]oxazol-2-yl)-2-(N-3-dimethylaminopropyl)aminoquin-
1
-
oxaline (18a), 0.144 g, yield 86 %, pale yellow solid, m.p. 166–167 °C, H NMR
(
400 MHz, CDCl ) δ 8.92 (s, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.76–7.70 (m, 3H),
3
7
.65–7.61 (m, 3H), 7.43–7.38 (m, 2H), 7.83–7.78 (m, 2H), 2.53–2.50 (m, 2H), 2.30
1
3
(
s, 6H), 2.03–1.93 (m, 2H). C NMR (100 MHz, CDCl ) δ 160.6, 150.7, 150.5, 143.5,
3
1
3
3
39.6, 136.1, 132.6, 132.1, 129.7, 128.8, 126.3, 126.1, 124.8, 121.0, 112.1, 57.6, 45.6,
+
9.4, 27.1. HRMS (ESI) m/z calcd for C H ClN O [M+H] 382.1429, found
2
0
21
5
82.1426.
4
.1.4.24. 3-(6-Methylbenzo[d]oxazol-2-yl)-2-(N-3-dimethylaminopropyl)aminoquin-
1
-
oxaline (19a), 0.078 g, yield 60 %, pale yellow solid, m.p. 121–123 °C, H NMR
(
400 MHz, CDCl ) δ 9.09 (s, 1H), 8.06–8.04 (m, 1H), 7.72–7.70 (m, 2H), 7.64–7.60
3
(m, 1H), 7.55 (s, 1H), 7.42–7.38 (m, 1H), 7.25 (d, J = 8.6 Hz, 1H), 3.84–3.79 (m, 2H),
1
3
2
1
1
.57–2.53 (m, 5H), 2.34 (s, 6H), 2.06–1.98 (m, 2H). C NMR (100 MHz, CDCl ) δ
3
59.5, 150.7, 150.6, 143.2, 138.7, 137.7, 136.1, 131.7, 129.6, 129.5, 126.6, 126.2,
24.6, 119.8, 111.6, 57.5, 45.5, 39.3, 27.0, 22.1. HRMS (ESI) m/z calcd for
+
C H N O [M+H] 362.1975, found 362.1970.
21
24
5
4
.1.4.25.
3-(Naphtho[1,2-d]oxazol-2-yl)-2-(N-3-dimethylaminopropyl)aminoquin-
1
-
oxaline (20a), 0.068 g, yield 39 %, pale yellow solid, m.p. 144–145 °C, H NMR
(
400 MHz, CDCl ) δ 9.19 (s, 1H), 8.55–8.54 (m, 1H), 8.07–7.98 (m, 2H), 7.89–7.86
3
(m, 2H), 7.74–7.72 (m, 2H), 7.64–7.59(m, 2H), 7.43–7.39 (m, 1H), 3.87–3.86 (m,
1
3
2
H), 2.67 (t, J = 6.9 Hz, 2H), 2.40 (s, 6H), 2.16–2.10 (m, 2H). C NMR (100 MHz,
CDCl ) δ 159.1, 150.5, 148.1, 143.1, 136.4, 136.2, 131.7, 131.4, 129.6, 129.5, 128.9,
3
1
28.2, 127.6, 126.2, 126.0, 124.7, 122.0, 111.4, 57.5, 45.4, 39.2, 27.1. HRMS (ESI)
+
m/z calcd for C H N O [M+H] 398.1976, found 398.1974.
2
4
24
5
2
6

ACCEPTED MANUSCRIPT
4
.1.4.26.
3-(5-Chlorobenzo[d]oxazol-2-yl)-2-(N-3-diethylaminopropyl)aminoquin-
1
-
oxaline (15g), 0.033 g, yield 18.5 %, pale yellow solid, m.p. 111–112 °C, H NMR
(
500 MHz, CDCl ) δ 8.86 (s, 1H), 8.04 (d, J =8.3 Hz, 1H), 7.81–7.80 (m, 1H),
3
7
.72–7.62 (m, 3H), 7.45–7.39 (m, 2H), 3.80–3.76 (m, 2H), 2.69–2.66 (m, 2H), 2.63
1
3
(
dd, J = 11.4, 5.7 Hz, 4H), 2.01–1.95 (m, 2H), 1.08 (t, J = 5.7 Hz, 6H). C NMR (125
MHz, CDCl ) δ 161.2, 150.7, 148.8, 143.6, 141.9, 136.1, 132.2, 130.8, 129.7, 128.7,
3
1
27.2, 126.3, 124.8, 120.3, 112.3, 50.5, 47.1, 39.5, 26.6, 11.7. HRMS (ESI) m/z calcd
+
for C H ClN O [M+H] 410.1742, found 410.1735.
2
2
25
5
4
.1.4.27.
3-(5-Methylbenzo[d]oxazol-2-yl)-2-(N-3-diethylaminopropyl)aminoquin-
1
-
oxaline (16g), 0.060 g, yield 35 %, pale yellow solid, m.p. 87–88 °C, H NMR (400
MHz, CDCl ) δ 9.08 (s, 1H), 8.06 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.65–
3
7
2
=
.63 (m, 3H), 7.41 (t, J = 9.5 Hz, 1H), 7.29–7.27 (m, 1H), 3.84–3.79 (m, 2H),
.84–2.80 (m, 2H), 2.76–2.74 (m, 4H), 2.52 (s, 3H), 2.09 (t, J = 7.0 Hz, 2H), 1.17 (t, J
1
3
7.1 Hz, 6H). C NMR (100 MHz, CDCl ) δ 159.9, 150.7, 148.6, 143.2, 141.0,
3
1
2
3
36.1, 135.3, 131.8, 129.7, 129.5, 128.2, 126.2, 124.8, 120.3, 111.0, 50.2, 46.9, 39.2,
+
5.8, 21.6, 11.0. HRMS (ESI) m/z calcd for C H N O [M+H] 390.2288, found
2
3
28
5
90.2276.
4
.1.4.28.
3-(5-(Tert-butyl)benzo[d]oxazol-2-yl)-2-(N-3-diethylaminopropyl)amino-
1
-
quinoxaline (17g), 0.129 g, yield 68 %, pale yellow oil, H NMR (400 MHz, CDCl )
3
δ 9.07 (t, J = 5.1 Hz, 1H), 8.04 (dd, J = 8.3, 1.0 Hz, 1H), 7.83–7.82 (m, 1H), 7.70 (dd,
J = 8.4, 0.9 Hz, 1H), 7.64 (d, J = 8.7, 1H), 7.61–7.57 (m, 1H), 7.52 (dd, J = 8.7, 1.9
Hz, 1H), 7.38–7.34 (m, 1H), 3.78–3.73 (m, 2H), 2.69–2.66 (m, 2H), 2.63 (q, J = 7.2
1
3
Hz, 4H), 2.01–1.94 (m, 2H), 1.40 (s, 9H), 1.08 (t, J = 7.2 Hz, 6H). C NMR (100
MHz, CDCl ) δ 160.1, 150.7, 148.8, 148.3, 143.3, 140.8, 136.0, 131.7, 129.6, 129.4,
3
1
26.2, 124.8, 124.5, 116.8, 110.7, 50.5, 47.0, 39.5, 35.0, 31.7, 26.6, 11.8. HRMS
+
(
ESI) m/z calcd for C H N O [M+H] 432.2758, found 432.2754.
2
6
34
5
4
.1.4.29.
3-(6-Chlorobenzo[d]oxazol-2-yl)-2-(N-3-diethylaminopropyl)aminoquin-
1
-
oxaline (18g), 0.118 g, yield 69 %, pale yellow solid, m.p. 77–78 °C, H NMR (400
MHz, CDCl ) δ 8.88 (s, 1H), 8.03 (t, J = 8.4 Hz, 1H), 7.76–7.71 (m, 3H), 7.67–7.62
3
(m, 1H), 7.44–7.39 (m, 2H), 3.81–3.76 (m, 2H), 2.68 (t, J = 7.2 Hz, 2H), 2.65–2.59
2
7

ACCEPTED MANUSCRIPT
1
3
(
m, 4H), 2.02–1.95 (m, 2H), 1.12–1.07 (m, 6H). C NMR (100 MHz, CDCl ) δ 160.6,
3
1
1
50.7, 150.5, 143.5, 139.6, 136.1, 132.6, 132.1, 129.7, 128.8, 126.3, 126.1, 124.8,
20.9, 112.1, 50.5, 47.1, 39.5, 26.6, 11.7. HRMS (ESI) m/z calcd for C H ClN O
2
2
25
5
+
[
M+H] 410.1724, found 410.1736.
4
.1.4.30.
3-(6-Methylbenzo[d]oxazol-2-yl)-2-(N-3-diethylaminopropyl)aminoquin-
1
-
oxaline (19g), 0.120 g, yield 70 %, pale yellow oil, H NMR (400 MHz, CDCl ) δ
3
9
7
8
2
.01 (t, J = 5.1 Hz, 1H), 8.03 (dd, J = 8.3, 1.0 Hz, 1H), 7.70 (dd, J = 8.4, 1.0 Hz, 1H),
.65–7.63 (m, 1H), 7.61–7.56 (m, 1H), 7.50 (s, 1H), 7.38–7.34 (m, 1H), 7.20 (dd, J =
.2, 0.8 Hz, 1H), 3.77–3.72 (m, 2H), 2.68–2.64 (m, 2H), 2.62 (q, J = 7.2 Hz, 4H),
1
3
.50 (s, 3H), 2.00–1.93 (m, 2H), 1.06 (t, J = 7.1 Hz, 6H). C NMR (100 MHz,
CDCl ) δ 159.5, 150.6, 150.5, 143.2, 138.6, 137.6, 136.0, 131.6, 129.6, 129.4, 126.6,
3
1
26.2, 124.5, 119.8, 111.5, 50.5, 47.0, 39.5, 26.5, 22.0, 11.7. HRMS (ESI) m/z calcd
+
for C H N O [M+H] 390.2288, found 390.2286.
2
3
28
5
4
.1.4.31. 3-(Naphtho[1,2-d]oxazol-2-yl)-2-(N-3-diethylaminopropyl)aminoquinoxaline
1
(
20g), 0.094 g, yield 50 %, pale yellow solid, m.p. 121–122 °C, H NMR (400 MHz,
CDCl ) δ 9.15 (s, 1H), 8.55–8.53 (m, 1H), 8.06 (d, J = 8.3 Hz, 1H), 8.01–8.00 (m,
3
1
3
H), 7.92–7.87 (m, 2H), 7.74–7.70 (m, 2H), 7.65–7.60 (m, 2H), 7.43–7.39 (m, 1H),
.84 (d, J = 5.8 Hz, 2H), 2.77 (t, J = 7.2 Hz, 2H), 2.69 (q, J = 7.2 Hz, 4H), 2.15–2.05
1
3
(
m, 2H), 1.10 (t, J = 7.2 Hz, 6H). C NMR (100 MHz, CDCl ) δ 159.2, 150.5, 148.1,
3
1
1
43.2, 136.4, 136.1, 131.6, 131.4, 129.6, 129.5, 128.9, 128.2, 127.5, 126.2, 126.0,
24.7, 122.0, 111.4, 50.8, 47.1, 39.6, 26.9, 11.7. HRMS (ESI) m/z calcd for
+
C H N O [M+H] 426.2289, found 426.2283.
26
28
5
4
.2. Biological activity
4
.2.1. Anti-proliferative activity
Cells were seeded in 96-well plates and incubated overnight in 180 µL of
medium containing 10% Fetal Bovine Serum (Gibco, USA). On the following day, 20
µL of compounds at different concentrations were added to each well and incubated
for 48 h. Then, 10 µL of 5 mg/mL MTT solution was added into each well and
incubated for 4 h at 37 °C. The culture medium was then removed, and 100 µL of
2
8