Structure-activity relationships and antiproliferative effects of 1,2,3,4-4H-quinoxaline derivatives as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bioorg.2021.104793

Colchicine binding site inhibitors (CBSIs) hold great potential for the treatment of various tumors and they can overcome multidrug resistance which the existing tubulin inhibitors such as paclitaxel and vinorelbine are faced with. Herein, we report the d

Full text of DOI:10.1016/j.bioorg.2021.104793

Bioorganic Chemistry 110 (2021) 104793
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Structure-activity relationships and antiproliferative effects of
1,2,3,4-4H-quinoxaline derivatives as tubulin polymerization inhibitors
Tingting Lianga, Xiaomin Zhoua, Lu Lua, Haiyang Donga, Yanan Zhanga, Yungen Xub,
,
,
,*
Jianguo Qia *, Yahong Zhanga *, Jianhong Wanga
a Key Laboratory of Natural Medicine and Immune-Engineering of Henan Province, Henan University Jinming Campus, Kaifeng 475004, Henan, China
b Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 211198, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Colchicine binding site inhibitors (CBSIs) hold great potential for the treatment of various tumors and they can
overcome multidrug resistance which the existing tubulin inhibitors such as paclitaxel and vinorelbine are faced
with. Herein, we report the design, synthesis and biological evaluation of a series of tetrahydro-quinoxaline
derivatives as colchicine binding site inhibitors. All the synthesized compounds were evaluated for their in
vitro antiproliferative activities against HT-29 and Hela cancer cell lines, and most of the target compounds
demonstrated moderate to strong activities towards two tumor cell lines. In addition, the structure-activity re-
lationships of these derivatives were also discussed. Among them, compounds 11a and 11b showed the most
potent activities. Moreover, compound 11a inhibited the tubulin polymerization in both cell-free and cellular
assays. Further profiling of compound 11a revealed that it arrested cell cycle in G2/M and induced cell apoptosis
in a dose-dependent manner. Furthermore, molecular docking study proved that compound 11a acted on the
colchicine binding site. Therefore, 11a is a promising candidate for the discovery of colchicine binding site
inhibitors.
Colchicine binding site
Inhibitors
Tetrahydro-quinoxaline derivatives
Antitumor activities
1. Introduction
the past decades, MTAs such as paclitaxel and vinca alkaloids have been
approved by the FDA for the treatment of tumors which show great
Microtubules are important components of the eukaryotic cytoskel-
eton, which are composed of -tubulin and β-tubulin subunits in a head-
success. However, their therapeutic applications are usually limited by
multidrug resistance.
α
to-tail arrangement [1–4]. And the dynamic equilibrium process of
polymerization / depolymerization is closely related to maintain cell
architecture [3,5,6] and regulate mitosis by forming spindle and sepa-
rating chromosomes [7–10]. Additionally, microtubules are also
involved in intracellular transport, organelle positioning, cell motility,
signaling control and so on [11,12]. Therefore, microtubules have been
one of the most attractive targets in the treatment of cancer, and dis-
rupting microtubule dynamics is an important strategy to develop novel
microtubule-targeting agents (MTAs) [13–15].Fig. 1
Colchicine binding site inhibitors (CBSIs) can not only perturb
mitosis and induce cell death, but also disrupt tumor blood vessels to
inhibit nutrients and oxygen delivery to the tumor tissues, acting as
vascular disrupting agents [18,19]. Moreover, accumulating evidence
suggests that CBSIs have been considered as candidates to overcome
multidrug resistance (MDR) for that CBSIs are not the substrates of the
MDR efflux pump [20,21]. Therefore, CBSIs gain more and more at-
tentions from medicinal chemists as new generation agents in cancer
chemotherapy. Colchicine, the first identified CBSI, suffers from high
toxicity and narrow therapeutic index, which limit its clinical use [22].
Combretastatins including CA-1, CA-2 and CA-4 show strong tubulin
polymerization inhibitory activities, but their clinical applications are
limited by low bioavailability and isomerization which generates the
less active trans isomer [23]. To address their shortcomings, many novel
CBSIs have been synthesized [11,16,24–26]. For example, Priego and
Based on the mechanisms of action, MTAs are generally divided into
two major classes: microtubule stabilizing agents and microtubule
destabilizing agents. According to their different binding sites, the
microtubule stabilizing agents act on the taxane and laulimalid binding
sites, while the microtubule destabilizing agents act on the vinca alka-
loids, colchicine, maytansine and pironetin binding sites [16,17]. Over
* Corresponding authors.
E-mail addresses: qijianguo@henu.edu.cn (J. Qi), zhangyahong_131@163.com (Y. Zhang), jhworg@126.com (J. Wang).
https://doi.org/10.1016/j.bioorg.2021.104793
Received 17 November 2020; Received in revised form 28 February 2021; Accepted 1 March 2021
Available online 5 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

T. Liang et al.
Bioorganic Chemistry 110 (2021) 104793
co-workers demonstrated the
α-methyl chalcone TUB091 as potent
2. Results and discussions
tubulin polymerization inhibitor with IC50 value being 1–10 nM against
cancer and endothelial cells [27]. Subsequently, based on conforma-
tional mimetics of TUB091, they constructed 1,2,3,4-tetrahydronaph-
thalen-2-yl aryl ketone (1) which exhibited potent antiproliferative
activity and arrested cell cycle in G2/M phase in 2018. Particularly, 1
showed equivalent colchicine site binding ability with combretastatin
A4 [28]. Recently, Bortolozzi et al. reported a 2-amino-3-(3′,4′,5′-tri-
methoxybenzoyl)-benzo[b]furan scaffold (2) as remarkable anti-
proliferative agent with IC50 being 5 pM against tumor cells, and they
revealed that the powerful antiproliferative activity was attributed to
the tubulin polymerization inhibition caused by binding to the colchi-
cine site [29]. To date, several CBSIs have been in the clinical trials for
treating various cancers. However, no CBSIs have been launched for the
clinical use [26,30]. Therefore, it is imperative to develop new
microtubule-targeting agents with novel structures that bind to the
colchicine binding site.
2.1. Chemistry
The target compounds were summarized as depicted in Scheme 1.
Commercially available material anilines 3b–3d were mono-substituted
with corresponding halides to give intermediates 4a–4j, which were
further reduced by hydrogenation with Pd/C to synthesize amines
5b–5k. Intermediate 5a was synthesized by nucleophilic substitution of
1,2-diaminobenzene 3a with corresponding halide. Then condensation
of intermediates 5a–5k with dimethyl oxalate gave intermediates
6a–6k, followed by reduction of intermediates 6a–6k to yield the target
compounds 7a–7k. Compound 7d was protected with (Boc)2O to obtain
the intermediate 8 and followed by debenzylation to provide interme-
diate 9. Condensation of 9 with acyl chlorides produced intermediates
10a–10l, which underwent deprotection to furnish the final compounds
11a–11l.
Previously, our group have reported 3,4-dihydroquinixalin-2(1H)-
one and 1,4-dihydroquinoxaline-2,3-dione derivatives as colchicine
binding site inhibitors [31,32]. Based on the previous studies, we
replaced the above scaffolds with 1,2,3,4-tetrahydro-quinoxaline scaf-
fold to find novel compounds with high antitumor activity and com-
plement the prior structural optimizations. Herein, we reported the
design, synthesis and biological evaluation of 1,2,3,4-tetrahydroqui-
noxaline derivatives. All target compounds were evaluated for their
antiproliferative activities against Hela and HT-29 cancer cell lines. The
most active derivative was assessed for its tubulin depolymerization
activity, the effects on cell cycle and cell death. The binding modes of
1,2,3,4-tetrahydroquinoxaline derivatives with tubulin through the
molecular docking study were also predicted.
2.2. Biological evaluations
2.2.1. In vitro antiproliferative activities
All synthetic target compounds were evaluated for their anti-
proliferative activities against Hela and HT-29 cancer cell lines using
MTT assay, compounds 12, 13, colchicine and CA-4 were chosen as
positive controls. As demonstrated in Table 1, most of the target com-
pounds showed antiproliferative activities against cancer cells with IC50
values under 15 μM.
Preliminary structure-activity relationships analysis was focused on
the following three directions: substituents on the tetrahydroquinoxa-
line ring (R1), the aromatic ring R2 and the linker. When the linker was
OH
N
O
O
O
OH
H
O
NH
O
N
H
N
O
H
O
O
O
O
O
O
OH
OH
H
N
O
O O
H
O
O
OH
O
O
Paclitaxel
OCH3
Vincristine
R
OH
H3CO
OCH3
OH
H3CO
OCH3
OCH3
O
OCH3
O
H3CO
OCH3
O
NH
OCH3
O
Combretastatin A-1 (CA-1): R = OH
Combretastatin A-4 (CA-4): R = H
Colchicine
Combretastatin A-2 (CA-2)
OCH3
H3CO
O
O
OCH3
CH3
O
OCH3
OCH3
H3CO
O
NH2
O
H3CO
OCH3
TUB091
NH2
NH2
OCH3
O
C2H5O
1
2
Fig. 1. Representative microtubule-targeting agents.
2

T. Liang et al.
Bioorganic Chemistry 110 (2021) 104793
H
N
H
N
R1
R1
O
O
R1
O2N
H2N
R1
O2N
HN
R1
H2N
HN
a
b
c
d
N
R2
N
R2
R2
R2
3b ~ 3d
4a ~ 4j
5a ~ 5k
6a ~ 6k
7a ~ 7k
a
H2N
H2N
3a
Boc
N
Boc
N
H
N
H
N
Boc
OMe
OMe
OMe
OMe
e
f
g
h
N
OMe
N
N
N
N
N
H
O
R2
11a ~ 11l
O
R2
7d
8
9
10a ~ 10l
Scheme 1. Reagents and conditions: (a) corresponding halides, KI, K2CO3, MeCN, 70 ◦C, 6 h; (b) 10% Pd/C, H2, MeOH, rt, 2 h; (c) dimethyl oxalate, 70 ◦C, 5 h; (d)
BH3-THF, anhydrous THF, 0 ◦C, 30 min then rt, 12 h; (e) (Boc)2O, TEA, DCM, 40 ◦C, 15 h; (f) 10% Pd/C, H2, MeOH, rt, 4 h; (g) corresponding acyl chlorides, DMAP,
anhydrous DCM, rt, 3 h; (h) CF3COOH, DCM, rt, 8 h.
methylene group, different substituents on the tetrahydroquinoxaline
ring (R1) were explored. As shown in Table 1, alkoxy groups at the R1
position showed better potency than hydrogen and electron-
withdrawing group fluorine, indicating that electron-donating sub-
stitutes at the R1 position could increase the inhibitory activities.
Meanwhile, compounds bearing methoxy were more potent than com-
pounds with ethoxy, demonstrating that the volume of R1 group was also
related with the inhibitory activity. Therefore, the optimal substitute of
R1 was methoxy group. Subsequently, different aromatic fragments at
the R2 position were explored. When R2 was phenyl group, variable
substituents at the C-4 position were investigated. As shown in Table 1,
7e and 7f with electron-withdrawing groups could reduce the inhibitory
activities, and 7g with electron-donating group could enhance the
inhibitory activity, compared with unsubstituted derivative 7d. Moving
the methoxy group to the C-3 position resulted derivative 7h which was
more potent than 7g. Replacing the phenyl with naphthyl led to com-
pound 7i which was less active than 7d. The ethoxy derivative 7j, which
was unsubstituted in phenyl group, was less active than 7k with 3,4,5-
trimethoxy in phenyl group, illustrating that introduction of electron-
donating group such as methoxy was favorable with the inhibitory
activity.
indicating that the hydrophobic interaction between the aromatic group
R2 and the tubulin played some roles, but the volume, conformation and
electronic effect of R2 were more important for antiproliferative
activities.
In summary, compounds 11a and 11b exhibited the greatest anti-
proliferative activities among all the synthetic target compounds.
Moreover, compounds 11a and 11b were equipotent to the previously
reported compounds 12 and 13 against Hela cells, and more active than
compounds 12 and 13 against HT-29 cells.
2.2.2. Tubulin polymerization inhibitory assay
Based on the antiproliferative activities, compound 11a was chosen
to evaluate its tubulin depolymerization activity in a tubulin polymeri-
zation inhibitory assay using purified tubulin. Meanwhile, colchicine
was used as the positive control. As shown in Fig. 2, compound 11a
exhibited dose-dependent tubulin polymerization inhibitory activity in a
colchicine-like manner, indicating that 11a was the microtubule-
depolymerization agent, and the inhibitory activity of 11a at 10 μM
was comparable to that of CA-4 at 5 μM.
2.2.3. Immunofluorescence analysis
Next, replacing the linker methylene group with carbonyl group
could significantly improve the antiproliferative activities of the target
compounds. Based on the above SAR, methoxy was chosen as R1 group,
and the investigation was focused on the R2 group. Compound 11a with
3,4,5-trimethoxyphenyl displayed the most antiproliferative activity
Next, immunofluorescence assay in Hela cells was conducted to
directly confirm that compound 11a could inhibit tubulin polymeriza-
tion dynamics in cellular assay. Hela cells were treated with 11a at three
concentrations (0.15, 0.20 and 0.30 μM) for 24 h, and then stained with
Hoechst 33,342 and anti-β-tubulin-Cy3 antibody. As shown in Fig. 3, in
the control group, the microtubules were filamentous and extended
throughout the cells to maintain the normal cellular morphology. On the
contrary, in the experimental groups, the microtubules decreased
significantly and solidified around the nucleus in the cells. These results
demonstrated that compound 11a disrupted the microtubule networks
in a dose-dependent manner.
with IC50 values ranging from 0.16 to 0.18 μM against Hela and HT-29
cell lines. Removing 4-OMe from 11a produced the derivative 11b,
which maintained activities against both cancer cell lines, implying that
4-OMe was not important for antiproliferative activity. Replacing the
methoxy group with the electron-withdrawing group (11c) resulted in a
dramatic decreased activity. Introducing the saturated or unsaturated
ring to the phenyl group resulted derivatives 11d–11j. However, most of
these compounds remarkably lost their activities except 11f and 11g.
11f with 4-methoxyl naphthyl exhibited ~5-fold reduced anti-
proliferative activity compared to that of 11a, and 11g bearing 6-quino-
linyl was 24-fold more active than 11h with 5-quinolinyl, and 11g was
only slightly less potent than 11a. Subsequently, the aromatic group R2
was then replaced with cyclohexyl or adamantly group. Unfortunately,
these modifications were unfavorable to the antiproliferative activities,
2.2.4. Cell cycle analysis
The flow cytometry analysis was performed to verify whether com-
pound 11a could obstruct the cell division and cause cell cycle arrest.
Hela cells were treated with compound 11a at 0.15, 0.20 and 0.25 μM
for 24 h, respectively. As presented in Fig. 4, compound 11a induced a
significant decrease in the proportion of cells in the G0/G1 phase and an
obvious increase in the proportion of cells in G2/M phase in a dose-
3

T. Liang et al.
Bioorganic Chemistry 110 (2021) 104793
Table 1
Table 1 (continued)
Compd.
R1
Linker
R2
MTT assay IC50
(μM)
a
Hela
HT-29
In vitro antiproliferative activities of all target compounds.
– –
CO
11j
OMe
8.94 ±
8.35 ±
Compd.
7a
R1
Linker
R2
MTT assay IC50
(
μ
M)
1.05
1.02
a
– –
CO
Hela
HT-29
11k
11l
OMe
OMe
3.54 ±
10.79 ±
0.24
1.73
–
–
–
– –
CH2
H
27.93 ±
35.38 ±
– –
CO
3.40
3.90
13.71 ±
14.77 ±
4.38
0.45
– –
CH2
7b
7c
F
>50
>50
>50
>50
12
0.126 ±
1.14 ±
0.015b
0.39
– –
CH2
F
–
–
–
– –
CH2
7d
7e
OMe
8.63 ±
27.14 ±
13
0.194 ±
0.510 ±
2.11
3.31
0.013c
0.036c
– –
CH2
OMe
12.20 ±
11.68 ±
3.70
0.73
– –
CH2
7f
OMe
OMe
OMe
OMe
12.92 ±
7.40 ±
1.99
1.74
–
–
–
– –
CH2
7g
7h
7i
6.00 ±
9.72 ±
2.74
0.39
Colchicine
CA-4
0.045 ±
0.012
0.25 ±
0.08
– –
CH2
4.17 ±
4.39 ±
0.013 ±
0.003
0.013 ±
0.001
0.57
0.53
– –
CH2
15.00 ±
17.40 ±
a
IC50 values were expressed as the mean ± SD of three independent
2.10
2.20
experiments.
b
c
–
–
– –
CH2
7j
OEt
>50
>50
Data were published in Ref. [31].
Data were published in Ref. [32].
– –
CH2
7k
OEt
10.32 ±
12.93 ±
1.23
2.68
dependent manner. When Hela cells were treated with 0.25 µM of
compound 11a, the number of cells in G2/M phase reached 78.3%.
These results indicated that compound 11a could arrest cell cycle at G2/
M phase to cause mitotic cell death or mitotic catastrophe.
– –
CO
11a
11b
11c
OMe
OMe
OMe
0.16 ±
0.18 ±
0.02
0.03
2.2.5. Cell apoptosis analysis
– –
CO
0.15 ±
0.20 ±
To determine the influence of compound 11a on cell death, the cell
apoptosis assay was performed in Hela cells. As shown in Fig. 5, com-
pound 11a induced an important increase in cell apoptosis compared
with the control group, in a dose-dependent manner. What’s more, the
0.02
0.02
– –
CO
11.70 ±
19.20 ±
1.92
2.40
number of apoptotic cells was more than 50% at 0.25 μM. These results
indicated that compound 11a could induce cell apoptosis to treat
– –
CO
11d
11e
OMe
OMe
13.40 ±
12.40 ±
cancers.
2.40
2.50
– –
CO
2.38 ±
2.12 ±
2.2.6. Cytotoxicity evaluation
0.28
0.30
Compound 11a was evaluated in vitro against human normal LO2
cells to estimate the potential cytotoxicity on non-tumor cell lines. As
demonstrated in Table 2, compound 11a showed lower toxicity toward
LO2 cells compared with CA-4.
– –
CO
11f
OMe
OMe
OMe
0.87 ±
1.05 ±
0.08
0.09
– –
CO
2.2.7. Molecular docking study
11g
11h
0.36 ±
0.43 ±
0.05
0.05
Molecular docking study was performed to understand the interac-
tion of compound 11a with tubulin. As predicted, 11a occupied the
colchicine binding site and the binding mode was consistent with that of
CA-4. As shown in Fig. 6, the 3,4,5-trimethoxyphenyl moiety was
accommodated in a hydrophobic pocket, the tetrahydroquinoxaline ring
was sandwiched between βN258 and βK352. Additionally, NH from
tetrahydroquinoxaline ring formed a hydrogen bond with carbonyl
– –
CO
8.57 ±
10.30 ±
2.27
1.98
– –
CO
11i
OMe
>50
>50
4

T. Liang et al.
Bioorganic Chemistry 110 (2021) 104793
Fig. 2. Effect of 11a (1, 5 and 10
μ
M) on tubulin polymerization in vitro and colchicine (5
μ
M) was used as the positive control and 0.1% DMSO was used as the blank
control. 11a (1, 5 or 10 μM), colchicine (5 μ
M) and 0.1% DMSO were added in a 96-well plate and then polymerizations were detected over 80 min at 37 ◦C.
Fig. 3. Images of immunofluorescence staining of microtubules in Hela cells treated with 11a (0.15, 0.20 or 0.30
33342 and anti-β-tubulin-Cy3 antibody.
μM) for 24 h. Cells were stained with Hoechst
group of
αT179.
3. Conclusions
In summary, we have reported the design and synthesis of tetrahy-
droquinoxaline derivatives as novel colchicine binding site inhibitors.
5

T. Liang et al.
Bioorganic Chemistry 110 (2021) 104793
Fig. 4. Compound 11a arrested cell cycle progression in a dose-dependent manner in Hela cells. (A) Hela cells were treated with 11a at 0.15, 0.20 and 0.25 μM for
24 h, then analyzed by flow cytometer. (B) The cell cycle distribution.
Fig. 5. Effects of compound 11a to induce apoptosis of Hela cells. (A) Hela cells were treated with 11a (0.15, 0.18 and 0.25 μM) and DMSO for 24 h, stained with
PITC AV and PI, and determined by the flow cytometric analysis. (B) The proportion of apoptotic cells.
illustrated that the carbonyl derivatives (11a–11l) were more potent
than the methylene derivatives (7a–7k), and the methoxy on the tet-
rahydroquinoxaline ring was important for the antiproliferative activity.
Among them, compounds 11a and 11b showed the strongest anti-
Table 2
Antiproliferative activity of 11a against human
normal LO2 cells
Comp.
IC50
(
μ
M) a
proliferative activities (11a: Hela IC50 = 0.16 ± 0.02
0.18 ± 0.03 M; 11b: Hela IC50 = 0.15 ± 0.02
0.02 M). The tubulin polymerization inhibitory assay and immuno-
μ
M, HT-29 IC50
=
11a
1.22 ± 0.09
0.33 ± 0.07b
μ
μM, HT-29 IC50 = 0.20 ±
CA-4
μ
a
fluorescence analysis suggested that compound 11a inhibited tubulin
polymerization and disturbed the dynamic equilibrium in a colchicine-
like manner, indicating 11a was microtubule depolymerization agent.
Further biological studies indicated that 11a was able to arrest cell cycle
in G2/M and induce cell death in a dose-dependent manner. Moreover,
11a exhibited low toxicity in normal LO2 cells in comparison to tumor
cells. Besides these, molecular docking study demonstrated that
IC50 values were expressed as the mean ± SD
of three independent experiments.
b
Data were reported in Ref [33].
The results of cellular assay demonstrated that some of the target
compounds showed moderate to strong antiproliferative activities
against Hela and HT-29 cell lines. Structure-activity relationships
6

T. Liang et al.
Bioorganic Chemistry 110 (2021) 104793
Fig. 6. (A) Proposed binding mode of compound 11a with the colchicine binding site (PDB: 5LYJ). (B) Overlap of the structures of 11a (green) and CA-4 (pink). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
compound 11a acted on the colchicine binding site. Therefore, com-
pound 11a was a promising drug candidate targeting colchicine binding
site for further development.
NaH2PO4 + 1000 mL H2O); temperature: 35 ◦C, flow rate, 1.0 mL/min;
UV wavelength, 254 nm), gradient elution: 0–1 min (methanol 25%),
1–5 min (methanol 25%-75%), 5–18 min (methanol 75%), 18–22 min
(methanol 75%-25%), 22–23 min (methanol 25%).
4. Experimental sections
4.1.2. Synthesis of 4-fluoro-2-nitro-N-(3,4,5-trimethoxybenzyl)aniline
(4a)
4.1. Chemistry
To a solution of 4-fluoro-2-nitroaniline (1.50 g, 9.61 mmol) in
acetonitrile (30 mL) was added anhydrous K2CO3 (3.19 g, 23.06 mmol),
KI (1.59 g, 9.61 mmol) and 5-(chloromethyl)-1,2,3-trimethoxybenzene
4.1.1. General materials and methods
All the materials were obtained commercially and used without
further purification unless noted otherwise. All solvents were analytical
purity. Reactions were monitored by thin layer chromatography (TLC)
by UV absorbance (254 nm). Flash column chromatography were per-
formed on silica gel (100–200 mesh, 200–300 mesh). Melting points
were determined by hot-stage microscope (SGW X-4) and the instrument
was uncorrected. NMR spectra were recorded on Bruker ADVANCE 300
(1H NMR: 300 MHz, 13C NMR: 75 MHz) and referenced to deuterium
dimethyl sulfoxide (DMSO‑d6) or deuterium chloroform (CDCl3). The
coupling constants J were given in Hz, and the abbreviations were used
to describe peak patterns where appropriate: singlet (s), doublet (d),
triplet (t), multiplet (m), broad resonances (br). High-resolution mass
spectrometry (HRMS) were acquired via a Thermo Scientific LTQ
Orbitrap XL mass spectrometer. The purities of all target compounds
were determined by reverse-phase HPLC using a Waters Alliance e2695
◦
(4.16 g, 19.22 mmol). The mixture was stirred at 70 C for 6 h. After
completion, the mixture was cooled to room temperature, and the sol-
vent was evaporated under reduced pressure. The crude product was
purified by column chromatography (PE:EA = 20:1) to obtain 4a (2.00
g, 61.9%) as an orange solid. m.p.: 97–98 ◦C. 1H NMR (300 MHz, CDCl3)
δ 8.26 (br, 1H), 7.91 (dd, J = 9.1, 2.9 Hz, 1H), 7.26–7.16 (m, 1H), 6.80
(dd, J = 9.4, 4.5 Hz, 1H), 6.56 (s, 2H), 4.46 (d, J = 5.4 Hz, 2H), 3.84 (s,
9H). MS (ESI) [M+H]+ 337.01.
Compounds 4b–4j and 5a were synthesized following a similar
method of compound 4a.
4.1.3. Synthesis of 4-fluoro-N-(3-methoxybenzyl)-2-nitroaniline (4b)
Orange-red oil (58.9%). 1H NMR (300 MHz, CDCl3) δ 8.34 (br, 1H),
7.92 (dd, J = 9.2, 3.0 Hz, 1H), 7.29 (t, J = 7.1 Hz, 1H), 7.17 (d, J = 7.4
Hz, 1H), 6.92 (d, J = 7.4 Hz, 1H), 6.85 (d, J = 9.0 Hz, 2H), 6.79 (d, J =
4.6 Hz, 1H), 4.52 (d, J = 5.7 Hz, 2H), 3.80 (s, 3H). MS (ESI) [M+H]+
series LC system (column, XBridge® C18, 4.6 mm × 150 mm, 5
μm;
mobile phase, methanol/buffer solution (3.56 g Na2HPO4 + 1.56 g
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T. Liang et al.
Bioorganic Chemistry 110 (2021) 104793
278.09.
4.1.13. Synthesis of 4-fluoro-N1-(3,4,5-trimethoxybenzyl)benzene-1,2-
diamine (5b)
4.1.4. Synthesis of N-benzyl-4-methoxy-2-nitroaniline (4c)
Orange solid (65.0%). m.p.: 94–95 ◦C. 1H NMR (300 MHz, CDCl3) δ
8.37 (s, 1H), 7.65 (d, J = 2.9 Hz, 1H), 7.42–7.28 (m, 5H), 7.09 (dd, J =
9.3, 2.8 Hz, 1H), 6.78 (d, J = 9.4 Hz, 1H), 4.55 (d, J = 5.8 Hz, 2H), 3.79
(s, 3H). MS (ESI) [M+H]+ 258.95.
To a solution of 4a (1.50 g, 4.46 mmol) in MeOH (25 mL) was added
10% Pd/C (0.23 g, 0.22 mmol), and the reaction mixture was stirred at
room temperature under H2 atmosphere. After completion, the mixture
was filtered with Celite, and the filtrate was concentrated under vacuum
to give 5b (0.95 g, 69.8%) as an orange solid. m.p.: 117–118 ◦C. 1H NMR
(300 MHz, CDCl3) δ 6.62 (s, 2H), 6.60–6.56 (m, 1H), 6.48 (d, J = 8.9 Hz,
2H), 4.18 (s, 2H), 3.85 (s, 9H), 3.56 (s, 2H), 3.30 (s, 1H). MS (ESI)
[M+H]+ 306.97.
4.1.5. Synthesis of N-(4-fluorobenzyl)-4-methoxy-2-nitroaniline (4d)
Red solid (78.5%). m.p.: 66–67 ◦C. 1H NMR (300 MHz, CDCl3) δ 8.33
(s, 1H), 7.65 (d, J = 2.9 Hz, 1H), 7.34–7.28 (m, 2H), 7.14–7.06 (m, 3H),
6.75 (d, J = 9.4 Hz, 1H), 4.52 (d, J = 5.7 Hz, 2H), 3.79 (s, 3H). MS (ESI)
[M+H]+ 275.13.
Compounds 5c–5k were synthesized following a similar method of
compound 5b.
4.1.14. Synthesis of 4-fluoro-N1-(3-methoxybenzyl)benzene-1,2-diamine
(5c)
4.1.6. Synthesis of N-(4-chlorobenzyl)-4-methoxy-2-nitroaniline (4e)
Red solid (75.3%). m.p.: 76–78 ◦C. 1H NMR (300 MHz, CDCl3) δ 8.34
(s, 1H), 7.65 (d, J = 3.0 Hz, 1H), 7.33 (d, J = 8.5 Hz, 2H), 7.27 (d, J =
6.5 Hz, 2H), 7.09 (dd, J = 9.3, 3.0 Hz, 1H), 6.71 (d, J = 9.3 Hz, 1H), 4.52
(d, J = 5.8 Hz, 2H), 3.79 (s, 3H). MS (ESI) [M+H]+ 293.45.
Reddish-brown solid (58.8%). m.p.: 108–109 ◦C. 1H NMR (300 MHz,
CDCl3) δ 7.29 (d, J = 7.8 Hz, 1H), 6.97 (d, J = 7.9 Hz, 1H), 6.94 (s, 1H),
6.84 (d, J = 8.2 Hz, 1H), 6.58 (dd, J = 8.3, 5.4 Hz, 1H), 6.53–6.38 (m,
2H), 4.23 (s, 2H), 3.81 (s, 3H), 3.57 (s, 2H), 3.33 (s, 1H). MS (ESI)
[M+H]+ 246.93.
4.1.7. Synthesis of 4-methoxy-N-(4-methoxybenzyl)-2-nitroaniline (4f)
Red solid (91.0%). m.p.: 75–76 ◦C. 1H NMR (300 MHz, CDCl3) δ 8.30
(s, 1H), 7.63 (d, J = 3.0 Hz, 1H), 7.27–7.26 (m, 1H), 7.25–7.23 (m, 1H),
7.09 (dd, J = 9.4, 3.0 Hz, 1H), 6.91–6.89 (m, 1H), 6.88–6.86 (m, 1H),
6.79 (d, J = 9.4 Hz, 1H), 4.46 (s, 2H), 3.80 (s, 3H), 3.78 (s, 3H). MS (ESI)
[M+H]+ 290.09.
4.1.15. Synthesis of N1-benzyl-4-methoxybenzene-1,2-diamine (5d)
Pale yellow oil (26.1%). 1H NMR (300 MHz, CDCl3) δ 7.44–7.27 (m,
5H), 6.63 (d, J = 8.5 Hz, 1H), 6.37 (s, 1H), 6.32 (d, J = 8.5 Hz, 1H), 4.24
(s, 2H), 3.73 (s, 3H), 3.55 (s, 2H), 3.19 (s, 1H). MS (ESI) [M+H]+
228.93.
4.1.8. Synthesis of 4-methoxy-N-(3-methoxybenzyl)-2-nitroaniline (4g)
Red solid (75.9%). m.p.: 65–66 ◦C. 1H NMR (300 MHz, CDCl3) δ 8.38
(s, 1H), 7.65 (s, 1H), 7.29 (t, J = 6.0 Hz, 1H), 7.09 (d, J = 9.4 Hz, 1H),
6.92 (d, J = 7.5 Hz, 1H), 6.87 (s, 1H), 6.83 (d, J = 8.1 Hz, 1H), 6.77 (d, J
= 9.4 Hz, 1H), 4.52 (d, J = 5.7 Hz, 2H), 3.80 (s, 3H), 3.79 (s, 3H). MS
(ESI) [M+H]+ 288.96.
4.1.16. Synthesis of N1-(4-fluorobenzyl)-4-methoxybenzene-1,2-diamine
(5e)
1
Red oil (35.8%). H NMR (300 MHz, CDCl3) δ 7.38–7.31 (m, 2H),
7.06–6.98 (m, 2H), 6.59 (d, J = 8.5 Hz, 1H), 6.37 (s, 1H), 6.32 (d, J =
8.4 Hz, 1H), 4.21 (s, 2H), 3.73 (s, 3H), 3.55 (s, 2H), 3.20 (s, 1H). MS
(ESI) [M+H]+ 246.94.
4.1.9. Synthesis of 4-methoxy-N-((6-methoxynaphthalen-2-yl)methyl)-2-
nitroaniline (4h)
4.1.17. Synthesis of N1-(4-chlorobenzyl)-4-methoxybenzene-1,2-diamine
(5f)
Red solid (74.0%). m.p.: 79–80 ◦C. 1H NMR (300 MHz, CDCl3) δ 8.45
(s, 1H), 7.77–7.61 (m, 4H), 7.41 (d, J = 8.5 Hz, 1H), 7.19–7.14 (m, 1H),
7.14 (s, 1H), 7.07 (d, J = 9.3 Hz, 1H), 6.83 (d, J = 9.4 Hz, 1H), 4.68 (s,
2H), 3.92 (s, 3H), 3.78 (s, 3H). MS (ESI) [M+H]+ 340.21.
White solid (34.9%). m.p.: 81–82 ◦C. 1H NMR (300 MHz, CDCl3) δ
7.31 (br, 4H), 6.56 (d, J = 8.5 Hz, 1H), 6.36 (s, 1H), 6.30 (d, J = 8.5 Hz,
1H), 4.22 (s, 2H), 3.73 (s, 3H), 3.55 (s, 2H), 3.22 (s, 1H). MS (ESI)
[M+H]+ 263.86.
4.1.10. Synthesis of N-benzyl-4-ethoxy-2-nitroaniline (4i)
4.1.18. Synthesis of 4-methoxy-N1-(4-methoxybenzyl)benzene-1,2-
diamine (5g)
Red solid (78.9%). m.p.: 65–66 ◦C. 1H NMR (300 MHz, CDCl3) δ 8.35
(s, 1H), 7.63 (s, 1H), 7.39–7.28 (m, 5H), 7.09 (d, J = 9.4 Hz, 1H), 6.77
(d, J = 9.3 Hz, 1H), 4.54 (d, J = 5.7 Hz, 2H), 3.99 (q, J = 7.0 Hz, 2H),
1.39 (t, J = 7.0 Hz, 3H). MS (ESI) [M+H]+ 274.12.
Pale pink solid (35.7%). m.p.: 87–88 ◦C. 1H NMR (300 MHz, CDCl3) δ
7.31 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 6.63 (d, J = 8.4 Hz,
1H), 6.36 (s, 1H), 6.33 (d, J = 8.4 Hz, 1H), 4.16 (s, 2H), 3.81 (s, 3H),
3.73 (s, 3H), 3.54 (s, 2H), 3.12 (s, 1H). MS (ESI) [M+H]+ 258.94.
4.1.11. Synthesis of 4-ethoxy-2-nitro-N-(3,4,5-trimethoxybenzyl)aniline
(4j)
4.1.19. Synthesis of 4-methoxy-N1-(3-methoxybenzyl)benzene-1,2-
diamine (5h)
Red solid (75.3%). m.p.: 100–101 ◦C. 1H NMR (300 MHz, CDCl3) δ
7.41 (s, 1H), 7.37 (s, 1H), 6.62 (s, 2H), 6.58 (d, J = 8.8 Hz, 1H), 6.40 (d,
J = 8.9 Hz, 1H), 3.94–3.91 (m, 4H), 3.84 (s, 3H), 3.76 (s, 3H), 3.69 (s,
6H). MS (ESI) [M+H]+ 362.07.
Red oil (45.2%). 1H NMR (300 MHz, CDCl3) δ 7.23 (t, J = 7.6 Hz,
1H), 6.97 (d, J = 6.0 Hz, 1H), 6.95 (s, 1H), 6.81 (d, J = 8.3 Hz, 1H), 6.60
(d, J = 8.4 Hz, 1H), 6.34 (s, 1H), 6.30 (d, J = 8.4 Hz, 1H), 4.19 (s, 2H),
3.78 (s, 3H), 3.71 (s, 3H), 3.42 (s, 3H). MS (ESI) [M+H]+ 258.93.
4.1.12. Synthesis of N1-(3,4,5-trimethoxybenzyl)benzene-1,2-diamine
(5a)
4.1.20. Synthesis of 4-methoxy-N1-((6-methoxynaphthalen-2-yl)methyl)
benzene-1,2-diamine (5i)
Following similar procedure to that described for preparing 4a,
compound 5a was obtained from benzene-1,2-diamine 3a (1.50 g,
13.87 mmol), anhydrous K2CO3 (4.60 g, 33.29 mmol), KI (2.30 g, 13.87
mmol) and 5-(chloromethyl)-1,2,3-trimethoxybenzene (6.01 g, 27.74
mmol) as a brown solid (54.3%). m.p.: 123–125 ◦C. 1H NMR (300 MHz,
CDCl3) δ 6.82 (td, J = 7.2, 1.2 Hz, 1H), 6.76–6.67 (m, 3H), 6.64 (s, 2H),
4.24 (s, 2H), 3.85 (s, 9H). HRMS (ESI) m/z: calcd for C16H20N2O3Na+
[M + Na]+ 311.13661, found 311.13608.
Reddish solid (60.6%). m.p.: 84–85 ◦C. 1H NMR (300 MHz, CDCl3) δ
7.79–7.69 (m, 3H), 7.49 (d, J = 8.3 Hz, 1H), 7.15 (d, J = 7.0 Hz, 2H),
6.67 (d, J = 8.5 Hz, 1H), 6.38 (s, 1H), 6.32 (d, J = 8.5 Hz, 1H), 4.37 (s,
2H), 3.93 (s, 3H), 3.73 (s, 3H), 3.56 (s, 2H), 3.20 (s, 1H). MS (ESI) [M +
1]+ 309.03.
4.1.21. Synthesis of N1-benzyl-4-ethoxybenzene-1,2-diamine (5j)
Reddish-brown oil (37.2%). 1H NMR (300 MHz, CDCl3) δ 7.42–7.27
(m, 5H), 6.62 (d, J = 8.6 Hz, 1H), 6.37 (s, 1H), 6.32 (d, J = 8.4 Hz, 1H),
8

T. Liang et al.
Bioorganic Chemistry 110 (2021) 104793
4.24 (s, 2H), 3.95 (q, J = 6.9 Hz, 2H), 3.53 (s, 2H), 3.19 (s, 1H), 1.37 (t, J
= 7.0 Hz, 3H). MS (ESI) [M+H]+ 242.94.
4.1.30. Synthesis of 6-methoxy-1-(3-methoxybenzyl)-1,4-dihydroquinoxa-
line-2,3-dione (6h)
White solid (59.3%). m.p.: 175–176 ◦C. 1H NMR (300 MHz, CDCl3) δ
11.36 (s, 1H), 7.25–7.19 (m, 1H), 7.07 (d, J = 8.8 Hz, 1H), 6.95–6.77 (m,
4H), 6.69 (d, J = 8.6 Hz, 1H), 5.41 (s, 2H), 3.81 (s, 3H), 3.77 (s, 3H). MS
(ESI) [M+H]+ 313.00.
4.1.22. Synthesis of 4-ethoxy-N1-(3,4,5-trimethoxybenzyl)benzene-1,2-
diamine (5k)
Red solid (73.8%). m.p.: 127–128 ◦C. 1H NMR (300 MHz, CDCl3) δ
6.64–6.60 (m, 3H), 6.38 (d, J = 2.6 Hz, 1H), 6.33 (dd, J = 8.5, 2.6 Hz,
1H), 4.16 (s, 2H), 3.95 (q, J = 7.0 Hz, 2H), 3.85 (s, 6H), 3.85 (s, 3H),
3.53 (s, 2H), 3.18 (s, 1H), 1.37 (t, J = 7.0 Hz, 3H). MS (ESI) [M+H]+
333.09.
4.1.31. Synthesis of 6-methoxy-1-((6-methoxynaphthalen-2-yl)methyl)-
1,4-dihydroquinoxaline-2,3-dione (6i)
White solid (44.8%). m.p.: 137–138 ◦C. 1H NMR (300 MHz, CDCl3) δ
11.74 (s, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.63 (s,
1H), 7.38 (d, J = 8.4 Hz, 1H), 7.12 (d, J = 9.4 Hz, 2H), 7.09 (s, 1H), 6.90
(s, 1H), 6.66 (d, J = 9.1 Hz, 1H), 5.55 (s, 2H), 3.89 (s, 3H), 3.76 (s, 3H).
MS (ESI) [M+H] + 361.03.
4.1.23. Synthesis of 1-(3,4,5-trimethoxybenzyl)-1,4-dihydroquinoxaline-
2,3-dione (6a)
5a (1.00 g, 3.47 mmol) and dimethyl oxalate (4.10 g, 34.68 mmol)
were added to 100 mL round-bottom flask, and the reaction mixture was
stirred at 70 ◦C for 5 h. Then the mixture was cooled to room temper-
ature, diluted with anhydrous ether (50 mL) and stirred for 30 min, then
the precipitate was filtered to obtain 6a (0.92 g, 77.3%) as a gray solid.
m.p.: 234–236 ◦C. 1H NMR (300 MHz, CDCl3) δ 11.35 (br, 1H), 7.37 (dd,
J = 7.2, 2.1 Hz, 1H), 7.27–7.26 (m, 3H), 6.54 (s, 2H), 5.40 (s, 2H), 3.81
(s, 9H). HRMS (ESI) m/z: calcd for C18H19N2O5Na+ [M + Na]+
365.11079, found 365.11108.
4.1.32. Synthesis of 1-benzyl-6-ethoxy-1,4-dihydroquinoxaline-2,3-dione
(6j)
White solid (53.8%). m.p.: 193–195 ◦C. 1H NMR (300 MHz, CDCl3) δ
11.92 (s, 1H), 7.39–7.26 (m, 5H), 7.04 (d, J = 9.2 Hz, 1H), 6.91 (s, 1H),
6.68 (d, J = 9.2 Hz, 1H), 5.44 (s, 2H), 4.01 (q, J = 6.5 Hz, 2H), 1.38 (t, J
= 6.7 Hz, 3H). MS (ESI) [M+H]+ 296.98.
Compounds 6b–6k were synthesized following a similar method of
compound 6a.
4.1.33. Synthesis of 6-ethoxy-1-(3,4,5-trimethoxybenzyl)-1,4-dihydroqui-
noxaline-2,3-dione (6k)
White solid (65.3%). m.p.: 258–260 ◦C. 1H NMR (300 MHz, CDCl3) δ
11.05 (s, 1H), 7.12 (d, J = 9.1 Hz, 1H), 6.81 (s, 1H), 6.73 (d, J = 9.1 Hz,
1H), 6.51 (s, 2H), 5.36 (s, 2H), 4.03 (q, J = 6.9 Hz, 2H), 3.81 (s, 9H),
1.41 (t, J = 7.0 Hz, 3H). MS (ESI) [M+H]+ 386.03.
4.1.24. Synthesis of 6-fluoro-1-(3,4,5-trimethoxybenzyl)-1,4-dihydroqui-
noxaline-2,3-dione (6b)
White solid (65.3%). m.p.: 223–224 ◦C. 1H NMR (300 MHz, CDCl3) δ
11.55 (s, 1H), 7.22–7.12 (m, 2H), 6.94–6.87 (m, 1H), 6.51 (s, 2H), 5.38
(s, 2H), 3.82 (s, 9H). MS (ESI) [M+H]+ 362.24.
4.1.34. Synthesis of 1-(3,4,5-trimethoxybenzyl)-1,2,3,4-
tetrahydroquinoxaline (7a)
4.1.25. Synthesis of 6-fluoro-1-(3-methoxybenzyl)-1,4-dihydroquinoxa-
line-2,3-dione (6c)
In a two-necked flask equipped with a dropping funnel under ni-
trogen, 6a (0.85 g, 2.48 mmol) was dissolved in anhydrous THF (15 mL),
and the mixture was cooled to 0 ◦C and stirred for 15 min. Then borane-
tetrahydrofuran complex (1 mol /L, 4.90 mL) was added to the mixture
dropwise during 30 min. The mixture reaction was then stirred at room
temperature overnight. Then the mixture was cooled to 0 ◦C and water
was added until no gas was formed and stirred for 20 min. The mixture
was extracted with DCM, and the organic layer was dried over anhy-
drous Na2SO4, filtered. The filtrate was concentrated and purified by
chromatography (PE:EA = 10:1) to afford 7a (0.64 g, 82.3%) as a white
solid. HPLC purity: 95.54%, retention time: 8.23 min. m.p.: 109–111 ◦C.
1H NMR (300 MHz, CDCl3) δ 6.64–6.57 (m, 3H), 6.54 (s, 3H), 4.35 (s,
2H), 3.84 (s, 3H), 3.82 (s, 6H), 3.44 (t, J = 4.8 Hz, 2H), 3.36 (t, J = 4.8
Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 153.6, 137.0, 135.7, 134.7, 134.2,
119.3, 118.2, 114.2, 112.3, 103.9, 61.0, 56.3, 55.9, 48.3, 41.3. HRMS
(ESI) m/z: calcd for C18H22N2O3Na+ [M+H]+ 337.15226, found
337.15244.
White solid (83.3%). m.p.: 174–175 ◦C. 1H NMR (300 MHz, CDCl3) δ
11.84 (s, 1H), 7.28 (d, J = 7.0 Hz, 1H), 7.19–7.09 (m, 2H), 6.86–6.81 (m,
4H), 5.43 (s, 2H), 3.77 (s, 3H). MS (ESI) [M+H]+ 302.12.
4.1.26. Synthesis of 1-benzyl-6-methoxy-1,4-dihydroquinoxaline-2,3-
dione (6d)
White solid (65.8%). m.p.: 142–143 ◦C. 1H NMR (300 MHz, CDCl3) δ
11.13 (s, 1H), 7.38–7.27 (m, 5H), 7.07 (d, J = 9.2 Hz, 1H), 6.82 (s, 1H),
6.70 (d, J = 9.1 Hz, 1H), 5.45 (s, 2H), 3.81 (s, 3H). MS (ESI) [M+H]+
281.98.
4.1.27. Synthesis of 1-(4-fluorobenzyl)-6-methoxy-1,4-
dihydroquinoxaline-2,3-dione (6e)
White solid (78.2%). m.p.: 152–153 ◦C. 1H NMR (300 MHz, CDCl3) δ
11.61 (s, 1H), 7.29 (d, J = 8.5 Hz, 2H), 7.03 (d, J = 8.5 Hz, 2H), 7.00 (d,
J = 8.8 Hz, 1H), 6.91 (s, 1H), 6.70 (d, J = 8.8 Hz, 1H), 5.39 (s, 2H), 3.81
(s, 3H). MS (ESI) [M+H]+ 300.96.
Compounds 7b–7k were synthesized following a similar method of
compound 7a.
4.1.28. Synthesis of 1-(4-chlorobenzyl)-6-methoxy-1,4-
dihydroquinoxaline-2,3-dione (6f)
4.1.35. Synthesis of 6-fluoro-1-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahy-
droquinoxaline (7b)
White solid (77.5%). m.p.: 125–126 ◦C. 1H NMR (300 MHz, CDCl3) δ
11.45 (s, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 7.00 (d,
J = 9.1 Hz, 1H), 6.90 (s, 1H), 6.69 (d, J = 8.6 Hz, 1H), 5.39 (s, 2H), 3.81
(s, 3H). MS (ESI) [M+H]+ 318.20.
Colorless oil (79.3%). HPLC purity: 96.05%, retention time: 8.04
min. 1H NMR (300 MHz, CDCl3) δ 6.54 (s, 2H), 6.46 (dd, J = 8.4, 5.5 Hz,
1H), 6.31–6.20 (m, 2H), 4.26 (s, 2H), 3.91 (br, 1H), 3.84 (s, 3H), 3.82 (s,
6H), 3.43 (s, 2H), 3.24 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 158.13,
155.02, 153.47, 136.78, 135.58 (d, JF-C = 10.2 Hz), 134.45, 131.47 (d,
JF-C = 1.7 Hz), 112.99 (d, JF-C = 9.0 Hz), 103.92, 103.56, 100.78 (d, JF-C
= 25.9 Hz), 60.89, 56.47, 56.11, 47.53, 41.09. HRMS (ESI) m/z calcd for
4.1.29. Synthesis of 6-methoxy-1-(4-methoxybenzyl)-1,4-dihydroquinoxa-
line-2,3-dione (6g)
White solid (69.2%). m.p.: 166–167 ◦C. 1H NMR (300 MHz, CDCl3) δ
11.06 (s, 1H), 7.22 (d, J = 8.6 Hz, 2H), 7.12 (d, J = 9.2 Hz, 1H), 6.85 (d,
J = 8.6 Hz, 2H), 6.80 (s, 1H), 6.71 (d, J = 9.2 Hz, 1H), 5.38 (s, 2H), 3.81
(s, 3H), 3.77 (s, 3H). MS (ESI) [M+H]+ 312.96.
C
18H22FN2O+3 [M+H]+ 333.16090, found 333.16098.
4.1.36. Synthesis of 6-fluoro-1-(3-methoxybenzyl)-1,2,3,4-tetrahydroqui-
noxaline (7c)
Colorless oil (61.4%). HPLC purity: 95.12%, retention time: 9.28
min. 1H NMR (300 MHz, CDCl3) δ 7.29–7.24 (m, 1H), 6.91 (d, J = 8.3
9

T. Liang et al.
Bioorganic Chemistry 110 (2021) 104793
Hz, 1H), 6.87 (s, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.44–6.38 (t, J = 9.0 Hz,
1H), 6.28 (d, J = 7.2 Hz, 1H), 6.23 (s, 1H), 4.33 (s, 2H), 3.84 (br, 1H),
3.79 (s, 3H), 3.45 (t, J = 4.0 Hz, 2H), 3.30 (t, J = 4.0 Hz, 2H). 13C NMR
6.35–6.03 (m, 2H), 4.45 (s, 2H), 3.92 (s, 3H), 3.71 (s, 3H), 3.45 (s, 2H),
3.28 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 157.62, 153.07, 133.93,
129.32, 129.10, 127.26, 126.47, 125.97, 118.93, 105.89, 55.70, 55.46,
48.07, 41.24, 29.85. HRMS (ESI) m/z calcd for C21H23N2O+2 [M+H]+
335.17540, found 335.17529.
(75 MHz, CDCl3) δ 160.02, 158.00, 154.90, 140.56, 135.44 (d, JF-C
=
10.1 Hz), 131.51 (d, JF-C = 1.7 Hz), 129.75, 119.56, 112.88, 112.69 (d,
JF-C = 9.1 Hz), 103.80 (d, JF-C = 21.8 Hz), 100.84 (d, JF-C = 25.9 Hz),
56.12, 55.25, 47.88, 41.11. HRMS (ESI) m/z calcd for C16H18FN2O+
[M+H]+ 273.13977, found 273.13995.
4.1.43. Synthesis of 1-benzyl-6-ethoxy-1,2,3,4-tetrahydroquinoxaline (7j)
Colorless oil (25.5%). HPLC purity: 96.89%, retention time: 7.96
1
min. H NMR (300 MHz, CDCl3) δ 7.36–7.30 (m, 4H), 7.26–7.19 (m,
4.1.37. Synthesis of 1-benzyl-6-methoxy-1,2,3,4-tetrahydroquinoxaline
(7d)
1H), 6.46 (d, J = 8.8 Hz, 1H), 6.24–6.09 (m, 2H), 4.33 (s, 2H), 3.90 (q, J
= 6.8 Hz, 2H), 3.41 (s, 2H), 3.25 (s, 2H), 1.34 (t, J = 7.0 Hz, 3H). 13
C
White solid (64.3%). HPLC purity: 95.26%, retention time: 8.11 min.
m.p.: 89–91 ◦C. 1H NMR (300 MHz, CDCl3) δ 7.36–7.29 (m, 4H),
7.26–7.20 (m, 1H), 6.48 (d, J = 8.7 Hz, 1H), 6.30–6.08 (m, 2H), 4.34 (s,
2H), 3.70 (s, 3H), 3.44 (s, 2H), 3.27 (s, 2H). 13C NMR (75 MHz, CDCl3) δ
152.94, 139.16, 135.60, 129.83, 128.65, 127.48, 127.00, 113.55,
102.96, 101.07, 56.52, 55.63, 48.06, 41.20. HRMS (ESI) m/z calcd for
NMR (75 MHz, CDCl3) δ 152.10, 139.13, 135.42, 129.78, 128.60,
127.41, 126.94, 113.49, 103.89, 101.74, 63.77, 56.43, 48.04, 41.18,
15.15. HRMS (ESI) m/z calcd for C17H21N2O+ [M+H]+ 269.16539,
found 269.16540.
4.1.44. Synthesis of 6-ethoxy-1-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahy-
droquinoxaline (7k)
C
16H19N2O+ [M+H]+ 255.14919, found 255.14946.
Colorless oil (63.8%). HPLC purity: 97.28%, retention time: 7.78
min. 1H NMR (300 MHz, CDCl3) δ 6.57 (s, 2H), 6.53 (d, J = 8.7 Hz, 1H),
6.34–6.10 (m, 2H), 4.25 (s, 2H), 4.01–3.88 (m, 2H), 3.84 (s, 3H), 3.83 (s,
6H), 3.43 (s, 2H), 3.22 (s, 2H), 1.35 (t, J = 7.0 Hz, 3H). 13C NMR (75
MHz, CDCl3) δ 153.42, 152.33, 136.80, 135.60, 134.91, 129.75, 113.85,
104.13, 103.95, 101.68, 63.78, 60.89, 56.88, 56.14, 47.77, 41.14,
15.06. HRMS (ESI) m/z calcd for C20H27N2O+4 [M+H]+ 359.19653,
found 359.19681.
4.1.38. Synthesis of 1-(4-fluorobenzyl)-6-methoxy-1,2,3,4-
tetrahydroquinoxaline (7e)
White solid (26.5%). HPLC purity: 97.21%, retention time: 8.82 min.
m.p.: 90–91 ◦C. 1H NMR (300 MHz, CDCl3) δ 7.33–7.25 (m, 2H), 7.00 (t,
J = 8.6 Hz, 2H), 6.45 (d, J = 8.8 Hz, 1H), 6.22–6.11 (m, 2H), 4.29 (s,
2H), 3.79 (s, 1H), 3.69 (s, 3H), 3.42 (s, 2H), 3.24 (s, 2H). 13C NMR (75
MHz, CDCl3) δ 163.58, 160.34, 153.00, 135.60, 129.59, 128.90 (d, JF-C
= 7.9 Hz), 115.43 (d, JF-C = 21.3 Hz), 113.59, 102.96, 101.06, 55.89,
55.63, 48.05, 41.19. HRMS (ESI) m/z calcd for C16H18FN2O+ [M+H]+
273.13977, found 273.13995.
4.1.45. Synthesis of tert-butyl 4-benzyl-7-methoxy-3,4-
dihydroquinoxaline-1(2H)-carboxylate (8)
To a solution of 7d (1.00 g, 3.92 mmol) in DCM (25 mL) was added
di-tert-butyl dicarbonate (1.72 g, 7.86 mmol) and TEA (0.80 g, 7.86
mmol), the mixture reaction was stirred at 40 ◦C for 15 h. Then the
reaction was cooled to room temperature, washed with water and dried
over anhydrous Na2SO4, filtered. The filtrate was concentrated and
purified by chromatography (PE:EA = 20:1) to afford 8 (0.90 g, 71.8%)
as a white solid. m.p.: 103–104 ◦C. 1H NMR (300 MHz, CDCl3) δ
7.38–7.26 (m, 4H), 7.25–7.15 (m, 2H), 6.60–6.51 (m, 2H), 4.45 (s, 2H),
3.83 (t, J = 5.2 Hz, 2H), 3.74 (s, 3H), 3.34 (t, J = 5.1 Hz, 2H), 1.54 (s,
9H). MS (ESI) [M+H]+ 355.11.
4.1.39. Synthesis of 1-(4-chlorobenzyl)-6-methoxy-1,2,3,4-
tetrahydroquinoxaline (7f)
White solid (27.8%). HPLC purity: 97.23%, retention time: 8.80 min.
m.p.: 108–110 ◦C. 1H NMR (300 MHz, CDCl3) δ 7.33–7.26 (m, 4H), 6.41
(d, J = 8.8 Hz, 1H), 6.23–6.09 (m, 2H), 4.30 (s, 2H), 3.77 (s, 1H), 3.70 (s,
3H), 3.44 (s, 2H), 3.26 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 153.04,
137.67, 135.55, 132.56, 129.41, 128.73, 113.57, 102.99, 101.06, 55.96,
55.58, 48.18, 41.14. HRMS (ESI) m/z calcd for C16H18ClN2O+ [M+H]+
289.11022, found 289.11069.
4.1.40. Synthesis of 6-methoxy-1-(4-methoxybenzyl)-1,2,3,4-tetrahydro-
quinoxaline (7g)
4.1.46. Synthesis of tert-butyl 7-methoxy-3,4-dihydroquinoxaline-1(2H)-
carboxylate (9)
Colorless oil (33.0%). HPLC purity: 95.97%, retention time: 8.48
min. 1H NMR (300 MHz, CDCl3) δ 7.24 (d, J = 8.6 Hz, 2H), 6.86 (d, J =
8.6 Hz, 2H), 6.51 (d, J = 8.7 Hz, 1H), 6.29–6.07 (m, 2H), 4.26 (s, 2H),
3.79 (s, 3H), 3.69 (s, 3H), 3.40 (s, 2H), 3.23 (s, 2H). 13C NMR (75 MHz,
CDCl3) δ 158.67, 152.88, 135.63, 130.94, 129.86, 128.69, 113.98,
113.63, 102.92, 100.97, 55.82, 55.58, 55.34, 47.64, 41.16. HRMS (ESI)
m/z calcd for C17H21N2O+2 [M+H]+ 285.15975, found 285.15967.
10% Pd/C (0.13 g, 0.12 mmol) was added to the solution of 8 (0.90 g,
2.54 mmol) in methanol (40 mL) and the mixture was stirred at room
temperature under H2 atmosphere. After completion, the mixture was
filtrated with Celite, and the filtrate was concentrated under vacuum
and purified by chromatography (PE:EA = 9:1) to give 9 (0.58 g, 86.4%)
as a white solid. m.p.: 70–72 ◦C. 1H NMR (300 MHz, CDCl3) δ 7.23 (s,
1H), 6.57–6.48 (m, 2H), 3.78 (t, J = 5.0 Hz, 2H), 3.74 (s, 3H), 3.36 (t, J
= 5.0 Hz, 2H), 1.53 (s, 9H). MS (ESI) [M+H]+ 264.11.
4.1.41. Synthesis of 6-methoxy-1-(3-methoxybenzyl)-1,2,3,4-tetrahydro-
quinoxaline (7h)
4.1.47. Synthesis of tert-butyl 7-methoxy-4-(3,4,5-trimethoxybenzoyl)-
3,4-dihydroquinoxaline-1(2H)-carboxylate (10a)
Colorless oil (32.1%). HPLC purity: 96.17%, retention time: 8.47
min. 1H NMR (300 MHz, CDCl3) δ 7.22 (d, J = 7.7 Hz, 1H), 6.94–6.87
(m, 2H), 6.79 (d, J = 8.2 Hz, 1H), 6.47 (d, J = 8.8 Hz, 1H), 6.29–6.02 (m,
2H), 4.30 (s, 2H), 3.78 (s, 3H), 3.69 (s, 3H), 3.41 (s, 2H), 3.25 (s, 2H).
13C NMR (75 MHz, CDCl3) δ 159.92, 152.81, 140.90, 140.84, 135.47,
129.58, 119.67, 113.47, 112.85, 112.26, 102.90, 100.95, 56.46, 55.57,
55.23, 48.02, 41.15. HRMS (ESI) m/z calcd for C17H21N2O+2 [M+H]+
285.15975, found 285.15976.
To 9 (0.11 g, 0.42 mmol) and 4-dimethylaminopyridine (0.078 g,
0.64 mmol) in anhydrous DCM (5 mL) was added 3,4,5-trimethoxyben-
zoyl chloride (0.15 g, 0.64 mmol) in DCM (2 mL), and the mixture was
stirred at room temperature for 3 h. After completion, the mixture was
washed with 2 N HCl, water and dried over anhydrous Na2SO4, filtered.
The filtrate was concentrated and purified by chromatography (PE:EA =
6:1) to afford 10a (0.16 g, 85.2%) as a yellow oil. 1H NMR (300 MHz,
CDCl3) δ 7.41 (s, 1H), 6.62 (s, 2H), 6.58 (d, J = 9.4 Hz, 1H), 6.39 (d, J =
8.8 Hz, 1H), 4.05 (t, J = 6.3 Hz, 2H), 3.93 (t, J = 6.4 Hz, 2H), 3.84 (s,
3H), 3.76 (s, 3H), 3.69 (s, 6H), 1.55 (s, 9H). MS (ESI) [M+H]+ 459.13.
Compounds 10b–10l were synthesized following a similar method of
compound 10a.
4.1.42. Synthesis of 6-methoxy-1-((6-methoxynaphthalen-2-yl)methyl)-
1,2,3,4-tetrahydroquinoxaline (7i)
White solid (48.7%). HPLC purity: 95.63%, retention time: 10.68
min. m.p.: 116–117 ◦C. 1H NMR (300 MHz, CDCl3) δ 7.74–7.67 (m, 3H),
7.43 (d, J = 8.4 Hz, 1H), 7.16–7.11 (m, 2H), 6.56 (d, J = 8.7 Hz, 1H),
10

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Bioorganic Chemistry 110 (2021) 104793
4.1.48. Synthesis of tert-butyl 4-(3,5-dimethoxybenzoyl)-7-methoxy-3,4-
dihydroquinoxaline-1(2H)-carboxylate (10b)
1.57 (s, 9H). MS (ESI) [M+H]+ 516.24.
Colorless oil (73.6%). 1H NMR (300 MHz, CDCl3) δ 7.46 (s, 1H),
6.78–6.59 (m, 1H), 6.54–6.49 (m, 2H), 6.47–6.43 (m, 1H), 6.38 (d, J =
9.1 Hz, 1H), 4.01 (t, J = 5.5 Hz, 2H), 3.91 (t, J = 5.5 Hz, 2H), 3.76 (s,
3H), 3.69 (s, 6H), 1.56 (s, 9H). MS (ESI) [M+H]+ 429.19.
4.1.57. Synthesis of tert-butyl 4-(cyclohexanecarbonyl)-7-methoxy-3,4-
dihydroquinoxaline-1(2H)-carboxylate (10k)
White solid (87.3%). m.p.: 80–81 ◦C. 1H NMR (300 MHz, CDCl3) δ
6.91 (d, J = 8.0 Hz, 1H), 6.26–6.19 (m, 2H), 3.90 (t, J = 5.2 Hz, 2H),
3.71 (s, 3H), 3.39 (t, J = 5.2 Hz, 2H), 2.93 (t, J = 11.0 Hz, 1H), 1.93–1.44
(m, 10H), 1.56 (s, 9H). MS (ESI) [M+H]+ 375.22.
4.1.49. Synthesis of tert-butyl 4-(4-chloro-3-nitrobenzoyl)-7-methoxy-3,4-
dihydroquinoxaline-1(2H)-carboxylate (10c)
Yellow solid (86.4%). m.p.: 61–64 ◦C. 1H NMR (300 MHz, CDCl3) δ
8.02 (s, 1H), 7.55 (s, 1H), 7.45–7.38 (m, 2H), 6.50–6.31 (m, 2H), 4.06 (t,
J = 6.2 Hz, 2H), 3.94 (t, J = 6.1 Hz, 2H), 3.77 (s, 3H), 1.58 (s, 9H). MS
(ESI) [M+H]+ 448.94.
4.1.58. Synthesis of tert-butyl 4-(adamantane-1-carbonyl)-7-methoxy-3,4-
dihydroquinoxaline-1(2H)-carboxylate (10l)
White solid (89.5%). m.p.: 143–145 ◦C. 1H NMR (300 MHz, CDCl3) δ
7.18–7.13 (m, 1H), 6.23–6.18 (m, 2H), 3.98 (t, J = 5.3 Hz, 2H), 3.70 (s,
3H), 3.41 (t, J = 5.4 Hz, 2H), 2.06–1.96 (m, 11H), 1.94–1.91 (m, 1H),
1.75–1.70 (m, 2H), 1.65–1.60 (m, 1H), 1.57 (s, 9H). MS (ESI) [M+H]+
427.27.
4.1.50. Synthesis of tert-butyl 4-(benzo[d][1,3]dioxole-5-carbonyl)-7-
methoxy-3,4-dihydroquinoxaline-1(2H)-carboxylate (10d)
Yellow oil (69.8%). 1H NMR (300 MHz, CDCl3) δ 7.58–7.48 (m, 1H),
6.96–6.90 (m, 2H), 6.69 (d, J = 8.4 Hz, 1H), 6.55 (d, J = 8.9 Hz, 1H),
6.37 (dd, J = 8.9, 2.7 Hz, 1H), 5.97 (s, 2H), 3.99 (d, J = 6.0 Hz, 2H), 3.90
(t, J = 5.8 Hz, 2H), 3.77 (s, 3H), 1.57 (s, 9H). MS (ESI) [M+H]+ 413.19.
4.1.59. Synthesis of (6-methoxy-3,4-dihydroquinoxalin-1(2H)-yl)(3,4,5-
trimethoxyphenyl)methanone (11a)
To a solution of 10a (0.060 g, 0.14 mmol) in DCM (5 mL) was added
trifluoroacetic acid (51 μL, 0.69 mmol), and the mixture was stirred
4.1.51. Synthesis of tert-butyl 4-(2,3-dichloro-1-methyl-1H-indole-5-
carbonyl)-7-methoxy-3,4-dihydroquinoxaline-1(2H)-carboxylate (10e)
White solid (78.6%). m.p.: 173–174 ◦C. 1H NMR (300 MHz, CDCl3) δ
7.80 (s, 1H), 7.53 (s, 1H), 7.20 (d, J = 7.1 Hz, 1H), 7.10 (d, J = 8.5 Hz,
1H), 6.49 (d, J = 8.2 Hz, 1H), 6.27 (d, J = 9.0 Hz, 1H), 4.06 (t, J = 6.1
Hz, 2H), 3.94 (t, J = 6.3 Hz, 2H), 3.74 (s, 3H), 3.73 (s, 3H), 1.59 (s, 9H).
MS (ESI) [M+H]+ 455.99.
overnight at room temperature. After completion, the mixture was
washed with water and brine, and then dried over Na2SO4, filtered. The
filtrate was concentrated and purified by chromatography (PE:EA = 6:1)
to afford 11a (0.044 g, 89.8%) as a white solid. HPLC purity: 95.80%,
retention time: 6.97 min. m.p.: 115–117 ◦C. 1H NMR (300 MHz, CDCl3) δ
6.68 (s, 2H), 6.65–6.24 (m, 1H), 6.14 (d, J = 2.7 Hz, 1H), 6.02 (d, J =
8.9 Hz, 1H), 3.96 (t, J = 5.2 Hz, 2H), 3.85 (s, 3H), 3.74 (s, 6H), 3.71 (s,
3H), 3.54 (t, J = 5.2 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 157.83,
152.79, 139.61, 137.92, 130.71, 125.93, 118.49, 106.34, 102.31,
100.10, 99.38, 60.93, 56.22, 56.15, 55.32, 42.72. HRMS (ESI) m/z calcd
for C19H23N2O+5 [M+H]+ 359.16015, found 359.16052.
4.1.52. Synthesis of tert-butyl 7-methoxy-4-(6-methoxy-2-naphthoyl)-3,4-
dihydroquinoxaline-1(2H)-carboxylate (10f)
Colorless oil (79.0%). 1H NMR (300 MHz, CDCl3) δ 7.99 (s, 1H), 7.72
(d, J = 9.0 Hz, 1H), 7.62–7.54 (m, 2H), 7.34 (d, J = 8.4 Hz, 1H), 7.15
(dd, J = 8.9, 2.4 Hz, 1H), 7.11–7.06 (m, 1H), 6.52 (d, J = 4.7 Hz, 1H),
6.25 (dd, J = 9.3, 2.7 Hz, 1H), 4.08 (t, J = 6.3 Hz, 2H), 3.96 (t, J = 6.1
Hz, 2H), 3.91 (s, 3H), 3.74 (s, 3H), 1.60 (s, 9H). MS (ESI) [M+H]+
450.18.
Compounds 11b–11l were synthesized following a similar method of
compound 11a.
4.1.60. Synthesis of (3,5-dimethoxyphenyl)(6-methoxy-3,4-
dihydroquinoxalin-1(2H)-yl)methanone (11b)
Brown oil (77.4%). HPLC purity: 96.65%, retention time: 7.15 min.
1H NMR (300 MHz, CDCl3) δ 6.57 (d, J = 2.3 Hz, 2H), 6.46 (t, J = 1.7 Hz,
1H), 6.12 (d, J = 2.6 Hz, 1H), 6.03 (s, 1H), 3.92 (d, J = 3.0 Hz, 2H), 3.72
(s, 6H), 3.70 (s, 3H), 3.51 (s, 1H), 3.49 (d, J = 3.0 Hz, 2H). 13C NMR (75
MHz, CDCl3) δ 160.61, 157.87, 138.04, 137.96, 125.88, 118.31, 118.20,
106.41, 102.55, 102.46, 99.45, 55.57, 55.37, 42.76, 29.80. HRMS (ESI)
m/z calcd for C18H21N2O+4 [M+H]+ 329.14958, found 329.14984.
4.1.53. Synthesis of tert-butyl 7-methoxy-4-(quinoline-6-carbonyl)-3,4-
dihydroquinoxaline-1(2H)-carboxylate (10g)
1
Colorless oil (72.0%). H NMR (300 MHz, CDCl3) δ 8.45–8.29 (m,
3H), 8.20–8.07 (m, 2H), 8.05–8.00 (m, 1H),7.99 (d, J = 8.7 Hz, 1H),
7.70 (dd, J = 7.9, 3.4 Hz, 1H), 7.48 (d, J = 7.4 Hz, 1H), 4.07 (t, J = 6.2
Hz, 2H), 3.97 (t, J = 6.2 Hz, 2H), 3.85 (s, 3H), 1.62 (s, 9H).
4.1.54. Synthesis of tert-butyl 7-methoxy-4-(quinoline-5-carbonyl)-3,4-
dihydroquinoxaline-1(2H)-carboxylate (10h)
4.1.61. Synthesis of (4-chloro-3-nitrophenyl)(6-methoxy-3,4-
dihydroquinoxalin-1(2H)-yl)methanone (11c)
Yellow oil (72.3%). 1H NMR (300 MHz, CDCl3) δ 8.95 (dd, J = 4.1,
1.6 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 8.02 (d, J = 1.6 Hz, 1H), 7.98 (d, J
= 8.8 Hz, 1H), 7.62 (dd, J = 8.8, 1.6 Hz, 1H), 7.60–7.56 (m, 1H), 7.43
(dd, J = 8.3, 4.2 Hz, 1H), 6.59–6.33 (m, 1H), 6.24 (dd, J = 8.9, 2.1 Hz,
1H), 4.10 (t, J = 6.2 Hz, 2H), 3.97 (t, J = 6.2 Hz, 2H), 3.74 (s, 3H), 1.60
(s, 9H). MS (ESI) [M+H]+ 420.12.
Yellow solid (78.3%). HPLC purity: 97.45%, retention time: 8.32
min. m.p.: 71–73 ◦C. 1H NMR (300 MHz, CDCl3) δ 7.99 (d, J = 1.9 Hz,
1H), 7.52 (d, J = 8.3 Hz, 1H), 7.46 (d, J = 8.3 Hz, 1H), 6.65–6.17 (m,
1H), 6.15 (d, J = 2.6 Hz, 1H), 6.03–5.89 (m, 1H), 3.97 (t, J = 5.7 Hz,
2H), 3.71 (s, 3H), 3.58 (t, J = 5.4 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ
164.41, 158.50, 147.54, 138.28, 135.82, 133.23, 131.51, 128.55,
126.31, 125.87, 117.23, 102.53, 99.60, 55.30, 42.63, 29.70. HRMS
4.1.55. Synthesis of tert-butyl 7-methoxy-4-(2-oxo-2H-chromene-3-
carbonyl)-3,4-dihydroquinoxaline-1(2H)-carboxylate (10i)
Yellow solid (75.7%). m.p.: 94–95 ◦C. 1H NMR (300 MHz, CDCl3) δ
7.87 (s, 1H), 7.64–7.47 (m, 3H), 7.34–7.25 (m, 2H), 6.73 (s, 1H), 6.35 (s,
1H), 4.03 (s, 2H), 3.94 (s, 2H), 3.74 (s, 3H), 1.57 (s, 9H). MS (ESI)
[M+H]+ 437.12.
(ESI) m/z calcd for
C
16H15ClN3O+4 [M+H]+ 348.07456, found
348.07483.
4.1.62. Synthesis of benzo[d][1,3]dioxol-5-yl(6-methoxy-3,4-
dihydroquinoxalin-1(2H)-yl)methanone (11d)
White oil (74.6%). HPLC purity: 96.32%, retention time: 7.20 min.
1H NMR (300 MHz, CDCl3) δ 6.98 (dd, J = 8.0, 1.6 Hz, 1H), 6.95 (d, J =
1.4 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H), 6.69–6.31 (m, 1H), 6.12 (d, J = 2.7
Hz, 1H), 6.01 (dd, J = 8.9, 2.3 Hz, 1H), 5.97 (s, 2H), 3.93 (t, J = 5.2 Hz,
2H), 3.70 (s, 3H), 3.51 (t, J = 5.2 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ
167.99, 157.80, 149.22, 147.41, 137.93, 129.79, 125.98, 123.85,
118.81, 109.51, 108.01, 102.67, 101.51, 99.47, 55.37, 42.92, 41.37.
4.1.56. Synthesis of tert-butyl 4-(6-bromo-2-oxo-2H-chromene-3-
carbonyl)-7-methoxy-3,4-dihydroquinoxaline-1(2H)-carboxylate (10j)
Yellow solid (82.5%). m.p.: 198–199 ◦C. 1H NMR (300 MHz, CDCl3) δ
7.78 (s, 1H), 7.70–7.57 (m, 3H), 7.23–7.12 (m, 1H), 6.77–6.63 (m, 1H),
6.42–6.26 (m, 1H), 4.11–3.97 (m, 2H), 3.97–3.88 (m, 2H), 3.75 (s, 3H),
11

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Bioorganic Chemistry 110 (2021) 104793
HRMS (ESI) m/z calcd for C17H17N2O+4 [M+H]+ 313.11828, found
4.1.68. Synthesis of 6-bromo-3-(6-methoxy-1,2,3,4-
313.11859.
tetrahydroquinoxaline-1-carbonyl)–2H-chromen-2-one (11j)
Yellow solid (79.8%). HPLC purity: 97.68%, retention time: 7.73
min. m.p.: 185–187 ◦C. 1H NMR (300 MHz, CDCl3) δ 7.90 (s, 1H),
7.67–7.59 (m, 2H), 7.17 (d, J = 8.6 Hz, 1H), 6.53 (d, J = 8.6 Hz, 1H),
6.12 (s, 1H), 5.90 (d, J = 8.3 Hz, 1H), 3.66 (s, 3H), 3.59 (s, 2H), 3.48 (s,
2H). 13C NMR (75 MHz, CDCl3) δ 162.95, 158.70, 156.48, 152.97,
142.02, 139.01, 135.27, 130.69, 127.60, 123.82, 119.79, 118.57,
117.29, 102.09, 99.41, 55.17, 50.91, 42.16, 40.04. HRMS (ESI) m/z
calcd for C19H16BrN2O+4 [M+H]+ 415.02880, found 415.02899.
4.1.63. Synthesis of (2,3-dichloro-1-methyl-1H-indol-5-yl)(6-methoxy-
3,4-dihydroquinoxalin-1(2H)-yl)methanone (11e)
Yellow solid (72.6%). HPLC purity: 98.10%, retention time: 9.53
min. m.p.: 157–158 ◦C. 1H NMR (300 MHz, CDCl3) δ 7.78 (s, 1H), 7.29
(d, J = 8.2 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 6.79–6.23 (m, 1H), 6.14 (d,
J = 2.7 Hz, 1H), 5.91 (d, J = 8.9 Hz, 1H), 3.98 (t, J = 5.2 Hz, 2H), 3.73 (s,
3H), 3.68 (s, 3H), 3.54 (t, J = 5.2 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ
169.08, 157.69, 138.00, 135.30, 128.74, 126.10, 124.23, 124.09,
123.84, 119.41, 119.01, 108.97, 103.40, 102.57, 99.48, 55.38, 42.95,
30.69, 29.83. HRMS (ESI) m/z calcd for C19H18Cl2N3O+2 [M+H]+
390.07706, found 390.07745.
4.1.69. Synthesis of cyclohexyl(6-methoxy-3,4-dihydroquinoxalin-1(2H)-
yl) methanone (11k)
White solid (63.6%). HPLC purity: 98.63%, retention time: 8.64 min.
m.p.: 104–105 ◦C. 1H NMR (300 MHz, CDCl3) δ 6.89–6.75 (m, 1H), 6.15
(dd, J = 8.7, 2.7 Hz, 1H), 6.07 (d, J = 2.4 Hz, 1H), 4.10 (br, 1H), 3.71 (t,
J = 5.0 Hz, 2H), 3.68 (s, 3H), 3.38–3.25 (m, 2H), 2.94–2.78 (m, 1H),
1.71–1.61 (m, 4H), 1.59–1.39 (m, 4H), 1.17–1.10 (m, 2H). 13C NMR (75
MHz, CDCl3) δ 176.09, 158.35, 139.12, 125.07, 118.38, 102.10, 99.33,
55.32, 42.79, 40.67, 39.07, 29.71, 25.80, 25.66. HRMS (ESI) m/z calcd
for C16H23N2O+2 [M+H]+ 275.17595, found 275.17618.
4.1.64. Synthesis of (6-methoxy-3,4-dihydroquinoxalin-1(2H)-yl)(6-
methoxynaph-thalen-2-yl)methanone (11f)
Yellow solid (78.3%). HPLC purity: 95.90%, retention time: 8.22
min. m.p.: 109–110 ◦C. 1H NMR (300 MHz, CDCl3) δ 7.98 (s, 1H), 7.72
(d, J = 8.9 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.42 (d, J = 8.6 Hz, 1H),
7.15 (dd, J = 8.8, 2.4 Hz, 1H), 7.12–7.07 (m, 1H), 6.55 (s, 1H), 6.15 (d, J
= 2.7 Hz, 1H), 5.91 (d, J = 9.0 Hz, 1H), 4.12 (s, 1H), 4.01 (t, J = 5.2 Hz,
2H), 3.92 (s, 3H), 3.68 (s, 3H), 3.57 (t, J = 5.2 Hz, 2H). 13C NMR (75
MHz, CDCl3) δ 168.75, 158.93, 157.80, 138.03, 135.50, 131.21, 130.40,
129.27, 128.23, 126.44, 126.34, 126.16, 119.47, 118.90, 105.81,
102.55, 99.58, 55.49, 55.37, 42.99, 29.85. HRMS (ESI) m/z calcd for
4.1.70. Synthesis of (adamantan-1-yl)(6-methoxy-3,4-dihydroquinoxalin-
1(2H)-yl)methanone (11l)
White solid (76.4%). HPLC purity: 98.13%, retention time: 10.35
min. m.p.: 173–174 ◦C. 1H NMR (300 MHz, CDCl3) δ 7.18 (d, J = 8.8 Hz,
1H), 6.21 (dd, J = 8.8, 2.7 Hz, 1H), 6.09 (d, J = 2.6 Hz, 1H), 4.00 (br,
1H), 3.90 (t, J = 5.0 Hz, 2H), 3.73 (s, 3H), 3.42 (t, J = 5.0 Hz, 2H),
2.06–1.98 (m, 9H), 1.75–1.67 (m, 6H). 13C NMR (75 MHz, CDCl3) δ
176.84, 157.97, 139.03, 127.02, 119.91, 102.22, 99.40, 55.32, 43.70,
43.41, 43.19, 39.45, 36.63, 28.55. HRMS (ESI) m/z calcd for
C
21H21N2O+3 [M+H]+ 349.15467, found 349.15494.
4.1.65. Synthesis of (6-methoxy-3,4-dihydroquinoxalin-1(2H)-yl)
(quinolin-8-yl) methanone (11g)
Yellow solid (75.4%). HPLC purity: 97.00%, retention time: 6.78
min. m.p.: 105–106 ◦C. 1H NMR (300 MHz, CDCl3) δ 9.02–8.91 (m, 1H),
8.16 (d, J = 8.0 Hz, 1H), 8.05 (s, 1H), 8.00 (d, J = 8.5 Hz, 1H), 7.67 (d, J
= 8.3 Hz, 1H), 7.43 (dd, J = 8.3, 4.2 Hz, 1H), 6.73–6.09 (m, 2H),
6.01–5.67 (m, 1H), 4.03 (s, 2H), 3.67 (s, 3H), 3.60 (s, 2H). 13C NMR (75
MHz, CDCl3) δ 157.93, 151.65, 148.50, 138.05, 136.83, 134.28, 129.23,
129.20, 129.12, 129.09, 128.98, 127.69, 126.04, 121.72, 102.30, 99.48,
55.25, 42.78, 28.42. HRMS (ESI) m/z calcd for C19H18N3O+2 [M+H]+
320.13935, found 320.13983.
C
20H27N2O+2 [M+H]+ 327.20725, found 327.20737.
4.2. Biological evaluations
4.2.1. Cell culture
Hela cells (human epithelial cervical cancer cell line) and HT-29 cells
(human intestinal epithelial cell line) were both obtained from the
American Type Culture Collection (ATCC, USA). Cultures were main-
tained in RPMI 1640 medium (HyClone Co., USA) containing with 10%
(v/v) fetal bovine serum (Sijiqing Biotechnology Co., China) under a
humidified atmosphere containing 5% (v/v) CO2 at 37 ◦C.
4.1.66. Synthesis of (6-methoxy-3,4-dihydroquinoxalin-1(2H)-yl)
(quinolin-6-yl) methanone (11h)
Yellow solid (71.5%). HPLC purity: 97.17%, retention time: 6.69
min. m.p.: 104–105 ◦C. 1H NMR (300 MHz, CDCl3) δ 8.95 (d, J = 2.7 Hz,
1H), 8.17 (d, J = 8.2 Hz, 1H), 8.05 (s, 1H), 8.01 (d, J = 8.6 Hz, 1H), 7.68
(d, J = 8.5 Hz, 1H), 7.44 (dd, J = 8.2, 4.2 Hz, 1H), 7.05–6.26 (m, 1H),
6.15 (d, J = 2.7 Hz, 1H), 5.86 (d, J = 8.9 Hz, 1H), 4.02 (t, J = 5.2 Hz,
2H), 3.67 (s, 3H), 3.59 (t, J = 5.2 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ
158.07, 151.60, 148.42, 138.20, 137.09, 134.48, 129.39, 129.15,
129.06, 127.81, 126.13, 121.82, 118.26, 102.45, 99.63, 55.35, 42.89,
29.83. HRMS (ESI) m/z calcd for C19H18N3O+2 [M+H]+ 320.13935,
found 320.13977.
4.2.2. Cell proliferation inhibitory assay
Cells were seeded in 96-well plates at a concentration of 4.5 × 103
cells per well, which were incubated at 37 ◦C, 5% CO2 for 24 h. After
incubation, the cells were treated with the tested compounds or positive
controls at different concentrations and incubated for further 72 h. Then
50 μ
L of MTT reagent was added per well, incubated at 37 ◦C for 4 h.
After incubation, MTT solution was discarded, and 100 μL DMSO was
added to dissolve the MTT formazan completely. The OD (optical den-
sity) was measured at a wavelength of 570 nm using a microplate reader.
The inhibition rate (%) = (ODcontrol – ODcompound) / (ODcontrol – ODblank
)
4.1.67. Synthesis of 3-(6-methoxy-1,2,3,4-tetrahydroquinoxaline-1-
carbonyl)–2H-chromen-2-one (11i)
× 100%. IC50 values were calculated with normalized dose-response
using Origin software.
Yellow solid (68.3%). HPLC purity: 96.61%, retention time: 6.81
min. m.p.: 131–133 ◦C. 1H NMR (300 MHz, CDCl3) δ 8.00 (s, 1H), 7.54
(d, J = 7.6 Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H), 6.57 (d, J = 9.3 Hz, 1H),
6.13 (s, 1H), 5.91 (d, J = 8.1 Hz, 1H), 3.76 (s, 2H), 3.67 (s, 3H), 3.61 (s,
2H). 13C NMR (75 MHz, CDCl3) δ 163.46, 158.55, 157.17, 154.16,
143.54, 139.00, 132.60, 128.59, 126.40, 124.72, 123.80, 118.29,
117.59, 116.81, 102.01, 99.38, 55.15, 42.18, 40.01. HRMS (ESI) m/z
calcd for C19H17N2O+4 [M+H]+ 337.11828, found 337.11847.
4.2.3. Tubulin polymerization inhibitory assay
Tubulin polymerization inhibitory assay was conducted using the
Tubulin Polymerization Assay Kit (BK011P, Cytoskeleton, USA) ac-
cording to the manufacturer. Compound 11a at various concentrations
and the positive control colchicine were added to 96-well plates and
incubated at 37 ◦C for 1 min. Then the tubulin reagent (2.0 mg/mL
porcine brain tubulin in 80 mM PIPES pH 6.9, 2.0 mM MgCl2, 0.5 mM
EGTA, 1.0 mM GTP and 15% glycerol) was added per well and mixed.
The mixture was monitored (excitation: 360 nm, emission: 450 nm)
every 60 s for 80 min using a multifunction microplate reader.
12

T. Liang et al.
Bioorganic Chemistry 110 (2021) 104793
4.2.4. Immunofluorescence analysis
org/10.1016/j.bioorg.2021.104793.
Hela cells were seeded into 96-well plates, which were incubated at
37 ◦C, 5% CO2 for 24 h. Then compound 11a at various concentrations
were added and incubated for further 24 h. After fixed in 3.7% para-
formaldehyde in the dark at 25 ◦C for 5 min, Hela cells were per-
meabilized with 0.1% Triton X-100 for 5 min and blocked with 1% BSA
for 1 h at 25 ◦C. Then these cells were incubated with the primary
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This work was financially supported by National Natural Science
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