Synthesis and biological evaluation of N-substituted 3-oxo-1,2,3,4-tetrahydro-quinoxaline-6-carboxylic acid derivatives as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.08.018

A series of novel N-substituted 3-oxo-1,2,3,4-tetrahydro-quinoxaline-6-carboxy- lic acid derivatives were synthesized and evaluated for their biological activities. Among all synthesized target compounds, 13d exhibited the most potent antiproliferative ac

Full text of DOI:10.1016/j.ejmech.2017.08.018

Accepted Manuscript
Synthesis and biological evaluation of N-substituted 3-oxo-1,2,3,4- tetrahydro-
quinoxaline-6-carboxylic acid derivatives as tubulin polymerization inhibitors
Jianguo Qi, Haiyang Dong, Jing Huang, Shufeng Zhang, Linqiang Niu, Yahong
Zhang, Jianhong Wang
PII:
S0223-5234(17)30621-9
10.1016/j.ejmech.2017.08.018
EJMECH 9662
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 October 2016
Revised Date: 5 August 2017
Accepted Date: 6 August 2017
Please cite this article as: J. Qi, H. Dong, J. Huang, S. Zhang, L. Niu, Y. Zhang, J. Wang, Synthesis and
biological evaluation of N-substituted 3-oxo-1,2,3,4- tetrahydro-quinoxaline-6-carboxylic acid derivatives
as tubulin polymerization inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.08.018.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and Biological Evaluation of N-substituted 3-oxo-1,2,3,4-
tetrahydro-quinoxaline-6-carboxylic Acid Derivatives as Tubulin
Polymerization Inhibitors
Jianguo Qi a, Haiyang Dong a, Jing Huang a, Shufeng Zhang a, Linqiang Niu a, Yahong
Zhang a, *, Jianhong Wang a, *
a Key Laboratory of Natural Medicine and Immuno-Engineering of Henan Province,
Henan University Jinming Campus, Kaifeng 475004, Henan, China
* Corresponding author
E-mail address: zhangyahong_131@163.com, jhworg@126.com
Abstract
A series of novel N-substituted 3-oxo-1,2,3,4-tetrahydro-quinoxaline-6-carboxy-
lic acid derivatives were synthesized and evaluated for their biological activities.
Among all synthesized target compounds, 13d exhibited the most potent
antiproliferative activity against HeLa, SMMC-7721, K562 cell line (IC50 = 0.126 µM,
0.071 µM, 0.164 µM, respectively). Furthermore, compound 13d inhibited tubulin
polymerization (IC50 = 3.97 µM), arrested cell cycle at the G2/M phase and induced
apoptosis. The binding mode at the colchicine binding site was also probed. These
studies provided a new molecular scaffold for the further development of antitumor
agents that target tubulin.
Keywords
Antiproliferative; Tubulin polymerization inhibitors; Cell cycle analysis; Cell
apoptosis; 3-Oxo-1,2,3,4-tetrahydro-quinoxaline-6-carboxylic acid derivatives.

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1. Introduction
Microtubules, which are dynamic polymers of αβ-tubulin, play an essential role
in mitosis, forming the dynamic spindle apparatus. Disruption of microtubule
dynamics prevents microtubule function and ultimately leads to cell death. This made
microtubules an important target for cancer chemotherapy. Microtubule-targeting
agents are divided into two groups, microtubule-stabilizing agents (paclitaxel,
laulimalide etc) and tubulin polymerization inhibitors (vinca alkaloids, colchicine etc).
These agents interact with tubulin through three major binding sites, vinca alkaloid-,
taxane- and colchicine-binding sites [1-3]. Recently, diverse small molecules that act
at the colchicine site on tubulin have come under intensive investigation. These
compounds not only display potent cytotoxicity against a wide variety of human
cancer cell lines but also show selective toxicity toward tumor endothelial cells
required for the growth of the cancer. Thus they represent a new class of vascular
disrupting agents which cause a significant shutdown of the blood vessels of tumors,
leading to cancer cell death via necrosis and apoptosis. Moreover, they show
promising ability to overcome multidrug resistance mediated by P-glycoprotein [4-14].
Therefore, this type of tubulin inhibitor might provide a new opportunity for cancer
therapy.
Currently, some drug candidates targeting at the colchicine site are in clinical
development. CA4-P (1a, Figure 1), the phosphate derivative prodrug of
combretastatin A-4 (CA-4, 1b, Figure 1), has entered clinical trials either alone or in
combination with other chemotherapeutic agents [15, 16]. However, the cis-stilbene
of CA-4 is prone to isomerisation into its inactive trans-form during storage and
administration. To avoid the stability problems of CA-4, many conformationally
restricted modification have been used [17-23]. Phenstatin (2, Figure 1) which is the
CA-4 analogue with the double bond of CA-4 being replaced by a carbonyl group
showed strong cytotoxicity and antitubulin activity similar to CA-4, but it is more
stable compared with CA-4 [24]. BNC-105P (3a, Figure 1), developed by Bionomics
(Australia), is a phosphorylated prodrug of BNC-105 (3b, Figure 1) and it entered
clinical trials in combination with everolimus for progressive metastatic clear cell

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renal cell carcinoma. It is the phenstatin analogue with the benzene ring (ring B) of
phenstatin being replaced by coumarone [25]. Based on BNC-105 and other
modification of CA-4 and phenstatin, we conclude that ring B could be replaced by
other heterocycles, such as chromane, indole etc [26-41].
Figure 1. CA-4 and its analogues targeting at the colchicine site of tubulin.
Quinoxalinone structure has good physico-chemical properties and drug-like
properties. Many bioactive compounds contain this moiety [42, 43]. The current study
was undertaken to investigate the in vitro antitumor activity of a novel modification of
phenstatin. The replacement of one phenyl ring with 3-oxo-1,2,3,4-tetrahydro-
quinoxaline-6-carboxylic acid and its derivatives were pursued. Herein we report the
synthesis and biological evaluation of a series of N-substituted 3-oxo-1,2,3,4-
tetrahydro-quinoxaline-6-carboxylic acid derivatives. Compound 13d was identified
with IC50 values ranging from 0.071 to 0.164 µM against the three cancer cell lines.
The effects on tubulin polymerization, cell cycle and apoptosis were also evaluated.
2. Results and discussion
2.1. Chemistry

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o
Scheme 1. Reagents and conditions: a) SOCl2, MeOH, 0-50 C; b) amines, EDCI, CH2Cl2, rt; c)
ethyl bromoacetate, Cs2CO3, 140 oC; d) 10% Pd/C, H2, MeOH, rt; e) benzyl chlorides, KI, K2CO3,
CH3CN, 70 oC; f) LiOH, THF, H2O, 40 oC; g) oxalyl chloride, PhMe, 70 oC; amines, K2CO3, THF,
rt; h) SOCl2, 70 oC; i) 12a-e, K2CO3, THF, rt.
Scheme 1 depicts the synthetic pathways used for the preparation of
quinoxalinone derivatives. Starting from the commercial compound 4-amino-3-
nitrobenzoic acid (4), the reaction with thionyl chloride in methanol and properly
substituted amines in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

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hydrochloride (EDCI) in CH2Cl2 provided the compounds 5a-5e (71.8% – 89.2%).
These latter compounds reacted with ethyl bromoacetate in the presence of Cs2CO3 to
afford compounds 6a-6e (44.3% – 58.3%). The key intermediates 7a-7e (68.8% –
82.4%) were synthesized via a “one pot” reduction of the nitro group using hydrogen
in the presence of palladium on charcoal and cyclization reaction of 6a-6e in
methanol. Compounds 8a-8c (42.3% – 67.8%) were prepared by nucleophilic
substitution of substituted benzyl chlorides with intermediates 7a-7e using K2CO3 as a
base. The subsequent hydrolysis of compound 8c using LiOH aqueous solution
furnished the desired compound 9 (72.6%). Compound 9 was converted into its acid
chloride with oxalyl chloride in toluene. Compounds 10a (73.2%) and 10b (62.4%)
were prepared by condensation of the acid chloride mentioned above with properly
substituted amines in the presence of K2CO3 in tetrahydrofuran (THF). Compounds
13a-13i (46.7% – 65.3%) were prepared by nucleophilic addition of intermediates
7a-7e with substitued benzoyl chlorides in the presence of K2CO3 in THF. The
benzoyl chlorides 12a-12e were gained by treatment of substituted benzoic acids with
thionyl chloride.
2.2. Biological evaluation
2.2.1. In vitro antiproliferative activity
The in vitro antiproliferative activities of the synthesized target compounds were
evaluated against HeLa (human epithelial cervical cancer), SMMC-7721(human
hepatoma cancer), and K562 (leukemia) cell lines using MTT assay. Doxorubicin and
CA-4 were chosen as reference drugs. The results are summarized in Table 1. The
antiproliferative activities of the compounds were expressed as the concentration of
compounds required for 50% inhibition of cell growth (IC50). IC50 values were
calculated from at least five different concentrations of test compounds.

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Table 1. The in vitro antiproliferative activities of synthesized target compounds (8a-8c, 9, 10a,
10b, 13a-13l, doxorubicin and CA-4 against three human cancer cell lines (HeLa, SMMC-7721
and K562)
Antiproliferative Activity ( IC50, µM)
Compound
HeLa
16.4±1.5
SMMC-7721
12.5±1.3
33.2±2.5
2.69±0.45
39.0±3.9
39.7±2.3
28.0±5.4
7.69±0.35
5.40±0.78
1.32±0.22
0.071±0.014
9.10±1.68
﹥50
K562
43.2±2.9
8a
8b
38.1±5.0
2.76±0.50
32.2±4.7
37.5±3.1
41.8±1.9
15.9±1.8
8.28±1.43
1.42±0.25
0.126±0.015
39.4±5.1
﹥50
43.6±0.7
3.42±0.85
35.9±8.5
43.0±7.2
42.6±4.5
9.80±2.17
7.72±2.70
3.00±1.05
0.164±0.005
45.3±3.5
﹥50
8c
9
10a
10b
13a
13b
13c
13d
13e
13f
13g
13h
13i
﹥50
43.1±7.6
40.6±1.7
34.4±3.1
37.9±8.3
34.9±7.9
39.2±7.6
1.59±0.27
0.0038±0.0020
﹥50
﹥50
﹥50
24.9±2.4
26.4±7.4
34.9±7.5
﹥50
42.9±4.0
﹥50
13j
13k
13l
﹥50
﹥50
doxorubicin
CA-4
1.82±0.31
0.013±0.003
0.89±0.17
0.013±0.001
As shown in Table 1, most of the synthesized compounds inhibited the growth of
the three cancer cells with IC50 values under 50 µM. The comparison of IC50 values of

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8a – 8c demonstrated that 8c which had 3,4,5-trimethoxybenzyl substitution on the
nitrogen atom of methyl 3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate had better
activity than 8a (4-methoxybenzyl) and 8b (3,4-dimethoxybenzyl). The activity
decreased by an order of magnitude when the methyl carboxylate of 8c was
hydrolyzed to carboxylic acid (9). Compound 10a or 10b with N-butyl or
N-cyclohexyl quinoxalinone-6-carboxamide substitued for quinoxalinone-6-
carboxylate of compound 8c showed comparable activity with compound 9.
Comparison of compounds 13b – 13d with compounds 8a – 8c revealed that the
replacement of benzyl group with benzoyl group led to a remarkable improvement in
antiproliferative activity of the corresponding compounds. The antiproliferative
activity of compounds 13b – 13d with different substituents on the benzene increased
in the following order: 13d (3,4,5-trimethoxy) > 13c (3,4-dimethoxy) > 13b
(4-methoxy). Compounds 13d and 13c were the two most potent compounds among
all the synthesized target compounds. Compound 13d exhibited better activity than
doxorubicin, but less than CA-4. Compound 13a with para-fluoro substituent on the
phenyl ring showed comparable activity with 13b (4-methoxy). Lengthening the
benzoyl moiety of 13d to phenylacetyl group (13e) caused a significant reduction in
antiproliferative activity in all cell lines. Replacement of methyl carboxylate on the
quinoxalinone with amide (13f-13l) led to weak activity.
2.2.2. In vitro inhibition of tubulin polymerization
Three compounds, including 8c, 13c and 13d, were selected to evaluate for their
ability to inhibit tubulin polymerization and they were employed at 20 µM in the
assay. CA-4 (2.5 µM) and paclitaxel (3 µM) were evaluated as reference compounds.
Compound 13d was employed at four different concentrations in order to determine
the IC50. In the assay, the IC50 was defined as the concentration of compound that
inhibited 50% the extent of assembly of 10 µM tubulin after 40 min incubation at 37
oC. As shown in figure 2A, 8c, 13c and 13d resulted in various degrees of inhibition
of tubulin polymerization compared with control (0.1% DMSO). Compound 13d
inhibited tubulin polymerization with an IC50 value of 3.97 µM.

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Figure 2. (A) Effects of 20 µM 8c, 13c and 13d on tubulin polymerization. Paclitaxel (3 µM) and
CA-4 (2.5 µM) were used as reference drugs, 0.1% DMSO as control. (B) Inhibition of tubulin
polymerization by 13d with different concentrations (1 µM, 5 µM, 10 µM, 50 µM).
2.2.3. Antimicrotubule effects in heLa cells
Figure 3. Fluorescence microscopic images of HeLa cells stained with Hoechst 33342 or
anti-α-tubulin-FITC antibody after treatment with 0.1% DMSO, 13d (0.10 µM, 0.15 µM, 0.20 µM)
or CA-4 (0.05 µM) for 24 h.
Compound 13d was selected to evaluate the effects on microtubule assembly in
the cell-based phenotypic assay. HeLa cells were treated with three different
concentrations (0.10 µM, 0.15 µM, 0.20 µM) of 13d or 0.05 µM CA-4 for 24 h. Cells

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were stained with Hoechst 33342 and anti-α-tubulin-FITC antibody. As shown in
figure 3, the cells without drug treatment exhibited normal filamentous microtubules
arrays, with microtubules extending to the peripheral region of the cells. The cells
treated with drugs exhibited reduced microtubule networks. Compound 13d induced a
dose-dependent collapse of the microtubule network.
2.2.4. Effect on cell cycle progression
Inhibition of tubulin polymerization leads to G2/M cell cycle arrest. Compound
13d was evaluated in dose-response experiments for study on cell cycle distribution.
HeLa cells were treated with four different concentrations (0.10 µM, 0.15 µM, 0.20
µM, 0.25 µM) of 13d for 24 h. As shown in Figure 4, compound 13d induced a
significant increase in the proportion of cells in the G2/M phase based on DNA
content at 0.15µM. Most cells accumulated in G2/M phase at 0.25 µM. It was
concluded that compound 13d caused G2/M cell cycle arrest in a concentration-
dependent manner.
Figure 4. (A) Flow cytometry analysis of HeLa cells treated with 0.1% DMSO, 0.10 µM, 0.15 µM,
0.20 µM or 0.25 µM compound 13d for 24 h. (B) The statistical graph of cell cycle distribution.
2.2.5. Compound 13d induce apoptosis
To investigate the mode of cell death induced by 13d, the annexin V/propidium

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iodide (PI) double staining assay was performed. HeLa cells were treated with three
different concentrations of 13d for 24 h. As depicted in figure 5, the four quadrants
represented live cells (annexin-V-/PI-), early apoptotic cells (annexin-V+/PI-), late
apoptotic cells (annexin-V+/PI+), and necrotic cells (annexin-V-/PI+). It was concluded
that 13d induced an accumulation of annexin-V positive cells in comparison with the
control group, in a concentration dependent manner. Cell shrinkage, membrane
blebbing and plasmatorrhexis that imply the cell apoptosis could be observed under
the microscope.
Figure 5. (A) Bi-parametric histograms of HeLa cells treated with 0.1% DMSO, 0.10 µM, 0.15
µM or 0.20 µM compound 13d for 24 h by flow cytometry after staining (annexin-V and PI). (B)
Percentage of cells found in the different regions of the histograms. (C) Images of cell
morphology under the microscope.
2.2.6. Competitive inhibition of colchicine binding to tubulin
To investigate the possible binding site of 13d, a fluorescence based competitive

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assay for the colchicine site was performed. Colchicine, which does not fluoresce,
exhibits marked fluorescence when bond to tubulin [44]. The changes in fluorescence
intensity of colchicine could be used as an index for 13d competition with colchicine
in tubulin binding [45, 46]. As shown in Figure 6, compound 13d decreased the
fluorescence intensity of colchicine-tubulin complex in a concentration-dependent
manner. Paclitaxel did not change the binding of colchicine to tubulin. These results
indicated that compound 13d binds at the colchicine site of tubulin.
Figure 6. Fluorescence based colchicine competitive binding assay. Tubulin (4 µM) was
co-incubated with indicated concentrations of paclitaxel or 13d for 1 h, then 10 µM colchicine was
added. Fluorescence values were normalized to control (DMSO).
2.3. Molecular modeling
Molecular docking was performed to study the binding mode of the most potent
compound 13d. Compound 13d was docked into the N-deacetyl-N-(2-mercaptoacetyl)
colchicine (DAMA-colchicine) binding site of the tubulin (PDB ID: 1SA0) using
surflex dock program (Sybyl-X 2.1) [47]. Some of the top 20 docking poses ranked by
total scores were shown in figure 7A. The binding orientation of compound 13d was
very similar to that of the cocrystallized DAMA-colchicine. As shown in figure 7B, a
hydrogen bond is present between the quinoxalinone of 13d and Thr 179 in the loop
of α-tubulin. The trimethoxyphenyl moiety occupied similar conformational space as
that of DAMA-colchicine and made nonpolar interaction with Cys 241, Leu 248, Ala
250, Leu 255, Ala 316 of β-tubulin.

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Figure 7. (A) Comparison between pose of cocrystallized DAMA-colchicine (green) with tubulin
and docking poses of 13d. (B) Proposed binding mode of 13d. Residues within 3.5 Å of 13d are
shown.
3. Conclusion
We have synthesized a series of N-substituted 3-oxo-1,2,3,4-tetrahydro-
quinoxaline-6-carboxylic acid derivatives with antiproliferative activity and inhibition
of tubulin polymerization activity. The methyl carboxylate on the quinoxalinone was
crucial for the antiproliferative activity. When this moiety was hydrolyzed to
carboxylate acid or replaced with amide, the activity decreases. The replacement of
benzyl group with benzoyl group led to a remarkable improvement of in vitro
antiproliferative activity. This led the discovery of 13d with IC50 values in the range
of 0.071 to 0.164 µM against the three cancer cell lines. Immunofluorescence assay
revealed that compound 13d induced mitotic arrest in HeLa cells through disruption
of microtubule dynamics. Compound 13d induced cell cycle arrest at the G2/M phase
and led to cell apoptosis. Compound 13d represented a novel lead compound of
N-substituted 3-oxo-1,2,3,4-tetrahydro-quinoxaline structure that paved the way for
the development of new promising anticancer agents.
4. Experimental section
4.1. Chemistry
4.1.1. General
All commercially obtained reagents and solvents were used as received.
Reactions were monitored by TLC with silica gel plates (0.25mm, indicator GF254,
Qingdao Haiyang Co. Ltd., China) under UV light. Column chromatography was

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performed with silica gel (100-200 mesh, 200-300 mesh, Qingdao Haiyang Co. Ltd.,
China). The melting points were measured on a hot-stage microscope (SGW X-4,
Shanghai Shenguang Instrument Co. Ltd., China) and were uncorrected. Mass spectra
were measured on a Bruker AmaZon SL mass spectrometer. HRMS spectra were
acquired on a Thermo Scientific LTQ Orbitrap XL mass spectrometer, and all the
errors were less than 3 ppm. 1H NMR spectra and 13C NMR spectra were acquired on
a Bruker DRX 300 NMR spectrometer using CDCl3 or CD3OD as solvent (unless
otherwise stated). Chemical shifts were reported in parts per million (ppm, δ) relative
13
to the solvent peak (1H, CDCl3 δ 7.26 ppm, CD3OD δ 3.31 ppm; C, CDCl3 δ 77.0
ppm, CD3OD δ 47.6 ppm). Coupling constants (J) were measured in hertz (Hz). The
infrared (IR) spectra were obtained using a Vertex 70 (Bruker, Germany)
Fourier-transform infrared spectrometer.
4.1.2. Procedure for synthesis of compound 5a
The commercial compound 4 (4.50 g, 24.7 mmol) was dissolved in methanol (80
o
mL). The reaction was cooled to 0 C, and thionyl chloride (5.38 mL, 74.1 mmol, 3
o
equiv) was added dropwise. The reaction mixture was stirred at 0 C for 30 min and
was heated to 50 oC for 4 h. The precipitate formed on cooling was collected by
filtration. The solvent was removed under reduced pressure. The residue was
dissolved with water and extracted with ethyl acetate. The organic layer was dried
over anhydrous Na2SO4, and the solvent was removed by evaporation. The yellow
solid was combined.
Methyl 4-amino-3-nitrobenzoate (5a). Yield: 89.2%, yellow solid, mp 181.9 –
o
1
182.7 C. H NMR (300 MHz, CDCl3) δ 8.85 (d, J = 1.9 Hz, 1H), 7.99 (dd, J = 8.8,
1.9 Hz, 1H), 6.83 (d, J = 8.8 Hz, 1H), 6.42 (brs, 2H), 3.90 (s, 3H). IR (KBr)
(νmax/cm-1): 3475, 3343, 3030, 2961, 2855, 1702, 1634, 1565, 1522, 1477, 1356, 1298,
1262, 1133, 832, 760. MS (ESI) [M+H] + 197.06.
4.1.3. General procedure for synthesis of compounds 5b-5e
To a solution of compound 4 (250 mg, 1.37 mmol) in dry CH2Cl2 (30 mL) were
added EDCI (314 mg, 1.64 mmol, 1.2 equiv) and the amine (1.37 mmol, 1 euqiv). The
reaction mixture was stirred at room temperature for 2 h. After completion of the

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reaction, the mixture was washed with water and dried over anhydrous Na2SO4. The
solvent was removed by evaporation to give the crude product, which was purified by
column chromatography.
4.1.3.1. 4-Amino-N-butyl-3-nitrobenzamide (5b). Yield: 84.0%, yellow solid, mp
o
1
162.8 – 164.6 C. H NMR (300 MHz, CDCl3) δ 8.47 (s, 1H), 7.90 (d, J = 8.7 Hz,
1H), 6.86 (d, J = 8.7 Hz, 1H), 6.35 (brs, 2H), 6.05 (brs, 1H), 3.42 – 3.49 (m, 2H), 1.58
– 1.67 (m, 2H), 1.35 – 1.49 (m, 2H), 0.97 (t, J = 7.3 Hz, 3H). IR (KBr) (νmax/cm-1):
3468, 3356, 3301, 2959, 2938, 2869, 1630, 1547, 1515, 1473, 1372, 1346, 840, 761.
MS (ESI) [M+H] + 238.08.
4.1.3.2. 4-Amino-N-cyclohexyl-3-nitrobenzamide (5c). Yield: 77.9%, yellow solid,
mp 205.3 – 207.6 oC. 1H NMR (300 MHz, CDCl3) δ 8.46 (s, 1H), 7.90 (d, J = 8.7 Hz,
1H), 6.86 (d, J = 8.7 Hz, 1H), 6.33 (brs, 2H), 5.80 – 5.93 (m, 1H), 3.89 – 4.01 (m, 1H),
1.97 – 2.08 (m, 2H), 1.73 – 1.82 (m, 2H), 1.61 – 1.71 (m, 1H), 1.35 – 1.50 (m, 2H),
1.20 – 1.30 (m, 3H). IR (KBr) (νmax/cm-1): 3493, 3380, 3266, 3080, 2930, 2857, 1633,
1552, 1515, 1336, 835, 759. MS (ESI) [M−1]− 262.12.
4.1.3.3. 4-Amino-3-nitro-N-phenylbenzamide (5d). Yield: 73.8%, yellow solid, mp
o
1
222.5 – 224.5 C. H NMR (300 MHz, CD3OD) δ 8.76 (s, 1H), 7.94 (d, J = 9.0 Hz,
1H), 7.65 (d, J = 8.0 Hz, 2H), 7.35 (t, J = 8.0 Hz, 2H), 7.13 (t, J = 7.4 Hz, 1H), 7.05
(d, J = 9.0 Hz, 1H). IR (KBr) (νmax/cm-1): 3459, 3391, 3352, 1630, 1547, 1510, 1417,
1358, 827, 753. MS (ESI) [M−1]− 256.08.
4.1.3.4. (4-Amino-3-nitrophenyl)(morpholino)methanone (5e). Yield: 71.8%,
o
1
yellow solid, mp 203.1 – 205.4 C. H NMR (300 MHz, CDCl3) δ 8.24 (s, 1H), 7.51
(d, J = 8.6 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 6.28 (brs, 2H), 3.71 (brs, 4H), 3.67 (brs,
4H). IR (KBr) (νmax/cm-1): 3464, 3302, 2918, 2864, 1621, 1559, 1517, 1475, 1347,
1254, 1072, 868, 755. MS (ESI) [M−1]− 250.06.
4.1.4. General procedure for synthesis of compounds 6a-6e
A mixture of compound 5 (1.53 mmol), Cs2CO3 (600 mg, 1.84 mmol, 1.2 equiv)
and ethyl bromoacetate (1.70 mL, 15.3 mmol, 10 equiv) was heated at 140 oC for 20 h.
After completion of the reaction, ethyl bromoacetate was removed by evaporation.
The residue was dissolved in CH2Cl2 and Cs2CO3 was removed by filtration. The

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solvent was removed by evaporation to give the crude product, which was then
purified by column chromatography.
4.1.4.1. Methyl 4-((2-ethoxy-2-oxoethyl)amino)-3-nitrobenzoate (6a). Yield:
o
1
58.3%, light yellow solid, mp 127.6 – 128.8 C. H NMR (300 MHz, CDCl3) δ 8.91
(d, J = 2.1 Hz, 1H), 8.75 (brs, 1H), 8.09 (dd, J = 9.0, 2.1 Hz, 1H), 6.72 (d, J = 9.0 Hz,
1H), 4.31 (q, J = 7.2 Hz, 2H), 4.14 (d, J = 5.2 Hz, 2H), 3.91 (s, 3H), 1.33 (t, J = 7.2
Hz, 3H). IR (KBr) (νmax/cm-1): 3345, 3098, 2985, 2955, 2851, 1740, 1708, 1628, 1570,
1532, 1438, 1373, 1162, 1132, 827, 763. MS (ESI) [M+H] + 283.09.
4.1.4.2. Ethyl 2-((4-(butylcarbamoyl)-2-nitrophenyl)amino)acetate (6b). Yield:
o
1
46.2%, light yellow solid, mp 152.8 – 154.0 C. H NMR (300 MHz, CDCl3) δ 8.66
(brs, 1H), 8.55 (s, 1H), 8.01 (d, J = 8.7 Hz, 1H), 6.76 (d, J = 8.7 Hz, 1H), 6.05 (brs,
1H), 4.31 (q, J = 7.2 Hz, 2H), 4.13 (d, J = 5.1 Hz, 2H), 3.40 – 3.51 (m, 2H), 1.57 –
1.67 (m, 2H), 1.37 – 1.49 (m, 2H), 1.33 (t, J = 7.2 Hz, 3H), 0.97 (t, J = 7.2 Hz, 3H).
IR (KBr) (νmax/cm-1): 3338, 3277, 3095, 2924, 2861, 1726, 1633, 1554, 1524, 1446,
1369, 1253, 1066, 1018, 835, 732. MS (ESI) [M−1]− 322.41.
4.1.4.3. Ethyl 2-((4-(cyclohexylcarbamoyl)-2-nitrophenyl)amino)acetate (6c).
Yield: 45.6%, light yellow solid, mp 173.5 – 175.0 oC. 1H NMR (300 MHz, CDCl3) δ
8.66 (brs, 1H), 8.53 (s, 1H), 8.00 (d, J = 8.7 Hz, 1H), 6.75 (d, J = 8.7 Hz, 1H), 5.90 (d,
J = 7.7 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H), 4.13 (d, J = 5.2 Hz, 2H), 3.90 – 4.01 (m,
1H), 1.99 – 2.07 (m, 2H), 1.73 – 1.82 (m, 2H), 1.63 – 1.71 (m, 1H), 1.39 – 1.51 (m,
2H), 1.23 – 1.33 (m, 6H). IR (KBr) (νmax/cm-1): 3425, 3355, 3283, 3085, 2930, 2857,
1741, 1630, 1524, 1369, 1232, 1072, 829, 758. MS (ESI) [M−1]− 348.44.
4.1.4.4. Ethyl 2-((2-nitro-4-(phenylcarbamoyl)phenyl)amino)acetate (6d). Yield:
o
1
46.5%, light yellow solid, mp 187.2 – 189.0 C. H NMR (300 MHz, CDCl3) δ 8.74
(brs, 1H), 8.71 (s, 1H), 8.09 (d, J = 8.7 Hz, 1H), 7.76 (brs, 1H), 7.64 (d, J = 7.5 Hz,
2H), 7.39 (t, J = 7.5 Hz, 2H), 7.17 (t, J = 7.5 Hz, 1H), 6.82 (d, J = 8.7 Hz, 1H), 4.32
(q, J = 7.2 Hz, 2H), 4.16 (d, J = 5.2 Hz, 2H), 1.34 (t, J = 7.2 Hz, 3H). IR (KBr)
(νmax/cm-1): 3389, 3337, 3283, 3062, 2922, 2854, 1730, 1637, 1522, 1440, 1391, 1320,
1247, 1068, 828, 754. MS (ESI) [M−1]− 342.28.
4.1.4.5. Ethyl 2-((4-(morpholine-4-carbonyl)-2-nitrophenyl)amino)acetate (6e).

ACCEPTED MANUSCRIPT
o
1
Yield: 44.3%, light yellow solid, mp 78.5 – 80.7 C. H NMR (300 MHz, CDCl3) δ
8.59 (brs, 1H), 8.33 (s, 1H), 7.62 (d, J = 8.7 Hz, 1H), 6.75 (d, J = 8.7 Hz, 1H), 4.31 (q,
J = 7.2 Hz, 2H), 4.12 (d, J = 5.2 Hz, 2H), 3.71 (brs, 4H), 3.67 (brs, 4H), 1.33 (t, J =
7.2 Hz, 3H). IR (KBr) (νmax/cm-1): 3441, 3369, 3108, 2976, 2920, 2857, 1741, 1633,
1564, 1530, 1456, 1355, 1227, 1067, 823, 755. MS (ESI) [M−1]− 336.54.
4.1.5. General procedure for synthesis of compounds 7a-7e
To a solution of compound 6 in methanol was added palladium-carbon catalyst
(10%, 0.05 equip). The reactor was purged with hydrogen. The reaction was carried
out under a hydrogen atmosphere at room temperature for 12 h. The palladium-carbon
was removed by suction filtration and the solvent was removed under reduced
pressure. The crude product was purified by column chromatography.
4.1.5.1. Methyl 3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxylate (7a). Yield:
82.4%, gray solid, mp > 280 oC. 1H NMR (300 MHz, CDCl3) δ 7.74 (brs, 1H), 7.60 (d,
J = 8.4 Hz, 1H), 7.38 (s, 1H), 6.64 (d, J = 8.4 Hz, 1H), 4.09 (s, 2H), 3.87 (s, 3H). IR
(KBr) (νmax/cm-1): 3317, 3254, 3104, 3040, 2953, 2847, 1672, 1610, 1550, 1504, 1444,
1398, 1295, 1222, 1111, 821, 771. MS (ESI) [M+H]+ 207.00.
4.1.5.2. N-butyl-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxamide (7b). Yield:
o
68.8%, gray solid, mp 211.9 – 213.3 C. 1H NMR (300 MHz, CD3OD) δ 7.32 (d, J =
8.1 Hz, 1H), 7.24 (s, 1H), 6.67 (d, J = 8.1 Hz, 1H), 3.92 (s, 2H), 3.32 – 3.37 (m, 2H),
1.54 – 1.63 (m, 2H), 1.35 – 1.44 (m, 2H), 0.96 (t, J = 7.2 Hz, 3H). IR (KBr)
(νmax/cm-1): 3340, 3096, 2963, 2873, 1696, 1602, 1521, 1456, 1389, 819, 762. MS
(ESI) [M−1]− 246.44.
4.1.5.3. N-cyclohexyl-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-carboxamide (7c).
Yield: 71.3%, gray solid, mp 268.8 – 270.8 oC. 1H NMR (300 MHz, CDCl3) δ 7.61 (s,
1H), 7.21 (d, J = 8.1 Hz, 1H), 6.64 (d, J = 8.1 Hz, 1H), 5.79 (brs, 1H), 4.05 (s, 2H),
3.91 – 3.99 (m, 1H), 1.99 – 2.06 (m, 2H), 1.70 – 1.80 (m, 2H), 1.62 – 1.67 (m, 1H),
1.40 – 1.47 (m, 2H), 1.18 – 1.24 (m, 3H). IR (KBr) (νmax/cm-1): 3392, 3291, 2929,
2854, 1678, 1634, 1541, 1454. MS (ESI) [M−1]− 272.22.
4.1.5.4. 3-Oxo-N-phenyl-1,2,3,4-tetrahydroquinoxaline-6-carboxamide
(7d).
o
1
Yield: 74.9%, gray solid, mp 260.9 – 262.5 C. H NMR (300 MHz, CD3OD) δ 7.62

ACCEPTED MANUSCRIPT
(d, J = 7.5 Hz, 2H), 7.49 (d, J = 8.1 Hz, 1H), 7.36 (s, 1H), 7.33 (t, J = 7.5, 2H), 7.11 (t,
J = 7.2 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 3.95 (s, 2H). IR (KBr) (νmax/cm-1): 3389,
2923, 2852, 1654, 1531, 1501, 820, 755. MS (ESI) [M−1]− 266.17.
4.1.5.5. 7-(Morpholine-4-carbonyl)-3,4-dihydroquinoxalin-2(1H)-one (7e). Yield:
70.2%, gray solid, mp 207.6 – 209.5 oC. 1H NMR (300 MHz, CDCl3) δ 7.85 (brs, 1H),
6.95 (d, J = 8.1 Hz, 1H), 6.88 (s, 1H), 6.64 (d, J = 8.1 Hz, 1H), 4.06 (brs, 1H), 4.03 (s,
2H), 3.69 (brs, 4H), 3.66 (brs, 4H). IR (KBr) (νmax/cm-1): 3279, 3209, 3041, 2966,
2923, 2857, 1679, 1612, 1499, 1274, 832, 770. MS (ESI) [M−1]− 260.14.
4.1.6. General procedure for synthesis of compounds 8a-8c
To a solution of compound 7 (30 mg, 0.145 mmol) in acetonitrile (2 mL) were
added anhydrous K2CO3 (24 mg, 0.174 mmol, 1.2 equiv), KI (12 mg, 0.073 mmol, 0.5
equiv) and substituted benzyl chloride (0.218 mmol, 1.5 equiv). The reaction mixture
was stirred at 70 oC for 6 h. After completion of the reaction, the mixture was filtered
and the solvent was removed by evaporation to give the crude product, which was
then purified by column chromatography.
4.1.6.1. Methyl
1-(4-methoxybenzyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-
o
1
carboxylate (8a). Yield: 42.3%, white solid, mp 217.0 – 219.0 C. H NMR (300
MHz, CDCl3) δ 8.48 (brs, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.44 (s, 1H), 7.19 (d, J = 8.1
Hz, 2H), 6.88 (d, J = 8.1 Hz, 2H), 6.76 (d, J = 8.4 Hz, 1H), 4.44 (s, 2H), 3.92 (s, 2H),
13
3.87 (s, 3H), 3.80 (s, 3H). C NMR (75 MHz, CDCl3) δ 166.7, 165.5, 159.3, 138.8,
128.8, 127.1, 126.7, 125.0, 119.9, 116.6, 114.4, 110.9, 55.4, 52.8, 51.9, 51.6. IR (KBr)
(νmax/cm-1): 3429, 3288, 3073, 2924, 2844, 1709, 1679, 1613, 1504, 1438, 1383, 1295,
1225, 1171, 1037, 816, 762. HRMS (ESI): calcd for C18H19N2O4 [M+H]+ 327.13393,
found 327.13388.
4.1.6.2. Methyl 1-(3,4-dimethoxybenzyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-
o
1
carboxylate (8b). Yield: 56.1%, white solid, mp 155.9 – 157.5 C. H NMR (300
MHz, CDCl3) δ 9.05 (brs, 1H), 7.65 (dd, J = 8.7, 1.8 Hz, 1H), 7.50 (d, J = 1.8 Hz, 1H),
6.82 – 6.79 (m, 4H), 4.43 (s, 2H), 3.92 (s, 2H), 3.87 (s, 6H), 3.83 (s, 3H).13C NMR
(75 MHz, CDCl3) δ 166.8, 165.8, 149.5, 148.7, 138.9, 127.6, 126.6, 125.2, 120.0,
119.9, 116.8, 111.4, 110.9, 110.5, 56.0, 53.2, 52.0, 51.6. IR (KBr) (νmax/cm-1): 3429,

ACCEPTED MANUSCRIPT
3288, 2951, 2842, 1700, 1615, 1511, 1302, 1263, 1225, 1142, 1025, 811, 764. HRMS
(ESI): calcd for C19H21N2O5 [M+H]+ 357.14450, found 357.14450.
4.1.6.3. Methyl 3-oxo-1-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroquinoxaline
o
1
-6-carboxylate (8c). Yield 67.8%, white solid, mp 188.9 – 191.0 C. H NMR (300
MHz, CDCl3) δ 8.02 (brs, 1H), 7.67 (dd, J = 8.4, 1.8 Hz, 1H), 7.43 (d, J = 1.8 Hz, 1H),
6.78 (d, J = 8.4 Hz, 1H), 6.47 (s, 2H), 4.42 (s, 2H), 3.93 (s, 2H), 3.88 (s, 3H), 3.84 (s,
3H), 3.81 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 166.7, 165.7, 153.8, 138.8, 137.5,
131.0, 126.7, 125.1, 120.2, 116.8, 111.0, 104.3, 60.9, 56.2, 53.8, 52.0, 51.8. IR (KBr)
(νmax/cm-1): 3429, 3266, 2948, 2839, 1696, 1613, 1503, 1459, 1302, 1220, 1162, 1124,
1005, 821, 766. HRMS (ESI): calcd for C20H23N2O6 [M+H]+ 387.15506, found
387.15512.
4.1.7. Procedure for synthesis of compound 9
To a solution of compound 8c (30 mg, 0.0776 mmol) in THF (2 mL)/H2O (1 mL)
was added anhydrous LiOH (3.7 mg, 0.155 mmol, 2 equiv). The reaction mixture was
o
stirred at 40 C for 10 h. The THF was removed by evaporation and the remaining
mixture was acidified with hydrochloric acid. The precipitate that formed on cooling
was collected by filtration without further purification.
3-Oxo-1-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroquinoxaline-6-carboxyli
o
1
c acid (9). Yield: 72.6%, white solid, mp 135.3 – 136.3 C. H NMR (300 MHz,
CDCl3) δ 11.95 (brs, 1H), 7.73 (s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 6.67 (d, J = 8.4 Hz,
1H), 6.51 (s, 2H), 4.46 (s, 2H), 4.04 (s, 2H), 3.84 (s, 9H). 13C NMR (75 MHz, CDCl3)
δ 170.3, 167.8, 153.8, 138.9, 137.5, 131.0, 127.2, 124.4, 119.0, 118.6, 109.9, 104.1,
60.9, 56.3, 53.9, 51.3. IR (KBr) (νmax/cm-1): 3421, 3067, 2937, 2843, 1690, 1609,
1501, 1459, 1232, 1126, 820, 772. HRMS (ESI): calcd for C19H21N2O6 [M+H]+
373.13941, found 373.13940.
4.1.8. General procedure for synthesis of compounds 10a, 10b
To a solution of compound 9 (25mg, 0.0671 mmol) in toluene (3 mL) was added
oxalyl chloride (57 µL, 0.671 mmol, 10 equiv). The reaction mixture was then stirred
o
at 70 C for 3 h. After completion of the reaction, toluene and the remaining oxalyl
chloride were removed by evaporation to give the crude product without further

ACCEPTED MANUSCRIPT
purification. To a solution of amine in anhydrous THF (1 mL) was added anhydrous
K2CO3 (28 mg, 0.201 mmol, 3 equiv). A mixture of the acyl chloride prepared in the
above step in anhydrous THF (1 mL) was added to the reaction mixture dropwise. The
mixture was stirred at room temperature for 20 min. After completion of the reaction,
the mixture was filtered and the solvent was removed by evaporation to give the crude
product, which was then purified by column chromatography.
4.1.8.1. N-butyl-3-oxo-1-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroquinoxaline
-6-carboxamide (10a). Yield 73.2%, white solid, mp 184.5 – 186.0 oC. 1H NMR (300
MHz, CDCl3) δ 8.72 (brs, 1H), 7.52 (s, 1H), 7.24 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 8.4
Hz, 1H), 6.48 (s, 2H), 6.06 (brs, 1H), 4.39 (s, 2H), 3.90 (s, 2H), 3.84 (s, 3H), 3.81 (s,
6H), 3.44 – 3.51 (m, 2H), 1.55 – 1.62 (m, 2H), 1.37 – 1.47 (m, 2H), 0.95 (t, J = 7.2 Hz,
3H).13C NMR (75 MHz, CDCl3) δ 166.6, 165.6, 153.7, 137.7, 137.5, 131.3, 126.0,
124.9, 122.2, 115.3, 111.1, 104.3, 60.9, 56.2, 53.9, 52.0, 39.9, 31.8, 20.2, 13.8. IR
(KBr) (νmax/cm-1): 3377, 3225, 3074, 2930, 2863, 1693, 1619, 1510, 1459, 1235, 1181,
1127, 817, 767. HRMS (ESI): calcd for C23H30N3O5 [M+H]+ 428.21800, found
428.21802.
4.1.8.2. N-cyclohexyl-3-oxo-1-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroquinoxa
line-6-carboxamide (10b). Yield: 62.4%, white solid, mp 167.1 – 169.0 oC. 1H NMR
(300 MHz, CDCl3) δ 8.80 (brs, 1H), 7.55 (d, J = 1.5 Hz, 1H), 7.24 (dd, J = 8.4, 1.5 Hz,
1H), 6.70 (d, J = 8.4 Hz, 1H), 6.48 (s, 2H), 5.91 (d, J = 8.1 Hz, 1H), 4.39 (s, 2H), 4.01
– 4.07 (m, 1H), 3.90 (s, 2H), 3.84 (s, 3H), 3.81 (s, 6H), 1.95 – 2.08 (m, 2H), 1.68 –
1.80 (m, 3H), 1.39 – 1.50 (m, 2H), 1.15 – 1.23 (m, 3H). 13C NMR (75 MHz, CDCl3) δ
165.7, 165.6, 153.7, 137.6, 137.5, 131.3, 126.1, 125.1, 122.1, 115.5, 111.0, 104.3,
60.9, 56.2, 53.9, 52.1, 48.7, 33.4, 25.6, 24.9. IR (KBr) (νmax/cm-1): 3425, 2931, 2854,
1689, 1620, 1508, 1458, 1383, 1231, 1126, 822, 765. HRMS (ESI): calcd for
C25H32N3O5 [M+H]+ 454.23365, found 454.23367.
4.1.9. General procedure for synthesis of compounds 12a-12e
A mixture of substituted benzoic acids and thionyl chloride (15 equiv) was heated
o
at 70 C for 3 h. After completion of the reaction, remaining thionyl chloride was
removed by evaporation to give the crude product without further purification.

ACCEPTED MANUSCRIPT
4.1.10. General procedure for synthesis of compounds 13a-13l
To a solution of compound 7 in anhydrous THF (1 mL) was added anhydrous
K2CO3 (2 equiv). A mixture of the acyl chloride (2 equiv) prepared in the above step
in anhydrous THF (1 mL) was added to the reaction mixture dropwise. The mixture
was stirred at room temperature for 20 h. After completion of the reaction, the mixture
was filtered and the solvent was removed by evaporation to give the crude product,
which was then purified by column chromatography.
4.1.10.1.
Methyl 1-(4-fluorobenzoyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline-6-
o
1
carboxylate (13a). Yield: 59.7 %, white solid, mp 239.6 – 241.5 C. H NMR (300
MHz, CDCl3) δ 9.31 (brs, 1H), 7.71 (d, J = 1.2 Hz, 1H), 7.39 – 7.53 (m, 3H), 7.02 (t,
J = 8.4 Hz, 2H), 6.70 (d, J = 8.4 Hz, 1H), 4.60 (s, 2H), 3.91 (s, 3H). 13C NMR (75
MHz, CDCl3) δ 168.2, 167.9, 165.9, 164.5 (J = 252), 131.4 (J = 8.25), 131.5, 129.9,
129.6 (J = 3.75), 127.8, 124.3, 124.2, 117.9, 115.9 (J = 21.7), 52.5, 47.6. IR (KBr)
(νmax/cm-1): 3422, 3074, 2957, 2925, 2857, 1713, 1664, 1606, 1497, 1440, 1347, 1296,
1231, 1153, 806, 763. HRMS (ESI): calcd for C17H14FN2O4 [M+H]+ 329.09321,
found 329.09311.
4.1.10.2.
Methyl 1-(4-methoxybenzoyl)-3-oxo-1,2,3,4-tetrahydroquinoxaline
-6-carboxylate (13b). Yield: 58.3 %, white solid, mp 205.5 – 207.2 oC. 1H NMR (300
MHz, CDCl3) δ 9.37 (brs, 1H), 7.70 (d, J = 1.8 Hz, 1H), 7.46 (dd, J = 8.4, 1.8 Hz, 1H),
7.39 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 6.73 (d, J = 8.4 Hz, 1H), 4.59 (s,
2H), 3.90 (s, 3H), 3.82 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 169.0, 168.2, 166.0,
162.3, 132.2, 131.3, 129.7, 127.3, 125.4, 124.3, 124.2, 117.8, 113.8, 55.4, 52.5, 47.7.
IR (KBr) (νmax/cm-1): 3431, 2924, 2852, 1711, 1664, 1607, 1498, 1440, 1345, 1296,
1245, 1168, 906, 843, 764. HRMS (ESI): calcd for C18H17N2O5 [M+H]+ 341.11320,
found 341.11310.
4.1.10.3.
Methyl
1-(3,4-dimethoxybenzoyl)-3-oxo-1,2,3,4-tetrahydro
o
quinoxaline-6-carboxylate (13c). Yield: 46.7 %, white solid, mp 217.0 – 218.8 C.
1H NMR (300 MHz, CDCl3) δ 9.39 (brs, 1H), 7.70 (d, J = 1.8 Hz, 1H), 7.47 (dd, J =
8.4, 1.8 Hz, 1H), 7.04 (d, J = 1.8 Hz, 1H), 6.95 (dd, J = 8.4, 1.8 Hz, 1H), 6.75 (d, J =
8.4 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 4.59 (s, 2H), 3.90 (s, 3H), 3.88 (s, 3H), 3.78 (s,

ACCEPTED MANUSCRIPT
3H).13C NMR (75 MHz, CDCl3) δ 169.0, 168.1, 166.0, 152.0, 148.9, 132.2, 129.6,
127.3, 125.4, 124.3, 124.2, 123.1, 117.8, 112.2, 110.3, 56.0, 52.5, 47.8. IR (KBr)
(νmax/cm-1): 3430, 3070, 2923, 2854, 1705, 1601, 1510, 1362, 1297, 1273, 1220, 1143,
824, 763. HRMS (ESI): calcd for C19H19N2O6 [M+H]+ 371.12376, found 371.12363.
4.1.10.4.
Methyl 3-oxo-1-(3,4,5-trimethoxybenzoyl)-1,2,3,4-tetrahydroquinoxa
o
1
line-6-carboxylate (13d). Yield 58.5 %, white solid, mp 92.0 – 94.6 C. H NMR
(300 MHz, CDCl3) δ 8.99 (brs, 1H), 7.67 (s, 1H), 7.51 (d, J = 8.1 Hz, 1H), 6.83 (d, J
= 8.1 Hz, 1H), 6.64 (s, 2H), 4.59 (s, 2H), 3.91 (s, 3H), 3.87 (s, 3H), 3.70 (s, 6H).13C
NMR (75 MHz, CDCl3) δ 168.9, 167.7, 165.9, 153.1, 141.0, 131.7, 129.6, 128.2,
127.5, 124.3, (2C) 117.6, 106.5, 61.1, 56.2, 52.5, 47.8. IR (KBr) (νmax/cm-1): 3429,
2927, 2853, 1705, 1591, 1494, 1422, 1353, 1298, 1221, 1124, 847, 763. HRMS (ESI):
calcd for C20H21N2O7 [M+H]+ 401.13433, found 401.13437.
4.1.10.5.
Methyl
3-oxo-1-(2-(3,4,5-trimethoxyphenyl)acetyl)-1,2,3,4-
tetrahydroquinoxaline-6-carboxylate (13e). Yield: 63.2 %, white solid, mp 188.0 –
181.9 oC. 1H NMR (300 MHz, CD3OD) δ 7.75 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 8.4 Hz,
1H), 7.51 (s, 1H), 6.26 (s, 2H), 4.45 (s, 2H), 3.92 (s, 2H), 3.90 (s, 3H), 3.71 (s, 6H),
13
3.68 (s, 3H). C NMR (75 MHz, CDCl3) δ 170.4, 165.8, 153.4, 137.0, 130.8, 129.3,
128.7, 124.6, 124.2 (2C), 117.9, 106.4, 105.6, 60.9, 56.1, 52.6, 41.4, 41.1. IR (KBr)
(νmax/cm-1): 3430, 3069, 2929, 2846, 1682, 1596, 1498, 1432, 1373, 1303, 1234, 1190,
1123, 831, 769. HRMS (ESI): calcd for C21H23N2O7 [M+H]+ 415.14998, found
415.14999.
4.1.10.6.
N-butyl-1-(3,4-dimethoxybenzoyl)-3-oxo-1,2,3,4-tetrahydro
quinoxaline-6-carboxamide (13f). Yield: 55.1 %, white solid, mp 224.5-226.7 oC. 1H
NMR (300 MHz, CD3OD) δ 7.50 (s, 1H), 7.20 (d, J = 8.4 Hz, 1H), 6.95 – 7.02 (m,
2H), 6.89 (d, J = 8.7 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 4.50 (s, 2H), 3.82 (s, 3H), 3.69
(s, 3H), 3.31 – 3.39 (m, 2H), 1.51 – 1.63 (m, 2H), 1.34 – 1.42 (m, 2H), 0.95 (t, J = 7.2
Hz, 3H). 13C NMR (75 MHz, CD3OD) δ 169.5, 168.1, 167.4, 152.0, 148.8, 132.1,
130.9, 130.4, 125.8, 124.0, 122.7, 120.5, 115.6, 112.2, 110.6, 55.0 (2C), 39.4, 31.2,
19.8, 12.8. IR (KBr) (νmax/cm-1): 3413, 3076, 2928, 2865, 1696, 1652, 1595, 1549,
1511, 1364, 1267, 1222, 1140, 826, 760. HRMS (ESI): calcd for C22H26N3O5 [M+H]+

ACCEPTED MANUSCRIPT
412.18670, found 412.18668.
4.1.10.7. N-butyl-3-oxo-1-(3,4,5-trimethoxybenzoyl)-1,2,3,4-tetrahydro
o
quinoxaline-6-carboxamide (13g). Yield: 65.3 %, white solid, mp 230.5 – 232.0 C.
1H NMR (300 MHz, CD3OD) δ 7.50 (s, 1H), 7.23 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 8.4
Hz, 1H), 6.70 (s, 2H), 4.51 (s, 2H), 3.77 (s, 3H), 3.69 (s, 6H), 3.32 – 3.37 (m, 2H),
1.52 – 1.62 (m, 2H), 1.34 – 1.44 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz,
MeOD) δ 169.4, 167.9, 167.4, 153.1, 140.5, 132.3, 131.0, 130.1, 129.0, 124.1, 120.5,
115.5, 106.3, 59.8, 55.2, 39.4, 31.2, 19.8, 12.7. IR (KBr) (νmax/cm-1): 3428, 2939,
2868, 1695, 1636, 1587, 1420, 1361, 1239, 1129, 845, 758. HRMS (ESI): calcd for
C23H28N3O6 [M+H]+ 442.19726, found 442.19727.
4.1.10.8.
N-cyclohexyl-3-oxo-1-(3,4,5-trimethoxybenzoyl)-1,2,3,4-tetrahydro
quinoxaline-6-carboxamide (13h). Yield: 54.6 %, white solid, mp 263.9 – 264.9 oC.
1H NMR (300 MHz, CDCl3) δ 9.07 (brs, 1H), 7.84 (s, 1H), 7.07 (d, J = 8.4 Hz, 1H),
6.83 (d, J = 8.4 Hz, 1H), 6.66 (s, 2H), 5.98 (d, J = 8.1 Hz, 1H), 4.56 (s, 2H), 4.01 –
4.10 (m, 1H), 3.88 (s, 3H), 3.73 (s, 6H), 1.99 – 2.08 (m, 2H), 1.64 – 1.80 (m, 3H),
1.24 – 1.53 (m, 2H), 1.25 – 1.29 (m, 3H). 13C NMR (75 MHz, CDCl3) δ 169.0, 167.0,
164.9, 153.2, 140.8, 132.0, 130.6, 130.2, 128.6, 124.0, 119.8, 116.8, 106.4, 61.0, 56.3,
49.1, 48.1, 33.2, 25.5, 24.8. IR (KBr) (νmax/cm-1): 3384, 3281, 2931, 2856, 1692, 1642,
1587, 1502, 1459, 1361, 1214, 1129, 1000, 843, 759. HRMS (ESI): calcd for
C25H30N3O6 [M+H]+ 468.21291, found 468.21289.
4.1.10.9.
1-(3,4-Dimethoxybenzoyl)-3-oxo-N-phenyl-1,2,3,4-tetrahydro
o
quinoxaline-6-carboxamide (13i). Yield: 51.5 %, white solid, mp 274.7 – 276.5 C.
1H NMR (300 MHz, CDCl3) δ 8.25 (brs, 1H), 7.75 (brs, 1H), 7.56 – 7.65 (m, 3H),
7.38 (t, J = 7.5 Hz, 2H), 7.15 – 7.25 (m, 2H), 7.07 (s, 1H), 6.97 (d, J = 8.4 Hz, 1H),
6.85 (d, J = 8.4 Hz, 1H), 6.76 (d, J = 8.1 Hz, 1H), 4.58 (s, 2H), 3.90 (s, 3H), 3.82 (s,
3H). 13C NMR (75 MHz, CDCl3) δ 167.2, 164.0 (2C), 152.0, 149.0, 137.5, 131.2,
129.2, 125.5 (2C), 125.0 (2C), 124.4, 123.0, 120.6, 120.2, 116.0, 112.1, 110.3, 56.0,
48.0. IR (KBr) (νmax/cm-1): 3426, 3297, 3073, 2924, 2854, 1691, 1658, 1595, 1513,
1445, 1369, 1328, 1267, 1142, 824, 759. HRMS (ESI): calcd for C24H22N3O5 [M+H]+
432.15540, found 432.15540.

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4.1.10.10. 3-Oxo-N-phenyl-1-(3,4,5-trimethoxybenzoyl)-1,2,3,4-tetrahydro
o
quinoxaline-6-carboxamide (13j). Yield: 55.3 %, white solid, mp 247.1 – 248.5 C.
1H NMR (300 MHz, CDCl3) δ 9.11 (brs, 1H), 8.07 (brs, 1H), 7.61 – 7.66 (m, 3H),
7.36 (t, J = 7.5 Hz, 2H), 7.27 (s, 1H), 7.15 (t, J = 7.5 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H),
13
6.67 (s, 2H), 4.53 (s, 2H), 3.88 (s, 3H), 3.73 (s, 6H). C NMR (75 MHz, CDCl3) δ
169.2, 167.3, 164.2, 153.2, 140.9, 137.6, 132.3, 130.5, 130.4, 129.2, 128.4, 125.0,
124.3, 120.8, 120.4, 116.3, 106.4, 61.0, 56.3, 48.2. IR (KBr) (νmax/cm-1): 3296, 3065,
2935, 2866, 1690, 1591, 1502, 1446, 1364, 1332, 1238, 1210, 1126, 824, 759. HRMS
(ESI): calcd for C25H24N3O6 [M+H]+ 462.16596, found 462.16595.
4.1.10.11. 4-(3,4-dimethoxybenzoyl)-7-(morpholine-4-carbonyl)-3,4-dihydro
o
1
quinoxalin-2(1H)-one (13k). Yield: 50.3 %, white solid, mp 204.0 – 205.1 C. H
NMR (300 MHz, CDCl3) δ 8.62 (brs, 1H), 7.11 (d, J = 13.8 Hz, 2H), 6.94 (d, J = 8.4
Hz, 1H), 6.83 (s, 2H), 6.74 (d, J = 8.4 Hz, 1H), 4.55 (s, 2H), 3.89 (s, 3H), 3.83 (s, 3H),
3.69 (brs, 4H), 3.54 – 3.58 (m, 4H).13C NMR (75 MHz, CDCl3) δ 169.0 (2C), 167.5,
151.9, 149.0, 132.5, 130.2, 129.6, 125.5, 124.2, 122.9, 121.6, 116.1, 112.1, 110.3,
66.8, 56.0 (2C), 48.1. IR (KBr) (νmax/cm-1): 3350, 3071, 2923, 2856, 1705, 1642, 1514,
1472, 1343, 1268, 1226, 1139, 1111, 1023, 764, 728. HRMS (ESI): calcd for
C22H24N3O6 [M+H]+ 426.16596, found 426.16595.
4.1.10.12.
7-(Morpholine-4-carbonyl)-4-(3,4,5-trimethoxybenzoyl)-3,4-dihydro
o
1
quinoxalin-2(1H)-one (13l). Yield 53.3 %, white solid, mp 205.5 – 207.0 C. H
NMR (300 MHz, CDCl3) δ 8.86 (brs, 1H), 7.16 (s, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.88
(d, J = 8.4 Hz, 1H), 6.66 (s, 2H), 4.54 (s, 2H), 3.87 (s, 3H), 3.74 (s, 6H), 3.69 (brs,
4H), 3.50 – 3.61 (m, 4H). 13C NMR (75 MHz, CDCl3) δ 169.0, 168.8, 167.4, 153.2,
141.0, 132.8, 130.4, 129.0, 128.4, 124.3, 121.5, 116.1, 106.4, 66.8, 61.0, 56.3, 48.3.
IR (KBr) (νmax/cm-1): 3429, 3075, 2924, 2855, 1707, 1666, 1588, 1521, 1464, 1352,
1243, 1141, 837, 782. HRMS (ESI): calcd for C23H26N3O7 [M+H]+ 456.17653, found
456.17651.
4.2. Biological evaluation
4.2.1. Cell lines and culture conditions
The human epithelial cervical cancer cell line HeLa, the human hepatoma cell

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line SMMC-7721 and the human leukemia cell line K562 were cultured in RPMI
1640 medium (HyClone Co., USA), supplemented with 10% fetal bovine serum
o
(Sijiqing Biotechnology Co., China), in 5% CO2 humidified air at 37 C. All the cell
lines were obtained from the American Type Culture Collection (ATCC, USA).
4.2.2. In vitro anti-proliferative activity
The in vitro anti-proliferative activities were determined with an MTT assay.
Compounds were dissolved in DMSO at 5 mmol/L and diluted with medium to obtain
the desired concentration. DMSO concentration in the medium was less than 0.1%.
Cells grown in the logarithmic phase were seeded into 96-well plates (4000-6000
cells/well). Twenty-four hours later, quadruplicate wells were untreated or treated
with different concentrations of drugs. After incubation for 72 h, 50 µL of MTT
solution (10 mg/mL in PBS) was added. Procedure A for the HeLa and SMMC-7721
cell lines: after 4 h of incubation, the medium was removed and replaced with 100 µM
DMSO. The plates were shaken for ten minutes and the optical density was measured
at a wavelength of 490 nm using a microplate reader (Powerwave XS, Biotek, USA).
Procedure B for the K562 cell line: after 4 h of incubation, 100 µM of extraction
solution (10% SDS - 5% isobutyl alcohol - 0.01 M HCl) was added. After incubation
for 12 h, the optical density was measured at a wavelength of 490 nm using the same
microplate reader. Percentages of cell proliferation inhibition versus drug
concentrations were plotted. The values shown in table 1 are the means and standard
deviations of at least three independent experiments.
4.2.3. Tubulin polymerization assay
Tubulin polymerization assay was conducted using a fluorescence-based tubulin
polymerization assay kit (BK011P, Cytoskeleton, USA) according to the manufacturer.
Compounds 8c, 13c and 13d were evaluated for their effect on tubulin polymerization.
Paclitaxel and CA-4 were used as references, 0.1% DMSO as vehicle control.
Compounds at different concentrations were added to the 96-well plate in duplicate.
Ater incubation at 37 oC for 1 min, the icy tubulin reaction mixture (2 mg/mL 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. The samples were mixed and tubulin assembly was monitored

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o
(excitation: 360 nm, emission: 450 nm) at 1 min intervals for 1 h at 37 C using
microplate reader (CLARIOstar, BMG Labtech Inc., Germany). The IC50 values were
calculated after 40 min.
4.2.4. Immunofluorescence microscopy
HeLa cells were seeded in a 96-well plate at 4000 cells per well and incubated for
24 h. Then the cells were treated with vehicle, 13d (0.1 µM, 0.15 µM, 0.2 µM) or
CA-4 (0.05 µM) for 24 h. The cells were fixed in 4% paraformaldehyde for 10 min at
o
4 C and permeabilized with 0.1% Triton X-100 in PBS for 5 min at 4 oC. After
washed with PBS, cells were blocked with 1% bovine serum albumin (BSA) in PBS
for 1h, and incubated with the primary antibody (Catalog No. BM1452, Boster Co.
Ltd., China, 1: 100 dilution in 2% BSA-PBS). After incubation for 12 h at 4 oC, the
cells were washed with PBS to remove unbound primary antibody, and incubated with
antimouse secondary antibody conjugated with FITC for 30 min at 37 oC (Catalog No.
BA1101, Boster Co. Ltd., China, 1: 100 dilution in 1% BSA-PBS). The cells were
washed with PBS to remove unbound secondary antibody, the nuclei were stained
with Hoechst 33342 and immunofluorescence was detected using a fluorescence
microscope (Leica TCS SP8, Germany).
4.2.5. Cell cycle analyses
HeLa cells were seeded in a 6-well plate at 1 × 105 cells per well and incubated
for 24 h. Then the cells were treated with vehicle (0.1% DMSO) or different
concentrations of 13d (0.1 µM, 0.15 µM, 0.2 µM, 0.25 µM) for 24 h. Cells were
harvested and fixed with ice-cold 70% ethanol at 4 oC for 12 h. Ethanol was removed
and the cells were washed with cold PBS. Then cells were incubated in 0.5 mL of
PBS containing 1 mg/mL Rnase for 30 min at 37 oC. Then the cells were stained with
o
50 µg/mL propidium iodide at 4 C in the dark for 30 min. The DNA contents of
20000 events were measured by flow cytometer (BD FACSVerse, USA).
4.2.6. Annexin-V assay
Annexin-V assay was conducted using a FITC Annexin-V apoptosis detection kit
I (Catalog No. 556547, BD PharmingenTM, USA). HeLa cells were seeded in a 6-well
plate (1 × 105 cells/well). After incubation for 24 h, the cells were treated with vehicle

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(0.1% DMSO) or various concentrations of 13d (0.1 µM, 0.15 µM, 0.2 µM) for 24 h.
The cells were harvested and washed with PBS, then the cells were stained with 5 µL
of annexin V-FITC and 5 µL PI in binding buffer (10 mM HEPES, 140 mM NaCl and
2.5 mM CaCl2 at pH 7.4) for 15 min at room temperture in the dark. The samples
were analyzed using the flow cytometer as mentioned above.
4.2.7. Colchicine competitive binding assay
Tubulin (4 µM, HTS03-A, Cytoskeleton, USA) in buffer containing 80 mM
PIPES (pH 6.9), 2 mM MgCl2 and 0.5 mM EGTA was incubated with different
concentrations (0.1 µM, 1 µM, 5 µM, 10 µM) of 13d or paclitaxel at 37 oC for 1 h in a
nontransparent black 96-well plate, with DMSO as control. Then, colchicine (10 µM)
o
was added to the mixture, which was incubated at 37 C for an additional 1 h. The
fluorescence intensity of the tubulin-colchicine complex (excitation: 340 nm,
emission: 435 nm) was measured using a microplate reader (CLARIOstar, BMG
Labtech Inc., Germany). Each experiment was performed independently with at least
three replicates and expressed as the mean ± SD.
4.3. Molecular modelling
The docking study was carried out using Sybyl-X 2.1. The crystal structure of
tubulin complexed with DAMA-colchicine was retrieved from PDB (1SA0). The 3D
structure of 13d was built using Sybyl-X 2.1 and minimized using tripos force field
and Gasteiger-Huckel charges. Powell’s method was employed with gradient
convergence criteria of 0.005 kcal/mol. The DAMA-colchicine structure was
extracted and the tubulin structure was prepared and minimized with Amber7 F99
force field. The binding site was constructed by the ligand protomol of surflex dock.
Then 13d was docked into the DAMA-colchicine binding site. The 20 final docked
conformations of 13d were ranked according to the total score. The docking mode
was chosen on the basis of total score.
Acknowledgements
The authors are grateful for the financial support from the National Natural
Science Foundation of China (No. 21272056, No. U1404819) and Program for

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Science Technology Innovation Talents in Universities of Henan Province
(16HASTIT029). In additon, this work was also supported by Key Scientific and
Technological Projects of Henan Province (Grant No. 132300410228) and Key
Scientific Research Program of the Higher Education Institutions of Henan Province
(Grant No. 15A350005, Grant No. 2011B310003).
Supplementary material
The 1H and 13C spectra of target compounds.
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