Activated carbon/Br?nsted acid-promoted aerobic benzylic oxidation under “on-water” condition: Green and efficient synthesis of 3-benzoylquinoxalinones as potent tubulin inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111894

Green chemistry is becoming the favored approach to preparing drug molecules in pharmaceutical industry. Herein, we developed a clean and efficient method to synthesize 3-benzoylquinoxalines via activated carbon promoted aerobic benzylic oxidation under “on-water” condition. Moreover, biological studies with this class of compounds reveal an antiproliferative profile. Further structure modifications are performed and the investigations exhibited that the most active 12a could inhibit the microtubule polymerization by binding to tubulin and thus induce multipolar mitosis, G2/M phase arrest, and apoptosis of cancer cells. In addition, molecular docking studies allow the rationalization of the pharmacodynamic properties observed. Our systematic studies provide not only guidance for applications of O₂/AC/H₂O system, but also a new scaffold targeting tubulin for antitumor agent discovery.

Full text of DOI:10.1016/j.ejmech.2019.111894

Journal Pre-proof
Activated carbon/Brønsted acid-promoted aerobic benzylic oxidation under “on-water”
condition: Green and efficient synthesis of 3-benzoylquinoxalinones as potent tubulin
inhibitors
Qi Guan, Lin Cong, Qing Wang, Changyue Yu, Kai Bao, Kai Zhou, Lan Wu, Weige
Zhang
PII:
S0223-5234(19)31046-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.111894
EJMECH 111894
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 October 2019
Revised Date: 31 October 2019
Accepted Date: 14 November 2019
Please cite this article as: Q. Guan, L. Cong, Q. Wang, C. Yu, K. Bao, K. Zhou, L. Wu, W. Zhang,
Activated carbon/Brønsted acid-promoted aerobic benzylic oxidation under “on-water” condition: Green
and efficient synthesis of 3-benzoylquinoxalinones as potent tubulin inhibitors, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111894.
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Activated Carbon/Brønsted Acid-Promoted Aerobic Benzylic Oxidation under
“On-Water” Condition: Green and Efficient Synthesis of
3-Benzoylquinoxalinones as Potent Tubulin Inhibitors
Qi Guan,a,d Lin Cong,a Qing Wang,b Changyue Yu,a Kai Bao,b Kai Zhou,d* Lan Wuc*
and Weige Zhang a*
a
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, China
b School of Wuya, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang
110016, China
c Department of Geratology, The First Affiliated Hospital, China Medical University, 155 Nanjing
North Street, Heping District, Shenyang 110001, China
d Department of Pharmacology, Northeast Pharmaceutical Group Co.,Ltd., 8 Kunming Lake Street,
Economic-Technological Development Area, Shenyang 110027, China
Abstract
Green chemistry is becoming the favored approach to preparing drug molecules in pharmaceutical
industry. Herein, we developed a clean and efficient method to synthesize 3-benzoylquinoxalines
via activated carbon promoted aerobic benzylic oxidation under “on-water” condition. Moreover,
biological studies with this class of compounds reveal an antiproliferative profile. Further
structure modifications are performed and the investigations exhibited that the most active 12a
could inhibit the microtubule polymerization by binding to tubulin and thus induce multipolar
mitosis, G2/M phase arrest, and apoptosis of cancer cells. In addition, molecular docking studies
allow the rationalization of the pharmacodynamic properties observed. Our systematic studies
provide not only guidance for applications of O2/AC/H2O system, but also a new scaffold
targeting tubulin for antitumor agent discovery.
Keywords: Green chemistry, on-water, 3-benzoylquinoxalinone, tubulin inhibitor, molecular
modelling.

1. Introduction
In the last decades, green chemistry gave medicinal chemists a new way of looking at the design
of drug molecules and the processes to make them that reduces or eliminates the impact on the
environment. Green chemistry principles have now become a strategic focus for pharmaceutical
industry. Benzylic oxidation is one of the most important synthetic transformations in organic
chemistry, as generated products may act as valuable building blocks for the manufacture of
pharmaceuticals.1 The development of oxidants for benzylic oxidation has therefore attracted
considerable interest. Therein, molecular oxygen is an ideal oxidant, as water is the sole
by-product. During the past few years, the benzylic oxidation of alkylaromatics to the
corresponding carbonyl compounds using molecular oxygen as oxidant has been reported
frequently, but organic solvents were generally used as the reaction medium.2-9 Recent trends in
organic synthesis involve reactions of water-insoluble organic compounds that take place under
“on-water” condition have received a great deal of attention because of their high efficiency and
operational convenience.10-12 However, benzylic oxidation via the direct utility of molecular
oxygen from air as oxidant under “on-water” condition, which is one of the requirements for the
realization of green chemical processes, remains a tremendous challenge in current organic
synthesis and industrial chemistry. This leads us to development of a novel catalytic process to
accomplish the green or greener synthesis.
Recent efforts in our laboratory revealed that activated carbon (AC) could promote the
aerobic oxidation of 2-benzylbenzimidazoles and 2-benzylbenzoxazoles to the corresponding
carbonyl products in the “on water” heterogeneous system (Scheme 1A).13 While the AC is found
to be an ideal catalytic system for the aerobic oxidation under on-water condition, both reaction
efficiency and substrate scope remain to be substantially improved. In our previous work, we also
have observed that the oxidation yield is related to the electric density at the benzylic position of
the substrate. This prompts us to verify whether the rule of computational electric density can be
used to search the substrate that is suitable for our approach.

Scheme 1. Our previous work and this work.
The molecular scaffolds of quinoxaline are of great interest in medicinal chemistry since they
are found in a large number of natural products and possess a wide range of biological
activities.14-20 Moreover, 3-benzoylquinoxalines, as an important structural motif, have been also
widely used as a synthon for the synthesis of bioactive products. General methods for the
preparation of 3-benzoylquinoxalines are oxidation of benzyl to corresponding carbonyl
compounds. Up to date, only few literatures have reported to achieve this transformation, and yet
all these methods have serious environmental impacts because of the required using
(super)stoichiometric quantities of Cr(VI) oxide as oxidant in organic solvent.21-23 We found that
the theoretically calculated electric density at the benzylic position of 3-benzylquinoxaline (3) was
well close with the data of 2-benzylbenzimidazole (1) and 2-benzylbenzoxazole (2) (Fig. 1).
Based on this result and our previous work, it is tempting to speculate that the O2/AC/H2O may be
a proper oxidation system for this oxidation transformation and we wish to explore the effect of
pH on this transformation.24 Even more intriguing, we noticed that 3-benzoylquinoxalinone
framework is as similar as some tubulin inhibitors, such as phenstatin (4),25 BNC-105P (5),26
BPR0L075 (6),27 and 7 (Fig. 2),28 which are receiving growing attention in anticancer drug
discovery. As a continuation of an ongoing research on the development of “green and sustainable”
methodologies for the synthesis of potential functional molecules, we herein wish to report a novel
strategy to extend the applications of AC promoted aerobic benzylic oxidation and develop a clean
method to synthesize 3-benzoylquinoxalines. This would not only offer potential biological
applications but also represent a significant contribution to green medicinal chemistry.

Figure 1. Connolly surface (probe radius = 1 Å) mapped with the molecular electrostatic potential (MEP),
quantum chemical calculations performed with Gaussian 09; Blue/red areas represent negative/positive
electrostatic potentials.
Figure 2. Structures of some tubulin inhibitors.
2. Results and Discussion
Chemistry and biological studies.
In order to validate our hypothesis, 3-benzylquinoxalin-2-ol (8a) was initially selected as the
model substrate to explore the predicted reactivity (Table 1). To our delight, the reaction
proceeded to afford selectively the desired product 9a in 83.4% yield when the suspension of AC
and the insoluble substrate 8a in water under oxygen atmosphere was stirred at 85 °C for 24 h
(Table 1, entry 3). To improve the efficiency of this transformation, various Brønsted-acids as
additives were also screened to examine their effects on the reaction. As shown in Table 1, we
were glad to see that the yield of 9a could be obviously improved to 71.9% after adding 10.0
equivalents of AcOH to the above aqueous solution at 85 °C for 4 h (Table 1, entry 4).
Replacement of AcOH with other acids, including HCOOH, H2SO4, TsOH and TFA, could also
accelerate the aerobic oxidation process (Table 1, entries 5‒8). Remarkably, the yield of 9a could
be improved to 94.4% in the presence of 10.0 equivalents of TFA (Table 1, entry 8). Further
optimization revealed that this TFA-promoted transformation could be performed using lower
additive loadings in aqueous solution (Table 1, entries 9‒11) while still giving 9a with a high yield
(>85%). Gratifyingly, increase of the reaction temperature to 100 °C led to completion of the
process in 4 h with 91.2% yield of 9a but no 9a′ was observed (Table 1, entry 13). Extending the
reaction time was unfavorable for the reaction (Table 1, entry 14). Based on these results, the

reaction could be directed cleanly to the desired products, 3-benzoylquinoxalines, by optimization
of the reaction conditions. Most importantly, this “on water” reaction is operationally
straightforward.
Table 1. Optimization of the Reaction Conditionsa
Entry
Additive (equiv)
Temperature (°C)
Time (h)
Ratio (9a′: 9a)c
1a
2b
3b
4b
5b
6b
7 b
8 b
9 b
10 b
11 b
12 b
13 b
14 b
none
85
85
85
85
85
85
85
85
85
85
85
70
100
100
4
4
24
4
4
4
4
4
4
4
4
4
4
6
NR
none
21%: 19.1%
4.5%: 83.4%
15.0%: 71.9%
9.8%: 76.9%
5%: 67.5%
5.2%: 90.4%
1.2%: 94.4%
0.9%: 87.6%
1.1%: 86.7%
1.4%: 85.8%
6.5%: 79.4%
0%: 91.2%
none
AcOH (10.0)
HCOOH (10.0)
H2SO4 (10.0)
TsOH (10.0)
TFA (10.0)
TFA (5.0)
TFA (3.0)
TFA (1.0)
TFA (1.0)
TFA (1.0)
TFA (1.0)
0%: 88.1%
b
aReaction conditions: 3-benzylquinoxalin-2-ol (0.2 mmol) in H2O (3.0 mL) under O2 atmosphere. Reaction
conditions: 3-benzylquinoxalin-2-ol (0.2 mmol), 50 wt % of AC, and additive in H2O (3.0 mL) under O2
c
atmosphere. Yields and the ratio of 9a′ to 9a were determined by HPLC analysis of the crude reaction mixture.
NR = no reaction.
Having established the optimal reaction conditions, we subsequently turned our focus to
explore the substrate scope of this protocol. The results are presented in Table 2. It was found that
AC-TFA pairing could effectively promote the aerobic oxidation of 3-benzylquinoxaline
derivatives to the corresponding carbonyl products in the “on water” heterogeneous system with
moderate to acceptable isolated yield (65‒85%). The presence of an electron-withdrawing group at
the phenyl ring somewhat lowered the yields (entries 3, 6, 7, 9 and 10). On the other hand, the
substrates with electron-donating groups at the 2-position of the quinoxaline ring showed higher
reactivity than those with electron-withdrawing groups on the same position (entries 8‒10 vs 5‒7).
Table 2 Substrate Scopea and Their Antiproliferative Activities in Vitrob

Entry
Product
9a
R1
H
R2
R3
H
Y
Yieldc (%)
IC50 (µM)
>30
1
H
OH
OH
OH
OH
Cl
71
80
77
65d
75
73
72
85
83
81
2
H
OCH3
Cl
H
>30
9b
9c
3
H
H
>30
4
OCH3
H
OCH3
OCH3
Cl
OCH3
H
0.47 ± 0.02
>30
9d
9e
5
6
H
H
Cl
>30
9f
7
H
Br
H
Cl
>30
9g
8
H
OCH3
Cl
H
OCH3
OCH3
OCH3
>30
9h
9i
9
H
H
>30
10
H
Br
H
>30
9j
aReaction conditions: 3-benzylquinoxalines (0.2 mmol), 50 wt % of AC, and TFA (0.2 mmol) in H2O (3.0 mL) at
100 °C for 4 h. bA549 cell line. cIsolated yield. dReaction conditions: 8d (0.2 mmol), 50 wt % of AC, and TFA (0.6
mmol) in H2O (3.0 mL) at 85 °C for 4 h.
Generally, the reaction process of AC-TFA promoted aerobic benzylic oxidation under
“on-water” condition is depicted in Scheme 2A. We speculate that the substrates are adsorbed on
the surface of the superhydrophobic porous activated carbon and the reaction is carried out at
air-liquid-solid joint interfaces where the hydrogen-bonding activation, absorption and
hydrophobic effects work together to achieve the accelerating effect. In addition, it was reported
that molecular oxygen could be activated and improved dissolution in aqueous TFA solution.24
Hence, a plausible mechanism for the formation of 3-benzoylquinoxalinones is proposed in
Scheme 2B. These two pathways might both involve the formation of peroxide (b) as a key
intermediate. Under the acidic conditions, b spontaneously evolved to the products c and f,
respectively. The hydroxyl product c could be further converted into the corresponding carbonyl
product f via oxidative dehydrogenation.

Gas phase
Solid phase
Adsorption phase
Water phase
Scheme 2. Proposed reaction model (A) and mechanism (B) of AC-TFA promoted aerobic benzylic oxidation
under “on-water” condition.
The screening of potential biological activity in synthetic or natural products is a significant
step in drug discovery and development process. Since they are structurally correlated with CSBIs,
the newly synthesized compounds 9a‒j were first screened for antiproliferative activities against
human lung adenocarcinoma cell line (A549). The result was shown in Table 2. As anticipated, 9d
showed potent antiproliferative activity and it suggested the essential role of 3,4,5-trimethoxy
substituents on the phenyl ring. This finding is consistent with results from most of the known
tubulin inhibitors in which the 3,4,5-trimethoxy substituted compound was most potent (Fig. 2).
To investigate the activity further, 9d was chosen as a lead compound for further structure
modification (Scheme 3 and 4). We retained the 3,4,5-trimethoxyphenyl group of 9d and mainly
focused on modifications to the substituent at the 2- and 3-positions of the quinoxaline ring. The
general synthesis of the modified derivatives of 9d is outlined in Schemes 1‒2. Firstly, chlorination
of 9d with neat POCl3 gave 10 in 91% yield,29 and 9d was converted into 11 by treatment with
Bu4NBr and P2O5 in refluxing toluene for 5 h in 75% yield.30 Subsequently, 11 was substituted
with different alcoholate or amino derivatives to form the corresponding products (12a‒n and
14a‒d). On the other hand, 9d reacted with the appropriately alkylating reagent in the presence of
KI and Na2CO3 in DMF, affording N-alkyl products (13a‒f).29

Scheme 3. Reagents and conditions: (i) POCl3, reflux, 3 h; (ii) P2O5, TBAB, toluene, reflux, 5 h; (iii) Na, ROH,
reflux, 0.5 h; (iv) R1X, Na2CO3, KI, DMF, 40~80 °C, 4 h.
Scheme 4. Reagents and conditions: (i) CH3NH2, THF, 50 °C, overnight; (ii) HY, microwave, 150 °C, 0.5 h.
Antiproliferative activity assays and SAR analysis. The synthesized compounds 10‒14 were
evaluated for their antiproliferative activities in three different human cancer cell lines (HT-1080,
SGC-7901 and A549). Combretastatin A-4 (CA-4), a well-known tubulin inhibitor, was included in
the assay as positive control. The results are summarized in Table 3. As shown in Table 3, 12a
displayed the most active compound. As compared to 12a, 12b had about 10-fold decrease in
activity likely due to its one-carbon longer alkoxy chain. Similarly, the longer and bulkier group at
2-postion of the quinoxaline ring, such as n-pentyloxy (12g), i-pentyloxy (12h), benzyloxy (12n)
and but-3-en-1-yloxy (12m), was tolerated but with a significant activity decline. Whereas
replacing the alkoxy chains with electron-withdrawing groups resulted in complete loss of
antiproliferative activities (11 and 12). Additionally, it is noted that the incorporation of a
comparatively polar amine did not increase the activity significantly (14a‒d). The activity
variation of the substituent at 2-postion of the quinoxaline ring suggests that the activity drop is
truly related to steric hindrance and electrical effect but not solubility. One interesting

phenomenon is that the modifications at 1-postion of the quinoxaline ring almost abrogated
activity (13a‒f).
Table 3. Growth Inhibition IC50 Values of Synthesized Quinoxaline Derivatives in Vitro
IC50 (µM)a
Compound
HT-1080
0.74 ± 0.05
>30
SGC-7901
0.88 ± 0.07
>30
A549
0.47 ± 0.02
>30
9d
10
>30
15.20 ± 1.35
0.24 ± 0.03
1.48 ± 0.11
0.66 ± 0.17
0.68 ± 0.08
4.58 ± 0.29
0.97 ± 0.04
3.43 ± 0.29
3.56 ± 0.18
0.84 ± 0.10
1.55 ± 0.22
1.81 ± 0.19
2.20 ±0.14
4.65 ± 0.26
4.46 ± 0.16
>30
>30
11
12a
12b
12c
12d
12e
12f
0.17 ± 0.01
1.60 ± 0.23
2.30 ± 0.31
2.06 ± 0.16
6.94 ± 0.35
2.13 ± 0.19
6.35 ± 0.42
6.13 ± 0.38
0.26 ± 0.07
1.90 ± 0.10
2.56 ± 0.13
1.18 ± 0.09
5.56 ± 0.36
6.50 ± 0.27
>30
0.28 ± 0.01
2.96 ± 0.16
3.23 ± 0.21
0.41 ± 0.05
2.09 ± 0.13
0.72 ± 0.06
2.43 ± 0.14
2.16 ± 0.12
1.28 ± 0.33
0.79 ± 0.15
1.32 ± 0.14
0.78 ± 0.06
0.82 ± 0.03
6.96 ± 0.44
>30
12g
12h
12i
12j
12k
12l
12m
12n
13a
13b
13c
13d
13e
13f
>30
>30
>30
>30
9.66 ± 0.92
>30
>30
10.86 ± 1.19
>30
>30
>30
>30
>30
>30
14.97 ± 1.06
8.67 ± 0.28
1.62 ± 0.17
1.28 ± 0.13
9.67 ± 0.44
0.012 ± 0.002
1.42 ± 0.22
6.07 ± 0.35
2.11 ± 0.21
25.83 ± 1.37
0.016 ± 0.003
7.35 ± 0.31
2.21 ± 0.29
0.68 ± 0.14
15.47 ± 1.05
0.024 ± 0.003
14a
14b
14c
14d
CA-4c
a
Bold values show IC50 of < 1 µM. IC50: Concentration of the compound (µM) producing 50% cell growth
inhibition after 72 h of drug exposure, as determined by the MTT assay. Each experiment was carried out in
b
triplicate. pIC50 values of A549 cell line. The pIC50 values are used 4.52 when the obtained IC50 is greater than
30µM. cUsed as a positive control.
Inhibition of Tubulin Polymerization. As we mentioned before, the quinoxaline derivatives were
originally desired to inhibit the tubulin polymerization and induce apoptosis. To validate this
biological target, the most active compound 12a was evaluated by microtubule dynamics assay in
parallel with taxol and CA-4 as reference. The result in this assay is shown in Fig. 3. At 1 µM, 12a

showed a weak antitubulin polymerization activity. When the concentration of 12a was increased
to 3.3 µM, a clear inhibition of polymerization was noted, and the rate of assembly as well as the
final amount of microtubules was lower than that in the control experiment. In contrast, CA-4 at 2
µM nearly completely inhibited tubulin polymerization. These data confirmed that the mechanism
of action of 12a was to inhibit tubulin polymerization.
Figure 3. Effect of 12a on microtubule dynamics.
Analysis of immunofluorescence staining. Next, we performed immunocytochemistry studies to
test effect of 12a on the cellular microtubule structure stained for DNA (blue) and α-tubulin
(green). Untreated cells were used as a negative control, and cells treated with CA-4 were used as
a positive control. As shown in Fig. 4, the microtubule network in HT-1080 and SGC-7901 cells
exhibited normal arrangement and organization in the absence of drug treatment. In contrast,
either 12a or CA-4 caused dramatic reduction of the interphase microtubule network wrapped
around the cell nucleus. These results indicated that 12a was like CA-4 as
a
microtubule-destabilizing agent.

Figure 4. Effects of 12a (0.34 µM) and CA-4 (0.032 µM) on the cellular microtubule network and microtubule
reassemble by immunofluorescence. HT-1080 and SGC-7901 cells were treated with 12a or CA-4 for 48 h, and
then direct microscopy detection of the fixed and stained cell was performed. The cellular microtubules were
stained with anti-α-tubulin-FITC specific antibodies (green). DNA was stained by 4′,6-diamidino-2-phenylindole
(DAPI, blue).
Cell cycle analysis. Because of the above results, 12a was also subjected to analysis for its effect
on cell cycle distribution by flow cytometry. HT-1080 cells were treated with different
concentrations of 12a for 12 and 24 h, respectively. The tested data are summarized in Fig. 5 and
Fig. 6. Cells treated with either 12a or CA-4 were observed an obvious G2/M phase arrest,
whereas control cells were mainly in the G0/G1 phase. The effect of 12a on cell cycle distribution
patterns was similar to the positive control CA-4, and it showed dose-dependent and
time-dependent clearly.
Figure 5. (A) Cell cycle distribution of HT-1080 cells with or without treatment of 12a for 12 h. (B) The statistical
histogram of the tested data.

Figure 6. (A) Cell cycle distribution of HT-1080 cells with or without treatment of 12a for 24 h. (B) The statistical
histogram of the tested data.
Molecular docking. To rationalize our experimental data from a structure-based perspective, a
series of molecular docking simulations of these new compounds in the colchicine site of tubulin
were performed by using a procedure reported previously.31, 32 As shown in Fig. 7A, 12a displayed
a binding conformation with low energy (-45.13 kcal/mol). It was found that the binding
conformation of 12a generated in the docking simulation was very similar to the conformation of
Phenstatin and the native ligand (Fig. 7D). Colchicine derivatives with groups substituted at the
methoxy positions can cross-link with Cys-β241.33 By examining the ligand interactions for the
docked compounds, it becomes apparent that the trimethoxyphenyl moieties of Phenstatin and 12a
were well overlapped with each other, and occupied a hydrophobic region in the binding site and
established a series of interactions with Cys-β241, Val-β238, and Leu-β255 (Fig. 7B). It has been
noted that, for many CA-4 analogues and Phenstatin, the 3-hydroxy-4-methoxyphenyl moiety was
essential for activity and the docked solution presented here indicated that the p-methoxy group on
the 3-hydroxy-4-methoxyphenyl of Phenstatin played a role in sterically interacting with the
Thr-α179 (Supporting Information, Fig. S1). For 12a, the quinoxaline moiety was well overlapped
with the 3-hydroxy-4-methoxyphenyl moiety of Phenstatin, and formed van der Waals forces with
the hydrophobic pocket near α-tubulin, and Asn-β258 formed a hydrogen bond with the nitrogen
atom at 1-position on the quinoxaline ring. The key hydrogen bond could stabilize the interaction
of 12a with the binding pocket, and this might explain why alkylation at this position showed a
dramatic loss in activity. From the results in Table 2, a larger group, such as 12e, 12g and 12n, at
2-position on the quinoxaline ring was unfavorable to activity. Indeed, the methoxy was the group
with optimal volume for interaction with the binding pocket as shown in Fig. 7C, and the docking

simulations of larger group compounds were not able to generate a reasonable binding pose. In
general, these docking results were in accordance with the data in the cell lines tested, and they
provided a possible structural justification of the SARs observed.
Figure 7. (A) Proposed binding for 12a (green) in the colchicine site. Residues within 3.5 Å of 12a are represented
as the thicker sticks. Hydrogen bonds (distance of < 3 Å) are shown as dotted green lines. (B) A interaction sketch
between 12a and its binding site. (C) The hydrophobicity surface on the receptor. (D) Superposition of 12a (green)
with the docking modes of CA-4 (gray) and co-crystallized DAMA-colchicine (purple).
3. Conclusion
In summary, a full account of approach for searching the proper substrate, which is suitable for
O2/AC/H2O system promoted aerobic benzylic oxidation, has been presented by analysis the
structure computationally. Use of 3-benzylquinoxalinones as substrates provides an experimental
proof, which may be applicable to our strategy. Data indicated that a Brønsted acid, especially
TFA, is able to significantly improve the efficiency of the reaction process and achieve high yield.
Among the resulting products, 9d exhibited excellent antiproliferative activity and was chosen to
structure modification. The in vitro immunofluorescence staining and tubulin polymerisation
assay demonstrated that 12a as an analogue of 9d inhibited tubulin polymerisation into
microtubules and caused microtubule destabilisation, which suggests that 12a is a novel tubulin
inhibitor. Further investigations are in progress in our laboratory to evaluate the strategy with a

broader range of substrates, and to synthesize more complex products that comprises privileged
scaffolds displaying diverse range of biological activities. We hope to provide more green
versions of the reactions for medicinal chemists to construct molecules in the led-seeking or
candidate-seeking stages of drug discovery.
4. Experimental section
4.1. Chemistry
All of reagents and solvents were purchased from chemical company. The AC was purchased
from Tokyo Chemical Industry (TCI) (catalogue number: C2194). 1H-NMR and 13C-NMR spectra
were tested in CDCl3 or d6-DMSO with TMS as the internal reference on a Bruker AVANCE 400
or 600 (1H at 400 or 600 MHz, 13C at 150 MHz). Mass spectra (MS) were measured on an Agilent
1100-sl mass spectrometer with an electrospray ionisation source from Agilent Co. Ltd. High
resolution accurate mass determinations (HRMS) for all of the final target compounds were
obtained on a Bruker Micromass Time of Flight mass spectrometer equipped with electrospray
ionisation (ESI). TLC analysis was used for determining the extent of reactions under UV light
(wavelength: 365 nm and 254 nm). Melting point was measured (uncorrected) on hot-stage
microscope (Beijing Taike, X-4).
4.1.1. General synthetic procedures for 3-benzoyl-2-methoxyquinoxaline (9a–j).
A mixture of substrate (8a–j) (0.2 mmol), trifluoroacetic acid (0.2 mmmol) and 50 wt % of
activated carbon in water (5 mL) was placed in a sealed tube (100 mL). The system was purged
with molecular oxygen and sealed. The reaction mixture was heated to 100°C and stirred for 4 h.
Upon completion, the reaction mixture was cooled to room temperature. After filtration of the
suspension and washing of the solids with EtOAc, the crude product was obtained by removal of
the solvent.
4.1.1.1. 3-(Hydroxy(phenyl)methyl)quinoxalin-2(1H)-one (9a′)
A mixture of 8a (0.2 mmol) and 50 wt % of activated carbon in water (5 mL) was placed in a
sealed tube (100 mL). The system was purged with molecular oxygen and sealed. The reaction
mixture was heated to 85°C and stirred for 4 h. Upon completion, the reaction mixture was cooled
to room temperature, filtered and washed with EtOAc. The removal of the solvent yielded a
residue that was purified by silica gel column chromatography (n-hexane: EtOAc=8:1 as eluent)
on silica gel to afford pure product. White solid (12% yield); Mp: 192–194°C; 1H NMR (600 MHz,

DMSO-d6): δ=12.38 (s, 1H), 7.72 (d, 1H, J=7.7 Hz), 7.47 (m, 1H), 7.27 (m, 2H), 6.65 (s, 2H),
4.05 (s, 2H), 3.72 (s, 6H), 3.61 (s, 3H); HRMS: calcd. for C15H13N2O2 [M+H]+ 253.0977, found
253.0979.
4.1.1.2. 3-Benzoylquinoxalin-2(1H)-one (9a)
1
Yellowish solid (71% yield); Mp: 179–181°C; H NMR (600 MHz, DMSO-d6): δ=12.38 (s, 1H),
7.72 (d, 1H, J=7.7 Hz), 7.47 (m, 1H), 7.27 (m, 2H), 6.65 (s, 2H), 4.05 (s, 2H), 3.72 (s, 6H), 3.61 (s,
3H); 13C NMR (150 MHz, DMSO-d6): δ=129.9, 156.7, 153.9, 135.1, 135.0, 133.2, 132.2, 131.7,
130.1 (×2), 129.6, 129.5 (×2), 124.3, 116.4; HRMS: calcd. for C15H10N2NaO5 [M+Na]+ 273.0640,
found 273.0638.
4.1.1.3. 3-Benzoylquinoxalin-2(1H)-one (9b)
1
Yellowish solid (80% yield); Mp: 244–245°C; H NMR (600 MHz, DMSO-d6): δ=12.68 (s, 1H),
7.93 (d, J=8.6 Hz, 2H), 7.81 (d, J=7.8 Hz, 1H), 7.63 (t, 1H), 7.38 (m, 2H), 7.07 (d, J=8.6 Hz, 2H),
3.8 (s, 3H); 13C-NMR (125 MHz, DMSO-d6): δ=191.2, 157.1, 164.8, 153.9, 133.1, 132.7 (×2),
131.9, 131.7, 129.5, 128.0, 124.3, 116.3 (×2), 114.8, 56.2; HRMS: calcd. for C16H11N2O3 [M–H]–
279.0770, found 279.0774.
4.1.1.4. 3-(4-Chlorobenzoyl)quinoxalin-2(1H)-one (9c)
1
Yellowish solid (77% yield); Mp: 242–244°C; H NMR (600 MHz, DMSO-d6): δ=8.41 (s, 1H),
7.98 (d, J=8.5 Hz, 2H), 7.79 (d, J=8.1 Hz, 1H), 7.62 (m, 3H), 7.45 (d, J=8.1 Hz, 1H), 7.34 (t, 1H);
13C-NMR (125 MHz, DMSO-d6): δ=191.8, 156.1, 153.8, 140.1, 133.8, 133.3, 133.4, 132.4, 132.0
(×2), 129.7, 129.6 (×2), 124.3, 116.4; HRMS: calcd. for C15H8ClN2O2 [M–H]– 283.0274, found
283.0270.
4.1.1.5. 3-(3,4,5-Trimethoxybenzoyl)quinoxalin-2(1H)-one (9d)
A mixture of substrate (8d) (0.2 mmol), trifluoroacetic acid (0.6 mmmol) and 50 wt % of activated
carbon in water (3 mL) was placed in a sealed tube (100 mL). The system was purged with
molecular oxygen and sealed. The reaction mixture was heated to 85°C and stirred for 4 h. Upon
completion, the reaction mixture was cooled to room temperature. After filtration of the
suspension and washing of the solids with EtOAc, the crude product was obtained by removal of
the solvent. Yellowish solid (83% yield); Yellowish solid (65% yield); Mp: 222–224°C; 1H NMR
(600 MHz, DMSO-d6): δ=12.78 (s, 1H), 7.82 (d, J=8.0 Hz, 1H), 7.64 (t, J=7.3 Hz, 1H), 7.40 (d,
J=8.0 Hz, 1H), 7.36 (t, J=7.3 Hz, 1H), 7.27 (s, 2H), 3.80 (s, 6H), 3.78 (s, 3H); 13C NMR (125

MHz, DMSO-d6): δ=191.6, 156.5, 153.9, 153.4 (×2), 143.7, 133.3, 132.1, 131.8, 130.4, 129.6,
124.2, 116.3, 107.9 (×2), 60.8, 56.8 (×2); HRMS: calcd. for C18H17N2O5 [M+H]+ 341.1137, found
341.1136.
4.1.1.6. (3-Chloroquinoxalin-2-yl)(4-methoxyphenyl)methanone (9e)
Yellowish solid (75% yield); Mp: 120–121°C; 1H NMR (600 MHz, CDCl3): δ=8.13 (m, 2H), 7.87
(m, 4H), 6.98 (d, J=8.7 Hz, 2H), 3.90 (s, 3H); 13C NMR (125 MHz, CDCl3): δ= 189.4, 164.9,
150.7, 142.0, 139.8, 132.9 (×3), 132.1, 130.9, 129.5, 128.5, 127.8, 114.2 (×2), 55.7; HRMS: calcd.
for C16H12ClN2O2 [M+H]+ 299.0587, found 299.0591.
4.1.1.7. (4-Chlorophenyl)(3-chloroquinoxalin-2-yl)methanone (9f)
Yellowish solid (73% yield); Mp: 143–146°C; 1H NMR (600 MHz, CDCl3): δ=8.14 (m, 2H), 7.92
(m, 1H), 7.88 (m, 3H), 7.50 (d, J=8.7 Hz, 2H); 13C NMR (125 MHz, CDCl3): δ=190.0, 149.6,
144.0, 142.2, 141.3, 139.6, 133.2, 132.5, 131.7 (×2), 131.1, 129.5, 129.3 (×2), 128.5; HRMS:
calcd. for C15H9Cl2N2O [M+H]+ 303.0092, found 303.0088.
4.1.1.8. (4-Bromophenyl)(3-chloroquinoxalin-2-yl)methanone (9g)
Yellowish solid (72% yield); Mp: 124–126°C; 1H NMR (600 MHz, CDCl3): δ=8.12 (m, 2H), 7.80
(m, 2H), 7.79 (d, J=8.7 Hz, 2H), 7.66 (d, J=8.7 Hz, 2H); 13C NMR (125MHz, CDCl3): δ=190.2,
149.5, 143.9, 142.2, 139.6, 133.5, 132.5, 132.3 (×2), 131.8 (×2), 131.1, 130.2, 129.5, 128.5;
HRMS: calcd. for C15H9BrClN2O [M+H]+ 346.9587, found 346.9589.
4.1.1.9. (4-Methoxyphenyl)(3-methoxyquinoxalin-2-yl)methanone (9h)
1
Yellowish solid (85% yield); Mp: 134–136°C; H NMR (600 MHz, CDCl3): δ=8.05 (dd, J=7.93,
J=0.59, 1H), 7.92 (d, J=8.8, 2H), 7.91 (d, J=7.9, 1H), 7.74 (m, 1H), 7.61 (m, 1H), 6.95 (d, J=8.8,
2H), 4.09 (s, 3H), 3.87 (s, 3H); 13C NMR (125 MHz, CDCl3): δ=190.5, 164.5, 155.5, 145.5, 140.8,
137.5, 132.8 (×2), 131.1, 129.3, 128.4, 127.2, 127.0, 114.0 (×2), 55.6, 54.2; HRMS: calcd. for
C17H15N2O3 [M+H]+ 295.1083, found 295.1087.
4.1.1.10. (4-Chlorophenyl)(3-methoxyquinoxalin-2-yl)methanone (9i)
1
Yellowish solid (83% yield); Mp: 140–142°C; H NMR (600 MHz, CDCl3): δ=8.05 (dd, J1=8.3
Hz, J2=1.0 Hz, 1H), 7.93 (dd, J1=8.3 Hz, J2=1.0 Hz, 1H), 7.90 (d, J=8.6 Hz, 2H), 7.77 (m, 1H),
7.63 (m, 1H), 7.46 (d, J=8.6 Hz, 2H), 4.10 (s, 3H); 13C NMR (125 MHz, CDCl3): δ=190.7, 155.5,
144.2, 141.0, 140.7, 137.5, 133.8, 131.7 (×2), 131.5, 129.4, 129.0 (×2), 127.4, 127.2, 54.2; HRMS:
calcd. for C16H12ClN2O2 [M+H]+ 299.0587, found 299.0588.

4.1.1.11. (4-Bromophenyl)(3-methoxyquinoxalin-2-yl)methanone (9j)
Yellowish solid (81% yield); Mp: 136–137°C; 1H NMR (600 MHz, CDCl3): δ=8.04 (d, J=8.2 Hz,
1H), 7.92 (d, J=8.2 Hz, 1H), 7.82 (d, J=8.37 Hz, 2H), 7.64 (d, J=8.4 Hz, 2H), 7.60 (m, 2H), 4.10
(s, 3H); 13C NMR (125 MHz, CDCl3): δ=190.9, 155.5, 144.1, 141.0, 137.5, 134.2, 132.0, 131.8
(×2), 131.5 (×2), 129.6, 129.4, 127.4, 127.2, 54.2; HRMS: calcd. for C16H12BrN2O2 [M+H]+
343.0082, found 343.0077.
4.1.1.12. (3-Chloroquinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (10)
1
Yellow solid (78% yield); Mp: 174–175°C; H NMR (400 MHz, CDCl3): δ=8.15 (m, 1H), 7.90
(m, 2H), 7.18 (m, 2H), 3.97 (s, 3H), 3.84 (m, 6H); 13C NMR (150 MHz, CDCl3): δ=190.1, 153.3
(×2), 150.2, 144.2 (×2), 142.1, 139.6, 132.3, 131.1, 129.7, 129.5, 128.5, 108.0 (×2), 61.1, 56.4
(×2); HRMS: calcd. for C18H16ClN2O4 [M+H]+ 359.0799, found 359.0796.
4.1.1.13. (3-Bromoquinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (11)
Yellowish solid (74% yield); Mp: 193–194°C; 1H NMR (400 MHz, CDCl3): δ=8.15 (m, 2H), 7.90
(m, 2H), 7.17 (s, 2H), 3.97 (s, 3H), 3.85 (s, 6H); 13C NMR (150 MHz, CDCl3): δ=190.7, 153.3
(×2), 151.8, 144.2, 143.0, 139.7, 135.5, 132.2, 131.2, 130.9, 129.5, 128.6, 108.0 (×2), 61.1, 56.4
(×2); HRMS: calcd. for C18H16BrN2O4 [M+H]+ 403.0293, found 403.0295.
4.1.2. General procedure for the synthesis of 12a–n.
In a round-bottomed flask, a mixture of sodium (12 mmol) and corresponding alcohols (12 mmol)
was dissolved in 10 mL of dry THF and heated at reflux for 0.5 h. The solution of
(3-Bromoquinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (11) (5 mmol) in dry THF (2 mL)
was added dropwise to the mixture, and the contents of the flask were stirred for 1 h at reflux.
Then water (30 mL) was added and the aqueous layer was extracted with EtOAc. The combined
organic extracts were dried with brine and Na2SO4. After filtration and the evaporation of the
solvent, the crude product was purified by column chromatography (n-hexane: EtOAc=7:1 as
eluent) on silica gel to afford pure product.
4.1.2.1. (3-Methoxyquinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12a)
1
Yellowish solid (86% yield); Mp: 135–137°C; H NMR (600 MHz, CDCl3): δ=7.94 (dd, 1H,
J1=1.5 Hz, J2=8.4 Hz), 7.69 (m, 1H), 7.43 (m, 2H), 7.26 (s, 2H), 3.94 (s, 3H), 3.87 (s, 6H), 3.76 (s,
3H); 13C NMR (125 MHz, CDCl3): δ=190.7, 155.6, 153.1 (×2), 144.7, 143.8, 140.9, 137.4, 131.3,
130.3, 129.3, 127.4, 127.1, 108.0 (×2), 61.0, 56.3 (×2), 54.2; HRMS: calcd. for C19H19N2O5

[M+H]+ 355.1294, found 355.1285.
4.1.2.2. (3-Ethoxyquinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12b)
1
Yellowish solid (82% yield); Mp: 132–134°C; H NMR (600 MHz, CDCl3): δ=8.05 (d, 1H,
J=11.6 Hz), 7.90 (d, 1H, J=8.2 Hz), 7.74 (m, 1H), 7.61 (m, 1H), 7.22 (s, 2H), 4.60 (m, 2H), 3.95
(s, 3H), 3.84 (s, 6H), 1.41 (t, 3H, J1=7.1 Hz, J2=14.2 Hz); 13C NMR (125 MHz, CDCl3): δ=190.8,
155.3, 153.2 (×2), 145.0, 143.7, 141.0, 137.4, 141.0, 137.4, 131.2, 130.5, 129.3, 127.2, 127.2,
107.9 (×2), 63.0, 31.0, 56.4 (×2), 14.3; HRMS: calcd. for C20H21N2O5 [M+H]+ 369.1450, found
369.1442.
4.1.2.3. (3-Propoxyquinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12c)
1
Yellowish solid (84% yield); Mp: 114–115°C; H NMR (600 MHz, CDCl3): δ=8.06 (dd, 1H,
J1=0.8 Hz, J2=8.2 Hz), 7.89 (m, 1H), 7.76 (m, 1H), 7.61 (m, 1H), 7.19 (s, 2H), 4.48 (t, 2H, J1=6.6
Hz, J2=13.1 Hz), 3.94 (s, 3H), 3.84 (s, 6H), 1.79 (m, 2H), 0.94 (t, 3H, J1=7.4 Hz, J2=14.9 Hz);
13C NMR (125 MHz, CDCl3): δ=190.9, 155.4, 153.2 (×2), 145.2, 143.7, 141.0, 137.5, 131.2,
130.6, 129.4, 127.2, 127.1, 107.8 (×2), 68.6, 61.1, 56.4 (×2), 22.0, 10.5; HRMS: calcd. for
C21H23N2O5 [M+H]+ 383.1607, found 383.1597.
4.1.2.4. (3-Isopropoxyquinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12d)
1
Yellow oil (81% yield); H NMR (600 MHz, CDCl3): δ=8.04 (m, 1H), 7.88 (d, 1H, J=8.3 Hz),
7.74 (m, 1H), 7.60 (m, 1H), 7.18 (s, 2H), 5.58 (m, 1H), 3.95 (s, 3H), 3.84 (s, 6H), 1.37 (d, 6H,
J=6.2 Hz); 13C NMR (125 MHz, CDCl3): δ=190.8, 154.6, 153.0 (×2), 145.4, 143.5, 140.9, 137.2,
131.0, 130.5, 129.1, 127.0, 126.9, 107.6 (×2), 70.0, 60.8, 56.2 (×2), 21.6 (×2); HRMS: calcd. for
C21H23N2O5 [M+H]+ 383.1607, found 383.1599.
4.1.2.5. (3-Butoxyquinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12e)
1
Yellowish solid (79% yield); Mp: 82–84°C; H NMR (600 MHz, CDCl3): δ=8.05 (1H, m), 7.90
(1H, d, J=8.0 Hz), 7.75 (1H, m), 7.61 (1H, m), 7.18 (2H, s), 4.52 (2H, t, J1=6.5 Hz, J2=13.1 Hz),
3.95 (3H, s), 3.84 (6H, s), 1.75 (2H, m), 1.38 (2H, m), 0.91 (3H, t, J1=7.4 Hz, J2=14.8 Hz); 13C
NMR (125 MHz, CDCl3): δ=191.0, 156.3, 153.2 (×2), 145.2, 143.8, 141.1, 136.8, 131.2, 130.7,
129.4, 127.2, 127.2, 106.5 (×2), 66.5, 60.9, 56.1 (×2), 31.0, 19.4, 13.9; HRMS: calcd. for
C21H23N2O5 [M+H]+ 397.1763, found 397.1758.
4.1.2.6. (3-Isobutoxyquinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12f)
Yellowish solid (82% yield); Mp: 94–95°C; 1H NMR (600 MHz, CDCl3): δ=8.06 (dd, 1H, J1=0.6

Hz, J2=8.2 Hz), 7.90 (d, 1H, J=7.9 Hz), 7.75 (m, 1H), 7.61 (m, 1H), 7.18 (s, 2H), 4.29 (d, 2H,
J=6.6 Hz), 3.94 (s, 3H), 3.84 (s, 6H), 2.07 (m, 1H), 0.93 (d, 1H, J=6.7 Hz); 13C NMR (125 MHz,
CDCl3): δ=191.0, 155.4, 153.2 (×2), 145.3, 143.7, 141.0, 137.6, 131.2, 130.7, 129.4, 127.2, 127.1,
107.7 (×2), 73.1, 61.0, 56.4 (×2), 27.8, 19.1 (×2); HRMS: calcd. for C22H25N2O5 [M+H]+
397.1763, found 397.1756.
4.1.2.7. (3-(Pentyloxy)quinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12g)
1
Yellowish solid (85% yield); Mp: 71–73°C; H NMR (600 MHz, CDCl3): δ= 8.06 (d, 1H, J=8.2
Hz), 7.90 (d, 1H, J=8.3 Hz), 7.76 (m, 1H), 7.61 (t, 1H, J1=7.2 Hz, J2=15.2 Hz), 7.18 (s, 2H), 4.51
(t, 2H, J1=6.6 Hz, J2=13.3 Hz), 3.94 (s, 3H), 3.83 (s, 6H), 1.76 (m, 2H), 1.31 (m, 4H), 0.86 (m,
3H); 13C NMR (125 MHz, CDCl3): δ=191.0, 155.4, 153.2 (×2), 145.2, 143.7, 141.0, 137.5, 131.2,
130.6, 129.4, 127.2, 127.1, 107.8 (×2), 67.1, 61.0, 56.4 (×2), 28.2, 28.0, 22.3, 14.0; HRMS: calcd.
for C23H27N2O5 [M+H]+ 411.1920, found 411.1909.
4.1.2.8. (3-(Isopentyloxy)quinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12h)
1
Yellowish solid (77% yield); Mp: 92–94°C; H NMR (600 MHz, CDCl3): δ=8.06 (d, 1H, J=8.2
Hz), 7.90 (d, 1H, J=8.3 Hz), 7.76 (t, 1H, J1=7.2 Hz, J2=15.3 Hz), 7.62 (t, 1H, J1=7.2 Hz, J2=15.2
Hz), 7.18 (s, 2H), 4.54 (t, 2H, J1=6.2 Hz, J1=12.4 Hz), 3.95 (s, 3H), 3.83 (s, 6H), 1.65 (m, 3H),
0.89 (d, 6H, J=5.8 Hz); 13C NMR (125 MHz, CDCl3): δ=191.0, 155.4, 153.2 (×2), 145.3, 143.7,
141.1, 137.6, 131.3, 130.7, 129.4, 127.3, 127.2, 107.8 (×2), 65.7, 61.1, 56.4 (×2), 37.3, 25.1, 22.6
(×2); HRMS: calcd. for C23H27N2O5 [M+H]+ 411.1920, found 411.1914.
4.1.2.9. (3-(Prop-2-yn-1-yloxy)quinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12i)
Yellowish solid (71% yield); Mp: 136–137°C; 1H NMR (600 MHz, CDCl3): δ=8.08 (d, 1H, J=8.0
Hz), 7.96 (d, 1H, J=8.1 Hz), 7.79 (t, 1H, J1=7.3 Hz, J2=14.3 Hz), 7.66 (m, 1H), 7.22 (s, 2H), 5.17
(s, 2H), 3.96 (s, 3H), 3.84, 2.45 (s, 1H); 13C NMR (125 MHz, CDCl3): δ=190.4, 153.9, 153.3 (×2),
144.7, 144.0, 140.6, 138.0, 131.6, 130.4, 129.5, 127.9, 127.4, 108.2 (×2), 78.2, 75.2, 61.1, 56.5
(×2), 61.1, 56.5 (×2), 54.2; HRMS: calcd. for C21H19N2O5 [M+H]+ 379.1294, found 379.1290.
4.1.2.10. (3-(Sec-butoxy)quinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12j)
1
Yellow oil (81% yield); H NMR (600 MHz, CDCl3): δ=8.04 (dd, 1H, J1=0.8 Hz, J2=8.4 Hz),
7.87 (dd, 1H, J1=0.6 Hz, J2=8.4 Hz), 7.73 (m, 1H Hz), 7.59 (m, 1H Hz), 7.17 (s, 2H), 5.40 (m, 1H),
3.94 (s, 3H), 3.83 (s, 6H), 1.71 (m, 1H), 1.64 (m, 1H), 1.35 (d, 3H, J=6.2 Hz), 0.88 (t, 3H, J1=7.4
Hz, J2=14.9 Hz); 13C NMR (125 MHz, CDCl3): δ=191.3, 155.1, 153.2 (×2), 145.8, 143.7, 141.1,

137.5, 131.1, 130.8, 129.4, 127.2, 127.1, 107.8 (×2), 74.8, 61.1, 56.5 (×2), 28.8, 19.2, 9.7; HRMS:
calcd. for C22H25N2O5 [M+H]+ 397.1763, found 397.1753.
4.1.2.11. (3-(Cyclopropylmethoxy)quinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12k)
Yellowish solid (74% yield); Mp: 133–138°C; 1H NMR (600 MHz, CDCl3): δ=8.05 (d, 1H, J=8.1
Hz), 7.88 (d, 1H, J=8.2 Hz), 7.75 (t, 1H, J1=7.6 Hz, J2=14.9 Hz), 7.61 (t, 1H, J1=7.6 Hz, J2=14.7
Hz), 7.21 (s, 2H), 4.38 (d, 2H, J=6.9 Hz), 3.95 (s, 3H), 3.84 (6H, s), 1.31 (s, 1H), 0.55 (d, 2H,
J=7.0 Hz), 0.33 (s, 2H); 13C NMR (125 MHz, CDCl3): δ=191.1, 155.5, 153.3 (×2), 145.3, 143.9,
141.1, 137.6, 131.3, 130.7, 129.5, 127.3, 127.2, 108.1 (×2), 71.7, 61.1, 56.5 (×2),9.9, 3.5 (×2);
HRMS: calcd. for C22H23N2O5 [M+H]+ 395.1607, found 395.1601.
4.1.2.12. (3-(Allyloxy)quinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12l)
Yellowish solid (76% yield); Mp: 105–107°C; 1H NMR (600 MHz, CDCl3): δ=8.06 (d, 1H, J=8.2
Hz), 7.91 (d, 1H, J=8.3 Hz), 7.77 (t, 1H, J1=7.2 Hz, J2=15.3 Hz), 7.63 (t, 1H, J1=7.3 Hz, J2=15.2
Hz), 7.21 (s, 2H), 6.06 (m, 1H), 5.37 (d, 1H, J=17.2 Hz), 5.24 (d, 1H, J=10.4 Hz), 5.05 (d, 1H,
J=5.2 Hz), 3.95 (s, 3H), 3.83 (s, 6H); 13C NMR (150 MHz, DMSO-d6): δ=191.6, 170.8, 156.4,
153.9, 153.4 (×2), 143.7, 133.3, 132.1, 132.0, 131.8, 130.3, 129.6, 129.1, 124.2, 116.3, 107.9, 60.7,
60.2, 56.7 (×2); HRMS: calcd. for C21H21N2O5 [M+H]+ 381.1450, found 381.1446.
4.1.2.13. (3-(But-3-en-1-yloxy)quinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12m)
Yellowish solid (71% yield); Mp: 67–70°C; 1H NMR (400 MHz, CDCl3): δ=8.07 (dd, 1H, J1=1.2
Hz, J2=7.9 Hz), 7.90 (dd, 1H, J1=1.0 Hz, J2=8.3 Hz), 7.76 (m, 1H), 7.62 (m, 1H), 7.18(s, 2H),
5.78 (m, 1H), 5.07 (m, 1H), 5.00 (m, 1H), 4.57 (t, 2H, J1=6.8 Hz, J2=13.5 Hz), 3.94 (s, 3H), 3.83
(s, 6H), 2.52 (m, 2H); 13C NMR (125 MHz, CDCl3): δ=190.8, 155.9, 153.1 (×2), 145.1, 143.7,
140.9, 136.8, 134.4, 131.2, 130.6, 129.2, 127.3, 117.1, 106.5 (×2), 65.7, 60.8, 56.1 (×2), 33.3;
HRMS: calcd. for C22H23N2O5 [M+H]+ 395.1607, found 395.1597.
4.1.2.14. (3-(benzyloxy)quinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (12n)
1
Yellowish solid (70% yield); Mp: 113–115°C; H NMR (400 MHz, CDCl3): δ=8.07 (dd, 1H,
J1=1.0 Hz, J2=8.2 Hz), 7.93 (dd, 1H, J1=0.9 Hz, J2=8.3 Hz), 7.77 (m, 1H), 7.63 (m, 1H), 7.38 (d,
2H, J=7.2 Hz), 7.30 (m, 3H), 7.17 (s, 2H), 5.60 (s, 2H), 3.94 (s, 3H), 3.79 (s, 6H); 13C NMR (150
MHz, CDCl3): δ=190.8, 171.3, 154.8, 153.1 (×2), 145.0, 143.6, 140.8, 137.7, 136.1, 131.3, 130.5,
129.3, 128.4 (×2), 128.0, 127.9 (×2), 127.4, 127.1, 107.8, 61.0, 56.3 (×2), 50.6; HRMS: calcd for
C25H23N2O5 [M+H]+ 431.1607, found 431.1599.

4.1.3. General procedure for the synthesis of 13a–f.
To a solution of anhydrous K2CO3 (0.28 g, 0.002 mol), KI (0.016 g, 0.0001 mol) and the
respective alkyl halide (0.002 mol) in DMF (5 mL) was added dropwise the solution of
(3-Hydroxyquinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (9d) (0.34 g, 0.001 mmol) in
DMF (0.5 mL) and the mixture was heated under stirring at 30–80°C for 3h. The reaction was
diluted with H2O (100 mL) then extracted with EtOAc (3×100 mL) and the combined organic
phases were washed with H2O (3×100 mL) and brine (50 mL), dried over MgSO4 and
concentrated in vacuo. The residue was purified by flash column chromatography (0-15% EtOAc
in n-hexane).
4.1.3.1. 1-Methyl-3-(3,4,5-trimethoxybenzoyl)quinoxalin-2(1H)-one (13a)
White solid (42% yield); Mp: 88–89°C; 1H NMR (600 MHz, DMSO-d6): δ=7.88 (dd, 1H, J1=1.2
Hz, J2=7.9 Hz), 7.77 (m, 1H), 7.70 (d, 1H, J=8.3 Hz), 7.46 (m, 1H), 7.27 (s, 2H), 3.79 (s, 6H),
3.78 (s, 3H), 3.67 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ=191.5, 155.0, 153.5 (×2), 153.4,
143.8, 132.4, 132.2, 130.4, 130.2, 124.3, 115.6, 108.0 (×2), 60.8, 56.8 (×2), 30.1; HRMS: calcd.
for C19H19N2O5 [M+H]+ 355.1294, found 355.1285.
4.1.3.2. 1-Ethyl-3-(3,4,5-trimethoxybenzoyl)quinoxalin-2(1H)-one (13b)
White solid (39% yield); Mp: 118–120°C; 1H NMR (600 MHz, CDCl3): δ=7.94 (d, 1H, J=7.9 Hz),
7.68 (t, 1H, J1=7.9 Hz, J2=15.5 Hz), 7.45 (d, 1H, J=8.5 Hz), 7.41 (t, 1H, J1=7.6 Hz, J2=15.2 Hz),
7.26 (s, 2H), 4.38 (q, 2H, J=7.1 Hz), 3.94 (s, 3H), 3.86 (s, 6H), 1.43 (t, 3H, J1=7.1 Hz, J2=14.3
Hz); 13C NMR (125 MHz, CDCl3): δ=190.7, 154.5, 153.2 (×2), 152.9, 144.0, 133.0, 132.5, 132.1,
131.3, 130.0, 124.1, 113.9, 107.8 (×2), 61.0, 56.5 (×2), 37.5, 12.5; HRMS: calcd. for C20H21N2O5
[M+H]+ 369.1450, found 369.1442.
4.1.3.3. 1-Propyl-3-(3,4,5-trimethoxybenzoyl)quinoxalin-2(1H)-one (13c)
1
White solid (37% yield); Mp: 135–136°C; H NMR (400 MHz, CDCl3): δ=7.95 (dd, 1H, J1=1.2
Hz, J2=8.3 Hz), 7.68 (m, 1H), 7.42 (m, 2H), 7.24 (s, 2H), 4.27 (t, 2H, J1=7.7 Hz, J2=15.5 Hz),
3.94 (s, 3H), 3.85 (s, 6H), 1.85 (m, 2H), 1.07 (t, 3H, J1=7.4 Hz, J2=14.8 Hz); 13C NMR (125 MHz,
CDCl3): δ=189.7, 153.5, 152.2 (×2), 152.1, 142.8, 132.1, 131.4, 130.9, 130.2, 129.0, 123.0, 113.0,
106.6 (×2), 59.9, 55.4 (×2), 42.8, 19.7, 10.3; HRMS: calcd. for C21H23N2O5 [M+H]+ 383.1607,
found 383.1597.
4.1.3.4. 1-Isopropyl-3-(3,4,5-trimethoxybenzoyl)quinoxalin-2(1H)-one (13d)

1
Colorless oil (37% yield); H NMR (400 MHz, CDCl3): δ=7.94 (d, 1H, J=8.0 Hz), 7.68 (d, 2H,
J=3.7 Hz), 7.40 (m, 1H), 7.25 (s, 2H), 5.44 (m, 1H), 3.94 (s, 3H), 3.86 (s, 6H), 1.70 (d, 6H, J=7.0
Hz); 13C NMR (150 MHz, CDCl3): δ=190.8, 154.6, 153.0 (×2), 145.4, 143.5, 140.9, 137.2, 131.0,
130.5, 129.1, 127.0, 126.9, 107.6 (×2), 70.0, 60.8, 56.2 (×2), 21.6 (×2); HRMS: calcd. for
C21H23N2O5 [M+H]+ 383.1607, found 383.1599.
4.1.3.5. 1-Butyl-3-(3,4,5-trimethoxybenzoyl)quinoxalin-2(1H)-one (13e)
1
White solid (40% yield); Mp: 142–144°C; H NMR (400 MHz, CDCl3): δ=7.95 (dd, 1H, J1=1.3
Hz, J2=7.9 Hz), 7.68 (m, 1H), 7.42 (m, 2H), 7.24 (s, 2H), 4.30 (t, 2H, J1=7.7 Hz, J2=15.5 Hz),
3.93 (s, 3H), 3.86 (s, 6H), 1.80 (m, 2H), 1.50 (m, 2H), 1.00 (t, 3H, J1=7.3 Hz, J2=14.7 Hz); 13C
NMR (125 MHz, CDCl3): δ=191.0, 155.4, 153.2 (×2), 149.2, 140.3, 137.6, 133.1, 129.4, 128.2,
126.8 (×2), 126.6, 107.9 (×2), 61.0, 56.4 (×2), 40.2, 30.7, 19.2, 13.8; HRMS: calcd. for
C21H23N2O5 [M+H]+ 397.1763, found 397.1758.
4.1.3.6. 1-Isobutyl-3-(3,4,5-trimethoxybenzoyl)quinoxalin-2(1H)-one (13f)
White solid (38% yield); Mp: 151–152°C; 1H NMR (400 MHz, CDCl3): δ=7.95 (d, 1H, J=8.0 Hz),
7.66 (t, 1H, J1=8.2 Hz, J2=16.1 Hz), 7.41 (m, 2H), 7.23 (s, 2H), 4.19 (d, 2H, J=7.4 Hz), 3.94 (s,
3H), 3.85 (s, 6H), 2.30 (m, 1H), 1.04 (d, 2H, J=6.6 Hz) ppm; 13C NMR (125 MHz, CDCl3):
δ=190.9, 156.2, 153.1 (×2), 149.3, 140.2, 138.5, 136.6, 133.3, 129.2, 128.4, 126.7, 126.3, 106.2
(×2), 72.8, 60.8, 56.0 (×2), 40.4, 28.0, 19.3 (×2); HRMS: calcd. for C22H25N2O5 [M+H]+ 397.1763,
found 397.1756.
4.1.4. The synthesis of 14a–d.
4.1.4.1. (3-(Methylamino)quinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (14a)
A mixture of (3-Bromoquinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (11) (500 mg, 1.24
mmol) and methylamine (2M solution in THF, 1.5 mL) in THF 13 (10 mL) was placed in a sealed
tube (100 mL). The system was sealed and the reaction mixture was heated to 50°C and stirred
overnight. Upon completion, the reaction mixture was cooled to room temperature. After removal
of the solution, the residue was purified by silica gel column chromatography (n-hexane:
EtOAc=5:1 as eluent) on silica gel to afford pure product. Brown solid (57% yield); Mp: 142–
1
144°C; H NMR (600 MHz, CDCl3): δ=7.95 (d, 1H, J=8.2 Hz), 7.78 (d, 1H, J=8.2 Hz), 7.61 (t,
1H, J=7.3 Hz), 7.44 (t, 1H, J=7.4 Hz), 6.55 (s, 2H), 3.84 (s, 3H), 3.80 (s, 6H), 3.79 (s, 1H), 2.98
(d, 6H, J=4.7 Hz); 13C NMR (150 MHz, CDCl3): δ=193.4, 152.8, 152.6 (×2), 142.8, 136.4, 135.1,

132.8, 131.9, 130.0 (×2), 126.2, 124.8, 109.2 (×2), 61.0, 56.3 (×2), 27.9; HRMS: calcd. for
C19H20N3O4 [M+H]+ 354.1454, found 354.1442.
4.1.4.2. (3-(Piperazin-1-yl)quinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (14b)
(3-Bromoquinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (11) (500 mg, 1.24 mmol) and
piperazine (112 mg, 1.30 mmol) were added to a microwave tube and heated at 150°C for 30
minutes. The mixture was diluted with water and the aqueous layer was extracted with EtOAc.
The combined organic layers were then dried with brine and Na2SO4. Evaporation of the solvent
1
yielded 380 mg (75% yield) of a light brown solid. Mp: 99-100°C; H NMR (400 MHz, CDCl3):
δ=7.92 (m, 1H), 7.79 (m, 1H), 7.67 (m, 1H), 7.47 (m, 1H), 7.31 (s, 1H), 3.97 (s, 3H), 3.87 (s, 6H),
3.54 (m, 4H), 2.92 (m, 4H); 13C NMR (150 MHz, CDCl3): δ=191.8, 167.7, 153.2 (×2), 143.9,
143.7, 141.6, 136.0, 131.3, 130.0, 129.1, 126.7, 126.1, 108.1 (×2), 61.0, 56.4 (×2), 49.0 (×2), 45.4
(×2); HRMS: calcd. for C22H25N4O5 [M+H]+ 409.1876, found 409.1867.
4.1.4.3. (3-Morpholinoquinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (14c)
1
Prepared according to procedure of 14b. Orange solid (81% yield); Mp: 112–114°C; H NMR
(600 MHz, CDCl3): δ=7.94 (m, 1H), 7.80 (m, 1H), 7.67 (m, 1H), 7.49 (m, 1H), 7.32 (s, 2H), 3.97
(s, 3H), 3.87 (s, 6H), 3.74 (m, 4H), 3.55 (m, 4H); 13C NMR (125 MHz, CDCl3): δ=191.6, 153.3
(×2), 151.7, 144.0, 143.9, 140.9, 136.1, 131.7, 130.0, 129.2, 126.4, 126.4, 108.3 (×2), 66.5 (×2),
61.1, 56.5 (×2), 48.7 (×2); HRMS: calcd. for C22H24N3O5 [M+H]+ 410.1716, found 410.1708.
4.1.4.3. (3-((4-methoxyphenyl)amino)quinoxalin-2-yl)(3,4,5-trimethoxyphenyl)methanone (11e)
Prepared according to procedure of 14b. Purple solid (66% yield); Mp: 156–157°C; 1H NMR (600
MHz, CDCl3): δ=10.06 (s, 1H), 7.91 (d, 1H, J=8.0 Hz), 7.81 (m, 3H), 7.70 (t, 1H, J1=9.0 Hz,
J2=14.5 Hz), 7.48 (m, 3H), 6.97 (d, 2H, J=8.8 Hz), 4.00 (s, 3H), 3.92 (s, 6H), 3.85 (s, 3H); 13C
NMR (125 MHz, CDCl3): δ=193.8, 155.9, 152.7 (×2), 149.8, 143.1, 143.0, 136.3, 135.9, 133.1,
132.4, 131.9, 130.0, 126.9, 125.9, 122.3 (×2), 114.3 (×2), 109.5 (×2), 61.2, 56.4 (×2), 55.7; HRMS:
calcd. for C25H24N3O5 [M+H]+ 446.1716, found 410.1709.
4.2. Biological evaluation
4.2.1. Cell culture
The SGC-7901, HT-1080 and A549 were cultured in RPMI-1640 medium containing 10%
FBS, 100 U/mL streptomycin and 100 U/mL penicillin at 37°C in a humidified atmosphere
containing 5% CO2. All cell lines were purchased from the American Type Culture Collection

(ATCC, Manassas, VA).
4.2.2. In vitro cell growth inhibitory assay.
The in vitro anti-proliferative activities of CA-4 (1a) and all of the target compounds were
assayed by conventional 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
method. In brief, cells were seeded into 96-well plates at a density of 1-3 × 104/well (depending on
the cell growth rate). 24 h later, cells were incubated with various concentrations of the test
compounds for 72 h. Then the drug-containing medium was removed and replaced with 100 µL of
fresh medium containing 5 mg/mL MTT solution. After 4 h of incubation, the medium with MTT
was removed, and 100 µL of dimethyl sulphoxide (DMSO) was added to each well. The plates
were gently agitated until the purple formazan crystals were dissolved, and the absorbance was
measured at 570 nm by a microplate spectrophotometer (MK3, Thermo, Germany). The data were
calculated and plotted as the per cent viability compared to the control. The 50% inhibitory
concentration (IC50) was defined as the concentration that reduced the absorbance of the untreated
wells by 50% of the vehicle in the MTT assay.
4.2.3. In vitro tubulin polymerization assay.
The effects of 12a and CA-4 on the polymerisation of tubulin were determined by employing
a fluorescence-based tubulin polymerization assay kit (BK011P, Cytoskeleton, USA) according to
the manufacturer’s protocol. The tubulin reaction mix contained 2 mg/mL porcine brain tubulin
(>99% pure), 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP, and 15% glycerol. First, 96-well plate
was incubated with 5 µL of inhibitors in various concentrations at 37°C for 1 min. Then 50 µL of
the tubulin reaction mix was added. The samples were mixed well, and tubulin assembly was
monitored (emission wavelength of 420 nm; excitation wavelength pf 360 nm) at 1 min intervals
for 90 min at 37°C using a plate reader (FASCalibur, BD Biosciences, USA). The IC50 values
were calculated after 20 min using the SPSS software.
4.2.4. Immunofluorescence staining
The SGC-7901 and HT-1080 cells were seeded in 24-well plate (with coverslips plated) at
density of 1 × 104 cells. After overnight adherence, they were exposed to 12a (0.34 µM) or CA-4
(0.032 µM) respectively, for 48 h. The coverslips were fixed in ice-cold methanol/acetic acid (3:1)
for 10 min and blocked with 3% bovine serum albumin for 20 min at room temperature. The
primary α-tubulin antibody (Santa Cruz, CA) was diluted (1:100) with 2% BSA in PBS and

incubated overnight at 4 °C. The cells were washed with PBS to remove unbound primary
antibody, and the cells were then incubated with FITC-conjugated antimouse secondary antibody
and diluted (1:1000) with 2% BSA in PBS for 3 h at 37 °C. The cells were washed with PBS to
remove unbound secondary antibody, the nuclei were stained with 4,6-diamino-2-phenolindol
dihydrochloride (DAPI) and immunofluorescence was then detected using a fluorescence
microscope (Olympus, Tokyo, Japan).
4.2.5. Cell cycle analysis
HT-1080 cells (8 × 104 cells) were incubated with various concentrations of CA-4, 12a or
0.05% DMSO for the indicated times. The cells were collected by centrifugation, washed with
PBS and fixed in ice-cold 70% ethanol. The fixed cells were harvested by centrifugation and
treated with RNase A at 37 °C for 30 min, and incubated with PI solution (Solarbio, China) at 4 °C
for 15 min. The samples were then analysed by FACScan flow cytometry (Becton-Dickinson,
Franklin Lakes, NJ, USA). The experiments were repeated at least three times.
4.3. Molecular modelling
The crystal structure of tubulin in complex with DAMA-colchicine (PDB: 1SA0) was used as
the template in the docking study. The protein structure was prepared using the Accelrys
Discovery Studio 3.0. After extracting the ligand, hydrogen atoms were added to the crystal.
Charges were added to biopolymer by CHARMm force field. Finally, 12a and CA-4 were docked
into the binding site using the CDOCKER protocol with the default settings.
Acknowledgement
We gratefully acknowledge the National Natural Science Foundation of China (81673293
and 81602969), Liaoning Revitalization Talents Program (XLYC1802072), Project funded by
China Postdoctoral Science Foundation (2018M641715), Shenyang Science & Technology
Bureau Item (17-231-1-41 and 18-014-4-01), and Plan for Development of Young Scholars of
Shenyang pharmaceutical university (ZQN2018002) for the generous financial support. This work
was also supported by the Program for Innovative Research Team of the Ministry of Education.
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1. A clean and green method to synthesize 3-benzoylquinoxalines under
“on-water” condition was developed.
2. As anticipated, compound 9d exerted potent antitumor efficacy.
3. Further structural optimization led to a promising compound 12a,
which is a novel tubulin inhibitor.
4. This work provides a green version for medicinal chemists to
assemble molecules in the seeking stage of drug discovery