Design, synthesis, biological evaluation and molecular docking study on peptidomimetic analogues of XK469

Article abstract of DOI:10.1016/j.ejmech.2016.08.010

XK469 is identified as a potent quinoxaline antineoplastic agent based on its significant clinical efficacy. It probably exerts its activity via DNA topoisomerase II (topo II) inhibition. To obtain more effective antineoplastic agents, a spectrum of peptidomimetic-type quinoxaline analogues of XK469 was herein designed, synthesized, and evaluated. Few compounds (e.g. 13a and 13b) exhibited obvious cytotoxicity indicated by in?vitro anti-proliferative assay. SAR investigation revealed that introducing of hydrophobic tert-butylamine or dodecylamine moiety at the 3-position of quinoxaline core is favorable for achieving a better anti-proliferative potency, while peptidomimetic derivatives only yielded moderate cytotoxicity. Compounds with improved anti-proliferative activities also demonstrated decent anti-metastatic potencies comparable with that of doxorubicin (Doxo) based on in?vivo mouse model study. The topo II-mediated kinetoplast DNA (kDNA) decatenation assay as well as molecular docking studies implicated that these compounds tend to be potent topo II inhibitors. Overall, compounds 13a and 13b, 13b in particular, standed out from various assessments and might be promising candidates for further chemical optimization.

Full text of DOI:10.1016/j.ejmech.2016.08.010

Accepted Manuscript
Design, synthesis, biological evaluation and molecular docking study on
peptidomimetic analogues of XK469
Qiao-Hong Xia, Wei Hu, Chen Li, Ji-Feng Wu, Liang Yang, Xue-Mei Han, Yue-Mao
Shen, Zhi-Yu Li, Xun Li
PII:
S0223-5234(16)30653-5
10.1016/j.ejmech.2016.08.010
EJMECH 8806
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 May 2016
Revised Date: 5 August 2016
Accepted Date: 6 August 2016
Please cite this article as: Q.-H. Xia, W. Hu, C. Li, J.-F. Wu, L. Yang, X.-M. Han, Y.-M. Shen, Z.-Y. Li, X.
Li, Design, synthesis, biological evaluation and molecular docking study on peptidomimetic analogues of
XK469, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.08.010.
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Graphical abstracts

ACCEPTED MANUSCRIPT
Design, synthesis, biological evaluation and molecular docking study on
peptidomimetic analogues of XK469
Qiao-Hong Xia1,#, Wei Hu2,# Chen Li3, Ji-Feng Wu4, Liang Yang1, Xue-Mei Han1, Yue-Mao Shen1,
Zhi-Yu Li5,*, Xun Li1,**
1 Department of Medicinal Chemistry, Key Laboratory of Chemistry and Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Shandong University, Ji’nan, Shandong 250012, P.R. China
2 State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Ji’nan, Shandong
250100, P.R. China
3 School of Bethune Medical Sciences, Jilin University, Changchun, P. R. China
4 Institute of Criminal Science and Technology, Ji’nan Public Security Bureau, Ji’nan, 250100, P. R. China
5 Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, Philadelphia, PA, 19104, USA
Abstract
XK469 is identified as a potent quinoxaline antineoplastic agent based on its significant clinical efficacy. It
probably exerts its activity via DNA topoisomerase II (topo II) inhibition. To obtain more effective
antineoplastic agents, a spectrum of peptidomimetic-type quinoxaline analogues of XK469 was herein
designed, synthesized, and evaluated. Few compounds (e.g. 13a and 13b) exhibited obvious cytotoxicity
indicated by in vitro anti-proliferative assay. SAR investigation revealed that introducing of hydrophobic
tert-butylamine or dodecylamine moiety at the 3-position of quinoxaline core is favorable for achieving a
better anti-proliferative potency, while peptidomimetic derivatives only yielded moderate cytotoxicity.
Compounds with improved anti-proliferative activities also demonstrated decent anti-metastatic potencies
comparable with that of doxorubicin (Doxo) based on in vivo mouse model study. The topo II-mediated
kinetoplast DNA (kDNA) decatenation assay as well as molecular docking studies implicated that these
compounds tend to be potent topo II inhibitors. Overall, compounds 13a and 13b, 13b in particular, standed
out from various assessments and might be promising candidates for further chemical optimization.
Keywords: XK469; quinoxaline peptidomimetic analogues; hydrophobic chain; topoisomerase II inhibitor;
antineoplastic agents.
#These two authors contributed equally to this manuscript. Corresponding addresses at:
*Zh.Li@usciences.edu; **tjulx2004@sdu.edu.cn (Li. X)

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1. Introduction
Almost all anticancer chemotherapeutics exert their therapeutic efficacies by killing fast dividing cells
and cause severe side effects. Cancer cells also quickly develop multidrug resistance (MDR) through
different mechanisms. The synthetic quinoxaline derivative XK469 (NSC 697887), or
(±)-2-[4-(7-chloro-2-quinoxaliny)oxy]phenoxypropionic acid, is a promising lead compound, due to its
significant therapeutic efficacy against a broad spectrum of solid tumors and high activity towards
numerous types of MDR cancers. More importantly, it exhibited relatively low toxicity and side effects,
compared with many conventional antineoplastic agents, such as camptothecin [1, 2].
Despite its significant antitumor potency, the exact biotarget(s) and action mechanism of XK469 have
not been clearly elucidated. At present, the proposed putative mechanisms are related to the induction of
G2-M cell cycle arrest by p53-dependent and -independent pathways [3], apoptosis regulation [4],
MEK/MAPK signaling pathway [5], inhibition of cyclin B1 ubiquitination [6], or mixed autophagy [7].
Recently, more evidences have supported that XK469 is more likely to function as a selective topo II
inhibitor, while has no or weak effect on topo I [8-10].
Sustained efforts from Horwitz’s group have clearly elaborated the structure-activity relationship
(SAR) of XR469 [11-15]. The parent structure of XK469 can be dissected into three parts (Fig. 1):
quinoxaline core (part A), hydroquinone linker (part B), and the lactic acid portion (part C). For part A, the
bicyclic quinoxaline framework generated the highest activity, while replacement of quinoxaline frame
with other rigid cyclic structures, in contrast, would lead to considerably decreased potency. As far as the
impact of substitution on quinoxaline ring was concerned, the 7-halogen substitution eventually gave the
best potency, with an activity order of F ≈ Cl ≈ Br > I, followed by compounds without any substituent at
this site. Other substituents, such as methyl, methoxyl, nitro, amino or azido, generated much less activities.
When halogen was added to other sites, all derivatives were inactive against cancer cells. For part B, either
a resorcinol or a catechol replacement resulted in the loss of antineoplastic activity, suggesting the
hydroquinone connecting motif is essential for maintaining the antineoplastic potency. For part C, although
R(+)- and S(−)-isomers have equal toxicity in animal tumor models, R(+) stereoisomer (NSC 698215) is
slightly more potent than S(−)-isomer (NSC 698216) [8]. Moreover, when carboxylic acid was replaced by
other groups, such as CONH2, CONHCH3, CON(CH3)2, CONHOH, CONHNH2, CN, or CN4H (tetrazole),
the corresponding derivatives displayed impaired antineoplastic activities, compared with that of free acid
counterparts. Only when carboxylic acid was converted into carboxylic ester or N,N-dimethylamide motif,
these derivatives exhibited slightly decreased bioactivities, implying an intact 2-oxypropionic acid frame is
a prerequisite for maintaining maximum antitumor potency as well as optimal water solubility. Collectively,
based on the SAR analysis of XK469, we can draw a conclusion that the quinoxaline ring with 7-halogen
substitution (or without substitution), hydroquinone mode, and R(+)-form 2-oxypropionic acid motif are
favorable contributors for achieving a higher antineoplastic efficacy.
“Enhancing protein backbone-binding” concept has been validated as an effective mean to combat

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drug resistance [16], which was thereby utilized in this work with an expectation of confronting MDR in
cancer cells. According to the 3D structure of topo II (PDB entry: 1ZXM), it clearly evinced that the
protein backbone possesses three important binding domains, via pocket I, II and III, as evinced in Fig. 1.
The binding pattern of XK469 with topo II revealed that the quinoxaline ring of XK469 (part A) locates in
pocket I, a relatively wide hydrophobic area, and the 1-N of XK469 forms a H-bonding interaction with the
lateral phenolic hydroxyl H of Tyr34, with a distance of 2.16Å. The polar carboxylic acid portion (part C)
inserts into pocket III, and the hydroxyl O, as an H-bond acceptor, interacts with the skeletal NHs of
Gly166 (2.58Å), Tyr165 (2.01Å) and Gly164 (1.82Å), respectively. In regard to the hydroquinone
connecting motif (part B), although the adjacent O of quinoxaline core orients toward the pocket II by
forming a H-bond with Ser149 (2.18Å), a hydrophobic side chain seems to be preferably introduced into
this relatively narrow cavity, resulting in enhanced protein backbone-binding effect via the formation of
effective hydrophobic interaction.
Fig. 1. Crystal structure of topo II (left) and binding mode of XK469 with topo II (right). H-bonding
interactions are represented as dot lines.
Hydrophobic tert-butyl chain was initially selected since it has been frequently found in topo II
inhibitor design. For instance, Kundu and co-workers have developed an array of 2-aryl-N-fused
aminoimidazole derivatives as potent topo II inhibitors [17]. SAR exploration suggested the
tert-butylamine group at the 3-position is crucial for retaining high affinity for topo II. Binding mode of the
most potent inhibitor (compound I, Fig. 2) against the ATPase domain of human topo IIα indicated that the
substituted 3-tert-butyl group not only provided a hydrophobic interaction through occupying a
hydrophobic pocket surrounded by Asn91, Asp94 and Arg98 residues, but participated in an additional
CH···O type contact with the side chains of Asp94 and Asn91. This weak H-bonding interaction was
presumed to offer an extra stability and make a positive contribution to the association of protein-ligand
adducts [18]. Moreover, other two tert-butyl-containing topo II inhibitors, quinoline aminopurine
compound II [19] and ATP-competitive purine analogue III [20], as shown in Fig. 2, were also proved to fit
well in the hydrophobic pocket and form effective hydrophobic contacts with surrounding amino acid

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residues.
Fig. 2. Examples of topo II inhibitors that contain hydrophobic tert-butyl functional group.
Recently, long-chain fatty acids (LCFAs), including dodecanoic acid (lauric acid), octadecanoic acid,
arachidic acid, etc, have emerged as powerful tools for constructing efficient drug delivery system in
conjunction with albumins [21-24]. Accordingly, LCFA carrier, such as human serum albumin (HSA) [22,
24], may efficiently deliver LCFA-conjugated antitumor drugs to tumor sites where payloads can be
released and exert their bioactivities, and achieve preferred pharmacokinetic profiles as well [25, 26]. In
addition, LCFAs are normally highly accumulated in cancer cells, in which LCFAs may not only serve as a
crucial energy resource for cell growth, but furnish essential substances for rapid proliferation,
development, and invasion [27, 28]. Therefore, LCFA chain is also chosen to fit the pocket II to for
enhanced topo II binding affinity as well as albumin-mediated drug delivery and formulation.
Based on the above SAR analysis and also as a long-standing interest in engaging in the research of
peptidomimetic-derived medicinal chemistry [29-38], we hereby aimed to exploit a novel series of
quinoxaline analogues of XK469 by incorporating these useful pharmacophoric groups and antitumor
inducements (e.g., tert-butyl and LCFA moieties) into a single peptidomimetic molecule for achieving
potent antineoplastic activity. Specifically, the design concepts of these novel XK469 analogues are
described as followings (Fig. 3): (i) The quinoxaline backbone (part A) is kept, and Cl and H atoms are
selected as R1 substituent at the 7-position. To better understand how the disposition of substituents on the
quinoxaline core affect the bioactivity, substitution at the 6-position was also explored; (ii) The R2 group at
the 3-position is attached with hydrophobic methyl, tert-butylamine, or dodecylamine fragments, aiming to
acquiring both effective hydrophobic contacts and possible enhanced affinity by penetrating into pocket II
of topo II. Besides, the basic amine motif in these substitutions is also believed to benefit the drug uptake of
cancer cells [39]; (iii) The essential hydroquinone connecting part (part B) remains unchanged, and the
naturally available
with an intention to avoid chiral synthesis or separation, and thus simplifying the synthetic procedures.
More importantly, the amino group of incorporated -propionic acid can readily react with another
L-propionic acid is directly incorporated to construct the R(+) free acid portion (part C),
L
carboxylic acid to build a desirable peptidomimetic function. As a surrogate for the O atom of XK469, the
integrated amide bond might produce better binding affinity for biotarget through offering potential
H-bonding donor (NH) and acceptor (C=O), and subsequently gain an elevated activity. Additionally, to
further expand the SAR investigation as well as biological scope of quinoxaline peptidomimetic derivatives,
we have explored the chemical diversification by introducing other
L-amino acids (e.g. leucine,

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phenylalanine, isoleucine, glycine), in addition to alanine.
Fig. 3. The design concepts of quinoxaline peptidomimetic analogues of XK469.
To test the above assumption, a spectrum of quinoxaline peptidomimetics were synthesized, and in
vitro and in vivo antitumor activities were consequently evaluated. Moreover, preliminary SAR
investigation, mechanism of action, and molecular modeling study of these derivatives were also examined.
All together, we hoped the present exploration on the XK469 analogues might provide a clue for further
development of diversified peptidomimetic derivates with antineoplastic potentials.
2. Results and discussion
2.1. Chemistry
To obtain series I compounds 5a-e & 6a-g (R2=methyl), the general synthetic steps outlined in Scheme
1 were adopted. Briefly, the condensation of commercially available o-phenylenediamine with ethyl
pyruvate led to the formation of quinoxalinone core 1. It should be noted that when 4-chloro
o-phenylenediamine was used as starting material, the reaction with ethyl pyruvate gave a mixture of two
positional isomers, 6-chloro-3-methylquinoxalin-2(1H)-one (1b) and
7-chloro-3-methylquinoxalin-2(1H)-one (1c), under the same reaction condition. The isomer 1b, which has
slightly lower polarity (higher Rf value on the TLC plate), can also be regioselectively prepared according
to our previously reported method [40]. After separation through flash column chromatography, the
chemical structures of these two isolated isomers (1b and 1c), can be easily distinguished based on
respective 1H NMR spectrum information (see Experimental section). Subsequent chlorination followed by
nucleophilic substitution by using methyl 4-hydroxybenzoate in the presence of K2CO3 gave the
O-substituted ester compound 3 with relatively high yield (>80%). After purification with conventional
column chromatography method, the ester intermediate 3 was then subjected to hydrolysis under alkaline
condition to generate the corresponding carboxylic acids 4a-c. This was followed by coupling with various
L-amino acid methyl ester hydrochlorides in the existence of isobutyl chloroformate and
N-methylmorpholine (NMM) in anhydrous THF to yield the quinoxaline peptidomimetic esters 5a-e. In
this reaction, apart from THF, other aprotic polar solvents, e.g. dichloromethane (CH2Cl2) and

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N,N-dimethylformamide (DMF), are possible alternatives. The quinoxaline peptidomimetic esters (5a-g)
were further subjected to hydrolysis process to afford the final products 6a-g.
Scheme 1. Synthetic procedures of quinoxaline peptidomimetic derivates 5a-e and 6a-g. Reagents and
conditions: (a) Ethyl pyruvate, anhydrous EtOH, rt; (b) POCl3, reflux; (c) K2CO3, DMF, 85oC; (d) LiOH,
Dioxane/H2O (3:1, v:v); (e) L-amino acid methyl ester hydrochloride, isobutyl chloroformate,
4-Methylmorpholine, anhydrous THF, 0oC to r.t.
The synthetic protocol of quinoxaline peptidomimetic derivatives 12a-b & 13a-h (R2= tert-butylamine
or dodecylamine moieties) is illustrated in Scheme 2. The condensation of unsubstituted
o-phenylenediamine with oxalic acid led to quinoxaline-2,3(1H,4H)-dione 7. After treating compound 7
with POCl3 under refluxing condition, the chlorinated intermediate 8 was obtained, which was subjected to
the subsequent nucleophilic substitution with methyl 4-hydroxybenzoate and alicyclic amines in the
presence of weak bases, K2CO3 and triethylamine (TEA), respectively, and was converted to the key ester
intermediates 10a and 10b. These esters were then treated by ester hydrolyzation, condensation, and
another round of ester hydrolyzation to afford the products 13a-h.

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H
NH2
NH2
N
O
O
N
N
8
Cl
Cl
O
HO
O
O
O
a
b
HO
N
H
OH
7
N
N
O
Cl
9
N
N
O
N
N
O
c
d
e
O
O
O
R2
R2
H
O
O
O
10a: R2=NH(CH2)11CH3
10b: R2=NHC(CH3)3
11a: R2=NH(CH2)11CH3
11b: R2=NHC(CH3)3
N
N
O
N
O
O
O
H
f
H
e
N
R2
O
N
N
R2
OH
R3
O
O
R3
12a: R2=NH(CH2)11CH3; R3=CH2C6H5
13a: R2=NH(CH2)11CH3; R3=CH2C6H5
2
R =NH(CH2)11CH3; R3=CH3
2
R =NH(CH2)11CH3; R3=CH3
12b:
13b:
12c: R2=NH(CH2)11CH3; R3=CH(CH3)CH2CH3
12d: R2=NHC(CH3)3; R3=H
13c: R2=NH(CH2)11CH3; R3=CH(CH3)CH2CH3
13d: R2=NHC(CH3)3; R3=H
2
3
2
3
12e:
R =NHC(CH3)3; R =CH3
13e:
R =NHC(CH3)3; R =CH3
12f: R2=NHC(CH3)3; R3=CH(CH3)CH2CH3
12g: R2=NHC(CH3)3; R3=CH2CH(CH3)2
13f: R2=NHC(CH3)3; R3=CH(CH3)CH2CH3
13g: R2=NHC(CH3)3; R3=CH2CH(CH3)2
2
3
2
3
12h:
R =NHC(CH3)3; R =CH2C6H5
13h:
R =NHC(CH3)3; R =CH2C6H5
Scheme 2. Synthetic procedures of quinoxaline peptidomimetic derivates 12a-h and 13a-h. Reagents and
conditions: (a) Conc. HCl, reflux; (b) POCl3, reflux; (c) K2CO3, DMF, 85oC; (d) R2NH2, TEA, DMSO,
75oC; (e) LiOH, Dioxane/H2O (3:1, v:v); (f) L-amino acid methyl ester hydrochloride, isobutyl
chloroformate, 4-Methylmorpholine, anhydrous THF, 0oC to r.t.
2.2. Anti-proliferative evaluation and structure-activity relationships (SARs) examination
Under the guidance of activity evaluation of topo II inhibitors [41], compounds 5a-e, 6a-g, 12a-b and
13a-h were initially screened for their anti-proliferative and cytotoxic efficacies against a panel of topo II
inhibitor-sensitive human tumor cell lines by MTT assay (Table 1). Topo II inhibitor Doxo was used as a
positive control.
In general, the vast majority of test compounds displayed moderate anti-proliferative activities against
tumor cell proliferation, except two ester compounds (12d and 12g) and one carboxylic acid compound
(13d) which did not exhibit obvious cytotoxicity. Besides, all carboxylic acid compounds (R4=H) showed
improved anti-proliferative activities compared with their precursor esters (R4=CH3, entries 5a-g vs. 6a-g,
12a-h vs. 13a-h). This result confirmed the essential role of the free acid unit of XK469 on maintaining
antitumor potency. It is likely that this free acid motif can provide more effective hydrogen-bonding
interactions with target protein, which may benefit the protein backbone-binding affinity. Generally, the
carboxylic acid compounds may enable potential oral bioavailability in comparison with the less-active
ester counterparts, largely due to better aqueous solubility.

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Among these compounds, when R2 group is fixed as a methyl group, the impact of R1 group on
quinoxaline core was subsequently examined. It indicated that Cl atom (entries 5b, 5e, 5g) did not
contribute to a dramatically elevated antitumor efficacy compared with XK469, implying halogen element
may not be a determining factor for the bioactivity of these compounds. However, changing halogen
substitution from the C-6 position to the C-7 position (entries 5e vs. 5g and 6e vs. 6g) resulted in
moderately improved activity, which is partially similar to the SAR of XK469. Following these clues, the
R1 group in compounds 12 and 13 was fixed as H atom, and then we turned our attention to the influence of
tert-butylamine or dodecylamine moiety.
In all series, compounds 13a and 13b that possess tert-butylamine or dodecylamine fragment at the
3-position of quinoxaline core generated better anti-proliferative potencies than Doxo, while 13f that
possesses tert-butylamine and L-propionic acid showed cytotoxicity comparable with that of control. These
results indicated the critical function of R2 substitution. Thus, to a certain extent, it proved our design
concept on XK467 analogues. However, compounds 12d, 12g, 13d and 13g with the same
pharmacophore-like groups, showed significantly impaired potencies. This result might be largely
attributed to the inappropriate size and/or orientation of aliphatic sec-butyl group of isoleucine, making
these four compounds cannot accommodate the active binding area of target protein. Likewise, other
compounds with sec-butyl group (entries 5c and 6c) also demonstrated reduced in vitro cytotoxicity.
Finally, when fixing the R3 group as a methyl or benzyl group, the efficacies of 13a and 13b (R2 =
dodecylamine fragment) are superior to that of entries 13e and 13h (R2 = tert-butylamine unit). A possible
explanation was that the flexibility of long chain dodecyl group made it be more suitable for fitting the
active binding pocket (pocket II) of the topo II. But it seemed that this finding cannot be applied to
compounds 13d and 13g, whose R2 substituent is a sec-butyl group. Although both compounds have weak
or even no activity, 13g with tert-butylamine portion resulted in a slightly increased activity compared with
the dodecylamine substituted counterpart 13d. This comparison suggests that the negative effect by
attaching a sec-butyl group to the R3 position.
In brief, in regard to the inhibitory effects, although almost all tested compounds exerted similar
cytotoxicity towards selected tumor cells in this study, most of them gave elevated anti-proliferative
activities than the prototype XK469 reported previously. According to the NCI anti-proliferative
assessment towards 60 tumor cells, the average IC50 value of XK469 is about 70µM [8]. Although the
quinoxaline peptidomimetic analogues tend to have similar action mechanism as that of XK469, they are
likely to yield better anti-proliferative efficacies. Among these XK469 derivatives, two compounds with
dodecylamine segment, 13a and 13b in particular, exhibited obviously improved cytotoxicity.
Table 1. Chemical structures and anti-proliferative activities of XK469 analogues.

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Compds R1
R2
R3
R4
MCF-7
K562
Hela
IC50(µM) a
38.43 ± 8.1
IC50(µM) a
45.54 ± 20.9
IC50(µM) a
32.20 ± 4.4**
H
CH3
CH3
CH3
CH3
CH3
5a
H
25.53 ± 0.34
22.63 ± 0.36
21.55 ± 0.37
5b
H
CH3
CH3
36.14 ± 3.8
38.26 ± 13.6
>50
5c
6-Cl CH3
6-Cl CH3
CH3
CH3 33.77 ± 2.1**
26.02 ± 3.8
42.51 ± 20.9
37.55 ± 0.32
5d
5e
CH3
22.75 ± 0.82
32.56 ± 0.32
7-Cl CH3
7-Cl CH3
CH3
CH3
25.52 ± 0.36
19.05 ± 0.1
27.53 ± 0.32
22.01 ± 2.0
28.76 ± 3.6*
18.64 ± 0.5**
5f
5g
H
H
CH3
CH3
CH3
H
H
25.55 ± 2.0
14.72 ± 0.49*
28.06 ± 2.6
22.31 ± 3.5
17.32 ± 1.6
6a
6b
19.51 ± 0.16
H
CH3
H
35.71 ± 1.5
30.50 ± 1.2
33.24 ± 2.1
6c
6-Cl CH3
6-Cl CH3
CH3
H
H
19.45 ± 3.7
17.41 ± 4.8**
20.85 ± 6.8*
21.33 ± 1.3
29.99 ± 8.0
25.02 ± 1.1
6d
6e
7-Cl CH3
7-Cl CH3
H
H
20.15 ± 3.2
15.11±6.6
21.51 ± 1.2*
14.52±2.8
20.21 ± 5.1
16.35 ± 0.4
6f
6g
H
NH(CH2)11CH3
CH3
33.43 ± 3.8
30.50 ± 0.1
34.44± 4.7**
12a
H
H
NH(CH2)11CH3
NH(CH2)11CH3
CH3
CH3 33.77 ± 2.1**
CH3 43.26 ± 3.8**
26.72 ± 8.3*
30.77 ± 9.1
28.48 ± 0.48
40.3 ± 1.3**
12b
12c
H
NH(CH2)11CH3
CH3
>50
>50
>50
12d
12e
12f
12g
H
H
H
NH(CH2)11CH3
NHC(CH3)3
NHC(CH3)3
H
CH3 38.43 ± 2.1**
CH3 33.44 ± 4.6**
42.23 ± 20.1
38.7 ± 5.3
>50
55.16 ± 8.5
31.9 ± 1.9*
>50
CH3
CH3
>50
H
NHC(CH3)3
CH3
>50
10.31 ± 1.7*
>50
12h

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H
NH(CH2)11CH3
H
6.79 ± 0.17
7.37 ± 0.14
9.19 ± 0.42
13a
H
H
NH(CH2)11CH3
NH(CH2)11CH3
CH3
H
H
4.11 ± 0.24
3.28 ± 0.28**
21.54 ± 0.31
6.24 ± 0.27
35.46 ± 3.7*
13b
13c
30.42 ± 0.68
H
NH(CH2)11CH3
H
>50
>50
>50
13d
H
H
H
NH(CH2)11CH3
NHC(CH3)3
NHC(CH3)3
H
H
H
H
29.44 ± 5.8** 26.41 ± 11.14
11.35 ± 0.37 18.34 ± 0.58*
23.43 ± 5.33*
13.36 ± 0.23
39.19 ± 3.8**
13e
13f
13g
CH3
40.32 ± 0.43
42.33 ± 0.46
H
NHC(CH3)3
H
25.34 ± 0.38
8.81 ± 0.11
15.22 ± 1.5* 20.50 ± 13.63
13.53 ± 1.3 11.50 ± 0.45
13h
Doxo
a Values are means of three experiments, standard deviation is given. * p < 0.01, ** p < 0.05 compared to the control
(Doxo).
2.3. Anti-metastatic activity in vivo
In vivo anti-metastatic evaluation was subsequently conducted. Human H22 hepatocarcinoma cells
metastatic cancer BALB/c mouse model was utilized following the protocol described by our previous
works [42, 43]. Five compounds revealing obvious anti-proliferative activities at cellular levels (6b, 6g,
13a, 13b, 13f) were subjected to the in vivo assessment through intragastric administration. The
anti-metastatic ability of these compounds was measured based on the inhibition of H22 cell localization in
the lungs.
As shown in Table 2, it demonstrated that the in vivo anti-tumor results using this model are in
accordance with that of in vitro anti-proliferative investigation. Moreover, an obvious weight loss was not
observed during continuous administration, suggesting mice could be continually experienced the
maximum tolerance dosage. However, all tested compounds exhibited lower inhibitory rates compared with
the control (Doxo, 59.89%), with the values of 33.71% (entry 13a), 42.47% (entry 13b), 27.60% (entry
13f), 14.77% (entry 6g) and 10.98% (entry 6b), respectively. This result might be attributed to the first-pass
effect by using intragastric administration, leading to ineffective absorption metabolized through
gastrointestinal tract. In this circumstance, developing more effective antitumor agents via structural
optimization or attempting other routes of administration (e.g. intravenous or intraperitoneal injection) is
expected to increase the metabolic stability and achieve improved bioavailability.
Another fascinating aspect that is connected to this in vivo evaluation is that relatively high levels of
XK469 (approximate 74.4 mg/kg) is tolerated to exert the therapeutic effects on tumor-bearing mice
reported previously [8], while the dosages used in this assay (10 mg/kg) is much lower than that of XK469 .

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This result alluded that these newly identified quinoxaline peptidomimetic derivates, 13a and 13b in
particular, which exhibited promising in vitro cytotoxicity and in vivo anti-metastatic activities, might
generate more potent curative efficacies than XK469. However, owing to the lower activity than the control
Doxo, further deliberate chemical optimization is still needed to generate more effective tool compounds
for tumor therapeutic screening.
Table 2. In vivo anti-metastatic effects of selected compounds.
Compds Survived
Body
Lung weight (g)
Metastasized nodes Inhibitory
mice (n)
weight (g)
22.46 ± 3.22
on lung surface (n)
18.23 ± 9.44**
rate (%)
10.98
10
0.238 ± 0.024
0.254 ± 0.033*
0.197 ± 0.025
0.230 ± 0.040*
0.211 ± 0.027
0.217 ± 0.021
0.287 ± 0.117
6b
6g
10
23.25 ± 4.14
20.85 ± 3.79
24.62 ± 3.82
21.84 ± 5.58
23.32 ± 1.80
20.17 ± 3.66
17.14 ± 6.11**
13.33 ± 4.14**
11.67 ± 6.73
14.56 ± 7.54*
8.11 ± 3.33
14.77
33.71
42.47
27.60
59.89
−
9(10)
9(10)
10
13a
13b
13f
9(10)
10
Doxo
Blank
20.11 ± 7.84
a The animal number in parentheses is the original number; b Blank control means 0.5% CMC-Na; *P<0.05; **P<0.01.
2.4. Topo II-mediated kinetoplast DNA (kDNA) decatenation assay
Given evidences have revealed that XK469 might selectively inhibit DNA topo II, rather than topo I,
the topo II-mediated kDNA decatenation assay was thereby implemented to validate the possible
mechanism of action of selected compounds, according to the formation of decatenated kDNA. Based on
the biochemical activity of topo II, inhibition of topo II activity leads to the accumulation of catenated
kDNA [44]. In this assay, two potent compounds (13a and 13b) that standed out from the in vitro and in
vivo screening were utilized. The validated topo II inhibitor, etoposide, was utilized as a positive control.
The results evinced that both inhibited the formation of decatenated kDNA examined by agarose gel
electrophoresis, as shown in Fig. 4. Especially, compound 13a gave the best kDNA decantation inhibition
and was approximate 4 times more active than the control. Another compound 13b showed comparable
potencies with the control. Dose-dependent aggregations of kDNA induced by two compounds were clearly
observed at four tested concentrations of 3.15, 12.5, 50 and 200 µM, higher than or comparable with that of
etoposide at the same concentrations. In short, these preliminary results indicated that these quinoxaline
peptidomimetic analogues of XK469 might work as topo II inhibitors, similar to the prototype XK469.

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Fig. 4. Selected compounds (13a and 13b) inhibited topo II-mediated kinetoplast DNA (kDNA)
decatenation to form minicircular DNA. Lane 14: Negative control without topo II; Lane 13: Positive
control that contains topo II but without inhibitors; Lanes 9 12: Topo II inhibitor etoposide (control) at four
tested concentrations of 3.15, 12.5, 50 and 200 µM; Lanes 1 8: Tested compounds at the same
concentrations.
2.5. Molecular docking study
Compound 13b was derived from XK469 and they showed comparable potencies, which was thereby
utilized as a model in molecular docking exploration. Structural configurations of prototype XK469 and
13b were initially optimized via ChemBio3D Ultra program, and their superimposed conformations were
compared via overlapping operation using Sybyl program. Figures 5(a) and 5(b) displayed their preferential
conformations. Fig. 5(c) exhibited that the skeletal backbones of two compounds adopted very similar
spatial orientation, suggesting identical affinity to the same spatial regions (pocket I, II and III).

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Fig. 5. The comparison of optimized configurations of XK469 (a) and 13b (b) via ChemBio3D Ultra
program, and superimposed conformations of XK469 (red) and 13b (gray) (c) signified similar
three-dimensional topology and binding affinity.
The interactions between 13b and topo II (PDB ID: 1ZXM) were further examined by molecular
docking program. As displayed in Fig. 6, 13b could commendably bind to the ATPase domain of topo II.
Both quinoxaline core and terminal carboxylic acid motif of 13b could fit well into the hydrophobic pocket
I and III of topo II, leading to possible enzymatic inhibition, consistent with their bioactivity results.
Moreover, the bulk and flexible long chain dodecyl group could undergo low-energy conformational
change to accommodate the long hydrophobic cavity of topo II (pocket II). In addition to these effective
hydrophobic interactions, several hydrogen-bonding contacts between 13b and key amino acids in the
ATPase domain of topo II also contribute to the enhanced protein backbone-binding affinity. Specifically,
the 1-N of 13b acts as an H-bonding acceptor to interact with the peripheral NH of the Asn91, with a
distance of 2.20Å. Besides, amide O of the peptidomimetic functional group of 13b also serves as an
H-bonding acceptor to bind with Ser149 residue (2.43Å). Finally, both carbonyl O and terminal hydroxyl O
of 13b work as H-bonding acceptors to interact with Asn91 (2.16Å), Ala167 (2.58Å and 1.83Å), and
Lys168 (2.74Å), respectively.

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Fig. 6. Binding pattern of compound 13b with the active binding pockets of topo II (PDB code: 1ZXM).
Comparing the binding modes of 13b and XK469, 13b not only provided more effective H-bonding
contacts, but also generated more hydrophobic interactions through well occupation of three hydrophobic
pockets (I, II and II) of topo II. As a result, 13b might be more potent than the prototype XK469 in
achieving potential anti-tumor drugs. Further biological assessments of 13a and 13b are underway and the
updated results will be reported in the near future.
3. Conclusion
In the present work, we report the design, synthesis, biological evaluation, biochemical assay, and
binding mode of novel quinoxaline peptidomimetic derivatives as potent antitumor agents. These
compounds exhibited obvious cytotoxicity against three tumor cell lines (MCF-7, K562 and Hela).
Subsequent in vivo anti-metastatic evaluation also supported the antitumor effects of these compounds,
especially compounds 13a and 13b. SAR exploration implicated that tert-butyl or dodecyl moiety at the
3-position of quinoxaline core was favorable to fit the ATPase pocket of topo II and contribute to improved
antineoplastic activities. The dodecyl moiety is proved to be more favored than that of tert-butylamine
motif, although both of these two motifs could lead to somehow improved anti-proliferative potencies
against selected cell lines. Moreover, the free carboxylic acid unit of XK469 is important to maintain the
antitumor activity. Additionally, topo II-mediated kDNA decatenation assay also preliminarily confirmed
that these derivatives are potent topo II inhibitors.
Lastly, molecular modeling of prototype XK469 and 13b offers a visual framework for understanding
the structural basis for their topo II inhibitory activities, and might reveal a general strategy for design and
screen more potent topo II inhibitors. In specific, 13b, a peptidomimetic analogue of XK469 with a flexible
long chain dodecyl group, adopted a very similar 3D topology and spatial orientation compared with
XK469. It implies that compounds reported in this paper might share similar mechanism of action with the
prototype XK469. The detailed mechanism of action of these derivatives is under investigation.

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Based on the biochemical and biological results, we envisioned that quinoxaline peptidomimetic
derivatives 13a and 13b might be promising candidates for further chemical optimization. In particular, the
introduced R2 hydrophobic dodecyl unit of these two compounds may not only be critical for elevated
antitumor activities through enhancing protein backbone binding interactions, but also contribute to
improved pharmacokinetic properties via albumin binding. Overall, we anticipated these identified
quinoxaline peptidomimetic derivatives may provide a clue for researchers to develop more diversified
compounds with improved antineoplastic activities.
4. Experimental section
4.1. Chemistry
4.1.1. General
All the starting materials including reagents and solvents, unless otherwise specified, were analytically
pure and obtained from commercial suppliers. They were used without further purification, when necessary,
were purified and dried by standard protocols. All reactions except those in aqueous media were carried out
by standard techniques for the exclusion of moisture. All reaction progress was monitored by thin-layer
chromatography (TLC) on 0.25-mm silica gel plates (grade 60, GF254) and visualized with UV light
(254nm), or iodine vapor. Flash chromatography was performed on silica gel (Yantai Muping Inc.,
300 400 mesh). Melting points were determined using X-6 digital display binocular microscope
1
(uncorrected). H NMR and 13C NMR spectra were recorded on a Brucker Advance 300 or DRX-400
spectrometer using CDCl3 or DMSO-d6 as solvents, TMS as an internal standard. Chemical shift values (δ)
are reported in parts per million (ppm) and the coupling constants (J) are given in hertz (Hz), and splitting
patterns are designated as follows: s, single; d, doublet; t, triplet; q, quartet; m, multiplet. Electrospray
ionization mass spectrometry (ESI−MS) data were collected on a Micromass Qtof-Micro LC−MS
instrument.
4.1.2. General synthetic procedure for the preparation of 3-methylquinoxalin-2(1H)-one (1)
o-Phenylenediamine (92.6 mmol, 10 g) was suspended in anhydrous ethanol (100 mL). The mixture
was then cooled in an ice bath. A solution of ethyl pyruvate (202.9 mmol, 23.54g) in anhydrous ethanol (20
mL) was added dropwise over a period of 20 min under stirring. The resulting solution was allowed to react
at room temperature for 6 h until the TLC showed the reaction has completed. The resulting precipitate was
filtered and washed with anhydrous ethanol (2 × 15 mL), and dried in vacuum to give the desire product 1a
(14.1 g, 95%) as white solid which was pure enough for further reaction. M.p: 241.0 242.7 oC; ESI-MS
m/z: 161.0 [M+H]+.
Using 4-chlorobenzene-1,2-diamine as starting material, the reaction with ethyl pyruvate under the
same condition with 1a gave two positional isomers, 6-chloro-3-methylquinoxalin-2(1H)-one (1b) and
7-chloro-3-methylquinoxalin-2(1H)-one (1c).
4.1.2.1.6-Chloro-3-methylquinoxalin-2(1H)-one (1b). Light yellow powder, yield: 54%, mp: 263.0 264.0

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1
oC. H NMR (400 MHz, DMSO-d6) δ: 12.36 (s, 1H, NH), 7.68 7.70 (d, J = 8.4 Hz, 1H, ArH), 7.27 7.30
(dd, J1 = 8.4 Hz, J2=2.3 Hz, 1H, ArH), 7.26 (d, J = 2.3 Hz, 1H, ArH), 2.39 (s, 3H, CH3); ESI-MS m/z:
195.3 [M+H]+.
4.1.2.2. 7-Chloro-3-methylquinoxalin-2(1H)-one (1c). Light yellow powder, yield: 43%, mp: 265.0 267.0
1
oC. H NMR (400 MHz, DMSO-d6, ppm) δ: 12.41 (s, 1H, NH), 7.74 7.75 (d, J = 2.3 Hz, 1H ArH),
7.51 7.53 (dd, J1 = 8.7 Hz, J2=2.3 Hz, 1H, ArH), 7.27 7.29 (d, J = 8.7 Hz, 1H, ArH), 2.41 (s, 3H, CH3);
ESI-MS m/z: 195.3 [M+H]+.
4.1.3. General procedure for 2-chloro-3-methyl substituted quinoxaline (2)
Compound 1a (28.1 mmol, 5.0 g) was dissolved in POCl3 (25 mL) at 0oC, the resulting solution was
allowed to reflux at 120oC for 5 h. After completion of reaction, the reaction was quenched with cooled
ammonia, which was further extracted with ethyl acetate (EtOAc, 2 × 15 mL). The organic phase was
separated and washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuum to
afford the crude product, which was purified by silica gel flas chromatography (PE : EA = 10:1, v:v) to
afford 5.0 g of 2a as a off-white solid, yield 90%. M.p.: 86.0 87.0 oC; ESI-MS m/z: 179.4 [M+H]+.
4.1.3.1. 2,6-Dichloro-3-methylquinoxaline (2b). White powder, yield: 89%, ESI-MS m/z: 213.8 [M+H]+;
mp: 128.2 129.3 oC.
4.1.3.2. 3,6-Dichloro-2-methylquinoxaline (2c). White powder, yield: 86%, ESI-MS m/z: 214.1 [M+H]+;
mp: 132.1 133.2 oC.
4.1.4. General procedure for methyl 4-((substituted 3-methylquinoxalin-2-yl)oxy)benzoate (3)
Methyl 4-hydroxybenzoate (42.1 mmol, 6.4 g) and K2CO3 (50.5 mmol, 5.3 g) were dissolved in DMF
(150 mL), the resulting solution was allowed to react at 85oC for 12 h. Compound 2a (23.5 mmol, 5.0 g)
was added in batches, and the resulting mixture was allowed to react for additional 12h at the same
condition. After completion of reaction, the reaction was quenched with cooled water (150 mL) and
extracted with EtOAc (2 × 100 mL). The combined organic phase was washed with brine, dried over
anhydrous MgSO4, filtered, and concentrated in vacuum to afford the crude product, which was purified by
silica gel flash chromatography (PE : EA = 50:1, v:v) to afford 6.6 g of 3a as oily semi- solid, yield 80%.
ESI-MS m/z: 295.4 [M+H]+.
4.1.4.1. Methyl 4-((6-chloro-3-methylquinoxalin-2-yl)oxy)benzoate (3b). Oily semi-solid, yield 68%;
ESI-MS m/z: 329.8 [M+H]+.
4.1.4.2. Methyl 4-((7-chloro-3-methylquinoxalin-2-yl)oxy)benzoate (3c). Oily semi-solid, yield 59%;
ESI-MS m/z: 329.6 [M+H]+.
4.1.5. General procedure for 4-((substituted 3-methylquinoxalin-2-yl)oxy)benzoic acid (4)
Methyl 4-((3-methylquinoxalin-2-yl)oxy)benzoate (8.0 mmol, 2.0 g) and LiOH (24.0 mmol, 0.85 g)
were suspended in a mixture of dioxane (15 mL) and water (5 mL) at 0oC, the resulting suspension was
reacted for 5h. After the removal of solvent, 20 mL of distilled water was added and the mixture was

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washed with EtOAc (2 × 10 mL). Then the combined water phase was acidified with 2N HCl and the
precipitate was collected, which was recrystallized with MeOH/H2O (3:1, v:v) to afford 1.8 g of the pure
product 4-((3-methylquinoxalin-2-yl)oxy)benzoic acid (4a) as white semi-solid. Yield 95%; 1H NMR (400
MHz, DMSO-d6) δ: 13.03 (s, 1H, COOH), 8.06 8.08 (d, J = 8.4 Hz, 2H, ArH), 7.99 (s, 1H, ArH), 7.66 (s,
3H, ArH), 7.45 7.47 (d, J = 8.4 Hz, 2H, ArH), 2.76 (s, 3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ:
166.66, 148.04, 139.02, 138.52, 131.09, 129.44, 127.80, 127.66, 126.81, 121.82, 20.28 ppm; ESI-MS m/z:
281.3 [M+H]+.
4.1.5.1. 4-((6-Chloro-3-methylquinoxalin-2-yl)oxy)benzoic acid (4b). Semi-solid, yield 90%; 1H NMR (400
MHz, DMSO-d6) δ: 13.04 (s, 1H, COOH), 8.06 8.08 (d, J = 8.0 Hz, 3H, ArH), 7.68 (s, 2H, ArH),
7.40 7.48 (m, 2H, ArH), 2.76 (s, 3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 167.13, 156.64,
149.65, 139.64, 137.30, 131.60, 130.29, 129.06, 126.69, 122.36, 100.03, 20.85 ppm; ESI-MS m/z: 315.5
[M+H]+.
4.1.5.2. 4-((7-Chloro-3-methylquinoxalin-2-yl)oxy)benzoic acid (4c). Semi-solid, yield 91%; 1H NMR (400
MHz, DMSO-d6) δ: 12.99 (s, 1H, COOH), 8.05 8.07 (d, 2H, J = 8.0 Hz, ArH), 7.99 8.01 (m, 1H, ArH),
7.67 7. 73 (m, 2H, ArH), 7.45 7.47(d, J = 8.0 Hz, 2H, ArH), 2.74 (s, 3H, CH3) ppm; 13C NMR (100 MHz,
DMSO-d6) δ: 167.18, 156.57, 156.26, 150.01, 140.10, 138.01, 132.86, 131.61, 130.30, 129.17, 128.47,
122.40, 120.44, 20.88 ppm; ESI-MS m/z: 315.7 [M+H]+.
4.1.6. General procedure for the synthesis of ester compound (5)
To a solution of ester compound 4a (1.8 mmol, 0.5 g) in anhydrous THF (15 mL) at -5oC was added
isobutyl chloroformate (2.2 mmol, 300 mg) and N-methylmorpholine (NMM, 2.2 mmol, 220 mg)
successively. After stirred at the same temperature for 30min, L-alanine methyl ester hydrochloride (2.0
mmol, 142 mg) was added in small portions. Then the mixture was stirred at room temperature for 4h until
the completion monitored by TLC, which was then concentrated and extracted with EtOAc (2 × 50 mL).
Layers were separated and the organc layer was washed in turn with 1N HCl (20 mL), saturated NaHCO3
(20mL) and brine (2 × 15 mL), dried over anhydrous Na2SO4, filtered, and evaporated in vacuo, and the
obtained crude compound was purified by flash chromatography (PE : EA = 5:1 to 4:1, v:v) to afford 370
mg of 5a as white solid, yield 58%. M.p. 161 162oC; 1H NMR (400 MHz, CDCl3) δ: 7.97 8.00 (dd, J1 =
6.0 Hz, J2 = 3.7 Hz, 1H ArH), 7.91 7.94 (d, J = 8.7 Hz, 2H, ArH), 7.67 7.70 (m, 1H, ArH), 7.58 7.61 (m,
2H, ArH), 7.35 7.36 (d, J = 8.7 Hz, 2H, ArH), 6.77 6.79 (d, J = 7.0 Hz, 1H, ArH), 4.80 4.88 (dd, J1 =
14.3, J2 = 7.2 Hz, 1H, CH), 3.82 (s, 3H, OCH3), 2.83 (s, 3H, CH3), 1.55 1.57 (d, J = 7.1 Hz, 3H, CH3).13C
NMR (100 MHz, CDCl3) δ: 173.74, 166.11, 155.75, 155.55, 147.85, 139.61, 139.22, 130.85, 129.29,
128.71, 128.07, 127.60, 127.31, 121.81, 52.65, 48.57, 20.62, 18.73 ppm; ESI-MS m/z: 366.5 [M+H]+.
The following compounds 5b-5g were prepared according to the general procedures as described for
the preparation of compound 5a.

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4.1.6.1. (S)-Methyl 4-methyl-2-(4-((3-methylquinoxalin-2-yl)oxy)benzamido)pentanoate (5b). White solid,
yield: 52%; m.p. = 166 169oC; 1H NMR (400 MHz, DMSO-d6) δ: 7.97 8.00 (dd, J1 = 8.0 Hz J2 = 4.0 Hz,
1H, ArH), 7.91 7.93 (d, J = 8.0 Hz, 2H, ArH), 7.68 7.69 (d, J = 4.0 Hz, 1H, ArH), 7.58 7.61 (dd, J1 = 8.0
Hz, J2 = 4.0 Hz, 2H, ArH), 7.35 7.37 (d, J = 8.0 Hz, 2H, ArH), 6.53 6.55 (d, J = 8.0 Hz, 1H, NH),
4.87 4.90 (m, 1H, CH-C=O), 3.79 (s, 3H, OCH3), 2.83 (s, 3H, CH3), 1.71 1.77 (t, J = 8.0 Hz, 2H, CH3),
1.67 1.69 (m, 1H, CH), 0.99 1.03 (m, 6H, 2×CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 173.85,
166.59, 156.00, 155.66, 148.02, 139.88, 139.40, 131.08, 129.42, 128.85, 128.25, 127.73, 127.48, 121.95,
52.56, 51.39, 42.19, 25.21, 23.00, 22.30, 21.17 ppm; ESI-MS m/z: 408.5 [M+H]+.
4.1.6.2. (2S,3R)-Methyl 3-methyl-2-(4-((3-methylquinoxalin-2-yl)oxy)benzamido)pentanoate (5c). White
solid, yield: 55%; m.p. = 172 173oC; 1H NMR (400 MHz, DMSO-d6) δ: 7.98 8.00 (m, 1H, ArH),
7.91 7.93 (d, J = 8.0 Hz, 2H, ArH), 7.69 7.71 (m, 1H, ArH), 7.58 7.61 (m, 2H, ArH), 7.36 7.38 (d, J =
8.0 Hz, 2H, ArH), 6.67 6.69 (d, J = 8.0 Hz, 1H, NH), 4.84 4.87 (m, 1H, CH-C=O), 3.80 (s, 3H, OCH3),
2.83 (s, 3H, CH3), 1.26 1.31 (m, 1H), 0.97 1.01 (m, 2H, CH2CH3), 0.91 0.93 (d, J = 8.0 Hz, 6H, 2×CH3),
ppm; 13C NMR (100 MHz, DMSO-d6) δ: 174.2, 170.8, 167.7, 158.8, 138.0, 137.7, 130.9, 129.4, 128.7,
128.2, 127.4, 127.1, 121.4, 57.6, 51.8, 38.2, 25.5, 17.1, 15.3, 12.1 ppm; ESI-MS m/z: 408.6 [M+H]+.
4.1.6.3. (S)-Methyl 2-(4-((6-chloro-3-methylquinoxalin-2-yl)oxy)benzamido)propanoate (5d). White solid,
yield: 51%; m.p. = 180 181oC; 1H NMR (400 MHz, DMSO-d6) δ: 7.92 7.96 (m, 3H, ArH), 7.61 7.63 (d, J
= 8.0 Hz, 1H, ArH), 7.53 7.55 (d, J = 8.0 Hz, 1H, ArH), 7.34 7.37 (m, 2H, ArH), 6.66 (d, J = 7.2 Hz, 1H,
NH), 4.28 4.30 (d, J = 8.0 Hz, 1H, CH-C=O), 3.82 (s, 3H, OCH3), 2.82 (s, 3H, CH3), 1.57 (d, J = 7.2 Hz,
3H, CHCH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 173.72, 166.02, 156.15, 155.39, 148.10, 139.74,
138.03, 134.99, 131.16, 129.20, 128.74, 128.38, 126.39, 121.90, 52.66, 48.58, 20.58, 18.72 ppm; ESI-MS
m/z: 400.3 [M+H]+.
4.1.6.4. (S)-Methyl 2-(4-((6-chloro-3-methylquinoxalin-2-yl)oxy)benzamido)-4-methylpentanoate (5e).
White solid, yield: 45%; m.p. = 156 158oC; 1H NMR (400 MHz, CDCl3) δ: 7.93 7.89 (t, J = 8.0 Hz, 3H,
ArH), 7.69 (s, 1H, ArH), 7.52 7.54 (d, J = 8.0 Hz, 1H, ArH), 7.33 7.35 (d, J = 8.0 Hz, 2H, ArH),
6.54 6.56 (d, J = 8.0 Hz, 1H, NH), 4.87 4.90 (m, 1H, CH), 3.79 (s, 3H, OCH3), 2.82 (s, 3H, CH3),
1.58 1.59 (m, 1H, CH), 1.24 1.31 (m, 2H, CH2), 0.99 1.03 (m, 6H, 2×CH3) ppm; 13C NMR (100 MHz,
CDCl3) δ: 173.65, 166.40, 153.31, 155.41, 148.07, 139.60, 138.06, 134.99, 130.2, 129.20, 128.71, 128.36,
126.39, 121.87, 52.40, 51.24, 42.02, 25.05, 22.84, 22.13, 20.53 ppm; ESI-MS: m/z: 442.5 [M+H]+.
4.1.6.5. (S)-Methyl 2-(4-((7-chloro-3-methylquinoxalin-2-yl)oxy)benzamido)-3-phenylpropanoate (5f).
White solid, yield: 40%; m.p. = 163 164oC; 1H NMR (400 MHz, CDCl3) δ: 7.87 7.88 (d, J = 2.0 Hz, 1H,
ArH), 7.82 7.85 (dd, J1 = 8.8 Hz, J2 = 2.5 Hz, 3H, ArH), 7.65 7.68 (dd, J1 = 8.8 Hz, J2 = 2.0 Hz, 1H,
ArH), 7.33 7.26 (m, 5H, ArH), 7.15 7.17(d, J = 6.8 Hz, 2H, ArH), 6.60 6.62 (d, J = 7.4 Hz, 1H, NH),
5.10 5.15 (dd, J1 = 13.0 Hz, J2= 5.6 Hz, 1H, CH-C=O), 3.79 (s, 3H, OCH3), 3.27 3.32 (m, 2H, CH2), 2.80
(s, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3) δ: 172.10, 166.14, 155.79, 155.50, 147.87, 139.61, 139.20,

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135.84, 130.79, 129.37, 129.31, 128.69, 128.53, 128.07, 127.63, 127.32, 127.27, 121.78, 53.58, 52.50,
37.95, 20.62 ppm; ESI-MS: m/z: 476.4 [M+H]+.
4.1.6.6. (S)-Methyl 2-(4-((7-chloro-3-methylquinoxalin-2-yl)oxy)benzamido)-4-methylpentanoate (5g).
White solid, yield: 40%; m.p. = 130 132oC; 1H NMR (400 MHz, DMSO-d6) δ: 7.97 7.98 (d, J = 4.0 Hz
1H, ArH), 7.91 7.93 (m, 2H, ArH), 7.60 7.63 (m, 1H, ArH), 7.53 7.55 (d, J = 8.0 Hz, 1H, ArH),
7.33 7.35 (d, J = 8.0 Hz, 2H, ArH), 6.54 6.56 (d, J = 8.0 Hz, 1H, NH), 4.89 4.90 (m, 1H, CH-C=O), 3.79
(s, 3H, OCH3), 2.82 (s, 3H, CH3), 1.33 1.34(m, 1H, CH), 1.25 1.28 (m, 2H, CH2), 0.99 1.03 (m, 6H, 2×
CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 173.68, 166.29, 155.95, 155.22, 149.20, 148.82, 139.87,
132.86, 131.43, 130.04, 128.71, 121.84, 52.42, 51.23, 42.01, 25.05, 22.84, 22.13, 20.61 ppm; ESI-MS: m/z:
442.5 [M+H]+.
4.1.7. General procedure for the synthesis of carboxylic acid compound (6)
These compounds were synthesized according to similar procedures with that of compound (4).
4.1.7.1. (S)-2-(4-((3-Methylquinoxalin-2-yl)oxy)benzamido)propanoic acid (6a). White solid, yield: 66%;
m.p. = 146 147oC; 1H NMR (400 MHz, DMSO-d6) δ: 12.57 (s, 1H, COOH), 8.73 8.74 (d, J = 4.0 Hz, 1H,
NH), 8.00 8.02 (t, J = 8.0 Hz, 3H, ArH), 7.65 (s, 3H, ArH), 7.42 7.45 (m, 2H, ArH), 4.42 4.46 (m, 1H,
CH-C=O), 2.76 (s, 3H, CH3), 1.41 1.42 (d, 3H, J =4.0 Hz, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ:
174.72, 165.87, 156.20, 155.59, 148.51, 139.43, 139.09, 131.60, 129.92, 129.63, 128.29, 128.06, 128.17,
127.28, 122.16, 48.83, 20.82, 17.53 ppm; ESI-MS: m/z: 350.4 [M-H]-.
4.1.7.2. (S)-4-Methyl-2-(4-((3-methylquinoxalin-2-yl)oxy)benzamido)pentanoic acid (6b). White solid,
yield: 82%; m.p. = 143 144oC; 1H NMR (400 MHz, DMSO-d6) δ: 12.61 (s, 1H, COOH), 8.67 (s, 1H, NH),
8.01 8.03 (d, J = 8.0 Hz, 3H, ArH), 7.65 (s, 3H, ArH), 7.43 7.45 (d, J = 8.0 Hz, 2H, ArH), 4.48 (s, 1H,
CH-C=O), 2.77 (s, 3H, CH3), 1.77 1.80 (d, J = 8.0 Hz, 2H, CH2), 1.61 (m, 1H, CH), 0.91 0.94 (m, 6H,
2×CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 174.77, 166.30, 156.22, 155.59, 148.48, 139.40, 139.08,
131.57, 129.91, 129.72, 128.28, 128.03, 127.26, 122.20, 51.40, 25.03, 23.49, 21.58, 20.83 ppm; ESI-MS:
m/z: 392.3 [M-H]-.
4.1.7.3. (2S,3R)-3-Methyl-2-(4-((3-methylquinoxalin-2-yl)oxy)benzamido)pentanoic acid (6c). White solid,
yield: 85%; m.p. = 156 157oC; 1H NMR (400 MHz, DMSO-d6) δ: 12.64 (s, 1H, COOH), 8.52 8.54 (d, J =
8.0 Hz, 1H, NH), 7.98 8.03 (m, 3H, ArH), 7.65 7.68 (m, 3H, ArH), 7.42 7.44 (d, J = 8.0 Hz, 2H, ArH),
4.34 4.38 (m, 1H, CH-C=O), 2.77 (s, 3H, CH3), 1.04 1.08 (m, 1H, CH), 0.95 0.97 (d, J = 8.0 Hz, 3H,
CH3), 0.84 0.93 (m, 5H, CH2CH3) ppm; 13C NMR (101 MHz, DMSO-d6) δ: 173.73, 166.61, 156.24,
155.57, 148.51, 139.40, 139.08, 131.62 ,129.90, 128.29, 128.04, 127.24, 122.12, 57.80, 36.16, 25.61, 20.84,
16.16, 11.54 ppm; ESI-MS: m/z: 392.7 [M-H]-.
4.1.7.4. (S)-2-(4-((6-Chloro-3-methylquinoxalin-2-yl)oxy)benzamido)propanoic acid (6d). White solid,
1
yield: 80%; m.p. = 133 135oC; H NMR (400 MHz, DMSO-d6) δ: 12.4 (s, 1H, COOH), 8.72 8.73 (d, J =
7.1 Hz, 1HꢀNH), 8.02 8.04 (d, J = 8.6 Hz, 2H, ArH), 7.98 8.00 (d, J = 8.9 Hz, 1H, ArH), 7.66 7.72 (m,

ACCEPTED MANUSCRIPT
2H, ArH), 7.44 7.45 (d, J = 8.4 Hz, 2H, ArH), 4.41 4.49 (m, 1H, CH-C=O), 2.75 (s, 3H, CH3), 1.42 1.44
(d, J = 7.3 Hz, 3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 174.72, 165.82, 156.80, 155.35, 149.23,
139.77, 138.01, 134.16, 131.70, 129.98, 129.67, 128.45, 126.15, 122.12, 48.79, 20.84, 17.47 ppm; ESI-MS:
m/z: 386.6 [M-H]-.
4.1.7.5. (S)-2-(4-((6-Chloro-3-methylquinoxalin-2-yl)oxy)benzamido)-4-methylpentanoic acid (6e). White
solid, yield: 80%; m.p. = 136 137oC; 1H NMR (400 MHz, DMSO-d6) δ: 12.54 (s, 1H, COOH), 8.65 8.67
(d, J = 8.0 Hz, 1H, NH), 7.99 8.04 (m, 3H, ArH), 7.74 (s, 1H, ArH), 7.67 7.69 (d, J = 8.0 Hz, 1H, ArH),
7.44 7.45 (d, J = 4.0 Hz, 2H, ArH), 4.47 (m, 1H, CH-C=O), 2.77 (s, 3H, CH3), 1.77 1.83 (m, 2H, CH2),
1.61 1.74 (m, 1H, CH), 0.90 0.95 (m, 6H, 2×CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 174.74,
166.27, 156.52, 155.40, 150.08, 139.70, 137.85, 132.04, 131.70, 130.25, 129.74, 129.01, 127.15, 122.21,
51.39, 25.02, 23.49, 21.58, 20.91 ppm; ESI-MS: m/z: 426.7 [M-H]-.
4.1.7.6. (2S,3R)-2-(4-((7-Chloro-3-methylquinoxalin-2-yl)oxy)benzamido)-3-methylpentanoic acid (6f).
1
White solid, yield: 88%; m.p. = 148 149oC; H NMR (400 MHz, DMSO-d6) δ: 13.01 (s, 1H, COOH),
8.66 8.70 (m, 1H, NH), 7.98 8.01 (d, J = 8.0 Hz, 1H, ArH), 7.91 7.93 (d, J = 8.0 Hz, 2H, ArH), 7.75 (s,
1H, ArH), 7.66 7.69 (d, J = 8.0 Hz, 1H, ArH), 7.40 7.43 (m, 2H, ArH), 7.26 7.32 (m, 4H, ArH),
7.19 7.20 (d, J = 3.2 Hz, 1H, ArH), 4.57 4.63 (t, J = 12.0 Hz, 1H, CH-C=O), 3.02 3.25 (m, 2H, CH2),
2.74 (s, 3H, CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 165.46, 156.67, 155.14, 149.40, 140.07, 139.45,
138.23, 132.32, 131.09, 130.08, 129.78, 129.38, 129.31, 128.41, 126.44, 122.67, 122.10, 55.71, 37.29,
20.88 ppm; ESI-MS: m/z: 426.4 [M-H]-.
4.1.7.7. (S)-2-(4-((7-Chloro-3-methylquinoxalin-2-yl)oxy)benzamido)-3-phenylpropanoic acid (6g). White
solid, yield: 82%; m.p. = 176 177oC; 1H NMR (400 MHz, DMSO-d6) δ: 12.60 (s, 1H, COOH), 8.66 8.68
(d, J = 8.0 Hz, 1H, NH), 8.01 8.07 (m, 3H, ArH), 7.67 (s, 2H, ArH), 7.43 7.45 (d, J = 8.0 Hz, 2H, ArH),
4.44 4.50 (m, 1H, CH-CH=O), 2.77 (s, 3H, CH3), 1.80 1.84 (t, J = 8.0 Hz, 2H, ArH), 1.75 1.79 (m, 1H,
CH), 1.71 1.74 (m, 3H, CH3), 1.23 1.30 (m, 3H, CH3) ppm; 13C NMR (100 Hz, DMSO-d6) δ: 174.63,
166.26, 156.49, 155.41, 139.74, 137.87, 131.73, 130.25, 129.71, 122.14, 51.39, 25.04, 23.45, 21.63, 20.88
ppm; ESI-MS: m/z: 460.7 [M-H]-.
4.1.8. Synthesis of quinoxaline-2,3(1H,4H)-dione (7)
A round-bottom flask charged with benzene-1,2-diamine (46.3 mmol, 5.0g) and oxalic acid (64.4
mmol, 5.8g), and 250 mL of distilled water was added in one portion. Then 4.5 ml of concentrated
hydrochloric acid was added to the suspension dropwise and the reaction mixture was refluxed for 8h.
Collected the obtained brown precipitate and washed with distilled water to afford 6.9 g of desired product,
yield: 92%. M.p. 241 242.7 oC; ESI-MS m/z: 161.0 [M+H]+.
4.1.9. Synthesis of 2,3-dichloroquinoxaline (8)
A solution of compound 7 (30.84 mmol, 5.0g) in phosphorus oxychloride (30 mL) was reacted under
refluxing condition for 5h, then the reaction was totally quenched by slowly pouring into cooled ammonia

ACCEPTED MANUSCRIPT
solution. The mixture was extracted with ethyl acetate (2×50 mL), the combined organic phase was washed
with brine (30 mL) and dried over anhydrous MgSO4. After evaporated in vacuo, the crude residue was
purified by silica gel flash chromatography (PE/EtOAc=10:1, v:v) to give 4.9 g of corresponding product
with 80% yield. M.p. 149 150 oC; ESI-MS m/z: 199.8 [M+H]+.
4.1.10. Synthesis of methyl 4-((3-chloroquinoxalin-2-yl)oxy)benzoate (9)
Methyl 4-hydroxybenzoate (3.8 g, 25 mmol) and K2CO3 (4.1 g, 30 mmol) were dissolved in 150 mL
of anhydrous DMF and the mixture was reacted at 85oC under nitrogen atmosphere. After 12h, compound 8
(4.98 g, 25 mmol) was added to the mixture in one portion and the reaction was allowed to react for
additional 12h at the same temperature until completion, as determined by TLC. The reaction was then
quenched by pouring into 250 mL of iced water and extracted with ethyl acetate (2 × 100 mL), the organic
phase was then washed successively with 5% citric acid (2 × 75 mL), saturated NaHCO3 (2 × 75 mL) and
brine (2 × 50 mL). The combined organic solvent was removed under vacuum, and the crude residue was
purified by flash chromatography (PE/EtOAc=50:1, v:v) to give 3.98 g of corresponding product as
semi-solid with 50% yield. ESI-MS m/z: 315.3 [M+H]+.
4.1.11. General procedure for the synthesis of methyl 4-((3-substituted quinoxalin-2-yl)oxy)benzoate (10)
Compound 9 (2.0 g, 6.4 mmol) was dissolved in 50 mL of anhydrous dimethyl sulfoxide, dodecyl
amine (1.2 g, 6.5 mmol) and triethylamine (Et3N, 1.5 g, 14.8 mmol) were added successively. The mixture
was reacted at 75oC for 12h and quenched with 150 mL of iced water. It was then extracted with ethyl
acetate (2 × 100 mL), washed with brine and dried over anhydrous Na2SO4. Filtered and concentrated under
vacuum condition, the crude residue was purified by flash chromatography (PE/EtOAc=100:1, v:v) to
produce the corresponding product.
4.1.11.1. Methyl 4-((3-(dodecylamino)quinoxalin-2-yl)oxy)benzoate (10a). Semi-solid, yield 43%, ESI-MS
m/z: 464.6 [M+H]+.
4.1.11.2. Methyl 4-((3-(tert-butylamino)quinoxalin-2-yl)oxy)benzoate (10b). Semi-solid, yield 51%,
ESI-MS m/z: 352.7 [M+H]+.
4.1.12. General procedure for 4-((3-substituted quinoxalin-2-yl)oxy)benzoic acid (11)
These compounds were synthesized according to similar procedures with that of compound (4).
4.1.12.1. 4-((3-(Dodecylamino)quinoxalin-2-yl)oxy)benzoic acid (11a). Semi-solid, yield 88%, ESI-MS m/z:
448.7 [M-H]-.
4.1.12.2.4-((3-(tert-Butylamino)quinoxalin-2-yl)oxy)benzoic acid (11b). Semi-solid, yield 84%, ESI-MS
m/z: 336.5 [M-H]-.
4.1.13. General procedure for the synthesis of(R)-substituted2-(4-((3-(substitutedamino)quinoxalin-2-yl)
oxy)benzamido)propanoate (12)
Compound 11a (0.45 mmol, 0.2 g) was dissolved in anhydrous THF (15 mL), then isobutyl
chloroformate (0.83 mmol, 107 µL) and N-methylmorpholine (0.85 mmol, 93 µL) were added successively.

ACCEPTED MANUSCRIPT
The temperature was maintained below -5oC in an ice-salt bath for 30 min. After the addition of
L-phenylalanine methyl ester hydrochloride (0.6 mmol, 120 mg) was added in several portions, the mixture
was allowed to warm to room temperature and stirred for another 4h under nitrogen atmosphere. The
solvent was removed under vacuum and partitioned between EtOAc (2 × 50 mL) and saturated NH4Cl
solution. The organic phase was separated and washed with saturated NaHCO3 (2 × 50 mL) and then with
brine (50 mL), dried over anhydrous MgSO4, filtered, and concentrated in vacuo to give the crude product,
which was further purified by flash chromatography (PE/EtOAc=25:1, v:v) to generate the pure product.
4.1.13.1.(S)-Methyl 2-(4-((3-(dodecylamino)quinoxalin-2-yl)oxy)benzamido)-3-phenylpropanoate (12a).
White solid, yield 68%, m.p. = 155 157oC; 1H NMR (400 MHz, CDCl3) δ: 7.82 7.84 (d, J = 8.7 Hz, 2H,
ArH), 7.68 7.70 (d, J = 9.2 Hz, 1H, ArH), 7.50 7.52 (d, J = 7.0 Hz, 1H, ArH), 7.42 7.46 (t, J = 7.7 Hz, 1H,
ArH), 7.33 7.35 (d, J = 8.7 Hz, 2H, ArH), 7.25 7.33 (dd, J1 = 14.2 Hz, J2=5.1 Hz, 4H, ArH), 7.15 7.17 (d,
J = 8.1 Hz, 2H, ArH), 6.59 6.61 (d, J = 7.6 Hz, 1H, NH-C=O), 5.58 5.61 (t, J = 5.4 Hz, 1H, NHCH2),
5.09 5.14 (m, 1H, CH-C=O), 3.67 (s, 3H, OCH3), 3.62 3.65 (m, 2H, NHCH2), 3.22 3.33 (m, 2H,CH2Ph),
1.24 1.28 (m, 20H, (CH2)10), 0.86 0.90 (t, J = 7.0 Hz, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3) δ:
172.06, 171.16, 166.10, 155.30, 147.37, 144.72, 140.15, 135.82, 134.20, 131.02, 129.36, 128.70, 128.68,
127.56, 127.27, 126.90, 125.43, 124.20, 121.91, 60.40, 53.59, 52.49, 41.03, 37.94, 31.92, 29.64, 29.59,
29.41, 29.36, 27.14, 22.70, 21.06, 14.21, 14.13 ppm; ESI-MS m/z: 611.6 [M+H]+.
4.1.13.2. (S)-Methyl 2-(4-((3-(dodecylamino)quinoxalin-2-yl)oxy)benzamido)propanoate (12b). White solid,
yield 52%, m.p. = 143 144oC; 1H NMR (400 MHz, CDCl3) δ: 8.93 (m, 1H, NH), 8.01 8.06 (m, 2H, ArH),
7.93 (m, 1H, ArH), 7.47 7.49 (d, J = 8.0 Hz, 3H, ArH), 7.40 7.44 (m, 1H, ArH), 7.29 7.32 (t, J = 8.0 Hz,
1H, ArH), 3.64 (s, 3H, OCH3), 3.40 3.43 (m, 1H, CHC=O), 1.66 1.70 (m, 2H, NHCH2), 1.35 1.40 (m, 3H,
NHCH(CH3)), 1.20 1.30 (m, 20H, (CH2)10), 0.80 0.83 (m, 3H, CH2CH3) ppm; 13C NMR (100 MHz,
CDCl3) δ: 173.84, 166.19, 155.45, 147.57, 144.88, 140.32, 134.40, 131.27, 128.83, 127.69, 127.06, 125.60,
124.34, 122.08, 52.78, 48.73, 41.18, 32.07, 29.82, 29.79, 29.74, 29.56, 29.50, 27.30, 22.84, 18.89, 14.26
ppm; ESI-MS m/z: 535.5 [M+H]+.
4.1.13.3. (2S,3R)-Methyl 2-(4-((3-(dodecylamino)quinoxalin-2-yl)oxy)benzamido)-3-methyl pentanoate
(12c). White solid, yield 50%, m.p. = 166 167oC; 1H NMR (400 MHz, CDCl3) δ: 7.90 7.92 (d, J = 8.0 Hz,
2H, ArH), 7.68 7.70 (d, J = 8.0 Hz, 1H, ArH), 7.50 7.52 (d, J = 8.0 Hz, 2H, ArH), 7.42 7.46 (t, J = 6.0 Hz,
1H, ArH), 7.36 7.38 (d, J = 6.0 Hz, 2H, ArH), 6.66 6.68 (d, J = 6.0 Hz, 1H, NHC=O), 5.60 (m, 1H,
NHCH2), 4.83 4.87 (m, 1H, CH-C=O), 3.80 (s, 3H, OCH3), 3.63 3.68 (m, 2H, NHCH2), 2.00 2.08 (m, 1H,
NHCHCHCH3), 1.74 1.76 (m, 2H, CHCH2CH3), 1.30 1.41 (m, 20H, (CH2)10), 0.97 1.01 (t, 6H, J = 6.0
Hz, 2×CH3) ppm; 13C NMR (100 MHz, CDCl3) δ: 172.79, 166.50, 155.42, 147.57, 144.88, 134.41, 131.50,
128.83, 127.69, 127.08, 125.61, 124.34, 122.10, 57.02, 52.38, 41.19, 38.50, 32.07, 29.79, 29.74, 29.56,
29.50, 27.30, 25.59, 22.84, 15.70, 14.26, 11.79 ppm; ESI-MS m/z: 577.8 [M+H]+.
4.1.13.4. Methyl 2-(4-((3-(tert-butylamino)quinoxalin-2-yl)oxy)benzamido)acetate (12d). White solid, yield

ACCEPTED MANUSCRIPT
62%, m.p. = 172 173oC; 1H NMR (400 MHz, CDCl3) δ: 7.90 7.93 (m, 2H, ArH), 7.67 7.69 (d, J = 8.0 Hz,
1H, ArH), 7.47 7.51 (t, J = 8.0 Hz, 2H, ArH), 7.29 7.43 (m, 3H, ArH), 6.72 (m, 1H, C=O-NH), 5.53 (s, 1H,
N=C-NH), 4.28 4.29 (d, J = 4.0 Hz, 2H, NHCH2C=O), 3.83 (s, 3H, OCH3), 1.25 1.34 (m, 9H, (CH3)3)
ppm; 13C NMR (100 MHz, CDCl3) δ: 170.56, 166.78, 155.93, 148.46, 145.93, 139.92, 134.61, 130.27,
128.73, 127.57, 126.42, 125.62, 124.54, 121.56, 52.53, 44.77, 41.80, 13.79 ppm; ESI-MS m/z: 409.7
[M+H]+.
4.1.13.5. (S)-Methyl 2-(4-((3-(tert-butylamino)quinoxalin-2-yl)oxy)benzamido)propanoate (12e). White
1
solid, yield 65%, m.p. =177 178oC; H NMR (400 MHz, CDCl3) δ: 7.90 7.94 (t, J = 8.0 Hz, 2H, ArH),
7.68 7.70 (d, J = 8.0 Hz, 1H, ArH), 7.31 7.37 (m, 3H, ArH), 7.42 7.52 (m, 2H, ArH), 6.81 6.82 (d, J =
4.0 Hz, 1H, C=O-NH), 4.81 4.88 (m, 1H, NHCHC=O), 3.82 (s, 3H, OCH3), 3.74 3.79 (m, 1H, N=C-NH),
1.55 1.63 (m, 9H, (CH3)3), 1.31 1.35 (m, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3) δ: 164.71, 155.51,
147.42, 143.81, 139.87, 133.67, 130.52, 128.90, 127.38, 126.59, 125. 85, 124.12, 122.15, 121.65, 52.02,
44.80, 28.73, 18.05, 13.78 ppm; ESI-MS m/z: 433.5 [M+H]+.
4.1.13.6. (2S,3R)-Methyl 2-(4-((3-(tert-butylamino)quinoxalin-2-yl)oxy)benzamido)-3-methyl pentanoate
(12f). White solid, yield 51%, m.p. =166 167oC; 1H NMR (400 MHz, CDCl3) δ: 7.82 7.84 (d, J = 8.6 Hz,
2H, ArH), 7.58 7.61 (d, J = 8.2 Hz, 1H, ArH), 7.39 7.41 (d, J = 7.0 Hz, 1H, ArH), 7.32 7.35 (t, J = 7.0 Hz,
1H, ArH), 7.24 7.28 (t, J = 7.5 Hz, 2H, ArH), 7.13 7.18 (m, 1H, ArH ), 6.65 66.67 (d, J = 8.2 Hz, 1H,
NHC=O), 5.46 (s, 1H, NHCCH3), 4.75 4.78 (m, 1H, CHC=O), 3.70 (s, 3H, OCH3), 1.58 (s, 9H, (CH3)3),
1.22 2.25 (t, J = 6.9 Hz, 2H, CHCHCH2), 1.17 (m, 1H, CHCHCH2), 0.88 0.92 (m, 6H, 2×CH3) ppm; 13
NMR (100 MHz, CDCl3) δ: 172.70, 166.43, 155.29, 147.41, 143.84, 139.88, 133.82, 131.21, 128.69,
127.32, 126.80, 125.80, 124.06, 122.05, 56.89, 52.27, 52.00, 38.28, 28.76, 25.42, 15.56, 11.66 ppm;
ESI-MS m/z: 465.8 [M+H]+.
C
4.1.13.7. (S)-Methyl 2-(4-((3-(tert-butylamino)quinoxalin-2-yl)oxy)benzamido)-4-methylpentanoate (12g).
White solid, yield 58%, m.p. =170 172oC; 1H NMR (400 MHz, CDCl3) δ: 7.80 7.83 (t, J = 5.8 Hz, 2H,
ArH), 7.57 7.59 (dd, J1 = 8.2 Hz, J2 = 0.9 Hz, 1H, ArH), 7.38 7.40 (dd, J1 = 8.0 Hz, J2 = 0.9 Hz, 1H, ArH),
7.31 7.34 (t, J = 7.6 Hz, 1H, ArH), 7.21 7.24 (m, 2H, ArH), 7.12 7.17 (m, 1H, ArH ), 6.72 6.74 (d, J =
6.3 Hz, 1H, C=ONH), 5.45 (s, 1H, N=C-NH), 4.77 4.82 (m, 1H, NHCHC=O), 3.69 (s, 3H, OCH3), 1.62 (d,
J = 2.9 Hz, 1H, CH2CH(CH3)2), 1.55 (m, 9H, (CH3)3), 1.21 (m, 2H, CHCH2CH), 0.90 (m, 6H, 2×CH3) ppm;
13C NMR (100 MHz, CDCl3) δ: 173.90, 166.48, 155.29, 147.43, 143.84, 139.87, 133.84, 130.96, 128.77,
127.30, 126.78, 125.80, 124.06 , 122.00 , 52.45, 52.29, 51.24, 41.73, 28.76, 25.03, 22.92, 22.03 ppm;
ESI-MS m/z: 465.6 [M+H]+.
4.1.13.8. (S)-Methyl 2-(4-((3-(tert-butylamino)quinoxalin-2-yl)oxy)benzamido)-3-phenylpropanoate (12h).
1
White solid, yield 65%, m.p. =182 183oC; H NMR (400 MHz, CDCl3) δ: 7.73 7.76 (t, J = 6.9 Hz, 2H,
ArH), 7.59 7.61 (d, J = 8.2 Hz, 1H, ArH), 7.40 7.43 (dd, J1 = 11.0 Hz, J2 = 3.1 Hz, 1H, ArH), 7.33 7.37
(m, 1H, ArH), 7.16 7.27 (m, 6H, ArH), 7.07 7.09 (d, J = 6.9 Hz, 2H, ArH), 6.53 6.55 (d, J = 6.0 Hz, 1H,

ACCEPTED MANUSCRIPT
NH-C=O), 5.42 5.48 (m, 1H, N=C-NH), 5.02 5.07 (m, 1H, NHCHC=O), 3.71 (s, 3H, OCH3), 3.16 3.27
(m, 2H, CH2C6H5), 1.22 1.54 (m, 9H, (CH3)3) ppm; 13C NMR (100 MHz, CDCl3) δ: 171.04, 165.09,
154.33, 146.29, 142.80, 138.83, 134.77, 132.74, 128.32, 127.62, 127.66, 126.30, 126.23, 125.75, 124.76,
123.03, 120.95, 120.49, 52.55, 51.47, 50.96, 36.89, 27.71 ppm; ESI-MS m/z: 499.1 [M+H]+.
4.1.14. General procedure for the preparation of (S)-2-(4-((3-substituted quinoxalin-2-yl)oxy)benzamido)-
2-substituted acetic acid (13a-h).
The synthetic procedure of compound 13 was carried out according to that of compounds (11a-b).
4.1.14.1. (S)-2-(4-((3-(Dodecylamino)quinoxalin-2-yl)oxy)benzamido)-3-phenylpropanoic acid (13a).
1
White crystal obtained from recrystallization (MeOH:H2O = 3:1, v:v), yield 93%, m.p. =139 141oC; H
NMR (400 MHz, DMSO-d6) δ: 8.60 8.61 (d, J = 6.0 Hz, 1H, O=C-NH), 7.89 7.91(d, J = 7.9 Hz, 2H,
ArH), 7.68 (s, 1H, NHCH2), 7.53 7.55 (d, J = 7.9 Hz, 1H, ArH), 7.38 7.42 (m, 4H, ArH), 7.24 7.33 (m,
4H, ArH), 7.17 7.22 (dd, J1 = 11.6 Hz, J2 =7.2 Hz, 2H ArH), 4.56 (s, 1H, NHCHCOOH), 3.05 3.08 (m,
2H, NHCH2), 1.66 (m, 2H, CH2Ar), 1.23 1.34 (m, 20H, (CH2)10), 0.84 (m, 3H, CH2CH3) ppm; 13C NMR
(100 MHz, DMSO-d6) δ: 173.82, 166.09, 155.30, 148.46, 145.26, 140.30, 138.87, 133.95, 129.55, 129.49,
128.63, 127.63, 126.75, 123.88, 122.26, 55.01, 40.74, 36.87, 31.76, 29.53, 29.49, 29.28, 29.18, 27.03,
22.56, 14.40 ppm; ESI-MS m/z: 595.5 [M-H]-.
4.1.14.2. (S)-2-(4-((3-(Dodecylamino)quinoxalin-2-yl)oxy)benzamido)propanoic acid (13b). White crystal
o
1
obtained from recrystallization (MeOH:H2O = 3:1, v:v), yield 89%; m.p. = 179 180 C; H NMR (400
MHz, DMSO-d6) δ: 12.46 (s, 1H, COOH), 8.75 8.73 (d, 1H, J = 6.0 Hz, CONH), 8.00 8.03 (d, J = 9.0 Hz,
2H, ArH), 7.64 7.68 (d, J = 12 Hz, 1H, ArH ), 7.54 7.57 (d, J = 9.0 Hz, 1H, ArH), 7.38 7.46 (m, 3H, ArH),
7.19 7.24 (m, 1H, ArH), 4.43 4.47 (m, 1H, NHCH2), 3.50 3.52 (m, 1H, CH), 1.67 1.71 (m, 3H, CHCH3),
1.35 1.43 (m, 22H, (CH2)11), 0.82 0.87 (m, 3H,CH2CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 175.38,
166.69, 156.08, 149.20, 146.04, 141.04, 134.70, 132.26, 130.31, 128.39, 127.46, 126.02, 124.65, 122.96,
49.39, 32.46, 30.23, 30.19, 30.15, 29.98, 29.87, 29.55, 27.73, 23.26, 18.10, 15.12 ppm; ESI-MS m/z: 519.6
[M-H]-.
4.1.14.3. (2S,3R)-2-(4-((3-(Dodecylamino)quinoxalin-2-yl)oxy)benzamido)-3-methylpentanoic acid (13c).
o
1
White crystal obtained from recrystallization (MeOH:H2O = 3:1, v:v), yield 92%; m.p. = 189 191 C; H
NMR (400 MHz, DMSO-d6) δ: 12.17 (s, 1H, COOH), 8.57 8.59 (d, J = 8.0 Hz, 1H, ArH), 8.02 8.04 (d, J
= 8.6 Hz, 2H, ArH), 7.87 (s, 1H, C=ONH), 7.43 7.47 (m, 4H, ArH), 7.28 7.32 (t, J = 7.5 Hz, 1H, ArH),
4.33 4.37 (t, J = 7.5 Hz, 1H, CHC=O), 3.63 (m, 2H, NHCH2), 1.98 (m, 1H, CH3CHCH2CH3), 1.63 1.72
(m, 2H, CH3CHCH2CH3), 1.23 1.38 (m, 20H, (CH2)10), 0.94 0.96 (d, J = 6.8 Hz, CH2CH2CH3, 3H),
0.82 0.90 (m, 6H, 2×CH3). 13C NMR (100 MHz, DMSO-d6) δ: 173.69, 166.57, 154.90, 149.05, 132.99,
132.00, 129.94, 129.11, 128.36, 126.94, 125.06, 122.16, 57.80, 36.11, 31.78, 29.56, 29.47, 29.29, 29.06,
28.52, 26.83, 25.60, 22.58, 16.16, 14.44, 11.53 ppm; ESI-MS m/z: 561.5 [M-H]-.
4.1.14.4. 2-(4-((3-(tert-Butylamino)quinoxalin-2-yl)oxy)benzamido)acetic acid (13d). White crystal

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o
1
obtained from recrystallization (MeOH:H2O = 3:1, v:v), yield 86%; m.p. = 158 161 C; H NMR (400
MHz, DMSO-d6) δ: 8.64 (s, 1H, C=ONHCH2), 7.74 7.76 (d, J = 8.1 Hz, 2H, ArH), 7.33 7.37 (d, J = 14.6
Hz, 1H, ArH), 7.16 7.22 (m, 4H, ArH), 7.00 7.04 (m, 1H, ArH), 3.70 3.72 (d, J = 5.2 Hz, 2H,
NHCH2COOH), 2.26 (s, 1H, N=C-NHC(CH3)3), 1.04 1.33 (m, 9H, 3×CH3) ppm; 13C NMR (100 MHz,
DMSO-d6) δ: 171.86, 166.31, 155.49, 149.10, 146.04, 139.70, 134.54, 131.29, 129.49, 127.98, 126.43,
125.66, 124.94, 122.03, 44.71, 28.80, 14.05 ppm; ESI-MS m/z: 393.2 [M-H]-.
4.1.14.5. (S)-2-(4-((3-(tert-Butylamino)quinoxalin-2-yl)oxy)benzamido)propanoic acid (13e). White crystal
o
1
obtained from recrystallization (MeOH:H2O = 3:1, v:v), yield 90%; m.p. = 135 137 C; H NMR (400
MHz, DMSO-d6) δ: 8.73 (s, 1H, NHC=O), 7.99 8.02 (t, J = 8.0 Hz, 2H, ArH), 7.58 7.60 (d, J = 8.0 Hz, 1H,
ArH), 7.41 7.44 (m, 4H, ArH), 7.24 (m, 1H, ArH), 4.43 (s, 1H, N=C-NHC(CH3)3), 3.72 (m, 1H,
NHCHCOOH), 1.58 (m, 9H, 3×CH3), 1.42 (m, 3H, CHCH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ:
175.47, 166.53, 155.94, 149.07, 145.21, 140.42, 134.44, 132.40, 130.22, 128.83, 127.44, 126.43, 125.05,
123.08, 122.59, 51.83, 42.64, 29.96, 18.35 ppm; ESI-MS m/z: 407.5 [M-H]-.
4.1.14.6. (2S,3R)-2-(4-((3-(tert-Butylamino)quinoxalin-2-yl)oxy)benzamido)-3-methylpentanoic acid (13f).
o
1
White crystal obtained from recrystallization (MeOH:H2O = 3:1, v:v), yield 80%; m.p. = 154 156 C; H
NMR (400 MHz, DMSO-d6) δ: 12.52 (s, 1H, COOH), 8.37 8.39 (d, J = 8.0 Hz, 1H, NHCHCOOH),
7.98 8.00 (d, J = 8.0 Hz, 2H, ArH), 7.57 7.59 (d, J = 8.0 Hz, 1H, ArH), 7.38 7.44 (m, 4H, ArH),
7.22 7.26 (m, 1H, ArH), 6.60 (s, 1H, N=C-NHC(CH3)3), 4.27 4.30 (s, 1H, CHC=O), 1.91 1.96 (m, 1H,
NHCHCOOH), 1.49 1.58 (m, 9H, 3×CH3), 1.23 1.32 (m, 2H, CH2), 0.80 0.94 (m, 6H, 2×CH3) ppm; 13C
NMR (100 MHz, DMSO-d6) δ: 154.32, 148.37, 144.00, 139.64, 133.99, 132.03, 129.70, 127.66, 126.69,
125.53, 122.02, 58.03, 51.41, 36.43, 28.76, 25.44, 16.78, 13.39, 11.43 ppm; ESI-MS m/z: 449.4 [M-H]-.
4.1.14.7. (S)-2-(4-((3-(tert-Butylamino)quinoxalin-2-yl)oxy)benzamido)-4-methylpentanoic acid (13g).
o
1
White crystal obtained from recrystallization (MeOH:H2O = 3:1, v:v), yield 81%; m.p. = 177 178 C; H
NMR (400 MHz, DMSO-d6) δ: 12.44 (s, 1H, COOH), 8.51 8.53 (d, J = 8.0 Hz, 1H, C=ONHCHCOOH),
7.99 8.01 (d, J = 8.0 Hz, 2H, ArH), 7.57 7.59 (d, 1H, J = 8.0 Hz, ArH), 7.38 7.44 (m, 4H, ArH),
7.21 7.25 (m, 1H, ArH), 6.59 (s, 1H, N=C-NHC(CH3)3), 4.40 (s, 1H, CHC=O), 1.72 1.77 (m, 2H, CH2),
1.48 1.64 (m, 9H, 3×CH3), 1.23 1.29 (m, 1H, CH), 0.83 0.93 (m, 6H, 2×CH3) ppm; 13C NMR (100 MHz,
DMSO-d6) δ: 165.87, 155.09, 144.46, 139.65, 133.69, 131.66, 131.36, 129.42, 127.64, 125.67, 124.31,
122.37, 52.10, 28.83, 25.10, 23.58, 21.94 ppm; ESI-MS m/z: 449.4 [M-H]-.
4.1.14.8. (S)-2-(4-((3-(tert-Butylamino)quinoxalin-2-yl)oxy)benzamido)-3-phenylpropanoic acid (13h).
o
1
White crystal obtained from recrystallization (MeOH:H2O = 3:1, v:v), yield 84%; m.p. = 168 180 C; H
NMR (400 MHz, DMSO-d6) δ: 8.28 (s, 1H, NH), 7.87 7.89 (d, J = 6.5 Hz, 2H, ArH), 7.57 7.61 (d, J = 7.9
Hz, 1H, ArH), 7.36 7.46 (m, 4H, ArH), 7.23 7.28 (m, 5H, ArH), 7.14 7.16 (d, J = 6.8 Hz, 1H, ArH), 4.49
(s, 1H, CHC=O), 3.70 3.72 (d, J = 8.0 Hz, 2H, CH2Ar), 3.08 (s, 1H, N=C-NHC(CH3)3), 1.25 1.57 (m, 9H,
3×CH3) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 173.87, 165.89, 160.99, 155.23, 148.36, 144.46, 139.66,

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134.53, 133.65, 131.34, 130.21, 129.54, 129.41, 128.66, 127.70, 126.80, 125.69, 124.33, 122.44, 60.24,
52.11, 36.83, 28.80 ppm; ESI-MS m/z: 483.1 [M-H]-.
4.2. Biological protocols
4.2.1. In vitro anti-proliferative assay
The tested chemicals were dissolved in dimethylsulfoxide (DMSO) or PBS and diluted to the desired
concentration with culture medium before using. The cell lines were maintained in RPMI-1640 medium
supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and incubated at 37°C in a
humidified incubator with 5% CO2. Cell proliferation was determined by the MTT
(3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) method. Briefly, cells were seeded in a
96-well plate (104 cells per well). After 4 h incubation, chemicals were subsequently added to wells to
achieve final concentration of 200, 150, 100, 50, and 10 µM. Control wells were prepared by addition of
culture medium. Treated cells were then incubated for 48 h. Once completion of incubation, 1% of 0.5
mg/mL MTT solution was added to each well and incubated for an additional 4 h. Formazan formed from
MTT was extracted by adding 100 µL of DMSO and mixed for another 15 min. The optical density was
measured using an enzyme-linked immunosorbent assay (ELISA) reader (Model 680, BIO-RAD) at 570
nm.
The growth inhibition rate was calculated as [(ODc−ODt)/(ODc−ODz)]×100%. In this formula, ODc
represents the OD values of the control group, ODt represents the OD values of the treating groups, and
ODz represents the OD values of the zero-setting groups. Three independent experiments with triplicated
samples were performed to achieve the cytotoxic results, and the IC50 values were calculated according to
inhibition ratios.
4.2.2. In vivo anti-metastatic experiment
Preparation of solution: Tested compounds were dissolved in 0.5% CMC-Na to prepare solution or
homogeneous suspension, with a final concentration of 2 mg/ml. If possible, ultrasonic device can be
utilized to promote dissolution.
Experimental animal model and grouping: Ascites was drawn from BALB/c mice bearing with
hepatocarcinoma 22 (H22) under aseptic condition, and diluted with physiological saline at 1:4. Final
concentration of tumor cells was 3 × 107 cells/ml. Then 50 healthy BALB/c mice weighing 18 22 g were
selected (available from Laboratory Animal Center, Shandong University), and 0.2 mL of hepatoma H22
cell suspension (6 × 106 cells) were inoculated via the caudal vein of the mice. After 24h, they were
weighted and randomly divided into 7 groups, 10 mice in each group (bisexual each half).
Drug dosages and methods: Animals of treatment groups were given tested compounds (6b, 6g, 13a,
13b, 13f and positive control Doxo) through intragastric administration, while the control group (0.5%
CMC-Na as a blank) was treated with the same volume of excipient by intragastric route, at a dose of 10

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mg/kg/day, 6 days/week for two consecutive weeks. On the twelfth day, all the surviving mice were
weighed and sacrificed for autopsy immediately. Lungs, livers, spleen with tumor nodes were removed and
weighed. Then lungs were placed in Bouin’s stationary solution (saturated 2,4,6-trinitrophenol
solution/formaldehyde/glacial acetic acid = 15:5:1). One day later, the number of metastasized nodes on
lung surface was counted under a microscope. The inhibitory rate (%) was calculated according to the
following formula:
Inhibitory rate (%) = {[(the average number of metastasized nodes of the control group) − (the
average number of metastasized nodes of the experimental group)] / (the average number of metastasized
nodes of control group)} × 100%.
4.3. Statistical analysis
Statistical significance was determined by the Student’s t-test after one-way ANOVA, without
correction for multiple comparisons. The limit of statistical significance was p < 0.05.
4.4. Topo II-mediated kDNA decatenation assay
Reaction buffer solution contains 50 mM tris(hydroxymethyl)aminomethane (Tris-HCl, pH 8.0), 0.5
mM dithiothreitol (DTT), 10 mM MgCl2, 2 mM ATP, and 20ꢀg/mL kDNA. Human topo IIα, 0.5 units
(TopoGen, TG2000H), was added right before the reaction. Indicated amount of tested compounds were
included in 20 µL of topo II reaction buffer. Mixtures were incubated for 15 min at 37oC, and then stopped
by adding 3ꢀL of stop solution containing 5% of SDS and 50% of glycerol. Electrophoresis was used to
separate the reaction products by running 1% agarose gel at 50 V for 50 minutes. Ethidium bromide-stained
DNA was visualized under UV light.
4.5. Molecular modeling procedure
The docking experiment was carried out based on the X-ray structure of topo IIα (PDB code: 1ZXM)
[45] and performed as follows: Selected compounds XK469 and 13b were constructed with a Sybyl/Sketch
module and optimized using Powell Energetic Gradiet method with a Tripos force field with the
convergence criterion set at 0.05 kcal/mol·Å, and assigned with Gasteiger-Hückel method. In this crystal
structure, Mg2+ ion and its two binding water molecules (W924, W931) were retained since it was assumed
that they played important roles in molecular recognition [46], and other docking parameters implied in the
program were kept as default.
Acknowledgment
This work was financially supported by the Natural Science Foundation of Shandong Province (No.
ZR2013HM101), China–Australia Centre for Health Sciences Research Program (No. 2015GJ07),
“ChangJiang Scholars and Innovative Research Team in University” Program (PCSIRT, No. IRT13028),
and also Shandong Innovation and Transformation of Achievements Grant (No. 2014ZZCX02601).
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