Synthesis of imidazo[1,2-a]quinoxalines by double Groebke reactions and inhibitory effects on radicals and DNA oxidation

Article abstract of DOI:10.1016/j.tet.2016.02.042

The o-phenylenediamine, aldehyde, and 2,4,4-trimethylpentan-2-yl isocyanide performed a Groebke 3CR to afford 2-aminoquinoxaline, which can react with an aldehyde and t-butyl isocyanide via another Groebke 3CR to give imidazo[1,2-a]quinoxaline. Exchanging two aldehydes in the sequential Groebke 3CR led to a couple of imidazo[1,2-a]quinoxaline isomer, in which the aldehyde moiety located at 2- or 4-position. The ferrocenyl group at 4-position in imidazo[1,2-a]quinoxaline was found to be active in trapping galvinoxyl radical, while the phenolic hydroxyl group at 2-position played a synergistic role with 4-ferrocenyl or 4-flavonyl group in scavenging 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH). In addition, 4-ferrocenyl with N,N-dimethylaminophenyl group at 2-position was able to quench 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS^+[rad]). Moreover, the combination of 4-ferrocenyl with 2-phenyl group (bearing para-N,N-dimethylamino or hydroxyl group) exhibited high inhibitory effect on DNA oxidation induced by 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH).

Full text of DOI:10.1016/j.tet.2016.02.042

Accepted Manuscript
Synthesis of Imidazo[1,2-a]quinoxalines by Double Groebke Reactions and Inhibitory
Effects on Radicals and DNA Oxidation
Jia-Feng Chen, Zai-Qun Liu
PII:
S0040-4020(16)30104-1
10.1016/j.tet.2016.02.042
TET 27512
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 14 December 2015
Revised Date: 17 February 2016
Accepted Date: 19 February 2016
Please cite this article as: Chen J-F, Liu Z-Q, Synthesis of Imidazo[1,2-a]quinoxalines by Double
Groebke Reactions and Inhibitory Effects on Radicals and DNA Oxidation, Tetrahedron (2016), doi:
10.1016/j.tet.2016.02.042.
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ACCEPTED MANUSCRIPT
Synthesis of Imidazo[1,2-a]quinoxalines by Double
Groebke Reactions and Inhibitory Effects on Radicals
and DNA Oxidation
Jia-Feng Chen, Zai-Qun Liu*
Department of Organic Chemistry, College of Chemistry, Jilin University, Changchun 130021, China.
CORRESPONDING AUTHOR FOOTNOTE
Zai-Qun Liu
Department of Organic Chemistry, College of Chemistry,
Jilin University
No.2519 Jiefang Road, Changchun 130021
China
Email: zaiqun-liu@jlu.edu.cn
1

ACCEPTED MANUSCRIPT
ABSTRACT
The o-phenylenediamine, aldehyde, and 2,4,4-trimethylpentan-2-yl isocyanide performed a
Groebke 3CR to afford 2-aminoquinoxaline, which can react with an aldehyde and t-butyl isocyanide
via another Groebke 3CR to give imidazo[1,2-a]quinoxaline. Exchanging two aldehydes in the
sequential Groebke 3CR led to a couple of imidazo[1,2-a]quinoxaline isomer, in which the aldehyde
moiety located at 2- or 4-position. The ferrocenyl group at 4-position in imidazo[1,2-a]quinoxaline was
found to be active in trapping galvinoxyl radical, while the phenolic hydroxyl group at 2-position
played a synergistic role with 4-ferrocenyl or 4-flavonyl group in scavenging 2,2’-diphenyl-1-
picrylhydrazyl radical (DPPH). In addition, 4-ferrocenyl with N,N-dimethylaminophenyl group at 2-
position was able to quench 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS+·).
Moreover, the combination of 4-ferrocenyl with 2-phenyl group (bearing para-N,N-dimethylamino or
hydroxyl group) exhibited high inhibitory effect on DNA oxidation induced by 2,2’-azobis(2-
amidinopropane hydrochloride) (AAPH).
KEYWORDS Groebke 3CR, N-rich polyheterocycles, radical-scavenging property, DNA oxidation.
2

ACCEPTED MANUSCRIPT
1. Introduction
Imidazoline-fused polyheterocycles are recently positioned at the convergence point in novel drug
discovery. 1,2 As shown in Scheme 1, the scaffold of imidazo[1,2-a]pyridine was usually employed in
3,4
5
the mesenchymal-epithelial transition factor (c-Met) inhibitor,
hepatitis C virus inhibitor, and
platelet-derived growth factor inhibitor6. In addition, an imidazoline linking with a thiadiazole was able
to inhibit the receptor kinase of transforming growth factor-β, 7 and imidazo[1,2-a]pyrazine combining
with coumarin can inhibit the growth of many kinds of tumor cells 8 because this structural moiety can
be readily recognized by the active sites in the related enzymes. 9
N
F
O
N
N
N
N
N
N
N
N
H
N
N
N
N
F
mesenchymal-epithelial transition (c-Met) inhibitor
F
HO
N
O
NH
Cl
O
N
N
N
N
O
N
O
CF3
N
O
platelet-derived growth inhibitor
hepatitis C virus inhibitor
Scheme 1 Recently found inhibitors containing imidazo[1,2-a]pyridine moiety.
Some methods have been applied to construct imidazo[1,2-a]-fused heterocycles. 10 For instance,
palladium-catalyzed Suzuki reaction led to an arylation at C-3 position of imidazo[1,2-a]pyridine. 11 On
the other hand, in the presence of N-iodosuccinimide, the reaction of 2-phenylacetaldehyde with 2-
aminopyridine can also reach the same target. 12 The 3-phenylpropiolaldehyde can be converted into the
imidazo moiety during the reaction with 2-aminopyridine in the case of CuI or Pd(OAc)2 being the
13
catalyst. Some small molecules such as alcohol, thiol, and amine can form ether, thioether, and
secondary amine as substituents in imidazo[1,2-a]pyridine. 14 Meanwhile, the nitrogen atom and C≡C in
3

ACCEPTED MANUSCRIPT
N-propargylaminopyridine may take place an intramolecular nucleophilic addition to afford
imidazo[1,2-a]pyridine scaffold. 15
In addition to the aforementioned methods, Groebke-Blackburn-Bienaymé reaction (also called
Groebke three-component-reaction, Groebke 3CR) offered a safety advantage for directly constructing
the imidazo[1,2-a] moiety by using α-aminopyridine, aldehyde, and isocyanide as the starting
materials.16 Owing to successful utilization of Groebke 3CR, we have prepared the ferrocenyl-appended
imidazo[1,2-a]pyridines and found that the introduction of ferrocenyl group into N-rich heterocycles
enhanced the inhibitory effects on radicals and DNA oxidation. 17 In addition to some efforts applied to
improve the reaction conditions of Groebke 3CR, 18,19,20 a previously reported method provided us with
much safe way to obtain 2-aminoquinoxaline by the Groebke 3CR with 2,4,4-trimethylpentan-2-yl
isocyanide being the precursor of amino group.21 The produced 2-aminoquinoxaline performed another
Groebke 3CR to generate imidazo[1,2-a]quinoxaline. The double Groebke 3CR procedure can be used
to change the position of the aldehyde moiety in the Groebke 3CR adduct (see equation (1)). In our
quest to discover novel inhibitors for DNA oxidation and radicals, as shown in Scheme 2, an
imidazo[1,2-a]quinoxaline library with 28 members was built up to evaluate the inhibitory effects on
2,2’-azobis(2-amidinopropane hydrochloride) (AAPH, R-N=N-R, R=-CMe2C(=NH)NH2)-induced
oxidation of DNA and on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS+·),
2,2’-diphenyl-1-picrylhydrazyl radical (DPPH), and galvinoxyl radicals, respectively.
4

ACCEPTED MANUSCRIPT
NO2
N
exchanging
positions
of two
Fe
Fe
N
5
N
N
N
N
4
N
3
N
groups
N
N
N
N
N
N
2
N
Fe
1
Fe
NH
NH
NH
NH
NH
NO2
4
(30%)
5
(20%)
2
(38%)
3
(35%)
1
(55%)
O
Br
Fe
Fe
N
Fe
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Fe
Fe
NH
NH
NH
NH
N
NH
O
Br
9
(37%)
10 (40%)
8
(36%)
7
(40%)
6
(33%)
N
N
OH
Fe
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Fe
NH
Fe
NH
NH
OH
NH
NH
N
11 (44%)
12 (28%)
15 (43%)
14 (40%)
13 (30%)
OH
Fe
N
Fe
N
Fe
N
N
N
N
N
N
N
N
O
N
N
N
N
Br
OH
Fe
N
NH
NH
NH
18 (39%)
OH
NH
NH
OH
20 (27%)
19 (29%)
16 (44%)
17 (41%)
Fe
N
N
N
N
O
O
O
3
N
2'
4
3'
N
N
4' O
O
N
N
1'
N
N
O
2
N
N
N
1NH
NH
H
H
O
O
Fe
21 (30%)
22 (34%)
23 (30%)
24 (37%)
OH
N
N
N
N
N
N
N
HN
N
N
HN
HO
O
N
N
O
O
O
O
O
N
N
N
NH
28 (25%)
O
NH
O
27 (22%)
26 (40%)
25 (34%)
Scheme 2 Structures of imidazo[1,2-a]quinoxalines synthesized in this work.
5

ACCEPTED MANUSCRIPT
2. Results and Discussion
2.1. Synthesis.
We herein applied a reported reaction condition to build up a series of imidazo[1,2-a]quinoxalines
(see Scheme 2). 21 As shown in equation (1), a Groebke 3CR took place among o-phenylenediamine,
aldehyde, and 2,4,4-trimethylpentan-2-yl isocyanide under concentrated HCl at room temperature. The
produced 1,4-dihydroquinoxaline performed an aromatization by using 2,3-dicyano-5,6-
dichlorobenzoquinone (DDQ) as the oxidant to afford quinoxaline. Subsequently, the amino group was
generated by hydrolyzing 2,4,4-trimethylpentan-2-yl group with HCl, leading to the formation of 2-
aminoquinoxaline as the reactant for the following Groebke 3CR. We still tested other catalysts for the
second Groebke 3CR with equation (2), (3), and (4) being model reactions (see Table 1).
C
O
H
N
R1
N
R1
THF, r.t.
N
N
R1
N
N
NH2
NH2
O
O
O
CN
CN
N
H
N
H
Cl
Cl
NH2
HCl, r.t.
N
H
R1
H
2-aminoquinoxaline
quinoxaline
O
1st Groebke 3CR
HCl, r.t.
O
(DDQ)
(1)
R2
H
Cat.
R1
N
CH3CN
reflux
exchanging R1CHO and R2CHO
in the 1st or 2nd Groebke 3CR for adjusting
C
N
N
N
positions of subsituents at 2- and 4-positions
2nd Groebke 3CR
N
R2
H
6

ACCEPTED MANUSCRIPT
O
N
Cat.
N
N
(2)
(3)
N
H
C
N
N
+
+
CH3CN
reflux
NH2
NH
1
O
H
N
Cat.
N
N
C
N
Fe
N
+
+
CH3CN
reflux
Fe
N
NH2
NH
2
O
Fe
N
Fe
Cat.
N
N
N
H
C
N
+
+
(4)
CH3CN
reflux
N
NH2
NH
3
Table 1 Screening catalysts in the second Groebke 3CR.
Yield (%)
Cat. a
Reaction (2)
15
Reaction (3)
-
Reaction (4)
-
CH3COOH
TsOH
40
12
48
53
44
65
60
78
35
-
-
AgOTf
10
15
13
5
Ce(OTf)3
NH4Cl
-
25
20
30
38
33
SbCl3
InCl3
20
35
40
(CH3)3SiCl
LaCl3
a
Equimolar amounts of both catalyst and all of the reactants.
7

ACCEPTED MANUSCRIPT
Some Bronsted and Lewis acids were able to catalyze the reaction for producing 1 in the refluxing
acetonitrile (see the column of reaction (2) in Table 1). Essentially, (CH3)3SiCl and LaCl3 were able to
generate 1 with satisfactory yields. 22 In the case of ferrocenyl carboxaldehyde-involved Groebke 3CR
25
(reaction (3)), no desired compound 2 was obtained when CH3COOH, 23 AgOTf, 24 and Ce(OTf)3
acted as catalysts, but other catalysts enabled the reaction (3) to afford 2 in yields of 20~38%. It
appeared that ferrocenyl carboxaldehyde was so susceptible that can readily be decomposed during the
reaction period even in the nitrogen atmosphere. It was reported that ferrocenyl carboxaldehyde was
well-tolerated towards LaCl3 in a solvent-free Groebke 3CR with 2-aminopyridine and cyclohexyl
isocyanide being reactants. 22 We herein employed LaCl3 to catalyze 2-aminoquinoxaline and t-butyl
isocyanide in the Groebke 3CR, and low yield of compound 2 (33%) may be owing to the low
reactivity of 2-aminoquinoxaline and low refluxing temperature. Attempts to perform the reaction (4)
by using these catalysts led to the same results, in which only (CH3)3SiCl and LaCl3 can result in
moderate yields (35% and 40%, respectively). Therefore, (CH3)3SiCl and LaCl3 were used in the
following synthesis of imidazo[1,2-a]quinoxalines.
It was clear that (CH3)3SiCl was not suitable for the hydroxyl-substituted aldehyde because it can
react with hydroxyl group. Thus, 12, 13, 16, 17, 19, 20, 27, and 28 were synthesized by utilizing LaCl3
as the catalyst in the refluxing ethanol, while other imidazo[1,2-a]quinoxalines were prepared by using
(CH3)3SiCl as the catalyst in the refluxing acetonitrile. Exceptionally, 22, 23, and 25 were obtained in
the media of chloroform, while 28 was obtained in the mixture of chloroform and ethanol (1:1, v:v)
because 2-(4-formylphenyl)chromen-4-one as the reactant cannot be dissolved in ethanol or acetonitrile
individually. In addition, low boiling point of these solvents cannot provide high temperature for the
reaction but can avoid the decomposition of temperature-sensitive reactants. Under the optimized
reaction condition (1 mmol (CH3)3SiCl or LaCl3, refluxing solvent), an aldehyde can be used in the first
or the second Groebke 3CR to afford the final product with the aldehyde moiety at 2- or 4-position in
imidazo[1,2-a]quinoxaline (see Scheme 2).
8

ACCEPTED MANUSCRIPT
2.2. Scavenging radicals.
The reaction of an antioxidant with 2,2’-diphenyl-1-picrylhydrazyl radical (DPPH, a nitrogen-
centered radical) followed the bimolecular kinetics as shown as equation (5). 26
d[DPPH]
(5)
r = k [DPPH] [antioxidant]
=
-
dt
The rate constant (
k
) can be calculated by the equation (5) when the reaction rate (
r
) along with the
concentrations of DPPH and the antioxidant were measured at a certain time-point. But the
concentrations of DPPH and the antioxidant at the beginning of the reaction (t = 0) were known, the
equation (5) can be transformed into equation (6), in which [DPPH]t=0 and [antioxidant]t=0 referred to
the concentrations of DPPH and the antioxidant at t = 0, and r0 was the reaction rate at the beginning of
the reaction.
r 0
= k [DPPH]t=0 [antioxidant]t=0
(6)
As we have reported a method for measuring r0, 27 the rate constants (
k
) of the reactions of imidazo[1,2-
a]quinoxalines with DPPH were achieved and shown in Scheme 3.
9

ACCEPTED MANUSCRIPT
0.08
0.06
0.04
0.02
0
0.068
DPPH
0.05
0.046
0.025
0.021
0.02
0.015
0.0058
0.013
0.013
0.01
0.0069
0.0053
0.0058
0.0041
0.0074
0.0032
0.0046
2
3
5
7
9
10 11 12 13 14 15 16 17 18 19 20 23 26 27
8.37
8
9
6
3
0
+.
6.17
ABTS
5.89
5.69
5.18
4.21
4.06
3.43
3.17
3.03
3.03
2.8
2.2
2.62
2.01
2.1
1.56
1.64
1.57
0.93
0.84
0.029
0.0021
0.74
0.66
0.0041
0.096
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Scheme 3 The rate constant (k) of imidazo[1,2-a]quinoxalines in quenching radicals.
10

ACCEPTED MANUSCRIPT
27
We have measured the k of Trolox in trapping DPPH (0.353×
103M-1s-1). The imidazo[1,2-
a]quinoxalines used herein were not as active as Trolox in quenching N-centered radical of DPPH, and
1, 4, 6, 8, 21, 22, 24, 25, and 28 did not show any activities. It was worth pointing out that the relatively
high k values of 13 (0.046× × ×
103M-1s-1), 20 (0.068 103M-1s-1), and 27 (0.050 103M-1s-1) indicated that
the phenolic hydroxyl group can provide hydrogen atom to be abstracted by N-centered radical, and the
ferrocenyl group in 13 and 20 can also provide electrons to quench DPPH. In addition, the ferrocenyl
group allowed 2 and 3 to exhibit lower activities towards DPPH (k of 2 and 3 were both 0.0032
1s-1) than 1 (no reaction with DPPH). Furthermore, the double ferrocenyl groups increased k value for
103M-1s-1, ~3-fold higher than that of 2 and 3. This is in agreement with our previous
observation on the benefit of ferrocenyl group for radical-scavenging property. 27 To be the isomer of 13,
103M-1s-1) than that of 13, indicating 2-ferrocenyl and 4-
×
103M-
18 to 0.01
×
12 did not exhibit higher k value (0.013
×
phenolic hydroxyl formed a powerful coordination for trapping N-centered radical, while diverse
positions (4-ferrocenyl and 2-phenolic hydroxyl) cannot play the same role. Recently, we found that
N,N-dimethylamino group in coumarin was active for trapping radicals, 28 and herein, high values of k
of 10 (0.021× ×
103M-1s-1) and 11 (0.025 103M-1s-1) also indicated that N,N-dimethylamino group in
imidazo[1,2-a]quinoxaline was still effective towards DPPH. Similar k values of 2 and 3 implied that
the ferrocenyl group at 2- or 4-positon cannot markedly influence the property of imidazo[1,2-
a]quinoxaline in trapping DPPH, but 23 and 24 also consisting of ferrocenyl group exhibited quite
different behaviors in this case. The k value of 23 (0.0041×
103M-1s-1) and no reaction of 24 with DPPH
revealed that 4-flavonyl group was able to improve the radical-scavenging property of 2-ferrocenyl
group. The comparison of k values of 27 (bearing 2-phenolic hydroxyl and 4-flavonyl group with
0.05×
103M-1s-1 of k) and 28 (bearing 2-flavonyl group and 4-phenolic hydroxyl, and no reaction with
DPPH) demonstrated the aforementioned rules. Moreover, exchanging positions of N,N-
dimethylaminophenyl and flavonyl groups generated 25 (bearing flavonyl group at 4-postion, and no
reaction with DPPH) and 26 (bearing flavonyl group at 2-postion with 0.0058
×
103M-1s-1 of k), in which
11

ACCEPTED MANUSCRIPT
4-flavonyl group was inactive for N,N-dimethylamino in trapping DPPH.
The galvinoxyl radical was employed to determine the ability of an antioxidant to trap O-centered
radical. As shown in Scheme 3, 2, 6, 8, 10, 12, 18, 19, 20, and 24 were able to react with galvinoxyl
radical, and the k values were lower than that of Trolox (1.70×
103M-1s-1). We herein found that 3, 5, 7,
9, 11, 13, 23 cannot react with galvinoxyl radical although both of them consisted of ferrocenyl group.
Thus, only 4-ferrocenyl group was active to quench O-centered radical. The double ferrocenyl groups
increased the k value of 18 (0.63× ×
103M-1s-1) ~2 fold higher than that of 2 (0.16 103M-1s-1). Thus, the
influence of the position of ferrocenyl group on trapping radicals can be identified by the reaction with
galvinoxyl radical.
The oxidation of ABTS salt generated ABTS+·, which can be used to test the ability of an
× ×
antioxidant to reduce radical. 29 Low k values of 1 (0.0041 103M-1s-1), 21 (0.0021 103M-1s-1), and 22
(0.029
×
103 M-1s-1) indicated that the skeleton of imidazo[1,2-a]quinoxaline emerged weak ability to
reduce ABTS+·. Despite of 14, 15, 16, 17, 25, 26, 27, and 28, the k values of other imidazo[1,2-
a]quinoxalines were all increased by the ferrocenyl group. We further compared k values of isomer
couples, i.e., 2 vs 3, 4 vs 5, 6 vs 7, 12 vs 13, 23 vs 24, and found that 2-ferrocenyl group was more
beneficial for the imidazo[1,2-a]quinoxaline to trap ABTS+· than 4-ferrocenyl group. An electron-
donating group such as -OCH3 (in 8 vs 9) and -N(CH3)2 (in 10 vs 11, 14 vs 15, 25 vs 26) at 4-phenyl
group played more active role for imidazo[1,2-a]quinoxaline in trapping ABTS+· than that at 2-position.
The comparison of k values of 16 vs 17, 27 vs 28, indicated that the phenolic hydroxyl group at 2-
position was benefit to trap ABTS+·. The 4-ferrocenyl group in conjunction with -OH or -N(CH3)2 in 2-
phenyl group increased the k of 12 and 10 to the highest values (8.37× ×
103M-1s-1 and 8.00 103M-1s-1,
respectively).
2.3. Inhibiting DNA oxidation.
12

ACCEPTED MANUSCRIPT
We attempted to use peroxyl radical (.OOCMe2C(=NH)NH2, deriving from the decomposition
30
of AAPH) to initiate the oxidation of DNA, during which the formed carbonyl species can be
spectroscopically detected at 535 nm after reacting with thiobarbituric acid (TBA). Thus, the oxidation
of DNA can be followed by determining the thiobarbituric acid reactive species (TBARS). 31 In the
blank experiment (see Figure 4S), the concentration of TBARS linearly increased with the reaction
period. The equation (7) indicated the relationship between the concentration of TBARS and the
reaction period (t), and the coefficient (d[TBARS]/dt = 5.63 nM.min-1) implied the rate of DNA
oxidation. 32
[TBARS (nM)] = 5.63 (± 0.28) t (min) + 3242.7 (± 162)
(7)
As shown in Figure 4S (in the supporting formation), the addition of 100
µM 1~9, and 21~24 actually
changed the slope of lines of [TBARS]~t, and the equations of [TBARS]~t were involved in Table 4S.
The d[TBARS]/dt was listed in Scheme 4. A low value of d[TBARS]/dt implied that the imidazo[1,2-
a]quinoxaline possessed high ability to decrease the oxidative rate of DNA. It can be found from
Scheme 4 that 2-ferrocenyl group together with 4-flavonyl (as 23) or 4-phenyl with electron-donating
group (as 9) exhibited relatively higher inhibitory effect on DNA oxidation than other compounds
contained in Scheme 4.
13

ACCEPTED MANUSCRIPT
N
N
HN
O
O
N
22
Fe
N
4.16 nM.min-1
N
N
N
O
N
N
O
NO2
Fe
Br
Fe
N
O
NH
Fe
N
NH
Br
O
N
N
N
N
N
N
6
21
N
N
N
O
N
N
N
N
3.66 nM.min-1
3.57 nM.min-1
N
H
Fe
NH
Fe
NH
NH
NH
NO2
Fe
9
23
5
4
7
Fe
2.58 nM.min-1
2.77 nM.min-1
4.29 nM.min-1
4.16 nM.min-1
4.22 nM.min-1
N
N
O
N
N
N
N
NH
2-ferrocenyl group with 4-flavonyl
or electron-donating group at 4-phenyl
O
NH
Fe
N
O
N
N
N
8
N
24
N
N
N
3.88 nM.min-1
N
3.63 nM.min-1
Fe
NH
NH
NH
4-ferrocenyl group with 2-flavonyl or 2-phenyl group
1
3
2
4.48 nM.min-1
4.20 nM.min-1
4.48 nM.min-1
blank
d[TBARS]/dt =
5.63 nM.min-1
electron-withdrawing group or no substituent
Scheme 4 The inhibitory effects of imidazo[1,2-a]quinoxalines without tinh generated.
14

ACCEPTED MANUSCRIPT
In addition to the aforementioned imidazo[1,2-a]quinoxalines, the addition of other compounds
can inhibit the DNA oxidation for a period as shown in Figure 5S (in the supporting formation). The
inhibition period (tinh) started from t = 0 min and finished at the cross-point of tangents for inhibitory
and oxidative periods. As shown in Figure 6S (in the supporting formation), tinh correlated linearly with
the concentration of imidazo[1,2-a]quinoxalines, and the equations of tinh~[imidazo[1,2-a]quinoxaline]
were collected in Table 5S. It has been proved that tinh was related proportionally to the concentration
of an antioxidant as shown as equation (8). 33
tinh = (n/Ri) [antioxidant]
(8)
Ri referred to the initiation rate of the radical-induced oxidation, while the stoichiometric factor (n)
stood for the number of the radical-propagation terminated by one molecule of the antioxidant. The
coefficient in equations of tinh~[imidazo[1,2-a]quinoxaline] (see Table 5S in the supporting formation)
was equivalent to n/Ri, in which the initiation rate (Ri) of DNA oxidation was assumed to be equal to
the radical-generation rate (Rg = (1.4
M.min−1) since both DNA sodium salt and AAPH were water-soluble compounds, and the
± × ×
0.2) × 10-6 [AAPH] s-1, viz., Ri = Rg = 1.4 10-6 40 mM.s-1 =
3.36
µ
34
radical attacked DNA at the same phase. Therefore, the n was the product of the coefficient in
tinh~[imidazo[1,2-a]quinoxaline] and Ri and outlined in Scheme 5.
9
7.46
7.12
stoichiometric factor (n)
5.24
6
3.7
3.33
3.36
3
0
1.98
1.88
1.78
1.78
1.65
1.55
1.44
1.48
1.24
10 11 12 13 14 15 16 17 18 19 20 25 26 27 28
Scheme 5 The stoichiometric factor (n) of imidazo[1,2-a]quinoxalines in inhibiting DNA oxidation.
15

ACCEPTED MANUSCRIPT
The inhibitory effects of 10~20 and 25~28 on AAPH-induced oxidation of DNA can also be
discussed based on n values. Among all of the tested compounds, 12 and 10 stood out as the most
efficacious compounds with 7.46 and 7.12 of n values, respectively. This is in line with the observation
in scavenging ABTS+. test, in which 4-ferrocenyl group in conjunction with -OH or (CH3)2N- in 2-
phenyl group were active to quench ABTS+.. Exchanging the order of the aforementioned functional
groups generated isomers 13 and 11 with high n values (5.24 and 3.33, respectively). The similar n
values of 19 and 20 (3.36 and 3.70, respectively) also implied that 4-ferrocenyl group was not as active
as that at 2-position. However, the double ferrocenyl groups did not allow 18 to exhibit strongly
inhibitory effect on AAPH-induced oxidation of DNA, and introducing phenyl or flavonyl group did
not enhance the n values of 14~17 and 25~28 (just ranging from 1.44 to 1.98, lower than that of
ferrocenyl-appended compounds). Trolox also cannot generate inhibitory period (tinh) and thus, the
stoichiometric factor (n) cannot be obtained to be the reference for the comparison with other
compounds. 27
With the oxidative potentials (E1/2) of 1 (1.13 V), 21 (1.12 V), and 22 (1.11 V) as the reference,
the introduction of ferrocenyl group into 4- or 2-position markedly decreased E1/2 of 2 and 3 (0.69 and
0.67 V, respectively) and of 23 and 24 (0.66 and 0.72 V, respectively). Thus, the benefit of ferrocenyl
group for increasing antioxidative effect was owing to decrease the oxidative potential for imidazo[1,2-
a]quinoxaline. The installation of phenolic hydroxyl group for ferrocenyl-appended imidazo[1,2-
a]quinoxalines did not further decrease E1/2 of 12 and 13 (0.60 and 0.65 V, respectively). However,
lower E1/2 of 27 and 28 (0.93 and 1.01 V, respectively) than that of the corresponding hydroxyl-free
compounds (21 and 22) still revealed the antioxidative role of phenolic hydroxyl group. Upon
structures of 27 and 28, replacing hydroxyl group by (CH3)2N- generated 25 and 26. The E1/2 to 0.69 V
(25) and 0.91 V (26) revealed that (CH3)2N- decreased the oxidative potential more efficiently than
hydroxyl group. Finally, comparing the k and n values of 25 and 26 with those of a previously reported
17
compound, ethyl 2-(2-(4-hydroxyphenyl)H-imidazo[1,2-a]pyridin-3-ylamino)acetate,
one can find
16

ACCEPTED MANUSCRIPT
that the antioxidative effects of the compounds in the present work were generally lower than that in
our previous report. The E1/2 of ethyl 2-(2-phenyl)H-imidazo[1,2-a]pyridin-3-ylamino)acetate (a
compound without phenolic hydroxyl group) was 0.50 V, 17 lower than that of 25 and 26 (0.69 and 0.91
V, respectively), indicating that the compounds in the present work were more difficult to be oxidized
than those in our previous report. But the flavonyl group made N,N-dimethylaminophenyl group exhibit
antioxidative effect in the imidazo[1,2-a]quinoxaline scaffold, which may be a novel candidate for the
screening of antioxidative drugs.
N
N
N
N
N
HN
N
O
O
O
N
O
N
N
NH
O
OH
N
NH
O
Ethyl 2-(2-(4-hydroxyphenyl)H-
imidazo[1,2-a]pyridin-3-ylamino)acetate
25
26
k
k
k
n
(ABTS+.) = 13.58 mM-1s-1
(DPPH) = 1.05 mM-1s-1
(galvinoxyl) = 0.31 mM-1s-1
= 5.70
(ABTS+.) = 0.096 mM-1s-1
(DPPH) = 0.00 mM-1s-1
(galvinoxyl) = 0.00 mM-1s-1
= 1.55
k
k
k
n
(ABTS+.) = 3.03 mM-1s-1
(DPPH) = 0.0058 mM-1s-1
(galvinoxyl) = 0.00 mM-1s-1
= 1.78
k
k
k
n
3. Conclusion
We applied a Groebke 3CR to prepare 2-aminoquinoxaline, which was the reactant for the
following Groebke 3CR to afford imidazo[1,2-a]quinoxaline. Some substituted benzaldehydes along
with ferrocenyl carboxaldehyde and 2-(4-formylphenyl)chromen-4-one took part in the aforementioned
sequential Groebke 3CR to give isomers with the aldehyde allocating 2- or 4-position in imidazo[1,2-
a]quinoxaline. The installation of a flavonyl group into imidazo[1,2-a]quinoxaline did not exhibit
additional activity towards either radicals or DNA oxidation, but 4-ferrocenyl group emerged as an
active feature for inhibiting DNA oxidation and radicals. The combination of 2-phenolic hydroxyl with
4-ferrocenyl increased antioxidative effectiveness of imidazo[1,2-a]quinoxaline. In the continued quest
for novel antioxidative drugs, ferrocenyl-appended imidazo[1,2-a]quinoxaline will offer a valuable
choice and thus be worth exploring in due course.
17

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4. Materials and Methods
4.1. Materials and Instrumentation.
AAPH and naked DNA sodium salt were products of ACROS Organics, Geel, Belgium, and
diammonium salt of 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS salt), DPPH, and
galvinoxyl radical were products of Fluka Chemie GmbH, Buchs, Switzerland. Solvents and reagents
used in the synthesis were obtained commercially and used as such unless noted otherwise. All of the
products were identified by 1H and 13C NMR spectroscopy (Bruker Avance III 400 MHz spectrometer)
and HRMS spectrometry (ESI as the ionization mode) equipped with HPLC (Agilent 1290-micrOTOF
Q II). The NMR spectra were provided in Supporting Information.
4.2. Preparation of 2-(4-formylphenyl)chromen-4-one and 2-aminoquinoxalines.
A mixture of 2-hydroxyacetophenone (0.01 mol) and p-phthalaldehyde (0.012 mol) was stirred in
50 mL of ethanol, and 0.5 mL of piperidine was added and refluxed under stirring for overnight. The
cooled mixture was purified on a silica gel column with CH2Cl2 being the eluent to afford chalcone.
Then, the chalcone was dissolved in dimethyl sulfoxide (DMSO), followed by adding I2 (0.01 equiv.).
The mixture was stirred at 110°C in N2 atmosphere for overnight and then poured into ice water to
collect solid product, which was further purified on a silica gel column with CH2Cl2 / ethyl acetate
being the eluent to afford 2-(4-formylphenyl)chromen-4-one.
To a 50 mL of methanolic solution of o-phenylenediamine (0.01 mol) was added the equimolar
aldehyde, 2,4,4-trimethylpentan-2-yl isocyanide, and concentrated HCl. The solution was stirred at
room temperature in N2 atmosphere for overnight. After methanol was evaporated, 50 mL of the
saturated aqueous solution of NaHCO3 was added to the mixture, and the aqueous layer was extracted
with CHCl3 and dried over anhydrous MgSO4. The combined organic solvent were removed under
vacuum, and the residue was dissolved in 50 mL of anhydrous tetrahydrofuran (THF), to which 1 equiv.
18

ACCEPTED MANUSCRIPT
of 2,3-dicyano-5,6-dichlorobenzoquinone (DDQ, dissolved in 10 mL of THF) was added. The above
mixture was stirred at room temperature for overnight, and the solvent was removed under vacuum.
The residue was purified on a silica gel column with CH2Cl2 being the eluent to give quinoxalines.
The quinoxaline (5 mmol) was dissolved in 4N HCl in dioxane (50 mL) and stirred at room
temperature for overnight. Then, the mixture was poured into ice water, and NaHCO3 was added until
pH > 7. The aqueous layer was extracted with CHCl3, and the combined organic layers were dried over
anhydrous MgSO4. Then, the organic solvent was removed under vacuum, and the residue was purified
on a silica gel column with CH2Cl2/ethyl acetatebeing the eluent to give 2-aminoquinoxaline.
4.3. Synthesis of imidazo[1,2-a]quinoxalines.
The acetonitrile solution (25 mL) of 2-aminoquinoxaline (1 mmol) and aldehyde (1 mmol) was
refluxed for 3 h and then cooled to room temperature. The catalyst (1 mmol (CH3)3SiCl or LaCl3) was
added and stirred at room temperature for 1 h. Then, tert-butyl isocyanide (1 mmol) was added, and the
mixture was refluxed for 36 h. After completion of the reaction as indicated by TLC, the mixture was
poured into NaHCO3 solution and extracted with CH2Cl2. The combined CH2Cl2 layers were dried over
anhydrous MgSO4 and then removed under vacuum. The residue was purified on a silica gel column to
give imidazo[1,2-a]quinoxalines 1-28.
tert-Butyl-(2,4-diphenyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (1). A white solid, yield: 215 mg
1
(55%). Rf = 0.37 (ethyl acetate : petroleum ether = 1 : 8, v:v), m.p.: 180-182°C. H NMR (400 MHz,
CDCl3)
δ: 9.68 (m, 1H), 8.77 (d, J = 7.2 Hz, 2H), 8.15 (s, 1H), 7.83 (d, J = 7.6 Hz, 2H), 7.58-7.52 (m,
13
5H), 7.46 (t, J = 7.6 Hz, 2H), 7.38(t, J = 7.2 Hz, 1H), 3.56 (s, 1H), 1.00 (s, 9H). C NMR (100 MHz,
CDCl3) δ: 150.2, 141.8, 137.0, 136.4, 135.1, 134.5, 130.5, 130.4, 130.3, 129.4, 128.7, 128.4, 128.0,
126.7, 126.0, 117.3, 57.8, 29.8. HRMS (ESI): m/z calcd for [M+H]+ of C26H24N4 393.2080, found:
393.2066.
19

ACCEPTED MANUSCRIPT
tert-Butyl-(2-phenyl-4-ferrocenyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (2). A red solid, yield:
175 mg (35%). Rf = 0.38 (ethyl acetate : petroleum ether = 1 : 8, v:v), m.p.: 192-194°C. 1H NMR (400
MHz, CDCl3)
δ: 9.58 (m, 1H), 8.05 (s, 1H), 7.90 (d, J = 7.6 Hz, 2H), 7.54-7.49 (m, 4H), 7.40 (m, 1H),
5.89 (s, 2H), 4.59 (s, 2H), 4.10 (s, 5H), 3.52 (s, 1H), 0.99 (s ,9H). 13C NMR (100 MHz, CDCl3)
δ
:
153.2, 141.1, 135.4, 134.1, 130.1, 129.3, 128.8, 128.7, 128.6, 127.8, 125.9, 125.5, 117.4, 71.2, 71.0,
69.9, 57.8, 29.8. HRMS (ESI): m/z calcd for [M+H]+ of C30H28FeN4 501.2055, found: 501.1701.
tert-Butyl-(2-ferrocenyl-4-phenyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (3). A yellow solid, yield:
190 mg (38%). Rf = 0.47 (ethyl acetate : petroleum ether = 1 : 8, v:v). m.p.: 215-217°C. 1H NMR (400
MHz, DMSO-d6)
δ
: 9.69 (d, J = 4.8 Hz, 1H), 8.90 (d, J = 4.4 Hz, 2H), 8.07 (d, J = 0.8 Hz, 1H), 7.70-
13
7.59 (m, 5H), 5.08 (s, 2H), 4.77 (s, 1H), 4.36 (s, 2H), 4.14 (s, 5H), 0.96 (s, 9H). C NMR (100 MHz,
DMSO-d6) δ: 149.2, 136.6, 136.4, 134.2, 130.3, 130.2, 130.0, 129.0, 128.1, 126.4, 125.7, 121.8, 117.2,
80.5, 69.7, 68.8, 68.5, 57.4, 29.8. HRMS (ESI): m/z calcd for [M+H]+ of C30H28FeN4 501.2055, found:
501.1717.
tert-Butyl-(2-(p-nitrophenyl)-4-ferrocenyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (4). A red solid,
yield: 164 mg (30%). Rf = 0.17 (ethyl acetate : petroleum ether = 1 : 8, v :v). m.p.: 254-256°C. 1H NMR
(400 MHz, DMSO-d6)
δ: 9.44 (m, 1H), 8.37 (d, J = 7.6 Hz, 2H), 8.17 (d, J = 7.6 Hz, 2H), 8.02 (m, 1H),
13
7.52 (m, 2H), 5.82 (s, 2H), 4.60 (s, 2H), 4.09 (s, 5H), 3.40 (s, 1H), 1.03 (s, 9H). C NMR (100 MHz,
DMSO-d6) δ: 153.5, 146.9, 142.0, 138.5, 137.4, 134.7, 130.8, 129.7, 128.8, 128.6, 126.3, 125.8, 123.9,
117.1, 79.9, 71.2, 70.8, 69.9, 58.1, 29.9. HRMS (ESI): m/z calcd for [M+H]+ of C30H27FeN5O2
546.1906, found: 546.1543.
tert-Butyl-(4-(p-nitrophenyl)-2-ferrocenyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (5). A red solid,
yield: 109 mg, 20 %. Rf = 0.34 (ethyl acetate : petroleum ether = 1 : 8, v:v). m.p.: 223-225°C. 1H NMR
(400 MHz, CDCl3)
δ: 9.61 (m, 1H), 9.17 (d, J = 8.8 Hz, 2H), 8.42 (d, J = 8.8 Hz, 2H), 8.13 (d, J = 8.8
Hz, 1H), 7.61-7.55 (m, 2H), 4.90 (s, 2H), 4.50 (s, 2H), 4.27 (s, 5H), 3.19 (s, 1H), 1.07 (s, 9H). 13C
20

ACCEPTED MANUSCRIPT
NMR (100 MHz, CDCl3)
δ: 148.6, 146.3, 142.5, 140.8, 136.5, 134.0, 131.1, 130.5, 130.3, 129.2, 127.4,
126.2, 123.2, 117.4, 70.0, 69.0, 57.6, 29.9. HRMS (ESI): m/z calcd for [M+H]+ of C30H27FeN5O2
546.1906, found: 546.1550.
tert-Butyl-(2-(p-bromophenyl)-4-ferrocenyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (6). A red
solid, yield: 191 mg (33%). Rf = 0.34 (ethyl acetate : petroleum ether = 1 : 8, v:v). m.p.: 215-217°C. 1H
NMR (400 MHz, CDCl3)
δ: 9.52 (m, 1H), 8.01 (m, 1H), 7.82 (m, 2H), 7.50 (m, 2H), 5.82 (s, 2H), 4.58
(s, 2H), 4.09 (s, 5H), 3.40 (s, 1H), 1.01(s, 9H). 13C NMR (100 MHz, CDCl3)
δ
: 153.2, 139.7, 137.3,
134.4, 134.2, 131.7, 130.0, 129.7, 129.5, 128.6, 125.9, 125.5, 121.7, 117.1, 80.1, 71.0, 70.7, 69.8, 57.7,
29.8. HRMS (ESI): m/z calcd for [M+H]+ of C30H27FeBrN4 579.0757, found: 579.0783.
tert-Butyl-(4-(p-bromophenyl)-2-ferrocenyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (7). A yellow
solid, yield: 231 mg (40%). Rf = 0.45 (ethyl acetate : petroleum ether = 1 : 8, v:v). m.p.: 237-239°C. 1H
NMR (400 MHz, CDCl3) δ: 8.59-8.57 (m, 1H), 8.87 (d, J = 8.4 Hz, 2H), 8.09 (d, J = 9.2 Hz, 1H), 7.73
(d, J = 8.4 Hz, 2H), 7.55-7.52 (m, 2H), 4.76 (s, 2H), 4.36 (s, 2H), 4.18 (s, 5H), 3.20 (s, 1H), 1.02 (s,
9H). 13C NMR (100 MHz, CDCl3)
δ
: 147.8, 140.3, 136.6, 135.4, 134.0, 131.9, 131.3, 130.1, 129.0,
126.6, 125.8, 124.9, 117.3, 80.4, 69.7, 68.8, 68.6, 57.5, 29.8. HRMS (ESI): m/z calcd for [M+H]+ of
C30H27FeBrN4 579.0757, found: 579.0780.
tert-Butyl-(2-(p-methoxyphenyl)-4-ferrocenyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (8). A red
solid, yield: 208 mg (36%). Rf = 0.20 (ethyl acetate : petroleum ether = 1 : 8, v:v). m.p.: 200-202°C. 1H
NMR (400 MHz, CDCl3)
δ: 9.54 (s, 1H), 8.04 (s, 1H), 7.83 (d, J = 7.6 Hz, 2H), 7.47 (s, 2H), 7.05-7.03
(m, 2H), 6.88 (s, 2H), 4.57 (s, 2H), 4.09 (s, 5H), 3.87 (s, 3H), 3.43 (s, 1H), 0.98 (s, 9H). 13C NMR (100
MHz, CDCl3) δ: 159.2, 152.9, 140.9, 137.0, 133.9, 129.7, 129.4, 129.2, 128.8, 127.9, 125.7, 125.4,
117.2, 114.0, 80.1, 71.0, 70.9, 69.9, 57.5, 55.4, 29.8. HRMS (ESI): m/z calcd for [M+H]+ of
C31H30FeON4 531.1875, found: 531.1825.
21

ACCEPTED MANUSCRIPT
tert-Butyl-(4-(p-methoxyphenyl)-2-ferrocenyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (9). A yellow
solid, yield: 196 mg (37%). Rf = 0.31 (ethyl acetate : petroleum ether = 1 : 8, v:v). m.p.: 230-232°C. 1H
NMR (400 MHz, CDCl3) δ: 9.57-9.55 (m, 1H), 8.96 (d, J = 8.8 Hz, 2H), 8.08 (s, 1H), 7.51-7.49 (m,
2H), 7.11 (d, J = 8.8 Hz, 2H), 4.82 (s, 2H), 4.40 (s, 2H), 4.23 (s, 5H), 3.93 (s, 3H), 3.18 (s, 1H), 1.03 (s,
9H). 13C NMR (100 MHz, CDCl3)
δ
: 161.5, 148.7, 140.1, 136.9, 134.3, 132.0, 123.0, 129.4, 128.9,
126.0, 125.7, 117.3, 113.6, 70.0, 69.0, 68.7, 57.4, 55.5, 29.9. HRMS (ESI): m/z calcd for [M+H]+ of
C31H30FeON4 531.1875, found: 531.1821.
tert-Butyl-(2-(p-dimethylaminophenyl)-4-ferrocenyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (10).
A red solid, yield: 217 mg (40%). Rf = 0.19 (ethyl acetate : petroleum ether = 1 : 8, v:v). m.p.: 207-
209°C. 1H NMR (400 MHz, CDCl3)
δ: 9.58-9.56 (m, 1H), 8.03 (s, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.48-
7.45 (m, 2H), 6.86 (d, J = 8.4 Hz, 2H), 5.89 (s, 2H), 4.56 (s, 2H), 4.09 (s, 5H), 3.47 (s, 1H), 3.05 (s,
6H), 1.01 (s, 9H). 13C NMR (100 MHz, CDCl3)
δ: 152.7, 150.0, 141.4, 137.1, 133.8, 129.2, 129.1,
129.0, 128.9, 125.5, 125.2, 123.3, 117.3, 112.3, 80.3, 70.9, 70.8, 69.8, 57.4, 40.5, 29.8. HRMS (ESI):
m/z calcd for [M+H]+ of C32H33FeN5 544.2164, found: 544.2128.
tert-Butyl-(4-(p-dimethylaminophenyl)-2-ferrocenyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (11).
A yellow solid, yield: 239 mg (44%). Rf = 0.26 (ethyl acetate : petroleum ether = 1 : 8, v:v). m.p.: 235-
1
237°C. H NMR (400 MHz, CDCl3)
δ
: 9.54-9.52 (m, 1H), 9.00-8.99 (m, 2H), 8.07 (s, 1H), 7.48-7.42
(m, 2H), 6.89 (d, J = 8.0 Hz, 2H), 4.79 (s, 2H), 4.35 (s, 2H), 4.21 (s, 5H), 3.17 (s, 1H), 3.10 (s, 6H),
1.02 (s, 9H). 13C NMR (100 MHz, CDCl3)
δ
: 152.0, 139.5, 137.3, 131.7, 129.7, 129.6, 128.8, 127.9,
125.5, 125.3, 121.9, 117.2, 111.6, 69.8, 68.9, 68.4, 57.4, 40.4, 29.9. HRMS (ESI): m/z calcd for
[M+H]+ of C32H33FeN5 544.2164, found: 544.2129.
p-(1-tert-Butylamino-4-ferrocenyl-imidazo[1,2-a]quinoxalin-2-yl)phenol (12). A red solid, yield:
144 mg (28%). Rf = 0.39 (ethyl acetate : petroleum ether = 1 : 2, v:v). m.p.: 195-197°C. 1H NMR (400
MHz, CDCl3)
δ
: 9.59-9.56 (m, 1H), 8.01-7.99 (m, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.49-7.47 (m, 2H),
22

ACCEPTED MANUSCRIPT
7.11 (d, J = 8.0 Hz, 2H), 5.84 (s, 2H), 5.54 (s, 1H), 4.55 (s, 2H), 4.08 (s, 5H), 3.46 (s, 1H), 1.00 (s, 9H).
13C NMR (100 MHz, CDCl3)
δ: 157.4, 152.7, 141.2, 137.0, 133.7, 129.6, 129.2, 129.0, 128.8, 126.3,
125.6, 125.4, 117.3, 116.0, 80.2, 77.2, 70.8, 70.7, 69.8, 52.8, 29.7. HRMS (ESI): m/z calcd for [M+H]+
of C30H28FeON4 517.1710, found: 517.1673.
p-(1-tert-Butylamino-2-ferrocenyl-imidazo[1,2-a]quinoxalin-4-yl)phenol (13). A red solid, yield:
155 mg (30%). Rf = 0.48 (ethyl acetate : petroleum ether = 1 : 2, v:v). m.p.: 244-246°C. 1H NMR (400
MHz, DMSO-d6) δ: 10.00 (s, 1H), 9.67-9.65 (m, 1H), 8.89 (d, J = 8.8 Hz, 2H), 8.01-7.97 (m, 1H),
7.65-7.54 (m, 2H), 6.99 (d, J = 8.8 Hz, 2H), 5.07 (s, 2H), 4.72 (s, 1H), 4.35 (s, 2H), 4.12 (s, 5H), 0.95
(s, 9H). 13C NMR (100 MHz, DMSO-d6)
δ
: 159.7, 147.3, 139.8, 136.1, 133.3, 131.7, 129.7, 129.3,
128.3, 127.1, 126.0, 125.6, 116.9, 114.9, 80.2, 69.2, 68.7, 68.0, 56.1, 29.4. HRMS (ESI): m/z calcd for
[M+H]+ of C30H28FeON4 517.1710, found: 517.1666.
tert-Butyl-[2-(p-dimethylaminophenyl)-4-phenyl-imidazo[1,2-a]quinoxalin-1-yl]-amine (14). A
yellow solid, yield: 174 mg (40%). Rf = 0.26 (ethyl acetate : petroleum ether = 1 : 6, v:v). m.p.: 210-
212°C. 1H NMR (400 MHz, CDCl3)
δ: 9.63-9.60 (m, 1H), 8.89 (d, J = 7.2 Hz, 2H), 8.10-8.06 (m, 1H),
7.85 (d, J = 7.2 Hz, 2H), 7.53-7.45 (m, 4H), 7.39-7.35 (m, 1H) 6.87 (d, J = 8.8 Hz, 2H), 3.54 (s, 1H),
3.08 (s, 6H), 0.99 (s, 9H). 13C NMR (100 MHz, CDCl3)
δ
: 150.2, 149.7, 142.3, 136.9, 136.6, 134.2,
130.3, 130.2, 130.1, 129.6, 129.4, 128.3, 126.4, 125.7, 123.0, 117.3, 112.4, 57.6, 40.6, 29.8. HRMS
(ESI): m/z calcd for [M+H]+ of C28H29N5 436.2502, found: 436.2418.
tert-Butyl-[4-(p-dimethylaminophenyl)-2-phenyl-imidazo[1,2-a]quinoxalin-1-yl]-amine (15). A
yellow solid, yield: 187 mg (43%). Rf = 0.29 (ethyl acetate : petroleum ether = 1 : 6, v:v). m.p.: 271-
273°C. 1H NMR (400 MHz, CDCl3)
(d, J = 8.4 Hz, 2H), 7.57-7.50 (m, 5H), 6.85 (s, 2H), 3.53 (s, 1H), 3.02 (s, 6H), 1.02 (s, 9H). 13C NMR
δ: 9.67-9.65 (m, 1H), 8.81-8.79 (m, 2H), 8.14-8.11 (m, 1H), 7.72
(100 MHz, CDCl3) δ: 152.0, 149.7, 141.3, 135.3, 134.6, 131.6, 130.2, 129.7, 129.0, 128.7, 128.6,
23

ACCEPTED MANUSCRIPT
127.8, 125.8, 125.5, 117.2, 111.7, 57.7, 40.4, 29.8. HRMS (ESI): m/z calcd for [M+H]+ of C28H29N5
436.2502, found: 436.2486.
p-(1-tert-Butylamino-4-phenyl-imidazo[1,2-a]quinoxalin-2-yl)phenol (16). A white solid, yield:
122 mg (44%). Rf = 0.30 (ethyl acetate : petroleum ether = 1 : 2, v:v). m.p.: 283-285°C. 1H NMR (400
MHz, DMSO-d6) δ: 9.77 (t, J = 8.0 Hz, 1H), 9.57 (s, 1H), 8.79-8.77 (m, 2H), 8.07 (d, J = 7.6 Hz, 1H),
7.91 (d, J = 8.4 Hz, 2H), 7.72-7.68 (m, 1H), 7.64-7.58 (m, 4H), 6.87 (d, J = 7.6 Hz, 2H), 4.98 (s, 1H),
0.95 (s, 9H). 13C NMR (100 MHz, DMSO-d6)
δ: 163.8, 157.7, 148.6, 142.2, 136.5, 133.6, 132.6, 130.7,
130.3, 129.3, 128.5, 127.4, 126.4, 125.9, 117.5, 116.3, 115.5, 56.8, 30.0. HRMS (ESI): m/z calcd for
[M+H]+ of C26H24N4O 409.2029, found: 409.2004.
p-(1-tert-Butylamino-2-phenyl-imidazo[1,2-a]quinoxalin-4-yl)phenol (17). A white solid, yield:
167 mg (41%). Rf = 0.40 (ethyl acetate : petroleum ether = 1 : 2, v:v). m.p.: 250-252°C. 1H NMR (400
MHz, DMSO-d6) δ: 10.01 (s, 1H), 9.76-9.73 (m, 1H), 8.79 (d, J = 8.8 Hz, 2H), 8.12-8.10 (m, 2H),
8.04-8.01 (m, 1H), 7.66-7.04 (m, 1H), 7.62-7.58 (m, 1H), 7.49 (t, J = 7.6 Hz, 2H), 7.41-7.37 (m, 1H),
13
6.97 (d, J = 8.8 Hz, 2H), 5.05 (s, 1H), 0.94 (s, 9H). C NMR (100 MHz, DMSO-d6)
δ: 160.3, 148.5,
141.4, 136.6, 135.2, 133.9, 132.2, 131.0, 129.9, 129.0, 128.6, 128.1, 127.3, 126.7, 126.5, 117.5, 115.4,
56.9, 29.9. HRMS (ESI): m/z calcd for [M+H]+ of C26H24N4O 409.2029, found: 409.2011.
tert-Butyl-[2,4-diferrocenyl-imidazo[1,2-a]quinoxalin-1-yl]-amine (18). A red solid, yield: 237 mg
1
(39%). Rf = 0.43 (ethyl acetate : petroleum ether = 1 : 8, v:v). m.p.: 244-246°C. H NMR (400 MHz,
CDCl3)
δ: 9.53-9.50 (m, 1H), 8.00-7.98 (m, 1H), 7.48-7.44 (m, 2H), 5.93 (s, 2H), 4.82 (s, 2H), 4.60 (s,
2H), 4.37 (s, 2H), 4.24 (s, 5H), 4.13 (s, 5H), 3.17 (s, 1H), 1.02 (s, 9H). 13C NMR (100 MHz, CDCl3)
δ:
152.5, 139.2, 137.2, 134.0, 129.2, 128.6, 125.4, 125.1, 117.2, 80.7, 80.4, 70.8, 70.5, 69.7, 69.6, 68.5,
68.3, 57.3, 29.8. HRMS (ESI): m/z calcd for [M+H]+ of C34H32Fe2N4 609.1405, found: 609.1348.
p-(1-tert-Butylamino-4-ferrocenyl-imidazo[1,2-a]quinoxalin-2-yl)-o-methoxyphenol (19). A red
solid, yield: 173 mg (29%). Rf = 0.64 (ethyl acetate : dichloromethane = 1 : 20, v:v). m.p.: 226-228°C.
24

ACCEPTED MANUSCRIPT
1H NMR (400 MHz, CDCl3)
δ
: 9.53-9.50 (m, 1H), 8.09 (s, 1H), 8.02-8.00 (m, 1H), 7.78 (d, J = 8.4 Hz,
1H), 7.51-7.48 (m, 2H), 7.16 (d, J = 8.4 Hz, 1H), 5.83 (s, 2H), 4.85 (s, 2H), 4.09 (s, 5H), 3.36 (s, 1H),
1.03 (s, 9H). 13C NMR (100 MHz, CDCl3)
δ
: 151.9, 137.3, 134.1, 131.9, 129.5, 129.4, 129.3, 129.2,
125.8, 125.5, 117.1, 116.1, 110.5, 80.1, 71.0, 70.7, 69.6, 57.6, 29.9. HRMS (ESI): m/z calcd for
[M+H]+ of C30H27BrFeN4O 596.0953, found: 596.0780.
p-(1-tert-Butylamino-4-ferrocenyl-imidazo[1,2-a]quinoxalin-2-yl)-o-bromophenol (20). A red
solid, yield: 148 mg (27%). Rf = 0.59 (ethyl acetate : dichloromethane = 1 : 20, v:v). m.p.: 207-209°C.
1H NMR (400 MHz, CDCl3)
δ: 9.57-9.54 (m, 1H), 8.04 (s, 1H), 7.50-7.48 (m, 3H), 7.33-7.31 (m, 1H),
7.06-7.03 (m, 1H), 5.86 (s, 2H), 5,78 (s, 1H), 4.58 (s, 2H), 4.10 (s, 5H), 4.05 (s, 3H), 3.46 (s, 1H), 1.00
(s, 9H). 13C NMR (100 MHz, CDCl3)
δ
: 152.9, 145.6, 141.0, 137.3, 133.9, 129.4, 128.8, 127.7, 125.7,
125.4, 121.5, 117.2, 114.4, 111.4, 80.3, 70.8, 70.7, 69.8, 57.6, 56.2, 29.8. HRMS (ESI): m/z calcd for
[M+H]+ of C31H30FeN4O2 547.1797, found: 547.1829.
tert-Butyl-(2-phenyl-4-flavonyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (21). A white solid, yield:
1
166 mg (25%). Rf = 0.31 (ethyl acetate : dichloromethane = 1 : 20, v:v). m.p.: 275-277°C. H NMR
(400 MHz, CDCl3) δ: 9.71-9.69 (m, 1H), 9.03 (d, J = 8.4 Hz, 2H), 8.34 (s, 1H), 8.27 (d, J = 7.6 Hz, 1H),
8.15 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 7.6 Hz, 2H), 7.76-7.72 (m, 1H), 7.64-7.61 (m, 3H), 7.52 (t, J = 7.2
13
Hz, 2H), 7.47-7.40 (m, 2H), 6.96 (s, 1H), 3.63 (s, 1H), 1.02 (s, 9H). C NMR (100 MHz, CDCl3)
δ:
178.5, 163.1, 156.3, 148.2, 142.1, 139.2, 136.6, 134.8, 134.2, 133.8, 133.0, 130.8, 130.4, 129.4, 128.7,
128.6, 128.1, 127.1, 126.2, 126.1, 125.7, 125.2, 124.1, 118.2, 117.3, 108.0, 57.8, 29.7. HRMS (ESI):
m/z calcd for [M+H]+ of C35H28N4O2 537.2291, found: 537.2283.
tert-Butyl-(2-flavonyl-4-phenyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (22). A white solid, yield:
1
160 mg (30%). Rf = 0.35 (ethyl acetate : dichloromethane = 1 : 20, v:v). m.p.: 263-265°C. H NMR
(400 MHz, CDCl3) δ: 9.58-9.56 (m, 1H), 8.80-8.78 (m, 2H), 8.25-8.23 (m, 2H), 8.07-8.00 (m, 4H),
7.73-7.69 (m, 1H), 7.59-7.53 (m, 6H), 7.42 (t, J = 8.0 Hz, 1H), 6.88 (s, 1H), 3.59 (s, 1H), 1.02 (s, 9H).
25

ACCEPTED MANUSCRIPT
13C NMR (100 MHz, CDCl3)
δ
: 178.4, 162.9, 156.2, 149.8, 140.7, 138.1, 134.4, 133.8, 131.2, 130.8,
130.7, 130.3, 130.0, 129.0, 128.9, 128.3, 126.9, 126.4, 126.3, 125.7, 125.3, 124.2, 118.1, 117.1, 107.5,
58.0, 29.8. HRMS (ESI): m/z calcd for [M+H]+ of C35H28N4O2 537.2291, found: 537.2252.
tert-Butyl-(2-ferrocenyl-4-flavonyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (23). A yellow solid,
1
yield: 193 mg (30%). Rf = 0.41 (ethyl acetate : dichloromethane = 1 : 20, v:v). m.p.: 233-235°C. H
NMR (400 MHz, CDCl3) δ: 9.62-9.60 (m, 1H), 9.14 (d, J = 7.6 Hz, 2H), 8.28 (d, J = 8.0 Hz, 2H), 8.18
(d, J = 7.6 Hz, 2H), 7.76-7.73 (m, 1H), 7.67-7.65 (m, 1H), 7.59-7.58 (m, 1H), 7.47-7.44 (m, 1H), 6.99
(s, 1H), 4.83 (s, 2H), 4.42 (s, 2H), 4.23 (s, 5H), 3.26 (s, 1H), 1.06 (s, 9H). 13C NMR (100 MHz, CDCl3)
δ
: 178.5, 163.2, 156.4, 147.5, 136.6, 134.2, 133.8, 132.9, 130.8, 130.3, 130.2, 129.1, 126.9, 126.0,
125.9, 125.8, 125.3, 124.1, 118.2, 117.3, 108.1, 100.0, 80.4, 69.8, 68.9, 68.7, 57.5, 29.8. HRMS (ESI):
m/z calcd for [M+H]+ of C39H32N4O2 645.1954, found: 645.1705.
tert-Butyl-(2-flavonyl-4-ferrocenyl-imidazo[1,2-a]quinoxalin-1-yl)-amine (24). A red solid, yield:
1
238 mg (37%). Rf = 0.41 (ethyl acetate : dichloromethane = 1 : 15, v:v). m.p.: 213-215°C. H NMR
(400 MHz, CDCl3) δ: 9.55-9.53 (m, 1H), 8.28 (d, J = 7.6 Hz, 1H), 8.17-8.10 (m, 4H), 8.04-8.02 (m,
1H), 7.76-7.72 (m, 1H), 7.64-7.62 (m, 1H), 7.53-7.44 (m, 1H), 6.94 (s, 1H), 5.86 (s, 2H), 4.61 (s, 2H),
4.11 (s, 5H), 3.49 (s, 1H), 1.05 (s, 9H). 13C NMR (100 MHz, CDCl3)
δ
: 178.5, 163.1, 156.3, 153.3,
139.5, 138.8, 137.3, 134.4, 133.8, 130.6, 130.4, 129.5, 128.8, 126.5, 126.0, 125.8, 125.6, 125.3, 124.1,
118.1, 117.2, 107.5, 80.1, 71.1, 70.8, 69.9, 57.9, 29.8. HRMS (ESI): m/z calcd for [M+H]+ of
C39H32N4O2 645.1954, found: 645.1712.
tert-Butyl-[2-(p-dimethylaminophenyl)-4-flavonyl-imidazo[1,2-a]quinoxalin-1-yl]-amine (25). A
yellow solid, yield: 197 mg (34%). Rf = 0.20 (ethyl acetate : dichloromethane = 1 : 15, v:v). m.p.: 280-
282°C. 1H NMR (400 MHz, CDCl3)
δ: 9.70-9.67 (m, 1H), 9.05 (d, J = 8.8 Hz, 2H), 8.28-8.25 (m, 1H),
8.17-8.12 (m, 3H), 7.77-7.71 (m, 3H), 7.64-7.56 (m, 3H), 7.46-7.43 (m, 1H), 6.96-6.89 (m, 3H), 3.55 (s,
1H), 3.05 (s, 6H), 1.02 (s, 9H). 13C NMR (100 MHz, CDCl3)
δ
: 178.5, 163.2, 156.3, 147.9, 142.4,
26

ACCEPTED MANUSCRIPT
139.6, 136.7, 134.0, 133.8, 132.7, 130.8, 130.3, 129.8, 129.3, 126.8, 126.0, 125.9, 125.7, 125.2, 124.1,
118.2, 117.3, 112.5, 107.9, 57.6, 40.6, 29.8. HRMS (ESI): m/z calcd for [M+H]+ of C37H33N5O2
580.2713, found: 580.2510.
tert-Butyl-[4-(p-dimethylaminophenyl)-2-flavonyl-imidazo[1,2-a]quinoxalin-1-yl]-amine (26). A
yellow solid, yield: 231 mg (40%). Rf = 0.25 (ethyl acetate : dichloromethane = 1 : 15, v:v). m.p.: 285-
287°C. 1H NMR (400 MHz, CDCl3)
δ: 9.56 (d, J = 8.0 Hz, 1H), 8.89 (d, J = 7.6 Hz, 2H), 8.28-8.26 (m,
1H), 8.10-8.05 (m, 5H), 7.75-7.71 (m, 1H), 7.62-7.60 (m, 1H), 7.55-7.43 (m, 3H), 6.92-6.87 (m, 3H),
3.49 (s, 1H), 3.09 (s, 6H), 1.04 (s, 9H). 13C NMR (100 MHz, CDCl3)
δ
: 178.5, 163.1, 156.3, 152.0,
149.6, 139.6, 138.6, 137.3, 134.9, 133.8, 131.5, 130.5, 129.8, 128.9, 126.3, 126.0, 125.7, 125.6, 125.2,
124.1, 124.0, 118.1, 117.0, 111.6, 107.4, 57.8, 40.3, 29.8. HRMS (ESI): m/z calcd for [M+H]+ of
C37H33N5O2 580.2713, found: 580.2662.
p-(1-tert-Butylamino-4-flavonyl-imidazo[1,2-a]quinoxalin-2-yl)phenol (27). A white solid, yield:
1
121 mg (22%). Rf = 0.25 (ethyl acetate : dichloromethane = 1 : 10, v:v). m.p.: 291-293°C. H NMR
(400 MHz, DMSO-d6) δ: 9.78 (d, J = 8.4 Hz, 1H), 9.60 (s, 1H), 9.04 (d, J = 8.8 Hz, 2H), 8.37 (d, J =
8.4 Hz, 2H), 8.14-8.09 (m, 2H), 7.95 (d, J = 7.6 Hz, 2H), 7.89-7.86 (m, 2H), 7.76-7.74 (m, 3H), 7.56-
13
7.54 (m, 1H), 6.89 (d, J = 8.4 Hz, 2H), 5.02 (s, 1H), 0.97 (s, 9H). C NMR (100 MHz, DMSO-d6)
δ:
177.6, 162.5, 156.2, 147.2, 142.3, 139.3, 136.4, 134.9, 133.6, 132.9, 132.0, 130.8, 130.5, 130.4, 129.4,
129.1, 127.8, 126.6, 126.5, 126.1, 125.7, 125.3, 123.9, 119.1, 115.5, 108.0, 56.9, 30.0. HRMS (ESI):
m/z calcd for [M+H]+ of C35H28N4O3 553.2240, found: 553.2051.
p-(1-tert-Butylamino-2-flavonyl-imidazo[1,2-a]quinoxalin-4-yl)phenol (28). A white solid, yield:
1
138 mg (25%). Rf = 0.28 (ethyl acetate : dichloromethane = 1 : 10, v:v). m.p.: 301-303°C. H NMR
(400 MHz, DMSO-d6) δ: 10.15 (s, 1H), 9.75-9.73 (m, 1H), 8.81 (d, J = 8.8 Hz, 2H), 8.40-8.37 (m, 2H),
8.50 (d, J = 8.8 Hz, 2H), 8.10-8.09 (m, 1H), 8.06-8.03 (m, 1H), 7.88-7.85 (m, 2H), 7.68 (t, J = 8.4 Hz,
1H), 7.62 (t, J = 8.0 Hz, 1H), 7.56-7.52 (m, 1H), 7.17 (s, 1H), 7.01 (d, J = 8.8 Hz, 2H), 5.24 (s, 1H),
27

ACCEPTED MANUSCRIPT
0.99 (s, 9H). 13C NMR (100 MHz, DMSO-d6)
δ: 177.6, 160.3, 156.2, 148.6, 140.1, 138.4, 136.7, 134.8,
134.2, 132.2, 131.8, 130.4, 130.0, 129.3, 129.1, 129.0, 127.2, 127.0, 126.7, 126.0, 125.3, 123.9, 119.1,
117.6, 115.5, 107.3, 57.2, 30.0. HRMS (ESI): m/z calcd for [M+H]+ of C35H28N4O3 553.2240, found:
553.2220.
4.4. Scavenging ABTS+·, DPPH, and galvinoxyl radicals.
Solutions of DPPH and galvinoxyl radicals were prepared by dissolving solids of DPPH and
galvinoxyl radical in ethanol directly. The absorbance was around 1.00 at 517 nm (for DPPH, εDPPH
=
4.09 × 103 M-1cm-1) and 428 nm (for galvinoxyl radical, εgalvinoxyl = 1.4 × 105 M-1cm-1), respectively.
The ABTS+· was generated in 2.0 mL of aqueous solution of 4.0 mM ABTS salt and 1.41 mM K2S2O8
after 20 h, and then, was diluted with 100 mL of ethanol. The absorbance of ABTS+· was also around
1.00 at 734 nm (εABTS = 1.6 × 104 M-1 cm-1). At the ambient temperature (25°C), a certain
+·
concentration of an imidazo[1,2-a]quinoxaline (dissolved in dimethyl sulfoxide (DMSO) as the stock
solution, 0.1 mL) was mixed with 1.9 mL of a radical solution, and the absorbance of the mixture was
immediately recorded and then measured with a certain time interval (see Supporting Information).
4.5 Inhibiting AAPH-induced oxidation of DNA.
AAPH and DNA sodium salt were dissolved in phosphate buffered solution (PBS: 8.1 mM
Na2HPO4, 1.9 mM NaH2PO4, 10.0
µM EDTA, pH=7.4) at room temperature. An imidazo[1,2-
a]quinoxaline was dissolved in DMSO as the stock solution. Then, DMSO solution of the imidazo[1,2-
a]quinoxaline was added to the mixture of DNA and AAPH with a certain final concentration.
Meanwhile, final concentrations of DNA and AAPH were kept at 2.0 mg/mL and 40.0 mM,
respectively. The mixture was dispatched into test tubes with each one containing 2.0 mL. All of the
28

ACCEPTED MANUSCRIPT
test tubes were incubated at 37°C for initiating the DNA oxidation. Three of them were taken out at
every 2 hours and cooled immediately, and 1.0 mL of thiobarbituric acid (TBA, 1.00 g of TBA and 0.40
g of NaOH dissolved in 100 mL of PBS) and 1.0 mL of 3.0% trichloroacetic acid aqueous solution
were added. The mixture was then heated in boiling water for 15 min and cooled to room temperature;
1.5 mL of n-butanol was added and shaken vigorously to extract thiobarbituric acid reactive species
(TBARS) for measuring the absorbance at 535 nm. The absorbance of TBARS was plotted vs the
reaction period (see Supporting Information).
4.6. Cyclic voltammetry.
The cyclic voltammetry (CV) was measured on a CHI 604C voltammetric analyzer with a
platinum button and a wire as the working and counter electrodes, respectively, along with a saturated
calomel electrode (SCE) as the reference electrode. A solution of 10 mL of CH2Cl2 (dehydration by
CaH2 in advance) consisting of ~1 mM corresponding compounds was scanned at the rate of 50 mV.s-1
with ferrocene as the internal reference and 0.1 M (n-C4H9)4NPF6 as the supporting electrolyte.
4.7. Statistical analysis.
All of the data were the average value from at least three independent measurements with the
experimental error within 10%. Equations were analyzed by one-way ANOVA in Origin 6.0
professional Software, and p<0.001 indicated a significance difference.
29