Antimicrobial evaluation and action mechanism of chalcone derivatives containing quinoxaline moiety

Article abstract of DOI:10.1007/s00706-019-02449-9

Abstract: A series of chalcone derivatives containing quinoxaline moieties were synthesized, and their antibacterial activities were evaluated. Antibacterial bioassays indicated that some of the compounds exhibited significant antibacterial activity against Xanthomonas axonopodis pv. Citri (Xac), Xanthomonas oryzae pv. oryzae (Xoo), and Ralstonia solanacearum (Rs) at the concentrations of 50 or 100?μg/cm³. 50% effective concentration (EC₅₀) values of (E)-3-(pyridin-2-yl)-1-[4-(quinoxalin-2-yloxy)phenyl]prop-2-en-1-one against Xac, Xoo, and Rs were 6.72, 15.17, and 9.29?μg/cm³, respectively, which were better than those of bismerthiazol (44.31, 42.46, and 62.36?μg/cm³, respectively). Scanning electron microscopy (SEM) was employed to analyze the mechanism of antibacterial action of that compound toward Xac. Graphical abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-019-02449-9

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-019-02449-9
ORIGINAL PAPER
Antimicrobial evaluation and action mechanism of chalcone
derivatives containing quinoxaline moiety
Rongjiao Xia1 · Tao Guo1 · Jun He1 · Mei Chen1 · Shijun Su1 · Shichun Jiang1 · Xu Tang1 · Ying Chen1 · Wei Xue1
Received: 11 March 2019 / Accepted: 13 May 2019
© Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract
A series of chalcone derivatives containing quinoxaline moieties were synthesized, and their antibacterial activities were
evaluated. Antibacterial bioassays indicated that some of the compounds exhibited signifcant antibacterial activity against
Xanthomonas axonopodis pv. Citri (Xac), Xanthomonas oryzae pv. oryzae (Xoo), and Ralstonia solanacearum (Rs) at the
concentrations of 50 or 100 μg/cm3. 50% efective concentration (EC50) values of (E)-3-(pyridin-2-yl)-1-[4-(quinoxalin-2-
yloxy)phenyl]prop-2-en-1-one against Xac, Xoo, and Rs were 6.72, 15.17, and 9.29 μg/cm3, respectively, which were bet-
ter than those of bismerthiazol (44.31, 42.46, and 62.36 μg/cm3, respectively). Scanning electron microscopy (SEM) was
employed to analyze the mechanism of antibacterial action of that compound toward Xac.
Graphical abstract
N
O
N
O
N
N
Ar
Ar
H
O
combinatorial
optimization
O
Chalcone
Exhibited superior bioactivities
against Xac, Xoo, and Rs
Keywords Synthesis · Chalcone · Quinoxaline · Antibacterial activity · Scanning electron microscopy
Introduction
Xanthomonas oryzae pv. oryzae (Xoo), and Ralstonia sola-
nacearum (Rs) [3]. Among them, plant diseases are one of
the most devastating diseases in some countries, leading
to the yields of rice to drop by 80% [4]. Controlling the
plant diseases is being a key challenge in the department of
agriculture. As one of the preventive measures toward these
bacterial diseases of plants, the application of traditional
pesticides is not very efcient due to their passive impact
on the environment as well as the increasing resistance of
plant pathogens [5, 6]. Therefore, searching for new, highly
efcient, and safe bactericides is still of great signifcance
in pesticide science.
One of the main causes of plant diseases is microbial infec-
tions, which leads to the damage of a large number of plants
and owing to this not only they possess a threat to the yield
of economic crops, but also afect human health seriously
[1, 2]. In recent years, commercial crops give rise to eco-
nomic loss due to Xanthomonas axonopodis pv. Citri (Xac),
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-019-02449-9) contains
supplementary material, which is available to authorized users.
As one of the essential natural products in medicinal
chemistry, chalcone was reported to exhibit extensive
biological activities such as antimicrobial [7, 8], anti-
inflammatory [9, 10], anticancer [11, 12], insecticidal
[13, 14], antiviral [15, 16], and antifungal [17, 18]. Nota-
bly, in recent years, chemists have reported an increasing
* Wei Xue
wxue@gzu.edu.cn
1
Key Laboratory of Green Pesticide and Agricultural
Bioengineering, Ministry of Education, Research
and Development Center for Fine Chemicals, Guizhou
University, Guiyang 550025, China
Vol.:(0123456789)
1 3

R. Xia et al.
number of chalcone derivatives with good antimicrobial
activities. Quinoxaline, which was regarded as an impor-
tant nitrogenous heterocycle, has drawn much attention
on account of its broad spectrum of biological activities,
including antimicrobial [19, 20], anticancer [21], and her-
bicidal [22]. However, studies on quinoxaline derivatives
with potent bactericidal activities were rarely reported
which necessitates investigations on their synthesis and
biological activities.
Results and discussion
The synthetic route to obtain the title compounds is shown in
Scheme 1, which exhibits the synthesis of a series of novel
chalcone derivatives containing quinoxaline fragments.
Condensation of the 1-(4-hydroxyphenyl)ethan-1-one with
diferent substituted aldehydes under ice-bath condition for
24 h led to intermediate 1 with yield 65–79%. Then, in the
waterless condition, 2-hydroxyquinoxaline was dissolved in
phosphorus oxychloride and DMF, stirred and refuxed at
90 °C for 1 h until the completion of the reaction. Following
this, it was poured into ice water and fltered to obtain white
solid that was intermediate 2 with yield 74%. Potassium car-
bonate as a base, acetonitrile as a solvent, intermediates 1
and 2 as the starting materials were mixed and stirred for
30 min and refuxed at 85 °C for 12 h. After the completion
of the reaction, the whole reaction system was diluted with
water and the resulting precipitate appeared in the continu-
ous stirring; the mixture was separated to obtain compound
3 with yield 42–75%, as displayed in Table 1.
Considering these above results, we speculated that
introducing quinoxaline fragment into chalcone might
generate novel lead compounds with greater antimicrobial
activities. Therefore, a series of novel chalcone deriva-
tives containing quinoxaline moieties were synthesized
(Scheme 1) and their antibacterial activities against Xac,
Xoo, and Rs were evaluated. Based on the excellent bac-
teriostatic activity of compound 3b against Xac, scanning
electron microscopy (SEM) was then performed.
Scheme 1
1 3

Antimicrobial evaluation and action mechanism of chalcone derivatives containing quinoxaline…
3d, 3o, and 3v at 50 μg/cm3 against Xac were 75, 62, 66,
Table 1 Physical properties of new compounds 3a–3x
and 62%, respectively, which exceeded the inhibition rates
of bismerthiazol (33%). The bioassay results indicated that
the antibacterial activities of compounds 3b, 3d, and 3v at
100 μg/cm3 against Xoo were 98, 91, and 82%, respectively,
which were excellent to bismerthiazol (72%). Compounds
3d and 3v at 50 μg/cm3 had best antibacterial activity against
Xoo, with the inhibition rates of 84 and 71%, respectively,
which were preferable to bismerthiazol (63%). The bioas-
say results indicated that the antibacterial activities of com-
pounds 3b, 3d, 3h, and 3i at 100 μg/cm3 against Rs were
90, 72, 72, and 87%, respectively, which were better than
bismerthiazol (53%). Compounds 3b, 3d, 3 h, 3i, and 3v at
50 μg/cm3 showed best antibacterial activity against Rs, with
the inhibition rates of 80, 60, 51, 57, and 55%, respectively,
which were superior to bismerthiazol (30%).
Compound Formula
Appearance Yield/% M.p./°C
2a
2b
3c
3d
3e
3f
3g
3h
3i
C24H18N2O2
White solid
White solid
Yellow solid 55
White solid
White solid
White solid
72
64
223–224
219–220
227–228
205–207
229–230
212–213
169–170
175–177
205–206
190–192
214–216
209–210
192–194
204–205
170–171
179–181
210–211
216–218
214–216
198–200
158–159
184–186
194–195
147–149
C22H15N3O2
C21H14N2O2S
C23H15N2O2Br
C23H16N2O2
C23H15N2O2Cl
C24H18N2O3
C22H15N3O2
C21H14N2O3
C23H15N2O2Br
C23H15N2O3Br
C24H18N2O3
46
50
53
Yellow solid 62
White solid
Gray solid
White solid
Yellow solid 66
Yellow solid 48
43
72
75
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
C23H14N2O2BrCl White solid
C22H14N3O2Cl
C21H13N2O2ClS
50
58
49
42
69
Gray solid
Gray solid
C23H14N2O2BrCl Gray solid
Structure–activity relationships (SAR)
of antibacterial activities
C23H15N2O2Cl
C23H14N2O2Cl2
C24H17N2O3Cl
C22H14N3O2Cl
C21H13N2O3Cl
C23H14N2O2BrCl Gray solid
C24H17N2O3Cl
C24H17N2O2Cl
Gray solid
Yellow solid 73
Gray solid
Gray solid
Gray solid
67
50
52
47
58
51
Antibacterial activity is related to the structure variations
of the title compounds. As indicated in Tables 2 and 3, the
inhibitory rates of most of the compounds against Xac were
superior as compared to using compounds against Xoo and
Rs. However, title compounds exhibited antibacterial activi-
ties which were signifcantly afected by varying diferent
substitutes R2 positions.
3t
3u
3v
3w
3x
Gray solid
Gray solid
For example, the designated compounds 3d (4-Br-Ph), 3j
(3-Br-Ph), 3p (4-Br-Ph), and 3v (3-Br-Ph) could efectively
enhance the antibacterial activity against Xac at 100 μg/cm3,
with inhibition rates of 88, 88, 62, and 70%, respectively. At
the same time, with the presence of the -Br-Ph group at the
R2 position, the corresponding compounds presented bet-
ter in vitro bioactivity against Xac, following the order of
3j (R1 = H, R2 = 3-Br-Ph)> 3d (R1 = H, R2 = 4-Br-Ph)> 3v
(R1 =Cl, R2 =3-Br-Ph)>3p (R1 =Cl, R2 =4-Br-Ph).
Based on the preliminary bioassays, EC50 values of the
selected compounds exhibited excellent activities against
Xac, Xoo, and Rs as summarized in Table 3. Compounds
3b, 3d, 3g, 3o, and 3q efectively inhibited Xac in vitro, with
EC50 values of 6.72, 24.16, 20.53, 28.77, and 18.25 µg/cm3,
respectively, which outperforms bismerthiazol (44.31 µg/
cm3). Meanwhile, compounds 3b, 3d, and 3v efectively
inhibited Xoo in vitro, with EC50 values of 15.17, 34.41,
and 28.84 µg/cm3, respectively, which were better compared
to bismerthiazol (42.46 µg/cm3). Compounds 3b, 3d, 3i, 3o,
and 3q showed signifcant antibacterial activities against Rs,
with EC50 values of 9.29, 30.02, 22.41, 20.63, and 32.38 µg/
cm3, respectively, which were preferable to bismerthiazol
(62.36 µg/cm3). Notably, the title compounds 3b and 3d
against Xac, Xoo, and Rs demonstrated better biological
activity compared to bismerthiazol.
The structures of all the target compounds 3a–3x were
characterized by 1H NMR, 13C NMR, and high-resolution
mass spectrometer (HRMS), and all the signals in the spectra
1
were in agreement with the proposed structure. From H
NMR spectra of compound 3a, using CDCl3 to display a
singlet at δ=8.7 ppm that corresponds to N=C functional-
ity, multiplet signals at 8.15–7.24 ppm indicate the presence
of protons in olefnic bonds and aromatic nuclei and also
a typical chemical shift at δ=198.27 ppm from 13C NMR
spectra confrmed the presence of C=O. The strong presence
of [M+H]+ ions indicates that the title compounds are in
steady state.
Antibacterial activity
The bacteriostatic activities of the compounds were evalu-
ated by the turbidimetric method [23, 24]. Some of the title
compounds exhibited considerable antibacterial activities
against Xac, Xoo, and Rs in vitro at 100 and 50 μg/cm3, and
the observed results are shown in Table 2.
For example, the inhibition rates of compounds 3b, 3d, 3i,
and 3o at 100 μg/cm3 against Xac were 92, 88, 78, and 85%,
respectively, which were superior compared to bismerthiazol
(54%). Meanwhile, the inhibition rates of compounds 3b,
1 3

R. Xia et al.
Table 2 Inhibition (%) efect of
title compounds against Xoo,
Xac, and Rs
Compound
Xac
100 μg/cm3
Xoo
100 μg/cm3
Rs
50 μg/cm3
50 μg/cm3
100 μg/cm3
50 μg/cm3
3a
52
92
50
88
67
56
64
73
78
88
56
63
16
40
85
62
67
44
55
53
53
70
50
52
54
49
75
45
62
60
46
54
50
57
50
54
51
16
31
66
28
55
30
42
26
52
62
47
44
33
47
98
35
91
50
63
63
54
50
67
66
76
61
71
69
70
73
71
69
76
67
82
73
60
72
14
46
23
84
46
47
16
9
41
40
30
44
50
57
63
66
65
66
41
60
57
71
55
12
63
45
90
24
72
47
34
31
72
87
44
45
62
63
30
65
46
68
52
51
62
48
57
31
59
53
40
80
10
60
17
28
25
51
57
22
31
30
61
13
30
44
23
40
49
52
42
55
25
49
30
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
Bismerthiazol
Table 3 EC50 values of target compounds against Xac, Xoo, and Rs
damage became more severe with an increase in the con-
centration of applied compound. It was worth noted that
without subjecting to any treatment with bacterial cells the
cell membrane was intact and smooth (Fig. 1a). At the same
time, the cell membranes began to wrinkle at 50 mg/cm3
(Fig. 1b), whereas when the concentration was 100 mg/cm3,
the cells membrane began to rupture (Fig. 1c). Therefore,
SEM study further confrmed that the compound 3b could
damage the bacterial cell membrane structure, resulting in
incomplete bacterial structure, and the purpose of steriliza-
tion had been achieved.
Compound
R1
R2
EC50/µg/cm3
Xac Xoo
6.72
Rs
9.29
30.02
–
–
–
22.41
20.63
32.38
–
3b
H
H
H
H
H
H
Cl
Cl
Cl
–
Pyridin-2-yl
4-Br-Ph
Ph
2-OCH3-Ph
Pyridin-3-yl
Furan-2-yl
Thiophen-2-yl
Ph
15.17
3d
24.16
39.65
20.53
33.92
30.92
28.77
18.25
34.64
44.31
34.41
–
–
–
–
–
–
3e
3g
3h
3i
3o
3q
3v
3-Br-Ph
–
28.84
42.46
Conclusion
Bismerthiazol
62.36
In summary, in the direction of obtaining novel and efcient
chalcone derivatives with better activities, a series of chalcone
derivatives containing quinoxaline fragments were designed
and evaluated for their antibacterial activities in vitro, against
Xanthomonas axonopodis pv. Citri (Xac), Xanthomonas ory-
zae pv. oryzae (Xoo), and Ralstonia solanacearum (Rs). It
has been observed that most of the compounds demonstrated
bacteriostatic action against plant bacteria. Among them, EC50
Scanning electron microscopy (SEM) studies
To study the mechanism of antibacterial action toward Xac,
scanning electron microscopy (SEM) analysis was carried
out based on the results of bacteriostatic activities of com-
pound 3b. It can be seen that the compound 3b causes cell
membrane wrinkle and damage, and the cell membrane
1 3

Antimicrobial evaluation and action mechanism of chalcone derivatives containing quinoxaline…
Fig. 1 SEM images for Xanthomonas axonopodis pv. Citri (Xac) after incubated in diferent concentrations of compound 3b a 0 mg/cm3, b
50 mg/cm3, and c100 mg/cm3. Scale bar for (a–c) are 2 μm
values of the compound 3b against Xac, Rs, and Xoo were
6.72, 15.17, and 9.29 μg/cm3, respectively, which were bet-
ter than those of bismerthiazol (44.31, 42.46, and 62.36 μg/
cm3, respectively). SEM analyses further proved bacterial cell
membrane damage due to the title compounds. Therefore, the
designed compounds should be further studied as potential
replacement templates in the search for novel antibacterial
agents.
refuxed at 90 °C for 1 h until the completion of the reaction.
Following this, it was poured into ice water, and fltered to
obtain a white solid compound 2a, with yield 74%.
General procedure for the synthesis of title
compounds 3a–3x
Based on the reported method [26, 27], potassium carbon-
ate as a base, acetonitrile as a solvent and intermediates 1a
and 2a as the starting materials were mixed and stirred for
30 min, and refuxed at 85 °C for 12 h. After the completion
of the reaction, the whole reaction system was diluted with
water and the resulting precipitate appeared in the continu-
ous stirring; the mixture was separated to obtain compound
3a, with yield 72%. By following the same procedure, the
title compounds 3b–3x were then obtained.
Experimental
The melting points were determined on an XT-4 binocular
microscopic melting point apparatus (Beijing Tech Instru-
1
ment Co, Beijing. China). H NMR and 13C NMR spec-
tra were obtained on a Bruker Ascend-400 spectrometer
(Bruker Optics, Switzerland), DMSO-d6 or CDCl3 was
used as a solvent, and TMS was used as an internal stand-
ard. Mass spectral studies were performed on a quadrupole
electrostatic feld orbitrap mass spectrograph. The chemical
reaction was monitored by thin-layer chromatography under
ultraviolet light of 254 nm. All the reagents and solutions
were of analytical reagent grade.
(E)‑1‑[4‑[(6‑Chloroquinoxalin‑2‑yl)oxy]phenyl]‑3‑(p‑tolyl)‑
prop‑2‑en‑1‑one (3a, C24H18N2O2) White solid; yield: 72%;
m.p.: 223–224 °C; 1H NMR (400 MHz, CDCl3): δ=8.74 (s,
1H, Ar–CH=), 8.15 (d, J=8.8 Hz, 2H, Ar(4-O)-2,6-H), 8.09
(dd, J=8.1, 1.5 Hz, 1H, Qu-3-H), 7.85 (d, J=15.7 Hz, 1H,
Qu-5-H), 7.81–7.77 (m, 1H, Qu-6-H), 7.67 (tdd, J=9.6, 7.0,
1.6 Hz, 2H, Qu-8-H, Ar–C=CH), 7.55 (t, J=12.1 Hz, 3H,
Ar(4-CH3)-2,6-H, Qu-7-H), 7.44 (d, J=8.8 Hz, 2H, Ar(4-
CH3)-3,5-H), 7.24 (d, J=8.0 Hz, 2H, Ar(4-O)-3,5-H), 2.40
(s, 3H, CH3) ppm; 13C NMR (101 MHz, CDCl3): δ=189.27,
156.44, 156.30, 145.08, 141.22, 139.92, 139.80, 139.15,
135.42, 132.14, 130.67, 130.40, 129.77, 129.05, 128.56,
127.90, 127.77, 121.35, 120.76, 21.59 ppm; HRMS (ESI):
m/z calcd. [M+H]+ 367.1441, found 367.1435.
General procedure for the synthesis of compounds
1a–1m
According to the synthetic method of the literature [25],
condensation of the 1-(4-hydroxyphenyl)ethan-1-one with
diferent substituted aldehydes under ice-bath condition for
24 h gives intermediate 1a–1m with yield 65–79%.
General procedure for the synthesis of compounds
2a, 2b
(E)‑3‑(Pyridin‑2‑yl)‑1‑[4‑(quinoxalin‑2‑yloxy)phenyl]‑
prop‑2‑en‑1‑one (3b, C22H15N3O2) White solid; yield: 64%;
m.p.: 219–220 °C; 1H NMR (400 MHz, DMSO-d6): δ=8.95
(s, 1H, Py-6-H), 8.71 (d, J = 4.4 Hz, 1H, Ar–C=CH),
In the waterless condition, 2-hydroxyquinoxaline was dis-
solved in phosphorus oxychloride and DMF, stirred and
1 3

R. Xia et al.
8.27 (d, J = 8.8 Hz, 2H, Ar–CH=, Ar(4-O)-2-H), 8.22 (d,
J=15.4 Hz, 1H, Ar(4-O)-6-H), 8.12 (d, J=7.4 Hz, 1H, Qu-
3-H), 7.97–7.94 (m, 1H, Qu-5-H), 7.92 (dd, J=7.8, 1.8 Hz,
1H, Qu-6-H), 7.79 (d, J= 2.8 Hz, 1H, Qu-8-H), 7.77 (dd,
J=2.6, 1.4 Hz, 2H, Qu-7-H, Py-3-H), 7.75 (d, J=3.3 Hz,
1H, Py-4-H), 7.58 (d, J=8.8 Hz, 2H, Py-5-H, Ar(4-O)-3-H),
7.48–7.44 (m, 1H, Ar(4-O)-5-H) ppm; 13C NMR (101 MHz,
DMSO-d6): δ = 188.79, 157.09, 156.81, 153.26, 150.54,
143.58, 140.27, 139.82, 139.56, 137.71, 134.96, 131.35,
131.15, 129.22, 128.54, 127.76, 125.55, 125.44, 125.36,
122.34 ppm; HRMS (ESI): m/z calcd. [M+H]+ 354.1237,
found 354.1232.
δ=188.47, 156.92, 156.86, 144.53, 140.26, 139.80, 139.58,
135.23, 135.16, 131.34, 131.14, 129.43, 129.41, 129.22,
128.50, 127.74, 122.42, 122.23, 122.10 ppm; HRMS (ESI):
m/z calcd. [M+H]+ 353.1285, found 353.1276.
(E)‑3‑(4‑Chlorophenyl)‑1‑[4‑(quinoxalin‑2‑yloxy)phenyl]‑
prop‑2‑en‑1‑one (3f, C23H15ClN2O2) White solid; yield:
53%; m.p.: 212–213 °C; 1H NMR (400 MHz, DMSO-d6):
δ = 8.94 (s, 1H, Ar–CH=), 8.33 (d, J = 8.7 Hz, 2H, Ar(4-
O)-2,6-H), 8.09 (dd, J=21.3, 12.0 Hz, 2H, Qu-3,5-H), 7.97
(d, J=8.5 Hz, 2H, Ar(4-Cl)-2.6-H), 7.81–7.78 (m, 1H, Qu-
6-H), 7.78–7.72 (m, 3H, Qu-8-H, Ar(4-Cl)-3.5-H), 7.56
(t, J = 8.6 Hz, 4H, Ar–C=CH, Qu-7-H, Ar(4-O)-3,5-H)
ppm; 13C NMR (101 MHz, DMSO-d6): δ=188.34, 156.99,
156.84, 143.03, 140.25, 139.81, 139.57, 135.61, 135.12,
134.15, 131.34, 131.18, 131.12, 129.44, 129.22, 128.50,
127.74, 123.14, 122.23 ppm; HRMS (ESI): m/z calcd.
[M+H]+ 387.0895, found 387.0889.
(E)‑1‑(4‑[Quinoxalin‑2‑yloxy)phenyl]‑3‑(thiophen‑2‑yl)‑
prop‑2‑en‑1‑one (3c, C21H14N2O2S) Yellow solid; yield:
1
55%; m.p.: 227–228 °C; H NMR (400 MHz, CDCl3):
δ=8.74 (s, 1H, Ar(4-O)-2-H), 8.14 (d, J=8.8 Hz, 2H, Ar(4-
O)-6-H, Qu-3-H), 8.09 (dd, J=8.1, 1.5 Hz, 1H, Th–CH=),
7.99 (d, J=15.3 Hz, 1H, Qu-5-H), 7.79 (dd, J=8.3, 1.4 Hz,
1H, Th-5-H), 7.71–7.63 (m, 2H, Th-3-H, Qu-6-H), 7.45 (d,
J=8.8 Hz, 3H, Qu-8-H, Th–C=CH, Qu-7-H), 7.40–7.35 (m,
2H, Qu-8-H, Th–C=CH, Qu-7-H), 7.10 (dd, J=5.0, 3.7 Hz,
1H, Ar(4-O)-5-H) ppm; 13C NMR (101 MHz, CDCl3):
δ=188.51, 156.51, 156.28, 140.39, 139.92, 139.79, 139.14,
137.35, 135.18, 132.27, 130.68, 130.34, 129.05, 128.94,
128.44, 127.91, 127.77, 121.38, 120.46 ppm; HRMS (ESI):
m/z calcd. [M+H]+ 359.0849, found 359.0844.
(E)‑3‑(2‑Methoxyphenyl)‑1‑[4‑(quinoxalin‑2‑yloxy)phenyl]‑
prop‑2‑en‑1‑one (3g, C24H18O3N2) Yellow solid; yield: 62%;
m.p.: 169–170 °C, 1H NMR (400 MHz, CDCl3): δ=8.75 (s,
1H, Ar–CH=), 8.19–8.14 (m, 3H, Ar(4-O)-2,6-H, Qu-3-H),
8.09 (d, J=9.6 Hz, 1H, Qu-5-H), 7.79 (d, J=9.5 Hz, 1H,
Qu-6-H), 7.71 (dd, J=7.5, 2.1 Hz, 1H, Qu-8-H), 7.68–7.66
(m, 1H, Ar(2-OCH3)-6-H), 7.64 (dd, J = 7.4, 2.1 Hz, 2H,
Qu-7-H, Ar(2-OCH3)-4-H), 7.44 (d, J = 8.8 Hz, 2H, Ar–
C=CH, Ar(2-OCH3)-3-H), 7.42–7.37 (m, 1H, Ar(4-O)-3-H),
7.01 (t, J=7.3 Hz, 1H, Ar(4-O)-5-H), 6.96 (d, J=8.5 Hz,
1H, Ar(2-OCH3)-5-H), 3.93 (s, 3H, CH3) ppm; 13C NMR
(101 MHz, CDCl3): δ = 189.87, 158.89, 156.35, 156.33,
140.64, 139.88, 139.82, 139.15, 135.59, 131.89, 130.69,
130.47, 129.39, 129.02, 127.90, 127.77, 123.88, 122.58,
121.29, 120.79, 111.28, 55.59 ppm; HRMS (ESI): m/z calcd.
[M+H]+ 383.1390, found 383.1382.
(E)‑3‑(4‑Bromophenyl)‑1‑[4‑(quinoxalin‑2‑yloxy)phenyl]‑
prop‑2‑en‑1‑one (3d, C23H15BrN2O2) White solid; yield:
1
46%; m.p.: 205–207 °C; H NMR (400 MHz, CDCl3):
δ=8.75 (s, 1H, Ar–CH=), 8.15 (d, J=8.7 Hz, 2H, Ar(4-O)-
2,6-H), 8.10 (d, J=9.5 Hz, 1H, Qu-3-H), 7.79 (dd, J=11.9,
3.7 Hz, 2H, Qu-5-H, Ar(4-Br)-3-H), 7.71 (d, J = 6.9 Hz,
1H, Ar(4-Br)-5-H), 7.65 (d, J=7.0 Hz, 1H, Qu-6-H), 7.58
(d, J=1.7 Hz, 1H, Qu-8-H), 7.54 (d, J=9.0 Hz, 3H, Ar(4-
Br)-2,6-H, Ar–C=CH), 7.50 (d, J = 6.8 Hz, 1H, Qu-7-H),
7.46 (d, J = 8.7 Hz, 2H, Ar(4-O)-3,5-H) ppm; 13C NMR
(101 MHz, CDCl3): δ = 188.90, 156.66, 156.24, 143.55,
139.90, 139.77, 139.11, 135.03, 133.76, 132.27, 130.74,
130.47, 129.86, 129.04, 127.98, 127.74, 124.94, 122.22,
121.44 ppm; HRMS (ESI): m/z calcd. [M+H]+ 431.0390,
found 431.0384.
(E)‑3‑(Pyridin‑3‑yl)‑1‑[4‑(quinoxalin‑2‑yloxy)phenyl]‑
prop‑2‑en‑1‑one (3h, C22H15N3O2) White solid; yield: 43%;
1
m.p.: 175–177 °C; H NMR (400 MHz, CDCl3): δ = 8.89
(d, J=2.0 Hz, 1H, Py-2-H), 8.75 (s, 1H, Py-6-H), 8.65 (dd,
J=4.8, 1.5 Hz, 1H, Py–CH=), 8.17 (d, J=8.8 Hz, 2H, Ar(4-
O)-2,6-H), 8.10 (dd, J=8.1, 1.5 Hz, 1H, Qu-3-H), 7.97 (d,
J=8.0 Hz, 1H, Py-4-H), 7.84 (d, J=15.8 Hz, 1H, Qu-5-H),
7.79 (dd, J=8.2, 1.4 Hz, 1H, Qu-6-H), 7.71–7.62 (m, 3H,
Qu-8-H, Py–C=CH, Qu-7-H), 7.47 (d, J =8.8 Hz, 2H, Py-
5-H, Ar(4-O)-3-H), 7.38 (dd, J=7.9, 4.8 Hz, 1H, Ar(4-O)-
5-H) ppm; 13C NMR (101 MHz, CDCl3): δ=188.46, 156.82,
156.20, 151.18, 149.99, 141.04, 139.97, 139.76, 139.10,
134.77, 134.66, 130.69, 130.68, 130.50, 129.07, 127.95,
127.74, 123.82, 123.61, 121.48 ppm; HRMS (ESI): m/z
calcd. [M+H]+ 354.1237, found 354.1228.
( E) ‑ 3 ‑ P h e ny l ‑ 1 ‑ [ 4 ‑ ( q u i n ox a l i n ‑ 2 ‑ y l ox y ) p h e ny l ] ‑
prop‑2‑en‑1‑one (3e, C23H16N2O2) White solid; yield: 50%;
m.p.: 229–230 °C; 1H NMR (400 MHz, DMSO-d6): δ=8.95
(s, 1H, Ar–CH=), 8.33 (d, J=8.7 Hz, 2H, Ar(4-O)-2,6-H),
8.12 (d, J=7.4 Hz, 1H, Qu-3-H), 8.03 (d, J=15.6 Hz, 1H,
Qu-5-H), 7.93 (dd, J= 6.5, 3.0 Hz, 2H, Qu-6,8-H), 7.83–
7.72 (m, 4H, Ar–C=CH, Qu-7-H, Ph-2-H, Ph-6-H), 7.57
(d, J = 8.7 Hz, 2H, Ph-3,5-H), 7.51–7.46 (m, 3H, Ph-4-H,
Ar(4-O)-3,5-H) ppm; 13C NMR (101 MHz, DMSO-d6):
1 3

Antimicrobial evaluation and action mechanism of chalcone derivatives containing quinoxaline…
(E)‑3‑(Furan‑2‑yl)‑1‑[4‑(quinoxalin‑2‑yloxy)phenyl]‑
prop‑2‑en‑1‑one (3i, C21H14N2O3) Gray solid; yield: 72%;
m.p.: 205–206 °C; 1H NMR (400 MHz, CDCl3): δ=8.74 (s,
1H, Fu-5-H), 8.18–8.14 (m, 2H, Ar(4-O)-2,6-H), 8.10 (dd,
J=8.1, 1.5 Hz, 1H, Qu-3-H), 7.80–7.77 (m, 1H, Fu–CH=),
7.70 (td, J =6.7, 3.4 Hz, 1H, Qu-5-H), 7.68–7.65 (m, 1H,
Qu-6-H), 7.65–7.62 (m, 1H, Qu-8-H), 7.55 (d, J =1.4 Hz,
1H, Fu-3-H), 7.50 (d, J=15.3 Hz, 1H, Fu–C=CH), 7.46–
7.42 (m, 2H, Qu-7-H, Ar(4-O)-3-H), 6.75 (d, J=3.4 Hz, 1H,
Ar(4-O)-5-H), 6.53 (dd, J=3.4, 1.8 Hz, 1H, Fu-4-H) ppm;
13C NMR (101 MHz, CDCl3): δ=188.50, 156.51, 156.29,
151.68, 145.01, 139.92, 139.80, 139.14, 135.23, 130.79,
130.67, 130.36, 129.04, 127.90, 127.77, 121.35, 119.02,
116.43, 112.75 ppm; HRMS (ESI): m/z calcd. [M + H]+
343.1077, found 343.1073.
Ar(4-OCH3)-2-H), 7.72–7.64 (m, 2H, Ar(4-OCH3)-6-H,
Qu-6-H), 7.62 (d, J = 8.8 Hz, 2H, Qu-8-H, Ar–C=CH),
7.45 (dd, J=12.2, 7.7 Hz, 3H, Qu-7-H, Ar(4-OCH3)-3,5-H),
6.95 (d, J=8.8 Hz, 2H, Ar(4-O)-3,5-H), 3.86 (s, 3H, CH3)
ppm; 13C NMR (101 MHz, DMSO-d6): δ=184.48, 157.02,
151.60, 151.57, 140.10, 135.15, 135.06, 134.39, 130.82,
125.91, 125.58, 125.56, 124.28, 123.13, 123.01, 122.85,
116.56, 114.70, 109.72, 50.70 ppm; HRMS (ESI): m/z calcd.
[M+H]+ 383.1390, found 383.1375.
(E)‑3‑(2‑Bromophenyl)‑1‑[4‑[(6‑chloroquinoxalin‑2‑yl)oxy]‑
phenyl]prop‑2‑en‑1‑one (3m, C23H14BrClN2O2) White solid;
yield: 50%; m.p.: 192–194 °C; 1H NMR (400 MHz, CDCl3):
δ = 8.76–8.72 (m, 1H, Ar–CH=), 8.16 (ddd, J = 8.0, 7.2,
5.8 Hz, 3H, Ar(4-O)-2,4-H, Qu-3-H), 8.08 (d, J = 2.3 Hz,
1H, Ar(2-Br)-3-H), 7.76–7.70 (m, 2H, Ar–C=CH, Ar(2-
Br)-5-H), 7.67–7.61 (m, 2H, Ar(2-Br)-4,6-H), 7.49–7.43
(m, 3H, Qu-5,7,8-H), 7.37 (t, J=7.6 Hz, 1H, Ar(4-O)-3-H),
7.31–7.26 (m, 1H, Ar(4-O)-3-H) ppm; 13C NMR (101 MHz,
CDCl3): δ=189.04, 156.40, 156.38, 143.39, 140.15, 140.07,
138.34, 135.15, 135.09, 133.63, 133.51, 131.49, 131.40,
130.57, 128.87, 128.10, 127.93, 127.74, 125.92, 124.82,
121.47 ppm; HRMS (ESI): m/z calcd. [M+H]+ 464.9999,
found 464.9985.
(E)‑3‑(3‑Bromophenyl)‑1‑[4‑(quinoxalin‑2‑yloxy)phenyl]‑
prop‑2‑en‑1‑one (3j, C23H15BrN2O2) White solid; yield: 75%;
m.p.: 190–192 °C; 1H NMR (400 MHz, CDCl3): δ=8.76 (s,
1H, Ar–CH=), 8.09 (dd, J=8.0, 1.6 Hz, 1H, Ar(4-O)-2-H),
7.96 (dd, J=4.9, 2.9 Hz, 2H, Ar(4-O)-6-H, Qu-3-H), 7.81–
7.77 (m, 2H, Qu-5-H, Qu-6-H), 7.75 (d, J= 3.3 Hz, 1H,
Qu-8-H), 7.71–7.67 (m, 1H, Ar–C=CH), 7.67–7.64 (m, 1H,
Qu-7-H), 7.62 (d, J=8.1 Hz, 1H, Ar(3-Br)-6-H), 7.57–7.54
(m, 2H, Ar(3-Br)-2,4-H), 7.51 (d, J = 15.7 Hz, 2H, Ar(3-
Br)-5-H, Ar(4-O)-3-H), 7.29 (t, J = 7.9 Hz, 1H, Ar(4-O)-
5-H) ppm; 13C NMR (101 MHz, CDCl3): δ=189.17, 156.62,
153.03, 143.55, 139.81, 139.54, 139.11, 136.84, 133.45,
130.90, 130.63, 130.53, 130.04, 129.02, 127.73, 127.37,
126.31, 125.56, 123.13, 122.96, 121.66 ppm; HRMS (ESI):
m/z calcd. [M+H]+ 431.0390, found 431.0372.
( E ) ‑ 1 ‑ [ 4 ‑ [ ( 6 ‑ C h l o r o q u i n o x a l i n ‑ 2 ‑ y l ) o x y ] ‑
p h e ny l ] ‑ 3 ‑ ( py r i d i n ‑ 2 ‑ y l ) p ro p ‑ 2 ‑ e n ‑ 1 ‑ o n e ( 3 n ,
C22H14ClN2O3) Gray solid; yield: 58%; m.p.: 204–205 °C;
1H NMR (400 MHz, CDCl3): δ=8.75 (s, 1H, Py-6-H), 8.70
(d, J = 3.9 Hz, 1H, Py–C=CH), 8.24 (d, J = 8.8 Hz, 2H,
Py–CH=, Ar(4-O)-2-H), 8.17 (d, J=15.2 Hz, 1H, Ar(4-O)-
6-H), 8.08 (d, J=2.3 Hz, 1H, Qu-3-H), 7.82 (d, J=15.2 Hz,
1H, Py-3-H), 7.77 (td, J=7.7, 1.8 Hz, 1H, Py-4-H), 7.72 (d,
J=8.9 Hz, 1H, Py-5-H), 7.64 (dd, J=8.9, 2.3 Hz, 1H, Qu-
5-H), 7.51 (d, J=7.8 Hz, 1H, Qu-7-H), 7.44 (d, J=8.8 Hz,
2H, Qu-8-H, Ar(4-O)-3-H), 7.32 (dd, J = 7.1, 4.2 Hz,
1H, Ar(4-O)-5-H) ppm; 13C NMR (101 MHz, CDCl3):
δ=189.12, 156.44, 156.39, 153.13, 150.23, 142.95, 140.11,
140.09, 138.34, 136.97, 135.12, 133.47, 131.50, 130.71,
128.90, 128.08, 125.59, 125.26, 124.56, 121.47 ppm; HRMS
(ESI): m/z calcd. [M+H]+ 388.0847, found 388.0837.
(E)‑3‑(2‑Bromophenyl)‑1‑[4‑(quinoxalin‑2‑yloxy)phenyl]‑
prop‑2‑en‑1‑one (3k, C23H15BrN2O3) Yellow solid; yield:
1
66%; m.p.: 214–216 °C; H NMR (400 MHz, CDCl3):
δ=8.74 (s, 1H, Ar–CH=), 8.16 (dd, J =12.2, 8.5 Hz, 3H,
Ar(4-O)-2,6-H, Qu-3-H), 8.08 (d, J=2.3 Hz, 1H, Qu-5-H),
7.73 (dd, J=13.1, 8.3 Hz, 2H, Qu-6,8-H), 7.64 (dd, J=12.2,
5.6 Hz, 2H, Qu-7-H, Ar(2-Br)-3-H), 7.45 (dd, J = 12.2,
7.3 Hz, 3H, Ar–C=CH, Ar(2-Br)-4,5-H), 7.37 (t, J=7.6 Hz,
1H, Ar(2-Br)-6-H), 7.26 (d, J=15.3 Hz, 2H, Ar(4-O)-3,5-H)
ppm; 13C NMR (101 MHz, CDCl3): δ = 189.13, 156.67,
156.25, 143.38, 139.91, 139.79, 139.11, 135.06, 134.93,
133.63, 131.43, 130.73, 130.61, 129.03, 127.97, 127.94,
127.76, 125.96, 124.79, 121.42, 115.60 ppm; HRMS (ESI):
m/z calcd. [M+H]+ 431.0390, found 431.0376.
( E ) ‑ 1 ‑ [ 4 ‑ [ ( 6 ‑ C h l o r o q u i n o x a l i n ‑ 2 ‑ y l ) o x y ] ‑
phenyl]‑3‑(thiophen‑2‑yl)prop ‑2‑ en‑1‑ one (3o,
C21H13ClN2O2S) Gray solid; yield: 49%; m.p.: 170–171 °C; 1H
NMR (400 MHz, CDCl3): δ=8.73 (s, 1H, Ar(4-O)-2,6-H),
8.13 (d, J=8.6 Hz, 2H, Qu-3-H), 8.07 (d, J=2.1 Hz, 1H, Th–
CH=), 7.98 (d, J=15.3 Hz, 1H, Th-5-H), 7.70 (d, J=8.9 Hz,
1H, Th-3-H), 7.62 (dd, J=8.9, 2.2 Hz, 1H,Th–C=CH), 7.43
(d, J=8.6 Hz, 3H, Th-4-H, Qu-5,7-H), 7.36 (d, J=15.0 Hz,
2H, Qu-8-H, Ar(4-O)-3-H), 7.12–7.08 (m, 1H, Ar(4-O)-
5-H) ppm; 13C NMR (101 MHz, CDCl3): δ=188.42, 156.43,
156.22, 140.37, 140.12, 140.08, 138.34, 137.37, 135.40,
(E)‑3‑(4‑Methoxyphenyl)‑1‑[4‑(quinoxalin‑2‑yloxy)phe‑
nyl]prop‑2‑en‑1‑one (3l, C214H18N2O3) Yellow solid; yield:
48%; m.p.: 209–210 °C; H NMR (400 MHz, DMSO-
d6): δ = 8.74 (s, 1H, Ar–CH=), 8.14 (d, J = 8.8 Hz, 2H,
Ar(4-O)-2,6-H), 8.09 (dd, J = 8.1, 1.5 Hz, 1H, Qu-3-H),
7.83 (d, J = 15.6 Hz, 1H, Qu-5-H), 7.80–7.77 (m, 1H,
1 3

R. Xia et al.
133.45, 132.20, 131.45, 130.31, 128.93, 128.86, 128.42,
128.09, 121.43, 120.45 ppm; HRMS (ESI): m/z calcd. [M+H]+
393.0459, found 393.0448.
1H, Ar(2-OCH3)-6-H), 7.71 (dd, J=7.5, 2.1 Hz, 1H, Ar(4-
O)-4-H), 7.68–7.60 (m, 3H, Ar–C=CH, Qu-5,7-H), 7.44
(d, J = 8.8 Hz, 3H, Qu-8-H, Ar(2-OCH3)-3-H, Ar(4-O)-
3-H), 7.03–6.94 (m, 2H, Ar(4-O)-5-H, Ar(2-OCH3)-5-H),
3.93 (s, 3H, CH3) ppm; 13C NMR (101 MHz, CDCl3):
δ=189.72, 158.90, 156.48, 156.05, 140.64, 140.10, 138.37,
135.82, 133.42, 131.86, 131.44, 130.43, 130.26, 129.36,
128.88, 128.08, 123.92, 122.58, 121.33, 120.80, 111.31,
55.59 ppm; HRMS (ESI): m/z calcd. [M + H]+ 417.1001,
found 417.0993.
(E)‑3‑(4‑Bromophenyl)‑1‑[4‑[(6‑chloroquinoxalin‑2‑yl)oxy]‑
phenyl]prop‑2‑en‑1‑one (3p, C23H14BrClN2O2) Gray solid;
yield: 42%; m.p.: 179–181 °C; 1H NMR (400 MHz, CDCl3):
δ=8.75 (s, 1H, Ar–CH=), 8.16 (d, J=8.8 Hz, 2H, Ar(4-O)-
2,6-H), 8.10–8.07 (m, 1H, Qu-3-H), 7.86 (d, J= 15.7 Hz,
1H, Ar(4-Br)-3-H), 7.71 (d, J=8.9 Hz, 1H, Ar(4-Br)-5-H),
7.69–7.66 (m, 2H, Ar(4-Br)-2,6-H), 7.65–7.62 (m, 1H, Ar–
C=CH), 7.57 (d, J = 15.7 Hz, 1H, Qu-5-H), 7.45 (s, 2H,
Qu-7,8-H), 7.43 (s, 2H, Ar(4-O)-3,5-H) ppm; 13C NMR
(101 MHz, CDCl3): δ = 189.14, 156.43, 156.22, 145.08,
140.11, 140.10, 138.34, 135.47, 134.84, 133.47, 131.49,
130.69, 130.45, 129.03, 128.88, 128.52, 128.09, 121.72,
121.47 ppm; HRMS (ESI): m/z calcd. [M+H]+ 464.9999,
found 464.9988.
( E ) ‑ 1 ‑ [ 4 ‑ [ ( 6 ‑ C h l o r o q u i n o x a l i n ‑ 2 ‑ y l ) o x y ] ‑
p h e ny l ] ‑ 3 ‑ ( p y r i d i n ‑ 3 ‑ y l ) p r o p ‑ 2 ‑ e n ‑ 1 ‑ o n e ( 3 t ,
C22H14ClN3O2) Gray solid; yield: 50%; m.p.: 198–200 °C;
1H NMR (400 MHz, CDCl3): δ = 8.89 (d, J = 1.7 Hz, 1H,
Py-2-H), 8.76 (s, 1H, Py-6-H), 8.65 (d, J=6.1 Hz, 1H, Py–
CH=), 8.17 (d, J = 8.8 Hz, 2H, Ar(4-O)-2,6-H), 8.09 (d,
J=2.3 Hz, 1H, Qu-3-H), 7.98 (d, J=8.0 Hz, 1H, Py-4-H),
7.85 (d, J = 15.8 Hz, 1H, Py–C=CH), 7.71 (d, J= 8.9 Hz,
1H, Py-5-H), 7.67–7.62 (m, 2H, Qu-5,7-H), 7.46 (d,
J = 8.8 Hz, 2H, Qu-8-H, Ar(4-O)-3-H), 7.39 (dd, J= 8.0,
4.8 Hz, 1H, Ar(4-O)-5-H) ppm; 13C NMR (101 MHz,
CDCl3): δ = 188.44, 156.48, 156.35, 151.25, 150.02,
141.17, 140.13, 140.08, 138.29, 134.97, 134.68, 133.53,
131.53, 130.62, 130.52, 128.85, 128.11, 123.85, 123.48,
121.59 ppm; HRMS (ESI): m/z calcd. [M+H]+ 388.0847,
found 388.0837.
(E)‑1‑[4‑[(6‑Chloroquinoxalin‑2‑yl)oxy]phenyl]‑3‑phenyl‑
prop‑2‑en‑1‑one (3q, C23H15ClN2O2) Gray solid; yield: 69%;
m.p.: 210–211 °C; 1H NMR (400 MHz, CDCl3): δ=8.75 (s,
1H, Ar–CH=), 8.15 (d, J=8.8 Hz, 2H, Ar(4-O)-2,6-H), 8.09
(d, J=2.3 Hz, 1H, Qu-3-H), 7.79 (d, J=15.7 Hz, 1H, Ar–
C=CH), 7.71 (d, J=8.9 Hz, 1H, Ph-2-H), 7.64 (dd, J=8.9,
2.3 Hz, 1H, Ph-6-H), 7.57 (d, J=6.9 Hz, 3H, Ph-3,4,5-H),
7.53 (d, J = 8.7 Hz, 3H, Qu-5,7,8-H), 7.44 (d, J= 8.8 Hz,
2H, Ar(4-O)-3,5-H) ppm; 13C NMR (101 MHz, CDCl3):
δ=188.82, 156.39, 156.34, 143.60, 140.13, 140.09, 138.32,
135.26, 133.75, 133.51, 132.29, 131.52, 130.45, 129.85,
128.86, 128.11, 124.96, 122.19, 121.52 ppm; HRMS (ESI):
m/z calcd. [M+H]+ 387.0895, found 387.0867.
(E)‑1‑[4‑[(6‑Chloroquinoxalin‑2‑yl)oxy]phenyl]‑3‑(furan‑2‑yl)‑
prop‑2‑en‑1‑one (3u, C21H13ClN2O3) Gray solid; yield: 52%;
1
m.p.: 158–159 °C; H NMR (400 MHz, CDCl3): δ = 8.75
(s, 1H, Fu-5-H), 8.17 (d, J= 8.8 Hz, 2H, Ar(4-O)-2,6-H),
8.08 (d, J=2.3 Hz, 1H, Qu-3-H), 7.71 (d, J=8.9 Hz, 1H,
Fu–CH=), 7.67–7.62 (m, 2H, Fu-3-H, Fu–C=CH), 7.53 (dd,
J = 20.1, 8.3 Hz, 2H, Qu-5,7-H), 7.43 (d, J= 8.8 Hz, 2H,
Qu-8-H, Ar(4-O)-3-H), 6.76 (d, J = 3.4 Hz, 1H, Ar(4-O)-
5-H), 6.54 (dd, J=3.4, 1.8 Hz, 1H, Fu-4-H) ppm; 13C NMR
(101 MHz, CDCl3): δ = 188.41, 156.44, 156.21, 151.68,
145.04, 140.10, 138.36, 135.45, 133.44, 131.46, 130.84,
130.35, 128.87, 128.09, 121.42, 118.99, 116.45, 112.76,
100.00 ppm; HRMS (ESI): m/z calcd. [M+H]+ 377.0688,
found 377.0678.
(E)‑3‑(4‑Chlorophenyl)‑1‑[4‑[(6‑chloroquinoxalin‑2‑yl)‑
oxy]phenyl]prop‑2‑en‑1‑one (3r, C23H14Cl2N2O2) Yellow
1
solid; yield: 73%; m.p.: 216-218 °C; H NMR (400 MHz,
CDCl3): δ=8.75 (s, 1H, Ar–CH=), 8.15 (d, J=8.7 Hz, 2H,
Ar(4-O)-2,6-H), 8.09 (d, J = 2.2 Hz, 1H, Qu-3-H), 7.81
(d, J = 15.7 Hz, 1H, Ar(4-Cl)-2-H), 7.71 (d, J = 8.9 Hz,
1H, Ar(4-Cl)-2-H), 7.65–7.62 (m, 1H, Ar(4-Cl)-3-H),
7.60 (d, J = 8.5 Hz, 2H, Ar(4-Cl)-5-H, Ar–C=CH), 7.55
(d, J = 15.7 Hz, 1H, Qu-5-H), 7.43 (dd, J = 12.7, 8.6 Hz,
4H, Qu-7,8-H, Ar(4-O)-3,5-H) ppm; 13C NMR (101 MHz,
CDCl3): δ=188.83, 156.39, 156.33, 143.54, 140.13, 140.09,
138.32, 136.59, 135.28, 133.51, 133.33, 131.52, 130.45,
129.65, 129.32, 128.86, 128.11, 122.10, 121.51 ppm; HRMS
(ESI): m/z calcd. [M+H]+ 421.0505, found 421.0489.
(E)‑3‑(3‑Bromophenyl)‑1‑[4‑[(6‑chloroquinoxalin‑2‑yl)oxy]‑
phenyl]prop‑2‑en‑1‑one (3v, C23H14BrClN2O2) Gray solid;
yield: 47%; m.p.: 184–186 °C; 1H NMR (400 MHz, CDCl3):
δ=8.75 (s, 1H, Ar–CH=), 8.16 (d, J=8.7 Hz, 2H, Ar(4-O)-
2,6-H), 8.08 (d, J=2.2 Hz, 1H, Qu-3-H), 7.83–7.73 (m, 2H,
Ar–C=CH, Ar(3-Br)-6-H), 7.71 (d, J = 8.9 Hz, 1H, Ar(3-
Br)-2-H), 7.63 (dd, J=8.9, 2.3 Hz, 1H, Ar(3-Br)-4-H), 7.59–
7.52 (m, 3H, Ar(3-Br)-5-H, Qu-5,7-H), 7.45 (d, J=8.7 Hz,
2H, Qu-8-H, Ar(4-O)-3-H), 7.31 (t, J=7.9 Hz, 1H, Ar(4-O)-
5-H) ppm; 13C NMR (101 MHz, CDCl3): δ=188.63, 156.40,
(E)‑1‑[4‑[(6‑Chloroquinoxalin‑2‑yl)oxy]phenyl]‑3‑(2‑meth‑
oxyphenyl)prop‑2‑en‑1‑one (3s, C24H17ClN2O3) Gray solid;
yield: 67%; m.p.: 214–216 °C; 1H NMR (400 MHz, CDCl3):
δ=8.75 (s, 1H, Ar–CH=), 8.15 (d, J=8.7 Hz, 2H, Ar(4-O)-
2,6-H), 8.09 (d, J=9.6 Hz, 1H, Qu-3-H), 7.79 (d, J=6.9 Hz,
1 3

Antimicrobial evaluation and action mechanism of chalcone derivatives containing quinoxaline…
[
156.37, 143.16, 140.12, 140.08, 138.31, 136.95, 135.13,
Inhibition rate (%) = (Corrected bacterial liquid OD value
133.51, 133.37, 131.51, 130.84, 130.53, 130.49, 128.86,
128.10, 127.35, 123.15, 122.88, 121.54 ppm; HRMS (ESI):
m/z calcd. [M+H]+ 464.9999, found 464.9989.
−Corrected OD value before adding bacteria)∕
]
Corrected bacterial liquid OD value × 100.
To obtain the EC50 values against Xac, Xoo, and Rs, some
compounds and the positive control were prepared at 100 μg/
cm3, 50 μg/cm3, 25 μg/cm3, 12.5 μg/cm3, and 6.25 μg/cm3
and their inhibition rates were calculated. Each experiment
was computed at least three times, and EC50 values were
determined by using SPSS17.0.
(E)‑1‑[4‑[(6‑Chloroquinoxalin‑2‑yl)oxy]phenyl]‑3‑(4‑meth‑
oxyphenyl)prop‑2‑en‑1‑one (3w, C24H17ClN2O3) Gray solid;
yield: 58%; m.p.: 194–195 °C; 1H NMR (400 MHz, CDCl3):
δ=8.74 (s, 1H, Ar–CH=), 8.11 (dd, J =24.1, 5.4 Hz, 3H,
Ar(4-O)-2,6-H, Qu-3-H), 7.84 (d, J= 15.6 Hz, 1H, Ar(4-
OCH3)-2-H), 7.71 (d, J = 8.9 Hz, 1H, Ar(4-OCH3)-6-H),
7.62 (d, J=8.7 Hz, 3H, Ar–C=CH, Qu-5,7-H), 7.48–7.41
(m, 3H, Qu-8-H, Ar(4-OCH3)-3,5-H)), 6.95 (d, J=8.7 Hz,
2H, Ar(4-O)-3,5-H)), 3.87 (s, 3H, CH3) ppm; 13C NMR
(101 MHz, CDCl3): δ = 189.16, 161.81, 156.47, 156.05,
144.91, 140.11, 140.10, 138.37, 135.80, 133.44, 131.46,
130.32, 130.30, 128.88, 128.08, 127.60, 121.38, 119.43,
114.49, 55.45 ppm; HRMS (ESI): m/z calcd. [M + H]+
417.1001, found 417.0989.
Scanning electron microscope sample
preparation
Xanthomonas axonopodis pv. Citri (Xac) cells (1.5 cm3)
incubated at the logarithmic phase were centrifuged and
washed three times with PBS (pH=7.1), and re-suspended
in 1.5 cm3 of PBS (pH = 7.1). Subsequently, bacteria Xac
was incubated with compound 3b at concentrations of 50 μg/
cm3 and 100 μg/cm3, and an equivalent volume of DMSO
(solvent control), were cultured at 28 °C in a constant tem-
perature shaking table 180 rpm/min for 8–10 h. After incu-
bation, these samples were washed three times with PBS
(pH=7.1) and the bacterial cells were fxed with 2.5% glu-
taraldehyde at 4 °C for 8 h and then dehydrated with difer-
ent concentrations of ethanol series of about 10 min/time.
Following dehydration, the samples were freeze-dried and
coated with gold and visualized using Nova NanoSEM 450.
(E)‑1‑[4‑[(6‑Chloroquinoxalin‑2‑yl)oxy]phenyl]‑3‑(m‑tolyl)‑
prop‑2‑en‑1‑one (3x, C24H15ClN2O2) Gray solid; yield: 51%;
1
m.p.: 147–149 °C; H NMR (400 MHz, CDCl3): δ = 8.74
(s, 1H, Ar–CH=), 8.16 (d, J=8.8 Hz, 2H, Ar(4-O)-2,6-H),
8.08 (d, J = 2.3 Hz, 1H, Qu-3-H), 7.84 (d, J = 15.7 Hz,
1H, Ar–C=CH), 7.71 (d, J = 8.9 Hz, 1H, Ar(4-CH3)-
6-H), 7.63 (dd, J=8.9, 2.3 Hz, 1H, Ar(4-CH3)-2-H), 7.55
(d, J = 15.7 Hz, 1H, Ar(4-CH3)-5-H), 7.45 (dd, J = 13.8,
7.9 Hz, 4H, Qu-5,7,8-H, Ar(4-O)-3-H), 7.32 (t, J=7.9 Hz,
1H, Ar(4-O)-5-H), 7.25 (d, J = 7.8 Hz, 1H, Ar(4-CH3)-
4-H), 2.41 (s, 3H, CH3) ppm; 13C NMR (101 MHz, CDCl3):
δ=189.17, 156.44, 156.20, 145.27, 140.12, 140.09, 138.70,
138.35, 135.55, 134.81, 133.47, 131.53, 131.48, 130.42,
129.09, 128.91, 128.87, 128.09, 125.77, 121.55, 121.43,
21.36 ppm; HRMS (ESI): m/z calcd. [M + H]+ 401.1051,
found 401.1036.
Acknowledgements We gratefully acknowledge the National Key
Research and Development Program of China (No. 2017YFD0200506)
and the Science and Technology Project of Guizhou Province (Nos.
20185781, 20171028).
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