Synthesis and Pesticidal Activities of New Quinoxalines

Article abstract of DOI:10.1021/acs.jafc.0c01042

Natural products are a source of many novel compounds with biological activity for the discovery of new pesticides and pharmaceuticals. Quinoxaline is a fused N-heterocycle in many natural products and synthetic compounds, and seven novel quinoxaline derivatives were designed and synthesized via three steps. Pesticidal activities of title quinoxaline derivatives were bioassayed. Most of these compounds had herbicidal, fungicidal, and insecticidal activities. The compounds 2-(6-methoxy-2-oxo-3-phenylquinoxalin-1(2H)-yl)acetonitrile (3f) and 1-allyl-6-methoxy-3-phenylquinoxalin-2(1H)-one (3g) were the most active herbicides and fungicides. Mode-of-action studies indicated that 3f is a protoprophyrinogen oxidase-inhibiting herbicide. Compound 3f also possessed broad-spectrum fungicidal activity against the plant pathogen Colletotrichum species. Some of these compounds also had insecticidal activity. Molecular docking and DFT analysis can potentially be used to design more active compounds.

Full text of DOI:10.1021/acs.jafc.0c01042

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Agricultural and Environmental Chemistry
Synthesis and Pesticidal Activities of New Quinoxalines
Xing-Hai Liu, Wei Yu, Li-Jing Min, David E. Wedge, Cheng-Xia Tan, Jian-Quan Weng, Hong-
Ke Wu, Dr. Charles L. Cantrell, Joanna Bajsa-Hischel, Xue-Wen Hua, and Stephen O. Duke
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c01042 • Publication Date (Web): 12 Jun 2020
Downloaded from pubs.acs.org on June 13, 2020
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Synthesis and Pesticidal Activities of New Quinoxalines
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Xing-Hai Liu1*, Wei Yu1, Li-Jing Min2, David E. Wedge3, Cheng-Xia Tan1, Jian-Quan Weng1,
Hong-Ke Wu1, Charles L. Cantrell3, Joanna Bajsa-Hirschel3, Xue-Wen Hua5, and Stephen O.
Duke4*
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1.College of Chemical Engineering, Research Institue of Pesticide, Zhejiang University of
Technology, Hangzhou, 310014, China
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2. Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province, College of Life
Science, Huzhou University, Huzhou Cent. Hosp., Huzhou, 313000, Zhejiang, China
3. Natural Products Utilization Research Unit, USDA-ARS University, MS 38677, USA
4. National Center for Natural Product Research(NCNPR), School of Pharmacy, University of
Mississippi, University, MS, 38677, USA
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5. College of Agriculture, Liaocheng University, 252000, Liaocheng, Shandong, China
Abstract: Natural products are a source of many novel compounds with biological activity for
discovery of new pesticides and pharmaceuticals. Quinoxaline is a fused N-heterocycle in many
natural products and synthetic compounds, seven novel quinoxaline derivatives were designed and
synthesized via three steps. Pesticidal activities of title quinoxaline derivatives were bioassayed.
Most of these compounds had herbicidal, fungicidal, and insecticidal activities. The compounds
2-(6-methoxy-2-oxo-3-phenylquinoxalin-1(2H)-yl)acetonitrile(3f)
and
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1-allyl-6-methoxy-3-phenylquinoxalin-2(1H)-one (3g) were the most active herbicides and
fungicides. Mode of action studies indicated that 3f is a protoprophyrinogen oxidase-inhibiting
herbicide. Compound 3f also possessed broad-spectrum fungicidal activity against the plant
pathogen Colletotrichum species. Some of these compounds also had insecticidal activity.
Molecular docking and DFT analysis can potentially be used to design more active compounds.
Keywords:Quinoxaline; herbicidal; fungicidal; insecticidal; DFT; molecular docking
INTRODUCTION
Pesticides are important tools to protect crops, increase yields and quality in agriculture. New
pesticide discovery can be from natural products. Benzopyrazine(quinoxaline) is a fused
N-heterocycle found in many natural compounds (Figure 1), such as the cyclic peptide triostin A,1
izumiphenazines A–C,2 hunanamycin A3,4, and vitamin B2.5,6 Quinomycins are a type of cyclic
oligopeptide antibiotic containing the quinoxaline group in the macrocycles. Izumiphenazines A-C
with the quinoxaline group were isolated from Streptomyces species. The natural product
hunanamycin A with a quinoxaline moiety, was isolated from Bacillus hunanensis. Currently,
synthetic quinoxaline derivatives possess a diversity of activities, such as antibacterial,7
anti-ebola,8 antileishmanial,9 antitubercular,10 antifungal,11 antimalarial,12 anticancer,13 and
antineoplastic activities14. The quinoxaline motif also exists infunctional materials, including
organic dyes15 and DNA cleavage agents.16-19
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OH
OH
O
N
O
HO
N
N
NH
HO
OH
O
N
N
N
O
HO
N
O
HO
OH
N
OH
OH
Vitamin B2
Izumiphenazine C
Hunanamycin A
N
H
N
O
NH
N
N
O
N
O
O
O
S
O
O
S
O
N
O
O
O
O
N
NH
HN
N
N
triostin A
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Figure 1 Some natural products with the quinoxaline moiety
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However, the quinoxaline(or pyrazine) moiety has had little application in industrial and
agricultural fields. Some patents20-22 reported the quinoxaline derivatives were used as herbicides
or herbicide safener. Thus far, only the herbicides diquat, quizalofop-p-methyl, and
quizalofop-p-tefuryl, the fungicide pyraziflumid, and the insecticide chinomethionat(Figure 2) are
commercial pesticides with the quinoxaline or pyrazine moiety.
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O
O
Cl
N
N
O
Cl
N
N
O
O
O
O
O
O
quizalofop-p-methyl
quizalofop-p-tefuryl
O
N
N
N
H
CF3
N
N
S
O
N
N
Br-
Br-
S
F
F
diquat
pyraziflumid
chinomethionat
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Figure 2 Representative pesticides with the pyrazine moiety
In our previous work, many nitrogen-containing heterocycles with pesticidal activity were
designed and synthesized.23-34 For the above reason, to discover active lead compounds, we
designed some quinoxaline compounds with a flexible side chain. Some of the synthetic
quinoxaline derivatives exhibited good herbicidal, fungicidal and mosquitocidal activity.
MATERIALS AND METHODS
Instruments.
Melting points(m.p.) were tested on an X-4 digital melting point instument(Gongyi, China) and
the temperature was uncorrected. FT-IR was measured on a Nocolet 670 fourier transform infrared
1
spectrometer(Thermofisher, USA). H NMR spectra were tested on an AP-500 nuclearꢀmagneticꢀ
resonanceꢀspectrometer(Bruker, Germany). ESI-HR-MS were tested on time-of-flight mass
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spectrometer (Agilent 6520, MA, USA or Jeol Accu TOF 4G, Tokyo, Japan). All the reagents
were freshly prepared or purchased from chemical company.
Synthesis
4-methoxybenzene-1,2-diamine(1)
To a mixture of 4-methoxy-2-nitroaniline (0.1 mol), NH2NH2·H2O (85%, 75 mL) in MeOH (40
mL), Raney Ni (0.4 mol) was added slowly, then refluxed. The residue Raney Ni was filtrated and
the solvent MeOH was evaporated to afford light brown crystal 1.
7-Methoxy-3-phenylquinoxalin-2(1H)-one(2)
To a solution of 4-methoxybenzene-1,2-diamine (0.1 mol) in EtOH (100 mL), methyl
2-oxo-2-phenylacetate (0.1 mol) was added and then further stirred for another 1 h. A yellow solid
was produced in good yield. 7-Methoxy-3-phenylquinoxalin-2(1H)-one, yield 95.6%, m.p.
180-183℃, 1H NMR(500MHz, CDCl3) δ: 8.43(m, 2H, Ph-H), 7.54(m, 3H, Ph-H), 7.41(d,
J=2.7Hz, 1H, Ph-H), 7.30 (s, 1H, Ph-H), 7.20 (d, J=2.7Hz, 1H, Ph-H), 3.92(s, 3H, OCH3).
General procedure for compounds 3a to 3g
7-Methoxy-3-phenylquinoxalin-2(1H)-one (1.26 g, 0.005 mol) and dried potassium carbonate (0.2
g, 0.0056 mol) was dissolved in DMF (15 mL) and stirred for 10 min, then different RCl (0.005
mol) was dropwised, and magnetic stirred for overnight. Then the reaction solution was poured
into ice H2O, a lot of solid was given, then filtered, recrystallized and dried to give 3a~3g in good
yields.
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O
O
NH2
NO2
NH2
N
Rany Ni
NH2NH2H2O
O
O
O
NH2
EtOH
O
N
H
O
1
2
N
RCl
K2CO3, DMF
O
N
R
O
3a~3g
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Scheme 1. The synthetic pathway of quinoxaline derivatives
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4-((7-methoxy-2-oxo-3-phenylquinoxalin-1(2H)-yl)methyl)benzonitrile 3a
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Yellow solid, m.p. 148-151℃, yield 62.3%, FT-IR (KBr, νmax, cm−1): 2922.24, 2225.96(C≡N),
1649.83(C=O), 1496.97, 1444.59, 1382.91, 1286.68, 1269.43, 1038.01, 809.97, 692.59, 545.98;
1H NMR (500 MHz, CDCl3) δ: 8.37-8.33 (m, 2H, Ph-H), 7.66-7.63 (m, 2H, Ph-H), 7.52-7.48 (m,
4H, Ph-H), 7.41 (d, J = 8.5 Hz, 2H, Ph-H), 7.12-7.08 (m, 1H, Ph-H), 6.95 (dd, J = 8.9, 2.5 Hz, 1H,
Ph-H), 5.60 (s, 2H, -CH2), 3.89 (s, 3H, -OMe); ESI-HRMS calcd for C23H17N3O2 368.1394, found
368.1399[M+H]+.
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96
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1-(4-(tert-butyl)benzyl)-7-methoxy-3-phenylquinoxalin-2(1H)-one 3b
99
Yellow solid, m.p. 86-89℃, yield 68.2%, FT-IR (KBr, νmax, cm−1): 2959.18, 1643.96(C=O),
1611.39, 1580.18, 1499.00, 1441.01, 1383.51, 1269.28, 1215.24, 1165.16, 1025.94, 812.85,
693.96, 600.44, 530.20; 1H NMR (500 MHz, CDCl3) δ: 8.38-8.34 (m, 2H, Ph-H), 7.49-7.45 (m,
3H, Ph-H), 7.42 (d, J = 2.9 Hz, 1H, Ph-H), 7.33-7.30 (m, 2H, Ph-H), 7.26-7.21 (m, 3H, Ph-H),
7.10 (dd, J = 9.2, 2.9 Hz, 1H, Ph-H), 5.52 (s, 2H, -CH2), 3.88 (s, 3H, -OMe), 1.27 (s, 9H, -Bu);
ESI-HRMS calcd for C26H26N2O2 399.2067, found 399.2066[M+H]+.
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1-(4-bromobenzyl)-7-methoxy-3-phenylquinoxalin-2(1H)-one 3c
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Yellow solid, m.p. 131-132℃, yield 72.4%, FT-IR (KBr, νmax, cm−1): 2926.92, 1649.98(C=O),
1572.67, 1490.75, 1444.94, 1384.19, 1266.04, 1210.18, 1174.08, 1070.73, 1032.79, 807.27,
756.52, 689.57, 622.97, 536.01, 477.70; 1H NMR (500 MHz, CDCl3) δ: 8.37-8.33 (m, 2H, Ph-H),
7.52-7.48 (m, 4H, Ph-H), 7.43 (d, J = 2.3 Hz, 2H, Ph-H), 7.17 (dd, J = 12.5, 4.0 Hz, 3H, Ph-H),
7.09 (dd, J = 9.2, 2.8 Hz, 1H, Ph-H), 5.50 (s, 2H, -CH2), 3.88 (s, 3H, -OMe); ESI-HRMS calcd for
C22H17BrN2O2 421.0546, found 421.0551 [M+H]+.
3-((7-methoxy-2-oxo-3-phenylquinoxalin-1(2H)-yl)methyl)benzonitrile 3d
Yellow solid, m.p. 157-159℃, yield 57.6%, FT-IR (KBr, νmax, cm−1): 2226.42(C≡N),
1647.55(C=O), 1610.21, 1496.28, 1441.92, 1383.41, 1285.65, 1244.28, 1161.20, 1038.81, 809.56,
692.91, 599.15, 544.89; 1H NMR (500 MHz, CDCl3) δ: 8.38-8.33 (m, 2H, Ph-H), 7.64-7.61 (m,
2H, Ph-H), 7.53-7.47 (m, 4H, Ph-H), 7.39 (d, J = 8.5 Hz, 2H, Ph-H), 7.12-7.06 (m, 2H, Ph-H),
5.60 (s, 2H, -CH2), 3.89 (s, 3H, -OMe); ESI-HRMS calcd for C23H17N3O2 368.1394, found
368.1398[M+H]+.
1-((2-chlorothiazol-5-yl)methyl)-7-methoxy-3-phenylquinoxalin-2(1H)-one 3e
Yellow solid, m.p. 158-160℃, yield 46.4%, FT-IR (KBr, νmax, cm−1): 2950.98, 1638.52(C=O),
1606.75, 1497.40, 1445.68, 1383.15, 1266.30, 1221.31, 1028.05, 927.23, 813.12, 693.36, 626.30,
1
540.82; H NMR (500 MHz, CDCl3) δ: 8.37-8.31 (m, 2H, Ph-H), 7.69 (s, 1H, thiazole-H),
7.55-7.50 (m, 4H, Ph-H), 7.37 (d, J = 9.2 Hz, 1H, Ph-H), 7.24 (dd, J = 9.1, 2.9 Hz, 1H, Ph-H),
5.56 (s, 2H, -CH2), 3.92 (s, 3H, -OMe); ESI-HRMS calcd for C19H14ClN3O2S 384.0568, found
384.0571 [M+H]+.
2-(7-methoxy-2-oxo-3-phenylquinoxalin-1(2H)-yl)acetonitrile 3f
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Yellow solid, m.p. 123-126℃, yield 45.1%, FT-IR (KBr, νmax, cm−1): 2921.19, 2259.19(C≡N),
1645.58(C=O), 1612.63, 1499.88, 1384.14, 1283.15, 1165.78, 1070.69, 801.90, 689.50, 622.17,
1
531.38; H NMR (500 MHz, CDCl3) δ: 8.34-8.30 (m, 2H, Ph-H), 7.53-7.49 (m, 4H, Ph-H), 7.28
(d, J = 1.1 Hz, 2H, Ph-H), 5.27 (s, 2H, -CH2), 3.94 (s, 3H, -OCH3); ESI-HRMS calcd for
C17H13N3O2 292.1081, found 292.1087 [M+H]+.
1-allyl-7-methoxy-3-phenylquinoxalin-2(1H)-one 3g
Yellow solid, m.p. 91-94℃, yield 52.3%, FT-IR (KBr, νmax, cm−1): 2922.09, 2851.01, 1636.25,
1612.38(C=O), 1572.77, 1495.47, 1413.09, 1383.33, 1288.92, 1268.37, 1055.61, 807.86, 688.34,
539.55; 1H NMR (500 MHz, CDCl3) δ: 8.36-8.32 (m, 2H, Ph-H) , 7.52-7.47 (m, 4H, Ph-H), 7.25
(d, J = 9.2 Hz, 1H, Ph-H), 7.18 (dd, J = 9.2, 2.9 Hz, 1H, Ph-H), 6.00-5.93 (m, 1H, -CH), 5.29-5.21
(m, 2H, =CH2), 5.00-4.97 (m, 2H, -CH2), 3.92 (s, 3H, -OCH3); ESI-HRMS calcd for C18H16N2O2
293.1285, found 293.1292 [M+H]+.
Herbicide Bioassays
Lettuce and Bentgrass
Previously published bioassays for herbicidal activity using Lactuca sativa L.(lettuce, Iceberg A
Crisphead cultivar from Burpee Seeds, Warminster, PA, USA) and Agrostis stolonifera L.
(bentgrass, Penncross variety from Turf-Seed, Inc. of Hubbard, OR, USA) were used.35 Briefly,
seeds of bentgrass and lettuce were surface sterilized for 10 min with a 0.5% to 1% (v/v) solution
of NaOCl, rinsed with deionized H2O, and then dried in a semi-sterile environment. A 1.5-cm
Whatman Grade 1 filter paper disc was putted into 24-well plate. Every wells contained 200 μL
control solution(deionized H2O, 200 μL), control solvent(deionized H2O, 180 μL+ test compound
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solvent, 20 μL) or samples (deionized H2O, 180 μL+ sample, 20 μL). Deionized H2O was putted
into the wells before solvent or samples. Samples were dissolved in CH3COCH3. Ten mg of
bentgrass seeds or five lettuce seeds or were putted into each 24-well for the bioassays, and then
sealed the plate by using Parafilm. These 24-well plates were incubated[(bentgrass, 12 days) and
(lettuce, 5 days)] in a CU-36L5 Percival Scientific (Perry, IA, USA) incubator. Temperature and
light conditions were continuous light (120 μmol∙s−1∙m−2) at 26 °C. Phytotoxicity of control
solvent or samples was quantitated 0-5 grade: 5 is no germination of the seeds, 4 is more than 50%
germination of the seeds, 3 is about 50% germination of the seeds, 2 is less than 50% germination
of the seeds, 1 is some stunting to the seedlings, but no inhibition of germination and 0 is no
effect. Each experiment was repeated twice.
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Duckweed
A previously published bioassay with duckweed (Lemna paucicostata Hegelm.) was used.36
A
single colony consisting of a mother and two daughter fronds was used to establish duckweed
stocks that were grown in Hoagland media (pH=5.5) in sterile jars with vented lids in a CU-36L5
Percival Scientific incubator. Hoagland media were changed every 2 to 3 days. Duckweed
experiments were carried out by using non-pyrogenic polystyrene 6-well plates (CoStar 3506,
Corning Incorporated). Each well contained H2O (50 μL), or CH3COCH3 (50 μL), or test
compound (50 μL) plus 4950 μL of the Hoagland’s media. The doubling time of frond area was
approximately 1 to 1.5 day. Then two, three-frond plants(four- to five-days-old) was inoculated
into each well and repeated 3 times. Growth was determined by image analysis of graphic
templates of the 6-well plates using LemnaTec (LemnaTec, Würselen, Germany) hardware and
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software. Total frond area, frond color (necrotic or chlorotic effects) and frond number were
recorded every day. R statistics software was used to determine IC50 values.37
Electrolyte-Leakage
The effect of pure compound 3f on plasma membrane stability was determinedon
cucumber-cotyledon discs using a previously published method.38 Under dim green light,
cucumber cotyledon discs (4 mm) were cut, and 50 discs were floated in Petri plates (sterile, 6 ×
1.5 cm, polystyrene) containing 4850 μL of 1 μM MES buffer (pH=6.5) with 2% sucrose plus
CH3COCH3 (150 μL) or test samples solution (150 μL). Compound 3f, 3g, the positive control
acifluorfen, clomazone and solvent control (CH3COCH3) was tested for three times at 10 μM, 33
μM, 100 μM, 333 μM and 1 mM. Acifluorfen causes rapid plasma-membrane leakage in the light
due to its activity as a protoporphyrinogen oxidase (PPO) inhbitor.38 At the beginning of the
experiment, 55 discs were placed in test tubes with buffer (5 mL). These tubes were placed in
o
boiling H2O for 15 min and then cooled to 20 C, after which electrical conductivity was
determined at 0-h. Then the dishes was placed under 18-h dark incubation. After that, electrical
conductivity was determined at 19, 20, 22, and 24 h under high light intensity condition. The
graphs of electrical conductivity vs time were drawn from these data. The maximal possible
electrical conductivity was tested by the measurement of the electrical-conductivity of the solution
in which the cotyledon discs were boiled.
Porphyrin-Dependent Activity
To determine if the leakage caused by 3f is due to production of a porphyrin, the experiment of
Lydon and Duke39 was conducted in which the porphyrin synthesis inhibitor gabaculine was used
with the herbicide. The PPO inhibitor acifluorfen was used as a positive control. Gabaculine itself
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has no significant effect in this bioassay. Reduction of the herbicide-caused electrolyte leakage by
gabaculine occurs with PPO inhibitors, such as acifluorfen.
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Fungicide Bioassay
Direct bioautography, a simple method to screen samples for fungicide activity,40 as used as a
fungicide bioassay. Standards at 2 mM in EtOH (95%) were spotted on plates in 8 µL droplets.
Pure test samples were prepared in 95% ethanol at 20 mg/mL and 8 µL of each was spotted on
plates. Compounds 3a~3g and the commercial fungicide captan were tested for fungicidal activity
against Colletotrichum gloeosporioides, Colletotrichum fragariae and Colletotrichum acutatum
(all strawberry anthracnose-causing plant pathogens) using direct overlay bioautography.
Following sample application and solvent evaporation, each TLC plate was sprayed spore
suspension of above three fungus (C. gloeosporioides, C. fragariae and C. acutatum) with the
concentration of 3.0 × 105 spores/mL, then the plate was incubated for 4 days at 26°C in a
moisture chamber with a 12-h photoperiod. Clear zones or diffuse zones of three fungal growth
inhibitions on the TLC plate indicated the effective of fungicidal activity for pure compound.
Mosquito Bioassay
Larval Aedes aegypti (Orlando strain) was provided by the USDA/ARS/CMAVE in Gainesville,
FL, US. This strain has never been supplemented with additional wild type mosquitoes since
initial collection in 1952 in Orlando, Florida. Production and maintenance procedures for mass
rearing had been reported previously.41,42
Larval Aedes aegypti activity was carried out following standard protocols and have been
described previously.43,44 Compounds 3a~3g were initially diluted in dimethyl sulfoxide or EtOH
to make 100 μg/μL stock solutions. Stocks of compounds 3a~3g were diluted to 200 μL for larval
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bioassays. At last, the larval bioassays was determiend against five 1st instar Ae. aegypti at 1, 0.5,
0.25 and 0.1 μg/μL, respectively. The negative controls was untreated while positive control used
was permethrin. The larval Aedes aegypti was judged as dead if it was no response to probing with
the tip of a pipette after 24 h. The experiments were done for three times on different days.
Molecular Docking
The structure of Nicotiana tabacum PPO (PDB ID: 1SEZ) was downloaded from protein data
bank (PDB). The protein crystal structure Nicotiana tabacum PPO and the small molecule
2-((7-methoxy-3-phenylquinoxalin-2-yl)oxy)acetonitrile 3f were prepared by standard methods
using the discovery studio 2.5. After molecular docking, the best binding mode was selected
according to the results of docking energy, compared with the ligand in the NtPPO.
Density Functional Theory Analysis
According to the bioassay results, high active compound 3f and low active compound 3c were
selected and drawn in Gaussview 5.0 and the two structures optimized using DFT-B3LYP/6-31G
methods in Gaussian 03 software.45 The two optimized geometries are at the minimal point on
the potential surface according to no imaginary frequencies.
RESULTS AND DISCUSSION
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233
Synthesis and Spectra
The synthetic route of quinoxaline derivatives is outlined in Scheme 1. Quinoxaline is an
234
235
236
important skeleton in many natural products. There are two classic synthetic methods. In our work,
the quinoxaline ring was synthesized using diamine and dione as reaction materials. In the process
of preparing final quinoxaline derivatives, we found two isomers. The main product is the O-alkyl
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product. The title seven quinoxaline derivatives were confirmed by FT-IR, 1H NMR and
1
238
239
240
241
HR-ESI-MS. In the H NMR spectra of synthesized quinoxaline derivatives, all the –CH2 proton
signals were found around 5 ppm. The signals around 3.9 ppm can be assigned as the proton of
OCH3 group. Meanwhile, all of the synthesized quinoxaline derivatives displayed [M+H]+ peak in
the HR-ESI-MS results, which is same as the theoretical values.
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Biological Activity
Herbicidal Activity
All the compounds were tested against lettuce and bentgrass at 1 mM. This bioassay determines
phytotoxicity of compounds against a dicotyledonous (lettuce) and a monocotyledonous
(bentgrass) plant species. At 1 mM, all the compounds had good phytotoxic effects. Compounds
3f and 3g, had the best activity against creeping bentgrass (ranking 4) and lettuce (ranking 5). The
leaf of lettuce was bleached by compound 3f. Compounds 3d and 3e had good activity against the
two species (ranking 4 on both). Compounds 3a, 3b and 3c had moderate activity against lettuce,
and weak activity against bentgrass. Among them, the herbicidal activity of compound 3f (ranking
5) and 3g (ranking 5) against the lettuce was the same as the positive control clomazone at 1 mM,
but a little weaker (ranking 4) than acifluorfen (ranking 5) against the bentgrass.
In the dose-response bioassays on duckweed, the two most active compounds against lettuce and
bentgrass (3f and 3g) were tested, and the IC50 values were 13.99±3.17 and 24.56±10.02 μM for
compounds 3f and 3g, respectively (Table 1). For the commercial bleach herbicide clomazone and
acifluorfen acid, the IC50 values were 126.10 ± 18.87 μM and 0.73 ± 0.09 μM, respectively. IC50
values of compounds 3f and 3g were better than the commercial carotenoid synthesis-inhibiting
herbicide clomazone (126.10 ± 18.87 μM) and weaker than the other commercial PPO inhibitor
herbicide acifluorfen (0.73 ± 0.09 μM) in the same bioassay. Although their modes of action are
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different, clomazone and acifluorfen both cause foliar bleaching. From the herbicidal activity
against lettuce and creeping bentgrass, it is obvious that the substituent R has a very large effect
on the activity. When the substitution is a substituted benzene ring, the herbicidal trend of the
compounds was alkyl>heterocycle>substituted benzene. Among these compounds, the CNCH2
provides the best activity and causes foliar bleaching. This may be because the CN group interacts
with a critical amino acid residue of PPO, because, in the simulated molecular docking, the CN
group interacted with Thr 176 via hydrogen bonding.
Table 1 The phytotoxicity of seven compounds against three plant species
No.
R
Lettuce Agrostis
Duckweed(IC50(μM))
3a
4-CNPhCH2
4-t-BuPhCH2
4-Br PhCH2
3-CNPhCH2
2-Cl-thiazole-5-CH2
CNCH2
3
3
1
1
1
3
3
4
4
5
5
-
3b
-
3c
2
-
3d
4
-
3e
3f
4
-
5*
5
13.99±3.17
24.56±10.02
126.10 ± 18.87
0.73 ± 0.09
3g
CH2=CHCH2
Clomazone
Acifluorfen
5
5
269
270
271
0 = no effect and 5 = no germination of the seeds.
*bleached
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Electrolyte-Leakage Assay
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PPO inhibitors all cause rapid loss of cellular electrolytes and bleaching of chloroplast pigments
by the photodynamic action of protoporphyin IX that accumulates when PPO is inhibited. Because
bleaching was observed with 3f in lettuce, an electrolyte leakage assay was done with acifluorfen
as a positive control. Compound 3f caused light-dependent electrolyte leakage (Figure 3). These
results are similar to those caused by commercial PPO inhibitor herbicides like acifluorfen. The
effects of 3f at 100 μM was similar to the effect of acifluorfen at 33 µM. The fact that the
compound 3f compromises membrane integrity indicates that the mechanism of action of this
compound is directly related to cell membrane damage like that of PPO inhibitors.
2000
Solvent control
Acifluorfen (33 M)
10 M
33 M
1900
1000
100 M
Boiled control
800
600
400
200
0
0
5
10
15
20
25
30
Time (hr)
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282
Figure 3. The electrolyte-leakage of cucumber cotyledon discs caused by compound 3f at different
concentrations. The arrow indicates exposure to light. Error bars are ± 1SE. Dotted line is
conductivity of boiled discs (maximum possible effect).
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Gabaculine inhibits the porphyrin pathway at an early stage, preventing the accumulation of
proporphyin IX caused by PPO inhibitors.39 Gabaculine reversed the effect of 3f and acifluorfen
on cucumber cotyledon disc leakage (Figure 4). Reversal was with acifluorfen was similar to that
reported previously.36 The results with gabaculine reversal indicate that 3f is a PPO inhibitor.
1000
Solvent control
Acifluorfen (33 M)
gabaculine (100 M)
Acifluorfen (33 μM) + gabaculine (100 μM)
3f (100 μM)
3f (100 μM) + gabaculine(33 μM)
800
600
400
200
0
-200
0
5
10
15
20
25
30
Time (hr)
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292
Figure 4. Effects of 100 µM gabaculine on the electrolyte leakage of cucumber cotyledon
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discs caused by 33 µM acifluorfen and 100 µM 3f. The arrow indicates exposure to light.
Molecular Docking with PPO
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From the electrolyte-leakage assay, the effects of compound 3f were like that of a PPO inhibitor,
so we conducted molecular modeling analysis of compound 3f with NtPPO. As shown in Figure
5a, there were four π–π interactions between FAD and the quinoxaline ring with the distances of
6.3 Å, 7.0 Å, 5.0 Å, 5.5 Å respectively; second, there were two strong hydrogen bonding
interactions (2.2 Å and 2.1 Å) between Thr176 of PPO and the N atom of the cyan group in the
compound 3f and Arg98 of PPO and the oxygen atom of the CH3O group in compound 3f,
respectively. From the cocrystal (Figure 5b), a strong hydrogen bond exists between Arg98 and
the oxygen atom of a carbonyl group of 3f. There are also π–π interactions between benzene ring
of PPO inhibitor and Phe392 in the cocrystal. From the herbicide assay, 3f caused leaf bleaching,
and gabaculine-reversible electrolyte-leakage, indicating that it is a PPO inhibitor. However, it is
weaker than the commercial PPO inhibitor acifluorfen. Its different activity may be attributable to
the different π–π interactions.
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304
305
306
307
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Figure 5. Simulated binding modes of compound 3f with NtPPO(a) and complex binding modes in
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co-crystal.
Fungicidal Activity
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With bioautographic methods, visible clear zones or diffuse zones observed after incubation and
the results were listed in Table 2 and Figure 6. All the seven quinoxaline compounds possessed
good fungicidal activity against strawberry anthracnose pathogens at the concentration tested,
especially compounds 3f and 3g (Table 2, Figure 6). The activity against three species is the same
as the positive control, captan, at 20 µg/mL. Compounds 3b and 3d produced both clear and
diffuse inhibition zones for the C. gloeosporioides and C. acutatum (Table 2). The diffuse zones
also had the good activity. From the fungicidal activity data, the trend of the compounds against
three fungi was C. acutatum>C. gloeosporioides>C. fragariae. Among these compounds, the
compounds with alkyl group still possessed the best activity against three fungi, whatever the clear
zone or diffuse zone. The (hetero-)aromatic substituted compounds exhibited weak activity against
C. fragariae. All the compounds displayed good activity against C. acutatum, except compound
3c, which is similar with the activity against C. gloeosporioides.
Table 2 The fungicidal activity against three fungi of seven compounds at 160 mg/spot
inhibitory-zone diameter (mm)
No.
C. acutatum
C. fragariae
C. gloeosporioides
3a
3b
3c
3d
3e
15
8/15a
6a
7
7
8
9/17a
6
6
9/17a
13
10
7
10/16a
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3f
3g
15
18
15
15
16
14
16
19
17
captan
Solvent
0
0
0
control(ethanol)
a
327
Clear zone/Diffuse zone beyond the clear zone
328
C. acutatum
3c
3a
3d
3e
3b
3f
3g
Diffuse zone
Clear zone
Diffuse zone
Clear zone
C. fragariae
3c
3a
3d
3e
3b
3f
3g
C. gloeosporioides
3c
3a
3d
3e
3b
3f
3g
Diffuse zone
Diffuse zone
Clear zone
Clear zone
329
330
331
332
333
Figure 6 The inhibitor zone of compound 3a-3g against three fungi.
Insecticidal Activity
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All of the compounds were tested for mosquito larvicidal activity (Table 3). Most of these
compounds provided excellent larvicidal activity (100%) at 1 μg/μL, except for compounds 3d
and 3f. When the concentration is at 0.5 μg/μL, the activities of compounds 3b and 3g are 100%,
and compounds 3c and 3e also possessed good mosquito larvicidal activity (>90%). Compounds
3a and 3f exhibited moderate activity (>60%). Compounds 3c, 3e, 3f and 3g maintained good
larvicidal activity (>60%), at 0.1 μg/μL. Negative controls produced no inseciticidal activity,
whereas permethrin was highly mosquito larvicidal activity against 1st instar ORL strain (Table 3).
From the larvicidal activity data, the trend of the seven quinoxaline compounds against larvae was
alkyl>heterocycle>substituted benzene. Among these compounds, the compounds with alkyl
group still possessed the best activity against larvae, while the heterocyclic substituted compound
and the 4-BrPhCH2 also exhibited good activity at 0.1 μg/μL. But they still displayed weaker
activity than that of positive control.
Table 3 The larvae activity against 1st instar ORL strain.
No.
3a
3b
3c
1 μg/μL
100
0.5 μg/μL 0.25 μg/μL 0.1 μg/μL
73.3 ± 30.6 66.7 ± 57.7 46.7 ± 41.6
100
100
86.7 ± 11.5 46.7 ± 41.6
100
93.3 ± 11.5 66.7 ± 30.6 60.0 ± 52.9
3d
3e
73.3 ± 30.6 33.3 ± 41.6 20.0 ± 34.6 26.7 ± 30.6
100 93.3 ± 11.5 73.3 ± 30.6 66.7 ± 57.7
3f
73.3 ± 46.2 66.7 ± 57.7 66.7 ± 57.7 60.0 ± 52.9
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3g
100
0
100
0
100
0
80.0 ± 34.6
Untreated
Permethrin
0
100
100
100
100
348
349
350
351
DFT(B3LYP) Calculation
HOMO, LUMO, energy gaps, ClogP and molecular total energy of low activity compound 3c and
high activity compound 3f are calculated using DFT method and the results are listed in Table 4.
LUMO
HOMO
3f
3c
352
353
Figure 7 The HOMO and LUMO of compounds 3c and 3f
354
355
356
357
358
Frontier molecular orbital can supply information about electrons transfer, especially between the
HOMO and LUMO. The two orbitals’ energy and the energy gap are important information for
mechanism of the molecules and target enzyme44-45. From Figure 7, the compounds 3c and 3f
contain two parts: the quinoxaline ring and CH2R-substituted group. For the compound 3c, the
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364
365
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HOMO is located on the quinoxaline ring, benezene ring, 4-bromobenzene ring and CH2 bridge,
But the LUMO is only located on the quinoxaline ring and CH2 bridge. Hence, electron transfer of
compound 3c is from the quinoxaline ring to the 4-bromobenzene ring via the CH2 bridge and the
energy gap is 0.14975 Hartree. For the compound 3f, the HOMO is mainly located on the
quinoxaline ring, benzene ring and CH2 bridge, while the LUMO is located on the quinoxaline
ring and CH2 bridge. The electron transition is on the quinoxaline ring and CH2 bridge, and the
CN group does not contribute to the electron transition and the energy gap is 0.15065 Hartree. The
total energies of the two compounds are different: compound 3c (-3679.73477399 Hartree) is
lower than compound 3f (-969.97326509 Hartree). Also the ClogP values of the two compounds
differ. The ClogP of compound 3f is 2.55843, while that of compound 3c is 5.30123. The ClogP
also indicated that the substituted group on the N-quinoxaline ring can influence the bioactivity.
Furthermore, the FMO provided meaningful information as to lead compound of this new family
of PPO inhibitor that will be useful to design more potential lead compounds in the future.
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373
Table 4. Total energy, HOMO, LUMO and energy gap, CLogP.
Energy
3c
3f
Etotal/Hartree b
EHOMO/Hartree
ELUMO/Hartree
-3679.73477399
-969.97326509
-0.21662
-0.06687
-0.22443
-0.07378
ΔE a/Hartree
0.14975
5.30123
0.15065
2.55843
CLogP
374
375
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393
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395
Acknowledgements
This work was supported by Zhejiang Provincial Natural Science Foundation of China (No.
LY19C140002), Research Fund of Department of Education of Zhejiang Province (Y201941832).
We thank Drs. James J. Becnel and Alden Estep for evaluating larvicidal activity and Solomon
Green, Jesse Linda Robertson and Robert Johnson for their technical assistance.
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