Luotonin A and Its Derivatives as Novel Antiviral and Antiphytopathogenic Fungus Agents

Article abstract of DOI:10.1021/acs.jafc.0c04278

Plant diseases caused by viruses and fungi have posed a serious threat to global agricultural production. The discovery of new leads based on natural products is an important way to innovate pesticides. In this work, natural product luotonin A was found to have good antiviral activity against tobacco mosaic virus (TMV) for the first time. A series of luotonin A derivatives were designed, synthesized, and evaluated for their antiviral activities and fungicidal activities systematically. Most compounds displayed better antiviral activities against TMV than commercial ribavirin. Compounds 9k, 12b, and 12d displayed about similar inhibitory effects as ningnanmycin (inhibitory rates of 55, 57, and 59% at 500 μg/mL for inactivation, curative, and protection activities in vivo, respectively), the best antiviral agent at present, and emerged as novel antiviral leads for further research. We selected 9k for further antiviral mechanism research via transmission electron microscopy and molecular docking, which revealed that compound 9k can interact with TMV coat protein through the hydrogen bond, leading to its polymerization, thus preventing virus assembly. Further fungicidal activity tests showed that these compounds also showed broad-spectrum fungicidal activities against 14 kinds of phytopathogenic fungi. Especially, compound 14 with a 100% antifungal effect against Botrytis cinereal emerged as a lead for further research. This work provides a reference for the development of agricultural active ingredients based on Chinese medicine plants.

Full text of DOI:10.1021/acs.jafc.0c04278

pubs.acs.org/JAFC
Article
Luotonin A and Its Derivatives as Novel Antiviral and
Antiphytopathogenic Fungus Agents
Yanan Hao, Kaihua Wang, Ziwen Wang,* Yuxiu Liu, Dejun Ma, and Qingmin Wang*
Cite This: https://dx.doi.org/10.1021/acs.jafc.0c04278
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Plant diseases caused by viruses and fungi have posed a serious threat to global agricultural production. The discovery
of new leads based on natural products is an important way to innovate pesticides. In this work, natural product luotonin A was
found to have good antiviral activity against tobacco mosaic virus (TMV) for the first time. A series of luotonin A derivatives were
designed, synthesized, and evaluated for their antiviral activities and fungicidal activities systematically. Most compounds displayed
better antiviral activities against TMV than commercial ribavirin. Compounds 9k, 12b, and 12d displayed about similar inhibitory
effects as ningnanmycin (inhibitory rates of 55, 57, and 59% at 500 μg/mL for inactivation, curative, and protection activities in vivo,
respectively), the best antiviral agent at present, and emerged as novel antiviral leads for further research. We selected 9k for further
antiviral mechanism research via transmission electron microscopy and molecular docking, which revealed that compound 9k can
interact with TMV coat protein through the hydrogen bond, leading to its polymerization, thus preventing virus assembly. Further
fungicidal activity tests showed that these compounds also showed broad-spectrum fungicidal activities against 14 kinds of
phytopathogenic fungi. Especially, compound 14 with a 100% antifungal effect against Botrytis cinereal emerged as a lead for further
research. This work provides a reference for the development of agricultural active ingredients based on Chinese medicine plants.
KEYWORDS: lead discovery, structure optimization, luotonin A, antiviral activity, fungicidal activity, mode of action
isolated from Peganum nigellastrum Bunge (a Chinese medicinal
plant) for the first time in 1997.15 Studies have shown that
luotonin A has certain inhibitory activity on topoisomerase I
and topoisomerase II.16−19 The unique molecular structure
and spectral biological activity of luotonin A have attracted
more and more attention of researchers. However, the
researchers mainly focus on the antitumor research of luotonin
A, rarely on the agricultural activity research at present. In
particular, there is no report on the study of antiplant virus
activity.
Our group has long been committed to the separation of
active components of traditional Chinese medicine plants and
the creation of antiviral agents based on the active ingredients.
We have discovered that Chinese medicine plant Cynanchum
komarovii AL has good antiviral activity against TMV, and the
active components were tylophora alkaloids, such as
tylophorine.20 However, the tylophorine content of the plants
is very low, and the molecule is light-sensitive and poorly
soluble. In efforts to overcome these disadvantages, various
tylophora alkaloids21 and their simplified salt forming and
sugar forming derivatives were designed and synthesized and
evaluated for their anti-TMV activities.22−24 A racemate of
INTRODUCTION
■
In the coming decades, the global demand for crops will
continue to grow, which is affected by the growth of
population and the improvement of per capita consumption.1
Also every day, a large area of normal land is becoming
marginal, which greatly reduces productivity and endangers
food security.2 We are facing one of the greatest challenges of
the 21st century: reducing the harm of agriculture to the
environment while meeting the growing food demand of
society.3 Plant diseases caused by viruses and fungi have posed
a serious threat to global agricultural production. Tobacco
mosaic virus (TMV), one of the most widely studied plant
pathogens in the world, has the most extensive host range of
more than 885 species in 65 families.4 However, there are few
commercially available antiviral agents. At present, the most
effective antiviral agent ningnanmycin has an inhibitory effect
of 50−60% at 500 μg/mL, and the most widely used antiviral
agent ribavirin has a less than 50% inhibitory effect at 500 μg/
mL.5 Therefore, it is urgent to develop novel and more
effective antiviral agents.
Natural products are the metabolites of animals and plants,
which have the advantages of biocompatibility, structural
diversity, and unique mechanism.6,7 The discovery of new
leads based on natural products is an important way to
innovate pesticides. There have been many studies on the
structural derivation of natural products in an attempt to find
highly active compounds.8−13 Quinazolinone and its deriva-
tives are the basic skeletons of about 150 natural alkaloids
isolated from plants, animals, and microorganisms.14 As a
representative molecule of quinazolone, luotonin A was
Received: July 6, 2020
Revised: July 29, 2020
Accepted: July 30, 2020
Published: July 30, 2020
https://dx.doi.org/10.1021/acs.jafc.0c04278
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
© XXXX American Chemical Society
A

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Figure 1. Design of luotonin A analogues.
134.2, 129.7, 128.8, 128.4, 126.6, 123.2, 121.0, 21.0. C16H14N3O2 [M
+ H]+, 280.1081; found, 280.1077.
tylophorine malate (designated NK007), an optimized
compound, is being industrialized as an anti-TMV candidate.
Luotonin A also has a five-ring planar structure, which is low
in content and poor in solubility. Based on our work
experience and the above findings, a series of luotonin A
derivatives were designed (Figure 1) and synthesized through
ring expansion and ring contraction strategies. The anti-TMV
and fungicidal activities and structure−activity relationships of
these compounds were systematically evaluated.
N-(3,4-Dimethoxyphenyl)-4-oxo-3,4-dihydroquinazoline-2-car-
boxamide (3c). It is obtained as a yellow green solid, yield 65%; mp
1
176−178 °C; H NMR (400 MHz, DMSO-d6): δ 12.45 (s, 1H),
10.66 (s, 1H), 8.21 (d, J = 7.7 Hz, 1H), 7.96−7.87 (m, 2H), 7.68−
7.62 (m, 1H), 7.58 (d, J = 2.3 Hz, 1H), 7.51 (dd, J = 8.7, 2.3 Hz, 1H),
6.98 (d, J = 8.8 Hz, 1H), 3.77 (d, J = 6.8 Hz, 6H). 13C NMR (100
MHz, DMSO-d6): δ 161.5, 158.0, 149.0, 147.4, 146.4, 146.3, 135.3,
131.5, 128.7, 128.3, 126.7, 123.1, 112.9, 112.2, 106.0, 56.1, 55.9.
C17H16N3O4 [M + H]+, 326.1135; found, 326.1131.
N-(4-Chlorophenyl)-4-oxo-3,4-dihydroquinazoline-2-carboxa-
mide (3d). It is obtained as a white solid, yield 54%; mp 294−296 °C;
1H NMR (400 MHz, DMSO-d6): δ 12.55 (s, 1H), 10.96 (s, 1H), 8.21
(d, J = 7.8 Hz, 1H), 7.97−7.90 (m, 4H), 7.66 (t, J = 6.7 Hz, 1H),
7.50−7.44 (m, 2H).
MATERIALS AND METHODS
■
Chemicals. The reagents were purchased from commercial
sources and were used as received. All anhydrous solvents were
dried and purified by standard techniques prior to use.
General Procedures for the Preparation of Compounds 4a−
4d. To a solution of compounds 3a−3d (28.3 mmol) in
dimethylformamide (DMF) (200 mL) was successively added
K2CO3 (4.3 g, 31.1 mmol) and propargyl bromide (2.7 mL, 31.1
mmol). The reaction mixture was stirred at room temperature
overnight and then poured into ice water. The resulting precipitates
were collected by filtration and purified by flash chromatography on a
silica gel using dichloromethane and petroleum ether (4:1, v/v) as the
eluent to give 4a−4d.
4-Oxo-N-phenyl-3-(prop-2-yn-1-yl)-3,4-dihydroquinazoline-2-
carboxamide (4a). It is obtained as a white solid, yield 78%; mp
175−177 °C; 1H NMR (400 MHz, DMSO-d6): δ 11.15 (s, 1H), 8.25
(d, J = 7.0 Hz, 1H), 7.99−7.92 (m, 1H), 7.85 (d, J = 8.0 Hz, 1H),
7.75 (d, J = 7.7 Hz, 2H), 7.69 (t, J = 7.5 Hz, 1H), 7.41 (t, J = 7.9 Hz,
2H), 7.19 (t, J = 7.4 Hz, 1H), 5.05 (d, J = 2.4 Hz, 2H), 3.35 (t, J = 2.4
Hz, 1H).
4-Oxo-3-(prop-2-yn-1-yl)-N-(p-tolyl)-3,4-dihydroquinazoline-2-
carboxamide (4b). It is obtained as a light pink solid, yield 70%; mp
178−180 °C; 1H NMR (400 MHz, DMSO-d6): δ 11.07 (s, 1H), 8.24
(d, J = 7.8 Hz, 1H), 7.95 (t, J = 7.6 Hz, 1H), 7.85 (d, J = 7.9 Hz, 1H),
7.69 (t, J = 7.6 Hz, 1H), 7.64 (d, J = 7.1 Hz, 2H), 7.21 (d, J = 7.4 Hz,
2H), 5.04 (s, 2H), 3.34 (s, 1H), 2.30 (s, 3H). C19H16N3O2 [M + H]+,
318.1237; found, 318.1233.
N-(3,4-Dimethoxyphenyl)-4-oxo-3-(prop-2-yn-1-yl)-3,4-dihydro-
quinazoline-2-carboxamide (4c). It is obtained as a yellow green
solid, yield 50%; mp 160−162 °C; 1H NMR (400 MHz, DMSO-d6):
δ 10.98 (s, 1H), 8.24 (d, J = 7.0 Hz, 1H), 7.95 (dd, J = 11.9, 4.8 Hz,
1H), 7.85 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.42 (d, J = 2.3
Hz, 1H), 7.32 (dd, J = 8.7, 2.3 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 5.06
(d, J = 2.3 Hz, 2H), 3.77 (d, J = 2.1 Hz, 6H), 3.34 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): δ 160.0, 159.1, 148.5, 148.3, 145.9, 145.7,
135.3, 131.0, 128.7, 127.7, 126.5, 120.9, 112.4, 111.9, 105.1, 78.5,
74.9, 55.7, 55.4, 33.1. C20H18N2O4 [M + H]+, 364.1292; found,
364.1288.
Instruments. The melting points of the compounds were tested
on an X-4 binocular microscope (Beijing Tech Instruments
Company). NMR spectra were obtained with a Bruker AV 400
spectrometer with either CDCl3, CD3OD, or DMSO-d6 as the solvent.
High-resolution mass spectra were obtained with an FT-ICR mass
spectrometer (Ionspec, 7.0 T). The in vitro TMV rod assembly
inhibition and 20S coat protein (CP) disk assembly inhibition were
tested via transmission electron microscopy (Tecnai G2 F20).
Preparation of Ethyl 4-Oxo-3,4-dihydroquinazoline-2-carboxy-
late (2). A mixture of anthranilamide (1, 2 g, 14.7 mmol) and diethyl
oxalate (50 mL) was refluxed at 185−186 °C for 5 h. Then, it was
cooled to room temperature and filtered to give compound 2: white
1
solid, yield 87.5%; mp 190−192 °C; H NMR (400 MHz, DMSO-
d6): δ 12.65 (s, 1H), 8.19 (dd, J = 7.9, 1.1 Hz, 1H), 7.94−7.88 (m,
1H), 7.86−7.82 (m, 1H), 7.69−7.62 (m, 1H), 4.39 (q, J = 7.1 Hz,
2H), 1.36 (t, J = 7.1 Hz, 3H).
General Procedures for the Preparation of Compounds 3a−
3d. To a solution of corresponding phenylamines (22 mmol) in
anhydrous dichloroethane (60 mL) was added 2 M AlMe3 (22 mL, 11
mmol) dropwise under an Ar atmosphere. The reaction mixture was
stirred at room temperature for 0.5 h and then added compound 2 (3
g, 13.76 mmol). The mixture was heated at reflux for 5 h and then
cooled to 0 °C and quenched by slow addition of 2 M HCl (20 mL)
and H2O (100 mL). Resulting precipitates were collected by filtration
and washed with water to give 3a−3d.
4-Oxo-N-phenyl-3,4-dihydroquinazoline-2-carboxamide (3a). It
1
is obtained as a white solid, yield 97%; mp 247−249 °C; H NMR
(400 MHz, DMSO-d6): δ 12.52 (s, 1H), 10.79 (s, 1H), 8.21 (d, J =
7.6 Hz, 1H), 7.97−7.86 (m, 4H), 7.66 (t, J = 7.2 Hz, 1H), 7.41 (t, J =
7.9 Hz, 2H), 7.18 (t, J = 7.4 Hz, 1H).
4-Oxo-N-(p-tolyl)-3,4-dihydroquinazoline-2-carboxamide (3b).
1
It is obtained as a light pink solid, yield 95%; mp 197−199 °C; H
NMR (400 MHz, DMSO-d6): δ 12.48 (s, 1H), 10.71 (s, 1H), 8.21 (d,
J = 7.8 Hz, 1H), 7.95−7.87 (m, 2H), 7.76 (d, J = 8.4 Hz, 2H), 7.65 (t,
J = 7.0 Hz, 1H), 7.21 (d, J = 8.4 Hz, 2H), 2.30 (s, 3H). 13C NMR
(100 MHz, DMSO-d6): δ 161.5, 158.3, 147.4, 146.4, 135.6, 135.3,
N-(4-Chlorophenyl)-4-oxo-3-(prop-2-yn-1-yl)-3,4-dihydroquina-
zoline-2-carboxamide (4d). It is obtained as a white solid, yield 54%;
1
mp 186−188 °C; H NMR (400 MHz, DMSO-d6): δ 11.30 (s, 1H),
B
https://dx.doi.org/10.1021/acs.jafc.0c04278
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
8.25 (d, J = 6.8 Hz, 1H), 7.99−7.93 (m, 1H), 7.88−7.83 (m, 1H),
7.83−7.77 (m, 2H), 7.70 (t, J = 7.6 Hz, 1H), 7.51−7.46 (m, 2H),
5.05 (d, J = 2.4 Hz, 2H), 3.35 (t, J = 2.5 Hz, 1H).
by H2O (50 mL). The resulting precipitates were collected by
filtration to give 9a−9m.
5,6-Dihydro-8H-quinazolino[4,3-b]quinazolin-8-one (9a). It is
1
obtained as a yellow solid, yield 63%; mp 178−180 °C; H NMR
General Procedures for the Preparation of Compounds 5a−
5d.25 To a solution of triphenylphosphine oxide (9.7 g, 34.8 mmol)
in anhydrous CH2Cl2 (300 mL) was added trifluoromethanesulfonic
anhydride (3 mL, 18 mmol) slowly at 0 °C under an Ar atmosphere.
After the mixture was stirred at 0 °C for 10 min, one of compounds
4a−4d (11.6 mmol) was added. The reaction mixture was stirred at
room temperature and quenched by addition of 10% aqueous
NaHCO3 solution. The resulting precipitates were collected by
filtration and washed with CHCl3 to give 5a−5d.
Quinolino[2′,3′:3,4]pyrrolo[2,1-b]quinazolin-11(13H)-one (5a).
It is obtained as a white solid, yield 78%; mp 208−210 °C; 1H
NMR (400 MHz, DMSO-d6): δ 8.78 (s, 1H), 8.32−8.27 (m, 2H),
8.18 (d, J = 8.1 Hz, 1H), 7.98−7.89 (m, 3H), 7.78 (t, J = 7.5 Hz, 1H),
7.68−7.62 (m, 1H), 5.33 (s, 2H). 13C NMR (100 MHz, DMSO-d6):
δ 160.2, 153.6, 152.0, 149.5, 148.8, 135.0, 132.3, 131.5, 133.0, 130.2,
129.0, 128.8, 128.5, 127.7, 126.4, 48.0.
(400 MHz, CD3OD): δ 8.27−8.16 (m, 2H), 7.81−7.76 (m, 1H), 7.71
(d, J = 8.0 Hz, 1H), 7.48−7.42 (m, 1H), 7.40−7.34 (m, 1H), 6.99−
6.92 (m, 1H), 6.88 (d, J = 8.1 Hz, 1H), 5.31 (s, 2H). 13C NMR (100
MHz, DMSO-d6): δ 159.8, 148.6, 148.4, 148.2, 135.1, 133.7, 127.7,
127.5, 126.8, 126.4, 120.8, 119.5, 116.6, 116.1, 52.9. HRMS (ESI):
calcd for C15H12N3O [M + H]+, 250.0975; found, 250.0974.
6-Methyl-5,6-dihydro-8H-quinazolino[4,3-b]quinazolin-8-one
(9b). It is obtained as a yellow solid, yield 65%; mp 116−118 °C; 1H
NMR (400 MHz, CD3OD): δ 8.27−8.16 (m, 2H), 7.83−7.76 (m,
1H), 7.72 (d, J = 8.1 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.40−7.33 (m,
1H), 6.90 (t, J = 7.6 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.32 (q, J = 6.1
Hz, 1H), 1.43 (d, J = 6.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d6):
δ 159.5, 148.1, 147.3, 145.7, 135.1, 134.1, 127.5, 127.4, 126.8, 126.4,
120.5, 118.9, 116.4, 115.1, 59.3, 19.9. HRMS (ESI): calcd for
C16H14N3O [M + H]+, 264.1131; found, 264.1131.
6-Cyclohexyl-5,6-dihydro-8H-quinazolino[4,3-b]quinazolin-8-
one (9c). It is obtained as a yellow solid, yield 83%; mp 235−237 °C;
1H NMR (400 MHz, DMSO-d6): δ 8.17 (d, J = 7.6 Hz, 2H), 7.83 (t, J
2-Methylquinolino[2′,3′:3,4]pyrrolo[2,1-b]quinazolin-11(13H)-
one (5b). It is obtained as a light red solid, yield 70%; mp 272−274
°C; 1H NMR (400 MHz, DMSO-d6): δ 8.66 (s, 1H), 8.30 (d, J = 7.8
Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 7.96−7.93 (m, 3H), 7.78−7.74 (m,
1H), 7.67−7.61 (m, 1H), 5.31 (s, 2H), 2.58 (s, 3H).
= 7.6 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.57−7.44 (m, 2H), 7.37 (t, J
= 7.6 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.84 (t, J = 7.5 Hz, 1H),
5.96−5.86 (m, 1H), 1.78 (d, J = 10.0 Hz, 2H), 1.69−1.44 (m, 3H),
1.22−1.11 (m, 1H), 1.11−0.88 (m, 5H). 13C NMR (100 MHz,
DMSO-d6): δ 160.2, 148.0, 147.6, 145.6, 135.2, 134.1, 127.5, 127.2,
126.3, 120.5, 118.6, 115.9, 115.8, 65.9, 28.7, 28.7, 26.0, 25.5, 25.5.
HRMS (ESI): calcd for C21H22N3O [M + H]+, 332.1757; found,
332.1757.
6-Cyclopropyl-5,6-dihydro-8H-quinazolino[4,3-b]quinazolin-8-
one (9d). It is obtained as a yellow solid, yield 97%; mp 98−100 °C;
1H NMR (400 MHz, CDCl3): δ 8.32−8.26 (m, 1H), 8.19 (d, J = 8.2
Hz, 1H), 7.74−7.64 (m, 2H), 7.42−7.31 (m, 2H), 6.88 (t, J = 7.6 Hz,
1H), 6.64 (d, J = 8.2 Hz, 1H), 5.45−5.38 (m, 1H), 3.60−3.50 (m,
1H), 3.38−3.29 (m, 1H), 3.28−3.18 (m, 1H), 2.22−1.93 (m, 3H).
13C NMR (100 MHz, CDCl3): δ 160.9, 146.9, 146.9, 144.5, 133.3,
132.5, 127.1, 126.1, 125.4, 125.0, 120.0, 118.0, 115.5, 111.8, 71.0,
44.6, 31.5, 19.8. HRMS (ESI): calcd for C18H16N3O [M + H]+,
290.1288; found, 290.1287.
6-Phenyl-5,6-dihydro-8H-quinazolino[4,3-b]quinazolin-8-one
(9e). It is obtained as a red brown solid, yield 71%; mp 188−190 °C;
1H NMR (400 MHz, DMSO-d6): δ 8.23−8.18 (m, 1H), 8.15−8.10
2,3-Dimethoxyquinolino[2′,3′:3,4]pyrrolo[2,1-b]quinazolin-11-
(13H)-one (5c). It is obtained as a yellow green solid, yield 76%; mp
1
280−282 °C; H NMR (400 MHz, DMSO-d6): δ 8.55 (s, 1H), 8.29
(d, J = 7.6 Hz, 1H), 7.97−7.88 (m, 2H), 7.68−7.60 (m, 2H), 7.54 (s,
1H), 5.28 (s, 2H), 4.00 (d, J = 10.1 Hz, 6H).
2-Chloroquinolino[2′,3′:3,4]pyrrolo[2,1-b]quinazolin-11(13H)-
one (5d). It is obtained as a white solid, yield 99%; mp >300 °C; 1H
NMR (400 MHz, DMSO-d6): δ 8.74 (s, 1H), 8.35 (d, J = 2.2 Hz,
1H), 8.31−8.28 (m, 2H), 7.97−7.90 (m, 3H), 7.68−7.62 (m, 1H),
5.33 (s, 2H).
Preparation of N-(2-Carbamoylphenyl)-2-nitrobenzamide (6).
To a solution of compound 2-aminobenzamide (5 g, 37 mmol) in
CHCl3 was added triethylamine (14 mL), 2-nitrobenzoyl chloride (7
mL, 52 mmol). The reaction mixture was stirred at room temperature
for 4 h, and the resulting precipitates were collected by filtration to
give 6: white solid, yield 80%; mp 195−197 °C; 1H NMR (400 MHz,
DMSO-d6): δ 12.52 (s, 1H), 8.48 (d, J = 8.2 Hz, 1H), 8.38 (brs, 1H),
8.11 (d, J = 8.1 Hz, 1H), 7.92−7.75 (m, 5H), 7.59 (t, J = 7.7 Hz, 1H),
7.23 (t, J = 7.6 Hz, 1H).
Preparation of 2-(2-Nitrophenyl) Quinazolin-4(3H)-one (7).
Compound 6 (4 g, 14 mmol) was dissolved in a solution of 10%
aqueous KOH solution (40 mL) and ethanol (20 mL). The mixture
was stirred at reflux for 5 h. After completion, the ethanol was
concentrated in vacuo, and the residue was extracted with ethyl
acetate, dried over anhydrous Na2SO4, filtered, and concentrated to
give 7: light brown solid, yield 92%; mp 178−180 °C; 1H NMR (400
MHz, DMSO-d6): δ 12.89 (s, 1H), 8.33−8.19 (m, 2H), 8.00−7.83
(m, 4H), 7.71 (d, J = 8.2 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6): δ 161.6, 151.8, 148.4, 147.4, 134.7, 133.9,
131.5, 131.4, 129.2, 127.3, 127.1, 125.9, 124.5, 121.1.
(m, 1H), 8.02 (d, J = 3.3 Hz, 1H), 7.91−7.85 (m, 1H), 7.75 (d, J =
8.0 Hz, 1H), 7.53 (t, J = 7.1 Hz, 1H), 7.37−7.30 (m, 1H), 7.30−7.21
(m, 4H), 7.18 (d, J = 6.8 Hz, 2H), 6.93 (d, J = 8.0 Hz, 1H), 6.82 (t, J
= 7.5 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): δ 159.7, 147.7,
147.3, 145.1, 139.4, 135.0, 133.7, 128.6, 128.2, 127.2, 126.9, 126.7,
126.2, 125.7, 119.9, 118.8, 115.9, 115.5, 62.5. HRMS (ESI): calcd for
C21H16N3O [M + H]+, 326.1288; found, 326.1288.
6-(2,4-Dimethoxyphenyl)-5,6-dihydro-8H-quinazolino[4,3-b]-
quinazolin-8-one (9f). It is obtained as a green solid, yield 65%; mp
193−195 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.20 (d, J = 7.7 Hz,
1H), 8.10 (d, J = 7.7 Hz, 1H), 7.86 (t, J = 7.1 Hz, 1H), 7.77 (d, J = 8.1
Hz, 1H), 7.48 (t, J = 7.4 Hz, 1H), 7.33 (d, J = 2.6 Hz, 1H), 7.31−7.24
(m, 2H), 6.89−6.78 (m, 2H), 6.61 (d, J = 1.9 Hz, 1H), 6.47 (d, J =
8.5 Hz, 1H), 6.29−6.22 (m, 1H), 3.90 (s, 3H), 3.65 (s, 3H). 13C
NMR (100 MHz, DMSO-d6): δ 160.6, 159.1, 157.4, 148.0, 147.7,
145.0, 134.9, 133.4, 127.2, 126.7, 126.5, 126.1, 125.8, 119.9, 119.5,
118.4, 115.8, 115.0, 104.0, 98.9, 59.2, 55.8, 55.1. HRMS (ESI): calcd
for C23H20N3O3 [M + H]+, 386.1499; found, 386.1500.
Preparation of 2-(2-Aminophenyl) Quinazolin-4(3H)-one (8). To
a solution of compound 7 (1.2 g, 4.5 mmol) in ethanol was added
SnCl2·2H2O (5.1 g, 22.5 mmol). The mixture was stirred at reflux for
6 h. After completion, the solvent was concentrated in vacuo, and the
residue was extracted with ethyl acetate, dried over anhydrous
Na2SO4, filtered, and concentrated to give 8: yellow solid, yield 80%;
1
mp 155−157 °C; H NMR (400 MHz, DMSO-d6): δ 12.18 (brs,
6-(3-Nitrophenyl)-5,6-dihydro-8H-quinazolino[4,3-b]quinazolin-
8-one (9g). It is obtained as a yellow solid, yield 85%; mp 183−185
°C; 1H NMR (400 MHz, CDCl3): δ 8.11 (t, J = 6.9 Hz, 2H), 7.81 (d,
J = 8.6 Hz, 2H), 7.58 (s, 2H), 7.33−7.18 (m, 4H), 7.16 (t, J = 7.5 Hz,
1H), 6.80 (t, J = 7.6 Hz, 1H), 6.67 (d, J = 8.0 Hz, 1H), 5.13 (s, 1H).
13C NMR (100 MHz, CDCl3): δ 160.8, 148.2, 147.9, 146.5, 146.2,
142.7, 135.0, 133.8, 127.9, 127.8, 127.3, 127.1, 126.6, 123.9, 121.6,
120.4, 118.2, 117.1, 62.8. HRMS (ESI): calcd for C21H15N4O3 [M +
H]+, 371.1139; found, 371.1140.
1H), 8.19 (d, J = 7.9 Hz, 1H), 7.88 (t, J = 7.6 Hz, 1H), 7.84−7.73 (m,
2H), 7.55 (t, J = 7.5 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 7.12 (brs, 1H),
6.89 (d, J = 8.3 Hz, 1H), 6.66 (t, J = 7.5 Hz, 1H). 13C NMR (100
MHz, DMSO-d6): δ 162.6, 154.1, 149.9, 148.5, 135.0, 132.3, 129.3,
127.4, 126.7, 126.2, 120.9, 117.1, 115.5, 112.8.
General Procedures for the Preparation of Compounds 9a−
9m. To a solution of compound 8 (500 mg, 2.11 mmol) in [BmIm]
Br (4 mL) was added I2 (27 mg, 0.11 mmol) and corresponding
aldehydes (2.11 mmol). After completion, the reaction was quenched
C
https://dx.doi.org/10.1021/acs.jafc.0c04278
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
6-(2,4-Dichlorophenyl)-5,6-dihydro-8H-quinazolino[4,3-b]-
quinazolin-8-one (9h). It is obtained as a yellow solid, yield 82%; mp
237−239 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.25 (d, J = 7.0 Hz,
1H), 8.09 (dd, J = 7.9, 1.0 Hz, 1H), 7.91−7.85 (m, 1H), 7.79 (d, J =
8.0 Hz, 1H), 7.77−7.70 (m, 2H), 7.54−7.47 (m, 1H), 7.38 (d, J = 3.8
Hz, 1H), 7.36−7.29 (m, 1H), 7.22 (dd, J = 8.4, 2.1 Hz, 1H), 6.93−
6.83 (m, 3H). 13C NMR (100 MHz, DMSO-d6): δ 159.2, 147.7,
147.3, 143.5, 135.8, 135.1, 133.9, 133.8, 132.4, 129.6, 127.7, 127.3,
127.1, 126.9, 126.4, 119.8, 119.2, 116.2, 115.1, 60.7. HRMS (ESI):
calcd for C21H14Cl2N3O [M + H]+, 394.0508; found, 394.0509.
6-(Pyridin-4-yl)-5,6-dihydro-8H-quinazolino[4,3-b]quinazolin-8-
one (9i). It is obtained as a yellow solid, yield 84%; mp 114−116 °C;
1H NMR (400 MHz, DMSO-d6): δ 8.54−8.43 (m, 2H), 8.23 (d, J =
7.2 Hz, 1H), 8.15−8.07 (m, 2H), 7.89 (t, J = 7.7 Hz, 1H), 7.77 (d, J =
8.1 Hz, 1H), 7.55 (t, J = 7.5 Hz, 1H), 7.36 (t, J = 7.1 Hz, 1H), 7.29
(d, J = 3.6 Hz, 1H), 7.17 (d, J = 5.3 Hz, 2H), 6.98 (d, J = 8.1 Hz, 1H),
6.92−6.81 (m, 1H). 13C NMR (100 MHz, DMSO-d6): δ 160.2, 150.5,
148.6, 148.2, 147.4, 145.3, 135.6, 134.3, 127.8, 127.4, 127.2, 126.9,
121.4, 120.3, 119.7, 116.6, 116.2, 62.2. HRMS (ESI): calcd for
C20H15N4O [M + H]+, 327.1240; found, 327.1239.
6-(1H-Imidazol-5-yl)-5,6-dihydro-8H-quinazolino[4,3-b]-
quinazolin-8-one (9j). It is obtained as a yellow solid, yield 72%; mp
211−243 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.20 (d, J = 7.8 Hz,
2H), 7.88 (t, J = 7.1 Hz, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 3.6
Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.26 (d, J =
3.7 Hz, 1H), 6.98−6.83 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ
159.6, 147.8, 147.5, 144.9, 144.4, 134.8, 133.2, 127.2, 126.8, 126.5,
126.0, 120.3, 119.3, 116.4, 116.3, 58.4. HRMS (ESI): calcd for
C18H14N5O3 [M + H]+, 316.1193; found, 316.1192.
7.9 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 7.87−7.76 (m, 4H), 7.65 (t, J =
6.8 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.50−7.38 (m, 3H). 13C NMR
(100 MHz, CDCl3): δ 159.8, 146.9, 146.6, 145.3, 142.0, 136.1, 135.7,
135.4, 133.7, 131.9, 129.8, 129.3, 129.0, 128.1, 127.6, 127.5, 127.3,
126.7, 126.2, 121.7, 119.8. HRMS (ESI): calcd for C21H12Cl2N3O [M
+ H]+, 392.0352; found, 392.0353.
Preparation of 8H-Quinazolino[4,3-b]quinazolin-8-one (9o). To
a solution of compound 8 (500 mg, 2.1 mmol) in toluene (50 mL)
was added DMF-dimethylacetal (500 mg, 4.2 mmol) and acetate (0.1
mL). The mixture was stirred at room temperature for 10 h. After
completion, the mixture was diluted with water and extracted with
methyl tert-butyl ether (3 × 50 mL). The combined organic phase
was washed with brine (3 × 100 mL), dried over anhydrous Na2SO4,
filtered, and concentrated to give 9o: yellow solid, yield 89%; mp
1
200−202 °C; H NMR (400 MHz, DMSO-d6): δ 9.29 (s, 1H), 8.72
(d, J = 7.9 Hz, 1H), 8.32 (d, J = 7.9 Hz, 1H), 7.97 (t, J = 7.6 Hz, 1H),
7.90 (t, J = 7.5 Hz, 1H), 7.84 (d, J = 7.9 Hz, 2H), 7.73 (t, J = 7.5 Hz,
1H), 7.60 (t, J = 7.5 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): δ
158.7, 147.1, 144.5, 142.9, 138.1, 135.9, 133.8, 128.9, 127.7, 127.3,
127.0, 126.5, 125.4, 121.2, 118.7. HRMS (ESI): calcd for C15H10N3O
[M + H]+, 248.0818; found, 248.0820.
General Procedures for the Preparation of Compounds
12a−12h, 14, 16.26 To a solution of corresponding isatins (3.4
mmol), corresponding 2-bromide pyridines or 2-bromthiazols or 2-
brombenzothiazoles (2.1 mmol) in dry THF (50 mL) was added
NaHCO3 (0.57 g, 6.8 mmol) and Cu(OAc)2·H2O (0.14 g, 0.68
mmol). The mixture was heated at reflux for 10 h. After completion,
the mixture was diluted with water and extracted with methyl tert-
butyl ether (3 × 50 mL). The combined organic phase was washed
with brine (3 × 100 mL), dried over anhydrous Na2SO4, filtered, and
concentrated. The residue was purified by flash chromatography on a
silica gel using petroleum ether and ethyl acetate (3:1, v/v) as the
eluent to give 12a−12h, 14, 16.
11H-Pyrido[2,1-b]quinazolin-11-one (12a). It is obtained as a
yellow solid, yield 84%; mp 208−210 °C; 1H NMR (400 MHz,
CDCl3): δ 8.85 (d, J = 7.2 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 7.87−
7.72 (m, 2H), 7.54−7.40 (m, 3H), 6.91−6.80 (m, 1H). 13C NMR
(100 MHz, CDCl3): δ 159.0, 148.6, 147.8, 135.1, 134.1, 127.4, 126.9,
126.7, 126.4, 125.3, 116.3, 112.5.
2-Fluoro-11H-pyrido[2,1-b]quinazolin-11-one (12b). It is ob-
tained as a yellow solid, yield 81%; mp 164−166 °C; 1H NMR
(400 MHz, CDCl3): δ 8.89 (d, J = 7.5 Hz, 1H), 8.10 (d, J = 8.6 Hz,
1H), 7.90−7.80 (m, 1H), 7.69−7.61 (m, 1H), 7.54 (s, 2H), 6.97−
6.86 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 160.9, 158.4, 158.4
(d, J = 3.8 Hz), 147.1 (d, J = 1.9 Hz), 145.4, 133.9, 129.5 (d, J = 8.1
Hz), 126.4 (d, J = 6.3 Hz), 124.5 (d, J = 25.1 Hz), 117.0 (d, J = 8.6
Hz), 112.9, 111.2 (d, J = 23.5 Hz).
2-Chloro-11H-pyrido[2,1-b]quinazolin-11-one (12c). It is ob-
tained as a yellow solid, yield 79%; mp 122−124 °C; 1H NMR
(400 MHz, CDCl3): δ 8.81 (d, J = 7.3 Hz, 1H), 8.35 (d, J = 2.1 Hz,
1H), 7.76−7.65 (m, 2H), 7.54−7.41 (m, 2H), 6.90−6.79 (m, 1H).
13C NMR (100 MHz, CDCl3): δ 157.1, 146.8, 146.1, 134.6, 133.4,
6-(5-Methylthiophen-2-yl)-5,6-dihydro-8H-quinazolino[4,3-b]-
quinazolin-8-one (9k). It is obtained as a brown solid, yield 88%; mp
1
153−155 °C; H NMR (400 MHz, DMSO-d6): δ 8.23−8.14 (m,
2H), 7.93 (d, J = 3.0 Hz, 1H), 7.88−7.82 (m, 1H), 7.73 (d, J = 8.1
Hz, 1H), 7.54−7.48 (m, 1H), 7.45−7.34 (m, 2H), 6.98 (d, J = 8.0 Hz,
1H), 6.93−6.85 (m, 1H), 6.74 (d, J = 3.4 Hz, 1H), 6.57−6.53 (m,
1H), 2.25 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 159.1, 147.6,
146.6, 145.0, 140.2, 139.3, 135.0, 133.8, 127.2, 126.9, 126.6, 126.2,
126.0, 124.6, 119.9, 119.1, 116.1, 115.3, 59.9, 14.7. HRMS (ESI):
calcd for C20H16N3OS [M + H]+, 346.1009; found, 346.1009.
6-(5-Bromofuran-2-yl)-5,6-dihydro-8H-quinazolino[4,3-b]-
quinazolin-8-one (9l). It is obtained as a brown solid, yield 78%; mp
1
168−170 °C; H NMR (400 MHz, CD3OD): δ 8.24 (d, J = 8.0 Hz,
2H), 7.86−7.80 (m, 1H), 7.76 (d, J = 8.1 Hz, 1H), 7.50 (t, J = 7.5 Hz,
1H), 7.38 (t, J = 7.1 Hz, 1H), 7.26 (s, 1H), 6.96−6.87 (m, 2H), 6.21
(d, J = 3.4 Hz, 1H), 6.05 (d, J = 3.3 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6): δ 159.6, 154.1, 148.1, 147.2, 145.2, 135.5, 134.1, 127.8,
127.4, 127.1, 126.8, 121.9, 120.4, 119.8, 116.4, 116.0, 113.0, 111.5,
58.2. HRMS (ESI): calcd for C19H13BrN3O2 [M + H]+, 394.0186;
found, 394.0185.
6-(4-Methylthiazol-5-yl)-5,6-dihydro-8H-quinazolino[4,3-b]-
quinazolin-8-one (9m). It is obtained as a brown solid, yield 78%;
1
mp 168−170 °C; H NMR (400 MHz, DMSO-d6): δ 8.62 (s, 1H),
8.26 (d, J = 7.0 Hz, 1H), 8.13 (dd, J = 8.0, 1.1 Hz, 1H), 7.88−7.82
(m, 1H), 7.78 (d, J = 3.0 Hz, 1H), 7.73 (d, J = 7.9 Hz, 1H), 7.54−
7.45 (m, 2H), 7.45−7.39 (m, 1H), 6.97 (dd, J = 11.2, 4.0 Hz, 1H),
6.88 (d, J = 7.9 Hz, 1H), 2.63 (s, 3H). 13C NMR (100 MHz, DMSO-
d6): δ 159.0, 151.1, 150.5, 147.4, 144.2, 135.1, 134.0, 129.2, 127.2,
127.0, 126.5, 119.8, 119.5, 116.3, 115.2, 58.2, 15.3. HRMS (ESI):
calcd for C19H15N4OS [M + H]+, 347.0961; found, 347.0959.
Preparation of 6-(2,4-Dichlorophenyl)-8H-quinazolino[4,3-b]-
quinazolin-8-one (9n). The compound 9h (600 mg, 2 mmol) was
dissolved in tetrahydrofuran (THF) solution (40 mL), and the
mixture was stirred at 100 °C under an O2 atmosphere. After
completion, the mixture was diluted with water and extracted with
methyl tert-butyl ether (3 × 50 mL). The combined organic phase
was washed with brine (3 × 100 mL), dried over anhydrous Na2SO4,
filtered, and concentrated. The residue was purified by flash
chromatography on a silica gel using petroleum ether and ethyl
acetate (10:1, v/v) as the eluent to give 9n: light yellow solid, yield
127.7, 125.7, 125.4, 125.3, 116.0, 112.0.
2-Methyl-11H-pyrido[2,1-b]quinazolin-11-one (12d). It is ob-
1
tained as a yellow solid, yield 80%; mp 135−137 °C; H NMR (400
MHz, CDCl3): δ 8.88 (d, J = 7.4 Hz, 1H), 8.25 (s, 1H), 7.75−7.65
(m, 2H), 7.55−7.45 (m, 2H), 6.85 (s, 1H), 2.54 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 158.9, 147.1, 146.7, 136.9, 135.4, 133.6, 126.7,
126.7, 126.3, 126.2, 116.1, 112.3, 21.4.
2-Methoxy-11H-pyrido[2,1-b]quinazolin-11-one (12e). It is ob-
1
tained as a yellow solid, yield 79%; mp 158−160 °C; H NMR (400
MHz, CDCl3): δ 8.85 (d, J = 7.4 Hz, 1H), 7.77−7.69 (m, 2H), 7.51−
7.40 (m, 3H), 6.87−6.80 (m, 1H), 3.94 (s, 3H). 13C NMR (100
MHz, CDCl3): δ 158.7, 157.4, 146.1, 143.7, 132.8, 128.7, 126.8,
126.4, 116.9, 112.6, 105.1, 55.9.
8-(Trifluoromethyl)-11H-pyrido[2,1-b]quinazolin-11-one (12f). It
1
is obtained as a yellow solid, yield 73%; mp 155−157 °C; H NMR
(400 MHz, CDCl3): δ 9.20 (s, 1H), 8.45 (d, J = 8.1 Hz, 1H), 7.89 (t,
J = 7.7 Hz, 1H), 7.80 (d, J = 8.3 Hz, 1H), 7.58−7.46 (m, 3H). 13C
NMR (100 MHz, CDCl3): δ 157.5, 147.1, 145.6, 134.7, 127.9, 126.7,
1
61%; mp 131−133 °C; H NMR (400 MHz, CDCl3): δ 8.82 (d, J =
D
https://dx.doi.org/10.1021/acs.jafc.0c04278
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Figure 2. Synthesis of compounds 5a−5d.
Figure 3. Synthesis of compounds 9a−9m.
126.5, 126.3, 125.6 (q, J = 5.8 Hz), 125.3, 121.7 (q, J = 342.5 Hz),
115.6, 115.5.
MHz, CDCl3): δ 9.05 (d, J = 8.5 Hz, 1H), 8.45 (d, J = 7.5 Hz, 1H),
7.82 (t, J = 7.1 Hz, 1H), 7.75−7.60 (m, 2H), 7.55−7.41 (m, 3H). 13C
NMR (100 MHz, CDCl3): δ 159.7, 155.9, 146.2, 135.1, 133.9, 126.1,
125.8, 125.7, 124.9, 124.8, 122.7, 120.7, 118.2, 117.6.
8-Bromo-11H-pyrido[2,1-b]quinazolin-11-one (12g). It is ob-
1
tained as a yellow solid, yield 76%; mp 162−164 °C; H NMR (400
Biological Assay. Each test was repeated three times at 25
1
MHz, CDCl3): δ 8.99 (d, J = 1.8 Hz, 1H), 8.44 (dd, J = 8.2, 1.1 Hz,
1H), 7.88−7.82 (m, 1H), 7.77 (d, J = 8.3 Hz, 1H), 7.54−7.46 (m,
2H), 7.38 (d, J = 9.6 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 158.0,
148.2, 145.9, 137.5, 135.3, 127.5, 127.4, 127.2, 126.6, 126.0, 116.3,
107.7.
°C. Active effect expressed in the percentage scale of 0−100 (0: no
activity; 100: total inhibited).
Specific steps for the anti-TMV and fungicidal activity tests and
mode of action studies were carried out using the literature
method,9,27 which is also seen in Supporting Information.
Ningnanmycin aqueous solution (2%) and 99% of ribavirin were
purchased from Beijing Hwrkchemical Co., Ltd.
8-Methyl-11H-pyrido[2,1-b]quinazolin-11-one (12h). It is ob-
1
tained as a yellow solid, yield 85%; mp 148−150 °C; H NMR (400
MHz, DMSO-d6): δ 8.63 (s, 1H), 8.31 (d, J = 8.1 Hz, 1H), 7.89 (t, J =
7.7 Hz, 1H), 7.74 (d, J = 8.3 Hz, 1H), 7.62 (d, J = 9.3 Hz, 1H), 7.54−
7.46 (m, 2H), 2.34 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 163.1,
153.3, 151.7, 143.3, 139.9, 131.8, 130.5, 130.0, 128.2, 127.8, 125.7,
120.8, 22.9.
RESULTS AND DISCUSSION
■
Chemistry. In order to investigate the effect of substituents
of quinoline ring on the biological activity, luotonin A (5a) and
the designed compounds 5b−5d were synthesized (Figure 2)
via the reported method with small modification.25 Quinazo-
linone 2 was synthesized by condensation of o-amino-
benzoamide with diethyl oxalate. Ammonolysis of 2 with
corresponding amines gave compounds 3a−3d. The key
intermediates 4a−4d were obtained by the reaction of
compounds 3a−3d with propargyl bromide. The Ph3PO-
5H-Thiazolo[2,3-b]quinazolin-5-one (14). It is obtained as a
yellow solid, yield 89%; mp 142−144 °C; 1H NMR (400 MHz,
CDCl3): δ 8.39 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 5.0 Hz, 1H), 7.81 (t, J
= 7.1 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 6.85
(d, J = 5.1 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 158.7, 158.1,
148.4, 135.0, 127.1, 126.2, 125.4, 121.2, 116.5, 109.5.
12H-Benzo[4,5]thiazolo[2,3-b]quinazolin-12-one (16). It is ob-
1
tained as a white solid, yield 87%; mp 188−190 °C; H NMR (400
E
https://dx.doi.org/10.1021/acs.jafc.0c04278
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Table 1. In Vivo Antiviral Activities of Compounds 5a−5d,
9a−9o, 12a−12h, 14, 16, Ningnanmycin, and Ribavirin
against TMV
Figure 4. Synthesis of compounds 9n and 9o.
Figure 5. Synthesis of compounds 12a−12h.
Figure 6. Synthesis of compounds 14 and 16.
catalyzed tandem cyclization of 4a−4d gave compounds 5a−
5d. Solubility has always been a barrier to the application of
polycyclic aromatic hydrocarbons. To improve the solubility of
luotonin A, the 6-aryl-5H-quinazolino[4,3-b]quinazolin-
8(6H)-one derivatives 9a−9m (Figure 3) were designed and
synthesized using ionic liquid as the solvent.28 Condensation
of o-aminobenzoamide with o-nitrobenzoyl chloride gave 6,
which underwent cyclization in the presence of KOH and gave
compound 7. Compound 7 was reduced and then condensed
with corresponding aldehydes to obtain compounds 9a−9m.
To explore the effect of N−H bond on biological activity, the
oxidation products 9n and 9o were synthesized (Figure 4).
Reducing the number of rings is the most effective way to
simplify the molecular structure. In order to study the number
of rings on the biological activity, we designed and synthesized
12a−12h, 14, and 16, a series of 3/4-membered ring
derivatives, by condensation of 2-bromo heterocycles with
isatin (Figures 5 and 6).26
a
Average of three replicates; all results are expressed as mean SD;
activity data with prominent were presented in pink bold with blue
color.
to crops, NYT 1965.1-2010), showed that the luotonin A and
its derivatives 5a−5d, 9a−9o, 12a−12h, 14, and 16 were safe
for testing on plants at 500 μg/mL.
Antiviral Activity In Vivo. The activities of luotonin A and
its derivatives 5a−5d, 9a−9o, 12a−12h, 14, and 16 against
TMV are listed in Table 1 with commercial viral inhibitors
ningnanmycin and ribavirin as controls. The inactive activities
Phytotoxic Activity. The phytotoxic activity tests
(according to the criterion of safety evaluation of pesticide
F
https://dx.doi.org/10.1021/acs.jafc.0c04278
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Figure 7. TMV rod assembly inhibition of compound 9k (200 nm
scale bar): (A) 20S CP disk + RNA, (B) 20S CP disk + RNA + 1/100
DMSO, (C) 20S CP disk + RNA + 10 μM 9k.
Figure 8. 20S CP disk assembly inhibition of compound 9k (100 nm
scale bar): (A) CP, (B) CP + 1/100 DMSO, (C) CP + 10 μM 9k.
of all the compounds were first tested at 500 μg/mL, and the
curative activities and protective activities of compounds with
good inactive activities (inactive effect > 40%) were further
tested.
As shown in Table 1, most of these compounds exhibited
better antiviral activities than ribavirin. What is more,
compounds 9i, 9k, 9l, 12b, and 12d showed about similar
level of antiviral activities with ningnanmycin, which may be
the most active antiviral agent at present. Natural product
luotonin A (5a) displayed similar antiviral activity (inhibitory
rates of 41, 45, and 36% at 500 μg/mL for inactivation,
curative, and protection activities in vivo, respectively) with
ribavirin (inhibitory rates of 40, 37, and 40% at 500 μg/mL for
inactivation, curative, and protection activities in vivo,
respectively). The effect of introducing push−pull electronic
groups into the A-ring on the activity is not obvious, and
compounds 5a, 5c, and 5d exhibited about similar level of
antiviral activities, and the activity of 5b is slightly lower. The
increase of solubility is beneficial to improve the biological
activity of luotonin A, and most of the four-membered fused
ring compounds 9a−9o showed better biological activities than
luotonin A. Among 9a−9o, compounds 9a−9d containing
hydrogen or alkyl groups at 6-postion showed better inhibitory
activities than luotonin A. The substitution of alkyl with the
benzene ring resulted in a significant decrease in activity
(inhibitory effect: 9a−9d > 9e). The introduction of electron-
Figure 9. Molecule docking results of compounds 9k (A), 12b (B),
and 12d (C) with TMV CP.
withdrawing group into the 6-position benzene ring is
beneficial for the improvement of activity (inhibitory effect:
9g and 9g ≈ 5a > 9e). The addition of pyridine, imidazole,
thiophene, or furan rings at 6-position is beneficial to the
activity improvement (inhibitory effect: 9f, 9i, 9j, 9k, 9l > 9a).
Compound 9m with the thiazole ring at 6-position showed less
activity than 9a. By comparing 9h and 9n aand 9a and 9o
(inhibitory effect: 9h > 9n, 9a > 9o), it can be found that the
N−H bond has a great influence on biological activity. Linear
quaternary-fused ring compound 16 showed the same level of
activity as luotonin A. By further reducing the number of fused
rings, the ternary-fused ring compounds 12a−12h and 14 were
obtained. Compounds 12a, 12b, and 12d with more simple
structures also exhibited significantly higher antiviral activities
than luotonin A. The activity of compound 12h is similar to
G
https://dx.doi.org/10.1021/acs.jafc.0c04278
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
H
https://dx.doi.org/10.1021/acs.jafc.0c04278
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
that of luotonin A. Compounds 12c, 12e, 12f, 12g, and 14
displayed significantly lower activity than luotonin A. From the
above activity data, it can be seen that for ternary-fused ring
compounds, hydrogen, fluorine, and methyl at the 2-position
are beneficial to the activity while the introduction of
substituents at 8-position is unfavorable to the activity. From
the above structure−activity relationship, it can be found that
small structural changes can lead to large changes in activity.
The optimized compounds 9k, 12b, and 12d may have
stronger interaction with virus particles, showing excellent
antiviral activity, which can be used as a new antiviral
candidate for further research. In order to confirm the above
conjecture, we further studied the mechanism of action.
Preliminary Mode of Action. We chose compound 9k to
study preliminary antiviral mechanism research using our
reported method.5 The test results show that the 20S CP disk
can be assembled with RNA to form rod-shaped TMV virus
particles with a length of about 300 nm (Figure 7A). A small
amount of dimethyl sulfoxide (DMSO) does not inhibit the
assembly of virus particles (Figure 7B). Compound 9k can
cause a reduction in the length and number of virus particles,
indicating that they can inhibit the assembly of viruses (Figure
7C). At the same time, we can see from Figure 7C that the
TMV particles are broken, fused, and aggregated, indicating
that compound 9k is likely to act on the TMV CP. We further
designed 20S CP disk assembly experiments to test the
interaction between 9k and 20S CP disk. The TMV protein
can form the disc structure (Figure 8A), and a small amount of
DMSO does not affect the formation process (Figure 8B).
Compound 9k does interact with TMV CP, leading to its
polymerization.
ring expansion and ring contraction strategies. The anti-TMV
and fungicidal activities and structure−activity relationships of
these compounds were systematically evaluated. The increase
of solubility is beneficial to improve the biological activity of
luotonin A, and most of the four-membered fused ring
compounds 9a−9o showed better biological activities than
luotonin A. For ternary-fused ring compounds, hydrogen,
fluorine, and methyl at the 2-position are beneficial to the
activity while the introduction of substituents at the 8-position
is unfavorable to the activity. The optimized compounds 9k,
12b, and 12d may have stronger interaction with virus
particles, showing excellent antiviral activity, which can be used
as a new antiviral candidate for further research. Further
antiviral mechanism research revealed that 9k can inhibit the
assembly of TMV particles by hydrogen bonding interaction
with TMV CP. These compounds also showed broad spectrum
inhibitory activities against all 14 plant pathogens. Structurally
simplified compound 14 with prominent fungicidal activity
emerged as a lead for further research. This work broadened
the biological activity spectrum of alkaloid luotonin A and laid
a foundation for the application of these compounds in plant
virus protection.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c04278.
Detailed bio-assay procedures and spectra data of
luotonin A and its derivatives 5a−5d, 9a−9o, 12a−
12h, 14, and 16 (PDF)
Molecular Docking. To further explore the interaction of
9k, 12b, 12d, and TMV CP (PDB code: 1EI7), we performed
molecular docking using AutoDock-vina 1.1.2.29 Compound
9k forms one conventional hydrogen bond with the active site
of TYR 139 at a distance of 3.4 c5 (Figure 9A). Compound
12b forms two conventional hydrogen bonds with amino acids
ASN 73 (2.2 c5) and GLY 137 (2.0 c5). Compound 12d forms
three conventional hydrogen bonds with amino acids ASN 73
(2.3 c5), GLY 137 (2.3 c5), and THR 136 (3.2 c5). The
molecular docking results indicate that these compounds
interact with CP through hydrogen bonding.
Fungicidal Activity. Luotonin A and its derivatives 5a−
5d, 9a−9o, 12a−12h, 14, and 16 were also evaluated for their
fungicidal activities with commercial fungicides, carbendazim,
chlorothalonil, and pyrimethanil, as controls (Table 2). At the
concentration of 50 μg/mL, luotonin A and its derivatives
showed broad spectrum inhibitory activity against all 14 plant
pathogens. As a whole, most of the derivatives showed good
inhibitory effect on Physalospora piricola, especially the
inhibitory rates of compounds 9g, 9m, 12e, and 12h were
over 80%. Compounds 9c and 9g exhibited higher fungicidal
activities than pyrimethanil against Rhizoctonia cerealis.
Compound 9k showed better inhibition effect than commer-
cial fungicides chlorothalonil, carbendazim, and pyrimethanil
on Phytophthora capsici with an inhibition rate of 89%. The
fungicidal activities of compounds 12b and 14 are higher than
that of carbendazim and pyrimethanil against Botrytis cinereal.
The ternary-fused ring compound 14 did not give good
antiviral activity, but its fungicidal activities are very prominent,
which can be used as a lead for further research.
AUTHOR INFORMATION
■
Corresponding Authors
Ziwen Wang − Tianjin Key Laboratory of Structure and
Performance for Functional Molecules, College of Chemistry,
Tianjin Normal University, Tianjin 300387, China;
orcid.org/0000-0001-9798-7021; Phone: 0086-22-
23766531; Email: hxxywzw@tjnu.edu.cn; Fax: 0086-22-
23766531
Qingmin Wang − State Key Laboratory of Elemento-Organic
Chemistry, Research Institute of Elemento-Organic Chemistry,
College of Chemistry, Collaborative Innovation Center of
Chemical Science and Engineering (Tianjin), Nankai
University, Tianjin 300071, China; orcid.org/0000-0002-
6062-3766; Phone: 0086-22-23503952; Email: wangqm@
nankai.edu.cn; Fax: 0086-22-23503952
Authors
Yanan Hao − Tianjin Key Laboratory of Structure and
Performance for Functional Molecules, College of Chemistry,
Tianjin Normal University, Tianjin 300387, China; State Key
Laboratory of Elemento-Organic Chemistry, Research Institute
of Elemento-Organic Chemistry, College of Chemistry,
Collaborative Innovation Center of Chemical Science and
Engineering (Tianjin), Nankai University, Tianjin 300071,
China
Kaihua Wang − State Key Laboratory of Elemento-Organic
Chemistry, Research Institute of Elemento-Organic Chemistry,
College of Chemistry, Collaborative Innovation Center of
Chemical Science and Engineering (Tianjin), Nankai
University, Tianjin 300071, China
In summary, based on our work experience, a series of
luotonin A derivatives were designed and synthesized through
I
https://dx.doi.org/10.1021/acs.jafc.0c04278
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
as novel agents against aplant virus. J. Agric. Food Chem. 2019, 67,
1795−1806.
(12) Wang, Y.; Zhang, J.; He, F.; Gan, X.; Song, B.; Hu, D. Design,
synthesis, bioactivity and mechanism of dithioacetal derivatives
containing dioxyether moiety. Bioorg. Med. Chem. Lett. 2019, 29,
2218−2223.
(13) Zhao, L.; Zhang, J.; Liu, T.; Mou, H.; Wei, C.; Hu, D.; Song, B.
Design, synthesis and antiviral activity of coumarin derivatives
containing dithioacetal structures. J. Agric. Food Chem. 2020, 68,
975−981.
(14) Shang, X.-F.; Morris-Natschke, S. L.; Liu, Y.-Q.; Guo, X.; Xu,
X.-S.; Goto, M.; Li, J.-C.; Yang, G.-Z.; Lee, K.-H. Biologically active
quinoline and quinazoline alkaloids part I. Med. Res. Rev. 2018, 38,
775−828.
(15) Nomura, T.; Ma, Z.-Z.; Hano, Y.; Chen, Y.-J. Two new
pyrroloquinazolinoquinoline alkaloids from Peganum nigellastrum.
Heterocycles 1997, 46, 541−546.
(16) Cagir, A.; Jones, S. H.; Gao, R.; Eisenhauer, B. M.; Hecht, S. M.
Luotonin A. A naturally occurring human DNA topoisomerase I
poison. J. Am. Chem. Soc. 2003, 125, 13628−13629.
(17) Cagir, A.; Eisenhauer, B. M.; Gao, R.; Thomas, S. J.; Hecht, S.
M. Synthesis and topoisomerase I inhibitory properties of luotonin A
analogues. Bioorg. Med. Chem. Lett. 2004, 12, 6287−6299.
(18) Ma, Z.; Hano, Y.; Nomura, T.; Chen, Y. Novel quinazoline−
quinoline alkaloids with cytotoxic and DNA topoisomerase II
inhibitory activities. Bioorg. Med. Chem. Lett. 2004, 14, 1193−1196.
(19) Almansour, A. I.; Arumugam, N.; Suresh Kumar, R.;
Yuxiu Liu − State Key Laboratory of Elemento-Organic
Chemistry, Research Institute of Elemento-Organic Chemistry,
College of Chemistry, Collaborative Innovation Center of
Chemical Science and Engineering (Tianjin), Nankai
University, Tianjin 300071, China
Dejun Ma − State Key Laboratory of Elemento-Organic
Chemistry, Research Institute of Elemento-Organic Chemistry,
College of Chemistry, Collaborative Innovation Center of
Chemical Science and Engineering (Tianjin), Nankai
University, Tianjin 300071, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c04278
Funding
This study was supported by the Natural Science Fund of
China (21772145, 21732002, 21772104) and Training
Program of Outstanding Youth Innovation Team of Tianjin
Normal University.
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Godfray, H. C. J.; Beddington, J. R.; Crute, I. R.; Haddad, L.;
Lawrence, D.; Muir, J. F.; Pretty, J.; Robinson, S.; Thomas, S. M.;
Toulmin, C. Food security: the challenge of feeding 9 billion people.
Science 2010, 327, 812−818.
́
Mahalingam, S. M.; Sau, S.; Bianchini, G.; Menendez, J. C.; Altaf,
M.; Ghabbour, H. A. Design, synthesis and antiproliferative activity of
decarbonyl luotonin analogues. Eur. J. Med. Chem. 2017, 138, 932−
941.
(2) Rodríguez, J. P.; Rahman, H.; Thushar, S.; Singh, R. K. Healthy
and resilient cereals and pseudo-cereals for marginal agriculture:
Molecular advances for improving nutrient bioavailability. Front.
Genet. 2020, 11, 49.
(3) Foley, J. A.; Ramankutty, N.; Brauman, K. A.; Cassidy, E. S.;
Gerber, J. S.; Johnston, M.; Mueller, N. D.; O’Connell, C.; Ray, D. K.;
West, P. C.; Balzer, C.; Bennett, E. M.; Carpenter, S. R.; Hill, J.;
(20) An, T.-y.; Huang, R.-q.; Yang, Z.; Zhang, D.-k.; Li, G.-r.; Yao,
Y.-c.; Gao, J. Alkaloids from cynanchum komarovii with inhibitory
activity against the tobacco mosaic virus. Phytochemistry 2001, 58,
1267−1269.
(21) Wang, Z.; Li, Z.; Wang, K.; Wang, Q. Efficient and chirally
specific synthesis of phenanthroindolizidine alkaloids by Parham-type
cycloacylation. Eur. J. Org. Chem. 2010, 2010, 292−299.
(22) Wang, Z.; Wei, P.; Liu, Y.; Wang, Q. D and E rings may not be
indispensable for antofine: discovery of phenanthrene and alkylamine
chain containing antofine derivatives as novel antiviral agents against
tobacco mosaic virus (TMV) based on interaction of antofine and
TMV RNA. J. Agric. Food Chem. 2014, 62, 10393−10404.
(23) Wang, K.; Su, B.; Wang, Z.; Wu, M.; Li, Z.; Hu, Y.; Fan, Z.; Mi,
N.; Wang, Q. Synthesis and antiviral activities of phenanthroindoli-
zidine alkaloids and their derivatives. J. Agric. Food Chem. 2010, 58,
2703−2709.
(24) Wu, M.; Han, G.; Meng, C.; Wang, Z.; Liu, Y.; Wang, Q.
Design, synthesis, and anti-tobacco mosaic virus (TMV) activity of
glycoconjugates of phenanthroindolizidines alkaloids. Mol. Divers.
2014, 18, 25−37.
(25) Zhou, H.-B.; Liu, G.-S.; Yao, Z.-J. Short and efficient total
synthesis of luotonin A and 22-hydroxyacuminatine using a common
cascade strategy. J. Org. Chem. 2007, 72, 6270−6272.
(26) Liu, M.; Shu, M.; Yao, C.; Yin, G.; Wang, D.; Huang, J.
Synthesis of pyrido-fused quinazolinone derivatives via copper-
catalyzed domino reaction. Org. Lett. 2016, 18, 824−827.
(27) Lv, P.; Chen, Y.; Shi, T.; Wu, X.; Li, Q. X.; Hua, R. Synthesis
and fungicidal activities of sanguinarine derivatives. Pestic. Biochem.
Phys. 2018, 147, 3−10.
̈
Monfreda, C.; Polasky, S.; Rockstrom, J.; Sheehan, J.; Siebert, S.;
Tilman, D.; Zaks, D. P. M. Solutions for a cultivated planet. Nature
2011, 478, 337−342.
(4) Ge, Y.-h.; Liu, K.-x.; Zhang, J.-x.; Mu, S.-z.; Hao, X.-j. The
limonoids and their antitobacco mosaic virus (TMV) activities from
Munronia unifoliolate Oliv. J. Agric. Food Chem. 2012, 60, 4289−
4295.
(5) Lu, A.; Wang, T.; Hui, H.; Wei, X.; Cui, W.; Zhou, C.; Li, H.;
Wang, Z.; Guo, J.; Ma, D.; Wang, Q. Natural products for drug
discovery: discovery of gramines as novel agents against a plant virus.
J. Agric. Food Chem. 2019, 67, 2148−2156.
(6) Rodrigues, T.; Reker, D.; Schneider, P.; Schneider, G. Counting
on natural products for drug design. Nat. Chem. 2016, 8, 531−541.
(7) Wu, W.; Tang, Y.; Yang, J.; Idehen, E.; Sang, S. Avenanthramide
aglycones and glucosides in oat bran: Chemical profile, levels in
commercial oat products, and cytotoxicity to human colon cancer
cells. J. Agric. Food Chem. 2018, 66, 8005−8014.
(8) Wang, T.; Yang, S.; Li, H.; Lu, A.; Wang, Z.; Yao, Y.; Wang, Q.
Discovery, structural optimization and mode of action of essramycin
alkaloid and its derivatives as anti-tobacco mosaic virus (TMV) and
anti-phytopathogenic-fungus agents. J. Agric. Food Chem. 2020, 68,
471−484.
(9) Guo, J.; Hao, Y.; Ji, X.; Wang, Z.; Liu, Y.; Ma, D.; Li, Y.; Pang,
H.; Ni, J.; Wang, Q. Optimization, structure-activity relationship and
mode of action of nortopsentin analogues containing thiazole and
oxazole moieties. J. Agric. Food Chem. 2019, 67, 10018−10031.
(10) Ji, X.; Wang, Z.; Dong, J.; Liu, Y.; Lu, A.; Wang, Q. Discovery
of topsentin alkaloids and their derivatives as novel antiviral and anti-
phytopathogenic fungus agents. J. Agric. Food Chem. 2016, 64, 9143−
9151.
(28) Ma, Y.-G.; Zhang, Y.; Feng, B.-B.; Wang, X.-S. Synthesis of 6-
aryl-5H-quinazolino [4, 3-b] quinazolin-8 (6H)-one derivatives in
ionic liquids catalyzed by iodine. Res. Chem. Intermed. 2016, 42,
1045−1055.
(29) Trott, O.; Olson, A. J. Autodock vina: Improving the speed and
accuracy of docking with a new scoring function, efficient
optimization, and multithreading. J. Comput. Chem. 2010, 31, 455−
461.
(11) Liu, B.; Li, R.; Li, Y.; Li, S.; Yu, J.; Zhao, B.; Liao, A.; Wang, Y.;
Wang, Z.; Lu, A.; Liu, Y.; Wang, Q. Discovery of pimprinine alkaloids
J
https://dx.doi.org/10.1021/acs.jafc.0c04278
J. Agric. Food Chem. XXXX, XXX, XXX−XXX