Discovery of Tryptanthrins as Novel Antiviral and Anti-Phytopathogenic-Fungus Agents

Article abstract of DOI:10.1021/acs.jafc.0c02101

Plant diseases seriously affect the yield and quality of crops and are difficult to control. Tryptanthrin and its derivatives (tryptanthrins) were synthesized and evaluated for their antiviral activities and fungicidal activities. We found that tryptanthrins have good antiviral activities against tobacco mosaic virus (TMV) for the first time. Most of the tryptanthrins showed higher anti-TMV activities than that of ribavirin (inhibitory rates of 40, 37, and 38% at 500 μg/mL for inactivation, curative, and protection activities in vivo, respectively). Compound 3n (inhibitory rates of 52, 49, and 54% at 500 μg/mL for inactivation, curative, and protection activities in vivo, respectively) and compound 14 (inhibitory rates of 51, 48, and 53% at 500 μg/mL for inactivation, curative, and protection activities in vivo, respectively) emerged as new antiviral lead compounds with excellent antiviral activities. Compound 16 was selected for further antiviral mechanism research, which revealed that compound 16 could inhibit virus assembly by decomposing 20S coat protein (CP) disk. Molecular docking results showed that compounds 3n and 14, which have higher antiviral activities in vivo than that of compound 16, do show stronger interaction with TMV CP. Further fungicidal activity tests showed that tryptanthrins displayed broad-spectrum fungicidal activities, especially for compound 16. These compounds showed good selectivity to Physalospora piricola. In the current study, a small molecular library of tryptanthrin was constructed and the bioactivity spectrum of these compounds was broadened, which lays a foundation for their application in plant protection.

Full text of DOI:10.1021/acs.jafc.0c02101

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Agricultural and Environmental Chemistry
Discovery of Tryptanthrins as Novel Antiviral
and Anti-phytopathogenic-fungus Agents
Yanan Hao, Jincheng Guo, Ziwen Wang, Yuxiu Liu, Yongqiang Li, Dejun Ma, and Qingmin Wang
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 01 May 2020
Downloaded from pubs.acs.org on May 2, 2020
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Page 1 of 36
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Discovery
of
Tryptanthrins
as
Novel
Antiviral
and
Anti-phytopathogenic-fungus Agents
†
†
†,‡,
†,
†
†
Yanan Hao , Jincheng Guo , Ziwen Wang *, Yuxiu Liu *, Yongqiang Li , Dejun Ma ,
†,
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;
‡Tianjin Key Laboratory of Structure and Performance for Functional Molecules, College of
Chemistry, Tianjin Normal University, Tianjin 300387, China;
*
To whom correspondence should be addressed. For Ziwen Wang, E-mail: hxxywzw@tjnu.edu.cn;
Phone: 0086-22-23766531; Fax: 0086-22-23766531; For Yuxiu Liu, E-mail: liuyuxiu@nankai.edu.cn;
Phone: 0086-22-23503792; Fax: 0086-22-23503792; For Qingmin Wang, E-mail:
wangqm@nankai.edu.cn; Phone: 0086-22-23503952; Fax: 0086-22-23503952.
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ABSTRACT: Plant diseases seriously affect the yield and quality of crops and are
difficult to control. Tryptanthrin and it’s derivatives (tryptanthrins) were synthesized and
evaluated for their antiviral activities and fungicidal activities. We found that
tryptanthrins have good antiviral activities against tobacco mosaic virus (TMV) for the
first time. Most of the tryptanthrins showed higher anti-TMV activities than ribavirin
(inhibitory rate: 40%, 37%, 38% at 500 μg/mL for inactivation, curative, and protection
activity in vivo, respectively). Compounds 3n (inhibitory rate: 52%, 49%, 54% at 500
μg/mL for inactivation, curative, and protection activity in vivo, respectively) and 14
(inhibitory rate: 51%, 48%, 53% at 500 μg/mL for inactivation, curative, and protection
activity in vivo, respectively) emerged as new antiviral lead compounds with excellent
antiviral activities. Compound 16 was selected for further antiviral mechanism research,
which revealed that 16 could inhibit virus assembly by decomposing 20S coat protein
(CP) disk. Molecular docking results showed that compounds 3n and 14, which have
higher antiviral activities in vivo than compound 16, do show stronger interaction with
TMV CP. Further fungicidal activity tests showed that tryptanthrins displayed
broad-spectrum fungicidal activities, especially for 16. These compounds showed good
selectivity to a Physalospora piricola. In current study, a small molecular library of
tryptanthrin was constructed and the bio-activity spectrum of these compounds was
broadened, which lays a foundation for their application in plant protection.
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KEYWORDS: lead discovery, structure optimization, tryptanthrin analogues, antiviral
activity, fungicidal activity, mode of action
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INTRODUCTION
Since the beginning of the 21st century, the total population of the world has
exceeded 7 billion. Study suggests that the population is expected to reach 11.3 billion by
the end of the century, and that the actual population will be much larger than currently
1
projected. Food is a basic human need, and meeting human demand for food while
2
maintaining healthy ecosystems is a fundamental challenge of this century. Plant
diseases, caused by viruses and fungi, seriously affect the yield and quality of crops and
are difficult to control. Tobacco mosaic virus (TMV) is one of the most widely studied
plant viruses, which can cause deformation and stunting of the leaves, flowers and fruits
3
of infected plants. Diseases caused by TMV are difficult to control because TMV shows
absolute parasitic and transmissibility to host cells and plants lack a complete immune
3
system. Although several commercial antiviral agents against TMV have been used,
efficient and practical varieties are few. The widely used antiviral agent ribavirin only
gave less than 50% anti-TMV effect at 500 μg/mL. The developing novel structure,
remarkable effect and environmentally friendly anti-TMV agents are needed urgently.3,4
On the other hand, the research on the mechanism of anti plant virus is not deep enough,
and it is difficult to design a new type of anti-virus agent based on target.5
Natural products are important source for development of novel pesticides with
unique mechanism due to their immense structural diversity and wide variety of
biological activities.6-8 Since Duggar and Armstrong found that pokeweed juice
9
(Phytolacca acinosa) could inhibit TMV in 1925, many other natural products and their
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derivatives with anti-TMV activities have been reported,10-15 however, there are only a
few reported economically viable antiviral chemicals available for practical application
in plant protection. Tryptanthrin, a kind of alkaloid with indolequinazolinone structure,
mainly exists in blue plants such as marblea and indigo.16,17 Studies revealed that
tryptanthrin and its derivatives have a wide range of biological activities, such as
1
8
19
20
anticancer activity, anti-inflammatory activity, insecticidal activity and anti-allergy
2
1
activity .
Our research group has long been committed to the discovery of new and efficient
antiviral lead compounds based on natural products. Considering the above findings and
our work experiences, we designed (Figure 1) and synthesized a series of tryptanthrin
derivatives. The antiviral activities, structure-activity relationship and mechanism of
these compounds were systematically evaluated. Moreover, the fungicidal activities of
these compounds were also tested.
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. Ribavirin was purchased from Beijing Hwrkchemical Company Limited with 99%
purity.
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 CDCl
3
, CD
3
OD or DMSO-d as the solvent.
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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 CP disk assembly inhibition
were tested via transmission electron microscopy (Tecnai G2 F20).
General Procedures for the Preparation of Compounds 3a–3i. A mixture of isatoic
anhydride (0.5 g, 3.4 mmol), corresponding isatin derivatives 2a–2i (3.4 mmol), and
triethylamine (2.4 mL, 17 mmol) in toluene (40 mL) was refluxed for 6 h. After cooling,
the reaction mixture was concentrated in vacuo. The resulting precipitates were filtrated.
The filtrate was taken into ethyl acetate (250 mL) and washed with brine (2 ×100 mL).
The organic phase was dried over anhydrous MgSO and evaporated under reduced
4
pressure. The residue was purified by flash column chromatography on silica gel (1:1
PE/EA) to give 3a–3i.
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Indolo[2,1-b]quinazoline-6,12-dione (3a). Yellow solid, yield 95%; mp 267−269 °C; H
NMR (400 MHz, DMSO-d
J = 3.8 Hz, 2H), 7.91 – 7.84 (m, 2H), 7.78 – 7.69 (m, 1H), 7.48 (t, J = 7.5 Hz, 1H). C
NMR (100 MHz, DMSO-d ) δ 182.4, 157.7, 146.4, 145.9, 145.0, 137.7, 135.1, 129.8,
6
) δ 8.47 (d, J = 7.9 Hz, 1H), 8.31 (d, J = 7.8 Hz, 1H), 7.94 (d,
1
3
6
126.9, 124.7, 123.2, 122.8, 117.0.
8-Fluoroindolo[2,1-b]quinazoline-6,12-dione (3b). Yellow solid, yield 95%; mp
1
282−284 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.52 – 8.45 (m, 1H), 8.32 (d, J = 7.8 Hz,
1H), 7.98 – 7.92 (m, 2H), 7.82 – 7.70 (m, 3H).
8-Chloroindolo[2,1-b]quinazoline-6,12-dione (3c). Yellow solid, yield 71%; mp
1
296−298 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.48 (d, J = 8.4 Hz, 1H), 8.34 (d, J = 7.8
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Hz, 1H), 8.02 –7.88 (m, 4H), 7.78 – 7.72 (m, 1H).
8-Bromoindolo[2,1-b]quinazoline-6,12-dione (3d). Yellow solid, yield 84%; mp
1
260−262 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.41 (d, J = 8.4 Hz, 1H), 8.33 (d, J = 7.8
Hz, 1H), 8.09 – 8.04 (m, 2H), 7.97 (d, J = 3.7 Hz, 2H), 7.78 – 7.73 (m, 1H).
8-Methoxyindolo[2,1-b]quinazoline-6,12-dione (3e). Brown solid, yield 75%; mp
1
268−270 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.75 – 8.67 (m, 2H), 8.56 (d, J = 2.2 Hz,
1H), 8.37 (d, J = 7.8 Hz, 1H), 8.00 (d, J = 3.8 Hz, 2H), 7.81 – 7.77 (m, 1H).
8-Nitroindolo[2,1-b]quinazoline-6,12-dione (3f). Red solid, yield 43%; mp 270−272 °C;
1H NMR (400 MHz, DMSO-d
) δ 8.37 (d, J = 8.5 Hz, 1H), 8.30 (d, J = 7.9 Hz, 1H), 7.93
6
(d, J = 3.7 Hz, 2H), 7.73 (dt, J = 8.2, 4.2 Hz, 1H), 7.45 – 7.39 (m, 2H), 3.87 (s, 3H).
8-Methylindolo[2,1-b]quinazoline-6,12-dione (3g). Yellow solid, yield 67%; mp
1
256−258 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.38 – 8.29 (m, 2H), 7.95 (d, J = 3.8 Hz,
2H), 7.78 – 7.71 (m, 1H), 7.71 – 7.63 (m, 2H), 2.42 (s, 3H).
7-Chloroindolo[2,1-b]quinazoline-6,12-dione (3h). Yellow solid, yield 90%; mp
1
250−252 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.48 (d, J = 7.8 Hz, 1H), 8.33 (d, J = 7.8
1
1
1
1
1
1
00
01
02
03
04
05
Hz, 1H), 7.97 (d, J = 3.7 Hz, 2H), 7.84 (t, J = 8.1 Hz, 1H), 7.79 – 7.72 (m, 1H), 7.52 (d, J
= 7.9 Hz, 1H).
7-Bromoindolo[2,1-b]quinazoline-6,12-dione (3i). Yellow solid, yield 90%; mp >
1
300 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.53 (d, J = 7.9 Hz, 1H), 8.33 (d, J = 7.9 Hz,
13
1H), 8.00 – 7.94 (m, 2H), 7.79 – 7.72 (m, 2H), 7.70 – 7.66 (m, 1H). C NMR (100 MHz,
3
CDCl ) δ 180.5, 158.1, 148.1 146.9, 145.1, 138.8, 135.8, 131.6, 130.4, 127.5, 123.6,
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121.1, 120.1, 116.6.
Preparation of 8-Hydroxyindolo[2,1-b]quinazoline-6,12-dione (3j). To a solution of
compound 3e (2 g, 7.2 mmol) in dry CH Cl (100 mL) was added AlCl (5 g, 37.6 mmol).
The mixture was refluxed under Ar atmosphere. After the reaction was completed, the
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resulting precipitates were collected by filtration in vacuo and were washed with CH
Cl
2 2
1
(3× 100 mL) to provide compound 3j: orange solid, yield 70%; mp > 300 °C; H NMR
(400 MHz, DMSO-d
6
) δ 10.25 – 10.21 (m, 1H), 8.28 (d, J = 6.6 Hz, 2H), 7.95 – 7.89 (m,
1
3
2H), 7.75 – 7.66 (m, 1H), 7.26 – 7.19 (m, 1H), 7.15 (s, 1H). C NMR (100 MHz,
DMSO-d
6
) δ 183.0, 157.6, 156.8, 146.9, 145.8, 139.1, 135.3, 130.3, 130.2, 127.2, 124.5,
+
123.9, 123.9, 118.8, 110.6. C15
H
9 2
N O
3
[M+H] 265.0608, found 265.0605.
Preparation of 8-Aminoindolo[2,1-b]quinazoline-6,12-dione (3k). To a solution of 3f
(1.0 g, 3.4 mmol) in ethanol was added SnCl ·2H O (3.8 g, 17 mmol), and then the
2
2
mixture was refluxed. After completion, the solvent was concentrated under reduced
pressure. Then the mixture diluted with 200 mL of EtOAc, and the organic phases were
washed with brine (3 × 100 mL). The organic layer was dried with MgSO
4
, filtrated, and
1
concentrated to provide compound 3k: black green solid, yield 83%; mp > 300 °C; H
NMR (400 MHz, DMSO-d ) δ 8.27 (d, J = 7.6 Hz, 1H), 8.14 (dd, J = 7.8, 1.2 Hz, 1H),
6
7.91 – 7.89 (m, 2H), 7.72 – 7.68 (m, 1H), 7.03 – 6.95 (m, 2H), 5.67 (brs, 2H).
General Procedures for the Preparation of Compounds 3l–3n. To a solution of 3h
(200 mg, 1.8 mmol) in NMP (5 mL) was added 6a–6c. and the mixture was stirred at
90 °C. After completion, the resulting precipitates were collected by filtration in vacuo
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and was washed with MeOH (3× 50 mL) to provide compounds 3l–3n.
7-Morpholinoindolo[2,1-b]quinazoline-6,12-dione (3l). Red solid, yield 90%; mp
1
265−267 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.30 (d, J = 7.9 Hz, 1H), 7.98 (d, J = 7.6
Hz, 1H), 7.93 – 7.89 (m, 2H), 7.72 – 7.64 (m, 2H), 6.97 (d, J = 8.6 Hz, 1H), 3.86 – 3.78
1
3
(m, 4H), 3.42 – 3.35 (m, 4H). C NMR (100 MHz, DMSO-d ) δ 178.64, 158.1, 151.6,
6
147.3, 147.1, 145.9, 139.1, 135.5, 130.0, 129.8, 127.4, 123.5, 116.0, 110.9, 108.1, 66.6,
+
51.1. C19H N O
16 3 3
[M+H] 334.1186, found 334.1182.
7-(4-Methylpiperazin-1-yl)indolo[2,1-b]quinazoline-6,12-dione (3m). Red solid, yield
1
85%; mp 272−274 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.30 (d, J = 7.7 Hz, 1H), 7.96 –
7.91 (m, 3H), 7.72– 7.68 (m, 1H), 7.67 – 7.62 (m, 1H), 6.97 (d, J = 8.6 Hz, 1H), 3.40 –
1
3
3.37 (m, 4H), 2.55 – 2.53 (m, 4H), 2.26 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 178.4,
6
157.6, 151.1, 146.8, 146.6, 145.2, 138.5, 135.0, 129.5, 129.2, 126.8, 122.9, 115.8, 110.2,
+
107.2, 54.5, 50.1, 45.7. C20H N O
19 4 2
[M+H] 347.1503, found 347.1499.
7-(Pyrrolidin-1-yl)indolo[2,1-b]quinazoline-6,12-dione (3n). Red solid, yield 87%; mp
1
234−236 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.29 (d, J = 7.6 Hz, 1H), 7.94 – 7.88 (m,
2H), 7.78 (d, J = 7.4 Hz, 1H), 7.72 – 7.65 (m, 1H), 7.55 – 7.48 (m, 1H), 6.79 (d, J = 8.8
1
3
Hz, 1H), 3.62 – 3.53 (m, 4H), 2.03 – 1.91 (m, 4H). C NMR (100 MHz, DMSO-d ) δ
6
176.9, 157.7, 147.9, 146.7, 145.9, 145.6, 137.4, 134.9, 129.3, 128.9, 126.8, 122.8, 114.2,
+
106.5, 104.1, 51.6, 25.2. C19H N O
16 3 2
[M+H] 318.1237, found 318.1235.
General Procedures for the Preparation of Compounds 5a–5d. To a solution of 3k
(300 mg, 1.14 mmol) in CH
2
Cl
2
was added triethylamine (0.1 mL). Then the solution
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was cooled to 0 C, corresponding acyl chlorides 4a–4d (2.4 mmol) was added dropwise.
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and the mixture was refluxed. After completion, the reaction was quenched with water,
and extracted with CH
2
Cl (3×100 mL). The organic phase was dried over anhydrous
2
MgSO and concentrated under reduced pressure to provide compounds 5a–5d.
4
N-(6,12-Dioxo-6,12-dihydroindolo[2,1-b]quinazolin-8-yl) acetamide (5a). Brown
1
solid, yield 76%; mp 226−228 °C; H NMR (400 MHz, DMSO-d
6
) δ 10.39 (s, 1H), 8.41
– 8.34 (m, 1H), 8.30 (d, J = 7.5 Hz, 1H), 8.20 (s, 1H), 8.00 – 7.90 (m, 2H), 7.89 – 7.82
1
3
(m, 1H), 7.77 – 7.68 (m, 1H), 2.10 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 182.9,
6
169.3, 157.8, 146.9, 145.7, 141.6, 138.6, 135.5, 130.3, 127.7, 127.3, 123.8, 123.0, 118.0,
114.7, 24.5.
4-(tert-Butyl)-N-((4-(tert-butyl)phenyl)sulfonyl)-N-(6,12-dioxo-6,12-dihydroindolo[2,
1-b]quinazolin-8-yl)benzenesulfonamide (5b). Red brown solid, yield 65%; mp
1
155−157 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.53 (t, J = 8.3 Hz, 1H), 8.33 (t, J = 6.8
Hz, 1H), 8.03 – 7.91 (m, 2H), 7.83 – 7.67 (m, 9H), 7.52 (t, J = 7.3 Hz, 1H), 7.30 (d, J =
1
3
6.2 Hz, 1H), 1.35 (d, J = 8.1 Hz, 18H). C NMR (100 MHz, DMSO-d ) δ 181.7, 158.8,
6
158.2, 147.0, 146.8, 145.5, 140.8, 135.9, 135.7, 132.1, 130.6, 128.6, 127.6, 127.1, 127.1,
+
123.7, 123.5, 118.6, 35.7, 31.2. C35
34 3
H N O
S
6 2
[M+H] 656.1884, found 656.1881.
N-(6,12-Dioxo-6,12-dihydroindolo[2,1-b]quinazolin-8-yl)thiophene-2-carboxamide
1
(5c). Red brown solid, yield 60%; mp > 300 °C; H NMR (400 MHz, DMSO-d
6
) δ 10.81
(s, 1H), 8.44 (d, J = 8.7 Hz, 1H), 8.38 – 8.30 (m, 2H), 8.25 – 8.15 (m, 2H), 7.99 – 7.93
1
3
(m, 2H), 7.91 (d, J = 5.0 Hz, 1H), 7.76 – 7.72 (m, 1H), 7.25 (t, J = 4.2 Hz, 1H). C NMR
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(100 MHz, DMSO-d
6
) δ 182.4, 160.3, 157.4, 146.4, 145.4, 141.5, 139.4, 137.7, 135.0,
132.4, 130.0, 129.9, 129.8, 128.7, 128.1, 126.9, 123.3, 122.5, 117.2, 115.7. C20
H
12
N
3
O S
3
+
[M+H] 374.0594, found 374.0597.
N-(6,12-Dioxo-6,12-dihydroindolo[2,1-b]quinazolin-8-yl)-N-(furan-2-carbonyl)furan
1
-2-carboxamide (5d). Yellow solid, yield 60%; mp 235−237 °C; H NMR (400 MHz,
DMSO-d
6
) δ 8.51 (d, J = 8.5 Hz, 1H), 8.33 (d, J = 7.8 Hz, 1H), 8.01 – 7.90 (m, 5H), 7.81
(dd, J = 8.5, 2.0 Hz, 1H), 7.79 – 7.70 (m, 1H), 7.28 (d, J = 3.5 Hz, 2H), 6.68 (d, J = 1.9
1
3
Hz, 2H). C NMR (100 MHz, DMSO-d ) δ 181.6, 160.5, 157.7, 148.0, 146.6, 146.4,
6
145.2, 144.9, 137.2, 136.6, 135.3, 130.0, 129.9, 127.0, 124.0, 123.5, 123.1, 120.4, 118.0,
+
14 3
112.8. C25H N O
6
[M+H] 452.0877, found 452.0877.
General Procedures for the Preparation of Compounds 8a–8b. A mixture of 3a (2.5 g,
10 mmol), corresponding hydroxylamines 7a–7b (50 mmol) in methanol (40 mL) was
refluxed. After cooling, the resulting precipitates were collected by filtration in vacuo and
washed with MeOH (3× 50 mL) to provide compounds 8a–8b.
(E)-6-(Hydroxyimino)indolo[2,1-b]quinazolin-12(6H)-one (8a). White solid, yield
1
65%; mp 282−284 °C; H NMR (400 MHz, DMSO-d
6
) δ 13.66 (s, 1H), 8.53 (d, J = 8.0
Hz, 1H), 8.37 (d, J = 7.6 Hz, 1H), 8.28 (d, J = 7.8 Hz, 1H), 7.89 (t, J = 7.6 Hz, 1H), 7.81
1
3
(d, J = 8.0 Hz, 1H), 7.63 (dt, J = 14.9, 7.6 Hz, 2H), 7.45 (t, J = 7.6 Hz, 1H). C NMR
(100 MHz, DMSO-d ) δ 158.5, 148.3, 146.9, 144.2, 139.3, 134.7, 132.1, 128.1, 127.5,
6
127.4, 126.6, 126.5, 121.5, 118.8, 116.2.
(E)-6-(Methoxyimino)indolo[2,1-b]quinazolin-12(6H)-one (8b). Yellow solid, yield
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62%; mp 207−209 °C; H NMR (400 MHz, DMSO-d ) δ 8.49 (s, 1H), 8.24 (d, J = 21.2
6
91
92
93
94
95
96
97
98
99
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09
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Hz, 2H), 7.86 (d, J = 24.0 Hz, 2H), 7.72 – 7.55 (m, 2H), 7.44 (s, 1H), 4.30 (s, 3H).
General Procedures for the Preparation of Compounds 10a–10b. To a solution of 3a
(500 mg, 2 mmol) in corresponding ketone (1.2 mmol) was added dimethylamine (0.33 g,
7.3 mmol). The mixture was stirred at room temperature for one day. Then the mixture
diluted with 200 mL of CH
2
2
Cl , and the organic phases were washed with brine (3× 100
mL). The organic layer was dried with MgSO
4
, filtrated, and concentrated in vacuo. The
residue was purified by flash chromatography on a silica gel using petroleum ether and
ethyl acetate (4:1, v/v) as the eluent to give 10a−10b.
6-Hydroxy-6-(2-oxo-2-phenylethyl)indolo[2,1-b]quinazolin-12(6H)-one (10a). Yellow
1
solid, yield 48%; mp 184−186 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.48 (d, J = 8.0 Hz,
1H), 8.35 (d, J = 7.8 Hz, 1H), 7.92 – 7.83 (m, 3H), 7.74 (d, J = 8.1 Hz, 1H), 7.70 (d, J =
7.4 Hz, 1H), 7.67 – 7.59 (m, 2H), 7.56 – 7.42 (m, 3H), 7.33 (t, J = 7.4 Hz, 1H), 6.62 (s,
1
3
1H), 4.56 (d, J = 18.3 Hz, 1H), 4.17 (d, J = 18.3 Hz, 1H). C NMR (100 MHz, DMSO-d )
6
δ 196.72, 161.5, 159.0, 147.1, 139.4, 135.5, 134.7, 134.1, 133.7, 129.7, 128.7, 128.0,
127.4, 127.3 126.5, 126.4, 123.7, 121.4, 116.0, 75.1, 47.7.
6-Hydroxy-6-(2-oxopropyl)indolo[2,1-b]quinazolin-12(6H)-one (10b). White solid,
1
yield 46%; mp 170−172 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.42 (d, J = 8.0 Hz, 1H),
8.34 – 8.29 (m, 1H), 7.92 – 7.85 (m, 1H), 7.78 – 7.74 (m, 1H), 7.66 – 7.59 (m, 2H), 7.54
– 7.47 (m, 1H), 7.40 – 7.32 (m, 1H), 6.46 (s, 1H), 3.82 (d, J = 17.9 Hz, 1H), 3.60 (d, J =
18.0 Hz, 1H), 1.98 (s, 3H).
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Preparation of 6-Ethyl-6-hydroxyindolo[2,1-b]quinazolin-12(6H)-one (11). To a
solution of 3a (1.0 g, 4.0 mmol) in dry THF (25 mL) was added 1 M CH
mL, 9 mmol) dropwise at -78 ºC. The reaction mixture was stirred for three hours and
NH Cl solution (1 mL) was added. After completion, the solvent were concentrated
under reduced pressure. Then adding NH Cl solution (20 mL) gave a solid which was
3
CH
2
MgBr (9
4
4
collected by filtration, and recrystallized from chloroform to give 11: white solid, yield
1
33%; mp 188−190 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.45 (d, J = 7.9 Hz, 1H), 8.30
(d, J = 7.8 Hz, 1H), 7.91 (t, J = 7.6 Hz, 1H), 7.86 – 7.80 (m, 1H), 7.66 – 7.60 (m, 2H),
7.54 (t, J = 7.8 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 6.36 (s, 1H), 2.21 – 2.11 (m, 2H), 0.57
1
3
(t, J = 7.4 Hz, 3H). C NMR (100 MHz, DMSO-d ) δ 161.7, 159.4, 147.5, 139.1, 135.3,
6
134.5, 130.1, 128.1, 127.9, 127.3, 126.9, 124.7, 121.8, 116.6, 78.6, 32.3, 8.2. C17
H
15
N
2
O
2
+
[M+H] 279.1128, found 279.1128.
General Procedures for the Preparation of Compounds 13a–13b. To a solution of 3a
(1 g, 3 mmol) in MeOH (50 mL) was added corresponding hydrazines 12a–12b, and the
mixture was stirred at 70 °C. After completion, the resulting precipitates were collected
by filtration in vacuo and was washed with MeOH (3 × 50 mL) to provide compounds
13a–13b.
(E)-6-(2-Phenylhydrazono)indolo[2,1-b]quinazolin-12(6H)-one (13a). Red solid, yield
1
89%; mp 195−197 °C; H NMR (400 MHz, DMSO-d
6
) δ 13.43 (s, 1H), 8.48 (d, J = 7.9
Hz, 1H), 8.35 (d, J = 7.8 Hz, 1H), 8.13 (d, J = 8.1 Hz, 1H), 7.99 – 7.90 (m, 2H), 7.69 –
1
3
7.60 (m, 3H), 7.54 (t, J = 7.9 Hz, 1H), 7.50 – 7.40 (m, 3H), 7.14 (t, J = 7.4 Hz, 1H). C
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NMR (100 MHz, CDCl
3
) δ 158.7, 146.8, 145.9, 142.6, 137.0, 134.3, 129.8, 128.3, 127.3,
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52
127.3, 127.2, 126.3, 126.2, 124.5, 123.5, 121.2, 119.0, 116.9, 114.5. C21
H
15
N
O [M+H]+
4
339.1240, found 339.1246.
(E)-6-(2-(Pyridin-2-yl)hydrazono)indolo[2,1-b]quinazolin-12(6H)-one (13b). Red
1
solid, yield 52%; mp 214−216 °C; H NMR (400 MHz, CDCl
3
) δ 13.40 (s, 1H), 8.59 (d,
J = 8.0 Hz, 1H), 8.46 (d, J = 7.7 Hz, 1H), 8.36 (d, J = 4.5 Hz, 1H), 7.99 (d, J = 8.1 Hz,
1H), 7.90 (d, J = 7.5 Hz, 1H), 7.82 (t, J = 7.6 Hz, 1H), 7.78 – 7.73 (m, 1H), 7.72 – 7.68
(m, 1H), 7.58 (t, J = 7.5 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.01 (t,
1
3
1H). C NMR (100 MHz, CDCl ) δ 158.7, 155.5, 148.2, 146.8, 145.7, 138.3, 137.9,
3
134.4, 129.3, 128.4, 128.1, 127.8, 127.2, 126.4, 124.2, 121.5, 119.5, 118.6, 117.0, 108.2.
+
C
20
H
14
N
5
O [M+H] 340.1193, found 340.1195.
Preparation of 6-Hydroxy-5a,6-dihydroindolo[2,1-b]quinazolin-12(5H)-one (14). To
a mixture of 3a (0.5 g, 2 mmol) and acetic acid (50 mL) was added NaBH (0.75 g, 20
4
mmol) in small portions in ice bath. The reaction mixture was allowed to warm to
ambient temperature and stir for 4 h. After completion, the mixture was poured into 100
mL of ice-water and extracted with EtOAc (3 × 150 mL). The combined organic phase
was washed with brine (3 × 50 mL), dried over anhydrous Na
2 4
SO , filtrated, and
1
concentrated in vacuo to give 14: yellow solid, yield 85%; mp 202−204 °C; H NMR
(400 MHz, DMSO-d ) δ 8.09 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.60 (s, 1H),
6
7.46 (d, J = 7.4 Hz, 1H), 7.40 – 7.34 (m, 2H), 7.16 (t, J = 7.4 Hz, 1H), 6.98 (d, J = 8.1 Hz,
1H), 6.87 (t, J = 7.5 Hz, 1H), 6.18 (d, J = 4.8 Hz, 1H), 5.30 – 5.22 (m, 1H) , 5.13 (d, J =
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6.6 Hz, 1H). C NMR (100 MHz, DMSO-d ) δ 160.5, 148.8, 140.3, 134.1, 132.7, 129.6,
6
128.2, 125.3, 124.4, 119.2, 117.0,116.0, 115.3, 78.4, 77.1.
Preparation of Indolo[2,1-b]quinazolin-12(5H)-one (15). Compound 14 (1.0 g, 4
mmol) was taken into polyphosphoric acid (20 mL). The mixture was heated under N for
2
1 h at 110 °C, then poured into ice-water and extracted with EtOAc (3 × 150 mL). The
combined organic phase was washed with brine (3 × 50 mL), dried over anhydrous
Na SO
2 4
, filtrated, and concentrated in vacuo to give 15: black solid, yield 82%; mp
1
265−267 °C; H NMR (400 MHz, DMSO-d
6
) δ 11.77 (s, 1H), 8.61 (d, J = 8.1 Hz, 1H),
8.17 (d, J = 7.6 Hz, 1H), 7.76 – 7.66 (m, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.38 – 7.26 (m,
1
3
2H), 7.19 – 7.14 (m, 2H), 6.04 (s, 1H). C NMR (100 MHz, DMSO-d ) δ 159.4, 140.9,
6
137.8, 135.3, 130.7, 129.6, 128.0, 124.6, 120.5, 119.9, 118.4, 115.8, 115.5, 111.6, 80.8.
+
C H N
15 11 2
O [M+H] 235.0866, found 235.0865.
Preparation of Indolo[2,1-b]quinazolin-6(12H)-one (16). Step 1: To a solution of 3a (2
g, 8 mmol) in ether (50 mL) was added LiAlH
4
(1 g, 24 mmol) at 0 °C. The reaction
o
mixture was refluxed overnight. The reaction was cooled to 0 C and quenched by slow
addition of H
phase was dried over anhydrous MgSO
red mixture. Step 2: To a solution of the red mixture (1.4 g) in dry CH
added MnO (0.7 g) at room temperature. The reaction mixture was stirred at room
temperature for 48 h. Then the mixture was poured into ice-water and extracted with
CH Cl (3 × 150 mL). The organic layer was dried with MgSO , filtrated, and
2
O (100 mL). The solution extracted with EtOAc (2×150). The organic
and concentrated under reduced pressure to give
Cl (50 mL) was
4
2
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2
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concentrated in vacuo. The residue was purified by flash chromatography on a silica gel
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using petroleum ether and ethyl acetate (3:1, v/v) as the eluent to give 16: red solid, yield
1
73%; mp 226−228 °C; H NMR (400 MHz, CDCl
3
) δ 7.70 (d, J = 7.5 Hz, 1H), 7.60 (t, J
= 7.7 Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.33 – 7.17 (m, 2H), 7.14 – 7.04 (m, 2H), 6.85 (d,
1
3
J = 7.9 Hz, 1H), 4.94 (s, 2H). C NMR (100 MHz, CDCl ) δ 184.7, 151.0, 146.2, 140.6,
3
137.7, 129.5, 129.1, 128.6, 126.7, 125.4, 122.5, 120.5, 119.6, 108.9, 42.9.
11H-Indeno[1,2-b]quinoxalin-11-one (19). A mixture of o-phenylenediamine (1.1 g, 10
mmol) and ninhydrin (1.8 g, 10 mmol) in ethanol/acetic acid (5:1, 36 mL) was refluxed
for 5 h. The reaction contents were cooled and the resulting precipitates were collected
1
by filtration in vacuo to give 19: yellow solid, yield 95%; mp 217−219 °C; H NMR (400
MHz, DMSO-d ) δ 8.20 – 8.11 (m, 2H), 8.08 (d, J = 7.8 Hz, 1H), 7.95 – 7.82 (m, 4H),
6
7.71 (t, J = 7.5 Hz, 1H).
Biological Assay. Each test was repeated three times at 25±1 °C. Active effect expressed
in 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
1
4
were carried out using literature method, also can be seen in Supporting Information.
RESULTS AND DISCUSSION
Chemistry.
In order to investigate the influence of the electronic effects on the biological activities
of these compounds, tryptanthrin (3a) and a series of it’s designed derivatives (3b−3i)
were synthesized via the reaction of isatoic anhydride with corresponding isatin
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derivatives (Figure 2).22 These compounds have rigid planar structure, and hydrogen
bond effect is also an important factor affecting biological activities for these compounds.
Compounds 3j, 3k and 5a−5d containing hydrogen bond donor and acceptor were
designed and synthesized to investigate the effect of hydrogen bond on the biological
activities of these compounds (Figures 3 and 4). In order to investigate the effect of
heterocyclic pharmacophore on biological activity, compounds 3l−3n were designed and
synthesized (Figure 5). Carbonyl group has a great influence on the electronic effect and
rigid planar structure of these compounds. Compounds 8a−8b, 10a−10b, 11, 13a−13b,
and 14−16 were designed and synthesized. The influence of 6 and 12 carbonyl groups on
the biological activities of these compounds was investigated. As shown in Figure 6, the
6-carbonyl group of tryptanthrin was reacted with hydroxylamines, ketones, Grignarg
reagent and hydrazines, respectively to give oximes 8a−8b, alcohols 10a−10b, 11 and
hydrazones 13a−13b. The 6-carbonyl group was reduced to hydroxyl by sodium
borohydride to obtain 14. Dehydroxylation of 14 gave 15. The 12-carbonyl group was
reduced to methylene by lithium tetrahydroaluminum to obtain 16. In order to further
investigate the influence of pyrimidinone ring on biological activity, compound 19
containing pyrazine structure was designed and synthesized (Figure 7).
Phytotoxic Activity.
The phytotoxic-activity tests (according to the criterion of safety evaluation of
pesticide to crops: NYT 1965.1-2010) showed that the tryptanthrins 3a−3n, 5a−5d,
8a−8b, 10a−10b, 11, 13a−13b, 14−16 and 19 were safe for testing on plants at 500
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Antiviral Activity in vivo. The activities of tryptanthrins 3a−3n, 5a−5d, 8a−8b,
10a−10b, 11, 13a−13b, 14−16 and 19 against TMV are listed in Table 1 with
commercial viral inhibitor ribavirin as control. The inactive activities of all the
compounds were first tested at 500 μg/mL, and the curative activities and protective
activities of the compounds with good inactive activities (inactive effect > 40%) were
further tested.
As can be seen from the table 1, most of the tryptanthrin derivatives showed good
anti-TMV activities, compounds 3e, 3g, 3i, 3j, 3k, 3l−3n, 5a, 5d, 8a, 10a, 13a, 14−16
and 19 displayed higher or similar inactive effects with ribavirin at 500 μg/mL,
especially for compounds 3n and 14. Natural product tryptanthrin (3a) showed 37%
inactive effect. The introduction of halogen into the 8-position of tryptanthrin has little
effect on its activity (inactive effect: 3a ≈ 3b−3d). Compound 3e with strong electron
absorbing group showed higher activity than tryptanthrin, which indicated that the
introduction of strong electron withdrawing group at 8-position is beneficial to biological
activity. Compound 3f with strong electron donating group exhibited similar level of
biological activity as tryptanthrin, however, compound 3g with medium electron
donating group showed higher biological activity than tryptanthrin. The above results
revealed that the electronic effect of 8-position substituents has a great influence on
biological activity. The activity of tryptanthrin decreased with the introduction of Cl at
7-position, but increased with the introduction of Br (inactive effect: 3i > 3a > 3h). The
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introduction of hydroxyl and amino groups at the 8-position of tryptanthrin is beneficial
to the increase of activity (inactive effect: 3k > 3j > 3a). Tryptanthrin derivatives with
heterocyclic rings at 7-position showed good bioactivity (inactive effect: 3l, 3m and 3n >
3a), in particular, 3n with tetrahydropyrrole ring exhibited significantly higher antiviral
activity than ribavirin. The 8-aminoacyl derivatives 5a−5d of 3k showed obvious
difference in activity (inactive effect: 5a > 3k > 5d > 5b ≈ 5c), which revealed that this
region is highly sensitive to electronic effects and steric hindrance. By comparing
compounds 8a, 8b, 10a, 10b, 11, 13a and 13b, it was found that the carbonyl group at
the 6-position of tryptanthrin was not irreplaceable (inactive effect: 13a > 8a ≈ 10a > 3a
≈ 11 ≈ 13b > 8b > 10b), among which compound 13a containing phenylhydrazine
structure displayed the best biological activity. In addition, the antiviral activities of the
reduction products (14, 15 and 16) of tryptanthrin were significantly higher than that of
tryptanthrin, especially for 14. Compound 19 with pyrazine structure showed higher
antiviral activity than compound 16 with pyrimidine structure. Compounds 3n and 14
emerged as new antiviral leads due to their excellent antiviral activities.
Preliminary Mode of Action.
Considering the physical and chemical properties and biological activity, we chose
compound 16 which displayed the similar activity as ribavirin to study the mode of
action using our reported method.5,14 And N′-(4-chlorobenzylidene)-(3S)-1-methyl-
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carbohydrazide (NK0209),
a
highly
effective virus inhibitor independently developed by our group, was used as control.23
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The test results showed that 20S CP could be assembled with TMV RNA into TMV
particles of about 300 nm in length, and DMSO had no effect on the assembly (Figures
8A and 8B). NK0209 and compound 16 can inhibit the assembly of the virus because a
decrease in the number of viruses and a short in their length can be observed (Figures 8C
and 8D). In Figure 8D, we can clearly see the broken and fused virus particles. We
further designed the interaction experiment between the 20S CP disk and 16 using our
reported method.5,14 The results showed that TMV CP could be incubated into 20S CP
disk (Figure 9A), and the addition of DMSO had no effect on the results (Figure 9B).
The control NK0209 can induce the aggregation and fusion of 20S CP disks (Figure 9C).
As can be seen in Figure 9D, compound 16 can cause the 20s protein disk to fuse and
then decompose.
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Molecular Docking. In order to further study the mechanism of the interaction between
tryptanthrins and TMV CP, we chose AutoDock-vina 1.1.2 for molecular docking.24
Compounds 3n, 14 and 16 were selected for molecular docking with TMV CP (PDB
code: 1EI7). Compound 3n forms one conventional hydrogen bond with the active site of
ARG 341 at a distance of 2.5 Å (Figure 10A). Compound 14 forms one conventional
hydrogen bond with the active site of ARG 341 at a distance of 2.2 Å (Figure 10B).
Compound 16 forms two conventional hydrogen bonds with SER 146 at a distance of 3.1
Å and 3.3 Å. As the control, NK0209 forms two conventional hydrogen bonds with
amino acids SER 147 (2.6 Å), GLN 257 (2.1 Å). Compounds 3n and 14, which have
higher antiviral activities in vivo than compound 16, do show stronger interaction with
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TMV CP.
Fungicidal Activity.
It’s also a very efficient method to find a new type of fungicidal lead compound based on
2
5
natural products. Tryptanthrins 3a−3n, 5a−5d, 8a−8b, 10a−10b, 11, 13a−13b, 14−16
and 19 were also evaluated for their fungicidal activities with commercial fungicides
carbendazim, chlorothalonil and pyrimethanil as controls.
Tryptanthrins exhibited broad-spectrum fungicidal activities against 14 kinds of
phytopathogenic fungi at 50 μg/mL (Table 2). On the whole, tryptanthrins showed
excellent fungicidal activities against Physalospora piricola. Most derivatives displayed
more than 60% inhibitory effects, especially, compounds 3c and 3k exhibited higher
fungicidal activities than pyrimethanil against Physalospora piricola. Compound 16
displayed higher fungicidal activity against Rhizoctonia cerealis than pyrimethanil. The
inhibitory effects of 3g, 5d, 16 and 19 were better than that of chlorothalonil and
carbendazim against Alternaria solani. In addition, the inhibition rates of most
compounds on Phytophthora capsici and Botrytis cinerea were higher than that of
chlorothalonil and carbendazim.
In summary, tryptanthrins 3a−3n, 5a−5d, 8a−8b, 10a−10b, 11, 13a−13b, 14−16 and
19 were synthesized and evaluated for their antiviral activities and fungicidal activities.
Most of these compounds displayed higher antiviral activities than ribavirin. Compounds
3n and 14 with significantly higher antiviral activities than ribavirin emerged as new
antiviral lead compounds for further research. A systematic study on the structure-activity
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relationship of these compounds was carried out. The preliminary mode of action studies
revealed that compound 16 can inhibit virus assembly by decomposing the 20s protein
disk. We further study the mechanism of the interaction between tryptanthrins and TMV
CP by molecular docking. Further fungicidal test revealed that these compounds
exhibited broad-spectrum fungicidal activities against 14 kinds of plant fungi at 50
μg/mL. Tryptanthrins showed excellent fungicidal activities against Physalospora
piricola. Compounds 3c and 3k exhibited higher fungicidal activities than pyrimethanil
against Physalospora piricola. Current research has laid a foundation for the application
of tryptanthrins in plant protection.
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ASSOCIATED CONTENT
Supporting Information
The detailed bio-assay procedures. The spectra data of tryptanthrins 3a−3n, 5a−5d,
8a−8b, 10a−10b, 11, 13a−13b, 14−16 and 19. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(Z.W.)
0086-22-23766531.
*(Y.L.) E-mail: liuyuxiu@nankai.edu.cn;
0086-22-23503792.
*(Q.W.) E-mail: wangqm@nankai.edu.cn. Phone: 0086-22-23503952. Fax:
E-mail:
hxxywzw@tjnu.edu.cn;
Phone:
0086-22-23766531;
Fax:
Phone:
0086-22-23503792;
Fax:
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0086-22-23503952.
Funding
This study was supported by Natural Science Fund of China (21772145, 21732002,
21772104).
Notes
The authors declare no competing financial interest.
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Figure Captions
Figure 1. Design of Tryptanthrin Analogues.
Figure 2. Synthesis of Compounds 3a–3i.
Figure 3. Synthesis of Compound 3j.
Figure 4. Synthesis of Compounds 3k and 5a–5d.
Figure 5. Synthesis of Compounds 3l–3n.
Figure 6. Synthesis of Compounds 8a–8b, 10a–10b, 11, 13a–13b and 14–16.
Figure 7. Synthesis of Compound 19.
Figure 8. TMV Rod Assembly Inhibition of Compound 16 and NK0209: (A) 20S CP
disk + RNA (200 nm scale bar), (B) 20S CP disk + RNA + 1/100 DMSO(100 nm scale
bar), (C) 20S CP disk + RNA + 10 μM NK0209 (100 nm scale bar), (D) 20S CP disk +
RNA + 10 μM 16 (200 nm scale bar).
Figure 9. 20S CP Disk Assembly Inhibition of Compound 16 and NK0209 (100 nm scale
bar): (A) CP, (B) CP + 1/100 DMSO, (C) CP +10 μM NK0209, (D) CP + 10 μM 16.
Figure 10. Molecule Docking Results of Compounds 3n (A), 14 (B), 16 (C) and
Ribavirin (D) with TMV CP.
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Table 1. In Vivo Antiviral Activities of Compounds 3a−3n, 5a−5d, 8a−8b, 10a−10b, 11,
1
3a−13b, 14−16, 19 and Ribavirin Against TMV.
concn
inactive effect
(%)a
curative effect
(%)a
protective effect
(%)a
compd
(
μg/mL)
3
a
500
500
37±1
39±3
—
—
—
—
3
b
3
c
500
500
31±2
31±5
46±4
8±2
—
—
—
—
3
d
5
1
00
00
39±4
13±1
—
47±2
18±1
—
3
e
3
f
500
35±1
44±2
14±1
32±1
45±3
16±1
41±4
8±2
5
1
00
00
42±2
7±1
—
35±4
0
3
g
3
h
500
—
5
1
5
1
5
1
00
00
00
00
00
00
37±3
11±2
32±3
0
43±1
7±1
47±3
11±1
39±2
0
3
i
3
j
46±2
15±3
47±2
11±1
3
k
5
1
5
1
5
1
5
1
00
00
00
00
00
00
00
00
44±4
0
46±3
5±1
43±5
9±1
3
l
45±1
14±1
52±2
21±1
49±1
21±2
42±4
11±1
49±5
20±1
47±2
17±3
48±1
19±2
54±4
24±3
40±1
13±1
3
m
3
n
5
a
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5
b
500
500
37±1
36±3
—
—
—
—
5
c
5
1
00
00
41±3
10±1
43±5
33±4
41±2
23±1
35±5
31±2
—
40±4
8±1
39±4
—
5
d
8
a
500
500
500
500
500
43±4
—
8
b
1
0a
34±2
—
37±1
—
1
0b
1
1
—
—
5
1
00
00
47±4
10±4
37±1
51±3
18±1
44±3
9±1
41±1
14±3
—
51±4
17±1
—
1
3a
1
3b
500
5
1
5
1
5
1
5
1
5
1
00
00
00
00
00
00
00
00
00
00
48±1
19±1
36±2
0
53±4
21±2
42±3
6±2
1
4
5
6
9
1
44±3
9±1
36±2
0
42±3
6±2
1
1
48±2
11±1
40±2
12±1
41±4
5±2
37±3
10±1
43±4
0
38±2
9±1
ribavirin
a
Average of three replicates; All results are expressed as mean ± SD; Activity Data with prominent were presented in
pink bold with blue color.
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Table 2. In Vitro Fungicidal Activities of Compounds 3a−3n, 5a−5d, 8a−8b, 10a−10b,
11, 13a−13b, 14−16, 19, Chlorothalonil, Carbendazim and Pyrimethanil against 14 Kinds
of Fungi.
a
Fungicidal activities (%) at 50 μg/mL
Compd
F.Cb
C.Hb
P.Pb
R.Cb
B.Mb
W.Ab F.Mb A.Sb F.Gb
P.Ib
P.Cb
S.Sb
B.Cb
R.Sb
3
a
20±1 30±2 70±3 60±1 28±2 29±2 20±1 31±2 8±2 13±1 42±2 13±1 16±1 4±1
20±2 30±1 79±1 61±2 36±4 29±2 20±1 25±2 23±2 63±3 65±3 25±2 16±1 39±2
20±1 19±2 85±2 46±1 23±2 26±1 17±2 31±1 12±1 25±2 19±1 38±2 13±2 35±1
15±1 22±2 76±2 54±2 23±3 29±2 27±1 13±2 12±2 25±1 7±2 38±1 13±1 39±2
3
b
3
c
3
d
3
e
15±1 56±2 79±2 48±2 23±2 21±3 27±2 13±2 4±1 38±1 10±2 13±2 6±2
4±1
3
f
25±2 30±1 79±1 67±1 41±1 31±2 20±2 31±2 27±2 63±2 55±1 50±2 34±1 82±2
18±1 22±2 70±2 44±2 31±1 36±2 27±1 50±2 31±1 50±1 3±1 50±2 19±1 59±2
43±2 31±1 71±1 60±2 36±2 52±3 47±2 25±2 6±1 50±1 19±2 85±3 46±2 5±1
3
3
g
h
3
i
15±1 30±2 79±2 37±1 23±2 26±3 27±2 13±1 19±2 31±2 10±1 13±2 9±1
4±1
3
j
15±2 30±2 70±1 34±1 18±2 33±1 27±2 13±1 19±2 38±2 3±1 38±3 9±2 29±1
23±1 52±3 89±2 67±2 28±1 29±2 50±1 25±2 12±1 38±2 23±2 13±1 25±2 20±2
15±2 26±2 49±2 51±2 18±1 26±1 20±1 31±2 23±2 13±2 16±2 50±1 19±2 61±2
33±1 26±1 60±3 34±2 44±2 41±2 37±2 25±1 19±1 63±3 74±2 38±2 19±2 67±1
20±2 19±2 60±2 58±1 81±2 33±3 33±2 19±2 8±2 13±1 13±3 38±2 31±1 20±2
15±1 22±1 77±2 42±2 31±1 29±1 17±1 13±2 8±2 25±2 10±1 13±1 13±2 14±1
10±2 19±2 60±1 37±1 39±2 31±2 23±2 19±3 4±1 13±2 7±1 38±2 13±2 6±1
3
k
3
l
3
m
3
n
a
b
5
5
5
c
8±1
19±1 45±2 38±2 10±2 21±2 33±3 25±1 8±1 25±1 29±2 13±1 19±1 20±2
5
8
8
d
a
b
19±2 45±2 55±2 23±2 19±1 30±1 17±1 50±2 35±2 33±2 4±1 28±2 36±2 10±1
23±1 22±2 70±3 57±2 36±2 36±2 37±1 31±1 23±1 25±2 29±2 13±1 16±2 29±2
35±2 67±1 55±1 66±1 21±2 33±1 50±2 31±2 19±2 25±3 16±2 25±1 31±2 35±1
13±1 19±2 60±2 39±2 23±1 19±2 17±2 13±2 19±2 38±1 10±1 38±2 19±3 58±2
10±2 22±2 66±2 29±2 13±2 26±2 27±1 13±1 39±1 13±2 10±2 38±1 19±2 26±1
30±1 33±2 79±2 66±1 36±2 38±1 40±2 25±2 19±2 6±1 16±2 38±2 16±1 17±2
1
0a
0b
1
11
1
3a
3b
13±2 19±1 59±1 32±2 18±1 24±2 33±3 6±2
4±1
6±2
7±1
6±1
6±2
6±1
1
10±1 70±2 35±1 23±2 26±2 33±1 13±1 15±2 13±2 26±2 38±2 19±1 39±2
0
1
4
5
33±2 33±2 62±1 35±2 23±2 36±1 33±2 38±2 23±2 75±1 71±2 63±2 31±2 53±1
20±1 26±1 76±2 52±1 15±1 21±2 33±2 31±2 8±1 25±2 3±1 13±1 13±2 26±2
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