Design, synthesis, and biological activities of aromatic gossypol schiff base derivatives

Article abstract of DOI:10.1021/jf504411g

A series of aromatic gossypol Schiff bases have been successfully synthesized via a feasible chemical modification. The antiviral activity against tobacco mosaic virus (TMV) of these gossypol Schiff bases has been tested for the first time. The bioassay studies indicated most of these derivatives exhibited excellent anti-TMV activity, in which o-trifluoromethylaniline Schiff base (19) displayed the best antiviral activities. Furthermore, compound 19 exhibited an eminent anti-TMV effect in the field and low toxicity to mice. These results suggest it is a promising candidate for the inhibitor of plant virus.

Full text of DOI:10.1021/jf504411g

Article
pubs.acs.org/JAFC
Design, Synthesis, and Biological Activities of Aromatic Gossypol
Schiff Base Derivatives
Ling Li,† Zheng Li,† Kailiang Wang,† Sheng Zhao,# Jiming Feng,# Jiarui Li,§ Peiwen Yang,§ Yuxiu Liu,†
Lizhong Wang,† Yongqiang Li,*,† Hui Shang,*,§ and Qingmin Wang*,†
†State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Collaborative Innovation
Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, People’s Republic of China
§Agricultural Environment and Resources Institute, Yunnan Academy of Agricultural Sciences, Yunnan 650205, People’s Republic of
China
#China Tobacco Yunnan Import and Export Company, Ltd., Yunnan 650031, People’s Republic of China
S
* Supporting Information
ABSTRACT: A series of aromatic gossypol Schiff bases have been successfully synthesized via a feasible chemical modification.
The antiviral activity against tobacco mosaic virus (TMV) of these gossypol Schiff bases has been tested for the first time. The
bioassay studies indicated most of these derivatives exhibited excellent anti-TMV activity, in which o-trifluoromethylaniline Schiff
base (19) displayed the best antiviral activities. Furthermore, compound 19 exhibited an eminent anti-TMV effect in the field and
low toxicity to mice. These results suggest it is a promising candidate for the inhibitor of plant virus.
KEYWORDS: gossypol, Schiff base, synthesis, anti-TMV activity, structure−activity relationships
agriculture. As a result, the field of TMV inhibitors still deserves
to be further exploited.
INTRODUCTION
■
The discovery of gossypol (1, Figure 1) has attracted
considerable attention due to its applications in antitumor
activity.1 Such a compound embedded in the cottonseed
pigment glands exists in three forms,2 which can be categorized
as the aldehyde tautomer 1a, the ketone (quinoid) tautomer
1b, and the lactol (hemiacetal) tautomer 1c (Figure 1). In most
cases, as a part of the plant’s defense system against pathogens
(Verticillium dahliae, Rhizoctonia solani, and Xanthomonas
campestris)3 and herbivorous insects such as Spodoptera species
and Heliothis species,4 gossypol exhibits multiple biological
properties,1d including spermicidal, antitumor, antiparasitic, and
antiprotozoan activities. It also reported to have antiviral
activity toward enveloped viruses such as HIV-1,5 H5N1,5 HSV-
2,6 and influenza virus7 rather than nonenveloped viruses, such
as poliovirus. However, a recent patent revealed that the
gossypol fatty acid reagent had antiviral activity against tobacco
mosaic virus (TMV),8 which is classified as a nonenveloped
virus.
The plant disease caused by TMV is found worldwide. It is
well-known that TMV can infect members of 36 plant families
and at least 400 individual species, including tobacco, tomato,
pepper, cucumber, and a number of ornamental flowers.9 TMV
disease can cause vast losses in agriculture. It is found that in
certain fields 90−100% of the plants showed mosaic or leaf
necrosis by harvesting time. Ribavirin is one of the most widely
used plant virus inhibitors, even though its inhibitory effects are
<50% at 500 μg/mL. Ningnanmycin, perhaps the most
successful registered antiplant viral agent, displayed a 56% in
vivo curative effect at 500 μg/mL. Recently, a number of
chemicals10 were reported to exhibit antiviral activities;
however, few of them have been utilized successfully in
Knowledge of the molecular mechanism of the biological
effects for gossypol is still limited. Some literature studies show
that gossypol exerts its pharmacological actions by an
interaction with biomolecular targets, such as enzymes, signal
transduction mediators, or membranes.11 Kovacic12 in 2003
discussed the mechanism of biological properties of gossypol as
well and pointed out that gossypol and its metabolites could
produce reactive oxygen species (ROS) through redox cycling
́
by electron transfer functions. Additionally, Bacso in 2011
reported that external application of ROS in tobacco may
control virus replication and symptom development.13 On the
basis of such interpretation, the antiviral activity of gossypol
toward a nonenveloped virus, TMV, can be inferred as being
relevant to ROS.
Numerous studies suggest gossypol is toxic to monogastric
animals, but its toxicity can be reduced while its therapeutic
effects are retained by modification of gossypol’s reactive
aldehyde groups.14 A few gossypol Schiff bases were
synthesized and tested for their biological activities including
antitumor, antimalaria, and interferon-inducing activities.15
However, reports of the anti-TMV activity of the gossypol
Schiff bases are rather rare, and no examples are documented in
the recent literature.5,15,16 Gossypol analogues with quinones
and conjugated imine groups have electron transfer function-
alities to generate ROS, which may control virus replication and
symptom development.13 Therefore, condensation products of
Received: September 13, 2014
Revised: October 24, 2014
Accepted: October 29, 2014
© XXXX American Chemical Society
A
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Figure 1. Structures of gossypol and its tautomeric forms.
Figure 2. Design of compounds 3−25.
solid was filtered, washed with ethanol, and recrystallized to give the
desired gossypol derivatives.
gossypol and aromatic amines are expected to adjust gossypol
to produce ROS and thus modulate the anti-TMV activity level.
(Figure 2) In this contribution, a family of aromatic gossypol
Schiff base derivatives has been synthesized and their anti-TMV
activities have been determined using both in vitro and in vivo
test methods. The structure−activity relationships of these
derivatives were discussed as well. Field trial and toxicity
evaluation were also conducted for the compound with high
anti-TMV activity primarily to assess its potential as an antivirus
candidate. Additionally, the synthesized derivatives were also
investigated for their potential as fungicidal, insecticidal, or
herbicidal reagents (see the Supporting Information).
General Procedure for the Synthesis of Aromatic Gossypol
Schiff Base Derivatives 11 and 23. A mixture of aromatic amine
(1.73 mmol), NaOH (2.59 mmol), and ethanol (50 mL) was stirred
and refluxed for 1 h. Then gossypol acetic acid (2, 0.50 g, 0.86 mmol)
was added, and the reaction liquid was refluxes for a further 5 h. The
reaction mixture was cooled to room temperature, and then the
resulting solid was filtered, washed with ethanol, and then recrystal-
lized to give the desired gossypol derivatives.
General Procedure for the Synthesis of Aromatic Gossypol
Schiff Bases 15−18 and 20. A solution of gossypol acetic acid (2,
0.50 g, 0.86 mmol) and aromatic amine (3.46 mmol) in ethanol (50
mL) was stirred and refluxed for 5 h and then the reaction mixture was
cooled to room temperature. The resulting solid was filtered, washed
with ethanol, and then recrystallized to give the desired gossypol
derivatives.
MATERIALS AND METHODS
■
General. Reagents were purchased from commercial sources and
were used as received. All anhydrous solvents were dried and purified
by standard techniques just before use. Reaction progress was
monitored by thin-layer chromatography on silica gel GF-254 with
detection by UV. Melting points were determined on an X-4 binocular
Data for 3: chocolate crystal recrystallized from benzene; yield,
95%; mp, 252−253 °C (lit.17 258−260 °C); H NMR (300 MHz,
1
CDCl3), δ 14.92 (d, J = 12.3 Hz, 2H), 10.18 (d, J = 12.3 Hz, 2H), 7.88
(s, 2H), 7.63 (s, 2H), 7.41−7.28 (m, 8H), 7.18 (t, J = 7.1 Hz, 2H),
5.76 (s, 2H), 3.79−3.67 (m, 2H), 2.16 (s, 6H), 1.58−1.53 (m, 12H);
13C NMR (75 MHz, CDCl3), δ 174.34, 154.14, 149.39, 147.00, 139.47,
132.76, 129.87, 129.78, 128.76, 125.88, 118.68, 118.21, 116.31, 114.46,
105.09, 27.57, 20.32, 20.29, 20.10; HRMS (ESI) calcd for C42H39N2O6
(M − H)− 667.2814, found 667.2812.
1
microscope melting point apparatus and are uncorrected. H NMR
and 13C NMR spectra were recorded on a Bruker Ascend 300/400
MHz spectrometer. Chemical shift values (δ) are given in parts per
million and downfield from internal tetramethylsilane. High-resolution
mass spectra (HRMS) were recorded on FT-ICR MS (Ionspec, 7.0 T).
General Procedure for the Synthesis of Aromatic Gossypol
Schiff Bases 3−10, 12−14, 19, 21, 22, 24, and 25. A solution of
gossypol acetic acid (2, 0.50 g, 0.86 mmol) and aromatic amine (1.73
mmol) in ethanol (50 mL) was stirred and refluxed for 5 h, and then
the reaction mixture was cooled to room temperature. The resulting
Data for 4: orange crystal recrystallized from chloroform; yield,
95%; mp, 246−248 °C; 1H NMR (400 MHz, CDCl3), δ 14.97 (d, J =
12.4 Hz, 2H), 10.15 (d, J = 12.4 Hz, 2H), 7.91 (s, 2H), 7.63 (s, 2H),
7.23−7.13 (m, 8H), 5.74 (s, 2H), 3.80−3.69 (m, 2H), 2.33 (s, 6H),
2.15 (s, 6H), 1.58−1.53 (m, 12H); 13C NMR (100 MHz, CDCl3), δ
B
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173.91, 154.21, 149.35, 147.03, 137.00, 135.95, 132.61, 130.42, 129.66,
128.49, 118.62, 118.14, 116.17, 114.49, 104.84, 27.55, 20.94, 20.35,
20.31, 20.13; HRMS (ESI) calcd for C44H43N2O6 (M − H)− 695.3127,
found 695.3139.
Hz, 2H), 7.81 (s, 2H), 7.63 (s, 2H), 7.31 (d, J = 8.6 Hz, 4H), 7.22 (d, J
= 8.6 Hz, 4H), 5.80 (s, 2H), 3.77−3.67 (m, 2H), 2.16 (s, 6H), 1.56−
1.53 (m, 12H); 13C NMR (100 MHz, CDCl3), δ 174.77, 153.99,
149.39, 146.89, 146.59, 138.27, 133.08, 129.97, 129.27, 122.66, 121.67,
119.30, 119.11, 118.84, 116.36, 114.22, 105.42, 27.59, 20.28, 20.24,
20.10; HRMS (ESI) calcd for C44H37F6N2O8 (M − H)− 835.2460,
found 835.2441.
Data for 5: dark orange crystal recrystallized from tetrahydrofuran
and ethanol; yield, 60%; mp, 272−274 °C (lit.18 287−289 °C); H
1
NMR (400 MHz, CDCl3), δ 15.08 (d, J = 12.0 Hz, 2H), 10.09 (d, J =
12.0 Hz, 2H), 7.90 (s, 2H), 7.63 (s, 2H), 7.24 (d, J = 9.0 Hz, 4H), 6.89
(d, J = 9.0 Hz, 4H), 5.75 (s, 2H), 3.79 (s, 6H), 3.77−3.70 (m, 2H),
2.15 (s, 6H), 1.57−1.54 (m, 12H); 13C NMR (100 MHz, CDCl3), δ
173.44, 157.94, 154.43, 149.30, 147.03, 132.82, 132.48, 129.54, 128.31,
119.65, 118.56, 116.09, 115.09, 114.50, 104.74, 55.59, 27.54, 20.37,
20.33, 20.13; HRMS (ESI) calcd for C44H45N2O8 (M + H)+ 729.3170,
found 729.3163.
Data for 13: gold solid; yield, 91%; mp, 262−263 °C (lit.19 268−
1
268 °C); H NMR (400 MHz, DMSO-d6), δ 14.67 (d, J = 12.7 Hz,
2H), 10.48 (s, 2H), 10.43 (d, J = 12.7 Hz, 2H), 8.68 (s, 2H), 8.21 (s,
2H), 7.51 (s, 2H), 7.33 (d, J = 8.1 Hz, 2H), 7.07−6.98 (m, 4H), 6.86
(t, J = 7.3 Hz, 2H), 3.78−3.70 (m, 2H), 2.00 (s, 6H), 1.49−1.44 (m,
12H); 13C NMR (100 MHz, DMSO-d6), δ 174.09, 150.90, 150.09,
147.37, 146.49, 132.15, 128.02, 127.73, 127.39, 125.84, 120.87, 119.94,
117.04, 115.99, 115.38, 114.71, 105.64, 26.56, 20.23, 20.21; HRMS
(ESI) m/z calcd for C42H39N2O8 (M − H)− 699.2712, found
699.2704.
Data for 6: orange crystal recrystallized from pyridine; yield, 79%;
mp, 241−243 °C (lit.19 291 °C); H NMR (400 MHz, DMSO-d6), δ
1
15.18 (d, J = 11.8 Hz, 2H), 10.30 (d, J = 11.8 Hz, 2H), 9.66 (s, 2H),
8.52 (s, 2H), 8.15 (s, 2H), 7.51 (s, 2H), 7.21 (d, J = 8.8 Hz, 4H), 6.83
(d, J = 8.8 Hz, 4H), 3.79−3.70 (m, 2H), 1.99 (s, 6H), 1.49−1.44 (m,
12H); 13C NMR (100 MHz, DMSO-d6), δ 172.52, 155.74, 153.64,
149.98, 146.16, 132.00, 131.27, 127.99, 127.47, 120.75, 119.36, 116.90,
116.46, 115.49, 104.98, 26.56, 20.27, 20.24, 20.21; HRMS (ESI) calcd
for C42H39N2O8 (M − H)− 699.2712, found 699.2712.
Data for 14: orange crystal recrystallized from pyridine; yield, 56%;
mp, 259−262 °C (lit.19 287 °C); H NMR (400 MHz, DMSO-d6), δ
1
14.86 (d, J = 12.0 Hz, 2H), 10.34 (d, J = 12.0 Hz, 2H), 9.82 (s, 2H),
8.57(s, 2H), 8.32 (s, 2H), 7.51 (s, 2H), 7.22 (t, J = 8.1 Hz, 2H), 6.78−
6.74 (m, 2H), 6.73 (t, J = 1.9 Hz, 2H), 6.63 (dd, J = 8.1, 1.9 Hz, 2H),
3.80−3.68 (m, 2H), 1.99 (s, 6H), 1.49−1.44 (m, 12H); 13C NMR
(100 MHz, DMSO-d6), δ 174.04, 158.87, 152.99, 150.23, 146.21,
140.50, 132.42, 130.91, 128.80, 127.75, 121.20, 117.08, 115.31, 112.80,
108.52, 105.50, 104.19, 26.61, 20.20, 20.18; HRMS (ESI) m/z calcd
for C42H39N2O8 (M − H)− 699.2712, found 699.2704.
Data for 7: yellow crystal recrystallized from pyridine and ethanol;
yield, 78%; mp, 266 °C (dec) (lit.20 203−204 °C); H NMR (400
1
MHz, DMSO-d6), δ 14.96 (d, J = 11.6 Hz, 2H), 10.31 (d, J = 11.6 Hz,
2H), 8.54 (s, 2H), 8.22 (s, 2H), 7.52 (s, 2H), 7.45−7.38 (m, 4H), 7.30
(t, J = 8.5 Hz, 4H), 3.79−3.71 (m, 2H), 2.00 (s, 6H), 1.49−1.43 (m,
12H); 13C NMR (100 MHz, DMSO-d6), δ 173.56, 160.93, 158.52,
153.87, 150.11, 146.10, 136.30, 132.46, 128.86, 127.77, 121.16, 119.83,
119.75, 117.10, 116.89, 116.66, 115.35, 105.61, 26.60, 20.22, 20.19;
HRMS (ESI) calcd for C42H37F2N2O6 (M -H)− 703.2625, found
703.2603.
1
Data for 15: yellow solid; yield, 76%; mp, 252−254 °C; H NMR
(400 MHz, CDCl3), δ 14.75 (d, J = 12.8 Hz, 2H), 10.20 (d, J = 12.8
Hz, 2H), 7.97 (s, 2H), 7.63 (s, 2H), 7.37 (d, J = 7.5 Hz, 2H), 7.14 (t, J
= 7.3 Hz, 2H), 6.98 (d, J = 7.8 Hz, 2H), 6.93 (t, J = 7.7 Hz, 2H), 5.76
(s, 2H), 4.03 (s, 6H), 3.78−3.69 (m, 2H), 2.15 (s, 6H), 1.58−1.52 (m,
12H); 13C NMR (100 MHz, CDCl3), δ 174.31, 152.40, 149.78,
149.39, 147.18, 132.64, 129.78, 128.83, 128.29, 126.13, 121.27, 118.61,
116.20, 115.50, 114.52, 111.49, 105.41, 56.19, 27.53, 20.33, 20.28,
20.12; HRMS (ESI) calcd for C44H43N2O8 (M − H)− 727.3025, found
727.3002.
Data for 8: yellow crystal recrystallized from chloroform and
ethanol; yield, 64%; mp, 286−287 °C (lit.19 302 °C); H NMR (400
1
MHz, CDCl3), δ 14.95 (d, J = 12.0 Hz, 2H), 10.09 (d, J = 12.0 Hz,
2H), 7.82 (s, 2H), 7.63 (s, 2H), 7.33 (d, J = 8.6 Hz, 4H), 7.23 (d, J =
8.6 Hz, 4H), 5.75 (s, 2H), 3.80−3.64 (m, 2H), 2.15 (s, 6H), 1.56−1.53
(m, 12H); 13C NMR (100 MHz, CDCl3), δ 174.65, 153.69, 149.37,
146.90, 138.13, 133.01, 131.13, 129.97, 129.91, 129.13, 119.29, 118.82,
116.29, 114.21, 105.30, 27.58, 20.28, 20.24, 20.10; HRMS (ESI) calcd
for C42H37Cl2N2O6 (M − H)− 735.2034, found 735.2034.
1
Data for 16: yellow solid; yield, 83%; mp, 266−268 °C; H NMR
(400 MHz, CDCl3), δ 14.82 (d, J = 12.2 Hz, 2H), 10.14 (d, J = 12.2
Hz, 2H), 7.87 (s, 2H), 7.63 (s, 2H), 7.26−7.23 (t, J = 8.0 Hz, 2H),
6.89 (dd, J = 8.0, 1.4 Hz, 2H), 6.81 (s, 2H), 6.71 (dd, J = 8.0, 1.4 Hz,
2H), 5.83 (s, 2H), 3.79 (s, 6H), 3.76−3.67 (m, 2H), 2.15 (s, 6H),
1.57−1.52 (m, 12H); 13C NMR (100 MHz, CDCl3), δ 174.33, 160.83,
154.04, 149.42, 146.94, 140.61, 132.82, 130.69, 129.73, 128.86, 118.66,
116.38, 114.40, 111.27, 110.48, 105.07, 104.17, 55.45, 27.56, 20.29,
20.13; HRMS (ESI) calcd for C44H43N2O8 (M − H)− 727.3025, found
727.3009.
Data for 9: orange-red crystal recrystallized from pyridine and
ethanol; yield, 81%; mp, 284−286 °C (lit.19 305−306 °C); H NMR
1
(400 MHz, CDCl3), δ 14.84 (d, J = 11.7 Hz, 2H), 10.12 (d, J = 11.7
Hz, 2H), 8.24 (d, J = 9.0 Hz, 4H), 7.69 (s, 2H), 7.64 (s, 2H), 7.36 (d, J
= 9.0 Hz, 4H), 5.82 (s, 2H), 3.75−3.66 (m, 2H), 2.17 (s, 6H), 1.58−
1.52 (m, 12H); 13C NMR (100 MHz, CDCl3), δ 176.46, 155.92,
151.73, 149.87, 149.67, 146.75, 137.66, 135.94, 134.01, 130.47, 125.94,
123.74, 117.51, 107.20, 106.02, 27.69, 20.19, 20.14, 20.12; HRMS
(ESI) calcd for C42H38N4NaO10 (M + Na)+ 781.2480, found 781.2486.
Data for 10: gold crystal recrystallized from pyridine and ethanol;
1
Data for 17: orange solid; yield, 97%; mp, 255−257 °C; H NMR
(400 MHz, CDCl3), δ 14.89 (d, J = 12.8 Hz, 2H), 10.13 (d, J = 12.8
Hz, 2H), 7.99 (s, 2H), 7.63 (s, 2H), 7.27 (d, J = 8.8 Hz, 2H), 6.54 (d, J
= 2.4 Hz, 2H), 6.44 (dd, J = 8.8, 2.4 Hz, 2H), 5.76 (s, 2H), 3.99 (s,
6H), 3.79 (s, 6H), 3.77−3.69 (m, 2H), 2.15 (s, 6H), 1.57−1.52 (m,
12H); 13C NMR (100 MHz, CDCl3), δ 173.35, 158.65, 152.58,
151.01, 149.33, 147.22, 132.35, 129.51, 127.85, 122.56, 118.46, 116.56,
116.06, 114.60, 105.06, 104.97, 99.27, 56.22, 55.63, 27.51, 20.38,
20.33, 20.14; HRMS (ESI) calcd for C46H47N2O10 (M − H)−
787.3236, found 787.3236.
1
yield, 79%; mp, 261−263 °C; H NMR (400 MHz, CDCl3), δ 14.89
(d, J = 11.2 Hz, 2H), 10.15 (d, J = 12.0 Hz, 2H), 7.77 (s, 2H), 7.65−
7.55 (m, 6H), 7.37 (d, J = 8.4 Hz, 4H), 5.75 (s, 2H), 3.78−3.68 (m,
2H), 2.16 (s, 6H), 1.58−1.55 (m, 12H); 13C NMR (100 MHz,
CDCl3), δ 175.51, 153.08, 149.89, 149.48, 146.85, 142.44, 140.87,
133.41, 130.20, 129.67, 127.21, 127.19, 127.15, 119.01, 117.90, 116.45,
105.87, 105.85, 27.62, 20.25, 20.19, 20.10; HRMS (ESI) calcd for
C44H38F6N2NaO6 (M + Na)+ 827.2526, found 827.2520.
1
Data for 18: gold solid; yield, 89%; mp, 234−235 °C; H NMR
(400 MHz, DMSO-d6), δ 14.62 (d, J = 11.6 Hz, 2H), 10.39 (d, J =
11.6 Hz, 2H), 8.72 (s, 2H), 8.35 (s, 2H), 7.52 (s, 2H), 7.12 (d, J = 6.8
Hz, 2H), 6.92 (s, 2H), 6.77 (d, J = 6.8 Hz, 2H), 3.90 (s, 6H), 3.69 (br
s, 8H), 1.98 (s, 6H), 1.46 (br s, 12H); 13C NMR (100 MHz, DMSO-
d6), δ 174.76, 154.00, 150.79, 150.35, 146.52, 143.23, 132.48, 129.00,
128.66, 128.00, 120.80, 117.14, 115.03, 113.19, 109.66, 106.21, 101.40,
56.49, 55.59, 26.61, 20.18; HRMS (ESI) calcd for C46H47N2O10 (M −
H)− 787.3236, found 787.3219.
Data for 11: purple solid; yield, 36%; mp, >300 °C; 1H NMR (400
MHz, DMSO-d6), δ 14.94 (d, J = 11.7 Hz, 2H), 10.41 (d, J = 11.7 Hz,
2H), 8.58 (s, 2H), 8.34 (s, 2H), 7.64 (d, J = 8.0 Hz, 4H), 7.52 (s, 2H),
7.29 (d, J = 8.0 Hz, 4H), 3.80−3.70 (m, 2H), 2.00 (s, 6H), 1.52−1.43
(m, 12H); 13C NMR (100 MHz, DMSO-d6), δ 174.71, 153.23, 146.68,
145.89, 139.81, 133.05, 129.55, 128.31, 127.85, 127.12, 121.85, 117.28,
115.75, 112.67, 106.31, 27.12, 20.72, 20.68; HRMS (ESI) calcd for
C42H39N2O12S2 (M − 2Na + H)− 827.1950, found 827.1928.
1
Data for 19: chocolate solid; yield, 43%; mp, 251−253 °C; H
NMR (400 MHz, CDCl3), δ 15.21 (d, J = 11.0 Hz, 2H), 10.10 (d, J =
11.0 Hz, 2H), 7.74 (s, 2H), 7.68 (d, J = 7.7 Hz, 2H), 7.62 (s, 2H), 7.53
(t, J = 7.7 Hz, 2H), 7.46 (d, J = 7.7 Hz, 2H), 7.26 (t, J = 7.7 Hz, 2H),
1
Data for 12: yellow solid; yield, 72%; mp, 251−253 °C; H NMR
(400 MHz, CDCl3), δ 14.94 (d, J = 12.0 Hz, 2H), 10.10 (d, J = 12.0
C
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Scheme 1. General Synthetic Route for Target Compounds 3−25
5.77 (s, 2H), 3.76−3.66 (m, 2H), 2.15 (s, 6H), 1.57−1.51 (m, 12H);
13C NMR (100 MHz, CDCl3), δ 174.94, 154.82, 149.50, 146.83,
138.56, 133.50, 133.34, 130.21, 129.54, 127.10, 127.05, 126.99, 126.94,
125.26, 125.00, 122.28, 121.15, 120.85, 119.03, 118.92, 116.41, 114.15,
106.43, 27.59, 20.23, 20.19, 20.12; HRMS (ESI) calcd for
C44H37F6N2O6 (M − H)− 803.2561, found 803.2533.
130.10, 124.12, 119.80, 119.13, 116.55, 112.09, 106.15, 27.65, 20.22,
20.20, 20.14.
Data for 23: yellow solid; yield, 65%; mp, >300 °C; 1H NMR (400
MHz, DMSO-d6), δ 14.93 (d, J = 12.2 Hz, 2H), 10.42 (d, J = 12.2 Hz,
2H), 8.66 (s, 2H), 8.62 (s, 2H), 7.69 (s, 2H), 7.48 (s, 2H), 7.43 (d, J =
8.5 Hz, 2H), 7.14 (d, J = 8.5 Hz, 2H), 3.97 (s, 6H), 3.80−3.68 (m,
2H), 1.97 (s, 6H), 1.51−1.41 (m, 12H); 13C NMR (100 MHz,
DMSO-d6), δ 174.48, 150.97, 150.68, 149.01, 146.23, 141.88, 132.34,
128.66, 127.72, 127.38, 123.09, 121.61, 116.97, 115.30, 112.34, 111.12,
106.29, 56.38, 26.61, 20.32, 20.22, 20.19; HRMS (ESI) calcd for
C44H43N2O14S2 (M − 2Na + H)− 887.2161, found 887.2121.
Data for 24: orange-red crystal recrystallized from pyridine and
Data for 20: yellow solid; yield, 81%; mp, 262−264 °C (lit.21 216−
217.5 °C); 1H NMR (400 MHz, CDCl3), δ 15.02 (d, J = 11.7 Hz, 2H),
10.14 (d, J = 11.7 Hz, 2H), 7.79 (s, 2H), 7.65 (s, 2H), 7.54−7.41 (m,
8H), 5.79 (s, 2H), 3.77−3.69 (m, 2H), 2.16 (s, 6H), 1.58−1.53 (m,
12H); 13C NMR (100 MHz, CDCl3), δ 175.19, 153.49, 149.49,
146.87, 140.23, 133.31, 133.11, 132.66, 132.32, 131.98, 130.54, 130.12,
129.55, 124.89, 122.18, 122.14, 122.10, 121.16, 118.97, 116.44, 114.91,
114.87, 114.11, 105.69, 27.63, 20.27, 20.24, 20.12; HRMS (ESI) calcd
for C44H37F6N2O6 (M − H)− 803.2561, found 903.2543.
ethanol; yield, 82%; mp, 281 °C (lit.19 302−303 °C); H NMR (400
1
MHz, CDCl3), δ 15.17 (d, J = 12.2 Hz, 2H), 10.32 (d, J = 12.2 Hz,
2H), 7.92 (s, 2H), 7.84 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 7.4 Hz, 4H),
7.69 (s, 2H), 7.67 (s, 2H), 7.51−7.38 (m, 6H), 5.85 (s, 2H), 3.82−
3.70 (m, 2H), 2.19 (s, 6H), 1.59−1.55 (m, 12H); 13C NMR (100
MHz, CDCl3), δ 174.36, 153.88, 149.81, 149.44, 147.01, 136.86,
133.82, 132.88, 131.46, 130.09, 129.82, 128.80, 127.85, 127.50, 127.25,
125.79, 118.74, 117.41, 116.31, 115.22, 105.33, 27.57, 20.33, 20.28,
20.14.
Data for 21: yellow crystal recrystallized from petroleum ether and
1
ethyl acetate; yield, 49%; mp, 221−223 °C; H NMR (400 MHz,
CDCl3), δ 15.12 (d, J = 11.6 Hz, 2H), 10.10 (d, J = 11.6 Hz, 2H),
7.72−7.62 (m, 10H), 5.85 (s, 2H), 3.89−3.58 (m, 2H), 2.17 (s, 6H),
1.57−1.51 (m, 12H); 13C NMR (100 MHz, CDCl3), δ 175.87, 152.50,
149.68, 146.72, 141.32, 133.99, 133.93, 133.66, 133.32, 130.49, 130.45,
124.15, 121.44, 119.22, 118.41, 117.71, 116.73, 113.77, 106.48, 27.71,
20.20, 20.17; HRMS (ESI) calcd for C46H35F12N2O6 (M − H)−
939.2309, found 939.2283.
Data for 25: orange-red solid; yield, 86%; mp, 280−281 °C (lit.19
1
283−285.5 °C); H NMR (400 MHz, CDCl3), δ 15.88 (d, J = 11.6
Hz, 2H), 10.36 (d, J = 11.6 Hz, 2H), 8.36 (d, J = 8.4 Hz, 2H), 7.99 (s,
2H), 7.91 (d, J = 8.4 Hz, 2H), 7.75−7.70 (m, 4H), 7.68 (s, 2H), 7.60
(t, J = 7.5 Hz, 2H), 7.52 (d, J = 7.4 Hz, 2H), 7.44 (t, J = 7.8 Hz, 2H),
5.86 (s, 2H), 3.85−3.73 (m, 2H), 2.20 (s, 6H), 1.60 (t, J = 6.1 Hz,
12H); 13C NMR (100 MHz, CDCl3), δ 174.30, 155.54, 149.84,
149.54, 147.03, 136.11, 135.94, 134.22, 132.86, 129.76, 128.93, 128.55,
127.34, 126.89, 126.38, 125.74, 125.59, 123.72, 121.22, 118.69, 116.31,
114.44, 113.69, 105.89, 27.59, 20.34, 20.29, 20.15.
Data for 22: orange-red crystal recrystallized from pyridine and
ethanol; yield, 62%; mp, 269−270 °C (lit.19 280−282 °C); H NMR
1
(400 MHz, CDCl3), δ 15.03 (d, J = 11.6 Hz, 2H), 10.16 (d, J = 11.6
Hz, 2H), 8.15 (s, 2H), 8.00 (d, J = 7.5 Hz, 2H), 7.73 (s, 2H), 7.66 (s,
2H), 7.62−7.50 (m, 4H), 5.79 (s, 2H), 3.76−3.70 (m, 2H), 2.17 (s,
6H), 1.56 (br s, 12H); 13C NMR (100 MHz, CDCl3), δ 175.68,
152.92, 149.84, 149.57, 149.37, 146.75, 141.03, 133.63, 130.80, 130.32,
D
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1
Figure 3. H NMR spectra of compound 5.
Biological Assay. The anti-TMV activity of all compounds was
tested using our previously reported methods.22
symmetrical protons should appear at the same position and
have identical coupling constants because of the axial symmetry
of the structure. The presence of doublet peaks with the
coupling constant of 12.0 Hz in the 1H NMR spectrum can be
attributed to the signals of HCC proton (j) and HNAr
proton (k). This behavior can be rationally explained by the
fact that two protons are adjacent and coupled with each other.
Antiviral Biological Assay. Compounds 1−25 were
initially tested for their phytotoxic activities against the test
plant, Nicotiana tabacum L. The results indicated that these
natural product-based compounds showed no phytotoxic
activity at 500 μg/mL. In other words, the compounds caused
no harm to the test plant; therefore, there would be no
interference with the determination of anti-TMV data.
The field trials were carried out in the trial area of the Institute of
Agricultural Environment and Resource, Yunnan Academy of
Agricultural Sciences, Kunming City, China, between June and July
2014. The formulation of compound 19 (1% emulsifiable concentrates
(EC)) was prepared in our research group. It was diluted to 750 L/ha
before use.
The rat oral gavage acute toxicity of pesticides was performed in Xu
He (Tianjin) Pharmaceutical Technology Co., Ltd. in May 2014.
Sixteen rats, half males and half females, were divided randomly into
four groups. The samples were respectively configured to 5 and 50
mg/mL.
RESULTS AND DISCUSSION
■
Chemistry. Gossypol (1) was obtained using our previously
reported method.23 Gossypol acetic acid (2) was purchased
from commercial sources. A series of Schiff bases of gossypol
(3−25) were prepared by treating 2 with the corresponding
amines in ethanol (Scheme 1). The Schiff bases had poor
solubility in ethanol, so they were simply collected by filtration.
Recrystallization of the products from suitable solvents could
give purer samples. Unfortunately, recrystallization was not fit
for compounds 15−18 and 20. However, they were obtained
via the employment of excess amine when the preparation was
repeated. For compounds 11 and 23, the sulfonic sodium salts
of corresponding amines were prepared from homologous
aminobenzenesulfonic acid and NaOH, for which were used 3
equiv of gossypol to ensure them a sodium sulfonate group. It is
worth mentioning that if there are multiple electron with-
drawing groups on the ortho and para positions of aromatic
amine, such as 2,4-dinitroaniline and 2-nitro-4-
(trifluoromethyl)aniline, the corresponding Schiff base of
gossypol could not be achieved but the dehydration product
of gossypol, anhydrogossypol,24 was obtained. All of our
synthetic gossypol Schiff bases exist in the enamine−enamine
form15,25 in solution as determined by 1H NMR. For
compound 5, for example, the structure can be interpreted by
the 1H NMR spectrum (Figure 3). The signal peaks of
The antiviral bioassay of the compounds against TMV was
carried out, and the antiviral results are listed in Tables 1 and 2.
To make a judgment on the antiviral potency of the synthesized
compounds, the commercially available plant virucide ribavir-
in24 was used as the control.
The in vitro anti-TMV activities of compounds 1−25 and the
contrast compound ribavirin are shown in Table 1. The results
indicate that these target compounds exhibit moderate to
excellent activities against TMV. Among these compounds,
gossypol (1) and gossypol acetic acid (2) displayed similar in
vitro anti-TMV activities (1, 32%; 2, 28%); however, they were
less effective than ribavirin (38%) at 500 μg/mL. In contrast,
except for 6, 22, and 24, other aromatic gossypol Schiff bases of
3−25 showed equivalent or even higher antiviral activities than
ribavirin (38%); especially compounds 5, 9, 10, 12, 15, 17, 19,
and 23 displayed obviously higher anti-TMV activities at a
concentration of 500 μg/mL, and the antiviral activities of
compounds 9 and 19 at 100 μg/mL even reach the level of
ribavirin at 500 μg/mL.
On comparison with the parent dianilinogossypol (3), except
for p-hydroxy compound 6, the activities of Schiff bases 4−12
with different substituent groups on the para position of aniline
are higher. However, they displayed distinctly different activity
levels against TMV in vitro. Compounds with methoxy (5),
E
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Table 1. In Vitro Antiviral Activity of Compounds 1−25 and
Ribavirin against TMV
Table 2. In Vivo Anti-TMV Activity of Compounds 1−25
and Ribavirin
in vitro
curative
in vitro
curative
in vivo activity
concn
concn
concn
(μg/mL)
inactivation
curative effect
protection
compd (μg/mL) effect (%)
compd
14
(μg/mL) effect (%)
compd
effect (%)
(%)
effect (%)
1
500
100
32
0
500
100
30
0
1
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
35
8
29
0
29
0
2
32
0
30
0
33
0
2
500
100
28
0
15
500
100
58
14
3
52
27
8
34
0
44
27
50
17
44
21
23
0
3
500
100
32
0
16
500
100
40
0
4
37
0
0
5
46
19
20
0
42
20
17
0
4
500
100
40
0
17
500
100
53
22
6
5
500
100
47
10
18
500
100
30
0
7
33
11
36
9
36
0
35
9
8
41
19
47
40
51
22
35
0
43
20
42
22
42
19
36
9
6
500
100
24
0
19
500
100
62
32
9
36
12
35
25
38
0
7
500
100
37
6
20
500
100
39
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
ribavirin
8
500
100
44
0
21
500
100
32
0
54
26
35
10
40
10
51
16
46
18
56
28
36
8
50
21
36
0
56
23
34
9
9
500
100
50
39
22
500
100
26
0
34
8
38
15
56
27
48
23
50
23
36
0
10
11
12
13
500
100
57
20
23
500
100
47
18
55
20
43
9
500
100
40
0
24
500
100
25
0
48
19
33
0
500
100
52
20
25
500
100
44
16
500
100
33
0
ribavirin
500
100
38
14
60
23
52
17
41
12
26
0
57
30
35
13
35
0
62
26
48
22
37
9
nitro (9), trifluoromethyl (10), and trifluoromethoxy (12)
showed excellent activities (48, 50, 57, and 52%, respectively).
To investigate the influence of the position and the number of
substituents on the activities, 13−23 were synthesized.
Compared with their para-substitutional substrates, correspond-
ing substituent groups on the ortho position were beneficial to
the increase of their anti-TMV activities, whereas the
substituent groups on meta positions were unfavorable to the
anti-TMV activities (MeO, 15 > 5 > 16; CF3, 19 > 10 >20;
NO2, 9 > 22). However, the substituent group of the hydroxyl
was an exception, as the o-hydroxyl compound 13 and m-
hydroxyl compound 14 had higher activities than p-hydroxyl
compound 6. Furthermore, the level of the anti-TMV activities
could not be improved by increasing the number of substituent
groups. Among methoxy-substituted compounds 5 and 15−18,
15 with only one substituent group on the ortho position
displayed the best inhibitory effect. However, 23 (18%) with o-
methoxy and m-sodium sulfonate substituent groups exhibited
better inhibitory effect than 11 (0%) and 15 (14%) at 100 μg/
23
0
34
0
46
17
37
8
43
13
28
0
38
15
30
0
39
10
38
12
37
19
37
17
43
16
40
18
mL. The Schiff bases of naphthylamines were synthesized as
well, and their anti-TMV activities were performed. Compound
25 (44% at 500 μg/mL), obtained by the condensation of
gossypol and 1-naphthylamine, displayed a better inhibitory
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effect than dianilinogossypol (3, 32% at 500 μg/mL). At
variance with 25, the activity of 2-naphthylamine Schiff base 24
(25% at 500 μg/mL) was relatively lower than that of 3.
Due to their well-performed behaviors of in vitro antiviral
activities against TMV, the in vivo antiviral activities of these
compounds deserve to be exploited further. As shown in Table
2, the majority of the compounds also displayed equivalent or
higher in vivo activities than the control ribavirin at a
concentration of 500 μg/mL. In particular, Schiff bases 12,
15, 17, and 19 exhibited obviously higher anti-TMV activities
than ribavirin at 500 μg/mL, of which compound 19 with an o-
trifluoromethyl group showed the best activity (inactivation
activity, 60%/500 μg/mL and 23%/100 μg/mL; curative
activity, 57%/500 μg/mL and 30%/100 μg/mL; and protection
activity, 62%/500 μg/mL and 26%/100 μg/mL), confirmed by
the in vivo inactivation tests, curative tests, and protection tests.
Additionally, the in vivo curative activities of these compounds
displayed structure−activity relationships similar to the
activities in vitro. More importantly, compound 9 with a p-
nitro group showed a better effect at 100 μg/mL (40%), which
is even higher than that of ribavirin at 500 μg/mL (37%).
Interestingly, the in vivo inactivation and protection activities
of dianilinogossypol (3) displayed excellent inhibitory effects
(inactivation activity, 52%/500 μg/mL and 27%/100 μg/mL;
protection activity, 44%/ 500 μg/mL and 27%/100 μg/mL).
However, only 12, 15, 17, 19, and 20 exhibited obviously better
in vivo curative and protection activities at 500 μg/mL than
parent dianilinogossypol (3). These results indicated the
introduction of trifluoromethoxy, methoxy, and trifluoromethyl
on the aromatic ring of aniline could increase the anti-TMV
activities level of parent dianilinogossypol. Further analysis of
the relationship between the positions of substituents and anti-
TMV activities indicated that the order of methoxy and
trifluoromethyl is ortho > meta > para position (15 > 16 > 5,
19 > 20 > 10). Other groups and the number of them displayed
similar SAR to anti-TMV activities in vitro. The derivatives of
naphthylamines (24 and 25) had lower inhibitory effect than
dianilinogossypol (3), but 1-naphthylamine (25) is relatively
higher than that of 2-naphthylamine (24).
Rat Oral Gavage Acute Toxicity of Gossypol and 19.
To balance the toxicity versus efficacy of compound 19, we next
explored the tolerated dose of 19 and gossypol using a group of
rats. They were treated with a single dose of 500 or 50 mg/kg
and observed for a period of 14 days, monitoring morbidity
(body weight loss) and mortality. All rats were alive after 14
days. The weight of the rats that were treated with gossypol at
500 mg/kg slightly declined over the first 6 days. There is no
obvious weight difference between any other two groups
(Figure 4). Therefore, gossypol and 19 display low toxicity to
rats, but the toxicity of 19 is lower.
Figure 4. Average weight of rats. Gossypol-L, dosage of gossypol is 50
mg/kg; gossypol-H, dosage of gossypol is 500 mg/kg; 19-L, dosage of
19 is 50 mg/kg; 19-H, dosage of 19 is 500 mg/kg.
In summary, to eliminate the toxicity caused by the aldehyde
group, retain the electron transfer functionalities to generate
ROS, and adjust the anti-TMV activity level of gossypol, a
series of aromatic gossypol Schiff bases 3−25 were prepared
and investigated for their anti-TMV activity for the first time.
The bioassay results indicated that most of these Schiff bases
showed moderate to excellent activities against TMV and were
more effective than gossypol (1) and gossypol acetic acid (2).
The introduction of methoxy, trifluoromethyl, or trifluorome-
thoxy groups, positioned ortho to amine of enamine, could
significantly increase the anti-TMV activity levels. Among all of
the compounds, compound 19 with an o-trifluoromethyl group
showed the best anti-TMV activities both in vitro and in vivo in
the laboratory. Furthermore, the compound at 10 g ai/ha in the
field trial had the same control effect as 5% amino glycan at 100
g ai/ha and 20% moroxydine hydrochloride−cupric acetate at
600 g ai/ha. More importantly, it showed lower toxicity to rats
than gossypol. Because of the easy preparation, good anti-TMV
activity, and low toxicity, the o-trifluoromethylaniline Schiff
base (19) is expected to be developed as a promising candidate
as an inhibitor of plant virus. Further studies on structural
optimization and mechanism of action are currently underway
in our laboratory.
Evaluation of Anti-TMV Activity of 19 in the Field.
Compound 19, which had exhibited the best anti-TMV activity
both in vitro and in vivo in the laboratory, was employed to
evaluate the antiviral activity in the field. A 1% EC solution of
19 was prepared and evaluated for the control of TMV in
Tengchong county of Yunnan province (southern China). The
results of the field trials are shown in Table 3, which indicated
that 19 at the amount of 10 g ai/ha had the same control effect
(about 66%) as 5% amino glycan at 100 g ai/ha and 20%
moroxydine hydrochloride−cupric acetate at 600 g ai/ha at 10
days after spraying. Therefore, it was suggested that 19 could be
considered as an alternative to the employed plant virus
inhibitor for further development on the basis of the field trials.
Table 3. Anti-TMV Effect in the Field in 2014
amount
(g ai/ha)
controlled effect
compd
(%)
19
10
50
66.61
83.88
94.56
65.70
66.57
ASSOCIATED CONTENT
■
100
100
600
S
* Supporting Information
5% amino glycan
Fungicidal, insecticidal, and herbicidal activities. This material is
available free of charge via the Internet at http://pubs.acs.org.
20% moroxydine hydrochloride−cupric
acetate
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AUTHOR INFORMATION
Corresponding Author
*(Q.W.) Phone: +86-(0)22-23503952. Fax: +86-(0)22-
23503952. E-mail: wang98h@263.net or wangqm@nankai.
edu.cn.
■
Funding
We are grateful to the National Natural Science Foundation of
China (21132003, 21121002, 21372131), the Specialized
Research Fund for the Doctoral Program of Higher Education
(20120031110010), and the Science Technology Plan Project
of China Tobacco Yunnan Co. (2012YN08) for generous
financial support for our programs.
Notes
The authors declare no competing financial interest.
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