First discovery of polycarpine, polycarpaurines A and C, and their derivatives as novel antiviral and antiphytopathogenic fungus agents

Article abstract of DOI:10.1021/acs.jafc.6b01415

Marine natural products polycarpine, polycarpaurines A and C, and their derivatives were designed, synthesized, and characterized on the basis of ¹H NMR and mass spectroscopy. The antiviral and antiphytopathogenic fungus activities of these alkaloids were first evaluated. Polycarpine derivative 1g displayed excellent in vivo antiviral activity against TMV (inactivation inhibitory effect, 57%/500 μg mL^-1 and 19%/100 μg mL^-1; curative inhibitory effect, 62%/500 μg mL^-1 and 23%/100 μg mL^-1; and protection inhibitory effect, 56%/500 μg mL^-1 and 29%/100 μg mL^-1), which is evidently higher than the activity of ribavirin (inactivation inhibitory effect, 37%/500 μg mL^-1 and 9%/100 μg mL^-1; curative inhibitory effect, 36%/500 μg mL^-1 and 13%/100 μg mL^-1; and protection inhibitory effect, 39%/500 μg mL^-1 and 17%/100 μg mL^-1), thus emerging as a new lead compound for antiviral research against TMV. Fungicidal testing in vitro showed that most of the compounds displayed good fungicidal activity against plant pathogenic fungi. Further in vivo fungicidal testing indicated that compounds 6a, 6f, and 8a-c displayed good fungicidal activity. Current results provide support for the development of polycarpine alkaloids as novel agrochemicals.

Full text of DOI:10.1021/acs.jafc.6b01415

Article
pubs.acs.org/JAFC
First Discovery of Polycarpine, Polycarpaurines A and C, and Their
Derivatives as Novel Antiviral and Antiphytopathogenic Fungus Agents
Pengbin Guo,† Ziwen Wang,*,‡ Gang Li,† Yuxiu Liu,† Yunfu Xie,‡ and Qingmin Wang*,†
‡Tianjin Key Laboratory of Structure and Performance for Functional Molecules, Key Laboratory of Inorganic−Organic Hybrid
Functional Materials Chemistry (Tianjin Normal University), Ministry of Education, College of Chemistry, Tianjin Normal
University, Tianjin 300387, China
†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, China
S
* Supporting Information
ABSTRACT: Marine natural products polycarpine, polycarpaurines A and C, and their derivatives were designed, synthesized,
1
and characterized on the basis of H NMR and mass spectroscopy. The antiviral and antiphytopathogenic fungus activities of
these alkaloids were first evaluated. Polycarpine derivative 1g displayed excellent in vivo antiviral activity against TMV
(inactivation inhibitory effect, 57%/500 μg mL−1 and 19%/100 μg mL−1; curative inhibitory effect, 62%/500 μg mL−1 and
23%/100 μg mL−1; and protection inhibitory effect, 56%/500 μg mL−1 and 29%/100 μg mL−1), which is evidently higher
than the activity of ribavirin (inactivation inhibitory effect, 37%/500 μg mL−1 and 9%/100 μg mL−1; curative inhibitory effect,
36%/500 μg mL−1 and 13%/100 μg mL−1; and protection inhibitory effect, 39%/500 μg mL−1 and 17%/100 μg mL−1), thus
emerging as a new lead compound for antiviral research against TMV. Fungicidal testing in vitro showed that most of the
compounds displayed good fungicidal activity against plant pathogenic fungi. Further in vivo fungicidal testing indicated that
compounds 6a, 6f, and 8a−c displayed good fungicidal activity. Current results provide support for the development of
polycarpine alkaloids as novel agrochemicals.
KEYWORDS: marine natural products, polycarpine, anti-TMV, antifungal activity
INTRODUCTION
■
The global population will exceed 9 billion people by 2050 from
the present 7 billion, which is associated with the demand for
increasing food production.1,2 Plant diseases, caused by fungal
and bacterial pathogens and viruses, are known as “plant cancer”.
These diseases can lead to severe losses to agriculture and
horticulture crops. Thus, new tools to control these diseases are
critically needed. On the other hand, many pesticides are losing
their effectiveness due to the evolution of pathogen resistance
Figure 1. Structures of alkaloids polycarpine (1a) and polycarpaurines A
(2) and C (3).
over the past decade. As such, more novel agrochemicals are
needed.3
For example, phenanthroindolizidine alkaloids with anti-TMV
activity also displayed good anti-HIV activity.10
Marine natural products have played a very important role in
In the discovery process of antiviral agents from natural
products using TMV as template test virus, phenanthroindoli-
zidine alkaloids,11 phenanthroquinolizidine alkaloids,12 and
beta-carboline alkaloids13 have been discovered as antiviral agents
against TMV. In current work, polycarpine, polycarpaurines A
and C, and their derivatives were systematically investigated for
their antiviral and antiphytopathogenic fungus activities.
the development of new drugs. The search for leads or scaffolds
from marine natural products started from the middle of the last
century.4 Nereistoxin, from the polychaeta worm Lumbrinereis
sp., is the first marine natural product with industrial use as a
highly effective, low toxicity, and low residue pesticide.5,6 With
the improvement of elucidation techniques for molecules and
chemical synthesis, more and more bioactive natural products
have been characterized from marine organisms. Polycarpine
(1a, Figure 1) was isolated from the ascidian Polycarpa aurata.7
As a promising marine natural product, polycarpine was
previously found to have antitumor activity.8,9 Polycarpaurines
A and C (2 and 3, Figure 1) are another two interesting marine
natural products, isolated from Indonesian ascidian.9
MATERIALS AND METHODS
■
Instruments. The melting points of the synthesized compounds
were determined on an X-4 binocular microscope (Beijing Tech
Instruments Co., Beijing, China). NMR spectra were obtained by using
Tobacco Mosaic Virus (TMV) can infect more than 400 plant
species, such as tobacco, potato, tomato, and cucumber. As
a well-studied plant virus, TMV can also be used as template
test virus to discover antiviral agents against other viruses.
Received: March 27, 2016
Revised: May 2, 2016
Accepted: May 9, 2016
Published: May 19, 2016
© 2016 American Chemical Society
4264
DOI: 10.1021/acs.jafc.6b01415
J. Agric. Food Chem. 2016, 64, 4264−4272

Journal of Agricultural and Food Chemistry
Article
Bruker AV 400/300 spectrometer. Chemical shifts (δ) are given in parts
per million (ppm) and were measured downfield from internal
tetramethylsilane. High-resolution mass spectra were obtained with an
FT-ICR MS spectrometer (Ionspec, 7.0 T).
3.04 mmol), and the mixture was stirred at room temperature for 1 h.
The mixture was concentrated, and the residue was recrystallized
with acetone (50 mL) to provide polycarpaurine C (3, 0.5 g, 72%) as a
yellow solid: mp 184 °C (dec); 1H NMR (400 MHz, DMSO-d6) δ 12.90
(s, 1H, NH), 7.88 (d, J = 8.9 Hz, 2H, Ar−H), 7.72 (s, 2H, NH2), 7.04
(d, J = 8.9 Hz, 2H, Ar−H), 3.81 (s, 3H, O−CH3), 3.50 (s, 3H, N−CH3).
General Procedures for the Preparation of Intermediates
4a−g. To a solution of imidazole amine 14 (1.0 g, 4.92 mmol) and
NEt3 (1.7 mL, 11.81 mmol) in dichloromethane (125 mL) was added
a solution of corresponding acyl chlorides (11.81 mmol) in dichloro-
methane (10 mL) drop by drop. The mixture was stirred for 30 min
at room temperature, washed with NaHCO3 solution (100 mL) and
brine (100 mL), dried over anhydrous MgSO4, and then filtered and
concentrated in vacuo. After purification by column chromatography
on silica gel, compound 4a was obtained. Compounds 4b−g were used
directly for the next step without purification.
Synthesis of 1-(4-Methoxyphenyl)ethanone (10). A mixture of
1-(4-hydroxyphenyl)ethanone (9, 25.0 g, 0.18 mol), K2CO3 (51.1 g,
0.37 mol), CH3I (28.4 g, 0.20 mol), and acetone (500 mL) was stirred
and heated to reflux for 2 h. Then the mixture was filtered and
evaporated. The residue was taken into CH2Cl2 (300 mL) and H2O
(300 mL) and separated. The aqueous layer was extracted with CH2Cl2
(60 mL × 2), and then the organic layer was dried over anhydrous
MgSO4 and evaporated. After purification by column chromatography
on silica gel, 1-(4-methoxyphenyl)ethanone (10, 24.6 g, 89%) was
obtained as a white solid: mp 33−35 °C; 1H NMR (400 MHz, CDCl3)
δ 7.94 (d, J = 8.8 Hz, 2H, Ar−H), 6.94 (d, J = 8.9 Hz, 2H, Ar−H), 3.87
(s, 3H, O−CH3), 2.56 (s, 3H, CH3CO).
N-Acetyl-N-(4-(4-methoxyphenyl)-1-methyl-1H-imidazol-2-
yl)acetamide (4a). White powder: yield 78%; mp 133−136 °C;
1H NMR (400 MHz, CDCl3) δ 7.63 (d, J = 8.9 Hz, 2H, Ar−H), 7.14
(s, 1H, CH), 6.91 (d, J = 8.9 Hz, 2H, Ar−H), 3.82 (s, 3H, O−CH3), 3.49
(s, 3H, N−CH3), 2.34 (s, 6H, CH3CO); 13C NMR (100 MHz, CDCl3)
δ 172.3, 158.9, 140.1, 138.9, 126.3, 126.0, 115.7, 114.0, 55.3, 32.3,
25.9; HRMS (ESI) calcd for C15H18N3O3 [M + H]+ 288.1343, found
288.1348.
General Procedures for the Preparation of Intermediates
5a−g. A solution of the corresponding compounds 4a−g (2.78 mmol),
TBAF (3.6 g, 13.90 mmol) and pyridine (1.1 g, 13.90 mmol) in THF
(100 mL) was stirred at room temperature for 3.5 h and concentrated.
The residue was taken into CH2Cl2 (150 mL), washed successively with
H2O (100 mL) and brine (100 mL), and then dried over anhydrous
MgSO4. After filtration and concentration, the residue was recrystallized
from acetone to give the corresponding compounds 5a−g.
Synthesis of 2-Bromo-1-(4-methoxyphenyl)ethanone (11).
To a solution of 1-(4-methoxyphenyl)ethanone (10, 24.0 g, 0.16 mol)
and concentrated HCl (0.2 mL) in MeOH (100 mL) was added a
solution of Br2 (9.8 mL, 0.19 mol) in MeOH (80 mL) drop by drop at
0 °C. The reaction mixture was stirred for 1 h at 0 °C and then warmed
to room temperature. After stirring for another 2 h at room temperature,
the mixture was concentrated in vacuo to 40 mL, kept at 0 °C for
3 h, and then filtered to give 2-bromo-1-(4-methoxyphenyl)ethanone
(11, 31.0 g, 85%) as a white solid: mp 70−73 °C; 1H NMR (300 MHz,
CDCl3) δ 7.97 (d, J = 8.9 Hz, 2H, Ar−H), 6.96 (d, J = 8.9 Hz, 2H,
Ar−H), 4.40 (s, 2H, CH2CO), 3.88 (s, 3H, O−CH3).
Synthesis of 1-(4-Methoxyphenyl)-2-(methylamino)-
ethanone Hydrochloride (12). To a solution of methylamine
(30% solution in absolute ethanol, 104 mL, 0.68 mol) in acetonitrile
(200 mL) was added dropwise a solution of 2-bromo-1-(4-
methoxyphenyl)ethanone (11, 31.0 g, 0.14 mol) in acetonitrile (150 mL)
and dichloromethane (50 mL) at −30 °C, and then the mixture was
stirred for 10 min. The mixture was deliquated with dichloromethane
(350 mL), washed successively with NaHCO3 solution (200 mL) and
H2O (200 mL), then dried over anhydrous MgSO4, and filtered. To the
filtrate was added a solution of HCl in ethyl acetate (239 mL, 1.125 M,
0.27 mol). The resulting mixture was stirred for 5 min at room
temperature and evaporated. The residue was recrystallized from
acetone (100 mL) to give compound 12 (17.1 g, 59%) as a white solid:
mp 196−199 °C; 1H NMR (400 MHz, CD3OD) δ 8.01 (d, J = 9.0 Hz,
2H, Ar−H), 7.08 (d, J = 9.0 Hz, 2H, Ar−H), 4.69 (s, 2H, CH2CO),
3.90 (s, 3H, O−CH3), 2.83 (s, 3H, CH3NH).
Synthesis of compound 14. To a solution of compound 12
(25.3 g, 0.12 mol) and 2-ethylisothiourea hydroiodide 13 (40.6 g,
0.18 mol)14 in H2O (350 mL) was added a solution of NaOH (16.4 g,
0.41 mol) in H2O (150 mL). The resulting mixture was stirred for 24 h at
room temperature and filtered to give compound 14 (18.2 g, 76%) as a
yellow powder: mp 143−145 °C; 1H NMR (400 MHz, CDCl3) δ 7.57
(d, J = 8.8 Hz, 2H, Ar−H), 6.88 (d, J = 8.8 Hz, 2H, Ar−H), 6.71 (s, 1H,
CH), 4.01 (s, 2H, NH2), 3.81 (s, 3H, O−CH3), 3.45 (s, 3H, N−CH3).
Synthesis of Polycarpine (1a). To a solution of compound 14
(4.0 g, 19.68 mmol) in acetic acid (200 mL) was added S2Cl2 (1.6 g,
11.81 mmol) and stirred for 10 h at room temperature. The mixture was
concentrated, and the residue was recrystallized with acetone (50 mL)
to obtain 1a (3.4 g, 63%) as a yellow powder: mp 206−208 °C (lit.8 mp
209−211 °C); 1H NMR (400 MHz, DMSO-d6) δ 13.43 (s, 2 H, HCl),
7.67 (s, 4 H, NH2), 7.46 (d, J = 8.4 Hz, 4 H, Ar−H), 7.01 (d, J = 8.9 Hz,
4 H, Ar−H), 3.89 (s, 3 H, OMe), 3.19 (s, 3 H, NMe).
N-(4-(4-Methoxyphenyl)-1-methyl-1H-imidazol-2-yl)-
acetamide (5a). White powder: yield 58%; mp 200−203 °C; 1H NMR
(400 MHz, CDCl3) δ 7.56 (d, J = 6.7 Hz, 2H, Ar−H), 6.97 (s, 1H, CH),
6.93 (d, J = 7.8 Hz, 2H, Ar−H), 3.85 (s, 3H, O−CH3), 3.60 (s, 3H,
N−CH3), 2.16 (s, 3H, CH3CO); 13C NMR (100 MHz, CDCl3) δ 171.9,
158.7, 141.0, 137.4, 126.4, 125.8, 114.4, 114.1, 55.3, 33.3, 23.1; HRMS
(ESI) calcd for C13H16N3O2+ [M + H]+ 246.1237, found 246.1242.
N-(4-(4-Methoxyphenyl)-1-methyl-1H-imidazol-2-yl)-
methanesulfonamide (5b). Light gray powder: yield 5%; mp 110−
1
113 °C; H NMR (400 MHz, CDCl3) δ 10.02 (s, 1H), 7.30 (d, J =
8.8 Hz, 2H, Ar−H), 6.93 (d, J = 8.8 Hz, 2H, Ar−H), 6.52 (s, 1H, CH),
3.83 (s, 3H, O−CH3), 3.42 (s, 3H, N−CH3), 3.03 (s, 3H, CH3SO2);
13C NMR (100 MHz, CDCl3) δ 159.7, 147.3, 125.6, 125.0, 120.0, 114.6,
109.6, 55.4, 42.4, 31.6; HRMS (ESI) calcd for C12H16N3O3S+ [M + H]+
282.0907, found 282.0907.
N-(4-(4-Methoxyphenyl)-1-methyl-1H-imidazol-2-yl)-
pivalamide (5c). White powder: yield 53%; mp 225−226 °C; 1H NMR
(400 MHz, CDCl3) δ 9.71 (s, 1H, NH), 7.50 (d, J = 8.7 Hz, 2H, Ar−H),
6.89 (s, 1H, CH), 6.88 (d, J = 8.7 Hz, 2H, Ar−H), 3.81 (s, 3H, O−CH3),
3.51 (s, 3H, N−CH3), 1.31 (s, 9H, C−CH3); 13C NMR (100 MHz,
CDCl3) δ 158.7, 125.8, 114.0, 55.3, 39.6, 32.8, 27.7; HRMS (ESI) calcd
for C16H22N3O2+ [M + H]+ 288.1707, found 288.1705.
N-(4-(4-Methoxyphenyl)-1-methyl-1H-imidazol-2-yl)-
benzamide (5d). White powder: yield 47%; mp 127−127 °C;
1H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 6.4 Hz, 2H, Ar−H),
7.48−7.41 (m, 5H, Ar−H), 6.95 (d, J = 8.7 Hz, 2H, Ar−H), 6.71 (s, 1H,
CH), 3.85 (s, 3H, O−CH3), 3.68 (s, 3H, N−CH3); 13C NMR
(100 MHz, CDCl3) δ 159.6, 137.8, 130.9, 128.7, 128.0, 125.6, 114.5,
+
110.0, 55.4, 31.8; HRMS (ESI) calcd for C18H18N3O2 [M + H]+
Synthesis of Polycarpaurine A (2). To a solution of compound 14
(4.0 g, 19.68 mmol) in acetic acid (200 mL) was added SCl2 (1.2 g,
11.81 mmol), and the mixture was stirred at room temperature for 10 h.
Then the mixture was concentrated, and the residue was recrystallized
with acetone (50 mL) to obtain polycarpaurine A (2, 2.1 g, 42%)
308.1394, found 308.1399.
N-(4-(4-Methoxyphenyl)-1-methyl-1H-imidazol-2-yl)-
benzenesulfonamide (5e). Light gray powder: yield 8%; mp 188−
192 °C; 1H NMR (400 MHz, CDCl3) δ 10.13 (s, 1H, NH), 7.96 (d, J =
7.7 Hz, 2H, Ar−H), 7.52−7.41 (m, 3H, Ar−H), 7.30 (d, J = 7.9 Hz, 2H,
Ar−H), 6.94 (d, J = 7.9 Hz, 2H, Ar−H), 6.52 (s, 1H, CH), 3.84 (s, 3H,
O−CH3), 3.42 (s, 3H, N−CH3); 13C NMR (100 MHz, CDCl3) δ 159.9,
147.2, 144.3, 131.3, 128.6, 125.8, 125.6, 125.1, 120.0, 114.7, 109.6, 55.4,
31.8; HRMS (ESI) calcd for C17H18N3O3S+ [M + H]+ 344.1063, found
344.1068.
1
as an orange red powder: mp 180 °C (dec); H NMR (400 MHz,
DMSO-d6) δ 13.65 (s, 2H, HCl), 7.81 (s, 4H, NH2), 7.69 (d, J = 8.8 Hz,
4H, Ar−H), 7.11 (d, J = 8.8 Hz, 4H, Ar−H), 3.84 (s, 6H, O−CH3),
2.90 (s, 6H, N−CH3).
Synthesis of Polycarpaurine C (3). To a solution of polycarpine
(1a, 0.5 g, 0.90 mmol) in H2O (50 mL) was added NaHSO3 (0.3 g,
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DOI: 10.1021/acs.jafc.6b01415
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Journal of Agricultural and Food Chemistry
Article
N-(4-(4-Methoxyphenyl)-1-methyl-1H-imidazol-2-yl)-
hexanamide (5f). White powder: yield 47%; mp 134−135 °C;
1H NMR (400 MHz, CDCl3) δ 7.52 (d, J = 8.6 Hz, 2H, Ar−H), 6.93
(s, 1H, CH), 6.89 (d, J = 8.6 Hz, 2H, Ar−H), 3.82 (s, 3H, O−CH3), 3.58
(s, 3H, N−CH3), 2.39 (t, J = 7.6 Hz, 2H, CH2CO), 1.71−1.59 (m, 2H,
COCH2CH2), 1.31−1.23 (m, 4H, CH2CH2CH3), 0.86 (t, J = 6.8 Hz,
3H, CH2CH3); 13C NMR (100 MHz, CDCl3) δ 175.2, 158.8, 141.2,
136.8, 126.1, 125.9, 114.1, 114.1, 55.3, 36.4, 33.4, 31.4, 25.3, 22.4, 13.9;
3.76 (s, 6H, O−CH3), 3.44 (s, 6H, N−CH3); 13C NMR (100 MHz,
DMSO-d6) δ 166.5, 159.7, 132.9, 132.2, 128.6, 128.4, 122.3, 114.0,
55.1, 31.1; HRMS (ESI) calcd for C36H33N6O4S2+ [M + H − 2HCl]+
677.1999, found 677.2003.
N,N′-(5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl-
1H-imidazole-5,2-diyl))dibenzenesulfonamide Dihydrochlor-
ide (6e). Yellow powder: yield 26%; mp 220−224 °C; 1H NMR
(400 MHz, DMSO-d6) δ 11.89 (s, 2H, HCl), 7.90 (d, J = 6.8 Hz, 4H,
Ar−H), 7.53−7.46 (m, 6H, Ar−H), 7.16 (d, J = 8.1 Hz, 4H, Ar−H), 6.92
(d, J = 8.6 Hz, 4H, Ar−H), 3.84 (s, 6H, O−CH3), 2.89 (s, 6H, N−CH3);
13C NMR (100 MHz, DMSO-d6) δ 160.6, 146.5, 145.0, 131.7,
+
HRMS (ESI) calcd for C17H24N3O2 [M + H]+ 302.1863, found
302.1871.
Ethyl 2-((4-(4-methoxyphenyl)-1-methyl-1H-imidazol-2-yl)-
amino)-2-oxoacetate (5g). White powder: yield 47%; mp 111−
113 °C; 1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 8.8 Hz, 2H, Ar−H),
6.96 (d, J = 8.8 Hz, 2H, Ar−H), 6.76 (s, 1H, CH), 4.36 (q, J = 7.1 Hz, 2H,
CH2CH3), 3.84 (s, 3H, O−CH3), 3.67 (s, 3H, N−CH3), 1.40 (t, J =
7.1 Hz, 3H, CH2CH3); 13C NMR (100 MHz, CDCl3) δ 166.0, 163.8,
160.0, 149.9, 126.4, 125.7, 120.1, 114.7, 110.4, 61.9, 55.4, 32.2, 14.2;
130.1, 129.1, 126.1, 118.6, 114.4, 114.1, 55.7, 28.9; HRMS (ESI) calcd
for C34H33N6O6S4+ [M + H − 2HCl]+ 749.1339, found 749.1333.
N,N′-(5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl-
1H-imidazole-5,2-diyl))dihexanamide Dihydrochloride (6f).
1
Yellow powder: yield 41%; mp 123−126 °C; H NMR (400 MHz,
CD3OD) δ 7.39 (d, J = 7.7 Hz, 4H, Ar−H), 6.93 (d, J = 7.7 Hz,
4H, Ar−H), 3.79 (s, 6H, O−CH3), 3.48 (s, 6H, N−CH3), 2.51 (t, J =
6.8 Hz, 4H, CH2CO), 1.69 (brs, 4H, COCH2CH2), 1.34 (brs, 8H,
CH2CH2CH3), 0.87 (brs, 6H, CH2CH3); 13C NMR (100 MHz,
CD3OD) δ 173.8, 162.8, 141.7, 139.1, 130.5, 118.2, 115.5, 56.3,
+
HRMS (ESI) calcd for C15H18N3O4 [M + H]+ 304.1292, found
304.1297.
General Procedures for the Preparation of Compounds
6a−g. To a solution of the corresponding compounds 5a−g
(3.67 mmol) in acetic acid (50 mL) was added S2Cl2 (2.20 mmol).
The mixture was stirred at room temperature for 10 h and then con-
centrated. The residue was dissolved in CH2Cl2 (150 mL), washed
successively with saturated aqueous NaHCO3 solution (100 mL) and
brine (100 mL), and then dried over anhydrous MgSO4. After filtration
and concentration, the residue was recrystallized from acetone (20 mL)
to provide the corresponding compounds 6a−g.
+
36.9, 32.4, 31.7, 25.2, 23.5, 14.3; HRMS (ESI) calcd for C34H45N6O4S2
[M + H − 2HCl]+ 665.2938, found 665.2952.
2,2′-((5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl-
1H-imidazole-5,2-diyl))bis(azanediyl))bis(2-oxoacetate) Dihy-
drochloride (6g). Yellow powder: yield 55%; mp 200−204 °C;
1H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 2H, HCl), 7.61 (brs, 4H,
Ar−H), 6.91 (d, J = 8.3 Hz, 4H, Ar−H), 4.32 (q, J = 6.6 Hz, 4H,
CH2CH3), 3.77 (s, 6H, O−CH3), 3.33 (s, 6H, N−CH3), 1.33 (t, J =
6.6 Hz, 6H, CH2CH3); 13C NMR (100 MHz, DMSO-d6) δ 159.7,
128.6, 114.2, 62.9, 55.5, 30.8, 14.3; HRMS (ESI) calcd for C30H33N6O8S2+
[M + H − 2HCl]+ 669.1796, found 669.1795.
(N,N′-(5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl-
1H-imidazole-5,2-diyl))diacetamide) Dihydrochloride (6a).
1
Yellow powder: yield 55%; mp 139−142 °C; H NMR (400 MHz,
DMSO-d6) δ 11.26 (s, 2H, HCl), 7.40 (d, J = 8.8 Hz, 4H, Ar−H), 6.95
(d, J = 8.8 Hz, 4H, Ar−H), 3.81 (s, 6H, O−CH3), 3.41 (s, 6H, N−CH3),
2.20 (s, 6H, CH3CO); 13C NMR (100 MHz, DMSO-d6) δ 170.0,
160.4, 141.5, 129.2, 114.3, 55.8, 31.8, 23.4; HRMS (ESI) calcd for
C26H29N6O4S2+ [M + H − 2HCl]+ 553.1686, found 553.1697.
General Procedures for the Preparation of Intermediates
7a−c. A mixture of compound 14 (2.46 mmol), K2CO3 (2.46 mmol)
and the corresponding aldehydes (9.84 mmol) in ethanol (60 mL) and
toluene (20 mL) was stirred at reflux for 8 h. Then the mixture was
filtered and evaporated in vacuo. The residue was taken into CH2Cl2
(200 mL), washed with brine (200 mL), dried over anhydrous MgSO4,
filtered, and evaporated. The residue was recrystallized from ethyl
acetate (10 mL) to obtain the corresponding intermediates 15a−c,
which was used for the next step directly. A mixture of 15a−c and
NaBH4 (2.95 mmol) was stirred for 2 h at 65 °C. After cooling to
room temperature, H2O (100 mL) and CH2Cl2 (200 mL) was added.
The mixture was separated and extracted with CH2Cl2 (60 mL × 2),
then dried over anhydrous MgSO4, and evaporated. After purification by
column chromatography on silica gel, the corresponding compounds
7a−c were obtained.
N,N′-(5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl-
1H-imidazole-5,2-diyl))dimethanesulfonamide Dihydrochlor-
ide (6b). Yellow powder: yield 20%; mp 218−220 °C; 1H NMR
(400 MHz, CDCl3) δ 7.28 (d, J = 8.4 Hz, 4H, Ar−H), 6.97 (d, J = 8.7 Hz,
4H, Ar−H), 3.92 (s, 6H, O−CH3), 3.22 (s, 6H, N−CH3), 2.93 (s, 6H);
13C NMR (100 MHz, CDCl3) δ 161.2, 146.5, 135.5, 127.3, 117.5, 114.8,
55.6, 42.6, 28.9; HRMS (ESI) calcd for C24H29N6O6S4+ [M + H − 2HCl]+
625.1026, found 625.1014.
N,N′-(5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl-
1H-imidazole-5,2-diyl))bis(2,2-dimethylpropanamide) Dihy-
drochloride (6c). Yellow powder: yield 52%; mp 150−152 °C;
1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 2H, HCl), 7.64 (d, J =
8.6 Hz, 4H, Ar−H), 6.97 (d, J = 8.7 Hz, 4H, Ar−H), 6.53 (s, 2H, NH),
3.78 (s, 6H, O−CH3), 3.28 (s, 6H, N−CH3), 1.28 (s, 18H, C(CH3)3);
13C NMR (100 MHz, DMSO-d6) δ 178.0, 159.6, 141.7, 128.5,
N-Benzyl-4-(4-methoxyphenyl)-1-methyl-1H-imidazol-2-
1
amine (7a). Yellow powder: yield 63%; mp 125−128 °C; H NMR
(400 MHz, CDCl3) δ 7.64 (d, J = 8.8 Hz, 2H, Ar−H), 7.46 (d, J = 7.2 Hz,
2H, Ar−H), 7.39−7.28 (m, 3H, Ar−H), 6.89 (d, J = 8.8 Hz, 2H,
Ar−H), 6.69 (s, 1H, CH), 4.63 (d, J = 5.8 Hz, 2H, CH2), 4.23 (brs,
1H, NH), 3.81 (s, 3H, O−CH3), 3.39 (s, 3H, N−CH3); 13C NMR
(100 MHz, CDCl3) δ 158.1, 149.6, 139.5, 136.9, 128.6, 128.3, 127.8,
127.5, 125.6, 113.9, 111.1, 55.3, 48.7, 31.1; HRMS (ESI) calcd for
C18H20N3O+ [M + H]+ 294.1601, found 294.1607.
122.4, 115.3, 114.0, 55.1, 39.2, 30.9, 26.8; HRMS (ESI) calcd for
C32H41N6O4S2+ [M + H − 2HCl]+ 637.2625, found 637.2634.
N,N′-(5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl-
1H-imidazole-5,2-diyl))dibenzamide Dihydrochloride (6d).
1
Yellow powder: yield 22%; mp 186−189 °C; H NMR (400 MHz,
DMSO-d6) δ 11.50 (s, 2H, HCl), 8.09 (d, J = 7.4 Hz, 4H, Ar−H),
7.71−7.57 (m, 12H, Ar−H and NH), 7.01 (d, J = 8.6 Hz, 4H),
N-(4-Methoxybenzyl)-4-(4-methoxyphenyl)-1-methyl-1H-
imidazol-2-amine (7b). Yellow powder: yield 49%; mp 144−147 °C;
Scheme 1. Synthesis of Compound 14
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Scheme 2. Synthesis of Polycarpine (1a) and Polycarpaurine A (2)
+
111.1, 55.32, 55.29, 48.2, 31.1; HRMS (ESI) calcd for C19H22N3O2
[M + H]+ 324.1707, found 324.1709.
Scheme 3. Synthesis of Polycarpaurine C (3)
4-(4-Methoxyphenyl)-1-methyl-N-(4-(trifluoromethyl)-
benzyl)-1H-imidazol-2-amine (7c). Yellow powder: yield 50%;
1
mp 113−116 °C; H NMR (400 MHz, CDCl3) δ 7.67−7.53 (m, 6H,
Ar−H), 6.88 (d, J = 8.8 Hz, 2H, Ar−H), 6.74 (s, 1H, CH), 4.66 (d, J =
6.0 Hz, 2H, CH2), 3.81 (s, 3H, O−CH3), 3.79 (s, 1H, NH), 3.40 (s, 3H,
N−CH3); 13C NMR (100 MHz, CDCl3) δ 158.2, 149.2, 143.7, 136.8,
129.4, 128.3, 126.4 (q, J = 210 Hz), 125.6, 125.4 (q, J = 3.8 Hz), 122.8,
113.9, 111.3, 55.3, 48.0, 31.1; HRMS (ESI) calcd for C19H19F3N3O+
[M + H]+ 362.1475, found 362.1482.
General Procedures for the Preparation of Compounds 8a−c.
The solution of corresponding compounds 7a−c (8.44 mmol) and
S2Cl2 (4.22 mmol) in AcOH (120 mL) was stirred at room temperature
for 10 h, then concentrated. The residue was taken into CH2Cl2
(200 mL), washed successively with saturated aqueous NaHCO3 solution
(200 mL) and brine (200 mL), then dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The residue was recrystallized from
acetone (30 mL) to provide the corresponding compounds 8a−c.
1H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 7.4 Hz, 2H, Ar−H), 7.37
(d, J = 7.4 Hz, 2H, Ar−H), 6.89 (d, J = 8.2 Hz, 4H, Ar−H), 6.72 (s, 1H,
CH), 4.51 (d, J = 5.1 Hz, 2H, CH2), 3.81 (s, 6H, O−CH3), 3.59 (s,
1H, NH), 3.36 (s, 3H, N−CH3); 13C NMR (100 MHz, CDCl3)
δ 159.0, 158.1, 149.6, 136.8, 131.6, 129.6, 127.8, 125.6, 114.0, 113.9,
Scheme 4. Synthesis of Compounds 4a−g, 5a−g, and 6a−g
Scheme 5. Synthesis of Compounds 15a−c, 7a−c, and 8a−c
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Scheme 6. Synthesis of Compounds 1b−g
5,5′-Disulfanediylbis(N-benzyl-4-(4-methoxyphenyl)-1-
methyl-1H-imidazol-2-amine) Dihydrochloride (8a). Brick red
powder: yield 58%; mp 152−154 °C; 1H NMR (400 MHz, DMSO-d6)
δ 13.20 (s, 2H, HCl), 8.69 (s, 2H, NH), 7.62 (d, J = 8.6 Hz, 4H, Ar−H),
7.44−7.33 (m, 8H, Ar−H), 7.30 (t, J = 6.7 Hz, 2H, Ar−H), 7.04 (d, J =
8.6 Hz, 4H, Ar−H), 4.96 (d, J = 15.0 Hz, 2H, CH2), 4.34 (d, J = 15.0 Hz,
2H, CH2), 3.89 (s, 6H, O−CH3), 3.26 (s, 6H, N−CH3); 13C NMR
(100 MHz, DMSO-d6) δ 160.4, 146.8, 137.2, 129.0, 128.3, 127.4,
127.4, 117.2, 113.6, 110.2, 55.5, 45.8, 29.5; HRMS (ESI) calcd for
C36H37N6O2S2+ [M + H − 2HCl]+ 649.2414, found 649.2428.
Table 1. In Vitro Antiviral Activity of Compounds Ribavirin
and the Compounds 1a−g, 2, 3, 4a, 5a−g, 6a−g, 7a−c, 8a−c,
and 14 against TMV
concn
nhibn rate
concn
inhibn rate
a
a
compd
(μg/mL)
(%)
compd
5g
(μg/mL)
(%)
1a
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
53
14
32
12
29
18
38
17
47
20
58
19
53
12
14
0
2
3
2
1
1
2
3
2
1
2
1
2
2
1
2
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
36
0
2
1b
1c
1d
1e
1f
6a
0
0
5,5′-Disulfanediylbis(N-(4-methoxybenzyl)-4-(4-methoxy-
6b
6c
19
0
1
2
3
2
2
3
2
phenyl)-1-methyl-1H-imidazol-2-amine) Dihydrochloride (8b).
1
Yellow powder: yield 34%; mp 152−154 °C; H NMR (400 MHz,
10
0
CD3OD) δ 7.61 (d, J = 8.8 Hz, 4H, Ar−H), 7.34 (d, J = 8.6 Hz, 4H,
Ar−H), 7.15 (d, J = 8.8 Hz, 4H, Ar−H), 6.98 (d, J = 8.6 Hz, 4H, Ar−H),
4.54 (d, J = 13.6 Hz, 2H, CH2), 4.33 (d, J = 14.3 Hz, 2H, CH2), 3.95 (s,
6H, O−CH3), 3.82 (s, 6H, O−CH3), 3.35 (s, 6H, N−CH3); 13C NMR
(100 MHz, CD3OD) δ 161.3, 159.8, 146.9, 136.9, 128.9, 128.7,
127.4, 117.4, 113.9, 54.9, 54.4, 46.4, 28.5; HRMS (ESI) calcd for
C38H41N6O4S2+ [M + H − 2HCl]+ 709.2625, found 709.2613.
5,5′-Disulfanediylbis(4-(4-methoxyphenyl)-1-methyl-N-(4-
(trifluoromethyl)benzyl)-1H-imidazol-2-amine) Dihydrochlor-
ide (8c). Gray powder: yield 27%; mp 153−155 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.27 (s, 2H, HCl), 8.28 (s, 2H, NH), 7.56 (d,
J = 7.1 Hz, 4H, Ar−H), 7.42 (d, J = 6.3 Hz, 4H, Ar−H), 7.33 (d, J =
7.3 Hz, 4H, Ar−H), 6.82 (d, J = 7.6 Hz, 4H, Ar−H), 4.84 (d, J = 17.2 Hz,
2H, CH2), 4.66 (d, J = 17.3 Hz, 2H, CH2), 3.71 (s, 6H, O−CH3), 3.27 (s,
6H, N−CH3); 13C NMR (100 MHz, DMSO-d6) δ 171.2, 160.0, 157.1,
139.9, 128.4, 128.3, 127.8, 127.5, 125.8, 124.6 (q, J = 3.6 Hz), 113.7,
88.4, 55.1, 43.4, 27.0; HRMS (ESI) calcd for C38H33F6N6O2S2+ [M − H
− 2HCl]+ 783.2005, found 783.2000.
6d
6e
27
0
18
0
1g
2
6f
32
0
6g
15
0
3
23
0
2
3
7a
12
0
4a
5a
5b
5c
5d
5e
5f
12
0
7b
7c
37
10
22
0
2
1
2
0
0
31
8
2
2
1
2
2
8a
29
0
1
2
1
1
General Procedures for the Preparation of Compounds
1b−g. A solution of polycarpine (1a, 0.5 g, 0.92 mmol) in MeOH
(5 mL), saturated aqueous NaHCO3 solution (30 mL), and CH2Cl2
(100 mL) was stirred for 30 min at room temperature. The mixture
was separated and extracted with CH2Cl2 (30 mL), then dried over
anhydrous Na2SO4, and filtered. To the filtrate was added dropwise a
solution of the corresponding acids (1.84 mmol) in methanol (100 mL).
The reaction mixture was stirred for 2 h at 40 °C, concentrated to 80 mL,
and then filtered to obtained the corresponding compounds 1b−g.
For 1b: red powder; yield 63%; mp 136−138 °C; 1H NMR
(400 MHz, DMSO-d6) δ 12.15 (brs, 2H, COOH), 9.59 (brs, 2H, OH),
7.54 (brs, 4H, NH2), 7.49 (d, J = 15.9 Hz, 2H, CH), 7.28 (s, 2H, Ar−H),
7.08 (d, J = 8.0 Hz, 2H, Ar−H), 6.83 (brs, 4H, Ar−H), 6.79 (d, J =
8.0 Hz, 2H, Ar−H), 6.37 (d, J = 15.9 Hz, 2H, CH), 6.13 (brs, 4H,
Ar−H), 3.81 (s, 6H, O−CH3), 3.75 (brs, 6H, O−CH3), 3.17 (brs, 6H,
N−CH3); 13C NMR (100 MHz, DMSO-d6) δ 168.7, 149.5, 148.4,
144.9, 126.3, 123.3, 116.3, 116.0, 111.6, 56.1, 55.5, 21.6.
28
0
8b
8c
41
0
10
0
27
0
31
0
14
0
0
15
0
ribavirin
41
10
2
1
a
Average of three replicates. All results are expressed as mean SD.
1
For 1c: yellow powder; yield 67%; mp 193 °C (dec); H NMR
(400 MHz, DMSO-d6) δ 7.50 (s, 4H, NH2), 6.81 (s, 4H, Ar−H), 6.23 (s,
4H, Ar−H), 4.26−4.18 (m, 1H, CH), 3.77 (s, 6H, O−CH3), 3.17 (s, 6H,
N−CH3), 2.60 (dd, J = 15.6, 5.5 Hz, 1H, CH2), 2.42 (dd, J = 15.6, 7.3 Hz,
1H, CH2).
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Table 2. In Vivo Antiviral Activity of Compounds Ribavirin and the Compounds 1a−g, 2, 3, 4a, 5a−g, 6a−g, 7a−c, 8a−c, and 14
against TMV
a
a
effect (%)
effect (%)
compd
concn (μg/mL)
inactivation
curative
protection
compd
5g
concn (μg/mL)
inactivation
curative
protection
1a
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
51
26
36
0
2
1
2
47
17
44
5
1
2
3
45
18
45
9
2
2
2
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
26
0
1
22
0
2
17
0
2
1b
1c
1d
1e
1f
6a
9
2
1
0
12
0
1
2
2
0
0
49
8
2
1
1
39
10
50
14
35
0
1
2
2
2
3
47
21
43
6
2
1
2
6b
6c
7
13
0
2
2
2
1
2
3
2
8
2
2
0
0
46
0
11
0
2
3
1
2
1
2
22
0
19
0
2
1
2
2
2
42
8
33
0
2
6d
6e
30
0
21
0
23
0
2
56
13
57
19
28
0
2
2
2
1
2
52
11
62
23
33
0
1
2
2
2
2
49
22
56
29
24
0
2
2
2
1
2
27
0
11
0
14
0
1g
2
6f
22
0
30
0
36
0
6g
10
0
18
0
6
2
0
3
30
0
2
2
1
15
0
1
3
2
34
0
2
2
2
7a
17
0
32
0
33
0
2
4a
5a
5b
5c
5d
5e
5f
18
0
27
0
15
0
7b
7c
42
16
32
0
1
2
2
46
10
28
0
2
3
2
49
25
37
0
2
2
2
16
0
13
0
25
0
47
20
9
2
2
40
16
12
0
2
2
1
43
10
19
0
2
2
2
8a
20
0
1
2
2
2
2
34
0
2
2
2
1
31
0
1
2
2
2
2
8b
8c
35
0
22
0
33
0
0
23
0
1
2
2
16
0
2
3
2
10
0
2
2
2
30
0
39
0
26
0
34
5
26
0
19
0
14
32
0
24
0
29
0
2
24
0
17
0
26
0
ribavirin
37
9
36
13
2
2
39
17
1
1
1
a
Average of three replicates. All results are expressed as mean SD.
1
For 1d: yellow powder; yield 48%; mp 187 °C (dec); H NMR
Antifungal Biological Assay. The test method of antifungal activity
of the synthesized compounds was described in previous work.15
(400 MHz, DMSO-d6) δ 7.50 (s, 4H, NH2), 6.81 (s, 4H, Ar−H), 6.24 (s,
4H, Ar−H), 4.26−4.13 (m, 1H, CH), 3.76 (s, 6H, O−CH3), 3.15 (s, 6H,
N−CH3), 2.60 (dd, J = 15.6, 5.4 Hz, 1H, CH2), 2.42 (dd, J = 15.6, 7.2 Hz,
1H, CH2).
RESULTS AND DISCUSSION
■
1
Chemistry. Although Novikov and coauthour have reported
the synthesis of polycarpine,8 the experimental details were
not given. As shown in Scheme 1, methylation and bromination
of acetophenone 9 gave bromide 11. The introduction of
methylamine group is a critical step as it is difficult to obtain
compound 12 as a pure solid. After condition optimization,
compound 12 was given with 59% yield over two steps using
acetone as a solvent for recrystallization. The detailed procedure
is given in Materials and Methods. Condensation of 12 with 13
gave the key intermediate 14. Oxidative coupling of 14 with S2Cl2
gave polycarpine (1a, Scheme 2). As shown in Scheme 2, using
SCl2 in place of S2Cl2 gave polycarpaurine A (2) in 42% yield.
Polycarpaurine C (3) can be obtained from polycarpine (1a) in
the presence of NaHSO3 (Scheme 3).
For 1e: yellow powder; yield 55%; mp 214 °C (dec); H NMR
(400 MHz, DMSO-d6) δ 7.50 (brs, 4H, NH2), 6.80 (brs, 4H, Ar−H),
6.62 (s, 2H, CH2), 6.33 (brs, 4H, Ar−H), 3.77 (s, 6H, O−CH3), 3.15 (s,
6H, N−CH3).
For 1f: yellow powder; yield 74%; mp 240−243 °C; 1H NMR
(400 MHz, DMSO-d6) δ 12.89 (s, 2H), 7.78 (s, 4H, NH2), 7.56 (d, J =
8.1 Hz, 4H, Ar−H), 7.42 (d, J = 8.6 Hz, 4H, Ar−H), 7.18 (d, J = 7.9 Hz,
4H, Ar−H), 6.98 (d, J = 8.9 Hz, 4H, Ar−H), 3.85 (s, 6H, O−CH3), 3.15
(s, 6H, N−CH3), 2.30 (s, 6H, CH3).
1
For 1g: yellow powder; yield 70%; mp 220 °C (dec); H NMR
(400 MHz, DMSO-d6) δ 13.72 (s, 2H, COOH), 7.90 (s, 4H, NH2), 7.42
(d, J = 8.5 Hz, 4H, Ar−H), 6.94 (d, J = 8.5 Hz, 4H, Ar−H), 3.86 (s, 6H,
O−CH3), 3.16 (s, 6H, N−CH3).
Biological Assay. The activity of synthesized compounds was tested
on representative test organisms. Each bioassay was replicated three times
at 25 1 °C. Percentage of mortalities was calculated according to a
percentage scale of 0−100 (0 means no activity, and 100 means total kill).
Antiviral Biological Assay. The synthesized compounds were tested
for their antiviral activity using TMV as template test virus on tobacco
(Nicotiana tabacum var. Xanthi nc). The test procedures and method
have been reported in our previous work.11,12
In order to investigate structure−activity relationship (SAR),
the derivatives 4−8 were designed and synthesized. As depicted
in Scheme 4, acetylation of 2-aminoimidazole 14 with cor-
responding acyl chlorides gave amides 4. As the impact of
imidazole ring, the acetylation only gave a mixture of amides 4
and 5 even at low temperature or with excess acyl chloride.
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Table 4. In Vivo Fungicidal Activity of the Compounds 1a−g, 2, 3, 4a, 5a−g, 6a−g, 7a−c, 8a−c, and 14 against 7 Kinds of Fungi
b
inhibition rate (%) /200 mg/kg
a
compd
P.C
12
R.C
10
B.G
40
S.S
16
R.S
13
B.C
12
C.C
29
1a
1b
1c
1d
1e
1f
2
2
1
1
2
2
2
3
2
2
3
2
2
1
2
2
2
1
2
2
2
2
1
2
2
2
2
3
2
2
2
1
2
1
2
2
2
1
2
2
1
8
2
2
0
20
10
0
9
2
8
2
1
7
1
12
17
29
14
29
17
19
14
29
29
33
19
29
22
29
22
19
29
17
14
17
29
14
22
19
19
33
29
22
21
75
8
20
0
16
23
20
5
1
2
2
8
12
12
21
12
18
19
18
19
21
24
12
26
12
19
12
21
12
26
19
7
2
2
3
2
2
2
2
1
2
1
2
3
2
2
3
2
2
2
1
13
17
4
3
2
19
13
13
13
8
3
2
2
3
0
0
2
2
0
0
2
1g
2
17
13
4
2
1
0
20
30
30
0
3
1
2
16
9
2
20
0
2
2
1
2
2
1
3
9
8
4a
5a
5b
5c
5d
5e
5f
38
13
25
25
21
17
25
21
42
17
21
25
17
17
13
21
25
21
42
42
25
11
100
2
1
2
1
2
2
3
2
2
1
2
1
2
2
3
2
2
2
1
1
2
2
0
12
16
34
11
16
16
12
16
7
2
2
2
3
2
3
2
2
13
17
29
13
8
2
3
2
1
0
0
0
0
0
0
0
0
3
2
50
0
0
6
0
13
13
9
2
3
5g
6a
6b
6c
6d
6e
6f
0
0
0
0
2
2
1
2
3
0
0
19
7
1
9
0
0
4
0
0
11
16
34
16
20
26
31
34
43
46
31
83
2
2
2
2
2
2
2
3
3
2
2
2
10
7
2
0
20
0
1
2
2
0
43
17
7
3
2
26
18
19
26
12
32
26
12
10
82
2
2
1
2
2
1
2
2
3
2
6g
7a
7b
7c
8a
8b
8c
14
0
0
0
0
1
0
0
22
33
28
30
15
12
81
2
1
2
1
2
2
1
0
30
10
20
0
3
2
2
0
0
0
0
10
100
3
c
azoxystrobin
100
a
P.C, Phytophthora capsici; R.C, Rhizoctonia cerealis; B.G, Blum eria graminis f. sp.tritici (wheat protection); S.S, Sclerotinia sclerotiorum (rape
protection); R.S, Rhizoctonia solani (rice protection); B.C, Botrytis cinerea. (cucumber protection); C.C, Corynespora cassiicola (cucumber protection).
b
c
Average of five replicates. The dilution of azoxystrobin is 1000 times.
Fortunately, amides 4 can be converted to 5 via tetra-n-
butylammonium fluoride. Oxidative coupling of 5 with S2Cl2
gave polycarpine derivatives 6. As shown in Scheme 5, reductive
amination of 2-aminoimidazole 14 with corresponding alde-
hydes gave compounds 7. Oxidative coupling of 7 with S2Cl2
gave polycarpine derivatives 8. In order to investigate the impact
of salification, polycarpine derivatives 1b−g were designed and
synthesized (Scheme 6).
Phytotoxic Activity. Phytotoxic activity of compounds 1−8
and 14 against the test plant was first tested. All of the tested
compounds are safe to the test plant at 500 μg/mL.
Antiviral Activity. The antiviral activity of compounds 1−8
and 14 against TMV was evaluated with commercial virucide
ribavirin as the control.
In Vitro Anti-TMV Activity. As shown in Table 1, most of the
synthesized compounds exhibited good antiviral activity,
especially for compounds 1a, 1f, and 1g with significantly
higher inhibitory effect than ribavirin, thus emerging as new lead
compounds for antiviral research. Among the salt derivatives
1a−g, tosylate 1f showed the best activity while (S)-malate 1c
displayed the lowest inhibitory effect. (R)-Malate 1d displayed
higher inhibitory effect than (S)-malate 1c. Corresponding acids
showed no activity. The solubility of compounds 1c, 1d, and 1f in
the tested solution are similar. The above results indicated that
the salt formation can impact the antiviral activity of these
compounds. The main difference between polycarpine (1a)
and polycarpaurine A (2) lies in the number of the S atom.
Polycarpine (1a) showed significantly higher inhibitory effect
than polycarpaurine A (2), which indicated that the two S atoms
are important for keeping high antiviral activity. The derivatives
4−8 and 14 displayed relatively lower inhibitory effect than
polycarpine (1a), which indicated that the −S−S− bond and
NH2 group are favorable to inhibitory effect.
In Vivo Anti-TMV Activity. The synthesized compounds also
showed similar or higher in vivo activity than the commercial
ribavirin (Table 2). Compound 1g displayed the best inhibitory
(inactivation inhibitory effect, 57%/500 μg mL−1 and 19%/
100 μg mL−1; curative inhibitory effect, 62%/500 μg mL−1and
23%/100 μg mL−1; and protection inhibitory effect, 56%/500 μg
mL−1 and 29%/100 μg mL−1), which is significantly higher than
the activity of ribavirin (inactivation inhibitory effect, 37%/
500 μg mL−1and 9%/100 μg mL−1; curative inhibitory effect,
36%/500 μg mL−1 and 13%/100 μg mL−1; and protection
inhibitory effect, 39%/500 μg mL−1 and 17%/100 μg mL−1).
Unlike in vitro antiviral assay results, compounds 5b and 7b
without sulfur also displayed higher activity than ribavirin.
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DOI: 10.1021/acs.jafc.6b01415
J. Agric. Food Chem. 2016, 64, 4264−4272

Journal of Agricultural and Food Chemistry
Article
Trifluoroacetate 1g exhibited a higher activity than 1f. The
modification of NH2 group also led to the decrease of inhibitory
effect (inhibitory effect: 1a > 6 and 8).
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In summary, marine natural products polycarpine and poly-
carpaurines A and C and their derivatives have been prepared and
first evaluated as antiviral and antiphytopathogenic fungus agents.
Polycarpine (1a) and derivatives 1f and 1g with significantly
higher antiviral activity than ribavirin emerged as new lead
compounds for antiviral research. The −S−S− bond and NH2
group are favorable for antiviral activity against TMV and
fungicidal activity against Fusarium graminearum and Sclerotinia
sclerotiorum. However, the modification of NH2 with acyl groups
(6a−g) is favorable to fungicidal activity against Physalospora
piricola and Rhizoctonia cerealis. Current results provide support
for the development of polycarpine alkaloids as novel agro-
chemicals.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.6b01415.
1H and 13C NMR and HRMS spectra of compounds 1−8,
10−12, and 14 (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*(Z.W.) E-mail: wangziwen2725@163.com. Phone: 0086-22-
23503792. Fax: 0086-22-23503792.
*(Q.W.) E-mail: wangqm@nankai.edu.cn. Phone: 0086-22-
23503952. Fax: 0086-22-23503952.
Funding
We gratefully acknowledge assistance from National Natural Science
Foundation of China (21132003, 21421062, 21372131, 21402142),
Tianjin Natural Science Foundation (15JCQNJC05600), Scientific
Research Foundation of Tianjin Normal University (5KQM110021,
5RL125), and Specialized Research Fund for the Doctoral
Program of Higher Education (20130031110017).
Notes
The authors declare no competing financial interest.
4272
DOI: 10.1021/acs.jafc.6b01415
J. Agric. Food Chem. 2016, 64, 4264−4272