Design, synthesis, and antiviral activity evaluation of phenanthrene-based antofine derivatives

Article abstract of DOI:10.1021/jf302746m

On the basis of our previous structure-activity relationship (SAR) and antiviral mechanism studies, a series of phenanthrene-based antofine derivatives (1-12 and 18-50) were designed targeting tobacco mosaic virus (TMV) RNA and synthesized and systematically evaluated for their antiviral activity against TMV. The bioassay results showed that most of these compounds exhibited good to excellent in vivo anti-TMV activity, of which compounds 19 and 27 displayed higher activity than commercial Ribavirin, thus emerging as potential inhibitors of plant virus. The novel concise structure provides another new template for antiviral studies.

Full text of DOI:10.1021/jf302746m

Article
pubs.acs.org/JAFC
Design, Synthesis, and Antiviral Activity Evaluation
of Phenanthrene-Based Antofine Derivatives
Ziwen Wang,† Peng Wei,† Xu Xizhi,‡ Yuxiu Liu,† Lizhong Wang,† and Qingmin Wang†,*
†State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Nankai University,
Tianjin 300071, People’s Republic of China
‡Jiangsu Lanfeng Biochemical Co., Ltd., JiangSu 221400, People’s Republic of China
S
* Supporting Information
ABSTRACT: On the basis of our previous structure−activity relationship (SAR) and antiviral mechanism studies, a series of
phenanthrene-based antofine derivatives (1−12 and 18−50) were designed targeting tobacco mosaic virus (TMV) RNA and synthesized
and systematically evaluated for their antiviral activity against TMV. The bioassay results showed that most of these compounds exhibited
good to excellent in vivo anti-TMV activity, of which compounds 19 and 27 displayed higher activity than commercial Ribavirin, thus
emerging as potential inhibitors of plant virus. The novel concise structure provides another new template for antiviral studies.
KEYWORDS: Phenanthrene-based antofine derivatives, antiviral activity, tobacco mosaic virus, structure−activity relationship, TMV,
SAR
Natural phenanthroindolizidine alkaloid antofine (Figure 1, Ia)
and its analogues [e.g., tylophorine (Ib) and deoxytylophor-
inine (Ic)] have been isolated primarily from the genera
Cynanchum, Pergularia, and Tylophora in the Asclepiadaceae
family.18 These compounds, commonly called tylophora
alkaloids, have been targets of synthesis and modification for
their significant cytotoxic activities.19
INTRODUCTION
■
As one of the most well-studied viruses, tobacco mosaic virus
(TMV) is known to infect members of 9 plant families and at
least 125 individual species, including tobacco, tomato, pepper,
cucumbers, and a number of ornamental flowers. The amount of
loss can vary from 5 to 90% depending upon the strain of TMV,
the total time of infection by TMV, the temperature during
disease development, and the presence of other diseases. It is
found that, in certain fields, 90−100% of the plants show mosic
or leaf necrosis by harvesting time. Therefore, this plant virus has
the name “plant cancer” and is difficult to control.1
As a successfully registered plantviral inhibitor, Ribavirin
(Figure 1) is widely used to prevent TMV disease.2 However, the
inhibitory effects of Ribavirin are less than 50% at 500 μg/mL. In
fact, there are no super chemical treatments that can absolutely
inhibit TMV once it has infected the plants. Because of the
unsatisfactory cure rate (30−60%) of common antiviral agents
(Ribavirin, Ningnanmycin, Virus A, etc.) and the economic loss
of tobacco, many efforts have been performed to develop novel,
potent, and structure concise antiviral agents. Some chemicals,
such as triazolyl compounds,3 thiadiazoles,4,5 pyrazole deriva-
tives,6,7 cyanoacrylate derivatives,8,9 α-aminophosphonate de-
rivatives,10,11 N-(pyrimidin-5-yl)-N′-phenylureas,12 and some
natural products,13,14 have been found to possess antiviral
activity. However, there are only a few reports on economically
viable antiviral chemicals available for application in agricul-
ture;15 thus, there still lies a great deal of scope for further
research in this field.
In the process of developing new potent plant virus inhibitors,
our research group first found that the extract from the aerial
parts of Cyanchum komarovii showed excellent antiviral activity
against TMV. Using a bioassay-directed fractionation approach,
the main active substances in C. komarovii were determined as
tylophorine alkaloids, in which antofine (Ia) presents a high
level.20 The other four alkaloids (Figure 2), 6-hydroxyl-2,3-
dimethoxyphenanthroindolizidine (Id), 7-demethoxytylophorine
N-oxide (Ie), 14-hydroxyantofine N-oxide (If), and 2,3-
dimethoxy-6-(3-oxobutyl)-7,9,10,11,11a,12-hexahydrobenzo-
[f ]pyrrolo[1,2-b]isoquinoline (Ig) were obtained at a lower
level. The bioassay results showed that antofine (Ia) and
6-hydroxyl-2,3-dimethoxyphenanthroindolizidine (Id) displayed
excellent antiviral activity.21 For example, the commercial
antiviral agents 2,4-dioxohexahydro-1,3,5-triazine (DHT) and
1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DADHT) and
moroxydine hydroxychloride copper acetate (Virus A) showed
50% inhibition at 500 μg/mL, whereas antofine (Ia) and
6-hydroxyl-2,3-dimethoxyphenanthroindolizidine (Id) has 63 and
70% inhibitory activity, respectively, even at the concentration
of 1.0 μg/mL, which was 10−100 times more active than any
reported plant virus inhibitors.21,22 Moreover, the structure−
activity relationship (SAR) studies showed that the presence
of free nitrogen in tertiary amine and phenanthrene ring are
essential for high antiviral activity.23−25
Natural product-based agrochemicals offer advantages that
they can sometimes be specific to a target species and have a
unique mode of action with low toxicity in mammals. Another
benefit is their ability to decompose rapidly, thereby reducing
their risk to the environment.16,17 An additional advantage is that
natural products can be a candidate that possesses the desirable
biological activities.
Received: March 12, 2012
Accepted: August 10, 2012
Published: August 10, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structures of Ribavirin and tylophora alkaloids Ia−Ic.
Figure 2. Chemical structures of tylophora alkaloids Id−Ig.
7.20 (s, 1H, Ar-H), 7.18 (s, 1H, Ar-H), 4.12 (s, 6H, OCH3), 4.02 (s, 3H,
OCH3), 4.00 (s, 3H, OCH3), 3.95 (m, 1H, NCH), 3.83 (m, 1H, NCH),
3.20 (m, 2H, NCH), 1.64−1.83 (m, 4H, NCH2CH2), 1.35−1.48 (m, 2H,
NCH2CH2CH2). Anal. Calcd for C24H27NO5: C, 70.40; H, 6.65; N,
3.42. Found: C, 70.41; H, 6.85; N, 3.46.
Further antiviral mechanism studies revealed that antofine has
a favorable interaction with the origin of TMV RNA (oriRNA),
likely exerting its virus inhibition by binding to oriRNA and
interfering with virus assembly initiation.26
On the basis of the above findings, a series of phenanthrene-
based antofine derivatives (1−12 and 18−50) were designed
targeting TMV RNA and synthesized and systematically
evaluated for their antiviral activity against TMV. Herein, we
report the recent research results about this work.
Synthesis of (S)-1-(2,3,6,7-Tetramethoxyphenanthrene-9-
carbonyl)pyrrolidine-2-carboxylic Acid (4). To the solution of
ester 3 (6.0 g, 13.2 mmol) in methanol (150 mL) was added 4 N NaOH
solution (100 mL). The reaction mixture was refluxed for 2 h and then
concentrated in vacuo to remove methanol. The residue was acidified to
a pH of 2 with 10% HCl at 0 °C and filtered to afford acid 4 (5.7 g, 98%
1
yield) as a white powder. mp = 248−250 °C. H NMR (400 MHz,
MATERIALS AND METHODS
■
DMSO-d6) δ: 12.86 (brs, 1H, CO2H), 8.07 (s, 1H, Ar-H), 8.04 (s, 1H,
Ar-H), 7.66 (s, 1H, Ar-H), 7.59 (s, 1H, Ar-H), 7.46 (s, 1H, Ar-H), 4.59
(dd, J = 4.9, 13.6 Hz, 1H, NCH), 4.06 (s, 6H, OCH3), 3.91 (s, 3H,
OCH3), 3.89 (s, 3H, OCH3), 3.12−3.23 (m, 2H, NCH2), 2.25−2.40 (m,
1H, NCHCH2), 1.91−2.03 (m, 1H, NCHCH2), 1.75−1.86 (m, 2H,
NCH2CH2). 13C NMR (100 MHz, DMSO-d6) δ: 173.7, 168.4, 149.8,
149.3, 149.1, 148.9, 131.3, 124.9, 124.3, 121.9, 121.4, 108.8, 105.7, 104.1,
103.7, 58.2, 56.0, 55.9, 55.6, 55.5, 48.3, 29.2, 24.5. Anal. Calcd
for C24H25NO7: C, 65.59; H, 5.73; N, 3.19. Found: C, 65.82; H, 5.96;
N, 3.40.
Synthetic Procedures. 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 GF254
with detection by ultraviolet (UV). Melting points were determined
using an X-4 binocular microscope melting point (mp) apparatus
(Beijing Tech Instruments Co., Beijing, China), and the thermometer
was uncorrected. 1H nuclear magnetic resonance (NMR) spectra were
obtained using Bruker AV 400, Bruker AV300, and a Varian Mercury
Plus 400 MHz spectrometer. Chemical shifts (δ) were given in parts per
million (ppm) and were measured downfield from internal
tetramethylsilane. 13C NMR spectra were recorded using Bruker AV
400 (100 MHz) and Bruker AV300 (75 MHz) with CDCl3 or
dimethylsulfoxide (DMSO)-d6 as a solvent. Chemical shifts (δ) were
reported in ppm using the solvent peak. Elemental analyses were
determined on a Yanaco C, H, N Corder MT-3 elemental analyzer.
High-resolution mass spectra were obtained with a Fourier transform
ion cyclotron resonance mass spectrometry (FT-ICR MS) spectrometer
(Ionspec, 7.0 T).
Synthesis of (S)-1-(2,3,6,7-Tetramethoxyphenanthrene-9-
carbonyl)pyrrolidine-2-carboxamide (5). To a stirred solution of
acid 4 (3.5 g, 8.0 mmol) and Et3N (0.8 g, 8.0 mmol) in tetrahydrofuran
(THF) (120 mL) was added ethyl chloroformate (2.0 g, 18.4 mmol)
at −15 °C. The mixture was stirred at −15 °C for 30 min, and then 25%
solution of NH3·H2O in H2O (5 mL, 32.0 mmol) was added dropwise.
Another 1 h later, the mixture was warmed to room temperature, stirred
for 12 h, and then concentrated in vacuo. The residue was taken into
CH2Cl2 (200 mL), washed with saturated aqueous NaHCO3 solution
(100 mL), H2O (100 mL), and brine (100 mL), then dried over MgSO4
anhydrous, and concentrated in vacuo to afford compound 5 (2.5 g,
Synthesis of Piperidin-1-yl(2,3,6,7-tetramethoxyphenanth-
ren-9-yl)methanone (2). To acid 1 (4.0 g, 11.7 mmol) was added
dropwise freshly distilled oxalyl chloride (50 mL) and dimethylforma-
mide (two drops) at 0 °C. The reaction mixture was then stirred at room
temperature for 1 h and refluxed for 3 h. The excess of oxalyl chloride
was removed under reduced pressure, and acyl chloride was used in the
next reaction without further purification.
The above acyl chloride was dissolved in CH2Cl2 (50 mL) and added
dropwise to a solution of piperidine (1.2 g, 14.0 mmol) and
triethylamine (2.8 g, 27.7 mmol) in CH2Cl2 (50 mL) at 0 °C. The
reaction mixture was warmed to room temperature, and stirring was
continued for 10 h. The organic phase was washed successively with 10%
aqueous hydrochloric acid and water, then dried over Na2SO4
anhydrous, filtered, and concentrated in vacuo. The residue was purified
by flash column chromatography on silica gel to give compound 2 (4.5 g,
94% yield) as a white powder. mp = 203−204 °C. 1H NMR (400 MHz,
CDCl3) δ: 7.80 (s, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.48 (s, 1H, Ar-H),
1
72%) as a white powder. mp = 206−208 °C. H NMR (400 MHz,
CDCl3) δ: 7.78 (s, 2H, Ar-H), 7.58 (s, 2H, Ar-H), 7.21 (s, 1H, Ar-H),
6.07 (s, 1H, NH2), 4.87 (s, 1H, NH2), 4.12 (s, 6H, OCH3), 4.04 (s, 3H,
OCH3), 4.00 (s, 3H, OCH3), 3.90−4.00 (m, 1H, NCH), 3.15−3.45
(m, 2H, NCH2), 1.70−2.35 (m, 4H, NCH2CH2CH2). Anal. Calcd
for C24H26N2O6: C, 65.74; H, 5.98; N, 6.39. Found: C, 65.82; H, 6.25;
N, 6.63.
Synthesis of (S)-1-(2,3,6,7-Tetramethoxyphenanthrene-9-
carbonyl)pyrrolidine-2-carbonitrile (6). To a stirred solution of
compound 5 (2.5 g, 5.7 mmol) and Et3N (2.6 g, 25.7 mmol) in CH2Cl2
(80 mL) was added trifluoroacetic anhydride (2.4 g, 11.4 mmol) at 0 °C
under N2. The mixture was warmed to room temperature for 10 h, then
washed with saturated aqueous NaHCO3 solution (100 mL), H2O (100
mL), and brine (100 mL), dried over MgSO4 anhydrous, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel to afford compound 6 (2.1 g, 88% yield)
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as a white powder. mp = 163−165 °C. 1H NMR (400 MHz, CDCl3) δ:
7.81 (s, 1H, Ar-H), 7.78 (s, 1H, Ar-H), 7.57 (s, 1H, Ar-H), 7.29 (s, 1H,
Ar-H), 7.20 (s, 1H, Ar-H), 5.11 (dd, J = 2.7, 7.3 Hz, 1H, NCH), 4.14 (s,
3H, OCH3), 4.13 (s, 3H, OCH3), 4.03 (s, 3H, OCH3), 4.01 (s, 3H,
OCH3), 3.26−4.02 (m, 2H, NCH2), 2.83−2.45 (m, 2H, NCHCH2),
1.96−2.22 (m, 2H, NCH2CH2). Anal. Calcd for C24H24N2O5: C, 68.56;
H, 5.75; N, 6.66. Found: C, 68.72; H, 5.96; N, 6.93.
NCH2CH2CH2). High-resolution mass spectrometry (HRMS) [electro-
spray ionization (ESI)] calcd for C25H30NO7 (M + H)+ 456.2017, found
456.2013.
Synthesis of (S)-2,3-Bis(3,4-dimethoxyphenyl)-1-(2-
(hydroxymethyl)pyrrolidin-1-yl)prop-2-en-1-one (12). To acid
10 (12.0 g, 34.9 mmol) was added freshly distilled oxalyl chloride (100
mL) at room temperature. The reaction mixture was then stirred for 4 h.
Then, the excess of oxalyl chloride was removed under reduced pressure,
and acyl chloride was used in the next reaction without further
purification.
The above acyl chloride was dissolved in CH2Cl2 (50 mL) and added
dropwise to a stirred solution of methyl L-prolinol (4.2 g, 41.6 mmol)
and Et3N (4.2 g, 41.6 mmol) in CH2Cl2 (100 mL) at −78 °C. The
reaction mixture was stirred for 3 h, then warmed to room temperature,
washed successively with 10% aqueous hydrochloric acid and water,
then dried over Na2SO4 anhydous, filtered, and concentrated in vacuo.
The residue was purified by flash column chromatography on silica gel
to give compound 12 (13.2 g, 89% yield) as a yellow oil. 1H NMR (300
MHz, CDCl3) δ: 6.65−7.40 (m, 7H, Ar-H, Ar-CH), 4.06−4.38 (m, 1H,
NCH), 3.88 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.74 (s, 3H, OCH3),
3.57 (s, 3H, OCH3), 3.64−3.73 (m, 2H, OCH2), 3.10−3.60 (m, 2H,
NCH2), 1.50−2.18 (m, 4H, NCH2CH2CH2). 13C NMR (100 MHz,
CDCl3) δ: 172.6, 149.1, 148.9, 148.8, 148.2, 136.1, 130.7, 128.0, 127.9,
123.0, 121.8, 112.4, 112.2, 111.4, 110.7, 66.7, 61.1, 55.9, 55.7, 55.4, 49.7,
28.2, 24.6. HRMS (ESI) calcd for C24H30NO6 (M + H)+ 428.2068,
found 428.2064.
Synthesis of (S)-1-(2,3,6,7-Tetramethoxyphenanthrene-9-
carbonyl)pyrrolidine-2-carbaldehyde (8). To a solution of oxalyl
chloride (0.8 g, 6.3 mmol) in CH2Cl2 (15 mL) was added dropwise the
solution of DMSO (1.3 g, 13.8 mmol) in CH2Cl2 (5 mL) at −78 °C. The
mixture was stirred for 15 min, and then the solution of alcohol 7 (2.0 g,
4.7 mmol) in CH2Cl2 (15 mL) was added dropwise. After stirring for 2 h,
the solution of Et3N (2.4 g, 23.8 mmol) in CH2Cl2 (5 mL) was added.
The mixture was warmed to room temperature, then washed with 5%
aqueous HCl solution (50 mL), H2O (50 mL), and brine (50 mL), dried
over MgSO4 anhydrous, and concentrated in vacuo. The residue was
purified by column chromatography on silica gel to afford compound 8
(1.4 g, 72% yield) as a white powder. mp = 121−123 °C. 1H NMR (400
MHz, CDCl3) δ: 9.83 (s, 1H, CHO), 7.78 (d, J = 3.0 Hz, 1H, Ar-H), 7.76
(d, J = 2.5 Hz, 1H, Ar-H), 7.63 (s, 1H, Ar-H), 7.56 (d, J = 2.6 Hz, 1H, Ar-
H), 7.17 (d, J = 3.2 Hz, 1H, Ar-H), 5.00 (dd, J = 2.4, 7.0 Hz, 1H, NCH),
4.12 (s, 6H, OCH3), 4.10 (s, 3H, OCH3), 4.01 (s, 3H, OCH3), 3.27−3.38
(m, 2H, NCH2), 2.07−2.32 (m, 2H, NCHCH2), 1.77−1.93 (m, 2H,
NCH2CH2). Anal. Calcd for C24H25NO6: C, 68.07; H, 5.95; N, 3.31.
Found: C, 68.22; H, 6.17; N, 3.53.
Synthesis of (S)-1-(tert-Butoxycarbonyl)pyrrolidine-2-car-
boxylic Acid (14). To a stirred solution of L-proline (30.0 g, 0.26
mol) and Et3N (48 mL, 0.35 mol) in CH2Cl2 (600 mL) was added
Boc2O at 0 °C. The reaction mixture was warmed to room temperature
and stirred for 12 h, and then 10% aqueous HCl solution (80 mL) was
added. The organic phase was separated, washed with brine (200 mL),
then dried over Na2SO4 anhydrous, filtered, and concentrated in vacuo.
The residue was recrystallized using ethyl acetate and petroleum ether to
afford acid 14 (52.3 g, 93% yield) as a white powder. mp = 134−135 °C
(literature27 mp = 135−137 °C). 1H NMR (400 MHz, CDCl3) δ: 9.20
(brs, 1H, CO2H), 4.23−4.35 (m, 1H, 2-H), 3.34−3.54 (m, 2H, 5-H),
2.04−2.27 (m, 2H, 3-H), 1.89−1.96 (m, 2H, 4-H), 1.44 [d, J = 22.6 Hz,
9H, C(CH3)3].
Synthesis of (S)-Methyl-1-(2,3-bis(3,4-dimethoxyphenyl)-
acryloyl)pyrrolidine-2-carboxylate (11). To acid 10 (12.0 g, 34.9
mmol) was added freshly distilled oxalyl chloride (100 mL) at room
temperature. The reaction mixture was then stirred for 4 h. Then, the
excess of oxalyl chloride was removed under reduced pressure, and acyl
chloride was used in the next reaction without further purification.
The above acyl chloride was dissolved in CH2Cl2 (50 mL) and added
dropwise to a stirred solution of methyl L-prolinate hydrochloride (5.6 g,
33.8 mmol), Et3N (6.8 g, 67.3 mmol), and 4-dimethylaminopyridine
(DMAP) (0.43 g, 3.5 mmol) in CH2Cl2 (100 mL) at 0 °C. The reaction
mixture was warmed to room temperature, and stirring was continued
for 4 h. The organic phase was washed successively with 10% aqueous
hydrochloric acid and water, then dried over Na2SO4 anhydrous, filtered,
and concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel to give compound 11 (11.9 g, 75% yield)
Synthesis of (S)-tert-Butyl-2-carbamoylpyrrolidine-1-carbox-
ylate (15). To a stirred solution of acid 14 (35.0 g, 0.16 mol) and Et3N
(16.5 g, 0.16 mol) in THF (300 mL) was added ethyl chloroformate
(40.0 g, 0.37 mol) at −10 °C. The mixture was stirred at −10 °C for 30
min, and then 25% solution of NH3·H2O in H2O (62 mL) was added
dropwise. Another 1 h later, the mixture was warmed to room
temperature, stirred for 12 h, and then concentrated in vacuo. The
residue was taken into CH2Cl2 (600 mL), washed with saturated
aqueous NaHCO3 solution (100 mL), H2O (100 mL), and brine
(100 mL), then dried over MgSO4 anhydrous, and concentrated in vacuo.
The residue was recrystallized using ether to afford compound 15 (33.5 g,
96%) as a white powder. mp = 106−107 °C (literature28 mp = 103.6−
1
as a yellow oil. H NMR (300 MHz, CDCl3) δ: 6.94 (s, 1H, Ar-H),
6.91 (s, 2H, Ar-H), 6.83 (s, 1H, Ar-H), 6.80 (s, 1H, Ar-H), 6.74 (s, 1H,
Ar-H), 6.70 (s, 1H, Ar-H), 4.57 (dd, J = 7.5, 5.3 Hz, 1H, NCH),
3.88 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.78 (s, 6H, OCH3), 3.57
(s, 3H, CO2CH3), 3.23−3.38 (m, 2H, NCH2), 1.73−2.30 (m, 4H,
Scheme 1. Synthesis of Compound 2
1
107.7 °C). H NMR (400 MHz, CDCl3) δ: 6.83 (s, 1H, NH), 6.13
(s, 1H, NH), 4.81−4.89 (m, 1H, 2-H), 4.18−4.29 (m, 1H, 5-H), 3.43−
3.48 (m, 1H, 5-H), 1.87−2.30 (m, 4H, 3,4-H), 1.43 [s, 9H, C(CH3)3].
Synthesis of (S)-tert-Butyl-2-cyanopyrrolidine-1-carboxylate
(16). To a stirred solution of compound 15 (20.0 g, 93.5 mmol) and
Et3N (42.0 g, 0.42 mol) in CH2Cl2 (300 mL) was added trifluoroacetic
anhydride (39.0 g, 0.19 mol) at 0 °C under N2. The mixture was warmed
Scheme 2. Synthesis of Compounds 4−6
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to room temperature for 10 h, then washed with saturated aqueous
NaHCO3 solution (100 mL), H2O (100 mL), and brine (100 mL), dried
over MgSO4 anhydrous, and concentrated in vacuo. The residue was
purified by column chromatography on silica gel to afford compound 16
(17.7 g, 97% yield) as a thick oil. 1H NMR (400 MHz, CDCl3) δ: 4.16−
4.38 (m, 1H, 2-H), 3.34−3.58 (m, 2H, 5-H), 1.87−2.24 (m, 4H, 3,4-H),
1.49 [s, 9H, C(CH3)3].
Synthesis of (S)-tert-Butyl-1-((10-bromo-2,3,6,7-tetrame-
thoxyphenanthren-9-yl)methyl)pyrrolidine-2-carbimidate
(19). The solution of compound 16 (1.4 g, 7 mmol) in TFA was stirred
for 2 h at 0 °C and then concentrated in vacuo to give crude product 17,
which was used in the next reaction without further purification.
The above crude product 17 was taken into acetonitrile (180 mL).
Then, to the solution was added K2CO3 (3.5 g, 25.5 mmol) and bromide
18 (3.0 g, 6.4 mmol). The stirred solution was refluxed for 8 h, then
cooled to room temperature, and concentrated in vacuo. The residue was
taken into CH2Cl2 (200 mL), washed with saturated aqueous NaHCO3
solution (100 mL), H2O (100 mL), and brine (100 mL), then dried over
MgSO4 anhydrous, and concentrated in vacuo. The residue was purified
by flash column chromatography on silica gel to give compound 19 (2.1
g, 68% yield) as a slight yellow powder. mp = 135−137 °C. 1H NMR
(400 MHz, CDCl3) δ: 7.80 (s, 1H, Ar-H), 7.75 (s, 1H, Ar-H), 7.71
(s, 1H, Ar-H), 7.61 (s, 1H, Ar-H), 6.59 (s, 1H, NH), 4.61 (d, J = 13.4 Hz,
1H, Ar-CH2), 4.38 (d, J = 13.4 Hz, 1H, Ar-CH2), 4.11 (s, 6H, OCH3),
4.06 (s, 6H, OCH3), 3.56 (dd, J = 2.8, 10.0 Hz, 1H, NCH), 3.37 (t, J =
7.8 Hz, 1H, NCH2), 2.87−2.93 (m, 1H, NCH2), 2.22−2.32 (m, 1H,
NCHCH2), 1.92−1.97 (m, 1H, NCHCH2), 1.83−1.88 (m, 1H, NCH2CH2),
1.65−1.75 (m, 1H, NCH2CH2), 0.55 [s, 9H, C(CH3)3]. Anal. Calcd
for C28H35BrN2O5: C, 60.11; H, 6.31; N, 5.01. Found: C, 60.20; H, 6.45;
N, 5.23.
Antiviral Biological Assay. The anti-TMV activity of the
synthesized compounds was tested using our previously reported
method.23
Antiviral Activity of Compounds against TMV in Vitro. Fresh leaf of
the 5−6 growth stage of tobacco inoculated by the juice-leaf rubbing
method (concentration of TMV is 5.88 × 10−2 μg/mL) was cut into
halves along the main vein. The halves were immersed into the solution
of 500 μg/mL of the compounds and double-distilled water for 20 min,
respectively, and then cultured at 25 °C for 72 h. Each compound was
replicated at least 3 times.
Protective Effect of Compounds against TMV in Vivo. The
compound solution was smeared on the left side, and the solvent served
as a control on the right side of growing Nicotiana tabacum L. leaves of the
same ages. The leaves were then inoculated with the virus after 12 h. A brush
was dipped in TMV of 6 × 10−3 mg/mL to inoculate the leaves, which were
previously scattered with silicon carbide. The leaves were then washed with
water and rubbed softly along the nervature once or twice. The local lesion
numbers appearing 3−4 days after inoculation were counted. There are
three replicates for each compound.
Scheme 3. Synthesis of Compound 8
Inactivation Effect of Compounds against TMV in Vivo. The virus
was inhibited by mixing with the compound solution at the same volume
for 30 min. The mixture was then inoculated on the left side of the leaves
of N. tabacum L., whereas the right side of the leaves was inoculated with
the mixture of solvent and the virus for control. The local lesion numbers
Scheme 4. Synthesis of Compounds 11 and 12
Scheme 5. Synthesis of Compound 19
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Figure 3. Chemical structures of compounds 9 and 20−50.
were recorded 3−4 days after inoculation. There are three replicates for
each compound.
chloroformate and NH3·H2O afforded amide 5. Dehydration of
compound 5 with trifluoroacetic anhydride (TFAA) gave nitrile
6 in 88% yield. Oxidation of alcohol 729 gave the aldehyde 8
in 72% yield (Scheme 3). To test the effect of the phenanthrene
ring on the anti-TMV activity, compounds 11 and 12 were
designed and synthesized. As shown in Scheme 4, condensation
of 2,3-bis(3,4-dimethoxyphenyl)acryloyl chloride (prepared by
chlorination of acid 1030 with oxalyl chloride) with L-proline
methyl ester hydrochloride in the presence of Et3N gave
compound 11 in 75% yield. The next reduction of ester 11 was
carried out in various conditions, but only the complex result was
obtained. At last, condensation of 2,3-bis(3,4-dimethoxyphenyl)-
acryloyl chloride with L-prolinol at −78 °C afforded alcohol 12 in
89% yield. During preparation of nitrile 31, the imidate ester 19
was obtained. As shown in Scheme 5, N-Boc protection of
L-proline (13) gave acid 14. Treatment of compound 14 with ethyl
chloroformate and NH3·H2O afforded amide 15. The amide 15
was conveniently dehydrated to nitrile 16 by action of TFAA. The
next deprotection in TFA gave a new structural rearranging com-
xpound 17, which coupling with bromide 18 afforded imidate
ester 19. Other phenanthrene-based antofine derivatives 9 and
Curative Effect of Compounds against TMV in Vivo. Growing leaves
of N. tabacum L. of the same ages were selected. TMV (concentration of
6.0 × 10−3 mg/mL) was dipped and inoculated on the whole leaves.
Then, the leaves were washed with water and dried. The compound
solution was smeared on the left side, and the solvent was smeared on
the right side for control. The local lesion numbers were then counted
and recorded 3−4 days after inoculation. There are three replicates for
each compound.
The in vitro and in vivo inhibition rates of the compound were then
calculated according to the following formula (“av” means average, and
controls were not treated with compound): inhibition rate (%) = [(av
local lesion number of control − av local lesion number of drug-
treated)/av local lesion number of control] × 100%.
RESULTS AND DISCUSSION
■
Chemistry. As shown in Scheme 1, condensation of the
9-phenanthrenecarbonyl chloride (prepared by chlorination of
9-phenanthrenecarboxylic acid 123 with oxalyl chloride) with
piperidine in the presence of Et3N gave compound 2 in 94%
yield. As depicted in Scheme 2, hydrolysis of ester 329 gave
acid 4 in 98% yield. Treatment of compound 4 with ethyl
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Journal of Agricultural and Food Chemistry
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20−50 (Figure 3) were prepared according to our previously
published literature.29−33
As shown in Table 1, the synthesized compounds also
exhibited good to excellent in vivo anti-TMV activity, especially
for compounds 19 and 27, which displayed higher antiviral
activity than Ribavirin, thus emerging as new lead compounds.
Aryl Amides 2−9, 11, and 12 and Acids 1 and 10. Aryl
amides 2, 4, 5, 7, and 9 exhibited almost similar antiviral activity,
which is slightly lower than Ribavirin. The difference between
compounds 3−9 lies in the substituents on the proline side. The
ester 3, nitrile 6, and aldehyde 8 showed relatively lower antiviral
activity, which indicated that the proline side of the designed
compounds may serve as a hydrogen donor. The bromide 9
displayed similar antiviral activity to alcohol 7. It seemed to be
that the bromide 9 serves as a prodrug of alcohol 7. To test the
effect of the phenanthrene ring on the antiviral activity,
compounds 11 and 12 were prepared. The bioassay results
indicated that the phenanthrene ring is essential for high antiviral
activity (antiviral effect: 3 > 11 and 7 > 12). A similar result was
also reported in our previous work.25 It ought to be mentioned
that the amino substituents are also important for maintaining
high antiviral activity. For instance, the acids 1 and 10 without
amino groups almost exhibited no antiviral activity.
Antiviral Activity. To make a judgment of the antiviral potency
of the phenanthrene-based antofine derivatives (1−12 and 18−50),
the commercial plant virucide Ribavirin was used as the control.
The first in vitro anti-TMV bioassay indicated that most of the
tested compounds displayed good antiviral activity, of which
compounds 19, 27, 44, and 47 exhibited higher inhibition than
Ribavirin (Table 1). Therefore, these compounds were
bioassayed further to investigate their antiviral activity in vivo.
Table 1. Antiviral Activity of Compounds 1−12 and 18−50
against TMV at 500 μg/mL
in vivo
in vitro inhibition
inactivation
effect (%)
curative
effect (%)
protection
effect (%)
compound
rate (%)
1
0
4.2
24.5
29.1
27.2
25.3
17.6
29.5
15.2
27.6
4.1
3.6
28.9
22.8
28.3
23.5
15.6
30.2
20.3
26.7
2.7
4.8
30.6
29.5
30
2
32.5
31.6
33.3
27
3
4
Arylmethylamines 19−50 and Bromide 18. In comparison
to corresponding aryl amides, the arylmethylamines displayed
lower or similar antiviral activity. However, most of the aryl-
methylamines showed good to excellent anti-TMV activity,
of which compounds 19 and 27 exhibited higher activity
than Ribavirin and emerged as potential inhibitors of plant
virus.
5
24.3
10.3
25.6
21.4
36.9
1.9
6
12.5
32.8
22.2
21.9
2.3
7
8
9
10
11
12
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Ribavirin
17.5
11.7
6.1
21.1
20.9
4.3
17.2
18.1
1.8
23.6
19.3
5.2
The mainly difference between compounds 19−23 and 25−27
lies in the substituents on the proline side. The imidate ester 19, acid
21, and amide 27 displayed relatively higher antiviral activity than
the others, which further indicated that the proline side of the
designed compounds may serve as a hydrogen donor. As the
enantiomer of amide 27, compound 28 showed a significantly lower
antiviral activity, which indicated that the stereo configuration plays
an important role for keeping high activity. The methoxy substituent
on the phenanthrene unit is important for maintaining high activity.
Removing the methoxyl at the 7 position of phenanthrene led to a
significant decrease in antiviral activity (antiviral effect: 30 < 27).
Changing the amide group to the nitrile group decreased the
antiviral activity (antiviral effect: 27 > 31 and 29 > 32), except for
compound 30 (antiviral effect: 30 < 33). Replacement of the amino
group by the diethylamino group decreased the antiviral activity for
compounds 27 and 29 and increased the antiviral activity for
compounds 28 and 30. The antiviral activity of compound 34 is
lower than that of compounds 35 and 36, which indicated that the
saturated pentaheterocycle is favorable for maintaining high activity.
Introduction of the bromine atom at the 4 position of the
phenanthrene ring increased the antiviral activity for compound 37
and decreased antiviral activity for compound 38. Introduction of
the bromine atom at the 5 position of the phenanthrene ring
increased the antiviral activity (antiviral effect: 41 < 43 and 42 < 44).
Replacement of the saturated pentaheterocycle by the saturated
hexaheterocycle decreased the antiviral activity for the R
configuration (antiviral effect: 36 > 46, 38 > 48, and 42 > 50)
and increased the antiviral activity for the S configuration
(antiviral effect: 41 < 49, 35 < 45, and 37 < 47). For saturated
pentaheterocycle compounds, the favorable antiviral config-
uration is R, except for compound 27. However, for saturated
hexaheterocycle compounds, the favorable antiviral configura-
tion is S. The bromide 18 without the amino group also exhibited
no antiviral activity.
39.7
14.2
32.4
15.9
32.1
38.4
19.6
20.4
44.3
10.5
35.5
0
36.6
27.1
33.7
17.4
20.3
30
40.3
16.8
25.6
23.5
16.3
35.2
27.8
13.5
36.6
12.2
30.7
5.6
42.2
23.5
35.3
25.2
21.1
35.9
33.3
10.2
39.5
15.3
32.4
0
21.4
17
42.1
0
12.3
14.5
10.3
27.7
12.6
20
24.7
20.9
22.7
18.2
17.2
33.3
12.5
35.6
19.6
22.4
23.5
35.7
32.5
42.6
19.7
23.5
39.2
21.6
30.7
12.1
38.5
18.2
18.9
20.5
10.6
21.6
30.2
8.9
33.3
24.5
15.3
13.1
25.3
27.6
16.8
27.1
15.3
20.3
18.9
33.1
26.1
39.2
28.5
21.7
37.2
19.6
34.3
21.3
34.2
22
21.3
23.7
26.9
17.8
10.4
15.7
27.6
20.7
30.2
21.5
13.7
28.6
23.7
25.2
15.4
32.1
31.6
22.3
13.3
14.2
31.4
23.5
35.8
26.3
14.2
31.5
14.9
26.4
17.6
34.5
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Telephone: 0086-22-23503952. Fax: 0086-22-23503952.
E-mail: wangqm@nankai.edu.cn.
■
Funding
We gratefully acknowledge financial assistance from the National
Key Project for Basic Research (2010CB126100), the National
Natural Science Foundation of China (21132003 and
21121002), and the China Postdoctoral Science Foundation
(2011M500519) and the National Key Technology Research and
Development Program (2011BAE06B02−17) and the Tianjin
Natural Science Foundation (11JCZDJC20500), we also thank
China Agricultural University to supply some of chemical reagents
and the National Key Technology Research and Development
Program (2012BAK25B03−3).
Notes
The authors declare no competing financial interest.
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