Design, synthesis, and anti-tobacco mosaic virus (TMV) activity of phenanthroindolizidines and their analogues

Article abstract of DOI:10.1021/jf303550a

On the basis of our previous structure-activity relationship (SAR) and antiviral mechanism studies, a series of phenanthroindolizidines and their analogues 3-20 were designed, targeting tobacco mosaic virus (TMV) RNA, synthesized, and systematically evaluated for their antiviral activity against TMV. The bioassay results showed that most of these compounds displayed good anti-TMV activity, and some of them exhibited higher antiviral activity than that of commercial Ningnanmycin (perhaps the most successful registered antiplant viral agent). Especially, (S)-deoxytylophorinine (5) with excellent anti-TMV activity (inactivation activity, 59.8%/500 μg mL^-1 and 40.3%/100 μg mL^-1; curative activity, 65.1%/500 μg mL ^-1 and 43.7%/100 μg mL^-1; and protection activity, 70.2%/500 μg mL^-1 and 51.3%/100 μg mL^-1) emerged as a potential inhibitor of the plant virus. Compound 20 exhibited a strong in vivo protection effect against TMV at 100 μg mL^-1, which indicated that phenanthroindolizidine analogues with a seven-membered D ring have a new and interesting structural scaffold and have great potential for further development as tobacco protection agents.

Full text of DOI:10.1021/jf303550a

Article
pubs.acs.org/JAFC
Design, Synthesis, and Anti-tobacco Mosaic Virus (TMV) Activity of
Phenanthroindolizidines and Their Analogues
Ziwen Wang, Peng Wei, 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
S
* Supporting Information
ABSTRACT: On the basis of our previous structure−activity relationship (SAR) and antiviral mechanism studies, a series of
phenanthroindolizidines and their analogues 3−20 were designed, targeting tobacco mosaic virus (TMV) RNA, synthesized, and
systematically evaluated for their antiviral activity against TMV. The bioassay results showed that most of these compounds
displayed good anti-TMV activity, and some of them exhibited higher antiviral activity than that of commercial Ningnanmycin
(perhaps the most successful registered antiplant viral agent). Especially, (S)-deoxytylophorinine (5) with excellent anti-TMV
activity (inactivation activity, 59.8%/500 μg mL−1 and 40.3%/100 μg mL−1; curative activity, 65.1%/500 μg mL−1 and 43.7%/100
μg mL−1; and protection activity, 70.2%/500 μg mL−1 and 51.3%/100 μg mL−1) emerged as a potential inhibitor of the plant
virus. Compound 20 exhibited a strong in vivo protection effect against TMV at 100 μg mL−1, which indicated that
phenanthroindolizidine analogues with a seven-membered D ring have a new and interesting structural scaffold and have great
potential for further development as tobacco protection agents.
KEYWORDS: Phenanthroindolizidines, diazepines, antiviral activity, tobacco mosaic virus, structure−activity relationship, TMV, SAR
advantage is that natural products can be candidates that
possess the desirable biological activities.
INTRODUCTION
■
Plant viruses cause a variety of detrimental effects on agriculture
and horticulture.1 Tobacco mosaic virus (TMV), one of the
most well-studied plant viruses, is known to infect more than
400 plant species belonging to 36 families, such as tobacco,
tomato, potato, and cucumber. 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.2−4
Natural phenanthroindolizidine alkaloid (R)-tylophorine (2)
and its analogues [e.g., (R)-antofine (4) and (R)-deoxytylo-
phorinine (6)] have been isolated primarily from the genera
Cynanchum, Pergularia, and Tylophora in the Asclepiadaceae
family.21 These compounds, commonly called tylophora
alkaloids, have been targets of synthesis and modification for
their significant cytotoxic activities.22
In a program aimed at screening of plants for biologically
active natural products as alternatives to conventional synthetic
antiviral agents, we first found that the alcohol extract of
Cynanchum komarovii displayed moderate antiviral activity
against TMV. Using a bioassay-directed fractionation approach,
the main active substances in C. komarovii were determined as
tylophorine alkaloids, in which (R)-antofine (4) presents a high
level.23 Moreover, another four alkaloids (Figure 2), 6-
hydroxyl-2,3-dimethoxyphenanthroindolizidine (I), 7-deme-
thoxytylophorine N-oxide (II), 14-hydroxyantofine N-oxide
(III), and 2,3-dimethoxy-6-(3-oxobutyl)-7,9,10,11,11a,12-
hexahydrobenzo[f ]pyrrolo[1,2-b]isoquinoline (IV), were ob-
tained at lower levels. Bioassay results showed that (R)-antofine
(4) and 6-hydroxyl-2,3-dimethoxyphenanthroindolizidine (I)
displayed excellent antiviral activity.24 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−1, whereas 6-
Ningnanmycin (Figure 1), perhaps the most successful
registered anti-plant viral agent, displayed 56.0% in vivo curative
effect at 500 μg mL−1. Ribavirin (Figure 1) is another widely
used plant viral inhibitor. Its inhibitory effects are also less than
50% at 500 μg mL−1. 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%) by
common antiviral agents (e.g., Ningnanmycin, Ribavirin, and
virus A) and economic loss of tobacco, much effort has been
directed toward the development of novel, potent, and
structurally concise antiviral agents. Some chemicals, such as
pyrazole derivatives,5,6 nucleotides,7 α-aminophosphonate
derivatives,8,9 3-acetonyl-3-hydroxyoxindole,10 triazolyl com-
pounds,11 oxidized polyamines,12 thiadiazoles,13 substituted
phenylureas,14 and some natural products,15−17 have been
found to possess antiviral activity. However, because there are
only a few reports on economically viable antiviral chemicals
available for application in agriculture,18 there is great potential
for further research in this field.
Natural-product-based antiviral agents offer advantages in
that they can sometimes be specific to a target species and often
have a unique mode of action with low mammalian toxicity.
Another benefit is their ability to decompose rapidly, thereby
reducing their risk to the environment.19,20 An additional
Received: August 15, 2012
Revised: September 22, 2012
Accepted: September 23, 2012
Published: October 4, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structures of Ningnanmycin, Ribavirin, and compounds 1−16.
Figure 2. Chemical structures of compounds I−IV.
Figure 3. Design of phenanthroindolizidines and their analogues.
hydroxyl-2,3-dimethoxyphenanthroindolizidine (I) has 70%
inhibitory activity, even at the concentration of 1.0 μg mL−1,
which was 10−100 times more active than any reported plant
virus inhibitor.24,25 Moreover, the structure−activity relation-
ship studies showed that most compounds of the antofine-
based library with structural diversity exhibited inhibitory
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8 h, the reaction was quenched with water (100 mL). The products
were extracted with methylene dichloride (3 × 50 mL). The combined
organic extracts were dried (MgSO4) and concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel
(50:1 CH2Cl2/CH3OH) to give alcohol 7 (0.45 g, 56%) as a slight
yellow powder. mp: 267−269 °C (dec) (literature30 mp: 270 °C).
[α]2D0 148.5 (c 2.0 in CHCl3). HRMS (ESI) calcd for C24H28NO5 (M +
H)+ 410.1962, found 410.1970.
(S)-Tylophorine (1). To a solution of 7 (1 g, 2 mmol) in
trifluoroacetic acid (30 mL) was added triethylsilane (0.64 g, 6 mmol),
and the resulting mixture was stirred at room temperature for 10 h in
the dark. The solvent was evaperated in vacuo, and the residue was
made basic with 10% aqueous solution of sodium carbonate. The
product was extracted with methylene dichloride (3 × 50 mL). The
combined organic extracts were dried (MgSO4) and concentrated in
vacuo to give the pure compound 1 (0.90 g, 94%) as a slight yellow
powder. mp: 282−284 °C (dec). [α]2D0 102 (c 1.0 in CHCl3)
[literature31 mp: 284−286 °C; [α]D20 73 (c 0.7 in CHCl3)]. ee: 97%
[flow rate of 1.0 mL/min for 75:25 n-hexane/i-propanol and 0.1%
triethylamine, at 41 bar and a 254 nm UV detector, with tR (major) =
10.38 min and tR (minor) = 13.32 min]. HRMS (ESI) calcd for
C24H28NO4 (M + H)+ 394.2013, found 394.2009.
(S)-1-[(9-Bromo-2,3,6,7-tetramethoxyphenanthren-10-yl)-
methyl]pyrrolidine-2-carboxamide (24). A solution of bromide 21
(5.00 g, 11 mmol), L-prolinamide (1.46 g, 13 mmol), and potassium
carbonate (2.20 g, 16 mmol) in DMF (150 mL) was refluxed for 8 h.
After cooling, the reaction mixture was concentrated in vacuo. The
residue was washed with brine to give carboxamide 24 (4.92 g, 92%) as
a white powder. mp: 252−253 °C (dec). [α]2D0 −14 (c 1.0 in CHCl3).
HRMS (ESI) calcd for C24H28BrN2O5 (M + H)+ 503.1176, found
503.1167.
(R)-1-[(9-Bromo-2,3,6,7-tetramethoxyphenanthren-10-yl)-
methyl]pyrrolidine-2-carboxamide (25). The analogue procedure for
the preparation of compound 24 was used. The bromide 21 (5.00 g,
11 mmol), D-prolinamide (1.46 g, 13 mmol), and potassium carbonate
(2.20 g, 16 mmol) gave compound 25 (4.87 g, 91%) as a white
powder. mp: 246−248 °C. [α]2D0 13 (c 1.0 in CHCl3). Other data are
the same as those of compound 24.
activity against TMV higher than that of commercial antiviral
agents26,27 and the presence of free nitrogen in tertiary amine
and phenanthrene ring was essential for high antiviral activity.28
Further antiviral mechanism studies revealed that antofine-
based alkaloids have 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.29
On the basis of the above findings, a series of
phenanthroindolizidines and their analogues (3−20) were
designed, targeting TMV RNA (Figure 3), synthesized, and
systematically evaluated for their antiviral activity against TMV.
MATERIALS AND METHODS
■
Synthetic Procedures. The melting points were determined using
an X-4 binocular microscope melting point apparatus (Beijing Tech
Instruments Co., Beijing, China), and the thermometer was
uncorrected. Mass spectra were obtained on a VG ZAB-HS instrument
spectrometer and a LCQ Advantage instrument spectrometer using
the electron ionization (EI) or fast atom bombardment (FAB) method
and electrospray ionization (ESI) method, respectively. High-
resolution mass spectrometry (HRMS) was obtained on Fourier
transform ion cyclotron resonance mass spectrometry (FT-ICR MS)
(Ionspec, 7.0 T). The enantiomeric excess (ee) of (S)-tylophorine (1)
and ( )-tylophorine was determined by high-performance liquid
chromatography (HPLC) on a chiralcel AD-H column using Agilent
1100 series, and the ee of compounds 17−20 was determined by
HPLC on a chiralcel AD-H column using Rheodyne 3725i-038 series.
Optical rotations were recorded on a Perkin-Elmer 341 MC
polarimeter. Chromatographic separations were carried out under
pressure on silica gel using flash column techniques. Reagents were
purchased from commercial sources and used as received. All
anhydrous solvent were dried and purified by standard techniques
just before use. Reaction progress was monitored by thin-layer
chromatography on silica gel GF254 with ultraviolet (UV) detection.
1H and 13C nuclear magnetic resonance (NMR) and infrared (IR) data
(S)-2,3,6,7-Tetramethoxy-10a,11,12,13-tetrahydro-9H-
phenanthro[9,10-e]pyrrolo[1,2-a][1,4]diazepin-10(15H)-one (17). A
mixture of carboxamide 24 (3.00 g, 6 mmol), Cs2CO3 (3.89 g, 12
mmol), CuI (0.45 g, 2 mmol), and N,N-dimethylglycine hydrochloride
(0.67 g, 5 mmol) in 1,4-dioxane (240 mL) was refluxed for 5 h under a
nitrogen atmosphere. After cooling, ethyl acetate (100 mL) was added
and the resulting mixture was filtrated through a short silica gel (100−
200 mesh) pad. The filtrate was concentrated, and the residue was
purified by flash column chromatography on silica gel (1:2 CH2Cl2/
EA) to give compound 17 (2.09 g, 83%) as a white powder. mp: 261−
263 °C (dec). [α]2D0 233 (c 1.0 in CHCl3). ee: more than 99% (flow
rate of 1.0 mL/min for 85:15 n-hexane/i-propanol and 0.1%
triethylamine, at 570−620 psi and a 254 nm UV detector, with tR =
16.88 min). HRMS (ESI) calcd for C24H27N2O5 (M + H)+ 423.1914,
found 423.1904.
of compounds 1, 7, 17, 19, and 22−24 were given in the Supporting
Information.
(S)-Methyl-1-[(9-bromo-2,3,6,7-tetramethoxyphenanthren-10-
yl)methyl]pyrrolidine-2-carboxylate (22). A mixture of bromide 21
(5.00 g, 11 mmol), methyl L-prolinate hydrochloride (2.64 g, 16
mmol), and potassium carbonate (4.40 g, 32 mmol) in N,N-
dimethylformamide (DMF) (150 mL) was refluxed for 8 h. After
cooling, the reaction mixture was concentrated in vacuo. The residue
was taken up in ethyl acetate (250 mL) and washed with brine (2 ×
100 mL). The organic phase was dried over anhydrous MgSO4 and
evaporated under reduced pressure. The residue was purified by flash
column chromatography on silica gel (3:1 PE/EA) to give carboxylate
22 (5.12 g, 93%) as a white powder. Melting point (mp): 227−229 °C
(dec). [α]D20 −9.5 (c 2.0 in CHCl3). HRMS (ESI) calcd for
C25H29BrNO6 (M + H)+ 518.1173, found 518.1166.
(R)-2,3,6,7-Tetramethoxy-10a,11,12,13-tetrahydro-9H-
phenanthro[9,10-e]pyrrolo[1,2-a][1,4]diazepin-10(15H)-one (18).
The analogue procedure for the preparation of compound 17 was
used. The carboxamide 25 (3.00 g, 6 mmol) gave compound 18 (2.11
g, 84%) as a white powder. mp: 254−256 °C. [α]2D0 −243 (c 1.0 in
CHCl3). ee: more than 99% (flow rate of 1.0 mL/min for 85:15 n-
hexane/i-propanol and 0.1% triethylamine, at 570−620 psi and a 254
nm UV detector, with tR = 13.96 min). Other data are the same as
those of compound 17.
(S)-2,3,6,7-Tetramethoxy-10,10a,11,12,13,15-hexahydro-9H-
phenanthro[9,10-e]pyrrolo[1,2-a][1,4]diazepine (19). A mixture of
compound 17 (0.50 g, 1 mmol) and LiAlH4 (0.14 g, 4 mmol) in THF
(150 mL) was refluxed for 4 h under a nitrogen atmosphere. After
cooling to 0 °C, water (3 mL) was added. The precipitated inorganic
salts were removed by filtration through Celite and washed with
chloroform (50 mL). The organic layer was dried (MgSO4) and
concentrated in vacuo to give compound 19 (0.46 g, 95%) as a slight
(S)-1-[(9-Bromo-2,3,6,7-tetramethoxyphenanthren-10-yl)-
methyl]pyrrolidine-2-carboxylic acid (23). To a solution of
carboxylate 22 (2.00 g, 4 mmol) in methanol (80 mL) was added a
2 N solution of potassium hydroxide (20 mL). The reaction mixture
was refluxed for 6 h and then made acidic with concentrated
hydrochloric acid. The product was extracted with CH2Cl2 (150 mL)
and crystallized from methanol to yield acid 23 (1.36 g, 84%) as a
slight yellow powder. mp: 238−240 °C (dec). [α]2D0 17 (c 1.0 in
CHCl3). HRMS (ESI) calcd for C24H27BrNO6 (M + H)+ 504.1016,
found 504.1016.
(13aS,14S)-14-Hydroxytylophorine (7). To a solution of acid 23
(1.00 g, 2 mmol) and tetramethylethylenediamine (TMEDA) (0.76 g,
7 mmol) in dry tetrahydrofuran (THF) (150 mL), n-BuLi (4.23 mL, 6
mmol) was added at −78 °C, and the resulting mixture was stirred at
this temperature for 4 h under a nitrogen atmosphere. Then, methanol
(30 mL) and sodium borohydride (0.75 g, 20 mmol) were added to
the mixture. After stirring at −40 °C for 1 h and room temperature for
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a
Scheme 1. Synthesis of the Target Compounds 1 and 7
a
Reagents and conditions: (a) methyl L-prolinate hydrochloride, K2CO3, DMF, reflux; (b) KOH aqueous, MeOH, reflux; (c) nBuLi, THF, TMEDA,
−78 °C; (d) MeOH, NaBH4, −40 °C to room temperature; and (e) Et3SiH, CF3COOH, room temperature.
a
Scheme 2. Synthesis of Compounds 17−20
a
Reagents and conditions: (a) L- or D-prolinamide, K2CO3, DMF, reflux; (b) CuI, DMGC, Cs2CO3, 1,4-dioxane, reflux; and (c) LiAlH4, THF, reflux.
yellow powder. mp: 167−169 °C. [α]2D0 −35 (c 1.0 in CHCl3). ee:
more than 99% (flow rate of 1.0 mL/min for 85:15 n-hexane/i-
propanol and 0.1% triethylamine, at 570−620 psi and a 254 nm UV
detector, with tR = 39.12 min). HRMS (ESI) calcd for C24H29N2O4
(M + H)+ 409.2122, found 409.2115.
mL−1 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.
(R)-2,3,6,7-Tetramethoxy-10,10a,11,12,13,15-hexahydro-9H-
phenanthro[9,10-e]pyrrolo[1,2-a][1,4]diazepine (20). The analogue
procedure for the preparation of compound 19 was used. The
compound 18 (0.50 g, 1 mmol) gave compound 20 (0.47 g, 97%) as a
slight yellow powder. mp: 179−181 °C. [α]2D0 47 (c 1.0 in CHCl3). ee:
more than 99% (flow rate of 1.0 mL/min for 85:15 n-hexane/i-
propanol and 0.1% triethylamine, at 570−620 psi and a 254 nm UV
detector, with tR = 27.70 min). Other data are the same as those of
compound 19.
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 var. Xanthi nc, whereas the right side of the
leaves was inoculated with the mixture of solvent and the virus for
control. The local lesion numbers were recorded 3−4 days after
inoculation. There are three replicates for each compound.
Curative Effect of Compounds against TMV in Vivo. Growing
leaves of N. tabacum var. Xanthi nc of the same ages were selected.
TMV (concentration of 6.0 × 10−3 mg mL−1) 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.
Antiviral Biological Assay. The anti-TMV activity of the
synthesized compounds was tested using our previously reported
method.27
Antiviral Activity of Compounds against TMV in Vitro. Fresh leaf
of the 5−6 growth stage of tobacco (Nicotiana tabacum var. Xanthi nc)
inoculated by the juice-leaf rubbing method (concentration of TMV is
5.88 × 10−2 μg mL−1) was cut into halves along the main vein. The
halves were immersed into the solution of 500 μg mL−1 of the
compounds and double-distilled water for 20 min 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 for growing N. tabacum var.
Xanthi nc 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
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%
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Table 1. In Vitro Antiviral Activity against TMV
compound
concentration (μg mL−1
)
inhibition rate (%)
compound
concentration (μg mL−1
)
inhibition rate (%)
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
60.2
30.5
52.8
27.4
71.2
50.0
56.5
32.9
62.4
34.7
51.2
23.0
30.0
0.0
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
15.3
0.0
3
14
15
16
17
18
19
20
38.4
15.3
29.8
0.0
4
5
34.8
4.6
6
32.2
20.0
50.0
18.5
40.4
23.1
54.1
36.9
49.7
21.1
40.0
12.5
71.4
30.4
7
8
9
43.5
21.6
45.2
29.6
47.7
33.1
48.1
30.9
10
11
12
13
(S)-tylophorine
(R)-tylophorine
Ribavirin
Ningnanmycin
RESULTS AND DISCUSSION
natural-product-based compounds showed no phytotoxic
activity at 500 μg mL−1.
■
Chemistry. Compounds 2−6 and 8−16 were prepared
using our previously reported methodology.32,33
In Vitro Anti-TMV Activity. The in vitro antiviral assay results
of all of the synthesized compounds were listed in Table 1. As
the control, Ribavirin exhibited a 40.0% inhibitory effect at 500
μg mL−1, whereas most of the synthesized compounds
exhibited higher antiviral activity than that of Ribavirin. The
commercial plant virucide Ningnanmycin, perhaps the most
successful registered antiplant viral agent, exhibited 71.4%
inhibitory effect at 500 μg mL−1 and 30.4% inhibitory effect at
100 μg mL−1. The optimum compound 5 exhibited nearly
equal inhibitory effect to Ningnanmycin at 500 μg mL−1. When
the concentration was lowered to 100 μg mL−1, compounds 3−
7 and 11−13 displayed nearly equal or higher inhibitory effect
than that of Ningnanmycin, especially for compound 5 with
50% inhibition rate emerging as a new lead. In comparison to
our previously reported (S)- and (R)-tylophorine (1 and 2), the
(S)- and (R)-antofine (3 and 4) displayed nearly equal
inhibitory effect, which indicated that the removal of the
methoxyl at the 7 position is tolerant. However, the removal of
methoxyl at the 2 position significantly increased the activity
(inhibition rate: 5 > 1; 6 > 2). The bioassay results also showed
that the 13aS configuration is the preferred antiviral
configuration for phenanthroindolizidine alkaloids (inhibition
rate: 1 > 2; 3 > 4; 5 > 6).
Introduction of a hydroxyl at the 14 position of
phenanthroindolizidines preserved or decreased the in vitro
antiviral activity (inhibition rate: 7 ≈ 1; 8 ≈ 2; 9 < 3; 10 < 4;
11 < 5; 12 ≈ 6). Introduction of a carbonyl group at the 9
position of (S)-tylophorine slightly decreased the antiviral
activity (inhibition rate: 13 < 1). A rigid indolizidine ring led to
a dramatic decrease of the inhibition rate (inhibition rate: 14 <
13). Opening the D ring or inserting an amino group into the
D ring decreased the antiviral activity (inhibition rate: 15−17
and 19 < 1; 18 and 20 < 2).
Synthesis of the Target Compounds 7 and 1. The
synthetic route of enantiopure (S)-tylophorine (1) was
illustrated in Scheme 1. The dibromide 2132 was coupled
with methyl L-prolinate hydrochloride33 to give ester 22 in 93%
yield. After hydrolysis, the ester 22 was converted to acid 23 in
84% yield. As a key step, the intramolecular cyclization reaction
that employs aryllithiums generated by lithium−halogen
exchange, known as the Parham cyclization reaction,34−36 was
successfully used to assemble the indolizidine ring in our
procedure, and the unstable ketone intermediate was directly
and stereoselectively reduced to the alcohol 7 to avoid
decomposition and racemization. The absolute configuration
of compound 7 was determined by comparison to authentic
sample DCB-3503.32 The alcohol 7 was reduced using
triethylsilane and trifluoroacetic acid to give (S)-tylophorine
(1) in 94% yield and 97% ee.
Synthesis of the Target Compounds 17−20. To explore
the effect of the D ring of phenanthroindolizidine alkaloids on
biological activity, diazepines 19 and 20 were prepared. As
shown in Scheme 2, the dibromide 21 was coupled with L- or D-
prolinamide37 to give the amides 24 and 25 in excellent yield.
CuI-catalyzed intramolecular C−N cross-coupling, known as
Ullmann−Goldberg condensation,38−40 was successfully used
to assemble the diazepine ring as a key step. Diazepinones 17
and 18 upon reduction afforded diazepines 19 and 20 in
excellent yields with more than 99% ee.
Antiviral Activity. The anti-TMV activity of compounds
3−20 was tested. The commercial plant virucides Ningnanmy-
cin and Ribavirin and our previously reported (S)- and (R)-
tylophorine27 were used as the controls.
Phytotoxic Activity. The phenanthroindolizidines and their
analogues 3−20 were first tested for their phytotoxic activity
against the test plant, and the results indicated that these
As shown in Table 1, most of the compounds displayed good
in vitro antiviral activity against TMV. Therefore, these
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compounds were bioassayed further to investigate their antiviral
activity in vivo.
In Vivo Anti-TMV Activity. As shown in Table 2, most of the
Just as the antiviral activity in vitro, (S)-phenanthroindolizi-
dine alkaloids 3 and 5 showed enhancement in vivo anti-TMV
activity compared to their corresponding enantiomers 4 and 6,
which indicated that the 13aS configuration is the preferred
antiviral configuration for phenanthroindolizidine alkaloids.
The phenanthrene substitution pattern greatly affects the in vivo
anti-TMV activity of the phenanthroindolizidines. Removal of
the methoxyl at the 2 position significantly increased the
activity (in vivo activity: 5 > 1; 6 > 2).
compounds also displayed higher in vivo activity than that of the
Table 2. In Vivo Antiviral Activity against TMV
concentration
inactivation
effect (%)
curative
effect (%)
protection
effect (%)
compound
(μg mL−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
51.1
37.2
42.7
15.3
59.8
40.3
48.9
23.6
57.8
32.9
49.2
26.7
24.8
0.0
55.5
26.7
46.0
19.4
65.1
43.7
44.7
24.5
61.9
30.6
43.4
32.8
19.3
0.0
56.4
28.4
45.5
20.4
70.2
51.3
60.5
31.7
63.7
20.0
53.9
26.5
21.2
0.0
3
Introduction of a hydroxy group at the 14 position of
phenanthroindolizidines greatly affects the in vivo anti-TMV
activity. The anti-TMV activity of compound 7 was increased in
comparison to (S)-tylophorine (1) at 500 μg mL−1 and slightly
decreased at 100 μg mL−1. The activity of compound 8 was
significantly increased in comparison to (R)-tylophorine (2).
The activity of compounds 10 and 12 were preserved in
comparison to compounds 4 and 6, respectively. The
introduction of a hydroxy group at the 14 position dramatically
decreased the activity levels for compounds 3 and 5 (in vivo
activity: 3 > 9; 5 > 11). It ought to be mentioned that the
(13aS,14S) configuration is the preferred antiviral configuration
for 14-hydroxytylophorine (in vivo activity: 7 > 8). In contrast,
the (13aR,14R) configuration is found to be the preferred
antiviral configuration for 14-hydroxyantofine (in vivo activity:
10 > 9) and 14-hydroxydeoxytylophorinine (in vivo activity: 12
> 11).
4
5
6
7
8
9
38.5
14.2
32.4
0.0
34.9
10.2
40.0
17.2
56.2
27.8
30.1
14.8
17.5
0.0
39.6
7.3
10
11
12
13
14
15
16
17
18
19
20
41.3
24.5
54.4
30.2
43.5
23.4
21.5
5.3
The rigid indolizidine ring and the introduction of a carbonyl
group at the 9 position both dramatically decreased the antiviral
activity (in vivo activity: 14 < 13 < 1). The D-ring-opened
analogues 15 and 16 also exhibited good anti-TMV activity at
500 μg mL−1, although the activity is lower than that of (S)-
tylophorine (1).
51.2
33.3
37.0
20.5
22.6
8.5
The new structural diazepine analogues 17−20 also exhibited
good anti-TMV activity, which is equal to or significantly higher
than that of Ribavirin, although the activity is lower than that of
(S)-tylophorine (1), except for the in vivo protection effect
(100 μg mL−1) of compound 20. The activity of compounds 19
and 20 is higher than that of compounds 17 and 18, which
indicated that the D ring of phenanthroindolizidine and the
unshared electron pair on nitrogen of diazepines 19 and 20 are
important for maintaining high anti-TMV activity. The in vivo
curative and protection effects (100 μg mL−1) of diazepine 20
are significantly higher than those of Ningnanmycin, which
indicated that phenanthroindolizidine derivatives with a seven-
membered D ring have a new structural scaffold and could have
great potential for further development as selective tobacco
protection agents.
In summary, a series of phenanthroindolizidines and their
analogues were prepared and systematically evaluated for their
antiviral activity against TMV. The bioassay results showed that
most of these compounds displayed good anti-TMV activity
and some of them exhibited higher antiviral activity than that of
commercial Ningnanmycin. The optimal compound (S)-
deoxytylophorinine (5) emerged as a potential inhibitor of
the plant virus. The 13aS configuration was found to be the
preferred antiviral configuration for these compounds. The
phenanthrene substitution pattern and the introduction of a
hydroxy group at the 14 position greatly affect the anti-TMV
activity. R-Diazepine 20 exhibited a strong in vivo protection
effect against TMV at 100 μg mL−1, which indicated that the
phenanthroindolizidine analogues with a seven-membered D
ring have a new structural scaffold for further development as
tobacco protection agents. Further studies on structural
31.3
9.6
30.1
6.2
43.3
18.9
39.6
13.1
29.7
7.6
25.2
0.0
25.8
0.0
32.9
22.2
34.9
21.2
42.5
20.3
31.5
12.6
50.5
34.2
39
31.5
0.0
35.7
18.9
44.6
22.7
44.6
28.7
53.8
37.0
44.2
26.4
36.7
8.2
36.4
19.7
53.6
28.5
46.5
33.7
56.8
28.6
47.6
22.5
38.9
7.5
(S)-tylophorine
(R)-tylophorine
Ribavirin
15.1
33.8
9.8
68.5
37.7
56.0
18.9
66.6
25.2
Ningnanmycin
control Ribavirin. (S)-Deoxytylophorinine (5) showed the best
in vivo anti-TMV activity (inactivation activity, 59.8%/500 μg
mL−1 and 40.3%/100 μg mL−1; curative activity, 65.1%/500 μg
mL−1 and 43.7%/100 μg mL−1; and protection activity, 70.2%/
500 μg mL−1 and 51.3%/100 μg mL−1), which is significantly
higher than that of Ningnanmycin (inactivation activity, 68.5%/
500 μg mL−1 and 37.7%/100 μg mL−1; curative activity, 56%/
500 μg mL−1 and 18.9%/100 μg mL−1; and protection activity,
66.6%/500 μg mL−1 and 25.2%/100 μg mL−1).
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Journal of Agricultural and Food Chemistry
Article
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S
* Supporting Information
1H and 13C NMR, IR, and HRMS spectra of compounds 1, 7,
17, 19, and 22−24. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Telephone/Fax: 0086-22-23503952. E-mail: wangqm@
nankai.edu.cn.
Funding
We gratefully acknowledge assistance from the National Key
Project for Basic Research (2010CB126100), the National
Natural Science Foundation of China (21132003 and
21121002), the China Postdoctoral Science Foundation
(2011M500519 and 2012T50207), the National Key Technol-
ogy Research and Development Program (2011BAE06B02-17),
and the Tianjin Natural Science Foundation
(11JCZDJC20500), and we also thank China Agricultural
University for supplying some of the chemical reagents and the
National Key Technology Research and Development Program
(2012BAK25B03-3).
(19) Qian, X. H.; Lee, P. W.; Cao, S. China: Forward to the green
pesticides via a basic research program. J. Agric. Food Chem. 2010, 58,
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Notes
The authors declare no competing financial interest.
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