Design, synthesis, and antiviral evaluation of phenanthrene-based tylophorine derivatives as potential antiviral agents

Article abstract of DOI:10.1021/jf103440s

A series of C9-substituted phenanthrene-based tylophorine derivatives (PBTs) were designed, synthesized, and first evaluated for their antiviral activities against tobacco mosaic virus (TMV). These compounds contain a phenanthrene core structure and can be synthesized some efficiently with excellent yields compared with tylophorine alkaloid. The bioassay results show that some of these compounds exhibited higher antiviral activity against TMV in vivo than tylophorine and commercial Ningnanmycin. Especially, compounds 3, 4, 9, 13, and 16 emerged as potential inhibitors of plant virus. These new findings demonstrate that these phenanthrene-based tylophorine derivatives (PBTs) represent another new template for antiviral studies and could be considered for novel therapy against plant virus infection.

Full text of DOI:10.1021/jf103440s

J. Agric. Food Chem. 2010, 58, 12337–12342 12337
DOI:10.1021/jf103440s
Design, Synthesis, and Antiviral Evaluation of Phenanthrene-
Based Tylophorine Derivatives as Potential Antiviral Agents
KAILIANG
WANG, YANNA
HU
, YUXIU
L
IU, N
A
M
I
, ZHIJIN
F
AN, Y
U
L
IU, AND
Q
INGMIN
WANG
*
State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic
Chemistry, Nankai University, Tianjin 300071, People’s Republic of China
A series of C9-substituted phenanthrene-based tylophorine derivatives (PBTs) were designed, syn-
thesized, and first evaluated for their antiviral activities against tobacco mosaic virus (TMV). These
compounds contain a phenanthrene core structure and can be synthesized some efficiently with
excellent yields compared with tylophorine alkaloid. The bioassay results show that some of these
compounds exhibited higher antiviral activity against TMV in vivo than tylophorine and commercial
Ningnanmycin. Especially, compounds 3, 4, 9, 13, and 16 emerged as potential inhibitors of plant
virus. These new findings demonstrate that these phenanthrene-based tylophorine derivatives (PBTs)
represent another new template for antiviral studies and could be considered for novel therapy
against plant virus infection.
KEYWORDS: Phenanthrene-based tylophorine derivatives; synthesis; antiviral activities; tobacco mosaic
virus; inhibitors
INTRODUCTION
evaluated as potential antitumor agents (5-7). To date, most of
the studies have been focused on anticancer activity in medicinal
formulation. However, relatively little is known about the anti-
viral activity of tylophora alkaloids in pesticide formulation, and
the antiviral activity of PBTs in pesticide formulation is virtually
unknown.
Plant viruses are unique in the deceptive simplicity of their
structure. However, this simplicity leads to a greater dependence
on the host, and a highly intricate relationship exists between the
two which complicates the strategic designs to control plant viruses
and the losses caused by them (1). The plant disease caused by
tobacco mosaic virus (TMV) is found worldwide. 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 on 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% ofthe plants showmosaicorleaf necrosis byharvesting
time. So, plant virus has the name of “plant cancer” and is dif-
ficult to control.
Natural phenanthroindolizidine alkaloid tylophorine (Figure 1)
and its analogues (e.g., antofine and deoxytylophorinine) have
been isolated primarily from the genera Cynanchum, Pergularia,
and Tylophora in the Asclepiadaceae family (2). These com-
pounds, commonly called tylophora alkaloids, have been targets
of synthesis and modification for their significant cytotoxic activ-
ities (3). Evaluation of tylophora alkaloids in the National Cancer
Institute’s antitumor screen showed a uniform and potent growth
inhibitory effect (GI50 = 10-8 M) against all 60 cell lines, with
notable selectivity toward several refractory cell lines, including
melanoma and lung tumor cell lines (4). Recently, polar phenan-
threne-based tylophorine derivatives (PBTs) were synthesized and
In our preliminary work, (-)-antofine was isolated from the
aerial parts of Cynanchum komarovii and was found to have good
antiviral activity against TMV in vitro (8). However, the con-
tent of natural antofine is especially low. Antofine also has the
drawbacks of being easily decomposed in light and poor water
solubility. All of these limited its application in plant protection.
To extend our research work on tylophora alkaloids as antiviral
agents against TMV, we designed and synthesized three repre-
sentative racemic alkaloids (tylophorine, antofine and deoxy-
tylophorinine) (9), two optically pure alkaloids ((S)-(þ)-tylo-
phorine and (R)-(-)-tylophorine) and a series of tylophorine salt
derivates for antiviral activity evaluation (10). Most of these com-
pounds exhibited good to excellent antiviralactivity against TMV,
and tylophorine salt derivatives also had better stability and better
water solubility in application than alkaloid itself. These findings
encouraged us to further study the optimal structure of the natural
product. In this article, we report our recent research results in the
design and synthesis of C9 substituted PBTs. Several N-contain-
ing cyclic and acyclic moieties were introduced at the C9 position
in order to explore and optimize the activity profiles of C9 sub-
stituted PBTs. In addition, we extended our in vivo antiviral
evaluation against TMV.
MATERIALS AND METHODS
*To whom correspondence should be addressed. Phone: þ86-022-
23499842. Fax: þ86-022-23499842. E-mail: wang98h@263.net, wangqm@
nankai.edu.cn.
Synthetic Procedures. Reagents were purchased from commercial
sources and were used as received. All anhydrous solvent were dried and
© 2010 American Chemical Society
Published on Web 11/08/2010
pubs.acs.org/JAFC

12338 J. Agric. Food Chem., Vol. 58, No. 23, 2010
Wang et al.
Figure 1. Chemical structures of tylophora alkaloids.
1
purified by standard techniques just befor use. Reaction progress was mon-
itored by thin-layer chromatography on silica gel GF-254 with detection
by UV. Melting points were determined on an X-4 binocular microscope
melting point apparatus (Beijing Tech Instruments Co., Beijing, China)
and are uncorrected. 1H NMR spectra were obtained at 400 MHz using a
Bruker AC-P 400 Chemical shift values (δ) are given in ppm and were
downfield from internal tetramethylsilane. High-resolution mass spectra
(HRMS) were recorded on FT-ICR MS (Ionspec, 7.0 T).
Data for 4: yield, 91.8%; mp 165-167 °C (lit. (12) 160-163 °C); H
NMR (400 MHz, CDCl3) δ 8.21 (s 1H), 7.80 (s, 1H), 7.78 (s, 1H), 7.47
(s, 1H), 7.19 (s, 1H), 4.57 (d, 2JHH=12.4 Hz, 1H), 4.12 (s, 9H), 4.02 (s, 3H),
2
3
3.69 (s, 3H), 3.60 (d, JHH=12.4 Hz, 1H), 3.27 (t, JHH=7.6 Hz, 1H),
2.80-2.90 (m, 1H), 2.29-2.39 (m, 1H), 2.12-2.25 (m, 1H), 1.90-2.05
(m, 1H), 1.68-1.87 (m, 2H); HRMS (ESI) m/z calcd for C25H30NO6 (M þ
H) 440.2068; found, 440.2065.
Data for 9: yield, 92.6%; mp 155-157 °C; 1H NMR (400 MHz, CDCl3)
δ 8.27 (s, 1H), 7.79 (s, 1H), 7.78 (s, 1H), 7.44 (s, 1H), 7.18 (s, 1H), 4.34
General Procedure for the Synthesis of Methyl Esters of Amino
Acid Hydrochlorides 2, 7, and 14. To a solution of amino acid (L-proline,
2
(d, JHH =12.4 Hz, 1H), 4.12 (s, 9H), 4.03 (s, 3H), 3.75 (s, 3H), 3.57-
(d, 2JHH=12.4 Hz, 1H), 3.10-3.19 (m, 1H), 2.81-2.94 (m, 1H), 2.06-2.17
(m, 1H), 1.73-1.93 (m, 2H), 1.56-1.69 (m, 1H), 1.32-1.52 (m, 3H);
HRMS (ESI) m/z calcd for C26H32NO6 (M þ H), 454.2224; found,
454.2226.
piperidine-2-carboxylic acid, or 6-aminohexanoic acid) (4 mmol) in dry
MeOH (4 mL) was added dropwise SOCl2 (0.4 mL) at -30 °C. The reac-
tionmixture was warmedto room temperature and heatedto reflux for 1 h.
Then the solvent was removed in vacuo, and the product was used in the
next reaction without further purification.
General Procedure for the Synthesis of N-(2,3,6,7-Tetramethoxy-
9-phenanthrylcarbonyl) Substituted Amino Acid Methyl Esters 3, 8,
and 15. To acid 1 (1.71 g, 0.005 mol) was added dropwise freshly distilled
oxalyl chloride (12.5 mL, 0.145 mol) and dimethylformamide (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.
General Procedure for the Synthesis of N-(2,3,6,7-Tetramethoxy-
9-phenanthrylmethyl) Substituted Amino Acids 5 and 10. A solution
of ester 4 or 9, 4 M NaOH, and MeOH (1:1) was refluxed for 4 h. The
solvents were concentrated, and 10 mL of water was added. The solution
was cooled to 0 °C and acidified with concentrated hydrochloric acid
(pH ≈1) to produce a white precipitate. The solid that separated was col-
lected to give 5 or 10 as a white solid.
1
Data for 5: yield, 96.8%; mp 201-203 °C (lit. (12) 203-205 °C); H
NMR (400 MHz, CDCl3) δ 7.74 (s 1H), 7.68 (s, 1H), 7.67 (s, 1H), 7.54
(s, 1H), 7.20 (s, 1H), 5.12 (d, 2JHH=13.2 Hz, 1H), 4.65 (d, 2JHH=13.2 Hz,
1H), 4.46-4.56 (m, 1H), 4.14 (s, 3H), 4.12 (s, 3H), 4.08 (s, 3H), 4.03 (s, 3H),
3.23-3.42 (m, 2H), 2.57-2.71 (m, 2H), 2.30-2.44 (m, 2H), 1.86-2.15
(m, 3H); HRMS (ESI) m/z calcd for C24H28NO6 (M þ H), 426.1911;
found, 426.1911.
The above acyl chloride was dissolved in CH2Cl2 (30 mL) and added
dropwise to a solution of the methyl esters of amino acid hydrochlorides
(0.005 mol) and triethylamine (1.21 g, 0.012 mol) in CH2Cl2 (20 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, dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by flash column chro-
matography on silica gel with EtOAc/petroleum ether as eluant.
Data for 10: yield, 96.5%; mp 186-188 °C; 1H NMR (400 MHz,
DMSO) δ 8.23 (s, 1H), 7.96 (s, 1H), 7.95 (s, 1H), 7.51 (s, 1H), 7.35 (s, 1H),
5.11 (s, 2H), 4.37-4.42 (m, 1H), 4.02 (s, 6H), 3.96 (s, 3H), 3.90 (s, 3H),
3.02-3.08 (m, 1H), 2.73-4.77 (m, 1H), 2.09-2.04 (m, 1H), 1.82-1.88 (m,
1H), 1.30-1.82 (m, 4H); HRMS (ESI) m/z calcd for C25H30NO6 (M þ H),
440.2068; found, 440.2065.
1
Data for 3: yield, 89.6%; mp 203-204 °C (lit. (12) 205-208 °C); H
NMR (400 MHz, CDCl3) δ 7.8 (s 1H), 7.8 (s, 1H), 7.47 (s, 1H), 7.58
(s, 1H), 7.2 (s, 1H), 4.83-4.85 (m, 1H), 4.13 (s, 6H), 4.10 (s, 3H), 4.03
(s, 3H), 3.84 (s, 3H), 3.39-3.41 (m, 1H), 2.24-2.30 (m, 1H), 2.32-2.42
(m, 1H), 2.08-2.10 (m, 1H),1.86-1.98 (m, 2H); HRMS (ESI) m/z calcd
for C25H28NO7 (M þ H), 454.1860; found, 454.1862.
General Procedure for the Synthesis of 6, 11, 12, and 16. To a
suspension of 5 (10, 1, or 15) (1 mmol) in 15 mL of dry THF was added
LiAlH4 (1 g) in portions at 0 °C. After addition, the reaction mixture was
refluxedfor 2 h and then cooled to 0 °C. The reaction mixture was quenched
with water carefully and extracted with CH2Cl2. The organic layer was
dried over Na2SO4 and concentrated to give the target compounds.
Data for 8: yield, 86.8%; mp 212-213 °C; 1H NMR (400 MHz,
CDCl3) δ 7.81 (s, 1H), 7.79 (s, 1H), 7.61 (s, 1H), 7.54 (s, 1H), 7.21 (s, 1H),
5.76-5.80 (m, 1H), 4.14 (s, 6H), 4.11 (s, 3H), 4.04 (s, 3H), 3.82 (s, 3H),
3.70-3.75 (m, 1H), 3.36-3.45 (m, 1H), 3.16-3.26 (m, 1H), 2.37-2.50
(m, 1H), 1.72-1.92 (m, 2H), 1.20-1.30 (m, 2H); HRMS (ESI) m/z calcd
for C26H29NO7Na (M þ Na), 490.1836; found, 490.1829.
1
Data for 6: yield, 96.5%; mp 221-222 °C (lit. (13) 223-225 °C); H
NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.78 (s, 1H), 7.60 (s, 1H), 7.54
(s, 1H), 7.20 (s, 1H), 4.50 (d, 1H), 4.12 (s, 6H), 4.09 (s, 3H), 4.04 (s, 3H),-
3.45-3.58 (m, 1H), 2.85-3.05 (m, 1H), 2.37-2.57 (m, 1H), 1.59-2.08
(m, 1H); HRMS (ESI) m/z calcd for C24H30NO5 (M þ H), 412.2118;
found, 412.2122.
Data for 15: yield, 85.6%; mp 158-159 °C; 1H NMR (400 MHz,
CDCl3) δ 7.78 (s, 1H), 7.79 (s, 1H), 7.76 (s, 1H), 7.71 (s, 1H), 7.22 (s, 1H),
6.15 (t, 3JHH=5.6 Hz, 1H), 4.13 (s, 3H), 4.12 (s, 3H), 4.04 (s, 3H), 4.03
Data for 11: yield, 92.5%; mp 219-221 °C; 1H NMR (400 MHz,
CDCl3) δ 7.82 (s, 1H), 7.78 (s, 1H), 7.73 (s, 1H), 7.52 (s, 1H), 7.20 (s, 1H),
3
(s, 3H), 3.67 (s, 3H), 3.55-3.60 (m, 2H), 2.37 (t, JHH =7.6 Hz, 2H),
1.68-1.79 (m, 4H), 1.45-1.55 (m, 2H); HRMS (ESI) m/z calcd for
C26H32NO7 (M þ H), 470.2173; found, 470.2169.
2
4.50 (d, JHH =12.8 Hz, 1H), 4.12 (s, 6H), 4.05 (s, 3H), 4.04 (s, 3H),
3.60-3.90 (m, 3H), 2.75-2.90 (m, 1H), 2.59-2.73 (m, 1H), 2.24-2.40
(m, 1H), 1.77-1.90 (m, 1H), 1.28-1.75 (m, 6H); HRMS (ESI) m/z calcd
for C25H32NO5 (M þ H), 426.2275; found, 426.2276.
General Procedure for the Synthesis of N-(2,3,6,7-Tetramethoxy-
9-phenanthrylmethyl) Substituted Amino Acid Methyl Esters 4
and 9. A solution of 0.01 mol of compound 3 or 8 and triethyloxonium
fluoroborate (0.011 mol) in 20 mL of dry CH2Cl2 was stirred for 20 h at
room temperature. The solvent was removed in vacuum, and the residue
was dissolved in 20 mL of ethanol. Sodium borohydride (0.95 g, 0.025 mol)
was added in small portions to the stirred solution at 0 °C and then stirred
at room temperature for 12 h. The solution was poured into 150 mL of
water, and the solid that formed was filtered.
Data for 12: yield, 96.5%; mp 181-182 °C (lit. (13) 185 °C); 1H NMR
(400 MHz, CDCl3) δ 7.81 (s, 1H), 7.75 (s, 1H), 7.56 (s, 1H), 7.54 (s, 1H),
7.18 (s, 1H), 5.11 (s, 2H), 4.13 (s, 3H), 4.12 (s, 3H), 4.06 (s, 3H), 4.02
(s, 3H).
Data for 16: yield, 87.3%; mp 128-130 °C; 1H NMR (400 MHz,
CDCl3) δ 7.86 (s, 1H), 7.79 (s, 1H), 7.76 (s, 1H), 7.70 (s, 1H), 7.21 (s, 1H),

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J. Agric. Food Chem., Vol. 58, No. 23, 2010 12339
Scheme 1. Synthesis of PBTs Starting from L-Proline
Scheme 2. Synthesis of PBTs Starting from Piperidine-2-carboxylic Acid
2
6.13 (br, 1H), 4.13 (s, 6H), 4.03 (s, 6H), 3.68 (t, JHH = 6.0 Hz, 2H),
7.81(s, 1H), 7.79 (s, 1H), 7.47(s, 1H), 7.20 (s, 1H), 4.13 (s, 6H), 4.05 (s, 3 H),
4.04 (s, 3 H), 3.88 (s, 2H), 3.69-3.78 (m, 1H), 2.80-2.90 (m, 2H), 2.25
(t, 3JHH=10.0 Hz, 2H), 1.84-1.94 (m, 2H), 1.50-1.63 (m, 4H); HRMS
(ESI) m/z calcd for C24H30NO5 (M þ H), 412.2118; found, 412.2122.
Antiviral Biological Assay. Purification of Tobacco Mosaic Virus.
Using Gooding’s method (14), the upper leaves of Nicotiana tabacum L.
inoculated with TMV were selected and ground in phosphate buffer and
then filtered through double-layer pledget. The filtrate was centrifuged at
10000g, treated with PEG twice, and centrifuged again. The whole exper-
iment was processed at 4 °C. Absorbance value was estimated at 260 nm by
ultraviolet spectrophotometer.
3.54-3.62 (m, 2H), 1.67-1.77 (m, 2H), 1.55-1.66 (m, 2H),1.42-1.60
(m, 4H); HRMS (ESI) m/z calcd for C25H32NO6 (M þ H), 441.2146;
found, 441.2143.
Synthesis of N-(2,3,6,7-Tetramethoxy-9-phenanthrylmethyl)-
piperidin-4-ol (13). Compound 12 (1.64 g, 5 mmol) was dissolved in
100 mL of CHCl3 and cooled to 0 °C. A solution of PBr3 (0.71 mL,
7.5 mmol) in 20 mL of CHCl3 was added dropwise under nitrogen. The
solutionwas then stirred at roomtemperature for 4 h, then pouredover ice,
and the two layers were separated. The organic phase was dried over Na2-
SO4, filtered, and concentrated in vacuo to afford a white solid. The solid
was then redissolved in 120 mL of DMF. 4-Hydroxypiperidine (0.71 g,
7.0 mmol) was added and stirred for 20 min. K2CO3 (1.0 g, 7.2 mmol) was
added, and the mixture was stirred at room temperature overnight. The
solution was then rotary evaporated, and the product was partitioned
between CHCl3 and H2O. The organic layer was dried over Na2SO4, fil-
tered, and concentrated to obtain a crude product. The crude product was
purified by flash column chromatography to give 1.73 g (84.0%) of 13 as a
white solid: mp 201-203 °C; 1H NMR (400 MHz, CDCl3) δ 7.95 (s 1H),
,
260nm
virus concn ¼ ðA260 ꢀ dilution ratioÞ=E0:1%
1cm
Protective Effect of Compounds against TMV in Vivo. The compound
solution was smeared on the left side while the solvent served as the control
on the right side of growing N. tabacum L. leaves of the same ages. The
leaves were then inoculated with the virus after 12 h. A brush was dipped in
tobacco mosaic virus of 6 ꢀ 10-3 mg/mL to inoculate the leaves, which

12340 J. Agric. Food Chem., Vol. 58, No. 23, 2010
Wang et al.
Scheme 3. Synthesis of PBTs Starting from 4-Hydroxypiperidine
Scheme 4. Synthesis of PBTs Starting from 6-Aminohexanoic Acid
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 (15). There are three replicates for each compound.
using PBr3 to obtain bromide, which was used without further puri-
fication. Nucleophilic substitution reaction of bromide with 4-hy-
droxypiperidine gave 13 in 84.0% yield (Scheme 3).
Antiviral Activity in Vivo. To make a judgment of the antiviral
potency of the C9-substituted phenanthrene-based tylophorine
derivatives (PBTs), the commercially available plant virucide
Ningnanmycin (17), perhaps the most successful registered anti-
plant viral agent, and tylophorine alkaloid were used as the con-
trols. The antiviral bioassayagainst TMV invivo is assayed by the
reported method (14,15), and the antiviral results of all the PBTs
against TMV are listed in Table 1. Compounds 3, 8, 9, 13, and 15,
containing five-membered ring, six-membered ring, and straight-
chain structures, exhibited higher protection activity (56.7%,
56.3%, 64.6%, 70.8%, and 54.6%, respectively) at 500 μg/mL
than tylophorine and Ningnanmycin at 500 μg/mL (53.3% and
48.7%, respectively). Compound 4, containing a five-membered
ring structure, showed the same protection activity level (38.3%)
as tylophorine at 100 μg/mL (37.9%). Compounds 4, 6, 10, and
13, containing five-membered ring and six-membered ring struc-
tures, showed higher inactivation activity (80.0%, 74.2%, 68.9%,
and 71.1%, respectively) at 500 μg/mL than tylophorine and
Ningnanmycin at 500 μg/mL (62.6% and 68.4%, respectively).
Compound 16, containing a straight-chain structure, showed the
same inactivation activity level (60.9%) as tylophorine and
Ningnanmycin at 500 μg/mL. In addition, compounds 4 and 9
showed higher inactivation activities (54.7% and 50.2%, respec-
tively) than Ningnanmycin (48.0%) at 100 μg/mL. Compound 16
alsoshowedhigherinactivation activity (57.8%) thantylophorine
and Ningnanmycin (56.4% and 48.0%, respectively) at 100 μg/mL.
For curative effect, compounds 3, 5, 15, and 16, containing five-
membered ring and straight-chain structures, exhibited higher
curative activity (64.6%, 54.1%, 65.9%, and 65.0%, respectively)
at 500 μg/mL than Ningnanmycin at 500 μg/mL (47.9%). In
addition, compounds 3, 15, and 16, containing simple five-
membered ring structure and straight-chain structures, exhibited
the same curative activity (64.6%, 65.9%, and 65.0%, respec-
tively) as tylophorine (65.9%) at 500 μg/mL. At 100 μg/mL,
compounds 15 and 16 also exhibited higher curative activity
(48.0% and 49.6%, respectively) than Ningnanmycin (39.0%).
In summary, a series of C9-substituted phenanthrene-based
tylophorine derivatives (PBTs) have been synthesized efficiently
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., while the right side of the leaves was inoculated with the mix-
ture of solvent and the virus for control. The local lesion numbers were
recorded 3-4 days after inoculation (15). There are three replicates for
each compound.
Curative Effect of Compounds against TMV in Vivo. Growing leaves of
N. tabacum L. of the same ages were selected. The tobacco mosaic virus
(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 com-
pound 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 (15). There are three replicates for
each compound. The in vivo inhibition rate of the compound was then
calculated according to the following formula (av means average, and con-
trols were not treated with compound).
inhibition rate ð%Þ ¼ ½ðav local lesion no: of control
- av local lesion no: of drug-treatedÞ=av local lesion no: of controlꢁ ꢀ 100%
RESULTS AND DISCUSSION
Chemistry. The PBTs can be efficiently synthesized by different
methods as shown in Schemes 1, 2, 3, and 4. Condensation of the
9-phenanthrenecarbonyl chloride (prepared by chlorination of
9-phenanthrenecarboxylic acid 1 (11,16) with oxalyl chloride) with
amino acid methyl ester hydrochlorides (2, 7, 14) in the presence
of triethylamine gave the amidoesters (3, 8, 15) in good yields
(Schemes 1, 2, and 4). The Borch reduction of 3 and 8, consisting
of an O-alkylation of the tertiary amide with triethyloxonium fluor-
oborate and later sodium borohydride reduction, led to the amino-
esters 4 and 9 in high yields. Hydrolysis of 4 and 9 to the corre-
sponding sodium salts with sodium hydroxide and acidification
with hydrochloric acid gave amino acids 5 and 10 in almost quan-
titative yields (Schemes 1 and 2). The reduction of 5, 10, 1, and 15
with lithium aluminumhydride ledto thealcohols 6, 11, 12, and16
in excellent yields. Lithium aluminum hydride reduction of carboxylic
acid 1 gave alcohol 12 in 96.5% yield. Alcohol 12 was brominated by

Article
J. Agric. Food Chem., Vol. 58, No. 23, 2010 12341
Table 1. In Vivo Antiviral Activities against TMV
in excellent yield compared with tylophorine alkaloid itself. The
in vivo antiviral bioassay showed that most of the PBTs exhib-
ited good to excellent inhibitory activity against TMV compared
to commercial Ningnanmycin. Especially, compounds 3, 4, 8, 9,
13, and 15 exhibited excellent protection activities. Compounds
4, 6, 9, 10, 13, and 16 exhibited excellent inactivation activities.
Compounds 3, 5, 15, and 16 exhibited excellent curative activities.
So, compounds 3, 4, 9, 13, and 16, containing five-membered ring,
six-membered ring, and straight-chain structures, respectively,
emerged as potential inhibitors of plant virus. Therefore, the pres-
ent work demonstrates that the antiviral activity against TMV
was maintained via the simplification of tylophorine alkaloid

12342 J. Agric. Food Chem., Vol. 58, No. 23, 2010
Wang et al.
€
structure. Thus, the findings demonstrate that the synthesized
PBTs represent a new template for antiviral studies.
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Received for review September 8, 2010. Revised manuscript received
October 21, 2010. Accepted October 26, 2010. We gratefully
acknowledge the National Key Project for Basic Research
(2010CB126100) and the National Natural Science Foundation of
China (20872072) and China Postdoctoral Science Foundation
(20090460057) for financial support.
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