Design, synthesis, antiviral activity, and structure-activity relationships (SARs) of two types of structurally novel phenanthroindo/quinolizidine analogues

Article abstract of DOI:10.1021/jf405562r

To investigate the influence of the variation of the original skeletons of natural phenanthroindo/quinolizidine alkaloids on antiviral activities, two types of structurally totally novel analogues 7a, 7b, 16a, and 16b were designed, synthesized, and evaluated against tobacco mosaic virus (TMV) for the first time. Bioassay results indicated that all four of the newly designed analogues showed good to excellent antiviral activities, among which analogue 16a dispalyed comparable activity with that of ningnanmycin, perhaps one of the most successful commercial antiviral agents, thus emerging as a potential inhibitor of plant virus and serving as a new lead for further optimization. Further structure-activity relationships are also discussed, demonstrating for the first time that the same changes of the original skeletons of phenanthroindolizidine and phenanthroquinolizidine exihibted totally different antiviral activities results, providing some original and useful information about the preferential conformation for maintaining high activities.

Full text of DOI:10.1021/jf405562r

Article
pubs.acs.org/JAFC
Design, Synthesis, Antiviral Activity, and Structure−Activity
Relationships (SARs) of Two Types of Structurally Novel
Phenanthroindo/quinolizidine Analogues
Bo Su,∥ Fazhong Chen,∥ Lizhong Wang, and Qingmin Wang*
State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Nankai University,
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China
S
* Supporting Information
ABSTRACT: To investigate the influence of the variation of the original skeletons of natural phenanthroindo/quinolizidine
alkaloids on antiviral activities, two types of structurally totally novel analogues 7a, 7b, 16a, and 16b were designed, synthesized,
and evaluated against tobacco mosaic virus (TMV) for the first time. Bioassay results indicated that all four of the newly designed
analogues showed good to excellent antiviral activities, among which analogue 16a dispalyed comparable activity with that of
ningnanmycin, perhaps one of the most successful commercial antiviral agents, thus emerging as a potential inhibitor of plant
virus and serving as a new lead for further optimization. Further structure−activity relationships are also discussed, demonstrating
for the first time that the same changes of the original skeletons of phenanthroindolizidine and phenanthroquinolizidine exihibted
totally different antiviral activities results, providing some original and useful information about the preferential conformation for
maintaining high activities.
KEYWORDS: phenanthroindolizidine alkaloid, phenanthroquinolizidine alkaloid, antiviral activity, tobacco mosaic virus,
structure−activity relationship, anti-TMV
Because of the great economic loss caused by TMV and the
unsatisfactory inhibitory effects (usually 30−60%) of these
antiviral agents, much effort has been made toward the
development of novel and high effective plant virucides.
Consequently, a number of chemicals, such as pyrazole
derivatives,3 nucleotides,4 α-aminophosphonate derivatives,5
3-acetonyl-3-hydroxyoxindole,6 triazolyl compounds,7 oxidized
polyamines,8 and substituted phenylureas,9 were reported to
possess antiviral activities, few of which have been applied
successfully in agriculture.
Compared with synthetic chemicals, natural product-based
antiviral agents have many advantages, such as low mammalian
toxicity, easy decomposition, friendly to environment, specific
to targeted species, unique mode of action, and so on.10,11
Phenanthroindo/quinolizdine alkaloids are a small family of
natural products isolated mainly from the Cynanchum,
Pergularia, and Tylophora species.12 They exhibit diverse
biological activities ranging from anticancer and anti-inflamma-
tory to antiamoebic and antilupus effects.13−17 In a program
aimed at screening of plants for biologically active natural
products as alternatives to conventional synthetic antiviral
agents, our group first found that the alcohol extract of
Cynanchum komarovii displayed moderate antiviral activity
against TMV.18 Further investigation demonstrated that the
main active compound was (R)-antofine (Figure 1). Antiviral
mechanism studies revealed that antofine has a favorable
INTRODUCTION
■
Tobacco mosaic virus (TMV) is one of the most well-studied
plant viruses worldwide, which can infect 9 plant families and at
least 125 individual species, such as tobacco, pepper, cucumber,
tomato, and many ornamental flowers.1 TMV is known as
“plant cancer” due to the fact that it is very difficult to control
and has brought great disasters to agriculture. The economic
loss caused by TMV is up to U.S. $100 million each year
worldwide.2
Ningnanmycin (Figure 1) as a commercial plant-virus
inhibitor is perhaps most effective against plant virus and
displays 56.0% in vivo curative effect at 500 μg/mL. Another
widely used antiplant viral agent is ribavirin (Figure 1), the
inhibitory effect of which is always <50% at 500 μg/mL.
Received: December 10, 2013
Revised: January 23, 2014
Accepted: January 27, 2014
Published: January 27, 2014
Figure 1. Chemical structures of ningnanmycin, ribavirin, and
representative phenanthroindo/quinolizidine alkaloids.
© 2014 American Chemical Society
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Journal of Agricultural and Food Chemistry
Article
8.33 (s, 1H), 7.80 (s, 1H), 7.77 (s, 1H), 7.32 (s, 1H), 4.44 (br, 1H),
4.15 (s, 3H), 4.13(s, 3H), 4.06 (s, 6H), 3.86 (d, J = 14.8 Hz, 1H), 3.40
(br, 2H), 3.10−2.98 (m, 1H), 2.13 (br, 1H), 1.89 (br, 3H), 1.48 (s,
9H); 13C NMR (100 MHz) δ 199.2, 156.7, 152.2, 151.0, 150.4, 149.0,
130.3, 130.14 126.0, 125.1, 124.9, 123.5, 109.5, 107.2, 105.9, 102.6,
81.2, 56.1, 56.0, 55.9, 55.8, 46.0, 44.3, 33.1, 28.6, 24.9; HRMS (ESI)
calcd for C29H36NO7 (M + H)+ 510.2486, found 510.2490.
interaction with the origin of TMV RNA (oriRNA), exhibiting
its virus inhibition by binding to oriRNA and interfering with
virus assembly initiation.19 Moreover, structure−activity
relationship (SAR) studies showed that most compounds of
the phenanthroindolizidine-based library with structural diver-
sity exhibited antiviral effect against TMV.
In previous work, the SAR studies mainly focused on (1)
substituted patterns of methoxyl group on the phenanthrene
ring, (2) the number of methoxyl groups on the phenanthrene
ring, (3) derivatation at the C-14 position, and (4) D ring
opened derivatives.20,21 However, there is no investigation on
the variation of basic skeletons of phenanthroindo/quinolizines,
which will largely change the conformation of the molecules.
To study the influence of the variation of basic skeletons and
conformations of these phenanthroindo/quinolizidine alkaloids,
two types of structurally novel phenanthroindo/quinolizidine
alkaloid analogues were designed, synthesized, and evaluated
for their antiviral activity against TMV. The SAR study of these
compounds against TMV is also discussed.
2-(2-(2,3,6,7-Tetramethoxyphenanthren-9-yl)ethyl)-
pyrrolidine 6a. To a solution of compound 5a (0.37 g, 0.73 mmol)
in EtOH (50 mL) was added NaBH4 (0.1 g, 2.6 mmol). The reaction
mixture was stirred at room temperature for 2 h and then quenched
with water. After evaporation, the aqueous layer was extracted with
CH2Cl2 (30 mL × 3). The combined organic phase was washed with
brine, dried over Na2SO4, and concentrated under reduced pressure.
Crude product was dissolved in CH2Cl2 (50 mL) without further
purification, and then Et3SiH (0.18 mL, 1.13 mmol) and CF3COOH
(0.33 mL, 4.4 mmol) were added. The reaction mixture was stirred at
room temperature overnight and then quenched with diluted aqueous
NaOH. After separation, the organic layer was washed with brine,
dried over Na2SO4, and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel to give
1
compound 6a (0.27 g, 93%) as a white solid: mp 224−226 °C; H
NMR (400 MHz, CDCl3) δ 7.63 (s, 1H), 7.56 (s, 1H), 7.30 (s, 1H),
7.23 (s, 1H), 7.04 (s, 1H), 4.08 (s, 6H), 4.02 (s, 3H), 3.97 (s, 3H),
3.40−3.30 (m, 1H), 3.28−3.22 (m, 1H), 3.19−3.05 (m, 2H), 2.99−
2.88 (m, 1H), 2.22−2.17 (m, 1H), 2.11−1.90 (m, 3H), 1.89−1.76 (m,
1H), 1.57−1.52 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 148.4,
148.4, 148.3, 130.9, 125.9, 124.7, 124.5, 123.2, 122.9, 107.9, 104.1,
102.9, 102.2, 60.6, 56.2, 55.8, 55.8, 44.5, 31.9, 30.8, 30.3, 23.6; HRMS
(ESI) calcd for C24H30NO4 (M + H)+ 396.2169, found 396.2168.
2,3,6,7-Tetramethoxy-11,12,13,13a,14,15-hexahydro-9H-
phenanthro[9,10-e]pyrrolo[1,2-a]azepine 7a. To a solution of
compound 6a (0.2 g, 0.51 mmol) in toluene (20 mL) were added
formaldehyde (1.0 mL, 30%) and CF3COOH (0.5 mL, 6.7 mmol). A
Dean−Stark trap topped with a reflux condenser was attached to the
reaction vessel, and the reaction mixture was heated at reflux. Seven
hours later, the solvent was evaporated under reduced pressure, and
then aqueous NaOH was added to the residue. The aqueous solution
was extracted with CH2Cl2 (30 mL × 3), and the combined organic
phase was washed with brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by column
chromatography on silica gel to give compound 7a (0.18 g, 87%) as
a light yellow solid: mp 178−180 °C; 1H NMR (400 MHz, CDCl3) δ
7.84 (s, 2H), 7.53 (s, 2H), 4.69 (s, 1H), 4.12 (s, 3H), 4.11 (s, 3H),
4.06 (s, 3H), 4.05(s, 3H), 3.88−3.83 (m, 1H), 3.65 (dd, J = 15.0, 7.5
Hz, 1H), 3.21 (s, 1H), 3.09 (t, J = 12.0 Hz, 1H), 2.69 (s, 2H), 2.22−
2.10 (m, 1H), 2.00 (s, 1H), 1.78 (s, 2H), 1.57−1.40 (m, 2H); 13C
NMR (100 MHz, CDCl3) δ 148.0, 147.8, 147.7, 147.5, 133.9, 127.4,
124.3, 123.8, 123.7, 122.9, 103.5, 103.2, 102.4, 102.3, 76.3, 76.0, 75.7,
55.0, 54.9, 54.8, 49.3, 30.2, 29.8, 28.7, 25.4, 19.9; HRMS (ESI) calcd
for C29H30NO4 (M + H)+ 408.2169, found 408.2169.
2-(2,3,6,7-Tetramethoxyphenanthren-9-yl)acetonitrile 9. To
a solution of compound 8 (7.2 g, 18.4 mmol) in DMF (150 mL) was
added NaCN (1.0 g, 20.4 mmol). The reaction was stirred at room
temperature for 5 h, and then solvent was evaporated under reduced
pressure. Water and CH2Cl2 were added to the residue, and, after
separation, the aqueous layer was extracted with CH2Cl2. The
combined organic phase was washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel to give compound 9 (4.2 g, 68%)
as a white solid: mp 198−200 °C; 1H NMR (400 MHz, CDCl3) δ 7.86
(s, 1H), 7.77 (s, 1H), 7.70 (s, 1H), 7.23 (s, 1H), 7.14 (s, 1H), 4.14 (s,
3H), 4.13 (s, 3H), 4.12 (s, 2H), 4.07 (s, 3H), 4.05 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 148.7, 148.3, 148.1, 148.0, 124.6, 123.9, 123.6,
123.4, 122.7, 120.3, 116.7, 107.2, 102.6, 102.3, 101.6, 55.1, 55.0, 54.9,
21.3; HRMS (ESI) calcd for C20H23N2O4 (M + NH4)+ 499.2551,
found499.2552.
MATERIALS AND METHODS
■
Instruments. 1H NMR spectra were obtained at 400 MHz using a
Bruker AC-P 400. Chemical shift values (δ) were given in parts per
million and were downfield from internal tetramethylsilane. High-
resolution mass spectra (HRMS) were recorded on an FT-ICR MS
(Ionspec, 7.0 T). Melting points were determined on an X-4 binocular
microscope melting point apparatus (Beijing Tech Instruments Co.,
Beijing, China) and were uncorrected. Reagents were purchased from
commercial sources and were used as received. All anhydrous solvents
were dried and purified according to standard techniques just before
use.
1-(2,3,6,7-Tetramethoxyphenanthren-9-yl)ethanone 3. To a
solution of phenanthryl carboxylic acid (6.84 g, 20 mmol) in THF
(100 mL) was added MeLi (28.8 mL, 1.6 M, 46 mmol) dropwise via a
syringe at −78 °C under an atmosphere of nitrogen. The reaction
mixture was stirred at this temperature for 30 min and then warmed to
room temperature. Two hours later, saturated aqueous NH4Cl (10
mL) was added to quench the reaction. After evaporation of THF, the
aqueous layer was extracted with CH2Cl2 (80 mL × 3). The combined
organic phase was washed sequentially with diluted aqueous HCl,
water, and brine, dried over MgSO4, filtered, and concentrated under
reduced pressure. Crude product was recrystallized from MeOH to
1
give compound 3 (5.2 g, 77%) as a white solid: mp 214−215 °C; H
NMR (400 MHz, DMSO) δ 8.44 (s, 1H), 8.41 (s, 1H), 7.98 (s, 1H),
7.95 (s, 1H), 7.53 (s, 1H), 4.07 (s, 3H), 4.04 (s, 3H), 3.94 (s, 3H),
3.89 (s, 3H), 2.77 (s, 3H); 13C NMR (100 MHz, DMSO) δ 201.0,
151.2, 148.9, 148.8, 148.8, 130.3, 129.5, 126.5, 124.9, 124.0, 122.4,
109.6, 106.8, 103.7, 103.3, 55.9, 55.7, 55.4, 55.1, 29.5; HRMS (ESI)
calcd for C20H20NO5Na (M + H)+ 363.1203, found 263.1205.
tert-Butyl 2-(2-Oxo-2-(2,3,6,7-tetramethoxyphenanthren-9-
yl)ethyl)pyrrolidine-1-carboxylate 5a. To a solution of compound
3 (0.68 g, 2.0 mmol) and i-Pr2NEt (0.38 g, 3.0 mmol) in CH2Cl2 (50
mL) was added TMSOTf (0.53 g, 2.4 mmol) in CH2Cl2 (10 mL)
dropwise. The reaction mixture was stirred at room temperature for 4
h and then quenched with water (30 mL). After separation, the
organic phase was washed sequentially with water and brine, dried over
MgSO4, filtered, and concentrated under reduced pressure. Crude
product 4 was dissolved in CH2Cl2 (50 mL) without further
purification, and then compound 2 (0.44 g, 2.18 mmol) was added.
BF3·Et2O (3.0 mL, 30%) in CH2Cl2 (10 mL) was added dropwise at
−78 °C. The reaction mixture was stirred at this temperature for 1 h,
warmed to room temperature, and then quenched with diluted
aqueous NaOH. After separation, the organic phase was washed with
water and brine, dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy on silica gel to give compound 5a (0.73 g, 72%) as a light yellow
2-(2,3,6,7-Tetramethoxyphenanthren-9-yl)ethanamine 10.
To a solution of compound 9 (0.3 g, 0.89 mmol) in THF (30 mL)
was added BH3 (2.7 mL, 1 M in THF) at 0 °C under an atmosphere of
1
solid: mp 147−149 °C; H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H),
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Article
nitrogen. Then, the reaction was heated at reflux for 3 h. After the
mixture had cooled to room temperature, diluted aqueous HCl was
added to quench the reaction. The mixture was heated at reflux for an
additional 0.5 h and then was cooled to room temperature. The
mixture was extracted with EtOAc, and the aqueous layer was basified
by aqueous NaOH to pH >7. The aqueous layer was extracted with
EtOAc and then washed with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure to give compound 10 (0.25 g,
67%) as a white solid: mp 148−149 °C;1H NMR (400 MHz, CDCl3)
δ 7.82 (s, 1H), 7.75 (s, 1H), 7.44 (s, 1H), 7.38 (s, 1H), 7.17 (s, 1H),
4.11 (s, 3H), 4.09 (s, 3H), 4.05 (s, 3H), 4.02 (s, 3H), 3.21 (m, 4H),
2.2 (br, 1H); 13C NMR (100 MHz, CDCl3) δ 148.9, 148.8, 148.6,
130.8, 126.2, 125.3, 125.0, 124.6, 123.7, 107.9, 104.6, 103.4, 102.7,
56.1, 56.0, 55.9, 55.8, 41.8, 37.1; HRMS (ESI) calcd for C20H24NO4
(M + H)+ 342.1700, found 342.1702.
4-Oxo-4-((2-(2,3,6,7-tetramethoxyphenanthren-9-yl)ethyl)-
amino)butanoic Acid 12a. To a solution of amine 10 (0.4 g, 1.17
mmol) in CHCl3 (30 mL) was added dihydrofuran-2,5-dione (0.23 g,
2.34 mmol). The reaction was stirred at room temperature for 2 h, and
then water (30 mL) was added and stirred for an additional 1 h. After
separation, the aqueous layer was extracted with CHCl3 (30 mL × 3).
The combined organic phase was dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel to give compound 12a (0.46 g,
89%) as a white solid: mp 203−204 °C; 1H NMR (400 MHz, DMSO)
δ 8.16 (t, J = 5.2 Hz, 1H), 8.03 (s, 1H), 7.97 (s, 1H), 7.65 (s, 1H), 7.47
(s, 1H), 7.33 (s, 1H), 4.03 (s, 3H), 4.01 (s, 3H), 3.98 (s, 3H), 3.89 (s,
3H), 3.42−3.36 (m, 2H), 3.13 (t, J = 8 Hz, 2H), 2.46 (d, J = 6.4 Hz,
2H), 2.35 (t, J = 6.8 Hz, 2H); 13C NMR (100 MHz, DMSO) δ 173.6,
171.1, 148.8, 148.7, 148.6, 148.5, 130.6, 125.8, 124.9, 124.5, 124.1,
123.3, 108.1, 105.0, 104.3, 103.7, 55.9, 55.8, 55.5, 55.3, 33.5, 30.1, 29.2;
HRMS (ESI) calcd for C24H28NO7 (M + H)+ 442.1860, found
442.1866.
1-(2-(2,3,6,7-Tetramethoxyphenanthren-9-yl)ethyl)-
pyrrolidine-2,5-dione 13a. To a solution of compound 12a (0.46 g,
1.04 mmol) in Ac2O (20 mL) was added AcONa (0.1 g). The reaction
was stirred at 70 °C for 2 h and then cooled to room temperature. The
reaction was quenched with water and stirred for an additional 1 h.
After separation, the aqueous phase was extracted with CH2Cl2 (3 ×
30 mL). The combined organic phase was dried over Na2SO4, filtered,
and concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel to give compound 13a (0.43 g,
98%) as a white solid: mp 262−263 °C; 1H NMR (400 MHz, CDCl3)
δ 7.82 (s, 1H), 7.76 (s, 1H), 7.75 (s, 1H), 7.50 (s, 1H), 7.17 (s, 1H),
4.18 (s, 3H), 4.13 (s, 3H), 4.11 (s, 3H), 4.02 (s, 3H), 3.83 (t, J = 8.6
Hz, 2H), 3.25 (t, J = 8.6 Hz, 2H), 2.74 (s, 4H);13C NMR (100 MHz,
CDCl3) δ 177.2, 149.02, 148.9, 129.4, 126.21, 125.2, 124.8, 124.0,
107.9, 104.7, 103.3, 102.8, 77.3, 77.0, 76.7, 56.2, 56.1, 56.0, 55.9, 39.2,
32.1, 28.2; HRMS (ESI) calcd for C24H26NO6 (M + H)+ 424.1755,
found 424.1754.
TMSI (0.44 g, 2.1 mmol) at −55 °C. The reaction was stirred for 1 h
and then quenched with diluted aqueous NaOH. After separation, the
aqueous layer was extracted with CH2Cl2 (3 × 30 mL). The combined
organic phase was dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy on silica gel to give compound 15a (0.3 g, 80%) as a light yellow
solid: mp 222−224 °C; 1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H),
7.83 (s, 1H), 7.29 (s, 1H), 7.25 (s, 1H), 5.45 (br, 1H), 4.66 (d, J =
11.7 Hz, 1H), 4.13 (s, 6H), 4.06 (s, 3H), 4.05 (s, 3H), 3.18 (br, 2H),
3.14−2.98 (m, 2H), 2.71 (br, 1H), 2.48 (br, 1H), 1.80 (br, 1H);13C
NMR (100 MHz, CDCl3) δ 149.1, 148.7, 148.6, 128.9, 125.9, 125.5,
124.3, 123.7, 123.2, 123.1, 104.6, 104.2, 103.7, 103.3, 56.2, 56.1, 56.0,
36.6, 29.7, 29.1, 26.6; HRMS (ESI) calcd for C24H26NO5 (M + H)+
408.1805, found 408.1805.
2,3,12,13-Tetramethoxy-5,6,8,9,10,10a-hexahydrodibenzo-
[f,h]pyrrolo[2,1-a]isoquinoline 16a. To a solution of compound
15a (0.29 g, 0.71 mmol) in THF (30 mL) was added LiAlH4 (50.0 mg,
1.3 mmol). The reaction mixture was heated at reflux for 2 h and then
quenched with diluted aqueous NaOH after cooling to room
temperature. After separation, the aqueous layer was extracted with
CH2Cl2 (3 × 30 mL). The combined organic phase was dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel to give
compound 16a (0.23 g, 82%) as a light yellow solid: mp 155−158 °C;
1H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.83 (s, 1H), 7.35 (s,
1H), 7.30 (s, 1H), 4.66 (t, J = 8.1 Hz, 1H), 4.12 (s, 6H), 4.05 (s, 6H),
3.25−3.16 (m, 2H), 3.15−3.04 (m, 4H), 2.73−2.69 (m, 1H), 2.04−
1.92 (m, 2H), 1.84−1.72 (m, 1H); 13C NMR (100 MHz, CDCl3) δ
147.8, 147.5, 147.3, 147.2, 129.3, 124.5, 124.2, 123.5, 122.9, 122.4,
104.1, 103.2, 102.4, 102.3, 58.8, 55.1, 55.0, 54.9, 54.8, 51.9, 44.2, 31.2,
23.4, 22.4; HRMS (ESI) calcd for C24H28NO4 (M + H)+ 394.2013,
found 394.2018.
2-(Piperidin-2-yl)-1-(2,3,6,7-tetramethoxyphenanthren-9-
yl)ethanone 5b. The synthetic procedure was similar to that of
compound 5a, and compound 5b was obtained as a light yellow solid
1
(73%): mp 203−205 °C; H NMR (400 MHz, DMSO) δ 8.55 (s,
1H), 8.45 (s, 1H), 8.07 (s, 1H), 8.03 (s, 1H), 7.64 (s, 1H), 4.09 (s,
3H), 4.06 (s, 3H), 3.95 (s, 3H), 3.90 (s, 3H), 3.85−3.77 (m, 1H), 3.69
(s, 1H), 3.57−3.55 (m, 1H), 3.26 (s, 2H), 2.97 (s, 1H), 1.94−1.91 (m,
1H), 1.77−1.75 (m, 4H); 13C NMR (100 MHz, DMSO) δ 200.1,
151.5, 149.1, 149.0, 148.8, 130.0, 128.9, 126.7, 125.0, 123.9, 122.3,
109.7, 106.7, 103.8, 103.4, 56.0, 55.8, 55.5, 55.2, 52.6, 44.3, 28.9, 22.6,
22.2, 14.0; HRMS (ESI) calcd for C25H30NO5 (M + H)+ 424.2118,
found 424.2123.
2-(2-(2,3,6,7-Tetramethoxyphenanthren-9-yl)ethyl)-
piperidine 6b. The synthetic procedure was similar to that of
compound 6a, and compound 6b was obtained as a white solid (88%):
mp 175−176 °C; 1H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.76 (s,
1H), 7.41 (s, 1H), 7.40 (s, 1H), 7.16 (s, 1H), 4.11 (s, 6H), 4.05 (s,
3H), 4.03 (s, 3H), 3.09 (t, J = 8.0 Hz, 3H), 2.71−2.59 (m, 2H), 1.89−
1.82 (m, 4H), 1.64−1.61 (m, 2H), 1.52−1.32 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 148.7, 148.5, 134.0, 126.4, 125.4, 124.9, 123.5,
107.9, 104.8, 103.4, 102.8, 77.3, 77.0, 76.7, 56.9, 56.1, 56.0, 55.9, 55.8,
47.2, 37.8, 33.0, 29.8, 26.7, 24.8; HRMS (ESI) calcd for C25H32NO4
(M + H)+ 410.2326, found 410.2331.
2,3,6,7-Tetramethoxy-9,11,12,13,14,14a,15,16-
octahydrophenanthro[9,10-e]pyrido[1,2-a]azepine 7b. The
synthetic procedure was similar to that of compound 7a, and
compound 7b was obtained as a white solid (68%): mp 216−218
°C; 1H NMR (400 MHz, CDCl3) δ 7.83 (s, 2H), 7.50 (s, 1H), 7.45 (s,
1H), 4.37 (d, J = 14.9 Hz, 1H), 4.16−4.02 (m, 13H), 3.56−3.53 (m,
1H), 3.30−3.19 (m, 1H), 3.06−3.04 (m, 1H), 2.60−2.41 (m, 2H),
2.01 (s, 1H), 1.68−1.60 (m, 5H), 1.49 (s, 1H), 1.34−1.31 (m, 1H);
13C NMR (100 MHz, CDCl3) δ 148.7, 148.6, 148.5, 148.3, 125.7,
5-Hydroxy-1-(2-(2,3,6,7-tetramethoxyphenanthren-9-yl)-
ethyl)pyrrolidin-2-one 14a. To a solution of compound 13a (0.48
g, 1.13 mmol) in CH2Cl2 (20 mL) was added LiEt3BH (1 M, 2.2 mL)
at −60 °C. The reaction mixture was stirred for 2 h and then quenched
with water. After separation, the aqueous layer was extracted with
CH2Cl2 (3 × 30 mL). The combined organic phase was dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel to give
1
compound 14a (0.44 g, 92%) as a white solid: mp 187−188 °C; H
NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.75 (s, 1H), 7.68 (s, 1H),
7.44 (s, 1H), 7.13 (s, 1H), 4.99 (s, 1H), 4.12 (s, 3H), 4.11(s, 3H), 4.10
(s, 3H), 4.06−4.02 (m, 1H), 4.00 (s, 3H), 3.82−3.72 (m, 1H), 3.67−
3.63 (m, 1H), 3.39−3.29 (m, 2H), 2.63−2.52 (m, 1H), 2.37−2.19 (m,
2H), 1.82−1.77 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 173.6,
148.1, 148.0, 147.9, 129.7, 125.2, 123.7, 122.9, 106.8, 103.9, 102.1,
101.8, 83.2, 55.2, 55.1, 55.0, 54.9, 40.7, 31.4, 27.9, 27.8; HRMS (ESI)
calcd for C24H28NO6 (M +H)+ 426.1911, found 426.1913.
124.2, 123.7, 104.6, 104.3, 103.4, 103.3, 94.5, 77.3, 77.0, 76.7, 56.8,
56.0, 55.9, 55.8, 55.7, 33.9, 33.5, 26.8, 26.5, 23.8; HRMS (ESI) calcd
for C26H32NO4 (M + H)+ 422.2326, found 422.2332.
2,3,12,13-Tetramethoxy-5,6,10,10a-tetrahydrodibenzo[f,h]-
pyrrolo[2,1-a]isoquinolin-8(9H)-one 15a. To a solution of
compound 14a (0.4 g, 0.94 mmol) in CH2Cl2 (20 mL) was added
5-Oxo-5-((2-(2,3,6,7-tetramethoxyphenanthren-9-yl)ethyl)-
amino)pentanoic acid 12b. The synthetic procedure was similar to
that of compound 12a, and compound 12b was obtained as a white
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Scheme 1. Synthetic Route for Compounds 7a and 7b
Scheme 2. Synthetic Route for Compounds 16a and 16b
solid (78%): mp 158−160 °C; 1H NMR (400 MHz, CDCl3) δ 7.80 (s,
1H), 7.74 (s, 1H), 7.54 (s, 1H), 7.37 (s, 1H), 7.14 (s, 1H), 5.92 (s,
1H), 4.10 (s, 3H), 4.09 (s, 3H), 4.08 (s, 3H), 4.01 (s, 3H), 3.67 (d, J =
6.8 Hz, 2H), 3.25 (t, J = 6.8 Hz, 2H), 2.39 (t, J = 6.8 Hz, 2H), 2.25 (t,
J = 7.2 Hz, 2H), 1.98−1.90 (m, 2H); 13C NMR (100 MHz, CDCl3) δ
176.0, 171.7, 147.9, 129.3, 123.5, 106.9, 103.8, 102.4, 101.8, 55.2, 55.1,
55.0, 54.9, 38.9, 34.3, 32.5, 28.9, 19.7, 15.3; HRMS (ESI) calcd for
C25H30NO4(M + H)+ 456.2017, found 456.2018.
1-(2-(2,3,6,7-Tetramethoxyphenanthren-9-yl)ethyl)-
piperidine-2,6-dione 13b. The synthetic procedure was similar to
that of compound 13a, and compound 13b was obtained as a white
solid (86%): mp 261−263 °C; 1H NMR (400 MHz, CDCl3) δ 7.89 (s,
1H), 7.83 (s, 1H), 7.78 (s, 1H), 7.54 (s, 1H), 7.19 (s, 1H), 4.21 (s,
3H), 4.13−4.08 (m, 8H), 4.03 (s, 3H), 3.28−3.16 (m, 2H), 2.71 (t, J =
8.4 Hz, 4H), 2.04−1.93 (m, 2H); 13C NMR (100 MHz, CDCl3) δ
171.5, 148.0, 147.9, 147.8, 147.7, 129.4, 125.3, 124.6, 124.2, 123.7,
122.9, 106.9, 104.3, 102.1, 101.7, 55.3, 55.1, 55.0, 54.9, 39.5, 31.8, 31.5,
16.2; HRMS (ESI) calcd for calcd for C25H27NO6Na+ (M + Na)+
460.1731, found 460.1735.
6-Hydroxy-1-(2-(2,3,6,7-tetramethoxyphenanthren-9-yl)-
ethyl)piperidin-2-one 14b. The synthetic procedure was similar to
that of compound 14a, and compound 14b was obtained as a white
solid (86%): mp 202−204 °C; 1H NMR (400 MHz, CDCl3) δ 7.81 (s,
1H), 7.80 (s, 1H), 7.76 (s, 1H), 7.45 (s, 1H), 7.14 (s, 1H), 4.74 (s,
1H), 4.18−4.09 (m, 9H), 4.01 (s, 3H), 3.91−3.81 (m, 1H), 3.71−3.64
(m, 1H), 3.41−3.35 (m, 2H), 2.56−2.46 (m, 1H), 2.40−2.27 (m, 2H),
1.99−1.89 (m, 1H), 1.82−1.73 (m, 1H), 1.73−1.65 (m, 2H); 13C
NMR (100 MHz, CDCl3) δ 170.4, 148.9, 148.9, 148.8, 148.8, 131.7,
126.3, 125.7, 124.8, 123.8, 107.8, 105.4, 103.2, 102.8, 81.1, 56.3, 56.1,
+
56.0, 55.9, 47.5, 32.6, 31.1, 15.9; HRMS (ESI) calcd for C25H30NO6
(M + H)+ 440.2068, found 440.2064.
2,3,13,14-Tetramethoxy-9,10,11,11a-tetrahydro-5H-
dibenzo[f,h]pyrido[2,1-a]isoquinolin-8(6H)-one 15b. The syn-
thetic procedure was similar to that of compound 15a, and compound
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1
15b was obtained as a white solid (80%): mp 153−155 °C; H NMR
(400 MHz, CDCl3) δ 7.85 (s, 1H), 7.82 (s, 1H), 7.32 (s, 1H), 7.21 (s,
1H), 5.35 (d, J = 10.8 Hz, 1H), 5.18 (dd, J = 12.4, 3.2 Hz, 1H), 4.13 (s,
6H), 4.05 (s, 3H), 4.04 (s, 3H), 3.19 (t, J = 16.0 Hz, 1H), 3.14−3.03
(m, 1H), 2.85−2.75 (m, 1H), 2.76−2.66 (m, 2H), 2.60−2.48 (m, 1H),
2.09−1.93 (m, 2H), 1.40−1.35 (m, 1H); 13C NMR (100 MHz,
CDCl3) δ 169.8, 149.2, 148.6, 148.53, 128.2, 127.6, 125.5, 124.5,
123.5, 123.3, 104.2, 104.1, 103.8, 103.3, 56.2, 56.1, 56.1, 56.0, 56.9,
38.0, 32.1, 31.6, 26.8, 20.0, 14.2; HRMS (ESI) calcd for C25H27NO5
(M + H)+ 422.1962, found 422.1959.
condensation of the aldehyde occurred quickly. To improve the
reactivity of the intermediate of the cyclization, we turned to an
alternative Bischler−Napieralski cyclization approach, which
employed highly reactive acyl iminium as key intermediate.
Imides 13 prepared from amine 10 and corresponding
anhydride were subjected to partial reduction, affording
compounds 14. The subsequent Bischler−Napieralski cycliza-
tion went smoothly when trimethyliodosilane was employed.
After reduction, isomerized phenanthroindo/quinolizidine
alkaloid analogues 16 were obtained in good yields.
Antiviral Activity. To make a judgment of the designed
structurally novel phenanthroindo/quinolizidine alkaloid ana-
logues 7a, 7b, 16a, and 16b, two commercially available
antiviral agents were used as control. To further investigate the
influence of the variation of the basic skeletons of the natural
alkaloids on the antiviral effects, corresponding tylophorine I
and phenanthroquinolizidine II (Figure 1) were also used for
comparison, which were synthesized using methods reported
previously by our group.24 The in vitro and in vivo (protection,
inactivation, and curative effect) antiviral results against TMV
of phenanthroindo/quinolizidine alkaloids I and II, analogues
7a, 7b, 16a, and 16b, ribavirin, and ningnanmycin are listed in
Table 1. All of the compounds were tested at both 500 and 100
μg/mL.
2,3,13,14-Tetramethoxy-6,8,9,10,11,11a-hexahydro-5H-
dibenzo[f,h]pyrido[2,1-a]isoquinoline 16b. The synthetic proce-
dure was similar to that of compound 16a, and compound 16b was
obtained as a white solid (60%): mp 89−92 °C; 1H NMR (300 MHz,
CDCl3) δ 7.83 (s, 1H), 7.82 (s, 1H),7.32 (s, 1H), 7.25 (s, 1H), 4.26
(d, J = 9.7 Hz, 1H), 4.11 (s, 6H), 4.04 (s, 3H), 4.03 (s, 3H), 3.47−3.39
(m, 1H), 3.28−3.25 (m, 1H), 3.21−3.13 (m, 2H), 2.93−2.87 (m, 1H),
2.21 (d, J = 11.9 Hz, 1H), 1.96−1.63 (m, 6H); 13C NMR (100 MHz,
CDCl3) δ 149.5, 149.3, 148.8, 131.1, 126.6, 126.5, 124.7, 124.5, 124.2,
105.4, 104.8, 104.3, 103.9, 60.5, 56.8, 56.7, 56.6, 56.5, 46.3, 29.2, 28.5,
26.6, 22.5; HRMS (ESI) calcd for C25H30NO4 (M + H)+ 408.2169,
found 408.2170.
Antiviral Biological Assay. The procedure of purifying TMV and
the method to test the anti-TMV activity of the synthesized
compounds were the same with those reported previously in the
literature.22
RESULTS AND DISCUSSION
■
Table 1. In Vitro and in Vivo Antiviral Activity of
Compounds 7a, 7b, 16a, and 16b against TMV
Chemistry. The synthesis of D-ring expanded phenan-
throindo/quinolizidine alkaloid analogues 7a and 7b is shown
in Scheme 1, which features a Mukaiyama−Aldol reaction and a
Pictet−Spengler cyclization. The precursors 2 of the
Mukaiyama−Aldol reaction were prepared from commercially
available lactams through literature protocols.23 Silylenol ether
4 was synthesized from acetophenanthrone 3, which was
obtained from readily available phenanthryl acid.24 After
extensive reaction screening, it was found that boron trifluoride
was the optimal Lewis acid for the desired Mukaiyama−Aldol
reactions, giving compounds 5 in good yields. Ketones 5 were
reduced to alcohols by sodium borohydride, followed by
deprotection and further reduction of the benzylic hydroxyl by
triethylsilicane under acidic conditions, furnishing amines 6
efficiently. It is worth noting that the following Pictet−Spengler
cyclization did not occur when concentrated hydrochloric acid
was used as catalyst in ethanol, which may be due to the energy
of the seven-membered ring transition state being too high.
When the reaction was performed in a high boiling point
solvent (toluene) using much stronger acid CF3COOH as
catalyst, the desired cyclized products 7 were obtained in good
yields.
inhibition rate (%)
in
concn
vitro
protection inactivation curative
compd
(μg/mL) effect
effect
effect
effect
I
500
100
38.5
15.3
40.4
18.2
45.2
17.5
42.6
13.1
7a
16a
II
500
100
43.8
20
46.5
21.6
47.3
20.3
42.5
10.6
500
100
60
54.1
24.1
55.8
26.7
58.6
30
30.6
500
100
67.5
32.4
65.8
32.1
64.2
30
68.9
34.7
7b
16b
500
100
54.7
27.6
48.3
26.9
52.6
28.9
50.7
21.7
500
100
37.7
10
42.9
17.5
41.7
18.3
39.5
12.4
The synthesis of the isomerized phenanthroindo/quinolizi-
dine analogues 16a and 16b is shown in Scheme 2, featuring a
Bischler−Napieralski cyclization. Using readily available
phenanthryl bromide 8 as starting material,25 intermediate 10
could be easily be prepared via sequential nucleophilic
substitution and reduction. With the phenanthrylethylamine
10 in hand, we initially attempted to construct the pentacyclic
skeleton through Pictet−Spengler cyclization/condensation
sequence. Unfortunately, although much effort was devoted
to the screening of reaction conditions, including catalysts,
solvents, and reaction temperature, all attempts to perform the
desired Pictet−Spengler cyclization failed. The main difficulty
was proposed to be the Pictet−Spengler cyclization, presum-
ably because reactivity of the key intermediate formed through
condensation of amine and aldehyde was so slow that self-
ribavirin
500
100
41.3
18.5
38.9
15.6
37.2
11.9
39.4
14.7
ningnanmycin
500
100
69.3
26.8
57.9
38.4
54.2
20.0
58.7
23.1
The first in vitro anti-TMV bioassay demonstrated that all of
the synthesized sturcturally novel analogues except for 16b
exhibted higher activity than ribavirin at both high (500 μg/
mL) and low (100 μg/mL) concentrations. Especially, although
compounds 16a and 7b showed only comparable inhibitory
effect with that of ningnanmycin at 500 μg/mL, they exhibited
higher antiviral activity at 100 μg/mL. Further in vivo anti-
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TMV bioassay indicated that all of the newly designed
analogues displayed protection and inactivation effects superior
to those of ribavirin at both concentrations, and all of them also
showed preferable curative effects except for 7a and 16b at 100
μg/mL. Compared with ningnanmycin, perhaps the most
effective commercial plant virucide, analogue 16a can also
exhibt higher inactivation effect at both concentrations, and 7b
and 16a showed inactivation and curative effects superior to
those of ningnanmycin at 100 μg/mL.
AUTHOR INFORMATION
■
Corresponding Author
*(Q.W.) Phone: +86-022-23503952. Fax: +86-022-23503952.
E-mail: wangqm@nankai.edu.cn.
Author Contributions
∥B.S. and F.C. contributed equally to this work.
Funding
We are grateful to the National Key Project for Basic Research
(2010CB126100), the National Natural Science Foundation of
China (21132003, 21121002, 21372131, 21002053), Tianjin
Natural Science Foundation (11JCZDJC20500), and Speci-
alized Research Fund for the Doctoral Program of Higher
Education (20130031110017).
The SARs demonstrated that phenanthroindolizidine ana-
logue 7a with the six-membered D-ring of natural alkaloid
expanded to seven-membered ring that showed higher in vitro,
protection, and inactivation effects than tylophorine I at both
concentrations. The isomerized analogue 16a also displayed
preferable antiviral effects both in vitro and in vivo relative to its
precursor I at both high and low concentrations. Then, we can
conclude that for natural phenanthroindolizidine alkaloid I, no
matter how the six-membered D-ring was expanded to seven-
membered or the fused pattern of D/E rings was changed,
analogues 7a and 16a showed superior antiviral activity to that
of the precursor I, which indicated that the original
conformation of the phenanthroindolizidine was not optimal
and deserved further optimization. However, no matter how
the D-ring was expanded from six-membered to seven-
membered or the fused pattern of D/E rings was changed,
both phenanthroquinolizidine analogues 7b and 16b exhibited
inferior inhibitory effects against TMV, indicating that the
original conformation of the phenanthroquinolizidine was a
relatively preferential conformation. Another interesting result
was that although six-membered E ring compound 7b showed
higher activity than five-membered E ring compound 7a, their
corresponding counterparts 16b and 16a displayed reverse
bioactive results, which suggested that the fused pattern of D/E
rings of phenanthroquinolizidine alkaloid also has a great effect
on its activity, and the original fused pattern is relatively
optimal.
In summary, two types of structurally totally novel analogues
of phenanthroindo/quinolizidines were synthesized and
evaluated for their antiviral activities aganist TMV for the first
time. The bioassay results indicated that most of the four
designed structural analogues showed good to excellent in vivo
anti-TMV activity, among which analogue 16a dispalyed
comparable activity with that of one of the most successful
commercial antiviral agents, ningnanmycin, thus emerging as a
potential inhibitor of plant virus. Further structure−activity
relationships demonstrated for the first time that the original
skeleton of the pentacyclic structure of phenanthroindolizidine
is not optimal, and further optimization for preferential
conformation is needed, whereas changes of the original
skeleton of the phenanthroquinolizidine descreased its antiviral
activity, indicating that the original conformation is a relative
preferential conformation. Further investigations on structual
optimization and mode of action are currently underway in our
laboratories.
Notes
The authors declare no competing financial interest.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1 H and 13 C NMR spectra of compounds 3−7, 9, 10, and 12−
16. This material is available free of charge via the Internet at
http://pubs.acs.org.
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