Article
J. Agric. Food Chem., Vol. 58, No. 5, 2010 2705
value: mp 282 °C dec; (lit. (9) mp 282-284 °C dec); [R]20D þ100° (CHCl3);
1H NMR (400 MHz, CDCl3) δ 7.84 (s, 1H), 7.83 (s, 1H), 7.32 (s, 1H), 7.17
(s, 1H), 4.64 (d, 2JHH = 14.4 Hz, 1H), 4.12 (s, 6H), 4.06 (s, 6H), 3.68 (d,
2JHH = 14.4 Hz, 1H), 3.47-3.51 (m, 1H), 3.33-3.40 (m, 1H), 2.87-2.95
(m, 1H), 2.42-2.54 (m, 2H), 2.20-2.30 (m, 1H), 1.73-2.10 (m, 3H).
Synthesis of (R)-(-)-Tylophorine. Following the same procedure as
for (S)-(þ)-tylophorine gave optically pure alkaloid (R)-(-)-tylophorine
Data for 20: mp 240 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.02
(s, 2H), 7.34 (s, 1H), 7.20 (s, 1H), 4.73 (d, 2JHH = 15.3 Hz, 1H), 4.07-4.15
(m, 1H), 4.03 (s, 6H), 3.94 (s, 6H), 3.42-3.52 (m, 2H), 2.53-2.95 (m, 5H),
2.37-2.44 (m, 1H), 2.21-2.34 (m, 1H), 1.88-2.05 (m, 2H), 1.66-1.82
(m, 1H).
Data for 21: mp 268 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.01
(s, 2H), 7.32 (s, 1H), 7.19 (s, 1H), 4.59 (d, 2JHH = 15.0 Hz, 1H), 4.02 (s,
2
with 96% ee value, [R]20 -93° (CHCl3). The optical purity of the
6H), 3.93 (s, 6H), 3.61 (d, JHH = 15.3 Hz, 1H), 3.34-3.40 (m, 2H),
D
synthesized alkaloids was determined via HPLC using an Agilent 1100
instrument with an AD-H column and gave 92 and 96% ee values,
respectively. Detection was conducted at 254 nm. n-Hexane/isopropa-
nol/triethylamine (75:25:0.025) was used as mobile phase at a flow rate of
1.0 mL/min.
2.76-2.85 (m, 1H), 2.41 (s, 6H), 2.12-2.28 (m, 1H), 1.59-1.97
(m, 3H).
Data for 22: mp 230 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.28
(s, 1H), 8.01 (s, 1H), 7.80 (s, 1H), 7.77 (s, 1H), 7.33 (s, 1H), 7.19 (s, 1H),
6.83 (s, 1H), 6.80 (s, 1H), 4.56 (d, 2JHH = 15.3 Hz, 1H), 4.02 (s, 6H), 3.93
2
General Procedure for the Preparation of Tylophorine Salt
Derivatives 10-25. To a solution of 1.0 mmol of inorganic acid or
organic acid in 50 mL of CHCl3 and 50 mL of CH3OH was added
1.0 mmol of racemic tylophorine in 50 mL of CHCl3 slowly under nitrogen
at 40-50 °C. The mixture was refluxed for 2 h and allowed to stand for 2 h;
the solvents were removed partially by rotary evaporation and filtered to
obtain the corresponding salt derivatives 10-25.
(s, 6H), 5.50 (d, JHH = 14.7 Hz, 1H), 3.32-3.41 (m, 2H), 2.70-2.85
(m, 1H), 2.33-2.45 (m, 2H), 2.10-2.25 (m, 1H), 1.57-2.00 (m, 3H).
.
Data for 23: mp 230 °C dec; 1H NMR (400 MHz, CDCl3) δ 7.81 (s,
2H), 7.28 (s, 1H), 7.11 (s, 1H), 4.71 (d, 2JHH = 14.8 Hz, 1H), 4.16-4.22 (m,
1H), 4.12 (s, 6H), 4.05 (s, 6H), 3.50-4.00 (m, 3H), 3.45-3.50 (m, 1H),
3.36-3.40 (m, 1H), 3.00-3.07 (m, 1H), 2.50-2.80 (m, 2H), 2.24-2.36 (m,
1H), 1.80-2.16 (m, 3H), 1.18-1.26 (m, 1H).
Data for 24: mp 241 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.07 (s,
2H), 7.20-7.37 (m, 4H), 6.39-6.42 (m, 2H), 5.00-5.37 (m, 2H),
4.44-4.70 (m, 1H), 4.05 (s, 6H), 3.97 (s, 6H), 3.50-3.80 (m, 3H),
3.08-3.23 (m, 1H), 1.80-2.30 (m, 4H).
Data for 10: mp 258 °C dec; 1H NMR (400 MHz, DMSO-d6) δ 8.04 (s,
1H), 8.03 (s, 1H), 7.33 (s, 1H), 7.17 (s, 1H), 5.00-5.15 (m, 1H), 4.33-4.50
(m, 1H), 4.03 (s, 6H), 3.94 (s, 6H), 3.40-3.83 (m, 3H), 3.15-3.30 (m, 2H),
2.30-2.45 (m, 1H), 1.86-2.18 (m, 3H).
1
Data for 11: mp 260 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.07
(s, 1H), 8.06 (s, 1H), 7.37 (s, 1H), 7.21 (s, 1H), 5.19-5.33 (m, 1H),
4.54-4.72 (m, 1H), 4.05 (s, 6H), 3.97(s, 6H), 3.64-3.91 (m, 3H), 3.15-3.48
(m, 2H), 2.53-2.64 (m, 1H), 1.86-2.31 (m, 3H).
Data for 25: mp 204-206 °C dec; H NMR (300 MHz, DMSO-d6)
δ 10.00-10.30 (br, 1H), 8.03-8.08 (m, 3H), 7.65-7.68 (m, 1H), 7.37
(s, 1H), 7.20 (s, 1H), 6.84 (d, 3JHH = 8.4 Hz, 1H), 5.24 (d, 3JHH = 15.6 Hz,
1H), 4.55-4.72 (m, 1H), 4.05 (s, 6H), 3.97 (s, 6H), 3.60-3.90 (m, 3H),
3.10-3.50 (m, 3H), 1.83-2.32 (m, 3H).
Data for 12: mp 240 °C dec; 1H NMR (400 MHz, DMSO-d6) δ 8.06 (s,
1H), 8.05 (s, 1H), 7.36 (s, 1H), 7.19 (s, 1H), 5.22-5.27 (m, 1H), 4.59-4.66
(m, 1H), 4.04 (s, 6H), 3.96 (s, 6H), 3.62-3.91 (m, 4H), 3.13-3.25 (m, 1H),
2.02-2.32 (m, 3H), 1.85-1.97 (m, 1H).
Antiviral Biological Assay. Purification of TMV
. Using
Gooding’s method (10), the upper leaves of Nicotiana tabacum
L. inoculated with TMV were selected and ground in phosphate buffer
and then filteredthrough double-layer pledget. The filtrate was centrifuged
at 10000g, treated with PEG twice, and centrifuged again. The whole
experiment was processed at 4 °C. Absorbance value was estimated at
260 nm by ultraviolet spectrophotometer.
Data for 13: mp 246 °C dec; 1H NMR (300 MHz, CDCl3) δ 8.02 (s,
1H), 8.00 (s, 1H), 7.82 (s, 2H), 7.45-7.51 (m, 1H), 7.30-7.40 (m, 3H), 7.14
(m, 1H), 4.86-4.91 (m, 1H), 4.12 (s, 3H), 4.11 (s, 3H), 4.06 (s, 3H), 4.04
(s, 3H), 3.97-4.00 (m, 1H), 3.65-3.76 (m, 1H), 3.38-3.48 (m, 1H), 3.17-
3.27 (m, 1H), 3.00-3.12 (m, 1H), 2.77-2.90 (m, 1H), 2.31-2.42 (m, 1H),
1.90-2.20 (m, 3.H).
virus concn ¼ ðA260 ꢀ dilution ratioÞ=E0:1%;260nm
1cm
Data for 14: mp 250 °C dec; 1H NMR (400 MHz, CDCl3) δ 7.80-7.85
(m, 2H), 7.21-7.26 (m, 1H), 7.02 (d, 1H), 5.37-5.57 (m, 1H), 4.78-4.93
(m, 1H), 4.44-4.60 (m, 1H), 4.04-4.15 (m, 12H), 3.55-3.65 (m, 1H),
3.32-3.44 (m, 1H), 3.08-3.20 (m, 2H), 2.66-2.69 (m, 1H), 2.48-2.56 (m,
1H), 2.37-2.46 (m, 1H), 2.13-2.30 (m, 2H), 1.89-1.91 (m, 1H), 1.64-
1.80 (m, 4H), 1.46-1.56 (m, 1H), 1.02-1.11 (m, 1H), 0.88 (s, 3H), 0.62
(s, 3H).
Antiviral Activity of Compounds against TMV in Vitro. In vitro
activity of the synthesized compounds against TMV was performed by the
conventional half-leaf method (11). 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 different concentra-
tions (see Table 1) of the compounds and double distilled water for 20 min,
respectively, and then cultured at 25 °C for 72 h. Every concentration for
each compound was replicated at least three times.
Data for 15: mp 225 °C dec; 1H NMR (400 MHz, DMSO-d6) δ 10.15
(br, 1H), 8.56 (s, 2H), 8.06 (s, 1H), 8.05 (s, 1H), 7.36 (s, 1H), 7.18 (s, 1H),
5.21-5.25 (m, 1H), 4.58-4.64 (m, 1H), 4.03 (s, 6H), 3.95 (s, 6H), 3.85-
3.91 (s, 1H), 3.63-3.78 (m, 3H), 3.32-3.45 (m, 1H), 3.12-3.26 (m, 1H),
2.05-2.25 (m, 2H), 1.84-1.98 (m, 1H).
Protective Effect of Compounds against TMV in Vivo. The
compound solution was smeared on the left side and the solvent serving
as 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 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 (12). There
are three replicates for each compound.
Data for 16: mp 260 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.00 (s,
2H), 7.47 (d, 3JHH = 15.9 Hz, 1H), 7.19-7.32 (m, 4H), 7.05-7.09 (m, 1H),
6.79 (d, 3JHH = 8.1 Hz, 1H), 6.35 (d, 3JHH = 15.9 Hz, 1H), 4.56 (d, 2JHH
= 15.6 Hz, 1H), 4.02 (s, 6H), 3.93 (s, 6H), 3.81 (s, 3H), 3.53 (d, 2JHH
=
15.6 Hz, 1H), 3.32-3.37 (m, 2H), 2.71-2.89 (m, 1H), 2.30-2.40 (m, 2H),
2.10-2.24 (m, 1H), 1.58-1.95 (m, 3H).
Data for 17: mp 260 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.03 (s,
2H), 7.35 (s, 1H), 7.20 (s, 1H), 4.75-4.81 (m, 1H), 4.20 (s, 4H), 4.07-4.14
(m, 1H), 4.03 (s, 6H), 3.94 (s, 6H), 3.48-3.54 (m, 2H), 2.67-3.00 (m, 3H),
2.21-2.35 (m, 1H), 1.67-2.03 (m, 3H).
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 were
recorded 3-4 days after inoculation (12). There are three replicates for
each compound.
Data for 18: mp 241 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.06
(s, 2H), 7.66 (d, 3JHH = 7.5 Hz, 1H), 7.37 (s, 1H), 7.22 (s, 2H), 6.64-6.71
(m, 2H), 4.98-5.16 (m, 1H), 4.28-4.52 (m, 1H), 4.04 (s, 6H), 3.96 (s, 6H),
3.60-3.84 (m, 2H), 3.33-3.50 (m, 1H), 3.06-3.26 (m, 2H), 2.33-2.47
(m, 1H), 1.82-2.18 (m, 3H).
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
Data for 19: mp 235 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 11.0 (br,
2H), 8.32 (s, 1H), 8.04 (s, 1H), 8.05 (s, 1H), 7.35 (s, 1H), 7.20 (s, 1H), 4.93
(d, 2JHH = 15.0 Hz, 1H), 4.12-4.29 (m, 1H), 4.04 (s, 6H), 3.95 (s, 6H),
3.50-3.70 (m, 2H), 2.90-3.27 (m, 3H), 2.58-2.67 (m, 4H), 2.27-2.44 (m,
1H), 1.73-2.13 (m, 3H).