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J. Nat. Prod. 2005, 68, 1071-1075
Phenanthroindolizidine Alkaloids from the Stems of Ficus septica
Amooru G. Damu,† Ping-Chung Kuo,†,| Li-Shian Shi,†,| Chia-Ying Li,† Chang-Sheng Kuoh,‡ Pei-Lin Wu,† andTian-Shung Wu*,†,§ Department of Chemistry, National Cheng Kung University, Tainan, Taiwan, Republic of China, Department of Life Science,National Cheng Kung University, Tainan, Taiwan, Republic of China, and National Research Institute of Chinese Medicine,Taipei, Taiwan, Republic of China In addition to six known phenanthroindolizidine alkaloids, eight new alkaloids, namely, ficuseptines B-D
(1-3), 10R,13aR-tylophorine N-oxide (4), 10R,13aR-tylocrebrine N-oxide (5), 10S,13aR-tylocrebrine N-oxide
(6), 10S,13aR-isotylocrebrine N-oxide (7), and 10S,13aS-isotylocrebrine N-oxide (8), were isolated from a
methanol extract of the stems of Ficus septica. The structures of the new compounds were elucidated by
means of spectroscopic data interpretation. Cytotoxicity of some of these alkaloids was assessed in vitro
using the HONE-1 and NUGC cell lines.
Ficus septica Burm. f. (Moraceae) is a subtropical tree, which occurs widely in low-altitude forests of Taiwan.1 This
species has been known for its detoxicant, purgative, and
emetic effects.1 The leaves of this plant have been used in
folk medicine to treat colds, fever, and fungal and bacterial
diseases.2-4 Several phenanthroindolizidine alkaloids, tri-
terpenoids, lignans, acetophenones, steroids, and long-
chain aliphatic compounds have been reported earlier from
the leaves and roots of F. septica.5-10 Members of the
phenanthroindolizidine alkaloid class are known to exhibit
pronounced cytotoxicity and antiamebic, antifungal, anti-
bacterial, and antiinflammatory activities and to also
inhibit enzymes involved in the synthesis of DNA and
proteins.7,11-25 In our ongoing investigations on cytotoxic
constituents from plants native to Taiwan, it was found
that the methanol extract of the stem of F. septica exhibited
potent cytotoxicity against the HONE-1 and NUGC cell
lines. Purification of the alkaloidal fractions of the metha-
nol extract by eluting over silica gel followed by HPLC
afforded eight new phenanthroindolizidine alkaloids (1-
8), along with six known analogues. In this paper, we report
the isolation and characterization of these new phenan-
throindolizidine alkaloids, which were found only as trace
constituents of F. septica stems.
Results and Discussion
consistent with the molecular formula C23H23NO4. Its UV Bioassay-guided fractionation and separation of metha- spectrum showed maxima at 255, 261, 281, 340, and 358 nolic extract of the stems of F. septica and subsequent nm, indicating a substituted phenanthrene ring system.28 HPLC purification gave eight new phenanthroindolizidine A set of ortho-coupled doublets (J ) 9.2 Hz) at δ 7.42 and alkaloids (1-8), together with six known compounds,
7.71 in 1H NMR spectrum was assigned to H-7 and H-8, tylophorine,26 tylocrebrine,5 isotylocrebrine,26 dehydroty- respectively, since the latter proton showed a NOE cor- lophorine,27 10S,13aR-tylophorine N-oxide,26 and 10S,- relation with H-9 at δ 4.58. The two aromatic singlets at 13aR-antofine N-oxide.16 Although a comparatively large δ 9.18 and 7.45 were attributed to para positions, H-4 and quantity of plant material was collected for this investiga- H-1, respectively, of ring A, based on the NOE interaction tion, these labile alkaloids were obtained in very low yields.
between H-1 and H-14 (δ 3.28). The 1H NMR spectrum Accordingly, it was not possible to obtain 13C NMR spec- of 1 also displayed signals corresponding to two methoxyl
troscopic data to aid in their structure elucidation in the groups at δ 3.88 (3H, s) and 4.01 (3H, s) and one set of methylenedioxy protons at δ 6.15 (2H, s). An intense Ficuseptine B (1), obtained as a colorless gum, showed
fragment ion peak was observed at m/z 308 in the EIMS, a molecular ion peak at m/z 377.1630 in its HREIMS, suggesting that these two methoxyls and the methylene-dioxy group are present in the phenanthrene moiety. The * To whom correspondence should be addressed. Tel: 886-6-2747538.
placement of one of the methoxyl groups on C-6 was Fax: 886-6-2740552. E-mail: tswu@mail.ncku.edu.tw.
supported by the NOE cross-peak between H-7 and a Department of Chemistry, National Cheng Kung University.
Department of Biotechnology, National Formosa methoxyl at δ 4.01. On the other hand, the methoxyl at δ University, Yunlin, Taiwan, Republic of China.
3.88 showed a NOE correlation between H-4 and OCH3-6, Department of Life Science, National Cheng Kung University.
§ National Research Institute of Chinese Medicine.
suggesting that it is attached to C-5. Accordingly, the 2005 American Chemical Society and American Society of Pharmacognosy Journal of Natural Products, 2005, Vol. 68, No. 7 Table 1. 1H NMR Data of Compounds 1-8
methylenedioxy group was placed at C-2 and C-3. The tions of H-1/H-14 (δ 3.35) and H-4/H-5. The stereochem- absolute stereochemistry of 1 was determined from the
istry of C-13a was determined as having the R configura- optical rotation and CD spectrum. Thus, phenanthroin- tion on the basis of the negative optical rotation value and dolizidine alkaloids with the R configuration at C-13a have a positive Cotton effect at 257 nm in the CD spectrum, and been found to exhibit a negative optical rotation and a thus H-13a was R oriented.29,30 Finally, the NOE between positive Cotton effect around 260 nm.29,30 Compound 1 was
H-13a and H-9R indicated the existence of a chair-like in agreement with this, as it showed a negative optical conformation for the piperidine ring.31 Thus, the structure rotation measured at the sodium-D line and a positive of 2 was elucidated as 6-methoxy-2,3-methylenedioxy-
Cotton effect at 259 nm in the CD spectrum. Thus, 1
13aR-phenanthroindolizidine, and it has been designated possesses the R configuration at C-13a. The presence of a NOE between H-13a and H-9R suggested that the piperi- Ficuseptine D (3) exhibited a molecular formula of
dine ring adopts a chair-like conformation.31 On the basis C23H25NO3, based on a molecular ion peak at m/z 363.1833 of the foregoing spectroscopic studies, the structure of in its HREIMS. Its UV absorption maxima at 252, 260, 280, compound 1 was established as 5,6-dimethoxy-2,3-meth-
342, and 351 nm suggested it to be a substituted phenan- ylenedioxy-13aR-phenanthroindolizidine, for which the threne derivative.28 The 1H NMR spectrum of 3 displayed
trivial name ficuseptine B was proposed, following a signals for three methoxyl groups at δ 3.92, 3.98, and 4.05 previous convention for these alkaloidal constituents of F. (each 3H, s). Since 3 showed a similar pattern of 1H NMR
signals due to the indolizidine moiety as observed for 2
Ficuseptine C (2) was obtained as colorless gum, and its
(Table 1), three methoxyl groups could be assigned in the UV absorption maxima were observed at 257, 282, and 340 phenanthrene moiety. This conclusion was supported by nm, very similar to those of trioxygenated phenanthrene an intense fragment ion peak at m/z 294 in the EIMS. The derivatives.28 It exhibited a molecular ion peak at m/z ortho-coupled doublets appearing at δ 7.49 and 7.83 (each 347.3513 in its HRFABMS, corresponding to the molecular 1H, d, J ) 8.4 Hz) were assigned to H-1 and H-2 of ring A, formula C22H21NO3, 30 amu less than that of 1. The
as a NOE correlation was observed between H-1 and H-14 observation of an intense fragment ion at m/z 278 in the (δ 3.32). A set of ABX pattern signals at δ 9.31 (1H, d, J ) EIMS due to a phenanthrene moiety suggested the pres- 2.8 Hz), 7.25 (1H, dd, J ) 7.2, 2.8 Hz), and 7.87 (1H, d, J ence of one methoxyl group and one methylenedioxy group, ) 7.2 Hz) were attributed to H-5, H-7, and H-8 of ring B instead of two methoxyl groups and one methylenedioxy on the basis of the NOEs of H-8 with H-9R (δ 4.58) and group as in 1. As expected, the 1H NMR spectrum also
H-7. The NOE correlation of the methoxyl protons at δ 3.92 displayed signals for methoxyl protons at δ 4.02 (3H, s) with H-7 and H-5 was used to determine the location of and methylenedioxy protons at δ 6.15 (2H, s). Since the the methoxyl group at C-6. The remaining two methoxyls proton signals of the B ring appeared as an ABX pattern at δ 3.98 and 4.05 were placed at C-3 and C-4, respectively, at δ 8.05 (1H, d, J ) 2.8 Hz, H-5), 7.86 (1H, d, J ) 8.8 Hz, as the former gave a NOE with H-2 and the latter with H-8), and 7.21 (1H, dd, J ) 8.8, 2.8 Hz, H-7), and NOEs of H-5. The negative optical rotation value and positive Cotton H-8/H-9R (δ 4.65), H-5/H-4 (δ 8.18) and the methoxyl effect at 259 nm in the CD spectrum of 3 supported the R
protons/H-5 and H-7 were observed, the methoxyl group configuration at C-13a.29,30 In addition, a NOE correlation was placed at C-6. The attachment of the methylenedioxy between H-13a and H-9R suggested a chair-like conforma- group at C-2 and C-3 was confirmed by the multiplicities tion for the piperidine ring.31 Accordingly, the structure of for H-1 and H-4 of ring A as two aromatic singlets at δ 3 (ficuseptine D) was concluded to be 3,4,6-trimethoxy-
7.43 (1H, s, H-1) and 8.18 (1H, s, H-4) and NOE correla- Phenanthroindolizidine Alkaloids from Ficus Journal of Natural Products, 2005, Vol. 68, No. 7 Alkaloid 4, obtained as yellow needles, was considered
OCH3-6; and H-8/H-7, H-9R. The positive CD curve at 261 to have the same molecular formula, C24H27NO5, as tylo- nm and a negative optical rotation value inferred the R phorine N-oxide, on the basis of its HREIMS, suggesting stereochemistry at C-13a and thus H-13a to be in an R it to be an isomer.15 The 1H NMR spectrum revealed the orientation.29,30 The resonance due to H-13a at δ 3.45 was presence of four methoxyl group signals at δ 4.02, 4.04 indicative of a trans-fused indolizidine ring junction.32,33 (each 3H, s) and 4.09 (6H, s). Since the UV spectroscopic pattern and 1H NMR signals and coupling patterns corre- (δ 3.65) by oxygen led to the assignment of the sponding to the phenanthrene moiety were similar to those configuration of the N-oxide group.16,33 Finally, the obser- of tylophorine N-oxide,15 the four methoxyl groups could vation of a NOE between H-13a and H-9R indicated that be located at C-2, C-3, C-6, and C-7. This was supported the piperidine ring adopted a chair-like conformation as by NOE cross-peaks between H-1/H-14R, H-14 , OCH3-2; in 4.31 Consequently, 6 was identified as 10S,13aR-tylo-
H-4/H-5, OCH3-3; OCH3-2/OCH3-3; OCH3-6/H-5, OCH3-7; and H-8/H-9R, H-9 , OCH3-7. Evidence for the presence of Alkaloid 7 was obtained as pale yellow needles. On the
an N-oxide unit was also suggested by the downfield shifts basis of the HREIMS data 7 was suggested to have the
of H-9R (δ 5.17), H-9 (δ 4.86), H-11R (δ 3.80), H-11 (δ same molecular formula, C24H27NO5, as isotylocrebrine 3.72), and H-13a (δ 3.94) and by the prominent [M - 16]+ N-oxide,15 indicating these alkaloids to be isomers. Accord- peak at m/z 393 in the mass spectrum of 4. The negative
ingly, two mutually coupled doublets (J ) 9.2 Hz) at δ 7.85 optical rotation measured at the sodium-D line and positive and 7.45 and two singlets at δ 9.30 and 6.98 in its 1H NMR Cotton effect at 256 nm in the CD spectrum for 4 inferred
spectrum were assigned to H-1, H-2, H-5, and H-8, respec- the C-13a R configuration.29,30 Thus, H-13a was oriented tively.15 Thus, four methoxyl groups at δ 4.02, 4.01, 3.92, in an R direction. The H-13a proton resonated at δ 3.94 and 3.99 could be placed, in turn, at C-3, C-4, C-6, and C-7.
and indicated a cis-fused ring junction of the indolizidine These assignments were confirmed by the correlations of moiety.32,33 Moreover, the strong deshielding of H-9R (δ H-1/H-14 ; H-2/OCH3-3; H-5/OCH3-4, OCH3-6; and H-8/ 5.17) and H-11R (δ 3.80) by oxygen supported the R OCH3-7, H-9R in a NOESY experiment. The positive Cotton configuration of the N-oxide group.16,33 The observation of effect at 279 nm in the CD spectrum of 7 and negative
NOE correlations between H-1 and H-14R, H-14 ; H-8 and optical rotation inferred the R configuration at C-13a.29,30 H-9R, H-9 ; H-9R and H-11R; and H-14R and H-13R in the Hence, H-13a was located with an R orientation. The NOESY spectrum suggested that the indolizidine moiety appearance of the H-13a signal at δ 3.51 led the ring adopted a boat-like conformation.31 Hence, 4 was charac-
junction configuration to be determined as trans32,33 for the terized as 10R,13aR-tylophorine N-oxide.
indolizidine unit. Moreover, the strong deshielding of H-9 Alkaloid 5 was assigned a molecular formula of C
δ 4.65) and H-11 (δ 3.68) by oxygen also inferred the configuration of the N-oxide group.16,33 The appearance of 5 from its HREIMS, one oxygen atom more than that of tylocrebrine, suggesting the presence of an N-oxide NOE cross-peaks between H-13a and H-9R was indicative functionality. 1H NMR multiplicities of the four aromatic of a chair-like conformation for the piperidine ring.31 The proton signals and four methoxyl groups similar to those structure of 7 was, therefore, defined as 10S,13aR-iso-
of tylocrebrine, and the fragment ion peak at m/z 324, due to the phenanthrene moiety in EIMS, allowed the four Alkaloid 8 was obtained as pale yellow needles. On the
methoxyl groups to be fixed at C-2, C-3, C-5, and C-6, basis of the molecular formula, C24H27NO5, as determined respectively. This was further supported by NOEs of H-1/ from the HREIMS, 8 was considered to be an isomer of 7.
H-14R, H-14 , OCH3-2; H-4/OCH3-3; OCH3-5, OCH3-6/H- Its UV spectrum showed absorptions at 245, 264, 285, 344, 7; and H-8/H-7, H-9R, H-9 . These assignments indicated and 360 nm and was practically superimposable on that that the extra oxygen must be present in the indolizidine of 7. In the 1H NMR spectrum, four methoxyls and four
moiety. Evidence for the presence of an N-oxide unit was aromatic proton signals were observed with the same also revealed by the significant [M - 16]+ ion peak at m/z multiplicities as those of 7, but differed in their indolizidine
393 and downfield shifts of H-9R (δ 5.13), H-9 (δ 4.81), protons (Table 1). The placement of four methoxyl groups H-11R (δ 3.72), H-11 (δ 3.65), and H-13a (δ 3.98) in the at C-3, C-4, C-6, and C-7 was determined from NOEs 1H NMR spectrum. The negative optical rotation value and between H-1/H-14 ; H-2/OCH3-3; H-5/OCH3-4, OCH3-6; positive Cotton effect at 275 nm in the CD spectrum and H-8/OCH3-7, H-9R. The positive specific rotation and established the R stereochemistry at C-13a, with H-13a in positive Cotton effect at 279 nm in the CD spectrum of 8
an R orientation.29,30 A strong deshielding of H-13a to δ confirmed the S configuration of C-13a16,26 and the 3.98 inferred a cis-fused ring junction of the indolizidine orientation of H-13a. The downfield shift of H-13a to δ 4.08 moiety.32,33 The R orientation of the N-oxide group was also inferred that the indolizidine ring junction should be in the indicated by a strong deshielding of H-9R and H-11R by cis32,33 configuration. Furthermore, the the oxygen atom.16,33 Similar NOE correlations between the N-oxide group was also inferred from a strong deshield- H-1 and H-14R, H-14 ; H-8 and H-9R, H-9 ; H-9R and H-11R; and H-14R and H-13R as in 4 suggested a boat-
appearance of NOE correlations between H-1 and H-14R, like conformation for the indolizidine moiety.31 Thus, 5 was
H-14 ; H-8 and H-9R, H-9 ; H-9R and H-11R; and H-14R concluded to be 10R,13aR-tylocrebrine N-oxide.
and H-13R in the NOESY spectrum indicated that the Alkaloid 6 was isolated as pale yellow needles. The
indolizidine moiety adopted a boat-like conformation.31 Therefore, it was concluded that the 8 is 10S,13aS-
IMS, was the same as that of 5, indicating these alkaloids
to be isomers. On the basis of the fact that 6 showed four
Compounds 6, 7, and tylophorine were tested in vitro
aromatic proton signals and four methoxyl signals in the for their cytotoxicity using HONE-1 and NUGC tumor cell 1H NMR spectrum similar to those of 5 (Table 1), the
lines.9,34 All three compounds exhibited strong cytotoxicity substituents were assigned to C-2, C-3, C-5, and C-6 as in against both HONE-1 and NUGC cell lines. The percent- 5. These assignments were supported by NOESY correla-
ages of inhibition observed for 6, 7, and tylophorine at 10
tions of H-1/H-14 , OCH3-2; H-4/OCH3-3, OCH3-5; H-7/ µM concentration against HONE-1 cell lines were 92%, Journal of Natural Products, 2005, Vol. 68, No. 7 87%, and 80%, respectively, whereas against NUGC cell 1711, 1683, 1511, 1467, 1424, 1396, 1286, 1010 cm-1; CD (c lines they exhibited 94%, 93%, and 85% inhibition, respec- tively, at the same concentration. Compound quantities MHz), see Table 1; EIMS m/z 377 [M]+ (43), 308 (100), 293 available did not permit the more formal determination of (34), 277 (15), 129 (10), 96 (13), 69 (20); HREIMS m/z 377.1630([M]+) (calcd for C Ficuseptine C (2): colorless gum; [R] 25 -
Experimental Section
MeOH); UV (MeOH) λmax (log ) 257 (3.46), 282 (3.42), 340(2.68) nm; IR (KBr) νmax 2925, 2853, 2375, 2311, 1715, 1710, General Experimental Procedures. Optical rotations
1683, 1548, 1514, 1454, 1412, 1391, 1371, 1212, 1035 cm-1; were measured using a JASCO DIP-370 digital polarimeter.
CD (c 1.09 × 10-4, MeOH) [θ] Circular dichroism (CD) and UV spectra were recorded at room 400 MHz), see Table 1; EIMS m/z 347 [M]+ (31), 278 (100), temperature on a JASCO J-700 spectropolarimeter and on a 264 (10), 248 (29), 175 (11), 153 (10), 129 (20), 95 (11), 69 (65); Hitachi UV-3210 spectrophotometer, respectively. IR spectra HREIMS m/z 347.3513 ([M]+) (calcd for C22H21NO3, 347.3515).
were obtained with a Shimadzu FT-IR DR-8011 spectropho- Ficuseptine D (3): colorless gum; [R] 25 -
tometer. NMR spectra were recorded on Bruker AMX-400, MeOH); UV (MeOH) λmax (log ) 252 (sh) (3.58), 260 (3.62), 280 AVANCE-300, and Varian Unity Plus 400 spectrometers.
(2.68), 342 (2.61), 351 (3.21) nm; IR (KBr) νmax 2923, 1679, Chemical shifts are shown in δ values (ppm) with tetrameth- 1668, 1659, 1600, 1514, 1455, 1410, 1398, 1282, 1010 cm-1; ylsilane as an internal standard. Mass spectra were measured CD (c 1.40 × 10-5, MeOH) [θ]259 3177; 1H NMR (acetone-d6, on a VG-70-250S spectrometer with EI or FAB ionization 400 MHz), see Table 1; EIMS m/z 363 [M]+ (40), 294 (100), (positive-ion mode). Column chromatography was performed 279 (18), 236 (10), 129 (11), 97 (15), 69 (21); HREIMS m/z363.1833 ([M]+) (calcd for C on silica gel (70-230 mesh, 230-400 mesh). Fractions were 10R,13aR-Tylophorine N-oxide (4): pale yellow needles;
monitored by TLC (Merck precoated Si gel 60 F254 plates), mp 210-220 °C (dec); [R] 25 -77.7° (c 0.019, MeOH); UV using UV light and Dragendorff’s reagent to visualize the (MeOH) λmax (log ) 241 (4.09), 258 (4.13), 287 (3.84), 303 (3.56), spots. High-performance liquid chromatography (HPLC) was 340 (2.52) nm; IR (KBr) νmax 2925, 1741, 1691, 1647, 1515, performed on a Shimadzu LC-10ATVP series pumping system 1463, 1427, 1315, 1257, 1153, 1039 cm-1; CD (c 4.55 × 10-5, equipped with a Shimadzu SPD-6AV UV-vis spectrophoto- metric detector at 275 nm, a Cosmosil packed column with 1; EIMS m/z 409 [M]+ (5), 393 (20), 392 (8), 337 (74), 319 (54), 5C18-AR-II Waters type (4.6 × 250 mm, 5 µm) and a 176 (32), 154 (100), 136 (76), 86 (20); HRFABMS m/z 410.4748 Lichrospher 100 RP-8 column (4.6 × 250 mm, 5 µm), and a ([M + H]+) (calcd for C24H28NO5, 410.4749).
10R,13aR-Tylocrebrine N-oxide (5): pale yellow needles;
Plant Material. The stems of F. septica were collected in
Tainan Hsien, Taiwan, Republic of China, in January 2000 (MeOH) λmax (log ) 244 (4.14), 265 (4.29), 276 (3.71), 310 (3.46) and were authenticated by Prof. C. S. Kuoh, Department of 320 (3.41) nm; IR (KBr) νmax 2941, 1741, 1706, 1562, 1515, Life Science, National Cheng Kung University. A voucher 1477, 1463, 1427, 1398, 1365, 1284, 1253, 1114, 1068, 1026 specimen (Wu 200000053) has been deposited in the Her- cm-1; CD (c 6.36 × 10-5, MeOH) [θ] barium of National Cheng Kung University, Tainan, Taiwan.
OD, 400 MHz), see Table 1; EIMS m/z 409 [M]+ (8), 393 (48), Extraction and Isolation. Dried and powdered stem of
392 (24), 337 (100), 324 (99), 176 (9), 154 (21), 86 (10); F. septica (46.5 kg) was refluxed with methanol (7 × 10 L) HRFABMS m/z 410.3916 ([M + H]+) (calcd for C24H28NO5, and filtered. A large amount of precipitate, asparagine (260 g),35 was formed during this process and filtered. The filtrate 10S,13aR-Tylocrebrine N-oxide (6): pale yellow needles;
was concentrated and suspended in water. Then the water solubles were partitioned with chloroform and n-butanol, (MeOH) λmax (log ) 245 (3.14), 264 (4.39), 279 (3.91), 285 (3.82), successively. The concentrated chloroform extract (80 g) was 307 (3.41), 319 (3.38) nm; IR (KBr) νmax 2937, 1741, 1606, 1515, subjected to open column chromatography over silica gel by 1463, 1396, 1286, 1255, 1211, 1114, 1035, 1026 cm-1; CD (c eluting with a stepwise gradient from 5% to 75% methanol in chloroform to afford 12 fractions. Fractions were monitored see Table 1; EIMS m/z 409 [M]+ (24), 393 (80), 337 (10), 324 by TLC using Dragendorff’s reagent to visualize the spots and (100), 154 (61), 136 (46), 86 (14); HRFABMS m/z 410.4416 tested for their cytotoxicity. Fractions 5-9 were considered to ([M + H]+) (calcd for C24H28NO5, 410.4413).
be active fractions, since they showed cytotoxicity against the 10S,13aR-Isotylocrebrine N-oxide (7): pale yellow needles;
HONE-1 and NUGC cell lines with the inhibition percentage values of 93% and 97%, 80% and 90%, 79% and 92%, 75% and (MeOH) λmax (log ) 245 (3.24), 263 (5.39), 283 (3.12), 287 (3.08), 84%, and 85% and 96%, respectively at 250 µM concentration.
309 (2.41) 344 (2.24), 359 (2.19) nm; IR (KBr) νmax 2943, 2842, Fraction 5 on repeated column chromatography over silica gel 1691, 1635, 1521, 1458, 1413, 1267, 1114, 1028 cm-1; CD (c and further purification by HPLC using 1 mL/min of MeOH- H2O-Et2NH (75:24.5:0.5) gave compounds 1 (1.0 mg, 0.0012%),
see Table 1; EIMS m/z 409 [M]+ (14), 393 (80), 324 (100), 307 2 (1.0 mg, 0.0012%), 3 (1.0 mg, 0.0012%), 4 (7.0 mg, 0.0087%),
(10),176 (25), 154 (61), 136 (46), 86 (14); HRFABMS m/z tylophorine (18.0 mg, 0.022%), tylocrebrine (1.0 mg, 0.0012%), 410.4961 ([M + H]+) (calcd for C24H28NO5, 410.4963).
isotylocrebrine (1.1 mg, 0.0013%), dehydrotylophorine (0.5 mg, 10S,13aS-Isotylocrebrine N-oxide (8): pale yellow needles;
0.00062%), and 10S,13aR-tylophorine N-oxide (1.5 mg, 0.0018%).
Similarly, repeated column chromatography and HPLC puri- (MeOH) λmax (log ) 245 (3.21), 264 (5.41), 285 (3.17), 305 (2.89) fication by using 1 mL/min of MeCN-H2O-Et2NH (20:79.5: 344 (2.21), 360 (2.14) nm; IR (KBr) νmax 2941, 2851, 1696, 1645, 0.5) of fraction 7 yielded compounds 6 (1.0 mg, 0.0012%) and
1562, 1463, 1255, 1114, 1024 cm-1; CD (c 5.01 × 10-5, MeOH) 8 (0.5 mg, 0.00062%). Fraction 8 was subjected to repeated
1128; 1H NMR (CD3OD, 400 MHz), see Table 1; EIMS column chromatography separation and then HPLC purifica- m/z 409 [M]+ (17), 393 (30), 324 (74), 307 (25),176 (12), 154 tion using 1 mL/min of MeCN-H2O-Et2NH (25:75:0.5) to (100), 136 (74), 118 (66); HRFABMS m/z 410.3446 ([M + H]+) obtain compounds 4 (1.9 mg, 0.0023%), 5 (2.6 mg, 0.0032%), 6
(2.6 mg, 0.0032%), 7 (5.9 mg, 0.0073%), 8 (2.5 mg, 0.0031%),
Cytotoxicity Assay. Two human cancer cell lines, NUGC
and 10S,13aR-antofine N-oxide (2.5 mg, 0.0031%). An attempt (gastric adenocarcinoma) and HONE-1 (nasopharyngeal car- to work up cytotoxic fractions 6 and 9 did not lead to the cinoma), were seeded in 96-well microliter plates at a density isolation of any additional alkaloids due to the lability of these of 6000/well in 10 µL of culture medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and Ficuseptine B (1): colorless gum; [R] 25 -
nonessential amino acid) and maintained at 37 °C in a MeOH); UV (MeOH) λmax (log ) 255 (3.80), 261 (3.91), 281 humidified incubator with 5% CO2. After an overnight adapta- (2.59), 340 (3.24), 358 (3.18) nm; IR (KBr) νmax 2924, 1745, tion period, test compounds (final concentration, 50 µg/mL) Phenanthroindolizidine Alkaloids from Ficus Journal of Natural Products, 2005, Vol. 68, No. 7 in serum-free medium were added to individual wells. Cells (14) Tanner, U.; Wiegrebe, W. Arch. Pharm. (Weinheim, Ger.) 1993, 326,
were treated with test compounds for 3 days. Cell viability (15) Abe, F.; Hirokawa, M.; Yamauchi, T.; Honda, K.; Hayashi, N.; Ishii, was determined by the 5-(3-carboxymethoxyphenyl)-2-(4,5- M.; Imagawa, S.; Iwahana, M. Chem. Pharm. Bull. 1998, 46, 767-
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