HETEROCYCLES

Paper | Regular issue | Vol. 83, No. 7, 2011, pp. 1603-1610
Received, 5th April, 2011, Accepted, 25th April, 2011, Published online, 11th May, 2011.
DOI: 10.3987/COM-11-12228

■ New Furanocoumarins from the Fruits of Melicope triphylla

Ken-ichi Nakashima, Masayoshi Oyama,* Tetsuro Ito, Hiroko Murata, and Munekazu Iinuma

Laboratory of Pharmacognosy, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan

Abstract

Four new furanocoumarins (1–4) were isolated from the fruits of Melicope triphylla (Rutaceae), together with two known coumarins (5, 6), nine flavonoids (7–15), two alkaloids (16, 17), and methyl p-geranyloxy-trans-cinnamate (18). The structures of the newly identified compounds were determined by extensive 1D- and 2D-NMR spectroscopic analyses to be linear-types of furanocoumarins bearing a hydroxyl or a hydroperoxy group on the geranyloxy side chain.

INTRODUCTION
Melicope triphylla (LAM.) MERR. is a rutaceous shrub that grows to 1.5–15 m high, and widely distributed throughout the Pacific islands including the Ryukyus in Japan.1 Continuous studies by Higa et al. have revealed the presence of polymethoxyflavonoids,2–4 furoquinoline alkaloids,5 and p-coumaric acid derivatives5 in the leaves of the plant. On the other hand, Wu et al. have worked on the root barks and found sesquiterpene lactones.6,7 However, no phytochemical studies of the fruits have been achieved. Herein we described on the structure elucidation of four new furanocoumarins, as well as fourteen known compounds, isolated from the fruits of M. triphylla.

RESULTS AND DISCUSSION
A new coumarin, named melicotriphyllin A (1), was obtained as yellow oil with absorption maxima at 312 and 270 nm in the UV spectrum. The HR-ESIMS gave an [M+Na]+ ion peak at m/z 407.1458, which established the quasi-molecular formula as C22H24O6Na (calc. 407.1465). The 1H-NMR spectrum showed two pairs of mutually coupled doublets at δH 6.26, 8.11 (1H each, d, J = 9.7 Hz) and δH 7.01, 7.63 (1H each, d, J = 2.4 Hz); the former protons were assignable to H-3 and H-4 in a coumarin skeleton, while the latter were regarded as H-2’ and H-3’ on a furan ring. A three-proton singlet resonated at δH 4.18 to be identified as a methoxy group that exhibited essential NOEs toward H-4 and H-3’ in the NOESY spectrum. Therefore, 1 was considered to be a linear furanocoumarin having a methoxy group at C-5.

The double quantum filtered COSY (DQF-COSY) spectrum inferred two segments [δH 4.86 (2H, d, J = 7.2 Hz), 5.60 (1H, br t, J = 7.2 Hz) for –OCH2CH=; δH 2.68 (2H, d, J = 6.6 Hz), 5.50 (1H, dt, J = 15.9, 6.6 Hz), 5.59 (1H, d, J = 15.9 Hz) for –CH2CH=CH–] and three methyl groups [δH 1.29 (6H, s, overlapping) and 1.64 (3H, s)]. The HMBC spectrum showed a side chain as –OCH2CH=C(CH3)CH2CH=CHC(CH3)2OH formed from a geranyl or a neryl group by the following correlations: H2-1”/C-3”; H-2”/C-4”, C-5”; H2-4”/C-7”; H-6”/C-3”, C-8”; and H-7”/C-9”(10”) (Figure 2). In the EIMS, the base ion peak at m/z 233 generated after the loss of C10H16O from the molecular ion also supported the presence of the side chain. In the NOESY spectrum, the key NOEs were observed between H2-1”/H3-5”, H-2”/H2-4” and H2-4”/H-7”, which indicated that the C10 side chain was originated from a geranyl group and the configurations were 2”E and 6”E. The C-8 substitution was demonstrated by a HMBC correlation between H2-1” (δH 4.86) and C-8 (δC 126.5). Thus, the structure of melicotriphyllin A was established as 1 (Figure 1).

Melicotriphyllin B (2), yellow oil, exhibited absorption bands at 313 and 269 nm in the UV spectrum. The quasi-molecular formula of 2 was confirmed to be C22H24O7Na (calc. 423.1414) based on the [M+Na]+ ion peak at m/z 423.1434 in the HR-ESIMS. This compound, then, bears one more oxygen atom than 1. The 1H-NMR spectrum of 2 was superimposed to that of 1, which implied that 2 was the similar furanocoumarin as 1 (Table 1). Comparison with both 13C-NMR spectral data, however, showed a significant downfield shift of the carbon signal at C-8” from δC 70.4 (1) to δC 82.0 (2) (Table 2), indicating that the hydroxyl group attaching to C-8” in 1 was converted into the hydroperoxy group in 2. The reduction of 2 with triphenylphosphine in MeOH at room temperature produced a compound identical to 1.8 Accordingly, the structure of melicotriphyllin B was determined as 2.

Melicotriphyllin C (3), obtained as optically inactive yellow oil, showed the same quasi-molecular formula for C22H24O6Na (calc. 407.1465) as 1 in the HR-ESIMS ([M+Na]+ at m/z 407.1488). The 1H- and 13C-NMR spectra had identifiable signals for a linear furanocoumarin framework (Tables 1 and 2). The location of a methoxy group was confirmed at C-5 by the NOESY correlations from MeO [δH 4.15 (3H, s)] to H-4 [δH 8.12 (1H, d, J = 9.7 Hz)] and H-3’ [δH 7.00 (1H, d, J = 2.4 Hz)]. Therefore, the structural difference between 1 and 3 was due to the geranyl-derived side chain. The 1H-NMR and DQF-COSY spectra exhibited a –OCH2CH= moiety [δH 4.87 (2H, m), 5.62 (1H, br t, J = 7.2 Hz)], a –CH2CH2CH(O)– moiety [δH 2.03 (2H, m), 1.58 (2H, m), 3.93 (1H, t, J = 6.8 Hz)], two methyl groups [δH 1.67, 1.71 (3H each, s)], and an olefinic methylene group [δH 4.94, 4.99 (1H each, br s)]. The side chain was established as –OCH2CH=C(CH3)CH2CH2CH(OH)C(CH3)=CH2 by the following HMBC correlations: H2-1”/C-3”; H-2”/C-4”, C-5”; H2-4”/C-7”; H2-6”/C-3”, C-8”; and H-7”/C-9”, C-10” (Figure 2). The configuration at C-2” was indicated to be E by the NOEs between H2-1”/H3-5” and H-2”/H2-4”. Consequently, the structure of melicotriphyllin C was confirmed as 3.
Melicotriphyllin D (4) was obtained as optically inactive yellow oil. The quasi-molecular formula was established as C22H24O7Na (calc. 423.1414) by an [M+Na]+ ion peak observed at m/z 423.1380 in the HR-ESIMS. Then, the structure relationship between 3 and 4 seemed identical to that between 1 and 2. The significant downfield shifts observed in the proton and carbon signals at C-7” from δH 3.93 and δC 75.1 (3) to δH 4.23 and δC 88.8 (4) demonstrated that a hydroperoxy group was substituted at C-7” in 4 instead of a hydroxyl group in 3. The idea was corroborated by the chemical conversion of 4 to 3 upon treatment with triphenylphosphine in the same manner as 2. Thus, the structure of melicotriphyllin D was determined to be 4. Since 3 and 4 were optically inactive, both were suggested to exist as racemic mixtures.
In addition, 8-geranyloxy-5-methoxypsoralen (5),9 phellopterin (6),10 5-demethylmelibentin (7),3,11 melibentin (8),11,12 melisimlexin (9),12,13 3,5,6,7,8,3’,4’-heptamethoxyflavone (10),14 3,5,8-trimethoxy- 6,7:3’,4’-bis(methylenedioxy)flavone (11),2,12 quercetin pentamethyl ether (12),3 gossypetin hexamethyl ether (13),3 4’-hydroxy-3,5,7,3’-tetramethoxyflavone (14),2 4’-hydroxy-3,5,7,8,3’-pentamethoxyflavone (15),3 kokusaginine (16),15 skimmianine (17),16 and methyl p-geranyloxy-trans-cinnamate (18)5 were isolated and identified by comparisons with the literatures.
All new compounds (1–4) were oxidative metabolites of 5 that was rarely found in natural sources and a characteristic component in the fruits of M. triphylla, although 6 was isolated from several rutaceous and apiaceous plants and common in every parts of this plant. And, geranyloxycoumarins possessing a hydroperoxy group such as 2 and 4 were yet discovered in genera of Phebalium17 and Clausena18 (Rutaceae). We also revealed that the fruits of M. triphylla abundantly contained 5, 8, 11, 17 and 18. Higa et al. have reported the presence of 18 in the leaves, however, the compound was apparently major in the fruits rather than the leaves based on our TLC analysis. From the chemotaxonomic standpoint of view,19–21 the genus Melicope may involve two chemical races depending on whether the primary components are polymethoxyflavones or prenyl acetophenones. Considering the previous and the present studies, M. triphylla would be a specimen classified into the polymethoxyflavone-rich race in the genus.

EXPERIMENTAL
General. 1H- and 13C-NMR spectra were measured on a JEOL JNM-AL-400 spectrometer equipped with a field gradient system (1H at 400 MHz and 13C at 100 MHz). Chemical shifts are given in δ values (ppm) relative to tetramethylsilane as an internal standard. EIMS (at 30 eV) and HR-ESIMS were obtained using a JEOL JMS-700T and a Shimadzu LCMS-IT-TOF spectrometers, respectively. UV spectra were recorded using a Shimadzu UV-3100 spectrophotometer (in MeOH solution). Optical rotations were recorded using a Jasco P-1020 polarimeter (in MeOH solution). Silica gel 60 (70–230 mesh, Merck) and Sephadex LH-20 (GE Healthcare) were used for column chromatography (CC). Vacuum liquid chromatography (VLC) was performed using silica gel 60H (Merck), while TLC analysis was performed using silica gel 60F254 (Merck) and silica gel RP-18F254S (Merck). Semi-preparative HPLC was performed on a Shimadzu LC-6AD liquid chromatography system equipped with a SCL-10A system controller, SIL-10A autoinjector, CTO-10A column oven, and SPD-10A UV-Vis detector, SPM-10Avp diode array detector, with the aid of Shiseido Capcell Pak C18 UG120 5 µm (10 mm i.d. x 250 mm).

Plant Material. The fruits of M. triphylla (1.7 Kg, dried weight) were collected at Ishigaki-jima in the Ryukyu Islands, Japan, in August 2009. A voucher specimen was deposited at Gifu Pharmaceutical University, Gifu, Japan.

Extraction and Isolation. The fruits were extracted with a 1:1 mixture of CHCl3 and MeOH at room temperature. The extract (220 g) was subjected to silica gel CC (Si CC) to afford 16 main fractions: Frs. 1, 2 (eluted with n-hexane–acetone, 20:1), Frs. 3, 4 (15:1), Fr. 5 (10:1), Fr. 6 (8:1), Frs. 7, 8 (6:1), Frs. 9, 10 (4:1), Frs. 11, 12 (2:1), Frs. 13, 14 (1:1), Frs. 15, 16 (0:1). Fr. 2 was separated by Si CC (n-hexane–EtOAc, 40:1 to 20:1) to yield 18 (746.3 mg). Fr. 4 was purified repetitively using Sephadex LH-20 CC (CHCl3–MeOH, 1:2), Sep-Pak tC18 cartridge (MeOH–H2O, 9:1), Si CC (CHCl3), and preparative HPLC (48% MeCN–H2O) to afford 2 (35.0 mg), 3 (12.9 mg), 4 (27.0 mg), and 5 (1.26 g). Fr. 5 was purified by Si CC (benzene–EtOAc, 7:2) and Sep-Pak tC18 cartridge (MeOH–H2O, 9:1) to yield 1 (23.4 mg), 6 (387.6 mg), and 7 (114.0 mg). Fr. 7 was dissolved in acetone to crystallize 8 (4.29 g). Fr. 9 was separated with the aid of Sephadex LH-20 CC (acetone), Sep-Pak tC18 cartridge (MeOH–H2O, 1:1), Si CC (c-hexane–EtOH, 5:1; benzene–EtOAc, 6:1), and VLC (benzene–acetone, 15:1) to provide 9 (143.4 mg), 10 (50.0 mg), and 16 (50.0 mg). Frs. 10 and 12 were dissolved in acetone to give 11 (1.57 g) and 17 (2.07 g) in crystal forms, respectively. Fr. 16 was separated by Si CC (CHCl3) and successive crystallization in EtOH to afford 12 (149.4 mg), 13 (26.2 mg), 14 (390.3 mg), and 15 (71.0 mg).

Melicotriphyllin A (4-methoxy-9-[[(2E,5E)-7-hydroxy-3,7-dimethyl-2,5-octadien-1-yl]oxy]-7H-furo- [3,2-g][1]benzopyran-7-one) (1): Yellow oil. UV (log ε, MeOH): 270 (4.19), 312 (4.00). 1H-NMR (CDCl3, 400 MHz) and 13C-NMR (CDCl3, 100 MHz): see Tables 1 and 2, respectively. HR-ESIMS (pos.): 407.1458 ([M+Na]+, C22H24O6Na+; calc. 407.1465). EIMS (%): 233 ([M+H-C10H16O]+, 100), 217 (94), 189 (43), 161 (21), 134 (54), 119 (46).

Melicotriphyllin B (4-methoxy-9-[[(2E,5E)-7-hydroperoxy-3,7-dimethyl-2,5-octadien-1-yl]oxy]-7H- furo[3,2-g][1]benzopyran-7-one) (2): Yellow oil. UV (log ε, MeOH): 269 (4.58), 313 (4.39). 1H-NMR (CDCl3, 400 MHz) and 13C-NMR (CDCl3, 100 MHz): see Tables 1 and 2, respectively. HR-ESIMS (pos.): 423.1434 ([M+Na]+, C22H24O7Na+; calc. 423.1414). EIMS (%): 232 ([M-C10H16O]+, 100), 217 (93), 189 (49), 161 (24), 135 (55), 134 (49), 119 (23).

Melicotriphyllin C (4-methoxy-9-[[(2E)-6-hydroxy-3,7-dimethyl-2,7-octadien-1-yl]oxy]-7H-furo- [3,2-g][1]benzopyran-7-one) (3): Yellow oil. [α]D 0° (c 0.1, MeOH). UV (log ε, MeOH): 271 (4.09), 313 (3.91). 1H-NMR (CDCl3, 400 MHz) and 13C-NMR (CDCl3, 100 MHz): see Tables 1 and 2, respectively. HR-ESIMS (pos.): 407.1488 ([M+Na]+, C22H24O6Na+; calc. 407.1465). EIMS (%): 232 ([M-C10H16O]+, 100), 217 (100), 189 (52), 161 (26).

Melicotriphyllin D (4-methoxy-9-[[(2E)-6-hydroperoxy-3,7-dimethyl-2,7-octadien-1-yl]oxy]-7H- furo[3,2-g][1]benzopyran-7-one) (4): Yellow oil. [α]D 0° (c 0.1, MeOH). UV (log ε, MeOH): 269 (4.11), 313 (3.92). 1H-NMR (CDCl3, 400 MHz) and 13C-NMR (CDCl3, 100 MHz): see Tables 1 and 2, respectively. HR-ESIMS (pos.): 423.1380 ([M+Na]+, C22H24O7Na+; calc. 423.1414). EIMS (%): 233 ([M+H-C10H16O]+, 100), 217 (100), 189 (60), 161 (28).

Reduction of 2 and 4 with triphenylphosphine. 2 (13.2 mg) was dissolved in MeOH (10 mL), and triphenylphosphine (13.3 mg) was added to the solution. The mixture was stirred for 4 hrs at room temperature, and then the solvent was evaporated off. The residue was purified by Sep-Pak tC18 cartridge (75% MeOH–H2O) to give 1 (7.6 mg). Equally, 4 (11.2 mg) and triphenylphosphine (11.3 mg) was treated as above. After purification, 3 (8.4 mg) was obtained.

References

1. T. G. Hartley, Allertonia, 2001, 8, 1.
2. M. Higa, Y. Miyagi, S. Yogi, and K. Hokama, Yakugaku Zasshi, 1987, 107, 954.
3. M. Higa, T. Ohshiro, K. Ogihara, and S. Yogi, Yakugaku Zasshi, 1990, 110, 822.
4. M. Higa, E. Nakadomari, M. Imamura, K. Ogihara, and T. Suzuka, Chem. Pharm. Bull., 2010, 58, 1339. CrossRef
5. M. Higa, S. Yogi, and K. Hokama, Bulletin of the College of Science, University of the Ryukyus, 1987, 43, 47.
6. T. S. Wu, T. T. Jong, W. M. Ju, A. T. McPhail, D. R. McPhail, and K. H. Lee, J. Chem. Soc., Chem. Commun., 1988, 956. CrossRef
7. T. T. Jong and T. S. Wu, J. Chem. Res. (S), 1989, 237.
8. K. Baba, K. Nakata, M. Taniguchi, T. Kido, and M. Kozawa, Phytochemistry, 1990, 29, 3907. CrossRef
9. K. Franke, A. Porzel, M. Masaoud, G. Adam, and J. Schmidt, Phytochemistry, 2001, 56, 611. CrossRef
10. O. Bergendorff, K. Dekermendjian, M. Nielsen, R. Shan, R. Witt, J. Ai, and O. Sterner, Phytochemistry, 1997, 44, 1121. CrossRef
11. E. Ritchie, W. C. Taylor, and S. T. K. Vautin, Aust. J. Chem., 1965, 18, 202.
12. T. T. Jong and T. S. Wu, Phytochemistry, 1989, 28, 245. CrossRef
13. L. H. Briggs and R. H. Locker, J. Chem. Soc., 1950, 2376. CrossRef
14. T. Kinoshita, Heterocycles, 1999, 50, 269. CrossRef
15. J. Pusset, J. L. Lopez, M. Pais, M. A. Neirabeyeh, and J. M. Veillon, Planta Med., 1991, 57, 153. CrossRef
16. A. Ahond, F. Picot, P. Potler, C. Poupat, and T. Sevenet, Phytochemistry, 1978, 17, 166. CrossRef
17. M. A. Rashid, A. I. Gray, and P. G. Watermann, J. Nat. Prod., 1992, 55, 851. CrossRef
18. C. Ito, S. Katsuno, and H. Furukawa, Chem. Pharm. Bull., 1998, 46, 341.
19. K. M. Ng, P. P. H. But, A. I. Gray, T. G. Hartley, Y. C. Kong, and P. G. Waterman, Biochem. Syst. Ecol., 1987, 15, 587. CrossRef
20. A. Adsersen, U. W. Smitt, H. T. Simonsen, S. B. Christensen, and J. W. Jaroszewski, Biochem. Syst. Ecol., 2007, 35, 447. CrossRef
21. A. A. Auzi, T. G. Hartley, and P. G. Waterman, Biochem. Syst. Ecol., 1997, 25, 611. CrossRef