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Sesquiterpene lactones from Smyrnium olusatrum
Phytochemistry 57 (2001) 1197–1200 www.elsevier.com/locate/phytochem
Sesquiterpene lactones from Smyrnium olusatrum Ali A. El-Gamal* Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt Received in revised form 12 January 2001
Abstract Fruits of Smyrnium olusatrum afforded three sesquiterpene lactones, namely, 1b,8b-dihydroxyeudesman-3,7(11)-dien-8a,12-olide, 1b,8b-dihydroxyeudesman-4(15),7(11)-dien-8a,l2-olide, and 1b,10a,4a,5b-diepoxy-6b-hydroxyglechoman-8a,12-olide. Four related known sesquiterpenes were also isolated and characterized. The structure elucidation of the isolated compounds was based primarily on 1D and 2D NMR analyses. The in vitro cytotoxicity of the extract and isolated compounds against P-388 mouse lymphoma cells will be also discussed. # 2001 Published by Elsevier Science Ltd. Keywords: Smyrnium olusatrum; Apiaceae; Sesquiterpenes; Eudesmen-8a,12-olide; Glechomanolides; Cytotoxic activity
1. Introduction Several sesquiterpene lactones, furanosesquiterpenes and oxepines have been isolated from members of the genus Smyrnium (Ulubelen et al., 1971, 1979, 1984, 1985; Bohlmann and Zdero, 1973; Ulubelen and Abdolmaleky, 1982; Goren et al., 1984; Ulubelen and Oksuz, 1984; Goren and Ulubelen, 1985, 1987a,b; Ulubelen and Goren, 1986; Molleken et al., 1998). The young shoots of Smyrnium olusatrum are cultivated and used as pot herb and edible salad in some areas in Libya. The plant is also used as antiscorbutic in Palestine (Jafri and El-Gadi, 1985). This study reports the isolation of three new sesquiterpene lactones 1, 2 and 5 in addition to four known ones 3, 4, 6 and 7 from the fruits of S. olusatrum collected from the Libyan desert. The cytotoxicity of the CH2Cl2 extract and the isolated compounds is also reported.
2. Results and discussion Repeated chromatography of the cytotoxic CH2Cl2 extract of the fruits of S. alusatrum on normal and C18 reversed phase silica gel afforded the new sesquiterpene lactones 1, 2 and 5, in addition to the four known sesquiterpenes 1b-acetoxyeudesman-4(15),7(11)-dien-8a,12-olide (3), 1b acetoxy, 8b-hydroxyeudesman-4(15),7(11)-dien* Tel.: +20-50-347496; fax: +20-50-347496. E-mail address: [email protected] (A.A. El-Gamal).
8a,12-olide (4) (Ulubelen et al, 1985), 1b,10a, 4a,5b diepoxyglechoman-8a,12-olide (6), previously reported as a semi-synthetic derivative (Stahl and Datta, 1972), and 1b,10a, 4a,5b-diepoxy-8a hydroxy-glechoman8a,12-olide (7) (Ulubelen et al., 1985). The EIMS of 1 displayed a molecular ion peak [M]+ at m/z 264, suggesting the molecular formula C15H20O4 and six degrees of unsaturation. The IR spectrum (KBr) of 1 showed an intense broad absorption band around 3400 cm 1, for hydroxyl groups, 1762 cm 1, for a,bunsaturated lactone functionality. The 13C and 1H NMR spectra of 1 (Table 1) were in agreement with a dihydroxy C-8,12 eudesmanolide skeleton for 1 (Ulubelen et al., 1985). The oxygenated doublet of doublets resonating at 3.78 (J=7.5 and 7.5) (Table 1), which correlated with the methine carbon resonating at 75.2, was assigned to H-1. This was based on the observed 3JHMBC coupling between C-1 and the methyl proton singlet resonating at 1.59 (H3-14) (Fig. 1). This was further supported by the observed COSY couplings between H-1 and both proton signals resonating at 2.45 and 2.30 (H2-2). The high J1,2 values suggested the a-configuration of H-1 and indicated its axial orientation (Ulubelen et al., 1985). Both H2-2 also displayed COSY couplings with the broad olefinic singlet resonating at 5.40 (H-3) which in turn displayed a 3J-HMBC coupling with the methyl carbon at 21.0 (C-15) (Fig. 1). The downfield quaternary carbon resonating at 105.1 was assigned to C-8. This was suggested by its 3JHMBC couplings with both proton signals resonating at 2.89 and 2.56 (H2-6). The carbonyl ester C-12 (C
0031-9422/01/$ - see front matter # 2001 Published by Elsevier Science Ltd. PII: S0031-9422(01)00211-4
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Table 1 13 C and 1H NMR spectral data of compounds 1a, 2a and 5b Position
1
2
C
H
1 2
75.2, d 33.4, t
3
122.1, d
4 5 6
133.5, s 49.2, d 24.4, t
7 8 9
162.1, s 105.1, s 47.9, t
10 11 12 13 14 15
39.5, 121.6, 172.7, 8.2, 10.5, 21.0,
1-OH 6-OH 8-OH
– – –
s s s q q q
3.78, 2.45, 2.30, 5.40, – 2.10, 2.89, 2.56, – – 3.33, 1.75, – – – 1.87, 1.59, 1.73,
C dd (7.5, 7.5) m m brs
brd (13.3) dd (13.1, 3.1) brt (13.2)
d (13.4) d (13.1)
3H, s 3H, s 3H, s
6.32, brs – 9.42, s
H
78.1, d 31.7, t 34.3, t 148.4, s 50.0, d 24.6, t 161.8, s 104.6, s 49.0, t 42.1, 121.5, 172.7, 8.2, 11.3, 107.6, – – –
s s s q q t
3.60, 1.91, 1.78, 2.35, 2.17, – 1.95, 2.75, – – 3.34, 1.81, – – – 1.83, 1.49, 4.96, 4.77, U – U
m m m m ddd (13.8, 5.2, 1.2) m m
d (13.4) d (13.4)
3H, s 3H, s d (1.2) d (1.2)
2c
5b
H
C
3.35, dd (11.5, 4.4) 1.70, m 1.60, brdd (12.5, 4.4) 2.33, ddd (11.5, 5.0, 3.0) 2.00, brdd (13.5, 5.2) – 2.1, brdd (13.5, 5.2) 2.62, dd (13.2, 3.4) 2.45, btr (13.2) – – 2.74, d (13.8) 1.50, d (13.8) – – – 1.80, 3H, s 1.00, 3H, s 4.89, s 4.56, s
H
69.0, d 22.8, t 37.1, t 60.0, s 65.7, d 68.8, d 156.6, s 78.6, d 38.7, t 57.8, 129.6, 172.6, 10.2, 17.4, 17.4, – – –
s s s q q q
2.74, 2.03, 1.40, 2.33, 1.38, – 3.19, 4.69, – 5.44, 2.97, 1.84, – – – 1.92, 1.08, 1.25,
brd (10.3) dd (13.9, 3.2) dd (13.9, 10.6, 3.2) m m d (6.5) dd (6.5, 2.9)
dd (5.4, 1.8) dd (15.9, 1.8) dd (15.9, 5.4)
3H, d (1.9)d 3H, s 3H, s
– 2.68, d (2.9) –
a In pyridine-d5, 400 MHz for 1H and 100 MHz for 13C NMR. Carbon multiplicities were determined by DEPT135 experiments. s=Quaternary, d=methine, t=methylene, q=methyl carbons, coupling constants (J) are in Hz, U=unobserved. b In CDCl3, 500 MHz for 1H and 125 MHz for 13C NMR. c In CDCl3, 400 MHz. d Long range with proton number 6 (Ulubelen and Goren, 1986).
Fig. 1. Important HMBC (solid lines) and NOESY (dashed lines) correlations of 1.
172.7) displayed a 3J-HMBC coupling with the proton methyl singlet resonating at 1.87 (H3-13). The assignment of the relative stereochemistry of 1 was further supported by NOESY data (Fig. 1) and the study of Drieding model. Thus, the b-oriented C-14 methyl singlet displayed NOESY correlations with both exchangeable proton singlets at 6.32 (C-1 OH) and 9.42 (C-8 OH), confirming their b-orientation. Likewise, the a-oriented H-1 was NOESY-correlated with the broad doublet resonating at 2.10 (H-5), suggesting their location on the same face of 1. Compound 1 was, therefore, identified as the new sesquiterpene 1b,8bdihydroxyeudesm - 3,7(11) - dien - 8a,12 - olide (1). It is
worth noting that the C-1 acetoxy derivative of 1 was previously reported from Smyrnium galaticum (Ulubelen and Goren, 1986). The EIMS of 2 displayed a molecular ion peak [M]+ at m/z 264, suggesting the same molecular formula of 1 (C15H20O4). The IR spectrum (KBr) of 2 showed a similar pattern to those of 1 with an additional absorption band at 890 cm 1, characteristic for an exocyclic methylene group. The 13C and 1H NMR spectral data of 2 (Table 1) were in agreement with those of 1 with the replacement of the 3,4 with 4,15 system. The two olefinic proton doublets resonating at 4.96 and 4.77 which correlated with the methylene carbon at 107.6 were assigned H2-15. Both protons displayed 3J-HMBC couplings with C-5 ( 50.0) and C-3 ( 34.3) and 2JHMBC coupling with the quaternary olefinic carbon at 148.8 (C-4). Therefore, 2 is the deacetylated derivative of compound 4. In fact, comparing the 1H NMR data of 2 in CDCl3 with those of 4 (Table 1) indicate that having significant difference of H1 proton being 3.35 (dd, J=11.5, 4.4 Hz) in 2 while it is 4.57 (dd, J=11.6, 4.7 Hz) in 4. Hence, compound 2 was proved to be 1b,8b-dihydroxyeudesm-4(15),7(11)-dien-8a,12-olide. The EIMS of 5 displayed a molecular ion peak [M]+ at m/z 280, suggesting the molecular formula C15H20O5 and six degrees of unsaturation. The IR spectrum (KBr)
A.A. El-Gamal / Phytochemistry 57 (2001) 1197–1200
of 5 showed an absorption band at 3637 cm 1 for hydroxyl group and 1756 cm 1 for a,b-unsaturated lactone functionality. The 13C and 1H NMR spectra of 5 (Table 1) suggested a monohydroxy C-8, 12 germacranolide skeleton for 5 (Stahl and Datta, 1972; Ulubelen et al., 1985). The oxygenated doublet of doublets resonating at 4.69 (J=6.5 and 2.9) (Table 1), which correlated with the methine carbon resonating at 68.8, was assigned to H-6. This was based on the observed COSY coupling between H-6 and the proton doublet resonating at 3.19 (H-5) and the exchangeable proton doublet at 2.68 (C-6 OH). The proton H-6 displayed 3 J-HMBC coupling with C-4 ( 60.0) and C-11 ( 129.6). A 2J-HMBC coupling was also observed between H-6 and C-7 ( 156.6). The proton doublet of doublets resonating at 5.44 which correlated with the oxygenated methine carbon at 78.6 was assigned H-8. This was based on its COSY coupling with the doublet of doublets resonating at 2.97 and 1.84 (H2-9). The methylene H2-9 displayed a 3J-HMBC coupling with C-14 methyl singlet (C 17.4). Once again, the assignments of the relative stereochemistry of 5 were based on NOESY data. Thus, the boriented C-14 methyl singlet displayed NOESY correlation with the proton doublet of doublets at 2.97 (H9b) which in turn showed cross peak with H-8, confirming their b-orientation. The exchangeable proton doublet at 2.68 showed NOESY correlation to the methyl singlet at 1.25 (H3-15), suggesting their borientation. Likewise, the a-oriented H-1 was NOESYcorrelated with the proton doublet resonating at 3.19 (H-5), suggesting their location on the same lower face of 5. Compound 5 was therefore identified as the new germacranolide 1b, 10a, 4a, 5b - diepoxy - 6b - hydroxyglechoman-8a,12-olide. The CH2Cl2 extract of the fruits of S. olusatrum exhibited significant in vitro cytotoxicity (IC50 9.0 mg ml 1) against P-388 mouse lymphoma cells. (Carmichael et al., 1987; Twentyman and Luscombe, 1987). However, the isolated compounds (1, 2, 3, 4, 6 and 7) showed marginal activity with IC50 60, 65, 42, 58, 94 and 88 mg ml 1, respectively.
1199
3. Experimental 3.1. General Mps: uncorrected, Yanagimoto MP-3 micro mp apparatus. 1H and 13C NMR: CDCl3 or pyridine with TMS as int. standard, Bruker AM 400 and Bruker AM 500 2D NMR (COSY, NOESY, HMQC and HMBC), EI–MS: 70 eV, Hitachi M-80 or VG Autospec. IR: KBr on a Jasco A-302 or Perkin Elmer 1710. UV: CH3OH, Hitachi 557. Prep. HPLC: Rp-18 column (10 mm, 302.15 cm i.d.). MPLC Rp-18 column (20 mm, 10 2.15 cm i.d.). Kusano Scientific Co. CC: Kieselgel 60, anal. TLC: silica gel 60F254 and RP-18 F254 (0.25 mm) precoated plates. Spots detection: UV light at 254 and/or spraying with 10% H2SO4. Cytoxicity bioassay: Tosoh MPR-A4i microplate reader. 3.2. Plant material The fruits of Smyrnium olusatrum were collected from the moist and shady places of the Gebel Akhdar area in Libya in 1996. The plant was identified by Professor Dr. Elgady, Faculty of Science, El-Faateh University, Tripoli, Libya. A voucher specimen is deposited in the Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. 3.3. Extraction and isolation Air dried powdered fruits (750 g) were extracted to exhaustion by hot 70% methyl alcohol. The methanolic extract was concentrated under reduced pressure to give about 100 g of the total extract (IC50=10.2 mg ml 1 to P-388 cell line). The extract was treated with a minimum volume of alcohol diluted with water and successively partitioned with pet. ether, CH2Cl2 and n-butanol. The CH2Cl2 extract (13 g, IC50 =9 mg ml 1 to P-388 cell line) was applied to the top of silica gel packed column (390 g) and eluted with pet. ether—EtOAc gradient. Similar fractions were combined and subjected either to direct crystallization or to further chromatographic purifica-
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tion on normal and C-18 reversed phase silica (using MPLC and HPLC). The CH2Cl2 extract afforded four main fractions, fractions 13–15, fraction 20, fractions 26–29 and fractions 30–35. Direct crystallization of fractions 13–15 and 20 gave compounds 3 (70 mg) and 4 (80 mg) respectively. Fractions 26–29 were further purified by using MPLC with H2O:CH3CN gradient to give compound 7 (60 mg). Fractions 30–35 were applied to the top of a silica gel packed column using CH2Cl2: MeOH gradient to give three main subfractions. Subfractions 6–8 afforded compound 6 (40 mg) on direct crystallization from MeOH while subfractions 1316 and 19–22 were further purified using HPLC on Rp18 column with CH3CN:H2O (20:80 and 30:70). Fractions 13–16 gave compound 7 (30 mg) while fractions 19–22 gave compounds 1, 2 and 5 (20, 30 and 3 mg respectively). 3.3.1. 1 ,8 -Dihydroxy eudesman 3,7(11)-dien 8 ,12 olide (1) White needle crystals from MeOH; mp 193–195 C, UV lmax MeOH, 215 IR v KBr 3400, 2976, 2933, 1762, 1898, 1471, 1353, 1323, 1241, 1042, 1007, 946. EI–MS m/z (rel. int.): 264 [M]+ (52) (calc. for C15H20O4), 246 [M+–H2O] (50), 228 (40), 213 (27), 194 (21), 166 (52), 126 (100), 121 (45), 105 (28), 91 (38), 69 (59), 53 (37). 1H NMR (400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-d5) Table 1. 3.3.2. 1 ,8 -Dihydroxy eudesman 4(15),7(11)-dien 8 ,12 olide (2) White prism crystals from MeOH; mp 178–180 C. UV lmax nm: 218 IR v KBr cm 1 3410, 2950, 1758, 1660, 1470, 1386, 1347, 890, 861. EI–MS m/z (rel. int.): 264 [M]+ (100) (calc. for C15H20O4), 246 [M–H2O]+ (55) 236 (75), 218 (38), 203 (47), 189 (37), 175 (52), 163 (81), 145 (56), 105 (47), 91 (60), 55 (62). 1 H NMR (400 MHz, pyridine-d5, CDCl3) and 13C NMR (100 MHz, pyridine -d5) Table 1. 3.3.3. 1 ,10 ;4 ,5 -Diepoxy 6- -hydroxy-glechoman8 , 12 olide (5) White needle crystals (3 mg) from MeOH; mp 165– 168 C. UV lmax nm: 217. IR v CHCl3 cm 1, 3637, 2906, 1756, 1034, 853, 765, 723. EI–MS m/z (rel. int.): 280 [M]+ (12) (calc. for C15H20O5), 262 [M–H2O]+(50), 251 (7), 233 (13), 205 (11), 181 (31), 150 (26), 126 (52), 111 (35), 97 (63), 83 (37), 69 (100), 53 (37). 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) Table 1.
Acknowledgements The author would like to thank Professors H. Itokawa and K. Takeya, Tokyo University of Pharmacy and Life Science, for the NMR analyses and Professor H. Abd El Fattah, Faculty of Pharmacy, Mansoura University, Egypt, for providing the plant material.
References Bohlmann, F., Zdero, C., 1973. Polyacetylenvenbindungen, 220. U¨ber die Inhaltsstoffe von Smyrnium olusatrum L. (Polyacetylenic compounds 220 on the constituents of Smyrnium olusatrum L. Chem. Ber. 106, 3614–3620. Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D., Mitchell, B., 1987. Evaluation of a tetrazolium-based semiautomated colorimeter assay: assessment of chemosensitivity testing. Cancer Res. 47 (4), 936–942. Goren, N., Ulubelen, A., 1985. Oxepine derivatives from the roots of Smyrnium rotundifolium. Phytochemistry 24 (12), 3051–3052. Goren, N., Ulubelen, A., 1987a. Glechomanolides and eudesmanolides from Smyrnium perfoliatum. Phytochemistry 26 (9), 2585–2587. Goren, N., Ulubelen, A., 1987b. Sesquiterpene lactones from the fruits of Smyrnium connatum. Phytochemistry 26 (9), 2633–2634. Goren, N., Ulubelen, A., Jakupovic, J., Bohlmann, F., Grenz, M., 1984. Sesquiterpene lactones from the fruits of Smyrnium rotundifolium. Phytochemistry 23 (10), 2281–2284. Jafri, S.M.H., El-Gadi, A., 1985. Flora of Libya. Al-Faateh University, Faculty of Science Publication Tripoli, 117, p. 30. Molleken, U., Sinnwell, V., Kubeczka, K.-H., 1998. The essential oil composition of the fruits from Smyrnium perfoliatum. Phytochemistry 47 (6), 1079–1083. Stahl, E., Datta, S.N., 1972. Neue sesquiterpenoide Inhaltsstoffe der Gundelrebe (Glechoma hederacea L). (New sesquiterpenoides of ground ivy (Glechoma hederacea L.). Liebigs Ann. Chem. 757, 23–32. Twentyman, P.R., Luscombe, M., 1987. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br. J. Cancer 56 (3), 279–285. Ulubelen, A., Abdolmaleky, H., 1982. Istanbulin D and istanbulin E, two new sesquiterpene lactones from Smyrnium creticum. Phytochemistry 21 (8), 2128–2129. Ulubelen, A., Ates, N., Nishida, T., 1979. Istanbulin C, a novel sesquiterpene lactone from Smyrnium connatum. Phytochemistry 18, 338. Ulubelen, A., Goren, N., 1986. Additional sesquiterpenoids from the fruits of Smyrnium galaticum. J. Nat. Prod. 49 (6), 1104–1105. Ulubelen, A., Goren, N., Bohlmann, F., Jakupovic, J., Grenz, M., Tanker, N., 1985. Sesquiterpene lactones from Smyrnium cordifolium. Phytochemistry 24 (6), 1305–1308. Ulubelen, A., Oksuz, S., 1984. Eremophilenolides as oxidative artifacts of furanoeremophilanes. J. Nat. Prod. 47 (1), 177–178. Ulubelen, A., Oksuz, S., Samek, Z., Holub, M., 1971. Sesquiterpene lactones from Smyrnium olusatrum L. roots. Tetrahedron Lett. 46, 4455–4456. Ulubelen, A., Oksuz, S., Tanker, N., 1984. Furanosesquiterpenes from the fruits of Smyrnium cordifolium. Phytochemistry 23 (8), 1793–1794.
Sesquiterpene lactones from Smyrnium olusatrum Ali A. El-Gamal* Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt Received in revised form 12 January 2001
Abstract Fruits of Smyrnium olusatrum afforded three sesquiterpene lactones, namely, 1b,8b-dihydroxyeudesman-3,7(11)-dien-8a,12-olide, 1b,8b-dihydroxyeudesman-4(15),7(11)-dien-8a,l2-olide, and 1b,10a,4a,5b-diepoxy-6b-hydroxyglechoman-8a,12-olide. Four related known sesquiterpenes were also isolated and characterized. The structure elucidation of the isolated compounds was based primarily on 1D and 2D NMR analyses. The in vitro cytotoxicity of the extract and isolated compounds against P-388 mouse lymphoma cells will be also discussed. # 2001 Published by Elsevier Science Ltd. Keywords: Smyrnium olusatrum; Apiaceae; Sesquiterpenes; Eudesmen-8a,12-olide; Glechomanolides; Cytotoxic activity
1. Introduction Several sesquiterpene lactones, furanosesquiterpenes and oxepines have been isolated from members of the genus Smyrnium (Ulubelen et al., 1971, 1979, 1984, 1985; Bohlmann and Zdero, 1973; Ulubelen and Abdolmaleky, 1982; Goren et al., 1984; Ulubelen and Oksuz, 1984; Goren and Ulubelen, 1985, 1987a,b; Ulubelen and Goren, 1986; Molleken et al., 1998). The young shoots of Smyrnium olusatrum are cultivated and used as pot herb and edible salad in some areas in Libya. The plant is also used as antiscorbutic in Palestine (Jafri and El-Gadi, 1985). This study reports the isolation of three new sesquiterpene lactones 1, 2 and 5 in addition to four known ones 3, 4, 6 and 7 from the fruits of S. olusatrum collected from the Libyan desert. The cytotoxicity of the CH2Cl2 extract and the isolated compounds is also reported.
2. Results and discussion Repeated chromatography of the cytotoxic CH2Cl2 extract of the fruits of S. alusatrum on normal and C18 reversed phase silica gel afforded the new sesquiterpene lactones 1, 2 and 5, in addition to the four known sesquiterpenes 1b-acetoxyeudesman-4(15),7(11)-dien-8a,12-olide (3), 1b acetoxy, 8b-hydroxyeudesman-4(15),7(11)-dien* Tel.: +20-50-347496; fax: +20-50-347496. E-mail address: [email protected] (A.A. El-Gamal).
8a,12-olide (4) (Ulubelen et al, 1985), 1b,10a, 4a,5b diepoxyglechoman-8a,12-olide (6), previously reported as a semi-synthetic derivative (Stahl and Datta, 1972), and 1b,10a, 4a,5b-diepoxy-8a hydroxy-glechoman8a,12-olide (7) (Ulubelen et al., 1985). The EIMS of 1 displayed a molecular ion peak [M]+ at m/z 264, suggesting the molecular formula C15H20O4 and six degrees of unsaturation. The IR spectrum (KBr) of 1 showed an intense broad absorption band around 3400 cm 1, for hydroxyl groups, 1762 cm 1, for a,bunsaturated lactone functionality. The 13C and 1H NMR spectra of 1 (Table 1) were in agreement with a dihydroxy C-8,12 eudesmanolide skeleton for 1 (Ulubelen et al., 1985). The oxygenated doublet of doublets resonating at 3.78 (J=7.5 and 7.5) (Table 1), which correlated with the methine carbon resonating at 75.2, was assigned to H-1. This was based on the observed 3JHMBC coupling between C-1 and the methyl proton singlet resonating at 1.59 (H3-14) (Fig. 1). This was further supported by the observed COSY couplings between H-1 and both proton signals resonating at 2.45 and 2.30 (H2-2). The high J1,2 values suggested the a-configuration of H-1 and indicated its axial orientation (Ulubelen et al., 1985). Both H2-2 also displayed COSY couplings with the broad olefinic singlet resonating at 5.40 (H-3) which in turn displayed a 3J-HMBC coupling with the methyl carbon at 21.0 (C-15) (Fig. 1). The downfield quaternary carbon resonating at 105.1 was assigned to C-8. This was suggested by its 3JHMBC couplings with both proton signals resonating at 2.89 and 2.56 (H2-6). The carbonyl ester C-12 (C
0031-9422/01/$ - see front matter # 2001 Published by Elsevier Science Ltd. PII: S0031-9422(01)00211-4
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Table 1 13 C and 1H NMR spectral data of compounds 1a, 2a and 5b Position
1
2
C
H
1 2
75.2, d 33.4, t
3
122.1, d
4 5 6
133.5, s 49.2, d 24.4, t
7 8 9
162.1, s 105.1, s 47.9, t
10 11 12 13 14 15
39.5, 121.6, 172.7, 8.2, 10.5, 21.0,
1-OH 6-OH 8-OH
– – –
s s s q q q
3.78, 2.45, 2.30, 5.40, – 2.10, 2.89, 2.56, – – 3.33, 1.75, – – – 1.87, 1.59, 1.73,
C dd (7.5, 7.5) m m brs
brd (13.3) dd (13.1, 3.1) brt (13.2)
d (13.4) d (13.1)
3H, s 3H, s 3H, s
6.32, brs – 9.42, s
H
78.1, d 31.7, t 34.3, t 148.4, s 50.0, d 24.6, t 161.8, s 104.6, s 49.0, t 42.1, 121.5, 172.7, 8.2, 11.3, 107.6, – – –
s s s q q t
3.60, 1.91, 1.78, 2.35, 2.17, – 1.95, 2.75, – – 3.34, 1.81, – – – 1.83, 1.49, 4.96, 4.77, U – U
m m m m ddd (13.8, 5.2, 1.2) m m
d (13.4) d (13.4)
3H, s 3H, s d (1.2) d (1.2)
2c
5b
H
C
3.35, dd (11.5, 4.4) 1.70, m 1.60, brdd (12.5, 4.4) 2.33, ddd (11.5, 5.0, 3.0) 2.00, brdd (13.5, 5.2) – 2.1, brdd (13.5, 5.2) 2.62, dd (13.2, 3.4) 2.45, btr (13.2) – – 2.74, d (13.8) 1.50, d (13.8) – – – 1.80, 3H, s 1.00, 3H, s 4.89, s 4.56, s
H
69.0, d 22.8, t 37.1, t 60.0, s 65.7, d 68.8, d 156.6, s 78.6, d 38.7, t 57.8, 129.6, 172.6, 10.2, 17.4, 17.4, – – –
s s s q q q
2.74, 2.03, 1.40, 2.33, 1.38, – 3.19, 4.69, – 5.44, 2.97, 1.84, – – – 1.92, 1.08, 1.25,
brd (10.3) dd (13.9, 3.2) dd (13.9, 10.6, 3.2) m m d (6.5) dd (6.5, 2.9)
dd (5.4, 1.8) dd (15.9, 1.8) dd (15.9, 5.4)
3H, d (1.9)d 3H, s 3H, s
– 2.68, d (2.9) –
a In pyridine-d5, 400 MHz for 1H and 100 MHz for 13C NMR. Carbon multiplicities were determined by DEPT135 experiments. s=Quaternary, d=methine, t=methylene, q=methyl carbons, coupling constants (J) are in Hz, U=unobserved. b In CDCl3, 500 MHz for 1H and 125 MHz for 13C NMR. c In CDCl3, 400 MHz. d Long range with proton number 6 (Ulubelen and Goren, 1986).
Fig. 1. Important HMBC (solid lines) and NOESY (dashed lines) correlations of 1.
172.7) displayed a 3J-HMBC coupling with the proton methyl singlet resonating at 1.87 (H3-13). The assignment of the relative stereochemistry of 1 was further supported by NOESY data (Fig. 1) and the study of Drieding model. Thus, the b-oriented C-14 methyl singlet displayed NOESY correlations with both exchangeable proton singlets at 6.32 (C-1 OH) and 9.42 (C-8 OH), confirming their b-orientation. Likewise, the a-oriented H-1 was NOESY-correlated with the broad doublet resonating at 2.10 (H-5), suggesting their location on the same face of 1. Compound 1 was, therefore, identified as the new sesquiterpene 1b,8bdihydroxyeudesm - 3,7(11) - dien - 8a,12 - olide (1). It is
worth noting that the C-1 acetoxy derivative of 1 was previously reported from Smyrnium galaticum (Ulubelen and Goren, 1986). The EIMS of 2 displayed a molecular ion peak [M]+ at m/z 264, suggesting the same molecular formula of 1 (C15H20O4). The IR spectrum (KBr) of 2 showed a similar pattern to those of 1 with an additional absorption band at 890 cm 1, characteristic for an exocyclic methylene group. The 13C and 1H NMR spectral data of 2 (Table 1) were in agreement with those of 1 with the replacement of the 3,4 with 4,15 system. The two olefinic proton doublets resonating at 4.96 and 4.77 which correlated with the methylene carbon at 107.6 were assigned H2-15. Both protons displayed 3J-HMBC couplings with C-5 ( 50.0) and C-3 ( 34.3) and 2JHMBC coupling with the quaternary olefinic carbon at 148.8 (C-4). Therefore, 2 is the deacetylated derivative of compound 4. In fact, comparing the 1H NMR data of 2 in CDCl3 with those of 4 (Table 1) indicate that having significant difference of H1 proton being 3.35 (dd, J=11.5, 4.4 Hz) in 2 while it is 4.57 (dd, J=11.6, 4.7 Hz) in 4. Hence, compound 2 was proved to be 1b,8b-dihydroxyeudesm-4(15),7(11)-dien-8a,12-olide. The EIMS of 5 displayed a molecular ion peak [M]+ at m/z 280, suggesting the molecular formula C15H20O5 and six degrees of unsaturation. The IR spectrum (KBr)
A.A. El-Gamal / Phytochemistry 57 (2001) 1197–1200
of 5 showed an absorption band at 3637 cm 1 for hydroxyl group and 1756 cm 1 for a,b-unsaturated lactone functionality. The 13C and 1H NMR spectra of 5 (Table 1) suggested a monohydroxy C-8, 12 germacranolide skeleton for 5 (Stahl and Datta, 1972; Ulubelen et al., 1985). The oxygenated doublet of doublets resonating at 4.69 (J=6.5 and 2.9) (Table 1), which correlated with the methine carbon resonating at 68.8, was assigned to H-6. This was based on the observed COSY coupling between H-6 and the proton doublet resonating at 3.19 (H-5) and the exchangeable proton doublet at 2.68 (C-6 OH). The proton H-6 displayed 3 J-HMBC coupling with C-4 ( 60.0) and C-11 ( 129.6). A 2J-HMBC coupling was also observed between H-6 and C-7 ( 156.6). The proton doublet of doublets resonating at 5.44 which correlated with the oxygenated methine carbon at 78.6 was assigned H-8. This was based on its COSY coupling with the doublet of doublets resonating at 2.97 and 1.84 (H2-9). The methylene H2-9 displayed a 3J-HMBC coupling with C-14 methyl singlet (C 17.4). Once again, the assignments of the relative stereochemistry of 5 were based on NOESY data. Thus, the boriented C-14 methyl singlet displayed NOESY correlation with the proton doublet of doublets at 2.97 (H9b) which in turn showed cross peak with H-8, confirming their b-orientation. The exchangeable proton doublet at 2.68 showed NOESY correlation to the methyl singlet at 1.25 (H3-15), suggesting their borientation. Likewise, the a-oriented H-1 was NOESYcorrelated with the proton doublet resonating at 3.19 (H-5), suggesting their location on the same lower face of 5. Compound 5 was therefore identified as the new germacranolide 1b, 10a, 4a, 5b - diepoxy - 6b - hydroxyglechoman-8a,12-olide. The CH2Cl2 extract of the fruits of S. olusatrum exhibited significant in vitro cytotoxicity (IC50 9.0 mg ml 1) against P-388 mouse lymphoma cells. (Carmichael et al., 1987; Twentyman and Luscombe, 1987). However, the isolated compounds (1, 2, 3, 4, 6 and 7) showed marginal activity with IC50 60, 65, 42, 58, 94 and 88 mg ml 1, respectively.
1199
3. Experimental 3.1. General Mps: uncorrected, Yanagimoto MP-3 micro mp apparatus. 1H and 13C NMR: CDCl3 or pyridine with TMS as int. standard, Bruker AM 400 and Bruker AM 500 2D NMR (COSY, NOESY, HMQC and HMBC), EI–MS: 70 eV, Hitachi M-80 or VG Autospec. IR: KBr on a Jasco A-302 or Perkin Elmer 1710. UV: CH3OH, Hitachi 557. Prep. HPLC: Rp-18 column (10 mm, 302.15 cm i.d.). MPLC Rp-18 column (20 mm, 10 2.15 cm i.d.). Kusano Scientific Co. CC: Kieselgel 60, anal. TLC: silica gel 60F254 and RP-18 F254 (0.25 mm) precoated plates. Spots detection: UV light at 254 and/or spraying with 10% H2SO4. Cytoxicity bioassay: Tosoh MPR-A4i microplate reader. 3.2. Plant material The fruits of Smyrnium olusatrum were collected from the moist and shady places of the Gebel Akhdar area in Libya in 1996. The plant was identified by Professor Dr. Elgady, Faculty of Science, El-Faateh University, Tripoli, Libya. A voucher specimen is deposited in the Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. 3.3. Extraction and isolation Air dried powdered fruits (750 g) were extracted to exhaustion by hot 70% methyl alcohol. The methanolic extract was concentrated under reduced pressure to give about 100 g of the total extract (IC50=10.2 mg ml 1 to P-388 cell line). The extract was treated with a minimum volume of alcohol diluted with water and successively partitioned with pet. ether, CH2Cl2 and n-butanol. The CH2Cl2 extract (13 g, IC50 =9 mg ml 1 to P-388 cell line) was applied to the top of silica gel packed column (390 g) and eluted with pet. ether—EtOAc gradient. Similar fractions were combined and subjected either to direct crystallization or to further chromatographic purifica-
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tion on normal and C-18 reversed phase silica (using MPLC and HPLC). The CH2Cl2 extract afforded four main fractions, fractions 13–15, fraction 20, fractions 26–29 and fractions 30–35. Direct crystallization of fractions 13–15 and 20 gave compounds 3 (70 mg) and 4 (80 mg) respectively. Fractions 26–29 were further purified by using MPLC with H2O:CH3CN gradient to give compound 7 (60 mg). Fractions 30–35 were applied to the top of a silica gel packed column using CH2Cl2: MeOH gradient to give three main subfractions. Subfractions 6–8 afforded compound 6 (40 mg) on direct crystallization from MeOH while subfractions 1316 and 19–22 were further purified using HPLC on Rp18 column with CH3CN:H2O (20:80 and 30:70). Fractions 13–16 gave compound 7 (30 mg) while fractions 19–22 gave compounds 1, 2 and 5 (20, 30 and 3 mg respectively). 3.3.1. 1 ,8 -Dihydroxy eudesman 3,7(11)-dien 8 ,12 olide (1) White needle crystals from MeOH; mp 193–195 C, UV lmax MeOH, 215 IR v KBr 3400, 2976, 2933, 1762, 1898, 1471, 1353, 1323, 1241, 1042, 1007, 946. EI–MS m/z (rel. int.): 264 [M]+ (52) (calc. for C15H20O4), 246 [M+–H2O] (50), 228 (40), 213 (27), 194 (21), 166 (52), 126 (100), 121 (45), 105 (28), 91 (38), 69 (59), 53 (37). 1H NMR (400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-d5) Table 1. 3.3.2. 1 ,8 -Dihydroxy eudesman 4(15),7(11)-dien 8 ,12 olide (2) White prism crystals from MeOH; mp 178–180 C. UV lmax nm: 218 IR v KBr cm 1 3410, 2950, 1758, 1660, 1470, 1386, 1347, 890, 861. EI–MS m/z (rel. int.): 264 [M]+ (100) (calc. for C15H20O4), 246 [M–H2O]+ (55) 236 (75), 218 (38), 203 (47), 189 (37), 175 (52), 163 (81), 145 (56), 105 (47), 91 (60), 55 (62). 1 H NMR (400 MHz, pyridine-d5, CDCl3) and 13C NMR (100 MHz, pyridine -d5) Table 1. 3.3.3. 1 ,10 ;4 ,5 -Diepoxy 6- -hydroxy-glechoman8 , 12 olide (5) White needle crystals (3 mg) from MeOH; mp 165– 168 C. UV lmax nm: 217. IR v CHCl3 cm 1, 3637, 2906, 1756, 1034, 853, 765, 723. EI–MS m/z (rel. int.): 280 [M]+ (12) (calc. for C15H20O5), 262 [M–H2O]+(50), 251 (7), 233 (13), 205 (11), 181 (31), 150 (26), 126 (52), 111 (35), 97 (63), 83 (37), 69 (100), 53 (37). 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) Table 1.
Acknowledgements The author would like to thank Professors H. Itokawa and K. Takeya, Tokyo University of Pharmacy and Life Science, for the NMR analyses and Professor H. Abd El Fattah, Faculty of Pharmacy, Mansoura University, Egypt, for providing the plant material.
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