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Carbazole alkaloids and coumarins from Clausena lansium roots
Phytochemistry Letters 5 (2012) 26–28
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Carbazole alkaloids and coumarins from Clausena lansium roots Wisanu Maneerat a, Thunwadee Ritthiwigrom b, Sarot Cheenpracha c, Surat Laphookhieo a,* a
Natural Products Research Laboratory, School of Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai 57100, Thailand Department of Chemistry, Faculty of Science, Chiang Mai University, Sutep, Muang, Chiang Mai 50200, Thailand c School of Science, University of Phayao, Maeka, Muang, Phayao 56000, Thailand b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 June 2011 Received in revised form 8 August 2011 Accepted 23 August 2011 Available online 6 September 2011
Two new carbazole alkaloids, mafaicheenamines D (1) and E (2), together with twelve known compounds (3–14) were isolated from the roots of Clausena lansium. Spectroscopic methods, including NMR, UV, IR, and MS spectral data were used for structural characterization. Some of isolates were evaluated for their cytotoxicity against three human cancer cell lines (KB, MCF-7, and NCI-H187). ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Clausena lansium Carbazole alkaloid Mafaicheenamine D Mafaicheenamine E Cytotoxicity Rutaceae
1. Introduction
2. Results and discussion
Clausena lansium or ‘‘mafaicheen’’ in local Thai name is a fruit tree belonging to the family of Rutaceae. Several parts of this plant have been used as traditional medicines in China, Taiwan, and Philippines for treatments of coughs, asthma, ulcers, and gastrointestinal diseases (Lin, 1989; Adebajo et al., 2009). Previous phytochemical investigations of Clausena genus, a numbers of bioactive compound especially carbazole alkaloids and coumarins had been reported (Rakash et al., 1978; Khan et al., 1983; Ngadjui et al., 1989; Li et al., 1991; Kumar et al., 1995; Milner et al., 1996; Ito et al., 1998, 2000). We have previous investigations of the twigs and seeds of C. lansium had resulted in seven new natural products including three carbazole alkaloids (Maneerat and Laphookhieo, 2010), two new coumarins (Maneerat et al., 2010), and two new amides (Maneerat et al., 2011). Herein, this paper reports further investigation of hexanes and acetone extracts from the roots of the same plant which led to the isolation and characterization of two new carbazole alkaloids (1 and 2) along with twelve known compounds (3–14) from the roots of C. lansium. The cytotoxicity against three human cancer cell lines, KB, MCF-7, and NCI-H187 of compounds 2, 4, 7, 8, and 14 was also reported.
The combination of hexanes and acetone extracts from airdried roots of C. lansium was purified by chromatographic techniques led to isolation of two new carbazole alkaloids (1 and 2), together with twelve known compounds including mafaicheenamine B (3) (Maneerat and Laphookhieo, 2010), O-demethylmurrayanine (4) (Ngadjui et al., 1989), murrayanine (5) (Li et al., 1991), indizoline (6) (Li et al., 1991), glycozoline (7) (Chakravarty et al., 1999), 3-formyl-6-methoxycarbazole (8) (Li et al., 1991), lansine (9) (Ma et al., 2005), glycozolidal (10) (Li et al., 1991), imperatorin (11) (Harkar et al., 1984), wampetin (12) (Khan et al., 1983), indicolactonediol (13) (Rakash et al., 1978), and umbelliferone (14) (Ngadjui et al., 1991) (Fig. 1). Mafaicheenamine D (1) was isolated as a yellow solid (mp 202.4–203.1 8C) and its molecular formula was determined as C19H18NO2 ([M + H]+ m/z 292.1332, calcd. for 292.1338) by ESI– TOF–MS. Its UV spectrum showed typical absorbance of carbazole alkaloids at lmax 246, 266, 278, 296, 303, 321, 334, and 357 nm (Ito et al., 1998, 2009; Maneerat and Laphookhieo, 2010), while the IR spectrum showed the absorption bands of NH (3286 cm1) and conjugated carbonyl (1675 cm1) functionalities. Analysis of its NMR spectral data including COSY, HMQC, and HMBC spectra, allowed unambiguous assignment of all proton and carbon signals. The 1H NMR displayed signals of NH at dH 8.39 (br s), a set of 1,2disubstituted benzene ring at dH 8.12 (1H, d, J = 7.6 Hz, H-5), 7.48 (1H, m, H-8), 7.47 (1H, m, H-7), and 7.29 (1H, m, H-6), and an aromatic proton at dH 8.33 (1H, s, H-4). These data suggested that 1
* Corresponding author. Tel.: +66 5391 6238; fax: +66 5391 6776. E-mail addresses: [email protected], [email protected] (S. Laphookhieo).
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.08.013
W. Maneerat et al. / Phytochemistry Letters 5 (2012) 26–28
27
Fig. 1. Structures of mafaicheenamine D (1) and E (2).
proton at dH 5.19 (1H, d, J = 9.2 Hz, H-20 )/dC 121.4 (C-20 ), two allyl methyl groups at dH 2.04 (3H, d, J = 1.2 Hz, H-40 )/dC 25.0 (C-40 ) and 1.85 (3H, d, J = 1.2 Hz, H-50 )/dC 17.7 (C-50 ), and dC 169.9 (lactone carbonyl)] instead of an exocylic a, b-unsaturated ketone moiety. Therefore, mafaicheenamine E was assigned to be 2, a structural isomer of furanoclausamine B isolated from the stems of Clausena anisata (Ito et al., 2009). Detailed assignments of the protons and carbons as well as HMBC correlations are shown in Table 1. Compounds 2, 4, 7, 8, and 14 were evaluated for their cytotoxicity against three human cancer cell lines including oral cancer (KB), breast cancer (MCF-7), and small cell lung cancer (NCIH187). Unfortunately, only mafaicheenamine E (2) exhibited cytotoxicity against MCF-7 cell line with IC50 value of 3.1 mg/mL whereas all the rest of compounds were found to be noncytotoxicity against all three human cancer cell lines. The above results suggested that mafaicheenamine E (2), newly isolated from C. lansium roots may have some interest for further studies with regard to selective anti-cancer activity against MCF-7 cell line.
was a 1,2,3-trisubstituted carbazole framework (Maneerat and Laphookhieo, 2010). The 1H NMR also contained a signal of methoxyl group at dH 4.16, a methylene group at dH 3.89 (2H, s, H10 ) and two allyl methyl groups at dH 2.51 (3H, s, H-40 ), and 2.07 (3H, s, H-50 ). The 13C NMR and DEPT spectral data indicated 19 signals including five methines (dC 126.6, 121.0, 120.3, 112.1, and 111.0), one methylene (dC 29.9), three methyls (dC 60.0, 24.4, and 20.3), one carbonyl (dC 193.3), and nine quaternary carbons (dC 148.1, 140.5, 139.9, 136.7, 133.3, 130.7, 125.5, 124.0, and 120.7). The above information suggested that 1 contained fused carbazole nucleus and cyclic ketone similar to those of mafaicheenamine C (Maneerat and Laphookhieo, 2010). The cyclic ketone was identified to be an exocylic a, b-unsaturated ketone which placed on C-2/C-3 due to the 3J HMBC correlations of H-4 (dH 8.33) and H-10 (dH 3.89) with carbonyl carbon (C-10, dC 193.3). The 4J HMBC correlation of CH3-40 (dH 2.51) with C-10 was also observed. Finally, the methoxyl group was located on C-1 by process of elimination and also supported with the HMBC correlations of OMe (dH 4.16) and a methylene group (dH 3.89, H-10 ) with C-1 (d 140.5). Therefore, mafaicheenamine D was indentified to the structure 1. Detailed assignments of the protons and carbons as well as HMBC correlations are shown in Table 1. Mafaicheenamine E (2) was obtained as a yellow solid (mp 209.2–212.1 8C), [a]27D – 14.3. The ESI–TOF–MS gave a pseudomolecular ion peak at [M + H]+ m/z 308.1281 (calcd. 308.1287) consistent with the molecular formula C19H18NO3. The 1H and 13C NMR spectral data of 2 were similar to those of 1 except compound 2 showed 1H and 13C NMR signals of g-lactone moiety [oxymethine proton at dH 6.41 (1H, d, J = 9.2 Hz, H-10 )/dC 75.9 (C-10 ), an olefinic
3. Experimental 3.1. General The optical rotation [a]D value was determined with a Bellingham & Stanley ADP400 polarimeter. UV–vis spectra were recorded with a Perkin-Elmer UV–vis spectrophotometer. The IR spectra were recorded using Perkin-Elmer FTS FT-IR spectrophotometer. The 1H and 13C NMR spectra were recorded by 400 MHz Bruker or 500 MHz Varian UNITY INOVA spectrometers.
Table 1 1 H and 13C NMR data of compounds 1 and 2. Position
1 2 3 4 4a 4b 5 6 7 8 8a 9a 10 10 20 30 40 50 1-OMe 9-NH a b
1a
2b 1
13
dC
dH (mult., J in Hz)
HMBC ( H !
140.5 133.3 120.7 112.1 124.0 125.5 121.0 120.3 126.6 111.0 139.9 136.7 193.3 29.9 130.7 148.1 24.4 20.3 60.0 –
– – – 8.33 – – 8.12 7.29 7.47 7.48 – – – 3.89 – – 2.51 2.07 4.16 8.39
– – – C-2, C-4a, C-9a, C-10 – – C-6, C-7, C-8a C-4b, C-7, C-8 C-5, C-6, C-8a C-4b, C-6, C-7 – – – C-1, C-2, C-10, C-20 – – C-10, C-20 , C-30 , C-50 C-20 , C-30 , C-40 C-1 –
(s)
(d, 7.6) (m) (m) (m)
(s)
(s) (s) (s) (br s)
Measured at 500 MHz NMR in CDCl3. Measured at 400 MHz NMR in acetone-d6.
C)
dC
dH (mult., J in Hz)
HMBC (1H ! 13C)
139.4 118.5 135.7 112.9 118.5 123.1 120.8 120.0 126.8 111.6 141.1 136.8 169.9 75.9 121.4 139.4 25.0 17.7 60.0 –
– – – 8.35 (s) – – 8.29 (d, 8.4) 7.29 (dd, 8.4 and 8.0) 7.50 (dd, 8.4 and 8.0) 7.61 (d, 8.4) – – – 6.41 (d, 9.2) 5.19 (d, 9.2) – 2.04 (d, 1.2) 1.85 (d, 1.2) 4.04 (s) 10.94 (br s)
– – – C-2, C-9a, C-10 – – C-8a, C-7 C-4a, C-8 C-5, C-8a C-4b, C-6 – – – C-1, C-3, C-10, C-20 C-40 , C-50 – C-20 , C-30 , C-50 C-20 , C-30 , C-40 C-1 –
28
W. Maneerat et al. / Phytochemistry Letters 5 (2012) 26–28
Tetramethylsilane (TMS) was used as internal reference. The MicroTOF, Bruker Daltonics mass spectrometer, was used to correct ESI–TOF–MS data. Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60 H (Merck, 5–40 mm) and silica gel 100 (Merck, 63–200 mm), respectively. Precoated plates of silica gel 60 F254 were used for analytical purposes. 3.2. Plant material The C. lansium roots were collected from Nan Province, northern Thailand, in April 2008. Botanical identification was achieved through comparison with a voucher specimen number QBG 25077 in the herbarium collection of Queen Sirikit Botanic Garden, Mae Rim, Chiang Mai, Thailand.
1730, 1612; 1H and 13C NMR spectroscopic data, see Table 1; ESI– TOF–MS (m/z): [M + H]+ 308.1281 (calcd. for C19H18NO3, 308.1287). 3.4. Cytotoxic assay The procedures for cytotoxic assay of three cancer cell lines including oral cavity cancer (KB), breast cancer (MCF-7) and small cell lung cancer (NCI-H187) were performed by resazurin microplate assay (REMA) which was a modified method of fluorescent dye for the mammalian cell cytotoxicity (Brien et al., 2000). The standard compound was doxorubicin which had IC50 values of 0.18, 1.25, and 0.07 mg/mL for cytotoxicity against KB, MCF-7, and NCI-H187 cell lines, respectively. Acknowledgements
3.3. Extraction and isolation Air-dried roots of C. lansium (2.93 kg) were extracted successively with hexanes and acetone over a period of 3 days at room temperature. The hexanes and acetone extracts (62.55 g) were combined. This extract upon standing at room temperature yielded a yellow crystal which was further washed with hexanes to give compound 6 (1.09 g). The remaining crude extract (61.46 g) was subjected to QCC over silica gel using a gradient of hexanes–EtOAc (100% hexanes to 100% EtOAc) to provide six fractions (A–H). Fraction D (1.03 g) was further separated by QCC with a gradient of EtOAc–hexanes (5% EtOAc–hexanes to 100% EtOAc) to afford seven subfraction (D1–D7). Subfraction D2 (86.1 mg) was performed by CC using 40% CH2Cl2–hexanes to yield compounds 5 (20.3 mg) and 11 (28.4 mg). Fraction D3 (307.6 mg) was separated by CC with 75% CH2Cl2–hexanes and followed by Sephadex-LH20 eluting with 100% MeOH gave compounds 3 (4.5 mg) and 9 (16.7 mg). Compounds 8 (33.9 mg) and 10 (30.5 mg) were obtained from repeated CC eluting with 80% CH2Cl2–hexanes and Sephadex-LH20 using 100% MeOH from subfraction D5 (169.7 mg) and D6 (135.4 mg), respectively. Fraction E (5.70 g) was isolated by QCC using a gradient of 10% EtOAc–hexanes to 100% EtOAc to provide five subfractions (E1–E5). Subfraction E2 (94.7 mg) was purified by CC with 30% CH2Cl2–hexanes to afford compound 7 (5.0 mg). Fraction E4 (1.35 g) was subjected to CC using 50% CH2Cl2–hexanes to give seven subfractions (E4a–E4g). Purification of subfraction E4b (14.6 mg) by CC with 10% EtOAc–hexanes yielded compounds 1 (2.3 mg) and 2 (6.9 mg). Compounds 14 (11.7 mg) and 4 (65.0 mg) was purified by Sephadex-LH20 using 100% MeOH from subfraction E4d (111.6 mg) and E4f (163.2 mg), respectively. Fraction G (7.94 g) was separated by QCC with a gradient of 20% EtOAc–hexanes to 100% EtOAc to afford four subfractions (G1–G4). Subfraction G3 (967.0 mg) was purified by CC with 10% EtOAc– CH2Cl2 to give compounds 12 (184.4 mg) and 13 (109.6 mg). 3.3.1. Mafaicheenamine D (1) Yellow solid; mp 202.4–203.1 8C; UV lmax MeOH (nm) (log e): 246 (3.67), 266 (3.79), 278 (3.90), 296 (3.64), 303 (3.66), 321 (3.26), 334 (3.16), 357 (3.03); IR (neat) nmax cm1: 3286, 1675, 1610; 1H and 13C NMR spectroscopic data, see Table 1; ESI–TOF–MS (m/z): [M + H]+ 292.1332 (calcd. for C19H18NO2, 292.1338). 3.3.2. Mafaicheenamine E (2) Yellow solid; mp 209.2–212.1 8C; [a]27D – 14.3 (c 0.012, MeOH); UV lmax MeOH (nm) (log e): 246 (3.97), 264 (4.12), 278 (4.27), 295 (3.76), 310 (3.59), 325 (3.48), 339 (3.29); IR (neat) nmax cm1: 3300,
We would like to thank the Thailand Research Fund through TRF research scholar to S.L. (Grant no. RSA5200811) and the Royal Golden Jubilee Ph.D. Program for a scholarship to W.M. (Grant no. PHD/0006/2552). We also thank Mae Fah Luang University for partial financial support and laboratory facilities. We appreciate to Mr. Nitirat Chimnoi, Chulabhorn Research Institute, Bangkok, for recording mass spectra and BIOTEC (Thailand) for cytotoxic assay. References Adebajo, A.C., Iwalewa, E.O., Obuotor, E.M., Ibikunle, G.F., Omisore, N.O., Adewunmi, C.O., Obaparusi, O.O., Klaes, M., Adetogun, G.E., Schmidt, T.J., Berspohl, E.J., 2009. Pharmacological properties of the extract and some isolated compounds of Clausena lansium stem bark: anti-trichomonal, antidiabetic, anti-inflammatory, hepatoprotective and antioxidant effects. J. Ethnopharmacol. 122, 10–19. Brien, J.O., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421–5426. Chakravarty, A.K., Sarkar, T., Masuda, K., Shiojima, K., 1999. Carbazole alkaloids from roots of Glycosmis arborea. Phytochemistry 50, 1263–1266. Harkar, S., Razdan, T.K., Waight, E.S., 1984. Steroids, chromone and coumarins from Angelica officinalis. Phytochemistry 23, 419–426. Ito, C., Katsuno, S., Furukawa, H., 1998. Structures of lansiumarin-A, -B, -C, three new furocoumarins from Clausena lansium. Chem. Pharm. Bull. 46, 341–343. Ito, C., Katsuno, S., Itoigawa, M., Ruangrungsi, N., Mukainaka, T., Okada, M., Kitagawa, Y., Tokuda, H., Nishino, H., Furukawa, H., 2000. New carbazole alkaloids from Clausena anisata with antitumor promoting activity. J. Nat. Prod. 63, 125–128. Ito, C., Itoigawa, M., Aizawa, K., Yoshida, K., Ruangrungsi, N., Furukawa, H., 2009. g-Lactone carbazoles from Clausena anisata. J. Nat. Prod. 72, 1202–1204. Khan, N.U., Navqi, S.W.I., Ishratullah, K., 1983. Wampetin, a furocoumarin from Clausena wampi. Phytochemistry 22, 2624–2625. Kumar, V., Vallipuram, K., Adebajo, A.C., Reisch, J., 1995. 1,7-Dihydroxy-3-formyl-1(30 -methyl-20 -butenyl)carbazole from Clausena lansium. Phytochemistry 40, 1563–1565. Li, W.S., McChesney, J.D., El-Feraly, F.S., 1991. Carbazole alkaloids from Clausena lansium. Phytochemistry 30, 343–346. Lin, J.H., 1989. Cinnamamide derivatives from Clausena lansium. Phytochemistry 28, 621–622. Ma, C., Case, R.J., Wang, Y., Zhang, H.J., Tan, G.T., Van Hung, N., Cuong, N.M., Franzblau, S.G., Soejarto, D.D., Fong, H.H., Pauli, G.F., 2005. Anti-tuberculosis constituents from the stem bark of Micromelum hirsutum. Planta Med. 71, 261–267. Maneerat, W., Laphookhieo, S., 2010. Antitumoral alkaloids from Clausena lansium. Heterocycles 81, 1261–1269. Maneerat, W., Prawat, U., Saewan, N., Laphookhieo, S., 2010. New coumarins from Clausena lansium twigs. J. Braz. Chem. Soc. 21, 665–668. Maneerat, W., Tha-in, S., Cheenpracha, S., Prawat, U., Laphookhieo, S., 2011. New amides from the seeds of Clausena lansium. J. Med. Plant. Res. 5, 2812–2815. Milner, P.H., Coates, N.J., Gilpin, M.L., Spear, S.R., 1996. SB-204900, a novel oxirane carboxamide from Clausena lansium. J. Nat. Prod. 59, 400–402. Ngadjui, B.T., Ayafor, J.F., Sondengam, B.L., Connolly, J.D., 1989. Quinolone and carbazole alkaloids from Clausena anisata. Phytochemistry 28, 1517–1519. Ngadjui, B.T., Mouncherou, S.M., Ayafor, J.F., Sondengam, B.L., Tillequin, F., 1991. Geranyl coumarins from Clausena anisata. Phytochemistry 30, 2809–2811. Rakash, D., Raj, K., Kapil, R.S., Popli, S.P., 1978. Coumarins from Clausena indica. Phytochemistry 17, 1194–1195.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Carbazole alkaloids and coumarins from Clausena lansium roots Wisanu Maneerat a, Thunwadee Ritthiwigrom b, Sarot Cheenpracha c, Surat Laphookhieo a,* a
Natural Products Research Laboratory, School of Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai 57100, Thailand Department of Chemistry, Faculty of Science, Chiang Mai University, Sutep, Muang, Chiang Mai 50200, Thailand c School of Science, University of Phayao, Maeka, Muang, Phayao 56000, Thailand b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 June 2011 Received in revised form 8 August 2011 Accepted 23 August 2011 Available online 6 September 2011
Two new carbazole alkaloids, mafaicheenamines D (1) and E (2), together with twelve known compounds (3–14) were isolated from the roots of Clausena lansium. Spectroscopic methods, including NMR, UV, IR, and MS spectral data were used for structural characterization. Some of isolates were evaluated for their cytotoxicity against three human cancer cell lines (KB, MCF-7, and NCI-H187). ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Clausena lansium Carbazole alkaloid Mafaicheenamine D Mafaicheenamine E Cytotoxicity Rutaceae
1. Introduction
2. Results and discussion
Clausena lansium or ‘‘mafaicheen’’ in local Thai name is a fruit tree belonging to the family of Rutaceae. Several parts of this plant have been used as traditional medicines in China, Taiwan, and Philippines for treatments of coughs, asthma, ulcers, and gastrointestinal diseases (Lin, 1989; Adebajo et al., 2009). Previous phytochemical investigations of Clausena genus, a numbers of bioactive compound especially carbazole alkaloids and coumarins had been reported (Rakash et al., 1978; Khan et al., 1983; Ngadjui et al., 1989; Li et al., 1991; Kumar et al., 1995; Milner et al., 1996; Ito et al., 1998, 2000). We have previous investigations of the twigs and seeds of C. lansium had resulted in seven new natural products including three carbazole alkaloids (Maneerat and Laphookhieo, 2010), two new coumarins (Maneerat et al., 2010), and two new amides (Maneerat et al., 2011). Herein, this paper reports further investigation of hexanes and acetone extracts from the roots of the same plant which led to the isolation and characterization of two new carbazole alkaloids (1 and 2) along with twelve known compounds (3–14) from the roots of C. lansium. The cytotoxicity against three human cancer cell lines, KB, MCF-7, and NCI-H187 of compounds 2, 4, 7, 8, and 14 was also reported.
The combination of hexanes and acetone extracts from airdried roots of C. lansium was purified by chromatographic techniques led to isolation of two new carbazole alkaloids (1 and 2), together with twelve known compounds including mafaicheenamine B (3) (Maneerat and Laphookhieo, 2010), O-demethylmurrayanine (4) (Ngadjui et al., 1989), murrayanine (5) (Li et al., 1991), indizoline (6) (Li et al., 1991), glycozoline (7) (Chakravarty et al., 1999), 3-formyl-6-methoxycarbazole (8) (Li et al., 1991), lansine (9) (Ma et al., 2005), glycozolidal (10) (Li et al., 1991), imperatorin (11) (Harkar et al., 1984), wampetin (12) (Khan et al., 1983), indicolactonediol (13) (Rakash et al., 1978), and umbelliferone (14) (Ngadjui et al., 1991) (Fig. 1). Mafaicheenamine D (1) was isolated as a yellow solid (mp 202.4–203.1 8C) and its molecular formula was determined as C19H18NO2 ([M + H]+ m/z 292.1332, calcd. for 292.1338) by ESI– TOF–MS. Its UV spectrum showed typical absorbance of carbazole alkaloids at lmax 246, 266, 278, 296, 303, 321, 334, and 357 nm (Ito et al., 1998, 2009; Maneerat and Laphookhieo, 2010), while the IR spectrum showed the absorption bands of NH (3286 cm1) and conjugated carbonyl (1675 cm1) functionalities. Analysis of its NMR spectral data including COSY, HMQC, and HMBC spectra, allowed unambiguous assignment of all proton and carbon signals. The 1H NMR displayed signals of NH at dH 8.39 (br s), a set of 1,2disubstituted benzene ring at dH 8.12 (1H, d, J = 7.6 Hz, H-5), 7.48 (1H, m, H-8), 7.47 (1H, m, H-7), and 7.29 (1H, m, H-6), and an aromatic proton at dH 8.33 (1H, s, H-4). These data suggested that 1
* Corresponding author. Tel.: +66 5391 6238; fax: +66 5391 6776. E-mail addresses: [email protected], [email protected] (S. Laphookhieo).
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.08.013
W. Maneerat et al. / Phytochemistry Letters 5 (2012) 26–28
27
Fig. 1. Structures of mafaicheenamine D (1) and E (2).
proton at dH 5.19 (1H, d, J = 9.2 Hz, H-20 )/dC 121.4 (C-20 ), two allyl methyl groups at dH 2.04 (3H, d, J = 1.2 Hz, H-40 )/dC 25.0 (C-40 ) and 1.85 (3H, d, J = 1.2 Hz, H-50 )/dC 17.7 (C-50 ), and dC 169.9 (lactone carbonyl)] instead of an exocylic a, b-unsaturated ketone moiety. Therefore, mafaicheenamine E was assigned to be 2, a structural isomer of furanoclausamine B isolated from the stems of Clausena anisata (Ito et al., 2009). Detailed assignments of the protons and carbons as well as HMBC correlations are shown in Table 1. Compounds 2, 4, 7, 8, and 14 were evaluated for their cytotoxicity against three human cancer cell lines including oral cancer (KB), breast cancer (MCF-7), and small cell lung cancer (NCIH187). Unfortunately, only mafaicheenamine E (2) exhibited cytotoxicity against MCF-7 cell line with IC50 value of 3.1 mg/mL whereas all the rest of compounds were found to be noncytotoxicity against all three human cancer cell lines. The above results suggested that mafaicheenamine E (2), newly isolated from C. lansium roots may have some interest for further studies with regard to selective anti-cancer activity against MCF-7 cell line.
was a 1,2,3-trisubstituted carbazole framework (Maneerat and Laphookhieo, 2010). The 1H NMR also contained a signal of methoxyl group at dH 4.16, a methylene group at dH 3.89 (2H, s, H10 ) and two allyl methyl groups at dH 2.51 (3H, s, H-40 ), and 2.07 (3H, s, H-50 ). The 13C NMR and DEPT spectral data indicated 19 signals including five methines (dC 126.6, 121.0, 120.3, 112.1, and 111.0), one methylene (dC 29.9), three methyls (dC 60.0, 24.4, and 20.3), one carbonyl (dC 193.3), and nine quaternary carbons (dC 148.1, 140.5, 139.9, 136.7, 133.3, 130.7, 125.5, 124.0, and 120.7). The above information suggested that 1 contained fused carbazole nucleus and cyclic ketone similar to those of mafaicheenamine C (Maneerat and Laphookhieo, 2010). The cyclic ketone was identified to be an exocylic a, b-unsaturated ketone which placed on C-2/C-3 due to the 3J HMBC correlations of H-4 (dH 8.33) and H-10 (dH 3.89) with carbonyl carbon (C-10, dC 193.3). The 4J HMBC correlation of CH3-40 (dH 2.51) with C-10 was also observed. Finally, the methoxyl group was located on C-1 by process of elimination and also supported with the HMBC correlations of OMe (dH 4.16) and a methylene group (dH 3.89, H-10 ) with C-1 (d 140.5). Therefore, mafaicheenamine D was indentified to the structure 1. Detailed assignments of the protons and carbons as well as HMBC correlations are shown in Table 1. Mafaicheenamine E (2) was obtained as a yellow solid (mp 209.2–212.1 8C), [a]27D – 14.3. The ESI–TOF–MS gave a pseudomolecular ion peak at [M + H]+ m/z 308.1281 (calcd. 308.1287) consistent with the molecular formula C19H18NO3. The 1H and 13C NMR spectral data of 2 were similar to those of 1 except compound 2 showed 1H and 13C NMR signals of g-lactone moiety [oxymethine proton at dH 6.41 (1H, d, J = 9.2 Hz, H-10 )/dC 75.9 (C-10 ), an olefinic
3. Experimental 3.1. General The optical rotation [a]D value was determined with a Bellingham & Stanley ADP400 polarimeter. UV–vis spectra were recorded with a Perkin-Elmer UV–vis spectrophotometer. The IR spectra were recorded using Perkin-Elmer FTS FT-IR spectrophotometer. The 1H and 13C NMR spectra were recorded by 400 MHz Bruker or 500 MHz Varian UNITY INOVA spectrometers.
Table 1 1 H and 13C NMR data of compounds 1 and 2. Position
1 2 3 4 4a 4b 5 6 7 8 8a 9a 10 10 20 30 40 50 1-OMe 9-NH a b
1a
2b 1
13
dC
dH (mult., J in Hz)
HMBC ( H !
140.5 133.3 120.7 112.1 124.0 125.5 121.0 120.3 126.6 111.0 139.9 136.7 193.3 29.9 130.7 148.1 24.4 20.3 60.0 –
– – – 8.33 – – 8.12 7.29 7.47 7.48 – – – 3.89 – – 2.51 2.07 4.16 8.39
– – – C-2, C-4a, C-9a, C-10 – – C-6, C-7, C-8a C-4b, C-7, C-8 C-5, C-6, C-8a C-4b, C-6, C-7 – – – C-1, C-2, C-10, C-20 – – C-10, C-20 , C-30 , C-50 C-20 , C-30 , C-40 C-1 –
(s)
(d, 7.6) (m) (m) (m)
(s)
(s) (s) (s) (br s)
Measured at 500 MHz NMR in CDCl3. Measured at 400 MHz NMR in acetone-d6.
C)
dC
dH (mult., J in Hz)
HMBC (1H ! 13C)
139.4 118.5 135.7 112.9 118.5 123.1 120.8 120.0 126.8 111.6 141.1 136.8 169.9 75.9 121.4 139.4 25.0 17.7 60.0 –
– – – 8.35 (s) – – 8.29 (d, 8.4) 7.29 (dd, 8.4 and 8.0) 7.50 (dd, 8.4 and 8.0) 7.61 (d, 8.4) – – – 6.41 (d, 9.2) 5.19 (d, 9.2) – 2.04 (d, 1.2) 1.85 (d, 1.2) 4.04 (s) 10.94 (br s)
– – – C-2, C-9a, C-10 – – C-8a, C-7 C-4a, C-8 C-5, C-8a C-4b, C-6 – – – C-1, C-3, C-10, C-20 C-40 , C-50 – C-20 , C-30 , C-50 C-20 , C-30 , C-40 C-1 –
28
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Tetramethylsilane (TMS) was used as internal reference. The MicroTOF, Bruker Daltonics mass spectrometer, was used to correct ESI–TOF–MS data. Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60 H (Merck, 5–40 mm) and silica gel 100 (Merck, 63–200 mm), respectively. Precoated plates of silica gel 60 F254 were used for analytical purposes. 3.2. Plant material The C. lansium roots were collected from Nan Province, northern Thailand, in April 2008. Botanical identification was achieved through comparison with a voucher specimen number QBG 25077 in the herbarium collection of Queen Sirikit Botanic Garden, Mae Rim, Chiang Mai, Thailand.
1730, 1612; 1H and 13C NMR spectroscopic data, see Table 1; ESI– TOF–MS (m/z): [M + H]+ 308.1281 (calcd. for C19H18NO3, 308.1287). 3.4. Cytotoxic assay The procedures for cytotoxic assay of three cancer cell lines including oral cavity cancer (KB), breast cancer (MCF-7) and small cell lung cancer (NCI-H187) were performed by resazurin microplate assay (REMA) which was a modified method of fluorescent dye for the mammalian cell cytotoxicity (Brien et al., 2000). The standard compound was doxorubicin which had IC50 values of 0.18, 1.25, and 0.07 mg/mL for cytotoxicity against KB, MCF-7, and NCI-H187 cell lines, respectively. Acknowledgements
3.3. Extraction and isolation Air-dried roots of C. lansium (2.93 kg) were extracted successively with hexanes and acetone over a period of 3 days at room temperature. The hexanes and acetone extracts (62.55 g) were combined. This extract upon standing at room temperature yielded a yellow crystal which was further washed with hexanes to give compound 6 (1.09 g). The remaining crude extract (61.46 g) was subjected to QCC over silica gel using a gradient of hexanes–EtOAc (100% hexanes to 100% EtOAc) to provide six fractions (A–H). Fraction D (1.03 g) was further separated by QCC with a gradient of EtOAc–hexanes (5% EtOAc–hexanes to 100% EtOAc) to afford seven subfraction (D1–D7). Subfraction D2 (86.1 mg) was performed by CC using 40% CH2Cl2–hexanes to yield compounds 5 (20.3 mg) and 11 (28.4 mg). Fraction D3 (307.6 mg) was separated by CC with 75% CH2Cl2–hexanes and followed by Sephadex-LH20 eluting with 100% MeOH gave compounds 3 (4.5 mg) and 9 (16.7 mg). Compounds 8 (33.9 mg) and 10 (30.5 mg) were obtained from repeated CC eluting with 80% CH2Cl2–hexanes and Sephadex-LH20 using 100% MeOH from subfraction D5 (169.7 mg) and D6 (135.4 mg), respectively. Fraction E (5.70 g) was isolated by QCC using a gradient of 10% EtOAc–hexanes to 100% EtOAc to provide five subfractions (E1–E5). Subfraction E2 (94.7 mg) was purified by CC with 30% CH2Cl2–hexanes to afford compound 7 (5.0 mg). Fraction E4 (1.35 g) was subjected to CC using 50% CH2Cl2–hexanes to give seven subfractions (E4a–E4g). Purification of subfraction E4b (14.6 mg) by CC with 10% EtOAc–hexanes yielded compounds 1 (2.3 mg) and 2 (6.9 mg). Compounds 14 (11.7 mg) and 4 (65.0 mg) was purified by Sephadex-LH20 using 100% MeOH from subfraction E4d (111.6 mg) and E4f (163.2 mg), respectively. Fraction G (7.94 g) was separated by QCC with a gradient of 20% EtOAc–hexanes to 100% EtOAc to afford four subfractions (G1–G4). Subfraction G3 (967.0 mg) was purified by CC with 10% EtOAc– CH2Cl2 to give compounds 12 (184.4 mg) and 13 (109.6 mg). 3.3.1. Mafaicheenamine D (1) Yellow solid; mp 202.4–203.1 8C; UV lmax MeOH (nm) (log e): 246 (3.67), 266 (3.79), 278 (3.90), 296 (3.64), 303 (3.66), 321 (3.26), 334 (3.16), 357 (3.03); IR (neat) nmax cm1: 3286, 1675, 1610; 1H and 13C NMR spectroscopic data, see Table 1; ESI–TOF–MS (m/z): [M + H]+ 292.1332 (calcd. for C19H18NO2, 292.1338). 3.3.2. Mafaicheenamine E (2) Yellow solid; mp 209.2–212.1 8C; [a]27D – 14.3 (c 0.012, MeOH); UV lmax MeOH (nm) (log e): 246 (3.97), 264 (4.12), 278 (4.27), 295 (3.76), 310 (3.59), 325 (3.48), 339 (3.29); IR (neat) nmax cm1: 3300,
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