- Email: [email protected]
Clausenawallines A and B, two new dimeric carbazole alkaloids from the roots of Clausena wallichii
Tetrahedron Letters 52 (2011) 3303–3305
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Clausenawallines A and B, two new dimeric carbazole alkaloids from the roots of Clausena wallichii Wisanu Maneerat a, Thunwadee Ritthiwigrom b, Sarot Cheenpracha c, Uma Prawat d, 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 d Department of Chemistry, Faculty of Science and Technology, Phuket Rajabhat University, Rassada, Muang, Phuket 83000, Thailand b
a r t i c l e
i n f o
Article history: Received 11 March 2011 Revised 2 April 2011 Accepted 15 April 2011 Available online 23 April 2011 Keywords: Clausena wallichii Carbazole alkaloid Clausenawalline A Clausenawalline B Anti-malaria Anti-TB Cytotoxicity
a b s t r a c t Two new dimeric carbazole alkaloids, clausenawallines A and B, were isolated from the roots of Clausena wallichii. Their structures were elucidated by spectroscopic methods. Clausenawalline A was evaluated for its biological activities [anti-malaria (IC50 2.46 lg/mL), anti-TB (MIC 12.50 lg/mL)] and cytotoxicity against three human cancer cell lines [KB (IC50 7.87 lg/mL), MCF7 (IC50 25.43 lg/mL), and NCI-H187 (IC50 10.97 lg/mL)]. Ó 2011 Elsevier Ltd. All rights reserved.
Carbazole alkaloids are major compounds found in Rutaceae plants, especially in the Clausena, Murraya, and Glycosmis genera.1 Many of these alkaloids showed interesting pharmacological activities including anti-cancer, anti-malaria, anti-bacteria, anti-TB, and anti-HIV activities.2 In a continuing search for bioactive metabolites from Clausena plants,3 we report herein the isolation and structure elucidation of two new dimeric carbazole alkaloids, clausenawallines A (1) and B (2), from the roots of Clausena wallichii. The anti-malarial activity against Plasmodium falciparum, anti-TB activity against Mycobacterium tuberculosis, and cytotoxicity against three human cancer cell lines, KB, MCF7, and NCI-H187 of compound 1 are also reported. The air-dried C. wallichii roots (1.02 kg), collected in Phrae Province, Northern Thailand, in June 2010, were extracted with acetone over a period of three days at room temperature.4 The acetone extract (18.68 g) was subjected to rapid column chromatography over silica gel eluting with a gradient of two solvents (hexanes and EtOAc) by gradually increasing the polarity (100% hexanes to 100% EtOAc). The eluents were collected and analyzed by TLC giving seven fractions (A–I). Fraction C (2.01 g) was further separated by repeated column chromatography with 20% EtOAc–hexanes and ⇑ Corresponding author. Tel.: +66 5391 6238; fax: +66 5391 6776. E-mail addresses: [email protected], [email protected] (S. Laphookhieo). 0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetlet.2011.04.064
20% CH2Cl2–hexanes, respectively, to afford clausenawalline A (1, 4.5 mg, 0.00044% yield).5 Fraction D (3.21 g) was subjected to rapid column chromatography eluting with a gradient system of EtOAc– hexanes (20% EtOAc–hexanes to 100% EtOAc) to provide seven subfractions (D1–D7). Clausenawalline B (2, 2.5 mg, 0.00024% yield)6 was derived from sub-fraction D3 (1.18 g) by column chromatography using Sephadex-LH20 eluting with 100% MeOH, followed by column chromatography eluting with 100% CH2Cl2. Clausenawalline A (1) was obtained as a brown solid, mp 203.7– 204.3 °C. The molecular formula, C36H32N2O4, was deduced from the ESI–TOF–MS spectrum which showed the pseudomolecular ion peak at m/z 557.2421 [M+H]+ (calcd for 557.2440). The IR spectrum showed OH and NH stretching bands at 3523 and 3302 cm1, respectively, and the UV spectrum showed typical absorbances at kmax 230, 273, 334, 379, and 391 nm indicative of the carbazole alkaloid skeleton in 1.3 The fragment ion [M+C18H16NO2+H]+ at m/z 279, representative of half the molecule, in the MS spectrum as well as 1H and 13C NMR spectral data suggested that the structure of 1 was a highly symmetrical carbazole alkaloid dimer.7 The 1 H NMR spectral data of 1 (Table 1) demonstrated the presence of NH and aromatic methyl groups at d 9.49 and 2.49, respectively. The methyl group was placed on C-3 because of the 2J and 3J HMBC correlations (Table 1) of this signal with C-2 (d 152.8), C-4 (d 123.6), and C-3 (d 117.7). The chemical shift of C-2 at d 152.8 suggested that this carbon was attached to a hydroxy group. A singlet
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W. Maneerat et al. / Tetrahedron Letters 52 (2011) 3303–3305
Table 1 NMR spectral data of clausenawalline A (1) in acetone-d6 at 400 MHz
Table 2 NMR spectral data of clausenawalline B (2) in CDCl3 at 500 MHz
Position
dH (mult., J in Hz)
dC
HMBC (1H ?
1 2 3 4 4a 4b 5 6 7 8 8a 9a 10 20 30 40 50 3-CH3 9-NH
— — — 8.03 — — — — 6.73 7.08 — — 7.42 5.95 — 1.50 1.50 2.49 9.49
110.9 152.8 117.7 123.6 116.4 119.5 114.7 146.6 113.6 111.9 135.9 141.0 120.7 131.4 74.9 26.9 27.0 16.5 —
— — — C-2, C-4b, 3-CH3 — — — — C-5, C-6, C-8a C-4b, C-6 — — C-4b, C-5, C-6, C-30 C-5, C-30 , C-40 , C-50 — C-20 , C-30 , C-50 C-20 , C-30 , C-40 C-2, C-3, C-4 C-4a, C-4b, C-8a, C-9a
(s)
(d, 8.8) (d, 8.8)
(d, 10.0) (d, 10.0) (s) (s) (s) (br s)
13
C)
aromatic proton at d 8.03 was assigned to H-4 and correlated with C-2 (d 152.8), C-4b (d 119.5), and 3-CH3 (d 16.5) in the HMBC spectrum. A set of ortho-coupled aromatic protons at d 7.08 (1H, d, J = 8.8 Hz) and 6.73 (1H, d, J = 8.8 Hz) were assigned as H-7 and H-8, respectively, and was supported by the 2J and 3J HMBC correlation of H-8 with C-4b (d 119.5) and C-6 (d 146.6). A pyran moiety showed resonances at d 7.42 (1H, d, J = 10.0 Hz, H-10 ), 5.95 (1H, d, J = 10.0 Hz, H-20 ), and 1.50 (6H, s, H-40 and H-50 ) which was placed at C-5 and C-6 due to the 2J and 3J correlations of H-10 (d 7.42) with C-4b (d 119.5), C-5 (d 114.7), C-6 (d 146.6), of H-7 (d 6.73) with C-5 (d 114.7), C-6 (d 146.6), C-8a (d 135.9), and of H-8 (d 7.08) with C-6 (d 146.6) and C-4b (d 119.5) in the HMBC spectrum. These findings, along with the presence of a low-field singlet due to proton H-4 (and H-400 , d 8.03) and the lack of protons at C-1 (and C-100 ) in the 1H NMR spectral data revealed the C-1/C-100 carbon–carbon linkage between the two carbazole moieties as in glycoborinine.8 The 3J HMBC correlation of the NH proton (d 9.49) with C-1 (d 110.9) also supported the assigned structure. A detailed assignment of the protons and carbons of 1 is shown in Table 1. Clausenawalline B (2) was also obtained as a brown solid, mp 214.3–215.2 °C. It showed a pseudomolecular ion peak at [M+H]+ m/z 507.1901 (calcd for C31H27N2O5, 507.1920) in the ESI–TOF– MS. The IR and UV spectra were similar to those of clausenawalline A suggesting the presence of a carbazole alkaloid skeleton in 2 as well. The 1H, 13C NMR, and HMBC spectral data (Table 2) established the unsymmetrical carbazole-dimer of clausenawalline B. The 1H NMR data showed two fragments, A and B (Figure 1), for the carbazole moieties. Fragment B was a 7-methoxyheptaphylline derivative9 which displayed a broad singlet for the NH at d 10.48, a chelated hydrogen at d 11.70 (20 -OH), a singlet due to a formyl proton at d 9.87 (CHO) and a singlet aromatic proton due to H-40 at d 8.27. The location of the formyl group was determined from HMBC correlations of CHO (d 9.87) with C-20 (d 158.1), C-40 (d 124.9), and C-30 (d 116.0). The prenyl moiety was evident from 1H NMR signals at d 5.41 (1H, tt, J = 6.8 and 1.3 Hz, H-200 ), 3.67 (2H, d, J = 6.8 Hz, H100 ), 1.85 (3H, s, H-400 ), and 1.70 (3H, s, H-500 ). This moiety was placed at C-10 because of the correlations of H-10 0 (d 3.67) with C20 (d 158.1), C-9a0 (d 146.2), and C-10 (d 110.2). The 1H NMR spectrum also showed a singlet aromatic proton at d 7.16 and the presence of a methoxy group at d 4.00 which were identified as H-80 and 70 -OMe, respectively, on the basis of the 2J and 3J correlations of C-70 (d 148.5) with H-80 (d 7.16) and 70 -OMe (d 4.00) in the HMBC spectrum. In fragment A, the 1H NMR spectral data displayed a set of four spin proton signals at d 7.38 (1H, d, J = 7.8 Hz, H-5), 7.12 (1H, ddd,
Position
dH (mult., J in Hz)
dC
HMBC (1H ?
13
1 2 3 4 4a 4b 5 6 7 8 8a 9a 10 20 30 40 4a0 4b0 50 60 70 80 8a0 9a0 10 0 20 0 30 0 40 0 50 0 2-OH 20 -OH 30 -CHO 60 -OH 70 -OMe 9-NH 90 -NH
7.17 (s) — — 7.96 (s) — — 7.38 (d, 7.8) 7.12 (ddd, 8.2, 7.8, 1.3) 6.69 (ddd, 8.2, 7.8, 1.3) 6.97 (d, 8.2) — — — — — 8.27 (s) — — — — — 7.16 (s) — — 3.67 (d, 6.8) 5.41 (tt, 6.8, 1.3) — 1.85 (s) 1.70 (s) 7.55 (br s) 11.70 (s) 9.87 (s) 6.95 (br s) 4.00 (s) 10.06 (br s) 10.48 (br s)
98.9 155.3 117.8 123.4 118.9 124.7 111.0 124.3 118.7 121.6 141.1 143.4 110.2 158.1 116.0 124.9 118.6 117.7 118.7 139.7 148.5 94.3 135.0 146.2 23.5 122.4 133.1 25.8 18.1 — — 196.7 — 56.6 — —
C-2, C-3, C-4a, C-9a — — C-3, C-9a, C-50 — — C-7, C-8a C-4b, C-8 C-5, C-6, C-8a C-4b, C-6, C-8a — — — — — C-20 , C-4a0 , C-9a0 , 30 -CHO — — — — — C-4b0 , C-70 , C-8a0 — — C-10 , C-20 , C-9a0 , C-30 0 C-10 , C-10 0 , C-40 0 , C-50 0 — C-20 0 , C-30 0 , C-50 0 C-20 0 , C-30 0 , C-40 0 — C-10 , C-20 , C-30 C-20 , C-30 , C-40 — C-70 C-4a, C-4b, C-8a, C-9a C-4a0 , C-4b0 , C-8a0 , C-9a0
C)
J = 8.2, 7.8, and 1.3 Hz, H-6), 6.97 (1H, d, J = 8.2 Hz, H-8), and 6.69 (1H, ddd, J = 8.2, 7.8, and 1.3 Hz, H-7)3b which were attributed to H-5, H-6, H-8, and H-7, respectively, and two singlets for the aromatic protons at d 7.17 and 7.96 were assigned to H-1 and H-4, respectively. The connectivity of both fragments was confirmed by HMBC correlations. The 2J and 3J HMBC correlations of H-4 (d 7.96) with C-3 (d 117.8) and C-50 (d 118.7) confirmed the carbon–carbon linkage of carbazole fragments A and B at C-3 and C50 , respectively, as well as the lack of proton signals for H-3 and H-50 in the 1H NMR spectrum. Detailed assignments of the protons and carbons of 2 are shown in Table 2. Clausena species are medicinal plants which are rich sources of carbazole alkaloids, however, they rarely produce dimeric carbazole alkaloids. Less than five carbazole dimers have been isolated from this genus. To the best of our knowledge, clausenawalline B is the first example of an unsymmetrical dimeric carbazole alkaloid isolated from Clausena plants. The C-1/C-10 0 carbon–carbon linkage of the symmetrical dimeric carbazole alkaloid of clausenawalline A is also the first example of this type from Clausena plants. Clausenawalline A was further evaluated for its biological activities including anti-malarial activity against P. falciparum, anti-TB activity against M. tuberculosis, and cytotoxicity against oral cavity cancer (KB), breast cancer (MCF7), and small cell lung cancer (NCIH187). Clausenawalline A exhibited strong anti-malarial activity against P. falciparum with an IC50 value of 2.46 lg/mL. Moderate activities were observed for anti-TB activity against M. tuberculosis (MIC 12.50 lg/mL) and cytotoxicity against KB (IC50 7.87 lg/mL) and NCI-H187 (IC50 10.97 lg/mL) cancer cell lines, whereas the cytotoxicity against the MCF7 (IC50 25.43 lg/mL) cancer cell line was weak. Compound 2 was not evaluated for its biological activity due to the small quantity of material obtained.
W. Maneerat et al. / Tetrahedron Letters 52 (2011) 3303–3305
3305
Figure 1. Structures of clausenwallines A (1) and B (2).
Acknowledgements This work was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant no. PHD/ 0006/2552) to W.M. and S.L. Mae Fah Luang University is acknowledged for partial financial support and laboratory facilities. We are indebted to Mr. Nitirat Chimnoi, Chulabhorn Research Institute, Bangkok, for recording mass spectra and the Bioassay Research Facility of BIOTEC (Thailand) for biological activity tests. References and notes 1. (a) Ito, C.; Katsuni, S.; Ohta, H.; Omura, M.; Kajiura, I.; Furukawa, H. Chem. Pharm. Bull. 1997, 45, 48–52; (b) Li, W. S.; McChesney, J. D.; El-Feraly, F. S. Phytochemistry 1991, 30, 343–346; (c) Ito, C.; Thoyama, Y.; Omura, M.; Kajiura, I.; Furukawa, H. Chem. Pharm. Bull. 2010, 41, 2096–2100; (d) Wu, T. S.; Wang, M. L.; Wu, P. L. Tetrahedron Lett. 1995, 36, 5385–5388. 2. (a) Kongkathip, N.; Kongkathip, B. Heterocycles 2009, 79, 121–144; (b) Rahman, M. M.; Gray, A. I. Phytochemistry 2005, 66, 1602–1606; (c) Thongthoom, T.; Songsiang, U.; Phaosiri, C.; Yenjai, C. Arch. Pharm. Res. 2010, 33, 675–680.
3. (a) Maneerat, W.; Prawat, U.; Saewan, N.; Laphookhieo, S. J. Braz. Chem. Soc. 2010, 21, 655–668; (b) Maneerat, W.; Laphookhieo, S. Heterocycles 2010, 81, 1261–1269; (c) Sripisut, T.; Laphookhieo, S. J. Asian Nat. Prod. Res. 2010, 12, 614– 617. 4. The plant was identified by Dr. Monthon Norsangsri and a voucher specimen (No. QBG 4533) has been deposited at the Herbarium of Queen Sirikit Botanic Garden, Mae Rim, Chiang Mai, Thailand. 5. Clausenawalline A (1): brown solid, mp 203.7–204.3 °C. UV (MeOH) kmax: 230, 273, 334, 379, 391 nm. IR (neat) mmax: 3523, 3302 cm1. 1H NMR (400 MHz, acetone-d6) and 13C NMR (100 MHz, acetone-d6) see Table 1. ESI–TOF–MS m/z 557.2421 [M+H]+ (calcd for C36H33N2O4, 557.2440). 6. Clausenawalline B (2): brown solid, mp 214.3–215.2 °C. UV (MeOH) kmax: 214, 236, 256, 306, 349, 538 nm. IR (neat) mmax: 3372, 1725, 1615 cm1. 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) see Table 2. ESI–TOF–MS m/z 507.1901 [M+H]+ (calcd for C31H27N2O5, 507.1920). 7. (a) Wu, T. S.; Huang, S. C.; Wu, P. L. Tetrahedron 1996, 37, 7819–7822; (b) Cuong, N. M.; Hung, T. Q.; Sung, T. V.; Taylor, W. C. Chem. Pharm. Bull. 2004, 52, 1175– 1178. 8. Chakravarty, A. K.; Sarkar, T.; Masuda, K.; Shiojima, K. Phytochemistry 1999, 50, 1263–1266. 9. Songsiang, U.; Thongthoom, T.; Boonyarat, C.; Yenjai, C. J. Nat. Prod. 2011, 74, 208–212.
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Clausenawallines A and B, two new dimeric carbazole alkaloids from the roots of Clausena wallichii Wisanu Maneerat a, Thunwadee Ritthiwigrom b, Sarot Cheenpracha c, Uma Prawat d, 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 d Department of Chemistry, Faculty of Science and Technology, Phuket Rajabhat University, Rassada, Muang, Phuket 83000, Thailand b
a r t i c l e
i n f o
Article history: Received 11 March 2011 Revised 2 April 2011 Accepted 15 April 2011 Available online 23 April 2011 Keywords: Clausena wallichii Carbazole alkaloid Clausenawalline A Clausenawalline B Anti-malaria Anti-TB Cytotoxicity
a b s t r a c t Two new dimeric carbazole alkaloids, clausenawallines A and B, were isolated from the roots of Clausena wallichii. Their structures were elucidated by spectroscopic methods. Clausenawalline A was evaluated for its biological activities [anti-malaria (IC50 2.46 lg/mL), anti-TB (MIC 12.50 lg/mL)] and cytotoxicity against three human cancer cell lines [KB (IC50 7.87 lg/mL), MCF7 (IC50 25.43 lg/mL), and NCI-H187 (IC50 10.97 lg/mL)]. Ó 2011 Elsevier Ltd. All rights reserved.
Carbazole alkaloids are major compounds found in Rutaceae plants, especially in the Clausena, Murraya, and Glycosmis genera.1 Many of these alkaloids showed interesting pharmacological activities including anti-cancer, anti-malaria, anti-bacteria, anti-TB, and anti-HIV activities.2 In a continuing search for bioactive metabolites from Clausena plants,3 we report herein the isolation and structure elucidation of two new dimeric carbazole alkaloids, clausenawallines A (1) and B (2), from the roots of Clausena wallichii. The anti-malarial activity against Plasmodium falciparum, anti-TB activity against Mycobacterium tuberculosis, and cytotoxicity against three human cancer cell lines, KB, MCF7, and NCI-H187 of compound 1 are also reported. The air-dried C. wallichii roots (1.02 kg), collected in Phrae Province, Northern Thailand, in June 2010, were extracted with acetone over a period of three days at room temperature.4 The acetone extract (18.68 g) was subjected to rapid column chromatography over silica gel eluting with a gradient of two solvents (hexanes and EtOAc) by gradually increasing the polarity (100% hexanes to 100% EtOAc). The eluents were collected and analyzed by TLC giving seven fractions (A–I). Fraction C (2.01 g) was further separated by repeated column chromatography with 20% EtOAc–hexanes and ⇑ Corresponding author. Tel.: +66 5391 6238; fax: +66 5391 6776. E-mail addresses: [email protected], [email protected] (S. Laphookhieo). 0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetlet.2011.04.064
20% CH2Cl2–hexanes, respectively, to afford clausenawalline A (1, 4.5 mg, 0.00044% yield).5 Fraction D (3.21 g) was subjected to rapid column chromatography eluting with a gradient system of EtOAc– hexanes (20% EtOAc–hexanes to 100% EtOAc) to provide seven subfractions (D1–D7). Clausenawalline B (2, 2.5 mg, 0.00024% yield)6 was derived from sub-fraction D3 (1.18 g) by column chromatography using Sephadex-LH20 eluting with 100% MeOH, followed by column chromatography eluting with 100% CH2Cl2. Clausenawalline A (1) was obtained as a brown solid, mp 203.7– 204.3 °C. The molecular formula, C36H32N2O4, was deduced from the ESI–TOF–MS spectrum which showed the pseudomolecular ion peak at m/z 557.2421 [M+H]+ (calcd for 557.2440). The IR spectrum showed OH and NH stretching bands at 3523 and 3302 cm1, respectively, and the UV spectrum showed typical absorbances at kmax 230, 273, 334, 379, and 391 nm indicative of the carbazole alkaloid skeleton in 1.3 The fragment ion [M+C18H16NO2+H]+ at m/z 279, representative of half the molecule, in the MS spectrum as well as 1H and 13C NMR spectral data suggested that the structure of 1 was a highly symmetrical carbazole alkaloid dimer.7 The 1 H NMR spectral data of 1 (Table 1) demonstrated the presence of NH and aromatic methyl groups at d 9.49 and 2.49, respectively. The methyl group was placed on C-3 because of the 2J and 3J HMBC correlations (Table 1) of this signal with C-2 (d 152.8), C-4 (d 123.6), and C-3 (d 117.7). The chemical shift of C-2 at d 152.8 suggested that this carbon was attached to a hydroxy group. A singlet
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W. Maneerat et al. / Tetrahedron Letters 52 (2011) 3303–3305
Table 1 NMR spectral data of clausenawalline A (1) in acetone-d6 at 400 MHz
Table 2 NMR spectral data of clausenawalline B (2) in CDCl3 at 500 MHz
Position
dH (mult., J in Hz)
dC
HMBC (1H ?
1 2 3 4 4a 4b 5 6 7 8 8a 9a 10 20 30 40 50 3-CH3 9-NH
— — — 8.03 — — — — 6.73 7.08 — — 7.42 5.95 — 1.50 1.50 2.49 9.49
110.9 152.8 117.7 123.6 116.4 119.5 114.7 146.6 113.6 111.9 135.9 141.0 120.7 131.4 74.9 26.9 27.0 16.5 —
— — — C-2, C-4b, 3-CH3 — — — — C-5, C-6, C-8a C-4b, C-6 — — C-4b, C-5, C-6, C-30 C-5, C-30 , C-40 , C-50 — C-20 , C-30 , C-50 C-20 , C-30 , C-40 C-2, C-3, C-4 C-4a, C-4b, C-8a, C-9a
(s)
(d, 8.8) (d, 8.8)
(d, 10.0) (d, 10.0) (s) (s) (s) (br s)
13
C)
aromatic proton at d 8.03 was assigned to H-4 and correlated with C-2 (d 152.8), C-4b (d 119.5), and 3-CH3 (d 16.5) in the HMBC spectrum. A set of ortho-coupled aromatic protons at d 7.08 (1H, d, J = 8.8 Hz) and 6.73 (1H, d, J = 8.8 Hz) were assigned as H-7 and H-8, respectively, and was supported by the 2J and 3J HMBC correlation of H-8 with C-4b (d 119.5) and C-6 (d 146.6). A pyran moiety showed resonances at d 7.42 (1H, d, J = 10.0 Hz, H-10 ), 5.95 (1H, d, J = 10.0 Hz, H-20 ), and 1.50 (6H, s, H-40 and H-50 ) which was placed at C-5 and C-6 due to the 2J and 3J correlations of H-10 (d 7.42) with C-4b (d 119.5), C-5 (d 114.7), C-6 (d 146.6), of H-7 (d 6.73) with C-5 (d 114.7), C-6 (d 146.6), C-8a (d 135.9), and of H-8 (d 7.08) with C-6 (d 146.6) and C-4b (d 119.5) in the HMBC spectrum. These findings, along with the presence of a low-field singlet due to proton H-4 (and H-400 , d 8.03) and the lack of protons at C-1 (and C-100 ) in the 1H NMR spectral data revealed the C-1/C-100 carbon–carbon linkage between the two carbazole moieties as in glycoborinine.8 The 3J HMBC correlation of the NH proton (d 9.49) with C-1 (d 110.9) also supported the assigned structure. A detailed assignment of the protons and carbons of 1 is shown in Table 1. Clausenawalline B (2) was also obtained as a brown solid, mp 214.3–215.2 °C. It showed a pseudomolecular ion peak at [M+H]+ m/z 507.1901 (calcd for C31H27N2O5, 507.1920) in the ESI–TOF– MS. The IR and UV spectra were similar to those of clausenawalline A suggesting the presence of a carbazole alkaloid skeleton in 2 as well. The 1H, 13C NMR, and HMBC spectral data (Table 2) established the unsymmetrical carbazole-dimer of clausenawalline B. The 1H NMR data showed two fragments, A and B (Figure 1), for the carbazole moieties. Fragment B was a 7-methoxyheptaphylline derivative9 which displayed a broad singlet for the NH at d 10.48, a chelated hydrogen at d 11.70 (20 -OH), a singlet due to a formyl proton at d 9.87 (CHO) and a singlet aromatic proton due to H-40 at d 8.27. The location of the formyl group was determined from HMBC correlations of CHO (d 9.87) with C-20 (d 158.1), C-40 (d 124.9), and C-30 (d 116.0). The prenyl moiety was evident from 1H NMR signals at d 5.41 (1H, tt, J = 6.8 and 1.3 Hz, H-200 ), 3.67 (2H, d, J = 6.8 Hz, H100 ), 1.85 (3H, s, H-400 ), and 1.70 (3H, s, H-500 ). This moiety was placed at C-10 because of the correlations of H-10 0 (d 3.67) with C20 (d 158.1), C-9a0 (d 146.2), and C-10 (d 110.2). The 1H NMR spectrum also showed a singlet aromatic proton at d 7.16 and the presence of a methoxy group at d 4.00 which were identified as H-80 and 70 -OMe, respectively, on the basis of the 2J and 3J correlations of C-70 (d 148.5) with H-80 (d 7.16) and 70 -OMe (d 4.00) in the HMBC spectrum. In fragment A, the 1H NMR spectral data displayed a set of four spin proton signals at d 7.38 (1H, d, J = 7.8 Hz, H-5), 7.12 (1H, ddd,
Position
dH (mult., J in Hz)
dC
HMBC (1H ?
13
1 2 3 4 4a 4b 5 6 7 8 8a 9a 10 20 30 40 4a0 4b0 50 60 70 80 8a0 9a0 10 0 20 0 30 0 40 0 50 0 2-OH 20 -OH 30 -CHO 60 -OH 70 -OMe 9-NH 90 -NH
7.17 (s) — — 7.96 (s) — — 7.38 (d, 7.8) 7.12 (ddd, 8.2, 7.8, 1.3) 6.69 (ddd, 8.2, 7.8, 1.3) 6.97 (d, 8.2) — — — — — 8.27 (s) — — — — — 7.16 (s) — — 3.67 (d, 6.8) 5.41 (tt, 6.8, 1.3) — 1.85 (s) 1.70 (s) 7.55 (br s) 11.70 (s) 9.87 (s) 6.95 (br s) 4.00 (s) 10.06 (br s) 10.48 (br s)
98.9 155.3 117.8 123.4 118.9 124.7 111.0 124.3 118.7 121.6 141.1 143.4 110.2 158.1 116.0 124.9 118.6 117.7 118.7 139.7 148.5 94.3 135.0 146.2 23.5 122.4 133.1 25.8 18.1 — — 196.7 — 56.6 — —
C-2, C-3, C-4a, C-9a — — C-3, C-9a, C-50 — — C-7, C-8a C-4b, C-8 C-5, C-6, C-8a C-4b, C-6, C-8a — — — — — C-20 , C-4a0 , C-9a0 , 30 -CHO — — — — — C-4b0 , C-70 , C-8a0 — — C-10 , C-20 , C-9a0 , C-30 0 C-10 , C-10 0 , C-40 0 , C-50 0 — C-20 0 , C-30 0 , C-50 0 C-20 0 , C-30 0 , C-40 0 — C-10 , C-20 , C-30 C-20 , C-30 , C-40 — C-70 C-4a, C-4b, C-8a, C-9a C-4a0 , C-4b0 , C-8a0 , C-9a0
C)
J = 8.2, 7.8, and 1.3 Hz, H-6), 6.97 (1H, d, J = 8.2 Hz, H-8), and 6.69 (1H, ddd, J = 8.2, 7.8, and 1.3 Hz, H-7)3b which were attributed to H-5, H-6, H-8, and H-7, respectively, and two singlets for the aromatic protons at d 7.17 and 7.96 were assigned to H-1 and H-4, respectively. The connectivity of both fragments was confirmed by HMBC correlations. The 2J and 3J HMBC correlations of H-4 (d 7.96) with C-3 (d 117.8) and C-50 (d 118.7) confirmed the carbon–carbon linkage of carbazole fragments A and B at C-3 and C50 , respectively, as well as the lack of proton signals for H-3 and H-50 in the 1H NMR spectrum. Detailed assignments of the protons and carbons of 2 are shown in Table 2. Clausena species are medicinal plants which are rich sources of carbazole alkaloids, however, they rarely produce dimeric carbazole alkaloids. Less than five carbazole dimers have been isolated from this genus. To the best of our knowledge, clausenawalline B is the first example of an unsymmetrical dimeric carbazole alkaloid isolated from Clausena plants. The C-1/C-10 0 carbon–carbon linkage of the symmetrical dimeric carbazole alkaloid of clausenawalline A is also the first example of this type from Clausena plants. Clausenawalline A was further evaluated for its biological activities including anti-malarial activity against P. falciparum, anti-TB activity against M. tuberculosis, and cytotoxicity against oral cavity cancer (KB), breast cancer (MCF7), and small cell lung cancer (NCIH187). Clausenawalline A exhibited strong anti-malarial activity against P. falciparum with an IC50 value of 2.46 lg/mL. Moderate activities were observed for anti-TB activity against M. tuberculosis (MIC 12.50 lg/mL) and cytotoxicity against KB (IC50 7.87 lg/mL) and NCI-H187 (IC50 10.97 lg/mL) cancer cell lines, whereas the cytotoxicity against the MCF7 (IC50 25.43 lg/mL) cancer cell line was weak. Compound 2 was not evaluated for its biological activity due to the small quantity of material obtained.
W. Maneerat et al. / Tetrahedron Letters 52 (2011) 3303–3305
3305
Figure 1. Structures of clausenwallines A (1) and B (2).
Acknowledgements This work was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant no. PHD/ 0006/2552) to W.M. and S.L. Mae Fah Luang University is acknowledged for partial financial support and laboratory facilities. We are indebted to Mr. Nitirat Chimnoi, Chulabhorn Research Institute, Bangkok, for recording mass spectra and the Bioassay Research Facility of BIOTEC (Thailand) for biological activity tests. References and notes 1. (a) Ito, C.; Katsuni, S.; Ohta, H.; Omura, M.; Kajiura, I.; Furukawa, H. Chem. Pharm. Bull. 1997, 45, 48–52; (b) Li, W. S.; McChesney, J. D.; El-Feraly, F. S. Phytochemistry 1991, 30, 343–346; (c) Ito, C.; Thoyama, Y.; Omura, M.; Kajiura, I.; Furukawa, H. Chem. Pharm. Bull. 2010, 41, 2096–2100; (d) Wu, T. S.; Wang, M. L.; Wu, P. L. Tetrahedron Lett. 1995, 36, 5385–5388. 2. (a) Kongkathip, N.; Kongkathip, B. Heterocycles 2009, 79, 121–144; (b) Rahman, M. M.; Gray, A. I. Phytochemistry 2005, 66, 1602–1606; (c) Thongthoom, T.; Songsiang, U.; Phaosiri, C.; Yenjai, C. Arch. Pharm. Res. 2010, 33, 675–680.
3. (a) Maneerat, W.; Prawat, U.; Saewan, N.; Laphookhieo, S. J. Braz. Chem. Soc. 2010, 21, 655–668; (b) Maneerat, W.; Laphookhieo, S. Heterocycles 2010, 81, 1261–1269; (c) Sripisut, T.; Laphookhieo, S. J. Asian Nat. Prod. Res. 2010, 12, 614– 617. 4. The plant was identified by Dr. Monthon Norsangsri and a voucher specimen (No. QBG 4533) has been deposited at the Herbarium of Queen Sirikit Botanic Garden, Mae Rim, Chiang Mai, Thailand. 5. Clausenawalline A (1): brown solid, mp 203.7–204.3 °C. UV (MeOH) kmax: 230, 273, 334, 379, 391 nm. IR (neat) mmax: 3523, 3302 cm1. 1H NMR (400 MHz, acetone-d6) and 13C NMR (100 MHz, acetone-d6) see Table 1. ESI–TOF–MS m/z 557.2421 [M+H]+ (calcd for C36H33N2O4, 557.2440). 6. Clausenawalline B (2): brown solid, mp 214.3–215.2 °C. UV (MeOH) kmax: 214, 236, 256, 306, 349, 538 nm. IR (neat) mmax: 3372, 1725, 1615 cm1. 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) see Table 2. ESI–TOF–MS m/z 507.1901 [M+H]+ (calcd for C31H27N2O5, 507.1920). 7. (a) Wu, T. S.; Huang, S. C.; Wu, P. L. Tetrahedron 1996, 37, 7819–7822; (b) Cuong, N. M.; Hung, T. Q.; Sung, T. V.; Taylor, W. C. Chem. Pharm. Bull. 2004, 52, 1175– 1178. 8. Chakravarty, A. K.; Sarkar, T.; Masuda, K.; Shiojima, K. Phytochemistry 1999, 50, 1263–1266. 9. Songsiang, U.; Thongthoom, T.; Boonyarat, C.; Yenjai, C. J. Nat. Prod. 2011, 74, 208–212.