Tetrahedron Letters 53 (2012) 1227–1230 Contents lists available at SciVerse ScienceDirect Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet Leucomidines A–C, novel alkaloids from Leuconotis griffithii Midori Motegi a, Alfarius Eko Nugroho a, Yusuke Hirasawa a, Takashi Arai a, A. Hamid A. Hadi b, Hiroshi Morita a,⇑ a b Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41, Shinagawa-ku, Tokyo 142-8501, Japan Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia a r t i c l e i n f o Article history: Received 24 November 2011 Revised 20 December 2011 Accepted 26 December 2011 Available online 2 January 2012 a b s t r a c t Two novel indole alkaloids and new quinazoline alkaloids, namely leucomidines A–C (1–3), were isolated from the barks of Leuconotis griffithii. The structures including the absolute configuration were elucidated on the basis of spectroscopic data, chemical means, and CD calculations. The quinazoline alkaloid, 3, is proposed to be derived from the same biogenetic precursor as the indole alkaloids 1 and 2. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: Indole alkaloid Quinazoline alkaloid Leuconotis griffithii Apocynaceae Leuconotis griffithii (Retz.) Gardner ex Thwaites is a member of the Apocynaceae family in Malaysia and Indonesia.1 The species of Leuconotis have been known to produce monoterpene indole alkaloids,2,3 whose skeletons are similar to those found in Alstonia and Kopsia species.4 The species of Alstonia, Kopsia, Hunteria, Tabernaemontana, and Leuconotis have been known to produce various alkaloids depending on the area where the plants were distributed.2–7 Recently, we isolated a new bisindole alkaloid, bisleuconothine A consisting of an eburnane–aspidosperma-type skeleton from the barks of L. griffithii.5 In our continuing search for structurally and biogenetically interesting alkaloids from tropical plants, we isolated three novel alkaloids, leucomidines A–C ( 1–3), together with leuconolam4 and O-methylleuconolam.4 In this Letter, the isolation and structure elucidation of 1–3 are described. Leucomidine A (1),8,9 yellowish amorphous solid, ½a24 +69 D (c 0.3, MeOH), showed molecular formula, C19H20N2O3, which was determined by HRESIMS [m/z 347.1380 (M+Na)+, D +0.1 mmu]. IR absorption bands were characteristic of amino or hydroxyl (3390 cm1) group and two carbonyl (1800 and 1730 cm1) groups. 1H and 13C NMR data (Table 1) suggested the presence of six sp3 methylenes, a methyl, two sp3 quaternary carbons, four sp2 methines, and six sp2 quaternary carbons. ⇑ Corresponding author. Tel./fax: +81 354985778. E-mail address: moritah@hoshi.ac.jp (H. Morita). 0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetlet.2011.12.116 The gross structure of 1 was deduced from extensive analyses of the two-dimensional NMR data, including the 1H–1H COSY, HSQC, and HMBC spectra in CD3OD (Fig. 1). The 1H–1H COSY and HSQC spectra revealed connectivity of four partial structures a (C-9–C12), b (C-3, C-14–C-15), c (C-16–C-17), and d (C-18–C-19), as 1228 M. Motegi et al. / Tetrahedron Letters 53 (2012) 1227–1230 Table 1 1 H (600 MHz) and 13 C (150 MHz) NMR data of leucomidines A–C (1–3) 1a 2b dH (J, Hz) 2 3a 3b 5 6 7 8 9 10 11 12 13 14a 14b 15a 15b 16a 16b 17a 17b 18 19a 19b 20 21 OMe a b dC 2.73 (1H, ddd, 13.1, 13.0, 4.2) 4.25 (1H, dd, 13.1, 4.9) 6.98 6.98 7.11 7.35 (1H, (1H, (1H, (1H, br s) d, 6.1) ddd, 8.1, 6.1, 2.1) d, 8.1) 1.66 1.53 1.74 1.73 2.91 (1H, (1H, (1H, (1H, (2H, m) br dd, 13.1, 3.1) dd, 9.1, 3.1) br s) m) 2.05 2.02 0.92 1.46 1.32 (1H, (1H, (3H, (1H, (1H, dt, 7.6, 3.2) ddd, 18.4, 13.9 10.1) t, 7.6) dq, 14.9, 7.6) dq, 14.9, 7.6) 141.8 40.7 154.1 161.1 103.6 125.4 117.6 121.5 123.0 112.7 138.3 20.7 26.6 19.9 29.5 7.6 23.1 42.7 96.1 3b dH (J, Hz) dC 2.98 (1H, ddd, 12.1, 7.9, 3.8) 4.30 (1H, br d, 12.1) 7.68 7.16 7.27 7.49 (1H, (1H, (1H, (1H, d, 8.2) br t, 8.2) br t, 8.2) d, 8.2) 1.60 1.69 1.59 1.76 2.60 2.74 2.18 (1H, (1H, (1H, (1H, (1H, (1H, (2H, m) m) m) br d, 11.7) m) m) t, 8.6) 0.58 (3H, t, 7.6) 0.67 (1H, dq, 14.5, 7.6) 1.12 (1H, dq, 14.5, 7.6) 4.36 (1H, s) 3.78 (3H, s) 175.3 40.1 162.8 127.6 135.2 128.2 121.4 122.5 124.8 114.1 142.5 21.2 30.1 29.2 32.7 7.6 23.7 40.2 63.8 52.4 dH (J, Hz) dC 175.4 49.0 4.01 (1H, m) 4.10 (1H, m) 8.17 7.47 7.78 7.64 (1H, (1H, (1H, (1H, dd, 8.2, 1.0) ddd, 8.2, 6.9, 1.3) ddd, 8.3, 6.9, 1.0) br d 8.3) 2.05 (2H, m) 1.85 1.99 2.30 2.54 2.12 2.27 0.88 1.76 2.11 (1H, (1H, (1H, (1H, (1H, (1H, (3H, (1H, (1H, ddd, 12.4, 6.9, 12.4, 8.3, 4.1) ddd, 15.8, 8.9, ddd, 15.8, 8.9, ddd, 14.8, 8.9, ddd, 14.8, 8.9, t, 7.6) dq, 14.8, 7.6) dq, 14.8, 7.6) 4.5) 29.3 6.1) 6.1) 6.1) 6.1) 29.1 3.41 (3H, s) In CD3OD. In CD3OD/CDCl3. Figure 1. Selected 2D NMR correlations for leucomidine A (1). Figure 3. Selected 2D NMR correlations for leucomidine B (2). Scheme 1. Reduction of leucomidine A (1) by LiAlH4. Figure 2. Selected NOESY correlations for leucomidine A (1, 20R⁄, 21S⁄). shown in Figure 1. The presence of an indole ring (C-2, C-7–C-13, and N-1) and the connectivity of partial structure c and the indole ring were revealed by the HMBC correlations of H-9 to C-7 and 164.0 120.5 126.8 127.5 135.0 127.7 148.3 19.7 Figure 4. Selected NOESY correlations for leucomidine B (2). 35.8 8.4 32.6 44.6 160.0 51.4 M. Motegi et al. / Tetrahedron Letters 53 (2012) 1227–1230 Figure 5. Selected 2D NMR correlations for leucomidine C (3). Figure 6. Calculated and experimental CD spectra of 1 (left scale) and 2 (right scale). C-13, H-10 and H-12 to C-8, H2-16 to C-7, and H2-17 to C-2. HMBC correlations of H3-18 to C-20, H2-19 to C-15, C-17, and C-21 established the connections among C-15, C-17, C-19, and C-21 through C-20. The HMBC cross-peaks of H2-3 to C-5 and C-21 and the chemical shift of C-3 (dC 40.7), C-5 (dC 154.1) and C-21 (dC 96.1) suggested the connection among C-3, C-5 and C-21 through a nitrogen atom. Based on the chemical shift of C-21 and the 1229 remaining carbonyl carbon (dC 161.1), the structure of 1 was assumed to be as shown in Figure 1. To clarify the structure, 1 was reduced by LiAlH4 (Scheme 1) to obtain 4 and 5 whose structures further support the proposed structure of 1 as shown in Figure 1.10 The lack of proton on C-5 and C-6 complicates the determination of the relative configuration of C-20 and C-21. Fortunately, the 20R⁄,21R⁄ and the 20R⁄,21S⁄ isomers have different stable conformation of the piperidine unit (boat and chair, respectively). The NOESY correlations and coupling constant data suggested that the piperidine unit in 1 took a chair conformation (Fig. 2), thus the relative configuration of 1 was deduced to be 20R⁄, 21S⁄. Leucomidine B (2),11 yellowish amorphous solid, ½a26 D 18 (c 0.3, CHCl3), showed molecular formula, C20H24N2O3, which was determined by HRESIMS [m/z 341.1840 (M+H)+, D 2.0 mmu]. IR absorption bands were characteristic of amino or hydroxyl (3400 cm1) and two carbonyl (1740 and 1670 cm1) groups. 1H and 13C NMR data (Table 1) suggested the presence of a sp3 methine, six sp3 methylenes, two methyls, a sp3 quaternary carbon, four sp2 methines, and six sp2 quaternary carbons. The planar structure of 2 was deduced from extensive analyses of the two-dimensional NMR data (1H–1H COSY, HSQC, and HMBC spectra, Fig. 3). The 1H–1H COSY and HSQC spectra revealed four partial structures a (C-9–C-12), b (C-3, C-14–C-15), c (C-18–C19), and d (C-16–C-17), as shown in Figure 3. HMBC correlations of H3-18 to C-20, H2-19 to C-17 and C-21, and H2-17 to C-15 suggested the connection of C-15, C-17, C-19, and C-21 through C-20. The presence of a piperidine ring system was deduced from the chemical shift of C-3 (dC 40.1) and C-21 (dC 63.8), and from the HMBC correlations of H-21 to C-3. HMBC correlations of H2-17 and H3-OMe to C-2 revealed the connection of a methoxy carbonyl group at C-16. Finally, the HMBC cross-peaks of H-21 to C-5, C-6, and C-7 were used to deduce the presence of a pyrolone ring system bridging the indole and the piperidine units. The relative configuration of 2 was determined by NOESY spectral data (Fig. 4). The b-orientation of H-21 and C-17 was suggested by NOESY correlations of H2-17/H-21. Leucomidine C (3),12 yellowish amorphous solid, ½a26 D +32 (c 0.3, CHCl3), showed molecular formula, C18H22N2O3, which was Figure 7. Plausible biogenetic pathway of 1–3. 1230 M. Motegi et al. / Tetrahedron Letters 53 (2012) 1227–1230 determined by HRESIMS [m/z 315.1685 (M+H)+, D 1.8 mmu]. IR absorption bands suggested the presence of two carbonyl (1740 and 1680 cm1) groups. 1H and 13C NMR data (Table 1) of 3 showed characteristic signals13 of a 2,3-disubstituted 4(3H)-quinazolinones ring [dC 164.0 (C-7), 120.5 (C-8), 126.8 (C-9), 127.5 (C-10), 135.0 (C-11), 127.7 (C-12), 148.3 (C-13) and 160.0 (C-21); dH 8.17 (H-9), 7.47 (H-10), 7.78 (H-11) and 7.64 (H-12)]. In addition to the quinazolinone ring, the 1H and 13C NMR data suggested the presence of one sp3 quaternary carbon, six sp3 methylenes, two methyls, and one sp2 quaternary carbon. Analyses of the 1H–1H COSY and HSQC spectra (Fig. 5) revealed the presence of partial structure b (C-3, C-14–C-15), c (C-18–C-19), and d (C-16–C-17), in addition to partial structure a (C-9–C-12) which is part of the quinazoline ring. HMBC correlations of H3-18 to C-20, H2-19 to C-15, C-17, and C-21 suggested the connection of C-15, C-17, C-19, and C-21 through C-20 while the HMBC correlation of H2-3 to C-21 and the chemical shift of C-3 indicated that C-3 is connected to N-4. Finally, the presence of a methoxy carbonyl group at C-16 was deduced from the HMBC correlation of H2-17 and H3-OMe to C-2. Thus, the structure of 3 was deduced to be as shown in Fig. 5. The absolute configuration of 1 and 2 were determined by comparing the calculated14 and the experimental CD spectra. As can be seen in Fig. 6, the calculated CD spectra of the 20R, 21S isomer of both compounds are similar to the experimental one. Thus, the absolute configurations of 1 and 2 were assigned as 20R, 21S. Biogenetically,1–3 are assumed to be derived from leuconolam4 (Fig. 7). Oxidation of C-6 and C-7 of leuconolam may give precursor A. 1 might be produced by ring closures of A, whereas hydrolysis of A followed by ring closures and reduction of OH on C-21 to H might give 2. Further oxidation of A followed by hydrolysis will give B, and 3 may be obtained from intermediate C after several steps. Considering the biogenetic route, though the absolute configuration of 3 could not be assigned by using CD calculation, the absolute configuration at C-20 of 3 is proposed to be R. Leucomidines A–C (1–3) were tested for cytotoxic activity against HL-60 cell line.15 All three compounds were found to be inactive (IC50 >100 lM). Acknowledgments This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, a grant from the Open Research Center Project and Takeda Science Foundation, and also a grant from HIR UM-MOHE (F000009-21001). References and notes 1. Whitmore, T. C. In Tree Flora Malaysia; Whitmore, T. C., Ed.; Academic Press: London, 1972; Vol. 2, pp 3–24. 2. Gan, C. Y.; Robinson, W. T.; Etoh, T.; Hayashi, M.; Komiyama, K.; Kam, T. S. Org. Lett. 2009, 11, 3962–3965. 3. Gan, C. Y.; Kam, T. S. Tetrahedron Lett. 2009, 50, 1059–1061. 4. Goh, S. H.; Ali, A. R. M.; Wong, W. H. Tetrahedron 1989, 45, 7899–7920. 5. Hirasawa, Y.; Shoji, T.; Arai, T.; Nugroho, A. E.; Deguchi, J.; Hosoya, T.; Uchiyama, N.; Goda, Y.; Awang, K.; Hadi, A. H. A.; Shiro, M.; Morita, H. Bioorg. Med. Chem. Lett. 2010, 20, 2021–2024. 6. Nugroho, A. E.; Hirasawa, Y.; Kawahara, N.; Goda, Y.; Awang, K.; Hadi, A. H. A.; Morita, H. J. Nat. Prod. 2009, 72, 1502–1506. 7. Hirasawa, Y.; Miyama, S.; Hosoya, T.; Koyama, K.; Rahman, A.; Kusumawati, I.; Zaini, N. C.; Morita, H. Org. Lett. 2009, 11, 5718–5721. 8. The barks of L. griffithii collected at Malaysia, were extracted with MeOH, and part (17 g) of the extract was treated with 3% tartaric acid (pH 2) and then partitioned with EtOAc. The EtOAc fraction was subjected to a silica gel column (hexane/EtOAc, CHCl3/MeOH) to give 27 fractions. Fraction containing 1 was further separated using a silica gel column (hexane/EtOAc, toluene/EtOAc) to give leucomidine A (1, 3.1 mg, 0.005%). While fraction containing 2 and 3 was further separated using an ODS silica gel column (MeOH/H2O) and ODS HPLC (MeOH/H2O) to give leucomidines B (2, 0.2 mg, 0.0003%) and C (3, 0.6 mg, 0.0009%). 9. Leucomidine A (1): yellowish amorphous solid; ½a24 D +69 (c 0.3, MeOH); IR (KBr) mmax 3390, 2950, 1800, and 1730 cm1; UV (MeOH) kmax 301 (e 2200), 250 (8100), and 216 (34100) nm; CD (MeOH) kmax 287 (De +0.64), 277 (0), 227 (+15.94), 218 (0), and 209 (8.97) nm; 1H and 13C NMR (Table 1); ESIMS (pos.) m/z 347 (M+Na)+; HRESITOFMS m/z 347.1380 (M+Na)+, calcd for C19H20N2O3Na 347.1379. 10. To a solution of 1 (2.0 mg) in THF (1 ml) was added LiAlH4 (5 mg). The mixture was allowed to stand at 80 °C for 3 h. After cooling, the mixture was partitioned between H2O and CHCl3. CHCl3 soluble materials were applied to a silica gel column (CHCl3/MeOH, 1:0?0:1) to give compounds 4 (0.7 mg) and 5 (0.6 mg). 4, 1H NMR (CD3OD, 600 MHz) 7.51 (d, 7.6; H-9), 7.29 (d, 7.6; H-12), 7.06 (t, 7.6; H-11), 7.04 (t, 7.6; H-10), 3.75 (br. s; H-21), 3.63 (m; H2-6), 3.47 (m; H-3a), 3.34 (m; H2-5), 2.93 (dd, 17.2, 7.2; H-16a), 2.79 (ddd, 17.2, 11.0, 7.2; H-16b), 2.68 (m; H-3b), 2.64 (m; H-17a), 1.96 (m; H-14a), 1.80 (br. d, 15.2; H15a), 1.77 (m; H-14b), 1.63 (br. t, 12.7; H-15b), 1.55 (dd, 13.0, 7.2; H-17b), 1.23 (dq, 14.1, 7.6; H-19a), 1.08 (dq, 14.1, 7.6; H-19b), and 0.80 (t, 7.6; H3-18); 13C NMR (CD3OD, 150 MHz) 138.4 (C-2), 138.3 (C-13), 130.3 (C-8), 122.1 (C-11), 120.6 (C-10), 118.6 (C-9), 111.9 (C-12), 103.6 (C-7), 66.3 (C-21), 58.7 (C-6), 57.8 (C-5), 53.8 (C-3), 38.1 (C-20), 34.6 (C-15), 30.7 (C-19), 24.9 (C-17), 21.3 (C-14), 20.8 (C-16), and 7.9 (C-18); ESIMS (pos.) m/z 299 (M+H)+. 5, 1H NMR (CD3OD, 600 MHz) 7.99 (d, 7.2; H-9), 7.41 (d, 7.2; H-12), 7.24 (td, 7.2, 1.4; H-11), 7.21 (td, 7.2, 1.4; H-10), 3.85 (dd, 15.8, 6.2; H-3a), 3.69 (ddd, 15.8, 10.7, 6.2; H-3b), 3.09 (ddd, 17.9, 12.7, 5.5; H-16a), 2.96 (ddd, 17.9, 5.2, 1.4; H-16b), 2.20 (ddd, 12.7, 5.2, 1.4; H-17a), 2.06 (m; H-14a), 2.06 (m; H-15a), 1.96 (td, 12.7, 5.5; H17b), 1.87 (m; H-14b), 1.78 (dq, 14.8, 7.2; H-19a), 1.73 (dq, 14.8, 7.2; H-19b), 1.54 (td, 14.5, 4.5; H-15b), and 0.98 (t, 7.2; H3-18); 13C NMR (CD3OD, 150 MHz) 176.3 (C-21), 52.0 (C-2), 139.7 (C-13), 125.3 (C-8), 124.6 (C-11), 123.2 (C-10), 120.9 (C-9), 113.3 (C-12), 107.6 (C-7), 46.7 (C-3), 39.5 (C-20), 33.5 (C-17), 28.8 (C-15), 26.9 (C-19), 21.2 (C-16), 18.3 (C-14), and 8.3 (C-18); ESIMS (pos.) m/z 253 (M+H)+. 11. Leucomidine B (2): yellowish amorphous solid; ½a26 D 18 (c 0.3, CHCl3); IR (KBr) mmax 3400, 1740, and 1670 cm1; UV (MeOH) kmax 298 (e 565), 222 (11250), and 202 (10800) nm; CD (MeOH) kmax 292 (De 0.57), 266 (0), 255 (+1.48), 225 (2.31), and 209 (0) nm; 1H and 13C NMR (Table 1); ESIMS (pos.) m/ z 341 (M+H)+; HRESITOFMS m/z 341.1840 (M+H)+, calcd for C20H25N2O3 341.1860. 12. Leucomidine C (3): yellowish amorphous solid; ½a26 D +32 (c 0.3, CHCl3); IR (KBr) mmax 1740 and 1680 cm1; UV (MeOH) kmax 305 (e 1700), 273 (3250), 226 (10100), and 204 (12200) nm; 1H and 13C NMR (Table 1); ESIMS (pos.) m/z 315 (M+H)+; HRESITOFMS m/z 315.1685 (M+H)+, calcd for C18H23N2O3 315.1703. 13. Larraufie, M.-H.; Courillon, C.; Ollivier, C.; Lacôte, E.; Malacria, M.; Fensterbank, L. J. Am. Chem. Soc. 2010, 132, 4381–4387. 14. (a) All CD calculations were done using Turbomole 6.3 at TDDFT B3LYP/TZVPP level of theory on RI-DFT B3LYP/TZVPP optimized geometries. The stable conformation used for the DFT geometry optimization was obtained through Monte Carlo conformational search in MacroModel 9.1 using MMFF94 force field for the relative molecular energy evaluation.; (b) TURBOMOLE V6.3, 2011, a development of University of Karlsruhe and Forschungszentrum Karlsruhe GmbH, 19892007, TURBOMOLE GmbH, since 2007; available from http:// www.turbomole.com.; (c) Eickorn, K.; Treutler, O.; Ohm, H.; Haser, M.; Ahlrichs, R. Chem. Phys. Lett. 1995, 240, 283–289; (d) Becke, A. D. Phys. Rev. A 1988, 38, 3098–3100; (e) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785–789; (f) Schafer, A.; Horn, H.; Ahlrichs, R. J. Chem. Phys. 1994, 100, 5829– 5835; MacroModel 9.1 (g) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440–467; (h) Halgren, T. J. Am. Chem. Soc. 1990, 112, 4710–4723; (i) Halgren, T. J. Am. Chem. Soc. 1992, 114, 7827–7843; (j) Halgren, T. J. Comput. Chem. 1996, 17, 490–519; (k) Halgren, T. J. Comput. Chem. 1996, 17, 520–552. 15. Wong, C. P.; Shimada, M.; Nagakura, Y.; Nugroho, A. E.; Hirasawa, Y.; Kaneda, T.; Awang, K.; Hadi, A. H. A.; Mohamad, K.; Shiro, M.; Morita, H. Chem. Pharm. Bull. 2011, 59, 407–411.