Bis-cephalezomines A–E from Cephalotaxus harringtonia var. nana

Tetrahedron 60 (2004) 7861–7868

Bis-cephalezomines A –E from Cephalotaxus harringtonia var. nana Miwa Yoshinaga, Hiroshi Morita, Tomoka Dota and Jun’ichi Kobayashi* Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Received 15 May 2004; revised 10 June 2004; accepted 14 June 2004 Available online 28 July 2004

Abstract—Five new dimeric Cephalotaxus alkaloids, bis-cephalezomines A –E (1 – 5), have been isolated from the leaves of Cephalotaxus harringtonia var. nana, and the structures and stereochemistry were elucidated on the basis of spectroscopic data including 2D NMR and FABMS/MS spectra, and chemical means. q 2004 Elsevier Ltd. All rights reserved.

Cephalotaxus alkaloids are a family of cytotoxic heterocyclic natural products with unique ring systems and elaborated by trees of the genus Cephalotaxus (Cephalotaxaceae), some of which showed antileukemic activity.1 Recently, we have isolated 12 new cytotoxic alkaloids, cephalezomines A,M,2 from the leaves of Cephalotaxus harringtonia var. nana and a novel pentacyclic alkaloid, cephalocyclidin A,3 from the fruits of the same plant. Our continuing search for structurally unique and biogenetically interesting Cephalotaxus alkaloids resulted in the isolation of five new dimeric alkaloids, bis-cephalezomines A –E (1– 5), consisting of two cephalotaxine skeletons from the leaves of C. harringtonia var. nana. In this paper we describe the isolation and structure elucidation of 1– 5. The leaves of C. harringtonia var. nana were extracted with MeOH, and the MeOH extract was partitioned between EtOAc and 3% tartaric acid. Water-soluble materials adjusted at pH 10 with sat. Na2CO3 aq. were partitioned with CHCl3. CHCl3-soluble materials were subjected to LH-20 column chromatography (CHCl3/MeOH, 1:1) followed by silica gel column chromatography (CHCl3/ MeOH, 30:1!MeOH), and then C18 HPLC (35 – 45% CH 3CN/0.1% TFA) to afford bis-cephalezomines A (1, 0.0001%), B (2, 0.0001%), C (3, 0.0001%), D (4, 0.00004%), and E (5, 0.00004%) together with cephalotaxidine4 (6, 0.0005%). Bis-cephalezomine A (1) showed the pseudomolecular ion peak at m/z 1089 (MþH)þ in the FABMS, and the Keywords: Cephalotaxus; Alkaloids; Dimer; Cytotoxicity; NMR; FABMS/MS. * Corresponding author. Tel.: þ81-11-706-4985; fax: þ81-11-706-4989; e-mail address: [email protected] 0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2004.06.070

molecular formula, C57H72N2O19, was established by HRFABMS [m/z 1089.4780, (MþH)þ, D 22.7 mmu]. IR absorptions implied the presence of hydroxyl, ester carbonyl, and amide carbonyl (3580, 1740, and 1650 cm21, respectively) functionalities. 1H and 13C NMR data (Table 1) revealed 57 carbon signals due to 15 sp2 and six sp3 quaternary carbons, six sp2 methines, six sp3 methines, 16 sp3 methylenes, and eight methyl groups. Among them, two sp3 methylene (dC 45.6; dH 2.60 and 2.68; dC 39.1; dH 3.04 and 3.83), two sp3 quaternary carbons (dC 71.5 and 70.0), one sp3 methine (dC 62.4; dH 3.29), and one sp2 quaternary carbon (dC 176.1) were ascribed to those bearing a nitrogen. Since 13 out of 23 elements of unsaturation were accounted for, 1 was inferred to possess 10 rings. The gross structure of 1 was elucidated by analyses of 2D NMR data including 1H – 1H COSY, HOHAHA, HMQC, and HMBC spectra in CD3OD (Fig. 1). Each pair of the observed 1H and 13C NMR signals seemed to be due to each half moiety (parts A and B) of a dimeric compound. In part A, connectivities of C-3 to C-4, C-6 – C-8, C-10 to C-11, and C-100 – C-300 were deduced from 1H – 1H COSY and HOHAHA correlations. In the HMBC spectrum, longrange 1H– 13C correlations (Fig. 1) indicated that 1 possessed a cephalotaxine-type framework. HMBC cross-peaks of H-300 to C-10 and C-100 , H3-OMe (50 ) to C-40 , and H-500 and H-600 to C-300 revealed the presence of the methyl ester of 2-hydroxy-2-(4hydroxyisohexyl)-butanedioic acid. The connectivity between C-3 and C-10 was indicated by HMBC correlations of H-3 and H-30 to C-10 . On the other hand, the corresponding 1H – 1H COSY, HOHAHA, and HMBC correlations were also observed for part B (Fig. 1). HMBC correlations of H-3000 to C-1000 and C-10000 , H3-OMe (5000 ) to C-4000 , and H-40000 and H-50000 to C-20000 revealed the presence of

7862

Table 1. 1H NMR data (dH) of bis-cephalezomines A –E (1–5) in CDCl3 at 300 K 1 Part A

a

2 Part B

a

3 Part B

Part A

4.68 s 5.90 d (9.4) 3.51 m 1.46 m 1.60 m

3.83 m

4.95 s 5.96 d (10.0) 3.71 d (10.0) 1.72 m 1.72 m 0.88 m 1.60 m 3.31 m 2.70 m

Part B

1(20) 3(22) 4(23) 6(25)a 6(25)b 7(26)a 7(26)b 8(27)b 10(29)a

4.96 s 5.95 d (10.0) 3.68 m 1.70 m 1.70 m 0.79 m 1.54 m 3.29 m 2.68 m

4.69 s 5.89 d (9.2) 3.52 m 1.46 m 1.60 m

3.83 m

4.97 s 5.94 d (10.1) 3.71 d (10.1) 1.70 m 1.70 m 0.79 m 1.53 m 3.29 m 2.68 m

10(29)b 11(30)a

2.60 m 2.31 m

3.04 m 2.54 m

2.59 m 2.33 m

3.05 m 2.54 m

2.61 m 2.34 m

11(30)b

3.06 m

3.17 m

3.05 m

3.17 m

3.07 m

14(33) 17(36) 18(37)a 18(37)b 19(38)Me 30 (3000 )a 30 (3000 )b 30 (3000 )Me 100 (10000 )a 100 (10000 )b

6.54 s 6.56 s 5.86 d (1.3) 5.97 d (1.3) 3.66 s 1.90 d (16.4) 2.24 d (16.4) 3.55 s 1.38 m 1.38 m

6.51 s 6.56 s 5.86 d (1.4) 5.89 d (1.4) 3.62 s 2.05 d (16.4) 2.34 d (16.4) 3.56 s 1.57 m 1.57 m

6.54 s 6.56 s 5.86 d (1.3) 5.97 d (1.3) 3.65 s 1.89 d (16.4) 2.26 d (16.4) 3.53 s 1.56 m 1.56 m

6.51 s 6.56 s 5.86 d (1.4) 5.88 d (1.4) 3.63 s 2.04 d (16.5) 2.31 d (16.5) 3.57 s 1.38 m 1.38 m

6.56 s 6.57 s 5.88 d (1.1) 6.00 d (1.2) 3.71 s 1.91 d (16.5) 2.28 d (16.5) 3.56 s 1.58 m 1.58 m

3.84 ddd (12.7, 12.7, 6.6) 3.10 m 2.56 ddd (12.2, 6.6, 2.5) 3.18 ddd (12.7, 12.2, 6.6) 6.53 s 6.57 s 5.88 d (1.1) 5.90 d (1.3) 3.67 s 2.07 d (16.5) 2.35 d (16.5) 3.59 s 1.58 m 1.58 m

200 200 300 400 500 600

1.17 m 1.37 m 1.38 m

1.52 m 1.52 m

1.56 m 1.56 m

1.11 m 1.35 m 1.32 m

1.60 m 1.60 m

1.60 m 1.60 m

1.13 s 1.12 s

1.14 s 1.12 s

1.15 s 1.14 s

1.16 s 1.14 s

a

(20000 )a (20000 )b (30000 ) (40000 ) (50000 ) (60000 )

1.16 s 1.16 s

2.68 m

Parts A and B indicate the partial structures as shown in Figure 1.

2.68 m

1.15 s 1.15 s

4.70 s 5.91 d (10.6) 3.51 d (10.6) 1.56 m 1.62 m 2.61 m

Part A

5 Part B

Part A

Part B

4.97 s 5.96 d (9.8) 3.69 d (10.0) 1.70 m 1.70 m 0.82 m 1.55 m 3.32 m 2.70 m

4.71 s 5.93 d (9.3) 3.53 m 1.50 m 1.57 m

3.86 m

4.95 s 5.95 d (9.8) 3.69 m 1.71 m 1.71 m 0.81 m 1.56 m 3.30 m 2.69 m

2.60 m 2.33 m

3.07 m 2.63 m

2.60 m 2.32 m

3.84 ddd (12.6, 12.6, 6.6) 3.06 m 2.55 m

3.07 m

3.19 m

3.09 m

3.18 m

6.55 s 6.60 s 5.88 s 5.99 s 3.67 s 1.92 d (16.4) 2.26 d (16.4) 3.58 s 1.42 m 1.42 m

6.52 s 6.58 s 5.83 s 5.90 s 3.64 s 3.48 d (8.2)

6.54 s 6.57 s 5.88 d (1.2) 5.99 d (1.2) 3.66 s 1.89 d (16.4) 2.26 d (16.4) 3.54 s 1.41 m 1.41 m

6.52 s 6.57 s 5.86 s 5.90 d (1.1) 3.63 s 2.06 d (16.4) 2.30 d (16.4) 3.57 s 1.41 m 1.41 m

0.95 m 0.95m 1.27 m 0.73 d (6.6) 0.75 d (6.6)

1.13m 1.41 m 1.35 m

1.14 m 1.37 m 1.37 m 1.18 s 1.18 s

2.70 m

3.57 s 1.49 m 1.85 ddd (13.4, 13.4, 4.0) 0.88 m 1.21 m 1.37 m 0.84 d (6.6) 0.82 d (6.6)

4.68 s 5.92 d (9.5) 3.52 m 1.49 m 1.60 m 2.69 m

1.16 s 1.17 s

M. Yoshinaga et al. / Tetrahedron 60 (2004) 7861–7868

Part A

4

M. Yoshinaga et al. / Tetrahedron 60 (2004) 7861–7868

Figure 1. Selected 2D NMR correlations for bis-cephalezomine A (1).

7863

7864

M. Yoshinaga et al. / Tetrahedron 60 (2004) 7861–7868

Figure 2. Fragmentation patterns observed in positive ion FABMS/MS spectrum of bis-cephalezomine A (1) (precursor ion m/z 544.6) and bis-cephalezomine B (2) (precursor ion m/z 530.6).

the methyl ester of 2-hydroxy-2-(3-hydroxyisopentyl)butanedioic acid. In addition, HMBC correlations for H2-29 and H-26 to C-27 (dC 176.1) suggested the presence of an amide carbonyl at C-27 such as cephalotaxinamide5 in the part B. The connection of C-8 to C-26 between parts A and B was provided by 1H – 1H COSY and HOHAHA correlations as shown in Figure 1. Thus, the gross structure of bis-cephalezomine A (1) was assigned as 1. Further evidence supporting the proposed structure of 1 was provided by tandem mass spectrometry through examination of the collision-induced dissociation (CID) mass spectrum of the (MþH)þ ions. The positive ion FABMS spectra of 1 showed the product ion peak (m/z 544) generated by fission at C-8 –C-26 bond between the two partial units A and B. In addition, the FABMS/MS fragmentation patterns corresponding to the part A indicated that the side chain at C-3 was the ester of 2-hydroxy-2-(4hydroxyisohexyl)-butanedioic acid (Fig. 2). The relative stereochemistry of 1 was deduced from ROESY correlations as shown in computer-generated 3D drawing (Fig. 3). The decacyclic core in 1 was elucidated to have the same relative stereochemistry as that of cephalotaxidine (6).4 The absolute configurations of the cyclic core of 1 were derived from chemical correlations as follows. Spectroscopic data including optical rotation of a hydro-

Figure 3. Selected ROESY correlations for bis-cephalezomine A (1). Two ester side chains are omitted for clarify.

lysate (7) prepared from 1 through hydrolysis with LiOH were identical with that derived from cephalotaxidine (6).4 The CD spectrum for the molybdate complex of each dicarboxy acid moiety (C-10 – C-40 and C-1000 – C-4000 ) derived from the acid hydrolysates of 1 showed a negative Cotton effect at 270 nm (D1270 21.1),6 indicating that the absolute configurations at C-20 and C-2000 of 1 were both R. The molecular formula of bis-cephalezomine B (2) was determined to be C57H72N2O19 by HRFABMS [m/z 1089.4812, (MþH)þ, D þ0.5 mmu], which was the same as that of 1. The IR spectrum implied the presence of hydroxyl (3580 cm21) and carbonyl (1740 and 1650 cm21) functionalities. 1H and 13C NMR data (Tables 1 and 2) suggested that 2 had the same cephalotaxine-type framework as 1. The 1H and 13C NMR signals observed for 2 were also close to those of 1. The positive ion FABMS spectra of 2 showed the product ion peak (m/z 530) generated by fission at C-8 – C-26 bond between two partial units A and B. In addition, FABMS/MS fragmentation patterns corresponding to the part A indicated that the side chain at C-3 was the ester of 2-hydroxy-2-(3-hydroxyisopentyl)-butanedioic acid (Fig. 2). Therefore, the side chains of parts A and B in 1 and 2 were reversed for each other. The CD spectrum (D1212 219.5, D1230 þ2.5, D1247 þ2.7, and D1291 23.3) of 2 showed Cotton effects similar to that (D1211 222.7, D1229 þ2.9, D1249 þ3.3, and D1292 23.8) of bis-cephalezomine A (1), and the hydrolysate of 2 was identical with that prepared from 1. Furthermore, the CD spectrum for the molybdate complex of the dicarboxy acid moiety (C-10 –C-40 and C-100 – C-600 ), which was derived from the acid hydrolysates of 2, showed a negative Cotton effect at 270 nm (D1270 21.0),6 indicating that the absolute configurations at C-20 and C-2000 of 2 were both R. Bis-cephalezomine C {3, [a]D 21188 (c 0.5, MeOH)} showed the pseudomolecular ion at m/z 1075 (MþH)þ, and the molecular formula, C56H70N2O19, was established by HRFABMS [m/z 1075.4738, (MþH)þ, D þ8.7 mmu]. IR absorptions were attributed to hydroxyl (3585 cm21) and carbonyl (1745 and 1665 cm21) groups, respectively. The FABMS spectrum of 3 showed a common fragment ion peak at m/z 530, characteristic for bis-cephalezomine B (2). 1 H and 13C NMR data (Tables 1 and 2) in one of the two partial units A and B corresponded well to those of isoharringtonine.7 The hydrolysate of 3 was identical with that prepared from bis-cephalezomine A (1), and the CD

M. Yoshinaga et al. / Tetrahedron 60 (2004) 7861–7868 Table 2.

13

C NMR data (dC) of bis-cephalezomines A –E (1–5) in CDCl3 at 300 K 1

1(20) 2(21) 3(22) 4(23) 5(24) 6(25) 7(26) 8(27) 10(29) 11(30) 12(31) 13(32) 14(33) 15(34) 16(35) 17(36) 18(37) 19(38) 10 (1000 ) 20 (2000 ) 30 (3000 ) 40 (4000 ) 30 (3000 )-Me 100 (10000 ) 200 (20000 ) 300 (30000 ) 400 (40000 ) 500 (50000 ) 600 (60000 ) a

7865

2

3

4

5

Part Aa

Part Ba

Part A

Part B

Part A

Part B

Part A

Part B

Part A

Part B

101.4 157.5 74.4 56.6 71.5 42.4 22.9 62.4 45.6 31.3 133.6 129.0 112.5 145.4 146.1 109.3 100.8 57.6 173.8 74.6 42.5 170.2 51.5 39.1 17.9 43.7 70.8 29.0 29.5

103.4 158.4 74.4 59.2 70.0 36.7 41.8 176.1 39.1 30.1 132.6 126.1 112.5 146.1 147.2 110.0 101.0 57.4 173.6 74.7 42.8 170.3 51.5 33.2 36.7 70.0 28.8 29.2

101.9 157.2 74.4 56.6 71.4 42.3 22.9 62.3 45.6 31.2 133.5 128.8 112.5 145.4 146.1 109.3 101.0 57.7 173.7 74.7 42.7 170.2 51.5 33.3 36.8 70.7 29.0 29.5

103.0 158.6 74.4 59.1 69.7 36.5 41.8 176.2 39.1 30.3 132.7 126.1 112.5 146.1 147.2 110.0 100.7 57.3 173.7 74.7 42.5 170.3 51.5 39.1 17.8 43.6 70.0 28.7 29.3

102.0 157.3 74.5 56.8 71.5 42.4 23.0 62.4 45.6 31.3 133.6 129.0 112.6 145.5 146.2 109.4 100.8 57.6 173.9 74.7 42.8 170.3 51.6 33.1 36.8 70.1 28.6 29.6

103.5 158.4 74.5 59.2 69.7 36.5 41.9 176.2 39.1 30.2 132.7 126.2 112.6 146.2 147.3 110.1 101.1 57.3 173.7 74.8 42.8 170.3 51.6 33.2 36.9 70.1 28.8 29.6

101.5 157.6 74.5 56.7 71.5 42.5 23.0 62.4 45.6 31.4 133.8 129.1 112.5 145.4 146.2 109.4 100.8 57.6 174.0 74.6 42.6 170.4 51.5 38.8 17.9 43.8 70.6 29.3 29.7

103.4 158.7 74.5 59.0 69.9 34.7 41.9 176.0 39.1 30.3 132.8 125.8 112.5 146.2 147.3 110.4 101.1 57.4 174.0 74.6 74.7 170.4 51.5 33.0 31.5 28.5 22.7 22.7

101.1 157.7 74.7 56.7 71.5 42.5 23.0 62.5 45.7 31.3 133.6 129.1 112.5 145.4 146.2 109.3 101.3 57.7 174.0 74.4 42.6 170.4 51.5 36.7 31.6 28.0 22.3 22.6

103.1 158.6 74.7 59.2 69.7 36.6 42.0 176.3 39.1 30.2 132.7 126.2 112.5 146.2 147.3 110.1 100.8 57.4 173.8 74.5 42.8 170.5 51.6 39.1 17.9 43.7 70.8 29.1 29.3

Parts A and B indicate the partial structures as shown in Figure 1.

spectrum for the molybdate complex of each dicarboxy acid moiety (C-10 – C-40 and C-100 – C-500 , and C-1000 –C-4000 and C-10000 –C-50000 ) derived from the acid hydrolysate showed a negative Cotton effect at 270 nm (D1270 21.1),6 indicating that the absolute configurations at C-20 and C-2000 of 3 were both R. Bis-cephalezomine D {4, [a]D 2868 (c 0.3, MeOH)} showed the pseudomolecular ion at m/z 1089 (MþH)þ, and the molecular formula, C57H72N2O19, was established by HRFABMS [m/z 1089.4830, (MþH)þ, D þ2.3 mmu]. IR absorptions were attributed to hydroxyl (3585 cm21) and carbonyl (1745 and 1645 cm21) groups, respectively. The FABMS spectrum of 4 showed a common fragment ion peak at m/z 544, characteristic for biscephalezomine A (1). 1 H and 13C NMR data (Tables 1 and 2) in one of the two parts corresponded well to those of isoharringtonine.7 Since the FABMS spectrum of acetate of 4 showed the same fragment ion peak at m/z 544 as 1, the side chains of parts A and B were elucidated to be 2-hydroxy-2-(4-hydroxyisohexyl)-butanedioic acid and 2,3-hydroxy-2-isopentylbutanedioic acid, respectively. To determine the absolute configuration at C-3000 , 4 was converted into its (S)- and (R)-2-methoxy-2-trifluoromethylphenylacetic acid (MTPA) esters. The values of Dd [d(S-MTPA ester) 2 d(R-MTPA ester)] obtained from the 1H NMR spectra of the MTPA esters suggested that the absolute configuration at C-3000 of 4 was S.8 Furthermore, the hydrolysate of 4 was identical with that prepared from bis-cephalezomine A (1) and the CD spectrum for the molybdate complex of each dicarboxy acid moiety (C-10 – C-40 and C-100 – C-500 ) derived from the acid hydrolysates of 4 showed a negative Cotton effect at

270 nm (D1270 21.1), indicating that the absolute configurations at C-20 and C-2000 of 4 were R.6,9 Therefore the absolute stereochemistry of 4 was elucidated to be 3S, 4S, 5R, 8S, 20 R, 22S, 23S, 24R, 26S, and 2000 R. Bis-cephalezomine E {5, [a]D 21468 (c 0.4, MeOH)} showed the pseudomolecular ion peak at m/z 1073 (MþH)þ and the molecular formula, C57H72N2O18, was established by HRFABMS [m/z 1073.4820, (MþH)þ, D 23.8 mmu], which was smaller than those of 1, 2, and 4 by one oxygen atom. IR absorptions implied the presence of hydroxyl (3585 cm21) and carbonyl (1745 and 1655 cm21) functionalities. In the FABMS spectra, 5 gave the fragment ion at m/z 514, which was smaller than that (m/z 530) of 2 by one oxygen atom. In the 1H NMR spectrum, two doublet methyl signals at d 0.73 and 0.75 (J¼6.6 Hz) were observed. Therefore, the side chain in the part A was elucidated to be 2-hydroxy-2-isopentylbutanedioic acid. Detailed analyses of the 1H – 1H COSY, HOHAHA, HMQC, and HMBC spectra of 5 led to the presence of a dimer with cephalotaxine-type backbone like 1– 4, and of the side chains at C-3 and C-22. The CD spectrum (D1216 219.3, D1229 þ6.2, D1248 þ6.9, and D1291 27.1) of 5 was similar to those of 1– 4. Furthermore, the hydrolysate of 4 was identical with that prepared from bis-cephalezomine A (1) and the molybdate complex of the dicarboxy acid moiety (C-10 –C-40 and C-100 –C-500 , and C-1000 –C-4000 and C-10000 – C-60000 ) derived from the acid hydrolysates of 5 showed a negative Cotton effect at 270 nm (D1270 21.3),6 indicating that the absolute configurations at C-20 and C-2000 were both R. A plausible biogenetic path for bis-cephalezomines A – E

7866

M. Yoshinaga et al. / Tetrahedron 60 (2004) 7861–7868

Scheme 1. A plausible biogenetic path of bis-cephalezomine B (2).

(1 –5) is proposed as shown in Scheme 1. A series of dimers might be derived by coupling of imine intermediate generated from the corresponding alkaloid such as homoharringtonine through Polonovski-type fragmentation,10 with the corresponding amide derivatives such as homoharringtonamide11 as shown in Scheme 1.

multiplication with squared cosine-bell windows shifted in both dimensions were performed prior to 2D Fourier transformation. FABMS was measured by using glycerol as a matrix.

Bis-cephalezomines A – E (1 – 5) are new dimeric Cephalotaxus alkaloids consisting of two cephalotaxinetype skeletons with different side chains at C-3. Biscephalezomines A – E (1 –5) exhibited cytotoxicity against murine lymphoma L1210 cells in vitro with IC50 values of 1.9, 1.9, 2.6, 3.1, and 3.7 mg/ml, respectively. These dimeric alkaloids exhibited relatively weak cytotoxicity, although each monomeric alkaloid corresponding to the parts A and B showed potent cytotoxicity.1

The leaves of Cephalotaxus harringtonia var. nana were collected in Sapporo (Hokkaido, Japan) in 2001. The botanical identification was made by Mr N. Yoshida, Health Sciences University of Hokkaido. A voucher specimen has been deposited in the herbarium of Hokkaido University.

1. Experimental 1.1. General methods 1 H and 2D NMR spectra were recorded on a 600 MHz spectrometer at 300 K, while 13C NMR spectra were measured on a 150 MHz spectrometer. The NMR samples of bis-cephalezomines A – E (1 – 5) were prepared by dissolving 1.0 mg each in 30 ml of CDCl3 in 2.5 mm micro cells (Shigemi Co. Ltd) and chemical shifts were recorded using residual CDCl3 (dH 7.26 and dC 77.0) as internal standards. Standard pulse sequences were employed for the 2D NMR experiments. COSY, HOHAHA, and NOESY spectra were measured with spectral widths of both dimensions of 4800 Hz, and 32 scans with two dummy scans were accumulated into 1 K data points for each of 256 t1 increments. NOESY and HOHAHA spectra in the phase sensitive mode were measured with a mixing time of 800 and 30 ms, respectively. For HMQC spectra in the phase sensitive mode and HMBC spectra, a total of 256 increments of 1 K data points were collected. For HMBC spectra with Z-axis PFG, a 50 ms delay time was used for long-range C –H coupling. Zero-filling to 1 K for F1 and

1.2. Material

1.3. Extraction and isolation The leaves of C. harringtonia var. nana(13 kg) were crashed and extracted with MeOH (20 L) three times and the extract (1016 g) was treated with 3% tartaric acid to adjust pH 2 and then partitioned with EtOAc. The aqueous layer was treated with sat. Na2CO3 aq. to adjust pH 10 and extracted with CHCl3 to give a crude alkaloidal fraction (18.3 g), in which a portion (3.1 g) was subjected to LH-20 column chromatography (CHCl3/MeOH, 1:1) followed by silica gel column chromatography (CHCl3/MeOH, 30:1! MeOH), and then C18 HPLC (35 – 45% CH3CN/0.1% TFA) to afford bis-cephalezomines A (1, 0.0001%), B (2, 0.0001%), C (3, 0.0001%), D (4, 0.00004%), and E (5, 0.00004%) together with cephalotaxidine (6, 0.0005%). 1.3.1. Bis-cephalezomine A (1). A colorless solid; [a]D 21518 (c 1.0, MeOH); IR (KBr) nmax 3585, 2960, 1740, 1650, 1490. 1370, 1225, and 1035 cm21; UV (MeOH) lmax 291 (1 6400) and 209 (34700); 1H and 13C NMR data (Tables 1 and 2, respectively); CD (MeOH) D1211 222.7, D1229 þ2.9, D1249 þ3.3, and D1292 23.8; FABMS m/z 1089 (MþH)þ; HRFABMS m/z 1089.4780 (MþH; calcd for C57H73N2O19, 1089.4807). 1.3.2. Bis-cephalezomine B (2). A colorless solid; [a]D 21268 (c 1.0, MeOH); IR (KBr) nmax 3580, 2960, 1740, 1650, 1490, 1370, 1225, and 1035 cm21; UV (MeOH) lmax

M. Yoshinaga et al. / Tetrahedron 60 (2004) 7861–7868

291 (1 5300) and 208 (28200); CD (MeOH) D1212 219.5, D1230 þ2.5, D1247 þ2.7, and D1291 23.3; 1H and 13C NMR data (Tables 1 and 2, respectively); FABMS m/z 1089 (MþH)þ; HRFABMS m/z 1089.4812 (MþH; calcd for C57H73N2O19, 1089.4807). 1.3.3. Bis-cephalezomine C (3). A colorless solid; [a]D 21188 (c 0.5, MeOH); IR (KBr) nmax 3585, 2925, 1745, 1665, 1220, and 1030 cm21; UV (MeOH) lmax 290 (1 4000) and 207 (27000); CD (MeOH) D1210 222.3, D1230 þ1.7, D1248 þ2.1, and D1290 22.8; 1H and 13C NMR data (Tables 1 and 2, respectively); FABMS m/z 1075 (MþH)þ; HRFABMS m/z 1075.4738 (MþH; calcd for C56H71N2O19, 1075.4651). 1.3.4. Bis-cephalezomine D (4). A colorless solid; [a]D 2868 (c 0.3, MeOH); IR (KBr) nmax 3585, 2920, 1745, 1645, 1035, and 720 cm21; UV (MeOH) lmax 290 (1 4800) and 207 (41000); CD (MeOH) D1210 28.2, D1231 þ0.2, D1248 þ0.3, and D1292 21.3; 1H and 13C NMR data (Tables 1 and 2, respectively); FABMS m/z 1089 (MþH)þ; HRFABMS m/z 1089.4830 (MþH; calcd for C57H73N2O19, 1089.4807). 1.3.5. Bis-cephalezomine E (5). A colorless solid; [a]D 21468 (c 0.4, MeOH); IR (KBr) nmax 3585, 2925, 1745, 1655, 1440, 1200, and 890 cm21; UV (MeOH) lmax 290 (1 4300) and 208 (33600); CD (MeOH) D1216 219.3, D1229 þ6.2, D1248 þ6.9, and D1291 27.1; 1H and 13C NMR data (Tables 1 and 2, respectively); FABMS m/z 1073 (MþH)þ; HRFABMS m/z 1073.4820 (MþH; calcd for C57H73N2O18, 1073.4858). 1.3.6. Molybdate complexes of hydrolysates of biscephalezomines A – E (1 –5) and cephalotaxidine (6). Each of bis-cephalezomines 1 –5 and cephalotaxidine (6) (0.5 mg) was hydrolyzed with 3 N HCl (1 ml) under reflux for 4 days. After cooling, 3 M NH4OH was added and the alkaline phase was extracted with CHCl3. Excess NH4OH was neutralized and the solvent was evaporated under reduced pressure. The residue was used directly in the preparation of solution for CD measurement, which contained 3 mM each hydrolysates and 2.7 mM Na molybdate. HCl and NaOH solution were added until pH 2.9 –3.1 was reached. Measurements of CD spectra were carried out in a 1 mm cell 5 days after the solution had been prepared. CD data: bis-cephaloezomines A (1, D1270 21.1), B (2, D1270 21.0), C (3, D1270 21.1), D (4, D1270 21.1), E (5, D1270 21.3), and cephalotaxidine (6, D1270 21.2). 1.3.7. Hydrolysis of bis-cephalezomines A – E. To each solution of bis-cephalezomines A – E (1– 5) (0.5 mg) in dioxane (0.2 ml) and H2O (0.2 ml) was added lithium hydroxide (1.1 mg). After the reaction mixture was stirred at 40 8C for 1 h, it was poured into water and was extracted with CHCl3 three times. The combined organic phase was washed with brine, dried over MgSO4 and evaporated to give hydrolysate (7) of 1 – 5 (0.4 mg) as an amorphous powder, whose spectral data and [a]D value were identical with those of that derived from cephalotaxidine by the same procedure. Compound 7: a colorless solid; [a]D 2848 (c 0.1, MeOH); IR (KBr) nmax 3585, 2920, 1655, 1560, and 1025 cm21; UV (MeOH) lmax 290 (1 3000) and 208

7867

(17,000); 1H NMR (CDCl3) d 4.85 (1H, s, H-1), 4.71 (1H, m, H-3), 3.59 (1H, d, 9.5, H-4), 1.76 (2H, m, H2-6), 0.88 (1H, m, Ha-7), 1.56 (1H, m, Hb-7), 3.34 (1H, m, H-8), 2.70 (1H, m, Ha-10), 2.60 (1H, m, Hb-10), 2.31 (1H, m, Ha-11), 3.22 (1H, m, Hb-11), 6.59 (1H, s, H-14), 6.63 (1H, s, H-17), 4.56 (1H, s, H-20), 4.62 (1H, d, 8.3, H-22), 3.31 (1H, d, 8.3, H-23), 1.48 (1H, m, Ha-25), 1.47 (1H, m, Hb-25), 2.60 (1H, m, H-26), 3.84 (1H, m, Ha-29), 3.22 (1H, m, Hb-29), 2.60 (1H, m, Ha-30), 3.22 (1H, m, Hb-30); FABMS m/z 643 (MþH)þ; HRFABMS m/z 643.2651 (MþH; calcd for C36H39N2O9, 643.2646). 1.3.8. (R)- and (S)-MTPA esters of bis-cephalezomine D (4). To a solution of 4 (0.5 mg) in CH2Cl2 (50 ml) was added (2)- or (þ)-MTPACl (1.1 ml), triethylamine (1.3 ml) and N,N-dimethylamino pyridine (0.2 mg). The mixture was allowed to stand at room temperature for 2 h. N,N-Dimethylamino-1,3-propandiamine (1.0 ml) was added, and after evaporation of solvent, the residue was applied to a silica gel column (CHCl3 – MeOH, 50:1!10:1) to give the (S)- or (R)-MTPA esters of 4 (0.6 mg, 87%). (R)-MTPA ester of 4. 1H NMR (CDCl3) d 4.97 (1H, s, H-1), 5.97 (1H, d, 9.5, H-3), 3.70 (1H, d, 9.5, H-4), 1.71 (2H, m, H-6), 0.85 (1H, m, Ha-7), 3.32 (1H, m, H-8), 2.71 (1H, m, Ha-10), 2.60 (1H, m, Hb-10), 2.35 (1H, m, Ha-11), 3.09 (1H, m, Hb-11), 6.56 (1H, s, H-14), 6.59 (1H, s, H-17), 5.89 (1H, s, Ha-18), 5.99 (1H, s, Hb-18), 3.67 (1H, s, H-19), 1.92 (1H, d, 16.7, Ha-30 ), 2.26 (1H, d, 16.3, Hb-30 ), 3.58 (3H, s, H-30 COOMe), 1.35 –1.45 (2H, m, H-100 ), 1.14 (1H, m, Ha-200 ), 1.35 –1.45 (1H, m, Hb-200 ), 1.35 – 1.45 (2H, m, H-300 ), 1.19 (3H, s, H-500 ), 1.18 (3H, s, H-600 ), 4.73 (1H, s, H-20), 5.96 (1H, m, H-22), 3.56 (1H, d, 10.3, H-23), 1.52 (2H, m, H-25), 2.71 (1H, m, H-26), 3.85 (1H, m, Ha-29), 3.09 (1H, m, Hb-29), 2.61 (1H, m, Ha-30), 3.18 (1H, m, Hb-30), 6.54 (1H, s, H-33), 6.59 (1H, s, H-36), 5.88 (1H, s, Ha-37), 5.91 (1H, s, Hb-33), 3.64 (3H, s, H-38), 3.52 (1H, m, Ha-3000 ), 1.34 (1H, m, H-10000 ), 0.91 (1H, m, Ha-20000 ), 1.18 (1H, m, Hb-20000 ), 1.38 (1H, m, H-30000 ), 0.80 (3H, d, 6.6, H-40000 ), 0.81 (3H, d, 6.6, H-50000 ), 7.42 – 7.60 (5H, m, H-MTPA); FABMS m/z 1306, (MþH)þ; HRFABMS m/z 1305.5160 (MþH; calcd for C67H80N2O21F3, 1305.5207). (S)-MTPA ester of 4. 1H NMR (CDCl3) d 4.98 (1H, s, H-1), 5.83 – 6.02 (1H, m, H-3), 3.68 (1H, m, H-4), 1.73 (2H, m, H-6), 0.76– 0.92 (1H, m, Ha-7), 3.35 (1H, m, H-8), 2.52– 2.75 (2H, m, H-10), 2.35 (1H, m, Ha-11), 3.11 (1H, m, Hb-11), 6.56 –6.69 (1H, m, H-14), 6.56 – 6.69 (1H, m, H-17), 5.83– 6.02 (2H, m, H-18), 3.66 (1H, s, H-19), 1.93 (1H, d, 16.5, Ha-30 ), 2.27 (1H, m, Hb-30 ), 3.59 (3H, s, H-30 COOMe), 1.35 –1.45 (2H, m, H-100 ), 1.14 (1H, m, Ha-200 ), 1.28 –1.42 (1H, m, Hb-200 ), 1.28 – 1.42 (2H, m, H-300 ), 1.19 (3H, s, H-500 ), 1.18 (3H, s, H-600 ), 4.72 (1H, s, H-20), 5.83– 6.02 (1H, m, H-22), 3.56 (1H, m, H-23), 1.53 (2H, m, H-25), 2.52– 2.75 (1H, m, H-26), 3.83 (1H, m, Ha-29), 3.11 (1H, m, Hb-29), 2.61 (1H, m, Ha-30), 3.21 (1H, m, Hb-30), 6.56 –6.69 (1H, m, H-33), 6.56 –6.69 (1H, m, H-36), 5.83 –6.02 (2H, m, H-37), 3.66 (3H, s, H-38), 3.49 (1H, m, Ha-3000 ), 1.30 (1H, m, H-10000 ), 0.88 (1H, m, Ha-20000 ), 1.10 (1H, m, Hb-20000 ), 1.25 (1H, m, H-30000 ), 0.69 (3H, d, 6.6, H-40000 ), 0.70 (3H, d, 6.6, H-50000 ), 7.40 – 7.64 (5H, m, H-MTPA); FABMS m/z 1306 (MþH)þ; HRFABMS m/z 1305.5160 (MþH; calcd for C67H80N2O21F3, 1305.5207).

7868

M. Yoshinaga et al. / Tetrahedron 60 (2004) 7861–7868

Acknowledgements The authors thank S. Oka and M. Kiuchi, Center for Instrumental Analysis, Hokkaido University, for measurements of FABMS and FABMS/MS, and N. Yoshida, Health Sciences University of Hokkaido, for botanical identification. 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.

References and notes 1. For reviews of the Cephalotaxus alkaloids, see: (a) Miah, M. A. J.; Hudlicky, T.; Reed, J. W. The Alkaloids; Cordell, G. A., Ed.; Academic: New York, 1998; Vol. 51, p 199. (b) Huang, L.; Xue, Z. The Alkaloids; Brossi, A., Ed.; Academic: New York, 1984; Vol. 23, p 157. 2. (a) Morita, H.; Arisaka, M.; Yoshida, N.; Kobayashi, J. Tetrahedron 2000, 56, 2929– 2934. (b) Morita, H.; Yoshinaga, M.; Kobayashi, J. Tetrahedron 2002, 58, 5489– 5495.

3. Kobayashi, J.; Yoshinaga, M.; Yoshida, N.; Shiro, M.; Morita, H. J. Org. Chem. 2002, 67, 2283– 2286. 4. Takano, I.; Yasuda, I.; Nishijima, M.; Hitotsuyanagi, Y.; Takeya, K.; Itokawa, H. Tetrahedron Lett. 1996, 37, 7053 –7054. 5. Zhi, X.; Lizhen, X.; Dihua, C.; Liang, H. Acta Pharm. Sinica. 1981, 16, 752– 756. 6. Brandange, S.; Josephson, S.; Vallen, S. Acta Chem. Scand. 1974, B28, 153– 156. 7. (a) Powell, R.; Weisleder, D.; Smith, C. R., Jr.; Rohwedder, W. K. Tetrahedron Lett. 1970, 815– 818. (b) Ipaktchi, T.; Weinreb, S. M. Tetrahedron Lett. 1973, 3895– 3898. 8. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092– 4096. 9. Brandange, S.; Josephson, S.; Vallen, S.; Powell, R. G. Acta Chem. Scand. 1974, B28, 1237– 1248. 10. Grierson, D. Org. React. 1990, 39, 85 – 295. 11. Cassady, J. M.; Cong, P. Z.; Cooks, R. G.; Roush, R.; Chang, C. J.; Powell, R. G. Yaoxue Xuebao 1988, 23, 351– 355.