Perhentidines A-C: Macroline-Macroline Bisindoles from Alstonia and the Absolute Configuration of Perhentinine and Macralstonine Siew-Huah Lim, † Yun-Yee Low, † Shin-Jowl Tan, † Kuan-Hon Lim, ‡ Noel F. Thomas, † and Toh-Seok Kam* ,† † Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia ‡ School of Pharmacy, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia * S Supporting Information ABSTRACT: Three new bisindole alkaloids of the macro- line-macroline type, perhentidines A-C(1-3), were isolated from the stem-bark extract of Alstonia macrophylla and Alstonia angustifolia. The structures of these alkaloids were established on the basis of NMR and MS analyses. The absolute configu- rations of perhentinine (4) and macralstonine (5) were estab- lished by X-ray diffraction analyses, which facilitated assign- ment of the configuration at C-20 in the regioisomeric bisindole alkaloids perhentidines A-C(1-3). A potentially useful method for the determination of the configuration at C-20 based on comparison of the NMR chemical shifts of the bisindoles and their acetate derivatives, in these and related bisindoles with similar constitution and branching of the monomeric units, is also presented. T he genus Alstonia (Apocynaceae), which is widely distributed in Southeast Asia, 1-3 is rich in alkaloids. 4-24 We previously reported the structure of perhentinine (4), a bisindole alkaloid constituted from the union of two macroline moieties from Alstonia macrophylla. 14 The structure was elucidated on the basis of the analysis of the NMR and MS data, which were however insufficient to assign the config- uration at C-20. In continuation of our ongoing studies of Malaysian Alstonia species, 12-21 we have isolated three additional bisindole alkaloids related to perhentinine from two Malayan Alstonia species, perhentidines A (1) and B (2) from A. macrophylla Wall and perhentidines A (1) and C (3) from A. angustifolia Wall. Perhentidine A (1) was obtained as a light yellowish oil with [α] 25 D -77 (c 0.40, CHCl 3 ). The IR spectrum showed bands at 3400, 1702, 1648, and 1617 cm -1 , due to the presence of OH, ketocarbonyl, and an α,β-unsaturated carbonyl group, respec- tively, while the UV spectrum showed absorption maxima at 231 and 286 nm, consistent with the presence of indole chromophores. The ESIMS of 1 showed a pseudomolecular ion [M + H] + at m/z 705, which analyzed for C 43 H 52 N 4 O 5 + H. The 13 C NMR spectrum (Table 1) showed a total of 43 resonances, comprising seven methyl, seven methylene, 16 methine, and 13 quaternary carbon atoms, in agreement with the molecular formula. The quaternary carbon resonance at δ C 212.9 is consistent with the presence of a ketocarbonyl, while the other quaternary carbon resonance at δ C 195.2 and the associated olefinic carbon signals at δ C 121.0 and 157.2, are consistent with the presence of an α,β-unsaturated carbonyl group. The unusual deshielding of the β-carbon at δ C 157.2 indicated oxygen substitution. In addition, two oxymethylene carbons were observed at δ C 66.8 and 67.6, the former due to a hydroxymethyl group, as shown by acetylation, which yielded an O-acetyl derivative (1a). The 1 H NMR data (Table 2) showed the presence of four aromatic hydrogens (δ H 7.16-7.56) associated with an unsubstituted indole moiety, a pair of AB doublets at δ H 6.75 and 7.22 associated with another indole moiety substituted at positions 11′ and 12′, a vinylic singlet at δ H 7.49 associated with a trisubstituted double bond, and a total of seven methyl singlets, corresponding to two N1-Me (δ H 3.58, 3.69), two N4- Me (δ H 2.36, 2.37), two acetyl methyls (δ H 1.55, 2.06), and an aromatic methoxy substituent (δ H 3.83, 11′-OMe). Since only six aromatic hydrogens were observed and both indolic nitro- gens are substituted, it is reasonable to conclude that the bisindole is branched from one of the aromatic carbon atoms of one monomer, with the adjacent position occupied by the methoxy substituent. The aromatic doublet at δ H 7.22 was assigned to H-9′ from its NOE with H-6′, while the placement of the methoxy substituent at C-11′ was confirmed by the ob- served NOE between H-10′ (δ H 6.75) and 11′-OMe (δ H 3.83). These assignments were further supported by the observed three-bond correlations from H-9′ to C-7′ and C-13′ and from H-9′ and 11′-OMe to C-11′ in the HMBC spectrum (Figure 1). These observations indicated C-12′ as the site of branching of the bisindole from this monomeric unit. The remaining part of this macroline half was assembled via the 2-D NMR data. The COSY spectrum revealed an NCHCH 2 CHCHCH 2 O fragment, which corresponds to the N-4′-C-3′-C-14′-C-15′-C-16′-C-17′-O unit of the lower macroline half. The observed three-bond correlations from the Received: February 14, 2012 Published: May 4, 2012 Article pubs.acs.org/jnp © 2012 American Chemical Society and American Society of Pharmacognosy 942 dx.doi.org/10.1021/np300120p | J. Nat. Prod. 2012, 75, 942-950