HETEROCYCLES

Short Paper | Special issue | Vol. 90, No. 1, 2015, pp. 601-606
Received, 13th February, 2014, Accepted, 26th February, 2014, Published online, 26th March, 2014.
DOI: 10.3987/COM-14-S(K)6

■ A New Indole Alkaloid from Voacanga grandifolia

Azusa Haseo, Alfarius Eko Nugroho, Yusuke Hirasawa, Toshio Kaneda, Osamu Shirota, Abdul Rahman, Idha Kusumawati, Noor Cholies Zaini, and Hiroshi Morita*

Faculty of Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan

Abstract

A new bisindole alkaloid, voacalgine F (1), has been isolated from the bark of Indonesian Voacanga grandifollia (Miq.) Rolfe. Its structure was elucidated on the basis of 1D and 2D-NMR data analysis.

Voacanga is a small genus of the Apocynaceae family consisting of 12 species. Species of this genus are distributed mainly in the tropical Africa and Malysia, and have been reported to contain vobasine, eburnane, iboga, and aspidosperma type of monoterpene indole alkaloids.1 Various activities have been reported for monoterpene indole alkaloids, such as cytotoxicity,2 anti-melanogenesis,3 anti-plasmodial,4 and vasorelaxant activities.5 In the search for new bioactive compounds from tropical plants,3,5-8 alkaloid constituents of V. grandifolia bark were investigated and a new bisindole alkaloid voacalgine F (1) was isolated together with voacamine,9-12 voacangine,9 voacanginehydroxyindolenine,13 and pagicerine.14 The isolation and structure elucidation of 1 are reported herein.

Voacalgine F (1) was obtained as yellow amorphous solid and the molecular formula was determined as C43H52N4O6 from the HRESIMS data (m/z 721.3978 [M+H]+, calcd for C43H53N4O6, 721.3965). The IR absorptions (3430 and 1720 cm–1) implied the presence of hydroxyl and carbonyl functionalities. Analysis of the 13C-NMR data (Table 1) showed that the chemical shift of 21 carbon signals is highly similar to the vobasine unit of voacamine, suggesting the presence of a vobasine unit in 1. The chemical shift of the other carbon signals is highly similar to that of voacangine hydroxyindolenine, except for downfield shift of C-9. These data suggested the structure of 1 as a new vobasine-iboga type of bisindole alkaloids as shown in Figure 1.

The planar structure of 1 was further confirmed by 2D NMR analysis (1H-1H COSY, HSQC and HMBC, Figure 1). Analysis of 1H-1H COSY and HSQC data revealed the presence of 7 partial structure (a–g). HMBC correlations of 7-OH to C-2, C-6, C-7 and C-8, H-11 to C-9 and C-13, and H-12 to C-8 and C-10 confirmed the presence of a 7-hydroxyindolenine moiety and the connection of C-6 and C-7. HMBC cross-peaks of H3-18 to C-20 suggested the connectivity of C-19 and C-20, and the HMBC correlations of H2-3 to C-5 and C-21, H2-19 to C-21, H-21 to C-2, C-5, C-16, C-17 and a carbonyl (δC 174.4), and a methyl (δH 3.73) to δC 174.4 completed the structure of the iboga unit. HMBC cross-peaks of H-3’ to C-7’, H2-6’ to C-2’, C-7’, and C-8’, H-9’ to C-8’ and C-13’, and H-12’ to C-8’ suggested the presence of an indole unit and the connectivity of partial structure f to the indole unit. HMBC cross-peaks of H3-18’ to C-20’, and H-19 to C15’ and C-21’ revealed the connectivity of partial structures f, g, and C-21’ through C-20’. HMBC correlations of a methyl (δH 2.61) to C-5’ and C-21’ established the connections between C-5’ and C-21’ through a nitrogen atom, and HMBC cross-peaks of H-16’ to C-17’, and another methyl (δH 2.49) to C-17’ suggested the presence of a methoxycarbonyl moiety at C-16’. Finally, the two units were confirmed to be connected by C-9 to C-3’ bond by the HMBC correlations of H-3’ to C-8 and C-10.

The relative configuration of 1 was assigned using the 1H-1H coupling constant values, 1H NMR chemical shift and NOESY correlations. The orientation of 7-OH and H-21 was assigned as α from the NOESY correlation 7-OH/H-21. The relative configuration C-14, C-16, C-20, C-21, C-3’ and C-5’ was assigned to be the same as in voacamine based on the NOESY correlations shown in Figure 2. The orientation of the methoxycarbonyl at C-16’ was deduced from the highly shielded 1H NMR chemical shift of the methoxy group (δH 2.49), and the configuration of the C-19’-C-20’ was determined to be E from the NOESY correlation of H-19’/H2-21’. Finally the relative configuration of the total molecule was deduced from the NOESY correlations of H-6a/NH and H-3’, 7-OH/H-3’ and H-14’a (Figure 3).

EXPERIMENTAL
General Experimental Procedures. Optical rotations were measured on a JASCO DIP-1000 automatic digital polarimeter. UV spectra were obtained on an Ultrospec 2100 pro spectrophotometer and IR spectra were recorded on a JASCO FT/IR-4100 spectrophotometer. High-resolution ESI MS were obtained on a LTQ Orbitrap XL (Thermo Scientific). 1H and 2D NMR spectra were recorded on a Bruker AV700 spectrometer and chemical shifts were referenced to the residual solvent peaks (δH 7.26 and δC 77.0 for chloroform-d). Standard pulse sequences were employed for the 2D NMR experiments.
Plant Material. The barks of V. grandifolia were collected at Purwodadi Botanical Garden, Indonesia in 2008. The botanical identification was made by Ms. Sri Wuryanti, Purwodadi Botanical Garden. A voucher specimen has been deposited in the herbarium at Purwodadi Botanical Garden, Pasuruan, Indonesia.
Extraction and Isolation. The dried and powdered bark of V. grandifolia (300 g) was extracted successively with MeOH. Part of the extract (17.0 g of 28.4 g) was dissolved in 3% aqueous tartaric acid (pH 2) and then partitioned with EtOAc. The aqueous layer was treated with saturated Na2CO3 (aq.) to pH 9 and was partitioned successively by CHCl3 and n-BuOH. Part of the CHCl3 soluble materials (5.0 g of 5.10 g) was subjected to an LH-20 column (CHCl3/MeOH 1:1) to obtain 12 fractions.
Fraction 7 was fractionated by amino silica gel column chromatography (n-hexane/EtOAc, 1:0∼1:1, CHCl3/MeOH, 0:1∼1:0) to obtain voacamine (100.8 mg, 0.032%). In addition, fraction eluted by CHCl3/MeOH (80:1) was further separated by ODS HPLC (Inertsil ODS-3, 5 µm, 10 x 250 mm; 35% MeCN in 0.1% aqueous HCO2H; flow rate 2 mL/min; UV detection at 254 nm) to obtain 1 (tr 30 min., 2.7 mg, 0.001%).
Fraction 11 was separated by repeated amino silica gel column chromatography (n-hexane/EtOAc, 1:0∼1:1, CHCl3/MeOH, 0:1∼1:0) and silica gel column chromatography (CHCl3/MeOH, 0:1∼1:0) to give voacangine (23.8 mg, 0.0043%), voacanginehydroxyindolenine (12.8 mg, 0.0079%), and pagicerine (3.6 mg, 0.0012%).
Voacalgine F (1): yellow amorphous solid; [α]D22 -132 (c 1.0, MeOH); IR (KBr) νmax 3430, 2940 and 1720 cm–1; UV (MeOH) λmax (ε) 225 (23400) and 290 (9000) nm; 1H and 13C NMR data (Table 1); ESIMS m/z 721 (M+H)+; HRESIMS m/z 721.3978 (M+H+; calcd for C43H53N4O6, 721.3965).

ACKNOWLEDGEMENTS
This work was supported by Grants in-Aid for Scientific Research from JSPS KAKENHI.

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