Indole Alkaloids from the Leaves of Nauclea officinalis

Three new indole alkaloids, named naucleamide G (1), and nauclealomide B and C (5 and 6), were isolated from the n-BuOH-soluble fraction of an EtOH extract of the leaves of Nauclea officinalis, together with three known alkaloids, paratunamide C (2), paratunamide D (3) and paratunamide A (4). The structures with absolute configurations of the new compounds were identified on the basis of 1D and 2D NMR, HRESIMS, acid hydrolysis and quantum chemical circular dichroism (CD) calculation. According to the structures of isolated indole alkaloids, their plausible biosynthetic pathway was deduced.


Introduction
Nauclea officinalis Pierre ex Pitard belongs to the genus Nauclea in family Rubiaceae, which is mainly distributed in Hainan, Guangxi, Guangdong and other provinces in the south of China.As a traditional Chinese medicine, the bark and twigs of N. officinalis is widely used for the treatment of colds, fever, acute tonsillitis, sore throat, conjunctivitis, enteritis, dysentery, eczema and other ailments [1].Previous phytochemical investigation had revealed that monoterpene indole alkaloids, which present a polycyclic skeleton with a tetrahydro-β-carboline ring, are the main components of this plant [2][3][4][5][6][7][8][9][10][11][12][13].The complex ring system and diversity of stereostructure of these monoterpene indole alkaloids have attracted great interest for phytochemical studies.As a part of our ongoing program to search for structurally unique alkaloids from the medicinal plants in southern China [5,13], we carried out the continuing investigation on the leaves of N. officinalis.In our present study, three new indole alkaloids, naucleamide G (1), and nauclealomide B and C (5 and 6), together with three known ones, paratunamide C (2), paratunamide D (3) and paratunamide A (4) (Figure 1), were isolated, among which oxindole alkaloids (2)(3)(4)(5)(6) were rarely reported from the Nauclea genus.In this paper, we describe the structure elucidation of these new alkaloids by means of NMR, HRESIMS, circular dichroism spectroscopy and computational analysis.
In the 1 H-NMR spectrum of 6, the coupling constants of 3 J 3,14a (J = 3.6 Hz) and 3 J 3,14b (J = 2.1 Hz) indicated the presence of α configuration of H-3.In the ROESY spectrum, the correlation between H-3 and H-9 was observed, which suggested that the carbonyl group at C-2 was above the C/D/E plane (Figure 3).Considering the biogenetic pathway for these indole alkaloids, the absolute configuration of C-15 was determined as S. Thus, the absolute configuration of 6 was elucidated as 3R, 7S, 15S, 20R, 21S.
In the 1 H-NMR spectrum of 6, the coupling constants of 3 J3,14a (J = 3.6 Hz) and 3 J3,14b (J = 2.1 Hz) indicated the presence of α configuration of H-3.In the ROESY spectrum, the correlation between H-3 and H-9 was observed, which suggested that the carbonyl group at C-2 was above the C/D/E plane (Figure 3).Considering the biogenetic pathway for these indole alkaloids, the absolute configuration Based on the structural features of compounds 1-6, which were all derived from L-tryptophan and secologanin, the plausible biosynthetic pathway for them was deduced (Scheme 1).

General Procedures
Optical rotations were measured on a JASCO P-1020 polarimeter (JASCO Corporation, Tokyo, Japan).UV spectra were recorded on a JASCO V-550 UV/VIS spectrophotometer (JASCO Corporation).A JASCO FT/IR-480 plus FT-IR spectrometer (JASCO Corporation) was used for scanning the IR spectra with KBr pellets. 1 H, 13 C, and 2D NMR spectra were recorded on a Bruker AV-400 spectrometer (Bruker Corporation, Rheinstetten, Germany).HR-ESI-MS data were conducted on an Agilent 6210 LC/MSD TOF mass spectrometer.CD spectra were obtained using a JASCO J-810 circular dichroism spectrometer.For column chromatography, silica gel (300-400 mesh; Qingdao Marine Chemical Group Corporation, Qingdao, China) and Sephadex LH-20 (Pharmacia, Pittsburgh, PA, USA) were used.TLC analyses were carried out using precoated silica gel GF 254 plates (Yantai Chemical Industry Research Institute, Yantai, China).Analytic high-performance liquid chromatography (HPLC) was performed on a SHIMADZU chromatography (SHIMADZU Corporation, Kyoto, Japan) equipped with an LC-20AD pump and an SPD-M20A diode-array detector (DAD) with an Inertsil ODS-3 column (4.6 mm ˆ250 mm, 5 µm).Preparative HPLC was carried out on a SHIMADZU instrument equipped with an LC-20AP pump and an SPD-20A (SHIMADZU Corporation) detector with a YMC-Pack ODS-A column (20 mm ˆ250 mm, 5 µm).

Plant Material
The leaves of N. officinalis were collected in Sanya City, Hainan Province, China, in July 2008, and authenticated by Professor Wei-ping Chen (Hainan Branch Institute of Medicinal Plants, Chinese biosynthetic pathway for all the alkaloids was proposed.Compound 1 with a tetrahydro-β-carboline ring originated from condensation of L-tryptophan with secologanin to give strictosidine; nevertheless, oxindole alkaloids (2-6) might be generated through another biosynthetic route.

Figure 1 .
Figure 1.Structures of compounds 1-6 isolated from N. officinalis, as well as structure of nauclealomide A mentioned in the text.

Figure 1 .
Figure 1.Structures of compounds 1-6 isolated from N. officinalis, as well as structure of nauclealomide A mentioned in the text.

Figure 4 .
Figure 4. Calculated and experimental CD spectra of 1.

Figure 4 .
Figure 4. Calculated and experimental CD spectra of 1.

Figure 4 .
Figure 4. Calculated and experimental CD spectra of 1.