Hybrid Monoterpenoid Indole Alkaloids Obtained as Artifacts from Rauvolfia tetraphylla

Abstract Five new hybrid monoterpenoid indole alkaloids bearing an unusual 2,2-dimethyl-4-oxopiperidin-6-yl moiety, namely rauvotetraphyllines F–H (1, 3, 4), 17-epi-rauvotetraphylline F (2) and 21-epi-rauvotetraphylline H (5), were isolated from the aerial parts of Rauvolfia tetraphylla. Their structures were established by extensive spectroscopic analysis. The new alkaloids were evaluated for their cytotoxicity in vitro against five human cancer cell lines. Graphical Abstract Electronic supplementary material The online version of this article (doi:10.1007/s13659-015-0074-2) contains supplementary material, which is available to authorized users.


Introduction
Rauvolfia genus of the Apocynaceae family, comprising about 60 species, is mainly distributed in America, Africa, Asia, and Oceania [1]. Plants of this genus are a rich source of monoterpenoid indole alkaloids, which have attracted great interests from biological and therapeutic aspects [2][3][4][5]. As part of a BioBioPha [http://www.chemlib.cn] objective to assemble a large-scale natural product library valuable in the discovery of new drug leads from nature, previous chemical study on the ethanolic extract of Rauvolfia tetraphylla had resulted in the isolation of five new indole alkaloids, rauvotetraphyllines A-E [6]. Further investigation of the remaining components led to the isolation of another five new alkaloids bearing an unusual 2,2dimethyl-4-oxopiperidin-6-yl moiety, rauvotetraphyllines F-H (1, 3,4), 17-epi-rauvotetraphylline F (2) and 21-epirauvotetraphylline H (5). The present paper describes the isolation, structure elucidation, and cytotoxic evaluation of the new compounds.

Results and Discussion
Compound 1, obtained as amorphous powder, possessed a molecular formula of C 31 H 41 N 3 O 7 , as evidenced by HR-ESI-MS (pos.) at m/z 568.3025 (calcd for C 31 H 42 N 3 O 7 , 568.3022), in combination with NMR spectra (Tables 1  and 3), requiring 13 degrees of unsaturation. In the UV spectrum, two characteristic maxima at 225 and 281 were detected, suggesting the existence of an unsubstituted indole chromophore [7]. The IR spectrum showed the presence of OH/NH (3404 cm -1 ) functionalities. The 1D-NMR spectra (Tables 1 and 3)   ,2-dimethyl-4-oxopiperidin-6-yl moiety by HMBC correlations ( Fig. 1) from H-17 to C-23, H-22 to C-23 and C-24, and H-24 to C-22, C-23, C-25, C-26, and C-27. The piperidinyl moiety was linked to C-16 through C-16-C-17 bond by HMBC correlations from H-16 to C-17 and C-22 and 1 H-1 H COSY correlation of H-16/H-17 (Fig. 1). The relative configuration of 1 was established by NMR analysis based on computer-generated 3D drawing with minimized energy by MM2 calculation (Fig. 2). ROESY correlations of H-16$H-6b/H-14b and H-5$H-21 suggested that 1 had the same stereochemistry as rauvotetraphylline B. The E-geometry of the ethylidene was indicated from ROESY correlations of H-15$Me-18 and H-19$H-21. The anti relationship of H-16 and H-17 was suggested by the large coupling constant (J 16,17 = 9.0 Hz), which could also be explained by that the molecule favors the conformation in which larger substituents are in the anti position. This was further supported by ROESY correlations of H-17$Me-18. The R* configuration of C-17 was implied by ROESY correlations of H-6b$H-22b and Me-18$Me-26 (Fig. 2). Another noteworthy observation is that the chemical shift of H-15 (d H 3.26) in 1 was relatively deshielded compared to that of H-15 (d H 2.79) in its 17-epimer 2 (vide infra). This is attributed to paramagnetic deshielding caused by the proximity of the NH nitrogen atom to H-15 (Fig. 2). Thus, the structure of 1 was established as shown and named rauvotetraphylline F. Compound 2, isolated as amorphous powder, had the same molecular formula as 1 based on HR-ESI-MS (pos.), showing a quasi-molecular ion peak at m/z 568.3031 (calcd for C 31 H 42 N 3 O 7 , 568.3022). The 1 H and 13 C NMR spectra of 2 (Tables 1 and 3) were very similar in all respects to those of 2 except for the chemical shifts of H-5, H-6b, and H-15 in the 1 H NMR spectrum. This discrepancy proved that compound 2 is a C-17 epimer of 1 while applying the same analysis carried out for 1. The paramagnetic deshielding experienced by H-15 in 1 was now experienced by H-5 and H-6b instead in 2 (Fig. 2), implying the S* configuration of C-17. This was further verified by strong ROESY correlations between H-22a and H-15/Me-18 and no correlation between Me-26 and Me-18. Therefore, the structure of 1 was elucidated as shown and named 17-epirauvotetraphylline F.  (Tables 1 and 3) were closely related to those of 1 except for the signals of a methylene group in 3 instead of an oxygenated methine group in 1, and the absence of a series of glucose resonances. The configuration of C-17 was designated as R* based on Me-18 showing ROESY correlation to Me-26, but no correlation to H-22. Consequently, the structure of 3 was determined and named rauvotetraphylline G.
Compound 4 was isolated as amorphous powder. Its molecular formula was determined as C 27 H 33 N 3 O 3 by positive HR-ESI-MS at m/z 448.2613 (calcd for C 27 H 34 N 3 O 3 , 448.2600). The 13 C NMR data (Table 3) were very similar to those of perakine [8]. The prominent difference between them was the aldehyde group in  Table 2). The proximity of the NH nitrogen atom to H-15 in 4 caused a marked downfield shift of H-15 (D = 0.30 ppm) (Fig. 4). Hence, the structure of 4 was assigned as shown and named rauvotetraphylline H. Compound 5, obtained as amorphous powder, had the same molecular formula as 4, possessing a quasi-molecular ion peak at m/z 448.2602 (calcd for C 27 H 34 N 3 O 3 , 448.2600). The 1 H and 13 C NMR spectra of 5 (Tables 2  and 3 (Fig. 4), revealing the S* configuration of C-21. This was further supported by significant ROESY correlation (Fig. 4) of H-22a$H-15 and no correlation of H-22a$H-19 or H-22b$Me-18. Therefore, the structure of 5 was elucidated as shown and named 21-epi-rauvotetraphylline H.
The contribution of artifacts on structural diversity of alkaloids from Rauvolfia species is not ignorable as acidic or basic conditions are often used during isolation process, in spite that many artifacts from this genus are generally  presented in literatures as naturally occurring compounds [9]. Considering that the presence of aldehyde group at C-16/C-20 is common for sarpagine/perakine type alkaloids [8,10,11], it's plausible to deduce that, like triacetonamine [12], a common artifact of plant extractions, the 2,2-dimethyl-4-oxopiperidine moiety might also be an artifact produced by reaction of aldehyde group with acetone/ammonia since the latter were used as eluents during the isolation procedures. These artifacts represent a unique type of sarpagine/perakine series bearing an unusual piperidine unit brought about by using common eluents. All of the isolated compounds were evaluated for their in vitro growth inhibitory effects against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW-480) with cisplatin and taxol serving as positive controls by the MTT method [13]. Regrettably, all tested compounds were inactive (IC 50 values [ 40 lM).

Plant Material
The aerial parts of Rauvolfia tetraphylla were collected in Xiaomenglun of Yunnan Province, China, in June 2010 and identified by Mr. Yu Chen of Kunming Institute of Botany, Chinese Academy of Sciences. The voucher specimen (No. BBP0234020RT) was deposited at BioBioPha Co., Ltd.