Three new indole alkaloids from Rauvolfia yunnanensis

One rare tetracyclic macroline-type indole alkaloid, named rauvoyunine A (1), and two new picraline-type alkaloids rauvoyunines B and C (2 and 3) were isolated from the aerial parts of Rauvolfia yunnanensis. Their structures were elucidated on the basis of extensive spectroscopic analysis. Compounds 2 and 3 were evaluated for their in vitro cytotoxicity against five human tumor cell lines. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-011-0023-7 and is accessible for authorized users.


Introduction
The plants of genus Rauvolfia, widely distributed in America, Africa, Asia, and Oceania, 1 are well-known rich sources of unique heterocyclic alkaloids with monoterpene indole skeletons. These alkaloids have attracted great interests from biological and therapeutic aspects, due to their anticancer, 2 antimalarial, 3 antihypertensive, 4 and sedative 5 properties.
Rauvolfia yunnanensis Tsiang, belonging to the Apocynaceae, is indigenous to southwestern China, and its roots are utilized locally for the remedy of scab 1 . Recently, several structurally interesting indole alkaloids were isolated from this plant. [6][7][8] As one part of our research program exploring bioactive monoterpene indole alkaloids from Chinese species of Rauvolfia, phytochemical analysis from the methanol extract of aerial parts of R. yunnanensis led to the isolation of three new alkaloids, rauvoyunines A-C (1-3). This paper reports the isolation and structure elucidation of new compounds and cytotoxic evaluation of selected compounds.

Results and Discussion
Compound 1, obtained as amorphous powder, has a molecular formula of C 21 H 28 N 2 O 3 based on HRESIMS (pos.), showing a quasi-molecular ion peak at m/z 357.2185 (calc. for C 21 H 29 N 2 O 3 , 357.2178). The UV spectrum showed absorptions at λ max 228, 282, and 303 nm typical of a substituted indole chromophore. 9 The IR spectrum showed absorptions at 3432 and 1628 cm -1 , attributed to hydroxy and olefin groups, respectively. The 1 H NMR spectrum (Table 1) showed signals for an aromatic AMX spin system at δ H 6.73 (dd, J = 8.7 and 2.3 Hz), 6.85 (d, J = 2.3 Hz), and 7.17 (d, J = 8.7 Hz), typical of an indole moiety substituted by a hydroxy group at 10 or 11 position, signals characteristic of an ethylidene group at δ H 1.44 (d, J = 6.8 Hz) and 5.58 (q, J = 6.8 Hz), singlets for Nmethyl groups at δ H 2.55 and 3.59, and resonances of two hydroxymethyl groups, one at δ H 3.75 (dd, J = 10.5 and 3.7 Hz) and 3.91 (dd, J = 10.5 and 5.5 Hz), and another at δ H 4.02 (2H, s). The 13 C NMR (DEPT) spectrum (Table 1)  , and three methyl groups (δ C 41.5, 29.3, and 13.1). The above-mentioned data, together with the presence of nine degrees of unsaturation suggested that 1 was an indole alkaloid with tetracyclic macroline skeleton. 10,11 This conclusion is supported by the HMBC (Figure 1) three-bond correlations of H-5 to C-3, C-15, and C-17, H-16 to C-6 and C-14, and H-15 to C-3, C-17, and C-19. In addition, the m-coupling doublet at δ H 6.85 (J = 2.3 Hz) showed an HMBC correlation with C-7 and a ROESY correlation with H-6β, indicating the hydroxyl at C-10. HMBC cross-peaks of H-18 to C-19 and C-20, and H-19 to C-15 and C-20 located the ethylidene side chain at C-20, and cross-peaks of H-17 to C-5, C-15, and C-16, and H-21 to C-15, C-19, and C-20 revealed that the two hydroxymethyl group was adjacent to C-16 and C-20, respectively. The position of each methyl group was confirmed by HMBC correlations from N(1)-Me to C-2 and C-13, and N(4)-Me to C-3 and C-5.
The relative stereochemistry of 1 was elucidated by ROESY experiments (Figure 1). The ROESY spectrum exhibited correlations of H-16↔H-6β and H-3↔N(4)-Me↔H-5, fixing the two bridgehead hydrogen atoms as α orientation, revealing *To whom correspondence should be addressed. E-mail: jkliu@mail.kib.ac.cn the stereochemistry of H-16 as β-oriented. The ROESY correlation between H-14α and the hydroxymethyl proton at C-20 indicated that C-20 was α-oriented and H-15 was βoriented. The ROESY correlations of Me-18↔H-15 and H-19↔H-21 confirmed that the ethylidene side chain had an Econfiguration. Consequently, the structure of compound 1 was determined as shown, named rauvoyunine A.
Compound 2 was obtained as amorphous powder. Its molecular formula was determined to be C 23 H 26 N 2 O 6 on the basis of HRESIMS (pos.), showing a quasi-molecular ion peak at m/z 427.1868 (calc. for C 23 H 27 N 2 O 6 , 427.1869). The UV spectrum showed maximum absorption at 292 nm, and the IR spectrum implied the presence of carbonyl (1738 cm -1 ) functionality. The 1 H NMR spectra (Table 1) showed the presence of three aromatic protons, an ethylidene side chain, a methyl ether, and an acetyl. The 13 C NMR spectra (Table 1) revealed 23 carbon signals due to six sp 2 quaternary carbons, four sp 2 methines, three sp 3 quaternary carbons, three sp 3 methines, four sp 3 methylenes, and three methyl groups. These spectroscopic features suggested that 2 had a picraline-type skeleton. 13,14 The three observed aromatic resonances at δ H 7.11 (d, J = 8.8 Hz), 6.20 (dd, J = 8.8, 2.2 Hz), and 6.19 (d, J = 2.2 Hz) combined with the fact that a hydroxylated carbon signal was detected at δ C 158.6 (s) suggested that an OH group located at C-10 or C-11. Finding HMBC correlation from the o-coupling doublet at δ H 7.11 (J = 8.8 Hz) to C-7 at δ C 53.2 (s) and ROESY correlation between the doublet and H-6α further confirmed placement of hydroxyl at C-11. Two geminally coupled proton signals at δ H 3.85 and 4.52 (each d, J = 11.1 Hz) showed correlations with an O-acetyl carbonyl carbon at δ C 171.9, indicating that an acetoxy group was located at C-17. An methoxy singlet at δ H 3.66 showed correlation to C-22 at δ C 173.7, allowing assignment of methyl ether at C-22 position. The ROESY cross-peak between H-17a and H-14β indicated that C-17 was β-oriented, which was further supported by the presence of an NMR signal of the acetyl methyl group at δ H 1.62 (the noticeable upfield shift being due to the shielding by the aromatic ring current). 15 ROESY cross-peaks of H-14α↔H-21α↔H-3 and H-21β↔H-5 revealed that the configuration of H-3 and H-5 were α and β, respectively. The ethylidene moity was assigned to be E-type since the correlations of H-18↔H-15 and H-19↔H-21 were observed. Therefore, the structure of 2 was unambiguously elucidated as 11-hydroxypicraline, named rauvoyunine B.
Compound 3, obtained as amorphous powder, possessed a molecular formula of C 32 H 36 N 2 O 9 , as evidenced by HRESIMS (pos.) at m/z 593.2496 (calc. for C 32 H 37 N 2 O 9 , 593.2499), in combination with 1 H and 13 C NMR (DEPT) spectra (Table 1), and appropriate for 16 degrees of unsaturation. The UV spectrum showed maximum absorption at 269 nm. Its IR spectrum revealed absorption bands of carbonyl (1722 cm -1 ). The 1 H NMR data showed signals of three aromatic protons at δ H 6.04 (br. d, J = 8.3 Hz), 6.26 (br. s), and 7.29 (d, J = 8.3 Hz), an ethylidene group at δ H 1.62 (d, J = 6.7 Hz) and 5.48 (q, J = 6.7 Hz), two methoxy groups at δ H 3.45 (s) and 3.64 (s), and a 3′,4′,5′-trimethoxybenzoyloxy unit, based on the presence of two aromatic protons at δ H 6.94 (2H, s) and three overlapped methoxy groups at δ H 3.87 (9H, s). The 13 C NMR (DEPT) data exhibited ten sp 2 quaternary carbons, six sp 2 methines, three sp 3 quaternary carbons, three sp 3 methines, four sp 3 methylenes, and six methyl groups. These spectroscopic features were closely related to those of 2, but it was evident that 3 had a trimethoxybenzoyloxy unit instead of the acetoxy moity at C-17 and a methoxyl group instead of the hydroxy group at C-11. This was also supported by HMBC correlations from H-17 to C-7′ and correlation from methoxy protons at δ H 3.45 (s) to C-11 at δ C 160.1. The ROESY cross-peaks of Me-18↔H-15 and H-19↔H-21 confirmed that the ethylidene possessed an Econfiguration. The cross-peak of 17a↔H-14β indicated that the stereochemistry of C-16 was identical to that of 2. Since the J 3 14 , J 14 15 , and J 5 6 values were essentially unchanged compared to 2, it is safe to deduce that the ring junction stereochemistry remain intact. Thus, the structure of 3 was established to be 11-methoxyburnamine-17-O-3′,4′,5′-trimethoxybenzoate and named as rauvoyunine C.

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-011-0023-7 and is accessible for authorized users.