Letter pubs.acs.org/OrgLett
Rauvomines A and B, Two Monoterpenoid Indole Alkaloids from Rauvolfia vomitoria Jun Zeng,† Dong-Bo Zhang,† Pan-Pan Zhou,† Qi-Li Zhang,‡ Lei Zhao,‡ Jian-Jun Chen,*,† and Kun Gao*,† †
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China ‡ Gansu University of Chinese Medicine, Lanzhou 730000, China S Supporting Information *
ABSTRACT: Two unusual normonoterpenoid indole alkaloids rauvomine A (1) and rauvomine B (2), together with two known compounds peraksine (3) and alstoyunine A (4), were isolated from the aerial parts of Rauvolf ia vomitoria. The structures with absolute configurations of 1 and 2 were elucidated by spectroscopic analysis, single-crystal X-ray diffraction, and electronic circular dichroism (ECD) calculations. Compound 2 is a novel C18 normonoterpenoid indole alkaloid with a substituted cyclopropane ring that forms an unusual 6/5/6/6/3/5 hexcyclic rearranged ring system. The plausible biogenetic pathways of 1 and 2 were proposed. Compound 2 exhibited significant anti-inflammatory activity.
Rauvolf ia vomitoria (Apocynaceae) is widely distributed in the tropical regions of Africa and Asia,1 and has been used in traditional folk medicine to treat a variety of ailments including fever, general weakness,2 gastrointestinal diseases,3 liver diseases, psychosis, pain, and cancers.4 We performed a phytochemical investigation on the aerial parts of Rauvolf ia vomitoria to afford two new monoterpenoid indole alkaloids rauvomine A (1) and rauvomine B (2). Compound 1 (Figures 1 and 2) is a new C18 sarpagine-type normonoterpenoid indole alkaloid with a chlorine atom. Interestingly, compound 2 (Figure 3) represents a novel 6/5/6/6/3/5-fused hexcyclos sarpagine-type normonoterpenoid indole alkaloid possessing a cyclopropane ring and C18 skeleton. In addition, two known monoterpenoid indole Figure 2. X-ray crystallographic structure of 1.
alkaloids peraksine5 (3) and alstoyunine A6 (4) were also obtained from this plant for the first time. This letter reports the isolation, structural characterization, and biological activities of these alkaloids, as well as their plausible biogenetic pathways (Scheme 1). Figure 1. Key (a) 1H−1H COSY (red), HMBC (H → C), and (b) NOESY correlations of 1. © 2017 American Chemical Society
Received: June 7, 2017 Published: July 18, 2017 3998
DOI: 10.1021/acs.orglett.7b01723 Org. Lett. 2017, 19, 3998−4001
Letter
Organic Letters Table 1. 1H and 13C NMR Data of 1 and 2a 1 δH (J in Hz) NH 2 3 5 6
Figure 3. Key (a) 1H−1H COSY (red), HMBC (H → C), and (b) NOESY correlations of 2.
7 8 9 10 11 12 13 14
Scheme 1. Hypothetical Biogenetic Pathways for Compounds 1−4
15 16 17 18 19 20
2 δC
7.84 s 4.10 br d (9.7) 3.98 m 3.08 dd (15.8, 4.9) 2.77 dd (15.8,1.4)
7.46 d (7.6) 7.09 dd (7.6, 7.6) 7.15 dd (7.6, 7.6) 7.29 d (7.6) 2.03 dd (13.2, 9.7) 1.87 ddd (13.2, 4.6, 2.0) 2.87 m 2.25 br d (8.1) 9.79 s 1.38 d (7.0) 3.37 qd (8.0, 7.0) 4.34 dd (8.0, 4.4)
δH (J in Hz)
δC
7.78 s 136.1 51.9 45.2 27.8 105.8 127.6 118.4 119.7 121.8 110.8 136.4 34.0 34.1 49.4 202.3 15.8 55.8 61.5
4.08 dd (10.3, 2.0) 4.19 d (7.4) 3.32 overlap 3.32 overlap
7.48 d (7.4) 7.08 dd (7.4, 7.4) 7.12 dd (7.4, 7.4) 7.26 d (7.4) 2.32 dd(13.2, 10.3) 1.70 ddd (13.2, 3.8, 2.0) 1.89 m 9.12 s 1.26 d (6.8) 3.29 overlap 2.10 d (8.4)
136.9 50.2 49.5 20.7 104.9 127.2 118.4 119.6 121.7 111.0 136.3 23.9 21.2 43.3 199.8 18.6 57.8 32.7
a1
H NMR (400 MHz) and 13C NMR (100 MHz), TMS, measured in CDCl3.
12), and 105.8 (C-7). Besides the indole ring signals (eight carbons), the alkaloid possessed one methyl (δC 15.8), two methylenes (δC 34.0, 27.8), six methines (δC 61.5, 55.8, 51.9, 49.4, 45.2, 34.1), and one aldehyde group (δC 202.3). The 1H−1H COSY spectrum showed an extended spin system CH−CH2−CH(−CH−CH−CH2)−CH−CH−CH3 (Figure 1). In the HMBC spectrum, correlative signals from δH 4.10 (1H, br d, 9.7 Hz, H-3) to the indole skeleton carbons δC 136.1 (C-2) and 105.8 (C-7) allowed an assignment of the CH (δC 51.9, C-3) adjacent to C-2. At the same time, the observed correlations of δH 3.08 (1H, dd, 15.8, 4.9 Hz, H-6a) and 2.77 (1H, dd, 15.8, 1.4 Hz, H-6b) with δC 136.1 (C-2), 105.8 (C-7), and 127.6 (C-8) indicated that the −CH2 (δC 27.8, C-6) was connected to the indole ring at C-7. Thus, the extended spin system can be readily attributed to the CH-3−CH2-14−CH15(−CH-16−CH-5−CH2-6)−CH-20−CH-19−CH3-18 unit. Aldehyde functional group was positioned at C-16 supported by the correlations of H-5, H-15, and H-16 with C-17 (δC 202.3), and the correlations of H-17 with C-5 and C-16 in the HMBC spectrum. Furthermore, the three-bond correlations from H-3 to C-5, from H-5 to C-3 and C-19, and from H-19 to C-3 and C-5 allowed that three carbons C-3 (δC 51.9), C-5 (δC 45.2), and C19 (δC 55.8) were fused by a nitrogen atom, and the chlorine atom only can be positioned at C-20 (δC 61.5). Thus, the gross structure of 1 was assigned (Figure 1). The relative configurations at the various stereogenic centers were deduced from the NOESY correlations (Figure 1). The NOESY interaction pairs of H-3/H-14α, H-3/H-19, H-14α/H20, and H-5/H-18 indicated that H-3, H-19, and H-20 were αoriented, and Cl-20 and CH3-18 were β-oriented in the sixmembered ring (C-3−C-14−C-15−C-20−C-19−N-4−C-3). A single-crystal X-ray diffraction analysis on the Mo Kα data (deposition number: CCDC 1546133) confirmed the structure of 1 (Figure 2). The value of the Flack parameter 0.00(10) and
The aerial parts of Rauvolf ia vomitoria (2.5 kg) were pulverized and extracted with methanol, and the extract (200 g) was partitioned between EtOAc and 5% H2SO4 solution (pH 3), the water-soluble material that was adjusted to pH 10 with 5% NaOH solution, which was extracted with CHCl3. The CHCl3 extract (20 g) was fractionated and purified by chromatography on MCI gel, silica gel, Sephadex LH-20, ODS, and preparative HPLC to afford compounds 1−4. Compound 1 (2.5 mg) was obtained as colorless crystal, [α]20 D +80 (c 0.9, CHCl3), and showed [M + H]+ ion peak at m/z 315.1264 and 317.1234 in its positive mode HR-ESI-MS, corresponding to the molecular formula C18H19ClN2O (calcd 315.1259 and 317.1229). The strong absorption bands in its IR spectrum denoted the presence of CO (1711 cm−1) and NH (3383 cm−1) groups in 1. The UV absorption of 1 at λmax 276 and 220 nm indicated an indole chromophore in it.7,8 Moreover, the signals of 1H and 13C NMR (DEPT) spectra (Table 1) displayed characteristics of the indole part at δH 7.46 (1H, d, 7.6 Hz, H-9), 7.29 (1H, d, 7.6 Hz, H-12), 7.15 (1H, dd, 7.6, 7.6 Hz, H-11), and 7.09 (1H, dd, 7.6, 7.6 Hz, H-10); δC 136.4 (C-13), 136.1 (C-2), 127.6 (C-8), 121.9 (C-11), 119.7 (C-10), 118.4 (C-9), 110.8 (C3999
DOI: 10.1021/acs.orglett.7b01723 Org. Lett. 2017, 19, 3998−4001
Letter
Organic Letters presence of a chlorine atom in 1 allowed the assignment of the (3S, 5S, 15R, 16R, 19S, 20S) absolute configuration.9 Rauvomine B (2) (1.5 mg) initially obtained as a white powder, [α]20 D −95.6 (c 0.8, CHCl3), had a molecular formula of C18H18N2O with 11 degrees of unsaturation as established by HR-ESI-MS at m/z 279.1498 [M + H]+ (calcd 279.1492). Its IR spectrum exhibited characteristic absorption bands of carbonyl group (1719 cm−1) and indole chromophore ring (1687, 1595, 1452 cm−1). The UV spectrum confirmed the presence of the indole chromophore ring due to λmax 279 and 225 nm. The signals of 1H NMR and 13C NMR (DEPT) displayed characteristics of the indole fragment at δH 7.48 (1H, d, 7.4 Hz, H-9), 7.26 (1H, d, 7.4 Hz, H-12), 7.12 (1H, dd, 7.4, 7.4 Hz, H-11), and 7.09 (1H, dd, 7.4, 7.4 Hz, H-10); δC 136.9 (C-2), 136.3 (C-13), 127.2 (C-8), 121.7 (C-11), 119.6 (C-10), 118.4 (C-9), 110.0 (C-12), and 104.9 (C-7).8 HSQC spectrum of 2 showed that the alkaloid possessed one methyl (δC 18.6), two methylenes (δC 23.9, 20.7), five methines (δC 57.8, 50.2, 49.5, 32.7, 21.2), one quaternary carbon (δC 43.3), and one aldehyde group (δC 199.8). The 1H−1H COSY cross signals revealed the connectivities of three partial structures −CH-9−CH-10−CH11−CH-12− (i), − CH−CH2− (ii), and −CH−CH2−CH− CH−CH−CH3 (iii) (Figure 3). In the HMBC spectrum, correlative signals from H-6 to the indole skeleton carbons [δC 136.9 (C-2), 104.9 (C-7), and 127.2 (C-8)] allowed an assignment of the −CH2 (δC 20.7, CH2−6) connected to the indole ring at C-7, and the subunit ii was assigned as CH-5−CH2-6. The correlations between H-3 with the indole skeleton carbons δC 136.9 (C-2) and 104.9 (C-7) suggested the −CH (δC 50.2, C-3) was adjacent to C-2, and fragment iii was readily attributed to C-2−CH-3−CH2-14−CH15−CH-20−CH-19−CH3-18. The observed HMBC crosspeaks of H-15 and H-20 to C-17, of H-6, H-14, H-15 and H19 to C-16, and of H-17 to C-5, C-15 and C-16 indicated that C5, C-15, C-17, and C-20 were linked by C-16. The 13C NMR chemical shifts of CH-15, C-16, and CH-20 were at δC 21.2, 43.3, and 32.7, respectively, and 3JH‑15,H‑20 was 8.4 Hz, which displayed diagnostic resonance of 1,1,2,3-tetrasubstituted cyclopropane expressly.10 Furthermore, in the HMBC spectrum, correlations from H-3 to C-5, from H-5 to C-3 and C-19, and from H-19 to C3 and C-5, indicated the connection of a nitrogen atom to C-3, C5, and C-19. All these evidence prompted the establishment of the planar structure of 2. The relative stereochemistry of 2 was fixed by NOESY as shown in Figure 3. The NOESY correlations of H−N/H-3, H-3/ H-19, H-3/H-14α, and H3-18/H-5 indicated H-3, H-19, and H20 were α-oriented and CH3-18 was β-oriented in the sixmembered ring (C-3−C-14−C-15−C-20−C-19−N-4−C-3). Interaction pairs H-17/H-15 and H-17/H-20 in the NOESY spectrum required H-15 and H-20 to be syn to each other in the tetrasubstituted cyclopropane ring. The calculation of the 13C NMR chemical shifts of 2 at the B3LYP/6-311G(d,p) level with GIAO method (in which the values of TMS at the B3LYP/6-311+G(2d,p) level with GIAO method were used as a reference)11 agreed well with the experimental data (Figure 4), a correlation coefficient (R2) of 0.9991 (Figure 5) with the largest outlier Δδ = 5.4 ppm (C-7)11 and the root-mean-square deviation (RMSD) 1.54 ppm,12 which further indicated that structure of 2 was rational. The optical rotation (OR) values of 2 and its enantiomer were calculated in the Gaussian 09 suite of programs. The OR values were predicted by B3LYP/6-311G(d,p) under IEFPCM model of solvation: for 2 it was −114.2, and for Pits enantiomer
Figure 4. Comparison of calculated 13C NMR chemical shifts [at the B3LYP/6-311G(d,p) GIAO level] for 2, with experimentally observed shifts.
Figure 5. Regression analysis of experimental versus calculated NMR chemical shifts of 2; linear fitting is shown as a line.
13
C
+114.2.11,12 The former was close to the experimental value of −95.6, which indicated the absolute configuration of 2 as given in Figure 2. Finally, the calculated electronic circular dichroism (ECD) spectrum for 2 using the time-dependent density functional theory (TDDFT) at the B3LYP/dgdzvp level with PCM in MeOH11,12 agreed with the experimental ECD spectrum (Figure 6), allowing an obvious assignment of the absolute structure to be 3S, 5S, 15S, 16R, 19S, 20R. The isolated four compounds were tested for their cytotoxicity against seven cancer cells SMMC-7721, HepG2, HT-29, SW480, A-549, HeLa, and MCF-7 according to the MTT method.
Figure 6. Experimental (black line) and calculated (red line) ECD spectra of 2 in MeOH and calculated (blue line) ECD spectrum of its enantiomer. 4000
DOI: 10.1021/acs.orglett.7b01723 Org. Lett. 2017, 19, 3998−4001
Letter
Organic Letters
(5) Arthur, H. R.; Johns, S. R.; Lamberton, J. A.; Loo, S. N. Aust. J. Chem. 1968, 21, 1399−1401. (6) Feng, T.; Li, Y.; Cai, X. H.; Gong, X.; Liu, Y. P.; Zhang, R. T.; Zhang, X. Y.; Tan, Q. G.; Luo, X. D. J. Nat. Prod. 2009, 72, 1836−1841. (7) Gan, C. Y.; Robinson, W. T.; Etoh, T.; Hayashi, M.; Komiyama, K.; Kam, T. S. Org. Lett. 2009, 11, 3962−3965. (8) Wong, S. P.; Chong, K. W.; Lim, K. H.; Lim, S. H.; Low, Y. Y.; Kam, T. S. Org. Lett. 2016, 18, 1618−1621. (9) Flack, H. D. Acta Crystallogr., Sect. A: Found. Crystallogr. 1983, A39, 876−881. (10) (a) Abraham, R. J.; Leonard, P.; Tormena, C. F. Magn. Reson. Chem. 2012, 50, 305−313. (b) Bishara, A.; Rudi, A.; Aknin, M.; Neumann, D.; Ben Califa, N.; Kashman, Y. Org. Lett. 2008, 10, 4307− 4309. (11) Hu, Z. X.; Shi, Y. M.; Wang, W. G.; Tang, J. W.; Zhou, M.; Du, X.; Zhang, Y. H.; Pu, J. X.; Sun, H. D. Org. Lett. 2016, 18, 2284−2287. (12) (a) Ditchfield, R. Mol. Phys. 1974, 27, 789−807. (b) Wolinski, K.; Hinton, J. F.; Pulay, P. J. Am. Chem. Soc. 1990, 112, 8251−8260. (c) Lodewyk, M. W.; Soldi, C.; Jones, P. B.; Olmstead, M. M.; Rita, J.; Shaw, J. T.; Tantillo, D. J. J. Am. Chem. Soc. 2012, 134, 18550−18553. (d) Toribio, A.; Bonfils, A.; Delannay, E.; Prost, E.; Harakat, D.; Henon, E.; Richard, B.; Litaudon, M.; Nuzillard, J. M.; Renault, J. H. Org. Lett. 2006, 8, 3825−3828. (13) Zhang, D. B.; Yu, D. G.; Sun, M.; Zhu, X. X.; Yao, X. J.; Zhou, S. Y.; Chen, J. J.; Gao, K. J. Nat. Prod. 2015, 78, 1253−1261. (14) Sun, L.; Chen, Y.; Rajendran, C.; Mueller, U.; Panjikar, S.; Wang, M.; Mindnich, R.; Rosenthal, C.; Penning, T. M.; Stöckigt, J. J. Biol. Chem. 2012, 287, 11213−11221. (15) Gao, Y.; Yu, A. L.; Li, G. T.; Hai, P.; Li, Y.; Liu, J. K.; Wang, F. Fitoterapia 2015, 107, 44−48. (16) Rosenthal, C.; Mueller, U.; Panjikar, S.; Sun, L.; Ruppert, M.; Zhao, Y.; Stöckigt, J. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2006, 62, 1286−1289. (17) Sun, L.; Ruppert, M.; Sheludko, Y.; Warzecha, H.; Zhao, Y.; Stöckigt, J. Plant Mol. Biol. 2008, 67, 455−467.
Unfortunately, no compound exhibited any appreciable inhibition against the cancer cells; only 4 showed weak cytotoxicities toward HT-29 (IC50 = 35.2 μM) and SW480 (IC50 = 45.3 μM) with auranofin positive control IC50 values of 2.5 and 3.9 μM, respectively. Additionally, the anti-inflammatory activities13 of four compounds (Table 2) were assessed. Table 2. Anti-inflammatory of Compounds 1−4 Isolated from Rauvolf ia vomitoria toward RAW 264.7 compd
1
2
3
4
celecoxib
IC50 (μM)
55.5
39.6
65.2
75.3
34.3
Compound 2 exhibited significant inhibition of 264.7 macrophages with an IC50 value of 39.6 μM, while the rest of the three compounds showed modest anti-inflammatory activities. The plausible biogenetic pathways of 1−4 were proposed as shown in Scheme 1. Transformation of tryptophan and secologanin to I was well established.14,15 Isomerization of I could lead to III by enolization,15 and III gave peraksine-type alkaloid 3 via partial reduction and intramolecular aldol condensation reaction,16,17 and III could also react with methanol to form 4. In view of this, we proposed that 1 and 2 were also derived from intermediate enol II, which was oxidized to form V. Then V was selectively reduced to the key intermediate VI by NADPH. Compound 1 was formed by substitution of the hydroxyl group in VI with chlorine atom (path a), whereas the intramolecular cyclization of VI could lead to compound 2 (path b).
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01723. Crystallographic data of 1 (CIF) Experimental procedures; 1D and 2D NMR, UV, CD, IR, and HRESIMS of rauvomines A (1) and B (2) (PDF)
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AUTHOR INFORMATION
Corresponding Authors
* E-mail:
[email protected]. * E-mail:
[email protected]. ORCID
Pan-Pan Zhou: 0000-0001-8111-8155 Kun Gao: 0000-0002-3856-3672 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Basic Research Programs (973) of China (2014CB138703) and the National Natural Science Foundation of China (31470421).
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REFERENCES
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DOI: 10.1021/acs.orglett.7b01723 Org. Lett. 2017, 19, 3998−4001
