Gelsekoumidines A and B: Two Pairs of Atropisomeric Bisindole

Sep 12, 2017 - Figure 2. Key NOESY correlations of 1a and 1b. Fortunately, crystals (1a) suitable for X-ray diffraction were obtained from the methano...
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Gelsekoumidines A and B: Two Pairs of Atropisomeric Bisindole Alkaloids from the Roots of Gelsemium elegans Wei Zhang,†,‡ Wei Xu,† Gui-Yang Wang,† Xue-Ying Gong,† Ni-Ping Li,† Lei Wang,*,†,‡ and Wen-Cai Ye*,†,‡ †

Institute of Traditional Chinese Medicine & Natural Products, and JNU-HKUST Joint Laboratory for Neuroscience & Innovative Drug Research, College of Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China ‡ Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, People’s Republic of China S Supporting Information *

ABSTRACT: Two pairs of atropisomeric bisindole alkaloids, gelsekoumidines A (1) and B (2), with a new carbon skeleton, were isolated from the roots of Gelsemium elegans. Compounds 1 and 2 represent the first examples of seco-koumine−gelsedine type alkaloids, which feature an unprecedented 20,21-seco-koumine scaffold fused with a gelsedine framework via a double bond. Their structures including absolute stereochemistry were elucidated by spectroscopic data, single-crystal X-ray diffraction, and electronic circular dichroism (ECD) calculation. A plausible biogenetic pathway for the new compounds is also proposed. Compound 2 exhibited a moderate inhibitory effect against nitric oxide (NO) production.

T

he plants of genus Gelsemium (Loganiaceae) are wellknown for producing structurally diverse monoterpenoid indole alkaloids (MIAs) with various biological activities.1 Previous phytochemical investigations on the plants of this genus resulted in the isolation of more than 140 MIAs named as Gelsemium alkaloids,1−3 some of which showed obvious biological effects, such as analgesic and antitumor activities.4,5 Their intriguing structures and potential bioactivities have inspired many chemists to develop strategies for their synthesis.6−9 Gelsemium elegans is a famous toxic plant that grows mainly in southeast Asia and southern China.10 In China, the roots of this plant have been used for a long time as folk medicine for the treatment of cancer, furuncle, and carbuncle.10 Our group had reported the isolation of a series of new Gelsemium alkaloids from the title plant.11−13 In our continuing research, two atropisomeric bisindole alkaloids with a new skeleton, gelsekoumidines A (1) and B (2) were isolated from the roots of the title plant. The two novel alkaloids represent the first examples of seco-koumine−gelsedine type bisindole alkaloids. Furthermore, 1 and 2 consist of a sterically compact polycyclic scaffold with 11 and ten stereocenters, respectively. Interestingly, 1 and 2 exist as two pairs of atropisomers (1a/1b and 2a/2b) caused by restricted rotation of an amide bond.11,14,15 Herein, we describe the structural elucidation, a hypothetical biosynthetic pathway, and the NO inhibitory activities of 1 and 2. © 2017 American Chemical Society

Gelskoumidine A (1) was obtained as light-yellow crystals. The molecular formula of 1 was determined to be C39H40N4O6 by its HR-ESI-MS data (m/z 661.3022 [M + H]+, calcd for C39H41N4O6: 661.3021). The UV spectrum of 1 showed an absorption maximum at 302 nm, which revealed the presence of conjugated systems. The IR spectrum indicated the presence of hydroxyl (3434 cm−1) and carbonyl (1725 cm−1) groups as well as a benzene ring (1618 and 1479 cm−1). Compound 1 exists as a pair of isomers (1a and 1b), as evident from their interconversion in solution (Figure S1). The 1H and 13C NMR spectra of 1 displayed two sets of signals in a ratio of approximately 3:5 (1a:1b).16 The 1H NMR spectrum for the isomer 1a showed the presence of two ortho-disubstituted benzene rings [δH 7.50 (1H, overlapped), 7.11 (1H, overlapped), 7.30 (1H, ddd, J = 7.7, 7.7, 0.9 Hz), and 6.95 (1H, br d, J = 7.7 Hz); and 7.59 (1H, Received: August 9, 2017 Published: September 12, 2017 5194

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basis of the analysis of the 1H−1H COSY, HSQC and HMBC spectra, the 1H and 13C NMR data of 1a were assigned as shown in Table 1. The 1H−1H COSY spectrum of 1a suggested the presence of five spin systems (H-9 to H-12, H-3 to H-6/H-17, H-9′ to H-12′, H-3′ to H-6′/H-17′, and H-18 to H-19), as shown in Figure 1. In the HMBC spectrum, correlations between H-9′ and C-7′/C-

overlapped), 7.51 (1H, overlapped), 7.50 (1H, overlapped), and 7.65 (1H, br d, J = 7.7 Hz)]; seven oxygenated methylene and methine protons [δH 4.46 (1H, overlapped), 4.37 (1H, overlapped), 3.58 (1H, br s), 4.33 (1H, d, J = 2.2 Hz), 5.10 (1H, br s), 4.46 (1H, overlapped), and 3.81 (1H, m)]; three methyl groups [δH 3.93 (3H, s), 3.06 (3H, s), and 1.95 (3H, s)]; two conjugated olefinic protons [δH 6.39 (1H, d, J = 11.6 Hz) and Table 1. 1H and 13C NMR Data of 1 (in CD3OD3, J in Hz) 1a δC

δH

2 3 5 6

186.9 72.5 54.1 41.2

7 8 9 10 11 12 13 14

64.3 141.9 125.9 127.9 129.6 122.5 154.5 33.6

15 16 17

29.7 40.5 66.3

18 19 20 21 22 2′ 3′ 5′ 6′

130.2 118.9 149.0 165.7 34.3 172.8 80.6 73.6 38.2

7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 17′

55.4 133.0 126.0 124.8 129.6 107.9 139.0 68.3 50.0 39.3 62.1

18′ 19′ 20′ 21′

13.5 135.2 179.4 64.0

− 5.10 br s 4.60 d (8.4) α 2.68 m β 2.53 m − − 7.59 7.51 7.50 7.65 br d (7.7) − α 3.17 m β 1.48 m 4.02 2.86 m α 4.46 β 3.81 m 7.09 d (11.6) 6.39 d (11.6) − 8.00 s 3.06 s − 3.58 br s 4.50 α 2.15 m β 2.55 − − 7.50 7.11 7.30 ddd (7.7, 7.7, 0.9) 6.95 br d (7.7) − 4.33 d (2.2) 3.36 d (8.5) 2.62 α 4.46 β 4.37 1.95 s − − 3.93 s

no.

a

1b a

δC

δ Ha

186.5 72.4 60.9 38.7

− 5.10 br s 4.00 m α 2.63 m β 2.57 m − − 7.59 7.51 7.50 7.65 br d (7.7) − α 3.16 m β 1.47 m 4.02 3.01 m α 4.47 β 3.83 m 7.08 d (11.6) 6.33 d (11.6) − 8.27 s 2.93 s − 3.59 br s 4.50 α 2.18 m β 2.55 − − 7.59 7.11 7.31 ddd (7.7, 7.7, 0.9) 6.96 br d (7.7) − 4.35 d (2.2) 3.43 d (8.5) 2.61 α 4.46 β 4.37 1.92 s − − 3.93 s

64.0 141.7 125.9 128.0 130.0 122.5 154.5 33.6 29.6 40.6 65.7 130.1 119.1 147.5 164.4 30.2 172.8 81.0 73.6 38.0 55.4 133.0 125.9 124.7 129.5 107.9 139.2 68.1 50.2 39.2 62.1 13.7 135.4 179.4 64.0

Figure 1. Key 1H−1H COSY and HMBC correlations of 1 and 2.

Figure 2. Key NOESY correlations of 1a and 1b.

11′/C-13′, between H-3′ and C-6′/C-8′/C-17′, between H-14′ and C-7′/C-16′, between H-5′ and C-20′, and between H-18′ and C-20′ confirmed the presence of a 14-hydroxygelsenicine moiety (part A, Figure 1).17 The remaining signals of 1a were similar to those of koumine,18 except that the signals of C-20 and CH2-21 in koumine were replaced with signals of a quaternary olefinic carbon (δC 149.0) and a formyl group (δH 8.00, δC 165.7) in 1a. This result suggested the presence of a 20, 21-seco-koumine moiety (part B, Figure 1). The HMBC cross peaks between H-10 and C-8/C-12, between H-3 and C-7/C-15, between H-5 and C-

Overlapped signals were reported without designating multiplicity.

7.09 (1H, d, J = 11.6 Hz)]; and a formyl proton [δH 8.00 (1H, s)]. The 13C NMR and DEPT spectra of 1a revealed the presence of 39 carbon signals including 2 carbonyls, 10 quaternary carbons, 5 methylenes, 19 methines, and 3 methyls. The above data suggested that 1a could be a dimeric Gelsemium alkaloid. On the 5195

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was in the Z configuration (Figure 2). Furthermore, the NOE cross peaks between H-5 and H-17β, and between H-15/H-16 and H-17α indicated the relative configurations of C-5, C-15, and C-16 in 1a were identical to those of known koumine-type alkaloids. In addition, the NOE correlations between H-18′ and H-19 as well as between H-18 and H-15/H-15′ indicated that the geometries of the two double bonds (C-18 and C-19′, and C-19 and C-20) and the intervening single bond (C-18 and C-19) were E,E and s-trans, respectively (Figure 2). Fortunately, crystals (1a) suitable for X-ray diffraction were obtained from the methanol solution of 1. The structure of 1a was established by X-ray diffraction analysis as showed in Figure 3. The partial double-bond character of the formamide C−N bond was further confirmed from the bond length of C21−N4 (1.352 Å).11 The final refinement of the Cu Kα data resulted in a small Flack parameter of 0.07 (15), allowing the assignment of absolute configuration of 1a. A comprehensive analysis of the 1D and 2D NMR data revealed that 1b had the same planar structure as 1a. The main differences between 1a and 1b involved resonances for CH-5, CH3-22, and CHO-21, which suggested that the two isomers varied only in the N4−C21 geometry. The NOESY spectrum of 1b exhibited obvious NOE correlations between H-21 and H-5/ H-16 (Figure 2), indicating that the N4−C21 bond of 1b was in the E configuration. The molecular formula of gelsekoumidine B (2) was determined to be C39H40N4O5 on the basis of its HR-ESI-MS data (m/z 645.3078 [M + H]+, calcd for C39H41N4O5: 645.3072). The 1H and 13C NMR spectra of 2 also showed two sets of signals, suggesting the presence of two isomers (2a and 2b). The NMR data of 2 (Table S1, see the SI) were very similar to those of 1, except for the absence of signals for CH-14′ and the presence of a methylene signal (δC 29.5 or 29.4), which suggested that the 14-hydroxygelsenicine unit (part A) in 1 was replaced with a gelsenicine unit in 2.19 This conclusion was further confirmed by an extensive analysis of the 1H−1H COSY, HSQC, HMBC, and NOESY spectra. The absolute configuration of 2 was verified by the quantum chemical ECD calculation using the TDDFT method (see the SI). The experimental ECD spectrum of 2 exhibited a negative Cotton effect at 299 (Δε −46.3) nm as well as positive Cotton effects at 228 (Δε +17.5) and 267 (Δε +15.5) nm, which were similar to those of compound 1 and the calculated ECD spectrum of

Figure 3. X-ray ORTEP drawing of 1a.

Figure 4. Calculated and experimental CD spectra of 2.

15/C-17/C-22, between H-18 and C-20, and between H-21 and C-5/C-22 further confirmed the structure of part B. The linkage of parts A and B through the C-18/C-19′ double bond was deduced from the HMBC correlations between H-18′ and C-18/ C-20′ as well as between H-18 and C-20′/C-20. Therefore, the planar structure of 1a was determined. Due to the presence of a formamide group, the interconversion between 1a and 1b arose from restricted rotation of the amide bond.11,14,15 The NOESY correlations between H-21 (δH 8.00) and H-22 (δH 3.06) revealed that the N4−C21 bond in 1a Scheme 1. Plausible Biosynthetic Pathway of 1, 2

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(3) Kitajima, M.; Kogure, N.; Yamaguchi, K.; Takayama, H.; Aimi, N. Org. Lett. 2003, 5, 2075−2078. (4) Kitajima, M.; Nakamura, T.; Kogure, N.; Ogawa, M.; Mitsuno, Y.; Ono, K.; Yano, S.; Aimi, N.; Takayama, H. J. Nat. Prod. 2006, 69, 715− 718. (5) Zhang, J. Y.; Gong, N.; Huang, J. L.; Guo, L. C.; Wang, Y. X. Pain 2013, 154, 2452−2462. (6) Harada, T.; Shimokawa, J.; Fukuyama, T. Org. Lett. 2016, 18, 4622−4625. (7) Diethelm, S.; Carreira, E. M. J. Am. Chem. Soc. 2013, 135, 8500− 8503. (8) Zhou, X.; Xiao, T.; Iwama, Y.; Qin, Y. Angew. Chem., Int. Ed. 2012, 51, 4909−4912. (9) Earley, W. G.; Jacobsen, J. E.; Madin, A.; Meier, G. P.; O’Donnell, C. J.; Oh, T.; Old, D. W.; Overman, L. E.; Sharp, M. J. J. Am. Chem. Soc. 2005, 127, 18046−18053. (10) Editorial Committee of Chinese Materia Medica. Chinese Materia Medica (Zhonghua Benchao); Shanghai Science and Technology: Shanghai, 1999; Vol. 17, pp 213−216. (11) Zhang, W.; Huang, X. J.; Zhang, S. Y.; Zhang, D. M.; Jiang, R. W.; Hu, J. Y.; Zhang, X. Q.; Wang, L.; Ye, W. C. J. Nat. Prod. 2015, 78, 2036− 2044. (12) Ouyang, S.; Wang, L.; Zhang, Q. W.; Wang, G. C.; Wang, Y.; Huang, X. J.; Zhang, X. Q.; Jiang, R. W.; Yao, X. S.; Che, C. T.; Ye, W. C. Tetrahedron 2011, 67, 4807−4813. (13) Zhang, W.; Zhang, S. Y.; Wang, G. Y.; Li, N. P.; Chen, M. F.; Gu, J. H.; Zhang, D. M.; Wang, L.; Ye, W. C. Fitoterapia 2017, 118, 112−117. (14) Che, Q.; Zhu, T. J.; Keyzers, R. A.; Liu, X. F.; Li, J.; Gu, Q. Q.; Li, D. H. J. Nat. Prod. 2013, 76, 759−763. (15) Aguirre, G.; Somanathan, R.; Hellberg, L. H.; Dwyer, T. J.; North, R. Magn. Reson. Chem. 2003, 41, 131−134. (16) The rotational barriers of the amide bond had been previously investigated by NMR experiments and DFT calculations, which revealed that the two atropisomers can interconvert rapidly at room temperature (see refs 11 and 14, 15). The 1H NMR experiments of 1 in different solvents (CDCl3, CDOD3, DMSO-d6, and pyridine-d5) were carried out (see Figure S15). The results indicated that (a) compound 1b is always the main product in different solvents (CDCl3, CDOD3, DMSO-d6, and pyridine-d5); (b) the ratio of 1a:1b in different solvents are slightly different (approximately 2:5 in CDCl3, and 3:5 in CDOD3, DMSO-d6 and pyridine-d5). Furthermore, dynamic HPLC analyses of 1a and 1b revealed the two atropisomers can interconvert rapidly at room temperature (see Figure S1). (17) Kobayashi, N.; Kobayashi, H.; Ishii, N.; Kitajima, M.; Wongseripipatana, S.; Takayama, H. Tetrahedron Lett. 2008, 49, 3638−3642. (18) Lin, L. Z.; Cordell, G. A.; Ni, C. Z.; Clardy, J. Phytochemistry 1990, 29, 965−968. (19) Lin, L. Z.; Cordel, G. A. J. Org. Chem. 1989, 54, 3199−3202. (20) Xu, Y. K.; Yang, L.; Liao, S. G.; Cao, P.; Wu, B.; Hu, H. B.; Guo, J.; Zhang, P. J. Nat. Prod. 2015, 78, 1511−1517. (21) O′Connor, S. E.; Maresh, J. J. Nat. Prod. Rep. 2006, 23, 532−547. (22) Han, J. J.; Bal, L.; Tao, Q. Q.; Yao, Y. J.; Liu, X. Z.; Yin, W. B.; Liu, H. W. Org. Lett. 2015, 17, 2538−2541. (23) Liu, C. P.; Xu, J. B.; Han, Y. S.; Wainberg, M. A.; Yue, J. M. Org. Lett. 2014, 16, 5478−5481. (24) Wang, X.; Xia, D.; Qin, W.; Zhou, R.; Zhou, X.; Zhou, Q.; Liu, W.; Dai, X.; Wang, H.; Wang, S.; Tan, L.; Zhang, D.; Song, H.; Liu, X. Y.; Qin, Y. Chem. 2017, 2, 803−816.

(3R,5S,7S,15R,16S,3′R,5′S,7′S,15′R,16′S)-2 (Figure 4). Hence, the absolute configuration of 2 was determined to be 3R,5S,7S,15R,16S,3′R,5′S,7′S,15′R,16′S. Compounds 1 and 2 represent the first examples of secokoumine−gelsedine type alkaloids. A plausible biosynthetic pathway for 1 and 2 is proposed (Scheme 1). The gelsedine-type alkaloids (14-hydroxygelsenicine and gelsenicine) and kouminetype one (21-hydroxykoumine20) were both generated from the common precursor strictosidine, which is derived from tryptamine and secologanin by strictosidine synthase- and strictosidine glucosidase-catalyzed condensations.21 The terminal double bond (C18C19) in hydroxykoumine could be oxidized to produce the intermediate i, which was followed by a basecatalyzed procedure involving C-20−C-21 cleavage and epoxide opening to afford ii.22,23 The oxidation of ii could lead to iii with an α, β-unsaturated aldehyde group, which could be combined with 14-hydroxygelsenicine or gelsenicine by aldol reaction to yield 1 or 2. The MIAs showed a wide range of biological activities.24 Accordingly, the novel alkaloids (1 and 2) were screened for multiple bioactivities. These compounds were found to be devoid of significant cytotoxic activities against A-549, HepG2, MCF-7, and MDA-MB-231 cancer cell lines at the concentration of 50 μM. On the other hand, compound 2 exhibited concentration-dependent inhibition of lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophage cells, with an IC50 value of 33.2 ± 2.1 μM (indomethacin was used as a positive control, IC50 = 23.1 ± 1.5 μM) (see the SI).

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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.7b02463. Detailed descriptions of the experimental procedure; UV, IR, MS, and NMR spectra for compounds 1, 2; ECD calculations for 1, 2 (PDF) X-ray data for 1a (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Wen-Cai Ye: 0000-0002-2810-1001 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Program for National Natural Science Foundation of China (Nos. U1401225, 81573307, 81273391), the Science and Technology Planning Project of Guangdong Province (No. 2016B030301004), and the Guangdong Natural Science Foundation for Distinguished Young Scholar (No. 2015A030306022).

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REFERENCES

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