Letter pubs.acs.org/OrgLett
Walrobsins A and B, Two Anti-inflammatory Limonoids from Root Barks of Walsura robusta Fa-Liang An, Dong-Mei Sun, Rui-Jun Li, Miao-Miao Zhou, Ming-Hua Yang, Yong Yin, Ling-Yi Kong,* and Jun Luo* Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China S Supporting Information *
ABSTRACT: Walrobsins A (1) and B (2), two limonoids featuring an unprecedented 5-oxatricyclo[5.4.11,4]hendecane ring system, were isolated from the root barks of Walsura robusta. Their structures, including their absolute configurations, were determined by analyses of HR-ESIMS, 1D/2D NMR, and X-ray crystallography. Compounds 1 and 2 possessed a stable hemiketal structure formed between the OH-11 and 3-carbonyl group in the hexatomic oxoheterocyclic ring. Compound 1 showed significant anti-inflammatory activity with an IC50 value of 7.8 μM and inhibited the expression of iNOS and IL-1β in a dose-dependent manner.
L
imonoids are a family of structurally diverse natural products with a wide range of biological activities and are used as a reliable source of pesticides and medicines.1 We were attracted to the limonoid, which is a class of highly oxidized tetranortriterpenoids, classified based on seco styles and cyclization patterns of rings A−D in the backbone.2 More than 1000 members of the limonoid family (with at least 35 types) have been isolated and reported since 1966. Not surprisingly, limonoids have attracted considerable interest for discovery programs, total syntheses, and bioactivity evaluations targeting various biological properties in recent years.3 The Walsura robusta, widely distributed in the Yunnan province of China, is known for its cytotoxic cedrelone-type limonoids.4 Previous chemical investigations on the genus Walsura led to an array of structurally diverse limonoids with interesting biological properties, such as cytotoxic activities, antimalarial activities, and 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitory effects.4a To discover new limonoids with a new structural scaffold and low cytotoxic activities, we investigated the chemical constituents of the root barks of W. robusta. In this study, walrobsins A (1) and B (2) (Figure 1), two novel limonoids with an unprecedented 5oxatricyclo[5.4.11,4]hendecane ring system in A1/A2/B rings, were isolated and identified. Their structures and absolute configurations were determined by comprehensive analyses of NMR, HRESIMS, and X-ray crystallography. Compound 1 was evaluated for anti-inflammatory effect in the lipopolysaccharide (LPS)-induced RAW264.7 cell line and showed significant activity with an IC50 value of 7.8 μM in a dose-independent manner. Herein, we described the isolation, identification, and biological evaluation of these two novel limonoids. © 2017 American Chemical Society
Figure 1. Structures of compounds 1 and 2.
Walrobsin A (1) was isolated as colorless crystals. A protonated molecular ion at m/z 567.2953 ([M + H]+, calcd for 567.2952) in its HR-ESI-MS spectrum indicated a molecular formula of C33H42O8 that was in accordance with the 13C NMR spectroscopic data, with corresponding degrees of hydrogen deficiency of 13. The 13C NMR spectrum (Table 1) resolved 33 carbon resonances corresponding to seven methyl, four methylene, 12 methines (five olefinic and three oxygenated), and ten quaternary carbons (one oxygenated, three olefinic, and three carbonyl) as distinguished by the HSQC spectrum (Figures S1−S4). A typical β-substituted furyl ring group (δH 7.43, 7.26, 6.34, each s, 1H; δC 124.2, 140.2, 111.3, 142.8), two singlet methyls (δH 1.69, 0.79; each s, 3H), one trisubstituted double bond (δH 5.93, brd s; δC 151.7, 126.3), and a tigloyl group (δH 7.00, m; 1.90, s; 1.88, d, J = 6.0 Hz) implied that compound 1 can be classified as a limonoid. These assigned fragments accounted in total for eight degrees of hydrogen deficiency together with a carbonyl group (δC 211.1), and an acetyl group (δH 2.16, s, 3H; δC 170.1, 21.3), Received: July 17, 2017 Published: August 18, 2017 4568
DOI: 10.1021/acs.orglett.7b02173 Org. Lett. 2017, 19, 4568−4571
Letter
Organic Letters Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) Data of Compounds 1−2 in CDCl3 1 δH
no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
5.25 t (3.0) 4.94 d (3.5)
2.46 m 1.99 m 2.82 td (14.0, 11.5) 2.33 m
1.94 d (6.0) 4.52 dt (7.5, 7.0) 2.31 m 1.82 m
5.93 brd s 2.82 ddd (15.0, 11.0, 2.0) 2.35 m 2.94 dd (11.0, 7.5)
2 δC 85.5 86.3 101.9 28.0 45.2 35.4 211.1 51.3 45.5 48.5 69.1 40.3 46.7 151.7 126.3 34.7 51.3
1
δH
δC
5.13 t (3.0) 4.86 d (3.5)
2.50 m 1.96 dd (11.5, 6.5) 2.75 dd (14.0, 11.5) 2.27 m
1.93 d (6.0) 4.43 dt (7.5, 7.0) 2.22 m 1.69 m
5.88 brd s 2.64 ddd (15.0, 11.0, 2.0) 2.35 m 2.89 dd (11.0, 7.5)
85.9 85.5
18 19
102.0 28.0 45.3 35.4 211.1 51.2 45.0 48.4 69.0 40.3
20 21 22 23 28 29 30 1′ 2′ 3′
46.7 151.7 126.3 34.6 51.2
4′ 5′ OAc OAc OH
2
δH
δC
δH
δC
0.79 s 2.46 m 2.33 m
20.0 37.1
0.73 s 2.41 m 2.24 m
19.7 37.0
no.
7.25 6.34 7.43 0.93 0.98 1.69
s s s d (7.0) d (7.0) s
7.00 m 1.83 d (6.0) 1.84 s 2.16 s 5.93 s
124.2 140.2 111.3 142.8 23.1 18.7 30.6 170.1 127.6 140.9 14.9 12.2 21.3 170.1
7.26 6.26 7.38 0.92 0.87 1.69
s s s d (7.0) d (7.0) s
1.70 2.50 1.50 0.92 1.18 2.11
m m m t (7.0) d (7.0) s
124.2 140.1 111.1 142.8 23.0 18.6 30.5 179.0 41.0 26.7 16.7 11.6 21.2 170.0
5.68 s
heterocyclic ring cannot be determined directly by the HMBC spectrum because of no valuable correlations for C-3. Fortunately, this uncertainty can be resolved by the assignments of relevant hydroxyl proton signals. The HMBC correlations from H-1 (δH 5.25) and H-2 (δH 4.94) to carbonyl signals at δC 170.1 and δC 170.1 indicated that OH-1 and OH-2 were acylated. Although no HMBC correlation between H-11 and C-3 can be observed, the sole possible location of the oxygen heterocyclic ring was built by the 3,11-oxide bridge. The stability of the hemiketal group may be due to the nearness between OH-3 and 1′-CO, which allows for hydrogen bond formation. Thus, the plane structure of 1 was delineated as an A-seco limonoid with an unprecedented 5-oxatricyclo[5.4.11,4]hendecane ring system architecture. The relative configuration of 1 was established by the analysis of its ROESY data. The ROESY correlations of H3-18/H-16α, H-23 suggested that the furyl ring and Me-18 were α-oriented. The ROESY correlations of H3-30/H-4, H-19b revealed that they were β-oriented in ring B. The ROESY cross peak between H-1 and H-9, and the correlation between H-2 and H-19a, indicated that H-1 and H-2 were in the trans-configuration in ring A and arbitrarily assigned as the α and β orientation, respectively. Compound 1 was recrystallized in the CH2Cl2/MeOH (1:1) mixture to yield prisms. The X-ray diffraction (Cu Kα) analysis (Figure 3) of the single crystal not only secured the 3,11-oxide bridge structure but also elucidated its stereoscopic centers as 2′E,1R,2S,3R,5S,8R,9R,10S,11S,13S,14E,17R (CCDC 1557716). Finally, the structure of 1 was resolved completely, featuring an unprecedented limonoid architecture with a unique central motif of 5-oxatricyclo[5.4.11,4]hendecane ring and was named as walrobsin A. Walrobsin B (2) showed similar 1H NMR and 13C NMR spectra as 1 with a molecular formula of C33H44O8 in HR-ESIMS data, which exhibited two daltons more than 1. The close similarity of its 1H and 13C NMR data (Table 1) to those of 1 indicated that 2 featured the same skeleton core. An additional doublet methyl and the absence of a singlet methyl in 1H NMR data indicated that the tigloyl group in 1 was substituted as a
which indicated 1 to be hexacyclic. In addition, two doublet methyls (δH 0.98, d, J = 7.0 Hz; 0.93, d, J = 7.0 Hz; each 3H) and a multiplet methane (δH 2.46, m, 1H) implied an isopropyl group in 1, which further indicated 1 was an A-seco limonoid with rare five-membered ring A1 constructed between C-3 and C-19. The connectivity of the structural fragments and the establishment of its planar structure were achieved by analysis of the 2D NMR correlations (Figure 2). The B, C, and D rings were readily established by comparison with those of several known limonoids.5 The appearance of a characteristic hemiketal carbon (δC 101.9), which is located at C-3 assigned by the HMBC correlations from H-1 and H2-19 to C-3, indicated that an oxygen heterocyclic ring appeared in accordance with one index of hydrogen deficiency. However, the location of oxygen
Figure 2. Key HMBC and ROESY correlations of 1. 4569
DOI: 10.1021/acs.orglett.7b02173 Org. Lett. 2017, 19, 4568−4571
Letter
Organic Letters
recognized as the origin, and the cleavage of ring A and the spirocyclic structure of rings A and B was conducted by the regiospecific in-cell free radical reaction between Me-19 and an α,β-unsaturated ketone. First, the Me-19 free radical was formed under the action of some enzymes in the plant cell. Second, this methyl radical attacks the C-3 carbonyl group and then leads to the cleavage of the C-3/4 bond and formation of a new C-3/19 bond. The formation of an oxygen heterocyclic ring (hemiketal formation) plays a vital role in the formation of the 5-oxatricyclo[5.4.11,4]hendecane ring of 1 and 2. In the bioactivity evaluations, compounds 1 and 2 did not show any cytoxicities in four tumor cell lines HepG2, HL-60, MCF-7, and HT-296 at 50 μM concentration. Further screening revealed that compound 1 exhibited significant antiinflammatory activity in the LPS-induced RAW264.7 cell line with IC50 7.95 ± 2.16 μM (L-NMMA as the positive control, IC50 30.6 ± 0.38 μM).7 Further research revealed that walrobsin A (1) can bind to iNOS enzyme in higher score (Figures S2 and S3), as judged by the reduction of NO production, the lower gene expression of iNOS and IL-1β, and the lower protein level of iNOS and COX-2 in the LPS-induced RAW 264.7 macrophages (Figure 5). These results reinforced its significance in the discovery effort of new anti-inflammatory agents.
Figure 3. X-ray crystal structures of walrobin A (1).
butanoate group in 2, which was consistent with its molecular formula. The planar structure and relative and absolute configurations were determined by X-ray diffraction (Cu Kα) of the single crystal (Figure 4), and its absolute configuration was assigned as 2′R,1R,2S,3R,5S,8R,9R,10S,11S,13S,14E,17R (CCDC 1557185).
Figure 5. Inhibition of 1 on the NO production, the gene expression of iNOS and IL-1β, and the proton expression of iNOS and COX-2 in LPS stimulated RAW 264.7 cells. Data were derived from three independent experiments and summarized as mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group. Figure 4. X-ray crystal structures of walrobin B (2).
In conclusion, walrobsins A and B, new limonoids featuring an unprecedented 5-oxatricyclo[5.4.11,4]hendecane ring system, were isolated from the root barks of W. robusta. Their structures were elucidated by HRESIMS, 1D/2D NMR, and
The hypothetical biosynthesis pathways of 1 and 2 are illustrated in Scheme 1. The cedrelone-type limonoid could be Scheme 1. Hypothetical Biosynthetic Pathways of 1 and 2
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DOI: 10.1021/acs.orglett.7b02173 Org. Lett. 2017, 19, 4568−4571
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Organic Letters
(6) An, F. L.; Wang, X. B.; Wang, H.; Li, Z. R.; Yang, M. H.; Luo, J.; Kong, L. Y. Sci. Rep. 2016, 6, 20045. (7) Li, R. J.; Gao, C. Y.; Guo, C.; Zhou, M. M.; Luo, J.; Kong, L. Y. Inflammation 2017, 40, 401−413.
single-crystal X-ray diffraction (Cu Kα). Walrobsin A (1) showed significant anti-inflammatory activity in the LPS induced RAW 264.7 cell line. Our study provides a new structural architecture of natural product that can be used in follow-up studies relevant to the development of antiinflammatory agents.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b02173. Details of isolation and biological experimental procedures; HRESIMS, UV, ECD, and NMR data for compounds 1 and 2 (PDF) Crystallographic data for 1 (CIF) Crystallographic data for 2 (CIF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (L.-Y.K.) *E-mail:
[email protected] (J.L.) ORCID
Ling-Yi Kong: 0000-0001-9712-2618 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support for this study is from the National Natural Science Foundation of China (31470416), the program for New Century Excellent Talents in University (NCET-20131035), the Outstanding Youth Fund of the Basic Research Program of Jiangsu Province (BK20160077), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63), and the Ph.D. Programs Foundation of Ministry of Education of China (20120096130002)
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REFERENCES
(1) (a) Tan, Q. G.; Luo, X. D. Chem. Rev. 2011, 111, 7437−7522. (b) Hill, R. A.; Connolly, J. D. Nat. Prod. Rep. 2017, 34, 90−122. (2) Li, W.; Shen, L.; Bruhn, T.; Pedpradab, P.; Wu, J.; Bringmann, G. Chem. - Eur. J. 2016, 22, 11719−11727. (3) (a) Lv, C.; Yan, X. H.; Tu, Q.; Di, Y. T.; Yuan, C. M.; Fang, X.; Ben-David, Y.; Xia, L.; Gong, J. X.; Shen, Y. M.; Yang, Z.; Hao, X. J. Angew. Chem., Int. Ed. 2016, 55, 7539−7543. (b) Mori, N.; Kitahara, T.; Mori, K.; Watanabe, H. Angew. Chem., Int. Ed. 2015, 54, 14920− 14923. (c) Schuppe, A. W.; Newhouse, T. R. J. Am. Chem. Soc. 2017, 139, 631−634. (d) An, F. L.; Luo, J.; Wang, X. B.; Yang, M. H.; Kong, L. Y. Org. Biomol. Chem. 2016, 14, 1231−1235. (e) An, F. L.; Luo, J.; Li, R. J.; Luo, J. G.; Wang, X. B.; Yang, M. H.; Yang, L.; Yao, H. Q.; Sun, H. B.; Chen, Y. J.; Kong, L. Y. Org. Lett. 2016, 18, 1924−1927. (4) (a) Wang, G. C.; Yu, J. H.; Shen, Y.; Leng, Y.; Zhang, H.; Yue, J. M. J. Nat. Prod. 2016, 79, 899−906. (b) Han, M. L.; Zhang, H.; Yang, S. P.; Yue, J. M. Org. Lett. 2012, 14, 486−489. (c) Yin, S.; Wang, X. N.; Fan, C. Q.; Liao, S. G.; Yue, J. M. Org. Lett. 2007, 9, 2353−2356. (d) Zhou, Z. W.; Yin, S.; Zhang, H. Y.; Fu, Y.; Yang, S. P.; Wang, X. N.; Wu, Y.; Tang, X. C.; Yue, J. M. Org. Lett. 2008, 10, 465−468. (e) Zhang, Y.; An, F. L.; Huang, S. S.; Yang, L.; Gu, Y. C.; Luo, J.; Kong, L. Y. Phytochemistry 2017, 136, 108−118. (5) (a) Nihei, K. I.; Asaka, Y.; Mine, Y.; Yamada, Y.; Iigo, M.; Yanagisawa, T.; Kubo, I. J. Nat. Prod. 2006, 69, 975−977. (b) Nihei, K. I.; Asaka, Y.; Mine, Y.; Kubo, I. J. Nat. Prod. 2005, 68, 244−247. 4571
DOI: 10.1021/acs.orglett.7b02173 Org. Lett. 2017, 19, 4568−4571
