Letter pubs.acs.org/OrgLett
Nicotabin A, a Sesquiterpenoid Derivative from Nicotiana tabacum Tao Feng,† Xue-Mei Li,‡ Jun He,† Hong-Lian Ai,† He-Ping Chen,† Xiao-Nian Li,§ Zheng-Hui Li,*,† and Ji-Kai Liu*,† †
School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan 430074, China Plant Protection and Quarantine Station of Dehong, Dehong 678400, China § State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China ‡
S Supporting Information *
ABSTRACT: Nicotabin A (1), a sesquiterpenoid derivative possessing a fused 5/6/5/5/5 ring system, was isolated from leaves of Nicotiana tabacum. The structure was elucidated by extensive spectroscopic methods and confirmed by single crystal X-ray diffraction. The plausible biosynthetic pathway for 1 was proposed. Compound 1 inhibited nitric oxide production in LPS-activated RAW264.7 macrophages with an IC50 of 22.1 μM. Nicotabin A (1)6 was isolated as colorless crystals (MeOH). Its molecular formula C21H28O7 was determined on the basis of the positive high-resolution ESI mass spectrum at m/z 415.1739 [M + Na]+ (calcd for C21H28O7Na: 415.1727), corresponding to eight degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3446 and 3361 cm−1), carbonyl (1774 cm−1), and olefinic (1647 cm−1) groups. Analyses of the 13C NMR spectrum, with the aid of DEPT and HSQC spectra, unlocked 21 carbon resonances attributable to three methyl, five methylene, six methine, and seven quaternary carbons (Table 1). Among the observed resonances, signals at δC149.9, 148.6, 116.9, 109.0, and 175.2 were assigned to two olefinic bonds and a carbonyl carbon, which occupied three degrees of unsaturation. These data suggest that 1 might be a terpene derivative with a pentacyclic ring system. The gross structure of 1 was established initially by analyses of 2D NMR spectra, in particular with HMBC and 1H−1H COSY data. Starting from signals at δC 148.6 (s, C-11) and 109.0 (t, C-12), a methyl-substituted terminal double bond was established on the basis of an HMBC correlation from δH 1.71 (3H, s, H-13) to C-11, whereas the key correlation from δH 2.51 (1H, m) to C-11 indicated a connection between C-7 and C-11. Analyses of the 1H−1H COSY spectrum revealed a partial structure of −CH2−CH−CH2−CH2−, indicating the connection of C-6/C-7/C-8/C-9. In addition, the signals for both H-6 and H-8 showed HMBC correlations to a quaternary carbon (δC 48.2, s, C-5). These data suggested that C-5, C-6, C7, C-8, and C-9 constructed five-membered carbon ring A (Figure 1). Further analyses of the 1H−1H COSY spectrum revealed another partial structure of CH3 −CH−CH−, corresponding to the connection of C-15/C-10/C-1 (Figure
Nicotiana tabacum, an annually grown herbaceous plant of the Solanaceae family that originated in the tropical Americas, is used as the main raw material for the tobacco industry and has a long history of worldwide cultivation.1,2 The aerial part of N. tabacum has also been used as a folk medicine for sedative, diaphoretic, anesthetic, and emetic purposes in many countries.3 Very few plant species have been subjected to such detailed chemical studies as the tobacco plant (N. tabucum L.), the obvious reasons being its economic importance and the health risks associated with its use.3,4 These studies have led to the identification of more than 2,500 constituents of tobacco to date, besides nicotine, which is the pharmacologically active principle, including terpenoids, steroids, lignans, flavonoids, phenylpropanoids, and biphenyls; in addition as many as 5,400 chemicals have been detected in tobacco and cigarette smoke.3,4 High altitude and extensive sunlight make much of the Yunnan province of China a special geographical environment, where tobacco is widely cultivated and always popular with people in China. As a matter of course, tobacco became a good resource for local chemists searching for new and bioactive natural products. In recent years, a series of chemicals had been isolated from Yunnan tobacco.5 Some of them possessed certain antitobacco mosaic virus (anti-TMV) activities,5c,h as well as cytotoxicities.5f Therefore, the adequate resources and a number of new isolates prompted us to explore the constituents of Yunnan tobacco comprehensively. We investigated the chemical constituents of N. tabacum leaves by a dereplication procedure, which resulted in the isolation of a novel compound, nicotabin A (1). Its structure was elucidated by extensive spectroscopic methods, as well as by single-crystal X-ray diffraction. Compound 1 possessed a fused 5/6/5/5/5 ring system, representing a totally new carbon skeleton of a sesquiterpenoid combined with a proposed C6 unit. Herein, we describe the isolation, structural elucidation, and biological evaluation of 1 and its biogenetic pathway. © 2017 American Chemical Society
Received: August 18, 2017 Published: September 7, 2017 5201
DOI: 10.1021/acs.orglett.7b02559 Org. Lett. 2017, 19, 5201−5203
Letter
Organic Letters Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for Nicotabin A (1) in Methanol-d4 δH
entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 17-OH 18-OH 21-OH
4.24 (1H, d, 4.8) 5.21 (1H, s)
2.28 1.49 2.51 1.82 1.82 2.07
(1H, (1H, (1H, (1H, (1H, (1H,
dd, 13.5, 7.5) m) m) m); 1.52 (1H, m) m); 1.71 (1H, m) qd, 7.1, 4.8)
4.71 1.71 1.82 0.95 5.40
(1H, (3H, (3H, (3H, (1H,
br s); 4.66 (1H, br s) s) s) d, 7.1) s)
4.00 3.61 5.05 5.73 3.84
(1H, (1H, (1H, (1H, (1H,
dd, 7.0, 2.9) m); 3.89 (1H, m) s) s) t, 5.8)
HMBC correlation to C-1. These data suggest that a substituted tetrahydrofuran ring C was established by C-1, C2, C-17, and C-16 (Figure 1). Analyses of 1H−1H COSY data revealed a structural fragment −CH−CH2−, corresponding to C-20/C-21. An HMBC correlation from H-16 to δC 87.1 (d, C20) and HMBC correlations from δH 4.00 (1H, dd, J = 7.0, 2.9 Hz, H-20) to δC 79.2 (s, C-18) and C-17 suggest that another substituted tetrahydrofuran ring D was formed by C-16, C-17, C-18, and C-20 (Figure 1). Thus far, only the carbonyl carbon at δC 175.2 (s, C-19) has not been assigned. In the HMBC spectrum, the proton of a hydroxy group at δH 5.73 (1H, s, OH-18), as well as H-20, showed key correlations to C-19, suggesting that the carbonyl carbon might be connected to C18. This information, together with analyses of mass data, NMR chemical shifts, and IR data at 1774 cm−1, suggested that the γlactone ring E was established by C-2, C-17, C-18, and C-19 (Figure 1). Compound 1 was therefore elucidated as a novel spirocyclic sesquiterpene derivative with an additional C6 moiety that constructed an adjacent 5/5/5 system. Preliminary analyses of ROESY data suggested that rings A and B possessed the same relative configuration as that of 3hydroxysolavetivone.7 In addition, the ROESY correlations of H-16/H-20 suggested that H-16 and H-20 were on the same side. A computational Chem3D structure of 1 indicated that the orientation of OH-17 and OH-18 should be on the same side with that of H-16. However, the ROESY spectrum could not identify the configuration of C-1 and C-2. Fortunately, after many attempts to crystallize 1 using different solvents, a single crystal of 1 was finally obtained from MeOH−H2O (9:1). Subsequent X-ray crystallographic analysis clarified not only the planar structure but also the absolute configuration of 1 (Figure 2).
δC 88.8, d 89.4, s 116.9, d 149.9, s 48.2, s 41.5, t 47.1, d 32.6, t 35.5, t 40.7, d 148.6, s 109.0, t 21.1, q 20.9, q 14.0, q 111.5, d 92.1, s 79.2, s 175.2, s 87.1, d 61.7, t
Figure 1. Key 2D NMR correlations of nicotabin A (1).
1), whereas the HMBC correlation from H-10 to C-5 suggested that the partial structure was connected to ring A by bond C10/C-5. In the HMBC spectrum, a singlet at δH 1.82 (3H, s, H14) for a methyl showed correlations to δC 149.9 (s, C-4) and C-5, suggesting that a methyl-substituted double bond was connected to C-5. Furthermore, two HMBC correlations from δH 4.24 (1H, d, J = 4.8 Hz, H-1) and 5.21 (1H, s, H-3) to δC 89.4 (s, C-2) were observed. These data suggest that C-1, C-2, C-3, C-4, C-5, and C-10 constructed six-membered ring B (Figure 1). The aforementioned data strongly suggest that compound 1 possesses rings A and B (C-1 to C-15) similar to those of 3-hydroxysolavetivone.7 Beyond the partial structures established as previously described, the remaining six carbons were highly oxygenated and responsible for three additional rings, whereas three of them were quaternary carbons, making structural elucidation difficult. In the 1H NMR spectrum, three signals for −OH groups were observed (Table 1). Among them, signals at δH 5.05 (1H, s, OH-17) showed key HMBC correlations to δC 92.1 (s, C-17), 111.5 (d, C-16), and C-2, indicating that C-17 might connect to C-2 and C-16, whereas the proton for a hemiacetal group at δH 5.40 (1H, s, H-16) showed a key
Figure 2. ORTEP diagram of nicotabin A (1).
Although nicotabin A (1) formally displayed a quite different skeleton, it is actually structurally combined by a sesquiterpenoid and a C6 unit. Splitting up the structure suggested that the C6 unit is probably related to the citric acid. Therefore, a hypothetical pathway for 1 is proposed as shown in Scheme 1. First, the main part of 1 should be derived from the known sesquiterpenoid 3-hydroxysolavetivone, a spirocyclic sesquiterpenoid reported previously from the same resource.7 Then, an important proposed intermediate that afforded the C6 unit was produced by the citric acid. This intermediate underwent nucleophilic additions with 3-hydroxysolavetivone to build a γlactone, as well as two furan rings sharing an acetal moiety. A number of natural products including sesquiterpenoids, flavones, phenyl derivatives, and alkaloids from Nicotiana species have been shown to possess certain anti-TMV activities or cytotoxicities.5 Therefore, on the basis of the resource origin of 1, we evaluated for its anti-TMV activity and cytotoxicity. 5202
DOI: 10.1021/acs.orglett.7b02559 Org. Lett. 2017, 19, 5201−5203
Letter
Organic Letters
Nationalities (CZZ17006). The authors thank Analytical & Measuring Centre, South-Central University for Nationalities, for the NMR measurements.
Scheme 1. Proposed Biosynthetic Pathway for 1
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However, there was no activity. In addition, we evaluated for its inhibitory activity against nitric oxide (NO) production in LPSactivated RAW264.7 macrophages. It was found to exhibit inhibitory activity against NO production in LPS-activated RAW264.7 macrophages with an IC50 of 22.1 μM (Positive control MG-132, IC50 = 0.17 μM). In conclusion, nicotabin A (1) isolated from leaves of Nicotiana tabacum has a new carbon skeleton furnishing a fused 5/6/5/5/5 ring system. The spirocyclic centers of C-2 and C-5, as well as multiple highly oxygenated carbons distributed in a rigid ring system, make the structure of nicotabin A (1) noteworthy. In addition, the proposed biosynthesis pathway as well as NO production inhibition makes nicotabin A (1) a good scaffold for further biosynthesis and pharmacological investigations.
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ASSOCIATED CONTENT
NOTE ADDED AFTER ASAP PUBLICATION The stereoconfiguration of C-1 in nicotabin A (compound 1) was depicted incorrectly in Graphical Abstract and Scheme 1. The graphics were corrected on September 18, 2017.
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b02559. General experimental procedures, plant materials, extraction and isolation, bioactivity assay, spectroscopic data (PDF) Crystallographic data for 1 (CIF)
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REFERENCES
(1) The Editorial Committee of the Administration Bureau of Flora of China in Flora of China; Science Press: Beijing, 2005; Vol. 67, p 152. (2) Hu, T. W.; Mao, Z. Tob. Control 2006, 15, 37−41. (3) Rodgman, A.; Perfetti, T. A. The Chemical Components of Tobacco and Tobacco Smoke, 2nd ed.; CRC Press: Boca Raton, FL, 2013. (4) Wahlberg, I.; Enzell, C. R. Nat. Prod. Rep. 1987, 4, 237−276. (5) Examples for recent chemical studies on N. tabacum: (a) Tan, J. L.; Chen, Z. Y.; Yang, G. Y.; Miao, M. M.; Chen, Y. K.; Li, T. F. Heterocycles 2011, 83, 2381−2385. (b) Gao, X. M.; Li, X. S.; Yang, X. Z.; Mu, H. X.; Chen, Y. K.; Yang, G. Y.; Hu, Q. F. Heterocycles 2012, 85, 147−153. (c) Chen, Z. Y.; Tan, J. N.; Yang, G. Y.; Miao, M. M.; Chen, Y. K.; Li, T. F. Phytochem. Lett. 2012, 5, 233−235. (d) Chen, J. X.; Leng, H. Q.; Duan, Y. X.; Zhao, W.; Yang, G. Y.; Guo, Y. D.; Chen, Y. K.; Hu, Q. F. Phytochem. Lett. 2013, 6, 144−147. (e) Li, L.; Shen, Q. P.; Liu, C. B.; Wang, Y.; Yao, J. J.; Zhang, T.; Zhang, F. M.; He, P.; Shi, X. X.; Liu, Z. H.; Miao, M. M.; Yang, G. Y. Phytochem. Lett. 2015, 13, 156−159. (f) Zhou, M.; Zhou, K.; Lou, J.; Wang, Y. D.; Dong, W.; Li, G. P.; Jian, Z. Y.; Du, G.; Yang, H. Y.; Li, X. M.; Hu, Q. F. Phytochem. Lett. 2015, 14, 226−229. (g) Zhang, F. M.; Xia, J. J.; Yang, P. S.; Shen, Q. P.; Liu, C. B.; He, P.; Wang, J. Q.; Liu, Z. H.; Ding, Z. T. Heterocycles 2016, 92, 1857−1863. (h) Shang, S. Z.; Zhao, W.; Tang, J. G.; Xu, X. M.; Sun, H. D.; Pu, J. X.; Liu, Z. H.; Miao, M. M.; Chen, Y. K.; Yang, G. Y. Fitoterapia 2016, 108, 1−4. (i) Yang, P. S.; Zhang, W.; Shen, X. F.; Wang, X. L.; Li, C.; Gong, X. W.; Zheng, X. D.; Zhu, D. L.; Wang, J. Q. Heterocycles 2016, 92, 1462−1467. (6) Physical data for nicotabin A (1). Colorless needles (MeOH), mp 201−203 °C; [α]25D −18.8 (c 0.10, MeOH); IR (KBr) νmax: 3446, 3361, 3242, 2947, 2885, 1774, 1647, 1454, 1407, 1378, 1242, 1209, 1097, 1072, 1044, 1007, 889; Positive ion HRESIMS m/z 415.1739 [M + Na]+ (calcd for C21H28O7Na: 415.1727). (7) Uegaki, R.; Fujimori, T.; Kubo, S.; Kato, K. Phytochemistry 1981, 20, 1567−1568.
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (Z.-H.L.). *E-mail:
[email protected] (J.-K.L.). ORCID
Tao Feng: 0000-0002-1977-9857 Zheng-Hui Li: 0000-0003-1284-0288 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (81561148013 and 81373289), the Key Projects of Technological Innovation of Hubei Province (No. 2016ACA138), and the Fundamental Research Funds for the Central University, South-Central University for 5203
DOI: 10.1021/acs.orglett.7b02559 Org. Lett. 2017, 19, 5201−5203
